Metalloporphyrin catalyzed oxidation of sunitinib and pazopanib, two

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Metalloporphyrin catalyzed oxidation of sunitinib and pazopanib, two anticancer tyrosine kinase inhibitors: evidences for new potentially toxic metabolites Marie-Noëlle PALUDETTO, Christian BIJANI, Florent PUISSET, Vania Bernardes-Genisson, Cécile ARELLANO, and Anne ROBERT J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00812 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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Journal of Medicinal Chemistry

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Metalloporphyrin catalyzed oxidation of sunitinib and pazopanib, two anticancer tyrosine kinase inhibitors: evidences for new potentially toxic metabolites.

Marie-Noëlle Paludetto,1,2,3,4 Christian Bijani,1 Florent Puisset,2,3,4 Vania BernardesGénisson,1,3 Cécile Arellano,2,3* Anne Robert1* 1

Laboratoire de Chimie de Coordination du CNRS (LCC-CNRS), Université de Toulouse,

205 route de Narbonne, BP 44099, 31077 Toulouse, Cedex 4, France. 2

Centre de Recherches en Cancérologie de Toulouse (CRCT), INSERM UMR1037,

Université de Toulouse, 2 avenue Hubert Curien, CS53717, 31037 Toulouse Cedex 1, France. 3

Université Paul Sabatier, Toulouse, France.

4

Pharmacie, Institut Claudius Regaud, IUCT-O, Toulouse, France.

*

Corresponding authors: Cécile Arellano, Anne Robert

Abstract

Oxidation of two tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib, using a chemical catalytic system able to mimic the cytochrome P450 type oxidation, allowed us to prepare and evidence putative reactive/toxic metabolites of these anticancer drugs. Among these metabolites, aromatic aldehyde derivatives were unambiguously characterized. Such biomimetic oxidation of TKI-type drugs was essential to facilitate the identification of low amounts of aldehydes generated from these TKIs when incubated with human liver microsomes (HLM), which are classical models of human hepatic metabolism. These TKI derivative aldehydes quickly react in vitro with amines. A similar reaction is expected to occur in vivo, and may be at the origin of the potentially severe hepatotoxicity of these TKIs. ACS Paragon Plus Environment


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Key words

Anticancer drugs, Biomimetic oxidation, Cytochrome P450, Metabolism, Metalloporphyrin, Oxidation.

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Introduction

Renal cell carcinoma (RCC) accounts for about 3% of adult malignancies and its incidence is increasing. RCC is inherently resistant to chemotherapy, radiations or hormone therapy.1 For localized RCC, nephrectomy is therefore the cornerstone of therapy. However, 40-45% of patients have locally advanced or metastatic RCC at the time of diagnosis. In addition, 2030% of patients undergoing surgery experience a local recurrence or subsequently develop metastatic disease. In these cases, a systemic therapy is required. For the last decade, tyrosine kinase inhibitors (TKIs) were considered as a very important class of anticancer medicines. TKIs are small chemical heterocyclic molecules with suitable chemical properties for oral administration, a real advantage in anticancer therapy. Among them, sunitinib and pazopanib target tumor angiogenesis by inhibition of the vascular endothelial growth factor receptor (VEGFR). These treatments have extended the median overall survival of patients with advanced RCC2,3 and are commonly used as first-line treatments.4,5 However, these drugs are also responsible for a severe hepatotoxicity occurring during the course of therapy with rare fatal cases of hepatic failure,6,7 whose mechanisms are poorly understood. Hepatic oxidation of sunitinib and pazopanib is primarily mediated by CYP3A4,8,9 giving rise to hydroxylated and N-dealkylated metabolites9,10 whose structures are not, at first glance, correlated to direct or indirect hepatotoxicity. Nevertheless, toxic species generated by drug metabolism likely have high reactivity.11 It is therefore possible that, when generated in vivo, such reactive/toxic metabolites would not exist in sufficiently large amount to be detected, due to their fast reaction with macromolecule targets, thus escaping from their individual detection and characterization. This was recently proposed for another TKI, erlotinib.12 In this context, the novel structural identification of the metabolites of sunitinib and pazopanib will help understand and manage the hepatotoxicity and drug-drug interactions induced by the TKI treatment. ACS Paragon Plus Environment


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The biomimetic oxidation of drugs by redox-active metalloporphyrin catalysts associated with a single oxygen atom donor [such as lithium hypochlorite, potassium hydrogen persulfate (KHSO5), magnesium monoperphthalate (MMPP)] has been reported to efficiently mimic the biotransformation performed in vivo by cytochromes P450s or peroxidases. In fact, these catalytic systems can efficiently oxidize a variety of substrates either in aqueous or organic solutions, and perform, amongst other transformations, hydroxylation of saturated structures, oxidation of aromatics or N-demethylation of tertiary amines, with a certain degree of regioselectivity.13-17 In these chemical catalytic conditions, it is possible to generate significant amounts of reactive metabolites that are not trapped by biological targets, and, consequently, can be more easily detected and characterized. These “in chimico conditions” should be considered as suitable tool for further in vivo investigations. In the present work, we investigated the biomimetic oxidation of sunitinib and pazopanib under various conditions, in order to identify potential reactive/toxic metabolites. This study allowed to isolate and characterize the aldehyde derivatives of sunitinib and pazopanib, which have not been identified up to now by NMR spectrometry. These metabolites are expected to readily react with endogenous amine functions, and can be consequently considered as potentially toxic entities. Moreover, these reactive aldehyde derivatives of sunitinib and pazopanib were also identified when sunitinib and pazopanib were oxidized by incubation with human liver microsomes (HLM), a classical tool for cytochrome P450 mediated metabolism studies.18 Our results indicate that P450-mediated oxidation of TKIs is expected to produce carboxaldehyde metabolites, that may escape detection in metabolism studies, due to their high reactivity, but should be considered as key intermediates to explain the hepatotoxicity of these drugs.


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Figure 1. Structures of the tyrosine kinase inhibitors (TKIs) sunitinib 1 and pazopanib 2.

Results and Discussion

The choice of the different catalysts and oxidants was based on their known abilities to mimic the reactions mediated in vivo by cytochrome P450 enzymes. The activation of metal complexes of porphyrins by single oxygen atom donors involves a highly electrophilic metaloxo as active species, which is reminiscent of cytochrome P450 active species generated in the presence of dioxygen associated with NADPH/P450 reductase.13 These metalloporphyrin based systems can oxidize a great variety of substrates, and provide similar oxidation products than those generated by P450/O2/P450 reductase. These biomimetic oxidation occurs in organic or aqueous solutions. In fact the active site of P450 enzymes is a highly hydrophobic pocket, and the P450 oxidation involving hydrophobic substrates is efficiently mimicked in organic solvents.16,17 So, the reaction can be tuned using different catalysts (different porphyrin ligands with manganese as redox active metal), and different single oxygen

atom

donors:

monoperoxyphthalate

meta-chloroperbenzoic

(MMPP),

potassium

acid

hydrogen

(mCPBA),

persulfate

magnesium

(KHSO5),

lithium

hypochlorite (LiOCl), peracetic acid (PAA). In addition, the reaction can be carried out in a homogeneous organic medium, or in a biphasic water/organic medium when the oxidant is water soluble (MMPP, KHSO5, LiOCl). In this latter case, a quaternary ammonium is added



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as a phase transfer catalyst to assist the transfer of the oxidant into the organic phase containing the catalyst and the substrate. In all cases, 4-t-butylpyridine was used as a cocatalyst.13,17 The general equation of the catalytic reactions and the metal complexes used as catalysts are depicted in Figure 2.

Figure 2. General scheme of catalytic oxidations and metalloporphyrins used as catalysts.

Catalytic chemical oxidation of sunitinib First of all, the oxidation of sunitinib carried out by KHSO5, MMPP or mCPBA, in the absence of metal catalyst, either in organic (CH2Cl2) or in biphasic (CH2Cl2/water/R4N+Cl–) conditions, provided sunitinib-N-oxide (1-N-oxide, Figure 3). This product was characterized by NMR. Upon oxidation of N18, the chemical shifts of H2C17 and H2C19 were drastically low field shifted with respect to the starting sunitinib base (∆δH = + 0.61-0.63 ppm, ∆δC = + 10.45-11.20 ppm in methanol-d4). The H2C16 and H3C20 were affected in a lower extent (∆δH = + 0.21-0.28 ppm, ∆δC = – 2.72-2.74 ppm). The 15N NMR signal of N18 was upfield shifted by ca. 80 ppm (δN = – 258.3 in sunitinib-N-oxide compared to – 338.6 ppm in sunitinib base). On the basis of mass spectrometry studies, sunitinib-N-oxide was detected as a minor metabolite in urines or feces of treated rats or monkeys, but not in humans.10 Noteworthy, as ACS Paragon Plus Environment

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already reported, the olefinic C3-C8 bond was slowly isomerized when sunitinib or sunitinib derivatives were kept in solution under ambient light.19 After two weeks in methanol, the Z/E ratio of sunitinib was 85/15, as evidenced by NMR (δH3C–C13 = 2.48 and 2.33 ppm for the Zand E-isomers, respectively; δC3 = 115.50 and 119.57 ppm, δC4 = 104.87 and 108.70 ppm, δC8 = 123.90 and 125.05 ppm, δC9 = 130.16 and 128.16 ppm, δC13 = 126.13 and 123.90, for the Zand E-isomers, respectively, in methanol-d4). Since the Z/E isomerization complicates the assignment of NMR spectra, all sunitinib solutions and reaction mixtures were kept protected from light. The catalytic oxidation of sunitinib was carried out using MnIII(Cl12TMP)Cl or MnIII(TDCPP)Cl associated with MMPP or KHSO5, at room temperature. TLC monitoring of the reactions showed that compounds more polar than the substrate were formed, and that these four different catalytic systems provided mixtures with the same products. As an example, when MnIII(TDCPP)Cl/KHSO5 was used (catalyst/oxidant/substrate mol ratio = 0.02/3/1), the reaction was quenched by addition of acetone after one hour, and purified over reverse phase C18 column. Despite chromatography did not allow to separate the different oxidation products, they were characterized as a mixture, by NMR in pyridined5. Along with some remaining starting material (67 mol%, even with longer reaction times, the substrate was never completely consumed in these reactions, probably due to the low excess of oxidant with respect to the substrate), three main oxidation products were identified (15 mol%, 12 mol% and 6 mol%). In all these products, the 1H and 13C NMR signals of the H3C–C11 (3.74/14.71 ppm in 1) disappeared, indicating that oxidation occurred at this particular methyl group. The more abundant product (1-CHO, Figure 3, 15 mol%) exhibited a 1

H NMR signal at 11.70 ppm correlated with a 13C NMR signal at 182.40 ppm, characteristic

of a carboxaldehyde function. C11 was detected at 133.78 ppm, shielded upfield by 4.67 ppm, with respect to 1 (138.45 ppm). The side chain of 1-CHO was very similar to that of 1, indicating that no oxidation occurred on the side chain (∆δ < 0.1 ppm for protons and < 0.7 ACS Paragon Plus Environment


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ppm for carbon atoms). In addition, a ROE correlation was detected between the aldehyde proton (11.70 ppm) and the HN15 detected at 9.86 ppm, thus confirming the structure of this aldehyde derivative of 1. In the NMR spectrum of the second oxidation product (12 mol%), the methyl-C11 was replaced by a methylene entity detected at 6.52 ppm and at 59.11 ppm in 1H and

13

C NMR

spectra, respectively and C11 was detected at 143.49 ppm (1-CH2OH, Figure 3, compared to 138.45 ppm for 1). Conversely, the H3C–C13 was detected at 3.83/12.33 ppm, very close to the corresponding chemical shifts of the substrate (3.78/12.10 ppm in 1). NMR data for C13 was not significantly modified (127.97 ppm in 1 and 127.73 ppm in 1-CH2OH), indicating that no reaction occurred at the methyl-C13 site. A ROE correlation was detected between the methylene at C11 (6.52 ppm) and the HN15 detected at 8.94 ppm. This feature is significant of the oxidation of the methyl-C11 to the alcohol HO–CH2–C11 (1-CH2OH, Figure 3). In addition, NMR detection of the complete side chain (HN15 to H3C20) allowed to rule out oxidation of the side chain. This product is the expected intermediate of oxidation of 1 to 1CHO.

Sunitinib 1 m/z = 399.2

3.74 / 14.71 138.45 8.61

17

19 20

H N 14

18 N

16

15

O

11

O

NH

2 3

3a

10 9

12 13

H N 7a 1

7 6 4 5

8 127.97

F

6.52 / 59.11

19 20

N 14

18 N

16

O

O

O

N

20

18

N H

13

O H N

F

3.75 / 11.45

OHC 9.86 H

H N

129.73

H N

11.7 / 182.4 133.78 O

1-CHO m/z = 413.2

NH 19

F

15

O

N

O


11

H N

O 137.34 NH

11

H N

F

O

F

4.52 / 59.4

6.09 / 68.5

NH

1-NHEt m/z = 371.2

8 127.73

CH3OH / H+

1-CH2OCH3 m/z = 429.2

H N

N

9

7 6 4 5

3.83 / 12.33

Catalytic oxidation

H N

2 1 3 3a

10

12 13

15

H N 7a

O

NH

11

17 8.94 H

3.78 / 12.10

1-N-oxide m/z = 415.2

143.49

HOH2C

MnIII(TDCPP)Cl KHSO5

Non catalytic KHSO5, MMPP oxidation or mCPBA

O

1-CH2OH m/z = 415.2

Catalytic oxidation

NH 10

N 14 12 13 O

9

8

F

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Figure 3. Catalytic oxidation of 1 in the presence of MnIII(TDCPP)Cl/KHSO5, resulting in oxidation at methyl-C11 (1-CH2OH, 1-CH2OCH3 and 1-CHO were characterized by NMR and mass spectrometry, partial NMR data in pyridine-d5 are reported on the structures) and in N18-desethylation (1-NHEt was detected by UHPLC-MS). Non-catalytic oxidation of 1 provided 1-N-oxide (given as comparison). The minor product detected by NMR (6 mol%) exhibited typical chemical shifts of a CH2– O–CH3 function at C11 (6.09/68.50 ppm for the methylene, 4.52/59.40 ppm for the methyl, 1CH2OCH3, Figure 3). The H3C–C13 was detected at 3.75/11.45 ppm, very close to the corresponding signals of the starting sunitinib. This compound, detected at m/z 429.2 amu (MH+) in mass spectrometry analyses, is likely to be the result of the methylation of 1CH2OH in the experimental conditions. The mechanism of its formation was not investigated further. In the LC-MS analysis of the metabolite mixture, the major products were detected with retention time values in the range 4-5 min in our conditions (Figure 4, trace a). The unreacted sunitinib was detected at 4.62 min (Figure 4, trace b). The extracted ionic current (XIC) m/z = 415 amu, assigned to the introduction of an oxygen atom in the molecule (+16 amu), displayed peaks at three different retention times (4.16, 4.36 and 4.70 min, Figure 4, trace c). This mass can be assigned either to the N-oxidation of the aliphatic N18, to the hydroxylation on the aliphatic side chain (metabolites hydroxylated at C16 or C20 have been detected in vivo10) or to a hydroxylation on the aromatic moiety. Oxidation on the side chain or on the aromatic scaffold could be differentiated by tandem mass spectrometry (MS/MS, MRM mode), using the transitions assigned to the cleavage of C17-N18 and C14-N15 bonds, respectively. The transitions 415→326 and 415→283, signatures of hydroxylation at C20 or N-oxidation at N18 on the side chain10 (Figure S1), gave rise to ionic currents at 4.16 and 4.70 min, and were not characterized further. The transitions 415→342 and 415→299, signatures of hydroxylation on the aromatic scaffold (Figure S1), gave rise to a single ionic current at ACS Paragon Plus Environment


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4.36 min. This product was therefore identified as the alcohol metabolite 1-CH2OH, which was characterized by NMR. The 1-CHO (XIC for m/z = 413) co-migrated with 1-CH2OH at 4.36 min (Figure 4, trace d). The molecular ion peak of the mass spectrum at 4.36 min (m/z = 413.14 amu) is consistent with oxidation of the methyl–C11 to an aldehyde function (+14 amu with respect to the spectrum of 1, m/z = 399.13, trace d, Insert). Tandem mass spectrometry spectra (MS/MS, MRM mode) for m/z = 413 produced main fragments at m/z = 340.1 and 297.1, assigned to the cleavage of C17-N18 and C14-N15 bonds (Figure S1), respectively, confirming that the introduction of the oxygen atom took place in the aromatic core and not at the amine side chain.


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11

4.62 4.36

a) TIC

4.69 4.16

4

4.47

4.2

4.4

4.6

4.8

5

Retention time, min MS at 4.62 min

4.62

b) XIC m/z = 399 Sunitinib 1

399.1

390

400

m/z

c) XIC m/z = 415 1-CH2OH 4.16

4.70 4.36

d) XIC m/z = 413 1-CHO

413.1

4.36 MS at 4.36 min

390

e) XIC m/z = 429 1-CH2OCH3

400

410

m/z 420

MS at 4.56 min 429.1

4.47 4.56

f) XIC m/z = 371 1-NHEt

425

4.2

4.4

m/z

435

MS at 4.28 min 371.1

4.28

360

4

430

4.6

365

370

4.8

375

m/z

380

5

Retention time, min

Figure 4. Catalytic oxidation of 1 by MnIII(TDCPP)Cl/KHSO5: UHPLC-MS of the reaction mixture. a) Total ionic current (TIC, scanning mode); b) Extracted ionic current (XIC) for m/z = 399, Insert: mass spectrum at 4.62 min, assigned to 1; c) XIC for m/z = 415; d) XIC for m/z = 413, Insert: mass spectrum at 4.36 min, assigned to 1-CHO; e) XIC for m/z = 429, Insert: mass spectrum at 4.56 min, assigned to 1-CH2OCH3; f) XIC for m/z = 371, Insert: mass spectrum at 4.28 min, assigned to 1-NHEt. ACS Paragon Plus Environment


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Derivatization of the aldehyde function, carried out by addition of methoxylamine in the mixture of sunitinib metabolites, resulted in complete disappearance of the extracted ionic current at m/z = 413, while the oxime derivative 1-CH=N–OCH3 was detected at 4.77 and 5.09 min (m/z = 442.3, Figure S2, trace a). The position of the oxime function was confirmed by the presence of peaks at the same retention time for 442→369 and 442→326 specific transitions in MRM MS/MS mode (Figure S2, traces b and c, respectively). The detection of two chromatographic peaks for this derivative can be tentatively assigned to Z/E isomerism of the oxime function. This result confirms both the presence of the aldehyde function on the aromatic moiety of sunitinib and its ability to readily react with primary amines. The chromatogram of the extracted ion (XIC) of the methylether derivative 1-CH2OCH3 (Figure 4, trace e) exhibited a similar profile than that of the parent alcohol, but with more retained peaks (Rt = 4.47, 4.56 and 4.70 min), all having a m/z value of 429.1 amu. By comparison with 1-CH2OH, the product eluted at 4.56 min is probably the methylether at C11. Noteworthy, the mono-N-desethyl sunitinib metabolite, which is the main human metabolite detectable in vivo,10 was also detected, partly overlapped by 1-CHO and 1-CH2OH (4.28 min, m/z = 371.1, Figure 4, trace f). This result indicates that the oxidation of sunitinib catalyzed by MnIII(TDCPP)Cl/KHSO5 was able to provide the main human metabolite of sunitinib. This catalytic system should therefore be considered a reliable model of in vivo P450 reactivity. Then, beside the oxidation of the amine side chain, the biomimetic oxidation of 1 using a metalloporphyrin catalyst was found to be regioselective at the α-pyrrolic position C11, the major oxidation products being the corresponding alcohol and aldehyde. Since aldehyde function is prone to react with amines in vitro, it is likely that, if the aldehyde is generated in vivo by P450 metabolism, it can further react with amine side chains of proteins and provide potentially toxic covalent sunitinib-protein adducts. ACS Paragon Plus Environment

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Human liver microsome induced oxidation of sunitinib Having in hands the aldehyde 1-CHO and the alcohol 1-CH2OH obtained by biomimetic oxidation, we decided to investigate the microsomal oxidation of sunitinib to check if these two drug oxidation products might be generated in human metabolization conditions. The identification, in biological conditions (microsomes), of these potential unknown metabolites, might be much easier with the available reference compounds that we characterized here for the first time. The results of the chemical oxidation of sunitinib 1 were compared to that obtained after incubation of 1 with human liver microsomes. These P450 rich subcellular fractions are indeed considered reliable models of drug hepatic metabolism.18 The supernatant of microsomal incubation media containing 1 were analyzed by UHPLC-MS. Results are reported in Figure 5. Along with unreacted 1 (Rt = 4.60 min, m/z = 399.4, trace a), and the N18-monodesethylated derivative (Rt = 4.27 min, m/z = 371.1, trace b), extracted ionic currents with m/z molecular ion values of 415 and 413 were detected at the same retention time as 1-CH2OH and 1-CHO of the catalytic biomimetic oxidation mixture (Rt = 4.36 min, Figure 5, traces c and f for 1-CHO and 1-CH2OH, respectively). Characterization of the 1CHO was confirmed by the transitions 413→340 (trace d) and 413→268 (trace e) in MRM mode, corresponding to the cleavage of the C17–N18 and C12–C14 bonds, respectively, while characterization of 1-CH2OH was assessed by the transitions 415→342 (trace g) and 415→299 (trace h) corresponding to the cleavage of the C17–N18 and C14–N15 bonds, respectively. This result formally confirmed the presence of the aldehyde derivative of 1 in the microsomal incubation media, along with the intermediate alcohol derivative. In addition, the MRM transitions 415→326 and 415→283 were detected at 4.15 and 4.69 min, indicating that incorporation of an oxygen atom in the amine aliphatic chain (hydroxylation or Noxidation) also occurred upon incubation of 1 with human liver microsomes (data not shown). ACS Paragon Plus Environment


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In addition, minor chromatographic peaks detected at 3.1 (m/z = 399.2) and 2.4 (m/z = 371.1) min were assigned to the E-stereoisomers of 1 and 1-NHEt, resulting from isomerization of the C3–C8 double bond under the reaction conditions (data not shown). Derivatization of 1-CHO produced by human liver microsomes was carried out by addition of methoxylamine in the incubation medium. This reaction resulted in the complete disappearance of the peak assigned to 1-CHO (m/z = 413), while the oxime derivative 1CH=N–OCH3 was detected at 4.74-4.77 min (m/z = 442, Figure S2, traces d and e). This result confirms the oxidation of 1 to an aldehyde derivative by human liver microsomes and the ability of this metabolite to react in the presence of primary amines.


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4.60

a) XIC m/z = 399 Sunitinib 1

b) XIC m/z = 371 1-NHEt 4.27

4

4.2

c) XIC m/z = 413 1-CHO

4.4 4.36

4.6 413.1

410

4

4.2

d) MRM 413→340

4.4

4.6

4.8 415.1

5

f) XIC m/z = 415 1-CH2OH

415.2

420 m/z

4.8

413.1

4.36

410

5

4

4.2

4.4

420

m/z

4.6

4.8

5

4.6

4.8

5

4.6

4.8

5

g) MRM 415→342

4.36

4.33

4.36

4

4.2

e) MRM 413→268

4.4 4.37

4.6

4.8

5

4

4.2 4.4 h) MRM 415→299 4.33

4

4.2

4.4

4.6

4.8

5

4

4.2

Retention time, min

4.4

Retention time, min

Figure 5. Oxidation of 1 catalyzed by human liver microsomes: UHPLC-MS of the reaction mixture. a) Extracted ionic current (XIC) for m/z = 399, assigned to 1; b) XIC for m/z = 371, assigned to 1-NHEt; c) XIC for m/z = 413, assigned to 1-CHO, Insert: mass spectrum at 4.36 min, assigned to the mixture of 1-CHO + 1-CH2OH; d) 413→340 MRM MS/MS transition; e) 413→268 MRM MS/MS transition; f) XIC for m/z = 415, assigned to 1-CH2OH, Insert: mass spectrum at 4.36 min, assigned to the mixture of 1-CH2OH + 1-CHO; g) 415→342 MRM MS/MS transition; h) 415→299 MRM MS/MS transition. ACS Paragon Plus Environment


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In conclusion, thanks to the characterization of oxidative derivatives of 1 obtained by the biomimetic oxidation system, it has been possible to unambiguously detect the carboxaldehyde 1-CHO after incubation of 1 with P450 rich human liver microsomes, a metabolite that has never been previously identified in animals or patients, probably because of its reactivity with primary amines. This aldehyde is therefore expected to be highly reactive with protein amines, and consequently should be considered as a potentially toxic metabolite. Since it can be generated by P450 in hepatocytes, this characterized metabolite may take a large part in the sunitinib hepatotoxicity. On the basis of mass spectrometry studies, hydroxylation of the aromatic core of sunitinib has previously been proposed as structure for a very minor fecal metabolite of 1 in humans.10 However, the regioselective hydroxylation at the α-pyrrolic position C11 was not reported.

Catalytic oxidation of pazopanib To the best of our knowledge, the characterization of pazopanib in vivo metabolites has only been reported in a single publication.9 After administration of [14C]pazopanib to ten patients, on the basis of HPLC-MS analyses, hydroxylations of the drug at C7 and C19 positions, along with N8-demethylation, have been proposed as P450 mediated phase I metabolism. In vitro, the drug was found stable under very drastic conditions: 3M HCl, 3M NaOH at 80 °C or 30% H2O2 at room temperature for 2 days.20 Pazopanib was susceptible only to photolytic conditions (UV light for three days at 200 W.h/m2).20 In these conditions, the oxidation of H3C–C7 to an aldehyde function was proposed on the basis of UHPLC-MS fragmentation patterns. However, these studies, carried out very far from any biologically relevant conditions, do not allow to propose relevant in vivo metabolism of this drug. Pazopanib is commercially available as a hydrochloride salt form, which is practically insoluble in aqueous media at pH above 4 and in many solvents except in DMSO or DMF, ACS Paragon Plus Environment

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which are not suitable for oxidation reactions. So, face to these limitations, we decided to use atypical solvents such as N-methylpyrrolidone and acetic acid. These solvents were able to solubilize pazopanib hydrochloride, a hydrosoluble oxidant and an organosoluble catalyst. For example, catalytic oxidation of pazopanib was carried out in acetic acid, using MnIII(TDCPP)Cl as catalyst, 4-t-butylpyridine as co-catalyst and MMPP as oxidant (catalyst/oxidant/substrate mol ratio = 0.02/3/1). The very poor solubility of the reaction products and the drug did not allow separation of the oxidation products. A preliminary study allowed to identify by mass spectrometry (using a direct introduction) compounds with m/z values of 438.5 amu, assigned to the starting pazopanib, and 454.4 and 452.5 amu, corresponding to an alcohol (M+16) and an aldehyde (M+14) derivative, respectively. A series of products with specific isotopic patterns of chlorinated derivatives was also detected. In this series, the m/z values of 472.4, 506.4, 488.3 and 486.3 amu were assigned to chloro- (M+34), dichloro- [M+(2x34)], chloroalcohol- (M+16+34) and chloroaldehyde (M+14+34) derivatives of pazopanib, respectively (data not shown). To go further into the characterization of these products, the mixture was then analyzed by NMR in pyridine-d5. In these conditions, eight products have been characterized in the reaction mixture. Unreacted starting material accounted for 42 mol% of the mixture and was completely characterized by 1H and

13

C NMR (Compound 2, Figure 6). The major product,

accounting for 27 mol%, was a derivative which had undergone oxidative chlorination at C14 (2-Cl, Figure 6). This feature has been evidenced by the disappearance of the aromatic H14 (at 7.09 ppm in 2) and by a 9 ppm downfield shift of C14 (98.59 ppm and 107.83 ppm in 2 and 2-Cl, respectively), along with a 4.6 ppm upfield shift of C9. This reaction is typically a chloroperoxidase-type reaction. It has been reported to occur in vivo in the presence of a hemin derivative associated to chloride anions.21 In two other products accounting for 9 mol% each, the H3C–C7 disappeared and was unambiguously replaced by an aldehyde function, with the CO detected at 180.00 ppm and ACS Paragon Plus Environment


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the HCO at 11.56 and 11.62 ppm (2-CHO, Figure 6). The H3C–N1 was also significantly downfield shifted upon oxidation at C7 (∆δ = + 0.44-0.52 ppm in proton and + 3.09-3.19 ppm in carbon-13). Complete assignment of all protons/carbons of these two metabolites cannot be achieved due to signal overlapping. However, taking into account that chlorination of pazopanib at C14 was evidenced (2-Cl), one can reasonably consider that one of these aldehydes is chlorinated at C14 and the other not. This proposal is consistent with the mass spectrometry data reported above. Two other products accounting for 6 mol% and 3 mol% were identified as alcohol derivatives resulting from the hydroxylation of H3C–C7, with a CH2OH detected at 6.39/54.44 ppm and 6.33/54.50 ppm, respectively (2-CH2OH, Figure 6). The C7 was downfield shifted with respect to 2 (∆δ = + 3.8 ppm). The H3C–N1 was downfield shifted compared to 2 (∆δ = + 0.28-0.33 ppm in proton and + 0.80-0.89 ppm in carbon-13) but in a lower extent than in 2-CHO. These alcohol derivatives are obviously intermediates in the oxidation of 2 to 2-CHO and, for the same reason as for the aldehyde derivatives, one of these alcohols is likely to be chlorinated at C14. In addition, the acetate esters of 2-CH2OH were characterized as two minor products (2CH2OCOCH3), with the carbonyl function detected at 171.20 ppm (2 + 2 mol%). These compounds are likely the result of the side reaction of 2-CH2OH (chlorinated or not at C14) with acetic acid used as solvent.


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3.56 / 10.39 8.78 / 121.80 121.30 133.05 8.04 / 121.25 6 6a 7 5.12 / 5 1N 143.64 4 2a 38.30 2 3 4.69 / N N8 39.46 8.84 / 164.23 116.00 149.18

Pazopanib 2 m/z = 438.2 42%

9.42 / 123.13 129.36 8.43 / 4.05 / 133.92 20.73 20 21 19 H2N 18 16 17

N10 11

15

S

N

27% 9.32 / 123.27 129.98 8.46 / 4.06 / 134.00 20.73 20 21 19 H2N 18 16 17

14 7.09 / 98.59

12 13 9.15 / 156.46

N

10.48 / H 119.72 11.61 141.50 144.27

O

9

2-Cl m/z = 472.1

O

O

S

O

10.40 / 119.84

8.68 / 120.77 122.38 8.13 / 122.92 6 6a 5 141.02 4 2a 3 4.73 / N8 41.64 159.62 9

N10 11

15

N H

Cl 14

2-CHO R= H Cl

O O

3.11 / 21.27 3.11 / 21.27 171.20 / 171.20

131.40 / 131.40 7 2

N

9

1N

N

N8

N 146.37

107.83

144.37

2-CH2OH

6a

134.65 5.07 / 38.50

N

R= H Cl

6.80 / 55.70 6.73 / 55.71

2

1N

11.84

2-CH2OCOCH3 4%

7

12 13 9.26 / 158.45

R= H Cl

m/z = 496.2 530.1a

3.48 / 10.48

R

m/z = 454.2 9% 488.1

121.30 / 122.20

6.39 / 54.44 6.33 / 54.50 6a

7 2

N

1N

N

N8 9

O

123.50 / 124.00

132.60 / 132.60 6a

5.45 / 39.19 5.40 / 39.10

14

7 2

N

N

9

1N

N

N8

R

14

N

11.56 / 180.00 / 180.00 11.62 H

OH 136.84 / 136.80

CH3COOH

5.34 / 39.10 5.38 / 39.10

m/z = 452.2 486.1 18%

5.56 / 41.49 5.64 / 41.39

R 14

N

Figure 6. Catalytic oxidation of 2 in the presence of MnIII(TDCPP)Cl/MMPP, resulting in chlorination at C14 providing 2-Cl, and oxidation at methyl-C7 providing 2-CH2OH, 2CH2OCOCH3 and 2-CHO. These metabolites were characterized by NMR and mass spectrometry. The chlorinated derivatives of 2-CH2OH, 2-CH2OCOCH3 and 2-CHO were also characterized by NMR (partial NMR data in pyridine-d5 are reported on the structures). a

Detected only in NMR.

Then, these products were characterized by UHPLC-MS. The XIC ionic currents of the expected m/z values are reported in Figure 7, along with the mass spectra. Under these conditions, pazopanib and its non-chlorinated metabolites were detected at 2.79, 2.56 and 2.82 min for 2, 2-CH2OH and 2-CHO, with R = H respectively (Figure 7, traces a-c). The monochlorinated series appeared at 3.55, 3.32 and 3.63 min for 2-Cl, 2-CH2OH and 2-CHO, with R = Cl respectively (traces d-f), with mass isotope profiles confirming the presence of a chorine atom in these molecules (see Figure 6 for the structures).



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Dichlorinated pazopanib was detected as a minor product (m/z = 506.2, co-eluted with 2CHO, R = Cl, Rt = 3.63 min, Figure 7, trace f).

a) XIC m/z = 438 Pazopanib 2

d) XIC m/z = 472 2-Cl MS at 2.79 min 438.3

2.79

430

2.4

2.6

2.8

3

b) XIC m/z = 454 2-CH2OH, R = H

3.2

440

3.4

m/z

3.6

m/z

450

3.8

4

2.4

2.6

3.55

480

2.8 2.9 3

3.2

e) XIC m/z = 488 2-CH2OH, R = Cl

MS at 2.56 min 454.3

2.56

MS at 3.55 min 472.3

3.4

3.6

MS at 3.32 min 488.2

3.32

480 450

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

2.4

2.6

2.8

f) XIC m/z = 486 2-CHO, R = Cl

2.8 2.9 3

3.2

490

450

3.4

3.6

3.2

3.4

MS at 3.63 min 486.2

3.6

3.8

4

3.8

4

3.63

506.2

480

2.6

3

MS at 2.82 min 438.3 452.3

440

2.4

m/z

460

c) XIC m/z = 452 2-CHO, R = H 2.82

3.8 3.9 4

m/z

3.8 3.9 4

Retention time, min

m/z 500

520

2.77

460

2.4

2.6

2.8

3

3.2

3.4

3.6

Retention time, min

Figure 7. Catalytic oxidation of 2 by MnIII(TDCPP)Cl/MMPP: UHPLC-MS of the reaction mixture. Traces a-c: Extracted ionic currents (XIC) for pazopanib 2, 2-CH2OH and 2-CHO, respectively. Traces d-f: XIC for the chlorinated derivatives 2-Cl, 2-CH2OH and 2-CHO, with R = Cl (see Figure 6 for the structures). In Inserts, the mass spectra of the assigned chromatographic peaks are reported.

Derivatization of the aldehyde function, carried out by addition of methoxylamine in the mixture of pazopanib oxidation products, resulted in complete disappearance of the extracted ionic current at m/z = 452, while the oxime derivative 2-CH=N–OCH3 was detected at 2.96 ACS Paragon Plus Environment

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and 3.11 min (m/z = 481.3, Figure S3, trace a). As for sunitinib, the detection of two chromatographic peaks for this derivative is likely due to Z/E isomerism of the oxime function. The oxime fragmentation produced the 481→449 and 481→368 transitions in MRM MS/MS mode (Figure S3, traces b and c, respectively). This result confirms both the presence of the aldehyde function on the aromatic moiety of pazopanib and its ability to react with primary amines. Then, the regioselective oxidation at C7, leading to an alcohol, which is over-oxidized to an aldehyde, was clearly evidenced using a biomimetic metalloporphyrin catalyst associated with a single oxygen atom donor. This result is consistent with the proposal of an alcohol at C7 as a pazopanib primary metabolite in humans.9 It is worth mentioning that the docking of pazopanib into the active cavity of CYP3A4 showed that the methyl group at C7 was in the close vicinity of the heme (the distance between the methyl hydrogen atom and the iron being 3.64 Å).22

Human liver microsome induced oxidation of pazopanib As reported above for sunitinib, we carried out pazopanib oxidation by human liver microsomes. After setting up of suitable UHPLC-MS analysis conditions using the previously characterized oxidation products of pazopanib, the supernatant of the microsomal incubation medium were then compared to the chemical oxidation products. After incubation of pazopanib 2 with human liver microsomes, unreacted 2 (XIC m/z = 438 at 2.81 min) and the hydroxylation product 2-CH2OH (XIC m/z = 454 and MRM transition 454→373 at 2.55 min) were detected in the supernatant of microsomal media (data not shown). More importantly, the aldehyde derivative 2-CHO was also unambiguously detected in microsomal incubation supernatants. The ionic currents due to the specific transitions 452→371, 452→343 and 452→328 assigned to 2-CHO (Rt = 2.84 min, Figure 8, traces a, b and c, respectively) were similar to that obtained with an authentic sample formerly prepared ACS Paragon Plus Environment


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by MnIII(TDCPP)Cl/MMPP catalytic oxidation (Rt = 2.82 min). The characterization of the metabolite 2-CHO was confirmed by addition of methoxylamine in the microsomal media. In these conditions, the MRM transition 481→449 assigned to the oxime derivative was detected at 3.09 min (Figure S3, trace d). The chlorinated derivative of pazopanib 2-Cl was also detected by UHPLC-MS of microsomal media. The ionic currents due to the transitions 472→436, 472→391 and 472→355 assigned to 2-Cl (Rt = 3.54-3.55 min) were fully consistent with the MnIII(TDCPP)Cl/MMPP biomimetic media (Figure 8, traces d, e and f, respectively). It should be noted that aromatic chlorination usually decreases solubility and increases the oxidative potential of the chlorinated fragment. These features may hinder further metabolism, thus preventing elimination of the drug and contributing to its toxicity.


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2-CHO, R=H 2.84

2-Cl, R=Cl d) MRM 472→436

a) MRM 452→371

2.4

2.6 2.822.8

3

3.2

3.4

3.6

.8

Mn(TDCPP)/ MMPP

2.4

2.6

2.8

2.84

3

3.2

3.4

3.6

3

3

2.4

2.6

3

2.8

2.84

3.4

3.6

3.2

3.4

3.6

.8

2.4

2.6

2.6

2.8 2.82

2.8

3

3.2

3.4

3

3.2

3.4

3.6 3.8 3.54

4

3.4

3.6

3.8

4

3.55

3

3.2

3.4

3.6 3.54 3.8

4

3

3.2

3.4

3.6

3.8

4

3.8

4

3.8

4

Mn(TDCPP)/ MMPP

3.55

Microsomes

3.6

Mn(TDCPP)/ MMPP

Retention time, min

3.2

f) MRM 472→355

c) MRM 452→328 Microsomes

2.4

3.4

Microsomes

3.2

Mn(TDCPP)/ MMPP

3.2

e) MRM 472→391

Microsomes

2.6 2.82 2.8

3

Mn(TDCPP)/ MMPP

b) MRM 452→343

2.4

3.55

Microsomes

Microsomes

.8

3

3.2

3.4

Mn(TDCPP)/ MMPP

3.6

3

3.2

3.4

3.6

3.54

3.6

Retention time, min

Figure 8. Comparison of specific MS/MS transitions (MRM mode) of 2-CHO (traces a-c) and 2-Cl (traces d-f) in the mixture of oxidation products of pazopanib 2, after oxidation by MnIII(TDCPP)Cl/MMPP or by human liver microsomes. R stands for the substituent at C14 (see Figure 6).

Conclusion ACS Paragon Plus Environment


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Using a biomimetic catalytic system, based on the association of a metalloporphyrin and a single oxygen atom donor, we prepared several oxidation products of sunitinib and pazopanib, which are predictive of the potential P450 metabolism of these tyrosine kinase inhibitors (TKIs) in vivo. Although pazopanib has been reported to be very recalcitrant to reaction, even under drastic chemical conditions, this biomimetic catalytic system allowed us to prepare, under mild conditions, and to characterize several oxidation and chlorination products of this drug. For both sunitinib and pazopanib, reactive aldehyde derivatives were characterized among other oxidation products. Then, with reference compounds in hands, the production of these reactive aldehydes was evidenced for both drugs upon human liver microsomes incubation, showing that metalloporphyrin-catalyzed oxidation can also be a reliable model of liver metabolism. Sunitinib and pazopanib oxidation to such reactive metabolites has not been experimentally documented yet, while aldehyde derivatives of several other TKIs have been proposed on the base of adduct derivatives identified in LC-MS.23 The reactivity of these metabolites, especially toward amines, may be responsible for the hepatotoxicity of these drugs. Moreover, sunitinib and pazopanib have been reported to inhibit several P450 isoenzymes.24 The putative reaction of carboxaldehyde metabolites of these drugs with the amine side chains of the P450 apoprotein may be responsible for the mechanism-based inhibition of these enzymes. Finally, it should be emphasized that the parallel oxidation of drugs by metalloporphyrin based biomimetic systems on one hand, and liver microsomes on the other hand, had not been reported yet. The identification of the same metabolites in both oxidation systems for both sunitinib and pazopanib gives a solid evidence for the biological relevancy of the biomimetic system.


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In addition, the preliminary experiments using the biomimetic catalytic system were an essential tool to identify the metabolites produced in microsomal incubation media. Clearly, if we had not prepared and characterized these P450 relevant oxidation products by metalloporphyrin catalyzed oxidation beforehand, the “fishing” of these reactive aldehyde in liver microsomal incubation media would not have gone so well. In addition, these studies strongly suggest that a way to reduce the hepatotoxicity of these two tyrosine kinase inhibitors, sunitinib and pazopanib, will be to change the oxidable methyl group at C11 or C7. A methyl substituent at C7 in pazopanib (and indazole analogues) exhibited good potency against all human VEGFR and also resulted in minimized CYP inhibition compared to other substituents.[25] In the sunitinib series, the C11 and C13-dimethyl pyrrole derivatives exhibited the highest activities.[26] In these conditions, replacement of a CH3 group by a CD3 is a way to considerably slow down metabolic conversion by kinetic isotope effect of deuterium, with minimum modification of the structure (and, therefore, of the activity) of the parent drug. Noteworthy, the first deuterated drug was approved by FDA in 2017.27

Experimental Section

Materials and instruments Sunitinib L-malate and pazopanib hydrochloride were purchased from Alsachim. All solvents and commercially available reagents were used without further purification. Sunitinib (free base, 1) was prepared from sunitinib L-malate. The manganese complexes of porphyrins were prepared as previously reported.

28

TLC was performed on neutral aluminium oxide 150 F254 plates (Merck).

Reverse phase chromatography was carried out on Sep-Pak®C18 classic cartridges (Waters). Human liver microsomes and incubation buffers were purchased from Corning (Ref 452161, 451220, 451200).



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NMR spectroscopy. 1H, 13C, 15N, 19F spectra were recorded at 293 K on a Bruker AV500 spectrometer that was equipped with a 5 mm broadband inverse triple-resonance probe with Z field gradients using methanol-d4 or pyridine-d5 as solvent. All chemical shifts were relative to tetramethylsilane for 1H and 13

C, to NO3- for 15N and to CCl3F for 19F.

Mass spectrometry. Direct introduction mass spectrometry analyses were performed on Qtof Premier (Waters) or on QTRAP 2000 (AB Sciex) mass spectrometers, in the positive ionization mode (ESI+) in the range m/z 80-700 amu. After dilution in water, samples were directly injected in water in the mass spectrometer without liquid chromatography system. The cone voltage was 30 or 50 V (Qtof) or 80 V (QTRAP) for MS. For direct introduction MS/MS analyses performed on Qtof Premier, the cone voltage was 30 V and collision energies 15 or 30 eV. LC/MS analyses. LC/MS analyses were performed on an UHPLC Waters Acquity system equipped with a Waters TQ-S micro mass spectrometer (triple quadrupole detector), an electrospray ionization source (ESI) and MassLynx™ software. The Acquity UPLC® BEH Shield RP18 (1.7 µm, 2.1 x 50 mm) column was used as a stationary phase. Eluent A was ammonium formate 5 mM pH 3.2 and eluent B was formic acid 0.1% (v/v) in acetonitrile. The flow rate was 0.4 mL/min and the column temperature was 30 °C. Eluting conditions for sunitinib were a linear gradient of A and B as follows: the percentage of B, initially set at 5%, increased to reach 15% (t = 0.5 min), 30% (t = 3 min), 35% (t = 3.5 min), stayed at 35% until 5.5 min, then increased again to reach 95% (t = 6.5 min), stayed at 95% until 7.5 min, then returned to initial conditions over 0.2 min. Afterward, the system was reequilibrated for 1.3 min before the following injection. Eluting conditions for pazopanib were a linear gradient of A and B as follows: the percentage of B, initially set at 5%, increased to reach 15% (t = 0.5 min), 45% (t = 2 min), 85% (t = 2.5 min), 95% (t = 3 min), stayed at 95% until 3.5 min, then returned to initial conditions over 0.2 min. Afterward, the system was re-equilibrated for 2.3 min before the following injection. The mass spectrometer was operated in positive ionization mode (ESI+), using cone voltages of 60 or 80 V, and collision energy of 30 eV. The spectra were acquired in Full Scan (m/z range 200-500 amu for sunitinib, 200-520 amu for pazopanib) and Multiple Reaction Monitoring (MRM) modes. MS/MS MRM transitions were as follows: for sunitinib 1: m/z 399→326, 283;

for 1-CH2OH: m/z 415→342, 299; for 1-CHO: m/z 413→340, 297, 268; for 1-CH=N–OCH3: ACS Paragon Plus Environment

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m/z 442→369, 326; for 1-NHEt: m/z 371→326, 283; for pazopanib 2: m/z 438→357, 341; for 2-Cl: m/z 472→436, 391, 355; for 2-CH2OH, R = H: m/z 454→436, 373; for 2-CHO, R = H: m/z 452→371, 343, 328; for 2-CH2OH, R = Cl: m/z 488→434, 407; for 2-CHO, R = Cl: m/z 486→405, 371; for 2-CH=N–OCH3: m/z 481→449, 368.

Chemical studies Neutralization of sunitinib L-malate. Sunitinib as a free base (1) was prepared by neutralization of the sunitinib salt form. Sunitinib L-malate (100 mg, 188 µmol) was dissolved in 40 mL of a NaOH aqueous solution (1 M) and extracted three times by 40 mL of CH2Cl2. The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo to yield the sunitinib free base (1, 74.8 mg, 99.6% recovery). The product is a mixture of isomers having Z and E configurations at C3–C8. For the sake of clarity, the NMR spectra of Z and E isomers are described separately. TLC (neutral Al2O3, CH2Cl2/CH3OH, 95/5, v/v): Rf = 0.75. 1H NMR (500 MHz, methanol-d4) for Z isomer: δ (ppm) = 7.60 (s, 1H, H8), 7.44 (m, 1H, H4), 6.88 (m, 2H, H6 and H7), 3.54 (t, 2H, H2C16), 2.83 (t, 2H, H2C17), 2.77 (q, 4H, 2 x H2C19), 2.52 (s, 3H, H3C–C11), 2.48 (s, 3H, H3C–C13), 1.16 (t, 6H, 2 x H3C20); for E isomer: δ (ppm) = 7.60 (s, 1H, H8), 7.43 (m, 1H, H4), 6.90 (m, 1H, H6), 6.88 (m, 1H, H7), 3.54 (t, 2H, H2C16), 2.83 (t, 2H, H2C17), 2.77 (q, 4H, 2 x H2C19), 2.52 (s, 3H, H3C–C11), 2.33 (s, 3H, H3C– C13), 1.16 (t, 6H, 2 x H3C20). 1H NMR (500 MHz, pyridine-d5) for Z isomer: δ (ppm) = 15.41 (1H, HN10), 13.41 (1H, HN1), 8.92 (1H, H8), 8.80 (1H, H7), 8.61 (1H, HN15), 8.16 (1H, H6), 8.11 (1H, H4), 4.81 (2H, H2C16), 3.78 (2H, H2C17), 3.78 (3H, H3C–C13), 3.74 (3H, H3C–C11), 3.61 (4H, 2 x H2C19), 2.09 (6H, 2 x H3C20). 13C NMR (125.72 MHz, methanol-d4) for Z isomer: δ (ppm) = 170.20 (C2), 167.27 (C14), 159.18 (C5), 136.70 (C11), 134.50 (C7a), 130.16 (C9), 127.20 (C3a), 126.13 (C13), 123.90 (C8), 119.45 (C12), 115.50 (C3), 112.40 (C6), 109.80 (C7), 104.87 (C4), 51.52 (C17), 48.73 (2 x C19), 36.52 (C16), 11.98 (H3C–C11), 10.00 (2 x C20), 9.27 (H3C–C13); for E isomer: δ (ppm) = 171.33 (C2), 134.50 (C7a), 128.16 (C9), 125.05 (C8), 123.90 (C13), 119.57 (C3), 119.19 (C12), 112.48 (C6), 109.80 (C7), 108.70 (C4), 51.60 (C17), 46.89 (2 x C19), 36.44 (C16), 12.00 (H3C–C11), 10.01 (2 x C20), 9.89 (H3C–C13). 13C NMR (125.72 MHz, pyridine-d5) for Z isomer: δ (ppm) = 172.00 (C2), 166.50 (C14), 160.26 (C5), 138.45 (C11), 136.70 (C7a), 131.21 (C9), 129.22 ACS Paragon Plus Environment


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(C3a), 127.97 (C13), 125.62 (C8), 122.10 (C12), 116.96 (C3), 113.76 (C6), 111.35 (C4), 106.99 (C7), 53.34 (C17), 47.92 (2 x C19), 38.65 (C16), 14.71 (H3C–C11), 13.04 (2 x C20), 12.10 (H3C–C13). 15N NMR (50.66 MHz, methanol-d4): δ (ppm) = –338.6 (N18), –218.0 (N10).

19

F NMR (376.31 MHz,

methanol-d4) for Z isomer: δ (ppm) = –124.11 (m); for E isomer: δ (ppm) = –124.00 (m). MS (ESI+/CH3OH): m/z = 399.2 (MH+); MS/MS fragments (30 eV): m/z = 326.1 [MH+ – (CH3CH2)2NH], m/z = 283.1 [MH+ – (CH3CH2)2N(CH2)2NH2]. Standard conditions for non-catalytic oxidations. Homogeneous conditions. Sunitinib (1, 4.0 mg, 10 µmol) was dissolved in CH2Cl2 (1 mL). Meta-chloroperbenzoic acid (mCPBA, 15 µmol) was then added. The solution was stirred at room temperature for 10 minutes and the reaction was quenched by addition of acetone (1 mL). The reaction mixture was then purified by column chromatography (neutral Al2O3, CH2Cl2/CH3OH, 100/0 to 90/10, v/v) to afford an orange solid. Quantitative yield. Biphasic conditions. In 1.15 mL of CH2Cl2 containing 4 mg of sunitinib (1, 10 µmol), benzyldimethyltetradecylammonium

chloride

(BDTAC,

4

µmol)

was

added.

Magnesium

monoperphthalate (MMPP, 15 µmol in 0.4 mL of water, i.e. 30 µmol of monoperphthalate) or potassium hydrogen persulfate (KHSO5, 30 µmol in 0.4 mL of water) was then added, and the reaction mixture was magnetically stirred for 30 minutes. The reaction mixture was purified by column chromatography (neutral Al2O3, CH2Cl2/CH3OH, 100/0 to 98/2, v/v). TLC (neutral Al2O3, CH2Cl2/CH3OH, 95/5, v/v): Rf = 0.43. 1H NMR (500 MHz, methanol-d4) for Z isomer: δ (ppm) = 7.58 (s, 1H, H8), 7.43 (m, 1H, H4), 6.88 (m, 1H, H6), 6.86 (m, 1H, H7), 3.82 (t, 2H, H2C16), 3.46 (t, 2H, H2C17), 3.38 (q, 4H, 2 x H2C19), 2.52 (s, 3H, H3C–C11), 2.48 (s, 3H, H3C– C13), 1.37 (t, 6H, 2 x H3C20); for E isomer: δ (ppm) = 7.60 (s, 1H, H8), 7.41 (m, 1H, H4), 6.96 (m, 1H, H6), 6.88 (m, 1H, H7), 3.82 (t, 2H, H2C16), 3.46 (t, 2H, H2C17), 3.38 (q, 4H, 2 x H2C19), 2.53 (s, 3H, H3C–C11), 2.33 (s, 3H, H3C–C13), 1.37 (t, 6H, 2 x H3C20). 13C NMR (125.72 MHz, methanol-d4) for Z isomer: δ (ppm) = 170.26 (C2), 167.23 (C14), 159.08 (C5), 136.93 (C11), 134.30 (C7a), 130.24 (C9), 127.21 (C3a), 126.16 (C13), 123.89 (C8), 118.79 (C12), 115.54 (C3), 112.42 (C6), 109.84 (C7), 105.00 (C4), 61.97 (C17), 59.93 (2 x C19), 33.80 (C16), 12.16 (H3C–C11), 9.49 (H3C–C13), 7.26 (2 x C20); for E isomer: δ (ppm) = 171.13 (C2), 137.50 (C11), 127.97 (C3a), 127.88 (C9), 124.99 (C8), 123.55 (C13), 119.80 (C3), 118.79 (C12), 113.89 (C6), 109.80 (C7), 108.60 (C4), 61.97 (C17), 59.93 ACS Paragon Plus Environment

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(2 x C19), 33.80 (C16), 11.65 (H3C–C11), 10.00 (H3C–C13), 7.26 (2 x C20). 15N NMR (50.66 MHz, methanol-d4): δ (ppm) = –258.3 (N18→O), –213.3 (N10). 19F NMR (376.31 MHz, methanol-d4) for Z isomer: δ (ppm) = –124.07 (m); for E isomer: δ (ppm) = –123.98 (m). MS (ESI+/CH3OH): m/z = 415.2 (MH+); MS/MS fragments (30 eV): m/z = 326.1 [MH+ – (CH3CH2)2NH(→O)], m/z = 283.1 [MH+ – (CH3CH2)2N→O(CH2)2NH2]. Standard conditions for catalytic oxidations. Sunitinib. Sunitinib (1, 12.0 mg, 30 µmol) was dissolved in CH2Cl2 (2 mL). MnIII(TDCPP)Cl (0.6 µmol in 0.3 mL of CH2Cl2), 4-t-butylpyridine (15 µmol in 0.15 mL of CH2Cl2), BDTAC (4.5 mg, 12 µmol) then KHSO5 (90 µmol in 0.4 mL of water) were added. The reaction mixture was stirred at room temperature. After 60 or 180 minutes, the reaction was stopped by addition of acetone (1 mL). The aqueous phase was then purified by column chromatography (reverse phase C18, H2O/CH3OH/0.5% NH4OH, 50/50 to 0/100, v/v). 1H NMR (500 MHz, pyridine-d5) for 1-CH2OH: δ (ppm) = 8.94 (s, 1H, HN15), 8.94 (s, 1H, H8), 8.83 (m, 1H, H7), 8.21 (m, 1H, H6), 8.10 (m, 1H, H4), 6.52 (s, 2H, HO-H2C–C11), 4.81 (t, 2H, H2C16), 3.87 (t, 2H, H2C17), 3.83 (s, 3H, H3C–C13), 3.61 (q, 4H, 2 x H2C19), 2.09 (t, 6H, 2 x H3C20); for 1-CHO: δ (ppm) = 11.70 (s, 1H, OHC–C11), 9.86 (s, 1H, HN15), 4.90 (t, 2H, H2C16), 3.86 (t, 2H, H2C17), 3.61 (q, 4H, 2 x H2C19), 2.09 (t, 6H, 2 x H3C20); for 1-CH2OCH3: δ (ppm) = 8.87 (s, 1H, H8), 6.09 (s, 2H, H3CO-H2C–C11), 4.52 (s, 3H, H3CO-H2C–C11), 3.75 (s, 3H, H3C–C13).

13

C NMR (125.72 MHz,

pyridine-d5) for 1-CH2OH: δ (ppm) = 171.95 (C2), 160.20 (C5), 143.49 (C11), 138.52 (C7a), 131.69 (C9), 127.73 (C13), 120.56 (C12), 117.88 (C3), 116.32 (C6), 111.39 (C4), 108.53 (C7), 59.11 (HOH2C–C11), 53.43 (C17), 48.00 (2 x C19), 38.80 (C16), 13.00 (2 x C20), 12.33 (H3C–C13); for 1CHO: δ (ppm) = 182.40 (OHC–C11), 133.78 (C11), 53.43 (C17), 48.00 (2 x C19), 39.29 (C16), 13.00 (2 x C20); for 1-CH2OCH3: δ (ppm) = 171.37 (C2), 137.34 (C11), 132.74 (C9), 129.73 (C13), 120.06 (C12), 68.50 (H3CO-H2C–C11), 59.40 (H3CO-H2C–C11), 11.45 (H3C–C13). MS (ESI+/CH3OH): m/z = 399.2 (MH+ for 1), 413.2 (MH+ for 1-CHO), 415.2 (MH+ for 1-CH2OH), 429.2 (MH+ for 1CH2OCH3), 432.2, 675.4. MS/MS fragments of the parent ion m/z = 413.2 (30 eV): m/z = 340.1 [MH+ – (CH3CH2)2NH], 297.1 [MH+ – CH3CH2)2N(CH2)2NH2], 296.1, 268.1, 240.1; MS/MS fragments of the parent ion m/z = 415.2 (30 eV): m/z = 342.1 [MH+ – (CH3CH2)2NH], 299.1 [MH+ – (CH3CH2)2N(CH2)2NH2]. Contaminated with a minor product oxidized on the side chain ACS Paragon Plus Environment


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(hydroxylation or N-oxidation) giving rise to fragments m/z = 326.1 [MH+ – (CH3CH2)2NH + O], 283.1 [MH+ – (CH3CH2)2N(CH2)2NH2 + O]. Pazopanib. Pazopanib hydrochloride (2, 4.7 mg, 10 µmol) was dissolved in acetic acid (the solvent was incrementally added under gentle heating until complete dissolution). MnIII(TDCPP)Cl (0.2 µmol in 0.1 mL of acetic acid), 4-t-butylpyridine (5 µmol in 0.05 mL of acetic acid) then MMPP (15 µmol, i.e. 30 µmol of monoperphthalate) were added. The reaction mixture was stirred at room temperature. After 90 minutes, the reaction was stopped by addition of acetone (1 mL). The crude mixture was then analyzed by mass spectrometry, NMR and LC-MS without further purification. 1H NMR (500 MHz, pyridine-d5) for 2: δ (ppm) = 11.61 (1H, HN15), 10.48 (1H, H17), 9.42 (1H, H21), 9.15 (1H, H13), 8.84 (1H, H3), 8.78 (1H, H6), 8.43 (1H, H20), 8.04 (1H, H5), 7.09 (1H, H14), 5.12 (3H, H3C–N1), 4.69 (3H, H3C–N8), 4.05 (3H, H3C–C19), 3.56 (3H, H3C–C7); for 2-Cl: δ (ppm) = 11.84 (1H, HN15), 10.40 (1H, H17), 9.32 (1H, H21), 9.26 (1H, H13), 8.68 (1H, H6), 8.46 (1H, H20), 8.13 (1H, H5), 5.07 (3H, H3C–N1), 4.73 (3H, H3C–N8), 4.06 (3H, H3C–C19), 3.48 (3H, H3C–C7); for 2-CH2OH, R14 = H or Cl: δ (ppm) = 6.33/6.39 (2H, HO-H2C-C7), 5.40/5.45 (3H, H3C–N1); for 2-CHO, R14 = H or Cl: δ (ppm) = 11.56/11.62 (1H, OHC–C7), 5.56/5.64 (3H, H3C–N1); for 2-CH2OCOCH3, R14 = H or Cl: δ (ppm) = 6.73/6.80 (2H, H3C-OC-O-H2C–C7), 5.34/5.38 (3H, H3C–N1), 3.11/3.11 (3H, H3C-OC-OH2C–C7). 13C NMR (125.72 MHz, pyridine-d5) for 2: δ (ppm) = 164.23 (C9), 156.46 (C13), 149.18 (C2a), 144.27 (C18), 143.64 (C4), 141.50 (C16), 133.92 (C20), 133.05 (C7), 129.36 (C19), 123.13 (C21), 121.80 (C6), 121.30 (C6a), 121.25 (C5), 119.72 (C17), 116.00 (C3), 98.59 (C14), 39.46 (H3C– N8), 38.30 (H3C–N1), 20.73 (H3C–C19), 10.39 (H3C–C7); for 2-Cl: δ (ppm) = 159.62 (C9), 158.45 (C13), 146.37 (C2a), 144.37 (C18), 141.02 (C4), 134.65 (C7), 134.00 (C20), 129.98 (C19), 123.27 (C21), 122.92 (C5), 122.38 (C6a), 120.77 (C6), 119.84 (C17), 107.83 (C14), 41.64 (H3C–N8), 38.50 (H3C–N1), 20.73 (H3C–C19), 10.48 (H3C–C7); for 2-CH2OH, R14 = H or Cl: δ (ppm) = 136.80/136.84 (C7), 121.30/122.20 (C6a), 54.44/54.50 (HO-H2C–C7), 39.10/39.19 (H3C–N1); for 2CHO, R14 = H or Cl: δ (ppm) = 180.00/180.00 (OHC–C7), 132.60/132.60 (C7), 123.50/124.00 (C6a), 41.39/41.49 (H3C–N1); for 2-CH2OCOCH3, R14 = H or Cl: δ (ppm) = 171.20/171.20 (H3C-OC-OH2C–C7),

131.40/131.40

(C7),

55.70/55.71

(H3C-OC-O-H2C–C7),

39.10/39.10

(H3C–N1),

21.27/21.27 (H3C-OC-O-H2C–C7). MS (ESI+/acetic acid): m/z = 438.50 (MH+), 452.50 (MH+ for 2ACS Paragon Plus Environment

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CHO), 454.40 (MH+ for 2-CH2OH), 472.40 (MH+ for 2-Cl), 486.30 (MH+ for 2-CHO, R = Cl), 488.30 (MH+ for 2-CH2OH, R = Cl), 496.40 (MH+ for 2-CH2OCOCH3) 506.40 (MH+ for a dichlorinated derivative of 2-CH2OH). Standard conditions for derivatization reactions. An aliquot of an aldehyde-containing medium (ca. 4 or 2 µmol of aldehyde derivative for sunitinib or pazopanib, respectively) was dissolved in H2O/CH3OH (0.5 mL, 80/20 v/v for sunitinib; 1.1 mL, 10/90 v/v for pazopanib). Sodium acetate (160 µmol in 0.05 or 0.2 mL of water for sunitinib or pazopanib, respectively) and methoxylamine (80 µmol in 0.05 or 0.2 mL of water for sunitinib or pazopanib, respectively) were then added. In both cases, the coupling reaction was accelerated by warming the mixture at +37 °C for 120 minutes. The crude reaction media were then analyzed by UHPLC-MS.

Biological studies Microsomal incubations were performed according to supplier protocol. An aliquot of human liver microsomes (final protein concentration = 0.5 mg/mL) was added in a mixture of NADPH regenerating solutions A (12.5 µL), B (2.5 µL) and 0.1 M phosphate buffer (pH = 7.4, final volume = 250 µL). After pre-incubation at 37 °C for 5 min, microsomal incubations were started by the addition of sunitinib or pazopanib solutions (final substrate concentration 10 or 50 µM in DMF/DMSO, final solvent concentration below 0.2%). Incubations were performed at 37 °C during 90-120 minutes and stopped by the addition of 250 µL of acetonitrile for protein precipitation. The incubation mixture was then centrifuged 20 min at 4000 g, at room temperature. After filtration (0.2 µm syringe filters), supernatants were analyzed by UHPLC-MS. Negative control incubations were performed in the absence of NADPH regenerating solutions. Positive control incubations were performed with another TKI, imatinib mesylate (final concentration 10 µM): the generation of the main imatinib metabolite, N-desmethylimatinib, upon HLM incubation was monitored by UHPLC-MS (data not shown).29 Derivatization of the aldehyde function was carried out by incubating human liver microsomes at 37 °C for 90 min with sunitinib or pazopanib (final substrate concentration 50 µM, final solvent concentration below 0.2%) in the presence of methoxylamine (final methoxylamine concentration 1 mM).



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Corresponding Author Information. Email addresses: [email protected], [email protected]

Acknowledgements. This work was supported by the CNRS, INSERM, and Institut Claudius Regaud, IUCT-O, Toulouse. Prof. Bernard Meunier, LCC-CNRS, Toulouse, is gratefully acknowledged for discussion about the manuscript. The Mass Spectrometry Core Facility of Institut de chimie de Toulouse (ICT) is gratefully acknowledged for direct introduction mass spectrometry analyses.

Abbreviations. BDTAC

Benzyldimethyltetradecylammonium chloride

CYP or P450

Cytochrome(s) P450

HLM

Human liver microsomes

mCPBA

meta-Chloroperbenzoic acid

MMPP

Magnesium monoperphthalate

MRM

Multiple reaction monitoring

MS/MS

Tandem mass spectrometry

PPA

Peracetic acid

RECC

Renal cell cancer

TKI

Tyrosine kinase inhibitors

Ancillary Information

Supporting Information. The Molecular Formula Strings (MFS), and Figures S1, S2, and S3 are provided as Supporting Information. ACS Paragon Plus Environment

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Figure S1. Mass spectrometry of sunitinib oxidation products. Assignment of m/z values detected in MRM MS/MS mode. Figure S2. UHPLC-MS of 1-CH=N–OCH3, the oxime derivatives of 1-CHO detected after oxidation of 1 by MnIII(TDCPP)Cl/KHSO5 or by human liver microsomes and derivatization

of

the

aldehyde

function

by

CH3ONH2.

Oxidation

by

MnIII(TDCPP)Cl/KHSO5: a) XIC for m/z = 442, Inserts: mass spectra of the products eluted at 4.77 and 5.09 min; b) MRM MS/MS 442→369 transition; c) MRM MS/MS 442→326 transition. Oxidation by human liver microsomes: d) XIC for m/z = 442; e) MRM MS/MS 442→326 transition. The structures of the products are depicted. Figure S3. UHPLC-MS of 2-CH=N–OCH3, the oxime derivatives of 2-CHO (R14 = H) detected after oxidation of 2 by MnIII(TDCPP)Cl/MMPP or by human liver microsomes and derivatization of the aldehyde function by CH3ONH2. Oxidation by MnIII(TDCPP)Cl/MMPP: a) XIC for m/z = 481, Inserts: mass spectra of the products eluted at 2.96 and 3.11 min; b) MRM MS/MS 481→449 transition; c) MRM MS/MS 481→368 transition. Oxidation by human liver microsomes: d) MRM MS/MS 481→368 transition. The proposed structures of the products are depicted.

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a) Meunier, B.; Robert, A.; Pratviel, G.; Bernadou, J. Metalloporphyrins in catalytic oxidations and oxidative DNA cleavage. In The Porphyrin Handbook, Kadish, K. M.; Smith, K. M.; Guilard R., Eds; Academic Press, 2000, vol. 4, pp 119-187. b) Meunier, B.; de Visser, S. P.; S. Shaik. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes. Chem. Rev. 2004, 104, 3947-3980. c) Huang, X.; Groves, J. T.

Oxygen

activation

and

radical

transformations

in

heme

proteins

and

metalloporphyrins. Chem. Rev. 2018, 118, 2491-2553. 14.

Bernadou, J.; Meunier, B. Biomimetic chemical catalysts in the oxidative activation of drugs. Adv. Synth. Catal. 2004, 346, 171-184.

15.

Vidal, M.; Bonnafous, M.; Defrance, S.; Loiseau, P.; Bernadou, J.; Meunier, B. Model systems for oxidative drug-metabolism studies - Catalytic behavior of water-soluble metalloporphyrins depends on both the intrinsic robustness of the catalyst and the nature of substrates. Drug Metab. Dispos. 1993, 21, 811-817.



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16.

Gaggero, N.; Robert, A.; Bernadou, J.; Meunier, B. Oxidation of SR 48117, an antagonist of vasopressin V1a receptors, by biomimetic catalysts based on metalloporphyrin or Schiff-base complexes. Bull. Soc. Chim. Fr. 1994, 131, 706-712.

17.

Nicolas, I.; Bijani, C.; Brasseur, D.; Pratviel, G.; Bernadou, J.; Robert, A. Metalloporphyrin-catalyzed hydroxylation of the N,N-dimethylamide function of the drug molecule SSR180575 to a stable N-methyl-N-carbinolamide. C. R. Chimie 2013, 16, 1002-1007.

18.

Cusack, K. P.; Koolman, H. F.; Lange, U. E. W.; Peltier, H. M.; Piel, I.; Vasudevan, A. Emerging technologies for metabolite generation and structural diversification. Bioorg. Med. Chem. Lett. 2013, 23, 5471-5483.

19.

Sistla, A.; Yang, W. L.; Shenoy, N. High-performance liquid chromatographic method for determination of reversible isomers of SU5416. J. Chromatogr. A 2006, 1110, 7380.

20.

Patel, P. N.; Kalariya, P. D.; Sharma, M.; Garg, P.; Talluri, M. V.; Gananadhamu, S.; Srinivasas, R. Characterization of forced degradation products of pazopanib hydrochloride by UHPLC-Q-TOF/MS and in silicotoxicity prediction. J. Mass. Spectrom. 2015, 50, 918-928.

21.

Robert, A.; Benoit-Vical, F.; Claparols, C.; Meunier, B. The antimalarial drug artemisinin alkylates heme in infected mice. Proc. Natl. Acad. Sci. USA 2005, 102, 13676-13680.

22.

Liu, X.-J.; Lu, H.; Sun, J.-X.; Wang, S.-R.; Mo, Y.-S.; Yang, X.-S.; Shi, B.-K. Metabolic behavior prediction of pazopanib by cytochrome P450 (CYP) 3A4 by molecular docking. Eur. J. Drug. Metab. Pharmacokinet. 2016, 41, 465-468.

23.

a) Liu, X.; Lu, Y.; Guan, X.; Dong, D.; Chavan, H.; Wang, J.; Zhang, Y.; Krishnamurthy, P.; Li, F. Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism. Biochem. Pharmacol. 2015, 97, 111-121. b) Attwa, ACS Paragon Plus Environment

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M. W.; Kadi, A. A.; Darwish, H. W.; Amer, S. M.; Alrabiah, H. A reliable and stable method for the determination of foretinib in human plasma by LC-MS/MS: application to metabolic stability investigation and excretion rate. Eur. J. Mass Spectrom. 2018, in press. Doi: 10.1177/1469066718768327. c) Schulz-Utermoehl, T.; Spear, M.; Pollard, C. R.; Pattison, C.; Rollison, H.; Sarda, S.; Ward, M.; Bushby, N.; Jordan, A.; Harrison, M. In vitro hepatic metabolism of cediranib, a potent vascular endothelial growth factor tyrosine kinase inhibitor: interspecies comparison and human enzymology. Drug Metab. Dispos. 2010, 38, 1688-1697. 24.

a) Sugiyama, M.; Fujita, K.-I.; Murayama, N.; Akiyama, Y.; Yamazaki, H.; Sasaki. Y. Sorafenib and sunitinib, two anticancer drugs, inhibit CYP3A4-mediated and activate CY3A5-mediated midazolam 1’-hydroxylation. Drug Metab. Dispos. 2011, 39, 757762. b) Keisner, S. V.; Shah, S. R. Pazopanib: the newest tyrosine kinase inhibitor for the treatment of advanced or metastatic renal cell carcinoma. Drugs 2011, 71, 443-454. c) Filppula, A. M.; Neuvonen, P. J.; Backman, J. T. In vitro assessment of timedependent inhibitory effects on CYP2C8 and CYP3A activity by fourteen protein kinase inhibitors. Drug Metab. Dispos. 2014, 42, 1202-1209.

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Harris, P. A.; Boloor, A.; Cheung, M.; Kumar, R.; Crosby, R. M.; Davis-Ward, R. G.; Epperly, A. H.; Hinkle, K. W.; Hunter, R. N.; Johnson, J. H.; Knick, V. B.; Laudeman, C. P.; Luttrell, D. K.; Mook, R. A.; Nolte, R. T.; Rudolph, S. K.; Szewczyk, J. R.; Truesdale, A. T.; Veal, J. M.; Wang, L.; Stafford, J. A. Discovery of 5-[[4-[(2,3Dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzenesulfonamide (Pazopanib), a novel and potent vascular endothelial growth factor receptor inhibitor. J. Med. Chem. 2008, 51, 4632-4640.

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a) Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMahon, G.; Tang, C. Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine kinases. J. Med. ACS Paragon Plus Environment


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Chem. 1998, 41, 2588-2603. b) Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui, J.; Fukuda, J. Y.; Chu, J.-Y.; Nematalla, A.; Wang, X.; Chen, H.; Sistla, A.; Luu, T. C.; Tang, F.; Wei, J.; Tang, C. Discovery of 5-[5-Fluoro-2-oxo-1,2- dihydroindol(3Z)-ylidenemethyl]-2,4-

dimethyl-1H-pyrrole-3-carboxylic

acid

(2-

diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase. J. Med. Chem. 2003, 46, 1116-1119. 27.

DeWitt, S. H. ; Maryanoff, B. E. Deuterated drug molecules : focus on FDA-approved deutetrabenazine. Biochemistry 2018, 57, 472-473.

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Paludetto, M.-N.; Puisset, F.; Le Louedec, F.; Allal, B.; Lafont, T.; Chatelut, E.; Arellano, C. Simultaneous monitoring of pazopanib and its metabolites by UPLC– MS/MS. J. Pharm. Biomed. Anal. 2018, 154, 373-383


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Table of contents graphic


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Sunitinib 1

20

19

18

N

17

O H N

16

H N

15

11 14

O

12

2 3

NH

10

9 13

7a

1

3a 8

Page 40 of 48

Pazopanib 2 7

5 4

6

N8

4 5

F

19 18

H 2N O

S O


N10

20

21 16 17

15

N H

11

9

14

12 13

N

6 3

6a

2a

7 2

1N

N

Page 41 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56


Substrate

Mn(Por)Cla / 4-tBu-pyridine single oxygen atom donorb

Oxidation, hydroxylation, N-dealkylation…

a Mn(Por)Cl

stands for a metalloporphyrin catalyst (see structures below),b the single oxygen atom donor is potassium hydrogen persulfate (KHSO5), hypochlorite, magnesium monoperphthalate (MMPP), peracetic acid (PAA) or meta-chloroperbenzoic acid (mCPBA).

MnIII(Por)Cl

R1 = R1

R2

R2 =

H

R2 N

Cl

N

MnIII

R1 N

R1

F R2

R2

R1

H Cl MnIII(TDCPP)Cl

MnIII(TMP)Cl

N

R2

R2 =

Cl

R2

R2

R1 =

Cl

R2

Cl MnIII(Cl

12TMP)Cl


F F

F F III Mn (PFPP)Cl

H


Sunitinib 1 m/z = 399.2

3.74 / O 14.71 138.45 8.61

20

2 3

10 H 9 N 14 8 12 13 127.97 16 15 O 3.78 / 12.10

17

19

11

NH

18 N

H N 7a 1 3a

4 5

F

KHSO5

O

O

17 8.94 H

19 20

H N

18 N

N H

O

H N

1-CHO m/z = 413.2 F

76 4 5

F

15

N

O


F

13

129.73

3.75 / 11.45

H N

11.7 / 182.4 133.78 O

OHC 9.86 H

11

H N

O 137.34 NH

O H N

3a

11

H N

N

NH 18

10

6.09 / 68.5

O

19

11

4.52 / 59.4

1-CH2OCH3 m/z = 429.2

O

2 3

NH

H N 7a 1

CH3OH / H+

F

1-NHEt m/z = 371.2

O

9 N 14 8 12 13 127.73 16 15 O 3.83 / 12.33

NH

20

143.49

Catalytic oxidation

H N

N

6.52 / 59.11

HOH2C

MnIII(TDCPP)Cl

Non catalytic KHSO5, MMPP oxidation or mCPBA

1-N-oxide m/z = 415.2

1-CH2OH m/z = 415.2

Catalytic oxidation

76

Page 42 of 48

NH

10

N 14 12 13 O

9

8

F

Page 43 of 48

a) TIC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

4.62Journal of Medicinal Chemistry

4.36

4.69 4.16

4

4.47

4.2

4.4

4.6

4.8

5

Retention time, min MS at 4.62 min

4.62

b) XIC m/z = 399 Sunitinib 1

399.1

390

4 c) XIC m/z 4.2= 415

1-CH2OH

4.4

4.6

m/z 410

400

4.8

5

4.8

413.1 5

4.16 4.70 4.36

4 d) XIC m/z 4.2= 413

4.4 4.36

4.6

1-CHO

MS at 4.36 min

390

4 e) XIC m/z 4.2= 429

4.4

400

410

4.6

4.8

MS at 4.56 min 429.1

1-CH2OCH3

m/z 420

5

4.47 410

4 f) XIC m/z4.2 = 371

1-NHEt

415

4.4 4.28

4.2

425

4.6

350

4

420

4.56

4.4

355

360

4.6

Retention time, min

430

m/z

435

440

4.8

5

MS at 4.28 min 371.1

365

370

375

m/z

380

ACS Paragon Plus Environment 4.8 5

420

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 44 of 48

Page 45 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.56 / 10.39 8.78 / 121.30 121.80 133.05 8.04 / 121.25 6 6a 7 5.12 / 5 1N 143.64 4 2a 38.30 2 4.69 / 3 N N 39.46 8 8.84 / 164.23 116.00 149.18 N10 9 14 7.09 / 98.59 11 13 9.15 / 156.46 15 12

Pazopanib 2 m/z = 438.2 42% 9.42 / 123.13 129.36 8.43 / 4.05 / 133.92 20.73 20 21 19 H 2N 18 16 17

N

S

m/z = 496.2 530.1a

4%

9.32 / 123.27 129.98 8.46 / 4.06 / 134.00 20.73 20 21 19 H 2N 18 16 17

S O

10.40 / 119.84

8.68 / 120.77 122.38

134.65 8.13 / 122.92 6 6a 7 5.07 / 5 1N 141.02 4 2a 38.50 2 4.73 / 3 N N8 41.64 146.37 159.62 Cl 9 N10 14 107.83 11 13 15 12 9.26 / 158.45

N H

O

3.11 / 21.27 3.11 / 21.27 171.20 / 171.20

131.40 / 131.40 7 2

N

N8 N

9

N

1N

R

N

11.84

2-CH2OH

2-CHO

R= H Cl

R= H Cl

m/z = 454.2 9% 488.1

121.30 / 122.20

6.39 / 54.44 6.33 / 54.50

CH3COOH

6a

N

9

132.60 / 132.60 1N

N

N8

O

123.50 / 124.00

7 2

5.34 / 39.10 5.38 / 39.10

m/z = 452.2 486.1 18% 11.56 / 180.00 / 180.00 11.62 H

OH 136.84 / 136.80

6a

3.48 / 10.48

144.37

O

6.80 / 55.70 6.73 / 55.71

27%

O

2-CH2OCOCH3 R= H Cl

m/z = 472.1

N

10.48 / H O 119.72 11.61 141.50 144.27

O

2-Cl

6a

5.45 / 39.19 5.40 / 39.10

2

14

N


N

9

N

R 14

1N

N

N8 R

14

7

5.56 / 41.49 5.64 / 41.39

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 46 of 48

Page 47 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56


2-CHO, R=H 2.84

2-Cl, R=Cl d) MRM 472→436

a) MRM 452→371

2.4

2.4

2.6 2.822.8

2.6

2.8

2.84

3

3.2

2.4 3.4

2.6 3.6

2.8 3.8

34

Mn(TDCPP)/ MMPP

3

3.2

3.4 2.4

3.6 2.6

3.8 2.8

34

3

2.4

2.6

3

2.8

2.84

2.4 3.4

2.6 3.6

2.8 3.8

2.4

2.6

2.6

2.8 2.82

3.2

2.4 3.4

2.6 3.6

2.8 3.8

2.8

3.2

3.4 2.4

Mn(TDCPP)/ MMPP

3

3.2

2.4 3.4

Retention time, min

4

3.6

3.8

4

3.55

34

3.2

3.4

3.6 3.54 3.8

4

34

3.2

3.4

3.6

3.8

4

3.8

4

3.8

4

f) MRM 472→355

c) MRM 452→328

3

3.4

Mn(TDCPP)/ MMPP

Microsomes

2.4

3.2

3.6 3.8 3.54

Microsomes

3.2

Mn(TDCPP)/ MMPP

3.4

e) MRM 472→391

Microsomes

2.6 2.82 2.8

3.2

Mn(TDCPP)/ MMPP

b) MRM 452→343

2.4

3.55

Microsomes

Microsomes

3.55

Microsomes

3.6 2.6

3.8 2.8

34

3.2

3.4

Mn(TDCPP)/ MMPP

2.6 3.6

2.8 3.8

34

3.2

3.4

3.6

3.54

3.6

Retention time, min ACS Paragon Plus Environment


Page 48 of 48

Anticancer tyrosine kinase inhibitors Sunitinib

O

H N

NH H N

N

F O N

Pazopanib N

S O

N H

Cytochromes P450

H

Reactive aromatic aldehydes

N

Human liver microsomes

N H 2N O

Biomimetic metalloporphyrin catalyzed oxidation

Hepatic toxicity

N


C

O

Metalloporphyrin catalyzed oxidation of sunitinib and pazopanib, two

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