Calix[4]arene α-aminophosphonic acids: Asymmetric synthesis and enantioselective inhibition of an alkaline phosphatase

Article abstract of DOI:10.1021/ol052469a

Chiral calix[4]arene α-aminophosphonic acids were obtained through diastereoselective Pudovik-type addition of sodium ethyl phosphites to the chiral calixarene imines, removal of chiral auxiliary groups, and mild dealkylation of phosphonate fragments. The

Full text of DOI:10.1021/ol052469a

ORGANIC
LETTERS
2006
Vol. 8, No. 4
549-552
Calix[4]arene
r-Aminophosphonic
Acids: Asymmetric Synthesis and
Enantioselective Inhibition of an
Alkaline Phosphatase
Sergiy Cherenok,† Andriy Vovk,*,‡ Iryna Muravyova,‡ Alexander Shivanyuk,†
Valery Kukhar,‡ Janusz Lipkowski,§ and Vitaly Kalchenko*,†
The Institute of Organic Chemistry, National Academy of Sciences of Ukraine,
Murmanska, 5, 02094, KyiV-94, Ukraine, The Institute of Bioorganic Chemistry and
Petrochemistry, National Academy of Sciences of Ukraine, Murmanska, 1, 02094,
KyiV-94, Ukraine, and The Institute of Physical Chemistry, Polish Academy of
Sciences, 01-224, Kasprzaka, 44, Warsaw, Poland
Vik@bpci.kieV.ua; VoVk@bpci.kieV.ua
Received October 12, 2005
ABSTRACT
Chiral calix[4]arene
the chiral calixarene imines, removal of chiral auxiliary groups, and mild dealkylation of phosphonate fragments. The diacids obtained show
inhibitory activity toward porcine kidney alkaline phosphatase that depends considerably on the absolute configuration of the -carbon atoms.
r-aminophosphonic acids were obtained through diastereoselective Pudovik-type addition of sodium ethyl phosphites to
r
Inhibition of nonspecific alkaline phosphatases is of signifi-
cant current medical interest1 since these enzymes catalyze
the hydrolysis of phosphate monoesters,2 which are involved
in a number of important biochemical pathways.
Recently, we have reported that calix[4]arenes bearing
fragments of methylenebisphosphonic acid at the wide rim
of the macrocycle are efficient inhibitors of calf intestine
alkaline phosphatase.3 Such an activity was explained by the
coordination of bidentate bisphosphonic residues to metal
ions in the enzyme active site. On the basis of these results,
it was hypothesized that calix[4]arenes functionalized with
chiral bidentate R-aminophosphonic acid residues4 would
possess a similarly enhanced inhibitory activity, which might
depend on the configuration of the chiral carbon atoms.
Herein we report the asymmetric synthesis of calix[4]arene
R-aminophosphonic acids and their efficient, enantioselective
inhibitory activity toward porcine kidney alkaline phos-
phatase (PKAP).
† The Institute of Organic Chemistry, NASU.
‡ The Institute of Bioorganic Chemistry and Petrochemistry, NASU.
§ The Institute of Physical Chemistry, PAS.
(1) (a) Boyan, B. D.; Schwartz, Z.; Lohmann, C. H.; Sylvia, V. L.;
Cochran, D. L.; Dean, D. D.; Puzas, J. E. J. Orthop. Res. 2003, 21, 638-
647. (b) Beck, G. R., Jr.; Sullivan, E. C.; Moran, E.; Zerler, B. J. Cell
Biochem. 1998, 68, 269-280. (c) Sanches de Medina, F.; Martinez-
Augustin, O.; Gonzalez, R.; Ballester, I.; Nieto, A.; Galvez, J.; Zarzuelo,
A. Biochem. Pharmacol. 2004, 68, 2317-2326.
(2) (a) Fennley, H. N. The Enzymes, 3rd ed.; Bouer, P. D., Ed., Academic
Press: New York, 1971; Vol. 4, pp 417-447. (b) Manes, T.; Hoylaerts,
M. F.; Muller, R.; Lottspeich, F., Holke, W.; Millan, J. L. J. Biol. Chem.
1998, 273, 23353-23360. (c) Nicolic-Hughes, I.; O’Brien, P. J.; Herschlag,
D. J. Am. Chem. Soc. 2005, 127, 9314-9315. (d) Myers, J. K.; Antonelli,
S. M.; Widlanski, T. S. J. Am. Chem. Soc. 1997, 119, 3163-3164.
The Pudovik reaction of (S)-imine 15 (Scheme 1) with an
(3) Vovk, A. I.; Kalchenko, V. I.; Cherenok, S. A.; Kukhar, V. P.;
Muzychka, O. V.; Lozynsky, M. O. Org. Biomol. Chem. 2004, 2, 3162-
3166.
(4) Aminophosphonic and Aminophosphinic Acids. Chemistry and Bio-
logical ActiVity; Kukhar, V. P., Hudson, H. R., Eds.; J. Wiley & Sons:
New York, 2000.
(5) Nakanishi, S.; Nakano, S.; Mochizuki, M.; Yoshioka, M. Jpn. Kokai
Tokkyo Koho 2001, 15.
10.1021/ol052469a CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/27/2006

and the other with quasicoplanar distal unsubstituted aromatic
rings. Unfortunately, the NMR spectra do not allow deter-
mination of which conformation is preferred in solution.
Scheme 1
(11) Synthesis of Aminophosphonates 6 and 7. Sodium (0.3 g, 13
mmol) was added to diethyl phosphite (5 mL) by portions (with caution!)
at room temperature. Calixarene monoimine 4 (4 mmol) or diimine 5 (2
mmol) was added to the resulting solution. The reaction mixture was stirred
at 10 °C for 12 h, quenched by water (100 mL), and extracted with
chloroform (3 × 50 mL). The chloroform extract was evaporated, and the
residue was washed with hexane and dried in vacuo. 5-(r-Phenylethyl-
aminodiethoxyphosphonylmethyl)-25,27-dipropoxycalix[4]arenes 6a,b.
Purified by flash column chromatography (silica, CHCl3/acetone 4:1) Rf )
0.5. [R]28D ) +12° (CHCl3) for 6a, [R]28D ) -12° (CHCl3) for 6b. Colorless
1
solids, yields 75-80%. Mp 119-120 °C. H NMR (300 MHz, CDCl3) δ:
0.85, 1.32 (two t, 3H+3H, J ) 7.5 Hz), 1.30, 1.31 (two t, J ) 7.5 Hz, each
3H), 1.33 (d, J ) 7.5 Hz, 3H), 2.01 (m, 4H), 3.30 (m, 1H), 3.32, 3.34 (two
d, J ) 13.2 Hz, each 2H), 3.75, 4.15 (two m, each 2H), 3.91 (d, J ) 11 Hz,
1H), 3.96, 3.98 (two t, J ) 7.5 Hz, each 2H), 4.24, 4.26, 4.28, 4.31 (four
d, J ) 13.2 Hz, each 1H), 6.80-7.40 (m, 11H), 8.40, 8.55 (two s, each
1H). 31P NMR (121 MHz, CDCl3) δ: 26.4; 5,17-Bis(r-phenylethylamino-
diethoxyphosphonylmethyl)-25,27-dipropoxycalix[4]arenes 7a,b. Purified
excess of sodium diethyl phosphite in diethylphosphite solu-
tion at 10 °C afforded R-aminophosphonate 2 in 98% yield
and 95% diastereomeric excess.6 The subsequent removal
of the phenylethyl group (Pd/C, H2) and the dealkylation of
the ester groups (Me3SiBr, MeOH) transformed compound
27 into acid 3.8 Single crystal X-ray analysis9 showed that
the R-carbon atom of aminophosphonate 2 has the (R)
configuration, which is in agreement with the empirical rule
for the Pudovik type addition to the R-phenylethylimines.4
Chiral iminocalix[4]arenes 4 and 5 were obtained in
preparative yields through the condensation of mono- or 1,3-
diformylcalix[4]arenes with (R)- or (S)-phenylethylamine.10
The ensuing diastereoselective addition of sodium diethyl
phosphite to the CdN bonds afforded aminophosphonates
6 and 7 in 60-80% yield and 75-85% diastereomeric excess
(Scheme 2).11
by column chromatography (silica, CHCl3/acetone 4:1) Rf ) 0.7. [R]28
)
D
+7° (CHCl3) for 7a, [R]28 ) -7° (CHCl3) for 7b. White solids, yields
D
60-65%. Mp 82-83 °C. 1H NMR (300 MHz, CDCl3) δ: 0.66, 1.32 (two
t, J ) 7.5 Hz, each 6H), 1.33 (m, 6H), 1.41 (d, J ) 7.5 Hz, 6H), 2.01 (m,
4H), 3.36 (m, 6H), 3.66, 3.81 (two m, each 2H), 3.92 (d, J ) 11 Hz, 2H),
3.96 (t, J ) 7.5 Hz, 4H), 4.09 (m, 4H), 4.26, 4.30 (two d, J ) 13.0 Hz,
each 2H), 6.67 (t, J ) 7.5 Hz, 2H), 6.81, 6.90 (two d, J ) 7.5 Hz, each
2H), 6.98, 7.11 (two s, each 2H), 7.25 (m, 10H), 8.26 (s, 2H). 31P NMR
(121 MHz, CDCl3) δ: 25.7.
(12) General Procedure for Synthesis of Aminophosphonates 8 and
9. Compound 6 (1.38 mmol) or 7 (1.07 mmol) was added to a suspension
of Pd/C (0.2 g) in methanol (25 mL). The reaction mixture was stirred
under hydrogen atmosphere at 40 °C for 72 h. The catalyst was filtered
off, and the solution was evaporated in vacuo. The residue was washed by
diethyl ether. 5-(Aminodiethoxyphosphonylmethy)-25,27-dipropoxycalix-
[4]arenes 8a,b. [R]28 ) +30° (CHCl3) for 8a, [R]28 ) -30° (CHCl3)
D
D
for 8b. White solids, yields 60-65%. Mp 95-97 °C. 1H NMR (300 MHz,
CDCl3) δ: 0.51, 0.62 (two t, J ) 7.5 Hz, each 2H), 1.31 (t, J ) 7.5 Hz,
6H), 2.01 (m, 4H), 3.55 (m, 4H), 3.96-3.98 (m, 4H), 4.28, 4.31 (two d, J
) 13.2 Hz, each 2H), 4.60 (d, J ) 11 Hz, 1H), 6.60 (t, J ) 7.5 Hz, 2H)
6.71 (t, J ) 7.5 Hz, 1H), 6.90-7.15 (m, 6H), 7.28, 7.30 (two s, each 1H),
8.41, 8.62 (two s, each 2H). 31P NMR (121 MHz, CDCl3) δ: 26.4.
Calculated for C39H48NO7P: C 69.52, H 7.18, N 2.08 P 4.60, found C 69.31,
H 7.26, N 2.15 P 4.46. m/z (FAB) 536.7 ([M - P(O)OEt2]+, 80%) 657.7
([M - NH2]+, 70%); 5,17-Bis(aminodiethoxyphosphonylmethyl)-25,27-
The chiral auxiliary groups of compounds 6 and 7 were
removed by catalytic hydrogenation (H2, Pd/C) to give
individual stereoisomers of calixarenes 8 and 9 in preparative
yields.12 The subsequent treatment of aminophosphonates 8
and 9 with Me3SiBr and methanol gave mono- and di-R-
aminophosphonic acids 10 and 11 in quantitative yields.13
The absolute configurations at the R-carbon atoms in
calixarenes 8-11 were assigned on the basis of studies on
the model compounds 2 and 3. (S)-Imines give (R)-amino
acids and vice versa (see above).
dipropoxycalix[4]arenes 9a,b. [R]28 ) -21° (CHCl3) for 9a, [R]28
)
D
D
+21° (CHCl3) for 9b. White solids, yields 85-90%. Mp 65-67 °C. 1H
NMR (300 MHz, CDCl3) δ: 0.88, 0.95 (two t, J ) 7.5 Hz, each 6H), 1.23
(t, J ) 7.5 Hz, 6H), 2.01 (m, 4H), 3.32 (d, J ) 13.0 Hz, 4H), 3.54, 3.75
(two m, each 2H), 3.90 (m, 8H), 4.04 (d, J ) 15.0 Hz, 2H), 4.22 (d, J )
13.0 Hz, 4H), 6.61 (t, J ) 7.2 Hz, 2H), 6.82, 6.84(two d, J ) 7.2 Hz, each
2H), 7.09, 7.12 (two s, each 2H) 8.18 (s, 2H). 31P NMR (CDCl3) δ: 24.9.
Calculated for C44H60N2O10P2: C 63.00, H 7.21, N 3.34 P 4.60, found C
62.89, H 7.15, N 3.42 P 4.68 (9a); found C 62.91, H 7.13, N 3.38 P 4.52
(9b).
1
The H NMR spectra of compounds 10 and 11 contain
AB doublets for the methylene protons of the bridges that
are characteristic of the C2V-symmetrical pinched cone
conformation.14 There are two possible pinched cone con-
formers of calixarenes 10 and 11: one with quasiparallel
(13) Synthesis of Aminophosphonic Acids 10 and 11. Bromotrimethyl-
silane (8-fold molar excess per phosphonate group) was added to a solution
of aminophosphonates 8 or 9 (1 mmol) in dry chloroform (5 mL). The
reaction mixture was stirred at room temperature for 30 h. Then the solvent
was evaporated under reduced pressure, and the residue was dissolved in
absolute methanol (15 mL). The reaction mixture was stirred at 50 °C for
2 h and then was evaporated in vacuo. The residue was dried in vacuo
(0.05 mm) for 10 h. 5-(Aminodihydroxyphosphonylmethyl)-25,27-
dipropoxycalix[4]arenes 10a,b. [R]28 ) +18° (CHCl3) for 10a, [R]28
(6) Diastereomeric excesses were calculated through integration of 31P
NMR signals of diastereomers.
(7) Sodium (0.3 g, 13 mmol) was added to diethyl phosphite (5 mL)
(caution!) at room temperature followed by compound 1 (4 mmol). The
reaction mixture was stirred at 10 °C for 12 h, quenched with water (100
mL), and extracted with chloroform (3 × 50 mL). The chloroform layer
was evaporated and the residue was washed with hexane and dried in vacuo.
(R,S)-R-Phenylethylaminodiethoxyphosphosphonylmethyl-4-hydroxy-
benzene 2. [R]20D ) +20° (CHCl3). Colorless solid, yield 96%. Mp 119-
120 °C. 1H NMR (300 MHz, CDCl3) δ: 1.16, 1.36 (two t, J ) 7.5 Hz,
each 3H), 1.30 (d, J ) 7.5 Hz, 3H), 3.80-4.20 (m, 6H), 6.64 (d, J ) 7.5
Hz, 2H), 7.08 (dd, J ) 7.5 Hz, J ) 2 Hz, 2H), 7.21-7.28 (m, 5H). 31P
NMR (121 MHz, CDCl3) δ: 25.01.
(8) This compound has been obtained earlier in a different way. See:
Lejczak, B.; Kafarski, P.; Makowiecka, E. Biochem. J. 1987, 242, 81-88.
(9) Lipkowski, J.; Cherenok, S.; Kalchenko, V. I. Unpublished results.
(10) (a) Solovyov, A. V.; Cherenok, S. A.; Tsymbal, I. F.; Failla, S.;
Consiglio, G.; Finocchiaro, P.; Kalchenko, V. I. Heteroat. Chem. 2001,
12, 58-67. (b) Arduini, A.; Fanni, S.; Manfredi, G.; Pochini, A.; Ungaro,
R.; Sicuri, A. R.; Ugozzoli, F. J. Org. Chem. 1995, 60, 1448-1453.
D
D
) -18° (CHCl3) for 10b. White solids, yields 90-95%. Mp 89 °C
(decomp). 1H NMR (300 MHz, DMSO-d6) δ: 1.35 (t, 6H, J ) 7.5 Hz,
6H), 2.05 (m, 4H), 3.33 (br m, 4H), 3.96-3.98 (m, 4H), 4.22 (d, J ) 11
Hz, 1H), 4.30 (br m, 4H), 6.62-6.71 (m, 3H), 6.92-7.17 (m, 6H), 7.28-
7.30 (br s, 2H), 8.39, 8.55 (two s, each 2H). 31P NMR (121 MHz, DMSO-
d6) δ: 15.75. Calculated for C35H40NO7P: C 68.06, H 6.53, N 2.27 P 5.01,
found C 68.31, H 6.64, N 2.16 P 4.91. m/z (FAB MASS) 536.7 ([M -
P(O)(OH)2]+, 80%) 601.6 ([M - NH2]+, 75%). 5,17-Bis(aminodihydroxy-
phosphonylmethyl)-25,27-dipropoxycalix[4]arenes 11a,b. [R]28D ) -12°
(CHCl3) for 11a, [R]28D ) +12° (CHCl3) for 11b. Colorless solids, yields
1
90-95%. Mp 95 °C (decomp). H NMR (300 MHz, DMSO-d6) δ: 1.24
(m, 6H), 2.01 (m, 4H), 3.32 (br m, 4H), 3.95 (br m, 4H), 4.07 (br d, J )
15 Hz, 2H), 4.19 (br m, 4H), 6.81 (br. m, 4H), 7.01(br m, 4H), 7.21 (br m,
4H), 8.61 (br s, 2H). 31P NMR (121 MHz, DMSO-d6) δ: 13.78. Calculated
for C36H44N2O10P2: C 59.50, H 6.10, N 3.85 P 8.52, found C 59.63, H
6.01, N 3.76, P 8.65 (11a); found C 59.41, H 6.15, N 3.79, P 8.61 (11b).
550
Org. Lett., Vol. 8, No. 4, 2006

Scheme 2
When assayed against the hydrolysis of p-nitrophenyl-
the stabilization of the enzyme-inhibitor complex by 1.2
kcal/mol. Such a reinforcement of the enzyme-inhibitor
interactions may be caused by multiple van der Waals
attractions between the bulky calixarene fragment and the
surface of the binding site.
phosphate by the porcine kidney alkaline phosphatase,
calixarenes 10 and 11 were found to be reversible inhibitors.
The influence of compounds 3, 10, and 11 on the enzyme
activity is in agreement with a mixed-type inhibition mech-
anism. The inhibition constants Ki and Ki′15 (Table 1) of
The inhibition of PKAP is sensitive to the absolute
configuration of the calixarene R-aminophosphonic acids.
The Ki value for isomer 10b is about two times smaller than
for its counterpart 10a. However in terms of free energy,
this difference (0.5 kcal/mol) is practically negligible. The
enantioselectivity of the inhibition is drastically increased
in the case of bis-R-aminophosphonic acids 11. Namely, the
(R,R) isomer 11a binds to PKAP about 50 times stronger
than the (S,S) isomer 11b, which corresponds to a difference
in free energy of 2.3 kcal/mol. Although the exact cause of
this enantioselectivity remains unrevealed, it seems plausible
that, at least partly, it can be attributed to the asymmetric
disposition of the amino acid residues around the binding
site, which may result in different sterical demands to the
binding of the enantiomeric inhibitors.
Table 1. Inhibition Constants (µM) for the Inhibition of PKAP
by Compounds 3, 10, and 11a and Free Energies (kcal/mol) for
the Formation of Enzyme-Inhibitor Complexes of PKAP and
BIAP
inhibitor Ki (PKAP) Ki′ (PKAP) ∆Gb (PKAP) ∆Gb (BIAP)
3
580 ( 110 6800 ( 840
-4.4
-5.6
-6.1
-7.8
-5.5
-4.5
-6.5
-6.6
-7.2
-6.4
10a (R)
10b (S)
73 ( 13
32 ( 5
540 ( 90
780 ( 100
130 ( 30
610 ( 210
11a (R,R) 1.7 ( 3
11b (S,S)
86 ( 11
a 0.1 M Tris-HCI buffer (pH 9), 296 K. b ∆G ) -RT ln(Ki-1).
Even after statistical correction, the Ki value for acid 10a
is 20 times higher than that for diacid 11a. This can be
attributed to the cooperative binding of the two R-amino-
phosphonate fragments of molecule 11a to the metal cations
and the arginine residue of the alkaline phosphatase binding
site.2,16 It should be noted that the free energy for the
formation of the enzyme-inhibitor complex with compound
10b is lower by 1 kcal/mol than the statistically corrected
calixarene 10a are considerably smaller than the values for
the model compound 3. This corresponds to an increase in
(14) Grootenhuis, P. D. J.; Kollman, P. A.; Groenen, L. C.; Reinhoudt,
D. N.; van Hummel, G. J.; Ugozzoli, F.; Andreetti, G. D. J. Am. Chem.
Soc. 1990, 112, 4165-4176.
(15) These are dissociation constants of enzyme-inhibitor and enzyme-
substrate-inhibitor complexes, respectively: Dixon, M.; Webb, E. C.
Enzymes; Mir: Moskow, 1982 (in Russian).
Org. Lett., Vol. 8, No. 4, 2006
551

value of the free energy in the case of 11b. This shows that
the attachment of the second (S)-R-aminophosphonate residue
to the calixarene scaffold contributes to the destabilization
of the enzyme-inhibitor complex. Similar effects have been
observed also for the binding of compounds 10 and 11 to
the enzyme-substrate complex, which is characterized by
inhibition constants Ki′ (Table 1).
As is seen from Table 1, compounds 10 and 11 display
almost identical affinities to bovine intestinal alkaline phos-
phatase (BIAP). This can be explained by the coordination
of only one R-aminophosphonate residue at the binding site.
The impact of the calixarene is more pronounced than in
the case of PKAP inhibition most probably as a result of
the better fit between the inhibitor and enzyme surfaces.
Surprisingly the enantioselectivity of the BIAP inhibition by
calixarenes 11a and 11b is not pronounced, with a difference
in free energy of only 0.8 kcal/mol. These results may reflect
subtle differences in the active sites of BIAP and PKAP.
In conclusion, the preorganization of two R-aminophos-
phonic acid fragments on the calix[4]arene platform results
in a significant increase in the inhibition of alkaline phos-
phatase. The strength of the enzyme-inhibitor interactions
is determined both by cooperative effects and the goodness
of the fit of the chiral inhibitor to the chiral space around
the binding site. To the best of our knowledge compound
11a is the first alkaline phosphatase inhibitor of R-amino-
phosphonic acid type that has an affinity for the enzyme in
the micromolar range.
Acknowledgment. This research was supported by the
National Academy of Sciences of Ukraine (Grants 5/4-B and
01/03-05) and the Polish Ministry of Science and Information
Society Technologies (Grant 4 T09A 068 25).
(16) Stec, B.; Holtz, K. M.; Kantrowitz, E. R. J. Mol. Biol. 2000, 299,
1303-1311.
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