Synthesis and RP HPLC studies of biologically active semicarbazides and their cyclic analogues 1,2,4-triazol-3-ones

Article abstract of DOI:10.1007/s00706-011-0715-z

The retention behaviour of semicarbazides and their cyclic analogues 1,2,4-triazol-3-ones, has been investigated by RP-8, RP-18 and IAM HPLC. The structures of new derivatives were proved by elemental analyses, IR, ¹H NMR and ¹³C NMR

Full text of DOI:10.1007/s00706-011-0715-z

Monatsh Chem (2012) 143:657–667
DOI 10.1007/s00706-011-0715-z
ORIGINAL PAPER
Synthesis and RP HPLC studies of biologically active
semicarbazides and their cyclic analogues 1,2,4-triazol-3-ones
•
Monika Pitucha Joanna Matysiak
•
Bogdan Senczyna
Received: 30 August 2011 / Accepted: 28 December 2011 / Published online: 28 January 2012
Ó The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract The retention behaviour of semicarbazides and
their cyclic analogues 1,2,4-triazol-3-ones, has been inves-
tigated by RP-8, RP-18 and IAM HPLC. The structures of
new derivatives were proved by elemental analyses, IR, 1H
NMR and 13C NMR spectroscopy. The compounds showed
regular retention behaviour in three chromatographic sys-
tems; their log k values decreased linearly with the increasing
concentration of an organic modifier in the mobile phase.
The ratio of the intercept (log kw) to the slope of compounds
is constant and the same for both groups of compounds on
C18 and IAM stationary phases. Differences between log kw
values from the octadecyl stationary phase of corresponding
cyclic and linear derivatives are constant, and they are
related to the mechanism of synthesis of 1,2,4-triazol-3-ones
from linear substrate semicarbazides, which was confirmed
by modelling studies. Good correlations between log kw
parameters obtained byRP-8or RP-18and determined by the
computational approach log P were found.
of biological activities [1–4], including antibacterial ones
[5, 6]. Examples of drugs bearing the 1,2,4-triazole residue
are the powerful azole antifungal agent fluconazole [7], as
well as the potent antiviral N-nucleoside ribavirin [8].
Their linear analogues, semicarbazides, commonly
applied in the 1,2,4-triazole synthesis, are also an important
class of compounds of diverse biological properties. They
have been studied as anticonvulsant [9], antitubercular [10]
and antinociceptive [11] agents. Additionally, the deriva-
tives having the heterocyclic ring exhibit antibacterial
activity, inhibiting growth of some gram-positive bacteria,
including B. cereus and M. luteus [12]. Some of them show
antinociceptive activity in a wide range of doses [13].
It is commonly known that biological activity of com-
pounds is a function of their lipophilicity. Reversed phase
(RP) chromatographic methods have been extensively
applied to determine this property of many bioactive
compounds [14, 15]. The use of alkyl-bonded phases in
liquid chromatography offers a convenient and highly
accurate method for the evaluation of the lipophilicity of a
large variety of compounds [16, 17]. As the HPLC sta-
tionary phase for the determination of phase affinity of
compounds, the immobilized artificial membrane (IAM) is
also used [18–20].
Keywords Semicarbazide ꢀ 1,2,4-Triazol-3-one ꢀ
RP HPLC ꢀ IAM chromatography ꢀ Lipophilicity
Introduction
A linear relationship between the retention parameters
(log k) and the concentration (u) of organic modifier
(acetonitrile, methanol, or others) in the aqueous mobile
Triazoles and their heterocyclic derivatives represent an
interesting class of compounds possessing a wide spectrum
´
phase described by the Soczewinski-Wachtmeister equa-
tion (Eq. 1) has to be established for a successful
chromatographic measurement of lipophilicity [21, 22].
M. Pitucha (&)
Department of Organic Chemistry,
Medical University of Lublin, Lublin, Poland
e-mail: monika.pitucha@umlub.pl
log k ¼ log kw þ Su
ð1Þ
log kw represents the retention factor of a solute with pure
water as the mobile phase; S is the slope of the regression
curve. This dependence allows for the extrapolation from
J. Matysiak ꢀ B. Senczyna
Department of Chemistry, University of Life Sciences in Lublin,
Lublin, Poland
123

658
M. Pitucha et al.
the data obtained for the water-organic modifier to water as
the mobile phase. On the basis of log k = f(u) relation-
ships, the lipophilicity parameter log kw and the specific
hydrophobic surface area S can be calculated. Lipophilicity
chromatographic descriptors obtained in this way help
explain differences in the bioactivity of similar structure
derivatives. They are usually used for QSAR model
building, prediction of biological activity of new structure
related analogues, and design and synthesis of more
effective new derivatives [23, 24].
well as their linear analogues, semicarbazides, were carried
out. The octyl, octadecyl and IAM stationary phases were
used. To explain differences in the retention behaviour of
compounds of both groups, molecular modeling was per-
formed. Log P values obtained from the computational
approach were used comparatively.
Results and discussion
In this work comparable chromatographic studies of
biologically active mono- and bis-1,2,4-triazol-3-ones as
The structures of compounds under consideration are pre-
sented in Scheme 1. They include linear semicarbazides
Scheme 1
R1
CH3
R2
R1
R2
N
1a, 1b
4-C2H5OC6H4-
9a, 9b
CH2=CHCH2-
N
CH3
N
N
N
N
2a, 2b
4-BrC6H4-
C2H5-
10a, 10b
11a, 11b
12a, 12b
13a, 13b
CH2=CHCH2-
Cyclohexyl-
CH3
N
3a, 3b
CH3
N
4a, 4b
C6H5CH2-
CH2=CHCH2-
4-C2H5OC6H4-
CH2=CHCH2-
CH3
N
5a, 5b
C6H5CH2-
S
6a, 6b
C6H5-
4-CH3C6H4-
C2H5-
14a, 14b
15a, 15b
CH3-
S
7a, 7b
C2H5-
S
8a, 8b
123

Synthesis and RP HPLC studies of biologically active semicarbazides
659
(a) and their cyclic analogues 3H-1,2,4-triazol-3-ones (b).
The compounds of structure b were obtained by conden-
sation reaction of a in alkaline medium. The process of
water molecule elimination from compounds 1a–13a or
two molecules from 14a and 15a gives the corresponding
3H-1,2,4-triazol-3-ones 1b–15b.
consisted of buffer mixed with various amounts of meth-
anol or acetonitrile to give pH 7.4. Linear relationships
were obtained between the log k and u in the mobile phase
in the whole studied ranges for all stationary phases.
Generally, for semicarbazide derivatives and IAM chro-
matography, a larger content of water in the mobile phase
can be used (Tables 1, 2). For some compounds log kw
values on IAM were determined experimentally. The
chromatograms of compound 12a are shown in Fig. 2.
The equations of the straight lines and statistics for three
techniques are reported in Tables 1 and 2. As follows from
the tables the absolute values of the slopes |S| are the highest
for the IAM stationary phase for both semicarbazides and
3H-1,2,4-triazol-3-ones. In the case of RP-8 and RP-18
chromatography, the log kw values for the linear structures
(1a–15a) are lower than for the cyclic ones (1b–15b).
Biagi et al. [25] found that for closely congeneric
compounds, the ratio of the intercept (log kw) to the slope
(S) in Eq. 1 is constant. The relationships between these
parameters of semicarbazides (a) for RP-8, 18 and IAM
chromatography are expressed by the following equations:
The substitution panel of compounds under consider-
ation includes heterocyclic, aryl, alkyl and alkenyl groups.
The structures of new derivatives were proved by ele-
mental analyses, IR, 1H NMR and 13C NMR spectroscopy.
The UV-Vis spectra in water-methanol solutions of dif-
ferent pH of compounds 13a and 13b are presented in Fig. 1.
They show that there are no significant changes in their
electronic structure at pH 2–8. As pH 7.4 (physiological) is
recommended for IAM chromatography measurements,
for comparison all chromatographic experiments were
performed at this pH.
The log k values of compounds were determined using
C-8, C-18 and IAM stationary phases. The mobile phases
log kwðC8ðaÞÞ ¼ 0:774 ðꢁ0:106Þ ðꢂSÞC8ðaÞ
ꢂ 1:295 ðꢁ0:383Þ n ¼ 15; r ¼ 0:896;
s ¼ 0:266; F ¼ 52:9; dfð1:13Þ
ð2Þ
ð3Þ
ð4Þ
log kwðC18ðaÞÞ ¼ 0:928 ðꢁ0:031Þ ðꢂSÞC18ðaÞ
ꢂ 0:940 ðꢁ0:049Þ n ¼ 15; r ¼ 0:992;
s ¼ 0:066; F ¼ 918:3; dfð1:13Þ
log kwðIAMðaÞÞ ¼ 0:407 ðꢁ0:051Þ ðꢂSÞIAMðaÞ
ꢂ 0:927 ðꢁ0:233Þ n ¼ 15; r ¼ 0:911;
s ¼ 0:284; F ¼ 63:3; dfð1:13Þ:
In the case of their cyclic analogues 3H-1,2,4-triazol-3-
ones (b), these relations are described by Eqs. 5–7:
log kwðC8ðbÞÞ ¼ 0:547 ðꢁ0:065Þ ðꢂSÞC8ðbÞ
ꢂ 0:288 ðꢁ0:286Þ n ¼ 15; r ¼ 0:918;
s ¼ 0:272; F ¼ 69:9; dfð1:13Þ
ð5Þ
ð6Þ
ð7Þ
log kwðC18ðbÞÞ ¼ 0:921 ðꢁ0:064Þ ðꢂSÞC18ðbÞ
ꢂ 0:963 ðꢁ0:145Þ n ¼ 15; r ¼ 0:969;
s ¼ 0:127; F ¼ 205:3; dfð1:13Þ
log kwðIAMðbÞÞ ¼ 0:387 ðꢁ0:074Þ ðꢂSÞIAMðbÞ
ꢂ 0:928 ðꢁ0:395Þ n ¼ 15; r ¼ 0:821;
s ¼ 0:313; F ¼ 27:1; dfð1:13Þ:
The best results were obtained for the C-18 stationary
phase (Eqs. 3, 6). However, in all cases congenerity of
compounds was confirmed. The slopes of Eqs. 2 and 5, 3
and 6, as well as 4 and 7 are similar. This may indicate the
Fig. 1 UV–Vis spectra of compounds 13a and 13b in methanol-
water solution of different pH
123

660
M. Pitucha et al.
Table 1 Chromatographic parameters: log kw (intercept) and -S (slope) of the linear dependences in Eq. 1 obtained by RP-8 and RP-18
chromatography
RP-8 HPLC
RP-18 HPLC
-S
Intercept
r2
u MeOH
n
-S
Intercept
r2
u MeOH
n
1a
3.978
3.847
3.449
3.728
2.967
3.371
4.872
2.919
2.948
2.293
3.725
3.422
3.386
4.398
4.401
4.140
4.153
3.301
3.841
3.539
3.601
5.124
3.637
3.417
2.711
4.591
4.829
3.793
6.725
6.305
1.946
2.144
1.022
1.717
0.797
1.563
2.443
0.799
0.721
0.581
1.783
1.682
1.234
1.705
1.998
2.350
2.434
1.324
2.139
1.550
1.987
2.893
1.661
1.551
0.874
2.172
2.244
1.923
3.038
3.311
0.970
0.979
0.984
0.981
0.991
0.992
0.984
0.991
0.990
0.990
0.977
0.985
0.992
0.970
0.930
0.972
0.978
0.973
0.974
0.981
0.975
0.959
0.980
0.979
0.975
0.981
0.981
0.973
0.979
0.954
0.70–0.25
0.70–0.35
0.70–0.20
0.70–0.25
0.55–0.10
0.70–0.20
0.55–0.30
0.70–0.10
0.70–0.10
0.70–0.10
0.70–0.30
0.70–0.25
0.70–0.10
0.70–0.20
0.70–0.25
0.70–0.35
0.70–0.35
0.70–0.20
0.70–0.25
0.55–0.20
0.80–0.30
0.70–0.40
0.70–0.20
0.70–0.20
0.70–0.10
0.70–0.30
0.70–0.30
0.70–0.30
0.70–0.30
0.70–0.35
7
5
6
7
9
7
6
9
9
9
6
7
9
8
7
5
5
7
5
7
7
6
8
8
9
6
6
6
6
5
1.880
2.279
1.155
2.065
0.910
1.959
2.365
0.987
1.257
0.854
1.452
1.506
1.451
0.590
1.406
2.392
2.623
1.353
2.507
1.591
2.670
3.268
1.758
1.757
1.767
1.905
2.331
2.211
1.974
2.821
0.783
1.279
0.089
0.871
-0.074
0.864
1.280
0.014
0.155
-0.154
0.479
0.471
0.446
-0.352
0.270
1.341
1.545
0.335
1.439
0.540
1.514
2.069
0.745
0.758
0.36
0.982
0.981
0.984
0.982
0.901
0.992
0.960
0.984
0.972
0.907
0.934
0.857
0.988
0.949
0.953
0.963
0.965
0.973
0.964
0.931
0.975
0.965
0.892
0.995
0.853
0.985
0.899
0.925
0.991
0.902
0.75–0.35
0.75–0.35
0.75–0.35
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.35
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.35
0.75–0.30
0.75–0.30
0.75–0.40
0.75–0.40
0.75–0.35
0.75–0.40
0.75–0.30
0.75–0.40
0.75–0.40
0.75–0.35
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.30
0.75–0.30
7
7
7
8
7
7
6
7
7
7
7
7
6
7
6
6
6
7
6
7
6
6
7
7
7
7
7
6
7
7
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
15b
0.862
0.977
1.126
0.792
1.531
same retention mechanism of both studied compounds in
these chromatographic systems. The relationships between
the intercept (log kw) and the slope (–S) of both groups of
compounds for three chromatographic systems are
presented in Figs. 3, 4 and 5.
log kwðIAMða;bÞÞ ¼ 0:373 ðꢁ0:035Þ ðꢂSÞIAMða;bÞ
s ¼ 0:256; F ¼ 113:2; df ð1:26Þ:
ꢂ 0:857 ðꢁ0:172Þ n ¼ 28; r ¼ 0:902;
ð10Þ
Compounds 2a and 2b are outliers in IAM
chromatography. The equations show that semicarbazides
and corresponding 3H-1,2,4-triazol-3-ones are a set of
congeneric compounds [25, 26]. Therefore, they were
analyzed together during further studies.
They are expressed by the following equations:
log kwðC8ða;bÞÞ ¼ 0:648 ðꢁ0:057Þ ðꢂSÞC8ða;bÞ
ꢂ 0:750 ðꢁ0:048Þ n ¼ 30; r ¼ 0:907;
s ¼ 0:294; F ¼ 130:2; df ð1:28Þ
ð8Þ
ð9Þ
The relationships between the log kw values obtained
by different chromatographic techniques are not very
powerful. It can be assumed that compounds 14 and 15
(with two semicarbazide moieties or two triazole rings)
are outliers. Under this assumption, the relationships
log kwðC18ða;bÞÞ ¼ 0:901 ðꢁ0:028Þ ðꢂSÞC18ða;bÞ
ꢂ 0:924 ðꢁ0:048Þ n ¼ 30; r ¼ 0:987;
s ¼ 0:097; F ¼ 1; 040:2; dfð1:28Þ
123

Synthesis and RP HPLC studies of biologically active semicarbazides
661
Table 2 Chromatographic parameters: log kw (intercept) and
-S (slope) of the linear dependences in Eq. 1 obtained by IAM
chromatography and log P values
IAM HPLC
log P
-S
Intercept
r2
u ACN
n
1a
5.094
5.358
3.514
4.077
2.458
4.626
5.061
2.453
2.783
2.581
5.227
4.588
3.318
6.727
7.091
5.878
5.519
4.228
5.211
4.062
4.761
5.900
4.274
3.782
3.994
5.374
5.924
4.593
6.902
7.684
1.257
1.847
0.067
0.825
0.097
1.230
1.522
0.003
0.101
0.206
1.137
0.740
0.318
1.459
1.725
1.543
1.705
0.441
1.391
0.513
1.243
1.760
0.712
0.584
0.158
0.985
1.056
0.935
1.322
1.913
0.995
0.993
0.901
0.997
0.955
0.995
0.995
0.868
0.993
0.965
0.997
0.993
0.986
0.998
0.996
0.996
0.996
0.985
0.998
0.995
0.998
0.997
0.995
0.908
0.998
0.986
0.994
0.996
0.994
0.997
0.30–0.05
0.30–0.10
0.40–0.15
0.30–0.00
0.25–0.00
0.50–0.15
0.30–0.05
0.30–0.00
0.30–0.00
0.30–0.00
0.30–0.05
0.30–0.00
0.30–0.00
0.30–0.05
0.30–0.05
0.30–0.05
0.30–0.10
0.40–0.15
0.30–0.05
0.30–0.00
0.50–0.15
0.30–0.10
0.30–0.00
0.30–0.00
0.30–0.00
0.30–0.05
0.30–0.00
0.30–0.00
0.30–0.05
0.30–0.10
6
5
6
7
6
8
6
7
7
7
6
7
6
6
6
6
5
6
6
7
8
5
7
7
7
6
7
7
6
5
0.91
1.53
-0.63
0.77
-0.27
1.90
2.39
0.58
0.09
-0.39
0.47
0.79
0.95
0.21
1.52
2.04
2.65
0.50
1.90
0.85
3.03
3.52
1.70
1.22
0.74
1.59
1.92
2.08
2.47
3.78
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
1b
Fig. 2 Chromatograms of compound 12a obtained by IAM chroma-
tography for different water contents in water/ACN mobile phase
The weakest correlation was found between the most
hydrophobic phase and the most polar one (Eq. 12).
For comparison, the theoretical values of log P were
calculated according to the fragmentation method intro-
duced by Crippen [27]. The analysis of both groups of
compounds gives the following equation:
2b
3b
4b
5b
6b
7b
log kwðC18Þ ¼ 0:451 ðꢁ0:058Þ log P þ 0:105 ðꢁ0:106Þ
n ¼ 26; r ¼ 0:937; s ¼ 0:201; F ¼ 174:1; dfð1:24Þ
8b
9b
ð14Þ
10b
11b
12b
13b
14b
15b
log kwðC8Þ ¼ 0:533 ðꢁ0:059Þ log P þ 1:061 ðꢁ0:106Þ
n ¼ 30; r ¼ 0:862; s ¼ 0:355; F ¼ 81:0; dfð1:28Þ:
ð15Þ
Compounds 14 and 15 are outliers in Eq. 14. In the case
of IAM chromatography very poor correlation was
obtained (r = 0.753, n = 30).
between the intercepts (log kw) are described in the fol-
lowing way:
Markedly different chromatographic behaviour of sol-
utes on the IAM chromatography is a result of some
differences in the stationary phase structure [28]. Gener-
ally, the correlation between the logarithms of the retention
factor determined on the IAM columns log kw(IAM) and the
reference parameter of hydrophobicity, log D as well as log
kw(C18) (logP) is not large [29]. However, the log kw(IAM)
values appeared to be a better predictor of bioactivity than
log D for several classes of drugs [29, 30].
log kwðC18ða;bÞÞ ¼ 0:853 ðꢁ0:071Þ log kwðC8ða;bÞÞ
ꢂ 0:654 ðꢁ0:126Þ n ¼ 26; r ¼ 0:926;
s ¼ 0:218; F ¼ 144:5; dfð1:24Þ
ð11Þ
log kwðC18ða;bÞÞ ¼ 0:692 ðꢁ0:102Þ log kwðIAMða;bÞÞ
þ 0:563 ðꢁ0:094Þ n ¼ 26; r ¼ 0:902;
s ¼ 0:249; F ¼ 105:1; dfð1:24Þ
ð12Þ
As the transformation of semicarbazides into 3H-1,2,4-
triazol-3-ones is associated with the elimination of water,
the differences between log kw(b) and log kw(a) values, D log
kw, were calculated. Table 3 shows that the lipophilicity
differences between the corresponding derivatives in RP-18
log kwðC8ða;bÞÞ ¼ 0:979 ðꢁ0:083Þ log kwðIAMða;bÞÞ
þ 0:832 ðꢁ0:085Þ n ¼ 26; r ¼ 0:924;
s ¼ 0:240; F ¼ 139:9; dfð1:24Þ:
ð13Þ
123

662
M. Pitucha et al.
Fig. 3 Relationship between
the intercept (log kw) and slopes
(-S) of both groups of
compounds (a and b) obtained
by RP-8 stationary phase
(Eq. 8)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
13b
12b
5b
6a
15a
14a
10b
9b
a
2b
13a
4b
1a
11b
10a
3a
6b
11a
5a
7b3b
1b
12a
8b
74aa
8a
9a
95%
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
-S (RP-8)
Fig. 4 Relationship between
the intercept (log kw) and slopes
(-S) of both groups of
compounds (a and b) obtained
by RP-18 stationary phase
(Eq. 9)
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
7b
2b
6b
15b
4b
1b
2a7a
13b
12b
4a
116ba
14b
1a
9b
8b
5b
11a
12a
13a
3b
10b
15a
9a
3a
8a
5a
10a
95%
14a
0.5
1.0
1.5
2.0
2.5
3.0
-S (RP-18)
chromatography, D log kw(C18), are in the range about
0.5–0.7. Higher values (about 1.2) for compounds 14 and 15
were found. They are formed by the elimination of two
water molecules from the linear substrates (Scheme 1), so
the lipophilicity differences are about twice as large. A
similar trend for RP-8 chromatography was observed
(Table 3). This finding reflects the synthesis mechanism of
compounds of structure b from a. At the same time it shows
that the elimination of water from a molecule reduces its
lipophilicity as well as absolute values of the specific
hydrophobic surface, and the ratio of the intercept (log kw) to
the slope (-S) of compounds is constant in both groups. The
obtained results are in accordance with the fragmental
method used for log P calculations [27]. The differences
123

Synthesis and RP HPLC studies of biologically active semicarbazides
663
Fig. 5 Relationship between
the intercept (log kw) and slopes
(-S) of both groups of
compounds (a and b) obtained
by IAM stationary phase
(Eq. 10)
2.0
1.5
1.0
0.5
0.0
-0.5
13b
5b
14a
15a
6a
2b
1a
10a
9b
13a
12b
4b
5a
10b
11b
11a
3a
3b
6b
7b
1b
12a
2a
9a
4a
7a
8b
8a
95%
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
-S (IAM)
7a and 7b are shown as representative analogues of the
analysed groups.
Table 3 The lipophilicity differences between the 3H-1,2,4-triazol-
3-ones and the corresponding semicarbazides (D log kw) obtained by
different chromatographic systems
As follows from Fig. 6 elimination of water from an
a molecule remarkably changes its shape, flexibility and
dipole moment, and decreases both the area and volume.
Of course, changes of individual compounds are also a
function of the substituents. The MEPs, the electrostatic
potential profile of compounds, show that the most nega-
tive potential of compound structure a is located on four
heteroatoms of the semicarbazide moiety (without taking
the substituent into account). In the cyclic analogues b it
covers mainly two oxygen atoms. The area and volume
reduction of b compared to a is associated with reduction
of the polar local area of molecules. It causes stronger
interactions with the hydrophobic octadecyl or octyl sta-
tionary phases and increases lipophilicity.
D log kaw(8)
D log kbw(C18)
D log kcw(IAM)
1a,b
2a,b
3a,b
4a,b
5a,b
6a,b
7a,b
8a,b
9a,b
10a,b
11a,b
12a,b
13a,b
14a,b
15a,b
a
0.404
0.290
0.301
0.422
0.754
0.423
0.450
0.862
0.830
0.293
0.389
0.563
0.689
1.333
1.313
0.558
0.266
0.246
0.568
0.614
0.650
0.789
0.731
0.603
0.514
0.383
0.506
0.680
1.144
1.261
0.286
-0.142
0.373
0.566
0.415
0.013
0.238
0.709
0.483
-0.048
-0.152
0.316
In conclusion, new semicarbazides and corresponding
1,2,4-triazol-3-one derivatives have been synthesized. In
RP-8, RP-18 and IAM chromatography, the dependences of
log k versus the concentration of an organic modifier (u) in
the mobile phase were linear. The constant ratio of the
intercept (log kw) to the slope (-S) of compounds showed
that the compounds of both groups are congeneric according
to Biagi. The differences in the retention behaviour of
compounds of structures b and a in C-18 and C-8 chroma-
tography reflect the synthesis method of 1,2,4-triazol-3-ones
from linear substrates connected with the elimination of
water from a molecule. The good correlation coefficient
between the log kw values obtained by octadecyl and octyl
phases suggests that these parameters can be used indepen-
dently in the evaluation of compounds under consideration.
0.617
-0.138
0.189
D log kw(C8) = log kw(C8(b)) - log kw(C8(a))
D log kw(C18) = log kw(C18(b)) - log kw(C18(a))
D log kw(IAM) = log kw(IAM(b)) - log kw(IAM(a))
b
c
between log P values of compound structures b and a are
equal 1.13 (1.12) for analogues 1–13 and 2.26 for 14 and 15.
The differences between the log kw(IAM(b)) and log kw(IAM(a))
values, D log kw(IAM), are varied (Table 3).
To compare the structures of compounds quantum
mechanical calculations were carried out with the semi-
empirical model at the PM3 basis set. In Fig. 6 compounds
123

664
M. Pitucha et al.
1-Methyl-1H-pyrrole-2-acetic acid 2-[(2-propenylamino)-
carbonyl]hydrazide (5a, C11H16N4O2)
Yield 1.96 g (83%); m.p.: 137–139 °C; IR (KBr):
m = 3,279, 3,192, 3,034, 1,701, 1,649, 1,444 cm-1 1H
;
NMR (300 MHz, DMSO-d6): d = 3.32 (s, 2H), 3.52 (s,
3H), 3.61–3.65 (m, 2H), 4.98–5.15 (m, 2H), 5.72–5.84 (m,
1H), 6.85–7.63 (m, 3H, pyrrole-H), 7.63 (s, NH), 7.78 (s,
NH), 9.60 (s, NH) ppm; 13C NMR (62.5 MHz, DMSO-d6):
d = 31.45 (CH2), 33.52 (CH3), 41.42 (CH2), 106.01 (CH),
107.84 (Carom), 114.40 (CH2), 121.82, 126.09, 136.28
(3 9 Carom), 157.91 (C=O), 169.26 (C=O) ppm.
Pyridine-2-carboxylic acid 2-[(2-propenylamino)-
carbonyl]hydrazide (9a, C10H12N4O2)
Yield 1.76 g (80%); m.p.: 195–197 °C; IR (KBr):
m = 3,261, 3,013, 2,814, 1,697, 1,651, 1,487 cm-1 1H
;
NMR (300 MHz, DMSO-d6): d = 3.63–3.72 (m, 2H),
4.98–5.18 (m, 2H), 5.71–5.86 (m, 1H), 6.59 (s, NH),
7.56–8.08 (m, 4H, pyridine-H), 8.72 (s, NH), 10.26 (s, NH)
ppm; 13C NMR (62.5 MHz, DMSO-d6): d = 13.31 (CH2),
61.74 (CH2), 113.06 (CH), 123.12, 130.03, 143.23, 148.01,
152.48 (5 9 Carom), 154.21 (C=O), 167.73 (C=O) ppm.
Pyridine-4-acetic acid 2-[(2-propenylamino)-
carbonyl]hydrazide (10a, C11H14N4O2)
Fig. 6 Optimised structures of 7a and 7b and the MEPs of the
electrostatic potential profile at -84 kJ mol-1
Yield 1.62 g (69%); m.p.: 130–131 °C; IR (KBr):
m = 3,309, 3,267, 2,960, 1,644, 1,598, 1,455 cm-1 1H
;
NMR (300 MHz, DMSO-d6): d = 3.49 (s, 2H), 3.56–3.70
(m, 2H), 5.00–5.15 (m, 2H), 5.72–5.85 (m, 1H), 6.53 (t,
J = 5.6 Hz, NH), 7.20–8.49 (m, 4H, pyridine-H), 7.85 (s,
NH), 9.82 (s, NH) ppm; 13C NMR (62.5 MHz, DMSO-d6):
d = 40.00 (CH2), 40.11 (CH2), 113.12 (CH2), 123.26
(CH), 123.82, 143.26, 147.78, 147.96 (5 9 Carom), 156.62
(C=O), 167.68 (C=O) ppm.
Experimental
Melting points were determined in a Fisher-Johns block.
Elemental analyses (C, H, N) were conducted using an
Elemental Analyser CHN; their results were found to be in
good agreement with the calculated values. IR spectra were
recorded from KBr discs using a Specord IR-75 spectro-
1
Propanoic acid 1,10-[2,20-[methylenebis(4,1-
phenyleneiminocarbonyl)]dihydrazide]
(15a, C21H26N6O4)
photometer. H NMR spectra were recorded on a Bruker
AC 300F instrument (300 MHz) in DMSO-d6 with TMS as
an internal standard. 13C NMR spectra were recorded on a
Bruker AC 250F instrument (62.5 MHz) in DMSO-d6 with
TMS as an internal standard. Chemicals were purchased
from Alfa-Aesar or Merck. The purity of the obtained
compounds was checked by TLC on aluminum oxide 60
F254 plates (Merck) in a CHCl3/C2H5OH (10:1 and 10:2)
solvent system with UV or iodine visualization. Com-
pounds 1–4, 6–8, 11a–13a and 14 were synthesized and
characterized earlier [31–35].
Yield 3.49 g (82%); m.p.: 356–358 °C; IR (KBr):
m = 3,304, 3,032, 2,977, 1,650, 1,463 cm-1 1H NMR
;
(300 MHz, DMSO-d6): d = 1.95–2.07 (m, 6H), 2.10–2.35
(m, 4H), 3.80 (s, 2H), 7.06–7.39 (m, 8H, phenyl-H), 7.90
(s, 2NH), 8.64 (s, 2NH), 9.54 (s, 2NH) ppm; 13C NMR
(62.5 MHz, DMSO-d6): d = 7.01 (2 9 CH3), 8.26 (2 9
CH2), 25.06 (CH2), 117.38, 118.72, 127.42, 133.80, 136.07
(12 9 Carom), 154.20 (2 9 C=O), 170.80 (2 9 C=O) ppm.
Synthesis of 4,5-disubstituted 2,4-dihydro-3H-1,2,4-
triazol-3-one derivatives
Synthesis of 1,4-disubstituted semicarbazides
To 10 mmol of appropriate carboxylic acid hydrazide dis-
solved in 10 cm3 of diethyl ether 10 mmol isocyanate was
added. The mixture was kept at room temperature for 24 h.
The precipitate was filtered and crystallized from ethanol.
A mixture of 10 mmol of semicarbazide derivatives a and
50 cm3 of 2% aqueous solution of sodium hydroxide was
refluxed for 20 h. After cooling, the solution was neutralized
123

Synthesis and RP HPLC studies of biologically active semicarbazides
665
ppm; 13C NMR (62.5 MHz, DMSO-d6): d = 14.49 (CH3),
31.23 (CH2), 63.36 (CH2), 114.86, 124.06, 124.96, 128.82,
144.42, 144.99, 149.22, 154.54 (12 9 Carom), 158.42
(C=O) ppm.
with dilute hydrochloric acid. The precipitate was filtered off
and then crystallized from ethanol.
2,4-Dihydro-5-[(1-methyl-1H-pyrrol-2-yl)methyl]-4-(2-
propenyl)-3H-1,2,4-triazol-3-one (5b, C11H14N4O)
Yield 1.65 g (76%); m.p.: 110–112 °C; IR (KBr):
2,4-Dihydro-5-(phenylmethyl)-4-(2-propenyl)-3H-1,2,4-
triazol-3-one (13b, C12H13N3O)
m = 3,283, 3,088, 3,015, 2,854, 1,697, 1,647, 1,461 cm-1
;
1H NMR (300 MHz, DMSO-d6): d = 3.49 (s, 3H), 3.83 (s,
2H), 4.12–4.14 (m, 2H), 4.92–5.13 (m, 2H), 5.66–5.83 (m,
1H), 5.88–6.66 (m, 3H, pyrrol-H), 11.52 (s, NH) ppm; 13C
NMR (62.5 MHz, DMSO-d6): d = 22.17 (CH2), 32.04
(CH3), 40.86 (CH2), 104.95 (CH), 106.51 (Carom), 115.14
(CH2), 121.14, 123.62, 131.36, 143.80 (5 9 Carom), 153.38
(C=O) ppm.
Yield 1.57 g (73%); m.p.: 105–108 °C; IR (KBr):
m = 3,143, 3,064, 2,780, 1,702, 1,641, 1,449 cm-1 1H
;
NMR (300 MHz, DMSO-d6): d = 3.30–3.45 (m, 2H), 4.10
(s, 2H), 4.66–5.05 (m, 2H), 5.40–5.70 (m, 1H), 6.93–7.32
(m, 5H, phenyl-H), 11.64 (s, NH) ppm; 13C NMR
(62.5 MHz, DMSO-d6): d = 31.44 (CH2), 40.05 (CH2),
116.55 (CH2), 126.85 (CH), 128.54, 128.64, 132.48,
135.19, 146.23 (7 9 Carom), 154.81 (C=O) ppm.
2,4-Dihydro-4-(2-propenyl)-5-(pyridin-2-yl)-3H-1,2,4-
triazol-3-one (9b, C10H10N4O)
4,40-(Methylenedi-4,1-phenylene)bis(5-ethyl-2,4-
dihydro-3H-1,2,4-triazol-3-one) (15b, C21H22N6O2)
Yield 2.61 g (68%); m.p.: 308–310 °C; IR (KBr):
Yield 1.37 g (68%); m.p.: 183–184 °C; IR (KBr):
m = 3,180, 3,014, 2,953, 2,814, 1,964, 1,640, 1,454 cm-1
;
m = 3,176, 3,066, 2,955, 1,698, 1,580, 1,417 cm-1 1H
;
1H NMR (300 MHz, DMSO-d6): d = 3.50–3.59 (m, 2H),
4.11–4.77 (m, 2H), 5.11–5.16 (m, 1H), 7.02–7.30 (m, 4H,
pyridine-H), 11.05 (s, NH) ppm; 13C NMR (62.5 MHz,
DMSO-d6): d = 39.00 (CH2), 42.29 (CH2), 114.71 (CH),
132.20, 136.00, 142.62, 145.61, 147.47 (6 9 Carom), 153.75
(C=O) ppm.
NMR (300 MHz, DMSO-d6): d = 1.00 (t, J = 4.6 Hz,
6H), 2.36 (q, J = 7.4 Hz, 4H), 3.32 (s, 2H), 7.05–7.94 (m,
8H, phenyl-H), 11.59 (s, 2NH) ppm; 13C NMR (62.5 MHz,
DMSO-d6): d = 8.30 (2 9 CH3), 17.92 (2 9 CH2), 39.05
(CH2), 126.05, 128.32, 139.98 (14 9 Carom), 146.54
(2 9 C=O) ppm.
2,4-Dihydro-4-(2-propenyl)-5-[(pyridin-4-yl)methyl]-3H-
1,2,4-triazol-3-one (10b, C11H12N4O)
UV-Vis spectroscopy
Yield 1.60 g (74%); m.p.: 255–257 °C; IR (KBr):
m = 3,180, 3,065, 2,790, 2,654, 1,703, 1,639, 1,457 cm-1
;
1H NMR (300 MHz, DMSO-d6): d = 4.25 (s, 2H),
4.21–4.27 (m, 2H), 4.93–5.09 (m, 2H), 5.71–5.84 (m, 1H),
7.95–8.88 (m, 4H, pyridine-H), 11.79 (s, NH) ppm; 13C
NMR (62.5 MHz, DMSO-d6): d = 29.80 (CH2), 39.05
(CH2), 40.81 (CH2), 115.45 (CH), 126.38, 131.16, 139.92,
142.93 (6 9 Carom), 154.29 (C=O) ppm.
UV-Vis spectra were recorded in water (buffer)-methanol
(1:1) solution on a UV-160A Shimadzu Spectrophotome-
ter. Quartz cuvettes (1 cm) were used for measurements.
Liquid chromatography
HPLC was carried out using a liquid chromatograph
(Knauer, Berlin, Germany) with a dual pump, a 20-mm3
simple injection valve and a UV–visible detector at
254 nm. The samples were prepared as solutions in
methanol.
4-Cyclohexyl-2,4-dihydro-5-[(pyridin-4-yl)methyl]-3H-
1,2,4-triazol-3-one (11b, C14H18N4O)
Yield 2.06 g (80%) m.p.: 237–238 °C; IR (KBr):
m = 3,231, 3,075, 2,916, 1,687, 1,564 cm-1 1H NMR
;
(300 MHz, DMSO-d6): d = 0.96–2.06 (m, 10H), 3.48–3.61
(m, 1H), 4.00 (s, 2H), 7.27–8.53 (m, 4H, pyridine-H), 11.48
(s, NH) ppm; 13C NMR (62.5 MHz, DMSO-d6): d = 24.53
(CH2), 25.14 (2 9 CH2), 28.97 (2 9 CH2), 30.94 (CH2),
53.97 (CH), 123.92, 144.96, 145.06, 149.74 (6 9 Carom),
154.69 (C=O) ppm.
RP-8 chromatography
A Hypersil MOS2 C8 (5 lm, 150 9 4.6 mm) column was
used as the stationary phase. The mobile phase consisted of
different volume mixtures of methanol and 20 mM acetate
buffer (KCl) as the aqueous phase to give pH 7.4. The
methanol concentration ranged from 0.10 to 0.70 at 0.1
(0.05) intervals. The flow rate was 1 cm3 min-1 at room
temperature. The retention time of an unretained solute (t0)
was determined by the injection of a small amount of
acetone dissolved in water.
4-(4-Ethoxyphenyl)-2,4-dihydro-5-[(pyridin-4-yl)methyl]-
3H-1,2,4-triazol-3-one (12b, C16H16N4O2)
Yield 2.10 g (71%); m.p.: 180–181 °C; IR (KBr):
m = 3,231, 3,074, 2,917, 1,678, 1,475 cm-1 1H NMR
;
(300 MHz, DMSO-d6): d = 1.32 (t, J = 6.9 Hz, 3H), 3.82
(s, 2H), 4.05 (q, J = 6.9 Hz, 2H), 6.94–7.07 (m, 4H,
phenyl-H), 7.14–8.41 (m, 4H, pyridine-H), 11.76 (s, NH)
123

666
M. Pitucha et al.
RP-18 chromatography
References
1. Aher NG, Pore VS, Mishra NN, Kumar A, Shukla PK, Sharma A,
Bhat MK (2009) Bioorg Med Chem Lett 19:759
2. Bhat AR, Bhat GV, Shenoy GG (2001) J Pharm Pharmacol
53:267
3. Trzaskos JM, Bowen WD, Shafiee A, Fischer RT, Gaylor JL
(1984) J Biol Chem 259:13402
4. Demirbas N, Ugurlouglu SA, Demirbas A (2002) Biorg Med
Chem 10:3717
5. Yu¨ksen H, Demirbas¸ A, Ikizler A, Johansson CB, Celik C, Ikizler
AA (1997) Arzneim Forsch 47:405
6. Hui XP, Zhang L, Zhang ZY, Wang Q, Wang F (2000) J Chin
Chem Soc 47:535
7. Trsukuda T, Shiratoi Y, Watanabe M, Ohtsuka H, Hattori K,
Shirai M, Shimma N (1998) Bioorg Med Chem Lett 8:1819
8. Witoaski JT, Robins RK, Sidwel RW, Simon LN (1972) J Med
Chem 15:1150
A Hypersil Gold C18 (3 lm, 100 9 3 mm) column was
used as the stationary phase. The mobile phase consisted of
different volume mixtures of methanol and 20 mM phos-
phate buffer as the aqueous phase to give pH 7.4. The
methanol concentration ranged from 0.30 to 0.75,
depending on the structure of compound, at 0.05 (0.1)
intervals. The flow rate was 0.5 cm3 min-1 at room tem-
perature. The retention time of an unretained solute (t0)
was determined by the injection of a small amount of
acetone dissolved in water.
IAM chromatography
9. Kritsanida M, Mouroutsou A, Marakos P, Pouli N, Papakon-
stantinou-Garoufalias S, Pannecougue C, Witvouw M, De Clerq
E (2002) Il Farmaco 57:253
´
10. Walczak K, Gondela A, Suwinski J (2004) Eur J Med Chem
39:849
A
Rexchrom IAM.PC.DD2 (12 lm, 100 9 4.6 mm,
˚
300 A) (Regis Technologies) column was used as the sta-
tionary phase. The mobile phase consisted of different
volume mixtures of acetonitrile and 20 mM phosphate
buffer as the aqueous phase to give pH 7.4 (0.02 M
KH2PO4, Na2HPO4, and 0.15 M KCl). The acetonitrile
concentration ranged from 0 to 0.50, depending on the
structure of compound, at 0.05 intervals. The flow rate was
1 cm3 min-1 at room temperature. The retention time of an
unretained solute (t0) was determined by the injection of a
small amount of citric acid dissolved in water [36, 37].
11. Amir M, Shikha K (2004) Eur J Med Chem 39:535
12. Shiradkar MR, Ahivaprasadh N (2006) Asian J Chem 18:319
´
13. Pitucha M, Chodkowska A, Maciejewski M, Jagiełło-Wojtowicz
E, Pachuta-Stec A (2010) Monatsh Chem 141:199
14. Valko K, Sleger P (1993) J Chromatogr A 669:346
15. Valko K (2004) J Chromatogr A 1037:299
16. Kaliszan R, Nasal A, Markuszewski MJ (2003) Anal Bioanal
Chem 377:803
17. Matysiak J, Niewiadomy A, Ma˛cik-Niewiadomy G (2000) Eur J
Pharm Sci 10:119
18. Ong S, Liu H, Qui X, Bhat G, Pidgeon C (1995) Anal Chem
67:755
Computational methods
19. Nasal A, Sznitowska M, Bucinski A, Kaliszan R (1995) J
Chromatogr A 692:83
20. Pidgeon C, Venkataram UV (1989) Anal Biochem 176:36
´
21. Soczewinski E, Wachtmeister CA (1962) J Chromatogr A 7:311
22. Snyder LR, Dolan JW, Gant JR (1979) J Chromatogr A 165:3
The 3D structure of each compound was built up from the
fragment library in the PC SPARTAN Pro ver 1.08
molecular modeling program, and it was fully geometry-
optimized at the semi-empirical PM3 level [38]. The
energy-minimized structure was subjected to conforma-
tional analysis implemented in SPARTAN. Conformational
analysis was carried out through systemic conformation
option. The conformers of the lowest energy were compared.
The log P values were calculated using ChemDraw Ultra
10.0 [39].
¨
23. Hollosy F, Lorand T, Orfi L, Eros D, Keri G, Idei M (2002) J
´
Chromatogr B 768:361
´
¨
24. Kaliszan
R (1987) Quantitative structure—chromatographic
retention relationship. Jon Wiley & Sons, New York
25. Biagi GL, Barbaro AM, Sapone A, Recanatini (1994) J Chro-
matogr A 669:246
26. Todeschini R, Consonni V (2009) Molecular descriptors for
chemoinformatics; methods and principles in medicinal chemis-
try, vol 41. Wiley-VCH, Weinheim
27. Ghose AK, Crippen GM (1987) J Chem Inf Comput Sci 27:21
28. Ward RS, Davies J, Hodges G, Roberts DW (2003) J Chromatogr
A 1007:67
Statistic analysis
29. Abraham MH, Chadha HS, Leitao RAE, Mitchell RC, Lambert
WJ, Kaliszan R, Nasal A, Haber P (1997) J Chromatogr A 766:35
30. Kaliszan R (1999) Trends Anal Chem 18:400
The coefficients in the regression equations were calculated
by the multiple regression analysis program Statistica,
version 7.1 [40]. Statistical significance of the regression
equation was tested by the correlation coefficient (r), the
standard error of estimate (s) and the variance ratio (F) at
specified degrees of freedom (df), n number of compounds.
´
31. Pitucha M, Polak B, Swieboda R, Kosikowska U, Malm A (2009)
Z Naturforsch 64b:570
32. Pitucha M, Olender A, Wujec M, Borowski P, Mardarowicz M
(2010) J Chin Soc 57:260
´
33. Pitucha M, Borowski P, Karczmarzyk Z, Fruzinski A (2009) J
Mol Struct 919:170
34. Ismail I, Imam I, El-Azeem AH, Abd A (1995) Chem Chem Eng
39:19
35. Pitucha M, Karczmarzyk Z, Kosikowska U, Malm A (2011) Z
Naturforsch 66b:505
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
123

Synthesis and RP HPLC studies of biologically active semicarbazides
667
36. Ward RS, Davies J, Hodges G, Roberts DW (2003) J Chromatogr
A 1007:67
37. Taillardat-Bertschinger A, Galland A, Carrupt PA, Testa B
(2002) J Chromatogr A 953:39
38. PC Spartan Pro (1999) Wavefunction Inc, California
39. ChemOffice (2006) CambridgeSoft Corporation, Cambridge
40. Statistica 7.1 (2005) StatSoft Inc, Tulsa
123