Tailoring the structure of aminobisphosphonates to target plant P5C reductase

Article abstract of DOI:10.1021/jf800029t

Using the structure of (3,5-dichlorophenyl)aminomethylenebisphosphonic acid as a lead compound, 25 new phosphonates were synthesized and evaluated as possible inhibitors of Arabidopsis thaliana δ¹-pyrroline-5- carboxylate (P5C) reductase. Deriv

Full text of DOI:10.1021/jf800029t

J. Agric. Food Chem. 2008, 56, 3193–3199 3193
Tailoring the Structure of Aminobisphosphonates To
Target Plant P5C Reductase
GIUSEPPE FORLANI,*,† ANDREA OCCHIPINTI,† ŁUKASZ BERLICKI,‡
GABRIELA DZIEDZIOŁA,‡ ANNA WIECZOREK,†,‡ AND PAWEŁ KAFARSKI
‡
Department of Biology and Evolution, University of Ferrara, Ferrara, Italy, and Department of
Bioorganic Chemistry, Wrocław University of Technology, Wrocław, Poland
Using the structure of (3,5-dichlorophenyl)aminomethylenebisphosphonic acid as a lead com-
pound, 25 new phosphonates were synthesized and evaluated as possible inhibitors of Arabidopsis
thaliana δ1-pyrroline-5-carboxylate (P5C) reductase. Derivatives substituted in the phenyl ring
retained the inhibitory potential, though to a different extent. On the contrary any variation in the
scaffold, i.e., the replacement of the second phosphonate moiety with a hydroxyl or an amino
residue, resulted in a significant loss of biological activity. The availability of several structures
capable of interfering with the catalytic mechanism in the micromolar to millimolar range allowed
a proper structure–activity relationship analysis, leading us to hypothesize about the steric and
electronic requirements for maintenance or enhancement of the inhibitory properties. Reversal
experiments with suspension cultured cells provided evidence for the occurrence of enzyme
inhibition in vivo. Because in higher plants the step catalyzed by P5C reductase is shared by all
pathways leading to proline synthesis, these compounds may be exploited for the design of new
substances endowed with herbicidal activity.
KEYWORDS: Derivatives of aminomethylenebisphosphonic acid; P5C reductase; proline synthesis;
structure–activity relationship analysis; amino acid biosynthesis inhibitors as herbicides
INTRODUCTION
required during floral development and pollen tube growth (9).
Thus, the enzymes involved in the anabolic pathway would be
greatly attractive as potential targets for new herbicides.
Plants are continually faced with several abiotic stress
conditions that may result in osmotic impairment. Either
temperatures below freezing point, drought, or substrate salinity
exert similar effects, and can cause water withdrawal, possibly
leading to cell dehydration (1). Even if the corresponding signal
transduction pathways might somewhat differ from each other,
the response of the plant cell is essentially the same (2).
Consistently, freezing tolerance is often accompanied by toler-
ance to dehydration (3). A pivotal role in counteracting the
effects of such fluctuations may be played by a rapid and
reversible increase in the concentration of small molecules such
as sugars, quaternary amines and amino acids, collectively
known as compatible solutes (4), among which the amino acid
proline has the widest distribution (5). Besides its obvious role
for protein and cell wall biosynthesis, in many higher plants
high intracellular levels of free proline are transiently ac-
cumulated to cope with hyperosmotic conditions (6), as well
as in response to the imposition of a wide range of biotic and
abiotic stresses (7). The synthesis of this amino acid seems also
to play a regulatory role promoting seed germination (8) and is
Two metabolic routes leading to proline synthesis have been
found in vascular plants. Under normoosmotic conditions and
nitrogen availability, proline production seems to proceed mainly
through ornithine, an intermediate in arginine biosynthesis,
whereas, under hyperosmotic stress and nitrogen deprivation,
it is synthesized directly from glutamate (6). The presence of
multiple pathways would hamper any attempt to induce proline
starvation through the inhibition of either of the enzymes that
catalyze the rate-limiting steps, allowing the plant to recover.
However, because the two pathways share the last reaction,
catalyzed by a δ1-pyrroline-5-carboxylate (P5C) reductase [EC
1.5.1.2] (5), this goal might be achieved through the develop-
ment of specific inhibitors of that enzyme.
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 envi-
ronmental pollution deriving from agricultural practice strictly
requires that such xenobiotics are endowed with low persistence,
thus being rapidly mineralized by the soil microflora. Intensive
efforts have been undertaken during past years to discover new
compounds with favorable environmental and safety features
to selectively control weeds. In the past decade, this aim has
* To whom correspondence should be addressed. Fax: (39)0532-
249761. E-mail: flg@unife.it.
† University of Ferrara.
‡ Wrocław University of Technology.
10.1021/jf800029t CCC: $40.75  2008 American Chemical Society
Published on Web 04/10/2008

3194 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Forlani et al.
Figure 1. Analogues of (3,5-dichlorophenyl)aminomethylenebisphosphonic acid (compound 1a) evaluated as possible inhibitors of plant P5C reductase.
been pursued with new strategies, switching from the classical
testing of chemicals for efficacy on whole plants toward the
use of in vitro assays against a given molecular target.
Aminoalkylphosphonic acids, structural analogues of amino
acids in which the carboxylic group is replaced by a phosphonic
or related moiety, have attracted increasing interest as lead
compounds for the development of new herbicides (10), also
because of their high susceptibility to degradation by soil
microorganisms (11, 12). High affinity of phosphonates toward
certain enzymes is the result of their ability to form either a net
of hydrogen bonds with amino acid residues or ionic interactions
with positively charged amino acids or metal ions that are
present in the active site. In several instances the structural
antagonism between amino acids or their biosynthetic interme-
diates and the phosphonic counterparts resulted in inhibition of
enzyme activity (13).
With the aim to identify 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 (14). In vitro assays with partially purified
enzymes from different plant sources showed that some of them
act by interfering with aromatic metabolism at the level of the
first enzyme in the shikimate pathway, 3-deoxy-D-arabino-
heptulosonate-7-phosphate synthase [EC 4.1.2.15] (15, 16).
Some others were found to interfere with nitrogen assimila-
tion through the inhibition of glutamine synthetase (GS [EC
6.3.1.2]) (17, 18). In a previous, blind screening, a series of
derivatives of aminomethylenebisphosphonic acid were evalu-
ated for the ability to inhibit P5C reductase, isolated from
Arabidopsis thaliana (A. thaliana) cultured cells. Three of them
were found to interfere with the catalytic mechanism in the
millimolar range, and one, namely, (3,5-dichlorophenyl)ami-
nomethylenebisphosphonic acid (compound 1a, Figure 1), was
active at micromolar concentrations. A thorough kinetic evalu-
ation coupled with a computer-assisted docking analysis pro-
vided information about its mechanism of action at the molecular
level (19). On the basis of these results, 25 new compounds
were synthesized by varying either the phenyl substituents or
the scaffold of the active molecule, i.e., by replacing one of the
two phosphonic groups of 1a with an amino or a hydroxyl
moiety. Here, we report the evaluation of the effectiveness of
these analogues in vitro against thale cress P5C reductase. In
vivo experiments with the most active compounds supported
the occurrence of proline starvation in treated plant cells. A
structure–activity relationship analysis allowed us to define steric
and electronic properties at the basis of the biological activity
against the selected molecular target.
MATERIALS AND METHODS
Chemistry. Unless otherwise indicated, chemicals were purchased
from Sigma-Aldrich or Merck Chemical Companies and were of
analytical grade. All obtained hydroxyphosphonic and aminophosphonic
acids were racemic.

New Bisphosphonate Inhibitors of Plant P5C Reductase
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3195
General Procedure for the Preparation of Aminomethylenebispho-
sphonic Acids (1) (20)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 for 12–16 h at 80 °C. The
reaction mixture was evaporated under reduced pressure to obtain the
corresponding crude ester that was dissolved in concentrated aqueous
hydrochloric acid and refluxed for 6 h. Then the volatile components
of the reaction mixture were evaporated under reduced pressure. The
solid residues (compounds 1a-h) were washed several times with
distilled water to obtain the pure products. The oily residues (compounds
1i,j) were crystallized from water/ethanol or water/methanol systems.
General Procedure for the Preparation of Hydroxyphosphonic Acids
(2) (21). A 1.83 mL aliquot of diethyl phosphite (1.96 g, 14.23 mmol)
was added under stirring to a mixture of magnesium oxide (1.0 g) and
the proper aromatic aldehyde (14.23 mmol) at room temperature. After
12 h dichloromethane (20 mL) was added, and the mixture was filtered
through celite. Then the solvent was evaporated under reduced pressure.
The light yellow residue was washed with distilled water to give the
pure ester, which was either refluxed for 6 h with 20% aqueous
hydrochloric acid (for compounds 2a-f) or stirred at room temperature
for 20 h with a 5-fold molar excess of bromotrimethylsilane in dry
dichloromethane (compound 2g), and then methanol was added.
Solvents were evaporated under reduced pressure. The oily residues
crystallized after 1–2 h, yielding pure solid products.
as previously described (19). A combination of ammonium sulfate
precipitation, negative chromatography at pH 7.5 on a DEAE-Sephacel
column, and anion-exchange chromatography at pH 10.0 on the same
column resulted in a 60-fold enrichment, with a 40% yield. Three
different enzyme preparations were used. The mean value for a specific
activity level in these samples was 46.2 ( 2.1 nkat mg-1. Proper checks
were done to rule out the presence in the final preparations 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 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.
Enzyme Assays. The physiological, forward reaction of P5C
reductase was measured by following the P5C-dependent oxidation of
NADH. The assay mixture contained 100 mM Tris-HCl buffer, pH
8.0, 0.1 mM MgCl2, 2 mM DL-P5C, and 0.25 mM NADH, in a final
volume of 1 mL. A limiting amount of enzyme (0.15–0.20 nkat) was
added to the prewarmed mixture, and the decrease in absorbance at
340 nm was determined at 35 °C for up to 10 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 by the method of Bradford (25), using
bovine serum albumin as the standard.
General Procedure for the Preparation of Aminophosphonic Acids
(3a-d) (22). A 0.72 g amount of ammonium formate (11.43 mmol)
and 2.0 g of acidic alumina were ground in a mortar until a fine,
homogeneous powder was obtained (5–10 min). Then the proper
aromatic aldehyde (11.43 mmol) was combined (solid aldehydes needed
to be ground before), and 1.50 mL of diethyl phosphite (1.58 g, 11.43
mmol) was added slowly. Following 12–16 h of vigorous stirring, the
reaction mixture was extracted with diethyl ether (250 mL). The solvent
was evaporated to dryness resulting in oily products, which were left
in a refrigerator for several days until the corresponding ester
crystallized. This was dissolved in dry dichloromethane (20 mL), and
a 5-fold molar excess of bromotrimethylsilane was added. After stirring
the mixture for 20 h at room temperature, methanol (5 mL) was added,
and 1 h later the mixture was evaporated under reduced pressure. The
oily residue crystallized after 1–2 h to give pure solid product.
General Procedure for the Preparation of Aminophosphonic Acids
(3e-i) (23). Acetamide (0.2 mol) was dissolved in acetic acid (40 mL)
and cooled in an ice bath. Then acetyl chloride (0.1 mol) was added
with cooling, and the formation of a crystalline byproduct was observed.
After 15 min the proper aldehyde (0.1 mol) was added, and the mixture
was kept for 30 min in an ice bath and left for a day at room
temperature. Then the mixture was cooled once more in an ice bath,
and phosphorus trichloride (0.1 mol) was added. The resulting mixture
was kept in the bath for 30 min, then allowed to warm to room
temperature, and finally heated for 1 h at 70–75 °C. Evaporation of
volatile components of the reaction mixture yielded an oily product,
which was refluxed for 8 h in concentrated aqueous hydrochloric acid
(100 mL). The acid was evaporated in vacuo, and the resulting product
was dissolved in ethanol (50 mL) and left until ammonium chloride
completely precipitated; then the latter was filtered off, and ethanol
was evaporated under reduced pressure. The obtained oily residue was
dissolved in ethanol (50 mL), and the aminophosphonate was precipi-
tated by the addition of pyridine and purified by recrystallization from
a water/ethanol system.
Enzyme Inhibition by Phosphonic Acids. P5C reductase inhibition
was evaluated by adding to the reaction mixture an appropriate dilution
of a 20 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
5 µM to 5 mM. At least three measurements were performed for each
dose. The concentrations causing 50% inhibition (IC50) of P5C reductase
activity were estimated utilizing the linear regression equation of
enzyme activity values, expressed as a percentage of untreated controls,
plotted against the logarithm of inhibitor concentration. At least three
concentrations in the rectilinear part of the resulting sigmoidal curve
were considered. Confidence limits of IC50 values were computed
according to Snedecor and Cochran (26).
In Vivo Inhibition and Reversal Experiments. To measure the
effect of some of the most active phosphonates on exponentially
growing cells, samples were withdrawn from the stock cultures in the
late exponential phase of growth and used to inoculate 100 mL culture
flasks to a density of 3.2–3.5 mg mL-1 (dry weight) in a final volume
of 25 mL. Filter-sterilized compounds (brought to pH 6.0 with KOH)
were added just after the density of the cell population reached 4.0 mg
mL-1 (dry weight). After a further 8 days of incubation, when untreated
controls reached the early stationary phase of growth, cells were
harvested by vacuum filtration, and the dry weight increase was
determined on each sample following oven drying at 90 ( 1 °C for
48 h. The same protocol was adopted for reversal experiments, where
inhibitor and amino acid supplements were added to the culture medium
simultaneously. Proline was added from a filter-sterilized 1.0 M solution.
In the case of glutamine, a freshly prepared 200 mM solution was used
to avoid spontaneous hydrolysis of the amide moiety. At least four
replicates were carried out for each treatment.
Structure–Activity Relationship Analysis. CoMFA 3D-QSAR
analysis was done within the QSAR module of Sybyl 6.9.1, Tripos
(Discovery Software, 2003), using default settings. Structures exhibiting
very low inhibitory activity (pIC50 < 1.50) were not used for
computations, as their mode of binding could be completely different
from that of highly active analogues. Additionally, another compound
that had been previously found to inhibit P5C reductase, namely, N-(3,5-
dibromo-6-methylpyridin-2-yl)aminomethylenebisphosphonic acid, was
also included in 3D-QSAR study. The structure of the most active
compound 1a was taken from the previous study (19). The other
inhibitor structures were constructed by appropriate modification of
the parent compound and minimization using the Tripos force field
and conjugate gradient minimizer. The charge for each atom in
minimized structure was computed using the MMF94 method. Mini-
mized structures were aligned by superimposition of phosphonate(s)
and aromatic ring atoms on the corresponding groups in the structure
of the lead compound 1a.
DL-P5C was synthesized by the periodate oxidation of δ-allo-
hydroxylysine and purified by cation-exchange chromatography on a
Dowex AG50 (200–400 mesh) column, as described in ref 24.
Plant Cell Culture and Growth Conditions. Suspension-cultured
cells 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 130 mL of MS medium with
0.3% (w/v) sucrose and 0.5 mg L-1 of both (2,4-dichlorophenoxy)acetic
acid and 6-benzylaminopurine. Subcultures were made every 10 days
by transferring 30 mL aliquots of the suspension to 100 mL of fresh
medium.
P5C Reductase Purification. The enzyme was partially purified
from cultured cells harvested in the early stationary phase of growth,

3196 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Forlani et al.
Table 1. Inhibition of Thale Cress P5C Reductase by Analogues of
(3,5-Dichlorophenyl)aminomethylenebisphosphonic Acid (Compound 1a)a
compound
IC50 (mM)
pIC50 (M)
1a
1b
1c
1d
1e
1f
1g
1h
1i
0.077 ( 0.011
0.102 ( 0.038
0.50 ( 0.19
0.73 ( 0.24
0.112 ( 0.059
0.268 ( 0.018
0.86 ( 0.37
1.07 ( 0.19
74.4 ( 50.6
533 ( 488
8.1 ( 2.1
4.114
3.991
3.301
3.137
3.951
3.572
3.066
2.971
1.128
0.273
2.092
2.149
2.292
2.310
2.854
2.237
1.342
2.337
1.975
2.086
1.449
0.449
2.180
-0.486
1.201
1.796
1j
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
3f
7.1 ( 2.8
5.1 ( 1.2
4.9 ( 1.2
Figure 2. Effect of the most effective inhibitors of plant P5C reductase
upon the growth of A. thaliana suspension cultured cells. The compounds
were added to the culture medium in the early exponential phase of growth.
After 8 days of incubation, when untreated controls reached the early
stationary phase, the resulting dry weight increase was determined and
expressed as a percent of that in the controls (6.15 ( 0.22 mg mL-1, n
) 8). Data are means ( SD over four replicates.
1.4 ( 0.4
5.8 ( 2.4
45.5 ( 29.1
4.6 ( 1.3
10.6 ( 6.1
8.2 ( 4.2
35.6 ( 31.7
356 ( 235
6.6 ( 4.8
3060 ( 2950
63 ( 44
16.0 ( 7.4
3g
3h
3i
exhibit the highest inhibitory potential. The comparison of the
effect of compounds with the same substituents at the aromatic
portion of the molecule (e.g., 1b versus 2d and 3a, or 1c versus
2f and 3c, respectively) revealed that their efficacy as inhibitors
is reduced by 1 order of magnitude when the second phospho-
nate moiety is replaced with either a hydroxyl or an amino
group. Bisphosphonates 1a-j are achiral, whereas the corre-
sponding hydroxyphosphonates and aminophosphonates are
recemic mixtures, yet the difference between their activity
largely exceeded that expected between a single, active com-
pound and the racemic mixture cointaining also the inactive
enantiomer. Threfore, the presence of two phosphonate residues
in the molecule seems to play a main role in its effectiveness.
As to the substituents in the phenyl ring, data did not provide
any plain pattern: neither the presence of one or two chlorines
nor their position in the phenyl ring seems to vary exceedingly
the resulting inhibitory potential. However, the occurrence of
only one chlorine at position 4, as in compounds 1g and 2g,
significantly reduced the ability to inhibit the enzyme. On the
other hand, results obtained with aminophosphonates confirmed
previous data on bisphosphonates (19), suggesting that the
replacement of chlorine with other substituents, either halogen
moieties (F, Br) or other groups (methyl, methoxyl), produces
a significant loss of activity. The same was true in the case of
compound 1h, in which the two chlorines in the lead compound
are replaced with methyl groups.
In Vivo Phytotoxicity of the Most Active Aminobisphos-
phonates and Its Reversal by Exogenously Supplied Amino
Acids. To investigate whether the interference of the most active
derivatives with P5C reductase activity may take place also in
vivo, and cause an actual reduction of proline biosynthesis that
could result in phytotoxic effects at the plant level, the growth
of A. thaliana suspension cultured cells was measured following
the addition to the culture medium of increasing concentrations
of compounds 1a,b,e. In fact several factors, including dif-
ferential membrane permeability/solubility, subcellular com-
partmentalization, and occurrence of metabolization/detoxifi-
cation reactions, can completely abolish inside the cell the
inhibitory potential found in vitro for a given compound. Results,
shown in Figure 2, indeed pointed out a significant reduction
of the dry weight increase in treated cultures that was propor-
tional to the dose added. Once again, the most remarkable effect
a Activity was evaluated as described under Materials and Methods either in
the absence or in the presence of a given phosphonate at concentrations ranging
from 0.005 to 5 mM. Each test was carried out in triplicate, and values were
expressed as the percentage of untreated controls. The concentrations causing
50% inhibition (IC50) of in vitro activity were estimated utilizing the linear regression
equation of the activity values plotted against the logarithm of inhibitor concentration.
Confidence limits were computed according to the method of Snedecor and Cochran
(26).
RESULTS AND DISCUSSION
In Vitro Activity of (3,5-Dichlorophenyl)aminomethyl-
enebisphosphonic Acid Derivatives against P5C Reductase.
Aiming at the development of new active principles targeting
the activity of plant P5C reductase, three groups of compounds
structurally related to a previously described (19) inhibitor of
the enzyme (compound 1a) were obtained: some bisphospho-
nates (1b-j), some hydroxyphosphonates (2), and some ami-
nophosphonates (3) (Figure 1). They were designed taking into
account the results of the previous screening of 26 different
bisphosphonates, most of which had been found to be com-
pletely ineffective (19), in order to investigate the effect of either
the number and the position of the substituents in the phenyl
ring, or the scaffold structure, i.e., the presence of two
phosphonic moieties. To evaluate these compounds, P5C
reductase was partially purified from suspension cultured cells
of A. thaliana by means of the protocol set up in the previous
work. The choice of such an experimental system allowed us
to obtain the large quantities of the enzyme that were required
to carry out hundreds of assays, in the absence of both interfering
activities and multiple enzyme forms. The presence of isoforms
with different susceptibility could in fact constitute a significant
drawback in assessing the efficacy of a given compound.
Moreover, cell suspension cultures represent a well-suited
system for verifying the occurrence of enzyme inhibition in vivo.
The activity of thale cress P5C reductase was measured in
the absence or in the presence of the 26 compounds in the
micromolar to millimolar range. Results, summarized in Table
1 as the concentrations causing 50% inhibition of enzyme
activity (IC50), clearly showed that bisphosphonate analogues

New Bisphosphonate Inhibitors of Plant P5C Reductase
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3197
Figure 4. Superimposition of selected representatives of bisphosphonic
(1a), hydroxyphosphonic (2a), and aminophosphonic (3a) acids.
Figure 3. Reversal of 1a toxicity by exogenously supplied amino acids.
The compounds (10 mM glutamine and 2 mM proline) were filter-sterilized
and added, either singly or in combination, to the culture medium in the
early exponential phase of growth, at the same time as the inhibitor (0.2
mM). After 8 days of incubation, the resulting dry weight increase was
determined and expressed as a percent of that in the untreated control
(6.22 ( 0.14 mg mL-1). Data are means ( SD over four replicates.
Significant differences between treated cultures and the corresponding
amino acid-fed controls are marked (/, P < 0.10; //, P < 0.05; ///, P
< 0.01; NS, not significant).
P5C reductase inhibition does occur in vivo and suggest that
the phytotoxicity of compound 1a may be due to inhibition of
key enzymes in both pathways for proline and glutamine
synthesis.
Structure–Activity Relationship Analysis. To shed some
further light on the relationships between the structure and the
activity of P5C reductase inhibitors, a comparative molecular
field analysis (CoMFA) was performed using the Sybyl program
(Tripos, Discovery Software). The structure of active inhibitors
was superimposed on that of the most potent compound 1a
(Figure 4), and molecular field was computed. When applied
to our data, the partial least squares method yielded high-
correlation coefficients [cross-validated q2 ) 0.69 (by leave-
one-out method) and non-cross-validated r2 ) 0.98, with six
principal components], confirming the statistical significance of
the analysis. The significance of non-cross-validated r2 was
proven by F-test, yielding F(n1 ) 6, n2 ) 13) ) 108.5.
Additionally, the leave-multiple-out method with five groups
was applied for testing the model. For 20 runs, mean q2 ) 0.70
and mean standard error of prediction 0.57 were obtained. The
3D-QSAR CoMFA map (Figure 5) showed regions of possible
steric (green ) favored, yellow ) unfavored) and electrostatic
(blue ) positively charged, red ) negatively charged) interac-
tions. The first phosphonic group is not marked, since it
represents the constant element in the whole set of inhibitors.
Three red areas indicate favorable, negatively charged groups:
one located, as expected, near the second phosphonate moiety
and two in the place of substituents of the phenyl ring (which
could correspond to the two chlorine atoms in compound 1a).
A blue area near the NH moiety of compounds 1 represents a
favorable, positively charged group. The CoMFA map does not
show any region covering the aromatic ring. However, data
suggest that the electron density in this part of the molecule is
important, as indicated by the comparison of the effectiveness
of compounds 1a,h, which are structurally, but not electronically,
similar. Whereas the former possesses electron-withdrawing
chlorine substituents, and exhibits a pIC50 ) 4.11, the latter
has electron-donating methyl groups and indeed shows a
biological activity that is more than 1 order of magnitude lower.
Thus, a low electron density on the aromatic ring seems to favor
increased activity.
was carried out by compound 1a, with a concentration causing
50% reduction of cell growth of 0.24 ( 0.04 mM, yet the
noteworthy difference in the effectiveness in vivo of the three
compounds seems poorly consistent with the almost negligible
diversity of their efficacy in vitro against the purified enzyme.
Even though a contribution of the above-mentioned factors
cannot be ruled out, such an apparent discrepancy may reflect
the ability to interfere with other target(s). In fact, compound
1a had been previously shown to inhibit also the activity of
glutamine synthetase, with an IC50 against rice GS of 33 ( 1
µM (18), and several of its analogues seem to share this
capability (29). However, this could also imply that the
phytotoxic effects shown in Figure 2 may be completely
unrelated with a block in proline biosynthetic pathways.
Direct evidence supporting the occurrence of P5C reductase
inhibition inside the cell might come from the measurement of
intracellular free amino acid pools. In some other cases, the
comparison of pools in treated versus untreated cells confirmed
the mode of action previously hypothesized for a given phos-
phonate on the basis of biochemical evidence in vitro (16-18).
However, in the current case this approach would not be
informative. Because of the concurrent reduction of glutamine/
glutamate synthesis caused by GS inhibition (18), a decrease
of proline synthesis could in fact occur even in the absence of
any direct intereference with the two anabolic pathways leading
to proline production (27). As an alternative, reversal experi-
ments may be carried out. The exogenous supply of the
product(s) of a metabolic route should result in a complete relief
of the effects caused by the inhibition of whatsoever step
upstream (e.g., see refs 14 and 28). The reversal of action of
compound 1a by feeding treated cells with amino acids was
therefore evaluated. Exogenously supplied glutamine and proline
were able to counteract completely the growth inhibition only
when supplied together. If provided singly, they actually
ameliorated the resulting increase in cell dry mass, but failed
in achieving full restoration of cell growth rate (Figure 3).
Overall, the results seem thus to strengthen the likelihood that
Interestingly, the presence in the 3D-QSAR CoMFA map of
regions describing steric features of the inhibitors is very limited
(Figure 5). Most likely, this is a consequence of the data set
used, which consists of compounds with similar structure.
However, the comparison of the results presented here with those
reported in the previous paper (19), where the binding of 1a in

3198 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Forlani et al.
Figure 5. Isosurfaces (regions of 20 or 80%-iles of StdDev*Coef values) generated by 3-D QSAR COMFA analysis of P5C reductase inhibitors and the
structure of the most potent inhibitor (1a). Green and yellow regions show positive and negative steric interactions, respectively, while blue and red ones
describe favorable interactions of positively and negatively charged groups. The correlation between predicted and actual values of pIC50 is presented
as an inset.
ACKNOWLEDGMENT
the active site of P5C reductase was investigated, strongly
suggests that steric demands are also substantial. These require-
ments result from the shape of the catalytic pocket, with which
the inhibitor interacts, that is relatively deep and tight.
The calculations were carried out using hardware and software
resources (including the Tripos programs) of the Supercomput-
ing and Networking Centre in Wrocław.
Several analogues of the previously described (19) compound
1a were herein analyzed for the ability of interfering with the
catalytic rate of plant P5C reductase. Derivatives substituted in
the phenyl ring retained a significant effectiveness, whereas the
replacement of the second phosphonate moiety with an amino
or a hydroxyl residue resulted in a strong reduction of the
inhibitory potential. Although the possibility that some further
modifications in the scaffold could increase their efficacy against
P5C reductase cannot be excluded, the presence of two
phosphonic groups seems a main structural feature to target the
active site of the enzyme. Experiments with plant cell suspension
cultures confirmed in vivo toxicity of the most active com-
pounds. Moreover, the reversal effect of exogenously supplied
amino acid suggested an actual occurrence of proline starvation
in treated cultures and strengthened the possibility that P5C
reductase inhibition does occur inside the cell. Notwithstanding
this, none of the tested analogues showed higher efficacy than
the lead compound, and all the most active derivatives bear
chlorine atoms in the phenyl ring. Being aromatic chlorinated
molecules, most likely endowed with high recalcitrancy, these
compounds themselves are not optimal candidates as new active
principles for weed control. However, a lower but significant
inhibitory potential was retained by compound 1h, in which
the two chlorine moieties of 1a are replaced with methyl groups.
Furthermore, the availability of several active structures allowed
a proper SAR analysis, one that provided us with useful
informations concerning the steric and electronic features that
are required to maintain the inhibitory effectiveness. Work is
currently in progress in our laboratories to accordingly design,
synthesize, and test nonchlorinated analogues, ones that would
combine the favorable steric features of 1h with the presence
of electron-withdrawing moieties that may result in a lower,
optimal electron density of the phenyl ring, as in 1a.
Supporting Information Available: Yields and spectral data
for compounds 1-3 (pdf). This material is available free of
charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
(1) Mahajan, S.; Tuteja, N. Cold, salinity and drought stresses: An
overview. Arch. Biochem. Biophys. 2005, 444, 139–158.
(2) Seki, M.; Kamei, A.; Yamaguchi-Shinozaki, K.; Shinozaki, K.
Molecular responses to drought, salinity and frost: Common and
different paths for plant protection. Curr. Opin. Biotechnol. 2003,
14, 194–199.
(3) Verslues, P. E.; Agarwal, M.; Katiyar-Agarwal, S.; Zhu, J. H.;
Zhu, J. K. Methods and concepts in quantifying resistance to
drought, salt and freezing, abiotic stresses that affect plant water
status. Plant J. 2006, 45, 523–539.
(4) Rathinasabapathi, B. Metabolic engineering for stress tolerance:
Installing osmo-protectant synthesis pathways. Ann. Bot. 2000,
86, 709–716.
(5) Kavi Kishor, P. B.; Sangam, S.; Amrutha, R. N.; Sri Laxmi, P.;
Naidu, K. R.; Rao, K. R. S. S.; Rao, S.; Reddy, K. J.; Theriappan,
P.; Sreenivasulu, N. Regulation of proline biosynthesis, degrada-
tion, uptake and transport in higher plants: Its implications in plant
growth and abiotic stress tolerance. Curr. Sci. 2005, 88, 424–
438.
(6) Yoshiba, Y.; Kiyosue, T.; Nakashima, K.; Yamaguchi-Shinozaki,
K.; Shinozaki, K. Regulation of levels of proline as an osmolyte
in plants under water stress. Plant Cell Physiol. 1997, 38, 1095–
1102.
(7) Hare, P. D.; Cress, W. A. Metabolic implications of stress-induced
proline accumulation in plants. Plant Growth Regul. 1997, 21,
79–102.
(8) Hare, P. D.; Cress, W. A.; van Staden, J. A regulatory role for
proline metabolism in stimulating Arabidopsis thaliana seed
germination. Plant Growth Regul. 2003, 39, 41–50.

New Bisphosphonate Inhibitors of Plant P5C Reductase
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3199
(9) Schwacke, R.; Grallath, S.; Breitkreuz, K. E.; Stransky, E.;
Stransky, H.; Frommer, W. B.; Rentsch, D. LeProT1, a transporter
for proline, glycine betaine, and γ-amino butyric acid in tomato
pollen. Plant Cell 1999, 11, 377–391.
(10) Kafarski, P.; Lejczak, B.; Forlani, G. Herbicidally active ami-
nomethylenebisphosphonic acids. Heteroatom Chem. 2000, 11,
449–453.
(11) Kafarski, P.; Lejczak, B.; Forlani, G. Biodegradation of pesticides
containing carbon-to-phosphorus bond. In Pesticide Biotransfor-
mation in Plants and Microorganisms; Hall, J. C., Hoagland, R. E.,
Zablotowicz, R. M., Eds.; ACS Symposium Series 777; American
Chemical Society: Washington, DC, 2001; pp 145–163.
(12) Quinn, J. P.; Kulakova, A. N.; Cooley, N. A.; McGrath, J. W.
New ways to break an old bond: The bacterial carbon-phosphorus
hydrolases and their role in biogeochemical phosphorus cycling.
EnViron. Microbiol. 2007, 9, 2392–2400.
(13) Berlicki, Ł.; Kafarski, P. The use of molecular modelling for
comparison of three possible modes of action of herbicidally active
derivatives of aminomethylene bisphosphonic acid. Pest. Biochem.
Physiol. 2002, 73, 94–103.
(14) Forlani, G.; Kafarski, P.; Lejczak, B.; Wieczorek, P. Mode of
action of herbicidal derivatives of aminomethylenebisphosphonic
acid. II. Reversal of herbicidal action by aromatic amino acids.
J. Plant Growth Regul. 1997, 16, 147–152.
(15) Forlani, G.; Lejczak, B.; Kafarski, P. N-pyridyl-aminomethylene-
bisphosphonic acids inhibit the first enzyme in the shikimate
pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase.
Pestic. Biochem. Physiol. 1996, 55, 180–188.
(16) Forlani, G.; Lejczak, B.; Kafarski, P. The herbicidally active
compound N-2-(6-methyl-pyridyl)-aminomethylene-bisphosphonic
acid inhibits in ViVo aromatic biosynthesis. J. Plant Growth Regul.
1999, 18, 73–79.
bisphosphonic acid inhibit plant glutamine synthetase. J. Agric.
Food Chem. 2004, 52, 3337–3344.
(19) Forlani, G.; Giberti, S.; Berlicki, Ù.; Petrollino, D.; Kafarski, P.
Plant P5C reductase as a new target for aminomethylenebispho-
sphonates. J. Agric. Food Chem. 2007, 55, 4340–4347.
(20) Soloducho, J. ; Gancarz, R.; Wieczorek, P. ; Korf, J. ; Hafner, J. ;
Lejczak, B.; Kafarski, P. Patent PL93-298436 93408, 1993.
(21) Kaboudin, B. A convenient synthesis of 1-aminophosphonates
from 1-hydroxyphosphonates. Tetrahedron Lett. 2003, 44, 1051–
1053.
(22) Kaboudin, B.; Nazari, R. Microwave-assisted synthesis of 1-ami-
noalkyl phosphonates under solvent-free conditions. Tetrahedron
Lett. 2001, 42, 8211–8213.
(23) Soroka, M.; Jaworska, D.; Szczêsny, Z. Synthesis of 1-aminoalkyl-
phosphonic acids via amidoalkylation of phosphorous acid by
N,N′alkylidene bis-amides. Liebigs Ann. Chem. 1990, 1153–1155.
(24) Forlani, G.; Scainelli, D.; Nielsen, E. ∆1-pyrroline-5-carboxylate
dehydrogenase from cultured cells of potato. Purification and
properties. Plant Physiol. 1997, 113, 1413–1418.
(25) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248–254.
(26) Snedecor, G. W.; Cochran, W. G. Statistical methods, 8th ed.;
The Iowa State University Press: Ames, IA, 1989.
(27) Brugiere, N.; Dubois, F.; Limami, A. M.; Lelandais, M.; Roux,
Y.; Sangwan, R. S.; Hirel, B. Glutamine synthetase in the phloem
plays a major role in controlling proline production. Plant Cell
1999, 11, 1995–2011.
(28) Grossmann, K.; Ehrhardt, T. On the mechanism of action and
selectivity of the corn herbicide topramezone: A new inhibitor of
4-hydroxyphenylpyruvate dioxygenase. Pest Manag. Sci. 2007,
63, 429–439.
(29) Occhipinti, A.; Forlani, G. Unpublished results.
(17) Forlani, G.; Lejczak, B.; Kafarski, P. The herbicidally active
compound N-2-(5-chloro-pyridyl)-aminomethylene-bisphosphonic
acid acts by inhibiting both glutamine and aromatic amino acid
biosynthesis. Aust. J. Plant Physiol. 2000, 27, 677–683.
(18) Obojska, A.; Berlicki, Ù.; Kafarski, P.; Lejczak, B.; Chicca, M.;
Forlani, G. Herbicidal pyridyl derivatives of aminomethylene-
Received for review January 4, 2008. Revised manuscript received
February 22, 2008. Accepted February 25, 2008. Support from the
University of Ferrara (Fondo d’Ateneo per la Ricerca 2007 and 2008)
is gratefully acknowledged.
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