Boronic Acid-Containing Proteasome Inhibitors: Alert to Potential

Article pubs.acs.org/crt

Boronic Acid-Containing Proteasome Inhibitors: Alert to Potential Pharmaceutical Bioactivation Austin C. Li,* Erya Yu, Steven C. Ring, and James P. Chovan Drug Metabolism and Pharmacokinetics, Teva Branded Pharmaceutical Products R&D, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States ABSTRACT: Medicinal chemists try to avoid certain organic functional groups, summarized in an ever-growing list, in order to avoid the potential bioactivation to reactive metabolites. To add to that alert list, we report herein that boronic acidcontaining compound structures, such as those found in proteasome inhibitors bortezomib and ixazomib, can become bioactivated to chemically reactive imine amide metabolites. Test compounds, ixazomib and bortezomib, were incubated in vitro using human liver fractions containing cytosol and microsomes (S9) under conventional conditions in the presence of GSH. Metabolites were then analyzed using LC-MSn with or without online hydrogen−deuterium exchange (HDX) liquid chromatography coupled with an LTQ-Orbitrap. The exact mass measurements of both the precursor and product ions were acquired through data dependent acquisition and compared with theoretical values of proposed fragment ions. Upon deboronation catalyzed by cytochrome P450 enzymes, both test compounds formed imine amide metabolites that were identified by high resolution exact mass measurements in both normal aqueous and HDX HPLC-MS analysis. GSH conjugates were also identified and were postulated as nucleophilic addition of GSH to the imine amide metabolites. All mass spectrometric and HDX measurements of these GSH conjugates proved that the GSH unit was added to the carbon atom of the imine amide partial structure, hence demonstrating the electrophilic property of these imine amide metabolites. The awareness of the formation of electrophilic imine amide metabolites from boronic acid-containing compounds, where the boron atom is bonded to a carbon atom adjacent to an amide nitrogen, should help in drug candidate design and optimization with regard to avoiding potential bioactivation.

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to facilitate oral availability. Ixazomib has been reported7,8 both in vitro and in vivo to completely hydrolyze and generate MLN2238, the biologically active component that contains a boronic acid unit just like the one in bortezomib. The metabolic profile of bortezomib was reported in 2005,6 and the results suggest that its principal biotransformation pathway is oxidative deboronation. Similar results were obtained for ixazomib in this study, suggesting that such deboronation is a characteristic of this structural class. However, the present article focuses not on that metabolic reaction but rather on the characterization of the chemical reactivity of imine amide metabolites that are formed after deboronation of each compound.

INTRODUCTION The potential for chemically reactive drug metabolites to cause drug-induced toxicities has been well documented,1,2 and molecular substructures that are prone to generate such metabolites have been identified. Lists of such structures have been compiled and are continually being updated for use by medicinal chemists as structures to avoid in order to reduce the risk of chemically induced idiosyncratic drug toxicity of drugs.3−5 To identify possible reactive drug metabolites, “trapping” techniques have been routinely used in the pharmaceutical industry employing nucleophilic reagents such as the tripeptide GSH or cyanide ion.2 The present article describes the results from the use of such a technique to test for the chemical reactivity of imine amides that are formed as metabolites of two proteasome inhibitors, bortezomib and ixazomib. To the knowledge of the authors, the reactivity of the imine amide substructure formed from these boronic acid-containing drugs has not been previously reported. Bortezomib and ixazomib have similar chemical structures (Scheme 1), are both potent proteasome inhibitors,6,7 and were or are being developed as anticancer therapeutics. Bortezomib has been approved by the U.S. Food and Drug Administration (FDA) and is currently marketed as Velcade, while ixazomib is still in development. Both compounds contain a boronic acid functionality on the carbon atom adjacent to an amide nitrogen, though the boronic acid group in ixazomib is derivatized, likely © 2013 American Chemical Society

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EXPERIMENTAL PROCEDURES

Chemicals and Reagents. Bortezomib was synthesized at the former Cephalon, Inc., facility, West Chester, PA, according to a published procedure.9 Ixazomib was purchased from Selleck Chemicals LLC (Houston, TX). Reduced GSH, ammonium acetate, and HPLCgrade acetonitrile (ACN) and formic acid were all from Sigma-Aldrich Chemical Co. (St. Louis, MO) or VWR International (West Chester, PA). Purified water was obtained from an in-house Milli-Q ultrapurification system (Millipore, Billerica, CT). Dideuterium monoxide (D2O) was purchased from Cambridge Isotope LaboReceived: January 23, 2013 Published: March 20, 2013 608

dx.doi.org/10.1021/tx400032n | Chem. Res. Toxicol. 2013, 26, 608−615

Chemical Research in Toxicology

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an electrospray interface. The chromatography was performed on a Varian Polaris 3 C18-A column (150 × 2.0 mm, 3 μm particle size, Varian, Palo Alto, CA) coupled with a guard cartridge (4 × 3 mm, Phenomenex, Palo Alto, CA). The same HPLC elution gradient was used to elute bortezomib and ixazomib and their metabolites. The gradient began at 10% mobile phase B (0.1% formic acid in ACN) and 90% mobile phase A (0.1% formic acid in water) and was held isocratic for 3 min, then linearly increased to 50% B within 30 min followed by a second linear increase to 90% B within 3 min. This mobile phase proportion (10% A−90% B) was maintained for 3 min before returning to initial conditions (ACN−water−formic acid, 10:90:0.1, v/ v/v) within 1 min. The column was re-equilibrated for 5 min prior to the next injection. The total mobile phase flow rate was 0.3 mL/min, and the column temperature was controlled at 35 °C. For hydrogen− deuterium exchange (HDX) runs, D2O was used to replace H2O in the preparation of mobile phase A. The mass spectrometer was tuned to the respective optimal conditions for bortezomib and ixazomib, and was operated under a data-dependent acquisition mode, which consisted of a survey full scan (250−1000 m/z) at a resolution of 30K and up to 4 dependent product ion scans at a resolution of 7.5K. The product ions were generated in collision-induced dissociation (CID) mode in the LTQ ion trap but were detected with the Orbitrap.

Scheme 1. Chemical Structures of Bortezomib, Ixazomib, and MLN2238

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RESULTS AND DISCUSSION Identification of Imine Amide Metabolites of Bortezomib and Ixazomib. Bortezomib (detected at m/z 367.19398 due to water loss in the ionization source) underwent deboronation and formed four metabolites that were detected as m/z 339.18184 at retention times of 21.02, 22.83, 28.91, and 30.08 min (Figure 1). The two eluted at RT 21.02 and 22.83 min were identified as carbinolamides, and their adduct ions of [M + Na]+ at m/z 379.17412 were also detected. They underwent water loss in the mass spectrometer ionization source (thermal degradation), and this was consistent with the previous literature.6 The other two metabolites, designated as B-1 and B-2 and eluting at 28.91 and 30.08 min, respectively, did not show adduct ions such as [M + Na]+ or [M + K]+. Both components showed nearly identical product ion profiles (Figure 2 for B-2 is representative), which supported the presence of a double bond in the same region of both metabolites. The previous literature6 suggested that the double bond was between two

ratories, Inc. (Cambridge, MA). Pooled male human liver S9 fractions were purchased from Human Biologics International (HBI, Phoenix, AZ). In-Vitro Incubation and Trapping of Chemically Reactive Metabolites. Both bortezomib and ixazomib were incubated at 10 μM with 1.0 mg/mL protein from human liver S9 fractions (containing cytosolic and microsomal fractions), an NADPHregeneration system, 1.0 mM reduced GSH, and 50 mM Na/K phosphate buffer (pH 7.4) containing 3.3 mM MgCl2 and 1 mM EDTA. The mixtures were incubated at 37 °C in a shaking water bath for 60 or 120 min before the reactions were stopped by adding 3 volumes of ice-cold ACN followed by centrifugation. The supernatants were removed, evaporated, and reconstituted in ACN−water−formic acid (10:90:0.1, v/v/v, the starting HPLC mobile phase). LC-MS/MS Methods. The LC-MS/MS system consisted of a Shimadzu Prominence HPLC system (Kyoto, Japan) and an LTQOrbitrap Mass Spectrometer (Thermo Scientific, Somerset, NJ) with

Figure 1. Extracted ion chromatograms of bortezomib imine amide metabolites B-1 and B-2, GSH conjugates B-3 and B-4, along with carbinolamide metabolites and bortezomib. 609



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Figure 2. Product ion spectrum of bortezomib imine amide B-2.

Upon HDX, both B-1 and B-2 increased 2 m/z units to 341.19376, indicating that both metabolites had only one exchangeable hydrogen atom (in addition to the charge) in their structures. Since the product ions, m/z 254 and 226, proved that the pyrazine-2-carboxamide partial structure (containing one exchangeable proton) was intact, the double bond formation had to be between the carbon and the nitrogen atoms (an imine) to satisfy all the mass spectrometric results observed in both full and product ion scans and both aqueous and HDX analysis. The fragmentation of B-2 is proposed in Scheme 2. This structure assignment was confirmed by product ion scan of both B-1 and B-2 after deuterium exchange (as shown for B-2, Table 2). The two imine amide metabolites are proposed to be E- and Z-isomers of the imine double bond. Similar incubation of ixazomib with human liver S9 fraction generated one imine amide metabolite, namely, I-1, along with MLN2238 (detected at m/z 343.07827 due to water loss in the ionization source) as reported previously (Figure 3). The metabolite (I-1) showed a protonated parent molecule of m/z 315.06598 at 20.61 min. The product ion spectrum (Figure 4) also supported the presence of a double bond in its structure. When analyzed using HDX, the metabolite increased 2 m/z

Scheme 2. Proposed MS Fragmentation of Bortezomib Imine Amide B-1

carbon atoms on the methylbutane unit, although it did not specify the exact position.

Figure 3. Extracted ion chromatograms of ixazomib imine amide metabolite I-1, GSH conjugates I-2 and I-3, and remaining MLN2238. 610



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Figure 4. Product ion spectrum of ixazomib imine amide I-1.

and m/z 201.98213 proved that the dicholorobenzamido-acetyl unit was intact, which had one exchangeable proton without modification, the formation of the double bond as a result of deboronation reduced the number of exchangeable protons, and therefore, the double bond must be an imine structure. Follow-up mass fragmentation of the metabolite I-1 after deuterium exchange confirmed that the one exchanged deuterium was associated with the dicholorobenzamido-acetyl unit and thus confirmed the imine amide structure formed due to deboronation. Scheme 3 shows the proposed fragmentation of I-1. Noteworthy is that, although bortezomib and MLN2238 share similar structures, particularly the boronic acid containing partial structure, MLN2238 was not observed to generate carbinolamide metabolites in the current work after repeated efforts. Identification of Imine Amide GSH Conjugates of Bortezomib and Ixazomib. Two GSH conjugates were detected for both bortezomib (17.11 min, B-3, and 18.46 min, B-4) (Figure 1) and ixazomib (16.95 min, I-2, and 17.49 min, I3) (Figure 3) after incubation. Upon CID (Figure 5), B-4 (m/z 646.26593) generated fragment ions at m/z 517, 376, and 308. Product ion m/z 517, due to a neutral loss of a glutamyl unit

Scheme 3. Proposed MS Fragmentation of Ixazomib Imine Amide I-1

units to 317.07770, indicating the presence of only one exchangeable proton. Since the product ions m/z 229.97711

Figure 5. Product ion spectrum of bortezomib GSH conjugate B-4. 611



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Figure 6. Product ion spectrum of ixazomib GSH conjugate I-2.

indicated that conjugation occurred at the methylbutane structural unit. With the identification of the imine amide metabolites (B-1 and B-2 of bortezomib) detailed above, the two GSH conjugates, B-3 and B-4, were rationalized to be conjugated between the sulfur atom in GSH and the carbon atom of the imine in B-1 or B-2. In HDX analysis, both B-3 and B-4 increased 9 m/z units to m/z 655.32025, indicating both B3 or B-4 had eight exchangeable protons, consistent with the proposed structure. Similar to B-4, I-2 and I-3 (m/z 622.14968) also fragmented upon CID (Figure 6 for I-2) showing the neutral loss of glutamyl (m/z 493, loss of 129 Da from the parent ion) that is characteristic of a GSH conjugate. The product ion m/z 376 was the same ion generated by B-4 mentioned above, based on the exact mass measurements as well as its fragment ions (Scheme 4 and Figures 5 and 6). Therefore, GSH also reacted with the methylbutane unit in I-1. I-2 increased 9 m/z units upon HDX, indicating eight exchangeable protons, further supporting the proposed structure. The proposed mass fragmentations of both B-4 and I-2 are presented in Scheme 4. Both B-4 and I-2 generated fragment ion m/z 308, indicating a protonated GSH, which was confirmed by the resultant MS3 fragment pattern (Figures 5 and 6). B-3 showed identical product ion fragmentation, exact mass measurements and HDX results compared to those of B-4. Similarly, I-3 patterns were also identical to I-2. The B-3/B-4 and I-2/I-3 pairs are proposed to be diastereomers formed due to the nonstereoselective addition of GSH to the double bond of the imine amides. With the identification of imine amide metabolites of both bortezomib and ixazomib, the formation of GSH conjugates was therefore proposed to be due to the electrophilic reactions of the imine amides to the nucleophilic reagent, GSH, as shown in Scheme 5. Detailed exact mass measurements of parent and product ions for bortezomib, ixazomib and their metabolites are listed in Table 1. Structure assignments were further confirmed by online HDX results, as presented in Table 2. The primary human hepatic enzymes responsible for the deboronation biotransformation of bortezomib were characterized as P450 3A4 and 2C19 along with lesser contributors 1A2, 2C9, and 2D6. 6,11 To understand the oxidative deboronation mechanism, particularly the observed near equal

Scheme 4. Proposed MS Fragmentation of Bortezomib and Ixazomib GSH Conjugates

Scheme 5. Proposed GSH Conjugate Formation Pathway for Bortezomib and Ixazomib

(129 Da), is characteristic of a GSH conjugate.10 The product ion m/z 376, based on the exact mass measurements, further indicated that the GSH was linked to the methylbutane unit. On MS3, this latter fragment underwent neutral loss of the glutamyl unit (129 Da) and generated m/z 247. This further 612



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Table 1. Exact Mass Measurements of Precursor and Product Ions of Imine Amide and GSH Conjugates of Bortezomib and Ixazomiba component

precursor ion

B-2

339

product ion 254 226 208

B-4

646 517 376 308 301 247 198

I-1

315 230 202 173

I-2

622 493 376 308 247 233 179

measured mass

theoretical mass

proposed formula

Δ mDa

Δ ppm

339.18184 254.09245 226.09749 208.08696 646.26593 517.22302 376.15359 308.09113 301.12125 247.11095 198.11240 315.06598 229.97711 201.98213 172.95563 622.14968 493.10669 376.15341 308.09097 247.10983 233.05904 179.04820

339.18155 254.09240 226.09749 208.08692 646.26536 517.22277 376.15368 308.09108 301.12165 247.11109 198.11247 315.06616 229.97701 201.98210 172.95555 622.14997 493.10735 376.15368 308.09108 247.11109 233.05905 179.04849

C19H23N4O2 C14H12N3O2 C13H12N3O C13H10N3 C29H40N7O8S C24H33N6O5S C15H26N3O6S C10H18N3O6S C13H21N2O4S C10H19N2O3S C10H16NO3 C14H17Cl2N2O2 C9H6Cl2NO2 C8H6Cl2NO C7H3Cl2O C24H34Cl2N5O8S C19H27Cl2N4O5S C15H26N3O6S C10H18N3O6S C10H19N2O3S C8H13N2O4S C5H11N2O3S

0.29 0.05 0.00 0.04 0.57 0.25 −0.09 0.05 −0.40 −0.14 −0.07 −0.18 0.09 0.03 0.08 −0.29 −0.66 −0.27 −0.11 −1.26 −0.01 −0.29

0.9 0.2 0.0 0.2 0.9 0.5 −0.2 0.2 −1.3 −0.6 −0.4 −0.6 0.4 0.1 0.5 −0.5 −1.3 −0.7 −0.4 −5.1 0.0 −1.6

Δ mDa = (measured mass − theoretical Mass) × 1000. Δ ppm = (mDa/theoretical mass) × 1000. Theoretical mass: Generated by ChemDraw Ultra 11.0.1, CambridgeSoft.

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Table 2. Exact Mass Measurements of Deuterated Precursor and Product Ions of Imine Amide and GSH Conjugates of Bortezomib and Ixazomib in Online HDX Analysisa component

precursor ion

B-2

341

product ion 255 227 209

B-3

655 524 382 316 305 251 200

I-1

317 231 203 173

I-2

631 500 382 316 251 238 185

measured mass

theoretical mass

proposed formula

Δ mDa

Δ ppm

341.19376 255.09808 227.10336 209.09264 655.32025 524.26587 382.19016 316.14078 305.14474 251.13553 200.12465 317.07770 230.98247 202.98778 172.95506 631.20447 500.15222 382.19101 316.14014 251.13594 238.09073 185.08565

341.19411 255.09868 227.10377 209.09320 655.32185 524.26670 382.19134 316.14130 305.14676 251.13620 200.12502 317.07871 230.98329 202.98837 172.95555 631.20646 500.15131 382.19134 316.14130 251.13620 238.09044 185.08615

C19H21D2N4O2 C14H11DN3O2 C13H11DN3O C13H9DN3 C29H31D9N7O8S C24H26D7N6O5S C15H20D6N3O6S C10H10D8N3O6S C13H17D4N2O4S C10H15D4N2O3S C10H14D2NO3 C14H15D2Cl2N2O2 C9H5DCl2NO2 C8H5DCl2NO C7H3Cl2O C24H25D9Cl2N5O8S C19H20D7Cl2N4O5S C15H20D6N3O6S C10H10D8N3O6S C10H15D4N2O3S C8H8D5N2O4S C5H5D6N2O3S

−0.35 −0.60 −0.41 −0.56 −1.60 −0.83 −1.18 −0.52 0.68 −0.67 −0.37 −1.01 −0.82 −0.59 −0.49 −1.99 0.91 −0.33 −1.16 −0.26 0.29 −0.50

−1.0 −2.4 −1.8 −2.7 −2.4 −1.6 −3.1 −1.6 2.2 −2.7 −1.8 −3.2 −3.6 −2.9 −2.8 −3.2 1.8 −0.9 −3.7 −1.0 1.2 −2.7

Δ mDa = (measured mass − theoretical mass) × 1000. Δ ppm = (mDa/theoretical mass) × 1000. Theoretical mass: Generated by ChemDraw Ultra 11.0.1, CambridgeSoft.

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mixtures of the two carbinolamide diastereomers, further research was conducted, and the investigators discovered that P450 2E1 also mediated the deboronation process.12 Another observation in the research was that the individual carbinolamides (M1 and M2) did not convert to the other in the

incubation media. This observation proved that an earlier interconvertion proposal via reversible dehydration mechanism (through an imine amide intermediate)13 was incorrect. Therefore, the possibility seems minimal that the imine amide metabolites detected in the current work were generated 613



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subsequently from carbinolamides via dehydration. Given the numbers of P450 enzymes involved in deboronation, multiple mechanisms are likely, including the putative carbon-centered radical intermediate.12 Nevertheless, whether the geometric isomers (E- and Z-) of B-1 and B-2 in the present work formed though this carbon-centered radical intermediate remains to be further investigated, in addition to the metabolism mechanism of ixazomib. Judged from the intensity of the extracted ion chromatograms presented in Figures 1 and 3, imine amide metabolites and their GSH conjugates generated from both drugs are at substantial levels, though they may have different ionization efficiencies from the parent drugs (MLN2238 for ixazomib). Severe hepatotoxicity due to bortezomib appears to be relatively rare compared with other drugs, as there are only a few clinical case reports of bortezomib-induced hepatotoxicity.14,15 This is probably because the dose is low (1.3 mg/m2 of skin surface16) and is consistent with the relationship between dose and adverse effects maintained in a previous review,3 despite the bioactivation described herein. The electrophilic nature of imine amides has been reported and utilized in the area of synthetic organic chemistry, though they were referred to as N-activated imines or N-acyl imines (i.e., imines bearing an electron-withdrawing group on the nitrogen atom).17,18 However, the use of such molecules in synthetic reactions is generally in the presence of catalysts and/ or at elevated temperatures rather than under the mild conditions utilized in the current study. Structurally, imine amides share similar properties with α,β-unsaturated ketones or esters known for their Michael-acceptor reactivity; hence, they may undergo Michael-type addition of nucleophiles. Being structurally related to an imine amide, a group of α,βunsaturated esters (acrylates, methacrylates, and propiolates) has been recently reported toxic to bacteria because of their electrophilic reactivity.19 Bioactivation of α,β-unsaturated structures has been documented for a number of compounds and/or metabolites, such as butadiene,3 valproic acid,3 2-(3chlorobenzyloxy)-6-(piperazinyl)pyrazine,20 and MB234 (a 1,3disubstituted piperazine).21 One note is that the α,βunsaturated reactive intermediate formed from MB234 also had an imine partial structure but that the nitrogen atom was not directly connected to the carbonyl carbon atom; therefore, the reported α,β-unsaturated structure was not imine amide. Also, nucleophiles were reported to attack the α carbon atom,21 whereas GSH attacked the β carbon atom of the imine amides in the current work. Although the reactive imine amides showed in this work were formed from a boronic acidcontaining proteasome inhibitor scaffold through the mechanism of oxidative deboronation, they may be formed through other metabolic mechanisms in other drug candidates as well.

Knowledge of the type of chemical structures that can potentially be bioactivated to reactive intermediates is important for medicinal chemists as they design and synthesize potential new drug candidates.22−24 Avoidance of such structures decreases the likelihood of failure of drug candidates during safety testing due to nonspecific chemical reactivity. Given the chemical reactivity of the imine amide group described in this article, attention should be paid to the potential formation of imine amide metabolites or intermediates of drug candidates.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 610-883-5616. Fax: 610-738-6377. E-mail: austin.li@ tevapharm.com. Funding

This research work was funded by Teva Branded Pharmaceutical Products R&D, Inc. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS HDX, hydrogen−deuterium exchange; ACN, acetonitrile; CID, collision induced dissociation REFERENCES

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CONCLUSIONS In conclusion, this article presents the electrophilic reactivity of imine amides that were trapped by the commonly used nucleophilic reagent, reduced GSH. These imine amides were formed during the biotransformation of both bortezomib and ixazomib via oxidative deboronation catalyzed by hepatic cytochrome P450 enzymes. By high resolution exact mass measurements and online HDX, the formation of imine amide metabolites and subsequent GSH-adducts was confirmed. These GSH-adducts clearly showed the electrophilic reactivity of the imine amide metabolites. 614



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Boronic Acid-Containing Proteasome Inhibitors: Alert to Potential

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