Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу
Aim. To develop a new kinetic spectrophotometric method for determining acetylcholinesterase (AChE) inhibitors – phenothiazine antipsychotic drugs (PhT) and their sulfoxide metabolites (S-oxides) without adding an exogenous catalyst to obtain a chromogenic agent.Materials and methods. The bases of S...
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| Published in: | Журнал органічної та фармацевтичної хімії |
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| Date: | 2022 |
| Volume: | 20 |
| Issue: | 1(77) |
| Pages: | 35-43 |
| ISSN: | 2518-1548 |
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| author | Kovalenko, Vladyslav S. Blazheyevskiy, Mykola Ye. Merzlikin, Serhii I. |
| author_facet | Kovalenko, Vladyslav S. Blazheyevskiy, Mykola Ye. Merzlikin, Serhii I. |
| author_institution_txt_mv | [
{
"author": "Vladyslav S. Kovalenko",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
},
{
"author": "Mykola Ye. Blazheyevskiy",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
},
{
"author": "Serhii I. Merzlikin",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
}
] |
| author_sort | Kovalenko, Vladyslav S. |
| baseUrl_str | https://ophcj.nuph.edu.ua/oai |
| collection | OJS |
| container_end_page | 43 |
| container_issue | 1(77) |
| container_start_page | 35 |
| container_title | Журнал органічної та фармацевтичної хімії |
| container_volume | 20 |
| datestamp_date | 2026-08-24T13:41:04Z |
| description | Aim. To develop a new kinetic spectrophotometric method for determining acetylcholinesterase (AChE) inhibitors – phenothiazine antipsychotic drugs (PhT) and their sulfoxide metabolites (S-oxides) without adding an exogenous catalyst to obtain a chromogenic agent.Materials and methods. The bases of S-oxides of promethazine (PMZ), chlorpromazine (CPM) and thioridazine (THZ) were obtained by oxidizing the corresponding PhT hydrochlorides with diperoxyadipic acid. The structure of the S-oxides of the corresponding PhT synthesized was proven by melting points, spectral characteristics (1H NMR and IR methods) and oscillopolarography results. 1H NMR spectra were recorded on a Varian XL-200 spectrometer. IR spectra were recorded within the range of 4000-400 cm–1 on a SPECORD M-80 spectrometer (Zeiss, Jena, Germany). To register polarograms, a “PO 03 CLA” oscillopolarograph with a three-electrode cell was used. The purity of S-oxides was determined by the high-performance liquid chromatography method on a Zorbax SB, C-18 (250 × 4.6) mm column. Measurements of absorbance of solutions were performed in a 1 cm cuvette on an Evolution 60S UV-Visible Thermo-Scientific Spectrophotometer (USA) (λ = 358 nm).Results and discussion. Acetylcholine (ACh) was found to mimic the activity of peroxidase; based on it, a spectrophotometric system containing ACh–H2O2–p-Ph for a sensitive and selective assessment of the AChE activity and determination of its inhibitors was developed. According to the plots of inhibition efficiency vs inhibitors concentration, the inhibiting ability of chlorpromazine, promethazine and thioridazine and their S-oxides was determined. The IC50 values of CPM, PMZ and THZ and their metabolites in relation to the AChE activity were estimated as 11 ng mL−1 (CPM) and 1.8 ng mL−1 (CPM S-oxide), 17 ng mL−1 (PMZ) and 2.5 ng mL−1 (PMZ S-oxide) and 27 ng mL−1 (THZ 2S,5S-dioxide). The results obtained indicate that S-oxides of the corresponding PhT are selective and potent inhibitors of AChE. The values of the inhibition efficiency obtained for S-oxides of PhT derivatives were an order of magnitude lower than those of the corresponding PhT derivatives.Conclusions. The spectrophotometric method proposed without the addition of other exogenous catalysts holds promise for the on-site determination of PhT antipsychotics and can be additionally used for sensory applications in areas related to environmental protection and food safety, as well as in the chemical-toxicological analysis. |
| doi_str_mv | 10.24959/ophcj.22.252351 |
| first_indexed | 2025-07-17T13:01:19Z |
| format | Article |
| fulltext |
ISSN 2308-8303 (Print) / 2518-1548 (Online) 35
Original research
http://ophcj.nuph.edu.ua
UDC 543.48:547.279.5:615.214.2
V. S. Kovalenko, M. Ye. Blazheyevskіy, S. I. Merzlikin
National University of Pharmacy of the Ministry of Health of Ukraine,
53, Pushkinska str., Kharkiv, 61002, Ukraine
A comparative study of the effect of phenothiazine derivatives
and their S-oxides on cholinesterase investigated by a new
kinetic spectrophotometric method
Abstract
Aim. To develop a new kinetic spectrophotometric method for determining acetylcholinesterase (AChE) inhibitors – pheno-
thiazine antipsychotic drugs (PhT) and their sulfoxide metabolites (S-oxides) without adding an exogenous catalyst to obtain
a chromogenic agent.
Materials and methods. The bases of S-oxides of promethazine (PMZ), chlorpromazine (CPM) and thioridazine (THZ) were
obtained by oxidizing the corresponding PhT hydrochlorides with diperoxyadipic acid. The structure of the S-oxides of the
corresponding PhT synthesized was proven by melting points, spectral characteristics (1H NMR and IR methods) and oscil-
lopolarography results. 1H NMR spectra were recorded on a Varian XL-200 spectrometer. IR spectra were recorded within the
range of 4000-400 cm–1 on a SPECORD M-80 spectrometer (Zeiss, Jena, Germany). To register polarograms, a “PO 03 CLA”
oscillopolarograph with a three-electrode cell was used. The purity of S-oxides was determined by the high-performance
liquid chromatography method on a Zorbax SB, C-18 (250 × 4.6) mm column. Measurements of absorbance of solutions were
performed in a 1 cm cuvette on an Evolution 60S UV-Visible Thermo-Scientific Spectrophotometer (USA) (λ = 358 nm).
Results and discussion. Acetylcholine (ACh) was found to mimic the activity of peroxidase; based on it, a spectrophoto-
metric system containing ACh–H2O2–p-phenetidine for a sensitive and selective assessment of the AChE activity and deter-
mination of its inhibitors was developed. According to the plots of inhibition efficiency vs inhibitors concentration, the
inhibiting ability of chlorpromazine, promethazine and thioridazine and their S-oxides was determined. The IC50 values of
CPM, PMZ and THZ and their metabolites in relation to the AChE activity were estimated as 11 ng mL−1 (CPM) and 1.8 ng mL−1
(CPM S-oxide), 17 ng mL−1 (PMZ) and 2.5 ng mL−1 (PMZ S-oxide) and 27 ng mL−1 (THZ 2S,5S-dioxide). The results obtained indi-
cate that S-oxides of the corresponding PhT are selective and potent inhibitors of AChE. The values of the inhibition efficiency
obtained for S-oxides of PhT derivatives were an order of magnitude lower than those of the corresponding PhT derivatives.
Conclusions. The spectrophotometric method proposed without the addition of other exogenous catalysts holds promise
for the on-site determination of PhT antipsychotics and can be additionally used for sensory applications in areas related to
environmental protection and food safety, as well as in the chemical-toxicological analysis.
Keywords: acetylcholine; acetylcholinesterase; phenothiazine derivatives; S-oxides; photometric methods of analysis
В. С. Коваленко, М. Є. Блажеєвський, С. І. Мерзлікін
Національний фармацевтичний університет Міністерства охорони здоров’я України,
вул. Пушкінська, 53, м. Харків, 61002, Україна
Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу
за допомогою нового кінетичного спектрофотометричного методу
Анотація
Мета. Розробити новий кінетико-спектрофотометричний метод визначення інгібіторів ацетилхолінестерази (АChЕ),
фенотіазинових антипсихотичних засобів (PhТ) та їх сульфоксидних метаболітів (S-оксидів) без додавання екзогенно-
го каталізатора для отримання хромогенного агента.
Матеріали та методи. Основи S-оксидів прометазину, хлорпромазину та тіоридазину синтезували шляхом окиснен-
ня відповідних гідрохлоридів PhТ дипероксиадипіновою кислотою. Будову синтезованих S-оксидів відповідних PhТ
доводили за температурами плавлення, спектральними характеристиками (1H ЯМР- та ІЧ-методи) та результатами
осцилополярографії. Спектри 1Н ЯМР записували на спектрометрі Varian XL-200. ІЧ-спектри реєстрували в діапазоні
ISSN 2308-8303 (Print) / 2518-1548 (Online) 36
Журнал органічної та фармацевтичної хімії 2022, 20 (1)
4000-400 см−1 на спектрометрі SPECORD M-80 (Zeiss, Jena, Німеччина). Для реєстрації полярограм використовували
осцилополярограф ПО, модель 03 ЦЛА з триелектродним елементом. Чистоту S-оксидів визначали методом ВЕРХ на
колонці Zorbax SB, C-18 (250 × 4,6) мм. Вимірювання поглинання випробуваних розчинів S-оксидів відповідних PhТ
проводили за довжини хвилі λ = 358 нм у кюветі завтовшки 1 см на спектрофотометрі Evolution 60S UV-Visible Thermo-
Scienticfic (США).
Результати та їх обговорення. З’ясовано, що ацетилхолін (ACh) імітує активність пероксидази, на основі чого було роз-
роблено спектрофотометричну систему, що містить ACh-H2O2-п-фенетидин для чутливого селективного оцінювання
активності АChЕ та визначення інгібіторів ферменту. Відповідно до графіків залежності інгібувальної здатності від
концентрації інгібіторів визначено інгібувальну здатність хлорпромазину, прометазину, тіоридазину та їх S-оксидів.
IC50 хлорпромазину, прометазину, тіоридазину та їх метаболітів щодо активності AChE оцінено так: 11 нг мл–1 – хлорпро-
мазин, 1,8 нг мл–1 – хлорпромазин S-оксид, 17 нг мл–1 – прометазин, 2,5 нг мл–1 – прометазин S-оксид та 27 нг мл–1 –
тіоридазин 2S,5S-діоксиду. Одержані результати свідчать про те, що S-оксиди відповідних PhТ є селективними та по-
тужними інгібіторами АChЕ. Отримані значення ефективності інгібування для S-оксидів похідних PhТ були на порядок
нижчими, ніж у відповідних похідних PhТ.
Висновки. Розроблена спектрофотометрична методика без додавання інших екзогенних каталізаторів має перспек-
тиви для визначення PhТ нейролептиків на місці і може бути додатково використана для сенсорних застосувань у сфе-
рах, пов’язаних із захистом навколишнього середовища та безпекою харчових продуктів, а також у хіміко-токсиколо-
гічному аналізі.
Ключові слова: ацетилхолін; ацетилхолінестераза; похідні фенотіазину; S-оксиди; фотометричні методи аналізу
Citation: Kovalenko, V. S.; Blazheyevskіy, M. Ye.; Merzlikin, S. I. A comparative study of the effect of phenothiazine derivatives and their
S-oxides on cholinesterase investigated by a new kinetic spectrophotometric method. Journal of Organic and Pharmaceutical Chemistry
2022, 20 (1), 35 – 43.
https://doi.org/10.24959/ophcj.22.252351
Received: 02 December 2021; Revised: 10 January 2022; Accepted: 23 January 2022
Copyright© 2022, V. S. Kovalenko, M. Ye. Blazheyevskіy, S. I. Merzlikin. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0).
Funding: The work is a part of the studies of the National University of Pharmacy on the topic “Organic synthesis and analysis
of biologically active compounds, drug development on the basis of synthetic substances” (the state registration No. 01144000943;
the research period 2019 – 2024).
Conflict of interests: The authors have no conflict of interests to declare.
■ Introduction
Acetylcholinesterase (AChE) is an important
enzyme in the central and peripheral nervous
system [1]. Its primary biological function is to
catalyze the breakdown of acetylcholine (ACh)
and some other choline esters that function as
neurotransmitters [2]. Nowadays, it is accepted
that Alzheimer’s disease (AD), common demen-
tia for older people worldwide, is related to a low
level of ACh in the hippocampus and cortex [3].
AChE inhibitors can penetrate the blood-brain
barrier and have been suggested to increase the
level of ACh to treat AD [4]. However, ACh ex-
cess results in neuromuscular paralysis or fatal
consequences [5].
Various analytical techniques, such as Ellman’s
method [6], electrochemical methods [7 – 10], the
liquid crystals-based method [11] and chemilu-
minescent and fluorescent methods [12 – 19], have
been developed for the AChE activity and screen-
ing of its inhibitors.
However, some of these methods suffer from
drawbacks of the time-consuming sample pre-
treatment, sophisticated instrument manipula-
tion or high costs. Moreover, it is reported that
Ellman’s method may lead to a false-positive ef-
fect [20].
Hence, developing a simple, highly sensitive
and selective method for probing the AChE ac-
tivity and screening for its potential inhibitors
is highly needed.
Photometric methods have attracted signifi-
cant attention because of their simplicity and low-
cost advantages. Moreover, they can circumvent
the relative complexity inherent in other detec-
tion methods by relying on unaided visual read-
outs instead of complicated instruments, which
is especially useful for on-site detection in real-
time [21, 22].
Earlier, we proposed a new kinetic spectro-
photometric method for determining the AСhE
activity. It consists of spectrophotometric meas-
urement of the rate of the ACh enzymatic hy-
drolysis through two conjugated reactions – pe-
rhydrolysis of the non-hydrolyzed ACh residue
with a hydrogen peroxide excess and oxidation
of the indicator substance p-phenetidine (p-Ph)
by peroxyacetic acid formed to 4,4’-azoxyphene-
tol (λmax = 358 nm, lgε 4.2) [23]. The rate of the
enzymatic hydrolysis of ACh was measured by
the tangent method using the linear part of the
ISSN 2308-8303 (Print) / 2518-1548 (Online) 37
Journal of Organic and Pharmaceutical Chemistry 2022, 20 (1)
kinetic curve in the light absorbance (A, 358 nm)
vs time (t, min) coordinates. The linear inversely
proportional dependence of the conditional reac-
tion rate on the enzyme concentration was ob-
served. The same principle has been used to quan-
tify compounds capable of inhibiting AChE en-
zyme as a change in the activity of AChE causes
a change in the amount of unreacted АСh in the
enzymatic hydrolysis reaction. The latter is quan-
tified by the kinetic-spectrophotometric method
as mentioned above (Scheme) [24].
■ Materials and methods
Acetylcholine chloride (pharmaceutical grade),
0.02 g per amp/5 mL, produced by “VECTOR”,
State Science Centre of Virology and Biotechno-
logy, Russia.
Disodium hydrogen phosphate dodecahyd-
rate, Na2HPO4∙12H2O (puriss. p.a.), produced by
“ReaChem”, Kharkiv, Ukraine.
A dry acetylcholinesterase enzyme from horse
serum – 80 mg per vial (VI class) with the known
specific activity of 27 AU mg-1 (according to the
certificate) produced by SMU “Biomed” (Russia).
The catalytic activity of 1 activity unit (AU) is
manifested by such an amount of this enzyme
preparation that converts 1 µmole of this sub-
strate in 1 min under given reaction conditions.
A stabilized hydrogen peroxide 30 – 40 % solu-
tion, puriss. p.a. (LLC Inter-Synthes, Boryslav,
Ukraine). The precise content of hydrogen per-
oxide was determined according to the State Phar-
macopoeia of Ukraine [25].
Chlorpromazine hydrochloride ≥ 98 % (TLC);
CAS 69-09-0, Sigma-Aldrich. C17H19ClN2S·HCl
(CPZ), 2-chloro-10-(3-dimethylaminopropyl)phe-
nothiazine hydrochloride.
Promethazine hydrochloride 98 %; CAS 58-33-3,
Sigma-Aldrich. C17H20N2S·HCl (PMZ), 10-[2-(di-
methylamino)propyl]phenothiazine hydrochlo-
ride.
Thioridazine hydrochloride ≥ 99 %; CAS 130-61-0,
Sigma-Aldrich. C21H26N2S2·HCl (THZ), 10-[2-(1-me-
thyl-2-piperidyl)ethyl]-2-(methylthio)-10H-phe-
nothiazine hydrochloride.
N
O Me
O
Me
Me
Me
acetylcholine
N
OHMe
Me
Me
choline
Me
O
OH
full
conversion
+AChE
partial
conversion
AChE + Inhibitor
N
OHMe
Me
Me
choline
Me
O
OH
unreacted
acetylcholine
acetic acid
+
acetic acid
H2O2
choline +
perhydrolysis
H2O2
perhydrolysis
OMe
NH2
OMe
N
N
O Me
O
p- idinephenet
4,4'-azoxyphenetol
�max 358 nm (lg 4.2)�
Me
O
OOH
peroxyacetic
acid
Scheme. An analytical system for the determination of AChE inhibitors using coupled reactions of ACh perhydrolysis and peroxyacid
oxidation of p-Ph as an indicator substance
ISSN 2308-8303 (Print) / 2518-1548 (Online) 38
Журнал органічної та фармацевтичної хімії 2022, 20 (1)
High purity double distilled water was used
in all experiments.
Chlorpromazine hydrochloride, promethazine
hydrochloride, thioridazine hydrochloride and thio-
ridazine 2S,5S-disulfoxide were obtained from
commercial sources and used without further pu-
rification.
The structures of the phenothiazine antipsy-
chotic drugs and the corresponding S-oxides are
given in Figure 1.
1H NMR spectra were recorded on a Varian
XL-200 Spectrometer (200 MHz) using DMSO-d6
as a solvent and TMS as an internal standard.
IR spectra were recorded within the range of
4000 – 400 cm–1 on a SPECORD M-80 spectrome-
ter (Zeiss, Jena, Germany) in KBr pellets (200 mg
of KBr and 2 mg of the test compounds).
Voltammograms were recorded on a PO 03 CLA
oscilllographic polarograph (Rostov-on-Don Re-
search Institute, Russia) in a three electrode ther-
mostated cell at 20 °C; the indicator microelec-
trode was a dropping mercury one, the reference
electrode was a saturated calomel electrode, and
the auxiliary electrode was a platinum electrode.
The potentials of peak maxima were measured
with a V7-21 digital voltmeter with the precision
of ±1 mV. Triangular-shaped polarizing voltage
was applied to the cell electrodes at a scanning
rate of = 0.5 V s–1. The potential was varied in
the range from –0.2 to –1.4 V. Dissolved oxygen
was removed from solutions by blowing purified
argon over 20 min. The polarography conditions
were selected in each case.
The pH measurements were performed with
a combined glass electrode (SP20B) together with
an EAL-1М3.1 reference standard silver chloride
electrode.
The measurements of absorbance (А) of solu-
tions were performed in a 1.0 cm cuvette on an Evo-
lution 60S UV-Visible Thermo-Scientific Spectro-
photometer (USA) against the buffer solution with
double distilled water (compensation solution).
The preparation procedure for the pro-
methazine S-oxide base
0.64 g (0.002 mol) of PMZ was dissolved in
15 mL of distilled water; 0.44 g (0.0025 mol) of
diperoxyadipic acid was added with stirring and
left at room temperature for 15 min. The mixture
was poured with 2 mL of 50 % sodium hydroxide
solution, and the resulting precipitate was ex-
tracted with diethyl ether (3 × 10 mL). The com-
bined organic phases were washed with water
cooled to 10 °C (3 × 20 mL), dried over anhydrous
sodium sulfate, and the solvent was evaporated
at room temperature. Acetone was added to the
residue, and the mixture was cooled. In 2 days,
white crystals precipitated; then they were fil-
tered and dried at room temperature. The yield
of PMZ S-oxide was close to the quantitative one.
Diperoxyadipic acid was obtained by the in-
teraction between hydrogen peroxide and adipic
acid in the presence of sulfuric acid according
to the known method [24]. Diperoxyadipic acid,
HO3C(CH2)4CO3H, 98 %, m. p. 114.5 °C (dec.), ac-
tive oxygen content, % (theor.), 17.5 (17.9).
Promethazine S-oxide base. M. p. 118-119 °C.
1H NMR (200 MHz, DMSO-d6), δ, ppm: 0.75 (3H, d,
CH3); 2.42 (6H, s, 2NCH3); 2.81 – 2.95 (1H, m, CH);
4.18 – 4.60 (2H, t, CH2); 7.38 – 7.92 (8H, m; ArH).
IR (KBr), ν, cm−1: 1028 (S=O).
CPZ S-oxide base was synthesized by the simi-
lar procedure as for PMZ S-oxide base.
Chlorpromazine S-oxide base. M. p. 111-112 °C.
1H NMR (200 MHz, DMSO-d6), δ, ppm: 1.82 (2H, q,
CH2); 2.15 (6H, s, 2 × NCH3); 2.35 (2H, t, CH3NCH2);
4.38 (2H, t, NCH2); 7.22 – 8.11 (7H, m, ArH).
Analysis of oxidation products of pheno-
thiazine derivatives by HPLC
In addition, the purity of S-oxides of CPZ and
PMZ was determined by the high-performance
liquid chromatography method on a Zorbax SB,
C-18 (250 × 4.6) mm column. The mobile phase was
the solution of camphorsulfonic acid (2.9 g L–1),
which pH was adjusted to 5.4 with 5 mol L–1
S
N
N
Me
Me
Cl
. HCl
chlorpromazine
hydrochloride
S
N
Me
. HCl
promethazine
hydrochloride
S
N SMe
. HCl
thioridazine
hydrochloride
N
Me Me N
Me
Figure 1. The structures of the phenothiazines tested
ISSN 2308-8303 (Print) / 2518-1548 (Online) 39
Journal of Organic and Pharmaceutical Chemistry 2022, 20 (1)
sodium hydroxide solution. The flow rate was
1 mL min–1. The volume of the injected sample
was 20 μL. Detection was performed by UV-spect-
rophotometry at 262 nm. The sulfoxides of PhT
derivatives studied were dissolved in the mobile
phase. The concentration of S-oxides, mg mL–1,
was: CPZ – 0.402, PMZ – 0.406. The percentage
of the active substance calculated by the norma-
lization method was: for CPZ S-oxide – 94.66 %,
S-oxide PMZ – 97.87 % with the purity of the pro-
minent peaks of 99.85 %, 99.84 %, respectively.
Oscillopolarograms (0.1 mol L–1 KCl, pH 5.5),
Ep
k (V): CPM S-oxide (1.8 × 10–5 mol L–1) –0.785,
–1.058 (Ep
A –0.935); PMZ S-oxide (1.8 × 10–5 mol L–1)
–1.140, –1.385 (Ep
A –1,13); THZ 2S,5S-disulfoxide
(1.8 × 10–5 mol L–1) –1.10, –1.295; (0.1 mol L–1 KCl,
0.01 M HCl), Ep
k (V): CPM S-oxide (1.8 × 10–5 mol L–1)
–0.865, –1.084 (Ep
A –0.938); PMZ S-oxide
(1.8 × 10–5 mol L–1) –0.937; THZ 2S,5S-disulfoxide
(1.8 × 10–5 mol L–1) –0.841, –0.975.
Preparation of the solutions
Stock solutions of PhT derivatives
Daily 5.0 × 10–3 mol L–1 of PhT derivatives stock
solutions were prepared by dissolving accurately
weighed samples of PhT derivatives in 0.01 mol L–1
HCl solution (THZ in 96 % ethanol) and diluting
the solution to 100 mL with double distilled wa-
ter in volumetric flasks. The corresponding ali-
quots of the stock solutions of PhT derivatives were
transferred into separate glass-stopper tubes.
Working standard solutions of PhT derivatives
Working standard solutions (WSS) of PhT de-
rivatives were prepared from the stock solutions
by the corresponding dilution with double distil-
late water or 0.01 M hydrochloric acid. All solu-
tions were stored at room temperature in a cool
dark place.
Preparation of 0.2 mol L–1 phosphate buffer
solution (pH 8.35)
Disodium hydrogen phosphate dodecahydrate
(35.75 g) was dissolved in a 500 mL flask using
double-distilled water. Then 19 mL of 0.1 mol L–1
solution of the hydrochloric acid solution was added.
The pH of the final solution was controlled by
potentiometry.
Preparation of 10 % hydrogen peroxide solution
The solution was prepared by the correspond-
ing high-test hydrogen peroxide dilution with dou-
ble-distilled water. The hydrogen peroxide con-
tent in 10 % working solution was determined by
the permanganatometric method.
Preparation of 1 % p-phenetidine hydrochlo-
ride solution
p-Phenetidine hydrochloride (p-Ph) was prepa-
red by dissolution of p-phenetidine in chloroform
followed by precipitation of the salt by gaseous
HCl. 1.00 g of p-phenetidine hydrochloride was
dissolved in 80 mL of double-distilled water in a
100 mL volumetric flask and diluted to the vol-
ume with the same solvent.
Preparation of acetylcholine chloride solution
The ampoule content (0.02 g of ACh) was dis-
solved in 20 mL of double-distilled water. The am-
poule was opened, 4.0 mL of water was pipetted
and added to the ampoule, then shaken until ace-
tylcholine was completely dissolved. Then the ACh
solution was transferred into a 20 mL volumet-
ric flask and diluted to the volume with double-
distilled water.
Preparation of acetylcholinesterase solution
The ampoule content of 80 mg of AChE was dis-
solved in 20 mL of double-distilled water. The am-
poule was opened, 4.0 mL of water was pipetted,
and shaken until AChE was completely dissolved.
Then the AChE solution was transferred into a
20 mL volumetric flask, and diluted to the volume
with double-distilled water.
The procedure generally recommended
The first part: 10.0 mL of 0.2 M phosphate
buffer solution (pH = 8.35) was transferred into
a 20 mL graduated test tube with a ground stop-
per, and 1.0 mL of 1 mg mL–1 ACh solution was
added. Then 1.6 mL of 10 % hydrogen peroxide
solution was added, and the stopwatch was started.
After that the solution was shaken thoroughly
and thermostated for 10 min. Then 0.5 mL of 1 %
p-Ph solution was added, and the solution was
diluted to the volume with double distilled wa-
ter. The stopwatch was switched on again, and
the solution was scanned photometrically every
minute over the period of 15 min (λ = 358 nm).
The solution containing only 0.2 M phosphate buf-
fer (10.0 mL, pH = 8.3) was used as a reference
solution. The rate of the reaction [(ACh + H2O2)
+ p-Ph] was determined as a slope of the kinetic
curve A vs t, (tgα0, min–1).
The second part: 10.0 mL of 0.2 M phosphate
buffer solution (pH = 8.35) was transferred into
a 20 mL graduated test tube with a ground stop-
per, and 1.0 mL of 1 mg mL–1 ACh solution was
added. After that a 0.5 mL accurate portion of
ChE was added, then 1.6 mL of 10 % hydrogen
peroxide solution was added while stirring, sha-
ken up thoroughly and kept for 10 min in a ther-
mostat. Then 0.5 mL of 1 % p-Ph solution was
added, and the mixture was diluted to the vol-
ume with double distilled water. The stopwatch
was switched on, and the solution was scanned
photometrically every minute over the period of
15 min (λ = 358 nm). The solution containing only
0.2 M phosphate buffer (10.0 mL, pH = 8.3) was
used as a reference solution. The rate of the reaction
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Журнал органічної та фармацевтичної хімії 2022, 20 (1)
[(ChE + ACh) + H2O2 + p-Ph] was determined as
a slope of the kinetic curve A vs t, (tgα, min–1).
The third part: 10.0 mL of 0.2 M of phosphate
buffer solution (pH = 8.35) was transferred into
a 20 mL graduated test tube with a ground stop-
per. The accurate volumes (from 0.40 to 3.20 mL)
of WSS of PhT derivatives (Inh) were added to the
tube. Then 0.5 mL of ChE was added while stir-
ring, the stopwatch was switched on, every so-
lution was shaken thoroughly and thermostated
for 10 min. After that 1.0 mL of 1 mg mL–1 ACh
solution was quickly added, and the stopwatch was
switched on, the content was shaken thoroughly
and thermostated for 10 min again. Then 1.6 mL
of 10 % hydrogen peroxide solution was added, the
tube was kept for 10 min in a thermostat, 0.5 mL
of 1 % p-Ph solution was added and diluted to the
volume with double distilled water. The stopwatch
was switched on, and the solution was scanned
photometrically every minute over the period of
15 min (λ = 358 nm). The buffer solution with
double distilled water was used as a reference
solution. The rate of the reaction [(ChE + Inh) +
ACh] +H2O2 + p-Ph] was determined as a slope
of the kinetic curve A vs t (tgαi, min−1).
Screening of AChE inhibitors
The inhibiting efficiency (IE, %) of the enzy-
matic hydrolysis of acetylcholine in the presence
of PhT derivatives was determined by the fol-
lowing equation:
IE (%) =
tgαi – tgα
tgα0 – tgα × 100 %
where tgαi (min–1) is the slope tangent of the linear
part of the kinetic curve in the A vs t coordinates
(a slope of the kinetic curve) for the reaction
[(ChE + Inh) + ACh] +H2O2 + p-Ph] in the pres-
ence of AChE and an inhibitor;
tgα0 is the slope tangent of the linear part of the
kinetic curve in the A vs t coordinates (a slope of
the kinetic curve) for the reaction [(ACh + H2O2)
+ p-Ph] in the absence of AChE and an inhibitor;
tgα is the slope tangent of the linear part of the
kinetic curve in the A vs t coordinates (a slope of
the kinetic curve) for the reaction [(ChE + ACh) +
H2O2 + p-Ph] in the presence of AChE and the
absence of an inhibitor.
■ Results and discussion
Figures 2 – 4 show the dependence of IE on
the concentration of PhT derivatives and their
S-oxides for the reaction [(ChE + Inh) + ACh] +
H2O2 + p-Ph]. The inhibition ability of CPZ, PMZ
and THZ and their metabolites is evaluated by
the IC50 value, which is the concentration of the
inhibitor needed for 50 % inhibition of the AChE
activity. The IC50 value of CPZ, PMZ and THZ
and their metabolites in relation to the AChE
activity was estimated as 11 ng mL−1 (CPM) and
1.8 ng mL−1 (CPM S-oxide), 17 ng mL−1 (PMZ)
and 2.5 ng mL−1 (PMZ S-oxide) and 27 ng mL−1
(THZ 2S,5S-dioxide) from the plots of IE versus the
concentration of inhibitors (Figures 2 – 4), which
were similar to the previous reports [27, 28].
Thus, to summarize, a new kinetic spectro-
photometric method for determining the activity
of AChE and its inhibitors of PhT antipsychotic
drugs and their sulfoxides metabolites is attrac-
tive because of its convenience, without adding
an exogenous catalyst to obtain a chromogenic
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80
IE
,
%
c, ng mL
–1
2
1
Figure 2. The effect of the CPZ (1) and its S-oxide (2) concentration on the AChE inhibition efficiency
ISSN 2308-8303 (Print) / 2518-1548 (Online) 41
Journal of Organic and Pharmaceutical Chemistry 2022, 20 (1)
agent, which can lead to complexity and inter-
ferences. In this study, for the first time we dis-
covered that ACh itself mimicked the activity of
peroxidase, and based on it, a simple and relia-
ble spectrophotometric system containing ACh–
H2O2–p-Ph for a sensitive and selective assess-
ment of the AChE activity and determination of
its inhibitors was developed.
PhT derivatives and their S-oxides inhibit the
activating action of AChE in the hydrolysis reac-
tion of acetylcholine. As seen, the values of the
inhibition efficiency IC50 obtained for sulfoxides
of the corresponding PhT derivatives were an or-
der of magnitude lower than those of the corre-
sponding PhT derivatives.
This result displays that the detection method
proposed provides a sensitive and rapid strategy
for screening AChE inhibitors.
It has also been demonstrated that this stra-
tegy can be applied to the determination of PhT
antipsychotic drugs in real samples. Meanwhile,
the sensor platform can also be implemented on
test sensors for fast PhT monitoring. Thus, this
extremely simple spectrophotometric strategy with-
out the addition of other exogenous catalysts holds
promise for the on-site determination of PhT anti-
psychotics and can be additionally used for sen-
sory applications in areas related to environmen-
tal protection and food safety, as well as in the
chemical-toxicological analysis.
0
10
20
30
40
50
60
70
80
90
0 10 20 30
IE
,
%
2
1
c, ng mL
–1
Figure 3. The effect of the PMZ (1) and its S-oxide (2) concentration on the AChE inhibition efficiency
0
10
20
30
40
50
60
70
80
0 10 20 30 40 50
IE
,
%
c, ng mL
–1
Figure 4. The effect of the THZ 2S,5S-dioxide concentration on the AChE inhibition efficiency
ISSN 2308-8303 (Print) / 2518-1548 (Online) 42
Журнал органічної та фармацевтичної хімії 2022, 20 (1)
■ Conclusions
A new kinetic spectrophotometric method for
determining AChE inhibitors – phenothiazine an-
tipsychotic drugs and their sulfoxide metabolites
without adding an exogenous catalyst to obtain
a chromogenic agent has been developed. The ACh
itself mimics the activity of peroxidase, and ba-
sed on it, a simple and reliable spectrophotometric
system containing ACh–H2O2–p-Ph for a sensi-
tive and selective assessment of the AChE activi-
ty and determination of its inhibitors has been
developed. The values of the inhibition efficiency
IC50 obtained for sulfoxides of the corresponding
PhT derivatives are an order of magnitude low-
er than those of the corresponding PhT deriva-
tives. It has also been demonstrated that this
strategy can be applied to the determination of
PhT antipsychotic drugs in real samples.
■ References
1. Li, Y.; Bai, H.; Li, C.; Shi, G. Colorimetric Assays for Acetylcholinesterase Activity and Inhibitor Screening Based on the Disassembly
− Assembly of a Water-Soluble Polythiophene Derivative. ACS Applied Materials & Interfaces 2011, 3 (4), 1306 – 1310. https://doi.
org/10.1021/am200101n.
2. Liao, D.; Chen, J.; Zhou, H.; Wang, Y.; Li, Y.; Yu, C. In Situ Formation of Metal Coordination Polymer: A Strategy for Fluorescence Turn-
On Assay of Acetylcholinesterase Activity and Inhibitor Screening. Anal. Chem. 2013, 85 (5), 2667 – 2672. https://doi.org/10.1021/
ac302971x.
3. Zhou, G.; Wang, F.; Wang, H.; Kambam, S.; Chen, X.; Yoon, J. Colorimetric and Fluorometric Assays Based on Conjugated Polydiacety-
lene Supramolecules for Screening Acetylcholinesterase and Its Inhibitors. ACS Applied Materials & Interfaces 2013, 5 (8), 3275 – 3280.
https://doi.org/10.1021/am400260y.
4. Lei, C.; Wang, Z.; Nie, Z.; Deng, H.; Hu, H.; Huang, Y.; Yao, S. Resurfaced Fluorescent Protein as a Sensing Platform for Label-Free Detec-
tion of Copper(II) Ion and Acetylcholinesterase Activity. Anal. Chem. 2015, 87 (3), 1974 – 1980. https://doi.org/10.1021/ac504390e.
5. Du, D.; Tao, Y.; Zhang, W.; Liu, D.; Li, H. Oxidative desorption of thiocholine assembled on core–shell Fe3O4/AuNPs magnetic nanocom-
posites for highly sensitive determination of acetylcholinesterase activity: An exposure biomarker of organophosphates. Biosens. Bio-
electron. 2011, 26 (10), 4231 – 4235. https://doi.org/10.1016/j.bios.2011.03.037.
6. Arduini, F.; Errico, I.; Amine, A.; Micheli, L.; Palleschi, G.; Moscone, D. Enzymatic Spectrophotometric Method for Aflatoxin B Detection
Based on Acetylcholinesterase Inhibition. Anal. Chem. 2007, 79 (9), 3409 – 3415. https://doi.org/10.1021/ac061819j.
7. Ju, K.-J.; Feng, J.-X.; Feng, J.-J.; Zhang, Q.-L.; Xu, T.-Q.; Wei, J.; Wang, A.-J. Biosensor for pesticide triazophos based on its inhibition of
acetylcholinesterase and using a glassy carbon electrode modified with coral-like gold nanostructures supported on reduced graphene
oxide. Microchimica Acta 2015, 182 (15), 2427 – 2434. https://doi.org/10.1007/s00604-015-1584-7.
8. Arduini, F.; Forchielli, M.; Amine, A.; Neagu, D.; Cacciotti, I.; Nanni, F.; Moscone, D.; Palleschi, G. Screen-printed biosensor modified
with carbon black nanoparticles for the determination of paraoxon based on the inhibition of butyrylcholinesterase. Microchimica
Acta 2015, 182 (3), 643 – 651. https://doi.org/10.1007/s00604-014-1370-y.
9. Fenzl, C.; Genslein, C.; Zöpfl, A.; Baeumner, A. J.; Hirsch, T. A photonic crystal based sensing scheme for acetylcholine and acetylcho-
linesterase inhibitors. Journal of Materials Chemistry B 2015, 3 (10), 2089 – 2095. https://doi.org/10.1039/C4TB01970A.
10. Chen, A.; Du, D.; Lin, Y. Highly Sensitive and Selective Immuno-Capture/Electrochemical Assay of Acetylcholinesterase Activity in Red Blood
Cells: A Biomarker of Exposure to Organophosphorus Pesticides and Nerve Agents. Environ. Sci. Technol. 2012, 46 (3), 1828 – 1833.
https://doi.org/10.1021/es202689u.
11. Wang, Y.; Hu, Q.; Guo, Y.; Yu, L. A cationic surfactant-decorated liquid crystal sensing platform for simple and sensitive detection of
acetylcholinesterase and its inhibitor. Biosens. Bioelectron. 2015, 72, 25 – 30. https://doi.org/10.1016/j.bios.2015.05.001.
12. Sabelle, S.; Renard, P.-Y.; Pecorella, K.; de Suzzoni-Dézard, S.; Créminon, C.; Grassi, J.; Mioskowski, C. Design and Synthesis of Chemilu-
minescent Probes for the Detection of Cholinesterase Activity. J. Am. Chem. Soc. 2002, 124 (17), 4874 – 4880. https://doi.org/10.1021/
ja0171299.
13. Gill, R.; Bahshi, L.; Freeman, R.; Willner, I. Optical Detection of Glucose and Acetylcholine Esterase Inhibitors by H2O2-Sensitive CdSe/
ZnS Quantum Dots. Angew. Chem. Int. Ed. 2008, 47 (9), 1676 – 1679. https://doi.org/10.1002/anie.200704794.
14. DiScenza, D. J.; Levine, M. Selective detection of non-aromatic pesticides via cyclodextrin-promoted fluorescence modulation. New J.
Chem. 2016, 40 (1), 789 – 793. https://doi.org/10.1039/C5NJ02357B.
15. Chen, G.; Feng, H.; Jiang, X.; Xu, J.; Pan, S.; Qian, Z. Redox-Controlled Fluorescent Nanoswitch Based on Reversible Disulfide and Its Appli-
cation in Butyrylcholinesterase Activity Assay. Anal. Chem. 2018, 90 (3), 1643 – 1651. https://doi.org/10.1021/acs.analchem.7b02976.
16. Liao, S.; Han, W.; Ding, H.; Xie, D.; Tan, H.; Yang, S.; Wu, Z.; Shen, G.; Yu, R. Modulated Dye Retention for the Signal-On Fluorometric
Determination of Acetylcholinesterase Inhibitor. Anal. Chem. 2013, 85 (10), 4968 – 4973. https://doi.org/10.1021/ac400865t.
17. He, T.; Qi, L.; Zhang, J.; Huang, Y.-L.; Zhang, Z.-Q. Enhanced graphene quantum dot fluorescence nanosensor for highly sensitive ace-
tylcholinesterase assay and inhibitor screening. Sensors and Actuators B: Chemical 2015, 215, 24 – 29. https://doi.org/10.1016/j.
snb.2015.03.043.
18. Long, Q.; Li, H.; Zhang, Y.; Yao, S. Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophospho-
rus pesticides. Biosens. Bioelectron. 2015, 68, 168 – 174. https://doi.org/10.1016/j.bios.2014.12.046.
19. Yi, Y.; Zhu, G.; Liu, C.; Huang, Y.; Zhang, Y.; Li, H.; Zhao, J.; Yao, S. A Label-Free Silicon Quantum Dots-Based Photoluminescence Sensor
for Ultrasensitive Detection of Pesticides. Anal. Chem. 2013, 85 (23), 11464 – 11470. https://doi.org/10.1021/ac403257p.
20. Wang, M.; Gu, X.; Zhang, G.; Zhang, D.; Zhu, D. Continuous Colorimetric Assay for Acetylcholinesterase and Inhibitor Screening with
Gold Nanoparticles. Langmuir 2009, 25 (4), 2504 – 2507. https://doi.org/10.1021/la803870v.
21. Chua, A.; Yean, C. Y.; Ravichandran, M.; Lim, B.; Lalitha, P. A rapid DNA biosensor for the molecular diagnosis of infectious disease.
Biosens. Bioelectron. 2011, 26 (9), 3825 – 3831. https://doi.org/10.1016/j.bios.2011.02.040.
ISSN 2308-8303 (Print) / 2518-1548 (Online) 43
Journal of Organic and Pharmaceutical Chemistry 2022, 20 (1)
22. Golub, E.; Freeman, R.; Willner, I. Hemin/G-Quadruplex-Catalyzed Aerobic Oxidation of Thiols to Disulfides: Application of the Process for
the Development of Sensors and Aptasensors and for Probing Acetylcholine Esterase Activity. Anal. Chem. 2013, 85 (24), 12126 – 12133.
https://doi.org/10.1021/ac403305k.
23. Blazheyevskіy, M. Y.; Koval’ska, O. V.; Diadchenko, V. V. A new method for determining the cholinesterase activity. Journal of Organic
and Pharmaceutical Chemistry 2021, 19 (2), 48 – 52. https://doi.org/10.24959/ophcj.21.234269.
24. Blazheyevskіy, M. Ye.; Koval’ska, O. V. The enzymatic method for the quantitative determination of benzalkonium chloride in the
antiseptic solution “CUTASEPT® F”. Journal of Organic and Pharmaceutical Chemistry 2021, 19 (4), 33 – 39. https://doi.org/10.24959/
ophcj.21.244359.
25. Derzhavna farmakopeia Ukrainy: v 3 tomakh, 2 vydannia [The State Pharmacopoeia of Ukraine: in 3 volumes, 2nd Ed., in Ukrainian];
State Enterprise “Ukrainian Scientific Pharmacopoeial Center for Quality of Medicines”: Kharkiv, 2015; Vol. 1.
26. Parker, W. E.; Witnauer, L. P.; Swern, D. Peroxides. IV. Aliphatic Diperacids. J. Am. Chem. Soc. 1957, 79 (8), 1929 – 1931. https://doi.
org/10.1021/ja01565a043.
27. Kunec-Vajic, E.; Bradamante, V.; Uroic, B. The effect of local anesthetics and phenotiazine derivatives on cholinesterase, investigated
by a chemiluminescence method. Acta pharmaceutica Jugoslavica 1985, 35 (2), 133 – 136.
28. Sanajou, S.; Nourhashemi, S.; Fallah, A.; Ercetin, T.; Sahin, M. F.; Gulcan, H. O. The investigation of the interaction of several antipsy-
chotic drugs with human cholinesterase enzymes. EMU Journal of Pharmaceutical Sciences 2018, 1 (1), 1 – 5.
Authors information:
Vladyslav S. Kovalenko, Postgraduate Student of the Analytical Chemistry and Analytical Toxicology Department, National University
of Pharmacy of the Ministry of Health of Ukraine; https://orcid.org/0000-0003-1504-1852.
Mykola Ye. Blazheyevskіy, D.Sc. in Chemistry, Professor of the Inorganic and Physical Chemistry Department, National University
of Pharmacy of the Ministry of Health of Ukraine; https://orcid.org/0000-0002-8032-347X.
Serhii I. Merzlikin (corresponding author), D.Sc. in Pharmacy, Professor of the Analytical Chemistry and Analytical Toxicology
Department, National University of Pharmacy of the Ministry of Health of Ukraine; https://orcid.org/0000-0001-8736-7689;
e-mail for correspondence: merzlikinserg07@gmail.com; tel. +380 96 7347893.
|
| id | oai:ojs.journals.uran.ua:article-252351 |
| institution | Journal of Organic and Pharmaceutical Chemistry |
| issn | 2518-1548 |
| keywords_txt_mv | |
| language | English |
| last_indexed | 2026-08-25T01:02:38Z |
| publishDate | 2022 |
| publisher | National University of Pharmacy |
| record_format | ojs |
| resource_txt_mv | ophcjnupheduua/11/60a2e547e3ab011ae08cf0f97b498711.pdf |
| spelling | oai:ojs.journals.uran.ua:article-2523512026-08-24T13:41:04Z A comparative study of the effect of phenothiazine derivatives and their S-oxides on cholinesterase investigated by a new kinetic spectrophotometric method Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу Kovalenko, Vladyslav S. Blazheyevskiy, Mykola Ye. Merzlikin, Serhii I. ацетилхолін ацетилхолінестераза похідні фенотіазину S-оксиди фотометричні методи аналізу acetylcholine acetylcholinesterase phenothiazine derivatives S-oxides photometric methods of analysis Aim. To develop a new kinetic spectrophotometric method for determining acetylcholinesterase (AChE) inhibitors – phenothiazine antipsychotic drugs (PhT) and their sulfoxide metabolites (S-oxides) without adding an exogenous catalyst to obtain a chromogenic agent.Materials and methods. The bases of S-oxides of promethazine (PMZ), chlorpromazine (CPM) and thioridazine (THZ) were obtained by oxidizing the corresponding PhT hydrochlorides with diperoxyadipic acid. The structure of the S-oxides of the corresponding PhT synthesized was proven by melting points, spectral characteristics (1H NMR and IR methods) and oscillopolarography results. 1H NMR spectra were recorded on a Varian XL-200 spectrometer. IR spectra were recorded within the range of 4000-400 cm–1 on a SPECORD M-80 spectrometer (Zeiss, Jena, Germany). To register polarograms, a “PO 03 CLA” oscillopolarograph with a three-electrode cell was used. The purity of S-oxides was determined by the high-performance liquid chromatography method on a Zorbax SB, C-18 (250 × 4.6) mm column. Measurements of absorbance of solutions were performed in a 1 cm cuvette on an Evolution 60S UV-Visible Thermo-Scientific Spectrophotometer (USA) (λ = 358 nm).Results and discussion. Acetylcholine (ACh) was found to mimic the activity of peroxidase; based on it, a spectrophotometric system containing ACh–H2O2–p-Ph for a sensitive and selective assessment of the AChE activity and determination of its inhibitors was developed. According to the plots of inhibition efficiency vs inhibitors concentration, the inhibiting ability of chlorpromazine, promethazine and thioridazine and their S-oxides was determined. The IC50 values of CPM, PMZ and THZ and their metabolites in relation to the AChE activity were estimated as 11 ng mL−1 (CPM) and 1.8 ng mL−1 (CPM S-oxide), 17 ng mL−1 (PMZ) and 2.5 ng mL−1 (PMZ S-oxide) and 27 ng mL−1 (THZ 2S,5S-dioxide). The results obtained indicate that S-oxides of the corresponding PhT are selective and potent inhibitors of AChE. The values of the inhibition efficiency obtained for S-oxides of PhT derivatives were an order of magnitude lower than those of the corresponding PhT derivatives.Conclusions. The spectrophotometric method proposed without the addition of other exogenous catalysts holds promise for the on-site determination of PhT antipsychotics and can be additionally used for sensory applications in areas related to environmental protection and food safety, as well as in the chemical-toxicological analysis. Мета. Розробити новий кінетико-спектрофотометричний метод визначення інгібіторів ацетилхолінестерази (АChЕ), фенотіазинових антипсихотичних засобів (PhТ) та їх сульфоксидних метаболітів (S-оксидів), без додавання екзогенного каталізатора для отримання хромогенного агента.Матеріали та методи. Основи S-оксидів прометазину, хлорпромазину та тіоридазину синтезували шляхом окиснення відповідних гідрохлоридів PhТ дипероксиадипіновою кислотою. Будову синтезованих S-оксидів відповідних PhТ доводили за температурами плавлення, спектральними характеристиками (1H ЯМР- та ІЧ-методи) та результатами осцилополярографії. Спектри 1Н ЯМР записували на спектрометрі Varian XL-200. ІЧ-спектри реєстрували в діапазоні 4000-400 см−1 на спектрометрі SPECORD M-80 (Zeiss, Jena, Німеччина). Для реєстрації полярограм використовували осцилополярограф ПО, модель 03 ЦЛА з триелектродним елементом. Чистоту S-оксидів визначали методом ВЕРХ на колонці Zorbax SB, C-18 (250 × 4,6) мм. Вимірювання поглинання випробуваних розчинів S-оксидів відповідних PhТ проводили за довжини хвилі λ = 358 нм у кюветі завтовшки 1 см на спектрофотометрі Evolution 60S UV-Visible Thermo-Scienticfic (США).Результати та їх обговорення. З’ясовано, що ацетилхолін (ACh) імітує активність пероксидази, на основі чого було розроблено спектрофотометричну систему, що містить ACh-H2O2-п-фенетидин для чутливого селективного оцінювання активності АChЕ та визначення інгібіторів ферменту. Відповідно до графіків залежності інгібувальної здатності від концентрації інгібіторів визначено інгібувальну здатність хлорпромазину, прометазину, тіоридазину та їх S-оксидів. IC50 хлорпромазину, прометазину, тіоридазину та їх метаболітів щодо активності AChE оцінено так: 11 нг мл−1 – хлорпромазин, 1,8 нг мл−1 – хлорпромазин S-оксид, 17 нг мл−1 – прометазин, 2,5 нг мл−1 – прометазин S-оксид та 27 нг мл−1 – тіоридазин 2S,5S-діоксиду. Одержані результати свідчать про те, що S-оксиди відповідних PhТ є селективними та потужними інгібіторами АChЕ. Отримані значення ефективності інгібування для S-оксидів похідних PhТ були на порядок нижчими, ніж у відповідних похідних PhТ.Висновки. Розроблена спектрофотометрична методика без додавання інших екзогенних каталізаторів має перспективи для визначення PhТ нейролептиків на місці і може бути додатково використана для сенсорних застосувань у сферах, пов’язаних із захистом навколишнього середовища та безпекою харчових продуктів, а також у хіміко-токсикологічному аналізі. National University of Pharmacy 2022-05-30 Article Article application/pdf https://ophcj.nuph.edu.ua/article/view/252351 10.24959/ophcj.22.252351 Journal of Organic and Pharmaceutical Chemistry; Vol. 20 No. 1(77) (2022); 35-43 Журнал органической и фармацевтической химии; Том 20 № 1(77) (2022); 35-43 Журнал органічної та фармацевтичної хімії; Том 20 № 1(77) (2022); 35-43 2518-1548 2308-8303 en https://ophcj.nuph.edu.ua/article/view/252351/254601 Copyright (c) 2022 Vladyslav S. Kovalenko, Mykola Ye. Blazheyevskiy, Serhii I. Merzlikin http://creativecommons.org/licenses/by/4.0 |
| spellingShingle | ацетилхолін ацетилхолінестераза похідні фенотіазину S-оксиди фотометричні методи аналізу Kovalenko, Vladyslav S. Blazheyevskiy, Mykola Ye. Merzlikin, Serhii I. Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу |
| title | Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу |
| title_alt | A comparative study of the effect of phenothiazine derivatives and their S-oxides on cholinesterase investigated by a new kinetic spectrophotometric method |
| title_full | Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу |
| title_fullStr | Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу |
| title_full_unstemmed | Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу |
| title_short | Порівняльне вивчення впливу похідних фенотіазину та їх S-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу |
| title_sort | порівняльне вивчення впливу похідних фенотіазину та їх s-оксидів на холінестеразу за допомогою нового кінетичного спектрофотометричного методу |
| topic | ацетилхолін ацетилхолінестераза похідні фенотіазину S-оксиди фотометричні методи аналізу |
| topic_facet | ацетилхолін ацетилхолінестераза похідні фенотіазину S-оксиди фотометричні методи аналізу acetylcholine acetylcholinesterase phenothiazine derivatives S-oxides photometric methods of analysis |
| url | https://ophcj.nuph.edu.ua/article/view/252351 |
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