Synthesis of novel N-(p-toluenesulfonyl)aminophosphonates and evaluation of their biological properties

Article abstract of DOI:10.1080/10426507.2018.1424712

The synthesis of a series of N-sulfonylaminophosphonates 2aA-2dC and evaluation of their cytotoxicity and ecotoxicity is described. N-sulfonylaminophsophonates 2aA-2dC were obtained by addition of dialkyl phosphites to azomethine bonds of sulfonimines 1a-

Full text of DOI:10.1080/10426507.2018.1424712

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Synthesis of novel N-(p-
toluenesulfonyl)aminophosphonates and
evaluation of their biological properties
Rafał Karpowicz, Jarosław Lewkowski, Małgorzata Stasiak, Aleksandra
Czopor, Paulina Tokarz, Adrianna Król, Diana Rogacz & Piotr Rychter
To cite this article: Rafał Karpowicz, Jarosław Lewkowski, Małgorzata Stasiak, Aleksandra
Czopor, Paulina Tokarz, Adrianna Król, Diana Rogacz & Piotr Rychter (2018): Synthesis of novel
N-(p-toluenesulfonyl)aminophosphonates and evaluation of their biological properties, Phosphorus,
Sulfur, and Silicon and the Related Elements, DOI: 10.1080/10426507.2018.1424712
To link to this article: https://doi.org/10.1080/10426507.2018.1424712
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Date: 06 January 2018, At: 01:29

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Synthesis of novel N-(p-toluenesulfonyl)aminophosphonates and evaluation of their
biological properties
Rafał Karpowicz,a Jarosław Lewkowski,a Małgorzata Stasiak,a Aleksandra Czopor,a Paulina
Tokarz,b Adrianna Król,b Diana Rogaczc and Piotr Rychterc
aDepartment of Organic Chemistry, Faculty of Chemistry, University of Łódź, Tamka 12, 91-403
b
Łódź, Poland; Department of Molecular Genetics, Faculty of Biology and Environmental
Protection, University of Łódź, Pomorska 141/143, 90-236 Łódź, Poland; cFaculty of
Mathematics and Natural Science, Jan Długosz University in Częstochowa, 13/15 Armii Krajowej
Av., 42-200 Częstochowa, Poland
CONTACT Jarosław Lewkowski  jlewkow@uni.lodz.pl and Rafał Karpowicz 
rafalkarpowicz@gmail.com
Abstract
The synthesis of a series of N-sulfonylaminophosphonates 2aA-2dC and evaluation of their
cytotoxicity and ecotoxicity is described.
N-sulfonylaminophsophonates 2aA-2dC were obtained by addition of dialkyl phosphites to
azomethine bonds of sulfonimines 1a-d in dichloromethane, and the reaction was mediated by
potassium carbonate. Sulfonimines 1a-d were synthesized by direct condensation of selected
aldehydes with p-toluenesulfonamide catalyzed by titanium(IV) ethoxide.
The cytotoxicity of compounds 2aB-2cB, 2aC-2cC, 2bA and 2dA against normal primary human
lymphocytes, non-small cell lung cancer cells A549, promyelocytic leukemia cells HL-60 and
glioblastoma astrocytoma cells U87 was found to be very low.
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The phytotoxicity of 2dC against monocotyledonous oat (A. sativa) and dicotyledonous radish
(R. sativus) was measured and the compound was found to be practically not toxic for two
model plants. Lack of toxicity for marine bacteria A. fischeri has been also detected. However,
the compound 2dC was found to be significantly toxic for freshwater, European crustaceans H.
incongruens (ostracods).
Keywords: Sulfonamidomethylphosphonates, aza-Pudovik reaction, cytotoxicity, ecotoxicology,
A. fischeri test, H. incogruens test
Introduction
Aminophosphonic derivatives are commonly known to have the potential biological activity and
this fact has been reported many times.1 Recently, several important papers have been
published proving that aminophosphonic and aminophosphinic derivatives show interesting
properties, which may be applied in medicine or pharmacy2 or in agrochemistry and pesticide
industry.3,4
A vast number of structure modifications has been made on aminophosphonates and among
them, N-sulfonylaminophosphonates (or sulfonamidophosphonates) constitute an important
group of compounds having biological potential. In 1977, Birum5 patented the preparation of
several of them and mentioned their biological activity. Much later, some authors reported
their intriguing properties as inhibitors of matrix metalloproteinases (MMPs),6,7 anti-HIV
agents8 or as agents inactivating the E. cloacae P99 -lactamase.9
Nobody should be astonished then that a number of papers dealing with the preparation of
sulfonylaminophosphonates has been published.1,2,10,11 Das et al.10 proposed the use of iodine
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as a catalyst, while the Chinese team,11 reported their synthesis in catalyst-free conditions.
Sulfonylaminophosphonates have been applied to the synthesis of chiral thiourea derivatives
bearing an -aminophosphonic moiety having the antiviral properties,12 or benzosultams
bearing an α-triaryl-substituted stereogenic center.13 Having that in mind, we performed
synthesis of a series of novel -sulfonylaminophosphonates 2aA-dC and investigated some of
their biological properties.
The REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) regulations
point out to the necessity of evaluating the toxicological behavior of new compounds in order
to predict their possible effect on environment and humans. Keeping in mind these
recommendations, the potentially bioactive, newly synthesized compounds 2bA-dC were then
investigated in the aspect of their cytotoxicity on human lymphocytes. By this occasion, their
cytotoxicity was also tested on several cancer cell lines. Finally, the selected, model compound
2dC was the subject of ecotoxicological assessments, i.e. evaluation of its phytotoxicity against
Raphanus sativus and Avena sativa as well as its ecotoxicity performed by means of commercial
biotests: the Microtox® system (using Aliivibrio fischeri bacteria) and OSTRACODTOXKIT F (using
Heterocypris incogruens crustaceans).
Results and Discussion
Preparation of N-sulfonylaminophosphonates 2bA-dC
Various methods for synthesis of sulfonamide phosphonates have been reported within last
decade. Das et al.10 reported their preparation by the addition of trimethylsilyldialkyl phosphite
to sulfonimines using iodine as catalyst. Palacios et al.14 performed the addition of dialkyl
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phosphites to sulfonimines in the presence of triethylamine. Onysko et al.15 suggested the
different approach, i.e. the reaction of arenesulfonyl chloride with N-unsubstituted
aminophosphonic acid esters. Pallikonda and Chakravarty16 have proposed the reactions of
sulfonamides with α-hydroxyphosphonates mediated by trifluoromethanesulfonic acid.
We decided to apply the addition of dialkyl phosphite to sulfonimines. The first step, however
was to obtain sulfonimines 1a-d. Numerous methods for preparation of these compounds have
been published and in each paper, the necessity of using a catalyst is stressed. Love et al.17
reported the use of tetraethyl orthosilicate, and later such catalysts as Montmorillonite K-1018
or iron trichloride19 were used. Ruano et al.20 proposed to prepare first N-sulfinylimines by
condensation of carbonyl compounds with sulfonamides catalyzed by titanium(IV) ethoxide21
and their subsequent oxidation with m-CPBA.
To our knowledge, the direct preparation of sulfonimines from aldehydes catalyzed by
titanium(IV) ethoxide was described only once,22 where authors reported the synthesis of 4-
methyl-N-(2-vinylbenzylidene)benzene-sulfonamide.
Basing on the above published methods, we performed the direct condensation of
p-toluenesulfonamide with selected aldehydes catalyzed by titanium(IV) ethoxide. Reactions
were carried out in dichloromethane. This method allowed to obtain the desired sulfonimines
in good yields and the high degree of purity. (Fig. 1)
[Insert Figure 1]
Such obtained sulfonimines 1a-d were used for the reaction with three model phosphites,
namely diethyl, diisopropyl and dibenzyl phosphites. The addition of phosphites to sulfonimines
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1a-d was mediated by potassium carbonate; reactions were carried out in dichloromethane.
The use of potassium carbonate as a promoter for the addition of dialkyl phosphites to
sulfonaldimines was described by Fan et al.23 as an example of an α C-H oxidation and
subsequent addition reactions of aryl sulfonamides. Such methodology allowed the desired
N-sulfonyl aminophosphonates 2bA-dC in a proper degree of purity and satisfactory yields to be
obtained. (Fig. 1)
1
The obtained sulfinamidophosphonates 2bA-dC were identified by the H, 13C and 31P NMR as
well as IR spectroscopy and ESI mass spectrometry. For all new compounds, elemental analysis
was made, in a case of known compounds values of melting points were compared with
literature data.
1H NMR spectra revealed the diagnostic doublet at over 4 ppm assigned to the proton at alpha
carbon atom with a characteristic coupling of a proton with a phosphorus nucleus. In some
cases, this proton coupled additionally to a proton at nitrogen atom and then , this signal
appeared as a doublet of doublets.
At 13C NMR spectra, a carbon nucleus directly linked to phosphorus gave a doublet at around 40
ppm and of a coupling constant over 150 Hz, which is also characteristic for aminophosphonic
systems.
N-sulfonylaminophosphonates 2bA-dC obtained in this way were subsequently investigated in
aspect of their biological properties.
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Cytotoxic activity of 2aB-2cB, 2aC-2cC, 2bA and 2dA
The synthesized aminophosphonates 2aB-2cB, 2aC-2cC, 2bA and 2dA were then tested in
aspect of their cytotoxicity. Compounds 2dB, 2dC and 2cA were not considered because of their
very low solubility in the medium used for cytotoxicity evaluation. The cytotoxic effect of newly
synthesized phosphonates 2aB-2cB, 2aC-2cC, 2bA and 2dA was investigated against normal
primary human lymphocytes (PBMCs) and three human cancer cell lines, namely the non-small
cell lung cancer cells A549, the promyelocytic leukemia cells HL-60 and the glioblastoma
astrocytoma cells U87 using standard resazurin reduction assays. This assay is based on the
reduction of resazurin to resorufin catalyzed by cellular mitochondrial dehydrogenases.24 The
amount of fluorescent resorufin obtained directly correlates with the number of live cells in a
culture. (Fig. 2)
[Insert Figure 2]
The activity of the tested compounds was studied in the concentration range from 1 nM to 10
µM. The compounds were prepared in relatively low concentrations due to their low solubility
in culture medium. Analysis carried out for eight N-sulfonylaminophosphonates (2aB-2cB, 2aC-
2cC, 2bA and 2dA) demonstrated no cytotoxic effect against PBMCs, A549, HL-60 and U87 cells
up to 1 µM and low cytotoxic effect at the concentration 10 µM – cell viability decreased
approximately to 80%. It would allow us to suggest that the studied
N-sulfonylaminophosphonates 2aB-2cB, 2aC-2cC, 2bA and 2dA are non-toxic probably due to
their low solubility in aqueous solutions. (Fig. 2)
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Evaluation of ecotoxicity of dibenzyl (ferrocenyl)(4-methylphenylsulfonamido)-
methylphosphonate (2dC)
Plant Growth Test – Germination, Growth Inhibition, Dry and Fresh Matter
Phytotoxicity of dibenzyl (ferrocenyl)(4-methylphenylsulfonamido)-methylphosphonate (2dC)
was tested on two model plants: monocotyledonous oat (Avena sativa) and dicotyledonous
radish (Raphanus sativus) according to OECD/OCDE. Terrestrial plant test: Seedling emergence
and seedling growth test.25
The aminophosphonate 2dC was selected because of its structural features, i.e. dibenzyl, N-
substituted ferrocene-derived aminophosphonate, as it was described in one of our previous
papers.26
The percentage growth inhibition (GI%) values of shoot height, root length and fresh matter of
both plants exposed on sulfonylaminophosphonate 2dC are presented in Table 1. Growth
inhibition of shoot height of both plants treated with dibenzyl (ferrocenyl)(4-
methylphenylsulfonamido)-methylphosphonate (2dC) is not very high. Regardless the kind of
examined plants, up to concentration 100 mg/kg of soil dry weight (s.d.w.), shoot growth
inhibition did not exceed 10%. At 100 mg/kg s.d.w., stronger impact of the compound 2dC on
oat was observed (8.5% vs. 5.2% respectively). Further increasing of aminophosphonate
concentration up to 200 mg/kg s.d.w. revealed larger difference in growth inhibition% of oat,
which increased almost twice (16.14 mg/kg) as compared to radish, the growth of which
inhibited slightly only (6.74 mg/kg).
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The increase of concentration of 2dC up to 400 mg/kg s.d.w. caused the reversal of tendency,
the significant rise of growth inhibition value of radish shoot height (34.5%) was observed with
simultaneous slight increase of oat shoot growth inhibition (18.2%). At higher concentrations
(800 and 1000 mg/kg s.d.w.), sulfonylaminophosphonate 2dC was distinctly much more
harmful for radish than to oat, as shoot growth inhibition for the former were 39.4 and 43.4%
respectively, while for oat – 21.5 and 27.6% respectively.
But it is to state that even at 1000 mg/kg s.d.w., dibenzyl (ferrocenyl)(4-methylphenyl-
sulfonamido)methylphosphonate (2dC) caused the shoot growth inhibition in less than 50% for
both plants. But it is worth mentioning that the higher negative impact on dicotyledonous
radish vs. monocotyledonous oat was observed. (Table 1)
[Insert Table 1]
Percentage changes of growth inhibition of roots was similar to GI% of shoot height (Table 1)
but the described above tendencies were not observed. Except for the 1-mg concentration of
the compound 2dC, it was definitely more harmful for radish than for oat regardless of the
concentration of 2dC. However, considering that, at 1000 mg/kg s.d.w., the studied compound
2dC caused the radish root growth inhibition in 32.4% only, it is to state that the evaluated
compound 2dC is very moderately toxic for roots of both plants.
The tendency of changes of fresh matter for both plants was similar to growth inhibition of
shoots of both tested plants (Table 1). Up to the concentration of 400 mg/kg s.d.w., the
compound 2dC was evidently more harmful for oat that for radish. The most significant
difference was observed at 100 mg/kg s.d.w. (16.5% for oat and 4% for radish fresh matter
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inhibition). Further increase of the concentration resulted in smaller and smaller difference in
toxicity between both plants and finally, at 800 mg/kg s.d.w., the tendency reversal was
observed. At 800 and 1000 mg/kg s.d.w., the evaluated aminophosphonate 2dC turned out to
be significantly more toxic for radish fresh matter than for oat one (53.2 and 63.2% inhibition
for radish and 29.1 and 35% for oat respectively). It requires to be stressed that the compound
2dC is seriously harmful for radish at 800 and 1000 mg/kg s.d.w.
The changes of percentage germination of tested plants are presented in Table 2. Comparing
the germination of both examined plants, it has been noticed that the sulfonyl-
aminophosphonate 2dC did not stopped germination and that germination inhibition was
extremely mere even at the highest tested concentration 1000 mg/kg of s.d.w.
Changes of effective concentration EC50, no observed effect concentration NOEC and lowest
observed effect concentration LOEC values are presented in Figure 3.
[Insert Figure 3]
[Insert Table 2]
The values of effective concentration EC50 for the evaluated compound 2dC have been found to
be much lower for radish, which clearly demonstrates that the studied
sulfonylaminophosphonate is significantly more toxic for radish than to oat. But this tendency
alters depending on the given inhibition factor.
Comparing the impact of 2dC on shoots and roots of both plants, it should be noted that the
tested compound is more harmful for shoots than for roots. EC50 values of the examined
compound 2dC for oat and radish shoots are 6755 and 1172 mg/kg s.d.w. respectively, while for
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roots: 16674 and 2971 mg/kg s.d.w. respectively. It clearly indicates that shoot of both plants is
more susceptible for the tested compound than root. Nevertheless, it is to stress that the ratio
EC50 for oat to EC50 for radish is in both cases similar (5.6 up to 5.8).
The fresh matter is significantly more susceptible for the compound 2dC than shoots and roots.
EC50 values are 4806 mg/kg s.d.w. for oat and 740 mg/kg s.d.w. for radish but their ratio is ca.
6.5, which pointed out to the selective action of 2dC on radish fresh matter.
Determined values of NOEC (no effect concentration) and LOEC (lowest effect concentration)
for the tested compound 2dC showed different tendency. Its NOEC and LOEC values for oat are
10 and 100 mg/kg of soil dry weight, while for radish, NOEC is 100 and LOEC – 200 mg/kg s.d.w.
This values show that less concentration (i.e. less amount) of dibenzyl (ferrocenyl)(4-
methylphenylsulfonamido)-methylphosphonate (2dC) is harmful for monocotyledonous oat
than for dicotyledonous radish. These data coincide well with above observation concerning
growth inhibition factors – at smaller concentration values of 2dC, inhibition of shoot and fresh
matter growth was observed to be greater for oat. At higher concentrations, the compound
2dC was found to be more effective (harmful) for radish.
Growing concentration of applied sulfonylaminophosphonate 2dC in soil caused the gradual
increase of dry weight value of both tested plants (Fig. 4). This phenomenon was described
previously by us26-28 and is caused by absorbing nutrients and water by plants through their
roots. In result, plant growth and development are strongly dependent on the concentration of
mineral nutrients available in the soil. At the beginning of plant growth, roots uptake water and
absorb the nutrients very intensively being simultaneously exposed on toxic agents in soil. In
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the measure of the exposition time, roots stop to develop and grow, the water absorption
become more and more difficult and the level of dry matter in a plant starts to increase. Such a
pattern during the exposure process of plants on contaminants was already described.29,30
[Insert Figure 4]
Changes of pigment levels
Growing concentration of the investigated sulfonamidophosphonate 2dC in soil resulted in the
decrease of the level of total chlorophyll in green parts of both tested plants (Fig. 5). The level
of carotenoids in oat seedlings treated with the sulfonylaminophosphonate 2dCa was very
symptomatic. For both tested plants, the growing concentration of the investigated compound
in soil provoked the constant decrease of the carotenoid amount in green parts of radish. Any
symptoms of chlorosis and necrosis of treated plants have been observed in digital photographs
(Fig. 6)
[Insert Figure 5]
[Insert Figure 6]
The results coincide with the determined level of plant pigments in stress conditions.31,32
Symptoms of chlorosis appearing on plant leaves (yellowing or whitening of normally green
plant tissue) indicate a decrease of the chlorophyll amount and are symptoms of disease
(chlorosis) or nutrient deficiency.
The regular ratio of chlorophyll level to carotenoid level is a crucial factor reflecting the proper
functionality of the photosynthetic system. Carotenoid pigments are critical for plant survival,
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therefore their composition and amount strongly depends on the physiological and pathological
conditions of living.33,34
It is well known that carotenoids play a distinct role in photoprotection of plants against
exposure on biotic or abiotic factors, however correlation of carotenoid level with stress
conditions is ambiguous.35,36 As far as in a case of (5-nitrofurfuryl)-derived aminophosphonic
esters, the level of carotenoids of oat seedlings has decreased,28 while pyrrole-derived
aminophosphonates caused an increase of the level of pigments.27
Microtox® assay
Results of the toxicity assessment using Aliivibrio fischeri Gram-negative bacteria as examined
organisms demonstrated a rather low toxicity of the tested compound 2dC. (Table 3, Fig. 7). In
comparison with the previously published toxicities of ferrocene-derived aminophosphonates
against A. fischeri,26 where EC50 values varied from 48 up to 235 mg/kg s.d.w, the toxicity of
sulfonylaminophosphonate 2dC characterised by EC50 = 767.5 mg/kg s.d.w. is significantly
smaller.
According to Hernando et al.,37 the toxicity categories based on the EC50 values are as follows:
“very toxic to aquatic organisms” (EC50  1 mg/L), “toxic” (EC50 in the range of 1–10 mg/L), and
“harmful” (EC50 in the range of 10–100 mg/L), which are established in the Directive 93/67/EEC.
Following these regulations, it is to stress that toxicity of the studied aminophosphonate 2dC
equaling 581.0 mg/L is outside the upper limit of harmfulness.
[Insert Table 3]
[Insert Figure 7]
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Ostracodtoxkit® Test
The changes of crustacean Heterocypris incongruens mortality are presented in Fig. 8. The
impact of sulfonamidophosphonate 2dC on ostracods at concentrations 10, 50, 100 and 250
mg/kg s.d.w. indicate in an evident way that the investigated compound is significantly toxic
against ostracods. This toxicity seems to be impressively high when compared with ostracod
mortality of previously published ferrocene-derived aminophosphonates.26 While the mortality
(%) of the latter do not exceed 10% at 10 mg/kg s.d.w. (at 50 mg/kg, mortality% oscillates
around 20%).26 The mortality of ostracod after treating them with 2dC is 35% at 10 mg/kg
s.d.w. and 65% at 50 mg/kg. It is to state that sulfonamidophosphonate 2dC is very toxic for H.
incongruens crustaceans.
[Insert Figure 8]
Growth inhibition is the second criteria of the toxic effect indicated by the Ostracodtoxkit F™
microbiotest. This parameter allows for the evaluation of the sub-lethal toxicity of sediments
(soil in this case). As usually, the growth inhibition is determined by comparing the size of
surviving ostracods living in the test sediment with the size of ostracods living in the non
treated sediment at the end of the test. Determination of the sub-lethal impact of sediment
toxicants is justified only for sediments, which do not cause a high ostracod mortality.
According to manual of Ostracodtoxkit® test, the growth inhibition should only be determined
for sediments, which mortality was found to be less than 30%. Therefore, the growth inhibition
was not chosen as a criterion of sub-lethal effects to determine toxicity not inducing substantial
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mortality in the test organisms, because the mortality, for each studied concentration was
higher than 30%.
Considering the contents of the above mentioned instruction, and the results of mortality of H.
incongruens on contact with the compound 2dC, the growth inhibition tests was not performed
as even 10-mg doses gave the mortality of 35%.
Summing
up,
ecotoxicological
nature
of
dibenzyl
(ferrocenyl)(4-
methylphenylsulfonamido)methylphosphonate (2dC) is ambiguous. The compound 2dC is not in
fact toxic for two tested terrestrial higher plants: monocotyledonous oat and dicotyledonous
radish. It is completely not toxic for Aliivibrio fischeri bacteria tested in a Microtox® system
using the MSPT method.
However, its toxicity against crustaceans Heterocypris incongruens (ostracods) is surprisingly
elevated in comparison with aminophosphonates bearing a ferrocene moiety.26 The compound
2dC, even in concentration of 10 mg/kg s.d.w. caused the 35%-mortality of these invertebrates;
increasing the concentration up to 50 mg/kg s.d.w. the compound resulted in 65%-mortality.
Conclusions
In this work, the synthesis of a series of N-sulfonylaminophsophonates 2bA-2dC and evaluation
of some of their biological properties, i.e. cytotoxicity and ecotoxicity is described. In the two-
step synthesis, i.e. preparation of sulfonimines 1a-d followed by addition of phosphites, imines
were synthesized by the direct condensation of p-toluenesulfonamide with model aldehydes
catalyzed by titanium(IV) tetraethoxide. To our knowledge, this approach has been used only
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once,22 where authors described exclusively one case of preparation of 4-methyl-N-(2-
vinylbenzylidene)benzene-sulfonamide.22
Imines 1a-d were subsequently converted to N-sulfonylaminophsophonates 2bA-2dC by
addition of model dialkyl phosphites to their azomethine bonds mediated by potassium
carbonate; reactions were carried out in dichloromethane. Potassium carbonate was applied
only once as a promoter for the addition of dialkyl phosphites to sulfonaldimines as an example
of an α C-H oxidation and subsequent addition reactions of aryl sulfonamides.23
The N-sulfonylaminophsophonates 2bA-2dC were investigated in terms of their cytotoxic
properties against PBMCs and three human cancer cell lines A549, HL-60 and U87. The
evaluation revealed that no cytotoxic effect against PBMCs, A549, HL-60 and U87 cells up to 1
µM was observed. At the concentration 10 µM low cytotoxic effect was noticed, as cell viability
decreased approximately to 80%. Higher concentrations could not be tested due to the low
solubility of tested compounds in aqueous solutions.
Ecotoxicological
evaluation
of
the
model
dibenzyl
(ferrocenyl)(4-
methylphenylsulfonamido)methyl-phosphonate (2dC) was performed in three aspects.
Phytotoxicity against two model plants: monocotyledonous oat (Avena sativa) and
dicotyledonous radish (Raphanus sativus) was performed according to the OECD/OCDE
procedure. Tests revealed that the investigated compound was nearly completely not toxic for
tested plants (EC50 = 4806 mg/kg s.d.w for oat and 740 mg/kg s.d.w for radish fresh matter). It is
however to emphasize that the lowest effective concentration for oat was found to be 100
mg/kg s.d.w.
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Toxicity of the sulfonamidophosphonate (2dC) for bacteria was performed on marine bacteria
Aliivibrio fischeri, using the Microtox® measuring system. The compound (2dC) was found to be
practically not toxic for these microorganisms (EC50 = 581.0 mg/L = 767.5 mg/kg s.d.w).
Dibenzyl (ferrocenyl)(4-methylphenylsulfonamido)methylphosphonate (2dC) was found to be
toxic for freshwater crustaceans Heterocypris incongruens. The mortality% for the lowest
applied concentration (10 mg/kg s.d.w.) was 35%, while at 100 mg/kg s.d.w., mortality reached
90%. In comparison with N-arylaminophsophonates bearing ferrocene moiety, which even at
250 mg/kg s.d.w., mortality did not exceed 75%, it is to state that the tested compound 2dC is
toxic for ostracods.
To conclude, N-sulfonylaminophsophonates from the investigated series could be claimed to be
toxic neither for cell lines (in vitro tests), nor for prokaryotes. However, it is toxic for higher
organisms such as crustaceans and therefore, one should handle this type of compounds with
care.
Experimental
Chemical synthesis
All solvents (POCh, Gliwice, Poland) were routinely distilled and dried prior to use. Amines,
diphenyl phosphite, as well as pyrrole-2-carboxaldehyde (Aldrich, Poznao, Poland) were used as
received. Melting points were measured on a MelTemp II apparatus and were not corrected.
NMR spectra were recorded on a Bruker Avance III 600 MHz (Billerica, MA, USA) operating at
600 MHz (1H NMR), 150 MHz (13C NMR) and 243 MHz (31P NMR). FT-IR spectra were recorded
on a Nexus FT-IR Thermo Nicolet. ESI-MS were recorded on a Varian 500-MS LC Ion Trap mass
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spectrometer (Palo Alto, CA, USA). Elemental analyses were carried out at the Laboratory of
Microanalysis, Faculty of Chemistry, University of Łódź, Poland.
Preparation of p-toluenesulfonimines 1a-d. General procedure.
To an appropriate aldehyde (2 mmol) dissolved in dichloromethane, p-toluenesulfonamide
(0.34 g, 2 mmol) and titanium(IV) ethoxide (0.9 g, 4 mmol) were added. A mixture was stirred at
room temperature for 48 hours then water (15 mL) was added and stirred for 5 min. Titanium
oxide precipitated, which was washed 15 times with small portions of dichloromethane and
finally with a small amount of water. The combined filtrates were placed in a separating funnel,
an organic layer was separated, dried with MgSO4 and evaporated. Obtained crude solids were
crystallized with hexane-ethyl acetate (6:2) to give pure imines 1a-d.
N-(4-methoxybenzylidene)-4-methylbenzenesulfonamide (1a).
Y = 0.53 g (91%). m.p. = 132–134°C (white powder), lit.38 131–132°C or lit.39 117°C. 1H NMR (600
3
3
MHz, CDCl3): δ 8.94 (s, CH=N, 1H); 7.88 (d, JHH = 8.7 Hz, p-C6H4, 2H); 7.87 (d, JHH = 8.0 Hz, p-
3
3
C6H4, 2H); 7.33 (d, JHH = 8.2 Hz, p-C6H4, 2H); 6.97 (d, JHH = 8.8 Hz, p-C6H4, 2H); 3.88 (s, OCH3,
3H); 2.43 (s, CH3, 3H).
N-(2- methoxybenzylidene)-4-methylbenzenesulfonamide (1b).
1
Y = 0.50 g (86%). m.p. = 111–113°C (white powder), lit.40 110–111°C. H NMR (600 MHz, CDCl3):
3
4
3
δ 9.55 (s, CH=N, 1H); 8.05 (dd, JHH = 7.8 Hz, JHH = 1.7 Hz, o-C6H4, 1H); 7.88 (d, JHH = 8.3 Hz, p-
3 (1)
3 (2)
4
3
C6H4, 2H); 7.55 (ddd, J
= J
= 8.7 Hz, JHH = 1,7 Hz, o-C6H4, 1H); 7.32 (d, JHH = 8,2 Hz, p-
HH
HH
C6H4, 2H); 6.95-6.99 (m, o-C6H4, 2H); 3.92 (s, OCH3, 3H); 2.43 (s, CH3, 3H).
N-Thienylidene-4-methylbenzenesulfonamide (1c).
17
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Y = 0.46 g (87%). m.p. = 125–127°C (yellow crystals), lit.41 99–100°C. 1H NMR (600 MHz, CDCl3):
3
3
δ 9.11 (s, CH=N, 1H); 7.87 (d, JHH = 8.3 Hz, p-C6H4, 2H); 7.77 (m, Hthioph, 2H); 7.33 (d, JHH = 8.1
Hz, p-C6H4, 2H); 7.20 (dd, 3JHH = 4.3 Hz, 3JHH = 8.7 Hz, Hthioph, 1H); 2.43 (s, CH3, 3H).
N-Ferrocenylidene-4-methylbenzenesulfonamide (1d)42-45
1
Y = 0.68 g (92%). m.p. = 135–137°C (orange powder). H NMR (600 MHz, CDCl3): δ 9.05 (s,
3
3
CH=N, 1H); 7.86 (d, JHH = 8.1 Hz, p-C6H4, 2H); 7.32 (d, JHH = 8.0 Hz, p-C6H4, 2H); 4.83 (m, CHfer,
2H); 4.71 (m, CHfer, 2H); 4.19 (s, Cp, 5H), 2.42 (s, CH3, 3H).
Elem. anal. Calcd for C18H17FeNO2S: C 58.87; H 4.67; N 3.81. Found: C 58.89; H 4.72; N 3.79.
Preparation of N-(p-toluenosulfonamido)methylphosphonic Acids Esters 2bA-dC. General
procedure.
To a corresponding sulfonimine (1 mmol) dissolved in dichloromethane, an appropriate
phosphite (4 mmol) and potassium carbonate (5 mmol) were added. The obtained mixture was
stirred at room temperature for 1 week (reaction progress was monitored by 31P NMR) and
after completion of the reaction, a small amount of saturated aqueous ammonium chloride was
added. An organic layer was separated and washed 3 times with water (3x5 mL) and once with
brine (5 mL), dried with MgSO4 and evaporated. Solid residue was dissolved in a small portion of
dichloromethane, and triple amount of hexane was added to precipitate pure
aminophosphonates 2bA-dC.
Diethyl (2-methoxyphenyl)(4-methylphenylsulfonamido)-methylphosphonate (2bA).
3
YIELDield = 0.17 g (40%). m.p. 175-177°C. 1H NMR (600 MHz, CDCl3): δ 7.46 (d, JHH = 8.2 Hz, p-
3
C6H4, 2H); 7.13-7.12 (m, o-C6H4, 1H); 7.10-7.07 (m, o-C6H4, 1H); 6.97 (d, JHH = 8.0 Hz, p-C6H4,
18
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3
3
3
2H); 6.72 (t, JHH = 7.5Hz, o-C6H4, 1H); 6.61 (d, JHH = 8.2Hz, o-C6H4, 1H); 6.02 (dd, JHH = 3.3Hz,
3
2
3JHH = 10.2Hz, NH, 1H); 5.19 (dd, JHH = 10.3Hz, JPH = 24.5Hz, CHP, 1H), 4.22-4.17 (m,
POCH2CH3, 2H); 3.92-3.85 (m, POCH2CH3, 1H); 3.74 (s, OCH3, 3H); 3.68-3.64 (m, POCH2CH3, 1H);
3
3
2.27 (s, CH3, 3H); 1.33 (t, JHH = 7.1Hz, POCH2CH3, 3H); 1.03 (t, JHH = 7.1Hz, POCH2CH3, 3H). 13C
NMR (150 MHz, CDCl3): δ 156.54 (d, 2JPC = 5.8Hz, Carom); 142.66 (Carom); 137.53 (Carom); 129.44 (s,
3
3JPC = 4.2Hz, Carom); 128.98 (d, JPC = 2.7Hz, Carom); 128.72 (Carom); 127.01 (Carom); 122.19 (Carom);
4
2
120.58 (d, JPC = 1.9Hz, Carom); 110.40 (Carom); 63.78 (d, JPC = 6.8Hz, POC); 63.32 (d, 2JPC = 6.8Hz,
1
3
POC); 55.50 (OCH3); 49.31 (d, JPC = 163,9Hz, PC); 21.29 (CH3); 16.43 (d, JPC = 5.7Hz, POCC);
3
16.01 (d, JPC = 5.6Hz, POCC). 31P NMR (243 MHz, CDCl3): δ 19.75. IR (KBr): 3432 (NH); 3155 (C-
Harom); 1061, 1497 (C=Carom); 1336 (S=O); 1239 (P=O); 1161 (S=O); 1060, 1023 (P-O-C). ESI-MS
m/z 428 (M+H+); 450 (M+Na); 466 (M+K). Elem. anal. Calcd for C19H26NO6PS: C 53.39; H 6.13; N
3.28. Found: C 53.32; H 6.05; N 3.16
Diethyl (2-thienyl)(4-methylphenylsulfonamido)-methylphosphonate (2cA).
Yield = 0.07 g (17%). m.p. 138-141°C. 1H NMR (600 MHz, CDCl3): δ 7.58 (d, 3JHH = 8.2 Hz, p-C6H4,
4
3
2H); 7.13-7.12 (m, p-C6H4 and Hthioph, 3H); 6.94 (m, Hthioph, 1H); 6.80 (dd, JHH = 0.8Hz, JHH
=
=
3
3
2
3
4.7Hz, Hthioph, 1H); 5.80 (dd, JHH = 4.6Hz, JPH = 9.7Hz, NH, 1H); 5.07 (dd, JPH 23.6Hz, JHH
9.3Hz, CHP, 1H); 4.26-4.18 (m, POCH2CH3, 2H); 4.04-4.00 (m, POCH2CH3, 1H); 3.83-3.79 (m,
3
3
POCH2CH3, 1H); 2.36 (s, CH3, 3H); 1.35 (t, JHH = 7.1Hz, POCH2CH3, 3H); 1.14 (t, JHH = 7.0Hz,
POCH2CH3, 3H). 13C NMR (150 MHz, CDCl3): δ 142.89 (Carom); 138.05 (Cthioph); 136.16 (Carom);
2
4
129.05 (Carom); 127.69 (d, JPC = 7.4Hz, Cthioph); 127.04 (Carom); 126.67 (d, JPC = 2Hz, Cthioph);
125.77 (d, 3JPC = 3.1Hz, Cthioph); 64.28 (d, 2JPC = 7.2Hz, POC); 63.81 (d, 2JPC = 7.0Hz, POC); 50.62 (d,
19
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1JPC = 162.9Hz, PCN); 21.39 (CH3); 16.40 (d, 2JPC = 5.6Hz, POCC); 16.11 (d, 2JPC = 5.7Hz, POCC). 31P
NMR (243 MHz, CDCl3): δ 18.01. IR (KBr): 3432 (NH); 3109 (C-Harom), 1598, 1460 (C=Carom); 1333
(S=O); 1234 (P=O); 1163 (S=O); 1055, 1020 (P-O-C). ESI-MS m/z 426 (M+Na); 442 (M+K). Elem.
anal. Calcd for C16H22NO5PS2: C 47.63; H 5.50; N 3.47. Found: C 47.89; H 5.63; N 3.28
Diethyl (ferrocenyl)(4-methylphenylsulfonamido)-methylphosphonate (2dA).
Yield = 0.25 g (49%). m.p. 173-175°C. 1H NMR (600 MHz, CDCl3): δ 7.84 (d, 3JHH = 8.2 Hz, p-C6H4,
2H); 7.31 (d, 3JHH = 8.0 Hz, p-C6H4, 2H); 5.10 (dd, 3JHH = 3.6Hz, 3JPH = 8.7Hz, NH, 1H); 4.62 (dd, 2JPH
= 18.7Hz, 3JHH = 9.1Hz, CHP, 1H); 4.26 (s, CHfer, 5H); 4.22 (m, CHfer, 1H); 4.17 (m, CHfer, 1H); 4.15-
4.14 (m, CHfer, 2H); 3.96-3.87 (m, POCH2CH3, 2H); 3.77-3.67 (m, POCH2CH3, 2H); 2.43 (s, CH3,
3H); 1.14 (t, 3JHH = 7.1Hz, POCH2CH3, 3H); 1.12 (t, 3JHH = 7.1Hz, POCH2CH3, 3H). 13C NMR (150
MHz, CDCl3): δ 142.85 (Carom); 139.37 (Carom); 129.31 (2x Carom); 127.00 (Carom); 84.22 (d, 2JPC =
6.9Hz, Cfer); 69.24 (Cfer); 68.15 (d, 3JPC = 3.4Hz, Cfer); 67.90 (Cfer); 67.83 (Cfer); 67.06 (d, 3JPC =
3.6Hz, Cfer); 63.06 (d, 2JPC = 6.8Hz, POC); 62.80 (d, 2JPC = 7.5Hz, POC); 50.58 (d, 1JPC = 158.9Hz,
PCN); 21.48 (CH3); 16.28 (d, 3JPC = 5.5Hz, POCC); 16.23 (d, 3JPC = 5.5Hz, POCC). 31P NMR (243
MHz, CDCl3): δ 19.69. IR (KBr): 3432 (NH); 3118 (C-Harom), 1598, 1476 (C=Carom); 1325 (S=O);
1231 (P=O); 1141 (S=O); 1055, 1027 (P-O-C). ESI-MS m/z 528.1 (M+Na); 544.1 (M+K). Elem.
anal. Calcd for C22H28FeNO5PS: C 52.29; H 5.58; N 2.77. Found: C 52.88; H 5.85; N 2.55
Diisopropyl (4-methoxyphenyl)(4-methylphenylsulfonamido)-methylphosphonate (2aB).
Yield = 0.29 g (64%). m.p. 139-140°C, lit14 130-131°C. 1H NMR (600 MHz, CDCl3): δ 7.43 (d, 3JHH
=
8.2Hz, p-C6H4, 2H); 7.10 (dd, 3JHH = 8.6Hz, 4JHH = 1.9Hz, p-C6H4, 2H); 6.94 (d, 3JHH = 7.9Hz, p-C6H4,
2H); 6.68-6.66 (m, NH, 1H); 6.57 (d, 3JHH = 8.5Hz, p-C6H4, 2H); 4.84-4.80 (m, OCH(CH3)2, 1H); 4.66
20
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(dd, 3JHH = 9.5Hz, 2JPH = 24.2Hz, CHP, 1H); 4.38-4.34 (m, OCH(CH3)2, 1H); 3.71 (s, OCH3, 3H); 2.26
(s, CH3, 3H); 1.36 (m, CH3, 6H); 1.21 (d, 3JHH = 6.2Hz, CH3, 3H); 0.82 (d, 3JHH = 6.2Hz, CH3, 3H). 13C
NMR (150 MHz, CDCl3): δ 159.16 (d, 4JPC = 2.2Hz, Carom); 142.30 (Carom); 138.36 (Carom); 129.75 (d,
2
3JPC = 5.8Hz, Carom); 128.75 (Carom); 127.09 (Carom); 126.09 (Carom); 113.34 (Carom); 72.65 (d, JPC
=
2
1
7.5Hz, POC); 72.38 (d, JPC = 7.1Hz, POC); 55.29 (d, JPC = 160.6Hz, PC); 55.22 (OCH3); 24.34 (d,
3
3
3
3JPC = 3.1Hz, POCC); 24.16 (d, JPC = 3.0Hz, POCC); 23.80 (d, JPC = 5.6Hz, POCC); 23.02 (d, JPC
=
6.1Hz, POCC); 21.27 (CH3). 31P NMR (243 MHz, CDCl3): δ 18.09. IR (KBr): 3421 (NH); 3136 (C-
Harom), 1612, 1515 (C=Carom); 1309 (S=O); 1235 (P=O); 1159 (S=O); 1037, 1007 (P-O-C). ESI-MS
m/z 478 (M+Na); 494 (M+K).
Diisopropyl (2-methoxyphenyl)(4-methylphenylsulfonamido)-methylphosphonate (2bB).
Yield = 0.07 g (16%). m.p. 210-212°C. 1H NMR (600 MHz, CDCl3): δ 7.44 (d, 3JHH = 8.1 Hz, p-C6H4,
2H); 7.12-7.10 (m, o-C6H4, 1H); 7.08-7.04 (m, o-C6H4, 1H); 6.94 (d, 3JHH = 8.1 Hz, p-C6H4, 2H); 6.69
3
3
3
(t, JHH = 7.5Hz, o-C6H4, 1H); 6.57 (d, JHH = 8.2Hz, o-C6H4, 1H); 5.89 (d, JHH = 6.4 Hz, NH, 1H);
3
2
5.14 (dd, JHH = 9.4Hz, JPH = 23.8Hz, CHP, 1H), 4.82-4.77 (m, POCH(CH3)2, 1H); 3.38-3.33 (m,
3
POCH(CH3)2, 1H); 3.72 (s, OCH3, 3H); 2.25 (s, CH3, 3H); 1.36 (d, JHH = 6.2Hz, POCH(CH3)2, 3H);
3
3
3
1.35 (d, JHH = 6.2Hz, POCH(CH3)2, 3H); 1.18 (d, JHH = 6.2Hz, POCH(CH3)2, 3H); 0.80 (d, JHH
=
2
6.2Hz, POCH(CH3)2, 3H). 13C NMR (150 MHz, CDCl3): δ 156.73 (d, JPC = 5.6 Hz, Carom); 142.75
3
3
(Carom); 137.36 (Carom); 129.33 (s, JPC = 4.2Hz, Carom); 128.88 (d, JPC = 2.9 Hz, Carom); 128.76
(Carom); 127.03 (Carom); 122.44 (Carom); 120.50 (d, 4JPC = 1.9 Hz, Carom); 110.49 (Carom); 72.35 (d, 2JPC
= 7.4 Hz, POC); 72.10 (d, 2JPC = 7.1 Hz, POC); 55.42 (OCH3); 50.30 (br s, PC); 24.26 (d, 3JPC = 3.2Hz,
POCC); 24.14 (d, 3JPC = 3.2Hz, POCC); 23.77 (d, 3JPC = 5.5Hz, POCC); 22.91 (d, 3JPC = 6.1Hz, POCC);
21
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21.29 (CH3). 31P NMR (243 MHz, CDCl3): δ 18.21. IR (KBr): 3432 (NH); 3149 (C-Harom); 1059,
1497 (C=Carom); 1336 (S=O); 1239 (P=O); 1161 (S=O); 1025 (P-O-C). ESI-MS m/z 478 (M+Na);
494 (M+K). Elem. anal. Calcd for C21H30NO6PS: C 55.37; H 6.64; N 3.07. Found: C 54.98; H 6.61 N
2.95.
Diisopropyl (2-thienyl)(4-methylphenylsulfonamido)-methylphosphonate (2cB).
Yield = 0.10 g (23%). m.p. 204-205°C. 1H NMR (600 MHz, CDCl3): δ 7.52 (d, 3JHH = 8.3 Hz, p-C6H4,
2H); 7.09-7.07 (m, p-C6H4 and Hthioph, 3H); 6.89-6.88 (m, Hthioph, 1H); 6.75 (dd, 3JHH = 4.0Hz, 3JHH
=
=
3
3
2
3
4.8Hz, Hthioph, 1H); 5.60 (dd, JHH = 9.1Hz, JPH = 5.2Hz, NH, 1H); 4.99 (dd, JPH = 23.9Hz, JHH
9.2Hz, CHP, 1H); 4.80-4.74 (m, OCH(CH3)2, 1H); 4.52-4.47 (m, OCH(CH3)2, 1H); 2.33 (s, CH3, 3H);
1.35 (d, 3JHH = 6.2Hz, CH3, 3H); 1.33 (d, 3JHH = 6.2Hz, CH3, 3H); 1.24 (d, 3JHH = 6.1Hz, CH3, 3H); 0.94
3
(d, JHH = 6.2Hz, CH3, 3H). 13C NMR (150 MHz, CDCl3): δ 143.08 (Carom); 137.79 (Carom); 136.68
2
4
(Carom); 129.18 (Carom); 127.53 (d, JPC = 7.6Hz, Cthioph); 127.05 (Carom); 126.51 (d, JPC = 1.9Hz,
3
2
2
Cthioph); 125.84 (d, JPC = 3.0Hz, Cthioph); 72.97 (d, JPC = 8.3Hz, POC); 72.92 (d, JPC = 8.3Hz, POC);
1
3
3
51.15 (d, JPC = 162.6Hz, PC); 24.21 (d, JPC = 3.4Hz, POCC); 24.15 (d, JPC = 3.1Hz, POCC); 23.70
3
3
(d, JPC = 5.5Hz, POCC); 23.08 (d, JPC = 5.6Hz, POCC); 21.40 (CH3). 31P NMR (243 MHz, CDCl3): δ
16.37. IR (KBr): 3424 (NH); 3131 (C-Harom), 1600, 1453 (C=Carom); 1335 (S=O); 1241 (P=O); 1163
(S=O); 1060, 992 (P-O-C). ESI-MS m/z 454 (M+Na); 470 (M+K). Elem. anal. Calcd for
C18H26NO5PS2: C 50.10; H 6.07; N 3.25. Found: C 50.09; H 6.04 N 3.19.
Diisopropyl (ferrocenyl)(4-methylphenylsulfonamido)-methylphosphonate (2dB).
Yield = 0.19 g (36%). m.p. 182-183°C. 1H NMR (600 MHz, CDCl3): δ 7.85 (d, 3JHH = 7.2Hz, p-C6H4,
2H); 7.31 (d, 3JHH = 7.5Hz, p-C6H4, 2H); 5.08 (dd, 3JHH = 4.0Hz, 3JPH = 8.9Hz, NH, 1H); 4.64 (dd, 2JPH
22
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= 19.4Hz, 3JHH = 8.9Hz, CHP, 1H); 4.51-4.42 (m, OCH(CH3)2, 2H); 4.28 (m, CHfer, 1H); 4.24 (s, CHfer,
3
5H); 4.19 (m, CHfer, 1H); 4.14-4.11 (m, CHfer, 2H); 2.43 (s, CH3, 3H); 1.20 (d, JHH = 5.8 Hz, CH3,
3H); 1.17 (d, 3JHH = 5.8Hz, CH3, 3H); 1.10 (d, 3JHH = 5.8Hz, CH3, 3H); 1.06 (d, 3JHH = 5.8Hz, CH3, 3H).
13C NMR (150 MHz, CDCl3): δ 143.16 (Carom); 139.09 (Carom); 129.44 (Carom); 127.18 (Carom); 84.61
(d, 2JPC = 6.5Hz, Cfer); 71.79 (d, 2JPC = 7.0Hz, POC); 71.76 (d, 2JPC = 7.2Hz, POC); 69.26 (Cfer); 68.25
(d, 3JPC = 4.4Hz, Cfer); 67.92 (Cfer); 67.72 (Cfer); 67.01 (d, 3JPC = 4.2Hz, Cfer); 51.26 (d, 1JPC = 158.6Hz,
CHP); 24.02 (d, 3JPC = 3.3Hz, POCC); 23.93 (d, 3JPC = 3.2Hz, POCC); 23.59 (d, 3JPC = 2.41Hz, POCC);
21.48 (CH3). 31P NMR (243 MHz, CDCl3): δ 17.66. IR (KBr): 3439 (NH); 3092 (C-Harom), 1630, 1598
(C=Carom); 1332 (S=O); 1237 (P=O); 1161 (S=O); 1047, 1036 (P-O-C). ESI-MS m/z 556 (M+Na); 572
(M+K). Elem. anal. Calcd for C24H32NO5PSFe: 54.04; H 6.05; N 2.63. Found: C 53.85; H 5.99; N
2.44.
Dibenzyl (4-methoxyphenyl)(4-methylphenylsulfonamido)-methylphosphonate (2aC).
1
3
Yield = 1.20 g (74%). m.p. 159-161°C. H NMR (600 MHz, CDCl3): δ 7.47 (d, JHH = 8.2Hz, ArH,
4
3
2H); 7.33-7.29 (m, ArH, 5H); 7.26-7.22 (m, ArH, 3H); 7.11 (dd, JHH = 2.0 Hz, JHH = 8.7 Hz, ArH,
3
3
2H); 7.104-7.03 (m, ArH, 2H); 6.96 (d, JHH = 8.1 Hz, ArH, 2H); 6.62 (d, JHH = 8.6 Hz, ArH, 2H);
3
3
2
6.31 (dd, JPH = 5.5Hz, JHH = 9.1Hz, NH, 1H); 5.08 and 5.01 (Part of ABX system, JHH = 11.8 Hz,
3
2
3JPH = 7.2 and 9.1 Hz, POCH, 1H); 4.82 (dd, JHH = 9.4 Hz, JPH = 23.7 Hz, CHP, 1H); 4.77 (Part of
3
ABX system, 3JPH = 7.4 Hz, 2JHH = 11.8 Hz, POCH, 1H); 4.52 (Part of ABX system, JPH = 8.8Hz, 2JHH
= 11.8 Hz, POCH, 1H); 3.73 (s, OCH3, 3H); 2.27 (s, CH3, 3H). 13C NMR (150 MHz, CDCl3): δ 159.49
2
3
(d, JPC = 2.1 Hz, Carom); 142.82 (Carom); 137.85 (Carom); 135.95 (d, JPC = 6.3 Hz, Carom); 135.70 (d,
3JPC = 5.7 Hz, Carom); 129.49 (d, 3JPC = 6.3 Hz, Carom); 129.06 (Carom); 128.56 (Carom); 12.44 (d, 3JPC
=
23
ACCEPTED MANUSCRIPT

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6.2Hz, Carom); 128.34 (Carom); 127.82 (Carom); 127.12 (Carom); 125.52 (Carom); 113.80 (Carom); 69.09
(d, 2JPC = 7.2Hz, POC); 68.70 (d, 2JPC = 7.0 Hz, POC); 55.24 (OCH3); 54.77 (d, 1JPC = 157.0 Hz, CPN);
21.34 (CH3). 31P NMR (243 MHz, CDCl3): δ 20.70. IR (KBr): 3432 (NH); 3115 (C-Harom), 1610, 1512
(C=Carom); 1308 (S=O); 1233 (P=O); 1161 (S=O); 1032, 1010 (P-O-C). ESI-MS m/z 550 (M-H+).
Elem. anal. Calcd for C29H30NO6PS: C 63.15; H 5.48; N 2.54. Found: C 62.85; H 5.28; N 2.48.
Dibenzyl (2-methoxyphenyl)(4-methylphenylsulfonamido)-methylphosphonate (2bC).
1
3
Yield = 0.34 g (62%). m.p. 121-122°C. H NMR (600 MHz, CDCl3): δ 7.48 (d, JHH = 8.3 Hz, ArH,
3
2H); 7.36-7.30 (m, ArH, 5H); 7.25-7.19 (m, ArH, 4H); 7.10-7.07 (m, ArH , 1H); 7.00 (d, JHH
=
3
3
6.7Hz, ArH, 2H); 6.91 (d, JHH = 8.0Hz, ArH, 2H); 6.71-6.69 (m, ArH, 1H); 6.59 (dd, JHH = 10.3Hz,
3JPH = 2.7Hz, NH, 1H); 6.56 (d, 3JHH = 8.3Hz, ArH, 1H), 5.39 (dd, 2JPH = 24.9Hz, 3JHH = 10.3Hz, CHP,
1H); 5.18-5.09 (Part of ABX system, 2JHH = 11.7 Hz, 3JPH = 7.0 and 8.7 Hz, POCH, 2H); 4.80 (Part of
2
3
2
ABX system, JHH = 11.8 Hz, JPH = 6.8 Hz, POCH, 1H); 4.55 (Part of ABX system, JHH = 11.8 Hz,
3JPH = 8.0 Hz, POCH, 1H); 3.60 (s, OCH3, 3H); 2.23 (s, CH3, 3H). 13C NMR (150 MHz, CDCl3): δ
3
3
156.54 (d, JPC = 5.7Hz, Carom); 142.75 (Carom); 137.46 (Carom); 136.22 (d, JPC = 5.9Hz, Carom);
135.94 (d, 3JPC = 6.4Hz, Carom); 129.50 (d, 3JPC = 4.5Hz, Carom); 129.16 (d, 4JPC = 2.3Hz, Carom); 128.8
(Carom); 128.51 (Carom); 128.34 (Carom); 128.31 (Carom); 128.12 (Carom); 127.56 (Carom); 127.04
(Carom); 121.96 (Carom); 120.67 (d, 4JPC = 1.9Hz, Carom); 110.53 (Carom); 69.08 (d, 2JPC = 7.3Hz, POC);
2
1
68.39 (d, JPC = 6.9Hz, POC); 55.36 (OCH3); 49.71 (d, JPC = 161.3Hz, PC); 21.32 (CH3). 31P NMR
(243 MHz, CDCl3): δ 20.67. IR (KBr): 3432 (NH); 3153 (C-Harom); 1051, 1495 (C=Carom); 1334 (S=O);
1247 (P=O); 1167 (S=O); 1017 (P-O-C). ESI-MS m/z 574 (M+Na); 590 (M+K). Elem. anal. Calcd for
C29H30NO6PS: C 63.15; H 5.48; N 2.54. Found: C 63.49; H 5.31; N 2.45.
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Dibenzyl (2-thienyl)(4-methylphenylsulfonamido)-methylphosphonate (2cC).
1
3
Yield = 0.30 g (55%). m.p. 115-117°C. H NMR (600 MHz, CDCl3): δ 7.55 (d, JHH = 8.2 Hz, ArH,
3
2H); 7.34-7.24 (m, ArH, 7H); 7.11-7.09 (m, ArH, 3H); 7.03 (d, JHH = 8.2 Hz, ArH, 2H); 6.96-6.95
2
3
3
3
(m, Hthioph, 1H); 6.76 (dd, JPH = 4.9Hz, JHH = 3.7Hz, Hthioph, 1H); 6.28 (dd, JHH = 9.4Hz, JPH
=
=
4.0Hz, NH, 1H); 5.15 (d, 2JPH = 23.9 and JHH = 9.5 Hz, CHP, 1H); 5.11 (Part of ABX system, JHH
3
2
3
2
3
11.6 Hz, JPH = 7.3 Hz, POCH, 1H); 5.03 (Part of ABX system, JHH = 11.6 Hz, JPH = 9.2Hz, POCH,
2
3
1H); 4.87 (Part of ABX system, JHH = 11.7Hz, JPH = 7.3Hz, POCH, 1H); 4.62 (Part of ABX system,
3
2JHH = 11.6Hz, JPH = 8.9Hz, POCH, 1H); 2.30 (CH3). 13C NMR (150 MHz, CDCl3): δ 143.05 (Carom);
3
3
137.79 (Carom); 137.86 (Carom); 135.99 (Carom); 135.85 (d, JPC = 6.1Hz, Carom); 135.62 (d, JPC
=
5.7Hz, Carom); 129.18 (Carom); 128.57 (Carom); 128.49 (d, 4JPC = 3.2Hz, Carom); 128.43 (Carom); 128.14
4
4
(Carom); 127.88 (d, JPC = 4.5Hz, Carom); 127.09 (Carom); 126.81 (d, JPC = 1.8Hz, Carom); 126.10 (d,
2
2
1
4JPC = 3.1Hz, Carom); 69.43 (d, JPC = 6.7Hz, POC); 69.02 (d, JPC = 6.7Hz, POC); 50.78 (d, JPC
=
163.0Hz, CPN); 21.42 (CH3). 31P NMR (243 MHz, CDCl3): δ 18.85. IR (KBr): 3432 (NH); 3152 (C-
Harom), 1599, 1451 (C=Carom); 1322 (S=O); 1237 (P=O); 1157 (S=O); 1061, 995 (P-O-C). ESI-MS m/z
550 (M+Na); 566 (M+K). Elem. anal. Calcd for C26H26NO5PS2: C 59.19; H 4.97; N 2.65. Found: C
59.08; H 4.84; N 2.64.
Dibenzyl (ferrocenyl)(4-methylphenylsulfonamido)-methylphosphonate (2dC).
1
3
Yield = 0.48 g (77%). m.p. 173-175°C. H NMR (600 MHz, CDCl3): δ 7.80 (d, JHH = 8.3Hz, ArH,
2H); 7.29-7.26 (m, ArH, 6H); 7.20 (d, 3JHH = 8.1Hz, ArH, 2H); 7.14-7.11 (m, ArH, 4H); 5.22 (dd, 3JPH
3
2
3
= 3.7Hz, JHH = 8.9Hz, NH, 1H); 4.82 (Part of ABX system, JHH = 11.8Hz, JPH = 7.8Hz, POCH, 1H);
2
4.77-4.71 (m, POCH, CHP, 2H); 4.55 (Part of ABX system, JHH = 11.6Hz, 3JPH = 8.9Hz, POCH, 1H);
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4.46 (Part of ABX system, 2JHH = 11.6 Hz, 3JPH = 10.0Hz, POC, 1H); 4.24 (s, C5H5, 5H); 4.24-4.23 (m,
CHfer, 1H); 4.19 (m, CHfer, 1H); 4.16-4.15 (m, CHfer, 2H); 2.36 (s, CH3, 3H). 13C NMR (150 MHz,
3
3
CDCl3): δ 143.16 (Carom); 138.82 (Carom); 136.00 (d, JPC = 5.3Hz, Carom); 135.87 (d, JPC = 5.5Hz,
3
Carom); 129.44 (Carom); 128.47 (Carom); 128.39 (d, JPC = 5.2Hz, Carom); 128.09 (Carom); 127.97
(Carom); 127.10 (Carom); 84.09 (d, 2JPC = 6.5Hz, Cfer); 69.30 (Cfer); 68.42 (d, 2JPC = 7.3Hz, POC); 68.30
3
2
3
1
(d, JPC = 4.3Hz, Cfer); 68.14 (d, JPC = 7.6Hz, POC); 66.96 (d, JPC = 3.4Hz, Cfer); 50.81 (d, JPC
=
158.8 Hz, CP); 21.51 (CH3). 31P NMR (243 MHz, CDCl3): δ 20.52. IR (KBr): 3432 (NH); 3104 (C-
Harom), 1625, 1599 (C=Carom); 1327 (S=O); 1234 (P=O); 1162 (S=O); 1020, 992 (P-O-C). ESI-MS m/z
652 (M+Na); 668 (M+K). Elem. anal. Calcd for C32H32FeNO5PS: C 61.06; H 5.12; N 2.23. Found: C
61.12; H 5.19; N 2.10.
Evaluation of cytotoxicity of compounds 2aB-2cB, 2aC-2cC, 2bA and 2dA
Preparation of drug stock and working solutions
Stock solutions of phosphonates were prepared at the concentration of 1 mM in DMSO. The
stock solutions of compounds were diluted in culture medium with the final concentration of
DMSO in culture medium lower than 1%.
Cell Culture
PBMCs were isolated by density gradient centrifugation over Histopaque 1077 (Sigma-Aldrich,
Poznan, Poland) from leukocyte buffy coat obtained from the Blood Donation Center in Lodz,
Poland as described by Böyum.46 A549 cell line was purchased from American Type Culture
Collection (ATCC, Rockville, MD, USA) and U87 from HPA Culture Collections (Sigma-Aldrich,
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Poznan, Polska). HL-60 was a kind gift from dr Adrianna Nowak from Łódź University of
Technology.
PBMCs were cultured in RPMI 1640 medium (Lonza, Basel, Switzerland) with 10% foetal bovine
serum (FBS, Biowest, Nuaillé, France), A549 cells were cultured in DMEM:F-12 1:1 mixture
medium (Lozna) with 10% FBS, HL-60 cells were maintained in IMDM medium (Lonza) with 15%
FBS and U87 cells were grown in EMEM (Sigma-Aldrich) with 10% FBS, 1% non-essential amino
acids (Sigma-Aldrich) and 1 mM sodium pyruvate (Sigma-Aldrich). All media were
supplemented with 100 units/ml penicillin (Lonza) and 100 μg/ml streptomycin (Lonza).
Cultures were maintained in an incubator with 5% CO2 at 100% humidity and 37 °C.
Resazurin cell viability assay
Cancer cells and PBMCs were plated in 96-well plates at a density of 1 × 104 or 5 × 104,
respectively, in 100 µl of culture medium. After overnight adaptation, the cells were exposed in
triplicate to different concentrations of studied compounds for subsequent 24 h. All
experiments included a negative control and additionally a solvent control.
The cytotoxic effect of studied compounds was evaluated with a system based on the weakly
fluorescent blue dye, resazurin, which upon reduction by living cells becomes highly red
fluorescent resorufin using In Vitro Toxicology Assay Kit (Sigma-Aldrich, Poznan, Poland).24 After
the incubation, resazurin dye solution was added in an amount equal to 10% of the culture
medium volume and the cells were incubated for another 3 h. Then the fluorescence intensity
was measured with the excitation and emission set at 530/25 nm and 590/35 nm, respectively
using a Synergy HT spectrophotometer (Biotek, Bad Friedrichshall, Germany). Relative cell
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viability (%) was expressed as a percentage relative to the untreated cells. GraphPad Prism
(version 5.01, GraphPad Software Inc., San Diego, CA, USA) was employed to produce graphs.
All results were presented as mean ± SEM.
Evaluation of ecotoxicity of dibenzyl (ferrocenyl)(4-methylphenylsulfonamido)-
methylphosphonate (2dC)
Phytotoxicity
The evaluation of phytotoxicity of the investigated aminophosphonate 2dC at applied
concentrations was made by comparing the germination, dry weight of control plants sprouts
(seedlings) with germination and of dry and fresh plants sprouts grown in the soil with an
admixture of given amounts of the tested compounds. In order to determine growth inhibition
of root and shoot of selected plants, height of shoot and length of root were measured as
described by Wang et al.47 The length of plants is defined as the length the tip of the longest
leaf to the base of culms, while root length is measured from the tip of longest root to the root-
shoot junction.
Inhibition ratio was calculated according to method given by Pawłowska and Biczak,48 namely:
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