Concise Synthesis of Pseudane IX, Its N-Oxide, and Novel Carboxamide Analogs with Antibacterial Activity

Abstract

A four-step synthesis of the natural product pseudane IX, starting from 3-oxododecanoic acid phenylamide and including only one chromatographic purification, was accomplished with an overall yield of 52%. The same synthetic sequence, but with a controlled partial reduction of a nitro group in the penultimate intermediate, led to the N-oxide of pseudane IX (NQNO). A shortened three-step variation of the synthesis allowed for the preparation of novel carboxamide analogs of the natural product. An agar diffusion assay against six different bacterial strains revealed significant antibacterial activity of the novel analogs against S. aureus at a concentration of 100 µg/mL. One of the novel compounds showed a remarkably broad spectrum of antibacterial activity, comparable to that of the positive control NQNO.

1. Introduction

The natural product pseudane IX, or 2-nonyl-4-quinolone, was isolated for the first time by Hays et al. from cultures of Pseudomonas aeruginosa [1] and its structure was determined later by Wells, who accomplished the first synthesis of this compound [2]. Since then, pseudane IX has been isolated from various Pseudomonas species [3,4] and, also, from plants such as Vepris ampody (Rutaceae) [5] and Ruta angustifolia [6]. An interesting spectrum of biological activities has been reported for pseudane IX, including swarming motility inhibition in Bacillus atrophaeus [7], activity against Plasmodium falciparum [4,8] and other protozoa [8], and inhibition of Candida albicans biofilm formation [9]. Notably, pseudane IX has shown remarkable antiviral activity against the hepatitis C virus, exceeding that of the standard drug ribavirin [6]. The N-oxide of pseudane IX (NQNO) is also a known bacterial metabolite with antibiotic activity [3,10,11]. The natural sources of pseudane IX do not offer convenient isolation and sufficiently large amounts of this compound. For this reason, most of the above-cited biological studies relied on synthetic pseudane IX. The only method used for this purpose until now is based on the classic Conrad–Limpach reaction, in which an enamino ester undergoes ring closure at 270 °C in refluxing diphenyl ether [2,7,8,9,11]. The overall yield of this approach is low, and the harsh conditions impose certain limitations on the preparation of functionalized analogs. The potential of pseudane IX as a lead compound in the search for novel antimicrobials has motivated us to attempt a new synthesis of this natural product, by applying our recently published method for the preparation of 2-alkyl-4-quinolones [12]. This method is operationally simpler than the classic approach, employs milder reaction conditions, and has the additional advantage of providing access to 3-carboxamide analogs.

2. Results and Discussion

In order to synthesize a few structural analogs of pseudane IX along with the targeted natural product, we first prepared three different amides of 3-oxododecanoic acid (1a–c). This was performed by acylation/deacetylation of the corresponding commercially available acetoacetamides, following our published method [13]. The β-keto amides 1 were then condensed with ethylamine, to provide enamines 2, which were directly subjected to acylation with 2-nitrobenzoyl chloride (Scheme 1). This way, good yields of the intermediates 3 were obtained (80–85%). Next, a reduction of the nitro group in intermediates 3 with Zn in CH₂Cl₂/HOAc was carried out. This was accompanied by spontaneous cyclization of the reduced intermediates to the corresponding 2-nonyl-4-quinolone-3-carboxamide derivatives 4 (R² = H). Compared with our previous experiments on similar substrates with shorter alkyl chains [12], the full conversion of 3 to 4 here was slightly slower and required a larger excess of the reducing agent. Alternatively, controlled partial reduction of the nitro group under Pd- or Pt-catalyzed hydrogenation conditions led to the N-hydroxy derivatives 5 (R² = OH). Compounds 5a,b were obtained by Pd-catalyzed transfer hydrogenation of 3a,b with ammonium formate, while compound 3c was hydrogenated with H₂ over DMSO-inhibited Pt/Al₂O₃ [14] to give 5c without concomitant reduction at the C–Cl bond. This way, a set of six new analogs of the natural product were prepared (Scheme 1,

Table 1. Isolated yield of products 4 and 5, obtained according to Scheme 1.

Product	R1	R2	Conditions	Yield (%)
4a	C6H5	H	A	84
4b	p-MeOC6H4	H	A	80
4c	p-ClC6H4	H	A	72
5a	C6H5	OH	B	70
5b	p-MeOC6H4	OH	B	70
5c	p-ClC6H4	OH	C	82

).

The synthesis of pseudane IX required one additional decarbamoylation step after the acylation stage (Scheme 2). In theory, any of the nitrobenzoylated intermediates 3 should be susceptible to such decarbamoylation and would give the same β-enamino ketone 6 upon heating in neat H₃PO₄ [15]. However, we only used intermediate 3a for this purpose as it offered the best atom economy and the lowest overall price of the synthesis. This reaction was carried out by stirring compound 3a in neat H₃PO₄ for 2 h at 60 °C. It should be noted that the time needed for the completion of the reaction was longer than previously reported by us for similar substrates with shorter alkyl chains [12]. The β-enamino ketone 6 was obtained in an 86% yield and its ¹H NMR spectrum in DMSO-d₆ indicated a mixture of Z/E isomers in a ratio of 85/15. In the last step, the β-enamino ketone 6 successfully underwent a reduction of the nitro group with Zn in CH₂Cl₂/HOAc and the ensuing spontaneous cyclization completed the synthesis of pseudane IX in 72% yield (7, Scheme 2). To stop the reduction at the N-oxide level, we carried out atmospheric pressure hydrogenation of the β-enamino ketone 6 with H₂ over DMSO-inhibited Pt/Al₂O₃ [14]. Under these conditions, the N-oxide of pseudane IX (8) was cleanly obtained in 70% yield.

Depending on the method of isolation, compound 8 was obtained in two distinctly different forms. When the crude product was only triturated with diethyl ether, it solidified as a white powder with good solubility in DMSO and a mp of 103–104 °C. The NMR spectrum of this material was taken in DMSO-d₆ at 25 °C and was indicative of a 4-quinolinol-N-oxide tautomeric form (Scheme 3), with the C3-H signal appearing at 7.08 ppm. On the other hand, when compound 8 passed through a silica gel column with diethyl ether as the eluent, it crystallized as colorless needles with a mp of 146–147 °C and a very poor DMSO solubility at 25 °C. Because of the poor solubility, the NMR spectrum in DMSO-d₆ had to be run at 70 °C, and this time it clearly indicated an N-hydroxy-4-quinolone tautomeric form, with the C3-H signal appearing at 5.97 ppm (See Supplementary Information). A similar change was not observed in compound 7, which was registered only as the 4-quinolone tautomer, regardless of the isolation method.

The antibacterial activity of the novel analogs 4 and 5 was tested at a concentration of 100 µg/mL against a set of six bacterial strains, using the agar diffusion method. The known natural compounds pseudane IX (7) and its N-oxide (8) were also included in the assay for comparison. The N-oxide 8 is known for its antibiotic activity [10,11] and served as the positive control. The observed inhibition zones (

Table 2. Antibacterial assay of the synthesized compounds at 100 µg/mL in DMSO, with 60 µL loading in 6 mm agar wells.

	Sterile Zone Diameter (mm) 1
Product	E. coli ATCC 25922	E. coli ATCC 8739	S. aureus ATCC 25923	S. aureus ATCC 6538	Enterococcus faecalis ATCC 29212	B. subtilis NBIMCC 1208
4a	-	-	19	-	-	-
4b	-	-	11	-	-	-
4c	-	-	9	-	-	9
5a	-	15	25	-	-	15
5b	-	-	20	-	15	15
5c	15	15	19	16	15	15
7	-	-	14	-	-	9
8	14	15	27	14	14	20

¹ Including 6 mm well diameter.

) indicated higher activity of the N-hydroxy derivatives 5 compared with that of their reduced counterparts 4, with S. aureus ATCC 25923 being the most susceptible among the studied bacteria. Notably, there was a sharp contrast in the susceptibility of the other assayed S. aureus strain (ATCC 6538), which was moderately inhibited by only one of the novel compounds (5c). Compound 5c was also the one with the broadest activity spectrum, inhibiting all of the studied bacteria and approaching the activity of the natural antibiotic 8. Against the resilient S. aureus strain (ATCC 6538), 5c even outperformed 8 with a slightly larger inhibition zone.

In conclusion, we have accomplished a convenient synthesis of two natural products with interesting biological profiles—pseudane IX and its N-oxide. The overall yield over four steps, starting from the phenylamide of 3-oxododecanoic acid and involving only one chromatographic purification, is 52% and 50% respectively. We have also demonstrated that the synthetic method used for this purpose allows for easy access to novel carboxamide analogs of these compounds. A broad antibacterial spectrum was observed in one of the newly obtained analogs, providing a lead for further structural optimization.

3. Materials and Methods

All reagents and solvents were purchased from Sigma-Aldrich, Darmstadt, Germany, and were used as supplied. Petrol refers to the 40–60 °C fraction, HOAc refers to acetic acid, and NMM refers to N-methylmorpholine. NMR spectra were run on a Bruker NEO 400 (400/100 MHz ¹H/¹³C) spectrometer. Chemical shifts (δ, ppm) are downfield from TMS. TLC was performed on aluminum-backed Silica gel 60 sheets (Merck) with KMnO₄ staining. Melting point measurements were performed 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). The agar diffusion antibacterial assay was carried out in LBG agar, in 6 mm wells, with 60 µL loading of 100 µg/mL solutions of the tested compounds in DMSO, according to a published protocol [16].

The starting amides of 3-oxododecanoic acid (1a–c) were prepared according to a previously published method [13], then the conversion of 1 to α-C-acylated β-enamino amides 3 was achieved according to another published general procedure [12].

• 3-Ethylamino-2-(2-nitrobenzoyl)-dodec-2-enoic acid phenylamide (3a): yellowish oil; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.83 (t, J = 7.0, 3H, CH₃), 1.12–1.24 (m, 12H, 6 × CH₂), 1.27 (t, J = 7.2, 3H, NHCH₂CH₃), 1.60 (m, 2H, βCH₂), 2.45 (m, 2H, αCH₂), 3.50 (m, 2H, NHCH₂CH₃), 6.94 (m, 1H, ArH), 7.15 (m, 2H, ArH), 7.27 (m, 2H, ArH), 7.50 (m, 2H, ArH), 7.65 (td, J = 7.5, J = 1.2, 1H, ArH), 7.96 (dd, J = 8.2, J = 1.0, 1H, ArH), 9.70 (br s, 1H, CONH), 11.62 (t, J = 5.6, 1H, NHCH₂CH₃); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.38, 15.77, 22.53, 27.94, 28.81, 29.05, 29.16, 29.51, 29.73, 31.65, 38.04, 107.58, 119.62, 123.62, 124.27, 128.78, 128.83, 129.84, 133.90, 137.70, 139.47, 146.46, 167.14, 169.33, 186.00; HRMS m/z (ES+): calcd. for C₂₇H₃₆N₃O₄⁺ [M+H]⁺ 466.2700, found 466.2704.
• 3-Ethylamino-2-(2-nitrobenzoyl)-dodec-2-enoic acid 4-methoxyphenylamide (3b): yellowish oil; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.82 (t, J = 7.0, 3H, CH₃), 1.12–1.24 (m, 12H, 6 × CH₂), 1.26 (t, J = 7.2, 3H, NHCH₂CH₃), 1.60 (m, 2H, βCH₂), 2.44 (m, 2H, αCH₂), 3.49 (m, 2H, NHCH₂CH₃), 3.66 (s, 3H, OCH₃), 6.73 (m, 2H, ArH), 7.15 (m, 2H, ArH), 7.50 (m, 2H, ArH), 7.66 (td, J = 7.5, J = 1.2, 1H, ArH), 7.96 (dd, J = 8.2, J = 0.9, ArH), 9.52 (br s, 1H, CONH), 11.60 (t, J = 5.5, 1H, NHCH₂CH₃); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.37, 15.78, 22.54, 27.95, 28.83, 29.08, 29.18, 29.54, 29.69, 31.67, 38.01, 55.52, 113.98, 121.24, 124.25, 128.82, 129.81, 132.60, 133.85, 137.72, 146.46, 155.72, 166.70, 169.20, 185.94; HRMS m/z (ES+): calcd. for C₂₈H₃₈N₃O₅⁺ [M+H]⁺ 496.2806, found 496.2801.
• 3-Ethylamino-2-(2-nitrobenzoyl)-dodec-2-enoic acid 4-chlorophenylamide (3c): yellowish oil; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.82 (t, J = 7.1, 3H, CH₃), 1.10–1.22 (m, 12H, 6 × CH₂), 1.27 (t, J = 7.2, 3H, NHCH₂CH₃), 1.59 (m, 2H, βCH₂), 2.44 (m, 2H, αCH₂), 3.50 (m, 2H, NHCH₂CH₃), 7.21 (m, 2H, ArH), 7.32 (m, 2H, ArH), 7.49 (m, 2H, ArH), 7.65 (m, 1H, ArH), 7.96 (m, 1H, ArH), 9.87 (br s, 1H, CONH), 11.62 (t, J = 5.6, 1H, NHCH₂CH₃); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.37, 15.77, 22.54, 27.86, 28.74, 29.07, 29.14, 29.43, 29.64, 31.65, 38.07, 120.91, 124.28, 127.19, 128.78, 129.88, 133.93, 137.57, 138.46, 146.40, 167.28, 186.02; HRMS m/z (ES+): calcd. for C₂₇H₃₅ClN₃O₄⁺ [M+H]⁺ 500.2311, found 500.2318.
• Synthesis of 3-Ethylamino-1-(2-nitrophenyl)-dodec-2-en-1-one (6): Intermediate 3a (466 mg, 1 mmol) was mixed with anhydrous H₃PO₄ (5–6 g) in a glass vial. The mixture was stirred intensely for two hours at 60 °C, then 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. The crude product obtained this way was sufficiently clean to be used directly in the next stage, without further purification. An analytically pure sample can be obtained by column chromatography on silica gel with Et₂O:Petrol (1:1) as the eluent, increasing polarity to Et₂O:Petrol (2:1). Yield: 298 mg yellowish oil (86%). The ¹H NMR spectra of 6 in DMSO-d₆ indicate a Z/E isomeric mixture in a ratio of 85:15. Only NMR signals corresponding to the major Z isomer are listed below. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.85 (t, J = 7.0, 3H, CH₃), 1.19 (t, J = 7.2, 3H, NHCH₂CH₃), 1.23–1.39 (m, 12H, 6 × CH₂), 1.53 (m, 2H, βCH₂), 2.34 (m, 2H, αCH₂), 3.39 (m, 2H, NHCH₂CH₃), 5.35 (s, 1H, CH), 7.57–7.70 (m, 3H, ArH), 7.81 (m, 1H, ArH), 10.94 (t, J = 5.6, 1H, NHCH₂CH₃); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.39, 15.72, 22.56, 27.78, 29.12, 29.17, 29.24, 29.35, 31.74, 37.61, 92.26, 124.12, 129.11, 130.65, 132.79, 136.94, 148.93, 170.38, 184.65; HRMS m/z (ES+): calcd. for C₂₀H₃₁N₂O₃⁺ [M+H]⁺ 347.2329, found 347.2331.
• Synthesis of compounds 4, 5, 7, and 8 by reductive cyclization of intermediates 3 and 6. General procedure A (preparation of products 4a–c and 7): Zn powder (2 g, prewashed with 1% HCl, water, and acetone) was added to the corresponding nitro-intermediate 3 or 6 (1 mmol), dissolved in a mixture of CH₂Cl₂ (30 mL) and acetic acid (4 mL). The heterogeneous 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 was 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 products crystallized upon trituration with Et₂O. Where necessary, further purification can be performed by column chromatography on silica gel with Et₂O as the eluent, increasing polarity to Et₂O:CH₃OH 20:1.
• General procedure B (preparation of products 5a and 5b): To the corresponding nitro-intermediate 3 (100 mg) in CH₃OH (10–15 mL), HCOONH₄ (300 mg) and Pd on charcoal (10 mg, 10 w% Pd) were added. The mixture was magnetically stirred for 90 min. at r.t., and the catalyst was removed by vacuum filtration through a pad of celite on a sintered glass funnel. The celite was rinsed thoroughly with methanol, and the solvent was removed from the filtrate under reduced pressure. Then, water (50 mL) was added to the solid residue and the product was extracted in CH₂Cl₂ (3 × 20 mL). The combined organic layers were dried with anhydrous sodium sulfate, the drying agent was filtered off, and the solvent was removed under reduced pressure. The products crystallized upon trituration with Et₂O. Where necessary, further purification can be performed by column chromatography on silica gel with Et₂O as the eluent, increasing polarity to Et₂O:CH₃OH 20:1.
• General procedure C (preparation of products 5c and 8): The corresponding nitro-intermediate 3 or 6 (0.5 mmol) was dissolved in isopropanol (IPA) (2 mL). Then, DMSO (0.013 g, 0.012 mL), n-butylamine (0.037 g, 0.050 mL), and 5 wt% Pt/Al₂O₃ (0.010 g) were added to the solution. The air in the reaction vessel was evacuated and replaced with H₂ with the help of a three-way stopper. The H₂ atmosphere was kept with a balloon for the next 24 h, while the reaction mixture was magnetically stirred at 25 °C. Then, the catalyst was filtered off through a pad of celite on a sintered glass funnel, with thorough rinsing with IPA. The IPA was removed from the filtrate on a rotary evaporator under reduced pressure. To remove any residual n-butylamine and DMSO, dilute aqueous HCl was added to the residue and the product was extracted in CH₂Cl₂ (2 × 30 mL). The combined organic layers were dried with anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the oily residue solidified upon trituration with diethyl ether, providing practically clean products. Analytically pure samples were obtained by column chromatography on a short silica gel plug, using Et₂O as the eluent.
• 2-Nonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid phenylamide (4a): white solid, mp 192–193 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.83 (t, J = 6.9, 3H, CH₃), 1.17–1.33 (m, 10H, 5 × CH₂), 1.39 (m, 2H, γCH₂), 1.72 (m, 2H, βCH₂), 3.16 (m, 2H, αCH₂), 7.06 (m, 1H, ArH), 7.33 (m, 2H, ArH), 7.43 (m, 1H, ArH), 7.64–7.76 (m, 4H, ArH), 8.23 (dd, J = 8.2, J = 1.1, 1H, ArH), 12.17 (br s, 1H, NH), 12.20 (s, 1H, NH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.40, 22.56, 29.14, 29.31, 29.52, 29.88, 31.74, 33.59, 112.38, 118.61, 120.06, 123.49, 124.84, 125.15, 125.88, 129.22, 133.08, 138.85, 139.81, 158.87, 164.58, 176.70; HRMS m/z (ES+): calcd. for C₂₅H₃₁N₂O₂⁺ [M+H]⁺ 391.2380, found 391.2387.
• 2-Nonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4-methoxyphenylamide (4b): white solid, mp 150–151 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.82 (t, J = 6.9, 3H, CH₃), 1.17–1.33 (m, 10H, 5 × CH₂), 1.38 (m, 2H, γCH₂), 1.72 (m, 2H, βCH₂), 3.15 (m, 2H, αCH₂), 3.74 (s, 3H, OCH₃), 6.90 (m, 2H, ArH), 7.42 (m, 1H, ArH), 7.63 (m, 3H, ArH), 7.73 (m, 1H, ArH), 8.21 (dd, J = 8.1, J = 1.1, 1H, ArH), 12.03 (s, 1H, NH), 12.12 (br s, 1H, NH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.40, 22.56, 29.15, 29.17, 29.33, 29.52, 29.91, 31.75, 33.56, 55.61, 112.54, 114.34, 118.60, 121.47, 124.75, 125.14, 125.85, 132.99, 133.03, 138.88, 155.55, 158.63, 164.16, 176.62; HRMS m/z (ES+): calcd. for C₂₆H₃₃N₂O₃⁺ [M+H]⁺ 421.2486, found 421.2481.
• 2-Nonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4-chlorophenylamide (4c): white solid, mp 172–173 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.83 (t, J = 6.9, 3H, CH₃), 1.17–1.32 (m, 10H, 5 × CH₂), 1.38 (m, 2H, γCH₂), 1.71 (m, 2H, βCH₂), 3.13 (m, 2H, αCH₂), 7.37 (m, 2H, ArH), 7.43 (m, 1H, ArH), 7.65 (m, 1H, ArH), 7.74 (m, 3H, ArH), 8.21 (dd, J = 8.2, J = 1.1, 1H, ArH), 12.19 (br s, 1H, NH), 12.30 (s, 1H, NH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.39, 22.56, 29.14, 29.32, 29.49, 29.84, 31.74, 33.54, 112.24, 118.65, 121.54, 124.89, 125.11, 125.84, 126.96, 129.09, 133.12, 138.74, 138.87, 158.89, 164.73, 176.64; HRMS m/z (ES+): calcd. for C₂₅H₃₀ClN₂O₂⁺ [M+H]⁺ 425.1990, found 425.1989.
• 1-Hydroxy-2-nonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid phenylamide (5a): white solid, mp 162–163 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.84 (t, J = 7.0, 3H, CH₃), 1.17–1.32 (m, 10H, 5 × CH₂), 1.39 (m, 2H, γCH₂), 1.76 (m, 2H, βCH₂), 3.12 (m, 2H, αCH₂), 7.07 (m, 1H, ArH), 7.33 (m, 2H, ArH), 7.49 (m, 1H, ArH), 7.71 (m, 2H, ArH), 7.83 (m, 1H, ArH), 7.93 (m, 1H, ArH), 8.25 (dd, J = 8.1, J = 1.2, 1H, ArH), 11.30 (s, 1H, NH), 12.10 (br s, 1H, OH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.41, 22.56, 28.44, 29.00, 29.15, 29.25, 29.58, 31.73, 115.05, 115.66, 119.90, 123.61, 124.96, 125.52, 125.97, 129.16, 133.33, 139.86, 139.95, 155.74, 164.60, 173.62; HRMS m/z (ES+): calcd. for C₂₅H₃₁N₂O₃⁺ [M+H]⁺ 407.2329, found 407.2324.
• 1-Hydroxy-2-nonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4-methoxyphenylamide (5b): white solid, mp 163–164 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.84 (t, J = 6.9, 3H, CH₃), 1.15–1.32 (m, 10H, 5 × CH₂), 1.38 (m, 2H, γCH₂), 1.75 (m, 2H, βCH₂), 3.11 (m, 2H, αCH₂), 3.74 (s, 3H, OCH₃), 6.91 (m, 2H, ArH), 7.47 (m, 1H, ArH), 7.63 (m, 2H, ArH), 7.82 (m, 1H, ArH), 7.92 (m, 1H, ArH), 8.24 (m, 1H, ArH), 11.15 (s, 1H, NH), 12.13 (br s, 1H, OH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.40, 22.57, 28.44, 29.01, 29.17, 29.27, 29.59, 31.74, 55.61, 114.28, 115.16, 115.64, 121.32, 124.87, 125.51, 125.95, 133.09, 133.23, 139.95, 155.63, 164.13, 173.57; HRMS m/z (ES+): calcd. for C₂₆H₃₃N₂O₄⁺ [M+H]⁺ 437.2435, found 437.2434.
• 1-Hydroxy-2-nonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4-chlorophenylamide (5c): white solid, mp 182–183 °C; ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.83 (t, J = 7.0, 3H, CH₃), 1.14–1.30 (m, 10H, 5 × CH₂), 1.38 (m, 2H, γCH₂), 1.75 (m, 2H, βCH₂), 3.09 (m, 2H, αCH₂), 7.38 (m, 2H, ArH), 7.49 (m, 1H, ArH), 7.75 (m, 2H, ArH), 7.83 (m, 1H, ArH), 7.93 (m, 1H, ArH), 8.24 (dd, J = 8.1, J = 1.2, 1H, ArH), 11.40 (s, 1H, NH), 12.13 (br s, 1H, OH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.40, 22.57, 28.37, 28.96, 29.16, 29.24, 29.50, 31.73, 114.93, 115.70, 121.36, 125.02, 125.50, 125.94, 127.14, 129.07, 133.39, 138.80, 139.97, 155.68, 164.77, 173.53; HRMS m/z (ES+): calcd. for C₂₅H₃₀ClN₂O₃⁺ [M+H]⁺ 441.1939, found 441.1932.
• 2-Nonyl-1H-quinolin-4-one (7): white solid, mp 135–136 °C (Lit. [2] mp 138–139 °C, Lit. [3] mp 134 °C); ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.83 (t, J = 6.7, 3H, CH₃), 1.18–1.36 (m, 12H, 6 × CH₂), 1.66 (m, 2H, βCH₂), 2.58 (m, 2H, αCH₂), 5.92 (s, 1H, C3-H), 7.27 (m, 1H, ArH), 7.54 (m, 1H, ArH), 7.61 (m, 1H, ArH), 8.04 (m, 1H, ArH), 11.50 (br s, 1H, NH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.39, 22.55, 28.81, 28.97, 29.12, 29.19, 29.35, 31.72, 33.72, 108.09, 118.33, 123.16, 125.10, 125.22, 131.87, 140.62, 154.02, 177.33; HRMS m/z (ES+): calcd. for C₁₈H₂₆NO⁺ [M+H]⁺ 272.2009, found 272.2007.
• 2-Nonyl-1-oxyquinolin-4-ol (8, quinolinol tautomer): white solid, mp 103–104 °C (Lit. [10] mp 148–149 °C, Lit. [3] mp 132 °C); ¹H-NMR (400 MHz, DMSO-d₆, δ ppm, J Hz): 0.84 (t, J = 6.8, 3H, CH₃), 1.17–1.43 (m, 12H, 6 × CH₂), 1.75 (m, 2H, βCH₂), 3.09 (m, 2H, αCH₂), 7.08 (s, 1H, C3-H), 7.77 (m, 1H, ArH), 8.07 (m, 1H, ArH), 8.25 (m, 1H, ArH), 8.30 (m, 1H, ArH); ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 14.40, 22.55, 27.55, 29.13, 29.15, 29.29, 31.73, 31.75, 105.48, 117.31, 121.35, 124.25, 127.70, 134.82, 140.03, 158.60, 167.31; HRMS m/z (ES+): calcd. for C₁₈H₂₆NO₂⁺ [M+H]⁺ 288.1958, found 288.1963.
• 1-Hydroxy-2-nonyl-(1H)-quinolin-4-one (8, quinolone tautomer): white solid, mp 146–147 °C (Lit. [10] mp 148–149 °C, Lit. [3] mp 132 °C); ¹H-NMR (400 MHz, DMSO-d₆, 70 °C, δ ppm, J Hz): 0.87 (t, J = 6.9, 3H, CH₃), 1.25–1.43 (m, 12H, 6 × CH₂), 1.70 (m, 2H, βCH₂), 2.77 (m, 2H, αCH₂), 5.97 (s, 1H, C3-H), 7.36 (m, 1H, ArH), 7.72 (m, 1H, ArH), 7.86 (m, 1H, ArH), 8.12 (m, 1H, ArH), 11.43 (br s, 1H, OH); ¹³C-NMR (100 MHz, DMSO-d₆, 70 °C, δ ppm): 14.22, 22.43, 27.77, 29.02, 29.08, 29.12, 29.25, 31.22, 31.66, 107.13, 115.45, 123.72, 125.30, 125.40, 132.12, 141.09, 153.83; HRMS m/z (ES+): calcd. for C₁₈H₂₆NO₂⁺ [M+H]⁺ 288.1958, found 288.1954.