Discovery of α-amidosulfones as potent and selective agonists of CB2: Synthesis, SAR, and pharmacokinetic properties

Article abstract of DOI:10.1016/j.bmcl.2008.11.026

A series of α-amidosulfones were found to be potent and selective agonists of CB₂. The discovery, synthesis, and structure-activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most p

Full text of DOI:10.1016/j.bmcl.2008.11.026

Bioorganic & Medicinal Chemistry Letters 19 (2009) 31–35
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of
a-amidosulfones as potent and selective agonists of CB2:
Synthesis, SAR, and pharmacokinetic properties
a
b
c
d
e
Isaac E. Marx a, , Erin F. DiMauro , Alan Cheng , Renee Emkey , Stephen A. Hitchcock , Liyue Huang ,
Ming Y. Huang f, Jason Human a, Josie H. Lee c, Xingwen Li e, Matthew W. Martin a, Ryan D. White a,
Robert T. Fremeau Jr. f, Vinod F. Patel a
*
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 Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
e Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
f Department of Neuroscience, 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:
A series of a-amidosulfones were found to be potent and selective agonists of CB2. The discovery, synthe-
Received 2 October 2008
Revised 4 November 2008
Accepted 10 November 2008
Available online 13 November 2008
sis, and structure–activity relationships of this series of agonists are reported. In addition, the pharmaco-
kinetic properties of the most promising compounds are profiled.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid G-protein coupled receptors
CB2 agonists
Since the isolation of
D
9-tetrahydrocannabinol (
D
9-THC), the
netic properties of a series of
tive agonists of CB2.
a-amidosulfones as potent and selec-
active ingredient in marijuana, and the subsequent discovery of
the cannabinoid class of G-protein coupled receptors1 (GPCRs)
there has been great interest in developing small molecules that
share THC’s analgesic properties without the undesired psychotro-
pic side effects.2 To date two subtypes of cannabinoid (CB) recep-
tors have been characterized and they are markedly different
both in their distribution within the body and in their pharmaco-
logical effects. The CB1 receptor is highly expressed in both the
central nervous system (CNS) and peripheral nervous system
(PNS), and CB1 agonism has been shown to induce hypothermia,
catalepsy, and motor dysfunction.2d,e,3 CB2 expression, on the other
hand, predominantly resides in the periphery, especially in im-
mune tissues, and CB2 agonism has been shown to be effective in
rodent pain models without CNS-related side effects.2d,e,4
Our internal screening efforts revealed that oxadiazole 16h
(Fig. 1) is a potent and selective CB2 agonist. In an effort to identify
CB2 agonists with alternate molecular scaffolds, we performed a
substructure search of the carbazole amide moiety of 1 against
our GPCR-preferred compound collection. The resulting virtual hits
were screened in a functional GTP-Europium binding assay7 and
we discovered that sulfone 2 was a potent and efficacious CB2 ago-
nist and was moderately (50-fold) selective over CB1 (Table 1, En-
try 1).8 Molecular modeling revealed that oxadiazole 1 and sulfone
2 adopted similar lowest energy conformations (Fig. 2), suggesting
these two molecules shared similar binding modes.9 To determine
whether the N-ethyl aminocarbazole moiety, which has known
genotoxicity issues,10 was critical for CB2 activity, we screened
Strong evidence suggests that CB1 accounts for most of the
undesired effects of dual CB agonists such as
D
9-THC.2a,d,3 Conse-
F
quently, selective CB2 agonism has become an attractive target
for the treatment of inflammatory and neuropathic pain.2d,5 Sev-
eral groups, including our own, have previously reported on the
synthesis and characterization of CB2 agonists.6 Herein we describe
the synthesis, structure–activity relationships, and pharmacoki-
N
O
H
N
N
Me
O
N
CB2 EC50 (Emax) = 0.111 µM (103%)
CB1 EC50 (Emax) = >2 µM (2%)
1
* Corresponding author. Tel.: +1 617 444 5136.
E-mail address: imarx@amgen.com (I.E. Marx).
Figure 1. Structure and activity of lead oxadiazole 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.026

32
I. E. Marx et al. / Bioorg. Med. Chem. Lett. 19 (2009) 31–35
Table 1
R
R
R
Initial phenylsulfone SAR (EC50
,
l
M)a
a
O
Cl
S
S
H
N
O
O
O
O
O
O
R
S
O
O
4
5
O
Compound
R
hCB2 EC50 (Emax %)
hCB1 EC50 (Emax %)
b, c
Me
R
2
0.008 (95)
0.390 (40)
N
H
N
d
HO
N
S
S
O
O
O
O
O
6
7
N
3
0.004 (31)
>10 (6)
Scheme 1. Synthesis of gem-dimethylsulfones. Reagents and conditions: (a)
ethylbromoacetate, Zn powder, sat. NH4Cl/THF, 110 °C, 48–81%; (b) MeI, K2CO3,
DMF, 40 °C, 80–95%; (c) LiOH, MeOH/THF/H2O, rt, 3 h, 87–99%; (d) 3-aminoquin-
oline, EDC, HOBt, DMF, rt, 17 h, 34–78%.
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.
compounds was determined by measuring the suppression of for-
skolin-induced cAMP production in CHO cells expressing the hu-
man CB1 or CB2 receptors.14,15 Introducing a trifluoromethyl
group to the 4-position of the phenyl ring (8) resulted in full effi-
cacy and good potency in the cellular assay but showed reduced
Table 2
SAR: variations on the 1-phenylsulfone group (EC50
,
l
M)a
H
N
R
N
S
O
O
O
Compound
R
GTP binding
hCB2 EC50 hCB1 EC50
(Emax %) (Emax %)
Whole cell cAMP
hCB2 EC50 hCB1 EC50
(Emax %) (Emax %)
Figure 2. Overlay of oxadiazole 1 (blue) and a-amidosulfone 2 (green), shown here
with a donor pharmacophore point (grey).
our compound collection for other amidosulfones. From the result-
ing hits, quinoline analogue 3 was found to be a potent agonist of
CB2, though only weakly efficacious, and did not show activity on
CB1 (Table 1, Entry 3). This result encouraged us to further investi-
gate the structure–activity relationship of this series of
compounds.
Despite our promising initial results, we were concerned that
the highly electron withdrawing nature of the phenylsulfone and
amide groups would leave the cyclopropyl group vulnerable to
nucleophilic ring opening in vivo.11 Our suspicions were confirmed
when a combination of metabolic and reactivity studies of com-
pound 3 revealed that glutathione opens the cyclopropyl ring in
a nucleophilic fashion.12 To eliminate this metabolic liability, we
chose to replace the cyclopropyl group with a gem-dimethyl moi-
ety, while simultaneously exploring the structure–activity rela-
tionship of the phenyl ring in order to find a more efficacious
analogue of 3.
The synthetic route to access the phenylsulfones is outlined in
Scheme 1. The starting sulfonyl chloride 4 was alkylated with eth-
ylbromoacetate via a modification of Zhang’s zinc-mediated cou-
pling reaction.13 The resulting sulfone ester 5 was bis-methylated
and hydrolyzed to afford carboxylic acid 6. The final compound
(7) was obtained by coupling 6 with 3-aminoquinoline under stan-
dard amide coupling conditions.
CF3
CF3
F
8
0.096 (88) 0.710 (86) 0.008 (96) 0.082 (93)
9
0.019 (94) 1.57 (71)
0.073 (94) >2 (41)
Cl
Cl
10
0.104
(108)
>10 (7)
ND
ND
ND
ND
O
F
11
12
0.148 (91) >10 (12)
Cl
0.109
(102)
2.78 (22)
0.033 (97) >2 (19)
Cl
13
0.057 (90) 1.68 (47)
0.013 (93) >1 (47)
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 (ND, data not determined).
The data for this initial series of molecules is shown in Table 2.
In addition to the GTP-binding assay, the cellular activity of the

I. E. Marx et al. / Bioorg. Med. Chem. Lett. 19 (2009) 31–35
33
Table 3
SAR: variations of the amide group (EC50, l
M)a
Cl
H
N
R
S
O
O
O
Compound
R
GTP binding
Whole cell cAMP
hCB2 EC50 (Emax %)
hCB1 EC50 (Emax %)
hCB2 EC50 (Emax %)
hCB1 EC50 (Emax %)
14
15
16
17
18
19
20
21
0.170 (89)
>10 (15)
0.035 (93)
>1 (21)
CF3
0.115 (77)
0.020 (102)
0.086 (77)
1.62 (44)
>10 (14)
>10 (14)
.700 (22)
>10 (7)
>10 (2)
>10 (0)
>10 (7)
>10 (2)
0.057 (93)
0.005 (96)
ND
>1 (19)
>2 (39)
ND
F3C
Cl
CF3
Br
Cl
OCF3
CN
ND
ND
Cl
Cl
ND
ND
>10 (0)
ND
ND
F
1.30 (76)
ND
ND
Cl
O
22
23
0.004 (101)
0.009 (102)
0.096 (82)
0.322 (80)
0.005 (101)
0.005 (99)
0.230 (98)
Cl
Me
S
0.331 (106)
N
24
25
26
27
28
3.86 (19)
>10 (4)
>10 (1)
ND
ND
Me
Me
N
N
O
S
>10 (7)
ND
ND
0.026 (104)
0.029 (99)
0.216 (99)
2.23 (37)
1.92 (57)
>10 (10)
0.025 (92)
0.011 (85)
ND
>2 (12)
>2 (23)
N
N
O
N
O
S
ND
N
Me
N
(continued on next page)

34
I. E. Marx et al. / Bioorg. Med. Chem. Lett. 19 (2009) 31–35
Table 3 (continued)
Compound
R
GTP binding
Whole cell cAMP
hCB2 EC50 (Emax %)
hCB1 EC50 (Emax %)
hCB2 EC50 (Emax %)
hCB1 EC50 (Emax %)
S
29
0.189 (81)
1.65 (48)
ND
ND
N
N
Me
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
(ND, data not determined).
Because of their superior potency and selectivity, we examined
the pharmacokinetic properties of compounds 16 and 26 (Tables 4
and 5). Both compounds had low intrinsic clearance (Clint) in hu-
man and rat liver microsomes. Compound 16 had high plasma pro-
tein binding (>99%), while compound 26 had lower plasma protein
binding across species (94%). To determine the in vivo pharmaco-
kinetic characteristics of 16 and 26, both compounds were dosed
iv and po to Sprague–Dawley rats (Table 5). Following intravenous
administration, compound 16 exhibited low clearance with a long
half-life (14 h) and a large volume of distribution. Compound 26
had moderate clearance (1.2 L/h/kg) and a much shorter half-life
and smaller volume of distribution when dosed iv. Upon oral dos-
ing compounds 16 and 26 were well absorbed in rat with an oral
bioavailability of 52% and 43%, respectively. In summary, both 16
and 26 displayed good pharmacokinetic properties for use in
in vivo pain models.
Table 4
In vitro pharmacokinetic parameters of 16 and 26
Compound
HLM CLint
l/min/mg)
RLM CLint
(ll/min/mg)
Human PPB (%)
Rat PPB (%)
(l
16
26
14
14
14
20
99.8
93.6
99.7
94.0
Table 5
In vivo pharmacokinetic parameters of 16 and 26 following administration in
Sprague–Dawley rats
Compound
iva
pob
Cmax
Cl
t1/2 (h)
Vss
(L/kg)
AUC0Àinf
tmax
%F
(L/h/kg)
(ng h/ml)
(ng/ml)
(h)
16
26
0.54
1.2
14
1.9
9.3
2.0
9678
3570
560
971
1.7
0.67
52
43
In conclusion, we have described a novel class of a-amidosulf-
a
n = 2 animals. Dosed at 0.5 mg/kg as a solution in DMSO.
n = 3 animals. Dosed at 10 mg/kg; 2% HPMC, 1% Tween 80 vehicle.
b
ones as potent and selective full agonists of CB2. Our efforts on this
series have afforded compounds that show high functional and cel-
lular activity on CB2, good selectivity against CB1, and have good
pharmacokinetic properties in rodents. Our evaluations of these
compounds for efficacy in in vivo pain models will be reported in
due course.
potency in the functional assay and only 10-fold selectivity over
CB1. Adding a chlorine in the 2-position dramatically improved
selectivity and increased functional potency, but reduced cellular
potency by 10-fold (9). Substituting the trifluoromethyl group with
a smaller electron withdrawing group and electron donating group
(10 and 11, respectively) greatly reduced CB2 activity. Moving
away from the 2,4-disubstituted phenyl rings, we next tried replac-
ing the trifluoromethyl group with smaller electron withdrawing
groups in the 4-position. A small group such as fluorine showed
good selectivity but only moderate CB2 potency (12). The optimal
moiety in the phenylsulfone region of the agonist was a 4-chloro-
phenyl group, which provided good potency and selectivity in both
the functional and cellular assays (13).
Based on these results, we chose to examine the p-chlorophe-
nylsulfones in greater detail. Importantly, when compound 13
was incubated with glutathione no nucleophilic addition was ob-
served, confirming our cyclopropyl activation hypothesis.16
We next explored the effect of the amide substituent on CB2
activity and selectivity, which is summarized in Table 3. A trifluo-
romethyl group at the 3 and 4-positions of the phenyl ring were
moderately tolerated (14 and 15), while 3,4-disubstituted ana-
logues showed good potency and selectivity (16 and 17). 2,4-
Disubstituted phenyl rings diminished agonist activity on both
CB1 and CB2 receptors (18 and 19), as did extending the phenyl
amides to benzyl amides (20 and 21). Biaryls were very potent
CB2 agonists but unfortunately were also potent full agonists of
CB1 (22 and 23). Five-membered heterocycles were also examined
and it was found that these compounds had strict size require-
ments with regards to their substitution patterns. Rings with
methyl substituents (24 and 25) were micromolar CB2 agonists,
while rings with larger tert-butyl substituents were highly potent
and selective (26 and 27). However, attaching other large groups
to the heterocycles such as non-planar heterocycles and phenyl
rings decreased CB2 potency (28 and 29).
Acknowledgments
We gratefully acknowledge Dan Waldon for metabolic support
and Mark Duggan for helpful discussion.
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7. Experimental procedure: Two membrane preparations (Bmax: 1.7 pmol/ml for
both) from Sf9 insect cells co-transfected with hCB1 or hCB2 and Gai3 were
assessed by monitoring binding of a non-hydrolysable GTP analogue, GTP-
Europium. Three micromolar of CP55940, a full agonist at both CB1 and CB2
receptors, defined 100% efficacy. The procedure was modified from
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–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 M 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
a
l
l
l
l
l
l
plate that came with the kit. The final DMSO concentration was 1%. The plate
was incubated at room temperature for 45 min followed by filtering and
washing twice with 300 ll of cold GTP wash buffer (supplied with the kit) on a
vacuum manifold device (Millipore, MAVM 096 0R). The plate was then read in
a fluorescent plate reader (Perkin-Elmer Envision) at 615 nm and data was
analyzed with ActivityBase.
15. For full descriptions of all assays, see Ref. 6h.
8. Emax was determined by measuring the maximum value of the dose–response
16. A metabolic analysis of 13 was also performed. Although oxidation of the
methyl group was observed it was a minor metabolite. The major metabolite
was a result of oxidation of the quinoline ring.
curve; 3
lM CP-55,940 (a potent dual CB1/CB2 agonist, purchased from Tocris
Bioscience) was used as a 100% efficacy control. See (a) Wiley, J. L.; Barret, R. L.;