Evaluation of antiviral activity 4-dimethylaminobenzaldehyde 2-hydroxybenzoyl-, nicotinoyl- and isonicotinoylhydrazones and their chelates with sncl4 on "phage-host" model

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Mechnikov National University

Evaluation of antiviral activity 4-dimethylaminobenzaldehyde 2-hydroxybenzoyl-, nicotinoyl- and isonicotinoylhydrazones and their chelates with sncl4 on "phage-host" model

Zinchenko O.Yu., Shmatkova N.V., Seyfullina I.Y.

Аннотация

Оценка противовирусной активности 4-диметиламинобензальдегида, 2-гидроксибензоил-, никотиноил- и изоникотиноилгидразонов и их хелатов с sncl4 на модели "фаг-хозяин"

Высокий уровень современных исследований различных типов координационных соединений биометаллов [1] с биолигандами свидетельствует об их широком применении в химиотерапии в качестве противоопухолевых, бактерицидных, противовирусных, противопаразитарных и других лекарственных препаратов [2-4].

Это объясняется особенностями структурной организации хелатов металлов как объектов исследования: их образование основано, по меньшей мере, на двух биологически активных компонентах (комплексообразователь - координационный центр) и различных формах органических молекул, содержащих N, O, S, связанных с ними по-разному, включая гипервалентные, би- или полидентат.

Следует отметить, что в последние десятилетия фармацевтический рынок был насыщен различными современными противовирусными препаратами, однако проблема профилактики и этиотропной терапии наиболее распространенных вирусных инфекций до сих пор остается нерешенной. На сегодняшний день одним из наиболее эффективных способов поиска противовирусных химиотерапевтических средств является их создание на основе биокомплексов металлов. Биологические эффекты различных соединений, относящихся к классу гидразонов, изучаются уже давно. Представители этой группы проявляют антибактериальную, противовоспалительную, противосудорожную, противопаразитарную и другие виды активности, в том числе противовирусную [5-7]. Поэтому их использование в качестве биолигандов в составе биокоординированных соединений является перспективным для разработки биологически активных молекул.

В последние годы пристальное внимание уделяется соединениям олова, которые проявляют антимикробную, противовирусную и противогрибковую активность [8-10]. Систематические исследования, проведенные нами для определения стратегии синтеза и структурообразования продуктов взаимодействия тетрахлорида олова с ароил- и пиридиноилгидразонами ароматических альдегидов, выявили их зависимость в ряду: состав - структура - свойства [11-14] (включая биологическую активность [15-19]).

Было установлено, что координация гидразонов с образованием Sn(IV) сопровождается изменением распределения электронной плотности, формы (кетон-енол) и конформации их молекул. В результате биологическая активность полученного биокоординационного соединения усиливается за счет синергизма его компонентов. На эту активность существенное влияние оказывает состав координационного узла в комплексе, наличие и расположение функциональных групп донорных центров в молекуле лиганда, который связан с Sn(IV) и свободными экзогелатами, способными к протонированию [15-19].

Данное исследование является продолжением упомянутого исследования. Его целью было определение и сравнение противовирусных свойств 2-гидроксибензоил-, никотиноил-, изоникотиноилгидразонов 4-диметиламинобензальдегида и их комплексов с SnCl4 и выявление факторов, влияющих на их проявление.

Поскольку эксперименты с вирусами человека и животных связаны с достаточно высоким риском для исследователя, длительным инкубационным периодом, значительными трудовыми и материальными затратами, целесообразно использовать относительно безопасные и дешевые модели "вирус/клетка-хозяин", которые включают в себя систему “фаг-хозяин” на начальном этапе. стадия скрининга [20, 21].

Вывод. Таким образом, было установлено, что все изученные соединения характеризуются достаточно высокой способностью снижать литическую активность стафилококкового бактериофага, что может свидетельствовать о потенциальной противовирусной активности.

При изучении антифагового эффекта I-III было обнаружено, что он возрастает в зависимости от гидразидного фрагмента в молекулах гидразонов (2-гидроксибензоил-, никотиноил-, изоникотиноил-), соответственно, ряда I < II < III (рис. 3, А-В).

Наиболее эффективным оказался изоникотиноилгидразон (III), в присутствии которого активность фага снижалась на 83%. По сравнению с гидразонами комплексы SnCl4 (IV-VI) характеризовались более высокой антифаговой активностью (89-99%), а максимальный эффект продемонстрировал комплекс V с никотиноилгидразоном (ингибирование на 99%) (рис. 3, А-В).

Это свидетельствует о синергизме компонентов всех комплексов: биологически активных гидразонов и SnCl4, образующих одинаковые координационные узлы в IV-VI группах. Их различие в активности, которое наблюдалось на примере исходных гидразонов, может быть объяснено различным составом гидразидного фрагмента в молекулах лиганда.

В комплексах V, VI, в отличие от IV, протонирован атом азота гидразидного фрагмента, а не альдегидного фрагмента (IV). Это сопровождалось изменением распределения электронной плотности в полученных молекулах, что способствовало повышению противовирусной активности комплексов в ряду IV < VI < V.

Кок и соавторы продемонстрировали корреляцию между противовирусной активностью растительных экстрактов против некоторых вирусов человека в культуре клеток MDCK и антифаговой активностью в модели бактериального фага MS2, доказав возможность использования таких моделей для первичного скрининга потенциальных противовирусных соединений [20]. Фаг MS2 относится к РНК-содержащим вирусам, в то время как известные фаги S. aureus относятся к отряду Caudovirales, которые являются ДНК-содержащими фагами [23-25]. Ряд этиологических возбудителей опасных заболеваний человека, таких как вирусы герпеса, папилломы и полиомавирусы, поксвирусы, имеют ДНК-геном. Следовательно, использование предложенной модели может оказаться целесообразным при определении активности новых соединений в отношении этих патогенов.

Наиболее активные комплексы с никотиноил- и изоникотиноилгидразонами могут быть рекомендованы для изучения активности в отношении вирусов млекопитающих на модели клеточной культуры.

Introduction

The high level of modern research of various types of coordination compounds of biometals [1] with bioligands indicates their widespread use in chemotherapy as antitumor, bactericidal, antiviral, antiparasitic and other pharmaceuticals [2-4].

This is explained by the peculiarities of the metal chelates structural organization as objects of study: their formation based on at least two biologically active components (complexing agent - a coordination center - and different forms of N, O, S-containing organic molecules, related to it in different ways including hypervalent, bi- or polydentate.

It should be noted that in recent decades the pharmaceutical market has been saturated with various latter-day antiviral drugs, but the problem of prevention and etiotropic therapy of the most common viral infections still remains unresolved. Today, one of the most effective ways to find antiviral chemotherapeutic agents is their creation on the base of metal biocomplexes. The biological effects of various compounds belonging to the hydrazones class have been studied for a long time. Representatives of this group demonstrate antibacterial, anti-inflammatory, anticonvulsant, antiparasitic and other types of activity, including antiviral [5-7]. Therefore, their use as bioligands in the composition of biocoordinated compounds is promising for the development of biologically active molecules.

In recent years, undiverted attention has been paid to tin compounds that show antimicrobial, antiviral and antifungal activity [8-10]. Systematic studies we carried out to determine the synthesis strategy and structure formation of the interaction products of tin tetrachloride with aroyl- and pyridinoylhydrazones of aromatic aldehydes revealed their dependence in the row: composition - structure - properties [11-14] (including biological activity [15-19]).

It was identified that coordination of hydrazones to Sn(IV) is accompanied by a change in the distribution of electron density, form (ketone-enol) and conformation of their molecules. As a result, the biological activity of the obtained biocoordination compound is intensified due to the synergism of its components. This activity is significantly influenced by the composition of the coordination node in the complex, the presence and location of functional groups of donor centers in the ligand molecule that is associated with Sn(IV) and vacant exohelates capable of protonation [15-19].

This study is the continuation of mentioned research. Its objective was to determine and compare the antiviral properties of 2-hydroxybenzoyl-, nicotinoyl-, isonicotinoylhydrazones of 4- dimethylaminobenzaldehyde and their complexes with SnCl4 and identification of factors influencing their display.

As far as the experiments with human and animal viruses are associated with a high enough risk for a researcher, long incubation period, significant labor and material costs, it is advisable to use relatively safe and cheap models of "virus / host cell", which include the “phage-host” system at the screening stage [20, 21].

Presentation of the main material

2-hydroxybenzoyl- (I), nicotinoyl- (II) and isonicotinoylhydrazones (III) of 4-dimethylaminobenzaldehyde, obtained by the condensation of hydrazides of 2-hydroxybenzoic, nicotinic and isonicotinic acids by general method [6, 7], as well as their complexes IV-VI with SnCl4, synthesized at the Department of Non-organic Chemistry and Chemical Education of Odessa National Mechnikov University by the interaction of SnCl4 with hydrazones in acetonitrile [11, 12] were used in the study.

Fig. 1. Structures of

Note: I - 2-hydroxybenzoylhydrazone of 4-dimethylaminobenzaldehyde, II - nicotinoylhydrazone of 4-dimethylaminobenzaldehyde, III - isonicotinoylhydrazone of 4-dimethylaminobenzaldehyde, IV - Sn(IV) complex with 2-hydroxybenzoylhydrazone of 4-dimethylaminobenzaldehyde, V - Sn(IV) complex with nicotinoylhydrazone of 4-dimethylaminobenzaldehyde, VI - Sn(IV) complex with isonicotinoylhydrazone of 4-dimethylaminobenzaldehyde.

The original hydrazones I-III and their complexes IV-VI were characterized by a set of elemental analysis, spectral, mass spectrometric, diffraction methods [11, 12]. Their structures are shown in Fig.1. Structure VI was proved by the X-ray diffraction of single crystals method and registered in the database of the Cambridge Bank [CIF files CCDC 1055248].

Complexes IV-VI belong to special zwitterionic compounds with the same enol tautomeric form of a bidentate coordinated ligand protonated on a vacant nitrogen atom. Its charge is compensated by a negative, focused on the coordination node {SnCEON}:

The antiphage activity of compounds I-VI was tested on a model system containing Staphylococcus aureus ATCC 25923 obtained from the culture collection of L. Hromashevskyi Institute of Epidemiology and Infectious Diseases NAMSU and polyvalent staphylococcal bacteriophage. Commercial preparation “Staphylococcal bacteriophage” (Biopharma, Ukraine) was used in used in the study.

To determine the antibacterial activity of compounds I-VI, working solutions containing were prepared and used to obtain two-fold dilutions of compounds in Hiss medium with final concentrations 25, 50 and 100 pM. Test tubes with compounds were autoclaved at 120 °С. Each concentration was tested in fiveplicates.

Overnight culture of S. aureus ATCC 25923 was used to prepare inoculum in sterile saline. The inoculum density was 2*10⁴ CFU/ml. Each test tube containing 1 ml of Hiss medium with studied compounds was inoculated by 50 pl of the obtained inoculum, so that the final concentration of cells was 1-10³ CFU / ml. antibacterial viral infection chelate biometal

The biomass accumulutaion by the test strain was determined by measuring the turbidity of the culture at a wavelength of 540 nm after 24 hours of incubation at 37 ⁰C. Intact culture of S. aureus, grown in the same conditions was used as control.

The antiphage activity of compounds I-VI was determined by a modified Gracia method of bacteriophage titration [22]. Phage suspension in saline (concentration 1,6*10⁹ PFU/ml) was previously incubated in the presence of 25, 50 and 100 pM of studied compounds at 37 0C during 1 h. 100 pl of this suspension and equal amount of S. aureus ATCC 25923 cell suspension were mixed with 1 ml of semi-solid nutrient agar and poured on agar plates in Petri dishes.

Each concentration of test compounds was tested in five replicates, and the experiment was repeated twice. Each experiment was accompanied by two controls: control of bacteriophage activity - option without the addition of compounds I-VI, control of bacterial culture growth - option without the addition of phage. Results were evaluated after 24 h of incubation at 37 0C by counting the PFU number on the bacterial lawn in experimental and control variants. Antiphage activity (A) was calculated as the percentage of inactivation by the formula:

A= (1 - No/Nk)x100 %,

where No is the number of negative colonies in the experiment, Nk is the number of negative colonies in the control.

The statistical significance of the differences was determined by the nonparametric Mann-Whitney test, estimating the probability of the obtained results at a significance level of at least 95% (p < 0.05).

The effect of studied compounds I-VI on the growth of a bacterial test strain. Compounds that are tested for antiphage properties have to be pre-tested for their possible inhibitory activity towards bacterial culture used as a phage-sensitive model.

Therefore, the first stage of our work was to determine the effect of aroylhydrazones (I-III) and their complexes (IV-VI) on the growth of S. aureus ATCC 25923 culture. It was found (Fig. 2) that most compounds in the concentrations used do not inhibit bacterial growth. Insignificant delay in biomass accumulation was observed only in the presence of 25 pM of complexes with nicotinoyl- (V) and isonicotinoylhydrazones (VI), but it did not exceed 20%.

Fig- 2The effect of I-VI on the growth of S. aureus ATCC 25923

Note: * - the difference is probable in comparison with the control (p <0,05).

Thus, it was determined that I-VI in selected concentrations can be tested for antiphage properties.

Influence of I-VI on lytic activity of polyvalent staphylococcal bacteriophage. The highest activity among hydrazones showed compound III, which caused significant inhibition of bacteriophage activity in all used concentrations (Fig. 3, C), with the greatest effect at 100 pM. In this case, the antiphage activity reached 83,7%, while at concentrations of 50 and 25 pM it decreased - 79.4% and 72.2% compared to the control, respectively.

Compound II showed the highest antiphage activity at the concentration of 50 pM. Phage inactivation was 35,8% compared to control. The change in concentrations (25 gM and 100 gM) reduced the activity, but did not significantly affect the respective values of phage activity - 50,3% and 52,2% (Fig. 3, B).

Compound I as well as compound III was active in all studied concentrations and demonstrated direct dose-dependent effect concentration, but inhibition of bacteriophage activity at concentrations of 25, 50 and 100 gM was only 48,5%, 57,6% and 59,1%, respectively (Fig. 3, A).

Fig. 3. Influence of I-VI on the activity of staphylococcal bacteriophage: A) I andIV, B) II and V, C) - III and VI.

Evaluation of the effect of compounds III-VI on the lytic activity of phage showed that the most potent antiphage effect was demonstrated by complex V with nicotinoylhydrazone: bacteriophage inhibition in the presence of 50 gM of this compound was 99,1% compared to control. Substance concentrations of 25 and 100 gM had a pronounced inhibitory effect - 98,2 and 96,4%, respectively (Fig. 3, B).

The same pattern was observed for complex IV, but in the opposite direction: antiphage activity was maximal (95,2%) at a substance concentration of 50 gM, and at concentrations of 25 and 100 gM - 86,3% and 88,1%, respectively (Fig. 3, A).

Compound VI showed an increase in antiphage activity with increasing concentration. Thus, at a concentration of 25 gM the antiphage activity was 81,6%, for concentrations of 50 and 100 gM - 85,8% and 89,2%, respectively (Fig. 3, C).

Conclusion

Thus, it was found that all studied compounds are characterized by a quite high ability to reduce the lytic activity of staphylococcal bacteriophage that can be evidence of potential antiviral activity.

When studying the antiphage effect of I-III, it was found that it increases depending on the hydrazide fragment in the molecules of hydrazones (2-hydroxybenzoyl-, nicotinoyl-, isonicotinoyl-), respectively, the series I < II < III (Fig. 3, A-C).

The most effective was isonicotinoylhydrazone (III), in the presence of which the activity of phage was reduced by 83%. In comparison with hydrazones, SnCl4 (IV-VI) complexes were characterized by higher antiphage activity (89-99%), and the maximum effect was shown by complex V with nicotinoylhydrazone (99% inhibition) (Fig. 3, A-C).

This indicates the synergism of the components of all complexes: biologically active hydrazones and SnCl4, forming the same coordination nodes in IV-VI. Their difference in activity which was observed on the example of the original hydrazones can be explained by different composition of the hydrazide fragment in the ligand molecules.

In complexes V, VI, in contrast to IV, the nitrogen atom of the hydrazide fragment, not the aldehyde fragment (IV), was protonated. This was accompanied by a change in the electron density distribution in the resulting molecules, which contributed to an increase in the antiviral activity of the complexes in the series IV < VI < V.

Cock and co-authors demonstrated a correlation between the antiviral activity of plant extracts against some human viruses in MDCK cell culture and the antiphage activity in the MS2-bacterial phage model, proving the feasibility of using such models for primary screening of potential antiviral compounds [20]. Phage MS2 belongs to RNA-containing viruses, while known phages of S. aureus belong to the order Caudovirales, which are DNA-containing phages [23-25]. A number of etiological agents of dangerous human diseases, such as herpesviruses, papilloma and polyomaviruses, poxviruses, have DNA genome. Therefore, the use of the proposed model may be appropriate in determining the activity of new compounds against these pathogens.

The most active complexes with nicotinoyl- and isonicotinoylhydrazones can be recommended to study the activity against mammalian viruses in a cell culture model.

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