Cytochrome P450-Mediated Epoxidation of 2-Aminothiazole-Based

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Cytochrome P450-Mediated Epoxidation of 2-Aminothiazole-Based AKT Inhibitors: Identification of Novel GSH Adducts and Reduction of Metabolic Activation through Structural Changes Guided by in Silico and in Vitro Screening Raju Subramanian,*,† Matthew R. Lee,# John G. Allen,‡ Matthew P. Bourbeau,‡ Christopher Fotsch,‡ Fang-Tsao Hong,‡ Seifu Tadesse,‡ Guomin Yao,‡ Chester C. Yuan,‡ Sekhar Surapaneni,†,§ Gary L. Skiles,† Xianghong Wang,‡ G. Erich Wohlhieter,‡ Qingping Zeng,‡,| Yihong Zhou,† Xiaochun Zhu,† and Chun Li†,⊥ Pharmacokinetics and Drug Metabolism and Chemistry Research and DiscoVery, Amgen Inc., One Amgen Center DriVe, M/S 30E-2-B, Thousand Oaks, California 91320 ReceiVed NoVember 16, 2009

A 2-aminothiazole derivative 1 was developed as a potential inhibitor of the oncology target AKT, a serine/threonine kinase. When incubated in rat and human liver microsomes in the presence of NADPH, 1 underwent significant metabolic activation on its 2-aminothiazole ring, leading to substantial covalent protein binding. Upon addition of glutathione, covalent binding was reduced significantly, and multiple glutathione adducts were detected. Novel metabolites from the in vitro incubates were characterized by LC-MS and NMR to discern the mechanism of bioactivation. An in silico model was developed based on the proposed mechanism and was employed to predict bioactivation in 23 structural analogues. The predictions were confirmed empirically for the bioactivation liability, in vitro, by LC-MS methods screening for glutathione incorporation. New compounds were identified with a low propensity for bioactivation. Introduction AKT, also known as protein kinase B (PKB),1 plays an important role in both normal and pathological signaling by the PI3K pathway. It is frequently activated in a number of human cancers (1-3) and, therefore, is an important target in oncology (4, 5). As part of a lead discovery program, compound 1 (Figure 1) was determined to be a potent and orally bioavailable inhibitor of AKT (6). However, compound 1 underwent significant NADPH-dependent metabolic activation of its constituent 2-amino-thiazole moiety, leading to substantial covalent binding in vitro. Covalent binding was considerably reduced upon inclusion of glutathione to the reaction mixture with concomitant formation of multiple glutathione adducts. In light of increasing literature evidence linking xenobioticinduced organ toxicity to drug bioactivation (7-10), metabolites were definitively characterized to determine the site and mechanism of bioactivation leading to glutathione adducts. An * To whom correspondence should be addressed. Tel: 805-447-6301. Fax: 805-375-9545. E-mail: [email protected]. † Pharmacokinetics and Drug Metabolism. ‡ Department of Medicinal Chemistry, Chemistry Research and Discovery. # Department of Molecular Structure, Chemistry Research and Discovery. § Current address: Drug Metabolism and Pharmacokinetics, Early Drug Development, Celgene Corporation, 86 Morris Ave., Summit, NJ 07901. | Current address: MannKind, 28903 North Avenue Paine, Valencia, CA 91355. ⊥ Current address: Metabolism and Pharmacokinetics, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, CA 92121. 1 Abbreviations: HPLC, high-performance liquid chromatography; LCESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; MS, mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond coherence; P450, cytochrome P450; GSH, L-glutathione; AKT (also known as PKB), protein kinase B; Q-TOF, quadrupole time-of-flight.

Figure 1. Structure of compound 1.

in silico model was developed based on the proposed bioactivation mechanism and was applied to predict glutathione adduct formation in structural analogues of 1. In silico predictions were confirmed with an in vitro screening effort based on glutathione incorporation utilizing LC-MS methods. These efforts led to identification of novel compounds (6, 11-14) that retained potency on the target and demonstrated ablation of glutathione adducts.

Materials and Methods Materials. Compounds 1-24 were synthesized at Amgen, and their stock solutions were prepared in acetonitrile (ACN) or DMSO. [14C]-1 and [14C]-2, both labeled at the C2-indolinone carbonyl carbon, were synthesized at Moravek Biochemicals Inc. (Brea, CA) with a specific activity of 57 mCi/mmol. All solvents were of analytical grade or higher. All other reagents including L-glutathione (GSH) were purchased from Sigma Aldrich (St. Louis, MO) and were of the highest quality available. Pooled human and male Sprague-Dawley rat liver microsomes (RLMs) were obtained from CellzDirect (Durham, NC). In Vitro Incubations. For metabolite profiling, rat and human liver microsomal incubations were conducted using 1 mg/mL protein, 10 µM test compound, 3 mM MgCl2, 10 mM GSH, and 1

10.1021/tx900414g  2010 American Chemical Society Published on Web 01/22/2010

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Figure 2. In vitro profile of 1 upon incubation in RLMs fortified with NADPH and GSH: (A) UV profile at 292 nm and (B) composite fullscan extracted ion chromatogram with M1 (m/z 704), M2 (m/z 244), M3 (m/z 704), and M4 (m/z 738).

mM NADPH at 37 °C for 60 min in 0.1 M potassium phosphate buffer at pH 7.4. Control incubations were also performed without GSH and/or NADPH. Incubations were stopped by adding an equal volume of ACN, and the mixtures were vortexed and centrifuged at 14000 rpm for 10 min. Supernatants were dried and reconstituted in 20% ACN/80% H2O/0.1% formic acid and used for LC-MS analysis. The in vitro conditions for the GSH adduct screening experiments were the same except that a 15 µM test compound was incubated for 30 min and supernatants were not concentrated prior to LC-MS analysis. Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS). Chromatographic separations for metabolite profiling were achieved using a reverse-phase high-performance liquid chromatography (HPLC) (Agilent 1100 system with a binary pump and a DAD detector; Agilent Technologies Inc., Wilmington, DE) on a C18 column (Luna; 3 µm, 2.0 mm × 100 mm; Phenomenex Inc., Torrance, CA) employing a gradient elution program at a flow rate of 200 µL/min. Mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in ACN (solvent B), and employed was the following solvent gradient program: 0-3 min, 95% A; 3-22 min, 95 to 35% A; 22-23 min, 35 A to 5% A; 23-25 min, 5% A; 25-26 min, 5-95% A; and 26-30 min, 95% A. The HPLC eluant was directly coupled to an ion trap mass spectrometer (LTQ; ThermoFisher Scientific, San Jose, CA). ESI-MS was performed in the positive ion mode using nitrogen as both a sheath and an auxiliary gas. The capillary temperature was 300 °C, the capillary voltage was 32 V, the source voltage was 5 kV, and the tube lens voltage was 90 V. Three scan events were used as follows: (1) m/z 160-1000 full-scan MS, (2) data-dependent scan MS2 on the most intense ion from the full-scan event, and (3) data-dependent scan MS3 on the most intense ion from the MS2 scan. The spectra were recorded using dynamic exclusion of previously analyzed ions for 10 s with two repeats and a repeat duration of 10 s. The MS2 and MS3 normalized collision energies were set to 35%. All data were processed in the Qual browser module of Xcalibur (ThermoFisher Scientific). UPLC-Quadrupole Time-of-Flight (Q-TOF) MS. Chromatographic separations for metabolite profiling were achieved via a reverse-phase UPLC (Acquity; Waters Corp., Milford, MA) on a T3 column (Acquity UPLC HSS 1.8 µm, 2.1 mm × 100 mm; Waters Corp.). The following solvent gradient was employed at a flow rate of 200 µL/min: 0-2 min, 95% A; 3-12 min, 95 to 5% A; 12-13 min, 5% A; 13-13.1 min, 5-95% A; and 13.1-14 min, 95% A. MS data were collected on a Q-TOF mass spectrometer (Q-TOF Premier; Waters Corp.) in the positive ion and V mode using nitrogen and argon as the API and collision gas, respectively.

Subramanian et al. Leucine enkephalin ([M + 1]+ ) 556.2771) was used for the lock mass. Two functions were employed as follows: (1) m/z 100-900 full scan and (2) m/z 100-900 full scan using a collision energy ramp from 10 to 30 V. All data were processed in MassLynx V4.1 (Waters Corp.). GSH Adducts Screening. Reverse-phase chromatographic separations were achieved on a C18 column (XTerra 3.5 µm, C18 2.1 mm × 50 mm; Waters Corp.) at a flow rate of 750 µL/min employing the following solvent gradient conditions: 0-1 min, 95% A; 1-3 min, 95 to 65% A; 3-5 min, 65 to 50% A; 5-6 min, 50 to 10% A; 6-7 min, 10% A; 7-8 min, 10-95% A; and 8-10 min, 95% A. ESI-MS data were collected in positive ion mode on a triple-quadrupole instrument (API-3000; Applied Biosystems, Toronto, Canada) by scanning for a neutral loss of 129 Da. Other MS conditions were as follows: nebulizer gas, 10 psi; curtain gas, 12 psi; ion spray voltage, 4.5 kV; source temperature, 300 °C; collision gas, 6 psi; declustering potential, 36 V; focusing potential, 220 V; entrance potential, 10 V; collision energy, 30 V; and collision cell exit potential, 15 V. Scale-up and Isolation of Metabolites. Scale-up reactions were conducted to isolate adequate amounts of the GSH adducts (M1, M3, and M4) and dihydro-imidazole (M2) metabolite for nuclear magnetic resonance spectroscopy (NMR) characterization. The GSH adduct scale-up reaction mixture contained 150 mL of potassium phosphate buffer (100 mM, pH 7.4), 20 mg of compound 1 dissolved in 100 µL of DMSO, pooled male RLM (1 mg/mL), NADPH (1 mM), and GSH (5 mM). The reaction volume was divided equally into five 50 mL tubes and incubated at 37 °C in a shaking water bath. At 90 min, an additional 2 mL of RLM (20 mg/mL) and 25 mg of NADPH were added to each reaction tube. The reaction was stopped at 4 h by adding 8 mL of cold ACN into each tube. The contents were then combined and diluted with 750 mL of water and loaded onto three preconditioned solidphase extraction cartridges (SPE, Oasis HLB, 35 cm3, 6 g, Waters Inc.). The contents of each SPE cartridge were eluted serially with 100 mL each of 10% MeOH/water, 20% MeOH/water, and 80% MeOH/water. The eluted volumes were checked by LC-MS for the presence of targeted metabolites, dried on a speedvac, reconstituted in 2 mL of ACN/H2O/0.1% formic acid, and subjected to a semipreparative HPLC inline with a PDA detector and a fraction collector (Agilent 1100 system). A reverse-phase column (YMCPack ODS-AQ, 5 µm, 100 mm × 10 mm; Waters Corp.) was employed at a flow rate of 5 mL/min under the following solvent gradient conditions: 0-15 min, 100 to 75% A; 15-17 min, 75 to 50% A; 17-17.5 min, 50 to 10% A; 17.5-22 min, 10% A; and 22-30 min, 100% A. Under the above conditions, GSH adducts M1, M3, and M4 eluted at 11.3, 14.3, and 15.5 min, respectively. M2 scale-up conditions were the same as above except that the reaction was performed in a 50 mL reaction volume with no GSH. The reaction was stopped at 3 h and quenched with 10 mL of ACN containing 20% acetic acid. The contents were then vortex mixed and centrifuged, and the supernatants were subjected to semipreparative reverse-phase HPLC inline with a MS detector (Quattro micro triple-quadrupole MS, Waters Corp.). Fractions were collected based on a selective ion monitoring of m/z 244 in positive ion mode. A reverse-phase column (X-Bridge C18, 5 µm, 150 mm × 10 mm; Waters Corp.) was employed at a flow rate of 10 mL/min under the following solvent gradient conditions: 0-3 min, 95% A; 3-17 min, 95 to 70% A; 17-18 min, 70 to 5% A; 18-19.5 min, 5% A; and 19.5-21 min, 5-95% A. Under the above conditions, M2 eluted at 14.8 min. NMR. Compound 1 and its metabolite isolates (M1-M3) were each dissolved in 160 µL of CD3OD and transferred to 3 mm tubes. NMR data were acquired on a 600 MHz spectrometer (Avance, Bruker Biospin, Billerica, MA) equipped with a cryoprobe (5 mm TCI probe, Bruker). A full complement of NMR data sets was acquired for each sample: 1D proton, 2D 1H-1H total correlation spectroscopy (TOCSY), 2D 1H-1H rotating Overhauser effect spectroscopy (ROESY), 2D multiplicity-edited 1H-13C heteronuclear single quantum coherence (HSQC), and 2D 1H-13C heteronuclear multiple bond coherence (HMBC).

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Table 1. LC-MS Data for 1 and Its Metabolites (M1-M4)a tR (min)

observed [M + H]+

calculated (mDa)c

LC-MSn fragments, m/z

M1

14.2

704.2104

704.2114 (-1.0)

M2 M3 M4

14.7 15.0 15.3

244.1078 704.2100 738.1965

244.1062 (1.6) 704.2114 (-1.4) 738.1992 (-2.7)

1

15.6

433.1306

433.1310 (-0.4)

MS2 on m/z 704: 575, 431, 244 MS3 on m/z 704 f 575: 500, 431, 332, 244 MS2 on m/z 244: 202, 184, 141, 84 same as M1 MS2 on m/z 738: 609, 592, 465, 431, 349, 332, 244 MS3 on m/z 738 f 609: 591, 465, 349, 332, 244 MS2 on m/z 433: 396, 376, 356, 269, 256, 244, 232 MS3 on m/z 433 f 396: 376, 356, 269, 255, 230

b

a Proposed MS fragment assignments are shown in Figure 3. milli-Daltons between the observed and the calculated mass.

b

Under HPLC conditions listed in the LC-ESI-MS/MS section.

c

Mass difference in

Figure 3. Proposed MS fragmentation assignments for 1 and its metabolites.

Covalent Binding. [14C]-1 and [14C]-2 (both 10 µM) were incubated in triplicate in RLM and HLM with or without NADPH (1 mM), and also with or without 10 mM GSH, at 37 °C for 60 min. The reactions were quenched with addition of one volume of ACN, vortex-mixed, and centrifuged. Covalently bound radioactivity to microsomal protein(s) was determined by employing the method described by Day et al. (15). Briefly, the protein pellets were collected and washed exhaustively to eliminate/minimize nonspecific binding. Radioactivity associated with washed proteins was determined by solubilizing the pellet in 1 M NaOH and counting on a liquid scintillation analyzer (TriCarb 2500, PerkinElmer Biosciences). Protein concentrations in the pellets were determined via the Lowry method using a Pierce assay kit. The final amount of radioactivity irreversibly bound to liver microsomal proteins was expressed as pmol of substrate per mg of protein per h. Epoxidation Calculations. Relative energies of epoxidation (∆∆Eepoxide) were computed quantum mechanically, according to the scheme shown in Table 6, using Gaussian 98 (16). Reactant and product geometries were optimized at the Hartree-Fock (RHF) level, utilizing the 6-31G* basis set. The calculated ∆∆Eepoxide values were based upon the RHF/6-31G* total energies at 0 K and were reported in kcal/mol, relative to the reference system established in compound 10 [i.e., ∆∆Eepoxide ) |∆Eepoxide (10) - ∆Eepoxide (test compound)|]. Because these energies did not include the source of oxidation, their absolute values were nonphysical but allowed cancellation of the ensuing systematic error in the reported ∆∆Eepoxide. With this type of system, the selection of the reference system is arbitrary.

Results Metabolite Profiling. Figure 2 displays the UV profile and extracted ion MS chromatograms of supernatants from incubation of compound 1 (Figure 1) in RLMs fortified with NADPH and GSH. In addition to the parent, four metabolite products

(labeled M1-M4) were observed at the retention times listed in Table 1. The same metabolites were also observed in human liver microsomal incubations fortified with GSH and NADPH and in the corresponding hepatocyte incubations. GSH adducts (M1, M3, and M4) were also observed in bile collected following oral administration of a single 2 mg/kg dose of [14C]1 to chronically bile duct-cannulated male Sprague-Dawley rats. Parallel in vitro and in vivo metabolite profiles were observed for analogue 2. Overall, oxidative metabolism was the major clearance pathway for both 1 and 2. Structural Characterization of Metabolites. The MS fragmentation patterns from 1 and its metabolites (M1-M4) are summarized in Table 1, and the proposed assignments for the observed fragments are shown in Figure 3. The available proton and carbon NMR data for 1, M1, M2, and M3 are listed in Table 2. M1. MS full-scan data showed a pseudomolecular ion, [M + H]+ at m/z 704, an addition of 271 Da to compound 1 [M + H]+ at m/z 433. The chemical formula derived from highresolution MS data indicated addition of GSH (307 Da) minus (S + 4H) (36 Da) to the parent molecule. MS2 fragmentation of m/z 704 ions revealed ions at m/z 575, which corresponded to a neutral loss of 129 Da, consistent with loss of pyroglutamic acid from the glutathione moiety. MS3 fragmentation on m/z 575 ions revealed ions at m/z 500 consistent with a neutral loss of glycine from glutathione moiety in the molecule. Other ions observed in the targeted MS2 and MS3 fragmentations were rationalized as shown in Figure 3. A full complement of NMR data was required to elucidate the structure of M1 adduct. When the 1D proton NMR spectrum of M1 (Figure 4A) was compared to that of 1 (not shown; chemical shifts listed in Table 2), all resonances except the thiazole proton (H15) from the parent molecule were observed.

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Table 2. Summary of Identifiable 1H and 13C Chemical Shifts (in ppm) and J Couplings (in Hz) for 1 and Its Metabolites (M1-M4) in CD3OD at 25 °Ca

a GS represents the glutathione group. Notations: gly, glycine; cys, cysteine; glu, γ-glutamic acid; §, overlapping peak; ¶, exchangeable protons; and ND, not determined. All chemicals shifts were internally referenced to solvent CD2HOD signal at δH 3.32/δC 47.8 ppm. J coupling notations: s, singlet; d, doublet; q, quartet; and m, multiplet.

Additionally, resonances anticipated from nonexchangeable protons in a GSH moiety were also observed (Figure 4A). Protons corresponding to methylenes (H8 and H11) and methine (H9) had undergone significant changes in both their chemical shifts and their scalar couplings (Table 2). M1 TOCSY and HSQC spectra (not shown) showed all anticipated cross-peaks and reaffirmed that the indolinone and CF3-phenyl moieties were unaffected. The ROESY spectrum for M1 was revealing and is shown in Figure 5A. Anticipated ROE cross-peaks were observed from phenyl protons H4/6 (7.12 ppm) to H8 (2.84 ppm), H9 (5.06 ppm), and H10 (3.72 ppm). New ROE crosspeaks were observed from indolinone protons H18 (7.25 ppm) and H21 (7.26 ppm) to H9 (5.06 ppm) and from H18 to one of the cysteinyl methylene protons (Hc′, 2.83 ppm) on the GSH moiety. A select expansion of the M1 HMBC spectrum is displayed in Figure 5B. HMBC correlations were revealed from Hc′ to C15 (126.3 ppm), H18/21 to C14 (129.2 ppm), and H10 to C12 (156.9 ppm). A summary of key ROESY and HMBC data for M1 is presented in Figure 5C. The ROESY and HMBC NMR data indicated that GSH conjugation had occurred on C15 of the parent thiazole ring. The combined MS and NMR data revealed an overall addition of GSH to 1 with the resulting intermediate having undergone a complex rearrangement to form M1 with the structure shown in Table 2.

M2. A full-scan MS spectrum revealed a protonated pseudomolecular ion at m/z 244, and high-resolution data indicated that the molecular formula was different from the MS2 m/z 244 ion observed for the parent molecule (Figure 3A). The MS2 fragmentation of m/z 244 displayed ions at m/z 202, 184 and 141, and 84. Some of the proposed fragment assignments matched those observed in the parent molecule (Figure 3B). A 1D proton spectrum of M2 indicated that the phenyl-CF3 protons were preserved, whereas the thiazole and indolinone moiety protons were absent. The 1D, TOCSY, and HSQC data sets showed that CH2 (8)-CH (9)-CH2 (10) were also intact and that the geminal protons on C10 were part of a ring system but those on C8 were not. The combined MS and NMR were fully consistent with the proposed structure of the substituted 2-amino-4,5-dihydroimidazole shown in Table 2. M3. A full-scan MS spectrum revealed a protonated pseudomolecular ion at m/z 704, and high-resolution data indicated that M3 was isobaric with M1. M3 also showed identical fragmentation patterns as M1 (Table 1), and the proposed fragment assignments were rationalized as shown in Figure 3D. MS data indicated that M3 was an isomer of M1, and a full complement of NMR data was required to characterize its structure. A 1D proton spectrum of M3 isolate is shown in Figure 4B. Analogous to M1, all anticipated peaks except the



Figure 4. One-dimensional 1H spectra of (A) M1 and (B) M3 GSH adducts. Atom labels are shown in Table 2.

Figure 5. M1 GSH adduct NMR: select expansions of (A) 2D 1H-1H ROESY and (B) 2D 1H-13C HMBC spectra. (C) Summary of key ROESY and HMBC data. Solid and dashed arrows represent the observed ROE and HMBC correlations, respectively.

thiazole proton (H15) from the parent molecule and peaks from a GSH addition were observed. Similar to M1, protons corresponding to methylenes (H8 and H11) and methine (H9) had undergone significant changes in both their chemical shifts and their scalar couplings (Table 2) as compared to the parent; however, the M1 and M3 chemical shift patterns were distinct. M3 TOCSY and HSQC spectra exhibited the anticipated crosspeaks and confirmed that the indolinone and CF3-phenyl moieties were unaffected. As in M1, the M3 ROESY and HMBC spectra were revealing and are displayed in Figures 6A

and 6B, respectively. M3 correlations similar to M1 were observed as follows: ROE cross-peaks were observed from H18 to both protons of Hc′ (2.95, 3.16 ppm); HMBC correlations were observed from both H18 and H21 (7.35 ppm) to C14 (129.4 ppm) and from geminal protons on cysteine (Hc′) to C15 (123.7). However, unlike in M1, ROE cross-peaks were observed from H18 (7.39 ppm) and H21 (7.35 ppm) to both geminal protons on C10 (3.85 and 4.24 ppm) for M3. HMBC correlations were observed from H10 (3.85 and 4.24 ppm) to C12 (155.7 ppm) and from H9 to C12 at a lower contour level

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Figure 6. M3 GSH adduct NMR: select expansions of (A) 2D 1H-1H ROESY and (B) 2D 1H-13C HMBC spectra. (C) Summary of key ROESY and HMBC data. Solid and dashed arrows represent the observed ROE and HMBC correlations, respectively.

Table 3. In Vitro Covalent Binding Characteristics for 1 and 2 covalent binding (mean ( SD; pmol equiv/mg protein/h) compound 1 2

RLM HLM RLM HLM

+NADPH

+NADPH + GSH

529 ( 22 454 ( 33 1091 ( 51 394 ( 14

139 ( 19 167 ( 23 288 ( 12 124 ( 10

(not shown). A summary of key ROESY and HMBC data for M3 is presented in Figure 6C. The combined MS and NMR data indicated that, as in M1, the GSH conjugation had occurred on C15 of the parent molecule for M3. However, the rearrangement was quite different from M1, and the combined data firmly supported the structure shown in Table 2. M4. A full-scan MS spectrum revealed a protonated pseudomolecular ion at m/z 738 indicating an addition of glutathionyl moiety (glutathione, 307 Da - 2H) to the parent molecule. The MS/MS data for M4 are listed in Table 1, and the proposed assignments are shown in Figure 3. Several diagnostic signatures of a glutathione addition to a thiazole ring were observed in the MS fragmentation patterns (Figure 3), and the MS data were fully consistent with a direct addition of a glutathione moiety on C15 of the thiazole ring in the parent molecule. Full-scale NMR characterization of M4 was not possible due to the small amount of material obtained from the scaled-up microsomal incubation. Covalent Binding. Compounds 1 and 2 both exhibited timeand NADPH-dependent irreversible binding to rat and human liver microsomal proteins (Table 3). The addition of glutathione in incubation mixtures resulted in 63 and 74% reduction in covalent binding for 1 and 2, respectively. GSH Adducts Screening. The 24 compounds listed in Tables 4 and 5 were screened by LC-ESI-MS/MS for the formation of rearranged GSH adducts (M1 and M3) and the direct GSH adduct (M4). Structural modifications in all parts of the molecules were studied. Compound 1 and its analogues listed in Table 4 all formed the rearranged and the direct GSH adducts. Compounds listed in Table 5 did not appear to form either the

direct or the rearranged GSH adducts. Identical results were confirmed using high-resolution UPLC-Q-TOF MS in full-scan positive ion mode. A subset of compounds in Table 5 were advanced as leads for the program (6). Epoxidation Calculations. The calculations of epoxide formation were highly predictive of GSH incorporation. Use of the system that was representative of compound 10 as the reference system led to energy differences less than or equal to zero (∆∆Eepoxide e 0) for all systems representing compounds that formed the rearranged GSH adducts; epoxide formation for all compounds in Table 4 was predicted to be more exothermic and more favorable versus compound 10. Moreover, use of the selected reference system also led to energy differences greater than zero (∆∆Eepoxide > 0) for all systems representing compounds that were observed not to form GSH adducts; epoxide formation for all compounds in Table 5 were predicted to be more endothermic and, hence, unfavorable versus compound 10. Thus, predictions based on epoxidation calculations were in agreement with the empirical evidence: Bioactivated compounds 1-12 (in Table 4) were predicted to form the epoxide and thus result in GSH adducts, whereas stable compounds 13-24 (in Table 5) were predicted not to form the epoxide.

Discussion Compound 1 readily underwent P450-mediated bioactivation via oxidation of its constituent thiazole moiety when incubated in rat or human liver microsomes in the presence of NADPH. Upon addition of GSH to the reaction mixtures, the covalent binding was ameliorated, presumably due at least in part to GSH trapping of the reactive intermediate(s). Three GSH adducts were detected (Figure 2) and characterized by application of MS and/ or NMR techniques. A proposed mechanism leading to the formation of metabolites M1-M4 is shown in Figure 7. All of the metabolties appear to be derived from a common epoxide intermediate I1, the formation of which was envisioned to occur via a P450catalyzed epoxidation of the C4-C5 bond (labeled C15 and C17, respectively, in Table 2) of the thiazole ring, analogous to literature precedents (17-19).

BioactiVation of 2-Aminothiazole-Based AKT Inhibitors Table 4. Compounds That Form the Direct (M4) and Rearranged GSH Adducts (M1 and M3)

The formation of M4 is proposed via pathway A, involving a nucleophilic attack of a glutathione moiety on the C4 (labeled C15, Figure 7) of the epoxidated thiazole moiety to form I2 followed by dehydration. Observation of a GSH direct adduction albeit on the C5 carbon on a C4-substituted 2-amino-thiazole has been reported via a different mechanism (20). Pathway B is proposed to follow hydrolysis of the I1 to afford a dihydrodiol (I3). The dihydrodiol (I3) could then undergo a ring scission to form the glyoxal (I4) and thiourea (I5) metabolites. The thiourea (I5) is then proposed to cyclize to form M2 with an accompanying loss of formal H2S. The formation of the rearranged GSH adducts M1 and M3 is proposed via pathway C involving attack of glutathione at C4 of the epoxidated thiazole ring in I1 followed by ring opening to give rise to a disubstituted thiourea I6, which could then cyclize by losing a molecule of H2S to form the 2-imidazoline I7. I8 is a tautomer


of I7, and an attack of an electron lone pair from either of the nitrogens (labeled N-11 and N-22 in Table 2) on the carbonyl carbon of the five member ring (labeled C14, Table 2), followed by loss of a water molecule, would lead to the isobaric M1 and M3 GSH adducts. Various screens have been reported in the literature for detecting metabolic activation of lead compounds formed in vitro or in vivo (8, 15, 21-23). The putative reactive intermediate is trapped via either a soft nucleophile (e.g., GSH or N-acetyl cysteine) or a hard nucleophile (e.g., cyanide as a trapping agent for an iminium intermediate), and the approximate extent of intermediate adduct formation is then reflective of the extent of bioactivation that would occur in the absence of the trapping nucleophile. MS methods have been particularly successful in detecting the resultant adducts (21, 22, 24-27). In the current study, GSH was employed as the trapping agent to screen for putative products arising from metabolic activation of compounds 1-24. The formation of GSH adducts was analyzed by two LC-MS methods, both employing a positive ion electrospray ionization technique, either by neutral loss scanning of 129 Da (loss of pyroglutamic acid from GSH) on a triple quadrupole spectrometer or for their accurate mass in a full-scan mode on a high-resolution Q-TOF mass spectrometer. Both MS techniques afforded the same results: If a GSH adduct was detected in the neutral loss 129 Da scan on the triple quadrupole instrument, it was also observed in the Q-TOF instrument; however, the sensitivity in the former was higher. Upon the observation that compound 1 underwent biotransformation via epoxide formation, medicinal chemistry efforts began to focus on analogues that would reduce the reactivity of the thiazole ring and thereby destabilize the formation of the epoxide I1 intermediate. Because the R1 functional groups listed in Table 6 were directly conjugated with the central azole ring through a biaryl bond, modulation of the electronic character of the R1 substituent was expected to directly influence that of the azole ring and the reactivity at the double bond that was proposed to undergo epoxidation. To that end, a significant effort was directed toward modifications of the R1 substituent, with initial chemistry efforts directed toward the introduction of fluorine atoms, resulting in compounds 4-6. This was specifically performed as an attempt to destabilize epoxide formation through inductive electron withdrawal, thereby decreasing the electron richness and overall reactivity of the conjugated thiazole. Table 6 shows that the predicted relative free energies of epoxide formation for systems representative of the fluorinated analogues 4-6 were indeed 0.1-0.3 kcal/mol less favorable than for the hydrogenated parents 1-3. However, as seen in Table 4, these fluorinated analogues did form GSH metabolites, indicating that the degree of destabilization of the epoxide intermediate was not sufficient to prevent GSH adduct formation. Another trend that could be observed among the R1 substituents was the effect of partial saturation of the R1 bicyclic moieties among the unsubstituted thiazoles (1-9 and 13). In general, the effects of methylene incorporation led to both stabilization of the epoxide formation and a concomitant formation of GSH adducts. Table 6 shows that among the fully aromatic R1 substituents, the isoquinoline (14 and 15) was most favorably predicted to form the epoxide; the cinnoline and quinazoline with an additional electron-withdrawing nitrogen were predicted to form the epoxide to lesser extents with less favorable ∆∆Eepoxide. In comparison, the partially saturated R1 substituents in general showed stabilization of epoxide formation by ca. 0.2-0.6 kcal/mol, when compared to the isoquinoline. While the partially saturated bicycles all contained a strongly

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Table 5. Compounds That Do Not Form the Direct and Rearranged GSH Adducts (M1-M3)

electron-withdrawing carbonyl atom, it was insulated from the aromatic ring by a methylene spacer in most cases (1-9), with all of them forming the GSH adducts. In the one instance (13) where the electron-withdrawing carbonyl was directly conjugated with the six-membered aryl ring and, equally important, para to the aryl carbon bearing the thiazole double bond, GSH adduct formation was not observed. The finding for 13 is not surprising, because the deactivating resonance-withdrawing effect of electron-withdrawing groups is known to occur at para (and ortho but not meta) positions. To summarize the trends at the R1 position in Table 6, substitution by electron-withdrawing fluorine atoms, as well as by electron-deficient aromatics at R1, decreased the electron richness of the biaryl system, thereby diminishing the extent of

epoxide formation. When the electron richness was decreased sufficiently, elimination of GSH adduct formation followed. In contrast to the R1 substituents, the R2 groups were separated from the five-membered azole ring by electronically insulating, nonconjugated methylene spacers and were not expected to influence azole reactivity. For this reason, R2 substituents were not explicitly included and were truncated to an N-methyl substituent in the epoxidation calculations, which in turn simplified the calculations while still capturing essential elements of the chemical system. Indeed, Tables 4 and 5 both indicate that R2 substitutions did not appear to affect formation of GSH adducts. Changes were also introduced in the center of the molecule, including functionalization at the 4-position and modification



Figure 7. Proposed mechanism for formation of metabolites M1-M4 from compound 1.

of the azole heterocycle itself. Predictably, GSH adducts were not observed for the thiadiazole analogues 18 and 19, which have a heteroatom at the 4-position and are thus unable to form I1. The R3 substituents introduced in compounds 20-24 also abolished GSH adduct formation. If, during the microsomal incubations, GSH incorporation is governed by kinetics, the R3 substituents may have sterically hindered access of the P450 enzyme and, hence, decreased the rate of epoxide formation to an extent that no GSH adducts were observed to form within the time scale of the experiment. Alternatively, if the epoxide formation reaches an equilibrium within the incubation time (30/ 60 min), the lack of GSH adduct formation may be explained by the lack of molecular recognition of the small molecule by the P450 enzyme resulting from a significant change in the overall shape of the molecules effected by R3 substituents, preventing the initial epoxide formation. Because of the inherent chemical instability of an epoxide ring under the employed experimental conditions, the putative epoxide intermediate was not observed directly. However, the differences in relative energies of epoxide formation (∆∆Eepoxide) for the various substituted thiazoles shown in Table 6 may represent relative positions of an equilibrium between the parent thiazoles and the corresponding epoxide derivatives within the oxidizing environment of the P450. Favorable exothermic thermodynamics for epoxide formation within the P450 active site would subsequently allow for ensuing glutathione adduction to intermediates I2 and I6 or, upon dissociation from the P450 active site, would subsequently lead to spontaneous formation to dihydrodiol I3. However, if the employed microsomal incubation time was not sufficient to reach an equilibrium for

epoxide formation, it can nevertheless be expected, in accordance with Hammond’s Postulate (28), that the calculated ∆∆Eepoxide reflects the relative kinetic facility with which the epoxide intermediate forms. It is important to note that our calculations probed the inherent electronic features induced by substituents on the substrate but not the effect of these substituents on binding to the enzyme(s) responsible for metabolism. This limitation would lead to computational ∆∆Eepoxide predictions that would be irrelevant when the substituents significantly modulated recognition of the small molecule by the metabolic machinery. The observed trends in bioactivation could not be explained by changes in lipophilicity; that is, if changes to the molecule resulted in substantial reduction in logD, binding to P450 active site could be affected, thus reducing/abolishing the formation of I1. The calculated logD values at pH 7.4 for compounds 1-24 are listed in Tables 4 and 5. The average logD values for 1-12 and 13-24 were 2.97 ( 0.56 and 2.72 ( 0.59, respectively. The P value comparing the two logD data sets was 0.4; thus, there was no statistical difference between the two groups in their calculated lipophilicity. To conclude, we have demonstrated that 2-aminothiazolecontaining compounds (1-24) may undergo metabolic activation depending on the ring substitution. Novel metabolites that were formed included GSH adducts and a dihydroimidazole product. Full structural characterization via MS and NMR was carried out to characterize the metabolites, and a mechanism for bioactivation via initial epoxide formation was proposed. On the basis of this mechanism, a predictive in silico model was developed centered on the favorability of epoxide formation, and it correlated well with observed GSH conjugation. Modeling

662


Table 6. In Silico Calculations Were Performed for Substructures Shown below To Predict the ∆∆Eepoxide Values for Different R1 Groups

predictions coupled with screening for GSH incorporation allowed rapid identification of lead compounds with reduced bioactivation liability. Acknowledgment. We thank Dr. Mark Grillo for generating the covalent binding data for compound 1, Dr. Greg Slatter for a thorough review of the manuscript, and Dr. Michael D. Bartberger for providing his expertise in discussions of the quantum mechanical epoxidation calculations. Supporting Information Available: High-resolution Q-TOF acquired MS2 spectrum of the GSH adducts M1, M3, and M4; full-scale 2D ROESY spectra of M1 and M3; and full-scale 2D HMBC spectra of M1 and M3. This material is available free of charge via the Internet at http://pubs.acs.org.

Subramanian et al.

References (1) Altomare, D. A., and Testa, J. R. (2005) Perturbations of the AKT signaling pathway in human cancer. Oncogene 24, 7455–7464. (2) Lindsley, C. W., Barnett, S. F., Layton, M. E., and Bilodeau, M. T. (2008) The PI3K/Akt pathway: recent progress in the development of ATP-competitive and allosteric Akt kinase inhibitors. Curr. Cancer Drug Targets 8, 7–18. (3) Dummler, B., and Hemmings, B. A. (2007) Physiological roles of PKB/Akt isoforms in development and disease. Biochem. Soc. Trans. 35, 231–235. (4) Luo, J., Manning, B. D., and Cantley, L. C. (2003) Targeting the PI3KAkt pathway in human cancer: rationale and promise. Cancer Cell 4, 257–262. (5) LoPiccolo, J., Granville, C. A., Gills, J. J., and Dennis, P. A. (2007) Targeting Akt in cancer therapy. Anti-Cancer Drugs 18, 861–874. (6) Zeng, Q., Allen, J. G., Bourbeau, M. P., Wang, X., Yao, G., Tadesse, S., Rider, J., Yuan, C., Hong, F. T., Lee, M. R., Zhang, S., Lofgren, J. A., Freeman, D. F., Yang, S., Li, C., Tominey, E., Huang, X., Hoffman, D., Yamane, H. K., Fotsch, C., Dominguez, C., Hungate, R., and Zhang, X. (2010) Azole-based inhibitors of AKT/PKB for the treatment of cancer. Bioorg. Med. Chem. Lett., in press. (7) Evans, D. C., and Baillie, T. A. (2005) Minimizing the potential for metabolic activation as an integral part of drug design. Curr. Opin. Drug DiscoVery DeV. 8, 44–50. (8) Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A., and Baillie, T. A. (2004) Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol. 17, 3–16. (9) Kalgutkar, A. S., Gardner, I., Obach, R. S., Shaffer, C. L., Callegari, E., Henne, K. R., Mutlib, A. E., Dalvie, D. K., Lee, J. S., Nakai, Y., O’Donnell, J. P., Boer, J., and Harriman, S. P. (2005) A comprehensive listing of bioactivation pathways of organic functional groups. Curr. Drug Metab. 6, 161–225. (10) Kalgutkar, A. S., and Soglia, J. R. (2005) Minimising the potential for metabolic activation in drug discovery. Expert Opin. Drug Metab. Toxicol. 1, 91–142. (11) Zeng, Q., Bourbeau, M. P., Wohlhieter, G. E., Yao, G., Monenschein, H., Rider, J. T., Lee, M. R., Zhang, S., Lofgren, J. A., Freeman, D. F., Li, C., Tominey, E., Huang, X., Hoffman, D., Yamane, H. K., Tasker, A. S., Dominguez, C., Viswanadhan, V. N., Hungate, R., and Zhang, X. (2010) 2-Aminothiadiazole inhibitors of AKT1 as potential cancer therapeutics. Bioorg. Med. Chem. Lett., in press. (12) Zeng, Q., Allen, J. G., Bourbeau, M. P., Dominguez, C., Fotsch, C. H., Han, N., Hong, F.-T., Huang, X., Lee, M. R., Li, A., Liu, Q., Rider, J. T., Tadesse, S., Tasker, A. S., Viswanadhan, V. N., Wang, X., Weiler, K. E., Wohlhieter, G. E., Yao, G., and Yuan, C. C. (2007) Thiazole compounds and methods of use. WO2007084391, pp 1319. (13) Zeng, Q., Yao, G., Wohlhieter, G. E., Viswanadhan, V. N., Tasker, A., Rider, J. T., Monenschein, H., Dominguez, C., and Bourbeau, M. P. (2006) Thiadiazole compounds and methods of use. WO6044860, pp 1-61. (14) Zeng, Q., Zhang, D., Yao, G., Wohlhieter, G. E., Wang, X., Rider, J., Reichelt, A., Monenschein, H., Hong, F.-T., Falsey, J. R., Dominguez, C., Bourbeau, M. P., and Allen, J. G. (2009) Heterocyclic modulators of PKB. WO2009011880, pp 1-249. (15) Day, S. H., Mao, A., White, R., Schulz-Utermoehl, T., Miller, R., and Beconi, M. G. (2005) A semi-automated method for measuring the potential for protein covalent binding in drug discovery. J. Pharmacol. Toxicol. Methods 52, 278–285. (16) Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Zakrzewski, V. G., Montgomery, J. A., Jr., Stratmann, R. E., Burant, J. C., Dapprich, S., Millam, J. M., Daniels, A. D., Kudin, K. N., Strain, M. C., Farkas, O., Tomasi, J., Barone, V., Cossi, M., Cammi, R., Mennucci, B., Pomelli, C., Adamo, C., Clifford, S., Ochterski, J., Petersson, G. A., Ayala, P. Y., Cui, Q., Morokuma, K., Rega, N., Salvador, P., Dannenberg, J. J., Malick, D. K., Rabuck, A. D., Raghavachari, K., Foresman, J. B., Cioslowski, J., Ortiz, J. V., Baboul, A. G., Stefanov, B. B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Gomperts, R., Martin, R. L., Fox, D. J., Keith, T., Al-Laham, M. A., Peng, C. Y., Nanayakkara, A., Challacombe, M., Gill, P. M. W., Johnson, B., Chen, W., Wong, M. W., Andres, J. L., Gonzalez, C., Head-Gordon, M., Replogle, E. S., Pople, J. A. (2002) Gaussian 98, Revision A.11.3, Gaussian, Inc., Pittsburgh, PA. (17) Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R. S., and O’Donnell, J. P. (2002) Biotransformation reactions of fivemembered aromatic heterocyclic rings. Chem. Res. Toxicol. 15, 269– 299.

BioactiVation of 2-Aminothiazole-Based AKT Inhibitors (18) Mizutani, T., Yoshida, K., and Kawazoe, S. (1994) Formation of toxic metabolites from thiabendazole and other thiazoles in mice. Identification of thioamides as ring cleavage products. Drug Metab. Dispos. 22, 750–755. (19) Obach, R. S., Kalgutkar, A. S., Ryder, T. F., and Walker, G. S. (2008) In vitro metabolism and covalent binding of enol-carboxamide derivatives and anti-inflammatory agents sudoxicam and meloxicam: insights into the hepatotoxicity of sudoxicam. Chem. Res. Toxicol. 21, 1890–1899. (20) Kalgutkar, A. S., Driscoll, J., Zhao, S. X., Walker, G. S., Shepard, R. M., Soglia, J. R., Atherton, J., Yu, L., Mutlib, A. E., Munchhof, M. J., Reiter, L. A., Jones, C. S., Doty, J. L., Trevena, K. A., Shaffer, C. L., and Ripp, S. L. (2007) A rational chemical intervention strategy to circumvent bioactivation liabilities associated with a nonpeptidyl thrombopoietin receptor agonist containing a 2-amino-4-arylthiazole motif. Chem. Res. Toxicol. 20, 1954–1965. (21) Argoti, D., Liang, L., Conteh, A., Chen, L., Bershas, D., Yu, C.-P., Vouros, P., and Yang, E. (2005) Cyanide trapping of iminium reactive intermediates followed by detection and structure identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chem. Res. Toxicol. 18, 1537–1544. (22) Ma, S., and Subramanian, R. (2006) Detecting and characterizing reactive metabolites by liquid chromatography/tandem mass spectrometry. J. Mass Spectrom. 41, 1121–1139.

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 663 (23) Soglia, J. R., Harriman, S. P., Zhao, S., Barberia, J., Cole, M. J., Boyd, J. G., and Contillo, L. G. (2004) The development of a higher throughput reactive intermediate screening assay incorporating microbore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent. J. Pharm. Biomed. Anal. 36, 105–116. (24) Baillie, T. A., and Davis, M. R. (1993) Mass spectrometry in the analysis of glutathione conjugates. Biol. Mass Spectrom. 22, 319–325. (25) Dieckhaus, C. M., Fernandez-Metzler, C. L., King, R., Krolikowski, P. H., and Baillie, T. A. (2005) Negative ion tandem mass spectrometry for the detection of glutathione conjugates. Chem. Res. Toxicol. 18, 630–638. (26) Jian, W., Yao, M., Zhang, D., and Zhu, M. (2009) Rapid detection and characterization of in vitro and urinary N-acetyl-L-cysteine conjugates using quadrupole-linear ion trap mass spectrometry and polarity switching. Chem. Res. Toxicol. 22, 1246–1255. (27) Zhu, M., Ma, L., Zhang, D., Ray, K., Zhao, W., Humphreys, W. G., Skiles, G., Sanders, M., and Zhang, H. (2006) Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data. Drug Metab. Dispos. 34, 1722–1733. (28) Hammond, G. S. (2002) A correlation of reaction rates. J. Am. Chem. Soc. 77, 334–338.

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Cytochrome P450-Mediated Epoxidation of 2-Aminothiazole-Based

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