Novel morpholine ketone analogs as potent histamine H3 receptor inverse agonists with wake activity

Article abstract of DOI:10.1016/j.bmcl.2012.01.004

Structure-activity relationship on a novel ketone class of H3R antagonists/inverse agonists is disclosed. Compound 4 showed excellent target potency, selectivity and brain penetration. Evaluation of antagonist 4 in the rat EEG/EMG model demonstrated robust wake activity thereby establishing preclinical proof of concept.

Full text of DOI:10.1016/j.bmcl.2012.01.004

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1546–1549
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel morpholine ketone analogs as potent histamine H3 receptor inverse
agonists with wake activity
⇑
Babu G. Sundar , Thomas R. Bailey, Derek Dunn, Greg A. Hostetler, Sankar Chatterjee, Edward R. Bacon,
Christoph Yue, Dominique Schweizer, Lisa D. Aimone, John A. Gruner, Jacquelyn Lyons, Rita Raddatz,
Brigitte Lesur
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:
Structure–activity relationship on a novel ketone class of H3R antagonists/inverse agonists is disclosed.
Compound 4 showed excellent target potency, selectivity and brain penetration. Evaluation of antagonist
4 in the rat EEG/EMG model demonstrated robust wake activity thereby establishing preclinical proof of
concept.
Received 8 December 2011
Revised 2 January 2012
Accepted 3 January 2012
Available online 10 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
H3R inverse agonist
Morpholine ketones
Rat EEG/EMG
Wake activity
The histamine H3 receptor (H3R) is a G-protein coupled receptor
(GPCR) target of wide interest due to its ability to affect various
central nervous system (CNS) functions. H3Rs are predominantly
expressed in presynaptic neurons, and function as inhibitory auto-
and hetero-receptors. Blockade of H3R can modulate the release of
ligand design came from the weak H3 activity shown by aplys-
amine-1, a marine natural product ( Fig. 2).6 Subsequently, the
phenoxypropyl amine moiety was incorporated into the H3 ligands
by various research groups.1,7 The diamine feature of aplysamine-1
was also used as a pharmacophore in the design of some of the H3
ligands.8 While the presence of dibasic moiety in the ligand pro-
vided very potent compounds, further testing revealed pharmaco-
kinetic and potential safety issues due to phospholipidosis.
One of the initial diamine H3 antagonists with wake promoting
activity, JNJ-5207852, was reported to induce phospholipidosis and
possess high brain residence time.9 Following this, a related wake
promoting compound (JNJ-7737782) with substantially reduced
pKa for the benzylamine moiety was reported (Fig. 2).10 For our dis-
covery project, ligands were designed with a keto group on the
phenethyl side of aplysamine-1 pharmacophore generating a novel
chemical class.
An initial compound from this ketone series, 1 had high affinity
for human H3 receptor with a Ki of 32 nM (Table 1). But replacement
of piperidine (pKa = 7) on the benzoyl side with morpholine
(pKa = 4.7) led to relatively weaker compounds (2 and 3 with Ki of
66 and 106 nM, respectively). By incorporating the (R)-2-methyl-
pyrrolidine moiety on the propyloxy tail, the H3 binding affinity
was significantly improved as illustrated by compound 4 with
human and rat Ki of 5.5 and 50 nM, respectively. Incorporation of
(S)-2-methylpyrrolidine (Ki of 100 nM) or the achiral 2,2-dimethyl-
pyrrolidine (Ki of >1000 nM) led to big drop in binding affinity. Both
(R)- and (S)-enantiomers of 2-methoxymethylpyrrolidine were also
not tolerated. As shown in Table 1, methoxymethyl analogs
a variety of neurotransmitters including histamine,
c-aminobu-
tyric acid (GABA), acetylcholine (ACh), norepinephrine (NE), sero-
tonin (5-HT) and dopamine (DA).1 As a result of their potential to
modulate multiple CNS functions, H3R antagonists are being clini-
cally evaluated for the treatment of CNS disorders including neuro-
pathic pain and deficits in sleep/wake and cognition (Fig. 1).2 The
benzazepine GSK-189254 advanced into clinical trials for Alzhei-
mer’s disease, pain and narcolepsy. BF-2649 advanced for cognitive
enhancement in schizophrenic patients, daytime sleepiness in
Parkinson’s disease and sleep apnea syndrome.3 Merck-Banyu
compound MK-0249, advanced for schizophrenia, showed insom-
nia as a clinical effect.4 We recently reported a novel series of pyri-
dazin-3-one H3R antagonists/inverse agonists which includes our
clinical compound CEP-26401 (irdabisant).5 In this Letter, we
report our work on an earlier chemical class, morpholine ketones,
which led to the identification of potent, selective and brain pene-
trant antagonist 4 establishing preclinical proof of concept in the
rat sleep/wake model.
Although an advanced knowledge of H3 ligand pharmacophore
is currently available, some of the early pharmacophore based
⇑
Corresponding author. Tel.: +1 610 455 1025.
E-mail address: bgsundar@yahoo.com (B.G. Sundar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.004

B. G. Sundar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1546–1549
1547
Me
N
O
H
3C
N
H
O
N
N
N
N
N
O
O
CF3
GSK-189254
MK-0249
Me
H
N
N
O
N
O
O
Cl
BF-2649
CEP-26401
Figure 1. Structures of clinical H3R antagonists.
N
N
N
O
O
N
O
JNJ-5207852
JNJ-7737782
Br
O
Me
O
N
O
N
N
O
N
C
C pK a: 9.1
Br
p
K a: 4.7
Aplysamine-1
Compound 4
Figure 2. Structures of preclinical H3R antagonists.
(compound 7 and 8) showed H3 affinities in the micromolar range
which may be attributed to a combination of sterics and reduction
in amine pKa (from 10.2 for 2-methyl pyrrolidine to 9.4 for the 2-
methoxymethyl pyrrolidine) of the propyloxyamine end. With (R)-
2-methylpyrrolidine being the best amine moiety on the propyloxy
side, the effect of substitution on the ketone side was briefly exam-
imidazoline-I2 and human Sigma-1, respectively). Based on the
favorable target H3R affinity, selectivity, in vitro metabolic stability
in liver microsomes and cytochrome P450 (CYP) inhibition selec-
tivity (IC50 >30 lM for 1A2, 2C9, 2C19, 2D6, 3A4), 4 was further
evaluated for pharmacokinetic properties in the rat (Table 3). Com-
pound 4 displayed an iv t of 2.3 h, clearance of 126 mL/min/kg,
½
ined (Table 2). Both
a
-methyl ketone 9 and dimethyl ketone 10
and oral bioavailability of 14% based on 6 h AUC data with high
brain partitioning (ip 1 h b/p = 30.7). The relatively superior brain
exposure compared to the plasma compartment prompted us to
conduct the brain concentration time course study. Due to com-
pound availability issue, racemate of 4 (human and rat Ki of 12
and 56 nM, respectively) was used for this study with the prior
knowledge that there was no rat pharmacokinetics difference be-
tween the racemate and either of the enantiomers 4 and 5. After
10 mg/kg ip dosing, the brain level of rac. 4 at 1 h, 4 h and 6 h time
intervals was determined to be 3930 592, 1609 167 and
955 41 ng/g, respectively. At 24 h post dose, compound rac. 4
was not detectable in the brain showing the lack of the undesired
brain accumulation for this compound.
Following the demonstration of potency, selectivity and brain
partitioning, compound 4 was further evaluated for wake promot-
ing activity in the rat.12 It has been shown that H3R antagonists
increase wakefulness at higher doses and receptor occupancy lev-
els than those required for the cognition activity.13 Wake promot-
ing activity was measured as previously described using male
Sprague Dawley rats surgically implanted for chronic recording
of electroencephalographic (EEG) and electromyographic (EMG)
signals.14 Compound 4 increased wake activity dose dependently
at 30 and 60 mg/kg ip based on cumulative time awake for 4 h
post dosing (4 h AUC) (Fig. 3). AUC values were 118 7, 163 8,
and 222 6 min at 10, 30, and 60 mg/kg, respectively versus
101 7 min for vehicle. At 60 mg/kg the treated animals were
awake 93% of the time up to 4 h post dosing, a 2.2-fold increase
in percent time awake over the vehicle treated animals. Both
maintained the affinity with Ki of 8 and 9 nM, respectively. Meso-di-
methyl morpholine analog 11 also displayed potent binding affinity
(Ki of 10 nM). Substitution of aryl ring at the ortho position to the
keto group by small substituent such as Me did not offer any
improvements. As shown by compound 12, ortho methyl substitu-
tion led to reduced the binding affinity (Ki = 19 and 244 nM) for hu-
man and rat receptors.
In vitro metabolic stability testing in liver microsomes showed
that compound 9 was stable across species with t of >40 min
½
(mouse, rat, dog and human). Despite our best efforts, the diaste-
reomers from compound 9 could not be separated. This may be
attributed to potential rapid epimerization of the chiral center
via enolization of keto functional group. The dimethyl ketone 10
displayed poor liver microsome stability for further advancement
in our discovery flow with t = >40 min for mouse but 9, 10 and
8 min for rat, dog and human, respectively. The unsubstituted
compound 4 was stable across all four tested species with t of
>40 min (mouse, rat, dog and human). Functionally, compound 4
½
½
showed potent antagonist activity and displayed full inverse ago-
nist activity in the [35S]GTP S hH3R assay5a,11 decreasing the basal
c
activity with an EC50 value of 5.5 nM.
Based upon target affinity and physical property data (pH7.4 sol-
ubility >145 lg/mL, ACD-logD = 0.26 and Tripos-PSA = 43.5), com-
pound 4 was further profiled for selectivity against a panel of 70
GPCRs, ion channels and enzymes (MDS Pharma Services, LeadPro-
filer). Compound 4 displayed >50% inhibition at 10
tion only for two out of 70 targets (67% and 78% against rat
lM concentra-

1548
B. G. Sundar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1546–1549
Table 1
Table 3
Aminomethyl ketone H3R binding data
Pharmacokinetic properties for 4 in rata,b
O
Rat PK parameters
iv
4
O
Y
t
(h)
2.3 0.6
27 11
½
X
N
Vd (L/kg)
CL (mL/min/kg)
126 28
Compound
X
C
Y
hH3Ra,b (Ki, nM)
rH3R (Ki, nM)
po
ip
6 h-AUC (ng/h/mL)
% F
109
5
14 0.7
1
N
32
174
1 h-Brain conc. (ng/g)
B/Pc
3768 899
30.7 3.2
2
3
O
O
N
106
66
594
440
a
Administration of 1 mg/mg iv, 5 mg/kg po and 10 mg/kg ip; data calculated
from 6 h AUC values.
N
b
iv formulation: 3% DMSO, 30% solutol, 67% phosphate buffered saline; po for-
mulation: 50% Tween 80, 40% propylene carbonate and 10% propylene glycol; ip
formulation: 0.5% methylcellulose, 0.2% Tween 80 in water.
4
5
6
O
O
O
5.4
50
c
N
B/P = Brain to plasma ratio.
100
885
>1000
N
N
>1000
OMe
OMe
7
8
O
O
> 3000
1170
> 3000
> 3000
N
N
a
[3H]NAMH binding in membranes prepared from cells transfected with human
Figure 3. Compound 4-induced wake promotion. Compound 4 was administered ip
to male rats chronically implanted with electrodes for recording EEG and EMG
activity. Cumulative Wake 4 h AUC values shown for each dose (mean + SEM, n = 7–
or rat H3R.
b
Ki values are an average of at least two determinations. The assay-to-assay
variation was typically within 2.5-fold.
⁄
8/group). p <0.05 Dunnett’s post hoc versus vehicle.
Table 2
dosing, nor any adverse EEG activity was evident at any dose.
EEG activity in the theta range (ꢀ6–9 Hz) was increased 2–3 fold
at 1 h after dosing with compound 4 at 30 and 60 mg/kg ip (AN-
OVA P = 0.018; Dunnett’s test P <0.05) (Fig. 4). Theta peak fre-
quency was unchanged (ANOVA, P >0.05). The increased theta
power is consistent with previous reports of H3R antagonist activ-
ity, and has been suggested to be indicative of pro-cognitive
activity.15
The synthesis of discussed compounds was accomplished as
previously described (Scheme 1).11 Commercially available
4-hydroxyaryl alkyl ketones 13 were readily O-alkylated with
3-bromo-1-chloropropane and crude chloroether product was sub-
Aminomethyl ketone H3R binding data
X
O
O
N
R
Compound
R
X
hH3Ra,b (Ki, nM)
rH3R (Ki, nM)
O
N
N
N
4
H
5.4
50
O
O
9
H
H
8
9
105
33
jected
a-keto bromination with elemental bromine. The selective
10
amination of bromoketone 14 with cyclic amines was readily
accomplished under mild conditions either at room temperature
or at 60 °C. With the intermediate amino ketones 15 in hand, sub-
stitution of the less reactive chloro group with basic amines was
accomplished via an in situ halogen exchange reaction completing
the synthesis of compounds 1–9 and 11–12. The dimethylketone
analog 10 was synthesized by methylation of 15 (R = Me, X = H)
O
O
11
12
N
N
H
10
19
78
Me
244
[3H]NAMH binding in membranes prepared from cells transfected with human
a
at the
a-keto position via enolate chemistry followed by
amination.11
or rat H3R.
b
Ki values are an average of at least two determinations. The assay-to-assay
In summary, a novel morpholine ketone class of H3 antagonists
was disclosed from which, compound 4 displaying excellent H3R
target potency, selectivity and rat brain penetration was identified.
Advanced into in vivo, compound 4 demonstrated potent wake-
promoting activity in the rat EEG/EMG model thereby establishing
preclinical proof of concept for this project. Compound 4 also
showed pronounced enhancement of theta EEG with no adverse
events.
variation was typically within 2.5-fold.
slow-wave sleep (SWS) and rapid eye movement sleep (REMS)
latencies were also significantly increased at 30 and 60 mg/kg ip
(data not shown). No increase in sleep following the enhanced
wake period (i.e. sleep rebound) was observed up to 22 h post

B. G. Sundar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1546–1549
1549
Figure 4. Compound 4-induced enhancement of theta EEG power. Theta EEG power (FFT amplitude, mV2/Hz) for time periods up to 4 h after dosing with compound 4 at 30
and 60 mg/kg ip versus vehicle.
X
X
X
O
O
O
a, b
c
O
Cl
OH
O
Cl
z
N
R
R
R
15
13
R = H, Me; X = H, Me
Br
14
Z = C, O
e, f
d
R = Me
X = H
X
Me
O
O
O
Y
O
N
z
N
R
O
N
Y = amine
10
1-9, 11-12
Scheme 1. Reagents and conditions: (a) K2CO3, Br(CH2)3Cl, acetone, 80 °C, 15 h, 70–88%. (b) Br2, Et2O, rt, 50–70%; (c) cyclic amine, DIPEA/EtOH, rt or K2CO3/CH3CN, 60 °C, 2–
4 h; (d) pyrrolidine or piperidine, NaI, K2CO3, CH3CN, 80 °C, 15 h (40–60%, 2 steps); (e) LDA, THF, MeI, -78 °C ? rt, 60%; (f) (R)-2-methylpyrrolidine, DIPEA, 2-butanone,
wave, 180 °C, 30 min, 87%.
l-
6. (a) Pompni, S.A; Gullo, V.P; Horan, A.C; Patel, M.G; Coval, S; U.S. Patent
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Apodeca, R.; Barbier, A. J.; Lovenberg, T. W.; Carruthers, N. I. Bioorg. Med. Chem.
Lett. 2006, 16, 897.
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