Synthesis of Norabietyl and Nordehydroabietyl Imidazolidine-2,4,5-Triones and Their Activity against Tyrosyl-DNA Phosphodiesterase 1

Abstract

New imidazolidine-2,4,5-triones with norabietic, nordehydroabietic, and adamantane substituents were synthesized by reacting oxalyl chloride and the corresponding ureas, providing good yields. Bioisosteric replacement of the ureide group with a parabanic acid fragment made it possible to increase the solubility of compounds and conduct biological studies. The compounds inhibit the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 in submicromolar concentrations. Cytotoxic concentrations were also studied on the glioblastoma cell line SNB19.

1. Introduction

Despite the variety of existing methods for treating cancer, the most commonly used treatment regimen in clinical practice consists of chemotherapy and radiation therapy. The action of cytostatic agents and ionizing radiation is aimed at damaging the DNA structure, and the highly efficient functioning of the DNA repair systems in cancer cells prevents the achievement of the desired result. The selective inhibition of DNA repair enzymes can lead to both increasing the effectiveness of the main therapy and overcoming resistance to it.

An enzyme that can serve as a molecular target in antitumor therapy is tyrosyl-DNA phosphodiesterase 1 (TDP1) [1]. TDP1 is a eukaryotic DNA repair enzyme that specifically cleaves the covalent bond between the 3′-phosphate group of DNA and the tyrosine associated with a small peptide remaining after proteolysis/denaturation of Top1 (topoisomerase 1) [2]. TDP1 plays a key role in repairing DNA damage caused by clinically used anticancer drugs such as topotecan and irinotecan [3]. Thus, selective inhibition of this enzyme can significantly enhance their effect [4]. In addition, TDP1 has broad substrate specificity, allowing it to remove various other 3′ blocking lesions. This enzyme is capable of removing from the 3′ end of DNA deoxyribonucleosides, ribonucleosides and their analogues used as antitumor and antiviral drugs, including acyclovir, cytarabine and zidovudine [5], as well as hydrolyzing 3′-phosphoglycolate groups [6,7], which result from oxidative DNA damage or damage by radiomimetic drugs such as bleomycin [8]. The substrates of this enzyme include, among other things, 3′-phosphoglycolates and 3′-apurinic/apyrimidinic sites (AP sites) formed in DNA under the action of ionizing radiation [9]. In addition to the ability to remove covalent adducts of Top1 and DNA, TDP1 can hydrolyze AP sites in DNA (formed during the BER process) and repair 3-DNA–peptide/protein cross-links [10].

A promising approach to the development of new TDP1 inhibitors is to carry out chemical modifications of natural molecules. Multiple classes of effective inhibitors have been found among derivatives of various plant metabolites (Figure 1). Conjugates of monoterpenoids and 3-arylcoumarins 1 and thiazolidinediones with thiophene and terpene substituents 2 are active in the lower micromolar range. The leaders in inhibitory characteristics are derivatives of usnic acid 3 and 4, which are active in nanomolar concentrations [11].

Previously, our team discovered a new class of effective inhibitors of the TDP1 repair enzyme—resin acid derivatives. Dehydroabietic and abietic acids are components of resins of coniferous plants; for example, a high acid content is found in the resin of Picea obovata [12]. Based on the diterpene compound dehydroabiethylamine, sets of ureas, thioureas, and biureas were synthesized and the resulting agents were shown to inhibit TDP1 in micromolar and submicromolar concentrations. For the first time, the synergistic effect of compound 5 with the alkylating agent temozolomide was studied on glioblastoma lines. The leading compound is able to improve its cytostatic properties by up to 40% (Figure 2) [13].

Attractive substance classes for the creation of new TDP1 inhibitors based on them are not only derivatives of dehydroabietic acid, but also other resin acids. Previously, our team obtained ureas with nordehydroabietic 6a–b and norabietic fragments 7a–b in the structure (Figure 2) [14]. The influence of the type of attachment of the adamantane fragment—in the first or second position of the scaffold—was studied. These compounds showed the ability to inhibit the DNA repair enzyme TDP1 in micromolar and submicromolar concentrations. However, further cellular experiments to study their cytostatic properties individually and in combinations could not be carried out due to their low solubility in water and organic solvents. To increase the solubility and bioavailability of certain classes of substances, a bioisosteric replacement of the ureide group with an imidazolidine-2,4,5-trione group can be used [15].

Imidazolidine-2,4,5-trione, or parabanic acid, is a natural metabolite and is formed in the body during the oxidation of uric acid. N-substituted derivatives of parabanic acid have found widespread use as inhibitors of important enzymes for the development of antitumor [16], antiepileptic [17], lipid-lowering [18] and antidiabetic therapies [19]. N-substituted derivatives of imidazolidine-2,4,5-triones have found use as effective inhibitors of soluble epoxide hydrolase (sEH) [20], acetylcholinesterase and butyrylcholinesterase [21]. Some trioxoimidazolidine compounds have demonstrated noticeable herbicidal properties [22]. Such a valuable heterocyclic structural block can be obtained on the basis of N-substituted ureas by reacting with oxalyl chloride [23,24] or diethyl oxalate under catalysis with dibutyltin oxide (DBTO) [25].

Thus, today, the creation of new tyrosyl-DNA phosphodiesterase inhibitors based on low-toxicity natural compounds is a promising approach to the development of multimodal approaches to anticancer therapy. Among these semisynthetic hybrid molecules, a significant number of compounds have been discovered that can enhance the effect of known chemotherapeutic drugs. The aim of the presented work was to synthesize new derivatives of abietic and dehydroabietic acids containing an adamantane fragment separated from the natural skeleton by an imidazolidine-2,4,5-trione linker and to study the biological properties of these agents.

2. Results

The starting substances for obtaining the target imidazolidine-2,4,5-triones were resin acids—abietic and dehydroabietic. According to the procedure described in the literature [14,26], unsymmetrical ureas with 1- and 2-adamantane fragments were synthesized. The imidazolidinetrione ring was obtained by reacting these ureas, 6a–b and 7a–b, with an excess of oxalyl chloride (Scheme 1). The reaction was carried out in methylene chloride with the addition of triethylamine for 1 h at room temperature. The target products were purified by means of column chromatography and isolated with good yields of 62–72%.

It is expected that this transformation will increase the solubility of derivatives with nordehydroabietic and abietic fragments and expand the possibility for in vitro studies.

Primary screening of the obtained compounds was carried out against the TDP1 enzyme. The inhibitory properties of all synthesized compounds were studied using a technique previously developed at the Novosibirsk Institute of Chemical Biology and Fundamental Medicine [27]. For all derivatives obtained, IC₅₀ values for the TDP1 enzyme were determined, presented in

Table 1. Inhibitory activity of novel compounds against TDP1 and their cytotoxicity.

Compound	IC50 * (TDP1), µM	CC50 ** (SNB19), µM
8a	0.52 ± 0.07	>100
8b	0.69 ± 0.07	>100
9a	0.78 ± 0.02	28.63 ± 1.16
9b	0.96 ± 0.04	>100
Furamidine	1.20 0.30	-

* IC₅₀—50% inhibiting concentration. ** CC₅₀—concentration causing death of 50% of cells.

. Cytotoxic CC₅₀ concentrations were determined using the standard MTT test [28] on the glioblastoma cell line SNB19.

For compounds with a nordehydroabietic fragment 9a–b, replacing the urea group with an imidazolidinetrione group led to a slight decrease in the inhibitory characteristics against the TDP1 enzyme (IC₅₀ = 0.78–0.96 µM for compounds 9a–b and IC₅₀ = 0.57–0.59 µM for ureas 7a–b). Meanwhile, the cyclic norabietine derivatives 8a–b turned out to be two to three times more active (IC₅₀ = 0.52–0.69 µM) than their ureide precursors 6a–b (IC₅₀ = 1.40–1.70 µM). The solubility of all new compounds in DMSO and other organic solvents turned out to be significantly higher, which made it possible to study their cytotoxic properties on the glioblastoma cell line SNB19. For three compounds, the CC₅₀ was above 100 µM, and only 9a showed noticeable toxicity. Thus, good inhibitory properties combined with low toxicity make this class of compounds interesting for further research. The introduction of parabanic acid fragments into target agents significantly increased the solubility of the substances, which is extremely important in the search for new effective biologically active compounds.

3. Materials and Methods

The ¹H and ¹³C NMR spectra in CDCl₃ were recorded on a Bruker AV-400 spectrometer (400.13 and 100.61 MHz, respectively). The residual signals of the solvent were used as references (δH 7.24, δC 76.90 for CDCl₃). High-resolution mass spectra were recorded on a Thermo Scientific DFS instrument in full scan mode over the m/z range of 0–500 by means of ionization with an electron impact of 70 eV, and direct introduction of samples. IR spectra were recorded on a Vector22 spectrometer (KBr). Melting points were determined on a Termosystem FP 900 instrument from Mettler Toledo. Thin-layer chromatography was performed on Silufol plates (UV-254). The TLC plates were visualized via exposure to ultraviolet light (254 and 365 nm). Merck silica gel (63−200 μm) was used for column chromatography. The atomic numbering in the compounds was provided for the assignment of signals in the NMR spectra and was different from the atomic numbering in the systematic name. Copies of NMR, IR and high-resolution mass spectra are presented in the Supplementary Materials.

General procedure for the synthesis of imidazolidine-2,4,5-triones.

Compounds 6a–b and 7a–b (0.2 g, 0.00045 mol) were dissolved in 30 mL of CH₂Cl₂ with the addition of 0.4 mL of triethylamine. Oxalyl chloride (0.00225 mol, 0.285 g) was added dropwise while stirring with a magnetic stirrer. The conversion was monitored by TLC. Upon completion of the reaction (1 h), the reaction mixture was washed with distilled water (3 × 20 mL), and the organic extract was dried over Na₂SO₄. Then, the solvent was evaporated on a rotary evaporator. The final product was purified using silica gel column chromatography using hexane/ethyl acetate (9:1) as an eluent.

• 1-(1-Adamantyl)-3-[(1R,4aR,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthren-1-yl]imidazolidine-2,4,5-trione (8a) Yield 62%, white powder. M.p. 144.6 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 5.74 (1H, s, H-14), 5.35 (1H, s, H-7), 0.98 and 0.97 (3H both, d, J_{16, 15} = 6.8, Me-16 and Me-17), 0.81 (3H, s, Me-18), 1.75 (3H, s, Me-19), 2.76–3.01 (1H, m, H-15), 2.28–2.50 (8H, m, 2H-24, 2H-25, 2H-26, H-5, H-6), 1.91–2.27 (7H, m, H-27, H-28, H-29, 2H-12, H-6, H-9), 1.50–1.87 (12H, m, 2H-30, 2H-31, 2H-32, 2H-2, H-11, H-1, 2H-3), 1.11–1.36 (2H, m, H-1, H-11). ¹³C NMR (100 MHz, CDCl₃, δ, ppm): 157.43, 156.23 and 154.60 (C-20, C-21 and C-22), 145.63 (C-13), 135.53 (C-8), 122.10 (C-14), 119.80 (C-7), 29.59 (C-27, C-28, C-29), 35.81 (C-30, C-31, C-32), 39.92 (C-24, C-25, C-26), 20.83 and 20.70 (Me-17 and Me-16), 21.26 (Me-18), 13.85 (Me-19), 34.75 (C-15), 50.62 (C-9), 45.44 (C-5), 18.80 (C-2), 22.54 (C-11), 24.44 (C-6), 27.28 (C-12), 35.29 (C-10), 37.26 (C-1), 35.96 (C-3), 68.20 (C-4), 62.19 (C-23). IR (KBr), ν, cm⁻¹: 3100–3600, 2915, 1726, 1356. Found, m/z: 504.3350 [M]⁺. (C₃₂H₄₄O₃N₂)⁺. Calculated, m/z: 504.3347.
• 1-(2-Adamantyl)-3-[(1R,4aR,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthren-1-yl]imidazolidine-2,4,5-trione (8b) Yield 64%, white powder. M.p. 111.4 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 5.74 (1H, s, H-14), 5.34 (1H, s, H-7), 4.14 (1H, s br, H-23), 2.82–3.00 (1H, m, H-15), 0.98 and 0.97 (3H both, d, J_{16, 15} = 6.8, Me-16 and Me-17), 0.81 (3H, s, Me-19), 1.77 (3H, s, Me-18), 2.39–2.55 (3H, m, H-5, H-24, H-25), 1.11–1.33 (2H, m, H-1a, H-11a), 2.01–2.28 (6H, m, 2H-26, 2H-28, H-1, H-6), 1.71–2.01 (12H, m, 2H-27, 2H-29, H-30, H-31, 2H-32, H-9, H-6, 2H-12), 1.58–1.71 (5H, m, H-11, 2H-2, 2H-3). ¹³C NMR (100 MHz, CDCl₃, δ, ppm): 157.77, 155.73 and 155.61 (C-20, C-21 and C-22), 145.66 (C-13), 135.58 (C-8), 122.02 (C-14), 119.65 (C-7), 20.71 (Me-17 and Me-16), 21.28 (Me-18), 13.87 (Me-19), 62.54 (C-23), 50.63 (C-9), 45.48 (C-5), 34.74 (C-15), 30.35 and 30.51 (C-24 and C-25), 27.17 and 26.45 (C-30 and C-31), 18.77 (C-2), 22.54 (C-11), 35.95 (C-3), 24.39 (C-6), 27.28 (C-12), 35.31 (C-10), 32.58 and 32.46 (C-26 and C-28), 68.28 (C-4), 37.25 (C-1), 37.89 and 37.94 (C-27 and C-29), 37.20 (C-32). IR (KBr), ν, cm⁻¹: 3100–3600, 2916, 1726, 1339. Found, m/z: 504.3353 [M]⁺. (C₃₂H₄₄O₃N₂)⁺. Calculated, m/z: 504.3347.
• 1-(1-Adamantyl)-3-[(1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]imidazolidine-2,4,5-trione (9a) Yield 70%, white powder. M.p. 96.8 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 7.14 (1H, d, J = 8.1, H-11), 6.98 (1H, d, J = 8.1, H-12), 6.86 (1H, s, H-14), 1.74 (3H, s, Me-18), 1.20 (6H, d, J = 6.9, Me-16 and Me-17), 1.21 (3H, s, Me-19), 2.61–2.95 (5H, m, H-5, H-15, 2H-7, H-3), 2.37 (6H, br s, H-24, H-25, H-26), 2.19–2.30 (1H, m, H-1), 2.05–2.19 (3H, m, H-27, H-28, H-29), 1.62–1.71 (6H, m, 2H-30, 2H-31, 2H-32), 1.72–1.95 (4H, m, H-3, 2H-2, H-6), 1.46–1.59 (2H, m, H-1, H-6). ¹³C NMR (100 MHz, CDCl₃, δ, ppm): 157.46, 156.24 and 154.62 (C-20, C-21 and C-22), 146.20 (C-9), 145.71 (C-13), 134.14 (C-8), 126.74 (C-14), 124.10 (C-11), 123.91 (C-12), 20.29 (Me-18), 23.83 and 23.86 (Me-17 and Me-16), 25.27 (Me-19), 33.31 (C-15), 44.60 (C-5), 29.54 (C-27, C-28, C-29), 19.13 (C-2), 19.93 (C-6), 29.66 (C-7), 35.77 (C-30, C-31, C-32), 39.89 (C-24, C-25, C-26), 69.24 (C-4), 38.44 (C-1), 36.80 and 35.10 (C-10 and C-3), 62.22 (C-23). IR (KBr), ν, cm⁻¹: 2912, 1725, 1352. Found, m/z: 502.3187 [M]⁺. (C₃₂H₄₂O₃N₂)⁺. Calculated, m/z: 502.3190.
• 1-(2-Adamantyl)-3-[(1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]imidazolidine-2,4,5-trione (9b) Yield 72%, white powder. M.p. 89.3 °C. ¹H NMR (400 MHz, CDCl₃, δ, ppm, J/Hz): 7.13 (1H, d, J = 8.2, H-11), 6.98 (1H, dd, J = 8.2, J = 1.7, H-12), 6.85 (1H, d, J = 1.7, H-14), 4.15 (1H, s br., H-23), 2.73–2.89 (4H, m, H-5, H-15, 2H-7), 2.65–2.73 (1H, m, H-3), 2.48–2.55 (2H, m, H-24, H-25), 2.21–2.29 (1H, m, H-1), 2.06–2.13 (2H, m, H-26, H-28), 1.71–1.98 (12H, m, 2H-27, 2H-29, H-30, H-31, 2H-32, H-3, 2H-2, H-6), 1.76 (3H, s, Me-18), 1.64–1.71 (2H, m, H-26, H-28), 1.48–1.60 (2H, m, H-1, H-6), 1.22 (3H, s, Me-19), 1.20 (6H, d, J = 6.9, Me-16 and Me-17). ¹³C NMR (100 MHz, CDCl₃, δ, ppm): 157.68, 155.77 and 155.52 (C-20, C-21 and C-22), 146.08 (C-9), 145.67 (C-13), 134.03 (C-8), 126.69 (C-14), 124.00 (C-11), 123.86 (C-12), 69.25 (C-4), 62.55 (C-23), 44.61 (C-5), 38.38 (C-10), 37.77 (C-27 and C-29), 37.11 (C-32), 36.75 (C-1), 35.12 (C-3), 33.26 (C-15), 32.43 and 32.48 (C-26 and C-28), 30.25 and 30.27 (C-24 and C-25), 29.52 (C-7), 27.07 and 26.37 (C-30 and C-31), 25.19 (Me-19), 23.79 (Me-17 and Me-16), 20.14 (Me-18), 19.84 (C-6), 19.06 (C-2). IR (KBr), ν, cm⁻¹: 2917, 1726, 1337. Found, m/z: 502.3188 [M]⁺. (C₃₂H₄₂O₃N₂)⁺. Calculated, m/z: 502.3190.