Functionalized 6-(Piperidin-1-yl)-8,9-Diphenyl Purines as Peripherally Restricted Inverse Agonists of the CB1 Receptor

Article abstract of DOI:10.1021/acs.jmedchem.9b00727

Peripherally restricted CB1 receptor antagonists may be useful in treating metabolic syndrome, diabetes, liver diseases, and gastrointestinal disorders. Clinical development of the centrally acting CB1 inverse agonist otenabant (1) was halted due to its p

Full text of DOI:10.1021/acs.jmedchem.9b00727

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Article
Functionalized 6-(Piperidin-1-yl)-8,9-Diphenyl Purines as
Peripherally Restricted Inverse Agonists of the CB1 Receptor
George Amato, Amruta Manke, Robert W. Wiethe, Vineetha Vasukuttan, Rodney
W. Snyder, Yun Lan Yueh, Ann M. Decker, Scott P Runyon, and Rangan Maitra
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00727 • Publication Date (Web): 11 Jun 2019
Downloaded from http://pubs.acs.org on June 11, 2019
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Functionalized 6-(Piperidin-1-yl)-8,9-Diphenyl Purines as
Peripherally Restricted Inverse Agonists of the CB1 Receptor
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#
#
George Amato , Amruta Manke , Robert Wiethe, Vineetha Vasukuttan, Rodney Snyder, Yun Lan
Yueh, Ann Decker, Scott Runyon and Rangan Maitra*
Discovery Science and Technology, RTI International, 3040 Cornwallis Rd., Research Triangle Park, NC
27709-2194, USA
* Corresponding author
#
Equal contributors
Key Words: CB1, cannabinoid, peripheral, antagonist, otenabant, purine, CB2, MDCK, blood
brain barrier
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ABSTRACT
Peripherally restricted CB1 receptor antagonists may be useful in treating metabolic syndrome,
diabetes, liver diseases and gastrointestinal disorders. Clinical development of the centrally acting CB1
inverse agonist otenabant (1) was halted due to its potential of producing adverse effects. SAR studies of 1
are reported herein with the objective of producing peripherally restricted analogs. Crystal structures of
hCB1 and docking studies with 1 indicate that the piperidine group could be functionalized at the 4-position
to access a binding pocket that can accommodate both polar and nonpolar groups. The piperidine is studied
as a linker, functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a urea connector. Orally
bioavailable and peripherally selective compounds have been produced that are potent inverse agonists of
hCB1 with exceptional selectivity for hCB1 over hCB2. Compound 38 blocked alcohol-induced liver
steatosis in mice and has good ADME properties for further development.
0
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1
. Introduction
The endocannabinoid system has many important physiological functions. There are two known
cannabinoid receptors – CB1 and CB2. These GPCRs are expressed in many different tissues. The CB1
receptor is highly expressed in the central nervous system (CNS) and in metabolically important tissues
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
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9
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1
including liver, skeletal muscles, adipocytes, and pancreas. In contrast, CB2 receptor expression is mainly
confined to immune cells.2, 3 Selective antagonism of peripheral CB1 receptors has potential therapeutic
applications in treating diabetes, metabolic syndrome, dyslipidemias and liver diseases such as steatosis or
4
-9
fatty liver disease. Antagonism of CB1 receptors in the CNS, however, is linked to psychiatric disorders,
precipitating the withdrawal of CNS-penetrating compounds such as otenabant (1) and rimonabant (2) from
clinical development.10 Presently, efforts are underway to develop compounds that selectively antagonize
8, 11-14
peripheral CB1 receptors.
These compounds by virtue of being CNS-sparing are not expected to
produce adverse effects seen with first-generation non tissue selective antagonists. Other approaches that
may not require restriction from the CNS include development of neutral orthosteric antagonists and
negative allosteric modulators.1
5, 16
O
N
Me
NH
N
N
Cl
O
N
N
N
N
N
NH2
Cl
Cl
NH
Cl
Et
Cl
1
, Otenabant
2, Rimonabant
(SR141716A)
(CP-945,598)
Figure 1. Examples of clinical CNS penetrating CB1 inverse agonists.
Identifying compounds that are both peripherally selective and orally bioavailable is possible by
carefully managing physiochemical properties. Analyses of past research and advanced compounds therein
suggest that a topological polar surface area (TPSA) of 80-140 Å, a molecular weight of 450-600 Da and
1
-3 hydrogen bond donors improve the likelihood of identifying such compounds.17, 18 To improve the
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potential for good drug-like properties, it is also desirable to have a cLogP of less than 5. Compounds with
a high cLogP may have poor aqueous solubility, which in turn can negatively impact absorption following
an oral dose. Compound 1 has several properties that in theory could restrict this compound to the periphery
1
9
including a TPSA of 102 Å, a cLogP of 4.4, 3 hydrogen bond donors and a molecular weight of >500 Da.
Compound 1 also has a polar and mildly basic 6-piperidinyl purine core that can aid in oral absorption.
0
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9
0
2
0
However, 1 can penetrate into the CNS due to intramolecular hydrogen bonding. It was hypothesized that
analogs of 1 with refined properties could lead to compounds that have limited penetration into the CNS.
We began such an effort with an investigation of isomeric replacements for the 4-aminopiperidine
group 21 and employment of the piperazine group as a replacement.22, 23 Early studies resulted in compounds
3-5, possessing excellent hCB1 potency (Ke ~ 20 nM) and good selectivity (>50 fold) against hCB2 (Figure
2
).21, 22, 24 These compounds are peripherally selective and orally bioavailable, but their ADME properties
were not optimized. Recent docking studies of 1 with the hCB1 crystal structure at the orthosteric binding
site indicate that functionalization of the piperidine group at the 4-position could result in access to a binding
pocket near the extracellular surface of the membrane.22, 25, 26 This binding pocket may accommodate polar
as well as nonpolar groups and differs significantly from hCB2.27, 28 By targeting this region, we believe it
is possible to identify compounds with better physical properties that have good hCB1 potency and
excellent selectivity versus hCB2.
O
Me
O
S
O
N
N
n-Bu
N
NH
N
N
N
N
NH
N
N
N
Cl
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
K hCB1: 8 nM
K hCB1: 19 nM
i
K hCB1: 6.2 nM
i
i
5
K hCB2: >10,000 nM
3
4
K hCB2: 1050 nM
i
K hCB2: 950 nM
i
i
Figure 2. Early purine based peripherally selective CB1 inverse agonists.
2.
Results and Discussion
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.1
Compound Design and Synthesis
N
R'
N
N
L R
L = NHCONH, NHCONHSO2,
R = Alkyl, Heteroalkyl, Aryl,
Heteroaryl
R' = H, Me
Ar = 4-ClPh, Heteroaryl
N
N
Ar
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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5
6
0
1
2
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8
9
0
1
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8
9
0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
6
Figure 3. General strategy for SAR studies
In this report, we disclose our findings in studying functionalization of the piperidine linker at the
4
-position with a urea or a sulfonyl urea connector (6, Figure 3). We focused on alkyl, heteroalkyl, aryl
and heteroaryl groups that are more likely to have good hCB1 potency. We also report on replacing the 4-
chlorophenyl group of the 6-purine position with heterocycles, to increase TPSA and reduce cLogP. Finally,
changing the R’ from hydrogen to methyl was looked at to adjust in vitro ADME properties and favor
higher hCB1 potency. Combined, these changes allowed us to design a variety of compounds with desirable
calculated properties that are favorable for both peripheral selectivity and oral absorption.
To prepare most of the targeted compounds, we used the 4-aminopiperidine 8a (Scheme 1) as a
key intermediate, the preparation of which was previously described.21 The 4-amino-4-methylpiperidine
analog (9) and analogs of 8a with replacements for the 4-chlorophenyl group of the purine ring were
prepared in a similar way as shown in Scheme 1 and detailed in the experimental section.
N
N
N
Me
NH2
N
Cl
N
N
NH2
N
N
N
a
N
N
Cl
NH2
N
b
N
N
N
N
Ar
Ar
OR
Cl
Cl
Cl
Cl
Cl
7(a-j)
8(a-j)
9
o
Scheme 1. Reagents and conditions: (a) (1) ArNH , 6 N HCl, n-BuOH, 80 C; (2) 2-ClPhCHO, 15%
2
o
FeCl /silica gel, dioxane, 95 C, then DDQ, CH Cl , rt; (b) (1) 4-N-Boc-aminopiperidine or 4-N-Boc-
3
2
2
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1
1
2
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2
2
2
2
3
3
3
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4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
o
o
amino-4-methylpiperidine, K CO , NMP, 80 C; (2) 6 N HCl, EtOH, 50 C. Ar = 4-chlorophenyl (7a, 8a),
2
3
4
-fluorophenyl (7b, 8b), 6-(trifluoromethyl)pyridin-3-yl (7c, 8c), 6-(difluoromethoxy)pyridin-3-yl (7d,
d).
8
0
1
2
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8
9
0
1
2
3
4
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7
8
9
0
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9
0
1
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3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
From intermediate 8a we prepared a variety of ureas using standard procedures as shown in Scheme
and provided in the experimental section. Intermediate 9 and analogs with a replacement for the 4-
2
chlorophenyl group of the purine ring were functionalized in the same way.
N
O
N
N
O
H
H
H
N
N
N
NRR'
N
N
NH2
N
N
N
2
N SO R
N
N
N
N
N
N
a or b
or c
d
Cl
Cl
Cl
Cl
Cl
Cl
8a
Scheme 2. Reagents and conditions: (a) (1) triphosgene, NaHCO , CH Cl , rt; (2) RR’NH, NEt , THF, rt;
3
2
2
3
(
b) (1) p-F-PhO CCl, NEt , DCE, rt; (2) RR’NH, dioxane, rt; (c) RNCO, NEt , THF, rt; (d) (1) p-NO -
2
3
3
2
PhO CCl, NEt , DCE, rt; (2) RSO NH , NaHMDS, THF, rt.
2
3
2
2
2
.2.
Pharmacological Characterization
All target compounds were evaluated in a FLIPR-based calcium mobilization assay for hCB1
activity (Tables 1-3) as previously reported.21, 29 In general, compounds displaying apparent antagonist
dissociation equilibrium constant (Ke) < 50 nM were further tested for affinity to hCB receptors using
3
radioligand displacement of [ H]CP55940 in purified membrane fractions overexpressing either hCB1 or
hCB2. Exceptions were made for certain interesting compounds. A selection of potent and selective
compounds were tested for potential peripheral selectivity by calculating % apical (A) to basal (B)
permeability in a MDCK-mdr1 monolayer permeability assay, which is predictive of brain penetration.30
Lower values are predictive of less CNS penetration. To assess metabolic stability, some compounds were
advanced to a human liver microsome (HLM) stability assay, determining half-life (HL) and clearance (Cl).
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Ureas are good linkers for compounds targeting peripheral selectivity, as they contain one or two
strong hydrogen bond donors and contribute three heteroatoms to assist in achieving higher TPSA values.
Table 1 contains in vitro data for a set of alkyl and aryl ureas. We previously reported that the n-propyl
urea 10 and other ureas with small aliphatic groups have hCB1 activity, but the potency and selectivity
versus hCB2 need further optimization.24 To take advantage of the binding site to which the piperidine ring
is neighboring, we tried larger groups that may result in an increase in both potency and selectivity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. In Vitro Data for CB1 Antagonists – Ureas
O
N
H
N
N
N
R
N
N
Cl
Cl
Ke
hCB1
nM+
Ki
hCB1
(nM+
Ki
hCB2
(nM+
MDCK-
mdr1
HLM
HL (mins);
Cl
Selectivity
Ki
CB2/CB1
#
1
R
(
A to B
a
a
b
SEM)
SEM)
SEM)
(%)
(µL/min/mg)
Otenabantc
n-PrNH
0.2
0.7
7700
11000
5
ND
ND
7
ND
ND
ND
ND
ND
ND
1
0d
130
5±3
28
150
1
1
n-PrNMe
0.9±0.2
1.1±0.6
0.8±0.3
ND
630±210
700
2500
750
ND
12
n-Pr(CF CH )N
1.2±0.6
6±2
2800±600
600±20
ND
ND
4
3
2
1
1
3
4
i-BuNMe
t-BuCH NH
270±130
ND
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
5
6
MeOCH CH NMe
13±7
0.26±0.03 2300±200
8800
ND
130
ND
ND
ND
2
2
N
190±110
7±4
ND
ND
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
N
1.2±0.8
400±36
330
ND
ND
1
1
8
9
O
N
340±60
25±14
ND
3±1
ND
ND
ND
1
ND
ND
c-PenNH
>10000
>3000
HL: 3
Cl: 381
NH
CF3
2
0
12±8
6.3±0.5
>10000
>1500
0.1
HL: 21
Cl: 58
21
c-HexNH
35±10
79±2
1.4±0.8
18±1
4600±600
>10000
3300
>500
0.3
F
F
HL: 163
Cl: 8
2
2
2
3
NH
ND
Me
NH
HL: 7
Cl: 178
F
F
0.4±0.3
63±37
5±3
7500±400
3200
1500
170
ND
0.2
2
2
4
5
c-PenCH NH
19±8
ND
2
±
1000
7
600
HL: 25
Cl: 50
c-HexCH NH
5±3
0.9±0
8400
384
0.1
2
±
4200
F
F
HL: 30
Cl: 42
26
CH NH
28±15
26±20
>10000
ND
2
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MeN
CH NH
ND
ND
ND
92
ND
ND
ND
2
±12000
F
HL: 150
Cl: 8
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
71±7
2.4±0.2
220±100
CH NH
2
HL: 123
Cl: 10
2
3
9
0
F
CH NH
19±6
30±7
3±1
7±0
>10000
>10000
>3000
>1400
ND
0.2
2
HL: 115
Cl: 11
PhNH
N
31
150±50
ND
ND
ND
ND
ND
NH
HL: 97
Cl: 13
3
2
2-F-PhNH
2,4-DiF-PhNH
3,4-DiF-PhNH
4-F-PhNH
58±7
4±1
4±1
14±4
ND
>10000
>10000
ND
>2500
>700
ND
0.1
0.1
ND
1.3
HL: 397
Cl: 3
33
3
3
4
5
290±80
78±20
ND
ND
22±9
6500±600
300
CF3
N
36
160±60
ND
ND
ND
ND
ND
NH
N
HL: 39
Cl: 32
3
3
7
8
37±16
26±9
51±32
22±3
>10000
>10000
>196
>263
ND
0.1
F3C
NH
N
HL: 423
Cl: 3
F2HCO
NH
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
HN
N
3
9
NH
270±60
ND
ND
ND
ND
ND
F3C
2
8000
8000
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
0
1
NH
ND
ND
ND
ND
ND
ND
ND
ND
ND
MeN
±
2-F-PhSO NH
280±130
380±40
2
a
3
Displacement was measured using [ H]CP55940 in HEK cell membrane preparations overexpressing
hCB1 or hCB2 receptors.
b% transported from the apical side (A) to the basal side (B).
cSee reference 21.
dSee reference 22.
ND: not done as compound did not meet progression criterion/criteria.
Satisfyingly, adding a methyl to the primary urea of 10 (Ke = 130 nM, Ki hCB2/hCB1 = 5), provides the
secondary urea 11 (Ke = 5 nM, Ki hCB2/hCB1 = 700) which showed an improvement in both hCB1 potency
and binding selectivity versus hCB2. Similar results were obtained with other secondary ureas (see 12, 13,
1
5 & 17). This prompted us to advance these compounds to the MDCK-mdr1 assay, in which we were
seeking an A to B transport of <1%, values that we found to favor peripheral selectivity for these purine-
based compounds. Unfortunately, the A to B values were generally >1%, indicating that they were unlikely
to achieve the desired level of peripheral selectivity. This was true even in the case of 15, which has a strong
hydrogen bond donor and favorable calculated physical properties (MW = 554, TPSA = 88, cLogP = 4.3).
It became evident to us, that for this class of compounds, two strong hydrogen bond donors would be needed
to favor exclusion from the CNS. Based on previous docking studies of 1 on the hCB1 receptor, we
anticipated that high polarity can be accepted close to the 4-position of the piperidine ring, where the
22
binding pocket is more polar. Groups attached to the 4-position that reach deeper into the pocket, however,
need to be less polar. This led us to focus on primary ureas that have a combination of better calculated
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physical properties and two strong hydrogen bond donors. We hypothesized that such compounds would
have good peripheral selectivity, reasonable ADME and acceptable potency towards hCB1.
Primary ureas with cycloalkyl groups retained good potency, affinity and selectivity. The
cyclohexyl urea 21, for example, has good hCB1 potency and affinity (Ke = 35 nM, Ki = 1.4 nM) and
excellent selectivity versus hCB2 (Ki hCB2/hCB1 = 3,300). In the MDCK-mdr1 assay, 21 and other
cycloalkyl ureas had values for A to B transport of <1%, indicating the potential for peripheral selectivity.
Several of these compounds were advanced to HLM stability studies, which revealed moderate clearance
values at best. The cyclohexyl urea 21 for example, has a HL of 21 minutes and a Cl of 58 µL/min/mg. This
led us to investigate fluorinated cycloalkyl groups as we hypothesized that the cycloalkyl group was
susceptible to oxidative metabolism. While fluorinated analogs 20 and 26 were still metabolically unstable,
the 4,4-difluorocyclohexyl urea 22 showed considerable improvement in the HLM assay with a HL of 270
minutes and a Cl of 5 µL/min/mg, meeting our goal of a HL >120 minutes and a Cl <10 µL/min/mg. We
attempted to improve the hCB1 potency and affinity of 22 (Ke = 79 nM, Ki = 18 nM) by adding a methyl
to the one position of the cyclohexyl ring. The resulting compound 23 showed a significant improvement
in hCB1 potency and affinity (Ke = 0.4 nM, Ki = 5 nM), but its stability in HLM was poor (HL = 7 minutes).
The benzyl ureas 28 and 29 were also tested and found to have good hCB1 binding (Ki = 2 nM and 3 nM,
respectively), good selectivity versus hCB2 (Ki hCB2/hCB1 ~92 and ~1600, respectively) and good HLM
stability (HL = 150 and 123 minutes, respectively).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Aryl ureas were pursued as options which would contain a more acidic urea hydrogen, a feature
which may favor peripheral restriction. The unsubstituted aryl urea 30 has good hCB1 potency (Ke = 30
nM, Ki = 7 nM), good selectivity versus hCB2 (Ki hCB2/hCB1 ~1400) and in the MDCK-mdr1 assay, has
an A to B transport ratio of 0.2%, indicating possible peripheral selectivity. The HLM stability was near
our goal, with a HL of 115 minutes and a Cl of 11 µL/min/mg. To improve HLM stability, fluorinated
analogs were pursued (32-35). The 2,4-difluorophenyl analog 33 had the desired profile. This compound
has good hCB1 potency (Ke = 4 nM, Ki = 14 nM), excellent selectivity versus hCB2 (Ki hCB2/hCB1
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
~
700), is predicted to be peripherally selective (A to B <0.1%) and has excellent HLM stability (HL = 397
minutes, Cl = 3 µL/min/mg). We also investigated heteroaryl ureas, as these have more favorable calculated
physical properties for solubility, which may result in bioavailability advantages. While most of these
compounds possess significantly less hCB1 potency, the substituted 3-pyridyl analogs 37 and 38 have good
hCB1 potency (Ke = 37 and 26 nM, respectively) and are highly selective versus hCB2 (~200-fold or greater
selectivity). The difluoromethoxy analog 38 performed well in the HLM stability assay, with a HL of 423
minutes and a Cl of 3 µL/min/mg. In the MDCK-mdr1 assay, 38 was predicted to be peripherally restricted
with an A to B transport ratio of 0.1%. The more acidic sulfonyl urea 41 was also investigated. This
compound has a TPSA of 122, a cLogP of 4.9 and two strong hydrogen bond donors, attractive properties
for a peripheral agent. Unfortunately, the hCB1 potency (Ke = 280 nM) was not good enough to progress
the compound.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Replacement of the 4-chlorophenyl in the purine 9-position with a heterocycle was of interest as
this may lead to more polar compounds with better pharmacokinetic properties. We investigated using the
difluoromethoxypyridyl and trifluoromethylpyridyl groups, which were previously shown to be satisfactory
replacements for the 4-chlorophenyl group when an amide or sulfonamide was used as the 4-piperidinyl
linker in place of a urea (unpublished results). For the ureas, however, these groups led to significant
reductions in hCB1 potency as shown in Table 2 (see 25 vs. 42 & 43; see 32 vs. 45 & 46).
Table 2. In Vitro Data for CB1 Antagonists – 4-Chlorophenyl Replacements
O
N
H
N
N
N
R
N
N
R'
Cl
Ke
hCB1
nM +
SEM)
Ki
hCB1
(nM+
Ki
hCB2
(nM+
Selectivity
Ki
CB2/CB1
#
R
R’
(
a
a
SEM)
SEM)
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2
4
5
2
c-HexCH NH
4-Cl-Ph
5±3
0.9±0
7600±4200
ND
8400
ND
2
N
c-HexCH NH
76±34
730±210
ND
2
F3C
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
4
3
c-HexCH NH
100±30
ND
ND
2
F2HCO
32
2-F-PhNH
2-F-PhNH
4-Cl-Ph
58±7
4±1
ND
>10000
ND
2500
ND
4
4
4
5
4-F-Ph
3800±1500
N
2-F-PhNH
2-F-PhNH
210±70
ND
ND
ND
ND
ND
ND
F3C
N
4
6
380±140
F2HCO
a
3
Displacement was measured using [ H]CP55940 in HEK cell membrane preparations overexpressing
hCB1 or hCB2 receptors.
ND: not done as compound did not meet progression criterion/criteria.
Introduction of an aliphatic group in the 4-position of the piperidine ring can enhance hCB1
potency.21 To enhance the activity of our molecules, while minimizing a reduction in polarity, we
investigated the effect of a methyl group on the hCB1 potency of our ureas (Table 3). In general, a trend
toward better Ke values was observed. Unfortunately, HLM stability was also generally lower. In one
noteworthy exception, 48, the methylated version of 22, showed an improvement in hCB1 Ke from 79 nM
to 2 nM. The selectivity versus hCB2 also remained high (Ki hCB2/hCB1 >550). Furthermore, this
compound retained good HLM stability (HL = 279 minutes). Another noteworthy example is 53, the
methylated version of the sulfonyl urea 41. The hCB1 Ke was again improved, from 280 nM to 7 nM. The
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HLM stability for this compound was checked and found to be just below our goal of 120 minutes. Analog
5
4 is a sulfamide with good hCB1 potency (Ke = 17 nM), but the selectivity versus hCB2 was only 82-fold.
Table 3. In Vitro Data for CB1 Antagonists – 4-Methyl Piperidines
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
Me
N R
N
N
H
N
N
Cl
Cl
Ke
hCB1
nM+
Ki
hCB1
(nM+
Ki hCB2 Selectivity
HLM
HL (mins)
#
R
(nM+
Ki
(
a
SEM)
CB2/CB1 Cl (µL/min/mg)
a
SEM)
SEM)
4
7
c-HexNHCO
7±5
13±2
>10000
>769
>588
ND
O
F
F
HL: 279
Cl: 5
48
N
H
2±1
17±3
15±1
>10000
F
O
4
9
14±7
1100±700
73
ND
CH N
2
H
HL: 77
Cl: 16
5
5
0
1
2-F-PhNHCO
1.6±1.1
9±4
4±2
6±1
>10000
>10000
>2500
>1600
HL: 49
Cl: 25
2,4-DiF-PhNHCO
O
N
HL: 51
Cl: 24
52
53
F2HCO
N
H
46±23
7±2
81±15
>10000
>10000
>120
>80
F
O
O
HL: 84
Cl: 15
120±40
S
N
H
O
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5
4
c-HexNHSO2
17±3
9±3
740±120
82
ND
a
3
Displacement was measured using [ H]CP55940 in HEK cell membrane preparations
overexpressing hCB1 or hCB2 receptors.
ND: not done as compound did not meet progression criterion/criteria.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
While several interesting compounds were identified, based on the obtained data, compounds 33
and 38 were advanced into further studies. Compounds 1 and 2 are inverse agonists. Compounds 33 and 38
were further characterized using the calcium assay to establish whether these compounds are neutral
2500
2000
1500
1000
#
2
#33
500
#38
Vehicle
0
-4
-2
0
2
4
6
log [ligand], nM
Figure 4. Compounds 33 and 38 are inverse agonists of hCB1. CHO-CB1 cells were loaded with
calcium indicator dye for 60 min as described in Methods. Cells were then stimulated by various
concentrations of each compound and fluorescence change recorded using FLIPR Tera (Molecular
Devices) instrument. Data is reported as Mean ± SEM from 3 independent measurements.
antagonists or inverse agonists of the hCB1 receptor. As shown in Figure 4, these compounds suppress the
basal signaling through hCB1 and are therefore inverse agonists. The EC for 33 and 38 were 765 and 718
5
0
nM, respectively compared to 585 nM for 2. Compound 33 showed 88% of Emax observed with 2, while 38
showed only a 34% change, closer to what might be described as a neutral antagonist.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We wanted to investigate whether these two lead compounds had the potential to significantly
induce CYP enzymes, which might pose a liability in chronic use and polypharmacy situations. Isoform-
specific CYP induction assays using real-time RT-PCR in HepaRG cells were performed to assess potential
of the two lead compounds as inducers of CYP3A4, CYP1A2 and CYP2B6. These three isoforms are
important within the context of human CYP-mediated drug metabolism of most common drugs. As
indicated in Table 5, marginal potential for CYP3A4 induction was noted with 33 whereas 38 showed little
to no activity. Some reported analogs of 1 were found to have hERG activity, which is a risk for
arrhythmias.19 These compounds were devoid of hERG channel interaction potential as assessed using
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
displacement of radiolabeled astemizole in a radioligand displacement assay (Table 5).31 19 Compound 38
was also characterized for off-target activity by testing at 10 M in the Eurofins SafetyScreen44TM Panel.
No off-target liability was detected after initial screening and follow-up studies (EC or IC >10 M on
5
0
50
all targets tested; data not shown).
Table 5. CYP induction potential and hERG interaction assessment
Compound
CYP1A2 induction*
CYP2B6 induction*
(% of phenobarbital)
CYP3A4 induction* hERG**
(% of omeprazole)
(% of rifampicin)
(Ki, µM)
33
<1
<1
<10
<10
33
>10
3
8
<10
>10
*
*
Data are expressed as % of positive control compound.
3
* Displacement was measured using [ H]astemizole in HEK cell membrane preparations overexpressing
hERG.
2
.3
Pharmacokinetic Studies
Compounds 33 and 38, with favorable in vitro properties, were tested in rodents to assess their
ability to be orally absorbed and kept out of the brain. Compounds were dosed to mice and rats in multiple
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time point studies to obtain plasma and brain maximum concentrations, which were used to calculate and
compare brain/plasma levels. The main goals were to achieve good plasma levels (>100 ng/mL after an
oral dose of 1 mg/kg or less) while keeping the ratio of maximum brain concentration to maximum plasma
concentration as low as possible, preferably less than 0.05. To facility efficacy studies, a plasma half-life
of at least 6 hours was targeted. As demonstrated in Table 6 (mouse PK) and Table 7 (rat PK), both lead
compounds demonstrated high oral absorption upon dosing, very low brain exposure and good hepatic
concentrations. Both compounds also possess a long plasma half-life and low clearance that would allow
for once daily oral dosing. Adjusted for dose, there was good correlation between PK data
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Mouse PK Data for CB1 Antagonists at 0.3 mg/kg PO
Plasma HL
Plasma
Max. Conc.
Liver
Max. Conc.
(ng/mg)
Brain
Max. Conc.
(ng/mg)
(hours)
Plasma Cl
(mL/h/kg)
a
a
a
#
Brain/
Plasma
b
(ng/mL)
HL: 12
Cl: 19
33
1233 at 1 h
940 at 2 h
205 at 2 h
338 at 4 h
17 at 2 h
10 at 4 h
0.013
0.011
HL: 11
Cl: 21
3
8
a. Time points used: 0.5, 1, 2, 4, 8 & 24 hours.
b. The maximum plasma and brain concentrations were used.
obtained in both species. The brain:plasma concentrations were very low in both species, accounting for
1% of peak plasma values in un-perfused tissues. Since the volume of blood in un-perfused mouse brain
~
3
2
is ~4-6% , these data indicate that these two lead compounds have little to no brain penetration.
Table 7. Rat PK Data for CB1 Antagonists at 1 mg/kg PO
Plasma
Max. Conc.
Liver
Max. Conc.
(ng/mg)
Brain
Max. Conc.
(ng/mg)
Plasma HL
(hours)
Plasma Cl
a
a
a
#
Brain/
Plasma
b
(ng/mL)
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
mL/h/kg)
HL: 12
Cl: 14
3
3
3,450 at 2 h
3,490 at 2 h
480 at 2 h
37 at 2 h
46 at 2 h
0.011
0.014
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HL: 9.9
Cl: 18
38
1020 at 4 h
a. Time points used: 0.5, 1, 2, 4, 8 & 24 hours.
b. The maximum plasma and brain concentrations were used.
2
.4.
Effect of 38 on Alcohol Induced Liver Steatosis
The inverse agonist 2 has been previously shown to reduce alcohol consumption as well as
alcoholic steatosis in preclinical models.33, 34 While the former effect is potentially through centrally
expressed CB1 receptors, our past studies confirm that hepatic CB1 receptors can be targeted to reduce
3
1
alcoholic steatosis. In alcoholic liver disease, paracrine activation of CB1 on liver hepatocytes by the full
agonist endocannabinoid molecule 2-arachidonoylglycerol (2-AG), promotes alcohol-induced steatosis
(AS) through a SREBP-1C regulated pathway.34 Efficacy studies of 38 in a mouse model of AS were
performed to assess effects on hepatic lipid accumulation. This compound was chosen over 33 due to its
overall better ADME profile including reduced CYP3A4 induction potential. Female C57BL6 mice were
maintained on a liquid Lieber-DeCarli diet containing ethanol or a matched control diet without alcohol for
four weeks. To mice consuming ethanol, vehicle or compound 38 (0.03, 0.1 and 0.3 mg/kg) was
administered by once daily oral gavage for the last two weeks of the study. Figure 5A shows representative
photomicrographs depicting lipid accumulation in mouse livers. In the livers of animals on the control diet
(
no ethanol), lipid accumulation was minimal, as revealed through Oil Red O (ORO) staining (left panel in
Figure 5A). The ethanol containing diet, in contrast, caused significant accumulation of lipid droplets
(center panel in Figure 5A). In the absence of drug treatment, histology and ORO staining showed that the
livers of ethanol diet-fed mice have microsteatosis and macrosteatosis. Administration of compound 38
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resulted in a significant reduction of hepatic lipid accumulation (right panel in Figure 5A). Quantification
of liver droplets revealed statistically significant reduction of steatosis in the livers of mice treated with 38
at the two higher doses. Normalized liver weights were also lower in these mice with a statistically
significant decrease noted in animals treated with the highest concentration of this agent. Collectively, these
data indicate that 38 is a promising candidate for alcoholic steatosis.
1
1
1
1
1
1
1
1
1
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0
A.
Control + Vehicle
Ethanol + Vehicle
Ethanol + #38 (0.3 mg/kg)
B.
150
3
2
1
0
*
100
*
*
50
0
Vehicle
Vehicle
+
Vehicle
38 [0.3 m #g 3/ k8 g [] 0.1 m 3g 8/ k [g0 ]. 03 mg/kg]
+ Vehicle
#
#38 [0.3 m #g 3/ k8 g [] 0.1 m 3g 8/ k [g0 ]. 03 mg/kg]
Control + Ethanol
+
+
Control + Ethanol
+
+
+
#
+ #
Ethanol Ethanol
Ethanol Ethanol
Ethanol
Ethanol
Figure 5. Oil Red O staining of liver sections indicates that compound 38 reduces steatosis. (A)
Representative liver sections from mice receiving control diet without ethanol + vehicle (left panel),
ethanol containing diet + vehicle (center panel) or ethanol containing diet + 38 (right panel). (B) Left:
Quantification (Image J software) of lipid droplets in liver sections indicates treatment with 38 resulted
in a statistically significant reduction of liver steatosis. Right: Reduced liver weights normalized to
brain weights in animals treated with 38. Data are reported as Mean + S.E.M. from 6-10 animals per
group. Statistical significance indicated as * (T-test, p<0.05 versus ethanol + vehicle).
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3
.
Conclusions
Targeting peripheral populations of CB1 receptors is an important strategy for metabolic diseases
as antagonism of this receptor in the CNS produces adverse psychological effects that could be life-
threatening including suicidal ideation. Thus, efforts are underway to produce peripherally restricted
antagonists of this receptor. Compound 1 is a selective CB1 inverse agonist developed by Pfizer, but its
clinical development was terminated once CNS-related adverse effects were noted.35 Recent docking
studies of 1 with crystal structures of hCB1 indicate that the 4-postion of the piperidine group could be
functionalized to access a binding pocket that may accommodate both polar and nonpolar groups. The same
binding pocket differs significantly from that of hCB2 and hence could be used to obtain highly selective
compounds. In this report, the piperidine linker was functionalized in the 4-position with alkyl, heteroalkyl,
aryl and heteroaryl groups using a urea connector (6), further establishing the SAR needed to identify better
compounds. Polarity was found to be best tolerated near to the 4-position of the piperidine ring, which is
supported by docking of 1 with the crystal structure of the hCB1 receptor.22 Compounds with a highly
polar primary urea connector at the four position of the purine piperidine were found to be potent hCB1
antagonists with excellent selectivity versus hCB2.
0
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0
Interesting compounds with requisite potency and selectivity were progressed into ADME studies.
These studies led to the identification of compounds 33 and 38 as lead molecules that had little to no
penetration across MDCK-mdr1 monolayers, long HLM half-lives, no hERG liability and little to no
induction of three important P450 isoforms. Pharmacokinetic assessments were performed in mice and rats.
High levels of peripheral restriction were observed with both compounds. Generally, <2% brain penetration
was noted compared to plasma in both rats and mice. More importantly, both compounds had low clearance
and long half-lives. These compounds are suitable for once daily oral dosing – an improvement over our
31
previously reported lead structure that requires BID dosing. The heteroaryl urea 38 was tested for efficacy
in a model of alcoholic liver injury. Treatment with this compound reduced hepatic steatosis induced by
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alcohol consumption. In summary, we have identified 38 as a potential clinical candidate pending further
IND-enabling studies.
4
.
Experimental
1
1
1
1
1
1
1
1
1
1
2
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0
1
2
3
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5
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7
8
9
0
1
2
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9
0
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9
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8
9
0
Chemistry General. Purity and characterization of compounds were established by a combination of MS,
1
NMR, HPLC and TLC analytical techniques, as described below. H spectra were recorded on a Bruker
Avance DPX-300 (300 MHz) spectrometer and were determined in chloroform-d (7.26 ppm) or methanol-
d4 (3.31 ppm) with TMS (0.00 ppm) or solvent peaks as the internal reference unless otherwise noted.
Chemical shifts are reported in ppm relative to the solvent signal and coupling constant (J) values are
reported in hertz (Hz). TLC was performed with EMD precoated silica gel 60 F254 plates. TLC spots were
visualized with UV light or I detection. Low-resolution mass spectra were obtained using a single
2
quadrupole PE Sciex API 150EX (ESI). Unless stated otherwise, all test compounds were at least 95% pure
as determined by HPLC. HPLC method: an Agilent-Varian system equipped with Prostar 210 pumps, a
Prostar 335 Diode UV detector and a Phenomenex Synergi 4 m Hydro RP 80A C18 250 x 4.6 mm column
using a 20-min gradient elution of 5-95% solvent B at 1 mL/min followed by 5 min at 95% solvent B
(solvent A, water with 0.1% TFA; solvent B, acetonitrile with 0.1% TFA and 5% water; absorbance
monitored at 220 and 254 nm).
General Procedure A: Ureas of 8 or 9 from Isocyanates. To a solution of 8 or 9 (0.2 mmol, 1 equiv) in
THF (1 mL) was added the isocyanate (0.24 mmol, 1.2 equiv), followed by TEA (0.24 mmol, 1.2 equiv).
The mixture was stirred at rt for 15 h. Water (0.4 mL) was added, followed by ethyl acetate (3 mL) and
then saturated NaHCO solution (0.8 mL). After 10 min, the aqueous layer was removed. Celite (600 mg)
3
was added to the organic layer and the solvent was evaporated. Flash chromatography using silica gel with
an EtOAc/hexanes gradient provided the purified urea.
General Procedure B: Ureas of 8 or 9 from Amines. To a solution of the 4-fluorophenyl carbamate of 8
or 9 (0.2 mmol; prepared using general procedure F) in dioxane (1 mL) was added the amine (0.6 mmol, 3
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1
1
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2
2
2
2
2
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3
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5
5
6
o
equiv). The mixture was heated at 80-85 C for 20 h. Ethyl acetate (3 mL) was added, followed by brine (1
mL) and 2 N NaOH (0.4 mL). After 10 min, the aqueous layer was removed, and the organic layer was
washed with 0.8 M NaHCO solution (1 mL). Celite (600 mg) was added to the organic layer and the solvent
3
evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient provided the purified
urea.
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
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9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedure C: Ureas of 8 or 9 from Amines. To an ice-cold solution of 8 or 9 (0.2 mmol, 1
equiv) in DCM (1 mL) was added NaHCO (0.6 mmol, 3 equiv), followed by saturated NaHCO solution
3
3
(0.2 mL). Triphosgene (0.2 mmol, 1 equiv) was added and after 10 min, the ice bath was removed, and the
mixture was stirred at rt for 1 h (gas evolution). Saturated NaHCO solution (0.8 mL) and water (0.4 mL)
3
were added. After 10 min, the aqueous layer was removed, and the organic layer dried with sodium sulfate
(
20 min). The mixture was filtered, toluene (0.5 ml) was added and the solvent evaporated. THF (1 mL)
was added, followed by the amine (0.4 mmol, 2 equiv) and then TEA (0.5 mmol, 2.5 equiv). The mixture
was stirred at rt for 15 h. Ethyl acetate (3 mL) was added, followed by saturated NaHCO solution (0.8 mL)
3
and water (0.4 mL). After 10 min, the aqueous layer was removed. Celite (600 mg) was added to the organic
layer and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes
gradient provided the purified urea.
General Procedure D: Ureas of 8 or 9 from Amines. To an ice-cold solution of the amine (0.4 mmol, 2
equiv) in DCM (1 mL) was added NaHCO (0.6 mmol, 3 equiv), followed by saturated NaHCO solution
3
3
(0.6 mL). Triphosgene (0.2 mmol, 1 equiv) was added and after 10 min, the ice bath was removed, and the
mixture was stirred at rt for 1 h (gas evolution). Saturated NaHCO solution (0.8 mL) and water (0.4 mL)
3
were added. After 10 min, the aqueous layer was removed, and the organic layer dried with sodium sulfate
(
20 min). The mixture was filtered, toluene (0.5 ml) was added and most of the solvent evaporated. THF (1
mL) was added, followed by 8 or 9 (0.2 mmol, 1 equiv) and then TEA (0.4 mmol, 2 equiv). The mixture
was stirred at rt for 15 h. Ethyl acetate (3 mL) was added, followed by saturated NaHCO solution (0.8 mL)
3
and water (0.4 mL). After 10 min, the aqueous layer was removed. Celite (600 mg) was added to the organic
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layer and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes
gradient provided the purified urea.
General Procedure E: Sulfonyl Ureas of 8 or 9 from Sulfonamides. To an ice-cold solution of the
sulfonamide (0.4 mmol, 2 equiv) in THF (1 ml) was added drop-wise 1 M NaHMDS in THF (0.44 mL, 2.2
equiv). After 10 min, the 4-nitrophenyl carbamate of 8 or 9 (0.2 mmol; prepared using general procedure
F) was added. After 10 min in the ice bath, the mixture was stirred at rt for 1 h. Brine (1 mL) was added,
followed by 2 N HCl (0.3 mL, 3 equiv). Ethyl acetate (3 mL) was added and after 10 min, the aqueous layer
was removed, and the organic layer was washed with brine (0.5 mL). Celite (600 mg) was added to the
organic layer and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes
gradient provided the purified sulfonyl urea.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
4
4
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4
4
4
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4
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedure F: Carbamates of 8 or 9 from Carbamoyl Chlorides. To an ice-cold solution of 8
or 9 (0.2 mmol, 1 equiv) in DCE (1 mL) was added TEA (0.24 mmol, 1.2 equiv), followed by slow addition
of the chloroformate (0.24 mmol, 1.2 equiv). The ice bath was removed and stirring continued for 2 h. Ethyl
acetate (3 mL) was added, followed by saturated NaHCO solution (0.8 mL) and water (0.4 mL). After 10
3
min, the aqueous layer was removed. Celite (600 mg) was added to the organic layer and the solvent
evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient provided the purified
carbamate.
General Procedure G: Preparation of 7 from Aryl Amines. To a heterogeneous mixture of 5-amino-
4
,6-dichloropyrimidine (1.6 g, 10 mmol) and an aryl amine (10 mmol, 1 equiv) in n-BuOH (20 mL) was
added 6 N HCl (0.84 mL, 0.5 equiv). The mixture was heated at 100 °C for 15 h. At rt, ethyl acetate (20
mL) and 2 N NaOH (8 mL) were added and the mixture stirred vigorously for 15 min. Additional ethyl
acetate (80 mL) and saturated NaHCO solution (40 mL) were added. The aqueous layer was removed, and
3
the organic layer washed with 0.8 M NaHCO solution (25 mL). Celite (10 g) and toluene (10 mL) were
3
added to the organic layer and most of the solvent evaporated. Purification by flash chromatography, using
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1
1
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EtOAc/hexanes, provided the intermediate 5-amino-4-arylamino-6-chloropyrimidine. To a solution of the
intermediate (10 mmol, 1 equiv) and 2-chlorobenzaldehyde (2.3 mL, 2 equiv) in dioxane (30 mL) was
added 15% FeCl /silica gel (6.0 g, 600 mg/mmol). The mixture was stirred at rt for 10 min and then at 95
3
°
C for 20 h. At rt, the mixture was filtered using a sintered glass funnel (sand on sodium sulfate on celite
with a medium frit) and washed with chloroform (30 mL). Toluene (3 mL) was added and most of the
solvent evaporated. The resulting residue was dissolved in CH Cl (30 mL) and cooled in an ice bath. DDQ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
(2.2 g, 1 equiv) was added and after 10 min, the ice bath was removed. The mixture was stirred at rt for 2
h and then most of the solvent was evaporated. Ethyl acetate (100 mL) was added and the resulting organic
solution washed with water (2x 50 mL). Celite (10 g) was added and the solvent evaporated. Flash
chromatography using silica gel with an EtOAc/hexanes gradient provided purified 7.
General Procedure H: Preparation of 8 or 9 from 7. To a solution of 7 (10 mmol, 1 equiv) and 4-N-
boc-aminopiperidine (2.4 g, 1.2 equiv; for intermediates 8) or 4-N-boc-amino-4-methylpiperidine (2.5 g,
1
.2 equiv; for intermediate 9) in NMP (20 mL) was added potassium carbonate (4.1 g, 3 equiv). The mixture
was stirred at rt for 15 min and then at 80 °C for 15 h. Ethyl acetate (100 mL) was added, followed by brine
40 mL) and water (20 mL). The organic layer was washed with brine/water (3/1, 2x30 mL). Celite (10 g)
(
was added to the organic layer and the solvent evaporated. Flash chromatography using silica gel with an
EtOAc/hexanes gradient provided the intermediate 9-aryl-8-(2-chlorophenyl)-6-(4-N-boc-aminopiperidin-
1-yl)-9H-purine (for intermediates 8) or 8-(2-chlorophenyl)-9-(4-chlorophenyl)-6-(4-N-boc-amino-4-
methylpiperidin-1-yl)-9H-purine (for intermediate 9). To a mixture of the intermediate (1 mmol, 1 equiv)
and ethanol (2 mL) was added 6 N HCl (1 mL). The mixture was stirred at rt for 10 min and then at 50 °C
for 3 h. The mixture was cooled in an ice bath and chloroform (6 mL) was added, followed by brine (2 mL).
6
N NaOH (1.1 mL) was added slowly and after 5 min, the bath was removed. After 10 min, the layers were
separated and the aqueous layer was saturated with sodium chloride. The aqueous layer was extracted with
chloroform (2x2 mL). The combined organic layers was dried (sodium sulfate for 20 min), filtered and
evaporated. Toluene (1 mL) was added and the solvent evaporated to provide 8 or 9.
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1
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-3-methyl-3-propylurea
(11). The title compound was prepared by the general procedure B to provide 53 mg (90%) of a white
1
crystalline solid, mp 218-219 °C. R = 0.32 (2%MeOH/80% EtOAc/hexanes; blue with UV). H NMR (300
f
MHz, CDCl ) δ 8.38 (s, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.31-7.46 (m, 5H), 7.20 (d, J = 8.5 Hz, 2H), 5.44 (br
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
s, 2H), 4.19 (d, J = 7.5 Hz, 1H), 3.95-4.13 (m, 1H), 3.25-3.44 (m, 2H), 3.20 (t, J = 7.4 Hz, 2H), 2.86 (s,
3
H), 2.11-2.22 (m, 2H), 1.39-1.60 (m, 4H), 0.90 (t, J = 7.4 Hz, 3H). MS (m/z) 538.3 (M+1). HPLC = >99%
at 19.81 min.
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-3-propyl-3-(2,2,2-
trifluoroethyl)urea (12). The title compound was prepared by the general procedure C to provide 60 mg
(83%) of a white crystalline solid, mp 212-213 °C. R = 0.48 (2%MeOH/60% EtOAc/hexanes; blue with
f
1
UV). H NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.30-7.45 (m, 5H), 7.20 (d, J =
3
8
3
.7 Hz, 2H), 5.38-5.50 (m, 2H), 4.38 (d, J = 7.5 Hz, 1H), 4.01-4.15 (m, 1H), 3.94 (q, J = 9.0 Hz, 2H), 3.24-
.40 (m, 2H), 3.20 (d, J = 7.37 Hz, 2H), 2.10-2.22 (m, 2H), 1.40-1.72 (m, 4H), 0.91 (t, J = 7.4 Hz, 3H). MS
(m/z) 606.6 (M+1). HPLC = >98% at 20.75 min.
1
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-3-methyl-3-(2-
methylpropyl)urea (13). The title compound was prepared by the general procedure B to provide 35 mg
(58%) of a white crystalline solid, mp 194-195 °C. R = 0.43 (2%MeOH/80% EtOAc/hexanes; blue with
f
1
UV). H NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.31-7.46 (m, 5H), 7.20 (d, J =
3
8.5 Hz, 2H), 5.44 (br s, 2H), 4.19 (d, J = 7.5 Hz, 1H), 3.95-4.13 (m, 1H), 3.25-3.44 (m, 2H), 3.05 (d, J =
7.4 Hz, 2H), 2.86 (s, 3H), 2.11-2.22 (m, 2H), 1.85-1.92 (m, 1 H), 1.39-1.60 (m, 2H), 0.91 (d, J = 7.4 Hz,
6H). MS (m/z) 552.5 (M+1). HPLC = 97% at 20.33 min.
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-3-(2,2-
dimethylpropyl)urea (14). The title compound was prepared by the general procedure B to provide 25 mg
(41%) of a white amorphous solid, mp 270-271 °C. R = 0.33 (2%MeOH/80% EtOAc/hexanes; blue with
f
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1
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2
2
2
2
2
2
2
2
3
3
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3
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3
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4
4
4
4
4
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5
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6
1
UV). H NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.24-7.43 (m, 5H), 7.19
3
(
d, J = 8.5 Hz, 2H), 5.42 (br s, 2H), 4.38 (t, J = 5.6 Hz, 1H), 4.27 (d, J = 7.5 Hz, 1H), 3.86-4.05
(m, 1H), 3.20-3.43 (m, 2H), 2.96 (d, J = 6.0 Hz, 2H), 2.02-2.26 (m, 2H), 1.35-1.56 (m, 2H),0.90
s, 9H). MS (m/z) 552.5 (M+1). HPLC = >98% at 19.79 min.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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0
(
1
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-3-(2-methoxyethyl)-3-
methylurea (15). The title compound was prepared by the general procedure B to provide 55 mg (90%) of
an off white crystalline solid, mp 193-194 °C. R = 0.08 (2%MeOH/80% EtOAc/hexanes; blue with UV).
f
1
H NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.29-7.42 (m, 5H), 7.20 (d, J = 8.7
3
Hz, 2H), 5.39 (d, J = 6.8 Hz, 1H), 5.28 (br s, 2H), 3.90-4.08 (m, 1H), 3.44-3.65 (m, 4H), 3.27-3.44 (m, 5H),
2
.93 (s, 3H), 2.00-2.25 (m, 2H), 1.39-1.58 (m, 2H). MS (m/z) 554.8 (M+1). HPLC = >99% at 19.09 min.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}pyrrolidine-1-
carboxamide (16). The title compound was prepared by the general procedure B to provide 53 mg (90%)
of a white amorphous solid, mp 134-136 °C. R = 0.17 (2% MeOH/80% EtOAc/hexanes; blue with UV).
f
1
H NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.51 (d, J = 6.59 Hz, 1H), 7.30-7.44 (m, 5H), 7.19 (d, J=8.67
3
Hz, 2H), 5.46 (br s, 2H), 3.92-4.22 (m, 3H), 3.19-3.52 (m, 5H), 2.07-2.22 (m, 2H), 1.69-1.88 (m, 2H), 1.37-
1
.59 (m, 2H). MS (m/z) 536.3 (M+1). HPLC = >99% at 19.36 min.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}piperidine-1-
carboxamide (17). The title compound was prepared by the general procedure B to provide 60 mg (99%)
1
of a white crystalline solid, mp 241-243 °C. R = 0.22 (2% MeOH/80% EtOAc/hexanes; blue with UV). H
f
NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.29-7.46 (m, 5H), 7.20 (d, J = 8.5 Hz,
3
2
1
H), 5.45 (br s, 2H), 4.31 (d, J = 7.4 Hz, 1H), 3.93-4.20 (m, 1H), 3.16-3.44 (m, 6H), 2.11-2.22 (m, 2H),
.40-1.75 (m, 8H). MS (m/z) 550.3 (M+1). HPLC = >99% at 19.93 min.
N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}morpholine-4-
carboxamide (18). The title compound was prepared by the general procedure B to provide 49 mg (81%)
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of a white crystalline solid, mp 210-211 °C. R = 0.14 (2% MeOH/80% EtOAc/hexanes; blue with UV). H
f
NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.50 (d, J = 6.59 Hz, 1H), 7.30-7.44 (m, 5H), 7.19 (d, J = 8.48 Hz,
3
2
H), 5.47 (br s, 2H), 4.30 (d, J = 7.35 Hz, 1H), 3.94-4.17 (m, 1H), 3.54-3.81 (m, 2H), 3.19-3.40 (m, 4H),
.07-2.26 (m, 2H), 1.35-1.58 (m, 2H). MS (m/z) 552.5 (M+1). HPLC = 96% at 18.12 min.
2
1
1
1
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1
1
1
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5
5
5
5
5
5
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0
1
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8
9
0
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1
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-3-cyclopentylurea (19).
The title compound was prepared by the general procedure A to provide 77 mg (100%) of a white crystalline
1
solid, mp 223-224 °C. R = 0.11 (1%MeOH/60% EtOAc/hexanes; blue with UV). H NMR (300 MHz,
f
CDCl ) δ 8.37 (s, 1H), 7.51 (d, J = 6.40 Hz, 1H), 7.29-7.45 (m, 5H), 7.19 (d, J = 8.48 Hz, 2H), 5.40 (br s,
3
1H), 4.22-4.49 (m, 2H), 3.79-4.09 (m, 2H), 3.25-3.40 (m, 2H), 2.04-2.25 (m, 2H), 1.85-2.03 (m, 2H), 1.54-
1.72 (m, 4H), 1.32-1.49 (m, 4H). MS (m/z) 550.1 (M+1). HPLC = 95% at 19.52 min.
1-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-3-[1-
(
trifluoromethyl)cyclopentyl]urea (20). The title compound was prepared by the general procedure D to
provide 32 mg (43%) of a white crystalline solid, mp 225-227 °C. R = 0.52 (2%MeOH/60%
f
1
EtOAc/hexanes; blue with UV). H NMR (300 MHz, CDCl ) δ 8.37 (s, 1H), 7.51 (d, J = 6.6 Hz, 1H), 7.30-
3
7
.45 (m, 5H), 7.19 (d, J = 8.7 Hz, 2H), 5.35-5.48 (m, 2H), 4.29-4.44 (m, 2H), 3.85-4.04 (m, 1H), 3.25-3.37
m, 2H), 2.01-2.24 (m, 6H), 1.68-1.93 (m, 4H), 1.35-1.55 (m, 2H). MS (m/z) 618.3 (M+1). HPLC = >98%
at 20.44 min.
(
3
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-cyclohexylurea (21).
The title compound was prepared by the general procedure B to provide 58 mg (93%) of a white amorphous
1
solid, mp 160-162 °C. R = 0.28 (2%MeOH/80% EtOAc/hexanes; blue with UV). H NMR (300 MHz,
f
CDCl ) δ 8.37 (s, 1H), 7.51 (d, J = 6.4 Hz, 1H), 7.29-7.45 (m, 5H), 7.19 (d, J = 8.3 Hz, 2H), 5.39 (br s, 2H),
3
4.20-4.43 (m, 2H), 3.96 (br s, 1H), 3.48 (br s, 1H), 3.25-3.41 (m, 2H), 2.01-2.24 (m, 2H), 1.84-2.01 (m,
2
1
H), 1.53-1.82 (m, 4H), 1.22-1.53 (m, 4H), 0.96-1.22 (m, 2H). MS (m/z) 564.3 (M+1). HPLC = >98% at
9.49 min.
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-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-(4,4-
difluorocyclohexyl)urea (22). The title compound was prepared by the general procedure B to provide 72
o
mg (100%) of an off white amorphous solid, mp 228-229 C. R = 0.21 (2% MeOH/60% EtOAc/hexanes;
f
1
blue with UV). H NMR (300 MHz, CDCl )  8.36 (s, 1H), 7.50 (d, J = 6.8 Hz, 1H), 7.29-7.43 (m, 5H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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8
9
0
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0
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.18 (d, J = 8.5 Hz, 2H), 5.38 (br s, 1H), 4.35-4.53 (m, 2H), 3.83-4.04 (m, 1H), 3.62-3.81 (m, 1H), 3.23-
3.42 (m, 2H), 1.67-2.22 (m, 8H), 1.32-1.59 (m, 4H). MS (m/z) 600.6 (M+1), 599.0 (M-1). HPLC = >99%
at 19.41 min.
3-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-(4,4-difluoro-1-
methylcyclohexyl)urea (23). The title compound was prepared by the general procedure C to provide 69
o
mg (94%) of an off white amorphous solid, mp 160-162 C. R = 0.30 (2% MeOH/60% EtOAc/hexanes;
f
1
blue with UV). H NMR (300 MHz, CDCl )  8.36 (s, 1H), 7.50 (d, J = 6.8 Hz, 1H), 7.29-7.44 (m, 5H),
3
7
3
.18 (d, J = 8.7 Hz, 2H), 5.38 (br s, 2H), 4.54 (d, J = 7.9 Hz, 1H), 4.31 (s, 1H), 3.80-4.00 (m, 1H), 3.20-
.40 (m, 2H), 2.04-2.22 (m, 4H), 1.73-1.95 (m, 4H), 1.54-1.73 (m, 1H), 1.40-1.53 (m, 2H), 1.38 (s, 3H).
MS (m/z) 614.4 (M+1). HPLC = >99% at 15.10 min.
3
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-
(cyclopentylmethyl)urea (24). The title compound was prepared by the general procedure B to provide 59
mg (95%) of a white amorphous solid, mp 135-137 °C. R = 0.27 (2% MeOH/80% EtOAc/hexanes; blue
f
1
with UV). H NMR (300 MHz, CDCl ) δ 8.36 (s, 1H), 7.50 (d, J = 6.2 Hz, 1H), 7.24-7.44 (m, 5H), 7.18 (d,
3
J = 8.1 Hz, 2H), 5.38 (br s, 2H), 4.43-4.78 (m, 2H), 3.96 (br s, 1H), 3.32 (br s, 2H), 2.95-3.17 (m, 2H),
1
.87-2.24 (m, 3H), 1.70 (br s, 2H), 1.34-1.63 (m, 6H), 1.04-1.32 (m, 2H). MS (m/z) 564.4 (M+1). HPLC =
98% at 20.24 min.
>
3
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-
(cyclohexylmethyl)urea (25). The title compound was prepared by the general procedure B to provide 65
mg (100%) of a white crystalline solid, mp 137-139 °C. R = 0.30 (2% MeOH/80% EtOAc/hexanes; blue
f
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with UV). H NMR (300 MHz, CDCl ) δ 8.36 (s, 1H), 7.50 (d, J = 6.2 Hz, 1H), 7.24-7.44 (m, 5H), 7.19 (d,
3
J = 8.1 Hz, 2H), 5.38 (br s, 2H), 4.43-4.78 (m, 2H), 3.96 (br s, 1H), 3.32 (br s, 2H), 2.90-3.17 (m, 2H),
1
5
.97-2.24 (m, 2H), 1.58-1.82 (m, 5H), 1.34-1.55 (m, 3H), 1.09-1.26 (m, 3H), 0.81-1.05 (m, 2H). MS (m/z)
78.5 (M+1). HPLC = 95% at 21.52 min.
1
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-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[(4,4-
difluorocyclohexyl)methyl]urea (26). The title compound was prepared by the general procedure C to
o
provide 64 mg (87%) of an off white amorphous solid, mp 135-137 C. R = 0.21 (2% MeOH/60%
f
1
EtOAc/hexanes; blue with UV). H NMR (300 MHz, CDCl )  8.37 (s, 1H), 7.50 (d, J = 6.8 Hz, 1H), 7.29-
3
7
3
1
.44 (m, 5H), 7.18 (d, J = 8.5 Hz, 2H), 5.39 (br s, 2H), 4.54 (t, J = 5.8 Hz, 1H), 4.38 (d, J = 7.9 Hz, 1H),
.82-4.04 (m, 1H), 3.19-3.45 (m, 2H), 3.08 (dd, J = 6.2, 6.2 Hz, 2H), 2.02-2.23 (m, 4H), 1.41-1.82 (m, 7H),
.18-1.35 (m, 2H). MS (m/z) 614.5 (M+1). HPLC = >99% at 14.70 min.
3
-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[(1-methylpiperidin-4-
yl)methyl]urea (27). The title compound was prepared by the general procedure B to provide 67 mg
100%) of a white amorphous solid, mp 121-122 °C. R = 0.10 (10% (20% NH OH/MeOH)/ EtOAc; blue
(
f
4
1
with UV). H NMR (300 MHz, CDCl ) δ 8.36 (s, 1H), 7.51 (d, J = 6.4 Hz, 1 H), 7.24-7.44 (m, 5H), 7.18
3
(
d, J = 7.9 Hz, 2H), 5.38 (br s, 2H), 4.67 (br s, 1H), 4.53 (d, J = 7.4 Hz, 1 H), 3.95 (br s, 1H), 3.32 (br s,
2
H), 3.06 (br s, 2H), 2.78-2.88 (m, 2H), 2.24 (s, 3H), 1.99-2.17 (m, 2H), 1.81-1.96 (m, 2H), 1.61-1.73 (m,
2H), 1.16-1.51 (m, 5H). MS (m/z) 593.4 (M+1). HPLC = 95% at 16.93 min.
3-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[(2-
fluorophenyl)methyl]urea (28). The title compound was prepared by the general procedure C to provide
71 mg (100%) of a white amorphous solid, mp 210-212 °C. R = 0.25 (2% MeOH/60% EtOAc/hexanes;
f
1
blue with UV). H NMR (300 MHz, CDCl ) δ 8.35 (s, 1H), 7.49 (d, J = 6.2 Hz, 1H), 7.25-7.43 (m, 7H),
3
7
.17 (d, J = 7.2 Hz, 2H), 7.00-7.10 (m, 1H), 6.86-7.00 (m, 1H), 5.30 (br s, 2H), 5.16-5.25 (m, 1H), 4.89 (d,
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