Dimethyl (2-Furyl)-N-(2-Methoxyphenyl)Aminomethylphosphonate Induces Apoptosis in Esophageal Squamous Cancer Cells. Structure Versus Activity of its Selected Analogs

Article abstract of DOI:10.1080/10426507.2014.965821

Cytotoxicities of several new aminophosphonic compounds bearing 2-furyl moiety 2b-d and 3a-c on KYSE 30, 150, and 270 esophageal cancer cell lines are reported here. The qualitative study on correlations between the structure and cytotoxic activity of fur

Full text of DOI:10.1080/10426507.2014.965821

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Dimethyl (2-Furyl)-N-(2-
Methoxyphenyl)Aminomethylphosphonate
Induces Apoptosis in Esophageal Squamous
Cancer Cells. Structure Versus Activity of its
Selected Analogs
Anna Agnieszka Klimczak, Agnieszka Matusiak, Jarosław Lewkowski, Jan
Bitner, Janusz Szemraj & Renata Kontek
To cite this article: Anna Agnieszka Klimczak, Agnieszka Matusiak, Jarosław
Lewkowski, Jan Bitner, Janusz Szemraj & Renata Kontek (2015) Dimethyl (2-Furyl)-N-(2-
Methoxyphenyl)Aminomethylphosphonate Induces Apoptosis in Esophageal Squamous
Cancer Cells. Structure Versus Activity of its Selected Analogs, Phosphorus, Sulfur, and Silicon
and the Related Elements, 190:7, 1088-1099, DOI: 10.1080/10426507.2014.965821
To link to this article: http://dx.doi.org/10.1080/10426507.2014.965821
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Date: 07 October 2015, At: 01:35

Phosphorus, Sulfur, and Silicon, 190:1088–1099, 2015
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2014.965821
DIMETHYL (2-FURYL)-N-(2-METHOXYPHENYL)
AMINOMETHYLPHOSPHONATE INDUCES APOPTOSIS IN
ESOPHAGEAL SQUAMOUS CANCER CELLS. STRUCTURE
VERSUS ACTIVITY OF ITS SELECTED ANALOGS
Anna Agnieszka Klimczak,1 Agnieszka Matusiak,2
Jarosław Lewkowski,2 Jan Bitner,3 Janusz Szemraj,1
and Renata Kontek4
1Department of Medicinal Biochemistry, Medical University of Ło´dz´, Mazowiecka,
Ło´dz´, Poland
2Department of Organic Chemistry, Faculty of Chemistry, University of Ło´dz´,
Tamka, Ło´dz´, Poland
3Independent Researcher
4Laboratory of Cytogenetics, Department of General Genetics, Molecular Biology
and Biotechnology, Faculty of Biology and Environmental Protection, University of
Ło´dz´, Banacha, Ło´dz´, Poland
GRAPHICAL ABSTRACT
Abstract Cytotoxicities of several new aminophosphonic compounds bearing 2-furyl moiety
2b-d and 3a-c on KYSE 30, 150, and 270 esophageal cancer cell lines are reported here. The
qualitative study on correlations between the structure and cytotoxic activity of furan-deriving
aminophosphonates has been performed.
Dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphosphonate (2a), which has been previ-
ously shown to have the interesting values of cytotoxicity on these esophageal cancer cell lines
was tested and found to induce apoptosis. The analyses of correlations between apoptosis,
necrosis, and viability were also performed.
Keywords Aminophosphonates; esophageal squamous cancer cells (ESCC); apoptosis; cyto-
toxicity; viability
Received 24 July 2014; accepted 2 September 2014.
Address correspondence to Professor Jaroslaw Lewkowski, Department of Organic Chemistry, Faculty of
Chemistry, University of Ło´dz´, Tamka 12, 91-403 Ło´dz´, Poland. E-mail: jlewkow@uni.lodz.pl or A. A. Klimczak,
Department of Medicinal Biochemistry, Medical University of Ło´dz´, Mazowiecka 6/8, 92-215 Ło´dz´, Poland. E-
mail: a.a.klimczak@gmail.com
1088

DIMETHYL (2-FURYL)-N-(2-METHOXYPHENYL)AMINOMETHYLPHOSPHONATE 1089
INTRODUCTION
Substituted furan derivatives are an important group of compounds for pharmaceu-
tical science1,2 with their most distinct example being an efficient antihistamine agent
– ranitidine.3 The antibacterial nifuroxazide,4 belonging to nitrofurfural-derived drugs is
still on use despite a rich variety of antibiotics, and originating from the same group ni-
furtimox5 is a valuable drug against Trypanosoma cruzii and is tested to to be applied
on other Trypanosoma protozoa.5–7 Some examples showing strong antifungal action of
2,5-diphenylfuran derivatives on Pneumocystis carinii were also reported.8
The acquisition of phosphonic analogues of natural amino acids9–11 prompted
scientists to perform biochemical studies, which confirmed that aminophosphonic and
aminophosphonous acids belong to the group of biologically active compounds.12
Aminophosphonic derivatives are therefore interesting for scientists in various fields
of biochemistry,13 pharmacology,14 and agriculture.15 They show interesting impact on
higher plants,16,17 they also show a high cytotoxicity toward some cancers .13 Recently, the
synthesis of several furan moiety-bearing aminophosphonates and their strong action on
leukemia18 and squamous esophageal cancer19 cell lines were reported.
Esophageal cancer occurs in two main subtypes, which are esophageal squamous
cell cancer (ESCC) and adenocarcinoma. These cellular types of cancer determine the
success in its treatment, but the statistical data are not optimistic.20 It affects more than
450,000 people globally each year with 380,000 deaths each year and the prognosis for
esophageal cancer is poor and survival is low, with 5-year survival rates usually less than
10%.21
Although, methods for suppressing esophageal cancer using the inactivation of certain
genes22 or inducing autophagy23 are described in literature, chemotherapy is one of the
most common approach and is adjuvant to surgery or, in some cases, it becomes almost
the only approach. The most common regimen, used for advanced esophagal cancer is the
combination of epirubicin, cisplatin, and fluorouracil.24 The most recent study demonstrated
encouraging antitumour activity for capecitabine combined with epirubicin and cisplatin.25
Nevertheless, all regimens giving clinically positive results are based on cisplatin, therefore
works on this simple molecule are still being developed.22
Due to its numerous adverse effects, ciplatin cannot be safely used in each case
and sometimes must be replaced by oxaliplatin or carboplatin, which not always provide
good therapeutic effect. Docetaxel was reported to have the positive therapeutic action on
ESCC,26 but it is still the area of approved anticancer drugs. The task of the high importance
is to look for new molecules of a potential chance to become efficient chemotherapeutic
agents.
For these reasons, we have reached for N-arylamino(2-furyl) methylphosphonates,
which demonstrated various degrees of cytotoxicity on human KYSE 30, 150, and 270
esophageal cancer cell lines (ESCC).19 In the present paper, we report cytotoxicities of new
aminophosphonates 2b-d and aminophosphonic acids 3a-c toward these cells and, on the
basis of these and previous19 results, we present preliminary conclusions on the structure
versus activity relationship of this group of compounds.
As dimethyl (2-furyl)-N-(2-methoxyphenyl)-aminomethylphosphonate (2a) demon-
strated the promising cytotoxic properties,19 we performed investigations in order to verify,
if the compound 2a is able to induce apoptosis of the studied cells.

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A. A. KLIMCZAK ET AL.
Scheme 1 Synthesis of studied aminophosphonates 2a-d and aminophosphonic acids 3a-c.
RESULTS AND DISCUSSION
Chemistry
This work is a continuation of our previous study, where we reported the synthesis
of new aminophosphonates bearing a furan moiety.19
Schiff bases 1a-e were synthesized following the previously published19,27,28 proce-
dures and their identity was confirmed by melting point measurement and/or the 1H NMR
spectroscopy and comparing the obtained data with literature reports.17,19,20,29–32
(2-Furyl)-N-arylaminomethylphosphonic acid esters (2b-d) were prepared based on
the previously published procedures,19,27,28 from a Schiff base 1b-c by the aza-Pudovik
reaction. Reactions of Schiff bases 1b-c with phosphites were carried out in acetonitrile with
a catalytic amount (two drops) of trifluoroacetic acid. (Scheme 1) The aminophosphonates
2b-c were synthesized for the first time and have never been described in literature. The
aminophosphonate 2d was obtained once33 on the route of the Kabachnik–Fields reaction,
while we synthesized it via the aza-Pudovik reaction.
The aminophosphonate 2a was obtained following the reported procedure19 and its
identity was confirmed by comparing with the authentic sample.
The identity of the aminophosphonate 2d was confirmed by 1H and 31P NMR spec-
troscopy and obtained data were compared with literature.19,33 The identity of newly syn-
thesized aminophosphonates 2b-c was confirmed by elemental analysis as well as by 1H,
1
13C, and 31P NMR spectroscopy. H, 13C, and 31P NMR spectra demonstrated diagnostic
peaks, which were discussed previously.19

DIMETHYL (2-FURYL)-N-(2-METHOXYPHENYL)AMINOMETHYLPHOSPHONATE 1091
Table 1 The fractions of living cells after 24 h incubation with aminophosphonates 2b-d and aminophosphonic
acids 3a-c
Aminophosphonic derivative
Cancer cell line
2b
2c
2d
3a
3b
3c
IC50 [μM]
>400
>400
KYSE 30
KYSE 150
KYSE 270
225.6 14.8
216 11.4
319. 19.3
367.3 20.5
320.4 21.2
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
Amino(2-furyl)methylphosphonic acids 3a-c were synthesized by the addition of
bis-(trimethylsilyl) phosphite to the azomethine bond of corresponding Schiff bases 1c-e
in dichloromethane (Scheme 1) by the method recently described by us.17
Their identity has been verified by means of the 1H, 13C, and 31P NMR spectroscopy
and the melting point measurements, which data were compared to authentic samples.17 The
1H and 13C NMR spectra of acids 3a-c showed diagnostic signals typical for aminophos-
phonic acids, such as a doublet of a HCP group or a doublet at around 50 ppm having
a coupling constant oscillating around 150 Hz corresponding to a CP bond, which was
described in details in the above mentioned paper.17 Spectra of all new compounds (1H,
13C, and 31P NMR) as well as of already known compounds (1H and 31P NMR) are collected
in Supplemental Materials (see Supplemental Materials Figures S1–S20).
Biological Activity
Biological activity of compounds 2a-d and 3a-c was determined by in vitro exper-
iments with three esophageal squamous cancer cell lines: KYSE 30,150, and 270. These
cell lines are described in literature.34–37
Cancer cell lines were cultivated without using antibiotic and/or antimycotic agents.
According to Freshney,38 routine use of antibiotics for cell culture is not recommended,
because they can mask contamination and, moreover, they can interfere with the metabolism
of sensitive cells.
Cytotoxicity MTT Assay of Aminophosphonic Derivatives 2a-b and 3a-c
In the MTT40,41 study of aminophosphonates 2b-d, aminophosphonic acids 3a-c,
400 μM was taken as a maximum concentration. Each experiment repeat was performed
on a different day. Each assay included a control with medium without chemicals and an
additional control of medium with 1% DMSO to test the solvent. Each chemical compound
had eight different concentrations in the 96-well plate with three repeats. There were at least
three repeats of each experiment and IC50 were calculated based on the control without any
chemical compound added (Table 1).
Experiments have shown that none of the three analyzed aminophosphonic acids:
(2-furyl)-N-(4-methylphenyl)aminomethylphosphonic acid (3a), (2-furyl)-N-(4-methoxy-
phenyl)-aminomethylphosphonic acid (3b) and (2-furyl)-N-benzhydrylaminomethylphos-
phonic acid (3c) had IC50 values higher than 400 μM (the highest concentration checked)
for the three cancer cell lines. Considering that acids with various N-substituents were used,

1092
A. A. KLIMCZAK ET AL.
Table 2 IC50 values of compounds for structure versus activity analysis
IC50 (μM)
Compound
KYSE 30
KYSE 150
KYSE 270
2a
4
2b
5
6
7
38.02 2.39
316.23 21.03
225.6 14.8
>400
251.19 15.41
380 21.35
>400
51.29 6.97
166 17.28
216 11.4
208.93 17.33
>400
77.43 3.68
223.87 15.51
319 19.3
204.84 8.90
>400
>400
>400
>400
>400
8
9
2c
2d
251.19 22.09
367.3 20.5
>400
199.52 16.85
320.4 21.2
>400
251.19 12.94
>400
>400
this suggests clearly that (2-furyl)aminomethylphosphonic acids are cytologically inactive
for ESCC (Table 1).
The similar results were obtained for diethyl (2-furyl)-N-(4-methoxyphenyl)amino-
methyl-phosphonate (2d) and for diethyl (2-furyl)-N-(3-methoxyphenyl)-aminomethylpho-
sphonate (2c). These aminophosphonates were found to be noncytotoxic for KYSE 30, 150,
and 270, because the IC50 values were either over 400 μM or much over 300 μM.
Dimethyl (2-furyl)-N-(3-methoxyphenyl)aminomethylphosphonate (2b) was found
to give its IC50 not much above 200 μM for KYSE 30 and 150, whereas for KYSE 270
results were slightly over 300 μM.
These supplementary data together with values reported in the previous paper19
allowed us to draw some conclusions concerning the structure versus cytotoxicological ac-
tivity relationship of dialkyl (2-furyl)-N-(methoxy- and methylphenyl)aminomethylphos-
phonates. For the reason of clarity, IC50 values for all compounds being compared are
collected in Table 2. The obtained results demonstrate clearly that diethyl (2-furyl)-N-
(methoxyphenyl)aminomethyl-phosphonates 2c-d independingly on the phenyl ring sub-
stitution are not cytotoxic for KYSE 30, 150, and 270 ESCC lines. Similar conclusions are
drawn for diphenyl (2-furyl)-N-(methylphenyl)aminomethylphosphonates 6-8, which are
found not to be cytoxic for the discussed cell lines. Therefore, it is to conclude that diphenyl
(2-furyl)amino-methylphosphonates cannot be considered as good candidates for potential
anticancer agents.
Much more promising results are observed for the dimethyl (2-furyl)-N-(methoxy-
phenyl)-aminomethylphosphonate series, which includes dimethyl (2-furyl)-N-(2-meth-
oxyphenyl)-aminomethylphosphonate (2a) with its IC50 much lower than 100 μM and
for KYSE 30 less than 40 μM. Although both other aminophosphonates demonstrated
much weaker cytotoxicity toward KYSE 30, 150, and 270 ESCC, these results show clearly
that dimethyl (2-furyl)-N-(methoxyphenyl)aminomethylphosphonates 2a-b, 4 are cytotoxic

DIMETHYL (2-FURYL)-N-(2-METHOXYPHENYL)AMINOMETHYLPHOSPHONATE 1093
Figure 1 Apoptosis after 2a treatment of KYSE 30, 150, and 270. Cells were incubated with different
concentrations of dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphosphonate (2a) for 24 h (mean SD;
n ≥ 3).
and that other dimethyl (2-furyl)-aminomethylphosphonates should show the same or better
cytotoxicity.
Although dibenzyl (2-furyl)-N-(3-methoxyphenyl)aminomethylphosphonate (9)
showed moderate cytotoxicity, the results are of interest, as the dibenzyl phosphonic
moiety seems to demonstrate its cytotoxicological activity and to be a potential target
for studies. Therefore, dimethyl and dibenzyl aminophosphonates bearing the furfuryl
moiety are good candidates to be investigated on other ESCC or even on esophageal
adenocarcinoma cells.
Cytotoxicity, Viability, and Apoptosis Assay of Dimethyl (2-furyl)-N-
(2-methoxyphenyl)aminomethylphosphonate (2a)
Cytotoxicity assay includes measurement of necrosis (numbers of dead cells) as well
as viability (numbers of live cells). To analyze viability of the cells and apoptotic activity
for dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphosphonate (2a), a commercial
kit for bioluminescence assays was used.
Because cytotoxicity of dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphos-
phonate (2a) has already been determined,19 to perform these assays three different concen-
trations of 2a (0.05, 0.1, and 0.15 μM) were selected basing on these data. Solutions were
prepared and added to wells, while fresh medium was added to control. After the incubation
the final measurements were done following the protocol manual.39 All readings for this
assays were done on a luminometer. Fluorescence for viability and necrosis was checked
with different filters: 400EX/505EM (viability) and 485EX/520EM (necrotic cells). In the end,
the measurement of luminescence was done to check caspase 3/7 activation which is a
symptom of apoptosis, as luminescence is proportional to the amount of caspase activity
present.39
Apoptosis assay has shown that as concentrations of the compound 2a grew (0.05, 0.1,
and 0.15 μM), the luminescence in KYSE 30, 150, and 270 increased. This observation
indicated clearly that dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphosphonate
(2a) induced an apoptotic process (Figure 1).

1094
A. A. KLIMCZAK ET AL.
Figure 2 Viability after 2a treatment of KYSE 30, 150, and 270. Cells were incubated with different
concentrations of dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphosphonate (2a) for 24 h (mean SD;
n ≥ 3).
The necrotic cells assay on these three cancer cell lines showed that the fluorescence
was growing in higher concentration of 2a. Viability analysis was performed in the same
concentrations of 2a as for the apoptosis assay and fluorescence was found to decrease for
KYSE 30, 150, and 270 with increasing concentration (Figures 2 and 3). Thus, viability
and necrosis values are inversely proportional, which is characteristic for most cytotoxicity
processes. In order to facilitate the comparison, necrosis and viability data of 2a for an
each cell line has been placed in one graph, where this tendency is clearly visible (see
Supplemental Materials Figures S21–S23).
Apoptosis and viability determined for 2a on KYSE 30, 150, and 270 were found
to be inversely proportional, which is a typical relationship too. Apoptosis and viability
were compared by placing the determined values of both quantities in one graph (see
Supplemental Materials Figures S24–S26). All these procedures were suggested within the
Apo-Tox-Glo Triplex Assay (Promega) protocol manual.39
Figure 3 Necrosis after 2a treatment of KYSE 30, 150, and 270. Cells were incubated with different
concentrations of dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphosphonate (2a) for 24 h (mean SD;
n ≥ 3).

DIMETHYL (2-FURYL)-N-(2-METHOXYPHENYL)AMINOMETHYLPHOSPHONATE 1095
It was then proved that dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphos-
phonate (2a) induces apoptosis of KYSE 30, 150, and 270 ESCC and, in view of their
promising IC50 values,19 further analysis of this compound, e.g., cell cycle analysis will be
done in the future.
CONCLUSIONS
In summary, dimethyl (2-furyl)-N-(2-methoxyphenyl)-aminomethylphosphonate
(2a) induces apoptotic process in KYSE 30, 150, and 270 ESCC. The comparison of
cytotoxicity versus viability as well as apoptosis versus viability fluorescence values for 2a
demonstrated the inverse proportionality for both pairs of values. The MTT cytotoxicity
test for three furfural-derived aminophosphonic acids 3a-c demonstrated clearly that
these compounds are not cytotoxic for KYSE 30, 150, and 270 ESCC. Results of tested
cytotoxicity of three aminophosphonates bearing the furfuryl moiety (2b-d) allowed to
perform the preliminary, qualitative analysis to find relationships between the structure of
furfural-derived N-aryl-substituted aminophosphonates and their cytotoxic activity. Results
of this analysis prompted us to conclude that the most potential compounds from among
this group are dibenzyl and dimethyl aminophosphonates and they will be tested on other
ESCC and even on other various cancer types. It is also to add that diphenyl and, especially
diethyl (2-furyl)aminomethylphosphonates showed no cytotoxic action on the KYSE 30,
150, and 270 ESCC. It is the evidence for the thesis that each type of cancer requires
individual attitude as diethyl (2-furyl)-N-(4-methylphenyl)-aminomethylphosphonate was
found18 to have the important cytotoxic action on chronic myeloid leukemia K-562 cells
and was satisfactory for other leukemia cells (LAMA-84 and HL-60).
EXPERIMENTAL
General
All solvents (POCh, Poland) were routinely distilled and dried prior to use. Amines,
dimethyl, diphenyl and dibenzyl phosphites, and furfural (Aldrich) were used as received.
NMR spectra were recorded on a Bruker Avance III 600 MHz operating at 600 MHz (1H
NMR), 150 MHz (13C NMR), and 243 MHz (31P NMR). TMS was used as the internal
standard for 1H NMR and phosphoric acid was used as the external standard for 31P NMR.
Elemental analyses were carried out at the Centre for Molecular and Macromolecular
Science of the Polish Academy of Science in Ło´dz´, Poland.
General Procedure for the Synthesis of Imines (1a-e)
Furfural (2.5 mmol, 0.24 g) was dissolved in methanol (15 mL) and to this solution,
aniline derivative 1a-e (2.5 mmol) was added. The mixture was stirred at room temperature
for 24 h, then solvent was evaporated to obtain almost pure Schiff base (1a-e).
1H NMR and melting point data of all the Schiff bases 1a-e are identical to those
previously described in the literature.17,19,20,29–32

1096
A. A. KLIMCZAK ET AL.
General Procedure for the Synthesis of (2-furyl)-Arylamino-
methylphosphonic Acid Esters (2a-d)
A Schiff base (1a-d) (4.5 mmol) was dissolved in acetonitrile and then phosphite
(4.5 mmol) was added followed by addition of two drops of trifluoroacetic acid. The
mixture was stirred at 80◦C during the day and at room temperature overnight. Reactions
lasted 72 h. Then, solvent was evaporated and crude products were chromatographed on
silica gel (AcOEt-hexane 4:1) to give pure aminophosphonates (2a-d).
1H and 31P NMR data of the aminophosphonate 2a19 are identical to those previously
described. Spectra are presented in Supporting Information.
Dimethyl (2-furyl)-N-(3-methoxyphenyl)aminomethylphosphonate 2b
1H NMR (CDCl3, 600 MHz): δ 7.40 (m, H5fur, 1H); 7.07 (t, J = 7.8 Hz, m-C6H4,
1H); 6.40 (d, J = 3.6 Hz, H3fur, 1H); 6.45 (m, m-C6H4, 2H); 6.27 (d, J = 7.8 Hz, m-C6H4,
1H); 6.23 (dd, J = 3.6 and 1.8 Hz, H3fur, 1H); 4.94 (dd, 2JPH = 23.4 Hz, 3JHH = 7.8 Hz,
CHP, 1H); 4.54 (d, J = 7.8 Hz, NH, 1H); 3.82 (d, 3JPH = 10.8 Hz, POCH3, 3H); 3.74 (s,
OCH3, 3H); 3.64 (d, 3JPH = 10.8 Hz, POCH3, 3H). 13C NMR (CDCl3, 150 MHz): δ 160.77
(Cipso); 149.07 (Cipso); 147.32 (d, 2JCP = 13.5 Hz, C2fur); 142.66 (d, 3JCP = 3.0 Hz, C3fur);
130.03 (Car); 110.84 (C5fur); 108.93 (d, 3JCP = 6.0 Hz, C4fur); 106.87 (Car); 104.39 (Car);
2
2
100.31 (Car); 55.09 (OCH3); 54.00 (d, JCP = 6.0 Hz, POC); 53.69 (d, JCP = 6.0 Hz,
POC); 49.92 (d, 1JCP = 156.0 Hz, POC). 31P NMR (CDCl3, 243 MHz): δ 22.46.
Elemental analysis: Calctd for C14H18NO5P: C, 54.02, H, 5.83, N, 4.50. Found: C,
53.79, H, 5.80, N, 4.64.
Diethyl (2-furyl)-N-(3-methoxyphenyl)aminomethylphosphonate 2c
3
4
fur
1H NMR (CDCl3, 600 MHz): δ 7.38 (dd, JHH = 1.8 Hz and JHH = 0.6 Hz, H5
,
1H); 7.05 (t, J = 7.8 Hz, m-C6H4, 1H); 6.39–6.37 (m, m-C6H4, 1H); 6.33–6.30 (m, m-C6H4,
H4fur, 3H); 6.28 (dd, 3JHH = 7.8 Hz and 4JHH = 1.8 Hz, m-C6H4, 1H); 6.22-6.21 (m, H3
,
fur
1H); 4.87 (dd, 2JPH = 24.0 Hz, 3JHH = 9.0 Hz, CHP, 1H); 4.53 (dd, J = 9.0 and 7.2 Hz,
NH, 1H); 4.21–4.13 (m, POCH2CH3, 2H); 4.09-4.03 (m, POCH2CH3, 1H); 3.91–3.84 (m,
3
POCH2CH3, 1H); 3.74 (s, OCH3, 3H); 1.34 and 1.24 (2t, JHH = 7.2 Hz, POCH2CH3,
2×3H). 13C NMR (CDCl3, 150 MHz): δ 160.75 (Cipso); 149.47 (d, 5JCP = 1.5 Hz, Cipso);
147.55 (d, 2JCP = 13.5 Hz, C2fur); 142.45 (d, 3JCP = 3.0 Hz, C3fur); 129.96 (Car); 110.78 (d,
5JCP = 1.5 Hz, C5fur); 108.73 (d, 3JCP = 7.5 Hz, C4fur); 106.86 (Car); 104.22 (Car); 100.14
(Car); 63.49 (d, 2JCP = 6.0 Hz, POC); 63.31 (d, 2JCP = 6.0 Hz, POC); 55.07 (OCH3); 50.33
1
3
3
(d, JCP = 159.0 Hz, POC); 16.42 (d, JCP = 4.5 Hz, POCC); 16.27 (d, JCP = 5.0 Hz,
POCC). 31P NMR (CDCl3, 243 MHz): δ 20.06.
Elemental analysis: Calctd for C16H22NO5P: C, 56.63, H, 6.53, N, 4.13. Found: C,
56.45, H, 6.48, N, 4.19.
Diethyl (2-furyl)-N-(4-methoxyphenyl)aminomethylphosphonate 2d33
1H NMR (CDCl3, 600 MHz): δ 7.38 (dd, 3JHH = 1.8 Hz and 4JHH = 0.6 Hz, H5
,
fur
3
4
1H); 6.74 and 6.62 (AA’XX’ system, JHH = 9.0 and JHH = 2.4 Hz, p-C6H4, 4H); 6.34
(d, J = 3.6 Hz, H3fur, 1H); 6.30 (dd, 3JHH = 1.8 and 3.6 Hz, H4fur, 1H); 4.80 (dd, 2JPH
=
3
23.4 Hz and JHH = 5.4 Hz, CHP, 1H); 4.25 (m, NH, 1H); 4.187 (m, POCH2CH3, 2H);

DIMETHYL (2-FURYL)-N-(2-METHOXYPHENYL)AMINOMETHYLPHOSPHONATE 1097
4.06 (m, POCH2CH3, 1H); 3.88 (m, POCH2CH3, 1H); 3.71 (s, OCH3, 3H); 1.31 and 1.21
(2t, 3JHH = 7.2 Hz, POCH2CH3, 2×3H). 31P NMR (243 MHz, CDCl3): δ 20.34.
General Procedure for Preparation of Aminophosphonic Acids (3a-c)
Furfural (2.5 mmol, 0.24 g) was dissolved in methanol (15 mL) and to this solution,
amine (2.5 mmol) was added. The mixture was stirred at room temperature for 24 h,
and obtained imine was used for further conversion without being isolated. To confirm
completion of the reaction, 1-mL sample was taken, evaproated and dissolved in CDCl3
and 1H NMR spectrum was recorded.
Diethyl phosphite (2.5 mmol) was dissolved in dry dichloromethane (10 mL) and to
this solution bromotrimethylsilane (6.8 mmol, 0.9 mL) was added dropwise for 10–15 min.
The mixture was stirred for 1 h at room temperature, a solution of an appropriate imine
(2.5 mmol) in dry dichloromethane (10 mL) was added and the mixture was refluxed for
4 h. Then, the reaction mixture was concentrated under reduced pressure and the residue
was dissolved in dry methanol and it was stirred for 30–45 min until precipitation of a solid,
which was filtered off and collected. In a case, if the solid did not precipitated, 10–20 mL
of propylene oxide was added and the mixture was refrigerated for 3–7 days and the solid
precipitate was collected by filtration.
1H, 13C, and 31P NMR and melting point data of aminophosphonic acids 3a-c are
identical to those previously described.17 1H and 31P NMR spectra are presented in Supple-
mentary Material.
Cytotoxic Activity
Materials. All chemicals and media for growing cell lines were ordered from
Sigma Aldrich. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and
dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich. To analyze viability,
necrosis, and apoptosis Apo-Tox-Glo Triplex Assay from Promega was used. This com-
mercial kit was chosen because it has the following benefits: all measurements are done in
the same well, it is easy to implement and it normalizes data by a built-in control, which
improves efficiency. The final concentration of DMSO, which was used to dilute tested
compounds was lower than 1% in media (chemicals were dissolved at first in DMSO, then
in growing media to achieve the final concentrations of < 1%). Fresh compounds were
weighed and diluted before each experiment.
Cell Line and Culture
The ESCC KYSE 30, 150, and 270 were ordered from the European Collection of
Cell Cultures (ECACC, UK). Cells were grown in a medium which included RPMI 1640
and Ham F12 in 1:1 ratio with 2 mM L-glutamine and 2% fetal bovine serum (FBS).
Standard conditions were used to culture cell lines (37◦C, v/v 5% CO2, relative humidity
100%) without antibiotics.
MTT Assay
The well-known MTT test was used to measure cytotoxicity.38,39 First, 100 μL of
cell solution (10 × 103 cells per mL) was added to each well of the 96-well plate. Wells on

1098
A. A. KLIMCZAK ET AL.
the edge of the plate were filled with 100 μL of phosphate-buffered saline (PBS) to reduce
evaporation. The plate was incubated in standard conditions for cell line incubation. After
24 h, the cells were incubated with eight different chemical concentrations (dissolved in
DMSO, the final concentrations of which was <1%). Then, 20 μL of 0.5 mg/mL MTT
solutions were added to wells. After 30 min in standard incubation conditions, the medium
with MTT was removed, and 100 μL of pure DMSO was added and then incubated for
20 min in RT. Then absorbance was recerded with 580 nm light on a GloMax Microplate
Reader (Promega). There were at least three repeats of each measurement on different days.
A control without chemicals was used to calculate IC50. To test the solvent and rule out
its possible toxicity or other influence on the cells, an additional control of medium with
1% DMSO was created for each experiment. Data was expressed as a percentage of the
controls.
Statistical Analysis
IC50 values were used to analyze cytotoxicity. Results were representative of at least
three independent experiments, each involving triplicate determinations. Excel 2010 soft-
ware and the IG50 in the “Origin” PC-program were used to calculate statistical deviations.
Supplemental Materials
Supplemental data for this article (NMR spectra of synthesized compounds and
graphs of viability/necrosis/apoptosis relationships) can be accessed on the publisher’s
website, www.tandfonline.com/gpss
REFERENCES
1. Ruppel, J. V.; Snyder, N. L.; Thompson, A. D.; Farnsworth T. W. In: J. J. Li (Ed.), Heterocyclic
Chemistry in Drug Discovery; Wiley: Hobocken, New Jersey, 2013; pp. 126-157.
2. Keay, B. A.; Dibble P. W. In: A. R. Katritzky, C. W. Rees, E. F. V. Scriven (Eds.), Comprehensive
Heterocyclic Chemistry 2, Vol. 2; Pergamon Press: Oxford, 1996; pp. 395-436.
3. Price, B. J.; Clitherow, J. W.; Bradshaw, J. US Patent 4128658, 1978.
4. Carron, M. C. E. US Patent 3290213, 1966.
5. Bisser, S.; N’Siesi, F.-X.; Lejon, V.; Preux, P.-M.; van Nieuwenhove, S.; Bilenge, C. M. M.;
Buscher, P. J. Infect. Dis. 2007, 195, 322-329.
6. Priotto, G.; Kasparian, S.; Mutombo, W.; Ngouama, D.; Ghorashian, S.; Arnold, U.; Ghabri, S.;
Baudin, E.; Buard, V.; Kazadi-Kyanza, S.; Ilunga, M.; Mutangala, W.; Pohlig, G.; Schmid, C.;
Karunakara, U.; Torreele, E.; Kande, V. Lancet 2009, 374, 56-64.
7. Zhou, L.; Stewart, G.; Rideau, E.; Westwood, N. J.; Smith, T. K. J. Med. Chem. 2013, 56,
796-806.
8. Boykin, D. W.; Kumar, A.; Xiao, G.; Wilson, W. D.; Bender, B. C.; Curdy, D. R.; Hall, J. E.;
Tidwell, R. R. J. Med. Chem. 1998, 41, 124-129.
9. Saito, J. B.; Egami, H.; Katsuki, T. J. Am. Chem. Soc. 2007, 129, 1978-1986.
10. Matveeva, E. D.; Podrugina, T. A.; Prisyajnoy, M. V.; Zefirov, N. S. Russ. Chem. Bull. Int. Ed.
2006, 55, 1209-1214.
11. Lewkowski, J.; Karpowicz, R. Heteroatom Chem. 2010, 21, 326-331.
12. Mastalerz, P.; Kafarski, P. In: V. P. Kukhar; H. R. Hudson (Eds.), Aminophosphonic and
Aminophosphinic Acids; John Wiley & Sons: New York, 2000; pp. 1-31.
13. Kafarski, P.; Lejczak, B. Curr. Med. Chem. Anti-Cancer Agents. 2001, 1, 301-312.

DIMETHYL (2-FURYL)-N-(2-METHOXYPHENYL)AMINOMETHYLPHOSPHONATE 1099
14. Kafarski, P.; Lejczak, B. Phosphorus Sulfur Silicon Relat. Elem. 1991, 63, 193-215.
15. Maier, L. Phosphorus Sulfur Silicon Relat. Elem. 1999, 144–146, 429-132.
16. Matusiak, A.; Lewkowski, J.; Rychter, P.; Biczak, R. J. Agric. Food Chem. 2013, 61, 7673-7678.
17. Forlani, G.; Lejczak, B.; Kafarski, P.; Wieczorek, P. J. Plant Growth Regul. 1997, 16, 147-152.
18. Kraicheva, I.; Bogomilova, A.; Tsacheva, I.; Momekov, G.; Troev, K. Eur. J. Med. Chem. 2009,
44, 3363-3367.
19. Klimczak, A. A.; Kuropatwa, A.; Lewkowski, J.; Szemraj, J. Med. Chem. Res. 2013, 22, 852-860.
20. Jemal, A.; Murray, T.; Ward, E.; Samuels, A.; Tiwari, R. C.; Ghafoor, A.; Feuer, E. J.; Thun, M.
J. CA Cancer J. Clin. 2005, 55, 10-30.
21. Boyle P.; Levin B. (Eds.), World Cancer Report; International Agency For Research on Cancer:
Lyon, 2008; pp. 338-343.
22. Xing, E. P.; Nie, Y.; Song, Y.; Yang, G.-Y.; Cai, Y. C.; Wang, L.-D.; Yang, C. S. Clin. Cancer
Res. 1999, 5, 2704-2713.
23. Liu, D.; Yang, Y.; Liu, Q.; Wang, J. Med. Oncol. 2011, 28, 105-111.
24. Ross, P.; Nicolson, M.; Cunningham, D.; Valle, J.; Seymour, M.; Harper, P.; Price, T.; Anderson,
H.; Iveson, T.; Hickish, T.; Lofts, F.; Norman, A. J. Clin. Oncol. 2002, 20, 1996-2004.
25. Rao, S.; Starling, N.; Cunningham, D.; Sumpter, K.; Gilligan, D.; Ruhstaller, T.; Valladares-
Ayerbes, M.; Wilke, H.; Archer, C.; Kurek, R.; Beadman, C.; Oates, J. Ann. Oncol. 2010, 21,
2213-2219.
26. Tanaka, E.; Hashimoto, Y.; Ito, T.; Kondo, K.; Higashiyama, M.; Tsunoda, S.; Ortiz, C.; Sakai,
Y.; Inazawa, J.; Shimada, Y. Clin. Cancer Res. 2007, 13, 1331-1340.
27. Cottier, L.; Descotes, G.; Gonera, G.; Grabowski, G.; Lewkowski, J.; Skowron´ski, R. Phosphorus
Sulfur Silicon Relat. Elem. 1996, 118, 181-188.
28. Lewkowski, J.; Rzez´niczak, M.; Skowron´ski, R. Org. Prep. Proced. Int. 2000, 32, 453-460.
29. Saito, S.; Hatanaka, K.; Yamamoto, H. Tetrahedron 2001, 57, 875-887.
30. Kiepo, Z.; Jakopcic, K. J. Chem. Eng. Data. 1985, 30, 235-237.
31. Ojima, I.; Habus, I.; Zhao, M.; Zucco, M.; Park, Y. H.; Sun, C. M.; Brigaud, T. Tetrahedron 1992,
48, 6985-7012.
32. Brunner, B.; Stogaitis, N.; Lautens, M. Org. Lett. 2006, 8, 3473-3476.
33. Wu, J.; Sun, W.; Xia, H. G.; Sun, X. Org. Biomol. Chem. 2006, 4, 1663-1666.
34. Tanaka, H.; Shimada, Y.; Harada, H.; Shinoda, M.; Hatooka, S.; Imamura, M.; Ishizaki, K.
Cancer Res. 1998, 58, 3429-3434.
35. Hou, Z.; Sang, S.; You, H.; Lee, M.-J.; Hong, J.; Chin, K.-V.; Yang, C. S. Cancer Res. 2005, 65,
8049-8056.
36. Chiu, P. K. Characterisation of the Anti-Cancer Effect of Gleditsia Sinesis in Esophageal Squa-
mous Cell Carcinoma; The Hong Kong Polytechnic University: Hong Kong, 2008.
37. Jing, Z.; Gong, L.; Xie, C.-Y.; Zhang, L.; Su, H.-F.; Deng, X.; Wu, S.-X. Radiother. Oncol. 2009,
93, 468-473.
38. Freshney, R. I. Culture of Animal Cells: A Manual of Basic Technique, 5th Ed.; John Wiley &
Sons, Inc.: New York, 2005; pp. 123.
39. ApoTox-GloTMTriplex Assay, Instructions for Use of Products G6320 and G6321, Promega
Corporation: Madison, USA.
40. Mosmann, T. J. Immunol. Methods 1983, 65, 55-63.
41. Sieuwerts, A. M.; Klijn, J. G.; Peters, H. A.; Foekens, J. A. Eur. J. Clin. Chem. Clin. Biochem.
1995, 33, 813-823.