The effect of new thiophene-derived aminophosphonic derivatives on growth of terrestrial plants: A seedling emergence and growth test

Article abstract of DOI:10.3390/molecules21060694

The aim of this work was to synthesize selected thiophene-derived aminophosphonic systems and evaluate the phytotoxicity of newly obtained products according to the OECD 208 Guideline. Seven new thiophene-derived N-substituted dimethyl aminomethylphosphonic acid esters 2a-h were synthesized by the addition of an appropriate phosphite to azomethine bond of starting Schiff bases 1a-h, and NMR spectroscopic properties of aminophosphonates were investigated. These eight compounds were analyzed in regard to their phytotoxicity towards two plants, radish (Raphanus sativus) and oat (Avena sativa). On the basis of the obtained results, it was found that tested aminophosphonates 2a-h showed an ecotoxicological impact against selected plants, albeit to various degrees.

Full text of DOI:10.3390/molecules21060694

molecules
Article
The Effect of New Thiophene-Derived
Aminophosphonic Derivatives on Growth of
Terrestrial Plants: A Seedling Emergence and
Growth Test
Jarosław Lewkowski 1,*, Zbigniew Malinowski 1, Agnieszka Matusiak 1, Marta Morawska 1,
Diana Rogacz 2 and Piotr Rychter 2,
1
*
Department of Organic Chemistry, Faculty of Chemistry, University of Łódz´, Tamka 12, Łódz´ 91-403, Poland;
zbigmal@uni.lodz.pl (Z.M.); agusk@poczta.fm (A.M.); martaem88@gmail.com (M.M.)
Faculty of Mathematics and Natural Science, Jan Długosz University in Cze˛stochowa,
13/15 Armii Krajowej Av., Cze˛stochowa 42-200, Poland; diana.rogacz@gmail.com
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.)
2
*
Academic Editor: György Keglevich
Received: 22 April 2016; Accepted: 23 May 2016; Published: 30 May 2016
Abstract: The aim of this work was to synthesize selected thiophene-derived aminophosphonic
systems and evaluate the phytotoxicity of newly obtained products according to the OECD 208
Guideline. Seven new thiophene-derived N-substituted dimethyl aminomethylphosphonic acid esters
2a
–
h
were synthesized by the addition of an appropriate phosphite to azomethine bond of starting
, and NMR spectroscopic properties of aminophosphonates were investigated.
Schiff bases 1a
–
h
These eight compounds were analyzed in regard to their phytotoxicity towards two plants, radish
(Raphanus sativus) and oat (Avena sativa). On the basis of the obtained results, it was found that
tested aminophosphonates 2a–h showed an ecotoxicological impact against selected plants, albeit to
various degrees.
Keywords: thiophene-derived aminophosphonates; phytotoxicity; growth inhibition; environmental
protection; ecotoxicology; OECD standard
1. Introduction
Nowadays, since production of food in agricultural areas is in the phase of intensive growth, the
use of herbicides is absolutely necessary to manipulate or control undesirable vegetation of plants.
Herbicides are used in landscapes throughout all over the world and are generally accepted as a
moderately safe compounds. However, widespread use or overdosage of herbicides over long periods
of time can result in residues in crops, soil, and land waters and, as a consequence, may lead to health
and environmental risks [
of a weed to the agent. Consequently, the weeds, while becoming resistant will not respond to the
herbicide’s active properties [ ]. With this respect, there is a necessity of designing new compounds
1–4]. Permanent applications of a given herbicide can result in resistance
5,6
with potential herbicidal properties.
Sulfur-containing compounds, which have been developed as agrochemicals for plant protection,
are currently being reviewed from the standpoint of their use and biological properties in each field of
fungicides, insecticides, or herbicides [7–9].
Sulfur-containing herbicides are successfully and widely used as a foliar (applied to weed foliage)
and a soil-applied herbicide absorbed by the root or shoot of emerging seedlings.
Biodegradability, photodegradation, persistence, soil mobility, and the accumulation in living
organisms of sulfur-containing herbicides are currently in extensive research phases [10–14].
Molecules 2016, 21, 694; doi:10.3390/molecules21060694
www.mdpi.com/journal/molecules

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Designing new sulfur-containing compounds with potential herbicidal activity in soil, their
persistence in soil must be taken into consideration. On the one side, herbicides, which are applied
to the soil, typically affect seed emergence or the growth of weed seedlings and must remain in the
soil to be effective. Unfortunately, on the other side, some herbicides like thifensulfuron-methyl can
persist in the environment for a long time, becoming harmful agents for the surrounding environment.
Therefore, planning the synthesis, it is difficult to strike a balance between the activity of a compound
and its biodegradability in soil [15].
Biological properties of thiophene-deriving aminophosphonates have been largely studied [16
Scientists found several of them to be promising plant protection agents [16] or to have rather strong
antimicrobial [17], cytotoxic [18], antifungal [19 20], or even antiviral [20] properties. Therefore,
working on new variations of methods for synthesis of aminophosphonic systems, chemists include
derivatives bearing thiophene moiety as examples [17 21 26]. It is to stress that all those methodologies
–20].
,
,
–
are mostly based on the Kabachnik–Fields reaction [27–29].
Working on our large project aimed at the search of a new class of herbicidal agents, we feel
obliged to perform tests of phytotoxicity of newly synthesized compounds, especially when these
compounds are aminophosphonic derivatives, which are known generally to have moderate-to-strong
phytotoxic action on higher plants [30–33].
Our previous investigation provided results demonstrating that aminophosphonic acids
bearing furfuryl moiety (derivatives of C-furfurylphosphonoglycine) are compounds of moderate
phytotoxicity being able to kill both monocotyledonous oat (Avena sativa) and dicotyledonous radish
(Raphanus sativus) with an amount of 100–200 mg in 1 kg of soil [34].
The presented research is concerned with a screening test of a synthesized series of
variously N-substituted dimethyl amino(2-thienyl)methylphosphonates (2a–g) and dibenzyl
N-furfurylamino(2-thienyl)methylphosphonate (2h) with respect to their phytotoxic properties. Our
assumption was that the potential hazard of thiophene-derived aminophosphonates is important
enough in the light of their possible applications and therefore should be investigated.
Apart from that, we performed the preliminary evaluation of aminophosphonates 2a–h as
potential soil-applied herbicides for agricultural/horticultural purposes. The potential effects of
herbicides strongly results from their mechanism of action and the way they are applied. Since
some types of herbicides are non-selective, which means the chemicals kill all classes of plants,
not only unwanted weeds, for the proposed experiment, but also two types of plants (mono- and
dicotyledonous) have been chosen as experimental objects.
2. Results and Discussion
2.1. Chemistry
Schiff bases 1a
–h were synthesized following the published and commonly known procedure of
simple mixing thiophene-2-carboxaldehyde with appropriate amine in methanol and stirring them
at room temperature for 24 h [35]. This procedure produced imines 1a
–
h
, which were isolated and
1
used for further conversions without any purification. H-NMR spectra were made only to verify
the identity, based on the 1H-NMR diagnostic singlet of a proton of the azomethine group (–CH=N–)
above 8 ppm [31,32].
Aminophosphonates 2a
modified procedure of a dimethyl or dibenzyl phosphite addition to the azomethine bond of
corresponding Schiff bases 1a in boiling toluene for 5 h. After the workup described in Section 3,
the resulting aminophosphonates 2a–h were obtained in 60%–70% yields (Scheme 1).
–h were synthesized basing on the aza-Pudovik reaction—a slightly
–
h

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Scheme 1. Synthesis of thiophene-derived aminophosphonates 2a–h.
2.2. Evaluation of Phytotoxicity of Aminophosphonates 2a–h
The rate of growth and development of plants as well as their quality, including increased number
and size of leaves and stems, is strongly dependent (as commonly known) on the ground composition.
Since plants have a high amount of water, its level depends on the contents of water in its environment,
so taking dry mass as a measure of plant growth seems to be more credible.
Water content is given as a percentage of the dry or fresh weight. To assess the phytotoxicity of the
analyzed compounds, the emergence and weight (dry and green) of control plant seedlings, with the
emergence and mass (dry and green) of plant seedlings growing on the soil with a given admixture of
the examined substances 2a–h, were determined and compared. Dry weight changes of tested plants
are shown in Figure 1. The visual assessment of any damage of tested plants concerns mainly the
degree of plant growth inhibition, which is evaluated by visible or other signs of necrosis of chlorosis.
Necrosis is the symptom of the death of plant tissues or organs caused by infection. Leaf and/or
stem deformation may also be observed, while chlorosis is an inhibition of chlorophyll biosynthesis,
resulting in yellowing of tissue.
2a
2b
2c
2a
2b
2c
250
200
150
100
50
0
200
400
600
800
1000
Concentration of compound (mg/kg of soil d.w.)
Figure 1. Changes of dry weight of treated plants expressed as percent to the value in untreated plants
(control plants = 100% of dry weight). Solid lines represent changes of oat dry weight. Dotted lines
represent changes of radish dry weight.

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Preliminary tests for aminophosphonates 2a
–
c
for both oat and radish have shown inhibitive
effects for plants. The dicotyledonous radish was noticed as the more sensitive plant when compared
to sprouts of oat. Comparing results of toxicity of aminophosphonates 2a for both types of tested
–
c
plants, it was noticed that Compounds 2a and 2b are more toxic as compared with 2c (Tables 1 and 2
Figures 1–4).
Table 1. Average changes (mean of three replicates) in basic parameters of the plant growth test for oat
(Avena sativa) treated with 2a–c. Least significant difference for samples (LSDS) and concentration
(LSDC) is given for each tested parameter. %F.M. refers to plant biomass (fresh weight) expressed as
percent of untreated control.
Sample Concentration in Soil
(mg/kg of Soil Dry Matter)
Emerged Seedlings
Number
% of
Germination
Fresh Matter
(g/pot)
% F.M.
0
1
10
100
200
400
800
1000
20
19
19
19
19
19
18
18
100
97
95
97
95
95
88
90
2.780
2.682
2.654
2.919
2.644
2.715
2.100
1.847
100
96
95
105
95
98
76
66
0
1
10
100
200
400
800
1000
20
19
19
19
19
20
18
18
100
95
97
97
97
98
92
92
2.780
2.740
2.663
2.729
2.651
2.661
2.021
1.659
100
99
96
98
95
96
73
60
0
1
10
100
200
400
800
1000
20
19
20
20
19
20
19
19
100
97
98
98
97
98
95
95
2.780
2.759
2.704
2.801
2.862
2.762
2.643
2.253
100
99
97
101
103
99
95
81
LSDS = 1
LSDC = 1
LSDS = 0.401
LSDC = 0.245
All the other results, to avoid confusion concerned with a large amount of data, have been placed in
Supplementary Materials. To describe results, moreover, the demonstrated values of no observed effect
concentration (NOEC) and lowest observed effect concentration (LOEC), changes in the fresh mass of plants were
expressed as % inhibition compared to control and presented as EC50 (Figure 2). Effective concentration EC50
was calculated using GraphPad Prism software (Version 7, GraphPad Software, Inc., La Jolla, CA 92037, USA).
Table 2. Average changes (mean of three replicates) in basic parameters of the plant growth test for
radish (Raphanus sativus) treated with 2a–c.
Sample Concentration in Soil
(mg/kg of Soil Dry Matter)
Emerged Seedlings
Number
% of
Germination
Fresh Matter
(g/pot)
% F.M.
0
1
10
100
200
400
800
1000
20
20
19
19
18
16
18
15
100
100
97
98
93
83
90
75
4.951
4.908
4.831
4.728
3.928
1.752
1.136
0.772
100
99
98
95
79
35
23
16

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Table 2. Cont.
Sample Concentration in Soil
(mg/kg of Soil Dry Matter)
Emerged Seedlings
Number
% of
Germination
Fresh Matter
(g/pot)
% F.M.
0
1
10
100
200
400
800
1000
20
20
19
19
19
17
17
15
100
100
95
97
97
86
88
78
4.951
4.803
4.843
4.728
3.928
2.142
1.546
1.106
100
97
98
95
79
43
31
22
0
1
10
100
200
400
800
1000
20
19
19
19
19
18
18
17
100
98
98
98
97
93
93
86
4.951
4.914
4.798
4.873
4.787
3.934
3.328
2.733
100
99
97
98
97
79
67
55
LSDS = 1
LSDC = 1
LSDS = 0.435
LSDC = 0.267
Least significant difference for samples (LSDS) and concentration (LSDC) is given for each tested parameter.
%F.M. refer to plant biomass (fresh weight) expressed as percent of untreated control.
2000
OAT RADISH
1523
1487
1600
1200
800
400
0
1442
1447
1284
1241
1192
1161
983.8
863.4
789.9
630.7
553.4
420.5
379.7
355.6
2a
2b
2c
2d
2e
2f
2g
2h
Compound
Figure 2. The EC50 values of fresh weight inhibition following exposure to tested compounds.
Figure 3. Cont.

Molecules 2016, 21, 694
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Figure 3. Digital photographs of oat treated with 2a–c (concentration in mg/kg of soil dry weight) on
the 14th day of growth.
Figure 4. Digital photographs of radish treated with 2a–c (concentration in mg/kg of soil dry weight)
on the 14th day of growth.

Molecules 2016, 21, 694
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N-methylphenyl aminophosphonates with ortho and meta substituted phenyl ring (2a, 2b)
have a significantly stronger influence on the inhibition of plant growth as compared with
N-para-methylphenyl aminophosphonate (2c). In a case of oat, the values of NOEC and LOEC
for Compounds 2a and 2b were 400 and 800 mg/kg of soil dry weight, respectively. These values
for Compound 2c were higher (NOEC and LOEC 800 and 1000 mg/kg d.w. of soil), indicating lower
toxicity and subsequently higher tolerance of oat against this substance (Figure 3).
At the highest concentration (1000 mg), a ca. 40% decrease in fresh matter of oat sprouts for
Compounds 2a and 2b when compared to control plants is observed (Table 1).
To make the description of the obtained results more readable and user-friendly, we only present
selected tables and figures to the main text here (Tables 1 and 2, Figures 1–4).
Tested substances were much more toxic for the seedlings of radish, resulting in a decrease in
NOEC and LOEC values. The first observed negative symptoms of tested 2a
radish were observed for the concentrations of 200 mg/kg of dry weight of soil (Samples 2a
of 400 mg/kg of dry weight of soil for Compound 2c (Figure 4). A very high drop of fresh weight of
radish seedling was noticed for all compounds (2a ), but aminophosphonates 2a and 2b were more
toxic. At the concentration of 1000 mg/kg of dry weight of soil, a decrease in radish fresh weight,
resulting from the action of Compounds 2a , was 16%, 22%, and 55%, respectively. High values
–
c
compounds against
,
2b) and
–c
–
c
of dry matter at higher concentrations of the tested substances in pots correspond with a stronger
inhibition of water uptake through the root system of plants (Figure 1). The increase in values of oat
dry matter when compared to control plants is not as high as in the case of radish, which indicates
better tolerance of oat roots against tested substances and moderately observed disturbances in the
water uptake process.
The number of emerged seedlings at higher concentrations of tested compounds also decreased
and was more visible for radish.
A plant growth test conducted for aminophosphonates bearing methoxy groups substituted in
position meta (2d) and para (2e) in a phenyl ring have proved their toxic impact against both types of
plants. Contrary to Compounds 2b–c substituted with meta-methyl and para-methylphenyl groups,
respectively, the examined N-m-methoxyphenyl derivative 2d was less toxic for both radish and oat
as compared with N-p-methoxyphenyl derivative 2e (Tables S1 and S2, Figures S1–S3). The NOEC
and LOEC values in the oat growth test for Compounds 2d were much higher (800 and 1000 mg
respectively) when compared to 2e (100 and 200 mg respectively). The same values of NOEC and
LOEC of Sample 2e were revealed for radish. Again, as in the case of Compounds 2a–c, the much
more sensitive plant was radish. In comparison, the yield of the fresh matter of oat at the highest
concentration (1000 mg) of 2d and 2e was 19% and 31% (respectively) lower compared to non-treated
plants. The yield of the fresh weight of radish at the highest concentration did not exceed 29% (for 2d
and 13% (for 2e) of fresh matter of control plants. The high increase in dry matter values point out the
dysfunction of roots (Figure S1).
)
Comparing the phytotoxicity results of aminophosphonates 2f, 2g, and 2h, Compound 2h was the
most toxic agent for both plants. NOEC and LOEC values of Compound 2h were the same for radish
and oat—40 and 80 mg/kg of dry matter, respectively. Among Compounds 2f and 2g, the former
had a higher negative impact on treated radish and oat. It was shown again that the more sensitive
specimen of plant was radish. A very low yield of fresh matter for radish at the highest concentration
(1000 mg) when compared to non-treated plants with simultaneously very high dry matter of plants
indicates a strong inhibition of plant growth (both sprouts and roots) (Tables S3–S5, Figures S4–S8)
It is worth comparing the plant growth of Samples 2a
derivative bearing the non-substituted phenyl ring. Sample 2f is much more toxic against oat
when compared to 2a In the case of radish, results are comparable to those obtained for
Samples 2a (the same values of NOEC and LOEC). Samples 2a and 2b, as well as N-benzyl
–c with 2f, the non-aromatic amine
–c.
,
b
derivative 2f, in the highest applied concentration (1000 mg/kg), harmfully influenced radish
seeds especially, while for oat, at the same concentration, Sample 2f (without substituted phenyl

Molecules 2016, 21, 694
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ring with methyl group) caused almost total inhibition of sprout germination. As a result,
contrary to dimethyl N-benzylamino(2-thienyl)methylphosphonate (2f), which kills both plants
in a non-selective way, dimethyl N-(2-methylphenyl)amino(2-thienyl)methylphosphonate (2a) and
dimethyl N-(3-methylphenyl)amino(2-thienyl)methylphosphonate (2b) were found to be selectively
toxic towards radish, slightly harming oat.
3. Materials and Methods
3.1. Chemistry
All solvents (POCh, Gliwice, Poland) were routinely distilled and dried prior to use. Amines,
dimethyl and dibenzyl phosphites, as well as 2-thiophenecarboxaldehyde (Aldrich, Poznan´, Poland)
were used as received. Melting points were measured on a MelTemp II apparatus (Bibby Scientific
Limited, Staffordshire, UK) 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). TMS was used as the internal standard for 1H- and 13C-NMR, and phosphoric acid was
used as the external standard for 31P-NMR. The following abbreviations were used for listing NMR
signals: s—singlet, d—doublet, dd—doublet of doublets, ddd—doublet of doublet of doublets, and
m—multiplet. Elemental analyses were carried out at the Centre for Molecular and Macromolecular
Science of the Polish Academy of Science in Łódz´, Poland.
General Procedures of Preparaing Amino(2-thienyl)methylphosphonates 2a–h
2-Thiophenecarboxaldehyde (1.12 g, 10 mmol) was dissolved in methanol (15 mL), and a solution
of an appropriate amine (10 mmol) in methanol (15 mL) was added. The obtained solution was stirred
at room temperature for 24 h. Then, a small portion of anhydrous potassium carbonate was added,
the mixture stirred for additional 15 min. Then, the inorganic salt was filtered off, and the filtrate was
evaporated to achieve imines in quantitative yields, which were used for the further reaction without
any purification.
Thus, the obtained imine (10 mmol) was dissolved in acetonitrile (15 mL), and an appropriate
phosphite (10 mmol) in acetonitrile (15 mL) was added. The mixture was refluxed during the day and
stirred at room temperature during the night. Total time of the reaction was 72 h. Then, the solvent
was evaporated, and the crude product was isolated and purified using various procedures.
Dimethyl aminophosphonates 2a–g were isolated as follows: crude products were dissolved
in DCM (30 mL), and the solution was washed 5 times with saturated aqueous sodium hydrogen
carbonate. After the usual workup, DCM was evaporated, and the product was purified by column
chromatography on silica gel using ethyl acetate-hexane in a 4:1 ratio.
Product 2h was chromatographed after evaporating acetonitrile, eluted using ethyl
acetate—hexane in a 3:2 ratio.
Dimethyl N-(2-methylphenyl)amino(2-thienyl)methylphosphonate (2a): Yield = 83% (2.58 g) yellow oil.
1H-NMR (CDCl3, 600 MHz):
δ
7.27–7.25 (m, H5thioph, 1H), 7.21–7.19 (m, H3thioph, 1H), 7.11 (d,
3JHH = 7.4 Hz, o-C6H4, 1H), 7.07 (ddd, 3J(1)
= 3J(2)
= 7.4 Hz and 4JHH = 1.7 Hz, o-C6H4, 1H), 7.01
HH
HH
(ddd, 3JHH = 5.0 and 4.2 Hz and 4JHH = 0.7 Hz, H4thioph, 1H), 6.74 (ddd, 3J(1)
= 3J(2)
= 7.4 Hz and
HH
HH
4JHH = 1.0 Hz, o-C6H4, 1H), 6.65 (d, 3JHH = 7.4 Hz, o-C6H4, 1H), 5.16 (d, 2JPH = 23.7 Hz, CHP, 1H), 3.82
(d, 3JPH = 10.6 Hz, POCH3, 3H), 3.67 (d, 3JPH = 10.6 Hz, POCH3, 3H), 2.28 (s, CH3, 3H). 13C-NMR
2
(CDCl3, 150 MHz):
δ
143.98 (d, JCP = 12.3 Hz, C2thioph), 139.81 (Carom), 130.41 (Carom), 127.19 (d,
J = 2.5 Hz, Cthioph), 127.03 (Carom), 126.19 (d, J = 7.2 Hz, Cthioph), 125.43 (d, J = 3.4 Hz, Cthioph), 123.39
(Carom), 118.86 (Carom), 111.48 (Carom), 54.17 (d, 2JCP = 7.0 Hz, POC), 53.88 (d, 2JCP = 7.3 Hz, POC),
51.84 (d, 1JCP = 157.8 Hz, PC), 17.47 (ArC). 31P-NMR (243 MHz, CDCl3):
δ
23.33. Elem. Anal. Calcd.
for C14H18NO3PS: C, 54.01, H, 5.83, N, 4.50. Found: C, 54.18, H, 5.97, N, 4.59.

Molecules 2016, 21, 694
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Dimethyl N-(3-methylphenyl)amino(2-thienyl)methylphosphonate (2b): Yield = 94% (2.92 g) yellow crystals,
˝
1
thioph
m.p.: 81–83 C. H-NMR (CDCl3, 600 MHz):
δ
7.26–7.25 (m, H5thioph, 1H), 7.21–7.20 (m, H3
,
1H), 7.07 (dd, JHH = 7.2 and 7.0 Hz, m-C6H4, 1H), 7.01 (m, H4thioph, 1H), 6.62 (d, JHH = 7.0 Hz,
m-C6H4, 1H), 6.55 (s, m-C6H4, 1H), 6.52 (dd, 3JHH = 7.2 Hz and 4JHH = 1.9 Hz, m-C6H4, 1H), 5.11 (d,
2JPH = 24.4 Hz, CHP, 1H), 3.82 (d, 3JPH = 10.4 Hz, POCH3, 3H), 3.65 (d, 3JPH = 10.8 Hz, POCH3, 3H),
3
3
2.28 (s, CH3, 3H). 13C-NMR (CDCl3, 150 MHz):
δ
145.94 (d, 2JCP = 13.1 Hz, C2thioph), 139.68 (Carom),
139.10 (Carom), 129.15 (Carom), 127.17 (d, J = 3.1 Hz, Cthioph), 126.25 (d, J = 7.3 Hz, Cthioph), 125.41 (d,
J = 4.0 Hz, Cthioph), 120.09 (Carom), 114.94 (Carom), 111.01 (Carom), 54.13 (d, 2JCP = 6.7 Hz, POC), 53.83
(d, 2JCP = 7.4 Hz, POC), 51.73 (d, 1JCP = 158.4 Hz, PC), 21.54 (ArC). 31P-NMR (243 MHz, CDCl3):
δ
23.20. Elem. Anal. Calcd. for C14H18NO3PS: C, 54.01, H, 5.83, N, 4.50. Found: C, 54.17, H, 5.79, N, 4.51.
Dimethyl N-(4-methylphenyl)amino(2-thienyl)methylphosphonate (2c): Yield = 54% (1.68 g) yellow crystals,
thioph
m.p.: 98–100 ˝C. 1H-NMR (CDCl3, 600 MHz):
δ
7.25–7.24 (m, H5thioph, 1H), 7.20–7.19 (m, H3
,
3
4
1H), 7.05–6.98 (m, H5thioph, p-C6H4, 3H), 6.64 (part of AA’XX’ system, JHH = 9.0 and JHH = 1.2
and 1.1 Hz, p-C6H4, 2H), 5.09 (d, JPH = 24.0 Hz, CHP, 1H), 3.82 (d, JPH = 10.8 Hz, POCH3, 3H),
2
3
3
3.65 (d, JPH = 10.8 Hz, POCH3, 3H), 2.24 (s, CH3, 3H). 13C-NMR (CDCl3, 150 MHz):
δ 143.63 (d,
2JCP = 13.4 Hz, C2thioph), 139.77 (Carom), 129.79 (Carom), 128.40 (Carom), 127.15 (d, J = 2.4 Hz, Cthioph),
2
126.26 (d, J = 7.4 Hz, Cthioph), 125.41 (d, J = 3.4 Hz, Cthioph), 114.25 (Carom), 54.13 (d, JCP = 7.1 Hz,
POC), 53.81 (d, 2JCP = 7.1 Hz, POC), 52.12 (d, 1JCP = 157.9 Hz, PC), 20.40 (ArC). 31P-NMR (243 MHz,
CDCl3):
δ 23.27. Elem. Anal. Calcd. for C14H18NO3PS: C, 54.01, H, 5.83, N, 4.50. Found: C, 54.13, H,
5.88, N, 4.63.
Dimethyl N-(3-methoxyphenyl)amino(2-thienyl)methylphosphonate (2d): Yield = 72% (2.35 g) yellow oil
1H-NMR (CDCl3, 600 MHz):
δ
7.26–7.25 (m, H5thioph, 1H), 7.20–7.19 (m, H3thioph, 1H), 7.08 (dd, J1 = J2
= 7.8 Hz, m-C6H4, 1H), 7.01–6.99 (m, H4thioph, 1H), 6.35–6.34 (m, m-C6H4, 1H), 6.33–6.31 (m, m-C6H4,
1H), 6.28–6.26 (m, m-C6H4, 1H), 5.10 (d, 2JPH = 24.0 Hz, CHP, 1H), 3.81 (d, 3JPH = 10.8 Hz, POCH3,
3H), 3.74 (s, OCH3, 3H), 3.65 (d, 3JPH = 10.8 Hz, POCH3, 3H). 13C-NMR (CDCl3, 150 MHz):
δ 160.77
(Carom), 147.34 (d, 2JCP = 13.5 Hz, C2thioph), 139.46 (Carom), 130.12 (Carom), 127.2 (d, J = 3.0 Hz, Cthioph),
126.3 (d, J = 6.9 Hz, Cthioph), 125.5 (d, J = 3.5 Hz, Cthioph), 106.91 (Carom), 104.38 (Carom), 100.23 (Carom),
55.08 (ArOC), 54.12 (d, 2JCP = 6.7 Hz, POC), 53.90 (d, 2JCP = 6.7 Hz, POC), 51.69 (d, 1JCP = 158.4 Hz,
PC).31P-NMR (243 MHz, CDCl3:
H, 6.08, N, 4.06. Found: C, 52.97, H, 5.96, N, 4.38.
δ
23.06. Elem. Anal. Calcd. for C14H18NO4PS
ˆ
1/5 C6H14: C, 52.98,
Dimethyl N-(4-methoxyphenyl)amino(2-thienyl)methylphosphonate (2e): Yield = 80% (2.62 g) yellow oil.
1H-NMR (CDCl3, 600 MHz):
δ
7.25–7.23 (m, H5thioph, 1H), 7.18–7.16 (m, H3thioph, 1H), 6.99–6.98 (m,
H4thioph, 1H), 6.75 and 6.66 (AA’XX’ system, 3JHH = 9.0 and 4JHH = 1.2 and 1.1 Hz, p-C6H4, 4H), 4.94
(d, 2JPH = 23.4 Hz, CHP, 1H), 4.54 (d, J = 8.4 Hz, NH, 1H), 3.81 (d, 3JPH = 10.8 Hz, POCH3, 3H), 3.72
(s, OCH3, 3H), 3.64 (d, 3JPH = 10.8 Hz, POCH3, 3H). 13C-NMR (CDCl3, 150 MHz):
δ 153.26 (Carom),
139.91 (d, 2JCP = 14.1 Hz, C2thioph), 139.73 (Carom), 127.1 (d, J = 2.8 Hz, Cthioph), 126.3 (d, J = 6.9 Hz,
Cthioph), 125.4 (d, J = 4.0 Hz, Cthioph), 115.69 (Carom), 114.83 (Carom), 55.63 (ArOC), 54.13 (d, 2JCP = 6.7 Hz,
POC), 53.81 (d, 2JCP = 6.7 Hz, POC), 52.85 (d, 1JCP = 158.4 Hz, PC). 31P-NMR (243 MHz, CDCl3):
δ
23.33. Elem. Anal. Calcd. for C14H18NO4PS
5.78, N, 4.26.
ˆ
1/6 C7H8: C, 53.16, H, 5.69, N, 4.09. Found: C, 53.21, H,
Dimethyl N-benzylamino(2-thienyl)methylphosphonate (2f): Yield = 57% (1.77 g) yellow oil. 1H-NMR
(CDCl3, 600 MHz): 7.26–7.20 (m, PhH, H5thioph, 5H), 7.18–7.15 (m, PhH, 1H), 7.02–7.01 (m, H3thioph, 1H),
6.93 (dd, 3JHH = 4.8 and 3.6 Hz, H4thioph, 1H), 4.24 (d, 2JPH = 23.8 Hz, CHP, 1H), 3.82 (d, 2JHH = 13.8 Hz,
3
2
CH2Ph, 1H), 3.68 (d, JPH = 10.2 Hz, POCH3, 3H), 3.57 (d, JHH = 13.8 Hz, CH2Ph, 1H), 3.55 (d,
3JPH = 10.2 Hz, POCH3, 3H). 13C-NMR (CDCl3, 150 MHz): 139.30 (d, 2JCP = 4.3 Hz, C2thioph), 138.99
δ
(Carom), 128.47 (d, J = 7.5 Hz, Cthioph), 128.38 (Carom), 128.23 (Carom), 127.28 (Carom), 126.97 (d, J = 3.5 Hz,
Cthioph), 125.68 (d, J = 3.3 Hz, Cthioph), 54.74 (d, 1JCP = 159.7 Hz, PC), 53.94 (d, 2JCP = 6.8 Hz, POC),

Molecules 2016, 21, 694
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53.63 (d, 2JCP = 6.8 Hz, POC), 51.24 (d, 3JCP = 15.6 Hz, PCC). 31P-NMR (243 MHz, CDCl3):
Elem. Anal. Calcd. for C14H18NO3PS: C, 54.01, H, 5.83, N, 4.50. Found: C, 53.77, H, 5.67, N, 4.46.
δ 23.98.
1
Dimethyl N-benzhydrylamino(2-thienyl)methylphosphonate (2g): Yield = 41% (1.59 g) yellow oil. H-NMR
thioph
(CDCl3, 600 MHz): 7.44–7.34 (m, PhH, H5thioph, 6H), 7.32–7.22 (m, PhH, 5H), 7.07–7.06 (m, H3
,
H4thioph, 2H), 4.91 (s, CH, 1H), 4.29 (d, 2JPH = 22.8 Hz, CHP, 1H), 3.91 (d, 3JPH = 10.8 Hz, POCH3, 3H),
3.64 (d, 3JPH = 10.8 Hz, POCH3, 3H), 2.57–2.48 (bs, NH, 1H). 13C-NMR (CDCl3, 150 MHz):
δ 139.36
(d, JCP = 6.4 Hz, C2thioph), 139.73 (Carom), 128.75 (Carom), 128.56 (Carom), 127.89 (Carom), 127.17 (d,
2
J = 7.9 Hz, Cthioph), 127.07 (d, J = 2.3 Hz, Cthioph), 125.72 (d, J = 3.4 Hz, Cthioph), 63.81 (d, 3JCP = 16.0 Hz,
2
2
1
PCC), 54.12 (d, JCP = 6.7 Hz, POC), 53.53 (d, JCP = 6.7 Hz, POC), 53.19 (d, JCP = 161.5 Hz, PC).
31P-NMR (243 MHz, CDCl3):
5.86, N, 3.45. Found: C, 63.54, H, 5.69, N, 3.75.
δ
24.32. Elem. Anal. Calcd. for C20H22NO3PS
ˆ
1/5 C7H8: C, 63.33, H,
1
Dibenzyl N-furfurylamino(2-thienyl)methylphosphonate (2h): Yield = 62% (2.81 g) yellow oil. H-NMR
thioph
(CDCl3, 600 MHz):
δ
7.36–7.32 (m, PhH, 10H), 7.25–7.23 (m, H5thioph, H5fur, 1H), 7.11–7.09 (m, H3
,
1H), 7.03–7.02 (m, H4thioph, 1H), 6.33–6.31 (m, H3fur, 1H), 6.15–6.14 (m, H4fur, 1H), 5.10–5.02 (Part AB
of ABX system, JPH = 7.5 and 8.8 Hz, JHH = 11.7 Hz, POCH2Ph, CHP, 2H), 5.00 (Part A of AMX
system, 3JPH = 7.2 and 2JHH = 11.8 Hz, POCH2Ph, 1H), 4.90 (Part M of AMX system, 3JPH = 8.2 and
2JHH = 11.8 Hz, POCH2Ph, 1H), 4.43 (d, 2JPH = 19.3 Hz, CHP, 1H), 3.92 and 3.69 (2d, 2JHH = 14.6 Hz,
3
2
CH2Fur, 3H). 13C-NMR (150 MHz, CDCl3):
δ 152.60 (Carom), 142.12 (Carom), 138.67 (d, J = 4.8 Hz,
Carom), 136.32 (d, 3JCP = 6.0 Hz, Carom), 136.19 (d, 3JCP = 6.3 Hz, Carom), 128.48 (d, J = 6.5 Hz, Carom),
128.34 (Carom), 128.28 (Carom), 128.00 (Carom), 127.84 (Carom), 127.36 (Carom), 127.30 (Carom), 126.92 (d,
J = 2.4 Hz, Carom), 125.82 (d, J = 3.5 Hz, Carom), 110.14 (Carom), 107.92 (Carom), 68.58 and 68.18 (2d,
2JCP = 40.6 Hz, POC), 55.11 (d, 1JCP = 159.8 Hz, PC), 43.72 (d, 3JCP = 16.4 Hz, NC). 31P-NMR (243 MHz,
1
CDCl3):
δ
22.19. Elem. Anal. Calcd. for C24H24NO4PS
ˆ
/10 CH2Cl2: C, 62.66, H, 5.28, .N, 3.03. Found:
C, 62.77, H, 5.03, N, 3.05.
3.2. Plant Growth Test of New Synthesized Compounds
The plant growth test of thiophene-derived aminophosphonates 2a–h was performed in
laboratory conditions following the OECD 208 Guideline Terrestrial Plants Growth Test for oat
(Avena sativa) as a monocotyledonous plant and radish (Raphanus sativus L. subvar. radicula Pers.),
a dicotyledonous plant.
According to the mentioned OECD 208 standard, the plant growth test of Compounds 2a–h was
carried out in sandy soil having the following parameters: granulometric composition of soil—77%
sand, 14% dust and loam, organic carbon content of approx. 1.2%, pH (KCl) equal to 6.3.
Tests were carried out in polypropylene pots (diameter of 90 mm and capacity of 300 cm3), which
were filled with the control soil or with the soil mixed with the tested Compounds 2a–h added at a
specific concentration. Twenty identical seeds of each of the selected plant species were sown into
the soil. Seeds originated from the same source. Plants were grown for 14 days under controlled
conditions: a constant humidity content at the level required for the plants (70% field water capacity),
˝
light intensity (7000 lux), and temperature (20
˘
2 C) were maintained. Then, seedlings were counted,
and the dry as well as fresh weight of the plants was determined. The parts of plants above the soil
surface were analyzed.
The performed plant growth was evaluated using a preliminary test; subsequently, the final test
was conducted afterward based on the obtained screening results. Preliminary tests were performed
to determine the range of concentrations of compounds affecting the soil quality; therefore, the used
concentrations of Samples 2a–h were as follows: 0 mg (control), 1 mg, 10 mg, 100 mg, and 1000 mg/kg
of soil dry weight.

Molecules 2016, 21, 694
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As already mentioned, based on the obtained results from preliminary test, the final, more precise
test to find values of the no observed effect concentration (NOEC) and the lowest observed effect
concentration (LOEC) of the compounds under study 2a–h was performed.
The evaluation of phytotoxicity of the studied aminophosphonates 2a–h at applied concentrations
was made by comparing the germination and (dry and fresh) weight of control plant 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.
˝
The dry weights of tested plants were measured after drying at 75 C until the constant weight.
The visual evaluation of phytotoxicity of aminophosphonates 2a at applied concentrations was
–h
performed by digital photography. Obtained pictures were analyzed in terms of any type of damage
to tested plants, such as their growth inhibition, chlorosis, and necrosis. Tests were carried out three
times for each sample.
The significance of the obtained results was evaluated using the analysis of variance (ANOVA).
The least significant difference (LSD) values at a confidence level of 95% were computed using the
Tukey test.
4. Conclusions
To conclude, the toxicity of N-methylphenyl aminophosphonates 2a–c was diagnosed to be
promising as a potential soil-applied herbicide, especially for their selectivity—they are evidently toxic
for dicotyledonous radish and not so harmful for monocotyledonous oat. Moreover, their herbicidal
efficiency is stronger for the N-methylphenyl aminophosphonates substituted with methyl group at
position ortho (2a) and meta (2b) in the phenyl ring.
Dimethyl N-benzylamino(2-thienyl)methylphosphonate (2f) exhibited non-selective, harmful
effects against both tested plants.
Among N-methoxyphenyl aminophosphonates 2d and 2e, the latter showed an inhibitive
effect especially against dicotyldenous plants. Its impact, however, was significantly weaker than
impact of 2a
,
b. This is to point out that the sole structural difference between N-methoxyphenyl
aminophosphonates 2d
,e
and N-methylphenyl aminophosphonates 2a is the substituent of the
–c
phenyl moiety, i.e., the methoxy and methyl group, respectively, which indicates that the type and
position of those groups in the phenyl ring play a key role in the toxicity effect against tested plants.
Among all tested samples, dibenzyl N-furfurylamino(2-thienyl)methylphosphonate (2h) was
found to be the most toxic for radish and oat but without significant selectivity. The investigation of
Compounds 2a,b, 2f, and 2h, such as their potential applications and mechanistic approaches, will
be continued.
Some thiophene-derived aminophosphonic derivatives were found to have various biological
properties (vide supra); therefore, important microbiological, herbicidal, and cytotoxic properties of
the studied aminophosphonic systems bearing thiophene moiety 2a–h cannot be excluded. If so, the
synthesis of such compounds for potential agrochemical or pharmacological application must be
taken into consideration from the environmental protection point of view. Our results call attention to
the necessity of further phytotoxicological investigation of any new synthesized aminophosphonic
derivatives bearing thiophene moiety.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/6/694/s1.
Acknowledgments: Studies were founded by the National Centre of Science of Polish State (NCN), grant no.
2014/13/B/NZ9/02418, where funds for publishing in open-access mode were scheduled. Special thanks are
addressed to Paulina Tomczyk and Justyna Biernacka, former M.Sc. students at the University of Łódz´, for their
fruitful assistance in the preparation of the studied compounds.
Author Contributions: J.L. and P.R. conceived and designed the experiments; A.M., Z.M., M.M., and D.R.
performed the experiments; J.L. and P.R. analyzed the data; J.L. and P.R. wrote the paper.

Molecules 2016, 21, 694
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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; or in the
decision to publish the results.
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Sample Availability: Samples of the compounds 2a–h are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).