Molecular tongs containing amino acid mimetic fragments: New inhibitors of wild-type and mutated HIV-1 protease dimerization

Article abstract of DOI:10.1021/jm060576k

We have designed, synthesized, and evaluated the inhibitory activity and metabolic stability of new peptidomimetic molecular tongs based on a naphthalene scaffold for inhibiting HIV-1 protease dimerization. Peptidomimetic motifs were inserted into one pep

Full text of DOI:10.1021/jm060576k

J. Med. Chem. 2006, 49, 4657-4664
4657
Molecular Tongs Containing Amino Acid Mimetic Fragments: New Inhibitors of Wild-Type
and Mutated HIV-1 Protease Dimerization
,†
Ludovic Bannwarth,#,† Albane Kessler, Ste´phanie Pe`the, Bruno Collinet,# Na¨ıma Merabet, Nicole Boggetto,# Sames Sicsic,
Miche`le Reboud-Ravaux,# and Sandrine Ongeri*,
UniVersite´ de ParissSud XI, IFR 141, Biocis, UMR-CNRS 8076, Faculte´ de Pharmacie, 5 Rue J. B. Cle´ment, F-92296 Chaˆtenay-Malabry
Cedex, France, and Laboratoire d’Enzymologie Mole´culaire et Fonctionnelle, FRE 2852, Institut Jacques Monod, CNRSsUniVersite´ Paris VI,
2 Place Jussieu, F-75251 Paris Cedex 05, France
ReceiVed May 15, 2006
We have designed, synthesized, and evaluated the inhibitory activity and metabolic stability of new
peptidomimetic molecular tongs based on a naphthalene scaffold for inhibiting HIV-1 protease dimerization.
Peptidomimetic motifs were inserted into one peptidic strand to make it resistant to proteolysis. The peptidic
character of the molecular tongs can be decreased without changing the way they inhibit dimerization.
Mutated HIV-1 proteases are also vulnerable to dimerization inhibitors, and the multimutated protease
ANAM-11 is twice as sensitive to the inhibitor compared to wild-type protease. Thus, the metabolic stability
of antidimeric molecular tongs can be increased without compromising their ability to inhibit wild-type and
mutated HIV-1 proteases in vitro.
Introduction
the efficiency of inhibitors.18,19 The interface peptides can also
be cross-linked with flexible,20 semirigid,21 or rigid spacers.22,23
A bicyclic guanidinium has also been successfully introduced
between a peptidic chain and a hydrophobic group.24 Our con-
tribution to the antidimer strategy was the synthesis of molecular
tongs based on a conformationally constrained scaffold attached
to two peptidic strands through two carboxypropyl links.
Inhibitors that use constrained scaffolds of naphthalene (com-
pound 1, Figure 1) or quinoline led to efficient antidimers (Kid
) 80 nM).22,23 The asymmetrical inhibitors (two different
peptidic arms) are more efficient than symmetrical ones (two
identical arms).23 This report describes the synthesis, enzyme
inhibitory activity against wild-type and mutated HIV-1 pro-
teases, and metabolic stability of the new asymmetrical molec-
ular tongs 2-6 containing amino acid mimetic fragments in
one strand with the other peptidic strand being VLV-OMe
(Figure 2). VLV-OMe is used because our previous studies
showed that this sequence led to the best dimerization inhibi-
tor.22,23 These new molecular tongs 2-6 were designed to have
less peptidic character and consequently to be less susceptible
to proteases in vivo. The peptidic character of a molecule can
be decreased by replacing one or more amino acids with groups
that mimic their hydrogen-bonding properties. The amino acids
of the molecular tongs that inhibited PR are believed to be
involved in hydrogen bond interactions with the N- and
C-termini of one PR monomer, favoring the formation of a
â-sheet structure and mimicking the four-strand â-sheet structure
of the PR dimer (Figure 1).22,23 Consequently, the groups
replacing amino acids should ideally provide the same array of
hydrogen-bonding groups as one edge of a peptidic â-strand.
In literature, Nowick and Sanderson have introduced a 5-amino-
2-methoxybenzamide group25 and a 3-amino-6-methylpyridin-
2(1H)-one group26 to potentially form hydrogen bonds such as
those of amino acids. We therefore designed new molecular
tongs with two amino acids of one strand replaced by a
peptidomimetic motif. The peptidomimetic Nowick motif was
inserted into molecular tongs 2 and 6, and the peptidomimetic
Sanderson motif was placed in molecular tongs 3 and 4 (Figure
2). In molecule 5, the peptidomimetic Sanderson motif was
simplified by omitting the methyl group in position 6 of the
HIV-1 protease (PR) is an important target for anti-AIDS
drugs because its inhibition results in the production of
uninfectious virus.1,2 The PR inhibitors used in highly active
antiretroviral therapy (HAART) act in conjunction with two
inhibitors of reverse transcriptase to reduce the virus load and
increase the number of CD4 lymphocytes in AIDS patients. The
most recent inhibitors, like tripanavir, have much less peptidic
character than the first commercially available inhibitor
saquinavir.3-6 However, the development of cross-resistance to
protease inhibitors is a serious limitation in long-term treatment
of AIDS patients.7,8 Several mutations located within or outside
the active site of PR have been observed and shown to lower
the affinity of the enzyme for inhibitors while not interfering
with its enzymatic profile. Although new peptidomimetic and
nonpeptidomimetic PR inhibitors are being developed by the
pharmaceutical industry to overcome this resistance, alternative
strategies for controlling HIV replication by inhibiting PR are
required. One possibility is to design inhibitors that prevent the
dimerization of PR or favor the dissociation of the dimer because
the enzyme is a homodimer. Protein-protein interactions in the
dimer include the antiparallel â-sheet formed by interdigitation
of the N- (residues 1-4) and C- (residues 96-99) terminal
strands of monomers (Figure 1), which contributes close to 75%
of the total Gibbs free energy of dimerization.9,10 This antipar-
allel â-sheet is an attractive target for overcoming resistance to
clinical drugs because its amino acid residues are highly
conserved in most HIV-1 and HIV-2 isolates.11-13 Blocking the
formation of the homodimer or disrupting it is an effective way
of inhibiting protease activity.14 Thus, all known inhibitors of
PR dimerization developed by rational design target this region.
C- and N-terminal mimetic peptides were the first dimerization
inhibitors to be developed.15-17 Suitable variation of the nature
of the peptidic sequence and addition of a lipophilic group to
the N-end favor targeting of the antiparallel â-sheet and improve
* To whom correspondence should be addressed. Phone: 33(0)-
146835743. Fax: 33(0)146835740. E-mail: sandrine.ongeri@cep.u-psud.fr.
# CNRSsUniversite´ de Paris VI.
† These authors contributed equally.
Universite´ de ParissSud XI.
10.1021/jm060576k CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/06/2006

4658 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Bannwarth et al.
Figure 1. (A) Diagram of the HIV-1 protease â-sheet. (B) Putative complex formed between HIV-1 monomer and compound 1.
Figure 2. Molecular tongs 2-6.
3-aminopyridin-2(1H)-one (Figure 2). We also identified the
most suitable position of the peptidomimetic unit within the
strand for active molecular tongs. Two cases were examined:
first, the mimic unit was attached directly to the carboxypropyl
link (molecules 2, 3, and 4); second, it was linked through a
Val unit (compounds 5 and 6). The results of building several
possible complexes using the Sybyl 7.0 molecular modeling
tools indicated that forming the postulated â-sheet structure
between the N- and C-termini of one PR monomer and molecule
5 or molecule 6 (Figure 3) is energetically possible, since the
correct array of alternating 10-member and 14-member rings
of hydrogen bonds is still present after minimization. Con-
versely, no stable â-sheet structure was formed with molecules
2-4. We synthesized and tested molecules 2-6 to confirm these
molecular models.
scribed by Nowick (Scheme 1).25e The protected benzoic acid
hydrazide 7 was acylated and the hydrazide 8 deprotected. The
resulting compound 9 was coupled to Z-valine using HBTU
and HOBT as coupling agents, with a good yield to give the
Z-protected compound 10, which was deprotected by hydro-
genolysis to give 11 (Scheme 1). The peptidomimetic strand
14 was obtained by condensation of N-protected (3-amino-6-
methyl-2-oxopyridin-1(2H)-yl)acetic acid 1226 with the dipeptide
Ile-Thr-NH2 using HBTU and by deprotection of the resulting
compound 13 (Scheme 2). Strand 17 containing two peptido-
mimetic units was obtained by condensation of the N-protected
Sanderson motif 1226 with the t-Bu ester 1526 of this same motif
in the presence of HBTU and by the N-deprotection of the
resulting compound 16 (Scheme 2). The peptidomimetic 22 was
synthesized from commercially available 3-nitropyridin-2(1H)-
one 18, which was successively N-alkylated by bromoacetamide,
hydrogenated into compound 20, condensed with Z-Val in the
presence of EDCI and HOBT, and N-deprotected by hydro-
genolysis (Scheme 3). The molecular tong 2 was synthesized
Chemistry
The peptidomimetic strand 11 was obtained in four steps from
the Boc-protected 5-amino-2-methoxybenzohydrazide 7 de-

Molecular Tongs
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4659
Scheme 2. Synthesis of Peptidomimetic Containing Strands 14
and 17a
a (a) H-IT-NH2, HBTU, Et3N, DMF, 48 h, room temp; (b) H2 10% Pd/
C, MeOH, 20 h, room temp; (c) HBTU, Et3N, DMF, 24 h, room temp; (d)
H2, 10% Pd/C, MeOH, 24 h, room temp.
Scheme 3. Synthesis of Peptidomimetic Containing Strand 22a
Figure 3. Putative complexes between HIV-1 protease monomer and
molecular tongs 5 and 6 based on molecular modeling.
a (a) BrCH2CONH2, K2CO3, THF, 16 h, 60 °C; (b) H2, 10% Pd/C,
MeOH, 4 h, room temp; (c) Z-V-OH, EDCI, HOBT, NMM, DMF, 48 h,
room temp; (d) H2, 10% Pd/C, MeOH, 20 h, room temp.
Scheme 1. Synthesis of Peptidomimetic Containing Strand 11a
Scheme 4. Synthesis of Molecular Tongs 2-6
a (a) Ac2O, THF, 40 min, 60 °C; (b) TFA, CH2Cl2, 3 h, room temp; (c)
Z-V-OH, HBTU, HOBT, DIPEA, DMF, 28 h, room temp; (d) H2, 10%
Pd/C, HCl/MeOH, DMF, room temp, 3 h.
by condensing 2323 with peptidomimetic 25, obtained by
N-deprotection of Nowick derivative 24,25e followed by hydro-
genation of the nitro group of the intermediate 26 (Scheme 4).
Finally, the molecular tongs 3-6 were synthesized directly, in
the presence of HBTU, by condensation of 2323 with peptido-
mimetic motifs (unprotected 17) or with strands containing
peptidomimetic motifs (11, 14, or 22) (Scheme 4).
a (a) 3.5 N HCl/MeOH, 24 h, room temp; (b) HBTU, HOBt, Et3N, DMF,
24 h, room temp; (c) H2, 10% Pd/C, MeOH, 24 h, room temp; (d) HBTU,
Et3N or DIPEA, X ) 11, 14, 17, or 22, DMF, 24-48 h, room temp.
Biological Results and Discussion
(100 mM sodium acetate, 1 mM EDTA, 1 M NaCl, 3% v/v
DMSO) prevented its study at concentrations higher than 5 µM
(28% inhibition). The mechanism of inhibition of PR by other
compounds was assessed using Zhang-Poorman kinetic analysis
in which plots of [E]0/xVi vs xVi, with xVi ) kexp[S]0, are
constructed16 in order to discriminate between inhibition of
The inhibitory activities of compounds 2-6 were assayed
against recombinant wild-type PR at pH 4.7 and 30 °C using a
fluorometric assay.22,23 Compound 5 was also assayed against
six recombinant mutated proteases. The poor solubility of
compound 2 in the experimental conditions for activity assays

4660 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Bannwarth et al.
Table 1. In Vitro Inhibition of Wild-Type and Mutated HIV-1
Proteases by Molecular Tongs (30 °C and pH 4.7) and Comparison with
Inhibition by Therapeutical Antiproteasesa
demethylated Sanderson peptidomimetic units can be used to
replace amino acids in the structures of the previously described
molecular tongs. The results are in agreement with molecular
modeling, indicating that the antidimer property depends on the
position of the mimic unit in the peptidomimetic strand. They
suggest that the flexibility of valine is required for the formation
of the hydrogen bonds in a four-strand antiparallel â-sheet,
implicating the N- and C-termini of the PR monomer. It was
essential to evaluate the action of our dimerization inhibitors
against the drug-resistant PR mutants we have produced. Some
active-site mutations are known to preferentially affect the
therapeutical antiproteases that bind to the active site: D30N
(nelfinavir),27 V82A (ritonavir),28 I50V (amprenavir).29 The
double-point mutation G48V/L90M affects saquinavir.30 Muta-
tion L90M is located at the terminal region, close to the
dimerization interface. Two multidrug-resistant mutants were
also used. The mutant MDR-HM31 contains six (L10I/M46I/
I54V/V82A/I84V/L90M) amino acid mutations located within
and outside the active site of the enzyme, while ANAM-1132
has 11 mutations (L10I/M36I/S37D/M46I/R57K/L63P/A71V/
G73S/I84V/L90M/I93L). Remarkably, the new pseudopeptidic
molecular tong 5 behaved as an antidimer inhibitor against single
and double mutated proteases and against the two multidrug-
resistant proteases (Table 1). It inhibited mutants D30N and
G48V/L90M as actively as wild-type PR (Kid,mut/Kid,WT ≈ 1.2)
and was even more active against some mutated proteases than
against wild-type PR (Kid,mut/Kid,WT ) 0.6 and 0.25 for V82A
and I50V, respectively). At the opposite, important decreases
in in vitro potency against the same mutated proteases were
observed for active site inhibitors (Table 1) whose activities
were reduced 15-fold (ritonavir/V82A),28 6-fold (nelfinavir/
D30N),27 83-fold (amprenavir/I50V),29 or even 1000-fold
(saquinavir/ G48V/L90M).30 Similar results were obtained with
multidrug-resistant proteases. For example, the affinity of
ANAM-11 protease for compound 5 was 2-fold greater (Kid )
200 nM) than that of wild-type PR (Kid ) 400 nM). In contrast,
the affinities of this mutated protease for indinavir, nelfinavir,
saquinavir, and ritonavir were about 2030, 2840, 4200, and
78 000 times lower than that of wild-type PR.32 This demon-
strates that the peptidomimetic molecular tong 5 still acts as an
efficient inhibitor of the dimerization of mutated proteases. It
inhibits in vitro 87-fold more actively ANAM-11 protease than
does ritonavir. The initial hypothesis suggesting that inhibitors
of PR dimerization are poorly sensitive to mutations is con-
firmed, as previously proposed by Chmielewski and collabora-
tors who developed dimerization inhibitors of HIV-1 PR by
cross-linking interface peptides with flexible spacers.33 Finally,
the stability of the peptide (1) and peptidomimetic (5 and 6)
molecular tongs in RPMI culture medium containing 20% fetal
calf serum was studied. The half-life of compound 1 was about
28 h and that of 5 about 41 h; no significant degradation of
compound 6 was observed after 33 h of incubation. The results
suggest that the metabolic stability has been improved by
inserting peptidomimetics into one arm of the molecular tong.
wild-type
(WT) or
mutated
Kic
Kid
Kid,mut
/
clinical
Ki,mut/
compd
proteaseb
(µM)c (µM)d Kid,WT antiprotease Ki,WT
1
2
3
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
WT
WT
WT
WT
WT
D30N
I50V
V82A
G48V/L90M
MDR-HM
MDR-HM
MDR-HM
MDR-HM
MDR-HM
ANAM-11
ANAM-11
ANAM-11
ANAM-11
WT
0.5622
Ie
9.7
8.4
Ie
0.4
0.5
0.1
0.24
0.5
4.8
4.8
4.8
4.8
4.8
0.2
0.2
0.2
0.2
0.2
1.2
0.25 amprenavir29
0.6
ritonavir28
1.25 saquinavir30
nelfinavir27
6
83
15
1000
210
85
12
nelfinavir31
amprenavir31
indinavir31
saquinavir31
ritonavir31
12
12
12
12
200
2000
1500
2840
2030
4200
78000
0.5
nelfinavir32
indinavir32
saquinavir32
ritonavir32
0.5
0.5
0.5
a Standard errors of initial rates are less than 5%. b Mutations in the active
site are in bold. c Competitive active site inhibition. d Dimerization inhibi-
tion. e 28% inhibition at 5 µM.
Figure 4. Plots of [E]0/xVi vs xVi for the hydrolysis of the fluorogenic
substrate DABCYL-γ-abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS
by HIV-1 PR WT at pH 4.7 and 30 °C in the absence (b) and presence
of 1 µM (9) and 2 µM (2) compound 5.
dimerization alone (parallel lines), competitive inhibition (altered
slopes and unaltered y-axis intercept), and mixed inhibition
(altered slopes and altered y-axis intercepts). In pure dimerization
inhibition, the inhibitor binds to the interface region, whereas a
competitive inhibitor binds to the active site and a mixed
inhibitor may bind to the active site as well as to the interfacial
region. The results are summarized in Table 1. Convergent lines
were obtained for compounds 3 and 4, demonstrating that they
act as active-site competitive inhibitors; parallel lines were
obtained with compounds 5 and 6, which is consistent with pure
dimerization inhibition (Figure 4). This result indicates that
replacing the tripeptide VLV (compound 1) with a pseudopep-
tidic strand composed of valine linked to the demethylated
Sanderson motif (compound 5) or the Nowick motif (compound
6) results in an antidimer inhibitor that is still fully active against
wild-type PR (Kid ) 0.4 µM for 5 and Kid ) 0.2 µM for 6,
compared to Kid ) 0.56 µM for 1). Thus, the Nowick and
Conclusion
Our previous finding that molecular tongs are inhibitors of
the PR interface22,23 prompted us to create proteolysis-resistant
inhibitors by replacing the peptidics strands with peptidomi-
metics. We replaced two amino acids in one strand of the
naphthalene-based molecular tongs by a peptidomimetic to
produce good inhibitors of PR dimerization. The resistance to
hydrolysis of these new molecular tongs is increased without
modifying their inhibitory potency. One of these dimerization
inhibitors was also evaluated against proteases with 1, 2, 6, and

Molecular Tongs
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4661
TFA (1.2 mL, 15.5 mmol) was then added dropwise at room
temperature. The reaction mixture was stirred under argon atmo-
sphere at room temperature for 3 h. Solvent was evaporated under
reduced pressure to yield a pale-yellow solid (210 mg, quantitative
yield). This crude product was used without any further purification
11 point mutations; it was as active (and in some cases more
active) on mutated proteases as on wild-type PR. This suggests
that peptidomimetic molecular tongs are candidates to success-
fully overcome the resistances presently encountered with
classical protease inhibitors. Our results also provide new
information on the mechanism of action of molecular tongs.
We postulated that the molecular tongs inhibit the dimerization
of PR via the formation of a four-strand antiparallel â-sheet
with one monomer of the protease.22,23 This study provides new
arguments for this because it confirms the molecular modeling
study. In contrast to molecules 2-4, hydrogen bonds can be
formed in molecular tongs 5 and 6 between the carbonyl group
of each carboxypropyl group and the N-terminal NH of the
Leu-5 and the NH of the C-terminal Phe-99, respectively
(Figures 1 and 3).
1
in the course of the synthesis. H NMR (MeOD-d4) δ 8.2 (d, J )
2.4 Hz, 1H), 7.7 (dd, J ) 9.0 Hz, 2.4 Hz, 1H), 7.2 (d, J ) 9.0 Hz,
1H), 4.0 (s, 3H), 2.1 (s, 3H); 13C NMR (MeOD-d4) δ 175.4, 170.1,
166.4, 154.1, 131.8, 125.1, 122.8, 111.7, 53.2, 22.0.
{1-[N′-(5-Acetylamino-2-methoxybenzoyl)hydrazinocarbonyl]-
2-methylpropyl}carbamic Acid Benzyl Ester 10. Compound 9
(209 mg, 0.62 mmol) and Z-V-OH (156 mg, 0.62 mmol) were
dissolved in DMF (20 mL). DIPEA (0.6 mL, 3.1 mmol) was then
added. HBTU (259 mg, 0.68 mmol) and HOBt (93 mg, 0.68 mmol)
were added to the reaction flask. The reaction mixture was stirred
under argon atmosphere atmosphere at room temperature for 24 h.
DMF was evaporated under reduced pressure and the residue was
dissolved in EtOAc (150 mL), washed with aqueous citric acid 5%
(50 mL), aqueous Na2CO3 10% (50 mL), and brine (50 mL). The
organic phase was dried over MgSO4 and concentrated under
reduced pressure. The crude solid was triturated in EtOAc, filtered
and dried under vacuum to yield a pale yellow powder: mp 227-
229°C (201 mg, 71% yield); 1H NMR (DMSO-d6) δ 10.3 (bs, 1H),
9.9 (bs, 2H), 7.9 (d, J ) 2.6 Hz, 1H), 7.8 (dd, J ) 9 Hz, 2.6 Hz,
1H), 7.3 (m, 5H), 7.1 (d, J ) 9.0 Hz, 1H), 5.0 (s, 2H), 4.0 (m,
1H), 3.8 (s, 3H), 2.0 (s, 3H), 1.9 (m, 1H), 0.9 (m, 6H); 13C NMR
(DMSO-d6) δ 169.3, 167.8, 155.9, 152.5, 149.4, 132.7, 132.5, 128.2,
127.6, 127.5, 124.8, 123.2, 121.2, 112.3, 65.3, 58.6, 56.1, 30.4,
23.6, 19.0, 18.1. Anal. (C23H28N4O6‚0.4H2O) C, H, N.
N-{3-[N′-(2-Amino-3-methylbutyryl)hydrazinocarbonyl]-4-
methoxyphenyl}acetamide 11. To a solution of 10 (140 mg, 0.62
mmol) in DMF (10 mL) was added Pd/C (28 mg, 20% mass). The
reaction flask was purged three times with hydrogen, and stirring
was maintained under hydrogen atmosphere at room temperature
for 3 h. The mixture was filtered through a mixture of Celite and
silica, and the cake was washed thoroughly with DMF (200 mL).
The filtrate was evaporated to dryness, and the residue was rinsed
with MeOH (20 mL) to yield the crude product as a white powder:
mp 193-195 °C (67 mg, 67% yield); 1H NMR (DMSO-d6) δ 10.1
(s, 1H), 10.0 (s, 1H), 9.8 (s, 1H), 8.3 (bs, 2H), 7.9 (d, J ) 2.7 Hz,
1H), 7.7 (dd, J ) 9.0 Hz, 2.7 Hz, 1H), 7.1 (d, J ) 9.0 Hz, 1H), 3.8
(s, 3H), 3.6 (d, J ) 5.7 Hz, 1H), 2.1 (m, 1H), 2.0 (s, 3H), 1.0 (d,
J ) 6.8 Hz, 6H); 13C NMR (DMSO-d6) δ 168.4, 166.4, 163.8,
152.9, 133.0, 121.5, 112.8, 56.6, 34.4, 30.2, 24.1, 18.5, 18.2
(1-{[1-(1-Carbamoyl-2-hydroxypropylcarbamoyl)-2-methyl-
butylcarbamoyl]methyl}-6-methyl-2-oxo-1,2-dihydropyridin-3-
yl)carbamic Acid Benzyl Ester 13. To a solution of 12 (150 mg,
0.47 mmol) in DMF (3 mL) was added HBTU (230 mg, 0.52 mmol)
and Et3N (0.05 mL, 0.52 mmol). The mixture was stirred for 20
min at room temperature. I-T-NH2 (110 mg, 0.47 mmol) was then
added, and the mixture was stirred at room temperature for 48 h.
DMF was evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 (50 mL), washed with H2O (10 mL), aqueous
1 N HCl (10 mL), and 20% aqueous NaOH (10 mL). The organic
phase was dried over MgSO4, filtrated, and concentrated under
reduced pressure. Trituration of the residue in Et2O (20 mL) and
filtration afforded the pure product as an off-white powder: mp
249-251 °C (150 mg, 60% yield); 1H NMR (DMSO-d6) δ 8.4 (d,
J ) 8.5 Hz, 1H), 8.3 (s, 1H), 7.7 (m, 2H), 7.3 (m, 5H), 7.0 (s, 2H),
6.1 (d, J ) 9.4 Hz, 1H), 5.1 (s, 2H), 4.9 (m, 2H), 4.7 (d, J ) 8.9
Hz, 1H), 4.2 (t, J ) 7.9 Hz, 1H), 4.1 (m, 1H), 4.0 (m, 1H), 2.2 (s,
3H), 1.8 (m, 1H), 1.4 (m, 1H), 1.1 (m, 1H), 1.0 (d, J ) 3.4 Hz,
3H), 0.8 (m, 6H); 13C NMR (DMSO-d6) δ 171.0, 170.0, 167.0,
166.0, 157.5, 140.0, 136.5, 129.0, 125.0, 123.0, 106.5, 67.0, 66.0,
59.0, 58.0, 48.0, 37.0, 25.0, 21.0, 16.0, 12.0. Anal. (C26H35N5O7)
C, H, N
Further studies on inhibitors of HIV-1 protease dimerization
containing peptidomimetics will focus on the synthesis of
molecular tongs with quinoline as the constrained scaffold, since
quinoline-based tongs with peptidic strands are also interfacial
inhibitors of the HIV-1 PR interface,23 and on the measurement
of their antiviral efficacy in cell culture assays.
Experimental Section
Synthesis. The usual solvents were purchased from commercial
sources. Dimethylformamide (DMF) was distilled on CaSO4.
Tetrahydrofuran (THF) was distilled on sodium/benzophenone, and
acetonitrile was distilled on CaCl2. TLC was performed on silica
gel 60F-250 (0.26 mm thickness) plates. The plates were visualized
with UV light (254 nm) or with a 3.5% solution of phosphomo-
lybdic acid in ethanol or with a solution of ninhydrin in ethanol.
Liquid chromatography was performed on Merck 60 silica gel
(230-400 mesh). 2-Hydroxy-3-nitropyridine 18, 2-bromoacetamide,
protected amino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetrameth-
yluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazol
(HOBT), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI)
were purchased from commercial sources. H-Val-Leu-Val-OMe,
H-Ile-Thr-NH2, 4-[7-(3-carboxypropoxy)naphthalen-2-yloxy]butyryl-
Val-Leu-Val-OMe 23,23 the Boc protected 5-amino-2-methoxy-
benzohydrazide 7,25e the Boc protected 2-methoxy-5-nitrobenzo-
hydrazide 24,25e 3-benzyloxycarbonylamino-6-methyl-1-methylene-
carboxy-2-pyridinone 12,26 and 3-amino-6-methyl-1-tert-butyl-
methylenecarboxy-2-pyridinone 1526 were prepared according to
published methods. Melting points were determined on a Kofler
melting point apparatus. NMR spectra were performed 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 the solvent. Chemical shifts δ are in ppm, and the
following abbreviations are used: singlet (s), doublet (d), doublet
doublet (dd), triplet (t), quadruplet (q), multiplet (m), broad doublet
(bd), broad triplet (bt), and broad singlet (bs). Mass spectra were
obtained using a Bruker Esquire electrospray ionization apparatus.
Element analyses (C, H, N) were performed on a Perkin-Elmer
CHN analyzer, model 2400, at the Microanalyses Service of the
Faculty of Pharmacy at Chaˆtenay-Malabry (France).
N′-(5-Acetylamino-2-methoxybenzoyl)hydrazinecarboxylic Acid
tert-Butyl Ester 8. Compound 7 (1.1 g, 3.9 mmol) was dissolved
in dry THF (15 mL), and the solution was heated to 60 °C. Acetic
anhydride (1.3 mL, 13.7 mmol) was added dropwise to this solution.
The reaction mixture was stirred for 40 min at 60 °C. THF was
evaporated under reduced pressure, and the crude product was dried
under vacuum to yield a white powder: mp 211-213 °C (1.24 g,
99% yield). This crude product was used without any further
purification in the course of the synthesis. 1H NMR (DMSO-d6) δ
9.9 (s, 1H), 9.6 (s, 1H), 8.9 (bs, 1H), 7.9 (s, 1H), 7.7 (d, J ) 8.4
Hz, 1H), 7.05 (d, J ) 8.4 Hz, 1H), 3.85 (s, 3H), 2.0 (s, 3H), 1.40
(s, 9H); 13C NMR (DMSO-d6) δ 168.3, 165.3, 155.6, 153.1, 132.9,
123.6, 121.7, 112.6, 79.4, 53.3, 28.4, 24.1
2-[2-(3-Amino-6-methyl-2-oxopyridin-1(2H)-yl)acetylamino]-
3-methylpentanoic Acid (1-Carbamoyl-2-hydroxypropyl)amide
14. To a solution of 13 (100 mg, 0.19 mmol) in MeOH (5 mL)
was added Pd/C (50 mg, 50% mass). The reaction flask was purged
three times with hydrogen, and stirring was maintained under
N-(3-Hydrazinocarbonyl-4-methoxyphenyl)acetamide 9. Com-
pound 8 (200 mg, 0.62 mmol) was suspended in CH2Cl2 (20 mL).

4662 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Bannwarth et al.
hydrogen atmosphere at room temperature for 24 h. The mixture
was filtered through a mixture of Celite and silica, and the cake
was washed thoroughly with MeOH (100 mL). The filtrate was
evaporated to dryness to yield a colorless oil. Trituration in Et2O
(20 mL) yielded an off-white powder: mp 104-106 °C (53 mg,
70% yield); 1H NMR (DMSO-d6) δ 8.4-8.2 (m, 3H), 7.7 (m, 2H),
7.0 (s, 2H), 6.0 (d, J ) 9.4 Hz, 1H), 4.8-4.7 (m, 3H), 4.2 (t, J )
8.4 Hz, 1H), 4.1 (m, 1H), 4.0 (m, 1H), 2.2 (s, 3H), 1.8 (m, 1H),
Hz, 1H), 6.0 (t, J ) 6.8 Hz, 1H), 5.0 (bs, 2H); 13C NMR (DMSO-
d6) δ 169.2, 157.5, 138.5, 126.1, 110.7, 105.9.
[1-(1-Carbamoylmethyl-2-oxo-1,2-dihydropyridin-3-ylcar-
bamoyl)-2-methylpropyl]carbamic Acid Benzyl Ester 21. To a
solution of Z-V-OH (75 mg, 0.3 mmol) in dry DMF (6 mL) were
added HOBt (40 mg, 0.3 mmol), EDCI (57 mg, 0.3 mmol), and
N-methylmorpholine (33 µL, 0.3 mmol). The solution was stirred
for 15 min, and then 2-(3-amino-2-oxopyridin-1(2H)-yl)acetamide
20 (50 mg, 0.3 mmol) was added. Stirring was maintained at room
temperature for 48 h. The solvent was evaporated under reduced
pressure, and the residue was purified by flash chromatography on
silica gel, eluting with CH2Cl2/MeOH, 95/5. The pure product was
obtained as a crystalline white solid: mp 217-219 °C (67 mg,
1.4 (m, 1H), 1.1 (m, 1H), 1.0 (d, J ) 3.4 Hz, 3H), 0.8 (m, 6H); 13
C
NMR (DMSO-d6) δ 171.5, 170.0, 167.0, 158.0, 140.0, 126.0, 123.0,
106.0, 67.0, 58.0, 57.0, 47.0, 38.0, 25.0, 20.0, 15.0, 11.0. Anal.
(C18H29N5O5) C, H, N
{3-[2-(3-Benzyloxycarbonylamino-6-methyl-2-oxopyridin-
1(2H)-yl)acetylamino]-6-methyl-2-oxopyridin-1(2H)-yl}acetic Acid
tert-Butyl Ester 16. To a solution of 12 (100 mg, 0.36 mmol) in
DMF (3 mL) was added HBTU (153 mg, 0.40 mmol) and Et3N
(0.14 mL, 0.40 mmol). The mixture was stirred for 20 min at room
temperature. Amine 15 (86 mg, 0.36 mmol) was then added, and
the mixture was stirred at room temperature for an additional 24 h.
DMF was evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 (50 mL), washed with H2O (20 mL), aqueous
1 N HCl (20 mL) and 20% aqueous NaOH (20 mL). The organic
phase was dried over MgSO4, filtered, and concentrated under
reduced pressure. Trituration of the residue in Et2O (20 mL) and
filtration afforded the pure product as an off-white powder: mp
1
56% yield); H NMR (DMSO-d6) δ 9.1 (s, 1H), 8.1 (dd, J ) 1.6
Hz, J ) 7.4 Hz, 1H), 7.7 (d, J ) 8.3 Hz, 1H), 7.5 (bs, 1H), 7.3-
7.2 (m, 6H), 7.1 (bs, 1H), 6.2 (t, J ) 7.1 Hz, 1H), 4.9 (s, 2H), 4.5
(s, 2H), 4.0 (dd, J ) 6.8, 8.0 Hz, 1H), 2.1-1.9 (m, 1H), 0.8 (d, J
) 6.7 Hz, 6H); 13C NMR (DMSO-d6) δ 171.3, 168.8, 157.1, 137.2,
128.7, 128.2, 128.1, 128.0, 127.8, 123.1, 122.3, 105.1, 65.9, 61.6,
51.6, 19.4, 18.3. Anal. (C20H24N4O5 ) C, H, N.
2-Amino-N-(1-carbamoylmethyl-2-oxo-1,2-dihydropyridin-3-
yl)-3-methylbutyramide 22. To a solution of 21 (250 mg, 0.6
mmol) in MeOH (15 mL) was added Pd/C (75 mg, 30% mass).
The reaction flask was flushed three times with hydrogen, and
stirring was maintained under hydrogen atmosphere at room
temperature for 20 h. The mixture was filtered through a mixture
of Celite and silica, and the cake was washed thoroughly with
MeOH (200 mL). The filtrate was evaporated to dryness to yield
1
237-239 °C (58 mg, 30% yield); H NMR (DMSO-d6) δ 8.3 (s,
1H), 8.7 (s, 1H), 8.1 (d, J ) 7.8 Hz, 1H), 7.8 (d, J ) 7.8 Hz, 1H),
7.4-7.3 (m, 5H), 6.2 (d, J ) 7.9 Hz, 2H), 5.2 (s, 2H), 5.0 (s, 2H),
4.8 (s, 2H), 2.2 (s, 6H), 1.4 (s, 9H); 13C NMR (DMSO-d6) δ 168.0,
167.0, 158.1, 154.0, 140.5, 137.0, 128.5-127.0, 126.0, 123.0, 124.1,
106.5, 82.0, 66.5, 48.5, 47.0, 28.3, 20.0. Anal. (C28H32N4O7‚
0.15H2O) C, H, N
1
a white solid: mp 177-179 °C (110 mg, 66% yield); H NMR
(DMSO-d6) δ 10.4-10.3 (m, 1H), 8.4 (dd, J ) 1.8, 7.4 Hz, 1H),
7.7 (bs, 1H), 7.3 (dd, J ) 1.8, 6.8 Hz, 1H), 7.2 (bs, 1H), 6.3 (t, J
) 7.1 Hz, 1H), 4.6 (s, 2H), 4.1 (dd, J ) 4.9, 10.1 Hz, 1H), 3.2 (d,
2H, J ) 4.8 Hz), 2.3-2.2 (m, 1H), 0.9 (d, 6H, J ) 6.9 Hz); 13C
NMR (DMSO-d6) δ 174.3, 168.7, 157.1, 143.4, 121.4, 109.4, 105.1,
60.6, 51.6, 31.1, 19.7, 16.6. Anal. (C12H18N4O3‚0.7H2O) C, H, N
2-Methoxy-5-nitrobenzoic Acid Hydrazide 25. A solution of
24 (1 g, 3.2 mmol) in MeOH/HCl (3.5 N) was stirred for 24 h at
room temperature. After evaporation of the solvent, the desired
hydrochloride salt was obtained as a white powder: mp 201-203
°C (688 mg, 87% yield); 1H NMR (DMSO-d6) δ 11.0 (s, 1H), 8.5
(m, 3H), 7.5 (d, J ) 7.5 Hz, 2H), 3.8 (s, 3H); 13C NMR (DMSO-
d6) δ 162.7, 162.3, 140.6, 129.3, 126.3, 121.0, 113.7, 57.6. Anal.
(C8H9N3O4‚HCl) C, H, N.
{3-[2-(3-Amino-6-methyl-2-oxopyridin-1(2H)-yl)acetylamino]-
6-methyl-2-oxopyridin-1(2H)-yl}acetic Acid tert-Butyl Ester 17.
To a solution of 16 (200 mg, 0.37 mmol) in MeOH (5 mL) was
added Pd/C (100 mg, 50% mass). The reaction flask was flushed
three times with hydrogen, and stirring was maintained under
hydrogen atmosphere at room temperature for 24 h. The mixture
was filtered through a mixture of Celite and silica, and the cake
was washed thoroughly with MeOH (200 mL). The filtrate was
evaporated to dryness to yield a pale-yellow oil. Trituration in Et2O
(20 mL) yielded a pale-yellow powder: mp 144-150 °C (98 mg,
1
66% yield); H NMR (DMSO-d6) δ 8.2 (m, 4H), 6.5 (d, J ) 7.2
Hz, 1H), 6.2 (d, J ) 7.7 Hz, 1H), 6.0 (d, J ) 7.3 Hz, 1H), 5.0 (s,
2H), 4.8 (s, 2H), 2.3 (s, 3H), 2.2 (s, 3H), 1.4 (s, 9H); 13C NMR
(DMSO-d6) δ 168.0, 167.0, 158.0, 140.0, 126.0, 123.0, 124.0, 105.0,
80.0, 47.8, 28.5, 20.5. Anal. (C20H26N4O5‚0.15H2O) C, H, N
2-(3-Nitro-2-oxopyridin-1(2H)-yl)acetamide 19. To a solution
of 3-nitropyridin-2(1H)-one 18 (0.35 g, 2.5 mmol) and 2-bromo-
acetamide (0.48 g, 3.0 mmol) in dry THF (12 mL) was added
K2CO3 (0.41 g, 3.0 mmol). The reaction mixture was refluxed with
stirring for 16 h. The resulting precipitate was filtered, and the
yellow solid was washed with MeOH (20 mL) and dried overnight
in a desiccator. Trituration in hot ethanol (20 mL) and filtration
afforded the pure product as a yellow powder: mp 200-202 °C
(405 mg, 82% yield); 1H NMR (DMSO-d6) δ 8.2 (dd, J ) 2.0, 7.7
Hz, 1H), 7.9 (dd, J ) 2.0, 6.6 Hz, 1H), 7.5 (bs, 1H), 7.1 (bs, 1H),
6.2 (dd, J ) 6.6, 7.7 Hz, 1H), 4.4 (s, 2H); 13C NMR (DMSO-d6)
δ 168.0, 154.1, 147.9, 139.5, 138., 103.3, 51.9
Synthesis of 26. To a solution of acid 23 (100 mg, 0.15 mmol)
and hydrazide 25 (37 mg, 0.15 mmol) in DMF (5 mL) under argon
atmosphere was added Et3N (23 µL, 0.16 mmol). HOBt (11 mg,
0.086 mmol) and HBTU (62 mg, 0.16 mmol) in solution in DMF
(1 mL) were then added, and the mixture was stirred at room
temperature for 24 h. DMF was evaporated under reduced pressure,
and the residue was dissolved in CH2Cl2 (50 mL). The reaction
product precipitated when the organic phase was washed with 5%
aqueous citric acid (10 mL). The crude solid was filtered and
washed successively with 10% aqueous Na2CO3 (10 mL) and water
(10 mL) to give an off-white powder: mp 159-161°C (90 mg,
70% yield); 1H NMR (DMSO-d6) δ 8.8 (s, 1H), 8.1-8.3 (m, 3H),
7.9 (m, 3H), 7.8 (m, 2H), 7.0-7.2 (m, 2H), 6.9 (d, 1H), 4.5-4.0
(m, 7H), 4.0 (s, 3H), 3.6 (s, 3H), 2.8-2.7 (m, 4H), 2.5-2.1 (m,
6H), 1.6 (m, 1H), 1.5 (m, 2H), 0.9 (m, 18H); 13C NMR (DMSO-
d6) δ 173.0, 172.8, 171.1, 163.5, 160.0, 156.0, 139.0, 135.1, 128.0,
127.0, 126.1, 123.0, 120.8, 115.0, 114.0, 104.5, 65.5, 57.0, 56.5,
56.0, 51.0, 51.0, 40.0, 31.5, 24.5, 28.0, 23.0, 22.5, 19.5; ESI+ MS
m/z 873.4 (M + Na)+. Anal. (C43H58N6O12‚2H2O) C, H, N
Synthesis of Molecular Tong 2. To a solution of 26 (100 mg,
0.11 mmol) in MeOH (7 mL) was added Pd/C (30 mg, 30% mass).
The reaction flask was flushed three times with hydrogen, and
stirring was maintained under hydrogen atmosphere at room
temperature for 20 h. The mixture was filtered through a mixture
of Celite and silica, and the cake was washed thoroughly with
MeOH (200 mL). The filtrate was evaporated to dryness to yield
an off-white solid: mp 170-180 °C (85 mg, 88% yield); 1H NMR
(DMSO-d6) δ 8.8 (s, 1H), 8.3-8.1 (m, 3H), 7.9 (m, 3H), 7.8 (m,
2-(3-Amino-2-oxopyridin-1(2H)-yl)acetamide 20. Compound
19 (0.55 g, 2.6 mmol) was suspended in MeOH (30 mL), and Pd/C
(110 mg, 20% mass) was added. The reaction flask was flushed
three times with hydrogen, and stirring was maintained under
hydrogen atmosphere at room temperature for 4 h. The mixture
was filtered through Celite, and the cake was washed thoroughly
with MeOH (100 mL). The filtrate was evaporated to dryness to
yield a crude brown powder, which was purified by flash chro-
matography on silica gel and eluted with CH2Cl2/MeOH/Et3N,
95/5/1. The pure product was obtained as a beige powder: mp 208-
1
210 °C (0.23 g, 53% yield); H NMR (DMSO-d6) δ 7.5 (bs,1H),
7.1 (bs,1H), 6.8 (dd, J ) 1.7, 6.8 Hz, 1H), 6.4 (dd, J ) 1.7, 6.8

Molecular Tongs
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4663
2H), 7.2-7.0 (m, 2H), 6.9 (d, 1H), 4.5-4.0 (m, 7H), 3.9 (s, 3H),
3.6 (s, 3H), 2.8-2.7 (m, 4H), 2.5-2.1 (m, 6H), 1.6 (m, 1H), 1.5
(m, 2H), 0.9 (m, 18H); 13C NMR (DMSO-d6) δ 173.0, 172.8, 171.5,
171.1, 163.5, 160.0, 156.0, 139.0, 135.1, 128.0, 127.0, 126.0, 123.0,
120.8, 115.0, 114.0, 104.5, 65.5, 57.0, 56.0, 51.0, 49.9, 39.0, 32.0,
29.5, 29.0, 23.8, 23.0, 23.0-22.0, 19.5; ESI+ MS m/z 843.5 (M +
Na)+. Anal. (C43H60N6O10‚2H2O) C, H, N
Synthesis of Molecular Tong 6. Acid 23 (96 mg, 0.15 mmol)
and amine 11 (43 mg, 0.13 mmol) were dissolved in DMF (10
mL), and DIPEA (0.11 mL, 0.66 mmol) was added. The reaction
mixture was stirred under argon atmosphere at room temperature
for 20 min. HBTU (56 mg, 0.15 mmol) and HOBt (20 mg, 0.15
mmol) were added to the reaction flask, and the solution was stirred
for 48 h at room temperature. DMF was evaporated under reduced
pressure, and the residue was dissolved in CH2Cl2 (50 mL). The
reaction product precipitated when the organic phase was washed
with 5% aqueous citric acid (10 mL). The crude solid was filtered
and washed successively with 10% aqueous Na2CO3 (10 mL) and
water (10 mL) to give 6 as a white powder: mp >266 °C (58 mg,
Synthesis of Molecular Tong 3. To a solution of acid 23 (166
mg, 0.25 mmol) and amine 14 (100 mg, 0.25 mmol) in DMF (5
mL) under argon atmosphere was added Et3N (38 µL, 0.28 mmol).
HBTU (106 mg, 0.28 mmol) in solution in DMF (1 mL) was then
added, and the mixture was stirred at room temperature for 24 h.
DMF was evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 (50 mL). The reaction product precipitated
when the organic phase was washed with 5% aqueous citric acid
(10 mL). The crude solid was filtered and washed successively with
10% aqueous Na2CO3 (10 mL) and water (10 mL) to give a yellow
1
50% yield); H NMR (DMSO-d6) δ 10.4 (bs, 1H), 10.0 (bd, 2H),
8.1-8.0 (m, 5H), 7.8 (dd, J ) 8.9, 2.7 Hz, 1H), 7.8 (bd, 2H), 7.2-
7.3 (m, 2H), 7.2 (d, J ) 9.1 Hz, 1H), 7.0-7.1 (m, 2H), 4.5-4.4
(m, 2H), 4.2-4.1 (m, 6H), 3.9 (s, 3H), 3.7 (s, 3H), 2.6-2.6 (m,
4H), 2.2-2.1 (m, 10H), 1.75-1.65 (m, 1H), 1.5 (t, J ) 7.2 Hz,
2H), 1.1-0.85 (m, 24 H); 13C NMR (DMSO-d6) δ 172.7, 171.9,
171.8, 171.5, 170.6, 168.1, 157.1, 152.8, 136.0, 132.5, 129.3, 124.1,
123.8, 121.5, 116.3, 112.7, 106.3, 67.4, 60.1, 57.5, 56.6, 56.4, 51.8,
51.2, 41.2, 31.9, 30.7, 29.0, 24.4, 23.0, 22.2, 19.0, 18.7, 18.5; ESI+
MS m/z 984.6 (M + Na)+. Anal. (C50H71N7O12‚2.5H2O ) C, H, N
Enzymatic Studies. The fluorogenic substrate DABCYL-γ-abu-
Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS {DABCYL, 4-(4′-di-
methylaminophenylazo)benzoyl; γ-abu, γ-amino butyric acid;
EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid)} was
purchased from Bachem (Germany). Other reagents and solvents
were purchased from commercial sources. The fluorescence and
absorbance measurements were performed using a Perkin-Elmer
LS 50B spectrofluorometer and a Uvikon 941 spectrophotometer,
respectively.
1
powder: mp 160-172°C (86 mg, 35% yield); H NMR (DMSO-
d6) δ 8.4 (bs, 1H), 8.2 (m, 2H), 7.9 (m, 3H), 7.8 (m, 2H), 7.7 (m,
2H), 7.1-7.0 (m, 2H), 7.0-6.9 (2s, 2H), 6.1 (d, J ) 9.4 Hz, 1H),
4.9 (bs, 2H), 4.7 (s, 2H), 4.4-4.0 (m, 7H), 4.0 (m, 1H), 3.6 (s,
3H), 2.8-2.7 (m, 4H), 2.4-2.1 (m, 9H), 1.8-1.7 (m, 1H), 1.6-
1.5 (m, 3H), 1.4-1.1 (m, 2H), 1.0 (d, J ) 3.0.4 Hz, 3H), 0.8 (m,
24H); 13C NMR (DMSO-d6) δ 173.0, 172.8, 171.1, 170.8, 171.0,
167.0, 157.5, 156.0, 140.0, 135.0, 127.0, 125.0, 123.0, 115.0, 106.5,
104.5, 67.0, 65.5, 59.0, 58.0, 57.0, 56.0, 51.0, 49.9, 48.0, 39.0, 37.0,
32.0, 29.5, 29.0, 25.0, 23.8, 23.0, 21.0, 19.5, 17.0, 16.0, 12.0; ESI+
MS m/z 1058.23 (M + Na)+. Anal. (C53H78N8O13‚3H2O) C, H, N
Synthesis of Molecular Tong 4. To a solution of acid 23 (100
mg, 0.15 mmol) and amine 17 (60 mg, 0.15 mmol) in DMF (5
mL) under argon atmosphere was added Et3N (22 µL, 0.16 mmol).
HBTU (113 mg, 0.30 mmol) in solution in DMF (1 mL) was then
added, and the mixture was stirred at room temperature for 24 h.
DMF was evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 (50 mL). The organic phase was washed with
5% aqueous citric acid (10 mL), 10% aqueous Na2CO3 (10 mL),
and water (10 mL). Trituration of the obtained yellow oil in Et2O
(20 mL) gave a yellow powder: mp 138-144°C (55 mg, 34%
Wild-Type and Mutated Proteases. The four PR mutants,
namely, D30N, I50V, V82A, and G48V/L90M, were constructed
with the Quickchange site-directed mutagenesis kit (Stratagene)
using the appropriate pairs of primers that confer the right
substitution in the nucleotide sequence. The plasmid pET9PRWT
bearing the wild-type sequence of the protease gene from the HIV
(BRU) DNA clone (subclone of λJ19) was used as a template for
the PCR amplification cycles. The resulting mutated plasmids were
sequenced (Genome Express) on both coding and noncoding strands
in order to check for the sole presence of the wanted mutation(s).
WT PR and D30N, I50V, V82A, and G48V/L90M mutants were
expressed in E. coli BL21(DE3) rosetta pLysS strain (Novagen) as
inclusion bodies. Subsequent purification in denaturing conditions
and refolding were performed according to Billich et al.34 Plasmids
encoding MDR-HM31 and ANAM-1132 mutants (generous gift of
Prof. E. Freire, Johns Hopkins University, Baltimore, MD) were
used to transform the E. coli BL21(DE3) rosetta pLysS strain, and
after expression both mutants were purified according to Muzammil
1
yield); H NMR (DMSO-d6) δ 8.1-7.9 (m, 5H), 7.7 (d, J ) 7.0
Hz, 2H), 7.2 (bs, 2H), 6.9 (d, J ) 7.0 Hz, 2H), 6.1 (bd, J ) 7.2
Hz, 2H), 4.7 (m, 4H), 4.4 (m, 1H), 4.2-4.1 (m, 2H), 4.1-4.0 (m,
4H), 3.6 (s, 3H), 2.4-2.2 (m, 4H), 2.2 (s, 6H), 2.2-1.9 (m, 6H),
1.7 (m, 1H), 1.4 (m, 2H), 1.4 (s, 9H), 0.9 (m, 18H); 13C NMR
(DMSO-d6) δ 172.9, 172.5, 171.7, 168.0, 158.0, 140.0, 129.0, 126.0,
123.0, 116.1, 106.1, 106.0, 80.0, 67.0, 58.0, 53.6, 51.5, 47.0, 41.5,
32.5, 31.0, 28.5, 25.6, 25.0, 20.0, 17.5; ESI+ MS m/z 1064.22 (M
+ Na)+. Anal. (C55H75N7O13‚3H2O) C, H, N
Synthesis of Molecular Tong 5. To a solution of acid 23 (50
mg, 0.076 mmol) and amine 22 (24 mg, 0.091 mmol) in DMF (5
mL) under argon atmosphere was added DIPEA (66 µL, 0.38
mmol). The mixture was stirred for 20 min at room temperature.
HOBt (11 mg, 0.086 mmol) and HBTU (33 mg, 0.086 mmol) in
solution in DMF (1 mL) were then added, and the mixture was
stirred at room temperature for 48 h. DMF was evaporated under
reduced pressure, and the residue was dissolved in CH2Cl2/MeOH,
90/10 (100 mL). The reaction product precipitated when the organic
phase was washed with 5% aqueous citric acid (20 mL). The crude
solid was filtered and washed successively with 10% aqueous
Na2CO3 (10 mL) and water (10 mL) to give a white powder: mp
32
et al.
Enzyme and Inhibition Assays. The enzymatic activities of
wild-type and mutated proteases were determined fluorometrically
using DABCYL-γ-abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS
(λex ) 340 nm; λem ) 490 nm) in 100 mM sodium acetate, 1 mM
EDTA, and 1 M NaCl at pH 4.7 and 30 °C.23 Substrate and
compounds were previously dissolved in DMSO, with the final
solvent concentration kept constant at 3% (v/v). A typical inhibition
experiment used 7.5 nM enzyme and 5.2 µM substrate. The IC50
values (inhibitor concentration leading to 50% inhibition) were
obtained by plotting the percent inhibition against inhibitor
concentration (0.5-28 µM) and fitting the experimental data to
1
210-217 °C (48 mg, 70% yield); H NMR (DMSO-d6) δ 9.1 (s,
1H), 8.2 (d, J ) 7.9 Hz, 1H), 8.1 (d, J ) 7.1 Hz, 1H), 8.0-7.9 (m,
3H), 7.7 (d, J ) 9.0 Hz, 2H), 7.6 (bs, 1H), 7.3 (d, J ) 6.7 Hz, 1H),
7.2 (bs, 1H), 7.1 (s, 2H), 6.9 (bd, J ) 9.0 Hz, 2H), 6.2 (t, J ) 7.1
Hz, 1H), 4.5 (s, 2H), 4.4-4.3 (m, 2H), 4.2-4.1 (m, 2H), 4.0 (m,
4H), 3.6 (s, 3H), 2.4-2.3 (m, 4H), 2.1-2.0 (m, 1H), 2.0-1.9 (m,
[I]0
% inhibition ) 100 ×
[I]0 + IC50
6H), 1.6 (m, 1H), 1.4 (t, J ) 4.8 Hz, 2H), 0.9-0.7 (m, 24 H); 13
C
NMR (DMSO-d6) δ 172.8, 172.6, 172.0, 171.4, 171.2, 168.8, 157.5,
156.5, 136.2, 133.5, 129.4, 128.4, 124.2, 122.9, 116.4, 106.7, 105.1,
67.4, 59.5, 58.3, 57.7, 52.1, 51.7, 51.3, 41.2, 32.0, 30.6, 30.3, 30.1,
25.4, 24.5, 23.4, 22.1, 18.6, 18.5; ESI+ MS m/z 928.6 (M + Na)+.
Anal. (C47H67N7O11‚1.5H2O ) C, H, N
The mechanism of inhibition and the corresponding kinetic
constants Kic (competitive inhibition) and Kid (dimerization inhibi-
tion) were determined using Zhang-Poorman kinetic analysis.16,22,23
Kinetics were carried out with a constant substrate concentra-
tion (5.2 µM) and at least five enzyme concentrations (4.7-43.5

4664 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
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nM). Inhibitor concentrations were 6 and 11 µM (compound 3),
4 and 6 µM (compound 4), 1 and 2 µM (compound 5), and 0.6
and 1.2 µM (compound 6). All experiments were performed in
triplicate.
Evaluation of Metabolic Stability. The stability of inhibitors
in RPMI culture medium containing 20% fetal calf serum was
assessed by studying their kinetics of breakdown at 37 °C for up
to 2 days. Incubation was terminated by adding ethanol. The mixture
was poured at 4 °C and centrifuged (10 000 rpm for 10 min).
Aliquots (20 µL) of the clear supernatant were injected onto the
RP-HPLC column (Waters 996). The degradation half-life was
obtained by a least-squares linear regression analysis of a plot of
the log[I] vs time using a minimum of five points.
Acknowledgment. The authors thank Prof. Ernesto Freire
of the Department of Biology and Biocalorimetry Center, The
Johns Hopkins University, Baltimore, MD, for his generous gift
of plasmids encoding MDR-HM and ANAM-11 mutants. L.B.
holds a doctoral fellowship from MNSER. The English text was
edited by Dr. Owen Parkes.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
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