N-Methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6- carboxamides as a novel class of cannabinoid receptors agonists with low CNS penetration

Article abstract of DOI:10.1016/j.bmcl.2012.04.128

Cannabinoid CB₁ receptor agonists exhibit potent analgesic effects in rodents and humans, but their clinical utility as analgesic drugs is often limited by centrally mediated side effects. We report herein the preparation of N-methyl-3-(tetrahy

Full text of DOI:10.1016/j.bmcl.2012.04.128

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3884–3889
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-
6-carboxamides as a novel class of cannabinoid receptors agonists
with low CNS penetration
Zhongyong Wei a, Hua Yang a, Ziping Liu a, Maxime Tremblay a, Shawn Johnstone a, Sara Béha a,
Shi Yi Yue a, Sanjay Srivastava a, Miroslaw J. Tomaszewski a, William Brown a, Christopher Walpole a,
Stéphane St-Onge b, Etienne Lessard c, Anne-Julie Archambault d, Thierry Groblewski b, Daniel Pagé a,
⇑
a Department of Medicinal Chemistry, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Saint-Laurent, Quebec, Canada H4S 1Z9
b Department of Molecular Pharmacology, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Saint-Laurent, Quebec, Canada H4S 1Z9
c Department of DMPK, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Saint-Laurent, Quebec, Canada H4S 1Z9
d Department of Bioscience, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Saint-Laurent, Quebec, Canada H4S 1Z9
a r t i c l e i n f o
a b s t r a c t
Article history:
Cannabinoid CB1 receptor agonists exhibit potent analgesic effects in rodents and humans, but their clin-
ical utility as analgesic drugs is often limited by centrally mediated side effects. We report herein the prep-
aration of N-methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxamides as a
novel class of hCB1/hCB2 dual agonists with attractive physicochemical properties. More specifically, (R)-
N,9-dimethyl-N-(4-(methylamino)-4-oxobutyl)-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-
carbazole-6-carboxamide, displayed an extremely low level of CNS penetration (Rat Cbr/Cplasma = 0.005
or 0.5%) and was devoid of CNS side effects during pharmaco-dynamic testing.
Received 30 March 2012
Revised 24 April 2012
Accepted 27 April 2012
Available online 4 May 2012
Keywords:
Cannabinoid
Tetrahydrocarbazoles
Pain
Ó 2012 Elsevier Ltd. All rights reserved.
Cannabinoid receptors belong to the super family of G-protein
coupled receptors (GPCRs) and two of them have been cloned
and characterized so far; CB1 and CB2.1,2 CB1 is widely expressed
in neurons in the brain3,4 and is responsible for most of the psycho-
active properties of cannabinoids. Even though it was recently
demonstrated that CB2 is expressed in the central nervous system
(CNS) under certain conditions,5 it is primarily abundant in spleno-
cytes and leukocytes2 and responsible for many of the immuno-
modulatory and anti-inflammatory effects of cannabis.6 CB1 is also
found in a variety of tissues including immune cells. A wide variety
of potential therapeutic applications associated with cannabinoid
ligands are currently under investigations, including eating disor-
ders,7 obesity,8 drug addictions9 and central immune functions.5
Cannabinoids and cannabinoid receptors have been implicated
in pain transduction and perception10 as well as neuroinflamma-
tion.11 Many published studies have demonstrated that cannabi-
noids, both CB1 and CB2 agonists, are potent analgesics not only
in a variety of preclinical pain models but also in the clinic.12–15
Unfortunately, the exploitation of the therapeutic potential of
CB1 agonists in man has been generally limited by their CNS side
effects, mostly associated with the CB1 receptors.16 Several studies
have established that CB2-selective agonists (relative to CB1) exhi-
bit efficacy in many rodent pain models and lack the CB1-mediated
HO
OH
O
N
C6H13
O
O
N
O
2, WIN55,212-2
1, HU-210
HO
O
O
OH
C4H9
O
O
C5H11
3, Ajulemic acid
4
⇑
Corresponding author. Tel.: +1 514 832 3200; fax: +1 514 832 3232.
E-mail address: daniel.page@astrazeneca.com (D. Pagé).
Figure 1. Several CB1 or CB1/CB2 dual agonists.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.128

Z. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3884–3889
3885
O
O
O
O
O
O
O
O
N
R"
N
N
R'
O
HO
b
a
core change
N
N
Me
N
N
SO2Et
14
13
O
6
5, AZ599
Figure 2. Scaffold-hopping around the c-carboline core.
O
R"
(S)-18,19,21
CNS effects at analgesic doses.17,18 Specifically, CB2-selective ago-
nists have shown efficacy in preclinical models of neuropathic
and inflammatory pain.19,20
N
R'
c
+
(R)-18,19,21
N
15-21
Another approach to effective and safe cannabinoid analgesics
would be to aim for the development of low brain penetrant CB1
agonists21–26 as analgesic drugs. This strategy, adopted by other
groups,25,26 demonstrated the reduction of CNS side effects by
peripherally restricted CB1 agonists while keeping their analgesic
effects. In clinical trials, topical administration of HU-210 (1,
Fig. 1) (a dual CB1/CB2 agonist) reportedly reduced thermal and
mechanical hyperalgesia and allodynia induced by capsaicin. No
psychoactive effects were observed.27 Ajulemic acid (3, Fig. 1), a
dimethylheptyl analogue of the 11-carboxylic acid metabolite of
THC with modest CB1/CB2 binding affinity, reportedly has a reduced
CNS penetration and showed promising efficacy and a better side
Scheme 2. Reagents and conditions: (a) NaOH, MeOH/H2O (88%); (b) R0R00NH,
HATU, DIPEA, DMF (60–90%); (c) chiral separation with ChiraPakÒ AD column,
Hexane/EtOH (60/40).
O
O
O
O
O
HO
a
N
N
effect profile in both animals and humans when compared to
D9-
O
(R)-13
(R)-14
THC.28,23 Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl) metha-
none (4, Fig. 1) was reported to be a CB1/CB2 dual agonist29 with
limited brain penetration, displaying good oral bioavailability and
potent antihyperalgesic activity in animal models. However, this
compound possesses poor physicochemical properties, including
an extremely poor solubility.
O
R"
N
R'
b
22-31
N
Our interest in the discovery and development of novel thera-
peutics for pain management prompted a high-throughput screen
Scheme 3. Reagents and conditions: (a) LiOH, dioxane/H2O (99%); (b) R0R00NH,
HATU, DIPEA, DMF (60–90%).
of our GPCR focused libraries. The c-carboline scaffold was identi-
fied in the efforts as a chemical series with attractive physicochem-
ical properties.30 Compound (5) (AZ599) exhibited binding affinities
on both human cannabinoid receptors (hCB1 Ki: 120 nM; hCB2 Ki:
9 nM) and was a CB1 full agonist (EC50: 49 nM, Emax: 120%) with good
present study, we report N-methyl-3-(tetrahydro-2H-pyran-4-yl)-
2,3,4,9-tetrahydro-1H-carbazole-6-carboxamides as a novel class
of hCB1/hCB2 dual agonists with very low CNS penetration.
solubility (>1000 lM) and low CNS penetration (Cbr/pl: 0.07). By
The synthesis of common intermediate (13) is outlined in
Scheme 1.31 Commercially available or readily prepared 1-(benzyl-
oxy)-4-bromobenzene (7) was treated with BuLi at ꢀ78 °C and
performing scaffold hopping from this series, we observed
compounds having a tetrahydrocarbazole core (6) exhibiting CB1
potency and desirable physico-chemical properties (Fig. 2). In the
O
O
O
O
Br
OH
e
d
c
a
b
OBn
O
OH
10
OBn
OH
7
11
9
8
O
O
O
O
(R)-13
(S)-13
+
O
f
O
g
N
H
N
13
12
Scheme 1. Reagents and conditions: (a) (i) BuLi, THF, ꢀ78 °C; (ii) tetrahydro-4H-pyran-4-one (67%). (b) H2, Pd/C, 3M HCl, THF/MeOH, (99%). (c) Rh/Al2O3 5%, MeOH, (100%).
(d) Bleach, TEMPO, CH2Cl2, (88%). (e) (i) 4-Hydrazinobenzoic acid hydrochloride, HCl, dioxane; (ii) MeOH, HCl (89%). (f) NaH/MeI THF (90%); (g) ChiralCelÒOD, Hexane/EtOH
(60/40).

3886
Z. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3884–3889
Table 1
Properties of selected N-methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxamides
O
O
R'R"N
N
Me
hCB1 Kia (nM)
hCB2 Kia (nM)
hCB1 EC50 (nM)
hClintc
(lL/min/mg)
hERG IC50
(
lM)
Solubilityd
(lM, pH 7.4)
a,b
Compound
R0R00N
2
4
46
35
120
2
19
9
112 (100%)
73 (112%)
49 (120%)
nd
nd
52
nd
nd
3.1
2
0
AZ599
>1030
H
N
N
N
15
16
17
18
19
20
21
7.1
nd
nd
nd
26.3
nd
nd
nd
16.2 (113%)
2.9 (114%)
nd
73
143
47
37
53
nd
27
>33
>33
>31
>33
>33
>33
>33
444
224
1
O
O
O
O
H
N
2.8
H
N
N
H
1440
44.5
7.3
H
N
N
N
95.3 (121%)
6.2 (106%)
nd
108
36
H
N
O
O
655
53.7
523
1550
N
H
N
H
N
N
78.9 (121%)
O
a
b
c
Values are means of at least three measurements.
Emax are reported in brackets.
Metabolic stability performed in human liver microsomes in presence of NADPH using test compounds at 1
Thermodynamic solubility measured in a pH 7.4 buffer using HPLC with UV detection for quantification; nd: not determined.
l
M.
d
followed by addition of tetrahydro-4H-pyran-4-one to give 4-[4-
(benzyloxy)phenyl]tetrahydro-2H-pyran-4-ol (8), which was re-
duced to 9 under acidic hydrogenation conditions.
shown in Scheme 3. (R)-13 was hydrolyzed under basic conditions
to (R)-9-methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-
1H-carbazole-6-carboxylic acid (R)-14, which was subjected to
amide coupling conditions to afford the desired amides 22–31.
4-(Tetrahydro-2H-pyran-4-yl)phenol was further reduced
under rhodium-catalyzed hydrogenation to 4-(tetrahydro-2H-pyr-
an-4-yl)cyclohexanol (10), which was oxidized to 11. 4-(Tetrahy-
dro-2H-pyran-4-yl)cyclohexanone was subjected to standard
Fischer indole synthesis condition with 4-hydrazinobenzoic acid
hydrochloride and followed by esterification to provide methyl
3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-
carboxylate (12). N-Methylation of 12 was done under basic condi-
tions to afford the desired product 13. Methyl 9-methyl-3-(tetrahy-
dro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxylate
was separated into its enantiomers (R)-13 and (S)-13, whose
configurations were established by vibrational circular dichroism
(VCD)32 and X-ray crystallography.
Synthesis of N-methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-
tetrahydro-1H-carbazole-6-carboxamides (15–21) is outlined in
Scheme 2. Compound 13 was hydrolyzed under basic conditions
to 9-methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-
carbazole-6-carboxylic acid 14, which was subjected to amide
coupling conditions using 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,
3-tetramethyluronium hexafluorophosphate (HATU) to afford the
desired amides 15–21. Compounds 18,19 and 21 were further
separated into their enantiomers (R)- & (S)-18, (R)- & (S)-19, and
(R)- & (S)-21.
During the efforts on the
c-carbolines, it was found that the
incorporation of an amide moiety provided compounds with full
CB1 agonist potency completely devoid of affinity for the hERG
potassium channel.
When this SAR was applied to the tetrahydrocarbazole series,
similar effects were found on the CB1 potency33 and hERG activity.
As shown in Table 1, linear chain amide derivative 15 exhibits very
potent hCB1 binding affinity (hCB1 Ki = 7.1 nM) and excellent hCB1
agonist activity (hCB1 EC50 = 16.2 nM) while its hERG IC50 is over
33 lM. As compared to the N-methyl amide 15, the N-ethyl deriv-
ative 16 is even more potent with increased hCB1 EC50 by over
5-fold. In the case of glycine amides, N-ethyl derivative 19 displays
a 15-fold increase in hCB1 EC50 as compared to its N-methyl analog
18. On the other hand, compound 17 displays very poor hCB1 bind-
ing affinity, which indicates that the NH has detrimental effects on
CB1 potency.
Chirality plays an important role in CB1 potency. Selected com-
pounds are presented in Table 2. Racemic 21 was separated into its
faster eluting (R)-enantiomer and slower eluting (S)-enantiomer
by preparative chiral HPLC separation. The stereochemical assign-
ments were established by X-ray analyses. (S)-21 is much less ac-
tive (hCB1 Ki = 576 nM). The more potent (R)-enantiomer (R)-21
demonstrated good hCB1 full agonist activity with extremely low
hERG activity. Racemates 18 & 19 were also separated by chiral
Synthesis
of
(R)-N-methyl-3-(tetrahydro-2H-pyran-4-yl)-
(22–31) is
2,3,4,9-tetrahydro-1H-carbazole-6-carboxamides

Z. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3884–3889
3887
Table 2
Chirality effects on CB1 potency
O
O
*
R'R"N
N
Me
a,b
Compound
R0R00N
hCB1 Kia (nM)
hCB2 Kia (nM)
hCB1 EC50 (nM)
hClintc
(l
L/min/mg)
hERG IC50
(lM)
Solubilityd
(lM, pH 7.4)
H
N
N
N
(R)-18
(S)-18
24.5
177
21.5
49.1
39.9 (116%)
446 (111%)
30
38
>33
>33
86
63
O
O
H
N
(R)-19
(S)-19
4.2
nd
nd
1.9 (106%)
36
nd
nd
nd
30
49
17.3
24.2 (106%)
H
N
N
(R)-21
(S)-21
29.3
576
15.5
nd
51.4 (116%)
nd
26
31
105
nd
1123
1070
O
a
b
c
Values are means of at least three measurements.
Emax are reported in brackets.
Metabolic stability performed in human liver microsomes in presence of NADPH using test compounds at 1
Thermodynamic solubility measured in a pH 7.4 buffer using HPLC with UV detection for quantification; nd: not determined.
lM.
d
Table 3
Properties of (R)-N-methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxamides
O
O
R'R"N
N
Me
hClintc
(l
L/min/mg)
Cbr/pl
hERG IC50
(lM)
Caco-2d (10ꢀ6 cm/s)
Solubilitye
(lM, pH 7.4)
a,b
Compound
R0R00N
hCB1 EC50 (nM)
2
4
112 (100%)
73 (112%)
49 (120%)
nd
nd
52
2.3
0.29
0.07
nd
nd
3.1
nd
nd
13
2
0
AZ599
>1030
H
N
N
N
(R)-18
(R)-19
(R)-21
22
39.9 (116%)
1.9 (106%)
51.4 (116%)
9.8 (107%)
12.9 (108%)
30
36
26
33
50
0.024
0.09
>33
nd
27
23
12
nd
nd
86
O
O
H
N
30
H
N
N
0.02
>33
nd
1120
71
O
H
N
F
F
N
0.068
0.02
O
O
H
N
N
23
nd
470
H
N
N
24
25
30.7 (113%)
43.7 (116%)
38
29
<0.02
0.019
>33
>33
nd
nd
760
O
HO
H
N
N
1670
O
F
O
HO
H
N
26
61.4 (118%)
27
0.025
>33
0.11
1310
N
O
(continued on next page)

3888
Z. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3884–3889
Table 3 (continued)
a,b
Compound
R0R00N
hCB1 EC50 (nM)
hClintc
21
(l
L/min/mg)
Cbr/pl
0.011
hERG IC50
>33
(lM)
Caco-2d (10ꢀ6 cm/s)
0.21
Solubilitye
1930
(lM, pH 7.4)
HO
H
N
27
78.7 (126%)
N
O
HO
H
N
28
29
101 (124%)
173 (120%)
15
17
<0.02
nd
>33
>33
0.15
nd
>3000
2490
N
O
H
N
N
O
HO
H
N
N
30
31
70.6 (110%)
291 (104%)
42
36
nd
nd
>33
>33
nd
nd
300
CN
O
H
N
O
N
1330
O
a
b
c
Values are means of at least three measurements.
Emax are reported in brackets.
Metabolic stability performed in human liver microsomes in presence of NADPH using test compounds at 1
Transwell assay: apparent permeability (apical to basolateral) across a Caco-2 cell monolayer measured under pH gradient pH 6.5–7.4 using test compound at 10
l
M.
d
l
M
applied on the apical side.
e
Thermodynamic solubility measured in a pH 7.4 buffer using HPLC with UV detection for quantification; nd: not determined.
Table 4
protein respectively). When considering the plasma protein bind-
ing in rat and binding to microsomal incubations (as calculated
by method published by Austin et al.35), the predicted clearance
(CL) was low (3.2 mL /min/kg) and in line with the observed
in vivo CL (2.8 mL/min/kg). Applying the same scaling method,
the predicted clearance in human would be approximately 2 mL/
min/kg, which is consistent with our expectation of an acceptable
bioavailability in man. The cytochrome P450 inhibitory potential of
(R)-21 was determined in human liver microsomes by use of the
major isoforms CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4,
in order to assess the potential likelihood of drug interactions.
Compound (R)-21 exhibited only moderate inhibition against
Pharmacokinetic Parameters of (R)-21 in Sprague–Dawley Ratsa
Intravenous administration (2
lmol/kg)
b
t
2.4 h
0.3 L/kg
2.8 mL/min/kg
1/2
VDss
CL
Intravenous administration (9
lmol/kg)
Tmax
Cmax
F (%)
2.2 h
6.3
lM
ꢁ100%
a
Values represent the means only for n = 3.
Terminal half-life.
b
CYP2C9 (IC50 = 7
isoforms.
lM) and was inactive (>20 lM) against the other
HPLC. In both cases, the (R)-enantiomers display better hCB1 ago-
nist activities (hCB1 EC50) by over 10-fold as compared to the cor-
responding (S)-enantiomers.
Compound (R)-21 showed a dose dependant reversal of heat
hyperalgesia in vivo when tested subcutaneously in the rat Carra-
Once the stereospecificity of the compounds on hCB1 had been
determined, efforts were focused on enantiomerically pure (R)-iso-
mers. The selected compounds are presented in Table 3. Various
groups are tolerated on the linear amide moiety, including F,
MeO, CN, and hydroxyl. All the compounds tested exhibited full
hCB1 agonist properties with very low hERG binding affinity for
the compounds tested and low CNS distributions. However, be-
cause of its overall pharmacokinetics and solubility profiles, (R)-
21 was preferred over the other analogues to be tested in vivo.
The pharmacokinetic profile of (R)-21 after oral and intravenous
administration to Sprague–Dawley rats is shown in Table 4. Oral
bioavailability was excellent (ꢁ100%), indicating that pre-systemic
first-pass metabolism is limited and gut permeability is not limit-
ing. The small steady-state volume of distribution (0.3 L/kg) and
moderate terminal half-life (2.4 h) after iv administration were
likely due to the low systemic clearance (2.8 mL/min/kg). Com-
bined with high affinity for plasma protein (97.9% bound), the fact
that (R)-21 is a good P-glycoprotein (Pgp) substrate (efflux ratio of
31 in MDR1-MDCK cells) resulted in a low CNS distribution as
demonstrated by in vivo microdialysis studies.34 Following oral
geenan inflammatory pain model at doses of 10, 30 and 100
kg.36 These doses resulted in total plasma concentrations of 6.9, 9.1
and 50.7
M, while none could be detected in the brain.37 These
observations further validate that peripherally restricted cannabi-
noid agonists can contribute to analgesia.
In summary, we have identified a new N-methyl-3-(tetrahydro-
2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxamide
series as potent mixed CB1/CB2 agonists with good physicochemi-
cal properties and low CNS penetration. Investigations into the
further development of these ligands are currently underway.
lmol/
l
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20. Quartiho, A.; Mata, H. P.; Ibrahim, M. M.; Vanderah, T. W.; Porreca, F.;
Makriyannis, A.; Malan, T. P., Jr. Anesthesiology 2003, 99, 955.
21. Kelly, S.; Jhaveri, M. D.; Sagar, D. R.; Kendall, D. A.; Chapman, V. Eur. J. Neurosci.
2003, 18, 2239.
22. Fride, E.; Feigin, C.; Ponde, D. E.; Breuer, A.; Hanus, L.; Arshavsky, N.;
Mechoulam, R. Eur. J. Pharmacol. 2004, 506, 179.
23. Dyson, A.; Peacock, M.; Chen, B.; Courade, J.-P.; Yaqoob, M.; Groarke, A.; Brain,
C.; Loong, Y.; Fox, A. Pain 2005, 116, 129.
24. Richardson, J. D.; Kilo, S.; Hargreaves, K. M. Pain 1998, 75, 111.
25. Adam, J. M.; Clark, J. K.; Davies, K.; Everett, K.; Fields, R.; Francis, S.; Jeremiah,
F.; Kiyoi, T.; Maidment, M.; Morrison, A.; Ratcliff, P.; Prosser, A.; Schulz, J.;
Wishart, G.; Baker, J.; Boyce, S.; Campbell, R.; Cottney, J. E.; Deehan, M.; Martin,
I. Bioorg. Med. Chem. Lett. 2012, 22, 2932.
26. Odan, M.; Ishizuka, N.; Hiramatsu, Y.; Inagaki, M.; Hashizume, H.; Fujii, Y.;
Mitsumori, S.; Morioka, Y.; Soga, M.; Deguchi, M.; Yasui, K.; Arimura, A. Bioorg.
Med. Chem. Lett. 2012, 22, 2894.
2.38–2.47 (m, 1H) 2.64–2.73 (m, 1H) 2.77–2.85 (m, 1H) 2.90 (dd, J = 16.02,
2.73 Hz, 1H) 3.42 (t, J = 11.72 Hz, 2H) 3.64 (s, 3H) 3.93 (s, 3H) 4.04 (dd,
J = 10.94, 3.52 Hz, 2H) 7.24 (d, J = 8.59Hz, 1H) 7.86 (dd, J = 8.59, 1.56 Hz, 1H)
8.23 (d, J = 1.17 Hz, 1H); [M+H]+ = 328.22; k0 = 3.20 (Chiral HPLC ChiralPakÒ AD,
40% ethanol/60% hexane). Compound (R)-21: 1H NMR (400 MHz, METHANOL-
D4) d ppm 1.37–1.50 (m, 2H) 1.52–1.61 (m, 3H) 1.77 (t, J = 12.70 Hz, 2H) 1.86
(s, 1H) 1.96 (s, 2H) 2.13–2.20 (m, 1H) 2.26 (s, 1H) 2.34–2.44 (m, 1H) 2.49 (s, 1H)
2.64–2.73 (m, 2H) 2.79–2.90 (m, 2H) 3.04 (s, 3H) 3.36–3.47 (m, 3H) 3.55 (s, 1H)
3.63 (s, 3H) 3.98 (dd, J = 10.94, 3.52 Hz, 2H) 7.13 (s, 1H) 7.31 (d, J = 8.20 Hz, 1H)
7.46 (s, 1H); [M+H]+ = 426.2; k0 = 3.20. (Chiral HPLC ChiralPakÒ AD, 40%
ethanol/60% hexane). Compound (S)-21: 1H NMR (400 MHz, METHANOL-D4) d
ppm 1.38–1.50 (m, 2H) 1.53–1.60 (m, 3H) 1.78 (t, J = 12.30 Hz, 2H) 1.85 (s, 1H)
1.96 (s, 2H) 2.13–2.20 (m, 1H) 2.26 (s, 1H) 2.39 (dd, J = 16.21, 6.84 Hz, 1H) 2.49
(s, 1H) 2.65–2.74 (m, 2H) 2.80–2.89 (m, 2H) 3.04 (s, 3H) 3.37–3.47 (m, 3H) 3.55
(s, 1H) 3.63 (s, 3H) 3.98 (dd, J = 11.33, 3.91 Hz, 2H) 7.12 (s, 1H) 7.31 (d,
J = 8.59 Hz, 1H) 7.46 (s, 1H); [M+H]+ = 426.2; k0 = 4.79 (Chiral HPLC ChiralPakÒ
AD, 40% ethanol/60% hexane).
32. A Monte Carlo molecular mechanics search of low energy conformers for the R
configuration of 13 was conducted using MacroModel within the Maestro
graphical interface (Schrödinger Inc.). The 16 lowest energy conformers
identified were used as starting points and minimized using density
functional theory (DFT) within Gaussian 03. Simulations of infrared and VCD
spectra for each conformer were generated using an in-house written program
to fit Lorentzian line shapes (10 cm-1 line width) to the computed spectra. In
this manner, direct comparisons between simulated and experimental spectra
were made.
33. Ki values were measured by displacement of the agonist 3H-CP55,940 binding
on membranes of Human Embryonic Kidney (HEK) 293s stable cell lines
transfected with the cloned human CB1 receptor. EC50 were measured by
GTPc
35S binding on membranes of Human Embryonic Kidney (HEK) 293 EBNA
stable cell line transfected with the cloned human CB1 receptor using
WIN55,212–2 (compound 2) as the reference agonist for Emax calculations.
34. Lessard, E.; Schroeder, P.; Ellis, A.; Yu, X. H.; Ducharme, J. Abstracts: Clinical
Pharmacology and Therapeutics IXth World Congress 2008 Can. J. Clin.
Pharmacol. 2008, 15(3), e550.
27. Rukwied, R.; Watkinson, A.; McGlone, F.; Dvorak, M. Pain 2003, 102, 283.
28. Salim, K.; Schneider, U.; Burstein, S.; Hoy, L.; Karst, M. Neuropharmacology
2005, 48, 1164.
29. Dziadulewicz, E. K.; Bevan, S. J.; Brain, C. T.; Coote, P. R.; Culshaw, A. J.; Davis, A.
J.; Edwards, L. J.; Fisher, A. J.; Fox, A. J.; Gentry, C.; Groarke, A.; Hart, T. W.;
Huber, W.; James, I. F.; Kesingland, A.; Vecchia, L. L.; Loong, Y.; Lyothier, I.;
McNair, K.; O’Farrell, C.; Peacock, M.; Portmann, R.; Schopfer, U.; Yaqoob, M.;
Zadrobilek, J. J. Med. Chem. 2007, 50, 3851.
35. Austin, R. P.; Barton, P.; Cockroft, S. L.; Wenlock, M. C.; Riley, R. J. Drug Metab.
Dispos. 2002, 30, 1497.
36. This study was conducted under a protocol that has been approved by an
ethical committee. The animals were kept and experiments were performed at
our main site (AZRDM: AstraZeneca R&D Montreal) or at a site which has
accreditation from: CCAC (Canadian Council on Animal Care), AAALAC
(Association for the Assessment and Accreditation of Laboratory Animal
Care) and/or approved by AZGVC (AstraZeneca Global Veterinary Council) for
study conduct.
37. The determination of the total plasma or brain concentration of (R)-21 was
performed by protein precipitation (after homogenization of brain samples),
followed by reversed-phase liquid chromatography and electrospray mass
spectrometry. Twenty microliters of biological samples from the CNS
experiments (plasma and brain) and blank matrix(ces) are added manually
30. Cheng, Y.-X.; Pourashraf, M.; Luo, X.; Srivastava, S.; Walpole, C.; Salois, D.; St-
Onge, S.; Payza, K.; Lessard, E.; Yu, X. H.; Tomaszewski, M. Bioorg. Med. Chem.
Lett. 2012, 22, 1619.
31. All products gave satisfactory analytical characterization showing purity >95%
as determined by HPLC using a Zorbax C-18 column (k = 215, 254 and 280 nm).
1H NMR spectra were obtained from
a
400 MHz Varian Unity Plus
spectrometer. Mass spectra were obtained on a Micromass Quattro micro API
or an Agilent 1100 Series LC/MSD instrument using loop injection. Selected
analytical characterizations; Compound 5 (AZ599): 1H NMR (METHANOL-D4) d
ppm 0.89 (d, J = 6.40 Hz, 3 H), 0.59–1.09 (m, 2H), 1.13 (t, J = 7.42 Hz, 3H), 1.47–
1.76 (m, 3H), 1.77–1.89 (m, 2H), 2.08 (d, J = 12.29 Hz, 2H), 2.70–2.83 (m, 1H),
2.96–3.08 (m, 1H), 3.29–3.47 (m, 5H), 3.42 (q, J = 7.25 Hz, 2H), 3.55–3.70 (m,
2H), 3.85–4.00 (m, 1H), 4.03 (dd, J = 11.65, 4.48 Hz, 2H), 4.34–4.70 (m, 3H),
7.32 (d, J = 8.70 Hz, 1H), 7.56 (s, 1H), 7.95 (d, J = 8.45 Hz, 1H); [M+H]+ 474.0;
Compound (R)-13: 1H NMR (CHLOROFORM-D) d ppm 1.42–1.50 (m, 1H) 1.50–
1.57 (m, 2H) 1.58–1.67 (m, 2H) 1.76 (d, J = 11.72 Hz, 2H) 2.12–2.18 (m, 1H)
into a 96 well plate. All the in vivo samples are precipitated by adding 60 lL of
ACN+. The plate is carefully capped, vortexed and centrifuged at 9000g for
30 min at 4 °C. The samples are then ready to be analyzed by LC-MS/MS. After
preparing an analytical standard in ACN+, serial dilutions are carried out in
order to prepare a calibration curve. The range of the calibration curve is 1.22
to 1000.