Research ArticleNEUROPHYSIOLOGY

Uncovering the psychoactivity of a cannabinoid from liverworts associated with a legal high

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Science Advances  24 Oct 2018:
Vol. 4, no. 10, eaat2166
DOI: 10.1126/sciadv.aat2166
  • Fig. 1 Phylogenetic separation of bryophytes and angiosperms and convergent evolution of the tetrahydrocannabinoid scaffold in (−)-trans-THC and (−)-cis-PET.

    Both cannabinoids act as partial agonists on CB1 receptors in vitro and in vivo. Ma, mega-annum. Photo credit: S. Fischer (D-CHAB, LOC, ETH Zurich).

  • Fig. 2 Stereodivergent synthesis of SNAr precursors.

    Reagents and conditions: (a) 1.0 equiv 1, 3.0 equiv 2, 3 mol% [{Ir(cod)Cl}2], 12 mol% (S)-L, 15 mol% (S)- or (R)-A, 5 mol% Zn(OTf)2, 1,2-dichloroethane (0.5 M), 25°C, 20 hours, for (S,R)-3: 59% yield, > 20:1 diastereomeric ratio (d.r.), >99% enantiomeric excess (e.e.), for (R,R)-3: 76% yield, > 12:1 d.r., >99% e.e.; (b) 5 mol% Grubbs II cat., CH2Cl2, 25°C, 16 hours; (c) 2.3 equiv NaClO2, 2.0 equiv NaH2PO4, 30 equiv 2-methyl-2-butene, tert-BuOH/H2O, 25°C; then 2.0 equiv Me3SiCHN2, C6H6/MeOH, 0°C, 90 min; (d) 8.0 equiv KOH, 3.9 equiv I2, MeOH, 0°C, 45 min; (e) 4.0 equiv MeMgBr, THF, 25°C, for (S,R)-4: 56% yield over three steps, for (R,R)-4: 56% yield over three steps; see the Supplementary Materials for structures of (S)-L, (S)-A, and (R)-A, as well as for further details.

  • Fig. 3 Stereodivergent total synthesis of (−)-cis-PET and (−)-trans-PET.

    Reagents and conditions: (f) 1.6 equiv KHMDS, THF, 65°C, 15 hours, for (S,R)-5: 78% yield, (R,R)-5: 82% yield; (g) 10.0 or 20.0 equiv NaSEt, DMF, 140°C, 16 hours, for (−)-cis-PET: 80% yield, (−)-trans-PET: 83% yield.

  • Fig. 4 CB1 receptor dependence of the pharmacological effects of Δ9-trans-THC and PET diastereoisomers in mice.

    (A) Hypothermia, (B) catalepsy, (C) hypolocomotion, and (D) analgesia elicited by Δ9-trans-THC (red), cis-PET (green), and trans-PET (blue) compared with vehicle control (white) in BALB/c male mice 1 hour after intraperitoneal injection. The pharmacological effects were fully blocked by the CB1 receptor antagonist rimonabant (SR1, gray columns). Doses are expressed in mg/kg. Data show means ± SD. Groups were compared with the vehicle-treated control group using a one-way analysis of variance (ANOVA) following Tukey’s post hoc test, n = 5 to 15 mice per group. ***P < 0.001, **P < 0.01, *P < 0.05 versus vehicle or as reported by the arches.

  • Fig. 5 Brain concentrations of Δ9-trans-THC and PET diastereoisomers and induced biochemical changes.

    LC-MS/MS quantification of (A) Δ9-trans-THC (red), cis-PET (green), and trans-PET (blue); (B) AEA; (C) 2-AG; (D) AA; (E) PGD2; and (F) PGE2 in the brain of male BALB/c mice 1 hour after intraperitoneal injection. The effects of PET diastereoisomers on PGD2 and PGE2 were inhibited by the CB1 receptor antagonist rimonabant (SR1). Doses are expressed in mg/kg. Data show means ± SD. Groups were compared with the vehicle-treated control group using a one-way ANOVA following Tukey’s post hoc test n = 5 to 15 mice per group. **P < 0.01, *P < 0.05 versus vehicle or as reported by the arches. ns, not significant.

  • Fig. 6 Effects of cis-PET and Δ9-trans-THC on a panel of 44 CNS targets (GPCRs, ion channels, nuclear receptors, membrane transporters, and enzymes).

    Data show the percentage of binding or protein activity (enzyme, transporter, and ion channels) at test concentrations of 10 μM (cis-PET, blue line) and 1 μM (cis-PET, orange line; Δ9-trans-THC, green line). Data presented are obtained from two independent experiments each performed in duplicate. For full names of abbreviations, see the Supplementary Materials. The 100% is intended as full enzymatic activity (enzymes), substrate transport (membrane transporter), receptor binding (GPCRs and nuclear receptors), and ion permeability (ion channels). The percentage is expressed as positive or negative values based on an increase or reduction of the target protein.

  • Table 1 Summary of binding interactions (Ki values) and functional activation ([35S]GTPγS binding assay) of THC and PET diastereoisomers on human CB1R and CB2R.

    Emax values represent the maximal [35S]GTPγS binding expressed as percentage of the vehicle-treated sample (100%). “Full” and “partial” in brackets stand for full and partial agonist.

    Receptor binding
    Ki values (nM, mean ± SD)
    Receptor activation
    EC50 (nM) and Emax (%) values (mean ± SD)
    CannabinoidCB1RCB2RCB1R (EC50)CB1R (Emax)CB2R (EC50)CB2R (Emax)
    CP55,9401.2 ± 0.50.7 ± 0.317 ± 9173 ± 6
    (full)
    1.9 ± 0.8194 ± 5
    (full)
    Δ9-trans-THC22 ± 1347 ± 1143 ± 30146 ± 3
    (partial)
    12 ± 7134 ± 4
    (partial)
    Δ9-cis-THC228 ± 4599 ± 29552 ± 123158 ± 6
    (partial)
    119 ± 69153 ± 5
    (partial)
    trans-PET127 ± 82126 ± 55171 ± 116142 ± 5
    (partial)
    478 ± 288116 ± 6
    (partial)
    cis-PET481 ± 125225 ± 61406 ± 175142 ± 5
    (partial)
    167 ± 136113 ± 4
    (partial)

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/10/eaat2166/DC1

    Fig. S1. Radioligand displacement assay using [3H]CP55,940 and membranes from CHO cells stably transfected with human CB1R and CB2R, respectively.

    Fig. S2. [35S]GTPγS binding curves for THC and PET stereoisomers on human cannabinoid receptors.

    Fig. S3. LC-MS/MS quantifications of the NAEs OEA, PEA, and LEA in the brain of BALB/c male mice 1 hour after intraperitoneal injection of cannabinoids.

    Fig. S4. LC-MS/MS quantification of Δ9-trans-THC, cis-PET, and trans-PET in plasma of BALB/c male mice 1 hour after intraperitoneal injection of cannabinoids.

    Fig. S5. Inhibition of COX-2 activity.

    Fig. S6. Representative LC-MS/MS reaction monitoring chromatogram of reference standards in mouse brain tissue showing LC separation of cis- and trans-PET.

    Table S1. Profiling of THC and PET diastereoisomers on endocannabinoid-degrading enzymes in vitro.

    Synthesis and characterization of products

    Preparation of allylic alcohol 1

    Preparation of (–)-cis-PET [(S,R)-PET]

    Preparation of (–)-trans-PET [(R,R)-PET]

    SFC traces

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Radioligand displacement assay using 3HCP55,940 and membranes from CHO cells stably transfected with human CB1R and CB2R, respectively.
    • Fig. S2. 35SGTPγS binding curves for THC and PET stereoisomers on human cannabinoid receptors.
    • Fig. S3. LC-MS/MS quantifications of the NAEs OEA, PEA, and LEA in the brain of BALB/c male mice 1 hour after intraperitoneal injection of cannabinoids.
    • Fig. S4. LC-MS/MS quantification of Δ9-trans-THC, cis-PET, and trans-PET in plasma of BALB/c male mice 1 hour after intraperitoneal injection of cannabinoids.
    • Fig. S5. Inhibition of COX-2 activity.
    • Fig. S6. Representative LC-MS/MS reaction monitoring chromatogram of reference standards in mouse brain tissue showing LC separation of cis- and trans-PET.
    • Table S1. Profiling of THC and PET diastereoisomers on endocannabinoid-degrading enzymes in vitro.
    • Synthesis and characterization of products
    • Preparation of allylic alcohol 1
    • Preparation of (–)-cis-PET (S,R)-PET
    • Preparation of (–)-trans-PET (R,R)-PET
    • SFC traces

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