Synthesis and evaluation of pyridazinone.phenethylamine derivatives as selective and orally bioavailable histamine H3 receptor antagonists with robust wake-promoting activity

Article abstract of DOI:10.1016/j.bmcl.2011.08.104

A series of pyridazinone-phenethylamine derivatives with moderate to low nanomolar affinity for rat and human H₃R are described. These analogs exhibited excellent selectivity and metabolic stability, with acceptable rat pharmacokinetic properties. In vivo, 7 and 11 demonstrated potent H₃R functional antagonism in the rat dipsogenia model and robust wake-promoting activity in the rat electroencephalogram/electromyography (EEG/EMG) model.

Full text of DOI:10.1016/j.bmcl.2011.08.104

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6362–6365
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of pyridazinone–phenethylamine derivatives
as selective and orally bioavailable histamine H3 receptor antagonists
with robust wake-promoting activity
⇑
Reddeppa reddy Dandu , John A. Gruner, Joanne R. Mathiasen, Lisa D. Aimone, Greg Hostetler,
Caitlyn Benfield, Robert J. Bendesky, Val R. Marcy, Rita Raddatz, 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:
A series of pyridazinone–phenethylamine derivatives with moderate to low nanomolar affinity for rat
and human H3R are described. These analogs exhibited excellent selectivity and metabolic stability, with
acceptable rat pharmacokinetic properties. In vivo, 7 and 11 demonstrated potent H3R functional antag-
onism in the rat dipsogenia model and robust wake-promoting activity in the rat electroencephalogram/
electromyography (EEG/EMG) model.
Received 29 June 2011
Revised 19 August 2011
Accepted 25 August 2011
Available online 1 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
H3R antagonists
Pyridazinone
(R)-2-Methylpyrrolidine
H3 receptor
Metabolic stability
Histamine acts via four G-protein coupled receptor subtypes
(H1R–H4R) and is implicated in a wide range of physiological and
patho-physiological functions.1 Antagonists that target the H1R
and H2R receptor subtypes, which are used in allergic conditions
and gastric acid disorders, respectively, have been highly success-
ful medicines for many years. The H4R subtype is mainly involved
in inflammatory and immuno-modulatory functions.2 The hista-
mine H3 receptor (H3R) located primarily in the brain functions
as an auto-receptor to modulate histamine release and as an inhib-
itory heteroreceptor, regulating the release of multiple neurotrans-
mitters including acetylcholine, dopamine, norepinephrine and
serotonin1 that are involved in attention, vigilance, and cognition.
Thus, H3R antagonists may have utility in addressing a variety of
CNS disorders associated with deficits in wakefulness, attention,
and cognition, including attention-deficit hyperactivity disorder
(ADHD), Alzheimer’s disease (AD), mild cognitive impairment,
and schizophrenia. The preliminary structure–activity relation-
ships (SAR) and characterization of 1 (CEP-26401, irdabisant) was
recently revealed3 and several additional compounds are under ac-
tive clinical investigation (Fig. 1).1g–i Modification of the aminopro-
pyloxy portion of 1 identified a new class of potent and selective
pyridazinone–phenethylamine analogs as H3R antagonists with ro-
bust in vivo wake promoting activity. In this Letter we present the
synthesis, SAR, selectivity, preliminary pharmacokinetic proper-
ties, and the in vivo activity of leads 7 and 11 in the rat dipsogenia
model and in rat EEG/EMG model of wakefulness.
The synthesis of pyridazinone–phenethylamine target 7 was
synthesized in six steps as illustrated in Scheme 1. The synthesis
of the key intermediate 34 commenced from commercially avail-
able phenethylacetate 2. Friedel–Craft acylation of 2 with succinic
anhydride and AlCl3 led to the key intermediate 3 in moderate
yield. Cyclocondensation of 3 with methyl hydrazine gave di-
hyro-pyridazinone derivative 4,3 which was easily oxidized with
Cu(II)Cl2 or SeO25 to furnish pyridazinone derivative 5 in excellent
yield and purity. Hydrolysis of 5 with K2CO3 in MeOH and water
produced phenethyl alcohol 6. The desired target 7 was synthe-
sized from alcohol 6 using MeSO2Cl and Et3N, followed by alkyl-
ation with (R)-2-methylpyrrolidine in the presence of K2CO3 in
moderate yield. In an analogous manner, analogs 8–14 were syn-
thesized using the appropriate hydrazine derivative and interme-
diate 3 via the sequence outlined for compound 7. Target 9 was
also synthesized using the synthetic sequence as described for
the compound 7, except pyrrolidine was used in the final N-alkyl-
ation step.
The pyridazinone–phenethylamine target compounds (7–14)
were tested using in vitro binding assays by displacement of
[3H]NAMH in membranes isolated from CHO cells transfected with
cloned human H3 or rat H3 receptors as illustrated in Table 1.3
Phenethylamine based H3 antagonists derived from the natural
⇑
Corresponding author.
E-mail address: rdandu@cephalon.com (R.r. Dandu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.104

R.r. Dandu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6362–6365
6363
Me
N
O
O
N
N
O
N
N
N
O
CF3
MK-0249
JNJ-311001074 (Bavisant)
F
O
N
Cl
O
N
N
F
H
BF2.649 (Pitolisant)
PF-03 654746
H
N
Me
N
O
O
N
1 CEP-26401 (Irdabisant)
Figure 1. Structures of H3R clinical antagonists.
O
a
b
HO
OAc
OAc
O
3
2
H3C
H3C
N N
O
H3C
N N
O
N N
H3C
N
O
c
d
OH
OAc
4
6
7
Scheme 1. Reagents and conditions: (a) succinic anhydride, AlCl3, 1,2-dichloroethane, 0–60 °C, 4 h, 50%; (b) MeNHNH2, 2-propanol, reflux, 2.5 h, 87%; (c) (i) Cu(II)Cl2, MeCN,
reflux, 4 h or SeO2, AcOH, 125 °C, 4 h, 87% ? 5; (ii) K2CO3, MeOH, H2O, rt to 50 °C, 1 h, 95%; (d) (i) MeSO2Cl, Et3N, CH2Cl2, 0 °C to rt, 3 h, 79%, (ii) (R)-2-methylpyrrolidine,
K2CO3, MeCN, 50 °C, 24–48 h, 44%.
hH4 receptor subtypes. Subsequently, compounds were tested for
the aqueous solubility (pH2 and pH7.4), in vitro microsomal stabil-
ity (rat, mouse, dog, and human), and inhibition of cytochrome
P450 isoforms (CYP1A2, 2C9, 2C19, 2D6, and 3A4). Selected com-
pounds were further screened in in vivo rat pharmacokinetic
experiments designed to evaluate iv intrinsic pharmacokinetic
Table 1
Pyridazinone–phenethylamine H3R binding data
R1
R2
N
N
N
O
parameters (t , Vd, and CL), oral bioavailability and brain penetra-
½
R1
R2
hH3R (Ki, nM)a
rH3R (Ki, nM)a
tion. Quality molecules at this stage were progressed into in vivo
efficacy models.
Changing the four atom phenoxypropyl of 1 to an ethyl linker
(analog 8) showed a 15-fold loss of hH3R affinity. However,
Compound
1
7
8
9
10
11
12
13
14
—
Me
H
Me
Phenyl
p-Fluoro-phenyl
p-Chloro-phenyl
Benzyl
—
2
7
7
(R)-Me
(R)-Me
H
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
58
43
135
42
48
26
18
242
31
83
34
13
8
Table 2
Rat pharmacokinetic profiles of 7 and 11a
7c
11c
5
47
2-Pyridyl
iv
AUC (ngꢀh/mL)
1647 405
0.5 0.1
1295 701
2.3 0.5
1
a
t
(h)
Ki values are an average of at least two determinations. The assay-to-assay
½
Vd (L/kg)
0.6 0.3
4.8 2.1
variation was typically within 2.5-fold.
CL (mL/min/kg)
12
4
21
7
product Conessine was recently reported by Arena.6 While explor-
ing the structure–activity relationships (SAR) of the pyridazinone–
phenethylamine series the (R)-2-methylpyrrolidine was fixed for
comparison based on earlier SAR optimization studies.3 The early
SAR exploration was focused at the R1 position of the pyridazinone
core to establish the discovery flow properties. In the discovery
flow, compounds meeting binding criteria (hH3R Ki <15 nM; rH3R
Ki <50 nM) were screened for selectivity against hH1, hH2, and
po
AUC (ngꢀh/mL)1
Cmax (ng/mL)
F (%)
B/Pb
28
18
<1
1
6
1733 106
255 14
27
2
6
0.3
11 0.3
a
b
c
Administration at 1 mg/mg iv and 5 mg/kg po
B/P = brain to plasma ratio 1 h post IP dosing.
iv formulation (3% DMSO, 30% solutol, 67% phosphate buffered saline); oral
formulation (50% Tween 80, 40% propylene carbonate and 10% propylene glycol).

6364
R.r. Dandu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6362–6365
A
B
*
240
*
*
240
180
120
60
180
*
120
60
0
*
0
Veh
10
30
Veh
3
10
30
Compound 7 (mg/kg, i.p.)
Compound 11 (mg/kg, i.p.)
Figure 2. Compounds 7- and 11-induced wake promotion. Cumulative wake 4 h AUC values following administration of vehicle (Veh), Compound 7 (A) or Compound 11 (B)
in rats chronically implanted with electrodes for recording EEG and EMG activity. Mean + SEM, n = 5–7/group. ⁄p <0.05, Dunnett’s test versus vehicle.
methylation of the N–H produced compound 7, which showed a 5-
fold improvement in hH3R affinity with a Ki value of 7 nM, but with
comparable rH3R affinity (Ki = 58 nM) to 8. Changing the (R)-2-
methylpyrrolidine of 7 to pyrrolidine 9 showed a 14-fold loss in
affinity. The N-phenyl 10 had Ki values of 34 and 42 nM for hH3R
and rH3R, respectively. Para substitution of the N-phenyl with flu-
oro 11 or chloro 12 showed 3–5-fold higher affinity compared to
10. The N-benzyl analog 13 was equipotent to the simple N-methyl
7 for hH3R, whereas the 2-pyridyl 14 showed equal affinity for
hH3R but weaker affinity for rH3R compared to 10. Based on the
initial binding SAR data, compounds 7 and 11 were selected for fur-
ther evaluation of selectivity, metabolic stability, cytochrome P450
inhibition, and rat pharmacokinetic parameters. Functionally, 7
and 11 showed potent antagonist activity and displayed full
inhibited RAMH-induced dipsogenia with ED50 values of 0.07 and
0.02 mg/kg, ip, respectively. This potent in vivo activity is consis-
tent with the potency and high CNS penetration of these
compounds.
Histaminergic neurons project from the tuberomamillary nu-
cleus to multiple brain regions involved in vigilance, attention
and sleep/wake. A number of H3R antagonists demonstrate wake
promoting activity in preclinical species8 and this effect has re-
cently been reported in clinical trials with the H3R antagonists
pitolisant and MK-0249.10 Compounds 7 and 11 were therefore
tested in the rat EEG/EMG model of wake promotion as previously
described (Fig. 2).3c,9 Compound 7 increased wake activity dose
dependently from 3–30 mg/kg ip based on 4 h AUC (P <0.001
ANOVA). Cumulative wake time at 10 mg/kg was 161 6 min, with
235 4 min wake achieved at 30 mg/kg ip by 4 h AUC values. At
30 mg/kg, waking was enhanced out to 4.5 h post dosing, and max-
imal cumulative wake surplus was ꢁ200 min reached at 7 h. No ef-
fect on sleep rebound was observed. At 30 mg/kg, 7 demonstrated
robust wake promotion, with the treated animals awake 98% of the
time up to 4 h post dose, a 68% increase in wake time over the vehi-
cle treated animals (Fig. 2). Compound 11 showed greater wake
activity at 10 mg/kg ip (207 5 min) compared to 7, and demon-
strated 94% wake time up to 4 h at 30 mg/kg ip (226 6 min)
(Fig. 2). At 30 mg/kg ip 11 was wake promoting for 7.5 h and no
sleep rebound was observed out to 22 h post dose. No adverse
EEG activity was noted for 7 or 11 at any dose. H3R antagonists
have been shown to produce robust effects in rodent wake models,
typically at doses higher than those in dipsogenia and cognition
models, corresponding to 80–90% receptor occupancy.9
inverse agonist activity in the [35S]GTP S hH3R binding assay3
c
N-Methyl 7 and 4-fluorophenyl 11 decreased basal activity with
EC50 values of 2 0.1 nM and 2.1 0.1 nM, respectively. Compound
7 exhibited favorable in vitro metabolic stability across species
in liver microsomes (t = mouse (25 min), rat (39 min), dog and
½
human (>40 min)) and had IC50 values of >30 lM for cytochrome
P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4. Furthermore, com-
pound 7 had excellent selectivity against hH1, hH2, and hH4 recep-
tor subtypes (<30% inhibition at 10 lM). In the rat, compound 7
exhibited acceptable rat iv pharmacokinetic properties (t = 0.5 h,
½
Vd = 0.5 L/kg, CL = 12 mL/min/kg). However, it demonstrated poor
oral exposure (F <1%) (Table 2).
Compound 11 displayed high hH3R affinity (Ki = 13 nM) and
moderate rat rH3R affinity (Ki = 48 nM). It showed excellent
in vitro microsomal stability (t >40 min in mouse, rat, dog, and hu-
½
man liver microsomes) and selectivity against cytochrome P450 iso-
In conclusion, design of a new series of pyridazinone–pheneth-
ylamine analogs and development of H3R structure–activity rela-
tionships identified two lead compounds with H3R target
potency, selectivity, metabolic stability, and rat pharmacokinetic
properties. Compounds 7 and 11 exhibited potent H3R functional
antagonist activity in vivo in the rat dipsogenia model and had ro-
bust wake-promoting activity in the rat EEG/EMG model. Com-
pound 11 demonstrated improved oral bioavailability (%F = 27)
compared to 7, however, ultimately due to hERG activity did not
advance further.
forms (IC50 >30
11 not only showed excellent selectivity for hH1, hH2, and hH4 recep-
tor subtypes (<30% inhibition at 10 M) and had good aqueous sol-
ubility (pH2/7.4 >125 g/mL), it also exhibited improved rat
lM, CYP1A2, 2C9, 2C19, 2D6, and 3A4). Compound
l
l
pharmacokinetic iv properties (t = 2.3 h, Vd = 4.8 L/kg, CL = 21 mL/
½
min/kg) and oral bioavailability (%F = 27, Cmax = 255 ng/mL) (Table
2). In addition, compound 11 had higher clogP (clogP = 4.1) and also
showed high brain exposure 1 h post a 10 mg/kg ip dose with a
brain/plasma ratio (B/P) = 11. Based on these overall profiles 7 and
11 were evaluated for in vivo activity in the rat dipsogenia model.
Administration of the H3R selective agonist, R-a-methylhista-
Acknowledgment
mine (RAMH), induces a transient increase in water drinking in
rats.7 This dipsogenic effect was used to measure in vivo H3R inhi-
bition following systemic administration of H3R antagonists, as
previously described.3 Compounds 7 and 11 dose-dependently
The authors thank Jamie Misler for excellent technical
assistance.

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