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Metabolic Activation of a 1,3-Disubstituted Piperazine Derivative: Evidence for a Novel Ring Contraction to an Imidazoline George A. Doss,*,† Randall R. Miller,† Zhoupeng Zhang,† Yohannes Teffera,† Ravi P. Nargund,‡ Brenda Palucki,‡ Min K. Park,‡ Yui S. Tang,† David C. Evans,† Thomas A. Baillie,† and Ralph A. Stearns† Departments of Drug Metabolism and Medicinal Chemistry, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065 Received October 18, 2004
MB243 (a 1,3-disubstituted piperazine) is a new, potent, and selective melanocortin receptor subtype-4 agonist with potential application in the treatment of obesity and/or erectile dysfunction. MB243 was observed to covalently bind extensively to liver microsomal proteins from rats and humans. In the presence of glutathione, two thioether adducts were detected in liver microsomal incubations by radiochromatography and LC/MS/MS analysis. These adducts were also formed when bile duct-cannulated rats were dosed with MB243. The two adducts were isolated, and their structures were determined by accurate mass MS/MS and NMR analyses. The proposed structures resulted from a novel contraction of the piperazine ring to yield a substituted imidazoline. A mechanism is proposed, which involves an initial six electron oxidation of the piperazine ring to form a reactive intermediate, which is trapped by glutathione. Hydrolysis of the glutamic acid residue followed by internal aminolysis by the cysteine amino group resulted in opening of the piperazine ring, which is followed by ring closure to an imidazoline. The resulting cysteinyl-glycine conjugate underwent subsequent hydrolysis of the glycine residue. Understanding of the mechanism of bioactivation led to the design of MB243 analogues that exhibited reduced covalent protein binding.
Introduction The formation of reactive intermediates by metabolism of xenobiotics represents a potential liability in drug discovery and development. Such intermediates can covalently bind to cellular proteins and enzymes, potentially leading to adverse reactions and/or toxicity (1-10). We recently disclosed the discovery of MB243 (Figure 1) as a potent, selective melanocortin receptor subtype-4 (MC4R) agonist for the potential treatment of obesity and/or erectile dysfunction (11). MB243 (a 1,3-disubstituted piperazine) unfortunately exhibited high levels of irreversible binding to liver microsomal proteins. To understand the cause(s) of this high binding, we studied the formation of thioether adducts formed by reaction of GSH or N-acetylcysteine (NAC)1 with possible electrophilic species resulting from the metabolic activation of MB243. We here describe the isolation and structure determination of these thioether adducts and propose a mechanism for the bioactivation of the piperazine ring that led to a novel ring contraction to an imidazoline derivative. On the basis of this mechanism, we were able to design analogues of MB243, which exhibited greatly diminished irreversible protein binding properties. * To whom correspondence should be addressed. Tel: 732-594-7089. Fax: 732-594-6645. E-mail:
[email protected]. † Department of Drug Metabolism. ‡ Department of Medicinal Chemistry. 1 Abbreviations: CID, collision-induced dissociation; Q-TOF, quadrupole time-of-flight; MS/MS, tandem mass spectrometry; NAC, N-acetylcysteine; TOCSY, total correlation NMR spectroscopy; HMQC, multiple quantum correlation NMR spectroscopy.
Figure 1. Structure of MB243 and its thioether metabolite adducts M1 and M2. The asterisk denotes the position of the tritium label.
Materials and Methods Chemicals and Reagents. The synthesis of MB243 has recently been described (11). [3H]MB243 was obtained by catalytic tritiation of the corresponding iodo precursor. The specific activity of the labeled derivative was 10 µCi/mg, and its radiochemical purity was better than 99.5%. All other chemicals were purchased from Sigma-Aldrich (Milwaukee, WI) unless otherwise specified. In Vitro Covalent Binding. Liver microsomes were prepared from male Sprague-Dawley rats and human subjects (pool of five) using literature methods (12), and protein was determined by the Lowry method (13). Radiolabeled compounds (10 µM) were incubated with rat and human liver microsomes (1 mL, 1 mg/mL protein) in phosphate buffer (0.1 M, pH 7.4,
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containing 1 mM EDTA). Incubations were carried out in the absence of NADPH as a negative control or in the presence of NADPH (1 mg/mL) with and without addition of GSH (5 mM). After 30 min at 37 °C, the proteins were precipitated by the addition of acetonitrile. The protein pellet was resolubilized in water and precipitated with ethanol at -20 °C. This procedure was repeated until no additional radioactivity could be extracted from the protein pellet (typically six iterations). The pellet then was solubilized by vortexing in 1 mL of 0.1 N NaOH until completely dissolved. An aliquot of the alkaline protein solution was neutralized with an equal volume of 0.1 N HCl, mixed with scintillation fluid cocktail (Scintisafe gel, Fisher Scientific), and counted for total radioactivity. Another aliquot was used to quantify the protein concentration using the BCA method (Pierce Biotechnology). Irreversible binding to microsomal proteins is expressed as pmol equiv/mg protein. Metabolites in Rat Bile. Three male Sprague-Dawley rats were anesthetized with pentobarbital, and their bile ducts were cannulated with PE-10 tubing. Control samples were collected 0-24 h after the surgery (15 min before treatment). Each rat was dosed with [3H]MB243 at 20 mg/kg by oral gavage using a 10 mg/mL aqueous solution. Bile was collected continuously on ice at 0-2, 2-4, 4-6, 6-8, and 8-24 h postdosing. An aliquot of the pooled bile was analyzed immediately and at different time intervals to assess the stability of the metabolites. The rest of the bile was kept frozen at -70 °C until further analysis. The care and use of animals was conducted according to all applicable regulations and guidelines, and the procedures were reviewed and approved by an Institutional Animal Care and Use Committee. LC/Tandem MS (MS/MS) Analysis. LC/MS/MS was carried out on a Perkin-Elmer SCIEX API 3000 tandem mass spectrometer (Toronto, Canada) interfaced to an HPLC system consisting of a Perkin-Elmer Series 200 quaternary pump and a Series 200 autosampler (Norwalk, CT). A heated nebulizer interface with positive ion detection was used. The source temperature was set at 200 °C, the ion spray voltage was set at 5 kV, the focusing potential was set at 310 V, and the entrance potential was set at -10 V. Nitrogen was used as a collision gas at a nominal setting of 4. For metabolite identification, pooled rat bile (15 µL) was loaded onto a MetaChem Polaris C18-A column (Torrance, CA, 4.6 mm × 250 mm, 3 µm). The flow rate was set at 1.0 mL/min with a 1:25 split to the ion source and a Packard Flow Scintillation Analyzer, respectively. The mobile phase consisted of solvent A (2 mM ammonium acetate in water-TFA, 100:0.1) and solvent B (2 mM ammonium acetate in acetonitrile-waterTFA, 90:10:0.1). The HPLC runs were programmed by a linear increase from 20 to 50% of solvent B in the first 30 min and from 50 to 90% of B in the next 10 min. The MS/MS spectra were recorded by collision-induced dissociation (CID) of MH+ species. Isolation of Adducts from Rat Bile. Pooled rat bile (P. O., 0-2 h, 1 mL) was mixed with 1 mL of aqueous solvent A (2 mM ammonium acetate, 0.1% TFA in H2O). The mixture was loaded onto a BondElut C18 solid phase extraction cartridge (Varian Chromatography Systems, Walnut Creek, CA). The cartridge was washed with 3 mL of water and then eluted with 5 mL of MeOH. The methanol solution was evaporated to dryness under N2 at room temperature. The residue was dissolved in 200 µL of MeOH-H2O (2:1). The sample (100 µL × 2) was separated by HPLC using the method described above, and radioactive fractions were collected. The fractions corresponding to metabolites of interest were pooled and were evaporated to dryness under N2 at room temperature. The purified samples were used for NMR analysis. Isolation of Adduct from in Vitro Incubations. Radiolabeled MB243 was incubated on a larger scale with rat liver microsomes, as described above, in the presence of GSH or NAC (5mM). Protein was precipitated with acetonitrile, and the supernatant was concentrated and subjected to preparative chromatography as described for the bile sample.
Doss et al. Table 1. Covalent Binding of MB243 to Microsomal Proteins (pmol equiv/mg Protein)a rat human a
+ NADPHb
+ NADPH + GSH
- NADPH
2153 ( 266 739 ( 212
356 124
11 8
After 30 min incubations. b Mean ( SD (n ) 3).
Figure 2. Radiochromatogram of rat bile following 20 mg/kg oral administration of [3H]MB243. Accurate Mass Measurements. The accurate mass experiments were carried out by using a Q-TOF-2 (quadrupole timeof-flight) mass spectrometer (Micromass, Beverley, MA) operated in electrospray positive ion mode with the cone voltage set at 42 V. CID was carried out by using argon as the collision gas with a collision energy of 40 V. Accurate mass measurements of the MH+ ions were conducted by using on-line HPLC/ TOF-MS with postcolumn addition of reserpine as a lock mass. An HP1100 HPLC system was used to separate the samples prior to analysis. A Keystone Hypersil BDS C8 (100 mm × 4.6 mm, 5 µm) column and a solvent system consisting of 1 mM ammonium acetate in water (B) and 90% acetonitrile in water (A) with 0.1% trifluoroacetic acid in both were used to effect separation. A 1 mL/min flow rate and a linear gradient of 40-90% A in 30 min were used. The HPLC effluent was split 1:5 with one part going to the MS. Samples were infused for acquiring accurate mass measurement of product ions. In this case, MH+ ion in each spectrum was used as the lock mass. NMR Analysis. NMR spectra were acquired on a Unity Inova 600 MHz spectrometer (Varian Inc., Palo Alto, CA). The isolated metabolites were dissolved in a CD3CN:D2O mixture (5:1) containing a trace of trifluoroacetic acid and analyzed using a 3 mm inverse detection microsample probe (Nalorac Corp., Martinez, CA) The parent compound was collected from a similar HPLC run and analyzed under the same conditions to minimize pH dependence. Total correlation NMR spectroscopy (TOCSY) (14) and multiple quantum correlation NMR spectroscopy (HMQC) (15) two-dimensional (2D) NMR experiments were carried out to obtain 1H-1H and 1H-13C correlation spectra, respectively.
Results and Discussion Covalent Binding to Proteins. When incubated with rat or human liver microsomes, MB243 exhibited high levels of covalent binding to proteins (Table 1). The binding was both time- and NADPH-dependent indicating that metabolic activation was taking place. Addition of GSH to the incubation medium caused an approximate 5-fold reduction in bound radioactivity, which indicated that an electrophilic reactive intermediate(s) was most likely being formed. To understand the nature of this bioactivation, we set out to determine the structure of the thioether adduct(s) formed.
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Figure 3. Partial 600 MHz proton NMR spectra of metabolites M1 (upper trace) and M2 (lower trace). Signals are doubled due to the presence of two rotamers (3:2 ratio) in solution.
Detection of Adducts. Analysis of incubation media for the presence of GSH adducts was first attempted by LC/MS/MS, scanning for constant neutral loss of 129 Da, corresponding to the loss of pyroglutamate, which is typical of GSH adducts (16, 17). This approach did not reveal any intact GSH adduct(s). Two radioactive peaks, however, were detected with MH+ ions at m/z 730 (M1) and 673 (M2). When NAC was used in the incubation instead of GSH as the trapping agent, only the m/z 673 adduct was observed. The same adducts were observed in bile from rats dosed with MB243. A representative radiochromatogram is shown in Figure 2. The adducts were unstable, but when kept cold under acidic conditions (pH ca. 4), they were sufficiently stable for isolation and structure determination. Structure Determination. The structures of the adducts were elucidated by detailed NMR and high-
resolution mass spectral analysis of isolated samples (Figures 3-5 and Tables 2-4). The molecular masses of the adducts M1 (729 Da) and M2 (672 Da) corresponded to the addition of the elements of cysteinylglycine and cysteine, respectively, to a reactive species derived from MB243. It thus appeared that the initial GSH adduct had undergone sequential peptide residue cleavage losing the γ-glutamyl followed by the glycyl residues. This observation was later confirmed by the 1H NMR spectra (Figure 3), which showed signals characteristic of the cysteine residue protons in both adducts and a signal at 3.92 ppm consistent with the glycine CH2 in the M1 adduct only. The initial NAC adduct similarly appeared to have lost the N-acetyl group to yield the same m/z 673 (M2) adduct. The reactive intermediate generated from MB243 was initially postulated to be an arene oxide formed at the
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Doss et al. Table 2. Proton NMR Data (600 MHz) for MB243 and Its Metabolitesa assignmentb a bc c d ec f g NMe Gly
M1
M2
MB243
4.24 dd, J ) 4.8, 11.9 Hz 3.50, 2.93 m 5.38 s 4.96 m 2.88 m 7.22 m, J ) 8.5, 5.5 Hz 7.02 m, J ) 8.8, 8.5 Hz 2.39 s 3.93 AB, J ∼ 18 Hz
3.87 dd, J ) 11.9, 4.7 Hz 2.85, 3.48 m 5.27 s 4.97 m 2.88 7.22 m, J ) 8.4, 5.6 Hz 7.02 m, J ) 8.8, 8.4 Hz 2.36 s
4.95 m 2.90 7.21 m, J ) 8.4, 5.4 Hz 7.03 m, J ) 8.8, 8.4 Hz 2.85 s
a In CD CN:D O 5:1 mixture. Two rotamers were present in a 3 2 3:2 ratio. Signals for the major rotamer are listed. b Refers to the annotation in Figure 3. c Overlapped with other signals but detected in TOCSY 2D NMR spectrum.
Figure 4. Product ion spectrum of metabolite M1 (MH+ m/z 730). The proposed assignments of fragment ions are supported by the accurate mass data (Table 3).
Table 3. Accurate Mass Measurements of Ions Formed by CID of MH+ of M1 (Figure 4) observed mass
calcd mass
ppm
formula
730.3743 655.3405 542.3511 464.1405 436.1464 276.1165 267.2437 248.1220
730.3762 655.3442 542.3506 464.1404 436.1455 276.1148 267.2436 248.1199
-2.6 -5.7 0.9 0.3 2.0 6.1 0.2 8.4
C36H53N7O6FS C34H48N6O4FS C30H45N5O3F C20H23N5O5FS C19H23N5O4FS C14H15N3O2F C16H31N2O C13H15N3OF
Table 4. Accurate Mass Measurements of Ions Formed by CID of MH+ of M2 (Figure 5)
Figure 5. Product ion spectrum of metabolite M2 (MH+ m/z 673). The proposed assignments of fragment ions are supported by the accurate mass data (Table 4).
para-fluorophenyl ring. Indeed, a reasonable mechanism could be proposed that would yield products with the molecular masses of the observed adducts. However, this hypothesis was quickly discarded as the proton NMR spectra of M1 and M2 showed unambiguously that the fluorophenyl ring was intact (Figure 3). On the basis of both the NMR spectra and the MS fragmentation patterns of M1 and M2, it became apparent that the piperazine moiety was the site of transformation. The molecular masses of M1 and M2 indicated that the bioactivation must have occurred by way of a six electron oxidation of MB243. An intense fragment ion at m/z 542 (corresponding to a neutral loss of 188 Da in M1 and 131 Da in M2) could only be accounted for by the loss of the thiol moiety plus one carbon from the piperazine ring. This carbon clearly was not the N-methyl group since its signal was intact in the proton NMR spectrum. Furthermore, the N-desmethyl analogue of MB243 formed similar adducts with the same fragmen-
observed mass
calcd mass
ppm
formula
673.3533 629.3653 542.3511 407.1183 379.1247 276.1145 267.2434 248.1190 242.0611
673.3547 629.3649 542.3506 407.1189 379.1240 276.1148 267.2436 248.1199 242.0599
-2.1 0.6 0.8 -1.5 1.9 1.3 -1.0 -3.8 4.9
C34H50N6O5FS C33H50N6O3FS C30H45N5O3F C18H20N4O4FS C17H20N4O3FS C14H15N3O2F C16H31N2O C13H15N3OF C9H12N3O3S
tation patterns except for a mass shift of 14 Da (data not shown). This important fragment led us to believe that a ring contraction had taken place in the piperazine moiety. The proton NMR spectra of both adducts showed a new signal (a singlet near 5.3 ppm) consistent with a CH group, which could be either olefinic or attached to two heteroatoms (e.g., a thioaminal proton). The latter was shown to be the case based on the 13C NMR chemical shift of that carbon (60.2 ppm) in the 2D HMQC NMR spectrum. On the basis of all of the above data, the structures of M1 and M2 were assigned as shown in Figure 1. Mechanism of Bioactivation. A plausible mechanism (Figure 6) for the formation of M1 and M2 involves oxidation of the piperazine ring to form an electrophilic conjugated imine-amide intermediate, which is trapped by GSH. The initial GSH adduct then undergoes hydrolysis of the glutamic acid residue, and the resulting free cysteinyl amino group attacks the thioaminal piperazine carbon causing opening of the piperazine ring and formation of a thiazolidine intermediate. The latter undergoes ring closure by loss of water to form the imidazoline adduct M1. Adduct M2 results from further cleavage of the glycine residue from M1. A similar mechanism can be written for the NAC adduct in which
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Figure 6. Proposed mechanism of bioactivation of MB243 and formation of adducts M1 and M2.
Figure 7. Improved analogues of MB243, which exhibited low covalent binding to protein. The numbers represent values for covalent binding in pmol equiv/mg protein after 30 min of incubation with rat liver microsomes in the presence of NADPH. MB243 was used as a positive control (2153 ( 266 pmol equiv/ mg protein). The R group is the same as in MB243.
hydrolysis of the acetyl group instead of the glutamyl residue results in M2. Although there are many drugs that contain a piperazine moiety and whose metabolism has been well-studied, to our knowledge there are no reports in which a piperazine ring has undergone this type of bioactivation and rearrangement. We are aware, however, of several other systems that form adducts in which the cysteinyl amino group is involved in further intramolecular reactions after the initial attack by the sulfhydryl group (18-21). Improved Analogues. Having elucidated the bioactivation of MB243, we undertook the synthesis of analogues substituted at the piperazine ring in an attempt to decrease bioactivation and thereby to reduce the level of irreversible binding to liver microsomal proteins (22). Various analogues with alkyl substitutions at the piperazine ring (Figure 7) were synthesized, radiolabeled, and tested for irreversible protein binding. These analogues all exhibited at least a 10-fold decrease in binding levels while maintaining the pharmacological potency and the selectivity of the original lead compound (22).
Conclusion We have described the structure determination of thioether adducts of an investigational drug candidate in which a piperazine ring underwent a novel bioactivation and rearrangement to a ring-contracted imidazoline. We proposed a mechanism for the adduct formation. We also have shown that by modifying the piperazine ring by alkyl substitution, it was possible to generate ana-
logues with a greatly reduced propensity to undergo metabolic bioactivation and bind irreversibly to liver microsomal proteins. The elucidation of this bioactivation pathway early in the drug discovery phase led to analogues, which should exhibit a decreased potential to cause drug-induced toxicities through the formation of chemically reactive metabolites.
Acknowledgment. We thank the Drug Metabolism animal support group for assistance with the rat bile duct cannulation.
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