Synthesis of new N-arylamino(2-furyl)methylphosphonic acid diesters, and in vitro evaluation of their cytotoxicity against esophageal cancer cells

Article abstract of DOI:10.1007/s00044-012-0065-3

N-Furfurylideneanilines and N-arylamino(2-furyl)methylphosphonates with tolyl and anisyl moieties were synthesized by the addition of phosphites to azomethine bond of corresponding Schiff bases and their NMR spectroscopic properties were investigated. The

Full text of DOI:10.1007/s00044-012-0065-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:852–860
DOI 10.1007/s00044-012-0065-3
ORIGINAL RESEARCH
Synthesis of new N-arylamino(2-furyl)methylphosphonic acid
diesters, and in vitro evaluation of their cytotoxicity against
esophageal cancer cells
•
Anna Agnieszka Klimczak Agnieszka Kuropatwa
•
•
Jarosław Lewkowski Janusz Szemraj
Received: 4 November 2011 / Accepted: 17 April 2012 / Published online: 15 May 2012
ꢀ Springer Science+Business Media, LLC 2012
Abstract N-Furfurylideneanilines and N-arylamino(2-
furyl)methylphosphonates with tolyl and anisyl moieties
were synthesized by the addition of phosphites to azome-
thine bond of corresponding Schiff bases and their NMR
spectroscopic properties were investigated. Then, they
were analyzed in the point of view of their influence on
KYSE 30, KYSE 150, and KYSE 270 esophageal cancer
cell lines and on immortalized esophageal cell line HET 1
A as a control group. Toxicity was evaluated by MTT
assay. Among 11 compounds, a few of them demonstrated
influence on cancer cells being neutral toward the control,
but only one aminophosphonate had IC50 lower than
100 lM and acted as a potential anticancer drug. Some
approaches to structure–activity relation were performed.
2,5-diphenylfuran derivatives with strong cytotoxicity
toward Pneumocystis carinii (Boykin et al., 1998). Their
importance derives also from their role as versatile syn-
thetic intermediates (Keay and Dibble, 1996).
The first preparation of the phosphonic analogs of nat-
ural amino acids (Fields, 1952; Saito et al., 2007; Matve-
eva et al., 2006) stimulated biochemical studies (Mastalerz
and Kafarski, 2000), which confirmed that aminophos-
phonic and aminophosphonous acids also belong to the
group of biologically active compounds. Aminophosphonic
acids are considered to be an interesting class of com-
pounds for scientists in various fields of chemistry, bio-
chemistry (Kafarski and Lejczak, 2001), pharmacology
(Kafarski and Lejczak, 1991), and agriculture (Maier,
1999). They show interesting influence on fungi and plant
growth in agriculture; they also show a high toxicity
toward some cancers (Kafarski and Lejczak, 2001).
Recently, the synthesis and the strong anti-leukemic action
of several aminophosphonates-bearing furan moieties was
reported (Kraicheva et al., 2009).
Keywords N-Arylamino(2-furyl)methylphosphonate ꢀ
Furfural Schiff base ꢀ Esophageal cancer cells ꢀ
Cytotoxicity
Introduction
Esophageal carcinoma causes serious problems over the
world as it is the sixth deadliest cancer in the world
(Bernard and Stewart, 2003; Kordek et al., 2007) as the
disease is very difficult to be treated because of the location
of the esophagus. Inefficient prophylaxis and detection of
this cancer is an additional problem. The treatment of most
patients with cancer is very difficult because it is usually
detected in later stages of development. High risk factors
cause this disease to appear in very specific places around
the globe. These areas include the ‘‘Asian Esophageal
Cancer Belt’’ that spreads from eastern Turkey through
Kazakhstan, Turkmenistan, Uzbekistan, Tajikistan, Iraq,
Iran, western and northern China, and Japan. Other regions
of the world with high esophageal cancer risk include south
western Africa, France, Bermudas and South America,
Substituted furan derivatives are important compounds
(Bosshard and Eugster, 1967), as exemplified by a series of
nitrofural-derived drugs (Merck, 2001) the efficient anti-
histamine agent ranitidine (Bradshaw et al., 1981) and
A. Kuropatwa ꢀ J. Lewkowski (&)
Department of Organic Chemistry, Faculty of Chemistry,
´ ´
University of Łodz, Tamka 12, 91-403 Lodz, Poland
e-mail: jlewkow@uni.lodz.pl
A. A. Klimczak (&) ꢀ J. Szemraj
Department of Medicinal Biochemistry, Medical University
´ ´
of Łodz, Mazowiecka 6/8, 92-215 Lodz, Poland
e-mail: a.a.klimczak@gmail.com
123

Med Chem Res (2013) 22:852–860
853
mainly with Brazil (Mun˜oz, 1993; Coleman et al., 1993;
Reed and Johnston, 1993).
procedures (Cottier et al., 1996a, b; Lewkowski et al., 2000).
Reactions of Schiff bases 2a–f with phosphites 3a–c were
carried out in acetonitrile with a catalytic amount (2 drops)
of trifluoroacetic acid. This catalytic amount of trifluoro-
acetic acid acts in the case of furan derivatives (Cottier et al.,
1996a, b) much better than Lewis acid catalysts such as
TaCl5 (Chandrasekhar et al., 2001) or MgClO4 (Wu et al.,
2006) (Scheme 1).
In treatment of esophageal cancer, chemotherapy or
chemoradiotherapy is important to be used simultaneously
with surgery. These treatments increase the patient’s
overall survivability and decrease tumor conditions. Drug
toxicity against healthy cells is also a very important issue
in chemotherapy. It is important to find new drugs that are
not toxic for normal cells and our research gives us a high
chance of finding one.
(2-Furyl)-N-arylaminomethylphosphonic acid esters
(4a–g) were purified by the column chromatography on
silica gel eluting with hexane–ethyl acetate (1:4) mixture to
receive pure products. Diphenyl (2-furyl)-(3-methyl-
phenyl)aminomethylphosphonate (4e) was contaminated
with a 3 % amount of unreacted diphenyl phosphite;
therefore, the product was dissolved in dichloromethane
and washed with 10 % NaHCO3 in water to remove a
phosphite. That is why, aminophosphonate 4e was crys-
tallized with an occluded molecule of dichloromethane,
which was demonstrated by results of elemental analysis.
The identity of aminophosphonates 4a–g was confirmed
Considering the above mentioned, in studies on various
aspects of aminophosphonic systems, we have focused our
attention on the synthesis of arylaminomethylphosphonates-
containing furan moiety, and evaluation of their potential
cytostatic action against esophageal cancer.
Results and discussion
Chemistry
1
by elemental analysis and H and 31P NMR spectroscopy.
Schiff bases 2a–f were synthesized by the previously pub-
lished (Cottier et al., 1996a, b) and commonly known pro-
cedure by simply mixing furfural with appropriate amine in
methanol and stirring them at room temperature for 24 h.
They were obtained in quantitative yield. Their identity was
confirmed by melting point measurement (Grammaticakis
and Texier, 1971; Kraicheva et al., 2009; Ojima et al., 1992)
and the 1H NMR spectroscopy (Kiepo and Jakopcic, 1985;
Saito et al., 2001; Zubkov et al., 2004) and comparing the
1H NMR spectra demonstrated several diagnostic peaks,
the signal of a proton situated at the asymmetric carbon
(CHP) appearing as a doublet of doublets as this proton
couples with a phosphorus atom as well as with the proton
of a secondary amino group (NH). The NH proton appears
as a doublet. O-methyl groups of phosphoryl moieties
appear as two doublets each for one OMe group demon-
strating their magnetic non-equivalence.
1
obtained measurements with literature data. The H NMR
Biological activity
revealed the diagnostic singlet of a proton of the azomethine
group (-CH=N–) and signals of furan protons.
Then, four 2-furaldimines 2a, 2c, 2e, and 2f and seven
(2-furyl)-N-arylaminomethylphosphonic acid esters 4a–g
have been analyzed. We would like to test the cytotoxicity
(2-Furyl)-N-arylaminomethylphosphonic acid esters
(4a–g) were prepared based on the previously published
Scheme 1 Preparation of
imines 2a–f and N-arylamino(2-
furyl)methylphosphonates 4a–g
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Med Chem Res (2013) 22:852–860
of these compounds toward esophageal cancer cell lines
compared to the control.
To perform this biological assay, four different cell lines
were taken: esophageal squamous cancer cell lines: KYSE
30, KYSE 150, KYSE 270, and SV-40. Immortalized
human esophageal epithelial cell line Het-1A was taken as
a control. All these lines were described in literature (Hou
et al., 2005; Chiu, 2008; Ji et al., 2010; Tanaka et al., 1998,
2007). The cell cytotoxicity was measured by means of
standard MTT assay (Mosmann, 1983; Sieuwerts et al.,
1995).
The investigation demonstrated that none of the ana-
lyzed chemical compounds had IC50 less than 400 lM for
Het-1A, so they could be considered as non toxic for
immortalized human esophageal epithelial cell line.
Unfortunately, testing esophageal cancer cell lines with
N-furfurylidene-p-anisidine (2c), N-furfurylidene-o-anisi-
dine (2a), and diphenyl (2-furyl)-N-(4-methylphenyl)am-
inomethylphosphonate (4f) demonstrated IC50 to be over
400 lM for all three cell lines. Similar results were
obtained for N-furfurylidene-p-toluidine (2f) and diphenyl
(2-furyl)-N-(3-methylphenyl)aminomethylphosphonate (4e),
so they cannot be considered as cytotoxic for any of studied
cancer cell lines. No highly promising results were
obtained for diphenyl (2-furyl)-N-(2-methylphenyl)amin-
omethylphosphonate (4d) and diphenyl (2-furyl)-N-(4-
methoxyphenyl)-aminomethylphosphonate (4c) because
aminophosphonate 4c turned out not to be cytotoxic for the
KYSE 30 line, but for KYSE 150 and KYSE 270, the IC50
was evaluated to be slightly over 200 lM (Table 1). The
aminophosphonate 4d demonstrated the IC50 to be less than
300 lM only for KYSE 30.
Fig. 1 Effect of aminophosphonate 4b on the survival of KYSE 30.
Cells were incubated with different concentrations of aminophosph-
onate 4a for 24 h and their survival was estimated by MTT test (mean
SD; n C 3)
the IC50 was found to be less than 300 lM but higher than
100 lM (Table 1), which cannot be considered as signifi-
cantly cytotoxic.
Much more interesting results were obtained for N-fur-
furylidene-m-toluidine (2e), which showed IC50 much less
than 200 lM and for KYSE 150 even 50.12 1.84 lM
(Table 1; Figs. 4, 5, 6).
The highest cytotoxic influence on cancer cell lines was
demonstrated by dimethyl (2-furyl)-N-(2-methoxyphenyl)
amino-methylphosphonate (4a), where IC50 was found to
be much less than 100 lM, i.e., 38.02 2.39 lM for the
KYSE 30, 51.29 6.97 lM for the KYSE 150, and finally
77.43 3.68 lM for the KYSE 270 cell line (Table 1;
Figs. 7, 8, 9).
For other compounds, results were much more interesting.
Dimethyl (2-furyl)-N-(4-methoxyphenyl)aminomethyl-phos-
phonate (4b) (Table 1; Figs. 1, 2, 3) and dibenzyl (2-furyl)-
N-(3-methoxyphenyl)-aminomethylphosphonate 4g, where
Considering that a limited number of compounds were
used for this investigation, it is too early to perform the real
Table 1 The fractions of living cells after 24 h incubation with imines 2a, 2c, 2e–f, and aminophosphonates 4a–g
Comp.
R1
R2
IC50 (lM)
KYSE-30
KYSE-150
KYSE-270
Het-1A
2a
2c
2e
2f
o-OCH3
p-OCH3
m-CH3
p-CH3
–
[400
[400
[400
[400
[400
[400
[400
[400
[400
[400
[400
[400
[400
[400
–
[400
[400
[400
–
158.49 17.23
[400
50.12 1.84
380.19 15.12
51.29 6.97
166 17.28
208.93 17.33
[400
111.2 7.48
398.11 23.40
77.43 3.68
223.87 15.51
204.84 8.90
[400
–
4a
4b
4c
4d
4e
4f
o-OCH3
p-OCH3
p-OCH3
o-CH3
CH3
CH3
Ph
Ph
Ph
Ph
CH2Ph
38.02 2.39
316.23 21.03
[400
251.19 15.41
380 21.35
[400
m-CH3
p-CH3
[400
[400
[400
199.52 16.85
[400
251.19 12.94
4g
m-OCH3
251.19 22.09
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Med Chem Res (2013) 22:852–860
855
Fig. 2 Effect of aminophosphonate 4b on the survival of KYSE 150.
Cells were incubated with different concentrations of aminophosph-
onate 4a for 24 h and their survival was estimated by MTT test (mean
SD; n C 3)
Fig. 4 Effect of imine 2e on the survival of KYSE 30. Cells were
incubated with different concentrations of imine 2e for 24 h and their
survival was estimated by MTT test (mean SD; n C 3)
Fig. 3 Effect of aminophosphonate 4b on the survival of KYSE 270.
Cells were incubated with different concentrations of aminophosph-
onate 4a for 24 h and their survival was estimated by MTT test (mean
SD; n C 3)
Fig. 5 Effect of imine 2e on the survival of KYSE 150. Cells were
incubated with different concentrations of imine 2e for 24 h and their
survival was estimated by MTT test (mean SD; n C 3)
structure vs. activity relationship (SAR) analysis. But,
some tendencies are visible. Tests carried out for four
diphenyl (2-furyl)-phenylaminomethylphosphonates 4c–f
demonstrated no antiproliferative activity (4e and 4f) or
very slight action (4c and 4d) toward esophageal squamous
cancer cell lines. It would allow us to suggest that am-
inophosphonic acid phenyl esters do not have the important
cytotoxic action, maybe due to their chemical instability.
Three tested Schiff bases N-furfurylidene-o-anisidine
(2a), N-furfurylidene-p-anisidine (2c), and N-furfurylid-
ene-p-toluidine (2f) have no cytotoxic influence on inves-
tigated cells. The negligible activity of imine 2f which
surprised us a bit in the light of Kraicheva’s paper
(Kraicheva et al., 2009), who has proven its high cyto-
toxicity toward human leukemia cells. But, it cannot be
stated that N-phenyl furfural Schiff bases are inactive for
this cancer cell lines because N-furfurylidene-m-toluidine
(2e) showed moderately interesting properties. Possibly,
the meta-methyl substitution plays a key role in its cyto-
toxicity, but it is too early to draw any conclusion. It is to
stress that the O,O0-dimethyl and O,O0-dibenzyl amino-
phosphonic esters 4a–b and 4g have interesting cytotoxic
action and that ortho-substitution seem to improve greatly
the cytotoxicity of aminophosphonates in study.
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Med Chem Res (2013) 22:852–860
Fig. 8 Effect of aminophosphonate 4a on the survival of KYSE 150.
Cells were incubated with different concentrations of aminophosph-
onate 4a for 24 h and their survival was estimated by MTT test (mean
SD; n C 3)
Fig. 6 Effect of imine 2e on the survival of KYSE 270. Cells were
incubated with different concentrations of imine 2e for 24 h and their
survival was estimated by MTT test (mean SD; n C 3)
Fig. 7 Effect of aminophosphonate 4a on the survival of KYSE 30.
Cells were incubated with different concentrations of aminophosph-
onate 4a for 24 h and their survival was estimated by MTT test (mean
SD; n C 3)
Fig. 9 Effect of aminophosphonate 4a on the survival of KYSE 270.
Cells were incubated with different concentrations of aminophosph-
onate 4a for 24 h and their survival was estimated by MTT test (mean
SD; n C 3)
Conclusion
aminomethylphosphonates bearing O,O0-dialkyl and
O,O0-dibenzyl groups will be also performed.
In conclusion, the obtained results showed that none of the
studied compounds are toxic for immortalized control,
human esophageal epithelial cell line HET-1A.
The Schiff base 2e as well as dimethyl aminophospho-
nates 4a, 4b and dibenzyl one 4g show some cytotoxicity
for all three esophageal squamous cancer cell lines (KYSE
30, KYSE 150, and KYSE 270). It is noteworthy that
dimethyl (2-furyl)-(2-methoxyphenyl)aminomethylphosph-
onate (4a) presented the best results (IC50 much less
than 100 lM and in one case less than 40 lM) and it seems
to be a perfect candidate for further in vivo analysis, which
is planned to be done. The SAR study of (2-furyl)
Experimental
Chemistry
All solvents (POCh, Poland) were routinely distilled and
dried before 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) and 243 MHz
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Med Chem Res (2013) 22:852–860
857
1
(31P NMR). TMS was used as the internal standard for H
NMR and phosphoric acid was used as the external stan-
dard for 31P NMR. Elemental analyses were carried out at
the Centre for Molecular and Macromolecular Science of
m-C6H4, 1H), 7.07–7.04 (m, m-C6H4, 3H), 6.95 (d,
,
J = 3.6 Hz, Hf3ur, 1H), 6.55 (dd, J = 3.6 and 1.8 Hz, Hf3ur
1H), 2.38 (s, OCH3, 3H).
´ ´
the Polish Academy of Science in Łodz, Poland.
N-Furfurylidene-p-toluidine 2f Quantitative yield (0.46 g),
mp = 35–38 ꢁC (Kraicheva et al., 2009), 41–42 ꢁC.
General procedure for the synthesis of 2a–f
1H NMR (CDCl3, 600 MHz): d 8.30 (s, CH=N, 1H),
7.59 (d, J = 1.8 Hz, H5fur, 1H), 7.19–7.15 (AA0BB0 system,
J = 9.0 Hz, p-C6H4, 4H), 6.92 (d, J = 3.6 Hz, Hf3ur, 1H),
6.54 (dd, J = 3.6 and 1.8 Hz, Hf3ur, 1H), 2.36 (s, CH3, 3H).
Furfural (2.5 mmol, 0.24 g) was dissolved in methanol
(15 ml) and to this solution aniline derivative 1a–f
(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 (2a–f).
General procedure for the synthesis of (2-furyl)-
arylaminomethylphosphonic acid esters 4a–g
N-Furfurylidene-o-anisidine 2a (Saito et al., 2001) Quan-
titative yield (0.50 g), dark yellow oil.
A Schiff base 2a–f (4.5 mmol) was dissolved in acetoni-
trile and then phosphite 3a–c (4.5 mmol) was added fol-
lowed by the addition of 2 drops of trifluoroacetic acid. The
mixture was stirred at 80 ꢁC during the day and at room
temperature overnight. Reactions lasted for 72 h. Then,
solvent was evaporated and crude products were chro-
matographed on silica gel (AcOEt–hexane 4:1) to give pure
aminophosphonates (4a–g).
1H NMR (CDCl3, 600 MHz): d 8.36 (s, CH=N, 1H),
7.63 (d, J = 1.8 Hz, Hf5ur, 1H), 7.21 (ddd, J = 9.0, 7.8 and
1.8 Hz, o-C6H4, 1H), 7.07 (dd, J = 7.2 and 1.8 Hz,
o-C6H4, 1H), 6.98 (m, o-C6H4, Hf3ur, 3H), 6.57 (dd, J = 3.6
and 1.8 Hz, Hf3ur, 1H), 3.91 (s, OCH3, 3H).
N-Furfurylidene-m-anisidine 2b (Kiepo and Jakopcic,
1985) Quantitative yield (0.50 g), yellow oil
Dimethyl (2-furyl)-N-(2-methoxyphenyl)aminomethylphos-
phonate 4a Y = 71 % (1.08 g), dark yellow oil.
1H NMR (CDCl3, 600 MHz): d 8.32 (s, CH=N, 1H),
7.64 (d, J = 1.8 Hz, H5fur, 1H), 7.30 (dd, J = 8.4 and
7.2 Hz, m-C6H4, 1H), 6.98 (d, J = 3.6 Hz, Hf3ur, 1H), 6.86
(d, J = 7.2 Hz, m-C6H4, 1H), 6.84 (s, m-C6H4, 1H), 6.82
(d, J = 7.2 Hz, m-C6H4, 1H), 6.58 (dd, J = 3.6 and
1.8 Hz, Hf3ur, 1H), 3.83 (s, OCH3, 3H).
1H NMR (CDCl3, 600 MHz): d 7.38 (d, J = 1.8 Hz,
Hf5ur, 1H), 6.71 (m, o-C6H4, Hf3ur, 3H), 7.07 (dd, J = 7.8
and 1.8 Hz, o-C6H4, 1H), 6.39 (m, o-C6H4, 1H), 6.57 (dd,
J = 3.6 and 1.8 Hz, Hf4ur, 1H), 5.04 (d, J = 7.8 Hz, NH,
1H), 4.94 (dd, 2JPH = 23.4 Hz, 3JHH = 7.8 Hz, CHP, 1H),
3.84 (s, OCH3, 3H), 3.80 (d, 3JPH = 10.8 Hz, POCH3, 3H),
3
N-Furfurylidene-p-anisidine 2c Quantitative yield (0.50 g),
mp = 60–64 ꢁC (Ojima et al., 1992), 68–70 ꢁC.
3.65 (d, JPH = 10.8 Hz, POCH3, 3H). 31P NMR
(243 MHz, CDCl3): d 22.60.
Elemental analysis: Calcd for C14H18NO5P: C, 54.02, H,
1H NMR (CDCl3, 600 MHz): d 8.30 (s, CH=N, 1H),
7.59 (d, J = 1.8 Hz, Hf5ur, 1H), 7.25 (d, J = 9.0 Hz,
p-C6H4, 2H), 6.92 (d, J = 9.0 Hz, p-C6H4, 2H), 6.91
(d, J = 3.6 Hz, Hf3ur, 1H), 6.54 (dd, J = 3.6 and 1.8 Hz,
Hf3ur, 1H), 3.82 (s, OCH3, 3H).
5.83, N, 4.50. Found: C, 54.29, H, 5.68, N, 4.78.
Dimethyl (2-furyl)-N-(4-methoxyphenyl)aminomethylphos-
phonate 4b Y = 63 % (0.98 g), dark yellow oil.
1H NMR (CDCl3, 600 MHz): d 7.38 (d, J = 1.8 Hz,
Hf5ur, 1H), 6.73 (d, J = 9.0 Hz, p-C6H4, 2H), 6.62 (d,
J = 9.0 Hz, p-C6H4, 2H), 6.35 (d, J = 3.6 Hz, Hf3ur, 1H),
6.31 (dd, J = 3.6 and 1.8 Hz, Hf3ur, 1H), 4.94 (dd,
N-Furfurylidene-o-toluidine 2d Quantitative yield (0.46 g),
mp = 50–53 ꢁC (Grammaticakis and Texier, 1971), 58 ꢁC.
1H NMR (CDCl3, 600 MHz): d 8.15 (s, CH=N, 1H),
7.60 (dd, J = 1.8 and 0.6 Hz, Hf5ur, 1H), 7.20 (ddd,
J = 9.0, 7.8 and 1.8 Hz, o-C6H4, 1H), 7.18 (dd, J = 7.8
and 1.8 Hz, o-C6H4, 1H), 7.11 (ddd, J = 9.0, 7.8 and
3
2JPH = 24.0 Hz, JHH = 8.4 Hz, CHP, 1H), 4.54 (d,
3
J = 8.4 Hz, NH, 1H), 3.81 (d, JPH = 10.8 Hz, POCH3,
3H), 3.70 (s, OCH3, 3H), 3.62 (d, 3JPH = 10.8 Hz, POCH3,
3H). 31P NMR (243 MHz, CDCl3): d 22.76.
Elemental analysis: Calcd for C14H18NO5P: C, 54.02, H,
1.8 Hz, o-C6H4, 1H), 6.94 (dd, J = 3.6 and 0.6 Hz, H3fur
,
1H), 6.89 (dd, J = 9.0 and 1.8 Hz, o-C6H4, 1H), 6.54 (dd,
J = 3.6 and 1.8 Hz, Hf3ur, 1H), 2.38 (s, OCH3, 3H).
5.83, N, 4.50. Found: C, 54.35, H, 5.98, N, 4.80.
N-Furfurylidene-m-toluidine 2e (Zubkov et al., 2004) Quan-
titative yield (0.46 g), dark yellow oil.
Diphenyl (2-furyl)-N-(4-methoxyphenyl)aminomethylphos-
phonate 4c Y = 96 % (2.08 g), mp = 77–79 ꢁC.
1H NMR (CDCl3, 600 MHz): d 7.36 (m, Hf5ur, 1H),
7.29–7.24 (m, PhH, 4H), 7.22 (t, J = 7.8 Hz, PhH, 1H),
1H NMR (CDCl3, 600 MHz): d 8.29 (s, CH=N, 1H),
7.61 (d, J = 1.8 Hz, Hf5ur, 1H), 7.27 (d, J = 7.8 Hz,
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Med Chem Res (2013) 22:852–860
4
7.16 (t, J = 7.8 Hz, PhH, 1H), 7.14–7.11 (m, PhH, 2H),
3
Hf3ur, 1H), 6.32 (d, JHH = 3.6 Hz, m-C6H4, 1H), 6.30 (dd,
3JHH = 7.8 Hz and 4JHH = 3.6 Hz, m-C6H4, 1H), 6.22 (dd,
3JHH = 7.8 Hz and 4JHH = 3.6 Hz, m-C6H4, 1H), 6.16 (m,
4
7.02 (dd, JHH = 7.8 Hz and JHH = 0.6 Hz, PhH, 2H),
6.75 and 6.66 (AA0XX0 system, J = 9.0 and 2.4 Hz,
3
2
p-C6H4, 4H), 6.44 (m, Hf3ur, 1H), 6.32 (t, JHH = 2.4 Hz,
Hf4ur, 1H), 5.07 (part of ABX system, JHH = 12.0 Hz,
Hf4ur, 1H), 5.15 (d, JPH = 24.0 Hz, CHP, 1H), 3.72 (s,
3JPH = 9.0 and 7.8 Hz, OCH2Ph, 2H), 4.97 and 4.79 (part
2
2
of AMX system, JHH = 12.0 Hz, JPH = 8.7 and 7.8 Hz,
3
OCH3, 3H). 31P NMR (243 MHz, CDCl3): d 12.73.
Elemental analysis: Calcd for C24H22NO5P: C, 66.20, H,
5.09, N, 3.22. Found: C, 66.15, H, 4.95, N, 3.27.
2
3
OCH2Ph, 2H), 4.91 (d, JPH = 24.0 Hz and JHH = 9 Hz,
CHP, 1H), 4.49 (d, J = 9.0 Hz, NH, 1H), 3.71 (s, OCH3,
3H). 31P NMR (243 MHz, CDCl3): d 21.67.
Diphenyl (2-furyl)-N-(2-methylphenyl)aminomethylphos-
phonate 4d Y = 99 % (2.07 g), dark yellow oil.
1H NMR (CDCl3, 600 MHz): d 7.42 (dd, 3JHH = 1.8 Hz
Elemental analysis: Calcd for C26H26NO5P: C, 67.38, H,
5.65, N, 3.02. Found: C, 67.48, H, 5.77, N, 3.02.
4
and JHH = 0.6 Hz, Hf5ur, 1H), 7.32–7.28 (m, PhH, 4H),
Biological assays
7.20–7.15 (m, PhH, 4H), 7.11–7.09 (m, o-C6H4, 2H), 7.07–
3
7.05 (m, PhH, 2H), 6.76 (ddd, JHH = 7.8 and 8.4 and
Cell line and culture
4JHH = 0.6 Hz, o-C6H4, 1H), 6.69 (d, J = 7.8 Hz, o-C6H4,
3
1H), 6.49 (m, Hf3ur, 1H), 6.36 (dd, JHH = 3.6 and 1.8 Hz,
The esophageal squamous cancer cell lines KYSE 30,
KYSE 150, and KYSE 270 were obtained from the Euro-
pean Collection of Cell Cultures (ECACC, UK). Cells were
grown in RPMI 1640 and Ham F12 in 1:1 ratio with 2 mM
L-glutamine and 2 % fetal bovine serum (FBS). Het-1A
(SV-40 immortalized human esophageal epithelial cell
line) was used as control. This cell line was obtained from
American Type Culture Collection (ATCC, USA) and was
cultured in Quantum 286. Cells were incubated in standard
conditions (37 ꢁC, v/v 5 % CO2, relative humidity 100 %)
without antibiotics.
Hf4ur, 1H), 5.33 (dd, 2JPH = 24.6 and 3JHH = 3.0 Hz, CHP,
1H), 4.47 (large s, NH, 1H), 2.19 (s, CH3, 3H). 31P NMR
(243 MHz, CDCl3): d 12.55.
Elemental analysis: Calcd for C24H22NO4P: C, 68.73, H,
5.29, N, 3.34. Found: C, 68.48, H, 5.02, N, 3.55.
Diphenyl (2-furyl)-N-(3-methylphenyl)aminomethylphos-
phonate 4e Y = 96 % (2.10 g), mp = 36–38 ꢁC.
1H NMR (CDCl3, 600 MHz): d 7.37 (m, Hf5ur, 1H),
7.29–7.22 (m, PhH, 4H), 7.17–7.10 (m, PhH, 4H), 7.06 (t,
3JHH = 7.8 Hz, m-C6H4, 1H), 7.03–7.01 (m, PhH, 2H),
3
6.61 (d, JHH = 7.2 Hz, m-C6H4, 1H), 6.52 (m, m-C6H4,
Materials
2H), 6.46 (d, 3JHH = 3.6 Hz, H3fur, 1H), 6.32 (m, Hf4ur, 1H),
5.28 (d, 2JPH = 24.0 Hz, CHP, 1H), 3.81 (large s, NH, 1H),
2.53 (s, CH3, 3H). 31P NMR (243 MHz, CDCl3): d 12.61.
Elemental analysis: Calcd for C24H22NO4P•2/3CH2Cl2:
C, 62.24, H, 4.94, N, 2.94. Found: C, 62.13, H, 5.11, N,
3.63.
All mediums and chemicals for cell growth were bought
from PAA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tet-
razolium bromide (MTT) and dimethyl sulfoxide (DMSO)
were from Sigma Aldrich. Tested compounds were freshly
prepared in DMSO and diluted in medium with the final
concentration lower than 0.5 %.
Diphenyl (2-furyl)-N-(4-methylphenyl)aminomethylphos-
phonate 4f Y = 99 % (2.08 g), mp = 118–119 ꢁC.
1H NMR (CDCl3, 600 MHz): d 7.38 (dd, 3JHH = 1.8 Hz
MTT assay
4
and JHH = 0.6 Hz, Hf5ur, 1H), 7.29–7.25 (m, PhH, 4H),
The cell cytotoxicity was measured with the MTT assay
(Mosmann, 1983; Sieuwerts et al., 1995). 100 ll of a
10,000 cells/ml solution was added to each well of the
96-well plate. The plate was incubated at 37 ꢁC in the dark.
After 24 h, the cells were incubated for another day with
eight different chemical concentrations. Wells on the edge
of the plate were filled with 100 ll of phosphate-buffered
saline (PBS). After incubation, the wells were washed with
PBS and 100 ll 0.5 mg/ml MTT solution was added for
3 h. Afterward the medium with MTT was removed, and
100 ll of pure DMSO was added. After 20 min, a colori-
metric reading was made with 580-nm light. Each experi-
ment repeat was performed on a different day. There were
at least three repeats of each experiment. IC50 was
7.18–7.12 (m, PhH, 4H), 7.03–7.01 (m, PhH, 2H), 6.98 (d,
J = 9.0 Hz, p-C6H4, 2H), 6.62 (d, J = 9.0 Hz, p-C6H4,
2H), 6.44 (m, H3fur, 1H), 6.32 (m, Hf4ur, 1H), 5.22 (d,
2JPH = 24.6 Hz, CHP, 1H), 4.47 (large s, NH, 1H), 2.23 (s,
CH3, 3H). 31P NMR (243 MHz, CDCl3): d 12.70.
Elemental analysis: Calcd for C24H22NO4P: C, 68.73, H,
5.29, N, 3.34. Found: C, 69.03, H, 5.32, N, 3.58.
Dibenzyl (2-furyl)-N-(3-methoxyphenyl)aminomethylphos-
phonate 4g Y = 98 % (2.12 g), mp = 42–44 ꢁC.
1H NMR (CDCl3, 600 MHz): d 7.36 (m, PhH, 1H), 7.29
(m, PhH, Hf5ur, 9H), 7.21 (m, PhH, 1H), 7.03 (t,
3
3JHH = 7.8 Hz, m-C6H4, 1H), 6.36 (d, JHH = 3.6 Hz,
123

Med Chem Res (2013) 22:852–860
859
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calculated based on the control without chemicals added.
An additional control of medium with 1 % DMSO was
used to test the solvent.
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Statistical analysis
To analyze cytotoxicity, IC50 values were used. All
experiments were examined by non-liner regression anal-
ysis (GraphPad Prism Software). A probability level of
0.05 was used for statistical validity.
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J (2007) Onkologia podre˛cznik dla studentow i lekarzy. Via
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Acknowledgments Authors wish to express special thanks to
´ ´
Mr Jan Bitner from the University of Łodz for his great assistance in
preparation of the final version of this manuscript.
Medica, Gdansk
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