Carbonylhydrazide-based molecular tongs inhibit wild-type and mutated HIV-1 protease dimerization

Article abstract of DOI:10.1021/jm300181j

We have designed and synthesized new molecular tongs based on a rigid naphthalene scaffold and evaluated their antidimer activity on HIV-1 protease (PR). We inserted carbonylhydrazide and oligohydrazide (azatide) fragments into their peptidomimetic arms t

Full text of DOI:10.1021/jm300181j

Article
pubs.acs.org/jmc
Carbonylhydrazide-Based Molecular Tongs Inhibit Wild-Type and
Mutated HIV‑1 Protease Dimerization
Laure Dufau,‡ Ana Sofia Marques Ressurreicao,† Roberto Fanelli,† Nadjib Kihal,† Anamaria Vidu,†
̧
̃
Thierry Milcent,† Jean-Louis Soulier,† Jordi Rodrigo,† Audrey Desvergne,‡ Karine Leblanc,†
Guillaume Bernadat,† Benoit Crousse,† Michele Reboud-Ravaux,*,‡ and Sandrine Ongeri*,†
̀
†UMR−CNRS 8076, Molec
rue J. B. Clement, 92296 Chat
‡UPMC−UR4, Enzymologie Molec
́
ules Fluoree
enay-Malabry Cedex, France
ulaire et Fonctionnelle, Case 256, 7 Quai St. Bernard, 75251 Paris Cedex 05, France
́ ́ ́ ́
s et Chimie Medicinale, LabEx LERMIT, Faculte de Pharmacie, Universite Paris-Sud 11, 5
́
̂
́
S
* Supporting Information
ABSTRACT: We have designed and synthesized new molecular tongs based on a rigid naphthalene scaffold and evaluated their
antidimer activity on HIV-1 protease (PR). We inserted carbonylhydrazide and oligohydrazide (azatide) fragments into their
peptidomimetic arms to reduce hydrophobicity and increase metabolic stability. These fragments are designed to disrupt the
protein−protein interactions by reproducing the hydrogen bond pattern found in the antiparallel β-sheet formed between the N-
and C-ends of the two monomers in the native PR. Kinetic analyses and fluorescent probe binding studies showed that several
molecular tongs can inhibit PR dimerization. The best nonpeptidic molecular tongs to date were obtained with an inhibition
constant Kid of 50 nM for PR and 80 nM for the multimutated protease ANAM-11. The PR inhibition was selective, the aspartic
proteases renin and pepsin were not inhibited.
free energy of PR dimerization.6 Moreover, these areas appear
INTRODUCTION
■
to be free of mutations.7 Dimerization inhibitors designed to
target the PR termini β-sheet interface can block the formation
of the homodimer or even disrupt it.8,9 The dimerization
inhibitors that target this β-sheet region are C-terminal and N-
terminal mimetic peptides,10−12 lipopeptides,13−15 bicyclic
guanidinium derivatives,16 and cross-linked peptides with
flexible17 or semirigid spacers.18 The recently approved PIs,
tipranavir and darunavir, are active against HIV-1 PR variants
that are resistant to many PIs and reportedly act as
conventional protease inhibitors. They also block protease
dimerization in cells but do not dissociate dimerized cellular
protease.19 Our strategy for inhibiting PR dimerization is to
design molecular tongs using a rigid spacer as this provides an
entropy benefit.20−23 We previously described the design of
organized molecular tongs based on a naphthalene or a
quinoline scaffold in which two peptide strands were attached
through a carboxypropyloxy linker to enable them to form an
The post-translational processing of virus polyproteins and the
subsequent generation of the structural and functional proteins
by human immunodeficiency virus type 1 (HIV-1) protease
(PR) are essential steps in the maturation of HIV.1 Inhibiting
PR results in the production of an uninfectious virus.2 The PR
inhibitors (PIs) used in highly active antiretroviral therapy
(HAART) target the PR active site where the two catalytic
aspartic residues are located. The emergence of resistance to
PIs due to several mutations within or outside the active site3,4
has led to a search for alternative strategies. One of these is to
prevent the essential dimerization process of PR because PR is
a homodimeric aspartyl protease (99 residues per monomer)
and each monomer contributes one catalytic aspartic residue.
The dimeric enzyme is active, whereas the monomer is inactive.
The PR homodimer is mainly stabilized by a four-stranded
antiparallel β-sheet that involves the N-termini (H-Pro(1)-
Gln(2)-Ile(3)-Thr(4)) and C-termini (Cys(95)-Thr(96)-
Leu(97)-Asn(98)-Phe(99)-OH) of both monomers. These
regions provide over 50% of the hydrogen bonds along the
dimer interface5 and contribute close to 75% of the total Gibbs
Received: February 9, 2012
Published: July 17, 2012
© 2012 American Chemical Society
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Figure 1. (A) Putative complexes between HIV-1 protease monomer and P1 peptide molecular tong; (B) putative complexes between HIV-1
protease monomer and dihydrazido peptidomimetic molecular tongs.
antiparallel β-sheet with the N- and C-termini of one HIV-1 PR
monomer (molecular tong P1 in Figure 1A). Identical peptide
strands led to efficient symmetrical antidimers, while non-
identical peptides gave asymmetrical antidimers.20,21 The next
step was to replace one or two peptide fragments by
peptidomimetic ones with keeping the same array of hydrogen
bonds.22,23 Inserting a 5-acetamido-2-methoxybenzohydrazide
group into one strand (while the other strand remained a
tripeptide) increased both the metabolic stability of the
molecular tongs and their ability to inhibit wild-type (PR)
and mutated HIV-1 proteases in vitro (Kid = 60 nM for PR and
100 nM for the multimutated protease ANAM-11).22,23 We
also inserted the 5-acetamido-2-methoxybenzydrazide group
into both strands to produce the first nonpeptide molecular
tongs.23 However, these molecules were less efficient inhibitors
of PR and mutated proteases (Kid up to 220 nM compared to
60 nM for the molecular tong with one tripeptide and one
peptidomimetic strands23). One drawback of our previous
peptide or peptidomimetic molecular tongs was that they were
completely insoluble in water. We are now concerned with
obtaining more efficient and more water-soluble nonpeptide
molecular tongs. This report describes the design and synthesis
of new molecular tongs containing the original carbon-
ylhydrazide (CONHNHCO) peptidomimetic fragments and
their ability to inhibit PR and two multimutated mutated HIV-1
proteases (Figure 2). These motifs were chosen because they
are isosteric elements that can replace amino acid residues and
introduce resistance to cleavage by peptidases.24,25 Oligohy-
drazide derivatives have also been used in the self-assembly of
hydrogen bond-mediated supramolecular systems to produce
well-established structures.26−28 However, the linear hydrogen-
bonding properties of carbonylhydrazide and oligohydrazide
peptidomimetics have not yet, to our knowledge, been
exploited in the design of protein ligands and protein−protein
interactions inhibitors. We postulated that these peptidomi-
metic elements would operate mainly by binding to the
antiparallel β-sheet formed between the N- and C-ends of
monomers and so disrupt dimers (Figure 1B). According to the
number of fragments introduced, they may also modulate the
balance between hydrophilicity and hydrophobicity and make
peptides resistant to proteolytic cleavage of the newly designed
molecular tongs. This report describes the most potent
nonpeptide molecular tongs obtained to date as dimerization
inhibitors of HIV-1 PR.
Figure 2. General structure of motifs inserted into one or two arms of
molecular tongs.
All the molecular tongs were based on a naphthalene scaffold
and the new strands, mimicking a tripeptidic sequence (except
for molecule 6), were anchored through a carboxypropyloxy
linker. We made several changes to the structures of the
strands. We first inserted one carbonylhydrazide motif by
linking two Val residues through a hydrazine bridge, thus
inverting the sense of the peptide arm (Figure 2). This new
peptidomimetic was introduced into one or two strands
(molecules 1 and 2, respectively, Figure 3). As our goal was
to decrease the peptide character and increase the water
solubility of the molecular tongs, we then introduced two
hydrazide moieties via an azatide (a sequence of aza amino
acids) by inserting two azaglycines into the peptidomimetic
arms (Figure 2). The azatide moieties were first directly
attached to the carboxypropyloxy link (in molecules 3−6,
Figure 3). A Val and a β-Ala amino acid residue were coupled at
the C-terminus of the arm in molecules 3 and 4 and in
molecule 5, respectively. Second, the azatide moieties were
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Figure 3. Structures of molecular tongs 1−10.
attached to the carboxypropyloxy link through an amino acid
residue at the N-terminus of the arm (in molecules 7−9, Figure
3). The azatide moiety was coupled to Val in molecule 7, β-Ala
in molecule 8, and the ε-amino group of an α-NH2 protected
Lys residue in molecule 9. The presence of the β-Ala residue
(8) or ε-amino-linked Lys residue (9) introduced flexibility in
the arm (Figure 3), as reported for molecular tongs having the
5-acetamido-2-methoxybenzohydrazide derivative unit.22,23 We
also designed a shorter dihydrazide-based peptidomimetic unit
in which the third terminal amino acid residue was replaced by
a methylamine group (molecule 6) (Figure 3). We also
evaluated the capacity of molecule 10, which possesses only
one peptidomimetic arm (Figure 3) to inhibit PR dimerization,
to confirm that only molecular tongs with two arms attached to
on the naphthalene scaffold are efficient inhibitors.
CHEMISTRY
■
The synthesis of the peptidomimetic 14 (to be introduced in
molecular tongs 1, 2, and 10) is described is Scheme 1 and
started with the preparation of the intermediate Z-Val-
NHNHBoc 11. 11 was obtained by coupling Z-Val-OH to
tert-butyl carbazate, using 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCI) and N-hydroxybenzotriazole (HOBt) as
coupling agents in good yield (88%) (Scheme 1). Hydro-
genolysis of the benzyl carbamate of 11 gave 12 in good yield
(96%). Acidic cleavage of the tert-butyl carbamate of 11 and its
coupling to N-Boc-Val-OH gave compound 13. Benzyl
carbamate hydrogenolysis of 13 gave the deprotected
peptidomimetic strand 14 with a satisfactory yield (71%)
(Scheme 1).
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a
Scheme 1. Synthesis of Peptidomimetics Containing Strands 12 and 14
a
(a) BocNHNH2, EDCI, HOBt, DIPEA, CH2Cl2, room temp; (b) H2, 10% Pd/C, MeOH, room temp; (c) HCl/dioxane 4 M, dioxane, room temp;
(d) N-Boc-Val-OH, EDCI, HOBt, DIPEA, DMF, room temp; (e) H2, 10% Pd/C, MeOH, room temp.
a
Scheme 2. Synthesis of Peptidomimetics Containing Strands 19, 21, and 23
a
(a) Pyridine, CH2Cl2, room temp; (b) H2NNH2·H2O, MeOH; (c) ClCO2Ph, pyridine, THF, room temp; (d) HCl·H-Val-NH2, Et3N, AcCN, room
temp; (e) TFA/CH2Cl2 (1:1, v/v), room temp; (f) HCl·NH2CH3, Et3N, AcCN, room temp; (g) HCl·H-β-Ala-OMe, Et3N, AcCN, room temp.
The procedure designed to obtain the new peptidomimetic
arms involved the synthesis of a dihydrazide motif 16 that was
common to all azatide-based arms (molecules 3−9). Thus,
compound 15 was prepared in 97% yield by reacting the
commercially available compounds tert-butyl carbazate and
phenyl chloroformate in dichloromethane in the presence of
pyridine (Scheme 2).29 Treatment of 15 with hydrazine hydrate
gave the intermediate 16 in 72% yield.29 Compound 16 was
then treated with phenyl chloroformate in THF, in the
presence of pyridine, to give 17 in good yield (91%).
Compound 17 was then coupled to HCl·H-Val-NH2 to give
the Boc-protected peptidomimetic arm 18 in good yield (93%).
Classical acid hydrolysis of 18 gave the new peptidomimetic
arm 19 (Scheme 2). Compound 17 was also coupled to methyl
amine to give 20 (yield 70%) and to HCl·H-β-Ala-OMe to give
22 (yield 67%). Acid cleavage of the tert-butyl carbamates gave
the new peptidomimetic arms 21 and 23 (Scheme 2).
OH, N-Boc-β-Ala-OH, or Nα-Boc-Nε-Z-Lys-OH, using O-
benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phos-
phate (HBTU) and HOBt as coupling agents to give
compounds 26, 28, or 30 in modest yields (46%, 12%, or
30%). These modest yields may be partly due to the
hydrophilic compounds 26, 28, and 30 remaining in the
aqueous phase during the workup of the coupling reaction and
consequently being very difficult to extract with organic
solvents. The acid hydrolysis of 26 and 28 gave the deprotected
peptidomimetic strands 27 and 29, and benzyl carbamate
hydrogenolysis of 30 gave the peptidomimetic strand 31
(Scheme 3).
The molecular tongs 1 was synthesized in three steps from 4-
[7-(3-carboxy-propoxy)naphthalene-2-yloxy]butyryl-Val-Leu-
Val-OMe 3221 (Scheme 4). 32 was first condensed with Val-
NHNHBoc 12 to form the intermediate 33 in 76% yield. tert-
Butyl carbamate cleavage of 33 was then followed by coupling
to N-Boc-Val-OH to give molecular tongs 1 in satisfactory yield
(55%). The molecular tongs 3 was synthesized directly by
condensing 4-[7-(3-carboxy-propoxy)naphthalene-2-yloxy]-
Compound 16 was acetylated to give 24 in good yield (96%)
(Scheme 3). Acid cleavage of the tert-butyl carbamate gave the
common intermediate 25, which was coupled to N-Boc-Val-
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a
Scheme 3. Synthesis of Peptidomimetics Containing Strands 27, 29, and 31
a
(a) Ac2O, THF, reflux; (b) HCl/dioxane 4M, dioxane, room temp; (c) N-Boc-Val-OH, HBTU, HOBt, DIPEA, DMF, room temp; (d) N-Boc-β-
Ala-OH, HBTU, HOBt, DIPEA, DMF, room temp; (e) Nα-Boc-Nε-Z-Lys-OH, HBTU, HOBt, DIPEA, DMF, room temp; (f) H2, 10% Pd/C,
MeOH, room temp.
a
Scheme 4. Synthesis of Molecular Tongs 1 and 3
a
(a) 12, HBTU, DIPEA, DMF, 5 days, room temp; (b) HCl/EtOH, DMF, room temp; (c) N-Boc-Val-OH, HBTU, Et3N, DMF, room temp; (d)
19, HBTU, HOBt, DIPEA, room temp.
butyryl-Val-Leu-Val-OMe 32,21 with the peptidomimetic 19 in
satisfactory yield (49%) (Scheme 4).
protocol (using HBTU and HOBt) to peptidomimetics 19,
21, 23, 27, 29, and 31, respectively, in satisfactory yields (40−
73%) (Scheme 5).
Molecular tongs 2 was synthesized from 4-((7-(3-carbox-
ypropoxy)-2-naphthyl)oxy)butanoic acid 3420 by a standard
coupling protocol (using HBTU and HOBt) to peptidomi-
metics 14 in 39% yield (Scheme 5). Molecule 10, which has
only one peptidomimetic arm, was obtained as a byproduct,
resulting from incomplete coupling, in 9% yield. The molecular
tongs 4−9 were synthesized from 4-((7-(3-carboxypropoxy)-2-
naphthyl)oxy)butanoic acid 3420 by a standard coupling
RESULTS AND DISCUSSION
■
We used a fluorimetric assay to test the capacity of compounds
1−10 to inhibit recombinant wild-type PR (PR) at the
optimum pH (4.7) and 30 °C.18,20−22 Compounds 5, 8, and
9 were not inhibitors at the highest tested concentration of 28
μM, whereas compounds 6 and 7 inhibited the enzyme poorly
(13 and 21% at 28 μM, respectively). In contrast, compounds
1−4 efficiently inhibited both PR and the two multimutated
proteases, MDR-HM and ANAM-11. The mutant MDR-HM30
contains six amino acid mutations located within and outside
the active site of the enzyme (Leu10Ile/Met46Ile/Ile54Val/
Val82Ala/Ile84Val/Leu90Met), while ANAM-1131 has 11
mutations: (Leu10Ile/Met36Ile/Ser37Asp/Met46Ile/
Arg57Lys/Leu63Pro/Ala71Val/Gly73Ser/Ile84Val/Leu90-
Met/Ile93Leu).
a
Scheme 5. Synthesis of Molecular Tongs 2 and 4−10
We used Zhang−Poorman kinetic analysis to identify the
mechanism of inhibition. Plots of [E]0/√vi versus √vi were
constructed, where vi were the initial rates. In all cases, parallel
lines were obtained, in agreement with dimerization inhibition
(Figure 4). The corresponding inhibition constants Kid are
a
(a) HBTU, HOBt, DIPEA, 14, DMF, room temp; (b) HBTU, HOBt,
DIPEA, 19 or 21 or 23 or 27 or 29 or 31, DMF, room temp.
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Figure 4. (A−C) Zhang kinetic analyses of PR, MDR-HM, and ANAM-11 inhibition by compounds 2 and 4 at pH 4.7 and 30 °C. (A) PR inhibition
■
■
■
▲
▲
■
▲
■
by 2.8 μM 2 ( ), 2 μM 2 ( ); (B) MDR-HM inhibition by 2.8 μM 2 ( ) and 1.9 μM 2 ( ); (C) ANAM-11 by 4.2 μM 2 ( ) and 2.8 μM 2 ( );
■
▲
▲
(D) PR inhibition by 17 μM 4 ( ), 11 μM 4 ( ); (E) MDR-HM inhibition by 8.5 μM 4 ( ) and 5.6 μM 4 ( ); (F) ANAM-11 by 22 μM 4 ( )
▲
●
and 17 μM 4 ( ). Enzyme activity was also always measured without inhibitor ( ).
shown in Table 1. The mechanism of inhibition was confirmed
for the four compounds using fluorescent probe binding. The
hydrophobic 1-anilino-8-naphthalene sulfonate (ANS) is
preferentially bound to the hydrophobic surfaces of proteins.32
We observed important enhancements of the fluorescence due
to ANS when compounds 1−3 and P1 were added to the
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hydrazide) arm (∼200 nM).22 They also show that the one-arm
molecule 10 is not an inhibitor, in agreement with our previous
results showing that there must be two peptide strands on the
molecular tongs to inhibit dimerization.20 Third, inhibition was
not affected by inserting an azatide moiety (diazaglycine) into
one arm directly linked to the carboxypropyloxy group while
the other arm was a tripeptide (Val-Leu-Val) (Kid of 150 nM
for 3 and 90 nM for 1). However, inhibitory activity was
decreased when azatide moieties were introduced in both arms
and directly linked to the carboxypropyloxy group (Kid of 700
nM for 4 and 50 nM for 2). Inserting an azatide moiety at the
C-terminus via a Val residue made the symmetric molecule 7
almost completely devoid of inhibitory activity (21% inhibition
at 28 μM). Similarly, further increasing the flexibility by
introducing a β-Ala residue or a Lys residue linked through its
ε-amino group completely suppressed inhibitory activity (8 and
9 relative to 4). The increased flexibility at the N-termini
destroyed all inhibitory activity in contrast to our previous
observation with 5-acetamido-2-methoxybenzohydrazide and 3-
amino-6-methylpyridine(1H)-one strands.22,23 The azatide
peptidomimetic unit is thus best directly attached to the
carboxypropyloxy linker (3, 4). Lastly, shortening both arms by
removing the C-terminal valinamide gave the poorly active
compound 6. Lengthening both arms with a β-Ala residue
linked to the C-terminal side (compound 5) gave a poorer
inhibitor. These two last observations are in accordance with
our previous results showing that a tripeptide sequence is better
than the shorter dipeptide or longer tetrapeptide sequen-
ces.20,21 The decreased inhibitory activity of molecular tongs 5
and 6 can be also ascribed to the lack of hydrophobic valine
side chain, while compound 4 has valine residues. We used
molecular modeling to estimate three physical properties of the
molecules, clogP, hydrophobic surface area, and hydrophilic
surface area (Table 1), in order to further correlate the
inhibitory activity with the hydropathic properties of the
molecules. A favorable influence of hydrophobicity on
inhibitory activity was observed as previously reported for
some molecular tongs.21,23
Table 1. Inhibition of PR and Mutated HIV-1 Proteases
MDR-HM and ANAM-11 by Compounds 1−10 (30 °C and
a
pH 4.7)
Kid (nM)
hydrophobic
hydrophilic
surface (Å2)
MDR- ANAM-
compd clogP surface (Å2)
PR
HM
11
P1
1
7.8
8.5
9.3
4.0
0.1
1467
1443
1391
1079
673
232
281
326
398
555
56020
nd
160
160
270
1600
nd
100
80
90
2
50
3
150
700
170
800
4
hydrophobic
surface (Å2)
hydrophilic
% inhibition at
compd clogP
surface (Å2)
28 μM
5
3.2
1.4
3.4
2.1
5.6
6.4
797
639
570
465
468
548
595
270
0
13
21
0
6
7
911
8
794
9
1304
924
0
10
0
a
Standard errors of initial rates are less than 5%. clogP and solvent
accessible surface areas are indicated. nd: not determined.
enzyme (less marked for 4) (Figure 5) but not when the active-
site inhibitors acetylpepstatin, saquinavir, or amprenavir were
The presence in the molecular tongs arms of carbon-
ylhydrazide (CONHNHCO) peptidomimetic fragments that
have particular hydrogen bonding properties led to a noticeable
increase of the inhibitory activity for compounds 1 and 2
compared to P1 (factors of 6.2 and 11.2 against PR), whereas
clogP was poorly increased (factors of 1.08 and 1.19,
respectively). Both inhibitory activity and cLogP were
improved for compound 3 (factor of 3.7 for PR inhibition
and 1.95 for cLogP). For the most soluble compound, 4 (≈ 0.3
mg/mL), the inhibitory activity was only decreased by a factor
of 1.25 whereas cLogP was improved by a factor of 78. These
data show that both activity and solubility can be increased
(compound 3). An important decrease of cLogP (factor of 78)
and hydrophobic surface (factor of 2.17) and increase in
hydrophilic surface (2.39) did not comprise the inhibitory
efficiency (compound 4). These data highlight the difficulty but
the possibility to find a compromise between hydrophilicity and
inhibitory activity.
Figure 5. ANS emission fluorescence (470 nm) measured at pH 4.7
and 25 °C. The excitation wavelength was 370 nm. PR (275 nM) was
△
preincubated in the absence of inhibitor ( ) or in the presence of
■
○
●
acetylpepstatin (×), saquinavir ( ), and compounds 1 ( ), 2 ( ), 3
◆
▼
), 4 ( ), and P1 ( ) prior to adding ANS. The final DMSO
▲
(
concentration was always 0.2% (v/v). Fluorescence intensity was
corrected for the fluorescence of the same concentrations of ANS and
inhibitor without enzyme.
added. Inhibitors 1−4 and the parent peptidic molecule P1
were bound to the unmasked hydrophobic surfaces of PR. This
is in agreement with the expected exposure of hydrophobic
interface area when the dimer is destabilized or dissociated.14,15
In contrast, active-site inhibitors changed the conformation
from semiclosed (dimer alone) to closed (active-site inhibitor/
dimer complex),15 leading to a smaller decrease in the ANS
fluorescence than that observed with the PR alone. The
experimental results summarized in Table 1 indicate that the
introduction of pseudopeptidic arms containing two Val
residues linked by an hydrazine bridge dramatically increased
the inhibitory potency (Kids of 90 nM for 1 and 50 nM for 2)
over those previously reported for the peptidic arm Val-Leu-Val
(P1, 560 nM)20 and to the Val-(5-acetamido-2-methoxybenzo-
Remarkably, the four new molecular tongs 1−4 behaved as
antidimers against the two multimutated proteases ANAM-11
and MDR-HM, which are analogous to the proteases found in
multiresistant viruses (Table 1). The inhibition of the two
mutated proteases by compounds 1−3 remained essentially
identical for a given mutated protease (for example, Kid of 2:
120 nM for MDR-HM and 80 nM for ANAM-11). Compound
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Figure 6. Putative complexes between PR monomer and the peptidomimetic molecular tong 4 (color by atom type): (A) one PR-monomer (ribbon
tube representation); (B) the C- and N-termini of one PR-monomer (green backbone).
4 was the poorest inhibitor of both PR and the mutated
proteases (Table 1).
monomer was higher for 4 than for 1 and 2 (13 against eight
and nine for compounds 1 and 2, respectively). However, as
indicated above, hydrophobic interactions are missing for
compound 4 to further stabilize the β-sheet structure.
Finally, we determined the stability of the symmetrical
molecular tongs 2 and 4 and asymmetrical molecular tongs 1
and 3 in RPMI culture medium containing 20% fetal calf serum
(Figure 4S in Supporting Information). The half-lives of
compounds 1 and 3 were about 24 h, which is very similar to
the 28 h half-life of the P1 peptide molecular tong bearing Val-
Leu-Val21 (Figure 1). Conversely, compounds 2 and 4 were not
significantly degraded after incubation for 48 h. These results
suggest that they are more resistant to hydrolysis than the
previously described peptide molecules and about the same as
those with 5-acetamido-2-methoxybenzohydrazide peptidomi-
metics (no significant degradation after 33 h).22 The metabolic
stability of the molecular tongs with two peptidomimetic arms
was also better than that of the asymmetrical molecules bearing
one tripeptidic arm.
We used molecular modeling to build several complexes that
might be formed between our new molecular tongs 1−9 and
the PR monomer and used them to correlate inhibitory activity
with structural characteristics. We used one monomer from the
crystallographic data for the dimeric PR and built molecular
tongs using the C- and N-terminal ends of the removed second
monomer of PR as templates. The designed carbonylhydrazide
peptidomimetics had an extended conformation compatible
with the formation of a β-sheet. The energy of the monomer−
molecular tongs complexes was minimized. This minimization
did not disrupt the postulated β-sheet structure formed
between the N- and C-termini of the remaining PR monomer
and molecular tongs 1−6. The carbonylhydrazide peptidomi-
metic arms still had an extended conformation. The PR−
molecular tongs complexes had an extensive network of
hydrogen bonds. The correct array of alternating 10-member
and 14-member rings involving two consecutive hydrogen
bonds was maintained after minimization (see Figure 6 and
Supporting Information). For example, there were still 13
hydrogen bonds in the complex with molecular tongs 4 (Figure
6). Most of them were with the C-terminal end (nine for
molecular tongs 4 and only five with the N-terminal end).
Conversely, there was no stable β-sheet structure with
molecular tongs 7−9 after minimization (see Supporting
Information). The biological results are in agreement with
the proposed complexes designed by molecular modeling;
molecules 1−4 interacted more strongly with the monomer
than did molecules 7−9. The absence of inhibitory activity of 5
and 6 compared to 4 may be explained by the lack of
hydrophobic amino acid side chains in 5 and 6 because
hydrophobic interactions are beneficial for stabilizing β-sheet
structures. Valine side chain(s) may reinforce the binding of
molecular tongs to the monomer ends and explain the
improved inhibitory efficiency of molecular tongs 1−4. The
same holds if we compare compound 4 (only one valine side
chain per arm) with compound 1 (two valine and one leucine
side chains in one arm, and two valine side chains in the other)
and with compound 2 (two valine side chains per arm). We
observed that the number of hydrogen bonds formed with the
inhibitor and the N- and C-termini of the remaining PR
Last, we evaluated the selectivity of molecules 1−4 toward
two other aspartic proteases, monomeric renin and pepsin.
Neither renin nor pepsin was inhibited by 10 μM
concentrations of compounds 1−4.
CONCLUSION
■
We have designed and synthesized a new series of naphthalene-
based molecular tongs containing carbonylhydrazide (CON-
HNHCO) and oligohydrazide (azatide) peptidomimetic side
arms. Their original hydrogen bonding properties made these
peptidomimetics good modulators of protein−protein inter-
actions involving β-sheet structures. Their scaffold gave the
molecular tongs some rigidity and forced the arms of the tongs
to have the extended structure essential for mimicking a β-
strand. As a proof-of-concept, we tested the ability of these
molecules to disrupt the PR termini β-sheet interface in order
to inhibit PR. The best of our nonpeptide molecular tongs
inhibited PR dimerization with an inhibition constant Kid of 50
nM, which is the best inhibition of HIV-1 PR dimerization
reported to date for molecular tongs. The azatide-based
scaffolds also increased water solubility. However, the inhibitory
potencies varied greatly according to the molecule structures,
indicating that there must be appropriate balances between
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Article
TR = 14.1 min, 97.5%; method 2, mixture A/B from 40/60 to 0/100
in 30 min, TR = 24.1 min, 97%. Anal. (C50H79N7O12·6H2O) C, H, N.
Synthesis of Molecular Tongs (2) and Molecule (10). Compound
14 (327 mg, 0.99 mmol, 2.2 equiv) was dissolved in DMF (5 mL) at 0
°C was mixed with compound 34 (150 mg, 0.45 mmol, 1 equiv),
DIPEA (145 mg, 0.99 mmol, 2.2 equiv), HOBt (134 mg, 0.99 mmol, 2
equiv), and HBTU (426 mg, 0.99 mmol, 2.2 equiv), and the mixture
was stirred for 48 h at room temperature. DMF was removed under
reduced pressure, and the resulting solid was washed successively with
CH2Cl2 (5 mL), 10% aqueous 10% citric acid (10 mL), water (10
mL), 10% aqueous K2CO3 (10 mL), and again with CH2Cl2 (5 mL).
Purification by HPLC gave products 2 (168 mg, 39%) and 10 (26 mg,
9%) as white solids. Product 2: mp 220−223 °C. 1H NMR (400 MHz,
DMSO-d6): δ 9.92 (s, 2H), 9.82 (s, 2H), 7.97 (d, J = 8.1 Hz, 2H), 7.70
(d, J = 8.6 Hz, 2H), 7.18 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.66 (d, J =
8.2 Hz, 2H), 4.22 (t, J = 8.1 Hz, 2H), 4.06 (t, J = 6.4 Hz, 4H), 3.81 (t,
J = 8.2 Hz, 2H), 2.37 (q, J = 6.9 Hz, 4H), 1.99 (t, J = 6.9 Hz, 4H), 1.94
(m, 2H), 1.90 (m, 2H), 1.37 (s, 18H), 0.92 (d, J = 6.5 Hz, 12H), 0.86
(d, J = 6.2 Hz, 12H). 13C NMR (100 MHz, DMSO-d6): δ 171.6, 170.2,
169.9, 157.0, 155.3, 135.7, 128.9, 123.7, 115.9, 106.1, 77.9, 67.0, 58.2,
56.1, 31.5, 30.6, 30.5, 28.2, 25.0, 19.1, 18.3. IR (neat): νmax 3221, 2036,
1605, 1478, 1209, 1169, 829, 651 cm−1. ESI+ MS m/z 979 [M + Na]+.
HPLC purity: method 1, mixture A/B from 60/40 to 0/100 in 20 min,
TR = 11.5 min, 96%; method 2, mixture A/B from 40/60 to 0/100 in
20 min, TR = 19.9 min, 99%. Anal. (C48H76N8O12·5H2O) C, H, N.
rigidity and flexibility, and between hydrophilicity and hydro-
phobicity, in order to obtain powerful inhibitors. The example
of the inhibitor 4 shows that water solubility does not
compromise inhibitory efficiency. The ability of these
proteolysis-resistant molecules to inhibit the multimutated
ANAM-11 highlights their potential to successfully overcome
the resistance to classical PI protease inhibitors presently
encountered.
EXPERIMENTAL SECTION
■
Chemistry. All solvents were purchased from commercial sources.
Dimethylformamide (DMF) was distilled over CaSO4, tetrahydrofuran
(THF) was distilled over sodium/benzophenone, and acetonitrile was
distilled over CaCl2. TLC was performed on 60F-250 silica gel (0.26
mm thick plates. Spots were visualized with UV light (254 nm), 3.5%
phosphomolybdic acid in ethanol, or with a solution of ninhydrin in
ethanol. Liquid chromatography was performed on Merck 60 silica
gels (230−400 mesh). Protected amino acids, O-benzotriazol-1-yl-
N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), and 1-
hydroxybenzotriazol (HOBT) were purchased from commercial
sources. 4-[7-(3-Carboxy-propoxy)naphthalene-2-yloxy]butyryl-Val-
Leu-Val-OMe 3221 and 4-((7-(3-carboxypropoxy)-2-naphthyl)oxy)-
butanoic acid 34,20 were prepared according to published methods.
Melting points were determined on a Kofler melting point apparatus.
NMR spectra were obtained on Ultrafield AVANCE 300 (1H, 300
MHz ; 13C, 75 MHz) or Bruker AVANCE 400 (1H, 400 MHz; 13C,
100 MHz) spectrometers. CDCl3 was used as solvent unless otherwise
stated. 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), doublet triplet (dt), and broad singlet
(bs). Mass spectra (APCI and ESI) were obtained using a Bruker
Esquire-LC apparatus at the SAMM (Faculty of Pharmacy at
1
Product 10: mp °C. H NMR (400 MHz, DMSO-d6): δ 11.90 (bs,
1H), 9.95 (s, 1H), 9.79 (s, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.70 (d, J =
8.6 Hz, 2H), 7.20 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 8.2 Hz,
1H), 4.25 (t, J = 8.1 Hz, 1H), 4.08 (t, J = 6.4 Hz, 4H), 3.81 (t, J = 8.2
Hz, 1H), 2.40 (m, 4H), 1.97 (m, 6H), 1.37 (s, 9H), 0.91 (d, J = 6.5
Hz, 6H), 0.86 (d, J = 6.2 Hz, 6H). 13C NMR (100 MHz, DMSO-d6): δ
171.6, 170.2, 169.9, 157.0, 155.3, 135.7, 128.9, 123.7, 115.9, 106.1,
77.9, 66.9, 66.5, 58.1, 56.1, 31.5, 30.5, 30.1, 28.1, 24.9, 24.2, 19.1, 18.3.
IR (neat): νmax 3221, 2036, 1605, 1478, 1209, 1169, 827, 652 cm−1.
ESI− MS m/z 643 [M − H]−. HPLC purity: method 1, mixture A/B
from 60/40 to 0/100 in 20 min, TR = 8.5 min, 100%. Anal.
(C33H48N4O9) C, H, N.
Chat
on a Perkin-Elmer CHN Analyzer 2400 at the Microanalyses Service of
the Faculty of Pharmacy at Chatenay-Malabry (France). Elemental
̂
enay-Malabry). Elemental analyses (C, H, N) were performed
̂
analysis data are included in the Supporting Information. The purity of
molecular tongs was determined by HPLC using a WATERS modular
system (quaternary pump + controller E600, UV detector PDA 2996,
autosampler 717). All molecular tongs tested in biological assays were
95% pure. HPLC conditions were as follows. Method 1: column
SUNFIRE, C18, 5 μm, 150 mm × 4.6 mm; mobile phase, a mixture of
(A) water (0.05% TFA) and (B) AcCN (0.05% TFA); room
temperature; flow rate 1 mL/min; detection at 254 nm. Method 2:
column SUNFIRE, C18, 5 μm, 150 mm × 4.6 mm; mobile phase a
mixture of (A) water (0.05% TFA) and (B) MeOH (0.05% TFA);
room temperature; flow rate 1 mL/min; detection at 235 nm.
Synthesis of Molecular Tongs (1). Product 33 (80 mg, 92 μmol, 1
equiv) was dissolved in DMF (4 mL), and 2 mL of 9 M HCl in
ethanol was added and the mixture stirred for 4 h. Then, Et3N (35 μL,
248 μmol, 2.7 equiv), HBTU (42 mg, 110 μmol, 1.2 equiv), and N-
Boc-Val-OH (20 mg, 91.8 μmol, 1 equiv) were added and the mixture
stirred again at room temperature for 5 days. The DMF was removed
under reduced pressure, and CH2Cl2 was added to the crude product
to precipitate a solid, which was washed successively with CH2Cl2,
aqueous 0.1 M HCl, aqueous 10% NaHCO3, brine, and again with
CH21Cl2 to yield 1 (49 mg, 55%) as a light-brown solid; mp 213-216
°C. H NMR (300 MHz, DMSO-d6): δ 9.92 (s, 1H), 9.82 (s, 1H),
9.69 (s, 1H), 8.72 (s, 1H), 7.91−8.01 (m, 3H), 7.71 (d, J = 8.7 Hz,
2H), 7.17 (s, 2H), 6.97 (d, J = 8.7 Hz, 2H), 4.35−4.43 (m, 1H), 4.12−
4.24 (m, 4H), 4.06 (t, J = 6.6 Hz, 4H), 3.61 (s, 3H), 2.33−2.43 (m,
4H), 1.90−2.06 (m, 8H), 1.54−1.66 (m, 1H), 1.44−1.49 (m, 2H),
1.38 (s, 9 H), 0.77−0.96 (m, 30 H). 13C NMR (75 MHz, DMSO-d6):
δ 172.1, 171.6, 171.4, 170.8, 170.1, 169.8, 156.9, 155.2, 135.6, 128.8,
123.6, 115.8, 106.0, 78.9, 66.8, 57.7, 57.2, 55.9, 51.5, 50.7, 40.6, 31.5,
31.4, 30.5, 30.1, 29.7, 27.9, 24.9, 24.9, 24.0, 22.8, 21.5, 19.1, 19.0, 18.7,
18.2, 18.1, 18.0. APCI+ MS m/z 970 [M + H]+. IR (neat): νmax 3277,
2963, 1743, 1633, 1537, 1386, 1209, 1163, 1021, 831, 668 cm−1.
HPLC purity: method 1, mixture A/B from 60/40 to 0/100 in 20 min,
Synthesis of Molecular Tongs (3). Compounds 19 (88 mg, 0.38
mmol) and 32 (230 mg, 0.35 mmol) were dissolved in DMF, to which
were added DIPEA (207 μL, 1.2 mmol, 3.5 equiv), HBTU (159 mg,
0.42 mmol, 1.2 equiv), and HOBt (52 mg, 0.38 mmol, 1.1 equiv), and
the mixture was stirred for 24 h at room temperature under an argon
atmosphere. The solvent was removed under reduced pressure to give
a beige oil. Trituration in EtOAc yielded a beige solid that was
collected on a filter and washed successively with EtOAc, CH2Cl2,
H2O, CH3OH, and Et2O to give 3 as a white powder (150 mg, 49%);
1
mp 218−220 °C. H NMR (400 MHz, DMSO-d6): δ 9.63 (s, 1H),
8.20 (br s, 2H), 7.92−7.97 (m, 3H), 7.78 (s, 1H), 7.71 (d, J = 9.3 Hz,
2H), 7.32 (bs, 1H), 7.17 (s, 2H), 7.05 (s, 1H), 6.98 (bs, 2H), 6.09 (d, J
= 7.2 Hz, 1H), 4.36−4.40 (m, 1H), 3.99−4.17 (m, 7H), 3.60 (s, 3H),
2.33−2.37 (m, 4H), 1.98−2.03 (m, 7H), 1.60 (bs, 1H), 1.44 (bs, 2H),
0.81−0.85 (m, 24H). 13C NMR (100 MHz, DMSO-d6): δ 173.5,
172.2, 171.7, 170.9, 158.3, 158.1, 157.0, 135.7, 129.0, 123.7, 115.9,
106.2, 66.9, 66.8, 57.8, 57.6, 57.3, 51.6, 50.8, 40.8, 31.6, 30.7, 30.3,
29.9, 29.7, 25.1, 24.4, 24.1, 23.0, 21.6, 19.3, 19.2, 18.8, 18.2, 18.1, 17.4.
IR (neat): νmax 3278, 2963, 1632, 1545, 1210 cm−1. ESI+ MS m/z 895
[M + Na]+, 911 [M + 39]+. HPLC purity: method 1, mixture A/B
from 60/40 to 0/100 in 20 min, TR = 8.1 min, 96%; method 2,
mixture A/B from 40/60 to 0/100 in 30 min, TR = 24.1 min, 97%.
Anal. (C42H65N9O11·1.5H2O) C, H, N.
Synthesis of Molecular Tongs (4). Same procedure as for 3 from 34
and 19. Product 4 was obtained as a white powder (174 mg, 56%); mp
1
181−185 °C. H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 2H), 8.20
(bs, 4H), 7.77 (s, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.32 (bs, 2H), 7.18 (s,
2H), 7.05 (s, 2H), 6.97 (d, J = 7.6 Hz, 2H), 6.08 (d, J = 8.4 Hz, 2H),
3.97−4.08 (m, 6H), 2.31−2.34 (m, 4H), 1.91−2.03 (m, 6H), 0.85 (d, J
= 6.7 Hz, 6H), 0.82 (d, J = 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO-
d6): δ 173.5, 171.8, 158.3, 158.1, 157.0, 135.7, 129.0, 123.7, 115.9,
106.2, 66.7, 57.6, 30.7, 29.6, 24.4, 19.3, 17.4. IR (neat): νmax 3252,
2967, 1657, 1516, 1211 cm−1. ESI+ MS m/z 783 [M + Na]+, 799 [M +
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K]+. HPLC purity: method 1, mixture A/B from 70/30 to 0/100 in 20
min, TR = 3.4 min, 100%; method 2, mixture A/B from 70/30 to 0/
100 in 20 min, TR = 15.6 min, 97%. Anal. (C32H48N12O10·1.5H2O) C,
H, N.
overnight at room temperature and then diluted with EtOAc (50 mL).
The organic phase was washed with 10% aqueous citric acid (5 mL),
water (10 mL), 10% aqueous K2CO3 (5 mL), and brine (5 mL). It was
then dried over MgSO4, filtered, and concentrated under reduced
pressure to give 11 as a white powder (2.5 g, 88%); mp 141−143 °C.
1H NMR (300 MHz, CD3OD): δ 7.43 (m, 5H), 5.12 (s, 2H), 4.08 (m,
1H), 2.10 (m, 1H), 1.47 (s, 9H), 1.03 (d, J = 7.0 Hz, 3H), 0.99 (d, J =
7.0 Hz, 3H). 13C NMR (75 MHz, CD3OD): δ = 173.7, 158.5, 157.4,
138.2, 129.6, 129.1, 128.9, 81.8, 67.8, 60.5, 32.3, 28.7, 19.9, 18.8. IR
(neat): νmax 3291, 2369, 1687, 1631, 1433, 1248, 1162, 1039, 742, 698
cm−1.; ESI+ MS m/z 388 [M + Na]+. Anal. (C18H27N3O5) C, H, N.
tert-Butyl N-[[(2S)-2-Amino-3-methyl-butanoyl]amino]-
carbamate (12). 11 (150 mg, 0.41 mmol) was dissolved in MeOH
(5 mL), and 10% Pd/C (15 mg, 10% mass) was added. The reaction
flask was flushed three times with hydrogen and stirred, under a
hydrogen atmosphere, for 24 h at room temperature. The mixture was
then filtered through a pad of Celite, and the cake was washed with
methanol (10 mL). The filtrate was evaporated under reduced
pressure to give 12 as a white solid (91 mg, 96%); mp 156−158 °C.
1H NMR (300 MHz, CDCl3): δ = 7.82 (bs, 1H), 7.35 (bs, 1H), 3.21
Synthesis of Molecular Tongs (5). Same procedure as for 3 from 34
and 21. Product 5 was obtained as a pale-gray powder (174 mg, 73%);
1
mp 177−187 °C. H NMR (400 MHz, DMSO-d6): δ 9.60 (s, 2H),
8.14 (bs, 4H), 7.70−7.72 (m, 4H), 7.19 (s, 2H), 6.97 (d, J = 8.4 Hz,
2H), 6.23 (br, 2H), 4.08 (br, 4H), 2.59 (s, 6H), 3.22−3.28 (m, 4H),
2.43−2.47 (m, 4H), 2.32 (t, J = 7.1 Hz, 4H), 2.02 (br, 4H). 13C NMR
(100 MHz, DMSO-d6): δ 171.9, 158.5, 158.1, 157.0, 135.7, 129.0,
123.7, 115.9, 106.2, 66.8, 51.3, 35.3, 34.3, 29.7, 24.5. IR (neat): νmax
3304, 2947, 1663, 1552, 1208 cm−1. ESI+ MS m/z 757 [M + Na]+.
HPLC purity: method 1, mixture A/B from 70/30 to 0/100 in 20 min,
TR = 4.1 min, 100%. Anal. (C30H42N10O12·2H2O) C, H, N.
Synthesis of Molecular Tongs (6). Same procedure as for 3 from 34
and 23. Product 6 was obtained as a white powder (98 mg, 55%); mp
1
206−210 °C. H NMR (400 MHz, DMSO-d6): δ 9.59 (s, 2H), 8.14
(bs, 4H), 7.63−7.72 (m, 4H), 7.19 (s, 2H), 6.96 (s, 2H), 6.09 (br,
2H), 4.08 (br, 4H), 2.57 (s, 6H), 2.33−2.42 (m, 4H), 2.01 (br, 4H).
13C NMR (100 MHz, DMSO-d6): δ 174.1, 159.2, 158.1, 156.9, 135.7,
128.9, 123.7, 115.9, 106.1, 66.6, 29.7, 26.2, 24.2. IR (neat): νmax 3280,
2947, 1664, 1515, 1210 cm−1. ESI+ MS m/z 613 [M + Na]+. HPLC
purity: method 1, mixture A/B from 80/20 to 0/100 in 30 min, TR =
11.7 min, 98%; method 2, mixture A/B from 40/60 to 0/100 in 30
min, TR = 24.1 min, 97%. Anal. (C24H34N10O8·2H2O) C, H, N.
Synthesis of Molecular Tongs (7). Same procedure as for 3 from 34
and 27. Product 7 was obtained as a brown solid (140 mg, 69%); mp
208−210 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.70 (s, 2H), 9.49 (s,
2H), 8.30 (s, 2H), 8.04 (s, 2H), 7.97 (d, J = 8.3 Hz, 2H), 7.70 (d, J =
8.9 Hz, 2H), 7.18 (s, 2H), 6.97 (dd, J = 8.8, 1.8 Hz, 2H), 4.20−4.15
(m, 2H), 4.08−4.04 (m, 4H), 2.38 (m, 4H), 2.00−1.97 (m, 6H), 1.80
(s, 6H), 0.90−0.85 (m, 12H). 13C NMR (100 MHz, DMSO-d6): δ
171.8, 170.8, 168.9, 157.2, 156.9, 135.7, 128.9, 123.6, 115.9, 106.0,
66.9, 56.3, 31.4, 30.4, 24.9, 20.5, 19.0, 18.3. IR (neat): νmax 3275, 1630,
(d, J = 7.3 Hz, 1H), 2.15 (m, 1H), 1.48 (s, 9H), 0.96 (d, J = 8.4 Hz,
3H), 0.83 (d, J = 8.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 173.7,
155.5, 81.5, 59.5, 31.3, 28.2, 19.4, 16.6. IR (neat): νmax 3205, 2256,
1694, 1214 cm −1. ESI+ MS m/z 254 [M + Na]+. Anal. C10H21N3O3
(%) C, H, N.
tert-Butyl N-[(1S)-1-[[[(2S)-2-(Benzyloxycarbonylamino)-3-meth-
yl-butanoyl]amino]carbamoyl]-2-methyl-propyl]carbamate (13).
Compound 11 (1.1 g, 3.01 mmol, 1 equiv) was dissolved in 4 M
HCl in dioxane (10 mL) at 0 °C and stirred at room temperature until
completely dissolved. The solvent was evaporated off, and the residue
was dissolved in DMF (6 mL). Then, N-Boc-Val-OH (0.74 g, 3.3
mmol, 1.1 equiv), DIPEA (1.75 g, 12.1 mmol, 4 equiv), HOBt (0.46 g,
3.3 mmol, 1.1 equiv), and EDCI (0.65 g, 3.3 mmol, 1.1 equiv) were
successively added and the reaction mixture was stirred at room
temperature for 24 h. DMF was removed under reduced pressure, and
the residue was dissolved in EtOAc (50 mL). The resulting organic
phase was washed successively with 10% aqueous citric acid (10 mL),
water (10 mL), 10% aqueous K2CO3 (10 mL), and brine (5 mL). The
organic phase was dried over MgSO4, filtered, and concentrated under
reduced pressure to yield 13 as a white solid (1.14 g, 82% over 2
+
1514, 1209 cm−1. APCI MS m/z 782 [M + Na]+. HPLC purity:
method 1, mixture A/B 60/40, TR = 4.1 min, 96%; Method 2, mixture
A/B from 50/50 to 20/80 in 20 min, TR = 5.88 min, 95%. Anal.
(C34H50N10O10·0.75H2O), C, H, N.
Synthesis of Molecular Tongs (8). Same procedure as for 3 from 34
and 29. Product 8 was obtained as a light-brown solid (53 mg, 40%);
1
steps); mp 172−175 °C. H NMR (300 MHz, DMSO-d6): δ 9.91 (s,
1
1H), 9.84 (s, 1H), 7.36 (m, 5H), 6.73 (s, 1H), 6.69 (s, 1H), 5.05 (s,
2H), 3.89 (m, 2H), 1.92 (m, 2H), 1.38 (s, 9H), 0.89 (m, 12H). 13C
NMR (75 MHz, DMSO-d6): δ 170.1, 169.9, 155.9, 155.3, 137.0, 128.3,
127.7, 127.6, 77.9, 65.35, 58.7, 58.1, 30.4, 30.3, 28.2, 19.1, 18.3. IR
(neat): νmax 3224, 1685, 1604, 1531, 1290, 1248, 1167, 1043 cm−1.
ESI+ MS m/z 487 [M + Na]+. Anal. C23H36N4O6 (%) C, H, N.
Benzyl N-[(1S)-1-[[[(2S)-2-Amino-3-methyl-butanoyl]amino]-
carbamoyl]-2-methyl-propyl] Carbamate (14). Same procedure as
for 12 from 13. Product 14 was obtained as a white solid (691 mg,
mp 225−227 °C. H NMR (400 MHz, DMSO-d6): δ 9.53 (bs, 4H),
8.24 (bs, 4H), 7.90 (bs, 2H), 7.70 (d, J = 8.9 Hz, 2H), 7.18 (s, 2H),
6.96 (dd, J = 8.9, 2 Hz, 2H), 4.05 (m, 4H), 3.27 (m, 4H), 2.27 (m,
8H), 2.00 (m, 4H), 1.80 (s, 6H). 13C NMR (100 MHz, DMSO-d6): δ
171.6, 170.2, 168.8, 157.5, 157.0, 135.8, 128.9, 123.7, 115.9, 106.1,
66.9, 35.1, 33.5, 31.7, 24.8, 20.6. IR (neat): νmax 3277, 2967, 1629,
1514, 1160 cm−1. ESI+ MS m/z 725 [M + Na]+, 741 [M + K]+ ]+.
HPLC purity: method 1, mixture A/B from 90/10 to 0/100, TR =
10.8 min, 100%; method 2, mixture A/B from 70/30 to 0/100 in 20
min, TR = 13 min, 100%. Anal. (C30H42N10O10·3H2O) C, H, N.
Synthesis of Molecular Tongs (9). Same procedure as for 3 from 34
and 31. Product 9 was obtained as a light-yellow solid (120 mg, 60%);
mp 157−159 °C. 1H NMR (300 MHz, CD3OD): δ 7.63 (d, J = 9 Hz,
2H), 7.12 (s, 2H), 6.94 (dd, J = 8.8, 2.1 Hz, 2H), 4.10 (m, 4H), 3.98
(t, J = 7.1 Hz, 2H), 3.19 (m, 4H), 2.42 (t, J = 7.3 Hz, 4H), 2.13 (m,
4H), 1.97 (s, 6H), 1.75 (m, 2H), 1.64 (m, 2H), 1.51 (m, 4H), 1.43 (s,
18H), 1.42 (m, 4H). 13C NMR (75 MHz, CD3OD): δ 175.6, 175.0,
173.1, 159.9, 158.8, 158.1, 137.5, 130.0, 125.8, 117.2, 107.3, 80.9, 68.2,
54.9, 40.1, 33.7, 32.4, 30.0, 28.7, 26.7, 24.0, 20.5. IR (neat): νmax 3215,
1642, 1542, 1212 cm−1. ESI+ MS m/z 1040 [M + Na]+ ]+. HPLC
purity: method 1, mixture A/B from 70/30 to 0/100, TR = 9.2 min,
100%; method 2, mixture A/B from 50/50 to 0/100 in 30 min, TR =
19.5 min, 100%. Anal. (C46H72N12O14·4H2O) C, H, N.
1
71%); mp 138−142 °C. H NMR (300 MHz, DMSO-d6): δ 9.93 (s,
1H), 9.82 (s, 1H), 6.61 (s, 1H), 3.93 (m, 2H), 2.01 (m, 2H), 1.48 (s,
9H), 0.92 (m, 12H). 13C NMR (75 MHz, DMSO-d6): δ 170.2, 169.9,
155.4, 77.4, 58.1, 57.9, 30.6, 30.2, 28.3, 19.2, 19.0. IR (neat): νmax 3276,
2912, 1677, 1607, 1275, 1186, 1089 cm −1. ESI+ MS m/z 387 [M +
Na]+. Anal. (C15H30N4O4·2H2O) C, H, N.
Phenyl N-(tert-Butoxycarbonylamino)carbamate (15). tert-Butyl
carbazate (2 g, 15.13 mmol) and pyridine (2.71 mL, 33.29 mmol, 2.2
equiv) were dissolved, with stirring, in CH2Cl2 (10 mL) and phenyl
chloroformate (2.08 mL, 16.64 mmol, 1.1 equiv) in CH2Cl2 (10 mL)
was added dropwise over 30 min at 0 °C. The mixture was stirred
overnight at room temperature, and CH2Cl2 was evaporated off under
reduced pressure. The residue was dissolved in EtOAc (400 mL) and
washed successively with 10% aqueous citric acid (50 mL), water (100
mL), 10% aqueous K2CO3 (50 mL), and water (50 mL). The organic
phase was dried over Na2SO4, filtered, and concentrated. The resulting
crude product was purified by flash chromatography on silica gel and
eluted with EtOAc/cyclohexane (20/80), to give 15 as a white powder
tert-Butyl N-[[(2S)-2-(Benzyloxycarbonylamino)-3-methyl-
butanoyl]amino]carbamate (11). N-Z-Val-OH (2 g, 7.97 mmol, 1
equiv) was dissolved in CH2Cl2 (30 mL) at 0 °C and to it were added
successively, DIPEA (3.1 g, 23.9 mmol, 3 equiv), tert-butyl carbazate
(1.16 g, 8.77 mmol, 1.1 equiv), HOBt (1.19 g, 8.77 mmol, 1.1 equiv),
and EDCI (1.68 g, 8.77 mmol, 1.1 equiv). This mixture was stirred
1
(3.68 g, 97%); mp 132−134 °C. H NMR (300 MHz, DMSO-d6): δ
7.36 (t, J = 7.7 Hz, 2H), 7.14−7.24 (m, 3H), 7.00 (bs, 1H), 6.50 (bs,
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1H), 1.49 (s, 9H). 13C NMR (75 MHz, DMSO-d6): δ 155.6, 155.3,
150.6, 129.4, 125.8, 121.4, 82.2, 28.2. IR (neat): νmax 3223, 2969, 1758,
1723, 1692, 1519, 1213, 1177, 1025 cm−1. ESI+ MS m/z 275 [M +
Na]+. Anal. (C12H16N2O4) C, H, N.
Methyl 3-[(Hydrazinecarbonylamino)carbamoylamino]-
propanoate (21). Same procedure as for 19 from 20. The
corresponding TFA salt of 21 was used in the next step without
further purification.
tert-Butyl N-[(Methylcarbamoylamino)carbamoylamino]-
carbamate (22). Same procedure as for 18 from 17 and
HCl·NH2CH3. The product 22 was obtained as a white solid (84
mg, 70%); mp 232−234 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.54
(bs, 1H), 8.05 (bs, 2H), 7.61 (s, 1H), 6.07 (bs, 1H), 2.56 (d, J = 4.5
Hz, 3H), 1.40 (s, 9H). 13C NMR (75 MHz, DMSO-d6): δ 159.1,
158.4, 155.9, 79.0, 28.1, 26.1. IR (neat): νmax 3272, 3106, 2936, 1665,
1474, 1159 cm−1. ESI+ MS m/z 270 [M + 23]+. Anal. (C8H17N5O4) C,
H, N.
tert-Butyl N-(Hydrazinecarbonylamino)carbamate (16). Com-
pound 15 (6.5 g, 25.77 mmol) and hydrazine monohydrate (8.14
mL, 167.5 mmol, 6.5 equiv) in methanol (100 mL) were heated under
reflux for 1.5 h, cooled to room temperature, and the solvent removed
under reduced pressure. The resulting oily residue was triturated in
petroleum ether, and the precipitate obtained was recrystallized from
EtOAc/petroleum ether to give 16 as a white solid (3.54 g, 72%); mp
1
134−136 °C. H NMR (300 MHz, DMSO-d6): δ 8.41 (bs, 1H), 7.80
(s, 1H), 7.34 (s, 1H), 4.04 (bs, 2H), 1.38 (s, 9H). 13C NMR (75 MHz,
DMSO-d6): δ 159.9, 155.9, 78.7, 28.1. IR (neat): νmax 3252, 2974,
1730, 1651, 1229, 1173 cm−1. ESI+ MS m/z 213 [M + Na]+. Anal.
(C6H14N4O3) C, H, N.
1-Amino-3-(methylcarbamoylamino)urea (23). Same procedure
as for 19 from 22. The TFA salt of 23 was used in the next step
without further purification.
tert-Butyl N-(Acetamidocarbamoylamino)carbamate (24). 16 (3
g, 15.7 mmol) was dissolved in dry THF (180 mL) with stirring, and
acetic anhydride (7.45 mL, 78.8 mmol, 5 equiv) was added dropwise.
The mixture was heated under reflux for 1.5 h, the solvent was
removed under reduced pressure, and the resulting residue was
triturated in CH2Cl2. The precipitate obtained was washed with
petroleum ether to yield compound 24 as a white solid (3.5 g, 96%);
Phenyl N-[(tert-Butoxycarbonylamino)carbamoylamino]-
carbamate (17). Pyridine (678 μL, 8.2 mmol, 2.5 equiv) was added
to a suspension of compound 16 (625 mg, 3.3 mmol) in THF (50
mL), and the mixture was stirred until a homogeneous solution was
obtained. The solution was then cooled to 0 °C, phenyl chloroformate
(458 μL, 3.6 mmol, 1.1 equiv) was added, and the mixture was stirred
for 15 min at room temperature. The solvent was evaporated off under
reduced pressure, and the residue was dissolved in EtOAc (50 mL).
The organic layer was washed sucessively with water (2 × 25 mL), 5%
aqueous citric acid (2 × 20 mL), and 10% aqueous K2CO3 (2 × 20
mL), dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash chromatography on silica
gel (EtOAc/cyclohexane, 80/20) to give product 17 as a white solid
1
mp 118−120 °C. H NMR (300 MHz, DMSO-d6): δ 9.46 (s, 1H),
8.51 (s, 1H), 8.09 (s, 1H), 8.07 (s, 1H), 1.80 (s, 3H), 1.39 (s, 9H). 13C
NMR (75 MHz, DMSO-d6): δ 168.7, 157.7, 155.8, 78.8, 28.05, 20.56.
IR (neat): νmax 3301, 1663, 1521, 1238, 1158 cm−1. ESI+ MS m/z 255
[M + Na]+. Anal. (C8H16N4O4·0.15H2O) C, H, N.
1-Acetamido-3-amino-urea (25). Compound 24 (88 mg, 0.38
mmol) was dissolved in 4 M HCl in dioxane (4 mL) and stirred at
room temperature for 2 h. The solvent was removed, and the HCl salt
of 25 was obtained as a white solid (64 mg, 100%) and used in the
next step without further purification.
1
(923 mg, 91%); mp 173−174 °C. H NMR (300 MHz, CDCl3): δ
7.81 (bs, 2H), 7.68 (bs, 1H), 7.09−7.31 (m, 5H), 6.99 (bs, 1H), 1.43
(s, 9H). 13C NMR (75 MHz, CDCl3): δ 158.7, 156.7, 155.8, 150.6,
129.3, 125.7, 121.4, 82.1, 28.1. IR (neat): νmax 3262, 3238, 1712, 1682,
1519, 1489, 1226, 1154 cm−1. ESI+ MS m/z 333 [M + Na]+. Anal.
(C13H18N4O5) C, H, N.
tert-Butyl N-[(1S)-1-[(Acetamidocarbamoylamino)carbamoyl]-2-
methyl-propyl]carbamate (26). 25 (163 mg, 0.97 mmol) and N-Boc-
Val-OH were dissolved in DMF (15 mL) with stirring at 0 °C, and
DIPEA (1.0 mL, 5.82 mmol, 10 equiv), HBTU (440 mg, 1.16 mmol,
1.2 equiv), and HOBt (145 mg, 1.07 mmol, 1.1 equiv) were added
successively. The reaction mixture was stirred under argon at room
temperature for 24 h. The DMF was removed under reduced pressure,
and the residue was dissolved in EtOAc (200 mL). The organic phase
was washed with water (2 × 50 mL) and concentrated under reduced
pressure. The aqueous phase was then concentrated and extracted
twice with EtOAc because compound 26 is extremely soluble in water.
The oily crude product was purified by flash chromatography on silica
gel (EtOAc 100% to EtOAc/CH3OH, 10/1) to give product 26 as a
tert-Butyl N-[[[(1S)-1-Carbamoyl-2-methyl-propyl]-
carbamoylamino]carbamoylamino] carbamate (18). Compound
17 (273 mg, 0.88 mmol) and HCl·H-Val-NH2 (151 mg, 0.97 mmol,
1.1 equiv) were dissolved in acetonitrile (17 mL), and triethylamine
(741 μL, 5.3 mmol, 6 equiv) was added to the mixture. The mixture
was stirred, under an argon atmosphere, for 3 days at room
temperature. The solvent was removed under reduced pressure, and
the resulting residue was purified by flash chromatography on silica gel
(EtOAc/CH3OH, 90/10) to give product 18 as a white solid (272 mg,
93%); mp 154−156 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.61 (bs,
1H), 8.13 (s, 2H), 7.77 (s, 1H), 7.37 (bs, 1H), 7.08 (s, 1H), 6.05 (d, J
= 8.4 Hz, 1H), 4.00 (dd, J = 8.4, 5.1 Hz, 1H), 1.95−2.02 (m, 1H), 1.41
(s, 9H), 0.87 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H). 13C NMR
(75 MHz, DMSO-d6): δ 173.5, 158.6, 158.1, 155.9, 79.0, 57.5, 30.6,
28.1, 19.3, 17.4. IR (neat): νmax 3259, 2969, 1658, 1529, 1241, 1158
cm−1. ESI+ MS m/z 355 [M + 23]+. Anal. (C12H24N6O5·0.2EtOAc) C,
H, N.
1
white solid (150 mg, 46%); mp 110−112 °C. H NMR (300 MHz,
DMSO-d6): δ 9.65 (br, 1H), 9.50 (s, 1H), 8.32 (br, 1H), 8.03 (s, 1H),
6.73 (d, J = 8.1 Hz, 1H), 3.79−3.74 (m, 1H), 1.95−1.87 (m, 1H), 1.80
(s, 3H), 1.37 (s, 9H), 0.89−0.83 (m, 6H). 13C NMR (75 MHz,
DMSO-d6): δ 171.1, 168.8, 157.2, 78.0, 58.2, 30.2, 28.1, 20.5, 19.0,
18.3. IR (neat): νmax 3261, 1668, 1507, 1244, 1161 cm−1. ESI+ MS m/z
354 [M + Na]+. HRMS calcd for C13H25N5O5 + Na (354.1753), found
354.1752.
(2S)-2-[(Hydrazinecarbonylamino)carbamoylamino]-3-methyl-
butanamide (19). 18 was dissolved in CH2Cl2 (0.13 M), an equal
volume of TFA was added, and the mixture was stirred at room
temperature for 3 h. The solvent was removed, and methanol was
added and immediately evaporated off, and then ether was added and
again immediately evaporated off to give the corresponding TFA salt
of 19. This was used in the next step without further purification.
Methyl 3-[[(tert-Butoxycarbonylamino)carbamoylamino]-
carbamoylamino]propanoate (20). Same procedure as for 18 from
17 and HCl·H-β-Ala-OCH3. The product 20 was obtained as a white
solid (274 mg, 67%); mp 122−124 °C. 1H NMR (300 MHz, DMSO-
d6): δ 8.54 (s, 1H), 8.05 (bs, 2H), 7.68 (s, 1H), 6.20 (br, 1H), 3.61 (s,
3H), 3.25 (dt, J = 11.7, 5.7 Hz, 2H), 2.45 (t, J = 5.7 Hz, 2H), 1.40 (s,
9H). 13C NMR (75 MHz, DMSO-d6): δ 172.0, 158.4, 155.9, 79.0,
51.3, 35.2, 34.3, 28.1. IR (neat): νmax 3270, 3106, 2936, 1666, 1542,
1-Acetamido-3-[[(2S)-2-amino-3-methyl-butanoyl]amino]urea
(27). Same procedure as for 25 from 26. The HCl salt of 27 was used
in the next step without further purification.
tert-Butyl N-[3-[2-(Acetamidocarbamoyl)hydrazino]-3-oxo-
propyl]carbamate (28). Same procedure as for 26 from 25 and
Boc-β-Ala-OH. The product 28 was obtained as a light-brown solid
1
(95 mg, 12%); mp 145−147 °C. H NMR (400 MHz, DMSO-d6): δ
9.54 (s, 1H), 9.49 (s, 1H), 8.21 (br, 2H), 6.77−6.74 (m, 1H), 3.17−
3.09 (m, 2H), 2.28−2.22 (m, 2H), 1.80 (s, 3H), 1.37 (s, 9H). 13C
NMR (100 MHz, DMSO-d6): δ 174.0, 173.2, 160.1, 158.3, 80.2, 37.6,
35.2, 28.7, 20.6. IR (neat): νmax = 3245, 1659, 1511, 1164. ESI− MS m/
z 302. Anal. (C11H21N5O5) C, H, N.
1-Acetamido-3-(3-aminopropanoylamino)urea (29). Same pro-
cedure as for 25 from 28. The HCl salt of 29 was used in the next step
without further purification.
1161 cm− 1
. +
ESI+ MS m/z 342 [M 23]+ . Anal.
(C11H21N5O6··0.15H2O) C, H, N.
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tert-Butyl N-[(1S)-1-[(Acetamidocarbamoylamino)carbamoyl]-5-
(phenoxycarbonylamino) pentyl]carbamate (30). Same procedure
as for 26 from 25 and Nα-Boc-Nε-Z-Lys-OH. The product 30 was
obtained as a white solid (88 mg, 30%); mp 129−131 °C. H NMR
340 nm; λem = 490 nm) in 100 mM sodium acetate, 1 mM EDTA,
and 1 M NaCl at pH 4.7 and 30 °C (final volume, 150 μL). The
substrate and the compound(s) were first dissolved in DMSO. The
final DMSO concentration was kept at 3% (v/v). The mechanism of
inhibition and the corresponding kinetic constants Kid (dimerization
inhibition) or Kic (competitive inhibition) were determined using
Zhang−Poorman kinetic analysis.11 Kinetic experiments were carried
out at a constant substrate concentration (5.2 μM) with at least six
enzyme concentrations (5.33−18.6 nM) and inhibitor concentrations
from 0.5 to 28 μM. The experimental data were fitted to the
appropriate equations according to ref 14. All experiments were
performed at least in triplicate.
Fluorescent Probe Binding. Hydrophobic ANS preferentially binds
to the hydrophobic surfaces of proteins. ANS emission spectra were
measured using an excitation wavelength of 370 nm and an emission
wavelength of 470 nm with bandwidths of 10 and 5 nm, respectively
(Perkin-Elmer LS 50B spectrofluorometer) as described in ref 21. PR
(350 nM) was dissolved in the assay buffer (100 mM sodium acetate, 1
M NaCl, 1 mM EDTA, pH 4.7) and incubated at 25 °C with or
without inhibitor (5 μM; acetylpeptsatin, saquinavir, compounds 1, 2,
3, 4, and P1). ANS (2 μL, final concentrations: 10−60 μM) was then
added. In all cases, the final DMSO concentration was 0.2% (v/v).
Each fluorescence measurement was made five times and averaged.
The intrinsic fluorescence of PR, acetylpepstatin, saquinavir, and
compounds 1, 2, 3, 4, and P1 was negligible under the experimental
conditions used.
Evaluation of Metabolic Stability. The stability of inhibitors in
RPMI (Roswell Park Memorial Institute medium) culture medium
containing 20% fetal calf serum was assessed by studying their kinetics
of breakdown at 37 °C for up to 2 days. Incubations were terminated
by adding ethanol. The mixture was poured at 4 °C and centrifuged
(10000 rpm for 10 min). Aliquots of the clear supernatant were
injected onto the HPLC column (Waters E600). The half-life of
breakdown was obtained by least-squares linear regression analysis of
the semilogarithmic plot of remaining inhibitor concentration versus
incubation time.
Inhibition of Renin and Pepsin. The proteolytic activity of renin
was determined fluorometrically using the fluorogenic substrate Arg-
Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys-
(DABCYL)-Arg (Km = 3.3 μM). Assays were performed in 50 mM
Tris−HCl, 100 mM NaCl, 1 mM EDTA, pH 8.0, and a substrate
concentration of 5 μM (λex= 340 nm, λem = 500 nm).
The proteolytic activity of pepsin from porcine gastric mucosa was
determined fluorometrically using the fluorogenic substrate Abz-Thr-
Ile-Nle-(p-NO2-Phe)-Gln-Arg-NH2 (Km = 13.3 μM). Assays were
performed in 10 mM NaHCOO, pH 3.5, and a substrate
concentration of 20 μM (λex= 337 nm, λem = 410 nm).
Molecular Modeling. Models of Molecular Tongs−PR Com-
plexes. Models of molecules 1−10 were built using CORINA 3.44
software33 and then energy minimized (by 2000 steps) using the
Powell method as implemented in SYBYL 8.0 software.34 Dihedral
angles were then adjusted so that each molecule was superimposed
over the backbone of chain B (subsequently removed) from the
crystallographic structure of HIV protease 7HVP (PDB accession
code).
The geometries of the resulting complexes were optimized by
energy minimization using the AMBER7 FF02 force field as
implemented in SYBYL to relax the structure and to remove steric
bumps. The minimizations were carried out by 1000 steps of steepest
descent followed by a conjugate gradient minimization until the rms
gradient of the potential was lower than 0.05 kcal·mol−1·Å−1. A
distance-dependent (ε = 4r) dielectric function was used. Analysis of
the complex models involved the study of the hydrogen bond network
and counting of favorable interactions.
Estimation of Accessible Surface Areas and clogP. Three-
dimensional coordinates for free compounds 1−10 and P1 generated
with CORINA software were energy-minimized with OPLS_2005
force field as implemented in MacroModel 9.9.35 Solvent-accessible
surface areas and clogP were calculated for these models with QikProp
3.4.36
1
(300 MHz, DMSO-d6): δ 9.65 (br, 1H), 9.49 (br, 1H), 8.32 (br, 1H),
8.04 (s, 1H), 7.37−7.32 (m, 5H), 7.24 (t, J = 5.2 Hz, 1H), 6.87 (d, J =
7.5 Hz, 1H), 5.00 (s, 2H), 3.92−3.85 (m, 1H), 2.99−2.93 (m, 2H),
1.80 (s, 3H), 1.61−1.47 (m, 2H), 1.40−1.24 (m, 13H). 13C NMR (75
MHz, DMSO-d6): δ 175.3. 173.3, 160.0, 159.1, 158.3, 138.6, 129.6,
129.1, 128.9, 81.0, 67.5, 55.1, 41.6, 32.6, 30.6, 28.9, 24.0, 20.7. IR
(neat): νmax 3307, 1665, 1522, 1246, 1167 cm−1. ESI− MS m/z 493 [M
− H]−; Anal. (C22H34N6O7·1H2O) C, H, N.
tert-Butyl N-[(1S)-1-[(Acetamidocarbamoylamino)carbamoyl]-5-
amino-pentyl]carbamate (31). Same procedure as for 12 from 30.
The product 31 was obtained as a white solid (164 mg, 100%); mp
1
150−160 °C. H NMR (300 MHz, CD3OD): δ 4.01 (t, J = 6.3 Hz,
1H), 2.69 (t, J = 6.7 Hz, 2H), 1.98 (s, 3H), 1.83−1.61 (m, 2H), 1.56−
1.45 (m, 13H). 13C NMR (75 MHz, CD3OD): δ 174.6, 173.1, 159.9,
158.2, 80.8, 55.0, 41.9, 32.6, 32.4, 28.7, 23.9, 20.5. IR (neat): νmax 3289,
2977, 1663, 1508, 1368, 1162 cm−1. ESI+ MS m/z 361 [M + H]+, 383
[M + Na]+.
Synthesis of Molecule (33). 32 (982 mg, 1.49 mmol, 1 equiv) was
dissolved, with stirring, in DMF (55 mL) and DIPEA (0.39 mL, 2.24
mmol, 1.5 equiv), 12 (449 mg, 1.94 mmol, 1.3 equiv) and HBTU (680
mg, 1.794 mmol, 1.2 equiv) were added successively. The mixture was
stirred at room temperature for 5 days, and the DMF was removed
under reduced pressure. A solid was precipitated from the oily crude
with CH2Cl2. This was washed successively with CH2Cl2, aqueous HCl
0.1 M, aqueous NaHCO3, brine, and CH2Cl2. Product 33 was
1
obtained as a white solid (986 mg, 76%); mp 220−222 °C. H NMR
(300 MHz, DMSO-d6): δ 9.70 (s, 1H), 8.73 (s, 1H), 8.28 (bs, 1H),
7.94−8.00 (m, 3H), 7.70 (d, J = 9.0 Hz, 2H), 7.17 (s, 2H), 6.97 (d, J =
9.0 Hz, 2H), 4.35−4.43 (m, 1H), 4.12−4.22 (m, 3H), 4.05 (t, J = 6.3
Hz, 4H), 3.60 (s, 3H), 2.32−2.41 (m, 4H), 1.89−2.05 (m, 7H), 1.53−
1.66 (m, 1H), 1.41−1.46 (m, 2H), 1.38 (s, 9 H), 0.80−0.90 (m, 24 H).
13C NMR (75 MHz, DMSO-d6): δ 172.1, 171.6, 171.5, 170.8, 170.6,
156.9, 155.0, 135.6, 128.8, 123.6, 115.8, 106.0, 78.9, 66.8, 57.7, 57.2,
55.9, 51.5, 50.7, 40.6, 31.5, 31.4, 30.6, 30.1, 29.8, 27.9, 24.9, 24.9, 24.0,
22.8, 21.5, 19.1, 18.7, 18.2, 18.1, 18.0. IR (neat): νmax 3279, 2956,
2365, 1740, 1635, 1535, 1210, 1162 cm −1. ESI+ MS m/z 894 [M +
Na]+. Anal. (C45H70N6O11·3H2O) C, H, N.
Enzymatic Studies. The fluorogenic substrate for PR and mutated
proteases was DABCYL-γ-abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-
EDANS [DABCYL, 4-(4′-dimethylaminophenylazo)benzoyl; γ-abu,
γ-amino butyric acid. EDANS, 5-[(2-aminoethyl)amino]naphthalene-
1-sulfonic acid)], was purchased from Bachem (Germany). 1-Anilino-
8-naphthalene sulfonate (ANS) was from Sigma-Aldrich, and
saquinavir and amprenavir were from the NIH (USA). The
fluorogenic pepsin substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-
Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg, the renin substrate Abz-Thr-
Ile-Nle-(p-NO2-Phe)-Gln-Arg-NH2, and recombinant renin and
pepsin were purchased from Sigma−Aldrich. Other reagents and
solvents were purchased from commercial sources. Absorbance
measurements were made with a Perkin-Elmer LS 50B spectropho-
tometer. Fluorescence intensities were measured in a BMG Fluostar
microplate reader.
Wild-Type and Mutated Proteases. The HIV-1 proteases (PR,
MDR-HM, and ANAM-1122) used in this study were expressed and
purified as described before.14 They were produced in Escherichia coli
using the expression vector pET-9 and the host bacterium strain
Rosetta (DE3)pLysS. The protease domain (PR) has five protective
mutations, Q7K, L33I, L63I to minimize the autoproteolysis of the
protease and C67A and C95A to prevent cysteine−thiol oxidation.
The multimutated HIV-1 proteases ANAM-11 and MDR-HM contain
11 mutations (L10I/M36I/S37D/M46I/R57K/L63P/A71 V/G73S/
I84 V/L90M/I93L) (ref 31) and six mutations (L10I/M46I/I54 V/
V82A/I84 V/L90M) (ref 30), respectively.
Enzyme and Inhibition Assays. The proteolytic activities of PR and
mutated proteases were determined fluorometrically using the
fluorogenic substrate DABCYL-γ-abu-SQNYPIVQ-EDANS (λex
=
6773
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Journal of Medicinal Chemistry
Article
E. Antivirals and antiviral strategies. Nature Rev. Microbiol. 2004, 2,
704−720.
ASSOCIATED CONTENT
■
S
* Supporting Information
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D. A.; Louis, J. M. Folded monomer of HIV-1 protease. J. Biol. Chem.
2001, 276, 49110−49116.
Putative complexes between the PR monomer and peptidomi-
metic molecular tongs 1−9. Purity: HPLC chromatograms of
molecular tongs 1−9 and analytical data of compounds.
Metabolic stability: HPLC chromatograms. This material is
available free of charge via the Internet at http://pubs.acs.org.
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(7) Gustchina, A.; Weber, I. T. Comparative analysis of the sequences
and structures of HIV-1 and HIV-2 proteases. Proteins 1991, 10, 325−
329.
AUTHOR INFORMATION
■
(8) (a) Boggetto, N.; Reboud-Ravaux, M. Dimerization inhibitors of
HIV-1 protease. Biol. Chem. 2002, 383, 1321−1324. (b) Banwarth, L.;
Reboud-Ravaux, M. An alternative strategy for inhibiting multidrug-
resistant mutants of the dimeric HIV-1 protease by targeting the
subunit interface. Biochem. Soc. Trans. 2007, 35, 551−554.
(9) Hamacher, K Relating sequence evolution of HIV1-protease to its
underlying molecular mechanics. Gene 2008, 422, 30−36.
(10) Schramm, H. J.; Nakashima, H.; Schramm, W.; Wakayama, H.;
Yamamoto, N. HIV-1 reproduction is inhibited by peptides derived
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(11) Zhang, Z. Y.; Poorman, R. A.; Maggiora, L. L.; Heinrikson, R. L.;
Kezdy, F. J. Dissociative inhibition of dimeric enzymes. Kinetic
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systematic evaluation of the inhibition of HIV-1 protease by its C-
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Corresponding Author
*For S.O.: phone, 33(0)-146835743; fax, 33(0)146835740; E-
mail, sandrine.ongeri@u-psud.fr. For M.R-R.: phone, 33(0)-
144275078; fax, 33(0)-144275140; E-mail, michele.reboud@
upmc.fr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Claire Troufflard (UMR 8076, Faculty of Pharmacy
at Chat
̂
enay-Malabry) for the NMR experiments, Christine
Philippe for their help with the syntheses, and Lixian Qin for
carrying some ANS experiments. Financial support for
A.S.M.R., A.V., and N.K. (Marie Curie Early Stage training
Fellowship of the European Community’s Sixth Framework
Programme: contract MESTCT-2004-515968) was provided
̀
by the European Community. The Ministere de la Recherche et
(13) Schramm, H. J.; de Rosny, E.; Reboud-Ravaux, M.; Buttner, J.;
Dick, A.; Schramm, W. Lipopeptides as dimerization inhibitors of
HIV-1 protease. Biol. Chem. 1999, 380, 593−596.
des Technologies (MRT) provided financial support for L.D.,
and the Italian Research Ministery provided finances for R.F.
We thank Prof. Ernesto Freire of the Department of Biology
and Biocalorimetry Center, Johns Hopkins University,
Baltimore, MD, for the generous gift of the plasmid encoding
the ANAM-11 mutant. Saquinavir was obtained through the
NIH AIDS Research and Reference Reagent Program, Division
of AIDS, NIAID, NIH. The English text was edited by Dr
Owen Parkes.
(14) Dumond, J.; Boggetto, N.; Schramm, H. J.; Schramm, W.;
Takahashi, M.; Reboud-Ravaux, M. Thyroxine-derivatives of lip-
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virus-1 protease. Biochem. Pharmacol. 2003, 65, 1097−1102.
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Jamart-Gregoire, B.; Pannecouque, C.; De Clercq, E.; Reboud-Ravaux,
M. Dimer disruption and monomer sequestration by alkyl tripeptides
are successful strategies for inhibiting wild-type and multidrug-resistant
mutated HIV-1 proteases. Biochemistry 2009, 48, 379−387.
(16) Breccia, P.; Boggetto, N.; Perez-Fernandez, R.; Van Gool, M.;
Takahashi, M.; Rene, L.; Prados, P.; Badet, B.; Reboud-Ravaux, M.; de
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(17) Zutshi, R.; Franciskovich, J.; Slultz, M.; Schwetizer, B.; Bishop,
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(18) Ulysse, L. G.; Chiemlevski, J. Restricting the flexibility of
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(19) Koh, Y.; Matsumi, S.; Das, D.; Amano, M.; Davis, D. A.; Li, J.;
Leschenko, S.; Baldridge, A.; Shioda, T.; Yarchoan, R.; Ghosh, A. K.;
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(20) Bouras, A.; Boggetto, N.; Benatalah, Z.; de Rosny, E.; Sicsic, S.;
Reboud-Ravaux, M. Design, synthesis, and evaluation of conforma-
tionally constrained tongs, new inhibitors of HIV-1 protease
dimerization. J. Med. Chem. 1999, 42, 957−962.
(21) Merabet, N.; Dumond, J.; Collinet, B.; Van Baelinghem, L.;
Boggetto, N.; Ongeri, S.; Ressad, F.; Reboud-Ravaux, M.; Sicsic, S.
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(22) Bannwarth, L.; Kessler, A.; Pethe, S.; Collinet, B.; Merabet, N.;
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ABBREVIATIONS USED
■
PR, HIV-1 protease; PIs, PR inhibitors; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide; HOBt, N-hydroxybenzo-
triazole; Boc, tert-butyloxycarbonyl; HBTU, O-benzotriazole-
N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate; ANS, 1-
anilino-8-naphthalene sulfonate; DABCYL, 4-(4′-
dimethylaminophenylazo)benzoyl; γ-abu, γ-amino butyric
acid; EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic
acid)}; EDTA, ethylenediaminetetraacetic acid; ANAM-11,
multimutated HIV-1 protease containing 11 mutations;
MDR-HM, multimutated HIV-1 protease containing six
mutations; rms, root-mean-square
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