Semisynthetic neoboutomellerone derivatives as ubiquitin-proteasome pathway inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.11.066

The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the assembly of a small library of analogues consisting of natural products isolated from the plant Neoboutonia melleri and of semisynthetic derivatives. As the tw

Full text of DOI:10.1016/j.bmc.2011.11.066

Bioorganic & Medicinal Chemistry 20 (2012) 819–831
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Semisynthetic neoboutomellerone derivatives as ubiquitin-proteasome
pathway inhibitors
Joséphine Beck a, Yves Guminski c, Christophe Long a, Laurence Marcourt a, Fadila Derguini a,
Fabien Plisson a, Antonio Grondin b, Isabelle Vandenberghe b, Stéphane Vispé b, Viviane Brel b,
Yannick Aussagues a, Frédéric Ausseil a, Paola B. Arimondo a, Georges Massiot a, François Sautel a,
⇑
,
Frédéric Cantagrel a,
⇑
a USR CNRS-Pierre Fabre No. 3388 ETaC, Centre de Recherche et Développement Pierre Fabre, 3 Avenue Hubert Curien, 31035 Toulouse Cedex 01, France
b Institut de Recherche Pierre Fabre, Centre de Recherche en Oncologie Experimentale, Centre de Recherche et Développement Pierre Fabre, 3 Avenue Hubert Curien,
31035 Toulouse Cedex 01, France
c Institut de Recherche Pierre Fabre, Division de Chimie Médicinale, Centre de Recherche Pierre Fabre, 17 rue Jean Moulin, 81106 Castres Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the
assembly of a small library of analogues consisting of natural products isolated from the plant Neobouto-
nia melleri and of semisynthetic derivatives. As the two enone systems (C23–C24a and C1–C3) and the
two hydroxyls groups (C22 and C26) of neoboutomellerones are required for activity, modifications were
focused on these functional groups. Biological evaluation by using a cellular assay for proteasome activity
provided clues regarding the mechanism of action of these natural products and synthetic derivatives.
Certain neoboutomellerone derivatives inhibited the proliferation of human WM-266-4 melanoma tumor
cells at submicromolar concentration and warrant evaluation as anticancer agents.
Received 6 October 2011
Revised 25 November 2011
Accepted 29 November 2011
Available online 8 December 2011
Keywords:
Cycloartane
Neoboutonia melleri
Proteasome
Ó 2011 Elsevier Ltd. All rights reserved.
Natural product
Anticancer agent
1. Introduction
neuropathy and thrombocytopenia.8–10 Therefore, there is a need
for proteasome inhibitors with other mechanisms of action and
A newly isolated natural cycloartane neoboutomellerone 1 was
recently identified as proteasome inhibitor.1 The ubiquitin-
proteasome pathway regulates the degradation of various proteins,
thus controlling biological processes such as signal transduction,
cell cycle progression, transcription, inflammation, and apopto-
sis.2,3 Proteins selected for degradation are marked by the attach-
ment of a polyubiquitin complex and then are recognized and
degraded by the proteasome. The 26S proteasome consists of a
20S proteolytic core and of two 19S regulatory caps. The 20S core
has three major catalytic activities: chymotrypsin-like (CTL), tryp-
sin-like (TL), and peptide-glutamyl-peptide-hydrolyzing (PGPH).
The ubiquitin-proteasome pathway is a target for cancer therapy,
and the proteasome inhibitor bortezomib (VelcadeÒ) has been ap-
proved by the FDA for the treatment of multiple myeloma and man-
tle cell lymphoma (Fig. 1).4–7 Despite clinical benefits, treatment
with bortezomib causes severe side effects such as peripheral
greater therapeutic indices.
Several other synthetic and natural inhibitors of the proteasome
have been described including
a
-amino acid boronates,11 epox-
omicin (isolated from Actinomycetes strains),12 and lactacystin (iso-
lated from Streptomyces)13–15 (Fig. 1). Bortezomib, epoxomicin,
and lactacystin are mainly inhibitors of the CTL activity; these
c
compound form covalent adducts with O -Thr1 in each of the three
catalytic sites. Synthetic epoxyketones have been developed in an
effort to decrease toxicity; carfilzomib16 and ONX-091217 are two
examples. Other naturals epoxyketones, TMC-86A and B from
Streptomyces sp. TC 1084, TMC-96 from Saccharothrix sp. TC
1094,18 and eponemycin isolated from Streptomyces hygroscopicus
No. P247-7 1,19 previously known for antibiotic and anti-angio-
genic activities, also have some activity against the proteasome.
Salinosporamide A is another potent inhibitor of the proteasome,
isolated from the marine actinomycete Salinispora tropica that is
currently in clinical trials.20,21
Syringolin A, isolated from the plant pathogen Pseudomonas
syringae pv. syringae, contains an a,b-unsaturated carbonyl moiety
⇑
Corresponding authors.
Tel.: +33 5 34506112; fax: +33 5 34503112 (F.S.);
tel.: +33 5 34506118; fax: +33 5 34503113 (F.C.).
E-mail addresses: francois.sautel@pierre-fabre.com (F. Sautel), frederic.cantagrel@
etac.cnrs.fr (F. Cantagrel).
that undergoes a Michael type addition with hydroxyl groups of
threonine residues in the three catalytic sites of the proteasome.22
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.066

820
J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
O
O
N
N
O
O
O
O
O
NH
H
N
O
H
N
NH
CO2H
OH
N
S
N
H
B
N
H
N
H
O
OH
O
O
HO
O
HO
Ph
OH
Bortezomib (PS-341)
Epoxomicin
Lactacystin
Ph
O
O
O
O
H
N
H
N
O
H
N
O
N
H
N
H
N
N
H
O
N
N
H
S
O
O
O
O
O
O
Ph
Carfilzomib
ONX 0912
Ph
O
O
O
OH
HN
O
H
N
N
N
H
H
O
HO
OH
O
O
OMe
OMe
NH
Syringolin A
Curcumin
H2N
O
O
O
O
H
Cl
N
OH
O
NH
O
H2N
N
H
H
H
O
COOH
O
7
O
O
O
OH
O
O
Salinosporamide A
Belactosin A
Fellutamide B
O
H2N
O
Me
HO
NH
S
O
H
N
HO
O
H
N
OH
HN
NH
HN
HN
O
O
HN
N
H
NH
O
CONH2
Me
O
O
H
O
H
N
HO
O
HN
N
OMe
H
O
O
O
HO
HN
O
N
H
H
Me
Argyrin A
TMC-95A
Celastrol
O
O
H
O
HO
O
H
O
H
O
O
O
Physalin B
Figure 1. Synthetic and natural proteasome inhibitors.
This family23 also includes compounds such as glidobactin and
cepafungin.24 TMC-95s25 (isolated from Apiospora montagnei Sacc.
TC 109326) and argyrin A (isolated from the myxobacterium Arch-
angium gephyra)27 are two cyclic peptides that strongly inhibit the
CTL activity. Curcumin28 (the major active ingredient of turmeric
(Curcuma longa) with its Schiff’s bases and associated copper com-
plexes29) and celastrol30 (a triterpene isolated from the root bark of
Tripterygium wildfordii) also inhibited the CTL activity of a purified
20S proteasome with micromolar IC50 values. Fellutamide was iso-
lated from fungus Penicillium fellutanum31 and was first described
as an inducer of nerve growth factor release.32 After achievement
of the total synthesis,33 fellutamide was shown to inhibit
proteasome catalytic activity.34 Belactosins, isolated from Strepto-
myces KY11780,35 and their hydrophobic derivatives KF33955
and KF4450436 show affinity for the catalytic subunits of the 26S
proteasome and inhibit growth of HeLa cells.37 Computational
modeling studies showed that both ketones of curcumin, the con-
jugated carbonyls of celastrol and syringolin, the epoxides of epox-
omicin and carfilzomib, and the aldehyde of fellutamide are highly
susceptible to a nucleophilic attack by the hydroxyl group of the
terminal threonine of the proteasome CTL subunit. All these com-
pounds target the proteasome catalytic sites.
In order to alleviate the observed side effects associated with
previously evaluated inhibitors of the catalytic activities of the

J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
821
20S proteasome, our research focused on the identification of
2.1. Modification of the enone system
The reactivity of these ,b unsaturated ketones can be exempli-
inhibitors of the ubiquitin-proteasome pathway in a cellular as-
say.38,39 Our assay system is a human DLD-1 colon cancer cell line
stably transfected with a gene encoding a ubiquitin-luciferase re-
porter protein, 4Ub-Luc. The 4Ub-Luc protein is efficiently targeted
by the ubiquitin-proteasome degradation pathway in this cell sys-
tem. Upon treatment of the cells with a proteasome inhibitor, the
reporter protein accumulates within the cells inducing a detectable
increase of bioluminescence. Physalin B was identified using this
assay.39 Physalin B induces a 19-fold increase of bioluminescence
a
fied by the reaction with primary hydroxylamines, chosen in order
to introduce a nitrogen atom and increase the diversity (Scheme
1). In these reactions, the ring A enone reacted more rapidly than
the one located in the side chain, and oximes 16, 19, and 20 were ob-
tained with some selectivity. Side products included the bis-adducts
17 and 21, for which a second addition occurred at C-24a in a Mi-
chael fashion. We also observed the formation of the methoxy ad-
duct 23. The mono-adduct on the side chain (18) with the less
hindered O-methyl hydroxylamine was obtained as a minor by-
product. The reactivity order may be explained by steric hindrance
around C-1, making the 1,4-addition more difficult, while hindrance
around C-23, associated with a default in planarity in the side chain
enone system, makes the corresponding centers less prone to nucle-
ophilic additions.
at 5
lM, whereas it inhibits cellular proteasomal CTL and PGPH
activities at 20 and 40
lM, respectively.
This cellular assay was adapted to a 96-well plate format, and a
high-throughput screening of over 12,000 natural products ex-
tracts and of 62,000 pure molecules led to the identification of
the natural product neoboutomellerone 1 as a potent inhibitor of
the ubiquitin-proteasome pathway. This new cycloartane,40 iso-
lated from the Euphorbiaceae Neoboutonia melleri,1 has two main
structural originalities (Fig. 2). Firstly, ring A possesses an enone
system and only one methyl group on position 4. Secondly, a
supernumary carbon substituent is present on the side chain of
the exo-methylene enone system (Fig. 2). Thirty related cycloar-
tanes were subsequently isolated from this plant; among them
was molecule 2, which lacked an acetate group on the alcohol C-22.
The two compounds 1 and 2 showed an interesting level of activ-
ity in the ubiquitin-proteasome reporter assay (a 48-fold increase of
2.2. Modification of the hydroxyl functional groups
The alcohols at C-22 and C-26 were substituted in order to en-
hance solubility relative to the parent compounds. Two strategies
were applied: In the first, the primary alcohol was oxidized to
the carboxylic acid, and in the second, an appendage containing
the desired functional group was conjugated (Scheme 2). In the
first strategy, neoboutomellerone 1 was oxidized to the corre-
sponding aldehyde 24 with the Dess–Martin periodinane reagent
and then converted into the acid 25 under Pinnick conditions.
The sequence proceeded smoothly, with moderate yields, without
reaction at the enones or migration of the C-24-C-24a double bond.
The second strategy was employed for addition of succinic anhy-
dride, which yielded acids 26 and 27, accompanied by some bis-ad-
bioluminescence at 1 lM). Their relatively high level of functional-
ization and their ready availability made them valuable candidates
for the starting points for semi-synthesis of additional neoboutom-
ellerone derivatives. A total of 47 analogues of the neoboutomeller-
ones were prepared to explore the roles of the
a,b-unsaturated
ketones, the substituents on the alcohol at C-26, and different acyl
groups on the alcohol at C-22.40 The high reactivity of these natural
products against acids, bases and nucleophiles prohibited some
obvious reactions. All the analogues were evaluated in the cellular
proteasome reporter assay in 4Ub-Luc DLD-1 cells and for inhibi-
tion of the CTL and PGPH catalytic activities. These analyses allowed
us to identify the structural elements responsible for proteasome
activity and to improve bioavailability, metabolic stability, and sol-
ubility relative to compounds 1 and 2. Finally, the cytotoxicity of se-
lected compounds was assessed in the WM-266-4 tumor cells, a cell
line derived from a human melanoma.
duct 28, when
2 was used as starting material. Succinate
derivatives were transformed into N-methyl glucamine salts to in-
crease the solubility in water.
In a similar fashion, the phosphate and sulfate derivatives were
prepared as described in Scheme 4. Synthesis of the sulfate deriv-
ative 29 from 1 was straightforward using the sulfur trioxide-pyr-
idine complex. Formation of the sulfate of compound 2 required
the use of the Burgess reagent and provided bis-sulfate 31 as a side
product of the monosulfate 30 (Scheme 3). Compound 30 was
2. Synthesis of neoboutomellerone derivatives
1
R1-NH2*HCl
Fifteen compounds (1–15)41 among the thirty previously de-
scribed natural products isolated from Neoboutonia melleri1 were
selected for a structure–activity relationship (SAR) study. The
semisynthetic syntheses began with the two most abundant com-
pounds from the plant, 1 and 2,42 and we aimed to improve activity
and solubility. The main concern was the polyfunctionality of these
compounds; each possessed three or four reactive centers: the
enones and the primary and secondary alcohols at C-26 and C-22.
AcONa, MeOH
R
AcO
AcO
OH
OH
H
H
O
O
OAc
H
OAc
C
D
+
B
A
H
X
X
H
H
16: X = N-OMe
62%
66%
17%
17%
17: X = N-OMe; R = N-OMe 8%
19: X =N-NHC(O)-NH2
20: X = N-OBn
24a
18: X = O;R = N-OMe
2%
R1O
21: X = N-OBn; R = N-OBn 17%
26
22: X = N-OH
21
18
25
20
23
OH
22
24
OMe
HO
E
19
12
H
17
11
O
27
C 13
14
OH
O
1
D
H
O
16
2
3
8
9
A
10 B
H
OAc
15
H
7
NaOH / MeOH
MeOH
O
30
1
5
O
4
O
H
6
H
28
R1 = Ac: 12
R1 = H:
23
Scheme 1. Modification of the enone systems.
Figure 2. Neoboutomellerones.

822
J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
Strategy 1
1
DMP
pyridine
O
O
AcO
AcO
H
OH
H
O
O
NaClO2
H
OAc
H
OAc
(CH3)2CO/H2O
O
H
O
H
24: 66%
25: 61%
Strategy 2
1 or 2
O
O
O
;DMAP
DCM
HO
HO
O
O
O
O
O
O
R
HO
O
O
O
O
H
H
O
OAc
+
O
OAc
H
H
O
O
H
H
28: 11% from 2
26: R = Ac
27: R = H
95%
66%
Scheme 2. Synthesis of acidic derivatives by two strategies.
O
S
O
OH
O
AcO
H
SO3*py
THF
O
O
1
H
OAc
H
29: 66%
O
O
OH
O
O
OH
O
S
HO
O
S
S
HO
H
O
O
O
H
Burgess' reagent
THF
O
O
OAc
2
H
OAc
+
H
O
O
H
H
30: 53%
31: 16%
Scheme 3. Synthesis of sulfate derivatives.
highly soluble in water and was purified by simple washing with
an organic solvent. The phosphate monoester 32, the phosphate
diethyl ester 33, and the phosphonate 34 were prepared in a
similar fashion. Phosphate derivative 32 was transformed into its
N-methyl glucamate to further increase solubility (Scheme 4).
Introduction of a nitrogen atom at the terminal position of the
side chain proved challenging, and most classical synthetic
strategies, including Mitsunobu, gave rise to conjugate addition.
Only reaction with a protected spermine was successful, yielding
derivative 41 in moderate yield (Scheme 5). Recourse to classical
methods of ester synthesis led to glycine derivatives 35–37 and
42 after Boc removal. The glycine–spermine synthon offered 40 in
10% yield after Boc deprotection. A facile decomposition into acry-
late 43 decreased the yield during preparation of the homologous

J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
823
AcO
H
the corresponding isocyanates were not easily available, the piper-
azine carbamates 50 and 51 were obtained by reaction of the alco-
hols with the hydrochloride salt of chlorocarbonylpiperazine
(Scheme 7).
O
OH
OH
P
O
O
POCl3, Et3N
1
H
OAc
32.NMG
DCM, lutidine
Ethers are generally stable derivatives under biological condi-
tions and termination of the side chain with an ether linkage was
desired. However, it was extremely difficult to convert the C-26
primary alcohol into an ether under basic conditions; saponifica-
tion of the acetate at C-16 and concomitant reaction with the side
chain acyloin system resulted under all conditions tested. The
methyl and allyl ethers, 52–54, were prepared with silver ion
activation of the corresponding halides, whereas acetal 55 was
synthesized using Hünig’s base (Scheme 8). The allyl ether 53,
which was prepared to open a route towards glycerol derivatives,
underwent over-oxidation under osmium catalysis to afford tetra-
ol 56 in 38% yield. Glycosylation under Schmidt conditions did
not provide the expected glycoside; orthoester 57 was obtained
instead.
A last series of compounds was prepared to determine the
importance of the acetate moiety on position C-22 to biological
activity, and several acyl groups were introduced at this position.
The starting material for these transformations was diacetate 58
prepared by acetylation of 2 using acetic anhydride. As expected,
these transformation led to triacetate 59, chloroacetate 60, N-Boc
glycinate 61, and succinate 62 (Scheme 9).
89% from 1
O
H
32
O
OEt
P
O
AcO
H
OEt
Cl-P(O)(OEt)2
DCM, lutidine
1
O
H
OAc
O
H
33: 11%
O
O
P
O
O
P
OH
OH
O
HO
AcO
H
OH
OH
1
CH3CN, DCC
O
OAc
H
O
H
34: 25%
Scheme 4. Synthesis of phosphate derivatives.
2.3. General observations
The purpose of this study was to enlarge the chemical diversity
around this natural scaffold and yields were not optimized at this
stage. It is worth noting that two major transformations could not
be performed due to neighboring group participation or to the
reactivity of the enones. Compounds with a basic nitrogen atom
at C-26 and compounds with various acid groups at C-16 are lack-
ing. For aza-analogues, we were unsuccessful in hydroxyl substitu-
tion by nitrogen because Michael type addition of nitrogenic
nucleophile onto the C23–C24a enone system. Concerning C-16,
all attempts to modify the acetate failed. Saponification of this
ester led spontaneously to cyclic compounds of O-16 with C-22
b-alanine derivative 36. The direct use of mono-acetate 2 in these
coupling reactions led to a mixture of the mono-adduct 44 and of
the di-adduct 45, but selectivity could be obtained by prior protec-
tion of the primary alcohol as a TBDMS ether. This sequence of pro-
tection/acylation/deprotection led to derivative 46, which formally
was another nitrogen substituted derivative of 1 (Scheme 6).
Carbamates were prepared in an analogous fashion using phe-
nyl-isocyanate or para-dimethylaminophenyl-isocyanate. Deriva-
tive 47, the phenylcarbamate derivative of 1, was obtained as a
crystalline solid suitable for X-ray crystallographic studies. Since
R1
O
O
R
O
H
O
H
OAc
O
H
35: R1 = Ac, R = CH2NMe2
36: R1 = H, R = CH2NMe2
37: R1 = Ac, R = CH2NHBoc
23% from 1
57% from 2
85% from 1
38: R1 = Ac, R = CH2CH2 NEt2
39: R1 = Ac, R = CH2Cl
15% from 1
14% from 1
42: R1 = Ac, R = CH2NH2
43: R1 = Ac, R = CH = CH2
24% from 36
quant.from 37
N
O
AcO
H
O
H
N
N
AcO
H
O
O
O
NBoc
H
OAc
O
NH
H
OAc
H2N
N
H
O
H
BocHN
N
O
Boc
H
40: 10% from 1 (2steps)
41: 12% from 1
Scheme 5. Synthesis of ester derivatives.

824
J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
NHBoc
O
NHBoc
O
O
O
BocHN
H
O
O
HO
H
H
O
O
+
OAc
H
OAc
O
O
H
H
NBoc-glycine
EDC, DMAP
DCM
45: 58%
44
: 36%
TBDMS
2
O
TBDMS
O
HO
H
BocHN
H
O
O
TBDMSOTf
imidazole, DCM
O
OAc
NBoc-glycine
H
H
O
OAc
EDC, DMAP
DCM
O
H
O
H
88%
64%
HF*py
Pyridine
O
BocHN
H
OH
O
H
O
OAc
O
H
46: 51%
Scheme 6. Synthesis of glycinate derivatives.
O
AcO
N
H
O
H
O
OAc
PhNCO, DMAP
Et3N, DCM
1
H
O
47: 47%
H
O
N
R
O
N
O
H
H
O
Me2N-(C6H4)NCO, DMAP
Et3N, DCM
1
2
or
H
OAc
O
48: R = Ac: 79%
H
49: R = H: 60%
O
R
O
O
Cl
N
O
Cl
N
H
NH
O
OAc
N
, K2CO3, TBAB,
CH3CN
1 or 2
H
O
50
51
: R = Ac: 29%
H
:
: R = H
35%
Scheme 7. Synthesis of carbamate derivatives.
or C-23. This was due to acyloin ketol shift observed in basic media.
Attempts to trap the enolate in situ with electrophile also failed.
The unexpected chemical reactivities are under investigation and
will be published in due course.
3. Proteasome inhibition
As shown previously, treatment of 4Ub-luc DLD-1 cells with
proteasome inhibitors increases the cellular concentration of the

J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
825
R1
were mainly over-oxidation (compound 5, oxidation on ring A;
compounds 6, 7, and 9, oxidation on ring B; compound 8, oxidation
on substituents) and reduction (compounds 3 and 4, hydrogena-
tion on ring A; compounds 11 and 12, reduction of the side chain).
These modifications did not increase the activity. Nevertheless,
compounds 4, 5, 9, 10, 11, and 12 showed an increased efficacy
at higher concentrations but a clear loss of potency relative to po-
tency compared to neoboutomellerones 1 and 2.
As described in the chemical section, neoboutomellerones were
modified on three different functions to increase solubility and bio-
logical activity. Firstly, the enones on ring A and on the side chain
were modified. Among these compounds, only the semi-carbazone
19 and hydroxime 22 showed a stronger efficacy (i.e., higher IF va-
lue) than the parent compounds 1 and 2, but a lower potency (i.e.,
at higher concentration). Their IF are respectively, 99 and 103 at
O
R
O
H
O
1
2
or
H
OAc
O
H
52: R1 = Ac, R = Me ;
77%
53: R1 = Ac, R = allyl ;
54: R1 = H, R = allyl ;
35% (with 41% of 1)
78%
59%
55: R1 = H, R = CH2OAc ;
Ac
O
OH
O
H
OH
O
OAc
NMO, OsO4
OH
53
H
5 lM. Compounds 16 and 20 had little or no activity. The addition
HO
O
of a nucleophile on the double bond of the enone of the side chain
had a dramatic effect and compounds 17, 18, 21, and 23 were
inactive.
Secondly, the primary hydroxyl group on position 26 was mod-
ified. To increase the solubility, acids (compounds 25–28), sulfates
(compounds 29–31), phosphates (compounds 32 and 33) and a
phosphonate (compound 34) were obtained. All acids forms were
inactive, including carboxylic acid 25. The phosphate diethyl ester
33 was as active as the parent neoboutomellerone, and succinates
H
56: 38%
OAc
OAc
OAc
O
OAc
OAc
O
Ac
O
O
OAc
O
Cl
OAc
Cl
O
O
Cl
H
NH
O
OAc
1
H
O
26 and 27 were more potent (up to IF 60 at 1 lM) than the parent.
H
57: 74%
The di-substitution, as for compound 28, of both hydroxyl groups
as succinate monoester resulted in the loss of activity. This could
be due to the fact that this compound with two negative charges
cannot penetrate the cellular membrane. Positively charged
compounds (35–38, 40–42, 44–45, 47–51) were also synthesized
and tested. The hydrochloride salt of the dimethylglycinate neo-
Scheme 8. Synthesis of ether derivatives.
protein 4Ub-Luc, since the protein is not degraded by the protea-
some pathway. This inhibition is thus measured as an increase of
the bioluminescence compared to untreated cells. This increase
of bioluminescence is expressed as an induction factor (IF). Results
are compared to a control, epoxomicin at 10À7 M, run in parallel
experiment; its relative IF is defined as 100. A molecule was con-
sidered active if the induction factor was equal or superior to 10.
This assay has the advantage of detecting molecules that penetrate
into the cells.
The results of analysis of selected neoboutomellerone analogues
in the proteasome inhibition assay are presented in Table 1. Gener-
ally the compounds had a ‘bell-shaped’ dose–response curve: At
higher concentrations the activity decreased, probably because of
cytotoxicity of the compounds. For the SAR study we considered
boutomellerone
1
(compound 35ÁHCl) was active (IF 66 at
0.5 M), confirming the importance of a positive charge. The
l
spermine derivative (compound 40 and 41) was not active,
whereas the primary amine analogue (compound 42) was. Carba-
mates 47–51 had higher activities than the parent compounds:
This could be due to the fact that these carbamate derivatives act
as prodrugs of neoboutomellerones 1 and 2, leading to an effec-
tively higher concentration of neoboutomellerone in cells.
Lastly, analogues substituted on position 22 of the triterpenic
skeleton were studied. Acetylation of position 22 did not affect the
activity in natural (3 compared to 4, 6–7, 11–12, and 13–14) and
synthetic compounds (26 compared to 27, 37–44, 48–49, 50–51,
58–59), in analogy with what observed for parent compounds 1
(acetylated) and 2 (non-acetylated). Interestingly, the presence of
a N-Boc glycinate in compound 45 increased the activity compared
to the acetate analogue 37 and to hydroxyl 43. The same was ob-
served in the series where position C26 was acetylated (compound
58 vs 62). Although the presence of an acetyl function (compound
the IF at 0.5 and 1 lM and compared activities of derivatives to
those of the natural leads 1 and 2.
Among the compounds isolated from the plant, the most active
were neoboutomellerones 1 and 2. The presence of the acetyl at C-
22 did not affect the activity. The natural modifications observed
O
R
OAc
OAc
HO
O
AcOAc (0.75 eq.)
H
H
2
O
OAc
O
OAc
Lutidine,
H
H
DMAP, DCM
O
O
H
H
58: 58%
59: R = H ;
60 :R = Cl ;
quant.from 2
97%
61: R = NHBoc,
77%
62: R = CH2COOH ;
78%
Scheme 9. Synthesis of C-22-substituted derivatives.

Table 1
Structures and biological evaluation of selected neoboutomellerone derivatives (the complete SAR table is found in the Supplementary data)
24
25
Me
26
Me
18
22
H
O
19
OAc
1
H
2
Me
A
B
3
H
6
Me
Compounds
C1
C3
C6–C7
C18
C22
C24
C25
C26
rIFa
10
(lM)
b
c
5
1
0.5
PGPH IC50
(l
M)
CTL IC50 (lM)
d
e
f
Epoxomicin
Lactacystin
Bortezomib
1
2
112
72
117
48
0.06
0.3
0.0009
23
0.006
0.07
0.0002
16
93
106
D
D
@O
@O
H,H
H,H
CH3
CH3
OAc
OH
@CH2
@CH2
a
a
CH2OH
CH2OH
4.4
2.9
37
28
2.1
1.4
45
51
42
O
H
H
15
D
@O
H,H
CH3
3.0
1.3
OAc
27ÁNMG
33
35ÁHCl
36ÁHCl
37
44
45
50ÁHCl
51ÁHCl
55
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
@O
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
OAc
OAc
OH
OAc
OH
C(O)CH2NHBoc
OAc
OH
OH
OH
OAc
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
@CH2
a
a
a
a
a
a
a
a
a
a
a
a
CH2OC(O)CH2CH2COOH
CH2OP(O)(OEt)2
CH2O C(O)CH2NMe2
CH2O C(O)CH2NMe2
CH2O C(O)CH2NHBoc
CH2O C(O)CH2NHBoc
CH2O C(O)CH2NHBoc
CH2OC(O)-pip-NMe
CH2OC(O)-pip-NMe
CH2O CH2OAc
5
2
18
16
11
34
43
47
33
4.2
4.5
34
29
48
21
3.4
16
46
43
50
17
14
4.8
47
68
70
40
26
11
64
69
69
2.6
3
66
1.3
47
61
91
19
2.7
4
10
16
17
1
1.4
3
6
52g
22
31g
17
>1000
>1000
38
16
32
31
45
43
100
23
92
>1000
21
13
39
22
31
35
50
4.3
8
12
1.7
1.7
1.1
23
76
2
58
59
60
61
CH2OAc
CH2OAc
CH2OAc
CH2OAc
C(O)CH2Cl
C(O)CH2NHBoc
C(O)CH2 CH2COOH
5
1.6
3
a
a
17
12
62ÁNMG
CH2OAc
10
a
b
c
d
e
f
rIF = relative IF in 4Ub-luc DLD-1 assay compared to epoxomicin.
Concentration in
Concentration in
l
l
M at which 50% of PGPH activity is inhibited (IC50).
M at which 50% of CTL activity is inhibited (IC50).
100 at 0.1
lM.
6.2 at 0.1
l
M.
133 at 0.1
lM.
g
Measured with 35.

J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
827
Table 2
was no or little difference between a hydroxyl and an acetate
group, but larger groups like glycine or chloroacetate enhanced
the activity. Most derivatives did not inhibit the cellular proteaso-
mal CTL or PGPH catalytic activities, suggesting that neoboutomell-
erone derivatives might interferes with components of the
proteasome pathway other than the protein degradation activity.
Cytotoxicity of neoboutomellerone analogues in WM-266-4 cells
lines at 72 h
a
Compounds
IC50
(
lM)
Epoxomicin
Bortezomib
1
2
0.0048
0.035
2.80
1.60
0.61
0.19
0.96
0.82
0.50
0.46
0.78
0.99
1.50
0.66
1.00
5. Experimental section
5.1. Chemistry
35ÁHCl
47
48
49
50ÁHCl
51ÁHCl
54
58
59
60
61
All solvents, including anhydrous solvents and reagents, were
purchased from Acros or Sigma–Aldrich. The NMR spectra were re-
corded on a Bruker Avance II spectrometer equipped with a 13C
cryoprobe at 500 MHz for 1H and 125 MHz for 13C. Chemical shifts
(d) were measured relative to acetonitrile-d3, or DMSO-d6 and are
expressed in ppm. Coupling constants (J) are expressed in Hertz.
High-resolution mass spectrometry (MS) was used to confirm
molecular formulas and was performed on a Bruker MicroTOF.
Compounds were analyzed on an LC/MS/UV/ESL system (Autopuri-
fy™, Waters Corporation). The system was controlled by MassLynx
4.1 and data were processed with OpenLynx. The HPLC system con-
sisted of a 2767 Autoinjector, a 2525 binary gradient module, a
Photodiode Array (PDA) 2996 detector, and a ZQ2000 single-quad-
rupole MS equipped with a Z-spray electrospray source. Positive
and negative modes were used simultaneously. MS instruments
settings were as follows: capillary voltage 3.2 kV; cone voltage
25 V; source temperature 120 °C; desolvation temperature
350 °C; desolvation gas flow 400 L/h; cone gas flow 50 L/h. The
multiplier was set at 650. The wavelength range used with the
PDA2996 was 210–500 nm. The evaporative light-scattering detec-
tor was a PL-ELS-1000 (Polymer Laboratories). A Synergi Six-Col-
umn Selector (Phenomenex) enabled the use of six different
columns. Three analytical columns were used for purity assess-
ments and the determination of compound solubility: SunFire
a
IC50 = concentration in
lM at which 50% inhibition of cell
proliferation is observed.
59) in comparison to the hydroxyl 58 did not alter the activity, the
presence of a chlorine atom (compound 60) or of an amino group
(compound 61) greatly increased efficacy and potency: Compound
61 showed the best activity with an IF of 76 at 0.5
charged succinate 62 showed an IF of 69 at 1 M.
In order to study the mechanism of action of the neoboutomell-
erone series, the most potent compounds were tested for inhibition
of the catalytic activities of the cellular proteasome. Levels of chy-
motrypsin (CTL) and peptidyl-glutamyl-peptide hydrolyzing
(PGPH) activities were measured using specific fluorogenic sub-
strates as previously described.43 No straightforward correlation
was observed between the induction of the luminescence signal
of the 4Ub-luc DLD-1 assay and the inhibition of the CTL or PGPH
catalytic activity. All compounds showed IC50 values higher than
lM. The negatively
l
10
epoxomicin, lactacystin, and bortezomib had IC50 values of 0.06,
0.3, and 0.0009 M, respectively, against PGPH and 0.006, 0.07,
and 0.0002 M, respectively, against CTL. These findings suggest
lM in the catalytic activity assays. The reference compounds
C18, 3.5
XBridge Shield C18, 3.5
l
m, 4.6 Â 50 mm; Atlantis dC18, 3
l
m, 4.6 Â 50; and
l
l
m, 4.6 Â 50 mm (all from Waters Corpo-
l
ration). Each column was used with a 4.6 Â 20 mm matching guard
column. Water (MilliQ purified) and acetonitrile with 0.1% (v/v)
formic acid were used as solvents A and B, respectively. Solvent
gradients of 5 min at a flow rate of 2 mL/min were applied after
a 0.5 min delay following the injection: SunFire 5–100% B; Atlantis
and Xbridge Shield 0–100% B. Washing and equilibration times
were 0.5 min and 0.75 min, respectively, for a total time of 7 min.
The extraction, isolation, and characterization of the natural
products 1-15 are described elsewhere.1,26
that the proteasome inhibition of the neoboutomellerone com-
pounds is not or is only partially mediated by the inhibition of
the catalytic activity of the proteasome. The three most potent
compounds in the ubiquitin-proteasome cellular assay, 61, 35,
and 60 (IFs at 0.5 lM of 76, 66, and 23, respectively) were poor
inhibitors in the PGPH and CTL catalytic assays.
The three most potent compounds in the cellular assay, 61,
35ÁHCl, and 60, and the active carbamates 47–51 were tested for
their cytotoxicity in human WM-266-4 metastatic melanoma cells
(Table 2). All compounds had similar IC50 values of around 1
with the exception of carbamate 47, which was more toxic (IC50
of 0.19 M).
lM
5.1.1. Preparation of compound 35 and its hydrochloride salt
To a stirred solution of DCC (3.5 mL, 0.1 mol/L, 0.35 mmol,
2 equiv) in CH2Cl2 were added N-dimethylglycine (36 mg,
0.35 mmol, 2 equiv), DMAP (2 mg, 0.018 mmol, 0.1 equiv), and 1
(100 mg, 0.18 mmol, 1 equiv). The mixture was stirred for 24 h at
room temperature. Then, the mixture was filtered and the filtrate
was washed with H2O and dried over Na2SO4. The product was
purified by chromatography on silica gel (elution: cyclohexane/
EtOAc, 3:7) to obtain the amine 35 as a translucent oil (26 mg,
23%). Hydrochloric acid (3 mL, 0.1 M) was added to 35 (26 mg,
0.0407 mmol), and the mixture was stirred at room temperature
for 1 h. The mixture was dried under high vacuum to furnish the
hydrochloride as a white light solid (28 mg, quant.).
l
4. Conclusions
Here we evaluated the activities of 15 natural products isolated
from N. melleri and the activities of 47 semisynthetic derivatives of
neoboutomellerones 1 and 2. Twelve analogues (26, 27, 33, 36, 38,
43, 45, 47, 52, 54, 55, 58) showed similar potencies in the cell-
based bioluminescence assay for proteasome inhibition as the par-
ent compounds 1 and 2, whereas compounds 35ÁHCl, 48, 49, 50, 51,
60, and 61 were more potent. The side chain and specially the exo
35: 1H NMR (500 MHz, CD3CN) d = 6.94 (1H, d, J = 10.1 Hz, H-1),
6.12 (1H, s, H-24a), 5.97 (1H, s, H-24a), 5.90 (1H, d, J = 9.8 Hz, H-2),
5.53 (1H, d, J = 2.1 Hz, H-22), 5.09 (1H, td, J = 7.6, 4.6 Hz, H-16),
4.00–4.15 (2H, m, 2 H-26), 3.10 (2H, s, H-26b), 3.02 (1H, sxt,
J = 6.9 Hz, H-25), 2.58 (1H, dqd, J = 11.0, 7.0, 2.1 Hz, H-20), 2.30
a,b-unsaturated ketone were essential for the activity. The enone
system on ring A conferred some activity. The protection of the
alcohol at C-26 increased the activity, presumably by increasing
the cellular stability. A variety of groups such as ester, carbamate,
and ether were acceptable substituents. At position C-22, there

828
J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
(1H, dd, J = 11.0, 7.3 Hz, H-17), 2.26 (6H, s, H-26e, H-26d), 2.13–
2.22 (2H, m, H-4, H-15), 2.09 (3H, s, H-22b), 2.03 (3H, s, H-16b),
1.95–2.06 (3H, m, H-5, H-8, H-11), 1.63–1.77 (3H, m, H-6, 2H-
12), 1.52–1.60 (1H, m, H-11), 1.41–1.50 (1H, m, H-7), 1.37 (1H,
dd, J = 14.2, 3.8 Hz, H-15), 1.24 (1H, d, J = 4.3 Hz, H-19), 1.19 (3H,
s, H-18), 1.15–1.22 (1H, m, H-7), 1.07 (3H, d, J = 7.3 Hz, H-27),
1.02 (3H, d, J = 6.7 Hz, H-28), 0.95 (3H, s, H-29), 0.89–0.99 (1H,
m, H-6), 0.85 (3H, d, J = 7.0 Hz, H-21), 0.58 (1H, d, J = 4.6 Hz, H-
19).13C NMR (126 MHz, CD3CN) d = 202.4 (C-3), 198.8 (C-23),
171.6 (C-22a), 171.5 (C-26a), 171.2 (C-16a), 155.5 (C-1), 149.1 (C-
24), 128.4 (C-2), 125.8 (C-24a), 78.3 (C-22), 76.7 (C-16), 67.7 (C-
26), 60.7 (C-26b), 51.3 (C-17), 48.4 (C-14), 47.6 (C-4), 46.9 (C-13),
46.7 (C-15), 45.3 (C-26d, 26e), 45.2 (C-8), 43.6 (C-5), 34.9 (C-25),
33.3 (C-20), 33.0 (C-12), 32.9 (C-10), 28.1 (C-11), 27.7 (C-19),
27.6, 27.2 (C-9), 24.3 (C-7, 6), 22.1 (C-16b), 20.9 (C-22b), 20.0 (C-
29), 18.3 (C-18), 17.3 (C-27), 13.3 (C-21), 11.3 (C-28). ESI-MS m/
z: 654.4 [M+H]+, 676.4 [M+Na]+. Purity (UV) = 100%, purity
(ESLD) = 99%.
(3H, s, H-29), 0.88–0.94 (1H, m, H-6), 0.86 (3H, d, J = 6.7 Hz, H-
21), 0.57 (1H, d, J = 4.3 Hz, H-19). 13C NMR (126 MHz, CD3CN)
d = 202.4 (C-3), 198.9 (C-23), 171.8 (C-22a), 171.3 (C-16a), 155.5
(C-1), 155.1 (C-26a), 149.4 (C-24), 148.8 (C-26e), 129.4 (C-26b),
128.5 (C-2), 125.5 (C-24a), 121.8 (C-26c, 26c), 114.2 (C-26d, 26d),
78.5 (C-22), 76.7 (C-16), 68.3 (C-26), 51.4 (C-17), 48.4 (C-14),
47.7 (C-4), 46.9 (C-13), 46.8 (C-15), 45.2 (C-8), 43.6 (C-5), 41.3
(C-26f, 26f), 35.3 (C-25), 33.4 (C-20), 33.0 (C-12), 33.0 (C-10),
28.1 (C-11), 27.7 (C-19), 27.3 (C-9), 24.3 (C-7, 6), 22.1 (C-16b),
21.0 (C-22b), 20.0 (C-29), 18.3 (C-18), 17.2 (C-27),13.4 (C-21),
11.3 (C-28). ESI-MS m/z: 731.5 [M+H]+, 753.4 [M+Na]+. Purity
(UV) = 100%, purity (ESLD) = 99%.
5.1.3. Preparation of compound 49
To a stirred solution of 2 (100 mg, 0.19 mmol) in CH2Cl2 (1 mL)
were added DMAP (12 mg, 0.095 mmol, 0.5 equiv), dimethylamin-
ophenyl isocyanate (76 mg, 0.475 mmol, 2.5 equiv), and triethyl-
amine (40 lL, 0.285 mmol, 1.5 equiv). After 22 h, the mixture
35ÁHCl: 1H NMR (500 MHz, D2O) d = 7.19 (1H, d, J = 9.8 Hz, H-1),
6.40 (1H, br s, H-24a), 6.28 (1H, s, H-24a), 5.94–6.06 (1H, m,
J = 5.5 Hz, H-2), 5.67 (1H, br s, H-22), 5.08 (1H, br s, H-16), 4.36
(1H, dd, J = 10.8, 5.6 Hz, H-26), 4.23 (1H, dd, J = 11.0, 7.9 Hz, H-
26), 4.11 (1H, d, J = 17.1 Hz, H-26b), 4.05 (1H, d, J = 17.4 Hz, H-
26b), 3.09–3.21 (1H, m, H-25), 2.67 (1H, br s, H-20), 2.29–2.42
(2H, m, H-4, H-17), 2.21 (3H, s, H-22b), 2.18–2.28 (1H, m, H-15),
2.15 (3H, s, H-16b), 1.95–2.12 (3H, m, H-5, H-8, 11), 1.71 (3H, br
s, H-6, 2H-12), 1.64 (1H, br s, H-11), 1.40–1.54 (2H, m, H-7, H-
15), 1.37 (1H, br s, H-19), 1.19 (3H, br s, H-18), 1.15–1.27 (1H, m,
H-7), 1.12 (3H, d, J = 7.0 Hz, H-27), 1.04 (3H, d, J = 6.7 Hz, H-28),
0.91–1.00 (1H, m, H-6), 0.95 (3H, br s, H-29), 0.88 (3H, d,
J = 6.4 Hz, H-21), 0.64 (1H, br s, H-19).13C NMR (126 MHz, D2O)
d = 174.8 (C-16a, 22a), 167.3 (C-26a), 160.6 (C-1), 147.9 (C-24),
129.2 (C-24a), 127.4 (C-2), 79.4 (C-22), 78.2 (C-16), 70.8 (C-26),
58.0 (C-26b), 51.1 (C-17), 48.2 (C-14), 47.4 (C-4), 46.7 (C-13),
46.1 (C-15), 44.7 (C-26e, 26d), 44.2 (C-8), 42.8 (C-5), 33.7 (C-20),
33.4 (C-10), 33.3 (C-25), 32.6 (C-12), 27.8 (C-11), 27.8 (C-9), 27.2
(C-19), 23.8 (C-6), 23.6 (C-7), 22.3 (C-16b), 21.0 (C-22b), 19.8 (C-
29), 17.9 (C-18), 16.7 (C-27), 13.3 (C-21), 11.1 (C-28). ESI-MS m/
z: 676.7 [M+Na]+. Purity (UV) = 96%, purity (ESLD) = 97%.
was diluted with EtOAc, and the organic solution was washed suc-
cessively with 4% HCl, NaHCO3, and brine. The organic solution was
dried over MgSO4 and concentrated under vacuum. The residue
was purified by chromatography on silica gel (elution: cyclohex-
ane/EtOAc, 7:3) to give 49 as a white solid (79 mg, 60%). 1H NMR
(500 MHz, CD3CN) d = 7.32 (1H, br s, H-41a), 7.19 (2H, br s, H-
26c), 6.93 (1H, d, J = 10.1 Hz, H-1), 6.71 (2H, d, J = 8.9 Hz, H-26d),
6.19 (1H, s, H-24aa), 6.09 (1H, d, J = 16.2 Hz, H-24ab), 5.89 (1H, d,
J = 9.8 Hz, H-2), 5.21 (1H, td, J = 7.5 Hz, J = 4.9 Hz, H-16), 4.72 (1H,
dd, J = 6.1 Hz, J = 1.5 Hz, H-22), 4.14 (1H, dd, J = 10.7 Hz, J = 6.5 Hz,
H-26), 4.08 (1H, dd, J = 10.6 Hz, J = 6.3 Hz, H-26), 3.53 (1H, d,
J = 6.2 Hz, H-30), 3.08 (1H, sxt, J = 6.9 Hz, H-25), 2.86 (6H, s, H-
26f), 2.40–2.51 (2H, m, H-20, H-17), 2.14–2.25 (2H, m, H-4, H-
15), 2.03–2.07 (1H, m, H-11), 2.02 (3H, s, H-16b), 1.97–2.01 (2H,
m, H-8a, H-5a), 1.60–1.72 (3H, m, H-6, 2H-12), 1.54 (1H, qd,
J = 8.8 Hz, J = 5.9 Hz, H-11), 1.42–1.48 (1H, m, H-7), 1.38 (1H, dd,
J = 14.6 Hz, J = 4.5 Hz, H-15), 1.24 (1H, d, J = 4.6 Hz, H-19), 1.19–
1.21 (1H, m, H-7), 1.17 (3H, s, H-18), 1.13 (3H, d, J = 7.0 Hz, H-
27), 1.03 (3H, d, J = 7.0 Hz, H-28), 0.96 (3H, s, H-29), 0.90–0.94
(1H, m, H-6), 0.65 (3H, d, J = 6.1 Hz, H-21), 0.57 (1H, d, J = 4.3 Hz,
H-19).13C NMR (126 MHz, CD3CN) d = 205.0 (C-23), 202.4 (C-3),
171.3 (C-16a), 155.6 (C-26a, 1), 155.2, 148.8 (C-26e), 148.2 (C-
24), 129.5 (C-26b), 128.4 (C-2), 127.3 (C-24a), 121.9 (C-26c, 26c),
114.2 (C-26d, 26d), 77.3 (C-22), 75.9 (C-16), 68.3 (C-26), 51.5 (C-
17), 48.4 (C-14), 47.7 (C-4), 46.9 (C-15), 46.7 (C-13), 45.4 (C-8),
43.7 (C-5), 41.3 (C-26f, 26f), 36.4 (C-25), 35.2 (C-20), 33.2 (C-12),
33.0 (C-10), 28.2 (C-11), 27.8 (C-19), 27.3 (C-9), 24.4 (C-7, 6),
22.1 (C-16b), 20.2 (C-29), 18.6 (C-18), 17.3 (C-27), 12.4 (C-21),
11.3 (C-28). ESI-MS m/z: 711.6 [M+Na]+, 1400.1 [2M+Na]+. Purity
(UV) = purity (ESLD) = 100%.
5.1.2. Preparation of compound 48
To a stirred solution of 1 (100 mg, 0.176 mmol) in CH2Cl2 (1 mL)
were added DMAP (11 mg, 0.080 mmol, 0.5 equiv), dimethylamin-
ophenyl isocyanate (43 mg, 0.264 mmol, 1.5 equiv), and triethyl-
amine (40 lL, 0.264 mmol, 1.5 equiv). The mixture was stirred at
room temperature for 24 h and one more equivalent of dimethyl-
aminophenyl isocyanate (29 mg, 0.176 mmol) was added. After
18 h, the mixture was diluted with EtOAc and the organic solution
was washed successively with 4% HCl, NaHCO3, and brine. The or-
ganic solution was dried over MgSO4 and concentrated under vac-
uum. The residue was purified by chromatography on silica gel
(elution: cyclohexane/EtOAc, 6:4) to give 48 as a white solid
(101 mg, 79%). 1H NMR (500 MHz, CD3CN) d = 7.32 (1H, br s, H-
44), 7.20 (2H, d, J = 8.2 Hz, H-26c), 6.94 (1H, d, J = 9.8 Hz, H-1),
6.71 (2H, d, J = 9.1 Hz, H-26d), 6.13 (1H, s, H-24aa), 5.99 (1H, s,
H-24ab), 5.90 (1H, d, J = 10.1 Hz, H-2), 5.55 (1H, d, J = 2.1 Hz, H-
22), 5.10 (1H, td, J = 7.6 Hz, J = 4.3 Hz, H-16), 4.11 (1H, dd,
J = 10.6 Hz, J = 7.1 Hz, H-26), 4.03 (1H, dd, J = 10.6 Hz, J = 6.3 Hz,
H-26), 3.05 (1H, sxt, J = 6.9 Hz, H-25), 2.86 (6H, s, H-26f), 2.56–
2.65 (1H, m, H-20), 2.30 (1H, dd, J = 11.0 Hz, J = 7.6 Hz, H-17),
2.14–2.22 (2H, m, H-4, H-15), 2.10 (3H, s, H-22b), 2.03 (3H, s, H-
16b), 1.96–2.01 (3H, m, H-11, H-8a, H-5a), 1.62–1.76 (3H, m, H-
6, 2H-12), 1.56 (1H, qd, J = 8.7 Hz, J = 6.3 Hz, H-11), 1.41–1.49
(1H, m, H-7), 1.37 (1H, dd, J = 13.9 Hz, J = 3.8 Hz, H-15), 1.24 (1H,
d, J = 4.3 Hz, H-19), 1.20–1.23 (1H, m, H-7), 1.18 (3H, s, H-18),
1.10 (3H, d, J = 7.0 Hz, H-27), 1.03 (3H, d, J = 7.0 Hz, H-28), 0.96
5.1.4. Preparation of compound 51
To a stirred solution of 2 (200 mg, 0.38 mmol) in CH3CN (1 mL)
were added K2CO3 (210 mg, 1.521 mmol, 4 equiv), 4-methylpipera-
zine carbonyl chloride hydrochloride (151 mg, 0.76 mmol, 2 equiv),
and TBAB (12 mg, 0.0352 mmol, 0.1 equiv). After 28 h, the mixture
was filtered and concentrated under vacuum. The residue was puri-
fied by chromatography on silica gel (elution: DCM/MeOH, 98:2 to
95:5). Two products were isolated as white solids: the expected car-
bamate (87 mg, 35%) and starting material 2 (96 mg, 48%). Hydro-
chloric acid (3 mL, 0.1 M) was added to the carbamate (69 mg,
0.106 mmol) and the mixture was stirred at room temperature for
1 h. The mixture was dried under high vacuum to furnish the
hydrochloride 51ÁHCl as a white fluffy solid (66 mg, 90%). 51: 1H
NMR (500 MHz, DMSO-d6) d = 10.30 (1H, br s, H-26e), 6.97 (1H, d,
J = 10.1 Hz, H-1), 6.10 (1H, s, H-24aa), 6.03 (1H, s, H-24ab), 5.90
(1H, d, J = 10.1 Hz, H-2), 5.15 (1H, dd, J = 7.4 Hz, J = 4.5 Hz, H-16),
4.79 (1H, d, J = 6.1 Hz, H-30), 4.60 (1H, d, J = 3.7 Hz, H-22),

J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
829
3.96–4.08 (4H, m, H-26, 26b), 3.34–3.42 (2H, m, H-26c), 3.09–3.21
(2H, m, H-26b), 2.89–3.03 (3H, m, H-25, 26c), 2.76 (3H, br s, H-26d),
2.29–2.39 (2H, m, H-20, H-17), 2.07–2.16 (2H, m, H-4, H-15), 2.02
(3H, s, H-16b), 1.93–2.00 (2H, m, H-11, H-8a), 1.90 (1H, td,
J = 12.5 Hz, J = 4.4 Hz, H-5a), 1.50–1.64 (4H, m, 2H-12, H-6, H-11),
1.37–1.44 (1H, m, H-7), 1.27–1.34 (1H, m, H-15), 1.24 (1H, d,
J = 4.3 Hz, H-19), 1.13–1.19 (1H, m, H-7), 1.11 (3H, s, H-18), 1.05
(3H, d, J = 7.0 Hz, H-27), 0.98 (3H, d, J = 6.7 Hz, H-28), 0.93 (2H, t,
J = 7.3 Hz, H-6), 0.90 (3H, s, H-29), 0.64 (3H, d, J = 6.4 Hz, H-21),
0.55–0.59 (1H, m, H-19). 13C NMR (126 MHz, DMSO-d6) d = 203.5
(C-23), 200.7 (C-3), 170.0 (C-16a), 154.8 (C-1), 154.0 (C-26a),
147.4 (C-24), 127.3 (C-2), 125.0 (C-24a), 75.3 (C-16), 74.2 (C-22),
68.5 (C-26), 51.9 (C-26c), 49.6 (C-17), 47.0 (C-14), 46.2 (C-4), 45.4
(C-15), 45.2 (C-13), 43.3 (C-8), 42.0 (C-26d, 5), 40.5 (C-26b), 34.4
(C-20), 33.2 (C-25), 31.8 (C-10, 12), 31.6, 26.7 (C-11), 26.3 (C-9),
26.0 (C-19), 22.9 (C-6), 22.8 (C-7), 21.5 (C-16b), 19.2 (C-29), 17.7
(C-18), 16.6 (C-27), 11.8 (C-21), 10.8 (C-28). ESI-MS m/z: 653.4
[M+H]+, 675.4 [M+Na]+. Purity (UV) = 95%, purity (ESLD) = 100%.
2.17–2.21 (1H, m, H-4), 2.16 (1H, m, H-15), 2.05–2.10 (1H, m, H-
11), 2.04 (3H, s, H-16b), 1.98–2.03 (2H, m, H-5a, H-8a), 1.97 (4H,
s, H-26b), 1.63–1.75 (4H, m, 2H-12, H-6), 1.53–1.61 (1H, m, H-
11), 1.41–1.49 (1H, m, H-7), 1.38 (1H, dd, J = 13.8 Hz, J = 4.2 Hz,
H-15), 1.25 (1H, d, J = 4.4 Hz, H-19), 1.19–1.21 (1H, m, H-7), 1.19
(3H, s, H-18), 1.08 (4H, d, J = 7.0 Hz, H-28), 1.02 (3H, d, J = 6.7 Hz,
H-27), 0.95 (3H, s, H-29), 0.90–0.94 (1H, m, H-6), 0.86 (3H, d,
J = 6.9 Hz, H-21), 0.57 (1H, d, J = 4.4 Hz, H-19). 13C NMR
(126 MHz, CD3CN) d = 202.4 (C-3), 197.7 (C-23), 171.8 (C-26a),
171.2 (C-16a), 168.2 (C-22a), 155.5 (C-1), 148.9 (C-24), 128.4 (C-
2), 126.4 (C-24a), 80.0 (C-22), 76.7 (C-16), 67.8 (C-26), 51.1 (C-
17), 48.4 (C-14), 47.6 (C-4), 46.9 (C-13), 46.7 (C-15), 45.1 (C-8),
43.5 (C-5), 42.1 (C-22b), 34.9 (C-25), 34.5 (C-10), 33.6 (C-20),
32.9 (C-12), 28.1 (C-11), 27.6 (C-19), 27.2 (C-9), 24.3 (C-6), 24.2
(C-7), 22.1 (C-16b), 21.1 (C-26b), 19.9 (C-29), 18.2 (C-18), 17.2
(C-27), 13.2 (C-21), 11.3 (C-28). ESI-MS m/z: 667.3 [M+Na]+,
1311.7 [2M+Na]+. Purity (UV) = 100%, purity (ESLD) = 100%.
5.1.7. Preparation of compound 61
5.1.5. Preparation of compound 58
To a stirred solution of 58 (66 mg 0.118 mmol) in CH2Cl2 (2 mL)
were added DCC (1.8 mL, 0.188 mmol, c = 0.1 mol/L, 1.6 equiv),
DMAP (1.5 mg, 0.012 mmol, 0.1 equiv), and N-Boc-glycine
(31 mg, 0.176 mmol, 1.5 equiv). The mixture was stirred for 3 h
at room temperature. Purification by flash chromatography (elu-
tion: cyclohexane/EtOAc, 90:10 to 50:50) gave 61 (44 mg, 52%) as
To a stirred solution of 2 (200 mg, 0.38 mmol) in CH2Cl2 (16 mL)
at 0 °C was added lutidine (45
lL, 0.38 mmol, 1 equiv), acetic anhy-
dride (27 L, 0.285 mmol, 0.75 equiv), and DMAP (5 mg,
l
0.038 mmol, 0.1 equiv). After 1 h, the mixture was treated with
water. The organic phase was successively washed with water,
CuSO4, water, and brine, dried over Na2SO4 and concentrated under
vacuum. The product was purified on silica gel chromatography
(elution: cyclohexane/EtOAc, 80:20) to obtain 58 (94.6 mg, 58 %)
as a white solid. 1H NMR (500 MHz, CD3CN) d = 6.94 (1H, d,
J = 10.1 Hz, H-1), 6.17 (1H, s, H-24aa), 6.04 (1H, d, J = 0.6 Hz, H-
24ab), 5.89 (1H, d, J = 9.8 Hz, H-2), 5.20 (1H, td, J = 7.5 Hz,
J = 4.6 Hz, H-16), 4.72 (1H, dd, J = 6.3 Hz, J = 1.7 Hz, H-22), 3.99–
4.10 (2H, m, H-26), 3.52 (1H, d, J = 6.1 Hz, H-30), 3.05 (1H, sxt,
J = 6.8 Hz, H-25), 2.37–2.49 (2H, m, H-20, 17), 2.22 (1H, dd,
J = 13.9 Hz, J = 7.8 Hz, H-15), 2.15–2.21 (1H, m, H-4), 2.03 (3H, s,
H-16b), 1.98–2.07 (2H, m, H-8, H-11), 1.97 (3H, s, H-26b), 1.94–
1.98 (1H, m, H-5), 1.60–1.74 (3H, m, H-6, 2H-12), 1.50–1.59 (1H,
m, H-11), 1.41–1.49 (1H, m, H-7), 1.38 (1H, dd, J = 13.6 Hz,
J = 4.4 Hz, H-15), 1.24 (1H, d, J = 4.6 Hz, H-19), 1.17–1.23 (1H, m,
H-7), 1.17 (3H, s, H-18), 1.09 (3H, d, J = 7.0 Hz, H-27), 1.03 (3H, d,
J = 7.0 Hz, H-28), 0.96 (3H, s, H-29), 0.94 (1H, qd, J = 12.8 Hz,
J = 4.0 Hz, H-6), 0.64 (3H, d, J = 6.1 Hz, H-21), 0.57 (1H, d,
J = 4.3 Hz, H-19). 13C RMN (126 MHz, CD3CN) d = 205.0 (C-23),
202.4 (C-3), 171.6 (C-26a), 171.3 (C-16a), 155.6 (C-1), 148.0 (C-
24), 128.4 (C-2), 127.4 (C-24a), 77.3 (C-16), 75.8 (C-22), 68.0 (C-
26), 51.4 (C-17), 48.3 (C-14), 47.6 (C-4), 46.9 (C-15), 46.7 (C-13),
45.3 (C-8), 43.6 (C-5), 36.4 (C-20), 34.7 (C-25), 33.1 (C-12), 32.9
(C-10), 28.1 (C-11), 27.7 (C-19), 27.2 (C-9), 24.3 (C-6), 24.3 (C-7),
22.1 (C-16b), 21.1 (C-26b), 20.1 (C-29), 18.5 (C-18), 17.3 (C-27),
12.3 (C-21), 11.3 (C-28). ESI-MS m/z: 591.3 [M+Na]+, 1159.7
[2M+Na]+. Purity (UV) = purity (ESLD) = 100%.
a
colorless oil. 1H NMR (500 MHz, CD3CN) d = 6.94 (1H, d,
J = 9.8 Hz, H-1), 6.12 (1H, s, H-24aa), 5.97 (1H, s, H-24ab), 5.90
(1H, d, J = 10.1 Hz, H-2), 5.62 (1H, br s, H-22c), 5.59 (1H, d,
J = 2.1 Hz, H-22), 5.08 (1H, td, J = 7.6 Hz, J = 4.6 Hz, H-16), 4.04
(1H, dd, J = 10.8 Hz, J = 6.4 Hz, H-26), 4.00 (1H, dd, J = 10.8 Hz,
J = 6.4 Hz, H-26), 3.94 (1H, dd, J = 17.7 Hz, J = 6.4 Hz, H-22b), 3.82
(1H, dd, J = 17.7 Hz, J = 6.2 Hz, H-22b), 3.01 (1H, sxt, J = 6.9 Hz, H-
25), 2.61 (1H, dtd, J = 13.9 Hz, J = 6.9 Hz, J = 2.2 Hz, H-20), 2.30
(1H, dd, J = 11.0 Hz, J = 7.6 Hz, H-17), 2.19 (1H, d, J = 6.7 Hz, H-4,
H-15), 2.15 (3H, s, H-26b), 2.04–2.06 (1H, m, H-11), 2.03 (3H, s,
H-16b), 2.00 (2H, dd, J = 6.9 Hz, J = 4.1 Hz, H-8a, H-5a), 1.64–1.75
(3H, m, 2H-12, H-6), 1.52–1.61 (1H, m, H-11), 1.43–1.47 (1H, m,
H-7), 1.41 (9H, s, H-22f, 22f, 22f), 1.37 (1H, dd, J = 13.9 Hz,
J = 4.1 Hz, H-15), 1.27 (1H, d, J = 3.7 Hz, H-7), 1.25 (1H, d,
J = 4.6 Hz, H-19), 1.18 (3H, s, H-18), 1.07 (3H, d, J = 7.0 Hz, H-27),
1.02 (3H, d, J = 7.0 Hz, H-28), 0.97 (3H, s, H-29), 0.91–0.96 (1H,
m, H-6), 0.85 (3H, d, J = 6.7 Hz, H-21), 0.57 (1H, d, J = 4.3 Hz, H-
19). 13C NMR (126 MHz, CD3CN) d = 202.4 (C-3), 198.4 (C-23),
171.6 (C-26a), 171.4 (C-22a), 171.2 (C-16a), 156.9 (C-22d), 155.5
(C-1), 149.0 (C-24), 128.4 (C-2), 126.0 (C-24a), 80.0 (C-22e), 78.8
(C-22), 76.7 (C-16), 67.8 (C-26), 51.2 (C-17), 48.4 (C-14), 47.6 (C-
4), 46.9 (C-13), 46.7 (C-15), 45.0 (C-8), 43.5 (C-5), 43.0 (C-22b),
34.9 (C-25), 33.6 (C-20), 33.0 (C-12), 33.0 (C-10), 28.6 (C-22f),
28.1 (C-11), 27.5 (C-19), 27.2 (C-9), 24.2 (C-7), 24.2 (C-6), 22.1
(C-16b), 21.1 (C-26b), 20.0 (C-29), 18.2 (C-18), 17.3 (C-27), 13.3
(C-21), 11.3 (C-28). ESI-MS m/z: 748.4 [M+Na]+, 1473.8 [2M+Na]+.
Purity (UV) = purity (ESLD) = 100%.
5.1.6. Preparation of compound 60
To a stirred solution of 57 (64 mg, 0.113 mmol) in CH2Cl2 (2 mL)
were added DCC (1.8 mL, 0.180 mmol, c = 0.1 mol/L, 1.6 equiv),
DMAP (1.4 mg, 0.011 mmol, 0.1 equiv), and chloroacetic acid
(16 mg, 0.169 mmol, 1.5 equiv). The mixture was stirred for 1 h
at room temperature. Purification by flash chromatography (elu-
tion: cyclohexane/EtOAc, 90:10 to 50:50) gave 59 (70 mg, 97%) as
5.2. Purity assessment
Samples were dissolved in dimethyl sulfoxide (DMSO) at con-
centrations of 5 or 10 mM. Injection volumes were 5 and 10 lL.
Mass spectrometric detection was used for molecular weight con-
firmation, whereas UV and ELSD data offered purity assessment.
a
white solid. 1H NMR (500 MHz, CD3CN) d = 6.94 (1H, d,
J = 10.0 Hz, H-1), 6.14 (1H, s, H-24aa), 6.01 (1H, s, H-24ab), 5.90
(1H, d, J = 10.0 Hz, H-2), 5.59 (1H, d, J = 2.1 Hz, H-22), 5.10 (1H,
td, J = 7.7 Hz, J = 4.4 Hz, H-16), 4.33 (1H, d, J = 15.1 Hz, H-22b),
4.25 (1H, d, J = 15.1 Hz, H-22b), 3.99–4.08 (3H, m, 2H-26), 3.02
(1H, sxt, J = 7.0 Hz, H-25), 2.63 (1H, dtd, J = 13.7 Hz, J = 7.1 Hz,
J = 2.3 Hz, H-20), 2.32 (1H, dd, J = 11.1 Hz, J = 7.6 Hz, H-17),
5.3. Determination of compound solubility
Kinetic solubility was determined in Dubelcco’s PBS at pH 7.2.
Stock compound (2
tiScreen Solubility Filter Plate (Millipore). Next, 198
PBS were added. The plate was mixed with shaking on an orbital
lL, 10 mM in DMSO) was dispensed into a Mul-
l
L of Dubelcco’s

830
J. Beck et al. / Bioorg. Med. Chem. 20 (2012) 819–831
plate shaker (900 rpm) at room temperature for 1.5 h. The solu-
tions were filtered and 160 L of filtrate were transferred to a
96-well plate then diluted with 40 L of DMSO. DMSO (200 L)
was dispensed into the MultiScreen Solubility Experiment Filter Plate
and the plate was mixed with shaking on an orbital plate shaker
(900 rpm) at room temperature for 15 min. Solutions were then fil-
tered into a 96-well plate. Solutions were analyzed by HPLC/MS/
algorithm provided by the GraphPad Software. Two independent
experiments were performed and results were expressed as aver-
age IC50 values (concentration of test compound that inhibits 50%
of cell proliferation).
l
l
l
Acknowledgments
UV. Quantitation was carried out against a 100
standard with the wavelength 254 nm. The solubility was calcu-
lated using the following formula: solubility ( M) = [((AUCfil-
trate  1.25)  100)/AUCstandard]. The % of the recovery was
calculated using the following formula: % recovery = [((AUCfil-
trate  1.25) + AUCDMSO filtrate)/AUCstandard]  100.
l
M-calibration
We thank Christian Bailly and Nicolas Guilbaud for their contin-
uous interest in this work. We thank Nicolas Molinier, Christophe
Menendez, Nathalie Chansard, Hocine Terki, Jérôme Filiol, and
Aline Stennevin for their excellent technical assistance. J.B. thanks
the CNRS for financial support.
l
Supplementary data
5.4. Biology
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.066.
5.4.1. Cell lines and culture
The human DLD-1 colon cancer cells were purchased from the
American Type Cell Culture Collection (ATCC). The generation of
stably transfected DLD-1 4Ub-Luc cell line was described else-
where.24 The engineered DLD-1 4Ub-Luc cells were cultured in
MEM medium (Gibco) supplemented with 5% heat-inactivated
References and notes
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DLD-1 4Ub-Luc cells were seeded at 104 cells/well in white 96-
well plates and incubated with indicated concentrations of test
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cence was read using a LB 960 Centro luminometer (Berthold). The
results were expressed as an induction factor (IF) which was the
ratio of accumulation of the proteasome targeted reporter protein
in treated cells versus untreated cells. In order to avoid variation of
the cells conditions, results were compared to control (epoxomicin,
at 10À7 M) run in parallel experiment, which induced a relative
induction factor of 100.
5.4.3. Inhibition of catalytic activity in DLD-1 4Ub-Luc cells
DLD-1 4Ub-Luc cells were seeded at 104 cells/well in 96-well
plates and incubated with indicated concentrations of proteasome
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