Project objectives

1. Investigation of the possibilities for modification of the carboxyl group of 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[3,2-c]pyridine-3-carboxylic acid and synthesis of amides and esters based on aromatic and aliphatic alcohols and amines, including those containing functional groups (sulfonyl, sulfonamide groups, amino groups, etc.). Optimization of experimental conditions to obtain these compounds in high yields and chemical purity, which is essential for their further development as pharmaceuticals.

2. Investigation of the possibilities for modification of the hydroxymethyl fragment and synthesis of the corresponding halogen derivatives, azides, and quaternary ammonium and phosphonium salts. Introduction of an azide group into the structure will enable the synthesis of compounds containing a triazole fragment via “click reaction” with various alkynes. The obtained phosphonium salts will be used as precursors for the introduction of a double bond into the structure of target compounds via the Wittig reaction with various carbonyl compounds. Optimization of experimental conditions aimed at obtaining these compounds in high yields and chemical purity will be carried out.

3. Investigation of the possibilities for modification of the double bond and synthesis (including stereoselective synthesis) of polycyclic compounds based on the 7-azacoumarin scaffold using 1,3-dipolar cycloaddition reactions. Optimization of experimental conditions to obtain the target polycycles in high yields and chemical purity.

4. Investigation of the cytotoxicity of the obtained compounds. Evaluation of cytotoxic activity will be performed on cultures of normal cell lines (Chang liver, RPMI 1788, WI-38 VA 13 subline 2RA) and tumor cell lines (A-549, M-Hela clone 11, Hep G2, PANC-1, T 98G – human glioblastoma, HL-60, HuTu 80). Known anticancer drugs Doxorubicin, 5-fluorouracil, and Tamoxifen will be used as reference compounds.

Expected results

During the first year of the project, 1,3-dipolar cycloaddition reactions of 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[3,2-c]pyridine-3-carboxylic acid with dipoles based on sarcosine, L- and D-proline, other amino acids, as well as ninhydrin, acenaphthenequinone, aceanthrenequinone, isatin, diacetyl, and dibenzoyl will be investigated. The influence of reaction conditions on the synthetic outcome will be established, and the effect of the structure of the 1,3-dipole on the course of the process will be determined. Based on the obtained data, the most optimal reaction conditions ensuring the highest yields of target compounds will be selected. As a result of these studies, in 2024, the first examples of polycyclic derivatives of 7-azacoumarin annelated with a pyrrolidine ring, including diastereo- and enantiomerically pure forms, will be obtained.

During the second year (2025) of the project, reactions of 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[3,2-c]pyridine-3-carboxylic acid and its amides with halogenating reagents (sulfuryl chloride, phosphorus oxychloride, thionyl chloride) will be studied, and derivatives of 5-chloromethyl-7-azacoumarin will be obtained. The interaction of these compounds with P- and N-nucleophiles (secondary amines, tertiary amines, triphenylphosphine) will be investigated, and new derivatives of 7-azacoumarin containing aminoalkyl, ammonium, phosphonium fragments, and an azide group will be obtained for the first time.

The possibility of using 7-azacoumarin derivatives containing a triphenylphosphonium group as starting compounds in the Wittig reaction will be investigated. Azide–alkyne cycloaddition reactions will be carried out using 7-azacoumarin derivatives containing an azide group. As a result, previously unknown 7-azacoumarin derivatives bearing a substituted vinyl group at the 5-position of the pyridine ring, as well as new derivatives modified with a substituted triazole fragment, will be obtained.

Amidation and esterification reactions of 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[3,2-c]pyridine-3-carboxylic acid with NH-substrates and alcohols, including structurally complex ones containing functional groups, will be studied. The most optimal methodologies for amidation and esterification, in terms of target product yield, will be selected. As a result, previously unknown functionally substituted esters and amides of 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[3,2-c]pyridine-3-carboxylic acid will be obtained.

The cytotoxicity of the obtained compounds will be evaluated against a series of normal cell lines (Chang liver, RPMI 1788, WI-38 VA 13 subline 2RA) and tumor cell lines (A-549, M-Hela clone 11, Hep G2, PANC-1, T 98G – human glioblastoma, HL-60, HuTu 80), using known anticancer drug – Doxorubicin, 5-fluorouracil, and Tamoxifen – as reference compounds. Based on the obtained data, structure–property relationships within the series of 7-azacoumarin derivatives will be established, and the most active lead compounds, promising for further research and practical application, will be identified.

1) The following will be published:

- At least 3 (three) articles and/or reviews in peer-reviewed scientific journals relevant to the project, indexed in the Science Citation Index Expanded and ranked in the 1st, 2nd, and/or 3rd quartiles by impact factor in the Web of Science database and/or having a CiteScore percentile of at least 60 in the Scopus database;

- At least 1 (one) article or review in a peer-reviewed international or domestic journal recommended by the Committee for Quality Assurance in Science and Higher Education.

The results obtained during the project will be published in 5 publications in peer-reviewed scientific journals in the project’s research area, including International Journal of Molecular Sciences (Q1, citation index 5.6), European Journal of Medicinal Chemistry (Q1, citation index 6.7), Journal of Organic Chemistry (Q1, citation index 3.6), Pharmaceuticals (Q2, citation index 4.6), and Molecules (Q2, citation index 4.6), or in other journals indexed in the Science Citation Index Expanded and ranked in Q1–Q3 by impact factor in the Web of Science database and/or having a CiteScore percentile of at least 60 in the Scopus database.

In addition, one article or review will be published in Bulletin of the Karaganda University – Chemistry or in other peer-reviewed international or domestic journals recommended by the Committee for Quality Assurance in Science and Higher Education, and the results will also be presented at international conferences.

2) The publication of monographs, books, and/or book chapters in foreign and/or domestic publishing houses is not planned.

3) Obtaining patents in foreign patent offices (European, American, Japanese), as well as in national or Eurasian patent offices: a patent of the Republic of Kazakhstan for an invention will be obtained.

4) The development of scientific, technical, and engineering documentation is not planned.

5) Dissemination of research results among potential users, the scientific community, and the general public: dissemination will be carried out through participation in international scientific conferences, as well as through national and regional mass media and social networks.

6) Other measurable results in accordance with the requirements of the tender documentation and the specifics of the project. The following are additionally specified in this section:

1) Field of application and target consumers of each expected result:
As a result of the implementation of the proposed project, an extensive library of previously undescribed 7-azacoumarin derivatives exhibiting high cytotoxicity toward cancer cell lines will be obtained. A structure–activity relationship analysis will be conducted, and lead compounds demonstrating high selectivity and low toxicity toward normal tissues and cells will be identified. This will serve as a basis for the further development of new types of antitumor agents and will contribute to the advancement of medicinal chemistry and pharmaceutical chemistry, as well as provide a reliable foundation for the practical implementation of new types of highly selective and low-toxicity antitumor agents.

2) Impact of the expected results on the development of the main scientific field and related areas of science and technology:
Pharmaceutical companies may become the primary target consumers of the project’s scientific results, as the obtained compounds are expected to exhibit a broad spectrum of biological activity, reduced toxicity, and the potential to compete with expensive foreign analogues.

3) Applicability and/or potential for commercialization of the obtained scientific results:
The obtained scientific results may be applied in the pharmaceutical industry and medicine as antitumor agents, as well as in the educational process for improving curricula, developing laboratory work, and organizing practical training for students at various levels.

4) Social, economic, environmental, scientific, technical, multiplicative, and/or other effects of the project results, with justification, including addressing existing regional challenges:
The results obtained during the implementation of this project will significantly expand current knowledge in the field of medicinal chemistry.

5) Other direct and indirect results of the project, including their qualitative and quantitative characteristics:
The results are planned to be presented at international and national conferences.

Results

The reactions of 1,3-dipolar cycloaddition of 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[3,2-c]pyridine-3-carboxylic acid with dipoles based on sarcosine, ninhydrin, acenaphthenequinone, and aceanthrenequinone were investigated. The influence of reaction conditions on the synthetic outcome was studied, and the effect of the structure of the 1,3-dipole on the course of the process was determined.

It was established that when the reaction was carried out in absolute ethanol under reflux in the ternary system 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[3,2-c]pyridine-3-carboxylic acid – sarcosine – ninhydrin, no formation of the target product was observed, and the starting acid was recovered unchanged. When the amino acid was replaced with L- or D-proline, the formation of the expected 1,3-dipolar cycloaddition products was also not observed.

It was established that the reaction of 7-azacoumarin-3-carboxylic acid with proline and ninhydrin proceeds via an alternative pathway, occurring simultaneously with preservation of the double bond in the pyran ring of 7-azacoumarin-3-carboxylic acid, its decarboxylation, opening of the pyrrolidine ring of proline, formation of an azomethine ylide (1,3-dipole), and subsequent formation of a piperidine ring substituted with a fragment of ninhydrin or aceanthrenequinone. The product yields were 78% (for the acid/proline/ninhydrin reaction) and 36% (for the acid/proline/aceanthrenequinone reaction).

The reliability of the obtained results is confirmed by the use of modern physicochemical methods: mass spectrometry (MALDI-TOF and ESI), IR spectroscopy, ¹H and ¹³C NMR spectroscopy, as well as elemental and X-ray crystallographic analysis.

¹H and ¹³C NMR spectra were recorded on Bruker AVANCE II-400 spectrometers operating at 400.1 MHz (¹H) and 100.6 MHz (¹³C), and on a Bruker Avance-600 spectrometer operating at 600.1 MHz (¹H) and 150.9 MHz (¹³C), referenced to the signals of residual protons of the deuterated solvent or carbon nuclei of the deuterated solvent (DMSO-d₆). IR spectra were recorded on a Bruker Vector 22 Fourier-transform spectrometer in the range of 400–4000 cm⁻¹. The samples were analyzed as KBr pellets. Elemental analysis was performed on a Carlo Erba EA 1108 instrument.

For product (A) based on ninhydrin, the MALDI mass spectrum shows a peak at m/z = 405.1 [M+H]⁺, while for product (B) based on aceanthrenequinone, a peak at m/z = 475.0 [M−H]⁻ is observed. In the ¹H NMR spectrum of product (A), the methylene group protons are observed as a multiplet at δ 4.49–4.57 ppm with an integral intensity of 2H. The hydroxyl group appears as a triplet at δ 5.39 ppm with an integral intensity of 1H and a spin–spin coupling constant J = 5.1 Hz. The methine proton of the pyridine ring appears as a singlet at δ 8.17 ppm with an integral intensity of 1H. The protons of the phenyl ring are observed as a multiplet at δ 7.78–7.81 ppm, and this region also includes the signal of the methine proton of the pyran ring. The methyl group appears as a singlet at δ 2.38 ppm with an integral intensity of 3H.

The methylene protons of the piperidine ring correspond to the following set of signals: δ 1.65–1.72 ppm (m, 1H), 1.82–1.86 ppm (m, 2H), 2.33–2.42 ppm (m, 1H), 2.91 ppm (d, 1H, J = 12.6 Hz), 3.28–3.34 ppm (m, 1H), 3.48 ppm (dd, 1H, J = 12.6, 3.2 Hz).

For product (A) based on ninhydrin, the MALDI mass spectrum shows a peak at m/z = 405.1 [M+H]⁺, while for product (B) based on aceanthrenequinone, a peak at m/z = 475.0 [M−H]⁻ is observed. In the ¹H NMR spectrum of product (A), the methylene group protons are observed as a multiplet at δ 4.49–4.57 ppm with an integral intensity of 2H. The hydroxyl group appears as a triplet at δ 5.39 ppm with an integral intensity of 1H and a spin–spin coupling constant J = 5.1 Hz. The methine proton of the pyridine ring appears as a singlet at δ 8.17 ppm with an integral intensity of 1H. The protons of the phenyl ring are observed as a multiplet at δ 7.78–7.81 ppm; this region also includes the signal of the methine proton of the pyran ring. The methyl group appears as a singlet at δ 2.38 ppm with an integral intensity of 3H.

The methylene protons of the piperidine ring correspond to the following set of signals: δ 1.65–1.72 ppm (m, 1H), 1.82–1.86 ppm (m, 2H), 2.33–2.42 ppm (m, 1H), 2.91 ppm (d, 1H, J = 12.6 Hz), 3.28–3.34 ppm (m, 1H), 3.48 ppm (dd, 1H, J = 12.6, 3.2 Hz).

Carrying out the reaction in a two-component system of 7-azacoumarin-3-carboxylic acid and a 1,3-dipole previously isolated as an individual compound from the reaction of proline with ninhydrin leads to a similar outcome.

Based on the obtained data, the most optimal reaction conditions were selected, namely performing the reaction under reflux in an alcohol medium, which ensures the highest yield of the target compounds. As a result of the conducted studies, in 2024 the first examples of polycyclic derivatives of 7-azacoumarin containing fragments of ninhydrin, acenaphthenequinone, and aceanthrenequinone were obtained.

The amount of financing