Elucidation of Racemization Process of Azaspirene Skeleton in

Nov 1, 2018 - Azaspirene and related congeners, which possess various biological activities, have a unique spirocyclic core structure. However, there ...
0 downloads 0 Views 2MB Size
Download PDF
Article Cite This: J. Org. Chem. 2018, 83, 14457−14464

pubs.acs.org/joc

Elucidation of Racemization Process of Azaspirene Skeleton in Neutral Aqueous Media Shun Hirasawa,† Ken Mukai,† Shinnosuke Sakai,† Shinnosuke Wakamori,†,⊥ Takahiro Hasegawa,† Kazunori Souma,‡ Nobuhiro Kanomata,*,†,‡ Narihito Ogawa,†,‡ Mamoru Aizawa,‡ and Makoto Emoto*,§ †

Department of Chemistry and Biochemistry, Waseda University, 3-4-1, Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan Department of Applied Chemistry, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan § Clinical Research Center and Division of Preventive Medicine, Fukuoka Sanno Hospital, International University of Health and Welfare, 3-6-45 Momochihama, Sawara-ku, Fukuoka 814-0001, Japan

J. Org. Chem. 2018.83:14457-14464. Downloaded from pubs.acs.org by STOCKHOLM UNIV on 12/18/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: Azaspirene and related congeners, which possess various biological activities, have a unique spirocyclic core structure. However, there are few studies on the chemical properties of (−)-azaspirene, despite the fact that it may provide important insights into unveiling the biosynthetic pathway. Here, we report a nine-step chemical synthesis of an azaspirene analogue with a new finding that the natural (−)-azaspirene skeleton easily racemizes in neutral aqueous media.

■

INTRODUCTION (−)-Azaspirene (1), isolated from the fungus Neosartorya sp. by Osada,1 possesses antiangiogenic properties2 that are widely recognized for their clinical usefulness in cancer, diabetic retinopathy, rheumatoid arthritis, and other angiogenesisrelated diseases.3−5 (−)-Azaspirene (1) is characterized by a highly oxygenated 1-oxa-7-azaspiro[4.4]non-2-ene-4,6-dione skeleton with benzyl and hexadienyl substituents. Structural similarities are found with other potent natural products,6−15 such as pseurotin A (2),16−20 pseurotin G (3),21 pseurotin A2,22,23 and cephalimysin A congeners.24−27 Cephalimysin A is one of the most potent natural products discovered to date, with strong cytotoxic activity (HL-60 cell line; IC50 9.5 nM).24 It was found from successful structure activity relationship (SAR) studies that 10-deoxypseurotin A, an analogue of 2, displays a stronger inhibition of immunoglobulin E production than 2 (IC50 0.066 versus 3.6 μM).28 As a result, many efficient short syntheses have been developed29 for azaspirene,30−33 pseurotins, synerazol, cephalimysins, berkeleyamide D, FD838, and analogues of these congeners. The biosynthetic pathway of compounds 1 and 2 was proposed by the Tamm,34 Turner,35 Keller,36 Tang,37−39 and Watanabe groups,37−40 as shown in Scheme 1. These reports revealed that (5S,9R)pseurotin A (2) is biosynthetically generated from (5S,9R)azaspirene (1). We hypothesized that (5R,9S)-pseurotin G (3) might be generated from (5R,9S)-azaspirene (1) in the same manner. Interestingly, Hayashi and co-workers observed the complete racemization during deprotection of enantiomerically © 2018 American Chemical Society

pure azaspirene precursor 4 in their total synthesis (Scheme 2A).30,41 To unveil the biosynthetic connection between (−)-1 and (+)-1, we began by studying the racemization mechanism with model compound 6a. Here, we report a racemization study of (5S,8R,9R)-6a at the C5, C8, and C9 stereogenic centers (Scheme 2B). To construct the spirocyclic scaffold, the model compound 6a was retrosynthetically disassembled into furanone 7 and isocyanate 8 (color-coded in green) based on the disconnection at the C5 carbon.

■

RESULTS AND DISCUSSION The synthesis of the azaspirene analogue 6a is shown in Scheme 3. Isocyanate 842 was obtained from commercially available α-acetamidocinnamic acid (9) by the following transformation sequence: esterification of 9, replacement of the N-Ac group with the N-Boc group giving 12,43 and isocyanate formation by using Kokotos protocol44 to afford 8 in good yields (Scheme 3A). Furan 18 was prepared by the following six-step sequence: Claisen condensation between 3pentanone (13) and benzyloxyacetyl chloride (14), removal of the Bn group, and spontaneous cyclization to afford furanone 7 in excellent yield (Scheme 3B). After coupling 7 with 8, the resulting amide was converted to furan 18 through a three-step functional group manipulation. The synthesis of rac-6a was completed by optimized sequential transformations. First, Received: August 28, 2018 Published: November 1, 2018 14457

DOI: 10.1021/acs.joc.8b02223 J. Org. Chem. 2018, 83, 14457−14464

Article

The Journal of Organic Chemistry Scheme 1. Proposed Biosynthesis of Pseurotin A (2) from (−)-Azaspirene (1),34−40 and Our Hypothesis for the Biosynthetic Pathway of Pseurotin G (3) from (+)-Azaspirene (1) via a Racemization Process

Scheme 3. Synthesis of (+)-(5R,8S,9S)-6a and (−)-(5S,8R,9R)-6a via Diastereomeric Resolution and Hydrolysisa

Scheme 2. Racemization of Azaspirene Analogue

a

Reagents and conditions: (a) EDC·HCl, 4-(dimethylamino)pyridine, MeOH, 96%; (b) (Boc)2O, NaH, THF; (c) NaOMe, MeOH, 50 °C, 85% (2 steps); (d) Tf2O, 2-Cl-pyridine, CH2Cl2, 95%; (e) LDA, then 14, THF, −78 °C, 81%; (f) H2, Pd/C, MeOH, 91%; (g) MeLi, then 8, THF, −40 °C; (h) MOMCl, N,N-diisopropylethylamine, CH2Cl2, 75% (2 steps); (i) LiAlH4, THF, 93%; (j) MnO2, THF, 50 °C, 90%; (k) 3 M HCl(aq), DMSO, 50 °C, then 3 M NaOH(aq), DMSO, 50 °C, 78%; (l) A, MNBA, 4-(dimethylamino)pyridine, Et3N, CH2Cl2, 44% for 19a and 40% for 19b; and (m) 3 M HCl(aq), THF, 50 °C, 96% for (+)-6a, >99% ee, 92% for (−)-6a, >99% ee. EDC·HCl = N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride; (Boc)2O = di-tert-butyl dicarbonate; Tf2O = trifluoromethanesulfonic anhydride; LDA = lithium diisopropylamide; MOMCl = chloromethyl methyl ether; and MNBA = 2-methyl-6-nitrobenzoic anhydride.

treatment in DMSO with aqueous hydrochloric acid solution removed the MOM group, followed by intramolecular aldol cyclization and subsequent hydration of the double bond, leading to a mixture of three diastereomers 6a−6c. Upon completion of the reaction, the resultant mixture was neutralized with aqueous NaOH solution, and diastereomers 6a−6c were consecutively heated at 50 °C for equilibration to provide thermodynamically favored rac-6a in 78% yield (see the following racemization study). The computed relative energies of four possible diastereomers 6a−6d45 indicated that 6a was the most stable in water.46 To separate the enantiomers, diastereomeric resolution and hydrolysis were conducted on rac-6a. After several attempts at diastereomeric resolution, we found that Shiina’s method47,48 for installation of a serine derivative as a chiral auxiliary and subsequent

hydrolysis of 19a/b were the most effective to supply enantiomerically pure (+)-6a and (−)-6a. The absolute configuration and stereostructure of (−)-6a were unequivocally confirmed by X-ray crystallographic analysis. 14458

DOI: 10.1021/acs.joc.8b02223 J. Org. Chem. 2018, 83, 14457−14464

Article

The Journal of Organic Chemistry

nearly racemic 20a (ca. 7% ee) along with partially racemized 20b (ca. 20% ee). Since there is no steric repulsion between the β-oriented hydroxy group at C9 and the benzyl group at C8, 20a is stable enough to be generated under thermodynamic conditions. As for (−)-6a, pure water instead of saline, DMSO addition, and increasing temperature all accelerated racemization, while forming other diastereoisomers 6b and 6c as minor side products. This is consistent with our experimental results in Scheme 3B (vide supra). It is worth noting, that no racemization was observed in toluene after 4 days, and other solvents, such as EtOH, MeOH, and even aqueous HCl solution (0.1−3 M), did not initiate any racemization (>99% ee for recovered (−)-6a).50 An alternate racemization process of (−)-6a could go through dehydration at the C8 position to induce the racemization via precursor 20b. However, this process was ruled out because hydrated intermediates were not observed during the isomerization of (5S,9S)-20b. This means that the dehydration process of 6a (i.e., elimination of a water molecule from 6a) is not involved in the racemization process. A postulated racemization mechanism is illustrated in Scheme 6. On the basis of our experimental results, we propose that hydration, or water solvation, on (−)-6a disrupts

In our synthetic strategy, we are also able to access 20b, which has a structure similar to that of Hayashi’s intermediate 5. Treatment of 18 in THF with aqueous HCl solution (1 M) cleaved the MOM group, and the following intramolecular aldol reaction gave rac-20a and rac-20b (Scheme 4). Both Scheme 4. Preparation of Synthetic Intermediate 20b and Determination of Its Stereochemistrya

a

Reagents and conditions: (a) 1 M HCl(aq), THF, rt, 74% for rac-20a, 9.3% for rac-20b; (b) HPLC resolution, (5S,9S)-20b, 95% ee; (c) (S)-MTPA, EDC·HCl, 4-(dimethylamino)pyridine, CH2Cl2, 84%; and (d) (R)-MTPA, EDC·HCl, 4-(dimethylamino)pyridine, CH2Cl2, 32%. (S)-MTPA = (S)-(−)-α-methoxy-α-trifluoromethylphenylacetic acid; (R)-MTPA = (R)-(+)-α-methoxy-α-trifluoromethylphenylacetic acid; and EDC·HCl = N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride.

Scheme 6. Proposed Racemization Mechanism in Aqueous Media

enantiomers of 20b were separated by chiral HPLC to afford (5S,9S)-20b. The absolute configuration at C9 of (5S,9S)-20b was confirmed by Mosher’s method.49 (S)- and (R)-MTPA esters 21 and 22 were prepared from (5S,9S)-20b. After the comparison of the difference between δ(S)‑21 and δ(R)‑22 [ppm] of 1H NMR (as shown in Figure S1), the absolute configuration at the C9 position was determined as the S-form. With (−)-6a and (5S,9S)-20b in hand, we investigated their stabilities in saline (Scheme 5). Surprisingly, complete racemization was observed in 6 days for a suspension of (−)-6a. On the other hand, the precursor (5S,9S)- 20b underwent both racemization and epimerization to afford Scheme 5. Racemization Study of (−)-6a and (5S,9S)-20ba

a

Reagents and conditions: (a) physiological saline suspension, rt, 6 d; and (b) physiological saline suspension, rt, 24 h. The ratio of diastereomers (20a:20b = 1:1.6). 14459

DOI: 10.1021/acs.joc.8b02223 J. Org. Chem. 2018, 83, 14457−14464

Article

The Journal of Organic Chemistry

mm. 1H NMR spectra were recorded at 400, 500, or 600 MHz. 13 C{1H} NMR spectra were recorded at 100, 125, or 150 MHz. Chemical shifts (δ) are reported in ppm relative to internal standard TMS (0 ppm) for 1H NMR and the residual solvent signal (δ 77.16 for 13C{1H} NMR in CDCl3). IR spectroscopic data were recorded using a diamond attenuated total reflectance (ATR) accessory. Signals are reported in reciprocal centimeters (cm−1). High-resolution mass spectra were measured with Thermo Fisher Scientific Exactive plus or JEOL JMS-T100CS AccuTOF. Optical rotations were recorded with a Na lamp (589 nm). X-ray data were collected with Cu Kα radiation. CYLview (Claude Legault, Université de Sherbrooke) was used for graphic rendering. Methyl Ester 10. α-Acetamidocinnamic acid (9) (4.00 g, 19.5 mmol) was dissolved in anhydrous MeOH (150 mL). The flask was evacuated until the solvent was observed to boil and backfilled three times with argon. N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (4.11 g, 21.4 mmol) and 4-(dimethylamino)pyridine (238 mg, 1.95 mmol) were added sequentially to the solution at room temperature. After 24 h of being stirred at room temperature, methanol was removed under reduced pressure, and H2O was added to the residue. The aqueous layer was extracted with EtOAc (×3), and the combined organic layers were washed with brine (×1), dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure to give the crude product. The residue was recrystallized from EtOAc to afford methyl ester 10 (4.12 g, 18.8 mmol) in 96% yield. TLC (hexanes:EtOAc 1:1): Rf = 0.15. White crystal. 1H NMR (400 MHz, CDCl3, δ): 2.15 (s, 3H), 3.86 (s, 3H), 6.98 (s, 1H), 7.30−7.70 (m, 6H). 13C{1H} NMR (100 MHz, CDCl3, δ): 23.3, 52.7, 124.5, 128.6, 129.5, 129.7, 132.5, 133.7, 165.9, 169.3. HRMS (ESI) (m/z): [M + Na]+ Calcd for C12H13NO3Na, 242.0788; found, 242.0788 (1H NMR data of 10 are identical to those of the compound reported previously).53 N-Boc Amide 12. Methyl ester 10 (4.12 g, 18.8 mmol) was dissolved in anhydrous THF (150 mL). The flask was evacuated until the solvent was observed to boil and backfilled three times with argon. NaH (55% dispersion in mineral oil, 2.46 g, 56.4 mmol) was added to the solution at 0 °C, after which the mixture was stirred for 1 h at room temperature. Di-tert-butyl dicarbonate (6.1 mL, 28 mmol) was added to the reaction mixture, the mixture was stirred for 1 h further before saturated aqueous NH4Cl was added. After the removal of THF under reduced pressure, the aqueous layer was extracted with EtOAc (×3). The combined organic layers were washed with brine (×1), dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure to give the crude 11, which was used in the next reaction without further purification. To a stirred solution of the crude 11 in anhydrous MeOH (100 mL) was added NaOMe (5.08 g, 94.0 mmol). After being stirred for 16 h at 50 °C, a saturated aqueous solution of NH4Cl was added to the reaction mixture. Under reduced pressure, MeOH was removed from the resultant solution, and the aqueous layer was extracted with EtOAc (×3). The combined organic layers were washed with brine (×1), dried over anhydrous MgSO4, filtered, and concentrated to give the crude product. The crude product was purified by flash column chromatography on silica gel (hexanes:EtOAc 2:1) to afford N-Boc amide 12 (4.41 g, 15.9 mmol) in 85% yield over 2 steps. TLC (hexanes:EtOAc 4:1): Rf = 0.31. White solid. 1H NMR (400 MHz, CDCl3, δ): 1.39 (s, 9H), 3.86 (s, 3H), 6.19 (brs, 1H), 7.25 (s, 1H), 7.28−7.40 (m, 3H), 7.54 (d, J = 7.2 Hz, 2H). 13C{1H} NMR (100 MHz, CDCl3, δ): 28.1, 52.6, 81.0, 124.7, 128.6, 129.2, 129.8, 130.2, 134.2, 152.9, 166.2. HRMS (ESI) (m/z): [M + Na]+ Calcd for C15H19NO4Na, 300.1206; found, 300.1207 (1H NMR data of 12 are identical to those of the compound reported previously).54 Isocyanate 8. To a solution of N-Boc amide 12 (1.20 g, 4.33 mmol) and 2-Cl-pyridine (1.6 mL, 17 mmol) in anhydrous CH2Cl2 (100 mL) was added Tf2O (1.45 mL, 8.65 mmol) in one portion at room temperature. After being stirred for 1 h, CH2Cl2 was removed under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes:EtOAc 4:1) to afford isocyanate 8 (832 mg, 4.10 mmol) in 95% yield as a colorless oil. TLC (hexanes:EtOAc 2:1): Rf = 0.61. 1H NMR (400 MHz, CDCl3,

the intramolecular hydrogen bond, resulting in acceleration of the retro-aldol reaction (Path A). Breaking the hydrogen bond to (−)-6a·2H2O triggers a retro-aldol reaction to afford furan Int-1, which undergoes an intramolecular aldol reaction to generate four diastereomers Int-2. Subsequent release of a hydroxyl group leads to the iminium cation rac-Int-3, and spontaneous hydration results in rac-6a−6c. In this sense, deprotonation from Int-3 to 20a and 20b is much slower than quenching Int-3 in neutral water. In contrast to 6a, rac-6b and -6c are thermodynamically less stable in water, so additional isomerization occurs to provide rac-6a via sequential retroaldol, aldol, dehydration, and hydration reactions under the equilibrium conditions. A retro-aldol reaction from Int-3 might be a complementary pathway for racemization. However, such reactive species surrounded by abundant water molecules would prefer to be quenched immediately to form hydrated products 6a−6c. Alternatively, treatment with HCl could protonate (−)-6a, resulting in the generation of cationic species Int-4 (Path B). The hydrogen bond, which is also stabilized by conjugation with the furanone ring, leaves a more positive charge on the C8 carbon of a hemiaminal moiety, which would energetically disfavor the retro-aldol reaction toward protonated Int-1. Antiangiogenic effects of azaspirene analogue 6a for HUVECs cocultured with FU-MMT-3 cells were previously reported.51 The IC50 values of each of these analogues (−)-6a and (+)-6a were determined as 10 μg/mL (31.5 μM).52 Both analogues (−)-6a and (+)-6a were also found to have equal antiangiogenic activity for suppression of tumor growth in vivo. In this study, racemization between (−)-6a and (+)-6a occurs in physiological saline suspension. These experimental results reported here could provide a rational explanation the equivalent biological activities of both enantiomers. The synthesis of azaspirene analogue rac-6a was accomplished in a convergent fashion, and their enantiomers were separated via diastereomeric resolution. Each enantiomer of 6a was found to racemize in neutral aqueous media, such as physiological saline suspension, by inversion of three contiguous stereogenic centers (C5, C8, and C9). These studies may inform the possible biosynthesis of (+)-azaspirene (1) or other isolated congeners related to azaspirene. Since structure activity relationship studies are in high demand for potent congeners, it is important to understand their chemical properties and synthesize their analogues. Efforts to understand the biosynthetic pathway and the racemization process will help to design azaspirene analogues for SAR studies. Further studies of racemization processes for azaspirene, pseurotin, cephalimysin, and other congeners are currently underway and will be reported in due course.

■

EXPERIMENTAL SECTION

General Methods. Saline (Otsuka normal saline or isotonic sodium chloride solution, pH 5.60) was purchased from Otsuka Pharmaceutical. Unless otherwise noted in the experimental procedures, all reactions sensitive to air or moisture were carried out under an argon atmosphere under anhydrous conditions. Analytical thin-layer chromatography (TLC) was performed using E. Merck silica gel 60 F254 precoated plates. Purification and isolation of products were performed using silica gel chromatography (both column and preparative thin-layer chromatography). Flash column chromatography was performed with either glass columns using 40− 50 μm silica gel 60N (Kanto Chemical Co., Inc.) or 60 μm PSQ60B (Fuji Silysia Chemical Ltd.). Preparative thin-layer chromatography was performed using Merck silica gel 60 F254 precoated plates, 0.5 14460

DOI: 10.1021/acs.joc.8b02223 J. Org. Chem. 2018, 83, 14457−14464

Article

The Journal of Organic Chemistry δ): 3.92 (s, 3H), 7.11 (s, 1H), 7.33−7.43 (m, 3H), 7.81−7.85 (m, 2H). 13C{1H} NMR (100 MHz, CDCl3, δ): 53.8, 121.7, 128.3, 128.8, 130.1, 130.4, 131.5, 133.0, 165.2. HRMS (ESI) (m/z): [M + H]+ Calcd for C11H10NO3, 204.0655; found, 204.0657 (1H NMR data of 8 are identical to those of the compound reported previously).54 Diketone 15. To a flame-dried flask with a stir bar was added iPr2NH (9.0 mL, 64 mmol) followed by anhydrous THF (80 mL). The solution was cooled to 0 °C under argon, and a solution of nBuLi (40 mL, 1.58 M in hexanes, 64 mmol) was added slowly with rapid mixing. The resultant yellow solution was then cooled to −20 °C, and 3-pentanone (13) (6.8 mL, 64 mmol) was added to the mixture. After being stirred for 1 h, benzyloxyacetyl chloride (14) (4.20 g, 22.9 mmol) was sequentially added to the mixture, and the solution was stirred for another 3 h at −78 °C. The reaction mixture was quenched by the addition of saturated aqueous NH4Cl, warmed at room temperature. After removal of THF under reduced pressure, the resulting mixture was extracted with EtOAc (×3). The combined organic layers were washed with brine (×1), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. Purification by flash column chromatography on silica gel (hexanes:EtOAc 6:1) provided an equilibrium mixture (15:enol isomers = 9:1) of diketone 15 and enol isomers (4.32 g, 18.5 mmol) in 81% yield as a colorless oil. 15 (major): 1H NMR (400 MHz, CDCl3, δ): 1.01 (t, J = 7.2 Hz, 3H), 1.26 (d, J = 7.2 Hz, 3H), 2.53 (q, J = 7.2 Hz, 2H), 3.90 (q, J = 7.2 Hz, 1H), 4.07 (s, 2H), 4.52 (s, 2H), 7.30−7.39 (m, 5H). 13C{1H} NMR (100 MHz, CDCl3, δ): 7.6, 12.0, 35.0, 56.1, 73.6, 74.3, 128.0, 128.2, 128.6, 136.9, 205.7, 207.9. HRMS (ESI) (m/z): [M + Na]+ Calcd for C14H18O3Na, 257.1148; found, 257.1154. Furanone 7. The equilibrium mixture of 15 and enol isomers (1.17 g, 5.00 mmol) and Pd/C (10 wt %, 160 mg) in anhydrous MeOH (20 mL) was stirred under an H2 atmosphere at room temperature for 1 h, and then it was filtered through a pad of Celite. The filtrate was concentrated, and the residue was purified by flash column chromatography on silica gel (hexanes:EtOAc 4:1) to afford furanone 7 (572 mg, 4.53 mmol) in 91% yield as a colorless oil. TLC (hexanes:EtOAc 2:1): Rf = 0.34. 1H NMR (400 MHz, CDCl3, δ): 1.23 (t, J = 7.6 Hz, 3H), 1.68 (s, 3H), 2.55 (q, J = 7.6 Hz, 2H), 4.44 (s, 2H). 13C{1H} NMR (100 MHz, CDCl3, δ): 5.2, 10.3, 22.4, 73.8, 110.5, 190.3, 203.5. HRMS (FAB) (m/z): [M + H]+ Calcd for C7H11O2, 127.0754; found, 127.0763. Furan 16. Furanone 7 (54.7 mg, 0.434 mmol) was dissolved in anhydrous THF (3.3 mL), and the solution was stirred at −78 °C under argon. MeLi (0.40 mL, 1.13 M in Et2O, 0.43 mmol) was added dropwise, and the mixture was stirred at −78 °C for 30 min. A solution of isocyanate 8 (88.1 mg, 0.434 mmol) in THF (1.0 mL) was then added over 10 min, and the reaction mixture was stirred at −78 °C for 18 h. After being warmed to −40 °C and stirred for another 2 h, the reaction mixture was quenched by saturated aqueous NH4Cl. After removal of THF under reduced pressure, the resulting aqueous layer was extracted with CH2Cl2 (×3), and the combined organic layers were washed with brine (×1), dried over MgSO4, filtered, and concentrated in vacuo. The residue was quickly passed through a pad of silica gel (hexanes:EtOAc 2:1) to afford amide (108 mg, 0.326 mmol) as a colorless oil. For the characterization of amide (unstable): TLC (hexanes:EtOAc 2:1): Rf = 0.34. HRMS (FAB) (m/z): [M + H]+ Calcd for C18H20NO5, 330.1336; found, 330.1354. Amide (534 mg, 1.62 mmol) was dissolved in anhydrous CH2Cl2 (16 mL) under argon, and to this solution was added N,N-diisopropylethylamine (1.10 mL, 6.49 mmol). MOMCl (0.250 mL, 3.24 mmol) was added dropwise to the reaction mixture, which was then stirred for 3 h. The reaction mixture was quenched by the addition of saturated aqueous NH4Cl, and the biphasic mixture was separated. The aqueous layer was additionally extracted with CH2Cl2 (×2). The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to give furan 16 (606 mg, 1.62 mmol) in 75% over 2 steps as a white solid. TLC (hexanes:EtOAc 2:1): Rf = 0.32. White solid. mp 82.2−82.8 °C. 1H NMR (400 MHz, CDCl3, δ): 1.23 (t, J = 7.6 Hz, 3H), 1.97 (s, 3H), 2.61 (q, J = 7.6 Hz, 2H), 3.49 (s, 3H), 3.86 (s, 3H), 5.26 (s, 2H), 7.30−7.37 (m, 4H), 7.52 (d, J = 6.8 Hz, 2H), 7.96 (s, 1H). 13C{1H} NMR (100 MHz, CDCl3, δ): 7.4,

12.3, 20.4, 52.8, 57.4, 99.5, 111.7, 124.2, 128.7, 129.4, 129.9, 131.2, 131.5, 134.1, 150.4, 155.6, 156.5, 166.1. HRMS (FAB) (m/z): [M + H]+ Calcd for C20H24NO6, 374.1598; found, 374.1598. Alcohol 17. A solution of the furan 16 (61.4 mg, 0.164 mmol) in anhydrous THF (0.5 mL) was added dropwise to a suspension of lithium aluminum hydride (17.3 mg, 0.457 mmol) in anhydrous THF (1.8 mL) at 0 °C, and the mixture was stirred at 0 °C for 45 min. After the reaction was complete, as judged by TLC analysis (ca. 45 min), the mixture was quenched by the sequential addition of H2O, 15 wt % aqueous NaOH, and H2O. The resulting mixture was stirred vigorously at room temperature until the solids turned white. After filtering the solution and washing the residue with EtOAc in portions (×3), the filtrate was concentrated in vacuo to give alcohol 17 (52.6 mg, 0.152 mmol) in 93% yield as a white solid. TLC (hexanes:EtOAc 2:1): Rf = 0.28. mp 64.1−65.7 °C. 1H NMR (400 MHz, CDCl3, δ): 1.17 (t, J = 7.6 Hz, 3H), 1.95 (s, 3H), 2.56 (q, J = 7.6 Hz, 2H), 3.31 (s, 3H), 4.45 (d, J = 7.6 Hz, 2H), 4.90 (t, J = 7.6 Hz, 1H), 5.02 (s, 2H), 5.92 (s, 1H), 7.27−7.31 (m, 1H), 7.35−7.43 (m, 4H), 8.76 (brs, 1H). 13C{1H} NMR (100 MHz, CDCl3, δ): 7.7, 12.0, 20.2, 57.2, 64.5, 98.3, 111.3, 114.1, 127.4, 128.7, 129.1, 131.3, 135.1, 136.7, 150.0, 156.2, 156.7. HRMS (FAB) (m/z): [M + H]+ Calcd for C19H24NO5, 346.1649; found, 346.1649. Aldehyde 18. Manganese(IV) oxide (7.86 g, 90.4 mmol) was added to a solution of the alcohol 17 (1.04 g, 3.01 mmol) in THF (45 mL), and the mixture was heated at 50 °C for 21 h. The resulting mixture was filtered through Celite, and the filtrate was concentrated in vacuo to afford aldehyde 18 (935 mg, 2.72 mmol) in 90% yield as a colorless oil. TLC (hexanes:EtOAc 2:1): Rf = 0.26. 1H NMR (400 MHz, CDCl3, δ): 1.25 (t, J = 7.6 Hz, 3H), 1.98 (s, 3H), 2.63 (q, J = 7.6 Hz, 2H), 3.52 (s, 3H), 5.27 (s, 2H), 7.03 (s, 1H), 7.34−7.40 (m, 3H), 7.50−7.56 (m, 2H), 8.29 (brs, 1H), 9.50 (s, 1H). 13C{1H} NMR (100 MHz, CDCl3, δ): 7.4, 12.3, 20.4, 57.5, 99.4, 111.8, 128.6, 130.2, 130.6, 131.5, 132.3, 134.3, 138.4, 150.7, 155.6, 155.9, 190.0. HRMS (FAB) (m/z): [M + H]+ Calcd for C19H22NO5, 344.1492; found, 344.1490. Azaspirene Analogue rac-6a. Aldehyde 18 (10.3 mg, 30.0 μmol) was dissolved in DMSO (50 μL) and HCl (950 μL, 3 M in H2O), and the solution was stirred for 16 h at 50 °C. After the consumption of aldehyde 18 was confirmed by TLC analysis of the reaction mixture, the pH value of the resulting solution was adjusted to around 7 by the addition of DMSO (50 μL) and NaOH (950 μL, 3 M in H2O). The reaction mixture was stirred for another 72 h at 50 °C, after which the mixture was extracted with preheated Et2O (×3). The combined organic layers were washed with brine (×1), dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexanes:EtOAc 2:1) to afford azaspirene analogue rac-6a (7.4 mg, 23 μmol) in 78% yield as a white solid. TLC (hexanes:EtOAc 1:1): Rf = 0.31. mp 145.5−146.2 °C. 1H NMR (500 MHz, CDCl3, δ): 1.30 (t, J = 7.5 Hz, 3H), 1.69 (s, 3H), 2.61−2.75 (m, 2H), 2.94 (d, J = 12.5 Hz, 2H), 3.27 (d, J = 13.5 Hz, 1H), 4.48 (d, J = 10.0 Hz, 1H), 5.87 (s, 1H), 6.45 (s, 1H), 7.27−7.39 (m, 5H). 13C{1H} NMR (100 MHz, CDCl3, δ): 5.6, 10.4, 22.9, 42.9, 74.5, 84.8, 93.6, 110.6, 127.8, 128.9, 130.5, 134.3, 164.6, 195.2, 199.9. IR (ATR, solid): 1741, 1679, 1608 cm−1. HRMS (FAB) (m/z): [M + H]+ Calcd for C17H20NO5, 318.1336; found, 318.1347. For Characterization of rac-6b. White solid. mp 73.7−75.6 °C. 1 H NMR (600 MHz, CDCl3, δ): 1.27 (t, J = 7.8 Hz, 3H), 1.72 (s, 3H), 2.64 (qd, J = 7.8, 1.8 Hz, 2H), 2.81 (d, J = 9.0 Hz, 1H), 3.18 (d, J = 13.8 Hz, 1H), 3.22 (m, 1H), 3.32 (d, J = 13.8 Hz, 1H), 4.41 (d, J = 9.0 Hz, 1H), 6.29 (brs, 1H), 7.30−7.39 (m, 5H). 13C{1H} NMR (150 MHz, CDCl3, δ): 0.1 (internal standard: TMS), 5.8, 10.7, 22.5, 43.9, 74.8, 85.9, 87.2, 111.5, 128.0, 129.2, 130.4, 134.1, 166.6, 190.5, 199.9. HRMS (FAB) (m/z): [M + H]+ Calcd for C17H20NO5, 318.1336; found, 318.1353. For Characterization of rac-6c. White solid. mp 74.9−76.6 °C. 1H NMR (400 MHz, CDCl3, δ): 1.33 (t, J = 7.6 Hz, 3H), 1.70 (s, 3H), 2.60 (d, J = 2.4 Hz, 1H), 2.28−2.80 (m, 2H), 3.20 (d, J = 1.2 Hz, 2H), 4.14 (m, 1H), 6.08 (t, J = 1.2 Hz, 1H), 6.26 (brs, 1H), 7.27− 7.44 (m, 5H). 13C{1H} NMR (100 MHz, CDCl3, δ): 5.7, 10.6, 22.8, 40.0, 75.4, 87.6, 91.0, 110.8, 127.3, 128.5, 131.1, 134.8, 165.6, 192.9, 14461

DOI: 10.1021/acs.joc.8b02223 J. Org. Chem. 2018, 83, 14457−14464

Article

The Journal of Organic Chemistry 200.7. HRMS (FAB) (m/z): [M + H]+ Calcd for C17H20NO5, 318.1336; found, 318.1337. Ester 19a and 19b. To a solution of N-Boc-O-benzyl-L-serine A (27.9 mg, 94.5 μmol) in CH2Cl2 (1.0 mL) were added MNBA (32.5 mg, 94.5 μmol), DMAP (1.2 mg, 9.5 μmol), and triethylamine (26 μL, 180 μmol). After being stirred for 30 min, a solution of azaspirene analogue rac-6a (10.0 mg, 31.5 μmol) in CH2Cl2 (1.0 mL) was added to the reaction mixture. After being stirred for another 1 h at room temperature, the mixture was quenched by saturated aqueous NH4Cl, and biphasic solution was extracted with CH2Cl2 (×3). The combined organic layers were washed with brine (×1), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by preparative-TLC (CHCl3:CH3CN 5:1) to give ester 19a (8.3 mg, 14.0 μmol) in 44% yield as a colorless oil, and ester 19b (7.5 mg, 12.6 μmol) in 40% yield as a colorless oil. Ester 19a. TLC (CHCl3:CH3CN 4:1): Rf = 0.64. 1H NMR (400 MHz, CDCl3, δ): 1.27 (t, J = 7.6 Hz, 3H), 1.45 (s, 9H), 1.69 (s, 3H), 2.55−2.73 (m, 2H), 2.89 (d, J = 14.0 Hz, 1H), 3.20 (d, J = 14.0 Hz, 1H), 3.71 (dd, J = 9.2, 2.8 Hz, 1H), 4.00 (dd, J = 9.2, 2.4 Hz, 1H), 4.46−4.52 (m, 1H), 4.52 (d, J = 12.0 Hz, 1H), 4.67 (d, J = 11.6 Hz, 1H), 5.43 (d, J = 9.2 Hz, 1H), 5.74 (s, 1H), 6.03 (s, 1H), 6.43−6.51 (m, 1H), 7.17−7.36 (m, 10H). 13C{1H} NMR (125 MHz, CDCl3, δ): 5.7, 10.3, 22.9, 28.4, 43.9, 54.0, 70.4, 73.6, 74.0, 80.4, 84.8, 90.9, 110.3, 127.7, 127.7, 128.0, 128.6, 128.8, 130.6, 134.2, 137.5, 155.6, 163.3, 170.1, 194.9, 198.8. [α]D29 +6.8 (c 0.15, CHCl3). CD (CH3CN): λext = 302.7 (Δε = −0.8), 270.7 (+0.6), 220.8 (−1.2). HRMS (ESI) (m/z): [M + Na]+ Calcd for C32H38N2O9Na, 617.2470; found, 617.2468. Ester 19b. TLC (CHCl3:CH3CN 4:1): Rf = 0.59. 1H NMR (400 MHz, CDCl3, δ): 1.15 (t, J = 7.6 Hz, 3H), 1.45 (s, 9H), 1.57 (s, 3H), 2.32−2.54 (m, 2H), 2.98 (d, J = 14.0 Hz, 1H), 3.22 (d, J = 14.0 Hz, 1H), 3.64 (dd, J = 9.6, 3.2 Hz, 1H), 3.78 (dd, J = 9.6, 2.8 Hz, 1H), 4.40 (d, J = 12.0 Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H), 4.43−4.49 (m, 1H), 5.31 (d, J = 8.4 Hz, 1H), 5.74 (s, 1H), 6.08 (s, 1H), 6.45−6.52 (m, 1H), 7.20−7.40 (m, 10H). 13C{1H} NMR (125 MHz, CDCl3): 5.6, 10.2, 22.6, 28.5, 43.9, 54.0, 70.3, 73.5, 73.8, 80.3, 84.5, 91.2, 109.9, 127.3, 127.8, 128.0, 128.6, 128.8, 130.7, 134.1, 137.5, 155.3, 163.4, 169.6, 194.7, 198.5. [α]D29 −19 (c 0.19, CHCl3). CD (CH3CN): λext = 282.4 (Δε = −1.3), 254.8 (+0.1), 223.9 (−1.8). HRMS (ESI) (m/z): [M + Na]+ Calcd for C32H38N2O9Na, 617.2470; found, 617.2468. Azaspirene Analogue (+)-6a. To a solution of 19a (4.3 mg, 7.2 μmol) in THF (0.8 mL) was added HCl (0.4 mL, 3 M in H2O), and the reaction mixture was stirred at 50 °C for 14 h. After the addition of NaHCO3, the resulting solution was extracted with EtOAc (×3). The combined organic layers were washed with brine (×1), dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexanes:EtOAc 2:1) to afford azaspirene analogue (+)-(5R,8S,9S)-6a (2.2 mg, 6.9 μmol) in 96% yield as a white solid, which was analyzed by HPLC using a chiral stationary phase for enantiomeric ratio determination (>99% ee). [α]D23 +106 (c 0.15, MeOH). HPLC conditions: CHIRALPAK ADH (Daicel Chemical Industries Ltd.), 0.46 cm × 25 cm, iPrOH:hexanes 1:3, 1.0 mL/min, room temperature, 13.1 min for (+)-6a, and 19.1 min for (−)-6a. Azaspirene Analogue (−)-6a. To a solution of 19b (3.3 mg, 5.6 μmol) in THF (0.6 mL) was added HCl (0.3 mL, 3 M in H2O), and the reaction mixture was stirred at 50 °C for 14 h. After the addition of NaHCO3, the resulting solution was extracted with EtOAc (×3). The combined organic layers were washed with brine (×1), dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexanes:EtOAc 2:1) to afford azaspirene analogue (−)-(5S,8R,9R)-6a (1.6 mg, 5.1 μmol) in 92% yield as a white solid, which was analyzed by HPLC using a chiral stationary phase for enantiomeric ratio determination (>99% ee). [α]D22 −106 (c 0.22, MeOH). Alcohol 20a and 20b. To a solution of aldehyde 18 (1.22 g, 3.56 mmol) in THF (71 mL) was added HCl (35.6 mL, 1 M in H2O), and the reaction mixture was stirred at room temperature for 20 min. After the addition of saturated aqueous NH4Cl, the resulting solution was

extracted with EtOAc (×3). The combined organic layers were washed with brine (×1), dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexanes:EtOAc 6:1 to 2:1) to afford alcohol rac-20a (787 mg, 2.63 mmol) in 74% yield as a white solid and alcohol rac-20b (99.0 mg, 0.331 μmol) in 9.3% yield as a white solid. Each enantiomer of rac-20b was separated by chiral HPLC to afford (5S,9S)-20b (95% ee) and (5R,9R)-20b (95% ee). HPLC conditions: CHIRALPAK ADH (Daicel Chemical Industries Ltd.), 0.46 cm × 25 cm, iPrOH:hexanes 1:5, 1.0 mL/min, room temperature, 10.2 min for (5R,9R)-20b, and 12.7 min for (5S,9S)-20b. rac-20a. TLC (hexanes:EtOAc 1:1): Rf = 0.26. White solid. mp 137.9−139.0 °C. 1H NMR (400 MHz, CDCl3, δ): 1.30 (t, J = 7.6 Hz, 3H), 1.73 (s, 3H), 2.46−2.75 (m, 3H), 5.02 (dd, J = 8.8, 1.6 Hz, 1H), 6.02 (s, 1H), 7.23−7.29 (m, 3H), 7.38 (t, J = 7.6 Hz, 2H), 8.01 (brs, 1H). 13C{1H} NMR (100 MHz, CDCl3, δ): 5.7, 10.7, 22.6, 72.2, 86.8, 106.1, 111.3, 127.4, 127.7, 129.2, 134.7, 135.9, 167.2, 191.0, 199.7. HRMS (FAB) (m/z): [M + H]+ Calcd for C17H18NO4, 300.1230; found, 300.1237. rac-20b. TLC (hexanes:EtOAc 1:1): Rf = 0.38. White solid. mp 175.5 °C (decomp.). 1H NMR (400 MHz, CDCl3, δ): 1.33 (t, J = 7.6 Hz, 3H), 1.70 (s, 3H), 2.61−2.77 (m, 2H), 2.92 (d, J = 12.4 Hz, 1H), 5.20 (dd, J = 12.4, 2.4 Hz, 1H), 5.98 (d, J = 2.0 Hz, 1H), 7.23−7.30 (m, 3H), 7.38 (t, J = 7.6 Hz, 2H), 7.88 (s, 1H). 13C{1H} NMR (150 MHz, CDCl3, δ): 5.6, 10.5, 22.7, 73.8, 91.5, 104.6, 110.3, 127.4, 127.7, 129.3, 134.8, 135.4, 165.3, 193.4, 198.0. HRMS (FAB) (m/z): [M + H]+ Calcd for C17H18NO4, 300.1230; found, 300.1243. MTPA Ester (S)-21. To a solution of (5S,9S)-20b (1.8 mg, 6.0 μmol) in CH2Cl2 (600 μL) were added (S)-(−)-α-methoxy-α(trifluoromethyl)phenylacetic acid (7.0 mg, 30 μmol), EDC·HCl (5.8 mg, 30 μmol), and DMAP (