Preliminary biological evaluations of new thalidomide analogues for multiple sclerosis application

Article abstract of DOI:10.1016/j.bmcl.2008.11.118

The present work deals with the synthesis of a new series of thalidomide derivatives for therapeutic applications. These compounds were evaluated in vitro on a human endothelial cell line EA.hy926 for their antiproliferative potential and in vivo on an experimental animal multiple sclerosis model called EAE as angiogenesis inhibitors. The preliminary results obtained on EAE assays seem to validate that anti-angiogenesis compounds could be promising tools for the treatment of MS.

Full text of DOI:10.1016/j.bmcl.2008.11.118

Bioorganic & Medicinal Chemistry Letters 19 (2009) 878–881
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preliminary biological evaluations of new thalidomide analogues
for multiple sclerosis application
a
a
Christiane Contino-Pépin a, , Audrey Parat , Sandrine Périno , Christine Lenoir b,c, Michel Vidal b,c
,
*
Hervé Galons b,c, Stephen Karlik d, Bernard Pucci a,
*
a Université d’Avignon, Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels, 33 rue Louis Pasteur, 84000 Avignon, France
b Université Paris Descartes, Faculté de Pharmacie and UFR Biomédicale, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, Paris F-75006, France
c Inserm U648, Paris F-75006, France
d University of Western Ontario, Department of Medicinal Imaging, LHSC-UC, 339 Windermere Road, London, Ont., Canada N6A 5A5
a r t i c l e i n f o
a b s t r a c t
Article history:
The present work deals with the synthesis of a new series of thalidomide derivatives for therapeutic
applications. These compounds were evaluated in vitro on a human endothelial cell line EA.hy926 for
their antiproliferative potential and in vivo on an experimental animal multiple sclerosis model called
EAE as angiogenesis inhibitors. The preliminary results obtained on EAE assays seem to validate that
anti-angiogenesis compounds could be promising tools for the treatment of MS.
Received 15 October 2008
Revised 26 November 2008
Accepted 27 November 2008
Available online 7 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Thalidomide
Multiple sclerosis
EAE
Angiogenesis inhibitors
Forty years ago J. Folkman hypothesized that angiogenesis, the
sprouting of new blood vessels from pre-existing ones, was a pre-
requisite phenomenon for a tumor’s supply in nutrients and oxy-
gen.1 Since then, various anti-angiogenesis strategies have been
investigated against tumor development. This extensive research
led to the recent FDA approval of the first anti-angiogenic agent
bevacizumab (Avastin, Genentech), a humanized anti-VEGF anti-
body, used in combination with chemotherapy for the treatment
of metastatic colorectal cancer.2 In the same field, vascular target-
ing strategies are currently being explored as promising tools for a
selective shutdown of any established tumor vasculature, followed
by rapid tumor cell death.3,4
In the course of our previous research, we focused on angiogen-
esis for oncological applications. We validated the therapeutic
interest of oligomeric prodrugs called ‘telomers’ bearing Ara-C
and endowed with RGD peptidic sequences for selective delivery
of the drug on angiogenic sites.5 Following these studies on angio-
genesis targeting and/or inhibition, we explored the anti-angiogen-
romolecule exhibiting a significant inhibition of corneal neovascu-
larization in a mouse model assay.7
Besides cancer therapy, other studies have revealed that patho-
logical angiogenesis occurs in a number of relevant diseases, such
as blinding ocular disorders,8 or chronic inflammatory diseases like
rheumatoid arthritis, atherosclerosis or psoriasis.9
Recently, angiogenesis has been implicated in the inflammatory
phase of multiple sclerosis (MS), a chronic autoimmune inflamma-
tory disease, and it may represent a target for therapeutic interven-
tion.10,11 Today the overall mechanisms by which thalidomide
exerts its anti-tumor activity still remain to be elucidated, but tha-
lidomide reduces the levels of angiogenic factors including TNF-
a,
VEGF and IL-6.12 Aside from its role in angiogenesis, TNF-
a
is an
inflammatory mediator also implicated in the pathogenesis of
cerebral endothelial damage in MS.13 The anti-angiogenic effect
of thalidomide and its negative feedback on TNF-
a production
prompted us to imagine new functionalized derivatives of thalido-
mide, some of which could subsequently be grafted on amphiphilic
molecular carriers, for intervention in MS.
esis potential of thalidomide, when grafted on
a telomeric
backbone. Indeed, thalidomide is a ‘re-discovered’ drug which
shows significant anti-angiogenic activity.6 Thus, our introduction
of multiple thalidomide units on a telomeric carrier led to a mac-
In the present work, we have undertaken several structural
development studies and obtained a series of thalidomide ana-
logues substituted on the phthalimide ring system (Scheme 1).
Some, like 3- or 4-nitro thalidomide,14,15 have been used as start-
ing materials for the preparation of new amino-thalidomide deriv-
atives. Considering the hydrophobic nature of thalidomide, we also
investigated the biological impact of derivatives containing a
* Corresponding authors. Tel.: +33 490144442; fax: +33 490144449.
E-mail addresses: christine.pepin@univ-avignon.fr (C. Contino-Pépin), bernard.
pucci@univ-avignon.fr (B. Pucci).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.118

C. Contino-Pépin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 878–881
879
C8F17
OH
BocHN
CO2H
CO2H
NH
O
O
C8F17
OH
OH
OH
OH
HO
O
N
H
NH(Z)
e)
OH
OH
O
HO
NH
O
O
O
OH
O
N
OH
OH
HO
OH
f)
O
N
N
H
4
a)
O
O
H
HO
OH
NH
5 (60%)
O
O
O
O
O
O
O
BocHN
d)
b, c)
NH
NH
NH
N
O
N
O
O
HO2C
F5PhO2C
O
O
1
(95%)
b, g)
2 (67%)
3 (78%)
O
O
O
O
O
O
NH
NH
NH
h)
b)
N
O
N
1
O
N
O
1
1
1
4
4
4
2
2
2
3
3
3
NH
NH
R
O
O
O
-O2CF3C+H3N
BocHN
6 (40%) R=3-NO2
7 (70%) R=4-NO2
-
3-NH(CH ) NH +CF3CO2
8 (98%)
:
9 (94%) :
3-NH(CH2)2NHBoc
4-NH(CH2)2NHBoc
10 (100%) :
11 (100%) :
2 2
3
j)
i)
-
4-NH(CH2)2NH3+CF3CO2
12 (15%) R=3-NH
(Actimid)
14 (70%) R=4-NH2
2
13 (21%) R=3-NHiPr
O
O
O
O
OH
k)
NH
l)
m)
b, n)
NH
CHO
O
BocHN
OH
BocHN
H2N
OH
N
O
OH
OH
OH
HO
N
O
OH
O
N
N
BocHN
O
15 (98%)
H
16 (61%)
H
N
O
HO
H
O
OH
18 (45%)
17 (70%)
Scheme 1. Synthesis of thalidomide analogues. Reagents and conditions: (a) CF3CONH2, TEA, HOBT, EDCÁCl, rt; (b) CF3CO2H/CH2Cl2 3:7, 0 °C to rt; (c) 4-carboxy phthalic
anhydride, TEA, THF, reflux, molecular sieves 4 Å; (d) HOPhF5, DCC, dioxane, rt; (e) H2/Pd/C, EtOH, rt; (f) DIEA, MeOH, rt; (g) 4-nitro or 3-nitro phthalic anhydrides, AcOH,
reflux, molecular sieves 4 Å; (h) BocNHCH2CHO, H2/Pd/C, THF/DMF (9:1), rt; (i) H2/Pd/C, acetone, rt; (j) H2/Pd/C, AcOH glacial, rt; (k) BocON, TEA, CH2Cl2, rt; (l) PCC, CH2Cl2,
célite, rt; (m) 7, H2/Pd/C, DMF/THF (9:1) rt; (n) lactobionolactone, TEA, 2-methoxyethanol, 60 °C.
hydrophilic moiety, such as lactobionamide (compound 18), or
grafting on a fluorinated amphiphilic carrier, which we have previ-
ously examined (compound 5).16 These new amphiphilic thalido-
mide derivatives should exhibit an increased bioavailability and a
better therapeutic index than the parent drug.
et al.,19 led to a mixture of the expected 3-amino-thalidomide or
‘Actimid’ 12 and of (N-isopropyl)-amino thalidomide 13 in low
yields. We assumed that in this last case the amino group formed
under these reductive conditions partially reacted with acetone
through a reductive amination to give 13. In an attempt to verify
this hypothesis and take advantage of this side reaction for the
preparation of new amino-thalidomide derivatives, we investi-
gated the condensation of commercial (BOC)-aminoethanal on ni-
tro-thalidomide under the same reductive conditions, using THF as
solvent. This reaction provided the N-(aminoethyl)-amino deriva-
tives 10 and 11 in good yields after hydrolysis of their correspond-
ing tert-butyloxycarbonyl protective groups. Due to the poor
chemical stability of previous (BOC)-aminoethanal, we lastly chose
to perform the same reaction with (BOC)-aminopropanal 16 ob-
tained in two steps from aminopropanol. The reductive amination
of 4-nitro thalidomide in the presence of compound 16, then pro-
vided compound 17. The amphiphilic amino thalidomide analogue
18 was prepared after acidic deprotection and subsequent conden-
sation of 17 with lactobionolactone.18
The anti-angiogenic activities of 4-carboxy thalidomide 2, and
compounds 6–11 were evaluated in vitro through their inhibitory
effect on the proliferation of
a human endothelial cell line
EA.hy926. Amphiphilic compounds 18 and 5, and some of their
intermediate analogues 2, 11, and 14 were screened in vivo on
one of the best-characterized experimental animal model for MS
study, namely the experimental autoimmune encephalomyelitis
(EAE) model.17 All the compounds were prepared by usual organic
synthesis methods, as shown in Scheme 1, and gave appropriate
analytical values.
Compounds 1, 2, 6, and 7 were obtained, as previously de-
scribed,7 from (Boc)-aminoglutarimide 1 upon phthalylation, with
phthalic anhydride derivatives: 4-carboxy, 3-nitro or 4-nitro
phthalic anhydride. The pentafluorophenyl ester of 4-carboxy tha-
lidomide (compound 3) was then obtained by condensation of pen-
tafluorophenol to compound 2 in dioxane. Consecutive coupling of
3 to the amphiphilic carrier 4 (prepared following our published
synthetic route)16 provided compound 5 in good yields.18
4-Nitro thalidomide 7 was reduced under catalytic hydrogena-
tion in glacial acetic acid to produce the 4-amino derivative 14, in
quantitative yield. Surprising, the same reaction when applied to
the 3-nitro thalidomide 6 in acetone as described by Muller
Although thalidomide is a prodrug requiring metabolic activa-
tion to exert an anti-angiogenic effect, it spontaneously hydrolyzes
at physiological pH to give a cascade of hydrolysis products.20
Some of them, as well as thalidomide, showed an in vitro inhibitory
activity against endothelial cells without the need for prior meta-
bolic activation.15,21–23
On the basis of these in vitro evaluations, some of our thalido-
mide analogues (compounds 2, 6–11) were first screened for their

880
C. Contino-Pépin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 878–881
ability to inhibit the proliferation of EA.hy926 cells, compared to
thalidomide used as a standard. The hybridoma EA.hy926 is a fu-
sion product between HUVEC and the epithelial cancer cell line
A549.24 This established cell line constitutes a valuable model to
evaluate a potential effect on endothelial cells.25 Moreover, its sus-
ceptibility to xenobiotics was recently shown to be comparable to
that of HUVEC and this model could constitute an alternative
material for routine cytotoxicity studies.26 In vitro evaluations
were performed on almost all thalidomide analogues except
amphiphilic derivatives 5 and 18, which were only used in the
in vivo experiments.
All products were tested in aqueous solution containing 0.01%
DMSO. In those conditions, all results are expressed comparatively
to the control using solvent alone and no drug. To determine an
IC50 value, a linearization of results is essential. Therefore, inhibi-
tion results were expressed as percent compared to control versus
the logarithm of the concentration used. Inhibition studies on
EA.hy926 are presented in Figure 1.
for MS utilization. For these in vivo experiments, we explored the
combination of thalidomide to hydrophilic or amphiphilic ligands
through the preparation of two new thalidomide derivatives: com-
pounds 5 and 18. These two amphiphilic compounds, derived
either from 4-carboxy thalidomide 2 (for which we previously re-
ported the anti-angiogenic behaviour7 on a mouse corneal model)
or from N-(aminopropyl)-amino thalidomide 17, were evaluated
on an experimental multiple sclerosis model and compared to
some of their intermediates (compounds 2, 11, and 14) and to free
thalidomide.
The effectiveness of these compounds was tested in the MOG
(35-55) peptide-induced experimental autoimmune encephalomy-
elitis (EAE) in C57BL/6J mice.28 Six- to eight-week-old mice (n = 34)
were immunized via a single flank injection of 200 lg of MOG (35–
55) peptide in CFA with 10 mg/ml added Mycobacterium tuberculo-
sis on day 0 and also received 200 ng pertussis toxin ip on days 0
and 2 post-immunization (pi). Animals were scored blindly as fol-
lows: 0, no change; 1, flaccid tail; 2, poor righting reflex; 3, one
hind limb paralyzed; 4, both hind limbs paralyzed; and 5,
moribund.
Five compounds (11, 7, 9, 10, and 14) exhibit an inhibitory
activity on EA.hy926 cells. Except compound 14, all present an
activity higher than thalidomide and two of them, 11 and 7, at a
one magnitude lower dose. The amino derivative 14 and thalido-
mide show the same inhibiting activity of endothelial cell
proliferation.
Dosing of thalidomide (100 mg/kg), thalidomide derivatives
(100 mg/kg thalidomide equivalent) or vehicle (0.5% CMC) com-
menced on d7 pi and continued for up to 15 days. Figure 2 illus-
trates the clinical progression for vehicle and treated mice. The
vehicle controls showed a gradual increase in mean clinical score
starting on d13 pi reaching a maximum on the day of sacrifice
(d22). Thalidomide treated mice also showed acute disease. Com-
pound 14 showed about the same clinical progress as the control
group. Surprisingly, the effects of compounds 5 and 11 were very
similar and accelerated the development of acute EAE. We do not
have data which would allow speculation on the mechanism for
disease acceleration with these two compounds, but they were
the only two that containing fluorinated side chains. 4-Carboxy
derivative 2, which was inert in vitro, produced diminished disease
in this acute model and we also observed a decrease in clinical
signs for amphiphilic N-(aminopropyl)-4-amino thalidomide
derivative 18. Thus, some compounds showed activity in vivo that
was not predicted from in vitro experiments.
Extrapolation of the dose–response curves gives the IC50 value
of each compound: thalidomide (equipotent to 14) and 10 have,
respectively, an IC50 around 200 and 150
41 M is found for derivative 9. The most active products 11 and
7 significantly inhibit endothelial cells, respectively, at concentra-
tions of 10 and 12 M. It has to be noted that at these concentra-
lM, whereas an IC50 of
l
l
tions no activity on cellular growth was found for other tested
products.
Some structure–activity relationships could be gained from
these results. Introduction of a substituent in the C4 position of
the aromatic ring seems to be required for inhibitory effect. In
the case of compound 10, we can speculate that the degree of free-
dom of the N-(aminoethyl)-amino group at the C3 position allows
a productive receptor-mediated interaction with the targeted cells.
However, among C4-substituted derivatives, 4-carboxy thalido-
mide derivative (compound 2) exhibits surprisingly no inhibitory
effect.
The influence of the electronic impact of the phthalimide ring
substituents is not clear at this stage. Indeed, the two more active
compounds 7 and 11, respectively, substituted by an electron-
withdrawing nitro group or by an electron-donating N-(amino-
ethyl)-amino group were equipotent in anti-angiogenic effect. A
similar reversal of biological activity between derivatives with
opposite electronic substituents was previously reported in the
literature.27
In conclusion, we report herein the synthesis of new thalido-
mide derivatives bearing either a carboxy or an aminoalkyl group
on their phthalimide ring. The in vitro and/or in vivo biological
activities of these compounds were evaluated. In vitro assays car-
ried out on EA.hy926 cells do not allow us to propose constructive
structure–activity relationships. Indeed, even if the C4 substitution
seems to be a prerequisite to the antiproliferative effect, N-amino-
4
vehicle (n=8)
2 (n=4)
In parallel to these preliminary in vitro assays, we investigated
the potential of a series of C-4 substituted thalidomide analogues
5 (n=4)
14 (n=5)
11 (n=4)
18 (n=3)
3
2
1
0
90
80
70
thalidomide (n=6)
thalidomide
60
50
40
30
20
10
0
14
9
10
11
7
treatment
0
5
10
15
20
25
0
0.5
1
1.5
2
2.5
day post immunization
Log (concentration %)
Figure 2. Mean clinical scores for EAE mice treated with vehicle, thalidomide and
compounds 2, 5, 11, 14, and 18.
Figure 1. Effect of thalidomide derivatives on the proliferation of EA.hy926 cells.

C. Contino-Pépin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 878–881
14. Melchert, M.; List, A. Int. J. Biochem. Cell Biol. 2007, 39, 1489.
881
propyl-3-amino derivative 10 shows a noticeable activity whereas
4-carboxy analogue 2 does not exhibit any effect. Such a negative
result of compound 2 was previously observed on a bovine capil-
lary endothelial cells model.7 Nevertheless, this 4-carboxy deriva-
tive showed an in vivo activity. Indeed, the second part of this
work focused on the effectiveness of our new thalidomide deriva-
tives to interfere with the progression of EAE. A series of amphi-
philic derivatives was evaluated and compared to free
thalidomide. Whereas the parent drug does not exhibit any effi-
ciency in decreasing the clinical signs of EAE when delivered in
0.5% CMC, 4-carboxy thalidomide 2 and amphiphilic thalidomide
analogue 18 show decreased clinical signs in this model. As regards
4-carboxy derivative, it has to be underlined that its combination
to a fluorinated amphiphilic carrier does not provide any positive
effect. On the other hand, the linkage of a lactobionolactone moiety
on N-(aminopropyl)-4-amino thalidomide 17, providing amphi-
philic compound 18, led to a promising activity in EAE model.
These preliminary results validate that angiogenesis is an inter-
esting target for the treatment of MS. Although all compounds
were derived from thalidomide, only compounds 2 and 18 showed
substantive in vivo activity and will be starting point for further
investigations. Indeed currently, no clear structure–activity rela-
tionships can yet be drawn from these in vitro and in vivo
assessments.
15. Noguchi, T.; Fujimoto, H.; Sano, H.; Miyajima, A.; Miyachi, H.; Hashimoto, Y.
Bioorg. Med. Chem. Lett. 2005, 15, 5509.
16. Périno, S.; Contino-Pépin, C.; Jasseron, S.; Rapp, M.; Maurizis, J. C.; Pucci, B.
Bioorg. Med. Chem. Lett. 2006, 16, 1111.
17. Steinman, L.; Zamvil, S. S. Ann. Neurol. 2006, 60, 12.
18. Compound 5: white powder (60%), mp 199.1–199.4 °C; [
D
a
]
+1.3 (c: 0.25,
20
DMSO), Anal. Calcd for C42H46F17N5O17Á2H2O: C 40.28, H 3.99, N 5.59, found: C
40.56, H 4.14, N 5.56; MS (FAB+): [thalidomide]+ = 257, [M+Na]+ = 1238. 1H
NMR (250 MHz, DMSO-d6, d): 11.18 (1H, s), 8.45 (1H, d, J = 7.47 Hz), 8.32 (1H,
s), 8.11 (1H, t), 8.07 (1H, d, J = 7.47 Hz), 7.71 (1H, m), 7.22 (1H, m), 5.47 (1H, m),
5.19–5.13 (2H, m), 4.98–4.80 (3H, m), 4.65–4.46 (4H, m), 4.36 (1H, d,
J = 4.6 Hz), 4.29–3.85 (8H, m), 3.73–3.44 (8H, m), 2.44–2.10 (6H, m), 1.59–
1.34 (6H, m). 13C NMR (250 MHz, DMSO-d6, d): 174.28; 173.57; 173.24;
173.21; 170.0; 167.14; 164.64; 140.71; 134.47; 129.63; 124.05; 122.14;
105.27; 86.32; 76.19; 73.68; 73.38; 72.04; 71.77; 71.58; 68.59; 62.66; 60.24;
52.58; 49.59; 40.85; 33.81; 31.38 (CH2Rf, t); 26.52; 24.94; 23.15; 22.39. 19F
NMR (250 MHz, DMSO-d6, d): À80.15; À113.36; À121.62; À122.42; À123.14;
D
À125.68. Compound 18: yellow powder (45%), mp 121.8 °C (dec); [
a
]
20 +6.33
(c: 0.66, CD3OD); MS (ESI) m/z: [M+H]+ = 671, [M+NH4]+ = 688, [M+Na]+ = 693,
[M+K]+ = 709. 1H NMR (250 MHz, CD3OD, d): 7.51 (1H, d, J = 8.4 Hz), 6.94 (1H,
s), 6.87 (1H, d, J = 8.4 Hz), 5.0 (1H, dd, J = 12.2 Hz), 4.52 (1H, d, J = 7.2 Hz), 4.29
(1H, m), 4.18 (1H, m), 3.82–3.27 (14H, m), 2.90–2.52 (3H, m), 2.07 (1H, m), 1.81
(2H, m). 13C NMR (250 MHz, CD3OD, d): d = 174.0; 173.3; 170.4; 168.4; 167.9;
154.6; 134.6; 124.8; 116.9; 115.4; 105.5; 104.4; 81.9; 75.8; 73.4; 72.6; 71.7;
71.4; 71.1; 69.0; 62.4; 61.3; 49.0; 40.0; 36.3; 30.8; 28.96; 28.2; 22.4.
19. Muller, G. W.; Chen, R.; Huang, S. Y.; Corral, L. G.; Wong, L. M.; Patterson, R. T.;
Chen, Y.; Kaplan, G.; Stirling, D. I. Bioorg. Med. Chem. Lett. 1999, 9, 1625.
20. Bauer, K. S.; Dixon, S. C.; Figg, W. D. Biochem. Pharmacol. 1998, 55, 1827.
21. Lepper, E. R.; Ng, S. S. W.; Gütschow, M.; Weiss, M.; Hauschildt, S.; Hecker, T.
K.; Luzzio, F. A.; Eger, K.; Figg, W. D. J. Med. Chem. 2004, 47, 2219.
22. Zhu, X.; Giordano, T.; Yu, Q. S.; Holloway, H. W.; Perry, T. A.; Lahiri, D. K.; Brossi,
A.; Greig, N. H. J. Med. Chem. 2003, 46, 5222.
References and notes
23. Komorowski, J.; Jerczyn´ ska, H.; Siejka, A.; Baran´ ska, P.; Lawnicka, H.;
Pawlowska, Z.; Stepien´ , H. Life Sci. 2006, 78, 2558.
24. Edgell, C. J.; McDonald, C. C.; Graham, J. B. Proc. Natl. Acad. Sci. U.S.A. 1983, 80,
3734.
25. Cell culture. EA.hy926 cells were serially cultured in DMEM (Invitrogen, Paisley,
UK) containing 15% heat-inactivated FCS, 150 U/mL penicillin, 150 U/mL
1. Folkman, J. N. Eng. J. Med. 1971, 285, 1182.
2. Ferrara, N.; Hillan, K. J.; Gerber, H. P.; Novotny, W. Nat. Rev. Drug Discov. 2004, 3,
391.
3. Trachsel, E.; Neri, D. Adv. Drug Deliv. Rev. 2006, 58, 735.
4. Rybak, J. N.; Trachsel, E.; Scheuermann, J.; Neri, D. ChemMedChem 2007, 2, 22.
5. Jasseron, S.; Contino-Pépin, C.; Maurizis, J. C.; Ollier, M.; Pucci, B. Eur. J. Med.
Chem. 2003, 38, 825.
6. D’Amato, R. J.; Loughnan, M. S.; Flynn, E.; Folkman, J. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 4082.
7. Périno, S.; Contino-Pépin, C.; Satchi-Fainaro, R.; Butterfield, C.; Pucci, B. Bioorg.
Med. Chem. Lett. 2004, 14, 421.
8. Kourlas, H.; Abrams, P. Clin. Ther. 2007, 29, 1850.
9. Pandya, N. M.; Dhalla, N. S.; Santani, D. D. Vasc. Pharmacol. 2006, 44, 265.
10. Kirk, S. L.; Karlik, S. J. J. Autoimmun. 2003, 21, 353.
11. Minagar, A.; Jy, W.; Jimenez, J. J.; Alexander, J. S. Neurol. Res. 2006, 28, 230.
12. Sleijfer, S.; Kruit, W. H. J.; Stoter, G. Eur. J. Cancer 2004, 40, 2377.
13. Sharief, M. K.; Thompson, E. J. J. Neuroimmunol. 1992, 38, 27.
streptomycin, 2 mM
L-glutamine, and 0.04% HAT (hypoxanthine,
aminopterin, thymine) supplement (all from Invitrogen, Paisley, UK) and
passaged weekly. For counting experiments, 30,000 cells were seeded on 6-
well plates at J0 and were treated with products at the appropriate dose 12 h
later. After 7 days of growing, cells were trypsinated and counted with a
counter coulter. The NIH3T3 cells were serially cultured in DMEM containing
15% heat-inactivated FCS, 150 U/mL penicillin, 150 U/mL streptomycin, 2 mM
L-glutamine and passaged weekly.
26. L’azou, B.; Fernandez, P.; Bareille, R.; Beneteau, M.; Bourget, C.; Cambar, J.;
Bordenave, L. Cell Biol. Toxicol. 2005, 21, 127.
27. Miyachi, H.; Azuma, A.; Ogasawara, A.; Uchimura, E.; Watanabe, N.; Kobayashi,
Y.; Kato, F.; Kato, M.; Hashimoto, Y. J. Med. Chem. 1997, 40, 2858.
28. Roscoe, W. A.; Kidder, G. M.; Karlik, S. J. Cell Commun. Adhes. 2007, 14, 57.