Concise Synthesis of Nannozinone A: An Unusual Dihydropyrrolopyrazinone Metabolite From Myxobacteria

Abstract

Nannozinone A is an unprecedented dihydropyrrolopyrazinone metabolite derived from myxobacterial Nannocystis pusilla strain MNa10913. In this paper, the synthesis of nannozinone A was disclosed for the first time. Nannozinone A was readily obtained in 6 steps and in 36% overall yield from commercially available N-Boc-propinal. Key feature of the synthesis includes intramolecular imine formation and subsequent spontaneous oxidation toward the unique dihydropyrrolopyrazinone skeleton. This concise synthetic route would be helpful to prepare natural dihydropyrrolopyrazinones and their derivatives.

Myxobacteria (also known as slime bacteria) are rod-shaped, Gram-negative bacteria that show unique features such as gliding motility on solid surfaces and formation of complex fruiting bodies. ¹ They have been considered as one of the proficient producers of structurally diverse and unique bioactive natural compounds that might contribute to the discovery of novel therapeutic molecules. ^2,3 Recently, Müller and his co-workers isolated unprecedent natural pyrazinones including nannozinone A (1) from a Myxobacteria Nannocytis pusilla, strain MNa10913. ⁴ It was revealed that these pyrazinones possess cytotoxic properties against several micro-organisms. For instance, 1 showed antifungal activity against Candida albicans with a Minimum Inhibitory Concentration (MIC) value of 33.3 µg/mL and moderate antibacterial activities against several Gram-positive bacteria. They also claimed that the dihyropyrrolpyrazinone core of 1 was a rarely observed scaffold in natural products. The promising therapeutic properties of 1 and its structural uniqueness strongly prompted to establish a concise synthetic route for 1 that can be utilized for further derivatization and biological studies.

As shown in Figure 1, it was envisioned that the pyrazinone moiety of nannozinone A (1) could be synthesized from glycinamide 2 via intramolecular condensation and subsequent oxidation of resulting dihydropyrazinone ring. The glycinamide 2, which comprised all the functional groups of 1, was expected to be synthesized from proline derivatives such as N-Boc-prolinal 3 via ketone formation followed by amidation.

Figure 1

Nannozinone A (1) and its retrosynthetic analysis.

As shown in Scheme 1, the synthesis of nannozinone A (1) was commenced with the preparation of ketone 5. Initially, nucleophilic addition of phenethyl magnesium bromide to corresponding Weinreb amide was attempted to prepare 5 directly, ⁵ but we could not obtain the desired compound. On the other hand, the ketone 5 was readily prepared by nucleophilic addition of the Grignard reagent to commercially available N-Boc-prolinal 3 and subsequent Swern oxidation of resulting alcohol 4 in moderate yields (87% and 85%, respectively). ⁶ Boc-deprotection of 5 with trifluoroacetic acid afforded secondary amine 6, which was subjected to amidation with Boc-glycine.

Scheme 1

Synthesis of nannozinone A (1).

To obtained amide 7 from amine 6, various reaction conditions were attempted such as amidation with Boc-glycine using 1-(3-dimethylaminopropyl)-N-ethylcabodiimide hydrochloride (EDC · HCl) ⁷ and condensations with corresponding acid chloride ⁸ or anhydride. ⁹ And it was observed that condensation with mixed anhydride led to maximum yield (75% in 2 steps) in the synthesis of amide 7. Boc-deprotection of 7 using hydrochloric acid afforded amine 8, which was subjected to intramolecular condensation to construct key dihydropyrrolopyrazinone skeleton. And without further oxidation steps, we could obtain the purposed nannozinone A (1) in moderate yield (66% in 2 steps) from crude 8 by heating in pyridine for 12 hours. ¹⁰ The spectral data of the synthesized 1 were consistent with the previously reported data. This result could be explained by spontaneous air oxidation ¹¹ of the dihydro-pyrazinone ring of intermediate 9 that led to the formation of the more stable aromatic pyrazinone ring of 1.

In summary, this paper describes a concise synthesis of nannozinone A (1), an unprecedented dihydropyrrolopyrazinone metabolite derived from myxobacterial Nannocystis pusilla strain MNa10913. Nannozinone A (1) was readily obtained in 6 steps, and in 36% overall yield from commercially available N-Boc-propinal. Key feature of the synthesis includes intramolecular imine formation and subsequent spontaneous oxidation toward pyrazinone skeleton. It would be helpful in the synthesis of derivatives of nannozinone A and construction of chemical library of dihydropyrrolopyrazinones.

Experimental

Unless noted otherwise, all starting materials and reagents were obtained commercially and were used without further purification. All solvents utilized for routine product isolation and chromatography were of reagent grade and glass distilled, and reaction flasks were dried at 100°C before use. Air- and moisture-sensitive reactions were performed under argon atmosphere. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck) with the indicated solvents. Thin-layer chromatography was performed using 0.25 mm silica gel plates (Merck). Mass spectra were obtained using an Agilent 6530 Quadrupole time of flight (Q-TOF) unit. Infrared spectra were recorded on a JASCO FT-IR-4200 spectrometer. UV-VIS spectra were obtained using a Shumadzu UV-1650. ¹H and ¹³C spectra were recorded on a Bruker ADVANCE 800 (800 MHz) spectrometer in deuteriochloroform (CDCl₃). Chemical shifts are expressed in parts per million (ppm, δ) downfield from tetramethylsilane and are referenced to the deuterated solvent. ¹H NMR data are reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; bs, broad singlet; and/or multiple resonances), number of protons, and coupling constant in Hertz (Hz).

Tert-Butyl 2-(1-Hydroxy-3-Phenylpropyl)pyrrolidine-1-Carboxylate (4)

To a solution of Boc-prolinal 3 (1.31 g, 6.58 mmol) in Tetrahydronfuran (THF; 22 mL), phenethyl magnesium chloride solution (1.0 M in THF, 7.2 mL) was added slowly at 0°C. After 90 minutes, the reaction mixture was quenched with water and diluted with EtOAc. The organic layer was washed with water and brine, dried over MgSO₄, and concentrated in vacuo. Purification of the residue via flash column chromatography on silica gel (EtOAc/n-hexane = 1:5) afforded diastereomeric mixture of alcohol 4 (57:43 d.r. [determined by isolation yields], 1.74 g, 87%) as a colorless oil.

Rf: 0.35, 0.45 (EtOAc/n-hexane = 1:2).

IR (thin film, neat): 3417, 2975, 1693, 1667, 1404, 1168 cm⁻¹.

¹H NMR (800 MHz, CDCl₃); diastereromer 4a: δ 7.33-7.27 (m, 2H, Ar-H), 7.25-7.21 (m, 2H, Ar-H), 7.18-7.21(m, 1H, Ar-H), 5.00 (bs, 1H, OH), 3.86-3.82 (m, 1H, CH), 3.48 (bs, 2H, -CH₂N), 3.28-3.25 (m, 1H, ArCH₂), 2.92-2.90 (m, 1H, ArCH₂), 2.74-2.70 (m, 1H, CH), 1.96-1.76 (m, 5H, CH₂), 1.67-1.65 (m, 1H, CH₂), 1.48-1.47 (m, 9H, tert-Bu); diastereromer 4b: δ 7.29-7.27 (m, 2H, Ar-H), 7.22-7.11 (m, 2H, Ar-H), 7.19-7.17 (m, 1H, Ar-H), 4.37 (bs, 1H, OH), 3.99-3.74 (m, 2H), 3.52-3.21(m, 2H), 2.98 (s, 1H), 2.67-2.64 (m, 1H, CH), 1.99-1.83 (m, 3H, CH₂), 1.74-1.69 (m, 1H, CH₂), 1.66-1.62 (m, 2H, CH₂), 1.46-1.38 (m, 9H, tert-Bu).

¹³C NMR (200 MHz, CDCl₃); diastereromer 4a: δ 156.8 (1C, C=O), 142.9 (1C, Ph), 128.6 (2C, Ph), 128.5 (2C, Ph), 125.8 (1C, Ph), 80. 2 (1C, OC(CH₃)₃), 73.5 (1C, CH), 63.4 (1C, CH), 48.3 (1C, NCH₂), 34.1 (1C, CH₂), 32.9 (1C, CH₂), 28.6 (3C, C(CH₃)₃, 28.2 (1C, CH₂), 24.4 (1C, CH₂).

High resolution mass spectroscopy (HRMS; Q-ToF): Calcd for C₁₈H₂₈NO₃ ⁺ (M+H⁺): 306.2064. Found: 306.2069.

Tert-Butyl 2-(3-Phenylpropanoyl)Pyrrolidine-1-Carboxylate (5)

To a solution of oxalyl chloride (0.4 mL, 4.26 mmol) in CH₂Cl₂ (16 mL), dimethyl Sulfoxide (0.6 mL, 8.51 mmol) was added at −78°C. After 30 minutes, a solution of alcohol 4 (650 mg, 2.13 mmol) in CH₂Cl₂ (5 mL) was added to the reaction mixture. After 2 hours, Et₃N (2.4 mL, 17.03 mmol) was added to the reaction mixture, and the reaction mixture was stirred for 4 hours at ambient temperature. The reaction mixture was quenched with saturated aqueous NaHCO₃ solution and diluted with CH₂Cl₂. The organic layer was washed with water and brine, dried over MgSO₄, and concentrated in vacuo. Purification of the residue via flash column chromatography on silica gel (EtOAc/n-hexane = 1:5) afforded ketone 5 (547 mg, 85%) as a yellow oil.

Rf: 0.35 (EtOAc/n-hexane = 1:4).

IR (thin film, neat): 2976, 1727, 1697, 1395, 1165, 701 cm⁻¹.

¹H NMR (800 MHz, CDCl₃, mixture of rotamers) δ 7.28-7.25 (m, 2H, Ar-H), 7.20-7.17 (m, Ar-H), 4.34-4.33 and 4.22-4.20 (m, 1H, -NCH), 3.53-3.40 (m, 2H, -NCH₂), 2.93-2.89 (m, 2H, ArCH₂), 2.81-2.68 (m, 2H, -CH₂-), 2.13-2.11 and 2.03-2.02 (m, 1H, -CH₂-), 1.81-1.78 (m, 2H, -COCH₂), 1.68-1.65 (m, 1H, -CH₂-), 1.46(s, 4H, tert-Bu), 1.38(s, 5H, tert-Bu).

¹³C NMR (200 MHz, CDCl₃, mixture of rotamers) δ 209.4/209.2 (1C, C=O), 154.8/154.0 (1C, C=O), 141.5/141.2 (1C, Ph), 128.7/128.6/128.5 (4C, Ph), 126.3/126.2 (1C, Ph), 80.3/80.0 (1C, OC(CH₃)₃), 65.5/65.0 (1C, NCHCO), 47.0/46.9 (1C, NCH₂), 41.0/40.3 (1C, COCH₂), 29.9/29.5 (1C, CH₂), 28.7/28.6 (1C, CH₂), 28.5/28.4 (3C, C(CH₃)₃, 24.5/23.8 (1C, CH₂).

HRMS (Q-ToF): Calcd for C₁₈H₂₆NO₃ ⁺ (M+H⁺): 304.1907. Found: 304.1914.

Tert-Butyl (2-Oxo-2-(2-(3-Phenylpropanoyl)Pyrrolidin-1-yl)Ethyl) Carbamate (7)

To a solution of Boc-pyrrolidine 3 (300 mg, 0.98 mmol) in CH₂Cl₂ (1 mL), trifluoroacetic acid (0.76 mL, 9.81 mmol) was added slowly at ambient temperature. After 1 hour, the reaction mixture was concentrated in vacuo. Then, the resulting amine 6 was used without further purification. To a solution of Boc-glycine (258 mg, 1.47 mmol) and Et₃N (0.24 mL, 1.77 mmol) in CH₂Cl₂ (1 mL), pivaloyl chloride (0.14 mL, 1.18 mmol) was added at 0°C. After 3 hours, a solution of amine 6 in CH₂Cl₂ (1 mL) and Et₃N (0.54 mL, 3.92 mmol) was added to the reaction mixture at same temperature. After being stirred at ambient temperature for 2 hours, the reaction mixture was quenched with saturated aqueous NaHCO₃ solution and diluted with CH₂Cl₂. The organic layer was washed with water and brine, dried over MgSO₄, and concentrated in vacuo. Purification of the residue via flash column chromatography on silica gel (EtOAc/n-hexane/CH₂Cl₂ = 1:2:0.1) afforded amide 7 (268 mg, 75%) as an amorphous yellowish solid.

MP: 101°C-104°C.

Rf: 0.15 (EtOAc/n-hexane = 1:2).

IR (neat): 3421, 3061, 2977, 1715, 1656, 1453, 1169 cm⁻¹.

¹H NMR (800 MHz, CDCl₃) δ 7.29-7.25 (m, 2H, Ar-H), 7.20-7.16 (m, 3H, Ar-H), 5.38 (bs, 1H, NHBoc), 4.59-4.57 (m, 1H, -NCH), 4.00-3.97 (m, 1H, -NCH₂), 3.91-3.88 (m, 1H, -NCH₂), 3.55-3.52 (m, 1H, -NCH₂), 3.46-3.43 (m, 1H, -NCH₂), 2.93-2.91 (m, 2H, ArCH₂), 2.88-2.86 (m, 1H, -CH₂-), 2.82-2.78 (m, 1H, -CH₂-), 2.05-2.02 (m, 1H, -CH₂-), 1.97-1.91 (m, 2H, -COCH₂), 1.72-1.69 (m, 1H, -CH₂-), 1.44-1.43 (m, 9H, tert-Bu).

¹³C NMR (200 MHz, CDCl₃) δ 207.4 (1C, C=O), 167.4 (1C, C=O), 155.9 (1C, C=O), 141.1 (1C, Ph), 128.6/128.6/128.5/128.5 (4C, Ph), 126.3 (1C, Ph), 79.8(1C, OC(CH₃)₃,), 64.9/64.8 (1C, NCHCO), 46.2 (1C, NCH₂), 43.1 (1C, COCH₂), 41.9 (1C, COCH₂), 29.5 (1C, CH₂), 28.5 (3C, C(CH₃)₃), 27.9/27.7 (1C, CH₂), 24.7 (1C, CH₂).

HRMS (Q-ToF): Calcd for C₂₀H₂₉N₂O₄ ⁺ (M+H⁺): 361.4615. Found: 361.4620.

Nannozinone A (1)

To a solution of N-Boc-ethylcarbamate 7 (30 mg, 83 μmol) in dioxane/EtOH (1:1, 1 mL), 4 N HCl solution (0.21 mL, 0.83 mmol) was added at ambient temperature. After being stirred for 8 hours at same temperature, the reaction mixture was concentrated in vacuo. Then, the resulting amine 8 was used without further purification. After dissolving the crude amine 8 in pyridine (0.4 mL), the reaction mixture was stirred for 12 hours at 60°C in an open air, quenched with water, and diluted with CH₂Cl₂. The organic layer was washed with 1 N HCl, water, and brine; dried over MgSO₄; and concentrated in vacuo. Purification of the residue via flash column chromatography on silica gel (EtOAc/n-hexane/CH₂Cl₂ = 2:1:0.1) afforded nannozinone A (1) (13.2 mg, 66%) as an amorphous yellow solid.

MP: 112°C-115°C.

Rf: 0.2 (EtOAc/n-hexane = 2:1).

IR (neat): 2925, 1657, 1596, 1171, 702 cm⁻¹.

UV (MeOH) λmax (log ε) 233 (4.03), 333 (3.78).

¹H NMR (800 MHz, CDCl₃) δ 8.05 (s, 1H, N=CH), 7.25-7.23 (m, 2H, Ar-H), 7.19-7.17 (m, 1H, Ar-H), 7.06-7.05 (m, 2H, Ar-H), 4.05 (t, 2H, J = 7.4 Hz, NCH₂), 2.95 (t, 2H, J = 7.3 Hz, ArCH₂), 2.80 (t, 2H, J = 7.3Hz, -CH₂-), 2.57 (t, 2H, J = 7.7 Hz, -CH₂-), 2.00 (quin, 2H, J = 7.6 Hz, -CH₂-).

¹³C NMR (200 MHz, CDCl₃) δ 155.5 (1C, C=O), 145.0 (1C, N=CH), 141.1 (1C, Ar), 139.4 (1C, NC=C), 130.3 (1C, NC=C), 128.7 (2C, Ar), 128.4 (2C, Ar), 126.1 (1C, Ar) , 48.8 (1C, NCH₂), 35.1 (1C, ArCH₂), 34.9 (1C, CH₂), 29.3 (1C, CH₂), 20.9 (1C, CH₂).

HRMS (Q-ToF): Calcd for C₁₅H₁₇N₂O⁺ (M+H⁺): 241.1335. Found: 241.1339.