Pharmacology of Cumyl-Carboxamide Synthetic Cannabinoid New Psychoactive Substances (NPS) CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-5F-PINACA, and Their Analogues

Article abstract of DOI:10.1021/acschemneuro.7b00267

Synthetic cannabinoids (SC) are the largest class of new psychoactive substances (NPS), and are increasingly associated with serious adverse effects. The majority of SC NPS are 1,3-disubstituted indoles and indazoles featuring a diversity of subunits at t

Full text of DOI:10.1021/acschemneuro.7b00267

Subscriber access provided by Purdue University Libraries
Letter
The pharmacology of cumyl-carboxamide synthetic cannabinoid
new psychoactive substances (NPS) CUMYL-BICA, CUMYL-
PICA, CUMYL-5F-PICA, CUMYL-5F-PINACA, and their analogues.
Mitchell Longworth, Samuel D. Banister, Rochelle Boyd, Richard
C. Kevin, Mark Connor, Iain S. McGregor, and Michael Kassiou
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 09 Aug 2017
Downloaded from http://pubs.acs.org on August 9, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
The pharmacology of cumyl-carboxamide synthetic cannabinoid new psychoactive
substances (NPS) CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-5F-
PINACA, and their analogues
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
56
57
58
59
60
Mitchell Longworth,a Samuel D. Banister,a,b Rochelle Boyd,c Richard C. Kevin,d Mark Connor,c
Iain S. McGregor,d Michael Kassioua*
aSchool of Chemistry, The University of Sydney, NSW 2006, Australia; bDepartment of
Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; cDepartment of
Biomedical Sciences, Macquarie University, NSW 2109, Australia; dSchool of Psychology, The
University of Sydney, NSW 2006, Australia
*Corresponding author
1
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 2 of 30
1
2
3
4
5
6
7
8
9
Abstract: Synthetic cannabinoids (SC) are the largest class of new psychoactive substances
(NPS), and are increasingly associated with serious adverse effects. The majority of SC NPS are
1,3ꢀdisubstituted indoles and indazoles featuring a diversity of subunits at the 1ꢀ and 3ꢀpositions.
Most recently, cumylꢀderived indoleꢀ and indazoleꢀ3ꢀcarboxamides have been detected by law
enforcement agencies and by emergency departments. Herein we describe the synthesis,
characterization, and pharmacology of SCs CUMYLꢀBICA, CUMYLꢀPICA, CUMYLꢀ5FꢀPICA,
CUMYLꢀPINACA, CUMYLꢀ5FꢀPINACA and related analogues. All cumylꢀderived SCs were
potent, efficacious agonists at CB1 (EC50 = 0.43–12.3 nM) and CB2 (EC50 = 11.3–122 nM)
receptors in a fluorometric assay of membrane potential, with selectivity for CB1 activation (3.1–
53 times over CB2). CUMYLꢀPICA and CUMYLꢀ5FꢀPICA were evaluated in rats using
biotelemetry, and induced hypothermia and bradycardia at doses of 1 mg/kg. Hypothermia was
reversed by pretreatment with a CB1, but not CB2, antagonist, confirming that cumylꢀderived
SCs are cannabimimetic in vivo.
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
56
57
58
59
60
Keywords: cannabinoid, CUMYLꢀBICA, CUMYLꢀPICA, CUMYLꢀPINACA, CUMYLꢀ5Fꢀ
PICA, CUMYLꢀ5FꢀPINACA
2
ACS Paragon Plus Environment

Page 3 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Introduction. Between 2008 and 2015, 644 new psychoactive substances (NPS) have been
reported to United Nations Office on Drugs and Crime (UNODC).1 Of these, synthetic
cannabinoids (SCs) are the largest pharmacological class, representing 32% of all reported NPS.1
The highly dynamic nature of the NPS market—influenced by user preference, law enforcements
efforts, and legal response—has produced hundreds of SCs of increasingly structural diversity,
with a general trend towards increased cannabinoid receptor potency.
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
56
57
58
59
60
Following the identification and subsequent prohibition of one of the earliest SCs, JWHꢀ018 (1,
Fig. 1), other 3ꢀacylindoles, such as XLRꢀ11 (2), emerged to replace it. Elaborated indole
analogues featuring a carboxylate (e.g. BBꢀ22, 3) or amide (SDBꢀ006, 4) linker in place of the
carbonyl group soon followed, as well as SCs based on other heteroaromatic cores, such as
indazole AKBꢀ48 (5). The most prevalent recent SCs are indoleꢀ and indazoleꢀ3ꢀcarboxamides
featuring pendant amino acid amides or esters, and various aliphatic, alicyclic, and aromatic
substituents at the 1ꢀposition, as exemplified by 5FꢀADB (6) and AMBꢀFUBINACA (7).
Figure 1. Representative synthetic cannabinoid new psychoactive substances.
3
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 4 of 30
1
2
3
4
5
6
7
8
9
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
56
57
58
59
60
The pharmacology and toxicology of many SCs has been explored, but new structural classes are
emerging every year.2ꢀ4 Most recently, SCs featuring a 2ꢀphenylpropanꢀ2ꢀyl (i.e. cumyl) group
(8–17, Fig. 2) have been reported around the world, however, these molecules originate from a
patent filed in New Zealand in 2013.5 CUMYLꢀ5FꢀPICA (10) was identified in Slovenia on 23
September 2014, along with CUMYLꢀBICA (8), and CUMYLꢀPICA (9).6 Consistent with the
trend for terminal fluorination of Nꢀpentyl SCs, CUMYLꢀ5FꢀPINACA (15) was identified in
Sweden several weeks later,6 and was reported to the UNODC Early Warning Advisory (EWA)
in Luxembourg and the United Kingdom in 2014.6 Very recently, occupational transdermal
exposure to CUMYLꢀPINACA was reported following a seizure by law enforcement officers in
4
ACS Paragon Plus Environment

Page 5 of 30
ACS Chemical Neuroscience
1
2
Slovenia.7 Despite their prevalence, to date there are relatively few reports in the literature
concerning the toxicological effects of these compounds.8
3
4
5
6
7
8
9
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
56
57
58
59
60
To provide analytical and pharmacological data on this emergent class of SCs, we have
systematically prepared a library of cumylꢀderived indoleꢀ and indazoleꢀ3ꢀcarboxamides
featuring the commonly encountered butyl, pentyl, 5ꢀfluoropentyl, 4ꢀfluorobenzyl, and
(cyclohexyl)methyl subunits at the 1ꢀposition (8–17).
Figure 2. Cumylꢀderived synthetic cannabinoids.
O
O
NH
NH
N
N
N
R
R
CUMYL-BICA (8; R = (CH2)3CH3)
CUMYL-BINACA (13; R = (CH2)3CH3)
9
14
CUMYL-PICA ( ; R = (CH2)4CH3)
CUMYL-PINACA ( ; R = (CH2)4CH3)
CUMYL-5F-PICA (10; R = (CH2)5F)
CUMYL-5F-PINACA (15; R = (CH2)5F)
11
16
CUMYL-FUBICA ( ; R = 4-FBn)
CUMYL-FUBINACA ( ; R = 4-FBn)
12
CUMYL-CHMICA ( ; R = CH2Cy)
17
CUMYL-CHMINACA ( ; R = CH2Cy)
Results and discussion
The synthesis of indoleꢀ (8–12) and indazoleꢀ3ꢀcarboxamides (13–17) is shown in Figures 3 and
4, respectively. As depicted in Figure 3, treatment of indole (18) with the appropriate alkyl
bromide, followed by trifluoroacetic anhydride, afforded 1ꢀalkylꢀ3ꢀ(trifluoroacetyl)indole
intermediates 19–23 from a convenient oneꢀpot procedure. Hydrolysis of the trifluoroacetyl
group by sodium hydroxide in refluxing aqueous methanol provided carboxylic acids 24–28,
5
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 6 of 30
1
2
3
4
which were subsequently coupled with cumylamine using EDC/HOBt, and furnished the desired
5
6
cumylꢀcarboxamides 8–12.
7
8
9
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
56
57
58
59
60
Figure 3. Synthesis of cumylꢀderived indoleꢀ3ꢀcarboxamides.a
aReagents and conditions: (a)(i) NaH, RBr, DMF, 0 °Cꢀrt, 1 h, (ii) (CF3CO)2O, rt, 1 h (72ꢀ94%);
(b) 1 M aq. NaOH, MeOH, reflux, 18 h (67ꢀ88%); (c) cumylamine, EDC·HCl, HOBt, DIPEA,
DMSO, rt, 14 h (67ꢀ80%).
The synthesis of indazoles 13–17 is outlined in Figure 4, beginning with alkylation of methyl
1Hꢀindazoleꢀ3ꢀcarboxylate (29). Previous studies had demonstrated the role of base in the
regiochemical outcome of this alkylation.9 In order to minimize formation of undesired 2ꢀalkylꢀ
2Hꢀindazoleꢀ3ꢀcarboxylate byꢀproducts, 29 was deprotonated with potassium tertꢀbutoxide, and
treated with the appropriate alkyl bromide to give almost exclusively the 1ꢀsubstituted 1Hꢀ
indazoleꢀ3ꢀcarboxylates 30–34. Saponification of the ester group was achieved using sodium
hydroxide in aqueous methanol, thereby providing 1ꢀalkylꢀ1Hꢀindazoleꢀ3ꢀcarboxylic acids 35–
39. Coupling of 35–39 with cumylamine using EDC/HOBt produced cumylꢀcarboxamide
indazoles 13–17.
6
ACS Paragon Plus Environment

Page 7 of 30
ACS Chemical Neuroscience
1
2
Figure 4. Synthesis of cumylꢀderived indazoleꢀ3ꢀcarboxamides.a
3
4
5
6
7
8
9
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
56
57
58
59
60
aReagents and conditions: (a)(i) KOtBu, RBr, THF, 0 °Cꢀrt, 48 h (65ꢀ84%%); (b) 1 M aq. NaOH,
MeOH, reflux, 18 h (81ꢀ91%); (c) cumylamine, EDC·HCl, HOBt, DIPEA, DMSO, rt, 14 h (71ꢀ
88%).
The synthesized indole and indazole cumylꢀcarboxamides 8–17 were screened at CB1 and CB2
receptors using a fluorometric imaging plate reader (FLIPR) assay to identify structureꢀactivity
relationships (SARs) for this class, and enable comparisons to other classes of prevalent SCs.
The activities of 8–17 at CB1 and CB2 were compared to the low efficacy CB1/2 ꢁ9ꢀ
tetrahydrocannabinol (ꢁ9ꢀTHC), and the efficacious CB1/2 agonist CP 55,940, in an assay of CB
receptorꢀdependent membrane hyperpolarization (Table 1). The assay used murine AtT20ꢀFlpIn
neuroblastoma cells stably expressing human CB1 or CB2 receptors. Activation of CB receptors
resulted in opening of endogenous G proteinꢀgated inwardly rectifying potassium channels
(GIRKs) that produced a hyperpolarization of the cells which was reflected in a decrease in the
fluorescence of a proprietary membrane potential dye.
7
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 8 of 30
1
2
The maximum effects of ꢁ9ꢀTHC and 8–17 were normalized to a maximally efficacious
concentration of CP 55,490. Data for each experiment were normalized to the change in
fluorescence produced by a maximally effective concentration of CP 55,940 (1 µM). CP 55,940
(1 µM) decreased fluorescence by 29.6 ± 0.9 % in CB1ꢀexpressing cells, and 29.7 ± 1.1 % in
CB2ꢀexpressing cells (n = 26 each). None of 8–17 produced a significant change in the
membrane potential of wild type AtTꢀ20 cells (n = 6 each, data not shown), which do not express
CB1 or CB2 receptors. Consistent with a predominant coupling of CB1 and CB2 receptors to
Gi/Go family G proteins, the effects of 8–17 were abolished by overnight treatment of the cells
with pertussis toxin (200 ng/mL), which blocks the coupling of GPCR to Gi/Go family G proteins
(n = 3 each, Table S2, Supporting Information).
3
4
5
6
7
8
9
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
56
57
58
59
60
Table 1. Functional activity of ꢁ9ꢀTHC, CP 55,940, JWHꢀ018, and novel SCs 10–25 at CB1 and
CB2 receptors.
CB1
sel.*
Compound
hCB1
hCB1
hCB2
hCB2
pEC50 ± SEM
(EC50, nM)
Max ± SEM
(% CP 55,940)
pEC50 ± SEM
(EC50, nM)
Max ± SEM
(%CP 55,940)
6.77 ± 0.05
(171)
32 ± 1 at 30
µM
∆9ꢀTHC
50 ± 11
100 ± 2
109 ± 5
116 ± 2
118 ± 2
112 ± 5
ꢀ
ꢀ
7.98 ± 0.04
(10.4)
7.64 ± 0.06
(22.9)
CP 55,490
92 ± 9
101 ± 7
102 ± 5
104 ± 5
92 ± 5
2.2
3.1
14
14
3.7
7.61 ± 0.20
(24.5)
CUMYLꢀBICA (8)
CUMYLꢀPICA (9)
8.10 ± 0.14 (8.0)
8.38 ± 0.04 (4.2)
7.23 ± 0.09
(58.4)
7.40 ± 0.10
(39.6)
CUMYLꢀ5FꢀPICA (10) 8.55 ± 0.05 (2.8)
7.91 ± 0.10
CUMYLꢀFUBICA (11)
(12.3)
7.34 ± 11 (45.5)
8
ACS Paragon Plus Environment

Page 9 of 30
ACS Chemical Neuroscience
1
2
3
4
5
CUMYLꢀCHMICA
8.02 ± 0.13 (9.5)
120 ± 5
6.91 ± 0.22 (122)
92 ± 9
13
(12)
6
7
8
9
7.92 ± 0.04
(12.0)
CUMYLꢀBINACA (13) 8.81 ± 0.10 (1.6)
CUMYLꢀPINACA (14) 8.64 ± 0.08 (2.3)
109 ± 3
116 ± 3
110 ± 3
89 ± 2
106 ± 6
87 ± 3
7.5
47
26
6.97 ± 0.13 (107)
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
56
57
58
59
60
CUMYLꢀ5FꢀPINACA
9.37 ± 0.06
(0.43)
7.95 ± 0.09
(11.3)
(15)
CUMYLꢀFUBINACA
7.63 ± 0.12
(23.7)
8.73 ± 0.06 (1.8)
8.76 ± 0.12 (1.7)
108 ± 3
112 ± 4
84 ± 5
84 ± 5
13
53
(16)
CUMYLꢀCHMINACA
7.05 ± 0.14
(90.0)
(17)
*CB1 selectivity expressed as the ratio of CB1 EC50 to CB2 EC50.
All cumylꢀderived SCs 8–17 activated CB1 and CB2 receptors. All compounds, except CUMYLꢀ
FUBICA (EC50 = 12.3 nM) had greater potency (0.43–9.5 nM) than CP 55,940 (10.4 nM) for
CB1 receptorꢀmediated activation of GIRK. Compounds 8–17 also had maximal effects that were
comparable to, or greater than, CP 55,940 at CB1 receptors (108–120%), indicating that these
SCs are high efficacy, full agonists. All SCs showed a preference for CB1 receptors over CB2
receptors, ranging from low (e.g. CUMYLꢀBICA, 8; 3.1 times) to moderate (e.g. CUMYLꢀ
CHMINACA, 17; 52.9 times).
The cumylꢀderived indazoles 13–17 (EC50 = 0.43–2.3 nM) were all more potent CB1 agonists
than indoles 8–12 (EC50 = 2.8–12.3 nM). No clear structureꢀactivity relationships were evident
for the nature of the 1ꢀsubstituent on CB1 potency in SCs 8–17, however, it is noteworthy that a
5ꢀfluoropentyl chain conferred the greatest potency within both the indole (10; EC50 = 2.8 nM)
and indazole (15; EC50 = 0.43 nM) series. Selectivity for CB1 over CB2 was greatest for cumylꢀ
9
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 10 of 30
1
2
3
4
5
6
7
8
9
derivatives featuring a pentyl, 5ꢀfluoropentyl, or (cyclohexyl)methyl group at the 1ꢀposition in
the indole (13 to 14 times) and indazole (26 to 53 times) series. Conversely, a butyl or 4ꢀ
fluorobenzyl group in the same position conferred lower levels of selectivity within the indole
(3.1 and 3.7 times, respectively) and indazole (7.5 and 13 times, respectively) SCs.
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
56
57
58
59
60
Having shown that 8–17 were potent and efficacious cannabimimetic agents in vitro, we sought
to demonstrate the activity of several of these cumylꢀderived SCs in vivo. The in vivo activity of
CUMYLꢀPICA (9) and CUMYLꢀ5FꢀPICA (10) were compared using biotelemetry in rats to
provide information regarding the activity of these newer SCs in a living system.
A recent study published as this work was being finalized reported the functional activity of a
subset of the compounds reported here in an assay of G protein activation (GTPγ
S binding).10
There are significant differences in the rank order of potency and CB1 selectivity reported
between the 2 studies (SI Table 2). Strikingly, we found CUMYLꢀ5FꢀPINACA to be 20 fold
more potent than CP 55,940 at CB1, while it was 20 times less potent than CP 55,940 in the
GTP
CB2, while in the GTP
The CP 55,940 selectivity for CB1 over CB2 was broadly similar in both studies – 2.2 fold for
GIRK, 0.65 fold for GTP S, and may reflect relative differences in receptor expression. It is also
γS assay. We also found that CUMYLꢀPINACA had a 50ꢀfold preference for CB1 over
γS assay, CUMYLꢀPINACA was reported as CB2 preferring by 10 fold.
γ
notable that in the GIRK assay CUMYLꢀPINACA, CUMYLꢀPICA and CUMYLꢀ5FꢀPICA has
maximal responses 15ꢀ20 % higher than those of CP 55,940, suggesting a higher efficacy. The
GTPγS assay is good for determining relative efficacy as the maximal response is usually
unconstrained, unfortunately the details of the relative maximal responses for these drugs were
10
ACS Paragon Plus Environment

Page 11 of 30
ACS Chemical Neuroscience
1
2
not reported by Asada et al.10 Overall, details of the GTP
γS assay are sparsely reported, without
3
4
5
6
7
8
even identification of the cell types used, but the striking differences in the results between the 2
assays may reflect an unappreciated signaling bias by this class of SCs. The assays are also very
different in that GIRK activation represents a transient maximal signal likely to be unaffected by
9
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
56
57
58
59
60
receptor regulation, while GTPγS assays are typically accumulation assays that are carried out
over extended time in broken cell preparations.
In rodents, crossꢀsubstitution of aroylindole SCs, like JWHꢀ018, and phytocannabinoid ꢁ9ꢀTHC
has been demonstrated, indicating that these cannabinoids produce similar pharmacological
effects despite structural heterogeneity.11ꢀ20 Cannabinoids induce bradycardia, hypothermia, and
hypolocomotion in mice and rats, and these physiological changes are common to functional
cannabinoids from disparate structural classes.21 We have previously determined the
hypothermic and bradycardic potencies of ꢁ9ꢀTHC and numerous structurally diverse SCs,
including JWHꢀ018, AMꢀ2201, URꢀ144, XLRꢀ11, APICA, STSꢀ135, PBꢀ22, 5FꢀPBꢀ22, ABꢀ
PINACA, and ABꢀFUBINACA in rats.22ꢀ26 The cannabimimetic activities of CUMYLꢀPICA and
CUMYLꢀ5FꢀPICA were assessed using radiotelemetry in male Long Evans rats, and the effects
of these SCs on body temperature (Fig. 5) and heart rate (Fig. 6) are presented below.
Rat body temperature from 1 hour prior to intraperitoneal (i.p.) injection to 6 hours post injection
of CUMYLꢀPICA and CUMYLꢀ5FꢀPICA are presented in 15 minute bins in Figure 5. The
dashed line on the figures represents the time of SC injection. Each SC was investigated using a
cohort of 4 rats, with a different cohort used for the two compounds. Doses were escalated from
11
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 12 of 30
1
2
3
4
5
6
0 mg/kg (baseline) to 0.1, 0.3, and 1.0 mg/kg for each compound with at least 2 washout days
with no injections between each dose.
7
8
9
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
56
57
58
59
60
Both CUMYLꢀPICA and CUMYLꢀ5FꢀPICA evoked a pronounced hypothermic effect at 1
mg/kg compared to vehicle during the 6 h immediately following injection (CUMYLꢀPICA P <
.001; CUMYLꢀ5FꢀPICA P < 0.0001; Fig. 5), analyzed via a twoꢀway mixedꢀmodel ANOVA. At
0.3 mg/kg, neither compound produced a statistically significant hypothermic effect over the 6 h
postꢀinjection, although there was some evidence of marginal, brief effects during the first hour.
The peak reduction in body temperature at 1 mg/kg was generally greater with CUMYLꢀ5Fꢀ
PICA (>3 °C) than CUMYLꢀPICA (>2 °C). When compared to 1 mg/kg of CUMYLꢀPICA, an
equivalent dose of CUMYLꢀ5FꢀPICA induced a longer hypothermic period, with mean core
body temperature returning to baseline roughly 4.5 h postꢀinjection, compared to approximately
2.5 h for CUMYLꢀPICA.
These results are in agreement with previously reported hypothermia produced by a single 3
mg/kg dose of CUMYLꢀPICA and CUMYLꢀ5FꢀPICA in rats, measured periodically via rectal
thermometer.27 Taken together, these results indicate that both compounds produce hypothermia
in rats in a doseꢀdependent manner at doses of 1 mg/kg and higher.
Figure 5. Effects of (a) CUMYLꢀPICA and (b) CUMYLꢀ5FꢀPICA on rat body temperature.
Dashed line denotes time of intraperitoneal injection. Each point represents the mean ± SEM for
four animals.
12
ACS Paragon Plus Environment

Page 13 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
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
56
57
58
59
60
Changes in heart rate effected by CUMYLꢀPICA and CUMYLꢀ5FꢀPICA are presented in Fig. 6.
Although heart rate data was more variable than body temperature data, analysis using a twoꢀ
way mixedꢀmodel ANOVA showed that CUMYLꢀPICA significantly reduced heartꢀrate over the
13
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 14 of 30
1
2
3
4
5
6
6 h immediately following doses of 0.3 mg/kg (P < 0.05) and 1 mg/kg (P < 0.01). CUMYLꢀ5Fꢀ
PICA significantly reduced heart rate following a dose of 1 mg/kg (P <.01) only.
7
8
9
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
56
57
58
59
60
Figure 6. Effects of (a) CUMYLꢀPICA and (b) CUMYLꢀ5FꢀPICA on rat heart rate. Dashed line
denotes time of intraperitoneal injection. Each point represents the mean ± SEM for four
animals.
14
ACS Paragon Plus Environment

Page 15 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
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
56
57
58
59
60
To confirm that the observed effects were mediated through CB1 or CB2 receptors, we assessed
the reversibility of the effects of CUMYLꢀPICA and CUMYLꢀ5FꢀPICA on body temperature in
rats following pretreatment with either CB1 receptor antagonist rimonabant (SR141716) or CB2
receptor antagonist SR144528 (Fig. 7). The hypothermic effect of 1 mg/kg CUMYLꢀPICA and
15
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 16 of 30
1
2
3
4
5
6
7
8
9
CUMYLꢀ5FꢀPICA was strongly attenuated by preꢀtreatment with rimonabant (all p < .001, Fig.
S21, Supporting Information). No attenuation of hypothermia was observed following preꢀ
treatment with SR144528 for either compound. This indicates a selective CB1 receptor
dependent hypothermic mechanism. We have reported similar CB1 receptor dependence for the
hypothermic effects of ABꢀPINACA, ABꢀFUBINACA, 5FꢀAMB, and MDMBꢀFUBINACA.24, 26
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
56
57
58
59
60
Figure 7. Effects of (a) 1 mg/kg CUMYLꢀPICA or (b) 1 mg/kg CUMYLꢀ5FꢀPICA on rat body
temperature following pretreatment (30 min prior) with vehicle solution (VEH), 3 mg/kg
rimonabant (CB1 antagonist), or 3 mg/kg SR144528 (CB2 antagonist). The first dashed line
denotes time of pretreatment injection (vehicle or antagonist). The second dashed line denotes
time of vehicle or SC injection. Each point represents the mean ± SEM for four animals.
16
ACS Paragon Plus Environment

Page 17 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
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
56
57
58
59
60
Conclusion: As the structural heterogeneity of SCs continues to increase, the rapid chemical and
pharmacological characterization of emergent analogues remains crucial to effective responses
by law enforcement agencies and policy makers. Here we have provided the systematic
evaluation of a series of Nꢀcumylꢀ1ꢀalkylꢀ1Hꢀind(az)oleꢀ3ꢀcarboxamide SCs of forensic interest,
17
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 18 of 30
1
2
3
4
5
6
7
8
9
including CUMYLꢀBICA, CUMYLꢀPICA, CUMYLꢀ5FꢀPICA, CUMYLꢀPINACA, CUMYLꢀ
5FꢀPINACA, and related analogues. The described synthetic method is general, and should
provide forensic chemists with access to newer cumylꢀderived analogues featuring alternative
substituents in the 1ꢀposition. All synthesized SCs were potent agonists of CB1 and CB2
receptors in a FLIPR membrane potential assay, thereby classifying them as functional
cannabinoids. As with earlier series of related SCs, the indazole analogues were generally more
potent CB1 agonists than the corresponding indole congeners. CUMYLꢀPICA and CUMYLꢀ5Fꢀ
PICA produced significant hypothermic and bradycardic effects in rats at doses of 1 mg/kg, and
the former were reversed by pretreatment with CB1 antagonist rimonabant (but not CB2
antagonist SR144528), suggesting CB1ꢀmediated effects. The in vitro and in vivo data indicate
that Nꢀcumylꢀ1ꢀalkylꢀ1Hꢀind(az)oleꢀ3ꢀcarboxamide SCs are potent cannabinoids with likely
central activity at CB1.
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
56
57
58
59
60
Materials and methods
General chemical synthesis details
The synthesis of 8–17 is shown in Figures 3 and 4. All reactions were performed under an
atmosphere of nitrogen or argon unless otherwise specified. Anhydrous tetrahydrofuran (THF),
methanol, acetonitrile, and dimethyl sulfoxide (DMSO) (SigmaAldrich, St. Louis, MO, USA)
were used as purchased. Commercially available chemicals (SigmaꢀAldrich) were used as
purchased. Analytical thinꢀlayer chromatography was performed using Merck aluminumꢀbacked
silica gel 60 F254 (0.2 mm) plates (Merck, Darmstadt, Germany), which were visualized using
18
ACS Paragon Plus Environment

Page 19 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
shortwave (254 nm) UV fluorescence. Flash chromatography was performed using Merck
Kieselgel 60 (230–400 mesh) silica gel. Melting point ranges (m.p.) were measured in open
capillaries using a Stuart SMP10 melting point apparatus (Bibby Scientific, Staffordshire, UK)
and are uncorrected. Nuclear magnetic resonance spectra were recorded at 300 K using either a
Bruker AVANCE DRX300 (300 MHz) spectrometer (Bruker, Bremen, Germany). The data for
1H NMR spectra are reported as chemical shift (δ ppm) relative to the residual protonated solvent
resonance, relative integral, multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet,
quat. = quartet, quin. = quintet, m = multiplet), coupling constants (J Hz) and assignment. The
data for 13C NMR spectra are reported as chemical shift (δ ppm) relative to residual solvent.
Assignment of signals was assisted by correlation spectroscopy (COSY), distortionless
enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC),
and heteronuclear multipleꢀbond correlation (HMBC) experiments where necessary. Lowꢀ
resolution mass spectra (LRMS) was recorded using electrospray ionization (ESI) recorded on a
Finnigan LCQ ion trap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA).
Highꢀresolution mass spectra (HRMS) were run on a Bruker 7T Apex Qe Fourier Transform Ion
Cyclotron resonance mass spectrometer equipped with an Apollo II ESI/APCI/MALDI Dual
source by the Mass Spectrometry Facility of the School of Chemistry at the University of
Sydney. IR absorption spectra were recorded on a Bruker ALPHA FTꢀIR spectrometer as solid
or thin film from ethanol, and the data are reported as vibrational frequencies (cm–1). Please see
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
56
57
58
59
60
1
13
the supplementary material for detailed chemical synthesis and H and C NMR spectra of all
final compounds.
19
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 20 of 30
1
2
3
4
5
6
7
8
9
General procedure A: Amidation of 1-substituted-1H-indole-3-carboxylic acids and 1-
substituted-1H-indazole-3-carboxylic acids (8–17). To a solution of the appropriate 1ꢀ
substitutedꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid or 1ꢀsubstitutedꢀ1Hꢀindazoleꢀ3ꢀcarboxylic acid (0.39
mmol), cumylamine (60 µL, 0.41 mmol, 1.05 equiv.), EDC·HCl (150 mg, 0.78 mmol, 2.0
equiv.) and HOBt (119 mg, 0.78 mmol, 2.0 equiv.) in DMSO (5 mL) was added DIPEA (340
µL, 1.95 mmol, 5.0 equiv.) dropwise and the mixture was stirred for 14 h. The reaction was
quenched by the addition of sat. aq. NaHCO3 (75 mL) and extracted with EtOAc (3 × 75 mL).
The combined organic layers were washed with brine (100 mL), dried (MgSO4), and evaporated
under reduced pressure. The pure amides were obtained following purification by flash
chromatography.
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
56
57
58
59
60
General procedure B: Synthesis of 1-(1-substituted-1H-indol-3-yl)-2,2,2-trifluoroethanones
(19–23). To a cooled (0 °C) suspension of sodium hydride (60% dispersion in mineral oil, 137
mg, 3.42 mmol, 2.0 equiv.) in DMF (6 mL) was added indole (200 mg, 1.71 mmol) portionwise
and the mixture stirred for 10 min. The mixture was treated dropwise with the appropriate
bromoalkane (1.80 mmol, 1.05 equiv.) and stirred at ambient temperature for 1 h. The cooled (0
°C) mixture was treated dropwise with trifluoroacetic anhydride (600 µL, 4.28 mmol, 2.5 equiv.)
and stirred at ambient temperature for 1 h. The reaction was poured into ice water (75 mL) and
extracted with CH2Cl2 (3 x 75 mL). The combined organic extracts were washed with H2O (100
mL), brine (100 mL), dried (MgSO4) and the solvent evaporated under reduced pressure. The
pure 1ꢀalkylꢀ3ꢀtrifluoroacetylindoles were obtained following purification by flash
chromatography.
20
ACS Paragon Plus Environment

Page 21 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
General procedure C: Synthesis of 1-substituted-1H-indole-3-carboxylic acids and 1-alkyl-
1H-indazole-3-carboxylic acids (24–28 and 35–39). To a solution of the appropriate 1ꢀ
substitutedꢀ3ꢀ(trifluoroacetyl)indole or methyl 1ꢀsubstittuedꢀ1Hꢀindazoleꢀ3ꢀcarboxylate (2.58
mmol) in MeOH (20 mL) was added 1 M aq. NaOH (3.87 mL, 3.87 mmol, 1.5 equiv.) and the
solution heated at reflux for 18 h. The mixture was cooled to ambient temperature, solvent was
evaporated in vacuo, and the mixture was poured onto sat. aq. NaHCO3 (75 mL). The aqueous
phase was washed with Et2O (75 mL) and the pH adjusted to 2 with 1 M aq. HCl. The aquoeus
phase was extracted with Et2O (3 × 75 mL) and the combined organic layers were washed with
brine (150 mL), dried (MgSO4) and concentrated in vacuo to give the crude products. Analytical
purity for 1ꢀsubstitutedꢀ1Hꢀindazoleꢀ3ꢀcarboxylic acids was achieved by recrystallization from iꢀ
PrOH.
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
56
57
58
59
60
General procedure D: Synthesis of methyl 1-substituted-1H-indazole-3-carboxylates (30–
34). To a cooled (0 °C) solution of methyl 1Hꢀindazoleꢀ3ꢀcarboxylate (29, 500 mg, 2.84 mmol)
in THF (15 mL) was added potassium tertꢀbutoxide (350 mg, 3.12 mmol, 1.1 equiv.), and the
mixture warmed to ambient temperature and stirred for 1 h. The cooled (0 °C) mixture was
treated dropwise with the appropriate bromoalkane (2.98 mmol, 1.05 equiv.) and stirred for 48 h.
The reaction was quenched by pouring onto H2O (100 mL) and the layers separated. The
aqueous phase was extracted with EtOAc (3 × 100 mL), and the combined organic layers were
washed with brine (150 mL), dried (MgSO4), and the solvent evaporated under reduced pressure.
The crude materials were purified by flash chromatography.
21
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 22 of 30
1
2
3
4
5
6
7
8
9
In vitro pharmacological evaluation of compounds 8–17. Mouse AtTꢀ20 neuroblastoma cells
stably transfected with human CB1 or human CB2 have been previously described26 and were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum
(FBS), 100 U penicillin/streptomycin, and 300 ꢂg/ml G418. Cells were passaged at 80%
confluency as required. Cells for assays were grown in 75 cm2 flasks and used at 90%
confluence. The day before the assay cells were detached from the flask with trypsin/EDTA
(SigmaꢀAldrich) and resuspended in 10 ml of Leibovitz’s Lꢀ15 media supplemented with 1%
FBS, 100 U penicillin/streptomycin and 15 mM glucose (membrane potential assay and Ca5
calcium assay). The cells were plated in volume of 90 ꢂl in black walled, clear bottomed 96ꢀwell
microplates (Corning, Oneonta, NY, USA) which had been precoated with polyꢀLꢀlysine
(SigmaꢀAldrich). Cells were incubated overnight at 37 °C in ambient CO2.
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
56
57
58
59
60
Membrane potential was measured using a FLIPR membrane potential assay kit (blue) from
Molecular Devices (Sunnyvale, CA, USA), as described previously 28. The dye was reconstituted
with assay buffer of composition (mM): NaCl 145, HEPES 22, Na2HPO4 0.338, NaHCO3 4.17,
KH2PO4 0.441, MgSO4 0.407, MgCl2 0.493, CaCl2 1.26, glucose 5.56, bovine serum albumin
(pH 7.4, osmolarity 315 ± 5). Prior to the assay, cells were loaded with 90 ꢂl/well of the dye
solution without removal of the Lꢀ15, giving an initial assay volume of 180 ꢂl/well. Plates were
then incubated at 37 °C at ambient CO2 for 60 min. Fluorescence was measured using a
FlexStation 3 (Molecular Devices) microplate reader with cells excited at a wavelength of 530
nm and emission measured at 565 nm. Baseline readings were taken every 2 s for at least 60 s, at
which time either drug or vehicle was added in a volume of 20 ꢂl. The background fluorescence
of cells without dye or dye without cells was negligible. Changes in fluorescence were expressed
22
ACS Paragon Plus Environment

Page 23 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
as a percentage of baseline fluorescence after subtraction of the changes produced by vehicle
addition. The final concentration of dimethyl sulfoxide was always 0.1%.
7
8
9
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
56
57
58
59
60
Data were analyzed with PRISM (GraphPad Software Inc., San Diego, CA), using fourꢀ
parameter nonlinear regression to fit concentrationꢀresponse curves. In all plates, a maximally
effective concentration of CP 55,940 (Cayman Chemical, Ann Arbor, MI, USA) was added to
allow for normalization between assays.
In vivo pharmacological evaluation of CUMYL-PICA and CUMYL-5F-PICA. Two cohorts
of 4 adult male Long Evans rats (Animal Resources Centre, Perth, Australia) initially weighing
between 174 and 192 g were used for biotelemetric assessment of body temperature and heart
rate changes following each compound. The rats were singly housed in an airꢀconditioned testing
room (22 ± 1 °C) on a 12 h reverse light/dark cycle (lights on from 21:00 to 09:00). Standard
rodent chow and water were provided ad libitum. All experiments were approved by The
University of Sydney Animal Ethics Committee.
Biotelemetry transmitters (TA11CTAꢀF40, Data Sciences International, St. Paul, MN) were
implanted as previously described.26 Briefly, following anaesthetization (isoflurane, 3%
induction, 2% maintenance) a biotelemetry transmitter (TA11CTAꢀF40, Data Sciences
International, St. Paul, MN) was placed in the peritoneal cavity according to the manufacturers
protocol. The wound was sutured closed and the rats were allowed one week of recovery before
data collection.
23
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 24 of 30
1
2
3
4
5
6
7
8
9
The rats were habituated over multiple days to injections of vehicle solution (5% EtOH, 5%
Tween 80, 90% physiological saline) at a set time of day (10:00 am). Each cohort then received
injections of each compound at the same time of day in an ascending dose sequence (0.1, 0.3, 1,
mg/kg). This ascending sequence reduces the risk posed to the animals in assessing hitherto
untested compounds. Two washout days were given between each dose.
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
56
57
58
59
60
For the antagonist studies (Fig. 7), a third and fourth cohort of drugꢀnaïve rats were used for each
compound (weight range 169–199 g). Each cohort received injections of either vehicle, CB1
antagonist (rimonabant, 3 mg/kg), or CB2 antagonist (SR144528, 3 mg/kg), followed by
CUMYLꢀPICA (1 mg/kg) or CUMYLꢀ5FꢀPICA (1 mg/kg). The order of treatments was
counterbalanced within each cohort. The vehicle or antagonist injections were given to rats 30
minutes prior to the CUMYLꢀPICA or 5FꢀCUMYLꢀPICA injections.
Data for heart rate and body temperature was gathered continuously at 1000 Hz and organized
into 15 minute bins using Dataquest A.R.T. software (version 4.3, Data Sciences International,
St. Paul, MN), and analysed using Prism (version 7.00, GraphPad Software Inc., San Diego,
CA). Hypothermic and bradycardic effects were analysed using a twoꢀway mixedꢀmodel
ANOVA. Main effects of each dose of CUMYLꢀPICA or CUMYLꢀ5FꢀPICA were compared to
vehicle using planned Dunnet’s contrasts.
For the antagonist studies, we calculated the area between baseline and drugꢀtreatment body
temperature curves for each rat as a measure of antagonist efficacy. Briefly, for any time point,
24
ACS Paragon Plus Environment

Page 25 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
the area between baseline data points (Bt) and drugꢀtreatment data points (Dt) and the subsequent
time points (Bt+1 and Dt+1) forms a trapezoid, the area of which can be calculated via the formula:
7
8
9
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
56
57
58
59
60
ꢄ
ꢈ
ꢅꢆ − ꢇꢆ + ꢄꢅꢆꢉꢊ − ꢇꢆꢉꢊ
ꢈ
ꢀꢁꢂꢃ =
2
The area between the vehicleꢀvehicle baseline and the vehicleꢀSC (i.e. vehicleꢀCUMYLꢀPICA or
vehicleꢀCUMYLꢀ5FꢀPICA), rimonabantꢀSC and SR144528ꢀSC treatments was calculated over a
6 h time period postꢀinjection of SC. These areas were analyzed using a oneꢀway repeated
measure ANOVA with planned Dunnet’s contrasts comparing the antagonist areas to the vehicleꢀ
SC area.
Acknowledgments
Work was supported by NHMRC Project Grant 1107088 awarded to M.K., I.S.M., and M.C.
Supporting Information
Table S1: Names, CAS numbers, and references for selected compounds (pp. 2–3).
Figures S1–S20:
Table S2: Hyperpolarization of AtTꢀ20 cells expressing CB
with and without pertussis toxin (PTX) pretreatment (pp. 24).
1
H and 13C NMR spectra for selected compounds (pp. 4–23).
1
or CB by CP 55,940 or 8ꢀ17
2
Figure S21: Mean area under the vehicleꢀvehicle baseline curve (AUC ± SEM) for body
temperature for CUMYLꢀPICA and CUMYLꢀ5FꢀPICA, following pretreatment with
antagonist), or SR144528 (CB
Table S3: Comparison of the potency of selected CUMYLꢀ drugs at human CB1 and CB2
receptors measured using activation of GIRK or stimulation of GTP
al., 2017) (pp.26).
vehicle, rimonabant (CB
1
2
antagonist) (pp. 25).
g
S binding (Asada et
25
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 26 of 30
1
2
3
4
References
5
6
7
8
1.
2.
UNODC, World Drug Report 2017.
Qian, Z.; Hua, Z.; Liu, C.; Jia, W., Four types of cannabimimetic indazole and indole
derivatives, ADBꢀBINACA, ABꢀFUBICA, ADBꢀFUBICA, and ABꢀBICA, identified as new
9
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
56
57
58
59
60
psychoactive substances. Forensic toxicology 2016, 34, 133ꢀ143.
3.
Shevyrin, V.; Melkozerov, V.; Eltsov, O.; Shafran, Y.; Morzherin, Y., Synthetic
cannabinoid 3ꢀbenzylꢀ5ꢀ[1ꢀ(2ꢀpyrrolidinꢀ1ꢀylethyl)ꢀ1Hꢀindolꢀ3ꢀyl]ꢀ1,2,4ꢀoxadiazole. The first
detection in illicit market of new psychoactive substances. Forensic Science International 2016,
259, 95ꢀ100.
4.
Adams, A. J.; Banister, S. D.; Irizarry, L.; Trecki, J.; Schwartz, M.; Gerona, R., "Zombie"
Outbreak Caused by the Synthetic Cannabinoid AMBꢀFUBINACA in New York. N Engl J Med
2017, 376 (3), 235ꢀ242.
5.
6.
Bowden, M. J.; Williamson, J. P. B. Cannabinoid Compounds. WO2014167530A1, 2014.
EMCDDA EMCDDA–Europol 2014 Annual Report on the implementation of Council
Decision 2005/387/JHA; 2015.
7. Dobaja, M.; Grenc, D.; Kozelj, G.; Brvar, M., Occupational transdermal poisoning with
synthetic cannabinoid cumylꢀPINACA. Clin Toxicol (Phila) 2017, 55 (3), 193ꢀ195.
8. Abouchedid, R.; Hudson, S.; Thurtle, N.; Yamamoto, T.; Ho, J. H.; Bailey, G.; Wood,
M.; Sadones, N.; Stove, C. P.; Dines, A.; Archer, J. R. H.; Wood, D. M.; Dargan, P. I.,
Analytical confirmation of synthetic cannabinoids in a cohort of 179 presentations with acute
recreational drug toxicity to an Emergency Department in London, UK in the first half of 2015.
Clin Toxicol (Phila) 2017, 55 (5), 338ꢀ345.
9.
Longworth, M.; Banister, S. D.; Mack, J. B. C.; Glass, M.; Connor, M.; Kassiou, M., The
2ꢀalkylꢀ2Hꢀindazole regioisomers of synthetic cannabinoids ABꢀCHMINACA, ABꢀFUBINACA,
ABꢀPINACA, and 5FꢀABꢀPINACA are possible manufacturing impurities with cannabimimetic
activities. Forensic toxicology 2016, 34 (2), 286ꢀ303.
10.
Asada, A.; Doi, T.; Tagami, T.; Takeda, A.; Satsuki, Y.; Kawaguchi, M.; Nakamura, A.;
Sawabe, Y., Cannabimimetic activities of cumyl carboxamideꢀtype synthetic cannabinoids.
Forensic toxicology 2017. doi 10.1007/s11419ꢀ017ꢀ0374ꢀ9.
26
ACS Paragon Plus Environment

Page 27 of 30
ACS Chemical Neuroscience
1
2
3
4
11.
Wiley, J. L.; Marusich, J. A.; Martin, B. R.; Huffman, J. W., 1ꢀPentylꢀ3ꢀ
5
6
7
8
phenylacetylindoles and JWHꢀ018 share in vivo cannabinoid profiles in mice. Drug Alcohol
Depen. 2012, 123 (1ꢀ3), 148ꢀ153.
12.
Wiley, J. L.; Marusich, J. A.; Lefever, T. W.; Grabenauer, M.; Moore, K. N.; Thomas, B.
9
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
56
57
58
59
60
F., Cannabinoids in disguise: ∆9ꢀtetrahydrocannabinolꢀlike effects of tetramethylcyclopropyl
ketone indoles. Neuropharmacology 2013, 75, 145ꢀ154.
13.
effects of compounds commonly found in K2/Spice. Behav. Pharmacol. 2014, 25 (8), 750ꢀ757.
14. Wiley, J. L.; Lefever, T. W.; Cortes, R. A.; Marusich, J. A., Crossꢀsubstitution of ∆9ꢀ
Gatch, M. B.; Forster, M. J., ∆9ꢀTetrahydrocannabinolꢀlike discriminative stimulus
tetrahydrocannabinol and JWHꢀ018 in drug discrimination in rats. Pharmacol. Biochem. Behav.
2014, 124, 123ꢀ128.
15.
cannabinoids found on the gray market. Behav. Pharmacol. 2015, 26 (5), 460ꢀ468.
16. Wiley, J. L.; Marusich, J. A.; Lefever, T. W.; Antonazzo, K. R.; Wallgren, M. T.; Cortes,
Gatch, M. B.; Forster, M. J., ∆9ꢀTetrahydrocannabinolꢀlike effects of novel synthetic
R. A.; Patel, P. R.; Grabenauer, M.; Moore, K. N.; Thomas, B. F., ABꢀCHMINACA, ABꢀ
PINACA, and FUBIMINA: Affinity and Potency of Novel Synthetic Cannabinoids in Producing
∆9ꢀTetrahydrocannabinolꢀLike Effects in Mice. J. Pharmacol. Exp. Ther. 2015, 354 (3), 328ꢀ
339.
17.
cannabinoids in mice and rats. Psychopharmacology (Berl.) 2016, 233 (10), 1901ꢀ1910.
18. Wiley, J. L.; Lefever, T. W.; Marusich, J. A.; Grabenauer, M.; Moore, K. N.; Huffman, J.
Gatch, M. B.; Forster, M. J., ∆(9)ꢀTetrahydrocannabinolꢀlike effects of novel synthetic
W.; Thomas, B. F., Evaluation of first generation synthetic cannabinoids on binding at nonꢀ
cannabinoid receptors and in a battery of in vivo assays in mice. Neuropharmacology 2016, 110
(Pt A), 143ꢀ153.
19.
Thomas, B. F.; Lefever, T. W.; Cortes, R. A.; Grabenauer, M.; Kovach, A. L.; Cox, A.
O.; Patel, P. R.; Pollard, G. T.; Marusich, J. A.; Kevin, R. C.; Gamage, T. F.; Wiley, J. L.,
Thermolytic Degradation of Synthetic Cannabinoids: Chemical Exposures and Pharmacological
Consequences. J. Pharmacol. Exp. Ther. 2017, 361 (1), 162ꢀ171.
20.
Wiley, J. L.; Lefever, T. W.; Marusich, J. A.; Craft, R. M., Comparison of the
discriminative stimulus and response rate effects of ∆9ꢀtetrahydrocannabinol and synthetic
cannabinoids in female and male rats. Drug Alcohol Depend 2017, 172, 51ꢀ59.
27
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 28 of 30
1
2
3
4
21.
Wiley, J. L.; Marusich, J. A.; Huffman, J. W., Moving around the molecule: Relationship
5
6
between chemical structure and in vivo activity of synthetic cannabinoids. Life Sci. 2014, 97 (1),
7
8
9
55ꢀ63.
22.
Banister, S. D.; Wilkinson, S. M.; Longworth, M.; Stuart, J.; Apetz, N.; English, K.;
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
56
57
58
59
60
Brooker, L.; Goebel, C.; Hibbs, D. E.; Glass, M.; Connor, M.; McGregor, I. S.; Kassiou, M., The
synthesis and pharmacological evaluation of adamantaneꢀderived indoles: cannabimimetic drugs
of abuse. ACS Chem. Neurosci. 2013, 4 (7), 1081ꢀ1092.
23.
Banister, S. D.; Stuart, J.; Kevin, R. C.; Edington, A.; Longworth, M.; Wilkinson, S. M.;
Beinat, C.; Buchanan, A. S.; Hibbs, D. E.; Glass, M.; Connor, M.; McGregor, I. S.; Kassiou, M.,
Effects of Bioisosteric Fluorine in Synthetic Cannabinoid Designer Drugs JWHꢀ018, AMꢀ2201,
URꢀ144, XLRꢀ11, PBꢀ22, 5FꢀPBꢀ22, APICA, and STSꢀ135. ACS Chem. Neurosci. 2015, 6 (8),
1445ꢀ1458.
24.
Banister, S. D.; Stuart, J.; Conroy, T.; Longworth, M.; Manohar, M.; Beinat, C.;
Wilkinson, S. M.; Kevin, R. C.; Hibbs, D. E.; Glass, M.; Connor, M.; McGregor, I. S.; Kassiou,
M., Structure–activity relationships of synthetic cannabinoid designer drug RCSꢀ4 and its
regioisomers and C4 homologues. Forensic toxicology 2015, 33 (2), 355ꢀ366.
25.
Banister, S. D.; Moir, M.; Stuart, J.; Kevin, R. C.; Wood, K. E.; Longworth, M.;
Wilkinson, S. M.; Beinat, C.; Buchanan, A. S.; Glass, M.; Connor, M.; McGregor, I. S.; Kassiou,
M., Pharmacology of Indole and Indazole Synthetic Cannabinoid Designer Drugs ABꢀ
FUBINACA, ADBꢀFUBINACA, ABꢀPINACA, ADBꢀPINACA, 5FꢀABꢀPINACA, 5FꢀADBꢀ
PINACA, ADBICA, and 5FꢀADBICA. ACS Chem Neurosci 2015, 6 (9), 1546ꢀ1559.
26.
Banister, S. D.; Longworth, M.; Kevin, R.; Sachdev, S.; Santiago, M.; Stuart, J.; Mack, J.
C.; Glass, M.; McGregor, I. S.; Connor, M.; Kassiou, M., Pharmacology of Valinate and tertꢀ
Leucinate Synthetic Cannabinoids 5FꢀAMBICA, 5FꢀAMB, 5FꢀADB, AMBꢀFUBINACA,
MDMBꢀFUBINACA, MDMBꢀCHMICA, and Their Analogues. ACS Chem. Neurosci. 2016, 7
(9), 1241ꢀ1254.
27.
Kevin, R. C.; Lefever, T. W.; Snyder, R. W.; Patel, P. R.; Fennell, T. R.; Wiley, J. L.;
McGregor, I. S.; Thomas, B. F., In vitro and in vivo pharmacokinetics and metabolism of
synthetic cannabinoids CUMYLꢀPICA and 5FꢀCUMYLꢀPICA. Forensic toxicology 2017, 1ꢀ15.
28
ACS Paragon Plus Environment

Page 29 of 30
ACS Chemical Neuroscience
1
2
3
4
28.
Knapman, A.; Santiago, M.; Du, Y. P.; Bennallack, P. R.; Christie, M. J.; Connor, M., A
5
6
7
8
continuous, fluorescenceꢀbased assay of µꢀopioid receptor activation in AtTꢀ20 cells. J Biomol
Screen 2013, 18 (3), 269ꢀ276.
9
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
56
57
58
59
60
29
ACS Paragon Plus Environment