Sulfide, Sulfoxide, and Sulfone Derivatives of Usnic Acid as Inhibitors of Human TDP1 and TDP2 Enzymes

Abstract

Tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) are important DNA repair enzymes that remove various adducts from the 3′- and 5′-ends of DNA, respectively. The suppression of the activity of these enzymes is considered as a promising adjuvant therapy for oncological diseases in combination with topoisomerase inhibitors. The simultaneous inhibition of TDP1 and TDP2 may result in greater antitumor effects, as these enzymes can mimic each other’s functions. We have previously shown that usnic acid-based sulfides can act as dual inhibitors, with TDP1 activity in the low micromolar range and their TDP2 at 1 mM. The oxidation of their sulfide moieties to sulfoxides led to an order of magnitude decrease in their cytotoxicity potential, while their TDP1 and TDP2 activity was preserved. In this work, we synthesized new series of usnic acid-based sulfides and their oxidized analogues, i.e., sulfoxides and sulfones, to systematically study these irregularities. The new compounds inhibit TDP1 with IC₅₀ values (the concentration of inhibitor required to reduce enzyme activity by half) in the 0.33–25 μM range. Most sulfides and some sulfoxides and sulfones inhibit TDP2 with an IC₅₀ = 138−421 μM. In addition, the most active compounds synergized (×4) with topotecan on the HeLa cell line as well as causing dose-dependent DNA damage, as confirmed by Comet assay. Sulfides with the 6-methylbenzoimidazol-2-yl substituent (8f, IC₅₀ = 0.33/138 μM, TDP1/2) and sulfones containing a pyridine-2-yl fragment (12k, IC₅₀ = 2/228 μM, TDP1/2) are the most potent derivatives and, therefore, are promising for further development.

1. Introduction

Oncological diseases are ranked second in mortality after cardiovascular pathology. One of the main reasons behind the resistance of cancer cells to chemotherapy is the action of the DNA repair system. Thus, attention has been drawn towards DNA repair enzymes due to their potential as adjunct targets. Tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) are DNA repair enzymes that are potential supplementary targets in combination with topoisomerase (TOP) inhibitors and other anticancer agents [1]. Topotecan and irinotecan are TOP1 inhibitors, and DNA damage caused by these agents is repaired by TDP1 [2,3,4]; etoposide and doxorubicin are TOP2 inhibitors, and damage to DNA caused by these agents is repaired by TDP2 [5,6,7]. TOP inhibitors act as stabilizers of covalent TOP/DNA complexes, leading to the accumulation of DNA strand breaks and consequent cell death [8,9,10]. Repairing such covalent adducts is a complex process that involves TDP at key stages and plays a significant role in the development of drug resistance. Thus, TDP1 and TDP2 are promising targets for the development of anticancer therapy in combination with TOP inhibitors, an important class of anticancer drugs [11,12]. Novel TDP1/2 inhibitors may also increase the treatment efficacy of other oncological diseases that involve DNA damage [13].

It was discovered that TDP1 and TDP2 can mimic each other’s functions, although with less efficiency [1,14,15,16], making the combined use of selective inhibitors or the creation of dual inhibitors desirable. It should be noted, however, that significant differences in the structures of the active centers of TDP1 and TDP2 makes the task of creating dual inhibitors challenging [11].

To date, many low-molecular-weight TDP1 inhibitors, 1–6 in Figure 1, have been developed, including derivatives of natural products [12] such as usnic acids, berberines, coumarins, and steroids, as well as synthetic inhibitors, such as isoquinolines [17,18,19,20] and thiazolidines [12,21]. Among the usnic acid derivatives, we discovered compounds with the general structure indicated for compound 7 in Figure 1, which can act as dual inhibitors of TDP1 (IC₅₀ = 0.3–0.5 µM) and TDP2 (7–11% at 500 µM) [22].

We have previously shown that usnic acid-based sulfides 8b and d (Figure 1) can act as dual inhibitors of TDP1 and TDP2, exhibiting inhibitory activity against TDP1 in the lower micromolar concentration range, as well as inhibiting TDP2 at a 1 mM concentration. Moreover, sulfides (8b and d, Figure 1) and sulfoxides (9b and d, Figure 1) are triple inhibitors of TDP1, TDP2, and PARP1 [23]. Unfortunately, most of them are cytotoxic, with CC₅₀ values in the inhibition range of TDP1. We have shown that oxidation of the sulfide moieties in 8b and 8d to the corresponding sulfoxides (9b and 9d) led to an order of magnitude decrease in their CC₅₀ values, while their inhibitory activity against TDP1 and TDP2 was preserved [23].

As a continuation of our efforts directed towards the synthesis of TDP1 and TDP2 dual inhibitors, we have synthesized several sulfides and their corresponding sulfoxide and sulfone derivatives that contain various R substituents. Of the sulfides, the most effective TDP1 inhibitors contain methyl- (8f) or methoxy-benzimidazole (8g) moieties. The general order relating to the ability of the studied products to inhibit TDP1 corresponds to sulfides > sulfoxides > sulfones. As for TDP2, the same compounds, 8f and 8g, are the most effective inhibitors. Regarding their cytotoxicity and their ability to enhance the cytotoxic effect of topotecan, no structure–function relationship could be elucidated. In general, the TDP1 inhibitory concentrations of the studied sulfones exceed their respective cytotoxic concentrations, so they are not suitable for further development. The most promising compounds are the sulfides bearing methyl- or methoxy-benzimidazole substituents.

2. Materials and Methods

2.1. Chemistry

¹H and ¹³C NMR spectra were recorded in CDCl₃ or DMSO-d₆ solution using solvent resonances (¹H 7.24 ppm, ¹³C 76.90 ppm and ¹H 2.50 ppm, ¹³C 39.50 ppm, respectively) as standards on a Bruker AV-400 spectrometer (Bruker Corporation, Karlsruhe, Germany; operating frequencies 400.13 MHz for ¹H and 100.61, for ¹³C). Mass spectra (ionizing-electron energy 70 eV) were measured in a DFS Thermo Scientific high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Macherey-Nagel silica gel (63–200 μ) was used for column chromatography. Thin-layer chromatography was performed on TLC Silica gel 60 plates (Merck KGaA, Darmstadt, Germany).

Starting materials, reagents, and solvents used for synthesis were purchased from Sigma Aldrich, Acros Organics, and AlfaAesar (95 99% pure). (+) usnic acid (UA) was obtained from Zhejiang Yixin Pharmaceutical Co., Ltd., (Lanxi, China). All chemicals were used as described unless otherwise noted. Reagent-grade solvents were redistilled prior to use. Bromousnic acid (BrUA) was synthesized according to the method used in the previous literature [23].

2.1.1. General Procedure for Synthesis of Sulfides 8a-m

Reaction of BrUA with thiols (general method): a weighed portion of KOH (1.1 mmol), MeOH (6 mL), and the appropriate thiol (1.1 mmol) were placed into a flask, stirred at room temperature for 10–15 min, treated with a solution of BrUA (1 mmol) in CH₂Cl₂ (2 mL), and stirred at room temperature for 2–3 h until the reaction was complete (TLC monitoring). The resulting mixture was washed with distilled H₂O (two times the volume), dried over MgSO₄, and concentrated. If necessary, the solid was chromatographed over silica gel using CH₂Cl₂ as the eluent. The ratio of diastereomers in the mixture was calculated from the integrated resonances of the H-4 proton.

The spectra of compound 8b corresponds to that seen in the literature [24]. The spectra of compounds 8c, d, i, e, j, k, and m also correspond to the literature [23].

(R)-2-Acetyl-6-(2-((4-fluorophenyl)thio)acetyl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 8a: yellow amorphous powder, yield 40% ¹H NMR (CDCl₃, δ) 1.74 (3H, s), 2.10 (3H, s), 2.65 (3H, s), 4.13 (2H, d, J 14.3 and d, J 14.3, AB-syst), 5.95 (1H, s), 6.97 (2H, m), 7.37 (2H, m), 11.11 (OH, s), 12.81 (OH, s), 18.84 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.8, 31.8, 45.3, 58.7, 98.4, 99.6, 104.1, 105.1, 109.5, 116.0 and 116.1, 128.9, 134.0 and 134.1, 154.3, 157.8, 161.4 and 163.4 (J_C–F 248), 164.1, 178.7, 191.5, 196.0, 197.7, 201.7. HRMS m/z 470.0825 (calcd for C₂₄H₁₉FO₇S, 470.0836).

(R)-2-Acetyl-6-(2-((4-bromophenyl)thio)acetyl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 8c: yellow amorphous powder, yield 55% ¹H NMR (CDCl₃, δ) 1.74 (3H, s), 2.09 (3H, s), 2.65 (3H, s), 4.19 (2H, d, J 14.3 and d, J 14.3, AB-syst), 5.93 (1H, s), 7.23 (2H, d, J 8.5), 7.37 (2H, d, J 8.5), 11.11 (OH, s), 12.77 (OH, s), 18.82 (OH, s). ¹³C NMR (CDCl₃, δ): 7.6, 27.9, 31.9, 44.3, 58.8, 98.5, 99.7, 104.1, 105.1, 109.6, 121.4, 133.5, 154.3, 157.9, 164.2, 178.6, 191.5, 195.8, 197.7, 201.7. HRMS m/z 530.0024 (calcd for C₂₄H₁₉BrO₇S, 530.0029).

(R)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-((5-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)dibenzo[b,d]furan-1(9bH)-one 8f: yellow amorphous powder, yield 56% ¹H NMR (CDCl₃, δ) 1.61 (3H, s), 2.01 (3H, s), 2.32 (3H, s), 2.61 (3H, s), 4.56 (2H, d, J 16.9 and d, J 16.9, AB-syst), 5.80 (1H, s), 6.87 (1H, d, J 8.3), 7.15 (1H, s), 7.26, (1H, d, J 8.3), 11.11 (OH, s), 11.41 (NH, s),12.61 (OH, s), 18.68 (OH, s). ¹³C NMR (CDCl₃, δ): 7.5, 21.5, 27.8, 31.8, 42.7, 58.6, 98.8, 100.0, 104.4, 105.1, 109.4, 113.9, 123.9, 132.4, 138.4, 148.0, 154.6, 158.3, 163.7, 178.2, 191.4, 195.2, 197.7, 201.7. HRMS m/z 506.1139 (calcd for C₂₆H₂₂N₂O₇S, 506.1142).

(R)-2-Acetyl-3,7,9-trihydroxy-6-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)acetyl)-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 8g: yellow amorphous powder, yield 71% ¹H NMR (CDCl₃, δ) 1.64 (3H, s), 2.03 (3H, s), 2.62 (3H, s), 3.74 (3H, s), 4.61 (2H, d, J 16.6 and d, J 16.6, AB-syst), 5.86 (1H, s), 6.70 (1H, d, J 8.8), 6.90 (1H, s), 7.30, (1H, d, J 8.8), 11.14 (OH, s), 12.64 (OH, s), 18.74 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.7, 29.6, 31.8, 42.8, 55.6, 58.6, 96.7, 98.8, 100.0, 104.4, 105.1, 109.4, 111.8, 116.5, 137.5, 147.3, 154.6, 156.3, 158.3, 163.7, 178.2, 191.4, 195.3, 197.7, 201.6. HRMS m/z 522.1094 (calcd for C₂₆H₂₂N₂O₈S, 522.1091).

(R)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-((1-methyl-1H-imidazol-2-yl)thio)acetyl)dibenzo[b,d]furan-1(9bH)-one 8h: yellow amorphous powder, yield 50% ¹Н NMR (CDCl₃, δ) 1.73 (3H, s), 2.07 (3H, s), 2.63 (3H, s), 3.62 (3H, s), 4.47 (2H, d, J 15.8 and d, J 15.8, AB-syst), 5.98 (1H, s), 6.90 (12H, d, J 1.1), 7.01, (1H, d, J 1.1), 11.10 (OH, s), 12.77 (OH, s), 18.77 (OH, s). ¹³C NMR (CDCl₃, δ): 7.5, 27.8, 31.9, 33.4, 44.9, 58.8, 98.7, 100.0, 104.2, 105.1, 109.4, 122.7, 129.6, 139.6, 154.6, 157.9, 163.8, 178.7, 191.5, 195.4, 197.9, 201.7. HRMS m/z 456.0979 (calcd for C₂₂H₂₀N₂O₇S, 456.0986).

(R)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-(pyridin-4-ylthio)acetyl)dibenzo[b,d]furan-1(9bH)-one 8l: yellow amorphous powder, yield 53% ¹H NMR (CDCl₃, δ) 1.76 (3H, s), 2.08 (3H, s), 2.65 (3H, s), 4.65 (2H, d and d, J 15.7, AB-syst), 5.98 (1H, s), 7.29 (2H, d, J 6.2), 8.39 (2H, d, J 6.2), 11.15 (OH, s), 12.68 (OH, s), 18.79 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.7, 31.8, 40.8, 58.7, 98.7, 99.6, 104.2, 105.1, 109.7, 120.7, 147.2, 149.2, 154.3, 158.2, 164.1, 178.3, 191.4, 194.7, 197.7, 201.7. HRMS m/z 453.0879 (calcd for C₂₃H₁₉NO₇S, 453.0877).

2.1.2. General Procedure for Synthesis of Sulfoxides 9b-f, j, k and m

Method A: a weighed portion of VO (acac)2 (0.5 mg) was dissolved in CH₂Cl₂ (5 mL), stirred at room temperature for 10–15 min, treated with the appropriate sulfide (8c or 8j, 0.215 mmol) in CH₂Cl₂ (3 mL) and H₂O₂ (0.027 mL, 30%), stirred for 3–4 h at room temperature until the reaction was finished, diluted with three times the volume of H₂O, and extracted with CH₂Cl₂. The extract was dried over MgSO₄, concentrated, and chromatographed over SiO₂ [CH₂Cl₂ + MeOH (1%) eluent]. The diastereomeric ratio of the mixture was 1:1 according to PMR spectra.

(9bR)-2-Acetyl-6-(2-((4-bromophenyl)sulfinyl)acetyl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 9b: yellow amorphous powder, yield 40% ¹H NMR (CDCl₃, δ) 1.70 (3H, s), 2.08 (3H, s), 2.64 (3H, s), 4.51 (2H, d and d, J 13.7, AB-syst) and 4.52 (2H, d and d, J 14.3, AB-syst), 5.97 (1H, s) and 5.94 (1H, s), 7.52 (1H, d, J 8.8), 7.62 (1H, d, J 8.8), 11.26 (OH, s), 12.59 (OH, s) and 12.63 (OH, s), 18.84 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.8, 31.8, 58.6, 68.6 and 68.8, 98.9, 101.5, 104.51, 105.1, 108.6, 125.8, 126.2, 132.4, 154.6, 158.9, 163.8, 178.1, 191.0, 191.4, 197.6, 201.8.

(9bR)-2-Acetyl-6-(2-((5-amino-1,3,4-thiadiazol-2-yl)sulfinyl)acetyl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 9j: yellow amorphous powder, yield 29% ¹H NMR (CDCl₃, δ) 1.683 and 1.69 (3H, s), 1.99 (3H, s), 2.59 (3H, s), 4.76 (d, J 14.1) and 4.96 (d, J 14.1)–2H, AB-syst, 4.82 (d, J 15.5) and 4.92 (d, J 15.5)–2H, AB-syst, 5.99 and 6.02 (1H, s), 6.65 (2H, NH₂), 11.21 and 11.22 (OH, s), 12.37 (OH, s), 18.71 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.7, 31.8, 58.7 and 60.3, 67.8 and 67.9, 99.2 and 99.3, 100.9 and 101.1, 104.7, 105.1, 109.6, 154.7, 159.1, 163.8, 171.9, 178.1, 190.8 and 190.9, 191.6, 197.7, 201.7.

Method B: weighed portions of VO (acac)2 (0.5 mg) and (R,E)-2,4-di-tert-butyl-6-(((1-hydroxy-3-phenylpropan-2-yl)imino)methyl)phenol (0.5 mg) were dissolved in CH₂Cl₂ (5 mL), stirred at room temperature for 10–15 min, treated with the appropriate sulfide (8b, 8d, 8g, 8k, or 8m, 0.215 mmol) in CH₂Cl₂ (3 mL) and H₂O₂ (0.027 mL, 30%), stirred for 3–4 h at room temperature until the reaction was finished, diluted with three times the volume of H₂O, and extracted with CH₂Cl₂. The extract was dried over MgSO₄, concentrated, and chromatographed over SiO₂ [CH₂Cl₂ + MeOH (1%) eluent].

The spectra of compounds 9b and 9d correspond to those in the literature [23].

(9bR)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-(pyridin-2-ylsulfinyl)acetyl)dibenzo[b,d]furan-1(9bH)-one 9k: yellow amorphous powder, yield 48% ¹Н NMR (CDCl₃, δ) 1.73 (3H, s), 2.07 (3H, s), 2.63 (3H, s), 4.36 (1H, m), 4.94 (1H, m), 5.96 (1H, s), 7.40 (1H, m), 7.96 (2H, m), 8.62 (1H, m), 11.19 (OH, s), 12.62 and 12.67 (OH, s and s), 18.82 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.8, 31.8, 58.7, 65.9 and 66.6, 98.7 and 98.9, 101.4 and 101.7, 104.3 and 105.1, 109.4 and 109.5, 120.3 and 120.4, 125.0, 138.1, 149.5, 154.7, 158.5 and 158.6, 163.7 and 163.8, 178.4, 191.5, 191.7 and 192.0, 197.7, 201.7.

(9bR)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-(pyrimidin-2-ylsulfinyl)acetyl)dibenzo[b,d]furan-1(9bH)-one 9m: yellow amorphous powder, yield 53% ¹H NMR (CDCl₃ + DMSO-d6, δ) 1.61 (3H, s), 1.91 (3H, s), 2.50 (3H, s), 4.47 (1H, m), 4.88 (1H,m), 5.84 (1H, s), 7.36 (1H, m), 8.77 (2H, m), 11.10 (OH, s), 12.40 and 12.42 (OH, s and s), 18.69 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.8, 31.9, 58.7, 64.4 and 65.3, 98.8 and 98.9, 101.3 and 101.8, 104.5, 105.1, 109.4 and 109.6, 122.0, 154.7 and 154.9, 158.5 and 158.6, 158.8, 163.7 and 163.8, 172.4 and 172.6, 178.4, 191.5, 191.4 and 191.7, 197.7, 201.8.

Method C: sulfide 8e or 8f (1 mmol) was dissolved in CH₂Cl₂ (5 mL) in an ice bath. The m-chloroperoxybenzoic acid (1 mmol) was added and the resulting solution was stirred for 30 min at 0 °C. After that, the mixture was treated with saturated sodium sulfite solution (3 mL) and stirred for 1 h at room temperature. Finally, the mixture was extracted with CH₂Cl₂, washed with water, dried over magnesium sulfate, and evaporated and chromatographed over SiO₂ [CH₂Cl₂ + MeOH (1%) eluent].

(9bR)-6-(2-((1H-Benzo[d]imidazol-2-yl)sulfinyl)acetyl)-2-acetyl-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 9e: yellow amorphous powder, yield 46% ¹H NMR (CDCl₃, δ) 1.58 (3H, s), 2.02 (3H, s), 2.61 (3H, s), 4.98 (2H, d and d, J 14.8, AB-syst), 4.94 (2H, d and d, J 14.8, AB-syst), 5.93 (1H, s), 5.98 (1H, s), 7.26 (1H, m), 7.48 (1H, m), 7.67 (1H, m), 11.19 (OH, s), 12.26 (OH, s), 12.43 (NH, s), 18.46 (OH, s). ¹³C NMR (CDCl₃, δ): 7.3, 29.2, 29.6, 58.4, 66.7, 99.3, 100.9, 104.6, 105.0, 109.5, 112.3, 120.3, 123.3, 124.8, 131.6, 134.4, 143.5, 151.3, 154.5, 159.0, 163.8, 178.0, 190.8, 191.3, 197.5, 201.7.

(9bR)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-((5-methyl-1H-benzo[d]imidazol-2-yl)sulfinyl)acetyl)dibenzo[b,d]furan-1(9bH)-one 9f: yellow amorphous powder, yield 34% ¹ H NMR (CDCl3, δ) 1.62 and 1.66 (3H, s), 2.07 and 2.08 (3H, s), 2.47 and 2.48 (3H, s), 2.66 and 2.68 (3H, s), 4.76 (d, J 15.4) and 5.21 (d, J 15.4)–AB-syst and 4.91 (d, J 15.0) and 5.08 (d, J 15.0)–AB-syst, 5.95 (1H, s) 5.99 (1H, s), 6.85–6.96 (1H, m), 7.38 (1H, s), 7.47–7.54 (1H, m), 9.78 (NH), 11.20 and 11.23 (OH, s), 12.51 (OH, s), 18.82 and 18.86 (OH, s). ¹³C NMR (CDCl₃, δ): 7.2 and 7.5, 21.0 and 21.2, 27.7, 31.2, 58.4, 65.2 and 65.9, 98.6 and 98.9, 101.3, 105.1 and 105.2, 105.3 and 105.4, 107.4 and 107.5, 108.1 and 109.1, 108.9 and 109.6, 120.8 and 123.1, 127.4 and 130.2, 129.3, 129.8 and 131.6, 152.5 and 152.6, 154.7 and 155.4, 157.5 and 157. 6, 162.2 and 162.3, 178.6 and 179.8, 191.3 and 191.4, 192.5 and 192.6, 197.7, 201.2.

2.1.3. General Procedure for Synthesis of Sulfones 12a-e, g, h, k and m

The corresponding sulfides, 8a–e, g, h, k, and m (1 mmol), were dissolved in CH₂Cl₂ (5 mL) in an ice bath. The m-chloroperoxybenzoic acid (3 mmol) was added and the resulting solution was stirred for 30 min at 0 °C. The mixture was then treated with saturated sodium sulfite solution (3 mL) and was stirred for 1 h at room temperature. Finally, the mixture was extracted with CH₂Cl₂, dried over magnesium sulfate, and evaporated and chromatographed over SiO₂ [CH₂Cl₂ + MeOH (1%) eluent].

(R)-2-Acetyl-6-(2-((4-fluorophenyl)sulfonyl)acetyl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 12a: yellow amorphous powder, yield 57% ¹ H NMR (CDCl₃, δ) 1.75 (3H, s), 2.08 (3H, s), 2.65 (3H, s), 4.80 (1H, d, J 13.7) and 4.89 (1H,d, J 13.7) –AB-syst, 6.04 (1H, s), 7.20 (2H, m), 7.87 (2H, m), 11.35 (OH, s), 12.48 (OH, s), 18.85 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.8, 31.8, 58.7, 66.6, 98.9, 101.7, 104.8, 105.1, 109.5, 116.3, 116.6, 131.3, 131.4, 135.0, 154.7, 159.2, 164.1, 164.3, 167.7, 178.0, 191.5, 191.7, 197.7, 201.8. HRMS m/z 502.0730 (calcd for C₂₄H₁₉O₉SF, 502.0728).

The spectrum of compound 12b corresponds to that in the literature [24].

(R)-2-Acetyl-6-(2-((4-bromophenyl)sulfonyl)acetyl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 12c: yellow amorphous powder, yield 55% ¹ H NMR (CDCl₃, δ) 1.72 (3H, s), 2.07 (3H, s), 2.64 (3H, s), 4.80 (1H, d, J 13.7) and 4.87 (1H,d, J 13.7) –AB-syst, 6.00 (1H, s), 7.64 (2H, d, J 8.5) and 7.69 (2H, d, J 8.5)–AB-syst, 11.33 (OH, s), 12.47 (OH, s), 18.83 (OH, s). ¹³C NMR (CDCl₃, δ): 7.5, 27.8, 31.9, 58.7, 66.5, 98.9, 101.7, 104.8, 105.2, 109.6, 129.6, 129.9, 132.4, 137.9, 154.7, 159.2, 164.2, 178.0, 188.1, 191.5, 197.7, 201.9. HRMS m/z 561.9930 (calcd for C₂₄H₁₉O₉SBr, 561.9928).

(R)-2-Acetyl-6-(2-(benzo[d]thiazol-2-ylsulfonyl)acetyl)-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 12d: yellow amorphous powder, yield 60% ¹H NMR (CDCl3, δ) 1.69 (3H, s), 2.06 (3H, s), 2.64 (3H, s), 5.25 (1H, d, J 14.7) and 5.31 (1H,d, J 14.7) –AB-syst, 6.02 (1H, s), 7.61 (2H, m), 7.99 (1H, m), 8.15 (1H, m), 11.31 (OH, s), 12.35 (OH, s), 18.84 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.7, 31.7, 58.6, 64.6, 99.0, 101.5, 104.7, 105.1, 109.7, 122.2, 125.4, 127.6, 128.2, 137.0, 152.3, 154.6, 159.2, 164.1, 170.6, 177.9, 187.2, 191.5, 197.6, 201.8. HRMS m/z 541.0498 (calcd for C₂₅H₁₉NO₉S₂, 541.0496).

(R)-2-Acetyl-3,7,9-trihydroxy-6-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)sulfonyl)acetyl)-8,9b-dimethyldibenzo[b,d]furan-1(9bH)-one 12g: yellow amorphous powder, yield 20% ¹H NMR (CDCl3, δ) 1.63 (3H, s), 2.05 (3H, s), 2.66 (3H, s), 3.83 (3H, s), 5.12 (1H, d, J 15.0) and 5.44 (1H,d, J 15.0)–AB-syst, 6.00 (1H, s), 6.96 (1H, d, J 7.5), 6.97 (1H, s), 7.56 (1H, d, J 7.5), 11.29 (OH, s), 12.37 (OH, s), 18.82 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.7, 31.8, 55.6, 58.4, 65.3, 99.2, 101.4, 104.7, 105.0, 109.5, 154.4, 159.2, 164.0, 175.2, 177.6, 187.7, 191.5, 197.5, 201.8. HRMS m/z 554.0995 (calcd for C₂₆H₂₂N₂O₁₀S, 554.0990).

(R)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-((1-methyl-1H-imidazol-2-yl)sulfonyl)acetyl)dibenzo[b,d]furan-1(9bH)-one 12h: yellow amorphous powder, yield 43% 1 H NMR (CDCl3, δ) 1.74 (3H, s), 2.05 (3H, s), 2.64 (3H, s), 3.95 (3H, s), 5.15 (2H, s), 6.05 (1H, s), 7.00 (1H, s), 7.55 (1H, s), 11.33 (OH, s), 12.38 (OH, s), 18.83 (OH, s). ¹³C NMR (CDCl₃, δ): 7.5, 27.8, 31.9, 58.7, 66.5, 98.9, 101.7, 104.8, 105.2, 109.6, 129.6, 129.9, 132.4, 137.9, 154.7, 159.2, 164.2, 178.0, 188.1, 191.5, 197.7, 201.9. HRMS m/z 488.0883 (calcd for C₂₂H₂₀N₂O₉S, 488.0884).

(R)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-(pyridin-2-ylsulfonyl)acetyl)dibenzo[b,d]furan-1(9bH)-one 12k: yellow amorphous powder, yield 52% ¹H NMR (CDCl3, δ) 1.75 (3H, s), 2.03 (3H, s), 2.64 (3H, s), 5.21 (2H, s), 6.05 (1H, s), 7.56 (1H, m), 7.95 (1H, m), 8.04 (1H, m), 8.72 (1H, m), 11.30 (OH, s), 12.35 (OH, s), 18.84 (OH, s). ¹³C NMR (CDCl₃, δ): 7.4, 27.8, 31.9, 58.7, 61.8, 99.0, 101.5, 104.7, 105.1, 109.5, 122.2, 127.5, 138.1, 150.0, 154.7, 156.9, 159.0, 164.0, 178.1, 188.7, 191.5, 197.7, 201.8. HRMS m/z 485.0773 (calcd for C₂₃H₁₉NO₉S, 485.0775).

(R)-2-Acetyl-3,7,9-trihydroxy-8,9b-dimethyl-6-(2-(pyrimidin-2-ylsulfonyl)acetyl)dibenzo[b,d]furan-1(9bH)-one 12l: yellow amorphous powder, yield 21% ¹H NMR (CDCl3, δ) 1.81 (3H, s), 2.07 (3H, s), 2.69 (3H, s), 5.38 (2H, s), 6.10 (1H, s), 7.62 (1H, t, J 4.8), 9.89 (2H, d, J 4.8), 11.36 (OH, s), 12.26 (OH, s), 18.88 (OH, s). ¹³C NMR (CDCl₃, δ): 7.3, 27.7, 31.8, 58.7, 61.1, 99.0, 101.1, 104.7, 105.1, 109.5, 123.7, 154.7, 158.5, 159.0, 163.9, 165.3, 178.1, 188.8, 191.5, 197.7, 201.8. HRMS m/z 486.0727 (calcd for C₂₂H₁₈N₂O₉S, 486.0728).

2.2. Biology

2.2.1. Real-Time Detection of TDP1 Activity

The oligonucleotide carrying a fluorophore at the 5′-end (5,6)-FAM and a fluorescence quencher Black Hole Quencher 1 (BHQ1) at the 3′-end (5′-FAM-AAC GTC AGG GTC TTC C-BHQ1–3′) was synthesized in the Laboratory of Nucleic Acid Chemistry at the Institute of Chemical Biology and Fundamental Medicine (Novosibirsk, Russia), and was used for TDP1 enzyme activity real-time fluorescence detection. A POLARstar OPTIMA fluorimeter (BMG LABTECH, GmbH, Ortenberg, Germany) was used to measure fluorescence [23].

The reaction mixtures were prepared to study the activity of TDP1 in a black 96-hole Corning™ plate. The reaction mixture (200 µL) contained a TDP1 buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 7 mM β-mercaptoethanol), 50 nM oligonucleotide, 1.5 nM TDP1, and various concentrations of the tested compounds. The inhibitors were dissolved in 100% DMSO. The reactions were initiated by the addition of purified TDP1. The reaction mixtures were incubated at a constant temperature of 26 °C in a POLARstar OPTIMA fluorimeter (BMG LABTECH, GmbH, Ortenberg, Germany). Fluorescence intensity was measured every 55 s for 8 min in three independent experiments. The average values of the half-maximal inhibitory concentration (IC₅₀), the concentration of the compound that inhibited 50% of the enzyme activity when compared to controls (samples with absence of an inhibitor), were determined using a six-point concentration–response curve. The IC₅₀ values were calculated as the average of at least three experiments ± standard deviation using MARS Data Analysis 2.0 (BMG LABTECH).

2.2.2. Gel-Based TDP2 Activity Assay

The oligonucleotide carrying a fluorophore at the 3′-end (5,6)-FAM) and a tyrosine residue at the 5′-end (5′-tyrosine-AAC GTC AGG GTC TTC C-FAM-3′) was synthesized to study the enzyme activity of TDP2 in polyacrylamide gel. The reaction mixture (10 µL) contained a TDP2 buffer (50 mM Tris-HCl, pH 8.0; 40 mM NaCl; 1 mM DTT, 0.05 mg/mL BSA), 8 mM MgCl₂, 100 nM oligonucleotide, 200 nM recombinant human TDP2, and various concentrations of the tested compounds. Reactions were initiated by the addition of TDP2 and incubated in the presence of an inhibitor for 15 min at 37 °C. Reactions were terminated by the addition of solution containing 90% formamide, 50 mm EDTA, and 0.025% of both bromophenol blue and xylen cyanol. Before using electrophoresis, the samples were heated at 97 °C for 5 min. The reaction products were separated by electrophoresis in a 20% denaturing PAGE with 7 M carbamide at a ratio of acrylamide to bisacrylamide of 19:1 [23]. Typhoon FLA 9500 phosphorimager (GE Healthcare, Boston, MA, USA) was used for gel scanning and imaging, and the data were analyzed with QuantityOne 4.6.7 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). IC₅₀ values were determined using OriginPro 8.6.0 (OriginLab, Northampton, MA, USA), and were calculated as the average of at least three experiments ± standard deviation.

2.2.3. MTT Test

Cytotoxicity of the compounds was examined against human cell lines HEK293A (human embryonic kidney), MRC-5 (human lung fibroblasts), and HeLa (cervical cancer) using the MTT test (Biomedica, Vienna, Austria) [25]. The HEK293A cell line was obtained from Thermofisher (ThermoFisher Scientific, Waltham, MA, USA). The HeLa cell line was obtained from the Russian Cell Culture Collection (RCCC) Institute of Cytology RAS, St. Petersburg, Russia. The MRC-5 cell line was provided by the Cell Culture Bank of the State Research Center for Virology and Biotechnology Vector, Novosibirsk, Russia. The cells were grown in DMEM/F12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), with 1x GlutaMAX (Gibco), 100 IU/mL penicillin, and 100 μg/mL streptomycin (MP Biomedicals, Santa Ana, CA, USA), and in the presence of 10% fetal bovine serum (Biolot, Ankara, Turkey) in a 5% CO₂ atmosphere. Cells were grown in the presence of 1% DMSO in the control wells. At 30–50% confluence, the tested compounds were added to the medium to a final DMSO concentration of 1%, and the cell culture was grown at 37 °C and 5% CO₂ for 3 days before MTT test. The values were normalized to their own control in each case. At least three independent experiments were carried out. The 50% cytotoxic concentration (CC₅₀) was defined as the compound concentration that reduced the cell viability by 50% when compared to the untreated controls, and was calculated as the average of at least three experiments ± standard deviation. The compound concentration that caused 50% cell growth inhibition was determined using OriginPro 8.6.0 software. The CI values were calculated with the CompuSyn version 1.0 software. The CI plot was determined for Tpc (0.5, 1, 2, 4, and 8 µM) and the TDP1 inhibitors (10 and 20 µM).

2.2.4. Alkaline Comet Assay

HeLa cells were grown in a 24-well plate to a concentration of 0.05 million/mL, then treated with the tested compounds and incubated for an hour. The cells in each well were treated with phosphate-buffered saline (PBS) (500 µL) and trypsin (30 µL for 5 min at 37 °C) and washed with 200 µL of PBS + 10% fetal bovine serum (FBS) to obtain a cell suspension.

The resulting cell suspension (30 µL) was mixed with 250 µL of a 1% solution of molten low melting-point (LMP) agarose (CertifiedTM LM Agarose; Bio-Rad, Hercules, CA, USA), and 250 µL of this mixture was transferred to the slides pre-coated with a 1% normal melting-point agarose (Agarose; Helicon) and left for several minutes at 4 °C for solidification. The resulting slides were incubated in a lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1% Triton, 5% DMSO, pH 10.0) for an hour at 4 °C. The slides were put in an electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 45 min at 4 °C. Electrophoresis was performed at 20 V and 450 mA for 10 min. After that, the slides were put in neutralization buffer (0.4 M Tris-HCl pH 7.5) for 5 min two times, washed twice with cold water, and stained with SYBR Green I (excitation maximum at 497 nm, emission maximum at 520 nm; Thermo Fisher Scientific, Waltham ,MA, USA). Finally, the slides were put in a 70% ethanol solution for 5 min and dried in a thermostat at 37 °C for 60 min.

The images were obtained using a CELENA© S (Logos Biosistems, Inc., Gyeonggi-do, Republic of Korea) digital microscope and analyzed in Comet analysis software version 1.3d (Trevigen, Inc., Gaithersburg, MD, USA). In all experiments, 2 slides were evaluated for each sample by counting at least 200 cells on the glass. The median value of the percentage of DNA in the tail was chosen as a parameter for measuring the level of DNA damage (100× the intensity of DNA fluorescence in the tail of the comet/the intensity of fluorescence of the total DNA of the comet).

2.3. Molecular Modeling and Chemical Space

The compounds were docked against the crystal structures of TDP1 (PDB ID: 6W7K, resolution 1.70 Å) [26] and TDP2 (PDB ID: 5J3S, resolution 3.40Å) [27] enzymes, which were obtained from the Protein Data Bank (PDB) [28,29]. The GOLD (v2020.2.0) software suite was used to prepare the crystal structures for docking, i.e., the hydrogen atoms were added, the water molecules were deleted, and the co-crystallised ligands were identified: 4-[(2-phenylimidazo [1,2-a]pyridin-3-yl)amino]benzene-1,2-dicarboxylic acid (TG7) for TDP1 and 2,4-dioxo-10-[3-(1H-tetrazol-5-yl)phenyl]-2,3,4,10-tetrahydropyrimido [4,5-b]quinolone-8-carbonitrile (6FQ) for TDP2. The Scigress version FQ 3.4.4 program [30] software suite was used to build the ligands, and the MM3 [31,32] force field was applied to identify the global minimum using the CONFLEX method [33] followed by structural optimization. The docking centers for TDP1 and TDP2 were defined as the positions of their co-crystallized TG7 and 6FQ ligands with 10 Å radius. Fifty docking runs were allowed for each ligand with default search efficiency (100%). The basic amino acids lysine and arginine were defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be deprotonated. The GoldScore (GS) [34] and ChemScore (CS) [35,36] ChemPLP (piecewise linear potential) [37] and ASP (Astex Statistical Potential) [38] scoring functions were implemented to predict the binding modes and relative binding energies of the ligands using the GOLD v2020.2.0 software suite.

The QikProp 6.2 [39] software package was used to calculate the molecular descriptors of the molecules. The reliability of QikProp for the calculated descriptors is established [40]. The known drug indexes (KDIs) were calculated from the molecular descriptors as described by Eurtivong and Reynisson [41]. For application in Excel, columns for each property were created and the following equations were used to derive the KDI numbers for each descriptor: KDI MW: = EXP(−((MW-371.76)²)/(2 × (112.76²))), KDI Log P: = EXP(−((LogP − 2.82⁾²)/(2 × (2.21²))), KDI HD: = EXP(−((HD − 1.88)²)/(2 × (1.7²))), KDI HA: = EXP(−((HA − 5.72)²)/(2 × (2.86²))), KDI RB = EXP(−((RB − 4.44)²)/(2 × (3.55²))), and KDI PSA: = EXP(−((PSA − 79.4)²)/(2 × (54.16²))). These equations could simply be copied into Excel and the descriptor name (e.g., MW) substituted with the value in the relevant column. To derive KDI_2A, this equation was used: = KDI MW + KDI LogP + KDI HD + KDI HA + KDI RB + KDI PSA; for KDI_2B: = KDI MW × KDI LogP × KDI HD × KDI HA × KDI RB × KDI PSA.

3. Results and Discussion

3.1. Chemistry

3.1.1. Sulfides

Compounds 8a–m were synthesized using a procedure that was described previously [24]. In the first step, bromination of (+) usnic acid (UA) by bromine in dioxane at room temperature gave the bromo-derivative BrUA. The subsequent reaction of BrUA with thiols or thiolates (generated with NaOH) in methanol at room temperature (for compounds 10a–m) resulted in the corresponding target sulfides 8a–m, obtained in yields of 40–94% (Scheme 1). Compounds 8c, d ,e, i, j, k, and m have been described in previous studies [23,24].

3.1.2. Sulfoxides

The synthesis of sulfoxides 9a–m was carried out by reacting the corresponding sulfides, 8a–m, with aqueous hydrogen peroxide (30%) as an oxidizing agent in the presence of vanadylacetylacetonate (VO (acac)₂) as a catalyst (Scheme 2). The reactions were carried out at room temperature with an excess (1.5–2 eq.) of hydrogen peroxide. Using these conditions, mixtures of diastereomeric sulfoxides 9c or 9j were formed in a 1:1 ratio (according to ¹H NMR spectra) with yields of 65% and 29%, respectively. For other compounds, the reaction proceeded slowly, not reaching 50% conversion even after a week or with higher temperature. To increase the conversion rate, we added chiral ligand 11 to the reaction mixture, which was previously selected for the chiral oxidation of sulfide 8b [24]. The use of the ligand significantly accelerated the reaction, which proceeded under these conditions over 3–4 h, and led, in some cases, to a change in the diastereomeric ratio of the sulfoxides that were obtained. Derivatives 9b, d, k, and m were isolated in 40–65% yields using these conditions. Compounds 9b and 9d have been described previously [23].

The target sulfoxides 9b–d, k, and m were isolated as mixtures of diastereomers. It is worth noting that the ratio of diastereomers depends on the structure of the substituent, reaching the highest values of de (diastereomeric excess) of 50% for 9b (

Table 1. Yields and diastereomeric excess (de) of compounds 9b-f, j, k, and m.

	9b	9c	9d	9e	9f	9j	9k	9m
Yield	40%	65%	50%	0%	0%	29%	48%	53%
de	50%	0%	10%	-	-	0%	5%	6%

).

Compounds 9e and 9f were not isolated in analytically pure forms, so oxidation was undertaken using an equimolar amount of meta-chloroperbenzoic acid in methylene chloride at 0 °C. After we conducted column chromatography, the sulfoxides were isolated in yields of 34% and 46%, respectively. Sulfoxides 9a, g, h, i, and l were not isolated.

3.1.3. Sulfones

Compounds 8a–m were oxidized to the corresponding sulfones, 12a–m, by reaction with a three-fold molar excess of meta-chloroperbenzoic acid in methylene chloride, with stirring and cooling of the reaction vessel in an ice bath (Scheme 3). Upon completion of the reaction, an aqueous solution of Na₂SO₃ was added to remove excess oxidizing agent. In several cases (sulfide 8i), oxidation under these conditions led to complex reaction mixtures containing, together with the initial sulfides, the corresponding sulfoxides, sulfones, and products of deeper oxidation that affected the usnic acid backbone [24]. The oxidation of 8e, f, and j resulted in the corresponding sulfoxides 9e, f, and j. An increase in the reaction time or temperature led to more complex mixtures of oxidation products. Targeted sulfones 12a–d, g, h, k, and m were isolated in 20–60% yields after column chromatography.

As a result of these synthetic endeavours, a series of 13 sulfides, 8 sulfoxides, and 8 sulfones was obtained for testing. It should be noted that, in a number of cases, it was not possible to isolate the target reaction products. In particular, in the case of the oxidation of sulfides 8g and 8h, it was not possible to isolate the target sulfoxides 9g and 9h due to rapid oxidation, which yielded the corresponding sulfones 12g and 12h.

In the case of compounds 8e, 8f, and 8j, when attempting to oxidate them to sulfones, the reaction stopped at the stage of sulfoxide formation, and further oxidation would probably proceed rather slowly. Attempts to increase the reaction yield by varying the temperature or time caused oxidation of the usnic acid moiety to take place, which is a known transformation [24]. A similar situation occurred for the oxidation of 8i. The increased complexity of the oxidation of the sulfur atom may be due to its increased accessibility in compounds 8e, f, j, and i, which corresponds to the results of other sulfides.

In the case of the oxidation of compounds 8a and 8l, structural rearrangements of the usnic acid backbone were observed using NMR spectroscopy, resulting in the formation of a large number of structural isomers that could not be separated chromatographically. Only compound 12a could be isolated using these conditions.

3.2. Biology

3.2.1. TDP1 and TDP2 Activity Inhibition

The derivatives were screened for the inhibition of TDP1 using a real-time activity assay to derive IC₅₀ values, and the inhibition of TDP2 activity by analysis of the reaction product in PAGE (polyacrylamide gel) [23]. Different concentration ranges were used for the TDP1 and TDP2 screens; hence, the IC₅₀ values derived for the two enzymes cannot be directly compared. The parent usnic acid does not inhibit TDP1 or TDP2 [23].

All of the sulfides described herein inhibit TDP1 in the submicromolar and micromolar concentration ranges (

Table 2. Concentration of half-maximal inhibition of TDP1 (upper) and TDP2 (lower), IC₅₀, µM.

Group	Element of structure (R)	Sulfide (8)	Sulfoxide (9)	Sulfone (12)
a		6.6 ± 0.8 >1000	not obtained	>50 >1000
b		2.2 ± 0.5 165 ± 5	1.4 ± 0.2 159 ± 41	20 ± 4 >1000
c		1.8 ± 0.5 380 ± 60	15 ± 4 150 ± 10	>50 421 ± 62
d		1.7 ± 0.6 >1000	2.1 ± 0.2 >1000	21 ± 5 >1000
e		2.4 ± 1.0 370 ± 30	4 ± 2 >1000	not obtained
f		0.33 ± 0.09 138 ± 25	5.0 ± 2.1 >1000	not obtained
g		0.4 ± 0.1 313 ± 8	not obtained	3.7 ± 1.9 332 ± 35
h		21 ± 5 >1000	not obtained	24 ± 3 >1000
i		25.2 ± 6.5 Nd *	not obtained	not obtained
j		4.3 ± 0.5 245 ± 40	21 + 5 >1000	not obtained
k		11.9 ± 0.4 >1000	25 + 4 >1000	2 ± 2 228 ± 43
l		1.5 ± 0.2 348 ± 74	not obtained	not obtained
m		16.9 ± 2.4 >1000	14.8 + 0.9 >1000	>50 >1000

* nd—not determined.

), in line with previous results [23] confirming their activity. The most potent compounds, 8f and 8g, contain a benzimidazole moiety with a methyl or methoxy substituent in the benzene ring (IC₅₀ 0.33 and 0.40 μM, respectively). It is worth noting that sulfoxide 10a and sulfides containing a benzothiazole and benzoxazole residue without substituents in the benzene ring (7e and g, described in Dyrkheeva et al. [23]), as well as the sulfide derivative of benzimidazole 8e from the present work, inhibited TDP1 with an IC₅₀ value of approximately 2 μM. Thus, the introduction of a substituent in the 5 position of the benzene ring leads to a significant (about five-fold) decrease in the IC₅₀ value.

The sulfoxides (9) showed comparable (9b, 9d, 9e, 9m) or lower (9c, 9f, 9j, 9k) levels of activity compared to their corresponding sulfides (8) against TDP1, without any apparent dependence on the structure of the heterocyclic moiety.

The sulfones (12) mostly had less TDP1 activity than their corresponding sulfides (8) and sulfoxides (9), except for 12k, containing a pyridine2-yl substituent, which was more active than its 8k and 9k analogs. Thus, the order of TDP1 activity was observed to be sulfide > sulfoxide > sulfone.

The sulfides tested herein mostly inhibited TDP2 in the range of 138–348 μM (Table 2); however, five compounds bearing different substituents (8a, 8d, 8h, 8k, 8m) did not show inhibitory activity up to a concentration of 1000 μM.

Of the sulfoxides, only two compounds with para-chloro- and para-bromo-phenyl substituents inhibited TDP2. It should be noted that similar potencies for TDP2 were shown by other usnic acid derivatives that also had a para-bromophenyl substituent [42].

Most of the series of sulfones (12) tested herein either did not show any significant TDP2 activity (12a, 12b, 12d, 12h, 12m) or retained the activity towards TDP2 that was inherent to their corresponding sulfides (IC₅₀ values of compounds 12c and 12g were 421 and 332 μM, respectively). An exception was compound 12k, which had a significantly higher activity compared to its parent sulfide 8k and its sulfoxide analog 9k.

It was concluded that the structure of the substituent bound to the sulfur atom of usnic acid derivatives more significantly affects the TDP2 inhibitory activity than the activity towards TDP1. It should also be noted that no clear relationship was observed between the oxidation state of the sulfur atom and TDP2 inhibition. In fact, all compounds that inhibited TDP2 at concentrations of 138–348 μM can be considered as dual inhibitors of both TDPs. They are type b compounds with a para-chlorophenyl fragment, 8c and 9c compounds with a para-bromophenyl substituent, benzimidazole derivatives 8e, 8f, 8g, 12g, aminothiadiazole 8j, and pyridines 12k and 8l.

The TDP1 inhibitory activity obtained for most members of the compound series 8, 9, and 12 is in the lower micromolar concentration range, which is comparable to the activity of most other TDP1 inhibitors described in the literature, including usnic acid derivatives 1 and 7, isoquinolines 2 and 5, coumarin 3, 4-thioxothiazolidin-2-on 6, and deoxycholic acid derivative 4 (Figure 1).

The TDP2 inhibitory activities of the obtained derivatives are low. However, these compounds represent a new type of dual inhibitors of TDP1 and TDP2, with only a few having been previously reported in the literature [12,20]. Compared to isoquinoline derivative 13 (Figure 2), compounds 8, 9, and 12 are more active against both TDP1 and TDP2. Furthermore, compared to the previously obtained usnic acid derivative 7 and deoxycholic acid derivative 14, the new derivatives 8, 9, and 12 exhibit comparable activity against TDP1, while being more active against TDP2.

3.2.2. Cell Viability and Synergy with Topotecan

The low intrinsic cytotoxicity of adjunctive drugs is crucial to not increasing the toxic burden of the body. Therefore, the cytotoxicity of the most potent inhibitors was assessed by MTT assay using non-cancerous HEK293A (human embryonic kidney) and MRC-5 (human lung fibroblast) cell lines, as well as the HeLa (cervical cancer) and A549 (lung cancer)) cell lines (

Table 3. Half-maximal cytotoxic concentrations of the most potent derivatives (CC₅₀, µM).

Group (R)	Cell Lines	Sulfide (8)	Sulfoxide (9)	Sulfone (12)
b	HEK293A	10 ± 2	>100	>100
	MRC5	13.4 ± 0.2	>100	>100
	HeLa	2.5 ± 0.5	27 ± 2	>100
	A549	27.1 ± 9.9	11.7 ± 3.6	5.3 ± 0.1
c	HEK293A	10 ± 2	6.8 ± 1.5	5.1 ± 0.1
	MRC5	8 ± 4	17.3 ± 5.1	8.8 ± 2.1
	HeLa	7 ± 1	14.5 ± 2.7	11.9 ± 1.5
	A549	40.5 ± 4.9	19.4 ± 4.4	5.8 ± 0.7
f	HEK293A	>100	>100	Not obtained
	MRC5	>100	>100
	HeLa	>100	>100
	A549	>100	>100
g	HEK293A	>100	Not obtained	>100
	MRC5	>100		>100
	HeLa	>100		36.5 ± 5.7
	A549	10.8 ± 2.6		30.1 ± 4.4
k	HEK293A	>100	>100	>100
	MRC5	>100	>100	>100
	HeLa	10.1 ± 0.2	32.1 ± 0.1	>100
	A549	12.1 ± 0.3	42.3 ± 2.9	62.1 ± 0.4

, Figure S55). The intrinsic cytotoxicity was quite high for the derivatives with a para-bromophenyl substituent (8c, 9c, and 12c), as evident from the CC₅₀ values for the cell lines, which were close to the corresponding TDP1 IC₅₀ values (see Table 2). However, compounds from the other groups were less toxic: for some derivatives, a tendency towards a decrease in cytotoxicity with an increase in the oxidation state of the sulfur atom was observed, which is consistent with our previous findings [23]. This trend is most notable for the derivatives with para-chlorophenyl and ortho-pyridine substituents (series 8b, 9b, and12b and 8k, 9k, and12k, respectively). Benzimidazole derivatives (series 8f, 9f, and 12f and 8g, 9g, 12g) are significantly less toxic than the other compounds for most cell types, although 12g, bearing the methoxybenzimidazole substituent, is moderately toxic for tumor cells. The lung cancer cells (A549) were sensitive to all studied compounds except for benzimidazole derivatives 8f and 12f. Non-cancerous cells were found to be insensitive to heterocyclic compounds of the f, g, and k series. The halogen-containing series b and c both exerted a toxic effect on a larger number of cell lines, including non-cancerous ones, compared to their non-halogenated counterparts.

Thus, the cytotoxicity of the studied compounds clearly depends on the nature of the substituent rather than on the oxidation state of the sulfur atom. No clear structure–function relationships could be found here.

In our previous work, we demonstrated that TDP1 inhibitors derived from usnic acid, when combined with topotecan, reduced tumor growth and metastasis in mice models [12]. The inhibitors in series b, f, g, and k, all with low inherent cytotoxic potentials (Table 3), were tested, and the non-cancerous HEK293A cells did not show any synergy (Figure S56); however, promising potentiation of the action of topotecan in the HeLa cancer cells was observed, except for with sulfoxide 9b (Figure 3 and

Table 4. CC₅₀ values (μM) of topotecan in HeLa cells in the presence of selected inhibitors.

Group	Sulfide (8)	Sulfoxide (9)	Sulfone (12)
b	0.6 ± 0.3	1.9 ± 1.3	0.6 ± 0.2
f	0.8 ± 0.1	0.6 ± 0.1	Not obtained
g	1.5 ± 0.3	Not obtained	0.6 ± 0.2
k	0.6 ± 0.1	0.6 ± 0.2	1.0 ± 0.7

). The CC₅₀ value for topotecan is 2.4 ± 0.7 μM.

It should be noted that sulfone 12b is a poor inhibitor of both TDP1 and TDP2 (Table 2) but enhances the action of topotecan (Table 4). Such a synergistic effect of poor TDP1 inhibition with topotecan was previously observed for nucleoside derivatives [43]. This is probably due to these compounds modulating other targets in the cell.

We derived the combination index (CI) for topotecan and 8f, 9f, and 12k in HeLa cells (Table S1), as this index is used to determine the degree of synergy between drugs [44]. All the CI values were lower than 1; thus, a clear sensitizing effect was seen. Furthermore, 8f showed a sensitizing effect in the cancer cell line A549. The other derivatives did not show significant CI values, were too cytotoxic to gain a plausible result, or were simply just weak TDP1 inhibitors.

3.2.3. Comet Assay Results

The Comet assay is a sensitive method for the detection of single- and double-strand breaks and alkaline labile sites in DNA [45]. Compounds 8f, 8g, and 12k were selected for testing since these ligands have good overall efficacy. Again, the HeLa cell line was used with topotecan. The percentage of DNA in the tails was only analyzed for the inhibitors, the inhibitors with 1% DMSO, topotecan with 1% DMSO, and topotecan in the presence of increasing concentrations of each inhibitor (Figure 4). All three inhibitors gave dose-dependent responses and exhibited increased DNA damage as compared to topotecan, with 8f and 12g being considerably more potent than 8g. However, 8g has the least intrinsic DNA damage potential as compared to 8f and 12g.

3.3. Molecular Modelling

Twenty-nine derivatives were docked into the binding sites of the TDP1 (PDB ID: 6W7K, resolution 1.70 Å) [26] and TDP2 (PDB ID: 5J3S, resolution 3.40Å) [27] enzymes. The robustness of the TDP1 docking scaffold has been previously established [46]. The scoring functions GoldScore (GS) [34], ChemScore (CS) [35,36], ChemPLP (piecewise linear potential) [37], and ASP (Astex Statistical Potential) [38] in the GOLD (v2020.2.0) docking algorithm were used. The GOLD docking suite is reported to be an excellent molecular modelling tool [47,48].

The binding scores for the TDP1 and TDP2 catalytic pockets are given in Table S2 and S3, respectively; all the ligands have reasonable values that indicate plausible binding. When the scores of the active ligands were correlated against their IC₅₀ values, only weak trends were observed for the ASP (R²–0.144), ChemPLP (R²–0.113), CS (R²–0.188), and GS (R²–0.036). No trends between the docking scores and the TDP2 IC₅₀ values were observed.

No dominant binding poses were predicted by the four scoring functions for either TDP1 or TDP2, i.e., different poses were predicted. The binding poses predicted by the ChemPLP scoring function [37] of 8f and 12k in TDP1 and 9c in TDP2 were investigated in detail. For TDP1, all the ligands overlapped with the co-crystallized ligands as well as occupying the binding pocket containing the catalytic His263 and His493 amino acid residues (see Figure 5a,c). The dibenzofuranone ring moiety (8f) overlaps with the co-crystallized ligand, whereas the benzoimidazole ring lies in an adjacent groove. A π–π stacking interaction was predicted between the dibenzofuranone ring and Trp590. The hydroxyl group in the 11′ position in the dibenzofuranone scaffold is predicted to form hydrogen bond interactions with His263 and Asn516 (see Figure 5b). 12k forms a hydrogen bond interaction between the hydroxyl group in the 2′ position of the dibenzofuranone ring and His263. There is also a hydrogen bond formed between His493 and the hydroxyl group in the 6′ position (see Figure 5d). In general, plausible poses are predicted between TDP1 and the ligands with various intramolecular interactions. Consistent and specific interaction between the sulfur moieties and TDP1 was not observed.

Molecular dynamics simulations have suggested that the TDP1 inhibitors occupy an allosteric binding pocket next to the catalytic site, as shown in Figure 4a,c [49]. Molecular modelling and structural activity relationship studies of usnic acid derivatives corroborate the existence of the allosteric site and its occupancy being beneficial to the overall binding efficacy [23]. Neither ligand is predicted to occupy the allosteric pocket, potentially explaining the relatively modest binding affinity that was observed.

For TDP2, 9c was used as an example. The conformation of 9c is shown in Figure 5: it was predicted to bind next to the co-crystallised ligand (see Figure 6a). The hydroxy group in the 11′ position of the dibenzofuranone ring forms a hydrogen bond with Asp348 and the para-bromophenyl forms a π–π stacking interaction with Trp297 and Phe315 (see Figure 6b). This is a plausible binding mode, but no specific intermolecular bonds were predicted for the sulfur containing group.

3.4. Chemical Space

The mainstream molecular descriptors MW (molecular weight), log P (water-octanol partition coefficient), HD (hydrogen bond donors), HA (hydrogen bond acceptors), PSA (polar surface area), and RB (rotatable bonds) were calculated for the 29 synthesized derivatives and are given in Table S4. The values of the molecular descriptors lie within a lead-like chemical space for HD, in lead- and drug-like spaces for log P, in a drug-like space for RB, and in a drug-like space and known drug space (KDS) for MW and HA. Finally, PSA spans lead-, drug-like spaces, KDS, and beyond, i.e., to very high values (for the definition of lead-like, drug-like, and KDS regions see reference [50] and Table S5). The TDP1 IC₅₀ values trend with log P (R²–0.202), with higher lipophilicity resulting in improved efficacy. No other trends nor correlations were seen between the IC₅₀ values and the molecular descriptors. MW, HA, and PSA all reach into a KDS, i.e., these are values are quite large; 26 derivatives have a PSA >140 Ų, with only three of the sulfides having lower values (8a, b, c). These high values need to be addressed in order for these compounds to have acceptable pharmacokinetic profiles.

The known drug indexes (KDIs) for the 29 ligands were calculated to gauge the balance of the molecular descriptors (MW, log P, HD, HA, PSA, and RB). This method is based on the analysis of drugs in clinical use, i.e., the statistical distribution of each descriptor is fitted to a Gaussian function and normalized to 1, resulting in a weighted index. Both the summation of the indexes (KDI_2a) and multiplication (KDI_2b) methods were used [42], as shown for KDI_2a in Equation (1) and for KDI_2b in Equation (2); the numerical results are given in Table S4.

KDI_2a = I_MW + I_{log P} + I_HD+ I_HA + I_RB + I_PSA

(1)

KDI_2b = I_MW × I_{log P} × I_HD× I_HA × I_RB × I_PSA

(2)

The KDI_2a values for the ligands range from 2.31 to 4.59, with a theoretical maximum of 6 and an average of 4.08 (±1.27) for the known drugs. The KDI_2b range is from 0.00 to 0.19, with a theoretical maximum of 1 and with a KDS average of 0.18 (±0.20). The oral bioavailability trends with both KDI_2a (R²–0.158) and KDI_2b (R²–0.084) [51]. Both indexes indicate that the ligands with higher values have reasonable biocompatibility; in particular, the sulfides (8) have reasonable KDI_2a/2b values, whereas the ligands with lower values, such as the sulfones (12), are unlikely to have good pharmacokinetic profiles. The sulfoxides (9) mostly lie between the 8 and 12 series.

4. Conclusions

TDP1 is an enzyme that plays a key role in repairing DNA damage caused by the presence of a TOP1 inhibitor, for example the anticancer drugs topotecan and irinotecan; thus, the action of TDP1 can suppress the effect of cancer chemotherapy. Some TOP1 inhibitors are quite toxic and so are not used in the first line of therapy, and care must be taken in the use of these inhibitors when titrating the dose required by the patient. The use of TDP1 inhibitors to enhance the effect of TOP1 inhibitors can increase the effectiveness of anticancer therapy, as well as reduce the dosage required for treatment. The possibility of using TDP1 inhibitors to sensitize tumors to the action of other anticancer drugs is an active research area [12,13].

In previous studies, cited within this report to provide context and comparisons, we discovered several usnic acid derivatives, readily synthesized from the naturally-occurring starting material, that were were effective TDP1 inhibitors. The usnic acid sulfide (thioether) and sulfoxide derivatives efficiently suppressed TDP1 activity, with IC₅₀ values in the 1 μM range [23]. In Dyrkheeva et al. [23], we found that the structure of the heterocyclic substituent affects the TDP1 inhibitory efficiency of these derivatives, and that sulfoxide usnic acid derivatives were less cytotoxic than their corresponding sulfide (thioether) analogs.

In this present work, a novel series of usnic acid derivatives of sulfides (8) (13 compounds), sulfoxides (9) (8 compounds), and sulfones (12) (8 compounds) were synthesized and their TDP1 and TDP2 inhibition activities were tested. These ligands inhibit TDP1 with IC₅₀ values in the range 0.33–25 μM. Amongst the studied sulfides, the most effective inhibitors of TDP1 were compounds containing methyl- (8f) or methoxy-benzimidazole (8g) modifications. Overall, for this library of compounds, the ability to inhibit TDP1 is dependent upon the oxidation state of the sulfur atom, in the order sulfides > sulfoxides > sulfones.

It is known that TDP1 and TDP2 seemingly operate co-operatively, and that the TDP2 action will increase should TDP1 be inhibited [1,14,15,16]. We also tested the ability of the synthesized compounds to inhibit TDP2. Most of the compounds that were screened inhibited TDP2 in the range 150–420 μM. The two lead compounds with the most potential as TDP1 inhibitors, 8f and 8g, were also observed to be amongst the most effective inhibitors of TDP2. In fact, derivatives from the three series corresponding to the oxidation state of the sulfur atom have broadly similar inhibition activity against both TDP1 and TDP2, as well as displaying potential as cytotoxic agents. Furthermore, selected TDP1 and TDP2 inhibitors were observed to significantly potentiate the TOP1 inhibition of topotecan in HeLa cells. Finally, a Comet assay highlighted that the individual compounds 8g, 8f, and 12k had little intrinsic DNA damage capacity, but did demonstrate a dose-dependent potentiation of topotecan activity. The most promising usnic acid analogues possess a methyl- or methoxy-benzimidazole substituent. Thus, in this work, we have increased the range of readily accessible usnic acid derivatives with sulfur atom modification which have significant potential in the fight against resistance to clinically used TOP1 inhibitors in cancer chemotherapy.