Abstract
Synthetic cannabinoids JWH-018, JWH-073 and AM2201 were metabolised by the fungus Cunninghamella elegans. In this article, data on individual metabolites of their retention times, mass accuracies, major product ions and structures indicated by product ions are presented. The data in this article is related to “Biotransformation of synthetic cannabinoids JWH-018, JWH-073 and AM2201 by Cunninghamella elegans” [1].
Specifications Table
| Subject area | Pharmacology |
| More specific subject area | Drug Metabolism |
| Type of data | Table |
| How data was acquired | Liquid chromatography -tandem mass spectrometry (Agilent 1290 LC system coupled to Agilent 6490 Triple Quadrupole mass spectrometer), high resolution quadrupole Time-of-Flight mass spectrometry (Agilent 6510 Accurate Mass QToF Mass Spectrometer) |
| Data format | Analysed |
| Experimental factors | Samples were extracted by dichloromethane. |
| Experimental features | Liquid chromatography-tandem mass spectrometry analysis of fungal metabolites of synthetic cannabinoids JWH-018, JWH-073 and AM2201 |
| Data source location | Sydney, Australia |
| Data accessibility | Data are available with this article |
Value of the data
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Chromatographic and mass spectrometric data on individual metabolites are provided for reference.
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Product ions indicative of the structures of metabolites are listed.
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The data can be compared with human or other in vitro metabolism.
1. Data
Table 1, Table 3, Table 5 list all the metabolites with biotransformation, retention time, observed accurate mass, formula and major product ions of JWH-018, JWH-073 and AM2201, respectively. Product ions representative of structures for JWH-018, JWH-073 and AM2201 metabolites are presented in Table 4, Table 6, respectively. Overlaid extracted ion chromatograms of all the metabolites of JWH-018, JWH-073 and AM2201 are shown in Fig. 2 of Ref. [1] with annotated metabolite identification (ID) names (Table 1, Table 2, Table 3, Table 4, Table 5, Table 6).
Table 1.
Metabolites of JWH-018 after C. elegans incubation.
| ID | Biotransformation | RT, min | m/z [M+H]+ | Mass accuracy (ppm) | Formula | Major product ions |
|---|---|---|---|---|---|---|
| Ma1 | Dihydrodiol formation+N-dealkylation | 8.0 | 306.1123 | −0.7 | C19H15NO3 | 143, 144, 171, 189 |
| Ma2 | Dihydrodiol formation+hydroxylation at pentyl side chain | 8.5 | 392.1852 | −1.0 | C24H25NO4 | 143, 144, 171, 189, 230, 374 |
| Ma3 | Dihydrodiol formation+hydroxylation at pentyl side chain | 9.2 | 392.1850 | −1.6 | C24H25NO4 | 143, 144, 171, 189, 230, 374 |
| Ma4 | Dihydrodiol formation+ketone formation at pentyl side chain | 9.3 | 390.1694 | −1.4 | C24H23NO4 | 143, 144, 171, 189, 228 |
| Ma5 | Dihydrodiol formation+ketone formation at pentyl side chain | 10.3 | 390.1695 | −1.1 | C24H23NO4 | 143, 144, 171, 189, 228 |
| Ma6 | Dihydroxylation at pentyl side chain and naphthalene moiety | 12.5 | 374.1745 | −1.5 | C24H23NO3 | 143, 144, 171, 230 |
| Ma7 | Dihydroxylation at pentyl side chain | 12.8 | 374.1745 | −1.6 | C24H23NO3 | 127, 144, 155, 246 |
| Ma8 | Dihydroxylation at pentyl side chain and naphthalene moiety | 13.1 | 374.1746 | −1.3 | C24H23NO3 | 143, 144, 171, 230 |
| Ma9 | Dihydroxylation at pentyl side chain and naphthalene moiety | 14.1 | 374.1746 | −1.3 | C24H23NO3 | 143, 144, 171, 230 |
| Ma10 | Ketone formation at pentyl side chain+hydroxylation at naphthalene moiety | 14.5 | 372.1590 | −1.0 | C24H21NO3 | 143, 144, 171, 228 |
| Ma11 | Dihydroxylation at pentyl side chain and naphthalene moiety | 14.9 | 374.1745 | −1.5 | C24H23NO3 | 143, 144, 171, 230 |
| Ma12 | Dihydrodiol formation | 16.1 | 376.1901 | −1.6 | C24H25NO3 | 143, 171, 189, 214 |
| Ma13 | Carboxylation (N-pentanoic acid)a | 16.4 | 372.1591 | −0.9 | C24H21NO3 | 127, 144 155 244 |
| Ma14 | Hydroxylation at pentyl side chain (5-hydroxypentyl)a | 17.2 | 358.1796 | −1.6 | C24H23NO2 | 127, 144, 155, 230 |
| Ma15 | Hydroxylation at pentyl side chain (4-hydroxypentyl)a | 17.6 | 358.1798 | −1.0 | C24H23NO2 | 127, 144, 155, 230 |
| Ma16 | Hydroxylation at pentyl side chain | 19.9 | 358.1798 | −1.1 | C24H23NO2 | 127, 144, 155, 230 |
| Ma17 | Ketone formation at pentyl side chain | 20.2 | 356.1640 | −1.6 | C24H21NO2 | 127, 144, 155, 228 |
| Ma18 | Ketone formation at pentyl side chain | 22.4 | 356.1641 | −1.2 | C24H21NO2 | 127, 144, 155, 228 |
| Ma19 | Hydroxylation at naphthalene moiety | 24.3 | 358.1797 | −1.3 | C24H23NO2 | 143, 171, 214 |
| Ma20 | Hydroxylation at naphthalene moiety | 24.8 | 358.1797 | −1.2 | C24H23NO2 | 143, 171, 214 |
| Ma21 | Dehydrogenation | 28.7 | 340.1692 | −1.1 | C24H21NO | 127, 144, 155, 212 |
| JWH-018 | 31.4 | 342.1851 | −0.5 | C24H23NO | 127, 144, 155, 214 |
Position confirmed by the use of reference standards.
Table 3.
Metabolites of JWH-073 after C. elegans incubation.
| ID | Biotransformation | RT, min | m/z [M+H]+ | Mass accuracy (ppm) | Formula | Major product ions |
|---|---|---|---|---|---|---|
| Mb1 | Dihydrodiol formation+N-dealkylation | 8.0 | 306.1123 | −0.5 | C19H15NO3 | 143, 144, 171 |
| Mb2 | Dihydrodiol formation+hydroxylation at butyl side chain | 8.2 | 378.1700 | −0.1 | C23H23NO4 | 143, 144, 171, 189, 216 |
| Mb3 | Dihydrodiol formation+hydroxylation at butyl side chain | 8.9 | 378.1690 | −2.6 | C23H23NO4 | 143, 144, 171, 189, 216 |
| Mb4 | Dihydrodiol formation+ketone formation at butyl side chain | 9.0 | 376.1543 | −0.1 | C23H21NO4 | 143, 144, 171, 189, 214 |
| Mb5 | Dihydroxylation at butyl side chain | 11.7 | 360.1594 | 0.0 | C23H21NO3 | 127, 144, 155, 232 |
| Mb6 | Dihydroxylation at butyl side chain and naphthalene moiety | 12.1 | 360.1595 | 0.1 | C23H21NO3 | 143, 144, 171, 216 |
| Mb7 | Dihydroxylation at butyl side chain and naphthalene moiety | 12.8 | 360.1594 | −0.1 | C23H21NO3 | 143, 144, 171, 216 |
| Mb8 | Dihydrodiol formation | 13.7 | 362.1751 | 0.1 | C23H23NO3 | 143, 144, 171, 189, 200 |
| Mb9 | Ketone formation at butyl side chain+hydroxylation at naphthalene moiety | 13.9 | 358.1438 | 0.0 | C23H19NO3 | 143, 144, 171, 214 |
| Mb10 | Ketone formation at butyl side chain+hydroxylation at naphthalene moiety | 14.5 | 358.1438 | 0.0 | C23H19NO3 | 143, 144, 171, 214 |
| Mb11 | Carboxylation (N-butanoic acid)a | 15.4 | 358.1437 | −0.1 | C23H19NO3 | 127, 144, 155, 230 |
| Mb12 | Hydroxylation at butyl side chain (4-hydroxybutyl)a | 15.7 | 344.1645 | −0.2 | C23H21NO2 | 127, 144, 155, 216 |
| Mb13 | Hydroxylation at butyl side chain (3-hydroxybutyl)a | 17.1 | 344.1646 | 0.3 | C23H21NO2 | 127, 144, 155, 216 |
| Mb14 | Ketone formation at butyl side chain | 19.5 | 342.1488 | 0.0 | C23H19NO2 | 127, 144, 155, 214 |
| Mb15 | Hydroxylation at naphthalene moiety | 21.5 | 344.1645 | −0.1 | C23H21NO2 | 143, 144, 171, 200 |
| Mb16 | Hydroxylation at naphthalene moiety | 22.0 | 344.1645 | 0.0 | C23H21NO2 | 143, 144, 171, 200 |
| Mb17 | Dehydrogenation | 25.7 | 326.1539 | −0.1 | C23H19NO | 127, 155, 198 |
| JWH-073 | 28.8 | 328.1697 | 0.3 | C23H21NO | 127, 144, 155, 200 |
Position confirmed by the use of reference standards.
Table 5.
Metabolites of AM2201 after C. elegans incubation.
| ID | Biotransformation | RT, min | m/z [M+H]+ | Mass accuracy (ppm) | Formula | Major product ions |
|---|---|---|---|---|---|---|
| Mc1 | Dihydrodiol formation+N-dealkylation | 8.0 | 306.1122 | −1.0 | C19H15NO3 | 143, 144, 171, 189 |
| Mc2 | Dihydroxylation at pentyl side chain and naphthalene moiety+glucosidation | 8.2 | 554.2185 | 0.0 | C30H32FNO8 | 143, 144, 171, 248, 392 |
| Mc3 | Dihydroxylation at pentyl side chain and naphthalene moiety+glucosidation | 8.4 | 554.2186 | 0.2 | C30H32FNO8 | 143, 144, 171, 248, 392 |
| Mc4 | Oxidative defluorination+dihydrodiol formation (JWH-018 dihydrodiol-hydroxy) | 8.5 | 392.1858 | 0.4 | C24H25NO4 | 143, 171, 189, 230 |
| Mc5 | Dihydrodiol formation+hydroxylation at pentyl side chain | 8.5 | 410.1764 | 0.6 | C24H24FNO4 | 143, 144, 171, 189, 248 |
| Mc6 | Dihydrodiol formation+hydroxylation at pentyl side chain | 8.7 | 410.1761 | −0.2 | C24H24FNO4 | 143, 144, 171, 189, 248 |
| Mc7 | Dihydroxylation at pentyl side chain and/or indole moiety+glucosidation | 9.0 | 554.2186 | 0.3 | C30H32FNO8 | 127, 155, 264, 392 |
| Mc8 | Trihydroxylation at pentyl side chain, indole moiety and naphthalene moiety | 9.3 | 408.1606 | 0.1 | C24H22FNO4 | 143, 160, 171, 264 |
| Mc9 | Dihydrodiol formation+dihydroxylation at indole moiety | 9.5 | 426.1712 | 0.1 | C24H24FNO5 | 143, 171, 176, 189, 264 |
| Mc10 | Dihydrodiol formation+ketone formation at pentyl side chain | 9.7 | 408.1606 | 0.0 | C24H22FNO4 | 143, 171, 189, 246 |
| Mc11 | Trihydroxylation at pentyl side chain, indole moiety and naphthalene moiety | 10.2 | 408.1606 | 0.0 | C24H22FNO4 | 143, 160, 171, 264 |
| Mc12 | Hydroxylation at indole moiety+glucosidation | 10.5 | 538.2235 | −0.1 | C30H32FNO7 | 127, 155, 160, 248, 376 |
| Mc13 | Dihydroxylation at pentyl side chain and/or indole moiety+glucosidation | 10.7 | 554.2183 | −0.3 | C30H32FNO8 | 127, 155, 264, 392 |
| Mc14 | Hydroxylation at naphthalene moiety+glucosidation | 10.8 | 538.2234 | −0.4 | C30H32FNO7 | 143, 144, 171, 232, 376 |
| Mc15 | Oxidative defluorination+hydroxylation at indole moiety (JWH-018 dihydroxy) | 11.1 | 374.1751 | 0.2 | C24H23NO3 | 127, 155, 160, 246 |
| Mc16 | Dihydroxylation at naphthalene+glucosidation | 11.5 | 554.2182 | −0.4 | C30H32FNO8 | 187, 232, 392 |
| Mc17 | Dihydroxylation at indole moiety and pentyl side chain | 11.5 | 392.1657 | 0.0 | C24H22FNO3 | 127, 155, 160, 264 |
| Mc18 | Oxidative defluorination+hydroxylation at indole moiety (JWH-018 dihydroxy) | 11.8 | 374.1748 | −0.8 | C24H23NO3 | 127, 155, 160, 246 |
| Mc19 | Dihydroxylation at indole moiety and pentyl side chain | 11.9 | 392.1656 | −0.2 | C24H22FNO3 | 127, 155, 160, 264 |
| Mc20 | Oxidative defluorination to carboxylic acid+hydroxylation at naphthalene moiety | 12.0 | 388.1547 | 1.0 | C24H21NO4 | 143, 144, 171, 244 |
| Mc21 | Oxidative defluorination+hydroxylation at pentyl side chain (JWH-018 dihydroxy) | 12.1 | 374.1751 | 0.2 | C24H23NO3 | 127, 144, 155, 246 |
| Mc22 | Dihydroxylation at indole moiety and pentyl side chain | 12.2 | 392.1658 | 0.5 | C24H22FNO3 | 127, 155, 160, 264 |
| Mc23 | Oxidative defluorination+hydroxylation at naphthalene moiety (JWH-018 dihydroxy) | 12.3 | 374.1749 | −0.4 | C24H23NO3 | 143, 144, 171, 230 |
| Mc24 | Dihydroxylation at pentyl side chain and naphthalene moiety | 12.5 | 392.1651 | 1.4 | C24H22FNO3 | 143, 144, 171, 248 |
| Mc25 | Dihydrodiol formation | 12.5 | 394.1812 | −0.3 | C24H24FNO3 | 143, 144, 171, 189, 232 |
| Mc26 | Oxidative defluorination to carboxylic acid+hydroxylation at naphthalene moiety | 12.7 | 388.1544 | 0.3 | C24H21NO4 | 143, 144, 171, 244 |
| Mc27 | Dihydroxylation at indole moiety and pentyl side chain | 12.7 | 392.1657 | 0.0 | C24H22FNO3 | 127, 155, 160, 264 |
| Mc28 | Oxidative defluorination+hydroxylation at naphthalene moiety (JWH-018 dihydroxy) | 12.9 | 374.1750 | −0.3 | C24H23NO3 | 143, 144, 171, 230 |
| Mc29 | Dihydroxylation at pentyl side chain and naphthalene moiety | 13.0 | 392.1655 | 0.3 | C24H22FNO3 | 143, 144, 171, 248 |
| Mc30 | Dihydrodiol formation | 13.5 | 394.1818 | 1.2 | C24H24FNO3 | 143, 144, 171, 189, 232 |
| Mc31 | Dihydroxylation at pentyl side chain and naphthalene moiety | 13.6 | 392.1655 | −0.3 | C24H22FNO3 | 143, 144, 171, 248 |
| Mc32 | Dihydroxylation at pentyl side chain and naphthalene moiety | 14.4 | 392.1659 | 0.6 | C24H22FNO3 | 143, 144, 171, 248 |
| Mc33 | Oxidative defluorination to carboxylic acid (JWH-018 N-pentanoic acid)a | 16.4 | 372.1592 | −0.7 | C24H21NO3 | 127, 144, 155, 244 |
| Mc34 | Oxidative defluorination (JWH-018 N-(5-hydroxypentyl))a | 17.1 | 358.1802 | 0.2 | C24H23NO2 | 127, 144, 155, 230 |
| Mc35 | Hydroxylation at pentyl side chain | 17.3 | 376.1709 | 0.4 | C24H22FNO2 | 127, 144, 155, 248 |
| Mc36 | Hydroxylation at indole moiety | 17.7 | 376.1707 | −0.2 | C24H22FNO2 | 127, 155, 160, 248 |
| Mc37 | Hydroxylation at pentyl side chain | 18.0 | 376.1708 | 0.1 | C24H22FNO2 | 127, 144, 155, 248 |
| Mc38 | Dihydroxylation at naphthalene moiety+sulfation | 18.7 | 472.1222 | −0.6 | C24H22FNO6S | 144, 158, 159, 186, 187, 232, 391, 392 |
| Mc39 | Hydroxylation at pentyl side chain | 18.8 | 376.1708 | 0.3 | C24H22FNO2 | 127, 144, 155, 248 |
| Mc40 | Hydroxylation at naphthalene moiety | 19.2 | 376.1709 | 0.6 | C24H22FNO2 | 143, 144, 171, 232 |
| Mc41 | Hydroxylation at naphthalene moiety | 19.8 | 376.1703 | −1.2 | C24H22FNO2 | 143, 144, 171, 232 |
| Mc42 | Dihydroxylation at naphthalene moiety+sulfation | 20.2 | 472.1224 | −0.1 | C24H22FNO6S | 144, 158, 159, 186, 187, 232, 391, 392 |
| Mc43 | Ketone formation at pentyl side chain | 20.4 | 374.1549 | −0.5 | C24H20FNO2 | 127, 144, 155, 246 |
| Mc44 | Hydroxylation at naphthalene moiety | 20.6 | 376.1706 | −0.3 | C24H22FNO2 | 143, 144, 171, 232 |
| Mc45 | Hydroxylation at naphthalene moiety | 21.3 | 376.1707 | −0.1 | C24H22FNO2 | 143, 144, 171, 232 |
| Mc46 | Dihydroxylation at indole moiety+sulfation | 22.0 | 472.1223 | −0.3 | C24H22FNO6S | 127, 155, 175, 176, 264, 391, 392 |
| AM2201 | 25.6 | 360.1759 | 0.2 | C24H22FNO | 127, 144, 155, 232 | |
| Mc47 | Defluorination+Demethylation (JWH-073)a | 28.8 | 328.1695 | −0.3 | C23H21NO | 127, 144, 155, 200 |
| Mc48 | Defluorination (JWH-018)a | 31.5 | 342.1853 | 0.0 | C24H23NO | 127, 144, 155, 214 |
Position confirmed by the use of reference standards.
Table 4.
Key diagnostic product ions and their tentative structures used in elucidating biotransformation pathways of JWH-073 after C. elegans incubation.
| Biotransformation | ID | Key diagnostic product ions (m/z) and tentative structures |
|---|---|---|
| Carboxylation at butyl side chain | Mb11 | 144: unchanged indole, 230: carboxylated butylindole |
| Dehydrogenation | Mb17 | 198: dehydrogenated N-butylindole |
| Dihydrodiol formation at naphthalene moiety | Mb8 | 189: naphthalene with dihydrodiol |
| Dihydrodiol formation at naphthalene moiety+hydroxylation at butyl side chain | Mb2, Mb3 | 144: unchanged indole, 189: naphthalene with dihydrodiol, 216: hydroxylated N-butylindole |
| Dihydrodiol formation at naphthalene moiety+ketone formation at butyl side chain | Mb4 | 144: unchanged indole, 189: naphthalene with dihydrodiol, 214: N-butylindole with ketone |
| Dihydrodiol formation at naphthalene moiety+N-dealkylation | Mb1 | 171: hydroxylated naphthalene (resulting from naphthalene with dihydrodiol [2]) |
| Dihydroxylation at butyl side chain | Mb5 | 144: unchanged indole, 232: dihydroxylated N-butylindole |
| Dihydroxylation at butyl chain and naphthalene moiety | M6, Mb7 | 144: unchanged indole, 171: hydroxylated naphthalene, 216: hydroxylated N-butylindole |
| Hydroxylation at butyl side chain | Mb12, Mb13 | 144: unchanged indole, 216: hydroxylated N-butylindole |
| Hydroxylation at naphthalene moiety | Mb15, Mb16 | 171: hydroxylated naphthalene |
| Ketone formation at butyl side chain | Mb14 | 144: unchanged indole, 214: N-butylindole with ketone |
| Ketone formation at butyl side chain+hydroxylation at naphthalene moiety | Mb9, Mb10 | 144: unchanged indole, 171: hydroxylated naphthalene, 214: N-butylindole with ketone |
Table 6.
Key diagnostic product ions and their tentative structures used in elucidating biotransformation pathways of AM2201 after C. elegans incubation. Square brackets indicate phase II metabolism.
| Biotransformation | ID | Key diagnostic product ions (m/z) and tentative structures |
|---|---|---|
| Defluorination (JWH-018) | Mc48 | 155: unchanged naphthalene, 214: unchanged N-pentylindole |
| Defluorination+Demethylation (JWH-073) | Mc47 | 155: unchanged naphthalene, 200: unchanged N- butylindole |
| Dihydrodiol formation at naphthalene moiety | Mc25, Mc30 | 189: naphthalene with dihydrodiol |
| Dihydrodiol formation at naphthalene moiety+dihydroxylation at indole moiety | Mc9 | 176: dihydroxylated indole, 189: naphthalene with dihydrodiol |
| Dihydrodiol formation at naphthalene moiety+hydroxylation at pentyl side chain | Mc5, Mc6 | 144: unchanged indole, 189: naphthalene with dihydrodiol, 248: hydroxylated N-fluoropentylindole |
| Dihydrodiol formation at naphthalene moiety+ketone formation at pentyl side chain | Mc10 | 144: unchanged indole, 189: naphthalene with dihydrodiol, 246: N-fluoropentylindole with ketone |
| Dihydrodiol formation at naphthalene moiety+N-dealkylation | Mc1 | 189: naphthalene with dihydrodiol |
| Dihydroxylation at indole moiety [+sulfation] | [Sulfate Mc46] | 176: dihydroxylation at indole, [392: dihydroxylated AM2201] |
| Dihydroxylation at indole moiety and pentyl side chain | Mc17, Mc19, Mc22, Mc27 | 160: hydroxylated indole, 264: dihydroxylated N-fluoropentylindole |
| Dihydroxylation at naphthalene moiety [+glucosidation and sulfation] | [Glucoside Mc16, sulfates Mc38, Mc42] | 187: dihydroxylated naphthalene, [392: dihydroxylated AM2201] |
| Dihydroxylation at naphthalene moiety and pentyl side chain [+glucosidation] | Mc24, Mc29, Mc31, Mc32, [glucosides Mc2, Mc3] | 144: unchanged indole, 171: hydroxylated naphthalene, 248: hydroxylated N-fluoropentylindole, [392: dihydroxylated AM2201] |
| Dihydroxylation at pentyl side chain and/or indole moiety [+glucosidation] | [glucosides Mc7, Mc13] | 264: dihydroxylated N-fluoropentylindole, [392: dihydroxylated AM2201] |
| Hydroxylation at indole moiety [+glucosidation] | Mc36, [glucoside Mc12] | 160: hydroxylated indole, 248: hydroxylated N-fluoropentylindole, [376: hydroxylated AM2201] |
| Hydroxylation at naphthalene moiety [+glucosidation] | Mc40, Mc41, Mc44, Mc45, [glucoside Mc14] | 171: hydroxylated naphthalene, [376: hydroxylated AM2201] |
| Hydroxylation at pentyl side chain | Mc35, Mc37 and Mc39 | 144: unchanged indole, 248: hydroxylated N-fluoropentylindole |
| Ketone formation at pentyl side chain | Mc43 | 144: unchanged indole, 246: N-fluoropentylindole with ketone |
| Oxidative defluorination (JWH-018 N-(5-hydroxypentyl)) | Mc34 | 144: unchanged indole, 230: hydroxylated N-pentylindole |
| Oxidative defluorination+dihydrodiol formation (JWH-018 dihydrodiol-hydroxy) | Mc4 | 189: naphthalene with dihydrodiol, 230: hydroxylated N-pentylindole |
| Oxidative defluorination+hydroxylation at indole moiety (JWH-018 dihydroxy) | Mc15, Mc18 | 160: hydroxylated indole, 246: dihydroxylated N-pentylindole |
| Oxidative defluorination+hydroxylation at naphthalene moiety (JWH-018 dihydroxy) | Mc23, Mc28 | 144: unchanged indole, 171: hydroxylated naphthalene, 230: hydroxylated N-pentylindole |
| Oxidative defluorination+hydroxylation at pentyl side chain (JWH-018 dihydroxy) | Mc21 | 144: unchanged indole, 246: dihydroxylated N-pentylindole |
| Oxidative defluorination to carboxylic acid (JWH-018 N-pentanoic acid) | Mc33 | 144: unchanged indole, 244: carboxylated pentylindole |
| Oxidative defluorination to carboxylic acid+hydroxylation at naphthalene moiety. | Mc20, Mc26 | 144: unchanged indole, 171: hydroxylated naphthalene, 244: carboxylated pentylindole |
| Trihydroxylation at indole moiety, naphthalene moiety and pentyl side chain | Mc8, Mc11 | 160: hydroxylated indole, 171: hydroxylated naphthalene, 264: dihydroxylated N-fluoropentylindole |
Table 2.
Key diagnostic product ions and their tentative structures used in elucidating biotransformation pathways of JWH-018 after C. elegans incubation.
| Biotransformation | ID | Key diagnostic product ions (m/z) and tentative structures |
|---|---|---|
| Carboxylation at pentyl side chain | Ma13 | 144: unchanged indole, 244: carboxylated pentylindole |
| Dehydrogenation at pentyl side chain | Ma21 | 144: unchanged indole, 212: dehydrogenated N-pentylindole |
| Dihydrodiol formation at naphthalene moiety | Ma12 | 189: naphthalene with dihydrodiol |
| Dihydrodiol formation at naphthalene moiety+hydroxylation at pentyl side chain | Ma2, Ma3 | 144: unchanged indole, 189: naphthalene with dihydrodiol, 230: hydroxylated N-pentylindole |
| Dihydrodiol formation at naphthalene moiety+ketone formation at pentyl side chain | Ma4, Ma5 | 189: naphthalene with dihydrodiol, 228: N-pentylindole with ketone |
| Dihydrodiol formation at naphthalene moiety+N-dealkylation | Ma1 | 189: naphthalene with dihydrodiol |
| Dihydroxylation at pentyl side chain | Ma7 | 144: unchanged indole, 246: dihydroxylated N-pentylindole |
| Dihydroxylation at pentyl side chain and naphthalene moiety | Ma6, Ma8, Ma9, Ma11 | 144: unchanged indole, 171: hydroxylated naphthalene, 230: hydroxylated N-pentylindole |
| Hydroxylation at naphthalene moiety | Ma19, Ma20 | 171: hydroxylated naphthalene |
| Hydroxylation at pentyl side chain | Ma14 – Ma16 | 144: unchanged indole, 230: hydroxylated N-pentylindole |
| Ketone formation at pentyl side chain | Ma17, Ma18 | 144: unchanged indole, 228: N-pentylindole with ketone |
| Ketone formation at pentyl side chain+hydroxylation at naphthalene moiety | Ma10 | 144: unchanged indole, 171: hydroxylated naphthalene, 228: N-pentylindole with ketone |
2. Experimental design, materials and methods
2.1. Chemicals
JWH-018 and JWH-073 were synthesized in-house following previously reported methods and characterized by mass spectrometry (MS) and 1D, 2D nuclear magnetic resonance (NMR) spectroscopy [3], [4]. AM2201 (purity 99.4%) was obtained from the National Measurement Institute (North Ryde, NSW, Australia). Reference standards JWH-018 N-(4-hydroxypentyl), JWH-018 N-(5-hydroxypentyl), JWH-018 N-pentanoic acid, JWH-073 N-(3-hydroxybutyl), JWH-073 N-(4-hydroxybutyl) and JWH-073 N-butanoic acid were obtained from PM separations (Capalaba, QLD, Australia). Reagent grade dichloromethane, methanol, KH2PO4, NaCl and LC grade acetonitrile and methanol were obtained from Chemsupply (Gilman, SA, Australia). Potato dextrose agar, glucose, peptone, and yeast extract were purchased from Oxoid Australia (Adelaide, SA, Australia).
2.2. Microbial culture and biotransformation conditions
Cultures of C. elegans ATCC 10028b (Cryosite Ltd, South Granville, NSW, Australia) were propagated on potato dextrose agar plates at 27 °C for 5 days. The mycelia from five plates were then transferred to 20 mL of sterile physiological saline solution and homogenized for 5 min. Approximately 3 mL aliquots of the homogenate were used to inoculate 250 mL Erlenmeyer flasks containing 100 ml of growth media. The cultures were incubated for 48 h at 26 °C on an Infors HT Multitron rotary shaker (in vitro Technologies, Noble Park North, VIC, Australia) operating at 180 rpm. After 48 h, 10 mg of JWH-018, JWH-073 or AM2201 dissolved in 0.5 mL of methanol was added to the culture and incubated for further 72 h [5]. Control experiments consisted of cultures without cannabinoids and flasks containing only media and cannabinoid [6], [7].
2.3. Extraction, isolation, and identification of metabolites
After 72 h of incubation, the contents of each flask, including the controls, were filtered through Buchner funnel into a separating funnel and extracted with three aliquots of dichloromethane (3×50 mL). The combined organic extracts were evaporated to dryness under vacuum at 40 °C using a Buchi rotary evaporator (in vitro Technologies, Noble Park North, VIC, Australia) and placed under high vacuum to remove traces of moisture. The residue was dissolved in acetonitrile to prepare 1 mg/mL stock solution and was filtered through 0.22 µM syringe filter before analysis. Cannabinoid parent drugs and metabolites were chromatographically separated using an Agilent Zorbax Eclipse XDB-C18 analytical column (150×4.6 mm, 5 μm). Mobile phases consisted of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). The gradient used consisted of 30% B (0 to 2 min), linear gradient from 30% B to 50% B (2 to 5 min), 50% B to 90% B (5 to 30 min, hold for 5 min) and 90% B to 30% B (35 to 40 min) run at 0.4 mL/min. MS data were acquired on an Agilent 6490 Triple Quadrupole mass spectrometer with an electrospray ionization source (ESI) source (positive ion mode), interfaced with an Agilent 1290 LC system. Samples prepared were injected in 2 µL volume to obtain full scan and product ion scan spectra. Product ion scan experiments were conducted on precursor ions that were presumed to be metabolites based on the comparison of full scan spectra of the samples and controls. A fragmentor voltage of 380 V with discrete collision energy of 10, 20, 30 and 40 eV (for product ion scan) was applied. The scanning mass range was set at m/z 100–1000 (scan time=500 ms). The sheath gas temperature and flow were set to 250 °C and 11 L/min, respectively. The capillary and nozzle voltages were 3000 V and 1500 V, respectively.
High resolution quadrupole Time-of-Flight mass spectrometry (HRQToFMS) experiments were carried out on an Agilent 6510 Accurate Mass QToF Mass Spectrometer, equipped with ESI source operated in positive ion mode, in order to determine accurate masses of the metabolites. The LC system and conditions used were the same as above. The following operation parameters were used: injection volume 2 µL (full scan) and 10 µL (product ion scan); capillary voltage 3500 V; nebulizer pressure 40 psi (275790 Pa); drying gas 10.0 L/min; gas temperature 350 °C; fragmentor voltage 160 V; collision energy 10, 20 and 40 eV; skimmer voltage 60 V. HRQToFMS accurate mass spectra were recorded across the range from m/z 100 to m/z 1000. The mass axis was calibrated using the mixture provided by the manufacturer over the m/z 50–3200 range. A second orthogonal sprayer with a reference solution was used as a continuous calibration using the following reference masses: m/z 121.0509 and m/z 922.0098. The chromatographic conditions and column used were same as described above. The controls were subjected to the same analysis. Analysis of the chromatographic and mass spectrometric data was conducted using MassHunter Workstation Software Qualitative Analysis (version B.06.00, Agilent). Peaks present in the fungus sample, but not in the controls, were manually identified and their fragmentation patterns and accurate masses were examined to identify the metabolites. The signal-to-noise ratio of all the identified metabolites was greater than 5.
Footnotes
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.dib.2016.02.039.
Appendix A. Supplementary material
Supplementary material
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