Oxidative ipso Substitution of 2,4-Difluoro-benzylphthalazines:Identification of a Rare Stable Quinone Methide and Subsequent

GSH Conjugate

Mithat Gunduz, Upendra A. Argikar, Amin Kamel, Kevin Colizza, Jennifer L. Bushee,Amanda Cirello, Franco Lombardo, and Shawn Harriman

Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts

Received April 13, 2012; accepted July 30, 2012

ABSTRACT:

In vitro metabolite identification and GSH trapping studies inhuman liver microsomes were conducted to understand thebioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluoro-benzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol],an inhibitor of the Hedgehog pathway. The results revealed theformation of a unique, stable quinone methide metabolite (M1)via ipso substitution of a fluorine atom and subsequent forma-tion of a GSH adduct (M2). The stability of this metabolite arises

from extensive resonance-stabilized conjugation of the substi-tuted benzylphthalazine moiety. Cytochrome P450 (P450) phe-notyping studies revealed that the formation of M1 and M2 wereNADPH-dependent and primarily catalyzed by CYP3A4 amongthe studied P450 isoforms. In summary, an unusual and stablequinone methide metabolite of compound 1 was identified, anda mechanism was proposed for its formation via an oxidativeipso substitution.

Introduction

Hedgehog signaling pathway was first discovered in the 1980s(Nusslein-Volhard and Wieschaus, 1980), and since then, small mol-ecule inhibitors of this pathway have been evaluated as anticancertherapeutic agents with compounds currently in preclinical develop-ment and clinical trials (Peukert and Miller-Moslin, 2010). Design andsynthesis of small molecule inhibitors of the Hedgehog pathway inearly drug discovery were reported previously (Miller-Moslin et al.,2009). The Hedgehog pathway has been implicated in carcinogenesis(Kiesslich and Neureiter, 2012; Raju and Pham, 2012) of varioustypes of cell lines such as translational cells, i.e., cells undergoingvarious stages of cell growth (Pignot et al., 2012) and organs such asgastrointestinal tract (Yang et al., 2012), etc. Lead optimization effortswithin the benzylphthalazine series for Hedgehog inhibitors led to thesynthesis of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol]. Although bioactivationcan arise via conjugative pathways (e.g., the formation of GSHconjugates, acyl glucuronides, and sulfation pathways) (Kalgutkar etal., 2005a; Argikar et al., 2011), most bioactivation reactions involveeither oxidation or reduction and can often be attributed to metabolismby cytochrome P450 (P450) and other oxidative enzymes. A com-monly employed chemical mediation strategy to reduce oxidativemetabolism via P450 is the addition of electron withdrawing groupssuch as fluoro, chloro, trifluoromethyl, etc., to the labile parts of the

molecule (Kalgutkar et al., 2007; Argikar et al., 2011). Compound 1was designed to include two fluorine atoms on the benzylphthalazinemoiety in an effort to reduce the electron density and subsequentlyreduce oxidative metabolism and, therefore, subsequent bioactivation.The objective of the present investigation was to understand thebioactivation potential of compound 1 by performing in vitro metab-olite identification and GSH trapping studies. Our results indicated anoxidative ipso substitution with the loss of a fluorine atom, an infre-quent biotransformation reaction. The end product of this reaction wasidentified as a resonance-stabilized quinone methide metabolite, withextensive conjugation between phenyl and phthalazine moietieslinked by a methylene group. Furthermore, we performed P450 phe-notyping studies to understand the enzyme responsible for this bio-transformation reaction.

Materials and Methods

Chemicals and Reagents. Compound 1 was synthesized at Novartis Insti-tutes for Biomedical Research (Cambridge, MA) as described previously(Miller-Moslin et al., 2009). Human liver microsomes (HLM) (20 mg/mlprotein content, pooled lot number 20) were purchased from CellzDirect(Durham, NC). GSH was purchased from Acros Organics (Fairlawn, NJ).Human Supersomes CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP1A2, andCYP2E1 (1000 pmol/ml) were purchased from BD Biosciences (San Jose,CA). Solvents and reagents were of mass spectrometry (MS) grade and werepurchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). NADPH wasobtained from MP Biomedicals (Solon, OH).

In Vitro Generation of Quinone Methide and GSH Adduct. The GSHtrapping experiment was conducted as per protocols reported previously (Kal-gutkar et al., 2005b). Incubations (1.0 ml each) were conducted in 2-ml DW-96well plates (Analytical Sales and Services, Inc., Pompton Plains, NJ) at 37°C

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http://dx.doi.org/10.1124/dmd.112.046268.

ABBREVIATIONS: P450, cytochrome P450; HLM, human liver microsomes; MS, mass spectrometry; LC, liquid chromatography; RF, radiofrequency; CID,collision-induced dissociation; amu, atomic mass unit; BMS-204352, (3S)-(�)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one.

1521-009X/12/4011-2074–2080$25.00DRUG METABOLISM AND DISPOSITION Vol. 40, No. 11Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics 46268/3798731DMD 40:2074–2080, 2012

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in a shaking heating block. The incubations contained HLM at a proteinconcentration of 1 mg/ml or human Supersomes at an enzyme concentration of100 pmol/ml in 0.1 M KH2PO4 buffer (pH 7.4), substrate (compound 1, 10�M) and MgCl2 (5 mM). The mixture was prewarmed for 2 min at 37°C, andthe reaction was started by adding NADPH (2 mM) and, 3 min later, GSH (5mM). The control experiments lacked either GSH or NADPH. At 0, 0.5, and1 h, aliquots (0.3 ml) were taken from the incubation and placed in equalvolumes of ice-cold acetonitrile to stop the reaction. The reaction mixture wasvortex-mixed and centrifuged for 5 min at 4200g. The resulting supernatantswere transferred to a clean, DW-96 plate (2 ml) with glass inserts (1 ml) forliquid chromatography (LC)/MSn analysis.

LC/MSn Method for Metabolite Identification. Sample analysis andmetabolite identification were conducted using a Thermo LTQ-Orbitrap massspectrometer (Thermo Fisher Scientific, Waltham, MA) capable of MSn scan-ning and accurate mass measurement interfaced with a 3� Ti high-perfor-mance LC pump and CTC PAL autosampler (LEAP Technologies, Carrboro,NC). The analytes (injected as 20-�l aliquots) were separated on a WatersSymmetry C18 analytical column (5 �m, 2.1 � 150 mm; Waters, Milford,MA) with a 35-min gradient elution method. Mobile phase A consisted of 10mM ammonium formate in MS-grade water with 0.1% formic acid. Mobilephase B consisted of MS-grade acetonitrile with 0.1% formic acid. The samplealiquots were eluted at a flow rate of 0.25 ml/min with 10% B over 5 min.Thereafter, the percentage of mobile phase B was gradually increased to 90%over 24 min. After the elution of compound 1 and its metabolites, the columnwas returned to 10% B over 2 min, where it was held for 3 min before the nextinjection. Positive ion mode electrospray ionization was performed at a cap-illary temperature of 375°C, a sheath gas flow rate of 35 ml/min, sourcevoltage of 4.20 kV, source current of 100 �A, capillary voltage of 35 V, andtube lens voltage equal to 100 V. Multipole radiofrequency (RF) amplifier wasset at 400 V peak to peak, along with multipole “0” offsets at �1.25 V and�4.75 V, lens “0” voltage at �6.50 V, lens “1” voltage at �13 V, gate lensoffset at �62 V, multipole “1” offset at �7 V, and front lens at �5 V. Theinstrument calibration was set at a multiple RF of 2824 Hz, main RF of 1187.5

Hz, and pulsed Q dissociation collision energy factor of 10. Fourier transfor-mation MS enabling accurate mass measurement was carried out at a massresolution of 60,000. Ion-trap MS and transformation MS-based data-depen-dent scan post-collision-induced dissociation (CID) and/or high-energy colli-sional dissociation were performed at a normalized collision energy of 35.Activation Q was set at 0.25 with an activation time of 30 ms.

Results

CID Product Ion Spectrum of Compound 1. Compound 1showed a protonated molecular ion [M � H]� at m/z 476. The LCretention time was approximately 14.8 min (Fig. 1). The CID production spectrum (Fig. 2) showed fragment ions at m/z 458, 298, 205, 187,161, and 127. Fragment ion at m/z 458 was formed by the loss ofwater from the m/z 476. Fragment ions at m/z 298 and 205 wereformed by cleavage of the piperazine moiety. Fragment ions at m/z187 and 161 were, respectively, derived from the dehydration and lossof a methyl group from fragment ion at m/z 205. Fragment ion at m/z127 was a result of cleavage from the 1-(2,4-difluorobenzyl)phthala-zine moiety.

Structural Elucidation of M1, Quinone Methide Metaboliteof Compound 1. M1 showed a protonated molecular ion [M �H]� at m/z 472, 4 atomic mass units (amu) lower than that ofcompound 1 (Fig. 3) with a retention time of approximately 17.5min (Fig. 1). The fragment ions at m/z 161 and 187 remained intact.The characteristic fragment ion at m/z 294, 4 amu lower than thatof the parent compound fragment ion at m/z 298, suggests elimi-nation of hydrogen fluoride and addition of an oxygen atom on the4-(2,4-difluorobenzyl)N-ethylphthalazin-1-amine moiety. Thissuggests the formation of a quinone methide metabolite (M1) ofcompound 1.

FIG. 1. Base peak chromatogram of compound 1 and its metabo-lites M1, M2, and M3 at [M � H]� of 476, 472, 779, and 795,respectively. Unmarked peaks are either unrelated to the parent, asconfirmed with accurate mass measurement and tandem MS inter-pretation or presence in 0 h of incubation.

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Structural Elucidation of M2 and M3 GSH Conjugate ofCompound 1. M2 showed the protonated molecular ion of m/zvalue 779, higher than compound 1 by 303 amu (Fig. 4). This wasdetected at a retention time of approximately 9.9 min (Fig. 1). Thefragment ion m/z 761 resulted from the loss of a water moleculefrom M2. The fragment ion m/z 743 resulted from the loss of twowater molecules. The fragment ion m/z 632 was the result of aneutral loss of pyroglutamic acid from fragment m/z 761. Fragmention m/z 488 corresponded to the formation of a thiol-containingcarbocation, possibly as a result of loss of the 2-(2-aminopropana-mide) acetic acid moiety from fragment ion m/z 632. Fragment ionm/z 328 was formed via modification of fragment ion m/z 298 fromthe parent spectrum reflecting the incorporation of sulfur and

oxygen and a loss of a fluorine. Electrospray ionization of M3 inpositive ion mode at a collision energy of 35 resulted in a proton-ated molecular ion of m/z value 795, 319 amu higher than that ofcompound 1 with a retention time of 9.5 min. On the basis of theM2 fragmentation pattern, M3 was identified as a hydroxylatedanalog of M2 (Fig. 5).

P450 Phenotyping. The formation of M1, M2, and M3 wasNADPH-dependent and primarily catalyzed by the CYP3A4 iso-form. Other P450 isoforms used in the assay did not catalyze thereaction leading to M1 and the subsequent GSH adducts. However,other oxidative metabolites were formed in the presence ofCYP2C9, CYP2C19, CYP2D6, CYP1A2, and CYP2E1 (data notshown).

FIG. 2. CID of compound 1 was obtainedat normalized collision energy of 35. Pro-tonated molecular ion [M � H]� is repre-sented by m/z 476 at approximately 5%signal intensity compared against the basepeak m/z 458. Other diagnostic ions includefragments resulting from cleavage of piper-azine moiety.

FIG. 3. CID of metabolite M1, the ipso-substituted quinone methide metabolite ofcompound 1. Fragment ion m/z 294 is char-acteristic of hydrogen fluoride eliminationand addition of an oxygen atom on the4-(2,4-difluorobenzyl)N-ethylphthalazin-1-amine moiety.

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Discussion

GSH trapping studies followed by structural elucidation via LC/MSn

revealed the formation of an unusual, stable quinone methide metabolite(M1) and two GSH conjugates, M2 and M3. The formation of M1 wasattributed to oxidative ipso substitution of a fluorine atom in compound 1 andsubsequently resulted in formation of a GSH adduct M2 (Fig. 4). Theaccurate mass measurement and the MSn mass spectra strongly supported theloss of hydrogen fluoride and incorporation of an oxygen atom. The othercalculated accurate masses for an empirical formula containing two fluorineatoms in the empirical formula did not support any other plausible structurefor M1. The mass accuracy was calculated as a mass shift in �ppm, and thevalues for compound 1, M1, M2, and M3 were 1.0, 3.0, 1.8, and 0.9,respectively. The possible mechanism for formation of M1 is shown in Fig. 6.Furthermore, these results provided direct evidence of the formation of anipso-substituted stable quinone methide, M1, as a precursor to the GSHconjugate M2. Taken together, these results also indicated that a fluorosubstitution on the benzylphthalazine moiety was insufficient to block theoxidation by P450 enzymes and led to the formation of a quinone methidemetabolite. The formation of M1, M2, and M3 was observed to beNADPH-dependent and was likely to proceed via either epoxidation or adirect hydrogen atom transfer mechanism at the benzylic carbon atom, asdepicted in Figs. 6, 7, and 8.

Quinones, quinone methides, or quinone imines are usually formed viaoxidation by P450 enzymes. In the process termed metabolic bioactiva-tion, such highly unstable electrophilic intermediates react with nucleo-philic groups of cellular macromolecules in their vicinity. A number of

drugs on the market such as nevirapine (Wen et al., 2009), tacrine (Woolfet al., 1993), (3S)-(�)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one (BMS-204352; MaxiPost)(Zhang et al., 2003), acolbifene (Liu et al., 2005), phencyclidine (Driscollet al., 2007), trimethoprim (Damsten et al., 2008), etc. are reported toundergo metabolic activation via formation of quinone methides. Qui-none methides have also been reported as reactive intermediates of anumber of xenobiotics including alkyl-substituted phenols such as p-cresol (Yan et al., 2005), eugenol (Thompson et al., 1990), polyphenolssuch as resveratrol (Steenwyk and Tan, 2010), catechols (Iverson et al.,1995), and bis-hydroxy toluene (Tajima et al., 1985; Bolton et al., 1990;Bolton and Thompson, 1991) and at times may even be formed byspontaneous rearrangement of o-quinones with 4-allylic substituents(Bolton et al., 1994).

The quinone methide metabolite identified in the present example wasfound to be stable enough to be separated and detected by LC/MS. M1may either be an ortho- or a para-quinone methide, as shown in Figs. 6and 7. We postulate that the stability of M1 arises from extensiveconjugation of the substituted benzylphthalazine moiety, leading to de-localization of electrons and resonance stabilization. In this case, M1 asdepicted may be the most contributing resonance form. M1 is hypothe-sized to be stabilized by resonance hybrids that include a structure withthe piperazinyl nitrogen atom (1N) of 5-fluoro-2-((4-(piperazin-1-ium-1-ylidene)phthalazin-1(4H)-ylidene)methyl)phenolate moiety bearing apositive charge and a corresponding phenolate anion with a neg-ative charge (Fig. 9). These factors contribute greatly toward the

FIG. 4. CID of metabolite M2 at m/z 779 is shown. The fragment ions m/z 761 and m/z 743 resulted from the losses of one and two water molecules, respectively. Fragmention m/z 632 corresponds to loss of pyroglutamate.

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stability of the identified quinone methide metabolite (M1). It is hypoth-esized that replacing the 1N atom of 4-(piperazin-1-yl)phthalazine moietywith a C atom may strongly decrease the stability of the correspondingquinone methide.

Some quinone methides have been shown to be more stable than others inthe literature. For example, a hydroxyl-quinone methide of bis-hydroxytoluene was 6 times more stable than its corresponding nonhydroxy analog inin vitro incubations containing GSH (Bolton et al., 1990). Ortho-quinone

FIG. 5. CID of metabolite M3 at m/z 795 resulted in diagnostic molecular ions of m/z 777 and m/z 648, corresponding to the loss of water and loss of pyroglutamate,respectively.

FIG. 6. Proposed metabolic pathway forformation of M1, a putative metabolite ofcompound 1 in HLM via arene oxide for-mation. M1 may possibly be a para- orortho-quinone methide metabolite afteripso substitution of a fluorine atom and aprecursor to M2 and M3.

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methides of 2-hydroxy estrone and 4-hydroxy estrone were shown to havehalf-lives of 42 s and 12 min, respectively (Iverson et al., 1996; Zhang et al.,1999). There have been reports of chemically stable quinone methide me-tabolites that could be isolated, identified, or even chemically synthesized.Two quinone methides formed as tertiary metabolites of tamoxifen have beenreported to be stable. A quinone methide (metabolite E) has been reported tohave a half-life of 4 h under physiological conditions (Fan and Bolton, 2001),

whereas 4-hydroxytamoxifen quinone methide has been reported to have ahalf-life of 3 h under physiological conditions (Fan et al., 2000). In the samestudy, 4-hydroxytamoxifen quinone methide formed upon bioactivation oftoremifene, another antiestrogen agent, was shown to have a physiologicalhalf-life of 1 h. In the present investigation, we were unable to isolate andpurify the quinone methide of compound 1 in quantities sufficient for con-ducting in vitro stability studies.

FIG. 7. Proposed metabolic pathway for for-mation of M1, a metabolite of compound 1 inHLM via hydrogen atom transfer mechanism.M1 may possibly be a para- or ortho-quinonemethide metabolite after ipso substitution of afluorine atom and a precursor to M2 and M3.

FIG. 8. Alternative metabolic pathway forformation of M2 and M3, metabolites ofcompound 1 in HLM.

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P450 phenotyping reactions were performed with CYP3A4, CYP2C9,CYP2C19, CYP2D6, CYP1A2, and CYP2E1 to understand the enzymeresponsible for the formation of M1. The formation of M1, M2, and M3 wasobserved to be NADPH-dependent and primarily catalyzed by CYP3A4.Other P450 isoforms—CYP2C9, CYP2C19, CYP2D6, CYP1A2, andCYP2E1—did not form M1 and the subsequent GSH adduct M2.

In summary, the current investigation revealed a rare, stable qui-none methide metabolite of compound 1 resulting from an oxidativeipso-substitution mechanism in HLM, catalyzed by CYP3A4. Thesestudies substantiate previous reports of oxidative ipso substitutionwith the loss of a fluorine atom and support the formation of aunusual, stable quinone methide metabolite. Furthermore, our dataalso support the theory that reducing electron density on heterocyclesby incorporation of an ortho- and/or para-fluoro substituent, a com-mon strategy used in drug discovery to block P450 activity, may notalways prevent oxidative metabolism.

Acknowledgments

We thank Joshua L. Johnson, Weislawa Maniara, Stefan Peukert, HongJiang, and Shari Bickford for helpful discussions.

Authorship Contributions

Participated in research design: Gunduz and Argikar.Conducted experiments: Gunduz, Colizza, Bushee, and Cirello.Contributed new reagents or analytic tools: Gunduz and Bushee.Performed data analysis: Gunduz.Wrote or contributed to the writing of the manuscript: Gunduz, Argikar,

Kamel, Colizza, Bushee, Cirello, Lombardo, and Harriman.

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Address correspondence to: Mithat Gunduz, Metabolism and Pharmacokinetics,Novartis Institutes for Biomedical Research, Inc., 250 Massachusetts Ave., Cambridge,MA 02139. E-mail: mithat.gunduz@novartis.com

FIG. 9. Resonance stabilization of quinone methide metabolite (M1).

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