Synthesis and Pharmacological Profiling of the Metabolites of

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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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ACS Chemical Neuroscience

Synthesis and Pharmacological profiling of the metabolites of synthetic cannabinoid drugs APICA, STS-135, ADB-PINACA, and 5F-ADB-PINACA

Mitchell Longworth,a# Mark Connor,b# Samuel D. Banister,c Michael Kassioua*

a

School 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

1 Environment ACS Paragon Plus


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Keywords: cannabinoid, THC, APICA, ADB-PINACA, metabolism Abstract: Synthetic cannabinoids (SCs) containing a 1-pentyl-1-H 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 ADBPINACA. 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 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.


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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 ∆9-tetrahydrocannabinol (∆9-THC, 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.2-4 While the metabolism of ∆9-THC 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,5-8 but attempts to understand the pharmacology of SC metabolites has been less systematic.

Unlike THC, which only contains one psychoactive metabolite (11-OH-THC, 2),9 it is hypothesized that the major oxidative metabolites of SCs retain cannabimimetic activity, contributing to the overall physiological profile of the drugs. JWH-018 (3) was one of the earliest recreational SCs detected,10 and the human urinary metabolites of JWH-018 were reported in 2010 at concentrations up to 83 ng/mL.11,12 Metabolic oxidation of JWH-018 was found to occur on the naphthalene and indole rings, as well as the pentyl chain. The terminally fluorinated analogue, AM-2201 (4), was found to generate common metabolites in some but not all cases. In humans, ω-hydroxy- (5-OH-JWH-018, 5), ω-1-hydroxy- (4-OHJWH-018, 6), and ω-carboxy-JWH-018 (5-COOH-JWH-018, 7) (Fig. 1) were found to be excreted in high concentrations (5 and 6 exclusively as glucuronic acid conjugates) following JWH-018 consumption.13 AM-2201 undergoes oxidative defluorination to give common metabolites 5 and 7, but not 6,14 as well as N-dealkylation to give naphthoylindole 8.15



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Figure 1. Selected cannabinoids and metabolites.

11

R

O

O

O

9 10

OH N

N

N 1

O

1

4

4

1

OH

5

R

5

O

5

JWH-018 (3; R = H) AM-2201 (4; R = F) 5-OH-JWH-018 (5; R = OH)

4 3

3

3

∆9-THC (1; R = H) 11-OH-THC (2; R = OH)

2

2

2

4-OH-JWH-018 (6)

OH

5-COOH-JWH-018 (7)

O O

O

NH 2

NH

NH

O N N

N

2

2

N H

1

1

3 5

8

4

4 3

R

APICA (9; R = H) STS-135 (10; R = F)

5

R

ADB-PINACA (11; R = H) 5F-ADB-PINACA (12; R = F)

Several phase I metabolites of JWH-018 and AM-2201, including common metabolite 5, possessed high affinity for CB1 receptors and exhibited efficacious agonist activity in vitro.16 Monohydroxylated JWH-018 metabolites also showed high affinity for human CB2 receptors.17 In contrast, the glucuronidated conjugate of 5-OH-JWH-018, a major human JWH-018 metabolite, was found to possess neutral antagonist activity at CB1 receptors.18 Additionally, when compared to ∆9-THC or CP 55,940, JWH-018 and 5-OH-JWH-018 are higher efficacy agonists in an assay of CB2 receptor inhibition of adenylyl cyclase (AC).17

In 2012, following the identification of numerous 3-acylindoles related to JWH-018, APICA (2NE1, SDB-001, 9) was reported as one of the first indole-3-carboxamide SCs, followed by its ω-fluorinated analogue, STS-135 (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 adamantane-derived SCs persist, and continue to be associated with serious adverse effects.22

The phase I metabolites of APICA and STS-135 have been described.23-24 As expected, oxidation of the N-alkyl 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 indole-3carboxamides but not 3-acylindoles,25 and 1-adamantylamine has been proposed as a simple APICA biomarker.26 Interestingly, 1-adamantylamine is itself a bioactive substance; the approved antiviral and anti-Parkinsonian drug amantadine.

ADB-PINACA (11) is a member of the recent, prevalent indazole-3-carboxamide SC class, and new variants continue to appear, including the anticipated 5-fluoro derivative 5F-ADBPINACA (12) (Fig. 1). ADB-PINACA exposure has been associated with severe adverse reactions, including neurotoxicity, cardiotoxicity, and death.27-28 Like APICA, ADBPINACA acts as a potent CB1 and CB2 receptor agonist in vitro and in vivo,29-30 but nothing is known of the pharmacology of its metabolites. The metabolism of closely related ABPINACA (differing by an isopropyl rather than tert-butyl side-chain) and its 5-fluorinated analogue (5F-AB-PINACA) was recently reported,31 enabling prediction of some likely ADB-PINACA metabolites. Another study in human liver microsomes confirmed that ABPINACA 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,34-36 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.38-39

The aim of the current work was to develop synthetic routes to several phase I metabolites of APICA, STS-135, ADB-PINACA, and 5F-ADB-PINACA and to characterize the pharmacology of these metabolites at human CB1 and CB2 receptors. Ultimately, we sought to elucidate whether apparent trends in the structure-activity relationships of acylindole SC metabolites translate to newer, prevalent indole- and indazole-3-carboxamide SC derivatives featuring a common 1-pentyl subunit.

Results and discussion: The synthesis of several phase I metabolites of APICA/STS-135 (13−18, Fig. 2) and ADB-PINACA/5F-ADB-PINACA (19−24), including diastereomers 19 and 20, was inspired by our previous synthetic routes to parent structures APICA/STS-135 and ADB-PINACA/5F-ADB-PINACA.29, 40

Figure 2. Representative phase I metabolites of APICA, STS-135, ADB-PINACA, and 5FADB-PINACA.


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O

O

NH

O NH

O NH

OH O

NH

NH O

N

N

N

N OH

N H

O OH

4-OH-APICA (13)

4-CO-APICA (14)

NH

O

5-OH-APICA (15)

O O

O

N

NH

NH 2

OH

NH

NH 2

NH

O

NH 2

O

NH

N

OH

N

N

N

O OH

(4R)-4-OH-ADB-PINACA (19) (4S)-4-OH-ADB-PINACA (20)

18

O O

N

N

3-Ad-OH-APICA (17)

O O

N

N

N

OH

5-COOH-APICA (16)

O

NH 2

NH

N

4-CO-ADB-PINACA (21)

5-OH-ADB-PINACA (22)

O

OH

5-COOH-ADB-PINACA (23)

24

The synthesis of 4-OH-APICA enantiomers 4R-13 and 4S-13, 4-CO-APICA (14), 5-OHAPICA (15), Ad-OH-APICA (17) are shown in Scheme 1. In short, it was envisaged that 1alkylation of the esters of 1H-indole- and 1H-indazole-3-carboxylic 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 one-pot N-alkylation 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 3hydroxy-1-aminoadamantane with HOBt and EDC provided 17, while 34–36 were reacted with 1-aminoadamantane 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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N-Dealkylated APICA metabolite, 18, and 5-COOH-APICA (16) were synthesized from indole-3-carboxylic acid (41), converting it to the corresponding acid chloride by treatment with oxalyl chloride, and reacting with 1-aminoadamantane to give 18. Alkylation of 18 with the methyl ester of 1-bromo-5-pentanoic acid (28), and subsequent hydrolysis upon work-up, provided 16.

Scheme 1. Synthesis of APICA metabolites 13, 14, 15, 16, 17 and 18.a

O

O

CF3

a

b

N H

O

OH

O

NH

d or e

N R

NH

g

N R

N R

N

OH 30: 31: 32: 33:

29

R = (CH 2)3C(O)CH3 R = (CH 2)3CH(OBn)CH3 R = (CH 2)5OBn R = (CH 2) 4CH3

34: 35: 36: 37: 38:

R = (CH 2)3C(O)CH3 R = (CH 2)3CH(OBn)CH3 R = (CH 2)5OBn R = (CH 2) 4CH3 R=H c

1

3 2

O

1

5

4

Br

O

3

= (CH 2) 3C(O)CH 3 = (CH 2) 3CH(OBn)CH3 = (CH 2) 5OBn =H = (CH 2) 4COOH

15

f

g

5

4

OH

Br 2

14: R 39: R 40: R 18: R 16: R

O

OBn

NH

O

O

NH

NH

h 26

25

N 1

5

3

Br

1

OBn 2

4

27

Br

5 2

N

N

O

3

OH

OR

O

4

28

17

(4R)-13 i

a

41: R = OAc (4S)-13: R = H

Reagents 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) 1-amino-3hydroxyadamantane hydrochloride, EDC, HOBt, DIPEA, DMSO, rt, 14 h, 41%; (d) 1aminoadamantane hydrochloride, PyBOP, DIPEA, DMSO, rt, 2 h, 46–97%; (e) for 48 only; (i) (COCl)2, DMF, 0 °C–rt, 1 h; (ii) 1-aminoadamantane, 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%.

The

synthesis

of

ADB-PINACA/5F-ADB-PINACA

metabolites

utilized

similar

methodologies described for APICA/STS-135 metabolites. The synthesis of diastereomers (4R)-4-OH-ADB-PINACA (19) and (4S)-4-OH-ADB-PINACA (20), as well as 4-CO-ADBPINACA (21), and 5-OH-ADB-PINACA (22), are depicted in Scheme 2. Regioselective 1alkylation of methyl 1H-indazole-3-methyl 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, (4R)-47, and 48 were coupled with L-tert-leucinamide to give 21, 49, and 50 respectively. Hydrogenolysis of O-benzyl 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 ADB-PINACA metabolites 19–22.a



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O O

O

O N

N H

a

O

OR' c

N N R

O O

NH 2

NH

NH

d

N N R

NH 2

N N

OH 42 b

43: R = (CH 2)3C(O)CH3; R' = CH3 (4R)-44: R = (CH 2) 3CH(OBn)CH3; R' = CH3 45: R = (CH 2)5OBn; R' = CH 3

21: R = (CH 2) 3C(O)CH3 49: R = (CH 2) 3CH(OBn)CH3 50: R = (CH 2) 5OBn

46: R = (CH 2)3CH(O)CH3; R' = H (4R)-47: R = (CH 2) 3CH(OBn)CH3; R' = H 48: R = (CH 2)5OBn; R' = H

22

d

O O

NH

O O

NH 2

NH

NH 2

e N

N

N

N OH

20

a

OR

f

51: R = OAc 19: R = H

Reagents and conditions: (a)(i) 25, 26, or 27, t-BuOK, 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) L-tert-leucinamide, 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 5-COOH-ADB-PINACA (23) required an orthogonal protecting group strategy and is depicted in Scheme 3. Indazole-3-carboxylic 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 L-tertleucinamide afforded 56, the hydrolysis of which furnished metabolite 23.


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Analogous to the preparation of 46, (4R)-47, and 48, alkylation of 42 with 1-bromopentane furnished 57, and subsequent hydrolysis gave 58. Coupling of 58 to methyl L-tert-leucinate using PyBOP afforded 59, which was subsequently hydrolyzed to give 24.

Scheme 3. Synthesis of ADB-PINACA/5F-ADB-PINACA metabolites 23 and 24.a O O

O

OR b

N

N

N

N

O 52: R = H 53: R = Bn

c

NH 2

NH

d

N

N H

a

O

OR

O

OR

O

54: R = Bn 55: R = H

56: R = Me 23: R = H

e

O O

O

O N

f

N

N H

g

e

57: R = Me 58: R = H

NH

OR

N

N

42

a

O

OR

N

e

59: R = Me 24: R = H

Reagents 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) L-tert-leucinamide·HCl, HOBt, EDC, DMF, r.t., 4 h, 73%; (e) 2 M aq. NaOH, MeOH, ∆, 2 h, 67–85%; (f) Br(CH2)4CH3, t-BuOK, THF, 0°C–rt, 48 h, 84%; (g) methyl Ltert-leucinate hydrochloride, PyBOP, DIPEA, DMSO, 0 °C–rt, 2 h, 76%.

The cannabimimetic activity of APICA and ADB-PINACA metabolites at CB1 and CB2 were compared to CP 55,940 and the parent compounds, with the results shown in Tables 1 and 2. AtT-20 neuroblastoma cells were transfected with CB1 or CB2, and the activities of CP



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55,940, APICA, ADB-PINACA and their respective metabolites were assessed using a fluorometric imaging reading plate reader (FLIPR) membrane potential assay. G proteingated inwardly rectifying K+ channels (GIRK) are expressed endogenously on AtT-20 cells which are activated by co-expressed CB1 and CB2 receptors.4 The effects of 9–24 were compared to CP-55,940, an efficacious CB1 and CB2 agonist, which produced a maximal decrease in fluorescence, corresponding to cellular hyperpolarization, of 26% ± 1% in AtT20-CB1 cells and 32% ± 2% in AtT-20-CB2 cells.

Table 1. Functional activity of CP 55,940, 14, and 16–21 at CB1 and CB2 receptors. Compound

hCB2

hCB1

CB2 sel.*

pEC50 ± SEM

Max ± SEM

pEC50 ± SEM

(EC50, nM)

(%CP 55,940)

(EC50, nM)

7.76 ± 0.05 (18)

-

7.66 ± 0.05 (22)

-

0.8

6.93 ± 0.09 (118)

109 ± 5

7.44 ± 0.09 (37)

92 ± 4

3.2

6.29 ± 0.05 (512)

109 ± 3

6.58 ± 0.20 (264)

113 ± 9

1.9

Max

±

SEM

(%CP 55,940)

CP 55,940

APICA (9)

(4R)-4-OH-APICA (4R-13)


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6.45 ± 0.05 (353)

106 ± 3

6.54 ± 0.07 (291)

109 ± 3

1.2

6.40 ± 0.07 (400)

100 ± 4

8.05 ± 0.11 (8.9)

99 ± 5

45

6.10 ± 0.13 (787)

99 ± 8

8.03 ± 0.11 (9.2)

94 ± 5

86

n.d.

82 (at 30 µM)

6.54 ± 0.16 (291)

89 ± 9

>34

7.63 ± 0.10 (24)

119 ± 5

8.14 ± 0.13 (7.3)

94 ± 4

3.3

(4S)-4-OH-APICA (4S-13)

4-CO-APICA (14)

5-OH-APICA (15)

5-COOH-APICA (16)

3-Ad-OH-APICA (17)



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5.44 ± 0.23 (3670)

95 ± 16

7.30 ± 0.08 (50)

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96 ± 4

73

18 *CB2 selectivity expressed as the ratio of CB1 EC50 to CB2 EC50. n.d. = not determined

In comparison to APICA itself (CB1 EC50 = 118 nM), all APICA metabolites, except for 17 (EC50 = 24 nM), showed reduced activity at CB1. The N-dealkylated 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.

3-Ad-OH-APICA 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, 5-COOH-APICA (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 CP-55,940, indicating they act as high efficacy agonists at CB2.

ADB-PINACA 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 5-COOHADB-PINACA (84% maximum response of CP-55,940), all metabolites showed a response comparable to ADB-PINACA at CB1. All metabolites except 5-COOH-ADB-PINACA exhibited similar functional activity (5.5 – 65 nM) and generally an increase in selectivity for CB2 compared to ADB-PINACA. 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 ADB-PINACA metabolites showed submaximal responses in compared to CP-55,940.

The concentration of APICA and ADB-PINACA 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 20-100 ng/ml (corresponding to a concentration of approximately 5-30 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

pEC50 ± SEM

Max ± SEM

pEC50 ± SEM

(EC50, nM)

(%CP 55,940)

(EC50, nM)


Max

CB2 sel.* ±

SEM

(%CP 55,940)


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7.76 ± 0.05 (18)

-

7.66 ± 0.05 (22)

-

0.8

8.91 ± 0.07 (1.3)

121 ± 5

8.59 ± 0.12 (2.6)

103 ± 6

0.5

7.85 ± 0.05 (14)

113 ± 4

7.73 ± 0.08 (19)

111 ± 4

0.8

7.77 ± 0.04 (17)

116 ± 4

7.89 ± 0.08 (13)

109 ± 4

0.8

7.19 ± 0.11 (65)

123 ± 6

7.98 ± 0.08 (11)

78 ± 3

5.9

CP 55,940

ADB-PINACA (11)

(4R)-4-OH-ADBPINACA (19)

(4S)-4-OH-ADBPINACA (20)

4-CO-ADBPINACA (21)


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7.24 ± 0.08 (58)

116 ± 5

8.26 ± 0.07 (5.5)

96 ± 4

11

>10 µM

84 (at 30 µM)

n.d.

81 (at 30 µM)

n.d.

6.48 ± 0.66 (333)

140 ± 40

7.19 ± 0.11 (65)

91 ± 4

5.1

5-OH-ADBPINACA (22)

5-COOH-ADBPINACA (23)

L-COOH-ADBPINACA (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 N-pentyl 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.

The preliminary findings from this work suggest that oxidation on the N-pentyl chain regardless of heterocyclic core generally results in a preference for CB2 functionality. Most notably were the 5-hydroxylated and 4-carbonylated 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 N-pentyl chain, causes a drastic decrease in cannabimimetic activity. These results suggest the N-pentyl 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. E-mail: [email protected]

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. 18 Environment ACS Paragon Plus

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Notes The authors declare no competing financial interest.

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 diphosphate-glucuronosyltransferase; AC, adenylate

cyclase;

HOBt,

dimethylaminopropyl)carbodiimide;

hydroxybenzotriazole; PyBOP,

EDC,

1-ethyl-3-(3benzotriazol-1-yl-

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 protein-gated inwardly rectifying K+ channels.

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105x50mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Synthesis and Pharmacological Profiling of the Metabolites of

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