(Z)-1,4-diamino-2-butene as a vector of boron, fluorine, or iodine for cancer therapy and imaging: synthesis and biological evaluation.

Article abstract of DOI:10.1016/S0968-0896(02)00147-5

Polyamine vectors are attractive for tumor targeting. We envisaged (Z)-1,4-diamino-2-butene (Z-DAB), an unsaturated analogue of putrescine as vector of (10)B, (18)F and (131)I for boron neutron capture therapy (BNCT), and tumor imaging by positron emission tomography or scintigraphy respectively. In the present work, the synthesis and characterization of new derivatives of Z-DAB were reported. Z-DAB was actively transported in cells via the polyamine transport system and converted into the spermidine analogue.(E)-2-iodo-1,4-diamino-2-butene (E-I-DAB) was not taken up by the polyamine transport system and may not be suitable for tumor imaging. In contrast, (Z)-2-[4-(5,5-dimethyl-dioxaborinan-2-yl)phenyl]methyl-1,4-diamino-2-butene (Z-4-Bbz-DAB) was a substrate of the transport system and allowed significant boron accumulation in 3LL cells. Its potential in BNCT will be evaluated.

Full text of DOI:10.1016/S0968-0896(02)00147-5

Bioorganic & Medicinal Chemistry 10 (2002) 2863–2871
(Z)-1,4-Diamino-2-butene as a Vector of Boron, Fluorine,
or Iodine for Cancer Therapy and Imaging:Synthesis and
Biological Evaluation
Benedicte Martin,a Francoise Posseme,b Caroline Le Barbier,b
Francois Carreaux,b Bertrand Carboni,b Nikolaus Seiler,a
Jacques-Philippe Moulinouxa and Jean-Guy Delcrosa,*
a
´
´
´
´
Groupe de Recherche en Therapeutiques Anticancereuses, CNRS FRE 2261, Faculte de Medecine,
´
2, Avenue du Pr. Leon Bernard, F35043 Rennes cedex, France
`
b
`
Synthese et Electrosynthese Organiques, UMR CNRS 6510, Institut de Chimie, Avenue du General Leclerc,
´ ´
F35042 Rennes cedex, France
Received 4 March 2002; accepted 7 May 2002
Abstract—Polyamine vectors are attractive for tumor targeting. We envisaged (Z)-1,4-diamino-2-butene (Z-DAB), an unsaturated
analogue of putrescine as vector of 10B, 18F and 131I for boron neutron capture therapy (BNCT), and tumor imaging by positron
emission tomography or scintigraphy respectively. In the present work, the synthesis and characterization of new derivatives of
Z-DAB were reported. Z-DAB was actively transported in cells via the polyamine transport system and converted into the sper-
midine analogue.(E)-2-iodo-1,4-diamino-2-butene (E-I-DAB) was not taken up by the polyamine transport system and may not be
suitable for tumor imaging. In contrast, (Z)-2-{4-(5,5-dimethyl-[1,3,2]dioxaborinan-2-yl)phenyl}methyl-1,4-diamino-2-butene
(Z-4-Bbz-DAB) was a substrate of the transport system and allowed significant boron accumulation in 3LL cells. Its potential in
BNCT will be evaluated. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
i The specificity of the polyamine transport system
is not stringent. A wide variety of polyamine
derivatives and analogues are able to use this
system.2
ii The activity of the polyamine transport system is
higher in transformed cells than in their normal
counterparts.3,4 In vivo, tissues with a high
transport rate, such as liver, and tissues with a
high demand for polyamines, such as tumors5
and prostate6 take upradiolabeled polyamines in
greater amounts than other tissues.
iii Depletion of intracellular polyamines, for exam-
ple by 2-(difluoromethyl)ornithine (DFMO), an
inactivator of ornithine decarboxylase, greatly
enhances polyamine uptake by various cell
lines.7ꢀ10 In vivo, the polyamine content of
tumor cells is more efficiently depleted by
DFMO than that of non- or slow-growing cells.
Putrescine, spermidine and spermine are ubiquitous low
molecular weight biogenic amines, essential for cell
growth and differentiation.1 In eukaryotic cells, com-
plex, highly regulated biosynthetic and catabolic path-
ways determine the intracellular pools of polyamines.
Cells are also equipped with active transport systems,
allowing them to take uppolyamines from the environ-
ment.2 The activity of the uptake system is under the
control of the intracellular free polyamine pool. Struc-
tural features, the enhanced requirement of tumor cells
for the polyamines, and the characteristics of the trans-
port system make the polyamines attractive vectors for
tumor targeting. The following aspects are important in
this regard:
As
a consequence, treatment with DFMO
increases the uptake rate of tumors, which read-
ily accumulate high amounts of polyamines.11ꢀ13
*Corresponding author. Tel.: +33-2-2323-4503; fax: +33-2-2323-
4608; e-mail: delcros@univ-rennes1.fr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00147-5

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B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
All these properties have led to the design of polyamine
derivatives for tumor targeting.2 In particular, the
polyamine transport system has been targeted for the
selective accumulation of various isotopes in cancer cells
for therapy or tumor imaging. BNCT is a method for
the treatment of brain tumors and melanomas. It
requires the accumulation of the boron isotope 10B
within the tumor cells and relies on the cytotoxic action
of energy-rich a particles formed by irradiation of the
10B isotope with low energy neutrons (for reviews, see
refs 14 and 15). o-Carborane cages attached to the pri-
mary or secondary amino groups of spermidine and
spermine have been synthesized for BNCT, but they
were too toxic for clinical use.16,17 Recently, we reported
the synthesis of boron-containing benzyl polyamines.18
These derivatives, which exhibit low cytotoxicity, affi-
nity for DNA, allow efficient boron accumulation in
tumor cells in vitro. They are potential candidates for
BNCT. Fluorinated putrescine or spermidine derivatives
were also designed and evaluated for Positron Emission
Tomography (PET), a non-invasive tumor-imaging mod-
ality.18ꢀ22 Finally, iodine-substituted diamines were stud-
ied for application in 125I scintigrap hy2.3,24
accumulation of the derivatives were studied in CHO
cells and in a polyamine transport deficient CHO
mutant cell line (CHO-MG).29 Studies were also per-
formed on 3LL cells (Lewis lung carcinoma) because of
our past experience with close related compounds on
this cell line18 and the availability of the in vivo model
in our laboratory.30
Results
Chemical syntheses
Synthesis of the target compound (Z)-1,4-diamino-
2-butene dihydrochloride (1, Z-DAB) is shown in
Scheme 1. The (Z)-1,4-dichloro-2-butene was employed
as starting material. This reagent reacted with sodium
azide at room temperature in DMSO to give the diazide
5 in quantitative yield. Due to the suspected instability
of this intermediate, it was used directly without further
purification in the next step. Reduction of the azido
groups by an in situ Staudinger reaction31 gave the
desired product 1 in 76% overall yield.
(Z)-1,4-Diamino-2-butene (Z-DAB, Fig. 1) is an unsat-
urated derivative of putrescine. Previous reports have
demonstrated that 1,4-diamino-2-butene (DAB, isomer
not specified) is a substrate of spermidine synthase,
which converts DAB into unsaturated spermidine.25,26
Only the (cis) isomer of unsaturated spermidine can be
converted into the spermine homologue by spermine
synthase.25,27 However, in contrast to spermidine or
spermine, none of the higher homologues of Z-DAB is
degraded along the retroconversion pathway, which
involves acetylation by spermidine/spermine N1-acetyl
transferase and oxidative splitting by polyamine
oxidase.27 DAB and its higher homologues reverse the
cytostatic effect of DFMO.25,27 Z-DAB competitively
inhibits the uptake of putrescine,28 which suggests that it
may use the polyamine transport system. Hence, in highly
proliferating cells treated with DFMO, Z-DAB may be
efficiently trapped via the polyamine transport system.
(E)-2-Iodo-1,4-diamino-2-butene dihydrochloride (2, E-I-
DAB),(E)-2-fluoro-1,4-diamino-2-butene dihydrochloride
(3, E-F-DAB), and (Z)-2-{4-(5,5-dimethyl-[1,3,2]dioxa-
borinan - 2 - yl)phenyl}methyl - 1,4 - diamino - 2 - butene
dihydrochloride (4, Z-4-Bbz-DAB) were synthesized by
Scheme 1. Results and conditions: (a) NaN3, DMSO, 25 ꢁC; (b) PPh3,
HCl, 25 ꢁC, 76%.
Because of its metabolic and transport properties,
Z-DAB is a potential tumor-targeting vector. New
boron-, fluorine-, and iodine-containing derivatives of
Z-DAB (Fig. 1) were designed to specifically target
tumor cells for BNCT, PET, and scintigraphy, respec-
tively. Biological properties of these derivatives are
reported with special focus on interactions with the
polyamine transport system and metabolic pathways of
the polyamines. In particular, the cytotoxicity and cell
Scheme 2. Results and conditons: (a) (Boc)2O, Et3N, THF, rt, 83%;
(b) HSnBu3, PdCl2(PPh3)2, THF, rt, 98%; (c) I2, CH2Cl2, rt, 72%;
(d) 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetra-
fluoroborate), CH3CN, rt, 40%; (e) BrCH2Ar, Pd(PPh3)4, toluene,
reflux, 51%; (f) HCl (12 N), EtOH, rt, 2, 88%, 3, 80%, 4, 87%.
Figure 1. Structure of Z-DAB derivatives.

B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
2865
a versatile route illustrated in Scheme 2 from 1,4-dia-
mino-2-butyne dihydrochloride. Protection of the
amino groups was performed with (Boc)2O in THF
containing 2 equiv of Et3N. Hydrostannation of 6 in the
presence of a catalytic amount of Pd(PPh3)4 at room
temperature, the key step of our synthesis, afforded the
corresponding vinylstannane 7 in a 95% yield.32 The
reaction is highly stereoselective, syn addition being
CHO than in CHO-MG cells (at least 2-fold higher). In
contrast, CHO and CHO-MG cells accumulated
equivalent amounts of E-I-DAB.
Effect on putrescine uptake by 3LL cells. In 3LL cells,
Z-DAB and Z-4-Bbz-DAB were weak inhibitors of
putrescine uptake with IC50 values of 250 and 535 mM,
respectively (Table 1). In contrast, up to 1 mM, E-I-
DAB had no effect on putrescine transport.
3
deduced from the small J coupling constant between
Sn and Ha (60 Hz). On reaction with I2 at room tem-
perature, 7 gave(E)-2-iodo-1,4-di-(tert-butyloxycarbonyl)-
amino-2-butene 8 in 72% yield with total conservation
of stereochemistry. Electrophilic fluorination of vinyl-
stannane 7 was performed using commercially available
1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane
bis(tetrafluoroborate).33(E)-2-Fluoro-1,4-di-(tert-butyl-
oxycarbonyl) amino-2-butene 9 was obtained in 40%
yield, accompanied by 20% of protonolysis of the
vinylstannane. Cross-coupling reaction of 7 with the
2,2-dimethylpropane-1,3-diol ester of the [4-bromo-
methyl)phenyl]boronic acid34 in the presence of palla-
dium catalyst provided the corresponding 2-substituted-
1,4-di-(tert-butyloxycarbonyl)amino-2-butene 10 in
51% yield. Deprotection of the geometrically pure
dehydroputrescines derivatives 8, 9, 10 was achieved
with aqueous concentrated hydrochloric acid in ethanol.
Effect on the growth of 3LL cells. The effects of Z-DAB
and its derivatives on the growth of 3LL cells were
similar to those observed on CHO cells. While Z-DAB
had very little effect on cell growth (about 25% inhibi-
tion at 1 mM), Z-4-Bbz-DAB and E-I-DAB inhibited
cell growth with IC50 values around 300 mM (Table 1).
At concentrations exceeding the IC50 values of Z-4-Bbz-
DAB and E-I-DAB, cells were swelling and granular
vacuoles developed in the cytoplasm. The same mor-
phological changes were observed in CHO cells.
The influence of DFMO on the cytotoxicity of Z-DAB
derivatives was studied by monitoring 3LL cell growth
after a 24 h pre-incubation with 5 mM DFMO, followed
by a 48 h exposure to the drugs. As shown in Fig. 2, all
derivatives, as free spermidine, antagonized DFMO-
induced cell growth arrest. They supported growth of
putrescine- and spermidine-depleted cells. Total rever-
sion of the DFMO effect required at least 50 times
higher concentrations of Z-DAB, than of spermidine.
Z-4-Bbz-DAB and E-I-DAB also antagonized the effect
of DFMO but less efficiently than Z-DAB.
Biological studies
Effect on the growth of CHO and polyamine transport
deficient CHO-MG cells. Upto 1 mM, Z-DAB had no
effect on the growth of CHO and CHO-MG cells.
Z-4-Bbz-DAB and E-I-DAB showed moderate cytotoxic
effects on CHO cells with IC50 values of 340 and 90 mM,
respectively (Table 1). CHO-MG cells were significantly
less sensitive to Z-4-Bbz-DAB (IC50 value around 1 mM)
than CHO cells. In contrast, E-I-DAB had similar growth
inhibitory effect on both CHO and CHO-MG cells.
Accumulation and metabolism of Z-DAB derivatives in
3LL cells. 3LL cells exposed for 24 h to Z-DAB accu-
mulated 2-fold higher amounts of the drug than after a
4 h incubation (Table 3). DFMO-pretreated cells
exposed to Z-DAB for 4 h accumulated 2-fold higher
amounts of the drug than control cells (p<0.05). After a
24 h exposure, drug levels of Z-DAB were similar in
DFMO-pretreated and control cells. Co-treatment with
an inhibitor of spermidine synthase [trans-4-methyl-
cyclohexylamine (4MCHA)]35,36 resulted in increased
intracellular levels of Z-DAB in both control and
DFMO-pretreated cells (p<0.05).
Accumulation in CHO and CHO-MG cells. Accumu-
lation of Z-DAB derivatives was studied after a 1- or
4-h exposure to 250 mM (Table 2). Intracellular levels of
Z-DAB and Z-4-Bbz-DAB were significantly higher in
Table 1. Inhibitory effects of (Z)-1,4-diaminobutene derivatives on
cell growth, polyamine uptake, and ODC activitya
HPLC chromatograms revealed the presence of two
Z-DAB metabolites (M1 and M2 with retention times of
20.7 and 25.3 min, respectively), which eluted close to
Cell growthb
ODCc
[
14C] putrescine
uptaked
3LL CHO CHO-MG
IC50 (mM)
IC50 (mM)
IC50 (mM)
Table 2. Accumulation of (Z)-1,4-diaminobutene derivatives in CHO
and in polyamine transport deficient CHO-MG cells
Z-DAB
Z-4-Bbz-DAB
E-I-DAB
>1000 >1000 >1000
30
>200e
80
260
535
>500f
298
380
338
87
1003*
108
CHO
CHO-MG
nmol/mg proteina
aAll data are expressed as mean (SD<20%).
bEffect on cell growth was measured after a 72-h incubation period.
cInhibition of ODC activity was determined in freshly prepared
extracts of kidneys of testosterone-stimulated DBA/2 mice.
dEffect of Z-DAB derivatives on putrescine uptake was determined in
3LL cells, after a 20-min incubation with Z-DAB derivatives and [14C]
putrescine (0.5 mCi; 10 mM).
Incubation timeb
Z-DAB
Z-4-Bbz-DAB
E-I-DAB
1 h
4 h
1 h
4 h
8.6ꢂ1.4
1.1ꢂ0.2
12.8ꢂ0.6
9.6ꢂ1.1
3.0ꢂ0.2
10.1ꢂ1.8
3.9ꢂ0.4* 4.5ꢂ1.1*
b.d.l.*
9.8ꢂ1.0
1.5ꢂ0.4*
8.4ꢂ1.7
aCompounds were determined by HPLC. All data are expressed as
meanꢂSD. b.d.l., below detection limit (<0.2 nmol/mg of protein).
bCells were treated with 250 mM derivative for 1 or 4 h.
eA 200-mM dose induced a 35% inhibition of ODC activity.
fA 500-mM dose provoked a 18% inhibition of putrescine uptake.
*Significantly different from values in CHO cells (p<0.05).
*Significantly different from values in CHO cells (p<0.05).

2866
B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
Figure 2. Effect of Z-DAB derivatives on the growth of 3LL cells in absence or after pretreatment with DFMO. 24 h after seeding, 5 mM DFMO
was added to the 3LL cell culture medium for 24 h, followed by 48-h exposure to spermidine or to a derivative of Z-DAB. The relative cell growth
rate was calculated from the value of cell growth of the corresponding control cells cultured in absence (OD540nm=2.01) or in presence
(OD540nm=0.57) of DFMO.
Table 3. Accumulation of (Z)-1,4-diaminobutene in 3LL cells and their effect on polyamine metabolism
Treatment
Time of
exposure (h)
Putrescinea
Spermidinea
Sperminea
Z-DABa
Z-DAB
derivativea,b
Peak 1c
Peak 2c
None
DFMOd
—
—
—
—
4
0.94
b.d.l.e
5.79
9.10
b.d.l.e
1.69
5.91
4.25
6.98
4.65
6.83
7.43
6.53
2.32
6.04
4.17
7.88
4.80
4.80
4.21
4.00
4.93
3.89
3.03
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4MCHAf
DFMOd/4MCHAf
Z-DABg
Z-DABg/DFMOd
Z-DABg
Z-DABg/DFMOd
Z-DABg/4MCHAf
Z-DABg/DFMOd/4MCHAf
Z-4-Bbz-DABh
Z-4-Bbz-DABh/DFMOd
Z-4-Bbz-DABh/4MCHAf
Z-4-Bbz-DABh/DFMOd/4MCHAf
E-I-DABg
E-I-DABg/DFMOd
E-I-DABg
E-I-DABg/DFMOd
b.d.l.e
0.20
0.19
b.d.l.e
9.07
1.49
—
—
3.56
b.d.l.e
67.09
117.40
171.14*
b.d.l.e
b.d.l.e
—
b.d.l.e
b.d.l.e
14.44
141.91*
b.d.l.e
b.d.l.e
—
4
7.07*
7.80
7.20
24
24
24
24
24
24
24
24
4
0.16
0.11
2.61
0.14
0.19
b.d.l.e
7.16
b.d.l.e
0.10
0.05
3.91
3.07
0.81
0.27
5.44
b.d.l.e
1.84
b.d.l.e
6.42
0.27
11.84
23.80*,y
Traces
Traces
n.m.j
Traces
b.d.l.e
b.d.l.e
b.d.l.e
b.d.l.e
1.61 (1.82)i
1.49 (1.49)i
1.53
1.70
8.15
8.72
6.93
8.05
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4
24
24
0.17
0.02
4.88
0.03
aData are mean of values expressed as nmol/mg protein measured by HPLC (SD<20%).
bZ-DAB derivative measured is Z-4-Bbz-DAB or E-I-DAB.
cData are expressed as area 103/mg protein of additional peaks detected by HPLC at 20.7 and 25.3 min, respectively, for peaks 1 and 2 (mean).
dCells were incubated with 5 mM DFMO for 24 h before derivative addition.
eBelow detection limit: <0.05 nmol/mg protein for putrescine and spermidine; <0.2 nmol/mg protein for Z-DAB and its derivatives; <5.103/mg protein for
peak 1 and 2.
fCells were incubated with 250 mM 4MCHA for 24 h before derivative addition.
gCells were treated with 250 mM derivative for 24 h.
hCells were treated with 138 mM derivative for 24 h.
iValues of boron measurement by ICP-AES method.
jNot measurable because of the broad putrescine peak.
*Significantly different from values in control cells (p<0.05); y significantly different from values in 4MCHA-treated cells (p<0.05).
spermidine and spermine (retention times of 20.6 and
25.1 min), respectively. M1 was detected as early as 4 h
after Z-DAB addition in DFMO-pretreated cells, but
not in control cells. After 24h, both M1 and M2 were
detected in 3LL cells. Higher amounts of these metabolites
were measured in cells pre-treated with DFMO (p<0.05).
4MCHA prevented the formation of both metabolites.
During a 4-h exposure, 3LL cells accumulated 2-fold
higher amounts of E-I-DAB compared with Z-DAB
( p<0.05). However, longer exposure did not result in
enhanced intracellular level of E-I-DAB. Cells pre-treated
with DFMO accumulated similar amounts than control
cells. HPLC chromatograms did not show any evidence
of metabolization of E-I-DAB.

B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
2867
Z-4-Bbz-DAB was poorly taken up by 3LL cells. After a
24-h exposure, 3LL cells accumulated less Z-4-Bbz-DAB
than Z-DAB. Neither DFMO nor 4MCHA affected the
accumulation of the derivative. Boron concentrations
determined by Inductively Coupled Plasma-Atomic
Emission Spectrometry (ICP-AES) were similar to the
amount of drug measured by HPLC. Traces of Z-DAB
were detected in all cells treated with Z-4-Bbz-DAB.
dine reduced the competition for spermidine synthase
and favored the conversion of the spermidine derivative.
In contrast, E-I-DAB and Z-4-Bbz-DAB did not cause
formation of detectable amounts of a new compound.
Previous work demonstrated the stringency of spermi-
dine synthase for the 1,4-butanediamine moiety.26
However, Sarhan and coll.25 showed that substituents
on the four-carbon chain were tolerated within certain
limits, with a better tolerance for substituents in the 2-
position. The bulky iodine atom and the boron-con-
taining benzyl moiety prevent interaction of the deriva-
tives with the synthase due to steric hindrance. In the
case of Z-4-Bbz-DAB, trace amounts of Z-DAB were
detected in 3LL cells, suggesting that the compound
may lose to some extent its boron-containing sub-
stituent. In a recent study, a similar loss was observed
from a derivative of spermidine with the same boron-
containing moiety attached to the central amino group
of spermidine.18 Previous studies have demonstrated
that Z-DAB was a substrate of diamine oxidase.37 We
also observed that this enzyme (data not shown) oxi-
dized the iodine-, but not the boron-containing deriva-
tive of Z-DAB. However, aminoguanidine, an inhibitor
of diamine oxidase, did not modify the cytotoxicity or
the accumulation of Z-DAB and E-I-DAB in 3LL cells
(data not shown).
Effect on polyamine pools. In 3LL cells, all three deriva-
tives induced a decrease of putrescine pools within 24 h
by approximately 80% of the control values, and a
reduction of spermidine levels between 40 and 60% of
control values (Table 3). Spermine levels were only
affected by treatment with E-I-DAB, which caused a
35% decrease.
DFMO-treated cells exposed for 24 h to Z-DAB exhib-
ited a dramatic decrease of their spermine pools, along
with a significant accumulation of spermidine. Addition
of E-I-DAB or Z-4-Bbz-DAB had no effect on the
polyamine pools of DFMO-treated cells.
Inhibition of spermidine synthase by 4MCHA in 3LL
cells led to a 6-fold increase of putrescine pools and a
5-fold decrease of spermidine levels. Addition of Z-DAB
to these 4MCHA-treated cells reduced amplification of
putrescine levels, while spermidine pools were more
profoundly diminished. Z-4-Bbz-DAB did not modify
the effect of 4MCHA in 3LL cells.
2. Polyamine patterns were modified in 3LL cells treated
with Z-DAB (Table 3). In agreement with previous
studies,38 Z-DAB was found to be a potent inhibitor of
ODC (Table 1) although less potent than the (E)-iso-
mer.39,40 Depletion of putrescine and spermidine pools
may result from ODC inhibition. However, a similar
effect was observed in cells treated with Z-4-Bbz-DAB, a
much weaker ODC inhibitor. This suggests that other
mechanisms may be involved in polyamine depletion.
Because of the polyamine-like structure of Z-DAB and
its derivatives down-regulation of the expression of
ODC by feedback mechanisms have to be considered.
Both Z-DAB and E-I-DAB inhibited ornithine dec-
arboxylase, with IC50 values of 30 and 80 mM, respec-
tively (Table 1). Although less efficient, Z-4-Bbz-DAB
also decreased ODC activity (35% inhibition at 200 mM).
Discussion
Three derivatives of Z-DAB carrying an atom of boron,
fluorine, or iodine, were synthesized for potential appli-
cation in cancer therapy or imaging. Biological studies
with the fluorinated derivative could not be performed
because of the instability of the drug. The metabolic and
transport properties of the other derivatives are dis-
cussed in comparison with the features of their parent
compound.
In DFMO-pretreated cells, the profound depletion of
spermine pools induced by Z-DAB can be explained by
the effective competition of Z-DAB and its spermidine
analogues with the natural polyamines for the syn-
thases. Such competitions also account for the presence
of elevated amounts of putrescine and spermidine in
DFMO-pretreated cells.
1. Z-DAB was significantly metabolized in 3LL cells.
Accumulation of Z-DAB was paralleled by the appear-
ance of two new compounds. It is known that both Z-
DAB and its spermidine homologue are substrates of
spermidine and spermine synthase, respectively, although
the rates of reaction are reduced and the KM are increased,
as compared with the natural substrates.25ꢀ27 In 3LL
cells, inhibition of spermidine synthase with 4MCHA
prevented the formation of both metabolites and
increased the intracellular levels of Z-DAB. Based on
these observations and on their chromatographic beha-
vior, the metabolites were tentatively identified as unsa-
turated spermidine and spermine analogues. In DFMO-
pretreated cells, the formation of the spermine analogue
was greatly enhanced because the depletion of spermi-
3. Z-DAB antagonized the growth inhibitory effect of
DFMO in 3LL cells, as has previously been described in
chick embryos.25 The partial repletion of putrescine and
spermidine pools by Z-DAB and its spermidine-like
aminopropylation derivative may contribute to the
reversion of the DFMO effect. However, it is known
that certain homologues of putrescine and spermidine
are able to reverse the growth arrest produced by expo-
sure of tumor cells to DFMO, probably by their ability
to occupy polyamine binding sites and prevent vital
processes of cell division.41,42 This property is likely to
be shared by Z-DAB and its spermidine analogue25,27 as
well as by Z-4-Bbz-DAB, which, in the absence of
putrescine and spermidine, also reversed efficiently the
cytostatic effect of DFMO. Presumably, the bulky

2868
B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
iodine atom diminishes the affinity of E-I–DAB for the
polyamine binding sites by steric hindrance as is evi-
denced by its poor ability to reverse the DFMO effect.
and spermidine pools by DFMO. This may restrain its
potential for BNCT. In vivo studies will be performed
to fully evaluate the clinical interest of Z-4-Bbz-DAB for
BNCT.
4. Previous studies have shown that Z-DAB is a weak
competitor of putrescine uptake by rat lung.28 Its low
affinity was imputed to the unsaturation, which reduces
the flexibility and the distance between the amino
groups of the diamine. Z-DAB was also found to be a
weak competitor for putrescine uptake in 3LL cells.
Substituents in the 2-position further reduced the affi-
nity of Z-DAB for the polyamine transport system.
However, despite their low affinity, Z-DAB and Z-4-
Bbz-DAB use the polyamine transport system as was
evidenced by the greater accumulation in CHO cells
than in CHO-MG mutant cells, which lack the poly-
amine transport system. The up-regulation of the poly-
amine transport system in polyamine-depleted 3LL cells
induced a 2-fold higher accumulation of Z-DAB after a
4 h-exposure. Because of the conversion of Z-DAB into
its spermidine and spermine analogues, the enhanced
accumulation was not obvious after a 24 h-exposure.
DFMO-induced upregulation of the transport activity
did not result in enhanced accumulation of Z-4-Bbz-
DAB. We have discussed earlier the possibility that this
compound may induce feedback regulatory mechanisms
leading to ODC inhibition. It is known that one reg-
ulatory pathway of polyamine transport involves the
induction of antizyme, which inhibits ODC but also the
activity of the polyamine uptake.43 Such effect could
antagonize the DFMO-induced upregulation of the
transport system and prevent a higher accumulation of
the derivative. In contrast to Z-DAB and its boron-
containing derivative, E-I-DAB was not transported by
the polyamine transport system probably because of the
steric hindrance of the iodine atom.
Experimental
Chemicals
Usual laboratory chemicals were purchased from Merck
(Darmstadt, Germany) or Sigma Chemical Co. (St
Louis, MO, USA) and used directly without further
purification unless otherwise specified. [14C] putrescine
(118 mCi/mmol) and [14C] ornithine (61 mCi/mmol)
were from Amersham France (Les Ulis, France) and
Pico-Fluor 15 from Packard Instr. Chemical Operations
(Groningen, The Netherlands). DFMO was obtained
from Ilex Oncology (San Antonio, TX, USA). Data are
given as mean valuesꢂSD of three or more experi-
ments. Comparisons between means were made using
the Student’s t-test assuming significance at p<0.05.
Chemical syntheses
General procedures. Tetrahydrofuran (THF) was dis-
tilled from deepblue solutions of sodium/benzophenone
ketyl prior to use. Methylene chloride was shaken with
concentrated H2SO4, dried over K2CO3, and distilled.
All melting points were determined on a Kofler appa-
ratus and are uncorrected. NMR spectra were recorded
on a 200- or 300-MHz spectrometer operating in the
Fourrier transform mode. 13C NMR spectra were
obtained with broadband proton decoupling. Chemical
shifts were expressed as d value with TMS (tetra-
methylsilane) or CCl3F (for 19F NMR) as internal
standard. For 11B NMR, the chemical shifts are in ppm
.
High tumor/normal tissue ratios are a requirement for
drugs used in BNCT as well as for probes used in
imaging. The rationale behind the synthesis of new (Z)-
1,4-diamino-2-butene derivatives was to developcom-
pounds sharing the ability of the natural polyamines to be
taken upby cells via the polyamine transport system. If
derivatives are substrates of this system, the DFMO-
induced upregulation of the activity of the polyamine
transport system should allow their preferential accu-
mulation in tumor cells. Z-DAB is a potential vector
because it shares transport properties with the natural
polyamines. However, the substitution on the 2-position
may induce profound alterations of its properties. An
iodine atom in 2-position abolished its ability to serve as
a substrate for the polyamine transport system. There-
fore, E-I-DAB is not useful for selective tumor imaging.
In contrast, substitution with the boron-containing
benzyl moiety did not prevent the ability of the deriva-
tive to use the polyamine transport system. Boron
delivery by Z-4-Bbz-DAB into 3LL cells (around 1.4 mg
of B/g of 3LL cells) was of the same order of magnitude
as that achieved by boronated N-benzyl polyamines, or
by borocaptate sodium (BSH) or (l)-p-borono-
phenylalanine (l-BPA), the two reference compounds
used in BNCT.18 Nevertheless, Z-4-Bbz-DAB accumu-
lation could not be enhanced by depletion of putrescine
relative to BF3 OEt2. HRMS were obtained with a
Varian MAT 311 mass spectrometer (Centre Regional
de Mesures Physiques de l’Ouest). Microanalyses were
performed at the Central Laboratory for Analysis,
CNRS, Lyon (France). Silica gel 60F254 was used for
column chromatography.
(Z)-1,4-Diazido-2-butene (5). To a stirred solution of
(Z)-1,4-dichloro-2-butene (0.5 mL, 4.75 mmol) in
DMSO (12 mL), NaN3 (2.77 g, 42.7 mmol) was added
and stirring was continued for 10 min at room tem-
perature. Water (10 mL) was added to the solution,
which was then extracted with AcOEt (3Â 15 mL). The
extract was dried (MgSO4), concentrated to afford
crude product 5 as a colorless oil (400 mg) which was
1
used without further purification. H NMR (200 MHz,
CDCl3) d 3.83 (m, 4H), 5.83 (m, 2H). 13C NMR
(50 MHz, CDCl3) d 46.9, 127.8.
(Z)-1,4-Diamino-2-butene dihydrochloride (Z-DAB, 1).
To a solution of the diazide 5 (230 mg) in 3 mL tetra-
hydrofuran was added a solution of triphenylphosphine
(0.88 g, 3.35 mmol) in tetrahydrofuran (2 mL) with stir-
ring. After 1 h at room temperature, the reaction mix-
ture was heated at 50 ꢁC for 3 h. An aqueous solution of
hydrochloric acid (1 mL, 3 M) was added and the tem-

B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
2869
perature was maintained at 50 ꢁC for another 1 h. The
solvents were removed in vacuo and the residue was
partitioned between methylene chloride (10 mL) and
water (10 mL). The aqueous phase was extracted with
methylene chloride (3 Â 10 mL). Removal of the water
under reduce pressure (40 ꢁC) gave the diamine dihy-
drochloride 1 as a colorless solid (200 mg, 76%), which
was recrystallized from a mixture of methanol–ether (1/
1). Mpof N,N0-dibenzoyl derivative: 177–178 ꢁC [lit,44
mp177.5–178.5 C]. H NMR (300 MHz, D2O) d 3.55
(d, J=5.5 Hz, 4H), 5.69 (t, J=5.5 Hz, 2H). 13C NMR
(75.5 MHz, D2O) d 38.7, 129.5. Anal. calcd for
C4H12N2Cl2: C, 30.21; H, 7.60; N, 17.61. Found: C,
30.25; H, 7.58; N, 17.63.
temperature, the reaction mixture was washed twice
with aqueous sodium bisulfite (10%) and then once with
5 mL of a 10% aqueous solution of potassium fluoride
to convert tributyltin iodide into tributyltin fluoride.45
The insoluble tributyltin fluoride was eliminated by fil-
tration, and the organic phase was decanted and dried
over magnesium sulfate. After evaporation of methylene
chloride, the residue was purified by flash chroma-
tography to afford the product 8 as a colorless oil
(52 mg, 72%). Rf=0.25 (ethyl acetate/heptane=2:8). 1H
NMR (500 MHz, CDCl3) d 1.43 (s, 9H), 1.46 (s, 9H),
3.80 (d, J=7.0 Hz, 2H), 4.18 (m, 2H), 5.15 (br s, 1H),
5.40 (br s, 1H), 6.38 (t, J=7.0 Hz, 1H). 13C NMR
(50 MHz, CDCl3) d 28.4, 39.3, 46.4, 79.4, 80.1, 125.9,
139.7, 155.6, 155.8. Anal. calcd for C24H25N2O4I: C,
54.15; H, 4.73; N, 5.26. Found: C, 54.18; H, 4.74; N,
5.27.
ꢁ
1
1,4-di-(tert-Butoxycarbonyl)amino-2-butyne (6). To a
stirred solution of 1,4-diamino-2-butyne dihydro-
chloride (2 g, 12.7 mmol) and Et3N (3.9 mL, 28 mmol) in
THF (13 mL) cooled to 0 ꢁC was added dropwise a
solution of (Boc)2O (6.11 g, 28 mmol) in THF (6 mL).
The stirring was continued for 12 h at room temperature
and the solvent was evaporated under vacuum. The
residue was taken upin Et 2O (100 mL) and the solution
was washed with a 0.01 N HCl solution (25 mL), then
with water (25 mL), and finally with a 5% NaHCO3
solution. The organic layer was dried over MgSO4, fil-
tered, and evaporated under vacuum to give 6 (3 g,
83%) as a colorless solid which was used without fur-
(E)-2-Fluoro-1,4-di-(tert-butyloxycarbonyl)amino-2-butene
(9). 1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]-
octane bis(tetrafluoroborate) (370 mg, 1.04 mol) was
added to a solution of vinyl stannane 7 (300 mg,
0.52 mmol) in dry CH3CN (6 mL) and the stirring was
continued at room temperature under argon atmo-
sphere. After 6 days, the yellowish mixture was poured
onto a plug of silica gel in a fritted glass funnel and
eluted with hexane. The fractions containing product
were combined and evaporated by rotary evaporator.
Flash chromatography on silica gel afforded 64 mg of
the product 9 as colorless oil (40%). Rf=0.20 (ethyl
ꢁ
1
ther purification. Mp 120–121 C; H NMR (200 MHz,
CDCl3) d 32;1.41 (s, 18H), 3.91 (d, J=5.4 Hz, 4H), 4.73
(bs, 2H); 13C NMR (50 MHz, CDCl3) d 28.3, 30.5, 79.2,
79.9, 155.4.
1
acetate/heptane=2:8). H NMR (200 MHz, CDCl3) d
1.42 (s, 9H), 1.44 (s, 9H), 3.73 (d, J=8.6 Hz, 2H), 3.88
(d, J=21.9 Hz, 2H), 5.11 (m, 2H), 5.30 (dt, J=8.6,
19.0 Hz, 1H). 13C NMR (50 MHz, CDCl3) d 28.4, 37.2,
37.9, 79.4, 80.1, 106.0 (J=21.5 Hz), 128.8 (J=245.7 Hz),
155.9. 19F NMR (282 MHz, CDCl3) d ꢀ107.73. Anal.
calcd for C14H25N2O4F: C, 55.25; H, 8.28; N, 9.20.
Found: C, 55.30; H, 8.32; N, 9.22.
(E) - 2 - (Tributylstannyl) - 1,4 - di(tert - butoxycarbonyl)-
amino-2-butene (7). To a THF solution (10 mL) of 6
(1 g, 3.52 mmol) containing PdCl2(PPh3)2 (70 mg,
0.02 equiv) was added tributyltin hydride (1.42 mL,
5.28 mmol) dropwise with a syringe over a period of ca.
5–10 min. The originally light yellow solution abruptly
turned orange-brown. After 30 min, an additional
0.2 equiv of tributyltin hydride was added, and stirring
was continued for about 10 min. THF was then evapo-
rated. The oily residue, which was now contaminated
with black palladium impurities, was purified by column
chromatography on silica gel (210–400 mesh), initially
with 100% heptane to remove tin byproducts, followed
by ethyl acetate/heptane (20/80). The desired vinyl-
stannane 7 was obtained as a colorless oil (1.98 g, 98%);
Rf=0.25 (ethyl acetate/heptane=1:9). 1H NMR
(200 MHz, CDCl3) d 0.89 (m, 18H), 1.27 (m, 9H), 1.44
(s, 9H), 1.45 (s, 9H), 3.80 (d, J=6.4 Hz, 2H), 3.94 (m,
2H), 4.75 (br s, 1H), 4.88 (br s, 1H), 5.63 [t, J=6.4 Hz,
1H, J(117SnH)=62 Hz, J(119SnH)=64 Hz]. 13C NMR
(50 MHz, CDCl3) d 9.7, 13.7, 27.4, 28.4, 29.1, 38.1, 42.0,
77.1, 77.7, 138.4, 145.0, 155.6, 155.9. Anal. calcd for
C26H52N2O4Sn: C, 54.27; H, 9.11; N, 4.87. Found: C,
54.30; H, 9.13; N, 4.90.
(Z)-2-{4-(5,5-Dimethyl-[1,3,2]dioxaborinan-2-yl)phenyl}-
methyl-1,4-di-(tert-butyloxycarbonyl)amino-2-butene (10).
To a schlenk was successively added 40 mg of tetra-
kis(triphenylphosphine)palladium (0.035 mmol), 16 mL
of dry toluene, 615 mg of 2-[4-(bromomethyl)phenyl]-
5,5-dimethyl-1,3,2-dioxaborane (1.73 mmol) and 1 g of
vinylstannane 7. The resulting solution was stirred at
reflux for 18 h, then cooled, partitioned between 30 mL
of ether and 30 mL of one-third saturated potassium
fluoride, and vigorously stirred for 30 min. The resulting
precipitate of tri-n-butylstannyl fluoride was removed
by gravity filtration, and the organic layer was sepa-
rated, washed with brine, and dried (MgSO4). The pro-
duct 10 was isolated by flash chromatography as
colorless oil (430 mg, 51%). Rf=0.3 (ethyl acetate/hep-
1
tane=3:7). H NMR (200 MHz, CDCl3) d 1.02 (s, 6H),
1.44 (s, 18H), 3.37 (s, 2H), 3.72 (d, J=6.2 Hz, 2H),
3.74–3.78 (m, 6H), 4.80 (br s, 1H), 5.15 (br s, 1H), 5.40
(m, 1H), 7.16 (d, J=7.6 Hz, 2H), 7.74 (d, J=7.6 Hz,
2H). 13C NMR (50 MHz, CDCl3) d 21.9, 28.4, 31.9,
37.6, 39.0, 42.2, 72.3, 79.0, 127.7, 128.3, 134.1,
139.0, 142.0, 156.0. Anal. calcd for C26H41N2O6B:
C, 63.94; H, 8.46; N, 5.74. Found: C, 63.93; H,
8.47; N, 5.77.
(E)-2-Iodo-1,4-di-(tert-butyloxycarbonyl)amino-2-butene
(8). A 0.17 M methylene chloride solution of iodine
(1 mL) was added, over a period of 15 min, into a stirred
solution of 100 mg (0.17 mmol) of 7 in methylene chlor-
ide (1 mL) under argon atmosphere. After 1 h at room

2870
B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
General procedure of amino-group deprotection
solution of N-tert-butoxycarbonyl derivative
a scintillation cocktail (Pico-Fluor 15). Protein content
was assayed on 0.1 mL of the cellular suspension by
reaction with the Folin Phenol reagent.46
A
(3 mmol) in ethanol (26 mL) was treated by a 12 M
aqueous solution of hydrochloric acid (6.4 mL) at room
temperature overnight. After removal of the solvent, the
mixture was diluted in water (10 mL) and washed with
ether. The aqueous solution was concentrated and dried
under high vacuum.
In vitro evaluation of drugs cytotoxicity/cytostasy. Cell
growth effect was assayed in 96-wells plates (Becton
Dickinson, Oxnard, CA, USA). Initial density was
1000 cells/well for 3LL, CHO and CHO-MG cells. Cell
growth rates were determined by measuring formazan
formation from 3-[4,5-dimethylthiazol-2-yl)]2,5-di-
phenyltetrazolium bromide (MTT) using a Titertek
Multiskan MCC/340 microreader (Labsystems, Life
Sciences Interactions, Cergy-Pontoise, France) for
absorbance measurements.47
(E)-2-Iodo-1,4-diamino-2-butene dihydrochloride (E-I-
1
DAB, 2). Hygroscopic colorless solid; 88%. H NMR
(200 MHz, D2O) d 3.61 (d, J=7.4 Hz, 2H), 3.91 (s, 2H),
6.59 (t, J=7.4 Hz, 1H). 13C NMR (50 MHz, D2O) d
38.5, 44.8, 96.2, 139.0. Anal. calcd for C4H11N2Cl2I: C,
16.86; H, 3.89; N, 9.83. Found: C, 16.92; H, 3.93; N, 9.87.
Accumulation of DAB derivatives in cells. Cells were
cultured in tissue culture flasks at an initial density of
5Â104 /mL. Unless stated otherwise, drugs were added
24 h after seeding. Cells were washed three times with
phosphate-buffered saline and harvested using trypsine/
EDTA.
(E)-2-Fluoro-1,4-diamino-2-butene dihydrochloride (E-F-
1
DAB, 3). Amorphous solid; 80%. H NMR (200 MHz,
D2O) d 3.52 (d, J=8.3 Hz, 2H), 3.79 (d, J=19.4 Hz,
2H), 5.52 (dt, J=8.3, 18.9 Hz, 1H). 13C NMR (50 MHz,
D2O) d 37.6, 38.0, 107.5 (J=246.2 Hz), 128.0. 19F NMR
(282 MHz, D2O) d ꢀ102.69. HRMS (EI) calcd for
C4H9N2F (M+) 104.0750, found 104.0751.
Determination of polyamines and of (Z)-DAB derivatives
by HPLC. Cells were disrupted by sonication in 0.5 mL
perchloric acid (0.2 N). After centrifugation (2500g), the
supernatant was used for HPLC analysis. Protein content
was determined on cell pellets according to Lowry et al.46
(Z)-2-{4-(5,5-Dimethyl-[1,3,2]dioxaborinan-2-yl)phenyl}-
methyl-1,4-diamino-2-butene dihydrochloride (Z-4-Bbz-
1
DAB, 4). Amorphous solid; 87%. H NMR (200 MHz,
D2O) d 0.84 (s, 6H), 3.36 (s, 4H), 3.60 (s, 2H), 3.67 (s,
2H), 3.78 (d, J=7.3 Hz, 2H), 5.69 (t, J=7.3 Hz, 1H).
13C NMR (50 MHz, D2O) d 20.7, 37.0, 37.7, 40.7, 68.4,
126.0, 129.2, 134.6, 138.1, 140.6. 11B NMR (96 MHz,
D2O) d 26.5. HRMS (EI) calcd for C16H25N2O2B (M+)
288.2009, found 288.2010.
Determination of polyamines, Z-DAB, and E-I-DAB.
Natural polyamines, Z-DAB, and E-I-DAB were sepa-
rated by HPLC, after reaction with dansyl chloride, as
previously described.48 The following gradient was used
for separation of the dansyl derivatives: 0 min (10% B);
15 min (50% B); 25 min (100% B); 29 min (100% B);
30 min (10% B); 33 min (10% B). Retention times of
Z–DAB, p utrescine,E-I-DAB, spermidine, and spermine
were 13.3, 14.0, 14.8, 20.6, and 25.1min, respectively.
Biological studies
Cells. 3LL murine Lewis lung carcinoma, CHO, and
CHO-MGR (provided by Dr. W. Flintoff, University of
Western Ontario) cells were cultured in RPMI 1640
medium (Eurobio, Les Ulis, France) supplemented with
10% fetal calf serum, 2 mM glutamine, streptomycin
(100 mg/mL) and penicillin (100 U/mL) (Biomerieux,
Marcy l’Etoile, France). Two mg/mL l-proline was
added in the culture medium for CHO-MGR cells. Cells
were grown at 37 ꢁC under a humidified 5% CO2 atmo-
sphere. As other diamines, Z-DAB and its derivatives
were not substrates of bovine serum amine oxidase
(BSAO). This allows the study of the drugs to be
carried out in culture medium with serum and without
the addition of aminoguanidine, an inhibitor of
BSAO.
Determination of polyamines and Z-4-Bbz-DAB. Poly-
amines and Z-4-Bbz-DAB were determined by separa-
tion of the ion pairs formed with n-octanesulfonic acid
on a reversed-phase column, reaction of the column
effluent with o-phtalaldehyde and N-acetylcysteine, and
monitoring of fluorescence intensity, as previously
described.49 Retention times of putrescine, Z-DAB,
Z–4-Bbz-DAB, spermidine and spermine were 14.9,
18.8, 22.0, and 26.2 min, respectively.
Boron measurements. 3LL cells were processed as pre-
viously described for boron analysis by ICP-AES.18
ICP-AES determinations of boron were done using the
249.773 nm line on a ICP atomic emission spectrometer
(JY 38 S, Jobin-Yvon ISA, Longjumeau, France).
Inhibition of putrescine uptake. 3LL cells were seeded in
12-well plates (Becton Dickinson, Oxnard, CA, USA)
24 h before the experiment (initial density=0.5 106 cells/
mL). Cells were then incubated for 20 min with 10 mM
[14C] putrescine (0.5 mCi) at 37 ꢁC (or 4 ꢁC for control of
passive transport) in the absence or presence of putres-
cine derivatives. After three washes with phosphate-
buffered saline, cells were dissolved in 0.5 mL of NaOH
0.1 N and radioactivity was determined on a 0.35 mL
aliquot of the cell suspension after mixing with 2 mL of
Determination of ODC activity. A previously estab-
lished [14C]CO2 trapping method was used with minor
modifications.48 Kidneys of testosterone-stimulated
DBA2 mice were homogenized in 20 volumes of ice-cold
Tris–HCl buffer (25 mM, pH 7.5) containing 1 mM
dithiothreitol, 0.1 mM EDTA and 0.02% Brij 35. After
a 15 min centrifugation at 25,000g, 190 mL aliquots of

B. Martin et al. / Bioorg. Med. Chem. 10 (2002) 2863–2871
2871
the supernatants were used per assay (total assay
volume 250 mL). Ornithine concentration per assay was
0.1 mmol (0.5 mCi). Blanks contained 5 mM DFMO in
order to inactivate ODC.
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We are indebted to Pr. W. Flintoff (University of Wes-
tern Ontario, London, Canada) for providing the CHO-
MG1 cells and to Pr. K. Samejima (Josai University,
Sakado, Saitama, Japan) for the generous gift of
4MCHA. We acknowledge the Ministere de la
Recherche et de la Technologie (MRT), the Centre
National de la Recherche Scientifique (CNRS), the
Association pour la Recherche sur le Cancer (A.R.C.),
the Ligue Nationale Contre le Cancer (L.N.C.C.—
Comites d’Ille-et-Vilaine, du Morbihan et des Cotes
d’Armor) and the Conseil Regional de Bretagne
(C.R.B.) for financial support and for fellowships to F.
Posseme (C.R.B.), B. Martin (L.N.C.C. and A.R.C.)
and C. Lebarbier (M.R.T.). Our sincere thanks are also
due to D. Desplanques and A. Papillon (Laboratoire
Interregional de la DGCCRF, Rennes, France) for giv-
ing us access to the ICP-atomic emission spectrometer
as well as for their appreciable advises and technical
assistance. We also gratefully acknowledge R. Havouis
for HPLC determinations.
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