Fosmidomycin analogues as inhibitors of monoterpenoid indole alkaloid production in Catharanthus roseus cells

Article abstract of DOI:10.1016/j.phytochem.2005.06.002

Substituted 3-[2-(diethoxyphosphoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)- ones were obtained by functionalization at 6-position with various substituents (aryl, vinyl, carbonyl chains) via reactions catalysed with palladium. We found that these new fosmidomycin analogues inhibited the accumulation of ajmalicine, a marker of monoterpenoid indole alkaloids production in plant cells. Some of them have greater inhibitory effect than fosmidomycin and fully inhibit alkaloid accumulation at the concentration of 100 μM.

Full text of DOI:10.1016/j.phytochem.2005.06.002

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 1797–1803
www.elsevier.com/locate/phytochem
Fosmidomycin analogues as inhibitors of monoterpenoid
indole alkaloid production in Catharanthus roseus cells
a
Zoia Mincheva , Martine Courtois , Franc¸oise Andreu ,
b
b
b
Marc Rideau , Marie-Claude Viaud-Massuard
a,
*
a
` ´
EA 3857, Laboratoire de Synthese et Physicochimie Organique et Therapeutique (SPOT), 31 Avenue Monge, 37200 Tours, France
´ ´ ´ ´
EA 2106 Biomolecules et Biotechnologies Vegetales, Universite de Tours, UFR Sciences Pharmaceutiques, 31 Avenue Monge, 37200 Tours, France
b
Received 10 February 2005; received in revised form 2 June 2005
Abstract
Substituted 3-[2-(diethoxyphosphoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-ones were obtained by functionalization at 6-position
with various substituents (aryl, vinyl, carbonyl chains) via reactions catalysed with palladium. We found that these new fosmido-
mycin analogues inhibited the accumulation of ajmalicine, a marker of monoterpenoid indole alkaloids production in plant cells.
Some of them have greater inhibitory effect than fosmidomycin and fully inhibit alkaloid accumulation at the concentration of
100 lM.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Catharanthus roseus; Apocynaceae; Madagascar periwinkle; Monoterpenoid indole alkaloids; Methylerythritol phosphate pathway;
Synthesis; Substituted oxazolo[4,5-b]pyridine-2(3H)-ones; Fosmidomycin analogues; Pd-catalysed reactions
1. Introduction
DXR as a complex with fosmidomycin in the presence
of NADPH and manganese has been elucidated (Steinb-
acher et al., 2003; Mac Sweeney et al., 2005).
The methylerythritol phosphate (MEP) pathway is a
newly discovered alternative biosynthetic route to iso-
pentenyl diphosphate (IPP) found to be present in many
bacteria, algae, plants and some protozoa (Rohmer,
´
1999; Rodrıguez-Concepcion and Boronat, 2002). The
second enzyme of the MEP pathway, 1-deoxy-D-xylu-
Catharanthus roseus (Madagascar periwinkle, Apo-
cynaceae) produces pharmaceutically valuable monot-
erpenoid indole alkaloids (MIAs). Among them, the
bisindole alkaloids vinblastine and vincristine are pow-
erful antitumor drugs whereas ajmalicine and serpentine
are used in the treatment of hypertension and circula-
tory disorders. MIAs were recently proven to originate
from the MEP pathway (Contin et al., 1998). Treating
C. roseus hairy roots or suspensions cells with fosmido-
mycin had no effect on the cell growth, but inhibited the
accumulation of MIA (Hong et al., 2003; Mincheva
et al., 2004). Similarly, some functionalized 4,5-dihyd-
roisoxazole derivatives (substrate analogues based on
fosmidomycin structure) obtained by one-pot synthesis
via nitrile oxides were shown to inhibit MIA produc-
tion without affecting the cell growth (Mincheva et al.,
2004).
´
lose-5-phosphate
reductoisomerase
(DXR;
EC
1.1.1.267), converts 1-deoxy-D-xylulose-5-phosphate
(DXP) into the branched compound 2C-methyl-D-eryth-
ritol-4-phosphate (MEP) (Takahashi et al., 1998). DXR
is a molecular target for fosmidomycin (Kuzuyama
et al., 1998; Altincicek et al., 2000; Mueller et al.,
2000; Proteau, 2004), and the crystal structure of E. coli
*
Corresponding author. Tel.: +33 247 36 72 27; fax: +33 247 36 72
29.
E-mail
address:
marie-claude.viaud-massuard@univ-tours.fr
(M.-C. Viaud-Massuard).
0031-9422/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.06.002

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Z. Mincheva et al. / Phytochemistry 66 (2005) 1797–1803
Oxazolopyridinones and their derivatives have been
O
O
P
H
(i)
investigated in numerous studies for their applications
as drugs and pesticides (Viaud et al., 1995; Viaud
et al., 1996). Investigating such compounds could lead
to the discovery of new representatives of this class hav-
ing valuable pharmacological properties. In another
study, we prepared fosmidomycin analogues containing
a benzoxazolone or oxazolopyridinones ring: none of
the 13 studied phosphonic esters and phosphonic acids
had effect on C. roseus cell growth, but four of the esters
exerted a significant inhibition of ajmalicine accumula-
tion: 45–85% at 125 lM (Courtois et al., 2004). It is
known that the activities of oxazolopyridinones depend
mainly on the nature of the substituents on the basic het-
erocyclic framework. This paper deals with the synthesis
of new fosmidomycin analogues via Pd-catalysed reac-
tions and their biological evaluation on the growth of
C. roseus cells and contents in MIAs.
P
OEt
Br
N
OEt
OEt
OEt
OH
1
2
(ii)
(iii)
O
P
H
N
OH
OH
OH
O
O
3
(iii)
H
N
P
OEt
O
OEt
OH
O
H
N
P
OH
diester of fosmidomycin
OH
OH
fosmidomycin
Scheme 1. Synthesis of precursors of the fosmidomycin. Reagents and
conditions: (i) NH2OHÆHCl/H2O–MeOH/NaOH, 0 °C; reflux 40–
45 °C. (ii) CH3COOH/HCl, reflux. (iii) HCOOH/(CH3CO)2O, 0 °C,
reflux 45 °C.
acetic–formic anhydride at room temperature gave 70%
yield of fosmidomycin as the monosodium salt. Similarly,
2 was also formylated to give diester of fosmidomycin.
In a second step, the inhibitory effect of compound 4
on the MIA production in C. roseus cells (see below) led
us to investigate the effects of the substituted 3-[2-(dieth-
oxyphosphoryl)-propyl]oxazolo[4,5-b]pyridine-2(3H)-
ones at 6-position (5a–d). We chose to investigate the
palladium catalysed cross-coupling (Tietze et al., 2004)
of 6-bromo-3-[2-(diethoxyphosphoryl)propyl]oxazolo
[4,5-b]pyridine-2(3H)-one (4) with the commercially
available boronic acids using Suzuki methodology (Suzuki,
1998; Miyaura and Suzuki, 1995). Compound 4 was cou-
pled directly with phenylboronic acid or 4-chlorophenyl
boronic acid under anhydrous conditions [3% Pd(PPh3)4,
1:1 N,N-dimethylformamide (DMF)–triethylamine (Et3N),
reflux for 24 h], (Thompson and Gaudino, 1984) to provide
the corresponding Suzuki coupled products 5a and 5b in
2. Results
2.1. Synthesis
In a first step, we prepared the following compounds:
fosmidomycin [3-(N-formyl-N-hydroxyamino)propyl-
phosphonic acid], diester of fosmidomycin [diethyl-3-
(N-formyl-N-hydroxy-amino)propylphosphosphonate]
and two precursors in the synthesis of fosmidomycin, i.e.
[diethyl-3-(N-hydroxyamino)propylphosphosphonate]
(2) and 3-(N-hydroxyamino)propyl phosphonic acid (3),
(see Scheme 1) (Kamiya et al., 1980; Hemmi et al., 1981;
Hemmi et al., 1982). The synthesis of 2 was carried out by
starting from diethyl-3-bromopropylphosphonate (1).
The ethyl groups of 2 were removed by hydrolysis with
concentrated hydrochloric acid–acetic acid under reflux
to give the hydroxylamine 3. Formylation of 3 with
O
EtO
EtO
P
N
O
P
OEt
OEt
(v)
N
N
N
H
N
N
NH2
OH
N
(i)
(iii)
(iv)
(ii)
R
O
O
O
O
O
O
Br
5
4
Compounds
R
5a
5c
5b
5d
O
Cl
OMe
Scheme 2. Synthesis of fosmidomycin analogues via palladium-catalysed reactions. Reagents and conditions: (i) CS2, reflux, EtOH/KOH/H2O;
(ii) KMnO4, CH3COOH; (iii) Br2/DMF; (iv) EtONa, Br(CH2)3PO(OEt)2, DMF, reflux; (v) 5a: PhB(OH)2, Pd(PPh3)4, DMF/Et3N, reflux 90 °C; 5b:
ClPhB(OH)2, Pd(PPh3)4, DMF/Et3N, reflux 90 °C; 5c: CH2@CHSnBu3, (Ph3P)2PdCl2, toluene, 110 °C; 5d: CH3OCOCH@CH2, Pd(OAc)2, P(o-tol)3,
DMF/Et3N, 130 °C.

Z. Mincheva et al. / Phytochemistry 66 (2005) 1797–1803
1799
high yield (Scheme 2). Synthesis of compound 5c was
performed according to Stilleꢀs reaction by using as key
reaction palladium (II) catalysed coupling of arylstannane
[tributyl(vinyl)tin] with
4 (Stille, 1986; Echavarren
and Stille, 1987). The Heck reaction has shown great
versatility in the construction of carbon–aryl bond (Heck,
1991). Although generally utilized in the formation of
cyclic or linear carbon-based systems, the Heck reaction
has been efficiently applied to heterocyclic ring system
(Link and Overman, 1998). Synthesis of compound
5d was performed in good yield according to Heck
methodology using palladium (II) acetate and tri-o-tolyl-
phosphine in DMF with Et3N as base (Viaud et al.,
1995).
Fig. 2. Effects of initial product 4 and the product 5d on alkaloid
accumulation in periwinkle cells. Standard errors of the mean of three
assays. C: control cells (n), DM: dry mass; growth cells (-m-).
2.2. Biological evaluation
2500
2000
1500
Fosmidomycin inhibited ajmalicine production
(chosen as a marker of MIA production) in a dose-
dependent manner (IC50 value, 10 lM) whereas the
diester of fosmidomycin doubled the accumulation of
ajmalicine (Fig. 1(a)). So, in a first time, we investigated
the biological effect of the precursors, ester 2 and acid 3
(Scheme 1). These two compounds had no effect on cell
growth: compound 2 did not affect MIA accumulation
(up to 125 lM), but 3 increased the alkaloid production
250
200
150
1000
500
0
100
50
0
C
5a
5b
F
Compounds
Fig. 3. Inhibition of alkaloid accumulation by compounds 5a, 5b and
fosmidomycin (F) at 50 lM. Standard errors of the mean of three
assays. C: control cells (n), DM: dry mass, growth cells (-m-).
up to 38% (Fig. 1(b)). These responses were not surpris-
ing in reference to our previous report (Mincheva et al.,
2004).
Then, four new products 5a–d, were synthesized
(Scheme 2) from 4, (Courtois et al., 2004). Compound
4 is a fosmidomycin analogue containing an oxazolopy-
ridinone ring. It inhibited alkaloid production by about
50% (IC50: 125 lM; Fig. 2).Compound 5d did not pos-
sess a better inhibitory activity than 4 (Fig. 2) but 5a
and 5b were more efficient (Fig. 3) with inhibitory effects
of 91% and 87%, respectively. We observed a complete
inhibition of ajmalicine accumulation at a concentration
of 100 lM. Clearly, the inhibitory activities of 5a and 5b
were better than the one fosmidomycin (inhibition 69%:
Fig. 3). Compound 5c was not tested due to its non-
solubility in culture medium.
3. Discussion and conclusions
Fosmidomycin is a phosphonic acid derivative origi-
nally isolated from Streptomyces lavandulae. This antibi-
otic had formerly been shown to have anti-bacterial
activity against most gram-negative bacteria (Mine
et al., 1980). Then, it was found to specifically inhibit
Fig. 1. (a) Effects of fosmidomycin (F) and diester of fosmidomycin
(DF) on alkaloid accumulation in periwinkle cells. (b) Effects of
intermediates 2 and 3 on alkaloid accumulation in periwinkle cells.
Standard errors of the mean of three assays. C: control cells (n), DM:
dry mass, growth cells (-m-).

1800
Z. Mincheva et al. / Phytochemistry 66 (2005) 1797–1803
the plant and bacteria DXR which catalyses the second
step of the MEP pathway (see references in Section 1),
and it is now extensively used as a tool to inhibit the bio-
synthesis of natural compounds coming from this path-
way, for example carotenoids (Laule et al., 2003), taxol
(Palazon et al., 2003) or various isoprenoids (Rodriguez-
Concepcion et al., 2004a). In agreement with data of
labelling studies showing that MIA derivate from the
MEP pathway (Contin et al., 1998), MIA production
is inhibited by fosmidomycin in both C. roseus hairy
roots (Hong et al., 2003) and suspension cells (Mincheva
et al., 2004, and this study).
(70 eV) apparatus. Analytical thin-layer chromatogra-
phy (TLC) was carried out on precoated plates (silica
gel, 60 F254, Merck), and spots were visualized under
UV light or by iodine vapor. Flash chromatography
was performed with Kieselgel 60 (230–400 mesh) silica
gel (Merck).
4.2. Reagents and chemicals
Organic solvents were purified when necessary
according to literature methods or purchased from
Aldrich Chimie. All solutions were dried over anhydrous
magnesium sulfate and evaporated on a Buchi rotatory
evaporator. All anhydrous reactions were performed in
over-dried glassware under an argon atmosphere. The
column chromatography solvents employed were dis-
tilled and solvent mixtures were reported as volume to
volume ratios. Starting materials were purchased from
Aldrich Chimie (St. Quentin-Fallavier, France) or Acros
(Noisy-le-Grand, France).
(Ph3P)2PdCl2, Pd(PPh3)4, Pd(OAc)2, P(o-tol)3 were
furnished by Aldrich. Oxazolo[4,5-b]pyridin-2(3H)-one
(Kalcheva and Peshakova, 1989) and 6-bromooxaz-
olo[4,5-b]pyridin-2(3H)-one (Viaud et al., 1995) were
prepared by following the reported procedures.
Diethyl-3-bromo-propylphosphonate was prepared by
method of Arbusov according to published procedure
(Germanaud et al., 1988). Literature procedures were
used for the preparation of 6-bromo-3-[2-(diethoxyphos-
phoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-one (Cour-
tois et al., 2004). Fosmidomyn, diester of fosmidomycin
and two precursors of the fosmidomycin 2 and 3 were
prepared according to published procedure (Kamiya
et al., 1980; Hemmi et al., 1981, 1982).
The Suzuki, Heck and Stille reactions used in the
present study to prepare new fosmidomycin analogues
were found to be efficient methods for generating com-
pounds 5a–d. It was reported that inhibiting the MEP
pathway of plants could be useful in the search of novel
antibiotics, antimalarials and herbicides (Lichtenthaler
et al., 2000). Using C. roseus suspension cells, we found
that the derivatives 5a and 5b are powerful inhibitors of
ajmalicine production. Such esters were more active
than fosmidomycin, and the presence of phenyl moiety
seems to be responsible of their strong inhibitory effect.
However, a definite conclusion cannot be drawn. The
MIA pathway is highly complex and we cannot exclude
that other steps downstream of the MEP pathway, or
even the regulation of the alkaloid biosynthesis might
be affected. Indeed, only the effect of the inhibitors on
DXR activity would give a definitive answer to this
question. Since we previously have characterized a
cDNA clone encoding DXR from C. roseus (Veau
et al., 2000) and that E. coli DXR cDNA is also avail-
able (Takahashi et al., 1998), we are presently preparing
recombinant proteins to test the effects of the inhibitors
and those previously synthesized (Mincheva et al., 2004;
Courtois et al., 2004). If a positive response is obtained,
the inhibitors could be of pharmacological interest be-
cause the MEP pathway is functional in the Apicom-
plexa Plasmodium falciparum. Fosmidomycin and
analogues prepared from this antibiotic are becoming
promising drugs against malaria (Jomaa et al., 1999;
Missinou et al., 2002; Lell et al., 2003; Phaosiri and Pro-
teau, 2004; Rodriguez-Concepcion, 2004b).
4.3. Synthesis of new fosmidomycin analogues
4.3.1. General procedure for Suzuki coupling
A stirred mixture of 150 mg (0.38 mmol) of 6-bromo-3-
[2-(diethoxyphosphoryl)propyl]oxazolo[4,5-b] pyridine-
2(3H)-one (4), 1.14 mmol phenylboronic acid (or 4-chlor-
ophenyl boronic acid), 22 mg (5 mol%) of Pd(PPh3)4 and
6 ml DMF–Et3N (1:1) was refluxed (90 °C) overnight un-
der a nitrogen atmosphere. The mixture was cooled to
room temperature and the solvents were distilled off under
reduced pressure. The residue was partitioned between
EtOAc and brine. The organic extracts were dried with
MgSO4 and concentrated under reduced pressure. Purifi-
cation of the crude product by column chromatography
on silica gel with CH2Cl2–MeOH (95:5) as eluent afforded
the desired compound 5a (or 5b) as a yellow oil.
4. Experimental
4.1. General
1H and 13C NMR spectra for all compounds were
recorded on a Bruker DPX 200 at 200.131 and
50.32 MHz, in CDCl3. The coupling constants are re-
corded in hertz (Hz) and the chemical shifts are reported
in parts per million (d, ppm) downfield from tetramethyl-
silane (TMS), which was used as an internal standard.
Mass spectra were recorded on a R 10-10 C Nermag
4.3.1.1. 6-Phenyl-3-[2-(diethoxyphosphoryl)propyl]oxazolo
[4,5-b]pyridine-2(3H)-one(5a). (132.0 mg, 88.6%). For
1H NMR (200 MHz, CDCl3) and 13C NMR (50 MHz,

Z. Mincheva et al. / Phytochemistry 66 (2005) 1797–1803
1801
CDCl3) assignments, see Tables 1 and 2; MS (IC with
NH3) m/z: 390.8 [M]+ for C19H23N2O5P.
gel column with CH2Cl2–MeOH (95:5) as eluent to give
the desired compound 5c as yellow oil (79.5 mg, 70.6%).
For 1H NMR (200 MHz, CDCl3) and 13C NMR
(50 MHz, CDCl3) assignments, see Tables 1 and 2; MS
(IC with NH3) m/z: 341.5 [M + 1]+ for C15H21N2O5P.
4.3.1.2. 6-(4-Chlorophenyl)-3-[2-(diethoxyphosphoryl)
propyl]oxazolo[4,5-b]pyridine-2(3H)-one (5b). (125.0 mg,
1
77.2%). For H NMR (200 MHz, CDCl3) and 13C NMR
(50 MHz, CDCl3) assignments, see Tables 1 and 2; MS
4.3.3. Synthesis of 3[[2-(diethoxyphosphoryl)propyl]
oxazolo[4,5-b]pyridin-6-yl]acrylic acid methyl ester
(5d)
(IC with NH3) m/z: 425.3 [M + 1]+ for C19H22ClN2O5P.
4.3.2. Synthesis of 6-vinyl-3-[2-(diethoxyphosphoryl)
propyl]oxazolo[4,5-b]pyridine-2(3H)-one (5c)
To a stirred solution of 6-bromo-3-[2-(diethoxyphos-
To a stirred solution of 6-bromo-3-[2-(diethoxyphos-
phoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-one
(4),
(713 mg, 1.81 mmol) in DMF (15 ml) methyl acrylate
(0.23 ml, 2.62 mmol), triethylamine (0.36 ml, 2.62
mmol), palladium diacetate (5 mg, 0.02 mmol) and tri-
o-tolyl-phosphine (26 mg, 0.08 mmol) were successively
added. The mixture was stirred at 130 °C under argon
overnight. After cooling, the residue obtained after con-
centration under vacuum was hydrolysed and extracted
with EtOAc. The organic layers were dried over MgSO4
and evaporated. The crude product was purified by
chromatography on a silica gel column with CH2Cl2–
MeOH (95:5) as eluent to give the desired compound
phoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-one
(4),
(130 mg, 0.33 mmol) in toluene (5 ml) vinyltributyltin
(0.21 ml, 0.73 mmol) and dichlorobis(triphenylphos-
phine)palladium (II) (10 mg, 0.014 mmol) were succes-
sively added and the mixture was refluxed at 110 °C
under argon overnight. After cooling, the solvent was
removed under pressure and residue obtained was
hydrolysed and extracted with EtOAc. The organic lay-
ers were dried over MgSO4 and evaporated. The crude
product was purified by chromatography on a silica
Table 1
1H NMR spectral data for compounds 5a–d (in CDCl3)
Position
5a
5b
5c
5d
2-(Diethoxyphosphoryl)propyl moiety
POCH2CH3
PCH2
PCH2CH2
(NCH2, POCH2CH3)a
1.28 t (7.00)
1.34 t (7.04)
1.81 m
2.20 m
1.29 t (7.04)
1.81 m
2.14 m
1.34 t (6.70)
1.81 m
2.14 m
1.85 m
2.15 m
4.05 m
4.10 m
4.04 m
4.08 m
Oxazolopiridinone moiety
Aromatic H7
Aromatic H5
b
b
7.50 s
8.03 s
7.61 s
8.20 s
8.28 s
8.30 d (1.78)
R moiety
7.39–7.69
Hphenyl
7.53–7.74
Hphenyl
5.32 d (10.91), Hvinyl
5.71 d (17.57), Hvinyl
6.70 dd (10.91; 17.57), Hvinyl
3.75 s, OCH3
6.41 d (16.00), CH
7.68 d (16.03), CH
a
Overlapped signals.
b
Pyridin lying between phenyl protons.
Table 2
13C NMR spectral data for compounds 5a–d (in CDCl3)
Position 5a
5b
5c
5d
2-(Diethoxyphosphoryl)propyl moiety
POCH2CH3
PCH2CH2
PCH2
16.77, 16.88 (5.5)
21.69
16.82, 16.93 (5.5)
21.73, 21.82 (4.5)
22.19, 25.04 (143.4)
41.88, 42.27 (19.6)
62.16, 62.29 (6.5)
16.73, 16.85 (6.0)
21.63, 21.72 (4.5)
22.08, 24.93 (143.4)
41.76, 42.14 (19.1)
62.10, 62.23 (6.5)
16.79, 16.91 (6.0)
21.64, 21.73 (4.5)
22.12, 24.97 (143.4)
41.96, 42.34 (19.1)
62.11, 62.23 (6.04)
22.13, 24.98 (143.4)
41.79, 42.17 (19.12)
62.09, 62.21 (6.0)
NCH2
POCH2CH3
Oxazolopiridinone moiety
Aromatic C
115.47–145.88
115.35–145.36
112.78, 115.53
132.54, 133.79, 142.67
153.76
113.4, 118.97
137.67, 141.15, 145.30
153.47, 167.12
Carbonyl (CO)
153.79
153.78
R moiety
Phenyl Ca
p-Chlorophenyl Ca
129.64 Cvinyl
145.40 Cvinyl
52.3, OCH3
126.47, 147.26, CH
a
Overlapped signals.

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Z. Mincheva et al. / Phytochemistry 66 (2005) 1797–1803
5d as yellow oil (587.0 mg, 81.5%). For 1H NMR
(200 MHz, CDCl3) and 13C NMR (50 MHz, CDCl3)
assignments, see Tables 1 and 2; MS (IC with NH3) m/
z: 398.8 [M + 1]+ for C17H23N2O7P.
triose phosphate/pyruvate pathway in a Catharanthus roseus cell
culture. FEBS Lett. 434, 413–416.
Courtois, M., Mincheva, Z., Andreu, F., Rideau, M., Viaud-Massu-
ard, M.C., 2004. Synthesis and biological evaluation with plant
cells of new fosmidomycin analogues containing a benzoxazolone
or oxazolopyridinone ring. J. Enzyme Inhib. Med. Chem. 19, 559–
565.
4.4. Plant material
Echavarren, A.M., Stille, J.K., 1987. Palladium-catalyzed coupling of
aryl triflates with organo-stannanes. J. Am. Chem. Soc. 109, 5478–
5482.
Madagascar periwinkle (C. roseus [L.] G. Don) cell
suspensions (line C20) were subcultured every week
(dilution rate 1:10) in the Gamborg et al. (1968) B5 med-
ium with 58 mM sucrose and 4.5 lM 2, 4-dichlorophe-
Gamborg, O.L., Miller, R.A., Ojima, R., 1968. Nutrients requirements
of suspension of cultures of Soybean root cells. Exp. Cell Res. 50,
151–158.
Germanaud, L., Brunel, S., Chevalier, Y., Le Perchec, P., 1988.
`
´
noxyacetic acid (Merillon et al., 1993). They were
` ´
Syntheses de phosphobeta¨ınes amphiphiles neuters a distances
intercharge variables. Bull. Soc. Chim. Fr. 4, 699–704.
maintained in 250 ml Erlenmeyer flasks containing
50 ml culture medium on a rotary shaker (100 rpm) at
24 °C, in darkness.
Heck, F.R., 1991. In: Trost, B.M., Fleming, I. (Eds.), Comprehensive
Organic Synthesis, 4. Pergamon Press, Oxford, pp. 833–863.
Hemmi, K., Takeno, H., Hashimoto, M., Kamiya, T., 1981. Studies on
phosphonic acid antibiotics. III. Structure and synthesis of (3-N-
acetyl-N-hydroxyamino)propylphosphonic acid (FR-900098) and
3-(N-acetyl-N-hydroxyamino)-2(R)-hydroxypropylphosphonic acid
(FR-33289). Chem. Pharm. Bull. 29, 646–650.
For experimental purpose, C. roseus cells were sub-
cultured in an alkaloid-inducing medium, i.e. a 2,4-D-
free B5 medium to which 5 lM zeatin were added on
the third day after subculture (Ouelhazi et al., 1993).
Treatments were performed at the third day: the com-
pounds to be tested were dissolved in MeOH–distilled
water. Then, the solutions were filter-sterilized (MIL-
LEX GV 0.22 lM, Millipore) before addition to the cul-
ture medium (final concentrations 50, 75, 100 and
125 lM). The cells were harvested at day 7 (vacuum fil-
tration) and freeze dried for ajmalicine and dry mass
quantitations.
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Alkaloids were extracted from 25 mg freeze-dried cells
´
with methanol, then separated by TLC (Merillon et al.,
1993). Ajmalicine, the chosen marker of alkaloid accu-
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(TLC Scanner III, Camag kex: 365 nm, kem > 400 nm).
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Acknowledgements
Laule, O., Furholz, A., Chang, H.S., Zhu, T., Wang, X., Heifetz, P.B.,
Gruissem, W., Lange, M., 2003. Crosstalk between cytosolic and
plastidial pathways of isoprenoid biosynthesis in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. USA 100, 6866–6871.
Lell, B., Ruangweerayut, R., Wiesner, J., Missinou, M.A., Schindler,
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We express our appreciation to the Le STUDIUM
´
(Agence Regionale de Recherche et dꢀAccueil Interna-
´
´
tional de Chercheurs Associes – Orleans, France) which
supported two years a research associate position for Z.
Mincheva. This work was also supported by the ‘‘Cons-
´ ´
eil Regional’’ (Region Centre, France).
Lichtenthaler, H.K., Zeidler, J., Schwender, J., Muller, C., 2000. The
¨
non-mevalonate isoprenoid biosynthesis of plants as a test system
for new herbicides and drugs against pathogenic bacteria and the
malaria parasite. Z. Naturforsch. C 55, 305–313.
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