Synthesis and biological evaluation of a new derivative of bevirimat that targets the Gag CA-SP1 cleavage site

Article abstract of DOI:10.1016/j.ejmech.2013.01.013

Bevirimat (2), the first-in-class HIV-1 maturation inhibitor, shows a low efficacy due essentially to the natural polymorphism of its target, the CA-SP1 junction. Moreover, its low hydrosolubility makes it difficult to study its interaction with the CA-SP1 junction. We have synthesized new derivatives of bevirimat by adding different hydrophilic substituents at the C-28 position to improve their hydrosolubility and perform the structural study of a complex by NMR. Synthesis of the new derivatives, the effect of substituents at the C-28 position and their hydrosolubility are discussed. The ability of these molecules to inhibit viral infection and their cytotoxicity is assessed. Compared to the well-known bevirimat (2), one of our compounds (16) shows a higher hydrosolubility associated with a 2.5 fold increase in activity, a higher selectivity index and a better antiviral profile. Moreover, for the first time a direct interaction between a derivative of bevirimat (16) and the domain CA-SP1-NC is shown by NMR. Information from this study should allow us to decipher the mechanism by which bevirimat inhibits HIV-1 maturation and how the natural polymorphism of the spacer peptide SP1 triggers resistance to inhibitors.

Full text of DOI:10.1016/j.ejmech.2013.01.013

European Journal of Medicinal Chemistry 62 (2013) 453e465
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of a new derivative of bevirimat
that targets the Gag CA-SP1 cleavage site
Pascale Coric , Serge Turcaud , Florence Souquet , Laurence Briant d, Bernard Gayc d, Jacques Royer b,
a
b
b
c
,
,
c
,
d
a,
*
Nathalie Chazal , Serge Bouaziz
Université Paris Descartes, Sorbonne Paris Cité, Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, 4 av. de l’Observatoire, Paris, France
Université Paris Descartes, Sorbonne Paris Cité, SSMIP-UMR 8638 CNRS, 4 avenue de l’Observatoire, Paris, France
Université Montpellier 1, Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé (CPBS), UMR 5236, CNRS, 1919 route de Mende F-34293 Montpellier, France
Université Montpellier 2, CPBS, F-34095 Montpellier, France
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 September 2012
Received in revised form
Bevirimat (2), the first-in-class HIV-1 maturation inhibitor, shows a low efficacy due essentially to the
natural polymorphism of its target, the CA-SP1 junction. Moreover, its low hydrosolubility makes it
difficult to study its interaction with the CA-SP1 junction. We have synthesized new derivatives of
bevirimat by adding different hydrophilic substituents at the C-28 position to improve their hydro-
solubility and perform the structural study of a complex by NMR. Synthesis of the new derivatives, the
effect of substituents at the C-28 position and their hydrosolubility are discussed. The ability of these
molecules to inhibit viral infection and their cytotoxicity is assessed. Compared to the well-known
bevirimat (2), one of our compounds (16) shows a higher hydrosolubility associated with a 2.5 fold
increase in activity, a higher selectivity index and a better antiviral profile. Moreover, for the first time
a direct interaction between a derivative of bevirimat (16) and the domain CA-SP1eNC is shown by NMR.
Information from this study should allow us to decipher the mechanism by which bevirimat inhibits
HIV-1 maturation and how the natural polymorphism of the spacer peptide SP1 triggers resistance to
inhibitors.
9
January 2013
Accepted 11 January 2013
Available online 19 January 2013
Keywords:
HIV maturation inhibitor
Betulinic acid
Bevirimat
DSB
CA-SP1 cleavage
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
p6 and two extra spacer peptides, SP1 and SP2 [6,7]. The first
cleavage occurs between SP1 and NC leading to the intermediate
Most of the anti-HIV-1 drug inhibitors target the reverse tran-
scriptase (RT) [1,2], the integrase (IN) [3,4] or the protease (PR) [1,5]
and cause the emergence of multidrug-resistant HIV strains. Gag
precursor (Pr55Gag) assembly and maturation are important steps
of the HIV-1 replication and constitute attractive targets for the
design of molecules competent to inhibit the retroviral cycle.
Pr55Gag is necessary and sufficient for HIV-1 particle assembly and
is cleaved during or after the assembly of the entire particle by PR in
four mature proteins, matrix (MA), capsid (CA), nucleocapsid (NC),
MA-CA-SP1, which is cleaved to MA and precursor CA-SP1 (p25).
The cleavage of p25, critical for the proper formation of mature and
infectious viral particles [8], occurs late in the virus life cycle to
generate the mature CA (p24) and SP1.
The region surrounding the CA-SP1 junction plays an important
role in the morphogenesis of the virion [9e14]. The mutation of
the residues within the CA-SP1 junction results in the production
of defective and noninfectious viral particles with aberrant cores
[12,15] demonstrating that the SP1 peptide is crucial both for HIV-1
assembly and maturation. Moreover, it has been shown that the
presence of the SP1 domain at the C-terminus of CA allows Pr55Gag
to multimerize [16]. Previous studies have shown that the
2
Abbreviations: Ac O, acetic anhydride; DEPT135, Distortionless Enhancement by
CA-SP1 junction adopts an a-helical conformation [17] confirmed
Polarization Transfer; DIEA, N,N-diisopropylethylamine; DMAP, 4-(dimethylamino)
pyridine; DQF-COSY, double quantum filtered-correlation spectroscopy; HMBC,
Heteronuclear Multiple Bond Correlation; HSQC, Heteronuclear Single Quantum
recently by electron cryotomography studies of immature HIV-1
virions [18]. The ability of SP1 to undergo a concentration-
dependent transition from a nearly unstructured conformation to
an a-helical one has suggested that SP1 behaves as a molecular
switch to prime assembly of HIV-1 Pr55Gag in the immature
lattice [19].
Correlation; n-Bu
tripyrrolodinophosphonium hexafluorophosphate; t-BuMe
thylsilyltrifluoromethanesulfonate; TOCSY, Total Correlation Spectroscopy.
4
NF, tetrabutylammonium fluoride; PyBOP, (benzotriazol-1-yloxy)
2
SiOTf, tert-butyldime-
*
Corresponding author. Tel.: þ33 0 1 53 73 95 78; fax: þ33 0 1 43 26 69 18.
E-mail address: serge.bouaziz@parisdescartes.fr (S. Bouaziz).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.013

454
P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
Derivatives of betulinic acid (BA, 1) (Fig. 1) retain antiviral prop-
Derivatives of bevirimat having a side chain at the C-28 posi-
tion, have been reported for their potent activity against HIV-1
replication and their interference with the processing of p25
[29e32]. Some of them block HIV-1 entry and constitute a new
class of bi-functional inhibitors with the potential to block both
entry and maturation. The introduction of basic substituents at the
C-28 position of bevirimat results in compounds with high anti-
HIV activity [33]. Molecules derived from betulin that target the
late stage of the replication have been proposed as anti-HIV agents
[34,35].
However, the nature of the interaction between Pr55Gag and
bevirimat remains unclear and the characterization of those in-
teractions may help to understand the mechanism underlying the
inhibitory effect of bevirimat. Moreover, the design of new matu-
ration inhibitors, derived from bevirimat, with improved efficiency
against both assembly and maturation may also throw light on the
mechanism. Such studies may also help to understand how the
natural polymorphism of the CA-SP1 junction enables the virus to
escape the effect of bevirimat. To address these queries and appre-
hend the mechanism by which bevirimat disrupts the maturation of
the virion, we have initiated structural studies of the complex by
NMR. Unfortunately, the structural study of both partners together
was complicated by the lack of solubility of bevirimat in water as
well as in the presence of organic solvents such as acetonitrile or
trifluoroethanol. The C-3 ester group within bevirimat is essential to
interact with its target during HIV-1 maturation [36]. Consequently,
novel bevirimat derivatives maintaining the dimethylsuccinyl
group at the C-3 position and various substituents at the C-28 po-
0
0
erties against HIV-1 [20e22]. The 3-O-(3 ,3 ꢀdimethylsuccinyl)-
betulinic acid derivative, also referred to as DSB, PA-457 or bevirimat
(
2) (Fig. 1) exhibits an anti-HIV activity in acutely infected H9 lym-
ꢀ
4
phocytes with EC50 values greater than 3.5 ꢁ 10
mM [20,23] and
blocks HIV-1 replication by interfering with the processing of CA-
SP1 [15,24e26]. In vivo, bevirimat inhibits Pr55Gag assembly and
VLP release in various cellular models, including mammalian cells as
well as baculovirus-infected cells (Sf9) [27]. Recent results using
cryo-electron tomography to examine the structures of HIV-1 viral
particles isolated from DSB-treated cells suggest that DSB inhibits
viral infectivity not only by blocking the CA-SP1 cleavage site but
also by stabilizing the immature lattice [27]. Recently, it was shown
that a molecule, PF-46396, structurally different from DSB had
a similar mode of action and that these two molecules interact dif-
ferently with the putative maturation inhibitor binding pocket [28].
sition, were synthesized to gain in hydrosolubility (Fig. 1): N-[(3
-(3-carboxy-3-methylbutanoyloxy)lup-20(29)-en-28-oyl]-glycine
12), N-[(3 )-3-(3-carboxy-3-methylbutanoyloxy)lup-20(29)-en-
b)-
3
(
b
6
2
2
8-oyl]-
)-3-(3-carboxy-3-methylbutanoyloxy)-lup-20(29)-en-28-oyl]-
-lysine (15), 4-({28-[(2-aminoethyl)amino]-28-oxolup-20,29-en-
b-alanine (13), N -[(1,1-dimethylethoxy)carbonyl, N -
[(3b
L
2
3
(
(
b
-yl}oxy)-2,2-dimethyl-4-oxobutanoic acid (16) and N -[(3
3-carboxy-3-methylbutanoyloxy)lup-20(29)-en-28-oyl]- -lysine
17). Our goal was not to obtain new derivatives of bevirimat
b)-3-
L
with higher activity against HIV replication but rather to get
more hydrosoluble molecules with the same activity against HIV
replication. Thus, we focused on the hydrosolubility of our
compounds and various hydrophilic functions were added at
the C-28 position of bevirimat to make them amenable to NMR
structural studies within a complex formed with peptides con-
taining the CA-SP1 junction.
This paper describes the synthesis, the characterization by
chemical analysis, NMR and Mass Spectroscopy (MS) of a new se-
ries of compounds. The polarity of these products was compared by
HPLC analysis, their antiviral activity assessed and their IC50 value
was determined. Their toxicity was controlled using models of
mammalian cells infected with the NL4.3 HIV-1 laboratory strain
and compared to bevirimat (2). One of our molecules (16) showed
a better polarity evaluated by HPLC and an antiviral profile with
a higher selectivity index. Moreover, for the first time, a direct
interaction between compound 16 and the peptide CA-SP1eNC was
proved by NMR. More recently, we showed that compound 16 was
able to generate an aberrant assembly of VLP, resulting in non-
enveloped, morula-like particles of 100 nm in diameter [37]. All
together, these data constitute a first step in the understanding of
the mechanism by which bevirimat inhibits HIV-1 assembly and
maturation as proposed in a previous model [27,38]. Several pat-
ents have been filed describing derivatives of bevirimat at the C-28
position. None of them corresponds to the new molecules that we
designed and synthesized except for one of them (WO 2006/
053255 A2) that is quoted but has never been synthesized and its
biological activity never tested against HIV-1.
Fig. 1. The structure of the betulinic acid (BA, 1), bevirimat (2) and derivatives
containing
stituents at the C-28 position. These molecules were synthesized to gain in hydro-
solubility: N-[(3 )-3-(3-carboxy-3-methylbutanoyloxy)lup-20(29)-en-28-oyl]-glycine
a dimethyl group at the C-3 position and various hydrophilic sub-
b
(
(
12),
N-[(3
b
)-3-(3-carboxy-3-methylbutanoyloxy)lup-20(29)-en-28-oyl]-
b
-alanine
6
2
13), N -[(1,1-dimethylethoxy)carbonyl, N -[(3
b)-3-(3-carboxy-3-methylbutanoyloxy)-
lup-20(29)-en-28-oyl]-L-lysine (15), 4-({28-[(2-aminoethyl)amino]-28-oxolup-20,29-
2
en-3
b
-yl}oxy)-2,2-dimethyl-4-oxobutanoic acid (16) and N -[(3
b)-3-(3-carboxy-3-
methylbutanoyloxy)lup-20(29)-en-28-oyl]-L-lysine (17).

P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
455
2
. Results
3D-QSAR and docking methods and made it possible to propose
new BA derivatives [45]. In the light of those results, the aim of our
work was, starting from the bevirimat drug, to propose a series of
more hydrosoluble molecules, by modifying the C-28 carboxylic
acid function, while conserving intact the C-3 dimethylsuccinyl
group required for HIV-1 maturation inhibition. Therefore, the side
chain at the C-28 position was modified by coupling different
substituents. As an example, the free acid glycine conjugate of BA
exhibits an increased hydrosolubility [48].
2.1. Chemistry
In the last fifteen years, many BA derivatives have been syn-
thesized with modifications at the C-3 hydroxyl [39], at the C-28
carboxylic acid [29,33e35]or at the C-19 isopropenyl groups along
with their resulting functional properties [20,23,40,41]. Two dis-
tinct classes of BA derivatives exhibit different activities against
HIV-1 according to the substitution. The BA derivatives in which the
C-3 hydroxyl group is acylated are efficient compounds against the
cleavage of the CA-SP1 junction leading to maturation inhibition.
Bevirimat (2) is the lead product of this derivative family (Fig. 1)
developed by the pharmaceutical company Panacos [39,40]. We
have synthesized this molecule, as previously described [20], with
slight modifications [41]. The molecules with a C-3 hydroxyl group
acylation have also been described to lead to assembly inhibition
First, the C-3 hydroxyl group (1, Scheme 1) was protected by the
acetyl group (3, Scheme 1), then various substituents were intro-
duced by peptide coupling with PyBOP and DIEA at the C-28 car-
boxylic acid. After the purification of the intermediates by flash
chromatography (4e7, Scheme 1), the saponification of the ester by
a 4 M sodium hydroxide solution was performed to furnish the
compounds with free hydroxyl group at the C-3 position (8e11,
Scheme 1). For compounds 8, 9 and 11 (Scheme 1) the sap-
onification was accompanied by a non-classical tert-butyl ester
cleavage of the coupled amino acid. The free hydroxyl derivatives
(8e11, Scheme 1) reacted with the 2,2-dimethylsuccinic anhydride
in pyridine, in the presence of DMAP, and provided a mixture of 3-
[41,42]. A second class of compounds produced by a modification of
the C-28 carboxylic acid amide obtained by introducing a hydro-
phobic side chain was also studied. This class of BA derivatives,
including the molecule named IC9564 was reported to inhibit HIV-
0
0
0
0
1
replication by blocking the viral entry step [21,22,32,43,44]. More
O-(2 ,2 -dimethylsuccinyl)- and 3-O-(3 ,3 -dimethylsuccinyl) BA
derivatives. The desired products (12e15, Scheme 1) were sepa-
rated by flash chromatography. Data from the literature show that
recently, a comprehensive study of the structureeactivity rela-
tionship of several BA derivatives has been achieved by using
Scheme 1. Synthesis of the derivatives of bevirimat.

456
P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
0
0
the 3-O-(3 ,3 -dimethylsuccinyl) betulinic acid was a more potent
0
0
anti-HIV-1 inhibitor than the 3-O-(2 ,2 -dimethylsuccinyl) betulinic
acid [20].
The N-Boc protection of product 14 (Scheme 1) was first
removed with tert-butyldimethylsilyltrifluoromethanesulfonate in
the presence of 2,6-lutidine to produce tert-butyldimethylsilyl
carbamates [46]. The removal of the N-tert-butyldimethylsilylox-
ycarbonyl group was then achieved by treatment with n-Bu
THF leading to compound 16. The N-Boc protection of product 15
Scheme 1) was cleaved with 6 M HCl in THF and compound 17 was
obtained after purification by flash chromatography.
4
NF in
(
2.2. Analysis of the polarity of the compounds by HPLC
As substitutions at the C28 position were performed to increase
the polarity of bevirimat and subsequently its hydrosolubility, while
keeping intact the function at the C-3 position, we assessed their
hydrosolubility by HPLC after purification. The compounds were
analyzed with an isocratic HPLC mode using MeOH (90%) in water as
solvent and a C18 column. In these conditions it was possible to
correlate the polarity of the compounds with their retention time
(Fig. 2). As an example, a glycine residue introduced at the C28 po-
sition in bevirimat (retention time: 17.890 min) divided by two the
retention time in compound 12 (retention time: 8.352 min). These
results are in keeping with those obtained previously on a derivative
of the betulinic acid [47]. The most interesting compounds were
found to be 16 and 17 with respectively an ethylenediamine and
a lysine group as substituent at the C28 position and a retention time
of 3.428 min and 2.295 min respectively determined by HPLC
analysis. The compounds wereclassified according totheir retention
time (or polarity) from the most hydrophilic compound to the least
hydrophilic one (17 > 16 > 12 > 13 > 1 > 2).
2.3. NMR
Our compounds were characterized by High Resolution Mass
Spectroscopy (HRMS) and by NMR. The complete assignment of the
1
13
H (Table 1) and C (Table 2) resonances was achieved by standard
D and 2D NMR experiments, the IUPAC convention was used to
1
1
1
number the carbon atoms. He H COSY (COrrelations Spectro-
scopY), TOCSY (TOtal Correlation SpectroscopY), 2D ( He C)-HSQC
1
13
1
13
(
Heteronuclear Single Quantum Correlation) and 2D ( He C)-
13
HMBC (Heteronuclear Multi Bond Correlation). The C spectra of
the six products (2, 12, 13, 15, 16 and 17) revealed 36 to 48 carbon
0
Fig. 2. The evaluation of the retention times of the different compounds (1, 2, 12, 13, 16
and 17) by HPLC analysis conducted on a Shimadzu LC-10ADvp with a Nucleodur
signals out of which 6 were assigned to the C-3 esterification (1 to
0
0
00
00
4
3
, two 5 methyl), 6 to the C-28 amide modifications (1 to 6 ) and
0 to the triterpenoid skeleton (Figures S1 to S5 in supplementary
4.6 mm ꢁ 250 mm C18 HTec 5
mm column and a Shimadzu SPD-M10Avp detector at
210 nm wavelength. The solvent system used was MeOH(0.1%TFA):water(0.1%
data).
Significant variations were observed when comparing the car-
bon chemical shifts of bevirimat and its derivatives with those
reported for the BA [48,49], mainly for the carbons C-2
TFA) ¼ 90:10 with an isocratic HPLC mode. In these conditions it was possible to
compare the water solubility of the compounds and classify them according to their
retention time from the most hydrophilic compound to the least hydrophilic one
17 > 16 > 12 > 13 > 1 > 2. Compound 15 has not been tested by HPLC since it cor-
responds to compound 17 with a N-Boc protection of the HNε of lysine. It is then
thought to be less hydrosoluble than 17.
(
Dd ¼ ꢀ4 ppm), C-3 (Dd ¼ þ3 ppm) and C-4 (Dd ¼ ꢀ1 ppm)
resulting from C-3 esterification and C-16 (Dd ¼ þ0.4 to þ1 ppm),
C-18 (Dd ¼ þ0.3 to þ1 ppm), C-20 (Dd ¼ þ0.5 to þ1 ppm), C-22
0
(
(
Dd ¼ þ0.4 to þ1.5 ppm), C-24 (Dd ¼ þ1 ppm) and C-28
Dd ¼ ꢀ1.6 ppm to þ0.5) consecutive to the C-28 amide mod-
signal strongly correlates with the H-5 proton signal for the 3-O-
0
0
(3 ,3 -dimethylsuccinyl) betulinic acid derivatives while the same
0
0
ification. Moreover, the carbon signal of C-24 was more perturbed
than that of C-23 because it is located above the plane of the first
ring A, as is the hydroxyl group at the C-3 position. All together,
these data support that the configuration of C-3 was well conserved
during the synthesis.
H-5 proton strongly correlates with the C-1 carbon signal for the
0
0
2-O-(2 ,2 -dimethylsuccinyl) betulinic acid derivatives. Addition-
1
13
ally, the 2D ( He C)-HMBC spectrum shows that the H-3 proton
signal correlates with the C-1 carbon signal and not with the C-4
0
0
one. These data validate the correct incorporation of the chain at
The opening of the 2,2-dimethylsuccinic anhydride during the
C-3 esterification synthesis leads to the formation of two com-
the C-3 position of the purified compounds. The correlations be-
0
0
tween the C-28 carbon signal and the NH and H-1 proton signals
for the five bevirimat derivatives prove the correct formation of the
amide.
1
13
pounds, which can be differentiated, on the 2D ( He C)-HMBC
0
experiment (data not shown). We observed that the C-4 carbon

P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
457
Table 1
1
H NMR spectral data for 2, 12, 13, 15, 16 in pyridine-d
5
and 17 in methanol-d
4
. o, overlapped signals.
2
12
13
15
16
17
d
H
d
H
d
H
d
H
d
H
d
H
1
2
3
0.91 1.56 (o)
1.66 1.8 (o)
4.76 (d) (o)
J ¼ 5.5 Hz
e
0.89 1.52 (o)
1.67 1.77 (o)
4.77 (d)
J ¼ 5 Hz
e
0.91 1.58 (o)
1.7 1.82 (o)
4.78 (o)
0.9 1.59 (o)
1.7 1.78 (o)
4.78 (dd)
J ¼ 5 Hz, 11.5 Hz
e
0.86 1.54 (o)
1.67 1.77 (o)
4.77 (o) d
0.99 1.72 (o)
1.6 1.68 (o)
4.44 (dd)
J ¼ 5 Hz, 10 Hz
e
4
5
e
e
0.77 (d)
J ¼ 11 Hz
1.42 (o)
1.32 (o)
e
0.77 (o)
0.74 (o)
0.82 (o)
0.77 (o)
0.82 (o)
6
7
8
9
1
1
1
1.3 1.4 (o)
1.37 (o)
e
1.3 1.4 (o)
1.33 (o)
e
1.36 1.46 (o)
1.44 (o)
e
1.3 1.4 (o)
1.30 (o)
e
1.44 1.52 (o)
1.38 1.45 (o)
e
1.29 (o)
e
1.3 (o)
e
1.3 (o)
e
1.30 (o)
e
1.28 (o)
e
1.36 (o)
e
0
1
2
1.2 1.3 (o)
1.15 1.94 (o)
1.15 1.3 (o)
1.15(o)
1.17 1.31 (o)
1.16 (o)
1.96 (m)
3.04 (m)
e
1.15 1.33 (o)
1.15
1.95 (o)
3.07 (m)
e
1.13 1.3 (o)
1.12(o) 1.92 (m)
1.26 1.43 (o)
1.04 1.7 (o)
1.93(m)
13
14
15
16
2.73 (m)
e
3.08 (m)
e
3.03 (m)
e
2.53 (o)
e
1.22 1.84 (o)
1.55(o) 2.63 (m)
1.26 2.03 (m)
1.67(o) 2.59(m)
1.2 1.85 (o)
1.58 (o) 2.44 (d)
J ¼ 13 Hz
e
1.32 1.89 (o)
1.67 (o) 2.58 (m)
1.17 1.7 (o)
1.64 (o) 2.67 (d)
J ¼ 12 Hz
e
1.18 1.49 (o)
1.56 (o) 2.17 (m)
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
7
8
9
0
1
2
3
4
5
6
7
8
9
e
e
e
e
1.75 (o)
3.55 (m)
e
1.74 (o)
3.66 (m)
e
1.7 (o)
3.64 (m)
e
1.73 (o)
3.64 (m)
e
1.7 (o)
3.61 (m)
e
1.64 (o)
3.06 (m)
e
1.56 2.26 (o)
1.56 2.25 (o)
0.97 (s)
0.92 (s)
0.73 (s)
1.02 (s)
1.05 (s)
e
1.54 2.38 (o)
1.59 2.39 (o)
0.96 (s)
0.92 (s)
0.73 (s)
1.12 (s)
1.06 (s)
e
1.5 (o) 2.24 (m)
1.52 (o) 2.14 (m)
0.97 (s)
0.94 (s)
0.74 (s)
1.08 (s)
1.04 (s)
e
1.53 (o) 2.34 (m)
1.54 (o) 2.43 (m)
0.97 (s)
0.96 (s)
0.76 (s)
1.09 (s)
1.08 (s)
e
1.48 2.14 (m)
1.55 2.25 (m)
0.97 (s)
0.93 (s)
0.75 (s)
1.03 (o)
1.03 (o)
e
1.35 1.9 (o)
1.47 1.93 (o)
0.87 (s)
0.85 (s)
0.92 (s)
0.96 (s)
1.01 (s)
e
4.78 (s) 4.95(s)
1.78 (s)
e
4.75 (s) 4.96(s)
1.78 (s)
e
4.77 (s) 4.94 (o)
1.81 (s)
e
4.77 (s) 4.92 (s)
1.78 (s)
e
4.75 (s) 4.93(s)
1.77 (s)
e
4.57 (s) 4.7 (s)
1.7 (s)
0
0
0
e
2.89 2.97 (2d)
J ¼ 15 Hz
e
2.91 2.95(2d)
J ¼ 15.5 Hz
e
2.94 3.00 (2d)(o)
J ¼ 15.5 Hz
e
2.90 2.98 (2d)
J ¼ 15.5 Hz
e
2.9 2.96 (2d)
J ¼ 15.5 Hz
e
2.55 2.62 (2d)
J ¼ 15.5 Hz
e
0
0
0
3
4
5
1
e
e
e
e
e
e
1.55 (o)
1.55 (o)
4.44 4.51(dd)
J ¼ 6 Hz, 17.5 Hz
1.55 (o)
3.88 4.00 (m)
1.55 (o)
5.17 (m)
1.55 (o)
3.87 3.81 (m)
1.25 (o)
4.3 (m)
0
0
0
0
2
3
4
5
6
3.00 (mo)
2.06 2.31 (m)
1.80 (o)
1.74 (o) 1.83
3.43 (m)
3.28 (m)
8.74 (o)
1.75 1.89 (m)
1.46 (o)
1.7
”
”
”
”
2.9 (t) J ¼ 8 Hz
e
e
e
e
NH
8.76 (t) J ¼ 5.5 Hz
8.33 (t) J ¼ 5.5 Hz
8.27 (d) J ¼ 8 Hz
7.1
Several remarks regarding the proton chemical shifts (Fig. 3) can
be made. The most important variation concerns the H-3 that is
less shielded (þ1.3 ppm) after the esterification of the ꢀhydroxyl
group. The chemical shifts of the H-1 protons ( and ), H-6 , H-9,
H-23 and the 3H-25 differ by 0.1e0.2 ppm. The supplementary
in the unstructured N- and the C-terminal extremities of the
peptide and were very sensitive to the solvent conditions. The
perturbation of the chemical shifts of the wild type junction is
represented as a histogram (Fig. 4C).
a
b
a
b
b
3
changes resulting from the C-28 amide modifications concern the
H-13 proton (þ0.3 ppm).
2.4. Bio assays
The most active compound (16) was tested by NMR in inter-
action with a wild type CA-SP1eNC peptide containing 48 amino
acids [17] and derived from the HIV-1 Pr55Gag sequence (Fig. 4A).
NOESY experiments were performed on the peptide in the pres-
ence or not of compound 16 and perturbation of the chemical
shifts of the junction CA-SP1-NC has been analyzed, demon-
strating that an interaction takes place between compound 16
and the wild type junction (Fig. 4B). The amino acids involved
in the interaction with compound 16 were identified within
SP1-(A366, M367, Q369, V370, N372 and I376) and in the NC
Novel bevirimat derivatives with higher solubility, containing
the dimethyl group at the C-3 position and various modifications at
the C-28 amide position were tested for their capacity to inhibit
HIV-1 assembly and/or maturation and consequences on viral
infectivity were determined. To this end, viruses produced by
transfection of the HIV-1 pNL-4-3 molecular clones in 293T cells
maintained in the presence of newly synthesized compounds 12,
13, 15, 16 and 17 were analyzed for their capacity to infect MAGIC-
5B cells in parallel with BA (1) and bevirimat (2). All compounds
displayed an IC50 value lower than that of BA, with compound 16
being more efficient than bevirimat at inhibiting HIV-1 infectivity.
The bioassay data are summarized in Table 3.
(
M378). Some amino acids showed their chemical shifts per-
turbed after the addition of compound 16 but they were located

458
P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
Table 2
that the C-3 dimethyl substitution of the succinyl side chain as well
as the carboxylic acid, were necessary for antiviral activity [50e52].
Although it is efficient in ex vivo bio assays, the use of bevirimat (2)
as an anti-HIV drug is hindered by its low efficacy due to the wide
natural polymorphism of the spacer peptide SP1 [53e58]. More-
over, bevirimat (2) was shown to have a low solubility in physio-
logical solvents as well as in organic solvent mixtures, preventing
any structural study by NMR of a complex with the CA-SP1eNC
domain previously characterized as an amphipathic helix [17].
We optimized the synthesis of triterpene derivatives by adding
different hydrophilic substituents at the C-28 position (12, 13, 15, 16
and 17). The new molecules showing a higher polarity according to
their retention time evaluated by HPLC, were characterized by NMR
and by HRMS, they were tested for their capacity to inhibit viral
infectivity and their cytotoxicity was evaluated (Table 3). We
showed that the essential maturation inhibitory activity of our
molecules, due to the dimethylsuccinyl group at the C-3 position,
was conserved for all the molecules and therefore our compounds
remained fully active against maturation and replication.
Moreover, the most active compound (16) was shown to interact
with the junction CA-SP1-NC by chemical shift perturbation NMR
experiments (Fig. 4B). The amino acids involved in this interaction
were identified in the domain SP1 (A366, M367, Q369, V370, N372,
I376) and in NC (M378). Our data confirm the results obtained
recently with photoaffinity analogs of bevirimat that were found to
crosslink to sequences overlapping the CA-SP1 junction and are
consistent with previous biochemical data on the effect of bevir-
imat on Pr55 Gag processing [59]. Thus, introduction of a constit-
uent containing an amino group at the C-28 position of bevirimat
made it possible to demonstrate by NMR for the first time a direct
interaction between a maturation inhibitor and its target within the
CA-SP1-NC junction. This NMR study was impossible to realize with
bevirimat because of its weak solubility in water.
1
3C spectral data for 2, 12, 13, 15, 16 in pyridine-d
5
4
and 17 in methanol-d .
2
12
13
15
16
17
d
C
d
C
d
C
d
C
d
C
d
C
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
1
2
3
4
5
6
39.0
24.6
81.3
38.5
56.1
18.85
35.0
41.45
51.05
37.7
21.6
26.4
39.0
43.25
30.7
33.3
57.0
50.1
48.2
39.0
24.6
81.4
38.5
56.0
18.9
35.0
41.6
51.2
37.7
21.6
26.6
38.1
43.2
30.3
34.0
56.4
51.0
47.7
39.0
24.6
81.4
38.5
56.1
18.9
35.0
41.6
51.2
37.75
21.7
26.6
38.2
43.2
30.3
34.0
56.3
51.1
47.7
39.0
24.6
81.4
38.5
56.1
19.0
35.15
41.6
51.2
37.75
21.7
26.6
38.1
43.2
30.3
34.2
56.5
51.1
47.7
39.1
24.6
81.3
38.5
56.1
19.0
35.0
41.5
51.3
37.7
21.6
26.5
38.1
43.15
30.3
34.0
56.5
51.2
47.7
152.2
31.8
39.0
28.5
17.4
16.75
16.9
15.2
177.7
110.2
20.0
172.1
45.7
39.8
24.8
82.8
39
57
19.4
35.7
42.2
52.1
38.4
22.3
27.2
39.2
43.7
30.9
34.4
57.4
51.4
48.3
152.4
32.1
39.3
28.7
17.2
17.0
17.1
15.2
178.9
110.2
19.8
173.1
45.8
41.5
180.8
26.2
54.0
32.7
24.05
28.2
40.7
177.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
151.8
31.6
38.0
152.2
31.8
39.1
28.5
17.3
16.7
17.0
15.3
177.9
110.3
20.0
172.1
45.7
41.3
179.8
26.7
42.4
152.2
31.8
39.0
28.6
17.4
16.7
16.9
15.2
177.2
110.2
20.0
172.0
45.7
41.3
179.8
26.7
36.7
35.7
152.2
31.9
38.8
28.6
17.4
16.7
17.0
15.3
177.7
110.2
20.0
172.1
45.7
41.3
179.8
26.4
53.0
32.4
24.5
30.6
41.4
176.6
28.5
17.35
16.65
16.8
15.3
179.3
110.5
19.9
172
45.7
41.3
179.8
26.7
0
0
0
0
0
0
41.4
179.9
26.7
41.05
42.2
0
0
0
0
0
0
0
0
0
0
0
0
The newly synthesized bevirimat derivatives were evaluated for
their capacity to inhibit HIV-1 replication by blocking the protease
(
PR)-mediated cleavage at the CA-SP1 junction. Only derivative 17
showed a moderate anti-HIV-1 activity with a selectivity index (SI)
of 11 and an IC50 of 4.33 M. However, we found that all compounds
2, 13, 15 and 16 exhibited a good anti-HIV-1 efficacy with an IC50
values of 0.160 M, 0.118 M, 0.170 M and 0.016 M and a SI of 309,
m
174.0
175.4
1
m
m
m
m
Tofurtherelucidate themechanism ofactionof these compounds,
411, 199 and 2118 respectively. These results should be compared to
those obtained in a very recent publication by Qian et al. [31] in
which a product with an IC50 of 0.0059 mM was studied.
viral samples used for infection assays were subjected to biochemical
characterization (Fig. 5). The analysis of the conversion of uncleaved
p25 (CA-SP1) to mature p24 (CA) revealed that compounds 2, 12, 13,
To elucidate the mechanism of action of our series of com-
pounds, HIV-1NL4.3 particles produced by transfection of 293T cells
maintained in the presence of the newly synthesized molecules
were subjected to characterization. Biochemical analysis of cell free
viral particles revealed that, derivatives 12, 15 and 16 function as
maturation inhibitors and partially inhibit the conversion of p25
(CA-SP1) to p24 (CA) as previously reported for BA and bevirimat.
These compounds result in a defective Pr55Gag processing and to
the production of morphologically abnormal and noninfectious
particles.
1
5 and 16 partially inhibit the CA-SP1 cleavage while compounds 1
and 17 do not. However, this biochemical approach is dependent on
the threshold of sensitivity of Western blot and we can not exclude
that the cleavage is inhibited for few molecules of CA-SP1.
To determine whether defect inp25 to p24 process induced by 1, 2,
2,13,15,16 and 17 affected virus morphology, an analysis by EM was
performed. In the absence of compounds, cells transfected with
pNL-4-3 produced virus particles with the classical mature mor-
phology characterized by the presence of condensed, conical cores
1
(
Fig. 6A). In contrast, viruses from cells treated with 2,12,13,15 and 16
Among all tested molecules, compound 16, displayed the most
potent anti-HIV-1 activity. It is important to note that this molecule,
unlike other derivatives designed in this work, is the only one that
does not come from the condensation with an amino acid at the C-
28 position. It seems that the presence of the carboxylic group,
introduced by the condensation of an amino acid at the C-28 po-
sition, decreases the activity of our compounds (12 and 13), com-
pared to the introduction of an amino group (16). Moreover the
distance between the triterpenic skeleton and the carboxylic
function may modulate this activity as shown by the comparison of
the results obtained with compounds 2, 12 and 13 (Table 3).
Compared to the well-known bevirimat, compound 16 showed an
displayed different phenotype with various distribution from aber-
rant, spherical and acentriccore to aberrant immature virions (Chazal,
N et al., in preparation) (Fig. 6CeG). Compounds 1 and 17 had almost
no capacity to inhibit viral maturation. Taken together, these results
indicate that 2, 12, 13, 15 and 16 positively block proper virion
maturation.
3
. Discussion
Very recently different modifications at the C-3 position of anti-
HIV triterpene-derived agents have been proposed and showed

P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
459
Fig. 3. One-dimensional 1H NMR spectra of bevirimat (2) and derivatives. A. Derivatives 2, 12, 13, 15 and 16 in pyridine ed5. B. Derivatives 2, 15 and 17 in MeOH-d4.
improved capacity to inhibit p25 maturation with a selectivity in-
dex which is twice that of bevirimat and represents an attractive
promising HIV-1 inhibitor lead for future clinical trials. The in vitro
efficacy of compound 16 against HIV-1 seems to be related to its
polarity and we assume that it is possible to improve the activity of
these molecules by increasing their polarity and subsequently their
hydrosolubility.
Since the solubility of our molecules has been increased by
adding hydrophilic functions at the C-28 position, the resolution of
a complex between the junction CA-SP1-NC and one of these new
molecules becomes feasible. The elucidation of such complexes
could help to elucidate the molecular bases required for the inhi-
bition of HIV-1 maturation by bevirimat and its derivatives. The
structure of that complex may help to explain how the natural
polymorphism and mutations in the spacer peptide SP1 lead to
resistance to maturation inhibitors [53e58].
4. Conclusions
Bevirimat derived compounds reported in the present study
display an increased hydrosolubility generated by the addition of
various hydrophilic substituents at the C-28 position. All the new
compounds still behave as maturation inhibitors but compound 16
showed a significant improvement in anti-HIV-1 activity compared
to the original bevirimat. Moreover, compound 16 was shown to
interact with the peptide CA-SP1eNC by NMR. This direct

460
P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
Fig. 4. Highlight of the interaction of the most active compound (16) with the wild type CA-SP1eNC domain. (A) Amino acid sequence of the wild type CA-SP1eNC domain used in
this study to test the interaction with compound 16. (B) 2D 1He H NOESY spectra showing the superimposition of the HNeH
1
a
regions of the wild type CA-SP1eNC peptide in the
absence (blue) and presence (magenta) of compound 16. The perturbation of the chemical shifts of the peptide by adding compound 16 are boxed and numbered on the spectra and
shows the strength of the interaction. Spectra were recorded at a pH of 3.8, in H O/TFE (70/30) and DMSO-d (1%) at 293 K. The amino acids undergoing the most important
perturbation of their chemical shifts were identified in the wild type domain and the variation of their perturbation of their chemical shifts has been reported on a histogram (C).
For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2
6
(
interaction between a maturation inhibitor and its target is
revealed for the first time by NMR and launches the field to the
design of new molecules based on the substitution at the C-28
position by hydrophilic groups. Beside the undeniable interest of
this property for future use as anti-HIV-1 molecules, it paves the
way to the resolution of complexes formed between derivatives of
bevirimat and CA-SP1eNC. The elucidation of such complexes will
help clarifying the mechanism accounting for HIV-1 maturation
inhibition by this class of compounds and will help to explain how
the natural polymorphism of the SP1 domain leads to the resistance
to maturation inhibitors. Bevirimat was launched by Panacos
Pharmaceuticals, Inc. [39,40] and later by Myriad Pharmaceuticals.
Recently, bevirimat succeeded in Phase IIa clinical trials and despite
the encouraging results obtained in Phase IIb clinical trials, the
development on this new class of anti-HIV drug was interrupted
principally because of the natural polymorphism of the CA-SP1
junction that leads to a natural resistance of the virus to matura-
tion inhibitors. The new compounds reported herein, represent
attractive and promising leads for future development of the next
generation of HIV-1 maturation inhibitors. Once their interaction

P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
461
Table 3
23.7; 25.5; 27.9; 28.0; 29.4; 30.8; 33.6; 34.3; 36.5; 37.1; 37. 6; 37.8;
8.3; 38.4; 40.7; 41.9; 42.5; 45.9; 46.7; 50.0; 50.5; 55.4; 55.7; 80.9;
82.0; 109.4; 150.9; 169.6; 171.0; 176.4.
a
Efficiency of HIV-1 infection inhibition by betulinic acid derivatives.
3
Compound
IC50
(
m
M)
CC50
4.52
31.0
49.5
48.5
33.9
33.9
48.7
(
m
M)
SIb
(
(
(
(
(
(
(
1)
2)
5.315
0.040
0.160
0.118
0.170
0.016
4.330
0.85
775.00
309.37
411.01
199.41
2118.75
11.24
5.1.2.2.
b
-Alanine, N-[(3
b
)-3-(acetyloxy)-28-oxolup-20(29)-en-28-
: 0.45 (toluene/acetone 10/0.
); Yield: 80% (300 mg); H NMR (400 MHz, C N): 0.80 (m, 4H,
eCH in 5, eCH in 25), 0.87e0.9 (m, 7H, eCH
1), 1.08 (1s, 3H, eCH in 27), 1.12 (1s, 3H, eCH
12)
13)
15)
16)
17)
yl]-, 1-(1,1-dimethylethyl) ester (5). R
5
f
1
5
D
5
d
3
3
in 23 and 24, eCH in
3
in 26), 1.18 (m, 1H, e
3
a
CH in 12), 1.19 (m, 1H, eCH in 11), 1.21 (m, 1H, eCH in 15), 1.33 (m,
H, eCH in 9), 1.34 (m, 1H, eCH in 11), 1.35 (m, 1H, eCH in 6), 1.42
(m, 2H, eCH in 7), 1.45 (m, 1H, eCH in 6), 1.49 (s, 9H, eCOOtBu), 1.5
m, 1H, eCH in 21), 1.54 (m, 1H, eCH in 22), 1.6 (m, 2H, eCH in 1 and
6), 1.65 (m, 1H, eCH in 2), 1.7 (m, 1H, eCH in 2), 1.74 (m, 1H, eCH in
5), 1.77 (s, 3H, eCH in 30), 1.94 (m, 1H, eCH in 12), 2.08 (s, 3H, e
COCH ), 2.13 (m, 1H, eCH in 22), 2.21 (m, 1H, eCH in 21), 2.4 (m, 1H,
eCH in 16), 2.77 (m,1H, eCH in 2 ), 2.85 (m,1H, eCH in 2 ), 3.08 (m,
The mean values (m) for the 50% inhibitory activity (IC50) of infection were given
1
as
m
M (m). The mean values (m) for cytotoxicity (CC50) were given as
mM (m).
b
The selectivity index (SI) represented the CC50/IC50 ratio.
(
1
1
with the CA-SP1 junction is fully characterized, these inhibitors will
serve as model to build new molecules conserving important
functions but not necessarily based on a triterpenic skeleton.
3
3
00
00
00
1
H, eCH in 13), 3.62 (m, 1H, eCH in 19), 3.7 (m, 1H, eCH in 1 ), 3.85
0
0
(
4
m, 1H, eCH in 1 ), 4.7 (dd, 1H, eCH in 3, J ¼ 4. 5 Hz and 11. 5 Hz),
5
5
5
. Material and methods
.78 (s, 1H, ]CH), 4.94 (s, 1H, ]CH), 8.35 (d, 1H, eCOeNHe.
13
J ¼ 7 Hz). C NMR (400 MHz, C
5
D
5
N):
d
15.2 (C27); 16.8 (C25);
.1. Chemistry
0
16.9 (C26); 17.2 (C24); 18.9 (C6); 20 (C30); 21.6 (C2 ); 21.66 (C11);
2
4.5 (C2); 26.6 (C12); 28.5 (3C, t-Bu and C23); 30.3 (C15); 31.8
.1.1. Generals
0
0
00
(C21); 34 (C16); 35.1 (C7); 36.3 (C2 ); 36.5 (C1 ); 37.7 (C10); 38
C13); 38.5 (C4); 39 (C22 and C1); 41.6 (C8); 43.2 (C14); 47.65 (C19);
BA, acetic anhydride, PyBOP, DIEA, DMAP, 2,2-dimethylsuccinic
(
anhydride, 2,6-lutidine, tert-butyldimethylsilyltrifluoromethane-
sulfonate were purchased from Aldrich. All amino acids, from Bachem
were O- Bu protected and the lysine side chain was protected as
N-Boc. The final compounds were characterized by H and C NMR
and high resolution mass spectrometry experiments (Bruker MicrO-
Tof-Q 2 system). Their purity was determined to be ꢂ 95% by ele-
mental analyses (C, H, N).
5
1
1.1 (C18); 51.3 (C9); 56.1 (C5); 56.3 (C17); 80.7 (Ct-Bu); 81.2 (C3);
t
0
10.2 (C29); 152.1 (C20); 171.1 (C1 ); 172.4 (C COOt-Bu); 177.4 (C28).
1
13
5
.1.2.3. N-[(3
b
)-3-acetyloxy-lup-20(29)-en-28-oyl]-1,1-dimethylethyl-
0.48 (toluene/acetone 90/10);
Yield: 86% (330 mg); H NMR (300 MHz, CDCl ): 0.77 (m, 1H), 0.83
s, 3H, eCH ), 0.84 (s, 6H, 2-CH ), 0.94 (s, 3H, eCH ), 0.96 (s, 3H, e
CH ), 1.09 (m, 1H), 1.10e1.41 (m, 8H), 1.47 (s, 9H, t-Bu), 1.49e1.65
m, 8H), 1.68 (s, 3H, eCH ), 1.70e1.80 (m, 3H), 1.98 (m, 3H), 2.04 (s,
), 2.45 (dt, 1H, J ¼ 3.1 Hz, J ¼ 12. 5 Hz), 3.13 (dt, 1H, J ¼ 4.
), 3.34 (m, 2H, eCH ), 4.46 (t, 1H,
(
2-aminoethyl)carbamate (6). R :
f
1
3
d
(
3
3
3
5
.1.2. General procedure for the synthesis of derivatives 4-7
3
(
3
PyBOP (0.31e0.47 g, 0.6e0.9 mmol) and the appropriate amine
3H, eCOCH
3
(
(
2
0.6e0.9 mmol) were successively added to a mixture of 3-O-Ac-BA
3) (200e300 mg, 0.4e0.6 mmol) and DIEA (0.28e0.42 mL, 1.6e
.4 mmol) in DMF (2e3 mL). The mixture was stirred for 72 h
and then diluted with CH Cl (50 mL), washed with aqueous citric
acid solution (10%), water, brine and dried over MgSO . The organic
layer was concentrated under vacuum and the residue was purified
by silica gel chromatography to provide the corresponding C28
intermediates.
4
Hz, J ¼ 11 Hz), 3.27 (m, 2H, eCH
2
2
J ¼ 7. 0 Hz), 4.59 (s, 1H, ¼CH), 4.74 (s, 1H, ¼CH), 5.04 (m, 1H, -NHe
COe), 6.33 (m, 1H, eCOeNHe).
2
2
4
6
2
5.1.2.4.
L-Lysine, N -[(1,1-dimethylethoxy)carbonyl]-, N -[(3b)-3-
(
(
(
7
1
acetyloxy)-28-oxolup-20(29)-en-28-yl]-, 1-(1,1-dimethylethyl) ester
1
7). R
400 MHz, C
H, eCH in 23 and 24, eCH in 1), 1.09 (2s, 6H, eCH
.16 (m, 1H, eCH in 11), 1.17 (m, 1H, eCH in 12), 1.29 (m, 1H, eCH in
5), 1.32 (m, 1H, eCH in 11), 1.33 (m, 1H, eCH in 9), 1.42 (m, 3H, eCH
f
: 0.28 (toluene/acetone 95/5); Yield: 73% (410 mg); H NMR
N): 0.80 (m, 4H, eCH in 5, eCH in 25), 0.90 (m,
in 27 and 26),
5
D
5
d
3
5
1
.1.2.1. Glycine, N-[(3
b
)-3-(acetyloxy)-28-oxolup-20(29)-en-28-yl]-,
: 0.38 (toluene/acetone 10/0.5);
): 0.75 (m, 1H, eCH
), 0.91 (s, 3H, eCH ), 0.95
), 1,09 (m, 2H), 1.10e1.45 (m, 11H), 1.47 (s, 12H, eCH , t-
Bu), 1.50e1.65 (m, 6H), 1.67 (s, 3H, eCH ), 1.75e2.0 (m, 3H), 2.03 (s,
), 2.45 (dt, 1H, J ¼ 3. 5 Hz, J ¼ 13 Hz), 3.10 (dt, 1H, J ¼ 3.
Hz, J ¼ 11 Hz), 3.89 (m, 2H, eCH ), 4.45 (t, 1H, J ¼ 7. 5 Hz), 4.58 (s,
H, ¼CH), 4.73 (s, 1H, ¼CH), 6.10 (t, 1H, J ¼ 5. 2 Hz). C NMR
): 14.6; 16.0; 16.2; 16.5; 18.2; 19.4; 20.9; 21.3;
3
3
-(1,1-dimethylethyl) ester (4). R
f
1
1
Yield: 64% (158 mg); H NMR (300 MHz, CDCl
in 5), 0.82 (s, 3H, eCH ), 0.83 (s, 3H, eCH
s, 3H, eCH
3
d
in 6 and 7), 1.48 (s, 9H, eCOOtBu and m, 1H, eCH in 6), 1.52 (m, 2H,
eCH in 21 and 22), 1.55 (s, 9H, eNHBoc), 1.58 (m, 2H, eCH in 1 and
3
3
3
(
3
3
2
(
1
), 1.69 (m, 2H, eCH
m, 1H, eCH in 2), 1.78 (s, 3H, eCH
.86 (m, 1H, eCH Lys), 1.92 (m, 1H, eCH in 12), 2.07 (m, 1H, eCH
Lys), 2.08 (s, 3H, eCOCH ), 2.30 (m,1H, eCH in 21), 2.33 (m,1H, eCH
2
g
Lys), 1.70 (m, 3H, eCH in 18, eCH
2d
Lys), 1.71
3
3
in 30), 1.80 (m, 1H, eCH in 15),
3
5
1
H, eCOCH
3
b
b
2
13
3
in 22), 2.54 (m, 1H, eCH in 16), 3.03 (m, 1H, eCH in 13), 3.40 (m, 2H,
eCH2ε Lys), 3.58 (m, 1H, eCH in 19), 4.7 (dd, 1H, eCH in 3, J ¼ 5 Hz
(
300 MHz, CDCl
3
d
a
and 11. 5 Hz), 4.75 (s, 1H, ]CH), 4.91 (m, 2H, ]CH and eCH Lys),
7
.55 (t, 1H, eNHeCOe J ¼ 5 Hz), 8.22 (d, 1H, eCOeNHe. J ¼ 8 Hz).
13
C NMR (400 MHz, C
5
D
5
N):
d
15.3 (C27); 16.7 (C25); 17 (C26); 17.2
0
(
(
(
(
(
(
C24); 19 (C6); 20 (C30); 21.6 (C2 ); 21.7 (C11); 24.3 (C
gLys); 24.5
C2); 26. 6 (C12); 28.4 (3C, t-Bu); 28.5 (C23); 29 (3C, Boc); 30.3
C15); 30.5(C Lys); 31.9 (C21); 32 (C Lys); 34 (C16); 35.1 (C7); 37.8
C10); 38.1 (C13); 38.5(C4); 38.65 (C22); 39 (C1); 41.3 (CεLys); 41.6
C8); 43.2 (C14); 47.6 (C19); 51.1 (C18); 51.3 (C9); 53.4 (C Lys); 56.1
d
b
a
C5); 56.4 (C17); 78.4 (CBoc); 81.2 (C3); 81.3 (Ct-Bu); 110.2 (C29);
Fig. 5. Effect of the compounds on the virus particle production and Pr55Gag pro-
cessing was analysed by western blot analysis. Note the accumulation of p25 in the
presence of 2, 12, 16, 15, 13 and 17.
0
152.1 (C20); 157.25 (C NHCOOt-Bu); 171.1 (C1 ); 173.4 (C COOt-Bu);
177.3 (C28).

462
P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
Fig. 6. Thin-section EM analysis of virions produced from BA, 2, 12, 16, 15, 13, 17-treated cells. 293T cells were transfected with pNL-4-3 and were not treated (A) or treated (B, C, D,
E, F, G, H) with BA, 2, 12, 16, 15, 13, 17. Two days post transfection, cells were fixed and analyzed by thin-section EM. Dashed arrow in A indicate mature, conical cores; arrows in B, C,
D, E, F, G, H indicate the crescent-shaped, electron-dense layer inside the viral membrane that results from inhibition of p25 processing. (Bar: z100 nm).
5
.1.3. General procedure for the synthesis of derivative 8e11
An aqueous NaOH (4 M, 0.75 mL) was added to the solution of
(C4); 42.6 (C8); 44.1 (C14); 48.7 (C19); 52.1 (C18); 52.4 (C9); 57.28
(C17); 57.34 (C5); 79.5 (C3); 110.1 (C29); 153.1 (C20); 177.5 (C3 );
178.1 (C28).
00
the above intermediates (0.3 mmol) in THF/MeOH (1/1.4 mL). After
stirring for 12 h at room temperature, the mixture was acidified
with 1 M HCl. The resulting precipitate was collected, washed with
water and dried over vacuum to yield the corresponding com-
pounds 8 and 9. For compounds 10 and 11, the mixture was
extracted with CH
water, brine and dried over MgSO
vacuum. The residue was purified by silica gel chromatography to
provide the corresponding derivatives.
5.1.3.3. N-[(3
b
)-3-hydroxy-lup-20(29)-en-28-oyl]-1,1-dimethylethyl-
: 0.48 (toluene/acetone 80/20);
Yield 67% (210 mg); H NMR (300 MHz, CDCl ): 0.70 (m, 1H, eCH
in 5), 0.77 (s, 3H, eCH ), 0.83 (s, 3H, eCH ), 0.94 (s, 3H, eCH ), 0.97
(s, 6H, 2-CH ), 1.09 (m, 1H), 1.10e1.40 (m, 9H), 1.45 (s, 9H, t-Bu),
1.50e1.65 (m, 7H), 1.69 (s, 3H, eCH ), 1.72 (m, 3H), 1.99 (m, 2H), 2.
46 (dt, 1H, J ¼ 3. 5 Hz, J ¼ 13 Hz), 3.20 (m, 6H), 3.20 (m, 1H), 4. 60 (s,
H, ]CH), 4. 74 (s, 1H, ]CH), 5.01 (m, 1H, eNHeCOe), 6.33 (m, 1H,
(2-aminoethyl)carbamate (10). R
f
1
3
d
2
Cl
2
(50 mL), the organic layer was washed with
and then concentrated under
3
3
3
4
3
3
1
5
.1.3.1. N-[(3
b
)-3-hydroxy-lup-20(29)-en-28-oyl]-glycine
(8). Yie
0.70 (m, 1H, eCH in 5), 0.77 (s,
), 0.93 (s, 3H, eCH ), 0.98 (s, 3H, eCH ),
), 1.09 (m, 1H), 1.10e1.65 (m, 20H), 1.70 (s, 3H, e
), 1.90 (m, 3H), 2.40 (dt, 1H, J ¼ 3.5 Hz, J ¼ 13 Hz), 3.10 (dt, 1H,
J ¼ 3.5 Hz, J ¼ 11 Hz), 3.20 (m,1H), 4. 05 (t, 2H, J ¼ 7.5 Hz), 4.61 (s, 1H,
CH), 4.75 (s, 1H, ]CH), 6.14 (t, 1H, J ¼ 5.2 Hz).
eCOeNHe).
1
ld: 83% (128 mg); H NMR (CDCl
3
0
CH
3
): d
6
2
3
H, eCH ), 0.83 (s, 3H, eCH
3
3
3
5.1.3.4. N -[(1,1-dimethylethoxy)carbonyl], N -[(3
b
)-3-hydroxy-lup-
.99 (s, 3H, eCH
3
20(29)-en-28-oyl]-
acetic acid 10/10/0.25); Yield 97% (277 mg); H NMR (400 MHz,
N): 0.86 (m, 4H, eCH in 5, eCH in 25), 1.02 (m, 1H, eCH in 1),
1.06 (s, 3H, eCH in 24), 1.10 (s, 3H, eCH in 27), 1.18 (s, 3H, eCH in
6), 1.20 (m, 1H, eCH in 12), 1.22 (m, 1H, eCH in 11), 1.26 (s, 3H, e
CH in 23), 1.35 (m, 1H, eCH in 15), 1.42 (m, 1H, eCH in 11), 1.43 (m,
1H, eCH in 9), 1.48 (m, 1H, eCH in 6), 1.50 (m, 2H, eCH in 7), 1.52
L
-lysine (11). R : 0.32 (n-heptane/ethyl acetate/
f
1
3
C
5
D
5
d
3
]
3
3
3
2
5.1.3.2. N-[(3
b
)-3-hydroxy-lup-20(29)-en-28-oyl]-
b
-alanine
(9).
3
1
Yield: 89% (141 mg); H NMR (400 MHz, C
CH in 5), 0.88 (m, 3H, eCH in 25), 0.99 (m, 1H, eCH in 1), 1.04 (s,
H, eCH in 24), 1.08 (s, 3H, eCH in 27), 1.16 (s, 3H, eCH in 26), 1.2
m, 1H, eCH in 12), 1.24 (m, 2H, eCH in 11 and eCH in 15), 1.27 (m,
H, eCH in 23), 1.41 (m, 3H, eCH in 6 and eCH in 7), 1.42 (m, 1H, e
CH in 9), 1.44 (m, 1H, eCH in 11), 1.5 (m, 2H, eCH in 21 and eCH in
2), 1.58 (m, 2H, eCH in 6 and eCH in 16), 1.68 (m, 1H, eCH in 1),
.72 (m, 1H, eCH in 18), 1.8 (s, 3H, eCH in 30), 1.86 (m, 1H, eCH in
5), 1.89 (m, 2H, eCH in 2), 1.98 (m, 1H, eCH in 12), 2.13 (m, 1H, e
CH in 22), 2.28 (m, 1H, eCH in 21), 2.44 (m, 1H, eCH in 16), 2.96 (m,
5
D
5
N):
d
0.86 (m, 1H, e
2
3
(m, 1H, eCH in 21), 1.53 (m, 1H, eCH in 22), 1.55 (s, 9H, eNHBoc),
1.61 (m, 1H, eCH in 6), 1.67 (m, 1H, eCH in 16), 1.70 (m, 1H, eCH
3
(
3
3
3
3
in 1), 1.75 (m, 1H, eCH in 18), 1.77 (s, 3H, eCH
2H, eCH Lys), 1.81 (m, 2H, eCH Lys), 1.88 (m, 2H, eCH
(m, 1H, eCH in 15), 1.96 (m, 1H, eCH in 12), 2.05 (m, 1H, eCH
2.30 (m, 1H, eCH Lys), 2.36 (m, 1H, eCH in 21), 2.45 (m, 1H, eCH in
3
in 30), 1.80 (m,
in 2), 1.91
Lys),
3
2
d
2
g
2
b
2
1
1
b
3
22), 2.58 (m, 1H, eCH in 16), 3.09 (m, 1H, eCH in 13), 3.43 (m, 2H, e
CH2ε Lys), 3.49 (m, 1H, eCH in 3), 3.65 (m, 1H, eCH in 19), 4.77 (s,
1H, ]CH in 29), 4.92 (s, 1H, ]CH in 29), 5.16 (m, 1H, eCH Lys), 7.58
a
0
0
13
2
3
H, eCH in 2 ), 3.05 (m, 1H, eCH in 13), 3.46 (m, 1H, eCH in 3),
(m, 1H, eNHeCOe), 8.25 (d, 1H, eCOeNH-, J ¼ 8 Hz). C NMR
(400 MHz, C N): 15.3 (C27); 16.85 (C24); 17 (C25); 17.1 (C26);
19.3 (C6); 20 (C30); 21.7 (C11); 24.5 (C Lys); 26.7 (C12); 28.8 (C2);
29.1 (3C, Boc); 29.2(C23); 30.4 (C15); 30.6 (C Lys); 31.9 (C21); 32.5
(C Lys); 34.2 (C16); 35.4 (C7); 38 (C10); 38.2 (C13); 38.8 (C22);
39.75 (C1); 40 (C4); 41.4 (CεLys); 41.7 (C8); 43.2 (C14); 47.7 (C19);
51.2 (C18); 51.6 (C9); 53 (C Lys); 56.4 (C5); 56.6 (C17); 78,4 (3CBoc);
00
.65 (m, 1H, eCH in 19), 3.93 (m, 1H, eCH in 1 ), 3.98 (m, 1H, eCH
5
D
5
d
0
0
in 1 ), 4.77 (s, 1H, ]CH), 4.93 (s, 1H, ]CH), 8.36 (d, 1H, eCOeNHe.
g
13
J ¼ 7 Hz). C NMR (400 MHz, C
5
D
5
N):
d
16. 2 (C27); 17.8 (C24);
d
17.86 (C25); 17.92 (C26); 20.2 (C6); 21 (C30); 22.6 (C11); 27.6 (C12);
b
2
9.7 (C2); 30 (C23); 31.2 (C15); 32.8 (C21); 35 (C16); 36.2 (C7); 37.4
0
0
00
(
C2 ); 37.9 (C1 ); 38.9 (C10); 39.2 (C13); 40.1 (C22); 40.6 (C1); 40.9
a

P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
463
7
8.6 (C3); 110.1 (C29); 152.2 (C20); 157.3 (C, NHCOOt-Bu); 176.6 (C,
5.1.4.3. 4-({28-[1,1-dimethylethyl-(2-aminoethyl)carbamate]-28-
oxolup-20,29-en-3 -yl}oxy)-2,2-dimethyl-4-oxobutanoic acid (14).
: 0.25 dichloromethane/methanol/acetic acid (10/0.2/0.01); Yield
COOH); 177.4 (C28).
b
R
f
1
5.1.4. General procedure for the synthesis of derivatives 12e15
82% (190 mg); H NMR (300 MHz, CDCl
0.77 (s, 3H, eCH ), 0.78 (s, 3H, eCH ), 0.79 (s, 3H, eCH
eCH ), 0.92 (s, 3H, eCH ), 1.10 (m, 1H), 1.26 (s, 6H), 1.27e1.40 (m,
6H), 1.42 (s, 9H, t-Bu), 1.42e1.63 (m, 7H), 1.69 (s, 3H, eCH ), 1.72 (m,
3
):
d
0.72 (m, 1H, eCH in 5),
A mixture of the above intermediates (8, 9, 10 or 11), 2,2-
3
3
3
), 0.89 (s, 3H,
dimethylsuccinic anhydride (10 equiv) and DMAP (1 equiv) in
anhydrous pyridine (20 mL/mmol) was refluxed overnight. The
mixture was then concentrated under vacuum and the residue was
chromatographed on silica gel to yield the derivatives 12, 13, 14 and
3
3
3
2H), 1.99 (m, 2H), 2.46 (m, 1H), 2.57 (m, 2H), 3.08 (m, 1H), 3.31 (m,
9H), 4.42 (m, 1H), 4.55 (s, 1H, ]CH), 4.69 (s, 1H, ]CH), 5.17 (m, 1H,
eNHeCOe), 5.28 (m, 1H, eCOeNHe).
15.
6
2
5
2
.1.4.1. N-[(3
8-oyl]-glycine (12). R
b
)-3-(3-carboxy-3-methylbutanoyloxy)lup-20(29)-en-
0.23 dichloromethane/methanol/acetic
5.1.4.4. N -[(1,1-dimethylethoxy)carbonyl, N -[(3
b
)-3-(3-carboxy-3-
0.31
f
:
methylbutanoyloxy)-lup-20(29)-en-28-oyl]- -lysine (15). R :
L
f
acid (10/0.1/0.02); Yield 58% (80 mg); HRMS (ESI) calcd for
n-heptane/ethyl acetate/acetic acid (10/10/0.25); Yield 36%
ꢀ
1
ꢀ
C
C
1
38
H
58N0
7
[M ꢀ H] 640.4219 found 640.4232; H NMR (400 MHz,
0.73 (s, 3H, eCH in 25), 0.77 (m, 1H, eCH in 5), 0.89 (m,
in 24), 0.96 (s, 3H, eCH in 23),
in 27), 1.12 (s, 3H, eCH in 26), 1.15 (m, 1H, eCH in
2), 1.15 (m, 1H, eCH in 11), 1.26 (m, 1H, eCH in 15), 1.30 (m, 2H, e
(106 mg); HRMS (ESI) calcd for C47
H
N):
75
N
d
2
0
7
[M ꢀ H] 811.5478 found
1
5
D
5
N):
d
3
811.5476; H NMR (400 MHz, C
5
D
5
0.76 (s, 3H, eCH
3
in 25), 0.82
3
in 24), 0.97
H, eCH in 1), 0.92 (s, 3H, eCH
3
3
(m,1H, eCH in 5), 0.9 (m,1H, eCH in 1), 0.96 (s, 3H, eCH
(s, 3H, eCH in 23), 1.08 (s, 3H, eCH
1.15 (m, 2H, eCH in 12 and eCH in 11), 1.3 (m, 1H, eCH in 9), 1.32 (m,
1H, eCH in 15), 1.33 (m, 1H, eCH in 11), 1.36e1.46 (m, 4H, eCH
in 6, eCH in 7), 1.53 (m, 1H, eCH in 21), 1.54 (m, 1H, eCH in 22),
1.55e1.59 (s, 3H, eCH
1.67e1.70 (m, 2H, eCH in 2, eCH in 16),1.70,1.73 (m,1H, eCH in 18),
1.78 (m,1H, eCH in 2),1.78 (s, 3H, eCH in 30),1.8 (m, 2H, eCH Lys),
1.83 (m, 1H, eCH Lys), 1.89 (m, 1H, eCH in 15), 1.95 (m, 1H, eCH in
12), 2.06 (m,1H, eCH Lys), 2.3 (m,1H, eCH Lys), 2.34 (m,1H, eCH
in 21), 2.43 (m, 1H, eCH in 22), 2.58 (m, 1H, eCH in 16), 2.9 (d, 1H,
1.06 (s, 3H, eCH
3
3
3
3 3
in 27), 1.09 (s, 3H, eCH in 26),
1
CH in 9, eCH in 11), 1.30e1.40 (m, 4H, eCH
2
in 6, eCH
2
in 7), 1.52
2
0
(
m, 1H, eCH in 1),1.54 (m, 1H, eCH in 21), 1.55 (s, 3H, eCH
3
in 5 ),
2
0
3
in 5 and s, 9H, eNHBoc and m,1H, eCH in 1),
1.59 ( m, 1H, eCH in 22), 1.67 (m, 2H, eCH in 2, eCH in 16), 1.74 (m,
1
3
H, eCH in 18), 1.77 (m, 1H, eCH in 2), 1.78 (s, 3H, eCH in 30), 1.93
(
2
m, 1H, eCH in 12), 2.03 (m, 1H, eCH in 15), 2.38 (m, 1H, eCH in
1), 2.39 ( m, 1H, eCH in 22), 2.59 ( m, 1H, eCH in 16), 2.91 (d, 1H,
3
2g
2d
0
0
eCH in 2 , J ¼ 15.5 Hz), 2.95 (d, 1H, eCH in 2 , J ¼ 15.5 Hz), 3.08 (m,
2b
2b
1
1
3
H, eCH in 13), 3.66 (m, 1H, eCH in 19),4.44e4.51 (dd, 2H, eCH
2
in
, J ¼ 6 Hz, J ¼ 17.5 Hz), 4.75 (s, 1H, eCH in 29), 4.77 (d, 1H, eCH in
, J ¼ 5 Hz), 4.96 (s, 1H, eCH in 29), 8.76 (t, 1H, -NHeCOe,
0
0
0
0
eCH in 2 , J ¼ 15.5 Hz), 2.98 (d,1H, eCH in 2 , J ¼ 15.5 Hz), 3.07 (m,1H,
eCH in 13), 3.43 (m, 2H, eCH2ε Lys), 3.63 (m, 1H, eCH in 19), 4.77 (s,
1H, eCH in 29), 4.78 (dd,1H, eCH in 3, J ¼ 5 Hz and J ¼ 11.5 Hz), 4.92
1
3
J ¼ 5.5 Hz). C NMR (400 MHz, C
5 5
D N): d 15.3 (C27); 16.7 (C25);
1
7 (C26); 17.3 (C24); 18.9 (C6); 20 (C30); 21.6 (C11); 24.6 (C2); 26.6
a
(s, 1H, eCH in 29), 5.17 (m, 1H, eCH Lys), 7.58 (m, 1H, -NHeCOeO),
0
13
(
(
(
(
(
(
C12); 26.7 (C5 ); 28.5 (C23); 30.3 (C15); 31.8 (C21); 34 (C16); 35
8.26 (d, 1H, eCOeNHe, J ¼ 8 Hz). C NMR (400 MHz, C
5 5
D N): d 15.3
C7); 37.7 (C10); 38.1 (C13); 38.5 (C4); 39 (C1); 39.1 (C22); 41.3
(C27); 16.7 (C25); 17 (C26); 17.4 (C24); 19 (C6); 20.0 (C30); 21.7
0
00
0
0
C3 ); 41.6 (C8); 42.4 (C1 ); 43.2 (C14); 45.6 (C2 ); 47.7 (C19); 51.0
C18); 51.2 (C9); 56.0 (C5); 56.4 (C17); 81.4 (C3); 110.3 (C29); 152.2
(C11); 24.5 (C
(3C, Boc); 30.3 (C15); 30.6 (C
(C16); 35.1 (C7); 37.75 (C10); 38.1 (C13); 38.5 (C4); 38.8 (C22); 39
gLys); 24.6 (C2); 26.4 (C5 ); 26.6 (C12); 28.6 (C23); 29.1
dLys); 31.9 (C21); 32.4 (CbLys); 34.2
0
00
0
C20); 172.1 (C1 ); 174 (C6 ); 177.9 (C28); 179.8 (C4 ). Anal.
$0.5H O) C, H, N.
0
0
C
38
H59NO
7
2
(C1); 41.3 (C3 ); 41.4 (CεLys); 41.6 (C8); 43.2 (C14); 45.7 (C2 ); 47.7
C19); 51.1 (C18); 51.2 (C9); 53 (C Lys) 56.1 (C5); 56.5 (C17); 78.4
(
a
0
00
5
.1.4.2. N-[(3
b
)-3-(3-carboxy-3-methylbutanoyloxy)lup-20(29)-en-
: 0.27 n-heptane/ethyl acetate/acetic acid
(COBoc); 81.4 (C3); 110.2 (C29); 152.2 (C20); 172.1 (C1 ); 176.6 (C6 );
0
28-oyl]- -alanine (13). R
b
f
177.7 (C28); 179.8 (C4 ). Anal. (C47
H
76
N
2
O
9
$2H
2
O) C, H, N.
(
[
[
10/10/0.5); Yield 2.62% (166 mg); HRMS (ESI) calcd for C39
H
60N0
7
ꢀ
M ꢀ H] 654.4375 found 654.4375, calcd for C39
H
59N0
7
Na
5.1.5. Procedure for synthesis of compound 4-({28-[(2-aminoethyl)
amino]-28-oxolup-20,29-en-3b-yl}oxy)-2,2-dimethyl-4-
oxobutanoic acid (16)
ꢀ
1
M ꢀ 2H þ Na] 676.4195 found 676.4186; H NMR (400 MHz,
N): 0.74 (m, 1H, eCH in 5 and s, 3H, eCH in 25), 0.91 (m, 1H,
eCH in 1), 0.94 (s, 3H, eCH in 24), 0.97 (s, 3H, eCH in 23), 1.04 (s,
H, eCH in 27), 1.08 (s, 3H, eCH in 26), 1.17 (m, 2H, eCH in 11 and
eCH in 12), 1.2 (m, 1H, eCH in 15), 1.30 (m, 2H, eCH in 9, eCH in 6),
.31 (m, 1H, eCH in 11), 1.33 (m, 2H, eCH in 7), 1.4 (m, 1H, eCH in 6)
C
5
D
5
d
3
3
3
tert-Butyldimethylsilyltrifluoromethanesulfonate
SiOTf, 150 L, 0.65 mmol) was added dropwise to a stirred solution
under argon of N-tert-Boc derivatives 14 (0.19 mg, 0.26 mmol) and
2,6-lutidine (91 L, 0.78 mmol) in dry CH Cl (1 mL). The reaction
(t-BuMe
2-
3
3
3
m
1
m
2
2
0
1
(
2
.52 (m, 2H, eCH in 21 and eCH in 22), 1.55 (s, 3H, eCH
m, 2H, eCH in 1 and eCH in 16), 1.7 (m, 2H, eCH in 18 and eCH in
), 1.81 (s, 3H, eCH in 30), 1.82 (m, 1H, eCH in 2), 1.85 (m, 1H, eCH
3
in 5 ), 1.58
mixture was stirred 30 min, quenched with saturated aqueous
ammonium chloride solution (2 mL). The mixture was diluted with
3
CH
dried over MgSO
the residue was used in the next step without purification. Tetra-
butylammonium fluoride (260 L, 1 M solution in THF, 0.26 mmol)
2
Cl
2
(5 mL), the organic layer was washed with water, brine and
in 15), 1.96 (m, 1H, eCH in 12), 2.16 (m, 1H, eCH in 22), 2.27 (m, 1H,
eCH in 21), 2.47 (m, 1H, eCH in 16), 2.94 (d, 1H, eCH in 2 ,
4
. The solvent was concentrated under vacuum and
0
0
J ¼ 15.5 Hz), 3.00 (d, 1H, eCH in 2 , J ¼ 15.5 Hz), 3.01 (m, 1H, eCH in
m
0
0
2
), 3.07 (m, 1H, eCH in 13), 3.67 (m, 1H, eCH in 19), 3.9 (m, 1H, e
was added to a stirred solution of the N-(tert-butyldimethylsily-
loxycarbonyl) derivatives in dry THF (0.5 mL) at room temperature.
The reaction mixture was stirred for 1 h, quenched with saturated
aqueous ammonium chloride solution (2 mL). The resulting pre-
cipitate was collected, washed with water and dried over vacuum to
00 00
2 2
in 1 ), 4.03 (m, 1H, eCH in 1 ) 4.79 (m, 2H, eCH in 3 and eCH
CH
in-29), 4.97 (s, 1H, eCH in 29), 8.33 (t, 1H, eNHeCOe, J ¼ 7 Hz).
NMR (400 MHz, C N): 15.2 (C27); 16.7 (C25); 16.9 (C26); 17.4
C24); 18.9 (C6); 20 (C30); 21.7 (C11); 24.6 (C2); 26.6 (C12); 26.7
13
C
5
D
5
d
(
(
(
(
(
(
(
0
C5 ); 28.6 (C23); 30.3 (C15); 31.8 (C21); 34 (C16); 35 (C7); 35.7
yield the corresponding compound 16 (Refer to Supporting data).
0
0
00
0
ꢀ
C2 ); 36.7 (C1 ); 37.7 (C10); 38.2 (C13); 38.5 (C4); 39 (C1); 39.1
Yield: 26% (45 mg); HRMS (ESI) calcd for C38
H
D
61
N
N):
2
0
d
5
[M ꢀ H]
0
1
C22); 41.3 (C3 ); 41.6 (C8); 43.2 (C14); 45.7 (C2 ); 47.7 (C19); 51.1
C18); 51.2 (C9); 56.1(C5); 56.3 (C17); 81.4 (C3); 110.2 (C29); 152.2
625.4586 found 625.4569; H NMR (400 MHz, C
eCH
3H, eCH
(s, 3H, eCH
5
5
0.75 (s, 3H,
3
in 25), 0.77 (m, 1H, eCH in 5), 0.86 (m, 1H, eCH in 1), 0.93 (s,
in 24), 0.97 (s, 3H, eCH in 23),1.03 (s, 3H, eCH in 27),1.03
in 26), 1.12 (m, 1H, eCH in 12), 1.13 (m, 1H, eCH in 11),
0
00
0
C20); 172 (C1 ); 175.4 (C6 ); 177.2 (C28); 179.8 (C4 ). Anal.
$0.5H O) C, H, N.
3
3
3
C
39
H61NO
7
2
3

464
P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
1
.17 (m, 1H, eCH in 15), 1.30 (m, 2H, eCH in 9, eCH in 11), 1.30e1.40
tubes. All chemical shifts are given in ppm (Fig. 3) and (Tables 1 and
2 and Figures S1 to S5 in supplementary data). The NMR spectra
were registered with spectral width of 4084.97 Hz and
(
m, 4H, eCH
2
in 6, eCH
2
in 7), 1.48 (m, 1H, eCH in 21), 1.54 (m, 1H,
0
eCH in 1), 1.55 (s, 3H, eCH
3
in 5 ), 1.55 (m, 1H, eCH in 22), 1.64e1.67
a
1
13
(
(
2
m, 2H, eCH in 2, eCH in 16),1.70 (m, 2H, eCH in 15, eCH in 18),1.77
m, 1H, eCH in 2), 1.77 (s, 3H, eCH in 30), 1.92 (m, 1H, eCH in 12),
.14 (m, 1H, eCH in 21), 2.25 (m, 1H, eCH in 22), 2.67 (m, 1H, eCH in
22,522.52 Hz for the H and C dimensions respectively. WALTZ
1
3
[60,61] pulse sequence was used for H decoupling. The DEPT135
experiment (Distortionless Enhancement by Polarization Transfer)
using polarization transfer from proton to other nuclei via one
covalent bond allowed the determination of the CH multiplicity.
n
0
0
1
6), 2.90 (d, 1H, eCH in 2 , J ¼ 15.5 Hz), 2.96 (d, 1H, eCH in 2 ,
0
0
J ¼ 15.5 Hz), 3.03 (m, 1H, eCH in 13), 3.28 (m, 2H, eCH
2
in 2 ), 3.61
00
(
m,1H, eCH in 19), 3.87e3.81 (m, 2H, eCH
2
in 1 ), 4.75 (s,1H, eCH in
All 2D experiments were performed using standard pulse
sequences with the Bruker software program XWIN-NMR vs. 3.0.
DQF-COSY and TOCSY spectra were recorded with 2048 points in
the F2 dimension and 512 increments in the F1 dimension. Each
increment was obtained with 40 scans, a spectral width of
4084.97 Hz, a recycling delay of 2 s and a mixing time of 80 ms were
used for the TOCSY experiments. The HSQC spectra were obtained
2
1
9), 4.77 (d, 1H, eCH in 3, J ¼ 5 Hz), 4.93 (s, 1H, eCH in 29), 8.74 (m,
H, eNHeCOe). 13C NMR (400 MHz, C
D
5
N): 15.2 (C27); 16.7
d
5
(
(
(
(
(
(
C25); 16.9 (C26); 17.4 (C24); 19 (C6); 20.0 (C30); 21.6 (C11); 24.6
C2); 26.5 (C12); 26.7 (C5 ); 28.5 (C23); 30.3 (C15); 31.8 (C21); 34.0
C16); 35.0 (C7); 37.7 (C10); 38.1 (C13); 38.5 (C4); 39.0 (C22); 39.1
C1); 41.0 (C1 ); 41.4 (C3 ); 41.5 (C8); 42.2 (C2 ); 43.1 (C14); 45.7
C2 ); 47.7 (C19); 51.2 (C18); 51.3 (C9); 56.1 (C5); 56.5 (C17); 81.3
C3); 110.2 (C29); 152.2 (C20); 172.1 (C1 ); 177.7 (C28); 179.9 (C4 ).
$3H O) C, H, N.
0
0
0
0
00
0
1
13
with the one-bond He C coupling constant set to 145 Hz, a GARP
0
0
13
C decoupling WALTZ [60,62], 2048 points in the F2 dimension and
Anal. (C38
H
62
N
2
O
5
2
256 increments in the F1 dimension, with a relaxation delay of 2 s.
The spectral width was set to 10,964.91 Hz and 4084.97 Hz in the
2
1
13
5
3
.1.6. Procedure for synthesis of compound N -[(3
-methylbutanoyloxy)lup-20(29)-en-28-oyl]- -lysine (17)
The N-Boc protection of product 15 was cleaved with 6 M HCl in
b
)-3-(3-carboxy-
F2 and F1 dimensions respectively. The 2D ( He C)-HMBC exper-
iments were performed with 40 scans for each of 256 F1 increments
and 2048 data points in F2. Spectral widths of 4084.97 Hz and
L
1
13
THF for 1 h. Compound 17 was obtained after purification by flash
chromatography. Yield: 45% (20 mg); HRMS (ESI) calcd for
22,522.52 Hz were used in the H and C dimensions respectively.
2D NMR spectra demonstrating the interaction between the CA-
SP1eNC junction and compound 16 were recorded at 293 K on
a 600 MHz Bruker Avance spectrometer equipped with a 5 mm
ꢀ
1
C
CD
3
0
1
H N
42 67 2
0
7
[M ꢀ H] 711.4954 found 711.4960; H NMR (400 MHz,
0.82 (m, 1H, eCH in 5), 0.85 (s, 3H, eCH in 24), 0.87 (s,
in 23), 0.92 (s, 3H, eCH in 25), 0.96 (s, 3H, eCH in 26),
.99 (m, 1H, eCH in 1), 1.01 (s, 3H, eCH in 27), 1.04 (m, 1H, eCH in
2), 1.18 (m, 1H, eCH in 15), 1.25 (s, 3H, eCH
in 11), 1.35 (m, 1H, eCH in 21), 1.36 (m, 1H, eCH in 9), 1.38e1.52 (m,
3
OD):
d
3
1
1
H, eCH
3
3
3
triple resonance inverse probe and a XYZ gradient unit. 2D He H
NOESY spectra were recorded for the peptide CA-SP1eNC (250 M)
in the absence and presence of compound 16 (550 M) and
superimposed, at a pH of 3.8, in H2O/TFE (70/30) mixture to pre-
vent aggregation, DMSO-d (1%) and a spectral width of 5296.6 Hz
3
m
0
3
in 5 ), 1.26 (m, 1H, eCH
m
9
3
H, eCH
2
in 6, eCH in 7, eCH in 11, eCH in 15, eCH in 22, eCH
0
), 1.56 (m, 1H, eCH in 16), 1.6 (m, 1H, eCH in 2), 1.64 (m, 1H, eCH
2
2
in
6
0
in both dimensions. Water suppression was achieved by using
excitation sculpting with a 2 ms selective Gaussian pulse at the
water signal frequency and a 250 ms NOE mixing time (Fig. 4B).
in 18), 1.68 (m, 1H, eCH in 2), 1.7 (m, 6H, eCH
3
in 30, eCH in 12, 2H,
0
0
00
eCH
1
(
2
in 4 ), 1.72 (m, 1H, eCH in 1), 1.75 ( m, 1H, eCH in 2 ), 1.89 (m,
H, eCH in 2 ), 1.9 (m, 1H, eCH in 21), 1.93 ( m, 1H, eCH in 22), 2.17
m, 1H, eCH in 16), 2.53 (m, 1H, eCH in 13), 2.55 (d, 1H, eCH in 2 ,
0
0
0
5.4. HIV-1 infection inhibition assays
0
J ¼ 15.5 Hz), 2.62 (d, 1H, eCH in 2 , J ¼ 15.5 Hz), 2.90 (t, 2H, eCH in
0
0
00
5
(
(
, J ¼ 8 Hz), 3.06 (m, 1H, eCH in 19), 4.3 (m, 1H, eCH in 1 ), 4.44
Infectivity of HIV-1 particles produced in the presence of in-
hibitors was carried out using MAGIC-5B indicator cells, which
dd, 1H, eCH in 3, J ¼ 5 Hz and J ¼ 10 Hz), 4.57 (s, 1H, eCH in 29), 4.7
13
s, 1H, eCH in 29), 7.1 (m, 1H, eNHeCOe). C NMR (400 MHz,
OD): 15.2 (C27); 17 (C25); 17.1 (C26); 17.2 (C24); 19.4 (C6);
9.8 (C30); 22.3 (C11); 24.05 (C3 ); 24.8 (C2); 26.2 (C5 ); 27.2 (C12);
stably express the b-galactosidase reporter gene cloned down-
4
CD
3
d
stream of the HIV-1 LTR promoter. Cells were plated at 8 ꢁ 10 cells
per well, in 24-well plates and exposed to virus-containing super-
natants produced by transfection of the HIV-1 molecular clone
(pNL-4.3) in 293T cells maintained in the presence or absence of
compounds 1, 2, 12, 13, 15, 16 and 17. Forty-eight hours post-
infection, viral infectivity was monitored by quantification of o-
00
0
1
2
3
(
(
(
00
00
8.2 (C4 ); 28.7 (C23); 30.9 (C15); 32.1 (C21); 32.7 (C2 ); 34.4 (C16);
5.7 (C7); 38.4 (C10); 39 (C4); 39.2 (C13); 39.3 (C22); 39.8 (C1); 40.7
00 0 0
C5 ); 41.5 (C3 ); 42.2 (C8); 43.7 (C14); 45.8 (C2 ); 48.3 (C19); 51.4
0
0
C18); 52.1 (C9); 54 (C1 ); 57 (C5); 57.4 (C17); 82.8 (C3); 110.2
C29); 152.4 (C20); 173.1 (C1 ); 177.3 (C6 ); 178.9 (C28); 180.8 (C4 ).
$2H O) C, H, N.
0
00
0
nitrophenyl
b-D-galactopyranoside hydrolysis from cell lysates (kit).
Anal. (C42
H
68
N
2
O
7
2
b-galactosidase activity was normalized according to the total
protein content in the cell lysate. The control wells containing the
virus and cells only (no virus or drug) were also prepared. The 50%
infection inhibition (IC50) was defined as the concentration of the
compound that reduced the HIV-1 infection level by 50% compared
to the untreated controls (Table 3).
5
.2. HPLC analysis
HPLC analysis were conducted using a Shimadzu LC-10ADvp
m column and
with a Nucleodur 4.6 mm ꢁ 250 mm C18 HTec 5
m
a Shimadzu SPD-M10Avp detector at 210 nm wavelength. The sol-
vent system used was MeOH(0.1%TFA):water (0.1%TFA) ¼ 90:10. The
isocratic HPLC mode was used and the flow rate was set to 1 mL/min.
5.5. Electron microscopy analysis
2
93Tcells were transfected with HIV-1 molecular clones (pNL-4-
3) using the JetPei transfection reagent (QBiogen) and maintained in
the presence or absence of 1, 2, 12, 13, 15, 16 and 17 (5 g/ml). Two
5
.3. NMR
m
1H NMR, 13C NMR, DEPT135, DQF-COSY, TOCSY, HSQC and HMBC
days after transfection, the cells were processed for thin-layer
spectra of compounds 2, 12, 13, 15, 16 and 17 (Fig. 1) were recorded
on Bruker AVANCE 400 NMR spectrometer operating at
00.13 MHz (SF) for H and 100.623 MHz (SF) for C with a 5 mm
inverse probe at room temperature. The concentration of the dif-
ferent compounds was 10 mM in 0.5 mL C N, in 5 mm NMR
electron microscopy as follows: the cells were fixed in situ with
2.5% glutaraldehyde in cacodylate buffer (pH 7.4) for 60 min at 4 C.
They were then post-fixed with 2% osmium tetroxide, washed in
cacodylate buffer containing 0.5% tanic acid, and embedded in epon
(Embed-812, Electron Microscopy Sciences Inc.). The sections were
ꢃ
a
1
13
4
5 5
D

P. Coric et al. / European Journal of Medicinal Chemistry 62 (2013) 453e465
465
counterstained with uranyl acetate and lead citrate and examined
with an Hitachi H7100 transmission electron microscope.
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[
[
[
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[
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[
clones (pNL-4-3) in 293T cells maintained in the presence or
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JetPei transfection reagent (QBiogen). Two days after the trans-
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h 30 min. Viral pellets were then suspended in RIPA lysis buffer
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[
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