Substituted phenoxypropyl-(R)-2-methylpyrrolidine aminomethyl ketones as histamine-3 receptor inverse agonists

Article abstract of DOI:10.1016/j.bmcl.2012.02.081

Optimization of a series of aminomethyl ketone diamine H₃R antagonists to reduce the brain exposure by lowering the pKa, led to molecules with improved pharmacokinetic properties. Compounds 9, 19, and 25 had high affinity for human H₃R and demonstrated in vivo H₃R functional activity in the rat dipsogenia model. Compound 9 displayed modest wake-promoting activity in the rat EEG/EMG model.

Full text of DOI:10.1016/j.bmcl.2012.02.081

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2807–2810
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted phenoxypropyl-(R)-2-methylpyrrolidine aminomethyl ketones
as histamine-3 receptor inverse agonists
⇑
Allison L. Zulli , Lisa D. Aimone, Joanne R. Mathiasen, John A. Gruner, Rita Raddatz, Edward R. Bacon,
Robert L. Hudkins
Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of a series of aminomethyl ketone diamine H3R antagonists to reduce the brain exposure by
lowering the pKa, led to molecules with improved pharmacokinetic properties. Compounds 9, 19, and 25
had high affinity for human H3R and demonstrated in vivo H3R functional activity in the rat dipsogenia
model. Compound 9 displayed modest wake-promoting activity in the rat EEG/EMG model.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 20 January 2012
Revised 22 February 2012
Accepted 23 February 2012
Available online 2 March 2012
Keywords:
Phenoxypropyl-(R)-2-methylpyrrolidine
CEP-26401 irdabisant
Histamine H3 receptor
Inverse agonist
Sleep-wake
Histamine elicits physiological responses mediated by four
G-protein coupled receptors (H1R–H4R) and exerts a variety of func-
tions in the central nervous system (CNS).1 The histamine H3 recep-
tor (H3R) functions in the brain both as an autoreceptor modulating
histamine release and as an inhibitory heteroreceptor regulating the
release of multiple neurotransmitters, including acetylcholine,
dopamine, norepinephrine and serotonin and is believed to be
involved in attention, sleep, and cognition.2,3 Clinically, H3R antag-
onists are of interest for potential treatment of multiple CNS disor-
ders associated with attention and cognitive deficits, including
sleep/wake disorders, attention-deficit hyperactivity disorder
(ADHD), Alzheimer’s disease (AD), mild cognitive impairment, and
the cognitive deficits in schizophrenia.4 Several clinical candidates
have been disclosed thus far including our phenoxypropyl pyrida-
zin-3-one 1a (CEP-26401, irdabisant) and its structure–activity
relationships (SAR).5
Our search for novel H3R antagonists for treating sleep-wake dis-
orders was initiated with a high-throughput screen that identified a
series of methyl ketone phenoxypropyl diamines 1c among the
group of hits. Although the phenoxypropylamine core is a well
established pharmacophore in the H3 field, compounds such as 1c
that contain the aminomethylketone substructure appeared to be
novel based on searches.6a,b Preliminary lead expansion of the series
identified 2 as an early lead compound (Fig. 1). Follow on synthesis
of each enantiomer revealed that the R-isomer (human H3R
Ki = 5.4 nM, rat H3Ki = 50 nM) was about 20-fold more potent than
the S-isomer and functionally a potent antagonist (Kb = 0.6 nM)
and full inverse agonist (EC50 = 5.5 nM) in the [35S]GTP
cS hH3R
binding assay.7 Further profiling to identify potential issues and
areas of improvement showed that R-2 had acceptable in vitro met-
abolic stability across species (t >40 min in mouse, rat, dog, mon-
½
key, and human), did not inhibit key cytochrome P450
isoenzymes (IC50 values >30
2C19, 2D6 and 3A4), and had high selectivity over hERG (21% inhi-
bition at 10 M; astemizole binding). It also showed high perme-
lM for inhibition of CYP 1A2, 2C9,
l
ability in Caco2, low plasma protein binding (<20% for rat and
human) and excellent selectivity (>1000-fold) for hH1, hH2, and
hH4 receptor subtypes and against a panel of 65 GPCRs, ion chan-
nels, and enzymes (<50% inhibition at 10 lM). However, issues with
early diamine compounds (e.g. 1b JNJ-5207852) reported from
Johnson & Johnson showed that they had exceptionally long bio-
half-life, brain residence time, and induced phospholipidosis.8 JNJ-
5207852 had potent H3 antagonism and was wake-promoting in
rats and mice. A series of rat pharmacokinetic experiments (PK) con-
ducted with R-2, showed that it also displayed very high clearance,
volume of distribution, and extremely high brain to plasma ratio (B/
P = 50) and brain residence time translating to very low plasma lev-
els following oral administration (Table 3). We report in this paper
an SAR study undertaken with the objective to lower the B/P ratio
and improve the PK by modulating the pKa of the western morpho-
line amine and reducing the dibasic structure of the molecule.
The synthesis of the key 2-bromo-1-[4-(3-chloropropoxy)-
phenyl]ethanone intermediate 5 required for the synthesis of each
⇑
Corresponding author.
E-mail address: azulli@cephalon.com (A.L. Zulli).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.081

2808
A. L. Zulli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2807–2810
H
Me
N
N
O
N
O
O
N
N
1b
1a CEP-26401
O
O
R
N
O
N
NR2
N
R
O
O
1c
2
Figure 1. Structures of H3R antagonists.
of the final analogs is outlined in Scheme 1.9 Alkylation of 1-(4-hy-
droxy-phenyl)-ethanone 3 with 1-bromo-3-chloro-propane gave
the chloro intermediate 4, followed by bromination to give 5. Com-
pounds 2 and 7–11 were synthesized by initially reacting 5 with the
amine (R2NH) (K2CO3, acetonitrile) to give the corresponding
amines 6, then displacement of the chloro with (R)-2-methyl-pyr-
rolidine to give the target compounds (Scheme 1). The piperazine
analogs 16–29 (Table 2) were synthesized in an analogous manner.
Starting with boc-piperazine 12, acylation with an acid chloride,
chloroformate, or methanesulfonyl chloride gave the corresponding
amides, carbamates, and methylsulfonamide 13, followed by boc-
deprotection to 14 (Scheme 2).
Table 1
Piperidine/pyrrolidine H3R binding dataa
O
R2N
O
N
b
Entry
2
NR2
hH3 (Ki nM)
rH3 (Ki nM)
pKa
The substituted phenoxypropyl-(R)-2-methylpyrrolidine ana-
logs were tested using in vitro binding assays by displacement of
O
5.4
50
5.15
5.37
N
[3H]N- -methylhistamine ([3H]NAMH) in membranes isolated
a
NC
from CHO cells transfected with cloned human H3 or rat H3 recep-
tors. The data is shown in Tables 1 and 2. The (R)-2-methylpyrroli-
dine was previously reported to have optimum potency and was
fixed, while modifications were made on the western region of the
molecule.10 The morpholine analog 2 showed high affinity for both
hH3R and rH3R and an acceptable overall profile, except for the
pharmacokinetic properties as described in the introduction. The
first approach to reduce the pKa was to incorporate electron-with-
drawing groups around piperidine or pyrrolidine moieties (Table
1). Cyano compound 7 and CF3 8 were both tolerated for binding
affinity, but did not significantly lower the amine pKa. The difluoro
analogs 9–11 had high H3R affinity with lower calculated pKa values
(calculated using the ACD method). The 4,4-difluoropiperidine
analog 9 (pKa = 3.75) displayed acceptable metabolic stability in li-
7
8
6.3
2.5
23
N
F
F
9.1
5.91
F
N
F
9
11
9
43
48
3.75
3.12
F
F
N
F
10
N
F
F
11
3.4
21
3.50
ver microsomes, and IC50 values >30 lM for inhibition of cyto-
chrome P450 enzymes (CYP 1A2, 2C9, 2C19, and 3A4). In a rat PK
experiment, 9 showed a slight improvement in the B/P ratio and
N
a
Ki values are an average of 2 or more determinations. The assay-to-assay var-
iation was typically within 2.5-fold.
clearance compared to 2, however the iv t was too short
½
b
pKa calculated using ACD.
(t = 0.5 h) (Table 3). The second approach to lower the pKa was
½
O
O
O
Br
a
b
OH
O
Cl
O
Cl
3
4
5
O
O
c
d
R2N
R2N
O
Cl
O
N
6
2, 7-11
Scheme 1. Reagents and conditions: (a) Br(CH2)3Cl, K2CO3, acetone, reflux, >90% (b) bromine, ether, 0 °C ? rt, 50% (c) R2NH, K2CO3, MeCN, 80 °C, >90% (d) (R)-2-methyl-
pyrrolidine.HCl, NaI, 2-butanone, reflux, 30%.

A. L. Zulli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2807–2810
2809
Table 2
Boc
N
Boc
N
H
N
Piperazine amide H3R binding dataa
a
b
R1
N
N
H
N
R1
13
R1
N
O
N
12
14
O
N
R1
c
N
O
N
O
R2
b
Entry
16
R1
hH3 (Ki nM)
rH3 (Ki nM)
pKa
15 R2 = Cl
16-29 R2 =
O
2.8
30
3.95
d
Me
/
/
N
O
17
18
2.1
3.4
17
26
3.37
3.33
CF3
Me
/
Scheme 2. Reagents and conditions: (a) acid chloride, chloroformate, or MeSO2Cl,
TEA, DCM, rt, >90% (b) HCl, DCM, rt, 40% (c) 5, K2CO3, MeCN, 80 °C, >90% (d) (R)-2-
methylpyrrolidine.HCl, NaI, 2-butanone, reflux, 30–50%.
O
S
/
O
O
Table 3
Pharmacokinetic properties in rata
19
20
1.9
0.6
14
3.93
3.82
/
R-2c
9c
0.5 0.06
2.2 0.5
53 13
19c
25c
iva
po
t1/2 (h)
Vd (L/kg)
2.3 0.6
27 11
1.2 0.1
1.4 0.03
14
689 191
165 30
2.6 0.4
5.4 1.6
24
619 45
128
22
O
CL (mL/min/kg)
AUC (ng h/mL)
Cmax (ng/mL)
F (%)
126 28
2
8
/
5.8
81
17
14
50
4
1
1
1
266
66
15
5
5
2
2
6
1
F
14
1.5 0.1
2
O
O
O
B/Pb
25
4.3 0.1
21
22
23
4.2
2
9
16
9.4
3.82
3.84
3.92
/
/
/
a
b
c
Administration at 1 mg/mg iv and 5 mg/kg po.
B/P = brain to plasma ratio.
iv Formulation (3% DMSO, 30% solutol, 67% phosphate buffered saline) oral
S
formulation (oral formulation (50% Tween 80, 40% propylene carbonate and 10%
propylene glycol).
O
N
0.9
(Table 2). The binding data revealed that large substitutions were
tolerated in this region of the molecule as each of these compounds
showed high binding affinity for both hH3R and rH3R with the aryl
and heteroaryl amide-piperazine analogs (19–24) showing single
digit nanomolar affinity. The 4-benzoyl-piperazine analog 19
(pKa = 3.93) displayed high affinity for human H3R (Ki = 1.9 nM),
O
/
24
1.2
4
3.82
S
IC50 values >30
lM for inhibition of cytochrome P450 enzymes,
O
and acceptable in vitro metabolic stability in liver microsomes (t
>40 min across species). The rat PK for 19 displayed improvement
in the B/P ratio (1.5), Vd, and clearance compared with morpholine
½
25
26
27
3.2
4
19
23
12
3.91
3.95
3.90
MeO
EtO
/
O
2
(Table 3). In comparison, 2-thiophen-yl-piperazine 21
/
(pKa = 3.82) also met the affinity criteria, but displayed unaccept-
able rat PK and low bioavailability and brain partitioning (%F = 7%;
B/P = 0.6). Similarly, acetyl 16, methylsulfonyl 18, 2-furanyl 22,
and N-methylpyrrole 23 also showed low B/P ratios <0.6. Subse-
quently, a series of carbamate-piperazine analogs (25–29, Table 2)
was synthesized to expand on the amide-piperazine SAR. Each of
these compounds displayed excellent binding affinity for both
hH3R and rH3R with the substituted aryl carbamate-piperazine ana-
logs 28 and 29 showing a 3–6 fold increase in affinity for rH3R over
the alkyl carbamates 25–27, suggesting a tolerance for bulkier
groups in that region. The methyl carbamate 25 (pKa = 3.91) was
O
4.6
iBuO
/
O
28
29
3.9
3
6.9
3.7
3.76
3.90
O
/
F
O
O
/
a
Ki values are an average of 2 or more determinations. The assay-to-assay var-
iation was typically within 2.5-fold.
further profiled and showed P450 enzyme IC50 values >30 lM and
acceptable in vitro metabolic stability (t >40 min across species).
b
pKa calculated using ACD.
½
In a rat PK experiment, 25 showed a longer intrinsic iv t with plas-
½
ma and brain exposure in the acceptable range for use in potential
CNS indications (brain concentration 6 h following a 5 mg/kg po do-
to replace the morpholine with a piperazine where the terminal
nitrogen was capped as an amide or carbamate to reduce the pKa
se = 1 lM; B/P = 4.3) (Table 3). Similar to 25, the ethyl carbamate 26

2810
A. L. Zulli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2807–2810
Dunnett’s test). At 30 mg/kg, wake was significantly elevated for
240
180
120
60
2.5 h after dosing compared to vehicle, averaging approximately
33% above vehicle for the first 4 h after dosing, and maximal cumu-
lative wake surplus was 46 19 min at 9 h after dosing. Compound
25 did not increase wake time up to 30 mg/kg ip. The estimated
brain concentration 1 h following a 10 mg/kg ip dose was 300-fold
above the rat H3R Ki value.
In summary, an SAR study to modulate the pKa of the western
amine identified 9, 19, and 25 with lower B/P ratios and improved
PK properties compared to 2. In vivo proof-of-concept studies
showed the compounds demonstrated H3R functional activity in
the rat dipsogenia model, with compound 9 displaying modest
wake-promoting activity in the rat while the others displayed
weak to no activity. Due to relatively weak in vivo efficacy, this ser-
ies was not advanced.
*
*
0
Vehicle
1.0
3.0
10
30
Compound 9 (mg/kg i.p.)
Figure 2. Compound 9-induced wake promotion. Cumulative wake 4 h AUC values
following administration of vehicle or compound 9 in rats chronically implanted
References and notes
with electrodes for recording EEG and EMG activity. Mean + SEM, n = 5–8/group. ⁄
P
<0.05, Dunnett’s test versus vehicle.
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was equipotent and had an acceptable rat PK profile (%F = 33, iv
= 2.1 h, CL = 16 mL/min/kg, Vd = 2.8 L/kg, B/P = 2.4). However,
t
½
the ethyl analog inhibited CYP 2D6 (IC50 = 5
ther profiled.
lM) and was not fur-
Compounds 9, 19, and 25 displayed potent antagonist activity
(Kb = 3.7, 0.8, and 0.4 nM) and full inverse agonist activity
(EC50 = 1.8, 1.0, and 0.9 nM), respectively in the [35S]GTP
cS hH3R
binding assay. Compounds 9, 19, and 25 were selected to advance
to in vivo proof-of-concept efficacy studies using the rat dipsogenia
model and EEG/EMG model of sleep/wake activity. Histamine and
the H3-selective agonist (R)-a-methylhistamine (RAMH) induce
the behavior of drinking water when administered in the rat
peripherally or centrally, an effect that is blocked by H3R antago-
nists.11 Compounds 19 and 25 dose-dependently inhibited
RAMH-induced dipsogenia with ED50 values of 0.43 and 0.54 mg/
kg ip, about 10-fold weaker than irdabisant. Compound 9 showed
significant inhibition of RAMH-induced dipsogenia at 1.0 mg/kg ip.
Following the demonstration of the potent in vivo H3R func-
tional activity in the brain, 9, 19, and 25 were further evaluated
for wake promoting activity in the rat. Wake promoting activity
was measured as previously described using male Sprague–Dawley
rats surgically implanted for chronic recording of EEG (electroen-
cephalographic) and EMG (electromyographic) signals.12 Wake
activity was evaluated during the normal quiet period of the rat
with dosing at 5 h after light on. Compound 9 produced a dose-re-
lated increase in cumulative wake activity for 4 h after dosing (4 h
AUC) (P <0.001 ANOVA) with significant increases at 10
(150 15 min) and 30 mg/kg ip (195 16 min) compared to vehi-
cle (85 9 min) (Fig. 2). At 30 mg/kg, wake activity was greater
than the vehicle group up to 3.5 h post dosing, and maximal cumu-
lative wake surplus (cumulative wake activity compared to the
vehicle group) was 113 18 min at 5 h. No evidence of sleep re-
bound was observed at any dose. Compound 19 was tested at 3,
10, and 30 mg/kg ip and was weakly active at 30 mg/kg ip by 4 h
AUC (127 9 vs 92 8 min for vehicle, P = 0.03, ANOVA; P <0.05,
12. (a) Edgar, D. M.; Seidel, W. F. J. Pharmacol. Exp. Ther. 1997, 283, 757–769; (b)
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