On the Reaction of 2-Alkanoylnaphthohydroquinones with Hydroxylamine: Access to Cytotoxic 2-(Hydroxyamino)-1,4-naphthoquinone and Their 3-(Hydroxyimino)alkyl Analogous

Quı́mica y Farmacia, Facultad de Ciencias de La Salud, Universidad Arturo Prat, Casilla 121, Iquique 1100000, Chile Bioanalysis and Pharmacology of Bioactive Lipids (BPBL), Louvain Drug Research Institute, Université Catholique de Louvain, 72 Avenue E. Mounier, BPBL 7201, Brussels 1200, Belgium Facultad de Farmacia y Bioquı́mica, Universidad Nacional de Trujillo, Trujillo 13011, Peru Research Group in Metabolism and Nutrition, Louvain Drug Research Institute, Université Catholique de Louvain, 73 Avenue E. Mounier, Brussels 1200, Belgium

The synthetic advantage of these acylhydroquinones [9][10][11][12][13][14] emerges from the coexistence of the hydroquinone and the ortho-hydroxyacylarene fragments in their structures. Within this group of acetylhydroquinones, the simplest naturally occurring member named quinacetophenone stands out by its property to inhibit the growth of diverse myeloma cells [15] and by its extensive use as a synthetic precursor of organic molecules such as chalcones, flavonoids, chromones, coumarins, quinones, and psoralens, relevant in medicinal chemistry [16].
In previous studies performed in our laboratory, two series of cytotoxic active acyl-containing quinoid compounds, V and VI, were prepared from acetylhydroquinones types I and III (R � alkyl and aryl) [3,4]. An interesting possibility to improve the cytotoxic activities of this series is to replace the corresponding acyl substituents with their oximes. Examples of this change in functionality to increase cytotoxic activity have been reported in cytotoxic acylcontaining natural products such as naringenin and flavonoid derivatives [17][18][19][20].
The abovementioned replacement approach requires precursors II and IV. Since access to oxime II from I (R � CH 3 ) has been reported [16], we focused our attention on the synthesis of oxime type IV from III.
Herein, we report preliminary results about the unexpected reactivity of 2-acyl-1,4-naphthohydroquinones III (R � n-alkyl) against hydroxylamine leading to the formation of 2-(hydroxyamino)-1,4-naphthoquinone and C-3 (hydroxyamino) alkyl derivatives rather than the corresponding oximes IV. The isolated novel quinoid compounds were evaluated for their in vitro cytotoxic activities on a panel of three human-derived tumor cell lines. In order to get a range of selectivity, such cytotoxic activities were compared to those obtained on nontumorigenic HEK-293 (human embryonic kidney cells).

General Information.
All the solvents and reagents were purchased from different companies, such as Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), and were used as supplied. Melting points (mp) were determined on a Stuart Scientific SMP3 (Staffordshire, UK) apparatus and are uncorrected. The IR spectra were recorded on an FT IR Bruker spectrophotometer, model Vector 22 (Bruker, Rheinstetten, Germany), using KBr disks, and the wave numbers are given in cm −1 . 1 H-and 13 C NMR spectra were recorded on a Bruker Ultrashield-300 instrument (Bruker, Ettlingen, Germany) in DMSO-d 6 at 300 and 75 MHz, respectively. Chemical shifts are expressed in ppm downfield relative to tetramethylsilane, and the coupling constants (J) are reported in Hertz. Data for the 1 H-NMR spectra are reported as follows: s � singlet, br s � broad singlet, d � doublet, t � triplet, q � quartet, m � multiplet, and the coupling constants (J) are in Hz. Bidimensional NMR techniques and distortion-less enhancement by polarization transfer (DEPT) were used for the signal assignment. Chemical shifts are expressed in ppm downfield relative to tetramethylsilane, and the coupling constants (J) are reported in Hertz. The HRMS data for all final compounds were obtained using an LTQ-Orbitrap mass spectrometer (Thermo-Fisher Scientific, Waltham, MA, USA) with the analysis performed using an atmospheric-pressure chemical ionization (APCI) source, operated in positive mode. Silica gel Merck 60 (70-230 mesh, from Merck) was used for preparative column chromatography and thin layer chromatography (TLC). Aluminum foil 60F 254 was used for analytical thin layer chromatography. The acylbenzohydroquinones (2)(3)(4)(5)(6)(7)(8)(9)(10)(11) were prepared according to a previously reported procedure [12].

General
Procedure for the Reaction of 2-Alkanoylnaphthohydroquinones with Hydroxylamine. Suspensions of hydroxylamine hydrochloride (2-equiv.), sodium acetate (2 equiv.), and methanol (20 mL) were stirred for 1 h at room temperature. 2-Acylnaphthohydroquinones (1-equiv.) were added to the methanolic solutions and the mixtures were refluxed for 20 h. The solvents were removed at reduced pressure and the residues were column chromatographed over silica gel (1/1 petroleum ether/ethyl acetate) to give the corresponding substituted naphthoquinones 12-19.    13 13

Cell Lines and Cell Cultures.
Briefly, human cancer cell lines DU-145 (prostate), MCF-7 (breast), T-24 (bladder), and nontumor HEK-293 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured at a density of 1-2 x 10 5 cells/mL in high-glucose Dulbecco's modified Eagle medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 μg/ mL). All cultures were kept at 37°C in 95% air/5% CO 2 at 100% humidity. The medium was changed at 48-72 h intervals. Phosphate-buffered saline (PBS) was purchased from Gibco. Cells were incubated for the indicated times at 37°C in the absence or the presence of quinones at various concentrations.

Cytotoxic
Assays. The cytotoxicity of quinones was assessed by following the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan blue [21]. Cells were seeded into 96-well plates at a density of 10,000 cells/well for 24 h and then further incubated for 48 h, with or without the quinone derivatives. Doxorubicin was used as a standard chemotherapeutic agent (positive control). Cells were then washed twice with warm PBS and incubated with MTT (0.5 mg/mL) for 2 hours at 37°C. Blue formazan crystals were solubilized by adding 100 μL DMSO/well, and the optical density of the colored solutions was subsequently read at 550 nm. Results are expressed as percentage of MTT reduction, compared to untreated control conditions. The IC 50 values were calculated using the GraphPad Prism software (San Diego, CA, USA).

Results and Discussion
Since the synthesis of acetylhydroquinone oxime II from hydroquinone I (R � CH 3 ) and hydroxylamine has been reported with high yield [16] and based on standard procedures to prepare acetophenone oximes [22], the reactivity of acetylhydroquinones III leading to the corresponding oximes IV was first explored. In this context, Scheme 1 shows the synthesis of the required precursor 2-acetylnaphthohydroquinone 2 from 1,4-naphthoquinone 1 and acetaldehyde, according to our previously reported procedure based on the solar photoacylation Friedel-Crafts reaction [12].
In this preliminary assay, acetylnaphthohydroquinone 2 was reacted with NH 2 OH in boiling methanol (Scheme 2). Workup of the reaction mixture provides an orange crystalline product, m.p. 141-142°C. The 1 H-NMR spectrum displays, at 11.16 ppm, a singlet (D 2 O exchangeable) for the hydroxyl proton of a ketoxime group [23]. The characteristic A 2 B 2 pattern signals for the aromatic protons of the 1,4naphthoquinone system appeared at 7.82 and 7.72 ppm. At ca. 7.56 ppm, there was a broad singlet signal for two protons (D 2 O exchangeable) assignable to the NHOH group and a singlet signal at 2.09 ppm for the proton of a methyl group. The 13 C NMR displays signals at 181.5 and 181.7 ppm for quinoid carbonyl carbons and for ketoxime carbon at 153.9 ppm [23]. Based on these spectral properties, HMBC correlation, and the high-resolution mass spectroscopy (HRMS), structure 12 was fully established for the new compound isolated in 58% yield.
This interesting and unexpected reaction of 2 with hydroxylamine to give 12 leads us to change our primary objective to prepare compounds type IV towards the substrate scope of the previously unreported one-pot reaction as potential general access to novel 2-(hydroxyamino)-3-(hydroxyimino)alkyl-1,4-naphthoquinones. In this context, compounds 3-12 were prepared, just like compound 2, by solar photoacylation of 1,4-naphthoquinone 1 with C 3 -C 11 linear aliphatic aldehydes according to Scheme 1.
The synthesized 2-acylnaphthohydroquinones 3-11 were reacted with hydroxylamine according to the aforementioned reaction conditions, and the products were isolated by column chromatography over silica gel. The results of the assays are summarized in Scheme 3. The structures of the new compounds 13-19 were fully established by 1 H and 13 C nuclear magnetic resonance (NMR), bidimensional nuclear magnetic resonance (2D-NMR), and high-resolution mass spectrometry (HRMS).
A tentative explanation was proposed for the reaction of compounds 2-8 with hydroxylamine to yield the respective disubstituted 1,4-naphthoquinones 12-18. Indeed, the course reaction explaining the formation of 12 from 2 is depicted in Scheme 4. To build such a hypothesis, the following points were taken into account: (a)Aerobic oxidation is chemically inert to acylnaphthohydroquinones 2-11. This assumption was confirmed by doing an experiment where compound 2 remained unchanged after boiling it for 20 hours in methanol under aerial conditions. This is likely because the hydroquinone system is strongly stabilized through the internal hydrogen bond between the carbonyl and 2hydroxy groups [24]. (b) The oxime formation process, involved in the reaction of carbonyl compounds with hydroxylamine, is reversible and occurs via a hemiaminal intermediate (tetrahedral intermediate) [25]. (c) The aerial oxidation of the hemiaminal and/or oxime intermediates prevents the reversal of the equilibria process to precursor 2, thus allowing the further oxidative amination of naphthoquinone intermediates to produce 12.
Regarding the formation of aminonaphthoquinone 19 from compounds 9-11 and hydroxylamine, a tentative course reaction is outlined in Scheme 5, starting from compound 10. Taking into account that the reaction of compounds 9-11 with hydroxylamine yields aminoquinone 19, resulting from C-C cleavage reactions and the efficient and single access to 19 from naphthoquinone I and hydroxylamine, no efforts were made in order to get additional pieces of evidence on these degradation reactions.
In light of our results, providing access to a new class of compounds 12-19 containing the oxime group and the anticancer aminonaphthoquinone pharmacophore as well [26,27], the in vitro cytotoxic evaluation of quinones was undertaken by using a panel of three human-derived tumor cell lines (Table 1). In order to get a range of selectivity, such cytotoxic activities were compared to those obtained on nontumorigenic HEK-293 (human embryonic kidney cells).
Cells were seeded at a density of 10 000 cells/well for 24 h into 96-well plates and then incubated for 48 h in the absence or presence of compounds. After washing, cells were further incubated with MTT (0.5 mg/mL) for 2 hours at 37°C. Blue   Table 1 shows moderate antiproliferative activities of 2-(hydroxyamino)naphthoquinone 19 and, to a lesser extent, its hydroxyimino-ethylnaphthoquinone analogue 12. Indeed, 19 was the most active compound in all cancer cell lines, but its effects are lower than those of doxorubicin. It should be underlined that doxorubicin IC 50 values are in agreement with those recently reported in the literature [28][29][30]. Regarding the mechanism of action of compound 19, it is unlike that which is the same as doxorubicin. Indeed, the cytotoxicity of the latter involves miscellaneous ways such as DNA adduct formation, DNA intercalation, inhibition of topoisomerase II, oxidative stress by ROS formation, and lipid peroxidation [31]. Moreover, the comparison of the cytotoxic activities of analogues 12-18 shows that the longer the linear aliphatic chain, the lower their potencies. It is also observed that the enlargement of the aliphatic chain from 12 to 18 somehow reduces the selectivity, given that cytotoxicity was similar in both nontumorous and cancer cells. Last, although the observed anticancer activities of the 12-18 series were rather low, it appears that bladder cancer cells were slightly more sensitive to that series when compared with breast cancer cells.

Data Availability
The data used to support this study are available from the corresponding author upon request and are included within supplementary materials.