КООРДИНАЦИОННЫЕ СОЕДИНЕНИЯ МЕДИ(II) С ПРОИЗВОДНЫМИ ТИОСЕМИКАРБАЗИДА В КАЧЕСТВЕ ИНГИБИТОРОВ СУПЕРОКСИДНЫХ РАДИКАЛОВ | Опубликовать статью ВАК, elibrary (НЭБ)

КООРДИНАЦИОННЫЕ СОЕДИНЕНИЯ МЕДИ(II) С ПРОИЗВОДНЫМИ ТИОСЕМИКАРБАЗИДА
В КАЧЕСТВЕ ИНГИБИТОРОВ СУПЕРОКСИДНЫХ РАДИКАЛОВ

Научная статья

Андронаке Л.^1, *, Гуля А.², Цапков В.³, Граур В.⁴, Пантя В.⁵, Швец И.⁶,
Матсовский В.⁷, Лысый Д.⁸, Ботнару М.⁹, Гудумак В.¹⁰

¹ ORCID: 0000-0002-8781-8037;

² ORCID: 0000-0003-2010-7959;

³ ORCID 0000-0003-1732-3116;

⁴ ORCID: 0000-0001-8153-2153;

⁵ ORCID: 0000-0002-8835-6612;

⁶ ORCID: 0000-0001-6059-1170;

⁷ ORCID: 0000-0001-8153-2153;

⁸ ORCID: 0000-0002-1141-1441;

⁹ ORCID: 0000-0003-4577-0466;

¹⁰ ORCID: 0000-0001-9773-1878;

^{1, 5, 6, 7, 8, 10} Государственный университет медицины и фармации им. Н. Тестемицану, Кишинёв, Молдова;

^{2, 3, 4, 9} Молдавский Государственный Университет, Кишинёв, Молдова

* Корреспондирующий автор (lilia.andronache[at]usmf.md)

Аннотация

Получены новые биологически активные координационные соединения меди(II) на основе замещенных 4-аллилтиосемикарбазонов 3-(фенил)-1- (пиридин-2-ил)проп-2-ен-1-онов. Установлено, что эти соединения проявляют сильные антирадикальные свойства при взаимодействии с супероксидным радикалом. Благодаря этому свойству полученные соединения могут найти широкое применение в медицине в качестве ингибиторов супероксидных радикалов в организме человека, предотвращая, таким образом, повреждение клеток и тканей, многофакторные заболевания, в том числе канцерогенез. Синтезируемые координационные соединения расширяют арсенал ингибиторов супероксид-радикалов, обладающих высокой биологической активностью. Обсуждается их возможное значение для разработки новых стратегий лечения заболеваний, связанных с гиперпродукцией супероксидных радикалов.

Ключевые слова: ингибиторы супероксид-радикалов, координационное соединение, тиосемикарбазоны.

COPPER(II) COORDINATION COMPOUNDS WITH THIOSEMICARBAZIDE DERIVATIVES
AS INHIBITORS OF SUPEROXIDE RADICALS

Research article

Andronake L.^1, *, Gulya A.², Tsapkov V.³, Graur V.⁴, Pantya V.⁵, Shvets I.⁶,
Matusovsky V.⁷, Lisii D.⁸, Botnaru M.⁹, Gudumak V.¹⁰

¹ ORCID: 0000-0002-8781-8037;

² ORCID: 0000-0003-2010-7959;

³ ORCID 0000-0003-1732-3116;

⁴ ORCID: 0000-0001-8153-2153;

⁵ ORCID: 0000-0002-8835-6612;

⁶ ORCID: 0000-0001-6059-1170;

⁷ ORCID: 0000-0001-8153-2153;

⁸ ORCID: 0000-0002-1141-1441;

⁹ ORCID: 0000-0003-4577-0466;

¹⁰ ORCID: 0000-0001-9773-1878;

^{1, 5, 6, 7, 8, 10} State University of Medicine and Pharmacy “Nicolae Testemitanu”, Chisinau, Republic of Moldova;

^{2, 3, 4, 9} Moldavian State University, Chisinau, Republic of Moldova

* Corresponding author (lilia.andronache[at]usmf.md)

Abstract

New biologically active coordination compounds of copper(II) based on substituted 3-(phenyl)-1-(pyridin-2-yl)prop-2-en-1-one 4-allylthiosemicarbazones have been obtained. It was found that these compounds exhibit strong antiradical properties when interacting with a superoxide radical. Due to this property, the obtained compounds can find wide application in medicine as inhibitors of superoxide radicals in the human body, thus preventing damage of cells and tissues, multifactorial diseases, including carcinogenesis. The synthesized coordination compounds expand the arsenal of superoxide radical inhibitors with high biological activity. Their possible significance for the development of new treatment strategies for diseases associated with the overproduction of superoxide radicals discussed.

Keywords: superoxide radical inhibitors, coordination compounds, thiosemicarbazones. 

Introduction

Monovalent reduction of molecular oxygen leads to the birth of the radical or superoxide anion (O₂^.-) – a product of cardinal importance that is involved in various chemical and biological systems [1], [2] and which today attracts special attention from scientists from various domains. Due to its high reactivity, superoxide radical is involved in multiple pathological conditions, such as acute and chronic inflammation, reperfusion lesions, metabolic disorders, cellular aging, multifactorial pathologies and carcinogenesis [3], [4], [5], [6].

Therefore, the inhibition of the superoxide radical (SR) represents a new contribution, because the substances with pronounced antiradical activity have a strong therapeutic effect, thus preventing their multiple harmful effects on the human body.

The purpose of the investigation is to obtain new copper coordination compounds (CC) with thiosemicarbazide derivatives, to evaluate their chemical properties, in particular, to investigate their influence on peroxidation processes with SR and to estimate and select the most active compounds that could be used for medication and prevention of pathologies caused by exacerbation of SR.

Material and methods

In the Laboratory “Advanced materials in the biopharmaceutical and technical field” of State University of Moldova, were obtained and examined the properties of a series the new biologically active CC with thiosemicarbazide derivatives, established their formulas, physico-chemical properties ( Scheme 1), but their influence on prooxidative processes with ROS, such as the SR not was evaluated [7], [8], [9].

SR scavenging activity was evaluated by spectrophotometric method [10], [11] with modifications and adapted for application to the Synergy H1 Multi-Mode hybrid microplate reader (BioTek Instruments, USA). First, the working dilutions of the tested compounds in DMSO solution (1.0; 10.0; 100.0 and 1000.0 µmol/l) was made ready for use. Next, 20 µl of each dilution of the tested compounds was introduced into the 96-well microplate wells and 180 µl of reaction mixture with 0.02 M phosphate buffer (pH 7.4), 0.1 mM NADH, and 0.09 mM nitro-blue tetrazolium (NBT) was added. Thus, the final concentration of the tested compounds was 0.1; 1.0; 10.0 and 100.0 µmol/l. Each dilution was prepared in duplicate. The control samples was prepared in duplicate in the same way as the test samples, but the dilutions of tested compounds was replaced with an equivalent amount of solution containing 0.02 M phosphate buffer (pH 7.4). The contents of microplate were shaken for 10-15 s and optical density (OD) was measured at 560 nm [OD₀]. Then, in each wells, 20 μl of 8.0 μM solution of phenazine metosulfate (PMS) was added, the microplate were shaken for 10-15 s and the samples was incubated at room temperature in the dark for 5 minutes and then optical density [OD₁] at 560 nm was appreciated again. The percentage of SR scavenging activity was calculated according to the equation:

SR scavenging activity (%) = [(OD₀ –OD₁)/OD₀ x 100];

where: OD₀ is the optical density of control compounds; OD₁ is the optical density of the tested compounds or standard and/or reference substances.

As a standard for establishing of SR scavenging activity was used a natural flavonol Quercetin (3,3′,4,5,6-pentahydroxyflavone) [12], [13].

The disadvantage of quercetin is that it has a low antiradical activity [semimaximal inhibition concentration (IC₅₀) is only 61.86 ± 2.5 μmol / L] and also this compound can cause serious medicinal side effects.

Among the synthetic CC with high described SR activity [14], the highest inhibitory effect was obtained in the case of bis (m₂-acetate-o)-bis {[N-prop-2-en-1-yl-N ‘- (pyridin-2-ylme thyl-idene) carbamo-hydrazonothioate] copper} dihydrate (pyridin-2-ylme thyl-idene) carbamo-hydrazonothioate] copper} dihydrate) (structural prototype).

This compound has the semimaximal SR inhibition concentration IC₅₀ = 0.35±0.07μmol / L [14].

Results and discussion

A series of new copper(II) CC on the base of substituted 3-(phenyl)-1-(pyridin-2-yl)prop-2-en-1-one 4-allylososemicarbazones with general formula:

I: R¹ = N(CH₃)₂, R² = H, X = Cl^–; II: R¹ = N(CH₃)₂, R² = H, X = NO₃^–;

III: R¹ = OCH₃, R² = H, X = Cl^–; IV: R¹ OCH₃, R² = H, X = NO₃^–;

V: R¹ = R² = OCH₃, X = Cl^–; VI: R¹ = R² = OCH₃, X = NO₃^–.

was synthesized and their physico-chemical and pharmacological properties, such as antimicrobial, antioxidant and antiproliferative activity have been described by us in a number of publications [8], [9], [15]. It was established that the II-VI CC possess a high SR activity with IC₅₀ values of 0.20-0.32 μmol / L, which is 193 – 309 times higher than the activity of quercetin, used in medicine as a standard for the assessment of SR scavenging activity and 1.1 – 1.8 times more efficient than the structural prototype (Table 1).

 

Table 1 – The anti-SR activity of the tested substance compared to quercetin and the structural prototype

№	Compound	IC50, μmol/L
	Quercetin (standard)	61,86±2,5
I	Bis(m2-chloro)-bis(N’-{3-[4-(dimethylamino)phenyl]-1-(pyridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamohydrazonothioato)-di-copper(II) (structural prototype)	0,35±0,07
II	Bis(m2-nitrato)-bis(N’-{3-[4-(dimethylamino)phenyl]-1-(pyridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamohydrazonotioato)-di-copper(II)	0,20±0,08
III	Bis(m2-chloro)-bis(N’-{3-[4-methoxyphenyl]-1-(pyridin-2-yl)prop-2-enyliden}-N-prop-2-en-1-ylcarbamohydrazonothioato)-di-copper(II)	0,28±0,07
IV	Bis(m2-nitrato)-bis(N’-{3-[4-methoxyphenyl]-1-(pyiridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ilcarbamohydrazonothioato)-di-copper(II)	0,32±0,12
V	Bis(m2-chloro)-bis(N’-{3-[3,4-dimethoxyphenyl]-1-(pyiridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamo-hidrazonothioato)-di-copper(II)	0,26±0,08
VI	Bis(m2-nitrato)-bis(N’-{3-[3,4-dimethoxyphenyl]-1-(pyridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamohydrazonothioato)-di-copper(II)	0,20±0,06

 

The detected property of the above indicated compounds II – VI are new, because their use as inhibitors of SR has not been known until now.

The comparative analysis of the CC II – VI with the structural prototype indicates that they not differ because they belong to the same classes of the chemical compounds and in the mentioned compounds a new chemical combination of already known chemical bonds have been made [15], [16].

The synthesis process of the indicated above I – VI CC is simple in execution, the yield is 70 – 77% compared to the one calculated theoretically. The synthesized complexes have a dark green color, are stable on contact with air, easily soluble in water and aliphatic alcohols and are soluble in dimethylformamide and dimethylsulfoxide, practically insoluble in ether [15], [16].

The mentioned CC I – VI was obtained by the template method at the interaction of hot ethanolic solutions (50-55^oC) with of copper chloride dihydrate (II) (complexes I, III and V) or copper (II) nitrate (II, IV, VI) with 4- allylthiosemicarbazide [N- (prop-2-en-1-yl) hydrazinecarbothioamide] and 3- [4- (dimethylamino) phenyl] -1- (pyridin-2-yl) prop-2-en-1-one (compounds I and II), 3- [4-methoxyphenyl] -1- (pyridin-2-yl) prop-2-en-1-one (compounds III and IV) or 3- [4-3,4-dimethoxyphenyl] -1 – (pyridin-2-yl) prop-2-en-1-one (compounds V and VI), which were taken in a molar ratio of 1: 1: 1.The reaction proceeds in 50-60 min according to the following scheme:

I: R¹ = N(CH₃)₂, R² = H, X = Cl^–; II: R¹ = N(CH₃)₂, R² = H, X = NO₃^–;

III: R¹ = OCH₃, R² = H, X = Cl^–; IV: R¹ OCH₃, R² = H, X = NO₃^–;

V: R¹ = R² = OCH₃, X = Cl^–; VI: R¹ = R² = OCH₃, X = NO₃^–

Fig. 1 – Scheme of the reaction

The mechanism of the present reaction is related that during the synthesis in the reaction mixture takes place the template condensation of 4-allylthosemicarbazide [N-(prop-2-en-1-yl)hydrazinecarbothioamide] with the corresponding chalcone {3- [4-(dimethyl-amino)phenyl]-1-(pyridin-2-yl)prop-2-en-1-one, 3- [4-methoxyphenyl]-1-(pyridin-2-yl)-prop-2-en-1-one or 3-[4-3,4-dimethoxyphenyl]-1-(pyridin-2-yl)prop-2-en-1-one} and the formation of the substituted thiosemicarbazone. The azomethines formed, in the presence of the pyridine nitrogen of the ligands, which fulfills the function of proton acceptor, deprotonate on the place of the thiol groups and coordinate at the copper(II) ion as monodeprotonated N,N,S-tridentate ligands. The fourth place in the inner sphere of the central atom is occupied by the chlorine or oxygen atom of the nitrate-ion composition of the neighboring molecule. In turn, in the neighboring molecule the fourth coordination place is occupied by the chlorine or oxygen atom of the nitrate ion in the first fragment of the complex [9], [15].

Example of obtaining bis(m₂-chloro)-bis(N’-{3-[4-(dimethylamino) phenyl]-1-(pyridin-2-yl)prop-2-enylidene}-N-prop-2-en-1-ylcarbamohydra-zonothioate)-di-copper(II) (compound I) [9], [15].

To the solution containing 10 mmol of copper(II) chloride dihydrate in 20 ml of ethanol, heated to 50-55 °C and constantly stirred with a magnetic stirrer, a solution containing 10 mmol of 4-allylthiosemicarbazide and 10 mmol of 3-[4-(dimethylamino)phenyl]-1-(pyridin-2-yl)prop-2-en-1-one in 50 ml of ethyl alcohol was added. After that, the reaction mixture was further heated with ascending refrigerant for 50-60 min. Upon cooling small dark green crystals were deposited, which were first filtered through a glass filter, washed with C₂H₅OH, ether and finally air dried [9], [15].

After analogous method, using as starting substances copper(II) nitrate trihydrate (compounds II, IV and VI) or copper(II) chloride dihydrate (compounds III and V) and 4-(2,6-dimethylphenyl)- (II), 4 – (2,5-dimethylphenyl) – (III and IV), 4- (3,4-dimethylphenyl) – (V) and 3- [4- (dimethylamino) phenyl] -1- (pyridin-2-yl) prop -2-en-1-one (compound II), 3- [4-methoxyphenyl] -1- (pyridin-2-yl) prop-2-en-1-one (compounds III and IV) or 3- [4 -3,4-dimethoxyphenyl] -1- (pyridin-2-yl) prop-2-en-1-one (compounds V and VI), taken in a 1: 1: 1 molar ratio, the compounds II-VI was obtined. Their chemical names and some physico-chemical characteristics are presented in tables 2 and 3 [9], [15].

 

Table 2 – Name and results of the chemical analysis of some new copper(II) CC with thiosemicarbaside derivatives

Compound	Chemical Name	The raw formula	Random %	Determined / calculated, %
				Cl	Cu	N	S
I	Bis(m2-chloro)-bis(N’-{3-[4-(dimethylamino)phenyl]-1-(pyridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamohydrazonothioato)-di-copper(II)	C40H44Cl2Cu2N10S2	71	7,41 / 7,65	13,47 / 13,71	14, 90 / 15,11	6,70 / 6,92
II	Bis(m2-nitrato)-bis(N’-{3-[4-(dimethylamino)phenyl]-1-(pyridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamohydrazonotioato)-di-copper(II)	C40H44Cu2N12O6S2	70	–	12,71 / 12,97	16,88 / 17,15	6,27 / 6,54
III	Bis(m2-chloro)-bis(N’-{3-[4-methoxyphenyl]-1-(pyridin-2-yl)prop-2-enyliden}-N-prop-2-en-1-ylcarbamohydrazonothioato)-di-copper(II)	C38H38Cl2Cu2N8O2S2	75	7,59 / 7,87	13,87 / 14,11	12,15 / 12,44	6,85 / 7,12
IV	Bis(m2-nitrato)-bis(N’-{3-[4-methoxyphenyl]-1-(pyiridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ilcarbamohydrazonothioato)-di-copper(II)	C38H38Cu2N10O8S2	72	–	13,07 / 13,32	14,40 / 14,68	6,49 / 6,72
V	Bis(m2-chloro)-bis(N’-{3-[3,4-dimethoxyphenyl]-1-(pyiridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamo-hidrazonothioato)-di-copper(II)	C40H42Cl2Cu2N8O4S2	77	7,17 / 7,38	12,85 / 13,23	11,37 / 11,66	6,40 / 6,67
VI	Bis(m2-nitrato)-bis(N’-{3-[3,4-dimethoxyphenyl]-1-(pyridin-2-yl)prop-2-en-yliden}-N-prop-2-en-1-ylcarbamohydrazonothioato)-di-copper(II)	C40H42Cu2N10O10S2	74	–	12,28 / 12,53	13,55 / 13,81	6,07 / 6,32

 

Table 3 – Some physico-chemical properties of new copper (II) CC with thiosemicarbaside derivatives

Compound	æ, Ω-1. cm2’. mol-1	μef., m. B. (293 K)	Some absorption bands (cm-1) present in IR spectra of compounds I – VI
			ν(OCH3)	ν(C=C)	ν(C=N)	ν(˃C=N-N=C˂)	δ(C-N)	ν(C-S)	ν(C-N)	ν(Cu-N), ν(Cu-S)
I	2	1,12	–	1650, 1645	1600, 1596	1570	1197, 1152	747	1030, 949	530, 462, 417
II	4	1,44	–	1652, 1642	1576, 1594	1562	1206, 1155	740	1025, 947	527, 451, 430
III	3	1,21	2830	1658, 1642	1605, 1597	1561	1201, 1152	750	1039, 953	520, 450, 422
IV	5	1,36	2832	1656, 1647	1575, 1593	1567	1204, 1160	742	1020, 945	530, 449, 412
V	3	1,20	2837	1652, 1644	1600, 1594	1572	1197, 1163	748	1032, 947	524, 450, 420
VI	5	1,38	2838	1654, 1646	1578, 1594	1568	1205, 1155	744	1025, 947	525, 452, 425

Note:  æ – molar conductivity in dimethylformamide (293 K)

 

Visual microscopic examination of the obtained CC demonstrates that they possess physical homogeneity. Due to the small size and absence of single-crystals of these complexes, the elemental analysis, IR spectroscopy and magnetochemistry method were used to determine their individuality and structure.

Based on the determination of the molar conductivity (æ) of CC I – VI in dimethylformamide, it was established (Table 2) that they are non-electrolyte [æ = 2-5 Ω^-1·cm²·mol^-1, 20 °C, c = 0.001 mol / L].

Magnetochemical study at room temperature (293 K) of the mentioned CC showed (Table 3) that they possess low effective magnetic moments (m_ef. = 1.1 – 1.4 m.B) compared to the spinal ones (S = ½), which demonstrates their polynuclear structure.

In order to determine the coordination of ligands to the copper(II) ion, a comparative analysis of the IR spectra of the tested CC with those of thiosemicarbazide, initial chalcones and copper complexes with 4-allythiosemicarbazones has been evaluated [9], [15].

It was was found (Table 3) that the thiosemicarbazones in complexes I – VI behave as monodeprotonized tridentate ligands, coordinating at the central ion through pyridine and azomethine nitrogen atoms and sulfur, forming two metallocycles of five atoms [15], [16].

Using of CC II-VI expands the arsenal of synthetic small molecular compounds with high SR scavenging activity.

As mentioned, repeated exposure to these radicals is considered a major cause of aging, neurodegenerative and inflammatory pathologies due to destruction of major cellular constituents such as DNA, proteins, carbohydrates and lipids.

The SR from a biochemical point of view, can be generated from two major sources: the mitochondrial respiratory chain and NADPH oxidase (an enzymatic complex found in the plasma membrane as well as in phagosocytes with a protective role in destruction of invasion of microorganisms). Overproduction of mitochondrial SR can contribute to DNA damage, metabolic oxidative stress, genomic instability, mutagenesis and, ultimately, tumorigenesis, as well as a multitude harmful actions on the body [16], [17], [18].

Mitochondria are a pivotal source of cellular ROS because they have several identified sites for the production of SR and hydrogen peroxide (H₂O₂) related to electron transport chain [19,20]; the excess plays a critical role in pathogenesis of many diseases, especially, ischemic heart disease, such as acute myocardial infarction during adaptive and pathological remodeling of the myocardium and vascular lesions during atherogenesis [21,22], as well as in Coronavirus infection (COVID-19) [23], [24], [25]. In this context, CC with high SR inhibition activity could offer notable benefits in fight against SARS-CoV-2 infection, so that in-depth studies in this direction have undeniable value and importance.

There is a lot of evidence that in-depth knowledge of pathogenesis of molecular mechanisms involved in COVID-19 will allow detection of effective treatment approaches, but also in assessing of prognosis and evolution of this particularly serious disease [26], [27].

Further research is needed to confirm the therapeutic utility of these bioactive compounds in various pathologies.

Финансирование Это исследование поддержано Государственной программой Республики Молдова (2020-2023) (исследовательский грант № 20.80009.5007.10).	Funding This study supported by the State Program (2020-2023) of the Republic of Moldova (research grant No. 20.80009.5007.10).
Конфликт интересов Не указан.	Conflict of Interest None declared.