`Vol. 255, No. 12, Issue of June 25, pp. 557~-5585, 1980
`Printed '" U.S. A.
`
`Self-catalyzed Inactivation of Hepatic Cytochrome P-450 by Ethynyl
`Substrates*
`
`(Received for publication, November 26, !979, and in revised form, February 20, 1980)
`
`Paul R. Ortiz de Montellano* and Kent L. Kunze
`From the Department of Pharmaceutical Chemistry, School of Pharmacy, and Liver Center. University of California, San
`Francisco, California 94143
`
`The following acetylenic substrates have been shown
`to mediate NADPH-dependent loss of cytochrome P-
`450 on incubation with hepatic microsomes from phe(cid:173)
`nobarbital-pretreated rats: 1-ethynylcyclohexanol, 1-
`ethynylcyclopentanol, 3-methyl-1-pentyn-3-ol, noreth(cid:173)
`isterone,
`(1-methoxycyclohexyl)acetylene, 3-(2,4-di(cid:173)
`chlorophenoxy)-1-propyne, 3-( 4-nitrophenoxy)-1-pro(cid:173)
`pyne, 3-phenoxy-1-propyne, 4-phenyl-1-butyne, 3-
`phenyl-1-propyne, cyclohexylacetylene, acetylene, 3-
`pentyn-2-ol, 4-methyl-2-octyn-4-ol, 2-hexyne, phenyla(cid:173)
`cetylene, and N-(1,1-dimethylpropynyl)-3,5-dichloro(cid:173)
`benzamide. A 10-fold higher nominal concentration of
`the last two agents was required to obtain the same
`degree of enzyme loss observed with the other agents
`at a 1 mM concentration. In vivo administration of
`acetylene gas and nine of the monosubstituted acety(cid:173)
`lenes led to accumulation of abnormal hepatic pig(cid:173)
`ments. Similar pigments were not observed in rats
`treated with disubstituted acetylenes. The pigments
`obtained with acetylene gas, norethisterone, and six
`other substrates, after isolation, methylation, and pu(cid:173)
`rification, exhibited essentially identical electronic ab(cid:173)
`sorption spectra. Field desorption mass spectrometric
`analysis of these eight pigments has established that
`each one gives a molecular ion which corresponds to
`the sum of the molecular weights of the dimethyl ester
`of protoporphyrin IX plus the substrate plus (probably)
`an oxygen atom. These results are used to formulate a
`destructive mechanism in which the enzyme prosthetic
`heme covalently binds to the substrate during at(cid:173)
`tempted metabolism of the triple bond.
`
`Cytochrome P-450 1 monooxygenases, a diverse group of
`enzymes related by similarities in their catalytic function and
`by the presence of a heme prosthetic group, dominate the
`hepatic metabolism of xenobiotic lipophilic substances (1-3).
`Each of these enzymes catalyzes reductive cleavage of molec(cid:173)
`ular oxygen with concomitant transfer of 1 of the oxygen
`
`• This work has been supported by National Institutes of Health
`Grants GM-25515 and P-50 AM-18520. The Berkeley Biomedical and
`Environmental Mass Spectrometry Resource, where mass spectra
`were obtained, is supported by National Institutes of Health Grant
`RR 00719. The Computer Graphics Laboratory, used in preparation
`of Scheme 2, is supported by National Institutes of Health Grant RR
`1081-021. The costs of publication of this article were defrayed in part
`by the payment of page charges. This article must therefore be hereby
`marked "advertisement" in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`:j: A Research Fellow of the Alfred P. Sloan Foundation.
`1 Cytochrome P-450 generally refers in this paper to the isozyrnes
`induced by phenobarbital, but occasionally is used in a generic sense
`to denote the class of hernoproteins whose reduced carbon monoxide
`spectra exhibit a maxima at around 450 nm.
`
`atoms to the substrate (1-3), although the observable conse(cid:173)
`quences of oxygen transfer depend on the precise nature of
`the substrate. In general, the established immediate outcome
`of cytochrome P-450-catalyzed oxygen transfer is (a) forma(cid:173)
`tion of a hydroxyl derivative by insertion into the bond
`between a hydrogen and a heavier atom; (b) formation of an
`epoxide by addition across a carbon-carbon double bond; or
`(c) formation of a dipolar oxide by combination with the free
`electron pair of a heteroatom (4, 5). These primary reactions,
`however, often yield unstable structures which undergo sec(cid:173)
`ondary chemical transformations to give final experimentally
`observed products (4, 5). The rearrangement of enzymatically
`formed aryl epoxides to phenolic derivatives is an example of
`such a secondary process (6).
`Although cytochrome P-450 enzymes involved in physiolog(cid:173)
`ical biosynthetic pathways usually exhibit high substrate spec(cid:173)
`ificity, those forms of the enzyme significantly engaged in
`xenobiotic metabolism are characterized by broad and over(cid:173)
`lapping substrate selectivities (7-10). The effectiveness of this
`metabolic apparatus, which copes with the wide range of
`substances to which an individual is environmentally exposed,
`is optimized through differential induction of cytochrome P-
`450 isozymes by lipophilic substrates (10, 11). However, the
`broad specificity and differential inducibility of these mono(cid:173)
`oxygenase enzymes render alterations in metabolism of one
`agent by another a relatively common phenomenon.
`Inhibition of the metabolism of one substance by another
`in order to enhance pharmacological activity has been com(cid:173)
`mercially exploited with the advent of insecticide synergists
`(12, 13). An impressive fraction of the substances shown to
`potentiate the action of insecticides is characterized by the
`presence of a monosubstituted acetylenic function. Among
`these active agents are aryl propargyl ethers (14-16), N-al(cid:173)
`kynyl phthalimides (17), alkynyl phosphate esters (12, 18),
`and alkynyl oxime ethers (12, 19). The mechanism by which
`these acetylenic substances alter insecticide metabolism and
`toxicity, however, has remained undefined except for the
`observation that in vivo administration of 3-(2,4,5-trichloro(cid:173)
`phenoxy)-1-propyne and 2-methylpropyl 2-propynyl phenyl(cid:173)
`phosphonate to mice causes a measurable decrease in hepatic
`cytochrome P-450 content (18).
`Research in a different biological arena has also provided
`evidence for altered drug metabolism due, in part, to the
`interaction of acetylenic groups with cytochrome P-450 en(cid:173)
`zymes. Synthetic 17 -ethynyl sterols, hormonally active con(cid:173)
`stituents of most birth control pills (20), have been found to
`inhibit oxidative drug metabolism (21-25). White and Miiller(cid:173)
`Eberhard (26) have reported that, at relatively high doses,
`ethynyl sterols specifically reduce hepatic cytochrome P-450
`and heme levels in rats. Norethisterone and ethynylestradiol,
`the two ethynylsterols examined, selectively affected the phe(cid:173)
`nobarbital-inducible isozymes (27) of cytochrome P-450. The
`
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`additional and highly significant observation was made that
`cytochrome P-450 loss was accompanied by accumulation of
`a hepatic "green pigment" not unlike that found by other
`investigators in rats treated with 2-isopropyl-4-pentenamide
`(28-30).
`Work in this laboratory has established that 2-isopropyl-4-
`pentenamide inactivates cytochrome P-450 by covalently
`binding to the heme prosthetic group during catalytic inter(cid:173)
`action with the enzyme (31-33). In a recent brief communi(cid:173)
`cation, we have also reported that radiolabeled norethisterone
`is covalently incorporated into the "green pigment" produced
`by treatment of rats with this sterol (34). Covalent attachment
`of the sterol to prosthetic heme, suggested by the radioisotopic
`data, was confirmed by the mass spectrometric molecular ion
`of the purified pigment, although the observed molecular ion
`could not be attributed to a specific molecular species (34).
`We now report an in-depth study of the interaction of acety(cid:173)
`lenes with cytochrome P-450 which demonstrates the gener(cid:173)
`ality of the process, unequivocally establishes the formation
`of substrate-heme adducts, and provides the basis for formu(cid:173)
`lation of a tentative reaction mechanism. Aspects of this study
`have been presented at several meetings (35-37).
`
`EXPERIMENTAL PROCEDURES
`Substrates and Reagents-The following substances, of the high(cid:173)
`est grade available, were obtained from the indicated commercial
`source and were used without further purification: 1-ethynylcyclo(cid:173)
`hexanol (A), 1-ethynylcyclopentanol (B), 3-methyl-1-pentyn-3-ol (C),
`3-pentyn-2-ol (0), 4-methyl-2-octyn-4-ol (P), 4-phenyl-1-butyne (1),
`and 2-hexyne (Q) (Farchan Division, Story Chemical Corp.); cyclo(cid:173)
`hexylacetylene (L) (Pfaltz-Bauer); phenylacetylene (K) (Aldrich
`Chemical Co.); and acetylene (M) (Matheson Chemical Co.). Litera(cid:173)
`ture procedures were used for the synthesis of (1-methoxycyclo(cid:173)
`hexyl)acetylene (E) (38), 3-phenyl-1-propyne (J) (39), 3-phenoxy-1-
`propyne (H) (14), 3-(2,4-dichlorophenoxy)-1-propyne (F) (14), and 3-
`(4-nitrophenoxy)-1-propyne (G) (14). Norethisterone (17-hydroxy-19-
`nor-17-pregn-4-en-20-yn-3-one, D) was generously provided by Syn(cid:173)
`tex Research, Palo Alto, Calif., and N-(1,1-dimethylpropynyl)-3,5-
`dichlorobenzamide (N, code No. RH-315) by Rohm and Haas Co.,
`Philadelphia, Pa. Solvents used for chromatographic purification of
`hepatic pigments were analytical grade and were distilled prior to use.
`In Vitro Assay of Cytochrome P-450 Inactivation-The general
`procedure used, based on that of the Hoffmann-La Roche group (40),
`has been described (33). Incubation mixtures contained, in addition
`to substrates, the following: microsomal protein (1 mg/ml), NADPH
`(1 mM), KCl (150 mM), and EDTA (1.5 mM), all in 0.1 M phosphate
`buffer (pH 7.4). Acetylenic substrates, added without solvent at a
`nominal 1 mM concentration, were preincubated with the microsomal
`suspension for 10 min to allow substrate equilibration before NADPH
`was added to initiate the reaction. Nominal 10 mM concentrations of
`phenylacetylene and N -( 1,1-dimethylpropynyl )-3,5-dichlorobenza(cid:173)
`mide were used since these agents caused only a marginal Joss of
`cytochrome P-450 at a 1 mM concentration. In all cases, control
`incubations were carried out in the absence of added substrates and,
`for each substrate, in the absence of NADPH. The loss of cytochrome
`P-450 in the absence of substrates was negligible, demonstrating that
`lipid peroxidation was effectively suppressed by the added EDT A.
`Isolation of Abnormal Hepatic Pigments-Sprague-Dawley male
`rats weighing 200 to 250 g were injected intra peritoneally once a day·
`for 4 days with an aqueous solution (80 mg/ml) of sodium phenobar(cid:173)
`bital (80 mg/kg dose). Acetylenic substrates were administered on the
`5th day. Norethisterone (25 mg/kg) was injected intraperitoneally in
`0.25 ml of trioctanoin after sonication of the mixture to disperse the
`sterol. All other solid agents (200 mg/kg) were injected intraperito(cid:173)
`neally as a solution in 150 11! of ethanol. Liquid agents (50 !'I! rat) were
`injected intraperitoneally without dilution in a solvent. Acetylene gas
`was administered by placing phenobarbital-pretreated rats for 4 h in
`a chamber through which a stream of air containing 10 to 15% (v/v)
`acetylene was passed at an approximate rate of 200 to 300 ml!min.
`Four hours after administration of acetylenic agents, rats were decap(cid:173)
`itated and their livers were perfused in situ with ice-cold 0.9% saline
`solution (100 rnl/liver). The pooled livers of rats treated with a
`common agent were homogenized in 5% (v/v) H"SO,/methanol (4
`ml/g of liver) and the homogenate was allowed to stand overnight at
`
`5579
`
`Inactivation of Cytochrome P-450 by Acetylenes
`4 ac in the dark. All manipulations were carried out in the dark since
`the hepatic pigments are photosensitive. The homogenate was ftltered
`to remove precipitated protein and equivalent volumes of water and
`chloroform were added. The brown chloroform layer was separated,
`washed with water until the washes were no longer acidic, and dried
`over anhydrous Na2SO,. After ftltration, 2 ml of a 0.5% solution of
`zinc acetate in chloroform was added before solvent removal at a
`rotary evaporator. The preparation of zinc complexes at this stage
`prevents the formation of artifactual metal complexes during purifi(cid:173)
`cation (32, 34). The oily brown residue obtained on solvent removal
`was chromatographed on preparative Silica Gel GF plates (Analtech,
`Inc.) using 3:1 chloroform/acetone as solvent. The zinc-complexed
`porphyrin pigments appear as visibly green, red-fluorescing bands
`with RF values of 0.5 to 0.8. One or two such bands were observed,
`depending on the substrate (see text). The pigment-containing frac(cid:173)
`tions were scraped from the plate and the pigment was extracted with
`acetone. The isolated zinc-complexed porphyrins were further puri(cid:173)
`fied by high pressure liquid chromatography on a Whatman Partisil
`10-PAC column using methanol/tetrahydrofuran 4:1 (v/v) as solvent
`(2 to 3 ml/min of flow rate). The column effluent was monitored with
`a variable wavelength detector set at 431 nm. After removal of an
`aliquot of the collected pigments for spectroscopic analysis, the puri(cid:173)
`fied zinc complexes were demetalated by standing in I ml of 5%
`H 2SO,/methanol for 15 min. Addition of equal volumes of water and
`chloroform, separation of the layers, washing of the chloroform frac(cid:173)
`tion with water until the washes were no longer acidic, drying over
`anhydrous sodium sulfate, and solvent removal yielded pure, metal(cid:173)
`free porphyrin pigments. The metal-free pigments were analyzed by
`high pressure liquid chromatography as previously described (31-34).
`In some instances, metal-free porphyrin pigments were reconverted
`into zinc complexes by the procedure given above.
`Spectroscopic Analysis of Purified Pigments-The electronic
`(UV -visible) absorption spectra of both the metal-free and zinc-com(cid:173)
`plexed pigments were determined in dilute chloroform solution on a
`Varian-Cary 118 spectrophotometer.
`A Kratos/ AEI MS-902 instrument with a field desorption source
`was utilized for the mass spectrometric studies. Operating conditions
`were the same as those previously reported (33). Except for noreth(cid:173)
`isterone, a mass spectrum was obtained for each isolated porphyrin
`both in its free-base and zinc-complexed form. Due to the already
`high molecular weight of the norethisterone-porphyrin adduct, it was
`only possible to obtain a mass spectrum for the metal-free form. In
`those instances where pigments were resolved into two components,
`electronic absorption and mass spectrometric data were independ(cid:173)
`ently obtained for each of the two components.
`In Vitro Pigment Formation-The formation of abnormal pig(cid:173)
`ments in vitro was established in a larger scale incubation of noreth(cid:173)
`isterone with the usual microsomal enzyme preparation. The incu(cid:173)
`bation mixture already described was used except for the following
`alterations in concentration: norethisterone (1 mM), NADPH (4 mM),
`and microsomal protein (4 mg/ml). The total incubation volume was
`40 rnl and the incubation time 40 min. At the end of the incubation,
`the microsomal protein was sedimented by ultracentrifugation
`(100,000 X g for I h). The microsomal pellet was added to 5 ml of 5%
`H 2SO,/methanol and was allowed to stand overnight. The pigment
`was extracted as already described. The electronic absorption spectra
`of the purified pigment, both as the free-base and as a zinc complex,
`were identical with those of the pigment obtained from in vivo
`experiments.
`
`RESULTS
`Sixteen acetylenic substrates have been incubated in this
`study with hepatic microsomes from phenobarbital-pretreated
`rats. A time-dependent decrease in measurable cytochrome
`P-450 content was caused by each of these substrates, in the
`presence of NADPH, under incubation conditions in which
`lipid peroxidation was suppressed (Fig. 1). No cytochrome P-
`450 was lost in the absence of added NADPH (data not
`shown). Although a quantitative structure-activity correlation
`is not possible due to the heterogeneous nature of the assay
`system and the differential solubility of the substrates, the
`data clearly show that all of the structures investigated exhibit
`the same order of magnitude activity except for phenylacety(cid:173)
`lene and N- ( 1,1-dimethyl-2-propynyl) -3,5-dichlorobenzamide.
`A 10-fold increase in concentration of these two substances
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`5580
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`Inactivation of Cytochrome P-450 by Acetylenes
`
`SUBSTRATE
`
`~
`
`PERCENT LOSS OF
`IN VIVO
`CYTOCHROME P-4&1
`30' PIGMENT
`10'
`20'
`J OJ 12 :t 6 24±7 34±1
`K Q-s
`L ex: 19±2 23±4 23±4
`
`YES
`
`18±3 2!5±2 27±2
`
`YES
`
`-
`
`PERCENT LOSS OF
`CY'J'OCtR)ME P-450
`IN VNO
`30' PIGMENT
`10 1
`20'
`
`SUBSTRATE
`
`A ex: 18±6 22±6 26±6
`
`YES
`
`B O<OH
`
`~
`
`18± 3 22±4 29±3
`
`YES
`
`~
`
`17
`
`24
`
`34
`
`-
`
`c ~OH
`D ~~ 20±5 25±2 27±3
`0 VOMe
`F 6'
`
`E
`
`22±5 29±6 30±6
`
`~
`
`c1
`
`o-"'~ 21±6 23±4 24±3
`
`G o20o.,..,....._~ 19± 10 23±11 30±11
`
`H
`
`Oo/"'..~ 25±3 31±4 34±2
`
`I ~~ 29±3 49±4 52±3
`
`YES
`
`M
`
`HCE CH
`
`24
`
`33
`
`40
`
`o
`Ci
`N ~NHX~ 19±5 30±2 32±3
`Cl
`OH
`A~ ...
`
`24±17 33±10 3!5±8
`
`0
`
`p ~~/
`HO
`
`3!5±4 4!5±3 47±1
`
`Q ~=-
`
`27:t3 34±5 36:t5
`
`-
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`-
`
`TOXIC
`
`NO
`
`NO
`
`Fw. L Destruction of cytochrome P-450 on incubation of
`acetylenic substrates with hepatic microsomes from pheno(cid:173)
`barbital-pretreated rats. Incubations were carried out as described
`under "Experimental Procedures." The data for norethisterone (D)
`are from Ref. 34 while the values for acetylene gas (M) are estimated
`from Ref. 41. The substrate concentration was nominally 1 mM except
`for phenylacetylene (K) and N-(1,1-dimethyl-2-propynyl)-3,5-dichlo(cid:173)
`robenzamide (N), which were examined at a 10 mM concentration
`since they caused only marginal cytochrome P-450 loss at the lower
`
`dose. The values given are averages of at least three determinations,
`with the exception that 3-methyl-1-pentyn-3-ol (C) was only assayed
`once. Standard deviations are given where applicable. Accumulation
`in vivo of an abnormal hepatic pigment in rats treated with the given
`agents (see "Experimental Procedures"') is indicated by a yes in the
`column marked «pigment." A no in this column indicates that pigment
`was looked for but was not found; a dash indicates that the experiment
`was not performed. Incubation time is given in minutes.
`
`was required to produce cytochrome P-450 losses equivalent
`to those observed with the other agents. Analogous data for
`acetylene gas, extrapolated from the graph reported by White
`(41), are included in the table for comparison.
`Ofthe 17 active agents in Fig. 1, 13 were selected for in vivo
`investigation. The livers of phenobarbital-pretreated rats were
`examined 4 h after administration of each of these 13 agents
`for the presence of abnormal ("green") pigments. Using the
`extraction and purification methods described under "Exper(cid:173)
`imental Procedures," characteristic abnormal pigments were
`found in rats treated with each of the 10 substrates bearing a
`terminal acetylenic function (Fig. 1). In contrast, no abnormal
`pigments were isolated by the standard procedure from rats
`treated with the three agents in which the acetylenic moiety
`was disubstituted, even though these agents caused in vitro
`loss of cytochrome P-450. In one of these three cases, that of
`3-pentyn-2-ol, this failure was due to the fact that rats receiv(cid:173)
`ing the usual 200 mg/kg dose of the agent died shortly after
`drug administration. It may be that this exceptional toxicity
`i'! related to the unique presence in this substrate of an
`oxidizable hydroxyl function adjacent to the acetylenic group,
`a pairing of functionalities which may lead to formation of a
`reactive carbonyl-conjugated acetylene. Large scale incuba(cid:173)
`tion of norethisterone with a microsomal enzyme preparation
`resulted in isolation of an abnormal pigment identical in its
`chromatographic and spectroscopic properties with that ob(cid:173)
`tained from in vivo administration of the drug.
`Abnormal hepatic pigments were analyzed by thin layer
`and high pressure liquid chromatography. The free-base form
`
`of 3 of the 10 pigments was resolved into two components by
`both of these techniques. The free-bases of the other seven
`pigments chromatographed as single bands, although the pres(cid:173)
`ence of unresolved isomers within these bands is suggested by
`frequently observed asymmetry in the high pressure liquid
`chromatography peaks. High pressure liquid chromatographic
`analysis of the zinc complexes was also performed, although
`these were not resolved into more than one peak even in those
`instances where two components were known to be present
`from the free-base chromatographic data. The high pressure
`liquid chromatographic analysis of a mixture of metal-free
`and zinc-complexed forms of the 3-phenoxy-1-propyne pig(cid:173)
`ment, a typical example, is given in Fig. 2. The three pigments
`which could be resolved into two components were those
`engendered by norethisterone (34), 1-ethynylcyclohexanol,
`and 1-ethynylcyclopentanol. These three substrates were the
`only ones with a tertiary hydroxyl group adjacent to a terminal
`ethynyl function. As already reported for norethisterone (34),
`the two components of each of the three pigments were not
`interconverted by complexation with zinc acetate and subse(cid:173)
`quent demetalation in 5% HzSO./methanol. Only the single
`component used as a starting material was obtained after thi'l
`reaction cycle. Nevertheless, even though not easily intercon(cid:173)
`vertible, the evidence to be outlined below strongly suggests
`that the two components of each of these pigments are in fact
`closely related isomeric structures.
`The electronic absorption spectrum of each of the purified
`pigments, and of each of the two components of a pigment
`when these were resolved, has been determined both in the
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`Inactivation of Cytochrome P-450 by Acetylenes
`
`5581
`
`0
`
`15
`
`10
`Time (min)
`FIG. 2. High pressure liquid chromatographic analysis of a
`mixture of the metal-free and zinc-eomplexed pigment isolated
`from rats treated with 3-phenoxy-1-propyne. A mixture of the
`metal-free and zinc-complexed pigment, obtained by addition of zinc
`acetate to pigment isolated as described under "Experimental Pro-
`was chromatographed on a Whatman Partisil 10-PAC col(cid:173)
`first solvent, hexane:tetrahydrofuran:methanol, 10:3:1, was
`umn.
`replaced at the point shown by an arrow with methanol:tetrahydro(cid:173)
`furan, 4:1. A 3 ml/min solvent flow rate was used. The detector was
`at 417 nm. The first peak is due to uncomplexed product and the
`second is due to the zinc complex.
`
`metal-free and zinc-complexed state. Within experimental
`tolerance (approximately ± 2 nm), the spectra of all of the
`uncomplexed pigments were essentially identical with two
`exceptions. The free-base of the pigment obtained with acet(cid:173)
`ylene gas exhibited a Soret band at 414 nm rather than at 420
`nm as in the spectra of the other pigments. This can be seen
`by comparing (Fig. 3) the spectrum of the 3-phenyl-1-propyne
`pigment, a ty}lical case, with that of the acetylene pigment.
`The second exception is provided by the phenylacetylene
`pigment, for which a reproducible free-base spectrum has not
`been obtained due to chemical instability of the chromophore.
`The spectra of the zinc complexes of the pigments were also
`essentially identical, in this case with no exceptions (Fig. 3).
`The zinc complex of the pigment obtained with phenylacety(cid:173)
`lene, in contrast to the free-base form, exhibited a stable
`spectrum which could not be differentiated from those of the
`other zinc-complexed pigments. Correlation of the spectra of
`both the metal-free and zinc-complexed forms of the pigments
`with the corresponding spectra of authentic porphyrins (42)
`leaves no doubt that the basic chromophore in the pigments
`is that of a porphyrin ring.
`Field desorption mass spectra were obtained for the free
`base of eight of the isolated pigments, including separate
`spectra for each of the two resolved components in the pig(cid:173)
`ments obtained with 1-ethynylcyclohexanol and 1-ethynylcy(cid:173)
`clopentanol. A mass spectrum was also obtained of the zinc
`complex of each of the pigments (Table I). The mass spectrum
`of the pigment produced by treatment with acetylene gas is
`reproduced in Fig. 4. In general, these spectra are character(cid:173)
`ized by the presence of a variable ratio molecular ion doublet
`and by a minimal amount of peaks due to molecular fragmen(cid:173)
`tation. The molecular ion doublet is due to desorption of
`protonated and unprotonated molecular species, the mono(cid:173)
`protonated form being favored by higher emitter currents in
`the mass spectrometer. Protonated and alkali metal-com(cid:173)
`plexed molecular ions are frequently observed in field desorp(cid:173)
`tion mass spectrometry (43). A sodium-complexed molecular
`ion was in fact observed for the norethisterone pigment (34).
`An instructive exception to the observation of a molecular ion
`doublet was provided by the 3-(2,4-dichlorophenoxy)-l-pro(cid:173)
`pyne pigment, which exhibited the molecular ion duster ex-
`
`600
`550
`500
`Wavelength (nm)
`FIG. 3. Electronic absorption spectra. Electronic (ultraviolet(cid:173)
`visible) absorption spectra in chloroform of (a) the free base of the
`pigment obtained with 3-phenyl-1-propyne (--); (b) the free base
`of the pigment obtained with acetylene gas(---); and (c) the zinc
`complex of the 3-phenyl-1-propyne pigment(·-'-·).
`
`TABLE I
`Analysis of isolated hepatic pigments by field desorption mass
`spectrometry
`The observed molecular ion for each purified pigment, after sub(cid:173)
`traction of the mass unit(s) due to the complexed proton or sodium
`in the table. These data are
`cation (shown in parentheses), is
`given both for the free-base and
`the zinc complex obtained on
`addition of zinc acetate. The principal peak in the zinc complex
`molecular ion
`corresponding to complexation with the major
`list.Bd.
`
`A (124)
`B (110)
`D (298)
`F (200)
`H (132)
`I
`(130)
`J (116)
`M (26)
`
`730
`716
`904
`806
`738
`736
`722
`632
`
`730 (H+)
`716(Wl
`886 (Na+)
`806(H')
`738(W)
`736(.1:1')
`722 (W)
`632(H')
`
`792(Wl
`778{W)
`
`868
`800
`798
`784
`694
`
`I~'
`
`450
`
`500
`
`600
`
`650
`
`550
`m;e
`FIG. 4. Field desorption mass spectrum of the hepatic pig(cid:173)
`ment isolated from phenobarbital-pretreated rats exposed to
`acetylene gas. Administration of the agent and isolation of the
`pigment are described under "Experimental Procedures." The prin(cid:173)
`cipal peak observed in the spectrum (m/e 633) corresponds to mon(cid:173)
`oprotonated molecular ion.
`
`Boehringer Ex. 2015
`Mylan v. Boehringer Ingelheim
`IPR2016-01564
`Page 4
`
`
`
`5582
`
`Inactivation of Cytochrome P-450 by Acetylenes
`
`pected of a mixture of protonated and unprotonated ions of a
`substance containing 2 chlorine atoms (44). The natural abun(cid:173)
`dance ratio of isotopes (45) was also reflected in the molecular
`ion patterns of the zinc complexes. Again, the zinc complex of
`the 3-(2,4-dichlorophenoxy)-1-propyne pigment gave an unu(cid:173)
`sually complex molecular ion pattern, as expected for a struc(cid:173)
`ture containing 1 zinc and 2 chlorine atoms. The molecular
`ion values tabulated in Table I correspond to the unproton(cid:173)
`ated molecular species. The values given for the zinc com(cid:173)
`plexes and for the chlorine-containing compound are those of
`the major peak in the molecular ion clusters and, thus, cor(cid:173)
`5Cl. In those cases where
`respond to the presence of 64Zn and :l
`a pigment was resolved into two components, both compo(cid:173)
`nents gave the same mass spectrometric data so that the value
`in Table I is valid for both components.
`
`DISCUSSION
`The importance of the acetylenic group in norethisterone
`and ethynylestradiol for the destructive interaction of these
`substrates with hepatic cytochrome P-450 was first noted by
`White and Miiller-Eberhard (26), who reported that destruc(cid:173)
`tive activity was lost on replacement of the ethynyl function
`with a hydrogen atom or an ethyl group. Our subsequent
`demonstration that the ethynyl function could also not be
`replaced by a vinyl moiety confirmed the specific role of the
`carbon-carbon triple bond in the destructive action of these
`hormonal steroids (34). The observation that nonsteroidal
`acetylenes also mediated the loss of cytochrome P-450 fur(cid:173)
`thermore demonstrated that the sterol framework was not an
`essential component of the destructive interaction (34, 41).
`The more extensive structure-activity study described here
`unequivocally establishes that the potential to interact de(cid:173)
`structively with cytochrome P-450 is an intrinsic property of
`the acetylenic group itself and that although the interaction
`can be attenuated or suppressed by the framework into which
`the triple bond is incorporated, no other substrate feature is
`essential for cytochrome P-450 destruction. Thus, this mono(cid:173)
`oxygenase system is efficiently destroyed by monosubstituted
`acetylenes in which the attached carbon is not only mono(cid:173)
`and di-, but also trisubstituted, a result which excludes de(cid:173)
`structive mechanisms based on formation of a delocalized
`radical by abstraction of an allylic hydrogen atom. The de(cid:173)
`structive activity of compounds such as 1-ethynylcyclohexane
`and phenylacetylene clearly shows that a vicinal hydroxyl
`function is also not required, although involvement of the
`hydroxyl group in oxidative metabolism of the triple bond in
`ethynyl sterols (46, 47) leaves open the possibility that it may
`intervene when present (34). Nevertheless, the lack of struc(cid:173)
`tural specificity implicit in the observation that all of the
`substrates listed in Fig. 1 destroyed cytochrome P-450 con(cid:173)
`vincingly argues that the nature of the structure bearing the
`acetylenic moiety is not a primary determinant of destructive
`activity, although the decreased effectiveness of phenyla(cid:173)
`cetylene and N-(1,1-dimethyl-2-propynyl)-3,5-dichlorobenza(cid:173)
`mide does show that the surrounding structure can interfere
`with the destructive process. The intrinsic activity of the
`acetylenic moiety is perhaps most strikingly demonstrated by
`the destructive action of acetylene itself (41), whereas the loss
`of cytochrome P-450 caused by the disubstituted acetylenes
`3-pentyn-2-ol, 4-methyl-2-octyn-4-ol, and 2-hexyne shows that
`it is the unsaturated bonds of the acetylenic group and not
`the acidic acetylenic proton which are responsible for the
`destructive interaction with cytochrome P-450, since in these
`substrates the acidic acetylenic proton is absent.
`The destruction of cytochrome P-450 by 2-isopropyl-4-pen(cid:173)
`requires
`tenamide, ethynyl sterols, and acetylene gas
`
`NADPH, oxygen, and catalytically competent enzyme (26, 41,
`48). However, the most characteristic feature of the inactiva(cid:173)
`tion of cytochrome P-450 by these particular agents, the
`feature which uniquely distinguishes their mechanism, is the
`accompanying loss of the enzyme prosthetic heme moiety and
`the accumulation of abnormal hepatic pigments derived from
`that moiety (26, 34, 41, 48). The destruction of cytochrome P-
`450 by monosubstituted acetylenes has been clearly shown by
`this study to be mediated by this mechanism, both by dem(cid:173)
`onstration that in every instance NADPH is required and,
`more definitively, that all of the new monosubstituted sub(cid:173)
`strates examined in vivo yielded hepatic pigments indistin(cid:173)
`guishable by absorption spectroscopy (Fig. 3) from that pre(cid:173)
`viously obtained with norethisterone (34). This identity in
`electronic absorption spectra, true both for the free-base and
`zinc-complexed forms of the pigments, was also valid for the
`two resolved components of the pigments obtained with
`norethisterone, 1-ethynylcyclohexanol, and 1-ethynylcyclo(cid:173)
`pentanol.
`The in vitro destruction of cytochrome P-450 was mediated
`equally well by monosubstituted and disubstituted acetylenes
`(Fig. 1). In contrast, however, abnormal hepatic pigments
`were only observed in rats which had been treated in vivo
`with monosubstituted acetylenes or with acetylene gas. Anal(cid:173)
`ogous hepatic pigments were not found in rats which had been
`treated with d