Glutathione-Dependent Metabolism of the Antitumor Agent Sulofenur

isocyanate (CPIC), which could carbamoylate biological macromolecules ... conjugate with glutathione (GSH) which would serve as a latent form of CPIC...
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Chem. Res. Toxicol. 2002, 15, 240-248

Glutathione-Dependent Metabolism of the Antitumor Agent Sulofenur. Evidence for the Formation of p-Chlorophenyl Isocyanate as a Reactive Intermediate Claudia M. Jochheim,† Margaret R. Davis,‡ Kathleen M. Baillie,§ William J. Ehlhardt,| and Thomas A. Baillie*,§ Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington 98195 Received September 21, 2001

The antitumor agent sulofenur (LY186641), which has shown promising activity against a wide range of cancers, causes hemolytic anemia and methemoglobinemia at dose-limiting toxicities. The antitumor and toxicological mechanism(s) of action of the drug is (are) not well understood, but unlike other antineoplastic agents, sulofenur does not interfere with DNA, RNA, or protein synthesis, or with polynucleotide function. In the present study, we evaluated the hypothesis that sulofenur undergoes bioactivation in vivo to generate p-chlorophenyl isocyanate (CPIC), which could carbamoylate biological macromolecules directly or form a conjugate with glutathione (GSH) which would serve as a latent form of CPIC. The objectives of this study, therefore, were to determine if the GSH and N-acetylcysteine conjugates of CPIC were excreted into bile and urine, respectively, after an i.p. dose of sulofenur to rats. In addition, the chemical stability and thiol exchange properties of these S-linked conjugates were determined. The results of this study indicate that sulofenur does undergo metabolism in vivo to yield the GSH conjugate of CPIC, and that this conjugation reaction is reversible and subject to thiol exchange in buffered aqueous solution (pH 7.4, 37 °C). In contrast, sulofenur itself was stable under these same conditions, even in the presence of GSH and glutathione-Stransferase (GST), thus raising the possibility that bioactivation of sulofenur is necessary for liberation of CPIC. These findings suggest that the generation of this isocyanate in vivo and subsequent carbamoylation of biological macromolecules may play a role in the toxicity and/or antitumor activity of sulofenur and related diarylsulfonylureas.

Introduction A variety of diarylsulfonylureas have been shown to exhibit antitumor activity in vivo in mouse tumor models (1, 2) and human tumor cloning systems (3-6). One of these compounds, sulofenur (N-(5-indanesulfonyl)-N′-(4chlorophenyl)urea, LY186641, Figure 1), was advanced to Phase I (7-10) and Phase II clinical trials for treatment of cancer of the kidney (11, 12), lung (13, 14), ovaries (15-17), stomach (18), and breast (19). One important clinical advantage of diarylsulfonylureas as orally active oncolytic agents is that they do not cause bone marrow suppression, which is the most common dose-limiting toxicity for most antineoplastic drugs. In fact, diarylsulfonylureas exhibit very little acute toxicity in adults (1), although dose-limiting toxicities are associated with methemoglobinemia and hemolytic anemia (7, 8, 18, 19). Unfortunately, in pediatric patients * Correspondence should be addressed to this author at WyethAyerst Pharmaceuticals, 500 Arcola Rd., S3414, Collegeville, PA 19426. Tel: 215-652-5326. Fax: 215-652-9427. † Present address: Immunex Corp., 51 University St., Seattle, WA 98101. ‡ Present address: Drug Safety and Metabolism, Wyeth Ayerst Research, CN-8000, Princeton, NJ 08543. § Present address: Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486. | Present address: Drug Metabolism, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.

with refractory malignant solid tumors, methemoglobinemia was observed at all doses during a Phase I clinical trial with sulofenur (20). Structure-activity relationship (SAR)1 studies have shown that the diarylsulfonylurea moiety (1) is required for cytotoxic activity, and this observation has been the basis upon which analogues have been synthesized, tested, and taken into clinical development. Interestingly, monoarylsulfonylureas such as the hypoglycemic agents tolbutamide, acetohexamide, and chlorpropamide do not exhibit antitumor activity, nor do the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl (Figure 1). While some diarylsulfonylureas produce mild hypoglycemic effects, this property does not correlate with their antitumor activity. Based on these observations, it was concluded that the mechanism by which diarylsulfonylureas elicit their antitumor effects must be different from those by which related sulfonylureas produce their characteristic hypoglycemic or herbicidal effects. Although the mechanism of the antitumor activity of diarylsulfonylureas remains unclear, it is believed to be 1 Abbreviations: CPIC, p-chlorophenyl isocyanate; d -DMSO, deu6 terated dimethyl sulfoxide; GST, glutathione-S-transferase; NAC, N-acetyl-L-cysteine; PIC, phenyl isocyanate; PrNCO, n-propyl isocyanate; SAR, structure-activity relationship; SCPC, S-(N-[p-chlorophenyl]carbamoyl)cysteine; SCPAC, S-(N-[p-chlorophenyl]carbamoyl)-Nacetyl-L-cysteine; SCPG, S-(N-[p-chlorophenyl]carbamoyl)glutathione; SPG, S-(N-phenylcarbamoyl)glutathione; TFA, trifluoroacetic acid.

10.1021/tx0155698 CCC: $22.00 © 2002 American Chemical Society Published on Web 01/24/2002

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Figure 2. General base-catalyzed decomposition of sulofenur to p-chlorophenyl isocyanate (CPIC), which may react with GSH to yield the corresponding conjugate (S-N-[p-chlorophenylcarbamoyl]glutathione, SCPG) or with water to afford p-chloroaniline. The identity of the base (‘B’), and any intermediates in the biological conversion of sulofenur to CPIC, remains to be established.

Figure 1. Chemical structures of compounds referred to in the text.

multifaceted and unlike that of other antineoplastic agents (1, 21-24). Diarylsulfonylureas do not interfere with macromolecular biosynthesis (i.e., DNA, RNA, or protein synthesis) or polynucleotide function (25). In vitro photoaffinity studies with a radiolabeled diarylsulfonylurea showed that the majority of the drug became localized in subcellular compartments: 52% in the mitochondria, 26% in the microsomal fraction, and only 6% in the nuclei (26). Also, this study reported that the drug alkylated 10 different proteins, 4 of which were mitochondrial proteins, through linkages that were reversible under mild conditions. Unfortunately, the investigators were unable to determine the identity of the drug-related species which was responsible for this binding, the specific proteins involved, or whether this process was associated with the antitumor and/or toxic properties of the drug. The metabolism of sulofenur has been examined in detail (27-29), where it was shown that oxidative metabolism occurs at the benzylic carbons of the indane ring. In addition, it was demonstrated that p-chloroaniline and its secondary metabolites, 2-amino-5-chlorophenyl sulfate and p-chlorooxanilic acid, were present in the urine of sulofenur-dosed mice, rats, monkeys, and humans. A second-generation diarylsulfonylurea, LY295501 (Figure 1), which currently is in Phase II clinical development, undergoes similar metabolism in vivo (30). However, this compound was appreciably more potent than sulofenur

(3, 4, 31) and showed classical radiomimetic dose-limiting toxicities in both preclinical (32) and clinical studies (33, 34). The mechanism by which diarylsulfonylureas give rise to p-chloroaniline is unknown. Since the carbonyl carbon of the diarylsulfonylurea linkage has limited electrophilic character, direct hydrolysis to give rise to p-chloroaniline, while possible, seems unlikely. In view of our findings that related nitrosourea and monoarylsulfonylurea derivatives undergo bioactivation to reactive isocyanate intermediates in vivo (35, 36), we considered the possibility that the diarylsulfonylurea linkage of sulofenur may degrade similarly (formally via base catalysis) to yield an isocyanate intermediate (p-chlorophenyl isocyanate; CPIC) and that this reactive, electrophilic species then undergoes hydrolysis to produce p-chloroaniline (Figure 2). If this were the case, it is likely that CPIC also would react with GSH, forming a carbamate thioester conjugate, and/or carbamoylate biological macromolecules, possibly leading to cellular toxicity. Baillie and Slatter (37) showed that conjugates of GSH that contain a carbamate thioester linkage are formed reversibly under physiological conditions and, consequently, GSH may serve as a “carrier” for reactive isocyanates in vivo. In this respect, rather than fulfilling a detoxification process, GSH conjugation serves to increase the effective lifetime of the isocyanate in vivo, thereby enhancing its ability to carbamoylate biological macromolecules and cause cellular toxicity. Interestingly, nonenzymatic bioactivation of the sulfonylurea chlorpropamide (and analogues thereof) to n-propyl isocyanate (PrNCO) was shown to be a prerequisite for inhibition of aldehyde dehydrogenase (38), and the S-linked GSH and cysteine conjugates of PrNCO also were shown to inhibit this enzyme (39). Similarly, the sulfonylurea tolbutamide undergoes bioactivation in vivo to generate reactive intermediates [n-butyl isocyanate and S-(nbutylcarbamoyl)glutathione] which are capable of carbamoylating and inhibiting glutathione reductase (36). The study presented here set out to investigate the role of CPIC as a potential reactive metabolite of sulofenur

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in the rat, with particular emphasis on the formation and properties of its conjugates with GSH and related thiols. In addition, the stability and susceptibility toward thiol exchange of the GSH adduct and the corresponding cysteine conjugate of CPIC were determined in aqueous, buffered media (pH 7.4) at 37 °C in an attempt to assess the intrinsic reactivity of these conjugates under physiological conditions.

Materials and Methods Materials. Sulofenur and LY188919 (>99% pure) were synthesized at Eli Lilly & Co., while the following materials were purchased from the Sigma Chemical Co. (St. Louis, MO): glutathione (reduced form, GSH), L-cysteine, p-chlorophenyl isocyanate (CPIC), phenyl isocyanate (PIC), and glutathioneS-transferase (GST). p-Chloroaniline and N-acetyl-L-cysteine (NAC) were obtained from the Aldrich Chemical Co. (Milwaukee, WI). The following compounds were prepared by synthesis, as described below: S-(N-[p-chlorophenyl]carbamoyl)glutathione (SCPG), S-(N-[p-chlorophenyl]carbamoyl)cysteine (SCPC), S-(N[p-chlorophenyl]carbamoyl)-N-acetyl-L-cysteine (SCPAC), and S-(N-phenylcarbamoyl)glutathione (SPG). All other chemicals were purchased from commercial sources and were of the highest purity available. Synthesis. 1. S-(N-[p-Chlorophenyl]carbamoyl)glutathione (SCPG), S-(N-[p-Chlorophenyl]carbamoyl)-N-acetylL-cysteine (SCPAC), and S-(N-Phenylcarbamoyl)glutathione (SPG). A solution of p-chlorophenyl isocyanate (1.0 mmol) in acetone (1 mL) was added over 30 min to a stirred solution of either GSH, N-acetyl-L-cysteine, or L-cysteine (0.5 mmol) in acetonitrile/water (7:3, v/v) under nitrogen at ambient temperature. After a period of 3 h, the solvent was removed under reduced pressure, and the desired products were purified by HPLC, as described below. The purified reaction products exhibited NMR, UV, and mass spectrometric characteristics fully consistent with their proposed structures, and these are summarized below. A sample of the internal standard, S(phenylcarbamoyl)glutathione (SPG), was obtained by a similar synthetic procedure, using phenyl isocyanate in place of pchlorophenyl isocyanate. A single product was obtained, which afforded a mass spectrum with the expected [M + H]+ ion at m/z 427. 2. SCPG. NMR (d6-DMSO): δ 1.89 (m, 2H, Glu-β-CH2), 2.31 (t, J ) 7 Hz, 2H, Glu-γ-CH2), 2.96 (dd, J ) 9 and 14 Hz, 1H, Cys-β-CH), 3.30-3.50 (m, 2H, Glu-R-H and Cys-β-CH′), 3.68 (d, 2H, J ) 5 Hz, Gly-CH2), 4.30-4.50 (m, 1H, Cys-R-H), 7.33 (d, 2H, J ) 9 Hz, Ar-H), 7.50 (d, 2H, J ) 9 Hz, Ar-H), 8.45 (d, J ) 9 Hz, 1H, Cys-NH), 8.61 (t, J ) 5 Hz, 1H, Gly-NH), and 10.50 (s, 1H, carbamoyl-NH). UV: λmax ) 254 nm. MS: m/z 461/463 ([M + H]+, 35Cl/37Cl). MS/MS (CID of m/z 461 ([M + H]+): m/z 386 ([M + H]+ - Gly), m/z 332 ([M + H]+ - γ-glutamyl moiety), m/z 229 (see Results and Figure 3), m/z 179 ([Cys-Gly + H]+). 3. SCPAC. NMR (d6-DMSO): δ 1.83 (s, 3H, CH3-CO), 3.03 (dd, 1H, J ) 9 and 14 Hz, Cys-β-CH), 3.37 (dd, 1H, J ) 9 and 14 Hz, Cys-β-CH′), 4.37 (m, 1H, Cys-R-H), 7.34 (d, J ) 9 Hz, 2H, Ar-H), 7.50 (d, J ) 9 Hz, 2H, Ar-H), 8.30 (d, J ) 8 Hz, 1H, Cys-NH), and 10.50 (s, 1H, carbamoyl-NH). UV: λmax ) 254 nm. MS: m/z 317/319 ([M + H]+, 35Cl/37Cl). MS/MS (CID of m/z 317 ([M + H]+): m/z 164 ([NAC + H]+), m/z 154 ([ClC6H4NCO + H]+), and m/z 122 ([Cys + H]+). 4. SCPC. NMR (d6-DMSO): δ 3.22 (dd, J ) 7 and 14 Hz, 1H, Cys-β-H), 3.49 (dd, J ) 4.5 and 14 Hz, 1H, Cys-β-H′), 3.59 (dd, J ) 4 and 7 Hz, 1H, Cys-R-H), 7.28-7.38 (m, 2H, Ar-H), 7.52-7.60 (m, 2H, Ar-H), and 10.38 (s, 1H, carbamoyl-NH). MS: m/z 275/277 ([M + H]+, 35Cl/37Cl). MS/MS (CID of m/z 275): m/z 229 ([M + H - HCO2H]+), 154 ([ClC6H4NCO + H]+), 126 ([ClC6H4NH3]+), 122 ([Cys + H]+), 105 (m/z 122 - NH3), and 76 (HSCH2CHNH2+). Biological Studies. Adult male Sprague Dawley rats (250300 g) were obtained from Charles River Laboratories (Wilm-

Jochheim et al. ington, MA). For studies on biliary metabolites, animals were anesthetized with an aqueous solution of urethane (1 g kg-1, i.p.) and equipped with a cannula in the common bile duct. Control bile then was collected over ascorbic acid for 30 min, following which the rats were administered sulofenur (100 mg kg-1 i.p., in corn oil). Bile was collected over ascorbic acid for 6 h and was stored at -80 °C until analyzed. For studies on urinary metabolites of SCPG, rats were dosed i.p. with SCPG (100 mg kg-1, given as a 10% acacia suspension, 500 µL). Urine was collected for 0-24 and 24-48 h over dry ice and kept at -70 °C until analyzed. Instrumentation and Analytical Methods. 1. Spectroscopy. All 1H NMR spectra were recorded at 300 MHz on a Varian VXR 300 spectrometer (Varian Associates, Palo Alto, CA) or a Nicolet QE300 instrument. Samples were dissolved in [2H6]dimethyl sulfoxide (d6-DMSO), and chemical shifts are expressed as parts per million (δ) downfield from tetramethylsilane. Signal multiplicities are reported as follows: s, d, t, q (quartet), dd (doublet of doublets), and m. Spectrophotometric assays were carried out using a Hewlett-Packard model 8451A diode array instrument. 2. HPLC Analysis. HPLC separations were carried out using a Beckman model 112 dual-pump system, equipped with a Hewlett-Packard 1040A photodiode array detector set to monitor absorbance at 260 nm. The mobile phase consisted of two components, viz., solvent ‘A’ [0.1% aqueous trifluoroacetic acid (TFA)] and solvent ‘B’ (0.1% TFA in acetonitrile). Purification of synthetic S-linked conjugates was performed using a Beckman C18 semipreparative column (25 cm × 10 mm i.d.; 5 µm) and a mobile phase consisting initially of solvent ‘A’ only, to which solvent ‘B’ was added at a rate of 2.5% min-1 throughout the analysis. Under these conditions, the conjugates exhibited retention times in the range of 20-25 min. HPLC analysis of the in vitro experiments was carried out in a similar fashion, except that a Beckman C18 analytical column (25 cm × 4.6 mm i.d.; 5 µm) was used with a mobile phase flow rate of 1.0 mL min-1 and a linear increase in solvent ‘B’ of 2% min-1. 3. Mass Spectrometry (MS) and Combined Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/ MS). All MS and LC-MS/MS analyses were carried out using a Perkin-Elmer Sciex API III triple quadrupole instrument equipped with an atmospheric pressure ionization source and an ionspray interface. The ionspray interface was maintained at 5 kV. High-purity air served as the nebulizing gas and was maintained at operating pressures of 40 psi for direct infusion and flow injection analyses, and 60 psi for LC-MS/MS analyses. Tandem mass spectrometry was based on CID of ions entering the rf-only quadrupole region, where argon was used as the target gas at a thickness of 3.4 × 1014 molecules cm-1. Full scan mass spectra of synthetic compounds and corresponding product ion spectra of [M + H]+ parent ions were recorded by direct infusion of samples into the mass spectrometer. Solutions containing ca. 10 µg of the sample were dissolved in methanol/ 1% aqueous formic acid (1:1, v/v; 1.0 mL) and infused at a flow rate of 5 µL min-1. Qualitative analysis of biological specimens was performed by LC-MS/MS, when specimens (50 µL) of crude bile or urine were injected onto a narrow-bore C18 HPLC column (150 mm × 2.0 mm i.d.) coupled to the mass spectrometer via a splitting tee. The mobile phase, which was supplied by an Applied Biosystems model 140B solvent delivery system at a constant flow rate of 200 µL min-1, consisted of solvent ‘A’ (0.06% aqueous TFA) and solvent ‘B’ (0.06% TFA in acetonitrile). Samples were injected at 100% solvent ‘A’ and, after 5 min, solvent ‘B’ was added to the mobile phase such that its proportion increased linearly at a rate of 1% min-1. The splitting tee divided the column effluent (split ratio 3:1) such that 50 µL min-1 was delivered to the mass spectrometer while the remainder was diverted to an Applied Biosystems model 785A UV-visible detector set to monitor absorbance at 260 nm. To detect the glutathione conjugate SCPG in bile, the mass spectrometer was operated in the constant neutral loss scanning mode, in which the two mass analyzers were scanned in unison

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Figure 3. Product ion mass spectrum of S-N-(p-chlorophenylcarbamoyl)glutathione (SCPG). The spectrum was obtained by CID of the 35Cl-containing [M + H]+ ion at m/z 461. Glutathione-S-transferase-Mediated Conjugation of Suwith a fixed mass offset of 129 Da (the neutral loss of the lofenur with Glutathione. Sulofenur (100 µM) was incubated glutamic acid residue of SCPG under CID conditions). For the in 100 mM phosphate buffer (pH 7.4) at 37 °C with rat liver detection of SCPAC in urine, the precursor ion scanning GST (30 µg, mixture of isozymes) for 2 min. GSH (500 µM) was technique was employed, in which the first mass analyzer was then added (3 mL final volume), and at time points of 4, 20, 40, scanned repetitively over the full m/z range while the second and 60 min, aliquots (100 µL) were taken and analyzed for was set to transmit only ions of m/z 164 (corresponding to [NAC SCPG by HPLC, as described above. + H]+). Quantitative measurements of SCPG in rat bile were perCarbamoylation of Free Thiols in Vitro by SCPG, formed by LC-MS/MS analysis. In these experiments, filtered SCPC, and Sulofenur. SCPG (50 µM) was incubated with specimens of bile (20 µL) were treated with internal standard cysteine (250 µM) in 100 mM phosphate buffer (pH 7.4) at 37 (SPG; 2.0 µg), and the resulting mixtures were diluted to a final °C (3 mL final volume). At various time points (0, 10, 20, 30, volume of 1.0 mL with methanol/1% aqueous formic acid (1:1, 60, 90, 120, 150, and 180 min), aliquots (100 µL) were removed, v/v). Quantitation of SCPG in bile was based on UV (λ ) 260 to which 1 µL of TFA was added to stop the reaction. The nm) and selected reaction monitoring MS/MS, in which the samples were kept frozen until analyzed by reversed-phase analyte and internal standard were detected by monitoring the HPLC (as described above for analysis of the decomposition of transitions m/z 461 f 179 (SCPG) and m/z 427 f 179 (SPG), SCPG and SCPC). The same procedure was employed for two corresponding to the detection of ions [M + H]+ f [Cys-Gly + additional experiments, to investigate thiol exchange between + H] in quadrupoles 1 and 3, respectively. A standard curve was the cysteine conjugate (SCPC) and free GSH, and between prepared by the addition to drug-free bile specimens of different sulofenur and free GSH. quantities of SCPG together with a fixed amount (2.0 µg) of the internal standard, SPG. Results Decomposition of Sulofenur, SCPG, and SCPC. Sulofenur (50 µM), SCPG (50 µM), and SCPC (50 µM) each were Identification of S-(N-4-Chlorophenylcarbamoyl)incubated in 100 mM phosphate buffer (pH 7.4) at 37 °C. At glutathione (SCPG) in Bile and S-(N-Acetyl-4-chlovarious time points, aliquots (100 µL) were removed, to which rophenylcarbamoyl)cysteine (SCPAC) in Urine fol1 µL of TFA was added immediately to stop the reaction. The samples were kept frozen until analyzed by reversed-phase lowing Administration of Sulofenur to Rats. Figure HPLC [C18 column, 5 µm particle size, 250 mm × 4.6 mm i.d., 3 shows the MS/MS product ion spectrum of synthetic mobile phase composed of solvent ‘A’ (0.1% aqueous TFA) and SCPG, which gave an [M + H]+ ion (35Cl-containing solvent ‘B’ (0.1% TFA in acetonitrile)]. The conjugates were species) at m/z 461. In addition to the parent ion, other eluted with a linear gradient (2.0% min-1 increase of solvent prominent ions include those at m/z 386, which corre‘B’ from 0% ‘B’) and monitored with a Hewlett-Packard model sponds to loss of the elements glycine, m/z 332, which 1040A diode array detector set to 260 nm. Sulofenur, SCPG, corresponds to cleavage of the γ-glutamyl bond, m/z 229, and SCPC were quantified relative to their external standard which is attributed to decarboxylated S-(p-chlorophenylcurve. The data obtained were subjected to nonlinear regression carbamoyl)cysteinimine, m/z 179, which corresponds to analysis (Enzfitter), and dissociation rates and half-lives were calculated accordingly. protonated cysteinylglycine, m/z 162, which is believed

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Figure 4. Reconstructed LC-MS/MS chromatograms from the analysis of synthetic SCPG, employing constant neutral loss scanning of 129 Da. Chromatograms are shown for the ‘total’ ion current (m/z 300-600) (A), and extracted ion currents for m/z 461 (B) and 463 (C).

to be protonated cysteinylglycine ketene, m/z 130, which corresponds to protonated glutamic acid, and m/z 76, which is protonated glycine. To verify the above assignments, the same sample was analyzed by MS/MS, with selection of the 37Cl-isotope (m/z 463) as the parent ion. Fragments that were 2 Da higher in mass than those observed in the 35Cl-isotope product ion spectrum were present at m/z 388 ([M + H]+ - Gly), m/z 334 ([M + H]+ - γ-glutamyl moiety), and m/z 231 [decarboxylated S-(pchlorophenylcarbamoyl)cysteinimine]. LC-MS/MS analysis of synthetic SCPG is shown in Figure 4. The upper chromatogram represents the total ion current chromatogram for m/z values between 300 and 600 with constant neutral loss scanning of 129 Da, which revealed only one peak (retention time of 21.4 min). The two lower chromatograms in Figure 4 were obtained by extraction of the ion currents for m/z 461 and 463 from this data set, depicting the elution from the HPLC column of the two molecular species of this conjugate (i.e., the [M + H]+ ions of the 35Cl- and 37Clcontaining isotopic forms of SCPG, respectively) in a relative abundance of 2.1:1. When this analytical method was applied to the analysis of bile from sulofenur-dosed rats, the reconstructed LC-MS/MS chromatograms shown in Figure 5 were obtained. These revealed a component that corresponded to SCPG, as indicated by the HPLC peak at a retention time of 22.1 min containing the two molecular species with [M + H]+ ions at m/z 461 and 463 in a relative abundance of 2.3:1. The corresponding NAC conjugate of CPIC was detected in urine of rats dosed with sulofenur and gave the expected [M + H]+ parent ions at m/z 317 (35Cl-SCPAC) and 319 (37ClSCPAC), again in the appropriate ratio (data not shown). These results confirm that the GSH and NAC conjugates of the proposed isocyanate, CPIC, are excreted into bile and urine after administration of sulofenur to rats. Quantitative Analysis of SCPG following Administration of Sulofenur. Quantitation of the glutathione

conjugate of CPIC excreted into bile of rats dosed with sulofenur was performed by LC-MS/MS analysis with SRM of the transition m/z 461 f 179. By this approach, it was found that SCPG accounted for only a very small fraction of the sulofenur dose (0.2 ( 0.03%) excreted in bile over 6 h, although the inherent instability of the conjugate (see below) may account for the low apparent recovery, especially in light of the basic environment in bile (pH 8.4). Studies with an Analogue of Sulofenur, Phenylsulfonyl-p-chlorophenylurea (LY188919; Figure 1). LY188919, an analogue of sulofenur containing a phenyl ring in place of the indane group of sulofenur and which also could give rise to CPIC, was dosed to rats, and bile was collected, as before, and examined for the presence of SCPG. Once again, although no quantitative analyses were performed, SRM LC-MS/MS revealed the presence of this glutathione conjugate, demonstrating that a structurally related diarylsulfonylurea also undergoes bioactivation in vivo to the electrophilic species, CPIC, which then forms a conjugate with GSH and is excreted into bile. Chemical Stability of Sulofenur, SCPG, and SCPC in Aqueous Solution. The parent compound, sulofenur, was found to be stable up to 6 h in aqueous solution (pH 7.4) at 37 °C (data not shown), suggesting that sulofenur most likely undergoes metabolic activation prior to release of CPIC. The S-linked conjugates of CPIC, on the other hand, decomposed rapidly under neutral aqueous conditions. Quantitative assessment of the rates of decomposition was obtained from HPLC measurements of substrates remaining over time, and the data were fit to a single-exponential decay model (Enzfitter) for both SCPG (Figure 6, panel A) and SCPC (Figure 6, panel B). Unimolecular decomposition rates (k) and half-lives (t1/2) were calculated as follows: k(SCPG) ) 0.10 min-1; t1/2(SCPG) ) 6.9 min; k(SCPC) ) 0.60 min-1; t1/2(SCPC) ) 1.2 min. Since decomposition of carbamate thioester

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Figure 5. Reconstructed LC-MS/MS chromatograms from the analysis of bile from sulofenur-dosed rats, employing constant neutral loss scanning of 129 Da. Chromatograms are shown for the ‘total’ ion current (m/z 300-600) (A), and extracted ion currents for m/z 461 (B) and 463 (C).

conjugates in aqueous media is known to take place primarily via a base-catalyzed elimination E1cb reaction (37), the GSH conjugate, SCPG, can be expected to be even more unstable in the basic environment of bile than in neutral solution. This may well account for the low recovery of SCPG in bile of sulofenur-dosed rats. Glutathione S-Transferase-Mediated Conjugation of Sulofenur to GSH in Vitro. Given that sulofenur itself was found to be stable toward decomposition at physiological pH and temperature, an experiment was performed to determine whether GSH conjugation could occur in the presence of GST. No adduct was detected after 1 h of incubating sulofenur with GSH in the presence of GST, although sulofenur once again was stable throughout the incubation. These data, together with the results described above, suggest that sulofenur requires bioactivation in vivo to CPIC before undergoing conjugation with GSH. Carbamoylation of Free Thiols by SCPG and SCPC in Vitro. The objective of this study was to determine if the S-linked conjugates SCPG and SCPC are subject to thiol exchange, as has been reported for other carbamate thioester derivatives (37). Incubation of SCPG with cysteine in aqueous solution (pH 7.4) at 37 °C rapidly yielded the corresponding cysteine conjugate, SCPC, whose formation was paralleled by a steady decline in the concentrations of SCPG (Figure 7, panel A). After 6 min, both conjugates followed single-order exponential decomposition kinetics. Most likely, depletion of substrate and product reflects elimination from each conjugate of CPIC, which undergoes hydrolysis and subsequent decarboxylation. Figure 7, panel B, shows a similar result obtained when the cysteine conjugate was incubated with GSH; rapid loss of SCPC was evident, with concomitant formation of SCPG. The only difference

Figure 6. In vitro decomposition of SCPG (A) and SCPC (B) in phosphate buffer, pH 7.4, at 37 °C. Open circles represent the regressional analysis of the decomposition data.

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Figure 7. In vitro thiol exchange reactions of SCPG with cysteine (A) and SCPC with GSH (B) in phosphate buffer, pH 7.4, at 37 °C. Square symbols denote SCPG, and diamonds denote SCPC.

observed in this incubation relative to the previous experiment was that thiol exchange and decomposition occurred more rapidly with SCPC, consistent with the higher reactivity observed with the cysteine conjugate (Figure 6).

Discussion General base-catalyzed decomposition of sulofenur theoretically could yield two isocyanate species, CPIC and indanesulfonyl isocyanate, each of which would be expected to undergo conjugation with GSH. However, the generation of CPIC would be favored because the indanesulfonyl moiety is a better leaving group than p-chloroaniline. Moreover, even though the sulfonamide proton in sulofenur is acidic (pKa ) 5.5), its abstraction most likely would lead to an anion stabilized by resonance through the indanesulfonyl group. In accordance with this reasoning, qualitative LC-MS/MS analyses of bile from sulofenur-dosed rats failed to reveal the putative GSH conjugate of indanesulfonyl isocyanate; instead, only the GSH adduct of CPIC was detected. Interestingly, the same conjugate (SCPG) also was detected in bile after rats were dosed with an analogue of sulofenur, LY188919 (Figure 1). Even though the fraction of the sulofenur dose excreted into bile over a 6 h period in the form of SCPG was low (0.2 ( 0.03%), the inherent instability of these S-linked

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conjugates in basic aqueous media indicates that GSH conjugation may be a more important route of metabolism from a quantitative standpoint than the above value suggests. No attempt was made in the present studies to detect and quantify the product of isocyanate hydrolysis, namely, p-chloroaniline, in the bile of rats dosed with any of the compounds of interest. However, this low molecular mass (127 Da) aniline derivative may serve as a useful indicator of metabolic flux through the isocyanate pathway, although it should be recognized that its excretion into bile would reflect the aggregate of direct hydrolysis and elimination of the parent diarylsulfonylureas. Also, no attempt was made in the present work to identify the fate of the indanesulfonamide portion of sulofenur that would have been liberated during decomposition of the drug to CPIC. However, characterization of products derived from this moiety may provide insight into the detailed mechanism by which sulofenur undergoes bioactivation, since it has been speculated that metabolism involving the proximal sulfonamide nitrogen may play a role in the biotransformation of sulfonylureas to isocyanates (38, 39). Previous findings, taken together with the results presented here, support the hypothesis that CPIC and its S-linked conjugates with GSH and cysteine may play a role in the antitumor and/or toxic properties of sulofenur. Thus, the synthetic S-linked conjugates of CPIC were shown to exhibit cytostatic properties in vitro when tested in GC3 and CCRF-CEM tumor cell lines (W. Ehlhardt, unpublished results). Interestingly, an analogue of sulofenur (DW2282, Figure 1), that, based upon its cyclic sulfonylurea structure, likely would not afford appreciable quantities of the reactive open-chain isocyanate tautomer, exhibited a distinct profile of anticancer properties compared to that of sulofenur. Thus, DW2282 was shown to inhibit protein and nucleic acid synthesis and to interfere with the cell cycle (6), whereas sulofenur lacks these characteristics and is believed to act on mitochondria as its cellular target (21-23, 26, 40-42). Characterization of the intracellular distribution and binding of a radiolabeled analogue of sulofenur revealed 10 different proteins, 4 of which were mitochondrial, which became alkylated upon exposure to the drug, and that the resulting adducts were reversible under mild conditions (26). More recently, Toth and co-workers (31) synthesized and evaluated a series of sulfonimidamide analogues of sulofenur for cytotoxicity, antitumor activity, and formation of 2-amino-5-chlorophenyl sulfate, a secondary metabolite of p-chloroaniline. These authors determined that only one enantiomer of the sulfonimidamide (Figure 1) exhibited antitumor activity in vivo and underwent metabolism to 2-amino-5-chlorophenyl sulfate (perhaps via CPIC), while its antipode lacked both properties. In conclusion, the data presented here suggest that sulofenur undergoes bioactivation in vivo to the reactive isocyanate CPIC which, in turn, is trapped in the form of a GSH adduct and processed further to the corresponding NAC conjugate. The GSH and cysteine conjugates are susceptible to thiol exchange reactions in vitro, raising the possibility that these species, in common with other carbamate thioester derivatives (35, 37), may serve as carbamoylating agents toward biomacromolecules. In this regard, conjugation with GSH may function to extend the effective biological lifetime of CPIC, as opposed to its more traditional role as a ‘detoxification’

Bioactivation of Sulofenur to p-Chlorophenyl Isocyanate

mechanism for electrophilic intermediates. Whether CPIC itself exerts oncolytic properties or contributes significantly to the toxicity of sulofenur remains to be established. Since many therapeutic agents contain the sulfonylurea moiety, determination of the contribution of this previously unrecognized pathway of sulfonylurea metabolism to the characteristic pharmacological and toxicological properties of the parent drug appears to represent a fruitful area for future investigations. Since both of the dose-limiting toxicities of sulofenur involve modifications of hemoglobin function, it would be valuable to study the effect(s) of CPIC and its S-linked conjugates on the structure and activity of this protein. Such investigations would seem warranted in light of a preliminary report by Jochheim et al. (43) that S-linked conjugates of CPIC carbamoylated human hemoglobin in vitro, while other workers (44, 45) have shown that inhaled diisocyanates lead to carbamoylation of hemoglobin systemically.

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