Synthesis and biological activity of 3-[Phenyl(1,3-thiazol-2-yl)-amino] propanoic acids and their derivatives

Article abstract of DOI:10.3390/molecules181215000

New N,N-disubstituted β-amino acids and their derivatives with thiazole, aromatic, and heterocyclic substituents were synthesized from N-phenyl-N-thiocarbamoyl-β-alanine by the Hantzsch method; derivatives with hydrazone fragments were also obtained. Some

Full text of DOI:10.3390/molecules181215000

Molecules 2013, 18, 15000-15018; doi:10.3390/molecules181215000
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molecules
ISSN 1420-3049
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Article
Synthesis and Biological Activity of 3-[Phenyl(1,3-thiazol-2-yl)-
amino]propanoic Acids and Their Derivatives
Vytautas Mickevičius 1,*, Aušra Voskienė 1, Ilona Jonuškienė 1, Ramūnė Kolosej 1,
Jūratė Šiugždaitė 2, Petras Rimantas Venskutonis 3, Rita Kazernavičiūtė 3, Zita Brazienė 4 and
Elena Jakienė 5
1
Department of Organic Chemistry, Kaunas University of Technology, Radvilėnų pl. 19,
LT-50254 Kaunas, Lithuania; E-Mails: ausravoskiene@yahoo.com (A.V.);
ilona.jonuskiene@ktu.lt (I.J.); ramune2004@gmail.com (R.K.)
2
Lithuanian University of Health Sciences, Veterinary Academy, Tilžės 18, LT-47181 Kaunas,
Lithuania; E-Mail: jurate.siugzdaite@lva.lt
3
Department of Food Technology, Kaunas University of Technology, Radvilėnų pl. 19, LT-50254
Kaunas, Lithuania; E-Mails: rimas.venskutonis@ktu.lt (P.R.V.); rita.kazernaviciute@ktu.lt (R.K.)
4
Lithuanian Research Centre for Agriculture and Forestry, Rumokai Experimental Station,
Klausučiai, 70462 Vilkaviškis Distr., Lithuania; E-Mail: zitamo421@gmail.com
5
Institute of Agricultural and Food Sciences, Aleksandras Stulginskis University, Studentų g. 11,
LT-53361 Akademija, Kaunor., Lithuania; E-Mail: elena.jakiene@asu.lt
* Author to whom correspondence should be addressed; E-Mail: vytautas.mickevicius@ktu.lt;
Tel.: +37-037-300-194; Fax: +37-037-300-152.
Received: 24 October 2013; in revised form: 3 December 2013 / Accepted: 3 December 2013 /
Published: 5 December 2013
Abstract: New N,N-disubstituted β-amino acids and their derivatives with thiazole,
aromatic, and heterocyclic substituents were synthesized from N-phenyl-N-thiocarbamoyl-
β-alanine by the Hantzsch method; derivatives with hydrazone fragments were also
obtained. Some of the synthesized compounds exhibited discrete antimicrobial activity, and
3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid was found to promote
rapeseed growth and to increase seed yield and oil content.
Keywords: N-phenyl-N-thiocarbamoyl-β-alanine; Hantzsch synthesis; thiazole; hydrazone;
biological activity

Molecules 2013, 18
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1. Introduction
Recently, special attention has been paid to β-amino acids as potential starting materials for
bio-organic, medicinal, and natural product chemistry [1]. Nitrogen-containing five- and six-membered
heterocyclic compounds and their derivatives, which can be easily synthesized in laboratories are
particularly important and often found in natural sources. Synthetic thiazole derivatives possess
various biological activities such as anti-inflammatory [2,3], antipyretic [4,5], antiviral [6],
anti-microbial [7–11], antifungal [12], and anticancer properties [13–16]. They have also been used as
anticoagulants and antiarrhythmics [16], antidepressants [17], in the treatment of Alzheimer's disease,
hypertension [18], schizophrenia [19], hepatitis C [20], allergies [21,22], HIV [23,24], diabetes [25,26],
and in functional dye synthesis [27,28]. The 2-aminothiazole structure provides herbicidal [6,29] and
antioxidant activities [30]. One of the most convenient methods of thiazole synthesis is the Hantzsch
synthesis, which is based on condensation of α-haloketones or aldehydes with thioamides or thiourea.
In our previous studies, N-aryl-N-thiocarbamoyl-β-alanines were used only for the synthesis of
1-substituted 2-thioxotetrahydro-4(1H)-pyrimidinones [31–33]. The aim of the present study was to
synthesize new potentially biologically active N,N-disubstituted β-amino acids and their derivatives with
thiazole, aromatic, heterocyclic fragments and to evaluate their chemical and biological properties.
2. Results and Discussion
2.1. Synthesis and Structural Peculiarities of New Compounds
3-[(4-Oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) was obtained from
N-phenyl-N-thiocarbamoyl-β-alanine (1) and monochloroacetic acid in different solvents (water, acetic
acid, DMF, ethanol), and by using different bases such as sodium acetate, sodium carbonate, and
triethylamine. Reactions in acetic acid and DMF were carried out at 90–100 °C and in ethanol and
water, at the mixture boiling temperature for 5 h.
The studies showed that the best yield was obtained when reactions were carried out in water in the
presence of sodium carbonate. Then the reaction mixture was acidified with acetic acid to pH 6.
In order to obtain more thiazole derivatives and to assess the influence of substituents on the biological
activity, a series of 3-{[5-substituted 4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic
acids 3 with aromatic and heterocyclic fragments was synthesized (Scheme 1) . The syntheses were
performed by condensation of dihydrothiazolone 2 and aromatic or heterocyclic aldehydes. Since the
starting compound 2 and the resulting products 3 are soluble in an alkaline medium, the reactions were
carried out in aqueous sodium carbonate solution in the presence of glycine as a bifunctional catalyst.
The target products from the reaction mixtures were separated by acidification of the aqueous solutions
with acetic acid to pH 6. The preliminary laboratory tests showed that the compounds 2 and 3 had growth
regulating effects for rapeseed (Brassica napus L.) and St. John’s wort (Hypericum perforatum L.) [34].
In the same way, the compound 4 was synthesized from dihydrothiazolone 2 and terephthaldehyde.

Molecules 2013, 18
Scheme 1. Synthesis of N-carboxyethylaminothiazoles and thiazolones 2–11.
15002
O
O
N
OH
N
OH
10
N
N
O
N
S
O
S
N
S
OH
O
OH
N
11
9
O
O
S
N
O
O
ii
R
ix
x
O
N
viii
O
3 a-i
OH
OH
O
i
vii
N
S
N
S
iii
N
N
OH
8
N
1
2
S
O
S
NH2
v
O
N
vi
iv
OH
O
O
O
N
OH
O
OH
4
OH
O
N
N
N
S
N
S
N
S
N
5
7
S
N
OH
CH3
6
CH3
O
O
CH3
e
g
a
c
d
b
f
i
h
Cl
NO2
R
Br
S
S
O
N
H
NO2
F
NO2
Reagents and Conditions: (i) ClCH2COOH, Na2CO3, H2O, reflux, 5 h, CH3COOH; (ii) RCHO,
NH2CH2COOH, Na2CO3, H2O, reflux, 5 h, CH3COOH; (iii) terephthalaldehyde, NH2CH2COOH, Na2CO3,
H2O, 5 h, CH3COOH; (iv) ClCH2CHO, H2O, reflux, 2 h, CH3COONa; (v) ClCH2COCH3,CO(CH3)2, reflux, 1 h,
CH3COONa; (vi) 3-chloro-2,4-pentanedione, CO(CH3)2, CH3COONa, reflux, 2 h; (vii) 2-bromoacetophenone,
CH3COONa, 2-propanol, reflux, 5 h; (viii) 2-bromo-2'-acetonaphthone, CH3COONa, 2-propanol, reflux, 4 h;
(ix) 3-(bromoacetyl)coumarin, CH3COONa, 2-propanol, reflux, 7 h; (x) 2,3-dichloro-1,4-naphthoquinone,
CH3COOH, CH3COONa, 80 °C, 24 h.
1
The structure of the synthesized compounds 3a–i was confirmed by elemental analysis, H-NMR,
1
13C-NMR and IR spectral data. For example, in the H-NMR spectrum of compound 3a, the CH2CO
and NCH2 group proton signals appear at 2.57 and 4.26 ppm, whereas aromatic and CH proton signals
are observed in the range of 7.32 to 7.63 ppm. In the 13C-NMR spectrum of compound 3a, the signals
of the CH2CO and NCH2 groups are observed at 34.57 and 51.44 ppm, respectively, whereas aromatic
and CH= group carbon signals are observed in the range 111.96–140.42 ppm. The combination of CS,
CN, CO, and COOH carbon signals are observed at 133.34, 171.94, 175.79, and 179.19 ppm
respectively. In the IR spectrum, the C=N absorption band is observed at 1,540 cm−1, two C=O bands
at 1,686 and 1,725 cm−1, and a band belonging to the OH group at 3,435 cm−1 are observed. The
formation of isomeric structures in these reactions was not observed. According to data published in
the literature, glycine used as a bifunctional catalyst gives a single isomer (Z) [35].

Molecules 2013, 18
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3-[Phenyl(1,3-thiazol-2-yl)amino]propanoic acid (5) was synthesized from compound 1 and
chloroacetaldehyde in refluxing water solution after 2 hours. A water-soluble amino acid
hydrochloride is formed, which is present in the base transferred by the addition of sodium acetate to
the reaction mixture. The structure of compound 5 is confirmed by its NMR spectral data: in the
1H-NMR spectrum two triplets at 2.62 and 4.11 ppm are assigned to the CH2CO and NCH2 group
protons, while two doublets at 6.71 and 7.18 ppm belong to the thiazole ring and the SCH NCH group
protons. Aromatic ring proton signals are observed at 7.31–7.50 ppm, and broad proton singlets of the
carboxyl group are detectable at 12.30 ppm.
Reactions of 1 with various haloketones were also performed, and a number of new thiazole
derivatives 6–11 with aliphatic, aromatic, or heterocyclic substituents were thus synthesized. The
compounds 6–10 were synthesized in acetone or 2-propanol at boiling temperature. Cyclic diketones—
quinones—are interesting both for their chemical properties and practical uses. Besides, many
compounds containing the quinone group are found in Nature. Various assays showed that these
compounds are characterized by a broad spectrum of biological activities [36]. A quinone fragment-
containing compound 11 in this work was synthesized from N-phenyl-N-thiocarbamoyl--alanine (1)
and 2,3-dichloro-1,4-naphthoquinone. The reaction was carried out in acetic acid at 80 °C for 24 h in
the presence of sodium acetate. Then the mixture was cooled and diluted with water. The synthesized
3-(N-(4,9-dihydro-4,9-dioxonaphtho[2,3-d]thiazol-2-yl)-N-phenylamino)propanoic acid (11) was
purified by dissolving it in aqueous sodium hydroxide solution, filtering, and then acidifying the
filtrate with acetic acid to pH 6.
The compound 8 was modified by introducing a hydrazone fragment into its molecule with the
porpose of increasing its antimicrobial properties (Scheme 2). Firstly, acid 8 was esterified with
methanol to obtain the corresponding ester 12, which afterwards was boiled in 2-propanol with
hydrazine monohydrate. The resulting acid hydrazide 13 was condensed with aromatic and
heterocyclic aldehydes. The NMR spectra of the synthesized hydrazones 14 showed that in DMSO-d6
solution they exist as a mixture of E/Z isomers due the presence of a CO-NH fragment in the molecule
and its hindered rotation around the CO-NH bond; however, the Z isomer was dominant.
Scheme 2. Synthesis of 3-[phenyl(4-phenyl-1,3-thiazol-2-yl)amino]propanoic acid
derivatives 12–14.
R
O
N
N
O
N
O
N
NH2
iii
O
N
CH3
ii
N
OH
i
N
O
H
H
N
S
N
S
N
S
N
S
12
13
14 b,c,i,j
8
j
c
b
i
NO2
R
NO2
Br
S
S
NO2
Reagents and Conditions: (i) CH3OH, H2SO4, reflux, 6 h; (ii) NH2NH2, 2-propanol, reflux, 7 h;
(iii) RCHO, 2-propanol, reflux, 5 h.

Molecules 2013, 18
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2.2. Antimicrobial Evaluation of Synthesized Compounds
The antibacterial activity of the compounds was determined against Gram-positive spore-forming
rods of Bacillus cereus (ATCC 11778), Gram-positive cocci of Staphylococcus (ATCC 9144),
Gram-negative rods of E. coli (ATCC 8739) and Pseudomonas aeruginosa (NCTC 6750) by the broth
and spread-plate methods. A range of concentrations for each compound was prepared according to the
experimental procedure mentioned in experimental section. The minimal inhibition concentration
(MIC, µg/mL) values are listed in Table 1.
Table 1. Antibacterial activity in vitro of the synthesized compounds (MIC, µg/mL).
Compound
Staphylococcus aureus Bacillus cereus Esherichia coli Pseudomonas aeruginosa
2
ni
250
250
250
250
250
250
250
ni
ni
ni
250
250
250
250
250
250
250
ni
ni
250
250
250
250
250
250
250
ni
3a
250
250
250
250
250
250
250
ni
3b
3c
3d
3e
3f
3g
3h
3i
250
ni
250
ni
250
ni
250
ni
5
6
250
250
250
1000
500
250
62.5
250
250
250
500
500
250
62.5
250
250
250
1000
500
250
62.5
250
250
250
ni
7
8
9
10
11
500
250
62.5
Oxytetracycline
ni—no inhibition. The minimum inhibitory concentrations were determined by the micro broth dilution method.
The MIC values of the samples were obtained in spread-plate tests, considering compound
concentrations demonstrating antibacterial activity. Antibacterial screening data revealed a prominent
inhibitory activity of compounds 3a–g, and 3i against the tested strains of S. aureus, B. cereus, E. coli
and P. aeruginosa (MIC = 250 µg/mL). The MIC value of 350 µg/mL was obtained against all bacteria
for compounds 3a, 3b, 3c, 3d, 3f, 3e, 3i, 6, 7, 8, and 11, while for compounds 10 and 9, depending on
bacterial species they were 500 or 1000 µg/mL. Compounds 2, 5 didn’t show antibacterial activity
against the test microorganisms. Based on these data, antibacterial activity of the investigated
compounds may be arranged in the following order:
Staphylococcus aureus:
3a ≈ 3b ≈ 3c ≈ 3d ≈ 3e ≈ 3f ≈ 3g ≈ 3i ≈ 6 ≈ 7 ≈ 8 ≈ 11 > 10 > 9
Increasing antibacterial activity

Molecules 2013, 18
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Bacillus cereus:
3a ≈ 3b ≈ 3c ≈ 3d ≈ 3e ≈ 3f ≈ 3g ≈ 3i ≈ 6 ≈ 7 ≈ 8 ≈ 11 > 9 ≈ 10
Increasing antibacterial activity
Esherichia coli:
3a ≈ 3b ≈ 3c ≈ 3d ≈ 3e ≈ 3f ≈ 3g ≈ 3i ≈ 6 ≈ 7 ≈ 8 ≈ 11 > 10 > 9
Increasing antibacterial activity
Pseudomonas aeruginosa:
3a ≈ 3b ≈ 3c ≈ 3d ≈ 3e ≈ 3f ≈ 3g ≈ 3i ≈ 6 ≈ 7 ≈ 8 ≈ 11 > 10
Increasing antibacterial activity
The minimum bactericidal values (MBC) are presented in Table 2. These data also revealed
pronounced inhibitory activity of some synthesized compounds against some bacteria species,
particulalry S. aureus and B. cereus at 250 µg/mL. For instance, the lowest MBC values
(250–500 µg/mL) were found for the compounds 3i, 3g, 11. However, the compounds 2, 3h, 5 didn’t
show any bactericidal activity against the tested microorganisms.
Table 2. Bactericidal activity in vitro of the synthesized compounds (MBC, µg/mL)
Compound
Staphylococcus aureus
Bacillus cereus Esherichia coli Pseudomonas aeruginosa
2
ni
500
500
500
500
350
500
350
ni
ni
1000
ni
ni
ni
3a
500
500
500
500
500
500
350
ni
500
1000
500
500
500
500
350
ni
3b
3c
1000
1000
1000
ni
3d
3e
3f
3g
350
ni
3h
3i
250
ni
500
500
ni
500
1000
ni
500
1000
ni
4
5
ni
6
500
500
250
1000
1000
250
ni
ni
500
500
500
1000
1000
250
1000
1000
1000
1000
250
500
500
500
ni
7
ni
8
ni
9
1000
1000
250
1000
1000
1000
1000
62.5
10
1000
250
1000
1000
1000
1000
250
11
14 b
14 c
ni
14 i
ni
14 j
ni
Oxytetracycline
62.5
ni—no inhibition; Minimum bactericidal concentrations were determined by using the spread plate method.

Molecules 2013, 18
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Based on these experiments, bactericidal activity of the investigated compounds may be arranged in
the following order:
Decreasing MBC Values against Staphylococcus aureus:
3i > 11 ≈ 8 > 3e ≈ 3g > 3a ≈ 3b ≈ 3c ≈ 3d ≈ 3f ≈ 6 ≈ 7 > 9 > 10
Increasing bactericidal activity
Decreasing MBC Values against Bacillus cereus:
11 > 3g > 3i ≈ 4 ≈ 3g > 3a ≈ 3c ≈ 3d ≈ 3e ≈ 9 ≈ 10 ≈ 14b ≈ 14c ≈ 14i ≈ 14j
Increasing bactericidal activity
Decreasing MBC Values against Esherichia coli:
11 > 3g > 3a ≈ 3b ≈ 3c ≈ 3d ≈ 3e ≈ 3f ≈ 3i ≈ 6 ≈ 7 ≈ 8 > 9 ≈ 10 ≈ 14b ≈ 14c ≈ 14i ≈ 14j
Increasing bactericidal activity
Decreasing MBC Values against Pseudomonas aeruginosa:
11 > 3g > 3a ≈ 3c ≈ 3d ≈ 3e ≈ 3f ≈ 3i ≈ 6 ≈ 7 ≈ 8 > 3b ≈ 10 ≈ 14b ≈ 14c ≈ 14i ≈ 14j
Increasing bactericidal activity
Among the all synthesized compounds, the highest antibacterial activity was exhibited by thiazole
compound 11 containing a naphthoquinone ring. Among compounds 3a–i, the highest antibacterial
activity was shown by the compounds with furan and bromothiophene substituents in their structure.
Aromatic substituents with different groups (NO2, F, Cl) didn’t show any inhibiting effect. Among
thiazole compounds 5–9, the highest antibacterial activity was exhibited by compound 8 with a phenyl
substituent in the 4-position of the thiazole ring.
Attempting to increase antimicrobial activity the structure of compound 8 was modified by
replacing the carboxy group by hydrazone and hydrazide derivatives with aromatic and heterocyclic
substituents, however, the results obtained suggest that modification of the carboxy group decreases
the antimicrobial activity.
It may thus be concluded that the antimicrobial activity of the tested compounds depends on their
structural peculiarities. 3-(N-(4,9-dihydro-4,9-dioxonaphtho[2,3-d]thiazol-2-yl)-N-phenylamino)propanoic
acid (11), 3-(N-(5-((furan-2-yl)methylene)-4,5-dihydro-4-oxothiazol-2-yl)-N-phenylamino)propanoic acid
(3g), and 3-(N-(5-((5-bromothiophen-2-yl)methylene)-4,5-dihydro-4-oxothiazol-2-yl)-N-phenyl-
amino)propanoic acid (3i) were found to exhibit a promising antibacterial activity.
2.3. Evaluation of Synthesized Compounds on Rapeseed Yield and Biochemical Content
The effect of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) on the
rapeseed yield and biochemical content is presented in Table 3. When rapeseed seedlings were sprayed
with solutions of 2 of various concentrations, the yield of seeds ranged from 2.07 to 2.44 t/ha, i.e.,
19%–40% higher in comparison with the control sample. The highest seed yield was obtained when
rapeseed seedlings were sprayed with 150 mg/L of the test compound solution. Other quality
characteristics were also affected by spraying rapeseed seedlings with the test compound; for instance,
the highest mass of 1,000 seeds (4.17 g) was determined when 25 mg/L were applied, whereas at the
same concentration the oil content increased by 13.3%–39.3% in comparison with the control sample.

Molecules 2013, 18
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Table 3. The effect of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic
acid (2) on rapeseed yield and biochemical content.
The concentration of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2), mg/L
0
25
50
75
100
125
150
Yield, t/ha
Seed mass (per
1,000 seeds), g
Oil content, kg/t
Protein content,
mg/100g
1.74 ± 0.11
2.07 ± 0.23 2.21 ± 0.08 2.11 ± 0.28 2.20 ± 0.08 2.41 ± 0.11 2.44 ± 0.20
4.17 ± 0.10 4.02 ± 0.12 3.81 ± 0.16 3.96 ± 0.09 3.85 ± 0.14 3.94 ± 0.21
283.4 ± 8.3 271.8 ± 7.5 334.3 ± 5.7 301.2 ± 4.9 281.8 ± 7.9 296.7 ± 6.5
3.94 ± 0.16
239.9 ± 1.7
15.9 ± 0.1
4.53 ± 0.02
27.1 ± 0.3
30.7 ± 0.3
31.2 ± 0.3
30.3 ± 0.3
22.8 ± 0.4
22.5 ± 0.2
Ash, %
4.53 ± 0.05 4.48 ± 0.04 4.23 ± 0.10 4.16 ± 0.10 4.07 ± 0.14 4.10 ± 0.13
Application of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) increased
both the content of oil and protein. The highest content of oil (334 kg/t) and protein (31.2 mg/100g)
was obtained upon using the 75 mg/L concentration of the study compound solution. The content of
protein varied from 15.9 to 31.2 mg/100 g. When rapeseed seedlings were sprayed with 75 mg/L of the
test compound solution, protein content was two times higher in comparison with the control sample.
The effect of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) on the
composition of fatty acids in rapeseed is presented in Table 4. The results showed that the variation of
oleic acid content was insignificant (−0.09 to +0.77%) in comparison with the control sample. When
rapeseed had been sprayed with 50 mg/L of the aforementioned solution, the highest content of oleic
acid (61.51%) was obtained in the seeds. The content of saturated fatty acids (palmitic and stearic)
varied insignificantly in comparison with the control sample. When rapeseed had been sprayed with
the test compound solutions at various concentrations, some increase in the content of linolenic acid
was observed; however, it was insignificant. Upon spraying rapeseed seedlings with 75 mg/L of the
study compound solution, the highest content of linoleic acid (20.96%) was obtained in the seeds.
Table 4. The effect of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic
acid (2) on the content of fatty acids in rapeseed.
The concentration of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2), mg/L
25 50 75 100 125 150
Fatty acid,
%
0
Palmitic
Stearic
5.11 ± 0.05 4.57 ± 0.06 4.79 ± 0.22 4.59 ± 0.07 5.23 ± 0.31 4.90 ± 0.09 4.76 ± 0.03
2.11 ± 0.08 2.02 ± 0.02 2.07 ± 0.01 2.02 ± 0.01 2.05 ± 0.01 2.04 ± 0.01 2.03 ± 0.01
61.04 ± 0.26 61.07 ± 0.09 61.51 ± 0.03 61.28 ± 0.02 60.98 ± 0.08 61.21 ± 0.18 61.41 ± 0.00
20.55 ± 0.13 20.83 ± 0.09 20.61 ± 0.01 20.96 ± 0.06 20.93 ± 0.02 20.84 ± 0.05 20.85 ± 0.5
1.02 ± 0.08 1.09 ± 0.03 1.07 ± 0.04 1.05 ± 0.01 0.93 ± 0.07 1.02 ± 0.02 0.99 ± 0.01
8.49 ± 0.03 8.75 ± 0.02 8.54 ± 0.02 8.66 ± 0.02 8.67 ± 0.01 8.64 ± 0.01 8.60 ± 0.02
Oleic
Linoleic
Eicosenoic
Linolenic
Summarizing the data of the field experiments spraying rapeseed seedlings with different
concentrations of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) solutions
resulted in the following:
(a) 100 mg/L: the highest number of pods and siliques was obtained;
(b) 150 mg/L: the highest seed yield was obtained;
(c) 25 mg/L: the highest seed mass was obtained.

Molecules 2013, 18
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During osmotic stress plants accumulate in the cytoplasm of their cells organic osmolytes such as
proline, valine, isoleucine, ectoine, aspartic acid, betaine, glucose, fructose, sucrose, fructans,
mannitol, pinitol, and myo-inositol (inositol) [37]. The regulating effect of compound 2 on rapeseed
could be explained by the properties of typical structure osmolytes: they could exist in the zwitterionic
form (Scheme 3).
Scheme 3. The possible forms of compound 2.
O
O
+
-
N
OH
NH
O
S
N
S
N
O
O
3. Experimental
3.1. General
The starting materials and solvents were obtained from Sigma-Aldrich Chemie GmbH (Munich,
Germany) and Fluka (Buchs, Switzerland) and were used without further purification. The methods
used to follow the reactions were TLC and NMR. The NMR spectra were recorded on a Varian Unity
Inova (300 MHz) spectrometer (Varian, Inc.,Palo Alto, CA, USA) and Bruker Avance III/400 (400 MHz).
Chemical shifts are expressed as , ppm relative to TMS. The J constants are given in Hz. The IR
spectra (, cm−1) were recorded on a Perkin–Elmer BX FT–IR spectrometer (Perkin–Elmer
Inc.,Waltham, MA, USA) using KBr tablets. Mass spectra were obtained on a Waters ZQ 2000
spectrometer (Waters, Eschborn, Germany) using the electrospray ionization (ESI) mode and operating
at 25 V. Elemental analyses were performed with a CE-440 elemental analyzer (Exeter Analytical Inc.,
Chelmsford, MA, USA). Melting points were determined with a B-540 Melting Point Analyzer (Büchi
Corporation, New Castle, DE, USA) and are uncorrected. TLC was performed using Merck, Silica gel
60 F254 (Kieselgel 60 F254) plates.
3-[(4-Oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic Acid (2). A mixture of thioureido
acid 1 (2.24 g, 0.01 mol), chloroacetic acid (1.04 g, 0.011 mol), sodium carbonate (1.16 g, 0.011 mol)
and water (30 mL) was refluxed for 5 h. Then the mixture was acidified with acetic acid (6 mL) to pH
6. The precipitate was filtered off, washed with water, and crystallized. Yield 1.78 g, 68%; light yellow
crystals m.p. 195–196 °C (from 2-propanol) (198–199 °C) [38]; IR (KBr):  (cm−1) 3426 (OH), 1723,
1
1659 (CO); H-NMR (DMSO-d6):  2.58 (t, J = 7.4 Hz, 2H, CH2CO), 3.92 (s, 2H, SCH2), 4.17 (t,
13
J = 7.3 Hz, 2H, NCH2), 7.41–7.57 (m, 5H, HAr), 12.42 (s, 1H, OH); C-NMR (DMSO-d6):  31.9
(CH2CO), 40.5 (SCH2), 49.9 (NCH2), 128.0, 129.6, 129.8, 140.1 (CAr), 171.9, 183.1 (CO), 186.9
(C=N); Anal. Calcd for C12H12N2O3S: C, 54.53%; H, 4.58%; N, 10.60%; Found: C, 54.69%; H,
4.56%; N, 10.68%.

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3.2. General Procedure for Preparation of 3-{[(5Z)-5-Substituted 4-oxo-4,5-dihydro-1,3-thiazol-2-
yl](phenyl)amino}propanoic Acids 3a–i
A mixture of compound 2 (0.52 g, 2 mmol), the corresponding aldehyde (2.2 mmol), glycine (0.15 g,
2 mmol), and sodium carbonate (0.21 g, 2 mmol), water (6 mL) was refluxed for 5 h. Then the reaction
mixture was diluted with water (50 mL) and acidified with acetic acid to pH 6. The precipitate was
filtered and washed with water. Purification was performed by dissolving the crystals in 10% aqueous
NaOH (100 mL), filtering, and acidifying the filtrate with acetic acid to pH 6 (the procedure was
repeated 2 times).
3-{[(5Z)-5-Benzylidene-4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic
Acid
(3a).
Yellow crystals, (0.52 g, 76%), m.p. 175–176 °C; IR (KBr):  (cm−1) 3435 (OH), 1725, 1686 (CO),
1540 (CN); 1H-NMR (DMSO-d6):  2.57 (t, J = 7.4 Hz, 2H, CH2CO), 4.26 (t, J = 7.5 Hz, 2H, NCH2),
7.32–7.63 (m, 11H, HAr+CH); 13C-NMR (DMSO-d6):  32.1 (CH2CO), 50.1 (NCH2), 127.9, 128.9,
129.1, 129.5, 129.7, 130.1, 133.3, 139.6 (CAr, Cthiazole, CH), 172.0, 175.8 (CO), 179.2 (C=N); Anal.
Calcd for C19H16N2O3S: C, 64.76%; H, 4.58%; N, 7.95%; Found: C, 64.44%; H, 4.63%; N, 8.04%.
3-{[(5Z)-5-(4-Nitrobenzylidene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic Acid (3b).
Yellow crystals, (0.57 g, 73%), m.p. 208–209 °C; IR (KBr):  (cm−1) 3058 (OH), 1729, 1699 (CO),
1539 (CN); 1H-NMR (DMSO-d6):  2.34 (t, J = 7.8 Hz, 2H, CH2CO), 4.20 (t, J = 7.9 Hz, 2H, NCH2),
13
7.32–7.64 (m, 10 H, HAr+CH); C-NMR (DMSO-d6):  34.5 (CH2CO), 52.0 (NCH2), 115.7, 124.2,
127.8, 128.1, 130.3, 133.3, 139.6, 140.4, 147.0 (CAr, Cthiazole, CH), 172.2, 175.9 (CO), 179.0 (C=N); Anal.
Calcd for C19H15N3O5S: C, 57.42%; H, 3.80%; N, 10.57%; Found: C, 56.94%; H, 3.75%; N, 10.86%.
3-{[(5Z)-5-(3-Nitrobenzylidene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic Acid (3c).
Yellow crystals, (0.50 g, 64%), m.p. 150–151 °C; IR (KBr):  (cm−1) 3074 (OH), 1731, 1709 (CO),
1531 (CN); 1H-NMR (DMSO-d6):  2.59 (t, J = 7.5 Hz, 2H, CH2CO), 4.28 (t, J = 7.5 Hz, 2H, NCH2),
7.55–8.43 (m, 10H, HAr+CH); 13C-NMR (DMSO-d6):  32.7 (CH2CO), 50.9 (NCH2), 124.4, 128.5,
128.6, 130.6, 131.3, 132.4, 135.4, 135.9, 140.2, 148.6 (CAr, Cthiazole, CH), 172.6, 176.2 (CO), 179.6
(C=N); Anal. Calcd for C19H15N3O5S: C, 57.42%; H, 3.80%; N, 10.57%; Found: C, 57.39%; H,
3.77%; N, 10.51%.
3-{[(5Z)-5-(2-Chloro-4-nitrobenzylidene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic
Acid (3d). Yellow crystals, (0.53 g, 62%), m.p. 155–156 °C; IR (KBr):  (cm−1) 3071 (OH), 1735,
1698 (CO), 1529 (CN); 1H-NMR (DMSO-d6):  2.63 (t, J = 7.4 Hz, 2H, CH2CO), 4.30 (t, J = 7.4 Hz,
2H, NCH2), 7.53–8.22 (m, 9H, HAr+CH); 13C-NMR (DMSO-d6):  31.9 (CH2CO), 50.4 (NCH2),
122.9, 123.6, 125.4, 128.2, 130.2, 130.3, 131.6, 133.4, 135.3, 139.6, 140.3, 146.6 (CAr, Cthiazole, CH),
172.0, 175.9 (CO), 178.6 (C=N); Anal. Calcd for C19H14ClN3O5S: C, 52.84%; H, 3.27%; N, 9.73%;
Found: C, 52.78%; H, 3.33%; N, 9.78%.
3-{[(5Z)-5-(4-Fluorobenzylidene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic Acid (3e).
Yellow crystals, (0.62 g, 85%), m.p. 167–168 °C; IR (KBr):  (cm−1) 3050 (OH), 1736, 1701 (CO),
1508 (CN); 1H-NMR (DMSO-d6):  2.61 (t, J = 7.2 Hz, 2H, CH2CO), 4.10 (t, J = 7.5 Hz, 2H, NCH2),

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6.71–7.52 (m, 10H, HAr+CH), 12.29 (OH); 13C-NMR (DMSO-d6):  32.7 (CH2CO), 50.5 (NCH2),
116.1, 116.4, 128.1, 128.8, 129.1, 129.9, 130.0, 131.7, 139.9, 160.7 (CAr, Cthiazole, CH) 172.47, 175.8
(CO), 179.4 (C=N); Anal. Calcd for C19H15FN2O3S: C, 61.61%; H, 4.08%; N, 7.56%; Found: C,
61.77%; H, 3.91%; N, 7.48%.
3-{[(5Z)-4-oxo-5-(1H-Pyrrol-2-ylmethylene)-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic
Acid (3f). Yellow crystals, (0.46 g, 69%), m.p. 248–249 °C; IR (KBr):  (cm−1) 3064 (OH), 1715, 1673
1
(CO), 1530 (CN); H-NMR (DMSO-d6):  2.28 (t, J = 8.3 Hz, 2H, CH2CO), 4.15 (t, J = 8.2 Hz, 2H,
13
NCH2), 6.03–7.66 (m, 9H, HAr, CH, CHpyrrole), 11.79 (NH); C-NMR (DMSO-d6):  35.4 (CH2CO),
52.0 (NCH2), 111.2, 111.9, 120.5, 122.1, 123.3, 127.6, 128.3, 129.6, 130.0, 140.6 (CAr, Cthiazole, CH,
Cpyrrole) 174.3, 174.7 (CO), 180.1 (C=N); Anal. Calcd for C17H15N3O3S: C, 59.81%; H, 4.43%; N,
12.31%; Found: C, 59.75%; H, 4.39%; N, 12.22%.
3-{[(5Z)-5-(2-Furylmethylene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic Acid (3g).
Brown crystals, (0.41 g, 61%), m.p. 139–140 °C; IR (KBr):  (cm−1) 3050 (OH), 1727, 1682 (CO),
1532 (CN); 1H-NMR (DMSO-d6):  2.61 (t, J = 7.4 Hz, 2H, CH2CO), 4.25 (t, J = 7.4 Hz, 2H, NCH2),
6.61–7.91 (m, 9H, HAr, CH, Hfuran); 13C-NMR (DMSO-d6):  32.0 (CH2CO), 49.8 (NCH2), 113.3,
117.0, 117.2, 126.4, 128.3, 130.0, 130.1, 140.0, 146.6, 149.5 (CAr, Cthiazole, CH, Cfuran), 172.1, 176.8
(CO), 179.4 (C=N); Anal. Calcd for C17H14N2O4S: C, 59.64%; H, 4.12%; N, 8.18%; Found: C, 59.58%
H, 4.21%; N, 8.22%.
3-{[(5Z)-4-Oxo-5-(2-thienylmethylene)-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic Acid (3h).
Yellow crystals (0.48 g, 68%), m.p. 137–138 °C; IR (KBr):  (cm−1) 3057 (OH), 1726, 1678 (CO),
1542 (CN); 1H-NMR (DMSO-d6):  2.62 (t, J = 7.4 Hz, 2H, CH2CO), 4.26 (t, J = 7.4 Hz, 2H, NCH2),
13
7.16–7.89 (m, 9H, HAr+CH, Hthiophene); C-NMR (DMSO-d6):  32.0 (CH2CO), 50.0 (NCH2), 123.5,
127.2, 128.3, 128.9, 130.0, 130.1, 131.5, 133.5, 138.4, 140.0 (CAr, Cthiazole, CH, Cthiophene), 172.1, 175.3
(CO), 179.3 (C=N); Anal. Calcd for C17H14N2O3S2: C, 56.96%; H, 3.94%; N, 7.82%; Found: C,
57.02%; H, 3.88%; N, 7.89%.
3-[{(5Z)-5-[(5-Bromo-2-thienyl)methylene]-4-oxo-4,5-dihydro-1,3-thiazol-2-yl}(phenyl)amino]propanoic
Acid (3i). Yellow crystals (0.47 g, 55%), m.p. 143–144 °C; IR (KBr):  (cm−1) 3007 (OH), 1723, 1682
1
(CO), 1538 (CN); H-NMR (DMSO-d6):  2.57 (t, J = 7.5 Hz, 2H, CH2CO), 4.25 (t, J = 7.5 Hz, 2H,
13
NCH2), 7.28–7.84 (m, 8H, HAr, CH, Hthiophene); C-NMR (DMSO-d6):  32.4 (CH2CO), 50.4 (NCH2),
116.9, 122.41, 124.4, 128.0, 128.2, 130.1, 132.3, 133.8, 139.9, 140.2 (CAr, Cthiazole, CH, Cthiophene),
172.4, 174.8 (CO), 179.0 (C=N); Anal. Calcd for C17H13BrN2O3S2: C, 46.69%; H, 3.00%; N, 6.41%;
Found: C, 46.77%; H, 2.97%; N, 6.50%.
3-({5-(4-{[2-[(3-Hydroxy-3-oxopropyl)aniline]-4-oxo-1,3-thiazol-5-yliden]methyl}phenyl)methyliden]-
4-oxo-4,5-dihydro-1,3-thiazol-2-yl}aniline)propanoic Acid (4). A mixture of compound 2 (0.61 g,
2.3 mmol), terephthalaldehyde (0.15 g, 1.14 mmol), glycine (0.17 g, 2.3 mmol), sodium carbonate
(0.49 g, 4.6 mmol) and water (7 mL) was refluxed for 5 h. Then the reaction mixture was diluted with
water (50 mL) and acidified with acetic acid to pH 6. The precipitate was filtered and washed with
water. Purification was performed by dissolving the crystals in 2% aqueous KOH (100 mL), filtering,

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and acidifying the filtrate with acetic acid to pH 6. Yellow crystals, (0.34 g, 27%), m.p. 269–270 °C;
IR (KBr):  (cm−1) 3023 (OH), 1729, 1710, 1694, 1688 (CO), 1532, 1538 (CN); 1H-NMR (DMSO-d6):
 2.61 (t, J = 7.2 Hz, 4H, CH2CO), 4.27 (t, J = 7.1 Hz, 4 H, NCH2), 7.46–7.65 (m, 16H, CAr, CH);
Anal. Calcd for C32H26N4O6S2: C, 61.33%; H, 4.18%; N, 8.94%; Found: C, 61.30%; H, 4.07%; N, 8.88%.
3-[Phenyl(1,3-thiazol-2-yl)amino]propanoic Acid (5). A mixture of the thioureido acid 2 (1.12 g, 5 mmol),
chloroacetaldehyde 50% aqueous solution (0.79 g, 0.01 mol), water (20 mL) was refluxed for 2 h. Then
sodium acetate (0.82 g, 0.01 mol) was added and the mixture was stirred for 5 min. The precipitate was
filtered and washed with water. Purification was performed by dissolving the crystals in 10% aqueous
sodium carbonate (75 mL), filtering, and acidifying the filtrate with acetic acid to pH 6 (the procedure
was repeated 2 times). Yellow crystals, (0.50 g, 42%), m.p. 144–145 °C; IR (KBr):  (cm−1) 3076
1
(OH), 1717 (CO), 1514 (CN); H-NMR (DMSO-d6): δ 2.62 (t, J = 7.2 Hz, 2H, CH2CO), 4.11 (t,
J = 7.5 Hz, 2H, NCH2), 6.71 (d, J = 3.6 Hz, 1H, NCH), 7.18 (d, J = 3.6 Hz, 1H, SCH), 7.31–7.50 (m,
13
5H, HAr), 12.30 (br s, 1H, OH); C-NMR (DMSO-d6): δ 32.2 (CH2CO), 48.5 (NCH2), 108.1, 126.6,
127.2, 130.0, 139.1, 144.8 (CAr, Cthiazole) 169.5 (CO), 172.5 (C=N); Anal. Calcd for C12H12N2O2S: C,
58.05%; H, 4.87%; N, 11.28%; Found: C, 58.11%; H, 4.93%; N, 11.31%.
3-[(4-Methyl-1,3-thiazol-2-yl)(phenyl)amino]propanoic Acid (6). A mixture of thioureido acid 1 (0.56 g,
2.5 mmol), chloroacetone (0.29 g, 3 mmol) in acetone (10 mL) was refluxed for 1 h and diluted with
water (50 mL). Then sodium acetate (0.98 g, 12 mmol) was added, and the mixture was stirred for 5 min.
The precipitate was filtered, and washed with water. Yellow crystals (0.52 g, 79%), m.p. 149–150 °C
1
(from acetone-water mixture); IR (KBr):  (cm−1) 3128 (OH), 1712 (CO), 1515 (CN); H-NMR
(DMSO-d6):  2.16 (s, 3H, CH3), 2.60 (t, J = 7.3 Hz, 2H, CH2CO), 4.09 (t, J = 7.4 Hz, 2H, NCH2),
6.26 (s, 1H, SCH), 7.30–7.49 (m, 5H, HAr), 12.29 (br s, 1H, OH); 13C-NMR (DMSO-d6):  17.4 (CH3),
32.3 (CH2CO), 48.3 (NCH2), 102.1, 126.8, 127.2, 129.9, 144.5, 148.3 (CAr, Cthiazole); 168.67 (CO),
172.6 (C=N); Anal. Calcd for C13H14N2O2S: C, 59.52%; H, 5.38%; N, 10.68%; Found: C, 59.47%; H,
5.45%; N, 10.55%.
3-[(5-Acetyl-4-methyl-1,3-thiazol-2-yl)(phenyl)amino]propanoic Acid (7). A mixture of thioureido acid
1 (1.0 g, 4.5 mmol), 3-chloro-2,4-pentanedione (0.76 g, 5.6 mmol), acetone (10 mL) was refluxed for 2 h
and diluted with water (50 mL). Then sodium acetate ((1.8 g, 22 mmol) was added, and the mixture
was stirred for 5 min. The precipitate was filtered and washed with water. Purification was performed
by dissolving the crystals in 10% aqueous sodium hydroxide (50 mL), filtering, and acidifying the
filtrate with acetic acid to pH 6. Light yellow crystals, (0.84 g, 92%), m.p. 175–176 °C; IR (KBr): 
1
(cm−1) 1721, 1700 (CO), 1576 (C=N); H-NMR (DMSO-d6):  2.31 (s, 3H, CH3), 2.48 (s, 3H, CH3),
2.51 (t, J = 7.2 Hz, 2H, CH2CO), 4.15 (t, J = 7.3 Hz, 2H, NCH2), 7.44–7.53 (m, 6H, HAr, Hthiazole), 12.36
(s, 1H, OH); Anal. Calcd for C15H16N2O3S: C, 59.19%; H, 5.30%; N, 9.20%; Found: C, 59.26%; H,
5.15%; N, 9.11%.
3-[Phenyl(4-phenyl-1,3-thiazol-2-yl)amino]propanoic Acid (8). A mixture of thioureido acid 2 (1.12 g,
5 mmol), 2-bromoacetophenone (0.99 g, 5 mmol), sodium acetate (0.82 g, 10 mmol), 2-propanol (10 mL)
was refluxed for 5 h. The mixture was diluted with water (30 mL). The precipitate was filtered and
washed with water. Purification was performed by dissolving the crystals in 10% aqueous KOH (30 mL),

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filtering, and acidifying the filtrate with acetic acid to pH 6. Yellow crystals, (1.56 g, 96%), m.p. 101–
1
102 °C; IR (KBr):  (cm−1) 3416 (OH), 1712 (CO), 1508 (CN); H-NMR (DMSO-d6):  2.66 (t, J =
7.3 Hz, 2H, CH2CO), 4.19 (t, J = 7.5 Hz, 2H, NCH2), 7.13 (s, 1H, SCH), 7.26–7.88 (m, 10H, HAr);
13C-NMR (DMSO-d6):  32.8 (CH2CO), 48.9 (NCH2), 102.9, 125.6, 126.7, 127.3, 127.5, 128.5, 129.9,
134.6, 150.3, 144.6 (CAr, Cthiazole), 168.7 (CO), 173.0 (C=N); Anal. Calcd for C18H16N2O2S: C, 66.64%;
H, 4.97%; N, 8.64%; Found: C, 66.60%; H, 4.83%; N, 8.57%.
3-{[4-(2-Naphthyl)-1,3-thiazol-2-yl](phenyl)amino}propanoic Acid (9). A mixture of thioureido acid 2
(1.12 g, 5 mmol), 2-bromoacetonaphthone (1.25 g, 5 mmol), 2-propanol (10 mL), sodium acetate (0.82 g,
10 mmol) was refluxed for 4 h. The mixture was diluted with water. The precipitate was filtered and
washed with water. Purification was performed by dissolving the crystals in 10% aqueous K2CO3 (50 mL),
filtering, and acidifying the filtrate with acetic acid to pH 6. Yellow crystals, (1.52 g, 82%), m.p.
1
143–144 °C; IR (KBr):  (cm−1) 3112 (OH), 1741 (CO), 1511 (CN); H-NMR (DMSO-d6):  2.67 (t,
J = 7.4 Hz, 2H, CH2CO), 4.25 (t, J = 7.4 Hz, 2H, NCH2), 7.30 (s, 1H, SCH), 7.48–7.88 (m, 12H, HAr);
Anal. Calcd for C22H18N2O2S: C, 70.57%; H, 4.85%; N, 7.48%; Found: C, 70.41%; H, 4.92%;
N, 7.39%.
3-{[4-(2-Oxo-2H-chromen-3-yl)-1,3-thiazol-2-yl](phenyl)amino}propanoic Acid (10). A mixture of
thioureido acid 2 (1.12 g, 5 mmol), 3-(bromoacetyl)coumarin (1.34 g, 5 mmol), sodium acetate (0.82 g,
10 mmol), 2-propanol (10 mL) was refluxed for 7 h. After cooling, the precipitate was filtered and
washed with water. Purification was performed by dissolving the crystals in 10% aqueous KOH (50 mL),
filtering, and acidifying the filtrate with acetic acid to pH 6. Light brown crystals (1.68 g, 86%), m.p.
1
172–173 °C; IR (KBr):  (cm−1) 3137 (OH), 1717, 1709 (CO), 1536 (CN); H-NMR (DMSO-d6): 
2.65 (t, J = 7.3 Hz, 2H, CH2CO), 4.23 (t, J = 7.3 Hz, 2H, NCH2), 7.34–7.89 (m, 10H, HAr), 8.64 (s, 1H,
SCH); Anal. Calcd for C21H18N2O4S: C, 63.95%; H, 4.60%; N, 7.10%; Found: C, 64.11%; H, 4.66%;
N, 7.08%.
3-(N-(4,9-Dihydro-4,9-dioxonaphtho[2,3-d]thiazol-2-yl)-N-phenylamino)propanoic Acid (11).
A
mixture of thioureido acid 2 (2.24 g, 0.01 mol), 2,3-dichloro-1,4-naphthoquinone (2.72 g, 12 mmol),
sodium acetate (2.95 g, 36 mmol), acetic acid (50 mL) was stirred at 80 °C for 24 h. After cooling
mixture was diluted with water (100 mL). The precipitate formed was filtered and washed with water.
Purification was performed by dissolving the crystals in 10% aqueous KOH (350 mL), filtering, and
acidifying the filtrate with acetic acid to pH 6. Claret crystals (2.38 g, 62%), m.p. 185 °C (decomp.);
1
IR (KBr):  (cm−1) 3063 (OH), 1712, 1634, 1622 (CO), 1527 (CN); H-NMR (DMSO-d6):  2.71 (t,
J = 7.1 Hz, 2H, CH2CO), 4.53 (t, J = 7.1 Hz, 2H, NCH2), 7.43–7.91 (m, 9H, HAr), 12.19 (br s, 1H,
13
OH); C-NMR (DMSO-d6):  32.1 (CH2CO), 49.1 (NCH2), 120.6, 125.8, 127.0, 128.7, 129.3, 130.5,
131.7, 135.0, 142.6, 160.1 169.8 (CAr, Cthiazole), 172.1, 175.3, 179.8 (CO); Anal. Calcd for
C20H14N2O4S: C, 63.48%; H, 3.73%; N, 7.40%; Found: C, 63.36%; H, 3.62%; N, 7.44%.
Methyl 3-[phenyl(4-phenyl-1,3-thiazol-2-yl)amino]propanoate (12). A mixture of 3-(N-phenyl-N-(4-
phenylthiazol-2-yl)amino)propanoic acid (8) (8.08 g, 0.025 mol), methanol (150 mL), and H2SO4 (2 mL)
was refluxed for 6 h. Then the solvent was evaporated. The precipitated product was neutralized with
5% sodium bicarbonate solution and extracted with diethyl ether (3 times with 100 mL); then the ether

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1
was evaporated. Oil, (7.5 g, 89%), Rf = 0.7; IR (KBr):  (cm−1) 1737 (CO), 1532 (CN); H-NMR
(DMSO-d6):  2.89 (t, J = 7.2 Hz, 2H, CH2CO), 3.61 (s, 3H, CH3), 4.35 (t, J = 7.2 Hz, 2H, NCH2),
13
6.67 (s, 1H, SCH), 7.24–7.90 (m, 10H, HAr); C-NMR (DMSO-d6):  32.9 (CH2CO), 49.1 (NCH2),
51.8 (CH3), 101.9, 126.1, 127.1, 127.6, 127.7, 128.6, 130.2, 135.1, 145.1, 151.5 (CAr, Cthiazole), 169.4
(CO), 172.4 (C=N); Anal. Calcd for C19H18N2O2S: C, 67.43%; H, 5.36%; N, 8.28%; Found: C,
67.35%; H, 5.42%; N, 8.19%.
3-[Phenyl(4-phenyl-1,3-thiazol-2-yl)amino]propanehydrazide (13). A mixture of ester 12 (7.2 g, 0.02 mol),
hydrazine monohydrate (2.07 g, 0.04 mol) was refluxed in 2-propanol (20 mL) for 7 h. The reaction
mixture was cooled, the solvent was evaporated, and the oilseed substance was purified by
chromatography on a silica gel 60 column (methanol–chloroform, 1:20), Rf 0.6. Oil, (5.1 g, 71%), IR
1
(KBr):  (cm−1) 3283, 3060 (NH, NH2), 1661 (CO), 1532 (CN); H-NMR (DMSO-d6):  2.71 (t,
J = 6.7 Hz, 2H, CH2CO), 3.53–4.06 (br s, 2H, NH2), 4.34 (t, J = 6.7 Hz, 2H, NCH2), 6.67 (s, 1H,
SCH), 7.26–7.88 (m, 10H, HAr), 8.06 (s, 1H, NH); 13C-NMR (DMSO-d6):  33.6 (CH2CO), 49.4
(NCH2), 102.3, 126.1, 126.9, 127.8, 127.9, 128.8, 130.3, 134.8, 144.7, 151.3 (CAr, Cthiazole), 170.1
(CO), 171.7 (C=N); Anal. Calcd for C18H18N4OS: C, 63.88%; H, 5.36%; N, 16.56%; Found: C,
63.72%; H, 5.45%; N, 16.59%.
3.3. General Synthesis of Hydrazones 14 b, c, i, j
A mixture of hydrazide 13 (1.01 g, 3 mmol), the corresponding aldehyde (4 mmol) and 2-propanol
(6 mL) was refluxed for 5 h. After cooling, the precipitated compound was filtered, washed with
2-propanol, and crystallized.
3-[(4-Phenyl-1,3-thiazol-2-yl)(phenyl)amino]-N'-(4-nitrophenyl)methylene]propanehydrazide (14b).
Yellow crystals, (1.19 g, 84%), m.p. 218–219 °C (from 1,4-dioxane); IR (KBr):  (cm−1) 3082 (NH),
1
1676 (CO), 1541, 1514 (CN); H-NMR (DMSO-d6):  (E/Z, 40/60): 2.76, 3.18 (2 t, J = 7.1 Hz, 2H,
CH2CO), 4.27–4.38 (m, 2H, NCH2), 7.13–8.30 (m, 15H, HAr, Htiazole), 8.25, 8.30 (2 s, 1H, N=CH),
11.70, 11.78 (1 H, 2 s, NHN); Anal. Calcd for C25H21N5O3S: C, 63.68%; H, 4.49%; N, 14.85%;
Found: C, 63.55%; H, 14.77%; N, 8.50%.
3-[(4-Phenyl-1,3-thiazol-2-yl)(phenyl)amino]-N'-(3-nitrophenyl)methylene]propanehydrazide (14c).
Red-yellow crystals, (1.15 g, 81%), m.p. 147–148 °C (from 1,4-dioxane); IR (KBr):  (cm−1) 3109
(NH), 1681 (CO), 1532, 1515 (CN); 1H-NMR (DMSO-d6):  (E/Z, 40/60): 2.75, 3.15 (2 t, J = 7.1 Hz,
2H, CH2CO), 4.27–4.39 (m, 2H, NCH2), 7.10–8.36 (m, 15H, HAr, Hthiazole), 8.47, 8.50 (2 s, 1H,
N=CH), 11.62, 11.73 (2 s, 1H, NHN); Anal. Calcd for C25H21N5O3S: C, 63.68%; H, 4.49%; N,
14.85%; Found: C, 66.79%; H, 4.61%; N, 14.80%.
N'-[(5-Bromo-2-thienyl)methylene]-3-[(4-phenyl-1,3-thiazol-2-yl)(phenyl)amino]propanehydrazide
(14i). Grey crystals, (1.1 g, 72%), m.p. 144–145 °C (from 1,4-dioxane); IR (KBr):  (cm−1) 3057
(NH), 1674 (CO), 1533, 1514 (CN); 1H-NMR (DMSO-d6):  (E/Z, 40/60): 2.69, 3.04 (2 t, J = 7.1 Hz,
2H, CH2CO), 4.23–4.34 (m, 2H, NCH2), 7.09–7.93 (m, 13H, HAr, Htiazole, Hthiophene), 8.04, 8.28 (2 s,

Molecules 2013, 18
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1H, N=CH), 11.43, 11.49 (2 s, 1H, NNH); Anal. Calcd for C23H19BrN4OS2: C, 54.01%; H, 3.74%; N,
10.95%; Found: C, 54.15%; H, 3.82%; N, 11.03%.
N'-[(5-Nitro-2-thienyl)methylene]-3-[(4-phenyl-1,3-thiazol-2-yl)(phenyl)amino]propanehydrazide
(14j). Orange crystals, (0.98 g, 70%), m.p. 208–209 °C (from 1,4-dioxane); IR (KBr):  (cm−1) 3065
(NH), 1672 (CO), 1541, 1518 (CN); 1H-NMR (DMSO-d6):  (E/Z, 40/60): 2.74, 3.08 (2 t, J = 7.0 Hz,
2H, CH2CO), 4.25–4.37 (m, 2H, NCH2), 7.11–8.11 (m, 13H, HAr, Hthiazole, Hthiophene), 8.12, 8.38 (2 s,
1H, N=CH), 11.79, 11.83 (2 s, 1H, NNH); Anal. Calcd for C23H19N5O3S2: C, 57.85%; H, 4.01%; N,
14.66%; Found: C, 57.77%; H, 4.14%; N, 14.71%.
3.4. Antimicrobial Activity
The following bacteria strains were used: gram-negative rods of Escherichia coli (ATCC 8739),
Pseudomonas aeruginosa (NCTC 6750), gram-positive cocci of Staphylococcus aureus (ATCC 9144)
and gram-positive spore forming rods of Bacillus cereus (ATCC 11778). Tryptic soy agar (TSA) and
tryptic soy broth (TSB) were used for bacteria cultivation and antibacterial activity tests.
Antibacterial activity of the compounds was determined by testing their different concentrations
against B. cereus, S. aureus, P. aeruginosa and E. coli bacteria by the broth-dilution and spread plate
methods [39,40]. A range of concentrations, 1000, 500, 350, 250, and 125 µg/mL were prepared for
each sample. They were streaked out on TSA plates and incubated at 37 °C for 24 h. A representative
colony was placed in 5 mL of TBS and incubated at 37 °C for 24 h.
B. cereus, S. aureus, E. coli, and P. aeruginosa cultures containing 108 CFU/mL (colony-forming
units corresponding to McFarland’s 0.5) were diluted with TSB and used for the antibacterial test. The
test organisms (100 µL) were added to each tube and incubated at 37 °C for 24 h. At the end of this
period, a small amount of the diluted mixture (different materials) from each tube was pulled out and
spread on TSA. The plates were incubated at 37 °C for 48 h. The growth of bacterial cells was
observed on agar plates. The lowest concentration of the bacterial material at which no growth was
observed was considered as the minimum bactericidal concentration (MBC) value [41]. The
antibacterial compound oxytetracycline inoculated with each test bacterium in the tubes and plates was
used as a control. The growth of the test bacteria was observed in all plates as positive controls.
3.5. Field Trials and Rapeseed Biochemical Ontent
The influence of N-substituted β-alanine derivatives on the growth of rapeseed (Brassica napus L.)
and St. John wort (Hypericum perforatum L.) in vitro was explored. According to the results, 3-[(4-
oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) was selected, because it had the
most remarkable positive effect on the growth of rapeseed and St. John wort in vitro [34]. Following
the data obtained during the in vitro experiments, compound 2 was selected for the field trials.
Experiments with 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid on spring
rapeseed (Brassica napus L.) of the SW Landmark variety were carried out in field trials in 2012 at the
Lithuanian Research Centre for Agriculture and Forestry branch Rumokai experimental station. Spring
rapeseed was sprayed with the study compound solutions (concentrations 25–150 mg/L) before
flowering. For each field, 1 L of the solution was used. The soil of the experimental field was

Molecules 2013, 18
15015
carbonaceous shallow luvisols (Calc or epihypogleyic luvisol IDg8-k (LVg-w-cc) with the following
charateristics: pHKCl 6.7, humus 1.82%, Ntotal 0.12%, 308 mg kg−1 of mobile P2O5, 296 mg kg−1 of
mobile K2O. Soil samples were collected on 25.04.2012. Barley was the preceding crop. The size of
the initial and accounted fields was 2.7 × 11 m and 2.2 × 10 m, respectively. Experiments with
rapeseed were performed in April–August. The average air temperature ranged from 7.9 to 19.3 °C,
the rainfall ranged from 44.4 to 118.4 mm per month, and during the experiment it was 361.1 mm. The
rainfall was by 11.1 mm higher than the average multi-annual precipitation. Biometric measurements
were carried out after harvesting. The moisture of rape seeds was 8.5%. The oil content in rape seeds
was measured by extraction with hexane for 3 hours in a Soxhlet apparatus (Behr Labor-Technik,
Dusseldorf, Germany). Fatty acid composition was analysed with a HRGC 5300 Mega Series gas
chromatograph Carlo Erba Strumentazione (Milano, Italy). The protein content in rape seeds was
measured by the Bradford method.
4. Conclusions
The N-phenyl-N-thiocarbamoyl-β-alanine was used for the first time for the synthesis of
biologically active 2-aminothiazole derivatives. By a new method of synthesizing the target structures
we have established a convenient way to produce a number of compounds, which were used for
biological testing. Some of the synthesized compounds exhibited promising antimicrobial activity, and
3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) was found to promote the
growth, to increase seed yield and oil content in rapeseed.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2013 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 license
(http://creativecommons.org/licenses/by/3.0/).