1-Aminobenzocyclobutene-1-phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia-Lyase

Article abstract of DOI:10.1002/cbdv.201600488

Five new geminal aminocycloalkanephosphonic acids (4 – 8) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia-lyase (PAL). Within the set of compounds which are related to 2-aminoindane-2-phosphonic acid (AIP, 3), a known powerful inhibitor of PAL, racemic 1-aminobenzocyclobutene-1-phosphonic acid (4), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine-derived anthocyanin synthesis in buckwheat.

Full text of DOI:10.1002/cbdv.201600488

Received Date: 22-Dec-2016
Revised Date: 02-Feb-2017
Accepted Date: 03-Feb-2017
Article Type: Full Paper
1-Aminobenzocyclobutene-1-phosphonic acid and related compounds as
inhibitors of phenylalanine ammonia-lyase
Jerzy Zoń* and Piotr Miziak†
Department of Thermodynamics, Theory of Machine and Thermal Systems; Faculty of
Mechanical and Power Engineering; Wrocław University of Science and Technology; 27
Wybrzeże Wyspiańskiego St.; 50-370 Wrocław; Poland
* Corresponding author; e-mail: jerzy.zon@pwr.edu.pl; phone: +48 71 320 3910; fax: +48 71 328 3818.
† Present address: Chojny 14; 62-600 Koło, Poland.
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201600488
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Abstract:
Five new geminal aminocycloalkanephosphonic acids (4-8) containing both an aromatic ring
and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat
phenylalanine ammonia-lyase (PAL). Within the set of compounds which are related to 2-
aminoindane-2-phosphonic acid (AIP, 3), a known powerful inhibitor of PAL, racemic 1-
aminobenzocyclobutene-1-phosphonic acid (4), was six times weaker than AIP as an in vitro
inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of
phenylalanine-derived anthocyanin synthesis in buckwheat.
Keywords:
8-aminobicyclo[4.3.0]nona-1,3,5-triene-8-phosphonic acid, 7-aminobicyclo[4.2.0]octa-1,3,5-triene-7-
phosphonic acid, PAL inhibitor
Introduction
Phenylalanine ammonia-lyase (PAL, EC 4.1.3.24) is the first committed enzyme of the
plant phenylpropanoid pathway and catalyzes the formation of (E)-cinnamic acid from (S)-
phenylalanine by the elimination of ammonia. (E)-Cinnamic acid is the precursor of a wide
array of metabolites involved in mechanical support, signalling, pigmentation, and
protection against biotic and abiotic stress.[1-4] The elimination of ammonia from (S)-
phenylalanine proceeds with participation of 4-methylideneimidazole-5-one (MIO) as the
cofactor of the enzyme, which is formed autocatalytically from an internal Ala-Ser-Gly
tripeptide segment[5]; further details of the mechanism of the reaction have recently been
published.[6-7] Tyrosine (TAL, EC 4.3.1.23) and phenylalanine/tyrosine (PAL/TAL, EC 4.3.1.25)
ammonia lyases are closely related enzymes with different substrate specificities and occur
in bacteria (TAL) and yeasts or monocotyledonous plants (PAL/TAL), respectively.[8] (S)-2-
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Aminooxy-3-phenylpropionic acid (AOPP, 1, Fig.1) was identified as a strong inhibitor of PAL
both in vitro and in vivo.[9] Years later, the substitution of the carboxylic group by a
phosphonic group in (S)-phenylalanine provided a simple and successful approach to the
design of a new strong PAL inhibitor, namely (R)-(-)-1-amino-2-phenylethylphosphonic acid
(2, Fig.1).[10-11] A substantial improvement of the inhibitory activity of 2 was achieved by
restricting its conformational flexibility, giving rise to 2-aminoindane-2-phosphonic acid (3,
Fig.1, AIP, IUPAC nomenclature: 8-aminobicyclo[4.3.0]nona-1,3,5-triene-8-phosphonic acid),
which is a somewhat less powerful inhibitor of PAL in vitro than AOPP (1), but to date is still
the most potent inhibitor available for in vivo inhibition of PAL.[12] The chemically and
biologically stable AIP (3) has become a valuable tool in the study of the functions of
phenylpropanoids in vivo or in reducing the concentrations of unwanted phenolic
compounds in plant tissues. Notably, in going from 2 to 3, the chirality of the molecule was
abolished. It was shown to inhibit the heterologously expressed PAL-1 isozyme from parsley
through a slow-binding mechanism.[13] It is worth mentioning than AIP (3) only weakly
inhibits of (S)-phenylalanine deamination by tyrosine ammonia lyase (TAL) from
Rhodobacter cupsulatus.[14] Experimental and ab initio calculated structures of 2-
aminoindane-2-phosphonic acid were bonded in silico to the model of the enzyme
structure.[15] We have studied the inhibitory activities of the derivatives of 2-aminoindane-2-
phosphonic acid substituted at the benzene ring[16-17] and the cyclopentane ring[17]
,
however, these compounds were all less inhibitory than 3. Next we decided to combine
aromatic rings with various cycloalkanes possessing an amino and phosphonic group in a
geminal position, and test their inhibitory activity on buckwheat PAL both in vitro and in
vivo. Here, we describe the synthesis and preliminary evaluation of five such new geminal
aminoalkylphosphonic acids (4-9, Fig. 1). The studied compounds contain an aromatic ring
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system (benzene, naphthalene or biphenyl) and a cycloalkane (cyclobutane, cyclopentane,
cyclohexane or cycloheptane) as either fused (3-6) or bridged (7-8) rings. Compound 9 does
not contain a cycloalkane ring but can be considered an open chain analogue of AIP (3).
Results and Discussion
Synthesis
The geminal aminoalkylphosphonic acids 4-9 (Fig. 1) were synthesized along diverse routes
(Scheme 1). Thus, the racemic compounds 4 and 6 were obtained from the ketones 11 and 12
respectively, by one-pot procedure (Scheme 1).[18] Earlier, we had exploited the transformation of
substituted benzaldehyde into substituted 1-aminobenzylphosphonic acids.[19] This would appear to
be a short route towards the synthesis of aminoalkylphosphonic acids; however, it requires the
laborious purification (usually through ion-exchange chromatography) of the crude products, and
may not lead to the desired product, as indicated by our unsuccessful attempts with 1-indanone.
Racemic compound 5, an isomer of compound 3, was obtained from N-1-
indanylidenebenzhydrylamine, as described previously.[20]
Lastly, compounds 7-9 were obtained using a multistep route consisting of the dialkylation of triethyl
phosphonoacetate (16) by various dibromides 13-14 or bromide 15, the regioselective hydrolysis,
the Curtius degradation and finally total hydrolysis (Scheme 1). This route was formerly used by us to
synthesize some derivatives of 2-aminoindane-2-phosphonic acid.[16-17]
The obtained geminal aminoalkylphosphonic acids (4-9) were crystallized, after which they were
characterized analytically and spectroscopically (see Experimental Section).
Inhibition of Buckwheat Phenylalanine Ammonia-Lyase in vitro and in vivo
The analytically pure geminal aminoalkylphosphonic acids (4-9) were tested as inhibitors of
PAL activity in crude extracts from buckwheat hypocotyls (in vitro),[11] as well as inhibitors of the
light-induced biosynthesis of (E)-cinnamic acid-derived anthocyanins in buckwheat hypocotyls (in
vivo).[21] All compounds were initially tested in both systems at 1 mM concentration, and then, for
promising inhibitors, the inhibitor constant (Ki) and IC50 values were determined in vitro and in vivo,
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respectively. These values were then compared with those obtained previously for 2-aminoindane-2-
phosphonic acid (3, AIP)[12] and (R)-(-)-1-amino-2-phenylethylphosphonic acid (2)[11] and its racemic
form ( (±)-2).[11] The results are presented in Table 1. Within the set of the newly synthesized geminal
aminoalkylphosphonic acids, (±)-1-aminobenzocyclobutene-1-phosphonic acid (4, IUPAC
nomenclature: 7-aminobicyclo[4.2.0]octa-1,3,5-triene-7-phosphonic acid) is an exceptional amino
acid due to its more rigid structure than that of 2-aminoindane-2-phosphonic acid (3) and all other
compounds in Table 1. It was a more potent inhibitor of anthocyanin synthesis (4, IC50=0.14 μM)
than AIP (3, IC50=0.83 μM), while the situation was reversed for PAL inhibition (4, Ki=0.32 μM; 3,
Ki=0.047 μM). It had previously been observed that another PAL inhibitor, (S)-2-aminooxy-3-
phenylpropionic acid (AOPP, 1, Fig.1), is a far more potent inhibitor of PAL in vitro than AIP, while AIP
has a higher in vivo activity.[12] This may be related to differential uptake and stability of the
inhibitors in in vivo conditions. Compound 4 was tested as a racemic mixture, and the relative
contribution of the two enantiomers to the inhibition cannot be easily assessed. In the case of the
conformationally flexible 1-amino-2-phenylethylphosphonic acid, the Ki value of the (R)-(-)
enantiomer [(R)-(-)-2] was about eight times lower than that of the (S)-(+) enantiomer [(S)-(+)-2],[11]
but this factor cannot simply be adopted for 4 with its rigid structure. In any case, we may assume
that the Ki of the R enantiomer of 4 [the same configuration as for (S)-phenylalanine] is close to half
the value of the Ki of the racemic mixture of 4 (0.32 µM), but very unlikely to be in the range of the Ki
of 3 (0.047 µM). In any case, inhibitors 3, 4 (as the racemate) and even hypothetically (R)-4 (as the
enantiomer), may be suitable tools in the exploration of the active site of PAL.[22-23]
Conclusion
Racemic 1-aminobenzocyclobutene-1-phosphonic acid (7-aminobicyclo[4.2.0]octa-1,3,5-
triene-7-phosphonic acid) with respect to both its inhibitory power and rigid structure is an
exceptional member of the PAL inhibitor library. The enantiomers of 1-aminobenzocyclobutene-1-
phosphonic acid must now be prepared and their inhibitory activities compared. They will be
valuable tools in exploring the active site of the parsley phenylalanine ammonia-lyase with its known
three-dimensional structure, and furthermore, may provide directions towards the synthesis of even
more powerful PAL inhibitors.
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Experimental
1H NMR spectra were recorded at 300 and 600 MHz; 31P{1H} NMR at 121 and 243 MHz; and
13C{1H} 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 an H2O signal. Chemical shifts (σ) are
provided in ppm. Melting points were determined using a Boëtius apparatus and were not
corrected. TLC was carried out on commercially available pre-coated plates (Fluka silica gel on TLC-
PET foils with or without fluorescent indicator 254 nm).
(±)-1-Aminoindane-1-phosphonic acid[20] (5) and 1-oxobenzocyclobutene[24] (11) were
obtained according to literature procedures. Commercially available benzyl bromide (15) and 2-
tetralone (12) were used.
The inhibition constants (Ki) for buckwheat PAL, and IC50 values of inhibition of anthocyanin
production in illuminated buckwheat hypocotyls were determined as described previously. [11] [21]
1,8-Bis(bromomethyl)naphthalene (13). 1,8-Bis(bromomethyl)naphthalene (13) was obtained from
commercially available 1,8-naphthalic anhydride in two steps. Thus according to a modified
literature procedure 1,8-bis(hydroxymethyl)naphthalene was synthesized.[25] 1,8-Naphthalic
anhydride was added in three portions to a suspension of LiAlH4 (8.42 g; 0.198 mol) in dry diethyl
ether (150 cm3), under a nitrogen atmosphere. The reaction mixture was refluxed for 13 hours. The
solvent was evaporated and ethyl acetate (40 cm3) and then 5% HCl (300 cm3) were added slowly to
the residue. The solution was acidified with concentrated hydrochloric acid to pH ca. 1. The
precipitate of 1,8-bis(hydroxymethyl)naphthalene was filtered off and crystallised from ethanol: 9.29
1
g; 58 %; mp 158 – 159ºC (lit.[25] mp 160 – 161ºC). H NMR (300 MHz, CDCl3): δ 5.13 (2H, s, CH2O);
3
3
5.15 (2H, s, CH2O); 5.19 (1H, s, OH); 5.21 (1H, s, OH); 7.38 (2H, dd, JHH=7.2 Hz, JHH=7.2 Hz, C10H6);
7.56 (2H, dd, 3JHH=7.0 Hz, 4JHH=1.1 Hz, C10H6); 7.76 (2H, m, 3JHH=8.2 Hz, 4JHH=1.3 Hz, C10H6).
In the next step, 1,8-bis(bromomethyl)naphthalene (13) was obtained according to the modified
procedure.[26] PBr3 (11 cm3; 0.113 mol) in dry diethyl ether (10 cm3) was added dropwise to a
solution of 1,8-bis(hydroxymethyl)naphthalene (6.10 g; 0,0323 mol) in dry diethyl ether (140 cm3) at
–10 to -2ºC over the course of 45 min. The mixture was stirred for another 10 minutes at about -2ºC.
Then the mixture was refluxed for 7 h and then poured onto crushed ice (250 cm3), after which
chloroform (150 cm3) was added. The organic layer was separated and the aqueous layer was
additionally extracted with chloroform (50 cm3). The combined organic extracts were washed with a
saturated aqueous solution of NaHCO3 (2x100 cm3) and dried with MgSO4. The organic solvent was
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evaporated to give 1,8-bis(bromomethyl)naphthalene (13): 9.33 g; 92 %; mp 132 - 133ºC (lit.[26] mp
131 – 132ºC). 1H NMR (300 MHz, CDCl3): δ 5.29 (4H, s, CH2Br); 7.44 (2H, dd, 3JHH=7.5 Hz; 3JHH=7.8 Hz,
C10H6); 7.61 (2H, dd, 3JHH=7.1 Hz, 4JHH=1.1 Hz; C10H6); 7.87 (2H, dd, 3JHH=8.2 Hz, 4JHH=1.0 Hz, C10H6).
2,2’-Bis-(bromomethyl)biphenyl (14). 2,2’-Bis-(bromomethyl)biphenyl (14) was synthesized from
biphenyl-2,2`-dicarboxylic acid. The first step involved the reduction of biphenyl-2,2`-dicarboxylic
acid to 2,2’-bis(hydroksymethyl)biphenyl using a borane-methyl sulfide complex.[27-28] A solution of a
borane-methyl sulfide complex (23.5 cm3; 0.248 mol) in dry tetrahydrofuran (15.0 cm3) was added
dropwise at -6ºC to a solution of biphenyl-2,2`-dicarboxylic acid (20.0 g; 0.0826 mol) in dry
tetrahydrofuran (290 cm3), under nitrogen, over a period of 20 min. Cooling and stirring were
continued for another 10 min. Next, the reaction mixture was gradually warmed to 55ºC and kept at
55ºC for 20 h while stirring. After cooling down to 20ºC, 1 M NaOH (170 cm3) was added and the
solution stirred for the next 1 h. The solution was concentrated to half of the initial volume and the
precipitate of 2,2’-bis(hydroxymethyl)biphenyl was filtered off. The diol was crystallized from ethyl
1
acetate: 15.67 g; 88 %; mp 111 – 112ºC (lit.[29] mp 109 - 111ºC). H NMR (300 MHz, CDCl3): δ 3.13
(2H, s, OH); 4.20 – 4.29 (4H, m, CH2O); 7.07 (2H, d, 3JHH=5.9 Hz, C6H4); 7.24 – 7.35 (4H, m, C6H4); 7.39
(2H, d, 3JHH=1.4 Hz, C6H4).
2,2’-Bis-(bromomethyl)biphenyl (14) was synthesized using a similar procedure as in the case of the
synthesis of 1,8-bis(bromomethyl)naphthalene (13), starting from 2,2’-bis(hydroksymethyl)biphenyl
(9.76 g; 0.0455 mol). 2,2’-Bis-(bromomethyl)biphenyl (14) was crystallized from ethanol: 12.14 g; 78
%; mp 85 – 88ºC (lit.[30] mp 82 – 83ºC). 1H NMR (300 MHz, CDCl3): δ 4.18 (2H, d, 2JHH=10.0 Hz, CH2Br);
2
3
4
4.33 (2H, d, JHH=10.0 Hz, CH2Br); 7.26 (2H, dd, JHH=7.4 Hz, JHH=2.3 Hz, C6H4); 7.33 – 7.43 (4H, m,
C6H4); 7.54 (2H, dd, 3JHH=7.4 Hz, 4JHH=1.5 Hz, C6H4).
Synthesis of triethyl phosphonoacetate derivatives (17 – 19) according to the existing methods.[17]
Recently in the literature the synthesis of derivatives of diethyl malonate were obtained in other
conditions than in this work.[31-32] A solution of bromomethylbenzene derivatives 13 – 14 (0.02 mol)
or 15 (0.040 mol), triethyl phosphonoacetate (16, 5.2 cm3, 0.026 mol) and tetrabutylammonium
bromide (1.290 g, 0.004 mol) in toluene (20 cm3) were stirred and cooled to 14°C. Powdered sodium
hydroxide (2.4 g, 0.06 mol) was added in about 25 portions over a period of 5 h at a temperature of
14–18°C. The solution was stirred for additional 50 min at the same temperature. The reaction
mixture was filtered off to remove NaBr precipitate and the filtrate was washed with water (3x5 cm3;
to pH~7) and brine (2x5 cm3). The toluene layer was dried with MgSO4. Finally, the drying agent was
filtered off.
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Ethyl 6-diethoxyphosphoryl-2,3-dihydro-1H-phenalene-2-carboxylate (17). Toluene was
evaporated to dryness and the residue was crystallized from hexane to give 17: 2,74 g; 36%; mp 51 –
53 ºC. 1H NMR (300 MHz, CDCl3): δ 0.83 (3H, t, 3JHH=7.1 Hz, CH3C); 1.35 (6H, t, 3JHH=7.0 Hz, CH3C); 3.54
2
3
2
3
(2H, dd, JHH=15.3Hz, JHP=8.1 Hz, CH2CCH2); 3.77 (2H, dd, JHH=15.4 Hz, JHP=6.5 Hz, CH2CCH2); 3.88
3
(2H, q, JHH=7.1 Hz, CH2O); 4.16 – 4.25 (4H, m, CH2OCH2); 7.24 – 7.38 (4H, m, C10H6); 7.64 (2H, d,
3JHH=8.1 Hz, C10H6); 31P{H} NMR (121 MHz, CDCl3): δ 25.6 (s).
Ethyl 6-diethoxyphosphoryl-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-6-carboxylate (18). Toluene
was evaporated to dryness and the residue was purified by silica gel chromatography using ethyl
acetate as eluent to yield 18: 4.95 g; 61%; Rf = 0,32 (ethyl acetate); an oil. 1H NMR (300 MHz, CDCl3):
δ 1.23 (6H, t, 3JHH=7.1 Hz, CH3C); 1.29 (3H, t, 3JHH=7.1 Hz, CH3C); 2.90 – 3.25 (4H, m, CH2CCH2); 3.97 –
3
4.16 (4H, m, CH2O); 4.21 (2H, q, JHH=10.6 Hz, CH2O); 7.23 – 7.35 (8H, m, C12H8); 31P{H} NMR (121
MHz, CDCl3): δ 24.3 (s).
Ethyl 2,2-dibenzylo-2-(dietoxyphosphoryl)acetate (19). Toluene was then evaporated and the
residue was purified by silica gel chromatography using ethyl acetate to give 19: 2,24 g; 32%; Rf =
1
3
0.42 (ethyl acetate); an oil; H NMR (300 MHz, CDCl3): δ 1.10 (6H, t, JHH=7.0 Hz, CH3C); 1.23 (3H, t,
3JHH=7.1 Hz, CH3C); 3.16 – 3.26 (4H, m, CH2CCH.); 3.82 – 3.93 (2H, m, CH2O); 3.95 – 4.01 (2H, m,
3
CH2O); 4.19 (2H, q, JHH=10.7 Hz, CH2O); 7.14 – 7.28 (10H, m, C6H5); 31P{H} NMR (121 MHz, CDCl3): δ
25.8 (s).
Synthesis of diethyl phosphonoacetic acid derivatives (20 – 22) according to literature
procedure.[17] A solution of the respective esters (17 – 19, 0.01 mol) and sodium hydroxide (0.50 g,
0.0125 mol) in water (0.78 cm3) and 95% ethanol (7.6 cm3) was refluxed for about 18 h. The ethanol
was evaporated to dryness under reduced pressure. Water (21 cm3) was added to the residue which
was then extracted with diethyl ether (3x10 cm3), and the organic phase discarded. The aqueous
layer was acidified with concentrated hydrochloric acid to pH~1 and extracted with CH2Cl2 (3x18
cm3). The combined CH2Cl2 layers were washed with water (2x18 cm3), and brine (18 cm3), and then
dried with MgSO4, before evaporation of the solvent.
6-Diethoxyphosphoryl-2,3-dihydro-1H-phenalene-2-carboxylic acid (20): 3.31 g; 95%; mp 224 – 225
ºC. 1H NMR (300 MHz, CDCl3): δ 1.30 (6H, t, 3JHH=7.0 Hz; CH3); 3.41 (2H, dd, 2JHH=15.5 Hz; 3JHP=8.3 Hz;
2
3
CH2CCH2); 3.67 (2H, dd, JHH=15.6 Hz, JHP=6.3 Hz, CH2CCH2); 4.08 – 4.19 (4H, m, CH2O); 7.22 (2H, d,
3
3
3JHH=6.9 Hz, C6H4); 7.32 (2H; t, JHH=7.1 Hz, C10H6); 7.59 (2H, d, JHH=8.1 Hz, C10H6); 31P{H} NMR (121
MHz, CDCl3): δ 30.6 (s).
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6-Diethoxyphosphoryl-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-6-carboxylic acid (21). After
acidification of the aqueous layer with concentrated hydrochloric acid to pH~1, product 21
1
precipitated: 3.59 g; 96%; mp 195 – 196 ºC. H NMR (300 MHz, CDCl3): δ 1.13 – 1.22 (6H, m, CH3);
2.82 – 3.48 (4H, m, CH2CCH2); 4.12 – 4.19 (4H, m, CH2O); 7.21 – 7.35 (8H, m, C12H8); 31P{H} NMR (121
MHz, CDCl3): δ 25.7 (s).
2,2-Dibenzyl-2-(dietoxyphosphoryl)acetic acid (22): 3.24 g; 86%; mp 90 – 95 ºC. 1H NMR (300 MHz,
CDCl3): δ 1.08 – 1.25 (6H, m, CH3); 3.18 – 3.29 (4H, m, CH2CCH2); 3.94 – 4.18 (4H, m, CH2O); 7.18 –
7.29 (10H, m, C6H5); 31P{H} NMR (121 MHz, CDCl3): δ 27.8 (s).
Synthesis of the urethane derivatives of diethyl alkylphosphonate (23 – 25) according to our
previously published procedure.[17] A solution of the derivative of the respective carboxylic acid (20-
22, 0.01 mol), triethylamine (1.8 cm3, 0.013 mol), diphenylphosphoryl azide (DPPA, 2.8 cm3, 0.013
mol) and benzyl alcohol (1.3 cm3, 0.013 mol) in dry toluene (6.7 cm3) was refluxed for 8 h under
protection from moisture. CH2Cl2 (75 cm3) was added to the solution and the organic phase washed
with water (2 x 30 cm3), then with a saturated aqueous solution of NaHCO3 (2 x 38 cm3), and finally
with brine (2 x 38 cm3). The organic layer was then dried with MgSO4 and evaporated to dryness
under reduced pressure to give the crude product, which was purified by chromatography on silica
gel.
Diethyl 2-benzyloxycarbonylamino-2,3-dihydro-1H-phenalene-2-phosphonate (23): 1.45 g; 32%; Rf
= 0.36 (ethyl acetate); mp 140 – 141 ºC (crystallized from ethanol). 1H NMR (300 MHz, CDCl3): δ 1.35
(6H, t, 3JHH=3.7 Hz, CH3); 3.60 (2H, dd, 2JHH=15.6 Hz, 3JHP=3.9 Hz, CH2CCH2); 3.88 (2H, dd, 2JHH=15.6 Hz,
3JHP=7.2 Hz, CH2CCH2); 4.12 – 4.30 (4H, m, CH2O); 4.66 (1H, s, NH); 4.87 (2H, s, ArCH2O); 7.08 (2H, bs,
3
3
C10H6); 7.23 – 7.29 (5H, m, C6H5); 7.42 (2H, t, JHH=7.6 Hz, C10H6); 7.73 (2H, d, JHH=8.3 Hz, C10H6);
31P{H} NMR (121 MHz, CDCl3): δ 26.6 (s).
Diethyl 6-benzyloxycarbonylamino-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-6-phosphonate (24):
3.36 g; 70%; Rf = 0.38 (ethyl acetate); mp 153 – 154 ºC (ethyl acetate). 1H NMR (300 MHz, CDCl3): δ
1.23 (6H; t, 3JHH=7.0, CH3); 3.08 – 3.17 (4H, m, CH2CCH2); 3.98 – 4.17 (4H, m, CH2O); 5.03 (1H; s, NH);
5.10 (2H, s, ArCH2O); 7.26 – 7.45 (13H, m, C12H8 and C6H5); 31P{H} NMR (121 MHz, CDCl3): δ 27.1 (s).
Diethyl 1-benzyloxycarbonylamino-1,1-dibenzylmethylphosphonate (25): 2.36 g; oil; 49%; Rf = 0.51
(ethyl acetate). 1H NMR (300 MHz, CDCl3): δ 1.09 (6H, t, 3JHH=7,1 Hz, CH3); 3.12 (2H, dd, 2JHH=20.8 Hz,
3JHP=14.0 Hz, CH2CCH2); 3.67 (2H; dd, 2JHH=14.0 Hz, 3JHP=10.8 Hz, CH2CCH2); 3.81 – 4.01 (4H, m, CH2O);
5.01 (1H; d, 3JPH=6.3 Hz, NH); 5.13 (2H, s, ArCH2O); 7.18 – 7.24 (11H, m, ArCH2O and C6H5); 7.29 – 7.40
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(11H, m, ArCH2O and C6H5); 31P{H} NMR (121 MHz, CDCl3): δ 26.4 (s).
Acid hydrolysis of the urethane derivatives of phosphonates (23-25) according to our published
procedure.[17] A solution of diethyl N-benzyloxycarbonylaminophosphonate (23 – 25, 0.01 mol),
concentrated hydrochloric acid (30 cm3) and water (30 cm3) was refluxed for 20 h. The solution was
extracted with diethyl ether (3x60 cm3). The organic layers were discarded. The water layer was
boiled with activated charcoal and evaporated to dryness. The residue was dissolved twice in
methanol (80, 40 cm3) and twice evaporated to dryness. Propylene oxide was added (0.6 cm3). The
resulting precipitate was isolated by filtration and further purified.
2-Amino-2,3-dihydro-1H-phenalene-2-phosphonic acid (7). It was purified by washing with 1 M
hydrochloric acid and extraction with methanol. The aminophosphonic acid 7 was precipitate by
adding propylene oxide: 0.77 g; 29%; mp 237 – 238 ºC (decomposition). 1H NMR (300 MHz, DMSOd6-
DCl): δ 3.26 – 3.56 (4H, m, CH2CCH2); 7.02 – 7.38 (6H, m, C12H6); 31P{H} NMR (121 MHz, DMSOd6-DCl):
1
δ 18.1 (s); 13C NMR (75 MHz, DMSOd6- DCl): δ 30.73; 30.88; 48.91 (d, JPC=156.5 Hz, NCP); 120.28;
122.58 (d, 3JPC=4.6 Hz); 124.23; 128.0; 132.77; 137.48. Elemental analysis calc. for C13H14NO3P: N 5.32
and P 11.77%. Found: N 5.63 and P 11.30%.
6-Amino-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-6-phosphonic acid (8). It was precipitated twice
1
from methanol with propylene oxide: 1.16 g; 40%; mp 230 – 232 ºC. H NMR (300 MHz, DMSOd6-
DCl): δ 1.66 (2H, bm, CH2CCH2); 2.00 (2H, bm, CH2CCH2); 5.59 – 6.53 (8H, m, C12H8); 31P{H} NMR (121
MHz, DMSOd6-DCl): δ 18.1 (s); 13C NMR (75 MHz, DMSOd6-D2O-DCl): δ 32.70; 60.32 (d, 1JPC=151.3 Hz;
NCP); 124.77; 125.15; 125.42; 127.10; 127.50; 136.47. Elemental analysis calc. for C15H16NO3P: N
4.81 and P 10.71%. Found: N 5.10 and P 10.40%.
1-Amino-1,1-dibenzylmethylphosphonic acid (9): 1.74 g; 60%; mp 212 – 215 ºC. 1H NMR (300 MHz,
DMSOd6-DCl): δ 2.96 – 3.15 (4H, m, CH2CCH2); 7.14 – 7.24 (6H, m, C6H5); 7.31 – 7.34 (4H, m, C6H5);
31P{H} NMR (121 MHz, DMSOd6-DCl): δ 17.6 (s); 13C NMR (75 MHz, DMSOd6-DCl): δ 57.62 (d,
3
1JPC=150.7 Hz, NCP); 127.45; 128.49; 131.75; 134.42 (d, JPC=5.1 Hz). Elemental analysis calc. for
C15H18NO3P: N 4.81 and P 10.63%. Found: N 5.05 and P 10.21%.
Synthesis of (±)-gem-aminocykloalkylphosphonic acids 4 and 6 according to the literature
procedure.[12] A solution of 1-oxobenzocyclobutene[24] (11) (0.0245 mol) or 2-tetralone (12) (0.0245
mol) and acetamide (2.90 g, 0.0491 mol) in glacial acetic acid (15 cm3) was cooled to 5°C. At this
temperature acetyl chloride (1.75 cm3, 0.0245 mol) was added dropwise while stirring. The reaction
mixture was stirred at a temperature below 10 °C for 2 h. Phosphorus(III) chloride (2.14 cm3, 0.0245
mol) was added, and the solution was stirred at room temperature for the next 5 h. Next, the
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mixture was gradually heated to 60 °C for 1 h and then to 90 °C during the next 1 h. Volatile by-
products, including acetic acid, were evaporated under a water aspirator. The residue was refluxed
for 15 h with conc. hydrochloric acid (25 cm3) and water (25 cm3). The insoluble impurities were
filtered off and the filtrate was extracted with CH2Cl2 (2x20 cm3). The water phase was treated with a
pinch of charcoal and evaporated to dryness under water aspirator pressure. The residue was
extracted twice with methanol (20 cm3) and conc. hydrochloric acid (0.5 cm3). The combined extracts
were evaporated to dryness, the residue dissolved in minimum volume water and were subjected to
ion exchange chromatography as water solution (Dowex 50W H+ form; size of bed: diameter 2,5 cm,
length 38 cm). The column was eluated with water. The eluates were monitored using TLC (silica gel;
n-butanol-glacial acetic acid-pyridine-water=6:6:3:5 v/v/v/v; 1% ninhydrin in isopropanol; 130°C).
The collected fractions were evaporated under a water respirator pressure. The crude acids were
crystallized from water.
(±)-1-Aminobenzocyclobutene-1-phosphonic acid (4). 1.33 g; 25%; mp 181-184°C (decomposition;
crystallized from H2O-CH3OH=1/1v/v); Rf=0.77 (silica gel; n-butanol-glacial acetic acid-pyridine-
1
water=6:6:3:5 v/v/v/v; 1% ninhydrin in isopropanol; visualization at 130°C). H NMR (300 MHz, D2O-
DCl): δ 3.24 (1H, dd, 2JHH=15.1 Hz, 3JHP=4.0 Hz, CH2CP); 3.55 (1H, dd, 2JHH=15.1 Hz, 3JHP=7.0 Hz, CH2CP);
7.00 – 7.28 (4H, m, C6H4); 31P{H} NMR (121 MHz, D2O-DCl): δ 15.3 (s); 13C NMR (75 MHz, D2O-DCl): δ
38.80 (CH2); 57.56 (d, 1JPC=156.4 Hz; NCP); 122.59 (C6H4); 123.95 (C6H4); 128.46 (d, 3JPC=1.1 Hz; C6H4);
3
2
131.13 (C6H4); 138.92 (d, JPC=5.0 Hz; C6H4); 141.76 (d, JPC=10.9 Hz; C6H4); IR (KBr): νmax 751, 911,
1150, 1534, 1611, 2107, 2433, 2810, 3181 cm-1. Elemental analysis calc. for C8H12NO4P (as
monohydrate): N 6.45 and P 14.26%. Found: N 6.37 and P 14.23 %.
(±)-2-Amino-1,2,3,4-tetrahydronaphthalene-2-phosphonic acid (6). 1.79 g; 32%; mp 234-236°C
1
(crystallized from H2O). H NMR (600 MHz, D2O-DCl): δ 2.08-2.14 (1H, m, CH); 2.-2.15-2.20 (1H, m,
CH); 2.73-2.81 (1H, m, CH); 2.92-2.98 (2H, m, CH); 3.23-3.29 (1H, m, CH); 7.14-7.18 (4H, m, C6H4); 13
C
2
2
NMR (150 MHz, D2O-DCl): δ 23.32 (d, JHP=7.5 Hz, CH2); 26,39 (CH2); 33.14 (CH2); 53.93 (d, JHP=9.1
3
Hz); 126.65 (C6H); 127.03 (C6H); 128.80 (C6H); 129.58 (C6H); 131.07 (d, JHP=9.1 Hz, C6); 134.55 (d,
4JHP=1.5 Hz, C6); 31P{H} NMR (243 MHz, D2O-NaOD): δ 23.7 (s); IR (KBr): νmax 760, 892, 987, 1057,
1189, 1520, 1622, 2122, 2270, 2770, 3000, 3352 cm-1. Elemental analysis calc. for C10H14NO3P: N 6.16
and P 13.63%. Found: N 6.26 and P 14.05%.
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Acknowledgements
The authors would like to thank Professor Nikolaus Amrhein for hosting P.M. at the Institute of Plant
Sciences of ETH Zürich and Philippe Roy for introducing P.M. to the kinetics of PAL. J.Z. would like to
thank the Faculty of Mechanical and Power Engineering of the Wrocław University of Science and
Technology for financial support.
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Figure 1.
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Scheme 1.
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Table 1.
Inhibition of anthocyanin
synthesis
Inhibition of PAL
Compound
IC50 ± SD
Kia ± SD
[%]d
[%]d
[μM]
[μM]
0.32±0.03
n.d.
(±)-4
(±)-5
(±)-6
7
83.0±11.0
0.14±0.02
n.d.
n.d.
<50
<40
n.d.
1800±270
n.d.
<50
n.d.
16.4±2.4
8.1±1.2
6.5±1.0
97.3±14.0
n.d.
3.7±0.5
0.00±0.05
1.2±0.2
100.0±15.0
n.d.
n.d.
8
n.d.
n.d.
9
n.d.
n.d.
3
0.83±0.12b
50±8c
n.d.
0.047±0.004b
1.5±0.1c
2.6±0.2c
(R)-(-)-2
(±)-2
83.0±11.0c
n.d.
aFor comparison Km=45 μM of buckwheat PAL for (S)-phenylalanine.[11] The absolute configuration
of (S)-phenylalanine and (R)-1-amino-2-phenylethylphosphonic acid (1) are the same.
b Used as a positive control. Data quoted after Zoń and Amrhein[12]
c Data quoted after literature.[11]
.
dConcentration of studied compounds is 1 mM.
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