Attempted Synthesis of the Pseudomonas aeruginosa Metabolite 2-Benzyl-4(1H)-quinolone and Formation of 3-Methylamino-2-(2-nitrobenzoyl)-4H-naphthalen-1-one as an Unexpected Product

Abstract

The unusual reactivity of key enamine intermediates led to the formation of 3-methylamino-2-(2-nitrobenzoyl)-4H-naphthalen-1-one as an unexpected product in an attempted synthesis of the P. aeruginosa metabolite 2-benzyl-4(1H)-quinolone. Although the synthesis of the natural product has not been successful, this methodology allows for the easy preparation of novel derivatives carrying a carboxamide moiety at the C3 position.

1. Introduction

2-Alkyl-4-(1H)-quinolones are an important class of bacterial secondary metabolites produced by various Pseudomonas and Burkholderia species [1]. While some of them, especially those that are oxidized at the quinolone nitrogen, act as antibiotics and directly suppress competing microorganisms [2], others have the more intricate function of chemical signals, coordinating the group behavior of their producers [3]. Such cell population density-dependent regulation is known as “quorum sensing” and is a matter of great research interest, as it may hold the key to novel antibacterial therapies [4,5]. Since the pioneering works of Wells et al., who for the first time isolated alkylquinolones from cultures of Pseudomonas aeruginosa and determined their structure by synthesis, [6,7] many other bacterial 2-alkyl-4(1H)-quinolones with various substituents in the heterocyclic ring have been discovered and synthesized [1,8]. The structural diversity of this class of natural compounds is impressive, but most often, at the C2 position of the quinolone core, they feature linear carbon chains of varying lengths and degrees of unsaturation. 2-Benzyl-4(1H)-quinolone (Figure 1) is one of the more unusual representatives of this class, which was isolated from P. aeruginosa BD06-03 by Clark et al. in 2020 [9] and synthesized later by the same research group [10].

2. Results

We considered the P. aeruginosa metabolite 2-benzyl-4(1H)-quinolone to be an interesting target and a good testing ground for our recently published method for the preparation of 2-alkyl-4(1H)-quinolones (Scheme 1) [11]. At the core of this method are intermediates III, which are readily prepared from the corresponding β-keto amides I. Compounds III are susceptible to decarbamoylation upon heating in H₃PO₄, which we have observed in a number of examples to give the corresponding β-enaminoketones IV in high yields [11,12]. Finally, the reduction of the nitro group in IV is followed by spontaneous cyclization to the corresponding 2-alkyl-4(1H)-quinolone V, with the elimination of an amine (R³-NH₂). The type of the R³ substituent is not of decisive importance to this method, so it is most convenient to use an inexpensive lower aliphatic amine in this auxiliary role.

With this in mind, three different γ-phenyl β-keto amides 1a–c were prepared [13] and condensed with methylamine to provide the corresponding β-enamino amides 2a–c in quantitative yields (Scheme 2). The acylation of 2a–c with 2-nitrobenzoyl chloride proceeded smoothly and resulted in the corresponding intermediates 3a–c with 65–74% yield.

In the next stage, however, the attempted decarbamoylation of 3 in neat H₃PO₄ at 60 °C did not give the expected result with any of the intermediates 3a–c. In all three cases, cyclization to the same product 5 was observed, with the elimination of the corresponding amine (RNH₂) and no trace of the expected product 4 (Scheme 3). The yields of the unexpected product 3-methylamino-2-(2-nitrobenzoyl)-4H-naphthalen-1-one 5 were consistently found to be within the range of 73–78%, regardless of the R substituent. The structure of 5 was elucidated on the basis of NMR and MS data (see Supplementary Materials) and could be a result of either an intramolecular Friedel–Crafts reaction or a 6π–electrocyclic process, occurring in a tautomer of 3, possessing the prerequisite conjugated π system. Interestingly, the protonated molecular ion of 5 was observed as the base peak in the mass spectra of intermediates 3a and 3c, with abundance exceeding that of the actual [M+H]⁺ ions of 3. The base peak for intermediate 3b corresponded to its own [M+H]⁺, but the protonated molecular ion of 5 was still present with high abundancy (50%), indicating that the cyclization depicted in Scheme 3 is also a favored process under the conditions of positive electrospray ionization.

Even though this unexpected reactivity of the benzyl-substituted intermediates 3 did not allow us to accomplish the planned synthesis of 2-benzyl-4(1H)-quinolone, we were still in the position to prepare derivatives of this natural product, carrying a carboxamide moiety at the C3 position of the quinolone core. This was easily achieved by the reduction of the nitro group in intermediates 3 with Zn in AcOH/CH₂Cl₂, which was followed by spontaneous cyclization to carboxamide derivatives 6 with 71–74% yield (Scheme 4).

In conclusion, we encountered an unexpected reactivity of benzyl-substituted intermediates 3, which limits the scope of a previously published methodology [11]. The methodology, however, still allowed 3-carboxamide derivatives of 2-benzyl-4(1H)-quinolone to be obtained in high yields. The unexpected product 3-methylamino-2-(2-nitrobenzoyl)-4H-naphthalen-1-one (5) was also obtained in a good yield, and probably other 3-alkylamino analogs of 5 are within reach by this route.

3. Materials and Methods

Compounds 1 were prepared according to our previously published method [13]. All other reagents and solvents were purchased from Sigma-Aldrich, Darmstadt, Germany, and were used as supplied. NMR spectra were generated on a Bruker NEO 400 (400/100 MHz ¹H/¹³C) spectrometer. Chemical shifts (δ, ppm) were downfield from TMS. TLC was performed on aluminum-backed Silica gel 60 sheets (Merck, Darmstadt, Germany) with KMnO₄ staining. Melting point measurements were carried out in capillary tubes on KRÜSS M5000 automatic mp meter and were not corrected. Mass spectral measurements were performed on a Thermo Fisher Scientific Q Exactive Plus high-resolution mass spectrometer with a heated electrospray ionization source (HESI-II). Petrol refers to the fraction 40–60 °C, NMM refers to N-methylmorpholine, and DMAP refers to 4-dimethylaminopyridine.

Synthesis of β-enaminoamides 2, general procedure: Methylamine (40% aq. solution, 0.45 mL, 5.2 mmol) was added to a solution of the corresponding β-keto amide 1 (5 mmol) in CH₂Cl₂ (15–20 mL). The mixture was magnetically stirred in a tightly closed vial for 4 h at r.t.; then, anhydrous sodium sulfate was added, and stirring was continued overnight at r.t. Then, the sulfate was filtered off, and the solvent was evaporated under reduced pressure. Compounds 2 crystallized upon trituration with a small volume of diethyl ether and were used in the next step without further purification.

3-Methylamino-4-phenylbut-2-enoic acid phenylamide (2a): m.p. 123–124 °C; ¹H-NMR (400 MHz, CDCl₃, δ ppm, J Hz): 2.83 (d, J = 5.3, 3H), 3.57 (s, 2H), 4.41 (s, 1H), 6.71 (br s, 1H), 7.03 (m, 1H), 7.27–7.31 (m, 5H), 7.36 (m, 2H), 7.47 (m, 2H), 9.24 (br s, 1H); ¹³C-NMR (100 MHz, CDCl₃, δ ppm): 29.53, 38.74, 86.19, 119.61, 122.82, 126.79, 128.72, 128.84, 136.51, 139.23, 162.60, 169.16; HRMS m/z (ES+): calcd. for C₁₇H₁₉N₂O⁺ [M+H]⁺ 267.1492, found 267.1490.

3-Methylamino-4-phenylbut-2-enoic acid 4-methoxyphenylamide (2b): m.p. 119–120 °C; ¹H-NMR (400 MHz, CDCl₃, δ ppm, J Hz): 2.82 (d, J = 5.3, 3H), 3.56 (s, 2H), 3.79 (s, 3H), 4.38 (s, 1H), 6.60 (br s, 1H), 6.84 (m, 2H), 7.28 (m, 3H), 7.35 (m, 4H), 9.20 (br s, 1H); ¹³C-NMR (100 MHz, CDCl₃, δ ppm): 29.51, 38.76, 55.49, 85.91, 114.07, 121.91, 126.74, 128.69, 129.56, 132.22, 136.61, 155.69, 162.21, 169.27; HRMS m/z (ES+): calcd. for C₁₈H₂₁N₂O₂⁺ [M+H]⁺ 297.1598, found 297.1594.

3-Methylamino-4-phenylbut-2-enoic acid 4-chlorophenylamide (2c): m.p. 110–111 °C; ¹H-NMR (400 MHz, CDCl₃, δ ppm, J Hz): 2.83 (d, J = 5.3, 3H), 3.57 (s, 2H), 4.37 (s, 1H), 6.70 (br s, 1H), 7.23 (m, 2H), 7.28 (m, 3H), 7.35 (m, 2H), 7.41 (m, 2H), 9.21 (br s, 1H); ¹³C-NMR (100 MHz, CDCl₃, δ ppm): 29.55, 38.70, 85.97, 120.59, 126.85, 127.52, 128.72, 128.75, 128.78, 136.34, 137.87, 163.02, 168.94; HRMS m/z (ES+): calcd. for C₁₇H₁₈ClN₂O⁺ [M+H]⁺ 301.1102, found 301.1107.

Synthesis of α-(2-nitrobenzoyl)-β-enaminoamides 3, general procedure [11]: 2-nitrobenzoyl chloride (0.13 mL, 1 mmol) was slowly added to a magnetically stirred solution containing the corresponding enaminoamide 2 (1 mmol), 4-methylmorpholine (0.11 mL, 1 mmol) and DMAP (24 mg, 0.2 mmol) in CH₂Cl₂ (20 mL). The mixture was left to stir for 2 h at r.t., and then it was transferred to a separatory funnel with an additional 30 mL of CH₂Cl₂ and washed with dilute aqueous (20:1) HCl. The aqueous layer was extracted again with 25 mL of CH₂Cl₂, the combined organic layers were dried with anhydrous sodium sulfate, the drying agent was removed by filtration, and the solvent was distilled off. The residue was purified by column chromatography on silica gel, using Et₂O–petrol 1:1 as the eluent.

3-Methylamino-2-(2-nitrobenzoyl)-4-phenylbut-2-enoic acid phenylamide (3a): m.p. 125–126 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 2.91 (d, J = 5.3, 3H), 3.93 (s, 2H), 6.93 (m, 1H), 7.14 (m, 2H), 7.25 (m, 3H), 7.37 (m, 4H), 7.50 (m, 1H), 7.59 (m, 1H), 7.69 (m, 1H), 8.00 (m 1H), 9.79 (br s, 1H), 11.49 (q, J = 5.3, 1H); ¹³C-NMR (100 MHz, DMSO-d6, δ ppm): 30.48, 35.68, 109.11, 119.71, 123.76, 124.31, 127.20, 128.88, 129.00, 129.12, 129.94, 134.09, 135.94, 137.75, 139.32, 146.34, 167.20, 167.36, 186.55; HRMS m/z (ES+): calcd. for C₂₄H₂₂N₃O₄⁺ [M+H]⁺ 416.1605, found 416.1605.

3-Methylamino-2-(2-nitrobenzoyl)-4-phenylbut-2-enoic acid 4-methoxyphenylamide (3b): m.p. 157–158 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 2.91 (d, J = 5.3, 3H), 3.64 (s, 3H), 3.91 (s, 2H), 6.71 (m, 2H), 7.09 (m, 2H), 7.26 (m, 1H), 7.35 (m, 2H), 7.40 (m, 2H), 7.52 (m, 1H), 7.58 (m, 1H), 7.69 (m, 1H), 8.00 (m, 1H), 9.61 (br s, 1H), 11.46 (q, J = 5.3, 1H); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 30.45, 35.64, 55.55, 114.02, 121.36, 124.29, 124.50, 127.18, 128.92, 129.02, 129.10, 129.44, 129.92, 131.12, 132.42, 133.98, 134.04, 135.99, 137.77, 146.33, 155.78, 166.77, 167.28, 186.48; HRMS m/z (ES+): calcd. for C₂₅H₂₄N₃O₅⁺ [M+H]⁺ 446.1710, found 446.1702.

3-Methylamino-2-(2-nitrobenzoyl)-4-phenylbut-2-enoic acid 4-chlorophenylamide (3c): m.p. 156–157 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 2.92 (d, J = 5.3, 3H), 3.91 (s, 2H), 7.19 (m, 2H), 7.25 (m, 3H), 7.35 (m, 4H), 7.50, (m, 1H), 7.57 (m, 1H), 7.69 (m, 1H), 7.99 (dd, J = 8.2, J = 1.0, 1H), 9.94 (br s, 1H), 11.48 (q, J = 5.3, 1H); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 30.51, 35.66, 109.00, 121.07, 124.32, 127.22, 127.29, 128.80, 128.89, 128.98, 129.12, 129.99, 134.11, 135.87, 137.63, 138.29, 146.27, 167.32, 167.40, 186.52; calcd. for C₂₄H₂₁ClN₃O₄⁺ [M+H]⁺ 450.1215, found 450.1220.

Synthesis of 3-methylamino-2-(2-nitrobenzoyl)-4H-naphthalen-1-one (5): Intermediate 3a, 3b, or 3c (1 mmol) was mixed with H₃PO₄ (4–5 g) in a glass vial. The mixture was heated to 60 °C and was magnetically stirred in the course of 2 h at this temperature. After the completion of the reaction, the vial was cooled to r.t. with tap water, and the contents were rinsed and poured into a separatory funnel with 50–70 mL of water. The product was extracted in CH₂Cl₂ (2×40 mL), the combined organic layers were dried (Na₂SO₄), and the solvent was removed under reduced pressure. Trituration and washing with a small volume of diethyl ether gave practically clean product 5 as a white solid: m.p. 174–175 °C; ¹H-NMR (400 MHz, CDCl₃, δ ppm, J Hz): 3.20 (d, J = 5.3, 3H, CH₃), 4.00 (s, 2H, CH₂), 7.19 (dd, J = 7.6, J = 1.2, 1H, C17-H), 7.28 (m, 1H, C6-H), 7.31 (m, 1H, C8-H), 7.45 (m, 1H, C7-H), 7.49 (m, 1H, C15-H), 7.65 (td, J = 7.5, J = 1.2, 1H, C16-H), 8.00 (dd, J = 7.9, J = 1.0, 1H, C9-H), 8.21 (dd, J = 8.3, J = 0.9, 1H, C14-H), 12.20 (br s, 1H, NH); ¹³C-NMR (100 MHz, CDCl₃, δ ppm): 30.14 (CH₃), 30.37 (CH₂, C4), 107.17 (C2), 123.93 (C14), 126.23 (C17), 126.90 (C9), 127.21 (C6), 127.51 (C8), 127.96 (C15), 131.84 (C7), 133.34 (C5), 134.11 (C16), 141.32 (C12), 144.94 (C13), 171.00 (C3), 181.11 (C1), 194.74 (C11); see Figure 2 for atom numbering; HRMS m/z (ES+): calcd. for C₁₈H₁₅N₂O₄⁺ [M+H]⁺ 323.1026, found 323.1025.

Synthesis of 2-Benzyl-4-quinolone-3-carboxamides 6, general procedure [11]: Zinc powder (1–1.5 g) was added to the corresponding nitro-intermediate 3 (1 mmol), dissolved in a mixture of CH₂Cl₂ (30 mL) and acetic acid (4 mL). The mixture was magnetically stirred for 24 h at r.t., and then the solids were filtered off with suction and rinsed thoroughly with CH₂Cl₂. The dichloromethane filtrate was transferred to a separatory funnel and extracted with water (50 mL) and then with a saturated aqueous solution of NaHCO₃ (25 mL). The organic phase was dried with anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed under reduced pressure. The crude products were purified by column chromatography on silica gel with Et₂O as the eluent, increasing polarity to Et₂O:MeOH 20:1 where necessary.

2-Benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid phenylamide (6a): m.p. 269–270 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 4.66 (s, 2H), 7.06 (m, 1H), 7.20 (m, 1H), 7.27–7.35 (m, 6H), 7.47 (m, 1H), 7.68 (m, 3H), 7.77 (m, 1H), 8.26 (m, 1H), 12.23 (s, 1H), 12.41 (br s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 38.28, 112.99, 118.84, 120.16, 123.63, 125.13, 125.29, 125.96, 126.90, 128.83, 128.88, 129.27, 133.33, 138.39, 139.00, 139.64, 156.15, 164.45, 176.93; HRMS m/z (ES+): calcd. for C₂₃H₁₉N₂O₂⁺ [M+H]⁺ 355.1441, found 355.1436.

2-Benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4-methoxyphenylamide (6b): m.p. 240–241 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 3.74 (s, 3H), 4.67 (s, 2H), 6.91 (m, 2H), 7.20 (m, 1H), 7.30 (m, 4H), 7.46 (m, 1H), 7.60 (m, 2H), 7.69 (m, 1H), 7.76 (m, 1H), 8.25 (dd, J = 9.4, J = 1.3, 1H), 12.07 (s, 1H), 12.36 (br s, 1H); ¹³C-NMR (100 MHz, DMSO-d6, δ ppm): 38.21, 55.63, 113.11, 114.39, 118.78, 121.59, 125.05, 125.27, 125.94, 126.88, 128.86, 132.84, 133.26, 138.45, 138.96, 155.64, 155.94, 164.01, 176.88; HRMS m/z (ES+): calcd. for C₂₄H₂₁N₂O₃⁺ [M+H]⁺ 385.1547, found 385.1550.

2-Benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4-chlorophenylamide (6c): m.p. 260–261 °C; ¹H-NMR (70 °C, 400 MHz, DMSO-d₆, δ ppm, J Hz): 4.70 (s, 1H), 7.20 (m, 1H), 7.25–7.37 (m, 6H), 7.47 (m, 1H), 7.70 (m, 3H), 7.76 (m, 1H), 8.28 (dd, J = 8.1, J = 1.1, 1H), 12.33 (s, 1H); ¹³C-NMR (70 °C, 100 MHz, DMSO-d₆, δ ppm): 38.37, 112.54, 118.82, 121.89, 125.13, 125.40, 125.99, 126.83, 127.25, 128.81, 129.05, 133.26, 138.35, 138.60, 139.02, 156.62, 164.57, 177.13; HRMS m/z (ES+): calcd. for C₂₃H₁₈ClN₂O₂⁺ [M+H]⁺ 389.1051, found 389.1046.