Synthesis of New Quinolinequinone Derivatives and Preliminary Exploration of their Cytotoxic Properties

Abstract

A series of 7-amino- and 7-acetamidoquinoline-5,8-diones with aryl substituents at the 2-position were synthesized, characterized and evaluated as potential NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor agents. The synthesis of lavendamycin analogs is illustrated. Metabolism studies demonstrated that 7-amino-analogues were generally better substrates for NQO1 than 7-amido-analogues as were compounds with smaller heteroaromatic substituents at the C-2 position. Surprisingly, only two compounds, 7-acetamido-2-(8’-quinolinyl)quinoline-5,8-dione (11) and 7-amino-2-(2-pyridinyl)quinoline-5,8-dione (23) showed selective cytotoxicity toward the NQO1-expressing MDA468-NQ16 breast cancer cells versus the NQO1-null MDA468-WT cells. For all other compounds, NQO1 protected against quinoline-5,8-dione cytotoxicity. Compound 22 showed a potent activity against human breast cancer cells expressing or not expressing NQO1 with IC50 values of respectively 190 nM and 140 nM and a low NQO1 mediated reduction rate, which suggests that the mode of action of 22 differs from lavendamycin and involves an unidentified target(s).

INTRODUCTION

Lavendamycin (Figure 1) is a quinolinequinone antibiotic with antitumor activity first isolated from Streptomyces lavendulae by Balitz et al in 1982.^1–2 It is structurally related to Streptonigrin which was first isolated from Streptomyces flocculus.^3–4 Streptonigrin is known for its potent cytotoxic properties, antitumor activity, and in vitro and in vivo antiviral properties and potent, broad spectrum antimicrobial properties. Although lavendamycin is not suitable for clinical use due to its toxicity, its analogs are less toxic and hence have potential as antitumor agents.⁵ Recent findings^6–11 suggest that some indolequinones and quinolinequinones are excellent substrates for the quinone reductase enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), and are selectively cytotoxic to cancer cell lines that overexpress NQO1. NQO1 is a ubiquitous flavoenzyme that catalyzes the 2-electron reduction of quinones to hydroquinones, and it is highly expressed in many solid tumors.¹² This forms the basis for the synthesis of novel quinolinequinones structurally related to lavendamycin as potential NQO1-directed antitumor agents.

Figure 1.

Natural quinolinequinone antibiotics

Behforouz et al. (1996)¹³ first demonstrated that 7-aminoquinoline-5,8-diones can be efficiently prepared from commercially available 8-hydroxy-2-methylquinoline. Fryatt and co-workers⁷ also showed that by starting with 6-methoxyquinoline, 6-methoxy-2-chloroquinoline-5,8-dione was prepared and subsequent palladium(0) catalyzed reaction with boronic acids gave novel quinoline quinones under reflux for 24 hours. Further, in 2004,¹⁴ arylboronic acids were shown to be more reactive than their counterparts, the arylpinacolboronate esters when reacted with indole bromides in Suzuki couplings under reflux. The lower reactivity was attributed to steric factors in the arylpinacolboronate esters. Also, 3-aryl-indazoles have been synthesized by the reaction of haloindazoles (3-bromoindazole and 3-iodoindazole) with aryl boronic acids under Pd(0) catalysis in Suzuki type cross couplings.¹⁵ The reaction times ranged from 1–18 hours under reflux conditions. In this present study we report a direct more efficient approach to 7-aminoquinoline quinones starting from commercially available 7-amino-8-hydroxyquinoline under microwave conditions where the reaction times are shorter. Computational, metabolism and cytotoxicity studies on the quinoline-5,8-diones are also described.

CHEMISTRY

The synthesis commenced with the nitration of 5-chloro-8-hydroxyquinoline under HNO₃/H₂SO₄ according to a procedure reported by Musser et al.¹⁶ to give the 5-chloro-7-nitro-8-hydroxyquinoline (1) in good yield (79%). Hydrogenation under Pd/C-catalysis at 40–50 psi not only reduced the nitro group to the free amine but also removed the chloride to provide the desired 7-amino-8-hydroxyquinoline (2) in excellent yield (99%). A direct approach to the amino alcohol 2 involves the heating of a mixture of 8-hydroxyquinoline and N-methyl-N-phenylhydrazine at 90°C, albeit very low yields were obtained.¹⁷Our attempt to synthesize the amino alcohol by heating in a microwave between 130–160°C did not improve the yield. Acetylation proceeded smoothly where both the amino and hydroxyl groups were protected. The resulting diacetylated product (3) was hydrolyzed in MeOH/H₂O under reflux to form 7-acetamido-8-hydroxyquinoline. Subsequent benzylation of the free hydroxyl was effected by reacting with BnBr/K₂CO₃ in DMF at 50°C for 24 hrs to give the 7-acetamido-8-benzyloxyquinoline (4) in 90% yield. Oxidation using mCPBA in 1,2-dichloroethane at rt for 48 hrs gave the N-oxide (5) in 82% yield.¹⁸ The key intermediate in the synthesis, the 2-chloro-7-acetamido-8-benzyloxyquinoline (6), was obtained in 62% yield by refuxing the N-oxide with POCl₃ in CHCl₃.¹⁹ The high regioselectivity of the reaction can be rationalized in terms of sterics as well as formation of an oxyphosphorane adduct anion in a rapid concerted mechanism.²⁰ We also attempted refluxing the N-oxide 5 with SO₂Cl₂ as reported in literature,⁹ but only resulted in massive decomposition of the starting material. Deprotection of the benzyl group was effected with BCl₃•SMe₂ in CH₂Cl₂ and subsequent oxidation using Fremy’s salt (potassium nitrosodisulfonate-(KO₃S)₂NO) gave the 7-acetamido-2-chloro-quinoline-5,8-dione (8) in 71% yield.⁷ The results are summarized in Scheme 1 below.

Scheme 1^a.

^aReagents and conditions: (i) HNO₃/H₂SO₄; (ii) H₂/Pd-C, MeOH, 40–50psi, overnight; (iii) CH₃COCl, DIEA, THF, 2hrs.; (iv) H₂O-MeOH, reflux, 1hr; (v) BnBr, K₂CO₃, DMF, 50°C, 24 hrs; (vi) mCPBA, ClCH₂CH₂Cl, 48hrs; (vii) POCl₃, CHCl₃, reflux, 2 hrs; (viii) BCl₃·SMe₂, CH₂Cl₂, overnight; (ix) Fremy's salt, rt, 1 hr; (x) RB(OH)₂, Pd(PPh₃)₄, 110–140°C, µW 20–25 min.

After the successful formation of the quinolinequinone 8, the stage was now set for Suzuki coupling chemistry. This was accomplished by reaction with different boronic acids under Pd(0) catalysis in a microwave as illustrated in Table 1 below. Generally, the reactions were complete within 20–30 minutes in good yields except for the arylboronate ester where only 27% of the product (16) was obtained. The mechanistic details of the reaction have been well studied in which case oxidative addition, transmetallation and reductive elimination being the most critical steps.²¹ Interestingly, the 7-amino-2-(2-pyridyl)quinoline-5,8-dione was prepared in 9 steps starting from 3-hydroxybenzoic acid where the key step was a Friedlander condensation of 2-acetylpyridine and 2-amino-3-benzyloxy-4-bromobenzaldehyde to give the 8-benzyloxy-7-bromo-2-(2’-pyridyl)quinoline.²² Although this seems an attractive strategy, the method lacks the flexibility needed to create a library of lavendamycin analogs.

Table 1.

Suzuki coupling products

R-X	Reaction conditions	R		Yield (%)
	Pd(PPh3)4, DME/Na2CO3 140°C, 20 min		(9)	70
	Pd(PPh3)4, DME/Na2CO3 120°C, 20 min		(10)	41
	Pd(PPh3)4, DME/Na2CO3 120°C, 20 min		(11)	51
	Pd(PPh3)4, DME/Na2CO3 110°C, 25 min		(12)	67
	*Pd(PPh3)4, p-dioxane 120°C, 20 min		(13)	71
	Pd(PPh3)4, DME/Na2CO3 110°C, 25 min		(14)	53
	Pd(PPh3)4, DME/Na2CO3 120°C, 20 min		(15)	42
	PdCl2(dppf), p-dioxane,K3PO4 120°C,30 min		(16)	27

^*Stille coupling reaction

The final step in the synthesis involved the removal of the acetate protecting group which was effected by reaction with H₂SO₄-MeOH at rt. The Boc-protected derivatives were also subjected to TFA/CH₂Cl₂ at rt for 2 hrs to provide the 7-acetamido derivatives.

ELECTROCHEMISTRY

Eletrochemistry was performed to compare the electrochemical behavior of the quinolinequinones with their reduction rates by NQO1, and the data are shown in Table 2. Tetrahydrofuran was used as the solvent for all compounds except 15, which was run in DMSO. The compounds were run against a Ag/AgCl electrode cathodic and anodic peak potentials, E_pc and E_pa, respectively, were measured at a potential sweep rate of 50 mV/sec, and the midpoint of the peak potentials was used to determine E_1/2 values, (E_pc + E_pa)/2. Unfortunately, many of the analogues did not show reversible electrochemistry, and in some cases, there were multiple reduction peaks. This makes interpretation of the numbers somewhat difficult, but some conclusions can be drawn. For instance, most of the acetylated quinolinequinones had a reduction peak between −1.08V and −1.18V, an indication that they are easier to reduce than the non-acetylated compounds due to the presence of this electron-withdrawing group. This is consistent with what we reported previously for lavendamycins. However, there was no correlation between reduction potentials and reduction rates by NQO1, in line with previous publications on this topic.^{6–8, 23, 24} This suggests that steric interactions are more likely to be predictive of substrate efficiency than reduction potentials.

Table 2.

Reduction rates^a and oxygen consumption^b as a result of quinoline-5,8-dione metabolism by recombinant human NQO1 and electrochemical reduction potentials vs ferrocene^c. Reduction rates were determined by spectrophotometric cytochrome c assay and oxygen uptake by oxygen electrode.

No.	R1	R2	Reduction rate by hNQO1 (µmol cyt c reduced /min/mg NQO1	Oxygen Consumption (µmol/min/mg NQO1)	Reduction Potential (E1/2(V) vs Fc)
9	CH3CO		4.5 +/− 0.5	5.2 +/− 1.0	−1.93 nrd
10	CH3CO		25 +/− 4	-	−1.17, −1.92 nr
11	CH3CO		7.0 +/− 0.3	-	−1.18, −1.68, −1.77
13	CH3CO		480 +/− 200	34 +/− 3	−1.90 nr
15	CH3CO		16 +/− 1	-	−1.08, −1.36, −1.58
17	CH3CO		2.8 +/− 0.2	-	−1.10, −1.60, −1.94
18	CH3CO		31 +/− 9	-	-
19	H		78 +/− 7	-	-
20	H		170 +/− 30	-	−1.99 nr
21	H		80 +/− 8	-	−1.84 nr
22	H		18 +/− 6	-	−1.53, −1.65
23	H		71 +/− 13	-	−1.85 nr
24	H		120 +/− 10	8.5 +/− 1.6	-

^aSpectrophotometric assay using cytochrome c as terminal electron acceptor (550 nm).

^bOxygen concentration monitored using an oxygen electrode.

^cE_1/2 values calculated as (E_pc+E_pa)/2 are average values from voltammograms recorded potential sweep rate of 50 mV/sec. E_pc = cathodic peak potential; E_pa = anodic peak potential.

^dnr = non-reversible, anodic peak only.

NMR SPECTROSCOPY AND SPECTROPHOTOMETRY

Complexation of zinc(II) triflate by compounds 13, 19, 22, and 23 was studied using ¹H NMR spectroscopy. No new peaks were observed in NMR spectra, indicating that free and complexed forms of zinc(II) triflate were in a rapid exchange relative to NMR timescale. The aromatic region of the NMR spectrum of compound 19 in THF-D₈ at room temperature is shown below in Figure 2.

Figure 2.

Aromatic proton region NMR spectra of 19 upon addition of increasing equivalent of Zn(SO₃CF₃)₂ in [²H₈]THF. Note the change in δ̣ of H-2’ and H-3 on addition of Zn²⁺. Equivalents of Zn²⁺: A=0, B=1, C=2, D=3, E=4, F=5 and G=10.

There was a small difference in chemical shifts of H-2’ (moving upfield) and H-3 (moving downfield) after addition of one equivalent zinc(II) triflate to the NMR solution (Table S1 and Figure 2) whereas the changes in δ of the other protons were barely noticeable. The biggest change in δ of H-2’ (−0.04 ppm) and H-3’ (+0.07 ppm) occurs after addition of ten equivalents of Zn(SO₃CF₃)₂. This suggests that weak binding occurs at low Zn²⁺ concentration.

In contrast, addition of only one equivalent of Zn(SO₃CF₃)₂ to compound 22 caused larger chemical shift variations of all the protons (Table S2 and Figure 3). Increasing the amount of Zn²⁺ (2–10 equiv.) added to compound 22 made little or no difference in δ afterwards (>0.01 ppm). This means that the quinoline derivative binds the Zn²⁺ more efficiently than compound 19 and only one equivalent of Zn²⁺ is enough to cause chemical shift variations.

Figure 3.

Aromatic protons region NMR spectra of 22 upon addition of increasing equivalent of Zn(SO₃CF₃)₂ in [²H₈]THF. Equivalents of Zn²⁺: A=0, B=1, C=2, D=3, E=4, F=5 and G=10.

Similar observations were made with compounds 23 (Table S3 and Figure 4) and 13 (Table S4). This is consistent with the results reported by Long and Harding²⁵ where they demonstrated that the 1:1 bipyridyl complex of streptonigrin was the major complex formed at room temperature by perfoming an NMR study in [²H₈]THF with addition of Zn²⁺. Titration of compound 23 with Zn²⁺ in a mixture of dimethylsulfoxide-methanol (1:3) was monitored by a spectrophotometer as reported in literature.²⁶ A plot of Δλ₃₅₅ against Zn²⁺ concentration gave a moderate affinity constant of 1.41×10⁴ M⁻¹ for compound 23 binding with Zn²⁺.

Figure 4.

Aromatic proton region NMR spectra of 23 upon addition of increasing equivalent of Zn(SO₃CF₃)₂ in [²H₈]THF. Equivalents of Zn²⁺: A=0, B=1, C=2, D=3, E=4, F=5 and G=10.

RESULTS AND DISCUSSION

Quinolinequinone metabolism by recombinant human NQO1 was examined using a spectrophotometric assay that employs cytochrome c as the terminal electron acceptor. Initial rates of reduction (µmol cytochrome c reduced/min/mg NQO1) were calculated from the linear portion (0–30 s) of the reaction graphs. The 7-acetamido-2-(2-pyridinyl) compound 13 displayed the highest reduction rate by NQO1 (Table 2), although it was the only acetylated analogue with a high reduction rate. In all other cases, 7-amino compounds had much higher reduction rates than corresponding 7-acetamido compounds with identical substituents at the quinoline-2-position. Although unusual, higher rates for acetylated analogues have been observed in other series.^{6, 11} With regard to the aromatic substituents at the quinoline-2-position, no clear trend in reduction rates was observed except that bulkier groups generally decreased reduction rates. Oxygen consumption is a measure of the ability of the reduced quinone (hydroquinone) to redox cycle following reduction by NQO1. This could lead to production of toxic reactive oxygen species and ultimately to cell death. Oxygen consumption was measured for select quinolinequinones, and the trend, if not the magnitude, mirrored the reduction rates. (Table 2).

Cell survival was measured using the MTT colorimetric assay. In previous work, we demonstrated that IC₅₀ values generated from standard clonogenic assays and MTT assays were positively correlated suggesting that the MTT assay is a reliable indicator of cytotoxicity.⁶ We utilized MDA-MB-468 human breast cancer cells stably transfected with human NQO1 cDNA (MDA468-NQ16) along with the non-transfected wild type cells (MDA468) to compare the cytotoxicity of the quinolinequinones (Table 3).²⁷

Table 3.

Cytotoxicity of quinoline-5,8-diones toward MDA468-WT (NQO1-deficient) and MDA468-NQ16 (NQO1-rich) human breast cancer cell lines.

No	R1	R2	IC50 (µM) MDA468-WT	IC50 (µM) MDA468-NQ16	Selectivity Ratio IC50 (WT)/ IC50 NQ16
9	CH3CO		1.7 +/− 0.8	2.4 +/− 1.9	0.73
10	CH3CO		3.3 +/− 0.1	6.3 +/− 0.2	0.52
11	CH3CO		0.80 +/− 0.33	0.64 +/− 0.41	1.2
13	CH3CO		0.53 +/− 0.27	2.2 +/− 0.5	0.24
17	CH3CO		7.4 +/− 5.0	19.1 +/− 5.9	0.39
19	H		5.3 +/− 0.8	17 +/− 5	0.31
20	H		5.6 +/− 1.3	15 +/− 2	0.37
21	H		4.8 +/− 0.9	10 +/− 1	0.47
22	H		0.14 +/− 0.02	0.19 +/− 0.04	0.75
23	H		19 +/− 12	5.3 +/− 2.1	3.5
24	H		4.5 +/− 1.9	17 +/− 2	0.26

Quinolinequinone cytotoxicity (IC₅₀) to MDA468 cells was generally in the single digit micromolar range following 2-h exposures with some in the high nanomolar range (11, 13, 22). Surprisingly, selectivity ratios [IC₅₀ (MDA468) / IC₅₀ (MDA468-NQ16)] were generally <1 meaning that the quinolinequinones were less cytotoxic to the NQO1-rich MDA468-NQ16 cells rather than more cytotoxic. This suggests that NQO1 was protective to the cells rather than functioning as an activating enzyme.²⁷ Only two compounds (11, 23) were selectively cytotoxic to the MDA468-NQ16 cells. The reason for the general absence of selective cytotoxicity with this particular series of compounds is unclear, but it is consistent with NQO1’s primary role as a detoxification enzyme.²⁷

Molecular docking of the quinolinequinones in the NQO1 active site was performed using Sybyl 8.1.1 and GOLD 5.1 for scoring. Three good NQO1 substrates (13, 20, 24) and three poor NQO1 substrates (9, 11, 17) were docked and scored using ChemPLP and ChemScore (Table 4). The highest scores representing a good fit for the model were found for 20 and 24 consistent with the metabolism data. The exception again was 13, which scored the lowest, but was the best substrate. Interatomic distances between quinolinequinone carbonyl groups and FAD N5 and His161 were shortest for 20, but all were within a reasonable distance for hydride transfer from FAD when the dynamic effects of the quinone-enzyme interaction are considered. Figure 5 shows possible docking conformations for 20 and 11 with NQO1. All quinolinequinones orient with the quinone ring above the FAD isoalloxazine ring as needed for hydride transfer.

Table 4.

Computational parameters for selected quinoline-5,8-diones.

No	R1	R2	ChemPLP	ChemScore	C=O8… NH5 (Ǻ)	C=O5… His161NE2 (Ǻ)
9	CH3CO		63.2	22.6	3.9	3.6
11	CH3CO		63.8	22.6	4.7	3.3
13	CH3CO		57.6	21.4	4.3	3.5
17	CH3CO		63.8	22.1	4.2	3.3
20	H		72.8	26.0	3.6	3.2
24	H		67.3	22.7	4.1	3.3

Figure 5.

Quinolinequinones docked in NQO1 active site: 20, cyan; 11, magenta; FAD, green.

The mechanism of action of lavendamycin and streptonigrin is not clearly understood. However previous studies demonstrated that quinone moieties are reduced by NQO1 to the corresponding hydroquinones which undergo auto-oxidation producing activated oxygen species including not only the semiquinone derivatives but also superoxide and hydroxyl radicals.²⁸ In addition, both streptonigrin and lavendamycin chelate divalent cationic metal ions. This property might confer to streptonigrin and lavendamycin the ability to shuttle iron cations into the cells which in turn can catalyze production of reactive oxygen species through a Fenton reaction. On the other hand, this chelation can result in depletion of intracellular cationic metals which might result in cell death.²⁹ Generation of the semiquinone radical, after reduction of the quinone to the hydroquinone followed by auto-oxidation, results in a decrease of activity in 9 compounds. The best NQO1 substrates are less active compounds (13, 20, 24) in NQO1 expressing cells. In contrast, poor NQO1 substrates such as compound 22 or 11, exhibit the best activity in both cancer cells expressing NQO1 and not expressing NQO1. According to the NMR experiments, the quinoline derivative 22 and compound 13 binds the Zn²⁺ more efficiently than compound 19; and only one equivalent of Zn²⁺ is enough to cause important chemical shift variations. Similar observations were made with compound 23, which was less cytotoxic than compound 22. Even though metal chelation by these compounds is still a plausible mechanism to explain their activity against breast cancer cells, another mode of action cannot be discarded. Most active compounds (11, 13 and 22) are potential tridentate ligands for metals. Compound 23 exhibits lower activity than the corresponding acetylated amino analogue 13. It was proposed that metals can assist tautomeric shift from the active quinone analogues to the quinoid analogue which has an isoelectronic structure with the biologically inactive azastreptonigrin.²⁴ This tautomeric shift can explain the decrease of activity of the amino derivative compared to the amido derivative. In our series of aryl substituted quinonequinolines the active molecule is the quinone derivatives and not the semiquinone derivatives. A similar mode of action than the bidentate metal ligands derivatives 8-hydroxyquinoline is currently under investigation.^{30, 31}

CONCLUSIONS

A ten-step synthetic scheme led to good yields for quinolinequinone analogs of lavendamycin projected as NQO1-directed antitumor agents. Unexpectedly, ten of eleven analogs demonstrated excellent cytotoxicity (IC₅₀ values of single digit micromolar or better) towards MDA468 breast cancer cells, but only two were selectively cytotoxic to NQO1-expressing MDA468-NQ16 cells. Compounds 22 and 11 are poor NQO1 substrates and exhibit the best activity against breast cancer cells. In our novel series of aryl substituted quinonequinolines the active molecule appears to be the quinone derivatives and not the semiquinone derivatives resulting from NQO1 reduction, suggesting that the mode of action of this novel series differs from lavendamycin and involves an unidentified target. Quinolinequinone derivatives 11, 13 and 22 cytotoxicities (IC50) to MDA468 cells were in the high nanomolar range. Our results seem to indicate that compounds 11, 13 and 22 effects could be also, at least partially, mediated by metal chelation. These aryl quinonequinoline derivatives represent a promising class of cytotoxic agents with a potential therapeutic value.

EXPERIMENTAL SECTION

Cell Culture

MDA-MB-468 (MDA468) human breast cancer cells and stably NQO1-transfected MDA468-NQ16³² were a gift from Dr. David Ross (University of Colorado-Denver, Denver, CO). MDA468 cells had no measurable NQO1 activiy whereas activity in MDA468-NQ16 cells was 1070 nmol/min/mg total cell protein using dichlorophenolindophenol (DCPIP) as the standard electron acceptor. Cells were grown in RPMI 1640 medium with L-glutamine and penicillin/streptomycin, and supplemented with 10% fetal bovine serum (FBS). Cell culture medium and supplements were obtained from Invitrogen (Carlsbad, CA). The cells were incubated at 37 °C under a humidified atmosphere containing 5% CO₂.

Spectrophotometric cytochrome c assay

Quinolinequinone reduction was monitored using a spectrophotometric assay in which the rate of reduction of cytochrome c was quantified at 550 nm. Briefly, the assay mixture contained cytochrome c (70 µM), NADH (1 mM), recombinant human NQO1 (0.1–10 µg) (gift from Dr. David Ross, University of Colorado-Denver, Denver, CO) and quinonlinequinones (25 µM) in a final volume of 1 mL Tris-HCl (25 mM, pH 7.4) containing 0.7 mg/mL BSA and 0.1% Tween-20. Reactions were carried out at room temperature and started by the addition of NADH. Rates of reduction were calculated from the initial linear part of the reaction curve (0–30 s) and results were expressed in terms of µmol of cytochrome c reduced/min/mg of NQO1 using a molar extinction coefficient of 21.1 mM⁻¹ cm⁻¹ for cytochrome c. All reactions were carried out at least in triplicate.

Oxygen Consumption

Oxygen concentration was monitored using a MI-730 Micro-Oxygen Electrode (Microelectrodes, Bedford, NH) with concentrations adjusted for temperature (25 °C). Assay mixtures contained 25 µM quinonlinequinones, 200 µM NADH and 1 µg/mL NQO1 in a 2 mL Tris-Hcl-BSA/Tween (0.1%) buffer system. Reactions were started with NADH and measured over 3 minute intervals at room temperature. All reactions were carried out in triplicate.

Electrochemistry

Cyclic Voltammagrams were collected for 10 analogues using a BAS CV-50W electrochemical analyzer using a standard 3 electrode cell. Experiments were performed using an Ag/AgCl reference electrode, a glossy carbon working electrode and a platinum wire auxiliary electrode. The reported potentials are referenced by the Ferrocene (0/+) couple in the solvent used, primarily THF, which occurs at +.60V vs. Ag/AgCl. The compounds were run at concentrations of 1mM in THF, except compound 15 which was run in DMSO, with a.1M concentration of tetrabutylammonium hexafluorophosphate as a supporting electrolyte. All samples were purged and run under an Ar atmosphere during the course of the experiment, and the electrodes were washed and wiped down between each sample. Each CV was collected at a sweep rate of 50mV/s in the potential range of 0V to −2V at room temperature of 21°C.

NMR spectroscopy

One-dimensional ¹H NMR spectra were recorded at room temperature on Bruker Avance IIITM spectrometer (The Woodlands, Texas) at 400 MHz using a 5-mm probe and a simple pulse-acquire sequence. Acquisition parameters consisted of spectral width of 4000 Hz with an acquisition time 3.98 s, number of scans of 128, and relaxation delay of 1 s. Complexes were prepared in a mixture of CDCl₃ and THF-D₈.

Cell Viability Assay

Growth inhibition was determined using the MTT colorimetric assay. Cells were plated in 96-well plates at a density of 10,000 cells/mL and allowed to attach overnight (16 h). Quinolinequinone solutions were applied in medium for 2 hours, removed and replaced with fresh medium, and the plates were incubated at 37 °C under a humidified atmosphere containing 5% CO₂ for 3–5 days. MTT (50 µg) was added and the cells were incubated for another 4 hours. Medium/MTT solutions were removed carefully by aspiration, the MTT formazan crystals were dissolved in 100 µL DMSO, and absorbance was determined on a plate reader at 560 nm. IC₅₀ values (concentration at which cell survival equals 50% of control) were determined from semi-log plots of percent of control vs. concentration. Selectivity ratios were defined as the IC₅₀ value for the MDA468 cell line divided by the IC₅₀ value for the MDA468-NQ16 cell line.

Molecular Modeling

For docking purposes, the crystallographic coordinates of the human NQO1 complex with 3-(hydroxymethyl)-5-(2-methylaziridin-1-yl)-1-methyl-2-phenylindole-4,7-dione (25) were obtained from the Brookhaven Database (PDB code 1H69³³ and resolution 1.86Ǻ) and was edited accordingly to provide a monomer of the protein. The protein complex was then minimized within Sybyl 7.3 (Tripos Ltd., St Louis) while holding all heavy atoms stationary. The ligand was then removed to leave the receptor complex which was used for the subsequent docking studies. For preparation of ligand structures, fragments from Sybyl 8.1.1 were used to construct the compounds and all symmetric compounds were prepared as monoanionic ligands. Ligands were subject to 1000 iterations of energy minimization using the Powell method with MMFF94s force field standard method. For computational docking, the GOLD 5.1 software was used in combination with the ChemPLP³⁴ scoring function (rescoring with ChemScore.³⁵ The active site was defined as being any volume within 8Ǻ of the quinone scaffold of 25 in its crystal pose in 1H69. Each GA run comprised using the default parameters of: 100000 genetic operations on an initial population of 100 members divided into five subpopulations with weights for crossover, mutation, and migration being set to 95, 95, and 10, respectively. GOLD allows a user-definable number of GA runs per ligand, each of which starts from a different orientation. For these experiments, the number of GA runs was set to 10, and scoring of the docked poses was performed with the ChemPLP scoring function using ChemScore rescore. Each GOLD run was saved and the strongest scoring binding pose of each ligand (subject to a rmsd default distance threshold of 1.5Ǻ) was compared to that of the reference ligand position observed in the crystal structure. The best output pose (orientations) of the ligands generated were analyzed based on its ChemPLP/ChemScore score, feasibility of hydride transfer process and H-bonding to the enzyme. The best pose(s) were visualized using PyMOL Molecular Graphics System version 1.3.

Chemistry

All moisture sensitive reactions were performed in an inert, dry atmosphere of argon in flame dried glassware. Air sensitive liquids were transferred via syringe or cannula through rubber septa. Reagent grade solvents were used for extraction and flash chromatography. THF was distilled from Na/benzophenone under argon; dichloromethane (CH₂Cl₂) and chloroform (CHCl₃) were distilled from CaH₂ under argon. All other reagents and solvents which were purchased from commercial sources, were used directly without further purification. The progress of reactions was checked by analytical thin-layer chromatography (Sorbent Technologies, Silica G TLC plates w/UV 254). The plates were visualized first with UV illumination followed by charring with ninhydrin (0.3% ninhydrin (w/v), 97:3 EtOH-AcOH). Flash column chromatography was performed using prepacked Biotage SNAP cartridges on a Biotage Isolera One instrument. Microwave reactions were performed using a Biotage Initiator instrument. The solvent compositions reported for all chromatographic separations are on a volume/volume (v/v) basis. ¹HNMR spectra were recorded at 400 or 500 MHz and are reported in parts per million (ppm) on the δ scale relative to tetramethylsilane as an internal standard. ¹³CNMR spectra were recorded at 100 or 125 MHz and are reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ 77.00). Melting points were determined on a Stuart melting point apparatus from Bibby Scientific Limited and are uncorrected. High Resolution mass spectrometry (HRMS) was performed on a Waters/Micromass LCT-TOF instrument. All compounds were more than 95% pure.

5-chloro-8-hydroxy-7-nitroquinoline (1)

This compound was prepared according to the literature¹² procedure to yield a yellow solid, 4.40 g (79%). M.p. 198–200°C, [lit.¹², m.p. 192–194°C]; ¹H NMR (500 MHz, DMSO) δ 9.09 (dd, J = 4.2, 0.5 Hz, 1H), 8.58 (dd, J = 8.5, 0.8 Hz, 1H), 8.18 (s, 1H), 7.94 (dd, J = 8.5, 4.3 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 150.5, 150.1, 139.9, 133.6, 132.3, 128.5, 125.9, 122.0, 117.9. HRMS (TOF MS ES+) for C₉H₆ClN₂O₃⁺ (MH+) calcd. 225.0067, found 225.0055.

7-Amino-8-hydroxyquinoline (2)

Compound 1 (2.4 g, 10.69 mmol) was placed in a hydrogenation apparatus equipped with a magnetic stir bar and methanol added. Pd/C (150 mg) in a small amount of MeOH (60 mL) was added and stirring commenced. H₂ gas was introduced at a pressure of 40–50 psi and reacted at rt overnight. TLC showed full conversion. The black solution was filtered using a celite pad and concentrated under reduced pressure to yield 2 as a black oil, 99% yield. ¹H NMR (500 MHz, CDCl₃) δ 8.66 (dd, J = 4.4, 1.6 Hz, 1H), 8.03 (dd, J = 8.2, 1.6 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 8.2, 4.4 Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 148.0, 137.9, 136.6, 136.1, 132.1, 122.4, 119.3, 118.5, 117.7. HRMS (TOF MS ES+) for C₉H₉N₂O⁺ (MH+) calcd. 161.0715, found 161.0707.

7-acetamido-8-acetyloxyquinoline (3)

Compound 2 (330 mg, 2.06 mmol) was dissolved in dried THF (10 mL) and DIEA added with stirring. AcCl (176 µL) in 1mL THF was added drop wise while stirring and reacted at rt for 2 hrs. Then concentrated under reduced pressure followed by redissolving in CH₂Cl₂ (20 mL) and water (10 mL). The two layers were allowed to partition and extracted 2× 20 mL CH₂Cl₂. The combined organic layers were dried over MgSO₄, filtered and concentrated under reduced pressure. Then purified on a Biotage SNAP cartridge (25 g) at a flow rate of 25 mL/min to yield an orange solid, 382 mg (76%); m.p. 151–153°C; ¹H NMR (500 MHz, CDCl₃) δ 8.85 (dd, J = 4.1, 1.3 Hz, 1H), 8.49 (d, J = 9.1 Hz, 1H), 8.13 (dd, J = 8.3, 1.5 Hz, 1H), 7.70 (d, J = 9.1 Hz, 1H), 7.67 (s, 1H), 7.36 (dd, J = 8.2, 4.2 Hz, 1H), 2.56 (s, 1H), 2.04 (s, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 169.7, 168.5, 150.6, 140.7, 135.8, 134.9, 130.8, 125.8, 125.6, 121.3, 120.6, 24.5, 21.0; HRMS (TOF MS ES+) for C₁₃H₁₃N₂O₃⁺ (MH+) calcd. 245.0926, found 245.0923.

7-acetamido-8-benzyloxyquinoline (4)

To a solution of 3 (1.2 g, 4.91 mmol) in MeOH (100 mL) was added water (10 mL) and the reaction stirred under reflux for 1 hr. The black solution was concentrated and in vacuo and flash chromatographed on a KP-Sil 100 g Biotage SNAP cartridge using MeOH: DCM as the solvent (0–5% MeOH). A white solid (0.9 g) obtained and used for the next step directly. R_f= 0.11 (5% MeOH:CH₂Cl₂).

To a solution of 7-acetamido-8-hydroxyquinoline (2.27 g, 11.23 mmol) in 40 mL DMF was added K₂CO₃ (2.33 g, 16.80 mmol) and BnBr (2 mL, 16.80 mmol) respectively. The reaction was stirred at 50°C for 24 hrs after which TLC showed almost all the starting material was consumed. The reaction mixture was diluted with 30 mL CH₂Cl₂, filtered with a pad of celite and concentrated under reduced pressure. The residue was loaded onto a 100 g Biotage SNAP cartridge by dissolving in a small amount of CH₂Cl₂ and eluted with EtOAc:heptane gradient (0–50%). Yield 2.95 g (90%) of a yellow oil was obtained. R_f= 0.50 (60% EtOAc:heptane). ¹H NMR (500 MHz, CDCl₃) δ 8.95 (dd, J = 4.2, 1.7 Hz, 1H), 8.58 (d, J = 9.0 Hz, 1H), 8.14 (dd, J = 8.3, 1.7 Hz, 1H), 7.77 (s, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.40 – 7.35 (m, 6H), 5.49 (s, 2H), 1.93 (s, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 168.3, 150.0, 142.0, 141.0, 137.4, 136.2, 132.0, 128.9, 128.8, 128.8, 126.0, 124.0, 120.0, 120.0, 77.3, 24.6. HRMS (TOF MS ES+) for C₁₈H₁₇N₂O₂⁺ (MH+) calcd. 293.1290, found 293.1264.

7-acetamido-8-(benzyloxy)quinoline-1-oxide (5)

The starting material (4) (428 mg, 1.46 mmol) was dissolved in 4.3 mL 1,2-dichloroethane with stirring. The mCPBA (340 mg, 1.76 mmol) was added (0.5 M) and the reaction stirred at rt for 48 hrs. TLC showed almost all the starting material was consumed. The precipitated mCPBA was filtered and washed with 5 mL 1, 2-dichloroethane. The filtrate was concentrated under reduced pressure and flash chromatographed on a KP-sil 100 g Biotage SNAP cartridge using a 5% MeOH: DCM gradient at a flow rate of 25 mL/min to yield a yellow solid, 373 mg (82%). M.p. 145–147°C; R_f 0.24 (5%MeOH:DCM). ¹H NMR (500 MHz, DMSO) δ 9.45 (s, 1H), 8.46 (d, J = 6.1 Hz, 1H), 8.20 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.58 – 7.50 (2H), 7.40 – 7.30 (aromatic, 4H). ¹³C NMR (126 MHz, DMSO) δ 168.9, 139.8, 138.1, 137.1, 136.4, 133.3, 129.8, 129.1, 128.1, 128.0, 124.7, 124.4, 120.8, 77.7, 23.8. HRMS (TOF MS ES+) for C₁₈H₁₇N₂O₃⁺ (MH+) calcd. 309.1239, found 309.1227.

7-acetamido-8-benzyloxy-2-chloroquinoline (6)

Phosphoryl chloride (280 µL, 3.0 mmol) in CHCl₃ (1.0 mL) was added to a stirred solution of the oxide 5 (770 mg, 2.50 mmol) in 21 mL CHCl₃ and stirred for 15 min. The mixture was then refluxed for 2 hrs, cooled and poured into ice (50 g) and the pH adjusted to 12 with NaOH (aq.). The aq. layer was extracted with 2 × 50 mL CH₂Cl₂, washed with 2 × 20 mL H₂O, dried over MgSO₄, filtered and concentrated under reduced pressure to yield a brown oil. Then purified on a HP-Sil 25 g Biotage SNAP cartridge using EtOAc:heptane gradient (0–50%) as the solvent. Yield 504 mg (62%) of an off-white solid was obtained. R_f= 0.58 (60% EtOAc:heptane); M.P. 92–94°C; ¹H NMR (500 MHz, CDCl₃) δ 8.60 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.81 (s, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.45 – 7.35 (m, 1H), 7.32 (d, J = 8.5 Hz, 1H), 5.48 (s, 1H), 1.96 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 168.4, 150.5, 141.4, 140.3, 139.0, 137.2, 133.0, 128.9, 128.8, 128.8, 124.3, 123.3, 121.1, 120.1, 77.4, 24.7. HRMS (TOF MS ES+) for C₁₈H₁₆ClN₂O₂⁺ (MH+) calcd. 327.0900, found 327.0936.

7-acetamido-2-chloro-8-hydroxyquinoline (7)

To a solution of 6 (330 mg, 1.01 mmol) in CH₂Cl₂ (10.1 mL) under an Ar atmosphere was added BCl₃•SMe₂ (10.1 mL) via a syringe and stirred at rt overnight. TLC showed the reaction was complete. The reaction was then quenched with saturated NaHCO₃(aq.) and extracted with 2x20 mL CH₂Cl₂. The organic layers were combined, dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified on 50 g KP-Sil Biotage SNAP cartridge using a MeOH: CH₂Cl₂ gradient (0–5% MeOH) at a flow rate of 25 mL/minute to give a yellow solid, 198 mg (82%). M.P. 176–178°C; R_f= 0.50 (5% MeOH:CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃) δ 8.60 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.82 (brs, 1H), 7.72 (s, 1H), 7.35 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 2.29 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.6, 149.7, 138.9, 138.2, 137.1, 124.4, 123.3, 121.5, 121.3, 118.0, 24.9. HRMS (TOF MS ES+) for C₁₁H₁₀ClN₂O₂⁺ (MH+) calcd. 237.0431, found 237.0424.

7-Acetamido-2-chloroquinoline-5,8-dione (8)

To a solution of 7 (300 mg, 1.27 mmol) in acetone (30 mL) was added a solution of Fremy’s salt in NaH₂PO₄ buffer (0.3 M, 30 mL) and the mixture stirred at rt for 1hr. A further solution of Fremy’s salt in the buffer (0.3M, 30 mL) was added and stirring continued for 2 hrs. The acetone was removed under reduced pressure and the residue extracted with 2 × 50 mL CH₂Cl₂. The CH₂Cl₂ phases were combined, dried over MgSO₄ and concentrated under reduced pressure. The residue was purified on a 25 g HP-Sil Biotage SNAp cartridge using EtOAc:heptanes gradient (0–60%) to obtain a yellow solid, 225 mg (71% over 2 steps); m.p. 224–226°C (decomposes into a black mass), R_f= 0.49 (60% EtOAc:heptane). ¹H NMR (500 MHz, CDCl₃) δ 8.41 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.97 (s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 2.34 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 183.4, 178.1, 169.5, 156.7, 145.9, 140.4, 137.2, 129.9, 128.0, 116.3, 25.1. HRMS (TOF MS ES+) for C₁₁H₈ClN₂O₃⁺ (MH+) calcd. 251.0223, found 250.0203.

General procedure for Suzuki coupling under microwave conditions

The 7-acetamido-2-chloroquinoline-5,8-dione 8 (21 mg, 0.08 mmol) was dissolved in 4 mL dimethoxyethane (DME) and degassed under reduced pressure. The palladium (0) catalyst, Pd(PPh₃)₄ (10 mg, 0084 mmol) was added and the solution degassed further. The mixture was stirred under Ar atmosphere for 10 minutes. Na₂CO₃ solution (0.2 mL, 2.0 M) was added followed by the boronic acid (0.126 mmol). The mixture was then heated using a Biotage microwave initiator at 110–140°C for 20 minutes. After cooling, TLC showed all the starting material was consumed. The reaction mixture was poured into DCM and washed with 2 × 10 mL water. Then dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified on HP-Sil 25 g Biotage SNAP cartridge using EtOAc:heptane gradient (0–50%) at a flow rate of 20 mL/min. For very polar products, MeOH:CH₂Cl₂ (0–10%MeOH) was used as solvent for purification.

7-acetamido-2-(4-(trifluoromethyl)phenyl)quinoline-5,8-dione (9)

Yield 21 mg (70%) of a yellow solid was obtained. R_f= 0.47 (50% EtOAc:heptane); m.p. 250°C(decomposes); ¹H NMR (500 MHz, CDCl₃) δ 8.53 (d, J = 8.2 Hz, 1H), 8.45 (s, 1H), 8.27 (d, J = 8.1 Hz, 2H), 8.17 (d, J = 8.2 Hz, 1H), 7.99 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 2.35 (s, 4H).¹³C NMR (126 MHz, CDCl₃) δ 184.1, 179.1, 169.5, 160.1, 146.1, 140.6, 135.7, 128.3, 128.0, 126.0, 126.0, 126.0, 125.3, 116.5, 25.2; HRMS (TOF MS ES+) for C₁₈H₁₂F₃NS₂O₃⁺(MH+) calcd. 361.0800, found 361.0834.

7-acetamido-2-(3-pyridinyl))quinoline-5,8-dione (10)

Yield 21 mg (41%) of a yellow solid obtained, R_f= 0.19 (5% MeOH:DCM); m.p. >300°C(decomposes); ¹H NMR (500 MHz, CDCl₃) δ 9.29 (s, 1H), 8.72 (d, J = 3.9 Hz, 1H), 8.56 (d, J = 8.2 Hz, 1H),8.55 (m, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.00 (s, 1H), 7.56 (dd, J = 8.0, 4.9 Hz, 1H), 2.35 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 184.2, 179.0, 170.4, 158.9, 150.7, 148.1, 146.1, 140.9, 135.7, 135.7, 128.1, 125.3, 116.6, 24.6 ; HRMS (TOF MS ES+) for C₁₆H₁₂N₃O₃⁺MH+) calcd. 294.0879, found 294.0914. 7-amino-2-(3-pyridinyl)quinoline-5,8-dione: 6 mg (12%) of a red solid was obtained. R_f= 0.13 (5% MeOH:DCM); m.p. 195–197°C (decomposes, turns black); ¹H NMR (500 MHz, CDCl₃) δ 9.29 (d, J = 1.7 Hz, 1H), 8.67 (dd, J = 4.9, 1.4 Hz, 1H), 8.58 (ddd, J = 8.0, 2.2, 1.7 Hz, 1H), 8.52 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 7.59 (ddd, J = 8.0, 4.9, 0.7 Hz, 1H), 6.07 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 181.9, 179.8, 157.0, 150.4, 149.6, 147.6, 146.3, 135.4, 135.0, 133.5, 129.4, 124.7, 123.8, 102.1. HRMS (TOF MS ES+) for C₁₄H₁₀N₃O₂⁺MH+) calcd. 252.0773, found 252.0795.

7-acetamido-2-(8’-quinolinyl)quinoline-5,8-dione (11)

Yield 31 mg (51%) of a yellow solid was obtained. R_f= 0.25 (70% EtOAc:heptane), crystallized from MeOH/CH₂Cl₂; m.p. 295°C (decomposes); ₁H NMR (500 MHz, CDCl₃) δ 8.93 (dd, J = 4.2, 1.8 Hz, 1H), 8.53 (d, J = 8.1 Hz, 1H), 8.48 (d, J = 8.1 Hz, 1H), 8.34 (dd, J = 8.3, 1.8 Hz, 1H), 8.22 (dd, J = 7.2, 1.4 Hz, 1H), 8.04 (dd, J = 8.2, 1.4 Hz, 1H), 8.00 (s, 1H), 7.76 (dd, J = 8.1, 7.3 Hz, 1H), 7.54 (dd, J = 8.3, 4.2 Hz, 1H), 2.34 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 184.8, 179.1, 170.9, 161.9, 150.5, 145.7, 145.1, 140.9, 136.7, 136.5, 133.3, 131.9, 131.7, 130.2, 128.4, 127.6, 126.3, 121.4, 116.5, 24.2. HRMS (TOF MS ES+) C₂₀H₁₄N₃O₃⁺ (MH+) calcd. 344.1035, found 344.1022.

7-acetamido-2-(2-(1-tert-butoxycarbonylindolyl))quinoline-5,8-dione (12)

Yield 63 mg (67%) of an orange solid was obtained. R_f= 0.40 (50% EtOAc:heptane); m.p. 191–193°C (decomposes); ¹H NMR (500 MHz, CDCl₃) δ 8.47 (s, 1H), 8.45 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.97 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.27 (dd, J = 9.1, 5.9 Hz, 1H), 6.98 (s, 1H), 2.33 (s, 3H), 1.41 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 184.2, 179.0, 169.6, 157.4, 149.7, 145.3, 140.4, 138.1, 137.5, 134.2, 128.6, 127.9, 127.6, 126.0, 123.3, 121.5, 116.5, 115.2, 114.0, 84.2, 27.8, 25.1. HRMS (TOF MS ES+) C₂₄H₂₂N₃O₅⁺(MH+) calcd. 432.1559, found 432.1568.

7-acetamido-2-(2-pyridinyl)quinoline-5,8-dione (13)

Yield 37 mg (71%) of a yellow solid was obtained. R_f= 0.19 (5% MeOH:CH2Cl2), crystallized from MeOH/CH₂Cl₂; m.p. 255–258°C (decomposes); ¹H NMR (500 MHz, DMSO) δ 10.08 (s, 1H), 8.78 (ddd, J = 4.8, 1.6, 0.8 Hz, 1H), 8.53 (d, J = 7.9 Hz, 1H), 8.46 (d, J = 8.2 Hz, 1H), 8.08 (td, J = 7.7, 1.8 Hz, 1H), 7.77 (s, 1H), 7.58 (ddd, J = 7.5, 4.7, 1.1 Hz, 1H), 2.28 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 184.6, 178.4, 171.5, 158.6, 153.6, 149.8, 146.4, 142.5, 137.8, 135.0, 128.5, 125.5, 124.6, 121.7, 115.3, 24.7. HRMS (TOF MS ES+) C₁₆H₁₂N₃O₃⁺ (MH+) calcd. 294.0879, found 294.0914.

7-acetamido-2-(2-(1-tert-butoxycarbonylpyrrolyl))quinoline-5,8-dione (14)

Yield 36 mg (53%) of a yellow solid was obtained. R_f= 0.30 (50% EtOAc:heptane); m.p. 191–193°C (decomposes), recrystallized from methanol; ¹H NMR (500 MHz, CDCl₃) δ 8.42 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.95 (s, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.42 (dd, J = 3.2, 1.7 Hz, 1H), 6.64 (dd, J = 3.4, 1.7 Hz, 1H), 6.29 (t, J = 3.3 Hz, 1H), 2.32 (s, 3H), 1.43 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 184.3, 179.2, 169.5, 156.9, 148.8, 145.3, 140.3, 134.0, 132.5, 128.0, 127.3, 125.5, 118.6, 116.4, 111.2, 84.4, 27.7, 25.1. HRMS (TOF MS ES+) C₂₀H₂₀N₃O₅⁺ (MH+) calcd. 382.1403, found 382.1381.

7-acetamido-2-(4-pyrazolyl))quinoline-5,8-dione (15)

Yield 31 mg (42%) of a brown solid was obtained. R_f= 0.33 (5% MeOH:CH₂Cl₂); m.p. 270°C (decomposes), recrystallized from methanol; ¹H NMR (500 MHz, DMSO) δ 13.34 (s, 1H), 9.97 (s, 1H), 8.55 (s, 1H), 8.25 (d, J = 8.2 Hz, 1H), 8.21 (s, 1H), 8.11 (d, J = 8.2 Hz, 1H), 7.69 (s, 1H), 2.26 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 184.6, 178.6, 171.4, 156.1, 146.6, 142.1, 134.2, 126.1, 123.8, 121.2, 115.1, 24.6. HRMS (TOF MS ES+) C₁₄H₁₁N₄O₃⁺ (MH+) calcd. 283.0831, found 283.0846.

7-acetamido-2-(3-(2-acetamido-pyridinyl))quinoline-5,8-dione (16)

The quinone 8 (71 mg, 0.28 mmol) was dissolved in 2 mL 1,4-dioxane and degassed under reduced pressure. PdCl₂(dppf) (20 mg), K₃PO₄ (238 mg) and the boronate were added and the solution degassed further. The mixture was stirred under Ar atmosphere for 10 minutes. The mixture was then heated heated using a Biotage microwave initiator at 120°C for 30 minutes. After cooling, the reaction mixture was poured into CH₂Cl₂ and washed with 2 × 10 mL water and extracted 2× 30 mL DCM. The combined organic phases were dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified on a HP-Sil 25 g Biotage SNAP cartridge using MeOH:CH₂Cl₂ gradient (0–5%) at a flow rate of 20 mL/min. Yield 23 mg (23%) of a brown solid was obtained. R_f= 0.32 (5% MeOH:CH₂Cl₂); m.p. 249°C (decomposes); ¹H NMR (500 MHz, DMSO) δ 10.82 (s, 1H), 10.04 (s, 1H), 9.17 (d, J = 2.5 Hz, 1H), 8.60 (dd, J = 8.8, 2.5 Hz, 1H), 8.45 (d, J = 8.3 Hz, 1H), 8.38 (d, J = 8.2 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 7.75 (s, 1H), 2.28 (s, 3H), 2.14 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 184.6, 178.5, 171.5, 169.7, 157.4, 153.6, 147.3, 146.6, 142.4, 137.0, 134.8, 128.0, 127.2, 124.1, 115.3, 113.0, 24.7, 24.0. HRMS (TOF MS ES+) C₁₈H₁₅N₄O₄⁺ (MH+) calcd. 351.1093, found 351.1064.

7-acetamido-2-(2-indolyl)quinoline-5,8-dione (17)

The starting material 12 (39 mg, 0.09 mmol) was dissolved in 2.5 mL CH₂Cl₂ and cooled to 0°C using an ice bath. Trifluoroacetic acid (140 µL) was the added dropwise and reacted at rt for 2 hrs. TLC showed full conversion. Then quenched with sat. NaHCO₃ (10 mL) and extracted 2×20 mL CH₂Cl₂. The organic layers were combined, dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified on a HP-Sil 25 g Biotage SNAP cartridge using EtOAc:heptane gradient (0–70%) at a flow rate of 20 mL/min. Yield 17 mg (59%) of a red solid was obtained after recrystallization from MeOH. M.p. 185°C, decomposes; R_f= 0.38 (70% EtOAc:heptane). ¹H NMR (500 MHz, CDCl₃) δ 8.35 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 7.92 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.23 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 2.34 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 184.2, 180.4, 170.5, 154.7, 145.4, 140.3, 137.8, 134.7, 134.4, 128.3, 126.6, 124.4, 124.4, 121.5, 120.2, 117.0, 111.7, 104.4, 24.4. HRMS (TOF MS ES+) C₁₉H₁₄N₃O₃⁺ (MH+) calcd. 332.1035, found 332.1030.

7-acetamido-2-(2-(pyrrolyl))quinoline-5,8-dione (18)

The starting material 14 (30 mg, 0.08 mmol) was dissolved in 3 mL CH₂Cl₂ and cooled to 0°C using an ice bath. Trifluoroacetic acid (150 µL) was the added dropwise and reacted at rt for 2 hrs. TLC showed full conversion. Then quenched with sat. NaHCO₃ (10 mL) and extracted 2×20 mL CH₂Cl₂. The organic layers were combined, dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified on a HP-Sil 25 g Biotage SNAP cartridge using EtOAc:heptane gradient (0–50%) at a flow rate of 20 mL/min. Yield 21 mg (93%) of a red solid was obtained after recrystallization from MeOH. M.P. 255°C, decomposes. R_f= 0.11 (50% EtOAc:heptane). ¹H NMR (500 MHz, DMSO) δ 11.65 (s, 1H), 9.95 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.69 (s, 1H), 7.07 – 7.04 (m, 2H), 6.28 – 6.22 (m, 1H), 2.27 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 184.6, 178.7, 171.4, 154.0, 146.6, 141.9, 133.9, 130.1, 125.3, 123.8, 121.8, 115.2, 111.9, 110.4, 24.7. HRMS (TOF MS ES+) C₁₅H₁₂N₃O₃⁺ (MH+) calcd. 282.0879, found 282.0909.

General procedure for removal of the acetate group with MeOH-H₂SO₄

To the starting material (0.1 mmol) in a 20 mL vial was added 175µL of H₂SO₄ in 3.0 mL MeOH and stirred at rt for 3 hrs. The red solution was then neutralized with 5 mL 5% NaHCO₃ (aq.) and extracted with 5 × 10 mL CH₂Cl₂. The combined organic extracts were dried over MgSO₄, filtered and concentrated under reduced pressure. Then purified on a HP-Sil 25 g Biotage SNAP cartridge using EtOAc:heptanes (0–70%) or MeOH:CH₂Cl₂ gradient (0–5%) at a flow rate of 20 mL/min.

7-Amino-2-(4-(trifluoromethyl)phenyl)quinoline-5,8-dione (19)

The general procedure was used to obtain 6.0 mg (67%) of a red solid; R_f= 0.38 (60% EtOAc:heptane); m.p. 151–153°C (decomposes, turns black); ¹H NMR (500 MHz, CDCl₃) δ 8.28 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 5.84 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 182.2, 180.1, 158.7, 150.3, 146.3, 140.6, 135.1, 129.4, 127.7, 125.5, 125.5, 125.1, 105.8, 102.4. HRMS (TOF MS ES+) C₁₆H₁₀F₃N₂O₂⁺ (MH+) calcd. 319.0694, found 319.0666.

7-amino-2-(3-pyridinyl)quinoline-5,8-dione (20)

The general procedure was used to obtain 10 mg (83%) of a red solid. R_f= 0.16 (5% MeOH:CH₂Cl₂); m.p. 195–197°C (decomposes, turns black). ¹H NMR (500 MHz, CDCl₃) δ 9.29 (d, J = 1.7 Hz, 1H), 8.67 (dd, J = 4.9, 1.4 Hz, 1H), 8.58 (ddd, J = 8.0, 2.2, 1.7 Hz, 1H), 8.52 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 7.59 (ddd, J = 8.0, 4.9, 0.7 Hz, 1H), 6.07 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 181.9, 179.8, 157.0, 150.4, 149.6, 147.6, 146.3, 135.4, 135.0, 133.5, 129.4, 124.7, 123.8, 102.1. HRMS (TOF MS ES+) for C₁₄H₁₀N₃O₂⁺MH+) calcd. 252.0773, found 252.0795.

7-amino-2-(2-indolyl)quinoline-5,8-dione (21)

The general procedure was used to obtain 19 mg (63%) of a dark-brown solid. R_f= 0.22 (70% EtOAc:heptane); m.p. 235°C decomposes. ¹H NMR (500 MHz, CDCl₃) δ 8.33 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 8.3 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.39 (s, 1H), 7.27 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.19 (s, 1H), 7.12 (td, J = 7.5, 0.8 Hz, 1H), 6.01 (s, 1H). ₁₃C NMR (126 MHz, CDCl₃) δ 182.6, 181.6, 153.6, 149.5, 145.9, 137.6, 135.0, 134.2, 128.3, 128.0, 124.1, 124.0, 121.3, 120.0, 111.7, 103.5, 102.9. HRMS (TOF MS ES+) for C₁₇H₁₂N₃O₂⁺ (MH+) calcd. 290.0930, found 290.0900.

7-amino-2-(8-quinolinyl)quinoline-5,8-dione (22)

The general procedure was used to obtain 55 mg (71%) of a brown solid. R_f= 0.29 (5% MeOH:CH₂Cl₂); m.p. 243–245°C, recrystallized from MeOH. ¹H NMR (500 MHz, CD₃OD) δ 8.92 (dd, J = 4.2, 1.8 Hz, 1H), 8.49 (d, J = 8.1 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H), 8.33 (dd, J = 8.3, 1.8 Hz, 1H), 8.21 (dd, J = 7.2, 1.5 Hz, 1H), 8.02 (dd, J = 8.2, 1.4 Hz, 1H), 7.75 (dd, J = 8.1, 7.3 Hz, 1H), 7.53 (dd, J = 8.3, 4.2 Hz, 1H), 6.06 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 182.8, 180.1, 160.5, 150.3, 150.2, 146.2, 145.2, 136.7, 136.7, 133.2, 131.6, 131.5, 129.8, 129.1, 128.4, 126.3, 121.2, 102.4. HRMS (TOF MS ES+) for C₁₈H₁₂N₃O₂⁺ (MH+) calcd. 302.0930, found 302.0939.

7-amino-2-(2-pyridinyl)quinoline-5,8-dione (23)

The general procedure was used to obtain 16 mg (76%) of a red solid. R_f= 0.25 (20% MeOH:CH₂Cl₂), recrystallized from MeOH. ¹H NMR (500 MHz, DMSO) δ 8.75 (d, J = 4.1 Hz, 1H), 8.72 (d, J = 8.2 Hz, 1H), 8.50 (d, J = 7.9 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H), 8.05 (t, J = 7.7 Hz, 1H), 7.58 – 7.53 (m, 1H), 5.89 (s, 1H). HRMS (TOF MS ES+) for C₁₄H₁₀N₃O₂⁺ (MH+) calcd. 252.0773 found 252.0749.

7-Amino-2-(2-pyrrolyl)quinoline-5,8-dione (24)

The general procedure was used to obtain 11 mg (78%) of a red solid. R_f= 0.37 (5% MeOH:CH₂Cl₂); m.p. 230°C (decomposes), recrystallized from MeOH. ¹H NMR (500 MHz, CDCl₃) δ 8.23 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 2.5, 1.3 Hz, 1H), 6.91 (dd, J = 3.7, 1.3 Hz, 1H), 6.32 (dd, J = 3.7, 2.6 Hz, 1H), 5.97 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 183.0, 181.7, 153.5, 149.4, 145.7, 133.9, 129.9, 126.6, 122.7, 122.3, 110.8, 110.3, 102.5. HRMS (TOF MS ES+) for C₁₃H₁₀N₃O₂⁺ (MH+) calc. 240.0773, found 240.0779.

ABBREVIATIONS

NQO1

NAD(P)H: quinone oxidoreductase 1

MeOH

Methanol

BnBr

Benzyl bromide

DME

1, 2-Dimethoxyethane

dppf

1,1’-Bis(diphenylphosphino)ferrocene

E_pc

cathodic peak potential

E_pa

anodic peak potential