Synthesis and evaluation of 4- and 5-pyridazin-3-one phenoxypropylamine analogues as histamine-3 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.04.028

A novel series of 4-pyridazin-3-one and 5-pyridazin-3-one analogues were designed and synthesized as H₃R antagonists. Structure-activity relationship revealed the 5-pyridazin-3-ones 8a and S-methyl 8b had excellent human and rat H₃R affinities, and acceptable pharmacokinetic properties. In vivo evaluation of 8a showed potent activity in the rat dipsogenia model and robust wake-promoting activity in the rat EEG/EMG model.

Full text of DOI:10.1016/j.bmc.2012.04.028

Bioorganic & Medicinal Chemistry 20 (2012) 3880–3886
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of 4- and 5-pyridazin-3-one phenoxypropylamine
analogues as histamine-3 receptor antagonists
⇑
Nadine C. Becknell , Jacquelyn A. Lyons, Lisa D. Aimone, Zeqi Huang, John A. Gruner,
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:
Received 18 November 2011
Revised 4 April 2012
Accepted 10 April 2012
Available online 20 April 2012
A novel series of 4-pyridazin-3-one and 5-pyridazin-3-one analogues were designed and synthesized as
H3R antagonists. Structure–activity relationship revealed the 5-pyridazin-3-ones 8a and S-methyl 8b had
excellent human and rat H3R affinities, and acceptable pharmacokinetic properties. In vivo evaluation of
8a showed potent activity in the rat dipsogenia model and robust wake-promoting activity in the rat EEG/
EMG model.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Histamine H3
H3R antagonists
CEP-26401
Pyridazinone
1. Introduction
2. Chemistry
Primarily located in the central nervous system (CNS), hista-
mine-3 receptors (H3R) function as regulators to modulate a wide
variety of neurotransmitters including histamine, acetylchloline,
dopamine, norepinephrine and serotonin.1–10 Consequently, H3R
antagonists have potential use for the treatment of a variety of
CNS diseases such as sleep and cognitive disorders, attention-deficit
hyperactivity (ADHD) and Alzheimer’s disease (AD).3–9 It is no sur-
prise that H3R has attracted a lot of attention from the pharmaceu-
tical industry in development of H3R ligands for the treatment of
these CNS disorders.3–9 We identified a novel class of pyridazin-3-
one H3R antagonists/inverse agonists with exceptional drug-like
properties, and in vivo profiles.11,12 6-{4-[3-(R)-2-methylpyrroli-
din-1-yl)propoxy]-phenyl-2H-pyridazin-3-one 1 (irdabisant; CEP-
26401) (Fig. 1) was selected as a clinical candidate and recently
completed phase I.12 During our H3 discovery project research, we
actively pursued a variety of structural core modifications.13–15
One strategy was to evaluate the various pyridazinone regiomers
and optimize the eastern portion of the core such as 2. This paper
describes the synthesis of 4- and 5-pyridazinone analogues 2 and
evaluation of their H3R binding structure–activity relationship
(SAR), pharmacokinetics (PK), selectivity and drug-like properties.
Synthesis of compounds 8a–e is shown in Scheme 1. Following
the literature procedure, 4,5-dichloro-2H-pyridazin-3-one 3 was
converted to 5-iodo-2H-pyridazin-3-one 4 in good yield.16,17 Pro-
tection of pyridazinone N–H with formaldehyde afforded 2-
hydroxymethyl-5-iodo-2H-pyridazin-3-one 5.16 Syntheses of tetra-
methyl-[1,3,2]dioxaborolanes 6 were described previously.12,13a,14
Substitution of mesylates 6 with various amines provided com-
pounds 7a, 7b. Palladium coupling of compounds 5 and 7a–e and
the subsequent loss of the hydroxymethyl group17 gave target com-
pounds 8a–e correspondingly.
N-Alkylation of pyridazinone 4 with bromomethyl methyl ether
provided compound 9 (Scheme 2). Palladium coupling of 9 and 7a
afforded compound 10. On the other hand, compounds 14–16 were
synthesized as shown in Scheme 3. N-Alkylation of pyridazinone 3
with bromomethyl methyl ether gave compound 11. Selective
methoxy displacement of either chloro in compound 11 could be
H
O
H
O
N
N
N
4
N
Me
R2
5
O
N
O
N
R1
(CH2)n
1
2
(Irdabisant; CEP-26401)
⇑
Corresponding author.
E-mail address: nadinebecknell@aol.com (N.C. Becknell).
Figure 1. Structures of pyridazin-3-one phenoxyproylamines H3R antagonists.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.028

N. C. Becknell et al. / Bioorg. Med. Chem. 20 (2012) 3880–3886
3881
O
O
O
Cl
Cl
a
b
HN
N
HN
N
HO
N
N
O
I
I
HN
N
3
4
5
d
R2
O
N
O
B
R1
O
B
c
(CH2)n
R2
O
O
8a R1 = H, R2 = Me, n = 1
O
N
8b R1 = S-Me, R2 = Me, n = 1
8c R1 = R-Me, R2 = Me, n = 1
8d R1 = S-Me, R2 = H, n = 2
8e R1 = R-Me, R2 = H, n = 2
R1
OMs
O
(CH2)n
R1
7a R1 = H, R2 = Me, n = 1
6a R1 = H
7b R1 = S-Me, R2 = Me, n = 1
7c R1 = R-Me, R2 = Me, n =1
7d R1 = S-Me, R2 = H, n = 2
7e R1 = R-Me, R2 = H, n =2
6b R1 = S-Me
6c R1 = R-Me
Scheme 1. Reagents and conditions: (a) 57% HI, H2O, 150 °C, 26 h, 46%; (b) 35% CH2O, H2O, 105 °C, 18 h, 84%; (c) (R)-2-methyl-pyrrolidine hydrochloride or piperidine, K2CO3,
CH3CN, reflux, 22 h, 48-71%; (d) tetrakis(triphenylphosphine)-palladium(0), K2CO3, DME-H2O (2:1), reflux, 48 h, 38–64%.
O
O
a
HN
N
MeO
N
N
O
I
I
4
9
MeO
N
N
b
Me
O
B
O
N
O
Me
10 (50%)
O
N
7a
Scheme 2. Reagents and conditions: (a) BrCH2OMe, iPr2NEt, CH2Cl2, rt, 22 h, 73%; (b) tetrakis(triphenylphosphine)palladium(0), K2CO3, DME-H2O (2:1), reflux, 4 h.
O
OMe
O
O
O
MeO
N
N
a
Cl
Cl
OMe
Cl
d
b
Cl
Cl
MeO
N
N
MeO
N
N
HN
N
Me
O
N
11
12
3
14 (44%)
c
MeO
H
N
O
N
O
O
e
d
N
N
Cl
MeO
N
Me
Me
N
OMe
OMe
OMe
O
N
O
N
13
16 (37%)
15 (27%)
Scheme 3. Reagents and conditions: (a) BrCH2OMe, iPr2NEt, CH2Cl2, rt, 22 h, 78%; (b) NaOMe, 1,4-dioxane, rt, 24 h, 94%; (c) NaOMe, MeOH, rt, 2 h, 98%; (d) 7a,
Tetrakis(triphenyl-phosphine)palladium(0), Na2CO3, DME-H2O (1:1), 100 °C, 24 h; (e) HCl, MeOH, 100 °C, 72 h.
accomplished by varying the solvent.18 Reaction of 11 with sodium
3. Results and discussion
methoxide in 1,4-dioxane provided 5-chloro-4-methoxy-2-
methoxymethyl-2H-pyridazin-3-one 12. Conversely, reaction of
11 with sodium methoxide in methanol afforded 4-chloro-5-meth-
oxy-2-methoxymethyl-2H-pyridazin-3-one 13. Palladium coupling
of 12 and 13 with 7a gave compounds 14 and 15, respectively. The
N-2-methoxymethyl was deprotected by heating 15 in an HCl
methanol solution to afford N–H 16. The attempt to remove N-2-
methoxymethyl in compound 14 was unsuccessful.
The H3R binding affinity SAR revealed that pyridazinone 5-regi-
omers showed, in general, high H3R affinity comparable with their
6-regiomers counterpart (Table 1).12 5-Regiomer 8a had compara-
ble H3R affinities as 1 (CEP-26401) in both human and rat as
disclosed previously.12 The effect of incorporating a chiral methyl
group on the propyl linker was investigated. Introduction of a
S-methyl on the propylamine side-chain 8b showed single digit

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N. C. Becknell et al. / Bioorg. Med. Chem. 20 (2012) 3880–3886
Table 1
H3R binding data for pyridazinones
Compound
Structure
hH3R Kia (nM)
rH3R Kia (nM)
H
O
N
N
1
2
3
7
Me
N
O
O
HN
N
8a
8b
9
4
Me
O
N
O
O
O
HN
N
3
Me
Me
O
O
O
O
N
N
N
N
Me
Me
Me
HN
N
8c
8d
8e
27
25
47
43
HN
N
34
O
HN
N
131
Me
O
O
MeO
MeO
N
N
10
14
4
5
14
13
Me
Me
O
N
N
OMe
N
N
O
MeO
N
N
O
15
10
20
19
62
Me
OMe
O
O
N
H
N
N
16
Me
OMe
N
O
a
Ki values are an average of 2 or more determinations. The assay-to-assay variation was typically within 2.5-fold.
nanomolar H3R affinities in both species with a eudismic ratio for R
over S-methyl (8c/8b) of 9 and 11 for hH3R and rH3R, respectively.
Replacement of the R-2-methylpyrrolidine with piperidine showed
a significant loss in H3R affinity. Similar to the R-2-methylpyrroli-
dine SAR, R-methyl piperidine 8e had weaker H3R affinities than
S-methyl 8d with a eudismic ratio of 2 and 4 for human and rat

N. C. Becknell et al. / Bioorg. Med. Chem. 20 (2012) 3880–3886
3883
Table 2
fraction in rat brain homogenate (45%), high permeability in the
Caco-2 assay, and high water solubility. Compound 8a was also
negative in the mini Ames assay, with and without metabolic acti-
vation. Based on it’s over all profiles 8a was evaluated for sleep-
wake activity in the rat EEG/EMG model.
Rat pharmacokinetics for 1, 8a, 8b and 10
112
8a12
8b
10
iv (1 mg/kg)
(h)
t
2.6
9.4
42
1.0 0.1
0.8 0.1
10
2.1 0.6
3.9 1.2
21
0.4 0.1
3.2 0.8
93 10
½
Vd (L/kg)
The rat dipsogenia model was used in the project as an in vivo
surrogate measure of H3R functional inhibition in the brain follow-
ing peripheral administration of compounds. Histamine and the
H3-selective agonist, RAMH induce water drinking in the rat when
administered either peripherally or centrally, an effect that is
blocked by H3R antagonists.19 Compound 8a showed potent func-
tional H3R antagonism in the rat dipsogenia model with an ED50 of
0.03 mg/kg, ip20 A number of H3R antagonists have been shown to
promote wake activity in preclinical species, including irdabisant
and its analogs.13,21–23 Compound 8a was tested in the rat EEG/
EMG model of wake promotion using rats surgically implanted
for chronic recording of EEG (electroencephalographic) and EMG
(electromyographic) signals.10,13,14,23 Compound 8a increased
wake activity dose dependently at 10 and 30 mg/kg ip based on
cumulative time awake for 4 h post dosing (4 h AUC) (Fig. 2).24
AUC values were 191 6 min at 10 mg/kg and 228 3 min at
30 mg/kg ip versus 76 6 min for vehicle. At 30 mg/kg the treated
animals were awake 95% of the time up to 4 h post dosing, a three-
fold increase in percent time awake over the vehicle treated ani-
mals. No increase in sleep following the enhanced wake period
(i.e. sleep rebound) was observed up to 22 h post dosing, nor was
any adverse EEG activity evident up to 30 mg/kg ip.
CL (mL/min/kg)
po (5 mg/kg)
AUC0ꢁt (ng h/mL)
Cmax (ng/mL)
%F
1
4
984
270
83
1292 41
401 126
22
762 63
187 19
24
42 19
53 10
6
3
1
2
B/Pa
2.6
1.8 0.4
6.2 1.9
ND
a
Based on the 6 h time point after 5 mg/kg po dosing.
240
***
***
180
120
60
0
Veh
10
30
Dose (mg/kg i.p.)
Figure 2. Compound 8a-induced wake promotion.24 Time awake for 4 h after
4. Conclusion
⁄⁄⁄
dosing with compound 8a at 10 and 30 mg/kg ip versus vehicle (Veh).
P <0.001
Dunnett’s test versus vehicle.
In summary, taken together the SAR revealed that the order of
H3R affinities for the pyridazinone regiomers was 6-regiomer ꢀ
5-regiomer > 4-regiomer. 5-Pyridazin-3-one 8a and 8b had single
digit nanomolar human H3R binding affinity with excellent selectiv-
ity and acceptable PK properties. In vivo 8a had potent H3R func-
tional activity in the rat dipsogenia model and showed robust
wake-promoting activity in the rat EEG/EMG model with no adverse
events.
H3R. Thus, 5-pyridazin-3-one regiomer combined with (R)-2-
methy-1-((S)-2-methyl-3-phenoxypropyl)pyrrolidine fragment 8b
was the most favorable for H3R affinities.
Next methoxy substitutions on the pyridazinone 2, 4 and 5-
positions were examined (Table 1). There was little effect on
hH3R affinity with methoxy substitution. For example, comparing
N-2-methoxymethyl 10 and 4-methoxy 14 had comparable hH3R
affinity with S-methyl 8b. And comparing 15 with 16 showed only
a 2-fold difference in H3R affinity. Thus, over all the order of affin-
ities for the pyridazinone regiomers was 6-regiomer ꢀ 5-regio-
mer > 4-regiomer.
5. Experimental section
5.1. General
Based on their H3R affinities, compounds 8a, S-methyl 8b and 10
were further profiled in the discovery flow. Compounds 8a, S-
methyl 8b and 10 had excellent selectivity over hH1R, hH2R and
hH4R subtypes with Ki values >10 lM. They had acceptable meta-
bolic stability in liver microsomes for human, mouse, rat, and mon-
All commercially available chemicals and solvents were used as
received. Compounds were purified by silica gel chromatography
using an ISCO apparatus with detection at 290 and 254 nm. Reverse
phase purification was accomplished using a Gilson 281 liquid han-
dler equipped with a Phenomenex C18 column (150 ꢂ 30 mm LD.,
key (t >40 min) and had low to no inhibition of cytochrome P450
½
S5 lM). Peak collection was triggered by UV detection at 215 and
enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) with IC50 values
>30 lM, indicating low potential for drug–drug interactions. Com-
254 nm. 1H NMR was obtained on a Bruker 400 MHz instrument
in the solvent indicated with tetramethylsilane as an internal stan-
dard. Coupling constants (J) are in hertz (Hz). Liquid chromatogra-
phy-mass spectrometry (LC/MS) was run on a Bruker Esquire 2000
ion trap LCMS. Compound purity was >96% determined by high
pressure liquid chromatography (HPLC) using a Zorbax RX-C8,
5 ꢂ 150 mm column, eluting with mixture of acetonitrile and water
containing 0.1% trifluoroacetic acid with a gradient of 10–100%.
Melting points were determined using a MEL-TEMP II and were
uncorrected. No attempts were made to optimize yields.
pounds 8a, S-methyl 8b and 10 were screened for PK properties in
the rat (Table 2). Compound 10 had poor PK properties with high
clearance, short half-life, low Cmax and AUC0ꢁt. Compound 8a had
acceptable rat PK properties but lower oral bioavailability and brain
to plasma ratio (B/P) compared with the 6-regiomer 1.12 S-methyl
8b had acceptable PK with moderate clearance (CL), Cmax and AUC
comparable with 8a. The volume distribution (Vd) and brain to
plasma ratio (B/P) for S-methyl 8b were much higher than 8a.
Compound 8a displayed potent antagonist and full inverse ago-
nist activity in the guanosine 50-(
binding assay.11,20 It showed excellent selectivity against a panel
70 GPCRs, ion channels and enzymes (<50% inhibition at 10 M;
MDS Pharma Services, LeadProfile screen) and hERG (IC50 = 10
c cS)
-thio)triphosphate ([35S]GTP
5.2. 5-Iodo-2H-pyridazin-3-one (4)
l
lM
A round bottom flask charged with 4,5-dichloro-2H-pyridazin-3-
one (1, 15.0 g, 90.9 mmol) and HI (57% in H2O, 100 mL, 0.80 mol)
was stirred at 140 °C for 24 h. After cooling to ambient temperature,
in a patch clamp assay). Compound 8a had low binding to plasma
proteins (mouse, rat, dog, and human 20–40%), high unbound

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N. C. Becknell et al. / Bioorg. Med. Chem. 20 (2012) 3880–3886
the black precipitate was collected by filtration and was washed
with H2O. This material was suspended in H2O (100 mL) and
Na2S2O3 was added in portions until the color turned pale yellow.
The solid material was collected by filtration and washed with
H2O. The crude material was dissolved in MeOH/CH2Cl2 (1:1,
300 mL) and filtered. The filtrate was concentrated and dried to give
12.9 g (64%) of 4 as brown solid. Mp 139–142 °C. 1H NMR (DMSO-d6
d): 7.54 (s, 1H), 8.08 (s, 1H), 13.25 (s, 1H). LCMS m/z: 222 (M).
at reflux for 20 h, the reaction was cooled to 80 °C and a small
amount of NaCNBH3 was added. The reaction was stirred for
5 min and then cooled to ambient temperature. The reaction was
diluted with MeOH–CH2Cl2 (1:3; 100 mL), washed with saturated
NaHCO3 solution, brine, dried (Na2SO4) and concentrated. The res-
idue was purified by column chromatography (10% MeOH in
CH2Cl2) to give 268 mg (51%) of 8b as yellow solid. Mp 154–
156 °C. 1H NMR (DMSO-d6, d): 1.01 (m, 6H), 1.29 (m, 1H), 1.64
(m, 2H), 1.87 (m, 1H), 2.05 (m, 2H), 2.19 (m, 1H), 2.27 (m, 1H),
2.55 (m, 1H), 3.04 (m, 1H), 3.94 (m, 2H), 7.06 (m, 3H), 7.79 (m,
2H), 8.29 (m, 1H), 13.01 (s, H). LCMS m/z: 328 (M+1). HPLC
(290 nm) tr 4.63 min, 99%.
5.3. 2-Hydroxymethyl-5-iodo-2H-pyridazin-3-one (5)
A round bottom flask charged with 4 (6.1 g, 27.6 mmol), form-
aldehyde (37% in H2O, 22 mL) and H2O (50 mL) was stirred at re-
flux for 18 h. After cooling to ambient temperature, the resulted
crystalline solid was collected by filtration, washed with a small
amount of H2O and dried to give 5.8 g (84%) of 5 as yellow crystal-
line solid. Mp 139–140 °C. 1H NMR (CDCl3, d): 4.15 (m, 1H), 5.50 (d,
2H, J = 8.5 Hz), 7.50 (d, 1H, J = 1.9 Hz), 7.95 (d, 1H, J = 1.9 Hz). LCMS
m/z: 222 (Mꢁ30).
Compounds 8c–e were synthesized using the methods for 8b.
5.9. 5-{4-[(R)-2-Methyl-3-((R)-2-methyl-pyrrolidin-1-yl)-propo-
xy]-phenyl}-2H-pyridazin-3-one (8c)
White solid. Yield 39%. Mp 130–133 °C. 1H NMR (DMSO-d6, d):
0.96 (d, 3H, J = 4.9 Hz), 1.01 (d, 3H, J = 6.3 Hz), 1.26 (m, 1H), 1.64
(m, 2H), 1.84 (m, 1H), 1.97 (m, 1H), 2.05 (m, 2H), 2.25 (m, 1H),
2.72 (m, 1H), 3.10 (m, 1H), 3.88 (m, 1H), 4.08 (m, 1H), 7.04 (m,
3H), 7.79 (m, 2H), 8.29 (m, 1H), 13.00 (s, 1H). LCMS m/z: 328
(M+1). HPLC (290 nm) tr 4.71 min, 99%.
5.4. (R)-2-Methyl-1-{(S)-2-methyl-3-[4-(4,4,5,5-tetramethyl-[1,
3,2]dioxaborolan-2-yl)-phenoxy]-propyl}-pyrrolidine (7b)
A round bottom flask charged with 6b (2.1 g, 5.6 mmol), (R)-2-
methyl-pyrrolidine hydrochloride (1.4 g, 11.1 mmol), K2CO3 (5.0 g,
36.2 mmol) and acetonitrile (35 mL) was stirred at reflux for 22 h.
After cooling to ambient temperature, the reaction was filtered and
the residue was concentrated. The produce was purification by col-
umn chromatography (2% MeOH in CH2Cl2) to give 900 mg (45%) of
7b. 1H NMR (DMSO-d6, d): 0.99 (m, 6H), 1.07 (m, 2H), 1.27 (s, 12H),
1.63 (m, 2H), 1.85 (m, 1H), 2.03 (m, 2H), 2.17 (m, 1H), 2.53 (m, 1H),
3.02 (m, 1H), 3.78–3.94 (m, 2H), 6.92 (m, 2H), 7.59 (m, 2H). LCMS
m/z: 360 (M+1).
5.10. 5-[4-((S)-2-Methyl-3-piperidin-1-yl-propoxy)-phenyl]-2H-
pyridazin-3-one (8d)
White solid. Yield 49%. Mp 145–148 °C. 1H NMR (DMSO-d6, d):
0.98 (d, 3H, J = 5.2 Hz), 1.38 (m, 2H), 1.50 (m, 4H), 2.13 (m, 2H),
2.30 (m, 5H), 3.87 (m, 1H), 4.02 (m, 1H), 7.06 (m, 3H), 7.79 (m,
2H), 8.29 (m, 1H), 13.00 (s, 1H). LCMS m/z: 328 (M+1). HPLC
(290 nm) tr 4.63 min, 99%.
Compounds 7c–e were synthesized using the methods for 7b.
5.11. 5-[4-((R)-2-Methyl-3-piperidin-1-yl-propoxy)-phenyl]-2H-
pyridazin-3-one (8e)
5.5. (R)-2-Methyl-1-{(R)-2-methyl-3-[4-(4,4,5,5-tetramethyl-[1,
3,2]dioxaborolan-2-yl)-phenoxy]-propyl}-pyrrolidine (7c)
White solid. Yield 40%. Mp 160–163 °C. 1H NMR (DMSO-d6, d):
0.98 (d, 3H, J = 4.9 Hz), 1.38 (m, 2H), 1.49 (m, 4H), 2.13 (m, 2H),
2.30 (m, 5H), 3.86 (m, 1H), 4.02 (m, 1H), 7.06 (m, 3H), 7.79 (m,
2H), 8.29 (m, 1H), 13.00 (s, 1H). LCMS m/z: 328 (M+1). HPLC
(290 nm) tr 4.67 min, 98%.
Yield 48%. 1H NMR (DMSO-d6, d): 0.95 (d, 3H, J = 5.7 Hz), 0.99 (d,
3H, J= 6.4 Hz), 1.07 (m, 2H), 1.27 (s, 12H), 1.63 (m, 2H), 1.84 (m, 1H),
2.03 (m, 2H), 2.24 (m, 1H), 2.70 (m, 1H), 3.09 (m, 1H), 3.81 (m, 1H),
4.03 (m, 1H), 6.90 (m, 2H), 7.59 (m, 2H). LCMS m/z: 360 (M+1).
5.12. 5-Iodo-2-methoxymethyl-2H-pyridazin-3-one (9)
5.6. 1-{(S)-2-Methyl-3-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaboro-
lan-2-yl)-phenoxy]-propyl}-piperidine (7d)
To a solution of 4 (2.8 g, 12.8 mmol), N,N-diisopropylethylamine
(3.3 mL, 19.2 mol), 4-dimethylaminopyridine (80 mg, 0.6 mmol)
and CH2Cl2 (30 mL) at 0 °C was added bromomethyl methyl ether
(2.13 mL, 25.6 mmol) and stirred at ambient temperature for
18 h. The reaction was filtered and the filtrate was concentrated.
The residue was purified by column chromatography (CH2Cl2) to
give 2.41 g (70%) of 9. 1H NMR (DMSO-d6, d): 3.31 (s, 3H), 5.25 (s,
2H), 7.65 (m, 1H), 8.17 (m, 1H). LCMS m/z: 267 (M+1).
Yield 62%. 1H NMR (DMSO-d6, d): 0.96 (d, 3H, J = 5.9 Hz), 1.07
(m, 1H), 1.27 (s, 12H), 1.37 (m, 2H), 1.48 (m, 4H), 2.10 (m, 2H),
2.28 (m, 4H), 3.78 (m, 1H), 3.97 (m, 1H), 6.92 (m, 2H), 7.59 (m,
2H). LCMS m/z: 360 (M+1).
5.7. 1-{(R)-2-Methyl-3-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaboro-
lan-2-yl)-phenoxy]-propyl}-piperidine (7e)
5.13. 2-Methoxymethyl-5-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-
propoxyl]-phenyl}-2H-pyridazin-3-one (10)
Yield 71%. 1H NMR (DMSO-d6, d): 0.96 (d, 3H, J = 5.9 Hz), 1.07
(m, 1H), 1.27 (s, 12H), 1.37 (m, 2H), 1.48 (m, 4H), 2.10 (m, 2H),
2.29 (m, 4H), 3.79 (m, 1H), 3.98 (m, 1H), 6.92 (m, 2H), 7.59 (m,
2H). LCMS m/z: 360 (M+1).
A round bottom flask charged with 9 (119 mg, 0.45 mol), 7a
(201 mg, 0.58 mmol), tetrakis(triphenylphosphine)palladium(0)
(52 mg, 0.045 mmol), K2CO3 (187 mg, 1.35 mmol), DME (12 mL)
and H2O (4 mL) was flashed with nitrogen for 15 min. After stirring
at 85 °C for 4 h, the reaction was cooled to ambient temperature
and concentrated. The residue was purified by column chromatog-
raphy (5% MeOH in CH2Cl2) to give 80 mg (39%) of 10 as white so-
lid. Mp 183–185 °C. 1H NMR (DMSO-d6, d): 1.36 (d, 2H, J = 6.4 Hz),
1.60 (m, 1H), 1.84–2.29 (m, 5H), 3.13 (m, 2H), 3.35 (s, 3H), 3.46 (m,
2H), 3.64 (m, 1H), 4.16 (m, 2H), 5.33 (s, 2H), 7.10 (m, 2H), 7.21 (m,
5.8. 5-{4-[(S)-2-Methyl-3-((R)-2-methyl-pyrrolidin-1-yl)-propo-
xy]-phenyl}-2H-pyridazin-3-one (8b)
A round bottom flask charged with 5 (0.50 g, 1.98 mol), 7b
(0.78 g, 2.18 mmol), tetrakis(triphenylphosphine)palladium(0)
(0.23 g, 0.20 mmol), K2CO3 (1.37 g, 9.92 mmol), DME (20 mL) and
H2O (20 mL) was flashed with nitrogen for 15 min. After stirring

N. C. Becknell et al. / Bioorg. Med. Chem. 20 (2012) 3880–3886
3885
1H), 7.87 (m, 2H), 8.41 (m, 1H). LCMS m/z: 358 (M+1). HPLC
(290 nm) tr 5.31 min, 98%.
stirring at 100 °C for 16 h, the reaction was cooled to ambient tem-
perature and extracted with CH2Cl2 (2 ꢂ 100 mL). The combined
organic layers were washed with 1 N HCl solution (30 mL), H2O,
brine, dried (Na2SO4) and concentrated. The residue was
purified by column chromatography (5–8% MeOH in CH2Cl2) to
give 1.08 g (27%, 2 steps) of 15 as off white solid. Mp 44–45 °C.
1H NMR (DMSO-d6, d): 0.77–2.36 (m, 10 H), 2.83–3.70 (m, 7H),
3.94 (s, 3H), 4.09 (m, 2H), 5.34 (s, 2H), 6.96 (m, 2H), 7.40 (m,
2H), 8.29 (s, 1H). LCMS m/z: 388 (M+1). HPLC (290 nm) tr
5.62 min, 96%.
5.14. 4,5-Dichloro-2-methoxymethyl-2H-pyridazin-3-one (11)
To a solution of 3 (10.0 g, 61.6 mmol), N,N-diisopropylethyl-
amine (15.8 mL, 90.9 mmol) and CH2Cl2 (80 mL) at 0 °C was added
bromomethyl methyl ether (7.50 mL, 94.8 mmol) slowly. After stir-
ring at ambient temperature for 18 h, the reaction was concentrated
and the residue was dissolved in small amount of CH2Cl2, then di-
luted with Et2O (300 mL). The reaction was filtered and the filtrate
was concentrated. The residue was purified by column chromatog-
raphy (1% MeOH in CH2Cl2) give 9.92 g (78%) of 11. 1H NMR (DMSO-
d6, d): 3.33 (s, 3H), 5.35 (s, 2H), 8.23 (s, 1H). LCMS m/z: 209 (M+1).
5.19. 5-Methoxy-4-{4-[3-((R)-2-methyl-pyrolidin-1-yl)-propo-
xy]-phenyl}-2H-pyridazin-3-one (16)
A round bottom flask charged with 15 (1.07 g (2.76 mmol), con-
centrated HCl (10 mL) and MeOH (10 mL) was stirred at 100 °C for
3 days. After cooling to ambient temperature, the reaction was
neutralized with 10 N NaOH solution to pH 7. The reaction was
washed with CH2Cl2 (100 mL) and the water layer was concen-
trated. The residue was dissolved in CH2Cl2 and filtered. The filtrate
was concentrated and the residue was purified by column chroma-
tography (15% MeOH in CH2Cl2) to give 350 mg (37%) of 16 as
white solid. Mp 135–140 °C. 1H NMR (DMSO-d6, d): 1.04–2.27
(m, 9H), 3.09 (m, 2H), 3.27-3.71 (m, 3H), 3.91 (s, 3H), 4.10 (m,
2H), 6.96 (m, 2H), 7.43 (m, 2H), 8.18 (s, 1H), 12.98 (s, 1H). LCMS
m/z: 344 (M+1). HPLC (290 nm) tr 4.47 min, 99%.
5.15. 5-Chloro-4-methoxy-2-methoxymethyl-2H-pyridazin-3-
one (12)
To a solution of 11 (2.00 g, 9.57 mmol) and 1,4-dioxane (30 mL)
was added sodium methoxide (1.0 g, 19.1 mmol) and then stirred
for 24 h. Additional 0.50 g of sodium methoxide was added and
stirring continued for 24 h. The reaction was quenched with H2O
(50 mL), then extracted with CH2Cl2 (2 ꢂ 100 mL). The combined
organic layers were washed with brine, dried (Na2SO4) and concen-
trated to give 1.79 g of 12 (91%). This material was used in the next
step without further purification. 1H NMR (DMSO-d6, d): 3.33 (s,
3H), 4.15 (s, 3H), 5.31 (s, 2H), 8.06 (s, 1H). LCMS m/z: 205 (M+1).
5.20. Rat H3 assays
5.16. 4-Methoxy-2-methoxymethyl-5-{4-[3-((R)-2-methyl-pyrr-
olidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one (14)
5.20.1. Cell line development and membrane preparation
The rat H3 receptor cDNA was PCR amplified from reverse-tran-
scribed RNA pooled from rat thalamus, hypothalamus, striatum and
prefrontal cortex with a sequence corresponding to bp #338-1672
of Genbank file #NM_053506, encoding the entire 445-amino-acid
rat histamine H3 receptor. This was engineered into the pIRES-neo3
mammalian expression vector, which was stably transfected into
the CHO-A3 cell line (Euroscreen, Belgium), followed by clonal
selection by limiting dilution. Cells were harvested and cell pellets
were frozen (ꢁ80 °C). Cell pellets were resuspended in 5 mM Tris–
HCl, pH 7.5 with 5 nM EDTA and a cocktail of protease inhibitors
(Complete Protease Inhibitor Tablets, Roche Diagnostics). Cells
were disrupted using a polytron cell homogenizer and the suspen-
sion was centrifuged at 1000ꢂg for 10 min at 4 °C. This membrane
pellet was washed in membrane buffer containing 50 mM Tris–HCl,
pH 7.5 with 0.6 mM EDTA, 5 mM MgCl2 and protease inhibitors,
recentrifuged as above and the final pellet resuspended in mem-
brane buffer plus 250 mM sucrose and frozen at ꢁ80 °C.
A round bottom flask charged with 12 (1.79 g, 8.75 mol), 7a
(3.32 g, 9.62 mmol), tetrakis(triphenylphosphine)palladium(0)
(1.00 g, 0.87 mmol), Na2CO3 (4.64 g, 43.7 mmol), DME (20 mL)
and H2O (20 mL) was flashed with nitrogen for 30 min. After stir-
ring at 90 °C for 17 h, the reaction was cooled to ambient temper-
ature and extracted with CH2Cl2 (2 ꢂ 100 mL). The combined
organic layers were washed with brine, dried (Na2SO4) and concen-
trated. The residue was purified by column chromatography (5% to
10% MeOH in CH2Cl2) to give 2.27 g (67%, 2 steps) of 14 as brown
oil. 1H NMR (DMSO-d6, d): 1.02 (d, 3H, J = 5.4 Hz), 1.30 (m, 1H),
1.65 (m, 2H), 1.88 (m, 3H), 1.99-2.38 (m, 3H), 2.93 (m, 1H), 3.10
(m, 1H), 3.37 (s, 3H), 3.97 (m, 3H), 4.08 (m, 2H), 5.35 (s, 2H),
7.05 (m, 2H), 7.58 (m, 2H), 8.03 (s, 1H). LCMS m/z: 388 (M+1). HPLC
(290 nm) tr 5.93 min, 98%.
5.17. 4-Chloro-5-methoxy-2-methoxymethyl-2H-pyridazin-3-
one (13)
5.20.2. Radioligand binding
To a solution of 11 (2.20 g, 10.5 mmol) in MeOH (50 mL) was
added sodium methoxide (1.14 g, 21.0 mmol) then stirred for
24 h. Additional 0.50 g of sodium methoxide was added and stirring
was continued for 24 h. The reaction was quenched with H2O
(30 mL), then extracted with CH2Cl2 (2 ꢂ 100 mL). The combined
organic layers were washed with brine, dried (Na2SO4) and concen-
trated give 2.12 g of 13 (92%). This material was used in the next
step without further purification. 1H NMR (DMSO-d6, d): 3.31 (s,
3H), 4.10 (s, 3H), 5.35 (s, 2H), 8.30 (s, 1H). LCMS m/z: 205 (M+1).
Membranes were resuspended in 50 mM Tris–HCl (pH 7.4),
5 mM MgCl2, 0.1% BSA. The membrane suspensions (10 lg protein
per well) were incubated in a 96 well microtiter plate with [3H]-N-
alpha-methylhistamine (approximately 1 nM final concentration),
test compounds at various concentrations (0.01 nM–30
scintillation proximity beads (Perkin Elmer, FlashBlueGPCR Scintil-
lating Beads) in a final volume of 80 l for 4 h at room temperature,
protected from light. Non-specific binding was determined in the
presence of 10 M clobenpropit. Radioligand bound to receptor,
lM) and
l
l
and therefore in proximity to the scintillation beads, was measured
5.18. 5-Methoxy-2-methoxymethyl-4-{4-[3-((R)-2-methyl-pyrr-
olidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one (15)
using a MicroBeta scintillation counter.
5.21. Human H3 assays
A round bottom flask charged with 13 (2.12 g, 8.75 mol), 7a
(3.94 g, 11.4 mmol), tetrakis(triphenylphosphine)palladium(0)
(1.20 g, 1.04 mmol), Na2CO3 (5.49 g, 51.8 mmol), DME (30 mL)
and H2O (30 mL) was flashed with nitrogen for 20 min. After
5.21.1. Membrane preparation
CHO cells stably expressing the human H3 receptor (GenBank:
NM_007232) were harvested and cell pellets were frozen

3886
N. C. Becknell et al. / Bioorg. Med. Chem. 20 (2012) 3880–3886
(ꢁ80 °C). Cell pellets were resuspended in 5 mM Tris–HCl, pH 7.5
with 5 nM EDTA and a cocktail of protease inhibitors (Complete
Protease Inhibitor Tablets, Roche Diagnostics). Cells were disrupted
using a polytron cell homogenizer and the suspension was centri-
fuged at 1000ꢂg for 10 min at 4 °C. The pellet was discarded and
the supernatant centrifuged at 40,000ꢂg for 30 min at 4 °C. This
membrane pellet was washed in membrane buffer containing
50 mM Tris–HCl, pH 7.5 with 0.6 mM EDTA, 5 mM MgCl2 and pro-
tease inhibitors, recentrifuged as above and the final pellet resus-
pended in membrane buffer plus 250 mM sucrose and frozen at
ꢁ80 °C.
prior to the initiation of the drinking trial period. Percent inhibition
of RAMH-induced drinking was calculated for each rat based on
normalization to the group mean RAMH-induced drinking using
the following equation: [100 ꢁ (Dr/(DgRAMH)) ꢂ 100], where Dr is
the amount of water an individual rat drinks and DgRAMH is the
group mean for the amount of water consumed by the RAMH-trea-
ted group. Group mean values for percent inhibition were then cal-
culated for each dosage group together with the associated
standard deviation and standard error of the mean. Treatment ef-
fects for percent inhibition vs RAMH-induced dipsogenia were
evaluated using a one-way ANOVA (GraphPad Prism 4). Dunnett’s
post hoc analysis was performed for multiple comparisons with
the RAMH group set as the control comparator.
5.21.2. Radioligand binding
The same method described above (5.20.2) was applied.
References and notes
5.22. Pharmacokinetics
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Adult male Sprague–Dawley rats (275–350 g; Charles River,
Kingston, NY) were used in the experiments. All animal usage
was approved by the Cephalon IUCAC. For routine compound
screening rats were dosed via the lateral rail vein at the indicated
dose for iv administration (3% DMSO, 30% Solutol, 67% phosphate
buffered saline or 100% saline) or via oral gavage (50% Tween 80,
40% propylene carbonate, and 10% propylene glycol, saline, or 2%
HCl–water) at the indicated dose. Rat were fasted overnight prior
to po administration. Serial blood samples was collected from the
lateral tail vein into heparinized collection tubes (approximately
0.25 mL) at seven sampling times over a 6 or 24 h period as indi-
cated. The plasma was separated by centrifugation, and the sample
was prepared for analysis HPLC/MS by protein precipitation with
acetonitrile. The plasma samples were analyzed for drug and inter-
nal standard via LC–MS/MS protocol. The pharmacokinetic param-
eters were calculated by a noncompartmental method using
WinNonlin software (Professional version 4.1, Pharsight Corpora-
tion, Palo Alto, CA, 1997). For experiments to detemine detailed
rat PK parameters, rats were administered 1 mg/kg iv and 3 mg/
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plasma concentrationꢁtime data (n = 12).
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5.23. Plasma protein binding
Test compounds were dissolved in DMSO and spiked into plas-
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(pH 7.4). The final concentration in plasma or PBS was 5 lM. The
16. Krajsovszky, G.; Károlyházy, L.; Riedl, Zs.; Csámpai, A.; Dunkel, P.; Lernyei, Á.;
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mixtrues were incubated in a 37 °C water bath with gentle shaking
for 1 h and then were loaded into the MultiScreen Ultracell-PPB
plate (Millipore Inc., Billerica, MA) that was centrifuged at 2000 g
for 45 min at 37 °C. The amount of compound present in the ultra-
filtrate was determined using LC/MS/MS. The percentage of drug
bound to plasma was calculated using mean peak area of test com-
pound in plasma ultrafiltrate (as free) and mean peak area of com-
pound in PBS buffer (as total). The results are the mean of duplicate
determinations.
19. Clapham, J.; Kilpatrick, G. J. Eur. J. Pharmacol. 1993, 232, 99.
20. The H3R SAR was developed using in vitro binding assays by displacement of
[3H]N- -methylhistamine ([3H]NAMH) in membranes isolated from CHO cells
a
transfected with cloned human H3 or rat H3 receptors. An H3R binding assay
using membranes prepared from rat cortex was used to compare the
recombinant rat assay to a native tissue.
21. Lin, J. S.; Dauvilliers, Y.; Arnulf, I.; Bastuji, H.; Anaclet, C.; Parmentier, R.;
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5.24. Rat dipsogenia model
22. James, L. M.; Iannone, R.; Palcza, J.; Renger, J. J.; Calder, N.; Cerchio, K.;
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Rat dipsogenia was conducted as previously described.11,19
RAMH-induced water intake was measured in Harlan Long Evans
rats (>300 g; Harlan, Dublin, VA, or Indianapolis, IN) for 30 min
beginning 20 min after administration of RAMH (10 mg/kg ip). Test
compound (in saline) was administered at the indicated times
24. Note in Methods: For dose response studies, all doses (including vehicle)
compared by ANOVA followed by Dunnett’s post-hoc multiple comparison test.