Synthesis and Cytotoxic Evaluation of N-Alkyl-2-halophenazin-1-ones

Abstract

In this study, the synthesis of N-alkyl-2-halophenazin-1-ones has been established. Six N-alkyl-2-halophenazin-1-ones, including WS-9659 B and marinocyanins A and B, were synthesized by the direct oxidative condensation of 4-halo-1,2,3-benzenetriol with the corresponding N-alkylbenzene-1,2-diamines. One of the most significant features of the present method is that it can be successfully applied to the synthesis of N-alkyl-2-chlorophenazin-1-ones. The traditional chlorination of N-alkyl-phenazin-1-ones with N-chlorosuccinimide selectively occurs at the 4-position to afford the undesired N-alkyl-4-chlorophenazin-1-ones. Our synthetic route successfully circumvents this problem, culminating in the first chemical synthesis of WS-9659 B. The cytotoxicity of six N-alkyl-2-halophenazin-1-ones and three N-alkylphenazin-1-ones against human promyelocytic leukemia HL-60, human lung cancer A549, and normal MRC-5 cells was evaluated. Among the compounds tested in this study, 2-chloropyocyanin possesses significant selectivity toward A549 cells. The cytotoxic evaluation provides structural insights into the potency and selectivity of these compounds for cancer cells.

Introduction

Phenazines are a large group of nitrogen-containing heterocycles. To date, more than 100 phenazine derivatives have been identified in nature and over 6000 phenazine compounds synthesized.¹⁻⁴N-Alkylphenazin-1-ones are a minor group of the phenazines. To the best of our knowledge, only four N-alkylphenazin-1-ones [pyocyanin,⁵⁻¹⁰ lavanducyanin (WS-9659 A),¹¹⁻¹⁵ phenazinomycin,^16,17 and 1-hydroxy-7-oxolavanducyanin¹⁸ and seven N-alkyl-2-halophenazin-1-ones (WS-9659 B¹²⁻¹⁴ and marinocyanins A-F^15,19) have been identified as natural products. In the present study, we focused on N-alkylphenazin-1-ones and N-alkyl-2-halophenazin-1-ones, which are depicted in Figure 1. Among them, pyocyanin (1a), lavanducyanin (WS-9659 A) (2a), WS-9659 B (2c), and marinocyanins A (2b) and B (3b) are natural products, which were isolated from Pseudomonas and Streptomyces species (Figure 1).^3,4 One of the simplest and best-known N-alkylphenazin-1-ones is pyocyanin (1a) isolated from Pseudomonas aeruginosa.^5,6 Pyocyanin shows several biological activities including antibacterial, antifungal, and cytotoxic activities.⁵ This compound plays an important role in biofilm formation and quorum sensing in P. aeruginosa.⁶⁻¹⁰ Lavanducyanin (2a), isolated from the Streptomyces sp., shows cytotoxic activities against mouse leukemia P388 and mouse lymphoid leukemia L1210 cells with half-maximal inhibitory concentration (IC₅₀) values of 0.09 and 0.10 μg/mL, respectively.¹¹ The same compound and its 2-chlorinated derivative, respectively, named as WS-9659 A (2a) and WS-9659 B (2c), were isolated from the Streptomyces sp. as testosterone 5α-reductase inhibitors with IC₅₀ values of 0.5 and 10 μM, respectively.¹²⁻¹⁴ Marinocyanins A (2b) and B (3b), 2-brominated derivatives, were isolated from a marine-derived Streptomyces sp. and actinomycete strains CNS-284 and CNY-960.^15,19 Compounds 2b and 3b inhibited TNF-α-induced NFκB activity (IC₅₀ values of 4.1 and 24.2 μM, respectively) and LPS-induced nitric oxide production (IC₅₀ values of >48.6 and 15.1 μM, respectively).¹⁵ They blocked PGE₂ production with greater efficacy (IC₅₀ values of 7.5 and 0.89 μM, respectively).¹⁵ Compounds 2b and 3b also showed strong cytotoxic activity against human colon carcinoma HCT116 cells with IC₅₀ values of 0.049 and 0.029 μM, respectively.¹⁹ In addition, both these compounds display modest antimicrobial activity against Staphylococcus aureus and the pathogenic yeast Candida albicans.¹⁹

Figure 1.

Structures of our target N-alkylphenazin-1-ones.

Because of these diverse biological activities, N-alkylphenazin-1-ones are attractive synthetic targets.^3,4,20−22 Our group recently reported the synthesis of 1a, 2a, 2b, 3a, and 3b.²² The oxidative condensation of pyrogallol with N-alkylbenzene-1,2-diamines (4, 5, and 6) under an oxygen atmosphere in 2-propanol afforded 1a, 2a, and 3a, respectively (Scheme 1). Bromination of the resultant phenazin-1-ones (2a and 3a) proceeded at the 2-position in a regioselective manner to afford 2b and 3b, respectively.

Scheme 1. Syntheses of Pyocyanin (1a), Lavanducyanin (2a), and Marinocyanins A (2b) and B (3b).

In the present study, we describe the synthesis of N-alkyl-2-halophenazin-1-ones by coupling 4-halo-1,2,3-benzenetriol with the corresponding N-alkylbenzene-1,2-diamines. The first synthesis of WS-9659 B (2c) was achieved by oxidative condensation of 4-chloro-1,2,3-benzenetriol with the corresponding N-alkylbenzene-1,2-diamine. Furthermore, the cytotoxic activities of synthetic N-alkylphenazin-1-ones and N-alkyl-2-halophenazin-1-ones were evaluated.

Results and Discussion

Halogenation of Pyocyanin and Lavanducyanin

To obtain 2-chlorinated N-alkylphenazin-1-ones, halogenation of pyocyanin (1a) was examined. A drastic change in the regioselectivity between bromination and chlorination was observed (Scheme 2). Bromination of 1a with N-bromosuccinimide (NBS) in CH₂Cl₂ afforded 2-bromopyocyanin (1b) in 51% yield. However, chlorination of 1a with N-chlorosuccinimide (NCS) gave 4-chloropyocyanin (7) in 65% yield. The position of the chlorine atom in 7 was confirmed by nuclear Overhauser effect spectroscopy (NOESY) correlations. NOESY correlations between H-2 (δ_H 6.47) and H-3 (δ_H 7.44) and between the H-6 (δ_H 7.40) and N-methyl group (δ_H 4.07) are observed (Figures 2, S32 and S33 in the Supporting Information). The difference in outcome can presumably be attributed to steric hindrance between the N-methyl group in 1a and the halogen atom. The van der Waals radius of a chlorine atom (1.75 Å) is smaller than that of a bromine atom (1.85 Å).^23,24 Consequently, electrophilic substitution with the sterically large bromine atom at the 4-postion is disfavored. The chlorination of lavanducyanin (2a) with NCS also proceeded at the 4-position in a regioselective manner to give 8 in 48% yield (Scheme 3). The position of the chlorine atom in 8 was also confirmed by NOESY correlations. NOESY correlations between H-2 (δ_H 6.49) and H-3 (δ_H 7.46) and between the H-6 (δ_H 7.27–7.25) and N-methylene group (δ_H 5.29) are observed (Figures 2 and S39 in the Supporting Information). Thus, an alternative methodology for the synthesis of WS-9659 B (2c) is needed.

Scheme 2. Selective Halogenation of Pyocyanin with NBS and NCS.

Figure 2.

Selected NOESY correlations observed in 7 and 8.

Scheme 3. Chlorination of Lavanducyanin (2a).

Oxidative Condensation of 4-Halo-1,2,3-benzenetriol with N-Alkylbenzene-1,2-diamines

To obtain N-alkyl-2-chlorophenazin-1-ones, reaction conditions for the oxidative condensation between the N-methylbenzene-1,2-diamine (4)²² and 4-chloro-1,2,3-benzenetriol (9)²⁵ were examined (Table 1). When 4 and 9 were reacted under an oxygen atmosphere, the desired 2-chloropyocyanin (1c) was obtained in 12% yield (entry 1). NOESY correlations between the H-4 (δ_H 6.27) and N-methyl group (δ_H 4.04) and between the H-6 (δ_H 8.10) and N-methyl group in 1c indicate the presence of the chlorine atom at the 2-position (Figures 3 and S11 in the Supporting Information). The use of 1,4-benzoquinone as an oxidant gave 1c in 10% yield (entry 2). When o-chloranil was used in this reaction, only a trace amount of 1c was obtained. These results indicate that molecular oxygen is the optimal oxidant. The addition of bases to the oxidative coupling with molecular oxygen was also examined (entries 4–6). The yield of 1c was decreased by addition of Na₂CO₃ or K₂CO₃ (entries 4 and 5, respectively) but slightly increased by addition of Cs₂CO₃ (entry 6). Other reaction conditions including the base, solvent, and temperature were also investigated but the yield of 1c was not improved. Thus, the entry 6 gave the optimal conditions.

Table 1. Oxidative Condensation of 4-Chloro-1,2,3-benzenetriol (9) with N-Methylbenzene-1,2-diamine (4)^a.

entry	oxidant	additive	yield (%)b
1c	O2		12
2d	1,4-benzoquinone		10
3d	o-chloranil		trace
4e	O2	Na2CO3	5
5e	O2	K2CO3	10
6e	O2	Cs2CO3	17

^aUnless otherwise noted, reactions were performed using 4 (0.246 mmol) and 9 (1.0 equiv) in 2-propanol (2.0 mL).

^bIsolated yield.

^cThe reaction was performed at room temperature (rt) under an oxygen atmosphere.

^dThe reaction was performed at 0 °C under an argon atmosphere using the oxidant (3.0 equiv) indicated.

^eThe reaction was performed at rt under an oxygen atmosphere using the additive (1.0 equiv) indicated.

Figure 3.

Selected NOESY correlations observed in 1c.

Using optimal conditions, several N-alkyl-2-halophenazin-1-ones were prepared (Table 2). The first synthesis of WS-5659 B (2c) was achieved by coupling between 5 and 9 (entry 1). The ¹H and ¹³C NMR spectroscopic data were identical to those reported for natural 2c.¹² Oxidative coupling of 9 with 6 gave 3c in 21% yield (entry 2). The desired oxidative coupling also proceeded using 4-bromo-1,2,3-benzenetriol (10)²⁶ as a coupling partner to give N-alkyl-2-bromophenazin-1-ones. The coupling between 4 and 10 afforded 2-bromopyocyanin (1b) in 18% yield (entry 3). Marinocyanins A (2b) and B (3b) were synthesized by oxidative coupling of 10 with 5 and 6, respectively (entries 4 and 5).

Table 2. Oxidative Condensation of 4-Halo-1,2,3-benzenetriols with N-Alkylbenzene-1,2-diamine^a.

entry	1,2-diamine	4-halo-1,2,3-benzenetriols	product	yield (%)b
1c	5	9	2c	20
2d	6	9	3c	21
3d	4	10	1b	18
4c	5	10	2b	10
5d	6	10	3b	15

^aUnless otherwise noted, reactions were performed at rt, with O₂ bubbling or under an oxygen atmosphere, using N-alkylbenzene-1,2-diamine (30.0 mg) and 4-halo-1,2,3-benzenetriols (1.0 equiv) in the presence of Cs₂CO₃ (1.0 equiv).

^bIsolated yield.

^cThe reaction was performed with oxygen bubbling.

^dThe reaction was performed under an oxygen atmosphere.

Evaluation of Cytotoxic Activity against Human Cancer Cells and Normal Cells

Initially, the cytotoxic activity of 1a, 1b, and 1c against human promyelocytic leukemia HL-60 cells, human lung adenocarcinoma A549 cells, and human normal lung fibroblast MRC-5cells was evaluated using the WST-8 method.²⁷ The concentrations of the compound required to reduce cell viability by 50% (IC₅₀) are summarized in Table 3. Table 3 also shows the values of the selectivity index (SI),²⁸⁻³⁰ which is defined as the ratio of the IC₅₀ value against normal cells to the corresponding IC₅₀ value against cancer cells of similar tissue. Because both A549 and MRC-5 are derived from the lung, the SI value in Table 3 is defined as the IC₅₀ value against normal MRC-5 cells divided by the IC₅₀ value against A549 cells. Generally, the compounds with a SI value higher than 2 are considered to give selective cytotoxicity against cancer cells.²⁸ SN-38, the active metabolite of irinotecan, was used as a positive control.³¹ The cytotoxicity against normal MRC-5 cells of 1a was less-potent than that against leukemia HL-60 cells and cancer A549 cells. The SI value of 1a (SI = 3.95) indicates a significant selectivity toward cancer cells. However, the cytotoxicity of 1b against normal MRC-5 cells was almost the same as that against leukemia HL-60 cells and cancer A549 cells. Among compounds 1a–c, the compound 1c exhibited the most potent and selective cytotoxicity against cancer A549 cells. At least 5.8-fold and 4.6-fold higher concentrations of 1c are required to induce cytotoxicity against MRC-5 cells, as compared to those required against A549 and HL-60 cells, respectively. The differential cytotoxicity of 1c was highly significant (SI = 5.80), suggesting that 1c is considered as a candidate for the development of chemotherapeutic agents. Pyocyanin (1a) has been reported to diffuse through the cell membrane and increase the intracellular ROS and Ca²⁺ levels.³² The presence of the chlorine atom at the 2-position in 1a may enhance the generation of ROSs or increase the chemical reactivity or binding affinity with the target molecules in A549 cells.

Table 3. Cytotoxic Activity of 1a–1c against Human Cancer Cells and Normal Cells (IC₅₀, μM) and Their Selective Indexes^a.

	IC50 (μM)
compounds	HL-60	A549	MRC-5	SIc
1a	5.82 ± 0.133	5.52 ± 0.280	21.8 ± 2.550	3.95
1b	3.07 ± 0.080	2.59 ± 0.056	3.36 ± 0.204	1.30
1c	3.56 ± 0.052	0.76 ± 0.065	4.41 ± 0.069	5.80
SN-38b	<0.05	0.038 ± 0.003	<0.1

^aThe concentration of the compound needed to reduce cell viability by 50% (IC₅₀) was determined using the WST-8 assay. IC₅₀ values are expressed as the mean ± SD of triplicate experiments.

^bSN-38 was used as a positive control.

^cThe SI is defined as the IC₅₀ value against normal MRC-5 cells divided by the IC₅₀ value against A549 cells.

Next, the cytotoxic activity of 2a–c and 3a–c against HL-60, A549, and MRC-5 cells was evaluated (Table 4). Although these compounds showed cytotoxicity against HL-60 cells, compounds 3a and 3c displayed weaker activity than the other compounds. Compounds 2a–c and 3a–c exhibited cytotoxicity against A549 and MRC-5 cells at concentrations lower than 1 μM. Unfortunately, no significant difference in the IC₅₀ values between these compounds was observed. The SI values of 2a–c and 3a–c are lower than 2. Selectivity against A549 cells was not observed. The displacement of the methyl group with the longer alkyl groups at the nitrogen atom in 1a and 1c decreased the selective cytotoxicity toward A549 cells.

Table 4. Cytotoxic Activity of 2 and 3 against Human Cancer Cells and Normal Cells (IC₅₀, μM) and Their Selective Indexes^a.

	IC50 (μM)
compounds	HL-60	A549	MRC-5	SIc
2a	0.36 ± 0.023	0.38 ± 0.003	0.68 ± 0.013	1.79
2b	0.27 ± 0.017	0.73 ± 0.015	0.60 ± 0.008	0.82
2c	0.23 ± 0.013	0.75 ± 0.006	0.37 ± 0.019	0.49
3a	2.59 ± 0.195	0.88 ± 0.023	0.87 ± 0.063	0.99
3b	0.67 ± 0.038	0.33 ± 0.010	0.54 ± 0.009	1.64
3c	2.20 ± 0.070	0.66 ± 0.016	0.66 ± 0.041	1.00
SN-38b	0.056 ± 0.002	0.052 ± 0.003	0.092 ± 0.001	1.77

^aThe concentration of the compound needed to reduce cell viability by 50% (IC₅₀) was determined using the WST-8 assay. IC₅₀ values are expressed as the mean ± SD of triplicate experiments.

^bSN-38 was used as a positive control.

^cThe SI is defined as the IC₅₀ value against normal MRC-5 cells divided by the IC₅₀ value against A549 cells.

Conclusions

In this study, the synthesis of N-alkyl-2-halophenazin-1-one was established. Six N-alkyl-2-halophenazin-1-ones, including WS-9659 B (2c) and marinocyanins A (2b) and B (3b), were synthesized by the oxidative condensation of 4-halo-1,2,3-benzenetriols with N-alkylbenzene-1,2-diamines. The present strategy enables the highly convergent and selective synthesis of N-alkyl-2-halophenazin-1-ones. In addition, the cytotoxicity of compounds 1a–c, 2a–c, and 3a–c against human leukemia HL-60 cells, cancer A549 cells, and normal MRC-5 cells was evaluated. Among the compounds tested in this study, 2-chloropyocyanin (1c) exhibited the most selective cytotoxicity against A549 cells. The high SI value of 1c (SI = 5.80) suggests that 1c is considered as a potential candidate for the development of chemotherapeutic agents. Halogenated phenazines have recently attracted increasing attention as a promising class of biofilm-eradicating agents that effectively target multiple persistent bacterial phenotypes.^33,34 Thus, halogenation of phenazines and phenazinones is one of the valuable approaches for improving their biological activities.³⁵ The present study will be invaluable not only for analyzing the molecular and cellular biological activities of N-alkylphenazin-1-ones and their halogenated derivatives but also in the design of more potent and selective cytotoxic derivatives against cancer cells. Additional biological studies to investigate the mechanism of action of these compounds are currently underway.

Experimental Section

General Information

All reactions sensitive to air or moisture were carried out under an argon atmosphere under anhydrous conditions, unless otherwise noted. Solvents and reagents were used without further purification unless otherwise noted. Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F₂₅₄ plates (0.25 mm, normal phase, Merck) and silica gel 60 RP-18 F_254S plates (0.25 mm, reversed phase, Merck). Normal-phase flash column chromatography was performed using silica gel 60 (particle size 40–63 μm; 230–400 mesh ASTM; SilicaFlash F60, SiliCycle Inc.). Reversed-phase flash column chromatography was performed using octadecylsilyl silica gel (C18, particle size 15–30 μm, FUJIFILM Wako Pure Chemical Co.). Melting point (M_p) data were determined using a Shimadzu MM-2 instrument and were uncorrected. IR spectra were recorded on a Horiba FT-720 spectrometer, using KBr pellets (solid). ¹H and proton-decoupled ¹³C (¹³C{1H}) NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 and 100 MHz, respectively), using chloroform-d (CDCl₃), dimethyl sulfoxide-d₆ (DMSO-d₆), and methanol-d₄ (CD₃OD) as the solvent. Chemical shift values are expressed in δ (ppm) relative to tetramethylsilane (TMS, δ 0.00 ppm) or the residual solvent resonance (CDCl₃, δ 77.0 ppm for ¹³C NMR; DMSO-d₆, δ 2.49 ppm for ¹H NMR and δ 39.7 ppm for ¹³C NMR; and CD₃OD, δ 3.30 ppm for ¹H NMR and δ 49.0 ppm for ¹³C NMR). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, and m = multiplet), coupling constants (J; Hz), and integration. Mass spectra were obtained using a Sciex X500R quadrupole time-of-flight (QTOF) high-resolution mass spectrometer using electrospray ionization (ESI). High purity levels of all synthetic compounds were verified using ¹H NMR spectra (see the Supporting Information). A549 cells were purchased from the Riken Cell Bank. HL-60 and MRC-5 cells were purchased from the National Institute of Biomedical Innovation, Health and Nutrition.

2-Bromo-5-methylphenazin-1(5H)-one (1b)

NBS (9.3 mg, 52.3 μmol) was added to a solution of 1a(22) (10.6 mg, 50.4 μmol) in CH₂Cl₂ (5.0 mL). The mixture was stirred at 0 °C for 1 h. The reaction was quenched by the addition of a saturated aqueous NaHCO₃ solution. The mixture was diluted with CHCl₃. After the layers were separated, the organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated to give a residue. The residue was purified by reversed-phase flash chromatography (MeOH/H₂O = 4/1) to give 1b (7.5 mg, 51%) as a dark-blue solid. Reversed-phase TLC (MeOH/H₂O = 4/1) Rf 0.48; mp 129–130 °C; IR (KBr) ν_max: 2958, 2914, 2848, 1724, 1620, 1560, 1493 cm^–1; ¹H NMR (400 MHz, DMSO-d₆): δ 8.24 (d, J = 8.1 Hz, 1H), 8.13–8.10 (m, 2H), 8.00 (t, J = 7.6 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 6.25 (d, J = 8.6 Hz, 1H), 4.04 (s, 3H); ¹³C{¹H} NMR (100 MHz, DMSO-d₆): δ 171.0, 145.4, 144.5, 136.4, 136.0, 135.2, 133.0, 132.7, 125.8, 116.0, 108.3, 91.9, 35.7; HRMS (ESI/QTOF) m/z: [M + H]⁺ calcd for C₁₃H₁₀⁷⁹BrN₂O, 288.9971; found, 288.9969.

4-Chloro-5-methylphenazin-1(5H)-one (7)

NCS (7.1 mg, 53.2 μmol) was added to a solution of 1a(22) (10.6 mg, 50.4 μmol) in CH₂Cl₂ (5.0 mL). The mixture was stirred at 0 °C for 2 h. The reaction was quenched by the addition of a saturated aqueous NaHCO₃ solution. The mixture was diluted with CHCl₃. After the layers were separated, the organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated to give a residue. The residue was purified by reversed-phase flash chromatography (MeOH/H₂O = 4/1) to give 7 (8.0 mg, 65%) as a dark-blue solid. TLC (CHCl₃/MeOH = 9/1) Rf 0.66; mp 125–127 °C; IR (KBr) ν_max: 3035, 2920, 1728, 1637, 1574, 1514, 1493 cm^–1; ¹H NMR (400 MHz, CDCl₃, TMS): δ 8.17 (dd, J = 8.1, 1.1 Hz, 1H), 7.74–7.70 (m, 1H), 7.44 (d, J = 10.0 Hz, 1H), 7.44–7.40 (m, 1H), 7.40 (d, J = 8.6 Hz, 1H), 6.47 (d, J = 10.0 Hz, 1H), 4.07 (s, 3H); ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 178.7, 146.9, 145.9, 136.0, 135.5, 134.6, 132.8, 132.7, 124.6, 119.1, 113.6, 100.3, 41.3; HRMS (ESI/QTOF) m/z: [M + H]⁺ calcd for C₁₃H₁₀³⁵ClN₂O, 245.0476; found, 245.0478.

4-Chloro-5-{(2,4,4-trimethylcyclohex-1-en-1-yl)methyl}phenazin-1(5H)-one (8)

NCS (4.5 mg, 33.7 μmol) was added to a solution of 2a(22) (10.6 mg, 31.9 μmol) in CH₂Cl₂ (3.0 mL). The mixture was stirred at 0 °C for 1.5 h. The reaction was quenched by the addition of a saturated aqueous NaHCO₃ solution. The mixture was diluted with CHCl₃. After the layers were separated, the organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated to give a residue. The residue was purified by reversed-phase flash chromatography (MeOH/H₂O = 4/1) to give 8 (5.6 mg, 48%) as a dark-blue solid. TLC (CHCl₃/MeOH = 9/1) Rf 0.47; mp 114–116 °C; IR (KBr) ν_max: 2954, 2920, 2850, 1724, 1716, 1643, 1603, 1514, 1469 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.18 (dd, J = 8.0, 1.4 Hz, 1H), 7.65–7.61 (m, 1H), 7.46 (d, J = 10.1 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.27–7.25* (m, 1H), 6.49 (d, J = 10.1 Hz, 1H), 5.29 (s, 2H), 1.81 (br s, 5H), 1.48 (br s, 2H), 1.16 (t, J = 6.4 Hz, 2H), 0.73 (s, 6H), (*this proton signal was assigned based on the HMQC spectrum.); ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 178.8, 146.9, 146.4, 136.1, 134.7, 134.0, 133.0 (2C), 130.3, 124.6, 123.2, 118.9, 114.5, 100.4, 54.2, 46.2, 35.0, 28.9, 27.9 (2C), 23.2, 19.3; HRMS (ESI/QTOF) m/z: [M + H]⁺ calcd for C₂₂H₂₄³⁵ClN₂O, 367.1572; found, 367.1579.

General Procedure for the Coupling between 4 and 9 under an Oxygen Atmosphere (Entries 1, 4, 5, and 6 in Table 1)

A solution of 4 (30.0 mg, 245.6 μmol) and 9 (1.0 equiv) in the absence or presence of the additive (1.0 equiv) indicated in Table 1 in 2-propanol (2.0 mL) was stirred under an O₂ atmosphere at rt in the dark until no further TLC changes were observed. The reaction was quenched by the addition of 1 M aqueous HCl solution. The mixture was diluted with CHCl₃. After the layers were separated, the aqueous layer was neutralized with 4 M aqueous NaOH solution. The aqueous layer was extracted with CHCl₃ twice. The combined organic layer was dried over Na₂SO₄ and concentrated to give a residue. The residue was purified by silica gel column chromatography and reversed-phase flash chromatography.

Following the general procedure, the reaction was performed using 4 (30.0 mg, 245.6 μmol) and 9 (39.8 mg, 247.9 μmol) in the presence of Cs₂CO₃ (81.0 mg, 248.6 μmol) for 4 days to give 1c (10.3 mg, 17%) as a dark-blue solid (entry 6). Reversed-phase TLC (MeOH/H₂O = 4/1) Rf 0.39; mp 124–125 °C; IR (KBr) ν_max: 3043, 2924, 1716, 1622, 1560 cm^–1; H NMR (400 MHz, DMSO-d₆): δ 8.24 (dd, J = 8.2, 1.2 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 8.02–7.98 (m, 1H), 7.96 (d, J = 8.7 Hz, 1H), 7.66 (td, J = 8.2, 1.2 Hz, 1H), 6.27 (d, J = 8.7 Hz, 1H), 4.04 (s, 3H); ¹³C{1H} NMR (100 MHz, DMSO-d₆): δ 170.8, 145.9, 141.2, 136.2, 136.0, 134.6, 133.0, 132.7, 125.8, 118.0, 115.9, 90.7, 35.6; HRMS (ESI/QTOF) m/z: [M + H]⁺ calcd for C₁₃H₁₀³⁵ClN₂O, 245.0476; found, 245.0476.

General Procedure for the Coupling between 4 and 9 Using the Oxidant (Entries 2 and 3 in Table 1)

A solution of 4 (245.6 μmol) and 9 (1.0 equiv) in the presence of the oxidant (3.0 equiv) indicated in Table 1 in 2-propanol (2.0 mL) was stirred at 0 °C until no further TLC changes were observed. The reaction was quenched by the addition of 1 M aqueous HCl solution. The mixture was diluted with CHCl₃. After the layers were separated, the aqueous layer was neutralized with 4 M aqueous NaOH solution. The aqueous layer was extracted with CHCl₃ twice. The combined organic layer was dried over Na₂SO₄ and concentrated to give a residue. The residue was purified by silica gel column chromatography and reversed-phase flash chromatography to yield 1c.

Following the general procedure, the reaction was performed using 4 (30.0 mg, 245.6 μmol), 9 (40.0 mg, 249.1 μmol), and 1,4-benzoquinone (79.6 mg, 736.4 μmol) for 1 h to give 1c (6.1 mg, 10%) (entry 2).

General Procedure of N-{(2,4,4-Trimethylcyclohex-1-en-1-yl)methyl}benzene-1,2-diamine (5) with 4-Halo-1,2,3-benzenetriols (General Procedure A; Entries 1, 4 in Table 2)

A solution of N-alkylbenzene-1,2-diamine (30.0 mg) and 4-halo-1,2,3-benzenetriols (1.0 equiv) in the presence of the Cs₂CO₃ (1.0 equiv) in 2-propanol (1.0 mL) was stirred and bubbled with O₂ from an O₂ balloon at rt in the dark until no further TLC changes were observed. The reaction was quenched by the addition of water. The mixture was diluted with CHCl₃. After the layers were separated, the organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated to give a residue. The residue was purified by silica gel column chromatography and reversed-phase flash chromatography.

Marinocyanin A (2b)

Following the general procedure A, the reaction was performed using 5 (30.0 mg, 122.8 μmol) and 10 (25.2 mg, 122.9 μmol) in the presence of Cs₂CO₃ (40.0 mg, 122.8 μmol) in 2-propanol with O₂ bubbling for 2 days. The residue was purified by silica gel column chromatography (CHCl₃/MeOH = 50/1) and reversed-phase flash chromatography (MeOH/H₂O = 4/1) to give 2b (5.0 mg, 10%) as a dark-blue solid. The structure of 2b was confirmed by comparison of the ¹H and ¹³C{¹H} NMR spectra with those reported for 2b.²²

WS-9659 B (2c)

Following the general procedure A, the reaction was performed using 5 (30.0 mg, 122.8 μmol) and 9 (19.9 mg, 123.9 μmol) in the presence of Cs₂CO₃ (40.2 mg, 123.4 μmol) in 2-propanol with O₂ bubbling for 2 days. The residue was purified by silica gel column chromatography (CHCl₃/MeOH = 50/1) and reversed-phase flash chromatography (MeOH/H₂O = 4/1) to give 2c (8.9 mg, 20%) as a dark-blue solid. Reversed-phase TLC (MeOH/H₂O = 4/1) Rf 0.43; mp 154–157 °C (lit. 152–153 °C);¹² IR (KBr) ν_max: 2918, 2848, 2362, 1699, 1626, 1604, 1562, 1495 cm^–1; ¹H NMR (400 MHz, CDCl₃): δ 8.40 (d, J = 7.8 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.57–7.52 (m, 2H), 5.89 (d, J = 8.4 Hz, 1H), 5.10 (br s, 2H), 1.91 (s, 3H), 1.88 (br s, 2H), 1.53–1.45 (br s 2H), 1.20 (t, J = 6.3 Hz, 2H), 0.83 (s, 6H); ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 172.7, 146.5, 140.9, 136.2, 135.4, 134.1, 133.8, 133.2, 131.5, 125.1, 120.9, 120.3, 113.6, 90.4, 50.1, 46.2, 34.8, 28.9, 28.0 (2C), 22.7, 19.5; HRMS (ESI/QTOF) m/z: [M + H]⁺ calcd for C₂₂H₂₄³⁵ClN₂O, 367.1572; found, 367.1568.

General Procedure of N-Methylbenzene-1,2-diamine (4) or N-(3-Methylbut-2-en-1-yl)benzene-1,2-diamine (6) with 4-Halo-1,2,3-benzenetriols (General Procedure B; Entries 2, 3, and 5 in Table 2)

A solution of N-alkylbenzene-1,2-diamine (30.0 mg) and 4-halo-1,2,3-benzenetriols (1.0 equiv) in the presence of Cs₂CO₃ (1.0 equiv) in 2-propanol was stirred under an O₂ atmosphere at rt in the dark until no further TLC changes were observed. The reaction was quenched by the addition of 1 M aqueous HCl solution. The mixture was diluted with CHCl₃. After the layers were separated, the aqueous layer was neutralized with 4 M aqueous NaOH solution. The aqueous layer was extracted with CHCl₃ twice. The combined organic layer was dried over Na₂SO₄ and concentrated to give a residue. The residue was purified by silica gel column chromatography or reversed-phase flash chromatography.

2-Bromo-5-methylphenazin-1(5H)-one (1b)

Following the general procedure B, the reaction was performed using 4 (30.0 mg, 245.6 μmol) and 10 (50.7 mg, 247.3 μmol) in the presence of Cs₂CO₃ (80.5 mg, 247.1 μmol) in 2-propanol (2.0 mL) under an O₂ atmosphere for 3 days. The residue was purified by reversed-phase flash chromatography (MeOH/H₂O = 2/1) to give 1b (12.7 mg, 18%) as a dark-blue solid.

Marinocyanin B (3b)

Following the general procedure B, the reaction was performed using 6 (30.0 mg, 170.2 μmol) and 10 (35.8 mg, 174.6 μmol) in the presence of Cs₂CO₃ (53.5 mg, 164.2 μmol) in 2-propanol (1.0 mL) under an O₂ atmosphere for 2 days. The residue was purified by silica gel column chromatography (CHCl₃/MeOH = 50/1) and reversed-phase flash chromatography (MeOH/H₂O = 4/1) to give 3b (8.5 mg, 15%) as a dark-blue solid. The structure of 3b was confirmed by comparison of the ¹H and ¹³C{¹H} NMR spectra with those reported for 3b.²²

2-Chloro-5-(3-methylbut-2-en-1-yl)phenazin-1(5H)-one (3c)

Following the general procedure B, the reaction was performed using 6 (30.0 mg, 170.2 μmol) and 9 (27.3 mg, 170.0 μmol) in the presence of Cs₂CO₃ (55.6 mg, 170.6 μmol) in 2-propanol (1.5 mL) under an O₂ atmosphere for 2 days. The residue was purified by reversed-phase flash chromatography (MeOH/H₂O = 2/1) to give 3c (10.6 mg, 21%) as a dark-blue solid. Reversed-phase TLC (MeOH/H₂O = 4/1) Rf 0.38; mp 145–149 °C; IR (KBr) ν_max: 3035, 2958, 2918, 2848, 1724, 1624, 1562, 1496 cm^–1; ¹H NMR (400 MHz, CD₃OD): δ 8.31 (d, J = 7.8 Hz, 1H), 8.07–8.03 (brm, 1H), 7.99–7.93 (m, 2H), 7.72 (t, J = 7.8 Hz, 1H), 6.41 (d, J = 8.7 Hz, 1H), 5.37 (br s, 2H), 5.21–5.18 (br s, 1H), 2.03 (s, 3H), 1.80 (d, J = 0.9 Hz, 3H); ¹³C{¹H} NMR (100 MHz, CD₃OD): δ 171.4, 146.4, 144.3, 140.4, 138.9, 138.1, 134.5, 134.0, 133.5, 128.0, 119.5, 117.3, 116.8, 93.8, 48.7*, 25.6, 18.7 (*this carbon signal was assigned based on the HMQC spectrum.); HRMS (ESI/QTOF) m/z: [M + H]⁺ calcd for C₁₇H₁₆³⁵ClN₂O, 299.0946; found, 299.0942.

Cell Lines and Culturing

A549 cells were cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HL-60 cells were cultured at 37 °C with 5% CO₂ in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS. MRC-5 cells were cultured in Minimum Essential Medium Eagle-alpha modification (α-MEM) without ribonucleosides and deoxyribonucleosides supplemented with 10% FBS.

Cytotoxicity Evaluation

Cell growth was evaluated using cell counting kit-8 according to the manufacturer’s instructions based on the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay.²⁷ For the assay, the adherent A549 and MRC-5 cells were cultured in a 96-well plate with each well containing 2000 cells in a total volume of 100 μL, and HL-60 cells were cultured in a 96-well plate with each well containing 5000 cells in a total volume of 100 μL. The concentration of DMSO in the cell cultures was 0.1–0.2% (v/v). The plates also included blank wells (0 cells) and control wells (2000 cells/100 μL for the adherent cells and 5000 cells/100 μL for HL-60 cells). A549 and MRC-5 cells were preincubated for 24 h before exposure to the test compounds. The plates were incubated with various concentrations of each compound for 72 h. At the end of incubation, 10 μL of the solution provided with the cell counting kit was then added, and the resulting mixture was incubated for 2 h at 37 °C. The absorbance values were then measured at 450 nm with a 96-well plate reader. Cell growth inhibition was evaluated as the ratio of the absorbance of the sample to that of the control.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c04253.

• ¹H, ¹³C{¹H}, and 2D NMR spectra for synthetic compounds (PDF)

Author Present Address

^† Shogo Kamo: Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.

Author Present Address

^‡ Shusuke Tomoshige: Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan.

The authors declare no competing financial interest.