Synthesis and structural study of some N-acyl-4-allylsemicarbazides and the product of their cyclization with a potential antimicrobial activity

Article abstract of DOI:10.1016/j.molstruc.2020.128552

In this paper semicarbazide (2a-2h) and 1,2,4-trazole (3a-3h) derivatives with allyl group were synthesized. All compounds were tested in vitro for their antimicrobial activity showing different activity to E. coli, S. aureus, S. epidermidis, M. smegmatis, M. phlei and M. tuberculosis H37Ra. The antimicrobial activity is showed 2g against S. epidermidis, 3g against E. coli and S. epidermidis and 3h against E. coli. The crystal structure of determinations of 2b, 2d, 3b, 3c and 3e were undertaken in order to confirm the synthesis pathway and identification of their tautomeric forms in the crystalline state. Theoretical calculations showed that the physico-chemicals (logP) and electronic properties (MEP distribution, energy localization of HOMO and LUMO orbitals) are related to observed antimicrobial activity of investigated compounds. The molecular docking study carried out for the most active against M. tuberculosis compound 3b using the M. tuberculosis cytochrome P450 CYP121 showed that this compound binds to the active site of P450 by hydrogen bonds via water molecule with the amino acid residue of Met86A and molecule of hem.

Full text of DOI:10.1016/j.molstruc.2020.128552

Journal of Molecular Structure 1219 (2020) 128552
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis and structural study of some N-acyl-4-allylsemicarbazides
and the product of their cyclization with a potential antimicrobial
activity
,
Monika Pitucha a *, Zbigniew Karczmarzyk b, Marta Swatko-Ossor c, Monika Drozd a,
Waldemar Wysocki b, Grazyna Ginalska c, Zofia Urbanczyk-Lipkowska d,
Dorota Kowalczuk e, Maja Morawiak d
a Independent Radiopharmacy Unit, Faculty of Pharmacy, Medical University, Chodzki 4A, 20-093, Lublin, Poland
b Faculty of Science, University of Natural Sciences and Humanities in Siedlce, 3-Maja 54, 08-110, Siedlce, Poland
c Department of Biochemistry and Biotechnology, Faculty of Pharmacy, Medical University, Chodzki 1, Lublin, 20-093, Poland
d Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224, Warsaw, Poland
e Department of Medicinal Chemistry, Faculty of Pharmacy, Medical University, Jaczewskiego 4, 20-090, Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper semicarbazide (2a-2h) and 1,2,4-trazole (3a-3h) derivatives with allyl group were syn-
thesized. All compounds were tested in vitro for their antimicrobial activity showing different activity to
E. coli, S. aureus, S. epidermidis, M. smegmatis, M. phlei and M. tuberculosis H37Ra. The antimicrobial ac-
tivity is showed 2g against S. epidermidis, 3g against E. coli and S. epidermidis and 3h against E. coli. The
crystal structure of determinations of 2b, 2d, 3b, 3c and 3e were undertaken in order to confirm the
synthesis pathway and identification of their tautomeric forms in the crystalline state. Theoretical cal-
culations showed that the physico-chemicals (logP) and electronic properties (MEP distribution, energy
localization of HOMO and LUMO orbitals) are related to observed antimicrobial activity of investigated
compounds. The molecular docking study carried out for the most active against M. tuberculosis com-
pound 3b using the M. tuberculosis cytochrome P450 CYP121 showed that this compound binds to the
active site of P450 by hydrogen bonds via water molecule with the amino acid residue of Met86A and
molecule of hem.
Received 6 January 2020
Received in revised form
26 May 2020
Accepted 27 May 2020
Available online 6 June 2020
Keywords:
Semicarbazide
Triazole
Structure
Theoretical calculation
© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Listeria monocytogenes derived from the popular Hummus
(chickpea dip) resulted in a significant reduction in their number.
Diseases caused by microorganisms are among the fastest
growing in the present times. Their ability to mutate means that
fewer and fewer drugs are able to eradicate them completely, so it is
important to look for new ones. The most important argument is
the high risk of new outbreaks, which makes it a serious, dangerous
problem. Although many compounds have potential antimicrobial
activity [1e7] our interest includes compounds having allyl group.
Allyl derivatives have wide applications in practice and bring
many benefits from their use. It has been proven that the use of allyl
isothiocyanate (AITC) against 5 strains of Salmonella enterica and
These results give hope for a significant reduction in the number of
listeriosis and salmonellosis in the future [8]. The use of the gas
form AITC in the case of fungal infections of maize in Brazil resulted
in a significant decrease in the commonly occurring strains Asper-
gillus parasiticus and Fusarium verticillioides, as well as a reduction
in the production of mycotoxins (B1, B2, G1, G2, B1 and B2 fumo-
nins). The safety of consumption will increase significantly and the
risk of gastrointestinal problems and minimization of the dose of
endotoxins consumed are reduced [9].
Allyl derivatives of thiocyanate were characterized for antitu-
berculosis effect. The synthesis of more than 40 derivatives of
various groups was performed, which were tested in vitro for non-
replicating and replicating Mycobacterium tuberculosis (Mth)
H37Rv strains. The results of these studies have enabled the se-
lection of good and very good activities against the replicating
* Corresponding author. Independent Radiopharmacy Unit, Faculty of Pharmacy,
ꢀ
Medical University of Lublin, Chodzki 4A, 20-093, Lublin, Poland.
E-mail address: monika.pitucha@umlub.pl (M. Pitucha).
https://doi.org/10.1016/j.molstruc.2020.128552
0022-2860/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

2
M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
forms of Mycobacterium tuberculosis (Mth) H37Rv [10]. Compounds
that have been shown to exhibit this effect were just allyl de-
rivatives of thiocyanates containing halophenyl (halo ¼ 2-Br, 4-Br,
4-Cl, 4-F), 4-methylphenyl and 2-naphthyl moieties [10]. Studies
were also conducted on a wider group of isocyanate derivatives,
which also confirmed that the whole group has a strong antimi-
crobial effect, however it can be distinguished that the allyl deriv-
ative has a stronger activity against Gram (ꢀ) bacteria compared to
the rest of the test substances, especially against Escherichia coli
[11].
The allyl group is also interesting due to its chemical properties.
The methylene bridge (eCH2e) between the vinyl group (-CH ¼
CH2) and the rest of the molecule and two carbons with different
hybridizations can have a significant effect on the molecular
structure.
3.63 (s, 2H, CH2), 3.64 (s, 3H, CH3), 3.65 (t, J ¼ 0.9 Hz, 2H, CH2),
4.98e5.15 (m, 2H), 5.76e5.82 (m, 1H, CH), 5.85e5.87 (m, 2H,
pyrrole-H), 6.50 (s, 1H, NH), 6.61e6.62 (m, 1H, pyrrole-H), 7.81 (s,
1H, NH), 9.63 (s, 1H, NH). 13C NMR (DMSO‑d6)
d: 29.42, 31.95, 42.65,
106.51, 108.34, 114.90, 122.33, 126.58, 136.83, 158.22, 169.78. IR:
v ¼ 3296 (NH), 3099 (¼CeH), 1698 (C]O), 1635 (C]C). LC-QTOF
MS (m/z): calculated monoisotopic mass: 236.123, measured
monoisotopic mass: 236.1277.
2.1.1.2. 4-Allyl-1-(pyridin-2-ylcarbonyl)semicarbazide
(2b).
Yield 87% (1.82 g), 80% [12]; m.p. 198-200 ꢁC. 1H NMR (DMSO‑d6)
d:
3.70 (t, J ¼ 1.8 Hz, 2H, CH2), 5.02e5.22 (m, 2H, CH2), 5.77e5.89 (m,
1H, CH), 6.60 (t, J ¼ 5.7, 1H, NH), 7.63e7.67 (m, 1H, pyridine-H);
8.00e8.05 (m, 3H, pyridine-H), 8.68 (d, J ¼ 1.2 Hz, 1H, NH), 10.23
(s, 1H, NH). 13C NMR (DMSO‑d6)
d: 42.02, 114.96, 122.78, 127.30,
In our work experimental and theoretical study of new 4-
allylsemicarbazide derivatives and their cyclic analogues tested
in vitro for antimicrobial activity were subjected using crystal
structure determination by X-ray diffraction, theoretical calcula-
tions at AM1 and DFT/B3LYP/6-311þþG(d,p) level and molecular
docking. An attempt to link the geometrical and electronic pa-
rameters of the molecules of investigated compounds and their
antimicrobial activity was undertake.
136.84, 138.18, 149.00, 149.98, 158.33, 164.18. IR: v ¼ 3222 (NH),
3086 (¼CeH), 1697 (C]O), 1636 (C]C). LC-QTOF MS (m/z):
calculated monoisotopic mass: 220.0960, measured monoisotopic
mass: 200.0963.
2.1.1.3. 4-Allyl-1-(pyridin-3-ylcarbonyl)semicarbazide
(2c).
: 3.72 (t,
Yield: 77% (1.61 g), m.p. 158-162 ꢁC. 1H NMR (DMSO‑d6)
d
J ¼ 4.0 Hz, 2H, CH2), 5.06e5.22 (m, 2H, CH2), 5.83e5.88 (m, 1H, CH),
6.88 (s, 1H, NH), 7.57e7.60 (m, 1H, pyridine-H), 8.28 (s, 1H,
pyridine-H), 8.78e8.80 (m, 2H, pyridine-H), 9.10 (s, 1H, NH), 10.41
2. Experimental
(s, 1H, NH).). 13C NMR (DMSO‑d6)
d: 41.95, 114.87, 123.92, 128.87,
2.1. Chemistry
129.35, 135.78, 148.54, 152.70, 158.55, 164.51. IR: v ¼ 3203 (NH),
3006 (¼CeH), 1693 (C]O), 1640 (C]C). LC-QTOF MS (m/z):
calculated monoisotopic mass: 220.0960, measured monoisotopic
mass: 220.0961.
All chemicals and solvents were purchased from AlfaAesar,
Sigma-Aldrich, POCH and Merck. Melting points were taken in
Gallenkamp Melting Point Apparatus (ref: WA11373) and were
uncorrected. The purity of the compounds was checked by thin-
layer chromatography on TLC on aluminum oxide 60 F245 plates.
Ethanol-chloroform mixture (10:1) or (8:2) were used as mobile
phase. The yield was calculated for product after purification. 1H
and 13C NMR spectra were obtained on Bruker AVANCE III 600 MHz
spectrometer. Tetramethylsilane was used as an internal standard
for proton and carbon NMR spectra. The IR spectra were recorded
on a Nicolet 6700 FT-IR Spectrometer spectrometer using ATR
diamond crystal and Omnic software version 8.2. The attenuated
total reflectance ATR-IR spectra were recorded over the range
4000-400 cmꢀ1.For accurate mass measurements an Agilent
Technologies liquid chromatograph 1290 coupled to an Agilent
Technologies 6550 iFunnel Q-TOF LC/MS equipped with Jet Stream
Technology Ion Source was employed. Flow injection analyses were
performed using 0.1% formic acid in water:acetonitrile (50:50 v/v),
mobile phase flow 0.5 ml min-1. Ions were acquired in positive
polarity. Mass correction was enabled, ions of m/z 121.0509 and
922.0098 were used as the reference ions. Instrument control, data
acquisition and analysis were performed using Agilent MassHunter
Workstation B.07 Software. Single-charged protonated ions for all
examined compounds were detected.
2.1.1.4. 4-Allyl-1-(pyridin-2-ylacetyl)semicarbazide (2d). Yield: 87%
(2.05 g); m.p. 136-138 ꢁC. 1HNMR (DMSO‑d6)
d: 3.65 (s, 2H, CH2),
3.69 (t, J ¼ 3.0 Hz, 2H, CH2), 5.01e5.18 (m, 2H, CH2), 5.75e5.88 (m,
1H, CH), 6.51 (s, 1H, NH), 7.23e7.32 (m, 4H, pyridine-H), 7.84 (s, 1H,
NH), 9.77 (s, 1H, NH). 13C NMR (DMSO‑d6)
d: 40.67, 114.94, 126.91,
128.67, 129.43, 129.62, 136.81, 158.42, 170.49. IR: v ¼ 3324 (NH),
3085 (¼CeH), 1651 (C]O), 1639 (C]C). LC-QTOF MS (m/z): calcu-
lated monoisotopic mass: 234.1117, measured monoisotopic mass:
234.1117.
2.1.1.5. 4-Allyl-1-(pyridin-4-ylacetyl)semicarbazide (2e). Yield: 69%
(1.62 g), 69% [12]; m.p. 130e131 ꢁC. 1H NMR (DMSO‑d6)
d: 3.50 (s,
2H, CH2), 3.66 (t, J ¼ 1.8 Hz, 2H, CH2), 5.00e5.14 (m, 2H, CH2),
5.76e5.82 (m, 1H, CH), 6.58 (s, 1H, NH), 7.23e7.32 (m, 2H, pyridine-
H), 7.88 (s, 1H, NH), 8.46e8.49 (m, 2H, pyridine-H), 9.85 (s, 1H, NH).
13C NMR (DMSO‑d6)
d: 23.47, 56.63, 114.93, 124.91, 136.62, 145.09,
149.98,158.54,169.58,174.66. IR: v ¼ 3152 (NH), 3015 (¼CeH),1697
(C]O), 1649 (C]C). LC-QTOF MS (m/z): calculated monoisotopic
mass: 234.1117, measured monoisotopic mass: 234.1120.
2.1.1.6. 4-Allyl-1-phenylacetylsemicarbazide
(2f). Yield:
d: 3.44 (s, 2H, CH2),
76%
Compounds 2a, 2b, 2e, 2f, 3a, 3b, 3e have been synthesized in
our laboratory and reported in literature [12,13].
(2.37 g); m.p. 117-119 ꢁC. 1H NMR (DMSO‑d6)
3.65 (t, J ¼ 1.8 Hz, 2H,CH2), 5.005.13 (m, 2H, CH2), 5.76e5.81 (m, 1H,
CH), 6.50 (s,1H, NH), 7.21e7.31 (m, 5H, phenyl-H), 7.82 (s, 1H, NH),
2.1.1. General procedure for the synthesis of 4-allyl-1-substituted
semicarbazide derivatives (2a-2h)
9.76 (s, 1H, NH). 13C NMR (DMSO‑d6)
d: 41.93, 42.06, 114.94, 126.71,
126.83, 128.50, 128.64, 129.41, 130.12, 136.25, 136.76, 170.52. IR:
v ¼ 3297 (NH), 3034 (¼CeH), 1692 (C]O), 1636 (C]C). LC-QTOF
MS (m/z): calculated monoisotopic mass: 233.1164, measured
monoisotopic mass: 233.1165.
A mixture of appropriate carboxylic acid hydrazide 1a-1h
(0.01 mol) and allylisocyanate (0.01 mol) in 15 ml diethyl ether was
kept for 48 h at room temperature. Obtained product was filtered
off and washed with diethyl ether and water. After drying semi-
carbazide derivatives were crystallized from ethanol.
2.1.1.7. 4-Allyl-1-(4-nitrophenylcarbonyl)semicarbazide
(2g).
: 3.68e3.71
Yield 76% (2.14 g); m.p.192-194 ꢁC. 1H NMR (DMSO‑d6)
d
2.1.1.1. 4-Allyl-1-(1-metyhylpyrrol-2-ylacetyl)semicarbazide
(2a).
(m, 2H, CH2), 4.99e5.21 (m, 2H, CH2), 5.77e5.89 (m,1H, CH), 6.88 (t,
1H, NH), 7.85 (d,1H, NH), 8.01e8.38 (m, 4H, phenyl-H), 10.51 (s, 1H,
Yield 88% (2.09 g), 83% [12]; m.p. 135e137 ꢁC. 1H NMR (DMSO‑d6)
d:

M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
3
NH). 13C NMR (DMSO‑d6)
d
: 41.99, 114.94, 123.97, 124.58, 129.64,
2.1.2.5. 4-allyl-3-pyridin-4-ylmethyl-1,2,4-triazoline-5-one
(3e).
136.33, 139.02, 149.77, 158.49, 165.35. IR: v ¼ 3281 (NH), 3012
(¼CeH), 1692 (C]O), 1640 (C]C). LC-QTOF MS (m/z): calculated
monoisotopic mass: 264.0859, measured monoisotopic mass:
264.0861.
Yield 80% (1.75 g), 74% [12]; m.p. 255e257 ꢁC. 1H NMR (DMSO‑d6)
d:
4.06 (s, 2H, CH2), 4.21e4.36 (m, 2H, CH2), 4.93e5.07 (m, 2H, CH2),
5.74e5.79 (m, 1H, CH), 8.00e8.04 (m, 2H, pyridine-H), 8.88e8.91
(m, 2H, pyridine-H), 11.82 (s, 1H, NH). 13C NMR (DMSO‑d6)
d: 31.15,
42.18, 116.80, 127.71, 132.63, 141.42, 144.33, 154.64, 155.61. IR:
v ¼ 3065 (NH), 2955 (¼CeH), 1701 (C]O), 1656 (C]C). LC-QTOF
MS (m/z): calculated monoisotopic mass: 216.1011, measured
monoisotopic mass: 216.1012.
2.1.1.8. 4-Allyl-1-(1-naphtylcarbonyl)semicarbazide (2h). Yield 78%
(2.11 g); m.p. 240-243 ꢁC. 1H NMR (DMSO‑d6)
d: 3.76 (t, J ¼ 1.8 Hz,
2H, CH2), 5.08e5.25 (m, 2H, CH2), 5.86e5.91 (m,1H, CH), 6.69 (s,1H,
NH), 7.57e8.12 (m, 7H, naphtyl-H), 8.39 (d,1H,NH),10.13 (s, 1H, NH).
13C NMR (DMSO‑d6)
d: 42.11, 115.01, 125.39, 126.12, 126.37, 127.24,
2.1.2.6. 4-allyl-3-benzyl-1,2,4-triazoline-5-one
(3f). Yield
d: 3.86e3.92 (m, 2H,
77%
(1.65 g); m.p. 105e107 ꢁC. 1H NMR (DMSO‑d6)
128.65,129.26,130.58,133.10,133.59, 136.64, 136.92,158.62,169.20.
IR: v ¼ 3233 (NH), 3013 (¼CeH), 1697 (C]O), 1637 (C]C). LC-QTOF
MS (m/z): calculated monoisotopic mass: 296.1164, measured
monoisotopic mass: 269.1167.
CH2), 4.06 (s, 2H, CH2), 4.88e5.05 (m, 2H,CH2), 5.63e5.67 (m, 1H,
CH), 7.22e7.34 (m, 5H, phenyl-H), 11.59 (s, 1H, NH). 13C NMR
(DMSO‑d6) d: 31.46, 42.12, 116.60, 126.92, 128.60, 128.71, 132.53,
135.24, 146.26, 154.84. IR: v ¼ 3155 (NH), 2994 (¼CeH), 1687 (C]
O), 1647 (C]C). LC-QTOF MS (m/z): calculated monoisotopic mass:
215.1059, measured monoisotopic mass: 215.1060.
2.1.2. General procedure for the synthesis of 4-allyl- 3-substituted
1,2,4-triazoline-5-one (3a-3h)
A mixture of semicarbazide (0.01 mol) and 50-60 ml aqueous
solution of sodium hydroxide (2%) was boiled for 10e15h. After
cooling, the solution was neutralized with dilute hydrochloric acid
(3 M). The precipitate was filtered off and then crystallized from
ethanol.
2.1.2.7. 4-allyl-3-(4-nitrophenyl)-1,2,4-triazoline-5-one
(3g).
Yield 76% (1.97 g); m.p. 192e194 ꢁC. 1H NMR (DMSO‑d6)
d:
4.20e4.26 (m, 2H, CH2), 4.93e5.07 (m, 2H, CH2), 5.73e5.80 (m, 1H,
CH), 7.95e7.97 (m, 2H, phenyl-H), 8.85e8.87 (m, 2H, phenyl-H),
11.78 (s, 1H, NH). 13C NMR (DMSO‑d6)
d: 31.14, 40.14, 42.18,
116.80, 127.64, 132.63, 141.61, 144.34, 154.64, 155.40. IR: v ¼ 3273
(NH), 2989 (¼CeH), 1668 (C]O), 1640 (C]C). LC-QTOF MS (m/z):
calculated monoisotopic mass: 246.0753, measured monoisotopic
mass: 246.0755.
2.1.2.1. 4-allyl-3-(1-methylpyrrol-2-yl)methyl-1,2,4-triazoline-5-one
(3a). Yield 79% (1.72 g), 76% [12]; m.p. 110e112 ꢁC. 1H NMR
(DMSO‑d6)
CH2), 5.10e5.12 (m, 2H, CH2), 5.69e5.76 (m, 1H, CH), 5.82e5.89 (m,
2H, pyrrole-H), 6.65e6.66 (m, 1H, pyrrole-H), 11.57 (s, 1H, NH). 13
NMR (DMSO‑d6) : 23.57, 33.44, 40.15, 42.05, 106.31, 107.87, 116.49,
d: 3.50 (s, 3H, CH3), 3.84 (s, 2H, CH2), 4.13e4.14 (m, 2H,
C
2.1.2.8. 4-allyl-3-naphtyl-1,2,4-triazoline-5-one (3h). Yield 79%
d
(2.09 g); m.p. 196e200 ꢁC. 1H NMR (DMSO‑d6)
d
: 3.66e3.68 (m, 2H,
CH2), 5.01e5.17 (m, 2H, CH2), 5.77e5.83 (m, 1H, CH), 8.03e8.35 (m,
7H, naphtyl-H), 10.45 (s, 1H, NH). 13C NMR (DMSO‑d6)
: 41.50,
122.46, 125.05, 132.83, 145.14, 154.80. IR: v ¼ 3333 (NH), 3100
(¼CeH), 1682 (C]O), 1551 (C]C). LC-QTOF MS (m/z): calculated
monoisotopic mass: 218.1168, measured monoisotopic mass:
218.1166.
d
114.44, 123.50, 124.11, 128.68, 129.16, 136.37, 138.51, 149.27, 158.00,
164.87. IR: v ¼ 3277 (NH), 3013 (¼CeH), 1668 (C]O), 1651 (C]C).
LC-QTOF MS (m/z): calculated monoisotopic mass: 251.1059,
measured monoisotopic mass: 251.1059.
2.1.2.2. 4-allyl-3-pyridin-2-yl-1,2,4-triazoline-5-one (3b). Yield 74%
(1.49 g), 68% [13]; m.p. 183-184 ꢁC. 1H NMR (DMSO‑d6)
d: 4.76e4.78
(m, 2H, CH2), 4.88e4.92 (m, 2H, CH2), 5.00e5.90 (m, 1H, CH),
7.47e7.49 (m, 1H, pyridine-H), 7.93e7.95 (m, 2H, pyridine-H),
8.64e8.65 (m, 1H, pyridine-H), 11.05 (s, 1H, NH). 13C NMR
2.2. Antimicrobial activity
The following bacterial strains were used in the study: Escher-
ichia coli ATCC 25992, Staphylococcus aureus sensitive to methicillin
MR3, Staphylococcus epidermidis ATCC 12228, Mycobacterium
smegmatis ATCC 20, and two strains donated by Scientific Research
Pulmonary Institute, Warsaw, Poland e Mycobacterium phlei and
Mycobacterium tuberculosis H37Ra. The well diffusion method was
employed to check the antibacterial activity of the tested sub-
stances. The 25 ml of Middlebrook 7H10 medium with ADC
enrichment (Becton-Dickinson) for Mycobacteria or 25 ml of
Muller-Hinton broth for other bacteria was poured into the sterile
Petri dishes diameter 100 mm and left to solidifying. The inoculum
was prepared by transferring single colonies from agar plates to
tubes with 4 ml of liquid medium. The obtained bacterial suspen-
sion was incubated for 24 h at 37 ꢁC. Afterwards, the bacterial
suspension was adjusted to a cell density corresponding to
1McFarland standard. In the case of mycobacteria, a small amount
of bacteria was scraped from the Lowenstein slants and transferred
to a liquid medium, followed by incubation at 37 ꢁC for about 10
days. After that time, the inoculum was suspended to a cell density
(DMSO‑d6) d: 44.13, 116.55, 122.10, 125.00, 124.12, 127.88, 144.47,
147.53, 149.36, 155.54. IR: v ¼ 3249 (NH), 3144 (¼CeH), 1707 (C]
O), 1653 (C]C). LC-QTOF MS (m/z): calculated monoisotopic mass:
202.0855, measured monoisotopic mass: 202.0856.
2.1.2.3. 4-allyl-3-pyridin-3-yl-1,2,4-triazoline-5-one (3c). Yield 77%
(1.55 g); m.p. 178e184 ꢁC; 1H NMR (DMSO‑d6)
d
: 3.68e3.88 (m, 2H,
CH2), 5.03e5.21 (m, 2H, CH2), 5.77e5.89 (m, 1H, CH), 6.83e8.38 (m,
3H, pyridine-H), 10.51 (s, 1H, NH).13C NMR (DMSO‑d6)
: 43.70,
d
114.90, 123.94, 129.60, 133.72, 136.81, 145.25, 148.57, 155.44, 165.32.
IR: v ¼ 3176 (NH), 3009 (¼CeH), 1692 (C]O), 1646 (C]C). LC-QTOF
MS (m/z): calculated monoisotopic mass: 202.0855, measured
monoisotopic mass: 202.0853.
2.1.2.4. 4-allyl-3-pyridin-2-ylmethyl-1,2,4-triazoline-5-one
(3d).
: 4.02 (s,
Yield 81% (1.75 g); m.p. 220e221 ꢁC. 1H NMR (DMSO‑d6)
d
2H, CH2), 4.13e4.14 (d, 2H, CH2), 4.86e5.04 (m, 2H, CH2), 5.66e5.72
(m, 1H, CH), 7.26e7.33 (m, 2H, pyridine-H), 7.74e7.77 (m, 1H,
pyridine-H), 8.48e8.49 (m, 1H, pyridine-H), 11.63 (s, 1H, NH).13
C
of 1 McFarland standard. Afterwards, a 200 ml of inoculum was
NMR (DMSO‑d6)
d
: 19.62, 56.46, 116.21, 122.72, 123.85, 133.10,
transferred to the plates and spread on the surface with a sterile
swab and next wells were cut using a sterile tip of 4 mm diameter.
The tested compounds were dissolved in DMSO to a concentration
137.43, 145.99, 149.66, 155.28, 155.94. IR: v ¼ 3168 (NH), 3068
(¼CeH), 1690 (C]O), 1646 (C]C). LC-QTOF MS (m/z): calculated
monoisotopic mass: 216.1011, measured monoisotopic mass:
216.1013.
of 5 mg/ml and aliquots containing 250
mg were poured into the
wells using a micropipette. The plates were incubated for 72 h at

4
M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
37 ꢁC for mycobacteria and 24h for other strains. Antibiotics
streptomycin and Amikacin were used as standards.
2.5. Molecular docking
Antibacterial activity of the compounds was examined in vitro
using the broth dilution method for determination of the Minimum
Inhibitory Concentration (MIC) to inhibit the growth of microor-
ganisms. The MIC was defined as the lowest concentration of the
compound which inhibited the growth of bacteria judged by lack of
turbidity in the vial. Streptomycin (Sigma, Chine) and amikacin
(Biodacine, Bioton, Poland) were used as the reference compounds,
for mycobacteria and other strains respectively. Stock solutions of
the substances with concentration of 20 mg/ml were prepared in
DMSO. The samples final concentration was in the range of
The crystal structure of Mycobacterium tuberculosis cytochrome
P450 CYP121 in complex with cyclodipeptide 3,6-bis[(4-
hydroxyphenyl)methyl]piperazine-2,5-dione (cYY) and molecule
of hem as a prostetic group obtained from Protein Data Bank (PDB
ID: 3G5H [23] was used in docking studies performed by the GOLD
Suite v.5.7.3 software [24]. The binding site of the native ligand cYY
in cytochrome P450 CYP121 was used as an active site for docked
molecule 3b after removing cYY from it. Six water molecules
(HOH685, HOH723, HOH728, HOH751, HOH810 and HOH944)
interacting with the ligand cYY in 3G5H were left at the active site
with the possibility of rotation and translation in the docking
procedure. The remaining water molecules were removed from the
protein structure. The docking back of cYY into binding site gave
the RMSD value of 0.227 Å for ChemPLP as scoring function
showing that the binding mode was successful. ChemPLP is
empirical fitness function optimized for pose prediction, which is
used to model the steric complementarity between protein and
ligand [24,25]. The selection of atoms in the active site within 6 Å of
original ligands was chosen and the docking simulation was
running with default parameter values of GOLD. Solutions and
protein-ligand interactions were analyzed using Hermes v.1.10.3
[24]. The initial molecular structure of 3b was obtained from DFT
calculation.
64e2048 m
g/mlꢀ1. Inocula dedicated for susceptibility testing were
prepared in the same manner as for well diffusion method.
Turbidity of the suspensions was evaluated using nephelometer BD
Phoenix Spec. Becton, Dickinson and Company USA. Then, 12 ml of
bacterial suspension was added to each tube containing the
appropriate dilution of analyzed substances. The vials were incu-
bated at 37 ꢁC. The observation was carried out for 48 h, and in the
case of mycobacteria
4 days for the fast growing strains
M. smegmatis and M. phlei and 10 days for M. tuberculosis H37Ra.
The control sample was a tube containing 2 ml of pure broth
inoculated with the same amount of bacteria and kept overnight at
4
ꢁC (as a standard to determine total inhibition). Control II - con-
tained the highest concentration of solvent used in the sample
(DMSO) in order to eliminate the effect of the lethal action of the
solvent. Control III
-
positive control of microbial growth
3. Results
(broth þ inoculate).
4-Allyl-1-substitued semicarbazide derivatives were synthe-
sized by the reaction of appropriate hydrazide with allyl isocyanate.
Hydrazides: 1-methylpyrrolacetic acid (1a) [26], 2-pyridine car-
boxylic acid (1b) [27], 2-pyridine acetic acid (1d) [28], 4-pyridine
acetic acid (1e) [29], phenyl acetic acid (1f) [30] and 4-
nitrophenyl carboxylic acid (1g) [31] were obtained by reacting
the corresponding ester with hydrazine hydrate. The nicotinic acid
hydrazide (1c) and 1-naphthalene carboxylic acid hydrazide (1h)
were purchased from the company. The semicarbazide 2d, 2g and
2h were new. Other compounds was obtained in our laboratory and
described earlier [12,32,33]. Next, semicarbazide derivatives (2a-
2h) were cyclized for the appropriate 1,2,4-triazole derivatives. The
reaction was carried out for 2 h under reflux in a 2% NaOH solution.
The cyclic products were precipitated using 3 M HCl (Scheme 1).
Compounds 3d, 3g, 3h are new, not described in the literature. We
have obtained known compounds with yield comparable or higher
than the literature data. The structure of the obtained compounds
was confirmed by spectral analysis, mass spectrometry and crys-
tallography analysis. Experimental data of all compounds are
described in Supplementary Material.
2.3. X-ray structure determinations
X-ray data of 2b and 2d were collected on the Bruker SMART
APEX II CCD diffractometer; CuK
T ¼ 296(2) K. X-ray data of 3b, 3c and 3e were collected on the
KUMA Diffraction KM-4 CCD diffractometer; MoK
¼ 0.71073 Å)
radiation,
scans, T ¼ 296(2) K. All structures were solved by direct
a
(l
¼ 1.54178 Å) radiation,
u scans,
a (l
u
methods using SHELXS97 [14] for 2b, 2d, 3b and 3c, and SIR92 [15]
for 3e and refined by full-matrix least-squares with SHELXL-2014/7
[14]. In all structures the H atoms were located by difference
Fourier synthesis and refined freely with isotropic displacement
parameters taken as 1.5 times those of the respective parent atoms.
All calculations were performed using WINGX version 1.64.05
package [16]. Crystal data, data collection and structure refinement
details are summarized in Table 1.
2.4. Theoretical calculations
Antimicrobial activity were performed for some 1,4-
disubstituted semicarbazide and corresponding 1,2,4-triazoline-5-
one derivatives with allyl group. All compounds were tested
in vitro against the strains of pathogenic bacteria: Escherichia coli,
Staphylococcus aureus, Staphylococcus aureus (sensitive to methi-
cillin MR3), Staphylococcus epidermidis, Mycobacterium smegmatis,
Mycobacterium phlei, Mycobacterium H37Ra. The study used the
methods of dilution and well diffusion. The MIC values (minimum
inhibitory concentration) and the zone of inhibition of microbial
growth were determined. The data obtained (Table 1) were
compared with the reference drugs (Streptomycin and Amikacin).
On the basis of the obtained results it can be concluded that the
four compounds exhibit antibacterial activity. Compound 2g
inhibited the growth of S. epidermidis only. A much wider spectrum
antibacterial activity was observed for the substance 3b. This
compounds inhibited growth all tested bacterial strains. Compound
3g exhibit antimicrobial activity against E. coli and S. epidermidis but
The energy, geometrical parameters (bond lengths, angles and
torsion angles) and electronic parameters (frontier orbitals, dipole
moments, NBO net charge distribution on the atoms and electro-
static potential distribution) for all structures 2ae2h and 3ae3h
were calculated with GAUSSIAN 03 [19] at the DFT/B3LYP level with
6e311þþG(d,p) basis set. The structures were fully optimized
without any symmetry constraints and the initial geometries were
built from the crystallographic data of 2b, 2d, 3b, 3c and 3e. Cal-
culations were carried out at the Academic Computer Centre in
Siedlce. The visualization of theoretical calculation results was
made using GaussView [20]. The partition coefficients, logP, were
calculated using QSAR properties procedure implemented in
HyperChem Professional package ver. 8.0.10 [21]. The logP value
was calculated by summing up all individual atom contributions to
lipophilicity of molecule [22].

M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
5
Table 1
Crystal data, data collection and structure refinement details.
Compound
2b
2d
3b
3c
3e
Chemical formula
Mr
C10H12N4O2
220.24
C11H14N4O2
234.26
C10H10N4O
202.22
C10H10N4O
202.22
C11H12N4O.HCl
252.70
Crystal system
Space group
Monoclinic
P2/n
Monoclinic
P21/c
Monoclinic
P21/n
Triclinic
Monoclinic
P21/n
P1
Temperature (K)
296(2)
296(2)
296(2)
296(2)
296(2)
a (Å)
b (Å)
c (Å)
a
b
g
12.5489(3)
4.5549(1)
19.6969(5)
94.763(9)
94.426(2)
90.753(10)
1122.50(5)
4
5.4629(1)
13.3161(3)
16.5566(3)
12.123(2)
5.6469(10)
14.894(3)
6.6765(9)
6.9666(7)
11.2627(12)
9.6110(11)
7.9508(9)
16.2977(19)
(ꢁ)
(ꢁ)
(ꢁ)
97.6(1)
100.112(19)
107.215(11)
97.550(11)
V (Å3)
Z
1192.75(4)
4
1.305
496
0.771
1003.7(3)
4
1.338
424
498.27(10)
2
1.348
212
1234.6(2)
4
1.360
528
Dcalc (Mg mꢀ3
F(000)
)
1.303
464
0.786
m
(mmꢀ1
)
0.093
0.093
0.299
Crystal size (mm)
Absorption correction
Tmin/Tmax
0.43 ꢂ 0.14 ꢂ 0.12
Multi-scan [17]
0.8220/0.9465
0.77 ꢂ 0.30 ꢂ 0.21
Multi-scan
0.5865/0.8537
0.60 ꢂ 0.40 ꢂ 0.20
Multi-scan [18] Multi-scan Multi-scan
0.8246/1.0000
0.60 ꢂ 0.40 ꢂ 0.40
0.40 ꢂ 0.30 ꢂ 0.30
6895/.0000
0.6440/1.0000
No. of reflections
measured
10984
9474
4067
3437
5105
independent
1942
2043
2308
2226
2821
observed [I > 2
s(I)]
1564
4.33e66.53
0.039
0.107
1.049
1959
5.40e66.52
0.036
0.102
1.046
2308
2.00e29.16
0.051
0.114
1.065
1584
2.94e28.87
0.053
0.144
1.029
2096
2.33e29.11
0.050
0.147
1.142
q
range (ꢁ)
R[F2 > 2 (F2)]
s
wR(F2)
GooF, S
Data/parameters
Extinction coefficient
Drmax
Drmin (eÅꢀ3
1942/182
0.0027(6)
þ0.164/-0.142
0.000
2043/200
0.0104(13)
þ0.112/-0.139
0.000
2308/167
0.25(4)
þ0.205/-0.232
0.000
2226/167
0.070(16)
þ0.229/-0.193
0.000
2821/193
None
/
)
þ0.368/-0.429
(D/s)
max
0.000
Scheme 1. Synthesis of 4-allyl-1-substituted semicarbazide (2a-2h) and 1,2,4-triazoline-5-one (3a-3h).
compound 3h inhibited growth E. coli. Only compound 3b showed
activity against Mycobacterium. Zones of inhibition for this de-
rivatives were higher or comparable to the standards (Amikacin or
Streptomycin). Of the substances tested, only substance 3b showed
against E. coli, S. aureus, S. MR3 and S. epidermidis exceeded the
value of 2 mg/ml, which allows to state that these strains are
insensitive to the test substances.
The crystal structure determinations of 2b, 2d, 3b, 3c and 3e
were undertaken in order confirm the synthesis pathway and
identification of their tautomeric forms in the crystalline state. The
anti-tuberculin activity (MIC ¼ 256
mg/ml) against all mycobacterial
strains tested. The determined MIC values for the test substances

6
M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
crystal and molecular structures of 4-allyl-5-benzyl-2,4-dihydro
[1,2,4]triazol-3-one, 3f, were reported earlier [33]. The compound
3e crystallizes as hydrochloride salt with one chloride anion and 4-
allyl-3-(4-pyridylmethyl)-1H-1,2,4-triazol-5-one cation protonated
at N54 pyridine atom. The structure and conformation of the
molecules 2b, 2d, 3b, 3c and 3e in the crystal are shown in Fig. 1.
In the crystalline state, the molecules 2b and 2d exist in N3eH/
N4eH/N6eH/O2/O5 amide tautomeric form, as evidenced by the
C2eO2 and C5eO5 bond lengths of 1.2205(19) and 1.2299(18) Å,
respectively, for 2b and 1.2150(15) and 1.2486(14) Å, respectively,
for 2d, typical for the carbonyl group and the positions of the H
atoms in the vicinity of all N atoms of carbonylsemicarbazide sys-
tem in the difference electron-density map. It should be noted, that
there are at least 10 possible tautomeric forms of the carbon-
ylsemicarbazide system [7] and we showed using theoretical cal-
culations at the ab initio/RHF/SCF/6-31G** level of theory, that the
tautomeric form observed for 2b and 2d in the crystalline state is
the most stable and only one existing for this system. The pyridine
ring in both investigated molecules is planar to within 0.004(2) Å in
2b and 0.0167(14) Å in 2d. The allylcarbonylsemicarbazide chain in
2b adopts a cis-trans-gauche-trans-trans-gauche-cis conformation
as is shown by the torsion angles N11eC12eC2eN3 ¼ ꢀ14.5(2)o,
C12eC1eC2eN3 ¼ ꢀ89.12(13)o, C1eC2eN3eN4 ¼ 174.24(10)o,
C2eN3eN4eC5 ¼ ꢀ78.07(15)o, N3eN4eC5eN6 ¼ ꢀ6.58(17)o,
N4eC5eN6eC7 ¼ ꢀ170.43(12)o, C5eN6eC7eC8 ¼ 92.02(17)o and
N6eC7eC8eC9 ¼ ꢀ1.4(4)o show, that the extended by one CH2
group allylsemicarbazide chain in 2d has a cis-gauche-trans-gauche-
cis-trans-gauche-cis conformation. This conformation is influenced
by intramolecular hydrogen bonds N6eH6‧‧‧N3 and N6eH6‧‧‧N11
(Fig. 1, Table 1S). The differences between conformations of mole-
cules 2b and 2d resulting from extension of allylsemicarbazide
chain are illustrated in Fig. 2a showing the overlay of both mole-
cules by fitting of urea systems.
In the central semicarbazide moiety the bond lengths C2eN3 of
1.346(2) Å in 2b and 1.3541(17) Å in 2d, N3eN4 of 1.3843(18) Å in
2b and 1.3869(13) Å in 2d, C5eN4 of 1.3739(19) Å in 2b and
1.3609(16) Å in 2d and C5eN6 of 1.3302(19) Å in 2b and 1.3337(16)
Å in 2d, which are intermediate between those for single and
double CeN and NeN bond lengths, are typical for a conjugated p-
electron system. The double bond length C8eC9 of 1.272(3) Å in 2b
and 1.282(3) Å in 2d is characteristic for the vinyl group (1.299(27)
Å) [34].
In the crystals, the molecules of the allyl[1,2,4]triazole de-
rivatives 3b, 3c and 3e, similarly as 3f, exist in N2eH/O31 amide
tautomeric form (Fig.1) as confirmed by the C3eO31 bond length of
1.237(2) Å for 3b, 1.226(2) Å for 3c and 1.232(3) Å for 3e typical for
the carbonyl group and the position of the H atoms in the vicinity of
N2 atom of the triazole ring. Theoretical calculations at the DFT/
B3LYP/6-311þþG(d,p) level show that this tautomeric form of
3ae3h is one existing form in the gaseous phase with a difference
in energy compared to the energy of an alternative N2/O31eH
iminol form in the range from 58.03 kJ/mol for 3a to 73.64 kJal/mol
for 3f (Table 3). It can be concluded that the population of the N2/
O31eH iminol tautomeric form in vacuum is below the threshold of
the detectability of conventional analytical methods and the
tautomeric equilibrium does not depend on the kind of substituent
at position 5 of 1,2,4-triazole ring. The molecules 3b, 3c and 3e
contain a 1,2,4-triazol-3-one system (closed semicarbazide system)
substituted by allyl group and different aromatic pyridin-2-yl in 3b,
pyridin-3-yl in 3c and pyridine-4-ylmethyl in 3e substituent in
positions 4 and 5 of 1,2,4-triazole ring, respectively. The triazole
and pyridine rings are planar respectively to within 0.0042(19) and
0.002(2) Å in 3b, 00.0072(17) and 0.009(3) Å in 3c and 0.011(3) and
0.004(3)Å in 3e. In 3b and 3c the nearly planar allyl substituent
adopts almost perpendicular gauche-cis conformation with respect
to the triazole ring with the torsion angles C3eN4eC41eC42 and
N4eC41eC42eC43 of 82.0(2) and 3.8(4)o in 3b and ꢀ90.8(2)
and ꢀ1.4(3)o in 3c. These torsion angles of ꢀ70.7(3) and 123.7(3)o,
respectively, in 3e show that the allyl substituent is not planar and
C12eC2eN3eN4
¼
ꢀ173.86(13)o, C2eN3eN4eC5
¼
69.6(2)o,
N3eN4eC5eN6 ¼ ꢀ166.72(13)o, N4eC5eN6eC7 ¼ ꢀ176.46(14)o,
C5eN6eC7eC8 ¼ 79.5(2)o and N6eC7eC8eC9 ¼ 3.7(4)o. This
conformation is stabilized by intramolecular hydrogen bond
N3eH3…N11 (Fig. 1; Table 1S (Supplementary materials)). The
torsion
angles
N11eC12eC1eC2
¼
6.68(16)o,
Fig. 2. Overlay of X-ray molecules by least-squares fitting of the atoms of (a) urea
systems of compounds 2b and 2d (RMS ¼ 0.0197 Å); (b) 1,2,4-triazole ring of molecules
3b, 3c and 3e (RMS ¼ 0.0092 Å).
Fig. 1. A view of the X-ray molecular structure of 2b, 2d, 3b, 3c and 3e with the atomic
labelling scheme (probability 30%).

M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
7
adopts the gauche-gauche conformation to the triazole ring. The
torsion angle N1eC5eC51eC56 of ꢀ4.3(3)o in 3b and ꢀ32.3(3)o in
3c shows that the pyridine substituent is nearly co-planar with
1,2,4-triazole ring in 3b, while it is slightly distorted from co-
planarity in 3c. In 3e the pyridine-4-ylmethyl substituent has a
cis-gauche conformation as is confirmed by the torsion angles
N1eC5eC51eC61 and C5eC51eC61eC62 of 1.9(4) and 87.5(3)o,
respectively. The comparing of conformations of molecules 3b, 3c
and 3e is presented in Fig. 2b showing the overlay of all analyzed
molecules by fitting of 1,2,4-triazole ring.
The molecular packing in the crystals of 2b, and 2d is influenced
by NeH‧‧‧X (X ¼ N, O) intermolecular hydrogen bonds involving
proton-donating amino groups and proton-accepting O-carbonyl,
and N-pyridine atoms characteristic for tautomeric forms existing
in the crystal state of investigated compounds (Fig. 1S and Table 1S)
[35e37]. This is confirmed by strong intermolecular hydrogen
bonds NeH‧‧‧O between the N and O atoms of the semicarbazide
chain in 2b and 2d and between the N and O atoms of the triazo-
line-5-one system in 3b and 3c with the short N‧‧‧O and H‧‧‧O dis-
tances, which are in the range from 2.768(2) Å in 3b to 2.9011(14) Å
in 2d and from 1.83(2) Å in 3b to 2.10(2) Å in 2b.
In the crystal structure of 2b molecules form double molecular
chains parallel to Y direction through intermolecular hydrogen
bond N3eH3‧‧‧N11 linking molecules related by 2 axis and bifur-
cated hydrogen bonds N4eH4‧‧‧O5 and N6eH6‧‧‧O5 involving
molecules related by b translation. In the crystal structure of 2d the
intermolecular N4eH4‧‧‧O5 hydrogen bond links inversion-related
molecules into molecular dimers. These dimers form double mo-
lecular chains parallel to a crystallographic axis via intermolecular
N3eH3‧‧‧O5 hydrogen bond. In the unit-cell packing of 3b, 3c and
3e the NeH‧‧‧Y (Y ¼ O, Cl) and CeH‧‧‧Z (Z ¼ O, N) intermolecular
hydrogen bonds are presented (Fig. 1S; Table 1S). In the crystal
structure of 3b molecules related by 21 axis are joined into mo-
lecular chains in b direction through N2eH2‧‧‧O31 hydrogen bonds.
In the crystal of 3c the net of the hydrogen bonds formed by dim-
mers of inversion-related molecules involved in the N2eH2‧‧‧O31,
C55eH55‧‧‧O31 and C54eH54‧‧‧N53 hydrogen bonds.
C5eN6eC7eC8 for 2b, 2c, 2g, 2h result from large rotation freedom
of allylsemicarbazde chain on its single bonds.
The calculated torsion angles describing the conformation of the
molecules 3ae3h are showed in Table 3S (Supplementary mate-
rials). It is worth to nothing that the conformation of the allile chain
in 3e obtained in the minimum of energy differs from that observed
in the crystal but it is very similar to those calculated for other
compounds in this series.
The NBO net charge on the atoms in the allylsemicarbazde part
of the molecules 2ae2h and allyl[1,2,4]triazolone system of the
molecules 3ae3h calculated at the DFT/B3LYP/6-311þþG(d,p) level
is presented in Table 4S. It can be seen that atomic charges are very
similar in all analyzed molecules and they are independent of the
presence of CH2 group and the type of substituent on the side of
carbonyl group in the allylsemicarbazde chain and the allyl[1,2,4]
triazole ring in 2ae2h and 3ae3h, respectively. Relatively large
negative charges are observed at heteroatoms, carbon atom of
methylene group in 2a, 2d, 2e and 2f and 3a, 3d, 3e and 3f, and
carbon atoms of the vinyl group, while large positive charges are
present at other carbon atoms, which leads to strong polarization of
certain bonds in allylsemicarbazde and allyl[1,2,4]triazole systems.
It should be added, that very similar results were obtained for some
semicarbazides, for which the results of theoretical calculations
were reported previously.
The dipole moments of molecules 2ae2h (Table 4; Fig. 2S) vary
from 2.034 D for 2b to 7.757 D for 2d and the vectors of dipole
moments have differentiated spatial orientation being roughly
perpendicular (2a, 2b, 2d, 2e, 2f and 2h) or parallel (2c and 2g) to
the allylsemicarbazide chain. The dipole moments of molecules
3ae3h (Table 4; Fig. 3S) are less varied than those observed for
2ae2h, having the maximum value of 4.704 D for 3b and minimum
value of 2.375 D for 3g. The vectors of dipole moments have similar
spatial orientation for 3ae3f and 3h and they are directed from the
1,2,4-triazol-5-one system to the aromatic substituent of the tri-
azole ring. The direction of the dipole moment vector for 3g is
opposite to that observed for other compounds in the series, which
results from the large negative net charge on the oxygen atoms of
the nitro group. It should be noted, that the cyclization of the
semicarbazide system in 2ae2h into the 1,2,4-triazolone system in
3ae3h does not change the qualitative charge distribution on the
atoms of this systems. Moreover, antibacterial active compounds 2g
has relatively large dipole moments with its vector directed along
extended allylsemicarbazide chain from the center of gravity of
negative charge localized within nitrophenyl substituent to the
center of gravity of positive charge localized within allyl group.
The Molecular Electrostatic Potential (MEP) is useful molecular
property to study the chemical reactivity of molecules and relation
between structure and biological activity. The MEP distribution in
the plane perpendicular to the direction defined by C]C double
bond in allyl group for molecules 2ae2h is shown in Fig. 3. Local
minima of MEP are mainly located near heteroatoms (large nega-
tive charge) as the possible reactive nucleophilic sites. For mole-
cules 2ae2f and 2h the additional two small minima of MEP are
observed on both sides of a vinyl bond, while for the molecule 2g
only one minimum occurs near this bond. This effect can be due to
strong electron-withdrawing properties of the nitro group in
phenyl substituent in 2g causing a movement of electrons from the
allyl group in the direction of phenyl ring probably through the
space. The MEP distribution in the plane of 1,2,4-triazole ring for
3ae3h is presented in Fig. 4. Two deep local minima of MEP near
the N and O atoms of the 1,2,4-triazol-5-one system are observed
for all investigated compounds. Additional local minimum occurs
in the vicinity of oxygen atoms of the nitro group in 3g, nitrogen
atoms of pyrrolyl in 3a and pyridyl substituent in 3b, 3c, 3d and 3e
and benzene and naphthalene rings in 3f and 3h, respectively.
The molecules of 3e form molecular chains along crystallo-
graphic direction via strong N2eH2‧‧‧Cl and N64eH64‧‧‧Cl
hydrogen bonds with Cl anion as an proton acceptor.
Theoretical calculations were aimed to determine structural and
electronic parameters of molecules 2a-2h and 3be3h and to try to
connect them with antibacterial activity of investigated com-
pounds. Octanol-water partition coefficient (logP) as an index of
lipophilicity was calculated for all investigated molecules (Table 4).
Its values show, that the compounds 2ae2f, 2h, 3ae3f and 3h
with logP in the range from 0.17 for 2a to 4.12 for 3h exhibit affinity
to organic phase, while the compounds 2g inhibiting the growth of
S. epidermidis with logP of ꢀ2.70 and 3g with antimicrobial activity
against E. coli and S. epidermidis with logP of ꢀ0.79, have hydro-
philic properties. This different behaviour of these two compounds
results from the presence of polar-hydrophilic NO2 nitro group in
their molecules.
View of the molecules 1ae8a and 1be8b in conformation ob-
tained after energy minimization and geometrical parameters
optimization at DFT/B3LYP/6-311þþG(d,p) level are shown in
Figs. 2S and 3S, respectively (Supplementary materials). The
calculated conformations of allylsemicarbazde chain in 2b, 2c, 2g,
2h and 2a, 2d, 2e, 2f very similar to those obtained from X-ray
analysis of 2b and 2d, respectively. The respective calculated torsion
angles compared with experimental values obtained using X-ray
analysis for 2b and 2d are presented in Table 2S (Supplementary
materials). The observed differences in calculated and experi-
mental values of torsion angles N11eC12eC1eC2 and
C2eN3eC4eC5 for 2a, 2d, 2e, 2f and C2eN3eC4eC5 and

8
M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
Fig. 3. The contour maps of molecular electrostatic potential for 2ae2h in the plane perpendicular to the C]C double bond in the allyl group; the red lines represent regions with
the negative electrostatic potential and yellow lines represent regions with the positive electrostatic potential.
In order to explain the reactivity differences and antibacterial
activity of investigated compounds the frontier orbitals, as the
reactivity descriptors for molecules 2ae2h and 3ae3h were
calculated. The energies of HOMO, LUMO and their orbital energy
gaps for all investigated compounds calculated at DFT/B3LYP/6-
311þþG(d,p) level are given in Table 4. It can be seen, that the
HOMO-LUMO energy gap of 354.626 kJ/mol for 2g and 342.234 kJ/
mol for 3g is significantly smaller than the energy gaps for other
investigated molecules being in the range from 104.360 kcal/mol
for 2h to 136.871 kcal/mol for 2f and from 415.643 kJ/mol in 3h to
549.228 kJ/mol in 3f. This is due to relatively low energy level of
LUMO orbital for 2g and 3g with the nitrophenyl substituent in
comparison to energy of this orbital in remaining molecules.
Low energy of LUMO orbital and small HOMO-LUMO energy gap
for 2g and 3g indicate a high reactivity of these compounds and
those may be one of the reason of its antibacterial activity. The plots
of HOMO and LUMO orbitals are shown in Figs. 4S and 5S
(Supplementary materials) for 2ae2h and 3ae3h, respectively.
For all molecules 2ae8h the LUMO orbital is localized mainly on the
aromatic substituent of allylsemicarbazide system, while the
localization of HOMO orbital depends on the kind of this substit-
uent. For 2b and 2c HOMO is concentrated on the whole molecule,
for 2a and 2h, similarly as LUMO, the HOMO is localized on aromatic
substituent and for 2de2g this frontal orbital extends on allylace-
tylsemicarbazide system.
More differentiated distribution of the frontier orbitals is
observed for molecules 3ae3h. The LUMO orbital is concentrated
on aromatic substituent of 1,2,4-triazole ring and 1,2,4-triazole ring
and its aromatic substituent in 3de3f and 3b, 3c, 3g and 3h,
respectively, while for 3a this orbital is distributed on allyl group.
The HOMO orbital has very similar distribution in the same groups
of compounds, as LUMO orbital, being localized at 1,2,4-triazole
ring and its aromatic substituent in 3b, 3c, 3g and 3h, 1,2,4-
triazole ring and partly at allyl group in 3de3f and aromatic

M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
9
Table 2
Table 4
Antimicrobial activity of semicarbazide and 1,2,4-triazol-5-one derivatives defined
by dilution method and microdilution method.
The total energies, E (a.u.), energies of HOMO and LUMO orbitals, EHOMO and ELUMO
(kJ/mol.),
Egap ¼ ELUMOeEHOMO (kJ/mol.), dipole moments, Dm (D), calculated at DFT/
6-311þþG(d,p) level and calculated logP for 2ae2h and 3ae3h.
D
Diameter of the growth inhibition zone (mm)
No
E
EHOMO
ELUMO
D
Egap
Dm
logP
E.c.
S.a.
S.M.
S.e.
M.s.
M.p.
M.H.
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
3e
3f
ꢀ798.46588
ꢀ757.94419
ꢀ757.93675
ꢀ797.27190
ꢀ797.26483
ꢀ781.22595
ꢀ946.46064
ꢀ895.57110
ꢀ721.99743
ꢀ681.48077
ꢀ681.47525
ꢀ720.80214
ꢀ720.79916
ꢀ704.75867
ꢀ869.99806
ꢀ819.10963
ꢀ606.122
ꢀ676.145
ꢀ672.469
ꢀ664.960
ꢀ695.442
ꢀ684.362
ꢀ663.752
ꢀ605.781
ꢀ560.964
ꢀ607.383
ꢀ622.269
ꢀ611.006
ꢀ630.120
ꢀ612.555
ꢀ650.231
ꢀ592.864
ꢀ65.454
540.668
496.535
503.939
500.315
526.570
572.674
354.626
436.647
502.625
431.685
453.187
498.451
514.178
549.228
342.234
415.643
5.948
2.034
2.515
7.757
4.389
6.393
6.463
2.275
3.040
4.704
2.589
4.114
3.319
3.343
2.375
3.600
0.17
0.77
0.83
0.70
0.77
1.15
ꢀ2.70
2.22
2.07
2.67
2.74
2.60
2.67
3.05
ꢀ0.79
4.12
2g
3b
3g
3h
e
e
e
22
17
31
e
e
e
e
ꢀ179.610
ꢀ168.530
ꢀ164.645
ꢀ168.872
ꢀ111.688
ꢀ309.126
ꢀ169.134
ꢀ58.339
15
11
9
35
e
14
e
15
e
21
e
21
e
22
e
e
e
e
e
e
Amikacin
Streptomycin
34
e
34
e
40
e
e
e
e
24
23
25
MIC (mM)
2g
e
e
e
2048
7692
256
1266
2048
8317
e
e
e
e
ꢀ175.698
ꢀ169.082
ꢀ112.555
ꢀ115.942
ꢀ63.327
3b
3g
3h
256
1266
2048
8317
2048
8150
<2
2048
10128
e
2048
10128
e
256
1266
e
256
256
1266
e
1266
e
3g
3h
ꢀ307.997
ꢀ177.221
e
e
e
e
e
e
e
e
Amikacin
<2
<4
e
<4
<7
e
<2
<4
e
<4
e
molecular docking crystallizes as a complex with cyclodipeptide
cYY and molecule of hem as a prostetic group Fig. 6S (Supple-
mentary materials). The molecular docking study performed for 3b
showed that this ligand bind to the active site of the cytochrome
P450 interacting through a water molecule using intermolecular
hydrogen bonds O‧‧‧HeOeH‧‧‧N and O‧‧‧HeOeH‧‧‧O connecting a
carbonyl oxygen atom of a 1,2,4-triazole ring with the amino acid
residue of Met86A and molecule of hem, respectively. A view of 3b
in the binding site of cytochrome P450 is showed in Fig. 5.
Streptomycin
64
110
128
220
128
220
E.c. - Escherichia coli; S.a.- Staphylococcus aureus; S.M. - Staphylococcus aureus
(sensitive to methicillin MR3); S.e.-Staphylococcus epidermidis; M.s-Mycobacte-
rium smegmatis; M.p.-Mycobacterium phlei; M.H.- Mycobacterium H37Ra.
Table 3
The total energies of amide N2eH/O31 (A) and iminol N2/O31eH (B) tautomeric
forms, EA/EB (a.u.), dipole moments, Dm (D), and
DFT/6-311þþG(d,p) level for 3ae3h.
D
E ¼ EB-EA (kJ/mol) calculated at
It should be noted, that the difference in the conformation of 3b
in active site of cytochrome 450 and crystalline state is observed,
which is connected with high flexibility of the allyl chain Fig. 6.
No
Tautomeric form
Dm [D]
EA/EB [a.u.]
D
E ¼ EB-EA [kJ/mol]
3a
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
3,0401
2,6811
4,7045
3,3102
2,5892
3,1792
4,1141
4,1979
3,3187
6,7971
3,3428
4,6269
2,3748
6,7949
3,5999
4,4713
ꢀ721,99742628
ꢀ721,97532000
ꢀ681,48076645
ꢀ681,45684486
ꢀ681,47525810
ꢀ681,45005449
ꢀ720,80214548
ꢀ720,77800713
ꢀ720,79915775
ꢀ720,77158151
ꢀ704,75867421
ꢀ704,73063225
ꢀ869,99806476
ꢀ869,97316438
ꢀ819,10969332
ꢀ819,08439309
58.03
62.80
66.19
63.39
72.38
73.64
65.40
66.44
4. Conclusions
3b
3c
3d
3e
3f
The 4-allyl semicarbazide derivatives 2a-2h and the product of
their cyclization 3a-3h were synthesized. All compounds were
tested in vitro for their antimicrobial activity. The most active was
compound 3b, for which the growth inhibition zone against
M. smegmatis M. phlei and M.H37Ra was comparable with the
standard Streptomycin. The antimicrobial activity also showed 2g
against S. epidermidis, 3g against E. coli and S. epidermidis and 3h
against E. coli. The crystal structure determinations of 2b, 2d, 3b, 3c
and 3e were undertaken in order to confirm the synthesis pathway
and identification of their tautomeric forms in the crystalline state.
The X-ray analysis exhibited that 2b and 2d and 3b, 3c and 3e exist
in N3eH/N4eH/N6eH/O2/O5 and N2eH/O31 amide tautomeric
forms, respectively, and theoretical calculations confirmed that
these tautomeric forms are the most stable and one existing in the
gaseous phase. The conformation and molecular packing in the
crystal of the molecules are strongly influenced by NeH‧‧‧X (X ¼ N,
O) intra- and intermolecular hydrogen bonds closely related to
tautomeric form observed in crystalline state. Theoretical calcula-
tions showed that the physico-chemicals (logP) and electronic
properties (MEP distribution, energy localization of HOMO and
LUMO orbitals) can be related to observed antimicrobial activity of
investigated compounds, especially for 2g and 3g with the nitro
group in para position of aromatic substituent of semicarbazide
chain and 1,2,4-triazol-5-one system, respectively. The molecular
docking study carried out for the most active against Mycobacte-
rium tuberculosis compound 3b using the Mycobacterium tubercu-
losis cytochrome P450 CYP121 as molecular target showed that this
compound binds to the active site of P450 by hydrogen bonds via
water molecule with the amino acid residue of Met86A and
molecule of hem.
3g
3h
substituent in 3a. It can be concluded, that the energy and distri-
bution of the frontier orbitals is strongly influenced by kind of ar-
omatic substituent of semicarbazide chain or 1,2,4-triazole ring for
both series 2ae2h and 3ae3h of investigated compounds.
Theoretical calculations showed that the compounds 2g and 3g
with the nitro group in para position of aromatic substituent have
slightly different physico-chemicals and electronic properties
compared to other analyzed compounds. Visible differences in
bioavailability, stability and reactivity of these two compounds can
be related to their observed antibacterial activity.
The most active against Mycobacterium tuberculosis compound
3b was tested in silico using molecular docking procedure to the
Mycobacterium tuberculosis cytochrome P450 CYP121. An enzyme
P450 CYP121 is of particular interest as a molecular target in a novel
antibiotic strategy, because the triazole drugs as fungal antibiotics
(e.g. fluconazole) and possessing potent antimycobacterial activity
are its inhibitors [38]. The cytochrome P450 CYP121 used in

10
M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
Fig. 4. The contour maps of molecular electrostatic potential for 3ae3h in the plane of 1,2,4-triazol-5-one system; the red lines represent regions with the negative electrostatic
potential and yellow lines represent regions with the positive electrostatic potential.
Declaration of competing interest
Drozd: Methodology. Waldemar Wysocki: Investigation Visuali-
zation. Grazyna Ginalska: Writing - review & editing. Zofia
Urbanczyk-Lipkowska: Project of X-ray experiment. Dorota
Kowalczuk: Validation. Maja Morawiak: Performed X-ray
experiments.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Credit authorship statement
Declaration of competing interest
Monika Pitucha: Conceptualization, Supervision. Zbigniew
Karczmarzyk: Conceptualization of the structural part Software.
Marta Swatko-Ossor: Formal analysis, Methodology. Monika
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

M. Pitucha et al. / Journal of Molecular Structure 1219 (2020) 128552
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Acknowledgements
The results of the research carried out in part under the research
theme No. 9/S/91 were financed from the science grant granted by
the Ministry of Science and Higher Education. The authors thank
prof. Emilia Fornal (Department of Pathophysiology, Medical Uni-
versity of Lublin, Poland) for LC/QTOF data acquisition and analysis.
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