A one step/one pot synthesis of N,N-bis(phosphonomethyl)amino acids and their effects on adipogenic and osteogenic differentiation of human mesenchymal stem cells

Article abstract of DOI:10.1016/j.bmc.2009.03.039

The one pot reaction of amino acids with diethylphosphite and formaldehyde yielded N,N-bis(phosphonomethyl)amino acids. This synthetic route does not require harsh reagents to cleave the ester group. The molecular structures of the new compounds were dete

Full text of DOI:10.1016/j.bmc.2009.03.039

Bioorganic & Medicinal Chemistry 17 (2009) 3388–3393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A one step/one pot synthesis of N,N-bis(phosphonomethyl)amino acids
and their effects on adipogenic and osteogenic differentiation of human
mesenchymal stem cells
a,
b
b
Johanna Kasser a, Alexey A. Nazarov a, , Christian G. Hartinger , Brigitte Wdziekonski , Christian Dani ,
*
*
Maxim L. Kuznetsov c, Vladimir B. Arion a, Bernhard K. Keppler a
a University of Vienna, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria
b Institute of Developmental Biology and Cancer, CNRS UMR 6543, Université Nice Sophia-Antipolis, Nice, France
c Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Technical University of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The one pot reaction of amino acids with diethylphosphite and formaldehyde yielded N,N-bis(phospho-
nomethyl)amino acids. This synthetic route does not require harsh reagents to cleave the ester group. The
molecular structures of the new compounds were determined by X-ray diffraction methods. By employ-
ing DFT calculations the hydrolysis of the intermediate phosphonic esters to the respective acids could be
Received 11 November 2008
Revised 13 March 2009
Accepted 20 March 2009
Available online 26 March 2009
explained by the decreasing P–OEt bond strength for C -bisalkylated amino acids. Biological evaluation
a
on the adipogenic and osteogenic differentiation of mesenchymal stem cells revealed no modification
of the adipocyte differentiation, but inhibition of osteoblast formation at concentrations without detect-
able cytotoxicity.
Dedicated to Eduard E. Nifant’ev on the
occasion of his 75th birthday
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Bisphosphonates
Crystal structure analysis
DFT calculations
Synthesis
Human adipose-derived stem cells
1. Introduction
Fotemustine) activity are known.10 Bisphosphonates were also
developed with activity against malaria, toxoplasmosis and leish-
Compounds containing phosphonate groups were found to ex-
hibit important pharmacological properties and are widely used
in the treatment of various diseases.1,2 The big majority of phos-
phonates and bisphosphonates are applied in the treatment of
osteoporosis (e.g., sodium alendronate) and hypercalcemia (caused
by cancer-related increased bone resorption), for calcium regula-
tion (e.g., disodium clodronate, etidronic acid), and inhibition of
bone resorption (e.g., sodium ibandronate, sodium risedronate,
disodium tiludronate),3–10 which is caused by the affinity of the
bisphosphonates to hydroxylapatite and Ca2+ ions. Osteoporosis
is a wide-spread disease in the elder population and affects up to
50% of females and up to 15% of males.11 The fragility of the bones
leads to a high risk of fractures and the quality of life decreases
significantly.
maniasis in vitro and in vivo and some were observed to inhibit
specifically biosynthetic routes in plants and protozoans.12,13 Other
examples are N-(phosphonomethyl)glycine (Glyphosate), and N,N-
(bisphosphonomethyl)glycine (Glyphosine) which are used world-
wide as plant growth regulators and herbicides.14 They typically
affect the shikimate biosynthesis pathway for the formation of aro-
matic amino acids and flavonoids; Glyphosate inhibits the conver-
sion shikimat-3-phosphate and phosphoenolpyruvate to 5-
enolpyruvylshikimat-3-phosphate and inorganic phosphate, and
the biosynthesis of proteins.15
The idea of exploiting the high affinity of bisphosphonates to
Ca2+ led to the development of platinum coordination com-
pounds conjugated to bisphosphonates via a chelating group.
Platinum coordination compounds hold an important position
in cancer chemotherapy,16,17 and phosphorus-containing ligands
were used for linking metal centers to organic moieties.18–21
Osteotropic bisphosphonomethyl platinum(II) complexes showed
remarkable activity against osteosarcoma and bone metasta-
ses.22 The type of DNA adducts formed by this compound are
similar to cisplatin and carboplatin, due to the release of the
Furthermore, drugs with antiviral (e.g., sodium foscarnet,
Cidofovir), antibiotic (e.g., Fosfomycin) and antineoplastic (e.g.,
* Corresponding authors. Fax: +43 1 4277 9526 (C.G.H.).
E-mail addresses: alex.nazarov@univie.ac.at (A.A. Nazarov), christian.hartinger@
univie.ac.at (C.G. Hartinger).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.039

J. Kasser et al. / Bioorg. Med. Chem. 17 (2009) 3388–3393
3389
bisphosphonates and interaction with nucleobases, which is
2.2.1. N,N-Bis(phosphonomethyl)-2-amino-2-methylpropionic
acid 1a
fastened by Ca2+ ions.23,24 In vivo experiments revealed that
the Pt complexes in combination with the antimetastatic agent
razoxane suppressed the tumor growth of an osteosarcoma and
reduced metastases.25
Using the general procedure, 2-amino-2-methylpropionic acid
(1.44 g, 14 mmol) was reacted with diethylphosphite (3.61 mL,
28 mmol) and formaldehyde solution (4.28 mL, 56 mmol). Yield:
2.14 g (52%), colorless crystals. Mp 194–196 °C; 1H NMR (D2O,
Herein, we present a new way to synthesize aminobisphosponic
2
acids bearing a bisalkyl-substituted C atom in a one step/one pot
400.13 MHz) d = 1.57 (s, 6H, CH3), 3.43 (d, JHP = 13.0 Hz, 4H, CH2)
a
procedure. The compounds have been characterized by standard
methods, including X-ray diffraction analysis for establishing the
structures. DFT calculations explain the hydrolysis of phosphonic
esters to the corresponding acids. The compounds were tested on
their biological activity on the adipogenic and osteogenic differen-
tiation of mesenchymal stem cells isolated from human adipose
tissue (hMADS cells).
ppm; 13C NMR (D2O, 100.63 MHz) d = 20.0 (CH3), 50.8
(J = 133.0 Hz, CH2), 73.9 (3J = 5.0 Hz, C(CH3)2), 174.2 (CO) ppm;
31P NMR (D2O, 161.98 MHz) d = 9.6 ppm. Anal. Calcd for
C6H15NO8P2ꢀH2O: C, 23.31; H, 5.54; N, 4.53; P, 20.04. Found: C,
23.37; H, 5.27; N, 4.40; P, 19.77. ESI-MS (neg): m/z 289.9 [MꢁH]ꢁ.
2.2.2. N,N-Bis(phosphonomethyl)-1-amino-1-cyclohexane
carboxylic acid 1b
Using the general procedure, 1-amino-1-cyclohexanecarboxylic
acid (2.00 g, 14 mmol) was reacted with diethylphosphite
(3.61 mL, 28 mmol) and formaldehyde solution (4.28 mL,
56 mmol). Yield: 1.53 g (33%), colorless crystals. Mp 187–189 °C;
1H NMR (D2O, 500.10 MHz) d = 1.05–1.20 (m, 1H, CH2), 1.36–1.50
(m, 2H, CH2), 1.58 (m, 1H, CH2), 1.65–1.80 (m, 4H, CH2), 2.17 (m,
2. Experimental
2.1. General
Analytical grade materials were obtained from commercial
suppliers and used without further purification. Melting points
were determined with a Büchi B-540 apparatus and are uncor-
rected. NMR spectra were recorded on a Bruker Avance DPX-
400 spectrometer at 400.13 MHz (1H), 100.63 MHz (13C{1H}) and
2
2H, CH2), 3.44 (d, JHP = 13.1 Hz, 4H, N(CH2)2) ppm; 13C NMR
(DMSO-d6, 125.75 MHz) d = 23.5 (CH2), 25.7 (CH2), 34.0 (CH2),
51.5 (3J = 4.0 Hz, J = 159.0 Hz), 67.0 (3J = 8.8 Hz, 3J = 9.7 Hz), 174.4
(CO) ppm; 31P NMR (D2O, 202.44 MHz) d = 8.9 ppm; Anal. Calcd
for C9H19NO8P2ꢀH2O: C, 30.95; H, 6.06; N, 4.01. Found: C, 31.03;
H, 5.64; N, 3.94. ESI-MS (neg): m/z 330.0 [MꢁH]ꢁ.
161.98 MHz
( a Bruker FT-NMR spectrometer
31P{1H}) or on
Avance IIITM 500 MHz at 500.10 MHz (1H), 125.75 MHz
1
1
(
13Cf Hg) and 202.44 MHz (31Pf Hg) at 298 K in D2O or DMSO-
d6. Electrospray ionization mass spectra were recorded on a Bru-
ker esquire3000 in negative ion mode. Elemental analyses were
performed by the Laboratory for Elemental Analysis of the Faculty
of Chemistry, University of Vienna, with a Perkin Elmer 2400 CHN
Elemental Analyzer. The elemental analysis of P was performed
using the following procedure:26 Molybdate forms a complex
with ortho-phosphate which can be reduced to a polymeric spe-
cies exhibiting a characteristic blue color (‘molybdenum blue’)
suitable for colorimetric analysis. In a first step the sample (2–
2.2.3. N,N-Bis(phosphonomethyl)-1-amino-1-cyclopentane
carboxylic acid 1c
Using the general procedure, 1-amino-1-cyclopentanecarboxy-
lic acid (1.00 g, 7.7 mmol) was reacted with diethylphosphite
(2.00 mL, 15.4 mmol) and formaldehyde solution (2.35 mL,
30.8 mmol). Yield: 0.80 g (33%), colorless crystals. Mp 184–
186 °C; 1H NMR (D2O, 400.13 MHz) d = 1.72–1.86 (m, 4H, CH2),
2.08–2.18 (m, 2H, CH2), 2.19–2.29 (m, 2H, CH2), 3.45 (d, 4H,
2JHP = 11.0 Hz, N(CH2)2) ppm; 13C NMR (D2O, 100.63 MHz)
d = 25.6 (CH2), 33.6 (CH2), 52.7 (J = 130.0 Hz, N(CH2)2), 83.2
(3J = 5.0 Hz, C(CH2)2), 174.5 (CO) ppm; 31P NMR (D2O,
161.98 MHz) d = 9.9 ppm; Anal. Calcd for C8H17NO8P2ꢀH2O: C,
28.67; H, 5.71; N, 4.18; P, 18.48. Found: C, 28.77; H, 5.48; N,
4.07; P, 18.70. ESI-MS (neg): m/z: 316.3 [MꢁH]ꢁ.
5 mg) is digested by addition of 750
ll of concentrated sulfuric
acid and heating for an hour. After cooling, 750
l
l of fuming nitric
acid are added and the mixture is kept boiling for another half an
hour until no more NOx fume is released. After cooling, the digest
is diluted to ca. 25 ml and titrated against phenolphthaleine, filled
up to the volumetric mark and 5.0, 10.0 or 25.0 ml of this analyte
solution are used for colorimetric determination. An aliquot of the
analyte solution is transferred to a 50.0 ml volumetric flask.
2.5 ml of 2.5% ammonium molybdate in 5 M sulfuric acid and
5.0 ml of 0.25% p-methylaminophenol in 15% sodium pyrosulfite
are added and the flask is filled up close to the volumetric mark.
Molybdenum blue forms at 40 °C in a water bath within 7 min.
Then the solution is cooled down to room temperature and at a
maximum of 30 min prior to the analysis the volumetric flask is
filled up to the mark with water. Absorption was measured with
an HP 8253 diode array UV–vis spectrophotometer (k = 820 nm)
using water as reference. Calibration is done using triphenylphos-
phine as analytical standard which is treated in the same way as
the samples.
2.3. Crystal structure data
X-ray diffraction measurements were performed on a Nonius
Kappa CCD diffractometer (1b) or on a Bruker X8 APEX II CCD dif-
fractometer (1a and 1c). A single crystal was mounted on a goni-
ometer head at 40, 30 and 40 mm from the detector, and 841,
455, and 1009 frames were measured, each for 30, 10, and 60 s
over 1, 1.5 and 1° scan width. The data were processed using the
DENZO-SMN and SAINT software packages.27 Crystal data, data collec-
tion parameters, and structure refinement details are given in
Table 1. The structures were solved by direct methods and refined
by full-matrix least-squares techniques. Non-hydrogen atoms
were refined with anisotropic displacement parameters. H atoms
were inserted at calculated positions and refined with a riding
model. The atoms C4 and C5 of the cyclopentane ring, and the
water molecule in 1c were found to be disordered over two
positions with S.O.F. 0.86 to 0.14. The following computer pro-
grams were used: structure solution, SHELXS-97;28 refinement,
SHELXL-97;28 molecular diagrams, ORTEP-3;29 computer, Pentium IV;
scattering factors.30 The crystal structures have been deposited
at the Cambridge Crystallographic Data Centre and allocated
the deposition numbers CCDC 708525 (1a), 708526 (1b) and
708527(1c).
2.2. General procedure for synthesis
The amino acid (1 equiv) was suspended in diethylphosphite
(2 equiv) and 36% aqueous formaldehyde solution (4 equiv) was
added dropwise at room temperature. The suspension was stirred
for 1 h at room temperature and refluxed for 17 h. The acid crystal-
lized from the mother liquor within 24 h after cooling to room
temperature. The crystals were collected, washed with ethanol
and recrystallized from water.

3390
J. Kasser et al. / Bioorg. Med. Chem. 17 (2009) 3388–3393
Table 1
3. Results and discussion
3.1. Synthesis
Selected crystallographic data for 1a–c
1a
1b
1c
Empirical formula
fw (g molꢁ1
C6H15NO8P2ꢀH2O
309.15
296(2)
C9H19NO8P2ꢀH2O
349.21
120(2)
C8H17NO8P2ꢀH2O
335.18
296(2)
Different synthetic routes to aminophosphonic acids have been
reported in the literature. The most frequently used are the Man-
nich-type Moedritzer–Irani-reaction and the Kabachnik–Fields-
reaction,36–38 the former involving the reaction of formaldehyde,
)
Temperature (K)
Crystal size
Color, habit
Crystal system
Space group
a (Å)
0.19 ꢂ 0.06 ꢂ 0.04 0.14 ꢂ 0.26 ꢂ 0.34 0.57 ꢂ 0.48 ꢂ 0.31
Colourless, plate
Monoclinic
P21/n
Colourless, block
Monoclinic
P21/n
11.188(2)
7.043(1)
18.576(4)
90.96(3)
1463.5(5)
4
1.585
3.41
736
3.09–26.00
Colourless, block
Monoclinic
P21/n
11.1652(2)
6.8742(1)
18.2364(4)
93.650(1)
1396.84(4)
4
1.594
3.54
704
3.17–30.00
phosphonic acid and
to yield
-aminomethylphosphonic acids.36 Kabachnik and Fields
introduced the synthesis of -aminomethylphosphonate from
a-amino acids in aqueous hydrochloric acid
11.1277(5)
6.7162(3)
17.1227(7)
99.303(3)
1262.85(10)
4
1.626
3.84
648
2.41–31.78
a
b (Å)
C (Å)
a
aldehydes or ketones, amines and dialkylphosphites in a one pot-
reaction.39,40 The phosphonic acids, required for binding to bone
material and for exhibiting activity on osteoclasts,41,42 can be ob-
tained by cleavage of the ester groups.43,44
b (°)
V (Å3)
Z
Dcalcd (g cmꢁ3
)
l
(cmꢁ1
F(0 0 0)
)
The conversion of different a-amino acids to the corresponding
a
-aminobis(methylphosphonic acids) was reported in the litera-
h range for data
collection (°)
h Range
k Range
l Range
ture,45,46 but only the reaction with glycine yielded pure product.36
Even changing from glycine to alanine provides mainly a decompo-
ꢁ16, 16
ꢁ9, 6
ꢁ13, 13
ꢁ8, 8
ꢁ15, 15
ꢁ9, 9
sition product due to N–C cleavage. Such unique behavior of gly-
a
ꢁ25, 24
4277
ꢁ22, 22
2877
ꢁ25, 25
4078
cine in this process compared to other amino acids may be
No. refls. used in
refinement
No. parameters
Rint
accounted for by the highest N–C bond energy in glycine. Indeed,
a
quantum chemical calculations at the DFT level of theory (see
173
209
214
0.0340
0.0389
0.1133
1.032
0.0121
0.0288
0.0853
1.036
0.0225
0.0291
0.0921
1.064
Computational Details) indicate that the vertical N–C bond energy
a
a
R1
in glycine (310.8 kcal/mol) is by 28.4 and 48.1 kcal/mol higher than
in alanine and 2-amino-2-methyl-propionic acid, respectively.
Meanwhile, when changing to amino acids having no protons at
b
wR2
GOFc
P
P
a
R1
=
||Fo| ꢁ |Fc||/ |Fo|.
the C , such as 1-amino-1-cyclohexane-carboxylic acid, 1-amino-
a
P
P
wR2 = { [w (F2o ꢁ F2c )2]/ [w(Fo2)2]}1/2
.
b
c
1-cyclopentane-carboxylic acid or 2-amino-2-methyl-propionic
acid, and using diethylphosphite as P source the corresponding
acids 1a–c have been obtained in a one pot/one step procedure
involving the hydrolysis of the ester groups, probably catalyzed
by the carboxylic acid functionality (Scheme 1). Notably, no by-
products were observed and the compounds were isolated in good
yields after crystallization from the reaction mixture and charac-
terized by means of 1H, 13C and 31P NMR spectroscopy and single
crystal X-ray diffraction analysis.
P
GOF = { [w(F2o ꢁ Fc2)2]/(n ꢁ p)}1/2, where n is the number of reflections and p is
the total number of parameters refined.
2.4. Adipocytes/osteoblast differentiation
hMADS3 cells31 have been induced to undergo differentiation in
a culture medium allowing differentiation into both adipocytes
and osteoblasts, as previously described by Zaragosi et al.32 in
the absence (control adipocytes/osteoblasts differentiation) or in
the presence of 10ꢁ7 M or 3 ꢂ 10ꢁ7 M of 1a–c or the reference sub-
stance sodium alendronate trihydrate (Sigma). Fourteen days after,
cells were fixed and stained with Oil red O to reveal adipocytes and
with Von Kossa to reveal calcium deposition secreted by osteo-
blasts. Adipocyte differentiation has been quantified by GPDH
activity (a specific marker of adipocytes) and osteoblast differenti-
ation by ALP activity (an osteogenic-specific marker).
The reaction progress was followed by 31P NMR and several
groups of signals were observed (Fig. 1), which were assigned to
esters (20–26 ppm), monophosphonates (13–20 ppm), diethyl-
phosphite and its hydrolysis products (9–11 ppm) and the bisphos-
phonic acids (ca. 10 ppm). Normally the Kabachnik–Fields-reaction
leads to the synthesis of phosphonic esters which can be further
converted into the acids, with the mildest method being micro-
wave-assisted hydrolysis with trimethylsilyl bromide.44 This pro-
cedure should lead to a single signal assignable to the ester,
however in the present case the ester bonds were cleaved which
leads initially to a mixture of different di- and monoesters and fi-
nally to the acid as the main product. Exceptions are such amino
2.5. Computational details
The full geometry optimization of all structures has been car-
ried out at the DFT level of theory using Becke’s three-parameter
hybrid exchange functional33 in combination with the gradient-
corrected correlation functional of Lee, Yang and Parr34 (B3LYP)
with the help of the GAUSSIAN-0335 program package. Symmetry
operations were not applied. The geometry optimization was car-
ried out using the 6-31G(d) basis set followed by single-point cal-
culations of the total energies at the 6-311+G(d,p) basis set. The
Hessian matrix was calculated analytically for all optimized struc-
tures in order to prove the location of correct minima (no ‘imagi-
nary’ frequencies). The starting geometries for the optimizations
were based on the experimental X-ray structures of 1a–c (this
work). Vertical bond energies were calculated as a difference of
the sum of the total energies of fragments with unrelaxed geome-
try and the total energy of the corresponding amino acid, amino-
phosphonic acid or ester of aminophosphonic acid. For
aminophosphonic acids and their esters, energies of a single P–
OH and P–OEt bond, respectively, have been calculated.
OH
R
R
R
O
OH
O
HPO3Et2
R
N
HCHO / H2O
H2N
Et2O3P PO3Et2
not isolated
OH
R
1a
1b
1c
1d
R = CH3
R
O
R = cyclohexane
R = cyclopentane
R = H
N
H2O3P PO3H2
1a-c
Scheme 1. Synthetic route to N,N-bis(phosphonomethyl) amino carboxylic acids.

J. Kasser et al. / Bioorg. Med. Chem. 17 (2009) 3388–3393
3391
Figure 1. Time-course of the reaction progress of 2-amino-2-methylpropionic acid,
diethylphosphite and formaldehyde followed by 31P{1H} NMR.
acids having C –H bonds which form the respective diethylphos-
a
phonic esters. A relative ability of phosphonic esters towards
hydrolysis is mostly determined by the relative energies of the
P–OEt bonds breaking in the ester and P–OH bonds forming the
acid. The calculated (at DFT level) vertical P–OEt bond energy
Figure 2. The structures of the zwitter ionic forms of the molecules in 1a–c with
thermal ellipsoids drawn at 50% probability level showing the intramolecular
hydrogen bonding interactions N1–Hꢀ ꢀ ꢀO5 [N1–H 0.91, Hꢀ ꢀ ꢀO5 2.235, N1ꢀ ꢀ ꢀO5
2.857 Å, N1–Hꢀ ꢀ ꢀO5 125.13°] (1a), bifurcated N1–Hꢀ ꢀ ꢀO5 and N1–Hꢀ ꢀ ꢀO8 [N1–H
0.93, Hꢀ ꢀ ꢀO5 2.223, N1ꢀ ꢀ ꢀO5 2.865 Å, N1–Hꢀ ꢀ ꢀO5 125.46°; Hꢀ ꢀ ꢀO8 2.567, N1ꢀ ꢀ ꢀO8
3.091 Å, N1–Hꢀ ꢀ ꢀO8 116.09°] (1b), and N1–Hꢀ ꢀ ꢀO5 [N1–H 0.91, Hꢀ ꢀ ꢀO5 2.277,
N1ꢀ ꢀ ꢀO5 2.889 Å, N1–Hꢀ ꢀ ꢀO5 124.26°] (1c), and disorder in the cyclopentane ring of
1c. Selected bond distances (Å) and torsion angles (deg): C4–O1 1.2048(18), C4–O2
1.3067(19), P1–O3 1.5295(12), P1–O4 1.5483(12), P1–O5 1.4952(11), P2–O6
1.5133(11), P2–O7 1.4915(12), P2–O8 1.5579(11), N1–C2–C4–O1 –39.76(19), O5–
P1–C5–N1 –4.01(13)° (1a), C7–O1 1.3083(19), C7–O2 1.2102(19), P1–O3
1.5529(11), P1–O4 1.5336(11), P1–O5 1.4931(11), P2–O6 1.4876(11), P2–O7
1.5640(11), P2–O8 1.5129(11), N1–C1–C7–O2 55.31(16), O5–P1–C8–N1 7.02(11)°
(1b), C1–O1 1.3034(14), C1–O2 1.2016(16), P1–O3 1.5475(9), P1–O4 1.5278(9), P1–
O5 1.4902(9), P2–O6 1.4879(9), P2–O7 1.5575(8), P2–O8 1.5093(9), O2–C1–C2–N1
40.99(14), O5–P1–C7–N1 11.39(9)° (1c).
(EP–OEt
)
in phosphonic esters decreases along the row
1d0 > 1a0 > 1c0 > 1b0 (243.9, 236.6, 232.6 and 231.0 kcal/mol,
respectively) whereas the P–OH bond energy (EP–OH) in acids 1a–
d varies insignificantly (245.7, 248.3, 246.9 and 246.6 kcal/mol,
correspondingly). The difference EP–OEt ꢁ EP–OH is only
ꢁ2.7 kcal/mol for glycine derivatives but reaches (ꢁ9.1)–
(ꢁ15.9) kcal/mol for other derivatives, which correlates well with
experimental observations.
3.2. Crystal structure analysis
The results of X-ray diffraction studies of 1a–c are shown in Fig-
ure 2. Selected bond distances (Å) and bond angles (deg) are
quoted in the legend to Figure 2. All three compounds crystallized
as racemates in the monoclinic space group P21/n with one mole-
cule of the corresponding N,N-bis(phosphonomethyl)amino acid in
zwitterionic form and one molecule of water in the asymmetric
unit. The non-equivalence of two phosphonate groups (one of
which is singly deprotonated) and the protonation of the central
3.3. Effects of N,N-bis(phosphonomethyl)amino acids on
adipogenic and osteogenic differentiation of mesenchymal
stem cells isolated from human adipose tissue (hMADS cells)
N1 atom result in chirality of the latter. The angle N–C –COOH in
The effects of 1a–c and sodium alendronate (also known as
FOSAMAXÒ) were investigated on the differentiation of hMADS
cells. The control experiment shows that without addition of com-
pounds adipocytes (lipid droplets in red) and osteoblasts (black
dots) were generated (Fig. 3). The addition of compounds 1a–c
did not modify the adipocyte differentiation, but induced the inhi-
bition of osteoblast formation at 10ꢁ7 M and 3 ꢂ 10ꢁ7 M as
a
1a–c is significantly smaller than that in glycine-derived bis(phos-
phonate) 2 (Table 2)47 and the ideal tetrahedral angle because of
the presence of sterically more demanding substituents at the C
a
atom. The P–CH2 and CH2–N bond lengths are largely unaffected
by the introduced substituents at C , while the N–C bonds of
a
a
1a–c are slightly longer than in 2.

3392
J. Kasser et al. / Bioorg. Med. Chem. 17 (2009) 3388–3393
observed at 3 ꢂ 10ꢁ7 M and this result confirmed the cytotoxic ef-
fect of this compound, as also reported on rat osteoblasts.48
As a positive effect of bisphosphonates on differentiation of
osteoblasts has been reported only when used at low concentra-
tion, a series of experiments was conducted at concentrations from
10ꢁ7 M to 10ꢁ10 M. Both the toxic effect of alendronate and the
inhibitory effect of the compounds were lost when cells were
maintained in the presence of 10ꢁ8 M or lower concentrations
(Fig. 5).
Table 2
Selected bond lengths (Å) and angles (°) for 1a–c and the glycine derivative 2
1a
1b
1c
Glycine, 2a
C –COOH
1.542(2)
1.5454(19)
1.5448(17)
1.5208(17)
1.5195(17)
1.8205(15)
1.8226(15)
103.82(11)
113.34(10)
112.59(10)
112.57(10)
112.00(9)
1.5371(15)
1.5288(13)
1.5126(12)
1.5162(13)
1.8184(11)
1.8222(11)
104.79(8)
112.74(8)
110.27(8)
113.13(7)
115.27(7)
1.506(4)
1.493(4)
1.503(4)
1.513(4)
1.830(3)
1.827(3)
111.0(3)
112.9(2)
111.6(2)
112.3(2)
116.7(2)
a
N–C
1.5451(19)
1.5150(18)
1.5119(19)
1.8219(15)
1.8243(15)
103.53(11)
113.27(11)
111.86(11)
112.55(9)
114.66(9)
a
CH2–N
P–CH2
N–C –COOH
a
CH2–N–C
a
4. Conclusions
P–CH2–N
Amino acids were reacted with diethylphosphite and formalde-
hyde to give N,N-bis(phosphonomethyl)amino acids in a one step/
one pot procedure, including the cleavage of the ester bonds. This
behavior was explained by DFT calculations revealing lower P–OEt
a
From Ref. 48.
revealed by the absence of Von Kossa staining, and importantly, no
cytotoxic effect was detectable. In contrast, alendronate elicited a
cytotoxic effect detectable at 10ꢁ7 M and more pronounced at
3 ꢂ 10ꢁ7 M.
bond strength than for compounds having C –H bonds. Further-
a
more, when C –H bond-containing amino acids (with the excep-
a
tion of glycine) were reacted to the corresponding N,N-
bis(phosphonomethyl)amino acids,
a by-product was formed,
Monitoring the GPDH activity revealed no effect of 1a–c on adi-
pogenesis (Fig. 4), but the ALP activity revealed inhibition of osteo-
genesis. In the case of alendronate no GPDH and ALP activity was
which is explained by a weakend C –N bond. The synthesized
a
compounds were characterized by spectroscopic methods and ele-
mental analysis and their 3D structures were established by X-ray
Figure 3. Effects of 1a–c and the positive control sodium alendronate on the differentiation of hMADS cells and untreated cells as control.
Figure 4. Influence of 1a–c on the GPDH and the ALP activity.

J. Kasser et al. / Bioorg. Med. Chem. 17 (2009) 3388–3393
3393
Figure 5. Concentration dependent influence of 1a–c on the ALP activity.
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Acknowledgements
The authors indebted to the University of Vienna, the Austrian
Council for Research and Technology Development, the FFG—Aus-
trian Research Promotion Agency (Project FA 526003), and COST
D39 for financial support. B.W. and C.D. were supported by the
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thank the computer center of the University of Vienna for technical
support and computer time at the Linux-PC cluster Schroedinger
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