Synthesis of Novel Diterpenic Peptides via the Ugi Reaction and Their Anticancer Activities

: Novel diterpenic peptide derivatives were synthesized via the Ugi four-component reaction at ambient temperature. The protocol employed a reaction between formaldehyde, benzyl amine, the corresponding diterpene acid, and ethyl 2-isocyanoacetate. The anticancer properties of the compounds were studied in vitro


Introduction
Despite significant progress in the development of therapeutic and diagnostic tools, cancer remains the leading cause of death worldwide [1]. The existing side effects of most anticancer drugs, such as resistance and toxicity, determine the need to search for and create effective anticancer drugs that can overcome these problems. At the same time, the use of biologically active substances of plant origin in the treatment of many diseases is becoming more and more relevant every year. One of the promising classes of natural compounds with a wide spectrum of biological activity [2][3][4] comprises the abietane diterpenoids, metabolites of the pine gum Pinus Silvestris, which have recently attracted considerable attention due to their anticancer potential [5,6]. There are many examples in the literature of the ability of diterpene acids and their derivatives to inhibit the proliferation of classical and atypical multidrug resistant tumor cells [7]. Thus, nitrile derivatives of levopimaric acid provided antiproliferative effect toward the Jurkat, K562, U937, and HeLa tumor cell cultures and they were effective inducers of tumor cell apoptosis affecting the S and G2 phases of the cell cycle [8]. Abietic acid abrogated tumor necrosis factor-α-induced phosphorylation of IκB kinase and inhibited the translocation of nuclear factor-κB [9]. Moreover, abietic acid triggered both G 2 /M cell arrest and subG 0 -G 1 subpopulation in MCF-7 and induced overexpression of key apoptotic genes [10]. In addition, abietic acid and its derivatives induced overexpression of the cytochrome C protein and significantly reduced carcinogenesis and chemotherapy resistance [11]. The multidirectional biological action of abietane diterpenoids, combined with their low toxicity and polyactivity, are their important advantages in the development of drugs for the prevention and treatment of malignant tumors [12,13].
Multicomponent reactions (MCRs) are of great interest to researchers involved in the targeted synthesis of substances with potential biological activity and high bioavailability due to the simplicity of their practical implementation and the wide range of the reaction product structures [14]. Isocyanide-based multicomponent reactions, including the Ugi four-component reaction (U4CR) [15], are among the brightest examples of MCRs, demonstrating a high degree of diversity of molecular products and universality of application in combinatorial chemistry and especially in drug discovery [16,17]. The possibility of using the U4CR for the synthesis of important cytotoxic agents that cause necrosis of cancer cells through apoptosis from terpene and steroid derivatives was shown [18]. For example, 2 of 5 ecdysteroid peptides obtained via the U4CR from 6-amino-20-hydroxyecdysone exhibited cytotoxic activity against leukemia T-cells [19]. Oleanolic and maslinic acid acylaminocarboxamides inhibited the growth A2780 ovarian carcinoma cells in the micromolar range of concentrations [20]. The U4CR products of the diterpene dehydroabietylamide demonstrated cytotoxicity in vitro toward human tumor cells A375, A2780, HT29, and MCF7, as well as nonmalignant mouse fibroblasts NIH 3T3 [21].
The present paper describes the synthesis of novel peptide derivatives via the U4CR using levopimaric acid diene adducts as the carboxyl component and the study of their anticancer properties in vitro.

Chemistry
The Ugi reaction is an efficient condensation of a carbonyl compound, a primary or secondary amine, an isocyanide, and a carboxylic acid in the presence of an acid catalyst to form a dipeptoid structure. The reaction is usually carried out in aprotic solvents at room or lower temperatures in one step [15]. The undoubted advantages of this type of reaction is its economy and efficiency, and importantly, its environmental friendliness from the point of view of "green chemistry", since a reduction in the number of stages leads to a decrease in the solvents used, and as a result, to a decrease in chemical waste.
Previously, in the U4CR, we used the available diterpene acids, abietic acid and diene adducts of levopimaric acid as the key building blocks. Their interaction with paraformaldehyde, 2,6-dimethoxyphenylisocyanide, as well as with methyl esters of glycine, L-phenylalanine, L-tyrosine or n-butylamine as amino components, led to the production of abietane diterpene derivatives with high antiviral activity against influenza A virus and SARS-CoV-2 pseudovirus [17]. We applied this methodology to the synthesis of new dipeptide derivatives with antitumor activity, when as starting materials, we used 2,3-dihydroquinopimaric 1 [22] and maleopimaric 2 [23] acids. The peptide derivatives were synthesized via the U4CR involving benzyl amine, paraformaldehyde, diterpenic acid, and ethyl 2-isocyanoacetate at ambient conditions for 5 days (Scheme 1).
Molbank 2023, 2023, x FOR PEER REVIEW 2 demonstrating a high degree of diversity of molecular products and universality of ap cation in combinatorial chemistry and especially in drug discovery [16,17]. The possib of using the U4CR for the synthesis of important cytotoxic agents that cause necros cancer cells through apoptosis from terpene and steroid derivatives was shown [18]. example, ecdysteroid peptides obtained via the U4CR from 6-amino-20-hydroxyecdys exhibited cytotoxic activity against leukemia T-cells [19]. Oleanolic and maslinic acid ylaminocarboxamides inhibited the growth A2780 ovarian carcinoma cells in the cromolar range of concentrations [20]. The U4CR products of the diterpene dehydroa tylamide demonstrated cytotoxicity in vitro toward human tumor cells A375, A2 HT29, and MCF7, as well as nonmalignant mouse fibroblasts NIH 3T3 [21]. The present paper describes the synthesis of novel peptide derivatives via the U using levopimaric acid diene adducts as the carboxyl component and the study of t anticancer properties in vitro.

Chemistry
The Ugi reaction is an efficient condensation of a carbonyl compound, a primar secondary amine, an isocyanide, and a carboxylic acid in the presence of an acid cata to form a dipeptoid structure. The reaction is usually carried out in aprotic solven room or lower temperatures in one step [15]. The undoubted advantages of this typ reaction is its economy and efficiency, and importantly, its environmental friendli from the point of view of "green chemistry", since a reduction in the number of st leads to a decrease in the solvents used, and as a result, to a decrease in chemical was Previously, in the U4CR, we used the available diterpene acids, abietic acid and d adducts of levopimaric acid as the key building blocks. Their interaction with parafo aldehyde, 2,6-dimethoxyphenylisocyanide, as well as with methyl esters of glycine, L-p nylalanine, L-tyrosine or n-butylamine as amino components, led to the production o ietane diterpene derivatives with high antiviral activity against influenza A virus SARS-CoV-2 pseudovirus [17]. We applied this methodology to the synthesis of new peptide derivatives with antitumor activity, when as starting materials, we used 2,3-d droquinopimaric 1 [22] and maleopimaric 2 [23] acids. The peptide derivatives were thesized via the U4CR involving benzyl amine, paraformaldehyde, diterpenic acid, ethyl 2-isocyanoacetate at ambient conditions for 5 days (Scheme 1). Scheme 1. Synthesis of dihydroquinopimaric and maleopimaric peptide derivatives. Scheme 1. Synthesis of dihydroquinopimaric and maleopimaric peptide derivatives.
The structure of the products was confirmed by NMR, IR, and HRMS spectroscopy (see Supplementary Material Figures S1 and S2). In the 13 C NMR spectra of the compounds 3 and 4, the CON-, CONH-and COO-carbon atom signals appeared at the δ 179.99/179.89, 170.13/169.67 and 169.42/169.28 ppm, respectively. The 1 H NMR spectra contained NH atom signals as broadened signals at the δ 6.69/6.65 ppm. The aromatic proton signals of the benzyl substituent resonated at the δ 7.42-7.12 ppm. The characteristic signals of the ethyl methyl atom protons were appeared as singlets at the δ 1.22 ppm. The mass spectra of the compounds 3 and 4 exhibited molecular ions peaks corresponding to the molecular masses of the compounds and confirmed the identity and purity of the products.

Biological Assay
The antitumor activity (cytotoxicity) screening was performed for the synthesized abietane

Materials and Methods
The spectra were recorded at the Center for the Collective Use "Chemistry" of the Ufa Institute of Chemistry of the UFRC RAS and RCCU "Agidel" of the UFRC RAS. The 1 H and 13 C NMR spectra were recorded on a "Bruker Avance III" (Bruker, Billerica, MA, USA, 500 and 125.5 MHz, respectively, δ, ppm, Hz) in CDCl 3 , internal standard-tetramethylsilane. The mass spectra were obtained on a liquid high-resolution chromatograph-mass spectrometer Agilent LC/Q-TOF 6530 (Santa Clara, CA, USA). The melting points were detected on a microtable "Rapido PHMK05" (Nagema, Dresden, Germany). The optical rotations were measured on a polarimeter Perkin-Elmer 241 MC (PerkinElmer, Waltham, MA, USA) at a tube length of 1 dm. The elemental analysis was performed on a Euro EA-3000 CHNS analyzer (Eurovector, Milan, Italy), and the main standard was acetanilide. The thin-layer chromatography analyses were performed on Sorbfil plates (Sorbpolimer, Krasnodar, Russia) using the solvent system chloroform-ethyl acetate, 40:1. The substances were detected by a 10% solution of sulfuric acid solution with subsequent heating at 100-120 • C for 2-3 min. All the chemicals were of reagent grade (Sigma-Aldrich, St. Louis, MO, USA). The dihydroquinopimaric 1 and maleopimaric 2 acids were synthesized as described in [22,23].

General Procedure
Paraformaldehyde (0.03 g, 1 mmol) was suspended in 10-20 mL dry methanol, followed by the addition of benzyl amine (0.13 g, 1.2 mmol). The suspension was stirred for 2 h at room temperature. Then, 1 mmol diterpenic acid 1 (0.41 g) or 2 (0.40 g) and the ethyl 2-isocyanoacetate (0.11 g, 1 mmol) were added, and the solution was stirred for an additional 5 days. The reaction mixture was poured into aqueous HCl (2 M) and the precipitate formed was filtered off, washed until neutral, and dried in air. The residue was purified via column chromatography using petroleum ether: ethyl acetate (10:1→3:1) as eluent.

Anticancer Assay
The experimental procedure is described in the Supplementary Materials.

Conclusions
With the participation of paraformaldehyde, benzyl amine, ethyl 2-isocyanoacetate and 2,3-dihydroquinopimaric or maleopimaric acids, the corresponding bisamide derivatives were synthesized via the Ugi reaction. The anticancer properties of the compounds were studied in vitro.

Supplementary Materials:
The following supporting information can be downloaded online. Figure S1: 1 H NMR and 13 C NMR spectra of compound 3 (CDCl 3 ); Figure S2: 1 H NMR and 13 C NMR spectra of compound 4 (CDCl 3 ); Figure S3: Anticancer screening data of compound 3 at single dose assay; Figure S4: Anticancer screening data of compound 4 at single dose assay.