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

Authors: Jaime A. Valderrama; Andrea Pérez-Herrera; Giulio G. Muccioli; Edmundo A. Venegas-Casanova; Rafael Jara-Aguilar; Pedro Buc Calderon; Julio Benites
Journal: Journal of Chemistry
 (2022)

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## Abstract

Oximes are known for their anti-inflammatory, antimicrobial, antioxidant, and anticancer activities. Frequently, modification of biologically active carbonyl compounds into oximes leads to increased activity. The present study reports the reactivity of 2-alkanoylnaphthohydroquinones against hydroxylamine under aerial conditions. Results show that, depending on the structure of the hydroquinones, the reaction proceeds through two different chemical pathways to produce 2-(hydroxyamino)-1,4-naphthoquinone and their C-3 (hydroxyimino)alkyl derivatives. Both the formation of the quinoid compounds under aerial oxidation and C-C cleavage reactions of hemiaminal intermediates are discussed. In vitro screening of the substituted 1,4-naphthoquinones on a panel of cancer cells reveals moderate cytotoxic activities. Compound19, 2-(hydroxyamino)-1,4-naphthoquinone, stands out by its anticancer potency against prostate cancer cells as shown by the lowest IC50 value (8.08 μM) and the best selectivity index (3.90).


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## Body

## 1. Introduction

2-Acylated-1,4-benzo- and 1,4-naphthohydroquinonesI and III (Figure 1) are of current interest by their use as synthetic building blocks for carbo- and heterocyclic compounds endowed with cytotoxic and antiproliferative effects in various human cancer cells, as well as antifungal activities [1–8]. The synthetic advantage of these acylhydroquinones [9–14] 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].Figure 1
Structure of compoundsI–VI.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 acyl-containing natural products such as naringenin and flavonoid derivatives [17–20].The abovementioned replacement approach requires precursorsII and IV. Since access to oxime II from I (R = CH3) 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-naphthohydroquinonesIII (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).


## 2. Materials and Methods

### 2.1. 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. 1H- and 13C NMR spectra were recorded on a Bruker Ultrashield-300 instrument (Bruker, Ettlingen, Germany) in DMSO-d6 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 1H-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 60F254 was used for analytical thin layer chromatography. The acylbenzohydroquinones (2–11) were prepared according to a previously reported procedure [12].


### 2.2. 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 naphthoquinones12–19.


#### 2.2.1. 2-(Hydroxyamino)-3-(hydroxyimino)methyl-1,4-naphthoquinone12

Prepared from2 (200 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 58% yield (140.3 mg, 0.57 mmol) as orange solid mp: 141–142°C; IR (KBr) νmax cm−1: 3467, 3416, 1638, 1618; 1H-NMR (300 MHz, DMSO-d6): δ 11.16 (s, 1H, OH), 7.96 (d, 2H, J = 7.7 Hz, 2H, arom), 7.82 (t, 1H, J = 7.5 Hz, arom), 7.72 (t, 1H, J = 7.4 Hz, arom), 7.56 (br s, 2H, OH + NH), 2.09 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.5, 180.7, 153.9, 147.0, 135.0, 133.3, 132.5, 130.0, 125.8, 125.6, 109.5, 15.5. HRMS (APCI): (M + H)+ Calcd for C12H10N2O4: 246.06406; found 246.07111.


#### 2.2.2. 2-(Hydroxyamino)-3-(hydroxyimino)propyl-1,4-naphthoquinone13

Prepared from3 (228 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 60% yield (161.8 mg, 0.59 mmol) as orange solid mp: 148–149°C. IR (KBr) νmax cm−1: 3473 (OH), 3413 (NH), 1638 (C=O), 1618 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.07 (s, 1H, OH), 7.96 (d, 2H, J = 8.8 Hz, H-arom), 7.83 (t, 1H, J = 8.1 Hz, H-arom), 7.73 (t, 1H, J = 8.1 Hz, H-arom), 7.55 (br s, 2H, OH + NH), 2.63 (dd, J = 8.8, 6.7 Hz, 2H, HO-N=C-CH2-CH2-CH3), 1.46 (h, 2H, J = 7.3 Hz, HO-N=C-CH2-CH2-CH3), 0.84 (t, 3H, J = 7.4 Hz,, HO-N=C-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.49, 180.69, 157.01, 147.53, 135.03, 133.13, 132.46, 129.99, 125.79, 125.66, 109.12, 29.89, 19.03, 14.43. HRMS (APCI): (M + H)+ Calcd for C14H14N2O4: 274.09536; found 274.09844.


#### 2.2.3. 2-(Hydroxyamino)-3-(hydroxyimino)butyl-1,4-naphthoquinone14

Prepared from4 (244 mg, 1.0 mmol) and hydroxylamine (2-equiv.), isolated into 58%, yield (167.2 mg, 0.58 mmol) as orange solid mp: 157–158°C. IR (KBr) νmax cm−1: 3452 (OH), 3450 (NH), 1638 (C=O), 1619 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.06 (s, 1H, OH), 7.96 (d, 2H, J = 8.9 Hz, H-arom), 7.83 (t, 1H, J = 7.7 Hz, H-arom), 7.73 (t, 1H, J = 7.4 Hz, H-arom), 7.53 (br s, 2H, OH + NH), 2.65 (m, 2H, HO-N=C-CH2-CH2-CH2-CH3), 1.41 (q, 2H, J = 7.3 Hz, HO-N=C-CH2-CH2-CH2-CH3), 1.24 (dq, 2H, J = 14.4, 7.1 Hz, HO-N=C-CH2-CH2-CH2-CH3), 0.82 (t, 3H, J = 7.2 Hz, HO-N=C-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.51, 180.69, 157.10, 147.55, 135.05, 133.12, 132.48, 130.00, 125.80, 125.68, 109.14, 27.72, 27.60, 22.64, 13.91. HRMS (APCI): (M + H)+ Calcd for C15H16N2O4: 288.1110; found 288.1010.


#### 2.2.4. 2-(Hydroxyamino)-3-(hydroxyimino)pentyl-1,4-naphthoquinone15

Prepared from5 (258 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 65% yield (193.5 mg, 0.64 mmol) as orange solid mp: 160–161°C. IR (KBr) νmax cm−1: 3473 (OH), 3413 (NH), 1638 (C=O), 1617 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.06 (s, 1H, OH), 7.96 (d, 2H, J = 7.7 Hz, H-arom), 7.83 (t, 1H, J = 7.5 Hz, H-arom), 7.73 (t, 1H, J = 7.5 Hz, H-arom), 7.53 (br s, 2H, OH + NH), 2.64 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH3), 1.44 (q, 2H, J = 6.9 Hz, HO-N=C-CH2-CH2-CH2-CH2-CH3), 1.22 (m, 4H, HO-N=C-CH2-CH2-CH2-CH2-CH3), 0.80 (t, 3H, J = 6.6 Hz, HO-N=C-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.51, 180.71, 157.17, 147.57, 135.08, 133.14, 132.50, 130.00, 125.82, 125.70, 109.12, 31.68, 27.78, 25.15, 21.96, 13.97. HRMS (APCI): (M + H)+ Calcd for C16H18N2O4: 302.12666; found 302.12123.


#### 2.2.5. 2-(Hydroxyamino)-3-(hydroxyimino)hexyl-1,4-naphthoquinone16

Prepared from6 (272 mg, 1.0 mmol) and hydroxylamine (2-equiv.), isolated in 55% yield (174.1 mg, 0.55 mmol) as orange solid mp. 162–163°C. IR (KBr) νmax cm−1: 3473 (OH), 3414 (NH), 1638 (C=O), 1619 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.05 (s, 1H, OH), 7.96 (d, 2H, J = 8.8 Hz, H-arom), 7.82 (td, 1H, J = 7.7; 1.1 Hz, H-arom), 7.72 (td, 1H, J = 7.7; 1.1 Hz, H-arom), 7.54 (br s, 2H, OH + NH), 2.64 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 1.41 (q, J = 7.4, 6.9 Hz, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 1.18 (m, 6H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 0.78 (t, J = 6.6 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.50, 180.69, 157.17, 147.54, 135.05, 133.13, 132.48, 129.99, 125.80, 125.67, 109.14, 31.09, 29.09, 27.78, 25.45, 22.07, 13.97. HRMS (APCI): (M + H)+ Calcd for C17H20N2O4: 316.14231; found 316.14799.


#### 2.2.6. 2-(Hydroxyamino)-3-(hydroxyimino)heptyl-1,4-naphthoquinone17

Prepared from7 (287 mg, 1.0 mmol) and hydroxylamine (2-equiv.). Isolated in 72% yield (234.6 mg, 0.71 mmol) as orange solid mp. 165–166°C. IR (KBr) νmax cm−1: 3470 (OH), 3416 (NH), 1638 (C=O), 1618 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.11 (s, 1H, OH), 7.95 (d, 2H, J = 7.9 Hz, H-arom), 7.81 (td, 1H, J = 7.6, 1.3 Hz, H-arom), 7.71 (td, 1H, J = 7.5, 1.4 Hz, H-arom), 7.48 (br s, 2H, OH + NH), 2.63 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.40 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.15 (m, 8H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.75 (t, J = 6.8 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.66, 180.93, 157.35, 147.67, 135.27, 133.24, 132.69, 130.12, 125.96, 125.86, 109.35, 31.38, 29.46, 28.64, 27.86, 25.60, 22.25, 14.13; HRMS (APCI): (M + H)+ Calcd for C18H22N2O4: 330.15796; found 330.16101.


#### 2.2.7. 2-(Hydroxyamino)-3-(hydroxyimino)octyl-1,4-naphthoquinone18

Prepared from8 (300 mg, 0.99 mmol) and hydroxylamine (2-equiv.) to yield quinone 18 (192.8 mg, 0.56 mmol, 57%), orange solid mp. 169–170°C. IR (KBr) νmax cm−1: 3472 (OH), 3416 (NH), 1638 (C=O), 1618 (C=O). 1H-NMR (300 MHz, DMSO-d6): δ 11.11 (s, 1H, OH), 7.95 (d, 2H, J = 7.7 Hz, H-arom), 7.82 (td, 1H, J = 7.6, 1.1 Hz, H-arom), 7.72 (td, 1H, J = 7.5, 1.2 Hz, H-arom), 7.47 (br s, 2H, OH + NH), 2.63 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.40 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.14 (m, 10H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.76 (t, J = 6.5 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3). 13C NMR (75 MHz, DMSO-d6) δ 181.67, 180.95, 157.36, 147.68, 135.30, 133.25, 132.73, 130.13, 125.98, 125.88, 109.37, 31.41, 29.44, 28.89, 28.77, 27.80, 25.56, 22.28, 14.14. HRMS (APCI): (M + H)+ Calcd for C19H24N2O4: 344.17361; found 344.17398.


#### 2.2.8. 2-(Hydroxyamino)-1,4-naphthoquinone19

Prepared from10 (328 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 72% yield (134.3 mg, 0.71 mmol) as orange solid mp. 142–143°C. IR (KBr) νmax cm−1: 3467, 3417, 1638, 1618; 1H-NMR (300 MHz, DMSO-d6): δ 7.92 (ddd, 2H, J = 13.9, 7.6, 1.4 Hz, arom), 7.80 (td, 1H, J = 7.5, 1.1 Hz, arom), 7.70 (td, 1H, J = 7.4, 1.5 Hz, arom), 7.22 (br s, 2H, OH + NH), 5.81 (s, 1H, 2-H). 13C NMR (75 MHz, DMSO-d6): δ 182.0, 181.8, 150.5, 134.7, 133.2, 132.2, 130.4, 125.8, 125.3, 102.3. HRMS (APCI): (M + H)+ Calcd for C10H7NO3: 189.04259; found 189.04899.


### 2.3. 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 105 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% CO2 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.


### 2.4. 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 IC50 values were calculated using the GraphPad Prism software (San Diego, CA, USA).


## 2.1. 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. 1H- and 13C NMR spectra were recorded on a Bruker Ultrashield-300 instrument (Bruker, Ettlingen, Germany) in DMSO-d6 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 1H-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 60F254 was used for analytical thin layer chromatography. The acylbenzohydroquinones (2–11) were prepared according to a previously reported procedure [12].


## 2.2. 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 naphthoquinones12–19.


### 2.2.1. 2-(Hydroxyamino)-3-(hydroxyimino)methyl-1,4-naphthoquinone12

Prepared from2 (200 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 58% yield (140.3 mg, 0.57 mmol) as orange solid mp: 141–142°C; IR (KBr) νmax cm−1: 3467, 3416, 1638, 1618; 1H-NMR (300 MHz, DMSO-d6): δ 11.16 (s, 1H, OH), 7.96 (d, 2H, J = 7.7 Hz, 2H, arom), 7.82 (t, 1H, J = 7.5 Hz, arom), 7.72 (t, 1H, J = 7.4 Hz, arom), 7.56 (br s, 2H, OH + NH), 2.09 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.5, 180.7, 153.9, 147.0, 135.0, 133.3, 132.5, 130.0, 125.8, 125.6, 109.5, 15.5. HRMS (APCI): (M + H)+ Calcd for C12H10N2O4: 246.06406; found 246.07111.


### 2.2.2. 2-(Hydroxyamino)-3-(hydroxyimino)propyl-1,4-naphthoquinone13

Prepared from3 (228 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 60% yield (161.8 mg, 0.59 mmol) as orange solid mp: 148–149°C. IR (KBr) νmax cm−1: 3473 (OH), 3413 (NH), 1638 (C=O), 1618 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.07 (s, 1H, OH), 7.96 (d, 2H, J = 8.8 Hz, H-arom), 7.83 (t, 1H, J = 8.1 Hz, H-arom), 7.73 (t, 1H, J = 8.1 Hz, H-arom), 7.55 (br s, 2H, OH + NH), 2.63 (dd, J = 8.8, 6.7 Hz, 2H, HO-N=C-CH2-CH2-CH3), 1.46 (h, 2H, J = 7.3 Hz, HO-N=C-CH2-CH2-CH3), 0.84 (t, 3H, J = 7.4 Hz,, HO-N=C-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.49, 180.69, 157.01, 147.53, 135.03, 133.13, 132.46, 129.99, 125.79, 125.66, 109.12, 29.89, 19.03, 14.43. HRMS (APCI): (M + H)+ Calcd for C14H14N2O4: 274.09536; found 274.09844.


### 2.2.3. 2-(Hydroxyamino)-3-(hydroxyimino)butyl-1,4-naphthoquinone14

Prepared from4 (244 mg, 1.0 mmol) and hydroxylamine (2-equiv.), isolated into 58%, yield (167.2 mg, 0.58 mmol) as orange solid mp: 157–158°C. IR (KBr) νmax cm−1: 3452 (OH), 3450 (NH), 1638 (C=O), 1619 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.06 (s, 1H, OH), 7.96 (d, 2H, J = 8.9 Hz, H-arom), 7.83 (t, 1H, J = 7.7 Hz, H-arom), 7.73 (t, 1H, J = 7.4 Hz, H-arom), 7.53 (br s, 2H, OH + NH), 2.65 (m, 2H, HO-N=C-CH2-CH2-CH2-CH3), 1.41 (q, 2H, J = 7.3 Hz, HO-N=C-CH2-CH2-CH2-CH3), 1.24 (dq, 2H, J = 14.4, 7.1 Hz, HO-N=C-CH2-CH2-CH2-CH3), 0.82 (t, 3H, J = 7.2 Hz, HO-N=C-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.51, 180.69, 157.10, 147.55, 135.05, 133.12, 132.48, 130.00, 125.80, 125.68, 109.14, 27.72, 27.60, 22.64, 13.91. HRMS (APCI): (M + H)+ Calcd for C15H16N2O4: 288.1110; found 288.1010.


### 2.2.4. 2-(Hydroxyamino)-3-(hydroxyimino)pentyl-1,4-naphthoquinone15

Prepared from5 (258 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 65% yield (193.5 mg, 0.64 mmol) as orange solid mp: 160–161°C. IR (KBr) νmax cm−1: 3473 (OH), 3413 (NH), 1638 (C=O), 1617 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.06 (s, 1H, OH), 7.96 (d, 2H, J = 7.7 Hz, H-arom), 7.83 (t, 1H, J = 7.5 Hz, H-arom), 7.73 (t, 1H, J = 7.5 Hz, H-arom), 7.53 (br s, 2H, OH + NH), 2.64 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH3), 1.44 (q, 2H, J = 6.9 Hz, HO-N=C-CH2-CH2-CH2-CH2-CH3), 1.22 (m, 4H, HO-N=C-CH2-CH2-CH2-CH2-CH3), 0.80 (t, 3H, J = 6.6 Hz, HO-N=C-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.51, 180.71, 157.17, 147.57, 135.08, 133.14, 132.50, 130.00, 125.82, 125.70, 109.12, 31.68, 27.78, 25.15, 21.96, 13.97. HRMS (APCI): (M + H)+ Calcd for C16H18N2O4: 302.12666; found 302.12123.


### 2.2.5. 2-(Hydroxyamino)-3-(hydroxyimino)hexyl-1,4-naphthoquinone16

Prepared from6 (272 mg, 1.0 mmol) and hydroxylamine (2-equiv.), isolated in 55% yield (174.1 mg, 0.55 mmol) as orange solid mp. 162–163°C. IR (KBr) νmax cm−1: 3473 (OH), 3414 (NH), 1638 (C=O), 1619 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.05 (s, 1H, OH), 7.96 (d, 2H, J = 8.8 Hz, H-arom), 7.82 (td, 1H, J = 7.7; 1.1 Hz, H-arom), 7.72 (td, 1H, J = 7.7; 1.1 Hz, H-arom), 7.54 (br s, 2H, OH + NH), 2.64 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 1.41 (q, J = 7.4, 6.9 Hz, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 1.18 (m, 6H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 0.78 (t, J = 6.6 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.50, 180.69, 157.17, 147.54, 135.05, 133.13, 132.48, 129.99, 125.80, 125.67, 109.14, 31.09, 29.09, 27.78, 25.45, 22.07, 13.97. HRMS (APCI): (M + H)+ Calcd for C17H20N2O4: 316.14231; found 316.14799.


### 2.2.6. 2-(Hydroxyamino)-3-(hydroxyimino)heptyl-1,4-naphthoquinone17

Prepared from7 (287 mg, 1.0 mmol) and hydroxylamine (2-equiv.). Isolated in 72% yield (234.6 mg, 0.71 mmol) as orange solid mp. 165–166°C. IR (KBr) νmax cm−1: 3470 (OH), 3416 (NH), 1638 (C=O), 1618 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.11 (s, 1H, OH), 7.95 (d, 2H, J = 7.9 Hz, H-arom), 7.81 (td, 1H, J = 7.6, 1.3 Hz, H-arom), 7.71 (td, 1H, J = 7.5, 1.4 Hz, H-arom), 7.48 (br s, 2H, OH + NH), 2.63 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.40 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.15 (m, 8H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.75 (t, J = 6.8 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.66, 180.93, 157.35, 147.67, 135.27, 133.24, 132.69, 130.12, 125.96, 125.86, 109.35, 31.38, 29.46, 28.64, 27.86, 25.60, 22.25, 14.13; HRMS (APCI): (M + H)+ Calcd for C18H22N2O4: 330.15796; found 330.16101.


### 2.2.7. 2-(Hydroxyamino)-3-(hydroxyimino)octyl-1,4-naphthoquinone18

Prepared from8 (300 mg, 0.99 mmol) and hydroxylamine (2-equiv.) to yield quinone 18 (192.8 mg, 0.56 mmol, 57%), orange solid mp. 169–170°C. IR (KBr) νmax cm−1: 3472 (OH), 3416 (NH), 1638 (C=O), 1618 (C=O). 1H-NMR (300 MHz, DMSO-d6): δ 11.11 (s, 1H, OH), 7.95 (d, 2H, J = 7.7 Hz, H-arom), 7.82 (td, 1H, J = 7.6, 1.1 Hz, H-arom), 7.72 (td, 1H, J = 7.5, 1.2 Hz, H-arom), 7.47 (br s, 2H, OH + NH), 2.63 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.40 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.14 (m, 10H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.76 (t, J = 6.5 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3). 13C NMR (75 MHz, DMSO-d6) δ 181.67, 180.95, 157.36, 147.68, 135.30, 133.25, 132.73, 130.13, 125.98, 125.88, 109.37, 31.41, 29.44, 28.89, 28.77, 27.80, 25.56, 22.28, 14.14. HRMS (APCI): (M + H)+ Calcd for C19H24N2O4: 344.17361; found 344.17398.


### 2.2.8. 2-(Hydroxyamino)-1,4-naphthoquinone19

Prepared from10 (328 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 72% yield (134.3 mg, 0.71 mmol) as orange solid mp. 142–143°C. IR (KBr) νmax cm−1: 3467, 3417, 1638, 1618; 1H-NMR (300 MHz, DMSO-d6): δ 7.92 (ddd, 2H, J = 13.9, 7.6, 1.4 Hz, arom), 7.80 (td, 1H, J = 7.5, 1.1 Hz, arom), 7.70 (td, 1H, J = 7.4, 1.5 Hz, arom), 7.22 (br s, 2H, OH + NH), 5.81 (s, 1H, 2-H). 13C NMR (75 MHz, DMSO-d6): δ 182.0, 181.8, 150.5, 134.7, 133.2, 132.2, 130.4, 125.8, 125.3, 102.3. HRMS (APCI): (M + H)+ Calcd for C10H7NO3: 189.04259; found 189.04899.


## 2.2.1. 2-(Hydroxyamino)-3-(hydroxyimino)methyl-1,4-naphthoquinone12

Prepared from2 (200 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 58% yield (140.3 mg, 0.57 mmol) as orange solid mp: 141–142°C; IR (KBr) νmax cm−1: 3467, 3416, 1638, 1618; 1H-NMR (300 MHz, DMSO-d6): δ 11.16 (s, 1H, OH), 7.96 (d, 2H, J = 7.7 Hz, 2H, arom), 7.82 (t, 1H, J = 7.5 Hz, arom), 7.72 (t, 1H, J = 7.4 Hz, arom), 7.56 (br s, 2H, OH + NH), 2.09 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.5, 180.7, 153.9, 147.0, 135.0, 133.3, 132.5, 130.0, 125.8, 125.6, 109.5, 15.5. HRMS (APCI): (M + H)+ Calcd for C12H10N2O4: 246.06406; found 246.07111.


## 2.2.2. 2-(Hydroxyamino)-3-(hydroxyimino)propyl-1,4-naphthoquinone13

Prepared from3 (228 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 60% yield (161.8 mg, 0.59 mmol) as orange solid mp: 148–149°C. IR (KBr) νmax cm−1: 3473 (OH), 3413 (NH), 1638 (C=O), 1618 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.07 (s, 1H, OH), 7.96 (d, 2H, J = 8.8 Hz, H-arom), 7.83 (t, 1H, J = 8.1 Hz, H-arom), 7.73 (t, 1H, J = 8.1 Hz, H-arom), 7.55 (br s, 2H, OH + NH), 2.63 (dd, J = 8.8, 6.7 Hz, 2H, HO-N=C-CH2-CH2-CH3), 1.46 (h, 2H, J = 7.3 Hz, HO-N=C-CH2-CH2-CH3), 0.84 (t, 3H, J = 7.4 Hz,, HO-N=C-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.49, 180.69, 157.01, 147.53, 135.03, 133.13, 132.46, 129.99, 125.79, 125.66, 109.12, 29.89, 19.03, 14.43. HRMS (APCI): (M + H)+ Calcd for C14H14N2O4: 274.09536; found 274.09844.


## 2.2.3. 2-(Hydroxyamino)-3-(hydroxyimino)butyl-1,4-naphthoquinone14

Prepared from4 (244 mg, 1.0 mmol) and hydroxylamine (2-equiv.), isolated into 58%, yield (167.2 mg, 0.58 mmol) as orange solid mp: 157–158°C. IR (KBr) νmax cm−1: 3452 (OH), 3450 (NH), 1638 (C=O), 1619 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.06 (s, 1H, OH), 7.96 (d, 2H, J = 8.9 Hz, H-arom), 7.83 (t, 1H, J = 7.7 Hz, H-arom), 7.73 (t, 1H, J = 7.4 Hz, H-arom), 7.53 (br s, 2H, OH + NH), 2.65 (m, 2H, HO-N=C-CH2-CH2-CH2-CH3), 1.41 (q, 2H, J = 7.3 Hz, HO-N=C-CH2-CH2-CH2-CH3), 1.24 (dq, 2H, J = 14.4, 7.1 Hz, HO-N=C-CH2-CH2-CH2-CH3), 0.82 (t, 3H, J = 7.2 Hz, HO-N=C-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.51, 180.69, 157.10, 147.55, 135.05, 133.12, 132.48, 130.00, 125.80, 125.68, 109.14, 27.72, 27.60, 22.64, 13.91. HRMS (APCI): (M + H)+ Calcd for C15H16N2O4: 288.1110; found 288.1010.


## 2.2.4. 2-(Hydroxyamino)-3-(hydroxyimino)pentyl-1,4-naphthoquinone15

Prepared from5 (258 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 65% yield (193.5 mg, 0.64 mmol) as orange solid mp: 160–161°C. IR (KBr) νmax cm−1: 3473 (OH), 3413 (NH), 1638 (C=O), 1617 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.06 (s, 1H, OH), 7.96 (d, 2H, J = 7.7 Hz, H-arom), 7.83 (t, 1H, J = 7.5 Hz, H-arom), 7.73 (t, 1H, J = 7.5 Hz, H-arom), 7.53 (br s, 2H, OH + NH), 2.64 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH3), 1.44 (q, 2H, J = 6.9 Hz, HO-N=C-CH2-CH2-CH2-CH2-CH3), 1.22 (m, 4H, HO-N=C-CH2-CH2-CH2-CH2-CH3), 0.80 (t, 3H, J = 6.6 Hz, HO-N=C-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.51, 180.71, 157.17, 147.57, 135.08, 133.14, 132.50, 130.00, 125.82, 125.70, 109.12, 31.68, 27.78, 25.15, 21.96, 13.97. HRMS (APCI): (M + H)+ Calcd for C16H18N2O4: 302.12666; found 302.12123.


## 2.2.5. 2-(Hydroxyamino)-3-(hydroxyimino)hexyl-1,4-naphthoquinone16

Prepared from6 (272 mg, 1.0 mmol) and hydroxylamine (2-equiv.), isolated in 55% yield (174.1 mg, 0.55 mmol) as orange solid mp. 162–163°C. IR (KBr) νmax cm−1: 3473 (OH), 3414 (NH), 1638 (C=O), 1619 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.05 (s, 1H, OH), 7.96 (d, 2H, J = 8.8 Hz, H-arom), 7.82 (td, 1H, J = 7.7; 1.1 Hz, H-arom), 7.72 (td, 1H, J = 7.7; 1.1 Hz, H-arom), 7.54 (br s, 2H, OH + NH), 2.64 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 1.41 (q, J = 7.4, 6.9 Hz, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 1.18 (m, 6H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3), 0.78 (t, J = 6.6 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6): δ 181.50, 180.69, 157.17, 147.54, 135.05, 133.13, 132.48, 129.99, 125.80, 125.67, 109.14, 31.09, 29.09, 27.78, 25.45, 22.07, 13.97. HRMS (APCI): (M + H)+ Calcd for C17H20N2O4: 316.14231; found 316.14799.


## 2.2.6. 2-(Hydroxyamino)-3-(hydroxyimino)heptyl-1,4-naphthoquinone17

Prepared from7 (287 mg, 1.0 mmol) and hydroxylamine (2-equiv.). Isolated in 72% yield (234.6 mg, 0.71 mmol) as orange solid mp. 165–166°C. IR (KBr) νmax cm−1: 3470 (OH), 3416 (NH), 1638 (C=O), 1618 (C=O); 1H-NMR (300 MHz, DMSO-d6): δ 11.11 (s, 1H, OH), 7.95 (d, 2H, J = 7.9 Hz, H-arom), 7.81 (td, 1H, J = 7.6, 1.3 Hz, H-arom), 7.71 (td, 1H, J = 7.5, 1.4 Hz, H-arom), 7.48 (br s, 2H, OH + NH), 2.63 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.40 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.15 (m, 8H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.75 (t, J = 6.8 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH3); 13C NMR (75 MHz, DMSO-d6) δ 181.66, 180.93, 157.35, 147.67, 135.27, 133.24, 132.69, 130.12, 125.96, 125.86, 109.35, 31.38, 29.46, 28.64, 27.86, 25.60, 22.25, 14.13; HRMS (APCI): (M + H)+ Calcd for C18H22N2O4: 330.15796; found 330.16101.


## 2.2.7. 2-(Hydroxyamino)-3-(hydroxyimino)octyl-1,4-naphthoquinone18

Prepared from8 (300 mg, 0.99 mmol) and hydroxylamine (2-equiv.) to yield quinone 18 (192.8 mg, 0.56 mmol, 57%), orange solid mp. 169–170°C. IR (KBr) νmax cm−1: 3472 (OH), 3416 (NH), 1638 (C=O), 1618 (C=O). 1H-NMR (300 MHz, DMSO-d6): δ 11.11 (s, 1H, OH), 7.95 (d, 2H, J = 7.7 Hz, H-arom), 7.82 (td, 1H, J = 7.6, 1.1 Hz, H-arom), 7.72 (td, 1H, J = 7.5, 1.2 Hz, H-arom), 7.47 (br s, 2H, OH + NH), 2.63 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.40 (m, 2H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 1.14 (m, 10H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.76 (t, J = 6.5 Hz, 3H, HO-N=C-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3). 13C NMR (75 MHz, DMSO-d6) δ 181.67, 180.95, 157.36, 147.68, 135.30, 133.25, 132.73, 130.13, 125.98, 125.88, 109.37, 31.41, 29.44, 28.89, 28.77, 27.80, 25.56, 22.28, 14.14. HRMS (APCI): (M + H)+ Calcd for C19H24N2O4: 344.17361; found 344.17398.


## 2.2.8. 2-(Hydroxyamino)-1,4-naphthoquinone19

Prepared from10 (328 mg, 0.99 mmol) and hydroxylamine (2-equiv.), isolated in 72% yield (134.3 mg, 0.71 mmol) as orange solid mp. 142–143°C. IR (KBr) νmax cm−1: 3467, 3417, 1638, 1618; 1H-NMR (300 MHz, DMSO-d6): δ 7.92 (ddd, 2H, J = 13.9, 7.6, 1.4 Hz, arom), 7.80 (td, 1H, J = 7.5, 1.1 Hz, arom), 7.70 (td, 1H, J = 7.4, 1.5 Hz, arom), 7.22 (br s, 2H, OH + NH), 5.81 (s, 1H, 2-H). 13C NMR (75 MHz, DMSO-d6): δ 182.0, 181.8, 150.5, 134.7, 133.2, 132.2, 130.4, 125.8, 125.3, 102.3. HRMS (APCI): (M + H)+ Calcd for C10H7NO3: 189.04259; found 189.04899.


## 2.3. 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 105 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% CO2 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.


## 2.4. 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 IC50 values were calculated using the GraphPad Prism software (San Diego, CA, USA).


## 3. Results and Discussion

Since the synthesis of acetylhydroquinone oximeII from hydroquinone I (R = CH3) 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].Scheme 1
Synthesis of 2-acetylnaphthohydroquinones2–11.In this preliminary assay, acetylnaphthohydroquinone2 was reacted with NH2OH in boiling methanol (Scheme 2). Workup of the reaction mixture provides an orange crystalline product, m.p. 141–142°C. The 1H-NMR spectrum displays, at 11.16 ppm, a singlet (D2O exchangeable) for the hydroxyl proton of a ketoxime group [23]. The characteristic A2B2 pattern signals for the aromatic protons of the 1,4-naphthoquinone system appeared at 7.82 and 7.72 ppm. At ca. 7.56 ppm, there was a broad singlet signal for two protons (D2O exchangeable) assignable to the NHOH group and a singlet signal at 2.09 ppm for the proton of a methyl group. The 13C 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.Scheme 2
Reaction of 2-acetylhydroquinone2 with NH2OH.This interesting and unexpected reaction of2 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 C3-C11 linear aliphatic aldehydes according to Scheme 1.The synthesized 2-acylnaphthohydroquinones3–11 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 1H and 13C nuclear magnetic resonance (NMR), bidimensional nuclear magnetic resonance (2D-NMR), and high-resolution mass spectrometry (HRMS).Scheme 3
Reaction of compounds3–11 with hydroxylamine.Interestingly, the results reveal that the reaction of compounds3–11 with hydroxylamine proceed, as that described for analogue 2, to yield the respective 2-(hydroxyamino)-3-(hydroxyimino)alkyl-1,4-naphthoquinones 13–18. Nevertheless, the reaction of compounds 9–11, with hydroxylamine, holding longer hydrocarbon chains than analogue 8, takes place in a quite different manner to give, in all the cases, the same product 2-(hydroxyamino)-1,4-naphthoquinone 19.A tentative explanation was proposed for the reaction of compounds2–8 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 2-hydroxy 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.Scheme 4
Plausible course reaction of2 with NH2OH to 12.Regarding the formation of aminonaphthoquinone19 from compounds 9–11 and hydroxylamine, a tentative course reaction is outlined in Scheme 5, starting from compound 10. The observed C-C cleavage is likely taking place through a retroaldol-type reaction, releasing the internal energy of the sterically crowded hemiaminal intermediate, giving rise to aminoquinone 19, along with hydroxamic acid C10H21CONHOH. The last step (1–19) of this reaction mechanism was suggested to occur through an oxidative amination of intermediate naphthoquinone 1 with hydroxylamine. Such an assumption was confirmed through an experiment where naphthoquinone I was reacted with hydroxylamine, in methanol at room temperature, to give aminoquinone 19 in 88% yield.Scheme 5
Plausible course reaction of10 with NH2OH to 19.Taking into account that the reaction of compounds9–11 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 compounds12–19 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).Table 1
In vitro cytotoxic activity of 12–19 on DU-145 (prostate), MCF-7 (breast), T-24 (bladder) cancer cell lines, and HEK-293 human embryonic kidney cells (nontumor fibroblasts).
IC50 ± SEM (μM)aNo.DU-145MCF-7T-24HEK-2931224.92 ± 0.0326.30 ± 0.3418.47 ± 0.8032.38 ± 1.651332.37 ± 1.2748.55 ± 3.2530.24 ± 0.9048.57 ± 1.921484.25 ± 1.6658.60 ± 6.1835.30 ± 3.4033.32 ± 3.711565.62 ± 4.5091.02 ± 3.6862.45 ± 3.3881.39 ± 2.1816>100>10069.52 ± 6.50>1001760.30 ± 3.8990.01 ± 2.2686.06 ± 1.1962.92 ± 0.6818>100>10070.03 ± 6.57>100198.08 ± 0.4213.59 ± 1.0315.89 ± 3.0431.53 ± 7.94Db0.93 ± 0.060.33 ± 0.050.65 ± 0.074.27 ± 0.34Cells 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 formazan crystals were solubilized by adding 100µl DMSO/well, and the optical density of colored solutions was subsequently read at 550 nm. Results are expressed as % of MTT reduction compared to untreated control conditions. The IC50 values were calculated using the GraphPad Prism software (San Diego, CA, USA). In table, adata represent the mean average values ± SEM for three separate experiments. bD, doxorubicin.Table1 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 IC50 values are in agreement with those recently reported in the literature [28–30]. 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.


## 4. Conclusions

In summary, we report a novel and straightforward route to antiproliferative 2-(hydroxyamino)-3-(hydroxyimino)alkyl-1,4-naphthoquinones12–18 and 2-(hydroxyamino)-1,4-naphthoquinone 19 from 2-alkanoylnaphthohydroquinones 2–11 and hydroxylamine. The study demonstrates that the access to 2,3-disubstituted 1,4-naphthoquinones depends on the length of the linear alkyl substituent (Cn) of the hydroquinone substrates since over C8, the reaction of 2-alkanoylnaphthohydroquinones such as 9–11 with hydroxylamine yields 2-(hydroxyamino)-1,4-naphthoquinone 19. This compound has moderate anticancer activity. Studies on suitable methods to prepare 2-(hydroxyimino)alkyl-naphthohydroquinones are in progress.



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*Source: 7664037-2022-08-23.xml*