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¹ Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Surrey, United Kingdom and ² Cyclacel Ltd., Dundee, United Kingdom

Requests for reprints: Florence I. Raynaud, Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom. Phone: 44-20-8722-4212; Fax: 44-20-8770-7899. E-mail: Florence.Raynaud{at}icr.ac.uk

	Abstract

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R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 µg/mL (28 µmol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N⁹-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC for R-roscovitine (38µmol/L/h) than for COOH-R-roscovitine (174 µmol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d₉) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d₉ or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d₉ was decreased by 24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d₉ remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d₉, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways.

	Introduction

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Mammalian cell division is tightly regulated by the activation of the cyclin-dependent kinase (CDK) family of cell cycle regulatory proteins (1). This regulation is required for the processes that govern cell proliferation and to allow DNA replication and mitosis to occur in proper sequence and at the correct time. Activation of the CDKs is controlled by various signal transduction pathways and requires formation of a complex consisting of the catalytic CDK unit and the appropriate regulatory cyclin subunit (2). Once activated, these cyclin-CDK complexes in turn regulate other factors (e.g., phosphorylation of the retinoblastoma protein) leading to an orderly progression through the cell cycle. Several such cyclin-CDK complexes have been identified and these were shown to regulate various points of the cell cycle (1). Control of the cell cycle is commonly deregulated in human cancers, for example, by mutation or altered expression of CDKs, cyclins, and their regulatory molecules (2). Because of this, synthetic inhibitors of CDKs may provide important new cancer treatments that are more selective than many of the cytotoxic drugs currently in use (2–4).

Previous reports have described the discovery of 2,6,9-trisubstituted purines that were effective at inhibiting the activity of several protein kinase classes, including the CDK family and especially CDK2 (5–8). Since the first report (5) of the selective inhibition of CDK2 by the 2,6,9-trisubstituted purine olomoucine (Fig. 1), there has been substantial progress in the development of more potent analogues. Among these, CYC202 (seliciclib), a pure and chirally defined form (9) of R-roscovitine (refs. 8, 10; Fig. 1), was chosen for development from a large set of 2,6,9-trisubstituted purine analogues (11) and is currently undergoing phase II clinical trials (12, 13).

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Chemical structures of olomoucine (OLO), R-roscovitine (ROS), COOH-R-roscovitine (COOH-ROS), and bohemine (BOH).

	Materials and Methods

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General
SKF-525A, NADPH, and formic acid (96% ACS grade) were purchased from Sigma-Aldrich (Gillingham, Dorset, United Kingdom). High-performance liquid chromatography–grade methanol was purchased from Laserchrom (Rochester, Kent, United Kingdom). Olomoucine and R-roscovitine were prepared as described previously (89). Schemes for the synthesis of other compounds used in this study are given in Fig. 2. Protein kinase assays were done as described previously (12).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Scheme for synthesis of compounds used in this study. a, BnNH2, iPr2NEt, n-butanol, 95°C, 4 hours. b, iPr-Br (for 3a) or iso-C3D7Br (for 3b), K2CO3, DMA, room temperature, 24 hours. C, 8 (for 4a) or NH2CH(CH2CH3)COOH (for 4b), DBU, NMP, 160°C, 1 hour (racemization during reaction). d, DIEA, n-butanol, DMSO, CH3CH2C(NH2)CH2OH, 140°C, 48 hours. e, DMA, N-bromosuccinimide, room temperature, 16 hours. f, DMSO, NaOH aqueous, 140°C, 32 hours. g, LiAlD4, monoglyme, 95°C, 16 hours. h, 9, DIEA, n-butanol, DMSO, 140°C, 48 h.

Benzyl-(2-fluoro-9-isopropyl-9H-purin-6-yl)amine (3a). To a stirred solution of 2 (0.83 g, 1 eq, 3.41 mmol) in dimethylacetamide (10 mL) at room temperature under an argon atmosphere was added powdered, anhydrous K₂CO₃ (2.35 g, 5 eq, 17.00 mmol) followed by 2-bromopropane (3.2 mL, 10 eq, 34.08 mmol). The reaction mixture was stirred at room temperature for 48 hours, when thin-layer chromatography with CH₂Cl₂/Et₂O/MeOH (55:40:5) indicated that the reaction had gone to completion. The solvent was evaporated in vacuo, and the residue was partitioned between EtOAc (100 mL) and water (200 mL). The aqueous phase was extracted with more EtOAc (2 x 50 mL), and the combined organic phases were washed with brine (50 mL), dried (MgSO₄), and evaporated in vacuo. The residue was purified by silica gel column chromatography eluted with CHCl₃ to afford 3a as a white solid (0.78 g, 80%). Melting point 158-160°C. ¹H NMR (d₆-DMSO, 250 MHz): 1.49 (2 x s, 6H, CH(CH₃)₂), 4.64 (m, 3H, -CH(CH₃)₂ + -HNCH₂-Bz), 7.26 (m, 5H, Bz), 8.25 (s, 1H, -N = CH-N-), 8.90 (brs, 1H, -HNCH₂-Bz). FABMS m/z: 286 ([M + H]⁺, 100), 242 (17), 176 (22), 154 (65), 136 (60). Accurate mass (M + H): actual: 286.1468, measured: 286.1462. Microanalysis (expected/measured): C₁₅H₁₆N₅F: C: 63.14:63.08, H: 5.65:5.58, N: 24.54:24.46.

Benzyl-(2-fluoro-9-[²H₇]isopropyl-9H-purin-6-yl)amine (3b). Methodology as for 3a but using 2-bromopropane-d₇ (1.00 g, 1.95 eq, 7.69 mmol). White solid (0.63 g, 55%). Melting point 154-155°C. ¹H NMR (d₆-DMSO, 250 MHz): 4.63 (d, 2H, J = 5.48 Hz), 7.24-7.33 (m, 5H), 8.22 (s, 1H), 8.82 (brs, 1H). FABMS m/z: 293 ([M + H]⁺, 100), 286 (25), 154 (40), 138 (14), 133 (75), 107 (12). Accurate mass (M + H): actual: 293.1907, measured: 293.1894. Microanalysis (expected/measured): C: 61.62:61.49, H: 5.67:5.51, N: 23.95:23.82.

2-(6-Benzylamino-9-isopropyl-9H-purin-2-ylamino) butyric acid (4a, COOH-R-Roscovitine). To a solution of 3a (151 mg, 0.05 mmol) in 1-methyl-2-pyrrolidinone (5 mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (1.5 mL) was added (R)-(–)-2-aminobutyric acid (8, 99% ee/GLC: 1.03 g, 10 mmol), and the mixture was stirred under nitrogen at 160°C for 1 hour. After cooling, the mixture was diluted with 10% aqueous citric acid and CH₂Cl₂ (25 mL each). The phases were separated, and the organic fraction was extracted with brine (2 x 10 mL), dried over MgSO₄, filtered, and evaporated. The residue was fractionated by preparative reverse-phase high-performance liquid chromatography. Appropriate fractions were pooled and lyophilized to afford 4a (137 mg, 74%) as an amorphous off-white solid. ¹H NMR (d₆-DMSO, 300 MHz) 0.95 (t, J = 7.3 Hz, 3H, CH₂CH₃); 1.51 (d, J = 6.7 Hz, 2H, CH₂CH₃); 1.78 (m, J = 7.3 Hz, 2H, CH₂CH₃); 4.27 (m, 1H, CHCH₂); 4.64 (septet, J = 6.7 Hz, 1H, CH(CH₃)₂); 4.69 (m, 2H, CH₂Ph); 7.26-7.41 (m, 5H, ArH). FABMS accurate mass [M + H]⁺: found 369.2033, calculated for C₁₉H₂₅N₆O₂ 369.2039.

2-(6-Benzylamino-9-[²H₇]isopropyl-9H-purin-2-ylamino)butyric Acid (4b, COOH-R-Roscovitine-d₇). Methodology as for 4a but using 3b (0.12 g, 1 eq, 0.41 mmol); white solid (0.081 g, 53%). Melting point 86-88°C. ¹H NMR (d₆-DMSO, 250 MHz): 0.91 (t, 3H, J = 7.35 Hz), 1.38-1.74 (m, 2H), 4.09-4.24 (m, 1H), 4.32-4.86 (m, 3H), 6.39 (m, 1H), 7.15-7.40 (m, 5H), 7.79 (brs, 2H), 12.21 (brs, 1H). FABMS m/z: 398 ([M + Na]⁺, 100), 376 ([M + H]⁺, 27), 330 (10), 176 (10), 136 (7). Accurate mass (M + Na): actual: 398.2298, measured: 398.2312. Microanalysis (expected/measured): C: 60.78:60.16, H: 6.57:6.51, N: 22.38:21.50.

(R)-2-(6-Benzylamino-9-isopropyl-9H-purin-2-ylamino)butan-1-ol (5a; R-Roscovitine). To a stirred solution of 3a (0.50 g, 1 eq, 1.75 mmol) in n-butanol/DMSO (10 mL, 4:1) at room temperature under an argon atmosphere was added diisopropylethylamine (3.0 mL, 9.82 eq, 17.22 mmol) followed by (R)-(–)-2-aminobutan-1-ol (1.6 mL, 10 eq, 17.0 mmol). The reaction mixture was placed in a preheated oil bath at 140°C and was stirred at this temperature for 48 hours, when thin-layer chromatography with CH₂Cl₂/Et₂O/MeOH (55:40:5) indicated that the reaction had gone to completion. The reaction mixture was allowed to cool to room temperature, and the solvent was evaporated in vacuo. The residue was partitioned between CH₂Cl₂ (100 mL) and water (150 mL), the aqueous phase was extracted with more CH₂Cl₂ (2 x 50 mL), and the combined organic phase was washed with brine (50 mL), dried (MgSO₄), and evaporated in vacuo. The residue was purified by gradient column chromatography on silica gel eluted with CH₂Cl₂/Et₂O/MeOH (50:50:0–50:50:1) to afford 5a as a white solid (0.58 g, 93%). Melting point 104-105°C. ¹H NMR (d₆-DMSO, 250MHz): 0.82 (t, 3H, J = 7.33 Hz, -NHCH (CH₂CH₃)CH₂OH), 1.45 (m, 8H, -NHCH(CH₂CH₃)CH₂OH + -CH(CH₃)₂), 3.30-3.42 (m, 2H, -NHCH(CH₂CH₃)CH₂OH), 3.70 (m, 1H, -NHCH(CH₂CH₃)CH₂OH), 4.51 (m, 4H, -HNCH₂-Bz +, OH + -CH (CH₃)₂), 5.80 (d, 2H, J = 8.37 Hz, -NHCH(CH₂CH₃)CH₂OH), 7.17-7.35 (m, 5H, Bz), 7.76 (brs, 2H, -N = CH-N- + -HNCH₂-Bz). FABMS m/z: 355 ([M + H]⁺, 100), 323 (52), 154 (10), 134 (14). Accurate mass (M + H): actual: 355.2246, measured: 355.2260. Microanalysis (expected/measured): C₁₉H₂₆N₆O: C: 64.38:64.21, H: 7.39:7.44, N: 23.71:23.37.

(R)-2-(6-Benzylamino-8-bromo-9-isopropyl-9H-purin-2-ylamino)butan-1-ol (6). To a stirred solution of 5a (0.10 g, 1 eq, 0.28 mmol) in dimethylacetamide (2.5 mL) at room temperature under an argon atmosphere was added N-bromosuccinimide (0.06 g, 1.15 eq, 0.33 mmol). The reaction mixture was stirred in the dark at room temperature for 16 hours, when thin-layer chromatography with CH₂Cl₂/Et₂O/MeOH (55:40:5) indicated that the reaction had gone to completion. The reaction mixture was cooled to 0°C, and sodium hydrosulfite solution (10% in water, 5 mL) was added. The solvent was evaporated in vacuo, and the residue was partitioned between EtOAc (50 mL) and saturated aqueous NaHCO₃ solution (50 mL), the aqueous phase was extracted with more EtOAc (2 x 20 mL), and the combined organic phase was washed with brine (20 mL), dried (MgSO₄), and evaporated in vacuo. The residue was purified by column chromatography on silica gel eluted with CHCl₃ to afford 6 as a white solid (0.12 g, 98%).Melting point 54-56°C. ¹H NMR (d₆-DMSO, 250 MHz): 0.82 (t, 3H, J = 7.28 Hz, -NHCH(CH₂CH₃)CH₂OH), 1.42-1.57 (m, 8H, -NHCH(CH₂CH₃)CH₂OH + -CH(CH₃)₂), 3.30-3.43 (m, 2H, -NHCH(CH₂CH₃)CH₂OH), 3.72 (m, 1H,-NHCH(CH₂CH₃)CH₂OH), 4.61 (m, 4H, -HNCH₂-Bz +, OH + -CH(CH₃)₂), 5.92 (d, 2H, J = 7.95 Hz, -NHCH(CH₂CH₃)CH₂OH), 7.18-7.33 (m, 5H, Bz), 7.95 (brs, 1H, -HNCH₂-Bz). FABMS m/z: 433 ([M + H]⁺, 100), 403 (45), 176 (25), 154 (74), 136 (73), 107 (40). Accurate mass (M + H): actual: 433.1351, measured: 433.1336. Microanalysis (expected/measured): C₁₉H₂₅N₆OBr: C: 52.66:52.77, H: 5.81:5.59, N: 19.39:18.15.

2-(R)-6-Benzylamino-2-(1-hydroxymethyl-propylamino)-9-isopropyl-7,9-dihydro-purin-8-one (7, C8-oxo-R-Roscovitine). To a stirred suspension of 6 (0.03 g, 1 eq, 0.07 mmol) in DMSO (1 mL) at room temperature in a pressure vessel was added NaOH solution (50% w/v in water; 5 mL). The vessel was sealed and placed in a preheated oil bath at 140°C, and the vessel contents were stirred at this temperature for 32 hours. The reaction mixture was allowed to cool to room temperature, and the pH was adjusted to 7.0 with citric acid solution (10% in water). This was extracted with EtOAc (3 x 50 mL), and the combined organic phase was washed with brine (20 mL), dried (MgSO₄), and evaporated in vacuo. The residue was purified by gradient column chromatography on silica gel eluted with CH₂Cl₂/Et₂O/MeOH (50:50:0–50:50:15) to afford 7 as a white solid (3.4 mg, 13%). Melting point 68-70°C. ¹H NMR (d₆-DMSO, 250 MHz): 0.83 (t, 3H, J = 7.63 Hz, -NHCH(CH₂CH₃)CH₂OH), 1.42-1.57 (m, 8H, -NHCH(CH₂CH₃)CH₂OH + -CH(CH₃)₂), 3.31-3.40 (m, 2H, -NHCH(CH₂CH₃)CH₂OH), 3.72 (m, 1H,-NHCH(CH₂CH₃)CH₂OH), 4.50-4.60 (m, 4H, -HNCH₂-Bz +, OH + -CH(CH₃)₂), 5.60 (d, 2H, J= 8.84 Hz, -NHCH(CH₂CH₃)CH₂OH),7.18-7.36 (m, 5H, Bz). FABMS m/z: 371 ([M + H]⁺, 100), 339 (40), 323 (20), 286 (17), 133 (55), 113 (10). Accurate mass (M+H):actual: 371.2195, measured: 371.2206.

[1-²H₂]-(R)-(-)-2-Amino-butan-1-ol (9). A solution of LiAlD₄ (1.22 g, 1.5 eq, 29.05 mmol) in anhydrous monoglyme (40 mL) cooled to 0°C was added to a stirred suspension of (R)-(=)-2-aminobutyric acid (8, 2.0 g, 1 eq, 19.39 mmol) in anhydrous monoglyme (20 mL) at 0°C under an atmosphere of argon. When addition was complete, the reaction mixture was allowed to reach room temperature over 20 minutes, placed in a preheated oil bath at 95°C, and refluxed at this temperature under argon for 16 hours. The solvent was evaporated in vacuo, and the residue was refluxed in acetonitrile (60 mL) for 3 hours. The solution was allowed to cool to room temperature, the precipitate was removed by filtration and washed with more acetonitrile (3 x 30 mL), and the combined filtrate was evaporated in vacuo to afford 9 as a pale yellow oil (1.34 g, 76%). ¹H NMR (d₆-DMSO, 250 MHz): 0.84 (t, 3H, J = 7.35 Hz), 1.03-1.53 (m, 2H), 2.42-2.53 (m, 1H).

[1-²H₂]-2-(6-Benzylamino-9-[²H₇]isopropyl-9H-purin-2-ylamino)-butan-1-ol (5b, R-Roscovitine-d₉). Methodology as for 5a but using 3b (0.58 g, 1 eq, 1.98 mmol) and [1-²H₂]-(R)-(–)-2-amino-butan-1-ol (9, 1.1 g, 6 eq, 12.06 mmol). White solid (0.36 g, 51%). Melting point 102-103°C. ¹H NMR (d₆-DMSO, 250 MHz): 0.82 (t, 3H, J = 7.38 Hz), 1.29-1.63 (m, 2H), 3.72-3.80 (m, 1H), 4.40-4.85 (m, 3H), 5.83 (d, 1H, J = 8.50 Hz), 7.12-7.36 (m, 5H), 7.77 (brs, 2H). FABMS m/z: 364 ([M + H]⁺, 100), 330 (28), 240 (8), 154 (25), 136 (20). Accurate mass (M + H): actual: 364.2811, measured: 364.2805. Microanalysis (expected/measured): C: 62.78:62.87, H, 7.38:7.34, N: 23.12:22.82.

In vitro Kinase Assays
Protein kinase assays were carried out by measurement of incorporation of radioactive phosphate from ATP into appropriate polypeptide substrates by purified recombinant human protein kinases and kinase complexes as described previously (12). Assays were done using 96-well plates and appropriate assay buffers [typically 25 mmol/L -glycerophosphate, 20 mmol/L MOPS, 5 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L Na₃VO₃ (pH 7.4)] into which were added 2 to 4 µg of active enzyme with appropriate substrates. The reactions were initiated by addition of Mg/ATP mix (15 mmol/L MgCl₂ and 100 mol/L ATP with 30–50 kBq per well of [-³²P]ATP), and mixtures were incubated as required at 30°C. Reactions were stopped on ice followed by filtration through p81 filter plates or GF/C filter plates (Whatman Polyfiltronics, Kent, United Kingdom). After washing thrice with 75 mmol/L aqueous orthophosphoric acid, plates were dried, scintillant was added and incorporated radioactivity was measured in a scintillation counter (TopCount, Packard Instruments, Pangbourne, Berks, United Kingdom). Compounds for kinase assay were made up as 10 mmol/L stocks in DMSO and diluted into 10% DMSO in assay buffer. Data were analyzed using curve-fitting software (GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego, CA) to determine IC₅₀ values (concentration of test compound that inhibits kinase activity by 50%).

In vitro Metabolism Studies
Mouse hepatic S9 fraction and microsomes were purchased from Tebu-bio Ltd. (Peterborough, United Kingdom). S9 fraction or microsomes (100 µL of a solution of 1 mg protein/mL in PBS) were mixed with either R-roscovitine (100 µL of 100 µg/mL solution) or a mixture of R-roscovitine and R-roscovitine-d₉ (100 µL solution containing 50 µg/mL of each) and 200 µL PBS. For inhibitor studies, SKF-525A was added to either S9 fraction or microsomes at a final concentration of 1 mmol/L. Reaction was started by the addition of 100µL NADPH (5 mmol/L). Following incubation at 37°C for 0, 15, and 60 minutes, the reaction was terminated by the addition of methanol (1.5 mL) and thoroughly mixed. Following centrifugation, the supernatant was transferred to clean tubes. Control incubations, prepared by replacing either microsomes or drug with an equivalent volume of PBS, were included and treated as described above.

In vivo Studies
Before use, female BALB/c^– mice (Charles River, Margate, United Kingdom) were housed in a maximizer positive, individually vented caging system (Thoren, Hazleton, PA) and allowed food (Lillico, Betchwork, Surrey, United Kingdom) and tap water ad libitum. All animal procedures complied with local and national (United Kingdom Coordinating Committee for Cancer Research) guidelines for animal welfare (16).

Mice were given 100 mg/kg i.v. in the tail vein (0.1 mL/kg) of either R-roscovitine, R-roscovitine-d₉, or a 1:1 mixture of R-roscovitine and R-roscovitine-d₉ (i.e., 50 mg/kg of each). For plasma pharmacokinetic studies, blood was sampled by cardiac puncture under transient anesthesia with halothane at 0, 0.25, 0.5, 1, 2, 4, 6, and 24 hours with three animals for each time point. All samples were then stored frozen at –20°C until analyzed. Levels of parent compound(s) and metabolites were determined in samples prepared for pharmacokinetic analysis. Metabolites were identified in 0.25-hour plasma samples, whereas plasma samples from vehicle-treated mice were used as controls.

In separate experiments to quantify urinary excretion of R-roscovitine and the major metabolite COOH-R-roscovitine, mice were given 50 mg/kg i.v. (n = 4), i.p. (n = 2), or p.o. (n = 2) R-roscovitine. Animals were kept individually in metabowls for 24 hours, and food and water were provided ad libitum. To ensure urination, mice were given 0.1 mL saline i.p., and urine was collected into labeled tubes. Cages were then washed with double-distilled water (1 mL), which was collected into the same tube as the urine, and stored frozen at –20°C until analyzed.

Sample Preparation
Identification of Metabolites of R-Roscovitine and R-Roscovitine-d₉ in Mouse Plasma, Urine, and Microsomal Incubations. Pooled 0.25-hour mouse plasma samples from treated animals were used for liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to obtain collision-induced dissociation spectra of metabolites to aid identification. For these studies, aliquots (33 µL) of plasma from three animals were combined and precipitated with methanol (300 µL), mixed, and centrifuged, and the supernatant was transferred to clean tubes. Further details of the analyses are given below. Aliquots (400 µL) of the microsome incubations treated as described above were mixed with olomoucine (20 µL of a 10 µmol/L solution) as an internal standard and then analyzed by full-scan LC/MS (details below).

Quantification of R-Roscovitine, COOH-R-Roscovitine, R-Roscovitine-d₉, and COOH-R-Roscovitine-d₇ in Mouse Plasma and Urine Samples. Calibration curves of the four analytes were prepared by dilution of stock solutions (2 mmol/L in DMSO) in methanol to provide a series of calibrant stocks. These were spiked (10 µL of each calibrant stock into either 100 µL control mouse plasma or urine) to give a calibration range of 50 to 25,000 nmol/L in plasma. Additional stocks were prepared and used to spike control plasma (100 µL) to provide quality-control samples at nominal concentrations of 150, 1,500, and 15,000 nmol/L. In circumstances where urinary analyte levels were outside the calibration range, urine samples were diluted (1:10, 1:100, or 1:1,000) and analyzed against calibration curves, and quality-control samples were prepared in suitably diluted control urine. Calibrants, quality-control, and test plasma or urine samples (100 µL) from treated mice were all added to olomoucine (20 µL of 10 µmol/L solution) as internal standard and mixed. All samples were then treated with methanol (400 µL) to precipitate proteins and salts and centrifuged, and the supernatant was transferred to clean tubes for subsequent analysis. Details of LC/MS equipment and conditions are given below.

Liquid Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry/Tandem Mass Spectrometry Analysis
Qualitative Analysis. Metabolite detection and LC/MS/MS product ion spectra studies were done using a Thermoseparations (Manchester, United Kingdom) AS3000 autosampler, P4000 quaternary pump, and UV1000 detector set to 254 nm attached to a Thermofinnigan (Hemel Hempstead, United Kingdom) LCQ ion trap mass spectrometer. Samples were injected into a Supelco LC-ABZ, 50 x 4.6 mm, 5 µm column (Supelco, Inc., Supelco Park, Bellefonte, PA). The mobile phase consisted of 0.1% formic acid (A) and methanol (B). The gradient started with 90:10 (A/B, v/v), which was held isocratically for 0.5 minute, followed by a linear increase to 10:90 (A/B, v/v) over 6 minutes and maintained at these conditions for a further 3.5 minutes; a flow rate of 1 mL/min was used throughout the analysis.

Eluant from the UV detector passed, without splitting, into a standard LCQ electrospray source operated in positive mode. Mass spectrometer conditions were sheath gas 80, auxiliary gas 20 (both arbitrary units), capillary voltage 4.5 kV, and heated capillary temperature 250°C. The mass range was 50 to 650. Scan time was controlled by the ion trap and was set to a maximum injection time of 200 ms or the time required to inject 2 x 10⁸ ions; for each scan, the system automatically used whichever time was reached first.

For MS/MS analysis, spectra were obtained by setting the ion to be retained in the ion trap and the ion energy (usually 30–50 V) used for fragmentation. Helium gas, continually present as damping gas in the trap, served as collision gas at the higher fragmentation voltages.

Quantitative Analysis. Multiple reaction monitoring LC/MS/MS analysis used a Waters (Watford, United Kingdom) high-performance liquid chromatography system consisting of a WISP 717 autosampler and 600 MS quaternary pump and controller. Samples were injected into the column as described in Qualitative Analysis, and the same mobile phase was used. However, a different gradient was employed with a lower mobile-phase flow rate to enhance sensitivity. The gradient (flow rate 0.6 mL/min) started with 80:20 (A/B, v/v), which was held isocratically for 0.5 minute followed by a linear increase to 10:90 (A/B, v/v) over 6 minutes and maintained at these conditions for a further 3.5 minutes.

Column eluant entered a Thermofinnigan TSQ700 triple-stage mass spectrometer without splitting via a standard Thermofinnigan electrospray source operated in positive ion mode. MS conditions were sheath gas 70 p.s.i., auxiliary gas 15 (arbitrary units), capillary voltage 4.5 kV, and heated capillary 250°C. Multiple reaction monitoring fragmentation was achieved using an argon pressure of 1.5 mTorr and collision offset energy of –30 eV; electron multiplier gain was set to 1,500 to 1,800 V.

The following transitions were used for all analyses: R-roscovitine m/z 355 to 241, R-roscovitine-d₉ m/z 364 to 250, COOH-R-roscovitine m/z 369 to 255, COOH-R-roscovitine-d₇ m/z 376 to 232, and olomoucine m/z 299 to 91.

	Results

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Characterization of Plasma Metabolites of R-Roscovitine
Detection of Metabolites in Plasma Samples from Mice Given R-Roscovitine and R-Roscovitine-d₉. Plasma metabolites of R-roscovitine were located by comparison of data from control and treated samples subjected to LC/MS analysis. MS ions that could not be detected in control samples but were present in plasma from treated mice were identified. Metabolites were identified as being derived from R-roscovitine if they could not be detected in plasma samples treated with R-roscovitine-d₉ and vice versa.

Metabolites were identified in plasma extracts from mice given 1:1 mixture of R-roscovitine and R-roscovitine-d₉. Extracted ion traces of these metabolite ion pairs are shown in Fig. 3. The mass difference between the lower mass ion (derived from R-roscovitine) and the higher mass ion (derived from R-roscovitine-d₉) of each pair indicates the number of deuterium atoms remaining in these metabolites. These traces show that several metabolites were identified for which the number of deuterium atoms present in each metabolite could be determined (see Table 1).

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Extracted ion traces for plasma metabolites of R-roscovitine and R-roscovitine-d9.

Interpretation of MS/MS Spectra of R-Roscovitine and Its Metabolites. R-roscovitine and its metabolites show a characteristic neutral loss of 42 Da derived from the isopropyl group, which is lost as propene. The hydroxyalkyl group of R-roscovitine and some hydroxylated metabolites can undergo several different fragmentation reactions and may lose water (neutral loss of 18 Da), methanol (loss of 32 Da), or the entire hydroxyalkyl moiety. Neutral loss of these hydroxyalkyl moieties gives characteristic mass differences of 72 Da (loss of hydroxybutene) and 88 Da (loss of dihydroxybutene). Sequential fragmentation reactions are indicated by the neutral loss of 60 Da seen in several spectra attributable to the initial loss of water (18 Da) and then a propene group (42 Da) or vice versa. These reactions are indicated in the product ion spectra list of Table 1 as the sum of several smaller neutral losses when these can be assigned. Another important loss seen for some metabolites is a fragment of 46 Da. This derives from neutral loss of formic acid (HCOOH) from a carboxylic acid moiety of the modified purine C2 substituent of R-roscovitine.

Although odd electron reactions are uncommon in collision-induced dissociation MS/MS spectra, they can still occur (17). R-roscovitine and its metabolites show an apparent neutral loss of 91 Da. This is difficult to explain if it is a true neutral loss and is probably due to formation of an energetically stable benzyl radical (18) and a positively charged ion.

Identification of R-Roscovitine Metabolites from MS/MS Spectra and Deuterated Analogue Experiments.

R-Roscovitine. The compound eluting at 4.74 minutes has the same retention time and same spectrum as R-roscovitine. R-roscovitine shows major fragmentation ions at m/z 337 (loss of water, –18 Da), 313 (loss propene from isopropyl group, –42 Da,), 295 (loss of 18 + 42 Da), 283 (loss hydroxybutene moiety from C2 substituent, –72 Da), and 241 (loss of 42 + 72 Da). Other fragment ions seen included m/z 233 (122 Da), 192 (163 Da), and 91 (benzylic ion).

M1: m/z 313. This ion is 42 Da less than R-roscovitine, which suggests loss of the isopropyl group attached to N⁹ of the purine nucleus. The selected ion trace for this metabolite derived from R-roscovitine-d₉ (see Fig 3, m/z 315 trace) is only 2 a.m.u. higher than the ion derived from R-roscovitine; this can be explained by loss of the d₇-isopropyl group. The MS/MS spectrum (Table 1) shows neutral losses of 18 Da (m/z 295), 32 Da (m/z 281), and 72 Da (m/z 241) from the molecular ion, which could arise from loss of water, methanol, and 1-hydoxybutene, respectively. Formation of these neutral moieties from the C2 substituent, and the lack of any ions or neutrals from the isopropyl group, support identification of this metabolite as N⁹-desisopropyl-R-roscovitine.

M2: m/z 353. This metabolite has a coeluting analoguederived from R-roscovitine-d₉ with a m/z of 361, suggesting loss of one deuterium atom. The MS/MS spectrum (Table 1; Fig. 4A) shows neutral losses of 42 Da (forming isopropenyl [CH₂ = CHCH₃] group) producing an ion at m/z 311, 70, and 72 (different rearrangements of hydroxybutyl group) producing ions at m/z 283 and 281 together with loss of 91 Da (benzyl radical) producing an ion at m/z 262. The spectrum of deuterated analogue with a m/z of 361 forms similar ions (see Fig. 4B), but the masses are different due to the presence of deuterium. For example, the ion m/z 313 is derived from loss of a deuterated isopropenyl moiety (CD₂ = CDCD₃) of mass 48 Da, leaving one of the deuteriums of the d₇-isopropyl group attached to the purine nucleus of the m/z 313 ion. This and the presence of the other deuterium in the hydroxybutyl group account for the 2 Da mass difference of this ion from the m/z 311 ion seen for the m/z 353 metabolite derived from R-roscovitine. The ions in the R-roscovitine-d₉ spectrum at m/z 288 and 290 (neutral losses of 72 and 70 Da, respectively) may be due to equivalent rearrangements of the hydroxybutyl moiety but with migration of the remaining deuterium atom to the C2-amino nitrogen atom. The spectrum also shows a loss of 91 Da to give an ion of m/z 270. This is probably due to loss of a benzyl radical, which suggests any metabolic change has not occurred in the benzyl moiety itself. Comparison of the ions and neutral losses for the corresponding metabolites derived from R-roscovitine and R-roscovitine-d₉ suggests that this metabolite is an aldehyde produced by oxidation of the hydroxybutyl carbinol.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 4. MS/MS spectra of metabolite M2 identified in plasma 0.25 hour following i.v. administration of 50 mg/kg (A) R-roscovitine and (B) R-roscovitine-d9.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 5. MS/MS spectra of (A) synthetic COOH-R-roscovitine and (B) corresponding metabolite detected in plasma.

M4: m/z 371. The MS/MS spectrum for the first of these metabolites contains the following ions: m/z 353 (–18 Da, loss of water), 329 (–42 Da, loss of the propenyl group from the N⁹ group), 311 (–60 Da, from sequential loss of 18 and 42 Da), 283 (–88 Da, loss of dihydroxybutene from C2 substituent, which contains additional oxygen; i.e., 72 + 16 Da), and 91. These data suggest that the metabolite in question is derived from oxidation of R-roscovitine in the hydroxybutyl group. Although the precise position of this hydroxylation cannot be ascertained from these results, the presence of nine deuterium atoms in the metabolite derived from R-roscovitine-d₉ suggests that hydroxylation has occurred on part of the hydroxybutyl side chain in which deuterium was not incorporated.

M5: m/z 371. This is another hydroxylated metabolite. Although the MS/MS spectrum of this metabolite is more complex, it has many of the same ions as M4. It is probably derived from oxidation at a different carbon atom of the hydroxybutyl group of R-roscovitine to that of M4. Once again, the presence of nine deuterium atoms in the R-roscovitine-d₉-derived analogue indicates that hydroxylation has not occurred at a deuterium-substituted carbon atom.

M6: m/z 371. This is the third hydroxylated metabolite. The MS/MS spectrum shows m/z 353 (–18 Da, loss of water), 329 (–42 Da, loss of propene), 299 (–72 Da, loss of hydroxybutene), 280 (–91 Da, benzyl radical), 257 (loss of both propene and hydroxybutene moieties, 42 +72 Da), and 91. Many of the neutral losses seen in this spectrum are the same as those of R-roscovitine, and the product ions are 16 Da heavier. Oxidation at C8 of the purine core of R-roscovitine would account for these findings. The identity of this metabolite was confirmed by synthesis of authentic C8-oxo-R-roscovitine as described in Materials and Methods. The synthetic compound had the same retention time and mass spectrum (see Fig. 6A) as M6 (Fig. 6B).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 6. MS/MS spectra of (A) synthetic C8-oxo-R-roscovitine and (B) corresponding metabolite detected in plasma.

Microsomal Metabolism of R-Roscovitine and R-Roscovitine-d₉
After LC/MS analysis of microsomal incubations, peak areas of internal standard, parent compound, and metabolites were obtained from the appropriate extracted ion traces. Metabolite levels were then expressed as peak area ratios of analyte/internal standard, and the results were plotted against time. Microsomal metabolism of both R-roscovitine and R-roscovitine-d₉ was rapid and extensive. Results for the parent compound and six phase I metabolites described earlier are shown in Table 2. Of these metabolites, COOH-R-roscovitine and C8-oxo-R-roscovitine had the highest peak area ratios. Although the corresponding ratio for the aldehyde intermediate was comparatively high in the 15-minute incubation, the levels were 4- to 5-fold lower in the 1-hour incubations. This is presumably due to rapid further metabolism of the aldehyde to COOH-R-roscovitine.

Comparative Kinase Inhibitory Activities of R-Roscovitine, COOH-R-Roscovitine, and C8-oxo-R-Roscovitine
Table 4 shows the IC₅₀ values for the inhibition by R-roscovitine, COOH-R-roscovitine, and C8-oxo-R-roscovitine of selected kinases. The results show that COOH-R-roscovitine is a considerably less potent inhibitor of both CDK2 (26-fold) and CDK4 (11-fold) compared withR-roscovitine. Thus, metabolism to the carboxylic acid represents a deactivation pathway. Conversion of R-roscovitine to the C8-oxo-R-roscovitine metabolite is also a deactivation pathway for CDK2 (35 times less active) but not for CDK4, where IC₅₀ values are 14.6 and 17 µmol/L for R-roscovitine and C8-oxo-R-roscovitine, respectively.

0-

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Plasma pharmacokinetic profiles for R-roscovitine, R-roscovitine-d9, COOH-R-roscovitine, and COOH-R-roscovitine-d7 in mice after i.v. administration of R-roscovitine and R-roscovitine-d9 (1:1) at a total dose of 100 mg/kg.

	Discussion

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View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Putative scheme for the metabolism of R-roscovitine.

Metabolic conversion of xenobiotics that contain a primary aliphatic alcohol group to carboxylic acids has been observed on many occasions (20). Either initial conversion of R-roscovitine to an aldehyde or direct conversion to the carboxylic acid is mediated by a NADPH-dependent system. In either case, the low levels of the R-roscovitine-derived aldehyde metabolite detected and its apparent rapid further oxidation to the carboxylate (Tables 2 and 3) suggest that pharmacologic complications arising from this potentially reactive metabolite are unlikely to be significant. Chmela et al. (21) suggested that a cytosolic class 1 alcohol dehydrogenase was responsible for conversion of bohemine, a structural analogue of R-roscovitine (see Fig. 1), to an analogous carboxylic acid metabolite. We have found that metabolism of R-roscovitine is primarily microsomal, is inhibited in the presence of the CYP inhibitor SKF-525A (19), and is NADPH dependent. This suggests that metabolism of R-roscovitine, at least in mouse microsomes, is principally CYP mediated. A subsequent study (22) showed that bohemine carboxylation was sensitive to certain CYP enzyme inhibitors and concluded that CYP2A and CYP3A contributed substantially to this biotransformation. In the case of R-roscovitine, it remains to be determined which CYP enzymes are responsible for the formation of the major metabolite COOH-R-roscovitine.

Oxidative N-dealkylation is a metabolic pathway seen with many xenobiotics and is usually due to activity of the microsomal mixed function oxidase system (20). Our results show that the isopropyl group of R-roscovitine is susceptible to N-dealkylation. However, this is a relatively minor metabolic pathway for these substituted purines as shown by the low levels of the dealkylated metabolite produced in microsomal incubations of R-roscovitine.

Another route of metabolism observed with R-roscovitine was hydroxylation, especially at the C8 position of the purine nucleus. Metabolism of purine analogues at this position has been seen for several compounds, including caffeine and theophylline (23). Several enzyme systems may be involved in this reaction, including xanthine oxidase, aldehyde oxidase, and CYP mixed function oxidase. Studies have shown that these enzymes have different substrate specificities and subcellular localizations. Xanthine and aldehyde oxidase are present in the soluble cytosol, unlike the microsomal mixed function oxidase. As C8 hydroxylation of R-roscovitine occurs in both microsomes and S9 fraction (which contains microsomal enzymes) is NADPH dependent but does not seem to be inhibited by SKF-525A (see Table 3), it is possible that more than one enzyme system may be responsible for this metabolic conversion.

Literature reports of the metabolism of O⁶-benzylguanine, which shares some structural features with the 2,6,9-trisubstituted purines, have shown that C8 hydroxylation is an important metabolic pathway in mice, rats, and man (24–28). In vitro experiments have shown hepatic formation of O⁶-benzyl-8-oxoguanine is primarily microsomal and NADPH dependent in both rats and man (2425). Further studies are required to elucidate, in more detail, the role of different enzyme systems in the metabolism of R-roscovitine.

Carbon-deuterium bonds are well known to be harder to break than equivalent carbon-hydrogen bonds. The difference in energy requirements has the effect of slowing some reaction processes; this is known as the deuterium isotope effect (18). For enzymatic reactions, this slowing effect can be detected by changes in the rate and extent of product formation. A 2- to 5-fold change in the rate of some metabolic pathways has been shown previously where carbon-deuterium bonds are involved in the metabolic process (29). For this reason, we synthesized the deuterated analogue R-roscovitine-d₉ and compared its quantitative and qualitative metabolism with that of R-roscovitine. The difference in formation of metabolites seen between R-roscovitine and R-roscovitine-d₉ can mostly be explained by the presence of deuterium leading to metabolic switching between alternative pathways. Interestingly, formation of R-roscovitine aldehyde did not seem to be inhibited by the presence of deuterium, although breaking of a carbon-deuterium bond was involved in this reaction (Table 2, column M2). However, formation of COOH-R-roscovitine from the aldehyde, which involves the breaking of another carbon-deuterium bond, was inhibited (Table 2, column M3). Comparison of the peak area ratios for the analogues of this metabolite shows that 14% less COOH-R-roscovitine-d₇ was formed from R-roscovitine-d₉ compared with COOH-R-roscovitine derived from R-roscovitine after incubation for 1 hour.

That inhibition of this major metabolic pathway by the presence of deuterium led to metabolic switching was shown by increased formation of several minor metabolites, most notably C8-oxo-R-roscovitine-d₉, for which peak area ratios were double those seen for the corresponding R-roscovitine metabolite (Table 2, column M6). One additional metabolite showed reduced formation from R-roscovitine-d₉ and is derived from breakage of a carbon-deuterium bond; this was N-d₇-desisopropyl-R-roscovitine (Table 2, column M1). About 33% of this metabolite were formed from R-roscovitine-d₉ compared with R-roscovitine.

The pharmacokinetic profile of R-roscovitine following i.v. administration confirms that clearance is mainly a result of metabolism to COOH-R-roscovitine. Although the levels of the latter were slightly decreased when R-roscovitine-d₉ was given, this did not result in an appreciable increase in either C_max or AUC for the parent compound. A major change in pharmacokinetics was not expected for R-roscovitine-d₉ compared with R-roscovitine given that the rate of conversion to the carboxylate was not greatly decreased. In addition, the fact that levels of the hydroxylated metabolites were increased (data not shown) suggests that metabolic switching, as described above for microsomal incubations, did occur in the mouse. Unfortunately, the major hydroxylated metabolite (C8-oxo-R-roscovitine) is less active at inhibiting CDK2 than R-roscovitine and so would not be expected to result in an overall improvement in activity in vivo of R-roscovitine-d₉ compared with R-roscovitine.

We have reported previously that plasma clearance of R-roscovitine is lower than that of the analogue bohemine (0.14 and 1.27 L/h, respectively; ref. 11). Studies on the metabolism of bohemine identified glycosidation as an important metabolic pathway (21, 22). Furthermore, a recent report (30) suggests that the aliphatic hydroxyl groups present in olomoucine, bohemine, and R-roscovitine can be conjugated with glycoside donors, especially UDP-glucose and UDP-glucuronic acid. Glucuronidation of bohemine was detected in mouse, rat, monkey, and human microsomes in the presence of UDP-glycosyl donors, whereas extensive glucosidation seemed to be confined to mouse microsomes. Furthermore, the order of susceptibility to both glucosidation and glucuronidation in mouse microsomes was bohemine >> R-roscovitine > olomoucine (30). Although detailed phase II microsomal incubations have not been carried out, glycosilated and glucuronidation products were only detected as traces in mice urine and represented <2% of the administered dose.

The high plasma clearance observed together with the high levels of carboxylic acid formed that we observed in the mouse were confirmed in subsequent studies in human healthy volunteers and were predictive of both parent and metabolite levels seen in humans (14). The presence of minor metabolites, however, was not examined in the human studies.

Metabolism studies provide important information for a modern drug development program. Identification of major metabolic pathways of candidate drugs can help drive drug design, especially when metabolism is an important factor affecting bioavailability or clearance. Traditionally, identification of metabolites has relied on (a) extraction of potential metabolites from biological fluids, such as plasma and urine; (b) identification by mass spectrometry; (c) chemical synthesis and characterization of all potential metabolite standards by spectroscopic means; and (d) coelution of metabolites and standards following chromatographic separation. However, with the need for speedy provision of metabolism data to facilitate drug design, insufficient time or resources usually limit the amount of interpretable data available. We have attempted to overcome some of these problems by using deuterium-labeled analogues of R-roscovitine. Mass differences of metabolites derived from R-roscovitine and R-roscovitine-d₉ were used to determine the number of deuterium ions present in the metabolites and to help elucidate the major metabolic pathways.

In conclusion, we have characterized the major mouse urinary metabolite of R-roscovitine as the carboxylic acid analogue and identified several other metabolites of this compound. Metabolism is both rapid and extensive, with COOH-R-roscovitine accounting for the majority of urinary metabolites. These data suggest that metabolism is an important factor affecting the plasma clearance of R-roscovitine. Metabolism to the carboxylic acid is a deactivation pathway, because this metabolite is a less potent CDK inhibitor than the parent compound.

	Acknowledgments

 
We thank Dr. Ted Mc Donald for valuable discussions.

	Footnotes

 
Grant support: Cancer Research UK and Cyclacel Ltd. D. Lane is a Gibb Fellow and P. Workman is a Life Fellow of Cancer Research UK.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with U.S.C. Section 1734 solely to indicate this fact.

Received 7/29/04; revised 11/ 3/04; accepted 11/10/04.

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