Reactive intermediates in copanlisib metabolism identified by LC-MS/MS: phase I metabolic profiling

Copanlisib (CNB; Aliqopa™) is a novel, intravenous phosphoinositide 3-kinase inhibitor used to treat various solid and hematological malignancies. CNB was recently approved by the U.S. FDA to treat adults that relapsed after two preceding systemic therapies. Using LC-MS/MS, we screened for the in vitro metabolites of CNB formed in human liver microsomes (HLMs) and probed for the generation of reactive electrophiles using methoxyamine and potassium cyanide as nucleophiles to capture reactive electrophiles by forming stable adducts that are suitable for identification by LC-MS/MS. Seven CNB phase I metabolites generated by oxidation, hydroxylation, oxidative dealkylation, reduction, and N-oxidation were identified. In addition, four reactive electrophiles, 2 aldehydes and 2 iminium ions, were identified, and a prediction of the corresponding bioactivation mechanism is presented. The formation of reactive metabolites may be associated with the side effects reported for CNB. To our knowledge, this is the first report on the detailed structural characterization of reactive intermediates generated in CNB metabolism.


Introduction
Phosphatidylinositol 3-kinase (PI3K) is an important target in the clinical management of different types of cancer as its biological activity is fundamental to interpreting how extracellular stimulation translates into intracellular signaling reactions, including cell survival and growth. The overexpression of PI3K isoforms is associated with a poor prognosis and considered as a major cause of relapse and cancer resistance in B-cell malignancies such as follicular lymphoma. [1][2][3] Gene mutation encoding phosphatase and tensin homolog (PTEN) and PI3K are responsible for reversing PI3K phosphorylation and are considered to be among the most commonly observed solid tumor alterations. [3][4][5] Therefore, there are many promising candidate PI3K inhibitor drugs currently under clinical evaluation for the treatment of a variety of blood cancers and solid tumors. 3,6,7 Idelalisib, a PI3Kd-selective inhibitor, has been approved for indolent non-Hodgkin's lymphoma treatment 8 but has no clinical benet in solid tumors.
Copanlisib (CNB) is a potent class I PI3K inhibitor with preferential activity against p110a and p110d than p110b and p110g. CNB is a novel, intravenous PI3K inhibitor used to treat different solid and hematological malignancies. 9,10 CNB induces tumor cell death such as apoptosis, and inhibits primary malignant B cell proliferation and tumor growth in the preclinical xenogra tumor models of malignant B cell. 11 CNB (Aliqopa™) is approved by the U.S. Food and Drug Administration for the treatment of adults who relapsed aer two prior treatments with systemic therapies. 12 Common toxic side effects of CNB include weakness, hypertension, hyperglycemia, diarrhea, nausea, low levels of specic white blood cells (leukopenia, neutropenia), low levels of blood platelets, and lower respiratory tract infections. 13 Metabolic detoxication involves pathways that transform endogenous compounds and xenobiotics into more hydrophilic species to facilitate excretion from the human body. The generated metabolites are oen less toxic than the parent molecules but in some cases, bioactivation may occur and promote reactive electrophile formation which leads to more toxic metabolites. [14][15][16] Reactive electrophiles are electron decient and can modify proteins and DNA by forming covalent bonds; this is considered the rst step in drug-mediated organ toxicities. 17,18 Verifying reactive metabolite production is a critical task in the study of drug-induced toxicity. Reactive metabolites are oen formed by phase-I metabolic reactions and cannot be directly characterized because of their transient nature. Instead, a trapping agent can be used to capture reactive intermediates via the formation of stable adducts that can be identied by mass spectrometry. 19,20 The structure of CNB, 2-amino-N-{7-methoxy-8-[3-(4 morpholinyl)propoxy]-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl}-5pyrimidinecarboxamide ( Fig. 1), contains a morpholine moiety (cyclic tertiary amine ring) that can undergo bioactivation by iminium ion generation or oxidative dealkylation to form aldehyde intermediates. [21][22][23][24] Glutathione and its derivatives are highly nucleophilic and react poorly with strong electrophiles. 25 However, the iminium ion and aldehydes are electrophiles that can be trapped using potassium cyanide and methoxyamine, respectively. 14,21,22 The adducts formed by nucleophilic-electrophilic interactions are considered stable and can be separated and identied by LC-MS/MS. [19][20][21]26,27 We hypothesized that these reactive intermediates are potentially involved in the reported side effects of CNB.
Literature review showed a single article that described the pharmacokinetics and disposition of CNB in human volunteers, 28 without addressing the structural identication of the formed metabolites or reactive intermediate formation. Therefore, the aim of the current work is to identify the bioactivation pathways of CNB resulting in reactive intermediates as can only be captured via in vitro experiments. While in vivo, these compounds have the capability to bind covalently to DNA and protein molecules and become undetectable. 17,20,26,29 2. Chemicals and methods

Chemicals
All chemical and solvents used were of analytical grade. CNB was procured from Med Chem. Express (USA). Acetonitrile, methoxyamine, ammonium formate, pooled human liver microsomes (HLMs, M0567) potassium cyanide and formic acid were procured from Sigma-Aldrich (USA). HPLC grade water (H 2 O) was purchased from Milli-Q plus system (USA).

Chromatographic conditions
Parameters used for chromatographic separation of the HLM incubation mixture are shown in Table 1.

HLM incubation
Several concentrations from 5 to 30 mM were analyzed; however, the only change observed was an increase in the concentration of metabolites to allow easier identication. Screening for in vitro metabolites of CNB was performed by incubating 30 mM CNB with 1.0 mg mL À1 HLMs in phosphate buffer (50 mM, pH 7.4) and 3.3 mM MgCl 2 . Incubation time and temperature were 2 h and 37 C, respectively. The samples were incubated in a shaking water bath. The CNB metabolic reactions were initiated by adding NADPH (1.0 mM) and stopped by adding 2 mL ice-cold acetonitrile. Protein precipitates were removed by centrifugation at 9000g (15 min, 4 C) and the supernatants  were evaporated then reconstituted in the mobile phase. Aliquots of 10 mL of each reconstituted sample were analyzed using LC-MS/MS system. [30][31][32] Replicates of blank sample were analyzed at the same run to conrm the absence of the proposed metabolites or adducts (ESI Fig. S11-S20 †).

Identication of CNB reactive metabolites
Full MS scans and extracted ion chromatograms of select m/z peaks were used to identify in vitro metabolites from the incubation chromatograms; molecular ions served as precursor ions (PIs) for fragmentation into daughter ions (DIs). Fragmentation (F) patterns were used to characterize in vitro metabolites and reactive intermediates generated by CNB metabolism. The same HLM incubation assay was repeated in the presence of methoxyamine or potassium cyanide to trap bioactive electrophiles. Reactions were performed in triplicate.

Fragmentation analysis of CNB
The CNB precursor ion peak (PIP) appeared at 24.4 min. F of PI at m/z 481 resulted in two DRs at m/z 128 and m/z 100, representing alkyl morpholine rings (Fig. 2).

Identication of CNB in vitro metabolites and reactive intermediates
Puried extracts recovered from HLM assays were subjected to LC triple-quadrupole MS (LC-QqQ MS) from which, three new phase I metabolites (M499, M483a, and M497a) and 4 reactive metabolites were identied. Six metabolites were produced from seven phase I metabolic reactions namely, (a-hydroxylation, a-oxidation, reduction, oxidative dealkylation, and Noxidation. In addition, two cyano and 2 methoxyamine adducts were identied (Table 2).   Fig. 3A and B).
The F of M497a resulted in three DRs at m/z 479, m/z 358, and m/z 128 (Fig. 3A), all containing the alkyl morpholine ring. The DR at m/z 128 suggested the absence of a metabolic change in the morpholine moiety. DRs revealed that M497a is a hydroxylated form of CNB with a 2,3-dihydroimidazole ring matching the DR at m/z 358 (Fig. 3A).
The F of M497b resulted in three DRs at m/z 479, m/z 144, and m/z 126 (Fig. 3B). The DR at m/z 142 represented the alkyl hydroxyl morpholine ring produced by single bond cleavage which matches the DR at m/z 126. Hydroxylation was predicted to occur in the a-position of the morpholine nitrogen (N) atom (Fig. 3B).  Ring cleavage and dealkylation were predicted to occur at the morpholine ring (Fig. 5).   Fig. 6A and B).
F of M483a resulted in three DRs at m/z 361, m/z 128 and m/z 100 (Fig. 6A), which represents the alkyl morpholine ring produced by single bond cleavage. DR at m/z 128 suggested that no metabolic change occurred at the morpholine ring. DRs revealed that M483a is a reduced form of CNB at the 2,3-dihydroimidazole ring (Fig. 6A).
F of M483b resulted in three DRs at m/z 354, m/z 233, and m/z 130 (Fig. 6B). DR at m/z 130 suggested that metabolic changes occurred in the morpholine ring matching the other DR at m/z 354. Ring cleavage and oxidative dealkylation were predicted to occur at the morpholine ring (Fig. 6B).
3.2.5. Identication of M499 phase I metabolite of CNB. M499 PIP appeared as [M + H] + (m/z 499) at 22.7 min. F of PI at m/z 499 resulted in DRs at m/z 128 and m/z 100, indicating the absence of a metabolic reaction at the morpholine ring. DR at m/z 361 suggested that reduction occurred in the dihydroimidazole ring whereas N-oxidation occurred at the pyrimidine ring (Fig. 7).

Reactive metabolites
Two cyano and 2 methoxyl adducts were identied aer incubating CNB with HLMs in the presence of trapping agents.      This journal is © The Royal Society of Chemistry 2019 dealkylation and oxidative opening of the morpholine ring, resulting in oxime with an aldehyde (Fig. 11).

Mechanism of CNB bioactivation
The generation of M506 and M508 cyanide adducts conrmed the formation of iminium intermediates, and hydroxylation of the piperazine ring in CNB followed by the loss of a water molecule created unstable iminium electrophiles that could be captured by a cyanide nucleophile to form a stable adduct (Scheme 1). The mechanism of the formation of the iminium intermediate and CNB bioactivation have been previously studied in cyclic tertiary amine-containing drugs. [33][34][35][36][37][38] The formation of M439 and M512 conrmed the production of aldehyde intermediates in CNB metabolism. The aldehyde electrophiles were formed by oxidative dealkylation Scheme 1 Proposed mechanism for the formation of iminium intermediates in CNB metabolism and the potential trapping strategy.
Scheme 2 Proposed mechanism of aldehyde generation. and captured with methoxyamine-forming oxime (M439 and M512). Oxidative dealkylation of the morpholine group formed an aldehyde captured by methoxyamine-forming M439. The opening of the morpholine ring by oxidative dealkylation resulted in aldehyde entrapment via M512 formation. Both oximes were stable, and were identied using LC-MS/MS (Scheme 2). Aldehyde formation in morpholine group-containing drugs has been described previously. 39,40

Conclusions
Seven phase I metabolites, 2 cyano adducts, and 2 methoxyamine adducts of CNB were detected (Fig. 12). All pathways for the reactive metabolites depended on the morpholine group of CNB, which may be associated with the side effects caused by CNB. These results have provided relevant groundwork for further investigations on CNB toxicity. Proling of phase I metabolites is very crucial as it may be the next generation drugs. Understanding the bioactivation pathways is crucial to identify bioactive so spots. Isosteric replacement or steric hindrance groups at these locations may block bioactivation and retain the pharmacological activity. Therefore, using the aforementioned data will help to develop next-generation drugs with less adverse effects.

Conflicts of interest
The authors declare no conict of interest.