U-89843 - ACS Publications - American Chemical Society

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Chem. Res. Toxicol. 1996, 9, 1230-1239

Articles Bioactivation of 6,7-Dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to Reactive Intermediates That Bind Covalently to Macromolecules and Produce Genotoxicity1 Zhiyang Zhao,*,† Kenneth A. Koeplinger,† Guy E. Padbury,† Michael J. Hauer,† Gordon L. Bundy,‡ Lee S. Banitt,‡ Theresa M. Schwartz,‡ David C. Zimmermann,‡ Philip R. Harbach,§ Judy K. Mayo,§ and C. Sidney Aaron§ Drug Metabolism Research, Discovery Chemistry, and Investigative Toxicology, Pharmacia & Upjohn, Inc., 301 Henrietta Street, Kalamazoo, Michigan 49001 Received June 5, 1996X

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and Nacetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

Introduction The increasing implication of reactive oxygen species in a variety of pathological conditions suggests the potential utility of reactive oxygen inhibitors and scavengers for prevention and treatment of these disorders (1, 2). The novel pyrrolo[2,3-d]pyrimidine antioxidants are a family of important biologically active molecules currently under evaluation for potential treatment of diseases which are mediated by reactive oxygen species (3, 4). The 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) is a potent member of this lipid peroxidation inhibitor family and has shown promising activity in modulating inflammatory processes involved in asthma (3). Further development of U-89843 * To whom correspondence should be addressed. † Drug Metabolism Research ‡ Discovery Chemistry. § Investigative Toxicology. X Abstract published in Advance ACS Abstracts, October 1, 1996.

S0893-228x(96)00092-6 CCC: $12.00

as a drug candidate was discontinued due partially to its positive response in the in vitro unscheduled DNA synthesis (UDS)2 assay. The observed in vitro genotoxicity of U-89843 prompted us to investigate further the possible mechanisms for the outcome with hope of designing a compound with a better toxicological profile. We reported previously that U-89843 underwent cytochrome P450 (P450) catalyzed oxidation in the rat, both in vivo and in vitro, to form the corresponding C-6 hydroxymethyl metabolite (U-97924), which was oxidized further to the C-6 formyl (U-97865) and the C-6 carboxyl metabolites of U-89843 (5). The 1 The author would like to dedicate this publication to Professor Neal Castagnoli, Jr., on the occasion of his 60th birthday. 2 Abbreviations: UDS, unscheduled DNA synthesis; P450, cytochrome P450; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; 2-AAF, 2-(acetylamino)fluorene; FBS, fetal bovine serum; ICDH, isocitric dehydrogenase; TdR, thymidine; PB-CI-LC/MS, particle beam-chemical ionization-liquid chromatography/mass spectrometry; PCI, positive ion chemical ionization; APCI, atmospheric pressure chemical ionization; NAC, N-acetylcysteine.

© 1996 American Chemical Society

Bioactivation of Pyrrolopyrimidine (U-89843)

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1231

Scheme 1. In Vitro and in Vivo Metabolic Pathways for U-89843 in the Male Rat

corresponding aldehyde and the acid were secondary metabolites derived from the sequential alcohol dehydrogenase and aldehyde dehydrogenase catalyzed oxidation of the carbinol and formyl intermediates, respectively (Scheme 1). Evidence supporting the chemical reactivity of U-97924, the major primary hydroxylated metabolite of U-89843, as an electrophile and its ability to bind covalently to intracellular macromolecules is presented. The mechanistic insights gained have led to the design of analogs of U-89843 which do not exhibit genotoxicity in the in vitro UDS assay.

Materials and Methods Chemicals and Reagents. U-89843, U-97924, U-98140, and U-107643 were synthesized at Pharmacia & Upjohn, Inc. (Kalamazoo, MI). [14C]U-89843 with a specific activity of 30.77 µCi/mg (9.91 mCi/mmol) was also synthesized by Pharmacia & Upjohn, Inc., with labeling on the pyrimidine ring at the C-2 position. For some studies the specific activity of the [14C]U89843 was diluted with unlabeled U-89843 to yield a specific activity of 9.5 µCi/mg. Isocitrate (Na salt), β-NADP+ (Na salt), β-NAD+ (Na salt), Sigma Trizma buffer, and isocitric dehydrogenase (ICDH) were purchased from Sigma Chemical Co. (St. Louis, MO). The 2-(acetylamino)fluorene (2-AAF) used as the positive control in UDS assay was obtained from Aldrich Chemical Co. (Milwaukee, WI). Fetal bovine serum (FBS) was obtained from Sterile Systems (Logan, UT). [3H]Thymidine ([3H]TdR, specific activity 40-60 Ci/mmol at 1 mCi/mL) was obtained from Amersham (Arlington Heights, IL). Kodak NTB-2 liquid autoradiography emulsion was obtained from International Biotechnology (New Haven, CT). Other chemicals were obtained from commercial sources and were of reagent, analytical, or HPLC grade as appropriate. Instrumentation. The HPLC systems used consisted either of Perkin Elmer (PE) components (a PE Series 410 quaternary gradient HPLC pump, PE ISS-200 autosampler, PE LC-235C diode array detector) or a Hewlett Packard HP1090M integrated liquid chromatograph. A Zorbax RX C8 HPLC column (4.6 mm i.d. × 25 cm) was used for all separations. Data were transmitted to a Harris computer system via a PE Nelson 900 Series Interface. Radiolabeled material was analyzed using a Radiomatic Flo-one A-515 detector placed in-line after a PE LC-235 diode array detector. The Radiomatic detector’s data acquisition, data storage, and reporting were controlled on an IBMcompatible computer (386) with Version 1.1 Radiomatic Flo-one/ Data for Windows software. An isocratic mobile phase (A/B, 17/83 v/v) was used with a flow rate of 1.0 mL/min. Eluent A was 0.5% (v/v) triethylamine adjusted to pH 5.0 with acetic acid, and eluent B was 90/10 (v/v, before mixing) acetonitrile/water. The detection wavelength was 245 nm. For radiochemical detection, 1 part mobile phase was mixed dynamically (postcolumn) with 3 parts Radiomatic Flo-Scint II liquid scintillation cocktail and the [14C] detection window was set at 0-156 keV. Particle beam-chemical ionization-liquid chromatography/ mass spectrometry (PB-CI-LC/MS) was performed on a Finnigan 4500 mass spectrometer (Finnigan-Mat, San Jose, CA) equipped with a thermal pneumatic nebulizer coupled with a momentum separator (built at Pharmacia & Upjohn, Inc., based on the

design of Extrel Corp., Pittsburgh, PA). The instrument was operated in the positive ion chemical ionization (PCI) mode using ammonia as the moderating gas. The chemical ionization gas pressure was optimized by the appearance of the m/z 52 ion (an ammonia cluster ion) since direct measurement of gas pressure is not possible. The electron energy of the ion region was 70 eV. The conversion dynode potential was adjusted to 6 keV. The particle beam parameters were controlled by Vestec thermospray electronics (Vestec, Houston, TX) with tip and expansion region temperatures of 180 and 80 °C, respectively. The helium flow rate was not measured directly. However, to achieve an appropriate flow rate through the nebulizer assembly, the head pressure was set to 150 psi, allowing the flow rate to be regulated by the 0.360 × 0.075 mm fused silica capillary column housed in a 0.020 in. stainless steel tube. Data were collected by a Vector II system (Teknivent, Maryland, MO) scanning at 3 s per scan over the range 60-700 amu. PB-EILC/MS was performed on the same instrument, and the conditions were identical to those just described. HPLC was performed on a system consisting of an PE ISS 100 autoinjector interfaced to a PE 410 pump with SEC-4 solvent environment control (Perkin Elmer, Norwalk, CT) and a Waters 490 MS UV detector (Waters, Milford, MA). LC conditions were the same as described above except the flow rate was 0.5 mL/min. Atmospheric pressure chemical ionization-liquid chromatography/mass spectrometry (APCI-LC/MS) was performed on a Finnigan TSQ 7000 mass spectrometer. The LC system consisted of a Hewlett Packard (Hewlett Packard, Naperville, IL) 1050 Series pump and autosampler and a Thar (Thar Designs Inc., Pittsburgh, PA) two position actuator. Chromatographic separation conditions were the same as described above with a flow rate of 1 mL/min. Mass spectrometric analysis was performed on a Finnigan-MAT (Finnigan-MAT, San Jose, CA) TSQ 7000 triple quadrupole mass spectrometer directly coupled to the LC system via a Finnigan atmospheric pressure ionization (API) source operated in the atmospheric pressure chemical ionization (APCI) mode. Data were collected on a DEC 3000 Model 300X computer running OSF/1 version 2.0 as the operating system. The mass spectrometer was controlled using Instrument Control Language (ICL) version 8.0, and the data were processed using Interactive Chemical Information System (ICIS) version 8.1.1 software. Tuning of the APCI source and MS was accomplished by infusing a solution containing 1 mg/mL of U-89843 into the mobile phase flow path post-column via a T connector at a rate of 5 µL/min using a Harvard syringe pump. The mass spectrometer was operated in the Q1MS mode for positive ions scanning from 100 to 1000 amu in 3 s. The conversion dynode and electron multiplier were set to 15 keV and 1450 V, respectively. The APCI vaporizer and capillary temperatures were 450 and 250 °C, respectively. The corona was set to 5 µA, and nitrogen was employed as a drying gas at a sheath pressure of 70 psi. The mass spectrometer was tuned to unit resolution. The HPLC flow to the mass spectrometer was diverted to waste for 2 min post-injection, thus allowing the very polar components to be diverted to waste which prevents fouling of the APCI interface. Automated control of the valve switching device was accomplished using an ICL procedure which toggled the state of the digital output 1 and 2 relays. FAB/MS Conditions. Samples were analyzed by FAB/MS on a Finnigan TSQ-70 triple quadrupole (mass range 2-4000)

1232 Chem. Res. Toxicol., Vol. 9, No. 8, 1996 mass spectrometer equipped with a 8 keV Xe saddle field gun. Glycerol was used as matrix, and masses were scanned between 50 and 800 amu. GC/MS Conditions. GC/MS analyses were performed on a HP Model 5890A gas chromatograph connected to an HP 5970 EI mass spectrometer with a DB-5 (J & W) capillary column (12 m × 0.25 mm) and a HP series computer. A temperature program of 100 °C for 1 min followed by 25 °C/min to 250 °C was used. 1H NMR spectra were recorded at 200 MHz with Varian Model XL 200 spectrometer and at 300 MHz with a Bruker Model AM-300 spectrometer.

S9 Fraction and Microsomes. S9 (supernatant of 9000g centrifugation) and microsomal fractions were prepared from saline rinsed whole livers, freshly obtained and pooled from 30 untreated male Crl:CD BR Sprague-Dawley rats (250-300 g; Charles River Labs, Portage, MI) following standard procedures (6). Briefly, rat liver pieces in 4 × volume/weight 1.15% potassium chloride (pH 7.4) and 10 mM EDTA were minced and homogenized. The homogenate was centrifuged at 10000g for 10 min at 4 °C. The resulting supernatant (S9 fraction) was centrifuged at 209000g for 42 min at 4 °C. The supernatant (cytosol) was separated, and the microsomal pellet was rehomogenized in 100 mM sodium pyrophosphate (pH 7.4) with 1 mM EDTA and centrifuged again at 209000g for 42 min (4 °C). The final microsomal pellet was resuspended in 20% glycerol solution (20 mM potassium phosphate buffer, pH 7.4; 20% w/v glycerol; and 0.1 mM EDTA). Microsomal protein concentrations were determined by the bicinchoninic acid method (7) (Pierce Chemical, Rockford, IL) and standardized relative to bovine serum albumin. Total P450 content was measured by absorption at 450 nm of the dithionite-reduced CO difference spectrum using E450-490 ) 91 mM-1 cm-1 (8). Microsomes and S9 fraction were stored below -65 °C until use. Microsomal Incubations. Typical microsomal metabolism studies of U-89843 were conducted in an incubation system consisting of 0.5 µM of total cytochrome P450 from rat liver microsomes with a final volume of 1 mL in 50 mM Tris-HCl buffer with 0.1 mM EDTA (pH 7.4) at 37 °C. U-89843 (5 mM) was added in 10 µL of methanol for a final concentration 50 µM. After preincubation at 37 °C for 2 min, the reaction was initiated by addition of 100 µL of an NADPH generating system (5 units of ICDH, 10 mM NADP+) to maintain a steady state concentration of 1 mM NADPH in the incubation mixture. After 5, 10, 15, and 20 min shaking at 37 °C, 200 µL aliquots of the incubation mixtures were quenched by the addition of an equal volume of acetonitrile, and the resulting mixtures were centrifuged at 4 °C for 5 min (Eppendorf centrifuge 5402, Rotor F-4518-11, 15800g). The supernatants were analyzed directly by HPLC (100 µL injections). Control incubations were conducted in the absence of either drug or the NADPH generating system. UDS Assay. Male Fischer-344 rats (approximately 280-350 g) were received from Charles River Laboratories (Kingston, NY) and were used in all experiments. All procedures in this study are in compliance with the Animal Welfare Act Regulations, 9 CFR Parts 1, 2, and 3, and with the Guide for the Care and Use of Laboratory Animals, DHEW Publication (NIH) 85-23, 1985. Strain differences warranted using Fischer-344 rats in all UDS testing (9). Rats were housed in polypropylene cages with hardwood-chip bedding and maintained on a 12 h light/12 h dark cycle. Rats received Purine Rodent Chow No. 5001, Ralston Purina Co. (St. Louis, MO), and water ad libitum. The procedures for primary hepatocyte isolation, treatment, and autoradiography have been described (9-11) and are similar to those of Butterworth et al. (12). Briefly, hepatocytes were dissociated from rat liver by collagenase perfusion, placed in monolayer culture, and incubated in the presence of the test article and [3H]TdR for 18-20 h, which is the standard dose duration for this assay. The cultures were fixed and mounted on microscope slides, and DNA repair (unscheduled DNA synthesis, UDS) was evaluated by measuring the incorporation of [3H]TdR by autoradiography.

Zhao et al. U-89843 was tested at 8 doses in the first experiment and at 6 doses in the second experiment. U-107634 was tested at 8 doses in a single experiment. Doses were limited by solubility. The positive control, 2-AAF, was tested at 0.3 µg/mL. All doses were tested in duplicate cultures. Two slides were scored per dose, 30 cells per slide. Net grain counts were calculated by subtracting the highest of two cytoplasmic grain counts from the nuclear grain count (11). Statistics which were routinely calculated include mean net grains (NG), slide-to-slide standard error (SE), and proportion of cells in repair (>5 NG). In addition, regression analysis was performed on the NG values (as well as the ranked NG values) to test for a dose-response (13). Dunnett’s t-test (14 ) was used to compare treated groups with the negative control group. Furthermore, the proportion of cells in repair in the treated groups was tested for a dose-response using a modified Jonckheere test (15). The criteria for evaluation of the UDS assay fall into two categories, namely, those bearing on the acceptability of the assay and those bearing on the definition of a positive or negative response. The UDS assay is considered acceptable if the solvent control group yields e0 NG and if the positive control group exceeds 10 NG. Furthermore, at least three doses must be scored in each experiment. A test compound is considered as positive if the UDS net grain count of any tested concentration is g5 NG in both the preliminary and replicate assays and if the percentage of cells in repair (% IR) is g10. The result is considered either inconclusive or potentially weakly positive if the highest UDS net grain count for any tested concentration is between 0 and 5 NG. The results of the UDS assay are considered negative if all tested concentrations produce NG counts of e0 NG and if testing is carried out to the limit of solubility or cytotoxicity or 3.0 mg/mL. In Vitro Covalent Binding to Microsomal Protein. Incubations were run in glass tubes following previously reported procedures with minor modifications (16). The incubation system consisted of 1 mg of protein from either rat, dog, monkey, or human liver microsomes (above) in a final volume of 1 mL of 50 mM Tris-HCl buffer with 0.1 mM EDTA (pH 7.4). [14C]U-89843 was added in 10 µL of methanol for a final concentration 50 µM (9.32 µCi/mg). After preincubating at 37 °C for 2 min, the reaction was started by the addition of 100 µL of an NADPH generating system. Control incubations were conducted by either omitting drug or the NADPH generating system. After shaking for 20 min at 37 °C, the incubations were quenched by the addition of an equal volume (1 mL) of acetonitrile. Trichloroacetic acid was not used as the quench solvent in the experiment because the resulting acidic conditions increased the content of covalent binding. The tubes were vortex mixed and centrifuged at 4 °C (1000g for 15 min). The supernatants were removed and aliquots were counted by liquid scintillation and analyzed by HPLC (100 µL injections). Pellets containing the microsomal protein from the centrifugation step were resuspended in 2.0 mL of CH3CN and recentrifuged and the supernatants discarded. The procedure was repeated three more times with 2.0 mL of 50% CH3CN, 2 mL of CH3CN, and another 2 mL of CH3CN. The supernatants from the last washing process were checked for radioactivity by liquid scintillation counting to ensure they were at approximately background levels. The protein pellets were allowed to dry overnight in a fume hood, then were solubilized by the addition of 1.0 mL of 0.1 N NaOH, and heated for about 30 min at 60 °C. The protein content of the solubilized pellets was determined by the bicinchoninic acid method (7). Covalently bound [14C]U-89843 equivalents were determined by liquid scintillation counting (100 µL of the solubilized pellets in 20 mL of ULTIMA GOLD cocktail and counted for 10 min). Data are expressed as nmol of [14C]U-89843 equivalents bound/mg of protein. Inhibition of in Vitro Covalent Binding by GSH. Microsomal protein covalent binding inhibition experiments were performed by the addition of either 0.5, 1, or 5 mM GSH to the standard rat liver microsomal incubation media. Microsomal proteins were washed as described previously except for the use

Bioactivation of Pyrrolopyrimidine (U-89843) of water instead of 50% aqueous CH3CN in the second washing. Changing the wash solvent ensured complete removal of residual GSH since GSH is more soluble in water than in organic solvents. In Vitro Covalent Binding to DNA. The measurement of drug related radioactive materials covalently bound to genomic DNA was performed in a manner analogous to reported procedures (16, 17). A stock solution of calf thymus DNA was prepared by dissolution overnight at 65 °C in TE buffer (15 mM Tris buffer, 1 mM EDTA, pH 8.0). [14C]U-89843 (specific activity 30.8 µCi/mg; 9.91 mCi/mmol, 50 µM) was incubated with 0.5 mg/mL untreated rat liver microsomal protein and 2 mg/mL calf thymus DNA (Sigma) and 0, 1, or 5 mM reduced glutathione (GSH) in 100 mM pH 7.4 potassium phosphate buffer. Incubations were carried out at 37 °C for 30 min. Metabolic reactions were started with the addition of an NADPH generating system (NADP+/ICDH). Control incubations were carried out in the absence of the NADPH generating system. Reactions were stopped by placing the samples on ice followed by the addition of 5.0 mL of DNA ISOLATOR (Genosys Biotechnologies, Inc., The Woodlands, TX, Genomic DNA isolation reagent, Catalog No. DNA-ISO-050). A volume of 1.0 mL of chloroform was added, and the tubes were inverted repeatably to allow mixing and then were placed on ice for 5 min. The samples were centrifuged at 2200g at 4 °C for 5 min, and the aqueous (top) layers were transferred to clean tubes (200-300 µL was left above protein interface to avoid contamination). A volume of 2.5 mL of isopropyl alcohol was added to the tubes containing the aqueous layers to precipitate DNA. The samples were placed on ice for 10 min. The precipitated DNA was transferred to clean tubes containing 1.5 mL of 70% aqueous ethanol. The tubes were mixed gently by inversion to wash the DNA and then placed on ice. The wash was decanted, and the DNA was washed twice more with 1.5 mL portions of 70% aqueous ethanol. Following the final wash, the DNA was air-dried for 30 min under a nitrogen stream. DNA in the samples was redissolved in 0.5 mL portions of TE buffer (pH 8.0). Samples were heated at 65 °C for 5 min to effect DNA dissolution. A260 and A260/A280 spectrophotometric measurements on aliquots of the DNA solutions were made to determine the DNA concentrations and assess purity, respectively. Aliquots (400 µL) of the DNA solutions were added to scintillation vials along with 10 mL of ULTIMA GOLD scintillation cocktail (Packard), and the solutions were counted on a liquid scintillation counter. The radioactivity (dpm/mL), µg/mL DNA calculated form the A260 reading, and specific activity (9.91 mCi/mmol) were used to calculate the pmol equivalents of U-89843 bound per mg of DNA for each sample. Inhibition of U-97924 Dimerization by Methanol, GSH, or N-Acetylcysteine. Inhibition of U-97924 dimerization by GSH or N-acetylcysteine was conducted in acidic aqueous acetonitrile (apparent pH 4) at room temperature with addition of either GSH or N-acetylcysteine. The corresponding products were analyzed by FAB/MS. Inhibition by MeOH was carried out in acidic aqueous methanol (pH 4) at room temperature, and the corresponding methoxy adduct was analyzed by GC/ MSD. Synthesis. 3-Bromo-2-hydroxypropyl Benzoate (6). A flame-dried, three-neck 500 mL round bottom flask was charged with commercially available 3-bromo-1,2-propanediol (11.4 g, 73.5 mmol) in pyridine (100 mL). The solution was cooled to -15 °C, and benzoyl chloride (10.3g, 73.5 mmol) was added at a rate of 2 mL/h. Following the addition, the reaction was allowed to warm to room temperature. After 28 h the solution was concentrated in vacuo, and a solution of the residue in CH2Cl2 was washed with 5% H2SO4 (3 × 50 mL), saturated NaHCO3 (2 × 50 mL), and brine (1 × 50 mL). The organic layer was dried (MgSO4), filtered, and concentrated in vacuo to give the desired ester as a colorless oil (14.6 g, 76.0%): 1H NMR (CDCl3) δ 8.04-8.07 (m, 2H), 7.57-7.62 (m, 1H), 7.41-7.49 (m, 2H), 4.47-4.50 (m, 2H), 4.19-4.24 (m, 1H), 3.52-3.65 (m, 2H), 2.66 (d, 1H).

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1233 3-Bromo-2-oxopropyl Benzoate (7). A flame-dried, threeneck 500 mL round bottom flask was charged with benzoate 6 (14.5 g, 56.0 mmol), dicyclohexylcarbodiimide (21.6 g, 10.5 mmol), DMSO (5 mL), pyridine (0.75 mL), and diethyl ether (150 mL). The stirred solution was cooled to -5 °C, and trifluoroacetic acid (0.75 mL) was added. The reaction mixture was allowed to warm and was maintained at 18 °C by using an ice bath to control a brief exotherm at 20 min into the reaction. Then, the reaction mixture was stirred for 1 h and recooled to 0 °C. Oxalic acid (6 g) in CH3OH (12 mL) was added with diethyl ether. The reaction mixture was filtered, and filtrate was concentrated in vacuo to yield a yellow oil. Hexane (800 mL) was added to the oil, and the resulting mixture was heated to reflux. The hot hexane solution was allowed to cool, and the resulting white crystalline solid was removed by filtration, washed with chilled hexane, and dried under high vacuum at room temperature to afford the desired product (6.4 g, 44.5%): 1H NMR (CDCl ) δ 8.08-8.11 (m, 2H), 7.59-7.65 (m, 1H), 7.463 7.50 (m, 2H), 5.15 (s, 2H), 4.03 (s, 2H); IR (mull) cm-1 2953, 1747, 1715, 1281; MS (EI) m/z 163, 122, 105, 77. Bis[7-methyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl]methane (5). A flame-dried 100 mL round bottom flask was charged with pyrimidine intermediate 8 (0.96 g, 3.89 mmol) in acetonitrile (50 mL). The solution was cooled to 0 °C, and diisopropylethylamine (0.87 mL, 4.98 mmol) was added to the solution followed by the bromoketone 7 (1.0 g, 3.89 mmol). The solution was allowed to warm to room temperature and stirred overnight. The reaction mixture was heated to reflux for 2 h, cooled to room temperature, and filtered, and the cake was washed with CH3CN. A 1H NMR spectrum of the solid revealed the absence of phenyl proton signals. The solid was combined with the mother liquor and chromatographed on a silica Prep-Pak eluting with 5/2/3 EtOAc/hexane/CH2Cl2. After 8 column volumes, a small peak was collected and concentrated in vacuo to give 50 mg of a tan solid which was identified as dimer U-98140: 1H NMR (CDCl3) δ 5.98 (s, 2H), 3.98 (s, 2H), 3.67-3.72 (m, 8H), 3.57-3.61 (m, 8H); 13C NMR (CDCl3) δ 157.8, 155.1, 154.4, 128.4, 99.4, 95.5, 47.4, 46.5, 27.8, 25.6, 25.3; MS (FAB) m/z 555 (MH+), 554 (M+), 284, 270. 4-(1-Methylhydrazino)-2,6-di-1-pyrrolidinylpyrimidine (19). A suspension of 51 g of chloro intermediate 18 (4) in 320 mL of methylhydrazine (Aldrich) was heated at reflux under nitrogen for 4 h. Reaction mixture become a homogeneous light yellow solution as it was heated. The mixture was then cooled to 0 °C, diluted with 350 mL of ice water, and filtered. The solid product was washed with cold water (3 × 250 mL) and then dried, first by pulling dry nitrogen through the funnel, then at 25 °C, 0.02 mm for 24 h. The product weighed 52.5 g (99% yield): mp 128-130 °C; IR (mull) cm-1 2954, 2925, 2855, 1573, 1553, 1469, 1452, 1346; 1H NMR (CDCl3) δ 5.30 (s, 1H, CH), 4.19 (s, 2H, NH2), 3.55-3.51 (t, 4H, NCH2), 3.49-3.33 (m, 4H, NCH2), 3.16 (s, 3H, CH3), 1.94-1.87 (m, 8H, CH2); 13C NMR (CDCl3) δ 165.6, 162.1, 159.9, 71.6, 46.1, 45.9, 39.7, 25.5, 25.2; MS m/z 262 (M+), 246, 234, 218, 206, 189, 177, 165, 148, 131, 121, 110, 70, 55; exact mass calcd for C13H22N6 m/z 262.1906, found m/z 262.1915. 7-Methyl-6-(trifluoromethyl)-2,4-dipyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (20). A 250 mL, three-neck round bottom flask was charged with hydrazine intermediate 19 (5.007 g, 19.08 mmol) in 125 mL of diphenyl ether. The suspension was treated with 1,1,1-trifluoroacetone (3.5 mL, 39.11 mol) at room temperature. The reaction mixture was then placed under nitrogen and heated to 245 °C. After 3 h at 245 °C, the reaction mixture was cooled and chromatographed directly on 900 g of 230-400 mesh silica gel. The column was packed with hexane and eluted with 3200 mL of hexane, 2000 mL of 10% EtOAc/ hexane, 2000 mL of 20% EtOAc/hexane, and 4000 mL of 50% EtOAc/hexane. An initial fraction of 3200 mL was collected followed by 40 mL fraction. Fractions 66-100 contained the desired trifluoromethyl analog and were combined, thereby affording 3.7 g (58%) of the product as a light tan solid: 1H NMR (CDCl3) δ 6.76 (s, 1H), 3.74 (m, 7H), 3.61 (m, 4H), 2.00-1.92 (m, 8H); MS m/z 339 (M+), 311, 284, 270, 70; TLC (silica gel

1234 Chem. Res. Toxicol., Vol. 9, No. 8, 1996

Zhao et al.

U-97924, which was further oxidized by P450 and alcohol dehydrogenase to the corresponding C-6 formyl analog U-97865 (5). A representative HPLC tracing of metabolite profile obtained from a U-89843 rat liver microsomal incubation is shown in Figure 1. Samples of supernatant from the U-89843 in vitro UDS assay were analyzed by HPLC to determine if the corresponding metabolites were formed in this test system. The analysis results are listed in Table 1. Two major metabolites U-97924 and U-97865 found in rat both in the in vivo and in vitro (5) were also observed in the supernatant of the UDS assay test system. In addition, three late eluting peaks at retention times of about 12, 23, and 27 min, designated as D1, D2, and D3, respectively, were also observed by UV detection. The first two peaks (D1 and D2) were also found to be degradation products generated from pure U-97924 in acidic aqueous solution at room temperature (see below). Analysis of the D2 component by LC/MS (CI) showed a protonated molecular ion at m/z 555 (MH+). Since the parent compound U-89843 contains 5 nitrogen atoms in the molecule with molecular ion at m/z 285, the even number of the molecular ion (M+, m/z 554) indicated D2 contained an even number of nitrogen atoms and was nearly double the mass of U-89843, suggesting a possible dimeric structure. Spectroscopic analysis revealed that D2 has a essentially identical UV spectrum to that of U-89843. The dimeric structure (5) was therefore proposed for this late eluting component D2 (Scheme 2). In an attempt to confirm this proposal, the synthesis of 5 was undertaken (Scheme 2). Selective monobenzoylation of commercially available 3-bromo-1,2-propanediol to form 3-bromo-2-hydroxypropyl benzoate (6) followed by Swern oxidation of the carbinol afforded the corresponding key R-bromoketone 7. Treatment of the available pyrimidine intermediate 8 (3) with bromoketone 7 afforded the symmetrical dimeric product 5 (U-98140) via the intermediate 9. The structure of U-98140 was assigned unambiguously on the basis of mass spectral and NMR data (see experiments). The HPLC retention time and UV spectrum of synthetic 5 were identical with the corresponding data obtained for dimer D2. Therefore, the final structure of this dimeric product is assigned as bis[7-methyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl]methane (U-98140). The UV spectrum of peak D1 also was similar to the spectrum of U-89843. Analysis of D1 by LC/MS (ESI) showed a protonated molecular ion at m/z 585 (MH+), an addition of 30 amu compared to the molecular ion of D2 (MH+, m/z 555), with a base fragmentation peak at m/z 284 which corresponds to the mass of U-89843 minus a proton and a weak ion at m/z 316 corresponding to the mass of U-89843 minus a proton and plus a unit of formaldehyde. The mass difference between D1 and D2 of 30 amu can be rationalized in terms of a unit of

Figure 1. Representative HPLC chromatogram with UV detection of U-89843 metabolite profile from hepatic microsomal incubations (peak 1 ) U-97924; peak 2 ) U-89843; peak 3 ) U-97865; peak 4 ) dimer 1; peak 5 ) dimer 2; and peak 6 ) dimer 3). GF) R ) 0.46 (10% EtOAc/hexane), UV visualization. This thermal Fischer-type indolization was very sensitive to the reaction temperature. At lower temperatures, a considerable amount of the intermediate hydrazone was recovered, while at higher temperatures the desired product was not stable. The free base (2.10 g, 6.18 mmol) in 100 mL of EtOAc at room temperature was treated with a solution of 2.9 M HCl/EtOAc (2.3 mL, 6.67 mmol). The solution remained homogeneous for 1-2 min after which the desired HCl salt precipitated. The salt was recrystallized from methanol/ethyl acetate and dried (0.05 mm, 40 °C, 18 h). The white crystals weighed 1.06 g (45%) and melted at 182-183 °C. Anal. Calcd for C16H21ClF3N5: C, 51.13; H, 5.63; Cl, 9.43; N, 18.63. Found (corrected for 1.96% H2O and 0.07% EtOAc): C, 51.06; H, 5.66; Cl, 9.18; N, 18.61.

Results and Discussion As a part of our investigations in the development of a new drug candidate, U-89843 was evaluated in the in vitro unscheduled DNA synthesis (UDS) assay. The principal role of the UDS assay is to assess the potential of mutagenic hazard of the compound or its corresponding metabolites. It was found that U-89843 induced a positive UDS dose response with net grains per nucleus (NG) at 30 µg/mL as high as +29.41 and +28.62 in the first and second experiments, respectively. The net grains per nucleus data as well as the % IR (percent of cells in repair, >5 NG) data showed a statistically significant concentration response. The no-effect concentration level was 3 µg/mL. Previous results in the male Sprague-Dawley rat suggested that U-89843 was metabolized by P450 2C11 to give the corresponding C-6 hydroxymethyl analog

Table 1. HPLC Analysis Results of UDS Assay Supernatant

a

20-min samples

20-h samples

U-89843 (µg/mL)

U-97924

U-97865

“dimers”

U-97924

U-97865

“dimers”

100a 30a 10 3 1 0.3 0.1 0.03

+ + + + + + NDc NDc

NDb ND ND ND ND ND NDc NDc

ND ND ND ND ND ND NDc NDc

+ + + +c +c +c NDc NDc



At these substrate concentrations, UDS assays were positive. b ND ) not detected. c The substrate (U-89943) was not detected.



Scheme 2. Synthetic Pathway for the Metabolite 5 (Dimer 2) of U-89843

Scheme 3. Proposed Mechanism for the Formation of Dimers (Dimer 1, Dimer 2, and Dimer 3)

formaldehyde. This fact plus the presence of fragment ions at m/z 284 and 316 suggest the symmetrical ether bis[7-methyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl]methyl ether (10) for the structure of D1 (Scheme 3). D3 also had an nearly identical UV spectrum as U-89843. However, this component was not observed as a degradation product derived from pure U-97924 in acidic aqueous solution at room temperature (see below). The analysis of D3 by LC/MS (ESI) showed a protonated molecular ion at m/z 569 (MH+), which was equivalent to the addition of 14 amu to the molecular ion of D2 (MH+, m/z 555). The major fragment peak in the mass spectrum m/z 298 corresponds to the fragmentation ion of U-89843 minus a proton and plus a unit of methylene. The similar retention time compared to D2 and the presence of a base peak at m/z 298 led us to assign the structure of D3 as an unsymmetrical dimer 11 (Scheme 3). The proposed mechanisms for the formation of D1, D2, and D3 are illustrated in Scheme 3. The hydroxylated

metabolite U-97924 can undergo dehydration to form a reactive iminium electrophile 12. This process is a vinylogous equivalent to the well-known equilibrium between the R-carbinolamines and iminium ions of alicyclic amines (18). The iminium intermediate can undergo nucleophilic attack at the exomethylene position by a second molecule of U-97924 or U-89843. The formation of the D2 dimer may proceed by attack at exomethylene of 12 by the C-6 carbon atom of U-97924. The resulting intermediate 13 can be converted to 5 (U-98140) by loss a molecule of formaldehyde. Analogous dimeric product formation from (hydroxymethyl)indole has also been reported in the literature (19, 20). The ether product 10 may be formed from nucleophilic attack of the oxygen atom present in U-97924 at the exo-methylene of the iminium species. The unsymmetrical dimeric product 11 may result from attack of U-89843 through its C-5 carbon atom at the exo-methylene of the iminium species 12. The resulting intermediate 14 will be converted to the product by loss of a proton.


Figure 2. Representative HPLC chromatogram with UV detection of dimeric product formation from U-97924 (A) in 50/ 50, MeCN/0.01 M HCl, (B) inhibition by methanol in 50/50, MeOH/0.01 M HCl, (C) N-acetylcysteine (NAC) in 50/50, MeCN/ 0.01 M HCl, and (D) glutathione (GSH) in 50/50, MeCN/0.01 M HCl (peak 1 ) GSH; peak 2 ) GSH adduct; peak 3 ) NAC; peak 4 ) NAC adduct; peak 5 ) U-97924; peak 6 ) methanol adduct; peak 7 ) dimer 1; peak 8 ) dimer 2).

The formation of the dimeric products was observed by HPLC from the incubations of U-89843 with hepatic microsomes. Two dimers (D1 and D2) were also generated from pure U-97924 in weakly acidic aqueous acetonitrile solutions at room temperature, presumably by the pathways shown in Scheme 3. These considerations led us to postulate that the genotoxicity of U-89843 might be mediated by the carbinol metabolite through covalent DNA modification resulting from nucleophilic attack on the electrophilic iminium species. To investigate the postulated formation of a reactive iminium intermediate, an experiment was designed to trap the putative intermediate using methanol as the

Zhao et al.

nucleophile. As discussed earlier, pure synthetic U-97924 was spontaneously converted to D1 and D2 (U-98140) under acidic aqueous acetonitrile conditions. In experiments where the acetonitrile was replaced with methanol, dimer formation was dramatically inhibited, and a new chromatographic peak was observed in the reaction mixtures at retention time 6.7 min (Figure 2). The new component, which had a UV spectrum similar to that of U-97924, was further analyzed by GC/MS (EI). Consistent with expected 6-CH2OCH3 methoxy adduct of U-97924, the molecular ion of the new component was observed at m/z 315, an addition of 14 amu to the molecular ion of U-97924 (m/z 301, M+). The fragmentation pattern of methoxy analog 15 was similar to that of U-97924 (5), which also suggested that the structural assignment for this methoxy adduct (Scheme 4) was reasonable. Reduced glutathione (GSH) was added to the acidic acetonitrile solution of synthetic U-97924 as a nucleophile in an attempt to trap possible reactive intermediate(s). The formation of the dimers (D1 and D2) also was inhibited under these conditions. A new component (tR ) 2.3 min), more polar than U-97924 and less polar than GSH, was observed by HPLC (Figure 2). The UV spectrum of this new component was similar to that of the starting material U-97924. The mixture was analyzed by FAB/MS and was found to contain a species with a parent ion at m/z 591 (MH+) which is consistent with the expected adduct (m/z 590, M+) of GSH with the reactive iminium intermediate derived from U-97924. The daughter ion observed at m/z 308 resulted from the fragmentation of the parent ion to form protonated GSH. The proposed structure of the GSH adduct (16) is illustrated in Scheme 4. A similar GSH adduct from the reactive methylene iminium intermediate has been reported for 3-methylindole (21, 22).

Scheme 4. Proposed Mechanism for the Formation of U-97924 Derived Covalent Adducts


Figure 3. [14C]U-89843 Metabolism in liver microsomes from rat, dog, monkey, and human: Microsomal protein covalent binding to drug related radioactive materials (A); enzymatic activity of formation of U-97924 (B).

In analogous experiments where N-acetylcysteine was used as the trapping reagent, the formation of a new chromatographic component (tR ) 3.5 min) was observed (Figure 2). This component, presumably the corresponding covalently bound adduct of U-97924 and N-acetylcysteine, had a UV spectrum similar to that of U-97924. Therefore, the new peak was assigned as the corresponding S-C N-acetylcysteine adduct (17) (Scheme 4). This type of adduct has previously been reported for the methylene iminium metabolite of 3-methylindole (23). The peaks at approximately 2.2 min from both the GSH and N-acetylcysteine trapping experiments are GSH and N-acetylcysteine standards, respectively. The formation of the dimeric species D1 and D2 of U-97924 was also inhibited under these conditions. These results confirm that U-97924, or the iminium ion derived from it, is reactive toward both sulfur and oxygen nucleophiles.


An in vitro microsomal protein binding assay was conducted to evaluate potential metabolism mediated covalent binding of the parent drug to biologically relevant nucleophiles. [14C]U-89843 (50 µM, 9.32 µCi/ mg) was incubated with hepatic microsomes obtained from rat, dog, monkey, and human in the presence of an NADPH generating system for 20 min. Control samples were run under the same conditions without NADPH. Under the conditions of the assay, NADPH-dependent covalent binding of [14C]U-89843 related materials was observed in hepatic microsomes of all four species (Figure 3A). Qualitatively, SDS-PAGE with autoradiographic analysis of covalent adduct indicated the involvement of a variety of microsomal proteins (data not shown). The highest NADPH-dependent binding was observed with the rat hepatic microsomal preparation, while the lowest binding was found in human liver microsomal preparation no. 15. The two human microsomal samples were selected because human no. 5 exhibited high P450 3A4 activity while human no. 15 exhibited low P450 3A4 activity. Previous studies with human hepatic microsomes (5) have demonstrated that the rates of U-89843 metabolism and primary metabolite (U-97924) formation were linearly correlated with testosterone 6β-hydroxylase activity, an indicator of possible metabolism of U-89843 by P450 3A4 in human (Figure 3B). The ratio of bound equivalents in different hepatic microsomal preparation (Figure 3A) was correlated with metabolic enzyme activities as measured by for formation of U-97924 (Figure 3B). An in vitro experiment designed to inhibit the covalent protein binding to U-89843 metabolites was conducted with GSH which is present in liver at high (5-10 mM) concentration (24). [14C]U-89843 (50 µM, 9.32 µCi/mg) was incubated in rat hepatic microsomes in the presence of 0, 0.5, 1, and 5 mM of GSH and in the presence and absence of NADPH. The covalent binding of drug related radioactive materials to microsomal proteins was inversely related to the concentration of GSH presented in the incubation. At 0.5 mM GSH, microsomal protein covalent binding of [14C]U-89843 was less than 3 nmol equiv/mg of protein corresponding to an inhibition of 80% relative to the positive control (Figure 4A). At 5 mM GSH concentration, the microsomal protein covalent binding was only slightly higher than the negative control (absence of NADPH). Analysis of the incubation supernatant by HPLC showed a peak which had the same retention time and UV spectrum as that of the U-97924GSH adduct identified in the chemical trapping experiments. The quantity of U-97924 in the supernatant from the incubation samples, with or without the addition of GSH, was similar based on HPLC analysis, indicating that addition of GSH at selected concentrations had not inhibited P450 enzyme activity. Binding of reactive metabolite to DNA also provides a useful index of the formation of electrophiles and is likely to be more relevant to the positive UDS assay result. Calf thymus DNA has been used in many in vitro binding experiments (16). Using an NADPH supplemented rat liver microsomal preparation drug derived radioactivity covalently bound to calf thymus DNA was found to be ∼10 pmol/mg of DNA (Figure 4B). The covalent binding of radioactive materials to DNA was inhibited in a concentration-dependent fashion by glutathione. At 5 mM concentration of GSH, the covalent binding of radioactive materials to DNA was almost completely blocked.


Zhao et al.

The metabolic fate of this trifluoromethyl analog was examined in the rat liver microsomes and its mutagenic potential in the in vitro UDS assay. This compound was metabolically stable under the identical incubation conditions used for U-89843. The in vitro UDS assay results also were negative for U-107634 at a concentration of 60 µg/mL. The carbon-fluorine bonds are much stronger than the corresponding carbon-hydrogen bonds, and consequently replacement of hydrogen by fluorine on the C-6 methyl group will block hydroxylation at this site, thus precluding the possibility of forming metabolic products by this pathway. These findings further support the proposed mechanism that metabolism-dependent covalent binding of U-89843 to macromolecules proceeds through the formation of the reactive iminium species generated by dehydration of hydroxylated metabolite U-97924. We therefore conclude that C-6 methyl hydroxylation of U-89843 is associated with the observed mutagenicity of this compound in the in vitro UDS assay. The observed reactivity and postulated mutagenicity of the (hydroxymethyl)pyrrole metabolite (U-97924) of U-89843 appear to be consistent with the reported reactivity and toxicity of (hydroxymethyl)furans (25, 26), (hydroxymethyl)indoles (27), and related pyrrolizidine alkaloids (28).

Figure 4. Inhibition of drug related radioactive materials’ covalent binding to macromolecules by glutathione: inhibition of covalent binding to microsomal protein (A); inhibition of covalent binding to DNA (B).

The evidence that U-89843 undergoes metabolic activation leading to covalent modification of DNA may be linked to the in vitro UDS assay results. To explore further the possible link between metabolism and genotoxicity, the trifluoromethyl analog U-107634 was prepared (Scheme 5). Treatment of the chloro intermediate 18 (4) with methylhydrazine afforded the corresponding methylhydrazine derivative 19. Reaction of 19 with 1,1,1-trifluoroacetone generated the desired trifluoromethyl analog 20 of U-89843.

In summary, these studies have led to the characterization of the reactivity of the hydroxymethyl metabolite U-97924 formed by the P450 catalyzed bioactivation of U-89843. Mechanistically, the reactivity of U-97924 can be explained by its dehydration to form the electrophilic methylene iminium species (Scheme 4). The dimerization was significantly inhibited by addition of methanol, glutathione, and N-acetylcysteine, and characterization of corresponding methanol, glutathione, and N-acetylcysteine adducts supports the proposed reactive iminium species derived from U-97924. Dramatic reduction in metabolism-dependent covalent binding of U-89843 derived radioactivity to liver microsomal protein was observed in the presence of added glutathione. The major metabolite, U-97924, was detected in the supernatant fraction of the in vitro UDS assay. This metabolite is capable of serving as an electrophile via the reactive methylene iminium species. These reactive species appear to mediate mutagenicity of U-89843 observed in the in vitro UDS assay. Replacement of the C-6 methyl group of U-89843 by a trifluoromethyl group (U-107634) eliminated the corresponding metabolic susceptibility. The negative responses obtained for U-107634 in the in vitro UDS assay support the proposal that hydroxylation at the C-6 methyl group plays a key role in the observed mutagenicity of U-89843. The mechanism for the formation of U-97924, either via nitrogen oxidation to form the iminium ion as the first step (29) or by initial hydrogen atom abstraction to directly form the hydroxylated metabolite, needs to be evaluated.

Scheme 5. Synthetic Pathway for the Trifluoromethyl Analog (U-107634)


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U-89843 - ACS Publications - American Chemical Society

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