Blocking Alcoholic Steatosis in Mice with a Peripherally Restricted Purine Antagonist of the Type 1 Cannabinoid Receptor

Article abstract of DOI:10.1021/acs.jmedchem.7b01820

Type 1 cannabinoid receptor (CB1) antagonists have demonstrated promise for the treatment of obesity, liver disease, metabolic syndrome, and dyslipidemias. However, the inhibition of CB1 receptors in the central nervous system can produce adverse effects, including depression, anxiety, and suicidal ideation. Efforts are now underway to produce peripherally restricted CB1 antagonists to circumvent CNS-associated undesirable effects. In this study, a series of analogues were explored in which the 4-aminopiperidine group of compound 2 was replaced with aryl- and heteroaryl-substituted piperazine groups both with and without a spacer. This resulted in mildly basic, potent antagonists of human CB1 (hCB1). The 2-chlorobenzyl piperazine, 25, was found to be potent (K_i = 8 nM); to be >1000-fold selective for hCB1 over hCB2; to have no hERG liability; and to possess favorable ADME properties including high oral absorption and negligible CNS penetration. Compound 25 was tested in a mouse model of alcohol-induced liver steatosis and found to be efficacious. Taken together, 25 represents an exciting lead compound for further clinical development or refinement.

Full text of DOI:10.1021/acs.jmedchem.7b01820

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A peripherally restricted purine antagonist of type 1
cannabinoid (CB1) receptor blocks alcoholic steatosis in mice
George Amato, Amruta Manke, Danni L. Harris, Robert W. Wiethe, Vineetha Vasukuttan, Rodney W. Snyder,
Timothy W. Lefever, Ricardo Cortes, Yanan Zhang, Shaobin Wang, Scott P Runyon, and Rangan Maitra
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01820 • Publication Date (Web): 24 Apr 2018
Downloaded from http://pubs.acs.org on April 24, 2018
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A peripherally restricted purine antagonist of type 1 cannabinoid (CB1)
receptor blocks alcoholic steatosis in mice
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#
#
George S. Amato , Amruta Manke , Danni L. Harris, Robert W. Wiethe, Vineetha Vasukuttan,
Rodney W. Snyder, Timothy W. Lefever, Ricardo Cortes, Yanan Zhang, Shaobin Wang, Scott P.
Runyon and Rangan Maitra*
Discovery Science and Technology, RTI International, 3040 Cornwallis Rd., Research Triangle Park, NC
2
7709-2194, USA
*
#
Corresponding author
Equal contributors
Key Words: CB1, cannabinoid, peripheral, antagonist, otenabant, purine, alcohol, steatosis,
CB2, MDCK, blood brain barrier, ADME
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Abstract
Type 1 cannabinoid receptor (CB1) antagonists have demonstrated promise for the treatment of obesity,
liver disease, metabolic syndrome and dyslipidemias. However, inhibition of CB1 receptors in the central
nervous system can produce adverse effects including depression, anxiety and suicidal ideation. Efforts
are now underway to produce peripherally restricted CB1 antagonists to circumvent CNSꢀassociated
undesirable effects. In this study, a series of analogs were explored in which the 4ꢀaminopiperidine group
of 2 was replaced with aryl and heteroaryl substituted piperazine groups both with and without a spacer.
This resulted in mildly basic, potent antagonists of human CB1 (hCB1). The 2ꢀchlorobenzyl piperazine
0
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5 was found to be potent (Ki = 8 nM), >1000ꢀfold selective for hCB1 over hCB2, possess favorable
ADME properties including no hERG liability, high oral absorption, and negligible CNS penetration.
Compound 25 was tested in a mouse model of alcohol induced liver steatosis and found to be efficacious.
Taken together, 25 represents an exciting lead compound for further clinical development or refinement.
2
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Introduction
The endocannabinoid (EC) system, comprised of endocannabinoids, receptors, transporters and
1ꢀ3
enzymes, is known to play a role in a large variety of physiological processes. Selective modulation of
the EC system has potential therapeutic applications in a broad range of medical conditions. Human
cannabinoid receptors hCB1 and hCB2 are G proteinꢀcoupled receptors (GPCRs) whose primary function
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is to activate G proteins (G ). The hCB1 receptor is present throughout the body and is highly expressed
i/o
in the central nervous system (CNS). In contrast, hCB2 is primarily expressed in the immune system and
1
nominally expressed in the CNS. Selective attenuation of peripheral hCB1 receptor activity is a
promising approach for treating diabetes, metabolic syndrome, dyslipidemias and liver diseases such as
4
, 5
nonꢀalcoholic steatohepatitis (NASH) and alcoholic liver disease. Rimonabant (1, Figure 1), a CNSꢀ
penetrant hCB1 receptor inverse agonist developed by SanofiꢀAventis, was clinically approved for the
©
treatment of obesity in Europe (marketed as Accomplia ). It had to be withdrawn, however, because of
adverse psychiatric episodes in some patients, including depression and suicidal ideation, resulting from
6
inhibition of hCB1 activity in the CNS. The adverse side effects seen with 1 precipitated withdrawal of
other CNSꢀpenetrating hCB1 antagonists from clinical development, including otenabant (2), a compound
developed by Pfizer. Currently, efforts are underway to produce peripherally selective CB1 antagonists
7
, 8
that have limited brain penetration. Of particular note among these are candidate compounds JD5037
9
ꢀ11
and TM38837. The former is reportedly undergoing INDꢀenabling studies while the later was tested in
humans to demonstrate peripheral restriction but its current development status is unknown. A thiophene
12
carboxamide analog of 1 has also been approved for phase 1 studies by USꢀFDA.
Our previous approach to limit CNS exposure using analogs of 2 focused on enhancing
topological polar surface area (TPSA) while eliminating the ability of the primary amide and ethyl amine
to form a hydrogen bonding pair, which has been proposed as the molecular basis for the high CNS
1
3, 14
permeability of 2.
Using this strategy, we reported the discovery of a peripherally selective analog 3
(
Figure 1). Compound 3 possessed excellent potency, reasonable hCB1 selectivity (~50ꢀfold over hCB2),
and most importantly, limited brain penetration (~5ꢀ7% of plasma concentration). Unfortunately,
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compound 3 suffered from limited aqueous solubility. Therefore, we continued to refine and produce
analogs of 3 with better ADME properties, while attempting to further reduce brain penetration. We
envisioned that reꢀintroduction of groups capable of being ionized at low pH would have the benefit of
providing compounds with improved solubility in the stomach and upper gastrointestinal (GI) tract while
still maintaining the potency and peripheral selectivity observed with 3.
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Compound 2 contains a 4,4ꢀdisubstituted piperidine in the 6ꢀposition of the purine core.
Conversion of the 4ꢀ4ꢀdisubstituted piperidine into a Nꢀsubstituted piperazine afforded compound 4 that
had good solubility in simulated gastric fluid but also possessed significant activity at hERG channels,
15
which is associated with a risk of LQT syndrome. In addition, a close analog of compound 4 (2,4ꢀ
dichlorophenyl instead of 2ꢀchlorophenyl in the 8ꢀposition of the purine ring) was reported to have
peripheral selectivity, despite having no hydrogen bond donating groups. This led us to examine analogs
of 2 in which the purine ring was functionalized with a piperazine that was then decorated with aryl or
heteroaryl groups, both with and without intervening linkers. In this report, we describe our efforts to
make analogs of 3 that resulted in 25 with good potency, peripheral selectivity, oral bioavailability,
ADME and pharmacokinetic properties. Compound 25 also showed efficacy in a murine model of
alcoholꢀinduced liver steatosis.
Figure 1. Examples of clinical CB1 antagonists (1, 2) and lead scaffolds 3 and 4.
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Chemical Synthesis
The targeted compounds were prepared as shown in Scheme 1. The chloride 5 was prepared as
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previously reported and then reacted with commercially available piperazines to provide the target
compounds in generally good yield (typically >50%).
o
Scheme 1. Reagents and conditions: (a) NEt , EtOH, 80 C, O/N.
3
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Results and Discussion
SAR studies of CB1 antagonists
All target compounds were evaluated in a FLIPRꢀbased calcium mobilization assay for hCB1
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3,14
activity (Tables 1 & 2) as has been described in our previous publications.
In general, those that
demonstrated apparent antagonist dissociation equilibrium constant (Ke) < 50 nM were further tested for
3
affinity to hCB1 and hCB2 receptors using radioligand displacement of [ H]CP55940 in purified
membrane fractions overexpressing either hCB1 or hCB2. Results were expressed as a selectivity ratio
(
hCB2/hCB1). The most potent and hCB1 subtype selective compounds were tested for peripheral
selectivity by calculating % apical (A) to basal (B) in an MDCKꢀmdr1 permeability assay, which is
1
6
predictive of brain penetration. Compounds that show limited transport from A to B were identified as
desirable for peripheral selectivity.
In Table 1, in vitro data for a set of aryl and heteroaryl compounds are reported wherein the aryl
ring was directly connected to the piperazine nitrogen. For comparison purposes, the methyl and
cyclohexyl analogs were included. The methyl compound 6 demonstrated a Ke = 120 nM in the calcium
hCB1 assay, but the cyclohexyl compound 7 was about 10ꢀfold more potent and had reasonable
selectivity for hCB1 (~60ꢀfold) in the binding assays. Compound 7, however, had ~2% permeability in
the MDCKꢀmdr1 assay, which was deemed unacceptable for further development as typically <1%
permeability is considered acceptable for further development. Phenyl compound 8 was even more
potent, with a Ke = 5 nM. It was also much more selective for hCB1 over hCB2 in the binding assay
(
>5000ꢀfold selective for hCB1) than the cyclohexyl compound 7. Phenyl analog 8 performed well in the
MDCKꢀmdr1 assay with no discernible
Table 1. In vitro data for CB1 antagonists
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9
0
Ke hCB1
(nM)
Ki hCB1
(nM)
Ki hCB2
(nM)
Selectivity
Ki CB2/CB1
MDCK-mdr1
A to B (%)
#
2
6
7
8
R
Otenabant
Me
a
a
b
c
0.2
120
17
5
0.7
7700
11000
d
3
cꢀHex
Ph
2
2
110
64
2
0
>10000
>5000
9
13
2
1400
700
0.06
1
0
1
1200
1
2ꢀCNꢀPh
7
2
2
>10000
5000
>5000
2500
0.02
0
12
2ꢀMeOꢀPh
3ꢀMeOꢀPh
3ꢀHOꢀPh
4ꢀFꢀPh
16
25
48
3
1
1
1
3
4
5
25
2
>10000
5100
>400
2600
0.08
2
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1
6
7
4ꢀCNꢀPh
4ꢀMeOꢀPh
4ꢀHOꢀPh
4
2
3
>10000
>10000
>5000
>3000
0
1
16
0.05
0
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0
18
1400
a
3
Displacement was measured using [ H]CP55940 in CHO cell membrane preparations overexpressing
hCB1 or hCB2 receptors.
b
Percent transported from the apical side (A) to the basal side (B).
c
See Griffith et al. Ref. 11
d
See Hadcock et al. Ref. 18
transport, which predicted limited in vivo CNS penetration. This led us to investigate a broader set of
compounds. Interestingly, the more polar 2ꢀpyridyl compound 9 retained similar hCB1 potency (Ke = 13
nM) and selectivity as seen with phenyl analog 8 and also showed limited permeability in the MDCKꢀ
mdr1 assay (% AꢀB = 0.06). The 4ꢀpyridyl compound 10, however, was much less potent at hCB1 (Ke =
1200 nM). Similarly, the 3ꢀhydroxy compound 14 had reasonably good potency (Ke = 48 nM), while the
4ꢀhydroxy compound 18 was only weakly active in the hCB1 functional calcium assay (Ke = 1400 nM).
It appeared that increasing polarity in the 4ꢀposition was not well tolerated. In general, nonpolar
substitutions such as fluoro, cyano and methoxy groups were well tolerated in either the ortho, meta or
para positions (15-17) as demonstrated in Table 1. These compounds when tested in the MDCKꢀmdr1
assay performed well with several demonstrating <1% A to B transport, which is predictive of limited in
vivo brain penetration. The 2ꢀfluoroꢀ and 2ꢀchlorophenylpiperazines were also prepared, but these had
issues with solubility, that resulted in precipitation from assay buffer, suggesting that solubility could be a
limiting factor for certain aryl piperazines. We expected that adding an alkyl spacer would improve
solubility by increasing the basicity of the piperazine and decreasing the rigidity of the piperazine
substituent. This idea was explored in a series of analogs reported in Table 2.
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Table 2. In vitro data for CB1 antagonists
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
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8
9
0
1
2
3
4
5
6
7
8
9
0
Selectivity
Ki
CB2/CB1
Ke hCB1 Ki hCB1
Ki hCB2
(nM)
MDCK-mdr1
A to B (%)
#
R
a
a
b
(
nM)
(nM)
1
9
0
Bn
17
4
430
110
2
2
2
4
37
6
1.1
7
1300
1200
0
1
2
460
1700
1100
66
77
92
22
12
4
4
23
0.5
2
2
2
4
5
6
0.9
14
3
7
8
3
2300
330
2
0
2
>10000
>10000
>1200
>3300
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6
2
2
2
7
8
9
6
5
4
5
4600
1200
>2000
>5000
>3300
>10000
0
1
2
3
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5
6
7
8
9
0
1
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0
1
2
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9
0
>10000
>10000
>10000
>10000
4
2
30
21
7
3
0.2
0
3
1
0.9
32
33
1
3
4
1
>10000
>10000
>10000
>10000
>2500
>10000
>1200
>800
0
0
0
0
3
3
4
5
5
8
15
12
3
3
6
7
42
12
8
990
120
150
7
24
3600
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PhCH
2
CH
2
11
2
2100
630
1000
52
0
3
270
12
1
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0
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a
3
Displacement was measured using [ H]CP55940 in CHO cell membrane preparations overexpressing
hCB1 or hCB2 receptors.
b
Percent transported from the apical side (A) to the basal side (B).
Installing an alkyl spacer between the piperazine and the aryl group provided compounds that
were more basic and predicted to be generally more soluble (Table 2). The unsubstituted benzyl
compound 19 was both potent (Ke = 17 nM) and selective for hCB1 (~110ꢀfold). Alpha substitution to the
nitrogen with a methyl, resulted in improved potency and selectivity (20, Ke = 4 nM, ~1200ꢀfold
selectivity). Adding an alpha cyano group to increase polarity resulted in compound 21, which still had
reasonable potency (Ke = 37 nM), but the selectivity for hCB1 was reduced (~60ꢀfold) compared to 19 or
2
0. Ortho, meta and para substituents were well tolerated among the benzyl series (24-35). Increasing the
spacer by one carbon provides phenethyl compound 38, which retained good potency (Ke = 11 nM) and
was even more selective (2100ꢀfold) than benzyl compound 19. A note of interest was that when the
linker was one carbon, a 4ꢀpyridyl group was well tolerated at hCB1 (22 and 23), but not well tolerated
when a twoꢀcarbon linker was used (39). Five membered heterocycles with a one carbon linker (36 and
3
7) were also found to be potent (Ke = 42 and 12 nM respectively). Several of these compounds (25, 30-
35, 38) showed no discernable penetration from the apical to basal side and were deemed suitable for
further evaluation. The heterocyclic analogs, however, displayed enhanced permeability in this assay (22,
2
3 and 36) and therefore were removed from further consideration.
Computational modeling and assessment of hCB1 antagonists
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Subsequent to the completion of this project, several crystal structures of smallꢀmolecule
17, 18
19
antagonist
and agonist bound hCB1 were solved. This afforded us the opportunity to evaluate our
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Figure 2. Docked poses for Table 1 compounds 2-18 in hCB1 (PDB-ID: 5TGZ). (A) Compounds
from Table 1 in their lowest MMGBSA docked poses show a common binding mode regarding the
diphenyl purines. The alternately substituted piperazine substituents interact with residues at the top of
the helical bundle and can extend into the solventꢀaccessible loopꢀdomain, (B) Docked poses of Table 1
compounds with protein environment hidden emphasizing the alternately substituted piperazine region.
SAR using the recently published crystal structures. Our initial strategy was to reproduce the crystal
structure poses for AM6538 using Autodock VINA and AMBER16 molecular mechanics generalized
Born surface area (MMGBSA) rescoring approaches as described in the experimental section and
supplementary materials (S1).
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Having validated our approach with known crystal structures, we evaluated binding modes for
compounds prepared during this investigation. Figure 2 shows the lowest MMGBSA/docked poses for
Table 1 compounds in the hCB1 receptor environment with the protein visible (Figure 2A) and hidden
(
Figure 2B). The panel illustrates that no residues occlude one face of the 4ꢀcyanophenyl in the 5TGZ
1
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crystal structure coordinates (AMBER16 model). This highlights that alteration of the piperazine
substituent preserves positioning of the core scaffold and illustrates that variations in MMGBSA free
energy scores are due primarily to interactions at the top of the TM helices. These substitutions are in an
Figure 3. Overall positioning of analog modification from Table 1. (A) Compound 16 in its lowest
MMGBSA scored pose with residues within 4ꢀangstroms of the ligand displayed in hCB1 (PDBꢀID:
5TGZ). (B) Compound 16 is partially solvent accessible as can be seen by the water contacting the
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environment that is partially solvent exposed as shown in Figure 3A. In this context, the alternately
substituted piperazines can make favorable contacts with polar residues (e.g. HIS 178/Ser 123/Gln 116 to
name a few) in addition to some extracellular solvent. As such, the ligand environment is quite
hydrophobic in the diaryl region of the ligands but of mixed character in the region of SAR variation.
Figure 3B shows an early molecular dynamics snapshot of 16 in its docked (lowest MMGBSA) pose
with the hCB1-complex simulation in a membrane (1,2ꢀDioleoylꢀsnꢀglyceroꢀ3ꢀphosphocholineꢀDOPC)
KCl/H2O environment. During course of the initial 50ꢀ100 nanoseconds of simulation, water diffuses into
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the extracellular/Nꢀterminal/loop region and by the extension of the SAR region into the extracellular
milieu and water interacts with the SAR region of the ligands in Tables 1 and 2. Even at short timescales
water can percolate in from the DOPC/K+/Clꢀ/water hCB1 environment and contact the extracellular
faces of the substituted piperazines.
1
1
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Figure 4. Factors affecting antagonist potency. (A) MMGBSA scores for compounds in Table 1 vs.
lnKe. (B) DFT calculated solvation energy vs. lnKe.
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Correlating computed MMGBSA free energetics of Table 1 ligands in complex with the hCB1
receptor with functional antagonism (lnKe) values demonstrated a moderate Pearson correlation (r = 0.61)
(
Figure 4A). The correlation is modest, as indicated by the Pearson r value, but captures a reasonable
antagonist potency ranking. This moderate Pearson correlation suggested that other factors may be
contributing to the variation in potency besides those included in the MMGBSA free energy rescoring
terms (e.g. configurational entropy/TꢀS).
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Figure 4B demonstrates that ligands in Table 1 make contact the extracellular aqueous
environment and suggests exposure of the ligands to the more heavily aqueous environment at the
extracellular face may influence antagonist potency. Figure 4B correlates lnKe versus quantum
chemically derived solvation values for Table 1 ligands. Again, a reasonably good Pearson correlation (r
=
0.7) demonstrates the importance of desolvation energetics of the ligands upon the overall free energy
estimated by the MMPBSA values.
The pattern exhibited throughout the SAR with polar substituents added to the aryl piperazines,
both with and without –CH spacers, is a competition between the cost of (partially) desolvating the
2
ligand and finding complimentary hCB1ꢀorthosteric site interactions given the position of the substitution.
In cases where one introduces a polar substituent giving rise to a large desolvation cost, but that
substituent does not find a large compensatory interaction (e.g. hydrogen bonding) in the binding site, one
will have a diminished binding and a larger Ke (lnKe). In cases, where a polar substituent is both present
and appears in an accessible configuration to a polar binding site sidechain partner, low Ke values are
obtained. In the case of a modest size 4ꢀF substitution in compound 15, with a computed B3LYP
solvation energy of only ꢀ13.1 kcal/mol, one does not have to find a polar partner to compensate for
modest desolvation cost, and notably the Ke value for 15 (4ꢀFꢀPhenyl) is not very different than 8
(
Phenyl) with Ke values of 3 and 5 nM, respectively. Compound 16 introduces a polar cyano group at the
ꢀ position of the aryl ring which has a sizable ꢀ17.3 kcal/mol solvation energy (optimized DFT estimate),
4
but as Figure 3A shows, this substituent extends well into the region entering the extracellular domain
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above the TM region with some exposure to Gln 115 and 116 and makes ample contact with diffusing
solvent above the DOPC membrane and consequently there is neither a high desolvation cost and it can
find partial exposure to polar Gln groups at the top of Helix 1. This compound is predicted to have
modest affinity as noted in our MMGBSA.
1
1
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Compound 25 achieves its inhibitory potential (Ke = 14 nM) by partial burial of its nonꢀpolar 2ꢀ
chloro phenyl into a favorable π stacking /hydrophobic interaction environment (HIS178/Phe174) at the
top of helix 2. The desolvation costs of its terminal substituent is modest (ꢀ14.9kcal/mol predicted
solvation energy) and it has some solvent exposure (TOC graphic). While these facets lead to decreased
inhibitory potency compared to its parent compound 2 (Ke = 0.2nM), with a more polar terminal
substituent, it achieves greater selectivity against hCB2 with ample discrimination against penetration of
the BBB.
In summary, the SAR in Tables 1 and 2 are readily comprehensible utilizing the recent antagonist
crystal structures and standard computational approaches. The alternately substituted piperazine groups
evaluated in this manuscript, do not occupy a buried hydrophobic pocket but extend into a region at the
top of the transmembrane helices that interact with the aqueous environment. The lnKe versus binding
free energy trends are a compensatory balance between polar hCB1 receptor interactions, and solvent
exposed energetics.
Compound 25 is an inverse agonist of hCB1.
Compound 25 was further characterized using the calcium mobilization assay to establish
whether this compound was a neutral antagonist or an inverse agonist of the hCB1 receptor. As indicated
in Figure 5, 25 was an inverse agonist of hCB1 with an EC of ~462 nM. Compound 1 was used in this
50
assay as a positive control. Maximal response of 25 was ~75% of 1.
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Figure 5. Compound 25 is an inverse agonist of hCB1. CHOꢀhCB1 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 the fluorescence change recorded using a FLIPR Tera (Molecular Devices) plate reader for 60
sec. Cells stimulated with vehicle in buffer alone served as a negative control. Compound 1 served as a positive
control for this experiment.
hERG activity, stability in human microsomes and aqueous solubility determination
Table 3. hERG activity, microsomal stability and solubility of 25
hERG activity
>
10 µM
3
(
[ H]astemizole displacement)
Halfꢀlife: 90 minutes
Human hepatic microsomal stability
Aqueous solubility
Clearance: 14 mL/min/kg
pH 1.6: >200 ꢁM
pH 7.4: <0.4 ꢁM
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The methyl piperazine 6 was previously reported to have undesirable activity in a hERG binding
15
assay and hence, we wanted to evaluate the most favorable compounds obtained from this study. We
3
20
evaluated 25 in a [ H]astemizole competition binding assay using HEKꢀhERG cell membranes.
Compound 25 was found to have Ki >10 µM (Table 3), thus this compound is unlikely to cause LQT
syndrome via blockade of hERG. The other benzyl piperazines 23 and 32 were also tested and like 25,
these did not demonstrate significant hERG interaction either (Ki>10 µM, data not shown).
1
1
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Stability of a compound in human liver microsomes is often predictive of human PK. As such, 25
was evaluated in human liver microsomes for stability and was found to have a halfꢀlife of 90 minutes and
a clearance of 14 mL/min/kg (Table 3). Thus, 25 appeared to be suitable for further evaluation in vivo.
Similarly, aqueous solubility of a compound can be predictive of absorption and distribution and more
soluble compounds are often better suited for clinical development. A kinetic solubility assay was utilized
to assess solubility of 25 (Table 3). The calculated aqueous solubility of 25 at an acidic pH of 1.6 was
excellent (>200 ꢁM), but it was poor at neutral pH (<0.4 ꢁM; pH 7.4). This was expected for a basic, but
lipophilic compound. Thus, 25 was deemed to possess good ADME properties and was advanced to in
vivo studies along with certain other candidates.
Pharmacokinetic studies
Select compounds were tested in rodents to assess their ability to be orally absorbed and kept out
of the brain. Initial studies entailed cassette dosing of rodents by oral gavage and consequently measuring
the brain and plasma levels of the parent compound at 2 hr postꢀdose (data not shown). This triage
strategy enabled us to quickly narrow down the list of candidate compounds and focus on those that had a
brain/plasma ratio of <0.10 and reasonable oral absorption. The chloroꢀsubstituted piperazine 25
performed well in initial studies and was evaluated in extended pharmacokinetic (PK) studies. For these
studies, C57BL6 mice (~10 weeks old) were orally administered a single dose of the compound. Plasma,
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whole liver and whole brain were taken from respective unꢀperfused mice at 0.5, 1, 2, 4, 8 and 24 hr postꢀ
dose.
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Table 4. Pharmacokinetic properties of 25
Dose
Tmax (hr)
Cmax*
T (hr)
1/2
Ratio
Ratio
po
Plasma, brain, liver Plasma, brain, liver Plasma, brain, liver Brain:Plasma Brain:Liver
mg/kg
1
0
0.5, 0.5, 0.5
507, 17, 2081
5.4, 11.9, 5.3
0.03
0.008
*Plasma concentration expressed as ng/ml, brain and liver as ng/g
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The amount of parent compound was quantitated in each tissue using LCꢀMS. As demonstrated in Table
4
and Figure 6, piperazine 25 performed well in these PK studies. It was rapidly absorbed upon oral
dosing, producing high levels of plasma and liver concentrations. The plasma halfꢀlife of this compound
was 5.4 hr at the dosing concentration (Table 5), indicating that the drug was suitable for in vivo
evaluation for efficacy studies. In addition, hepatic concentrations at various timeꢀpoints were also
favorable with a peak concentration of 2081 ng/mg at 0.5 hr postꢀdosing and a halfꢀlife of 5.3 hr (Table 4,
Figure 6). The maximum unꢀperfused brain/plasma ratio for this compound was 0.03, suggestive of very
little brain penetration (Table 4). The other compounds tested either had higher levels of brain
concentration compared to 25 or were poorly absorbed upon oral dosing (data not shown). Further, a 7ꢀ
day repeat dosing PK study of 25 (Supporting Materials, Table S2) indicated that this compound was not
accumulating in the brain upon repeat dosing. In sharp contrast, PK analyses of 1 indicated that this
1
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reference compound had significant brain exposure upon oral dosing in mice (Supporting Materials,
Figure S4) with higher brain concentration as compared to plasma at certain timeꢀpoints.
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Figure 6. Pharmacokinetic evaluation of compound 25. C57BL6 mice (10 weeks) were orally
dosed with 25 suspended in a suitable vehicle as described in Methods. Animals were sacrificed at
various time points and tissues were collected. LCꢀMS was used to quantitate the tissue concentration
of 25 at each timeꢀpoint. Four mice were sacrificed at each timeꢀpoint for this study.
Mouse Hypothermia Study
In addition to showing low brain levels with PK experiments, it is important to establish that
compounds of interest do not affect a centrally mediated property of CB1 agonists, thus minimizing the
risk of adverse effects. It is known that the CB1 agonist CP55940 induces CNSꢀmediated reduction of
21
22
body temperature in rodents. The CNS penetrating CB1 antagonist 2 is known to block this effect.
A
study of compound 25 in the rodent hypothermia assay showed little to no block of temperature drop
when dosed orally at 10 mg/kg in mice (Figure 7). These data confirmed that the low brain penetration of
2
5 was not sufficient at blocking centrally located CB1 receptor mediated hypothermia (Figure 7). The
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parent compound 2 (30 mg/kg), however, blocked hypothermia as expected when used as a positive
control compound.
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Figure 7. Mouse hypothermia study of 25 and 2. Female C57BL6 mice (10 weeks) were dosed
with test agent (2, 25, or vehicle) by oral gavage as indicated in Methods. Thirty minutes after dosing
with inhibitor, animals were administered CP55940 by an ip injection. Rectal temperature data were
collected at regular intervals. Compound 2 blocked hypothermia induced by CP55940 (Twoꢀway
ANOVA, p<0.05) compared to animals treated with vehicle only but this effect was absent in animals
dosed with 25.
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Effect of 25 on Alcohol Induced Liver Steatosis
Past studies had indicated that paracrine activation of CB1 on liver hepatocytes by 2ꢀ
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Figure 8. Oil Red O staining of liver sections indicate reduction of steatosis upon treatment with
compound 25. (Top) Representative liver sections from mice receiving control diet without ethanol +
vehicle (left panel), ethanol containing diet + vehicle (center panel) or ethanol containing diet + 25 (right
panel). (Bottom) Quantification (Image J software) of lipid droplets in liver sections indicated
statistically significant reduction of liver steatosis upon treatment with 25 (ANOVA, *, p<0.001 vs
control + vehicle; #, p<0.01 vs ethanol + vehicle).
arachidonoylglycerol (2ꢀAG), secreted by hepatic stellate cells (HSC) during alcoholic liver injury,
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promoted hepatic lipid accumulation and alcoholic steatosis (AS).
The prototypical CB1 inverse
agonist 1 was effective in reducing AS by blocking transcription of lipogenic genes activated by SREBꢀ
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C. Therefore, in vivo efficacy studies were undertaken in a mouse model of AS to assess whether 25
would be efficacious in blocking disease progression/development. Female C57BL6 mice were
maintained on a liquid LieberꢀDeCarli diet containing ethanol or a matched control diet without alcohol
for 4 weeks. Compound 25 or vehicle was administered to these rodents as described in Methods for the
last 2 weeks. Figure 8 shows representative photomicrographs depicting lipid accumulation in liver slices
of mice from various treatment groups. Lipid accumulation in livers of animals on the control diet (no
ethanol) was minimal, as revealed through Oil Red O (ORO) staining (left panel in Figure 8). In contrast,
the ethanol containing diet caused significant accumulation of lipid droplets as expected (center panel in
Figure 8). Liver histology and ORO staining analyses showed obvious microsteatosis and macrosteatosis
in the livers of all ethanol dietꢀfed mice compared to control dietꢀfed mice in the absence of drug
treatment. Using ethanol dietꢀfed mice, administration of compound 25 at a dose of 1.25 mg/kg twice
daily by oral gavage significantly reduced hepatic lipid accumulation (right panel in Figure 8) compared
to the vehicle control group (center panel in Figure 8). Additionally, evaluation of the brains of mice
exposed to 25 at the end of this study indicated that there was no significant accumulation of the
compound upon twiceꢀdaily repeat oral dosing for 14 days (Supporting Materials, S3). These findings
support the efficacy of test compound 25 in limiting the progression of disease in a murine model of AS.
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Summary and conclusions
Peripherally restricted hCB1 antagonists are of interest to investigate their therapeutic value while
minimizing their risk of adverse brain mediated psychiatric disorders. Such compounds could become
important tools in treating diabetes, metabolic syndrome, dyslipidemias and liver diseases. While several
7
groups are actively working in this area, an advanced clinical candidate is yet to emerge. In this study,
we investigated a series of compounds based on 2 in which the 4ꢀaminopiperidine group was replaced
with a smaller piperazine group. The piperazine was used as a mildly basic linker to access a lipophilic
binding site via functionalization with aryl or heteroaryl groups with and without a linker. Potent hCB1
antagonists/inverse agonists with excellent selectivity versus hCB2 were discovered. Positive MDCKꢀ
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mdr1 results lead us to test several compounds for peripheral restriction in rodents after oral dosing.
Varying levels of peripheral restriction was observed. The 2ꢀchlorobenzyl piperazine 25 was found to
have good peripheral restriction, while attaining good plasma and hepatic levels. It is interesting to note
that compound 25 bears neither a Hꢀbond donating group nor another additional polar group. The source
of the peripheral restriction is probably due to the presence of a basic amine in combination with steric
factors. Compound 25 performed as hoped in a pharmacological model of CB1 mediated CNS activity,
failing to block hypothermia induced by a prototypical CB1 agonist in mice. This compound also had
several other favorable properties including good solubility at acidic pH, lack of hERG interaction, and
low clearance in human liver microsomes. Perhaps most interestingly, this compound was effective in
reducing AS in a mouse model of liver injury induced by feeding animals an ethanol containing liquid
diet. This compound and its analogs may be suitable for further clinical development. Over the years,
several groups including ours have reported on the development of peripherally restricted CB1 receptor
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antagonists. The initial probeꢀlike molecules are now being refined in each successive generation to
produce more mature lead structures that have favorable ADME and “drugꢀlikeness”. The series of
investigations reported in this paper provide support for further development of this class of compounds.
Experimental
Chemistry General. Purity and characterization of compounds were established by a combination of MS,
1
NMR, HPLC and TLC analytical techniques described below. H spectra were recorded on a Bruker
Avance DPXꢀ300 (300 MHz) spectrometer and were determined in chloroformꢀd (7.26 ppm) or
methanolꢀd (3.31 ppm) with tetramethylsilane (0.00 ppm) or solvent peaks as the internal reference
4
unless otherwise noted. Chemical shifts are reported in ppm relative to the solvent signal and coupling
constant (J) values are reported in hertz (Hz). Thinꢀlayer chromatography (TLC) was performed on EMD
precoated silica gel 60 F254 plates. TLC spots were visualized with UV light or I detection. Lowꢀ
2
resolution mass spectra were obtained using a single quadrupole PE Sciex API 150EX (ESI). Unless
stated otherwise, all test compounds were at least 95% pure as determined by HPLC. HPLC method: an
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AgilentꢀVarian system equipped with Prostar 210 pumps, a Prostar 335 Diode UV detector and a
Phenomenex Synergi 4 ꢁm Hydro RP 80A C18 250x4.6 mm column using a 20ꢀminute gradient elution
of 5ꢀ95% solvent B at 1 mL/min followed by 5 minutes 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 280 nm).
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Testing for Pan-assay Interference (PAIN). The compounds synthesized were considered low risk for
15
PAIN as they are analogs of a previously wellꢀcharacterized diphenyl purine scaffold specific for hCB1.
Additionally, compounds were manually inspected to identify structural similarities related to known
2
4ꢀ26
PAIN compounds
and all compounds were tested using the calcium mobilization assay in parental
cells without hCB1 to ensure specificity of action.
General Procedure for Reacting Piperazines with 5. To a solution of the chloropurine 5 (93.9 mg, 0.25
mmol) in EtOH (5 ml) was added TEA (3 equiv, 0.10 ml) and the appropriate piperazine (1.25 equiv, 0.31
o
mmol). The reaction vessel was capped and the mixture stirred at 78 C overnight. The resulting mixture
was concentrated and chromatographed on a 4 g silica gel column using a 0ꢀ100% EtOAc/hexanes
gradient. TLC and MS were used to identify the fractions with the desired product. The fractions were
concentrated and dried under high vacuum overnight, thus providing the product as a white or offꢀwhite
solid.
8
-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-(4-methylpiperazin-1-yl)-9H-purine
(6).
The
title
o
1
compound was prepared by the general method to provide 32 mg (29%) of a solid, mp 217ꢀ219 C. H
NMR (300 MHz, CDCl ) δ 8.39 (s, 1H), 7.49ꢀ7.56 (m, 1H), 7.29ꢀ7.44 (m, 5H), 7.15ꢀ7.23 (m, 2H), 4.40
3
(
br s, 4H), 2.58 (t, J = 5.0 Hz, 4H), 2.36 (s, 3H). MS (m/z) 439.3 (M+1). HPLC = 99% at 16.91 min.
8
-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-(4-cyclohexylpiperazin-1-yl)-9H-purine (7). The title
o
1
compound was prepared by the general method to provide 25 mg (61%) of a solid, mp 174ꢀ176 C. H
NMR (300 MHz, CDCl ) δ 8.38 (s, 1H), 7.49ꢀ7.56 (m, 1H), 7.29ꢀ7.43 (m, 5H), 7.15ꢀ7.24 (m, 2H), 4.37
3
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(
br s, 4H), 2.63ꢀ2.83 (m, 4H), 2.32 (br s, 1H), 1.71ꢀ1.97 (m, 4H), 1.64 (d, J = 11.7 Hz, 1H), 1.04ꢀ1.33 (m,
5
H). MS (m/z) 507.1 (M+1). HPLC = >99% at 19.12 min.
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-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-(4-phenylpiperazin-1-yl)-9H-purine
(8).
The
title
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compound was prepared by the general method to provide 54 mg (59%) of a solid, mp 199ꢀ201 C. H
NMR (300 MHz, CDCl ) δ 8.42 (s, 1H), 7.48ꢀ7.57 (m, 1H), 7.26ꢀ7.45 (m, 7H), 7.15ꢀ7.24 (m, 2H), 6.99
3
(d, J = 7.9 Hz, 2H), 6.90 (t, J = 7.3 Hz, 1H), 4.54 (br s, 4H), 3.25ꢀ3.47 (m, 4H). MS (m/z) 501.6 (M+1).
HPLC = >99% at 22.47 min.
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-(4-pyridin-2-ylpiperazin-1-yl)-9H-purine (9). The title
o
1
compound was prepared by the general method to provide 31 mg (45%) of a solid, mp 203ꢀ204 C. H
NMR (300 MHz, CDCl ) δ 8.42 (s, 1H), 8.18ꢀ8.29 (m, 1H), 7.47ꢀ7.58 (m, 2H), 7.30ꢀ7.42 (m, 5H), 7.15ꢀ
3
7
9
.24 (m, 2H), 6.61ꢀ6.76 (m, 2H), 4.51 (br s, 4H), 3.66ꢀ3.82 (m, 4H). MS (m/z) 502.6 (M+1). HPLC =
9% at 18.05 min.
8
-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-(4-pyridin-4-ylpiperazin-1-yl)-9H-purine (10). The title
o
1
compound was prepared by the general method to provide 59 mg (47%) of a solid, mp 155ꢀ157 C. H
NMR (300 MHz, CDCl ) δ 8.42 (s, 1H), 8.31 (d, J = 6.4 Hz, 2H), 7.47ꢀ7.57 (m, 1H), 7.30ꢀ7.45 (m, 5H),
3
7
.21 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 6.5 Hz, 2H), 4.52 (br s, 4H), 3.38ꢀ3.70 (m, 4H). MS (m/z) 502.6
(
M+1). HPLC = >99% at 17.87 min.
2
-{4-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperazin-1-yl}benzonitrile (11). The
o
title compound was prepared by the general method to provide 59 mg (54%) of a solid, mp 168ꢀ169 C.
1
H NMR (300 MHz, CDCl ) δ 8.42 (s, 1H), 7.60 (dd, J = 1.5, 7.9 Hz, 1H), 7.46ꢀ7.57 (m, 2H), 7.29ꢀ7.44
3
(
m, 5H), 7.16ꢀ7.25 (m, 2H), 6.98ꢀ7.11 (m, 2H), 4.60 (br s, 4H), 3.26ꢀ3.48 (m, 4H). MS (m/z) 526.5
(
M+1). HPLC = >99% at 23.85 min.
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-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-[4-(2-methoxyphenyl)piperazin-1-yl]-9H-purine (12). The
o
title compound was prepared by the general method to provide 23 mg (17%) of a solid, mp 177ꢀ179 C.
1
H NMR (300 MHz, CDCl ) δ 8.41 (s, 1H), 7.49ꢀ7.59 (m, 1H), 7.30ꢀ7.41 (m, 5H), 7.17ꢀ7.24 (m, 2H),
3
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7.00ꢀ7.09 (m, 1H), 6.85ꢀ6.99 (m, 3H), 4.57 (br s, 4H), 3.90 (s, 3H), 3.14ꢀ3.31 (m, 4H). MS (m/z) 531.5
(M+1). HPLC = 99% at 20.81 min.
8-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-[4-(3-methoxyphenyl)piperazin-1-yl]-9H-purine (13). The
o
title compound was prepared by the general method to provide 77 mg (85%) of a solid, mp = 171ꢀ172 C.
1
H NMR (300 MHz, CDCl ) δ 8.41 (s, 1H), 7.49ꢀ7.58 (m, 1H), 7.30ꢀ7.43 (m, 5H), 7.15ꢀ7.24 (m, 3H),
3
6
.60 (dd, J = 2.0, 8.0 Hz, 1H), 6.52 (t, J = 2.3 Hz, 1H), 6.46 (dd, J = 2.0, 8.0 Hz, 1H), 4.53 (br s, 4H),
.80 (s, 3H), 3.29ꢀ3.42 (m, 4H), 1.58 (s, 2H). MS (m/z) 531.4 (M+1). HPLC = 95% at 22.73 min.
3
3
-{4-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperazin-1-yl}phenol (14). The title
o
1
compound was prepared by the general method to provide 22 mg (33%) of a solid, mp 281ꢀ283 C. H
NMR (300 MHz, CDCl ) δ 8.42 (s, 1H), 7.50ꢀ7.57 (m, 1H), 7.30ꢀ7.45 (m, 5H), 7.17ꢀ7.24 (m, 2H), 7.13
3
(
t, J = 8.1 Hz, 1H), 6.56 (dd, J = 1.9, 8.2 Hz, 1H), 6.43 (t, J = 2.2 Hz, 1H), 6.35 (dd, J = 2.0, 7.9 Hz, 1H),
5
.11 (br s, 1H), 4.52 (br s, 4H), 3.24ꢀ3.43 (m, 4H). MS (m/z) 517.4 (M+1). HPLC = >99% at 19.57 min.
8
-(2-Chlorophenyl)-9-(4-chlorophenyl)-6-[4-(4-fluorophenyl)piperazin-1-yl]-9H-purine (15). The
o
title compound was prepared by the general method to provide 22 mg (17%) of a solid, mp 180ꢀ182 C.
1
H NMR (300 MHz, CDCl ) δ 8.42 (s, 1H), 7.49ꢀ7.58 (m, 1H), 7.30ꢀ7.45 (m, 5H), 7.16ꢀ7.24 (m, 2H),
3
6.88ꢀ7.07 (m, 4H), 4.53 (br s, 4H), 3.17ꢀ3.35 (m, 4H). MS (m/z) 519.6 (M+1). HPLC = >99% at 23.31
min.
4
-{4-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperazin-1-yl}benzonitrile (16). The
o
title compound was prepared by the general method to provide 24 mg (30%) of a solid, mp 253ꢀ254 C.
1
H NMR (300 MHz, CDCl ) δ 8.43 (s, 1H), 7.47ꢀ7.59 (m, 3H), 7.28ꢀ7.46 (m, 5H), 7.14ꢀ7.24 (m, 2H),
3
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