Highly potent and selective cannabinoid receptor 2 agonists: Initial hit optimization of an adamantyl hit series identified from high-through-put screening

Article abstract of DOI:10.1016/j.bmcl.2013.01.044

A series of highly potent & selective adamantane derived CB2 agonists was identified in a high-throughput screen. A SAR was established and physicochemical properties were significantly improved. This was accompanied by potency of the compounds on the Q63R variant and varying β-arrestin data which will support the insight into their relevance for the in vivo situation.

Full text of DOI:10.1016/j.bmcl.2013.01.044

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1177–1181
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Highly potent and selective cannabinoid receptor 2 agonists: Initial
hit optimization of an adamantyl hit series identified from
high-through-put screening
b
a
b
a
Matthias Nettekoven a, , Jürgen Fingerle , Uwe Grether , Sabine Grüner , Atsushi Kimbara ,
Bernd Püllmann a, Mark Rogers-Evans a, Stephan Röver a, Franz Schuler c, Tanja Schulz-Gasch a,
Christoph Ullmer b
⇑
a F. Hoffmann-La Roche Ltd, Pharma Research & Early Development, Medicinal Chemistry, Grenzacher Str. 124, 4070 Basel, Switzerland
b F. Hoffmann-La Roche Ltd, Pharma Research & Early Development, Discovery Biology, Grenzacher Str. 124, 4070 Basel, Switzerland
c F. Hoffmann-La Roche Ltd, Pharma Research & Early Development, DMPK, Grenzacher Str. 124, 4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of highly potent & selective adamantane derived CB2 agonists was identified in a high-through-
put screen. A SAR was established and physicochemical properties were significantly improved. This was
accompanied by potency of the compounds on the Q63R variant and varying b-arrestin data which will
support the insight into their relevance for the in vivo situation.
Received 21 December 2012
Revised 9 January 2013
Accepted 12 January 2013
Available online 23 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid receptor
Adamantane
b-Arrestin
Hit optimization
Throughout evolution the mammalian body has developed
numerous protective mechanisms to prevent and limit tissue
injury caused by various types of neuronal as well as non-neuronal
insults. Lipid signaling through activation of cannabinoid 2 (CB2)
receptors is thought to be an important part of this protective
machinery.1 Inflammation/tissue injury causes a rapid increase in
local endocannabinoid levels which leads to a fast modulation of
signaling pathways in immune and other cells. It has been reported
that endocannabinoids, endocannbinoid-like and/or synthetic CB2
receptor agonists positively affect a large number of pathological
conditions, spanning from cardiovascular,2 over gastrointestinal,3
liver,4 kidney,5 lung,6 neurodegenerative7 and psychiatric8 disor-
ders to pain,9 cancer,10 bone,11 reproductive system12 and skin
pathologies.13 Prototypical CB2 receptor agonists range from end-
ogeneous ligands such as anandamide (AEA)14 and 2-arachidonoyl
and possess different selectivity against the cannabinoid receptor
1 (CB1).18
With the goal to identify new CB2 receptor agonist hit struc-
tures, a high-throughput screen was performed. Various hit clusters
were found. One cluster entailed a considerable number of adaman-
tyl-derivatives with more than 30 members suggesting a specific
interaction of this moiety with the CB2 receptor. A very preliminary
SAR could be deduced from that data set. In general, all compounds
showed high selectivity towards the CB1 receptor with consider-
ably potency range at the CB2 receptor. Representative examples
are shown in Table 1.
The majority of compounds in this cluster were amide based
derivatives, however also carbamoylthioyl-amides were found, like
example 1 with an EC50 of 1.4 lM at the CB2 receptor and being
inactive at the CB1 receptor. Heteroaryl derivatives were active
as well, exemplified with thiazolyl-derivative 2 which was already
active in the high nano-molar range. Substituted aryl-amide deriv-
atives yielded a very preliminary SAR. The substitution on the aryl-
moiety seemed to be determining the activity at the CB2 receptor.
Substituents in the 4-position (like in example 3) yielded com-
glycerol (2-AG)15 over the plant-derived
D9-tetrahydrocannabinol
((À)-
D
9-THC)14 to exogenous cannabinoid type agonists such as
HU-308,14 JWH-13316 and HU-9104 as well as even newer non-
cannabinoid type agonists which are described in several recent
overviews.17 These agonists have various degrees of activities
pounds active in the
lM range. The nature of the substituent in
the 2-position influenced the activity considerably. Derivative 4
was active with 100 nM at the CB2 receptor, compound 5 was
active in the low nano-molar range (EC50 = 15 nM) and both
⇑
Corresponding author.
E-mail address: matthias.nettekoven@roche.com (M. Nettekoven).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.044

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M. Nettekoven et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1177–1181
Table 1
Initial SAR from representative members of the adamantyl hit cluster19
H
H
O
CO2Et
H
H
N
N
N
H
N
H
N
N
N
N
R
S
O
O
O
O
OEt
O
S
O
CO2Me
No.
hCB2: cAMP EC50
hCB2: b-arrestin EC50
1
2
3
4
5
6
(
l
M)
(lM)
1.4
0.5
>10
0.6
0.9
>10
1.2
—
>10
0.1
—
>10
0.015
0.076
>10
>10
—
>10
hCB1: cAMP EC50
(
lM)
clogP, MW
4.0, 306
3.5/327
4.4/299
4.1/297
4.7/313
4.2/313
compounds had EC50s well above 10
l
M at the CB1 receptor. Shift-
There is a nice match of pharmacophoric features of the hydro-
phobic adamantane, the aromatic moiety as well as donor/acceptor
features of the amide and inverse amide, respectively. The vector at
the aromatic moiety has the same directionality. Thus, the confor-
mation of the HTS hit is determined through intra-molecular H-
bond interaction and therefore suggested the replacement of the
anthranilic acid moiety through bicyclic systems offering opportu-
nities for optimization of these CB2 receptor agonists. To prove this
initial hypothesis, adamantane concept compounds with varying
bi-cyclic amide substitutions were designed. The synthesis entails
a one-step transformation of an activated adamantane acid deriv-
ative with the respective amine moiety to access novel derivatives
(8–11) which were profiled under various aspects; that is, CB2
function and binding, additionally CB2 b-arrestin function and pre-
liminary molecular properties (i.e., lipohilicity (logD), solubility
(LYSA), permeability (Pe) and stability in human microsomes
(MAB-maximal achievable bioavailability)24 (Table 2).25 Further-
more, functional activity on the Q63R variation of the CB2 receptor
was assessed. This relevant polymorphism is suggested to be
associated, for example, with liver damage in obese children,26
increases of risk of celiac disease,27 and with the risk for
schizophrenia28 and therefore might have significant impact on
the efficacy of CB2 receptor agonist derived drugs for the human
situation.
As expected from our ‘modeling hypothesis’ the tetrahydro-
quinoline is a good replacement for the anthranilic acid moiety. Al-
ready the un-substituted compound 8 was active in the nano-mo-
lar range on functional CB2 with high selectivity for CB1.29 Micro-
molar activity in the binding assay was observed, the compound
was though inactive on human CB2 b-arrestin. In the CB2 human
Q63R variant the compound was as well active in the nano-molar
range. Preliminary molecular properties assessment confirmed the
compound to be insoluble with a MAB value of 23% in human
microsomes. Further substitution of the tetrahydroquinoline with
a methoxy functionality yielded derivative 9 which was even more
ing the ester moiety to position 3 yielded inactive derivative 6. The
ability of CB2 agonists to recruit b-arrestin, which ultimately leads
to the steric inhibition of G protein coupling and termination of
signaling independent of G protein classes, was determined. The
fact that various partial agonists might constitutively stimulate
receptor activation without causing b-arrestin recruitment might
be considered as
a crucial parameter for the translation of
in vitro potency into in vivo efficacy.20 Compounds 1 and 2 showed
an EC50 in the b-arrestin assay in the high nano-molar range and
derivative
5 was active with an EC50 of 76 nM. Calculated
molecular properties of these derivatives revealed in general a
low molecular weight of ꢀ300 Da and clogP values ꢀ4. This ada-
mantyl-cluster offered a preliminary SAR on the human CB2 and
CB1 cAMP read-out and on the activation potential of the human
CB2 b-arrestin. Several derivatives in this adamantyl-cluster dis-
played high ligand efficiencies21 with clogP values generally ꢀ4
or above. JWH-133 and HU-910 are both highly lipophilic com-
pounds (clogP: 7.99 and 8.79, respectively) and active in the cAMP
assay in the low nano-molar range (4.3 nM and 5.3 nM, respec-
tively) which corroborated our confidence into the HTS derived
adamantyl-cluster being a valuable starting point for optimization.
Adamantane groups are well known moieties in approved drugs
(i.e., Amantadine or Vildagliptin) useful in the treatment of dis-
eases related to the CNS as well as diseases to be treated peripher-
ally.22 The adamantane moiety, optimally substituted, can entail
favorable properties like high bioavailability, good metabolic sta-
bility and half-life. Literature analysis23 yielded a competitor com-
pound 7, an analog of a clinically evaluated derivative, which was
reported with sub-nano molar activity at the CB2 receptor to have
some selectivity for the CB1 receptor. Superimposition and fitting
into the homology model of the hit structure 5 and adamantane
derivative 7 yielded similar specific interactions thus even further
supporting our confidence in this hit-cluster in combination with
CB2 agonism (Fig. 1).
O
O
O
H
N
N
H
N
O
N
5
7
competitor compound
CB2 Ki: 0.15 nM
Roche HTS hit
N
CB1 Ki: 6.8 nM
O
Figure 1. Superimposition of compound 5 (orange) and 7 (green) in the binding site of a CB2 homology model.

M. Nettekoven et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1177–1181
1179
Table 2
Properties of adamantane concept compounds 8–11
O
O
O
O
R
N
N
N
N
O
No.
8
9
10
11
h/m CB2: cAMP EC50
hCB2 Q63R var. EC50
(
l
M)
M)
0.072/0.126
0.055
0.027/0.023
0.078
0.0018/0.028
—
0.02/0.02
0.024
(l
h/m CB2: Ki (
hCB2: b-arrestin EC50
l
M)
0.79/4.3
>10
0.77/5.2
1.3
0.99/>10
—
0.35/1.5
0.9
(
lM)
h/m CB1: cAMP EC50
(lM)
>10/>10
—/<1
Precip.
23
>10/>10
3.79, 10
3.3, 4.6
12
>10/5
4.09, 17
0.7/0.9
6
>10/>10
3.82, 97
4.4, 5.7
5
logD, LYSA (lg/mL)
Pe (10À6 cm/s), % acc
MAB h %
Table 3
Properties of adamantane compounds 12–15
N
R
N
N
N
N
O
No.
12
13
14
15
>10/>10
h/m CB2: cAMP EC50
hCB2 Q63R var. EC50
(
l
M)
M)
0.029/0.022
0.071
0.023/0.026
—
0.02/0.007
—
(l
h/m CB2: Ki (
hCB2: b-arrestin EC50
l
M)
0.5/2.8
>10
0.46/>10
0.22
0.5/0.9
0.6
(
lM)
h/m CB1: cAMP EC50
(lM)
>10/>10
4.09, <1
0, 0
>10/>10
—/<1
0.4, 2
45
>10/>10
—, <1
0.5, 14
51
logD, LYSA (lg/mL)
Pe (10À6 cm/s), % acc
MAB h %
10
potent at the CB2 receptor maintaining the selectivity versus CB1
and similarly active in the binding assay as derivative 8. The b-arr-
estin value was improved to 1.3 lM while the activity at the Q63R
poorly soluble though highly permeable. Microsomal stability
was greatly improved to human MAB values around 50%. This ini-
tiated a more in depth profiling of these two bi-cyclic systems as
adamantyl derivatives with either dihydo-indole or iso-indoline
moieties were not systematically unstable in human microsomes.
This investigation was complemented by the result that seven-
membered bi-cyclic systemes like tetrahydro-1H-benzo[b]azepine
derivatives were inactive 15. A broader array of dihydro-indole
derivatives was synthesized to establish a more in depth SAR also
thereby establishing the general molecular property profile. Repre-
sentative examples (16–20) are listed in Table 4.
variant was maintained. The methoxy group rendered the com-
pound 9 less lipophilic and more soluble with good permeation
properties. Metabolic stability though was decreased. Further elon-
gation of this vector towards an ethoxy- (compound 10) and also
methoxyethoxy-substituent (compound 11) contributed further
to the preliminary SAR. Compounds 10 and 11 were active in the
nano-molar range at CB2 with excellent selectivity versus CB1.
Binding data in the micro-molar range and a b-arrestin/Q63R
variant value for 11 of 0.9/0.024
l
M, respectively were found.
Compounds which are substituted in the 2-position of the dihy-
dro-indole moiety are generally inactive on the CB2 receptor.
Substituting the 3-position (like in example 16) yielded com-
pounds which were active and selective with binding values in
the micro-molar range. Activity on the Q63R variant was found
in the nano-molar range. Substituting the 4-position had the most
pronounced effect on CB2 agonism (while keeping the selectivity).
Highly potent and selective compounds, like derivative 17 were
identified. Substituting position 5, 6 or 7 led as well to potent
and selective compounds (18–20). Depending on the nature of
the substituent EC50 on the CB2 receptor were found to be below
100 nM, keeping high selectivity towards the CB1 receptor. Binding
Lipophilicity values of around 4 paired with some solubility and
low metabolic stability required improvement of this sub-series.
Our initial hypothesis was substantiated with values from
the shown concept compounds which led to further establishing
an SAR around this series. Ring sizes and nature of the bi-cyclic
amide-substituent was investigated. The results are shown in
Table 3.
Tetrahydro-quinoxaline derivatives, exemplified with com-
pound 12, were potent, selective and showed binding activities
versus CB2 in the micro-molar range. Compound 12 was inactive
in the b-arrestin assay though maintained its nano-molar activity
in the Q63R CB2 variant in the nano-molar range. Molecular prop-
erties of these derivatives in general revealed high lipophilicity,
low solubility and almost no permeability. In human microsomes
compound 12 was only weakly stable. Investigating the ring-size
potential of these bi-cycles showed that dihydro-indole derivative
13 and iso-indoline derivative 14 had very similar profiles. Already
when unsubstituted they were active at the CB2 receptor in the
low nano-molar range with high selectivity for the CB1 receptor.
Binding affinities were in the low micro-molar range as were the
b-arrestin values. Derivatives 13 and 14 were rather lipophilic,
values as well as b-arrestin values varied in the low
lM range.
Q63R variant values were always in the low nM range. Compounds
with such profiles that is, highly potent on CB2 and the Q63R var-
iant with high selectivity versus CB1 and varying values in the b-
arrestin recruitment assay will be used to assess the relevance of
CB2 receptor desensitization for the respective pharmacodynamics
effects in in vivo models. Physicochemical properties for all deriv-
atives were generally very similar, that is, highly lipophilic, poorly
soluble with varying permeation ability. The MAB in human micro-
somes though was pleasingly in the 40–50% range.

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M. Nettekoven et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1177–1181
Table 4
Properties of adamantane compounds 16–20
R
O
N
N
N
N
F
N
F
F
F
No.
16
17
18
19
20
h/m CB2: cAMP EC50
hCB2 Q63R var. EC50
(
l
M)
M)
0.016/0.04
0.036
0.5/4.9
0.9
0.004/0.007
0.011
0.17/1.3
0.29
0.0046/0.009
—
0.2/2.4
0.2
0.015/0.0029
0.049
2.5/2.8
0.75
0.055/0.05
0.081
0.6/>10
1.6
(
l
h/m CB2: Ki (
l
M)
hCB2: b-arrestin EC50
(
l
M)
h/m CB1: cAMP EC50
(lM)
>10/>10
—, <1
0, 0
>10/>10
—, <1
Precip.
46
>10/>6
—, <1
2.3, 4.7
52
>10/>10
—, <1
Precip.
36
>10/>10
—, <1
Precip.
51
logD, LYSA (lg/mL)
Pe (10À6 cm/s), % acc
MAB h %
39
Table 5
Properties of adamantane compounds 21–24
nor
O
OH
NH
N
N
N
O
N
O
O
No.
21
22
23
24
h/m CB2: cAMP EC50
hCB2 Q63R var. EC50
(
l
M)
M)
0.022/0.019
0.027
0.27/0.4
0.7
0.022/0.051
—
0.5/>10
0.9
0.007/0.02
0.011
0.32/2.2
0.16
0.49/1
0.531
—
(
l
h/m CB2: Ki (
l
M)
hCB2: b-arrestin EC50
(
l
M)
2.6
h/m CB1: cAMP EC50
(lM)
>10/>10
4.26, <1
12, 26
37
>10/>10
3.7, <1
1.9, 0.5
55
>10/>10
—, <1
0.1, 5
>10
logD, LYSA (
lg/mL)
2.77, >318
10.3, 25
90
Pe (10À6 cm/s), % acc
MAB h %
37
To further broaden the SAR of the adamantyl-derivatives and
influence the physicochemical properties new derivatives were de-
signed in which the nature of the linkage between the adamantane
and the substituent as well as the nature of any adamantane sub-
stitution was investigated. Representative examples (21–24) are
shown in Table 5.
In summary, a series of highly potent & selective adamantyl de-
rived CB2 agonists was identified in a high-throughput screen.
Conformational analysis of the most active derivatives suggested
the replacement of the anthranilic acid ester moiety with bi-cyclic
systems. This was studied and a SAR around various bi-cycles was
established. Dihydro-indole and isoindoline derived adamantane
derivatives were discovered and a more in depth SAR was estab-
lished. Vectors for further optimization were found, physicochem-
ical properties were significantly improved and room for further
improvement was discovered. This was accompanied by potency
of the compounds on the Q63R variant and varying b-arrestin data
which will support the insight into their relevance for the in vivo
situation.
In adamantane-derivative 21 the amide linkage was changed to a
urea moiety and the compound was still active in the low nano-mo-
lar range on CB2/CB2 Q63R variant and selective versus the CB1
receptor. Binding and b-arrestin values were found in the micro-
molar range. Urea derivatives in general showed an improved abil-
ity to permeate through membranes as indicated by the high Pe va-
lue of 12 for compound 21. Microsomal stability was maintained in
the 40% range. Introduction of a nor-adamantane moiety was well
tolerated and yielded compound 22 with a very similar profile as
in the respective adamantane derivative 13. This offers now a new
and unexplored vector for diversification. Shifting the amide moiety
to the 2-position of the adamantane scaffold furnished very potent
derivatives, exemplified with compound 23. The profile is again
very similar to the one of the isomeric adamantane derivative 13
offering another vector for diversification and differentiation. Sub-
stitution of the adamantane scaffold with solubilizing groups like
hydroxyl yielded derivatives, as exemplified with compound 24,
with preliminarily promising in vitro values. The physicochemical
properties profile was improved considerably that is, in compound
24 the lipophilicity was reduced by a factor 10–100 and the solubil-
ity was greatly enhanced. This combined with high permeation and
high human microsomal bioavailability potential opened up new,
unexplored and promising vectors for further derivatisation in the
optimization phase of this adamantane hit cluster.
Acknowledgments
The technical assistance of Astride Schnöbelen, Tanja Minz and
Anja Osterwald is greatly acknowledged.
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exemplified with the preparation procedure of 8:
(0.18 mmol) 1,2,3,4-tetrahydroquinoline, 37.3 mg
A
mixture of 24.5 mg
(0.188 mmol)
19. (a) cAMP assays were performed using CHO cells expressing human CB1 and
human CB2 receptors variants as described in Ullmer et al. (Br. J. Pharmacol.
2012, 167, 1448).; (b) b-Arrestin recruitment assays were performed according
to the instructions of the manufacturer (DiscoveRx, Fremont, CA).; (c)
Radioligand binding assays were performed with membranes prepared from
cells expressing human or mouse CB2 receptors using 3H-CP55940 (Perkin
Elmer) as radioligand. EC50’s or Ki were calculated from a single experiment
using triplicates of 10 different concentration of compound.
1-adamatanecarbonyl chloride and 44.4 mg (0.34 mmol) DIPEA in 1 mL DCM
was shaken at room temperature for 4 h. The mixture was evaporated,
dissolved in DMF and subjected to column chromatography on reversed phase
eluting with a gradient formed from acetonitrile, water and formic acid to yield
after evaporation of the product containing fractions 34.9 mg (64%) of the title
compound as white solid. MS (m/e): 296.3 (MH+).