Synthesis, spectral characterization of several novel pyrene-derived aminophosphonates and their ecotoxicological evaluation using heterocypris incongruens and vibrio fisheri tests

Article abstract of DOI:10.3390/molecules21070936

: Four diphenyl pyrene-derived aminophosphonates were synthesized. Attempts were made to synthesize diphenyl N-(R)-α-methylbenzylamino(pyren-1-yl)methylphosphonate (3e) in order to obtain the chiral aminophosphonate bearing a pyrene moiety. Because these attempts failed, dimethyl and dibenzyl N-(R)-α-methylbenzyl substituted aminophosphonates 4 and 5 were synthesized and the predominant diastereoisomer of dimethyl aminophosphonate 4 was isolated. The resolution of the diastereomeric mixture of 5 failed. Aminophosphonates 3a-d and the predominant diastereoisomer of 4 were investigated in terms of their ecotoxicity using tests performed on the ostracode Heterocypris incongruens and the fluorescent bacterium Vibrio fisheri. The tests confirmed the moderate-to-high ecotoxicity of aminophosphonates 3a-d and 4, but no evident correlation between the structure and toxicity has been found.

Full text of DOI:10.3390/molecules21070936

molecules
Article
Synthesis, Spectral Characterization of Several Novel
Pyrene-Derived Aminophosphonates and Their
Ecotoxicological Evaluation Using
Heterocypris incongruens and Vibrio fisheri Tests
Jarosław Lewkowski 1,*, Maria Rodriguez Moya 1, Marta Chmielak 1,†, Diana Rogacz 2,
Kamila Lewicka 2 and Piotr Rychter 2,
*
1
Department of Organic Chemistry, Faculty of Chemistry, University of Łódz´, Tamka 12, 91-403 Łódz´, Poland;
MRM_chem@wp.pl (M.R.M.); martachmielak1@gmail.com (M.C.)
2
Institute of Chemistry, Environmental Protection and Biotechnology, Jan Długosz University in
Cze˛stochowa, 13/15 Armii Krajowej Av., 42-200 Cze˛stochowa, Poland; diana.rogacz@gmail.com (D.R.);
lewickakamilla@gmail.com (K.L.)
*
Correspondence: jlewkow@uni.lodz.pl (J.L.); p.rychter@ajd.czest.pl (P.R.);
Tel.: +48-426-355-751 (J.L.); +48-343-615-154 (P.R.)
†
M.Sc. student at the Faculty of Chemistry, University of Łódz´ (Academic Year 2015–2016).
Academic Editor: György Keglevich
Received: 26 June 2016; Accepted: 14 July 2016; Published: 19 July 2016
Abstract: Four diphenyl pyrene-derived aminophosphonates were synthesized. Attempts were
made to synthesize diphenyl N-(R)-
order to obtain the chiral aminophosphonate bearing a pyrene moiety. Because these attempts
failed, dimethyl and dibenzyl N-(R)- -methylbenzyl substituted aminophosphonates and were
synthesized and the predominant diastereoisomer of dimethyl aminophosphonate was isolated. The
resolution of the diastereomeric mixture of failed. Aminophosphonates 3a and the predominant
diastereoisomer of were investigated in terms of their ecotoxicity using tests performed on the
ostracode Heterocypris incongruens and the fluorescent bacterium Vibrio fisheri. The tests confirmed the
α-methylbenzylamino(pyren-1-yl)methylphosphonate (3e) in
α
4
5
4
5
–d
4
moderate-to-high ecotoxicity of aminophosphonates 3a–d and 4, but no evident correlation between
the structure and toxicity has been found.
Keywords: pyrene-1-carboxaldehyde; aminophosphonates; ecotoxicity; ostracode test; Vibrio fisheri test
1. Introduction
Aminophosphonic derivatives are well known for their biological activity. They are described
as effective inhibitors of various enzymes and, therefore they find applications as crop protective [
1],
antibacterial [ ] or anticancer [
2
3] agents. Analogues of C-phenylphosphonoglycine, have been
demonstrated to show activity in various fields as herbicides and plant growth regulators [
4],
agrochemical fungicides [5,6] and glutamate receptor agonists and antagonists [7].
In a sort of logical consequence, a lot of compounds from this group have been synthesized,
including those bearing benzene and its polycyclic aromatic analogues [ ], five- and six-membered
heteroaromatic rings [ 10], and even the ferrocene [11] moiety. Their syntheses are generally based on
8
9
,
the Kabachnik-Fields and the aza-Pudovik reactions [12–14].
In contrast, aminophosphonic derivatives of 1-pyrene, i.e., amino(1-pyrene)methylphosphonic
compounds have been described only twice in the chemical literature. Hudson et al. [15] reported
the synthesis of diethyl N-benzhydrylamino(1-pyrene)methyl phosphonate, and Jayaprakash and
co-workers [16] described the synthesis of a series of N-aryl substituted, diethyl amino(1-pyrene)methyl
phosphonates and results of preliminary studies on their fluorescence properties.
Molecules 2016, 21, 936; doi:10.3390/molecules21070936
www.mdpi.com/journal/molecules

Molecules 2016, 21, 936
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We have tried to fill this void and very recently have published results of our studies on the
synthesis, photophysical properties and cytotoxic action of a series of -aminophosphonates bearing
α
pyren-1-yl moieties [17]. They demonstrated moderate-to-satisfactory cytotoxicities against two colon
cancer cell lines. Such interesting biological properties may result in potential future applications and
their biological hazards should be evaluated using typical biotoxicological tests.
Herein, we wish to report the synthesis of a new series of N-substituted-C-(pyren-1-yl)-
phosphonoglycine derivatives and their ecotoxicological properties evaluated using ostracode
(Heterocypris incongruens) and fluorescence bacteria Vibrio fisheri tests.
Nowadays, ecotoxicology faces the challenge of evaluating and predicting the effects of a variety
of chemicals on aquatic species and ecosystems. Since a chemical pollutant may follow various routes
from a source to the target, there is an urgent need to follow their potential ecotoxicological impact
using different biotests [18–21].
Toxkit microbiotests are presently used in many countries worldwide for biomonitoring of
contaminated waters and soils. These small-scale bioassays are becoming more and more popular
in laboratories all over the world because of their various useful advantages, such as high degree
of standardization and precision, simple testing procedures which are very clearly documented
down to the last detail, operation with small sample volumes, the repeatability as well as giving
comparable results to equivalent ISO and OECD full-scale tests. However, the major advantage of
Toxkit microbiotests in comparison to conventional bioassays is that the tested microorganisms are
incorporated in the kits in a dormant or immobilized form, from which they can be hatched or activated
“on demand” prior to carrying out the toxicity tests [22].
The Microtox® system test with the luminescent bacterium Vibrio fischeri and the crustacean
Heterocypris incongruens, when used as bioindicator allows one to monitor potential toxicity of
soil or water contaminants in a rapid, simple and inexpensive way. This bioanalytical technique
allows one to overcome some disadvantages of the traditional physicochemical methods such as
concentration of toxic substances below analytical limits or potential chemical interactions leading
to additive, synergistic or antagonistic effect which cannot be identified using the abovementioned
methods. The Vibrio fischeri bioassay can be applied in all types of water, including surface and ground
water, wastewater, drilling sump fluids and many other aqueous solutions [23]. Both Vibrio fischeri
and Heterocypris incongruens bioassay tests have been already successfully used for ecotoxicity
determination of various pharmaceuticals, pesticides, ionic liquids, detergents, heavy metals, road dust,
eluates and organic extracts of wastes, etc. [24–30].
As it was mentioned above, the aim of the study was the preliminary evaluation of the potential
toxicity of newly synthesized aminophosphonates 3a–d and 4, if introduced to the environment.
In order to perform it, the Microtox® test using the bioluminescent bacterium Vibrio fischeri
was employed to examine the acute toxicity of sediment eluates loaded with tested compounds.
Due to the cost effective, simple operation and time-savings, the biotest based on the bioluminescence
inhibition of Vibrio fischeri is probably the most widely applied bacterial test used for environmental
pollution monitoring.
However, due to fact that the obtained substances are almost insoluble in water, the Microtox®
solid phase test (MSPT), as a one of the most popular tests for sediment toxicity assessments due to its
simplicity, reproducibility, ecological relevance and sensitivity, has been used [26,31–33].
Additionally, the standardized crustacean biotest with Heterocypris incongruens was selected
as a representative benthic invertebrate species for screening the toxicity of sediments containing
the tested substances. Crustaceans are promising representative species of the benthic environment
as they spend their lives swimming in water and in direct contact with the sediment. Therefore,
the Ostracodtoxkit® test has been used for toxicity evaluation. The toxicity test using mentioned
freshwater ostracodes is a sub-chronic static test that exposes individuals to whole sediments over a
period of 6 days, the endpoints being mortality and growth changes [34].

Molecules 2016, 21, 936
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2. Results and Discussion
2.1. Synthesis of Aminophosphonic Derivatives 3a–e, 4 and 5 Bearing 1-Pyrene Moieties
Schiff bases 1a
simple mixing pyrene-1-carboxaldehyde with an appropriate amine in hexane (in a case of p-anisidine,
p-toluidine and aniline) or methanol (for benzylamine and (R)- -methylbenzylamine) and stirring
them at room temperature for 24 h; the reaction with p-anisidine required 48 h [14]. This procedure
–e were synthesized following the published and commonly known procedure by
α
gave imines 1a–e, which were isolated and used for further conversions without any purification.
1H-NMR spectra were recorded only to verify the identity, based on the diagnostic 1H-NMR singlet
corresponding to the proton of the azomethine group (–CH=N–) above 8 ppm [17].
Aminophosphonates 3a
i.e., the addition of diphenyl phosphite to imines 1a
–
d
,
4
and
5
were synthesized basing on the aza-Pudovik reaction,
. However, important modifications were
–
d
necessary to be introduced, whereby reactions were carried out in a small amount of dichloromethane
and diphenyl phosphite was used in three-fold excess. The reaction mixtures were heated for
5 min then stirred for 24 h at room temperature. After workup as described in the Experimental
Section, the resulting aminophosphonates 3a–d were obtained in 60%–70% yields (Scheme 1).
They were purified by column chromatography on neutral aluminum oxide, because diphenyl
amino(1-pyrene)methylphosphonates 3a–d decomposed on silica gel.
OPh
O
OPh
OPh
P
O
P OPh
H
2A
N R
HN R
a: R = C6H5
b: R = 4-(CH3O)C6H4
c: R = 4-CH3C6H4
d: R = CH2C6H5
1a-e
3a-e
e: R = (R)-CH(CH3)C6H5
OR'
P OR'
O
H
2B-C
OR'
O
OR'
P
Ph
HN
2B, 4: R' = CH3
2C, 5: R' = CH2C6H5
4, 5
Scheme 1. Synthesis of N-substituted amino(1-pyrene)methylphosphonates 3a–e, 4 and 5.
The identity of synthesized compounds was verified by means of 1H-, 31P- and 13C-NMR as well
as by FT-IR spectroscopy. The proton NMR spectra of diphenyl pyrene-derived aminophosphonates
3a–d showed the characteristic signals of all compounds, i.e., the doublet corresponding to the proton
from the CH-P bond and the system of signals attributable to the pyrene ring protons. The 13C-NMR
spectra revealed the presence of two doublets at around 150 ppm with a coupling constant close
to 10 Hz, which are attributed to the phenyl ring carbons from the P-O-C groups. Doublets in the
50–55 ppm range with coupling constants between 150 and 160 Hz are easily recognizable as C-P bond
signals. IR bands at around 1250 cm´1 correspond to the bending vibrations of P=O bonds, while
the presence of aromatic carbon-carbon bonds was confirmed by characteristic bands at around 1600,
1570, 1500 and 1450 cm´1. Scans of NMR spectra are collected in the Supplementary Materials as
Figures S1–S7.

Molecules 2016, 21, 936
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In order to obtain the chiral, non-racemic diphenyl ester of a phosphonoglycine derivative
bearing a pyren-1-yl moiety, the synthesis of diphenyl N-(R)- -methylbenzylamino(pyren-1-yl)-
α
methylphosphonate (3e) was performed using the method described above. The resulting mixture
contained two diastereomers in a 100:15 ratio, but all attempts to purify the product by column
chromatography on neutral aluminum oxide caused extensive decomposition of the product. From
among available adsorbents, silica gel was totally inconvenient (vide supra), and chromatography on
microcrystalline cellulose with chloroform allowed us to get rid of impurities only roughly, i.e., crude
diphenyl N-(R)-α-methylbenzylamino(pyren-1-yl)methylphosphonate (3e) was obtained.
Thus, in order to investigate the stereochemistry of phosphite addition to the azomethine
bond of pyrene-1-carboxaldimine 1e, addition of dimethyl and dibenzylphosphites has been
performed. Dimethyl N-(R)-α-methylbenzylamino(pyren-1-yl)methylphosphonate (4) was obtained as
a mixture of two diastereoisomers in a 100:13 ratio. We succeeded in separation of the predominant
diastereoisomer using column chromatography on silica gel. Its absolute configuration however could
not be determined, because the predominant isomer of aminophosphonate
thus an X-ray study could not be performed. Isolation of the minor diastereoisomer of
always obtained in a mixture with the major one.
4
is a dense oily liquid,
failed, it was
4
Dibenzyl N-(R)-α-methylbenzylamino(pyren-1-yl)methylphosphonate (5) was obtained as
a mixture of two diastereoisomers in a 100:26 ratio. Unfortunately, the obtained mixture of
diastereoisomers was not separable by column chromatography, as all applied solvent systems led to
mixtures with different ratios of isomers. However, the crude diastereomeric mixture of
5 was purified
by column chromatography on silica gel to give satisfactory elemental analysis results.
Thus the obtained diphenyl aminophosphonates 3a–d and the isolated predominant
diastereoisomer of were tested in terms of their ecotoxicity using two popular ecotoxicological
4
tests, the ostracode Heterocypris incongruens and fluorescence bacteria Vibrio fisheri.
2.2. Ecotoxicological Properties of Aminophosphonates 3a–d and 4
2.2.1. Microtox Toxicity Assay
Bioassays are the complementary tool for characterizing the biological effects and hazards of
contaminated sediments or soils. During the solid phase Microtox® assay the bioluminescent bacteria
are exposed to a sediment suspension and their response reflects totally the action of toxic agents along
with synergists and antagonists present in a given sample. The effects on light emission are evaluated
in the liquid phase that remains after removal of the sediments by filtration. Effective concentration
(EC50) values (Table 1) were calculated from the concentration–response curves (Figure 1).
Obtained EC50 values ranged between 92 and 533 mg/kg of dry weight of soil, depending
on the tested compound. Among the compounds 3a–c with aromatic substituents on the amine
groups, diphenyl N-(4-methoxyphenyl)amino(pyren-1-yl)methylphosphonate (3b) was found to
be the most toxic, with the lowest EC50 value (92.63 mg/kg). Much less harmful action against
Vibrio fischeri was found for the N-(4-methylphenyl) derivative 3c (EC50 = 214.5 mg/kg), while diphenyl
N-phenylamino(pyren-1-yl)methylphosphonate (3a) demonstrated nearly a lack of toxicity. Thus,
the presence of a substituent at the phenyl group linked to nitrogen seems to be crucial in terms of
toxicological properties of the studied compounds 3a–c. The results also showed that a methoxy
substituent increases the toxicity of the compound 3b in comparison with the methyl substituted
aminophosphonate 3c.
Among the aminophosphonates 3d and
groups, results were indeed surprising. Diphenyl N-benzylamino(pyren-1-yl)methylphosphonate
3d) with a N-benzyl substituent demonstrated five times higher toxicity (EC50 = 102.7 mg/kg) as
compared to the N-(R)- -methylbenzylamino derivative (EC50 = 533.1 mg/kg). Therefore we can
4 having the amino groups substituted with non-aromatic
(
α
4
conclude that the presence of a methyl substituent in the benzyl moiety dramatically decreases the
toxicity of compound 4.

Molecules 2016, 21, 936
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Table 1. Microtox® EC50 values (mg/kg of soil dry weight) of exposure of the luminescent marine
bacteria Vibrio fischeri to tested aminophosphonates 3a and , with respective 95% confidence limits
(in brackets) obtained in the fit of the data.
–d
4
Compounds
EC50 (Lower Limit; Upper Limit)
Coefficient of Determination (R2)
3a
3b
3c
3d
4
495.9 (203.4; 1209)
92.63 (65.27; 131.4)
214.5 (165.0; 279.0)
102.7 (81.83; 128.9)
533.1 (439.4; 646.8)
0.7632
0.8912
0.9452
0.9571
0.9516
Figure 1. The EC50 graded dose-response curves for the five tested compounds 3a–d and 4. Overlapped
curves are plotted by the Microtox® Analyzer 500 software. Error bars represent standard deviation
error (SD, n = 3 determinations).
2.2.2. Ostracod Test Kit
Ostracodtoxkit FTM is the very first sediment-contact microbiotest with a crustacean test species
for the assessment of the total toxicity of sediments, hence covering the toxic hazards of both dissolved
and not dissolved pollutants. Considering the very low solubility of synthesized aminophosphonates
3a–d and 4, it seemed justified to evaluate reliably their potential toxicity.
The first criterion of the effect on tested crustaceans is the percent of mortality. Toxicity
was expressed in % of mortality, i.e., the lethal effect of the sample on the number of ostracods
compared with the control plate. As it was expected, the mortality of ostracodes increased with
growing concentration of tested substance in soil (Figure 2). The concentration 500 mg/kg of soil
was found to be lethal for all samples. However, among all tested compounds, N-benzylamino
(pyren-1-yl)methylphosphonate (3d) demonstrated the highest toxicity at every concentration when
compared to other samples (with the sole exception of aminophosphonate
4 at a concentration of

Molecules 2016, 21, 936
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100 mg/kg of soil). Even at a concentration of 10 mg/kg of dry weight of soil, compound 3d caused
10% mortality of crustaceans, while other samples at this concentration did not show any mortal effect.
Figure 2. The mortality (%) of Heterocypris incongruens living in soil contaminated with pyrene-derived
aminophosphonates 3a–d and 4 at concentrations: 10, 50, 100 and 500 mg/kg of soil dry weight.
Error bars represent standard deviation error (SD, n = 6 determinations).
Growth inhibition is the second criterion of the toxic effect indicated by the Ostracodtoxkit F
™
microbiotest. This criterion allows one to evaluate the sub-lethal toxicity of sediments. 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 reference 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. It is advised that the growth inhibition should only be determined for sediments
which mortality was found to be less than 30%. Therefore, the growth inhibition was chosen as
a criterion of sub-lethal effects to determine toxicity not inducing substantial mortality in the test
organisms. Consequently, measurements of lengths were only performed when the mortality was less
than 30%. In this respect, the growth inhibition rates have not been measured for aminophosphonates
3c, 3d and 4 at concentrations of 100, 250 and 500 mg per kg of soil dry weight. Crustaceans were more
resistant against compounds 3a and 3b and their growth inhibition reached ca. 20% for a concentration
of 100 mg/kg of soil, nevertheless it was not measured at 250 and 500 mg per kg of soil dry weight.
Values of growth inhibition for compounds 3c, 3d and 4 at 50 mg/kg of soil concentration varied
between 16% and 23% (Figure 3).
Figure 3. Growth inhibition (%) of Heterocypris incongruens living in soil contaminated with
pyrene-derived aminophosphonates 3a and at concentrations of 10, 50, 100 and 500 mg/kg of soil
dry weight. Error bars represent standard deviation error (SD, n = 6 determinations).
–
d
4

Molecules 2016, 21, 936
7 of 14
Comparison of results of luminescence inhibition for Vibrio fischeri tests with growth inhibition
of crustaceans for all concentration of compounds did not show any reasonable relationship.
Only diphenyl N-benzylamino(pyren-1-yl)methylphosphonate (3d), which was strongly toxic for
Vibrio fischeri (EC50 ~103 mg/kg of soil) also revealed the highest mortality of ostracods even at a
concentration of 10 mg/kg of soil.
However, diphenyl N-(4-methoxyphenyl)amino(pyren-1-yl) methylphosphonate (3b) that
induced a very strong luminescence inhibition in V. fischeri was only moderately toxic for crustaceans
considering both the mortality and the growth inhibition index. On the other hand, diphenyl
N-phenylamino(pyren-1-yl)methylphosphonate (3a), being nearly non-toxic for V. fischeri (EC50 close
to 500 mg/kg of soil) is also very weakly toxic for ostracods (with respect to both criteria, the mortality
and the growth inhibition).
Thus, based on the obtained results, Vibrio fischeri bacteria were very sensitive to
aminophosphonates 3b and 3d, since relatively low effective concentration (EC50) values were
determined, while for ostracods, the highest negative impact considering both mortality and inhibition
growth have been found for diphenyl N-benzylamino(pyren-1-yl)methylphosphonate (3d). Therefore,
being the most toxic against both tested organisms, the aminophosphonate 3d should be considered as
a potentially hazardous compound for environment, which may be toxic against many other living
species. Its high toxicity may be related with its N-benzyl group, which is more flexible than other
substituents and may bind more easily to enzymes or receptors of the tested organisms. Nevertheless,
this is only a very preliminary hypothesis, which requires further verification.
The usefulness of bioassays for the assessment of areas affected by phosphate industry wastes
has already been reported and discussed [35,36]. Authors performing risk assessments in such
contaminated areas have proven that the development of sensitive and observable organisms as
bio-indicators for monitoring and management of the environmental pollution is a very useful tool
to complement chemical analyses. Biological analysis allows one to reflect the harmful effects of
contaminants on organisms and provides data to reveal the mechanisms of the toxic effects including
formation, development and removal of contaminants.
Since the soil is generally the reservoir for agrochemical residues, the evaluation of any xenobiotics
including pesticides using a simple battery of assay tests is strongly needed to control level and fate
of their residues. In a successful study focused on the ecotoxicological evaluation of select pesticides
(chlorpyrifos, glyphosate, vinclozolin, endosulfan) present in agricultural soils, Antunes et al have
reported the use of standard aquatic bioassays for testing of soil eluates [30].
Our obtained effective concentration values are very close to the inhibition concentration (IC50)
values for herbicides examined by Joly et al. [28]. Toxicity values of the pesticides S-metolachlor,
nicosulfuron and benoxacor were 178.4, 167.8 and 93.3 mg/L, respectively. In this respect, the toxicity
of the first two herbicides are close to that of the tested aminophosphonate 3c, while the last IC50 value
matches the values of compounds 3b and 3d. Nevertheless, the action of aminophosphonates 3a
and against V. fischeri demonstrated much lower toxicity when compared to the herbicides tested by
the mentioned authors.
–d
4
Ostracodtoxit tests using Heterocypris incongruens are also a very useful tool for environmental
toxicity determination. Results related to mortality and growth inhibition of ostracods correlated with
various types of polycyclic aromatic hydrocarbons (PAHs) have been reported by Cvancarova et al. [37]
They found that H. incongruens was strongly sensitive to bioavailable PAHs, while luminescence
inhibition of V. fischeri varied widely between ca. 15%–90% depending on the type of soil examined.
Oleszczuk and Hollert [38], in their studies devoted to determination of the influence of different
soils on sewage sludge toxicity, have found that the mortality values of Heterocypris incongruens ranged
from 0.26% to 11.5% depending on the sludge tested [39]. Growth inhibition values ranged from 10.7%
to 36.2% depending on the type of sludge. Płaza et al. [39] applied Microtox®, Ostracodtoxkit F™,
to assay bioremediation processes in soils heavily contaminated with petroleum. The test species
.

Molecules 2016, 21, 936
8 of 14
demonstrated varying sensitivity to soils and the effects on test organisms exposed to tested soils
correlated with the soil contaminants’ concentrations [39].
Results of toxicity against H. incongruens obtained for the studied aminophosphonates 3a
–d
and
4 were at a similar level. The mortality varied from ca. 5% until 70% for the concentration
range 10–100 mg per kg of soil. At higher concentrations, the tested compounds were practically
lethal for crustaceans. The growth inhibition varied from 16% up to 23% for a concentration of
50 mg per kg of soil. All the data presented above lead us to stress the necessity of handling
pyrene-derived aminophosphonic derivatives with special care, as they may be potentially hazardous
for the environment.
3. Materials and Methods
3.1. Chemistry
All solvents (POCh, Gliwice, Poland) were applied routinely and dried prior to use. Commercial
reagents (Aldrich, Poznan´, Poland) were generally used as received. NMR spectra were recorded
on an Avance III 600 MHz apparatus (Bruker, Billerica, MA, USA) operating at 600 MHz (1H-NMR),
150 MHz (13C-NMR) and 243 MHz (31P-NMR). IR spectra were recorded on a Nexus FT-IR
spectrometer (Thermo Nicolet, Waltham, MA, USA). Rotations were measured using a 241 MC
polarimeter (Perkin-Elmer, Waltham, MA, USA). Melting points were measured using a MelTemp
II apparatus (Bibby Scientific Limited, Staffordshire, UK), in a capillary and were not corrected.
Elemental analyses were performed in the Laboratory of Microanalysis, the Center of Molecular and
Macromolecular Studies PAS in Łódz´ and the Laboratory of Molecular Spectroscopy at the Faculty of
Chemistry, University of Łódz´.
3.1.1. General Procedure for Synthesis of Phosphonates 3a–d, 4 and 5
Equimolar quantities of 1-pyrenecarboxaldehyde and amine (2 mmol each) were dissolved in
hexane (1a–c) or methanol (1d–e) (20 mL) and stirred at reflux for 24 h (1b–c, 1d–e) or 48 h (1a).
The reaction was monitored by 1H-NMR and after the completion, the solvent was removed under
reduced pressure, and obtained products were used directly for further conversion. Imine (1 mmol)
and the appropriate phosphite 2A–C (3 mmol or 5–6 mmol in a case of dimethyl phosphite due to the
low solubility of the imine in it) were placed in a 25 mL round-bottom flask, which was heated with
stirring on a water bath for 5 min until all of the imine was dissolved in the phosphite. In the case
of derivatives 3a–d, dichloromethane (2 mL) was added in order to improve the solubility. Then the
mixture was stirred at room temperature for 24 h. The crude aminophosphonates were purified by
column chromatography on neutral aluminum oxide using chloroform as eluent.
Diphenyl˝ N-phenylamino(pyren-1-yl)methylphosphonate (3a). Y = 0.41 g (76%), yellow solid, m.p.
184–185 C. 1H-NMR (CDCl3):
δ
8.38 (d, 3JHH = 9.3 Hz, Hpyr, 1H); 8.28 (dd, 3JHH = 8.0 and
4JHH = 2.6 Hz, Hpyr, 1H); 8.20 (t, Hpyr 3JHH = 8.0 Hz, 2H); 8.12 (d, 3JHH = 8.0 Hz, Hpyr, 1H);
,
8.10 (d, 3JHH = 9.2 Hz, Hpyr, 1H); 8.07–8.06 (m, Hpyr, 1H); 8.04–8.01 (m, Hpyr, 2H); 7.30–7.29
3 HH
3 HH(1)
3 HH(2)
(m, HPh, 5H); 6.94 (t, J
= 7.4 Hz, HPh, 1H); 6.84 (dd, J
= J
= 7.8 Hz, HPh, 2H); 6.79
(app. d, 3JHH = 8.5 Hz, p-C6H4, 2H); 6.67 (d, J
= 7.4 Hz, HPh, 2H); 6.46 (app. d, J
= 8.5 Hz,
3 HH
3 HH
p-C6H4, 2H); 5.92 (d, 2JPH = 24.1 Hz, CHP, 1H); 5.10–5.08 (m, POCH2, 2H); 4.65 and 4.22 (Part of
AMX system, JHH = 11.6 Hz and JPH = 7.8 and 8.5 Hz, POCH2, 2H). 13C-NMR (CDCl3):
δ 150.7
2
3
(d, 2JCP = 9.9 Hz,
C
POC); 150.3 (d, 2JCP = 9.5 Hz, CPOC); 146.1 (d, 3JCP = 15.27 Hz, CPh); 131.6 (Cpyr);
131.5 (d, 4JCP = 3.1 Hz, Cpyr); 130.8 (Cpyr); 130.2 (Cpyr) 130.0 (CPh); 129.6 (Cpyr); 129.5 (d, 4JCP = 2.0 Hz,
CPh); 128.7 (Cpyr); 128.6 (d, 4JCP = 2.1 Hz, Cpyr); 127.9 (Cpyr); 127.7 (Cpyr); 127.4 (Cpyr); 127.0 (Cpyr);
4
126.8 (Cpyr); 126.3 (Cpyr); 125.8 (CPh); 125.7 (CPh); 125.6 (d, JCP = 3.4 Hz, Cpyr); 125.5 (Cpyr); 125.2
3
3
(Cpyr); 125.0 (Cpyr); 122.1 (Cpyr); 120.9 (d, JCP = 4.0 Hz, CPh); 119.9 (d, JCP = 4.4 Hz, CPh); 119.1
1
(CPh); 114.2 (CPh); 52.2 (d, JCP = 154.6 Hz, PC). 31P-NMR (CDCl3):
δ 16.16. IR (KBr): 3313 (νCH);
3037; 2923 (CH3); 1600, 1581; 1524, 1483 (C=C); 1249 (P=O); 1214; 1185; 1062; 1021 (P-O); 941; 853;

Molecules 2016, 21, 936
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824; 622.Elemental analysis: Calcd. for C35H26NO3P: C, 77.91; H, 4.86; N, 2.60. Found: C, 77.76, H,
4.86, N, 2.79.
Diphenyl N-(4-methoxyphenyl)amino(pyren-1-yl)methylphosphonate (3b). Y = 0.3 g (52%), yellow solid, m.p.
161 ˝C. 1H-NMR (CDCl3):
δ
8.49 (d, 3JHH = 9.2 Hz, Hpyr, 1H); 8.34 (dd, 3JHH = 7.7 and 4JHH = 1.9 Hz,
Hpyr, 1H); 8.24-8.21 (m, Hpyr, 3H); 8.14 (d, Hpyr, 3JHH = 8.1 Hz, 1H); 8.08 (d, 3JHH = 8.9 Hz, Hpyr, 1H);
8.04 (d, 3JHH = 7.7 Hz, Hpyr, 1H); 8.02–8.01 (m, Hpyr, 2H); 7.31–7.28 (m, HPh, 2H); 7.19–7.16 (m, HPh, 3H);
6.95–6.92 (m, HPh, 2H); 6.89–6.87 (m, HPh, 1H); 6.62–6.58 (m, HPh, p-C6H4, 6H); 6.23 (d, 2JPH = 24.4 Hz,
CHP, 1H); 3.61 (s, CH3, 3H). 13C-NMR (CDCl3):
δ
153.2 (d, 2JCP = 5.4 Hz, Cpara); 150.7 (d, 2JCP = 9.9 Hz,
C
POC); 150.3 (d, 2JCP = 9.7 Hz, CPOC); 131.5 (Cpyr); 131.2 (d, 4JCP = 3.2 Hz, Cpyr); 130.8 (Cpyr); 130.0 (CPh);
129.7 (Cpyr); 129.6 (d, 3JCP = 7.2 Hz, Cpyr); 129.5 (CPh); 128.8 (Cpyr); 128.6 (Cpyr); 127.9 (Cpyr); 127.7
(Cpyr); 126.3 (CPh); 125.8 (CPh); 125.6 (Cpyr); 125.6 (Cpyr); 125.5 (Cpyr); 125.2 (d, 4JCP = 1.8 Hz, Cpyr);
125.0 (Cpyr); 124.9 (Cpyr); 122.2 (Cpyr); 121.0 (d, 3JCP = 4.2 Hz, CPh); 120.0 (d, 4JCP = 4.4 Hz, CPh); 115.7
(d, 4JCP = 9.7 Hz, CPh); 115.1 (Cpara); 55.7 (CAr-OC); 53.0 (d, 1JCP = 154.3 Hz, PC). 31P-NMR (CDCl3):
δ 16.32. IR (KBr): 3383 (νNH); 3294 (νCH); 3037; 2958 (CH3); 1591, 1511, 1486; 1451 (C=C); 1245 (P=O);
1211; 1068; 1024 (P-O); 941; 834; 815; 618. Elemental analysis: Calcd. for C36H28NO4P: C, 75.91; H, 4.95;
N, 2.46. Found: C, 75.76, H, 5.06, N, 2.54.
Diphenyl N-(4-methylphenyl)amino(pyren-1-yl)methylphosphonate (3c). Y = 0.44 g (79%), yellow solid,
˝
1
3 HH
3 HH
m.p. 181–182 C. H-NMR (CDCl3):
δ
8.51 (d, J
= 9.3 Hz, Hpyr, 1H); 8.34 (dd, J
= 8.2
4
and JHH = 2.5 Hz, Hpyr, 1H); 8.25-8.21 (m, Hpyr, 3H); 8.13 (d, Hpyr
(d, 3JHH = 8.9 Hz, Hpyr, 1H); 8.04 (t, J
,
3JHH = 8.2 Hz, 1H); 8.08
3 HH
3 HH
= 7.6 Hz, Hpyr, 1H); 8.01 (d, J
= 8.8 Hz, Hpyr, 2H);
7.31–7.29 (m, HPh, 2H); 7.20–7.15 (m, HPh, 3H); 7.05–7.02 (m, HPh, 2H); 6.94–6.91 (m, HPh, 2H); 6.88-6.86
(m, HPh, 1H); 6.68-6.64 (m, HPh, 3H); 6.58–6.57 (m, HPh, 2H); 6.31 (d, 2JPH = 24.6 Hz, CHP, 1H). 13C-NMR
(CDCl3):
δ
150.7 (d, 2JCP = 9.8 Hz, CPOC); 150.3 (d, 2JCP = 9.7 Hz, CPOC); 143.7 (d, 2JCP = 15.9 Hz, Cpara);
131.6 (Cpyr); 131.2 (d, 4JCP = 3.2 Hz, Cpyr); 130.8 (Cpyr); 130.0 (CPh); 129.8 (Cpyr); 129.6 (d, 3JCP = 7.3 Hz,
4
Cpyr); 129.5 (Cpara); 128.8 (d, JCP = 2.1 Hz, Cpyr); 128.6 (Cpyr); 128.3 (Cpyr); 127.8 (Cpara); 127.7
(d, 4JCP = 1.6 Hz, Cpyr); 126.3 (Cpyr); 125.8 (Cpyr); 125.6 (CPh); 125.5 (d, 4JCP = 2.3 Hz, Cpyr); 125.5 (CPh);
125.2 (d, 4JCP = 1.9 Hz, Cpyr); 125.0 (Cpyr); 124.9 (Cpyr); 122.2 (Cpyr); 121.0 (d, 3JCP = 4.0 Hz, CPh); 120.0
(d, 3JCP = 4.4 Hz, CPh); 114.3 (Cpara); 52.5 (d, 1JCP = 155.1 Hz, PC); 20.5 (CAr-OC). 31P-NMR (CDCl3):
δ 15.23. IR (KBr): 3303 (νCH); 2923 (CH3); 1591, 1489, 1458 (C=C); 1268 (P=O); 1024 (P-O); 926, 840;
770; 688. Elemental analysis: Calcd. for C36H28NO3P: C, 78.11; H, 5.10; N, 2.53. Found: C, 77.86;
H, 5.31; N, 2.47.
Diphenyl˝ N-benzylamino(pyren-1-yl)methylphosphonate (3d). Y = 0.43 g (77%), yellow solid, m.p.
118–119 C. 1H-NMR (CDCl3):
δ
8.51–8.49 (m, Hpyr, 1H); 8.26 (d, 3JHH = 8.0 Hz, Hpyr, 1H); 8.24–8.21
3 HH
(m, Hpyr, 3H); 8.13–8.09 (m, Hpyr, 3H); 8.04 (t, J
= 7.6 Hz, Hpyr, 1H); 7.29–7.25 (m, HPh, 5H);
7.20–7.19 (m, HPh, 2H); 7.15–7.13 (m, HPh, 3H); 7.02–7.00 (m, HPh, 2H); 6.93 (app. t, 3JHH = 7.4 Hz,
HPh, 1H); 6.73 (app. d, 3JHH = 8.2 Hz, HPh, 2H); 5.58 (d, JPH = 20.8 Hz, CHP, 1H); 3.93
2
(d, 3JHH = 13.3 Hz, NCH2, 1H); 3.66 (d, J
= 13.3 Hz, NCH2, 1H). 13C-NMR (CDCl3):
C
δ
151.0
3 HH
(d, 2JCP = 9.9 Hz, CPOC); 150.5 (d, 2JCP = 9.7 Hz,
POC); 139.1 (CPh); 131.6 (Cpyr); 131.4 (d, 4JCP = 3.0 Hz,
Cpyr); 130.8 (Cpyr); 130.4 (Cpyr); 130.3 (Cpyr) 130.0 (Cpyr); 129.9 (Cpyr); 129.8 (CPh); 129.5 (CPh); 128.8
(CPh); 128.6 (CPh); 128.2 (Cpyr); 128.0 (Cpyr); 127.7 (d, 4JCP = 1.5 Hz, Cpyr); 127.5 (Cpyr); 126.3 (Cpyr);
125.7 (CPh); 125.5 (d, 4JCP = 2.8 Hz, Cpyr); 125.4 (CPh); 125.2 (Cpyr); 125.0 (Cpyr); 122.7 (Cpyr); 120.8
(d, 3JCP = 4.3 Hz, CPh); 120.3 (d, 3JCP = 4.3 Hz, CPh); 51.6 (d, 3JCP = 17.8 Hz, PCNC). 31P-NMR (CDCl3):
δ
16.22. IR (KBr): 3386 (νNH); 3041, 2917 (CH3); 1616, 1587, 1518, 1451 (C=C); 1245 (P=O); 1066; 1024
(P-O); 941, 843; 777; 618. Elemental analysis: Calcd. for C36H28NO3P: C, 78.11; H, 5.10; N, 2.53. Found:
C, 77.81; H, 5.24; N, 2.40.
3.1.2. Dimethyl N-(R)-α-Methylbenzylamino(pyren-1-yl)methylphosphonate (4)
The reaction was carried out as it is described above, but the post-reaction mixture was dissolved
in dichloromethane and washed three times with a saturated aqueous sodium bicarbonate solution. The

Molecules 2016, 21, 936
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organic layer was dried, the solvent was removed in vacuo and the residual oil was purified by column
chromatography on silica gel with chloroform as eluent. The procedure gave 440 mg of as a dense
4
yellow oil. Isolated predominant diastereomer: Y = 0.4 g (92%, after column chromatography), dense yellow
“
20
˝
48.3 (c = 0.1, CHCl3). H-NMR (CDCl3): δ
8.29–8.27 (m, Hpyr, 1H); 8.22 (d, 3JHH = 8.0 Hz,
1
oil. αsD
=
´
Hpyr, 1H); 8.21–8.18 (m, Hpyr, 3H); 8.10–8.06 (m, Hpyr, 3H); 8.02 (t, 3JHH = 7.6 Hz, Hpyr, 1H); 7.25–7.23
(m, PhH, 2H); 7.22–7.20 (m, PhH, 2H); 7.19–7,16 (m, PhH, 1H); 5.33 (d, 2JPH = 21.1 Hz, CHP, 1H);
3 HH
3
3.91 (q, J
POCH3, 3H); 1.39 (d, J
= 6.5 Hz, CHPh, 1H); 3.86 (d, JPH = 10.5 Hz, POCH3, 3H); 3.26 (d, 3JPH = 10.5 Hz,
3 HH
= 6.5 Hz, CHCH3, 3H). 13C-NMR (CDCl3):
δ 145.2 (CPh); 131.6 (Cpyr);
131.1 (d, 4JCP = 2.3 Hz, Cpyr); 130.8 (Cpyr); 130.3 (Cpyr); 129.4 (d, 3JCP = 7.7 Hz, Cpyr); 128.6 (CPh); 128.2
(Cpyr); 127.8 (Cpyr); 127.7 (Cpyr); 127.3 (CPh); 127.0 (CPh); 126.2 (Cpyr); 126.1 (Cpyr); 125.6 (Cpyr); 125.4
3
(d, 4JCP = 2.8 Hz, Cpyr); 125.3 (Cpyr); 125.1 (Cpyr); 125.0 (Cpyr); 55.8 (d, JCP = 11.9 Hz, PCNC); 54.0
2
2
(d, JCP = 7.4 Hz, POC); 53.6 (d, JCP = 7.2 Hz, POC); 29.9 (Caliph); 27.8 (Caliph). 31P-NMR (CDCl3):
δ
26.70. IR (KBr): 3440 (νNH); 3309 (νCH); 2949 (CH2); 1600, 1584, 1511, 1454 (C=C); 1242 (P=O);
1055; 1027 (P-O); 846; 815; 761; 609. Elemental analysis: Calcd. for C27H26NO3P: C, 73.12; H, 5.91;
N, 3.16. Found: C, 73.15, H, 5.72, N, 2.96. Mixture of diastereomers—the minor diastereomer is denoted
with an asterisk. 1H-NMR (CDCl3):
δ 8.29–8.28 (m, Hpyr, 1H); 8.23–8.22 (m, Hpyr, 1H); 8.21–8.17
(m, Hpyr, 3H); 8.17–8.16* (m, Hpyr, 1H); 8.10–8.06 (m, Hpyr, 3H); 8.03–8.00 (m, Hpyr, 1H); 7.95–7.94*
(m, Hpyr, 1H); 7.30–7.26* (m, PhH); 7.24–7.22 (m, PhH, 2H); 7.22–7.20 (m, PhH, 2H); 7.19–7.16 (m, PhH,
1H); 7.12–7.10 (m, PhH); 5.33 (d, 2JPH = 21.0 Hz, CHP, 1H); 4.98* (d, 2JPH = 21.7 Hz, CHP, 1H); 3.91
(q, 3JHH = 6.5 Hz, CHPh, 1H); 3.86 (d, 3JPH = 10.6 Hz, POCH3, 3H); 3.83* (d, 3JPH = 10.5 Hz, POCH3,
3H); 3.50* (q, 3JHH = 6.6Hz, CHPh, 1H); 3.26 (d, 3JPH = 10.4 Hz, POCH3, 3H); 3.23* (d, 3JPH = 10.4 Hz,
3 HH
POCH3, 3H); 1.39 (d, 3JHH = 6.5 Hz, CHCH3, 3H); 1.34* (d, J
= 6.6 Hz, CHCH3, 3H). 31P-NMR
(CDCl3):
δ 26.70 and 26.18* (10:4). Elemental analysis: Calctd for C27H26NO3P: C, 73.12; H, 5.91; N,
3.16. Found: C, 73.17, H, 5.85, N, 3.10.
3.1.3. Dibenzyl N-(R)-α-Methylbenzylamino(pyren-1-yl)methylphosphonate (5)
The procedure was identical as in a case of aminophosphonate 4. Mixture of diastereoisomers—the
predominant diastereoisomer is denoted with an asterisk (13C-NMR data are quoted with two decimal
digits due to the large number of signals situated very close one to another). Y = 0.12 g, (20%, after
1
column chromatography), dense yellow oil (mixture). H-NMR (CDCl3):
δ 8.21–8.20 (m, Hpyr, 2H);
8.16* (d, 3JHH = 7.5 Hz, Hpyr, 1H); 8.15–8.12 (m, Hpyr); 8.10–8.06 (m, Hpyr, 4H); 8.03–8.01 (m, Hpyr
,
3 HH
1H); 7.98* (d, J
= 9.3 Hz, Hpyr, 1H); 7.38–7.36 (m, PhH, 2H); 7.34–7.32 (m, PhH, 3H); 7.22–7.17
(m, PhH, 5H); 6.97–6.94* (m, PhH, 2H); 6.92–6.90 (m, PhH); 6.89–6.86* (m, PhH, 2H); 6.83–6.81
(m, PhH); 6.76–6.74 (m, PhH, 2H); 6.68–6.66 (m, PhH); 5.38* (d, 2JPH = 21.0 Hz, CHP, 1H); 5.26* and
5.10* (Part of ABX system, 2JHH = 11.8 and 3JPH = 8.6 and 7.0 Hz, OCH2Ph, 2H); 5.24–5.22 (m, CHP);
5.13–5.03 (m, OCH2Ph); 4.72* and 4.39* (part of AMX system, 2JHH = 11.8 and 3JPH = 8.4 and 7.3 Hz,
OCH2Ph, 2H); 4.67-4.64 (m, OCH2Ph); 4.40–4.35 (m, OCH2Ph); 3.93* (q, 3JHH = 6.4 Hz, CHPh, 1H); 1.33*
(d, 3JHH = 6.4 Hz, CHCH3, 3H); 1.30–1.29 (m, CHCH3). 13C-NMR (CDCl3):
δ 145.21 (CAr); 144.28 (CAr);
136.83 (CAr); 136.79 (CAr); 136.01 and 135.98 (CAr); 131.58 (CAr); 131.06 and 131.05 (CAr); 130.87 (CAr);
130.41 (CAr); 129.57 and 129.52 (CAr); 128.67 and 128.66 (CAr); 128.63 (CAr); 128.45 (CAr); 128.41 (CAr);
128.12 (CAr); 128.03 (CAr); 127.98 (CAr); 127.85 (CAr); 127.75, 127.74, 127.71 and 127.70 (CAr); 127.49 and
127.48 (CAr); 127.43 (CAr); 127.29 (CAr); 126.98 (CAr); 126.13 (CAr); 125.49 (CAr); 125.40 (CAr); 125.32 and
125.30 (CAr); 125.19 (CAr); 125.16 (CAr); 125.09 (CAr); 125.00 (CAr); 124.97 (CAr); 68.79 and 68.75 (POC);
68.64 and 68.60 (POC); 68.34 and 68.30 (POC); 68.10 and 68.06 (POC); 55.94 (Caliph); 55.86 (Caliph); 55.58
(Caliph); 55.46 (Caliph); 24.92 (Caliph); 22.85 (Caliph). 31P-NMR (CDCl3):
δ 25.31*; 24.64 (4:1).Elemental
analysis: Calcd. for C39H34NO3P: C, 78.64; H, 5.75; N, 2.35. Found: C, 78.48, H, 5.96, N, 2.16.
3.1.4. Diphenyl N-(R)-α-Methylbenzylamino(pyren-1-yl)methylphosphonate (3e)
To a solution of the imine 1e (1mmol) and diphenyl phosphite (1 mmol) in dichloromethane
(ca. 2 mL) was slowly added BF3 OEt2 (24 mg, 0.67 mmol) via syringe. The mixture was stirred at
¨

Molecules 2016, 21, 936
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room temperature for 24 h while monitoring the course of the reaction using 31P-NMR spectroscopy
or TLC. The solvent was removed in vacuo and the residual oil was roughly purified by column
chromatography on microcrystalline cellulose with chloroform as eluent to give 3e as a brown oil in
0.9 g (72%) yield of a crude product. Further attempts to purify and to analyze the product resulted in
its decomposition.
3.2. Toxicity Tests
3.2.1. Microtox® Toxicity Assay
The method is based on the analysis of light emission reduction of luminescent bacteria
(
Vibrio fischeri) under toxic stress. The tests were carried out in a Microtox® M500 analyzer according
to the 1992 Microtox® Manual. The Microtox® Solid-Phase Test (MSPT) was adopted to report of
Doe et al. [31]. The MSPT procedure allows the test organisms to come direct contact with the solid
sample in an aqueous suspension of the test sample. Thus it is possible to detect toxicity which is due
to the insoluble solids that are not in the solution.
All materials and reagents were purchased from MODERNWATER (New Castle, DE, USA).
Toxicity was determined by using the marine luminescent bacterium, Vibrio fischeri, naturally adapted
to a saline environment. Briefly, bacteria were regenerated with 1 mL of Reconstitution Solution
(0.01%) and placed in the reagent well of the Microtox®. A suspension of 7 g of the sediment was
prepared in 35 mL of a Solid Phase Diluent (3.5% NaCl) and was magnetically stirred for 10 min. Then
a series of dilutions were made and bacteria (approx. 1
ˆ
106 cell mL´1 per assay) were exposed to
¨
these dilutions and to a blank (3.5% NaCl solution) for 20 min. Next, after filtration, the light output
of supernatants containing exposed bacteria was measured after 5 min with a Microtox® Analyzer
500. Inhibition was calculated as the concentration of compound loaded to sediment (mg/L) that
caused a 50% reduction in the light emitted by the bacteria, and EC50 along with 95% confidence limit
determined by the software provided with the Analyzer.
3.2.2. Ostracod Test Kit
Ecotoxicity evaluation of synthesized compounds was performed in a short term contact test
using Ostracodtoxkit F™ provided by MicroBiotests Inc. (Gent, Belgium). This direct sediment contact
bioassay was performed in multiwell test plates using neonates of the benthic ostracod crustacean
Heterocypris incongruens hatched from cysts [35]. After 6 days of contact with the sediment (or soil) the
percentage mortality and the growth of the crustaceans were determined and compared to the results
obtained in a non-treated reference sediment (soil).
Briefly, according to manual of Ostracodtoxkit test, the cysts (Heterocypris incongruens) were
transferred into˝a Petri dish filled with 10 mL standard fresh water (reconstituted water) and were
incubated at 25 C for 52 h under continuous illumination (approx. 3000–4000 lux).
After 48 h of cysts incubation, pre-feeding of the freshly hatched ostracods was performed
with algae (spirulina-powder) provided in the test kit. Next, after hatching, before feeding with
algal food suspension, the length measurements of ostracod neonates has been done. Algae
(Selenastrum capricornutum) used as feed in the test plate were reconstituted according to the
manufacturer’s procedure. Each well of a test plate was filled in the following order: 2 mL standard
freshwater, 2 ˆ 500 µL of sediment (soil) treated and non-treated for comparison (blank), 2 mL already
prepared algal suspension, 10 ostracods. The test plates were covered with Parafilm® and closed by a
˝
lid. Then multiwall plates were incubated at 25 C in darkness for 6 days. After 6 days of exposure, the
ostracods have been recovered from the multiwells to determine the percentage mortality. To calculate
the growth inhibition of survived organisms, their length measurements have been also done. Mortality
of test organisms was determined in six replicates. The measurement of length was carried out by
means of a micrometric strip placed on the bottom of a glass microscope plate. Growth inhibition (GI)
of H. incongruens in the test sediment was calculated as follows:

Molecules 2016, 21, 936
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% growth inhibition “ 100 ´ rpgrowth in test sed.{growth in ref. sed.q ˆ 100s
Statistical differences between variables were analyzed with ANOVA.
(1)
4. Conclusions
To conclude, a series of diphenyl pyrene-derived aminophosphonates 3a
–d, 4 and 5 has
been prepared. Our previous study [17] revealed that pyrene-derived aminophosphonates have
interesting cytotoxic properties against colon cancer cell lines, therefore, the ecotoxicity of the
newly synthesized compounds has been evaluated using the crustacean Heterocypris incongruens
and bacterium Vibrio fischeri biotests. The evaluation demonstrated clearly that all compounds
3a–d and 4 are toxic in a moderate to high degree and that diphenyl N-benzylamino(pyren-1-yl)
methylphosphonate (3d) shows the highest degree of ecotoxicity. It has been also revealed that a weak
correlation between the toxicity against H. incongruens and against V. fischeri has been found and the
structure-toxicity correlation is rather problematic.
Although no evident correlations between the structure of the tested compounds and their toxicity
have been observed, the combination of the different bioassays—the Microtox® and Ostracodtoxkit F
™
tests—has been proved to be a very useful tool for determining and comparing the potential toxicities
of examined substances 3a–d and 4 as well as for the assessment of their toxicity interactions.
Supplementary Materials: The following are available online at: http://www.mdpi.com/1420-3049/21/7/936/s1,
scans of 1H, 13C and 31P NMR of compounds 3a–d, 4 and 5.
Acknowledgments: We thank the Polish National Centre of Science (NCN) for funding the work in the framework
of the grant no. 2014/13/B/NZ9/02418. Within the framework of this grant, funds for covering the costs to
publish in open access were scheduled.
Author Contributions: Jarosław Lewkowski and Piotr Rychter conceived and designed the experiments;
Maria Rodriguez Moya and Marta Chmielak performed the chemical experiments; Diana Rogacz and Kamila
Lewicka performed the ecotoxicological experiments; Jarosław Lewkowski and Piotr Rychter analyzed the
data (Jarosław Lewkowski chemistry, Piotr Rychter. ecotoxicity); Jarosław Lewkowski and Piotr Rychter wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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Sample Availability: Samples of the compounds 2a–d, 4 and 5 are available from the authors.
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