Synthesis and pharmacological investigation of azaphthalazinone human histamine H1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.08.032

5-Aza, 6-aza, 7-aza and 8-aza-phthalazinone, and 5,8-diazaphthalazinone templates were synthesised by stereoselective routes starting from the appropriate pyridine/pyrazine dicarboxylic acids by activation with CDI, reaction with 4-chlorophenyl acetate ester enolate to give a β-ketoester, which was hydrolysed, and decarboxylated. The resulting ketone was condensed with hydrazine to form the azaphthalazinone core. The azaphthalazinone cores were alkylated with N-Boc-D-prolinol at N-2 by Mitsunobu reaction, de-protected, and then alkylated at the pyrrolidine nitrogen to provide the target H ₁ receptor antagonists. All four mono-azaphthalazinone series had higher affinity (pK_i) for the human H₁ receptor than azelastine, but were not as potent as the parent non-aza phthalazinone. The 5,8-diazaphthalazinone was equipotent with azelastine. The least potent series were the 7-azaphthalazinones, whereas the 5-azaphthalazinones were the most lipophilic. The more hydrophilic series were the 8-aza series. Replacement of the N-methyl substituent on the pyrrolidine with the n-butyl group caused an increase in potency (pA₂) and a corresponding increase in lipophilicity. Introduction of a β-ether oxygen in the n-butyl analogues (2-methoxyethyl group) decreased the H₁ pA₂ slightly, and increased the selectivity against hERG. The duration of action in vitro was longer in the 6-azaphthalazinone series. The more potent and selective 6-azaphthalazinone core was used to append an H₃ receptor antagonist fragment, and to convert the series into the long acting single-ligand, dual H₁ H₃ receptor antagonist 44. The pharmacological profile of 44 was very similar to our intranasal clinical candidate 1.

Full text of DOI:10.1016/j.bmc.2012.08.032

Bioorganic & Medicinal Chemistry 20 (2012) 6097–6108
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological investigation of azaphthalazinone human
histamine H1 receptor antagonists
Panayiotis A. Procopiou a, , Christopher Browning b, Paul M. Gore a, Sean M. Lynn c, Stephen A. Richards c,
⇑
Robert J. Slack b, Steven L. Sollis a
a Medicinal Chemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
b Respiratory Biology, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
c Analytical Chemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Aza, 6-aza, 7-aza and 8-aza-phthalazinone, and 5,8-diazaphthalazinone templates were synthesised by
stereoselective routes starting from the appropriate pyridine/pyrazine dicarboxylic acids by activation
with CDI, reaction with 4-chlorophenyl acetate ester enolate to give a bÀketoester, which was hydroly-
sed, and decarboxylated. The resulting ketone was condensed with hydrazine to form the azaphthalazi-
none core. The azaphthalazinone cores were alkylated with N-Boc-D-prolinol at N-2 by Mitsunobu
reaction, de-protected, and then alkylated at the pyrrolidine nitrogen to provide the target H1 receptor
antagonists. All four mono-azaphthalazinone series had higher affinity (pKi) for the human H1 receptor
than azelastine, but were not as potent as the parent non-aza phthalazinone. The 5,8-diazaphthalazinone
was equipotent with azelastine. The least potent series were the 7-azaphthalazinones, whereas the
5-azaphthalazinones were the most lipophilic. The more hydrophilic series were the 8-aza series.
Replacement of the N-methyl substituent on the pyrrolidine with the n-butyl group caused an increase
in potency (pA2) and a corresponding increase in lipophilicity. Introduction of a b-ether oxygen in the
n-butyl analogues (2-methoxyethyl group) decreased the H1 pA2 slightly, and increased the selectivity
against hERG. The duration of action in vitro was longer in the 6-azaphthalazinone series. The more
potent and selective 6-azaphthalazinone core was used to append an H3 receptor antagonist fragment,
and to convert the series into the long acting single-ligand, dual H1 H3 receptor antagonist 44. The phar-
macological profile of 44 was very similar to our intranasal clinical candidate 1.
Received 8 June 2012
Revised 14 August 2012
Accepted 16 August 2012
Available online 31 August 2012
Keywords:
H1 receptor antagonist
Phthalazinone
SAR
Allergic rhinitis
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
as a result of nasal congestion, which in turn leads to reduced
performance at work and school.3
Allergic rhinitis, also known as ‘hay fever’ affects between 10%
and 25% of the world’s population and has shown a steady increase
in prevalence during the last 40 years.1 The prevalence of allergic
rhinitis may be significantly underestimated because of misdiag-
nosis, underdiagnosis and failure of patients to seek medical atten-
tion. There are two types of allergic rhinitis, seasonal and
perennial. The symptoms of seasonal allergic rhinitis include at
the early stage nasal itching, irritation and sneezing, and at the late
stage rhinorrhoea and nasal congestion. The symptoms of peren-
nial allergic rhinitis are similar, however, nasal congestion may
be more pronounced. Either type of allergic rhinitis may also cause
other symptoms such as irritation of the throat and/or eyes, epiph-
ora and oedema around the eyes.2 In addition to the classical
symptoms it is now recognised that allergic rhinitis has a signifi-
cant impact on quality of life, such as social life, sleep disturbance
Allergic rhinitis and other allergic conditions are associated
with the release of histamine from various cell types, but particu-
larly mast cells. The physiological effects of histamine are medi-
ated by four major G-protein-coupled receptors, termed H1, H2,
H3, and H4, which differ in their expression, signal transduction
and histamine-binding characteristics.4 H1 receptors are widely
distributed throughout the CNS and periphery, and play a critical
role in regulating inflammatory responses and CNS activity, such
as wakefulness. H1 antagonists, also known as H1 blockers or anti-
histamines, are the most commonly used first-line medication for
allergic rhinitis.5,6 First generation H1 receptor antagonists were
introduced 70 years ago and although they were effective, caused
sedation attributed to their ability to blockade H1 receptors in
the CNS. Second generation H1 receptor antagonists were devel-
oped with reduced CNS penetration and a corresponding reduction
in the sedative side-effects. Oral second generation H1 receptor
antagonists such as cetirizine, desloratadine, fexofenadine, lorata-
dine and levocetirizine (Chart 1)7 are effective in treating all the
⇑
Corresponding author. Tel.: +44 1438 762883; fax: +44 1438 768302
E-mail address: pan.a.procopiou@gsk.com (P.A. Procopiou).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.032

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P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
lower logD in order to improve its selectivity profile. In the present
Cl
Cl
paper we report the synthesis and pharmacological profile of the
regioisomeric 5-aza, 6-aza, 7-aza, and 8-aza-phthalazinone ana-
logues of 2, together with the 5,8-diazaphthalazinone analogue,
and the combination of the most potent and selective 6-aza core
with an H3 antagonist fragment to give a novel H1 H3 dual
antagonist.
N
N
N
N
R
O
O
OH
2. Chemistry
(rac)-cetirizine
(R)-cetirizine = levocetirizine
R = -CO2Et, loratadine
R = -H, desloratadine
The preparation of analogues containing a nitrogen atom at the
6- or 7-position of phthalazinone 2 was initially achieved using the
non-regiospecific route outlined in Scheme 1, which is analogous
to the route used for the preparation of 2.
O
OH
N
This route involved reaction of 3,4-pyridinedicarboxylic anhy-
dride (3) with 4-chlorophenylacetic acid (4) to give a 7:1 mixture
of regioisomeric lactones 5 and 6 in 20% yield. The major isomer’s
structure was confirmed by NOE and shown to be 5. Irradiation of
the olefinic proton at 7.18 ppm (1H, s) produced a positive NOE to
the aromatic doublet at 7.85 ppm (2H, d, J 8.5 Hz) and to the pyr-
idine 5-H at 8.10 ppm (1H, dd, J 5.5 and 1 Hz) (Fig. 1). The mixture
of regioisomeric lactones 5 and 6 was treated with hydrazine and
heated in ethanol to 90 °C for 1 h. The resulting regioisomeric mix-
ture of phthalazinones 7 and 8 was separated by column chroma-
tography on silica gel. The major product 7 was obtained in 36%
yield, and the minor isomer 8 in 2%. The phthalazinone 7 was alkyl-
N
N
N
CO2H
Cl
OH
fexofenadine
azelastine
Chart 1. Representative H1 receptor antagonists used clinically.
symptoms of allergic rhinitis, apart from the nasal congestion.
Hence they are often used in combination with -adrenergic ago-
nist decongestants such as pseudoephedrine. However, the use of
-adrenergic agonists is limited due to their potential to produce
hypertension, agitation and insomnia. In addition to the oral anti-
histamines there are topical treatments available, such as azelas-
tine nasal spray, which has been shown to benefit patients who
have not responded adequately to loratadine and fexofenadine,
and is significantly more efficacious than cetirizine and levocabas-
tine in patients with seasonal allergic rhinitis.8,9
Azelastine (Chart 1), which was originally developed for oral
administration, is marketed as a racemic mixture. It is about ten-
times more potent than the first-generation H1 antagonist chlor-
pheniramine10 and is oxidatively metabolised to an active metab-
olite, desmethylazelastine. Adverse effects of azelastine nasal
spray include bitter taste (20%), somnolence (11%) and nasal burn-
ing (4%).8,10 Azelastine was resolved and the potency of its enanti-
omers was reported to be the same as that of the racemic
mixture,11 however, there are no reports associating the adverse
effects of azelastine with one of its enantiomers. The expanding
role of H1 antihistamines has recently been reviewed.12 We have
recently published our efforts in optimising phthalazinone-based
histamine H1 receptor antagonists and on the discovery of H1
and H3 receptor single-ligand antagonists, culminating in the iden-
tification of 1 as a development candidate for the treatment of
allergic rhinitis suitable for intranasal administration (Chart 2).13
Compound 2, our optimised histamine H1 receptor antagonist
had a measured logD of 3.0, which was higher than azelastine
(logD 2.3), we were therefore interested in analogues of 2 with a
ated with N-Boc-D-prolinol by Mitsunobu coupling to give 9 in 43%
a
yield, and then de-protected by treatment with 4 M hydrogen chlo-
ride in 1,4-dioxane to give the secondary amine 10 in 54% yield.
The latter compound was converted to the tertiary amine 11 using
Eschweiler-Clarke alkylation in 62% yield. In a similar manner the
regioisomeric phthalazinone 8 was converted to the tertiary amine
14, via 12 and 13. The preparation of phthalazinone 8 as described
in Scheme 1 provided sufficient material for initial biological
screening, however, compound 8 was obtained as a minor compo-
nent in a mixture, which required chromatographic separation, and
was isolated in very low yield. For this reason an alternative, reg-
ioselective route to phthalazinone 8 was investigated, and is out-
lined in Scheme 2. 3,4-Pyridinedicarboxylic anhydride (3) was
regioselectively converted to the 4-monomethyl ester 15 according
to the method of Joule.14 The carboxylic acid of 15 was activated by
treatment with carbonyl di-imidazole (CDI), treated in situ with
sodium hydride, followed by tert-butyl 4-chlorophenylacetate to
give the b-ketoester 16 in 75%, as a mixture of keto-enol tautomers.
The tert-butyl ester was cleaved, after treatment with trifluoroace-
tic acid, and the resulting acid spontaneously decarboxylated, to
give the ketone 17 in 73% yield. The synthesis of 8 was completed
by reaction of keto-ester 17 with hydrazine in refluxing ethanol
and acetic acid to give the required phthalazinone 8, free of its reg-
ioisomer 7, in 78% yield.
a
In addition to the N-methyl analogues 14 and 2, the N-butyl
analogues 18a and 19a were prepared by reductive alkylation of
the parent secondary amines 13 and 20 with n-butyraldehyde
and sodium triacetoxyborohydride (Scheme 3). Furthermore the
N-(2-methoxyethyl) analogues 18b and 19b were prepared by
O
8
O
N
N
2
N
N
N
4
N
O
5
N
1
2
Cl
Cl
Chart 2. GSK’s intranasal histamine H1H3 dual receptor antagonist, and phthalazinone H1 receptor antagonist template.

P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
6099
O
O
O
N
+
a
b
O
O
N
N
O
5
6
OH
O
Cl
Cl
3
O
Cl
4
O
O
R
O
O
O
N
10 R = H
11 R = Me
e
N
N
N
c
N
NH
N
N
N
N
N
d
9
7
Cl
Cl
Cl
N
O
O
O
O
O
R
N
N
NH
N
f
N
N
13 R = H
14 R = Me
N
d
e
N
N
N
12
8
Cl
Cl
Cl
Scheme 1. Reagents and conditions: (a) NaOAc, AcOH, 230 °C, 20%; (b) NH2NH2ÁH2SO4, NaOH, EtOH, 90 °C; (c) N-Boc-
D
-prolinol, PPh3, i-PrO2CNNCO2Pr-i, THF, À15 to 20 °C,
43%; (d) 4 M HCl–dioxane; (e) HCHO, HCO2H, 100 °C; (f) N-Boc- -prolinol, PPh3, tert-BuO2CNNCO2Bu-tert, THF, À10 to 20 °C, 100%.
D
hydrazine to give phthalazinone 26. Its structure was confirmed
by NOE from the benzylic CH2 at 4.31 ppm to the 5-H of the 8-
azaphthalazinone ring at 8.35 ppm, and to the aromatic protons
of the chlorophenyl ring at 7.34 ppm (Fig. 3). Phthalazinone 26
O
2
3
4
N
6
O
5
was alkylated with N-Boc-D-prolinol by Mitsunobu coupling, and
Cl
then de-protected using TFA to give 27. Eschweiler-Clarke methyl-
ation of 27 gave the methyl analogue 28, whereas reductive alkyl-
ation with butyraldehyde gave the n-butyl analogue 29a. Reaction
of 27 with 2-methoxyethyl bromide provided the ether analogue
29b. The structure of 28 was confirmed by HMBC correlations
and shown in Figure 2. The 5-aza analogues were prepared from
24 in a similar manner to give the methyl analogue 32. The n-butyl
analogue 33a was obtained by alkylation of 31 with n-butyl bro-
mide, and the ether analogue 33b from 2-methoxyethyl bromide.
The structure of phthalazinone 32 was confirmed by HMBC corre-
lations between 8-H of the 5-azaphthalazinone ring to the carbonyl
(Fig. 2).
The synthesis of the 5,8-diaza analogues is outlined in Scheme
5. Pyrazinedicarboxylic anhydride (34) was treated with methanol
to give the mono-methyl ester 35,16 and the resulting monocar-
boxylic acid was activated with CDI and treated with methyl 4-
chlorophenylacetate in the presence of sodium hydride to give a
3:1 mixture of the ester 36 and the corresponding carboxylic acid
37. The mixture of 36 and 37 was hydrolysed and decarboxylated
with HCl to give the ketone 38, which was treated with hydrazine
Figure 1. Key NOESY (H M H) confirming the structure of regioisomer 5.
alkylation of 13 and 20 with 2-methoxyethyl bromide in the pres-
ence of potassium carbonate in DMF in a microwave oven at
150 °C.
The 5-aza and 8-aza regioisomers were prepared in a similar
way to the 6-aza and 7-aza analogues according to Scheme 4.
2,3-Dicarboxylic anhydride (21) was converted to the 2-mono-
ethyl ester 22,15 its structure was confirmed by HMBC correlations
(Fig. 2), the free 3-carboxylic acid was activated with CDI and then
treated in situ with the enolate of methyl 4-chlorophenyl acetate
in DMF to give a mixture of b-keto-ester 23 and the carboxylic acid
24. Both compounds were obtained as mixtures of keto–enol tau-
tomers containing in addition a small amount of the respective
regioisomer arising from addition of the enolate to the ethyl ester
instead of the imidazolide. The keto-ester 23 was hydrolysed in
aqueous 5 M HCl at 90 °C and spontaneously decarboxylated to
give the corresponding ketone 25. The latter was treated with
O
O
O
O
O
CO2Me
N
O
e
f
N
a
b,c,d
8
3
N
O
CO2H
15
O
Cl
Cl
16
17
Scheme 2. Reagents and conditions: (a) NaOMe, MeOH, THF, À70 °C, 45%; (b) CDI, DMF; (c) NaH; (d) tert-butyl 4-chlorophenylacetate, 75%; (e) TFA, DCM, 73%; (f)
NH2NH2ÁH2O, EtOH, AcOH, reflux, 78%.

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P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
O
O
O
O
X
N
N
N
N
N
N
N
N
N
N
N
O
N
a or b
OH
N
13
O
18a X = CH2
18b X = O
Cl
Cl
Cl
32
22
28
Figure 2. Key HMBC (H ? C) correlations confirming the structure of compounds
22, 28 and 32.
O
O
X
H
N
N
N
N
a or b
N
N
to provide the phthalazinone 39. Mitsunobu coupling, followed by
TFA cleavage of the resulting N-Boc-protected amine 40 gave the
secondary amine 41, which was methylated by the Eschweiler-
Clarke procedure to provide the target tertiary amine 42.
Finally the 6-aza analogue of the dual H1 H3 receptor antagonist
1 was prepared by alkylation of 13 with methanesulfonate 4313 in
acetonitrile in the presence of sodium bicarbonate to give 44
(Scheme 6).
19a X = CH2
19b X = O
20
Cl
Cl
Scheme 3. Reagents and conditions: (a) n-PrCHO, DCM, AcOH, NaBH(OAc)3; (b) 2-
MeOCH2CH2Br, K2CO3, DMF, microwave irradiation, 150 °C.
O
O
N
O
O
O
O
OH
O
N
N
b, c
a
O
N
O
OH
O
O
O
22
O
21
O
O
23
24
Cl
Cl
O
O
O
N
N
H
OH
O
NH
N
N
N
N
d
e
f, g
h
23
N
25
26
27
Cl
Cl
Cl
O
O
X
N
N
N
N
N
N
N
i or j
N
27
28
29a X = CH2
29b X = O
Cl
Cl
O
O
O
H
N
NH
N
d, e
f, g
N
N
h
N
N
24
N
N
N
N
30
31
32
Cl
Cl
Cl
O
X
N
k or j
N
N
31
N
33a X = CH2
33b X = O
Cl
Scheme 4. Reagents and conditions: (a) EtOH, 53%; (b) CDI, DMF, 50 °C; (c) NaH, methyl 4-chlorophenylacetate, 5 °C; (d) 5 M HCl, 90 °C; (e) NH2NH2ÁH2SO4, NaOH, EtOH,
90 °C; (f) N-Boc- -prolinol, PPh3, tert-BuO2CNNCO2Bu-tert, 20 °C; (g) TFA, DCM, 20 °C; (h) HCHO, HCO2H, 95 °C; (i) n-PrCHO, NaBH(OAc)3, 20 °C; (j) 2-MeOCH2CH2Br, K2CO3,
DMF, 150 °C; (k) n-BuBr, K2CO3, DMF, 100 °C, microwave, 52%.
D

P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
6101
O
O
respectively. The 5,8-diaza analogue 42 was equipotent with
azelastine (pKi 8.9), but more hydrophilic (logD7.4 1.7, azelastine
logD7.4 2.3). Furthermore 42 was more selective than azelastine
over the human H3 receptor, and the adrenergic receptors a1A
N
NH
N
NH
N
N
and
a1B. Agonists of the latter receptors act on smooth muscle
causing constriction. The 5-, 6-, 7- and 8-aza analogues 32, 14,
11 and 28, respectively were more potent than azelastine, and less
lipophilic than the parent phthalazinone analogue 2. Analogues 14,
28 and 32 had similar H1 receptor antagonist activity and were
more potent than 11, however, all four N-methyl analogues were
less potent than 2. Similar findings were observed with the N-butyl
analogues of the azaphthalazinones, the 6-aza analogue 18a being
slightly more potent than the other N-butyl analogues. The logD of
the azaphthalazinones varied with the position of the nitrogen
atom in the aromatic ring, the more lipophilic being the 5-aza ana-
logue 32 (logD7.4 2.7), whereas the 8-aza analogue 28 was the least
lipophilic (logD7.4 1.7). The N-butyl analogues displayed a similar
trend in logD, the 5-aza analogue 33a having the highest value
(3.4) and the 8-aza analogue 29a the lowest (2.8). All of the above
azaphthalazinone analogues were more selective than azelastine
towards the H3 receptor. Azaphthalazinones were slightly less
selective than 2 towards the H3 receptor, however, they were still
between 2.7 and 3.2log units more selective for H1 over H3 recep-
tor, and at least 10-fold more selective against other receptors,
such as the adrenergic a1A and a1B receptors. Azelastine and 2
exhibited high affinity for the hERG channel in the defetilide bind-
ing assay (7.0 and 6.7, respectively). In the N-methyl series the
26
30
Cl
Cl
Figure 3. Key NOESY (H M H) confirming structures of phthalazinones 26 and 30.
3. Results and discussion
The H1 receptor affinity of compounds was evaluated in vitro
using recombinant human histamine H1 receptor in intact CHO
cells by means of plate-based calcium imaging. Inhibition of ago-
nist-induced cellular calcium mobilisation was monitored using
the calcium sensing dye Fluo-4AM in a FLIPR instrument.17 The
compounds were also evaluated in membranes from CHO cells
transfected with cloned human histamine H3 receptors for their
ability to reduce histamine stimulated GTP-c-S binding as deter-
mined by Scintillation Proximity detection. The adrenergic a1A
and a1B receptor affinity of the test compounds was assessed in
intact Ratl fibroblast cells by means of plate-based calcium imag-
ing. The hERG activity was measured in 3H-dofetilide radioligand
binding assay, and the data from all the above screens are summa-
rised in Table 1. Azelastine and the phthalazinone 2 were used as
reference compounds with affinities (pKi) for H1 of 8.9 and 9.8,
O
O
N
N
O
N
N
O
OH
O
O
O
a
b, c
N
N
N
N
O
+
O
OH
O
O
O
O
O
g
O
34
35
36
37
Cl
Cl
O
O
O
O
N
N
O
N
OH
O
NH
N
N
N
N
N
d
e
f
N
N
38
39
40
Cl
O
Cl
Cl
O
H
N
N
N
N
N
N
h
N
N
N
N
41
42
Cl
Cl
Scheme 5. Reagents and conditions: (a) MeOH, 20 °C, 75%; (b) CDI, DMF, 61 °C; (c) NaH, methyl 4-chlorophenylacetate, 5 °C, 99%; (d) 5 M HCl, H2O, dioxane, 100 °C; (e)
NH2NH2ÁH2O, NaOH, 95 °C, 99%; (f) N-Boc-
D
-prolinol, tert-BuO2CNNCO2Bu-tert, PPh3, THF, 20 °C; (g) TFA, DCM, 20 °C; (h) HCHO, HCO2H, 95 °C, 47%.
O
O
MsO
N
N
N
N
43
N
O
N
13
44
Cl
Scheme 6. Reagents and conditions: NaHCO3, MeCN, 80 °C, 6 days, 19%.

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P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
Table 1
Antagonist affinitya of target compounds at the human H1 receptor (determined by fluorescence imaging plate reader), human H3 receptor (determined by a functional GTP
c
[S]-
assay), affinity at the human
binding affinity.
a1A and a1B receptors (determined in intact fibroblast cells by means of plate-based calcium imaging), measured logD at pH 7.4, and dofetilide hERG
Compound
H1 pKi (n)
H3 pKi (n)
a1A pKi (n)
a1B pKi (n)
LogD7.4
hERG pIC50
Azelastine
2
19a
19b
11
8.92 0.02 (364)
9.79 0.08 (10)
9.04 0.05 (4)
9.29 0.07 (6)
9.16 0.07 (6)
9.46 0.08 (13)
9.40 0.09 (16)
9.60 0.05 (8)
9.45 0.06 (6)
9.2 0.1 (4)
6.83 0.05 (56)
6.36 0.02 (6)
6.36 0.00 (4)
6.4 0.1 (6)
6.4 0.1 (6)
6.4 0.0 (11)
6.2 0.0 (12)
6.2 0.0 (7)
6.6 0.1 (6)
6.2 0.0 (4)
6.2 0.0 (6)
6.5 0.1 (5)
6.2 0.1 (13)
6.2 0.0 (4)
6.2 0.0 (4)
9.25 0.04 (7)
9.6 0.0 (33)
7.3 0.0 (145)
7.5 0.3 (14)
7.8 0.1 (4)
7.7 0.3 (3)
7.7 0.1 (4)
7.9 0.2 (9)
8.2 0.1 (8)
7.9 0.1 (5)
6.2 0.0 (3)
7.0 0.3 (2)
6.5 0.1 (3)
6.7 0.1 (3)
7.3 0.1 (8)
6.8 0.1 (2)
6.4 0.0 (2)
7.5 0.2 (3)
7.4 0.0 (17)
7.3 0.0 (97)
8.0 0.3 (12)
7.8 0.1 (4)
7.9 0.1 (3)
6.9 0.2 (4)
7.6 0.2 (8)
8.1 0.1 (8)
8.0 0.1 (3)
6.8 0.2 (3)
7.0 0.3 (2)
6.5 0.2 (3)
7.3 0.1 (4)
7.7 0.1 (9)
7.4 0.1 (2)
6.6 0.2 (2)
7.4 0.1 (3)
7.5 0.1 (14)
2.3
3.0
3.1
3.3
2.2
1.9
3.2
2.7
1.7
2.8
2.1
2.7
3.4
2.9
1.7
—
7.0
6.7
7.8
6.6
5.9
5.6
7.3
5.9
5.6
7.1
5.3
6.8
8.4
7.2
5.9
6.8
7.3
14
18a
18b
28
29a
29b
32
33a
33b
42
9.0 0.1 (18)
9.5 0.1 (6)
9.2 0.1 (15)
9.27 0.08 (4)
8.89 0.06 (4)
7.76 0.05 (9)
8.0 0.1 (36)
44
1
3.2
a
Shows mean SEM (where applicable) of estimated functional pKi for n <3 the SEM is the SD. n = number of experiments
Table 2
regioisomers with the lowest hERG affinity were the 6-azaphthala-
zinone 14 and 8-aza analogue 28. A similar trend was observed in
the N-butyl series, where the 5-azaphthalazinone 33a had the
highest affinity for hERG, whereas the 6-aza (18a) and 8-azaphtha-
lazinone (29a) had the lowest hERG affinity. One way of reducing
further hERG liability is to reduce the basicity of the amino group
by introducing a b-oxygen. Compounds 18b, 19b, 29b and 33b
were found to have significantly reduced hERG affinity by between
1.2 and 1.9log units. The series with the lowest hERG liability were
again the 6-aza and 8-aza regioisomers 18b and 29b.
Antagonist pA2 affinity at the human H1 receptor, determined by fluorescence
imaging plate reader, and in vitro duration
Compound
pA2 SEMa
n
Wash-out
Azelastine
2
19a
11
9.7 0.1
10.0 0.1
10.4 0.3
8.6 0.4
9.5 0.1
10.0 0.1
9.6 0.1
9.3 0.1
9.5 0.3
9.1 0.1
9.5 0.1
9.8 0.1
8.4 0.1
9.0 0.1
9.1 0.1
19
4
7
2
7
10
12
8
2
6
6
8
3
17
11
Reference
Faster
—
—
No difference
No difference
No difference
—
—
Faster
—
No difference
—
Slower
Slower
14
18a
18b
28
29a
29b
32
33
42
44
1
In addition to the assays reported above, a more precise, lower
throughput, modified version of the human H1 FLIPR assay was
run, which provided apparent pA2 values. Antagonist pA2 values
were determined by generating histamine concentration–response
curves either in the absence or presence of a single concentration
of antagonist (100 nM) at 30 min incubation. The histamine con-
centration–response curves were analysed using a four-parameter
logistic equation to determine the mid-point (EC50) of the curve.
Antagonist pA2 values were calculated using the equation pA2 =
log(DR À 1) À log[B] where DR, the dose ratio, is the EC50 in the
presence of antagonist divided by the EC50 for the control curve,
and [B] is the molar concentration of the antagonist tested. The
data for azaphthalazinones are summarised in Table 2 and com-
pared to azelastine (pA2 9.7), and to the phthalazinones 2 and
19a (pA2 10.0 and 10.4, respectively. Duration of action in vitro
was determined in the FLIPR assay by incubation of adherent
CHO cells with antagonist for 30 min, followed by washing, and
then by repeat histamine challenges at intervals of 90 and
270 min at 37 °C. Agonist dose ratios were converted to receptor
occupancies, which were plotted against time. A measure of dura-
tion was obtained from the gradient of the percent receptor occu-
pancy versus time plot. Results were statistically analysed and
related to azelastine in the same assay, and expressed as slower,
no-difference or faster wash-out than azelastine, with slower
wash-out equating to longer duration of action. The 5- and 6-
azaphthalazinone analogues were more potent than the 7- and
8-aza analogues. Furthermore the 5-azaphthalazinone 33a and 6-
aza 14 exhibited duration similar to that of azelastine, and were
longer acting than the parent phthalazinone 2. The 5-azaphthalazi-
none analogues were more lipophilic and had the highest liability
for the hERG channel and were therefore rejected. In contrast the
6-aza series was the most potent, selective, and longer acting,
and was chosen for appending the H3 fragment used in the dual
a
All pA2 values taken from curve shifts generated at 30 min incubation time and
with 100 nM antagonist. Table 2 shows mean pA2 SEM for n <3 the SEM is the SD.
n = number of experiments.
pharmacology single-ligand antagonist 1 to convert the series into
a dual H1 H3 receptor antagonist.13 Compound 44 had a similar H1
potency to 1 (Tables 1 and 2) and only slightly reduced pKi for the
H3 receptor. The selectivity of 44 against the adrenergic a1A and
a1B receptors was similar to 1, however, the selectivity of 44 over
hERG was better than 1. Furthermore both 1 and 44 had longer
duration of action than azelastine. Appendage of the H3 fragment
to other H1 antagonists, such as 18b, would be expected to provide
additional single-ligand dual H1 H3 antagonists as potential back-
up compounds to 1. Further to our report that 1 was selected for
further development,13 the compound progressed to phase I and
II safety and efficacy studies in seasonal allergic rhinitis. Single-
dose intranasal suspensions of the napadisylate salt of 1 (0.22,
1.10 mg) failed to demonstrate clinically significant attenuation
of symptoms of allergic rhinitis induced in an environmental aller-
gen challenge chamber.19 Three-day repeat dosing of a 1 mg solu-
tion of the dihydrochloride salt of 1 demonstrated a statistically
significant attenuation of nasal symptoms, but this was less than
cetirizine, which was used as a reference. Furthermore the dihy-
drochloride salt of 1 was poorly tolerated by 87% of the subjects
who reported nasal discomfort. Since combined H1 H3 antagonism
did not demonstrate sufficient differentiation from H1 antagonism

P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
6103
in reducing nasal congestion, no further work was carried out in
these series.
biological assays was examined by LCMS analysis and was found
to be P95%, unless otherwise specified.
5.1.1. 11-[(4-Chlorophenyl)methyl]pyrido[3,4-d]pyridazin-
4(3H)-one (7) and 4-[(4-chlorophenyl)methyl]pyrido[3,4-
d]pyridazin-1(2H)-one (8)
4. Conclusion
The regioselective synthesis and pharmacological profile of
5-aza, 6-aza, 7-aza, and 8-aza-phthalazinone analogues of 4-[(4-
chlorophenyl)methyl]-2-{[(2R)-1-methyl-2-pyrrolidinyl]methyl}-
1(2H)-phthalazinone, the optimised (R-enantiomer) histamine H1
receptor antagonist 2, together with the 5,8-diazaphthalazinone
analogue are reported. All four mono-azaphthalazinone series had
higher affinity for the human H1 receptor than azelastine, but were
not as potent as the parent phthalazinone antagonist 2. The 5,8-
diazaphthalazinone was equipotent with azelastine. The most
potent H1 antagonists were found to be the 5- and 6-aza series.
Introduction of nitrogen in the phthalazinone core brought down
the lipophilicity of the analogues with the most hydrophilic series
being the 6- and 8-aza series, which were also more selective over
hERG. Introduction of an ether b-oxygen brought the hERG affinity
of the 6-aza and 8-aza analogues further down, especially so for the
8-aza series. The 6-azaphthalazinone core, which was found to be
the more potent and selective template, was used to append an
H3 receptor antagonist fragment and to provide the long acting
single-ligand dual H1 H3 receptor antagonist 44. The profile of 44
was very similar to our intranasal development candidate 1
(GSK1004723), which unfortunately failed to demonstrate suffi-
cient differentiation of H3 over and above H1 antagonism in
reducing nasal congestion, and hence this series was not
progressed.13,18,19
A mixture of solid 4-chlorophenylacetic acid (20.9 g, 122 mmol)
and 3,4-pyridinedicarboxylic anhydride (3) (18.3 g, 122 mmol) was
treated with sodium acetate (400 mg, 4.8 mmol) and acetic acid
(7 mL, 122 mmol). The mixture was heated to 230 °C in a Dean-
Stark apparatus for 3.75 h. The mixture was allowed to cool to
room temperature, and then partially purified by filtration on silica
(250 g) eluting with ethyl acetateÀcyclohexane, followed by ethyl
acetate to give a 7:1 mixture of (1Z)-1-[(4-chlorophenyl)methyli-
dene]furo[3,4-c]pyridin-3(1H)-one (5) and (3Z)-3-[(4-chloro-
phenyl)methylidene]furo[3,4-c]pyridin-1(3H)-one (6) (6.26 g,
20%): LCMS rt = 3.32 min, 89%, ES+ve m/z 258/260 (M+H)+; 1H
NMR d (DMSO-d6) major isomer 9.21 (1H, s, pyridine 2-H), 8.98
(1H, d, J 5 Hz, pyridine 6-H), 8.10 (1H, d, J 5 Hz, pyridine 5-H),
7.85 (2H, d, J 8.5 Hz, aromatic 2-H and 6-H), 7.58 (2H, d, J 8.5 Hz,
aromatic 3-H and 5-H), 7.18 (1H, s, olefinic). On irradiation of the
olefinic proton at 7.18 a NOE was observed to the two aromatic
protons doublet at 7.85 and to the 5-H doublet at 8.10, confirming
that the major isomer was (1Z)-1-[(4-chlorophenyl)methyli-
dene]furo[3,4-c]pyridin-3(1H)-one (5). The 7:1 mixture of regioi-
somers (5.87 g, 22.8 mmol) was dissolved in ethanol (50 mL) and
treated with hydrazine sulfate (3.56 g, 27.3 mmol) and 1 M sodium
hydroxide (54 mL). The mixture was heated to 90 °C for 1 h, al-
lowed to cool to room temperature, and diluted with water
(250 mL). The precipitated solid was collected by filtration, washed
with water (50 mL), dissolved in acetone, and re-evaporated to
give a red solid (4.46 g, 72%). A portion (3.2 g) was purified by chro-
matography on silica (750 g) eluting with a gradient of 10À100%
ethyl acetateÀdichloromethane, followed by 10% metha-
nolÀdichloromethane to give the minor isomer 4-[(4-chloro-
phenyl)methyl]pyrido[3,4-d]pyridazin-1(2H)-one (8) (100 mg,
2%): LCMS rt = 2.71 min, 90%, ES+ve m/z 272/274 (M+H)+; 1H
NMR d (DMSO-d6) 12.90 (1H, s), 9.33 (1H, s), 8.95 (1H, d, J 5 Hz),
8.08 (1H, d, J 5 Hz), 7.37 (4H, s), 4.40 (2H, s), (contains 4% of the
major isomer), and 1-[(4-chlorophenyl)methyl]pyrido[3,4-d]pyri-
dazin-4(3H)-one (7) (2.22 g, 36%): LCMS rt = 2.73 min, 99%, ES+ve
m/z 272/274 (M+H)+; 1H NMR d (DMSO-d6) 12.92 (1H, s), 9.45
(1H, s), 8.99 (1H, d, J 5.5 Hz), 7.81 (1H, d, J 5.5 Hz), 7.35 (4H, s),
4.30 (2H, s) (contains 6% of the minor isomer).
5. Experimental
5.1. Chemistry
Organic solutions were dried over anhydrous Na2SO4 or MgSO4.
TLC was performed on Merck 0.25 mm Kieselgel 60 F254 plates.
Products were visualised under UV light and/or by staining with
aqueous KMnO4 solution. LCMS analysis was conducted on a Supe-
lcosil LCABZ+PLUS column (3.3 cm  4.6 mm id) eluting with 0.1%
formic acid and 0.01 M ammonium acetate in water (solvent A),
and 0.05% formic acid and 5% water in acetonitrile (solvent B),
using the following elution gradient 0.0À0.7 min 0% B,
0.7À4.2 min 100% B, 4.2–5.3 min 0% B, 5.3–5.5 min 0% B at a flow
rate of 3 mL minÀ1. The mass spectra were recorded on a Fisons
VG Platform spectrometer using electrospray positive and negative
mode (ES+ve and ESÀve). Column chromatography was performed
on Flashmaster II. The Flashmaster II is an automated multi-user
flash chromatography system, available from Argonaut Technolo-
gies Ltd, which utilizes disposable, normal phase, SPE cartridges
(2–100 g). Mass-directed auto-preparative HPLC (MDAP) was con-
ducted on a Waters FractionLynx system comprising of a Waters
600 pump with extended pump heads, Waters 2700 auto-sampler,
Waters 996 diode array and Gilson 202 fraction collector on a
10 cm  2.54 cm id ABZ+ column, eluting with 0.1% formic acid
in water (solvent A) and 0.1% formic acid in acetonitrile (solvent
B), using an appropriate elution gradient over 15 min at a flow rate
of 20 mL minÀ1 and detecting at 200À320 nm at room tempera-
ture. Mass spectra were recorded on Micromass ZMD mass spec-
trometer using electro spray positive and negative mode,
alternate scans. The software used was MassLynx 3.5 with Open-
Lynx and FractionLynx options. 1H NMR spectra were recorded at
400 MHz. The chemical shifts are expressed in ppm relative to tet-
ramethylsilane. High resolution positive ion mass spectra were ac-
quired on a Micromass Q-Tof 2 hybrid quadrupole time-of-flight
mass spectrometer. The purity of all compounds screened in the
5.1.2. 1-[(4-Chlorophenyl)methyl]-3-[(2R)-2-pyrrolidinylmeth
yl]pyrido[3,4-d]pyridazin-4(3H)-one (10)
A solution of triphenylphosphine (1.57 g, 6.0 mmol) in anhy-
drous THF (10 mL) was cooled to À20 °C and di-isopropyl azodicar-
boxylate (1.0 mL, 5.1 mmol) was added. The resulting suspension
was stirred at À15 °C for ten minutes and then treated with a sus-
pension of N-BOC-D-prolinol (636 mg, 3.16 mmol) and 1-[(4-chlo-
rophenyl)methyl]pyrido[3,4-d]pyridazin-4(3H)-one (7) (592 mg,
2.17 mmol) in THF (12 mL). The mixture was allowed to warm to
20 °C and stirred for 18 h. The reaction mixture was treated with
methanol (10 mL) and solvent was removed under reduced pres-
sure. The residue was purified by chromatography on a silica car-
tridge (100 g) on Flashmaster eluting with a gradient of 0À100%
ethyl acetateÀcyclohexane over 60 min to give the BOC-protected
product 9 (625 mg, 43%): MS ES+ve m/z 455/457 (M+H)+. The prod-
uct (625 mg, 1.37 mmol) was dissolved in dioxane (5 mL), treated
with 4 M hydrogen chloride in dioxane (10 mL) and stirred at
20 °C for 1 h. The suspension was concentrated under reduced
pressure; the residue was dissolved in methanol and applied to a
methanol pre-conditioned SCX-2 ion-exchange cartridge. The
cartridge was washed with methanol and then eluted with 10%

6104
P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
aqueous ammonia in methanol. The ammoniacal fraction was con-
centrated under reduced pressure and the residue was purified by
chromatography on a silica cartridge (100 g) on Flashmaster elut-
ing with a gradient of 0À30% [(methanol containing 1% triethyl-
amine) À dichloromethane] over 60 min to give 10 (413 mg,
54%): LCMS rt = 2.26 min, 92%, ES+ve m/z 355/357 (M+H)+; 1H
NMR d (DMSO-d6) 9.50 (1H, s), 9.00 (1H, d, J 5.5 Hz), 7.81 (1H, d,
J 5.5 Hz), 7.44 (2H, d, J 8 Hz), 7.36 (2H, d, J 8 Hz), 4.45 (2H, m),
4.36 (2H, m), 3.87 (1H, m), 3.31–3.20 (2H, m), 3.18–3.08 (1H, m),
2.18–2.06 (1H, m), 2.03–1.84 (2H, m), 1.81–1.69 (1H, m).
evaporated in vacuo to give 16 (2.94 g, 75%, mixture of keto- and
enol-forms) as a pale brown oil: LCMS rt = 3.41 min, 46% and
3.63 min, 48%, ES+ve m/z 390/392 (M+H)+; 1H NMR d (CDCl3)
13.47 (0.5H, s), 8.81 (0.5H, d, J 4.5 Hz), 8.71 (0.5H, s), 8.59 (0.5H,
d, J 5 Hz), 8.29 (0.5H, s), 7.71 (0.5H, d, J 5 Hz), 7.59 (0.5H, d, J
5 Hz), 7.35 (2H, s), 7.27 (2H, s), 7.08 (1H, d, J 8 Hz), 7.00 (1H, d, J
8 Hz), 5.17 (0.5H, s), 3.96 (1.5H, s), 3.91 (1.5H, s), 1.50 (4.5H, s),
1.44 (4.5H, s).
5.1.6. Methyl 3-[(4-chlorophenyl)acetyl]-4-pyridinecarboxylate
(17)
5.1.3. 1-[(4-Chlorophenyl)methyl]-3-{[(2R)-1-methyl-2-
A solution of 16 (2.94 g, 7.5 mmol) in dry dichloromethane
(12 mL) was treated with trifluoroacetic acid (5 mL) and the mix-
ture was stirred at room temperature under nitrogen for 20 h.
The solvent was removed under reduced pressure and the residue
was dissolved in dichloromethane (5 mL). This was applied to a sil-
ica cartridge (100 g) and eluted with a gradient of 0À100% ethyl
acetate in cyclohexane over 60 min. The required fractions were
combined and evaporated under reduced pressure to give 17
(1.59 g, 73%) as a pale orange oil: MS ES+ve m/z 290/292 (M+H)+;
1H NMR d (CDCl3) 8.82 (1H, d, J 5 Hz), 8.66 (1H, s), 7.72 (1H, d, J
5 Hz), 7.32 (2H, d, J 8 Hz), 7.21 (2H, d, J 8 Hz), 4.15 (2H, s), 3.96
(3H, s)
pyrrolidinyl]methyl}pyrido[3,4-d]pyridazin-4(3H)-one (11)
A solution of 10 (145 mg, 0.4 mmol) in formic acid (1.85 mL)
was treated with formaline solution (37% w/v, 3.38 mL) and the
mixture was heated to 100 °C for 4 h. The reaction mixture was
cooled to room temperature and partitioned between dichloro-
methane (20 mL) and 2 M hydrochloric acid (20 mL). The phases
were separated and the aqueous was washed with dichlorometh-
ane (20 mL). The aqueous phase was basified with 2 M sodium
hydroxide (40 mL) and extracted with dichloromethane (20 mL).
All the organic extracts were combined and concentrated under re-
duced pressure. The residue was purified by chromatography on a
silica cartridge (100 g) on Flashmaster eluting with a gradient of
0À15% [(methanol containing 1% triethylamine)Àdichlorometh-
ane] over 60 min to give 11 (92 mg, 62%) as a white solid: LCMS
rt = 2.24 min, 94%, ES+ve m/z 369/371 (M+H)+; 1H NMR (CD3OD)
9.54 (1H, s), 8.90 (1H, d, J 5.5 Hz), 7.78 (1H, d, J 5.5 Hz), 7.36–
7.26 (4H, m), 4.43 (1H, dd, J 13, 4 Hz), 4.34 (2H, s), 4.16 (1H, dd, J
13, 8 Hz), 3.11–3.03 (1H, m), 2.81–2.72 (1H, m), 2.42 (3H, s),
2.34–2.23 (1H, m), 1.97–1.84 (1H, m), 1.85–1.68 (3H, m).
5.1.7. 4-[(4-Chlorophenyl)methyl]pyrido[3,4-d]pyridazin-1(2H)-
one (8)
A solution of 17 (1.59 g, 5.5 mmol) in ethanol (60 mL) was trea-
ted with hydrazine hydrate (0.3 mL, 6 mmol) and a few drops of
acetic acid. The reaction mixture was heated at reflux for 3 h.
The reaction mixture was allowed to cool, the solid was collected
by filtration, washed with ethanol (10 mL), and dried in vacuo to
give 8 (1.17 g, 78%) as a white solid: LCMS rt = 2.73 min, 98%, ES+ve
m/z 272/274 (M+H)+; 1H NMR d (DMSO-d6) 12.90 (1H, s), 9.33 (1H,
s), 8.95 (1H, d, J 5 Hz), 8.08 (1H, d, J 5 Hz), 7.37 (4H, s), 4.40 (2H, s).
5.1.4. 4-[(Methyloxy)carbonyl]-3-pyridinecarboxylic acid (15)14
A suspension of 3,4-pyridinedicarboxylic anhydride (3) (26.7 g,
180 mmol) in dry tetrahydrofuran (250 mL) was treated at À70 °C
under nitrogen with a suspension of sodium methoxide (11.2 g,
2.01 mol) in dry methanol (50 mL). The reaction mixture was al-
lowed to warm to room temperature and stirred for 18 h. The sol-
vents were removed under reduced pressure and the residue was
dissolved in water (350 mL). This solution was acidified to pH 2
using concentrated hydrochloric acid. The resultant solid was col-
lected by filtration, washed with water, and dried in vacuo at 45 °C
to give 15 (14.6 g, 45%) as a white solid: LCMS rt = 0.98 min, ES+ve
m/z 182 (M+H)+; 1H NMR d (DMSO-d6) 13.80 (1H, br), 9.02 (1H, s),
8.87 (1H, d, J 5 Hz), 7.63 (1H, d, J 5 Hz), 3.84 (3H, s).
5.1.8. 4-[(4-Chlorophenyl)methyl]-2-[(2R)-2-pyrrolidinylmeth
yl]pyrido[3,4-d]pyridazin-1(2H)-one (13)
A solution of triphenylphosphine (10.42 g, 40 mmol) in anhy-
drous tetrahydrofuran (80 mL) was treated with a solution of
di-tert-butyl azodicarboxylate (8.38 g, 36 mmol) in anhydrous tet-
rahydrofuran (60 mL) at À10 °C. The solution was allowed to warm
to 15 °C and then cooled to 0–5 °C. To the slight suspension was
added a suspension of 8 (5.98 g, 22 mmol) and N-Boc-D-prolinol
(5.14 g, 25.6 mmol) in anhydrous tetrahydrofuran (100 mL). The
suspension was allowed to warm to ambient temperature and stir-
red for 23 h. The solvent was removed in vacuo to leave an oil
(30 g): LCMS rt = 3.48 min, ES+ve m/z 455/457. The crude product
(30 g) was dissolved in 1,4-dioxane (80 mL), treated with 4 M
hydrogen chloride in 1,4-dioxane (80 mL, 320 mmol), and stirred
at ambient temperature for 5 h. The solvent was removed under
reduced pressure and the residue was partitioned between 1 M
aqueous hydrochloric acid (400 mL) and ethyl acetate (200 mL).
The phases were separated and the aqueous phase washed with
ethyl acetate (200 mL). The combined organic extracts were
washed with 1 M aqueous hydrochloric acid (200 mL). The com-
bined aqueous extracts were basified to pH 10 using 2 M aqueous
sodium hydroxide (350 mL) and the resulting suspension extracted
with ethyl acetate (2 Â 400 mL, 1 Â 200 mL). The combined organ-
ic extracts were concentrated under reduced pressure to give 13
(7.83 g, 100%): LCMS rt = 2.15 min, 99%, ES+ve m/z 355/357
(M+H)+; 1H NMR d (CD3OD) 9.25 (1H, s), 8.91 (1H, d, J 5 Hz), 8.20
(1H, d, J 5 Hz), 7.38–7.28 (4H, m), 4.43 (2H, s), 4.33–4.21 (2H, m),
3.63–3.52 (1H, m), 3.04–2.94 (1H, m), 2.84–2.75 (1H, m), 1.98–
1.72 (3H, m), 1.62–1.49 (1H, m).
5.1.5. Methyl 3-{2-(4-chlorophenyl)-3-[(1,1-dimethylethyl)oxy]-
3-oxopropanoyl}-4-pyridinecarboxylate (16)
To a solution of 15 (1.81 g, 10 mmol) in dry dimethylformamide
(90 mL) under nitrogen was added carbonyl diimidazole (1.7 g,
10.5 mmol). The reaction mixture was heated at 50 °C for 90 min
and then cooled to À5 °C in an ice-salt bath. To this was added
tert-butyl 4-chlorophenylacetate (2.38 g, 10.5 mmol), followed by
portion wise addition of sodium hydride (60% dispersion in min-
eral oil, 1.4 g, 35 mmol) over 15 min. The reaction mixture was
stirred at À5 °C for 10 min and then warmed to room temperature.
After 2 h the reaction mixture was poured into a saturated solution
of ammonium chloride (100 mL), and extracted with ethyl acetate
(3 Â 100 mL). The combined organic solutions were washed with
water (2 Â 100 mL) and brine (2 Â 100 mL). The organic phase
was dried (MgSO4) and the solvent removed in vacuo. The residue
was dissolved in dichloromethane (5 mL) and applied to a silica
cartridge (100 g). This was eluted using a gradient of 0À50% ethyl
acetate in cyclohexane over 60 min. The required fractions were

P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
6105
5.1.9. 4-[(4-Chlorophenyl)methyl]-2-{[(2R)-1-methyl-2-
3.28–3.19 (1H, m), 3.15–3.04 (1H, m), 2.31 (1H, q, J 8 Hz), 2.20–
2.07 (1H, m), 2.06–1.92 (2H, m), 1.72 (2H, quint, J 7.5 Hz), 1.52–
1.36 (2H, m), 0.96 (3H, t, J 7.5 Hz).
pyrrolidinyl]methyl}pyrido[3,4-d]pyridazin-1(2H)-one (14)
A solution of 13 (39 mg, 0.11 mmol) in formic acid (0.5 mL) was
treated with 37% formaldehyde solution in water and methanol
(0.91 mL, 11 mmol) and the mixture was heated to 100 °C for
4.5 h. The reaction mixture was partitioned between DCM
(10 mL) and 2 M HCl solution (10 mL). The aqueous phase was fur-
ther extracted with DCM (10 mL), and then basified with 2 M
NaOH solution and extracted with DCM (2 Â 10 mL). All the organ-
ic extracts were combined and concentrated under reduced pres-
sure. The residue was purified by column chromatography on a
silica cartridge (20 g) on Flashmaster eluting with 0–50% MeOH
in DCM over 30 min. Appropriate fractions were combined and
evaporated under reduced pressure to give 14 (26 mg, 64%): LCMS
rt = 2.25 min, 98%, ES+ve m/z 369/371 (M+H)+; 1H NMR (CD3OD)
9.25 (1H, s), 8.91 (1H, d, J 5 Hz), 8.23–8.17 (1H, m), 7.37–7.26
(4H, m), 4.43 (1H, dd, J 13, 4 Hz), 4.43 (2H, s), 4.17 (1H, dd, J 13,
8 Hz), 3.07 (1H, ddd, J 9, 6, 3 Hz), 2.82–2.73 (1H, m), 2.43 (3H, s),
2.36–2.24 (1H, m), 1.97–1.86 (1H, m), 1.85–1.71 (3H, m).
5.1.13. 4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[2-(methyloxy)
ethyl]-2-pyrrolidinyl}methyl)-1(2H)-phthalazinone
trifluoroacetate salt (19b)
Was prepared in a similar way to 18b: MS ES+ve m/z 412/414
(M+H)+; 1H NMR d (CD3OD) 8.41 (1H, dd, J 7.5, 2 Hz), 7.99 (1H,
dd, J 7.5, 2 Hz), 7.93–7.84 (2H, m), 7.34–7.28 (4H, m), 4.62 (2H, d,
J 5 Hz), 4.39 (2H, s), 4.09–4.00 (1H, m), 3.78–3.65 (4H, m), 3.34
(3H, s), 3.39–3.32 (1H, m), 3.30–3.25 (1H, m), 2.36 2.26 (1H, m),
2.18–1.95 (3H, m).
5.1.14. 2-[(Ethyloxy)carbonyl]-3-pyridinecarboxylic acid (22)
A
suspension of 2,3-pyridinedicarboxylic anhydride (21)
(19.3 g, 0.13 mol) in EtOH (100 mL) was heated to reflux for 2 h.
The clear solution was allowed to cool to 20 °C overnight. The reac-
tion mixture was concentrated under reduced pressure and the
residual solid was triturated in ether to give 22 (13.36 g, 53%) as
a white crystalline solid: LCMS rt = 1.53 min, 99%, ES+ve m/z 196
(M+H)+; 1H NMR d (DMSO-d6) 13.72 (1H, br s, CO2H), 8.77 (1H,
dd, J 5, 1.5 Hz, 6-H), 8.29 (1H, dd, J 8, 1.5 Hz, 4-H), 7.67 (1H, dd, J
8, 5 Hz, 5-H), 4.33 (2H, q, J 7 Hz, –OCH2CH3), 1.31 (3H, t, J 7 Hz, –
OCH2CH3); 13C NMR d (DMSO-d6) 166.2 (CO2Et), 165.9 (CO2H),
151.8 (6-C), 151.45 (2-C), 137.84 (4-C), 125.50 (3-C), 125.06 (5-
C), 61.26 (OCH2), 13.66 (OCH2CH3). HMBC correlations: from 4.33
(q) to 166.2 (CO2Et); from 8.29 (4-H) to 165.9 (CO2H).
5.1.10. 2-{[(2R)-1-Butyl-2-pyrrolidinyl]methyl}-4-[(4-
chlorophenyl)methyl]pyrido[3,4-d]pyridazin-1(2H)-one formic
acid salt (18a)
A solution of 13 (35 mg, 0.1 mmol) in DCM (0.9 mL) and acetic
acid (0.1 mL) was treated with n-butyraldehyde (16 mg,
0.22 mmol) and the mixture was stirred for 30 min before it was
treated with triacetoxyborohydride (45 mg, 0.21 mmol). The reac-
tion mixture was stirred overnight at room temperature and then
was acidified with 2 M hydrochloric acid, neutralised with sodium
bicarbonate solution, and extracted with DCM. The organic solu-
tion was dried (hydrophobic frit) and evaporated under reduced
pressure. The residue was dissolved in methanol and purified by
MDAP to give 18a (29 mg, 63%): MS ES+ve m/z 411/413 (M+H)+;
1H NMR d (CD3OD) 9.30 (1H, s), 8.96 (1H, d, J 5 Hz), 8.44 (1H, s),
8.23 (1H, d, J 5 Hz), 7.38–7.29 (4H, m), 4.63–4.50 (2H, m), 4.45
(2H, s), 3.75 (1H, s), 3.68–3.56 (1H, m), 3.45–3.33 (1H, m), 3.12–
3.01 (1H, m), 3.10–2.89 (1H, m), 2.28–2.15 (1H, m), 2.10–2.02
(1H, m), 2.02–1.89 (2H, m), 1.69 (2H, quint, J 7.8 Hz), 1.46–1.32
(2H, m), 0.96 (3H, t, J 7.4 Hz).
5.1.15. Ethyl 3-[2-(4-chlorophenyl)-3-(methyloxy)-3-
oxopropanoyl]-2-pyridinecarboxylate (23) and 2-[2-(4-
chlorophenyl)-3-(methyloxy)-3-oxopropanoyl]-3-
pyridinecarboxylic acid (24)
A solution of 22 (1.95 g, 10 mmol) in DMF (10 mL) was treated
with carbonyl diimidazole (1.70 g, 10.5 mmol) portionwise under
nitrogen at 50 °C. Gas evolution occurs as soon as CDI was added,
and the mixture was heated overnight at 53 °C. LCMS indicated
completion of carboxylic acid activation (rt = 0.69 min, 83%, ES+ve
m/z 246 (M+H)+ for the imidazolide). The reaction mixture was
cooled in ice-salt and treated with methyl 4-chlorophenylacetate
(2.03 g, 11 mmol) under nitrogen, followed by sodium hydride
(60% oil dispersion; 1.4 g, 35 mmol). As soon as the first portion
of NaH was added the colour of the reaction mixture changed from
colourless to red. The mixture was stirred at À10 °C for 2 h, and
then allowed to warm to 5 °C and stirred for a further 2 h. The mix-
ture was carefully quenched with 2 M HCl solution (18 mL,
26 mmol) and extracted with ethyl acetate. The organic solution
was washed with brine, dried (MgSO4), and evaporated under re-
duced pressure to give an oil (2.59 g). This was dissolved in dichlo-
romethane and purified by column chromatography on a silica
(100 g) cartridge on Flashmaster eluting with a gradient of 0–
100% ethyl acetateÀcyclohexane over 60 min. Fractions eluting
with rt between 32 min and 45 min were combined and evapo-
rated to give a yellow oil: a mixture containing ethyl 3-[2-(4-chlo-
rophenyl)-3-(methyloxy)-3-oxopropanoyl]-2-pyridinecarboxylate
(23) as the major component (1.2 g, 33%). LCMS rt = 3.10 min, 33%,
and rt = 3.21 min, 53%, ES+ve m/z 360/362 (M+H)+; 1H NMR d
(CDCl3) 13.2 (1H, s), 8.77 (1H, dd, minor), 8.59 (1H, dd, major),
7.66 (1H, dd, minor), 7.48 (1H, dd, minor), 7.34 (m), 7.22 (1H, dd,
major), 7.12 (2H, d, J 8 Hz), 7.05 (2H, d, J 8 Hz), 5.28 (1H, s), 4.48
and 4.46 (2H, 2 overlapping q, J 7 Hz), 4.01 (m), 3.80 (3H, s, major),
3.72 (3H, s, minor), 1.45 (3H, m).
5.1.11. 4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[2-(methyloxy)
ethyl]-2-pyrrolidinyl}methyl)pyrido[3,4-d]pyridazin-1(2H)-one
(18b)
A suspension of 13 (28 mg, 0.08 mmol) and potassium carbon-
ate (22 mg, 0.16 mmol) in DMF (1 mL) was treated with 2-
methoxyethyl bromide (22 mg, 0.16 mmol) and the mixture was
heated to 150 °C for 15 min in a microwave oven. The reaction
mixture was filtered and the filtrate was loaded onto a SCX-2
ion-exchange column (10 g) eluting with MeOH and then with a
10% aqueous ammonia solution in MeOH. The ammoniacal frac-
tions were combined and concentrated under nitrogen in a blow-
down unit. The residue was purified by MDAP to give 18b
(8.1 mg, 25%) as a gum: LCMS rt = 2.35 min, 87%, ES+ve m/z 413/
415 (M+H)+; 1H NMR d (CD3OD) 9.25 (1H, s), 8.91 (1H, d, J 5 Hz),
8.20 (1H, d, J 5 Hz), 7.38–7.28 (4H, m), 4.43 (2H, s), 4.35 (1H, dd,
J 13, 4 Hz), 4.12 (1H, dd, J 13, 8 Hz), 3.57–3.46 (2H, m), 3.30 (3H,
s), 3.21–3.13 (1H, m), 3.09–2.98 (2H, m), 2.62–2.54 (1H, m),
2.39–2.30 (1H, m), 1.90–1.73 (4H, m).
5.1.12. 2-{[(2R)-1-Butyl-2-pyrrolidinyl]methyl}-4-[(4-
chlorophenyl)methyl]-1(2H)-phthalazinone (19a)
Was prepared in a similar way to 18a (57.6 mg, 70%): LCMS
rt = 2.60 min, 100%, ES+ve m/z 410/412 (M+H)+; 1H NMR d (CD3OD)
8.46–8.38 (1H, m), 7.99 (1H, br d, J 7.5 Hz), 7.89 (2H, s), 7.36–7.26
(4H, m), 4.69 (1H, dd, J 14, 5 Hz), 4.60 (1H, dd, J 14, 5 Hz), 4.45–4.32
(2H, m), 3.95 (1H, d, J 5 Hz), 3.76–3.65 (1H, m), 3.55–3.41 (1H, m),
The highly coloured aqueous phase and brine washings were
combined and evaporated under reduced pressure. The resulting
brown solid was washed with ethyl acetateÀmethanol (4:1,
100 mL) and the filtrate was evaporated under reduced pressure

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P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
to give a mixture containing crude 2-[2-(4-chlorophenyl)-3-(meth-
yloxy)-3-oxopropanoyl]-3-pyridinecarboxylic acid (24) as a brown
oil (1.3 g): LCMS rt = 0.89 min, 33%, ES+ve m/z 334/336 (M+H)+.
5.1.18. 5-[(4-Chlorophenyl)methyl]-7-{[(2R)-1-methyl-2-
pyrrolidinyl]methyl}pyrido[2,3-d]pyridazin-8(7H)-one formate
salt (28)
Was prepared by Eschweiler-Clarke methylation in a similar
way to 14: LCMS rt = 2.19 min, 98%, ES+ve m/z 369/371 (M+H)+;
1H NMR d (CD3OD) 9.04 (1H, dd, J 4.5, 1 Hz, 7-H), 8.46 (1H, s,
HCO2H), 8.40 (1H, dd, J 8.3, 1 Hz, 5-H), 7.86 (1H, dd, J 8.3, 4.5 Hz,
6-H), 7.35–7.28 (4H, m, Ar), 4.60 (1H, dd, J 14, 5 Hz, NCH2CH),
4.53 (1H, dd, J 14, 5.8 Hz, NCH2CH), 4.38 (2H, s, CH2Ar), 3.61–
3.45 (2H, m, CHNMe and one of MeNCH2), 3.00–2.88 (1H, m, one
of MeNCH2), 2.84 (3H, s, -NCH3), 2.28–2.14 (1H, m, one of
CHCH2CH2), 2.08–1.89 (3H, m, one of CHCH2CH2 and CHCH2CH2).
5.1.16. 5-[(4-Chlorophenyl)methyl]pyrido[2,3-d]pyridazin-
8(7H)-one (26)
The crude 23 (1.2 g, 3.3 mmol) was suspended in 5 M aqueous
hydrochloric acid (15 mL, 75 mmol) and heated to 90 °C with vig-
orous stirring overnight. LCMS rt = 0.85 min, 60%, ES+ve m/z 276/
278 (M+H)+ for 3-[(4-chlorophenyl)acetyl]-2-pyridinecarboxylic
acid and 1.23 min, 27%, ES+ve m/z 258/260 (M+H)+ for the corre-
sponding lactone. Hydrazine sulfate (430 mg, 3 mmol) was added
to the reaction mixture, followed by sodium hydroxide (40 mL)
and the mixture was heated to 90 °C for 1 h. Additional hydrazine
sulfate (260 mg, 2 mmol) and ethanol (10 mL) were added and
heated to 90 °C for 2.5 h. The mixture was allowed to cool to room
temperature and the precipitated solid was collected by filtration,
dried in vacuo at 60 °C to give a 5:1 mixture of 8-azaphthazinone
(26) and 5-azaphthalazinone (30) (300 mg, 33%) as a white solid:
LCMS rt = 0.87 min, 79%, and rt = 1.01 min, 15%, ES+ve m/z 272/
274 (M+H)+; 1H NMR d (DMSO-d6) major 12.83 (1H, s, NH), 9.03
(1H, dd, J 4.5, 1.5 Hz, 7-H), 8.35 (1H, dd, J 8.5, 1.5 Hz, 5-H), 7.86
(1H, dd, J 8.5, 4.5 Hz, 6-H), 7.34 (4H, m, Ar), 4.31 (2H, s, ArCH2–),
NOE was observed from singlet at 4.31 to multiplet at 7.34 and
dd at 8.35; minor 12.83 (1H, s, NH), 9.14 (1H, dd, J 4.5, 1.5 Hz, 6-
H), 8.59 (1H, dd, J 8.5, 1.5 Hz, 8-H), 7.90 (1H, dd, overlaps with ma-
jor isomer’s 7.87 dd), 7.34 (4H, m, Ar), 4.36 (2H, s, ArCH2-), NOE
5.1.19. 7-{[(2R)-1-Butyl-2-pyrrolidinyl]methyl}-5-[(4-
chlorophenyl)methyl]pyrido[2,3-d]pyridazin-8(7H)-one
trifluoroacetic acid salt (29a)
Was obtained in a similar way to 18a: LCMS rt = 2.47 min, 97%,
ES+ve m/z 411/413 (M+H)+; 1H NMR d (CD3OD) 9.08 (1H, dd, J 4.3,
1.0 Hz), 8.43 (1H, dd, J 8.4, 1.4 Hz), 7.89 (1H, dd, J 8.4, 4.5 Hz), 7.36–
7.28 (4H, m), 4.73 (1H, dd, J 14.5, 5 Hz), 4.63 (1H, dd, J 14.5, 5 Hz),
4.40 (2H, s), 4.01–3.91 (1H, m), 3.78–3.70 (1H, m), 3.63–3.51 (1H,
m), 3.28–3.20 (1H, m), 3.14–3.05 (1H, m), 2.39–2.28 (1H, m),
2.21–2.07 (1H, m), 2.06–1.95 (2H, m), 1.79–1.68 (2H, m), 1.48–
1.36 (2H, m), 0.97 (3H, t, J 7.4 Hz).
5.1.20. 5-[(4-Chlorophenyl)methyl]-7-({(2R)-1-[2-
(methyloxy)ethyl]-2-pyrrolidinyl}methyl)pyrido[2,3-
d]pyridazin-8(7H)-one formate salt (29b)
Was prepared in a similar way to 18b MS ES+ve m/z 413/415
(M+H)+; 1H NMR d (CD3OD) 9.07 (1H, br d, J 4.5 Hz), 8.42 (1H, br
d, J 8 Hz), 8.28 (1H, br s), 7.88 (1H, dd, J 8, 4.5 Hz), 7.36–7.29
(4H, m), 4.68–4.56 (2H, m), 4.40 (2H, s), 4.02–3.94 (1H, m), 3.77–
3.60 (4H, m), 3.36 (3H, s), 3.35–3.28 (2H, obscured by CHD2OD),
3.28–3.19 (1H, m), 2.34–2.24 (1H, m), 2.16–1.95 (3H, m).
was observed from singlet at 4.36 to
rt = 10.77 min, found 272.0596. C14H11ClN3O requires 272.0591
and rt = 13.01 min, found 272.0602. 14H11ClN3O requires
m at 7.34; LCHRMS
C
272.0591. The filtrate was concentrated to half-volume under re-
duced pressure whereupon a solid precipitated. The solid was col-
lected by filtration to give additional quantity of 26 (255 mg, 28%)
LCMS rt = 0.87 min 76%, and rt = 1.01 min, 6%, ES+ve m/z 272/274
(M+H)+; 1H NMR d (DMSO-d6) 12.83 (1H, s), 9.03 (1H, dd, J 4.5,
1 Hz), 8.35 (1H, dd, J 8, 1 Hz), 7.86 (1H, dd, J 8, 4.5 Hz), 7.37À7.31
(4H, m), 4.31 (2H, s).
5.1.21. 8-[(4-Chlorophenyl)methyl]pyrido[2,3-d]pyridazin-
5(6H)-one (30)
Was prepared in a procedure similar to that for 26 from crude
2-[2-(4-chlorophenyl)-3-(methyloxy)-3-oxopropanoyl]-3-pyridin-
ecarboxylic acid 24 (1.3 g) to give a 4:1 mixture of 5- and 8-azaph-
thalazinone (30:26): LCMS rt = 0.87 min, 20% and rt = 1.01 min,
80%, ES+ve m/z 272/274 (M+H)+; 1H NMR d (DMSO-d6) 12.8 (1H,
s), 9.15 (1H, dd, J 5, 2 Hz), 8.60 (1H, dd, J 8, 2 Hz), 7.85 (1H, dd, J
8, 5 Hz), 7.32 (4H, m), 4.36 (2H, s).
5.1.17. 5-[(4-Chlorophenyl)methyl]-7-[(2R)-2-
pyrrolidinylmethyl]pyrido[2,3-d]pyridazin-8(7H)-one (27)
The solution of 8-azaphthalazinone (26) (538 mg, 2 mmol), N-
Boc-D-prolinol (478 mg, 2.38 mmol), triphenylphosphine (1.05 g,
4 mmol) in THF (12 mL) was treated with di-tert-butyl azodicar-
boxylate (920 mg, 4 mmol) at room temperature under nitrogen.
The mixture was stirred for 1.5 h and then concentrated under re-
duced pressure. The residue was purified by chromatography on a
silica cartridge (100 g) on Flashmaster eluting with a gradient of
0À25% methanol-dichloromethane over 20 min. Appropriate frac-
tions were combined and evaporated under reduced pressure to
give N-BOC-27 (1.055 g). LCMS rt = 1.23 min, 85%, ES+ve m/z 455/
457 (M+H)+. This product was dissolved in dichloromethane
(15 mL) and treated with TFA (10 mL). The mixture was stirred
for 20 min and then concentrated under reduced pressure. The
residue was dissolved in methanol and applied to a methanol
pre-conditioned SCX-2 ion-exchange cartridge (50 g). The cartridge
was washed with methanol and then eluted with 10% aqueous
ammonia in methanol. The ammoniacal fractions were combined
and evaporated under reduced pressure to give 5-[(4-chloro-
phenyl)methyl]-7-[(2R)-2-pyrrolidinylmethyl]pyrido[2,3-d]pyri-
dazin-8(7H)-one (27) (560 mg, 79%): LCMS rt = 0.75 min, 95%,
ES+ve m/z 355/357 (M+H)+; 1H NMR d (CD3OD) 9.01 (1H, dd, J 4,
1 Hz), 8.34 (1H, dd, J 8, 1 Hz), 7.82 (1H, dd, J 8, 4 Hz), 7.30 (4H,
m), 4.36 (2H, s), 4.35–4.30 (2H, m), 3.66–3.58 (1H, m), 3.06–
2.99 (1H, m), 2.86–2.79 (1H, m), 2.00–1.75 (3H, m), 1.65–1.55
(1H, m).
5.1.22. 8-[(4-Chlorophenyl)methyl]-6-[(2R)-2-
pyrrolidinylmethyl]pyrido[2,3-d]pyridazin-5(6H)-one (31)
MS ES+ve m/z 355/357 (M+H)+; 1H NMR d (CDCl3) 9.06 (1H, dd, J
4.5, 1.5 Hz), 8.67 (1H, br d, J 8 Hz), 7.65 (1H, dd, J 8, 4.5 Hz), 7.37
(2H, d, J 8 Hz), 7.25 (2H, d, J 8 Hz), 4.43 (2H, s), 4.38–4.30 (2H,
m), 3.84–3.73 (1H, m), 3.11–2.95 (2H, m), 1.90–1.75 (3H, m),
1.63–1.52 (1H, m).
5.1.23. 8-[(4-Chlorophenyl)methyl]-6-{[(2R)-1-methyl-2-
pyrrolidinyl]methyl}pyrido[2,3-d]pyridazin-5(6H)-one (32)
Was prepared by Eschweiler-Clarke methylation in a similar
way to 14: LCMS rt = 2.40 min, 98%, ES+ve m/z 369/371 (M+H)+;
1H NMR d (CD3OD) 9.12 (1H, dd, J 4.5, 1.5 Hz, 6-H), 8.67 (1H, dd,
J 8.2, 1.5 Hz, 8-H), 7.81 (1H, dd, J 8.2, 4.5 Hz, 7-H), 7.37 (2H, d, J
8.3 Hz, aromatic 2-H and 6-H), 7.25 (2H, d, J 8.3 Hz, aromatic 3-H
and 5-H), 4.47 (1H, dd, J 13.5, 4.5 Hz, one of NCH2), 4.45 (2H, s,
ArCH2), 4.31 (1H, dd, J 13.5, 7.0 Hz, one of NCH2CH), 3.28–3.21
(1H, m, one of MeNCH2), 3.15–3.04 (1H, m, one of NCH2CH), 2.59
(3H, s, NCH3), 2.58–2.53 (1H, m, one of MeNCH2), 2.06–1.96 (1H,
m, one of CHCH2CH2), 1.91–1.79 (3H, m, one of CHCH2CH2 and

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CHCH2CH2); 13C NMR d (101 MHz, CD3OD) 161.6, 156.8, 150.3,
146.6, 138.5, 136.3, 133.5, 132.1, 129.5, 127.9, 125.4, 67.1, 58.2,
53.7, 41.2, 37.1, 29.8, 22.9.
5.1.28. 8-[(4-Chlorophenyl)methyl]pyrazino[2,3-d]pyridazin-
5(6H)-one (39)
The mixture from the previous experiment (3.62 g) was sus-
pended in 5 M HCl (20 mL) and heated to 100 °C for 20 min. The es-
ter did not dissolve by this time, so 4 M HCl in dioxane (8 mL) was
added and heating was continued for a further 3.5 h. LCMS
rt = 0.77 min, 52%, ES+ve m/z 277/279 (M+H)+ for keto-acid 38,
then allowed to cool to room temperature and treated with sodium
hydroxide (5.28 g, 132 mmol) cautiously. Hydrazine monohydrate
(0.49 mL, 10 mmol) was added to the mixture and heated to
95 °C for 20 min. More hydrazine monohydrate (0.2 mL, 4 mmol)
and sodium hydroxide (80 mg, 2 mmol) were added and heating
was continued for a further 1.5 h. The mixture was concentrated
under reduced pressure, the residue was extracted with ethyl ace-
tate, and the filtrate evaporated to a gum. The gum was triturated
in ether to give 39 as a glass (2.8 g): LCMS rt = 0.91 min, 72%, ES+ve
m/z 273/275 (M+H)+; 1H NMR d (DMSO-d6) 13.0 (1H, br), 9.18 (1H,
d, J 2 Hz), 9.12 (1H, d, J 2 Hz), 7.32 (4H, s), 4.35 (2H, s).
5.1.24. 6-{[(2R)-1-Butyl-2-pyrrolidinyl]methyl}-8-[(4-
chlorophenyl)methyl]pyrido[2,3-d]pyridazin-5(6H)-one (33a)
A solution of 31 (19 mg, 0.054 mmol) in DMF (0.5 mL) was trea-
ted with potassium carbonate (18 mg, 0.13 mmol) and butyl bro-
mide (0.009 mL, 0.08 mmol) and the mixture was heated for
15 min at 100 °C in a microwave oven. A further portion of butyl
bromide (0.009 mL, 0.08 mmol) was added and the mixture was
heated for a further 15 min at 100 °C. The reaction mixture was ap-
plied to a methanol pre-conditioned SCX-2 ion-exchange column
(10 g) and washed with methanol, followed by elution with 10%
aqueous ammonia in methanol. The ammoniacal fractions were
concentrated under reduced pressure and the residue was purified
by MDAP to give 33a (11.6 mg, 52%): LCMS rt = 2.61 min, 98%,
ES+ve m/z 411/413 (M+H)+; 1H NMR d (CD3OD) 9.15 (1H, dd, J
4.5, 1.8 Hz), 8.70 (1H, dd, J 8.0, 1.5 Hz), 7.83 (1H, dd, J 8.0,
4.5 Hz), 7.37 (2H, d, J 8.3 Hz), 7.25 (2H, d, J 8.5 Hz), 4.53 (2H, d, J
5.3 Hz), 4.50–4.42 (2H, m), 3.74–3.65 (1H, m), 3.61–3.53 (1H, m),
3.37–3.33 (1H, m), 3.04–2.96 (1H, m), 2.94–2.85 (1H, m), 2.22–
2.12 (1H, m), 2.08–1.88 (3H, m), 1.69–1.60 (2H, m), 1.42–1.31
(2H, m), 0.93 (3H, t, J 7.4 Hz).
5.1.29. 8-[(4-Chlorophenyl)methyl]-6-[(2R)-2-
pyrrolidinylmethyl]pyrazino[2,3-d]pyridazin-5(6H)-one (41)
Was obtained by a procedure similar to that described for 13:
LCMS rt = 0.78 min, 97%, ES+ve m/z 356/358 (M+H)+; 1H NMR d
(CD3OD) 9.15 (1H, d, J 2 Hz), 9.07 (1H, d, J 2 Hz), 7.38 (2H, d, J
8 Hz), 4.25 (2H, d, J 8 Hz), 4.50–4.40 (4H, m), 3.89–3.82 (1H, m),
3.28–3.22 (1H, m), 2.20–1.72 (3H, m).
5.1.25. 8-[(4-Chlorophenyl)methyl]-6-({(2R)-1-[2-
(methyloxy)ethyl]-2-pyrrolidinyl}methyl)pyrido[2,3-
d]pyridazin-5(6H)-one formate salt (33b)
5.1.30. 8-[(4-Chlorophenyl)methyl]-6-{[(2R)-1-methyl-2-
pyrrolidinyl]methyl}pyrazino[2,3-d]pyridazin-5(6H)-one (42)
LCMS rt = 2.27 min, 92%, ES+ve m/z 370/372 (M+H)+; 1H NMR d
(CD3OD) 9.17 (1H, d, J 2 Hz), 9.08 (1H, d, J 2 Hz), 8.37 (1H, br s),7.37
(2H, d, J 8.3 Hz), 7.27 (2H, d, J 8 Hz), 4.68–4.57 (2H, m), 4.52–4.42
(2H, m), 3.77–3.68 (1H, m), 3.65–3.57 (1H, m), 3.14–3.05 (1H, m),
2.92 (3H, s), 2.32–2.23 (1H, m), 2.12–1.97 (3H, m).
Was prepared in a similar way to 18b: MS ES+ve m/z 413/415
(M+H)+; 1H NMR d (CD3OD) 9.14 (1H, dd, J 4.5, 2 Hz), 8.69 (1H,
dd, J 8, 2 Hz), 8.48 (1H, br s), 7.82 (1H, dd, J 8, 4.5 Hz), 7.37 (2H,
d, J 8 Hz), 7.25 (2H, d, J 8 Hz), 4.51 (1H, dd, J 14, 5 Hz), 4.46 (2H,
s), 4.39 (1H, dd, J 14, 7 Hz), 3.70–3.55 (3H, m), 3.54–3.46 (1H, m),
3.45–3.37 (1H, m), 3.33 (3H, s), 3.08–3.00 (1H, m), 2.97–2.84 (1H,
m), 2.15–2.04 (1H, m), 2.01–1.85 (3H, m).
5.1.31. 4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-
5.1.26. 13-[(Methyloxy)carbonyl]-2-pyrazinecarboxylic acid
(35)16
(hexahydro-1H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-
pyrrolidinyl}methyl)pyrido[3,4-d]pyridazin-1(2H)-one (44)
A mixture of 13 (54 mg, 0.15 mmol), 4-(4-{[3-(hexahydro-1H-
azepin-1-yl)propyl]oxy}phenyl)butyl methanesulfonate (43)13
(78 mg, 0.20 mmol) and sodium bicarbonate (28 mg, 0.33 mmol)
in MeCN (3 mL) was heated at 80 °C with stirring for 6 days under
a nitrogen atmosphere. The cooled reaction mixture was filtered,
and the filtrate was concentrated in vacuo. The residue was parti-
tioned between water and DCM, and the organic phase separated
using a hydrophobic frit. The aqueous phase was diluted with brine
and further extracted with DCM (Â6). The combined organic ex-
tracts were concentrated in vacuo and the residue was purified
by MDAP. The relevant fractions were concentrated, to give two
batches of material, each containing a different impurity. Each of
these batches was further separately purified by chromatography
on silica [2 g, eluted with 2–4% (10% aqueous ammonia in
MeOH)ÀDCM]. Concentration of the appropriate fractions from
these two purifications gave 44 (18 mg, 19%): LCMS rt = 2.37 min,
100%, ES+ve m/z 643/645 (M+H)+ and 322/323 (M/2+H)+; 1H
NMR d (DMSO-d6) 9.23 (1H, s), 8.89 (1H, d, J 5.5 Hz), 8.17 (1H, d,
J 5 Hz), 7.33–7.27 (4H, m), 7.00 (2H, d, J 8 Hz), 6.76 (2H, d, J
8 Hz), 4.37 (2H, s), 4.30 (1H, dd, J 13, 4.5 Hz), 4.11 (1H, dd, J 13,
8 Hz), 3.94 (2H, t, J 6 Hz), 3.14–3.07 (1H, m), 3.01À2.94 (1H, m),
2.85–2.75 (1H, m), 2.73–2.65 (6H, m), 2.48 (2H, t, J 6.5 Hz), 2.35–
2.20 (4H, m), 1.97–1.89 (2H, m), 1.88–1.58 (12H, m), 1.57–1.45
(4H, m); 1H NMR d (CD3OD) 9.23 (1H, s), 8.89 (1H, d, J 5.3 Hz),
8.17 (1H, d, J 5.5 Hz), 7.33–7.26 (4H, m), 7.00 (2 H, d, J 8.5 Hz),
6.77 (2H, d, J 8.5 Hz), 4.38 (2H, s), 4.31 (1H, dd, J 13.0, 4.5 Hz),
4.11 (1H, dd, J 13.0, 8.0 Hz), 3.94 (2H, t, J 6.1 Hz), 3.15–3.07 (1H,
m), 2.98 (1 H, dd, J 7.8, 4.8 Hz), 2.84–2.76 (1H, m), 2.74–2.65 (6
2,3-Pyrazinedicarboxylic anhydride (34) (15.3 g, 102 mmol)
was dissolved in methanol (100 mL) and the solution was stood
at room temperature for 3 days. The solvent was removed under
reduced pressure and the residue was crystallised from ethyl ace-
tate to give 35 (13.85 g, 75%): MS ES+ve m/z 183 (M+H)+; 1H NMR d
(DMSO-d6) 8.91 (1H, d, J 2 Hz), 8.90 (1H, d, J 2 Hz), 3.90 (3H, s).
5.1.27. Methyl 3-[2-(4-chlorophenyl)-3-(methyloxy)-3-
oxopropanoyl]-2-pyrazinecarboxylate (36)
A solution of 35 (1.89 g, 10.4 mmol) in DMF (10 mL) was treated
portionwise with CDI (1.8 g, 11 mmol) and heated to 61 °C under
nitrogen. A further portion of CDI (0.2 g, 1.2 mmol) was added after
2.5 h, the mixture was heated for 2 h, and then allowed to cool to
room temperature overnight. Methyl 4-chlorophenylacetate
(2.2 g, 12 mmol) was added to the mixture, followed by DMF
(2 mL) and the mixture was cooled in an ice-salt bath. Sodium hy-
dride (60% oil dispersion; 1.4 g, 35 mmol) was added portionwise
and the mixture was stirred for 1.5 h. Saturated aqueous ammo-
nium chloride solution (50 mL) was added to the reaction mixture
and the product was extracted with ethyl acetate (25 mL Â 3).
The organic extracts were washed with aqueous NH4Cl, 2 M HCl,
water, and twice with brine, dried (MgSO4) and evaporated to give
a 3:1 mixture of methyl 3-[2-(4-chlorophenyl)-3-(methyloxy)-3-
oxopropanoyl]-2-pyrazinecarboxylate (36) and 3-[2-(4-chloro-
phenyl)-3-(methyloxy)-3-oxopropanoyl]-2-pyrazinecarboxylic
acid (37) (3.62 g) LCMS rt = 0.79 min, 22%, ES+ve m/z 335/337
(M+H)+ for acid, and rt = 1.10 min, 78%, ES+ve m/z 349/351
(M+H)+ ester.

6108
P. A. Procopiou et al. / Bioorg. Med. Chem. 20 (2012) 6097–6108
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1.89–1.44 (16 H, m).5.9 Biological assays have been reported
previously18,20
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Acknowledgments
We thank Dr. Sadie Vile, Mrs. Karen M. L. Morriss, and Miss Jen-
nifer M. Buckley for technical assistance, Dr. Bill Leavens for the
HRMS, Ms. Linda J. Russell for the duration studies, and Dr. Keith
Biggadike for his comments on the manuscript.
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