Synthesis of SMZ derivatives and investigation of effects on germination, root, and plant growth of Arabidopsis thaliana L.

Article abstract of DOI:10.3906/kim-1901-27

A series of sulfonamide derivatives were synthesized by reactions with various functional groups containing benzenesulfonyl chlorides and aniline derivatives under different substitution reaction conditions. The structures of SMZ derivatives were confirmed with melting point, FT-IR, ¹ H NMR, ¹³ C NMR, and LC-MS/MS techniques. In order to investigate the cytotoxic effects of these derivatives, we used a model plant species. The synthesized compounds (S1–S5) and sulfamethazine (SMZ) as a positive control were applied to Arabidopsis thaliana seeds. Our results indicated that S3 and S4 induced shorter roots and lower wet weight in plants. Plants treated with S2 and S5 showed no growth effects, similar to the untreated control group, while S1 slightly reduced root length and wet weight. These results suggest that S3 and the newly synthesized S4 derivatives have potential for use as herbicides since they possess cytotoxic effects on A. thaliana plants.

Full text of DOI:10.3906/kim-1901-27

Turk J Chem
Turkish Journal of Chemistry
(2019) 43: 1123 – 1133
© TÜBİTAK
http://journals.tubitak.gov.tr/chem/
doi:10.3906/kim-1901-27
Research Article
Synthesis of SMZ derivatives and investigation of effects on germination, root,
and plant growth of Arabidopsis thaliana L.
1
1
2
3
Tuğba GÜNGÖR ,, Ferah CÖMERT ÖNDER ,, Fatih SEZER ,, Aslıhan ÖZBİLEN ,,
2
1,∗
Kemal Melih TAŞKIN ,, Mehmet AY
,
1
Department of Chemistry, Faculty of Sciences and Arts, Çanakkale Onsekiz Mart University, Çanakkale, Turkey
2
Department of Molecular Biology and Genetics, Faculty of Sciences and Arts, Çanakkale Onsekiz Mart University,
Çanakkale, Turkey
3
Department of Biology, Faculty of Sciences and Arts, Çanakkale Onsekiz Mart University, Çanakkale, Turkey
Received: 12.01.2019
•
Accepted/Published Online: 10.06.2019
•
Final Version: 06.08.2019
Abstract: A series of sulfonamide derivatives were synthesized by reactions with various functional groups containing
benzenesulfonyl chlorides and aniline derivatives under different substitution reaction conditions. The structures of SMZ
1
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derivatives were confirmed with melting point, FT-IR, H NMR, C NMR, and LC-MS/MS techniques. In order to
investigate the cytotoxic effects of these derivatives, we used a model plant species. The synthesized compounds (S1–S5)
and sulfamethazine (SMZ) as a positive control were applied to Arabidopsis thaliana seeds. Our results indicated that S3
and S4 induced shorter roots and lower wet weight in plants. Plants treated with S2 and S5 showed no growth effects,
similar to the untreated control group, while S1 slightly reduced root length and wet weight. These results suggest that
S3 and the newly synthesized S4 derivatives have potential for use as herbicides since they possess cytotoxic effects on
A. thaliana plants.
Key words: SMZ derivatives, Arabidopsis thaliana L., herbicide, sulfonamide, synthesis
1. Introduction
Sulfonamides are well-known structures with various applications in medicinal chemistry and biological activity.
The most important implementation of sulfa drugs (e.g., sulfamethoxazole and sulfisoxazole) is to inhibit the
growth and multiplication of bacteria. Sulfonamides are also used as carbonic anhydrase inhibitors, matrix
metalloproteinase inhibitors, and antiallergy, antiinflammatory, antiviral, antifungal, antimalarial, anticancer,
antiarthritis, and antiemphysema drugs [1–5].
Sulfamethazine (SMZ) (4-amino-N -(4,6-dimethylpyrimidine-2-yl)benzenesulfonamide), known as sul-
fadimidine, sulfadimerazine, or sulfadimezine, is a broad-spectrum antibiotic used to treat bronchitis, prostate,
and urinary infections. SMZ is also used as veterinary antibiotic. Its presence in animal excreta and manure,
uptake by plants, and distribution in soil have been researched [6,7]. Interestingly, sulfamethazine can also
suppress DNA methylation in plants by impairing folate synthesis [8]. This phenomenon decreases the levels of
the universal methyl donor, S-adenosyl methionine, which is used by most methyltransferases [9]. DNA methyl-
transferase enzymes are responsible for the transfer of a methyl group of S-adenosylmethionine to the cytosine
residues in DNA [10]. DNA methylation is a well-known epigenetic marker and regulates various developmental
∗Correspondence: mehmetay06@comu.edu.tr
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GÜNGÖR et al./Turk J Chem
stages in plants such as vernalization, embryogenesis and seed viability, apical dominance, plant size, leaf size
and shape, fertility, and root length [11–15]. Several studies indicated that root length and fresh weight were
reduced when Arabidopsis thaliana plants were hypomethylated [8,13]. A. thaliana is a model organism for
plant genetics and molecular biology studies. A. thaliana was chosen in this study since it has a short life span,
grows easily in laboratory conditions, and has the potential to reveal main issues in plant biological structure
and functions [16].
Therefore, in this study, we synthesized and characterized some SMZ derivatives (S1–S5) and investigated
the cytotoxic effects of these compounds on A. thaliana plants (Figure 1). Our results indicated that S3 and
S4 are toxic at higher concentrations and may reduce the root length and fresh weight if applied at lower
concentrations. However, other SMZ derivatives (S1, S2, and S5) did not have a significant effect on plant
growth.
Figure 1. Structure of SMZ and synthesized sulfonamide derivatives (S1-S5).
2. Results and discussion
2.1. Chemistry
As shown in Figure 1, sulfamethazine (SMZ), which was used as a standard in our study, is a sulfonamide
derivative consisting of two aromatic rings, 4-aminophenyl and dimethylpyrimidine. We aimed to synthesize
some modified SMZ derivatives, investigate their herbicide properties on A. thaliana plants, and compare the
results with the standard SMZ. For this purpose, compounds S1–S5 were designed to have nitrophenyl or
chlorophenyl instead of a pyrimidine ring. Also, methyl, nitro, amino, and dibutylamino groups were used in
the 4-position of the phenyl ring. Unlike others, S4 and S5 have two nitro groups at 3,5-positions in the phenyl
ring (Figure 1).
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The S1–S3 derivatives were prepared with 47%–72% yield by heating 4-methyl/4-nitro benzenesulfonyl
chloride and corresponding nitroaniline derivatives in basic conditions (Figure 2). S1 was synthesized according
to the literature [17]. S2 was obtained from TosCl and 4-nitroaniline using NaH as a base in DMF under modified
literature conditions. Reaction of 3-nitroaniline and 4-nitrobenzenesulfonyl chloride in DMF containing pyridine
at 40–50 ◦ C for 3 h led to S3 with 72% yield [18]. To obtain S4, first 4-amino-3,5-dinitrobenzenesulfonyl
chloride as an intermediate was prepared from 2,6-dinitroaniline and chlorosulfonic acid at 100 ◦ C for 2 h.
Then, after stirring this intermediate and 4-nitroaniline in DMA as a solvent at room temperature for 48
h, target sulfonamide S4 was obtained with 42% yield. S5 was synthesized in five steps. The first two
steps consisted of sulfonylation of chlorobenzene and nitration of 4-chloro benzenesulfonic acid with potassium
nitrate, respectively [19]. The following two steps were the substitution of dibutylamine and potassium 4-chloro-
3,5-dinitro benzenesulfonate and the chlorination reaction of sulfonate by using PCl5 at room temperature
conditions, leading to 4-(dibutylamino)-3,5-dinitrobenzenesulfonyl chloride [20]. Finally, S5 was prepared by
the substitution reaction of the obtained intermediate and 4-chloroaniline in pyridine as a solvent at 50 ◦ C for
24 h as shown in Figure 2.
Figure 2. Synthesis of sulfonamide derivatives (S1-S5).
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All compounds were characterized by melting point, FT-IR, H NMR,
C NMR, and LC-MS/MS
analyses (see Section 3 and Supplementary information). Observations of the characteristic N-H stretching
peak of sulfonamide at 3251–3356 cm−1 in the FT-IR spectra and the –NH singlet peak of sulfonamide between
1
8.79 and 11.33 ppm in the H NMR spectra are the greatest evidence of the formation of the final sulfonamide
1
products. In the H NMR spectrum of compound S3, six aromatic hydrogens were observed at 7.73–8.88 ppm
and a singlet sulfonamide N-H peak was observed at 11.13 ppm. Ten different aromatic carbons were obtained
13
in the 114.08–154.49 ppm range with C NMR.
While three different aromatic hydrogen signals at 7.31–8.69 ppm and a NH2 singlet signal on ring A at
1
8.74 ppm were observed in the H NMR spectrum of compound S4, the NH proton of sulfonamide was found
at higher chemical shifts at 11.33 ppm. Additionally, eight different aromatic carbons of S4 were observed in
13
1
the 119.13–143.82 ppm range in the C NMR spectrum. In the H NMR spectrum of compound S5, the CH3
chemical shift of the butyl chain was observed at 0.90 ppm as a triplet peak. Also, two multiplet CH2 peaks
of butyl were determined at 1.36 and 1.59 ppm, respectively, and the N-CH2 peak has a chemical shift at 3.44
ppm. Doublet and doublet of doublets peaks of He and Hf were observed at 7.18 and 8.20 ppm, respectively.
Aromatic hydrogen signals on ring A and the sulfonamide N-H signal overlapped at 8.80 ppm with 2 +
1 integrations. There are eight different aromatic carbons in the 115.66–148.55 ppm range and four different
13
aliphatic carbons in the 14.06–43.00 ppm range in the C NMR spectrum of compound S5. All peaks of other
functional groups such as nitro, aromatic, or aliphatic on FT-IR and signals of proton/carbons in NMR spectra
confirm the structures of the sulfonamide derivatives. Molecular weights of compounds S3–S5 were confirmed
with LC-MS/MS analysis in negative mode (see Supplementary information).
2.2. Cytotoxic effects of the SMZ derivatives
To compare the cytotoxic effects of the SMZ derivatives, we used SMZ as a positive control. First, we treated
seeds with 50, 25, and 1 µM SMZ and cultured them in half-strength Murashige and Skoog medium for 15
days. These treatments promoted a high frequency of seed germination within 7 days in culture. Seedlings
initially appeared healthy and produced green cotyledons and roots. During the following days, the leaves did
not appear, and seedlings became white. We analyzed the root lengths and the fresh weight of seedlings after
15 days of culture.
Our results showed that SMZ treatment decreased the root length compared with untreated control seeds.
The control seeds (without SMZ) produced 18.66 ꢀ 3.57 mm roots. Meanwhile, seedlings treated with 50 µM
SMZ produced 2.80 ꢀ 0.34 mm roots, 25 µM SMZ induced 3.10 ꢀ 0.28, and 1 µM SMZ produced 5.04 ꢀ 0.94
mm roots. The mean weight was measured as 0.6569 mg per untreated control plant. However, plants treated
with 50, 25, or 1 µM SMZ produced 0.25, 0.33, and 0.184 mg fresh weight, respectively. Therefore, these results
indicated that higher levels of SMZ (50 µM) suppressed growth due to some cytotoxic effects (Figure 3) [8].
However, the same concentrations (1, 25, and 50 µM) of SMZ derivatives produced different root growth and
biomass production patterns.
Derivative S1 promoted a high frequency of seed germination for all treatments. Even though the
seedlings treated with 1 µM S1 were pale green after 2 weeks of culture, higher levels of S1 (25 µM) produced
healthy plants with many root hairs (Figure 3). While seedlings treated with 50 µM S1 produced 9.37 mm
roots, 25 µM S1 induced 15.6 ꢀ 2.6 mm and 1 µM S1 produced 20.5 ꢀ 5.5 mm root lengths (Figure 4).
Furthermore, seedlings treated with 50, 25, or 1 µM S1 produced 0.512, 0.4, and 0.672 mg fresh weight,
respectively (Figure 5).
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Figure 3. A. thaliana seedlings 15 days after treatments.
Figure 4. Root lengths of A. thaliana plants after 15 days of treatment with SMZ derivatives.
S2 caused high germination rates for all treatments. Although the seedlings treated with 50 µM S2 were
pale green after 2 weeks in culture, 25 µM and 1 µM S2 produced healthy plants with many root hairs (Figure
3). Seedlings treated with 50 µM S2 produced 16.13 ꢀ 2.30 mm roots, 25 µM S2 induced 16.23 ꢀ 3.88 mm,
and 1 µM S2 produced 15.16 ꢀ 4.08 mm root lengths (Figure 4). Also, seedlings treated with 50, 25, or 1 µM
S2 produced 0.253, 0.440, and 0.398 mg fresh weight, respectively (Figure 5).
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Figure 5. Mean weight of one plant 15 days after transfer to germination medium.
Although plants treated with 1 µM compound S3 were yellow in color and died after 2 weeks of culture,
at 25 µM and 50 µM plants were white and died earlier in culture (Figure 3). Mean root lengths of S3-treated
plants were 5.63 ꢀ 1.03 at 1 µM, 5.28 ꢀ 1.42 at 25 µM, and 6.61 ꢀ 3.14 at 50 µM (Figure 4). Also, seedlings
treated with 50, 25, or 1 µM S3 produced 0.052, 0.04, and 0.045 mg fresh weight, respectively (Figure 5).
Compound S4-treated seedlings died after 2 weeks in culture at all concentrations (Figure 3). Meanwhile,
seedlings treated with 50 µM S4 produced 4.89 ꢀ 0.92 mm roots, 25 µM S4 induced 3.92 ꢀ 1.36 mm, and 1
µM S4 produced 4.67 ꢀ 0.67 mm roots (Figure 4). Also, seedlings treated with 50, 25, or 1 µM S4 produced
0.051, 0.051, and 0.219 mg fresh weight, respectively (Figure 5).
The SMZ derivative S5 also promoted a high frequency of seed germination for all treatments. Although
the seedlings treated with 50 µM S5 were yellow-green after 2 weeks of culture, 25 µM and 1 µM S5 produced
healthy plants with many root hairs (Figure 3). S5 showed similar root growth pattern to the control group
with means of 15.53 ꢀ 4.61 mm at 1 µM, 15.70 ꢀ 3.86 mm at 25 µM, and 18.44 ꢀ 5.97 mm at 50 µM (Figure
4). S5 treatment resulted in 0.749 mg at 50 µM, 0.636 mg at 25 µM, and 0.795 mg at 1 µM fresh weight,
which is similar to the control group (Figure 5).
2.3. Conclusions
A series of sulfonamide derivatives (S1–S5) were synthesized under different reaction conditions and charac-
terized by various spectral methods. With the results obtained from this study, we conclude that among all
SMZ derivatives, only S3 and S4 that have a 4-nitro (S3) or 4-amino-3,5-dinitro (S4) on phenyl ring A and
contain an m- or p-nitro group on ring B showed similar results to SMZ. Plants grown in Murashige and Skoog
medium supplemented with S3 and S4 were bleached. Both root length and fresh weight of plants treated
with S3 and S4 derivatives showed statistically significant decreases compared to the control group without
treatment. S1 treatment results showed dose-dependent effects on plants, with higher concentrations reducing
the root lengths. Also, at the highest concentration S1 was not as effective as SMZ, S3, or S4. S2 and S5
did not affect root growth or plant biomass. When the experimental results were interpreted structurally, it
was determined that both S1 and S2 have a methyl group at the p-position of ring A and positions (o- or
p-) of the nitro group on ring B causing effectiveness at higher concentrations for compound S1 (o-nitro) and
ineffectiveness for compound S2 (p-nitro). Also, compound S5 with bulky dibutyl groups on the nitrogen atom
on ring A and the chloro atom on ring B had no effect on A. thaliana plants.
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In our study, we aimed to analyze cytotoxic effects of synthesized SMZ derivatives on A. thaliana plants
as a model. Several SMZ derivatives were synthesized with various effects on plants, including S3 and S4, which
are promising. SMZ is also routinely used as a veterinary drug and was found to accumulate in animal tissues
such as muscles and also urine, blood, and milk [21–26]. Therefore, there are concerns about SMZ pollution
in soil and plants, as well. The removal of SMZ from soil is another concern and a subject of several studies
[27–29]. The SMZ derivatives synthesized in this study may overcome these problems. However, further studies
on these compounds are required to assess their potential.
3. Experimental
3.1. Chemistry
3.1.1. General Information
All chemicals were purchased from Sigma-Aldrich and Merck and used as supplied without further purification.
SMZ was also obtained from Sigma-Aldrich (S6256). Synthesis of the compounds was monitored by TLC
on 0.25-mm silica gel plates (60F254) and visualized with UV light and/or KMnO4 stain. Melting points
were uncorrected with an X-4 melting-point apparatus. Infrared spectra were obtained with a PerkinElmer
Spectrum 100 FTIR spectrophotometer using ATR techniques. Frequencies are given in cm−1 and only selected
1
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absorbances are provided. H NMR and C NMR spectra were recorded at 500 and 125 MHz or 400 and 100
MHz in deuterated DMSO. Chemical shifts (δ) are expressed in parts per million (ppm), the coupling constants
(J) are expressed in hertz (Hz), and tetramethylsilane (TMS) was used as an internal standard. The following
abbreviations were used to specify peaks: s, singlet; d, doublet; t, triplet; q, quadruplet; quint, quintuplet; sext,
sextuplet; m, multiplet; and dd, double doublet.
3.1.2. Preparation of sulfonamide derivatives (S1–S5)
3.1.2.1. 4-Methyl-N-(2-nitrophenyl)benzenesulfonamide (S1)
Compound S1 was synthesized from 2-nitroaniline and 4-methylbenzenesulfonyl chloride (TosCl) according to
the described procedure [17]. Yellow crystal; yield: 63%; mp 112–113 ◦ C (lit. mp 112–113 ◦ C) [17]; IR (ATR;
ϑ/cm−1): 3275, 3123, 3104, 3084, 1609, 1599, 1522, 1484, 1382, 1343, 1273, 1213, 1163, 1143, 1089, 1039, 914,
1
847, 815, 779, 739, 704, 661; H NMR (500 MHz, DMSO-d6 , ppm): δ 10.21 (s, 1H, -NH), 7.92 (d, 1H, j =8.2
13
Hz), 7.61 (m, 2H + 1H), 7.36 (m, 2H + 1H), 7.27 (d, 1H, j =8.24 Hz), 2.36 (s, 3H); C NMR (DMSO-d6 ,
125 MHz, ppm): δ 144.24, 143.48, 136.79, 134.65, 130.82, 130.24, 127.29, 126.62, 125.93, 125.72, 21.46.
3.1.2.2. 4-Methyl-N-(4-nitrophenyl)benzenesulfonamide (S2)
DMF solution (10 mL) of 4-nitroaniline (1.0 g, 7.24 mmol) was added dropwise to NaH solution (0.35 g,
7.96 mmol, 1.1 eq., 55% oil dispersion) in 5 mL of DMF and stirred at room temperature for 3 h. 4-
Methylbenzenesulfonyl chloride (1.37 g, 7.24 mmol) was added to the reaction mixture and refluxed for 16
h. After completion of the reaction, the mixture was cooled to room temperature and poured into cold water
(200 mL). The resulting precipitate was filtered, washed with water, and dried at room temperature. The crude
product was purified by crystallization with ethanol. Yellow crystal; 1.0 g; yield: 47%; mp 190–191 ◦ C (lit. mp
191 ◦ C) [30,31]; IR (ATR; ϑ/cm−1): 3333, 3119, 3086, 3041, 1594, 1521, 1495, 1464, 1338, 1291, 1235, 1152,
1
1089, 906, 854, 750, 692, 663; H NMR (500 MHz, DMSO-d6 , ppm): δ 11.22 (s, 1H), 8.13 (d, 2H, j =9.27
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13
Hz), 7.765 (d, 2H, j =8.36 Hz), 7.395 (d, 2H,j =8.40 Hz), 7.31 (d, 2H, j =9.23 Hz), 2.35 (s, 3H); C NMR
(DMSO-d6 , 125 MHz, ppm): δ 144.90, 144.46, 142.83, 136.63, 130.43, 127.19, 125.79, 118.32, 21.41.
3.1.2.3. 4-Nitro-N-(3-nitrophenyl)benzenesulfonamide (S3)
DMF solution (10 mL) of 3-nitroaniline (0.3 g; 2.17 mmol) was added dropwise to a mixture of 5 mL of DMF
solution of 4-nitrobenzenesulfonyl chloride (0.48 g; 2.17 mmol) and pyridine (0.21 g; 2.65 mmol; 1.2 eq.). The
reaction mixture was heated at 40–50 ◦ C for 3 h. After this time, the mixture was poured into cold water (100
mL). The obtained solid was filtered and dried. The crude product was purified by crystallization with ethanol
to obtain the corresponding sulfonamide. Cream solid; 0.5 g; yield: 72%; mp 268 ◦ C. IR (ATR; ϑ/cm−1):
3251, 3160, 3110, 3094, 3043, 1603, 1518, 1473, 1428, 1343, 1241, 1176, 1118, 1026, 1003, 890, 863, 821, 739,
1
678; H NMR (600 MHz, DMSO-d6 , ppm): δ 11.13 (s, 1H), 8.88 (m, broad, 1H), 8.31 (s, 1H), 8.15 (m, broad,
13
2H), 8.08 (m, broad, 1H), 7.81 (m, broad, 2H), 7.73 (m, broad, 1H); C NMR (DMSO-d6 , 150 MHz, ppm):
δ 154.49, 148.84, 147.68, 139.22, 131.53, 127.32, 125.63, 123.75, 120.72, 114.08; LC-MS/MS (negative mode):
m/z calcd. for C12 H9 N3 O6 S: 323.28; found: 322.00.
3.1.2.4. 4-Amino-3,5-dinitro-N-(4-nitrophenyl)benzenesulfonamide (S4)
The starting material for S4 (4-amino-3,5-dinitrobenzenesulfonyl chloride) was prepared from 2,6-dinitroaniline
and chlorosulfonic acid according to the reported method [32]. Yellow solid; mp 154–157 ◦ C. IR (ATR;
ϑ/cm−1): 3443, 3334, 3097, 1632, 1555, 1530, 1448, 1419, 1367, 1275, 1172, 1139, 1095, 1045, 918, 898,
775, 726. This solid (0.14 g; 0.49 mmol) was dissolved in 3 mL of DMA and 4-nitroaniline (69 mg; 0.49 mmol)
was added to the reaction medium. The resulting mixture was stirred at room temperature for 48 h. After
completion of the reaction, 30 mL of ethyl acetate was added to the medium. The organic phase was washed
with water (2 × 50 mL), brined, and dried over Na2 SO4 . The solvent was evaporated under reduced pressure.
The residue was purified by washing with diethyl ether to give S4 as an orange solid. 80 mg; yield: 42%; mp
274–275 ◦ C. IR (ATR; ϑ/cm−1): 3437, 3329, 3196, 3092, 3077, 1624, 1596, 1511, 1494, 1339, 1249, 1168, 1133,
1
1104, 1045, 896, 849, 824, 776, 729, 693; H NMR (500 MHz, DMSO-d6 , ppm): δ 11.33 (s, 1H), 8.74 (s, 2H),
13
8.69 (s, 2H), 8.135 (d, 2H, j =9.05 Hz), 7.315 (d, 2H,j =9.10 Hz); C NMR (DMSO-d6 , 125 MHz, ppm):
δ 143.82, 143.48, 143.22, 135.29, 131.48, 125.91, 123.09, 119.13; LC-MS/MS (negative mode): m/z calcd. for
C12 H9 N5 O8 S: 383.29; found: 381.00.
ı
ı
3.1.2.5. 4-(N ,N -Dibutylamino)-3,5-dinitro-N-(4-chlorophenyl)benzenesulfonamide (S5)
Potassium 4-chloro-3,5-dinitrobenzenesulfonate was synthesized from chlorobenzene by the reactions of sulfony-
lation and then nitration [19]. This compound was converted to 4-(dibutylamino)-3,5-dinitrobenzenesulfonyl
chloride in two steps according to the reported method [20]. The obtained intermediate (0.6 g; 1.52 mmol) and
4-chloroaniline (0.68 g; 5.33 mmol; 3.5 eq.) were dissolved in 30 mL of pyridine and heated at 50 ◦ C for 24 h.
After completion of the reaction, excess pyridine was distilled at reduced pressure and the residue was poured
into cold water (50 mL). The mixture was extracted with ethyl acetate (3 × 40 mL) and the organic layer was
washed with water (50 mL). The ethyl acetate phase was dried over sodium sulfate, filtered, and concentrated
in vacuo. The crude product was purified by column chromatography over silica gel (0% to 30% methanol in
a chloroform gradient) and preparative thin-layer chromatography CHCl3 :hexane (100:1) to give S5 as bright
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yellow crystal. 0.3 g; yield: 41%; mp 83–84 ◦ C. IR (ATR; ϑ/cm−1): 3356, 3109, 2960, 2932, 2872, 1618, 1586,
1
1520, 1494, 1419, 1330, 1311, 1267, 1225, 1111, 1071, 1049, 922, 831, 744, 714; H NMR (600 MHz, DMSO-d6 ,
ppm): δ 8,805 (d, 2H,j =2.71 Hz), 8.79 (s, 1H), 8.20 (dd, 2H,j =9.59 and 2.25 Hz), 7.185 (d, 2H, j =9.65 Hz),
3.44 (q, 4H,j =6.78 Hz), 1.58 (quint, 4H,j =7.32 Hz), 1.36 (sext, 4H, j =7.46 Hz), 0.90 (t, 6H,j =7.40 Hz);
13
C NMR (DMSO-d6 , 150 MHz, ppm): δ 148.55, 135.02, 130.35, 129.98, 124.07, 115.66, 43.00, 30.60, 19.93,
14.06; LC-MS/MS (negative mode): m/z calcd. for C20 H25 ClN4 O6 S: 484.95; found: 483.00.
3.2. Plant material
In this study, Arabidopsis thaliana Columbia ecotype seeds were kindly provided by the University of Bath,
Department of Biology and Biochemistry, and used to test the cytotoxic effects of the synthesized and charac-
terized SMZ derivatives (S1–S5) on plants. For this purpose, first of all, the seeds were surface-sterilized with
6% sodium hypochlorite for 10 min and washed 3 times with sterile distilled water. Secondly, half-strength
Murashige and Skoog plant growth medium was prepared with 30 g/L sucrose and 8 g/L agar. Then S1–S5
and SMZ were added to the media respectively. Applications were performed with 1 µM, 25 µM, and 50 µM
concentrations for each group. A control group, which did not include any plant growth regulators, was also
used. Afterwards, seeds were transferred to the media in sterile petri dishes and incubated in the dark at 4
◦ C for 2 days to break the seed dormancy. At the end of the second day, these petri dishes were transferred
to a plant growth chamber. Germinating seeds were kept in 16:8 light conditions at 21 ◦ C and 70% relative
humidity for 15 days.
3.3. Phenotypic and statistical analysis
A. thaliana Columbia ecotype seeds were germinated on Murashige and Skoog media containing S1–S5 and
SMZ and a plant growth regulator-free Murashige and Skoog medium to determine and compare the cytotoxic
effects of the synthesized SMZ derivatives. Fifteen days after the germination, the root length and the fresh
weight of the seedlings in all groups were measured. A minimum of 37 and a maximum of 50 plant roots were
measured for different groups. One-way analysis of variance (ANOVA) tests and Tukey’s honestly significant
differences tests were used to assess significant differences within each group of root length data. Mean fresh
weight values were calculated for each group and used to prepare graphs for fresh weight/application.
Acknowledgment
A part of this study was financially supported by a research grant from the Scientific and Technological Research
Council of Turkey (TÜBİTAK Grant No. 110T754).
References
1. Supuran CT. Drug interaction considerations in the therapeutic use of carbonic anhydrase inhibitors. Expert
Opinion on Drug Metabolism & Toxicology 2016; 12 (4): 423-431. doi: 10.1517/17425255.2016.1154534
2. Ammazzalorso A, Filippis B, Giampietro L, Amoroso R. N -acylsulfonamides: Synthetic routes and biological
potential in medicinal chemistry. Chemical Biology & Drug Design 2017; 90 (6): 1094-1105. doi: 10.1111/cbdd.13043
3. Han T, Goralski M, Gaskill N, Capota E, Kim J et al. Anticancer sulfonamides target splicing by inducing RBM39
degradation via recruitment to DCAF15. Science 2017; 356 (6336): 397. doi: 10.1126/science.aal3755
1131

GÜNGÖR et al./Turk J Chem
4. Vermelho AB, Capaci GR, Rodrigues IA, Cardoso VS, Mazotto AM et al. Carbonic anhydrases from Trypanosoma
and Leishmania as anti-protozoan drug targets. Bioorganic & Medicinal Chemistry 2017; 25 (5): 1543-1555. doi:
10.1016/j.bmc.2017.01.034
5. Adhikari N, Mukherjee A, Saha A, Jha T. Arylsulfonamides and selectivity of matrix metalloproteinase-2: an
overview. European Journal of Medicinal Chemistry 2017; 129: 72-109. doi: 10.1016/j.ejmech.2017.02.014
6. Tasho RP, Cho JY. Veterinary antibiotics in animal waste, its distribution in soil and uptake by plants: a review.
Science of the Total Environment 2016; 563-564: 366-376. doi: 10.1016/j.scitotenv.2016.04.140
7. Du L, Liu W. Occurrence, fate, and ecotoxicity of antibiotics in agro-ecosystems. A review. Agronomy for Sustain-
able Development 2012; 32: 309-327. doi: 10.1007/s13593-011-0062-9
8. Zhang H, Deng X, Miki D, Cutler S, La H et al. Sulfamethazine suppresses epigenetic silencing in Arabidopsis by
impairing folate synthesis. Plant Cell 2012; 24 (3): 1230-1241. doi: 10.1105/tpc.112.096149
9. Loenen WA. S-adenosylmethionine: jack of all trades and master of everything? Biochemical Society Transactions
2006; 34 (2): 330-333. doi: 10.1042/BST0340330
10. Bird AP. Use of restriction enzymes to study eukaryotic DNA methylation: II. The symmetry of methylated sites
supports semi-conservative copying of the methylation pattern. Journal of Molecular Biology 1978; 118 (1): 49-60.
doi: 10.1016/0022-2836(78)90243-7
11. Dennis ES, Finnegan EJ, Bilodeau P, Chaudhury A, Genger R et al. Vernalization and the initiation of flowering.
Seminars in Cell and Developmental Biology 1996; 7: 441-448. doi: 10.1006/scdb.1996.0055
12. Xiao W, Custard KD, Brown RC, Lemmon BE, Harada JJ et al. DNA methylation is critical for Arabidopsis
embryogenesis and seed viability. Plant Cell Online 2006; 18: 805-814. doi: 10.1105/tpc.105.038836
13. Finnegan EJ, Peacock WJ, Dennis ES. Reduced DNA methylation in Arabidopsis thaliana results in abnormal
plant development. Proceedings of the National Academy of Sciences of the USA 1996; 93 (16): 8449-8454. doi:
10.1073/pnas.93.16.8449
14. Kakutani T, Jeddeloz JA, Richards EJ. Characterization of an Arabidopsis thaliana DNA hypomethylation mutant.
Nucleic Acids Research 1995; 23 (1): 130-137. doi: 10.1093/nar/23.1.130
15. Ronemus MJ, Galbiati M, Ticknor C, Chen J, Dellaporta SL. Demethylation-induced developmental pleiotropy in
Arabidopsis. Science 1996; 273 (5275): 654-657. doi: 10.1126/science.273.5275.654
16. Meinke DW, Cherry JM, Dean C, Rounsley SD, Koornneef M. Arabidopsis thaliana: a model plant for genome
analysis. Science 1998; 282 (5389): 662-682. doi: 10.1126/science.282.5389.662
17. Purushottamachar P, Aakanksha K, Tadas S, Robert DB, Lalji KG et al. Potent anti-prostate cancer agents derived
from a novel androgen receptor down-regulating agent. Bioorganic & Medicinal Chemistry 2008; 16: 3519-3529.
doi: 10.1016/j.bmc.2008.02.031
18. Shimotori H, Yanagida H, Enomoto Y, Igarashi K, Yoshinari M et al. Evaluation of benzenesulfonanilide derivatives
for the control of crucifers clubroot. Journal of Pesticide Science 1996; 21: 31-35. doi: 10.1584/jpestics.21.31
19. Furniss BS, Hannaford AJ, Smith PWG, Tatchell AR. Vogel’s Textbook of Practical Organic Chemistry. 4th ed.
London, UK: Longman, 1978.
20. George TG, Johnsamuel J, Delfín DA, Yakovich A, Mukherjee M et al. Antikinetoplastid antimitotic activity and
metabolic stability of dinitroaniline sulfonamides and benzamides. Bioorganic & Medicinal Chemistry 2006; 14
(16): 5699-5710. doi: 10.1016/j.bmc.2006.04.017
21. Sternesjo A, Mellgren C, Bjorck L. Determination of sulfamethazine residues in milk by a surface plasmon
resonance-based biosensor assay. Analytical Biochemistry 1995; 226 (1): 175-181. doi: 10.1006/abio.1995.1206
1132

GÜNGÖR et al./Turk J Chem
22. Aust MO, Godlinski F, Travis GR, Hao X, McAllister TA et al. Distribution of sulfamethazine, chlortetracycline
and tylosin in manure and soil of Canadian feedlots after subtherapeutic use in cattle. Environmental Pollution
2008; 156 (3): 1243-1251. doi: 10.1016/j.envpol.2008.03.011
23. Dixon-Holland DE, Katz SE. Competitive direct enzyme-linked immunosorbent assay for detection of sulfamet-
hazine residues in swine urine and muscle tissue. Journal of the Association of Official Analytical Chemists 1988;
71: 1137-1140.
24. Bevill RF, Sharma RM, Meachum SH, Wozniak SC, Bourne DW et al. Disposition of sulfonamides in food-
producing animals: concentrations of sulfamethazine and its metabolites in plasma, urine, and tissues of lambs
following intravenous administration. American Journal of Veterinary Research 1977; 38 (7): 973-977.
25. Fleeker JR, Lovett LJ. Enzyme Immunoassay for screening sulfamethazine residues in swine blood. Journal of the
Association of Official Analytical Chemists 1985; 68: 172-174.
26. Zayas-Blanco F, Garcıꢀa-Falcón MS, Simal-Gándara J. Determination of sulfa methazine in milk by solid phase
extraction and liquid chromatographic separation with ultraviolet detection. Food Control 2004; 15: 375-378.
doi:10.1016/S0956-7135(03)00100-2
27. Dolliver H, Kumar K, Gupta S. Sulfamethazine uptake by plants from manure-amended soil. Journal of Environ-
mental Quality 2007; 36 (4): 1224-1230. doi: 10.2134/jeq2006.0266
28. Rajapaksha AU, Chen SS, Tsang DCW, Zhang M, Vithanage M et al. Engineered/designer biochar for contaminant
removal/immobilization from soil and water: potential and implication of biochar modification. Chemosphere 2016;
148: 276-291. doi: 10.1016/j.chemosphere.2016.01.043
29. Topp E, Chapman R, Devers-Lamrani M, Hartmann A, Marti R et al. Accelerated biodegradation of veterinary
antibiotics in agricultural soil following long-term exposure, and isolation of a sulfamethazine-degrading sp. Journal
of Environmental Quality 2013; 42 (1): 173-178. doi:10.2134/jeq2012.0162
30. Jammi S, Sakthivel S, Rout L, Mukherjee T, Mandal S et al. CuO nanoparticles catalyzed C-N, C-O, and C-
S cross-coupling reactions: scope and mechanism. Journal of Organic Chemistry 2009; 74 (5): 1971-1976. doi:
10.1021/jo8024253
31. Bunce RA, Smith CL, Knight CL. N -(Nitrophenyl) benzamide and benzenesulfonamide derivatives by nucle-
ophilic aromatic substitution. Organic Preparations and Procedures International 2004; 36 (10): 482-487. doi:
10.1080/00304940409356635
32. Sato K, Takahagi H, Yoshikawa T, Morimoto S, Takai T et al. Discovery of a novel series of N -Phenylindoline-5-
sulfonamide derivatives as potent, selective, and orally bioavailable acyl CoA:monoacylglycerol acyltransferase-2
inhibitors. Journal of Medicinal Chemistry 2015; 58 (9): 3892-3909. doi: 10.1021/acs.jmedchem.5b00178
1133

Supplementary Information (SI)
Figure S1. 1H NMR spectra of S1.
Figure S2. 13C NMR spectra of S1.
1

Figure S3. 1H NMR spectra of S2.
Figure S4. 13C NMR spectra of S2.
2

Figure S5. 1H NMR spectra of S3.
Figure S6. 13C NMR spectra of S3.
3

202
H
O
S
N
NO2
96
88
80
72
64
56
48
40
32
24
16
8
O
O2N
m/z= 323.28 g/mol
S3
227
159
322
249
215
276
293
176
160
180
200
220
240
260
280
300
320
340
m/z
Figure S7. MS spectra of S3.
Figure S8. 1H NMR spectra of S4.
4

Figure S9. 13C NMR spectra of S4.
381
H
O
S
N
96
88
80
72
64
56
48
40
32
24
16
8
O2N
H2N
O
NO2
NO2
338
m/z= 383.29 g/mol
359
S4
228
194
258
244
282
163
327
320
180
200
220
240
260
280
m/z
300
340
360
380
400
Figure S10. MS spectra of S4.
5

Figure S11. 1H NMR spectra of S5.
Figure S12. 13C NMR spectra of S5.
6

227
H
O
S
159
N
O2N
N
96
88
80
72
64
56
48
40
32
24
16
8
O
Cl
NO2
m/z= 484.95 g/mol
S5
383
266
198
293
344
395
448
483
160
180
200
220
240
260
280
300
320
m/z
340
360
380
400
420
440
460
480
Figure S13. MS spectra of S5.
7