2.2. Metabolite Identification of 25B-NBF
The incubation of 25B-NBF with human hepatocytes for 2 h at 37 °C resulted in the formation of 33 metabolites from 25B-NBF. Representative extracted ion chromatograms of 25B-NBF and its metabolites are presented in
Figure 2, and the retention times, molecular formulae, exact molecular ions ([M + H]
+), mass accuracies, fragment ions, and biotransformation are summarized in
Table 1.
25B-NBF produced an [M + H]
+ ion at
m/
z 368.0656 and the isotope ion at
m/
z 370.0635 with similar intensity to the [M + H]
+ ion owing to the characteristic isotope pattern of bromine. 25B-NBF produced three characteristic fragment ions at
m/
z 243.0015 [2-(4-bromo-2,5-dimethoxyphenyl)ethan-1-ylium],
m/
z 227.9780 [4-bromo-2,5-dimethoxyphenyl) methylium], and
m/
z 109.0448 [(2-fluorophenyl)methylium], which served as a marker of fragment ions for metabolite identification (
Figure 3A).
M1–M4 produced an [M + H]
+ ion at
m/
z 384.0605, 15.9949 amu higher than that of 25B-NBF, indicating that they were hydroxy-25B-NBF. M1–M4 produced product ions at
m/
z 260.0280,
m/
z 243.0015,
m/
z 227.9780, and
m/
z 125.0399 [(hydroxy-2-fluorophenyl)methylium], and therefore, M1–M4 were formed via hydroxylation at the fluorobenzyl moiety but the accurate hydroxylation position of each metabolite was not determined (
Figure 3B).
M5 and M6 showed an [M + H]
+ ion at
m/
z 354.0499, 14.0157 amu lower than 25B-NBF, indicating that they were
O-demethyl-25B-NBF. Based on the product ions at
m/
z 228.9859 and
m/
z 213.9624, which were 14 amu lower than
m/
z 243.0015 and
m/
z 227.9780 ions of 25B-NBF, respectively, M5 and M6 appeared to be
O-demethyl-25B-NBF (
Figure 3C). However, the accurate demethylation position of M5 and M6 could not be determined because of the absence of authentic standards.
The [M + H]
+ ion of M7 was observed at
m/
z 340.0343, 28.0313 amu lower than 25B-NBF, indicating bis-
O-demethyl-25B-NBF, which was supported by the characteristic product ions at
m/
z 214.9703 [2-(4-bromo-2,5-dihydroxyphenyl)ethan-1-ylium] and
m/
z 109.0451 (
Figure 3D).
M8 was identified as 2C-B [2-(4-bromo-2,5-dimethoxyphenyl)ethanamine] on the basis of its [M + H]
+ ion at
m/
z 260.0281 and product ions at
m/
z 243.0014 (loss of NH
3 from the [M + H]
+ ion) and
m/
z 227.9780 (
Figure 3E).
M9 produced an [M + H]
+ ion at
m/
z 544.0977, 176.0321 amu higher than 25B-NBF, and a product ion at
m/
z 368.0292 (25B-NBF ion due to loss of glucuronic acid), suggesting that it was 25B-NBF
N-glucuronide (
Figure 3F).
M10 produced an [M + H]
+ ion at
m/
z 487.0697, 119.0041 amu higher than 25B-NBF, and a product ion at
m/
z 243.0014 and
m/
z 227.9780, suggesting that M10 was a 25B-NBF cysteine conjugate. However, the accurate cysteine conjugation position was not identified (
Figure S1A).
M11–M17 showed an [M + H]
+ ion at
m/
z 370.0449, 1.9793 amu higher than 25B-NBF, suggesting that they were products of monohydroxylation and
O-demethylation. Six metabolites (M11–M15, M17) produced the characteristic product ions at
m/
z 245.0046 (loss of fluorobenzyl moiety from [M + H]
+ ion),
m/
z 228.9859,
m/
z 142.0663 (loss of 2-(4-bromo-hydroxymethoxyphenyl)ethane from [M + H]
+ ion), and
m/
z 125.0398 (
Figure S1B), indicating that M11–M15, and M17 were formed by
O-demethylation at the dimethoxyphenyl moiety and hydroxylation at the fluorobenzyl moiety of 25B-NBF. M16 produced product ions at
m/
z 228.9859,
m/
z 214.9702, and
m/
z 109.0451 (
Figure S1C), indicating that M16 was formed by
O-demethylation and hydroxylation at the amino group of 25B-NBF. The exact positions of hydroxylation and
O-demethylation for M11–M17 could not be identified.
M18–M20 produced an [M + H]
+ ion at
m/
z 560.0926, 192.0270 amu higher than 25B-NBF, and product ions at
m/
z 384.0605 (loss of glucuronic acid from [M + H]
+ ion),
m/
z 243.0015, and
m/
z 125.0399, suggesting that M18-M20 may be hydroxy-25B-NBF glucuronide via monohydroxylation at the fluorobenzyl moiety and glucuronidation (
Figure S2A). The exact location of hydroxylation and glucuronidation could not be determined.
M21–M23 produced [M + H]
+ ion at
m/
z 546.0769 and the product ions at
m/
z 370.0449 (loss of glucuronyl moiety from the [M + H]
+ ion), suggesting that they were hydroxy-
O-demethyl-25B-NBF glucuronides. M21 and M23 also produced the product ions at
m/
z 352.0345 (loss of H
2O from
m/
z 370.0449),
m/
z 228.9859,
m/
z 214.9702, and
m/
z 109.0448, suggesting glucuronidation of M16 (
Figure S2B). M22 produced product ions at
m/
z 422.0442 (loss of hydroxyflourobenzyl moiety from the [M + H]
+ ion),
m/
z 246.0124 (loss of glucuronosyl moiety from
m/
z 422.0442),
m/
z 228.9861, and
m/
z 125.0399 (
Figure S2C).
M24–M26 produced an [M + H]
+ ion at
m/
z 450.0017, 79.9568 amu higher than the [M + H]
+ ion of hydroxy-
O-demethyl-25B-NBF, and the product ions at
m/
z 370.0449 and
m/
z 228.9861, suggesting sulfation of hydroxy-
O-demethyl-25B-NBF. M24 and M25 produced the product ion at
m/
z 125.0399 (
Figure S3A), whereas M26 showed a
m/
z 109.0451 ion (
Figure S3B), indicating that the positions of hydroxylation in M24 and M25 were different from those of M26.
M27 and M28 produced an [M + H]
+ ion at
m/
z 246.0124, 14.0157 amu lower than M8, indicating
O-demethyl-2C-B, and these metabolites were confirmed by the presence of a product ion at
m/
z 228.9858 (loss of NH
3) and
m/
z 213.9597 (
Figure S3C).
M29 produced an [M + H]
+ ion at
m/
z 530.0820 and product ions at
m/
z 354.0499 (loss of glucuronyl moiety from [M + H]
+ ion) and
m/
z 228.9859, indicating that M29 was
O-demethyl-25B-NBF glucuronide (
Figure S4A).
M30 and M31 produced an [M + H]
+ ion at
m/
z 434.0068 and the product ions at
m/
z 354.0499 (loss of sulfate from the [M + H]
+ ion),
m/
z 308.9427 [loss of (2-fluorophenyl)methanamine from the [M + H]
+ ion),
m/
z 228.9859 (loss of sulfate from
m/
z 308.9427), and
m/
z 109.0451 (
Figure S4B), indicating that M30 and M31 were
O-demethyl-25B-NBF sulfates formed by sulfation at the OH group.
M32 produced an [M + H]
+ ion at
m/
z 516.0664 and product ions at
m/
z 340.0341 (loss of glucuronyl moiety from the [M + H]
+ ion),
m/
z 214.9701, and
m/
z 109.0451, indicating that M32 was bis-
O-demethyl-25B-NBF (M7) glucuronide (
Figure S4C).
M33 produced an [M + H]
+ ion at
m/
z 302.0386 and product ions at
m/
z 260.0280 (loss of acetyl moiety from the [M + H]
+ ion),
m/
z 243.0014 (loss of NH
3 from the [M + H]
+ ion) and
m/
z 227.9781, indicating that M33 was acetyl-2C-B (
Figure S4D).
2.3. Screening of CYPs and UGTs Responsible for the Metabolism of 25B-NBF
To characterize CYP enzymes involved in 25B-NBF metabolism, 25B-NBF (10 µM) was incubated with major human cDNA-expressed CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, or CYP3A5 in the presence of reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). The metabolism of 25B-NBF was mediated by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2J2, and CYP3A4 (
Figure 4). Some metabolites such as M4, M9, M10, M13, M14, M15, and M17 were not detected after incubation of 25B-NBF with recombinant CYP enzyme incubates.
UGT enzymes responsible for the glucuronidation of 25B-NBF were investigated with incubation of 25B-NBF in major human cDNA-expressed UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, and UGT2B15 in the presence of UDPGA. The formation of 25B-NBF glucuronide (M9) was solely mediated by UGT2B7 (
Figure 4). However, other glucuronides of phase I metabolites of 25B-NBF could not be identified using recombinant UGT enzymes because of no availability of authentic metabolite standards.