Synthesis and microcalorimetric determination of the bioactivities of a new Schiff base and its bismuth(III) complex derived from o-vanillin and 2,6-pyridinediamine

Article abstract of DOI:10.1007/s10973-016-5892-x

In this work, a new Schiff base [N,N′-bis(2-hydroxy-3-methoxyphenylmethylidene)-2,6-pyridinediamine, C₂₁H₁₉N₃O₄] and its bismuth(III) complex [Bi(C₂₁H₁₇N₃O₄)]Cl·2H₂O were synthesized with o-vanillin and 2,6-pyridinediamine in ethanol and THF solvent, respectively. The compositions and structures of the two synthetic compounds were characterized by elemental analysis, chemical analysis, spectrum analysis (including MS, FT-IR, NMR, and UV–Vis), and thermogravimetric analysis. The thermogenic curves for the growth metabolism of Helicobacter pylori (H. pylori) and Schizosaccharomyces pombe (S. pombe) treated by different concentrations of the two synthetic compounds were determined by isothermal heat conduction microcalorimetry at 37.00 and 32.00?°C, respectively. Based on the thermogenic curves, some important thermokinetic parameters including the microbial growth rate constant (k), inhibition ratio (I), and half inhibition concentration (IC₅₀) were calculated. The obtained results revealed that the Schiff base stimulated the growth of H. pylori, while the complex inhibited its growth. By contrast, both the Schiff base and the complex inhibited the growth of S. pombe, but the inhibitory effect of the complex was stronger than that of the Schiff base.

Full text of DOI:10.1007/s10973-016-5892-x

J Therm Anal Calorim
DOI 10.1007/s10973-016-5892-x
Synthesis and microcalorimetric determination of the bioactivities
of a new Schiff base and its bismuth(III) complex derived
from o-vanillin and 2,6-pyridinediamine
Xu Li1 Chuan-Hua Li1 Jian-Hong Jiang1 Hui-Wen Gu2 De-Liang Wei1
•
•
•
•
•
Li-Juan Ye1 Ji-Lin Hu1 Sheng-Xiong Xiao1 Dong-Cai Guo3 Xia Li1
Hui Zhang1 Qiang-Guo Li1
•
•
•
•
•
•
Received: 28 June 2016 / Accepted: 1 October 2016
´
´
Ó Akademiai Kiado, Budapest, Hungary 2016
Abstract In this work, a new Schiff base [N,N0-bis(2-hy-
droxy-3-methoxyphenylmethylidene)-2,6-pyridinediamine,
C21H19N3O4] and its bismuth(III) complex [Bi(C21H17N3-
O4)]ClÁ2H2O were synthesized with o-vanillin and 2,6-
pyridinediamine in ethanol and THF solvent, respectively.
The compositions and structures of the two synthetic
compounds were characterized by elemental analysis,
chemical analysis, spectrum analysis (including MS, FT-
IR, NMR, and UV–Vis), and thermogravimetric analysis.
The thermogenic curves for the growth metabolism of
Helicobacter pylori (H. pylori) and Schizosaccharomyces
pombe (S. pombe) treated by different concentrations of the
two synthetic compounds were determined by isothermal
heat conduction microcalorimetry at 37.00 and 32.00 °C,
respectively. Based on the thermogenic curves, some
important thermokinetic parameters including the micro-
bial growth rate constant (k), inhibition ratio (I), and half
inhibition concentration (IC50) were calculated. The
obtained results revealed that the Schiff base stimulated the
growth of H. pylori, while the complex inhibited its
growth. By contrast, both the Schiff base and the complex
inhibited the growth of S. pombe, but the inhibitory effect
of the complex was stronger than that of the Schiff base.
Keywords Microcalorimetry Á Bioactivities Á
Thermokinetics Á Schiff base bismuth(III) complex Á
H. pylori Á S. pombe
Introduction
Electronic supplementary material The online version of this
article (doi:10.1007/s10973-016-5892-x) contains supplementary
material, which is available to authorized users.
Microcalorimetry provides a general analytical tool for the
characterization of the microbial growth process. It has
been extensively used to investigate drug and the microbial
cell interaction and has furnished much useful information
[1–6]. One of the most prominent features of the microbial
growth process is the production of heat. If the heat is
monitored by a microcalorimeter, much useful information
(both qualitative and quantitative) may be obtained. Each
type of microbial has a unique thermogenic curve, as
recorded by the microcalorimeter, under a defined set of
growth conditions. Any chemicals that can modify the
metabolic growth processes involved in cell will change
the thermogenic curve obtained from the microcalorimeter.
Based on the thermogenic curves, not only thermodynamic
data but also thermokinetic data can be obtained. Many
studies have demonstrated that the test results obtained
from microcalorimetry are in agreement with those of
conventional bioassay methods such as the agar cup
& Hui-Wen Gu
gugo@yangtzeu.edu.cn
& Qiang-Guo Li
liqiangguo@163.com
1
Hunan Provincial Key Laboratory of Xiangnan Rare-Precious
Metals Compounds and Applications, College of Chemistry
Biology and Environmental Engineering, Xiangnan
University, Chenzhou 423043, Hunan Province, People’s
Republic of China
2
College of Chemistry and Environmental Engineering,
Yangtze University, Jingzhou 434023, Hubei Province,
People’s Republic of China
3
State Key Laboratory of Chemo/Biosensing and
Chemometrics, College of Chemistry and Chemical
Engineering, Hunan University,
Changsha 410082, Hunan Province, People’s Republic of
China
123

X. Li et al.
method, the cylinder diffusion method, the paper disk
diffusion assay, and the broth microdilution method [7, 8].
Owing to its good sensitivity, accuracy, and reproducibil-
ity, microcalorimetry is being accepted by more and more
researchers, and has been gradually used in new drug dis-
covery [9, 10].
and broad application prospects. In recent years,
diaminopyridine-based Valen Schiff bases and their coor-
dination compounds containing 3d and 4f block metal ions
have been widely studied [39–41], However, Valen Schiff
bases coordination compounds containing main group
metal elements, especially bismuth element, are still less
investigated [41].
Valen Schiff bases are a type of compounds derived
from (modified) o-vanillin and various amines via the
condensation reaction. At present, the studies of Valen
Schiff bases and their metal coordination compounds
mainly focused on their functions as catalysts, antioxidants,
and luminescent materials, while their antibacterial activ-
ities have been less investigated. In fact, on the one hand,
the antibacterial activity of o-vanillin is better than that of
salicylaldehyde [11, 12]. What’s more, the antibacterial
activities of some Valen Schiff bases are also superior to
those of salicylaldehyde Schiff bases [13, 14]. On the other
hand, Valen Schiff bases exhibit very strong coordination
abilities due to the existence of ‘‘O’’ and ‘‘N’’ atoms in
their molecules [15]. Accordingly, more species of metal
coordination compounds with plentiful structures can be
synthesized from the Valen Schiff bases. In our previous
works, some new Valen Schiff bases and their metal
coordination compounds were designed and synthesized
[15–22]. Their bioactivities on living cells were studied by
microcalorimetry [19–22]. The preliminary results indi-
cated that these synthetic compounds really possessed good
bioactivities [19–22].
In this work, a new Schiff base [N,N0-bis(2-hydroxy-3-
methoxyphenylmethylidene)-2,6-pyridinediamine, C21H19
N3O4] and its bismuth(III) complex [Bi(C21H17N3O4)]-
ClÁ2H2O were synthesized via the condensation reaction of
2,6-pyridinediamine and o-vanillin in a 1:2 molar ratio. Ele-
mental analysis, chemical analysis, spectrum analysis (in-
cluding MS, FT-IR, NMR, and UV–Vis), and ther-
mogravimetric analysis were employed to characterize their
compositionsand structures. Then, H. pyloriandS. pombe cell
lines were chosen as ideal biological models to assess the
bioactivities of the two synthetic compounds by isothermal
heat conduction microcalorimetry. As a basic research, the
aim of this study was to provide some useful thermokinetic
data for the further development and application of Valen
Schiff bases and their bismuth(III) complexes.
Experimental
Chemicals and reagents
Bismuth is the heaviest stable element locating at the
junction of metal and nonmetal elements in the periodic
table, which has a ground-state electron configuration [Xe]
4f145d106s26p3. It exhibits the characteristics of non-toxi-
city and low radioactivity and is recognized as the only
green heavy metal in nature [23]. Although China abounds
in bismuth resources, the development of bismuth-refined
products is facing a huge challenge [24–26]. Owing to
large ionic radius and strong deformability, bismuth(III)
could overcome steric hindrance and accept multiple pair
of electrons, forming compounds with different coordina-
tion numbers. The coordination number of bismuth(III)
could be up to 10. At present, the catalytic and biological
medicinal properties of bismuth(III) complexes have
attracted more and more attention [27–32]. Various bis-
muth(III) compounds have been used for medicinal pur-
poses for more than 200 years, which have a significant
effect on the treatment of a variety of microbial infections
such as syphilis, diarrhea, gastritis colitis, ulcer, and H.
pylori [33, 34]. Meanwhile, some bismuth(III) compounds
with good bioactivities can effectively inhibit the growth
and reproduction of cancer cells [35–38]. From the stand-
point of ‘‘Green Chemistry’’ and ‘‘Sustainable Develop-
ment’’, design and synthesis of new bismuth(III)-
containing compounds will have important scientific values
o-Vanillin (C8H8O3, 99.0 %), 2,6-pyridinediamine
(C5H7N3, 99.0 %), tetrahydrofuran (THF, 99.0 %), and
ethanol (EtOH, 99.5 %) were purchased from the Sino-
pharm Chemical Reagent Co., Ltd. (Shanghai, China).
Dimethyl sulfoxide (DMSO, [99.5 %) and N,N-dimethyl
formamide (DMF, [99.5 %) were bought from Tianjin
Guangfu Chemical Research Institutes (Tianjin, China).
Bismuth chloride (BiCl3, 99 %) was obtained from Tianjin
Jingke Chemical Research Institutes (Tianjin, China). All
other chemicals including sodium chloride (NaCl, 99.5 %),
silver nitrate (AgNO3, 99.8 %), and EDTA (99.0 %) were
purchased from the Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). The water used was double-distilled.
All chemicals and reagents were used as received without
further purification.
Physical measurements
The elemental analysis of C, H, and N was carried out on a
PerkinElmer 2400 elemental analyzer. The contents of
Bi3? and Cl- in Schiff base bismuth(III) complex were
determined by the EDTA titration and Mohr method,
respectively. Mass spectra were determined using a
Thermo Finnigan MAT95XP high-resolution mass spec-
trometer with methanol as solvent. FT-IR spectra
123

Synthesis and microcalorimetric determination of the bioactivities of a new Schiff base and…
(400–4000 cm-1) were measured using a Thermo Nicolet
Avatar 360 Fourier transform IR spectrometer with a KBr
pellet. 1H and 13C NMR spectra were recorded on a Bruker
400 MHz advance nuclear magnetic resonance spectrom-
eter with DMSO-d6 as solvent and trimethyl silicane
(TMS) as internal standard. All absorption spectra were
measured by a Hitachi U-3010 UV–Vis spectrophotometer.
The crystallization water content of the complex was
determined by a Shimadzu DTG-60 thermogravimetric/
differential thermal analyzer (TG/DTA) in flowing air with
a heating rate of 10 °C min-1 between 20 and 1200 °C.
The molar conductance was determined by a DDS-12DW
conductivity meter (Shanghai Lida Instrument Factory,
China). Microcalorimetric measurements were taken on a
3116-2/3239 TAM Air isothermal heat conduction
microcalorimeter (Thermometric AB, Sweden), which was
equipped with eight twin calorimetric channels with one
side for the samples and the other for a static reference. The
principles and structures of the microcalorimeter have been
described at length in our previous work [42].
green to yellow, and finally became red. The reaction was
stirred for 6 h and then allowed to settle overnight,
resulting in plenty of nacarat precipitates. Subsequently,
the precipitates were filtrated and recrystallized with
methanol–methylene chloride mixed solution (1:1, v/v).
The product was dried under vacuum at 50 °C until the
mass of the crystals was constant, with a yield of 70 %.
The synthetic scheme of the Schiff base is outlined in
Fig. 1.
Synthesis of the Schiff base bismuth(III) complex
A 50-mL round-bottom flask was used as the reactor.
Firstly, 0.5 mmol (0.0189 g) Schiff base was dissolved in a
50-mL round-bottom flask with 20 mL THF, then the
solution was heated to keep a constant temperature of
55 °C, and stirred using a magnetic stirrer until the solid
was dissolved completely. Subsequently, 0.5 mmol
(0.0158 g) BiCl3 was dissolved with 5 mL THF and added
dropwise to THF solution of the Schiff base. The mixture
was heated with stirring and refluxing. As the reaction
progressed, plenty of brown precipitates were generated.
After continuously reacted for 3 h, the resulting mixtures
were transferred to a clean beaker to settle and cool. The
precipitates were filtrated and washed with hot methanol
for several times. The filter cake was dried, and finally,
dark red powdered solid bismuth(III) complex was
obtained, with a yield of 68 %. The synthetic scheme of the
Schiff base bismuth(III) complex is outlined in Fig. 2.
Cell lines and culture conditions
H. pylori (ATCC43504) was provided by Shanghai Beinuo
Biological Technology Co., Ltd. The compositionofH. pylori
culture medium (HB8647) was 10.0 g proteose peptone,
10.0 g beef brain infusion, 9.0 g beef heart infusion, 5.0 g
NaCl, 2.0 g glucose, 2.5 g Na2HPO4, pH = 7.4 0.2, at
25 °C. 3.85 g HB8647 was dissolved in 93 mL distilled
water, heated, and stirred until dissolution. Then, the resulting
solution was sterilized under high pressure at 121 °C for
30 min. The stock solution of HB8647-HP was prepared by
adding 7 mL sterile defibrinated sheep blood and 1 HP bac-
teriostatic agent to the above HB8647 solution.
Measurement of thermogenic curves of H. pylori
and S. pombe cells treated by the Schiff base and its
bismuth(III) complex
S. pombe (ACCC20047) was provided by the Agricul-
tural Culture Collection of China. It was grown in the
Edinburgh minimal medium (EMM). 1 L of EEM (natural
pH) contained 3 g K2HPO4, 2.2 g Na2HPO4, 5 g NH4Cl,
20 g glucose, 20 mL inorganic salt, 1.0 mL vitamin, and
0.1 mL minerals. Before use, the EEM medium was ster-
ilized under high pressure at 121 °C for 30 min.
The thermogenic curves for the growth metabolism of H. pylori
and S. pombe cells treated by different concentrations of the
Schiff base and its bismuth(III) complex were determined by a
3116-2/3239 TAM Air isothermal heat conduction
microcalorimeter, respectively. The optimum temperature of
growth metabolism of H. pylori and S. pombe is 37.00 and
32.00 °C, respectively. Steps of the determination were sum-
marized as follows: The microcalorimeter was adjusted to
37.00 °C (or 32.00 °C), and the measurement was taken with
the ampoule method. Baselines were taken before each mea-
surement, and the microcalorimeter was calibrated electrically.
When the system had gained a stable baseline, 5 mL HB8647-
HP (or EMM-sterilized) culture medium was added to the
sterilized sample ampoules. H. pylori (or S. pombe) cell lines
Synthesis of the Schiff base
The Schiff base, N,N0-bis(2-hydroxy-3-methoxyphenyl-
methylidene)-2,6-pyridinediamine, was prepared according
to the literature [41, 43]. The synthetic process can be
briefly summarized as follows: 1 mmol (0.1091 g) 2,6-
pyridinediamine and 2 mmol (0.3043 g) o-vanillin were
dissolved in 15 mL ethanol, respectively. Then, the ethanol
solution of 2,6-pyridinediamine was added dropwise to the
ethanol solution of o-vanillin at 55 °C. As the reaction
progressed, the color of the mixture changed from light
were inoculated with an initial density of 1 9 106 cells mL-1
.
The compounds at different concentrations were added to the
cell suspension, respectively. All the ampoules containing the
cell suspension of H. pylori (or S. pombe) were sealed up and
put in 8-channel calorimeter block. The data were collected
123

X. Li et al.
Fig. 1 Synthetic scheme of the
Schiff base
CHO
OH
N
N
N
Ethanol, 55 °C
2
–2H2O
OH
HO
N
NH2
NH2
O
O
O
N
N
N
N
N
N
THF
Bi
2HCl
2H2O
Cl
BiCl3
+2H2O
O
O
OH
HO
O
O
O
O
Fig. 2 Synthetic scheme of the Schiff base bismuth(III) complex
1
continuously by the dedicated software package. All the
microcalorimetric experiments were repeated three times, and
the obtained results were identical.
mass spectra, FT-IR, H and 13C NMR spectra, UV–Vis
spectra, and TG–DTA analysis. The corresponding data for
the Schiff base and its bismuth(III) complex are summa-
rized in Tables S1–S6 in the Supplementary Materials,
respectively.
Results and discussion
It can be seen from Table S1 that the elemental and
chemical analysis data are in a good accordance with their
theoretical values. Based on these results, we can deduce
the compositions of the Schiff base and its bismuth(III)
complex were C21H19N3O4 (abbreviated as H2L,
L = C21H17N3O4) and [Bi(C21H17N3O4)]ClÁ2H2O (abbre-
viated as [Bi(L)]Cl Á 2H2O), respectively, which will be
further characterized by MS, FI-IR, NMR, UV–Vis, and
TG–DTA analysis.
Characterization of the Schiff base and its
bismuth(III) complex
The Schiff base was a nacarat powder with a melting point of
219 1 °C. It is very stable in the atmosphere. The powder
can be easily soluble in some organic solvents such as
dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), N,N-
dimethyl formamide (DMF), and acetone, but insoluble in
water, methanol, dichloromethane, trichloromethane, n-
hexane, benzene, toluene, and ethyl acetate. The molar
conductance of Schiff base in DMSO at 25 °C was deter-
mined to be 1.29 S cm2 mol-1, indicating that the Schiff
base is a nonelectrolyte and exists as a neutral molecule in
DMSO solvent.
The results of mass spectra of the Schiff base and its
bismuth(III) complex (see Table S1) demonstrated that the
M? = [H2L]? = 377.1
and
M? = [[Bi(L)]ClÁ2H2-
O]? = 655.5 for positive EI source, respectively. These
experimental results are consistent with their theoretical
molecular
weight
M[H2L] = 377.4
and
M[[Bi(L)]Cl Á 2H2O] = 655.9, which further confirmed the
composition of the Schiff base and the complex.
The Schiff base bismuth(III) complex was a red brown
powder with a melting point of 261 1 °C. The dissolubility
experiment indicated that the complex cannot be dissolved in
water, methanol, dichloromethane, trichloromethane, n-hex-
ane, benzene, toluene, ethyl acetate, acetone, THF, and die-
thyl ether, while it can be dissolved in DMSO and DMF,
especially easily dissolved in the mixed solution of tri-
chloromethane and DMSO (v/v = 1:1). The molar conduc-
tance of the complex in DMSO at 25 °C was determined to be
65.45 S cm2 mol-1, indicating that the complex is an elec-
trolyte in DMSO solvent.
As can be seen from Table S2, there are four strong
absorption peaks appearing at 1604, 1561, 1456, and
1254 cm-1 in the FT-IR spectra of the Schiff base H2L,
corresponding to the m(C=N) stretching vibration of
azomethine group [44], m(C=N) stretching vibration of
pyridine group, m(C–N) stretching vibration of azomethine
group [45], and m(C–O) stretching vibration of phenolic
hydroxyl group, respectively. After the complex formation,
the peak assigned to m(C=N) stretching vibration of
azomethine group has moved to a high wave number
1611 cm-1, Dm = (mcomplex-mSchiff base) = 7 cm-1, which
indicated that the nitrogen atoms of azomethine group in
The Schiff base and its bismuth(III) complex were
characterized by elemental analysis, chemical analysis,
123

Synthesis and microcalorimetric determination of the bioactivities of a new Schiff base and…
the complex involved in the coordination with the bis-
muth(III) ion Bi3? [40]. What is more, the m(C–O) phenolic
hydroxyl group of the free Schiff base exhibited a strong
absorption peak at 1254 cm-1. However, this absorption
peak appeared at 1243 cm-1 for the complex, which was
an indication of C–O–Bi3? bond formation and provided
further evidence for coordination via the deprotonated
phenolic oxygen atoms. The absorption peak attributed to
m(C=N) stretching vibration of pyridine group disappeared
after the complex formation, which could confirm that the
nitrogen atom of pyridine group in the complex partici-
pated in the coordination. The wide m(O–H) stretching
vibration for the Schiff base and complex appeared at 3376
and 3352 cm-1, respectively. The reason could be due to
the hydrogen bonding between the phenolic hydroxyl
groups of the free Schiff base or the complex containing
water molecules, which was consistent with the results of
elemental analysis and the thermogravimetric analysis.
As can be seen from Table S6, the thermal decompo-
sition and mass losses of bismuth(III) complex can be
divided into two steps. The first step was from 45 to
198 °C with a mass loss of 5.49 %, which was due to the
removal of 2 mol H2O, and consistent with the theoretical
value 5.48 %. The composition of the remainder was
[Bi(C21H17N3O4)]Cl. The second step ranges from 198 to
743.5 °C with a mass loss of 64.22 %, which corre-
sponded to the loss of 1 mol of the ligand (C21H17N3O4)
and 1 mol of Cl-, and was consistent with the theoretical
value 62.65 %. The composition of the remainder was
Bi2O3 (or BiO1.5).
Based on the above results of elemental analysis,
1
chemical analysis, mass spectra, FI-IR, H and 13C NMR
data, UV–Vis spectra, and TG–DTA analysis, we can
conclude that the Schiff base and its bismuth(III) complex
have a composition of C22H24N4O5 and [Bi(C21H17N3-
O4)]ClÁ2H2O. The chemical structures and 3D structures of
the two compounds are shown in Figs. 3 and Fig. 4.
1
As can be seen from Table S3 and Table S4, the H
NMR peaks of the complex were very similar to those of
the Schiff base except the disappearance of two hydroxyl
protons at d 13.13 ppm, which indicated that the two
hydroxyl groups from the Schiff base participated in the
coordination to form the complex. This result can be fur-
Thermokinetics of H. pylori and S. pombe cells
treated by the Schiff base and its bismuth(III)
complex
1
ther confirmed by the following NMR analysis. The H
NMR spectrum of the Schiff base showed a singlet at
9.57 ppm due to the imine protons, multiplet in the range
6.88–8.11 ppm due to the aromatic protons. 13C NMR
spectrum of the Schiff base showed at 165.10 (or 164.39)
ppm due to the imine carbon, at 56.21 (or 56.10) ppm due
to OCH3 carbon, and at 176.3 ppm due to the pyridine
carbon.
The growth rate constant (k) of H. pylori and S. pombe
Microcalorimetry was applied to evaluate the bioactivities
of the Schiff base and its bismuth(III) complex on the
growth metabolism of H. pylori and S. pombe. The ther-
mogenic curves (power–time curves) for the growth
metabolism of H. pylori and S. pombe cells treated by
different concentrations of Schiff base and its bismuth(III)
complex were recorded on a TAM Air isothermal heat
conduction microcalorimeter at 37.00 and 32.00 °C,
respectively. All microcalorimetric experiments were
repeated three times. The measured thermogenic curves for
the growth metabolism of H. pylori and S. pombe are
illustrated in Figs. 5 and 6, respectively.
As can be seen from Table S5, Schiff base showed a
wide and strong absorption peak 382 nm due to the p–p
conjunction and n–p* transition between the lone pair
electrons of nitrogen atom in C=N group and the big p
bond of benzene ring. Besides, there is another strong
absorption peak at 280 nm, which was attributed to the p–p
conjugation and n–p* transition between the lone pair
electrons of the phenolic hydroxyl oxygen atom and ben-
zene ring. For the absorption peak at 248 nm, it was
resulted from the p–p* transition of the big conjugated
bond in the benzene ring. The experimental results showed
that the product is a Schiff base. After the formation of the
bismuth(III) complex, the two n-to-p* transition absorption
bands have shifted, which indicated that the phenolic
hydroxyl oxygen atom and the nitrogen atom of C=N are
As can be seen from Figs. 5 and 6, the shapes of the
thermogenic curves for the growth metabolism of H. pylori
(or S. pombe) are similar to each other except the differ-
ences in peak positions and heights caused by different
concentrations of Schiff base and bismuth(III) complex.
The process of the growth metabolism of H. pylori (or
S. pombe) can be abstracted as a mathematical model as
shown in the insert map of Figs. 5a and 6a, respectively.
The thermogenic curves could be divided into four phases,
i.e., a lag phase (AB), a log phase (BC), a stationary phase
(CD), and a decline phase (DE). During the log phase, the
thermogenic curves obeyed the following exponential
equation:
involved in coordination to the bismuth(III) ion Bi3?
.
Meanwhile, the absorption bands at 382 and 280 nm have
blue-shifted to 343 and 266 nm, respectively. The tentative
structure for the complex is shown in Fig. 4. The water
molecules may be in the lattice.
123

X. Li et al.
10
(a)
(b)
O
N
C
H
11
12
9
7
13
8
N
N
N
5
15
6
14
19
4
3
16
17
1
OH
HO
18
2
O
O
21
20
Fig. 3 Chemical structure a and 3D structure b of the Schiff base
(b)
(a)
O
N
10
9
8
11
12
C
H
Bi
Cl
7
13
19
5
N
15
N
N
6
14 16
4
Bi
1
2H2O
Cl
O
O
3
17
18
2
O
O
21
20
Fig. 4 Chemical structure a and 3D structure b of the Schiff base bismuth(III) complex
(a)
0.0030
(b) 0.0030
0.0030
0.0025
1
2
C
D
3
4
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
1 0.0020
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
3
0.0015
7
0.0010
0.0005
0.0000
5
5
6
B
E
A
8
6
4
50,000
1,00,000
1,50,000
t/s
7
8
2
0
50,000
1,00,000
1,50,000
2,00,000
0
40,000
80,000
1,20,000
t/s
t/s
Fig. 5 Thermogenic curves of the growth of H. pylori treated by the
Schiff base a and its bismuth(III) complex b at 37.00 °C; a concen-
tration c/(mmol L-1) of the Schiff base corresponding to curves 1–8:
0.0000, 0.3911, 0.7822, 1.1575, 1.5645, 1.9556, 2.3467, 2.7378;
b concentration c/(mmol L-1) of the complex corresponding to
curves 1–8: 0.0000, 0.0830, 0.1245, 0.1660, 0.2074, 0.2489, 0.2904,
0.3319
123

Synthesis and microcalorimetric determination of the bioactivities of a new Schiff base and…
(b)
(a)
0.0018
0.0018
0.0018
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
D
C
0.0015
0.0012
0.0009
0.0006
0.0003
0.0000
1
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
1
2
3
A
2
3
E
B
0
40,000 80,000 1,20,000 1,60,000
4
4
5
t/s
6
5
7
6
8
7
8
80,000
t/s
0
40,000
80,000
1,20,000
1,60,000
0
40,000
1,20,000
1,60,000
t/s
Fig. 6 Thermogenic curves of the growth of S. pombe treated by the
Schiff base a and its bismuth(III) complex b at 32.00 °C; a concen-
tration c/(mmol L-1) of the Schiff base corresponding to curves 1–8:
0.0000, 0.4059, 0.6089, 0.8118, 1.0148, 1.2178, 1.4207, 1.6237; b
concentration c/(mmol L-1) of the complex corresponding to curves
1–8: 0.0000, 0.0253, 0.0507, 0.0760, 0.1014, 0.1267, 0.1520, 0.1774
base and the complex, which revealed that both the Schiff
base and the complex possessed inhibitory effects on the
growth metabolism of S. pombe.
nt ¼ n0 exp½kðt À t0ފ
ð1Þ
where t is the time after the start of logarithmic growth
phase, t0 is the start time of logarithmic growth phase, nt
and n0 are the germ or cell number at time t and t0,
respectively. If the power output of each germ or cell was
one w, then
Inhibition ratio (I) and half inhibition concentration (IC50)
The inhibition ratio (I) of the growth metabolism of H.
pylori and S. pombe treated by drugs was defined as
follows:
ntw ¼ n0w exp½kðt À t0ފ
If define Pt ¼ ntw; P0 ¼ n0w, then
Pt ¼ P0 exp½kðt À t0ފ
ð2Þ
ð3Þ
ð4Þ
ð5Þ
I ¼ ðk0 À kcÞ=k0 Â 100 %
ð6Þ
or
where k0 is the control rate constant (without any drug
inhibition) of H. pylori or S. pombe and kc is the growth
rate constant of H. pylori or S. pombe treated by an inhi-
bitor with a concentration of c. When the inhibition ratio
(I) was 50 %, the corresponding drug concentration was
called as the half inhibition concentration (IC50). The
values of (I) and (IC50) are shown in Table 1 and Table 2,
respectively.
ln Pt ¼ ln P0 þ kt À kt0
or
ln Pt ¼ A þ kt
Eq. (5) is a linear equation, where Pt is the heat output
power of the H. pylori or S. pombe cell at time t, and k is
the growth rate constant of the H. pylori or S. pomb cell at
specified conditions, whose size represented growth speed.
Using this equation, the growth rate constant k of H. pylori
and S. pomb can be calculated, and the results are shown in
Table 1 and Table 2, respectively.
The results showed that the inhibition ratio I increases
with the increase in concentration of the Schiff base, but no
rules, for the H. pylori, while the inhibition ratio I decreased
with the increase in concentration of the complex for the
H. pylori. In other words, the Schiff base stimulated the
growth of the H. pylori, but the complex possessed inhibi-
tory effects on the growth of H. pylori. The half inhibi-
tion concentration (IC50) of the complex was about
0.1812 mmol L-1. Based on this phenomenon, we inferred
that the structure factors which govern antimicrobial
activities strongly depend on the central metal ion.
By contrast, both the Schiff base and the complex pos-
sessed significantly inhibitory effects on the growth
From Fig. 5 and Table 1, it could be seen that the growth
rate constant (k) increases with the increase in concentration
of the Schiff base, but no rules. With regard to the complex,
the growth rate constant (k) decreased with the increase in
concentration. These results indicated the free Schiff base
stimulated the growth of the H. pylori, while the complex
inhibited its growth. By contrast, it could be seen from Fig. 6
and Table 2 that the growth rate constant (k) of S. pombe
decreased with the increase in concentrations of the Schiff
123

X. Li et al.
Table 1 Thermokinetic parameters of the growth of H. pylori treated by different concentrations of Schiff base and complex at 37.00 °C
Compound
ca/mmol L-1
kb/s-1
Ic/%
ICd50/mmol L-1
The Schiff base
0.0000
0.3911
0.7822
1.1575
1.5645
1.9556
2.3467
2.7378
0.0000
0.0830
0.1245
0.1660
0.2074
0.2489
0.2904
0.3319
1.55 9 10-4 1.15 9 10-7 e
1.82 9 10-4 2.00 9 10-7
1.98 9 10-4 2.00 9 10-7
1.87 9 10-4 1.83 9 10-7
1.86 9 10-4 3.67 9 10-7
1.71 9 10-4 1.67 9 10-7
1.92 9 10-4 4.67 9 10-7
1.58 9 10-4 2.33 9 10-7
1.56 9 10-4 1.57 9 10-7
1.37 9 10-4 1.43 9 10-7
1.06 9 10-4 9.67 9 10-8
8.27 9 10-5 8.50 9 10-8
7.05 9 10-5 5.33 9 10-8
4.70 9 10-5 2.33 9 10-8
3.80 9 10-5 2.17 9 10-8
3.45 9 10-5 2.00 9 10-8
0.00
–
-14.99
-27.74
-20.75
-20.22
-10.54
-23.55
-1.61
0.00
The complex
0.1812
13.33
32.59
47.51
55.24
70.16
75.87
78.10
a
b
The concentration; the growth rate constant of H. pylori; the inhibition ratio; the half inhibition concentration; mean
c
d
e
SD., n = 3
Table 2 Thermokinetic parameters of the growth of S. pomb treated by different concentrations of Schiff base and complex at 32.00 °C
Compound
ca/mmol L-1
kb/s-1
Ic/%
ICd50/mmol L-1
The Schiff base
0.0000
0.4059
0.6089
0.8118
1.0148
1.2178
1.4207
1.6237
0.0000
0.0253
0.0507
0.0760
0.1014
0.1267
0.1520
0.1774
5.42 9 10-5 2.83 9 10-8 e
5.10 9 10-5 3.51 9 10-8
4.22 9 10-5 2.36 9 10-8
2.67 9 10-5 2.64 9 10-8
1.55 9 10-5 5.92 9 10-9
6.02 9 10-6 6.75 9 10-9
3.41 9 10-6 1.20 9 10-8
–
6.32 9 10-5 5.72 9 10-8
6.15 9 10-5 5.06 9 10-8
5.74 9 10-5 3.43 9 10-8
5.28 9 10-5 2.85 9 10-8
4.43 9 10-5 2.74 9 10-8
3.60 9 10-5 1.67 9 10-8
2.42 9 10-5 1.62 9 10-8
9.32 9 10-6 9.66 9 10-9
0.00
0.8054
5.90
22.14
50.74
71.40
88.89
93.71
100
The complex
0.00
2.69
0.1362
9.18
16.45
29.91
43.04
61.71
85.25
a
b
The concentration; the growth rate constant of S. pomb; the inhibition ratio; the half inhibition concentration; mean
c
d
e
SD., n = 3
metabolism of S. pombe cell lines. The values of IC50 of
the Schiff base and the complex on the growth metabolism
of S. pombe cell lines were found to be 0.8054 and
0.1362 mmol L-1, respectively. The inhibitory ability of
the two compounds on the growth metabolism of the S.
pombe has been observed to decrease according to the
following order: the complex [ the Schiff base.
Conclusions
In this work, the synthesis and bioactivities of a new Schiff
base and its bismuth(III) complex have been reported. The
bioactivities of the two synthetic compounds on H. pylori
and S. pombe cell lines were determined by isothermal heat
conduction microcalorimetry. The preliminary results
123

Synthesis and microcalorimetric determination of the bioactivities of a new Schiff base and…
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two compounds on the growth metabolism of S. pombe cell
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Acknowledgments The authors would like to acknowledge the
financial supports from the National Natural Science Foundation of
China (Grant Nos. 21273190 and 20973145), the Science and Tech-
nology Department Foundation of Hunan Province (Grant No.
2014TT2026), and the Educational Committee Foundation of Hunan
Province (Grant Nos. 15C1272, 15A175, and 14A134). Prof. Qiang-
Guo Li (E-mail: liqiangguo@163.com) is the chief-director of these
fund projects.
Compliance with ethical standards
Conflict of interest The authors declare no competing financial
interest.
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