Novel α-aminophosphonic acids. Design, characterization, and biological activity

Article abstract of DOI:10.1016/j.bmc.2005.11.002

Novel α-aminophosphonic acids are synthesized reacting 1,3-oxazolidin-2-one derivatives with formaldehyde and phosphorus trichloride. Treatment of N-(phosphonomethyl)oxazolidinones with aqueous NaOH gave the expected α-aminophosphonic acids. The oxidation

Full text of DOI:10.1016/j.bmc.2005.11.002

Bioorganic & Medicinal Chemistry 14 (2006) 2190–2196
Novel a-aminophosphonic acids. Design, characterization,
and biological activity
a
b
a
Emilia Naydenova, Margarita Topashka-Ancheva, Petar Todorov,
c
d,
*
Ts. Yordanova and Kolio Troev
a
University of Chemical Technology and Metallurgy, Department of Organic Chemistry, Sofia 1756, Bulgaria
b
Institute of Zoology, Bulgarian Academy of Sciences, ‘‘Tzar Osvoboditel’’ 1, Sofia 1000, Bulgaria
c
Sofia University, Faculty of Biology, ‘‘Dragan Tsankov’’ 8, Sofia 1164, Bulgaria
d
Institute of Polymers, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria
Received 28 July 2005; revised 26 October 2005; accepted 2 November 2005
Available online 2 December 2005
Abstract—Novel a-aminophosphonic acids are synthesized reacting 1,3-oxazolidin-2-one derivatives with formaldehyde and phos-
phorus trichloride. Treatment of N-(phosphonomethyl)oxazolidinones with aqueous NaOH gave the expected a-aminophosphonic
acids. The oxidation of (2-hydroxy-1,1-dimethylethylamino)methyl phosphonic acid in the presence of CdO and water resulted in
1
13
31
N-phosphonomethyl-2-methyl-1-propanoic acid. Their structures were proved by means of IR, H, C, and P NMR spectrosco-
py. The genotoxic, clastogenic, and antiproliferative effects of newly synthesized original aminophosphonic acids were investigated
for the first time.
Ó 2005 Elsevier Ltd. All rights reserved.
1
. Introduction
effective in inhibiting test-tube growth of Plasmodium
falciparum, the parasite that causes malaria.
Additionally, it has the same effect on related types
of single-celled parasites such as Toxoplasma and
Cryptosporidium which cause opportunistic infections
Aminophosphonic acids constitute an important class
of biologically active compounds, and their synthesis
has been a focus of considerable attention in synthetic
organic chemistry as well as in medicinal chemistry.
These acids are considered to be structural analogues
of the corresponding amino acids, thus acting as com-
petitive inhibitors and they can act as false substrates
during the course of amino acid metabolism. In addi-
tion, aminophosphonic acids express other forms of
biological activity including antibiotics and enzyme
1
7
in AIDS patients. Moreover, it has been found that
the N-(phosphonomethyl) glycines are especially effec-
tive in suppressing the growth of cancer, tumor, virus,
or bacteria. A pharmaceutical composition for treat-
ment of mammals, warm-blooded animals, and hu-
mans, comprising a pharmaceutical carrier and an
effective amount of a chemotherapeutic agent and anti-
cancer compound selected from the group consisting of
1
inhibiting effects. Numerous applications have been
found for aminophosphonic acids: in the design of en-
1
8
N-(phosphonomethyl) glycine derivatives.
2
–5
6
zyme inhibitors, as plant growth regulators, as anti-
7
8
bacterial agents, as neuroactive compounds, and as
9
Consequently, development of new methods for the syn-
thesis of a-aminophosphonic acids is an active area of
research and many methods are now available: Kabach-
–13
herbicides, antifungal agents,
1
and anti-HIV
agents. The herbicidal activity of N-(phosphonometh-
4
1
5
16
19
20
yl) glycine is reported by Baird et al. Another study
demonstrated that the N-(phosphonomethyl) glycine is
nik and Medved and Fields have discovered the first
method for the preparation of a-aminophosphonic acids
reacting ammonia or amine, dialkyl H-phosphonate,
2
1
and aldehydes; the reaction of dialkyl H-phosphonates
22
Keywords: Aminophosphonic acids; N-(Phosphonomethyl) glycine;
2-Oxazolidinone derivatives; Chromosome aberrations; Cell prolifer-
ation; Clastogenic effects.
or phosphorous acid with imines afforded also a-
aminophosphonic acids. a-Aminophosphonic acids are
obtained reacting amides, or 2-oxazolidinone deriva-
2
3
*
Corresponding author. Fax: +35928700309; e-mail: ktroev@
polymer.bas.bg
2
4
tives, with formaldehyde and phosphorus trichloride.
0
968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.002

E. Naydenova et al. / Bioorg. Med. Chem. 14 (2006) 2190–2196
2191
a-Aminophosphonic acids bearing heterocyclic, aromat-
for the P-CH protons. These data confirm the formation
2
2
5,26
13
of P–C bond. The data from the C{H} NMR spectrum
ic rings such as furane, anthracene,
azole, imidazole, and pyridine are described.
thiophene, pyr-
2
7
also confirm this conclusion. The doublet for 2a at
4
1
2.45 ppm with J
= 154.0 Hz and at 38.96 ppm with
P,H
1
There are no available data on the genotoxic and anti-
proliferative effects of the investigated aminophosphonic
acids, but many studies concerning the genotoxic effects
of N-phosphonomethyl glycine (glyphosate) are carried
out. The obtained results are rather contradictory.
JP,H = 157.57 Hz for 2b has to be assigned to the carbon
atom connected to phosphorus P-CH . The triplet in the
2
3
1
2
P NMR spectrum at 17.32 ppm with J
= 12.20 Hz
= 11.17 Hz can be as-
P,H
2
and that at 17.70 ppm with J
P,H
signed to the phosphorus atoms of 2a and 2b, respectively.
Treatment of 2 with aqueous NaOH gave the expected
a-aminophosphonic acids 3. The hydrolysis of oxazolidi-
none ring is accompanied by decarboxylation.
Assignment of structures 3a and b was made on the basis
This article is a continuation of our study on the
synthesis of a-aminophosphonic acids. Herein we de-
scribe the synthesis of novel a-aminophosphonic acids
reacting phosphorus trichloride with formaldehyde
and 1,3-oxazolidin-2-one derivatives. For the total
characterization of these newly synthesized derivates
of N-phosphonomethyl glycine it is very important
and appropriate to investigate their genotoxic and
antiproliferative effects. The present study is the first
proof of genotoxic and antiproliferative effects of
some original aminophosphonates.
1
13
of H and C NMR spectral data reported in the Exper-
8
2
imental part. The oxidation of 3b in the presence of CdO
2
9
and water results in N-phosphonomethyl-2-methyl-1-
propanoic acid 4. In the C{H} NMR spectrum of 4,
1
3
there is no signal at 64.26 ppm for the carbon atom of
the CH -OH group. The new signal at 173.40 ppm can
2
be assigned to the carbonyl carbon atom of the carboxylic
group, which is formed as a result of the oxidation of
CH OH group. IR spectroscopy study confirms the struc-
2
ture of the above-mentioned compounds. Further studies
for the preparation of N-(phosphonomethyl) glycine
derivatives are in progress.
2
. Results and discussion
2
.1. Chemistry
2.2. Biological activity
In our present study, we used the procedure, described
2
3
by Pikl and Engelmann, according to which as starting
compounds for the preparation of the aminophosphonic
acids are used phosphorus trichloride, formaldehyde,
and 2-oxazolidinone derivatives 1a and 1b. The synthet-
ic route is shown in Scheme 1.
The obtained results of the induced chromosome aberra-
tions, frequency in bone marrow mice cells after 2a, 2b,
3a, 3b, and 4 treatments are presented consequently in
Table 1. (5-Methyl-2-oxo-1,3-oxazolidin-3-yl)methyl-
phosphonic acid (2a), 10 mg/kg (24th hour) provoked
4.33% chromosome aberrations, predominantly centro-
In the first step of the interaction, N-(phosphonometh-
yl)oxazolidinones 2 was obtained in good yield and their
formation were confirmed by H, C{H}, P NMR,
mer/centromeric fusions (c/c). On the 48th hour the clas-
togenic effect of the injected compound slightly
decreased. In the experimental mice groups treated with
100 mg/kg, the percentages of aberrant bone marrow
cells were higher than those injected with 2a, 10 mg/kg,
especially on the 48th hour groups (p < 0.01). In the
higher concentration group, there was no significant dif-
1
13
31
1
and IR spectroscopy. The doublet in the H NMR spec-
= 10.4 Hz and at
= 11.2 Hz for 2b can be assigned
2
trum of 2a at 3.48 ppm with J
P,H
2
.32 ppm with J
3
P,H
H
th
ference between the samples on the 24 and 48th hour.
O
O
1
.
O
O
Hundred milligrams per kilogram 2a concentration en-
sures enough amounts of active molecules and provokes
the same percentage of aberrations on the 48th hour too.
C
C
OH
OH
+
H
O
NH
X
O
N
CH2
X
P
2
. PCI3
H
Z
Y
H
Z
Y
After the (4,4-dimethyl-2-oxo-1,3-oxazolidin-3-yl)meth-
ylphosphonic acid (2b) administration, the percentages
of cells with aberration for the two applied concentra-
tions and for the samples from 24th and 48th hours were
slightly different but the differences in the calculated
values were not significant (p > 0.05).
1
1
a X=Y=H; Z=CH3
2a X=Y=H; Z=CH3
b X=Y=CH ; Z=H
2b X=Y=CH ; Z=H
3
3
O
X Z
HO
HO
+ NaOH/H O
2
P CH2 NH C C OH
Y
H
A more detailed analysis has showed that 2b (10 mg/kg,
3
a X=Y=H; Z=CH3
NaOH
2
CdO
2
(
4th and 48th hour) induced predominantly c/c fusions
4.33 ± 0.61 and 4 ± 0.52%, respectively). The percent-
age of breaks and fragments was significantly lower
1 ± 0.43%) (24th hour) and 0.6 ± 0.27 (48th hour).
3
b X=Y=CH ; Z=H
H O
3
+
(
O
X
C
Y
+
NaOH/H2O/CdO
HO
HO
P CH2 NH
COOH
There was no difference in the percentage of chromo-
some aberrations between 24th and 48th hour after 2b,
4a X=Y=CH3
100 mg/kg injection. On the 24th hour highly predomi-
nated c/c fusions (p < 0.001).
Scheme 1. Synthesis of aminophosphonic acids.

2192
E. Naydenova et al. / Bioorg. Med. Chem. 14 (2006) 2190–2196
Table 1. Frequencies of chromosome aberrations and mitotic activity in affected C57Bl mouse bone marrow cells after ip treatment with novel a-
aminophosphonic acids
Compound and
doses
Interval Analyzed metaphases
(h)
Type of aberrations
Breaks Fragments Exchanges
c/c t/t c/t
Metaphases Cells with
Mitotic index,
ꢀ
with more
than two
aberrations
aberrations, & (X ± SE)
ꢀ
% (X ± SE)
2
2
2
2
3
3
3
3
4
4
a, 10 mg/kg
a, 100 mg/kg
b, 10 mg/kg
b, 100 mg/kg
a, 10 mg/kg
a, 100 mg/kg
b, 10 mg/kg
b, 100 mg/kg
, 10 mg/kg
24
220
300
300
250
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
150
200
2
0
2
2
2
2
0
1
3
0
2
3
2
2
3
3
3
2
4
1
10
0
0
0
0
1
1
0
0
1
2
0
5
3
0
0
4
0
0
1
4
0
13
0
7
9
2
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
0
0
0
0
0
0
0
2
0
0
36
0
4.33 ± 0.97
2.67 ± 0.42
6.25 ± 0.62
5.20 ± 0.48
6.00 ± 1.03
4.67 ± 0.67
4.00 ± 0.52
5.33 ± 0.84
6.00 ± 0.73
5.67 ± 0.80
6.64 ± 0.86
6.67 ± 0.67
9.42 ± 1.02
7.09 ± 0.50
6.69 ± 0.50
5.12 ± 0.34
5.13 ± 0.76
5.99 ± 0.41
5.76 ± 0.56
6.15 ± 0.73
5.43 ± 0.31
7.10 ± 0.47
5.21 ± 0.49
5.91 ± 0.54
48
24
8
48
24
10
13
12
12
14
11
17
16
14
7
48
24
48
24
48
24
48
24
3.00 ± 0.45 11.92 ± 0.39
2.67 ± 0.42 12.35 ± 0.66
48
24
6
11
6
5.00 ± 0.68
3.00 ± 0.45
8.53 ± 0.91
5.2 ± 0.54
48
24
6
3.00 ± 0.45 10.39 ± 0.65
2.67 ± 0.42 10.96 ± 0.19
48
24
5
, 100 mg/kg
4
4.00 ± 0.52
2.67 ± 0.42
46.67 ± 3.03
1.00 ± 0.57
5.18 ± 0.42
5.25 ± 0.96
5.49 ± 0.19
17.3 ± 1.00
4
8
7
Mitomycin C, 3.5 mg/kg 24
.9% NaCl 24
11
2
0
c/c, centromer/centromeric fusions; t/t, telomer/telomeric fusions; c/t, centromer/telomeric fusions.
(
2-Hydroxypropylamino)methylphosphonic acid (3a) at
a concentration of 10 mg/kg caused the presence of
± 0.73% (24th hour) and 5.67 ± 0.8% (48th hour) bone
the control groups, except for 3b and 4, 10 mg/kg. This
percentage was significantly higher than that of negative
control and significantly lower than that of positive con-
trol—Mitomycin C (p < 0.01). These results are evidence
for some of the clastogenic effects of the studied amino-
phosphonic acids. Furthermore, no dispersed metaphases
were observed on the slides of the aminophosphonic acid
experimental groups in comparison with those of the
Mitomycin C positive control.
6
marrow cells with aberrant metaphases. Significant differ-
ences between treated groups injected with 10–100 mg/kg
at the two time span intervals were not detected (p > 0.05).
The values of c/c fusions were close to those obtained for
2
a. The number of cells with breaks and fragments was
significantly lower. Metaphases with more than two aber-
rant chromosomes were also observed.
Some other conclusions about the relatively low genotox-
icity of the studied aminophosphonic acids could be
drawn using the data of the correlation between the differ-
ent types of aberrations. The investigated aminophos-
phonic acids in the two applied concentrations induced
c/c fusions predominantly. The number of metaphase
plates with chromosome breaks and fragments was signif-
icantly lower. For a comparison, in the negative control
group cells with breaks and fragments were not found,
but in the positive Mitomycin C control the percentage
was very high up to 71.3% (or 29.33% of the total number
of analyzed cells).
In the experimental mice samples treated with (2-Hy-
droxy-1,1-dimethylethylamino)methylphosphonic acid
(
3b), 10 mg/kg (24th hour), a very low percentage of
chromosome aberrations was obtained. In the groups,
treated with 100 mg/kg 3b, the value of aberrant mitoses
on the 24th hour significantly increased (p < 0.05).
N-Phosphonomethyl-2-methyl-1-propanoic acid (4) at
1
0 mg/kg induced 3.0 ± 0.45% chromosome aberrations
at the 24th hour and 2.67 ± 0.42% at the 48th hour
p>0.05). A tendency to higher incidences of chromosome
(
changes—c/c fusions, specific for 2b and 3a, was also
followed.
It can be summarized that all the five investigated
aminophosphonic acids possess moderate clastogenic ef-
fects. The analysis of the type of chromosome aberra-
tions obtained after the treatment of the investigated
compounds showed that in all the experimental groups
centromeric fusions predominated, with the exception
of the 4 100 mg/kg (24th hour) group, where the values
of chromosome breaks were significantly higher in all
variants. These results allowed a hypothesis that newly
synthesized aminophosphonic acids damage predomi-
There were no significant differences between the per-
centage of chromosome aberrations in the experimental
groups, treated with 10 and 100 mg/kg. The higher dose
applied induced only 4 ± 0.52% on the 24th hour and
2
.67 ± 0.42% on the 48th hour.
Generally, in the most treated groups the percentage of
aberrant metaphases differed significantly from that of

E. Naydenova et al. / Bioorg. Med. Chem. 14 (2006) 2190–2196
2193
nantly the centromeric chromosome regions. This might
be facilitated by the telocentric nature of mice chromo-
somes, which allows centromer/centromeric recombina-
tions between non-homologous chromosomes without
altering the normal mice genome.
Metaphase analysis showed that the changes of the
chromosome structure in the bone marrow cells of the
treated animals were predominantly c/c fusions and
rarely breaks and fragments. These results suggest that
the investigated compounds affect the centromeric chro-
mosome regions, which allow centromer/centromeric
recombinations between non-homologous chromosomes
without altering the normal mice genome.
The effect of the newly synthesized aminophosphonic
acids on the proliferative activity of bone marrow cells
was evaluated by the mitotic index parameter. The
obtained results are presented in Table 1.
The correlation between the moderate clastogenic effect
and the low values of mitotic index (MI), obtained after
the treatment with 2a and 2b, 100 mg/kg, 3a and 4
100 mg/kg is an interesting fact. We consider further
detailed investigations on the experimental tumor
models in vivo and in vitro as appropriate.
The mitotic index values (MI) of bone marrow cells in
the groups, treated with 2a in a dose of 10 mg/kg, slight-
ly decreased especially on the 24th hour after treatment.
Compound 2a at a dose of 100 mg/kg had a strong anti-
proliferative affect and the values of MI were 6.69 ± 0.50
and 5.12 ± 0.34&, respectively.
4. Experimental
After a 10 mg/kg 2b treatment, the mitotic activity on the
2
4th hour (5.13 ± 0.76&) was close to that on the 48th
hour (5.99 ± 0.41&). The data about the effect of 2b
00 mg/kg were closely similar to those after a 10 mg/kg
Melting points (mp) were determined on a Koffler
microscope and are uncorrected. The infrared (IR) spec-
tra in KBr were recorded on a Perkin-Elmer Model 1600
Series FTIR instrument. The purity of the products was
checked by TLC on precoated plates of Silica gel 60 F254
1
treatment.
Significant differences between the mitotic index values
obtained for the investigated bone marrow cell popula-
tions at the 24th and 48th hours, treated with the two
applied 3a concentrations, were not found. MI varied
from 5.21 ± 0.49 to 7.10 ± 0.47&.
(Merck) using a mobile phase n-BuOH/AcOH/H O,
2
3:1:1. Spots on TLS chromatograms were detected by
chlorine/o-toluidine reaction. The microanalyses were
performed on a Perkin-Elmer elemental analyzer. All
1
13
31
compoundsÕ characterization with H, C, and
NMR spectra was recorded on a Bruker DRX 250
P
Compounds 4 and 3b in dose 10 mg/kg had a compara-
tively low expressed antiproliferative effect—from 10.39
to 12.35&. The higher concentration in both applied
compounds (4 and 3b) suppressed significantly the cell
division in bone marrow cells (8.53& for 3b on the
spectrometer.
4.1. General methods
Phosphorus trichloride, paraformaldehyde, 1-amino-2-
propanol, 2-amino-2-methyl-1-propanol, and 1,3-dioxo-
lan-2-one (ethylene carbonate) were purchased from
Aldrich and used without further purification. 2-
Oxazolidinone derivatives were synthesized by reacting
an equimolar amount of ethylene carbonate with amino
2
4th hour to 5.18& for 4 on the 24th hour).
All the five investigated aminophosphonic acids on bone
marrow mice test system induced a statistically signifi-
cant (p < 0.05) decrease of the proliferative activity.
2
9
alcohol at temperatures below 10 °C . After that, the
reaction mixture allowed to stand at 60 °C for 6 h.
The resulting substituted 2-oxazolidinones were isolated
by vacuum distillation. All compound characterization
3. Conclusion
1
13
31
We describe the synthesis of novel a-aminophosphonic
acids reacting 1,3-oxazolidin-2-one derivatives with
formaldehyde and phosphorus trichloride.
with H, C, and P NMR spectra was recorded on a
Bruker DRX 250 spectrometer.
4.2. 5-Methyl-1,3-oxazolidin-2-one (1a)
The clastogenic and antiproliferative effects of the newly
synthesized original aminophosphonic acids were inves-
tigated for the first time.
The titled compound was obtained reacting 1,3-dioxo-
lan-2-on with 1-amino-2-propanol. Bp 175–77 °C/
0
.05 mmHg. Yield 87%; solid product. H NMR
3
The studied compoundsdid not possessclearexpressed rela-
tionship Ôdose–effectÕ (high percentage of aberrations after
higher dose applied) in their clastogenic effects as this rela-
tionship is specific for the alkylating agent Mitomycin C.
(250 MHz, CD Cl): d = 1.45 (d, JH,H = 3.8 Hz, 3H,
3
CH ), 3.65–3.71 (m, 2H, N-CH ), 4.73–4.81 (m, 1H,
C{H} NMR
3
2
1
3
CH), 6.8 (br s, 1H, NH) ppm.
(250 MHz, CDCl ): d = 20.84 (CH ), 47.78 (N-CH ),
3
3
2
73.90 (CH), 160.91 (C@O) ppm.
Comparatively, low percentage of bone marrow meta-
phases with chromosome aberrations and the lack of
aberrant metaphase plates with disintegrated chromo-
somes and dispersed chromatin are evidence of the
moderate clastogenic effect of the newly synthesized
aminophosphonic acids.
4.3. 4,4-Dimethyl-1,3-oxazolidin-2-one (1b)
The titled compound was obtained reacting 1,3-dioxo-
lan-2-on with 2-amino-2-methyl-1-propanol. Bp 95 °C/
0.05 mmHg, mp 48–51 °C. Yield 85%; solid product.

2
1
194
E. Naydenova et al. / Bioorg. Med. Chem. 14 (2006) 2190–2196
H NMR (250 MHz, CD Cl): d = 1.23 (s, 6H, CH ), 3.9
H O, 3:1:1) = 0.40. IR (KBr) 2986–2920 (C–H), 2315
2
3
3
1
s, 2H, O-CH ), 6.8 (br s, 1H, NH) ppm. C{H} NMR
3
+
(
(NH ) 1687 (C@O), 1304, 1246 (P@O), 1158, 1064,
2
À1
1012 (C–O–C) cm . H NMR (250 MHz, CDCl ):
3
1
(
250 MHz, CDCl ): d = 24.82 (CH ), 59.3 C4 atom,
3
3
2
7
3.90 (O-CH ), 159.91 (C@O) ppm.
d = 1.23 (s, 6H, CH ), 3.30 (d, 2H, JH,H = 11.2 Hz,
2
3
P-CH ), 3.90 (s, 2H, O-CH ), 7.90 (br s, 2H, P-OH)
2
2
1
3
4.4. (5-Methyl-2-oxo-1,3-oxazolidin-3-yl)methylphos-
phonic acid (2a)
ppm.
(CH ), 38.96 (d, J
C{H} NMR (250 MHz, CDCl ): d = 24.82
3
1
= 154.0 Hz, P-CH ), 59.30 (C4),
3
P,H 2
3
1
7
5.12 (C5), 157.86 (C@O) ppm. P NMR (250 MHz,
2 31
5
-Methyl-1,3-oxazolidin-2-one (8.66 g, 0.085 mol) and
DMSO): d = 17.70 (t,
NMR (250 MHz, D O): d = 21.53 (t, J
J
= 11.17 Hz) ppm.
P
P,H
2
paraformaldehyde (2.57 g, 0.085 mol) were added in a
four-necked round-bottomed flask equipped with a
magnetic stirrer, reflux condenser, thermometer, drop-
ping funnel, and argon inert. At vigorous stirring glacial
acetic acid 42.6 mL was added dropwise. A white sus-
pension formed. The reaction mixture was refluxed
= 11.10 Hz)
P,H
2
ppm. Anal. Calcd for C H NO P (209.14): C, 34.46%;
H, 5.78%; N, 6.70%; P, 14.81%. Found: C, 34.18%; H,
5.65%; N, 6.48%; P, 14.20%.
6
12
5
4.6. (2-Hydroxypropylamino)methylphosphonic acid (3a)
(
approximately 115 °C) for 45 min, after which it be-
came a clear solution. Then the temperature was low-
ered to 20 °C and 7.5 mL phosphorus trichloride was
added dropwise. During and after the addition, hydro-
gen chloride evolved. The reaction mixture was refluxed
(5-Methyl-2-oxo-1,3-oxazolidin-3-yl)methylphosphonic
acid (2a) (5.0 g, 0.025 mol), NaOH (10.12 g, 0.25 mol) in
85.6 mL H O were placed in a three-necked round-bot-
2
tomed flask equipped with a magnetic stirrer, reflux con-
denser, and thermometer. The reaction mixture was
heated at 140 °C for 7.30 h. Subsequently, the reaction
mixture was treated with Dowex 50WX8-200 in order
to exchange the sodium cations by hydrogen-ion ex-
change. The crude mixture was purified by crystalliza-
tion from water. Yield, 2.75 g (63%); mp 198–200 °C;
R (n-BuOH/AcOH/H O, 3:1:1) = 0.21.
(
approximately 118 °C). After 2 h refluxing, 48.5 mL
water (distilled) was added. After 1 h and 30 min reflux-
ing, the reaction mixture was concentrated under re-
duced pressure. The crude mixture was purified by
crystallization from the mixture ethyl acetate/ethyl
alcohol 2:1. Yield 9.62 g (57.5%); mp 160–162 °C; R
f
(
n-BuOH/AcOH/H O, 3:1:1) = 0.37.
2
f
2
+
IR (KBr) 3400–3000 (NH and OH, br), 1718 (C@O),
IR (KBr) 3413–3000 (NH and OH, br), 1579 (NH), 1312
À1
1
À1
1
1
309, 1268 (P@O), 1074, 975 (C–O–C) cm . H NMR
(P@O), 1097, 1064 (C–O, CH-OH) cm
.
H NMR
= 6.4 Hz, 6H, CH3),
3
3
(
CH ), 3.24 (t, 1H, J
250 MHz, CDCl ): d = 1.32 (d,
J
= 7.8 Hz, N-CH ) and 3.78 (t,
= 6.2 Hz, 3H,
(250 MHz, D O): d = 1.18 (d, J
2 H,H
3
3
H,H
3
2.61 (d, JP,H = 13.0 Hz, 2H, P-CH ), 3.95 (sex,
2
3
H,H
2
3
H, J
2
3
13
JH,H = 6.3 Hz, 1H, CH-OH) ppm. C{H} NMR
1
2
= 8.4 Hz, N-CH ), 3.48 (d, J = 10.4 Hz,
P,H
H,H
2
3
H, P-CH ), 4.60 (sex, 1H, J
= 7.8 Hz, O-CH), 9.23
(250 MHz, D O): d = 20.0 (CH3), 47.50 (d,
2
H,H
2
1
3
1
(
br s, 2H, P-OH) ppm. C{H} NMR (250 MHz, CDCl ):
3
d = 21.17 (CH ), 42.45 (d, JP,H = 154.0 Hz, P-CH ),
2.21 (N-CH ), 70.72 (O-CH), 158.15 (C@O) ppm.
NMR (250 MHz, DMSO): d = 17.32 (t, J
ppm. P NMR (250 MHz, D O): d = 20.65 (t,
JP,H = 10.47 Hz) ppm. Anal. Calcd for C H NO P
JP,C = 136.30 Hz, P-CH ) 57.25 (N-CH ), 66.03 (CH-
OH) ppm. P NMR (250 MHz, D O): d = 9.90 ppm.
2
2
1
31
3
2
2
3
1
5
P
= 12.2 Hz)
Anal. Calcd for C H NO P (169.12): C, 28.41%; H,
7.15%; N, 8.28%; P, 18.32%. Found: C, 28.31%; H,
7.09%; N, 8.19%; P, 18.17%.
2
4
12
4
2
P,H
3
1
2
2
5
10
5
(195.11): C, 30.78%; H, 5.17%; N, 7.18%; P, 15.87%.
Found: C, 30.48%; H, 5.02%; N, 7.05%; P, 15.23%.
4.7. (2-Hydroxy-1,1-dimethylethylamino)methylphos-
phonic acid (3b)
4.5. (4,4-Dimethyl-2-oxo-1,3-oxazolidin-3-yl)methylphos-
phonic acid (2b)
(4,4-Dimethyl-2-oxo-1,3-oxazolidin-3-yl)methylphos-
phonic acid (2b) (1.5 g, 0.007 mol), NaOH (4.72 g,
0
.118 mol), and 23.6 mL H O were placed in a three-
2
4
,4-Dimethyl-1,3-oxazolidin-2-one (9.00 g, 0.078 mol)
necked round-bottomed flask equipped with a magnetic
stirrer, reflux condenser, and thermometer. The reaction
mixture was heated at 130 °C. Subsequently, the reac-
tion mixture was treated with Dowex 50WX8-200 in
order to exchange the sodium cations by hydrogen-ion
exchange. The crude mixture was purified by crystalliza-
tion from water. Yield 1.15 g (88%) mp = 130–132 °C;
R (n-BuOH/AcOH/H O, 3:1:1) = 0.50.
and paraformaldehyde (2.34 g, 0.078 mol) were added
in a four-necked round-bottomed flask equipped with
a magnetic stirrer, reflux condenser, thermometer, drop-
ping funnel, and argon inert. At vigorous stirring glacial
acetic acid 38.76 mL was added dropwise. The reaction
mixture was refluxed (approximately 115 °C) for 45 min,
after which it became a clear solution. Then the temper-
ature was lowered to 20 °C and 6.8 mL phosphorus tri-
chloride was added dropwise. During and after the
addition, hydrogen chloride evolved. The reaction mix-
ture was refluxed (approximately 118 °C). After 2 h
refluxing, 44 mL water (distilled) was added. After 1 h
and 30 min refluxing, the reaction mixture was concen-
trated under reduced pressure. The crude mixture was
purified by crystallization from ethyl alcohol. Yield
f
2
IR (KBr) 3413–3000 (NH and OH, br), 1631 (NH), 1269
H NMR
À1
1
(P@O), 1080 (C–O, CH -OH) cm
.
2
(250 MHz, D O): d = 1.34 (s, 6H, CH ), 3.05 (d,
2
3
2
JP,H = 13.5 Hz, 2H, P-CH ), 3.64 (s, 2H, CH -OH)
C{H} 1NMR (250 MHz, D O): d = 19.72
2
2
1
3
ppm.
(CH ), 38.20 (d, J = 132.23 Hz, P-CH ), 44.7 (s, tert
2
3
P,C
2
3
C), 64.26 (CH -OH) ppm. P NMR (250 MHz, D O):
1
2
2
1
1.5 g (70.3%); mp 157–158 °C; R (n-BuOH/AcOH/
d = 9.81 ppm. Anal. Calcd for C H NO P (183.14): C,
5 14 4
f

E. Naydenova et al. / Bioorg. Med. Chem. 14 (2006) 2190–2196
2195
3
3
2.79%; H, 7.71%; N, 7.65%; P, 16.91%. Found: C,
2.54%; H, 7.69%; N, 7.22%; P, 16.47%.
4.10. Statistical analysis
Three-way analysis of variance (ANOVA) with fixed ef-
fects, followed by two-group StudentÕs t test and post
hoc pairwise comparison test of Dunnett with a control,
was performed using BMDP4V, BMDP3D, and
BMDP7D programs. Statistical significance is ex-
pressed as ***p < 0.001; **p < 0.01; *p < 0.05; p > 0.05
(not significant). Unless otherwise stated eight animals
were used per group.
4
.8. N-Phosphonomethyl-2-methyl-1-propanoic acid (4)
To a 100 mL stainless steel autoclave equipped with mag-
netic stirrer and pressure gauge was introduced (4,4-
dimethyl-2-oxo-1,3-oxazolidin-3-yl)methylphosphonic
acid (2b) (4.0 g, 0.019 mol), NaOH (3.8 g, 0.095 mol) in
3
0
2
3.0 mL H O and 0.24 g CdO. The reaction mixture was
2
heated at 230 °C for 8.30 h. The reaction solution was
cooled to 5 °C and filtered. The filtrate was decarboxylat-
ed by adding 6.75 mL HCl. At this time copious gas evo-
lution resulted. Subsequently, the reaction mixture was
treated with Dowex 50WX8-200 in order to exchange
the sodium cations by hydrogen-ion exchange. The crude
mixture was purified by crystallization from water. Yield
Acknowledgment
This study was supported by Grant ÔBYX-12Õ from the
Ministry of Education and Science.
1
.95 g (52%); mp = 166–168 °C; R (n-BuOH/AcOH/
f
H O, 3:1:1) = 0.44. IR (KBr) 3400–3000 (NH and OH,
br), 1620, 1404 (COO ), 1253, 1288 (P@O), cm
2
References and notes
À
À1
.
1
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3
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13
= 13.9 Hz, 2H, P-CH ) ppm. C{H} NMR
(
(
P,H 2
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