Synthesis and evaluation of effective inhibitors of plant δ1-pyrroline-5-carboxylate reductase

Article abstract of DOI:10.1021/jf401234s

Analogues of previously studied phenyl-substituted aminomethylene- bisphosphonic acids were synthesized and evaluated as inhibitors of Arabidopsis thaliana δ¹-pyrroline-5-carboxylate reductase. With the aim of improving their effectiveness, two main modifications were introduced into the inhibitory scaffold: the aminomethylenebisphosphonic moiety was replaced with a hydroxymethylenebisphosphonic group, and the length of the molecule was increased by replacing the methylene linker with an ethylidene chain. In addition, chlorine atoms in the phenyl ring were replaced with various other substituents. Most of the studied derivatives showed activity in the micromolar to millimolar range, with two of them being more effective than the lead compound, with concentrations inhibiting 50% of enzyme activity as low as 50 μM. Experimental evidence supporting the ability of these inhibitors to interfere with proline synthesis in vivo is also shown.

Full text of DOI:10.1021/jf401234s

Article
pubs.acs.org/JAFC
Synthesis and Evaluation of Effective Inhibitors of Plant δ1‑Pyrroline-
5-carboxylate Reductase
Giuseppe Forlani,*,† Łukasz Berlicki,‡ Mattia Duo,† Gabriela Dziędzioła,‡ Samuele Giberti,†
̀
Michele Bertazzini,† and Paweł Kafarski‡
†Department of Life Science and Biotechnology, University of Ferrara, via L. Borsari 46, I-44121 Ferrara, Italy
‡Department of Bioorganic Chemistry, Wrocław University of Technology, Wybrzeze Wyspian
́
skiego 27, PL50-370 Wrocław, Poland
̇
ABSTRACT: Analogues of previously studied phenyl-substituted aminomethylene-bisphosphonic acids were synthesized and
evaluated as inhibitors of Arabidopsis thaliana δ1-pyrroline-5-carboxylate reductase. With the aim of improving their effectiveness,
two main modifications were introduced into the inhibitory scaffold: the aminomethylenebisphosphonic moiety was replaced
with a hydroxymethylenebisphosphonic group, and the length of the molecule was increased by replacing the methylene linker
with an ethylidene chain. In addition, chlorine atoms in the phenyl ring were replaced with various other substituents. Most of
the studied derivatives showed activity in the micromolar to millimolar range, with two of them being more effective than the
lead compound, with concentrations inhibiting 50% of enzyme activity as low as 50 μM. Experimental evidence supporting the
ability of these inhibitors to interfere with proline synthesis in vivo is also shown.
KEYWORDS: amino acid biosynthesis inhibitors as herbicides, aminomethylene-bisphosphonic acid derivatives, P5C reductase,
proline synthesis
temperature stress, implying a role in stress tolerance and
osmoregulation.7 The poor interest for the development of
proline-targeting active principles is most likely due to the
redundancy of proline synthesis. Proline can in fact be
produced from either glutamate or ornithine,8 the glutamate
route usually being the main pathway.9 In the presence of two
pathways, the inhibition of either of the enzymes that catalyze
the rate-limiting steps would be ineffective, because proline
starvation would not be achieved and the cell would be allowed
to recover. Moreover, in some soil and plant-associated bacteria
a stereospecific and irreversible conversion of L-ornithine to L-
proline may be accomplished in a single step by an ornithine
cyclodeaminase [EC 4.3.1.12].10 However, because the latter
has never been convincingly identified in plants, and the
glutamate and the ornithine pathways share the last reaction
catalyzed by a δ1-pyrroline-5-carboxylate (P5C) reductase [EC
1.5.1.2], proline starvation might be achieved through the
development of specific inhibitors of this enzyme.
With the aim of identifying new active principles, we
previously evaluated several groups of aminophosphonates,
most of which exerted remarkable phytotoxic effects at both the
plant and cell culture levels.11 In a preliminary screening, a
series of phenyl derivatives of aminomethylenebisphosphonic
acid (compounds 1, Figure 1) were evaluated for the ability to
inhibit P5C reductase, isolated from Arabidopsis thaliana
cultured cells. Three of them were found to interfere with
the catalytic mechanism in the millimolar range, and one,
namely, 3,5-dichlorophenylaminomethylenebisphosphonic acid,
was active at micromolar levels.12 On the contrary, their pyridyl
INTRODUCTION
■
Modern agrochemicals should have a favorable combination of
properties, including high levels of herbicidal activity, low
application rates, crop tolerance, and low levels of toxicity to
mammals. Moreover, increasing public concern for the
environmental pollution deriving from agricultural practice
strictly requires that active ingredients are endowed with low
persistence, thus being rapidly mineralized by the soil
microflora. Intensive efforts have been undertaken during the
past decades to discover new compounds with suitable
environmental and safety features to selectively control
weeds. However, the unfavorable economics of herbicide
development, mainly due to increasing regulatory requirements,
is greatly limiting industrial discovery programs.1 On the other
hand, because of a high selective pressure for weeds, the rapid
diffusion of resistant biotypes that usually occurs within a few
years after their introduction2 is dramatically reducing the
commercial life of most active principles. Moreover, several
herbicides that are currently in use came from a small set of
molecular scaffolds, whose functional lifetime has been
extended by synthetic tailoring. Because the members of
these families of compounds share the same target at the
molecular level, the emergence and diffusion of cross-resistance
is rapidly occurring.3 Therefore, the discovery of new scaffolds
is required, or new herbicide targets should be identified.4 In
this perspective, inhibitors of enzymes that catalyze key
reactions in amino acid metabolism could represent promising
new leads for weed control.5
From this point of view, little attention has been paid to date
to proline synthesis. Proline plays an important role in protein
structure, uniquely contributing to protein folding and
stability.6 Moreover, in a wide variety of higher plants a rapid
and reversible increase in the intracellular concentration of free
proline has been shown in response to either osmotic or
Received: March 19, 2013
Revised: May 24, 2013
Accepted: June 24, 2013
Published: June 24, 2013
© 2013 American Chemical Society
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Figure 2. Substitution patterns of the phenyl ring in amino-
methylenebisphosphonates 1a−m, hydroxymethylenebisphosphonates
5b−g, hydroxyethylidenebisphosphonates 6b−e, and aminoethylide-
nebisphosphonates 7a−h evaluated in the present work.
1H (300.13 MHz or 600.58 MHz) and 31P NMR (121.51 MHz or
243.12 MHz) spectra were recorded on Bruker AVANCE DRX 300
MHz or Bruker AVANCE 600 MHz using D2O solutions with TMS as
Figure 1. Analogues of phenyl-substituted aminomethylenebisphos-
phonic acids (compounds 1), the scaffold of which has been evaluated
in previous studies (compounds 212 and compounds 3 and 413) and in
the present work (compounds 5, 6, and 7) aiming at the development
of effective inhibitors of plant P5C reductase.
an external reference for 1H NMR and phosphoric acid (85%) for 31
P
NMR spectra. Chemical shifts (δ) are reported in parts per million,
and coupling constants (J) are reported in hertz. Data for compounds
1a−e have been already reported in the preceding study.13
General Procedure for the Preparation of Aminomethyle-
nebisphosphonic Acids (1). The appropriate amine (12.35 mmol),
1.35 mL of triethyl orthoformate (1.31 g, 12.35 mmol), and 3.20 mL
of diethyl phosphite (3.41 g, 24.70 mmol) were heated under reflux for
12−16 h. The volatile components of the reaction mixture were
evaporated under reduced pressure to obtain the corresponding crude
ester, which was dissolved in concentrated hydrochloric acid and
refluxed for 6 h. Then the volatile components of the reaction mixture
were evaporated under reduced pressure. The solid residues were
washed several times with distilled water to obtain the pure products.
3,5-Di(trifluoromethyl)phenylaminomethylenebisphosphonic
acid (1f): yield 30%; 1H NMR (300 MHz, D2O) δ 7.19 (s, 1H, ArH),
analogues 2 were substantially ineffective.12 A thorough kinetic
evaluation coupled with a computer-assisted docking analysis
provided information about its mechanism of action at the
molecular level, showing that it inhibits the enzyme non-
competitively with respect to NAD(P)H and uncompetitively
with respect to P5C.12 On the basis of these results, 25 new
compounds were synthesized by varying either the phenyl
substituents or the scaffold of the active molecule, that is, by
removing the amino group and replacing one of the two
phosphonic groups with an amino (compounds 3) or a
hydroxyl (compounds 4) moiety. Derivatives substituted in the
phenyl ring retained the inhibitory potential, although to a
different extent. All of the most active substances had two
chloride substituents, and none showed higher effectiveness
than the lead compound, 1a. Conversely, both variations in the
scaffold resulted in a substantial loss of biological activity.13 The
availability of several structures active in the micromolar to
millimolar range permitted a proper structure−activity relation-
ship analysis, allowing us to hypothesize about the steric and
electronic requirements for enhancement of the inhibitory
properties. On this basis, 22 new derivatives were designed,
either introducing new electron-withdrawing substituents in the
phenyl ring (compounds 1f−m, Figure 2) or modifying the
structure (compounds 5) and/or the length (compounds 6 and
7) of the scaffold. Here we report on the synthesis and
evaluation of the inhibitory potential of these derivatives against
plant P5C reductase.
2
7.15 (s, 2H, ArH), 4.20 (t, 1H, JH−P = 21.4, CHP2); 31P NMR δ
16.36.
4-Benzylphenylaminomethylenebisphosphonic acid (1g): yield
61%; 1H NMR (300 MHz, D2O) δ 7.21−7.04 (m, 5H, ArH), 6.91 (d,
3
3
2H, JH−H = 8.2, ArH), 6.51 (d, 2H, JH−H = 8.2, ArH), 3.67 (s, 2H,
CH2), 3.36 (t, 1H, JH−P = 19.8, CHP2); 31P NMR δ 17.24.
2
5,6,7,8-Tetrahydro-2-naphthylaminomethylenebisphosphonic
acid (1h): yield 18%; 1H NMR (300 MHz, D2O) δ 6.69 (d, 1H, 3JH−H
= 8.3, ArH), 6.32 (d, 1H, 3JH−H = 10.0, ArH), 6.26 (s, 1H, ArH), 3.28
2
(t, 1H, JH−P = 19.9, CHP2) 2.45 (bs, 2H, CH2), 2.37 (bs, 2H, CH2),
1.49 (bs, 4H, 2 × CH2); 31P NMR δ 17.47.
1
Indan-5-ylaminomethylenebisphosphonic acid (1i): yield 4%; H
3
NMR (300 MHz, D2O + NaOD) δ 6.91 (d, 1H, JH−H = 8.1, ArH),
6.53 (s, 1H, ArH), 6.40 (d, 1H, 3JH−H = 8.1, ArH), 3.37 (t, 1H, 2JH−P
=
19.7, CHP2), 2.67−2.57 (m, 4H, 2 × CH2), 1.88−1.79 (m, 2H, CH2);
31P NMR δ 17.60.
3,5-Dibromophenylaminomethylenebisphosphonic acid (1j):
1
Yield 46%. HNMR (300 MHz, D2O) (δ) 6.75 (s, 1H, ArH), 6.61
2
(s, 2H, ArH), 3.31 (t, 1H, JH−P = 25.1, CHP2); 31PNMR δ 16.29.
3,5-Difluorophenylaminomethylenebisphosphonic acid (1k):
1
3
yield 38%; H NMR (300 MHz, DMSO) δ 6.43 (d, 2H, JHF = 9.0,
Ar), 6.19 (t, 1H, 3JHF = 9.4, Ar), 3.91 (t, 1H, 2JH−P = 21.5); 31P NMR δ
15.62.
MATERIALS AND METHODS
■
Chemistry. Unless indicated otherwise, chemicals were purchased
from Sigma-Aldrich or Merck Chemical Companies and were of
analytical grade. DL-P5C was synthesized by the periodate oxidation
of δ-allo-hydroxylysine and purified by cation-exchange chromatog-
raphy on a Dowex AG50 (200−400 mesh) column, as described in the
literature.14
3-Carbamoylophenylaminomethylenebisphosphonic acid (1l). 3-
Carboxyphenylaminomethylenebisphosphonic acid (0.5 g, 1.61
mmol), which was obtained using the above general procedure, and
an excess of thionyl chloride were heated under reflux for 30 min. The
volatile components of the reaction mixture were removed under
reduced pressure. Then concentrated NH3 aq was added and heated
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under reflux for 15 min. The excess of reagent was removed under
ArH), 6.81 (t, 2H, 3JH−H = 8.0, ArH), 3.46 (td, 2H, 3JH−P = 13.9, 3JH−H
1
= 6.6, CH2CHP2), 1.90 (tt, 1H, 3JH−P = 20.7, 3JH−H = 6.6, CHP2); 31
NMR δ 19.20.
P
reduced pressure. Yield 0.48 g (96%); H NMR (300 MHz, D2O +
3
NaOD) δ 7.07 (t, 1H, JH−H = 7.6, ArH), 7.02 (s, 1H, ArH), 6.93 (d,
3
3
1H, JH−H = 7.6, ArH), 6.73 (d, 1H, JH−H = 7.9, ArH), 3.62 (t, 1H,
2-(3,5-Dimethylphenylamino)ethylidenebisphosphonic acid (7e):
2JH−P = 19.3, CHP2); 31P NMR δ 13.49.
1
yield 45%; H NMR (300 MHz, D2O) δ 6.50 (s, 2H, ArH), 6.47 (s,
1H, ArH), 3.41 (td, 2H, 3JH−P = 14.0, 3JH−H = 6.8, CH2CHP2), 2.17 (s,
3-Chlorophenylaminomethylenebisphosphonic acid (1m): yield
1
3
3
3
80%; H NMR (300 MHz, D2O) δ 6.95 (t, 1H, JH−H = 8.1, ArH),
6H, 2 × CH3), 1.88 (tt, 1H, JH−P = 20.9, JH−H = 6.8, CHP2); 31P
6.58 (s, 1H, ArH), 6.47 (d, 1H, 3JH−H = 8.3, ArH), 6.40 (d, 1H, 3JH−H
NMR δ 19.38.
= 7.8, ArH), 3.38 (t, 1H, JH−P = 19.6, CHP2); 31P NMR δ 16.54.
2
2-(3,5-Di(trifluoromethyl)phenylamino)ethylidenebisphosphonic
acid (7f): yield 60%; 1H NMR (300 MHz, D2O) δ 7.18 (s, 1H, ArH),
7.14 (s, 2H, ArH), 3.36 (td, 2H, 3JH−P = 13.9, 3JH−H = 6.6, CH2CHP),
General Procedure for the Preparation of Hydroxybi-
sphosphonic Acids (5 and 6). The appropriate carboxylic acid
(7.85 mmol) and thionyl chloride (6.8 mL, 47.1 mmol) were refluxed
for 1 h. The volatile portion of the reaction mixture was evaporated
under reduced pressure to give crude acid chloride. The solution of
obtained acid chloride in dry THF was added dropwise to a solution of
tris(trimethylsilyl)phosphite (5.25 mL, 15.9 mmol) under nitrogen.
The reaction mixture was stirred for 1 h at room temperature.
Subsequently, methanol was added and stirring was continued for 1 h.
Solvents were removed under reduced pressure, and residues were
washed with diethyl ether. When necessary, the product was purified
by RP-HPLC (water/acetonitrile 0.1% TFA).
1.85 (tt, 1H, JH−P = 20.9, JH−H = 6.8, CHP2); 31P NMR δ 18.21.
2-(4-Benzylphenylamino)ethylidenebisphosphonic acid (7g):
yield 18%; 1H NMR (300 MHz, D2O) δ 7.24−7.08 (m, 5H,
3
3
3
3
ArHCH2), 7.03 (d, 2H, JH−H = 8.3, ArH), 6.72 (d, 2H, JH−H = 8.4,
3
3
ArH), 3.76 (s, 2H, ArCH2), 3.26 (td, 2H, JH−P = 13.9, JH−H = 6.7,
CH2CHP2), 1.81 (tt, 1H, 3JH−P = 14.0, 3JH−H = 6.9, CHP2); 31P NMR
δ 19.27.
2 - ( 5 , 6 , 7 , 8 - T e t r a h y d r o - 2 - n a p h t h y l a m i n o ) -
ethylidenebisphosphonic acid (7h): yield 23%; 1H NMR (600 MHz,
3
3
D2O) δ 6.84 (d, 1H, JH−H = 8.1, ArH), 6.53 (d, 1H, JH−H = 8.2,
3
3
2,3-Dichlorophenylhydroxymethylenebisphosphonic acid (5b):
ArH), 6.52 (s, 1H, ArH), 3.20 (td, 2H, JH−P = 14.0, JH−H = 6.7,
CH2CHP2), 2.52 (m, 2H, CH2), 2.46 (m, 2H, CH2), 1.76 (tt, 1H,
3JH−P = 20.8, 3JH−H = 6.6, CHP2), 1.66 (m, 4H, 2 × CH2); 31P NMR δ
19.30.
1
3
yield 27%; H NMR (300 MHz, D2O) δ 7.80 (d, 1H, JH−H = 7.5,
ArH), 7.36 (d, 1H, 3JH−H = 7.9, ArH), 7.11 (t, 1H, 3JH−H = 8.1, ArH);
31P NMR δ 16.14.
3,5-Dimethylphenylhydroxymethylenebisphosphonic acid (5e):
Plant Cell Culture and Growth Conditions. Cell suspension
cultures of A. thaliana Heynh., ecotype Columbia, were grown at 24
1 °C on a rotary shaker (100 rpm) under dim (<50 μmol m−2 s−1)
light in 500 mL Erlenmeyer flasks containing 125 mL of Murashige
and Skoog medium with 0.3% (w/v) sucrose and 0.5 mg L−1 of both
2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Subcultures
were made every 7 days by transferring 25 mL aliquots of the
suspension to 100 mL of fresh medium.
1
yield 98%; H NMR (300 MHz, D2O) δ 7.19 (s, 2H, ArH), 6.85 (s,
1H, ArH), 2.11 (s, 6H, 2 × CH3); 31P NMR δ 16.96.
4-Benzylphenylhydroxymethylenebisphosphonic acid (5g): yield
1
3
85%; H NMR (300 MHz, D2O) δ 7.54 (d, 2H, JH−H = 8.6, ArH),
7.23−7.07 (m, 7H, ArH), 3.84 (s, 2H, CH2); 31P NMR δ 17.12.
2-(2,3-Dichlorophenyl)-1-hydroxyethylidenebisphosphonic acid
1
3
(6b): yield 65%; H NMR (300 MHz, D2O) δ 7.42 (d, 1H, JH−H
=
3
3
7.8, ArH), 7.32 (d, 1H, JH−H = 7.3, ArH), 7.08 (t, 1H, JH−H = 7.9,
P5C Reductase Purification and Assay. The enzyme was
partially purified from cultured cells harvested in the early stationary
phase of growth as previously described,12 with minor modifications. A
combination of ammonium sulfate precipitation, negative chromatog-
raphy on a DEAE-Sephacel column equilibrated at pH 7.5, and anion-
exchange chromatography at pH 10.0 on the same column resulted in
a 70-fold enrichment, with a 35% yield. The mean value for specific
ArH), 3.48 (t, 2H, JH−P = 13.5, CH2); 31P NMR δ 18.90.
3
2-(4-Chlorophenyl)-1-hydroxyethylidenebisphosphonic acid (6c):
1
3
yield 51%; H NMR (300 MHz, D2O) δ 7.25 (d, 2H, JH−H = 8.5,
ArH), 7.19 (d, 2H, 3JH−H = 8.5, ArH), 3.17 (t, 2H, 3JH−P = 13.3, CH2);
31P NMR δ 19.12.
2-(2,6-Dichlorophenyl)-1-hydroxyethylidenebisphosphonic acid
1
3
(6d): yield 30%; H NMR (300 MHz, D2O) δ 7.18 (d, 2H, JH−H
=
activity level in these samples was 122.7
3.9 nkat mg−1. Proper
3
3
8.1, ArH), 7.02 (t, 1H, JH−H = 8.0, ArH), 3.60 (t, 2H, JH‑P = 14.0,
checks were done to rule out the presence of other enzymes able to
use the same substrates and/or further metabolize enzyme products
(i.e., P5C dehydrogenase, EC 1.5.1.12). Active fractions were filter-
sterilized (0.22 μm) and stored at 4 °C until used for biochemical
determinations. Under these conditions, P5C reductase activity was
found to be stable for at least 2 months.
The physiological forward reaction of P5C reductase was measured
by following the P5C-dependent oxidation of NADH. The assay
mixture contained 100 mM Hepes−KOH buffer, pH 7.75, 0.1 mM
MgCl2, 2 mM DL-P5C, and 0.25 mM NADH, in a final volume of 0.2
mL. A limiting amount of enzyme (0.03−0.04 nkat) was added to the
prewarmed mixture, and the decrease in absorbance at 340 nm was
determined at 35 °C for up to 5 min by continuous monitoring of the
sample against blanks from which P5C had been omitted. Activity was
determined from the initial linear rate, with the assumption of an
extinction coefficient of 6220 M−1 cm−1. Protein concentration was
determined according to the method of Bradford,15 using bovine
serum albumin as the standard.
Enzyme Inhibition by Bisphosphonic Acids. P5C reductase
inhibition was evaluated by adding to the reaction mixture an
appropriate dilution of a 10 mM solution of a given compound,
brought to pH 7.5−8.0 with KOH, so as to obtain the desired final
concentration, ranging from 1 μM to 1 mM. Three to five different
doses in the active range were evaluated, and at least three
measurements were performed for each dose. The concentrations
causing 50% inhibition (IC50) of P5C reductase activity, and
confidence intervals were estimated by nonlinear regression analysis
of enzyme activity values, expressed as percentage of untreated
CH2); 31P NMR δ 18.67.
2-(3,5-Dimethylphenyl)-1-hydroxyethylidenebisphosphonic acid
(6e): yield 34%; 1H NMR (300 MHz, D2O) δ 6.92 (s, 2H, ArH), 6.85
(s, 1H, ArH), 3.13 (t, 2H, 3JH−P = 13.6, CH2), 2.14 (s, 6H, 2 × CH3);
31P NMR δ 19.46.
General Procedure for the Preparation of Aminoethylenebi-
sphosphonic Acids (7). Tetraethyl ethylidenebisphosphonate (3
mmol) and the appropriate amine (3 mmol) were dissolved in dry
THF. The reaction mixture was stirred for 24 h at room temperature,
and solvents were evaporated at reduced pressure. The obtained ester
was purified with the use of column chromatography on silica gel
(ethyl acetate/hexanes). Subsequently, the ester was dissolved in dry
acetonitrile, and trimethylsilyl bromide (6 equiv) was added. The
mixture was stirred for 12 h. Then methanol was added, and the
mixture was stirred for 2 h. Volatile components were removed under
reduced pressure. When necessary, the product was purified by RP-
HPLC (water/acetonitrile 0.1% TFA).
2-(3,5-Dichlorophenylamino)ethylidenebisphosphonic acid (7a):
1
yield 48%; H NMR (300 MHz, D2O) δ 6.71 (s, 3H, ArH), 3.33 (td,
3
3
3
2H, JH−P = 14.0, JH−H = 6.7, CH2CHP2), 1.88 (tt, 1H, JH−P = 21.0,
3JH−H = 6.8, CHP2); 31P NMR δ 18.92.
2-(2,3-Dichlorophenylamino)ethylidenebisphosphonic acid (7b):
yield 3%; 1H NMR (300 MHz, D2O) δ 7.06 (t, 1H, 3JH−H = 8.5, ArH),
3
3
6.75 (d, 1H, JH−H = 9.1, ArH), 6.77 (d, 1H, JH−H = 9.1, ArH), 3.32
3
3
3
(td, 2H, JH−P = 14.2, JH−H = 6.5, CH2CHP2), 1.90 (tt, 1H, JH−P
=
20.5, JH−H = 6.5, CHP2); 31P NMR δ 18.92.
3
2-(2,6-Dichlorophenylamino)ethylidenebisphosphonic acid (7d):
1
3
yield 28%; H NMR (300 MHz, D2O) δ 7.23 (d, 2H, JH−H = 8.0,
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controls, using Prism 5 for Windows, version 5.01 (GraphPad
Software).
In Vivo Effects on Proline Biosynthesis. To evaluate bi-
sphosphonate effect upon proline synthesis in exponentially growing
cells, samples were withdrawn from the stock cultures in the late
exponential phase of growth and used to inoculate 250 mL culture
flasks to a density of 1.0−1.2 mg mL−1 (dry weight) in a final volume
of 60 mL. Filter-sterilized compounds (brought to pH 6.0 with KOH)
were added just after the density of cell population reached 1.4 mg
mL−1 (dry weight). After a further 5−7 days of incubation, when
untreated controls approached the early stationary phase of growth,
cells were harvested by vacuum filtration, resuspended in 1 mL g−1 of a
3% (w/v) solution of 5-sulfosalicylic acid, and homogenized in a
Teflon-in-glass Potter homogenizer by 20 strokes. After centrifugation
for 3 min at 12000g, the supernatant was mixed with the same volume
of o-phthaldialdehyde solution [0.5 M in 0.5 M sodium borate buffer,
pH 10.0, containing 0.5 M β-mercaptoethanol and 10% (v/v)
methanol]. After exactly 60 s, 20 μL of derivatized samples was
injected onto a 4.6 × 250 mm Zorbax ODS column (Rockland
Technologies, Newport, DE, USA) equilibrated with 59% solvent A
[50 mM sodium phosphate−50 mM sodium acetate buffer, pH 7.5,
containing 2% (v/v) of both methanol and tetrahydrofuran] and 41%
solvent B (65% methanol). Elution proceeded at a flow rate of 60 mL
h−1 using a computer-controlled (Data System 450; Kontron,
Munchen, Germany) complex gradient from 41 to 100% solvent B
as described previously,16 monitoring the eluate at 340 nm. Peaks were
integrated by area, with variation coefficients ranging from 0.8 to 3.2%.
At least three replicates were carried out for each treatment (biological
replicates), and two analyses were carried out for each extract
(technical replicates). Proline and total amino acid content were
quantified by using the acid ninhydrin method.14 P5C was measured
by reaction with o-aminobenzaldehyde, as previously described.17
Figure 3. Synthesis of the four groups of bisphosphonic acids
evaluated in the present work. Reagents and conditions: (a) reflux for
6 h; (b) HClaq or TMSBr and then MeOH; (c) stirring for 24 h; (d)
TMSBr and then MeOH; (e) stirring for 1 h; (f) MeOH.
anolysis).18 The addition of amine to the double bond of
tetraethyl ethenylidenebisphosphonates resulted in tetraethyl 2-
aminoethylene-1,1-bisphosphonates, which were deprotected to
acids 7.19 Hydroxybisphosphonic acids (5 and 6) were
synthesized from appropriate acyl chlorides and tris-
(trimethylsilyl)phosphate, followed by methanolysis of the
obtained esters.20
Activity of Bisphosphonate Derivatives against P5C
Reductase. To evaluate the ability of these compounds to
interfere with the activity of plant P5C reductase, the protein
was partially purified from suspension cultured cells of A.
thaliana, a model species in which only one gene coding for this
enzyme is present.7 The occurrence of multiple enzyme forms
with different susceptibilities could in fact represent a significant
drawback in assessing the efficacy of a given compound. The
activity of Arabidopsis P5C reductase was measured in the
absence or in the presence of the 22 new derivatives in the
micromolar to millimolar range. Because slight differences in
sensitivity may derive from small variations in enzyme
purification level and absolute enzyme amount, the five (1a−
e) most effective aminomethylenebisphosphonates evaluated in
the previous study13 were also included in the experimental
plan as reference compounds. Results, summarized in Table 1
as the concentrations causing 50% inhibition of enzyme activity
(IC50), showed that, with the only exception of compound 7h,
all of the derivatives are capable of inhibiting the enzyme when
present at millimolar levels. This denotes the correctness of the
steric and the electronic features previously hypothesized13 to
maintain the inhibitory potential. The comparison of the effect
of compounds with the same substituents at the aromatic
portion of the molecule (Figure 4) revealed that either the
increase of the chain length in compounds 7 or the replacement
of the aminomethylenebisphosphonic group with a hydroxy-
methylene-bisphosphonic moiety in compounds 5 does not
exceedingly vary the effectiveness of the lead compounds 1. On
the contrary, the presence of both variations was found to be
detrimental, because all 1-hydroxyethylidenebisphosphonic
derivatives 6 were less effective than their counterparts 1. As
to the substituents in the phenyl ring, several electron-
withdrawing moieties conferred equipotency with the two
chlorines in reference compounds 1a, 1b, and 1d. Two
derivatives, namely, compounds 1j and 7a, showed a
significantly higher effectiveness than the lead compound 1a,
thereby proving the validity of the design scheme.
Results were expressed as nmol (g FW)−1, and means
standard
deviation over replications are presented. Data were analyzed by using
standard statistical procedures for analysis of variance and t test. When
differences are reported, they are at the 99% confidence level (P <
0.01).
RESULTS AND DISCUSSION
■
Design and Synthesis of Bisphosphonate Derivatives.
A previous screening13 of bisphosphonic acid derivatives against
P5C reductase allowed us to hypothesize on the structural
features required for high inhibitory activity, that is, the
presence of two phosphonic groups, a phenyl ring, and a
hydrogen bond donor. Four different scaffolds showing these
features were taken into account (Figure 1). In this study, the
previously explored N-phenylaminomethylenebisphosphonic
acid core (1) was extended to 2-phenylaminoethylidenebi-
sphosphonic acid (7). In addition, the hydrogen bond donor
secondary amine fragment was replaced by an isoelectronic
hydroxyl substituent, and hydroxybisphosphonic acids 5 and 6
were envisaged. Various substitution patterns of phenyl ring
(Figure 2) were also considered: (1) different positioning of
chlorine atoms (derivatives a−d and m); (2) the presence of
both electron-withdrawing and -donating groups in 3,5-
positions of the phenyl ring (derivatives a, e, f, j, k); (3)
substituents that substantially increase the phenyl ring volume
(g, h, i); (4) a carbamoyl substituent similar to that present in
the molecule of NAD, because bisphosphonates are expected to
act as analogues of the dinucleotide.
All compounds were synthesized using published method-
ologies (Figure 3). Aminomethylenebisphosphonates were
obtained in a tricomponent reaction of amine, ethyl
orthoformate, and diethylphosphite and subsequent hydrolysis
of tetraethyl esters leading to the requested acids (with HClaq
or via transesterification to trimethylsilyl esters and meth-
In Vivo Effects of the Most Active Bisphosphonate on
Proline Synthesis. Cell suspension cultures represent a well
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suitable system to verify the occurrence of enzyme inhibition in
vivo. To investigate whether the interference of the most active
derivatives with P5C reductase activity may take place also
inside the cell and cause an actual reduction of proline
biosynthesis that could result in phytotoxic effects at the plant
level, the culture medium of A. thaliana suspension cultured
cells was supplemented with micromolar concentrations of
compound 1j. As a consequence, cell growth was reduced (data
not shown), with a concentration inhibiting by 50% the dry
weight increase of 132 7 μM. On this basis, the intracellular
levels of proline, P5C, and free amino acids were determined in
samples treated with 100 and 200 μM compound 1j. Results
(Table 2) showed that in cells treated with the lower dose of
Table 1. Inhibition of Arabidopsis P5C Reductase by
Analogues of 3,5-
Dichlorophenylaminomethylenebisphosphonic Acid (1a)
a
compd
IC50 (mM)
pIC50 (M)
1a
1b
1c
1d
1e
1f
0.134 0.009
0.335 0.010
0.970 0.066
0.450 0.017
1.245 0.093
0.413 0.018
0.852 0.120
0.684 0.043
1.887 0.501
0.054 0.003
0.719 0.090
0.547 0.039
1.618 0.185
1.187 0.058
1.409 0.118
0.325 0.012
0.488 0.021
2.071 0.303
1.639 0.459
1.914 0.254
0.082 0.003
0.431 0.025
0.763 0.092
0.386 0.018
0.463 0.033
0.488 0.042
ineffective
3.873
3.475
3.013
3.347
2.905
3.384
3.070
3.165
2.724
4.268
3.143
3.262
2.791
2.926
2.851
3.488
3.312
2.684
2.785
2.718
4.086
3.366
3.117
3.413
3.334
3.312
<2
1g
1h
1i
a
1j
Table 2. Free Amino Acid, Proline, and P5C Levels in A.
1k
1l
thaliana Cultured Cells Treated with Micromolar
Concentrations of Compound 1j
1m
5b
5e
5g
6b
6c
6d
6e
7a
7b
7d
7e
7f
treatment
control
100 μM
200 μM
compd
(μmol (g FW)−1
)
(μmol (g FW)−1
)
(μmol (g FW)−1
)
Asp
Glu
Asn
Ser
0.343 0.009
0.794 0.022
0.228 0.018
0.547 0.028
1.027 0.022
0.060 0.005
0.257 0.012
0.787 0.062
0.105 0.000
0.502 0.018
0.340 0.013
0.054 0.003
0.173 0.006
0.391 0.018
0.069 0.014
0.392 0.018
0.250 0.015
0.059 0.002
Gln
Arg + Gly +
Thr
Ala + GABA +
Tyr
0.606 0.027
0.384 0.029
0.155 0.006
Trp
Met
Val
0.035 0.001
0.004 0.001
0.266 0.018
0.065 0.004
0.034 0.001
0.037 0.002
0.035 0.001
0.175 0.005
0.010 0.002
0.022 0.000
0.002 0.000
0.230 0.011
0.032 0.001
0.033 0.002
0.033 0.003
0.036 0.004
0.132 0.002
0.008 0.001
0.012 0.000
0.003 0.000
0.117 0.000
0.016 0.003
0.016 0.001
0.018 0.001
0.032 0.001
0.116 0.006
0.029 0.003
7g
7h
a
Activity was evaluated as described under Materials and Methods
Phe
Ile
either in the absence or in the presence of a given bisphosphonate at
concentrations ranging from 0.001 to 1 mM. Each test was carried out
in triplicate, and values were expressed as percentage of untreated
controls. The concentrations causing 50% inhibition (IC50) of in vitro
activity and confidence limits were estimated by nonlinear regression
analysis utilizing the software GraphPad Prism (version 5).
Leu
Lys
Pro
P5C
a
Cell suspension cultures were treated with compound 1j at 100 and
200 μM, causing growth reductions of 17.2 1.8 and 92.7 1.6%,
respectively. Free amino acid content was evaluated 6 days after the
treatment by RP-HPLC following derivatization with oPDA, as
described under Materials and Methods. Pro was measured by the
acid ninhydrin method, and P5C was quantified by reaction with o-
aminobenzaldehyde. Three independent replications were done for
each treatment, and means SE over replicates are reported.
the bisphosphonate, the synthesis of glutamine is the most
affected, with a 67% reduction of the level of this amino acid
with respect to controls. This most likely relies on the ability of
compound 1j to inhibit also the activity of plant glutamine
synthetase (data not shown), as previously reported for several
other bisphosphonates 1.21 The inhibition of glutamine
synthesis, in turn, affected the level of other amino acids,
mainly asparagine (−44%). Proline levels were also reduced,
although to a lower extent (−25%). However, such a reduction
took place even if the level of the proline direct precursor,
glutamate, was unaffected. Therefore, the possibility that the
lowered free proline may be an indirect consequence of the
inhibition of glutamine synthetase seems unlikely. Moreover, in
cells treated with the higher concentration of the bi-
sphosphonate, where the level of most amino acids was
strongly reduced, a significant increase of the concentration of
Figure 4. Comparison between the effectiveness of aminomethylenebi-
sphosphonic acids 1 and their analogues 5, 6, and 7. The inverses of
the logarithm of the concentrations causing 50% inhibition of enzyme
activity (pIC50) were plotted against each other. The 1:1 line indicates
equipotency. A point above the line shows that a given analogue is
more effective than its aminomethylenebisphosphonic counterpart
showing the same substitution pattern in the phenyl ring and vice versa
for a point below the line.
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P5C was evident, providing direct proof of the inhibition of the
enzyme. To the best of our knowledge, this is the first report
showing increased P5C levels as the result of the treatment with
compounds able to inhibit P5C reductase. Such compounds
might thus represent a useful tool to investigate the highly
debated22,23 molecular mechanisms underlying the noxious
effects induced in plants by the exogenous supply of
proline.17,24 Even though the relative weight of the inhibition
of P5C reductase versus that of glutamine synthetase in
determining the phytotoxicity of phosphonates still awaits to be
elucidated, the present data confirm that P5C reductase
inhibition occurs inside the plant cell.
The results herein reported represent a further step toward
the development of new active principles for weed control
targeting P5C reductase. Several new derivatives were found to
be active in the micromolar range. Some of them do not
contain halogen substituents and should be more easily
mineralized by soil microorganisms. The most effective
compound was shown to be able to interfere with the synthesis
of proline in vivo. However, when several of the same
aminomethylenebisphosphonates 1 were recently evaluated as
inhibitors of a bacterial P5C reductase, the susceptibility of the
enzyme from Streptococcus pyogenes was found to be strikingly
higher than that of the plant enzyme.25 In all cases IC50 values
were 2−3 orders of magnitude lower that those found in the
present study (Figure 5). This seems to imply the occurrence of
AUTHOR INFORMATION
Corresponding Author
*(G.F.) E-mail: flg@unife.it.
Funding
Support from the University of Ferrara (Fondo d’Ateneo per la
Ricerca 2010 and 2011) and from the National Science Centre,
Poland (Grant 2011/01/B/ST5/00843), is gratefully acknowl-
edged.
■
Notes
The authors declare no competing financial interest.
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