Synthesis and antiproliferative activity of ligerin and new fumagillin analogs against osteosarcoma

Article abstract of DOI:10.1016/j.ejmech.2014.04.012

Ligerin (1) is a natural chlorinated merosesquiterpenoid related to fumagillin (2) exhibiting a selective antiproliferative activity against osteosarcoma cell lines and an in vivo antitumor activity in a murine model. Semisynthesis of ligerin analogs was performed in order to study the effects of the C3-spiroepoxide substitution by a halogenated moiety together with the modulation of the C6 chain. Results showed that all derivatives exhibited an in vitro antiproliferative activity against osteosarcoma cell lines and that chlorohydrin compounds were equally or more active than their spiroepoxy analogs. Among semisynthetic analogs, the parent compound 1 was the best candidate for further studies since it exhibited higher or equivalent activity compared to TNP470 (3) against SaOS2 and MG63 human osteosarcoma cells with a four times weaker toxicity against HFF2 human fibroblasts. Quantitative videomicroscopy analysis was conducted and allowed a better understanding of the mechanism of its antiproliferative activity.

Full text of DOI:10.1016/j.ejmech.2014.04.012

European Journal of Medicinal Chemistry 79 (2014) 244e250
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of ligerin and new fumagillin
analogs against osteosarcoma
,
,
Elodie Blanchet a b, Marieke Vansteelandt a 1, Ronan Le Bot b, Maxim Egorov b,
Yann Guitton a, Yves François Pouchus a, Olivier Grovel a
,
*
a University of Nantes, Faculty of Pharmacy, MMS-EA260, Nantes F-44000, France
b Atlanthera, Atlantic Bone Screen, Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Ligerin (1) is a natural chlorinated merosesquiterpenoid related to fumagillin (2) exhibiting a selective
antiproliferative activity against osteosarcoma cell lines and an in vivo antitumor activity in a murine
model. Semisynthesis of ligerin analogs was performed in order to study the effects of the C3-
spiroepoxide substitution by a halogenated moiety together with the modulation of the C6 chain. Re-
sults showed that all derivatives exhibited an in vitro antiproliferative activity against osteosarcoma cell
lines and that chlorohydrin compounds were equally or more active than their spiroepoxy analogs.
Among semisynthetic analogs, the parent compound 1 was the best candidate for further studies since it
exhibited higher or equivalent activity compared to TNP470 (3) against SaOS2 and MG63 human oste-
osarcoma cells with a four times weaker toxicity against HFF2 human fibroblasts. Quantitative video-
microscopy analysis was conducted and allowed a better understanding of the mechanism of its
antiproliferative activity.
Received 14 February 2014
Received in revised form
31 March 2014
Accepted 4 April 2014
Available online 5 April 2014
Keywords:
Ligerin
Fumagillin
Osteosarcoma
Antiproliferative
Cytostatic
Ó 2014 Elsevier Masson SAS. All rights reserved.
Marine-derived Penicillium
1. Introduction
[3,4]. First studied and used in clinical medicine for its antimi-
crobial activity in human and veterinary health [5], this molecule
Despite its low incidence (around 300 new cases/year in
Europe), osteosarcoma is the most frequent malignant primary
bone tumor. It affects predominantly children, teenagers and young
adults and accounts for 8.9% of cancer-related deaths in children
[1,2]. No satisfactory treatment is currently available and no sig-
nificant improvement in prognosis has been noticed since the
advent of combined chemotherapy in the 90’s. The 5-year survival
rate for patients diagnosed with osteosarcoma is still remaining
between 60% and 70% without metastasis [2]. Current therapy
consists in combining surgery and multiagents chemotherapy
(neoadjuvant and post-surgery) based principally on methotrexate,
ifosfamide, doxorubicin and cisplatin treatments. All these drugs
induce significant side effects, highlighting the need to improve
current treatment strategies.
has known a renewed interest since 1990 because of its anti-
angiogenic properties [6]. Given the high toxicity of this com-
pound, many syntheses of derivatives were done and led to the
synthesis of the first clinically developed analog: the 6-O-chlor-
oacetylcarbamoyl-fumagillol named TNP470 (3). This compound
presented a strong activity against adenocarcinoma but its clinical
development was stopped in phase II trial due to its neurotoxicity
[7e10]. The instability and the toxicity of TNP-470 are likely due,
at least in part, to the presence of three functional groups chem-
ically labile or metabolically unstable, the two epoxides (the spi-
roepoxide and the 10-20 epoxide on the C4 side chain) and the
chloroacetyl moiety at C6.
The mechanism of action of this class of compounds remains not
fully resolved, but the methionine aminopeptidase-2 (MetAP2) has
been identified as their main molecular target. This metal-
loprotease is responsible for the removal of the methionine residue
from newly synthetized polypeptides, allowing their further myr-
istoylation and functionalization. Inhibition of this enzyme would
cause cell-cycle arrest. Most of these sesquiterpenes inhibit selec-
tively and irreversibly the MetAP2 through insertion of the C4 chain
in the active pocket, mimicking the terminal part of native proteins,
and via the formation of a covalent bond between the C-7 of the
Fumagillin (2) is a merosesquiterpene isolated for the first time
in 1949 from a crude extract of a strain of Aspergillus fumigatus
* Corresponding author.
E-mail address: olivier.grovel@univ-nantes.fr (O. Grovel).
Present address: University of Toulouse III, Faculty of Pharmaceutical Sciences,
1
UMR 152 IRD/UPS Pharma-DEV, F-31062 Toulouse Cedex 9, France.
http://dx.doi.org/10.1016/j.ejmech.2014.04.012
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

E. Blanchet et al. / European Journal of Medicinal Chemistry 79 (2014) 244e250
245
spiroepoxide group and a nitrogen of the 231-histidine residue of
the enzyme [11].
(4a) were synthesized together with their respective chlorohydrin
analogs, ligerin (1) and 7-chloro-fumagillol (4) (Scheme 1). Given
that only two brominated compounds have been described in the
literature in the fumagillin series [21], the synthesis of a bromo-
hydrin analog of ligerin was also performed (5). In a second time,
some ligerin analogs with different C6 moieties were synthesized,
by introducing a heteroatom or a benzene ring in the side chain or
by extending the length of the carboxylic acid side chain.
For that purpose, a first step consisted in preparing 4a, the
saponification product of (þ)-fumagillin. Instead of the classical
two steps process consisting in a first purification of the fumagillin
dicyclohexylamine salt contained in a commercial product (Fumidil
BÒ) followed by the hydrolysis of the C-6 ester, a one-step reaction
was developed. In this way, the entire commercial preparation was
directly submitted to alkaline hydrolysis using 0.5 N NaOH, allow-
ing to purify 4a from the reaction mix by liquid/liquid partition
after acidification. From 4a, each structural modulation was ob-
tained with the same semisynthetic approach. Compound 4a was
esterified using different anhydrides to afford compounds 1a, 6a,
7a, 8a. To obtain the chlorohydrin or bromohydrin analogs (com-
pounds 1, 5e8), a halogenation step was further performed using
the corresponding halogenous salt, i.e. LiCl or LiBr. The structures of
all compounds were confirmed by 1H and 13C NMR analyses.
In 2005, Rodeschini [12] defined the role of the two epoxides for
the binding of fumagillin analogs to the active site of MetAP2 which
involves two water molecules, a binuclear metal center and the
231-histidin residue [13]. As the removal of the 10-20 epoxide
doesn’t impact the activity of the molecule, it would most likely be
involved in the orientation of the side chain for the recognition by
the enzyme [14]. Griffith et al. defined the importance of the second
epoxide, demonstrating that the removal of the ring epoxide
dramatically lowered the activity of fumagillol against MetAP2 [15].
Since its first discovery and use in medicine, structural modifica-
tions on the sesquiterpene backbone concerned mainly the C4-
branched chain in order to improve the affinity for the hydropho-
bic channel surrounding the catalytic pocket [16]. Among dozens of
synthetic derivatives of fumagillin, two analogs have recently been
undergoing development in phase I clinical trials. CKD-732 is
studied for the treatment of refractory solid cancer [17] and in
combination with capecitabine and oxaliplatine for the treatment
of metastatic colorectal cancer in patients who progressed on
chemotherapy based on irinotecan [18]. PPI-2458 is evaluated for
the treatment of non-Hodgkin lymphomas and several solid tu-
mors [19]. These two compounds corroborate the interest of this
chemical family in the search for new anticancer drugs.
In the course of our search for new drugs against osteosarcoma,
ligerin (1), a new natural compound related to fumagillin (2), has
previously been isolated from a marine-derived strain of Penicillium
sp. (Fig. 1). Ligerin, the 3-hydroxy, 3-chloromethylene, 6-(3-
carboxy-1-oxopropyl)-fumagillol was the second natural product
exhibiting a halogenated moiety in place of the common spiroep-
oxide. Assayed against various murine cell lines, it exhibited a se-
lective antiproliferative activity against osteosarcoma compared to
non-tumor cells [20].
This work was focused on the semisynthesis of new halogen-
ohydrin analogs related to ligerin, in order to investigate the impact
of halogen atoms such as chlorine or bromine on the anti-
proliferative activity compared to their spiroepoxide analogs.
Structural modulations of C6-side chain were also explored,
maintaining the terminal carboxylic acid moiety. Bioactivities as
well as selectivity of all compounds were evaluated using in vitro
assays against both murine and human osteosarcoma and non-
tumor cell lines and were compared to TNP470 (3) and reference
anticancer compounds. Further studies on the ligerin bioactivity
were also conducted, in order to get a better understanding of its
antiproliferative mechanism.
2.2. Pharmacology
2.2.1. Antiproliferative activity
The antiproliferative activity of halogenated compounds (1, 4
and 5) and their respective spiroepoxide analogs (1a, 4a) was tested
in vitro against two osteosarcoma cell lines: one murine (POS-1)
and one human (SaOS2).
As shown in Fig. 2, compounds 1a and 1 were more active than
4a and 4 as no IC50 could be measured in the range of 0.40e
2100 nM for these last compounds. This first result confirms that a
C6 side-chain is required for a high activity against these cancer
cells [22]. The C7 halogen substitution was shown to have a weaker
influence on the cytotoxicity. The activity of the chlorohydrins and
their respective spiroepoxide analogs was found to be equivalent
except for 1 against SaOS2 cell line which was more active than its
spiroepoxide analog 1a and in the same manner for 4 and 4a
against POS-1 cells. Similar activity was also obtained for 3 and its
halogenohydrin derivative against the two osteosarcoma cell lines.
Nonetheless, substitution of the chlorine atom by a bromine atom
decreased the activity as 5 exhibited an intermediate activity
compared to 1 and other analogs. IC50 of 5 against POS-1 and SaOS2
cells were respectively 272 nM and 801 nM whereas IC50 of 1 were
78 nM and 137 nM.
2. Results and discussions
In literature, the two epoxides have often been described as
essential for the binding to MetAP2, the target enzyme of this class
of compounds [12], but the impact of the C3 epoxide opening on
the activity remains unclear. In this way, it has been reported that
compounds for which the spiroepoxide was opened were less
active than their intact analog. For instance, the C3-
methylthiomethyl derivative of 3 was over 100 times less active
than the parent compound on both MetAP2 inhibition and HUVEC
assays [23], and halogenation of the spiroepoxide of fumagillin
derivatives led to a 10 fold decrease of their activity on the enzyme
[24]. On the contrary, Hayashi et al. have shown that chlovalicin, a
natural chlorinated analog in the fumagillol series, was 3 times
more active than its epoxidized form ovalicin [25]. More recently, in
a in vitro metabolization study, Arico-Muendel et al. have shown
that, after exposure of the fumagillin analog PPI-2458 to an acidic
treatment mimicking the stomach milieu, some metabolites
formed were chlorohydrin analogs. These compounds were found
as effective as their epoxide precursor on a MetAP2 inhibition assay
2.1. Chemistry
In order to evaluate the effect of the spiroepoxide opening and
its substitution by a halogenomethylene moiety on the cytotoxicity
of this chemical series, 6-O-succinylfumagillol (1a) and fumagillol
Fig. 1. Structures of ligerin (1), fumagillin (2) and TNP470 (3).

246
E. Blanchet et al. / European Journal of Medicinal Chemistry 79 (2014) 244e250
Scheme 1. Synthetic routes for compounds 1, 5e8. Reagents and conditions: a) 0.5 N NaoH, rt, 18 h; b) anhydride, DMAP/dry pyridine, rt, 24 h; c) LiCl or LiBr, THF, acetic acid, rt,
24 h.
Fig. 2. Effects of compounds 1, 1a, 4, 4a and 5 against POS-1 (a) or SaOS2 (b) cell proliferation after 72 h exposure. Cell viability was measured by XTT assay. Data are mean ꢀ SD of
triplicate experiments.
and against HUVEC proliferation [19], as we observed for 1a and 1
against osteosarcoma cells. Because of a rapid re-cyclization
observed in rat plasma to afford the initial epoxide form they hy-
pothesized that chlorohydrins lack intrinsic activity [19]. In the
present study, the observation of a lower cytotoxicity for the bro-
mohydrin analog 5 should tend to envisage that the C7 methylene
substitution has an effective involvement in the activity of these
compounds. Nevertheless, a molecular docking study performed
with 1 and 5 using the 3-dimensional structure of the human
MetAP2/fumagillin complex (PDB code: 1Boa) [11] revealed that
nor a chlorine nor a bromine atom can prevent the interaction of
the C7 methylene with the His231 residue of MetAP2, as it has been
described for 2 and 3 [11] (data not shown).
the different IC50 observed for the three synthetized compounds
were cell line-dependent, no clear structureeactivity relationship
could be deduced.
For determination of selectivity against cancer cells and toxicity
assessment, activities against osteosarcoma cells were compared
with cytotoxicity against non-tumor fibroblastic cell lines. All
Table 1
Antiproliferative activity of compounds 1, 3, 5e8 and reference anticancer molecules
against three tumor cell lines (one murine POS1, and two human SaOS2 and MG63)
and two non-tumor cell lines (one murine L929 and one human HFF2), expressed as
IC50 [nM] and SI (selectivity index).
In a second time, the modulation of the C6 moiety was explored.
Cytotoxicity of compounds 6e8 was evaluated against a panel of
three osteosarcoma cell lines, one murine (POS-1) and two human
(SaOS2 and MG63) and two non-tumor fibroblasts L929 (murine)
and HFF2 (human) (Table 1). All compounds displayed an anti-
proliferative activity associated with a dose effect against osteo-
sarcoma cells, even if IC50 could not be reached for compounds 5e8
against MG63 and for 8 against SaOS2 (maximum 42e48% inhibi-
tion). For all tested compound except 7, activity was higher against
murine than human cell lines.
Results showed that all derivatives exhibited a lower activity
compared to the parent compound 1, whatever the cell line. For
example, against POS-1 compounds 6e8 were found to be 3 to 17
times less active than 1. Thus, the introduction of a heteroatom or a
benzene ring in the succinyl moiety or the lengthening of the car-
bon chain could not improve the antiproliferative activity of 1. As
Compound
Murine cell lines
IC50 (nM)
Human cell lines
IC50 (nM)a
SIb
SIb
POS-1
L929
SaOS2
MG63
HFF2
1
3
5
6
7
8
78
2
>2300
>2300
>2100
>2300
>2300
>2100
521
>29
137
508
801
863
629
>2100
52
1459
1521
>2100
>2300
>2300
>2100
NT
>2300
1979
>2100
>2300
>2300
>2100
NT
NT
NT
NT
NT
>17
4
>3
>3
>4
>961
>8
>10
>2
>9
5
272
234
1394
230
95
Paclitaxel
Vinscristine
Doxorubicin
Irinotecan
Fludarabine
75
419
6
4
1
3
11
NT
43
161
48
NT
6300
5700
6500
17,500
NT
NT
NT
NT
a
NT: Non Tested.
b
The selectivity index (SI) was calculated as the ratio between the IC50 values on
normal cells versus cancer cells from the same origin (L929/POS-1; HFF2/SaOS2).

E. Blanchet et al. / European Journal of Medicinal Chemistry 79 (2014) 244e250
247
compounds induced a higher inhibition of cancer cell proliferation
versus the non-tumor one from the same origin (murine or human).
In this way, none of the compounds showed a sufficient activity
allowing to establish an IC50 value against L929, and a maximum of
40% of inhibition of cell viability was attained in the range of con-
centrations tested. Furthermore, all compounds were at least 3
times more potent against human cancer cells than against HFF2.
The selectivity index of compounds 6e8 was compared to 1 by
calculating the ratio between the IC50 values against normal cells
versus cancer cells from the same origin, highlighting that ligerin
seems to be the most selective compound against osteosarcoma
cells, with SI found over 29 and 17 against murine and human cell
lines respectively.
increased from 17 h for negative control to 22 h for the ligerin-
treated cells. Videomicroscopy also allowed the observation of
the effects induced by the tested compounds on cell shape and
movements. In normal conditions, POS-1 cells are continuously
exhibiting unilateral movements, and they require an initial contact
between them to engage their division. In the presence of 1, cell
movements were dramatically perturbed and cell divisions were
observed even when cells were isolated, without any previous
contact with other cells. However, no morphological modifications
were noticed on osteosarcoma cells, contrary to what occurred on
L929 which exhibited a rounded form in place of their characteristic
fibroblastic shape (Fig. 4). Then, at the tested concentration, 1
exhibited a selective activity against osteosarcoma. As it was
observed for apomine, this activity was characterized as cytostatic.
As among all compounds tested, 1 appeared to be the more
potent analog, its antiproliferative activity and selectivity for oste-
osarcoma were compared to the phase II engaged drug candidate,
TNP-470 (3) the 6-O-chloroacetylcarbamoyl semisynthetic deriva-
tive of fumagillol. Against murine osteosarcoma, 3 was found to be
more potent than 1 with IC50 values of 2 and 78 nM, respectively.
On the contrary, 1 exhibited a higher activity against human SaOS2
(IC50 of 137 and 801 nM for 1 and 3 respectively), whereas the two
compounds exhibited a similar activity against MG63 with a
maximal diminution of cell viability of 55% within the range of
concentrations tested. Against the non-tumor L929 and HFF2 cell
lines, the cytotoxicity of 1 was weaker than 3 in the range 0.30e
230 nM. Although 3 showed a high selectivity against murine cells
(SI > 961), 1 exhibited a better ratio against human cell lines, with a
selectivity index 4 times higher than the one of 3. For instance, at a
concentration of 30 nM, the decrease of MG63 and SaOS2 viability
was 30e35% for both compounds, whereas the proliferation of
HFF2 fibroblasts was inhibited of 15% and 43% for 1 and 3
respectively.
3. Conclusion
In conclusion, new analogs of the fumagillin-related natural
compound ligerin 1 were semisynthesized using a simplified three-
step process and were investigated for their antiproliferative ac-
tivity against osteosarcoma and normal cell lines. Results showed
that both halogenohydrin and spiroepoxy compounds exhibited an
antiproliferative activity against murine or human cancer cell lines
higher than against normal fibroblastic cells. Chlorohydrins 1 and 4
were found to be at least as active as their epoxy derivatives against
SaOS2 and POS1 osteosarcoma cell lines, whereas the bromohydrin
5 showed a reduced activity, emphasizing the role of the C7 sub-
stituents. Compared to synthetized compounds and the reference
drug candidate 3,1 exhibited a higher activity against human SaOS2
and MG63 cancer cells together with a lower cytotoxicity against
normal cells. Furthermore, 1 was evaluated as more than 70 times
more active than irinotecan and fludarabine and as potent as
vincristine and paclitaxel against POS-1 cells. Pharmacological in-
vestigations highlighted that 1 exerted its antiproliferative activity
through a cytostatic mechanism. Further studies will be performed
in order to better understand the mechanism of action of 1 at the
molecular level.
Furthermore, 1 was found to be at least 70 times more active
than irinotecan and fludarabine against POS-1 and showed equi-
potent efficacy with vincristine and paclitaxel. Compared to the five
anticancer drugs tested, the selectivity index of 1 was higher
against murine cell lines. For instance, 1 presented a selectivity
index at least 7 times higher than the one of the most active drug
doxorubicin.
4. Experimental protocols
2.2.2. Effect of ligerin on cell replication/viability
4.1. Chemistry
Further studies were performed to determine whether 1
exhibited a cytotoxic or a cytostatic activity. After 72 h exposure to
1, viable POS-1 and L929 cells were counted using a trypan blue dye
exclusion assay. Two positive controls were used, apomine as
reference compound for cytostatic activity and staurosporin for
cytotoxicity. Contrary to the staurosporin treatment which induced
immediate cell death against POS-1, 81% of cells were found to be
4.1.1. General
Reagents were purchased from SigmaeAldrich (Saint-Quentin
Fallavier, France). Solvents from Carlo Erba SDS (Val de Reuil,
France) were distilled before use. Fumidil BÒ was obtained from
Ceva Santé Animale (Libourne, France). Progress of reactions was
monitored by thin layer chromatography on Silica Gel plates
AlugramÒ Xtra SIL G/UV254 (MachereyeNagel, Hoerdt, France). 1D
and 2D NMR spectra were recorded in CDCl3 on a Bruker 500 MHz
spectrometer fitted with a TCI cryoprobe. Chemical shift are
living cells after a contact with 0.7 mM of 1, a concentration which
induced 54% inhibition of POS-1 proliferation. This result was
similar to the proportion of living cells in the assay performed with
the solvent negative control (82% living cells) and slightly higher
than with apomine (67%). On L929, equivalent percentages were
obtained for 1, apomine and negative control.
The effect of ligerin on the cell division duration and on the
morphology of L929 and POS-1 cells was evaluated thanks to a time
lapse analysis using quantitative videomicroscopy performed with
a reversed microscope and a camera taking a picture of each well of
a 24-well plate every 10 min. Each experiment, i.e. negative control,
staurosporine, apomine and 1 was performed in triplicate. Cells
were then counted and growth curves were constructed as shown
expressed in
d (ppm) with TMS as internal standard and coupling
constants in Hertz. HRMS analyses were performed with an IT-TOF
mass spectrometer composed of an ESI ion source and a hybrid Ion
Trap-Time-Of-Flight mass analyzer (Shimadzu, Kyoto, Japan).
4.1.2. Semisynthesis
Each semisynthetic approach started from 2. The first step
consisted of an alkaline hydrolysis of 2 contained in the commercial
product Fumidil BÒ. For that purpose, 50 g of Fumidil BÒ were
dissolved in 1 L of NaOH (0.5 N) and the reaction was stirred at
room temperature during 18 h. 5% citric acid was added before
extraction with diethyl ether. Then, organic phase was dried with
NaHCO3/anhydrous Na2SO4 and concentrated under vacuum, to
give 4a (yellow oil, 609 mg). The second step consisted of the
in Fig. 3. With a treatment consisting of 0.7 mM of ligerin, POS-1
proliferation was slowed down and the doubling time of cell pop-
ulation was twice longer, 30 h instead of 15 h for negative control.
This effect was weaker against L929, as the cell cycle duration

248
E. Blanchet et al. / European Journal of Medicinal Chemistry 79 (2014) 244e250
Fig. 3. Effect of 0.7
m
M 1 on cell viability and cell proliferation of POS-1 (a, b) and L929 (c, d) cells. a, c Trypan blue exclusion assay after 72 h incubation (values ꢀSD of four
independent experiments). b, d Growth curves of cells observed every 10 min during 2 days by quantitative videomicroscopy. Red line average number of cells exposed to 1, (orange
area: ꢀSD of triplicates); blue line average number of cells exposed to negative control, (green area: ꢀSD of triplicates). Apomine concentration 10
mM. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
esterification of 4a. This compound (600 mg, 2.12 mmol, 1 eq) was
dissolved with DMAP (dimethylaminopyridine, 330 mg, 2.75 mmol,
1.30 eq), succinic anhydride (827 mg, 8.28 mmol, 3.9 eq) in 4 mL of
anhydrous pyridine. The reaction was stirred at room temperature
during 24 h. The reaction mix was extracted with AcOEt and
washed with water. The organic phase was then extracted with a
saturated solution of NaHCO3 before the acidification until pH4
with NaH2PO4. The mixture was finally extracted with AcOEt and
dried using Na2SO4anh before concentration under vacuum to
obtain compound 1a (yellow oil, 620 mg). This compound (600 mg,
Fig. 4. Morphological changes of POS-1 (left) and L929 (right) cells after 0, 24, 48, 72 h exposure to negative control, 0.7
magnification).
mM 1, 10
mM apomine, 1
mM staurosporin (Gꢁ10

E. Blanchet et al. / European Journal of Medicinal Chemistry 79 (2014) 244e250
249
1.56 mmol, 1 eq) was dissolved with LiCl (285 mg, 6.80 mmol, 4.35
eq) in 5 mL of THF at 0 ꢂC and acetic acid (450
L, 486 mg, 8.1 mmol,
(CH3); 20.4 (CH2); 22.7 (CH3); 23.5 (CH2); 25.9 (CH3); 27.4 (CH2);
29.0 (CH2); 33.6 (2CH2); 43.1 (CH); 50.0 (CH2); 56.6 (CH3); 62.3
(CH); 64.0 (C); 65.8 (CH); 76.7 (C); 78.6 (CH); 118.1 (CH); 134.8 (C);
172.6 (C); 176.1 (C).
m
5.2 eq) was then added. Mixture was warmed to room temperature
and the reaction was stirred during 24 h. Ligerin (1) (colorless oil,
401 mg) was purified by liquid chromatography on a silica gel
column (elution by cyclohexane and CH2Cl2/MeOH 10:1 (v/v)).
Compounds 5e8 were synthesized with the same semisynthetic
approach; only the anhydride used for the esterification step and
the halogenous salt for the last step changed (succinic anhydride
and LiBr for 5, glutaric anhydride and LiCl for 6, diglycolic anhydride
and LiCl for 7, phthalic anhydride and LiCl for 8). For compound 4,
the halogenation step with LiCl was performed with 4a. Compound
3 was synthesized according to the method described by Marui
et al. [26].
4.1.2.6. 7-Chloro-6-[[(2-carboxymethoxy)acetyl]oxy]-fumagillol (7).
Colorless oil; HRESIMS m/z [MþNa]þ 457.1596 (calcd for
C
20H31ClO8Na, 457.1600, D d: 1.35 &
0.85 ppm); 1H NMR (CDCl3)
1.98 (m; 2H); 1.48 (s; 3H); 1.68 (s; 3H); 1.75 (s; 3H); 1.82e1.86 (m;
2H); 2.18 & 2.46 (m; 2H); 2.39 (m; 1H); 2.96 (t; J ¼ 5.5 Hz; 1H); 3.29
(s; 3H); 3.34 & 3.85 (d; J ¼ 10 Hz; 2H); 3.48 (s; 1H); 4.12e4.45 (m;
4H); 5.18 (t; J ¼ 7.5 Hz; 1H); 5.58 (s; 1H); 13C NMR (CDCl3)
d: 17.8
(CH3); 22.7 (CH3); 23.4 (CH2); 25.9 (CH3); 27.4 (CH2); 29.0 (CH2);
43.1 (CH); 50.3 (CH2); 56.9 (CH3); 62.2 (CH); 63.3 (C); 66.8 (CH);
67.5 (CH2); 68.3 (CH2); 76.0 (C); 78.4 (CH); 118.0 (CH); 134.8 (C);
169.4 (C); 176.2 (C).
4.1.2.1. 6-(3-Carboxy-1-oxopropyl)-fumagillol (1a). Colorless oil;
HRESIMS m/z [MþNa]þ 405.1896 (calcd for C20H30O7Na, 405.1884,
D
3.0 ppm); 1H NMR (CDCl3)
d: 1.10 & 2.05 (m; 2H); 1.2 (s; 3H); 1.68
4.1.2.7. 7-Chloro-6-[(2-carboxybenzoyl)oxy]-fumagillol
(8).
(s; 3H); 1.76 (s; 3H); 1.82e1.86 (m; 2H); 1.98 (m; 1H); 2.17 & 2.39
(m; 2H); 2.56 & 2.96 (d; J ¼ 10 Hz; 2H); 2.63 (t; J ¼ 5.5 Hz; 1H); 2.71
(s; 4H); 3.40 (s; 3H); 3.63 (m; 1H); 5.19 (t; J ¼ 7.0 Hz; 1H); 5.68 (s;
Colorless oil; HRESIMS m/z [MþNa]þ 489.1655 (calcd for
C
24H31ClO7Na, 489.1651, D d: 1.48 &
0.82 ppm); 1H NMR (CDCl3)
1.93 (m; 2H); 1.51 (s; 3H); 1.68 (s; 3H); 1.76 (s; 3H); 1.82e1.93 (m;
2H); 2.19 & 2.47 (m; 2H); 2.23 (m; 1H); 2.98 (t; J ¼ 5.5 Hz; 1H); 3.39
(m; 1H); 3.39 (s; 3H); 3.57 & 3.90 (d; J ¼ 10 Hz; 2H); 5.21 (t;
J ¼ 7.0 Hz; 1H); 5.77 (s; 1H); 7.58 (d; J ¼ 8 Hz; 4H); 7.84 (d; J ¼ 8 Hz;
1H); 13C NMR (CDCl3)
d: 18.3 (CH3); 26.0 (CH2); 26.1 (CH3); 27.7
(CH2); 29.7 (CH2); 29.7 (CH2); 29.7 (CH2); 48.1 (CH); 51.2 (CH2); 57.1
(CH3); 59.6(C); 59.7 (C); 61.5 (CH); 67.2 (CH); 79.2 (CH); 118.7 (CH);
135.4 (C); 14.0 (CH3); 171.9 (C); 175.7 (C).
4H); 13C NMR (CDCl3)
d: 17.8 (CH3); 22.7 (CH3); 23.2 (CH2); 25.8
(CH3); 27.3 (CH2); 29.1 (CH2); 43.4 (CH); 50.4 (CH2); 56.8 (CH3); 62.4
(CH); 64.1 (C); 67.6 (CH); 76.0 (C); 78.7 (CH); 118.4 (CH); 128.8 (CH);
129.0 (CH); 129.6 (C); 130.9 (CH); 131.2 (CH); 134.8 (C); 134.9 (C);
167.4 (C); 171.8 (C).
4.1.2.2. Ligerin (1). Colorless oil; HRESIMS m/z [MþNa]þ 441.1656
(calcd for C20H31ClO7Na, 441.1651,
D d:
1.3 ppm); 1H NMR (CDCl3)
1.40 & 1.96 (m; 2H); 1.51 (s; 3H); 1.68 (s; 3H); 1.76 (s; 3H); 1.82e1.86
(m; 2H); 2.19 & 2.47 (m; 2H); 2.39 (m; 1H); 2.73 (s; 4H); 2.98 (t;
J ¼ 5.5 Hz; 1H); 3.28 (s; 3H); 3.3 (m; 1H); 3.52 & 3.85 (d; J ¼ 10 Hz;
4.2. Biological activities
2H); 5.21 (t; J ¼ 7.0 Hz; 1H); 5.52 (s; 1H); 13C NMR (CDCl3)
d: 17.9
(CH3); 22.2 (CH3); 23.4 (CH2); 25.8 (CH3); 27.4 (CH2); 29.3 (2CH2);
43.2 (CH); 50.3 (CH2); 56.6 (CH3); 62.3 (CH); 64.0 (C); 66.4 (CH);
76.7 (C); 78.4 (CH); 118.2 (CH); 134.8 (C); 171.5 (C); 176.9 (C).
4.2.1. Cell lines and cell culture
Three cancer cell lines POS-1 (murine osteosarcoma), MG63 and
SaOS2 (human osteosarcomas ATCC number CRL-1427 and HTB-
85), and two non-tumor cell lines, L929 (murine fibroblasts, ATCC
number CCL-1) and HFF2 (human fibroblasts) were cultured. L929
and POS-1 cell lines were grown in RPMI 1640 medium (GibcoÒ,
Grand Island, New York) supplemented with 5% (v/v) of fetal bovine
serum. MG63, SaOS2 and HFF2 cell lines were grown in DMEM
medium (GibcoÒ Grand Island, New York) supplemented with fetal
bovine serum 10% (v/v). All the cell lines were maintained in culture
at 37 ꢂC in an atmosphere of 5% CO2.
4.1.2.3. 7-Chloro-fumagillol (4). Colorless oil; HRESIMS m/z
[MþNa]þ 341.1502 (calcd for C16H27ClO4Na, 341.1490,
D 3.74 ppm);
1H NMR (CDCl3)
d: 1.36 & 2.10 (m; 2H); 1.48 (s; 3H); 1.67 (s; 3H);
1.74 (s; 3H); 1.82e1.86 (m; 2H); 2.18 & 2.45 (m; 2H); 2.40 (m; 1H);
2.98 (t; J ¼ 5 Hz; 1H); 3.33 (m; 1H); 3.35 (s; 3H); 3.51 & 3.81 (d;
J ¼ 10.8 Hz; 2H); 4.22 (s; 1H); 5.20 (t; J ¼ 6.8 Hz; 1H); 13C NMR
(CDCl3) d: 18.2 (CH3); 22.5 (CH3); 23.6 (CH2); 26.1 (CH3); 27.8 (CH2);
30.0 (CH2); 42.6 (CH); 51.0 (CH2); 56.6 (CH3); 62.5 (CH); 63.6 (C);
64.2 (CH); 76.5 (C); 80.8 (CH); 118.6 (CH); 135.1 (C).
4.2.2. XTT assay
In order to evaluate the antiproliferative activity of the semi-
4.1.2.4. 7-Bromo-6-(3-carboxy-1-oxopropyl)-fumagillol
(5).
synthetic compounds, cells were sown in 96-well plates (100 mL/
Colorless oil; HRESIMS m/z [MþNa]þ 485.1133 (calcd for
well at a density of 1$104 cells/mL for POS-1, HFF2 and L929, 1.5$104
for SaOS2 and MG63). After 24 h incubation at 37 ꢂC with 5% CO2 to
allow cell attachment, and renewal of culture medium, cells were
exposed to various concentrations of tested compounds for 72 h.
Cytotoxic activity was determined by colorimetric method using
tetrazolium salts. An XTT solution (sodium 30-[1-[(phenylamino)-
carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sul-
fonic acid hydrate, Roche Applied Science) was added in each well
C
20H31BrO7Na, 485.1145, D d: 1.48 &
2.47 ppm); 1H NMR (CDCl3)
1.99 (m; 2H); 1.513 (s; 3H); 1.68 (s; 3H); 1.76 (s; 3H); 1.81 (m; 2H);
2.19 & 2.48 (m; 2H); 2.48 (m; 1H); 2.74 (s; 4H); 2.99 (t; J ¼ 5.5 Hz;
1H); 3.29 (m; 1H); 3.29 (s; 3H); 3.50 & 3.75 (d; J ¼ 10 Hz; 2H); 5.20
(t; J ¼ 7.0 Hz; 1H); 5.51 (s; 1H); 13C NMR (CDCl3)
d: 17.6 (CH3); 22.0
(CH3); 23.3 (CH2); 25.5 (CH3); 27.0 (CH2); 28.8 (CH2); 28.8 (CH2);
29.9 (CH2); 39.9 (CH2); 43.7 (CH); 56.7 (CH3); 62.3 (CH); 64.0 (C);
66.1 (CH); 78.7 (C); 78.7 (CH); 118.2 (CH); 135.4 (C); 171.4 (C); 177.5
(C).
(50 m
L/well) and plates were incubated for 4 h at 37 ꢂC, 5% CO2.
Absorption at 450 nm was measured with a plate reader (Wallace
Victor 2Ô 1420 MultiLabel Counter Perkin ElmerÒ). IC50 were
determined as the concentration that inhibited cell viability by 50%.
4.1.2.5. 7-Chloro-6-(4-carboxy-1-oxobutoxy)-fumagillol
(6).
Colorless oil; HRESIMS m/z [MþNa]þ 455.1811 (calcd for
C
21H33ClO7Na, 455.1807,
D
0.88 ppm); 1H NMR (CDCl3)
d
: 1.40 &
4.2.3. Quantitative videomicroscopy
2.03 (m; 2H); 1.51 (s; 3H); 1.68 (s; 3H); 1.80 (s; 3H); 1.83 (m; 2H);
2.03 & 2.20 (m; 2H); 2.20 (m; 1H); 2.42e2.56 (m; 6H); 2.98 (t;
J ¼ 5.5 Hz; 1H); 3.31 (s; 3H); 3.32 (m; 1H); 3.51 & 3.7 (d; J ¼ 10 Hz;
The cell growth was evaluated thanks to a time lapse experi-
ment using quantitative videomicroscopy. Cells were grown in 24-
well plates. A negative control and two positive controls (apomine
and staurosporin treatment) were used. Assays were performed in
2H); 5.20 (t; J ¼ 7.3 Hz; 1H); 5.52 (s; 1H); 13C NMR (CDCl3)
d: 17.9

250
E. Blanchet et al. / European Journal of Medicinal Chemistry 79 (2014) 244e250
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mM ligerin, 10 mM apomine and 1 mM staur-
osporin. The plates were incubated at 37 ꢂC in a 5% CO2 atmosphere.
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Acknowledgment
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The authors want to acknowledge the French National Associ-
ation of Research and Technology for a Ph.D. grant for E. Blanchet.
Authors also thank Fabien Petit and Marie-Claude Boumard for
technical assistance, Sandrine Pottier and Arnaud Bondon from
UMR CNRS 6290, PRISM, Université de Rennes 1 for NMR spectra
acquisition, and P. Hulin from IFR26 PICell platform, Université de
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Appendix A. Supplementary data
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