1521-009X/44/5/634–646$25.00 http://dx.doi.org/10.1124/dmd.116.069310DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:634–646, May 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Biosynthesis of Fluorinated Analogs of Drugs Using HumanCytochrome P450 Enzymes Followed by Deoxyfluorination and

Quantitative Nuclear Magnetic Resonance Spectroscopy to ImproveMetabolic Stability s

R. Scott Obach, Gregory S. Walker, and Michael A. Brodney

Pharmacokinetics, Pharmacodynamics, and Drug Metabolism (R.S.O., G.S.W.) and Worldwide Medicinal Chemistry (M.A.B.), PfizerInc., Groton, Connecticut and Cambridge, Massachusetts

Received January 5, 2016; accepted February 24, 2016

ABSTRACT

Replacement of hydrogen with fluorine is a useful drug design strategywhen decreases in cytochrome P450 (P450) metabolic lability areneeded. In this paper, a facile two-step method of inserting fluorineintometabolically labile sites of drugmolecules is described that utilizesless than 1 mg of starting material and quantitative NMR spectroscopyto ascertain the structures and concentrations of products. In the firststep, hydroxyl metabolites are biosynthesized using human P450enzymes, and in the second step these metabolites are subjectedto deoxyfluorination using diethylaminosulfur trifluoride (DAST).The method is demonstrated using midazolam, celecoxib, ramelteon,and risperidone as examples and CYP3A5, 2C9, 1A2, and 2D6 tocatalyze the hydroxylations. The drugs and their fluoro analogs were

tested formetabolic lability. 9-Fluororisperidone and 49-fluorocelecoxibwere 16 and 4 times more metabolically stable than risperidone andcelecoxib, respectively, and 2-fluororamelteon and ramelteonweremetabolized at the same rate. 19-Fluoromidazolamwasmetabolized atthe same rate as midazolam by CYP3A4 but was more stable inCYP3A5 incubations. The P450-catalyzed sites of metabolism of thefluorine-containing analogswere determined. Some of themetabolitesarose via metabolism at the fluorine-substituted carbon, wherein thefluorine was lost to yield aldehydes. In summary, this method offers anapproach whereby fluorine can be substituted in metabolically labilesites, and the products can be tested to determine whether anenhancement in metabolic stability was obtained.

Introduction

Drug design can have many simultaneous challenges of optimizingtarget potency, selectivity, dispositional properties, and safety. Amongdispositional properties, rapid metabolic intrinsic clearance is a com-monly encountered problem that can result in unacceptable projecteddosing regimens (required dose level is too high owing to high clearanceand first-pass extraction, and/or dosing frequency is too high owing toshort half-life), as well as a greater potential for pharmacologically activeor toxic metabolites. Cytochrome P450-mediated metabolism is themost frequently encountered reason for excessively high intrinsicclearance. Common strategies to mitigate high metabolic turnoverfrom P450-mediated clearance include optimizing physicochemicalproperties such as lipophilicity, molecular weight, and/or pKa of basicamines. Additional strategies to reduce metabolic clearance involveblocking or modifying a metabolic soft-spot by the use of chemicalisosteres or groups that modify the electronics or sterics at a metabolicsite. By introducing specific design elements proximal to or in place of asite of rapid P450-catalyzed metabolism, the intrinsic clearance canbe attenuated by decreasing metabolic rate or changing the site ofmetabolism. Molecular properties and specific structural entities willimpact binding of molecules to P450 enzymes, and the subsequent

orientation and chemical reactivity of specific positions on thesemolecules dictates the observed metabolic products. Since the P450catalytic mechanism for aliphatic hydroxylation involves abstraction ofa hydrogen atom (Groves, 2005), replacing a rapidly abstracted hydrogenwith a halogen atom such as fluorine is a commonly employed strategy toreduce clearance.Incorporating a fluorine in drug design is a powerful strategy to

improve compound properties (Purser, et al., 2008; Gillis, et al., 2015;Murphy and Sandford, 2015). The small size and electronegativity offluorine can impart dramatic changes on compound conformation,permeability, pKa, potency, and metabolism. Specifically, fluorinationat a site of P450 metabolism may result in either a decrease in metabolicrate or a change in the site of metabolism, thus fluorine substitution is acommonly used approach to modulate drug metabolism. Therefore, anability to easily carry out fluorinations of lead structures at metabolicsites would be a welcome technique to rapidly assess whether such asubstitution would have a beneficial impact in drug research projects.Reagents to carry out deoxyfluorination reactions have been described(Al-Maharik and O’Hagan, 2011), with one of the oldest and bestdescribed being diethylaminosulfur trifluoride (DAST; Hudlický,1988). DAST reactions can be done at ambient conditions in aproticsolvents and, as we show in this work, can be done in low volume atmicrogram scale. Although not proven, a proposed mechanism fordeoxyfluorination reactions using DAST begins with reaction of analiphatic alcohol group with the sulfur and displacement of fluoride as

dx.doi.org/10.1124/dmd.116.069310.s This article has supplemental material available at dmd.aspetjournals.

org.

ABBREVIATIONS: DAST, diethylaminosulfur trifluoride; DMSO, dimethylsulfoxide; HPLC, high-performance liquid chromatography; HSQC,heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; P450, cytochrome P450.

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HF. This intermediate can undergo either subsequent SN2 displacementof the alkyl group by the released fluoride or a dissociative SN1mechanism and recombination with fluoride to yield the alkyl fluorideproduct (Baptista, et al., 2006). Overall, this approach would representan alternative to classic total synthesis approaches.In a recent report we described a method for generating nanomole

quantities of drug metabolites (Walker, et al., 2014). Employment ofcryomicroprobe 1H–nuclear magnetic resonance (NMR) spectroscopyto characterize the isolated metabolites in both a qualitative and

quantitative fashion yielded solutions of drug metabolites of knownconcentration and structure, and these solutions could then be useful forbiologic testing, or used as authentic standard stock solutions for high-performance liquid chromatography (HPLC)–mass spectroscopy (MS)bioanalysis methods, among other applications. While NMR has beenhistorically viewed as a qualitative technique useful for structuralcharacterization, it is also a valuable tool for quantitation. A significantadvantage NMR holds over other analytical techniques (i.e., LC-MS,LC-UV) is its uniform response that is independent of structure. As an

Fig. 1. Reaction schemes for P450-catalyzed biosynthetic hydroxylation and deoxyfluorination of four drugs.

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example, the response for a given hydrogen in a steroid from a 1 mMsolution should be equal to the response for a given hydrogen in aglucuronide from a 1 mM solution. This uniform response allows anNMR instrument to be calibrated with a known compound and quantitatesamples in the absence of authentic standards (Walker, et al., 2011).In this report, we describe a facile method whereby drugs that are

rapidly metabolized by P450 enzymes can be converted into analogsfluorinated at the site of enzymatic hydroxylation using a hybridprocedure of biosynthesis, chemical synthesis, and quantitative NMRspectroscopy (Fig. 1). A previous report by Rentmeister et al. (2009)coupled the use of a bacterially derived panel of P450 enzymes andsubsequent deoxyfluorination to yield fluorinated derivatives on ascale of tens of milligrams. In the present work, synthesis is done atnanoscale, so that fluorinated analogs can be quickly prepared for thepurpose of testing whether fluorine substitution yields an improve-ment in metabolic lability. Hydroxy metabolites are prepared bybiosynthesis using the very P450 enzymes responsible for the highlability and then subjected to nanomole-scale deoxyfluorinationusing DAST. The fluorinated analogs are isolated and analyzed byquantitative NMR spectroscopy to determine the structure andconcentration of the stock solution. The material can then be tested forbiologic activity and metabolic lability. By using the same enzymesresponsible for rapid metabolism, the likelihood that the fluorinatedanalog ultimately generated will have improved metabolic stability willbe greater. The method is described herein using four well known drugsas examples.

Materials and Methods

Materials. Drugs were obtained from the following vendors: midazolam(Cerilliant, Round Rock, TX), celecoxib and ramelteon (Pfizer, Groton, CT),and risperidone (Sigma-Aldrich, St. Louis, MO). Heterologously expressedrecombinant human P450 enzymes in microsomes from Sf9 cells were customprepared under contract by Panvera (Madison, WI). Pooled human livermicrosomes from 50 donors of both sexes were prepared under contract by BDGentest (Woburn, MA). Hexadeuterated dimethylsulfoxide (DMSO) was fromCambridge Isotope Laboratories (Tewksbury, MA). NADPH and DAST (1 Min CH2Cl2) were from Sigma-Aldrich. Note that DAST is a caustic chemicaland should be used with appropriate personal protective equipment in alaboratory chemical fume hood.

General Biosynthesis and Fluorination Procedure. Substrates (20 mM)were incubated with recombinant P450 enzymes (1–2 nmol), NADPH (1.3 mM),andMgCl2 (3.3 mM) in 40ml potassium phosphate buffer (100 mM, pH 7.5) in ashaking water bath maintained at 37�C. Incubation times varied from 40 minutesto 2 hours. Incubations were terminated with addition of CH3CN (40 ml) and theprecipitated material was removed by spinning the mixtures in a centrifuge for5 minutes at 1700g. The supernatant was partially evaporated in a vacuumcentrifuge (Genevac, Gardiner, NY) for 2 hours. To the remaining mixture wasadded 0.5 ml neat formic acid, 0.5 ml CH3CN, and water to a final volume of50 ml. This mixture was spun in a centrifuge at 40,000g for 30 minutes. The clearsupernatant was applied to an HPLC column (Polaris C18, 4.6� 250 mm; 5-mmparticle size) through a Jasco HPLC pump at a rate of 0.8 ml/min. After the entire

sample was applied, an additional ;5 ml of mobile phase (0.1% formic acidcontaining 1% CH3CN) was pumped through the system. The column wasmoved to a ThermoFisher LTQ HPLC-MS system (Thermo Fisher Scientific,Sunnyvale, CA) containing a photodiode array detector, and a mobile phasegradient was applied to elute material of interest. The mobile phase comprised0.1% formic acid in water and CH3CN and was run at a flow rate of 0.8 ml/min.The gradient began by using a dummy injection of water. The eluent passedthrough the photodiode array detector scanning from 200–400 nm and then to asplitter (ratio was approximately 15:1) with the larger portion going to a LEAPTechnologies fraction collector (Leap Technologies, Carrboro, NC). Fractionswere collected every 20 seconds, generally over a total run time of 60–80minutes. The remainder was introduced into the mass spectrometer operated inthe positive ion mode.MS1 andMS2 data were collected with source temperatureand potential settings optimized for signal and fragmentation for each parentdrug. Fractions containing hydroxyl metabolites of interest were analyzed on aThermoFisher Orbitrap Elite UHPLC-UV-MS system containing an Acquitycolumn (HSS T3 C18, 2.1 � 100 mm, 1.7-mm particle size) using the mobilephases described above at a flow rate of 0.4 ml/min and an injection volume of5 ml. Fractions containing single peaks by UV and the desired protonatedmolecular ions were combined and evaporated by vacuum centrifugation in15-ml conical polypropylene centrifuge tubes.

To the dried residues was added 0.15 ml of 150 mM DAST in CH2Cl2. Thetubes were capped and initially mixed, and then allowed to stand at roomtemperature for 2 hours. The reaction mixtures were processed by addition of1 M Na2CO3 in water (0.5 ml) and extraction with ethyl acetate (2 � 2 ml). Thesolvent was removed by vacuum centrifugation and the residue was reconstitutedin 20 ml CH3CN and 80 ml 0.1% HCOOH. This material was injected onto theaforementioned Polaris C18 column, ThermoFisher LTQHPLC-UV-MS systemwith LEAP Technologies fraction collector. Fractions were collected every 20seconds and those containing fluorinated analogs of interest were analyzed on theaforementioned ThermoFisher Orbitrap Elite UHPLC-UV-MS system. Fractionswere pooled as appropriate and the solvent was removed by vacuum centrifuga-tion. Specific details of reaction and HPLC-MS conditions for each drug are listedin the Supplemental Information.

Quantitative Nuclear Magnetic Resonance Analysis. Dried residuescontaining fluorinated analogs were dissolved in 0.04 ml of DMSO-d6“100%” (Cambridge Isotope Laboratories, Andover, MA) and placed in a1.7-mm NMR tube in a dry argon atmosphere. 1H and 13C spectra werereferenced using residual DMSO-d6 (

1H d = 2.50 ppm relative to TMS, d = 0.00,13C d =3 9.50 ppm relative to TMS, d = 0.00). NMR spectra were recorded on anAvance 600 MHz (Bruker BioSpin Corporation, Billerica, MA) controlled byTopspin V3.2 and equipped with either a 1.7-mm TCI CryoProbe or a 5-mmBBO CryoProbe (Bruker BioSpin). One-dimensional 1H spectra were recordedusing an approximate sweep width of 8400 Hz and a total recycle time ofapproximately 7 seconds. The one-dimensional 19F data were recorded using thestandard pulse sequence (zgfhigqn.2) provided by Bruker. One-dimensional 19Fspectra were recorded using an approximate sweep width of 11,000 Hz and atotal recycle time of approximately 7.5 seconds. The resulting time-averaged freeinduction decays were transformed using an exponential line broadening of1.0 Hz to enhance signal to noise. The two-dimensional data were recorded usingthe standard pulse sequences provided by Bruker. Postacquisition data process-ing was performed with either TopSpin V3.2 or MestReNova V8.1 (MestrelabResearch, Santiago de Compostela, Spain)

Metabolic Lability Experiments. Drugs and their fluorinated analogs at asubstrate concentration of 0.2 mMwere incubated with human liver microsomes(0.2–2.0 mg/ml depending on the test compound) or recombinant P450 enzymes(10–100 pmol/ml, depending on the test compound), NADPH (1.3 mM), andMgCl2 (3.3 mM) in an incubation volume of 1 ml potassium phosphate buffer(100 mM, pH 7.45) at 37�C. Incubations were commenced with the addition ofNADPH, and aliquots of 0.1 ml were removed at 0, 2, 5, 10, 15, 20, 40, and60minutes and added to 0.5mlCH3CNcontaining clozapine (0.04mM)as an internalstandard. Terminated incubation mixtures were spun in a centrifuge for 5 minutes at1700g, the supernatant was removed in a vacuum centrifuge, and residues werereconstituted in 0.2 ml 1% HCOOH in 15% CH3CN and analyzed by HPLC-MS.

Tenmicroliters of supernatant were injected onto an Acquity column (HSS T3C18, 2.1� 100mm, 1.7-mmparticle size) coupled with a ThermoFisher OrbitrapElite UHPLC-MS system. The mobile phase consisted of 0.1% formic acid inwater (A) and acetonitrile (B) at a flow rate of 0.4 ml/min. Initial mobile phase

TABLE 1

Fluorinated analogs of four drugs generated by biosynthesis of hydroxyl metabolitesand deoxyfluorination by DAST

Parent DrugBiosynthesisEnzyme

FluorinatedProduct

Stock ConcentrationObtained

mM

Midazolam CYP3A5 19-Fluoromidazolam 1.35Ramelteon CYP1A2 2-Fluororamelteon 0.26Celecoxib CYP2C9 49-Fluorocelecoxib 2.32Risperidone CYP2D6 9-Fluororisperidone 0.96

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conditions were at 10% B, increased linearly to 50% B at 2 minutes, then to 95%B at 3.2 minutes, held at this composition for 1.5 minutes, followed by re-equilibration at initial conditions for 0.8 minutes. The eluent was introduced intothe ion source operated in the positive ion mode at a temperature of 350�C,capillary temperature of 300�C, and sheath gas and auxiliary gas settings at 25.Scanning was done fromm/z 250 to 450 at a resolution setting of 30,000. Analytepeak areas were determined by integration of ion currents of protonatedmolecular ions to a range of 5 ppm. Analyte response was corrected by theinternal standard response, and the peak area ratios were normalized to thatmeasured at the t = 0 sampling point.

The decline in peak area ratio was plotted over the incubation time, and thefirst order rate constant was determined using the data that showed a log-linearrelationship using GraphPad Prism (v6.03; GraphPad Software, San Diego, CA).Intrinsic clearance was calculated as:

CLint ¼ �k½E� ð1Þ

where [E] is the concentration of microsomal protein or P450 used in theincubation and -k is the negative slope of the decline of natural log substrateconcentration versus incubation time plot.

Fig. 2. High resolution MSn, 1H-NMR, and19F-NMR spectra for 19-fluoromidazolam.

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Biotransformation of Fluorinated Analogs. Fluorinated analogs (10 mM)were incubated with recombinant P450 enzymes (10 pmol/ml CYP3A4 and3A5 with 19-fluoromidazolam, 25 pmol/ml CYP1A2 for 2-fluororamelteon,50 pmol/ml CYP2C9 for 49-fluorocelecoxib, 100 pmol/ml CYP2D6 for9-fluororisperidone), NADPH (1.3 mM), and MgCl2 (3.3 mM) in an incubationvolume of 0.4 ml potassium phosphate buffer (100 mM, pH 7.45) at 37�C. Thestock solutions of fluorinated analogs in DMSO-d6 were evaporated in aGenevac to remove the solvent prior to metabolic incubations and the incubationbuffer was added to the tube and sonicated for 10 minutes. Incubations werecarried out for 30 minutes, terminated by addition of 2 ml CH3CN, and spun in acentrifuge at 1700g for 5 minutes to remove precipitated protein. The supernatantwas evaporated in a Genevac and reconstituted in 0.08 ml 1% HCOOH foranalysis by UHPLC-UV-MS using the aforementioned column and system.

Results

Biosynthesis of Hydroxy Metabolites, Fluorination, and StructureDetermination

Four drugs were successfully converted to their fluorine-containinganalogs using human P450 enzymes and DAST-mediated deoxyfluori-nation (Table 1). A description of each preparation follows.Fluoromidazolam. Incubation of midazolam with CYP3A5 yielded

hydroxymidazolam, which was purified and subjected to deoxy-fluorination by DAST. The resulting fluoro analog of midazolamwas isolated by HPLC and analyzed by high resolution massspectrometry and NMR spectroscopy. The protonated molecular

Fig. 3. High resolution MSn, 1H-NMR, and19F-NMR spectra for 2-fluororamelteon.

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ion of m/z 344.0757 was consistent with an empirical formula ofC18H13N3ClF4 (–1.0 ppm; Fig. 2). As midazolam is well known to behydroxylated at the 19- and 4-positions by CYP3A enzymes (Gorski,et al., 1994), the most likely sites of fluorine substitution are these twopositions. However, the major fragment ions resulted from simplelosses of chlorine, HF, and methylamine and thus were not informativefor structure assignment. Analysis by NMR spectroscopy provided datato assign the site of fluorination on the 19 position (Fig. 2). The 1Hspectrum of the isolated fluoromidazolam contained no methylresonance and contained a set of new resonances integrating to one

hydrogen each at d5.34 (dd, J = 50.1, 11.8 Hz) and d5.70 (dd, J = 50.1,11.8 Hz), Fig. 2. These new resonances are assigned as themethylene of the 19 position of the fluoromidazolam. This structureis additionally supported by the 1H-13C heteronuclear singlequantum coherence (HSQC) data (Supplemental Figs. 1 and 2). As withthe 1H spectrum there is no evidence of a methyl. Furthermore the newresonances at d5.34 and d5.70 can be correlated to a carbon at d76.5,which is consistent with a CH2F moiety. All of this data, as well asall other data collected, supports the proposed fluorination at the19-position.

Fig. 4. High resolution MSn, 1H-NMR, and19F-NMR spectra for 49-fluorocelecoxib.

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Fluororamelteon. Incubation of ramelteon with CYP1A2 generatedthree hydroxyl metabolites; however, after reaction with DAST onlyone fluorinated analog was obtained with a protonated molecular ionof m/z 278.1550, consistent with an empirical formula of C16H21NO2F(–0.3 ppm, Fig. 3). Fragment ions showed that the site of fluorinationwas on the propanamide, which is consistent with a known majormetabolite of ramelteon generated by CYP1A2 (Obach and Ryder,2010). Analysis by NMR spectroscopy demonstrated that the site offluorination is on the 2-position. In the 1H spectrum of the isolatedfluororamelteon, the methyl group is assigned to a set of resonancesintegrating to three hydrogens at d1.43 (dd, J = 24.5, 6.7 Hz), Fig. 3. In

addition to these resonances, a methine resonance at d4.99 (dq, J = 49.7,7.3) can be correlated through the 1H-13C HSQC data to a carbon atd88.8 Both the 1H and 13C shifts are consistent with a R-CHF-CH3

moiety (Supplemental Figs. 3 and 4). 2-Hydroxyramelteon is reportedas the major metabolite of ramelteon and the stereochemistry at position2 is reported as the (S)-isomer (Nishiyama, et al., 2014). From the one-and two-dimensional NMR spectral data it appears that there was asingle 2-fluororamelteon diastereoisomer, and HPLC analysis alsoshows a single peak for the material. Depending on the substrate, theDAST reaction can proceed through either an SN1 mechanism thatwould result in a mixture of stereoisomers or an SN2 reaction that would

Fig. 5. High resolution MSn, 1H-NMR, and19F-NMR spectra for 9-fluororisperidone.

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result in a single isomer of inverted configuration from the hydroxylprecursor. Since there was a single isomer of 2-fluororamelteon made, itis proposed that it is the single stereoisomer of R-configuration at the2-position.Fluorocelecoxib. Celecoxib was incubated with CYP2C9, which is

known to yield a hydroxyl metabolite on the tolyl methyl position(Tang, et al., 2000). Deoxyfluorination of the isolated hydroxycelecoxibmetabolite yielded a fluorinated analog with a protonated molecular ionof m/z 400.0738, indicative of an empirical formula of C17H14N3O2SF4(0.2 ppm). The mass spectral fragmentation pattern was not veryinformative regarding structure assignment as most fragments merelyreflected sequential losses of HF (Fig. 4); however, as there is only onealiphatic site for oxidation and DAST does not deoxyfluorinatephenols, there is only one possible site for modification. This wasconfirmed with NMR spectroscopy, wherein the 1H spectrum of theisolated fluorocelecoxib contained no methyl resonance and containeda set of new resonances integrating to one hydrogen each at d5.42 (s)and d5.50 (s) (Fig. 4). These new resonances are assigned as themethylene of the 4 position of the fluorocelecoxib. This structure isadditionally supported by the 1H-13C HSQC data (Supplemental Figs. 5and 6). As with the 1H spectrum there is no evidence of a methyl.Furthermore, the new resonances at d5.42 and d5.50 can be correlatedto a carbon at d84.0, which is consistent with a CH2F moiety. All ofthis data, as well as all other data collected, supports the proposedfluorination at the 4 position.Fluororisperidone. Risperidone was incubated with CYP2D6 and

the isolated hydroxyrisperidone intermediate was subjected to deoxy-fluorination with DAST. The subsequent fluororisperidone product hada protonated molecular ion of m/z 429.2094, consistent with anempirical formula of C23H27N4O2F2 (–0.6 ppm). The fragment ion atm/z 209 and its subsequent loss of HF suggests that the site offluorination is on the tetrahydropyridopyrimidone portion of risper-idone (Fig. 5). Subsequent analysis by NMR showed that the site offluorination is on the 9-position, which is consistent with the known siteof hydroxylation of risperidone (Mannens et al., 1993; Fang, et al.,1999). In the 1H spectrum of the isolated fluororisperidone the set ofresonances at d5.35 (dt, J = 48.2, 4.8 Hz) is assigned as the 9 positionCHF. In addition to these resonances, the methine resonance at d5.35can be correlated through the 1H-13C HSQC data to a carbon at d87.4Both the 1H and 13C shifts are consistent with a R-CHF-CH2-R moiety.All of this data, as well as all other data collected (Supplemental Figs. 7and 8), supports the proposed fluorination at the C9 position. CYP2D6has been reported to generate only the R-hydroxy stereoisomer atposition 9 (Yasui-Furukori, et al., 2001), while the DAST reaction hasthe potential to generate a mixture of fluoro stereoisomers (via SN1reaction) or a single stereoisomer with inverted structure (via SN2reaction). The NMR spectral and chromatographic data stronglysuggest that a single enantiomer was generated, and on the basis ofthe mechanism of reaction, the S-isomer was proposed.

Metabolic Lability

The four fluorinated analogs were incubated with the P450 enzymeslargely responsible for metabolism of the respective nonfluorinatedanalogs, and the CLint data obtained were compared to determinethe impact, if any, of fluorine substitution at the metabolically labilesite. Data are listed in Table 2. In two instances (midazolam versus19-fluoromidazolamwithCYP3A4 and ramelteon versus 2-fluororamelteonwith CYP1A2) there was minimal to no observed difference inmetabolic lability, whereas in the other three there was a markeddecrease in metabolism with fluorination. 9-Fluororisperidoneshowed the most marked decrease in lability compared with itsnonfluoro counterpart with a 16-fold decrease in metabolism byCYP2D6. Fluorocelecoxib showed an almost 4-fold decrease in meta-bolic lability catalyzed by CYP2C9. Interestingly, 19-fluoromidazolamwas metabolized as quickly as midazolam by CYP3A4 but in CYP3A5it was 3-fold more stable. Overall the data indicate that the impact offluorine substitution at a site of P450-catalyzed hydroxylation is notpredictable and may be governed by other physicochemical propertiessuch as lipophilicity. The data also support using this approach to modifyparticular P450s responsible for metabolism as a strategy to attenuatedrug-drug interactions or overreliance on an enzyme known to be subjectto genetic polymorphism.Comparison of metabolic lability in pooled human liver micro-

somes offered some differences from the individual P450 enzymes. Forexample, 49-fluorocelecoxib was actually metabolized faster than cele-coxib and the large impact that fluorine substitution had on CYP2D6catalyzed hydroxylation of risperidone was considerably diminished inhuman liver microsomes (Table 3). 2-Fluororamelteon was also metabo-lized faster than ramelteon in human liver microsomes, whereas themetabolism of 19-fluoromidazolam was slightly slower than midazolam.

Biotransformation of Fluorinated Analogs

The four fluorinated analogs were incubated with recombinant P450enzymes to provide insight into how biotransformation reactionscontribute to changes in metabolic lability. The quantities of metabo-lites generated were too low to yield NMR data, so structures ofmetabolites were proposed fromMS data. Comparison was made to theknown metabolite profiles for the nonfluorinated analogs. Highresolution mass spectrometric data for the metabolites of the fluorinatedanalogs is in the Supplemental Information.Biotransformation of 19-Fluoromidazolam by CYP3A4 and

3A5. 19-Fluoromidazolam was metabolized by both CYP3A4 and 3A5to a similar array of metabolites with a proposed scheme in Fig. 6.Metabolism on the 19-carbon yielded metabolites that lost the fluorine—presumably via generation of an initial unstable halohydrin that lostfluoride to yield the aldehyde metabolite (peak 7; m/z 340.0649) that wassubsequently reduced to 19-hydroxymidazolam (peak 1;m/z 342.0801) oroxidized to the carboxylic acid (peak 4; m/z 356.0597). There was also a

TABLE 2

Comparison of metabolic lability in recombinant human P450 enzymes betweendrugs and their fluorinated analogs

Compound EnzymeCLint (ml/min per picomole P450)

Parent Drug Fluorinated Analog Stability Factor

Midazolam CYP3A4 23.4 (0.7) 17.7 (0.7) 1.3Midazolam CYP3A5 45.9 (1.0) 15.3 (0.5) 3.0Ramelteon CYP1A2 27.3 (6.4) 28.8 (5.1) 0.95Celecoxib CYP2C9 3.0 (0.4) 0.78 (0.15) 3.8Risperidone CYP2D6 5.6 (0.3) 0.34 (0.37) 16

Values in parentheses are standard errors.

TABLE 3

Comparison of metabolic lability in pooled human liver microsomes between drugsand their fluorinated analogs

Compound

CLint

(ml/min per milligram of protein)Stability Factor

Parent Drug Fluorinated Analog

Midazolam 691 (38) 507 (23) 1.4Ramelteon 63 (18) 95 (10) 0.66Celecoxib 23 (5) 34 (4) 0.67Risperidone 40 (1) 27 (5) 1.5

Values in parentheses are standard errors.

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large peak in the CYP3A5 incubation, where one carbon atom was lost(peak 2;m/z 312.0697), and is hypothesized to arise via decarboxylation ofpeak 4. This is an unprecedented pathway and would require furtherexploration to confirm or refute this proposed pathway and mechanism.The other initial pathway yielded putative ring-opened metabolites thatlost a nitrogen atom (peak 8; m/z 361.0545), similar to those seen formidazolam (Song and Abramson, 1993). These are proposed to arise viainitial 4-hydroxylation that ring-opens to the imine/aldehyde, withthe imine hydrolyzing to the ketone. Thus, the sites of metabolism of19-fluoromidazolam by CYP3A are largely the same as those formidazolam itself (Heizmann and Ziegler, 1981; Gorski, et al., 1994).Biotransformation of 2-Fluororamelteon by CYP1A2. 2-Fluo-

roramelteon was metabolized by CYP1A2 at the same rate asramelteon. There were five peaks in the HPLC-UV chromatogramfrom the incubation extract (Fig. 7) of a similar pattern observed forramelteon metabolism by CYP1A2 (Obach and Ryder, 2010). A pair ofinterconverting hydroxyl diastereomers (peak 1) were observed thathave base peaks of a sodium adduct (m/z 316.1317) and in-sourcedehydration (m/z 276.1393), with fragmentation that suggests hydrox-ylation on the indenofuran system. The other major hydroxyl metab-olite (peak 3; m/z 294.1499) has substitution on the aromatic ringsuggested by the fragmentation pattern and small red shift in UVwavelength absorption maximum. The third metabolite (peak 4) had aprotonated molecular ion of m/z 276.1592, indicating the replacement

of fluorine with oxygen, and this metabolite had the same retention timeand mass spectral fragmentation as 2-hydroxyramelteon. The fourth(peak 2) arose via hydroxylation and a loss of 2 H (m/z 292.1244) andno further data were gathered. The proposed metabolic pathways forfluororamelteon are in Fig. 7.Biotransformation of 49-Fluorocelecoxib by CYP2C9. 49-Fluo-

rocelecoxib was metabolized by CYP2C9 at the position of thefluoromethyl. Three metabolites were observed (Fig. 8) and areproposed to be the methyl alcohol (peak 1), aldehyde (peak 3), andcarboxylic acid (peak 2). High resolution mass spectra yieldedprotonated molecular ions of m/z 398.0776, 396.0623, and 412.0577,all of which indicate the loss of one of the fluorines. Hydroxylation ofthe fluoromethyl group would result in elimination of the fluoride fromthe halohydrin to yield the aldehyde, which can undergo subsequentreduction or further oxidation to the alcohol and carboxylic acid,respectively. The proposed metabolism scheme is shown in Fig. 8.Biotransformation of 9-Fluororisperidone by CYP2D6.

9-Fluororisperidone was metabolized by CYP2D6 to two metabolites(Fig. 9): a major one with a protonated molecular ion of m/z 445.2044consistent with the addition of an oxygen atom (peak 2) and a minor oneatm/z 427.2142 indicating the replacement of a fluorine with a hydroxyl(peak 1). The exact site of hydroxylation in peak 2 cannot be determinedfrom MS data alone; however, the fragmentation pattern is consistentwith the hydroxyl being introduced to one of the three aliphatic carbons

Fig. 6. HPLC-UV traces for CYP3A4 and 3A519-fluoromidazolam incubation extracts andproposed metabolic pathways. Peak P refersto unchanged parent. Peak 1 matched 19-hydroxymidazolam. High resolution mass spec-tral data for these metabolites are included inSupplemental Figs. 9–16.

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on the terahydropyridopyrimidone portion. The other metabolite was amatch with 9-hydroxyrisperidone. The proposed metabolic scheme for9-fluororisperidone is shown in Fig. 9.

Discussion

Fluorine is an important atom in drug design, and in this report wehave described a facile method to introduce fluorine into drugmolecules at sites that are prone to hydroxylation by human P450enzymes. The quantities of fluorinated products generated are small(nanomoles); however, the use of quantitative NMR spectroscopyreduces the material required to verify the structure and permits aconcentrated stock solution to be obtained that can be used for in vitrobiologic testing and drug metabolism experiments. Four drugs wellknown to undergo metabolism by different human P450 enzymes wereselected to demonstrate this method, and the impact of fluorinesubstitution was evaluated in follow up metabolic lability and metaboliteidentification experiments.For the four drugs tested, fluorination had different impacts on

metabolic lability. 9-Fluororisperidone and 49 -fluorocelecoxib wereconsiderably more stable in CYP2D6 and CYP2C9 incubations, respec-tively, than their nonfluorinated counterparts, whereas 2-fluororamelteonwas metabolized at a comparable rate as ramelteon by CYP1A2. It is

interesting to note that for risperidone and celecoxib, metabolism byCYP2D6 and CYP2C9 primarily occurs at a single site and when thissite possesses fluorine, the rate of metabolism is reduced. The CYP2D6mediated hydroxylation that did occur for 9-fluororisperidone occurredat a nearby site; also, a small amount of oxidative defluorination ofthe 9-position was observed. The metabolism of 49-fluorocelecoxibby CYP2C9 still occurred at the same carbon as in celecoxib (albeit atone-fourth the rate), and the resulting metabolites lost the fluorine. It ismost likely that one of the remaining two H-atoms on the methyl isabstracted and the hydroxy intermediate that is generated sponta-neously loses fluoride to yield the observed aldehyde. The case of2-fluororamelteon is different in that CYP1A2 catalyzes hydroxylationat multiple sites on ramelteon (Obach and Ryder, 2010) and thusblocking only one of these with a fluorine atom does not yield adecrease in metabolic lability. The CYP1A2-generated metaboliteprofile of 2-fluororamelteon was similar to that of ramelteon itself,albeit metabolism at the 2-position was less. There was a small amountof oxidative defluorination of 2-fluororamelteon. Overall, this suggeststhat this approach for reducing metabolism would work best forcompounds that are metabolized rapidly, but at a single site. Forcompounds already metabolized at multiple sites, fluorine substitutionmay only result in “metabolic switching,” wherein blocking a site ofmetabolism only results in a change to an alternate position but no

Fig. 7. HPLC-UV trace for the CYP1A2 2-fluororamelteon incubation extract and pro-posed metabolic pathways. Peak P refers tounchanged parent. Peak 4 matched 2-hydrox-yramelteon by retention time and mass spectrum.High resolution mass spectral data for thesemetabolites are included in Supplemental Figures17–19.

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decrease in overall rate of metabolism. However, these same effectswere not necessarily noted in human liver microsomes. 49-Fluorocele-coxib was metabolized more rapidly than celecoxib, and the markeddifference between 9-fluororisperidone and risperidone with CYP2D6was substantially lower in liver microsomes. It is probable that otherP450 enzymes contribute to the metabolism of the fluoro analogs,increasing the rate. Preliminary investigation has shown that49-fluorocelecoxib is metabolized by CYP2D6 and CYP2C19,whereas fluororisperidone is metabolized by CYP3A4 by N-dealkylationat the piperidine (unpublished observations). Ramelteon is metab-olized by CYP1A2, 2C19, and 3A4, and it is possible that thefluorinated analog is a better substrate for CYP2C19 and/or 3A4.Thus it cannot be assumed that fluorination will always result in anoverall decrease in metabolic lability.The case of 19-fluoromidazolam was interesting in that fluorination

yielded a different impact on CYP3A4- versus 3A5-mediated metab-olism. The rate by the former was largely unaffected, but for the latterthere was a 3-fold decrease. The sites of metabolism for both enzymes(i.e., 19- and 4-positions) appeared to be the same, and similar tothose for midazolam itself. Like the other fluoro analogs, there wassome oxidative defluorination observed for 19-fluoromidazolam. Also,

19-fluoromidazolam was shown to lose the 19-CH2F group entirely andthis may occur via an initial oxidative defluorination and furtheroxidation at this position that can result in overall C–C bond cleavage.We have proposed that this occurs via the carboxy metabolite (which isnot formed in the absence of fluorine at that position), but more in-depthwork would be needed to support or refute this proposal.The procedure described can yield fluorinated analogs of drugs

within a couple of days, which will almost always be faster thanredesigning a synthetic scheme for preparing fluorinated analogs of alead compound. Also, the method has the potential to yield multiplyfluorinated analogs if the P450-generated metabolite is either multiplyhydroxylated and/or a ketone/aldehyde, and this has been observed forother compounds to which this method has applied (data on file).However, there are practical limitations to the approach, notwithstandingthe very low quantities generated and the need for relatively expensivecryomicroprobe NMR instrumentation. Hydroxy compounds generatedenzymatically need to be able to withstand the DAST reaction, whichcan cause dehydration of some alcohols instead of deoxyfluorination.Also, very importantly, DAST works best on aliphatic alcohols,especially benzylic alcohols like three of the four examples used inthis paper, and it does not deoxyfluorinate phenols. Future efforts are

Fig. 8. HPLC-UV trace for the CYP2C949-fluorocelecoxib incubation extract andproposed metabolic pathways. Peak P refers tounchanged parent and the peak marked Xdesignates nondrug related material. Peak 1matched 49-hydroxycelecoxib by retentiontime and mass spectrum. High resolutionmass spectral data for these metabolites areincluded in Supplemental Figures 20–22.

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aimed toward expanding the range of metabolites that can be convertedto their fluoro substituted analogs, exploring other fluorination reagentsfor this purpose (Al-Maharik and O’Hagan, 2011; Tang, et al., 2011;Fujimoto and Ritter, 2015; Nielsen, et al., 2015), investigating theutility of other sources of P450 activity (e.g., liver microsomes) for theenzymatic portion of the procedure, and working toward generatingmultiply fluorinated analogs from secondary metabolites possessingmore than one hydroxyl group. Furthermore, the methodology de-scribed in the paper has utility in diversification of lead molecules inorder to impact other properties critical in drug discovery, such aspotency, selectivity, DDI, and distribution properties. Application ofthese methods to a contemporary medicinal chemistry program isunderway and will be reported in due course.

Acknowledgments

The authors acknowledge Raman Sharma for NMR analysis of otherunpublished fluorination products made previously using this approach.

Authorship ContributionsParticipated in research design: Obach, Walker, Brodney.Conducted experiments: Obach, Walker.Performed data analysis: Obach, Walker.

Wrote or contributed to the writing of the manuscript: Obach, Walker,Brodney.

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Address correspondence to: Dr. R. Scott Obach, Pfizer Inc., Eastern Point Road,Groton, CT 06340. r.scott.obach@pfizer.com

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