Inhibition of angiogenesis by THAM-derived cotelomers endowed with thalidomide moieties

Article abstract of DOI:10.1016/j.bmcl.2003.10.045

The synthesis of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived cotelomer endowed with thalidomide units and a preliminary assessment of its biological activity are described. 4-Carboxy thalidomide and 4-(N-acryloyl) lysine thalidomide derivatives were prepared. The polymerization of these compounds with THAM in the presence of octanethiol as transfer reagent provided a water-soluble telomer bearing several thalidomide units. The ability of this telomer to inhibit angiogenesis in a mouse model of corneal neovascularization was compared to 4-carboxy thalidomide and thalidomide. A significant inhibition in area of neovascularization stimulated by a bFGF pellet was observed only in the mice treated with the telomer.

Full text of DOI:10.1016/j.bmcl.2003.10.045

Bioorganic & Medicinal Chemistry Letters 14 (2004) 421–425
Inhibition of angiogenesis by THAM-derived cotelomers
endowed with thalidomide moieties
Sandrine Perino,a Christiane Contino-Pepin,a,* Ronit Satchi-Fainaro,b
Catherine Butterfieldb and Bernard Puccia,*
a
` ´ ´
Laboratoire de Chimie Bioorganique et des Systemes Moleculaires Vectoriels, Faculte des Sciences,
33, rue Louis Pasteur, 84000, Avignon, France
bHarvard Medical School, Surgical Research, Children’s Hospital, 300 Longwood Avenue, Boston MA 02115, USA
Received 16 July 2003; revised 1 October 2003; accepted 27 October 2003
Abstract—The synthesis of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived cotelomer endowed with thalidomide units
and a preliminary assessment of its biological activity are described. 4-Carboxy thalidomide and 4-(N-acryloyl) lysine thalidomide
derivatives were prepared. The polymerization of these compounds with THAM in the presence of octanethiol as transfer reagent
provided a water-soluble telomer bearing several thalidomide units. The ability of this telomer to inhibit angiogenesis in a mouse
model of corneal neovascularization was compared to 4-carboxy thalidomide and thalidomide. A significant inhibition in area of
neovascularization stimulated by a bFGF pellet was observed only in the mice treated with the telomer.
# 2003 Elsevier Ltd. All rights reserved.
Thalidomide, a hypnosedative drug introduced in the
1950s, was first launched to prevent nausea in pregnant
women. Its use was abruptly halted in 1961 with the
discovery of its potential for causing severe birth defects
in children whose mothers were exposed to the drug
during the first trimester of pregnancy.1À3
growth factor (bFGF) in the rabbit cornea and vascular
endothelial growth factor (VEGF) in the corneas of
mice.10À12 Moreover for a few years, numerous metabolites
or thalidomide derived compounds have been assayed to
better understand the thalidomide activity and to specify
their potentialities in medical oncology, notably as inhibi-
tors
of
angiogenesis,
TNF-a
production
or
However, numerous attractive properties of thalidomide
have been observed since then. It was approved in 1998
by the Food and Drug Administration for the short-
term treatment of cutaneous manifestations of moderate
to severe erythema nodosum leprosum, a complication
of leprosy (Hansen’s disease).4,5 Recently, it was descri-
bed as a promising drug in the treatment of a number of
cancers (in particular for multiple myeloma
therapy)1,6À8 and inflammatory- or immune-based com-
plications of HIV infection and other diseases.9
cyclooxygenase13À16 or for the suppression of liver injury.17
In the course of our previous research, we focused on
the phenomenon of angiogenesis, hypothesized 30 years
ago to be an absolute requirement for the growth and
metastasis of solid tumors.18À20 We validated the ther-
apeutic potential of oligomeric prodrugs of Ara-C called
‘telomers’ bearing RGD peptidic sequences known to be
selectively recognized by specific receptors over-
expressed on angiogenic sites.21À23 Moreover, we
obtained interesting biological results with telomeric
carriers endowed with chemotherapeutics like Ara-C or
5-Fluorouracil24,25 which prompted us to apply this
concept of drug delivery to the design of macro-
molecular derivatives of thalidomide.
In the field of medical oncology, one of its most pro-
mising properties is its antiangiogenic effect. It has been
shown that thalidomide can inhibit angiogenesis
induced by angiogenic cytokines such as basic fibroblast
We predicted that the introduction of multiple thalido-
mide units on a telomeric carrier through an appropriate
peptidic spacer arm would provide a macromolecule
with antiangiogenic properties. Such a conjugate could
Keywords: Thalidomide; Telomers; Angiogenesis; Prodrug.
* Corresponding authors. Tel.: +33-490-144442; fax: +33-490-144449
(B.P); tel.: +33-490-144435; fax: +33-490-144449 (C.C.-P.); e-mail:
bernard.pucci@univ-avignon.fr; christine.contino@univ-avignon.fr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.045

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S. Perino et al. / Bioorg. Med. Chem. Lett. 14 (2004) 421–425
422
be used for the specific delivery of antitumoral agents by
the enhanced permeability and retention (EPR) effect.26
In this paper, we describe the synthesis of a functiona-
lized derivative of thalidomide (1) used for the prepa-
lysine residue was introduced as a spacer arm during the
preparation of the thalidomide derived monomer (Fig.
2). The synthesis of N-g-acrylamido methyl lysinate was
first achieved with 76% yield by coupling acryloyl
chloride with N-a-t-butyloxycarbonyl methyl lysinate in
methylene chloride at 0 ꢀC in the presence of N,N die-
thylisopropylamine. The acid hydrolysis of the tert-
butyloxycarbonyl protective group was then quantitat-
ively performed by using a trifluoroacetic/methylene
chloride (1/1) mixture-providing compound 2. Penta-
fluorophenyl ester of 4-carboxy thalidomide was then
prepared, with a yield of 78%, by condensing penta-
fluorophenol on compound 1 in dioxane. The reaction
was carried out at room temperature in the presence of
dicyclohexylcarbodiimide. Consecutive coupling of this
active ester with N-g-acryloyl methyl lysinate was per-
formed in pyridine in the presence of catalytic amounts
of diazabycyclooctane (DABCO) at room temperature,
providing monomer 4 (F=111 ꢀC, 75% yield). This
monomer gave satisfactory elemental analysis and was
fully characterized by NMR spectroscopy and mass
spectra.
ration of
a polymerizable monomer (4) bearing
thalidomide grafted through a lysine spacer. The
telomeric compound (A) resulting from the cotelo-
merization of this monomer (4) with tris(hydroxy-
methyl)acrylamido methane (5) (THAM) was evaluated
as a potential inhibitor of angiogenesis on a chicken
chorioallantoic membrane (CAM) assay and a mouse
corneal micropocket assay.
1. Synthesis
The polymerization of THAM moieties needs the graft-
ing of a suitable polymerizable functionality onto thali-
domide which would not inhibit its biological activity.
Towards that aim, we introduced a carboxy function in
the 4-position of thalidomide aromatic group, such a
localized modification should not modify the activity of
this compound. The first stage of this synthesis was
based on the condensation of N-tert-(butyloxycarbonyl)
glutamic acid with trifluoroacetamide.27 The acidolysis
of the tert-(butyloxycarbonyl) protective group followed
by the condensation of 1,2,4-benzenetricarboxilic anhy-
dride, according to the technique proposed by Robin et
al.,28 led to acid thalidomide derivative 1 with an overall
yield of 60% (Fig. 1).
The preparation and physico-chemical data of THAM
co-monomer 5 were previously reported.29
Telomerization experiments (Fig. 3) were carried out by
refluxing compounds 4 and 5 in methanol under a
nitrogen atmosphere in the presence of octanethiol as
transfer reagent and AIBN as radical initiator. The
AIBN concentration was roughly ten times lower than
that of the telogen. The number average degree of
polymerization (DPn), equal to the amount of repeating
units (x+y), depends on the (telogen)/(monomers) ratio
adjusted through both starting materials and experi-
mental conditions.30
In order to insure a maximum degree of freedom to the
active principle grafted onto the polymer backbone, a
The proportions of monomers 4 and 5 and octanethiol
used are reported in Table 1. The telomerization was
continued until the complete disappearance of the
monomers.
Figure 1. Synthesis of an analogue of thalidomide bearing an acid
.
function in 4-position. (a) CF3CONH2, TEA, HOBT, EDC Cl,
CH2Cl2; (b) TFA/CH2Cl2; (c) 1,2,4-benzenetricarboxilic anhydride,
TEA, THF, molecular sieves 4 A.
Telomer A was purified by chromatography through a
Sephadex LH20 column (eluent: ethanol/methylene
Figure 2. Synthesis of monomer 4 derived from thalidomide. (a)
HOPhF5, DCC, dioxane; (b) CH2Cl2, DIEA; (c) TFA/CH2Cl2; (d)
Pyridine, DABCO.
Figure 3. Synthesis of THAM derived telomeric carriers bearing
thalidomide moieties.

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S. Perino et al. / Bioorg. Med. Chem. Lett. 14 (2004) 421–425
423
chloride 1/1) and was obtained as a white powder (56%
yield) fully soluble in water. The structure of this cote-
lomer, that is, the relative proportions of each THAM
(x) and thalidomide residues (y) moiety in the cotelo-
mer, and the DPn of the macromolecule were deter-
Male C57 Bl/6 mice were anesthetized with 300 mL of
2.5% Avertin. The eyes were topically anesthetized with
0.5% proparacaine HCl (Ophthetic, Allergan). Using an
operating microscope (Zeiss, Oberkochen, Germany), a
central, intrastromal linear keratotomy (approximately
0.6 mm length) was performed with a 30ꢀ micro-knife
(Xomed, Florida). The eye was then proptosed using
forceps. Using a modified von Graefe knife no. 3
(2Â30 mm), a micropocket was dissected toward the
temporal limbus. For bFGF-containing pellets, the
pocket was extended to 1.0 mm of the temporal limbus.
A single pellet was placed on the corneal surface at the
base of the pocket with jeweler’s forceps, and, using one
arm of the forceps, the pellet was advanced to the tem-
poral end of the pocket. Antibiotic ointment (ery-
thromycin) was applied once to the operated eye. The
same procedure was repeated on the other eye. Control
group was injected intraperitoneally (ip) daily with
250 mL 0.45% methyl cellulose. Treated groups were
injected ip with 50 mg/kg daily equivalent dose of thali-
domide. Each group consisted of both eyes of 6 mice.
1
mined in H NMR (solvent: DMSO-d6) by comparing
the peaks area assigned to the terminal methyl signal in
the hydrocarbon tail (d 0.9 ppm, 3H), to the thalidomide
imide proton (d 11.2ppm, yH), and to methylene pro-
tons of THAM (d 5.0 ppm, 6xH). The telomer A was
composed of roughly 4 thalidomide units and 36
THAM moieties affording the water solubility to the
macromolecule.
2. Biological assays
2.1. Bovine capillary endothelial (BCE)
Cell Proliferation Assay Bovine capillary endothelial
cells were obtained and grown as previously described.31
A cell suspension (30,000 cells/mL) was made in
Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% bovine calf serum (BCS) (Life
Technologies, Inc.), 0.29 mg/mL l-glutamine, 100 units/
mL penicillin and 100 mg/mL streptomycin (1% GPS)
(Gibco), plated onto gelatinized 24-well culture plates
(0.5 mL/well), and incubated (37 ꢀC, 10% CO2) for 24 h.
The media was replaced with 0.25 mL of DMEM with
5% BCS and 1% GPS and the test sample was applied.
Cells were challenged with thalidomide, compound 1
and telomer A (200 mM down to 0.02 mM). After 20 min
of incubation, media and bFGF were added to obtain a
final volume of 0.5 mL of DMEM with 5% BCS, 1%
GPS, and 1 ng/mL bFGF. After 72h, cells were
dispersed in trypsin, resuspended in Hematall (Fisher
Scientific, Pittsburgh, PA), and counted by Coulter
counter.
The eyes were examined by slit lamp biomicroscopy on
postoperative day 6 after pellet implantation. Mice were
anesthetized with isofluorane and the maximum vessel
length (VL) of the neovascularization zone, extending
from the base of the limbal vascular plexus toward the
pellet, was measured with a linear reticule through the slit
lamp. The contiguous circumferential zone of neo-
vascularization was measured as clock hour with a 3600
reticule (where 300 of arc equals 1 clock hour). The
percentage of inhibition of vessel growth was calculated
as the area of neovascularization in the treated mice (T)
Table 1. Physico-chemical data of cotelomer A. The concentrations
of each monomer 4 or 5 used are expressed in relation to the octa-
nethiol concentration
Initial conditions
[monomer]/[Telogen]
Telomer
structures
2.2. Chick chorioallantoic membrane (CAM) assays
Comp
Tel A
4
5
y (Thal)
4
z (THAM)
36
DPn
40
Chick chorioallantoic membrane (CAM) assays were
performed as described.32,33 Briefly, three-day-old ferti-
lized white Leghorn eggs (Spafas, Norwich, CT) were
cracked, and embryos with intact yolks were placed in
100 Â 20 mm Petri dishes. After three days of incu-
bation (37 ꢀC and 3% CO2), a carboxymethylcellulose
(Fisher Scientific, Fair Lawn, NJ) disc containing thali-
domide or telomer A (1 mg and 10 mg) was applied to the
CAM of individual embryos.
5.4
19
After 48 h of incubation, embryos and CAMs were
observed by stereomicroscope. The effects on the devel-
oping vasculature were recorded at 48 h after implant-
ation of the 0.5% carboxymethylcellulose pellet
containing various drugs.
2.3. Murine corneal neovascularization
Pellets containing basic fibroblast growth factor bFGF
(80 ng/pellet) were made of the slow-release polymer
Hydron (polyhydroxyethylmethacrylate [polyHEMA])
as previously described.34
Figure 4. Representative CAM at day 4 after implantation of carbox-
ymethylcellulose pellets as Control (a) or containing thalidomide (b),
compound 1 (c) or telomer A (d). None of the compounds show sig-
nificantly less neovascularization.

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424
reduced. However, this fact is not taken into considera-
tion when variants are put into formula for calculating
the area. Due to the lack of an objective grading scale,
these results are not presented. Thalidomide did not
inhibit corneal neovascularization at the dose of 50 mg/
kg. However, inhibition was seen at a teratogenic dose
(200 mg/kg) of thalidomide.11 No treated animal
demonstrated any signs of sedation, toxicity or weight
loss.
In summary, we have shown that acid thalidomide
derivative 1 and telomer A were more potent than tha-
lidomide at equivalent doses in an in vivo angiogenesis
assay. Evaluation of the in vivo antitumor activity of
these compounds is warranted.
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Figure 5. Representative corneas at day 6 after implantation of bFGF
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