Design and synthesis of pyridazinone-based 5-HT2C agonists

Article abstract of DOI:10.1016/j.bmcl.2009.07.136

The SAR of a series of pyridazinone derived 5-HT_2C agonists has been explored and resulted in identification of a compound with excellent levels of 5-HT_2C functional agonism and selectivity over 5-HT_2A and 5-HT_2B

Full text of DOI:10.1016/j.bmcl.2009.07.136

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5791–5795
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of pyridazinone-based 5-HT2C agonists
a
a
a
a
a
Charlotte M. N. Allerton a, , Mark D. Andrews , Julian Blagg , David Ellis , Edel Evrard , Martin P. Green ,
*
Kevin K.-C. Liu c, Gordon McMurray b, Michael Ralph a, Vivienne Sanderson a, Robin Ward b, Lesa Watson a
a Department of Discovery Chemistry, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT139NJ, UK
b Department of Discovery Biology, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT139NJ, UK
c Department of Discovery Chemistry, Pfizer Global Research and Development, LA Jolla, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The SAR of a series of pyridazinone derived 5-HT2C agonists has been explored and resulted in identifica-
tion of a compound with excellent levels of 5-HT2C functional agonism and selectivity over 5-HT2A and 5-
HT2B. This compound displayed good in vivo efficacy in pre-clinical models of stress urinary incontinence,
despite having physiochemical properties commensurate with impaired CNS penetration.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 30 June 2009
Revised 27 July 2009
Accepted 28 July 2009
Available online 6 August 2009
Keywords:
5-HT2C
Agonist
Pyridazinone
CNS penetration
Serotonin (5-HT) is a neurotransmitter which plays an important
role in mood, sleep, emesis, sexuality and appetite.1 5-HT receptors
mediate the effects of serotonin. There are many sub-types of 5-HT
receptors and the processes they mediate are quite diverse. The 5-
HT2C receptor is found primarily in the brain, and 5-HT2C agonists
have the potential for treating a number of conditions including
obesity, schizophrenia, sexual dysfunction and urinary inconti-
nence.2 Our aim was to identify potent 5-HT2C agonists, with selec-
tivity over 5-HT2A and 5-HT2B and good CNS penetration, for
potential use in the treatment of urinary incontinence and sexual
dysfunction. The selective serotonin and noradrenaline reuptake
inhibitor Duloxetine has shown efficacy in reducing episodes of
stress urinary incontinence (SUI) in controlled clinical trials.3 This
effect has been hypothesized to be at least partly due to increases
in serotonergic and noradrenergic stimulation of neurons within
Onuf’s nucleus, a key site within the sacral spinal cord which is
known to be involved in contraction of the external urethral sphinc-
ter.4 Interestingly pre-clinical studies have indicated a role for 5-
HT2C receptors in these pathways. In particular 5-HT2C receptor
agonism appears to increase urethral tone in multiple species, sug-
gesting potential utility in the treatment of SUI.5
is associated with hallucinogenic and cardiovascular effects, while
5-HT2B agonism has been linked to potentially fatal heart valvulop-
athy in man.8
Our SAR studies commenced with the pyrazine lead compound
1, which originated from in-house work optimizing the known
non-selective 5-HT2C agonist meta-chlorophenyl piperidine (m-
CPP) (Fig. 1).9 Compound 1 is a potent and CNS penetrant 5-HT2C
agonist with moderate selectivity over the 5-HT2A sub-type but
poor selectivity over 5-HT2B. In addition, compound 1 was muta-
genic in the in vitro AMES assay.10
SAR studies were commenced to look at a range of alternative
templates with the aim of identifying a series with an improved
in vitro functional selectivity profile over compound 1 and no
mutagenic activity. The pyridazinone template as exemplified by
compound 2 was identified as meeting the above requirements.
Compound 2 is a partial 5-HT2C agonist with excellent functional
selectivity over the 5-HT2B receptor sub-type, and displayed no
mutagenic activity in the AMES assay. In addition the compound
showed improved metabolic stability due to the reduced lipophil-
icity, but this also resulted in impaired CNS penetration (free plas-
ma concentration:CSF 5:1). Hence SAR work commenced with the
aim of maintaining the selectivity seen with compound 2, while
minimizing the potential dose required to achieve efficacy in
man, by enhancing potency and preferably also improving CNS
penetration. In addition the SAR studies were focused on determin-
ing the optimal lipophilicity range to enable good CNS penetration,
while maintaining metabolic stability.
Similarly pre-clinical studies have highlighted a role for 5-HT2C
agonism in the control of bladder function6 and in sexual function
in both males and females.7 Achieving functional selectivity over
the 5-HT2A and 5-HT2B receptor sub-types was a key challenge
for the project, and particularly important since 5-HT2A agonism
The SAR within the pyridazinone series was limited, with the
majority of substitutions resulting in a complete loss of 5-HT2C
* Corresponding author. Tel.: +44 1304644550.
E-mail address: charlotte.allerton@pfizer.com (C.M.N. Allerton).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.136

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C. M. N. Allerton et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5791–5795
Cl
Cl
Cl
O
N
O
O
N
N
N
N
N
N
N
H
N
H
N
H
mCPP
1
2
5-HT2C EC50 (Emax) 170 nM (71%)
5-HT2A EC50 (Emax) 167 nM (58%)
5-HT2B EC50 (Emax) 125 nM (35%)
5-HT2C EC50 (Emax) 10 nM (69%)
5-HT2A EC50 (Emax) 308 nM (56%)
5-HT2B EC50 (Emax) 187 nM (42%)
LogD7.4 = 2.2; HLM=94 µl/min/mg
MDCK efflux ratio = 2
5-HT2C EC50(Emax) 164 nM (58%)
5-HT2A EC50(Emax) 254 nM (41%)
5-HT2B >10000 nM
LogD7.4 = 0.6; HLM<7 µl/min/mg;
MDCK efflux ratio = 5; free plasma: CSF 5:1
Figure 1. Structures and activity data for lead compounds m-CPP, 1 and 2.
Table 2
agonism. Substitution off the pyridazinone ring with aliphatic
groups, including formation of a bicyclic ring, resulted in a loss of
5-HT2C functional agonism (Table 1). R-Me substitution was toler-
ated off the piperidine ring adjacent to the substituted nitrogen (6,
Table 2), but homologation to an ethyl group resulted in a reduc-
tion of activity (8).
SAR for compounds 6–10, investigating substituents off the piperazine ring
Cl
O
O
N
N
S-Me substitution off the ethyloxy side chain adjacent to the
pyridazinone nitrogen resulted in good 5-HT2C agonism (12, Table
3), while all other substitutions resulted in a loss of in vitro effi-
cacy. Ortho substituents on the phenyl ring were found to be pref-
erable for 5-HT2C functional agonsim over meta and para
substitution, although only a trifluromethyl group resulted in sub
100 nM potency (compound 21, Table 4). Hence only compounds
6, 12 and 21 displayed 5-HT2C functional agonism worthy of fur-
ther investigation.
The most encouraging compounds (compounds 6, 12 and 21,
Table 5), all showed greater potency in the 5-HT2C functional assay
than the lead compound 2, although they all displayed similar lev-
els of partial intrinisic efficacy. In addition compound 24, which
combined the favorable R-Me piperazine substituent with the
ortho-CF3 phenyl substituent, showed the highest 5-HT2C func-
tional potency. The increases in functional potency seen with these
compounds was achieved through an increase in binding affinity
for the 5-HT2C receptor, as demonstrated by the 5-HT2C binding
data (Table 5).
As stated earlier, achieving selectivity over the 5-HT2A and 5-
HT2B sub-types was a key goal for the project in order to avoid
the associated cardiovascular and hallucinogenic side effects. Com-
pounds 6, 12 and 24 (Table 5) showed low levels of selectivity vs 5-
HT2A, although benchmarking using an in vitro 5-HT2A human
platelet aggregation biomarker showed increased selectivity.14
Compound 12 also displayed some functional agonist efficacy at
the 5-HT2B receptor which was considered to be a key risk for
the compound, while compounds 6, 21 and 24 showed excellent
N
X
Y
N
H
X
Y
5-HT2C EC50 nM11 (Emax%)
6
7
8
9
10
R-Me
S-Me
R-Et
C@O
H
H
H
H
H
25 (51)
>10,000
407 (41)
>9980
S-Me
>10,000
Table 3
SAR generated via substituents off the alkyl side chain
X
Cl
O
O
N
N
Y
N
N
H
X
Y
5-HT2C EC50 nM11 (Emax%)
11
12
13
14
R-Me
S-Me
H
H
H
R-Me
S-Me
>1920 (55)
25 (77)
858 (53)
360 (87)
H
selectivity over this sub-type. This selectivity was further con-
firmed using a human colon in vitro biomarker.15
Table 1
SAR for compounds 3–5, investigating substituents off the pyridazinone ring
Due to their excellent potency and selectivity, compounds 6 and
24 were progressed. Both compounds displayed similar in vitro
ADME data to the lead compound 2 (Table 6) indicating that the
compounds should have low hepatic clearance in man, but that im-
paired CNS penetration would be expected. The project felt that it
was unrealistic to look to increase CNS penetration through incor-
porating further lipophilicity since based on prior SAR this would
compromise the excellent selectivity and metabolic stability pro-
file of the compounds. Compound 24 was selected for further pro-
filing since the project felt it had the best balance of potency,
selectivity and ADME properties. It was screened for off-target
pharmacology against a panel of receptors, enzymes and ion chan-
Cl
O
X
Y
O
N
N
N
N
H
X
Y
5-HT2C EC50 (nM)11 (Emax%)
3
4
5
Me
H
>10,000
>10,000
510 (31)
–(CH2)3
H
Me
nels (CEREP, BioprintTM) and found to have sub 1
lM binding

C. M. N. Allerton et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5791–5795
5793
Table 4
Table 6
SAR for compounds 2, 15–23, investigating substituents off the phenyl ring
In vitro ADME data on compounds 2, 6 and 24
O
X
X
O
O
O
N
Y
z
N
N
N
N
Me
N
N
H
N
H
X
Y
Z
5-HT2C EC50 nM11 (Emax%)
X
5-HT2C EC50 nM11
(Emax%)
MDCK efflux
ratio
Log D7.4
HLM (
l
l/min/mg)
2
15
16
17
18
19
20
21
22
23
Cl
H
H
H
H
H
Cl
H
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
164 (58)
>10,000
>10,000
>10,000
>2130
114 (69)
>4550 (36)
47 (45)
2
6
24
Cl
Cl
CF3
341 (55)
25 (51)
47 (45)
5
6
3
0.6
0.9
1.4
<7
9.8
15.3
Cl
CN
Me
CF3
OMe
Br
Cl
>10,000
101 (34)
Cl
N
O
R1
R1
R2
R1
N
N
NH
N
25
R2
b, PG=Bn
N
b,c PG=Boc R2
a
N
N
R3
R4
N
R3
Cl
+
affinity for the human 5-HT1B, 5-HT1D 5-HT6, D2 and GABAA recep-
tors. On follow-up using in vitro functional assays, this pharmacol-
ogy was found not to be significant. Additionally this compound
was found to have low activity at the potassium hERG channel
H
N
R3
R4
N
R4
26
27
28
PG
PG
N
PG
(IC50 = 3.7 lM). Initial studies in dogs (n = 3) showed a sustained
O
O
R1
R2
R1
(>20%) increase in peak urethral pressure during continuous i.v.
infusion of compound 24, at mean free plasma concentrations of
52 nM ( 17 nM).16 Further studies are required to fully elucidate
the pharmacodynamic effects of this compound.
O
O
Ar
Ar
N
N
N
e, PG=Bn
f, PG=Boc
N
R2
d
N
N
R3
R4
N
R3
R4
The main route used to synthesise the pyridazinones described
in this paper is shown in Scheme 1. Mono halo displacement on the
appropriate dichloro pyridazine template 2517 with benzyl or Boc
protected piperazine 26 was followed by aqueous acetic acid med-
iated hydrolysis to give the pyridazinone core in high yield. These
hydrolytic conditions also resulted in removal of the Boc protecting
group, if present, which was then reintroduced by treatment with
Boc anhydride. Incorporation of the side chain via alkylation, fol-
lowed by deprotection led to the desired products in good yield.
Compound 24 was made on multigram scale in an overall yield
of 30% using the method outlined in Scheme 1.
N
H
29
30
PG
Scheme 1. General synthesis of pyridazinones. PG = BOC or Bn. Reagents and
conditions: (a) toluene, Et3N, reflux (46–83%); (b) AcOH, reflux (42–80%); (c) Boc2O,
CH2Cl2 (90–95%); (d) NaHMDS, LiBr, aryloxyethyl bromide or chloride, DMF, 60 °C
(50–86%); (e) ACE–Cl, iPr2NEt, DCM then MeOH, reflux (58–85%); (f) 4 M HCl in
dioxan (85–98%).
t
ylphosphine and butyl azodicarboxylate), followed by treatment
with HCl in dioxan then gave the desired pyridazinones.
Pyridazinone 5 was synthesised in two steps from chloropyrid-
azinone 3418 via N-alkylation with o-chlorophenoxyethyl bromide,
followed by reaction with piperazine in tert-butanol at 120 °C
(Scheme 3).
An alternative route was used to synthesise a range of pyridaz-
inones in parallel (Scheme 2). Alkylation of the Boc protected
pyridazinones 31 with ethylene carbonate gave the key alcohol
intermediates 32. Treatment of the alcohol 32 with phenols under
modified Mitsunobu conditions (using polymer supported triphen-
Table 5
Selectivity and binding data for compounds 2, 6, 12, 21 and 24
z
O
N
Y
O
N
N
X
N
H
X
Y
Z
5-HT2C EC50 nM11 (Emax%)
5-HT2C Ki nM12
5-HT2A EC50 nM13 (Emax%)
5-HT2B EC50 nM13 (Emax%)
2
6
12
21
24
H
Me
H
H
Me
H
H
Me
H
H
Cl
Cl
Cl
CF3
CF3
164 (58)
25 (51)
25 (77)
47 (45)
11 (51)
1600
325
728
424
89
254 (41)
114 (42)
65 (69)
1090 (49)
41 (61)
>10,000
>10,000
506 (30)
>10,000
>2610

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C. M. N. Allerton et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5791–5795
O
O
O
O
O
Cl
O
N
OH
O
OH
O
NH
N
N
N
N
N
Ar
NH
N
N
N
N
N
b, c
a
a
b,c
N
N
R3
N
N
R3
R4
N
R3
R4
N
N
N
N
O
O
O
R4
N
H
41
42
13 (R)
14 (S)
N
H
Boc
Boc
Boc
31
Boc
32
33
Scheme 6. Synthesis of pyridazinones 13 and 14. Reagents: (a) (R) or (S)-propylene
oxide, DCM, water, benzyltriethylammonium chloride (70–80%); (b) polymer
supported PPh3, tbutyl azodicarboxylate, phenol, DCM (20–40%); (c) 2 M HCl in
MeOH (70–90%).
Scheme 2. Alternative pyridazinone synthesis. Reagents and conditions: (a) KOH,
DMF, 110 °C (59–70%); (b) phenol, polymer supported PPh3, tbutyl azodicarboxyl-
ate, DCM; (c) 4 M HCl in dioxan (40–75% over two steps).
with DIBAL to give the alcohol 40. Mitsunobu reaction followed
by Boc deprotection then gave 11 or 12.
Compounds 13 and 14 were synthesised from 41 by reaction
with the appropriate enantiomer of propylene oxide under phase
transfer conditions to give alcohol 42, followed by Mitsunobu reac-
tion and Boc deprotections (Scheme 6).
In summary, extensive SAR exploration of the pyridazinone
template resulted in identification of compound 24, as a potent,
partial 5-HT2C functional agonist with excellent selectivity over
5-HT2B and other aminergic GPCRs. This compound resulted from
optimization of the lead compound 2, through designing partial
agonist compounds with increased binding affinity at the 5-HT2C
receptor. However, compound 24 did not have physiochemical
properties commensurate with enhanced brain penetration com-
pared to compound 2. Despite this, compound 24 demonstrated
good in vivo efficacy in pre-clinical models of stress urinary urge
incontinence as a result of its excellent potency. In addition, com-
pound 24 was clean in the AMES assay. Compound 24 was pro-
gressed for further studies, the results of which will be published
in due course.
O
O
Cl
O
a, b
NH
N
N
N
Cl
N
34
5
N
H
Scheme 3. Synthesis of pyridazinone 5. Reagents and conditions: (a) NaHMDS, LiBr,
o-chlorophenoxyethyl bromide, 60 °C (70–80%); (b) piperazine, tBuOH, 120 °C
(reactivial), 3 days (10–20%).
Pyridazinone 9 was synthesised from chloropyridazinone 35 by
alkylation with o-chlorophenoxyethyl bromide followed by Buch-
wald–Hartwig amidation and Boc deprotection, albeit in low over-
all yield (Scheme 4).
Pyridazinones 11 and 12 were synthesized from 37 by alkyl-
ation with the appropriate enantiomer of the triflate of methyl lac-
tate (Scheme 5). Exchange of the nitrogen protecting group from
benzyl to Boc was then followed by reduction of the methyl ester
Acknowledgments
O
Cl
O
O
O
Cl
N
N
Thanks to Susan Cole, Neil Attkins and Rob Webster of the Phar-
macokinetics, Dynamics and Metabolism department for generat-
ing ADME data and for useful discussions. Thanks to William
McCarte and Nunzio Sciammetta for useful input into the synthesis
of key compounds. Thanks also to Eugene Gbekor, Simeon Ramsey
and Lara Sanders for generating biology data on these compounds.
O
b,c
NH
N
a
N
N
N
O
Cl
Cl
N
H
35
36
9
Scheme 4. Synthesis of pyridazinone 9. Reagents and conditions: (a) NaHMDS, LiBr,
o-chlorophenoxyethyl bromide, 60 °C (53%); (b) 3-oxo-piperazine-1-carboxylic acid
tert-butyl ester, Pd(OAc)2, Cs2CO3, Xantphos, toluene, reflux; (c) 4 M HCl in dioxan
(10% over two steps).
References and notes
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O
O
O
3. Millard, R. J.; Moore, K.; Rencken, R.; Yalcin, I.; Bump, R. C. BJU Int. 2004, 93,
311.
4. Thor, K. B. Urology 2003, 62, 3.
NH
N
N
N
CO2Me
N
N
CO2Me
a
b,c,d
5. Conlon, K.; Miner, W.; Christy, C.; McCleary, S.; Brinkman, H.; Rees, H.;
McMurray, G. Program No. 48.14. 2005, Neuroscience Abstract Viewer.
Washington, DC: Society for Neuroscience, 2005.
N
N
N
N
TfO
CO2Me
(R) or (S)
N
N
6. McMurray, G.; Miner, W. FASEB J. 2005, 19, A536.
37
38
39
7. Kimura, Y.; Naitou, Y.; Wanibuchi, F.; Yamaguchi, T. J. Urol. 2006, 175, 1953;
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Bn
Bn
Boc
Cl
O
O
OH
O
N
N
N
N
e
f,g
N
N
40
11 (R)
12 (S)
N
N
H
11. Activity at the 5-HT2C receptor was initially evaluated by measuring the ability
to induce a fluorescent based calcium mobilization signal in a FLIPR assay
employing recombinant CHO K1 cells expressing the human 5-HT2C receptor.
Andrews, M.; Blagg, J.; Brennan, P.; Fish, P. V.; Roberts, L.; Storer, R. I.; Whitlock,
G. A. WO patent 117169, 2008.
Boc
Scheme 5. Synthesis of pyridazinones 11 and 12. Reagents: (a) NaH, THF, À40 °C
(78%); (b) ACE–Cl, proton sponge, DCM; (c) MeOH (d) Boc2O, iPr2NEt CH2Cl2 (96%
over three steps); (e) DIBAL, toluene, THF (11%); (f) phenol, polymer supported
PPh3, tbutyl azodicarboxylate, DCM; (c) 4 M HCl in dioxan (22% over two steps).
12. Compounds were tested for their ability to inhibit binding of [3H]-meselurgine
at the human 5-HT2C receptor utilizing SPA technology and cellular membrane

C. M. N. Allerton et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5791–5795
5795
preparations generated from recombinant Swiss 3T3 cells. Andrews, M.; Blagg,
J.; Brennan, P.; Fish, P. V.; Roberts, L.; Storer, R. I.; Whitlock, G. A. WO patent
117169, 2008.
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jpet.109.154963v1 .
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measured in FLIPR assays employing Swiss 3T3 cells expressing the
recombinant human 5-HT2A receptor or recombinant human 5-HT2B receptor.
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17. The dichloropyridazine required for the synthesis of
4 was synthesised
according to: Horning, R. H.; Amstutz, E. D. J. Org. Chem. 1955, 20, 707.
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