Alkoxy and Enediyne Derivatives Containing 1,4-Benzoquinone Subunits—Synthesis and Antitumor Activity

The compounds produced by a living organism are most commonly as medicinal agents and starting materials for the preparation of new semi-synthetic derivatives. One of the largest groups of natural compounds consists of products containing a 1,4-benzoquinone subunit. This fragment occurs in three enediyne antibiotics, dynemicin A, deoxydynemicin A, and uncilamicin, which exhibit high biological activity. A series of alkoxy derivatives containing 1,4-naphthoquinone, 5,8-quinolinedione, and 2-methyl-5,8-quinolinedione moieties was synthesized. Moreover, the 1,4-benzoquinone subunit was contacted with an enediyne fragment. All obtained compounds were characterized by spectroscopy and spectrometry methods. The resulting alkane, alkene, alkyne and enediyne derivatives were tested as antitumor agents. They showed high cytotoxic activity depending on the type of 1,4-benzoquinone subunit and the employed tumor cell lines. The synthesized derivatives fulfill the Lipinski Rule of Five and have low permeability through the blood–brain barrier.


Introduction
Nature is a main source of novel compounds useful as medicinal agents and starting materials for the preparation of semi-synthetic derivatives [1][2][3][4][5][6][7]. The isolated products exhibit a broad spectrum of pharmacological effects, including antioxidant, anticancer, antibacterial, antiviral, and anti-inflammatory activities [2,[5][6][7]. The derivatives containing a 1,4-benzoquinone subunit constitute one of the largest and diverse groups of secondary metabolites characterized by a broad range of biological properties [8,9]. The mechanism of their action has not been completely explained. However, a 1,4-benzoquinone unit plays an important role in oxidation processes. It should be noted that this moiety catalyzes oxidation of the intra-and extra-mitochondrial nicotinamide adenine dinucleotide phosphate (NAD(P)H), and the by-products of this process are reactive oxygen species that induce cell death [1,10,11]. The group of natural compounds that contains 1,4-benzoquinone also includes geldanamycin, mitomycin, bolinaquinone, and ubiquinone. Often, this subunit is connected with other fragments creating 1,4-naphthoquinone, anthracycline, and 5,8-quinolinedione, which occur in many chemotherapeutic drugs, such as plubmagin, anthracyclines, and streptonigrin. Unfortunately, the high toxicity of the antibiotics limits their use in the treatment of cancer [12][13][14][15][16].
A second interesting group of natural products have been isolated from bacteria enediyne antibiotics [3,5]. These natural products contain a unique nine-or ten-membered unsaturated ring composed of two triple bonds connected through a double bond. The compounds are characterized by an unusual mechanism of action. In the Bergman cyclization, the enediyne ring was converted to a 1,4-diradical, which interacts with DNA and causes breaks in single-and double-stranded molecules [3,5,17]. So far, eleven compounds belonging to the enediyne family have been discovered. Three of them, dynemicin A, deoxydynemicin A, and uncilamicin, contain a 1,4-benzoquinone moiety ( Figure 1). These compounds affect the DNA cleavage on two difference routes, directly by reduction of the anthraquinone ring and opening of the epoxide ring and indirectly by the formation of a highly reactive 1,4-diradical intermediate from the (Z)-enediyne group [18][19][20]. Thereby, natural conjugates of the enediyne ring and the 1,4-benzoquinone subunit show high activity against Gram-positive and Gram-negative bacteria and antitumor activity against a broad spectrum of human and murine cancer cell lines [2,3,17,[21][22][23]. Unlike the other enediyne antibiotics, dynemicin A exhibits low toxicity [22,23]. A second interesting group of natural products have been isolated from bacteria enediyne antibiotics [3,5]. These natural products contain a unique nine-or ten-membered unsaturated ring composed of two triple bonds connected through a double bond. The compounds are characterized by an unusual mechanism of action. In the Bergman cyclization, the enediyne ring was converted to a 1,4-diradical, which interacts with DNA and causes breaks in single-and double-stranded molecules [3,5,17]. So far, eleven compounds belonging to the enediyne family have been discovered. Three of them, dynemicin A, deoxydynemicin A, and uncilamicin, contain a 1,4-benzoquinone moiety ( Figure 1). These compounds affect the DNA cleavage on two difference routes, directly by reduction of the anthraquinone ring and opening of the epoxide ring and indirectly by the formation of a highly reactive 1,4-diradical intermediate from the (Z)-enediyne group [18][19][20]. Thereby, natural conjugates of the enediyne ring and the 1,4-benzoquinone subunit show high activity against Gram-positive and Gramnegative bacteria and antitumor activity against a broad spectrum of human and murine cancer cell lines [2,3,17,[21][22][23]. Unlike the other enediyne antibiotics, dynemicin A exhibits low toxicity [22,23]. Continuing our research on synthesis and anticancer activity of alkoxy derivatives of the 5,8quinolinedione moiety [24,25], alkoxy compounds containing 1,4-naphthoquinone and 2-methyl-5,8quinolinedione subunit were received. Moreover, the connection of the 1,4-benzoquinone fragment with a 1,5-diyne-3-ene unit allows for the obtainment of enediyne products containing 1,4naphthoquinone, 5,8-quinolinedione, and 2-methylo-5,8-quinolinedione moieties. For all synthesized derivatives, the antitumor activity against three human cancer cell lines was determined and the pharmacological parameters were calculated.
A condensation of the 1,4-benzoquinones 1-3 with Compound 22 in the presence of potassium carbonate and tetrahydrofuran led to the acyclic enediyne derivatives 23-25. On the other hand, the change of solvent from tetrahydrofuran to dimethyl sulfoxide allowed for the obtainment of the cyclic compounds 26-28 (Scheme 2). All compounds were purified by flash chromatography. The 7-alkoxy derivatives 2-12 and 6,7-dialkoxy compounds 13-21 were obtained with 53-76% and 41-72% yields, respectively. The structures of the synthesized derivatives 4-21 were confirmed on the basis of 1 H-and 13 C-NMR, IR, and HR-MS spectra.
A condensation of the 1,4-benzoquinones 1-3 with Compound 22 in the presence of potassium carbonate and tetrahydrofuran led to the acyclic enediyne derivatives 23-25. On the other hand, the change of solvent from tetrahydrofuran to dimethyl sulfoxide allowed for the obtainment of the cyclic compounds 26-28 (Scheme 2).
The major products of the reactions were separated by flash chromatography to yield the pure derivatives 23-28 in 35-51% yields. Chemical structures of the enediyne compounds 23-25 were elucidated with the aid of 1 H-, 13 C-NMR, IR spectroscopy, and HR-MS analysis.

Antiproliferative Activity
The obtained compounds 1-28 were evaluated for cytostatic activity against three human cancer cell lines: melanoma (C-32), glioblastoma (SNB-19), breast cancer (MDA-MB-231), and normal fibroblasts (HFF-1) cell lines. The results are presented in Tables 1 and 2. Moreover, the selectivity index (SI), expressed as the ratio of IC50 of a normal fibroblast to IC50 of the corresponding cancer cell line, was calculated (Table S1; Supplementary Materials). The major products of the reactions were separated by flash chromatography to yield the pure derivatives 23-28 in 35-51% yields. Chemical structures of the enediyne compounds 23-25 were elucidated with the aid of 1 H-, 13 C-NMR, IR spectroscopy, and HR-MS analysis.
The second group of the synthesized compounds consisted of the 5,8-quinolinedione derivatives 7-12 and 16-21. Comparing the activity of the compounds containing 5,8-quinolinedione (7-9 and 16-18) and 2-methyl-5,8-quinolinedione (10-12 and 19-21), it was shown that the introduction of a methyl group at C-2 position of 5,8-quinolinedione unit led to a decrease in the antiproliferative activity against all tested cancer cell lines. The general trend of the antitumor activity showed that the introduction of alkoxy substituent to the 2-methyl-5,8-quinolinedione moiety led to a reduction of cytotoxicity against all cancer cell lines compared with 6,7-dichloro-2-methyl-5,8-quinolinedione (2). One could notice that the derivatives of 2-methyl-5,8-quinolinedione were characterized by a lower selectivity of action (Table S1; Supplementary Materials).
The analysis of the structure-activity relationship showed that the activity of alkoxy derivatives containing the 1,4-benzoquinone subunit depended on the nitrogen atom at the C-1 position and the group at the C-2 position.
The cyclic compounds containing 1,4-naphthoquinone (26)  Physicochemical parameters such as lipophilicity (clog P), molecular mass (M), topological polar surface area (PSA), the number of donors (nHD), the acceptors (nHA) of the hydrogen bond, and the number of rotatable bonds (nRTB) were used to assess absorption, distribution, metabolism, and excretion of the compound in a biological system [28][29][30]. An important property of the newly synthesized compounds is their penetration through the blood-brain barrier (BBB). Determination of log BB (drug penetration through the BBB) allows for predicting their neurotoxicity. All parameters were calculated using ACD/iLab software, and the values are presented in Table S2 (Supplementary Materials).
The obtained compounds 1-28 fulfill the Lipinski rules. The ACD/iLab software calculated the number of donors (nHD) of the hydrogen bond depending on the amounts of hydroxyl and amine groups in a compound. One might notice that the nHD number could be higher, because in the derivatives containing the 5,8-quinolinedione subunit, a weak hydrogen bond between the C-H group as a donor and the oxygen atom as an acceptor of the hydrogen bond was observed [24,25,31,32]. The topological polar surface area (PSA) obtained for the discussed compounds is in the range of 34.14-76.50 Å 2 , which determined a high oral bioavailability [33]. According to the literature [34,35], if the log BB value is higher than 0.3, the compounds pass the BBB rapidly; however, a value lower than −1 indicates poor distribution in the blood-brain barrier. The log BB values for the studied compounds were lower than 0.3, suggesting that they could have a low permeability through the blood-brain barrier. Moreover, three of them, 15, 18, and 21, had a log BB value lower than −1, so they had no access to the central nervous system.

Chemistry
Melting points were established on an Electrothermal IA 9300 melting point apparatus. The 1 Hand 13 C-NMR spectra were determined using a Bruker Avance 600 spectrometer (Bruker, Billerica, MA, USA) in CDCl 3 ; chemical shifts (δ) are reported in ppm and J values in Hz. The peak multiplicity is designated as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), doublet of triplets (dt), and multiplet (m). High-resolution mass spectral analysis was measured on a Bruker Impact II instrument (Bruker). Infrared spectra were recorded on an IRAffinity-1 Shimadzu spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Thin layer chromatography (TLC) was performed on silica gel 60 254F plates (Merck, Darmstadt, Germany) using a mixture of chloroform and ethanol (40:1 or 15:1, v/v) as an eluent. The spots were visualized by UV light (254 nm) and iodine. All new compounds were purified by flash chromatography (Reveleris Flash system, Grace technologies, Ellicott City, MD, USA). The column for flash chromatography was filled silica gel (Grace Technologies, Ellicott City, MD, USA). As a mobile phase was used chloroform-ethanol (40:1 or 15:1, v/v). It was isocratically eluted at a flow rate of 2.0 mL/min. The effluent was monitored by UV detector (254 nm) and peak fractions were collected according to the elution profile.

General Procedure for the Synthesis of Dialkoxy Derivatives 13-21
A mixture of 1,4-benzoquinone 1-3 (0.441 mmol), potassium carbonate (0.125 g, 0.909 mmol), and alcohol (2.2 eqv., 0.970 mmol) in dry dimethyl sulfoxide (1 mL) was stirred at room temperature for 3 h. Subsequently, CH 2 Cl 2 was added to the reaction mixture and extracted with water. The organic layer was dried over Na 2 SO 4 . After filtration, the solvents were removed by evaporation under reduced pressure. The residue was purified by silica gel chromatography (chloroform/ethanol, 40:1, v/v) to yield pure products 13-21.   Figure S9).

WST-1 Test
WST-1 colorimetric assay for cell proliferation (Roche Diagnostics GmbH, Mannheim, Germany, reagent kit cat. 11644807001) is based on the viable cells' ability to cleave the bright red-colored stable tetrazolium salt WST-1 to dark red soluble formazan. This bioreduction occurs under the influence of mitochondrial dehydrogenases (depends mostly on production of NAD(P)H in viable cells). The amount of formazan dye formed correlates directly with the number of metabolically active cells in the culture and is measured by absorbance (λ = 450 nm) following 1 h incubation of cells with the reagent.

Conclusions
In this study, we identified antiproliferative agents among a series of mono and dialkoxy derivatives containing 1,4-naphthoquinone, 5,8-quionolinedione, or 2-methyl-5,8-quinolinedione subunits. The general trend of the antitumor activity showed that the introduction of an alkoxy substituent to the 1,4-benzoquinone moiety leads to an increase in the activity against all tested cancer cell lines. In the series of enediyne compounds, the best activity against melanoma (C-32) and glioblastoma (SNB-19) cell lines was exhibited by 6-chloro-7-(8-hydroxy-4-octen-2,6-diynyloxy)-5,8quinolinedione. The analysis of the calculated physicochemical parameters suggested that the synthesized derivatives were characterized by a high oral bioavailability and a low pass through blood-brain barrier.