Synthesis of constrained benzocinnolinone analogues of CEP-26401 (irdabisant) as potent, selective histamine H3 receptor inverse agonists

Article abstract of DOI:10.1016/j.bmcl.2012.04.001

A novel class of benzocinnolinones analogs of irdabisant were designed and synthesized as histamine H3R antagonists/inverse agonists. Modifications to the pyridazinone portion of the core and linker led to the identification of molecules with excellent target potency and selectivity with improved rat pharmacokinetic properties and reduced potential hERG liabilities.

Full text of DOI:10.1016/j.bmcl.2012.04.001

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4198–4202
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of constrained benzocinnolinone analogues of CEP-26401 (irdabisant)
as potent, selective histamine H3 receptor inverse agonists
⇑
Kurt A. Josef , Lisa D. Aimone, Jacquelyn Lyons, 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 novel class of benzocinnolinones analogs of irdabisant were designed and synthesized as histamine
H3R antagonists/inverse agonists. Modifications to the pyridazinone portion of the core and linker led
to the identification of molecules with excellent target potency and selectivity with improved rat phar-
macokinetic properties and reduced potential hERG liabilities.
Received 8 December 2011
Revised 23 March 2012
Accepted 2 April 2012
Available online 26 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Histamine
H3 antagonist
Inverse agonist
Benzocinnolinone
Histamine has been established as a modulator of neuronal
activity with at least four subtypes of the G-protein coupled recep-
tors (H1R–H4R) reported.1 Of these, the H3 receptor is predomi-
nately expressed in the CNS, while H1, H2, and H4 receptors are
more widely distributed in both central and peripheral tissues.
The H1 and H2 receptors have been successfully targeted with
pharmacological agents to treat allergic response and control gas-
tric acid secretion, respectively, while the H4 receptor is predomi-
nately expressed in inflammatory cells, suggesting its critical role
in the regulation of inflammatory and immune responses.2 In the
CNS, H3 receptors are involved in presynaptic regulation of the re-
lease of neurotransmitters such as acetylcholine (ACh), dopamine
(DA), norepinephrine (NE), and serotonin (5-HT) making this
receptor an attractive target for indications such as attention-
deficit hyperactivity disorder (ADHD), Alzheimer’s disease (AD),
mild cognitive impairment and schizophrenia.1c–j The H3 receptor
demonstrates a high degree of constitutive or spontaneous activity
(receptor is active in the absence of agonist stimulation) in vitro
and in vivo, therefore, ligands to the receptor can display agonist,
neutral antagonist or inverse agonist effects. Initial work in the
field focused on imidazole analogs of the natural ligand histamine,
but has evolved into a search for amine-based compounds with
We have identified a novel class of pyridazin-3-one H3R antago-
nists/inverse agonists,3 and recently disclosed the structure–activ-
ity relationships (SAR) and characterization of clinical compound 1
(CEP-26401, irdabisant).4 As part of our H3 discovery project study-
ing the SAR around 1, we actively pursued a variety of structural
core modifications.5 One strategy, which is the focus of this
paper, was to develop a synthesis and evaluate the effect of
constraining the left-hand (western region) A–B rings by linking
the pyridazinone C5 and A-ring phenyl of irdabisant to form
constrained 2H-benzo[h]cinnolin-3-ones, or the pyridazinone C6
to form 3H-benzo[f]cinnolin-2-ones.
Scheme 1 outlines the general procedure for the synthesis of
N2-substituted benzocinnolinones 3–6. The aldol condensation
product of 6-methoxy-1-tetralone with glyoxylic acid monohy-
drate in refluxing acetic acid was isolated in 30% yield. This was
subsequently cyclized with the N-substituted hydrazines in isopro-
panol to give the 5,6-dihydro-2H-benzo[h]cinnolin-3-one interme-
diates 2 in 30–50% yield.6 Demethylation with boron tribromide in
cold dichloromethane, followed by alkylation with 1-bromo-3-
chloro-propane, and then substitution with (R)-2-methylpyrroli-
dine (3–5) or piperidine (6) gave the targets 3–6 in 8% to 10%
overall yield for the three steps. The N2-H analogs of both the
5,6-dihydrocinnolinone and the 4,4a,5,6-tetrahydrocinnolinone,
as illustrated in Scheme 2, were synthesized via the chloropropyl
ether intermediate 7 obtained from the alkylation of 6-hydroxy-
1-tetralone with1-bromo-3-chloro-propane in a 73% yield. Cyclo-
condensation providing the dihydro target 8 was achieved in a
similar manner as described above, except that the intermediate
aldol product was not isolated. Condensation with glyoxylic acid
in refluxing acetic acid proceeded for 2 h, was then cooled, and
more drug-like properties. These scaffolds usually contain
a
tertiary-amine linked to a lipophilic aryl group as a pharmaco-
phore that can be broadly modified to give a diversity of structural
classes yet still possess a binding affinity to the H3 receptor. Several
H3R candidates have advanced into clinical evaluation (Fig. 1).1h–j
⇑
Corresponding author.
E-mail address: kurtajosef@verizon.net (K.A. Josef).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.001

K. A. Josef et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4198–4202
4199
Me
O
N
O
O
N
N
N
H
N
N
Me
Cl
O
CF3
MK-0249
GSK-189254
O
F
N
O
N
N
H
F
BF2.649
H
N
Me
PF03654746
N
O
N
O
1 CEP-26401
Figure 1. Structures of clinical H3R clinical antagonists.
R
N
R
N
O
O
N
O
N
a,b
c,d,e
8-10%
10-15%
amine
O
O
O
2
3 R = Me, amine = (R)-2-Me-pyrrolidine
4 R = iPr, amine = (R)-2-Me-pyrrolidine
5 R = Ph, amine = (R)-2-Me-pyrrolidine
6 R = CH2Ph, amine = piperidine
Scheme 1. Reagents and conditions: (a) glyoxylic acid monohydrate, AcOH, 120 °C; (b) H2NNHR, iPrOH, 110 °C; (c) BBr3, CH2Cl2, 0–25 °C; (d) Br(CH2)3Cl, K2CO3, ACN, 100 °C;
(e) (R)-2-methylpyrrolidine or piperidine, K2CO3, NaI, ACN, 100 °C.
H
O
N
O
O
N
b, c
a
73%
51%
OH
Cl
N
O
O
7
8
c
65%
H
N
O
O
N
d
33%
N
O
N
O
9
10
Scheme 2. Reagents and conditions: (a) Br(CH2)3Cl, K2CO3, ACN, 100 °C; (b) glyoxylic acid monohydrate, AcOH, 120 °C; hydrazine monohydrate, 90 °C;
(c) (R)-2-methylpyrrolidine, K2CO3, NaI, ACN, 100 °C; (d) glyoxylic acid monohydrate, AcOH, 120 °C; zinc dust, 100 °C; hydrazine monohydrate, 90 °C.
O
hydrazine monohydrate was added directly to the reaction to give
H
the benzocinnolinone in 51% yield. Subsequent substitution with
(R)-2-methylpyrrolidine gave target 8. In the case of the tetrahydro
analog 10, the chloropropylether 7 was not stable to a later reduc-
tion step and was therefore first converted to the (R)-2-methylpyr-
rolidyl-propoxy-tetralone 9 in 65% yield. This intermediate was
transformed into target 10 using a single reaction vessel in which
a stepwise addition of glyoxylic acid, followed by two equivalents
of zinc dust,7 and then hydrazine monohydrate, all in refluxing ace-
tic acid, gave a 33% yield of 10. The isomeric 5,6-dihydro-3H-
benzo[f]cinnolin-2-one analog 11 was synthesized with an overall
yield of 4% using similar procedures described in Schemes 1 and 2,
but starting with the 6-methoxy-2-tetralone (Scheme 3). The
cyclobutyl-piperidinyl target 12 was obtained by first coupling 6-
N
N
5 steps
O
4% overall
O
N
O
11
Scheme 3. Reagents and conditions: glyoxylic acid monohydrate, AcOH, 120 °C;
hydrazine monohydrate, 90 °C; BBr3, CH2Cl2, 0–25 °C; Br(CH2)3Cl, K2CO3, ACN,
100 °C; (R)-2-methylpyrrolidine, K2CO3, NaI, ACN, 100 °C.
hydroxy-1-tetralone and N-boc-4-hydroxypiperidine using DEAD
and triphenylphosphine in cold tetrahydrofuran (70% yield,
Scheme 4). Conversion to the tetrahydro-benzocinnolinone using

4200
K. A. Josef et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4198–4202
H
N
H
N
O
O
O
N
N
boc
a, b
c, d
N
N
55%
25%
OH
O
O
13
12
Scheme 4. Reagents and conditions: (a) DEAD, PPh3, N-Boc-4-OH-piperadine, THF, 0–25 °C; (b) glyoxylic acid monohydrate, AcOH, 120 °C; zinc dust, 100 °C; hydrazine
monohydrate, 90 °C; (c) TFA, 25 °C; (d) cyclobutanone, Na(CN)BH3, MeOH, DMF, AcOH, 60 °C.
Table 1
Pyridazin-3-one central ring and linker H3R binding data
Entry
Structure
hH3 (Ki nM)
rH3 (Ki nM)
clogP
Me
O
O
O
O
N
N
N
N
3
3.6 0.3
8.3a
3.3
O
O
O
O
N
N
N
N
iPr
N
4
5
4.2 0.7
17
23
6
2
4.1
4.8
Ph
N
16
4
CH2Ph
N
N
6
105 44
140 25
5.2
H
O
O
H
O
N
N
8
4.3 1.1
14
3
2.7
2.6
O
O
O
O
N
N
N
H
N
N
10
15
2
53 10
O
H
N
N
11
6.2 0.6
19
4
2.5
1.9
N
N
12
17 0.4
26 10
N
a
n = 2; for all others, n = 3–5.

K. A. Josef et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4198–4202
4201
the one pot reductive cyclocondensation procedure described in
Scheme 2 gave intermediate 13 in 35% yield. Partial de-protection
of the piperidine moiety occurred under the refluxing acetic acid
conditions. Complete removal of the Boc group was achieved in tri-
fluoroacetic acid at ambient temperature for 2 h. The subsequent
reductive amination with cyclobutanone and sodium cyanoboro-
hydride in dimethylformamide and methanol gave 12 in 55% yield.
All target compounds 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.3,4 In
the series of 1 and its analogs,4,5 we investigated substitution
around the central phenyl ring and the pyridazinone head group.
It was hypothesized that some restricted rotation around these
two aryl ring systems may enhance the activity and further im-
prove the selectivity of the series. As previously described, H3R
binding affinity was not significantly affected by substitution of
increasing steric bulk at the N2 position.4 This SAR trend was also
reflected in the benzocinnoline series, Table 1. With the clogP, a
measure of the lipophilicity, of the minimally substituted 8 already
at 2.7, increasing the size of the N2-R group, even to the methyl
analog 3, resulted in clogP values over 3.0. These higher clogP
numbers and corresponding higher molecular weights can de-
crease the ligand efficiency (LE) and the ligand lipophilic efficiency
(LLE)8 as demonstrated in the N2-R set of 8 (N–H; LLE = 5.70), 3 (N-
methyl; LLE = 5.14), and 4 (N-isopropyl; LLE = 4.28). Elevated clogP
values have also been shown to enhance hERG activity, the propen-
sity for high tissue distribution, and the induction of phospholipi-
dosis.9 Binding affinity was fairly consistent for N–H and lower
alkyl substitution, with minimal differences for hH3R and rH3R be-
tween the N–H (8), N-methyl (3) and N-isopropyl (4) analogs.
Selectivity of these compounds compared to the histamine recep-
tor isoforms hH1, hH2, and hH4 was high with less than 40% inhibi-
analog 8 also displayed poor in vitro metabolic stability and oral
bioavailability in rat, and was not suitable for further advancement
into in vivo testing. The tetrahydro-benzocinnolinone analog 10
showed slightly weaker binding affinity (hH3R Ki = 15 nM and
rH3R Ki = 53 nM) compared with 3 or 8 and there was little differ-
ence in the iv PK properties of the dihydro and tetrahydro analogs
8 and 10 (Table 2). Encouragingly, there was a modest increase in
bioavailability (F = 11%) for 10. At this stage, based on its profile, no
attempt was made to isolate the diastereomers of the tetrahydro
benzocinnolinone 10. The regiomeric 5,6-dihydro-3H-benzo[f]-
cinnolin-2-one 11 showed comparable H3R affinity (hH3R Ki =
6.2 nM, rH3R Ki = 19 nM) with the 5,6-dihydro-2H-benzo[h]- cinn-
olin-3-one 8. Compound 11 also demonstrated good selectivity for
hH3R compared to the other hHistamine receptor subtypes (hH1R,
hH2R, and hH4R) with less than 45% inhibition of radioligand bind-
ing at 10 lM concentration. In an attempt to improve the PK profile
and advanced compounds from the benzocinnolinone series into
in vivo proof of concept studies, the (R)-2-methylpyrrolidyl-propyl
moiety was switched to the constrained N-cyclobutylpiperidine 12.
Compound 12 had essentially equivalent binding affinity (hH3R
Ki = 17 nM and rH3R Ki = 26 nM) compared to its tetrahydro coun-
terpart 10. Further, compound 12 showed an acceptable in vitro
metabolic stability across species (t >40 min) and a clean CYP
½
inhibition selectivity profile (IC50 >30 lM). Based on the in vitro
data, compound 12, as a mixture of diastereomers, was screened
in the rat for PK properties and showed improved oral exposure
and Cmax values (F = 44%; Table 2). However, the iv profile showed
a high volume of distribution (Vd = 6.6) and short t consistent
½
with the high clearance. The high Vd for compound 12 also was re-
flected in high brain exposure with a 5 mg/kg oral dose producing
brain concentrations of 780 nM 6 h post and a high brain to plasma
ratio (B/P) of 5.2.10 Compared to the phenoxypropyl core, the
tion of radioligand binding at 10
lM concentration (>1000-fold
piperidine chemotype had a lower calculated clogP of 1.9
selectivity). Binding affinity differences between the N2-phenyl 5
and N2-benzyl 6 analogs were likely due to the change in the
amine moiety from (R)-2-methylpyrrolidine (5) to the piperidine
(6) as seen previously.4 The methyl analog 3 was screened for po-
tential hERG liabilities using the astemizole binding assay (MDS
(clogD7.4 = 0.7), which was with a key component in our earlier
disclosed relationship between clogP and hERG activity.4 Com-
pound 12 showed weak hERG activity with an IC50 = 18
lM in
the patch clamp assay (MDS Pharma Services, PatchExpress).
In summary, the tricyclic benzocinnolinone pyridazinone core
was designed and synthesized showing high H3R binding affinity
with excellent selectivity against the H1R, H2R and H4R subtype.
Modification to the linker/amine region of the pharmacophore re-
sulted in 12, which showed improved metabolic stability and rat
pharmacokinetics following oral administration. Presently, meth-
ods were developed to scale up 12 for separation and evaluation
of the individual isomers.
Pharma Services) and showed 34% inhibition at 10
tion. Functionally, showed potent antagonist activity (K
b,app = 0.4 nM) and displayed full inverse agonist activity in the
35S]GTP S hH3R binding assay3,4 by decreasing basal activity with
an EC50 = 1.7 nM. Compound 3 had IC50 values >30 M for inhibi-
lM concentra-
3
[
c
l
tion of cytochrome P450 enzymes CYP1A2, 2C9, 2C19, 2D6 and
3A4, indicating minimal potential for drug–drug interactions.
Unfortunately, when screened in the rat for pharmacokinetic (PK)
properties compound 3 showed unacceptable iv intrinsic proper-
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iva
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