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Saint-Petersburg University

Manuscript copyright

Mikolaichuk Olga Vladislavovna

SYNTHESIS AND STUDY OF THE PROPERTIES OF NEW MATERIALS WITH ANTI-TUMOUR ACTIVITY BASED ON POLYNITROGENIC HETEROCYCLES

Scientific specialty 1.4.16. Medical chemistry

The dissertation is submitted for the degree of Candidate of Chemical Sciences

Academic supervisors:

Doctor of Biological Sciences,

Sharoyko Vladimir Vladimirovich

Doctor of Chemical Sciences,

Semenov Konstantin Nikolaevich

Saint Petersburg

2023

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	CONTENTS
LIST OF ABBREVIATIONS .....................................................................................................	147
INTRODUCTION .......................................................................................................................	148
CHAPTER 1. LITERATURE REVIEW.....................................................................................	153

1.METHODS FOR THE SYNTHESIS OF TRI-SUBSTITUTED 1,3,5-

TRIAZINES WITH ANTI-TUMOUR ACTIVITY........................................................								153
1.1. Cyclisation reactions......................................................................................								153
1.2.	Reactions	of	nucleophilic		substitution	of	chlorine atoms	in
2,4,6-trichloro-1,3,5-triazine derivatives.........................................................................								159
1.3.	Preparation	of	hybrid	polynitrogenous		heterocyclic derivatives		of
1,3,5-triazine ....................................................................................................................								165
CHAPTER 2. NANOFORM OF DRUGS ..................................................................................								172
CHAPTER 3. RESULTS AND DISCUSSION					..........................................................................			178
3.1. Synthesis of compound 1.57..........................................................................								178
3.2. Synthesis of hybrid triazinyltetrazole ............................................................								179
3.2.1. Synthesis of starting tetrazole- ................................containing derivatives								179
3.3. Synthesis and identification of the .................................GO-1.57 conjugate								182
3.4. Study of the physicochemical properties of aqueous solutions of
compound 1.57 ................................................................................................................								187
3.4.1. Density of aqueous solutions of ........................................compound 1.57								188
3.4.2. Viscosity of aqueous solutions ...............................of the compound 1.57								190
3.4.3. Refractions of aqueous solutions ............................of the compound 1.57								193
3.4.4. Mathematical description of T-C dependences of density, viscosity and
refractive index of aqueous solutions of compound ................................................1.57								196
3.4.5. Solubility of compound 1.57 in ........................................................water								199
3.4.6. Distribution of compound 1.57 ...............in the n-octanol – water system								199
3.4.7.	Study of	the	stability	of	aqueous solutions of		compound 1.57	by
NMR spectroscopy ..........................................................................................................								200
3.5. Biocompatibility of compound 1.57 ..............................................................								203
3.5.1. Haemocompatibility ...................................................................................								203
3.5.2. Study of binding to DNA and HSA............................................................								206
3.5.3. Antioxidant activity ....................................................................................								213
3.5.4. Genotoxicity of compound 1.57 .................................................................								219

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3.5.5. Cytotoxicity of compound 1.57 ..................................................................		222
3.5.6. Mitochondrial potential ..............................................................................		223
3.6. Study of the physicochemical properties of aqueous solutions of
compound 3.6 ..................................................................................................................		224
3.6.1. Stability of compound 3.6 in aqueous solutions.........................................		224
3.7. Compound 3.6 biocompatibility study ..........................................................		224
3.7.1. Haemocompatibility study..........................................................................		224
3.7.2. Study of the binding of compound 3.6 to DNA and HSA .........................		227
3.7.3. Antiradical activity of compound 3.6 .........................................................		234
3.7.4. Compound 3.6 cytotoxicity ........................................................................		237
3.7.5. Effect of compound 3.6 on HIF-1α stabilisation ........................................		238
3.8. Biocompatibility of non-covalent conjugate based on GO		and
compound 1.57 ................................................................................................................		239
3.8.1. Haemocompatibility study..........................................................................		239
3.8.2. Antioxidant activity of GO-1.57 conjugate ................................................		242
3.8.3. Genotoxicity ...............................................................................................		245
3.8.4. Study of thermodynamic parameters of GO-1.57 binding to HSA by
calorimetric method .........................................................................................................		248
3.8.5. Cytotoxicity of the GO-1.57 conjugate ......................................................		248
3.8.6. Study of the mechanisms of endocytosis of the GO-1.57 conjugate..........		249
CHAPTER 4. EXPERIMENTAL PART ....................................................................................		251
4.1	Synthesis of 1,3,5-triazine derivatives ...............................................................	253
4.2	Synthesis of tetrazole derivatives ......................................................................	255
4.3	Synthesis of triazinyltetrazoles .......................................................................	257
4.4	Study of the stability of compounds 1.57 and 3.6 ..........................................	258
4.5	GO synthesis...................................................................................................	259
4.6	Synthesis of GO conjugate with 2,4,6-trisubstituted-1,3,5-triazine 1.57 .......	259
4.7	Biocompatibility study ...................................................................................	260
MAIN RESULTS AND CONCLUSIONS .................................................................................		269
BIBLIOGRAPHY .......................................................................................................................		270

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LIST OF ABBREVIATIONS

DNA — Deoxyribonucleic acid

RNA — Ribonucleic acid

IC50 — Half maximal inhibition concentration DMF — N, N-Dimethylformamide

Glu 30 — Glutamic acid

HIV-1 — Human immunodeficiency virus

GO — Graphene oxide

HSA — Human serum albumin

PBS — Phosphate-buffered saline

THF — Tetrahydrofuran

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INTRODUCTION

Relevance of the research topic

To date, drug anticancer therapy is represented by three areas: chemotherapy, targeted therapy and immunotherapy. Chemotherapy is a non-specific treatment that uses chemicals that inhibit cell proliferation by affecting cellular DNA, RNA, receptors, metabolism, and cytoskeletal components [1]. Due to the difficulty of obtaining targeted and immune drugs (this fact significantly increases their cost), chemotherapy is still a relevant approach, since cytostatics are low molecular weight chemicals that are much easier to synthesise. The main disadvantage of chemotherapy is the lack of specificity of cytostatics to tumour cells, which leads to the development of a wide range of toxic side effects, among which are increased fatigue, alopecia, aplastic anaemia and thrombocytopenia, immunodeficiency, neuropathy, development of skin ulcers, impaired cognitive and reproductive functions, diarrhoea, nausea, loss of appetite. It is worth noting the development of resistance to therapy, which necessitates the search for new chemicals with cytostatic properties.

Thus, the problem of the synthesis of new biologically active substances and the creation of new drugs on their basis for the treatment of oncological diseases is one of the most important tasks.

Purpose and tasks of the work

The aim of the work is to create new promising materials based on 1,3,5-triazines and tetrazoles, as well as graphene oxide to reduce systemic toxicity and regulate bioactivity.

To achieve this goal, the following tasks were set:

1)creation of new promising biologically active compounds of the 1,3,5-triazine series with antitumour activity;

2)synthesis of a nanoform of the leader compound with graphene oxide;

3)study of the physicochemical properties of the obtained compounds of the 1,3,5-triazine series;

4)in vitro testing of obtained compounds and conjugate.

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Scientific novelty and practical significance

1.Synthesis of new heterocyclic compounds based on 1,3,5-triazine, including those containing both triazine and tetrazolyl fragments, has been carried out.

2.For the first time, a non-covalent conjugate containing graphene oxide enriched with oxygen-containing functional groups and a cytostatic preparation based on 1,3,5-triazine (the degree of loading of the cytostatic drug is 68 %) was synthesised and characterised.

3.A comprehensive study of the physicochemical properties of aqueous solutions of a cytostatic drug based on 1,3,5-triazine was carried out, including the study of stability, solubility, distribution coefficient, density, and viscosity.

4.The biocompatibility of the synthesised materials was studied: haemocompatibility, antioxidant properties, genotoxicity.

5.The cytotoxicity of the obtained compounds was studied on various cell lines.

Reliability and approbation of research results

The results were published in six papers in peer-reviewed scientific journals indexed in the Scopus and Web of Science databases and presented at six international and allRussian scientific conferences.

List of publications

Mikolaichuk O., Sharoyko V., Popova E., Protas A., Fonin A., et. al. Biocompatibility and bioactivity study of a cytostatic drug belonging to the group of alkylating agents of the triazine derivative class // J. Mol. Liq. – 2021. – Vol. 343. – P. 117630.

2.Mikolaichuk O., Popova E., Protas A., Rakipov I., Nerukh D., et. al. A cytostatic drug from the class of triazine derivatives: Its properties in aqueous solutions, cytotoxicity, and therapeutic activity // J. Mol. Liq. – 2022. – Vol. 356. – P. 119043.

3.Mikolaichuk O., Sharoyko V., Popova E., Protas A., Fonin A., et. al. Synthesis, study of interaction with DNA and antitumour activity of a new tetrazole-containing derivative of 2-amino-4,6-di(aziridin-1-yl)-1,3,5-triazine // Russian Chemical Bulletin. – 2022. – No.

5.– 1050.

4.Molchanov O., Maistrenko D., Granov D., Vasina L., Popova A., Vasilevskaya I.,

Mikolaichuk O., et. al. Biomarkers and potential targets for immune and cellular therapy in triple negative breast cancer // Cell Ther Transplant. – 2022. – Vol. 11(2). – P. 18.

150

5. Mikolaichuk O., Popova E., Protas A., Shemchuk O., Vasina L. Study of biocompatibility, cytotoxic activity in vitro of a tetrazole-containing derivative of 2-amino- 4,6-di(aziridin-1-yl)-1,3,5-triazine // Biochem Biophys Res Commun. // 2022. – Vol. 629.

– P. 176.

6. Sharoyko V., Mikolaichuk O., Shemchuk O., Abdelhalim A. O. E., Potanin A., et. al. Novel non-covalent conjugate based on graphene oxide and alkylating agent from 1,3,5- triazine class // J. Mol. Liq. – 2023. – Vol. 372. – P. 121203.

List of conferences

1. Proceedings of the 5th Russian Conference on Medicinal Chemistry with international participation ‘MedChem-Russia 2021’ October 5–8, 2021. O. V. Mikolaichuk, A. V. Protas, E. A. Popova, A. M. Malkova, V. A. Ostrovsky, A. A. Bogdanova, Yu. N. Pavlyukova, N. T. Shmanyova, Yu. A. Nashchekina, V. V. Sharoyko, K. N. Semenov

‘Derivatives of 5-phenyltetrazol-2-ylacetic acid as key reagents in the synthesis of biologically active substances exhibiting antitumour activity’, Volgograd, Russian Federation, 2021.

2.Proceedings of the XXIV International Medical and Biological Conference of Young Researchers ‘Fundamental Science and Clinical Medicine – Man and His Health’, April 2021. O. V. Mikolaichuk, A. V. Protas, G. O. Yuryev, K. N. Semenov ‘Study of the interaction of a 1,3,5-triazine derivative with DNA’, St. Petersburg, Russian Federation, 2021.

3.Materials of the LXXXII scientific and practical conference with international participation ‘Topical issues of experimental and clinical medicine — 2021’ April 24, 2021. O. V. Mikolaichuk, A. V. Protas, E. A. Popova ‘Synthesis and study of the properties of new hybrid heterocyclic systems based on 1,3,5-triazines’, St. Petersburg, Russian Federation, 2021.

4.Proceedings of the All-Russian Scientific Conference ‘Markovnikov Readings: Organic Chemistry from Markovnikov to the Present Day’ October 8–11, 2021. O. V. Mikolaichuk, A. V. Protas, E. A. Popova, A. A. Spiridonova, O. Ya. Volkova, A. M. Malkova, V. V. Sharoyko, K. N. Semenov ‘Synthesis, structure and biological activity of some 1,3,5- triazine derivatives’, Sochi, Russian Federation, 2021.

5.Proceedings of the All-Russian Congress ‘KOST-2021’ on the chemistry of heterocyclic compounds October 12–16, 2021. O. V. Mikolaichuk, A. V. Protas, E. A. Popova, A. A.

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