Inhibitors of phenylalanine ammonia-lyase: Substituted derivatives of 2-aminoindane-2-phosphonic acid and 1-aminobenzylphosphonic acid

Article abstract of DOI:10.1016/j.phytochem.2006.11.022

Six derivatives of 2-aminoindane-2-phosphonic acid and 1-aminobenzylphosphonic acid were synthesized. The compounds were tested both as inhibitors of buckwheat phenylalanine ammonia-lyase (in vitro) and as inhibitors of anthocyanin biosynthesis (in vivo). (±)-2-Amino-4-bromoindane-2-phosphonic acid was found to be the strongest inhibitor from investigated compounds. The results obtained are a basis for design of phenylalanine ammonia-lyase inhibitors useful in the enzyme structure studies in photo labelling experiments.

Full text of DOI:10.1016/j.phytochem.2006.11.022

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 407–415
www.elsevier.com/locate/phytochem
Inhibitors of phenylalanine ammonia-lyase: Substituted
derivatives of 2-aminoindane-2-phosphonic acid and
1-aminobenzylphosphonic acid
a
a,
b
a
Piotr Miziak , Jerzy Zon *, Nikolaus Amrhein , Roman Gancarz
´
a
Faculty of Chemistry, Department of Medicinal Chemistry and Microbiology, Wrocław University of Technology, Wybrzez_e Wyspian´skiego
27, 50-370 Wrocław, Poland
b
Institute of Plant Sciences, ETH Zu¨rich, CH-8092 Zu¨rich, Switzerland
Received 19 September 2006; received in revised form 9 November 2006
Available online 9 January 2007
Abstract
Six derivatives of 2-aminoindane-2-phosphonic acid and 1-aminobenzylphosphonic acid were synthesized. The compounds were
tested both as inhibitors of buckwheat phenylalanine ammonia-lyase (in vitro) and as inhibitors of anthocyanin biosynthesis
(in vivo). ( )-2-Amino-4-bromoindane-2-phosphonic acid was found to be the strongest inhibitor from investigated compounds. The
results obtained are a basis for design of phenylalanine ammonia-lyase inhibitors useful in the enzyme structure studies in photo labelling
experiments.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Phenylalanine ammonia-lyase; Inhibitor; 2-Aminoindane-2-phosphonic acid; 2-Amino-4-bromoindane-2-phosphonic acid; (+)-1-Amino-30,40-
dichlorobenzylphosphonic acid
´
´
1. Introduction
lyase (Zon and Amrhein, 1992; Appert et al., 2003; Zon
et al., 2004, 2002). Recently, 5-substituted derivatives of
2-aminoindane-2-phosphonic acid have been synthesized
Phenylalanine ammonia-lyase (PAL, EC 4.1.3.5) is a
´
plant enzyme which catalysis elimination of ammonia from
and tested (Zon et al., 2005). Here, we describe the synthe-
´
(S)-phenylalanine (Poppe and Retey, 2005; Pilbak et al.,
sis of new derivatives of AIP (1–4, Fig. 1) as well as deriv-
atives of 1-aminobenzylphosphonic acid (5 and 6, Fig. 1).
The obtained compounds were evaluated as inhibitors of
buckwheat PAL as well as inhibitors of anthocyanin bio-
2006). The enzyme plays an important role in secondary
metabolite pathways (Croteau et al., 2000). In spite of
much recent work on the structure and catalytic mecha-
nism of PAL, the precise binding site of phenylalanine in
´
synthesis in buckwheat hypocotyls (Zon and Amrhein,
´
´
the enzyme is not known (Poppe and Retey, 2005; Pilbak
1992; Zon et al., 2002, 2005).
et al., 2006). Inhibitors of PAL containing a photoaffinity
group may therefore be useful tools in studies of PAL
structure and function.
2. Results and discussion
2-Aminoindane-2-phosphonic acid (AIP) and (+)-1-
amino-30,40-dichlorobenzylphosphonic acid have been
found to be potent inhibitors of phenylalanine ammonia-
The derivatives of 2-aminoindane-2-phosphonic acid
(1–4, Fig. 1) were synthesized from derivatives of 1,2-
bis(bromomethyl)benzene (7–9 and 19, Scheme 1) by
´
known methods (Zon, 1979). First, 4-substituted deriva-
*
Corresponding author. Tel.: +48 71 320 32 60; fax: +48 71 328 40 64.
tives of ethyl 2-(diethoxyphosphoryl)indane-2-carboxylates
´
E-mail address: jerzy.zon@pwr.wroc.pl (J. Zon).
0031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.11.022

408
P. Miziak et al. / Phytochemistry 68 (2007) 407–415
X
Br
PO3H-
PO3H-
Br
PO3H-
PO3H-
( ) -
( ) -
( ) -
+
X
+
NH3
+
NH3
NH3
Br
+
NH3
Y
Br
1 X= OH; 2 X = Br
5 X = Cl, Y = NO2;
6 X = H, Y = C6H5CO
3
4
Fig. 1. The structures of synthesized and evaluated compounds in this work.
X
Y
X
Y
COOC2H5
(i)
(ii)
COOC2H5
Br
Br
( ) -
+
PO(OC2H5)2
PO(OC2H5)2
10 X = MeO, Y = H
11 X = Br, Y = H
12 X = H, Y = CH3
7 X = MeO, Y = H
8 X = Br, Y = H
9 X = H, Y = CH3
X
Y
X
Y
X
Y
(iii)
COOH
+
NHCOOCH2C6H5
PO(OC2H5)2
(iv)
NH3
PO3H-
( ) -
( ) -
( ) -
PO(OC2H5)2
13 X = MeO, Y = H
14 X = Br, Y = H
15 X = H, Y = CH3
16 X = MeO, Y = H
17 X = Br, Y = H
18 X = H, Y = CH3
2 X = Br, Y = H
3 X = H, Y =
CH3
Br
Br
Br
COOC2H5
Br
Br
Br
Br
COOC2H5
PO(OC2H5)2
Br
Br
(v)
(ii)
COOH
Br
Br
+
PO(OC2H5)2
PO(OC2H5)2
Br
Br
Br
20
19
21
Br
Br
(iii)
Br
Br
NHCOOCH2C6H5
PO(OC2H5)2
+
(vii)
Br
Br
NH3
PO3H-
Br
Br
22
4
O
OH
OH
NHCOOCH2C6H5
PO(OC2H5)2
(vi)
+
(iv)
NHCOOCH2C6H5
PO(OC2H5)2
NH3
( ) -
( ) -
( ) -
PO3H-
16
1
Scheme 1. Reagents and conditions: (i) NaOH, C6H5CH3, (C4H9)4NBr, 14ꢁ ! RT; (ii) NaOH, H2O, reflux; (iii) C6H5CH3, (C6H5)2P(O)N3, C6H5CH2OH,
reflux; (iv) conc. HCl, H2O, reflux, 20 h; (v) THF, NaH, RT and then heated to reflux; (vi) CH2Cl2, BBr3, O ꢁC ! RT; (vii) 40% HBr in water–
CH3COOH, reflux, 10 h.
(10–11 and 20, Scheme 1) were synthesized. These com-
pounds (10–12 and 20) were obtained with lower yields
than the corresponding 5-substituted ethyl 2-(diethoxy-
carboxylates (10–12 and 20, Scheme 1) were hydrolysed
with high yield to the corresponding carboxylic acids (13–
15 and 21, Scheme 1). Next, after the Curtius degradation
of the carboxylic acids, the urethane-phosphosphonates
(16–18 and 22, Scheme 1) were formed. Finally, the com-
plete deprotection of the functionality gave the derivatives
of 2-aminoindane-2-phosphonic acids (1–4, Scheme 1).
´
phosphoryl)indane-2-carboxylates (Zon et al., 2005). Ethyl
2-(diethoxyphosphoryl)-1-methylindane-2-carboxylate (12)
was obtained in medium yield as a single diastereoisomer,
as determined by 31P NMR. In the second step, the ethyl

P. Miziak et al. / Phytochemistry 68 (2007) 407–415
409
Table 1
The derivatives of substituted 1-aminobenzylphos-
phonic acid (5–6, Fig. 1) were obtained in a modified
Green’s et al. hydrophosphonylation procedure using
diphenylmethylamine (benzhydrylamine) as a source of
the amino group (Green et al., 1994). Pure diethyl 1-(diphe-
nylmethylamino)benzylphosphonates (27 and 28, Scheme
2) were completely deprotected in a simple acidic hydroly-
sis to yield the corresponding aminophosphonic acids 5
and 6 (Scheme 2).
The six new derivatives of 2-aminoindane-2-phosphonic
acid and 1-aminobenzylphosphonic acid (1–6, Fig. 1) were
evaluated as inhibitors of buckwheat phenylalanine ammo-
nia-lyase and as well as inhibitors of anthocyanin biosyn-
In vitro (Ki) and in vivo (IC50) inhibitory activity of obtained amino-
phosphonic acids (1–6)
Compound
Ki, buckwheat PALa (lM)
IC50 anthocyaninb (lM)
1
1.95
0.09
8.64
n.d.c
7.27
n.d.c
0.08e
60.26
33.88
851.14
n.d.d
19.95
n.d.c
2
3
4
5
6
AIP
1.5e
a
Standard deviations for Ki values were in the range of 5–8%.
Standard deviations for IC50 values were in the range of 10–15%.
Inhibition about 90% for the 1 mM inhibitor concentration.
Inhibition about 70% for the 1 mM inhibitor concentration.
AIP is the strongest in vivo PAL inhibitor and was used in this studies
b
c
d
e
´
thesis in buckwheat hypocotyls (Zon and Amrhein, 1992;
´
Zon et al., 2002, 2005). The results are presented in Table 1.
´
as standard. The data were taken from the literature (Zon and Amrhein,
1992).
2-Amino-4-bromoindane-2-phosphonic acid (2) was the
strongest inhibitor both in PAL and anthocyanin tests. In
general 4-substituted derivatives of AIP are stronger inhib-
itors of PAL than corresponding 5-substituted compounds,
however IC50 is not changed. For example the inhibition
constant of 2-amino-4-bromoindane-2-phosphonic acid
(2, Ki = 0.09 lM, IC50 = 33.88 lM, Table 1) is one order
of magnitude lower than that for 2-amino-5-bromoin-
dane-2-phosphonic acid (Ki = 0.57 lM and IC50 = 39.8
A ratio of inhibition constants for enantiomers of any 4-
substituted derivatives of 2-aminoindane-2-phosphonic
acid (KSi =KRi ) are expected to be different by one order of
magnitude since for conformationally labile enantiomers
of 1-amino-2-phenylethylphosphonic acid we have found
it to be at that range (Laber et al., 1986; KSi ¼ 11:6 lM;
KRi ¼ 1:5 lM; KiS=KRi ꢀ 8).
´
lM) (Zon et al., 2005). A similar tendency was observed
for hydroxy substituted derivatives of 2-aminoindane-2-
phosphonic acid based on comparison of corresponding
data for 2-amino-4-hydroxyindane-2-phosphonic acid (1,
Table 1) with 2-amino-5-hydroxyindane-2-phosphonic acid
3. Conclusions
´
The results suggest that 4-substituted are promising
derivatives of 2-aminoindane-2-phosphonic acid contain-
ing a photoreactive group may lead to phenylalanine
ammonia-lyase inhibitors suitable for photoaffinity label-
ling of the enzyme and they are more promising than cor-
responding 5-substituted derivatives. In the line of
derivatives of 1-aminobenzylphosphonic acid we cannot
yet formulate similar conclusion as the above.
from the reference (Zon et al., 2005).
1-Amino-40-chloro-30-nitrobenzylphosphonic acid (5) is
a weaker inhibitor of PAL compared to compound 2 by
two orders of magnitude. The lower inhibitory activities
for compounds 4 and 6 can be explained by the presence
of too bulky substituents at the benzene ring, as our earlier
studies indicated that the PAL hydrophobic pocket is of
´
limited space (Zon et al., 2002).
(C6H5)2HCHN
( ) -
PO(OC2H5)2
(iii)
(C6H5)2HCN
CHO
(i)
(ii)
H2NCH(C6H5)2
+
Y
Y
Y
X
X
X
25 X = Cl, Y = NO2
26 X = H, Y = C6H5CO
23 X = Cl, Y = NO2
24 X = H, Y = C6H5CO
27 X = Cl, Y = NO2
28 X = H, Y = C6H5CO
+H3N
PO3H-
( ) -
Y
X
5 X = Cl, Y = NO2
6 X = H, Y = C6H5CO
Scheme 2. Reagents and conditions: (i) CH2Cl2, K2CO3, RT, 3 h; (ii) (C2H5O)2P(O)H, 100 ꢁC, 2 h; (iii) conc. HCl, H2O, reflux, 20 h.

410
P. Miziak et al. / Phytochemistry 68 (2007) 407–415
4. Experimental
during 20 min under a nitrogen atmosphere. Cooling and
stirring were continued for another 10 min. Then the cool-
ing bath was removed and the reaction mixture was grad-
ually warmed to 55 ꢁC and kept at 55 ꢁC during 13 h
while stirring. After cooling down to 20 ꢁC, 1 M NaOH
(360 ml) was added and the solution was stirred during
the next 1 h. Finally the solution was concentrated to 1/3
of its starting volume on a vacuum evaporator. The residue
was extracted with ethyl acetate (3 · 60 ml). The combined
extracts were washed with brine (60 ml) and dried with
MgSO4. The solution was evaporated to obtain 1,2-
bis(hydroxymethyl)-3-methoxybenzene. The crude product
was crystallized from CHCl3 to give the pure diol: 1.41 g,
20%, mp 94–95 ꢁC (lit. Brewster and Jones, 1969; mp
92–93 ꢁC). Then 3-methoxy-1,2-bis(bromomethyl)benzene
(7) was obtained from 3-methoxy-1,2-bis(hydroxymethyl)-
benzene by modified literature procedures (Tolbert and
Haubrich, 1994; Oshakawa et al., 1997). To a solution of
3-methoxy-1,2-bis(hydroxymethyl)benzene (1.50 g, 8.92
mmol) in dry diethyl ether (55 ml) at 4–8 ꢁC, PBr3
(3.0 ml, 0.0312 mol) in dry diethyl ether (7 ml) was added
dropwise during 20 min. The mixture was heated at reflux
for 9 h and then poured into crushed ice (100 ml). The
ether layer was separated and the aqueous layer was
additionally extracted with diethyl ether (3 · 15 ml). The
combined ether extracts were washed with brine (25 ml)
and dried with Na2SO4. The ether was evaporated to give
1,2-bis(bromomethyl)-3-methoxybenzene (7): 2.23 g, 85%,
mp 80–81 ꢁC ((Oshakawa et al., 1997) mp 78–79 ꢁC).
1H NMR spectra were recorded at 300 MHz; 31P NMR
at 121 MHz; 13C NMR at 75 MHz on Bruker instruments.
For samples dissolved in CDCl3 the reference signal was
CHCl3: whereas for measurements in D2O–DCl it was
the external H2O. Melting points were determined using
a Boe¨tius apparatus and are uncorrected. Elemental
analyses were performed by the Laboratory of Elemental
Analysis of the Institute Organic Chemistry, Biochemistry
and Biotechnology, Wrocław. TLC was carried out on
commercially available pre-coated plates (Fluka silica gel
on TLC-PET foils with or without fluorescent indicator
254 nm).
The inhibition constants (Ki) for buckwheat PAL and
the concentrations of the compounds inhibiting anthocya-
nin production in illuminated buckwheat hypocotyls by
50% (IC50values) were determined as described previously
´
´
(Zon and Amrhein, 1992; Zon et al., 2002, 2005; Laber
et al., 1986).
4.1. 1,2-Bis(bromomethyl)-3-methoxybenzene (7)
1,2-Bis(bromomethyl)-3-methoxybenzene (7) was obtained
from commercially available 2,3-dimethylphenol. To a
solution of 2,3-dimethylphenol (100 g, 0.818 mol), dimethyl
sulfate (155 ml, 1.636 mol) and tetrabutylammonium
hydrogensulfate (22.79 g, 0.0818 mol) in acetone (500 ml)
and solid K2CO3 (339.2 g, 2.454 mol) was added gradually
while stirring. After initial exothermic reaction stirring
under reflux was continued for 24 h. The acetone solution
was filtered off and the precipitate washed with acetone
(3 · 350 ml). Acetone from the washings was evaporated
to yield an oily residue, which was dissolved in CHCl3
(150 ml) and consecutively washed with 1 M NaOH
(50 ml and 30 ml), water (2 · 50 ml), and brine (2 · 50 ml)
and then dried over MgSO4. The dry CHCl3 solution was
evaporated and the residue was distilled under reduced
pressure to give 2,3-dimethylanisole: 81.8 g, 74%, bp
86–88 ꢁC (42 mm Hg). Then 3-methoxyphthalic acid was
obtained from 2,3-dimethylanisole by permanganate
oxidation according to a modified literature procedure
(Wentzel et al., 2000). To a suspension of 2,3-dimethylani-
sole (10.0 g, 0.073 mol), t-BuOH (120 ml), water (295 ml)
and solid KMnO4 (73.89 g, 0.467 mol) was added. The sus-
pension was refluxed for 2 h. The precipitate of MnO2 was
filtered off and washed with hot water (2 · 40 ml). The
combined filtrates were acidified to pH ꢁ 1 with concen-
trated HCl and a precipitate of 3-methoxyphthalic acid
was filtered off and washed with CHCl3 to give product:
5.80 g, 40%, mp 156–159 ꢁC (Beard and Hauser, 1960;
mp 169.5–170.5 ꢁC). To reduce 3-methoxyphthalic acid
with the borane-methyl sulfide complex (Fujita and Mori,
2001) a solution of 3-methoxyphthalic acid (8.40 g,
0.0428 mol) in dry tetrahydrofuran (140 ml) was added at
ꢂ6 ꢁC to a solution of the borane-methyl sulfide complex
(12.2 ml, 0.13 mol) in dry tetrahydrofuran (12 ml) dropwise
4.2. 3-Bromo-1,2-bis(bromomethyl)benzene (8)
3-Bromo-1,2-bis(bromomethyl)benzene
(8)
was
obtained according to a modified literature procedure
(Kreher and Herd, 1988). A suspension of 3-bromo-o-
xylene (5.0 g, 0.027 mol), N-bromosuccinimide (9.62 g,
0.054 mol) and azoisobutyronitrile (0.126 g, 0.77 mmol) in
dry CCl4 (26 ml) was gradually warmed to boiling point
and kept under reflux for 0.5 h. The precipitate of succini-
mide was filtered off and the solution was evaporated to
dryness to give 8: 8.15 g, 88%, mp 44–48 ꢁC.
4.3. 1-(10-Bromoethyl)-2-bromomethylbenzene (9)
1-(10-Bromoethyl)-2-bromomethylbenzene
(9)
was
obtained from 2-acetylbenzoic acid (Cannone et al., 1988;
Berner, 1982). To a suspension of LiAlH4 (3.3226 g,
0.088 mol) in dry (135 ml), cooled to ꢂ1 ꢁC, was added
during 80 min a solution of 2-acetylbenzoic acid (9.06 g,
0.055 mol) in THF (20 ml). The mixture was stirred during
10 min then warmed to room temperature and left over-
night. The resulting suspension was cooled to 0 ꢁC and a
saturated solution of NH4Cl (120 ml) was added. Addi-
tional diethyl ether (240 ml) was added. Layers of the mix-
ture were separated and the water layer was washed with
diethyl ether (50 ml). All organic layers were combined,
washed with brine (3 · 100 ml) and dried with MgSO4.

P. Miziak et al. / Phytochemistry 68 (2007) 407–415
411
The solvent was evaporated to dryness and the residue was
crystallized from t-BuOMe to give 1-(10-hydroxyethyl)-2-
hydroxymethylbenzene: 2.40 g, 30%, mp 66–67 ꢁC ((Ber-
CCH2); 3.78– 4.15 (5H, m, CH2OP and CHC); 4.25 (2H,
3
q, JHH = 8.3 Hz, CH2O); 7.12–7.28 (4H, m, C6H4);
31P{1H} NMR spectral data (121 MHz, CDCl3): d 28.34 (s).
1
ner, 1982) mp 71 ꢁC); H NMR spectral data (300 MHz,
3
CDCl3): d 1.47 (3H, d, JHH = 6.5 Hz, CH3); 3.32 (2H,
4.5. General procedure for the synthesis of substituted
2-(diethoxyphosphoryl)indane-2-carboxylic acid (13–15)
3
bs, OH); 4.50 (1H, d, JHH = 12.1 Hz, CH2O); 4.66 (1H,
3
3
d, JHH = 12.1 Hz, CH2O); 5.03 (1H, q, JHH = 6.5 Hz,
3
CHO); 7.18–7.28 (3H, m, C6H4); 7.38 (1H, d, JHH
=
A solution of ethyl 2-(diethoxyphosphoryl)indane-2-
carboxylates (10–12, 0.01 mol), sodium hydroxide (0.50 g,
0.0125 mol) in water (0.78 ml) and 95% ethanol (7.6 ml)
was heated at reflux for about 18 h. The ethanol was evap-
orated to dryness under reduced pressure. Water (21 ml)
was added to the residue and extracted with diethyl ether
(3 · 10 ml), which was discarded. The aqueous layer was
acidified with concentrated hydrochloric acid to pH ꢁ 1
and extracted with CH2Cl2 (3 · 18 ml). The combined
CH2Cl2 layers were washed with water (2 · 18 ml), and
brine (18 ml), then dried with MgSO4, before evaporation
of the CH2Cl2.
7.5 Hz, C6H4).
Using a similar procedure as for the synthesis of
1,2-bis(bromomethyl)-3-methoxybenzene (7), 1-(10-bromo-
ethyl)-2-bromomethylbenzene (9) was obtained from
1-(10-hydroxyethyl)-2-hydroxymethylbenzene
(2.40 g,
0.0162 mol): 3.66 g, 81%. 1H NMR spectral data (300
3
MHz, CDCl3): d 2.04 (3H, d, JHH = 6.8 Hz, CH3); 4.44
2
2
(1H, d, JHH = 9.5 Hz, CH2Br); 4.73 (1H, d, JHH = 10.6
3
Hz, CH2Br); 5.55 (1H, q, JHH = 6.8 Hz; CH(Br)C); 7.20–
7.33 (3H, m, C6H4); 7.57 (1H, d, 3JHH = 7.7 Hz, C6H4).
4.4. General procedure for the synthesis of substituted ethyl
2-(diethoxyphosphoryl)indane-2-carboxylate (10–12) (Zon´
et al., 2005)
Data of 13: 1.97 g; 60%; 1H NMR spectral data
3
(300 MHz, CDCl3): d 1.22 (6H, 2xt, JHH = 6.7 Hz; CH3);
3.37–3.55 (2H, m, CH2O); 3.62–3.71 (2H, m, CH2O); 3.77
(3H, s, CH3O); 4.06–4.19 (4H, m, CH2CCH2); 6.63 (1H, d,
3
A solution of derivatives of 1,2-bis(bromomethyl)ben-
zene (0.02 mol), triethyl phosphonoacetate (5.2 ml,
0.026 mol) and tetrabutylammonium bromide (1.290 g,
0.004 mol) in toluene (20 ml) was stirred and cooled to
14 ꢁC. Powdered sodium hydroxide (2.4 g, 0.06 mol) was
added in about 25 portions during 5 h at a temperature
of 14–18 ꢁC. The solution was stirred additionally for
50 min at the same temperature. The reaction mixture
was filtered off to remove NaBr precipitate and the filtrate
was washed with water (3 · 5 ml; to pH ꢁ 7) and brine
(2 · 5 ml). The toluene layer was dried with MgSO4. The
toluene was then evaporated and the residue was purified
by silica gel chromatography.
3JHH = 8.1 Hz, C6H3); 6.76 (1H, d, JHH = 7.4 Hz, C6H3);
3
3
7.10 (1H, dd, JHH = 7.8 Hz, JHH = 7.8 Hz, C6H3);
31P{1H} NMR spectral data (121 MHz, CDCl3): d 28.59 (s).
Data of 14: 2.79 g; 74%; 1H NMR spectral data
(300 MHz, CDCl3): d 1.21–1.28 (6H, m, CH3); 3.45–3.82
(4H, m, CH2CCH2); 4.08–4.25 (4H, m, CH2O); 6.97–7.30
(3H, m, C6H3); 31P{1H} NMR spectral data (121 MHz,
CDCl3): d 27.70 (s).
Data of 15: 2.50 g; 80%; mp 127–130 ꢁC; 1H NMR spec-
3
tral data (300 MHz, CDCl3): d 1.12 (6H, t, JHH = 7.0 Hz,
3
CH3); 1.31 (3H, d, JHH = 7.1 Hz, CH3); 3.26 (1H, m,
CH2O); 3.65–3.76 (1H, m, CH2O); 3.79–3.92 (1H, m,
CH2O); 3.94–4.02 (3H, m, CH2CCH2); 4.04–4.17 (1H, m,
CH2O); 7.04–7.14 (4H, m, C6H4); 31P{1H} NMR spectral
data (121 MHz, CDCl3): d 29.96 (s).
1
Data of 10: 0.91 g; 13%; Rf = 0.34 (ethyl acetate); H
NMR spectral data (300 MHz, CDCl3): d 1.21–1.28 (9H,
m, CH3C); 3.42–3.74 (4H, m, CH2CCH2); 3.78 (3H, s,
CH3O); 4.02–4.23 (6H, m, CH2O); 6.63 (1H, d,
4.6. General procedure for the synthesis of substituted of
diethyl 2-(benzyloxycarbonylamino)indane-2-phosphonate
(16–18)
3
3JHH = 8.1 Hz, C6H3); 6.77 (1H, d, JHH = 7.5 Hz, C6H3);
3
3
7.11 (1H, dd, JHH = 7.8 Hz, JHH = 7.8 Hz, C6H3);
31P{1H} NMR spectral data (121 MHz, CDCl3): d 27.45 (s).
1
Data of 11: 1.12 g; 14%; Rf = 0.39 (ethyl acetate); H
A solution of the substituted derivative of 2-(diethoxy-
phosphoryl)indane-2-carboxylic acids (13-15, 0.01 mol),
triethylamine (1.8 ml, 0.013 mol), diphenylphosphoryl
azide (DPPA, 2.8 ml, 0.013 mol), benzyl alcohol (1.3 ml,
0.013 mol) in dry toluene (6.7 ml) was refluxed for 8 h
under protection from moisture. CH2Cl2 (75 ml) was added
to the solution and washed with water (2 · 30 ml), then
NMR spectral data (300 MHz, CDCl3): d 1.23–1.28 (9H,
m, CH3C); 3.49–3.82 (4H, m, CH2CCH2); 4.02–4.23 (6H,
3
3
m, CH2O); 7.00 (1H, dd, JHH = 7.6 Hz, JHH = 7.6 Hz,
3
C6H3); 7.09 (1H, d, JHH = 7.1 Hz, C6H3); 7.28 (1H, dd,
3JHH = 7.7 Hz, JHH = 0.5 Hz, C6H3); 31P{1H} NMR
4
spectral data (121 MHz, CDCl3): d 26.74 (s).
1
Data of 12: 2.25 g; 33%; Rf = 0.31 (ethyl acetate); H
with
a
saturated aqueous solution of NaHCO3
NMR spectral data (300 MHz, CDCl3): d 1.17 (3H, dxt,
(2 · 38 ml), and finally with brine (2 · 38 ml). The organic
layer was then dried with MgSO4 and evaporated to dry-
ness under reduced pressure to give the crude product,
which was purified by chromatography on silica gel.
Data of 16: 2.51 g; 58%; Rf = 0.33 (ethyl acetate); H
NMR spectral data (300 MHz, CDCl3): d 1.19–1.26 (6H,
4
3JHH = 7.1 Hz, JHP = 0.4 Hz, CH3C); 1.24 (3H, dxt,
4
3JHH = 6.4 Hz, JHP = 0.3 Hz, CH3C); 1.28 (3H, t,
3
3JHH = 7.2 Hz, CH3C); 1.32 (3H, d, JHH = 7.1 Hz,
2
3
1
CH3C); 3.44 (1H, dd, JHH = 16.3 Hz, JHP = 16.4 Hz,
2
3
CCH2); 3.67 (1H, dd, JHH = 16.3 Hz, JHP = 16.1 Hz,

412
P. Miziak et al. / Phytochemistry 68 (2007) 407–415
3
m, CH3); 3.36–3.64 (4H, m, CH2CCH2); 3.78 (3H, s,
CH3O); 4.02–4.13 (4H, m, CH2O); 5.01 (2H, s, ArCH2O);
3JHH = 7.6 Hz, JHH = 7.6 Hz, C6H3); 31P{1H} NMR
spectral data (121 MHz, D2O–DCl): d 17.06 (s); 13C
NMR spectral data (75 MHz, D2O–DCl): d 37.10, 40.42,
3
5.05 (1H, bs, NH); 6.66 (1H, d, JHH = 7.4 Hz, C6H3);
3
1
6.78 (1H, d, JHH = 7.4 Hz, C6H3); 7.13 (1H, dd,
61.02 (d, JPC = 152.9 Hz, NCP), 114.09, 117.27, 124.85
(d, JPC = 9.0 Hz), 129.55, 141.23 (d, JPC = 9.2 Hz), 152.26.
3
3JHH = 7.8 Hz, JHH = 7.8 Hz, C6H3); 7.25–7.34 (5H, m,
C6H5); 31P{1H} NMR spectral data (121 MHz, CDCl3): d
28.12 (s).
4.8. Procedure for the synthesis of substituted
2-aminoindane-2-phosphonic acids (2–3)
1
Data of 17: 1.64 g; 34%; Rf = 0.44 (ethyl acetate); H
NMR spectral data (300 MHz, CDCl3): d 1.20–1.28 (6H,
m, CH3); 3.46–3.76 (4H, m, CH2CCH2); 4.02–4.14 (4H,
m, CH2O); 5.02 (2H, s, ArCH2O); 5.16 (1H, d,
3JPH = 4.1 Hz, NH); 6.99–7.10 (2H, m, C6H3and C6H5);
7.24–7.36 (6H, m, C6H3 and C6H5); 31P{1H} NMR spectral
data (121 MHz, CDCl3): d 27.56 (s).
A solution of the substituted derivative of diethyl 2-
(benzyloxycarbonylamino)indane-2-phosphonate (17 and
18, 0.01 mol), concentrated hydrochloric acid (30 ml) and
water (30 ml) was refluxed for 20 h. The solution was
extracted with diethyl ether (3 · 60 ml). The water layer
was boiled with activated charcoal and evaporated to dry-
ness. The residue was dissolved twice in methanol (80 ml
and 40 ml) and twice evaporated to dryness. Propylene
oxide was added (0.6 ml). The resulting precipitate was iso-
lated by filtration and was used in the biological tests.
Data of 2 as hydrochloride: 1.74 g; 53%; mp 238–240 ꢁC;
1H NMR spectral data (300 MHz, D2O–DCl): d 2.54 (1H,
Data of 18: 1.16 g; 28%; Rf = 0.44 (ethyl acetate); mp
114–115 ꢁC (ethyl acetate); 1H NMR spectral data
(300 MHz, CDCl3): d 1.18–1.29 (6H, m, CH3); 1.36 (3H;
3
d, JHH = 7.1 Hz, CH3C); 3.42–3.52 (1H, m, CHCCH2);
3.73–3.82 (1H, m, CHCCH2); 4.04–4.15 (5H, m, CHCCH2
2
and CH2OP); 4.92 (1H, d, JHH = 12.2 Hz, ArCH2); 4.99
3
3
(1H, d, JPH = 2.7 Hz, NH); 5.04 (1H, d, JHH = 12.2 Hz,
ArCH2); 7.09–7.13 (1H, m, C6H4); 7.15–7.18 (3H, m,
C6H4 and C6H5); 7.26–7.33 (5H, m, C6H4 and C6H5);
31P{1H} NMR spectral data (121 MHz, CDCl3): d 28.14 (s).
2
3
dd, JHH = 24.0 Hz, JHP = 5.6 Hz, CH2CCH2); 2.61 (1H,
2
3
dd, JHH = 21.0 Hz, JHP = 5.9 Hz, CH2CCH2); 2.88 (1H,
2
3
dd, JHH = 32.1 Hz, JHP = 12.6 Hz, CH2CCH2); 2.93
(1H, dd, JHH = 31.9 Hz, JHP = 13.7 Hz, CH2CCH2);
2
3
3
4.7. ( )-2-Amino-4-hydroxyindane-2-phosphonic acid (1)
6.42 (1H, t, JHH = 7.7 Hz, C6H3); 6.53 (1H, d,
3JHH = 7.4 Hz, C6H3); 6.69 (1H, d, JHH = 7.8 Hz, C6H3);
3
2-Amino-4-hydroxyindane-2-phosphonic acid (1) was
obtained by a two-step deprotecting procedure from
diethyl 2-(benzyloxycarbonylamino)-4-methoxyindane-2-
phosphonate (16). To a stirred solution of compound 16
(0.778 g, 1.795 mmol) in dry CH2Cl2 (1.8 ml), cooled in
an ice-NaCl bath for 30 min under an N2 atmosphere, a
solution ꢁ1 M of BBr3 (0.69 ml, 7,8 mmol) in CH2Cl2
(7.2 ml) was added over the course of 5 min. The solution
was stirred until the ice had melted, and was then left over-
night. The reaction mixture was cooled to 0 ꢁC and water
(15 ml) was added dropwise. The mixture was warmed to
room temperature and evaporated to dryness. The residue
was dissolved in water (16 ml) and again evaporated.
Water (21 ml) and concentrated hydrochloric acid (21 ml)
were added to the residue, and the mixture was refluxed
for 20 h. The solution was washed with diethyl ether
(2 · 22 ml) and evaporated to dryness. The water layer
was treated with charcoal and then evaporated to dryness.
The residue was dissolved in 0.05 M HCl and purified by
ion exchange chromatography on Dowex 50W (eluent:
0.05 M HCl). Fractions containing 2-amino-4-hydroxyin-
dane-2-phosphonic acid were identified based on TLC
(Rf = 0.36, silicagel; butanol–acetic acid–water=3:1:1 v/v/
v, ninhydrin). The selected fractions were combined and
evaporated yielding 2-amino-4-hydroxyindane-2-phos-
phonic acid (1) as hydrochloride: 0.086 g; 18%; mp 320–
330 ꢁC (decomposition); 1H NMR spectral data
(300 MHz, D2O–DCl): d 3.02–3.22 (2H, m, CH2CCH2);
3.41–3.62 (2H, m, CH2CCH2); 6.74 (1H, d, 3JHH = 8.0 Hz,
31P{1H} NMR spectral data (121 MHz, D2O–DCl): d
18.64 (s); 13C NMR spectral data (75 MHz,D2O–DCl): d
1
39.45; 39.96, 57.93 (d, JPC = 160.8 Hz, NCP), 118.04,
3
123.04, 128.79, 129.66, 137.22 (d, JPC = 9.3 Hz), 138.58
3
(d, JPC = 8.6 Hz).
Data of 3 as hydrochloride: 0.11 g; 45%; mp 231–231 ꢁC;
1H NMR spectral data (300 MHz, D2O–DCl): d 1.06 (3H,
d, 3JHH = 7.3 Hz, CH3); 2.75–2.81 (1H, d, 3JHH = 17.2 Hz,
CH2CCH); 3.10–3.24 (1H, m, CH2CCH); 3.38–3.46 (1H,
m, CH2CCH); 6.87–6.92 (4H, m, C6H4); 31P{1H} NMR
spectral data (121 MHz, CDCl3): d 19.44 (s); 13C NMR
spectral data (75 MHz, D2O–DCl): d 11.68, 39.15, 43.04,
1
64.0 (d, JPC = 158.7 Hz, NCP), 123.69, 124.67, 127.75,
3
3
127.77, 136.66 (d, JPC = 10.9 Hz), 142.60 (d, JPC
=
11.6 Hz).
4.9. 1,2,3,4-Tetrabromo-5,6-bis(bromomethyl)benzene (19)
1,2,3,4-Tetrabromo-5,6-bis(bromomethyl)benzene was
obtained according to a modified literature procedure
(Kreher and Herd, 1988). A solution of 3,4,5,6-tetrab-
romo-o-xylene (16.6 g, 0.0393 mol), N-bromosuccinimide
(14.34 g, 0.0806 mol) and azobisisobutyronitrile (0.20 g,
1.2 mmol) in dry CCl4 (26 ml) was gradually warmed to
boiling point and kept under reflux for 2.5 h. The precipi-
tate of succinimide was filtered off from the hot solution
and filtrate was cooled to room temperature. 1,2,3,4-Tet-
rabromo-5,6-bis(bromomethyl)benzene (19) was filtered
off: 16.9 g; 74%; mp 165–167 ꢁC (Kreher and Herd, 1988;
mp 158–160 ꢁC).
3
C6H3); 6.88 (1H, d, JHH = 7.5 Hz, C6H3); 7.15 (1H, dd,

P. Miziak et al. / Phytochemistry 68 (2007) 407–415
413
4.10. Ethyl 2-(diethoxyphosphoryl)-4,5,6,7-
tetrabromoindane-2-carboxylate (20)
3.64–3.69 (4H, m, CH2CCH2); 4.04–4.22 (4H, m, CH2O);
3
5.01 (2H, s, ArCH2O); 5.36 (1H, d, JPH = 6.3 Hz, NH);
7.25–7.35 (5H, m, C6H5); 31P{1H} NMR spectral data
(121 MHz, CDCl3): d 26.86 (s).
To a suspension of NaH (80% suspension in mineral oil,
19 mmol) in dry THF (30 ml), under N2, a solution of
1,2,3,4-tetrabromo-5,6-bis(bromomethyl)benzene
(19,
4.13. 2-Amino-4,5,6,7-tetrabromoindane-2-phosphonic acid
(4)
5.0 g, 0.0086 mol) and triethyl phosphonoacetate (1.93 g,
0.0086 mol) in dry THF (45 ml) was added dropwise. The
mixture was stirred for 30 min at room temperature, then
refluxed for 4 h and finally was evaporated to dryness.
The residue was taken up in CH2Cl2 (60 ml), and the solu-
tion extracted: water (50 ml), 2 M HCl (25 ml), saturated
solution of NaHCO3 (25 ml), brine (2 · 25) and dried with
MgSO4. The solvent was evaporated to dryness and the res-
idue extracted with diethyl ether. The ether extract was
concentrated and the product left to crystallise. Yield 20:
1.50 g; 27%; mp 116–118 ꢁC; 1H NMR spectral data
(300 MHz, CDCl3): d 1.21–1.31 (9H, m, CH3C); 3.59–
3.86 (4H, m, CH2CCH2); 4.08–4.26 (6H, m, CH2O);
31P{1H} NMR spectral data (121 MHz, CDCl3): d 25.55
(s).
A suspension of diethyl 2-(benzyloxycarbonylamino)-
4,5,6,7-tetrabromoindane-2-phosphonate
(22,
0.20 g,
0.28 mmol) in glacial acetic acid (3 ml) and a solution of
40% HBr in water (6 ml) were refluxed for 10 h. The
solution was evaporated to dryness. To the residue water
was added (5 ml) and evaporated to dryness. This was done
again to give 2-amino-4,5,6,7-tetrabromoindane-2-phos-
phonic acids (4) as hydrobromide: 0.13 g; 77%; mp 228–
1
229 ꢁC; H NMR spectral data (300 MHz, DMSO–DCl):
d 3.26–3.35 (2H, m, CH2CCH2); 3.52–3.69 (2H, m,
CH2CCH2); 31P{1H} NMR spectral data (121 MHz,
DMSO–DCl): d 17.65 (s); 13C NMR spectral data
1
(75 MHz, DMSO–DCl):
d
44.84, 56.50 (d, JPC
=
3
154.4 Hz, NCP), 121.91, 126.82, 142.44 (d, JPC = 7.5 Hz).
4.11. 2-(Diethoxyphosphoryl)-4,5,6,7-tetrabromoindane-2-
carboxylic acid (21)
4.14. 3-Benzoylbenzaldehyde (24)
A solution of ethyl 2-(diethoxyphosphoryl)-4,5,6,7-tet-
rabromoindane-2-carboxylate (20, 1.12 g, 1.74 mmol),
sodium hydroxide (0.084 g, 3.09 mmol) solution in water
(0.14 ml) and 95% ethanol (1.4 ml) was refluxed for about
20 h. The solvents were evaporated under reduced pressure.
Then water (3.5 ml) was added to the residue and the
resulting suspension acidified with concentrated hydrochlo-
ric acid to pH ꢁ 1. The precipitate was filtered off to give
3-Benzoylbenzaldehyde was obtained from 3-(bromom-
ethyl)benzophenone according to a modified literature pro-
cedure (Meerpoel et al., 1991). The latter was obtained by
bromination of 3-methylbenzophenone according to the lit-
erature procedure (Stephenson, 1963).
A solution of Na (0.097 g, 4.2 mmol) and 2-nitropro-
pane (0.36 ml, 4.0 mmol) in absolute MeOH (60 ml) under
N2 was stirred for 40 min. 3-(Bromomethyl)benzophenone
was added and the solution left for 24 h. The mixture was
poured into 10% aqueous solution of NH4Cl (110 ml), stir-
red for 25 min and washed with CH2Cl2 (3 · 50 ml). The
combined organic layer was dried with MgSO4. The sol-
vent was evaporated to dryness and the residue was crystal-
lized from hexane to yield 24: 0.46 g; 61%; mp 91–99 ꢁC; 1H
NMR spectral data (300 MHz, CDCl3): d 7.45–8.48 (9H,
m, C6H5 and C6H4), 10.00 (1H, s, CHO).
1
21: 1.04 g; 97%; mp 235–236 ꢁC; H NMR spectral data
3
(300 MHz, CDCl3–DMSO): d 0.83 (6H, t, JHH = 7.0 Hz,
CH3); 3.07–3.19 (2H, m, CH2O); 3.30–3.39 (2H, m,
CH2O); 3.64–3.73 (4H, m, CH2CCH2); 31P{1H} NMR
spectral data (121 MHz, CDCl3): d 30.74 (s).
4.12. Diethyl 2-(benzyloxycarbonylamino)-4,5,6,7-
tetrabromoindane-2-phosphonate (22)
A solution of the 2-(diethoxyphosphoryl)-4,5,6,7-tetrab-
romoindane-2-carboxylic acid (21, 1.0 g, 1.63 mmol),
diphenylphosphoryl azide (DPPA, 0.46 ml, 2.2 mmol), tri-
ethylamine (0.3 ml, 2.2 mmol), benzyl alcohol (0.2 ml,
2.2 mol) in dry toluene (2.2 ml) was refluxed for 6 h under
protection from moisture. CH2Cl2 (12 ml) was then added
to the solution, which was then washed with: water
(2 · 5 ml), a saturated aqueous solution of NaHCO3
(2 · 5 ml), brine (2 · 5 ml). The organic layer was dried
with MgSO4 and then evaporated to dryness under reduced
pressure to give the crude product, which was treated with
ethyl acetate (2 ml) and the solution obtained discarded.
The residue was crystallized from ethanol to yield 22:
0.45 g; 38%; mp 200–201 ꢁC; 1H NMR spectral data
4.15. Derivatives of benzylidenebenzhydrylamine (25, 26)
A solution of aldehyde (23 or 24, 0.027 mol) and ben-
zhydrylamine (4.65 ml, 0.027 mol), CH2Cl2 (14 ml) was
stirred at room temperature for 3 h. K2CO3 (7.1 g,
0.051 mol) was added and the mixture left overnight. The
K2CO3 was filtered off and the solution evaporated to dry-
ness under reduced pressure:
Data of 25: 6.94 g; 73%; mp 81–82 ꢁC (ethanol).
Data of 26: the residue was washed with hexane; 5.27 g;
52%; mp 62–69 ꢁC; 1H NMR spectral data (300 MHz,
CDCl3): d 5.20 (1H, s, CH (Ar)2, Z-isomer); 5.61 (1H,
s, CH(Ar)2, E-isomer); 7.24–7.80 (19H, m, C6H5 and
C6H4); 7.81 (1H, s, CHN, Z-isomer); 7,81 (1H, s, CHN,
E-isomer).
3
(300 MHz, CDCl3): d 1.27 (6H, t, JHH = 7.0 Hz, CH3);

414
P. Miziak et al. / Phytochemistry 68 (2007) 407–415
4.16. Derivatives of diethyl 1-
benzhyrylaminebenzylphosphonate (27, 28)
work was partially supported by the Ministry of Science
and Information Society Technologies (Poland) and Wroc-
ław University of Technology.
Benzylidenebenzhydryloamine (25 or 26, 0.0168 mol)
and diethyl phosphite (2.6 ml, 0.02 mol) were heated for
2 h in ꢁ100 ꢁC.
References
Data of 27: This was crystallized from cyclohexane:
4.66 g; 58%; mp 98–99 ꢁC; 1H NMR spectral data
´
Appert, C., Zon, J., Amrhein, N., 2003. Kinetic analysis of the inhibition
3
of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid
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3
1.27 (3H, t, JHH = 7.1 Hz, CH3); 2.35 (1H, bs, NH);
3.88–4.00 (5H, m, CH2O and CHN); 4.63 (1H, s, CHAr2);
7.14–7.31 (10H, m, 2 · C6H5); 7.47 (2H, m, C6H3); 7.79
(1H, s, C6H3); 31P{1H} NMR spectral data (121 MHz,
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Data of 28: This was purified by chromatography on sil-
ica gel; 1.60 g; 18%; Rf = 0.27 (ethyl acetate); 1H NMR spec-
3
tral data (300 MHz, CDCl3): d 1.11 (3H, t, JHH = 7.2 Hz,
3
CH3); 1.32 (3H, t, JHH = 7.1 Hz, CH3), 1.63 (1H, bs,
3
NH); 3.99 (1H, d, JHP = 19.0 Hz, PCHN); 3.92–4.03 (2H,
m, CH2O); 4.12–4.22 (2H, m, CH2O); 4.69 (1H, s, CHAr2);
7.17–7.31 (10H, m, 2 · C6H5); 7.40–7.59 (5H, m, C6H5);
7.70–7.71 (1H, m, C6H3); 7.76–7.80 (3H, m, C6H4);
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A solution of substituted derivatives of 1-aminobenzyl-
phosphonic acid (27 or 28, 0.01 mol) in water (30 ml) and
concentrated hydrochloric acid (30 ml) was refluxed for
20 h. The solution was washed with diethyl ether
(3 · 60 ml), boiled with activated charcoal and evaporated
to dryness. The residue was dissolved in methanol (80 ml)
and evaporated to dryness. The residue was again dissolved
in methanol (40 ml) and propylene oxide added (0.6 ml).
The resulting precipitate was isolated by filtration.
1
Data of 5: 1.70 g; 64%; mp 240–241 ꢁC; H NMR spec-
tral data (300 MHz, D2O–DCl):
d
3.90 (1H, d,
3JHP = 17.3 Hz, PCH); 6.78 (2H, m, C6H3); 7.22 (1H, s,
C6H3); 31P{1H} NMR spectral data (121 MHz, D2O–
DCl): d 11.97 (s); 13C NMR spectral data (75 MHz,
1
D2O–DCl): d 50.58 (d, JPC =145.0 Hz, NCP), 124.92,
´
127.16, 130.71, 132.27, 132.64, 146.45.
Data of 6: 2.25 g; 77%; mp 255–257 ꢁC; H NMR spec-
tral data (300 MHz, D2O–DCl):
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3.47 (1H, d,
1JHP = 17.3 Hz, PCH); 6.07–6.12 (2H, m, C6H3 and
C6H5); 6.19–6.33 (5H, m, C6H3 and C6H5); 6.40–6.44
(2H, m, C6H3 and C6H5); 31P{1H} NMR spectral data
(121 MHz, D2O–DCl): d 13.25 (s); 13C NMR spectral data
(75 MHz, DMSO–D2O–DCl): d 50.88 (d, 1JPC = 147.3 Hz,
´
Poppe, L., Retey, J., 2005. Friedel–Crafts-type mechanism for the
enzymatic elimination of ammonia from histidine and phenylalanine.
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R.J.M., 2000. N-Hydroxyphthalimide/cobalt(II) catalyzed low tem-
perature benzylic oxidation using molecular oxygen. Tetrahedron 56,
7797–7803.
3
NCP), 127.50, 128.47, 129.23, 129.67 (d, JPC = 4.7 Hz),
130.19, 131.30, 131.36, 132.72, 134.91, 136.34, 198.62.
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