Novel N-(phosphonomethyl) glycine derivatives: Design, characterization and biological activity

Article abstract of DOI:10.1016/j.ejmech.2007.08.010

A series of Cα,α-disubstituted cyclic derivatives of N-(phosphonomethyl) glycine have been synthesized and characterized. They exhibited moderate clastogenicity, low antiproliferative activity on mice bone marrow cells and well expressed cytotoxicity against human tumor cell lines. The 8- and 12-membered cyclic analogs proved superior to the remaining compounds and were found to trigger apoptotic cell death in DOHH-2 cells. The latter compound caused 50% inhibition of the viability of hemobastose-derived cell lines at concentrations ranging from 20 to 67 μM.

Full text of DOI:10.1016/j.ejmech.2007.08.010

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1199e1205
http://www.elsevier.com/locate/ejmech
Original article
Novel N-(phosphonomethyl) glycine derivatives: Design,
characterization and biological activity
Emilia D. Naydenova a, Petar T. Todorov a, Margarita N. Topashka-Ancheva b,
Georgi Ts. Momekov c, Tsvetelina Z. Yordanova b, Spiro M. Konstantinov c,
Kolio D. Troev d,
*
a University of Chemical Technology and Metallurgy, Department of Organic Chemistry, Sofia 1756, Bulgaria
b Institute of Zoology, Bulgarian Academy of Sciences, ‘‘Tzar Osvoboditel’’ 1000, Sofia 1000, Bulgaria
c Laboratory of Experimental Chemotherapy, Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy,
Medical University e Sofia, Sofia 1000, Bulgaria
d Institute of Polymers, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria
Received 10 May 2007; received in revised form 17 July 2007; accepted 9 August 2007
Available online 11 September 2007
Abstract
A series of Ca,a-disubstituted cyclic derivatives of N-(phosphonomethyl) glycine have been synthesized and characterized. They exhibited
moderate clastogenicity, low antiproliferative activity on mice bone marrow cells and well expressed cytotoxicity against human tumor cell lines.
The 8- and 12-membered cyclic analogs proved superior to the remaining compounds and were found to trigger apoptotic cell death in DOHH-2
cells. The latter compound caused 50% inhibition of the viability of hemobastose-derived cell lines at concentrations ranging from 20 to 67 mM.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Aminophosphonic acids; N-(Phosphonomethyl) glycine derivatives; Chromosome aberrations; Cell proliferation; Clastogenic effects; Cytotoxic activity
1. Introduction
compounds [5]. The most important a-aminophosphonic acids
are N-(phosphonomethyl) glycine and its derivatives, which
have been found to exert prominent antineoplastic, antiviral
and antibacterial effects [6e11]. Kabachnik and Medved [12],
and Fields [13] have discovered the first method for the prep-
aration of a-aminophosphonic acids. The impressive array of
applications has recently stimulated considerable effort towards
the synthesis of a-aminophosphonic acids and many methods
are now available [14e20]. Novel a-aminophosphonic acids
with moderate clastogenic effect were synthesized reacting
1,3-oxazolidin-2-one derivatives with formaldehyde and phos-
phorus trichloride [21]. One-pot reaction is used to prepare a-
aminoalkylphosphonic acid in good yield via ethyl carbamate,
aldehyde and dichlorophosphites [22]. Optically active amino-
phosphonic acids have found widespread application in medic-
inal chemistry and pharmaceutical science [23]. Highly effective
solvent-free and catalyst-free microwave-assisted synthesis of
a-aminophosphonates was shown [24,25]. The recent advance
Aminophosphonic acids are considered to be an important
class of amino acid mimetics. They have reached a position
of eminence in the field of, or in the research works aimed
at discovery, understanding, and modification of physiological
processes in living organisms [1e3]. a-Aminophosphonic
acids are found to compete effectively with their amino acid
counterparts for binding to enzyme active centers or other cel-
lular targets [4]. This, together with their low mammalian tox-
icity makes the a-aminophosphonic acids an important class
of antimetabolites and a potential source of medicinal lead
* Corresponding author. Tel.: þ359 2 979 2203; fax: þ359 2 870 0309.
E-mail addresses: e_naydenova@abv.bg (E.D. Naydenova), pepi_37@abv.
bg (P.T. Todorov), topashka.mn@lycos.com (M.N. Topashka-Ancheva),
gmomekov@gmail.com (G.Ts. Momekov), ktroev@polymer.bas.bg (K.D.
Troev).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.08.010

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E.D. Naydenova et al. / European Journal of Medicinal Chemistry 43 (2008) 1199e1205
in synthesis, stereochemistry and biological activity of a-ami-
nophosphonic acid and their esters is reported by Song and
Jiang [26]. Cyclic or heterocyclic rings introduced into the
molecular skeleton increase its rigidity and modify electronic
effects. Thus in recent years, many cyclic a-aminophosphonic
acids or aminophosphonates have been prepared [27]. Some
cyclic N-(phoshonomethyl) glycine derivatives were prepared
from cycloalkaneaminocarboxylic acids and their biological
activity was studied [28e30].
Taking into account the fact that the aminocyclopentane-
l-carboxylic acid, cycloleucin, exerts antineoplastic effects
[31], we synthesized a series of new 1-(dimethoxyphosphono-
methylamino) cycloalkanecarboxylic acids with 5-, 6-, 7-, 8-
and 12-membered rings and studied their biological activity
including the role of the ring size in the molecule.
As most of the antineoplastic agents interfere with cell di-
vision and are often genotoxic, the determination of their clas-
togenic and antiproliferative potential against normal cells is
an inevitable component of the preliminary screening pro-
grams for identification of possible anticancer drugs. Thus
we sought to evaluate the ability of the novel compounds to
induce chromosomal aberrations and to inhibit the prolifera-
tion of murine bone marrow cells, following 24 or 48 h expo-
sure. The cytotoxic activity of the compounds was evaluated in
a panel of human tumor cell lines, whereby the clinically uti-
lized drug cisplatin was used as positive control. The cytotox-
icity determination was extended against the human
embryonal kidney cell line 293T chosen as a model of normal,
non-malignant cellular population.
alkaline hydrolysis to the cycloaminoacid. The synthetic path-
way followed to obtain the novel N-(phosphonomethyl) glycine
derivatives 1ce5c is reported in Scheme 1.
1
In the O NMR spectra of all newly synthesized a-amino-
phosphonic acids doublets are present at: 2.51 ppm with
2
2JPeH ¼ 14.7 Hz for 1c; 2.47 ppm with JPeH ¼ 14.69 Hz for
2
2c; 2.50 ppm with JPeH ¼ 15.1 Hz for 3c; 2.70 ppm with
2
2JPeH ¼ 14.8 Hz for 4c; 3.16 ppm with JPeH ¼ 13.22 Hz for 5c
which can be assigned for the PeCH2 protons (see Section 4).
The doublets in the 13C{1H} NMR spectra at: 39.8 ppm with
1
1JPeC ¼ 146.0 Hz for 1c; 42.0 ppm with JPeC ¼ 144.2 Hz
1
for 2c; 38.8 ppm with JPeC ¼ 145.7 Hz for 3c; 39.3 ppm
1
1
with JPeC ¼ 144.7 Hz for 4c; 39.0 ppm with JPeC
¼
153.39 Hz for 5c can be assigned to the carbon atom con-
nected to phosphorus (PeCH2). The signals in the 31P{1H}
NMR spectra are between 22.17 and 27.41 ppm which repre-
sent a triplet with 3JPeH ¼ 12.3 Hz, characteristic for the phos-
phorus atom of a-aminophosphonic acids. The spectral data
including elemental analysis are reported in Section 4.
2.2. Biology
2.2.1. Clastogenic effect
Cytogenetical investigations were carried out on C57Bl
murine bone marrow metaphase plates following i.p. treatment
with the respective compounds in concentrations of 10 and
100 mg/kg body weight. Structural chromosome aberrations
(breaks and fragments) and intrachromosome exchanges (cen-
tromere/centromeric and telomere/telomeric fusions) were re-
ported. The results of this analysis are presented in Table 1.
The data analysis showed that the highest percentage of meta-
phases with aberrations (10.0 ꢀ 1.73) was scored in the cells
of the experimental animals treated with 100 mg/kg 5c at
the 48th hour after administration. The slightest clastogenic
effect was observed in 3s. The differences between the exper-
imental groups were within the statistical error limits
( p > 0.005). All the other compounds showed moderate clas-
togenic effect. Dose depending effect was not detected. Differ-
ences were observed with respect to the types of aberrations.
Compound 1c provoked the highest amount of breaks and
fragments (about half of the whole amount of the aberrations
scored). Centromere/centromeric fusions were prevalent in
the bone marrow cell slides of the other experimental groups’
slides. These centromere/centromeric fusions are part of the
interchromosome exchange groups known as Robetsonian
2. Results and discussion
2.1. Chemistry
1-[(Dimethoxyphosphono)methylamino]cycloalkanecarbox-
ylic acids with 5-, 6-, 7-, 8- and 12-membered rings 1ce5c
were prepared with good yields via KabachnikeFields reac-
tion. The corresponding aminocycloalkanecarboxylic acid
was treated with paraformaldehyde in the presence of triethyl-
amine in methanol. When the reaction mixture became homo-
geneous, dimethyl hydrogen phosphonate was added. The
Ca,a-disubsituted amino acids Ac5c, Ac6c, Ac7c, Ac8c and
Ac12c (1be5b) were prepared following the procedure, which
involves the formation of cycloalkanespiro-5-hydantoins
(lae5a) from the corresponding cyclic ketones, followed by
O
COOH
COOH
NH2
NH
iii
O
P
i
ii
(CH2)n
(CH2)n
(CH2)n
(CH2)n
OCH3
OCH3
O
NH CH2
HN
O
1 n = 0
2 n = 1
comp. a
comp. b
comp. c
n = 2
3
4
n = 3
n = 7
5
Scheme 1. General synthetic pathway. Reagents and conditions: (i) KCN, (NH4)2CO3, NH4OH, C2H5OH, H2O, 6 h, then concentrated HCl; (ii) Ba(OH)2$8H2O/
H2O, 3.5 h, then (NH4)2CO3; (iii) HCHO, CH3OH, (C2H5)3N, 45 min, (C2H5)3N, 3.5 h, then dimethyl hydrogen phosphonate, 3.5 h.

E.D. Naydenova et al. / European Journal of Medicinal Chemistry 43 (2008) 1199e1205
1201
Table 1
Frequencies of chromosome aberrations in affected mouse bone marrow cells after i.p. treatment of a-aminophosphonic compounds
Sample
Interval (h)
Number of analyzed
metaphases
Type of aberrations
br fr
Percentage of cells with
aberrations ðX ꢀ SEÞ
Mitotic index
ðX ꢀ SEÞ (%)
c/c
t/t
1c
10 mg/kg
24
48
24
48
350
350
300
250
7
1
9
13
10
8
0
1
0
2
4.85 ꢀ 0.40
5.77 ꢀ 0.63
7.0 ꢀ 0.92
6.40 ꢀ 0.40
10.39 ꢀ 1.13
10.66 ꢀ 0.92
10.97 ꢀ 1.18
8.33 ꢀ 0.72
6
8
6
0
3
0
100 mg/kg
2c
3c
4c
5c
10 mg/kg
24
48
24
48
300
300
350
350
4
6
0
1
1
0
11
9
0
0
0
0
5.33 ꢀ 1.11
5.33 ꢀ 0.42
8.34 ꢀ 0.61
7.42 ꢀ 0.84
7.27 ꢀ 0.98
6.71 ꢀ 1.07
7.15 ꢀ 0.78
9.23 ꢀ 1.2
100 mg/kg
7
14
20
12
10 mg/kg
24
48
24
48
300
300
350
273
3
0
7
4
0
0
0
0
7
3
3
3
0
0
0
0
2.85 ꢀ 0.40
1.00 ꢀ 0.68
3.33 ꢀ 1.12
2.72 ꢀ 1.01
14.37 ꢀ 1.14
9.58 ꢀ 1.43
13.24 ꢀ 1.69
12.00 ꢀ 0.41
100 mg/kg
10 mg/kg
24
48
24
48
350
300
350
300
2
0
4
3
1
1
3
0
10
14
11
12
0
0
1
0
3.71 ꢀ 0.52
4.67 ꢀ 0.67
5.43 ꢀ 0.37
5.00 ꢀ 0.45
11.35 ꢀ 0.85
13.95 ꢀ 0.93
13.06 ꢀ 0.99
9.67 ꢀ 1.09
100 mg/kg
10 mg/kg
24
48
24
48
300
300
350
250
4
2
4
6
2
1
0
6
11
12
11
11
0
0
0
2
5.57 ꢀ 0.13
5.0 ꢀ 0.85
4.29 ꢀ 0.73
10.0 ꢀ 1.73
11.13 ꢀ 0.58
8.6 ꢀ 1.29
100 mg/kg
11.82 ꢀ 0.67
12.82 ꢀ 0.48
Mitomycin C
3.5 mg/kg
24
150
10
13
11
2
0
46.67 ꢀ 3.03
1.0 ꢀ 0.57
5.49 ꢀ 0.19
17.3 ꢀ 2.49
0.9% NaCl
br e breaks; fr e fragments; c/c e centromere/centromeric fusions; t/t e telomere/telomeric fusions.
translocations. These translocations change the groups of con-
necting, but do not drastically change the amount of the chro-
mosome material in the affected cell. Comparing the results
obtained under the influence of the newly synthesized amino-
phosphonic acids (Table 1) to the clastogenic effect of the pos-
itive control Mit. C, we find that the new compounds break the
chromosomes’ entity to a much smaller extent. After Mit. C
administration 46% chromosome aberrations were encoun-
tered, whereby the prevalent types of aberrations were breaks,
fragments, non-reciprocal translocations and pericentric
inversions.
2-[(phosphonomethyl)amino]propanoic acid which could be
considered as an analog of the herein described compounds,
demonstrated similar low genotoxic effect, comparable to
that of compound 3c.
2.2.3. Cytotoxic effect
The cytotoxic activity of the tested aminophosphonates was
evaluated by the MTT-dye reduction assay in a panel of six tu-
mor cell lines of human origin. The IC50 values were deter-
mined as the concentration of tested agents producing 50%
decrease of cell survival. Table 2 summarizes the cytotoxicity
data derived following a 72 h continuous exposure.
2.2.2. Antiproliferative effect
As evident from the results obtained, the tested amino-
phosphonates demonstrated cytotoxic effects, whereby the
leukemic cell lines HL-60, LAMA-84, K-562 and the non-
Hodgkin lymphoma DOHH-2 were found to be more respon-
sive than HD-MY-Z and EJ cells.
Throughout the spectrum of tumor models the 1-[(dime-
thoxyphosphono)methylamino]cyclododecanecarboxylic acid
5c proved to be the most active cytotoxic agent amongst the
newly synthesized aminophosphonates, causing 50% inhibi-
tion of cell viability at low micromolar concentrations. 1-[(Di-
methoxyphosphono)methylamino]cyclooctanecarboxylic acid
4c was characterized by somewhat lower activity, whereas
The proliferative activity of bone marrow cell populations
was determined by evaluation of their mitotic index. The
most prominent inhibition of the normal bone marrow cell
proliferation was encountered with compound 2c. It caused
about 65% reduction in the number of mitoses compared to
the untreated control. Compound 3c possessed the weakest
antiproliferative effect in vivo. Compounds 1c, 4c and 5c
showed similar antiproliferative effect e at a rate of about
36% suppression of cell division. Generally these data are in
line with the results of our previous study of non-cyclic
aminophosphonates.
Moreover,
compound
2-methyl-

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E.D. Naydenova et al. / European Journal of Medicinal Chemistry 43 (2008) 1199e1205
Table 2
Cytotoxic activitya of tested compounds in a panel of human tumor cell linesb
Tested compd.
IC50 (mM)c
HL-60
LAMA-84
K-562
DOHH-2
HD-MY-Z
EJ
293T
1c
2c
157.4
136.2
85.9
73.5
41.4
5.2
162.2
98.6
91.2
71.4
67.3
14.4
>200
128.3
100.8
79.1
144.7
84.2
88.1
37.4
20.1
7.6
>200
164.5
168.7
127.4
67.3
>200
>200
>200
>200
116.1
3c
4c
151.4
115.4
110.7
9.7
176.2
159.2
12.4
5c
Cisplatin
47.1
21.1
11.2
a
Determined by MTT-dye reduction assay after 72 h exposure.
Cell line origin: HL-60, acute promyelocyte leukemia; LAMA-84 and K-562, chronic myeloid leukemia; DOHH-2, non-Hodgkin lymphoma; HD-MY-Z,
b
Hodgkin lymphoma; EJ, urinary bladder cancer; 293T, human embryonal kidney.
c
Means from 8 separate measurements.
the further decrease in the molecular weight of 1-[(dimethox-
yphosphono)methylamino]cycloalkanecarboxylic acid moiety
was consistent with a progressive loss of activity. Conversely
2c and 3c were found to be practically equipotent with IC50
values being up to ca. 2e3 fold higher as compared to those
of 5c. Compound 1c displayed the least pronounced cytotoxic
effects, showing only marginal activity in K-562, HD-MY-Z
and EJ cells.
The cytotoxicity determination was extended to the human
embryonal kidney cell line 293T, chosen as representative for
a normal, non-malignant cellular population. As evident from
the IC50 values obtained the aminophosphonic acids were far
less cytotoxic against 293T cells as compared to the tumor
cell lines. Thus the most active compounds 4c and 5c caused
50% inhibition of 293T cells at substantially higher concentra-
tions in comparison to their effects upon malignant cells,
whereas the other aminophosphonates exerted only marginal
cytotoxicity against the kidney cell line. In a dissimilar fashion
the reference cytotoxic agent cisplatin exhibited significant cy-
totoxicity against 293T cells, comparable to its effects in the
panel of human tumor cell lines.
potential, combined with low clastogenicity. Current data
show that the increase of lipophilicity and the steric bulk, con-
sistent with the enlargement of the cycloalkane ring size in the
molecule of the 1-[(dimethoxyphosphono)methylamino]cy-
cloalkanecarboxylic acids appears to be a crucial prerequisite
for optimal activity. The results obtained revealed that the in-
creased number of methylene groups in the ring resulted in
a marked augmentation of the cytotoxic activity. It should be
pointed out that there was no correlation between the geno-
toxic effects of tested compounds against the bone marrow
cells and their cytotoxic activity upon human tumor cell lines.
Thus the most active cytotoxic agents 4c and 5c were found to
be only moderately clastogenic. Moreover, these compounds
as well as all the newly synthesized phosphonates exerted
far less pronounced cytotoxicity against the 293T human em-
bryonal kidney cells. These discrepancies between the cyto-
toxic/antiproliferative potential of 4c and 5c against tumor
cells, on one hand, and embryonal kidney cells, on the other
hand, could be largely attributed to their ability to trigger ap-
optotic cell death at low micromolar concentrations as evi-
denced by the established DNA-fragmentation in DOHH-2
cells. Moreover, aminophosphonic acids have been found to
accumulate more intensively in malignant cells than in normal
2.2.4. Induction of apoptosis
In order to elucidate the mechanisms implicated in the cy-
totoxicity of tested aminophosphonates we evaluated the abil-
ity of the most active analogs 4c and 5c to induce apoptosis in
DOHH-2 cells. To meet this objective we monitored the frag-
mentation of genomic DNA e a key hallmark feature of apo-
ptosis, using a commercially available ELISA-kit. As evident
from the data presented in Fig. 1 the 24 h treatment with both
compounds led to significant increase of the levels of histone-
associated oligonucleosomal DNA-fragments in DOHH-2 cell.
At equipotent concentrations the more cytotoxic compound 5c
proved to be the superior inducer of apoptosis as well.
3. Conclusion
Novel 1-[(dimethoxyphosphono)methylamino]cycloalkane-
carboxylic acids (lce5c) representing N-(phosphonomethyl)
glycine derivatives, were obtained via KabachnikeFields reac-
tion. The results from this study unambiguously indicate that
the newly synthesized aminophosphonates exert antineoplastic
Fig. 1. Cytosolic enrichment of DOHH-2 with oligonucleosomal DNA-
fragments, following 24 h exposure to equitoxic concentrations of 4c (gray
columns) or 5c (white columns) versus the untreated control (black column).
The results are arithmetic means (ꢀSD) of three independent experiments.

E.D. Naydenova et al. / European Journal of Medicinal Chemistry 43 (2008) 1199e1205
1203
cells, which could contribute to the established selective cyto-
toxicity [31].
The cytotoxicity of compounds 4c and 5c against tumor cells
in low micromolar concentrations, added to their moderate gen-
otoxic potential in vivo, gives us reason to consider both agents
as potential lead compounds for further investigations.
1.39e1.63 (m, 22H, CH2). 13C{1H} NMR (62.90 MHz,
D2O),
d
in ppm: 21.0 3,11SO2; 23.5 7SO2; 27.7
4,5,6,8,9,10SO2; 34.3 2,12SO2; 61.6 (eCe); 184.2 C]O.
4.1.3. General procedure for the preparation of
1-[(dimethoxyphosphono)methylamino]-
cycloalkanecarboxylic acids (1ce5c)
4. Experimental
Paraformaldehyde
(0.40 g,
0.0129 mol),
methanol
(20.20 ml), and triethylamine (0.14 ml) were put into
a three-necked flask equipped with a condenser, magnetic stir-
rer, thermometer and dropping funnel and inert argon. The re-
action mixture was heated to reflux temperature and held for
45 min, after which it became a clear solution. Aminocycloal-
kane-l-carboxylic acid (0.0083 mol) and triethylamine
(1.70 ml) were added to this solution. The suspension was
heated at 65e70 ꢁC and after 3.5 h it became a clear solution.
Dimethyl hydrogen phosphonate 0.883 ml (1.059 g,
0.0094 mol) was added to this solution for approximately
10 min. This reaction mixture was heated at 65e70 ꢁC and
held there for 3.5 h, after which it was cooled to room temper-
ature and concentrated under reduced pressure. Compounds
(1ce5c) were obtained from the methanol solution, after re-
moving the non-reacted aminocycloalkane-l-carboxylic acid
having lower solubility in methanol than the products (1ce5c).
Cycloalkanone, dimethyl hydrogen phosphonate, parafor-
maldehyde, and solvents were purchased from Fluka and
Merck, and used without further purification.
Melting points (mp) were determined on Koffler microscope
and were uncorrected. The infrared (IR) spectra in KBr were re-
corded on a PerkineElmer Model 1600 Series FTIR instrument.
The purity of the products was checked by TLC on pre-coated
plates of Silica gel 60 F254 (Merck) using a mobile phase n-
BuOH:AcOH:H2O, 3:1:1. Spots on TLC chromatograms were
detected by chlorine/o-tolidine reaction. Microanalyses were
1
performed on PerkineElmer elemental analyzer. H, 13C and
31P NMR spectra were determined by means of a Bruker
DRX 250 spectrometer and referenced to the solvent. 13C
NMR spectra were fully decoupled. Chemical shifts are re-
ported in d values (ppm), and J values are reported in hertz (Hz).
Compounds 1ae5a were prepared according to Bucherere
Lieb reaction [32,33].
4.1.3.1. 1-[(Dimethoxyphosphono)methylamino]cyclopentane-
carboxylic acid (1c). White solid, 68.0% yield, mp ¼ 218e
220 ꢁS, Rf ¼ 0.4; IR (KBr, cmꢂ1): 3399 (NH), 2966e2877
(CeH), 1674 (C]O), 1303, 1255 (P]O), 1177, 1042 (Re
4.1. Synthesis
1
4.1.1. General procedure for the preparation of
1-aminocycloalkanecarboxylic acids (1be4b)
OeC). H NMR (250.13 MHz, D2O), d in ppm: 1.44e1.90
2
(m, 8H, CH2); 2.51 (d, 2H, JPeH ¼ 14.7 Hz, PeCH2); 3.44
A suspension of 0.0227 mol cycloalkanespiro-5-hydantoins
(1ae4a), 11.4 g (0.0665 mol) Ba(OH)2 and 70 ml water was
poured into a stainless steel autoclave equipped with a mag-
netic stirrer and a pressure gauge. The reaction mixture was
heated at 170 ꢁS for 3.5 h. The reaction solution was cooled
to room temperature, diluted with distilled water and filtered
to remove BaCO3. (NH4)2CO3 (2.6 g, 0.0270 mol) was added
to the clear filtrate aiming at precipitating BaCO3 and then fil-
tered again. The clear filtrate was concentrated under reduced
pressure and colorless crystal forms of the 1-aminocycloalka-
necarboxylic acids (1be4b) were obtained. The crude mixture
was purified by crystallization from water.
(d, 6H, 3JPeH ¼ 11 Hz, OeCH3). 13C{1H} NMR
(62.90 MHz, D2O), d in ppm: 24.9 SO2; 36.9 SO2; 39.8 (d,
2
1JPeC ¼ 146.0 Hz, PeCH2); 57.9 (d, JPeC ¼ 11.5 Hz, Oe
CH3); 72.4 eCe; 183.7 C]O. 31P{1H} NMR (242.94 MHz,
D2O), d in ppm: 24.48. Anal. Calcd for C9H18NO5P
(251.22): C, 43.03%; H, 7.22%; N, 5.58%; P, 12.33%; found:
C, 42.99%; H, 7.17%; N, 5.52%; P, 12.30%.
4.1.3.2. 1-[(Dimethoxyphosphono)methylamino]cyclohexane-
carboxylic acid (2c). White solid, 58.3% yield, mp ¼ 246e
248 ꢁS, Rf ¼ 0.38; IR (KBr, cmꢂ1): 3369 (NH), 2942e2854
(CeH), 1610 (C]O), 1296, 1245 (P]O), 1091, 1054 (Re
1
OeC). H NMR (250.13 MHz, D2O), d in ppm: 1.28e1.96
2
4.1.2. 1-Aminocyclododecanecarboxylic acid (5b)
(m, 10H, CH2); 2.47 (d, 2H, JPeH ¼ 14.69 Hz, PeCH2);
3
A suspension of 4.00 g 1,3-diazaspiro[4.11] hexadecane-
2,4-dione (0.0158 mol) and 80 ml 1.25 N NaOH was poured
into a stainless steel autoclave equipped with a magnetic stir-
rer and a pressure gauge. The reaction mixture was heated at
195 ꢁS for 2.5 h. The reaction solution was cooled down to
room temperature and diluted with distilled water and neutral-
ized by concentrated HCl. The residue from 1-aminocyclodo-
decanecarboxylic acid was filtered and washed with hot
methanol.
2.89 (d, 6H, JPeH ¼ 6.6 Hz, PeCH3). 13C{1H} NMR
4
(62.90 MHz, D2O), d in ppm: 22.7 CH2; 25.3 3,5SO2; 33.3
2,6SO2; 42.0 (d, 1JPeC ¼ 144.2 Hz, PeCH2); 48.8 (d, 2JPeC
¼
7.5 Hz, OeCH3); 64.2 eCe; 182.8 C]O. 31P{1H} NMR
(242.94 MHz, D2O), d in ppm: 22.17. Anal. Calcd for
C10H20NO5P (265.24): C, 45.28%; H, 7.60%; N, 5.28%; P,
11.68%; found: C, 45.21%; H, 7.58%; N, 5.22%; P, 11.61%.
4.1.3.3. 1-[(Dimethoxyphosphono)methylamino]cycloheptane-
carboxylic acid (3c). White solid, 62.1% yield, mp ¼ 209e
211 ꢁS, Rf ¼ 0.45; IR (KBr, cmꢂ1): 3117 (NH), 2929e2854
(CeH), 1611 (C]O), 1280, 1235 (P]O), 1169, 1041
Yield 90.27%, mp ¼ 280 ꢁS, Rf ¼ 0.63; IR (KBr, cmꢂ1):
3467 (NH), 2933e2848 (CeH), 1654, 1473 (NHþ3 ), 1526,
1405 (SPPꢂ). 1H NMR (250.13 MHz, D2O), d in ppm:

1204
E.D. Naydenova et al. / European Journal of Medicinal Chemistry 43 (2008) 1199e1205
1
(ReOeC). H NMR (250.13 MHz, D2O), d in ppm: 1.41e
(non-Hodgkin lymphoma), HD-MY-Z (Hodgkin lymphoma)
and 293T (human embryonal kidney) were supplied by DSMZ
GmbH, Germany; the urinary bladder carcinoma EJ originated
from the American Type Cell Culture, USA. Cells were cul-
tured routinely in a controlled environment: 37 ꢁC in 5%
CO2 humidified atmosphere. The human embryonal kidney
293T cells were cultured in Dulbecco’s modified MEM me-
dium, whereas all other cell lines were maintained in RPMI
1640; growth media were supplemented with 2 mM L-gluta-
mine and 10% fetal calf serum. All cell lines were subcultured
twice weekly to maintain continuous logarithmic growth.
2
1.98 (m, 12H); 2.50 (d, 2H, JPeH ¼ 15.1 Hz, PeCH2); 3.47
(d, 6H, 3JPeH ¼ 6.6 Hz, OeCH3). 13C{1H} NMR
(62.90 MHz, D2O), d in ppm: 22.6 4,5SO2; 29.2 3,6SO2;
37.2 2,7SO2; 38.8 (d, JPeC ¼ 145.7 Hz, PeCH2); 51.4 (d,
1
2JPeC ¼ 5.7 Hz, OeCH3); 67.9 eCe; 183.3 C]O. 31P{1H}
NMR (242.94 MHz, D2O), d in ppm: 24.84. Anal. Calcd for
C11H22NO5P (279.27): C, 47.31%; H, 7.94%; N, 5.02%; P,
11.09%; found: C, 47.21%; H, 7.90%; N, 5.01%; P, 11.03%.
4.1.3.4. 1-[(Dimethoxyphosphono)methylamino]cyclooctane-
carboxylic acid (4c). White solid, 54.8% yield, mp ¼ 206e
208 ꢁS, Rf ¼ 0.46; IR (KBr, cmꢂ1): 3140 (NH), 2921e2849
(CeH), 1734 (C]O), 1305, 1212 (P]O), 1051 (ReOeC).
1H NMR (250.13 MHz, D2O), d in ppm: 1.47e2.10 (m,
4.2.3. Cytotoxicity assay
Cell survival was evaluated by using the MTT-dye reduction
assay, which is based on the ability of viable cells to metabolize
a yellow tetrazolium salt to violet formazan product that can be
detected spectrophotometrically. The assay was carried out as
previously described [34] with minor modifications [35]. Expo-
nentially growing cells were plated in 96-well sterile plates at
a density of 104 cells/well in 100 ml of medium and were incu-
bated for 24 h. Thereafter the tested compounds were applied
in concentrations ranging from 0.195 to 200 mM. After a 72 h
continuous exposure 10 ml aliquots from a 5 mg/ml MTT
solution were added to each well and the plates were further
incubated for 4 h at 37 ꢁC in a humidified 5% CO2 atmosphere.
The formazan crystals yielded were solubilized by addition
of HCOOH (5%) acidified DMSO. The MTT-formazan absor-
bance was read on a Labexim LMR-1 multiplate reader.
2
14H); 2.70 (d, 2H, JPeH ¼ 14.8 Hz, PeCH2); 3.52 (d, 6H,
3JPeH ¼ 7.3 Hz, OeCH3). 13C{1H} NMR (62.90 MHz,
D2O), d in ppm: 22.1 SO2; 24.7 4,6SO2; 27.8 3,7SO2; 32.2
5
2,8SO2; 39.3 (d, JPeC ¼ 144.7 Hz, PeCH2); 52.0 (d,
1
2JPeC ¼ 5.3 Hz, OeCH3); 68.1 eCe; 183.3 C]O. 31P{1H}
NMR (242.94 MHz, D2O), d in ppm: 23.94. Anal. Calcd for
C12H24NO5P (293.29): C, 49.14%; H, 8.25%; N, 4.78%; P,
10.56%; found: C, 49.03%; H, 8.19%; N, 4.75%; P, 10.46%.
4.1.3.5. 1-[(Dimethoxyphosphono)methylamino]cyclododeca-
necarboxylic acid (5c). White solid, 63.6% yield,
mp ¼ 221e223 ꢁS, Rf ¼ 0.57; IR (KBr, cmꢂ1): 3448 (NH),
2935e2862 (CeH), 1706 (C]O), 1353, 1235 (P]O), 1158,
1052 (ReOeC). 1H NMR (250.13 MHz, D2O), d in ppm:
2
1.23e1.71 (m, 22H, CH2); 3.16 (d, 2H, JPeH ¼ 13.22 Hz,
4.2.4. Apoptosis assay
PeCH2); 3.84 (d, 6H, JPeH ¼ 11 Hz, PeCH3). 13C{1H}
The characteristic for apoptosis mono- and oligonucleoso-
mal fragmentation of genomic DNA was detected using
‘Cell Death Detection’ ELISA-kit (Roche Diagnostics,
Germany). Cytosolic fractions of 1 ꢃ 104 cells per group
(treated or untreated) served as antigen source in a sandwich
ELISA, utilizing primary anti-histone antibody-coated micro-
plate and a secondary peroxidase-conjugated anti DNA-anti-
body. The photometric immunoassay for histone-associated
DNA-fragments was executed according to the manufacturer’s
instructions at 405 nm, using ELISA reader (Labexim LMR-
1). The results were expressed as the oligonucleosome enrich-
ment factor (representing a ratio of the absorption in the
treated versus the untreated control samples).
3
NMR (62.90 MHz, D2O), d in ppm: 21.7 3,11SO2; 24.3
7SO2; 28.4 4,5,6,8,9,10SO2; 31.1 2,12SO2; 39.0 (d, JPeC
¼
1
2
153.39 Hz, PeCH2); 56.8 (d, JPeC ¼ 6 Hz, OeCH3); 72.9
eCe; 181.5 C]O. DEPT (62.90 MHz, D2O), d in ppm:
21.7 3,11SO2; 24.3 SO2; 28.4 4,5,6,8,9,10SO2; 31.1 2,12SO2;
7
39.0 (d, PeCH2); 56.7 OeCH3. 31P{1H} NMR
(242.94 MHz, D2O), d in ppm: 27.41. Anal. Calcd for
C16H32NO5P (349.40): C, 55.00%; H, 9.23%; N, 4.01%; P,
8.86%; found: C, 54.87%; H, 9.11%; N, 4.00%; P, 8.72%.
4.2. Biological assays
4.2.1. In vitro assays. Chemicals, solutions and
other materials
4.2.5. Cytogenetical method
The cell culture flasks and the 96-well microplates were
provided by NUNCLON (Denmark). MTT, FCS and cisplatin
were purchased from Sigma Co. The stock solutions of tested
compounds (20 mM) were freshly prepared in DMSO, and
stored at 4 ꢁC, protected from light for a maximum period
of 1 week. The serial dilutions of tested compounds were pre-
pared just before use. In the final dilutions obtained the con-
centrations of DMSO never exceeded 1%.
The cytogenetical investigation was conducted as described
by Preston et al. [36]. Inbred male and female C57Bl mice,
weighing 20.0 ꢀ 1.5 g were kept at standard conditions at
20 ꢁC and 12 h light/dark cycle, having free access to food
and water. The compounds 1ce5c were administered i.p. in
doses of 10 and 100 mg/kg. Mitomycin C (Kyowa) 3.5 mg/kg
was used as a positive control. The negative control animals
were injected only with 0.9% NaCl.
Bone marrow chromosome aberration assay was performed
on groups of animals each one consisting of 3 males and 3 fe-
males treated with the compound studied, and 5 pure control an-
imals. The animals were injected i.p. with colchicine at a dose of
4.2.2. Cell lines and culture conditions
The cell lines HL-60 (acute promyelocyte leukemia),
LAMA-84 and K-562 (chronic myeloid leukemia), DOHH-2

E.D. Naydenova et al. / European Journal of Medicinal Chemistry 43 (2008) 1199e1205
1205
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