Toward the first nonpeptidic molecular tong inhibitor of wild-type and mutated HIV-1 protease dimerization

Article abstract of DOI:10.1002/cmdc.201000308

Herein we describe the synthesis and HIV-1 protease (PR) inhibitory activity of 16 new peptidomimetic molecular tongs with a naphthalene scaffold. Their peptidic character was progressively decreased. Two of these molecules exhibited the best dimerization

Full text of DOI:10.1002/cmdc.201000308

DOI: 10.1002/cmdc.201000308
Toward the First Nonpeptidic Molecular Tong Inhibitor of
Wild-Type and Mutated HIV-1 Protease Dimerization
Anamaria Vidu,[a, c] Laure Dufau,[b] Ludovic Bannwarth,[b] Jean-Louis Soulier,[a] Sames Sicsic,[a]
Umberto Piarulli,[c] Michꢀle Reboud-Ravaux,*[b] and Sandrine Ongeri*[a]
Herein we describe the synthesis and HIV-1 protease (PR) in-
hibitory activity of 16 new peptidomimetic molecular tongs
with a naphthalene scaffold. Their peptidic character was pro-
gressively decreased. Two of these molecules exhibited the
best dimerization inhibition activity toward HIV-1 wild-type
and multimutated ANAM-11 proteases obtained to date for
this class of molecules (~40 nm for wild-type PR and 100 nm
for ANAM-11 PR). Although the peptidic character of one mo-
lecular tong was completely suppressed, the mechanism of in-
hibition and inhibitory potency toward both proteases were
maintained.
Introduction
Human immunodeficiency virus type 1 (HIV-1) protease (HIV-
PR) is the enzyme responsible for post-translational processing
of viral polyproteins and subsequent generation of the struc-
tural and functional proteins essential for viral replication.[1] As
such, HIV-PR is a major target for antiretroviral therapies. HIV-
PR is a homodimeric aspartyl protease, with two monomers of
99 residues each. The enzyme active site is located at the
bottom of a cavity within the dimer interface. Access to this
active site is monitored by the b hairpin flaps. HIV-PR is active
only in its dimeric form; because each monomer contributes
one of the two catalytic aspartic residues (Asp25), dissociation
of the enzyme homodimer results in a complete loss of catalyt-
ic activity. The protease homodimer is mainly stabilized by a
four-stranded antiparallel b sheet involving both the N- and C-
termini of each monomer (H-Pro1-Gln2-Ile3-Thr4 and Cys95-
Thr96-Leu97-Asn98-Phe99-OH). This region appears to be
highly conserved in HIV-1 isolates.[2] More than 50% of the hy-
drogen bonds along the dimer interface involve terminal resi-
dues 1–4 and 96–99 at the antiparallel b sheet.[3] The contacts
in this region contribute to 75% of the total Gibbs free energy
of HIV-PR dimerization.[4] Many HIV-PR inhibitors (PIs) have
been developed which target the active site. However, several
mutations located within or outside of the HIV-PR active site
result in lower affinity towards inhibitors,[5,6] and alternative
strategies to circumvent the cross-resistance of HIV-PR inhibi-
tors are crucially needed. Recently approved PIs, tipranavir and
darunavir, are active against HIV-1 protease variants resistant
to multiple PIs. Data obtained using a FRET-based HIV-1 ex-
pression assay suggested that these molecules act as conven-
tional protease inhibitors. They also block protease dimeriza-
tion in cells, but do not dissociate already dimerized cellular
proteases.[7] The protease termini b sheet interface has been
explored as a dimerization inhibition target, and known inhibi-
tors that target this region are able to block or disrupt the for-
mation of the homodimer.[8] C- and N-terminal mimetic pep-
tides[9–11] and lipopeptides[12–14] have proven to be efficient PR
dimerization inhibitors. A bicyclic guanidinium group has been
introduced between the peptidic and lipophilic moieties,[15]
and the interface peptides have also been cross-linked with
flexible,[16] semi-rigid,[17] or rigid spacers.[18–20]
Our strategy involved the synthesis of conformationally con-
strained, scaffold-based molecular tongs, attached to two pep-
tidic strands by carboxypropyl linkers (Figure 1). The two pepti-
dic strands were able to be suitably oriented by the scaffold,
allowing putative formation of an antiparallel b sheet with the
C-terminal end of one HIV-1 PR monomer and resulting in an
entropic benefit (Figure 1).[18] Naphthalene or quinoline scaf-
folds with symmetrical or asymmetrical peptidic strands were
efficient antidimers (Kid up to 80 nm).[18,19] The introduction of
amino acid mimetic fragments, specifically a 5-amino-2-me-
thoxybenzamide or a 3-amino-6-methylpyridin(1H)-one group
in a single strand, increased the metabolic stability of the mo-
lecular tongs without compromising their ability to inhibit
wild-type and mutated HIV-1 proteases in vitro.[20] These
groups were selected to replace two amino acids, as they were
hypothesized to provide the same array of hydrogen bonding
groups as one edge of a peptidic b strand. Our previous results
[a] Dr. A. Vidu, Dr. J.-L. Soulier, Prof. S. Sicsic, Dr. S. Ongeri
Molꢀcules Fluorꢀes et Chimie Mꢀdicinale, BioCIS UMR-CNRS 8076, IPSIT
Universitꢀ Paris-Sud 11, Facultꢀ de Pharmacie
5 rue Jean-Baptiste Clꢀment, 92296, Chꢁtenay-Malabry Cedex (France)
Fax: (+33)146835740
E-mail: Sandrine.Ongeri@u-psud.fr
[b] L. Dufau, Dr. L. Bannwarth, Prof. M. Reboud-Ravaux
UPMC-UR4, Enzymologie Molꢀculaire et Fonctionnelle
7 Quai St Bernard 75252 Paris Cedex 05 (France)
Fax: (+33)144-275-140
E-mail: michele.reboud@upmc.fr
[c] Dr. A. Vidu, Prof. U. Piarulli
Universitꢂ degli Studi dell’Insubria, Dipartimento di Scienze Chimiche e Am-
bientali, Via Valleggio, 11, 22100 Como (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000308.
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S. Ongeri, M. Reboud-Ravaux, et al.
MED
We also report the first nonpeptidic molecular tong 15 that
acts as an antidimer.
Results and Discussion
Chemistry
Protected peptidomimetic strand 18 was obtained by coupling
2-(3-amino-2-oxopyridin-1-(2H)-yl)acetamide 17[20] to N-Boc-N-
Z-Lys-OH, using HBTU and HOBt as coupling agents, in good
yield (Scheme 1). Compound 18 was selectively deprotected at
the a-nitrogen by acidic hydrolysis (TFA in CH2Cl2) to afford 19,
or at the e-nitrogen by hydrogenolysis to give 20, both in ex-
cellent yields (Scheme 1).
Figure 1. Schematic representation of a putative HIV-1 monomer–molecular
tong complex.
suggest that the flexibility of
valine is required for formation
of the hydrogen bonds in a four-
stranded antiparallel b sheet,
which involves both the N- and
C-termini of the PR monomer.[20]
This report describes the syn-
thesis and enzyme inhibitory ac-
tivity against wild-type and mu-
tated HIV-1 proteases of new
molecular tongs with amino acid
mimetic fragments in one (mole-
cules 1–6, Figure 2) or two (mol-
ecules 7–16, Figure 2) strands.
We decreased the peptidic char-
acter of the molecular tongs by
introducing
peptidomimetic
fragments, first replacing two
and then three amino acids in
one or both strands. The 5-
amino-2-methoxybenzamide or
3-amino-6-methylpyridin(1H)-one
groups were first attached to the
carboxypropyl linker through a
valine or lysine residue for mole-
cules 1–2 and 7–12. In order to
totally suppress the peptidic
character of the strands, and to
increase flexibility between the
scaffold and the mimetic unit,
the amino acid residue (valine or
lysine) was replaced by a longer
and more flexible moiety. The
mimetic unit was attached to
the carboxypropyl linker through
the e-amino group of a lysine
residue (molecules 3–6 and 13–
16). We describe herein the
most potent antidimeric molecu-
lar tongs 1 and 2 obtained to
date for this class of molecules. Figure 2. Molecular tongs 1–16.
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Inhibitors of HIV-1 Protease Dimerization
(Scheme 4). Cleavage of the N-
Boc group of compounds 13
and 15 gave molecular tongs 14
and 16, respectively, while hy-
drogenolysis of 9 and 11 afford-
ed molecular tongs 10 and 12,
respectively (Scheme 4).
Biology
Scheme 1. Synthesis of peptidomimetic-containing strands 19 and 20. a) N-Boc-N-Z-K-OH, HBTU, HOBt, DIPEA,
DMF, 48 h, room temperature; b) TFA, CH2Cl2, 2 h, room temperature; c) H2, 10% Pd/C, MeOH, 12 h, room temper-
ature.
The inhibitory activities of com-
pounds 1–16 were assayed
against recombinant wild-type
PR (WT-PR) at pH 4.7 and 308C using a fluorimetric assay.[18–20]
The most efficient inhibitors were also assayed against the
multidrug-resistant mutated protease ANAM-11. Zhang–Poor-
man kinetic analyses were used to characterize the mechanism
of inhibition as dimerization alone (parallel lines), competitive
inhibition (altered slopes and unaltered y-axis intercepts), and
mixed inhibition (altered slopes
Protected peptidomimetic strand 22 was obtained from N-
(3-hydrazinocarbonyl-4-methoxyphenyl)acetamide (21),[20] fol-
lowing the same procedure as described for 18 (Scheme 2).
Compound 22 was deprotected by acidic hydrolysis to give
23, or by hydrogenolysis to give 24, both in good yields
(Scheme 2).
and y-axis intercepts). The results
are summarized in Table 1. Paral-
lel lines were obtained for com-
pounds 1–6, 8, and 15, demon-
strating that they act purely as
dimerization inhibitors (Figure 3).
Compounds 7, 9, 11, and 13 ex-
hibited mixed inhibition. Weak
inhibitory effects were observed
with compounds 10, 12, 14, and
16 (28–30% at 14 mm for 12, or
28 mm for 10, 14, and 16).
Scheme 2. Synthesis of peptidomimetic-containing strands 23 and 24: a) N-Boc-N-Z-K-OH, HBTU, HOBt, DIPEA,
DMF, 24 h, room temperature; b) TFA, CH2Cl2, 1 h, room temperature; c) H2, 10% Pd/C, MeOH, 12 h, room temper-
ature.
Molecular tongs 1–3 and 5 were synthesized by
condensation of 4-[7-(3-carboxypropoxy)naphthalen-
2-yloxy]butyryl-Val-Leu-Val-OMe (25)[19] with peptido-
mimetics 19, 23, 20, and 24, respectively, in satisfac-
tory yields (Scheme 3). Acidic cleavage of the tert-
butyl carbamate group of compounds 3 and 5 gave
molecular tongs 4 and 6, respectively (Scheme 3).
Molecular tongs 7–9, 11, 13, and 15 were synthe-
sized from 4-{[7-(3-carboxypropoxy)-2-naphthyl]oxy}-
butanoic acid (26)[18] by coupling with respective
peptidomimetics 27, 28, 19, 23, 20, and 24
Scheme 4. Synthesis of molecular tongs 7–16. a) HBTU, HOBt, DIPEA, 19, 20, 23, 24, 27,
or 28, DMF, 48 h, room temperature; b) for 14 or 16: 13 or 15, TFA, CH2Cl2, 30 min, room
temperature; c) for 10 or 12: 9 or 11, H2, 10% Pd/C, MeOH/DMF, 12 h, room tempera-
ture.
Based on the observed results, it was noted that replace-
ment of the valine residue by an N-protected lysine (asymmet-
rical molecular tongs 1 and 2) dramatically increases the inhibi-
tory potency (40 nm for 1 relative to 400 nm for the valine ana-
logue,[20] and 60 nm for 2 relative to 200 nm for the valine ana-
logue[20]). The symmetric molecular tongs 7, 9, and 11 are fairly
Scheme 3. Synthesis of molecular tongs 1–6. a) HBTU, HOBt, DIPEA, 19, 20,
23, or 24, DMF, 48 h, room temperature; b) for 4 or 6: 3 or 5, TFA, CH2Cl2,
30 min, room temperature.
efficient inhibitors that act through a mixed inhibition mecha-
nism. For example, 11 exhibits both a dimerization inhibition
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S. Ongeri, M. Reboud-Ravaux, et al.
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tion and inhibitory efficacy (15 vs 13). Deprotection of the
amino moiety of the strands noticeably decreased the inhibito-
ry potency (10 vs. 9, 12 vs. 11, 14 vs. 13, and 16 vs. 15), con-
firming a favorable influence of the hydrophobic character of
the tong arms on inhibitory activity.[18,19] Remarkably, new
pseudopeptidic molecular tongs 1, 2, 5, and 15 behaved as an-
tidimers against the multimutated protease ANAM-11, which is
analogous to a protease found in multi-drug resistant viruses.
In contrast, the affinity of ritonavir for this mutated protease[21]
was about 78000-fold lower than for wild-type PR, demonstrat-
ing that the peptidomimetic molecular tongs 1, 2, 5, and 15
can still efficiently inhibit dimerization of mutated proteases.
Compound 2 exhibits 20-fold higher in vitro activity toward
ANAM-11 protease than does ritonavir.[21] This confirms that in-
hibitors of PR dimerization are insensitive to protease muta-
tions.
Table 1. In vitro inhibition of wild-type PR and ANAM-11 by molecular
tongs (308C, pH 4.7).
Compd
WT PR or ANAM-11
K
ic [mm][a]
Kid [mm][b]
1
WT
ANAM-11
WT
ANAM-11
WT
0.04
0.25
0.06
0.1
1.2
4.5
2
3
4
5
WT
WT
0.4
ANAM-11
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
ANAM-11
WT
1.36
3
6
7
8
0.550
0.2
0.053
0.220
0.12
9
[c]
10
11
12
13
14
15
–
0.15
0.053
[c]
–
31
17
–
[c]
Conclusions
0.28
2
[c]
Molecular tongs, containing a naphthalene scaffold and pepti-
dic side arms, were reported to be inhibitors of the PR inter-
face.[18,19] Replacement of two amino acids by peptidomimetics
in one strand of these molecular tongs led to proteolysis-resist-
ant inhibitors.[20] We replaced the remaining valine residue by a
more hydrophobic and flexible side chain of the Cbz-protected
lysine residue to afford the most potent molecular tong inhibi-
tor of PR dimerization (~40 nm). In addition, replacement of
two amino acids by peptidomimetics in both strands of these
symmetrical molecular tongs predominantly resulted in a
mixed mechanism of inhibition (competitive inhibition and an-
tidimeric activity). The replacement of three amino acids by
nonpeptidic fragments in each of two strands of the molecular
tongs resulted in the first potent nonpeptidic protease inhibi-
tor able to dissociate the mature protease dimer. In this way,
we demonstrated that total suppression of peptidic character
within this class of molecules does not alter their mechanism
of action.
16
–
[a] Competitive active site inhibition; [b] dimerization inhibition;
[c] percent inhibition at 28 mm: 28 (10), 30 (14 and 16); at 14 mm: 30 (12).
Standard errors of initial rates are <5%.
Four dimerization inhibitors were also evaluated against the
ANAM-11 protease, which contains 11 mutations, and were
found to be equally active toward both ANAM-11 and wild-
type PR. We corroborated previous findings that peptidomi-
metic molecular tongs are candidates for successfully overcom-
ing the resistance presently encountered with classical pro-
tease inhibitors. Deprotection of amino groups in the strands
noticeably decreased the inhibitory potency of these mole-
cules. Further studies of PR dimerization inhibitors containing
peptidomimetics will focus on the synthesis of molecular
tongs with greater hydrophilic character.
p
p
Figure 3. Plots of [E]0/ vi vs vi for the hydrolysis of the fluorogenic sub-
strate DABCYL-g-abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS by wild-type
*
HIV-1 PR at pH 4.7 and 308C in the absence ( ) and presence of compound
15 at 9.25 mm ( ) and 7.08 mm ( ).
~
&
component (Kid =53 nm) and a slight competitive inhibition
component (Kic =150 nm). Increasing both the length and flexi-
bility of the nonpeptidic strand in asymmetrical molecular
tong 3 has a detrimental effect on the inhibitory potency (3 vs.
1). In contrast, a simultaneous increase in both length and flex-
ibility of the nonpeptidic strand in symmetrical molecular tong
15 has a favorable effect on inhibitory potency and results in
purely dimerization inhibition activity (15 vs. 11) although the
peptidic character of molecular tong 15 was completely sup-
pressed. The hydrazide motif favors both dimerization inhibi-
Experimental Section
Synthesis
Common solvents were purchased from commercial sources. N,N-
Dimethylformamide (DMF) was distilled over CaSO4, tetrahydrofur-
an (THF) was distilled over sodium/benzophenone, and CH3CN was
distilled over CaCl2. TLC was performed on silica gel 60 F250
(0.26 mm thickness) plates. The plates were visualized with UV
1902
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Inhibitors of HIV-1 Protease Dimerization
light (l 254 nm), or with a 3.5% solution of phosphomolybdic acid
or ninhydrin in EtOH. Liquid chromatography was performed on
Merck 60 silica gel (230–400 mesh). Protected amino acids O-ben-
[5-Amino-5-(1-carbamoylmethyl-2-oxo-1,2-dihydropyridin-3-yl-
carbamoyl)pentyl]carbamic acid tert-butyl ester (20). Pd/C 10%
(53 mg, 20% mass) was added to a solution of 18 (264 mg,
0.5 mmol) in MeOH (25 mL). The reaction flask was purged three
times with hydrogen, and stirring was maintained under hydrogen
atmosphere at room temperature for 12 h. After filtration through
Celite, the cake was washed with MeOH (200 mL), and the filtrate
was concentrated to afford a solid that was triturated with cyclo-
hexane and petroleum ether to yield 20 as a white solid: mp: 146–
1488C (190 mg, 96% yield); 1H NMR: d=9.2 (s, 1H), 8.4 (d, J=
7.3 Hz, 1H), 7.6 (s, 1H), 7.1 (d, J=7.3 Hz, 1H), 6.9 (s, 1H), 5.9 (bs,
1H), 5.4 (bs, 1H), 6.2 (t, J=7.3 Hz, 1H), 4.6 (s, 2H), 4.4 (m, 1H), 2.7
(m, 2H), 1.9 (m, 1H), 1.7 (m, 1H), 1.4–1.2 (m, 13H); 13C NMR: d=
171.7, 168.9, 156.6, 156.2, 131.3, 127.8, 123.2, 107.3, 78.4, 54.0, 53.3,
41.4, 32.4, 30.0, 28.4, 22.8; ESI+ MS m/z: 418 [M+Na]+; HRMS calcd
for C18H29N5O5+Na: 418.2066, found: 418.2078.
zotriazol-1-yl-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
(HBTU) and 1-hydroxybenzotriazol (HOBT) were purchased from
commercial sources. H-Val-Leu-Val-OMe,[19] 4-[7-(3-carboxypropoxy)-
naphtalen-2-yloxy]butyryl-Val-Leu-Val-OMe (25),[19] 2-(3-amino-2-
oxo-2H-pyridin-1-yl)acetamide (17),[20] N-(3-hydrazinocarbonyl-4-
methoxyphenyl)acetamide (21),[20] 4-{[7-(3-carboxypropoxy)-2-naph-
tyl]oxy}butanoic acid (26),[18] 2-amino-N-(1-carbamoylmethyl-2-oxo-
1,2-dihydropyridin-3-yl)-3-methylbutyramide (27)[20] and N-{3-[N’-(2-
amino-3-methylbutyryl)hydrazinocarbonyl]-4-methoxyphenyl}aceta-
mide (28)[20] were prepared according to published methods. Melt-
ing points were determined on a Kofler melting point apparatus.
NMR spectra were performed on a Ultrafield AVANCE 300 (1H,
300 MHz; 13C, 75 MHz) or a Bruker AVANCE 400 (1H, 400 MHz; 13C,
100 MHz). Unless otherwise stated, CDCl3 was used as solvent.
Chemical shifts (d) are in ppm, using the following abbreviations:
singlet (s), doublet (d), doublet doublet (dd), triplet (t), quintuplet
(quint), multiplet (m), broad multiplet (bm), and broad singlet (bs).
Mass spectra were obtained using a Bruker Esquire electrospray
ionization apparatus at the SAMM (Faculty of Pharmacy at Chꢂte-
nay-Malabry, France). Elemental analyses (C, H, N) were performed
on a PerkinElmer CHN Analyser 2400 at the Microanalyses Service
of the Faculty of Pharmacy at Chꢂtenay-Malabry (France). Elemen-
tal analysis data are included in the Supporting Information.
{6-[N’-(5-Acetylamino-2-methoxybenzoyl)hydrazino]-5-benzyl-
oxycarbonylamino-6-oxohexyl}carbamic acid tert-butyl ester
(22). Compound 22 was synthesized from 21, following the same
procedure described for 18, and was obtained as a white solid in
73% yield: mp: 136–1388C; 1H NMR: d=11.9 (s, 1H), 11.3 (d, J=
7.0 Hz, 1H), 8.8 (s, 1H), 8.4 (d, J=9.0 Hz, 1H), 7.9 (s, 1H), 7.3 (m,
5H), 7.0 (d, J=9.0 Hz, 1H), 5.6 (d, J=7.5 Hz, 1H), 5.0 (s, 2H), 4.9 (m,
1H), 4.7 (s, 1H), 4.0 (s, 3H), 3.0 (m, 2H), 2.2 (s, 3H), 1.8 (m, 1H), 1.7
(m, 1H), 1.4 (s, 9H), 1.4–1.3 (m, 4H); 13C NMR: d=169.0, 166.3,
158.6, 156.3, 155.6, 153.6, 136.6, 133.1, 128.4, 128.0, 126.1, 122.5,
117.8, 112.4, 79.8, 66.5, 56.5, 52.0, 40.7, 34.0, 29.5, 28.6, 24.6, 21.9;
ESI+ MS m/z: 608 [M+Na]+; Anal. (C, H, N): C29H39N5O8.
[5-Benzyloxycarbonylamino-5-(1-carbamoylmethyl-2-oxo-1,2-di-
hydropyridin-3-ylcarbamoyl)pentyl]carbamic
acid
tert-butyl
ester (18). Compound 17 (436 mg, 2.61 mmol) and Na-Boc-Ne-Z-Lys
(1.09 g, 2.87 mmol) were dissolved in DMF (20 mL). DIPEA (1.1 mL,
6.53 mmol), HBTU (1.19 g, 3.13 mmol), and HOBt (388 mg,
2.87 mmol) were successively added to the reaction mixture. The
reaction mixture was stirred under argon at room temperature for
48 h. DMF was evaporated under reduced pressure and the residue
was dissolved in EtOAc (200 mL). The organic phase was washed
with 10% aqueous citric acid (50 mL), H2O (100 mL), 10% aqueous
K2CO3 (50 mL), and brine (25 mL). The organic phase was dried
over Na2SO4, filtered and concentrated under reduced pressure.
The resulting crude product was purified by flash chromatography
on silica gel and eluted with EtOAc/MeOH (95:5) to give 18 as a
white solid: mp: 192–1938C (818 mg, 79% yield); 1H NMR
([D6]DMSO): d=9.1 (s, 1H), 8.2 (dd, J=1.3, 7.3 Hz, 1H), 7.6 (s, 1H),
7.6–7.3 (m, 6H,), 7.2 (s, 1H), 6.2 (t, J=7.3 Hz, 1H), 5.0 (s, 2H), 4.6 (s,
2H), 4.0 (m, 1H), 3.0 (q, J=6 Hz, 2H), 1.7 (m, 1H), 1.6 (m, 1H), 1.4–
1.3 (m, 13H); 13C NMR ([D6]DMSO): d=171.8, 168.3, 156.7, 156.1,
155.7, 137.3, 133.0, 128.3, 128.0, 127.7, 122.0, 104.7, 78.5, 65.1, 55.4,
51.2, 40.1, 30.8, 29.0, 28.1, 22.9; ESI+ MS m/z: 553 [M+Na]+, 568
[M+39]+; Anal. (C, H, N): C26H35N5O7.
{6-[N’-(5-Acetylamino-2-methoxybenzoyl)hydrazino]-5-amino-6-
oxohexyl}carbamic acid benzyl ester (23). TFA (4 mL) was added
to a solution of 22 (350 mg, 0.60 mmol) in dry CH2Cl2 (4 mL). The
mixture stirred at room temperature for 1 h, then the solvent was
evaporated under reduced pressure. The crude product was crys-
tallized from MeOH/Et2O to yield compound 23 as a light pink
solid: mp: 140–1428C (315 mg, 88% yield); 1H NMR ([D6]DMSO):
d=10.9 (s, 1H), 10.2 (s, 1H), 10.0 (s, 1H), 8.2 (bs, 2H), 8.0 (s, 1H),
7.7 (d, J=9.0 Hz, 1H), 7.4–7.3 (m, 6H), 7.1 (d, J=9.0 Hz, 1H), 5.0 (s,
2H), 3.8 (m, 4H, H11), 3.0 (m, 2H), 2.0 (s, 3H), 1.8 (m, 2H), 1.5–1.4
(m, 4H); 13C NMR ([D4]MeOH): d=171.6, 169.1, 158.6, 166.4, 159.1,
155.7, 133.5, 129.5, 129.0, 128.7, 127.0, 124.4, 120.9, 113.5, 67.4,
56.9, 53.4, 41.2, 32.2, 30.5, 23.6, 22.9; APCI+ MS m/z: 487 [M+H]+;
Anal. (C, H, N): C26H32N5O6·CF3COOH·1.5H2O.
{6-[N’-(5-Acetylamino-2-methoxybenzoyl)hydrazino]5-amino-6-
oxohexyl}carbamic acid tert-butyl ester (24). Pd/C 10% (23 mg,
20% mass) was added to a solution of 22 (150 mg, 0.26 mmol) in
MeOH (15 mL). The reaction flask was purged three times with hy-
drogen, and stirring was maintained under hydrogen atmosphere
at room temperature for 12 h. The mixture was filtered through
celite, and the cake was washed with MeOH (200 mL). The filtrate
was evaporated to dryness to yield crude product 24 as a colorless
[5-Amino-5-(1-carbamoylmethyl-2-oxo-1,2-dihydropyridin-3-yl-
carbamoyl)pentyl]carbamic acid benzyl ester in trifluoroacetic
acid (19). Trifluoroacetic acid (TFA; 5 mL) was added to a solution
of 18 (300 mg, 0.57 mmol) in dry CH2Cl2 (5 mL). The mixture was
stirred at room temperature for 1.5 h, then the solvent was evapo-
rated under reduced pressure. The crude product was crystallized
from MeOH/Et2O to yield 19 as a beige solid: mp: 124–1268C
1
oil (111 mg, 96% yield): H NMR: d=9.3 (s, 1H), 8.9 (s, 1H), 8.3 (s,
2H), 7.9 (m, 1H), 6.9 (m, 1H), 5.6 (m, 1H), 4.6 (m, 2H), 4.1–3.9 (m,
4H), 2.3–2.2 (m, 5H), 1.8 (bm, 2H), 1.5–1.3 (m, 13H); 13C NMR: d=
169.2, 169.1, 158.5, 155.6, 153.7, 132.9, 125.9, 122.7, 118.2, 112.0,
79.9, 56.47, 52.3, 52.2, 50.2, 27.0, 28.4, 24.4, 22.2; ESI+ MS m/z: 452
[M+H]+; HRMS calcd for C21H33N5O6+H: 452.2509, found: 452.2526.
1
(248 mg, 100% yield); H NMR ([D6]DMSO): d=10.0 (s, 1H), 8.3–8.2
(m, 4H), 7.6 (s, 1H), 7.4–7.2 (m, 7H), 6.3 (t, J=7.2 Hz, 1H), 5.0 (s,
2H), 4.6 (s, 2H), 4.26 (m, 1H), 2.98 (m, 2H), 1.74 (m, 2H) 1.4–1.3 (m,
4H); 13C NMR (CD3OD) d=171.4, 169.2, 159.15, 156.5, 135.1, 129.5,
129.3, 129.0, 128.8, 127.3, 107.3, 67.4, 55.0, 52.9, 41.2, 32.4, 30.5,
23.0; ESI+ MS m/z: 430 [M+H]+; HRMS calcd for C21H27N5O5+H:
430.2090, found: 430.2112.
Synthesis of molecular tong 1. Compounds 25 (200 mg,
0.3 mmol) and 19 (195 mg, 0.36 mmol) were dissolved in DMF
(10 mL). DIPEA (0.37 mL, 2.1 mmol), HBTU (284 mg, 0.75 mmol),
and HOBt (89 mg, 0.66 mmol) were successively added to the reac-
tion mixture, which stirred under argon at room temperature for
ChemMedChem 2010, 5, 1899 – 1906
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1903

S. Ongeri, M. Reboud-Ravaux, et al.
MED
48 h. DMF was evaporated under reduced pressure, and the resi-
due was washed successively with EtOAc, CH2Cl2, MeOH, H2O
(100 mL), 10% aqueous citric acid (50 mL), 10% aqueous K2CO3
(50 mL), and H2O again (50 mL). Product 1 was obtained as a white
30.6, 30.3, 29.2, 25.5, 25.4, 24.5, 23.4, 22.3, 22.1, 19.6, 19.3, 18.6;
ESI+ MS m/z: 936 [M+H]+, 953 [M+Na]+; Anal. (C, H, N):
C48H70N8O11·CF3COOH.
Synthesis of molecular tong 5. Tong 5 was synthesized from 24,
following the same procedure as described for 1 from 25, and was
1
solid: mp: 233–2358C (277 mg, 86%); H NMR ([D6]DMSO): d=9.2
(s, 1H), 8.3 (d, J=7.3 Hz, 1H), 8.2 (d, J=7.2 Hz, 1H), 8.0–7.9 (m,
3H), 7.7 (d, J=8.9 Hz, 2H), 7.6 (s, 1H), 7.3–7.1 (m, 6H), 7.2 (s, 2H),
7.1 (s, 2H), 7.0 (d, J=8.9 Hz, 2H), 6.2 (t, J=7.2 Hz, 1H), 5.0 (s, 2H),
4.5 (s, 2H), 4.4–4.3 (m, 2H), 4.1 (q, J=7.2 Hz, 2H), 4.0 (q, J=6.5 Hz,
4H), 3.6 (s, 3H), 2.9 (q, J=6.1 Hz, 2H), 2.4–2.3 (m, 4H), 2.2–2.0 (m,
6H), 1.8–1.7 (m, 1H), 1.6–1.5 (m, 2H), 1.5–1.3 (m, 4H), 1.3 (m, 2H),
0.9–0.8 (m, 18H); 13C NMR ([D6]DMSO): d=172.3, 172.2, 171.7,
171.3, 170.9, 168.3, 157.0, 156.7, 156.1, 137.3, 135.7, 133.1, 128.9,
128.3, 128.1, 127.7, 123.7, 122.3, 115.9, 106.1, 104.7, 66.9, 65.1, 57.8,
57.3, 53.7, 51.6, 51.3, 50.8, 40.7, 40.1, 31.6, 30.7, 30.3, 29.9, 29.1,
25.0, 24.9, 24.1, 22.9, 22.8, 21.6, 19.2, 18.8, 18.2, 18.1; ESI+ MS m/z:
1092 [M+Na]+, 1108 [M+K]+; HRMS (calcd for C56H76N8O13+Na:
1092.2376, found: 1091.5454; Anal. (C, H, N): C56H76N8O13.
1
obtained as a white solid in 53% yield: mp: 150–1568C; H NMR:
d=11.0 (bs, 1H), 9.2 (bs, 1H), 8.5 (d, J=9 Hz, 1H), 8.4 (bs, 1H), 8.0
(s, 1H), 7.9 (bs, 1H,), 7.58 (d, J=8.8 Hz, 2H), 7.2 (bs, 1H), 7.2–6.9
(m, 5H), 6.1–5.8 (m, 2H), 4.8 (m, 1H), 4.7 (m, 1H), 4.6 (m, 1H), 4.5
(m, 1H), 4.1 (m, 4H), 4.0 (s, 3H), 3.7 (s, 3H), 3.1 (m, 2H), 2.6 (m,
1H), 2.4 (m, 4H), 2.3 (m, 1H), 2.1–2.2 (m, 8H„ 1.9 (m, 1H), 1.6–1.5
(m, 5H), 1.4 (s, 9H), 1.3 (m, 2H), 0.9–0.8 (m, 18H); 13C NMR: d=
172.6, 172.1, 172.1, 172.0–171.9, 171.7, 168.8, 167.6, 167.4, 157.5,
157.4, 153.4, 134.9, 133.5, 129.1, 125.3, 124.3, 122.3, 116.5, 116.2,
118.0, 106.4, 106.2, 79.5, 66.9, 66.7, 57.9, 57.4, 56.5, 52.8, 52.0, 51.6,
40.9, 39.1, 32.6, 31.9, 31.0, 29.3, 28.8, 28.3, 25.1, 24.8, 24.2, 22.6,
22.4, 19.0, 18.6, 17.8; ESI+ MS m/z: 1114 [M+Na]+; Anal. (C, H, N):
C56H82N8O14.
Synthesis of molecular tong 2. Tong 2 was synthesized from 23,
following the same procedure as described for 1 from 25, and was
obtained as a white solid in 63% yield: mp: 199–2018C; H NMR
Synthesis of molecular tong 6. TFA (2 mL, 26 mmol) was added to
a solution of 5 (100 mg, 0.09 mmol) in dry CH2Cl2 (2 mL). The mix-
ture stirred for 2 h, then the solvent was evaporated under re-
duced pressure. Azeotropic removal of excess TFA was carried out
using toluene. The crude product was recrystallized from MeOH/
Et2O to give product 6 as a light pink solid: mp: 136–1398C
1
([D6]DMSO): d=10.3 (bs, 1H), 10.1 (bs, 1H), 9.9 (s, 1H), 8.0 (d, J=
7.1 Hz, 1H), 8.0–7.9 (m, 4H), 7.76 (dd, J=2.4, 9.0 Hz, 1H), 7.7 (d, J=
8.8 Hz, 2H), 7.4–7.3 (m, 5H), 7.2 (t, J=5.4 Hz, 1H), 7.2 (m, 2H), 7.1
(d, J=9.0 Hz, 1H), 7.0 (m, 2H), 5.0 (s, 2H), 4.4 (q, J=7.8 Hz, 2H),
4.16 (q, J=7.8 Hz, 2H), 4.04 (m, 4H), 3.84 (s, 3H), 3.60 (s, 3H), 3.0
(q, J=6.2 Hz, 2H), 2.3 (t, J=7.0 Hz, 4H), 2.0–1.9 (m, 9H), 1.7 (m,
1H), 1.6 (m, 2H), 1.5–1.4 (m, 4H), 1.3 (m, 2H), 0.9–0.8 (m, 18H);
13C NMR ([D6]DMSO): d=172.2, 171.7, 171.5, 170.9, 168.0, 163.3,
157.0, 156.1, 152.6, 137.3, 135.8, 132.6, 129.0, 128.3, 127.7, 123.7,
123.2, 121.3, 121.2, 115.9, 112.4, 106.1, 67.0, 66.9, 65.1, 57.8, 57.3,
56.1, 51.6, 51.1, 50.8, 40.7, 40.1, 32.1, 31.6, 30.2, 29.9, 29.2, 25.0,
24.9, 24.1, 23.8, 22.9, 22.6, 21.6, 19.2, 18.8, 18.2, 18.1; ESI+ MS m/z:
1148 [M+Na]+, 1164 [M+K]+; Anal. (C, H, N): C59H80N8O14.
1
(79 mg, 78%); H NMR ([D6]DMSO): d=10.1 (bs, 1H), 9.9 (bs, 2H),
8.0–7.7 (m, 8H), 7.2–7.0 (m, 3H), 6.9 (s, 2H), 4.4 (bs, 1H), 4.1–4.0
(m, 6H), 3.8 (m, 4H), 3.6 (s, 3H), 3.0 (m, 2H), 2.5–2.4 (m, 4H), 2.0
(m, 10H), 1.78 (m, 2H), 1.6–1.4 (m, 8H), 0.9–0.7 (m, 18H); 13C NMR
([D6]DMSO): d=172.2, 171.7, 171.4, 170.9, 168.0, 157.0, 152.6,
135.7, 132.6, 128.9, 124.5, 123.7, 121.3, 115.9, 112.5, 106.1, 66.9,
57.8, 57.2, 56.2, 51.6, 51.1, 50.8, 40.7, 32.0, 31.8, 31.6, 30.2, 29.8,
25.0, 24.9, 24.1, 23.8, 22.9, 21.6, 21.4, 19.16, 18.8, 18.2, 18.1; ESIꢀ MS
m/z: 990 [MꢀH]+; Anal. (C, H, N): (C51H74N8O12·CF3COOH·1.5H2O.
Synthesis of molecular tong 7. Tong 7 was synthesized from 27,
following the same procedure as described for 1 from 25, and was
obtained as a white solid in 54% yield: mp: 191–1968C; H NMR
Synthesis of molecular tong 3. Tong 3 was synthesized from 20,
following the same procedure as described for 1 from 25, and was
obtained as a white solid in 57% yield: mp: 172–1768C; H NMR
1
1
([D6]DMSO): d=9.16 (s, 1H), 8.25 (d, J=8.0 Hz, 1H), 8.20 (d, J=
7.0 Hz, 1H), 7.69 (d, J=8.9 Hz, 1H), 7.62 (s, 1H), 7.31 (d, J=7.0 Hz,
1H), 7.21 (s, 1H), 7.16 (s, 1H), 6.97 (d, J=8.9 Hz, 1H), 6.23 (t, J=
7.0 Hz, 1H), 4.56 (s, 2H), 4.37 (t, J=8 Hz, 1H), 4.06 (t, J=7 Hz, 2H),
2.43 (t, J=6.6 Hz, 2H), 2.11 (m, 1H), 2.02 (m, 2H), 0.89 (d, J=
6.4 Hz, 6H); 13C NMR (100 MHz, [D6]DMSO): d=172.3, 170.7, 168.3,
157.0, 156.7, 135.7, 133.2, 128.9, 128.0, 123.7, 122.5, 115.9, 106.2,
104.6, 66.9, 58.9, 51.3, 31.6, 29.7, 25.0, 19.2, 18.0; ESI+ MS m/z: 851
[M+Na]+, 867 [M+K]+; Anal. (C, H, N): C42H52N8O10·2H2O.
([D6]DMSO): d=9.2 (s, 1H), 8.2 (d, J=7.5, 1H), 7.9 (m, 3H), 7.8 (s,
1H), 7.7 (d, J=8.8, 2H), 7.6 (s, 1H), 7.4 (d, J=6.4, 1H), 7.3 (d, J=
7.0, 1H), 7.2 (m, 3H), 7.0 (d, J=9.0, 2H), 6.2 (t, J=6.7, 1H), 4.6 (s,
2H), 4.38 (m, 1H), 4.2–4.1 (m, 2H), 4.1–4.0 (m, 5H), 3.6 (s, 3H), 3.0
(m, 2H), 2.4–2.2 (m, 4H), 2.0–1.9 (m, 6H), 1.7–1.6 (m, 1H), 1.6–1.5
(m, 2H), 1.4–1.2 (m, 15H), 0.9–0.7 (m, 18H); 13C NMR ([D6]DMSO):
d=172.6, 172.3, 172.1, 171.9, 171.4, 168.7, 157.5, 157.2, 136.2,
133.4, 129.4, 128.5, 124.2, 122.5, 116.4, 106.7, 105.2, 79.0, 67.4, 58.3,
57.7, 55.9, 52.0, 51.7, 51.3, 41.2, 38.7, 32.3, 32.1, 31.2, 30.6, 30.3,
29.2, 28.6, 25.5, 25.4, 24.5, 23.5, 23.4, 22.1, 19.6, 19.3, 18.6; ESIꢀ MS
m/z 1034 [MꢀH]+; Anal. (C53H78N8O13·3H2O) C, H, N.
Synthesis of molecular tong 8. Tong 8 was synthesized from 28,
following the same procedure as described for 1 from 25, and was
1
obtained as a white solid in 89% yield: mp: 224–2468C; H NMR
Synthesis of molecular tong 4. TFA (2 mL, 26 mmol) was added to
a solution of 3 (100 mg, 0.1 mmol) in dry CH2Cl2 (2 mL). The mix-
ture was stirred for 30 min, then the solvent was evaporated under
reduced pressure. Azeotropic removal of excess TFA was carried
out using toluene. The crude product was recrystallized from
MeOH/Et2O to give 4 as a white solid: mp: 180–1828C (100 mg,
(400 MHz, [D6]DMSO): d=10.32 (s, 1H), 9.93 (s, 2H), 8.00 (d, J=
9.0 Hz, 1H), 7.94 (s, 1H), 7.74 (dd, J=2.7, 9.0 Hz, 1H), 7.70 (d, J=
9.0 Hz, 1H), 7.18 (bd, J=2 Hz, 1H), 7.09 (d, J=9.0 Hz, 1H), 6.97 (dd,
J=2.0, 9.0 Hz, 1H), 4.33 (m, 1H), 4.05 (t, J=6.5 Hz, 2H), 3.84 (s,
3H), 2.39 (m, 2H), 2.01 (s, 3H), 1.98 (m, 3H), 0.95 (d, J=6.0 Hz, 3H),
0.90 (d, J=6.0 Hz, 3H); 13C NMR (100 MHz, [D6]DMSO): d=171.6,
169.5, 168.0, 163.3, 157.0, 152.6, 135.7, 132.6, 128.9, 123.7, 123.3,
121.3, 121.1, 115.9, 112.4, 106.1, 66.9, 56.1, 31.5, 30.7, 25.0, 19.2,
18.3; IR (cmꢀ1): 3269, 1635, 1605, 1542, 1489, 1210 (CꢀO); ESI+ MS
m/z: 964 [M+Na]+, 980 [M+K]+; Anal. (C, H, N): C48H60N8O12·1.5H2O.
1
99%); H NMR ([D6]DMSO): d=10.0 (s, 1H), 8.2 (d, J=7.2, 1H), 8.0–
7.8 (m, 5H), 7.7 (d, J=8.9, 2H), 7.6 (s, 1H), 7.4 (d, J=6.8, 1H), 7.2
(m, 3H), 7.0 (m, 1H), 6.3 (t, J=7.1, 1H), 4.6 (s, 2H), 4.4 (m, 1H), 4.2–
4.0 (m, 7H), 3.6 (s, 3H), 3.1 (s, 2H), 2.4–2.2 (m, 4H), 2.0–1.8 (m, 6H),
1.8–1.6 (m, 3H), 1.6–1.4 (m, 6H), 0.9–0.8 (m, 18H); 13C NMR
([D6]DMSO): d=172.6, 172.2, 172.1, 171.8, 171.4, 168.7, 157.5,
157.2, 136.2, 134.5, 129.4, 128.2, 124.2, 122.5, 116.3, 106.7, 104.9,
67.4, 58.4, 57.7, 53.4, 52.0, 51.7, 51.4, 41.2, 38.6, 32.3, 32.1, 31.9,
Synthesis of molecular tong 9. Tong 9 was synthesized from 19,
following the same procedure as described for 1 from 25, and was
1
obtained as a white solid in 82% yield: mp: 183–1858C; H NMR
1904
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1899 – 1906

Inhibitors of HIV-1 Protease Dimerization
(400 MHz, [D6]DMSO): d=9,19 (s, 1H), 8.36 (d, J=7.3 Hz, 1H), 8.19
(dd, J=1.2, 7.1 Hz, 1H), 7.69 (d, J=8.9 Hz, 1H), 7.61 (s, 1H), 7.34–
7.29 (m, 6H), 7.20 (s, 1H), 7.16 (s, 1H), 6.97 (dd, J=2.2, 8.9 Hz, 1H),
6.23 (t, J=7.1 Hz, 1H), 5.00 (s, 2H), 4.56 (s, 2H), 4.40 (bm, 1H), 4.06
(t, J=6.3 Hz, 2H), 2.96 (q, J=6.3 Hz, 2H), 2.39 (t, J=6.4 Hz, 2H),
2.02 (quint, J=6.4 Hz, 2H), 1.74 (m, 1H), 1.58 (m, 1H), 1.39 (m, 2H),
1.32 (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=172.3, 171.3, 168.3,
157.0, 156.7, 156.1, 137.3, 135.7, 133.1, 133, 128.9, 128.3, 128.1,
127.7, 123.7, 122.3, 115.9, 106.2, 104.7, 66.9, 65.1, 53.7, 51.3, 40.1,
31.7, 30.7, 29.1, 24.9, 22.8; IR (cmꢀ1): 3303, 1676, 1647, 1515, 1210;
ESI+ MS m/z: 1178 [M+Na]+; Anal. (C, H, N): C60H70N10O14·2.5H2O.
ESI+
C50H66N10O12·2CF3COOH·3.5H2O.
MS
m/z:
1000
[M+H]+;
Anal.
(C,
H,
N):
Synthesis of molecular tong 13. Tong 13 was synthesized from
20, following the same procedure as described for 1 from 25, and
was obtained as a white solid in 69% yield: mp: 149–1518C;
1H NMR (400 MHz, [D6]DMSO): d=9.15 (s, 1H), 8.21 (d, J=7.0 Hz,
1H), 7.83 (t, J=5.2, 1H), 7.70 (d, J=8.8 Hz, 1H), 7.61 (s, 1H), 7.37
(d, J=7.0 Hz, 1H), 7.30 (d, J=7.0 Hz, 1H), 7.20 (s, 1H), 7.18 (s, 1H),
6.95 (dd, J=1.6, 8.8 Hz, 1H), 6.23 (t, J=7.0 Hz, 1H), 4.57 (s, 2H),
4.04 (m, 3H), 3.03 (bq, J=5.8 Hz, 2H), 2.25 (t, J=7.0, 2H), 1.98 (q,
J=7.0 Hz, 2H), 1.71 (m, 1H), 1.55 (m, 1H), 1.38 (s, 9H), 1.38 ꢀ1.31
(m, 4H); 13C NMR (100 MHz, [D6]DMSO): d=171.8, 171.4, 168.3,
157.0, 156.7, 155.7, 135.8, 133.0, 129.0, 128.0, 123.7, 122.1, 115.9,
106.1, 104.7, 78.5, 67.0, 55.4, 51.2, 38.3, 31.8, 30.8, 28.8, 28.2, 24.9,
23.1; IR (cmꢀ1): 3314, 2933, 1684, 1645, 1514, 1369, 1252, 1210,
1163; ESIꢀ MS m/z: 1086 [Mꢀ1]+. Anal (C, H, N):
C54H76N10O14·1.5H2O.
Synthesis of molecular tong 10. Pd/C 10% (12 mg, 20% mass)
was added to a solution of 9 (65 mg, 0.06 mmol) in MeOH (20 mL)
and DMF (2 mL). The reaction flask was purged three times with
hydrogen, and stirring was maintained under hydrogen atmos-
phere at room temperature for 12 h. The mixture was filtered
through Celite, and the cake was washed with MeOH (200 mL).
The filtrate was concentrated under reduced pressure and the re-
sulting residue was dissolved in MeOH. TFA (0.02 mL, 0.26 mmol,
4.3 equiv) was added, and the mixture stirred for 10 min before
evaporation of the solvent under reduced pressure. The crude
product was recrystallized from MeOH/Et2O to obtain 10 as a
beige solid: mp: 176–1788C (45 mg, 72%). Rf =0 (EtOAc/MeOH/
NH4OH, 79:20:1); 1H NMR (400 MHz, [D6]DMSO): d=9.21 (s, 1H),
8.39 (d, J=8.0 Hz, 1H), 8.27 (bs, 3H), 8.19 (d, J=8.0 Hz, 1H), 7.71
(d, J=8.8, 1H), 7.63 (s, 1H), 7.32 (d, J=8.0 Hz, 1H), 7.20 (s, 1H),
7.18 (s, 1H), 6.97 (d, J=8.8 Hz, 1H), 6.24 (t, J=8.0 Hz, 1H), 4.57 (s,
2H), 4.46 (bm, 1H), 4.06 (m, 2H), 2.97 (m, 2H), 2.40 (t, J=7.2 Hz,
2H), 2.03 (m, 2H), 1.77 (m, 1H), 1.60 (m, 1H), 1.40–1.33 (m, 4H);
13C NMR (100 MHz, [D6]DMSO): d=172.0, 170.1, 168.3, 156.2, 135.7,
133.6, 129.0, 128.7, 123.7, 122.6, 115.7, 105.9, 104.8, 66.7, 47.7, 51.0,
31.6, 30.7, 32.0, 24.8, 22.6; IR (cmꢀ1): 3338, 1644, 1514, 1200, 1130;
ESI+ MS m/z: 888 [M+H]+, 910 [M+Na]+; Anal. (C, H, N):
C44H48N10O10·2CF3COOH ·1.5H2O.
Synthesis of molecular tong 14. Tong 14 was synthesized from
13, following the same procedure as described for 4, and was ob-
tained as a white solid in 99% yield: mp: 148 ꢀ1508C; 1H NMR
(400 MHz, [D6]DMSO): d=9.98 (s, 1H), 8.21 (d, J=1.5 Hz, 1H), 8.17
(s, 3H), 7.82 (t, J=5.3 Hz, 1H), 7.68 (d, J=9.0 Hz, 1H), 7.64 (s, 1H),
7.37 (dd, J=1.5, 7.0 Hz, 1H), 7.20 (s, 1H), 7.16 (d, J=2.0, 1H), 6.94
(dd, J=2.2, 9.0 Hz, 1H), 6.25 (t, J=7.0 Hz, 1H), 4.57 (s, 2H), 4.24 (t,
J=6.0 Hz, 1H), 4.02 (t, J=6.0 Hz, 2H), 3.02 (q, J=7.0 Hz, 2H), 2.23
(t, J=7.0 Hz, 2H), 1.95 (quint, J=7.0 Hz, 2H), 1.73 (m, 2H), 1.39 (q,
J=7.0 Hz, 2H), 1.33 (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
171.4, 168.5, 168.3, 157.0, 156.7, 135.7, 134.2, 129, 127.7, 125.0,
123.6, 116.0, 106.1, 104.4, 67.0, 52.6, 51.2, 38.2, 31.8, 31.1, 28.7,
24.9, 21.7; IR (cmꢀ1): 3262, 2968, 1633, 1514, 1200, 1130; ESI+ MS
m/z: 888 [M+H]+, 910 [M+Na]+; Anal. (C, H, N):
C44H58N10O10·2CF3COOH·2H2O.
Synthesis of molecular tong 15. Tong 15 was synthesized from
24, following the same procedure as described for 1 from 25, and
was obtained as a white solid in 67% yield: mp: 164–1708C;
1H NMR (400 MHz, CDCl3): d=11.85 (bs, 1H), 11.25 (bs, 1H), 8.92 (s,
1H), 8.40 (bm, 1H), 7.95 (s, 1H), 7.54 (d, J=8.6 Hz, 1H), 6.97 (d, J=
8.9 Hz, 1H), 6.89 (s, 1H), 6.86 (d, J=8.6 Hz, 1H), 5.86 (bs, 1H), 5.66
(d, J=7.8 Hz, 1H), 4.88 (bm, 1H), 4.00 (m, 5H), 3.08 (m, 2H), 2.27
(m, 2H), 2.19 (s, 3H), 2.09 (m, 2H), 1.80 (m, 1H), 1.71 (m, 1H), 1.45
(s, 9H), 1.34–1.32 (m, 4H); 13C NMR (100 MHz, [D6]DMSO): d=172.4,
171.0, 169.2, 167.0, 157.3, 155.6, 153.6, 135.8, 133.1, 129.0, 126.2,
124.2, 122.6, 117.9, 116.1, 112.1, 106.3, 79.8, 66.7, 56.6, 52.1, 39.3,
34.0, 32.7, 29.2, 28.4, 25.1, 24.5, 22.1; IR (cmꢀ1): 3286, 2936, 1631,
1493, 1251, 1161; ESI+ MS m/z: 1222 [M+Na]+; Anal. (C, H, N):
C60H82N10O16·4H2O.
Synthesis of molecular tong 11. Tong 11 was synthesized from
23, following the same procedure as described for 1 from 25, and
was obtained as a white solid in 74% yield: mp: 188–1908C;
1H NMR (400 MHz, [D6]DMSO): d=10.33 (s, 1H), 9.93 (m, 2H), 8.06
(d, J=8.1 Hz, 1H), 7.94 (d, J=2.6, 1H), 7.76 (dd, J=2.6, 9.0 Hz, 1H),
7.69 (d, J=9.0 Hz, 1H), 7.35–7.31 (m, 5H), 7.21 (bs, 1H), 7.18 (s,
1H), 7.09 (d, J=9.0 Hz, 1H), 6.96 (dd, J=2.0, 9.0 Hz, 1H), 5.00 (s,
2H), 4.41 (bm, 1H), 4.04 (t, J=6.1, 2H), 3.84 (s, 3H), 2.98 (q, J=
6.1 Hz, 2H), 2.36 (t, J=7.2 Hz, 2H), 2.01 (s, 3H), 1.98 (m, 2H), 1.69
(bm, 1H), 1.58 (bm, 1H), 1.40 (m, 2H), 1.34 (m, 2H); 13C NMR
(100 MHz, [D6]DMSO): d=171.6, 170.2, 168.0, 163.1, 157.0, 156.1,
152.7, 137.3, 135.8, 132.6, 128.9, 128.3, 127.7, 123.7, 123.4, 121.3,
121.0, 115.9, 112.4, 106.2, 66.9, 65.1, 56.1, 50.9, 40.1, 32.0, 31.6, 29.1,
24.9, 23.8, 22.6; IR (cmꢀ1): 3261, 1686, 1605, 1539, 1252, 1210,
1023; ESI+ MS m/z: 1290 [M+Na]+, 1306 [M+K]+; Anal. (C, H, N):
C66H78N10O16·1.5H2O.
Synthesis of molecular tong 16. Tong 16 was synthesized from
15, following the same procedure as described for 4, and was ob-
tained as a white solid in 92% yield: mp: 169–1718C; 1H NMR
(400 MHz, [D6]DMSO): d=10.13 (bs, 1H), 9.98 (bs, 1H), 8.69 (bs,
1H), 7.97 (d, J=2.5 Hz, 1H), 7.86 (t, J=5.1 Hz, 1H), 7.72 (dd, J=2.5,
9.0 Hz, 1H), 7.70 (d, J=8.7 Hz, 1H), 7.16 (s, 1H), 7.12 (d, J=9.0 Hz,
1H), 6.94 (dd, J=2.1, 8.7 Hz, 1H), 4.04 (t, J=6.2 Hz, 2H), 3.85 (s,
3H), 3.83 (m, 1H), 3.06 (m, 2H), 2.28 (t, J=7.0 Hz, 2H), 2.01 (s, 3H),
1.99 (m, 2H), 1.78 (bm, 2H), 1.43 (m, 4H); 13C NMR (100 MHz,
[D6]DMSO): d=171.5, 168.1, 167.2, 163.5, 157.0, 152.6, 135.8, 132.7,
129.0, 123.7, 123.5, 121.2, 121.0, 115.9, 112.5, 106.1, 67.0, 56.2, 51.2,
38.2, 31.9, 31.0, 28.7, 25.0, 23.8, 21.5; IR (cmꢀ1): 3316, 2948, 1632,
1490, 1252, 1133, 1016; ESI+ MS m/z: 1000 [M+1]+; Anal. (C, H, N):
C50H66N10O12·2CF3COOH ·2H2O.
Synthesis of molecular tong 12. Tong 12 was synthesized from
11, following the same procedure as described for 10 from 9, and
was obtained as a white solid in 69% yield: mp: 139–1418C;
1H NMR (400 MHz, [D6]DMSO): d=10.35 (s, 1H), 9.97 (d, J=8.9 Hz,
2H), 8.51 (bs, 3H), 8.13 (d, J=7.3 Hz, 1H), 7.98 (s, 1H), 7.70 (d, J=
8.4 Hz, 2H), 7.19 (s, 1H), 7.10 (d, J=8.8 Hz, 1H), 6.98 (d, J=7.9 Hz,
1H), 4.43 (m, 1H), 4.06 (m, 2H), 3.83 (s, 3H), 2.84 (m, 2H), 2.36 (m,
2H), 2.01 (s, 3H), 1.72 (m, 2H), 1.57 (m, 1H), 1.38 (m, 2H), 1.29–
1.23 (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=171.7, 170.2, 168.0,
163.3, 158.5–157.6 (q, J=32 Hz), 157.0, 152.7, 135.8, 132.6, 129.0,
123.7, 123.4, 121.3, 121.0, 118.8, 115.9, 112.4, 106.2, 66.9, 56.1, 50.7,
48.0, 31.7, 31.6, 29.0, 24.9, 23.8, 22.2; IR (cmꢀ1): 1648, 1513, 1128;
ChemMedChem 2010, 5, 1899 – 1906
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1905

S. Ongeri, M. Reboud-Ravaux, et al.
MED
Enzymatic studies
Keywords: dimerization · HIV-1 · inhibitors · peptidomimetics ·
proteases
The fluorogenic substrate DABCYL-g-abu-Ser-Gln-Asn-Tyr-Pro-Ile-
Val-Gln-EDANS {DABCYL: 4-(4’-dimethylaminophenylazo)benzoyl; g-
abu: g-amino butyric acid; EDANS: 5-[(2-aminoethyl)amino]naph-
thalene-1-sulfonic acid)} was purchased from Bachem (Germany).
Other reagents and solvents were purchased from various com-
mercial sources as well. Absorbance measurements were obtained
using a spectrofluorimeter PerkinElmer LS50B. Fluorescence intensi-
ties were measured using a BMG Fluostar microplate reader.
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Acknowledgements
[19] N. Merabet, J. Dumond, B. Collinet, L. Van Baelinghem, N. Boggetto, S.
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4664.
We thank Claire Troufflard for NMR experiments, the European
Community for financial support for A.V. (Marie Curie Early Stage
Training Fellowship of the European Community’s Sixth Frame-
work Programme, contract MESTCT-2004-515968), the Ministꢃre
de la Recherche et des Technologies (MRT), and the Agence Na-
tionale sur la Recherche contre le Sida (ANRS) for financial sup-
port for L.D. and L.B., respectively. We also thank Prof. Ernesto
Freire of the Department of Biology and Biocalorimetry Center,
Johns Hopkins University, Baltimore, MD (USA), for the generous
gift of the plasmid encoding the ANAM-11 mutant.
[21] S. Muzammil, P. Ross, E. Freire, Biochemistry 2003, 42, 631–638.
Received: July 23, 2010
Revised: September 9, 2010
Published online on October 8, 2010
1906
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ChemMedChem 2010, 5, 1899 – 1906