Synthesis, antiproliferative activity and genotoxicity of novel anthracene-containing aminophosphonates and a new anthracene-derived Schiff base

Article abstract of DOI:10.1016/j.bmc.2011.11.024

A new Schiff base, 9-anthrylidene-furfurylamine and three novel anthracene-containing α-aminophosphonates, [N-methyl(dimethoxyphosphonyl)- 1-(9-anthryl)]-p-toluidine, [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]-p- toluidine and [N-methyl(diethoxyphosphon

Full text of DOI:10.1016/j.bmc.2011.11.024

Bioorganic & Medicinal Chemistry 20 (2012) 117–124
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antiproliferative activity and genotoxicity of novel
anthracene-containing aminophosphonates and a new
anthracene-derived Schiff base
I. Kraicheva a, , I. Tsacheva a, E. Vodenicharova a, E. Tashev a, T. Tosheva a, A. Kril b, M. Topashka-Ancheva c,
⇑
I. Iliev b, Ts. Gerasimova c, K. Troev a
a Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 103A, 1113 Sofia, Bulgaria
b Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 25, 1113 Sofia, Bulgaria
c Institute of Biodiversity and Ecosystems Research, Bulgarian Academy of Sciences, 2 Gagarin Str., 1113 Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
A new Schiff base, 9-anthrylidene-furfurylamine and three novel anthracene-containing
a-aminophos-
Received 12 August 2011
Revised 10 November 2011
Accepted 12 November 2011
Available online 20 November 2011
phonates, [N-methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine, [N-methyl(diethoxyphospho-
nyl)-1-(9-anthryl)]-p-toluidine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were
synthesized. The compounds have been characterized by elemental analysis, TLC, IR, NMR and fluorescent
spectra. The aminophosphonates and their synthetic precursors were tested for in vitro antitumor activ-
ity on a panel of seven human epithelial cancer cell lines. Safety testing was performed both in vitro (3T3
NRU test) and in vivo on ICR mice for genotoxicity and antiproliferative activity. 9-Anthrylidene-furfuryl-
amine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were most potent cytotoxic
agents towards colon carcinoma cell line HT-29. The latter compound exhibited also antiproliferative
activity to HBL-100, MDA-MB-231 and 647-V cells. The aminophosphonate [N-methyl(dimethoxyphos-
phonyl)-1-(9-anthryl)]-p-toluidine and its synthetic precursor 9-anthrylidene-p-toluidine were found
to be cytotoxic to HBL-100 and HT-29 tumor cell lines, respectively. Moderate genotoxic and antiprolif-
erative activity in vivo and low toxicity to Balb/c 3T3 (clone 31) mouse embryo cells were observed for all
tested compounds. The subcellular distribution of two tested compounds in a tumor cell culture system
was also studied.
Keywords:
Aminophosphonic acids
Schiff bases
NMR
Antitumor activity
Cytotoxicity
Genotoxicity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
transport through cellular membranes.7,8 Bisaminophosphonates
inhibit osteoclast-mediated bone resorption, delay the progression
Aminophosphonic acid derivatives have been found to posses
various important biological activities that enable wide range
applications of these compounds in the pharmacological and agro-
of bone metastases and exert marked apoptotic and antiprolifera-
tive effect on tumor cells.9 Numerous of these compounds are used
as haptens for the generation of catalytic antibodies, antibiotics,
bone seeking radiopharmaceuticals and therapeuticals.10–12 Some
authors suggest that the biological activity of the aminophospho-
nates is correlated to their lipophilicity.13,14
chemical fields.1–4
a-Aminophosphonates are quite attractive com-
pounds in the development of potential drugs against several
metabolic disorders because of their structural similarity to the nat-
ural
a
-aminocarboxylic acids.2,3 Thus, due to the tetrahedral config-
Anthracene-bearing a-aminophosphonates might be of particu-
uration at phosphorus, aminophosphonates can act as analogues of
the transition state in enzymatic reaction and therefore they are
widely used in the design of the enzyme inhibitors.5,6 Aminophos-
phonates can suppress bacterial and viral growth and enhance
lar interest in the design of new antitumor therapeuticals consider-
ing the fact that the DNA-intercalating anthracene-derived planar
structure is the main pharmacophoric fragment of some cytostatic
drugs, which have found clinical applications for the treatment of
human cancers.15,16 Highly active antimitotic anthracene-based
agents, inhibit tubulin polymerization.17–19 For some of these
anthracene-containing substances have been reported to display
strong antiproliferative activity against several tumor cell lines,
including multidrug resistant phenotypes.20 In addition, fluores-
cent anthracenoyl crown ethers are used as a sensitive probe for
the solid phase transitions of phosphatidylcholines.21 Therefore,
⇑
Corresponding author. Tel.: +359 2 979 6633; fax: +359 2 870 0309.
E-mail addresses: kraicheva@yahoo.com (I. Kraicheva), ivelina_772000@yahoo.
com (I. Tsacheva), elitcapetrova@abv.bg (E. Vodenicharova), tashev@polymer.bas.bg
(E. Tashev), ttosheva@polymer.bas.bg (T. Tosheva), antonkrill@yahoo.com (A. Kril),
topashka.mn@lycos.com (M. Topashka-Ancheva), taparsky@abv.bg (I. Iliev), cvetij@
yahoo.com (Ts. Gerasimova), ktroev@polymer.bas.bg (K. Troev).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.024

118
I. Kraicheva et al. / Bioorg. Med. Chem. 20 (2012) 117–124
the fluorescent properties of anthracene-based aminophospho-
nates could find valuable bioanalytical application in studying
the subcellular distribution and binding in normal and tumor cells.
To the best of our knowledge, only a few examples of anthra-
cene-derived aminophosphonates are described in the litera-
ture.22–24 The addition of dialkyl (or diaryl) phosphites to Schiff
bases is a convenient method for the preparation of aminophosph-
onate derivatives.22,24–26 In this work we report on the synthesis,
in vitro antitumor activity and in vitro and in vivo safety evaluation
of new anthracene-derived Schiff base and three novel anthracene-
containing aminophosphonates. Data about the cytotoxic potential
of an earlier described anthracene-containing Schiff base27 are also
presented. In addition, we report data about subcellular distribu-
tion of two of the tested compounds in a tumor cell culture system.
13C{1H} NMR spectra of the aminophosphonates also confirm the
P–C bond formation. The doublets with large coupling constants
154.1 (3), 153.2 (4) and 158.4 (5) Hz have to be assigned to the car-
bon atom connected to the phosphorus. The assignment of the
anthracene proton and carbon signals of the compounds 1–5 is
based on the analysis of their 2D spectra and literature data.23,28,29
The 31P{1H} NMR spectra of the aminophosphonates reveal one
singlet at 27.69 (3), 25.25 (4) and 25.80 (5) ppm. The fluorescent
emission of the compounds 1–5 was registered and the maxima
appear in the region 318–442 nm of the spectra.
2.2. In vitro antitumor activity
The Schiff bases 1 and 2 and the aminophosphonates 3–5 were
tested for cytotoxicity against a panel of seven cancer cell lines rep-
resentative of some important types of human tumors. All com-
pounds exerted concentration-dependent antiproliferative effects
after 24 h exposure which enabled the construction of concentra-
tion–response curves (not shown) and the calculation of the corre-
sponding IC50 values summarized in Table 1. As evident from the
cytotoxicity data the Schiff base 2 and the corresponding amin-
ophosphonate 5 proved to be the most potent cytotoxic agents to-
wards colon carcinoma cell line HT-29, which implies that the
presence of both anthracene ring and furan ring is an important
prerequisite for optimal activity for these compounds. This is fur-
ther supported by the fact that compound 5 showed two times
higher activity to HBL-100 cells than the referent anticancer drug
Doxorubicin. In addition, the cytotoxic potential of the same com-
pound was comparable to that of the positive control substance,
used in the experiments, when tested on cell lines MDA-MB-231
(highly metastatic carcinoma of the breast) and 647-V (bladder
carcinoma). The other Schiff base 1 and its aminophosphonate
derivative 3 were found to be cytotoxic to colon carcinoma cells
(HT-29 line) and HBL-100 cell line, respectively. All compounds
were generally less active as compared to the referent anticancer
drug Doxorubicin after testing on cell cultures from the cancer cell
lines MCF-7, HepG2 and HeLa (Table 1).
2. Results and discussion
2.1. Chemistry
The Schiff bases, 9-anthrylidene-p-toluidine (1) and 9-anthry-
lidene-furfurylamine (2) were prepared by condensation of 9-
anthracenecarboxaldehyde with p-toluidine and furfurylamine,
respectively. Three novel
a-aminophosphonic acid diesters,
bearing anthracene ring, were synthesized: [N-methyl(dimethoxy-
phosphonyl)-1-(9-anthryl)]-p-toluidine (3), [N-methyl(diethoxy-
phosphonyl)-1-(9-anthryl)]-p-toluidine
(4)
and
[N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine (5).
The aminophosphonates were obtained through addition of di-
methyl phosphite and diethyl phosphite to the Schiff bases 1 and
2 according to the Scheme 1. The reaction of dialkyl phosphites
to the Schiff base 1 was carried out without catalyst. The products
3 and 4 were obtained as yellow fluorescing crystalline solids. The
addition of diethyl phosphite to the imine 2 was performed using
CdI2 as a catalyst. The compound 5 was prepared as an oil and puri-
fied by column chromatography on silica gel. Thin layer chroma-
tography (TLC) gave one spot for each of the compounds 1–5.
The formation of the aminophosphonate structures was confirmed
by IR and NMR spectroscopy. The 1H NMR spectra of the amino-
phosphonates exhibit a doublet signal at 6.37 (3), 6.35 (4) and
5.73 (5) ppm, which can be assigned for the CHP proton. The NH
proton signal of 3–5 is observed as a broad singlet. The data from
The results obtained imply that compounds 2 and 5 could be
considered as promising leads for further development of agents
active in chemotherapy of malignant breast and colon disease.
2.3. In vitro safety testing
The results from the validated Balb/c 3T3 (clone 31) Neutral Red
Uptake Assay (3T3 NRU test) revealed dose-dependent cytotoxic
activity of compounds 1–5 (Fig. 1). The aminophosphonate 4 and
the starting Schiff base 1 appeared to be less toxic than other
tested compounds with lowest toxic doses of 1.36 and 0.15 mg/ml,
respectively. The Schiff base 2 and the corresponding amin-
ophosphonate 5 showed statistically significant (p <0.001) cytotox-
icity in a wide concentration range (1–0.07 mg/ml) to mouse
embryo fibroblastic cells, compared to untreated control cell cul-
tures. The aminophosphonate 3 also induced significant cytotoxic
effect (p <0.001) within the concentration range tested. Compared
to the cytotoxic effect on Balb/c 3T3 cells of the positive control
substance sodium dodecyl sulphate, however, the cytotoxicity of
the tested compounds was comparable (2 and 5), two-fold lower
(1 and 3) and over 10-fold (4) lower (Fig. 1).
RO
RO
HN
P
X
N
X
+
P
O
H
O
OR
RO
1, 2
3 - 5
1: X =
3: X =
4: X =
5: X =
, R = CH
CH
CH
CH
3
3
3
3
H C
2
X =
2:
, R = C H
2 5
O
2.4. Clastogenic and antiproliferative activity
, R = C H
2 5
H C
2
O
The results obtained about the induced frequency of chromo-
some aberrations in bone marrow cells of ICR mice after treatment
Scheme 1. Synthesis of
a-aminophosphonic acid diesters 3–5. Reagents and
with the Schiff bases 1 and 2 and the
a-aminophosphonates 3–5
conditions: 3, 9-anthrylidene-p-toluidine, dimethyl phosphite, diethyl ether, rt,
13 h; 4, 9-anthrylidene-p-toluidine, diethyl phosphite, benzene, reflux, 14 h; 5, 9-
anthrylidene-furfurylamine, diethyl phosphite, benzene, CdI2, reflux, 6 h.
are presented in Tables 2 and 3. The Schiff base 1 exhibited a mod-
erate clastogenic effect, varying from 5.25 0.53% to 6.57 1.04%.

I. Kraicheva et al. / Bioorg. Med. Chem. 20 (2012) 117–124
119
Table 1
Comparative cytotoxic activity of compounds 1–5 versus referent substance Doxorubicin in a panel of human tumor cell lines after 24 h treatment (MTT-dye reduction assay)
Tumor cell lines
Mean IC50 values (mg/ml)a
2
1
3
4
5
Doxorubicin
MCF-7
0.45 0.017
0.12 0.007
0.31 0.027
0.18 0.011
0.08 0.003
0.13 0.004
0.17 0.004
0.45 0.024
0.88 0.023
0.02 0.006
0.83 0.025
>2
>2
>2
0.26 0.014
0.10 0.003
0.07 0.001
0.07 0.001
0.11 0.002
0.11 0.001
0.07 0.001
0.12 0.001
<0.068
<0.068
0.14 0.011
<0.068
0.58 0.013
<0.068
MDA-MB-231
HBL-100
HepG2
HT-29
647-V
0.45 0.142
0.46 0.018
0.12 0.005
0.20 0.012
0.08 0.002
0.36 0.008
0.73 0.023
>2
>2
>2
0.60 0.021
>2
HeLa
1.94 0.06
<0.068
a
Values are means standard deviation from three consecutive experiments.
100
75
50
25
0
100
***
***
2
1
***
75
50
25
0
***
***
***
***
***
***
***
***
***
***
***
C
1
0.68 0.46 0.32 0.22 0.15 0.1 0.07
Concentration (mg/ml)
C
1
0.68 0.46 0.32 0.22 0.15 0.1 0.07
Concentration (mg/ml)
5
***
100
75
50
25
0
3
***
*** ***
***
100
75
50
25
0
***
***
***
***
***
***
***
***
***
***
***
C
2
1.36 0.93 0.63 0.43 0.29 0.2 0.14
Concentration (mg/ml)
C
1
0.68 0.46 0.32 0.22 0.15 0.1 0.07
Concentration (mg/ml)
100
75
50
25
0
100
75
50
25
0
Sodium dodecyl sulphate
4
***
***
***
***
***
***
***
***
***
***
C
0.52 0.37 0.27 0.19 0.14 0.1 0.07 0.05
Concentration (mg/ml)
C
2
1.36 0.93 0.63 0.43 0.29 0.2 0.14
Concentration (mg/ml)
Figure 1. In vitro cytotoxicity of compounds 1–5 on cultures from cell line Balb/c 3T3, clone 31 (3T3 NRU test). C, vehicle treated cell cultures (negative control); sodium
dodecyl sulphate, positive control substance; ⁄⁄⁄ p <0.001, compared to negative control.

120
I. Kraicheva et al. / Bioorg. Med. Chem. 20 (2012) 117–124
Table 2
Clastogenic effect and proliferative activity of bone marrow cells of ICR line laboratory mice after ip treatment with Schiff base 1 and the corresponding aminophosphonates 3
and 4
Compounds
and doses
Time after
treatment (h) metaphases scored
Number of
Type of chromosome aberrations
Percentage of cells Statistical significance Mitotic index Statistical
with aberrations
(X m)
(‰) (X m)
significance
Breaks Fragments Rearrangements
a
b
c
d
a
b
c
d
c/c
t/t
c/t
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
*** ***
***
(1) 10 mg/kg
(1) 100 mg/kg
(3) 10 mg/kg
(3) 100 mg/kg
(4) 10 mg/kg
(4) 100 mg/kg
24
48
24
48
24
48
24
48
24
48
24
48
400
400
350
400
400
400
400
400
400
400
400
400
200
400
700
500
6
3
5
7
2
6
6
6
5
9
7
8
17
17
4
5
5
8
7
1
3
2
4
3
9
10
9
1
2
1
1
0
0
2
0
1
1
2
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5.25 0.53
5.00 0.38
6.57 1.04
6.5 0.73
4.0 0.38
4.75 0.37
5.5 0.5
6.0 0.84
4.75 0.65
6.00 0.75
8.5 0.73
8.00 0.53
30.5 2.36
15.8 0.81
1.14 0.34
0.6 0.3
12.63 1.02
8.45 1.82
10.66 1.42
11.69 1.98
12.60 0.77
15.64 1.38
11.97 1.,08
14.11 1.64
13.41 1.14
11.86 1.24
16.08 1.16
11.78 1.28
5.49 0.19
7.29 0.34
20.06 1.38
16.88 0.56
*** ***
*
*
11
13
12
12
14
10
10
14
9
*** ***
***
**
*** ***
***
**
*** **
**
**
4
***
**
*
**
11
14
30
24
0
**
***
***
Mit. C 3.5 mg/kg 24
7
20
4
48
Control 0.9% NaCl 24
48
***
***
***
***
0
0
3
c/c, Centromeric/centromeric fusion; t/t, telomere/telomeric fusion; c/t, centromere/telomeric fusion.
a, Compared to Mitomycin C; b, compared to control; c, compared to dose 100 mg/kg; d, compared to 1.
Statistics: Student t-test.
*
p <0.05.
p <0.01.
p <0.001.
**
***
Dose-dependent effect was not detected. The values for the aber-
rant metaphases in the samples obtained after treatment with
Schiff base 1 at doses 10 and 100 mg/kg body weight did not signif-
icantly differ (p >0.05). The corresponding aminophosphonate 3
had lower clastogenic effect than the Schiff base 1, but the differ-
ences did not reach statistical significance. Main types of structural
alterations in chromosomes that have been observed in slides ana-
lyzed were as follows: breaks and fragments—35.6 5.63% and
centromere/centromeric fusions—62.17 5.72%. The quantity of
metaphases with chromosome breaks and fragments in the bone
marrow cells of the experimental animals treated with the amin-
ophosphonate 3 were lower than those in animals treated with
the Schiff base 1 (Table 2). The compound 4 at a dose100 mg/kg
after 24 h exposure induced significantly higher percentage of
chromosome aberrations (p <0.01), compared to the lower concen-
tration applied (10 mg/kg) and the quantity of bone marrow cells
with damaged chromosomes reached 8.5 0.73%. Significant dif-
ferences in the number of aberrant mitoses were not detected in
samples obtained at 24th and 48th hour after ip application of 4.
Both doses of this aminophosphonate ensured enough amounts
of active molecules to provoke the same percentage of aberrations
at 48th hour post inoculation (p.i.). The compound 2 showed clas-
togenic effects, ranging from 4.43% to 5.5%. These values were sig-
nificantly lower (p <0.001) compared with the values calculated
after treatment of experimental animals with the alkylating agent
Mitomycin C. An increase in the yield of chromosomal aberrations
in samples examined after injection into laboratory mice of the
higher dose of the compound 2 (100 mg/kg) was not found
(p >0.05). The clastogenic effect of the aminophosphonate 5 was
similar to that of the parent compound 2. The values ranged from
4.75% metaphases with chromosomal aberrations in samples ob-
tained 24 h after treatment of ICR mice with a dose 10 mg/kg to
6.25% in 48th hour p.i. samples. The difference was statistically
insignificant (p >0.05). The main type of chromosome aberrations
provoked were centromere/centromeric fusions (c/c). This type of
aberrations represented 68.76 3.13% of all reported aberrations
in the analyzed slides. For comparison, treatment with Mitomycin
C had resulted only in 24.9% damaged metaphases with c/c fusions,
while metaphase plates with breaks and fragments were 75.1%
(Table 3).
The data reflecting the antiproliferative activity of test sub-
stances, expressed through changes in mitotic index values in bone
marrow cell population are presented in Tables 2 and 3. All sub-
stances applied in the experiments showed lower antiproliferative
effect on the normal bone marrow cells than the positive control
Mitomycin C with a level of statistical significance ranging from
p <0.01 to 0.001. Mitotic index values ranged from 8.45‰, ob-
served in experimental animals treated with the Schiff base 1
(10 mg/kg, 48 h after treatment) to 16.08‰ in the experimental
group injected with the corresponding aminophosphonate 4 (Table
2). It was also found that the compounds 2 and 3 48 h after ip
administration did not inhibited cell division in the bone marrow
cell population.
In summary, the compounds did not induce clear expressed
‘dose-effect’ clastogenic activities, in contrast to the alkylating
agent Mitomycin C. The low percentage of bone marrow meta-
phases with chromosome aberrations and the lack of aberrant
metaphase plates with disintegrated chromosomes and dis-
persed chromatin is an evidence of the moderate clastogenic ef-
fect of the tested substances. The main types of chromosome
structure alterations in the ICR mice bone marrow cells were
c/c fusions, breaks and fragments, which suggests that the
tested compounds affect the centromeric chromosome regions,
which allow centromere/centromeric recombinations between
non-homologous chromosomes. Along with the moderate clasto-
genic activity of the compounds an inhibition of cell division
was observed, but the antiproliferative effects were fairly lower
than that of the alkylating anticancer drug Mitomycin C, indica-
tive of
a lesser damage to proliferating bone marrow cell
population.
The data from in vivo experiments are in a good correlation
with the data on the cytotoxic effect of tested Schiff bases and their
aminophosphonate derivatives on mouse embryo fibroblastic cell
line Balb/c 3T3 (clone 31). It can be speculated that these com-
pounds have lower toxic effect on normal cells as compared to
anticancer and cytotoxic agents.

I. Kraicheva et al. / Bioorg. Med. Chem. 20 (2012) 117–124
121
Table 3
Clastogenic effect and proliferative activity of ICR mice bone marrow cells after ip application of the compounds 2 and 5
Compounds
and doses
Time after
treatment
(h)
Number of
metaphases
scored
Type of chromosome aberrations
Percentage of
cells
with aberrations
(X m)
Statistical
significance
Mitotic
index
(‰) (X m)
Statistical
significance
Breaks Fragments Rearrangements
a
b
c
d
a
b
c
d
c/c
t/t
c/t
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
**
***
***
***
(2) 10 mg/kg
(2) 100 mg/kg
(5) 10 mg/kg
24
48
24
48
24
48
400
400
400
400
400
400
200
400
700
500
1
3
4
1
4
3
1
4
3
4
15
12
13
17
17
12
7
20
4
3
0
0
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
4.75 0.84
4.43 0.70
5.50 0.5
5.5 0.5
6.25 0.80
4.75 0.37
30.5 2.36
15.8 0.81
1.14 0.34
0.6 0.3
11.26 1.04
10.18 0.67
12.43 1.31
15.17 0.89
15.09 1.19
10.07 0.99
5.49 0.19
7.29 0.34
20.06 1.38
16.88 0.56
***
***
***
***
**
**
*
***
***
***
7
2
Mit. C 3.5 mg/kg 24
48
Control 0.9%
NaCl
17
17
4
30
24
0
***
***
***
***
24
48
0
0
c/c, Centromeric/centromeric fusion; t/t, telomere/telomeric fusion; c/t, centromere/telomeric fusion.
a, Compared to Mitomycin C; b, compared to control; c, compared to dose 100 mg/kg; d, 5 compared to 2.
Statistics: Student t-test.
*
p <0.05.
p <0.01.
p <0.001.
**
***
Figure 2. Fluorescence microscopy of cytoplasmic distribution of Schiff base 1 (A) and nuclear accumulation of aminophosphonate 4 (B) in HBL-100 cells.
2.5. Fluorescent studies
cursor—Schiff base 2. The Schiff base 1, bearing only an anthracene
ring and its aminophosphonate derivative 3 were found to be cyto-
toxic to colon carcinoma cells (HT-29 line) and HBL-100 cell line,
respectively. The other aminophosphonate 4, obtained from the
same Schiff base (1) and diethyl phosphite, was less active against
all tested cancer cell lines, as compared to the referent anticancer
drug Doxorubicin. The results obtained underline the importance
of the simultaneous presence of three pharmacophoric fragments
in the same molecule—an anthracene moiety, a furan ring and an
aminophosphonate group, which is an important prerequisite for
higher antitumor activity.
The fluorescent properties of the anthracene-containing com-
pounds 1 and 4 were used to study their subcellular distribution
in HBL-100 model cell culture system after 24 h exposure to non-
toxic concentrations of the substances. The fluorescent signal of
Schiff base 1 was observed mainly in the cytoplasm of tumor cells.
In contrast, the most intensive fluorescence after application of
aminophosphonate 4 was found in the nuclei and nuclear mem-
branes of HBL-100 cells (Fig. 2).
In vitro and in vivo safety testing revealed that the compounds
exert lower toxicity to normal cells, as compared to well known
anticancer and cytotoxic agents. Therefore, the novel substances
are promising for future work on the development of agents active
in chemotherapy of malignant breast and colon disease. Moreover,
the fluorescent properties of anthracene ring allow adequate and
precise studies on the cellular uptake and intracellular distribution
of the novel compounds in malignant and normal cells.
3. Conclusion
A new anthracene-derived Schiff base 9-anthrylidene-furfuryl-
amine 2 and three novel a-aminophosphonic acid diesters, bearing
anthracene moiety—[N-methyl(dimethoxyphosphonyl)-1-(9-an-
thryl)]-p-toluidine 3, [N-methyl(diethoxyphosphonyl)-1-(9-an-
thryl)]-p-toluidine
4 and [N-methyl(diethoxyphosphonyl)-1-(9-
anthryl)]furfurylamine 5, were synthesized and characterized.
The aminophosphonates and their precursors were evaluated for
in vitro antitumor activity on a panel of seven human epithelial
cancer cell lines. Two of the compounds (2 and 5), combining in
their molecules an anthracene residue and a furan ring, showed
optimal antiproliferative activity to human tumor cells from colon
carcinoma and from malignant tumors of the breast and urinary
bladder. Moreover, the aminophosphonate 5 exhibited higher
activity against all tested cancer cell lines, than its synthetic pre-
4. Experimental
Dimethyl phosphite (Sigma Aldrich Chemie GmbH, Steinheim,
Germany) and diethyl phosphite (Fluka Chemie Ag, Buchs, Switzer-
land) were purified by vacuum distillation. 9-Anthracenecarboxal-
dehyde, p-toluidine and furfurylamine were purchased from Fluka.
All solvents were freshly distilled prior to use. The melting points

122
I. Kraicheva et al. / Bioorg. Med. Chem. 20 (2012) 117–124
of the compounds were determined on a Kofler microscope and are
uncorrected. IR spectra were taken on a IRAffinity-1 spectropho-
tometer. 1H, 13C{1H} and 2D NMR spectra in CDCl3 (compounds
1–4) and 1H NMR spectrum in CD3OD (compound 2) were recorded
on a Bruker DRX-250 spectrometer at rt and tetramethylsilane
(TMS) as an internal standard. 1H, 13C and 2D NMR spectra in CDCl3
of compound 5 were recorded on an Avans 600 MHz spectrometer
at rt and TMS as an internal standard. 31P{1H} NMR spectra in
CDCl3 were taken on a Bruker DRX-250 spectrometer using 85%
H3PO4 as an external standard. The thin layer chromatography
was performed on Merk Silica gel 60 F254 at room temperature.
The samples were applied as methanolic solutions and the chro-
matograms were developed ascendingly in the eluting system
diethyl ether:hexane = 4:1. The spots were detected under UV
light. Column chromatography: For purification of single product
molecules, the crude reaction mixture was separated by normal
pressure liquid chromatography in a 2 cm ꢀ 35 cm column, con-
taining 50 g silica gel 60 particle size 0.063–0.2 mm (70–230 mesh,
Fluka), with mobile phase diethyl ether: 1,4-dioxane (4:1 (v/v).
Fractions of 2 ml were collected and analyzed by thin layer chro-
matography (TLC). The desired products were separately pooled
and the solvent evaporated under reduced pressure. Fluorescent
spectra were recorded on a Jasko fluorimeter 6600.
tion mixture was stirred at ambient temperature for 13 h. The yel-
low precipitate obtained was recrystallized from methanol. Yield:
1.53 g (74%); mp 179
24H24NO3P: C, 71.11; H, 5.93; N, 3.46; P, 7.65. Found: C, 70.88;
H, 5.67; N, 3.39; P, 7.51. IR (neat), NH); 1232
(cmꢁ1): 3327 (
- 180 °C; Rf = 0.51. Anal. calcd for
C
m
m
(mP@O); 1161, 1020 (mPOMe). Fluorescent spectrum: Ex 295 nm—
kmax = 326 and 345 nm; Ex 325 nm—kmax = 403 and 421 nm. 1H
NMR (CDCl3), d (ppm), JHH (Hz), JPH (Hz): 9.06 (d, 3J = 9.0, 1H, Ant-
hH-
a a
), 8.47 (d, 3J = 8.4, 1H, AnthH- ), 8.46 (d, 6J = 2.8, 1H, AnthH-
10), 8.08 (d, 3J = 8.4, 1H, AnthH-
a
), 8.00 (d, 3J = 8.9, 1H, AnthH-
a),
7.57 (m, 4H, AnthH-b), 6.79 (m, 2H, ArH-30,50), 6.46 (m, 2H, ArH-
20,60), 6.37 (d, 2J = 27.0, 1H, CHP), 4.68 (br s, 1H, NH), 3.90 and
3.21 (2d, 3J = 10.8 and 10.6, 6H, OCH3), 2.11(s, 3H, ArCH3).
13C{1H} NMR (CDCl3), d (ppm), JPC (Hz): 144.58 (d, 3J = 13.2, ArC-
10), 129.88 (AnthC-
a
), 129.62 (ArC-30,50), 129.24 (AnthC-
a),
5
129.06 (d, J = 4.5, AnthC-10), 127.51 (ArC-40), 127.03 (AnthC-b),
126.30 (AnthC-
(AnthC-b), 122.53 (AnthC-
a), 126.09 (AnthC-b), 125.14 (AnthC-b), 124.72
a
), 113.61 (ArC-20,60), 53.68 and 53.54
(2d, 2J = 6.9 and 6.9, OCH3), 52.91 (d, 1J = 154.1, CHP), 20.23
(ArCH3). 31P{1H} NMR (CDCl3), d (ppm): 27.69.
4.1.2.2. [N-Methyl(diethoxyphosphonyl)-1-(9-anthryl)]-p-tolui-
dine (4).
9-Anthrylidene-p-toluidine (1) (2.16 g, 7.3 mmol)
and diethyl phosphite (1.01 g, 7.3 mmol) were dissolved in ben-
zene (15 ml) and placed in a flask equipped with magnetic stirrer
and a reflux condenser. The reaction mixture was refluxed for
14 h with stirring. After removal of the benzene in vacuum, the
crude product was recrystallized from ethyl alcohol. Yield: 2.16 g
(68%) ; mp 133–134 °C; Rf = 0.36. Anal. calcd for C26H28NO3P: C,
72.06; H, 6.47; N, 3.23; P, 7.16. Found: C, 71.79; H, 6.72; N, 3.21;
4.1. Synthesis
4.1.1. Schiff bases (1 and 2)
4.1.1.1. 9-Anthrylidene-p-toluidine (1).
from 9-anthracenecarboxaldehyde and p-toluidine according to
Prot,27 using diethyl ether as solvent, instead of benzene, conduct-
ing the reaction at room temperature and recrystallized from ethyl
alcohol. Yield: 82%; mp 114–115 °C (literature mp 106–107 °C);
It was prepared
P, 6.88. IR (neat),
POEt). Fluorescent spectrum: Ex 290 nm—kmax = 318, 348, 362,
and 403 nm. 1H NMR (CDCl3), d (ppm), JHH (Hz), JPH (Hz): 9.09 (d,
3J = 9.0, 1H, AnthH- ), 8.50 (d, 3J = 9.0, 1H, AnthH-
), 8.45 (d,
6J = 2.9, 1H, AnthH-10), 8.07 (d, 3J = 8.4, 1H, AnthH-
), 7.98 (d,
), 7.56 (m, 4H, AnthH-b), 6.79 (m, 2H, ArH-
m mNH); 1238 (mP@O); 1156, 1026
(cmꢁ1): 3315 (
(m
Rf = 0.90. IR (neat),
m
(cmꢁ1): 1622 (
m
C@N); 1610, 1587, 1520,
1440 (
m
C@C). Fluorescent spectrum: Ex 306 nm—kmax = 348 nm.
a
a
1H NMR (CDCl3), d (ppm): 9.68 (s, 1H, CH@N), 8.75 (m, 2H, AnthH),
8.53 (s, 1H, AnthH-10), 8.03 (m, 2H, AnthH), 7.55 (m, 4H, AnthH),
7.36 (m, 4H, ArH); 2.47 (s, 3H, ArCH3). 13C{1H} NMR (CDCl3), d
(ppm): 158.94 (CH@N), 130.38 (AnthC-10), 129.87 (ArC), 128.95,
127.10, 125.31,124.77 (AnthC), 120.94 (ArC), 21.03 (ArCH3).
a
3J = 8.5, 1H, AnthH-
a
30,50), 6.45 (m, 2H, ArH-20,60), 6.35 (d, 2J = 27.1, 1H, CHP), 4.53 (br
s, 1H, NH), 4.27, 3.76 and 3.32 (3 m, 4H, OCH2), 2.11 (s, 3H, ArCH3),
1.38 and 0.69 (2td, 3J = 7.1 and 7.1, 4J = 0.3 and 0.6, 6H, CH3).
13C{1H} NMR (CDCl3), d (ppm), JPC (Hz): 144.76 (d, 3J = 13.1, ArC-
4.1.1.2. 9-Anthrylidene-furfurylamine (2).
9-Anthracene-
10), 129.74 (AnthC-
a
), 129.51 (ArC-30,50), 129.09 (AnthC-
a),
5
carboxaldehyde (2.20 g, 10.7 mmol) dissolved in diethyl ether
(150 ml) and furfurylamine (1.04 g, 10.7 mmol) were mixed and
stirred at an ambient temperature for 15 h. Then diethyl ether
was removed in vacuum and the crude product was recrystallized
from petroleum ether. Yield: 2.19 g (72%); mp 84–85 °C; Rf = 0.87.
Anal. calcd for C20H15NO: C, 84.21; H, 5.26; N, 4.91. Found: C,
128.84 (d, J = 4.5, AnthC-10), 127.25 (ArC-40), 126.75 (AnthC-b),
126.47 (d, J = 1.9, AnthC-
124.62 (AnthC-b), 122.74 (AnthC-
a
), 125.83 (AnthC-b), 125.03 (AnthC-b),
a
), 113.50 (ArC-20,60), 63.21 and
62.96 (2d, 2J = 7.1 and 7.0, OCH2), 53.23 (d, 1J = 153.2, CHP), 20.16
(ArCH3), 16.48 and 15.71 (2d, 3J = 5.8 and 5.8, CH3). 31P{1H} NMR
(CDCl3), d (ppm): 25.25.
83.91; H, 5.16; N, 5.06. IR (neat)
1519, 1440 C@C); 1016 COC). Fluorescent spectrum: Ex
m mC@N); 1558,
(cmꢁ1): 1627 (
(
m
(
m
4.1.2.3. [N-Methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfuryl-
365 nm—kmax = 426 and 442 nm. 1H NMR (CDCl3), d (ppm), JHH
(Hz): 9.48 (t, 4J = 1.4, 1H, CH@N), 8.51 (m, 2H, AnthH), 8.48 (s,
1H, AnthH-10), 8.07 (m, 2H, AnthH), 7.51 (m, 5H, AnthH, FurH-
5), 6.44 (2 pseudo-s, 2H, FurH-3,4), 5.11 (d, 4J = 1.4, 2H, CH2Fur).
1H NMR (CD3OD), d (ppm), JHH (Hz): 9.43 (t, 4J = 1.2, 1H, CH@N),
8.52 (s, 1H, AnthH-10), 8.28 (m, 2H, AnthH), 8.00 (m, 2H, AnthH),
7.58 (dd, 3J = 1.8, 4J = 1.0, 1H, FurH-5), 7.49 (m, 4H, AnthH), 6.47
and 6.46 (2 pseudo-s, 2H, FurH-3,4); 5.07 (d, 4J = 1.3, 2H, CH2Fur).
13C{1H} NMR (CDCl3), d (ppm): 162.43 (CH@N), 152.18 (FurC-2),
142.32 (FurC-5), 129.47 (AnthC-10), 128.78, 126.70, 125.20,
124.72 (AnthC), 110.49 (FurC-4), 107.82 (FurC-3), 58.36 (CH2Fur).
amine (5).
Diethyl phosphite (1.67 g, 12.1 mmol) dissolved in
dry benzene (15 ml) and CdI2 (0.09 g, 0.25 mmol) were placed in a
flask, equipped with magnetic stirrer, a thermometer, an inlet for
inert gas and reflux condenser. After stirring for a half an hour at
rt for dissolving the catalyst to the reaction mixture was added
9-anthrylidene-furfurylamine (2) (3.45 g, 12.1 mmol), dissolved
in dry benzene (10 ml). The mixture was refluxed for 6 h with stir-
ring. Then benzene was removed in vacuum and the residue was
purified on Silica gel 60 (230–400 mesh) using diethyl ether–hex-
ane = 4:1 and adding 10–50% methanol step by step to give a pure
product. Yield: 3.38 g (66%); oil; Rf = 0.27. Anal. calcd for
C
24H26NO4P: C, 68.09; H, 6.15; N, 3.31; P, 7.33. Found: C, 67.88;
H, 6.01; N, 3.22; P, 7.07. IR (neat), NH); 1230
(cm-1): 3302 (
P@O); 1163, 1008 ( POEt COC). Fluorescent spectrum: Ex 242 nm—
kmax = 396, 418, and 441 nm. 1H NMR (CDCl3), d (ppm), JHH (Hz),
JPH (Hz): 9.36 (d, 3J = 9.0, 1H, AnthH- ), 8.44 (d, 6J = 2.6, 1H, Ant-
hH-10), 8.12 (d, 3J = 8.9, 1H, AnthH- ), 8.00 (d, 3J = 7.8, 1H, Ant-
4.1.2. Aminophosphonates (3–5)
m
m
4.1.2.1.
toluidine
[N-Methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-
(3). 9-Anthrylidene-p-toluidine (1) (1.51 g,
(m
m
,
5.1 mmol) was dissolved in diethyl ether (40 ml) and dimethyl
phosphite (0.56 g, 5.1 mmol) was added to the solution. The reac-
a
a

I. Kraicheva et al. / Bioorg. Med. Chem. 20 (2012) 117–124
123
hH-
a
), 7.99 (d, 3J = 8.3, 1H, AnthH-
a
), 7.49 (m, 4H, AnthH-b), 7.24
tration rage was applied (from 2 to 0.0681 mg/ml; dilution factor
of 6 10 = 1.47) and after treatment with Neutral Red medium,
washing and application of the fixative (Ethanol/Acetic acid) the
p
(dd, 3J = 1.9, 4J = 0.8, 1H, FurH-5), 6.20 (dd, 3J = 3.2 and 1.9, 1H,
FurH-4), 5.89 (dd, 3J = 3.2, 4J = 0.7, 1H, FurH-3), 5.73 (d, 2J = 23.9,
1H, CHP), 4.07 (m, 2H, OCH2), 3.77 (m, 2H, OCH2, CH2Fur), 3.50
(m, 2H, OCH2, CH2Fur), 3.21 (br s, 1H, NH), 1.26 and 0.80 (2td,
3J = 7.1 and 7.1, 4J = 0.4 and 0.5, 6H, CH3). 13C{1H} NMR (CDCl3), d
(ppm), JPC (Hz): 152.82 (FurC-2), 141.91 (FurC-5), 129.30 (AnthC-
absorption was measured on
540 nm.
a TECAN microplate reader at
The statistical analysis included application of One-way ANOVA
followed by Bonferroni’s post hoc test. p <0.05 was accepted as the
lowest level of statistical significance.
a
), 128.83 (d, 5J = 4.5, AnthC-10), 128.79 (AnthC-
hC- ), 126.09 (AnthC-b), 125.40 (AnthC-b), 125.20 (AnthC-b),
124.56 (AnthC-b), 123.59 (AnthC-
a), 127.48 (Ant-
a
a), 109.95 (FurC-4), 107.96
4.2.3. Cytogenetical method
(FurC-3), 62.69 and 62.64 (2d, 2J = 7.4 and 7.6, OCH2), 54.80 (d,
1J = 158.4, CHP), 44.27 (d, 3J = 15.9, FurCH2), 16.38 and 15.91 (2d,
3J = 6.1 and 5.7, CH3). 31P{1H} NMR (CDCl3), d (ppm): 25.80.
The cytogenetical investigation was conducted as described by
Preston et al.34 Male and female ICR mice, weighing 20 1.5 g were
kept at standard conditions ꢁ20 °C, 12 h light/dark cycle; food and
water were available ad libitum. All the compounds investigated
were administered ip at doses of 10 and 100 mg/kg. Mitomycin C
(Kyowa) 3.5 mg/kg was used as a positive control substance. Group
of animals injected with 0.9% saline were used as a negative control.
Bone marrow chromosome aberration assay was performed on
seven groups of animals. The experimental groups consisted of four
males and four females treated with the studied compounds and
control groups consisted of 10 animals each. The experimental
and control groups of animals were injected ip with colchicine at
a dose of 0.4 mg/kg, 24 and 48 h after the administration of the ap-
plied chemicals and 1 h prior isolation of the bone marrow cells. All
mice were euthanized by deep anaesthesia with diethyl ether.
Bone marrow cells were flushed from the femur and hypotonized
in 0.075 M KCl at 37 °C for 20 min. Thereafter the cells were fixed
in methanol–acetic acid (3:1), dropped on cold slides, air dried and
stained with 5% Giemsa solution (Sigma Diagnostic). At least 50
well-spread metaphases were analyzed per experimental animal
at random. Mitotic indices were determined by counting the num-
ber of dividing cells among 1500 cells per animal. The frequencies
of abnormalities and the mitotic index were determined for each
animal and then the mean standard error of mean for each group
was calculated. Student’s t-test was applied for statistical analysis.
Statistical significance was expressed as ⁄⁄⁄ p <0.001; ⁄⁄ p <0.01;
⁄ p <0.05; p >0.05—not significant).
4.2. Biological assays
4.2.1. In vitro antitumor activity
The antitumor activity testing was performed on cell cultures
from several human cancer cell lines using the standard MTT-dye
reduction assay, described by Mosmann.30 Cell lines from ductal
carcinoma of the breast (MCF-7 and MDA-MB-231—with low and
high metastatic potential, respectively), HBL-100 line (colostrum-
derived myoepithelial cells, expressing polyoma virus large T-anti-
gen), bladder carcinoma (647-V), hepatocellular carcinoma
(HepG2), colon carcinoma (HT-29) and the CL HeLa—cervical carci-
noma were used in all experiments. The cell lines were routinely
grown as monolayers in 75 cm2 tissue culture flasks (Orange Scien-
tific), in either RPMI 1640 medium (ELTA 90, Ltd) (MCF-7 and HBL-
100 lines) or high-glucose (4.5‰) Dulbecco’s modified minimal
essential medium (DMEM) (ELTA 90, Ltd), supplemented in both
cases with 10% fetal calf serum (Sigma) and antibiotics in usual
concentrations. Cultures were maintained at 37.5 °C in a humidi-
fied atmosphere and 5% CO2. Cells were plated at a density of
1 ꢀ 104 cells in 100 ml culture medium in each well of 96-well
flat-bottomed microplates and allowed to adhere for 24 h before
treatment with test compounds in DMSO solution, further diluted
in phosphate-buffered saline (PBS) to reach the desired test con-
centrations. A concentration range from 1 to 0.0681 mg/ml (dilu-
p
tion factor of 6 10 = 1.47) was applied for 24 h. The DMSO
4.2.4. Fluorescence studies
concentration never exceeded 1% (v/v). The referent antineoplastic
drug Doxorubicin hydrochloride (Lemery) was used as a commer-
cially available sterile dosage form for clinical application and used
after the appropriate dilution in phosphate-buffered saline. All
experiments were performed in triplicate. The MTT-formazan
absorption was registered using a microplate reader (TECAN, Sun-
rise TM, Groedig/Salzburg, Austria) at 580 nm. Cytotoxic activities
were expressed as IC50 values (concentrations required for 50%
inhibition of cell growth), calculated using non-linear regression
analysis (GraphPad Prizm5 Software). There was a good reproduc-
ibility between replicate wells with standard errors below 10%.
Student’s t-test was applied and value of p <0.05 was accepted as
the lowest level of statistical significance.
HBL-100 cells (1 ꢀ 105/ml) were seeded on sterile 12 ring diag-
nostic slides (Thermo Scientific), allowed to adhere overnight at
37.5 °C in a humidified atmosphere under 5% CO2 and treated with
non-toxic concentrations of the Schiff base 1 and the amin-
ophosphonate 4 for 24 h. After fixation in cold (ꢁ20 °C) acetone
the slides were air-dried, covered and examined with Leica DM
5000 B (Wetzlar, Germany) fluorescent microscope, equipped with
a digital camera and the appropriate Leica Software.
Acknowledgement
Thanks are due to Bulgarian National Science Fund of Ministry
of Education and Science for the financial support: contract DTK-
02/34(2009).
4.2.2. Cytotoxicity testing (3T3 NRU test)
The cytotoxicity testing was performed as described by Boren-
freund and Puerner31 and the latest modification32 of the validated
Balb/c 3T3 (clone 31) Neutral Red Uptake Assay (3T3 NRU test)33
for cytotoxicity/phototoxicity testing. BALB/c 3T3, clone 31 mouse
embryo cells were grown as monolayers in 75 cm2 tissue culture
flasks in low-glucose (1‰) DMEM (ELTA 90, Ltd), supplemented
with 10% fetal bovine serum and antibiotics. Cultures were main-
tained at 37.5 °C in a humidified atmosphere under 5% CO2. Cells
were plated at a density of 1 ꢀ 104 cells in 100 ml culture medium
in each well of 96-well flat-bottomed microplates and allowed to
adhere for 24 h before treatment with test compounds, dissolved
in DMSO (ELTA 90, Ltd) and further diluted in PBS. A wide concen-
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