Design, synthesis, and biological evaluation of novel combretastatin A-4 thio derivatives as microtubule targeting agents
ABSTRACT: A series of novel combretastatin A-4 (CA-4) thio derivatives containing different molecular cores, namely α-phenylcinnamic acids (core 1), (Z)-stilbenes (core 2), 4,5- disubstituted oxazoles (core 3), and 4,5-disubstituted N-methylimidazoles (core 4), as cis- restricted analogues were designed and synthesized. They were selected with the use of a parallel virtual screening protocol including the generation of a virtual combinatorial library based on an elaborated synthesis protocol of CA-4 analogues. The selected compounds were evaluated for antiproliferative activity against a panel of six human cancer cell lines (A431, HeLa, MCF7, MDA-MB-231, A549 and SKOV) and two human non-cancer cell lines (HaCaT and CCD39Lu). Moreover, the effect of the test compounds on the inhibition of tubulin polymerization in vitro was estimated. In the series studied here, oxazole-bridged analogues exhibited the most potent antiproliferative activity. Compounds 23a, 23e, and 23i efficiently inhibited tubulin polymerization with IC50 values of 0.86, 1.05, and 0.85 µM, respectively. Thio derivative 23i, when compared to its oxygen analogue 23j, showed a 5-fold higher inhibitory impact on tubulin polymerization. Compounds 23e and 23i, which showed both best cytotoxic and antitubulin activity, were further studied in terms of their effect on cell cycle distribution and proapoptotic activity. Compound 23e induced a statistically significant block of the cell cycle at the G2/M phase in A431, HaCaT, HeLa, MCF-7, MDA-MB-231, and SKOV-3 cells to an extent comparable to that observed in CA-4. In HeLa and SKOV-3 cells incubated with 23i, a concentration-dependent block of the G2/M phase was observed. The proapoptotic effect of 23e and 23i in A431, HaCaT, MCF-7, MDA-MB-231, and SKOV-3 was demonstrated with ELISA assay and double staining with Annexin V-FITC/PI. The results indicated that compound 23e and 23i may serve as novel lead compounds in research on more effective anticancer agents.
1.Introduction
Microtubules are highly dynamic components of the cytoskeleton that consist of αβ-tubulin heterodimers and are involved in a wide range of various cellular functions, including maintenance of the cell structure, motility, intercellular transport, and cell division, where they are responsible for mitotic spindle formation and proper chromosomal separation [1–4]. The biological importance of microtubules in mitosis and cell division as well as their dynamic nature makes them a significant and intensively investigated molecular target for anticancer drugs [5– 11].Microtubule-interfering agents (MIAs), of which several are natural products, act via two different mechanisms of action leading to either the inhibition or enhancement of tubulin polymerization. Both effects impair microtubule dynamics and have an impact on cell proliferation. MIAs bind to one of the three major different binding sites on tubulin, i.e. the taxol binding site, colchicine-binding site, and vinca alkaloid domain [5,6]. Ligands which stimulate microtubule polymerization, known as microtubule stabilizers such as paclitaxel or epothilone, bind to the taxol binding site [12,13]. In turn, microtubule destabilizers (tubulin polymerization inhibitors) induce depolymerization of microtubules. In this group, different vinca alkaloids can be distinguished, such as vinblastine and vincristine which bind to the vinca domain [14,15], and a large class of ligands which interact with the colchicine-binding site, including colchicine (2), podophyllotoxin (3), steganacin (4), and combretastatins [16–20].
Combretastatins are a group of natural cis-stilbenes that were isolated from the bark of the African bush willow tree Combretum caffrum over 30 years ago [21–24]. The most active member of this group as described by Pettit et al. [25] is combretastatin A-4 (CA-4, 1a, Fig. 1), which presents remarkable biological activity that is manifested in strong inhibition of tubulin polymerization, potent in vitro cytotoxicity against a variety of human cancer cell lines, including multidrug resistant (MDR) cell lines overexpressing P-glycoprotein (Pgp), and in vivo efficacy as a vascular-disrupting agent (VDA) and antiangiogenic agent [25–29]. Its water- soluble prodrug, CA-4 disodium phosphate (CA-4P, 1b, Fig. 1), has shown promising results in numerous clinical trials in both monotherapy and in combination with various anticancer agents, exhibiting high efficacy in the treatment of anaplastic thyroid cancer and ovarian cancer [30,31].Promising results in anticancer therapy with CA-4’s unique and multidirectional activity as well as its relatively simple structure as a lead make extensive structural modification that will improve its natural anticancer properties possible. Meanwhile, numerous structure-activity relationship (SAR) studies of CA-4 have been conducted leading to the identification of functional groups and their positions that are important to ensure optimal interactions with the colchicine binding site and efficacious antimitotic activity [29,32–37]. In general, these modifications pertained to the A-ring, B-ring, and/or ethylene bridge. They showed the importance of the 3,4,5-timethoxy substitution pattern on the A-ring, 4-methoxy substituted B- ring, and the cis-configured double bond, which are fundamental for the inhibitory activity of tubulin polymerization.
Bioisosterism is a widely used strategy for the rational design of new drugs including anticancer agents, particularly for designing agents with optimal pharmacological properties [38]. The compounds of the studied series of CA-4 derivatives possess an atom of bivalent oxygen replaced by sulfur. Our previous studies on methylthio-trans-stilbenes as chemopreventive agents showed the high affinity of selected thio derivatives to human recombinant CYPs, particulary CYP1A1 and CYP1B1 [39–42]. Other authors showed that the introduction of a less electronegative sulfur atom instead of the oxygen atom led to a reduction of toxicity in HEK 293 cells (human embryonic kidney cell line) and thus could improve selectivity for cancer cell lines [43]. Further modification of the CA-4 molecule, as taken into account in the design of our series, was an improvement of the stability of the designed CA-4 analogues because the ethylene bridge in CA-4 is able to undergo cis-trans isomerization, which causes complete loss of cytotoxicity. This was possible by replacing the olefinic double bond with five- member heterocyclic rings such as oxazole and N-methylimidazole. Several groups of researchers have reported that this type of structural modification allows to avoid the stability problem and improves anticancer activity [44–54]. Such replacement allows to retain the correct geometric orientation of the phenyl rings of the CA-4 derivatives by placing them at an appropriate distance for optimal interaction with the colchicine-binding domain on tubulin.
In this paper we present the design, synthesis, and biological evaluation of a series of CA-4 thio derivatives with different molecular cores. The compounds were designed using a parallel virtual screening protocol including the generation of a virtual combinatorial library (VCL) based on an elaborated synthesis protocol of CA-4 analogues, 3-dimensional pharmacophore screening, two QSAR filters, docking to the colchicine binding site of tubulin, and a final ranking strategy. The selected derivatives were synthesized and evaluated for their effect on tubulin polymerization in vitro. Moreover, their antiproliferative activity was assessed in a panel of six human cancer (A431, HeLa, MCF7, MDA-MB-231, A549, SKOV3) and two non-cancer cell lines (HaCaT, CCD39Lu). The most potent compounds in the series 23e and 23i were further studied for cell cycle effects, apoptosis, and on a microtubule network.
2.Results and discussion
A protocol including combinatorial library generation and screening was developed and tested to support the design of CA-4 thio analogues (Fig. 2). Based on an elaborated synthetic protocol for sulfur analogues of CA-4 (Scheme 2-6), different substituted aromatic aldehydes (BB1) and phenylacetic acids (BB2) were selected from an in-house library and used to generate the VCL by iterative combination of selected reagents. The obtained VCL (1,159 cmpds) was processed by the parallel virtual screening (VS) protocol including a 3-dimensional pharmacophore filter, two QSAR models, and the post-docking scoring method (for details, seeTo evaluate the real efficiency of the methodology used here, we assumed that the compound inhibited tubulin polymerization when its IC50 was lower than 10 µM. Based on this assumption and the predicted VS scores, the ROC curve with VS score cut-offs was plotted (Fig. 3). It showed very good predictive power of the applied VS protocol (AUROC and BEDROC were 0.85 and 1.00, respectively).Interestingly, when the VS score cut-off was greater or equal to 4 the sensitivity (true positive rate) was 0.43 and precision was 1.00. However, a VS score cut-off greater or equal to 3is an optimal choice for virtual screening subjects because both sensitivity and precision are high (0.86 and 0.67, respectively).CA-4 (1a) was prepared using the two-step procedure described by Gaukroger and co- workers [34] using Perkin-type condensation between 3,4,5-trimethoxyphenylacetic acid (5) and isovanillin (6) and subsequent decarboxylation of the obtained α-phenylcinnamic acid (7) (Scheme 1).Preparation of the designed CA-4 derivatives differing in four molecular cores, namely α- phenylcinnamic acids (core 1, 14a-b), (Z)-stilbenes (core 2, 15a-b), 4,5-disubstituted oxazoles (core 3, 23a-j), and 4,5-disubstituted N-methylimidazoles (core 4, 24a-f), as cis-restricted analogues is illustrated in Schemes 2-6 following general procedures as detailed below and in the Experimental Section.
Bromobenzaldehyde (10) [55] was prepared from commercially available vanillin (8) by its bromination and subsequent methylation of the obtained bromovanillin (9) [56] with methyl iodide. Methylthiobenzaldehydes 6d [57], 8d [58], 9d, 11d [57] and 12d [59] were obtained in a multistep reaction starting from appropriately substituted hydroxybenzaldehydes 6, 8-9 and 11-12 (Scheme 2). In the first step, the obtained O-aryl thiocarbamates 6a [60], 8a [58], 9a, 11a [61] and 12a [59] were converted using a Newman-Kwart rearrangement to thecorresponding S-aryl thiocarbamates 6b, 8b [58], 9b, 11b [61] and 12b [59] which were then readily hydrolyzed using methanolic potassium hydroxide to thiophenols, and in a one-pot reaction converted to final products by subsequent methylation with methyl iodide or dimethyl sulfate. In the preparation of benzaldehydes 9d and 12d, an additional step of protecting the carbonyl group in S-aryl thiocarbamates 9b and 12b was required by the formation of cyclic acetals, 1,3-dioxolanes 9c and 12c. Their hydrolysis, methylation, and deprotection were conducted in a one-pot reaction which allowed to obtain methylthiobenzaldehydes 9d and 12d in good yields.(10% in MeOH), 3-7 h, 80 ºC; (i) Me2SO4, 1 h, 25-55ºC; (j) 5% HCl (pH=2-3), 2h, 55ºC, 67-98% (h, iand j).The synthesis of α-phenylcinnamic acids 14a-b [62,63] possessing core 1 and (Z)-stilbenes 15a-b [62,63] with core 2 (Scheme 3) was conducted in a manner similar to the preparation of CA-4. The geometries of the (Z)-stilbenes were confirmed by their characteristic 1H NMR coupling constants for olefinic protons of ca. 12–12.1 Hz.The appropriate substituted benzaldehydes obtained earlier, 10 and 12d, or commercially available 16, were reacted with freshly prepared p-toluenesulfinic acid and formamide, catalyzed by 10-camphorsulfonic acid (CSA) to give tosylmethyl formamides 17-19 [44,64].
Dehydration with POCl3 afforded the desired TosMICs 20-22. Preparation of N-methylimidazole-bridged analogues (Scheme 6) involved the first generation of aldimines resulting from the reaction of appropriate benzaldehydes and methylamine and then their condensation with TosMICs in the presence of anhydrous K2CO3.Preparation of heterocycle-based derivatives 23c, 23e, and 24c possessing the mercapto or hydroxy group involved synthesis of their S-aryl thiocarbamate or O-aryl thiocarbamate analogues followed by basic hydrolysis of the carbamate groups.Table 1 summarizes the cytotoxic effects of the obtained CA-4 analogues with each molecular core, namely -phenylcinnamic acids (14a-b), (Z)-stilbenes (15a-b), 4,5-disubstituted oxazoles (23a, 23c, 23e-j), and 4,5-disubstituted N-methylimidazoles (24a, 24c-f), against a panel of six human cancer cell lines (A431, HeLa, MCF7, MDA-MB-231, A549, and SKOV) and two human non-cancer cell lines (HaCaT and CCD39Lu), using CA-4 as the reference compound.Table 1. Cytotoxic activities of 14a-b, 15a-b, 23a, 23c, 23e-j, 24a, 24c-f and CA-4 against human cancer and non-cancer cell linesThe results listed in Table 1 indicate that cytotoxicity toward cancer cells varied between each molecular core. Compounds 14a-b as analogues possessing the α-phenylcinnamic acid core (core 1) were generally inactive against the cancer cell lines (IC50 > 20µM), except for MCF7 from estrogen-dependent breast cancer adenocarcinoma, toward which they displayed relatively high antiproliferative activity (IC50 values of 0.95-1.84 µM).In turn, compounds 15a-b containing the (Z)-stilbene scaffold (core 2) with the same substituents in the phenyl rings displayed slightly enhanced activity toward four cancer cell lines (A431, HeLa, MDA-MB-231, and SKOV3) with IC50 values of 3.61-14.47 µM, while activity against MCF7 cells was retained.
The oxazole-bridged CA-4 analogues (core 3) generally displayed the best antiproliferative activity from all of the tested compounds toward the cancer cell lines except for A549, toward which most of the compounds were inactive. The number and position of the substituents in the phenyl B-ring had a major influence on antiproliferative activity. Compounds with substituents at 3,4-position of the phenyl B-ring showed much higher activity than compound 23a with the – SCH3 group at the orto-position or compound 23g with substituents at 3,4,5-position in the phenyl B-ring. Replacement of the m-OCH3 group in the trimethoxyphenyl A-ring (compound 23f) with an electron withdrawing bromine atom (compound 23h) maintained antiproliferative activity.The most active compound, 23e, in this series and of all the tested compounds was over 10- fold more potent against HeLa cells than the reference compound CA-4 with an IC50 value of0.009 µM vs 0.11 µM, respectively. Interestingly, bioisosteric replacement of the oxygen atom with a sulfur atom (introduction of the m–SCH3 group in compound 23i instead of the m–OCH3 group in compound 23j) led to an increase in antiproliferative activity toward the A431 (28.7- fold), MCF7 (2.2-fold), and MDA-MB-231 (8.5-fold) cell lines. On the other hand, only compound 23j was active against the highly drug-resistant A549 lung adenocarcinoma with an IC50 value of 0.5 µM.N-methylimidazole-bridged CA-4 analogues (24a, 24e, and 24f) (core 4) were significantly less active than their corresponding oxazole analogues (23a, 23h, and 23i) when compared to the results of 23a vs 24a, 23h vs 24e, and 23i vs 24f, thus suggesting that replacement of the oxazole with the N-methylimidazole moiety was the major reason for the loss of activity in these compounds. Compound 24c as an imidazole-bridged analogue of 23e was only active in this series.
Because the toxicity of antitumor drugs toward normal tissues is a very important issue in chemotherapy, we evaluated the cytotoxic effects of these derivatives on normal human cells. For this purpose, all compounds were tested in vitro in human immortalized keratinocytes HaCaT and lung fibroblasts CCD39Lu. Based on the results, most of the compounds, with the exception of 24d, showed slight cytotoxicity on fibroblast cells (IC50 > 20 µM) in contrast to the reference compound CA-4 (IC50 of 0.009 µM). However, some of the tested derivatives (23c, 23e, 23f, 23h, 23i and 24c) were cytotoxic against immortalized human keratinocytes HaCaT (IC50 values of 0.32-1.28 µM verus 0.19 µM for CA-4).Finally, two of the most active compounds (23e and 23i) and six of the most sensitive cell lines (A431, HaCaT, HeLa, MCF-7, MDA-MB-231, and SKOV-3) were selected for further studies such as assessment of the cell cycle, tubulin staining (effect on microtubule dynamics), and apoptosis induction.Compounds 14a-b, 15a-b, 23a, 23c, 23e-j, 24a, and 24c-f were investigated for their effect on tubulin polymerization with the use of turbidimetric assay in vitro. The N-methylimidazole- bridged analogues, unlike oxazole-bridged analogues, were poor tubulin polymerization inhibitors (Table 2). In the series of oxazole-bridged analogues, compounds 23a, 23e, and 23i efficiently inhibited tubulin polymerization with an IC50 of 0.86, 1.05, and 0.85 µM, respectively (Table 2). CA-4 was used as a reference compound.Compound 23j as an oxygen analogue of 23i weakly inhibited tubulin polymerization with IC50 equal to 4.80 µM. It should be noted that this outcome is in line with predictions obtained in the post-docking scoring strategy combining Tanimoto and SIFt (for details, see Supplementary Information), where the sulfur analogue was scored higher than the oxygen analogue.2.3.3.Analysis of cell cycle and tubulin stainingIn this experiment, the effect of the tested compounds on the cell cycle was assayed in six selected cancer cell lines; camptothecin at a concentration of 50 nM was used as a positive control (Fig. 4).
Interestingly, very similar changes in cell cycle distribution were observed in the HeLa and SKOV-3 cell lines. Compound 23e induced a statistically significant concentration-independent block of SKOV-3 cells in G2/M, while a similarly massive G2/M block was observed after incubation with CA-4. In contrast, after incubation with compound 23i in HeLa and SKOV-3 cells, the concentration-dependent block of the G2/M phase was shown.A similar trend was observed in A431, HaCaT, MCF-7, and MA-MB-231 cells treated with compounds 23e, 23i, and CA-4. The tested compounds were also investigated for their inhibition of tubulin polymerization using immunostaining (Fig. 5).It is worth noting that besides disturbances in tubulin polymerization as shown in all of the tested cells, condensation of chromatin (stained with Hoechst 33358 dye) which is also typical for apoptosis, could be observed in the nuclei of cells incubated with 23i (Fig. 5). Perturbations in the cytoskeleton structure as well as micronuclei formation corresponded with the results of ELISA and flow cytometry studies, where clear differences between effects exerted on cells by 23e, CA-4, and, to a lesser extent, by 23i could also be observed.Induction of apoptosis in cells incubated with compounds 23e, 23i, and CA-4 was analyzed using two different methods. The ELISA assay measures the presence of markers for apoptotic cells, which was based on the quantitative detection of histone-associated DNA fragments in mono- and oligonucleosomes, while staining with Annexin V allows to detect phosphatidylserine externalization. Translocation of phosphatidylserine from the inner to the outer leaflet of the membrane is observed in intermediate stages of apoptosis and may be shown using Annexin V- FITC staining followed by flow cytometry. For cells incubated with 23e, 23i, and CA-4, a clear concentration-dependent statistically significant increase of Annexin V positive cells was observed (Fig. 6).Interestingly, in cells incubated with 23e, the highest level of histone-associated DNA fragments in mono- and oligonucleosomes was observed only at the lowest concentration of 23e and CA-4, while changes observed in cells incubated with 23i may be described as concentration-dependent (Fig. 7).independent experiments. Statistical significance between groups was assessed by Dunnett’s Multiple Comparison Test.
All treated groups were significantly different from control at p < 0.05.mean ± SD of three independent experiments. Statistical significance is indicated with asterisks: ***p < 0.001, **p < 0.01 and *p < 0.05 indicate a significant difference from the control.These results may suggest significant differences in the cellular uptake, metabolism, and cellular efflux of these cells as well as coexistence of different cellular signaling pathways stimulated by these compounds, although subsequent tests are necessary in order to fully explain these mechanisms.Docking studies revealed that the binding modes of the most active compounds (23e and 23i) are coherent with co-crystallized DAMA-colchicine (Fig. 8). The trisubstituted phenyl A-ring of 23e and 23i is buried in the β-subunit occupying the same position as the corresponding ring of colchicine.The thiol group of Cys241β forms a hydrogen bond with the sulfur or oxygen atom of the methylthio or methoxy group in para-position in 23e and 23i, respectively. In the most activecompound 23e, the -SCH3 group (ring A) is involved in hydrophobic interaction with the side chain of Leu255β, Ala316β, and Ile378β. The bromine atom in 23i is placed in a position that allows hydrophobic interactions with Cys241β, Ala250β, and Leu255β. Moreover, the -SCH3 substituent (23i, ring B) remains in close contact with Ala316β, Leu255β, and Met259β, thus making non-bonded SS interaction with the sulfur atom of Met259β possible. It should be noted that oxazole derivatives (23e and 23i) showed a similar binding mode with other cis- restricted analogues of CA-4 containing five-member aromatic heterocyclic rings, such as thiazoles [47], triazoles [46], and terazoles [48] with the trimethoxyphenyl ring placed in proximity of Cys241β.3.ConlusionsWe designed and synthesized a series of CA-4 thio derivatives containing different molecular cores, namely -phenylcinnamic acids (core 1), (Z)-stilbenes (core 2), 4,5-disubstituted oxazoles (core 3), and 4,5-disubstituted N-methylimidazoles (core 4) as cis-restricted analogues. Compounds for synthesis were selected with the use of a parallel virtual screening protocol which showed a good predictive power (AUROC and BEDROC values were 0.85 and 1.00, respectively).Biological evaluation of the tested compounds revealed the highest antiproliferative activities in the group of oxazole-bridged analogues (core 3) with the 3,4,5-trisubstituted phenyl A-ring and 3,4-disubstituted phenyl B-ring. The compounds 23e, 23f, 23h, 23i, and 24c (N- methylimidazole-bridged analogue) displayed pronounced cytotoxic activity against the cancer cell lines: A431, HeLa, and MDA-MB-231. Compound 23e was over 10-fold more potent in inhibiting HeLa cell proliferation with IC50 values of 0.009 µM than positive control CA-4. Additionally, the compounds 23a, 23e, and 23i were the most potent inhibitors of tubulinpolymerization determined in vitro. Compound 23i, a sulfur analogue of 23j, showed 5-fold higher inhibitory effectiveness on tubulin polymerization compared to 23j. The influence of exchange of oxygen to sulfur could be a promising subject for further studies.Moreover, compounds 23e and 23i, which showed both the highest cytotoxic and antitubulin activities, were further studied in terms of their effect on cell cycle distribution and proapoptotic activity. The impact of 23e and 23i on cell proliferation was cell line-dependent. A statistically significant block of the cell cycle at the G2/M phase was observed for compound 23e in A431, HaCaT, HeLa, MCF-7, MDA-MB-231, SKOV-3 cells, and for compound 23i in HeLa and SKOV-3 cells. The proapoptotic effect of 23e and 23i in studied cell lines was demonstrated with ELISA assay and double staining with Annexin V-FITC/PI. The results of our studies indicate that the CA-4 derivatives 23e and 23i may serve as novel lead compounds in research on more effective anticancer agents. 4.Experimental section All reagents and solvents were purchased from Sigma-Aldrich, Acros, Fluka, and POCH and were used as received. Reactions that involved air or moisture-sensitive reagents were performed in oven-dried glassware under an inert atmosphere of dry nitrogen with dried solvents, unless otherwise stated. The progress of all reactions was monitored on Merck precoated silica gel plates (with fluorescence indicator UV254) and visualized in UV light (λmax 254 or 365 nm). Melting points were determined in capillary tubes on a Stuart SMP10 micro melting point apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznań, using Bruker 400 (400 MHz for 1H and 101 MHz for 13C), Bruker 500 (500 MHz for 1H and 126 MHz for 13C), and Bruker 700 (700 MHz for 1H and 176 MHz for 13C) spectrometers with TMS as an internal standard in CDCl3 or DMSO-d6. Chemical shifts (δ) are quoted in parts per million (ppm) and are referred to as a solvent residual peak (CDCl3, δ 7.26 ppm for 1H and δ 77.0 ppm for 13C NMR; DMSO-d6 δ 2.5 ppm for 1H and δ 39.5 ppm for 13C NMR). Coupling δ constants (J) are quoted in Hertz (Hz) and peaks are listed as singlet (s), doublet (d), triplet (t), multiplet (m), doublet of doublets (dd), triplet of doublets (td), and broad signal (bs). Additional techniques (1H−1H COSY, HSQC, HMBC) were used to assist allocation. LRMS (EI) spectra were recorded on a Bruker 320MS/420GC mass spectrometer and HRMS (EI) spectra were recorded on an Intectra Mass AMD 402 or 604 mass spectrometer by the Advanced Chemical Equipment and Instrumentation Facility at the Faculty of Chemistry, Adam Mickiewicz University in Poznań. IR spectra were recorded on a Bruker FT-IR IFS 66v/s spectrometer by the Advanced Chemical Equipment and Instrumentation Facility at the Faculty of Chemistry, Adam Mickiewicz University in Poznań or on a Thermo Scientific Nicolet iS5 FT-IR spectrometer at the Institute of Mathematical and Natural Sciences, Department of Chemistry, State Higher Vocational School in Tarnów. Dry flash column chromatography was carried out on Merck silica gel 60, particle size 40−63 µm or 15-40 µm using EZSafe low-pressure columns. UV−vis spectra were recorded on a Hitachi UV/vis U-1900 spectrophotometer; λmax (log ε), nm. All final target compounds were characterized and determined to be at least >95% pure using a Waters ACQUITY UPLC H-class system equipped with a UV DAD and TQD Waters MS detector with electrospray ionization at the Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences in Kraków. LC analysis was performed using a ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1,7 µm) at a flow rate of 0.3 mL/min (20–100% aqueous CH3CN over 3 min, RMC-4630 100% CH3CN over 0.5 min, and 100–20% aqueous CH3CN over 2.5 min).