4,5-Dihydropyridazin-3-one derivatives as histamine H3 receptor inverse agonists

Article abstract of DOI:10.1016/j.bmcl.2011.11.037

H₃R structure-activity relationships for a new class of 4,5-dihydropyridazin-3-one H₃R antagonists/inverse agonists are disclosed. Modification of the 4,5-dihydropyridazinone moiety to block in vivo metabolism identified 4,4-dimethyl

Full text of DOI:10.1016/j.bmcl.2011.11.037

Bioorganic & Medicinal Chemistry Letters 22 (2012) 194–198
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4,5-Dihydropyridazin-3-one derivatives as histamine H3 receptor
inverse agonists
⇑
Robert L. Hudkins , Lisa D. Aimone, Reddeppa reddy Dandu, Derek Dunn, John A. Gruner, Zeqi Huang,
Kurt A. Josef, Jacquelyn A. Lyons, Joanne R. Mathiasen, Ming Tao, Allison L. Zulli, Rita Raddatz
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:
H3R structure–activity relationships for a new class of 4,5-dihydropyridazin-3-one H3R antagonists/
inverse agonists are disclosed. Modification of the 4,5-dihydropyridazinone moiety to block in vivo
metabolism identified 4,4-dimethyl-6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-4,5-dihy-
dro-2H-pyridazin-3-one 22 as a lead candidate demonstrating potent in vivo functional H3R antagonism
in the rat dipsogenia model and robust wake promoting activity in the rat EEG/EMG model.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 30 September 2011
Revised 8 November 2011
Accepted 9 November 2011
Available online 16 November 2011
Keywords:
Histamine H3 receptor
H3 inverse agonist
CEP-26401
4,5-Dihydropyridazin-3-one
Sleep-wake
The histamine H3 receptor (H3R) is localized primarily presyn-
aptically in the brain where it functions both as an autoreceptor
modulating histamine release and as an inhibitory heteroreceptor
regulating the release of multiple neurotransmitters, including
acetylcholine, dopamine, norepinephrine and serotonin thought
to be involved in attention, sleep and cognition.1 Activation of
the H3R results in the inhibition of neurotransmitter release, while
blockade of H3R by selective antagonists or inverse agonists would
reverse the histamine-mediated inhibition, leading to enhanced
neurotransmitter release. The discovery of H3R antagonists is of
current interest with potential for treating multiple CNS disorders
associated with attention and cognitive deficits, including deficits
in wakefulness, attention-deficit hyperactivity disorder (ADHD),
Alzheimer’s disease (AD), mild cognitive impairment, and
schizophrenia.1 Several clinical candidates (Ia–Ie) have advanced
into trials (Fig. 1).1a,f–l We identified and recently disclosed the pre-
liminary structure–activity relationships (SAR) for a novel class of
pyridazin-3-one H3R antagonists/inverse agonists and the profile
of clinical compound 1e (CEP-26401, irdabisant).2 A subsequent
series of papers disclosed further modifications to the aromatic
pyridazinone phenoxypropyl amine series exploring 4,5-substitu-
tion, 4,5 ring-fused pyridazinones, central ring and linker SAR,
and constrained amine replacements.3 A novel SAR direction inves-
tigated to identify potential back-up, second-generation H3R inhib-
itors to irdabisant, was to explore the non-aromatic 4,5-
dihydropyridazin-3-one phenoxypropyl core 2. In this paper, the
strategy, optimization and profile of
4,4-dimethyl-6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phe-
nyl}-4,5-dihydro-2H-pyridazin-3-one 22 is described.
a
lead candidate,
The N2-substituted 4,5-dihydropyridazin-3-one targets were
synthesized as outlined in Scheme 1.2,3a,4 The versatility of the syn-
thesis tolerates the amine side chain installed either before (Route
A) or after (Route B) construction of the 4,5-dihydropyridazinone
ring. To install the side chain first, the route commences with
O-demethylation of a 4-(4-methoxyphenyl)-4-oxobutyric acid (or
ester) 3 using BBr3 or 48% HBr, alkylation of the phenol to the
chloropropyl intermediate 4 and then amine substitution to give
intermediates 5. Cyclocondensation reaction of 5 with N-substi-
tuted hydrazines produced analogs 8–11, and 17–21 in good yield
and purity.5 In an analogous manner, targets 12–16 were synthe-
sized using route B (Scheme 1). Intermediate 3 was initially reacted
with an N-substituted hydrazine, then O-demethylated to produce
dihydro-phenol 6. Installation of the amine side chain via chloro 7
gave analogs 12–16. The synthesis of 4,4-dimethyl-6-{4-[3-((R)-2-
methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-4,5-dihydro-2H-pyri-
dazin-3-one 22 is outlined in Scheme 2. p-Anisyl magnesium
bromide 26 was reacted with 3,3-dimethylsuccinic anhydride to
give keto-acid 27, with excellent regio-purity, but in only moderate
yield. Demethylation and ethyl ester formation produced phenol
28 in 90% yield for the two steps. Installation of the (R)-2-methyl-
pyrrolidine propyl side chain to 29, followed by hydrazine cyclo-
condensation produced 22 in excellent yield and purity.
The synthesis of the 5,5-dimethyl-6-{4-[3-((R)-2-methyl-pyrr-
olidin-1-yl)-propoxy]-phenyl}-4,5-dihydro-2H-pyridazin-3-one reg-
iomer 23 is outlined in Scheme 3. Lithium chloride catalyzed
⇑
Corresponding author.
E-mail address: rhudkins@cephalon.com (R.L. Hudkins).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.037

R. L. Hudkins et al. / Bioorg. Med. Chem. Lett. 22 (2012) 194–198
195
N
Me
N
O
H
N
N
N
Me
CF3
O
O
O
N
Ia GSK-189254
Ib MK-0249
O
O
F
O
N
Et
N
N
H
N
N
F
Ic JNJ-31001074
Id PF03654746
R2
H
N
O
N
N
O
N
R4a
R4b
O
N
R5b
R5a
O
amine
1e CEP-26401 (Irdabisant)
2
4.5-Dihydropyridazin-3-one series
Figure 1. Structures of clinical candidates and 4,5-dihydropyridazin-3-one core.
R4
R4
O
O
a, b
EtO2C
EtO2C
R5
R5
Route A
OMe
O
Cl
4
3
d, a
Route B
c
R2
N
R4
O
O
N
EtO2C
R5
R4
amine
O
R5
OH
5
6
d
b
R2
N
R2
N
c
O
O
N
N
R4
R4
R5
R5
O
Cl
O
amine
7
8-21
Scheme 1. Reagents and conditions: (a) BBr3, DCM, 5 °C to rt, 70–95%; (b) K2CO3, Br(CH2)3Cl, acetone, 65 °C, 80–95%; (c) amine, K2CO3, CH3CN 80 °C, 20–70%; (d) R2NHNH2, 2-
propanol, reflux, 65–86%.
O
O
BrMg
b, c
a
HO2C
EtO2C
OMe
OMe
f
OH
26
27
28
O
H
N
N
O
d, e
EtO2C
N
O
O
N
29
22
Scheme 2. Reagents and conditions: (a) 3,3-dimethylsuccinic anhydride, THF, 0 °C to rt, 30%; (b) 48% HBr, reflux, 90%; (c) EtOH, H2SO4, 90%; (d) K2CO3, Br(CH2)3Cl, CH3CN,
65 °C, 97%; (e) (R)-2-methyl-pyrrolidine, KI, K2CO3, CH3CN, 80 °C, 67%; (f) NH2NH2, 2-propanol, reflux, 75%.
cyanosilation of benzyloxybenzaldehyde 30 produced silyated
cyanohydrin 31 in 93% yield.6 Alkylation of 31 with methyl
3-methyl-2-butenoate, followed by desilyation produced the
keto-ester intermediate 32 in 47% yield for the two steps. Debenzy-
lation and installation of the (R)-2-methylpyrrolidine propyl side
chain using standard conditions gave 33 in 65% yield for the three
steps. Last, hydrazine cyclocondensation produced 23 in excellent
yield. The dihydro 4-pyridyl 24 and the 5-(2-pyridyl) 25 were
synthesized as described previously.3a,4
The pyridazinone analogs were tested using in vitro binding as-
says by displacement of [3H]NAMH in membranes isolated from
CHO cells transfected with cloned human H3 or rat H3 receptors

196
R. L. Hudkins et al. / Bioorg. Med. Chem. Lett. 22 (2012) 194–198
O
OTMS
O
a
b
H
NC
MeO2C
OBn
OBn
OBn
30
31
32
H
N
N
O
O
c, d, e
f
MeO2C
O
N
O
N
33
23
Scheme 3. Reagents and conditions: (a) TMSCN, LiCl, THF, 93%; (b) (i) LDA, THF, 3-methyl-but-2-enoic acid methyl ester, À72 °C; (ii) TBAF, THF, 0 °C, 47%; (c) 10% Pd/C,
MeOH, HCl, 99%; (d) K2CO3, Br(CH2)3Cl, CH3CN, 65 °C, 75%; (e) (R)-2-methylpyrrolidine, KI, K2CO3, CH3CN, 80 °C, 86%; (d) NH2NH2, 2-propanol, reflux, 84%.
as shown in Table 1 and Table 2.2,3 The (R)-2-methylpyrrolidine
4,5-dihydro 8 showed essentially equivalent binding affinity for
Table 2
4,5-Pyridazin-3-one H3R binding dataa
R2
N
both the hH3R and rH3R as the aromatic pyridazinone 1e (Table 1).
The (S)-2-methylpyrrolidine isomer 9 had about 10-fold weaker
binding affinity for both hH3R and rH3R, a similar trend as observed
for 1e.2 Pyrrolidine 10, compared with (R)-2-methylpyrrolidine 8
showed a significant loss in affinity, indicating the (R)-2-methyl-
pyrrolidine was optimum for the 4,5-dihydropyridazinone series,
similar to the pyridazinone series.2 Substitution at the N2 position
accommodated significant steric bulk without affecting H3R bind-
ing affinity (see 11–13, 15–17), although increasing the size of
the R2 group greater than methyl (11) resulted in compounds with
O
N
R4a
R4b
R5b
R5a
R5a/R5b
O
amine
Entry
R4a/R4b
Me
Amine
hH3R (Ki nM)
rH3R (Ki nM)
18
H
1.9
5.6
6.8
4.7
4
N
N
19
19a
19b
H
H
H
Me
Me
Me
18
29
20
Table 1
Pyridazin-3-one H3R binding dataa
N
R2
N
O
N
N
HN
20
21
H
H
Me
Me
83
21
85
28
amine
rH3R (Ki nM)
O
HN
N
Entry
1e
R
Amine
hH3R (Ki nM)
cLogP
2
7.2
2.3
22
23
24
25
Me, Me
H
H
4.3
20
13
59
42
21
8
H
3.2
8.8
2.2
N
Me, Me
H
N
19
10
11
H
37
74
5
114
256
16
2.2
1.8
2.3
HN
HN
2-Pyr
H
8.8
9.9
N
H
2-Pyr
N
Me
N
N
b
R4a and R5b = H unless noted.
Ki values are an average of 2 or more determination. The assay-to-assay vari-
a
12
iPr
9
63
3.3
ation was typically within 2.5-fold
13
14
15
CH2CF3
CH2CF3
Ph
5
10
62
16
4.1
4.3
4.2
high clogP values. Analogous to the loss in affinity observed chang-
ing 8 to pyrrolidine 10, changing (R)-2-methylpyrrolidine 13 to
piperidine 14 resulted in a 6–8 fold loss in H3R affinity.
N
HN
38
10
Further discovery flow profiling was conducted with 6-{4-[3-
((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-4,5-dihydro-2H-
pyridazin-3-one 8. Compound 8 showed high affinity for both hu-
man and rat H3Rs (hH3R Ki = 3.2 0.8 nM, rH3R Ki = 8.8 1.6 nM)
N
N
N
16
17
Bn
2
25
42
4.5
2.7
and full inverse agonist activity in the [35S]GTP
cS hH3R binding as-
say with an EC50 = 0.7 nM.2,4 It displayed acceptable drug-like prop-
erties with low lipophilicity (clogP = 2.2), high permeability in the
Caco-2 assay (Papp = 11.4 Â 10À6 cm/sec) and high water solubility
(pH 2 and pH 7.4 >2 mg/mL). Compound 8 had acceptable in vitro
2-Pyr
12
bPyr = 2-pyridine.
a
metabolic stability across species in liver microsomes (t >40 min
½
Ki values are an average of 2 or more determination. The assay-to-assay vari-
ation was typically within 2.5-fold.
in mouse, rat, dog, monkey and human) and IC50 values >30 lM

R. L. Hudkins et al. / Bioorg. Med. Chem. Lett. 22 (2012) 194–198
197
Table 3
for inhibition of cytochrome P450 enzymes CYP1A2, 2C9 and 2C19
and >11 M for 2D6 and 3A4, indicating low potential for drug-drug
Pharmacokinetic properties in rata
l
interactions. Compound 8 showed excellent selectivity over hH1,
hH2, and hH4 receptor subtypes and a panel of 70 GPCRs, ion chan-
nels and enzymes (>1000-fold selectivity) where it showed inhibi-
8
19a
1.8 0.4
3.5 1.1
22
1356 70
301 16
19b
1.5 0.0
3.4 0.3
25
1327 142
274 35
52 10
22
1.9 0.3
4.7 0.9
27
837 46
189 13
iv t½ (h)
Vd (L/kg)
1.1 0.1
0.8 0.2
CL (mL/min/kg)
po AUC (ng h/mL)
Cmax (ng/mL)
F (%)
8.5 1.8
1828 126
376 19
2
1
3
tion only for the muscarinic M2 subtype (60%) at 10
concentration (MDS Pharma Services, LeadProfile screen). It also
showed acceptable hERG selectivity with an IC50 value >10 M in a
lM screening
l
19
1
40
4
38
3
B/Pb
3.1 0.3
1.8 0.1
1.9 0.9
2.4 0.1
patch-express assay. Compound 8 was screened in the rat for phar-
macokinetic properties and showed acceptable iv intrinsic proper-
a
Administration at 1 mg/mg iv and 5 mg/kg po; iv formulation (3% DMSO, 30%
ties (t = 1.1 h, CL = 8.5 mL/min/kg, Vd = 0.8 L/kg), however with
½
solutol, 67% phosphate buffered saline) oral formulation (50% Tween 80, 40% pro-
pylene carbonate and 10% propylene glycol).
lower than acceptable oral bioavailability (F = 19%) (Table 3). The
brain exposure and brain to plasma ratio (B/P) was in the acceptable
range for use in potential CNS indications (brain concentration 6 h
b
B/P = brain to plasma ratio measured 6 h post 10 mg/kg ip dose.
following a 5 mg/kg po dose = 2.2 lM; B/P = 3.1). Based on its overall
discovery profile 8 was screened in the rat dipsogenia model for
in vivo H3R functional inhibition in the brain following oral admin-
istration.2,3a,7 Compound 8 potently and dose-dependently inhib-
ited RAMH-induced dipsogenia with an ED50 value of 0.06 mg/kg
po Encouraged by the in vivo potency, further pharmacokinetic
studies were conducted in rat and dog designed to understand the
in vivo metabolic fate of the 4,5-dihydropyridazinone moiety. The
primary question was the potential for oxidation to the H3R active
pyridazinone metabolite 1e. Following oral administration of 8 in
the rat, low levels (ꢀ4%) of oxidized metabolite 1e were observed
in plasma. Following oral administration of 8 in the dog, plasma lev-
els of 1e were about 25–30% compared to plasma levels of 8. Based
on the formation of an active H3R metabolite, compound 8 was not
further advanced and the new objective going forward was to design
metabolically stable 4,5-dihydropyridazinones.
The initial strategy employed was to probe methyl substitution
on the 4- and 5-positions with the aim of blocking metabolic con-
version. The 4-methyl 18 and 5-methyl 19 modifications were
synthesized and found to be equally tolerated for H3R binding
affinity (Table 2). The pyrrolidine 20 and piperidine 21 analogs
of 19 were synthesized confirming the previous SAR that the
(R)-2-methylpyrrolidine was optimum. Substitution in the 4- or
5- position with larger groups, such as 2-pyridyl was also tolerated
(see 24 and 25).
H
N
H
N
O
O
N
N
O
N
O
N
19b
35
hH3R Ki = 2.9 nM
rH3R Ki = 12 nM
Figure 2. Structure of metabolite 35 formed from 19b.
Next, the 4,4-dimethyl 22 and 5,5-dimethyl 23 analogs were
synthesized, a maneuver that would block aromatic metabolite
formation and remove the chiral center. Profiling 4,4-dimethyl 22
showed it had high affinity for both hH3R (Ki = 4.3 1.0 nM) and
rH3R (Ki = 13 3 nM) equivalent with 8. Whereas the 5,5-dimethyl
regiomer 23 had about 5-fold weaker affinity (hH3R Ki = 20 6 nM
and rH3R Ki = 59 20 nM). Functionally, 22 showed potent antago-
nist activity (Kb,app = 0.18 0.03 nM) and displayed full inverse
agonist activity in the [35S]GTP
cS hH3R binding assay with an
EC50 of 0.73 0.21 nM. Based on target potency, acceptable
in vitro metabolic stability (t >40 min across species), cytochrome
P450 enzyme selectivity (IC50 values >30 lM) and solubility (pH2
and pH7.4 >2 mg/mL), 22 was profiled further for selectivity. Com-
pound 22 showed <30% inhibition against hH1, hH2, and hH4 sub-
types and a panel of 164 GPCRs, ion channels and enzymes at
½
Compound 19, as a mixture of diastereomers, was selected for
further discovery flow profiling and showed acceptable in vitro
metabolic stability across species in liver microsomes (t
>40 min) and IC50 values >30 lM for inhibition of cytochrome
P450 enzymes. Preliminary rat pharmacokinetic screening showed
10
LeadProfile and Spectrum screens). It also showed acceptable hERG
selectivity with an IC50 value of 20 M in the patch-express assay.
lM concentration (>1000-fold selective) (MDS Pharma Services,
½
l
it had acceptable iv intrinsic pharmacokinetic properties
The permeability of 22 was evaluated in the Caco-2 intestinal epi-
(t = 1.8 h, CL = 19 mL/min/kg, Vd = 3 L/kg), and oral bioavailability
thelial cell line where it was classified as high permeability
(Papp = 14
ratio (PDR) of 1.4, indicating minimal interaction with efflux trans-
porters such as P-gp. It had low plasma protein binding for rat
(28%), dog (16%), and human (26%). Compound 22 was evaluated
for pharmacokinetic properties in the rat (Table 3). It showed an
½
(F = 35%) that warranted separation of the isomers for further stud-
ies. 5-Methyl 19 was separated by chiral SCF chromatograph
(MeOH, 0.1% DEA, chira-cel OD-H 4.6 Â 250 cm, 2.5 mL/min) to
give peak 1 (rt = 11.1 min, 19a) and peak 2 (rt = 12.1 min, 19b).
Both isomers showed similar hH3R binding affinity (19a
Ki = 6.8 0.9 nM; 19b Ki = 4.7 0.5 nM), rH3R binding affinity
(19a Ki = 29 10 nM; 19b Ki = 20 5 nM) and >1000-fold
selectivity over hH1 and hH2 receptor subtypes. Functionally, both
isomers also showed potent antagonist activity (19a Kb,app = K-
0.9 Â 10À6 cm/sec) with a permeability directional
iv t of 1.9 h, clearance of 27 mL/min/kg, and Vd of 4.7 L/kg and
½
the oral bioavailability was 38% with acceptable brain exposure
(brain concentration 6 h following a 5 mg/kg po dose = 900 nM;
B/P = 2.4).
,
b app = 0.68 0.03 nM; 19b Kb,app = 0.54 0.09 nM) and full inverse
Compound 22 met all discovery flow criteria and was advanced
into in vivo evaluation using the rat dipsogenia model and the rat
EEG/EMG sleep-wake model.1i,7–9 Compound 22 potently and
dose-dependently inhibited RAMH-induced dipsogenia with an
ED50 value of 0.16 mg/kg po. Wake-promoting activity in the rat
was measured from 0.3 to 10 mg/kg ip as previously described
using rats surgically implanted for chronic recording of EEG (elec-
troencephalographic) and EMG (electromyographic) signals.3a,10
The cumulative wake time at 4 h after dosing was evaluated during
the normal quiet period of the rat where 22 dose-dependently in-
creased waking at 1, 3 and 10 mg/kg ip by 4 h AUC values (P <0.001
agonist activity in the [35S]GTP
cS hH3R binding assay with EC50
values of 1.3 0.2 and 1.4 0.2 nM, respectively. Also, the isomers
were not significantly different in in vitro metabolic stability (t
>40 min in mouse, rat, dog, monkey and human liver microsomes),
inhibition of cytochrome P450 enzymes (IC50 values >30 lM) or
pharmacokinetic properties in the rat (Table 3). However, the iso-
mers showed a different metabolite profile in vivo in the rat. Fol-
lowing administration of a 10 or 30 mg/kg po dose of 19a,
metabolite 353a was not observed in plasma, whereas 19b con-
verted to 35 (ꢀ7% of 19b levels) at 10 mg/kg po over 24 h (Fig. 2).
½

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R. L. Hudkins et al. / Bioorg. Med. Chem. Lett. 22 (2012) 194–198
chon, Nicole Lepallec, Isabelle Kanmacher, Véronique Agathon
and Mehran Yazdanian.
240
180
120
60
*
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0
Vehicle
0.3
1
3
10
Compound 22(mg/kg i.p.)
Figure 3. Compound 22-induced wake promotion. Cumulative wake 4 h AUC
values following administration of vehicle or compound 22 in rats chronically
implanted with electrodes for recording EEG and EMG activity. Mean + SEM,
n = 8–14/group. ⁄p <0.05, Dunnett’s test versus vehicle.
ANOVA). Wake was increased to 119 4 min and 127 8 min at 1
and 3 mg/kg, respectively, compared to vehicle (80 5 min) (P
<0.05, Dunnett’s test). At 10 mg/kg, wake activity was increased
to 201 8 min, maintaining over 90% time awake for the first 3
hours after dosing. Compound 22 thus demonstrated robust wake
promotion, with the drug treated group showing over 150% greater
4 h cumulative wake time at 10 mg/kg compared to the vehicle
group (Fig. 3). Maximal cumulative wake surplus (time awake rel-
ative to the vehicle group) at this dose began to plateau at about
130 min at 5 h and increased gradually until reaching a maximum
of 203 min at 17 h (data not shown). No sleep rebound was ob-
served at any dose.
In summary, optimization of the 4,5-dihydropyridazin-3-one
phenoxypropyl amine core led to the identification of new mole-
cules displaying excellent H3R target potency, selectivity and rat
pharmacokinetic properties. A design strategy to block in vivo
metabolism of the 4,5-dihydro moiety identified 4,4-dimethyl-6-
{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-4,5-dihy-
dro-2H-pyridazin-3-one 22 as a back-up candidate. Compound 22
was profiled in detail and met all discovery criteria, including dem-
onstration of potent functional H3R antagonism in vivo in the rat
dipsogenia model and robust wake activity in the rat EEG/EMG
model.
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Acknowledgments
The authors thank and acknowledge the assistance and support
of Emily Kordwitz, Mark Olsen, Curtis Haltwanger, Gilbert Moa-