Aryl 1,4-diazepane compounds as potent and selective CB2 agonists: Optimization of drug-like properties and target independent parameters

Article abstract of DOI:10.1016/j.bmcl.2011.05.068

A high throughput screening campaign identified aryl 1,4-diazepane compounds as potent and selective cannabinoid receptor 2 agonists as compared to cannabinoid receptor 1. This class of compounds suffered from poor drug-like parameters as well as low micr

Full text of DOI:10.1016/j.bmcl.2011.05.068

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4276–4280
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Aryl 1,4-diazepane compounds as potent and selective CB2 agonists:
Optimization of drug-like properties and target independent parameters
Renée Zindell a, , Edward R. Walker d, John Scott d, Patricia Amouzegh d, Lifen Wu a, Monika Ermann d,
⇑
David Thomson a, Micheal B. Fisher c, Cody Lee Fullenwider c, Heather Grbic c, Paul Kaplita c, Brian Linehan c,
Mita Patel c, Monica Patel c, Sabine Löbbe e, Svenja Block e, Claudia Albrecht e, Mark J. Gemkow e,
Daw-Tsun Shih b, Doris Riether a
a Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877-0368, USA
b Department of Inflammation and Immunology, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877-0368, USA
c Department of Drug Discovery Support, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877-0368, USA
d Evotec (UK) Ltd., 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, United Kingdom
e Evotec AG, Schnackenburgallee 114, 22525 Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A high throughput screening campaign identified aryl 1,4-diazepane compounds as potent and selective
cannabinoid receptor 2 agonists as compared to cannabinoid receptor 1. This class of compounds suffered
from poor drug-like parameters as well as low microsomal stability and poor solubility. Structure–
activity relationships are described with a focus on improving the drug-like parameters resulting in
compounds with improved solubility and permeability.
Received 14 April 2011
Revised 17 May 2011
Accepted 18 May 2011
Available online 27 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid receptor
CB2
1,4-Diazepane
Drug-like parameters
Permeability
The therapeutic use of cannabis has been known for several
centuries. However, its widespread use and acceptance has been
hampered by the psychoactive effects it elicits since they have
the potential for abuse. More recently, the identification of
CB2 selective agonists have been reported to be efficacious in
both in vitro and in vivo models of inflammation8–10 as well as
inflammatory and neuropathic pain.11–14 Some of these com-
pounds, shown in Figure 1, are GW40583315 and GW842166,16
with the latter having entered clinical trials for the treatment of
D
9-tetrahydrocannabinol as the active constituent of cannabis
allowed for the identification of the receptors mediating its effects.
These are G protein-coupled receptors cannabinoid receptor 1
(CB1) and 2 (CB2).1–3 The CB2 receptor is implicated in the
anti-inflammatory activity of cannabis as it is expressed primarily
in peripheral immune cells such as B-cells, monocytes, macro-
phages, and in organs such as the spleen, pancreas, thymus, lung,
and tonsil.4,5 The CB1 receptor is expressed primarily in the central
nervous system and is believed to be responsible for the undesir-
able effects such as ataxia, hypothermia, and euphoria. The highest
density of CB1 is found in the basal ganglia, cerebellum, and
hippocampus.6 CB2 shares 44% homology with CB1 which, coupled
with differences in receptor densities in various tissues, offers the
possibility to separate the immune-related effects of cannabinoids
from the psychoactive effects typically associated with CB1.7
O
O
CF3
N
Cl
N
H
N
O
Cl
N
N
H
N
O
Cl
O
Cl
GW842166
GW405833
O
O
S
O
N
N
Cl
N
O
N
H
Cl
A
B
⇑
Corresponding author. Tel.: +1 203 798 5419; fax: +1 203 791 6072.
E-mail address: renee.zindell@boehringer-ingelheim.com (R. Zindell).
Figure 1. Examples of CB2 selective agonists and modulators reported in the
literature.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.068

R. Zindell et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4276–4280
4277
inflammatory pain. The aryl sulfonamide A17 and morpholine scaf-
fold B18 are recent additions from our research. Herein we report
the identification and optimization of a class of diazepanes origi-
nating from a high throughput screen as potent and selective
CB2 agonists.
compounds 5–7 suggested that 5,6-fused ring systems could be
employed to improve the CB2 potency while maintaining similar
selectivity as compared to substituted five-membered heteroaro-
matic rings. In summary, these efforts resulted in a 300-fold range
of CB2 potency and 17- to >130-fold ranges in selectivity can be
achieved by manipulation of the R1 fragment of the diazepane.
Our attention next turned to R2 of the diazepane core again with
the goal to reduce the MW and c log P. We chose to keep the
isoxazole R1 substituent found in compound 2 due to the potency
it offered as we developed the SAR. The synthesis of these analogs
is depicted in Scheme 2. Commercially available Boc-protected dia-
zepane was converted to the carbamoyl chloride in situ employing
triphosgene followed by urea bond formation under basic condi-
tions. Subsequent deprotection of the Boc group afforded the
amine in quantitative yield which then underwent aromatic sub-
stitution to afford final compound. The aromatic substitution was
accomplished on either pyridyl fluorides or chlorides with various
bases and heating.
The high throughput screen employed a cellular assay which
measured the ability of a CB2 agonist to activate Gai, which in turn
inhibits cyclic adenosine monophosphate (cAMP) production. The
assay was performed in CB2 over-expressing CHO cells stimulated
with forskolin.19,20 Selectivity over CB1 was determined in a simi-
lar assay which used CB1 over-expressing CHO cells. These two as-
says were used to drive the structure–activity relationship
determinations described in this letter. Compound 1 was identified
in the manner described and displays CB2 cAMP EC50 of 154 nM
with 92% efficacy and greater than 130-fold selectivity over CB1
(Fig. 2). The compound suffers from a high human in vitro clear-
ance of >75% QH in human liver microsomes as well as poor solu-
bility of <0.1 l
g/mL at both pH 4.5 and 7.4.21 Additionally, the
compound has a relatively high molecular weight of almost 470
amu and c log P of 5.2. These measures of drug-likeness offered
room for improvement when considering Lipinski’s Rule of Five.
Initially, R1 of the diazepane core was explored to determine the
impact on potency and selectivity. We began our efforts by replac-
ing the dichlorophenyl with heteroaromatic rings which offer a de-
crease in c log P while offering the same pi-stacking interaction
potential. The synthesis of these analogs is depicted in Scheme
1.22 Commercially available 1-[3-chloro-5-(trifluoromethyl)pyri-
din-2-yl]-1,4-diazepane was acylated with the corresponding iso-
cyanate to afford compounds 2–4, and arylated under basic
conditions to afford analogs 5–7.23
Compound 2 carried the same R2 substitution, namely 2-chloro-
4-trifluoromethyl-substituted pyridine as the initial hit differing in
the R1 as a heteroaromatic which reduced molecular weight to 445
amu and c log P of 4 while improving potency. Compounds 8–10
were synthesized to understand the requirements for substituents
on the aromatic ring. The unsubstituted pyridyl 8 had no CB2 ago-
nist activity when measured up to 20
Incorporation of the chlorine in the ortho-position 9 built in agonist
activity without introducing measurable CB1 activity up to 20 M.
lM in the cellular assay.
l
Similarly CB2 agonist activity could be built into the pyridine with
para-substitution as demonstrated with the trifluoromethyl substi-
tuent 10 which offered more selectivity over CB1 as compared to 2.
Based on these four examples one may conclude the potency of the
heteroaromatic is driven by the substituent in the 4-position while
substituent in the 2-position contributes to the selectivity of the
molecule. Replacement of the triflouromethyl group with a more
polar cyano group 11 offered similar potency as compared to 10
with >1000-fold selectivity. Regioisomers of 11 were synthesized
depicted by compounds 12–14 and resulted in loss of CB2 potency
reinforcing the regioisomeric preference of the receptor. These re-
sults demonstrate that R2 modifications to the core offer very po-
tent compounds with no measurable activity against the CB1
Replacement of the 3,4-dichlorophenyl R1 residue with 3-tert-
butyl-5-aminoisoxazole in compound 2 resulted in a 30-fold
improvement in potency and a decrease in selectivity. This sug-
gested the receptor was tolerant of heteroaromatic groups and
was expanded upon in compounds 3 and 4. These compounds
maintain the lipophilic tert-butyl group in the same position and
vary the arrangement of heteroatoms in the aromatic ring. The im-
proved potency is maintained for both compounds, however their
selectivity over CB1 vary considerably. The thiazole maintains
selectivity similar to that measured for compound 2 however the
pyrazole 3 shows a significant improvement in this parameter.
One may hypothesize substituents in the 2-position with respect
to the core linkage have the potential to build in selectivity over
CB1. Heteroaromatic groups directly attached to the diazepane
core were also well tolerated as exemplified by compounds 5–7.
This type of amide bioisostere led to CB2 agonists for which CB1
selectivity was significantly eroded. Further comparison of
receptor up to 20 lM. They also highlight the importance of substi-
tuent placement for potency and selectivity. Compound 11 had a
decreased MW of 368 amu and c log P of 1.9, however, upon fur-
ther profiling revealed it had an improved yet moderate solubility
of 13 lg/mL at pH 7.4 and high human in vitro clearance of >75%
QH. In fact compounds exemplified, for which in vitro clearance
H
N
O
O
Cl
F
F
Cl
N
O
(i-iii)
N
O
N
N
O
Cl
F
N
N
H
N
N
H
N
H
iv
1
R2
Figure 2. Hit identified from a high throughput screen.
N
O
N
N
N
O
F
F
H
F
F
R1NCO
F
F
Cl
Cl
N
N
N
N
or
1
N
R1-X
R
Scheme 2. Reagents and conditions: (i) 3-Amino-5-tertbutylisoxazole, triphosgene,
Hunig’s Base, dichloromethane, room temperature, 100% yield; (ii) 4 N HCl/
dioxanes, dichloromethane, 100% yield; (iii) (a) NaHMDS, R2X = ArX, dimethylsulf-
oxide, 60 °C, 3–100% yield, X = F, Cl; (b) RCl, TEA, NMP, 80 °C, 17–20% yield; (c) RCl,
K2CO3, Cu, NMP, 80 °C, 3–17% yield.
N
H
Scheme 1. Reagents and conditions: R1NCO, Hunig’s Base, dichloromethane, room
temperature, 7–59% yield or R1X = ArX, NaHMDS, dimethylsulfoxide, 60 °C, 18 h,
70–100% yield wherein X = Cl, F.

4278
R. Zindell et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4276–4280
was obtained (compounds 2, 10, and 11), demonstrated similarly
high human in vitro clearance.
shown in Table 1, compounds 7 and 16–19. The syntheses de-
scribed in Schemes 1 and 2 were adapted to access these mole-
cules. Compound 7 was chosen as the starting point due to the
exquisite potency it offered. Replacement of the aromatic with a
small cycloalkyl substituent linked to the diazepane core via an
amide bond shown in compound 16 offered an improvement in
solubility and selectivity without much loss in potency as com-
pared to compound 7. Expanding upon this cycloalkyl, the tetrahy-
dropyran (THP) was synthesized as compound 17. Incorporation of
this heteroalkyl offered little change to the CB2 potency but greatly
Our concurrent efforts in the diazepane scaffold24 allowed frag-
ments such as the THP found in Figure 1 GW842166 and demon-
strated aliphatic amides and ureas have
a better aqueous
solubility profile as well as low clearance. Compound 15 shown
in Table 1 is one of the best compounds described in the amide/
urea type of molecules.
The aliphatic amides were adapted from the amide/urea series
targeting improvements in the solubility and clearance and are
Table 1
Diazepane SAR
R2
N
1
N
R
Compound
R2
R1
CB2 cAMP
EC50 (nM)
Efficacy (%)
CB1 EC50
CB2 EC50
/
HLM
(%QH)
Solubility pH 7.4
g/mL)
Caco-2 AB/BA
Molecular
weight (amu)
c log P
(
l
(cmsÀ1 Â 10À6
)
Cl
Cl
O
N
Cl
*
*
*
*
*
*
*
O
1
F
154
5
92
>130
80
>75
>75
>75
>75
>75
<0.1
0.6
10
1
468
446
459
462
440
447
453
5.49
F
F
F
F
F
F
F
N
N
H
*
*
F
Cl
O
N
N
H
2
3
4
5
6
7
F
106
100
99
4.04
3.71
4.74
4.81
5.72
6.13
N
F
Cl
O
O
N
S
N
H
*
*
F
8
800
67
N
F
Cl
N
N
H
F
30
N
F
Cl
N
S
N
*
*
F
30
95
17
N
F
Cl
N
S
F
180
0.51
87
38
N
Cl
F
Cl
O
*
N
F
107
33
0.1
N
F
*
*
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
8
9
>20,000
280
5
343
378
411
368
2.27
3.07
3.3
N
H
*
*
*
*
*
Cl
70
>71
N
H
N
*
F
F
N
N
10
11
98
253
>75
>75
0.4
13
N
H
F
*
20
103
>1000
1.9
N
H
N
*
N
N
H
12
13
>20,000
200
368
368
2.1
1.9
N
N
O
O
N
*
93
>100
N
H
*
N

R. Zindell et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4276–4280
4279
Table 1 (continued)
Compound
R2
R1
CB2 cAMP
EC50 (nM)
Efficacy (%)
CB1 EC50
CB2 EC50
/
HLM
(%QH)
Solubility pH 7.4
g/mL)
Caco-2 AB/BA
Molecular
weight (amu)
c log P
(
l
(cmsÀ1 Â 10À6
)
N
O
O
O
N
N
N
H
14
>20,000
368
1.9
*
*
N
O
O
15
16
67
2
104
100
>299
60
42
>96
26
7/42
378
384
0.14
4.69
*
N
H
O
*
*
O
O
N
>75
*
O
O
O
*
*
*
N
17
18
1
6
105
105
1770
753
>75
>75
>96
49/64
385
386
2.23
3.96
*
*
O
O
O
N
N
>113
O
O
N
O
N
*
19
6
107
683
>75
>75
>103
>93
435
2.99
S
O
O
S
*
*
Cl
20
21
62
99
>322
>133
62/71
380
380
1.82
1.82
N
*
*
O
O
O
O
S
150
100
N
Cl
S
*
*
N
22
>20,000
380
1.82
*
*
O
O
Cl
S
O
O
N
N
23
24
60
60
98
>333
>323
>75
>75
>96
>93
67/61
53/70
372
398
0.91
1.01
S
Cl
*
103
N
*
CF3
O
enhanced the selectivity profile while significantly improving the
aqueous solubility of the molecule. Encouraged by this result, the
morpholine 18 and thiomorpholine dioxide 19 were both targeted
resulting in similar profiles in terms of CB2 potency and solubility
with slight decrease in the CB1 selectivity as compared to 17.
While compound 17 demonstrates an improvement in aqueous
solubility as compared to compound 7, the human in vitro clear-
ance shows the same high rate as the other compounds reported
herein. The optimization effort described for the amide/urea diaze-
panes leading to compounds such as 15 focused on reducing the
in vitro human clearance by decreasing the lipophilicity. 15 has
an in vitro human clearance of <24% QH and a low c log P of 0.14.
However, subsequent profiling of compound 15 revealed bioavail-
ability of 3% in cynomolgous monkey which was hypothesized to
be a result of intestinal flux as suggested by the efflux ratio of 6
measured in caco-2 cells. (AB flux: 7 cm/s  10À6 and BA flux: 42
(cm/s  10À6).25
taining the THP group for its potency and solubilizing properties.
We also targeted compounds with reduced c log P in an effort to
manage the in vitro human clearance. Incorporation of the benzo-
thiazole 20 results in 60-fold loss of potency against the CB2 recep-
tor as compared to 17. This potency loss is significantly less than
the 360-fold loss seen in Table 1 between compounds 6 and 7. This
suggests the THP is much more tolerant of changes to R1 than the
substituted pyridine. The solubility and permeability of the mole-
cule were also maintained with this substitution. Expanding the
SAR to benzothiazole regioisomers 20 and 22 eroded or eliminated
the CB2 potency as measured to 20 lM. The ability of the THP ver-
sus substituted pyridine to tolerate right side variations is further
tested with compound 23. Replacement of the benzoxazole 7 and
16 with a thiadiazole 5 and 23 resulted in a 60-fold loss of CB2 po-
tency, irrespective of R2. The effect against CB1 is markedly im-
proved from 17- to >333-fold with the THP group. Compound 24
is another monocyclic heteroaromatic group which maintained po-
tency, selectivity, aqueous solubility, and showed improvement of
the permeability. Taken together, these molecules demonstrate po-
tent, selective CB2 agonists with good permeability and solubility
can be achieved. Further these molecules have molecular weights
below 400 amu and c log P in the range of 1–2 as compared to
the hit which has a molecular weight of almost 500 amu and a
c log P greater than 5. While the calculated drug-like parameters,
solubility and permeability were optimized, improvement to the
The benzoxazole 17 as compared to isoxazole 15 showed
improvement in this ratio as measured by flux across Caco-2 mon-
olayers and therefore offered an opportunity to address this issue.
We hypothesized the change in ratio may be attributed to the
change in hydrogen bonding pattern between the amide and amide
bond isostere.26 The amide carries a hydrogen bond donor whereas
the isostere does not. Based on this observation, we examined aro-
matic moieties directly attached to the diazepane core while main-

4280
R. Zindell et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4276–4280
18. Zindell, R.; Riether, D.; Bosanac, T.; Berry, A.; Gemkow, M. J.; Ebneth, A.; Löbbe,
S.; Raymond, E.; Thome, D.; Shih, D.-T.; Thomson, D. Bioorg. Med. Chem. Lett.
2009, 19, 1604.
19. CB2 and CB1 cAMP assays: CHO cells expressing human CB2 or CB1
(Euroscreen) were plated at a density of 5000 cells per well in 384-well
plates and incubated overnight at 37 °C. After removing the media, the cells
were treated with test compounds diluted in stimulation buffer containing
human in vitro clearance remained elusive despite reduction of the
lipophilicity of the molecules. We concluded that with this class of
molecules the in vitro human clearance is very challenging to ob-
tain without negatively impacting other parameters.
In summary, from the initial high throughput screen hit, modi-
fications to each side of the core were well tolerated and demon-
strate CB2 potency and selectivity profiles can be attenuated to
deliver desirable biological profiles. Expanding upon this work,
aqueous solubility and permeability were built into the molecules
via the THP moiety and amide bond bioisosteres, respectively.
While building these properties into the molecule the molecular
weight and lipophilicity were lowered to a more drug-like range.
Finally, molecules which maintain good CB2 agonist activity and
lack measurable CB1 agonist activity were achieved. These mole-
cules should lack the undesired effects associated with the CB1
receptor while maintaining analgesic and immunomodulatory ef-
fects attributed to CB2 agonists. The high in vitro human clearance
of the compounds precluded interest in obtaining their in vivo
characterization. These compounds offer a good starting point for
further optimization which will be reported in due course.
1 mM IBMX, 0.25% BSA and 10 lM forskolin. The assay was incubated for
30 min at 37 °C. Cells were lysed and the cAMP concentration was measured
using DiscoveRx XS+ cAMP kit, following the manufacturer’s protocol. The
maximal amount of cAMP produced by forskolin compared to the level of
cAMP inhibited by 200 nM CP-55940 (Tocris; catalog number 0949) is defined
as 100%. The EC50 value of each test compound was determined as the
concentration at which 50% of the forskolin-stimulated cAMP synthesis was
inhibited using a four-parameter logistic model.
20. Reproducibility of the cAMP assays is assessed using the control compound CP-
55940 (Tocris; catalog number 0949) which is tested twice on every assay
plate to rule out any plate artifacts. Efficacy at CB1 and CB2 is expressed as a
percentage relative to the efficacy of CP-55940. Each compound is tested in
triplicate at least three times with individual dilutions from the stock. The
results reported in the table are the mean values of the measurements and
individual values for the compounds do not differ by more than a factor of
three from the mean.
21. Solubility assay; Solubility of compounds is measured at pH 4.5 and pH 7.4
buffers. Six microliter of a 10 mM DMSO stock solution is spiked into pH 4.5
and 7.4 buffers targeting 50–200 lM final concentrations in buffers in
duplicate deep well plates. The DMSO content is 0.5%. The samples are
incubated for 16–18 h, filtered and analyzed using spectrophotometer. The
UV spectra of samples and reference are scanned from 230 to 500 nm.
Solubility is measured taking the ratio of area under the curve of reference
to sample.
Acknowledgment
The authors would like to thank Dr. Paige Mahaney for her time
and effort in proof reading the manuscript.
HLM assay: Test compounds were incubated in Duplicate Matrix
MultiScreen mintubes (Matrix Technologies, Hudson, NH) with liver
microsomes (Xenotech, Lenexa, KS). Each assay is performed in 50 mM
potassium phosphate buffer, pH 7.4, and 2.5 mM NADPH. Compounds were
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l
L
a
filtered through wells in 0.25 mm glass fiber filter plates by centrifugation at
3000 rpm for 5 min. Sample extracts were analyzed by LC–MS–MS to
determine parent compound levels. Percent loss of parent compound was
calculated from the peak area at each time point to determine the half-life
for test compounds (T1/2, min) and clearance (T1/2 expressed as percent
hepatic blood flow, %QH).
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25. Caco-2 assay description: Caco-2 cells (1.67 Â 105 cells/1 cm2 area) are seeded
on filter inserts (BD FalconÒ HTS Multiwell Insert system, 1.0
lm pore size, PET
filters) and cultured (DMEM) for 16–18 days. Compounds are dissolved in
appropriate solvent (like DMSO) into 10 mM stock solutions. Stock solutions
are diluted with Hanks’Balanced Salt Solution (GibcoÒ 14025-092)
supplemented with 10 mM HEPES, 20 mM glucose, (pH 7.2) to prepare the
transport solutions (10 lM compound, final DMSO 6 0.5%). The transport
solution is applied to the apical or basolateral donor side for measuring A–B or
B–A permeability (3 filter replicates), respectively. The receiver side contains
buffer containing equal solvent concentrations. Samples are collected at the
start and end of experiment from the donor and at various time intervals for up
to 1.5 h also from the receiver side for concentration measurement by
HPLCMS/MS. Sampled receiver volumes are replaced with fresh receiver
solution.
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Gemkow, M. J.; Kahrs, A. F.; Ebneth, A.; Löbbe, S.; Thome, D.; O’Shea, K.; Dinallo,
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