Structural modifications of N-arylamide oxadiazoles: Identification of N-arylpiperidine oxadiazoles as potent and selective agonists of CB2

Article abstract of DOI:10.1016/j.bmcl.2008.06.096

Structural modifications to the central portion of the N-arylamide oxadiazole scaffold led to the identification of N-arylpiperidine oxadiazoles as conformationally constrained analogs that offered improved stability and comparable potency and selectivity. The simple, modular scaffold allowed for the use of expeditious and divergent synthetic routes, which provided two-directional SAR in parallel. Several potent and selective agonists from this novel ligand class are described.

Full text of DOI:10.1016/j.bmcl.2008.06.096

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4267–4274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural modifications of N-arylamide oxadiazoles: Identification
of N-arylpiperidine oxadiazoles as potent and selective agonists of CB2
a
b
c
f
d
Erin F. DiMauro a, , John L. Buchanan , Alan Cheng , Renee Emkey , Stephen A. Hitchcock , Liyue Huang ,
Ming Y. Huang e, Brett Janosky d, Josie H. Lee c, Xingwen Li e, Matthew W. Martin a, Susan A. Tomlinson a,
Ryan D. White a, Xiao Mei Zheng a, Vinod F. Patel a, Robert T. Fremeau Jr. e
*
a Department of Medicinal Chemistry, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
b Department of Molecular Structure, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
c Department of HTS and Molecular Pharmacology, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
d Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
e Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
f Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural modifications to the central portion of the N-arylamide oxadiazole scaffold led to the identifi-
cation of N-arylpiperidine oxadiazoles as conformationally constrained analogs that offered improved
stability and comparable potency and selectivity. The simple, modular scaffold allowed for the use of
expeditious and divergent synthetic routes, which provided two-directional SAR in parallel. Several
potent and selective agonists from this novel ligand class are described.
Received 27 May 2008
Revised 26 June 2008
Accepted 30 June 2008
Available online 3 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid G-protein coupled receptors
CB2 agonists
Piperidine oxadiazoles
The cannabinoid G-protein coupled receptors, CB1 and CB2,
play important roles in the transduction and perception of pain.1
The contribution of CB1 to the modulation of antinociception has
been well established.1 However, agonism of CB1, which is pre-
dominantly expressed in the CNS, results in undesirable psycho-
tropic effects, sedation, and catalepsy.2 Selective agonism of CB2,
which is predominantly expressed in immune cells and tissues,
presents an opportunity for pain management without the unfa-
vorable CNS side effects. Indeed, a number of CB2-selective ago-
nists have shown efficacy in rodent models of inflammatory and
neuropathic pain at doses that do not cause sedation or locomo-
tor impairment.3
In accordance with our ongoing efforts to identify small mole-
cule agonists of CB2 for use as therapeutic agents in the treatment
of pain, we recently disclosed the N-arylamide oxadiazoles.4 This
series is represented by 1 (Scheme 1), which exhibits full agonism5
of the CB2 receptor at low nanomolar concentrations with >200-
fold selectivity over CB1 in a functional GTP-Eu binding assay (Ta-
ble 1).6 Unfortunately, 1 suffered from high clearance in vivo (rat
i.v. CL = 5.7 L/h/kg). High clearance was also observed for several
of its analogs, and hydrolysis of the amide bond was identified as
a major metabolic pathway in rats (Scheme 1).7
In an effort to improve the metabolic stability without compro-
mising the agonist potency and efficacy, we investigated reversed
amide 3 and propylamines 4–6 (Table 1).8 The reversed amide 3
proved to be significantly less potent and efficacious than 1. Next,
we explored the removal of the amide carbonyl leading to propyl-
amine 4,4 which was only 5-fold less potent and selective than the
corresponding amide (1). Unfortunately, 4 was metabolically
unstable in liver microsomes.7 Accordingly,
a-methyl substituents
were introduced to block the potential oxidative metabolism,
affording secondary and tertiary propylamines (( )-5 and 6). In
the case of the mono-methyl analog ( )-5 only a modest 7-fold loss
of potency was observed. The gem di-methyl analog 6 led to a
more significant 20-fold loss of potency and provided only a partial
agonist (Emax = 52%). Also, both compounds were still rapidly
metabolized in liver microsomes.
The data from propylamines 4–6 suggested that the carbonyl
functionality was not essential for potency and efficacy. Further-
more, the 20- to 100-fold selectivity afforded by 4–6 was unantic-
ipated since the vast majority of CB2-selective functional agonists
contain amide or sulfone moieties.3c–f,9 Encouraged by this discov-
ery, we sought to explore cyclic tethers that could presumably im-
prove the metabolic stability and intrinsic potency through
* Corresponding author. Tel.: +1 617 444 5189; fax: +1 617 621 3907.
E-mail address: edimauro@amgen.com (E.F. DiMauro).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.096

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E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4267–4274
Cl
N
Cl
N
O
O
NH2
+
N
F
F
N
N
H
N
HO
R
N
2
O
O
R
1: R = H
Scheme 1. Amide oxadiazoles and hydrolysis products.
Table 1
Functional GTP-Eu assay results for compounds 1–6 (EC50
,
l
M; Emax, %) and CLint. in rat and human liver microsomes (RLM, HLM)a
Cl
N
O
F
N
R
Compound
R
CB2 EC50 (Emax
)
CB1 EC50 (Emax
)
CLint. (lL/min/mg)
RLM
126
HLM
49
H
N
N
1
0.002 (115)
0.403 (51)
O
HO
N
2
3
NA (10)
NA (0.3)
—
—
—
—
O
O
N
H
1.55 (87)
NA (À1)
H
N
N
N
4
0.011 (104)
0.015 (112)
0.472 (103)
1.56 (69)
614
179
142
H
N
( )-5
>399
H
N
N
6
0.053 (52)
1.30 (70)
775
429
a
The results are expressed as the means SEM for n = 2–20 independent measurements, and were calculated in Prism by use of a logistic fit. Emax, % is given in parentheses
(NA, not active).
increased conformational rigidity (Table 2). The cyclopentyl amine
derivatives, ( )-7 and ( )-8, displayed decreased potency and effi-
cacy. However, the trans-cyclopropyl amine ( )-9, pyrrolidines 10
and 11, and piperidine 12a all showed promising functional activ-
ity and selectivity. Of these, 12a afforded the best combination of
potency, selectivity, and low intrinsic clearance in vitro.
In light of the in vitro profile of lead compound 12a, we set out
to explore the structure–activity relationships around this novel N-
arylpiperidine oxadiazole scaffold. Molecular modeling revealed
that 1 and 12a adopt similar low energy conformations (Fig. 1),10
suggesting that these two series of molecules share similar binding
modes, and may therefore exhibit parallel SAR.4

E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4267–4274
4269
Table 2
Functional GTP-Eu binding assay results for compounds ( )-7–12a (EC50
,
l
M; Emax, %) and CLint. in rat and human liver microsomes (RLM, HLM)a
Cl
N
O
F
N
R
N
Compound
( )-7
R
CB2 EC50 (Emax
0.465 (73)
1.60 (77)
)
CB1 EC50 (Emax
)
CLint. (lL/min/mg)
RLM
—
HLM
H
N
2.23 (39)
—
—
H
N
( )-(8
NA (4)
—
( )-(9
0.099 (110)
0.086 (116)
0.140 (109)
0.089 (101)
NA (8)
193
69
H
N
10
1.27 (46)
1.69 (24)
3.03 (36)
426
579
80
215
215
92
N
11
N
N
12a
a
The results are expressed as the means SEM for n = 2–20 independent measurements, and were calculated in Prism by use of a logistic fit. Emax, % is given in parentheses
(NA, not active).
Despite having steric bulk which was comparable to the lead
12a, compounds such as 12b with 3,4-di-substituted aryl substitu-
ents at R1 were significantly less potent (Table 3). Upon confirming
that the 3-quinoline isomer was clearly preferred to the 4-quino-
line isomer 12c, we quickly discovered that close structural ana-
logs of 3-quinoline afforded the best combination of potency and
selectivity (i.e., 12e–i).
The SAR around R2 revealed that the 2-Cl substituent was crit-
ical for potency (13a vs. 13b, 13c vs. 13d, Table 4). Although 13b
was 4-fold more potent than 12a, it was significantly less selective
and less stable in vitro. Although 13d was equi-potent to 12a and
metabolically stable, it was significantly less selective. The cyclo-
Figure 1. Overlay of amide oxadiazole 1 (green) and piperidine oxadiazole 12a
hexyl analog 13f had a favorable combination of potency, efficacy,
(purple).
and selectivity, but was still a sub-micromolar full agonist of CB1
with inferior selectivity (23-fold vs. 34-fold) and metabolic stabil-
ity to that of 12a. From this initial SAR evaluation, compound 12h
had the best overall potency, efficacy, selectivity, and metabolic
stability profile.
On the basis of potency, selectivity, and in vitro stability, se-
lected compounds were further studied in a human cell-based
cAMP assay. This assay monitors a distal signaling event from the
CB2 and CB1 receptors. Activation of CB2 and CB1 receptors results
in inhibition of adenylate cyclase. Compounds were assessed for
Taking advantage of a modular and divergent synthetic strat-
egy, we held the oxadiazole-aryl substituent (R2) constant as 2-
chloro-4-fluoro-phenyl, and varied the N-aryl substituent (R1, Ta-
ble 3). Concurrently, fixing the N-aryl substituent (R1) constant as
3-quinoline, we varied the oxadiazole substituent (R2, Table 4).11
Our initial survey of R groups was based on the SAR observed in
the N-aryl amide oxadiazole series and the overlay of low energy
conformations for the two scaffolds.

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E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4267–4274
Table 3
Functional GTP-Eu binding assay results for compounds 12b–i (EC50
,
l
M; Emax, %) and CLint. in rat and human liver microsomes (RLM, HLM)a
F
Cl
N
R1
N
N
O
Compound
R1
CB2 EC50 (Emax
)
CB1 EC50 (Emax
)
CLint. (lL/min/mg)
RLM
—
HLM
—
12b
0.376 (93)
NA (2)
Cl
N
CF3
12c
12d
2.86 (35)
NA (2)
—
—
—
—
N
0.340 (88)
NA (13)
N
N
12e
12f
12g
0.019 (113)
0.032 (108)
0.016 (106)
0.910 (60)
0.722 (93)
0.015 (34)
130
>399
54
95
380
23
N
N
F3C
N
12h
0.007 (120)
1.43 (55)
32
38
Cl
N
12i
0.003 (110)
0.419 (33)
18
28
OCF3
a
The results are expressed as the means SEM for n = 2–20 independent measurements and were calculated in Prism by use of a logistic fit. Emax, % is given in parentheses
(NA = not active).
their ability to inhibit a forskolin-induced increase in intracellular
cAMP (Table 5).12 Compounds 12a and 12h proved superior to ( )-
9 and 10 and comparable to 1 and 6 in potency, efficacy, and
selectivity.13
The pharmacokinetic parameters of 12h were assessed in
rats following both i.v. and oral administration (Table 6).
Compound 12h was metabolically stable in liver microsomes
and displayed low clearance and a long half-life. Following
oral administration, reasonable exposure was achieved despite
a low oral bioavailability of 2%. 12h has good permeability
and is not a substrate for PgP.14 We speculate that the low
bioavailability is due to poor oral absorption resulting from
poor solubility (0.01 N HCl < 1
lg/mL; PBS < 1 lg/mL; SIF 2 lg/
mL).15

E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4267–4274
4271
Table 4
Functional GTP-Eu binding assay results for compounds 13a–g (EC50
,
l
M; Emax, %) and CLint. in rat and human liver microsomes (RLM, HLM)a
R2
N
N
O
N
N
Compound
R2
CB2 EC50 (Emax
)
CB1 EC50 (Emax
)
CLint. (lL/min/mg)
RLM
—
HLM
13a
0.313 (53)
NA (8)
—
N
N
Cl
13b
13c
13d
13e
13f
0.024 (103)
1.78 (26)
0.096 (23)
2.62 (26)
0.951 (27)
NA (8)
187
—
251
—
Cl
Cl
Cl
F
0.080 (99)
1.55 (89)
43
58
—
—
0.026 (113)
1.44 (38)
0.600 (105)
NA (8)
185
—
283
—
13g
a
The results are expressed as the means SEM for n = 2–20 independent measurements, and were calculated in Prism by use of a logistic fit. Emax, % is given in parentheses
(NA, not active).
Table 5
Table 6
M; Emax, %)a
hCB1 EC50 (Emax)
Pharmacokinetic properties of 12h in male Sprague–Dawley ratsa
Cellular cAMP assay results for selected compounds (EC50, l
ivb
poc
Compound
hCB2 EC50 (Emax
)
1
6
( )-9
10
12a
12h
0.005 (98)
0.050 (93)
>2 (0)
>2 (23)
0.017 (95)
0.011 (102)
0.559 (49)
>2 (46)
>2 (1)
>2 (3)
>1 (17)
>2 (19)
CL
Vss
t1/2
0.039 L/h/kg
0.365 L/kg
8.9 h
%F
Cmax
AUC0 À 24h
2
682 ng/mL
5240 ng*h/mL
a
b
c
n = 3 animals per study.
Dosed intravenously at 0.5 mg/kg in DMSO.
Dosed orally at 10 mg/kg as a suspension in 2% HPMC/1% Tween80/97% water.
a
The results are expressed as the means SEM for n = 2–20 independent mea-
surements and were calculated in Prism by use of a logistic fit. Emax, % is given in
parentheses.
der reductive amination conditions, to afford propylamines such
as 4.4 For the synthesis of mono-methyl propylamine ( )-5, 4-
oxopentanoic acid 17 was condensed with N-hydroxybenzami-
dine 15 to afford ketone 18, which underwent a reductive ami-
nation with 3-aminoquinoline to provide ( )-5. The gem di-
methyl propylamine 6 was synthesized beginning with a con-
densation of N-hydroxybenzamidine 15 with anhydride 19 to
afford the carboxylic acid 20, which was converted to the ter-
tiary amine 21 under Curtius conditions. Amine 21 was then
coupled with 3-bromoquinoline using a Buchwald-Hartwig ami-
nation to afford 6.16
The synthetic routes to 1–11 are detailed in Schemes 2 and
3. Condensation of nitrile 14 and hydroxylamine afforded N-
hydroxybenzamidine 15, which was condensed with succinic
anhydride to afford the carboxylic acid 2 (Scheme 2). As previ-
ously described,4 N-arylamide oxadiazoles such as 1 were pre-
pared by the reaction of intermediate 2 with anilines such as
3-aminoquinoline. Alternatively, carboxylate 1 was converted
to the aldehyde 16, which was then reacted with anilines, un-

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E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4267–4274
F
HO
N
NC
Cl
N
O
b
a
H2N
O
Cl
N
F
Cl
F
HO
14
15
2
F
c
N
O
N
2
NH
O
Cl
N
d, e
1
F
F
N
N
N
O
f
NH
O
H
Cl
Cl
N
N
O
16
4
F
F
Cl
OH
O
N
N
O
N
h
g
O
O
NH
Cl
N
N
O
17
18
( )-5
F
F
N
O
N
N
k
R
i
NH
Cl
Cl
N
N
O
O
O
O
19
20: R = CO2H
21: R = NH2
6
j
Scheme 2. Reagents and conditions: (a) Hydroxylamine hydrochloride, Na2CO3, MeOH/Water (4:1), 95%; (b) Succinic anhydride, DMF, 120 °C, 98%; (c) PS-Carbodiimide,
HOBt, 3-aminoquinoline, CH2Cl2, 13%; (d) i—TMS-diazomethane, ii—DIBAL-H, CH2Cl2/MeOH, 66%; (e) Dess-Martin periodinane, CH2Cl2, 84%; (f) 3-aminoquinoline, NaBH4,
DCE, 40%; (g) 15, HOBt hydrate, DIPEA, DMF, 110 °C, 77% (h) 3-aminoquinoline, NaBH(OAc)3, DCE, 3%; (i) 15, DMF, 120 °C, 90%; (j) DPPA, NEt3, benzene, 47%; (k) 3-
bromoquinoline, Pd2dba3, NaOt-Bu, X-Phos, toluene, 60 °C, 3%.
The syntheses of ( )-7, ( )-8, and ( )-9 were accomplished using
oxadiazole formation and reductive amination sequences similar
to that applied in the synthesis of ( )-5 (Scheme 3). Pyrrolidines
10 and 11 were isolated by chiral resolution of the racemate ( )-
32, which was prepared from amine ( )-31 using Pd-Xantphos-cat-
alyzed Buchwald-Hartwig amination.16
Our synthetic approach to piperidine oxadiazoles allowed for fi-
nal stage modifications of either of the two aryl termini (Scheme
4). TBTU/HOBt-promoted reaction of 1-Boc-piperidine-4-carboxyl-
ate (33) with N-hydroxybenzamidine 15, followed by removal of
the Boc protecting group, afforded the penultimate piperidine oxa-
diazole 34. Finally, N-aryl groups (R1) were installed to afford 12a–
i using a Pd-Xantphos-catalyzed Buchwald-Hartwig amination.16
These amination conditions were similarly used to afford nitrile
36 from piperidine-4-carbonitrile (35) and 3-bromoquinoline.
After hydrolysis of the nitrile, the resulting acid 37 was condensed
with a variety of N-hydroxybenzamidines under the usual condi-
tions to afford 13a–g.11
In summary, structural modifications to the central portion of
the N-arylamide oxadiazole scaffold were investigated in an effort
to improve potency and metabolic stability through increased con-
formational constraint. These efforts led to the identification of N-
arylpiperidine oxadiazole 12a as a novel, potent, selective, full ago-
nist of CB2. The modular scaffold allowed for the use of expeditious
and divergent synthetic routes which provided two-directional
SAR in tandem. Our initial lead-optimization efforts have identified
12h as a highly potent, >200-fold selective, full agonist with prom-
ising pharmacokinetics in rodents. We believe that N-arylpiperi-
dine oxadiazoles with appropriate PKDM properties could be
developed as analgesic agents for the treatment of inflammatory
and neuropathic pain.
Acknowledgement
The authors are grateful to Yuan Cheng, and Jim Brown for help-
ful discussions.

E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4267–4274
4273
F
F
Cl
Cl
N
N
O
OH
O
H
N
N
N
O
b
F
a
a
O
N
O
( )-23
( )-22
( )-7
F
Cl
Cl
N
OH
O
H
N
b
N
N
O
N
O
N
O
O
( )-24
( )-8
( )-25
F
Cl
N
H
N
OEt
OR
a
O
c
N
N
O
HN
O
O
H
( )-26
( )-9
N
( )-27: R = Et
( )-28: R = H
d
F
N
F
OH
O
N
N
O
Cl
O
N
a
f
N
N
N
Cl
O
O
N
R
( ) 30: R = CO2tBu
( )-31: R = H
( )-32
10 and 11
g
e
( )-29
Scheme 3. Reagents and conditions: (a) 15, HOBt hydrate, DIPEA, DMF, 110 °C, 88% ( )-23, 65% ( )-25, 15% ( )-9, 49% ( )-3; (b) 3-aminoquinoline, NaBH(OAc)3, DCE, 5% ( )-7,
15% ( )-8; (c) 3-aminoquinoline, NaBH4, Ti(Oi-Pr)4, CH2Cl2, 100%; (d) LiOH, THF/MeOH, 75%; (e) TFA, CH2Cl2, 98%; (f) 3-bromoquinoline, Pd2dba3, NaOt-Bu, XantPhos, toluene,
100 °C, 60%; (g) chiral HPLC.
F
F
Cl
Cl
N
N
c
a, b
R1
N
N
CO2H
Boc
N
HN
N
N
O
O
34
33
35
12a-i
R2
N
N
O
d
f
HN
CN
N
N
R
N
36: R = CN
37: R = CO2H
13a-g
e
Scheme 4. Reagents and conditions: (a) 15, TBTU, HOBt, DIPEA, DMF, 100 °C, 55%; (b) TFA, 100%; (c) R1Br, Pd2(dba)3, Xantphos, NaOt-Bu, toluene, 100 °C, 13–57%; (d) 3-
bromoquinoline, Pd2(dba)3, Xantphos, NaOt-Bu, toluene, 100 °C, 90%; (e) H2SO4, H2O, 45%; (f) R2-N-hydroxybenzamidine, TBTU, HOBt, DIPEA, DMF, 100 °C, 21–34%.
2. (a) Campbell, F. A.; Tramer, M. R.; Carroll, D.; Reynolds, J. M.; Moore, R. A.;
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Huang, M.; Lee, J. H.; Lin, F. -F.; Martin, M. W.; Nguyen, H. Q.; Patel, V. F.;
Tomlinson, S. A.; White, R. D.; Xia, X.; Hitchcock, S. A. J. Med. Chem. in press..
5. Johnson, M. R.; Melvin, L. S. Full agonist activity was defined as Emax equal to
100% efficacy of CP 55,940, a known agonist of both CB1 and CB2 receptor. In
Cannabinioids as Therapeutic Agents; Mechoulam, R., Ed.; CRC Press: Boca Raton,
FL, 1986; p 121.
8. Compounds were considered ‘‘not active” if their Emax was less than 15% at
10 M concentration. In these cases an EC50 value was not calculated.
l
9. For other examples, see: (a) Omura, H.; Kawai, M.; Shima, A.; Iwata, Y.; Ito, F.;
Masuda, T.; Ohta, A.; Makita, N.; Omoto, K.; Sugimoto, H.; Kikuchi, A.; Iwata, H.;
Ando, K. Bioorg. Med. Chem. Lett. 2008, 18, 3310; (b) Verbist, B. M. P.; De Cleyn,
M. A. J.; Surkyn, M.; Fraiponts, E.; Aerssens, J.; Nijsen, M. J. M. A.; Gijsen, H. J. M.
Bioorg. Med. Chem. Lett. 2008, 18, 2574; (c) Ermann, M.; Riether, D.; Walker, E.
R.; Mushi, I. F.; Jenkins, J. E.; Noya-Marino, B.; Brewer, M. L.; Taylor, M. G.;
Amouzegh, P.; East, S. P.; Dymock, B. W.; Gemkow, M. J.; Kahrs, A. F.; Ebneth, A.;
Löbbe, S.; Thome, D.; O’Shea, K.; Dinallo, R.; Raymond, E.; Shih, D-T.; Thomson,
D. Bioorg. Med. Chem. Lett. 2008, 18, 1725; (d) Stern, E.; Muccioli, G. G.; Millet,
R.; Goossens, J-F.; Farce, A.; Chavatte, P.; Poupaert, J. H.; Lambert, D. M.;
Depreux, P.; Hénichart, J-P. J. Med. Chem. 2006, 49, 70; (e) Raitio, K. H.;
Savinainen, J. R.; Vespäläinen, J.; Latinen, J. T.; Poso, A.; Järvinen, T.; Nevalainen,
T. J. Med. Chem. 2006, 49, 2022; (f) Manera, C.; Benetti, V.; Castelli, M. P.;
Cavallini, T.; Lazzarotto, S.; Pibiri, F.; Saccomanni, G.; Tuccinardi, T.; Vannacci,
A.; Martinelli, A.; Ferrarini, P. L. J. Med. Chem. 2006, 49, 5947; (g) Murineddu, G.;
Lazzari, P.; Ruiu, S.; Sanna, A.; Loriga, G.; Manca, I.; Falzoi, M.; Dessi, C.; Curzo,
M. M.; Chelucci, G.; Pani, L.; Pinna, G. A. J. Med. Chem. 2006, 49, 7502; (h) Pagé,
D.; Yang, H.; Brown, W.; Walpole, C.; Fleurent, M.; Fyfe, M.; Gaudreault, F.; St-
Onge, S. Bioorg. Med. Chem. Lett. 2007, 17, 6183; (i) Lavey, B. J.; Kozlowski, J. A.;
Shankar, B. B.; Spitler, J. M.; Zhou, G.; Yang, D-Y.; Shu, Y.; Wong, M. K. C.; Wong,
S-C.; Shih, N-Y.; Wu, J.; McCombie, S. W.; Rizvi, R.; Wolin, R. L.; Lunn, C. A.
Bioorg. Med. Chem. Lett. 2007, 15, 783.
10. Low energy confirmations and overlay were calculated using FLAME: Cho, S. J.;
Sun, Y. J. Chem. Inf. Model. 2006, 46, 298–306. MMFF94 energies of
conformations are within 1 kcal/mol of lowest energy conformations.
6. The GTP-Eu binding assay is a proximal assay that measures activation of G-
protein coupled receptors by the ability of the associated G -protein to bind
a
GTP. Two membrane preparations (Bmax
insect cells co-transfected with human CB1 or CB2 and Gai3 were assessed
by monitoring binding of non-hydrolysable GTP analog, GTP-Europium.
M CP55940, a full agonist at both CB1 and CB2 receptors, defined 100%
efficacy. The procedure was modified from a commercial kit (Perkin Elmer,
AD-0167), in that 25 L of buffer A (50 mM Hepes, 0.1% BSA) containing a
certain concentration (0.01 nM to 10 M) of tested compound was added to
125 L of buffer B (50 mM Hepes, 0.1% BSA, 10 mM MgCl2, 150 mM NaCl, 5
uM GDP, 10 nM GTP-Europium, 0.0005% Saponin and CB1 (4.5 g/well) or
CB2 (14 g/well) membranes) in a 96-well filtration assay plate that came
with the kit. The final DMSO concentration was 1%. The plate was incubated
at rt for 45 min followed by filtering and washing twice with 300 L of cold
GTP wash buffer (supplied with the kit) on vacuum manifold device
:
1.7 pmol/mL for both) from Sf9
11. All products gave satisfactory analytical data as indicated by 1H NMR and
LCMS. Data for 12h are as follows: 1H NMR (400 MHz, DMSO-d6) d ppm 8.94 (d,
J = 2.8 Hz, 1H), 8.01 (dd, J = 8.7, 6.0 Hz, 1H), 7.98 (s, 1H), 7.87 (s, 1H), 7,73 (dd,
J = 8.8, 2.5 Hz, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.48–7.42 (2H), 3.96 (m, 2H), 3.48
(m, 1H), 3.13 (m, 2H), 2.27 (m, 2H), 2.01 (m, 2H); LCMS EI exact mass
calculated for C22H17Cl2FN4O 443.05005 m/z found (M+H+) 443.08362.
12. For cAMP modulation as a read-out of cannabinoid activity see (a) Kaminski, N.
E. Toxicol. Lett. 1998, 102–103, 59; (b) Herring, A. C.; Koh, W. S.; Kaminski, N. E.
Biochem. Pharmacol. 1998, 55, 1013; (c) Howlett, A. C.; Breivogel, C. S.; Childers,
S. R.; Deadwyler, S. A.; Hampson, R. E.; Porrino, L. J. Neuropharmacology 2004,
47, 345. For a complete description of cAMP assay conditions see Ref. Ref. 4.
a
3
l
l
l
l
l
l
l
13. In
IC50 = 0.5
other GPCRs (sst4,
NK1, -opiod, H1, M1).
a
hERG binding assay using radiolabeled dofetilite, 12 h had an
M. 12 h was highly selective (IC50 and EC50 >50 M) over several
1-adreno, b1-adreno, D2, 5HT1A, 5HT2B, CXR2, CCR2b,
a
l
l
(Millipore, MAVM 096 0R). The plate was then read in a fluorescent plate
reader (Perkin-Elmer Envision) at 615 nm and data were analyzed with
Activity Base.
a
l
14. Transport of 12h across LLC-PK1 cells at 5 lM in the presence of 0.1% BSA.
7. Following i.v. dosing of 0.5 mg/kg to male Sprague–Dawley rats, >50% of the
Wild-type: Papp (avg) = 9.95 Â 10À6 cm/s; Efflux ratio = 0.9. h-MDR1: Papp
(avg) = 10.65 Â 10À6 cm/s; Efflux ratio = 0.9.
dose was converted to
2 for several N-arylamide oxadiazoles and
N-arylpropylamine oxadiazoles. High levels of 2 were consistently observed
in plasma samples taken from pharmacodynamic studies of N-arylamide
oxadiazoles and N-arylpropylamine oxadiazoles in rodents.
15. Tan, H.; Semin, D.; Wacker, M.; Cheetham, J. JALA 2005, 10, 364.
16. Klingensmith, L. M.; Strieter, E. R.; Barder, T. E.; Buchwald, S. L. Organometallics
2006, 25, 82.