Determination of the Absolute Configuration of the Nabumetone Metabolite 4-(6-Methoxy-2-naphthyl)butan-2-ol Using the Chiral Derivatizing Agent, 1-Fluoroindan-1-carboxylic Acid

Abstract

The absolute configuration of (+)-4-(6-methoxy-2-naphthyl)butan-2-ol ((+)-MNBO), a nabumetone metabolite, was determined using 1-fluoroindan-1-carboxylic acid (FICA). Both enantiomers of the FICA methyl esters were derivatized to diastereomeric esters of (+)-MNBO by an ester exchange reaction. The results of ¹H- and ¹⁹F-NMR spectroscopy of the diastereomeric FICA esters of (+)-MNBO confirmed the absolute configuration of (+)-MNBO was (S).

Introduction

Nabumetone (1) is a nonsteroidal anti-inflammatory drug (NSAID) and a pro-drug that is metabolized to 6-methoxy-2-naphthylacetic acid, which is a potent inhibitor of prostaglandin synthesis.¹⁾ Another main metabolic pathway of nabumetone (1) is reduction of the ketone to both enantiomers of 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) (2)^2–4) (Fig. 1). Matsumoto et al. reported that the formation ratios of (−)-MNBO (2a) and (+)-MNBO (2b) were different in microsomes and the cytosol.²⁾ Therefore, elucidation of the absolute configuration of an enantiomer of MNBO (2) would provide insight into the metabolic pathway of nabumetone (1).

Fig. 1. Nabumetone (1) and MNBO (2)

X-Ray crystallographic analysis is the most reliable method for assigning the absolute stereochemistry. However, Akgun et al. reported that the crystal structure of (−)-MNBO (2a) was not suitable for this purpose.^5,6) There are several convenient and reliable methods that involve diastereomer formation with a chiral derivatizing agent (CDA) followed by NMR spectroscopy. Among them, the modified Mosher’s method with α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA) (3) is one of the most reliable and frequently used procedures. Because its concept is clear and it has a self-examination mechanism to know if the observed data can be available or if they should be abandoned.⁷⁾ We developed 1-fluoroindan-1-carboxylic acid (FICA) (4) as a CDA and the concept of the modified Mosher’s method was confirmed to be successfully applied to the FICA-based procedure using ¹H-NMR for known chiral secondary alcohols.^8,9) In the case of isomenthol, the FICA-based method using ¹H-NMR was applicable, however, the modified Mosher’s method was not.⁹⁾ Therefore, the FICA-based method is of broader applicability than the modified Mosher’s procedure. In addition, the signs of the Δδ_F values of the FICA esters showed correlations with the absolute configurations for all 13 alcohols examined.⁹⁾ By contrast, no consistent relationship was found with the corresponding MTPA esters^7,9) (Fig. 2).

Fig. 2. (S)-MTPA ((S)-3) and (R)-FICA ((R)-4)

We have elucidated the stereochemistry of (+)-MNBO (2b) to be (S) according to the modified Mosher’s method, but did not show the details.²⁾ Herein we report its full experimental details and the first application of the ¹H-NMR-based FICA method to the confirmation of absolute configuration of a metabolite of medicine. Moreover, we describe here that the ¹⁹F-NMR-based FICA method is promising for the determination of absolute stereochemistry of chiral secondary alcohols.

Results and Discussion

Nabumetone (1) was treated with sodium borohydride to give racemic (±)-MNBO (2) quantitatively. The enantioseparation of (±)-MNBO (2) was carried out by HPLC using a CHIRALPAK AS-H column. The first fraction gave (−)-MNBO (2a) ([α]_D²⁶ −11) while the second contained (+)-MNBO (2b) ([α]_D²⁷ +12) (Chart 1). HPLC analysis of the latter fraction gave results that were identical to those of the metabolite (+)-MNBO (2b).

Chart 1. Enantioseparation of (±)-MNBO (2)

The absolute configuration of (+)-MNBO (2b) was examined using the FICA-based method⁹⁾ and the modified Mosher’s method.⁷⁾

After (+)-MNBO (2b) was treated with n-butyl lithium (n-BuLi), (R)-FICA Me ester ((R)-5) and (S)-FICA Me ester ((S)-5) were added to the reaction mixture to give the (R)-FICA ester of (+)-MNBO (6) in 13% yield and the (S)-FICA ester of (+)-MNBO (7) in 49% yield, respectively (Chart 2).

Chart 2. Formation of (R)- and (S)-FICA Esters of (+)-MNBO (6) and (7)

The (S)-MTPA ester of (+)-MNBO (8) was obtained from the reaction of (+)-MNBO (2b) with (R)-MTPACl in 46% yield. Similarly, the (R)-MTPA ester of (+)-MNBO (9) was prepared from (S)-MTPACl in 84% yield (Chart 3).

Chart 3. Formation of (S)- and (R)-MTPA Esters of (+)-MNBO (8) and (9)

These four diastereomeric esters 6–9 were analyzed by ¹H- and ¹⁹F-NMR spectroscopy to determine the absolute stereochemistry by the FICA-based method and the modified Mosher’s method. We assigned as many of the proton signals in the esters as possible by H,H-correlation spectroscopy (COSY) and nuclear Overhauser effect (NOE) NMR techniques. Then, the Δδ_H values were calculated from δ_R–δ_S for the FICA esters and δ_S–δ_R for the MTPA esters.^7,9–11) The signs of the Δδ_H values were distributed symmetrically with respect to the planes bisecting the carbinyl proton and the ester carbonyl, which were designated as the FICA and MTPA planes, respectively. The Δδ_H values on the right side of FICA and MTPA planes were positive and those on the left side were negative. Accordingly, the absolute configuration of (+)-MNBO (2b) was determined to be (S) (Fig. 3).

Fig. 3. Δδ_H and Δδ_F Values of FICA and MTPA Esters of (+)-MNBO (6–9)

Moreover, according to the ¹⁹F-NMR-based FICA method,⁹⁾ the Δδ_F value calculated for the FICA esters of (+)-MNBO (6) and (7) using δ_R–δ_S was positive. Therefore, the absolute stereochemistry of (+)-MNBO (2b) was (S), which agreed with the result from ¹H-NMR analysis.

Conclusion

The absolute configuration of the nabumetone metabolite (+)-MNBO (2b) was confirmed to be (S) by the FICA-based method and the modified Mosher’s method using ¹H-NMR spectroscopy. Furthermore, the FICA ester of (+)-MNBO had a positive Δδ_F value, which agreed with the ¹H-NMR data. For all 14 alcohols examined, including (+)-MNBO (2b), the signs of Δδ_F values of the FICA esters were correlated with the absolute configurations. These results show that FICA (4) is a promising CDA for determination of the configurations of chiral secondary alcohols using both ¹H- and ¹⁹F-NMR spectroscopy.

Experimental

General Information

Melting points were measured with a micro melting point apparatus (Yanaco, Kyoto, Japan) and not corrected. Microanalyses were performed by the Microanalysis Center of Josai University (Saitama, Japan). Spectroscopic measurements were carried out with the following instruments: optical rotation, JASCO P1020 digital polarimeter (JASCO, Tokyo, Japan); IR, JASCO FT/IR-4200 (JASCO); MS, JEOL JMS-700 (JEOL, Tokyo, Japan); high resolution (HR)-MS, JEOL JMS-700 (JEOL); ¹H-NMR, JEOL ECA 500 (500 MHz) (JEOL); ¹³C-NMR, JEOL ECA 500 (125 MHz) (JEOL); and ¹⁹F-NMR, JEOL ECA 500 (470 MHz) (JEOL). All NMR experiments were conducted using solutions prepared in CDCl₃. The internal standard for ¹H- and ¹³C-NMR was Me₄Si (Merck, Darmstadt, Germany), and that for ¹⁹F-NMR was CFCl₃ (TCI, Tokyo, Japan).

(−)- and (+)-4-(6-Methoxy-2-naphthyl)butan-2-ol (2a) and (2b)

To a solution of 4-(6-methoxy-2-naphthyl)butan-2-one (1) (300 mg, 1.31 mmol) in EtOH (15 mL) under a nitrogen atmosphere at −10°C was added NaBH₄ (90.5 mg, 2.39 mmol). After stirring at −10°C for 3 h, the EtOH was evaporated under vacuum. Both CH₂Cl₂ (10 mL) and cold water (1 mL) were added to the residue. After stirring at room temperature for 30 min, the organic layer was washed with H₂O and dried over MgSO₄. The solvent was evaporated under vacuum to give the product 2. The enantiomers were separated by chiral HPLC using a Daicel CHIRALPAK AS-H column (25 × 1.0 cm i.d., Daicel Chemical Co., Osaka, Japan) with n-hexane–2-propanol–CH₃CO₂H (volume 90 : 10 : 0.1, flow rate 1.9 mL/min) as the eluent. The first fraction eluted at t₁ = 22 min gave the alcohol 2a ([α]_D²⁶ −10.6, c = 0.45 in CHCl₃) and the second eluted at t₂ = 33 min gave the alcohol 2b ([α]_D²⁷ +11.9, c = 0.51 in CHCl₃). The solvents of both fractions were evaporated under vacuum to give colorless crystals, respectively.⁵⁾

(S)-4-(6-Methoxy-2-naphthyl)-2-butyl (R)-1-Fluoroindan-1-carboxylate (6)

To a solution of (+)-4-(6-methoxy-2-naphthyl)butan-2-ol (2b) (43.9 mg, 0.186 mmol) in dry tetrahydrofuran (THF) (1 mL) at −5°C under a nitrogen atmosphere was added n-BuLi (1.55 mol/L in n-hexane, 0.18 mL, 0.28 mmol) dropwise. After stirring for 30 min, a solution of (R)-FICA Me ester ((R)-5) (36.2 mg, 0.186 mmol) in dry THF (2 mL) was added slowly to the mixture. After 1 h, the reaction was quenched with a saturated aqueous NH₄Cl solution (1 mL), and then water (5 mL) and Et₂O (14 mL) were added. The organic layer was separated, washed with 2% HCl (5 mL), water (5 mL), and a saturated aqueous NaHCO₃ solution (5 mL), and dried over MgSO₄. The solvent was evaporated under vacuum and the residue was purified by preparative TLC (n-hexane–AcOEt = 7 : 3) to give the product 6 (13%, 9.7 mg, 0.024 mmol) as a colorless solid and the original reactant 2b (65%, 28.0 mg, 0.122 mmol).

mp 87.8–89.0°C. ¹H-NMR (500 MHz, CDCl₃) δ: 1.26 (3H, d, J = 6.3 Hz, 1′-H), 1.88–1.95 (1H, m, 4′-H), 2.02–2.10 (1H, m, 4′-H), 2.49 (1H, dddd, J = 5.2, 8.6, 14.3, 22.9 Hz, 2-H), 2.71–2.87 (3H, m, 2-H, 3′-H), 3.04–3.11 (1H, m, 3-H), 3.17–3.23 (1H, m, 3-H), 3.91 (3H, s, –OCH₃), 5.10 (1H, qdd, J = 6.3, 6.3, 6.3 Hz, 2′-H), 7.10 (1H, d, J = 2.3 Hz, 5″-H), 7.12 (1H, dd, J = 2.3, 8.6 Hz, 7″-H), 7.25 (1H, dd, J = 1.8, 8.6 Hz, 3″-H), 7.28 (1H, t, J = 7.5 Hz, 6-H), 7.33 (1H, d, J = 7.5 Hz, 4-H), 7.39 (1H, t, J = 7.5 Hz, 5-H), 7.44 (1H, d, J = 7.5 Hz, 7-H), 7.49 (1H, s, 1″-H), 7.65 (1H, d, J = 8.6 Hz, 8″-H), 7.67 (1H, d, J = 7.5 Hz, 4″-H). ¹³C-NMR (125 MHz, CDCl₃) δ: 19.8, 30.2, 31.7, 36.2 (d, J = 22.7 Hz), 37.4, 55.3, 72.4, 101.9 (d, J = 169.3 Hz), 105.6, 118.8, 124.1, 125.2, 126.2, 126.9, 127.1, 127.5, 128.9, 129.0, 130.4, 133.0, 136.2, 139.5 (d, J = 23.8 Hz), 145.0, 157.2, 170.2 (d, J = 33.4 Hz). ¹⁹F-NMR (470 MHz, CDCl₃) δ: −139.30 (t, J = 25.2 Hz). IR (neat) cm⁻¹: 2922 (CH), 1725 (C=O). HR-MS (electrospray ionization (ESI), m/z): 392.1774 (Calcd for C₂₅H₂₅FO₃: 392.1788). Electron ionization (EI)-MS m/z: 392 (M⁺), 212. [α]_D²⁷ −3.0 (c = 0.89, CHCl₃).

(S)-4-(6-Methoxy-2-naphthyl)-2-butyl (S)-1-Fluoroindan-1-carboxylate (7)

To a solution of (+)-4-(6-methoxy-2-naphthyl)butan-2-ol (2b) (29.6 mg, 0.129 mmol) in dry THF (1 mL) at −5°C under a nitrogen atmosphere was added n-BuLi (1.55 mol/L in n-hexane, 0.12 mL, 0.19 mmol) dropwise. After stirring for 30 min, a solution of (S)-FICA Me ester ((S)-5) (25.0 mg, 0.129 mmol) in dry THF (2 mL) was added slowly to the mixture. After 1 h, the reaction was quenched with a saturated aqueous NH₄Cl solution (1 mL), and then water (5 mL) and Et₂O (14 mL) were added. The organic layer was separated, washed with 2% HCl (5 mL), water (5 mL), and a saturated aqueous NaHCO₃ solution (5 mL), and dried over MgSO₄. The solvent was evaporated under vacuum and the residue was purified by preparative TLC (n-hexane–AcOEt = 7 : 3) to give the product 7 (49% yield, 24.8 mg, 0.06 mmol) as a colorless solid and the original reactant 2b (41%, 12.2 mg, 0.05 mmol).

mp 91.2–92.6°C. ¹H-NMR (500 MHz, CDCl₃) δ: 1.33 (3H, d, J = 6.3 Hz, 1′-H), 1.82–1.98 (2H, m, 4′-H), 2.45–2.61 (3H, m, 2-H, 3′-H), 2.85 (1H, dddd, J = 5.2, 8.6, 13.7, 22.9 Hz, 2-H), 3.07–3.15 (1H, m, 3-H), 3.19–3.24 (1H, m, 3-H), 3.91 (3H, s, –OCH₃), 5.03–5.10 (1H, m, 2′-H), 7.09 (1H, d, J = 2.3 Hz, 5″-H), 7.12 (2H, dd, J = 2.3, 8.6 Hz, 3″-H, 7″-H), 7.30 (1H, t, J = 8.1 Hz, 6-H), 7.35 (1H, d, J = 8.0 Hz, 4-H), 7.37 (1H, s, 1″-H), 7.40 (1H, t, J = 7.5 Hz, 5-H), 7.48 (1H, d, J = 8.0 Hz, 7-H), 7.63 (1H, d, J = 8.0 Hz, 4″-H), 7.64 (1H, d, J = 9.2 Hz, 8″-H). ¹³C-NMR (125 MHz, CDCl₃) δ: 20.0, 30.3, 31.3, 36.2 (d, J = 22.7 Hz), 37.5, 55.2, 72.2, 101.9 (d, J = 190.8 Hz), 105.6, 118.7, 124.1, 125.2, 126.2, 126.8, 127.1, 127.5, 128.8, 129.0, 130.4, 133.0, 136.3, 139.6 (d, J = 20.3 Hz), 144.8, 157.2, 170.2 (d, J = 25.0 Hz). ¹⁹F-NMR (470 MHz, CDCl₃) δ: −139.86 (t, J = 23.0 Hz); IR (neat) cm⁻¹: 2923 (CH), 1718 (C=O). HR-MS (ESI, m/z): 392.1778 (Calcd for C₂₅H₂₅FO₃: 392.1788). EI-MS m/z: 392 (M⁺), 212. [α]_D²² +54.9 (c = 1.50, CHCl₃).

(S)-4-(6-Methoxy-2-naphthyl)-2-butyl (S)-α-Methoxy-α-(trifluoromethyl)phenylacetate (8)

To a solution of (+)-4-(6-methoxy-2-naphthyl)butan-2-ol (2b) (12.2 mg, 0.053 mmol) in dry CH₂Cl₂ (2 mL) under a nitrogen atmosphere at room temperature were added Et₃N (0.02 mL, 0.159 mmol), (R)-MTPACl (0.01 mL, 0.064 mmol) and a catalytic 4-(dimethylamino)pyridine (DMAP). After stirring at room temperature for 10 min, the mixture was extracted with Et₂O (2 × 7 mL). The organic layer was washed with a saturated aqueous NaHCO₃ solution and brine, and dried over MgSO₄. The solvent was evaporated under vacuum and the residue was purified by preparative TLC (n-hexane–AcOEt = 7 : 3) to give the product 8 (46%, 10.8 mg, 0.02 mmol) as a yellow oil and the original reactant 2b (42%, 5.1 mg, 0.02 mmol).

¹H-NMR (500 MHz, CDCl₃) δ: 1.33 (3H, d, J = 6.3 Hz, 1′-H), 1.91–1.98 (1H, m, 4′-H), 2.06–2.13 (1H, m, 4′-H), 2.71–2.84 (2H, m, 3′-H), 3.58 (3H, s, –OCH₃), 3.91 (3H, s, –OCH₃), 5.21 (1H, qdd, J = 6.3, 6.3, 6.3 Hz, 2′-H), 7.11 (1H, d, J = 2.9 Hz, 5″-H), 7.13 (1H, dd, J = 2.9, 8.6 Hz, 7″-H), 7.25 (1H, dd, J = 1.8, 8.6 Hz, 3″-H), 7.41–7.43 (3H, m, Ar-H), 7.50 (1H, s, 1″-H), 7.57 (2H, m, Ar-H), 7.66 (1H, d, J = 8.6 Hz, 8″-H), 7.67 (1H, d, J = 8.6 Hz, 4″-H). ¹³C-NMR (125 MHz, CDCl₃) δ: 19.5, 31.6, 37.4, 55.3, 55.4, 73.7, 84.7 (d, J = 27.4 Hz), 105.6, 118.8, 123.4 (q, J = 286.2 Hz), 126.2, 127.0, 127.4, 128.4, 128.9, 129.0, 129.6, 132.3, 133.1, 136.1, 157.3, 166.1. ¹⁹F-NMR (470 MHz, CDCl₃) δ: −71.83. IR (neat) cm⁻¹: 2934 (CH), 1741 (C=O), 1264 (C–O), 1165 (C–O). HR-MS (ESI, m/z): 446.1681 (Calcd for C₂₅H₂₅F₃O₄: 446.1705). EI-MS m/z: 446 (M⁺), 171. [α]_D²¹ −5.6 (c = 1.11, CHCl₃).

(S)-4-(6-Methoxy-2-naphthyl)-2-butyl (R)-α-Methoxy-α-(trifluoromethyl)phenylacetate (9)

To a solution of (+)-4-(6-methoxy-2-naphthyl)butan-2-ol (2b) (12.4 mg, 0.054 mmol) in dry CH₂Cl₂ (2 mL) under a nitrogen atmosphere at room temperature were added Et₃N (0.02 mL, 0.162 mmol), (S)-MTPACl (0.01 mL, 0.065 mmol), and a catalytic DMAP. After stirring at room temperature for 10 min, the mixture was extracted with Et₂O (2 × 7 mL). The organic layer was washed with a saturated aqueous NaHCO₃ solution and brine, and dried over MgSO₄. The solvent was evaporated under vacuum and the residue was purified by preparative TLC (n-hexane–AcOEt = 7 : 3) to give the product 9 (84%, 20.2 mg, 0.05 mmol) as a yellow oil.

¹H-NMR (500 MHz, CDCl₃) δ: 1.39 (3H; d, J = 6.3 Hz, 1′-H), 1.87–1.94 (1H, m, 4′-H), 1.98–2.05 (1H, m, 4′-H), 2.58–2.70 (2H, m, 3′-H), 3.60 (3H, s, –OCH₃), 3.90 (3H, s, –OCH₃), 5.17–5.24 (1H, m, 2′-H), 7.09 (1H, d, J = 2.3 Hz, 5″-H), 7.12 (1H, dd, J = 2.4, 8.6 Hz, 7″-H), 7.16 (1H, dd, J = 1.7, 8.6 Hz, 3″-H), 7.41–7.42 (3H, m, Ar-H), 7.42 (1H, s, 1″-H), 7.58–7.59 (2H, m, Ar-H), 7.64 (2H, d, J = 8.1 Hz, 4″-H, 8″-H). ¹³C-NMR (125 MHz, CDCl₃) δ: 19.9, 31.3, 37.4, 55.3, 55.4, 73.5, 84.4 (d, J = 28.6 Hz), 105.6, 118.8, 123.4 (q, J = 286.2 Hz), 126.2, 126.9, 127.2, 128.4, 127.5, 128.9, 129.0, 129.6, 132.6, 133.0, 136.2, 157.2, 166.1. ¹⁹F-NMR (470 MHz, CDCl₃) δ: −71.77. IR (neat) cm⁻¹: 2937 (CH), 1742 (C=O), 1264 (C–O), 1160 (C–O). HR-MS (ESI, m/z): 446.1691 (Calcd for C₂₅H₂₅F₃O₄: 446.1705). EI-MS m/z: 446 (M⁺), 171. [α]_D²⁶ +69.4 (c = 1.27, CHCl₃).

Conflict of Interest

The authors declare no conflict of interest.

References and Notes

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• 5) Akgun H., Balkan A., Berk B., Urgan H., Karaaslan E., Fabad J. Pharm. Sci., 26, 67–71 (2001).
• 6) Goudie reported that (S)-(+)-MNBO was obtained from the reaction of the corresponding dithiane with (S)-2-methyloxirane, followed by reductive desulfurization. However, details were not shown except for its melting point and specific rotation; Goudie, A. C., Ger. Offen. (1977), DE 2721207 A1 19771124.
• 7) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem. Soc., 113, 4092–4096 (1991).
• 8) Takahashi T., Kameda H., Kamei T., Ishizaki M., J. Fluor. Chem., 127, 760–768 (2006).
• 9) Takahashi T., Kameda H., Kamei T., Koyanagi J., Pichierri F., Omata K., Ishizaki M., Nakamura H., Tetrahedron Asymmetry, 24, 1001–1009 (2013).
• 10) When Ar, COOH, and F or CF₃ groups are arranged similarly on the stereogenic centers, the absolute configurations of FICA and MTPA are opposite because of priority of the substituents.
• 11) Δδ values are expressed in ppm unit.