Pharmacokinetic optimisation of novel indole-2-carboxamide cannabinoid CB1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.02.019

The pharmacokinetic based optimisation of a novel series of indole-2-carboxamide antagonists of the cannabinoid CB₁ receptor is disclosed. Compound 24 was found to be a highly potent and selective cannabinoid CB₁ antagonist with high

Full text of DOI:10.1016/j.bmcl.2011.02.019

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2034–2039
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pharmacokinetic optimisation of novel indole-2-carboxamide cannabinoid
CB1 antagonists
Phillip M. Cowley a, , James Baker c, Kirsteen I. Buchanan a, Ian Carlyle a, John K. Clark a, Thomas R. Clarkson a,
⇑
Maureen Deehan b, Darren Edwards a, Yasuko Kiyoi a, Iain Martin c, Dawn Osbourn c, Glenn Walker b,
Nick Ward c, Grant Wishart a
a Department of Chemistry, MSD, Newhouse, Lanarkshire ML1 5SH, UK
b Department of Molecular Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
c Department of Pharmacology, MSD, Newhouse, Lanarkshire ML1 5SH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The pharmacokinetic based optimisation of a novel series of indole-2-carboxamide antagonists of the
cannabinoid CB1 receptor is disclosed. Compound 24 was found to be a highly potent and selective can-
nabinoid CB1 antagonist with high predicted human oral bioavailability.
Received 17 December 2010
Revised 2 February 2011
Accepted 3 February 2011
Available online 19 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid
CB1
Antagonist
Indole
Pharmacokinetics
CYP3A4
Glucuronidation
The identification of cannabinoid CB1 and CB2 receptors and
their endogenous ligands in the early 1990s provided further
encouragement to the research community to identify novel li-
gands for these G-protein coupled receptors.1 In particular, the dis-
covery of rimonabant (SR141716A, 1) provided an important tool
with which to probe the therapeutic potential of selective cannab-
inoid CB1 receptor antagonists.2 Indeed, this compound was
approved for use in the treatment of obesity in Europe and other
territories. Subsequently, psychiatric adverse events were noted
in clinical use and the product was withdrawn from the market.
We have previously described an optimisation programme
leading to the discovery of novel cannabinoid CB1 receptor antag-
onists 2 and 3 (Fig. 1).3 Compound 3, in particular, was noted to
be a potent antagonist both in vitro and in vivo and was subse-
quently the subject of further studies. During this further profiling,
compound 3 was shown to undergo metabolism by both oxidation
(mediated by CYP3A4) and direct glucuronidation (Fig. 2). Rat he-
patic portal vein cannulation studies showed that both metabolites
were found in plasma from the hepatic portal vein following an
oral dose of 3 (data not shown). This suggested that gut wall
OH
O
N
N
H
N
N
NC
H
N
N
O
Cl
Cl
F3C
O
R
Cl
2 R=H
3 R=OCH3
1
Figure 1. Structures of rimonabant (1), 2 and 3.
metabolism by both P450 and UGT mechanisms, in addition to he-
patic metabolism, may limit oral bioavailability. The rate of metab-
olism by CYP3A4 was measured for several 5-chloro and 5-cyano
indole-2-carboxamides.4 For the 5-cyano indole-2-carboxamide
subseries a convincing relationship between the rate of CYP3A4
metabolism and c log P5 was identified (Fig. 3). Medicinal chemis-
try design strategies were employed to reduce the rate of metabo-
lism by balancing the lipophilicity of the series with the potential
⇑
Corresponding author.
E-mail address: phillip.cowley@tppgd.com (P.M. Cowley).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.019

P. M. Cowley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2034–2039
2035
OH
O
H
NC
N
N
O
OH
F3C
P450
H
N
O
NC
O
O
O
N
O
O
F3C
O
OH
O
H
N
O
NC
HO
OH
UGT
3
N
O
F3C
O
O
Figure 2. Metabolism of compound 3 in rat hepatocytes.
and NADPH-mediated oxidation, complementing previous reports
of raloxifene interactions with CYP3A4.15 Compound 3 showed
marked species differences in extent of P450 and UGT mediated
metabolism, with these species differences being reflected in the
in vivo PK data.
Compounds from the indole-2-carboxamide series were as-
sessed for potency as antagonists of the human CB1 receptor in a
CB1 reporter gene assay as described previously.16 The values of
pIC50 are reported as the means of at least two independent exper-
iments. Compounds were subsequently selected for screening in
human microsomes from gut and liver, primed for both P450 and
UGT pathways to provide an approximate ranking with respect
to predicted PK profile.
4.50
4.00
3.50
3.00
The synthesis of indole-2-carboxamide analogues of compound
3 is depicted in Scheme 1, all starting materials and reagents were
purchased from commercial sources unless otherwise stated. Ethyl
5-cyanoindole-2-carboxylate (4) was benzylated by deprotonation
with sodium hydride followed by addition of the appropriate ben-
zyl bromide to afford 5. Hydrolysis gave the acid (6), which was
converted to the amides (7–31) by coupling with the amine using
1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-[3-dimethylamino-
propyl)carbodiimide] hydrochloride (EDCI).
Synthesis of non-commercially available amines is described in
Scheme 2. 4-Amino-1,1,1-trifluoro-2-trifluoromethylbutan-2-ol
(34) was obtained from the reaction of the bis(trifluoro-
methyl)butyrate (32) in aqueous ammonia, followed by reduction
of the amide (33) with lithium aluminium hydride in tetrahydrofu-
ran. 1-(1-(Aminomethyl)cyclopentyl)ethanol (38) was prepared
from ethyl cyanoacetate (35) treated with sodium ethoxide and
1,4-dibromobutane to give ethyl 1-cyanocyclopentanecarboxylate
(36) which on treatment with (trimethylsilyl)methyl lithium fol-
lowed by addition of water gave 1-acetylpentanecarbonitrile (37).
Reduction with lithium aluminium hydride afforded the desired
1-(1-(aminomethyl) cyclopentyl)ethanol (38). 1-(2-Amino-
ethyl)cyclobutanol (41) was obtained from treating acetonitrile
with lithium diisopropylamide at À70 °C then addition of cyclobu-
tanone (39), the resulting cyano alcohol (40) was hydrogenated in
the presence of Raney nickel. Where the benzyl bromides were
not commercially available they were prepared from the appropri-
ate benzyl alcohol using PPh3 and CBr4.
1
2
3
4
5
6
clogP
5-Cl
5-CN
Compd 3
Figure 3. Plot of rate of CYP3A4 metabolism versus c log P for selected compounds
from the indole-2-carboxamide series of CB1 antagonists.
for blocking sites of metabolism. Compounds were ranked by
in vitro assessment of both intestinal and hepatic P450 and UGT
stability. The aim was to identify compounds with improved pre-
dicted human pharmacokinetics compared to 3 and to gain confi-
dence in these predictions with data across pre-clinical species.
The in vitro to in vivo extrapolation (IVIVE) of clearance by glu-
curonidation was known to be challenging, largely due to differ-
ences in experimental set-up influencing the rate of in vitro
glucuronidation, for example, the inhibitory effects of fatty acids
and enabling of compound access with alamethicin.6–8 To increase
confidence in IVIVE for a combined P450/UGT model9 (Fig. 4) incor-
porating intestinal and hepatic metabolism, predictions for 3 and
raloxifene (Evista, known to undergo extensive gut first pass
metabolism10,11) in a range of species were compared with in-
house pre-clinical and published clinical PK data. Subsequent to
these studies, alternative models for prediction of gut extraction
from in vitro data have been suggested in the literature, particu-
larly for compounds metabolised predominantly by CYP3A4.12–14
As demonstrated in Table 1, the combined UGT/P450 approach
predicted clearance and oral bioavailability with reasonable accu-
racy, suggesting that ranking compounds on the basis of such
in vitro stability data may be a useful approach. Interestingly,
raloxifene was shown to undergo both direct glucuronidation
From our earlier SAR exploration of this indole-2-carboxamide
series it was clear that the indole N-1 benzyl moiety is tolerant
to multiple substituents at the ortho and meta positions, although
there was a general preference for more lipophilic substituents.3 In
a first attempt to lower metabolic clearance whilst maintaining

2036
P. M. Cowley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2034–2039
Drug
Gut lumen
P450
UGT
Intestinal CLint = CLint (NADPH) + CLint (UDPGA)
fg = 1 – (CLin vivo,blood/Q gut mucosa
Drug
Drug
Intestinal wall
Hepatic portal vein
Liver
)
Hepatic CLint = CLint (NADPH) + CLint (UDPGA)
fh = 1 – (CLin vivo,blood/Qliver
P450
UGT
)
Systemic
circulation
F = fabs . fg . fh
Figure 4. Model for predicting gut and intestinal metabolism.
Table 1
Intrinsic clearances, predicted and observed pharmacokinetic data for raloxifene and 3 in a range of species
Compd
Raloxifene
3
Species
CLint
(l
L/min/mg)
CLpred (mL/min/kg)
Fpred (%)
CLobs (mL/min/kg)
Fobs (%)
Gut
Liver
NADPH
UGT
NADPH
UGT
Mouse
Rat
Dog
0
0
0
0
34
5
22
66
79
178
79
0
11
26
24
69
55
23
18
15
21
17
5
108
58
40
10
4
5
Human
346
21
2
Mouse
Rat
Dog
0
0
0
0
0
0
0
45
69
0
0
191
7
60
72
7
9
6
68
4
17
104
7
30
8
67
—
Human
68
69
31
19
—
CLint—in vitro intrinsic clearance (values of 0 indicate <6
CLpred—predicted in vivo plasma clearance.
CLobs—observed in vivo plasma clearance.
lL/min/mg).
F
F
pred—predicted oral bioavailability.
obs—observed oral bioavailability.
See Ref. 9 for details of the assays.
2
H
NC
N R
NC
NC
O
O
(a)
(c)
N
R
O
N
OEt
N
R
OR
1
1
H
4
7-31
5 R = Et
6 R = H
(b)
Scheme 1. Reagents and conditions: (a) R1Br, NaH, DMF, 60 °C; (b) KOH, EtOH/H2O, 60 °C; (c) R2NH2, EDCI, HOBt, Et3N, CH2Cl2, rt.
potency, subtle changes to substituents and lipophilicity in the
benzyl region were pursued (Table 2). 2-Ethoxy-5-trifluorometh-
oxy benzyl analogue 7 showed a seven-fold decrease in CB1 antag-
onist potency compared to compound 3 with no improvement in
intrinsic clearance. 3,5-Dichloro analogue 8 was also disappointing
showing a modest 4.5-fold decrease in CB1 potency and high
intrinsic clearance. Compounds 9–15 (c log P range 4.06–4.81),
of modifications to the amide and benzyl moieties were explored
(Table 3). Cycloalkyl derivatives 16–20 showed similar or de-
creased CB1 potency compared to 3, however no significant
improvement in metabolic stability was achieved. Adopting a more
direct approach by blocking the major site of P450 metabolism via
substitution of the carbon adjacent to the hydroxyl moiety resulted
in significant success. Chain shortened tertiary alcohols 21 and 22
showed significantly reduced glucuronidation and P450 mediated
metabolism upon comparison with 3, this was accompanied with
an order of magnitude decrease in CB1 receptor potency. 3-Hydro-
xy-3-methylbutyl analogue 23 showed a similar profile to 21 and
22. However, CB1 potency could be partially recovered via changes
exhibited similar or reduced lipophilicity to compound
3
(c log P = 4.76) and all showed high CB1 antagonist potency. How-
ever, intrinsic clearance again showed no improvement. It was evi-
dent that subtle changes to the benzyl region were failing to
decrease hepatic and gut wall metabolism, therefore, combinations

P. M. Cowley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2034–2039
CF3
OH
2037
O
CF3
OH
O
CF3
OH
(a)
(b)
O
H2N
H2N
F3C
F3C
F3C
32
33
34
O
N
N
O
(c)
(d)
(e)
O
O
H2N
OH
N
O
35
36
37
38
NH2
N
O
(f)
(g)
OH
OH
39
40
41
Scheme 2. Reagents and conditions: (a) NH4OH, MeOH, rt, 40 h; (b) LiAlH4, THF, 0 °C, 20 h; (c) Br(CH2)4Br, NaOEt, EtOH, 70 °C, 4 h; (d) TMSCH2Li, pentane, MeOH, 0 °C, 35 h;
(e) LiAlH4, THF, rt, 18 h; (f) MeCN, LDA, THF, À70 °C, 2 h; g) Raney Nickel, H2, EtOH, 5 bar, 50 °C, 20 h.
Table 2
Benzyl substituent SAR
OH
H
NC
N
N
O
R
Compd
R
CB1 pIC50
Gut CLint (NADPH)
Gut CLint (UGT)
Liver CLint (NADPH)
Liver CLint (UGT)
1
2
3
7
8
9
10
11
12
13
14
15
H
7.95
8.74
8.93
8.08
8.27
8.70
9.11
9.12
9.29
9.24
9.20
8.81
0
0
0
0
0
0
0
0
0
0
0
13
0
136
68
103
98
63
94
64
102
124
124
45
69
62
69
0
20
31
20
22
10
23
23
16
23
29
10
3-OCF3
2-OCH3, 5-OCF3
2-OCH2CH3, 5-OCF3
3-Cl, 5-Cl
2-OCH3, 5-Cl
2-OCH3, 5-Br
2-OCH3, 5-CH3
2-CH3, 5-CF3
2-CH3, 5-Cl
2-CH3, 5-CH3
3-OCH2CH2CH3
103
103
83
104
191
180
119
257
156
CLint in lL/min/mg (from duplicate assays with human microsomes)
in the benzyl moiety. Combination of the 3-hydroxy-3-methylbu-
tyl amide with a 2-methyl-5-trifluoromethyl substituted benzyl
resulted in compound 24 showing only a four-fold decrease in po-
tency compared to 3, yet maintaining the promising in vitro met-
abolic profile of analogues 21–23. Secondary alcohols 25 and 26
maintained high CB1 receptor potency, however P450 mediated
hepatic metabolism was significantly increased upon comparison
with the tertiary alcohols. Analogue 27 exhibited a reasonable pro-
file and high potency but cyclobutyl alcohols 28 and 29 together
with primary alcohol 30 showed hepatic P450 intrinsic clearances
in the same range as for compound 3. Highly lipophilic secondary
alcohol 31 unsurprisingly showed high intrinsic clearances.
Selected compounds with good predicted PK and high CB1
receptor potency were advanced to in vivo evaluation in a reversal
of mouse hypothermia induced by the cannabinoid agonist WIN
55,212-2 (Table 4).17 Compound 24 dosed orally showed potent
reversal of WIN 55,212-2 induced hypothermia comparable to that
of rimonabant (1) and the previously disclosed 3 (ID50s 24 = 1.16,
rimonabant
(ID50 = 9.67
(both with ID50 >10
(1) = 1.29,
mol/kg) was less potent and compounds 21 and 22
mol/kg) were inactive at the doses tested.
3 = 0.88
l
mol/kg).
Compound
23
l
l
The affinities of compounds at the cannabinoid receptors were
determined by radioligand binding experiments performed at least
in duplicate. Affinity for CB1 was measured by competition with
[3H]SR141716A binding to membranes prepared from CHO cells
expressing the human CB1 receptor. Affinity for CB2 was measured
by competition with [3H]CP55,940 binding to recombinant human
CB2 receptors expressed in Sf9 cell membranes. Compounds 21–24
all showed high CB1 receptor affinity. CB2 receptor affinity was
only measured for 24 which was found to be greater than 1000-
fold selective for the CB1 receptor versus the CB2 receptor.
The human microsomal stability screening identified 24 as hav-
ing a potentially superior PK profile than 3. As shown in Table 5,
the same in vitro models predicted in vivo PK reasonably well
across species and, most importantly, suggested that 24 should
have a much improved human PK profile. In human liver micro-

2038
P. M. Cowley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2034–2039
Table 3
Benzyl and amide substituent SAR
H
R1
NC
N
N
O
R2
Compd
16
R1
R2
CB1 pIC50
8.29
Gut CLint (NADPH)
Gut CLint (UGT)
Liver CLint (NADPH)
Liver CLint (UGT)
2-OCH3, 5-OCF3
2-OCH3, 5-CF3
2-OCH3, 5-tBu
0
0
0
51
49
26
139
155
188
18
7
OH
OH
OH
*
*
*
17
8.88
18
8.83
0
19
2-OCH3, 5-Cl
8.59
0
179
196
34
OH
OH
*
20
21
2-CH3, 5-F
8.31
7.65
0
0
175
8
190
0
49
0
*
*
2-OCH3, 5-OCF3
OH
22
23
24
25
2-OCH3, 5-CF3
3-OCF3
7.94
7.87
8.29
8.05
0
0
0
0
0
9
0
0
7
9
0
0
0
0
*
*
OH
OH
OH
OH
*
*
2-CH3, 5-CF3
3-OCF3
12
113
OH
*
26
27
28
3-OCF3
8.09
8.22
8.83
26
0
0
0
0
59
20
88
31
24
0
CF3
OH
*
*
*
*
*
3-OCF3
F3C CF3
OH
2-OCH3, 5-CF3
0
OH
OH
OH
29
30
3-OCF3
8.58
8.03
0
0
0
0
78
41
0
0
2-OCH3, 5-Br
31
3-OCF3
8.29
99
0
505
9
CLint in lL/min/mg (from duplicate assays with human microsomes).
somes oxidation was observed at three distinct sites on the mole-
cule, demonstrating that metabolism no longer occurred at the
alcohol. In wider selectivity profiling 24 showed no appreciable
Table 4
affinity (estimated pKi <5) at 10 lM for a panel of 63 molecular tar-
Reversal of WIN 55,212-2 induced hypothermia
gets comprising of GPCRs, ion channels, transporters, enzymes and
nuclear receptors.
Compd
Hypothermia ID50
lmol/kg (95% CI)
CB1 pKi
CB2 pKi
1
2
3
21
22
23
24
1.29 (0.40–4.17)
3.42 (0.97–12.04)
0.88 (0.58–1.34)
>10
9.03
9.10
9.40
8.26
8.12
8.30
8.36
6.07
<5.0
<5.0
ND
ND
ND
In summary, a pharmacokinetic based optimisation utilising
in vitro assays to assess tissue and species specific intrinsic
clearances has been employed. Compound 2418 has been identified
as a novel potent and selective CB1 antagonist. Furthermore, 24
showed reduced human intrinsic clearance compared to starting
point 3, leading to a significantly increased predicted human oral
bioavailability. The compound displayed reversal of the hypother-
mia induced by a cannabinoid agonist in vivo, with potency com-
parable to that of rimonabant.
>10
9.67 (2.91–32.10)
1.16 (0.23–5.93)
<5.0
ND = not determined.
CI = confidence interval.
ID = inhibitory dose.

P. M. Cowley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2034–2039
2039
Table 5
Intrinsic clearances, predicted and observed pharmacokinetic data for 24 in a range of species
Compd
Species
CLint
(
lL/min/mg)
CLpred (mL/min/kg)
Fpred (%)
CLobs (mL/min/kg)
Fobs (%)
Gut
Liver
NADPH
UGT
NADPH
UGT
24
Mouse
Rat
Dog
0
0
0
0
0
0
0
0
33
>280
17
0
53
> 78
14
14
< 8
52
10
200
15
31
2
42
—
>280
0
0
Human
12
7
65
—
following physiological parameters were used for prediction of human PK:
hepatic blood flow (Qh) 21 mL/min/kg, liver weight 21 g/kg, liver microsomal
protein 45 mg/g, intestinal mucosal blood flow 4.6 mL/min/kg, intestine weight
30 g/kg, intestinal microsomal protein 3 mg/g. Estimates of fraction absorbed
(Fabs) were P38% for compound 3 (rat HPV data), 100% for compound 24 (rat
HPV data) and 60% for raloxifene (Ref. 11).
Acknowledgements
We thank our Analytical Chemistry colleagues for structure and
purity determination.
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17. The antagonist or vehicle (5% mulgofen in saline, 10 mL/kg) was administered
p.o. 75 min before rectal temperature was measured. WIN 55,212-2 mesylate
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Clarkson, T. R.; Deehan, M.; Edwards, D.; Goodwin, R. R.; Jaap, D.; Kiyoi, Y.;
Mort, C.; Palin, R.; Prosser, A.; Walker, G.; Ward, N.; Wishart, G.; Young, T.
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4. Compounds were incubated at
1 lM with human recombinant CYP3A4
(50 pmol/mL, BD Biosciences) and 1 mM NADPH. Compound loss was
measured in duplicate time–course assays with detection using LC–MS/MS.
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#204 Claremont, CA 91711-4707, USA.
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temperature measurement. The 60 min pre-treatment with WIN 55,212-2
mesylate corresponded to the maximal hypothermia attained by this agonist.
Rectal temperature was measured using a metal probe with a Fluke 51 K/J
thermometer. The probe was covered in a lubricant (vaseline) and was inserted
approximately 1.5 cm into the rectum. The highest temperature stable for 10 s
was recorded. Following completion of the test animals were humanely
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9. Non-restricted well stirred model using intrinsic clearance data from the sum
of NADPH and UDPGA dependent pathways. Duplicate microsomal incubations
(hepatic microsomes prepared in-house for pre-clinical species, BD Biosciences
for human; small intestinal microsomes all from BioPredic (TCS Cellworks, UK))
18. Analytical data for compound 24: ESI-MS: m/z = 444.3 [M+H]+, 426.0 [MÀOH]+.
1H NMR (400 MHz, CDCl3) d: 8.03–8.05 (d, J = 1.6 Hz, 1H), 7.49 (dd, J = 1.5 and
8.7 Hz, 1H), 7.45 (broad, 1H), 7.41–7.44 (dd, J = 1.2 and 8.0 Hz, 1H), 7.38 (d,
J = 1.2 and 8.0 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.22 (dd, J = 1.5 and 8.7 Hz, 1H),
7.00 (s, 1H), 6.47 (s, 1H), 5.87 (s, 2H), 3.50–3.57 (m, 2H), 2.47 (s, 3H), 1.72 (m,
2H), 1.30 (s, 6H).
were performed with 0.5 mg protein/mL at
supplemented with either 1 mM NADPH at pH 7.4 (P450 assay) or 2 mM
UDPGA, 50 g/mL alamethicin, 1 mM MgCl2 at pH 7.1 (UGT assay). The
1 lM drug concentration
l