Determination of the lipophilicity of some new derivatives of semicarbazide and 1,2,4-Triazol-5-one with potential antibacterial activity

Article abstract of DOI:10.1515/znb-2009-0514

The chromatographic behavior of new derivatives of 1,2,4-triazol-5-one and semicarbazide was determined. The lipophilicity was confirmed by the use of a RP-TLC method. The partition coefficients were calculated by use of theoretical procedures. The correl

Full text of DOI:10.1515/znb-2009-0514

Determination of the Lipophilicity of Some New Derivatives of Semi-
carbazide and 1,2,4-Triazol-5-one with Potential Antibacterial Activity
a
b
c
d
´
Monika Pitucha , Beata Polak , Ryszard Swieboda , Urszula Kosikowska ,
and Anna Malmd
a Department of Organic Chemistry, Medical University, Staszica 6, 20-081 Lublin, Poland
b Department of Physical Chemistry, Medical University, Staszica 6, 20-081 Lublin, Poland
c Department of Inorganic Chemistry, Chair of Chemistry, Medical University, Staszica 6,
20-081 Lublin, Poland
d Department of Pharmaceutical Microbiology, Medical University, Chodzki 1, 20-093 Lublin,
Poland
Reprint requests to Dr. Monika Pitucha. E-mail: monika.pitucha@am.lublin.pl
Z. Naturforsch. 2009, 64b, 570 – 576; received February 15, 2009
The chromatographic behavior of new derivatives of 1,2,4-triazol-5-one and semicarbazide was de-
termined. The lipophilicity was confirmed by the use of a RP-TLC method. The partition coefficients
were calculated by use of theoretical procedures. The correlation between theoretical and experimen-
tal lipophilicity was determined. All obtained compounds were tested for their antimicrobial activity.
Key words: Semicarbazide, 1,2,4-Triazol-5-one, Lipophilicity, Antibacterial Activity
Introduction
a nonpolar, organic phase and water. It can be deter-
mined by the traditional “shake-flask” partition method
between n-octanol (or other organic compounds, im-
miscible with water) and water. Similar conditions of
determination can be achieved by the use of reversed
phase chromatography (both TLC and HPLC meth-
ods) [21– 23]. A linear relationship between the re-
tention parameters and the concentration (Φ) of an
organic modifier in the aqueous mobile phase has to
be established for a successful chromatographic mea-
surement of lipophilicity [24– 27]. The lipophilicity of
some biologically active compounds was experimen-
tally determined [28 – 32]. Some authors determined
the lipophilicity of a solute as the logPTLC coeffi-
cient (logPTLC = experimental lipophilicity obtained
for standards solutes in investigation condition). For
this purpose the RMW (RMW = intercept in equation
RM = RMW + aC; relative lipophilicity) values of in-
vestigated compounds and standard solutes were com-
pared [33 – 35]. The evaluation of the lipophilicity of
the solute with potential activity allows the estima-
tion of its biological activity in compliance with the
Hansch theory [36], and pertinent research refers to
Quantitative Structure Activity Relationships (QSAR)
[37 – 40].
Many people around the world suffer from micro-
bial infections, including opportunistic or recurrent
diseases associated usually with immunosuppressants.
In addition, the effectiveness of available antibacte-
rial agents is diminishing due to developing, increasing
and spreading of microbial resistance [1, 2]. Therefore,
synthesis of novel compounds of potential antimicro-
bial activity seems to be necessary. A great number
of 1,2,4-triazoles and 4,5-dihydro-1H-1,2,4-triazol-5-
ones have a wide range of biological activities such
as analgesic [3], antibacterial [4 – 7], fungicidal [8, 9],
anti-inflammatory [10], antiviral [11, 12], anticonvul-
sant [13], and antitumor [14, 15] properties. Due to
these properties, the triazole moiety is a structural ele-
ment of drugs such as fluconazole, itraconazole, voro-
zole, letrozole, and anastrozole [16 – 18].
It is known that the biological activity of the molecules
is related to their structure and physicochemical prop-
erties. The lipophilicity denotes the physical property
of a molecule which influences the transport of com-
pounds through the biological system. This property of
molecules could be used to characterize bioavailabil-
ity, distribution, activity, toxicity, and other processes
[19, 20]. Lipophilicity is usually measured by the parti-
The objective of the studies reported here is a chro-
tion coefficient of the investigated compound between matographic analysis of two series of newly synthe-
c
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M. Pitucha et al. · Some New Derivatives of Semicarbazide and 1,2,4-Triazol-5-one
571
R = 4-BrC6H4 (a), CH2C6H5 (b), 4-C2H5OC6H4 (c), C6H11 (d), C6H5 (e), C2H5 (f)
Scheme 1.
sized derivatives of 1,2,4-triazol-5-one and semicar-
bazide with potential antibacterial activity.
Results and Discussion
The new 3-(1-methyl-pyrrol-2-yl)-4-substituted4,5-
dihydro-1H-1,2,4-triazol-5-one derivatives 3a – f were
synthesized by cyclization of semicarbazide deriva-
tives 2a – f in alkaline medium (Scheme 1). They
Fig. 3. Relationships between RM of semicarbazide deriva-
tives and concentration of acetonitrile in the mobile phase.
Fig. 1. Relationships between RM of semicarbazide deriva-
tives and concentration of methanol in the mobile phase.
Fig. 4. Relationships between RM of 1,2,4-triazol-5-one
derivatives and concentration of acetonitrile in the mobile
phase.
were then characterized by elemental analyses, IR,
1H and 13C NMR spectroscopy, as well as by MS.
The lipophilicity of selected solutes (2a – f, 3a – c,
3e – f) was chromatographically determined. Experi-
mental data obtained by the RP-TLC method are pre-
sented as plots of RM (RM = log (1−R ) ) values vs. con-
F
R
centration of methanol or acetonitFrile (% v/v) in the
Fig. 2. Relationships between RM of 1,2,4-triazol-5-one
derivatives and concentration of methanol in the mobile mobile phase (Figs. 1 – 4). Linear relationships showed
phase.
the regular dependence of retention on the organic
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M. Pitucha et al. · Some New Derivatives of Semicarbazide and 1,2,4-Triazol-5-one
Table 1. Parameters of the RM = a + bC, where C is the con-
modifier concentration. A similar range of RM values
was determined for both kinds of solutes. For both
kinds of mobile phase polar modifiers, the sequence
of RM values for semicarbazide derivatives was (from
the biggest to the smallest values) as follows: 2a, 2b,
2e, 2d, 2c, 2f; for 1,2,4-triazol-5-one derivatives it was
as follows: 3a, 3c, 3b, 3e and 3f. The sequence of so-
lutes did not depend on the kind of the polar modi-
fier. However, the results indicate that for solutes of
centration of modifier (%; v/v) in the mobile phase. R2
=
correlation coefficient square of the equation.
Slope
Intercept
Methanol:
R2
RMW
−Φ
2a
2b
2c
2d
2e
−0.04
−0.03
−0.05
−0.05
−0.03
−0.02
−0.04
−0.03
−0.03
−0.03
−0.02
2.70
1.82
3.09
3.19
1.87
1.21
2.94
2.26
2.60
2.09
1.66
0.96
0.98
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
2.70
1.82
3.10
3.19
1.87
1.21
2.94
2.26
2.60
2.09
1.66
0.01
0.01
0.02
0.02
0.02
0.02
0.01
0.01
0.01
0.01
0.01
the semicarbazide series of investigated compounds, 2f
3a
3b
3c
3e
3f
the sequence changes when the concentration of the
polar modifier is increased. It was also noticed that
the highest RM values were found when methanol was
utilized as polar modifier. The sequence of solutes de-
pended on the kind of substituent. The smallest adsorp-
tion (the smallest RM values) in both groups of solutes
was determined for compounds with alkyl substituents,
the biggest adsorption was measured for compounds
with a bromophenyl group. Relationships of RM vs. or-
ganic modifier concentration (C) allowed to determine
the RMW and Φ values according to the Soczewinski-
Wachtmeister equation [41]:
Acetonitrile:
2a
2b
2c
2d
2e
2f
3a
3b
3c
3e
−0.03
−0.02
−0.02
−0.02
−0.01
−0.01
−0.02
−0.02
−0.02
−0.02
−0.02
2.01
1.25
1.22
0.92
0.51
0.41
1.51
1.35
1.42
1.42
1.19
0.99
0.98
0.99
0.94
0.96
0.96
0.99
0.98
0.98
0.99
0.99
2.1
0.02
0.02
0.02
0.02
0.03
0.03
0.02
0.02
0.02
0.02
0.02
1.25
1.22
0.91
0.51
0.41
1.51
1.35
1.42
1.42
1.19
RM = RMW + b C
(1) 3f
where C is the percentage of methanol in the mobile
phase and Φ is the ratio RMW/b.
with theoretically calculated lipophilicity coefficients.
The results of this comparison for each group of inves-
tigated solutes are presented in Table 3. The best corre-
lations for the 1,2,4-triazole group of solutes measured
as Pearson coefficient was achieved for logPKOWIN
and for ΦACN (0.9973) and for Pallas ANNLOG P
(0.9916) and for RMWMeOH. The correlations for the
semicarbazide group of solutes was poorer. However
the best results were achieved for MMP Q logP
Both coefficients could be used for lipophilicity
determination. The values of coefficients as well as
the other parameters of the linear correlation of RM
vs. organic modifier concentration are presented in
Table 1. RMW values are in the range from 0.409
to 3.186 for semicarbazide derivatives (solutes for the
semicarbazide group) and from 1.188 to 2.943 for
1,2,4-triazol-5-ones. Among the semicarbazide deriva-
tives, the smallest values were determined for so-
and ΦACN
.
The in vitro antimicrobial activities of compounds
lutes with an ethyl substituent (2f), and the highest 2a – f and 3a – f were evaluated using the agar-well dif-
value was found for a solute with a cyclohexyl sub- fusion method (at concentrations 1000 – 5000 mg L−1
stituent (2d, methanol as modifier). For 1,2,4-triazol- per well). Our results showed that all compounds were
5-one derivatives, the smallest RMW value was ob- inactive against four reference strains of fungi. Simi-
tained for alkyl-substituted solutes, the highest values lar results were obtained for two reference species of
for bromophenyl-substituted ones. The biggest values Staphylococcus spp. It was found that the tested com-
of coefficients was reached when methanol was used as pounds possess some antibacterial activity against the
mobile phase organic modifier. The second parameter reference strains of Gram-positive or Gram-negative
Φ was very low for both groups of solutes. For semi- bacteria as revealed by a complete or partial reduc-
carbazide derivatives it was in the range 0.013 – 0.035, tion (20– 80 %) of the growth around the wells with
for 1,2,4-triazol-5-one derivatives in the range 0.013– or without the clear zone, depending on the strains and
0.019. Theoretically determined lipophilicity parame- the concentration of the compound (1000 – 5000 mg
ters (logP) of the investigated solutes are given in Ta- L−1 per well); the zone diameter of growth inhibi-
ble 2. The RMW and Φ coefficients were compared tion was ≥ 12 – 15 mm. The antibacterial effect of
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M. Pitucha et al. · Some New Derivatives of Semicarbazide and 1,2,4-Triazol-5-one
Table 2. Theoretically determined logP coefficients of investigated solutes.
573
2a
2.36
1.98
2b
1.55
1.40
2c
1.98
1.77
2d
1.76
1.09
2e
1.55
1.40
2f
0.73
0.43
3a
2.47
3.20
3b
1.97
2.48
3c
2.10
2.99
3e
1.67
2.43
3f
0.77
1.52
Molinspiration
ALOGPS
logPKOWIN
2.32
1.61
2.58
1.14
1.74
1.53
2.23
1.43
0.60
1.63
0.02
0.74
0.70
1.09
1.10
1.20
1.98
0.79
1.31
0.91
1.71
1.90
2.01
2.01
0.60
2.01
0.58
1.41
1.43
0.64
1.68
0.19
0.78
0.70
1.09
0.61
0.18
0.96
3.45
2.32
3.14
2.15
2.43
2.65
3.53
2.86
1.52
2.13
1.24
1.60
1.89
2.67
3.14
1.92
2.53
1.79
2.00
2.04
2.88
2.56
1.29
2.24
1.20
1.42
1.83
2.67
1.64
0.49
0.95
0.10
0.56
0.50
1.22
Pallas ANNLOG P
Pallas ATOMIC6
Pallas CDI-REKKER
Pallas COMBINED
CHEM3D log P
CHEM3D Partition Coefficient
(Octanol/Water)
SciLogP
MMP Q logP
MMP log oct/wat
TITAN logP
HYPERCHEM
ACD/log P
−0.60
0.28
−0.14
0.44
5.88
−1.07
6.11
0.87
1.90
5.53
−1.69
5.59
0.11
1.11
5.85
−1.37
5.71
0.26
1.20
5.64
−1.33
5.47
5.51
−1.88
5.69
0.05
1.11
5.24
−2.46
5.05
5.75
2.73
5.60
2.00
2.55
1.75
5.50
2.59
5.33
1.24
1.85
1.13
5.73
2.63
5.08
1.38
1.85
0.36
5.33
2.11
5.08
1.17
1.76
0.74
4.80
1.01
4.40
−0.07
−0.79
−0.15
1.01
0.24
0.42
2.18
1.12
1.53
1.36
1.05
0.36
−0.48
the tested compounds showing some activity against Experimental Section
various species of bacteria assessed by the agar-well
diffusion method was further determined spectropho-
tometrically by the broth dilution method (31.25 to
500 mg L−1). According to our results, the most ef-
fective antibacterial compounds were compound 2a
(MIC = 500 mg L−1 for B. cereus ATCC 10876 and
P. mirabilis ATCC 12453 and about 84 % inhibition of
M. luteus ATCC 10240 growth at the same concentra-
tion), compound3c (MIC = 500 mg L−1 and about 40 –
50 % inhibition of the growth of E. coli ATCC 25922
in lower concentrations of this compound and by about
60 – 65 % inhibition of the growth of K. pneumoniae
Synthesis
All chemicals were purchased from Merck Co. or Lan-
caster (Gdan´sk, Poland) and used without further purifica-
tion. Melting points were determined in a Fisher-Johns block
and are presented withought correction. IR spectra were
recorded in KBr on a Perkin-Elmer 1725X FTIR spectrom-
eter. 1H and 13C NMR spectra were recorded on a Brucer
Avance 300 instrument in [D6]DMSO with TMS as inter-
nal standard. The mass spectra were obtained on a Finnigan
Trace DSQ spectrometer operating at 70 eV.
(1-Methyl-pyrrol-2-yl)acetic acid hydrazide (1) was pre-
pared from the reaction of the corresponding carboxylic acid
ester with hydrazine hydrate by the method described ear-
lier [42].
ATCC 13883 in concentrations 31.25– 500 mg L−1
)
and compound 3d (MIC = 500 mg L−1 for P. aerugi-
nosa ATCC 9027 and about 70 – 60 % inhibition of the
growth of K. pneumoniae ATCC 13883 at values rang-
ing from 31.25– 500 mg L−1).
Preparation of 1-[(1-methyl-pyrrol-2-yl)acetyl]-4-sub-
stituted semicarbazides 2a – c
A mixture of (1-methyl-pyrrol-2-yl)acetic acid hydrazide
1 (1.53 g, 10 mmol) and the appropriate isocyanate
(10 mmol) in 10 mL of N,N-dimethylacetamide was kept at
r. t. for 24 h, and then water (40 mL) was added. The pre-
cipitate was filtered off and crystallized from ethanol. The
physical constants of the products ar◦e given below.
Conclusion
The lipophilicity of newly obtained derivatives of
1,2,4-triazol-5-oneand semicarbazide was determined.
The relationship RM vs. C of a mobile phase polar mod-
ifier (methanol or acetonitrile) is linear and allowed
to calculate the lipophilicity coefficients. The small-
est values of lipophilicity were determined for com-
pounds with alkyl substituents in both groups of so-
lutes. Good correlation between theoretical and exper-
imental lipophilicity was achieved for the 1,2,4-triazol-
5-one series of solutes. The results obtained in the
study should be of value to further detailed studies on
the biological activity of all compounds.
Compound 2a: M. p. 200 – 201 C. – Yield 86 %. – IR
(KBr): ν = 3287, 3037, 2960, 1490, 1696 cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 3.49 (s, 2H, CH2), 3.54 (s,
3H, NCH3), 5.96 – 6.63 (m, 3H, Ar-H), 7.39 – 7.45 (m, 4H,
Ar-H), 8.13, 8.87, 9.79 (3 ×s, 3H, 3NH). – C14H15N4O2Br
(351.2): calcd. C 47.87, H 3.30, N 15.95; found C 47.83,
H 4.25, N 15.98.
◦
Compound 2b: M. p. 150 – 151 C. – Yield 76 %. – IR
(KBr): ν = 3251, 3029, 2974, 1494, 1682 cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 3.31 (s, 2H CH2), 3.43 (s, 3H,
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574
M. Pitucha et al. · Some New Derivatives of Semicarbazide and 1,2,4-Triazol-5-one
NCH3), 4.26 (s, 2H, CH2), 5.77 – 6.62 (m, 3H, Ar-H), 7.14 –
7.34 (m, 5H, Ar-H), 7.86, 8.80, 9.64 (3 × s, 3H, 3NH). –
C15H18N4O2 (286.3): calcd. C 62.92, H 6.33, N 19.56; found
C 62.88, H 6.39, N 19.49.
◦
Compound 2c: M. p. 192 – 193 C. – Yield 82 %. – IR
(KBr): ν = 3283, 3036, 2974, 1494, 1682 cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 1.29 (t, J = 7.0 Hz, 3H, CH3),
3.47 (s, 2H, CH2), 3.54 (s, 3H, NCH3), 3.96 (q, J = 6.9 Hz,
2H, CH2), 5.81 – 6.62 (m, 3H, Ar-H), 7.14 – 7.37 (m, 4H,
Ar-H), 7.94, 8.71, 9.74 (3 × s, 3H, 3NH). – C16H20N4O3
(316.3): calcd. C 60.74, H 6.37, N 17.71; found C 60.68,
H 6.41, N 17.67.
Preparation of 1-[(1-methyl-pyrrol-2-yl)acetyl]-4-sub-
stituted semicarbazides 2d – f
(1-Methyl-pyrrol-2-yl)acetic acid hydrazide 1 (1.53 g,
10 mmol) and the appropriate isocyanate (10 mmol) in 10 mL
of anhydrous diethyl ether were kept at r. t. for 24 h. Then the
formed compound was filtered off, washed with diethyl ether
and crystallized from ethanol. The physical constants of the
compounds prepared are given below.
◦
Compound 2c: M. p. 178 – 179 C. – Yield 81 %. – IR
(KBr): ν = 3321, 3014, 2930, 1495, 1682 cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 1.05 – 1.72 (m, 10H, 5CH2),
3.41 (s, 2H, CH2), 3.53 (s, 3H, NCH3), 3.61 – 3.69 (m,
1H, CH) 5.83 – 6.61 (m, 3H, Ar-H), 7.61, 8.78, 9.57 (3 × s,
3H, 3NH). – C14H22N4O2 (278.3): calcd. C 60.40, H 7.96,
N 20.12; found C 60.25, H 7.88, N 20.08.
◦
Compound 2e: M. p. 165 – 167 C. – Yield 84 %. – IR
Φ
(KBr): ν = 3306, 3116, 2938, 1446, 1695 cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 3.48 (s, 2H CH2), 3.55 (s,
3H, NCH3), 5.82 – 7.01 (m, 3H, Ar-H), 7.24 – 7.48 (m, 5H,
Ar-H), 8.03, 8.93, 9.78 (3 × s, 3H, 3NH). – C14H16N4O2
(272.3): calcd. C 61.75, H 5.92, N 20.57; found C 61.82,
H 5.89, N 20.63.
Compound 2f: M. p. 165 – 166 ◦C. – Yield 78 %. – IR
(KBr): ν = 3300, 3004, 2941, 1441, 1684 cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 0.99 (t, J = 7.1 Hz, 3H, CH3),
3.03 (q, J = 5.8 Hz, 2H, CH2), 3.35 (s, 2H, CH2), 3.45 (s, 3H,
NCH3), 5.79 – 6.61 (m, 3H, Ar-H), 8.07, 9.05, 9.70 (3 × s,
3H, 3NH). – C10H16N4O2 (224.2): calcd. C 53.55, H 7.19,
N 24.98; found C 53.48, H 7.28, N 25.01.
Preparation of 3-[(1-methyl-pyrrol-2-yl)methyl]-4-sub-
stituted-4,5-dihydro-1H-1,2,4-triazol-5-ones 3
A mixture of semicarbazide 2 (10 mmol) and 40 – 50 mL
of a 2 % (in the case of compound 2b 10 %) aqueous solu-
tion of sodium hydroxide was boiled for 15 – 20 h (2 h for
2b). After cooling, the solution was neutralized with dilute
hydrochloric acid. The precipitate was filtered off and then
crystallized from ethanol.
Φ
Φ
Φ
Φ
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M. Pitucha et al. · Some New Derivatives of Semicarbazide and 1,2,4-Triazol-5-one
575
◦
Compound 3a: M. p. 180 – 181 C. – Yield 89 %. – IR Chromatographic analysis of lipophilicity
(KBr): ν = 3065, 2948, 1492, 1704, 1572, 1492 cm−1. –
Thin-layer chromatography was performed on 10×10 cm
TLC plates (Merck, Darmstadt, Germany) precoated with
RP-18 F245 gel. Methanol-water or acetonitrile-water mix-
tures were used as mobile phases. The methanol or acetoni-
trile content was varied from 50 to 70 % (v/v) in 5 % steps.
Plates were developed to a distance of 9 cm at r. t. in a hor-
izontal DS chamber (Chromdes, Lublin, Poland) and after
drying visualized under λ = 254 nm UV light. Unfortunately
there was no possibility to visualize the solute 3d (lack of
spot under UV light). Each experiment was performed in
quadruplicate, coefficients of variation (CV) of experimen-
tally obtained RF values were below 6 %. RF values were
used to calculate RM by use of Eq. 2.
1H NMR (300 MHz, [D6]DMSO): δ = 3.38 (s, 3H, NCH3),
3.79 (s, 2H, CH2), 5.54 – 6.56 (m, 3H, Ar-H), 7.25 – 7.69
(m, 5H, Ar-H), 11.60 (s, 1H, NH). – C14H13N4OBr (333.2):
calcd. C 50.46, H 3.93, N 16.81; found C 50.58, H 3.78,
N 16.77.
◦
Compound 3b: M. p. 185 – 187 C. – Yield 80 %. – IR
(KBr): ν = 3084, 2938, 1448, 1709, 1572, 1493 cm−1. –
1H NMR (300 MHz, [D6]DMSO): δ = 3.44 (s, 3H, NCH3),
3.75 (s, 2H, CH2), 4.75 (s, 2H, CH2), 5.74 – 6.63 (m, 3H,
Ar-H), 7.13 – 7.34 (m, 5H, Ar-H), 11.62 (s, 1H, NH). –
13C NMR (300 MHz, [D6]DMSO): δ = 23.63 (CH2), 33.30
(CH3), 43.16 (CH2), 106.26, 107.80, 122.41, 124.77; 126.82
127.47, 128.62, 136.58 (all Ar-CH), 145.11 (C=N), 155.17
(C=O). – MS (EI, 70 eV): m/z (%) = 268 (35) [M+],
91 (100). – C15H16N4O (268.3): calcd. C 67.14, H 6.01,
N 20.88; found C 67.21, H 5.98, N 20.66.
ꢀ
ꢁ
1
RF
RM = log
−1
(2)
◦
The partition coefficients were calculated for the compounds
by use of different theoretical procedures: log PKOWIN, Pal-
las ANNLOG P, Pallas ATOMIC 6, Pallas CDI-REKKER,
Pallas COMBINED, CHEM 3D logP, CHEM 3D Par-
tition Coefficient (octanol/water), log P, SCILOG P Ap-
plication Version 3.0, TITAN logP (Ghose-Crippen), HY-
PECHEM, MOLINSPIRATION lipophilicity (coefficients were
determined by internet website [http://www.molinspiration.
com/cgi-bin/properties?textMode=1]).
Compound 3c: M. p. 183 – 184 C. – Yield 76 %. – IR
(KBr): ν = 3079, 2982, 1459, 1706, 1577, 1513 cm−1. –
1H NMR (300 MHz, [D6]DMSO): δ = 1.34 (t, J = 6.9 Hz,
3H, CH2), 3.36 (s, 3H, NCH3), 3.71 (s, 2H, CH2), 4.06 (q,
J = 7.0 Hz, 2H, CH2), 5.46 – 6.56 (m, 3H, Ar-H), 6.96 – 7.18
(m, 4H, Ar-H), 11.62 (s, 1H, NH). – C16H18N4O2 (298.3):
calcd. C 64.41, H 6.08, N 18.77; found C 64.68, H 6.28,
N 18.59.
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Compound 3d: M. p. 218 – 219 C. – Yield 89 %. – IR
(KBr): ν = 3068, 2942, 1451, 1704, 1574, 1514 cm−1. –
1H NMR (300 MHz, [D6]DMSO): δ = 1.06 – 2.06 (m, 10H,
5CH2), 3.50 (s, 3H, NCH3), 3.60 – 3.70 (m, 1H, CH), 3.91 (s,
2H, CH2), 5.82 – 6.66 (m, 3H, Ar-H), 11.34 (s, 1H, NH). –
C14H20N4O (260.3): calcd. C 64.58, H 7.74, N 21.52; found
C 64.71, H 7.49, N 21.62.
Antimicrobial activity
The newly synthesized compounds were screened for
their in vitro activity against 9 reference species of aero-
bic bacteria (5 Gram-positive: Staphylococcus epidermidis
ATCC 12228, Staphylococccus aureus ATCC 25923, Bacil-
lus subtilis ATCC 6633, Bacillus cereus ATCC 10876, Mi-
crococcus luteus ATCC 10240 and 4 Gram-negative: Es-
cherichia coli ATCC 25922, Klebsiella pneumoniae ATCC
13883, Proteus mirabilis ATCC 12453, Pseudomonas aerug-
inosa ATCC 9027), and 4 reference species of fungi (Can-
dida albicans ATCC 10231, Candida albicans ATCC 2091,
Candida parapsilosis ATCC 22019, Aspergillus niger ATCC
16404). The inoculum density was adjusted to 0.5 McFar-
land standard with sterile saline (0.85 % NaCl), and then the
suspensions were diluted 1 : 10 (for the agar well diffusion
◦
Compound 3e: M. p. 157 – 158 C. – Yield 80 %. – IR
(KBr): ν = 3078, 2921, 1456, 1705, 1579, 1500 cm−1. –
1H NMR (300 MHz, [D6]DMSO): δ = 3.36 (s, 3H, NCH3),
4.76 (s, 2H, CH2), 5.41 – 6.55 (m, 3H, Ar-H), 7.26 – 7.48
(m, 5H, Ar-H), 11.73 (s, 1H, NH). – 13C NMR (300 MHz,
[D6]DMSO): δ = 23.57 (CH2), 33.16 (CH3), 106.18, 107.46,
121.94, 125.04, 127.41, 128.50, 129.17, 132.83 (all Ar-CH),
144.99 (C=N), 154.32 (C=O). – MS (EI, 70 eV): m/z (%)
= 254 (59) [M+], 94 (100). – C14H14N4O (254.3): calcd.
C 66.13, H 5.54, N 22.03: found C 66.32, H 5.68, N 21.91.
Compound 3f: M. p. 150 – 151 ◦C. – Yield 90 %. – IR method) or 1 : 100 (for the broth dilution method) in Mueller-
(KBr): ν = 3070, 2930, 1470, 1703, 1572, 1520 cm−1. – Hinton broth without (for bacteria) or with 2 % glucose (for
1H NMR (300 MHz, [D6]DMSO): δ = 0.97 (t, J = 7.1 Hz, fungi). All stock solutions of the assayed compounds were
3H, CH3), 3.51 (q, J = 5.3 Hz, 2H, CH2), 3.74 (s, 3H, NCH3), prepared in 100 % dimethylsulfoxide (DMSO). Using the
3.91 (s, 2H, CH2), 6.83 – 6.67 (m, 3H, Ar-H), 11.47 (s, 1H, agar well diffusion method, microbial suspensions were put
NH). – 13C NMR (300 MHz, [D6]DMSO): δ = 13.86 (CH3), onto Mueller-Hinton agar without (for bacteria) or with 2 %
23.55 (CH2), 33.38 (CH3), 35.27 (CH2), 106.29, 107.74, glucose buffered at pH = 5.6 (for fungi). Then, a well was
122.37, 125.36 (all Ar-CH), 144.94 (C=N), 154.87 (C=O). – prepared in the plates with the help of a cork-borer (0.85 cm)
MS (EI, 70 eV): m/z (%) = 206 (43) [M+], 108 (100). – in the agar medium, and 80 µL of the tested compounds in
C10H14N4O (206.2): calcd. C 58.23, H 6.84, N 27.16; found concentrations of 1000 – 5000 mg L−1 were added to the
C 58.47, H 6.95, N 27.01.
wells. The same volumes of sterile 0.85 % NaCl or DMSO
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576
M. Pitucha et al. · Some New Derivatives of Semicarbazide and 1,2,4-Triazol-5-one
◦
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were used as a control. The plates were incubated at 37 C ing the broth dilution method. After incubation (37 C for
◦
for 18 h for bacteria and at 30 C for 48 h for Candida spp. 18 h) optical density (OD600) measurements were carried
or 5 d for Aspergillus niger, depending on the compound- out for bacterial cultures in broth medium containing 31.25
free growth control. After incubation microbial growth was to 500 mg L−1 of the compounds. The MIC (Minimal In-
determined by measuring the diameter of zones of growth hibitory Concentration) values, defined as the lowest concen-
inhibition. The antibacterial assay for the compounds, which tration of compound at which there is no visible growth of
had shown promising activity against bacterias assessed by the tested bacteria, were determined by comparison with the
the first method, were also tested spectrophotometrically us- growth of the control (compound-free) medium.
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