Synthesis and Pharmacological Profiling of the Metabolites of Synthetic Cannabinoid Drugs APICA, STS-135, ADB-PINACA, and 5F-ADB-PINACA

Article abstract of DOI:10.1021/acschemneuro.7b00116

Synthetic cannabinoids (SCs) containing a 1-pentyl-1-H substituted indole or indazole are abused around the world and are associated with an array of serious side effects. These compounds undergo extensive phase 1 metabolism after ingestion with little understanding whether these metabolites are contributing to the cannabimimetic activity of the drugs. This work presents the synthesis and pharmacological characterization of the major metabolites of two high concern SCs; APICA and ADB-PINACA. In a fluorometric assay of membrane potential, all metabolites that did not contain a carboxylic acid functionality retained potent activity at both the CB₁ (EC₅₀ = 14-787 nM) and CB₂ (EC₅₀ = 5.5-291 nM) receptors regardless of heterocyclic core or 3-carboxamide substituent. Of note were the 5-hydroxypentyl and 4-pentanone metabolites which showed significant increases in CB₂ functional selectivity. These results suggest that the metabolites of SCs potentially contribute to the overall pharmacological profile of these drugs.

Full text of DOI:10.1021/acschemneuro.7b00116

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Letter
Synthesis and Pharmacological profiling of the metabolites of synthetic
cannabinoid drugs APICA, STS-135, ADB-PINACA, and 5F-ADB-PINACA.
Mitchell Longworth, Mark Connor, Samuel D. Banister, and Michael Kassiou
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017
Downloaded from http://pubs.acs.org on June 3, 2017
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Synthesis and Pharmacological profiling of the metabolites of synthetic cannabinoid
drugs APICA, STS-135, ADB-PINACA, and 5F-ADB-PINACA
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Mitchell Longworth,a# Mark Connor,b# Samuel D. Banister,c Michael Kassioua*
aSchool of Chemistry, The University of Sydney, NSW 2006, Australia; bDepartment of
Biomedical Sciences, Macquarie University, NSW 2109, Australia; cDepartment of
Pathology, Stanford University School of Medicine, CA 94305, USA;
*Corresponding author
# Equal contribution
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Keywords: cannabinoid, THC, APICA, ADBꢀPINACA, metabolism
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Abstract: Synthetic cannabinoids (SCs) containing a 1ꢀpentylꢀ1ꢀH substituted indole or
indazole have found abuse around the world and are associated with an array of serious side
effects. These compounds are known to undergo extensive Phase 1 metabolism after
ingestion with little understanding whether these metabolites are contributing to the
cannabimimetic activity of the drugs. This work presents the synthesis and pharmacological
characterization of the major metabolites of two high concern SCs; APICA and ADBꢀ
PINACA. In a fluorometric assay of membrane potential, all metabolites that did not contain
a carboxylic acid functionality retained potent activity at both the CB1 (EC50 = 14 – 783 nM)
and CB2 (EC50 = 5.5 – 291 nM) receptors regardless of heterocyclic core or 3ꢀcarboxamide
substituent. Of note were the 5ꢀhydroxypentyl and 4ꢀpentanone metabolites which showed
significant increases in CB2 functional selectivity. These results suggest that the metabolites
of SCs potentially contribute to the overall pharmacological profile of these drugs.
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Introduction: As of July 2015, 96 countries and territories have reported more than 540
novel psychoactive substances (NPSs) to the United Nations Office on Drugs and Crime
(UNODC).1 Like the psychoactive phytocannabinoid ꢁ9ꢀtetrahydrocannabinol (ꢁ9ꢀTHC, 1,
Fig. 1), many SCs possess activity at cannabinoid type 1 and type 2 (CB1 and CB2) receptors,
and the in vitro and in vivo pharmacology of many of these SCs has been explored.2ꢀ4 While
the metabolism of ꢁ9ꢀTHC is well understood, the rate of emergence of novel SCs impedes
identification and pharmacological profiling of SCs and their metabolites. Forensic chemists
and toxicologists have incrementally increased our knowledge of SC metabolism and
developed methods for identification and quantitation of various SC metabolites,5ꢀ8 but
attempts to understand the pharmacology of SC metabolites has been less systematic.
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Unlike THC, which only contains one psychoactive metabolite (11ꢀOHꢀTHC, 2),9 it is
hypothesized that the major oxidative metabolites of SCs retain cannabimimetic activity,
contributing to the overall physiological profile of the drugs. JWHꢀ018 (3) was one of the
earliest recreational SCs detected,10 and the human urinary metabolites of JWHꢀ018 were
reported in 2010 at concentrations up to 83 ng/mL.11,12 Metabolic oxidation of JWHꢀ018 was
found to occur on the naphthalene and indole rings, as well as the pentyl chain. The
terminally fluorinated analogue, AMꢀ2201 (4), was found to generate common metabolites in
some but not all cases. In humans, ωꢀhydroxyꢀ (5ꢀOHꢀJWHꢀ018, 5), ωꢀ1ꢀhydroxyꢀ (4ꢀOHꢀ
JWHꢀ018, 6), and ωꢀcarboxyꢀJWHꢀ018 (5ꢀCOOHꢀJWHꢀ018, 7) (Fig. 1) were found to be
excreted in high concentrations (5 and 6 exclusively as glucuronic acid conjugates) following
JWHꢀ018 consumption.13 AMꢀ2201 undergoes oxidative defluorination to give common
metabolites 5 and 7, but not 6,14 as well as Nꢀdealkylation to give naphthoylindole 8.15
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Figure 1. Selected cannabinoids and metabolites.
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O
R
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OH
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N
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O
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OH
R
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JWH-018 (3; R = H)
AM-2201 (4; R = F)
5-OH-JWH-018 (5; R = OH)
ꢀ9-THC (1; R = H)
11-OH-THC (2; R = OH)
4-OH-JWH-018 (6)
5-COOH-JWH-018 (7)
O
O
NH2
O
NH
NH
O
N
N
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2
1
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R
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R
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APICA (9; R = H)
STS-135 (10; R = F)
ADB-PINACA(11; R = H)
5F-ADB-PINACA (12; R = F)
Several phase I metabolites of JWHꢀ018 and AMꢀ2201, including common metabolite 5,
possessed high affinity for CB1 receptors and exhibited efficacious agonist activity in vitro.16
Monohydroxylated JWHꢀ018 metabolites also showed high affinity for human CB2
receptors.17 In contrast, the glucuronidated conjugate of 5ꢀOHꢀJWHꢀ018, a major human
JWHꢀ018 metabolite, was found to possess neutral antagonist activity at CB1 receptors.18
Additionally, when compared to ꢁ9ꢀTHC or CP 55,940, JWHꢀ018 and 5ꢀOHꢀJWHꢀ018 are
higher efficacy agonists in an assay of CB2 receptor inhibition of adenylyl cyclase (AC).17
In 2012, following the identification of numerous 3ꢀacylindoles related to JWHꢀ018, APICA
(2NE1, SDBꢀ001, 9) was reported as one of the first indoleꢀ3ꢀcarboxamide SCs, followed by
its ωꢀfluorinated analogue, STSꢀ135 (10) (Fig.1).19 APICA was found to exert potent agonist
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activity at CB1 and CB2 receptors in vitro,20 and to possess cannabimimetic activity in vivo.21
Despite the emergence of newer SCs, APICA and related adamantaneꢀderived SCs persist,
and continue to be associated with serious adverse effects.22
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The phase I metabolites of APICA and STSꢀ135 have been described.23ꢀ24 As expected,
oxidation of the Nꢀalkyl group was prominent, and several monohydroxylated metabolites
were reported. Hydroxylation of the adamantane group was also extensive. Hydrolysis of the
carboxamide occurs for APICA, a transformation that appears general for indoleꢀ3ꢀ
carboxamides but not 3ꢀacylindoles,25 and 1ꢀadamantylamine has been proposed as a simple
APICA biomarker.26 Interestingly, 1ꢀadamantylamine is itself a bioactive substance; the
approved antiviral and antiꢀParkinsonian drug amantadine.
ADBꢀPINACA (11) is a member of the recent, prevalent indazoleꢀ3ꢀcarboxamide SC class,
and new variants continue to appear, including the anticipated 5ꢀfluoro derivative 5FꢀADBꢀ
PINACA (12) (Fig. 1). ADBꢀPINACA exposure has been associated with severe adverse
reactions, including neurotoxicity, cardiotoxicity, and death.27ꢀ28 Like APICA, ADBꢀ
PINACA acts as a potent CB1 and CB2 receptor agonist in vitro and in vivo,29ꢀ30 but nothing
is known of the pharmacology of its metabolites. The metabolism of closely related ABꢀ
PINACA (differing by an isopropyl rather than tertꢀbutyl sideꢀchain) and its 5ꢀfluorinated
analogue (5FꢀABꢀPINACA) was recently reported,31 enabling prediction of some likely
ADBꢀPINACA metabolites. Another study in human liver microsomes confirmed that ABꢀ
PINACA metabolism occurs mainly via hydroxylation of the pentyl chain,32 although
hydrolysis of the terminal primary amide has also been reported.33
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Several reviews of the metabolism of SCs have appeared, focusing predominantly on
methods for metabolite identification and quantitation in various matrices,34ꢀ36 however, there
is growing interest in understanding the pharmacology of SC metabolites more
systematically.37 Methods for the prediction of SC metabolites have shown good agreement
with in vitro results, indicating that proactive pharmacological profiling of predicted SC
metabolites is feasible for the latest SCs.38ꢀ39
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The aim of the current work was to develop synthetic routes to several phase I metabolites of
APICA, STSꢀ135, ADBꢀPINACA, and 5FꢀADBꢀPINACA and to characterize the
pharmacology of these metabolites at human CB1 and CB2 receptors. Ultimately, we sought
to elucidate whether apparent trends in the structureꢀactivity relationships of acylindole SC
metabolites translate to newer, prevalent indoleꢀ and indazoleꢀ3ꢀcarboxamide SC derivatives
featuring a common 1ꢀpentyl subunit.
Results and discussion: The synthesis of several phase I metabolites of APICA/STSꢀ135
(13−18, Fig. 2) and ADBꢀPINACA/5FꢀADBꢀPINACA (19−24), including diastereomers 19
and 20, was inspired by our previous synthetic routes to parent structures APICA/STSꢀ135
and ADBꢀPINACA/5FꢀADBꢀPINACA.29, 40
Figure 2. Representative phase I metabolites of APICA, STSꢀ135, ADBꢀPINACA, and 5Fꢀ
ADBꢀPINACA.
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OH
O
O
O
O
O
NH
NH
NH
NH
NH
O
NH
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N
N
N
N
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OH
O
H
OH
OH
5-OH-APICA (15)
O
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4-CO-APICA (14)
5-COOH-APICA (16)
3-Ad-OH-APICA (17)
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O
O
O
O
O
O
NH2
O
NH2
O
O
NH
O
NH2
NH2
OH
NH
NH
NH
NH
N
N
N
N
N
N
N
N
N
N
OH
O
OH
5-COOH-ADB-PINACA (23)
OH
5-OH-ADB-PINACA (22)
O
(4R)-4-OH-ADB-PINACA (19)
(4S)-4-OH-ADB-PINACA (20)
4-CO-ADB-PINACA (21)
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The synthesis of 4ꢀOHꢀAPICA enantiomers 4Rꢀ13 and 4Sꢀ13, 4ꢀCOꢀAPICA (14), 5ꢀOHꢀ
APICA (15), AdꢀOHꢀAPICA (17) are shown in Scheme 1. In short, it was envisaged that 1ꢀ
alkylation of the esters of 1Hꢀindoleꢀ and 1Hꢀindazoleꢀ3ꢀcarboxylic acids with suitably
protected alkyl bromides 25−27 would be followed by deprotection and chemoselective
coupling of the carboxylic acid with the appropriate amines.
A oneꢀpot Nꢀalkylation using the appropriate synthon, followed by addition of trifluoroacetic
anhydride to indole (29), provided 30–33, which were subsequently treated with hydroxide to
furnish carboxylic acids 34–37. The hydrolysis of the acetal protecting group to yield 30 can
likely be attributed to the generation of trifluoroacetic acid in situ. Coupling of 37 to 3ꢀ
hydroxyꢀ1ꢀaminoadamantane with HOBt and EDC provided 17, while 34–36 were reacted
with 1ꢀaminoadamantane in the presence of PyBOP to give 14, 38 and 39 respectively.
Hydrogenolysis of 38 and 39 afforded (4R)ꢀ13 and 15 respectively. Finally, a Mitsunobu
esterification of (4R)ꢀ13 gave the inverted acetate ester 40, saponification of which afforded
the (4S)ꢀ13 enantiomer.
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NꢀDealkylated APICA metabolite, 18, and 5ꢀCOOHꢀAPICA (16) were synthesized from
indoleꢀ3ꢀcarboxylic acid (41), converting it to the corresponding acid chloride by treatment
with oxalyl chloride, and reacting with 1ꢀaminoadamantane to give 18. Alkylation of 18 with
the methyl ester of 1ꢀbromoꢀ5ꢀpentanoic acid (28), and subsequent hydrolysis upon workꢀup,
provided 16.
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Scheme 1. Synthesis of APICA metabolites 13, 14, 15, 16, 17 and 18.a
O
O
O
O
CF3
OH
NH
NH
a
b
d or e
g
N
N
N
N
N
H
R
R
R
OH
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30: R = (CH2)3C(O)CH3
31: R = (CH2)3CH(OBn)CH3
32: R = (CH2)5OBn
34: R = (CH2)3C(O)CH3
14: R = (CH2)3C(O)CH3
39: R = (CH2)3CH(OBn)CH3
40: R = (CH2)5OBn
18: R = H
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35: R = (CH2)3CH(OBn)CH3
36: R = (CH2)5OBn
37: R = (CH2)4CH3
38: R = H
33: R = (CH2)4CH3
f
16: R = (CH2)4COOH
c
g
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OH
Br
Br
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O
O
O
O
OBn
NH
NH
NH
h
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25
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N
N
O
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3
5
1
3
OH
OR
Br
OBn
Br
O
5
2
4
2
4
(4R)-13
17
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28
41: R = OAc
(4S)-13: R = H
i
aReagents and conditions: (a)(i) 25, 26 or 37, NaH, DMF, 0 ºC–rt, 1 h; (ii) (CF3CO)2O, 0 ºC–
rt, 1 h, 60–94%; (b) 3 M aq. NaOH, MeOH, ∆, 14 h, 75–95%; (c) 1ꢀaminoꢀ3ꢀ
hydroxyadamantane hydrochloride, EDC, HOBt, DIPEA, DMSO, rt, 14 h, 41%; (d) 1ꢀ
aminoadamantane hydrochloride, PyBOP, DIPEA, DMSO, rt, 2 h, 46–97%; (e) for 48 only;
(i) (COCl)2, DMF, 0 °C–rt, 1 h; (ii) 1ꢀaminoadamantane, DIPEA, CH2Cl2, rt, 58%; (f)(i) 28,
NaH, DMF, 0 °C–rt; (ii) 2 M aq. NaOH, 1 h, 41%; (g) 10% Pd/C, H2(g), MeOH, rt, 2 h, 94–
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98%; (h) DIAD, PPh3, AcOH, THF, 0 ºC–rt, 16 h, 95%; (i) 1 M aq. NaOH, MeOH, rt, 30
min, 77%.
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The synthesis of ADBꢀPINACA/5FꢀADBꢀPINACA metabolites utilized similar
methodologies described for APICA/STSꢀ135 metabolites. The synthesis of diastereomers
(4R)ꢀ4ꢀOHꢀADBꢀPINACA (19) and (4S)ꢀ4ꢀOHꢀADBꢀPINACA (20), as well as 4ꢀCOꢀADBꢀ
PINACA (21), and 5ꢀOHꢀADBꢀPINACA (22), are depicted in Scheme 2. Regioselective 1ꢀ
alkylation of methyl 1Hꢀindazoleꢀ3ꢀmethyl carboxylate (42) with the appropriate synthons
25, 26 or 27, afforded 43, (4R)ꢀ44, and 45 respectively, followed by ester hydrolysis to
furnish the corresponding acids 46, (4R)ꢀ47, and 48. In the case of 43, additional reaction
time in the presence of aqueous acid was required for acetal hydrolysis. Carboxylic acids 46,
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(4R)ꢀ47, and 48 were coupled with Lꢀtertꢀleucinamide to give 21, 49, and 50 respectively.
Hydrogenolysis of Oꢀbenzyl protected alcohols 49 and 50 furnished 20 and 22 respectively.
As with the corresponding APICA metabolites, 20 was subjected to a Mitsunobu
esterification to invert the alcohol stereocenter yielding acetate 51, the hydrolysis of which
provided diastereomer 19.
Scheme 2. Synthesis of ADBꢀPINACA metabolites 19–22.a
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O
O
4
5
O
O
O
O
NH2
NH2
O
OR'
NH
NH
a
c
d
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8
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N
N
N
N
N
N
N
H
R
R
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OH
42
43: R = (CH2)3C(O)CH3; R' = CH3
(4R)-44: R = (CH2)3CH(OBn)CH3; R' = CH3
45: R = (CH2)5OBn; R' = CH3
21: R = (CH2)3C(O)CH3
49: R = (CH2)3CH(OBn)CH3
50: R = (CH2)5OBn
22
b
46: R = (CH2)3CH(O)CH3; R' = H
(4R)-47: R = (CH2)3CH(OBn)CH3; R' = H
48: R = (CH2)5OBn; R' = H
d
O
O
O
O
NH2
NH2
NH
NH
e
N
N
N
N
OH
OR
20
51: R = OAc
19: R = H
f
aReagents and conditions: (a)(i) 25, 26, or 27, tꢀBuOK, THF, 0 ºC–rt, 48 h; (ii) ∆, 24 h, 54–
84%; (b)(i) 3 M aq. NaOH, MeOH, ∆, 14 h, 71–90%; (ii) 1 M aq. HCl, MeOH, rt, 54% (43
only); (c) Lꢀtertꢀleucinamide, EDC, HOBt, DMSO, rt, 14 h, 64–84%; (d) 10% Pd/C, H2(g),
MeOH, rt, 2 h, 88–99%; (e) DIAD, PPh3, AcOH, THF, 0 ºC–rt, 16 h, 94%; (f) 1 M aq.
NaOH, MeOH, rt, 30 min, 99%.
The synthesis of 5ꢀCOOHꢀADBꢀPINACA (23) required an orthogonal protecting group
strategy and is depicted in Scheme 3. Indazoleꢀ3ꢀcarboxylic acid (52) was esterified with
benzyl alcohol using DCC to give 53. Regioselective alkylation of 53 with 28 afforded 54,
and was followed by hydrogenolysis to give carboxylic acid 55. Coupling acid 55 with
leucinamide afforded 56, the hydrolysis of which furnished metabolite 23.
Lꢀtertꢀ
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Analogous to the preparation of 46, (4R)ꢀ47, and 48, alkylation of 42 with 1ꢀbromopentane
5
6
7
8
furnished 57, and subsequent hydrolysis gave 58. Coupling of 58 to methyl
using PyBOP afforded 59, which was subsequently hydrolyzed to give 24.
Lꢀtertꢀleucinate
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10
11
12
13
14
15
16
17
18
19
20
21
22
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43
44
45
46
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51
52
53
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55
56
57
58
59
60
Scheme 3. Synthesis of ADBꢀPINACA/5FꢀADBꢀPINACA metabolites 23 and 24.a
O
O
O
O
NH2
OR
OR
NH
b
d
N
N
N
N
N
N
H
O
OR
O
O
52: R = H
53: R = Bn
54: R = Bn
55: R = H
56: R = Me
23: R = H
a
c
e
O
O
O
O
O
OR
NH
OR
f
g
N
N
N
N
N
H
N
42
57: R = Me
58: R = H
59: R = Me
24: R = H
e
e
aReagents and conditions: (a) BnOH, DCC, DMAP, CH2Cl2, r.t., 3 h, 74%; (b)
Br(CH2)4COOCH3, K2CO3, KI, CH3CN, ∆, 24 h, 50%; (c) 10% Pd/C, MeOH, H2(g), r.t., 2 h,
68%; (d) Lꢀtertꢀleucinamide·HCl, HOBt, EDC, DMF, r.t., 4 h, 73%; (e) 2 M aq. NaOH,
MeOH, ∆, 2 h, 67–85%; (f) Br(CH2)4CH3, tꢀBuOK, THF, 0°C–rt, 48 h, 84%; (g) methyl
tertꢀleucinate hydrochloride, PyBOP, DIPEA, DMSO, 0 °C–rt, 2 h, 76%.
Lꢀ
The cannabimimetic activity of APICA and ADBꢀPINACA metabolites at CB1 and CB2 were
compared to CP 55,940 and the parent compounds, with the results shown in Tables 1 and 2.
AtTꢀ20 neuroblastoma cells were transfected with CB1 or CB2, and the activities of CP
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55,940, APICA, ADBꢀPINACA and their respective metabolites were assessed using a
fluorometric imaging reading plate reader (FLIPR) membrane potential assay. G proteinꢀ
gated inwardly rectifying K+ channels (GIRK) are expressed endogenously on AtTꢀ20 cells
which are activated by coꢀexpressed CB1 and CB2 receptors.4 The effects of 9–24 were
compared to CPꢀ55,940, an efficacious CB1 and CB2 agonist, which produced a maximal
decrease in fluorescence, corresponding to cellular hyperpolarization, of 26% ± 1% in AtTꢀ
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14
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17
18
19
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23
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20ꢀCB1 cells and 32% ± 2% in AtTꢀ20ꢀCB2 cells.
Table 1. Functional activity of CP 55,940, 14, and 16–21 at CB1 and CB2 receptors.
Compound
hCB1
hCB2
CB2 sel.*
pEC50 ± SEM Max ± SEM pEC50 ± SEM Max
±
SEM
(EC50, nM)
(%CP 55,940)
(EC50, nM)
(%CP 55,940)
7.76 ± 0.05
(18)
7.66 ± 0.05
(22)
ꢀ
ꢀ
0.8
CP 55,940
6.93 ± 0.09
(118)
7.44 ± 0.09
(37)
109 ± 5
92 ± 4
3.2
APICA (9)
6.29 ± 0.05
(512)
6.58 ± 0.20
(264)
109 ± 3
113 ± 9
1.9
(4R)-4-OH-APICA
(4R-13)
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6.45 ± 0.05
6.54 ± 0.07
(291)
106 ± 3
109 ± 3
1.2
(353)
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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
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39
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41
42
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(4S)-4-OH-APICA
(4S-13)
6.40 ± 0.07
(400)
8.05 ± 0.11
(8.9)
100 ± 4
99 ± 5
45
4-CO-APICA (14)
6.10 ± 0.13
(787)
8.03 ± 0.11
(9.2)
99 ± 8
94 ± 5
86
5-OH-APICA (15)
82
6.54 ± 0.16
(291)
n.d.
89 ± 9
>34
(at 30 µM)
5-COOH-APICA
(16)
7.63 ± 0.10
(24)
8.14 ± 0.13
(7.3)
119 ± 5
94 ± 4
3.3
3-Ad-OH-APICA
(17)
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5.44 ± 0.23
(3670)
7.30 ± 0.08
(50)
95 ± 16
96 ± 4
73
8
18
*CB2 selectivity expressed as the ratio of CB1 EC50 to CB2 EC50. n.d. = not determined
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In comparison to APICA itself (CB1 EC50 = 118 nM), all APICA metabolites, except for 17
(EC50 = 24 nM), showed reduced activity at CB1. The Nꢀdealkylated metabolite (18, CB1
EC50 = 3673 nM) and terminally carboxylated metabolite (16, CB1 EC50 > 10 µM) showed
the most substantial decrease in potency. Despite varying potencies, all APICA metabolites
were shown to be highly efficacious CB1 agonists with 17 eliciting a greater response (119%)
than CP 55,940.
3ꢀAdꢀOHꢀAPICA also proved to be the most potent metabolite at CB2 (EC50 = 7.3 nM).
Interestingly, pentyl oxidized metabolites 14 and 15 exhibited potent activity at the CB2
receptor (EC50 = 8.9 and 9.2 nM respectively), exceeding that of APICA (CB2 EC50 = 37 nM)
and displaying a substantial increase in selectivity for CB2 (45 and 85 times more selective
for CB2 than CB1 respectively). In contrast, 4R-13 and 4S-13 showed lower efficacy (264 and
291 nM respectively) and selectivity (1.9 and 1.2 respectively) for CB2. Despite being less
efficacious, 5ꢀCOOHꢀAPICA (CB2 EC50 = 291 nM) and dealkylated APICA (CB2 EC50 = 50
nM) also displayed significant selectivity for the CB2 receptor (>34 and 73 respectively). The
hyperpolarization produced by each of the APICA metabolites was comparable to CPꢀ55,940,
indicating they act as high efficacy agonists at CB2.
ADBꢀPINACA metabolites showed similar results to their APICA counterparts. Despite
showing decreased potency compared to the parent compound (CB1 EC50 = 1.3 nM),
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metabolites 19–22, arising from oxidation of the pentyl chain, retained potent activity at CB1
(EC50 = 14–58 nM) while the carboxylic acid metabolites 23 and 24 showed greatly
diminished activity (CB1 EC50 = 333 nM and >10 µM respectively). Excluding 5ꢀCOOHꢀ
ADBꢀPINACA (84% maximum response of CPꢀ55,940), all metabolites showed a response
comparable to ADBꢀPINACA at CB1. All metabolites except 5ꢀCOOHꢀADBꢀPINACA
exhibited similar functional activity (5.5 – 65 nM) and generally an increase in selectivity for
CB2 compared to ADBꢀPINACA. Most notably were 21 and 22 (EC50 = 3.2 and 5.5 nM
respectively), which showed a reversal in receptor selectivity compared to the parent
compound (5.9 and 11 respectively). Hydroxylated metabolites 19, 20 and 22 proved to be
highly efficacious CB2 agonists whereas all other ADBꢀPINACA metabolites showed subꢀ
maximal responses in compared to CPꢀ55,940.
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
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57
58
59
60
The concentration of APICA and ADBꢀPINACA metabolites generated following ingestion
is unknown except for 2328, however, based on the findings that SC metabolites can often be
found in amounts in the range of 20ꢀ100 ng/ml (corresponding to a concentration of
approximately 5ꢀ30 nM for the compounds reported here),41 it is likely that some of the
metabolites will be exerting significant agonist actions on their own, and these may
contribute to persistent intoxication.
Table 2. Functional activity of CP 55,940 and 11, and 19–24 at CB1 and CB2 receptors.
Compound
hCB1
hCB2
CB2 sel.*
pEC50 ± SEM Max ± SEM pEC50 ± SEM Max
±
SEM
(EC50, nM)
(%CP 55,940)
(EC50, nM)
(%CP 55,940)
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7.76 ± 0.05
7.66 ± 0.05
(22)
ꢀ
ꢀ
0.8
(18)
8
9
CP 55,940
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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47
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53
54
55
56
57
58
59
60
8.91 ± 0.07
(1.3)
8.59 ± 0.12
(2.6)
121 ± 5
103 ± 6
0.5
ADB-PINACA (11)
7.85 ± 0.05
(14)
7.73 ± 0.08
(19)
113 ± 4
116 ± 4
123 ± 6
111 ± 4
109 ± 4
78 ± 3
0.8
0.8
5.9
(4R)-4-OH-ADB-
PINACA (19)
7.77 ± 0.04
(17)
7.89 ± 0.08
(13)
(4S)-4-OH-ADB-
PINACA (20)
7.19 ± 0.11
(65)
7.98 ± 0.08
(11)
4-CO-ADB-
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7.24 ± 0.08
8.26 ± 0.07
(5.5)
116 ± 5
96 ± 4
11
(58)
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5-OH-ADB-
PINACA (22)
84
81
>10 µM
n.d.
n.d.
(at 30 µM)
(at 30 µM)
5-COOH-ADB-
PINACA (23)
6.48 ± 0.66
(333)
7.19 ± 0.11
(65)
140 ± 40
91 ± 4
5.1
L-COOH-ADB-
PINACA (24)
*CB2 selectivity expressed as the ratio of CB1 EC50 to CB2 EC50. n.d. = not determined
This work presents an effective strategy to the synthesis of a range of Nꢀpentyl oxidized
metabolites of newer generation SC designer drugs. These synthetic approaches could be of
use to forensic chemists in the synthesis of chemical reference standards of major SCs. All
metabolites were also shown to retain some cannabimimetic activity in vitro as shown in a
FLIPR membrane potential assay, suggesting they could be contributing to the physiological
effects of the drugs. An intriguing approach to addressing the activity of synthetic
cannabinoids and metabolites is the recently reported luminescent assay of arrestin
recruitment by CB receptors expressed in HEK293 cells.42 Different cannabinoids are likely
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to recruit arrestin more or less well than they couple to inwardly rectifying K channels (the
basis of our assay),43 and it is possible that there will be absolute bias for one pathway over
the other, but the 2 approaches are complementary, and it would be intriguing to compare the
same compounds in the 2 systems.
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The preliminary findings from this work suggest that oxidation on the Nꢀpentyl chain
regardless of heterocyclic core generally results in a preference for CB2 functionality. Most
notably were the 5ꢀhydroxylated and 4ꢀcarbonylated metabolites, which showed significant
increases in CB2 selectivity for both series of compounds. It is also apparent the presence of a
carboxylic acid at the terminus of the Nꢀpentyl chain, causes a drastic decrease in
cannabimimetic activity. These results suggest the Nꢀpentyl oxidized metabolites of high
concern SCs potentially contribute to the overall pharmacological and toxicological profiles.
Further investigation into the biodistribution of these metabolites should provide further
insights as to whether they act in a synergistic manner with their parent compounds.
Supplementary Information: Full experimental detail for all synthesized compounds
including 1H and 13C NMR spectra of final compounds are included.
Corresponding Author
Michael Kassiou. Eꢀmail: michael.kassiou@sydney.edu.au
Author Contributions
M.K. and M.L. conceived and designed the project. M.L. and S.D.B carried out the organic
synthesis and analyzed all the compounds. M.C. carried out and analyzed the in vitro
experiments. M.L. and S.D.B. wrote the manuscript with the help of M.C. and M.K.
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Notes
7
8
The authors declare no competing financial interest.
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Acknowledgments
Work was supported by NHMRC Project Grant 1107088 awarded to M.K. and M.C.
The authors thank Dr. Nick Proschogo for HRMS measurements.
Abbreviations
SC, synthetic cannabinoid; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; NPS,
novel psychoactive substance; UGT, uridine diphosphateꢀglucuronosyltransferase; AC,
adenylate
cyclase;
HOBt,
hydroxybenzotriazole;
PyBOP,
EDC,
1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide;
benzotriazolꢀ1ꢀylꢀ
oxytripyrrolidinophosphonium hexafluorophosphate; DMF, dimethylformamide; DIPEA,
diisopropylethylamine; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; DIAD, diisopropyl
azodicarboxylate; DCC, N,N’ꢀdicyclohexylcarbodiimide; FLIPR, fluorometric imaging
reading plate reader; GIRK, G proteinꢀgated inwardly rectifying K+ channels.
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