Synthesis, characterization and anticancer evaluation of nitrogen-substituted 1-(3-aminoprop-1-ynyl)-4-hydroxyanthraquinone derivatives

Anthraquinones are of significant interest due to their biological activity, coloring properties, and synthetic applications. Here, we describe a mild and convenient method for modification of 1-ethynyl-4-hydroxyanthraquinone that was obtained from the Sonogashira cross-coupling reaction of 1-hydroxy-4-iodoanthraquinone with alkynes. The copper(I) catalyzed one-pot three-component reaction (A3-coupling) of the new 1-ethynyl-4-hydroxyanthraquinone with secondary amines and formaldehyde was the main approach for the synthesis of nitrogen-substituted 1-[3-(amino)prop-1-ynyl]-4-hydroxyanthraquinones. The influence of different substituents in the amine on reaction rate and yield has been evaluated. The cytotoxicity of 1-ethynyl-4-hydroxyanthraquinones was assessed using the conventional MTT assay. Among all compounds synthesized, anthraquinone-propargylamine derivatives 28, 29, 30, and 34 possess the most promising cytotoxic potential towards glioblastoma cancer cells; compounds 14 and 19 shown selectivity towards the prostate cancer cells DU-145, and 18, 24 — on breast cancer cell line MCF-7. The grown inhibition on these cancer cells by compounds 18 and 24 was comparable to those of standard drug Doxorubicin. Molecular modeling of new anthraquinone derivatives in DNA G-quadruplex binding sites was performed to help understand the observed SAR trends.


Introduction
The derivatives of anthracene-9,10-dione (anthraquinone) are of interest in many applications, in particular, are well known for their diverse and profound biological activities [1]. They are effective to treat tumors and cancers [2][3][4], and also used as antibacterial [5,6], antifungal, insecticidal [7], antimicrobial [8,9], and antidiabetic [10] agents. Antraquinones were widely studied for their structural evaluation and biological significance. Antracenedione drugs are known to exert their biological effects through interaction with DNA resulting in modification of its structure and hence inhibition of its replication. Anthraquinones mitoxantrone and ametantrone ( Fig. 1) are potent synthetic anticancer drugs that block DNA synthesis by inhibiting the function of DNA topoisomerases [11,12]. Several anthraquinone pharmacophores can realize their anticancer activity by affecting other molecular targets. For example, a new rhein-derived compound AQ-101 can target the oncogenic protein MDM2 (a member of the RING-fingertype family of E3 ubiquitin ligases) for degradation by binding to MDM2 and blocking the interaction between MDM2 and MDM4 which leads to MDM2 self-ubiquitination and degradation; this, in turn, causes p53 activation and cancer cell apoptosis, particularly in cancer cells expressing high levels of MDM2 [13]. Purpurin is a non-competitive inhibitor of adipocyte-derived leucine aminopeptidase (A-LAP) which plays a crucial role in angiogenesis [14]. Emodin is characterized as a significant inhibitor of cell proliferation, presumably via downregulation of ERCC1 (excision repair crosscomplementary 1) and DNA recombinase protein Rad51 [15]. 2,4-(Dibromo)emodine exert their anti-proliferative activity at least in part, by inhibition of ATP citrate lyase (ACL), which plays a critical role in generating cytosolic acetyl CoA [16]. Emodin and 2-chloroemodin are considered as potential targets of dioxygenases (ALKBH 2, 3 proteins, and FTO) overexpression blockers [17]. Purpurin [18] and emodin [19] (Fig.  1) are also assessed as monoamine oxidase isoforms inhibitors.
A series of studies have been focused on the optimization of efficacy and safety profile of anthraquinone-based compounds [20]. Of particular interest in the search of antitumor agents in the development of studies on the preparation of anthraquinone derivatives containing nitrogen substituent, including piperidine [21] and pyrimidine [22] fragments.
For the synthesis of nitrogen substituted anthraquinones, besides the routes for the construction of anthraquinone core (for example, of Friedel-Crafts condensations of benzene derivatives with functionalized phthalic anhydrides or phthaloyl dichlorides [23]), attention has been drawn to the development of C-N coupling processes, for example, the copper(0)-catalyzed Ullmann-type reaction of bromo/chloro anthraquinones with a variety of amines [4,[24][25][26][27] or the Pd-catalyzed Buchwald-Hartwig cross-coupling reaction of halogen-substituted anthraquinones [28,29].
In the framework of our studies dealing with the development of convenient routes to functionalization of 1hydroxyanthraquinone [30], we were interested in the preparation of anthraquinone derivatives, containing an aminopropargyl substituent in the anthraquinone core. Propargylamines found wide application in medicinal and pharmaceutical chemistry [31]. The copper(I) catalyzed onepot three-component reaction (A 3 -coupling) among aldehyde, amine and terminal alkyne has become a popular approach to synthesize propargylamines [32,33]. Therefore, the sequence of the Sonogashira cross-coupling reaction of 4-iodo-1hydroxyanthraquinone with alkynes and the copper-catalyzed Mannich reaction of 1-ethynyl-4-hydroxyanthraquinone with secondary amines and formaldehyde (37% aq. solution or paraform) (A 3 -coupling) was used as the main synthetic approach in this studies. Taken into account the interest in substituted anthraquinones as anticancer agents we evaluated the cytotoxicity of the synthesized compounds toward cancer cell lines in vitro. The molecular docking of new compounds to G-quadruplex DNA motifs was carried out.

Chemical synthesis
As the starting compound, we used 1-hydroxy-4-iodo-9,10anthraquinone 1, obtained by iodination of 1hydroxyanthraquinone with iodine and iodic acid in acetic acid in the presence of sodium acetate (yield 76%) [34]. The cross-coupling reaction of compound 1 with phenylacetylene 2 was used as the model reaction to optimize the conditions (Table 1 and Scheme 1). The cross-coupling reaction of 1 with 2 under standard Sonogashira reaction conditions [35] in the presence of dichlorobis(triphenylphosphine)palladium, triphenylphosphine, copper(I) iodide, and triethylamine as a base in benzene or toluene under reflux was unsuccessful (Table 1, entries 1, 2; Scheme 1). Carrying out the reaction in DMF and using an excess of triethylamine was found to be ineffective (Table 1, entry 3). An efficient way to improve the yield of the cross-coupling reaction products was the addition of tetraalkylammonium salts to the reaction mixture as proposed by Jeffery [36]. The ability to generate a stable form of the catalyst without the addition of stabilizing phosphine ligand as one of the roles of tetraalkylammonium salts was reported by Reetz et al. [37]. The reaction of 1-hydroxy-4-iodo-9,10-anthraquinone 1 with phenylacetylene 2 in the presence of dichloro-bis(triphenylphosphine)palladium, copper(I) iodide, triethylamine, and ammonium salts (Bu 4 NBr) (1 equiv.) in DMF by heating at 65°C (TLC-control) afforded the 4-phenyl-1-hydroxyanthraquinone 6 in 78% yield (Table 1, entry 4). We obtained a similar result when using 0.2 equiv. of Bu 4 NBr; the isolated yield of compound 6 composed to 82% (Table 1 and entry 5). In this condition, the reaction of 1-hydroxy-4-iodo-9,10-anthraquinone 1 with 4-methoxyphenylacetylene 3, 4-fluorophenylacetylene 4, or 4-acetylamino-3-ethoxycarbonyl-phenylacetylene 5 gave the corresponding 4-aryl-1-hydroxyanthraquinones 7-9 in the isolated yield 52, 46, 41% respectively.
The reaction of iodoanthraquinone 1 with trimethylsilylacetylene 10 in the found condition proceeded more smoothly afforded the coupling compound 11 in the yield of 83%. Desilylation by the action of tetrabutylammonium fluoride in methylene chloride afforded the corresponding terminal acetylene 12 (yield 74%). Thus, we proposed a simple and convenient three-step procedure for the synthesis of 1-ethynyl-4-hydroxyanthracene-9,10-dione 12 with an overall yield of about 49% from 1hydroxyanthraquinone.
For the synthesis of N-substituted 1-(3-aminopropargyl)-4-hydroxyanthraquinones we studied the Cu(I) iodidecatalyzed A 3 -coupling reaction between 1-ethynyl-4hydroxyanthracene-9,10-dione 12, formaldehyde, and different secondary amines. We found that the reaction of compound 12, formaldehyde (generated in situ from paraformaldehyde), and diethylamine 13 in dioxane in the presence of 0.02 equiv of copper(I) iodide at 65°C proceeds smoothly, and after 30 min the alkyne was almost consumed and the desired 1-(3-(diethylamino)prop-1-ynyl)-4-hydroxyanthracene-9,10-dione 14 was isolated in the yield 50% (Scheme 2). Copper(I) chloride was less effective with increasing the reaction time to 1.5 h and yield decreasing to 39%. Performing the reaction in the presence of copper(II) acetate monohydrate in dioxane led to a slightly improved  spectral analyzes were performed to prove the structures. 1 H-NMR, 13 C NMR, and mass spectral analysis were found to prove the expected structures. It is well known, that the reported reaction is thought to proceed through the alkyne activation forming a copper acetylide. After a nucleophilic addition on the intermediate formed by the reaction of formaldehyde and a secondary amine, the propargylamine derivative is obtained [32]. We performed also a "one-pot" deprotection-A 3 -coupling tandem procedure for obtaining compound. Successful versions of the copper(I)-catalyzed reaction of azides with trimethylsilylalkynes in the presence of an organic base we reported [38,39]. We found, that a simple mixing of 12, formalin, and amine 13 in the presence of catalytic quantities (0.05 equiv.) of Cu(OAc) 2 × H 2 O and Bu 4 NF (2 equiv.) in THF under argon flow gave the desired product 14 isolated in the yield 23%. No products of alkyne deprotection reaction (compound 12 or dimeric butadiyne) were observed in this condition.
All the new compounds exhibited satisfactory spectral data correlating with their structures. IR Spectrum of all final compounds 6-9, 11, 12, 14, 18-20, 23, 24, 28-30, 33, 34, 36 showed C ≡ C stretching vibration around 2100-2205 cm −1 . The structure of the synthesized 1-(3aminopropargyl)-4-hydroxyanthraquinones was clearly confirmed by the results of the NMR study. In particular, the 1 H NMR spectra of 14, 18-20, 23, 28, 29, 33, 34, 36 contain the resonance signals of the CH 2 group represented as a singlet of two protons at the range of 3.63-3.81 ppm. For these protons in the spectra for compounds 24 and 30, having a substituent at the α-position of the heterocyclic ring, the doublet AB system centered at 3.72 and 3.92 ppm (J = 17.6 Hz) or 3.41 and 3.52 ppm (J = 17.6 Hz) was observed. For the correct assignment of the chemical shifts of the signals of the carbon atoms of the core and the substituent, we used the data of 2D NMR spectroscopy (for example compound 30, Suppl. part). The optical rotation of compound 30 in comparison to those of compound 27 was also measured. All synthesized compounds had UV spectra characteristic of anthraquinones. For example, the spectrum of 6 had absorption bands with maxima at 258 (4. MTT assays were performed for quantitative evaluation of in vitro cytotoxicity [40]. Doxorubicin (DOX) is clinically used to treat cancer as a drug and has a very wide antitumor spectrum. Therefore we used this compound as the positive control. The results are presented in Table 2.
The SAR revealed that the substituent at the C-1 position of 4-hydroxyanthraquinones has a great influence on cytotoxicity. Compounds 6, 7, 8, 9 with arylethynyl substituent exhibited low or insignificant cytotoxicity with high GI 50 values on the cancer cell lines. Previously, we have reported that 4-hydroxyanthraquinones with 1-arylsubstituent have great anticancer potency [30]. Spacing the aromatic ring away from anthraquinone by an ethynyl group led to greatly decrease in the anticancer potency. In comparison, all the 1-(3-(N-substituted)aminoprop-1-ynyl)-4hydroxyanthraquinones 14, 18-20, 23, 24, 28-30, 33, 34, and 36 shown much more potent cytotoxicity with significantly lower GI 50 values on most of the tumor cells. Thus, we demonstrate that modification of 1-position of 4hydroxyanthraquinone with aminopropynyl substituent can significantly potentiate the anticancer activity. A remarkable increase in activity towards breast cancer cell line MCF-7 was observed for anthraquinones 18,24,29,36; the effect in this cell line was comparable to that of Doxorubicin. Among these compounds 18 (with a (dipropylamino)propargyl substituent) and 36 (with a cyclic (azocane-1yl) propargyl substituent) shown higher cytotoxicity also towards normal cell lines. Among all analogs, 4hydroxyanthraquinones 14 and 19 with diethylaminopropynyl or di-i-propylaminopropynyl substituent showed high potency against prostate cancer cell line DU-145 ( Table 2). The GI 50 value for those derivatives approaches that of the positive control, Doxorubicin. Compound 28, contains the cyclic 3-(piperidino), 29 with 3-(4-methyl)piperidino-, and 30 with 3-(2-anabasinyl)piperidino substituent in the propin-1-yl side-chain possess selective cytotoxicity towards glioblastoma cancer cells SNB-19 and U-87MG. Relative to Doxorubicin, these derivatives showed at least 4-10 fold less toxicity against normal cell lines. The synthesized 3-(pyrrolidino)propynylanthraquinone 23 was less active than 3-(piperidino)propynylanthraquinone 28 towards cancer cell lines. The N-methylpiperidinopropynyl-1-hydroxyanthraquinone 34 demonstrated higher cytotoxicity than the morpholinopropynyl-1-hydroxyanthraquinone 33 on cancer cells. All the obtained results suggested that the substituent at the nitrogen of 4-(aminopropargyl)-1hydroxyanthraquinones increased the cytotoxicity. There have been previous studies on the anticancer properties of aminoanthraquinones with the aminoalkylamino side chains at the 1-1,4-1,5-or 1,8-positions [41][42][43][44][45]. These reports suggest that the substitution by 1-(N,N-dimethylamino) ethylamine side chain led to optimal activity: compounds with 1-(N,N-dimethylamino)propylamine substituent shown The experimental results are given as the data average values obtained from three independently conducted experiments c Doxorubicin twofold decrease of cytotoxicity on cancer cell lines [44]. The modification in the chain second amine function also changed the cytotoxicity; compounds with morpholino (propylenamino)-substituted anthraquinones were less potent than compounds with piperidino-or pyrrolidino (propylenamino)-substituted anthraquinones against leukemia P388 cells [42] and compound with piperidinomoiety were more active that the pyrrolidino-substituted one in the ovarian cancer cell lines [45]. Herein we demonstrate that 3-position amine modification of 1-(3aminopropargyl)-substituent can also significantly potentiate the anticancer activity of hydroxyanthraquinone.
Modeling the interaction of new anthraquinones with G-quadruplex DNA motifs G-quadruplexes are widely represented in telomeric sequences, some promoter regions, as well as in the 5′-untranslated regions of mRNA. Stabilization of G-quadruplexes of telomeric sequences by small molecules promotes inhibition of telomerase, which exhibits increased activity in tumor cells. The binding of the G-quadruplexes of oncogenic promoters leads to suppression of their expression. Thus, the interaction of molecules with the G-quadruplexes of DNA in tumor cells can trigger their apoptosis and develop a cytotoxic effect. Gquadruplex is currently investigated as a target for contrasting unregulated cell proliferation, neurodegeneration, and viral replication [46]. Small molecules with an anthraquinone core [47], hetaryl fused anthracene-9,10-diones [48], and also Doxorubicin [49,50] were able to interact with Gquadruplexes and capable of forming four-strand structures. Recently, the anthracene-9-propargylamine scaffold was characterized as a new G-quadruplex ligand [51].
G-Quadruplex is a structure consisting of four nitrogenous guanine bases linked by eight hydrogen bonds. The π-systems of guanines are located in one plane, which creates the possibility of stacking interactions with planar condensed aromatic systems of small molecules. Naphthalene diimide compound MM41 stabilizes the G-quadruplex due to the interactions of its planar aromatic nucleus with the π-systems of the purine rings of all four guanines. The nitrogen atoms of the methylpiperazine and morpholine rings of branched substituents can be protonated, which makes it possible for the dynamic formation of hydrogen bonds with the oxygen atoms of phosphoric acid residues of neighboring nucleotides, depending on the position of the aromatic nucleus of the molecule above the plane of four guanines.
Blind docking of compounds in the G-quadruplex structure was performed for new compounds 9, 14, 34, and MM41 (Fig. S1, Supplementary part). All poses of MM41 obtained as a result of blind docking are located above the plane of the G-quadruplex near coordinates of the molecule from the XRD model (Fig. S1A). This is apparently due to the large size of the central planar scaffold and extended substituents of the MM41 molecule. New anthraquinone derivatives show more diverse possible binding. Some poses demonstrate the possibility of their penetration between the loop structures of the DNA sequence adjacent to the G-quadruplex (compounds 14 and 34, Fig. S1B, C). However, for all new anthraquinone derivatives studied (compounds 9, 14, and 34), poses are possible that are located above the plane of the Gquadruplex like MM41 inhibitor molecule.
The results of docking studies of compounds 6,7,8,9,12,14,18,19,20,23,24,28,29,30,33,34,36, and MM41 in the G-quadruplex binding site are listed in Table 3 and Fig. 2. The superposition of molecules over the plane of the G-quadruplex is given in Supplementary Part (Fig. S2). The presence of a rigid acetylene bond causes the only possible position of the linear part of the substituent in the plane of the anthraquinone nucleus in all new compounds.
The lowest estimated binding energy was observed for compound 14 (Table 3) with a compact branched aliphatic substituent. The nitrogen atom of the substituent is capable of shifting the electron density from the terminal carbon atoms, creating the possibility of a π-sigma interaction.
The anthraquinone nucleus of 14 can be more evenly located, interacting with all π-systems of guanines (Fig. 2B). The methylpiperazine ring of compound 34, apparently, causes more significant conformational difficulties, causing a a Value is not genuine binding energy but estimated docking score displacement of the anthraquinone nucleus, which in this case is able to interact only with two nitrogenous bases of the Gquadruplex (Fig. 2C). The binding energies for G-quadruplex complexes of compound 9 (−6.492 kcal/mol) were slightly higher than for other Sonogashira cross-coupling reaction products (6,7,8,12). In the case of compound 9, a more large and polar ethyl-2-acetamidobenzoate substituent causes a pronounced displacement of the entire molecule away from the central symmetry axis of the G-quadruplex due to the formation of hydrogen bonds and electrostatic interactions with the phosphate group of the neighboring nucleotide. It becomes possible to form a hydrogen bond between the proton of the hydroxyl group of the anthraquinone nucleus and the keto group of the purine ring of one of the guanines (Fig. 2D). Finally, the docking of compounds 14, 34, and 9 as well as for MM41 revealed a K+ binding of the core.

Conclusion
In summary, we developed a convenient method for the modification of anthraquinone derivatives that can be used for the modification of polyfunctionalized anthraquinones. Using this scheme, a series of 1-ethynyl-4-hydroxyanthraquinones and 1-(3-aminopropynyl)-4-hydroxy-anthraquinones were prepared for the first time.  Noncovalent interactions of molecules are shown by dotted lines: green-hydrogen bonds, orange-electrostatic interactions, purplestacking, and π-sigma interactions (CDCl 3 ) was used as a solvent, with residual CHCl 3 (δ H = 7.24 ppm) or CDCl 3 (δ C = 76.9 ppm) being employed as internal standards. NMR assignments were supported by using COSY, HMBC, and HSQC spectra if appropriate. In the description of the 1 H and 13 C-NMR spectra for all compounds, the anthraquinone skeleton and substituent atoms numeration system are given in structures 14 and 36 was used (Scheme 2). IR absorption spectra were obtained for neat thin films by using a Bruker Vector-22 spectrometer. UV spectra were obtained on an HP 8453 UV-Vis spectrometer (Hewlett-Packard, Waldbronn, Germany) in EtOH solutions ( [53]. The solvents (DMF, PhCH 3 , CHCl 3 , CH 2 Cl 2 , 1,4-dioxane), as well as Et 3 N, were purified according to standard methods. The purity of all compounds (at least 95% or more) was checked by TLC and HPLC-MS analysis. HPLC-MS was performed on an Agilent 1200 liquid chromatography and a hybrid time-of-flight quadrupole mass spectrometer micrOTOF-Q (Bruker).

Synthesis of 1-ethynyl-4-hydroxyanthraquinone (12)
A stirred solution of (trimethylsilyl)alkynyl substituted anthraquinone (11) (100 mg, 0.31 mmol) in dichloromethane (5 mL), was treated with a solution of tetrabutylammonium fluoride (0.163 mg, 0.62 mmol) in dichloromethane (5 mL) and the mixture was stirred at 20°C for 30 min. After completion based on TLC, the solvent was removed in vacuo. The solvent was removed under reduced pressure, and the residue was subjected to column chromatography (CCl 4 ) to give 57 mg (yield 74 %) of compound (12) as an orange powder.

Molecular modeling
Molecular modeling was carried out in the Schrodinger Maestro visualization environment using applications from the Schrodinger Small Molecule Drug Discovery Suite 2016-1 package [54]. Three-dimensional structures of the derivatives were obtained empirically in the LigPrep application using the OPLS3 force field [55]. For the calculations, the XRD model of intramolecular human telomeric DNA G-quadruplex bound by the naphthalene diimide compound [56] with PDB ID 3UYH (resolution 1, 95 Å) from Protein Data Bank was chosen. To model a possible mechanism of stabilization of G-quadruplex, molecular docking of new compounds was performed at the binding site of naphthalene diimide compound MM41 using Glide [57]. The search area for docking was selected according to the size of MM41. Docking was performed in comparison with the MM41 molecule. The threedimensional structure of MM41 was obtained in the Pub-Chem database and prepared in the LigPrep application. Non-covalent interactions of molecules in the binding site were visualized using Schrodinger Maestro and Biovia Discovery Studio Visualizer [58]. A blind docking procedure was performed in the full structure of the G-quadruplex using Autodock Vina [59].