Introduction

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Aromatic ring hydroxylation and N-dealkylation are the major oxidative pathways of propranolol (P¹) metabolism (Fig. 1). In humans, the ring hydroxylation process produces regioisomeric phenolic metabolites, primarily via 4'-hydroxylation, with much less 5'-hydroxylation, smaller amounts of the 7-hydroxylation product, and very, very small amounts of dihydroxylated products (Talaat and Nelson, 1988).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Major oxidative pathways of propranolol metabolism.

A variety of evidence indicates that aromatic hydroxylation of P is catalyzed predominantly by CYP2D6. This process cosegregates with debrisoquine/sparteine polymorphism in vivo (Lennard et al., 1984; Raghuram et al., 1984). Significant inhibition of liver microsomal hydroxylation of P by quinidine occurs in vitro, and strong correlations with CYP2D6 content and debrisoquine-4-hydroxylation (Masubuchi et al., 1994) and 2-hydroxylation of desipramine have been observed (Yoshimoto et al., 1995). Inhibition by an antibody to rat CYP2D1 was also reported (Masubuchi et al., 1994). Based on the lack of complete inhibition by quinidine and a positive intercept in the linear correlation with CYP2D6 content, other P450 isozymes might contribute in a minor way to aromatic hydroxylation. Pharmacokinetic evidence for participation by an additional enzyme(s) has been obtained from incubations in human liver microsomes (Marathe et al., 1994).

Results using recombinant CYP2D6 in yeast cells showed formation of 4'-OHP and 5'-OHP and minor amounts of DIP (desisopropylpropranolol), with aromatic hydroxylation rates on the enantiomers of P exceeding those of N-dealkylation by ca. 10-fold (Bichara et al., 1996). When the enantiomers of P were studied separately in microsomes from recombinant CYP2D6 in human lymphoblastoid cells, the rate of 4'-hydroxylation exceeded the rate of N-dealkylation by 3- to 9-fold, but both metabolic processes were observed (Yoshimoto et al., 1995). Similar results have been obtained with recombinant enzymes and in human liver microsomes (Otton et al., 1990; Rowland et al., 1996).

Site-directed mutagenesis studies have identified Asp301 as being important for substrate binding and orientation in the active site of CYP2D6 and suggested that this negatively charged Asp residue interacts ionically with the protonated amine nitrogen of the substrate (Ellis et al., 1995). The binding pockets around the heme in the wild type and in these Asp301 mutants have been explored by migration of aryl groups from -bonded aryl-iron complexes formed from aryl-substituted diazenes (Mackman et al., 1996). Migration of large aryl substituents to the A-pyrrole ring occurred in all of the mutants and the wild type, indicating that loss of activity in the Asp301 mutants is probably not due to major structural reorganization but to the loss of the ion-pairing interaction with the substrate (Mackman et al., 1996). Sequence alignments of CYP2D6 with CYP101 (P450_cam) and CYP102 (P450_BM3) place Asp301 within SRS-4 (Gotoh, 1992). Protein homology models of CYP2D6 based on the crystal structures for CYP101 and CYP102 place Asp301 in the I helix at an appropriate distance from the heme catalytic center to influence substrate binding (Koymans et al., 1993; Lewis et al., 1997).

Typical substrates for CYP2D6 are basic aliphatic amines, and often there is a flat hydrophobic region near the site(s) of oxidation. Pharmacophore models suggest the preferred sites of oxidation tend to be about 5 to 7 Å from the nitrogen (Koymans et al., 1992). NMR relaxation studies on codeine in the presence of CYP2D6, with its O-methyl group about 8 Å away from the basic nitrogen (de Groot et al., 1999a), suggest that it binds with the O-methyl group close to the heme iron (3.1 Å) (Modi et al., 1996). Imposing distance constraints, derived from the NMR studies on a protein homology model to generate a model of the codeine-CYP2D6 complex, positions the nitrogen atom of codeine close to Asp301 (Modi et al., 1996). Substrates with larger aryl or aralkyl groups, such as amitriptyline and imipramine, are oxidized primarily on the aromatic ring or adjacent to it, with smaller amounts of N-dealkylation. CYP2D6 catalyzed N-dealkylation occurs in many substrates, but the N-dealkylation pathway is usually minor. The few exceptions, such as N,N-dialkylamphetamines (Bach et al., 2000) and dexfenfluramine (Haritos et al., 1998), are similar in structure to amphetamine and have branching alpha to the nitrogen on the aliphatic side chain. However, CYP2D6 contributes to N-dealkylation in a small but significant way, especially where CYP1A2 activity is low (Rowland et al., 1996). A model for the binding of substrates that undergo N-dealkylation in which Phe 481 interacts with the aromatic ring of these substrates has been proposed (de Groot et al., 1999b).

The experimental data for regioselectivity of P metabolism in CYP2D6 suggest that the preferred binding orientation of P in the active site has the 4'-position close to the heme. This is consistent with the idea that interaction of the P amine nitrogen with Asp301 is directing the orientation of P in the active site of CYP2D6. P is considered to be a "7Å" substrate for CYP2D6, having a distance between the nitrogen and preferred site of oxidation of about 7.9 Å (de Groot et al., 1999a). It also fits the recently summarized CYP2D6 substrate profile (Lewis, 2000). Besides being a basic amine, P is relatively lipophilic with an aromatic ring, and it has potential hydrogen bonding groups at the amine nitrogen and the side chain hydroxyl group.

In this work, we report preparation of a series of mono-, di-, and trifluorinated analogs of P, along with some nonfluorinated steric congeners, with the goal of studying the influences of changes in structure on metabolism by CYP2D6 and by CYP1A2 (Upthagrove and Nelson, 2001). To separate basicity, lipophilicity, and steric effects, a series of amines having the 1"-substitution of one, two, and three fluorines in propranolol [fluoropropranolol (FP), difluoropropranolol (DFP), and trifluoropropranolol (TFP)] were examined. Additional congeners having isopropyl and t-butyl groups [1",1"-dimethylpropranolol (iPrMe) and 1",1",1"-trimethylpropranolol (tBuMe)], intended to approximate the size of the trifluoromethyl group, and one substituting a trifluoroethyl substituent for the N-isopropyl [trifluoroethylpropranolol (TFE)], providing a smaller trifluorinated analog, were included in the series. Two analogs that do not contain the -hydroxyl group, one related to P [deshydroxypropranolol (desOHP)] and one related to trifluoropropranolol [deshydroxytrifluoropropranolol (desOHTFP)], were also examined. We incubated each of the P analogs with baculovirus-insect cell-expressed human recombinant CYP2D6 and compared changes in observed Michaelis-Menten kinetic parameters to changes in physical chemical properties.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Instrumentation. NMR Spectroscopy. NMR spectra were acquired on a Varian VXR 300 spectrometer (Varian, Inc., Palo Alto, CA) or a Bruker AF-300 spectrometer (Bruker Instruments, Inc., Billerica, MA). ¹⁹F NMR spectra were calibrated using neat trifluoroethanol (Aldrich, Milwaukee, WI) as an external reference,  = 77.8 ppm relative to CFCl₃ (Everett, 1995).

Mass Spectrometry. ESI mass spectra were obtained using a Finnigan LCQ quadrupole ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with a capacitive electrospray ion source modification (Wang and Hackett, 1998) of the Finnigan API interface. Collision-induced dissociation was carried out in the mass analyzer on an ion selected from the mass spectrum, using the He gas present in the trap. The samples, either synthetic standards or metabolic product mixtures in CH₃OH, were infused directly via a syringe pump at a flow rate of 300 nl/min. The heated capillary was maintained at 160°C and the source voltage at 1.7 kV.

Synthesis. Propranolol-Related Substrates and Standards. Propranolol enantiomers (2S-P and 2R-P) were obtained from Sigma (St. Louis, MO). All other compounds were prepared in our laboratory. The fluorinated analogs FP, DFP, and TFP and the steric analogs iPrMe and tBuMe (Beedle et al., 1989) were prepared as their 2S-diastereomers from 2S-DIP (Cardillo et al., 1987). This amine was prepared from 2S-1,2-epoxy-3-(1-naphthoxy)propane by a reaction with ammonia (Powell et al., 1980), followed by reductive amination (sodium cyanoborohydride) of mono-, di-, trifluoroacetone, isopropyl methyl ketone, and t-butyl methyl ketone, respectively. 1,1-Difluoroacetone was prepared by a reaction of 1,1-dichloroacetone with potassium bifluoride (Shapiro et al., 1973; Hudlicky, 1976). Other ketones were obtained from Aldrich (Milwaukee, WI). Reductive amination (sodium cyanoborohydride) of trifluoroacetaldehyde mono methyl acetal (Aldrich) with 2S-DIP gave the N-trifluoroethyl analog TFE (Weglicki et al., 1998). 4'-OHP, 5'-OHP, and 7'-OHP standards were synthesized as previously reported (Oatis et al., 1981).

2S-DIP. 1-Naphthol (480 mg, 3.33 mmol) and sodium hydride suspension (80% in oil, 130 mg, 4.3 mmol) were combined in dry DMF (10 ml). After stirring at room temperature for 25 min, 2S-glycidyl tosylate (653 mg, 2.86 mmol) dissolved in dry DMF (5 ml) was added dropwise over 5 min. After stirring for 4 h at room temperature, the reaction mixture was partitioned between H₂O (50 ml) and Et₂O (50 ml). The aqueous layer was extracted with more Et₂O (50 ml). The combined Et₂O extracts were washed with aqueous 1 N NaOH (20 ml) and H₂O (10 ml), dried over MgSO₄, and the solvent evaporated to afford the intermediate epoxide, which was used without further purification.

2R-DIP. The 2R-enantiomer was prepared using 2R-glycidyl tosylate (649 mg, 2.85 mmol) and 1-naphthol (482 mg, 3.3 mmol) by the procedure described above for the 2S-enantiomer to yield 236 mg (38%) of 2R-DIP as a white solid. ¹H NMR (CDCl₃):  8.23 (1H, m, H-8'); 7.80 (1H, m, H-5'); 7.45 (3H, m, H-7', -6', -4'); 7.36 (1H, dd, H-3'); 6.83 (1H, d, H-2'); 4.16 (3H, m, H-3, -2); 3.02 (2H, m, H-1). ESI-MS/MS [MH]⁺ 218  [C₁₃H₁₁O]⁺ 183, [C₃H₈NO]⁺ 74.

1"-FP. 2S-DIP (49 mg, 0.22 mmol) and sodium cyanoborohydride (57 mg, 0.9 mmol) were dissolved in CH₃OH (1 ml). Monofluoroacetone (100 mg, 1.5 mmol) and acetic acid (~20 µl) were added to the reaction mixture. It was capped tightly and stirred at 100°C for 2 h. Methylene chloride (4 ml) was added, and the solution was washed with saturated aqueous Na₂CO₃ solution (1 ml) and H₂O (1 ml). After evaporation of the organic solvent, the amine was purified by flash column chromatography, affording 18 mg (18%) of FP. ¹H NMR (CDCl₃):  8.25 (1H, m, H-8'); 7.81 (1H, m, H-5'); 7.48 (3H, m, H-7', -6', -4'); 7.39 (1H, dd, H-3'); 6.82 (1H, d, H-2'); 4.40 (2H, m, CFH₂); 4.19 (3H, m, H-3, -2); 3.00 (3H, m, H-1, -2"); 1.15 (3H, d, 2"-CH₃). ¹⁹F NMR (CDCl₃):  224.3 (dt, ²J_HF = 51 Hz, ³J_HF = 19 Hz) and 224.5 (dt, ²J_HF = 51 Hz, ³J_HF = 19 Hz). ESI-MS/MS [MH]⁺ 278  [C₁₃H₁₁O]⁺ 183, [C₆H₁₃NOF]⁺ 134.

1",1"-DFP. ¹H NMR (CDCl₃):  8.24 (1H, m, H-8'); 7.81 (1H, m, H-5'); 7.47 (3H, m, H-7', -6', -4'); 7.39 (1H, dd, H-3'); 6.82 (1H, d, H-2'); 5.69 (1H, tm, CF₂H); 4.18 (3H, m, H-3, -2); 3.07 (3H, m, H-1, -2"); 1.19 (3H, d, 2"-CH₃). ¹⁹F NMR (CDCl₃):  124.29 and 124.43 (ddd, J_FF = 300 Hz, ²J_HF = 60 Hz, ³J_HF = 11 Hz) and 126.5 and 126.9 (ddd, J_FF = 300 Hz, ²J_HF = 60 Hz, ³J_HF = 12 Hz). ESI-MS/MS [MH]⁺ 296  [C₁₃H₁₁O]⁺ 183, [C₆H₁₂NOF₂]⁺ 152.

2S-1",1",1"-TFP. ¹H NMR (CDCl₃):  8.22 (1H, m, H-8'); 7.81 (1H, m, H-5'); 7.48 (3H, m, H-7', 6', 4'); 7.39 (1H, dd, H-3'); 6.82 (1H, d, H-2'); 4.19 (3H, m, H-3, 2); 3.10 (3H, m, H-1, 2"); 1.31 (3H, d, 2"-CH₃). ¹⁹F NMR (CDCl₃):  77.14 (³J_HF = 7 Hz) and 77.34 (³J_HF = 7 Hz). ESI-MS/MS [MH]⁺ 314  [C₁₃H₁₁O]⁺ 183, [C₆H₁₁NOF₃]⁺ 170.

2R-1",1",1"-TFP. ¹H NMR (CDCl₃):  8.22 (1H, m, H-8'); 7.81 (1H, m, H-5'); 7.48 (3H, m, H-7', -6', -4'); 7.39 (1H, dd, H-3'); 6.82 (1H, d, H-2'); 4.19 (3H, m, H-3, -2); 3.10 (3H, m, H-1, -2"); 1.31 (3H, d, 2"-CH₃). ¹⁹F NMR (CDCl₃):  77.14 (³J_HF = 7 Hz) and 77.34 (³J_HF = 7 Hz). ESI-MS/MS [MH]⁺ 314  [C₁₃H₁₁O]⁺ 183, [C₆H₁₁NOF₃]⁺ 170.

2S,1"R-1",1",1"-Trifluoropropranolol (2S,1"R-TFP). The hydrochloride salt of R-1,1,1-trifluoro-2-propylamine (2.8 g, 18.7 mmol) was prepared as previously reported (Soloshonok and Ono, 1997). Immediately before using, the free amine was prepared by extraction into cold Et₂O (40 ml) from cold aqueous 2 N NaOH (20 ml). After drying the cold amine solution over Na₂SO₄, the solution was added to dry basic alumina (35 g). 2S-1-naphthoxy-2,3-epoxypropane (883 mg, 4.4 mmol) in anhydrous Et₂O (10 ml) was added slowly to the stirring reaction mixture. The reaction mixture was stirred at room temperature for 20 h, then CH₃OH (150 ml) was added, and the mixture was stirred for another 4 h. At the end of this time, the reaction mixture was filtered through Celite, and the solvent was evaporated to leave the crude product. Purification by flash column chromatography on silica gel using CH₂Cl₂ afforded 28 mg (28%) of pure 2S,1"R-TFP. The optical purity of the product (92%) was determined by HPLC. The isomers were separated on a Chiralcel-OD column (250 × 4.6 mm; Chiral Technologies, Exton, PA) using 92% hexane and 8% isopropyl alcohol at a flow rate of 1 ml/min. Under these conditions, 2R,1"S-TFP and 2R,1"R-TFP coeluted at 11.6 min., 2S,1"R-TFP eluted at 30.7 min, and 2S,1"S-TFP eluted at 34.8 min. ¹H NMR (CDCl₃):  8.22 (1H, m, H-8'); 7.81 (1H, m, H-5'); 7.48 (3H, m, H-7', -6', -4'); 7.39 (1H, dd, H-3'); 6.82 (1H, d, H-2'); 4.19 (3H, m, H-3, -2); 3.10 (3H, m, H-1, -2"); 1.31 (3H, d, 2"-CH₃). ¹⁹F NMR (CDCl₃):  77.14 (³J_HF = 7 Hz) and 77.34 (³J_HF = 7 Hz). ESI-MS/MS [MH]⁺ 314  [C₁₃H₁₁O]⁺ 183, [C₆H₁₁NOF₃]⁺ 170.

iPrMe. 2S-DIP (591 mg, 2.7 mmol), 3-methyl-2-butanone (282 mg, 3.3 mmol), and p-toluenesulfonic acid (6 mg, 0.03 mmol) were dissolved in benzene (25 ml). The mixture was refluxed and H₂O collected in a Dean-Stark trap. Progress of the reaction was monitored by TLC on silica gel using solvent mixture A. After 62 h, the reaction was complete. The benzene was evaporated, and the residue was partitioned between CH₂Cl₂ (20 ml) and aqueous 2 N NaOH (10 ml). The aqueous layer was extracted with an additional 20 ml of CH₂Cl₂. The combined organic extracts were washed with H₂O (5 ml), dried over Na₂SO₄, and the solvent was evaporated. The residual oil and sodium cyanoborohydride (800 mg, 12.7 mmol) were dissolved in 20 ml of methanol. Acetic acid (~100 µl) was added to the mixture, which was then stirred at room temperature for 15 h. Aqueous 1 N NaOH (30 ml) was added to the reaction mixture, and the product was extracted into CH₂Cl₂. The organic phase was washed with H₂O, dried over Na₂SO₄, and the solvent evaporated to leave a brown oil. This oil was purified by flash column chromatography on silica gel eluting with a gradient of CH₂Cl₂ to 20% EtOAc, 4% triethylamine in CH₂Cl₂ to afford iPrMe as a white solid, 350 mg (54%). ¹H NMR (CDCl₃):  8.28 (1H, m, H-8'); 7.82 (1H, m, H-5'); 7.45 (4H, m, H-7', -6', -4', -3'); 6.87 (1H, d, H-2'); 4.19 (3H, m, H-3, H-2); 2.90 (2H, m, H-1); 1.77 (1H, m, H-2"); 1.08 (3H, d, 2"-CH₃); 0.95 [7H, m, H-1", 1"-(CH₃)₂]. ESI-MS/MS [MH]⁺ 288  [C₁₃H₁₆NO₂]⁺ 218, [C₁₃H₁₁O]⁺ 183.

tBuMe. ¹H NMR (CDCl₃):  8.24 (1H, m, H-8'); 7.80 (1H, m, H-5'); 7.45 (3H, m, H-7', -6', -4'); 7.33 (1H, dd, H-3'); 6.73, 6.71 (1H, 2d, H-2'); 4.40 to 4.10 (3H, m, H-3, -2); 3.80, 3.57 (2H, 2 m, H-1); 3.02, 2.87 (1H, 2 m, H-2"), 1.47, 1.43 (3H, 2d, 2"-CH₃); 1.17 [9H, s, 1"-(CH₃)₃]. ESI-MS/MS [MH]⁺ 302  [C₁₃H₁₆NO₂]⁺ 218, [C₁₃H₁₁O]⁺ 183.

TFE. 2S-DIP (550 mg, 4.61 mmol), trifluoroacetaldehyde methyl hemiacetal (628 mg, 4.83 mmol), and p-toluenesulfonic acid (15 mg, 0.08 mmol) were dissolved in benzene (30 ml). The mixture was refluxed and H₂O collected in a Dean-Stark trap. After 2.5 h, the benzene was evaporated and the residue partitioned between CH₂Cl₂ (20 ml) and aqueous 2 N NaOH (10 ml). The aqueous layer was extracted with an additional 20 ml of CH₂Cl₂. The combined organic extracts were washed with H₂O (5 ml), dried over Na₂SO₄, and evaporated. The residual oil and sodium cyanoborohydride (1.20 g, 19 mmol) were dissolved in CH₃OH (15 ml). Acetic acid (~100 µl) was added and the reaction mixture was allowed to stir at room temperature for 24 h. At the end of this time, CH₂Cl₂ (30 ml) and aqueous 1 N NaOH (20 ml) were added to the reaction mixture, and the product was extracted into CH₂Cl₂. The aqueous phase was extracted with additional CH₂Cl₂ (20 ml). The combined CH₂Cl₂ extracts were washed with H₂O (10 ml), dried over Na₂SO₄, and the solvent was evaporated to leave a tan solid. This solid was purified by flash column chromatography on silica gel with a stepwise gradient of CH₂Cl₂ to 20% EtOAc in CH₂Cl₂, affording 414 mg (30%) of TFE as a white solid. ¹H NMR (CDCl₃):  8.21 (1H, m, H-8'); 7.82 (1H, m, H-5'); 7.49 (3H, m, H-7', -6', -4'); 7.38 (1H, dd, H-3'); 6.83 (1H, d, H-2'); 4.21 (2H, m, H-3); 3.75 (1H, m, H-2); 3.36 (2H, q, H-2"); 3.20, 3.08 (2H, 2dd, H-1). ESI-MS/MS [MH]⁺ 300  [C₁₃H₁₁O]⁺ 183, [C₅H₉NOF₃]⁺ 156.

Deshydroxydesisopropylpropranolol (desOHDIP). Preparation of this primary amine was modified from the reported procedure (Glennon et al., 1989). 1-Naphthol (4.0 g, 28 mmol) and sodium hydride (80% in oil, 1.5 g, 50 mmol) were combined in DMF (25 ml) under a dry argon atmosphere. After 20 min at room temperature, N-(3-bromopropyl)phthalimide (5.0 g, 18.6 mmol) was added, and the reaction mixture heated to reflux. After 15 h, the DMF was evaporated, and the remaining residue was partitioned between 25 ml H₂O and 50 ml Et₂O. The aqueous phase was then extracted with CH₂Cl₂ (3 × 25 ml). The combined CH₂Cl₂ extracts were washed with H₂O, dried over MgSO₄, and solvent evaporated to yield a white crystalline solid, 285 mg (46%) of the naphthoxypropylphthalimide. Hydrazine (4.0 g, 125 mmol) in ethanol (15 ml) was added dropwise to a solution of the phthalimide (430 mg, 13 mmol) in ethanol (10 ml). The reaction mixture was then heated to reflux for 2 h. At the end of this time the precipitate was removed by filtration and extracted with hot ethanol (40 ml). The ethanol was evaporated from the filtrate and the residue was dissolved in 20 ml of CH₂Cl₂ and extracted with aqueous 1 N HCl (40 ml, and then 2 × 20 ml). After the combined aqueous layers were adjusted to pH 11 by addition of aqueous 6 N NaOH, the amine was extracted into CH₂Cl₂. The combined CH₂Cl₂ extracts were washed with H₂O, dried over Na₂SO₄, and evaporated affording 98 mg (38%) of desOHDIP. ¹H NMR (CDCl₃):  8.25 (1H, m, H-8'); 7.79 (1H, m, H-5'); 7.41 (4H, m, H-7', -6', -4', -3'); 6.79 (1H, d, H-2'); 4.19 (2H, t, H-3); 3.35 (2H, N-H), 3.07 (2H, t, H-1); 2.16 (2H, tt, H-2). ESI-MS/MS [MH]⁺ 202  [C₁₃H₁₃O]⁺ 185.

desOHP. Primary amine desOHDIP (402 mg, 2.0 mmol) and acetone (544 mg, 9.4 mmol) were dissolved in benzene (5 ml), and the mixture flushed with dry argon and stirred at room temperature for 46 h. At the end of this time, the benzene was evaporated and the imine reduced. It was dissolved in CH₃OH (10 ml), and sodium cyanoborohydride (500 mg, 8.0 mmol) and acetic acid (~100 µl) were added. After the mixture was stirred a room temperature for 8 h, the mixture was partitioned between CH₂Cl₂ (20 ml) and aqueous 1 N NaOH (20 ml). The aqueous layer was extracted with additional CH₂Cl₂ (2 × 10 ml), and the combined CH₂Cl₂ layers were washed with H₂O (5 ml), dried over Na₂SO₄, and the solvent was evaporated. The resulting oil was purified on a silica flash column and eluted with stepwise changes in solvent from CH₂Cl₂ to 50% EtOAc in CH₂Cl₂, affording 250 mg (51%) of desOHP. ¹H NMR (CDCl₃):  8.28 (1H, m, H-8'); 7.81 (1H, m, H-5'); 7.46 (4H, m, H-7', -6', -4', -3'); 6.83 (1H, d, H-2'); 4.22 (2H, t, H-3); 2.95 (2H, t, H-1); 2.90 (1H, septet, H-2"); 2.15 (2H, tt, H-1); 1.11 (6H, d H-1"). ESI-MS/MS [MH]⁺ 244  [C₁₃H₁₃O]⁺ 185, [C₆H₁₄N]⁺ 100.

desOHTFP. ¹H NMR (CDCl₃):  8.30 (1H, m, H-8'); 7.85 (1H, m, H-5'); 7.48 (4H, m, H-7', -6', -4', -3'); 6.85 (1H, d, H-2'); 4.25 (2H, t, H-3); 3.21 (1H, qq, H-2"); 3.05 (2H, t, H-1); 2.11 (2H, tt, H-2); 1.28 (3H, d, 2"-CH₃). ESI-MS/MS [MH]⁺ 298  [C₁₃H₁₃O]⁺ 185, [C₆H₁₁NF₃]⁺ 154.

4'-Hydroxydeshydroxypropranolol (4'-OHdesOHP). 4-Methoxy-1-naphthol (843 mg, 4.8 mmol) was dissolved in DMF (8 ml, dried over KOH). Sodium hydride (80% in oil, 500 mg, 16.6 mmol) was rinsed with petroleum ether and then transferred with 6 ml of dry DMF into the reaction vessel. After stirring for 20 min under a dry argon atmosphere, a solution of N-(3-bromopropyl)phthalimide (1.39 g, 5.2 mmol) in 10 ml of dry DMF was added and the reaction mixture and heated to reflux. The mixture was heated at reflux for 6 h, while monitoring the progress of the reaction by TLC (CH₂Cl₂). The solvent was evaporated, and the remaining residue was partitioned between 50 ml H₂O and 50 ml CH₂Cl₂. The aqueous phase was then extracted with CH₂Cl₂ (25 ml). The combined CH₂Cl₂ extracts were washed with H₂O, dried over MgSO₄, and the solvent evaporated to yield the phthalimide intermediate as a brown solid, 1.79 g (~100%).

4'-Hydroxytrifluoropropranolol (4'-OHTFP). The hydrochloride salt of 1,1,1-trifluoro-2-propylamine (908 mg, 6 mmol) (Ono et al., 1996) was converted to the free amine by extraction into cold Et₂O (15 ml) from cold aqueous 2 N NaOH (7 ml) immediately before use. After drying the cold amine solution over Na₂SO₄, the solution was added to dry basic alumina (12 g). 1,2-Epoxy-3-(4-methoxy-1-naphthoxy)propane (345 mg, 1.5 mmol) (Oatis et al., 1981) in anhydrous Et₂O (15 ml) was added slowly to the stirring reaction mixture. The reaction mixture was stirred at room temperature for 22 h, then methanol (70 ml) was added, and the mixture was stirred for another 4 h. At the end of this time, the reaction mixture was filtered through Celite, and the solvent was evaporated to leave the crude product. Purification by flash column chromatography on silica gel using CH₂Cl₂ afforded 120 mg (23%) of the intermediate 4'-O-methyl ether. The 4'-methoxy compound (44 mg, 0.13 mmol) and freshly prepared pyridine hydrochloride (196 mg) were heated to 180°C in a sealed vial for 4 h. The reaction mixture was partitioned between aqueous 1 N HCl (3 ml) and Et₂O (5 ml). The pH of the acid aqueous phase was adjusted to 7 by addition of saturated aqueous Na₂CO₃ solution, and the amine was extracted into Et₂O (3 × 5 ml). After drying over Na₂SO₄, the solvent was evaporated to leave the crude product. The product was purified by flash column chromatography on silica gel and eluted with a gradient from CH₂Cl₂ to 30% EtOAc in CH₂Cl₂ to afford 6 mg (14%) of 4'-OHTFP. ¹H NMR (CDCl₃):  8.16 (2H, m, H-8', -5'); 7.52 (2H, m, H-7', -6'); 6.71 (1H, d, H-3'); 6.62 (1H, d, H-2'); 4.12 (3H, m, H-3, -2); 3.23 (1H, m, H-2"); 3.07 (2H, m, H-1); 1.32 (3H, d, 2"-CH₃). ESI-MS/MS [MH]⁺ 314  [C₁₃H₁₁O₂]⁺ 199, [C₆H₁₁NOF₃]⁺ 170.

Propranolol O-Methyl Ether (MeO-P). Propranolol HCl (100 mg, 0.34 mmol), triethylamine (200 µl, 1.4 mmol), and di-t-butyl dicarbonate (73.8 mg, 0.34 mmol) were dissolved in 10 ml of CH₂Cl₂. The mixture was heated to reflux for two h. After evaporating the solvent and triethylamine, the residue was dissolved in CH₂Cl₂ (20 ml), washed with 0.5 N HCl (2 × 10 ml) and then H₂O (10 ml), dried over Na₂SO₄, and the solvent was evaporated. The remaining oil, the N-t-butyl carbamate of P (110 mg, 0.28 mmol), was dissolved in tetrahydrofuran (10 ml). Powdered KOH (78 mg, 1.34 mmol) and CH₃I (200 µl, 3.2 mmol) were added. The reaction mixture was sealed (septum) and stirred at room temperature for 15 h. Water (10 ml) and CH₂Cl₂ (10 ml) were added, and the CH₂Cl₂ layer was removed, washed with H₂O (5 ml), dried over Na₂SO₄, and the solvent evaporated. The remaining O-methyl ether carbamate was dissolved in a mixture of concentration HCl (1 ml) and EtOAc (4 ml) and then stirred at room temperature for 30 min. After making the solution alkaline by addition of aqueous 5 N NaOH, the EtOAc layer was removed, washed with H₂O, dried over Na₂SO₄, and the solvent was evaporated to yield MeO-P, 75 mg (80%). ¹H NMR (CDCl₃):  8.25 (1H, m, H-8'); 7.78 (1H, m, H-5'); 7.40 (4H, m, H-7', -6', -4', -3'); 6.81 (1H, d, H-2'); 4.20 (2H, m, H-3"); 3.89 (1H, m, H-2'); 3.58 (3H, s, OCH₃); 2.89 (3H, m, H-1, -2"); 1.10 [6H, d, 2"-(CH₃)₂]. ESI-MS/MS [MH]⁺ 274  [C₁₃H₁₁O]⁺ 183, [C₇H₁₆NO]⁺ 130.

Basicity and Lipophilicity. The pK_a values for our series of amines were determined at pION, Inc. (Cambridge, MA), using their validated potentiometric method (Avdeef et al., 1993). The log P and log D values for these compounds were also determined at pION, Inc., according to their validated potentiometric method (Avdeef, 1992; Slater et al., 1994).

Incubations in Insect Cell Expressed Human CYP2D6. Baculovirus-insect cell microsomes coexpressing human CYP2D6 and human P450 reductase were purchased from GENTEST (Woburn, MA). Duplicate incubation mixtures, having a final volume of 200 µl, contained 10 pmol of CYP2D6/ml, 100 mM phosphate buffer (pH 7.4), and 4 µl of substrate dissolved in a 1:1 mixture of CH₃OH and H₂O (final CH₃OH concentration, 1%). After preincubation at 37°C for 3 min, NADPH (Sigma) in phosphate buffer (final concentration, 1 mM) was added to initiate the reaction. After 2 min, the reaction was stopped by addition of 20 µl of 7% perchloric acid, and 5 mg of ascorbic acid was added to stabilize ring-hydroxylated metabolites. Substrate concentrations of 0.49, 1.95, 7.8, 31.25, and 125 µM were used. Preliminary incubations of P and TFP were performed to establish the linearity of metabolite formation with respect to time and the amount of enzyme.

HPLC Separation and Metabolite Quantitation. Following addition of the internal standard, incubation samples were vortexed and centrifuged to precipitate protein. One hundred microliters of the supernatant was analyzed by HPLC using a Hewlett-Packard 1100 LC system (Palo Alto, CA) with Chemstation instrument control and data analysis software. For all substrates, separation was achieved on a Microsorb-MV phenyl column (250 × 4.6 mm; Varian, Palo Alto, CA). Mobile phase gradients consisted of an aqueous phase containing 1% by volume triethylamine, 0.8% by volume phosphoric acid (to bring the pH to 2.2), and acetonitrile. The HPLC gradient used for metabolite separation from all substrates except FP and TFE was 25% (v/v) acetonitrile (75% aqueous buffer described above) for 10 min, then a linear increase to 70% acetonitrile over the next 10 min, an additional 2 min at 70% acetonitrile, then returned to 25% acetonitrile over the next 5 min. For metabolites from FP, the gradient was 20% acetonitrile (80% aqueous buffer) for 10 min, then a linear increase to 60% acetonitrile over the next 10 min, held at 60% acetonitrile for 3 min, and then returned to 20% acetonitrile over the next 4 min. For metabolites from TFE, the gradient was 20% acetonitrile (80% aqueous buffer) for 10 min, then a linear increase to 70% acetonitrile over the next 10 min, held at 70% acetonitrile for 2 min, and then returned to 20% acetonitrile over the next 5 min. The internal standards used were MeO-P for metabolites of P, FP, DFP, TFP, and tBuMe and P for metabolites of iPrMe, TFE, desOHP, and desOHTFP.

Metabolite Quantitation. DIP, 4'-OHP, 5'-OHP, 4'-OHTFP, desOHDIP, and 4'-hydroxydeshydroxypropranolol were quantitated by comparison of peak area ratios of these compounds formed metabolically with standard curves of peak area ratios of known amounts of synthetic standards. Amounts of other metabolites were estimated by comparison of the area ratios with standard curve ratios of 4'-OHP, 5'-OHP, or 4'-OHTFP.

Metabolite Identification. Fluorescence Excitation and Emission Spectra. Unknown metabolite peaks in the HPLC eluent were collected in silane-treated tubes. The fluorescence excitation and emission spectra of each fraction were obtained after transfer to a limited volume quartz cuvette.

Mass Spectra. Fraction collection was repeated, and combined fractions (total approximately 2 ml) were treated with 150 µl of aqueous saturated Na₂CO₃ solution, which was adequate to bring the eluent solution to approximately pH 8. The solutions were then extracted with 3 ml of EtOAc, vortexed for at least 15 s to mix, then centrifuged to separate the layers. The EtOAc layer was transferred to a silane-treated concentration tube, and the solvent evaporated using a stream of dry nitrogen and heat. The metabolite residues were dissolved in 50 µl of CH₃OH and 50 µl of H₂O. The metabolite residues dissolved in CH₃OH/H₂O were infused into a Finnigan LCQ mass spectrometer with a modified electrospray source at a rate of 300 nl/min. MS/MS spectra were obtained by collision-induced dissociation in the ion trap.

Calculation of Michaelis-Menten Kinetic Parameters. K_m and V_max for each of the enzyme-catalyzed reactions were determined by nonlinear fitting of initial velocity versus substrate concentration curves using the k.cat program (Biometallics, Inc., Princeton, NJ). The robust weighting option was used. All velocity values deviating from the calculated curve by less than 10 times the average residual were included in the fit. The rate constants (k_cat values) for metabolite formation were calculated by dividing the V_max values by the enzyme concentration. Substrate concentrations were corrected for substrate depletion by using the average of the initial substrate concentration and the final substrate concentration (Segel, 1975). Final substrate concentrations were calculated by subtracting the concentrations of all metabolites from the initial substrate concentrations.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Fluorinated and Steric Analogs of P and Standards. The propranolol-related compounds prepared for the metabolic studies are listed in Table 1. These include the 1"-mono-, di-, and tri-fluorinated derivatives (FP, DFP, TFP) and steric congeners with two and three additional 1"-methyl groups (iPrMe, tBuMe). Reductive amination of the corresponding ketones using a single enantiomer of DIP and sodium cyanoborohydride afforded the desired P analogs. TFE was prepared by a similar procedure, reductive amination of trifluoroacetaldehyde mono methyl acetal. Examples of these procedures are given under Materials and Methods. To examine the metabolism of a single enantiomer of TFP, its 2S,1"R-enantiomer was prepared from 2S-1-naphthoxy-2,3-epoxypropane (Klunder et al., 1986) and 2R-1,1,1-trifluoro-2-propylamine (Soloshonok and Ono, 1997), using alumina as a catalyst (Posner and Rogers, 1977a,b). Deshydroxy analogs desOHP and desOHTFP were also prepared by reductive amination of acetone and trifluoroacetone, respectively, using desOHDIP and sodium cyanoborohydride. Standards of 4'-hydroxylated metabolites of desOHP and TFP (4'OH-desOHP and 4'OH-TFP) were prepared to aid in the identification of metabolites, and MeO-P was prepared as an analytical standard. Methods for their preparation are also given under Materials and Methods.

View this table: [in this window] [in a new window]   TABLE 1 pKa values and lipophilicity parameters of propranolol analogsa

Basicity, Partition and Distribution Coefficients, and Steric Parameters. The pK_a values of the compounds in the series and data on their partition characteristics appear in Table 1. Octanol-water partition coefficients (log P) and distribution coefficients (log D) at physiological pH were determined. To provide an indication of the steric effect of adding fluorines and methyl groups, steric parameters from the literature are tabulated in Table 2.

View this table: [in this window] [in a new window]   TABLE 2 Steric substituent parameters for propranolol analogs

pK_a Values. The sequential addition of fluorines to the N-isopropyl substituent causes a regular decrease in the pK_a of the amine by 1.6 to 1.8 pK_a units per fluorine (Table 1), whereas the pK_a values of our nonfluorinated congeners iPrMe and tBuMe are similar to that of P. The pK_a values of the trifluoroethylamine TFE and TFP are nearly identical. The removal of the side chain -hydroxyl group from P causes an increase in pK_a from 9.53 in P to 10.25 in desOHP. Similarly, the pK_a of desOHTFP is higher than the pK_a of TFP (5.15 and 4.37, respectively). The lower pK_a in the amines having a -hydroxyl group reflects intramolecular hydrogen bonding of the hydroxyl group to the amine nitrogen, decreasing the electron density on the nitrogen.

Partition Coefficients (log P). The log P values for the fluorinated compounds, partition coefficients of the unionized amines, show small and nonsystematic changes. The monofluorinated compound FP has a slightly lower log P than does P, whereas the difluorinated compound DFP has a larger log P. The substitution of a larger nonfluorinated alkyl group for a methyl group, as in iPrMe or tBuMe, causes a larger increase in log P than does fluorine substitution (Table 1). N-Trifluoroethylamine TFE has a lower log P than either TFP or P, consistent with alkyl group size and branching, and has a larger effect on log P than fluorine substitution. The deshydroxy analogs desOHP and desOHTFP have higher log P values than their side chain hydroxyl-substituted counterparts P and TFP.

Distribution Coefficients (log D). The distribution coefficients (log D, 7.4) (Table 1) reflect partitioning of both ionized and nonionized species between octanol and water at physiological pH and provide a better estimate than log P of distribution between aqueous and nonaqueous phases under physiological conditions. Those compounds with higher pK_a values show lower log D values, reflecting lower partitioning of the protonated amine at neutral pH into the organic phase. Compounds with low pK_a values (e.g., trifluorinated amines being protonated to less than 1% at physiological pH) show log D values equal to their log P values, as expected.

Steric Parameters. A measure of the relative size of the substituents is given by the parameters in Table 3. The Taft steric parameter E_s (Taft, 1956) and the Charton van der Waals radius r_v (Charton  parameter modified by the addition of the van der Waals radius of the hydrogen atom) (Charton, 1975) is given for each of the substituents. Although fluorine is sometimes considered to be isosteric with hydrogen, it is somewhat larger. The generally accepted value for the van der Waals radius of fluorine is 1.47 Å, closer to oxygen at 1.52 Å than to hydrogen at 1.20 Å (Smart, 1994), and the sp³ C-F bond is longer than the sp³ C-H bond (1.40 Å versus 1.09 Å) (March, 1992). The addition of multiple fluorines can cause a significant increase in size of an alkyl group. These values show that the sizes of the isopropyl and t-butyl substituents in iPrMe and tBuMe bracket those of the trifluoromethyl group in TFP, being slightly smaller and slightly larger, respectively.

Identities of Metabolites Formed by Incubation with CYP2D6. Incubations of all the substrates in CYP2D6 resulted in formation of two metabolites with retention times shorter than that of the parent drug and small amounts of N-dealkylation product. Representative chromatograms showing the retention times of ring-hydroxylated metabolites from DFP and TFP are presented in Figs. 2 and 3. The collision-induced dissociation-mass spectra (also in Figs. 2 and 3) of the indicated peaks all clearly show [M + H]⁺ ions at m/z values corresponding to addition of an oxygen atom and fragment ions with correct m/z values for the hydroxylated ring-containing ion (usually the base peak; m/z = 199) and for the side chain ion, which had an m/z value corresponding to no addition of oxygen (Upthagrove et al., 1999).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Representative chromatograms showing the retention times of ring-hydroxylated metabolites from DFP. a, representative chromatogram of incubation of 2S-DFP with CYP2D6; b, MS/MS spectrum of m/z 312; c, fluorescence excitation (ex) and emission spectra (em), respectively, of the 5.3-min peak, identified as 5'-OHDFP; d, MS/MS spectrum of m/z 312; e, fluorescence excitation and emission spectra, respectively, of the 6.0-min peak, identified as 4'-OHDFP.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Representative chromatograms showing the retention times of ring-hydroxylated metabolites from TFP. a, representative chromatogram of incubation of 2S-TFP with CYP2D6; b, MS/MS spectrum of m/z 330; c, fluorescence excitation (ex) and emission (em) spectra, respectively, of the 8.9- to 9.1-min peak, identified as 5'-OHTFP diastereomers; d, MS/MS spectrum of m/z 330; e, fluorescence excitation and emission spectra, respectively, of the 9.9- to 10.1-min peak, identified as 4'-OHDFP diastereomers.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Fluorescence emission (em) and excitation (ex) spectra of P and TFP metabolite standards. a, 4'-OHP; b, 5'-OHP; c, 7'-OHP; d, 4'-OHTFP; e, DIP.

Metabolite Quantitation. The amounts of metabolites for which standards were available were calculated based on standard curves, which were linear in the range used. Comparison of the slopes of the standard curves indicates that 4'-OHP, 4'-OHTFP, and 4'-hydroxydeshydroxypropranolol have nearly identical responses under our fluorescence detection conditions. On this basis, we assumed that the 4'-hydroxylated metabolites from the other substrates would have similar responses, and the amounts of these metabolites could be estimated based on comparison to standard curves for the synthetic metabolite standards. Similarly, the amounts of the 5'-hydroxylated metabolites were estimated based on standard curves for 5'-OHP.

Kinetic Parameters. The K_m and k_cat values, determined from quantitation of each of the metabolites formed from incubations of increasing substrate concentrations with CYP2D6, are summarized in Tables 3 and 4. There are marked changes in K_m and k_cat/K_m over the series. In all cases, ring hydroxylation is preferred over N-dealkylation. As the number of fluorines increased from zero to three, the K_m increased in a regular manner. The TFP and TFE analogs exhibited at least 10-fold higher K_m values than P in our system. Only a small difference is observed between the 2R-P and 2S-P. Likewise, the TFP diastereomers show relatively little stereoselectivity in this process.

Correlations with Physicochemical Parameters. To obtain more quantitative information about the changes in the CYP2D6-catalyzed metabolism of these analogs, physicochemical parameters pK_a and log D (Table 1) were correlated with the metabolic kinetic parameters (Table 3). The K_m values cover a 370-fold range, and the summed k_cat terms vary only about 5-fold across the series. In Fig. 5, separate plots of log K_m and log catalytic efficiency versus pK_a of all the analogs are shown. There is a strong negative correlation of log K_m with pK_a for members of this series (r² = 0.80). Conversely, the log k_cat/K_m is correlated positively and strongly with pK_a (r² = 0.88). The more basic compounds have higher affinities (lower K_m values) and higher catalytic efficiencies.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Plot of log Km () and log kcat/Km () for CYP2D6-catalyzed metabolism of propranolol analogs versus pKa. Log Km = 0.34 pKa + 2.95; r2 = 0.80 (p < 0.05). Log kcat/Km = 0.30 pKa  1.16; r2 = 0.88 (p < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Plot of log Km () and log kcat/Km () for CYP2D6-catalyzed metabolism of propranolol analogs versus log D.  Log Km = 0.51 log D  0.86; r2 = 0.35 (p < 0.05). Log kcat/Km = 0.48 log D + 2.30; r2 = 0.43 (p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Physicochemical Parameters. The range in pK_a values (Table 1) among the fluorinated P analogs, about 5 pK_a units, is expected. A similar wide range of changes have been observed in series of -mono-, di-, and trifluorinated ethylamines (e.g., two series of -fluoroethyl-substituted normeperidine and normetazocine analgesics) in which basicity ranges from pK_a values higher than 9.0 to 3.0 and to 4.5, respectively (Reifenrath et al., 1980). In the simple -mono-, di-, and trifluorinated ethylamines the range is similar, ranging from pK_a 10.7 to 5.4 (Kluger and Hunt, 1984).

Metabolism. We observed similar catalytic efficiencies for metabolism of the P enantiomers (S/R ratio = 0.7), suggesting low stereoselectivity in the metabolism of P in CYP2D6. Our results are in general agreement with previous observations in lymphoblastoid cell-expressed CYP2D6 (Yoshimoto et al., 1995), yeast-expressed CYP2D6 (Bichara et al., 1996; Ching et al., 1996; Ellis et al., 2000), and human liver microsomes (von Bahr et al., 1982; Nelson and Shetty, 1986; Otton et al., 1990; Marathe et al., 1994).