Structure-activity relationship studies of SEN12333 analogues: Determination of the optimal requirements for binding affinities at α7 nAChRs through incorporation of known structural motifs

Article abstract of DOI:10.1016/j.ejmech.2015.03.025

Alpha7 nicotinic acetylcholine receptors (nAChRs) have implications in the regulation of cognitive processes such as memory and attention and have been identified as a promising therapeutic target for the treatment of the cognitive deficits associated with schizophrenia and Alzheimer's disease (AD). Structure affinity relationship studies of the previously described α7 agonist SEN12333 (8), have resulted in the identification of compound 45, a potent and selective agonist of the α7 nAChR with enhanced affinity and improved physicochemical properties over the parent compound (SEN12333, 8).

Full text of DOI:10.1016/j.ejmech.2015.03.025

European Journal of Medicinal Chemistry 95 (2015) 277e301
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationship studies of SEN12333 analogues:
Determination of the optimal requirements for binding affinities at
nAChRs through incorporation of known structural motifs
a7
,
Corinne Beinat a b, Tristan Reekie a, Samuel D. Banister b, James O'Brien-Brown a,
Teresa Xie c, Thao T. Olson c, Yingxian Xiao c, Andrew Harvey d, Susan O'Connor d,
, f, *
Carolyn Coles d, Anton Grishin d, Peter Kolesik d, John Tsanaktsidis e, Michael Kassiou a
a School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
b Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305, USA
c Department of Pharmacology and Physiology, Georgetown University, Washington, DC 20057, USA
d Bionomics Limited, Thebarton, SA 5031, Australia
e CSIRO Materials Science & Engineering, Ian Wark Laboratory, Bayview Avenue, Clayton, Victoria 3168, Australia
f Faculty of Health Sciences, The University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Alpha7 nicotinic acetylcholine receptors (nAChRs) have implications in the regulation of cognitive pro-
cesses such as memory and attention and have been identified as a promising therapeutic target for the
treatment of the cognitive deficits associated with schizophrenia and Alzheimer's disease (AD). Structure
Received 30 November 2014
Received in revised form
11 March 2015
Accepted 12 March 2015
Available online 14 March 2015
affinity relationship studies of the previously described
identification of compound 45, a potent and selective agonist of the
improved physicochemical properties over the parent compound (SEN12333, 8).
© 2015 Elsevier Masson SAS. All rights reserved.
a
7 agonist SEN12333 (8), have resulted in the
a7 nAChR with enhanced affinity and
Keywords:
a
7 nicotinic receptors
Acetylcholine receptor
CNS
Structureeactivity relationships
1. Introduction
and AD are both chronic conditions with intense and devastating
symptoms; modulators of the 7 nAChR have been extensively
a
Neuronal nicotinic acetylcholine receptors (nAChRs) are a family
of ligand-gated cation channels that have generated extensive in-
terest as potential therapeutic targets for the treatment of cognitive
disorders [1e3]. There exist multiple subtypes of nAChRs where
each subtype is formed from either a homo- or heteropentameric
studied for the treatment of the cognitive deficits associated with
these pathologies [4,6,9e11].
Schizophrenia is a disease defined by positive (hallucinations,
delusions) and negative symptoms (reduced affect, low motivation,
social withdrawal, disorganized thoughts) and cognitive impair-
ments (learning and memory deficits, decreased attention) [12].
While positive symptoms are somewhat controlled with current
medications, cognitive deficits and negative symptoms largely
remain untreated. Examinations of the post-mortem brains of
combination of twelve possible subunits:
a2ea10 and b2eb4. The
homomeric 7 nAChR is one of the most commonly expressed
a
nicotinic receptor subtypes in the human brain and is found in high
levels of expression in regions associated with learning and
memory such as the cerebral cortex and the hippocampus [4e6].
Experimental evidence supports the involvement of this receptor in
schizophrenia and Alzheimer's disease (AD) [7,8]. Schizophrenia
schizophrenic patients have revealed a reduction in
expression and concomitantly a reduction in the density of
nAChR protein [13e15]. Furthermore, polymorphisms within the
7 nAChR subunit gene CHRNA7 have been associated with the
a7 mRNA
a7
a
auditory gating deficits in schizophrenia, a defect ordinarily
normalized by nicotine and possibly underlying the high incidence
of tobacco use among schizophrenic patients [16e18]. Selective a7
* Corresponding author. School of Chemistry, The University of Sydney, Sydney,
NSW 2006, Australia.
E-mail address: michael.kassiou@sydney.edu.au (M. Kassiou).
http://dx.doi.org/10.1016/j.ejmech.2015.03.025
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

278
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
nAChR agonists have shown excellent in vivo efficacy in the
normalization of auditory gating in rats, indicating potential utility
in the treatment of the cognitive deficits associated with schizo-
phrenia [4,19,20].
nAChRs. It displays over 250-fold selectivity for a7 over other
nAChR subtypes and negligible affinity for 100 other receptors
[34,35]. Phencyclidine induced cognitive deficits were shown to be
normalized following SSR180711 (3) administration in mice which
A reduction in the expression of
been observed in the hippocampus of AD patients [21,22]. A
component of -Amyloid (A ) peptides, the characteristic neuritic
plaques of AD thought to contribute to neurodegeneration, was
found to interact with the 7 nAChR with picomolar affinity [23,24].
Exogenous nAChR agonist nicotine exhibits protective effects
against the neurotoxicity of A peptides, and this neuroprotection
can be blocked through selective antagonism of 7 nAChRs [25].
Furthermore, utilization of siRNA transfection to inhibit 7 mRNA
and protein expression, further exacerbated the toxicity of A
peptides in neuroblastoma SH-SY5Y cells [26]. As is consistent with
such findings, selective activation of 7 nAChRs attenuated A
a
7 nAChR protein has similarly
further supports the role of a7 nAChR agonists in treating CDS and
AD [36,37]. SSR180711 (3) successfully entered Phase II clinical
trials, however, studies were discontinued due to cataract devel-
opment in subjects and a narrow therapeutic index. Additional
b
b
a
examples of a7 nAChR agonists entering clinical trials for the
treatment of CDS include TC-5619 (4, Phase II), ABT-107 (5, Phase I),
and EVP-6124 (6, Phase III) [6,38e40]. Despite showing initial
success, TC-5619 (4) and ABT-107 (5) have since been withdrawn
from the clinic, however, EVP-6124 (6) has successfully progressed
b
a
a
b
to Phase III clinical trials and suggests that
safely targeted in the clinic. It is evident that known
a
7 nAChR agonist can be
7 ligands
a
a
b
-
display relatively little structural diversity and have likely been
developed through optimization of a common chemotype. The
majority of these ligands therefore possess the same cross-
reactivity with other sites, such as activity at hERG channels.
High-throughput screening by Siena Biotech and Wyeth led to
the discovery of piperazine 7 (Fig. 2) as a novel chemotype with
induced cell death, and suggests a therapeutic application for
nAChR agonists in the treatment of AD [25,26].
a7
Whilst
a
7 nAChR is a valid target with therapeutic application in
7 nAChR
schizophrenia and AD, few structural classes of selective
a
agonists have been identified, and reported ligands are primarily
centred on anabaseine, quinuclidine, or diazabicyclic scaffolds [27].
weak partial agonist activity at a7 nAChRs, further investigation of
One of the initial functionally
partial agonist, dimethoxybenzylidene anabaseine (DMXB-A, 1,
Fig. 1), exhibiting only micromolar potency at 7 nAChRs with off-
target activity at 2 nAChRs and 5-HT3 receptors [28,29]. In proof
a
7-selective agents identified was
the piperazine, biaryl, and amide regions of compound 7 culmi-
nated in the discovery of SEN12333 (8) [41]. SEN12333 (8) is a
a
potent and selective
a7 nAChR agonist, displaying exceptional
a
4
b
selectivity for 7 over other nAChR subtypes, 5-HT3 receptors, and
a
of concept trials for non-smoking schizophrenic patients, DMXB-A
(1) exhibited an improvement in various neurocognitive measures
and has progressed to Phase II studies, further providing a foun-
hERG channels [41,42]. Aside from its favourable in vitro profile,
SEN12333 also exhibited acceptable bioavailability and good brain
penetration in rats [42]. Initial investigations of SEN12333 in ani-
mal models of episodic memory unveiled its ability to reverse both
scopolamine- and MK-801-induced amnesia [41,42].
dation for the therapeutic use of
a7 agonists [2,30].
In relation to the pharmaceutical industry, various
a
7 nAChR
agonists have entered clinical trials for use in the treatment of
cognitive deficits of schizophrenia (CDS). An early example dis-
closed by AstraZeneca was the quinuclidine-derived spiro-oxazo-
lidinone, AR-R17779 (2), a potent full agonist with several hundred-
The development of SEN12333 (8) was centred on simultaneous
improvement of potency and drug-like properties. Accordingly,
only limited structureeactivity relationships are evident for this
class of a7 nAChR ligands. Modification of the morpholine func-
fold in vitro selectivity for rat
a
7 over rat
a
4
b
2 nAChRs [31]. Phar-
tionality contained within compound 8 revealed branched acyclic
amines to result in decreased potency while small, aliphatic aza-
cycles appear to be optimal, however, few examples of these are
reported [41]. Brief exploration into the replacement of the aryla-
macological profiling of AR-R17779 (2) in vivo demonstrated im-
provements in learning and memory in several rat models, in
accordance with the expected cognition-enhancing properties of
selective
a7 nAChR agonists [32,33]. SR180711 (3) was reported by
nilide with other aromatic moieties showed little effect on
a7
Sanofi-Aventis as a potent partial agonist of recombinant human
a
7
nAChR activity, however, biaryls were generally preferred over
Fig. 1. Selected a7 nAChR agonists evaluated in preclinical and clinical studies.

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
279
Fig. 2. Activity of SEN12333 (8) and its parent structure 7, at a7 nAChRs in a fluorescence assay [40].
mono-aromatic groups [41]. Previous work within our laboratory
examining the effect of contracted or elongated carbon chain
linkers have identified the 4-carbon chain linker of compound 8 to
be optimal for binding and functional activity at a7 nAChRs [43].
Further work by Haydar and co-workers examines the effect on
the first ring of the biaryl system in SEN12333 (8) and details the
development of a novel series of ligands (Series 3) which was
developed from two known, previously studied series (Series 1 and
2), as shown in Fig. 3 [44]. Series 1 is representative of SEN12333
(8), whilst Series 2 described replacement of the amide moiety with
urea functionality [45]. Series 2 generally exhibits enhanced po-
Fig. 4. The structure of compound 9 and its activity against
and patch clamp (EC50) assays [45].
a
7 nAChR in binding (Ki)
tency at
demonstrated low selectivity particularly against the homologous
3 receptor, unfavourable hERG activity and substantial cyto-
chrome P450 inhibition [45]. The development of Series 3 resulted
in compounds with a good level of potency, improved selectivity, an
optimal hERG profile and no cytochrome P450 inhibition [44]. Se-
ries 3 retains two hydrogen bond donors and therefore encom-
passes the possibility of additional interactions when compared to
Series 1 and 2 [44].
a7 nAChRs as compared to Series 1, however, it also
be achieved by synthesizing hybrids of SEN12333 (8) through
integration of functionalities present within compound 9 in addi-
tion to those seen in other known a7 nAChR agonists. This would
allow us to not only determine which pharmacophoric units were
optimal for the binding of SEN12333, but also to identify the
optimal functionalities possible for binding of compound 9 and
ascertain if the functionalities could be interchanged whilst main-
taining affinity and selectivity.
a
The most striking difference between SEN12333 (8) and com-
pound 9 appeared to be the choice of heterocycle comprising the
basic centre of the molecule. Therefore this portion of the molecule
was selected as the starting point for further SAR study with the
synthesis of analogues 10e17 achieving this goal. Morpholine was
replaced by small azacycles piperidine, 1-methylpiperazine,
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane and 2-oxa-6-azaspiro[3.3]
heptane. These azacycles were chosen to represent a stepwise
progression from the heterocycle contained within SEN12333 (8,
morpholine) to that contained within compound 9 ((1S,4S)-2,5-
diazabicyclo[2.2.1]heptane). The 2-oxa-6-azaspiro[3.3]heptane
moiety was also included due to its known structural similarities to
morpholine and favourable physicochemical properties [47].
At the same point in time, compound 9 (Fig. 4) was identified as
a potent and selective a7 nAChR agonist [46]. Structureeactivity
relationship (SAR) studies revealed the 5-N-methyl, heteroaryl
linker and the nature of terminal aryl group to be critical for affinity
and agonist activity at a7 nAChRs.
The differences in the general scaffolds of SEN12333 (8) and
compound 9 were noticed. It was postulated that compound 9 was
somewhat of a truncated hybrid of SEN12333; whereby removal of
the carbon chain linker and amide bond of SEN12333 (8) would
result in a compound of high similarity to compound 9 (Fig. 5).
Herein we report our continued research into analogues of
SEN12333 (8) to expand the SAR profile and identify more
favourable pharmacophoric units for increased affinity. This was to
Fig. 3. Series 1, 2 and 3 with pharmacophoric elements: basic centre (blue), carbon chain (black), central linker (green) and biaryl system (red). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

280
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
Fig. 5. Similarities between SEN12333 (8) and compound 9.
Additionally, it offers the potential for varied hydrogen bonding
interactions. Due to the convergent nature of their synthesis, with
the phenyl pyridyl scaffold being constructed in the final step, we
took the opportunity to prepare an analogous set of biphenyl-
containing compounds. The two scaffolds allowed a simultaneous
examination of the effect of the presence of the pyridine nitrogen
within the terminal aromatic ring.
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane is also engaged with Tyr210,
Trp77 and Tyr115. The essential bridged di-sulfide residues Cys212
and Cys213 are also shown to interact with the carbonyl func-
tionality of compound 14.
2.2. Synthetic chemistry and in vitro studies
The syntheses of analogues 10e17 are depicted in Scheme 1 and
involved the initial conversion of 5-bromovaleric acid (18) to the
corresponding acid chloride and subsequent treatment with 4-
bromoaniline in the presence of triethylamine to give desired ani-
lide 19 (91%). This last compound, in the presence of catalytic io-
dide, could be used to alkylate the appropriate amine to afford
compounds 20e24 in good yields (51e93%). Morpholine, piperi-
dine and 1-methylpiperazine were all readily available, however
amine 25 was synthesized according to procedures previously
described by our laboratory [52] and 2-oxa-6-azaspiro[3.3]heptane
was synthesized according to procedures previously described by
Carreira and co-workers [47]. Subjecting bromoarenes to Suzuki
cross-coupling with either 3-pyridylboronic acid or phenylboronic
acid gave the desired ligands 10e17 (70e90%). SEN12333 (8) was
synthesized for direct comparison with the new analogues.
An alternative synthesis was also proposed in order to prepare
the reverse-amide analogues of the newly synthesized compounds
(Scheme 2). This began by alkylating the appropriate amine with
bromide 28 to yield amines 29e31 (74e91%) followed by hydrazi-
nolysis and amide bond formation to give compounds 32e34
(35e66% over two steps). Final Suzuki cross-coupling with either 3-
pyridylboronic acid or phenylboronic acid gave the desired retro-
amide ligands 37e42 (55e91%).
2. Results and discussion
2.1. Molecular modelling studies
While the
a7 X-ray crystal structure is yet to be determined,
crystals of 7 agonists bound to the extracellular domain of the
a
homologous acetylcholine binding protein (AChBP) [48] have pro-
vided the template for the construction of homology models. Res-
idues conserved from Torpedo marmorata at the a7 nAChR binding
site include aromatic amino acids Tyr94 (loop A), Trp152 and
Tyr154 (loop B), Tyr191 and Tyr199 (loop C) and Trp56 and Gln58
(loop D). Bridged cysteine residues Cys193 and Cys194 (loop C) are
only present within
a subunits and are known to interact with
nicotinic ligands in the co-crystal of AChBP [48,49]. Molecular
modelling methods [43] were applied to SEN12333 (8) and com-
pound 14 in order to predict the key interactions and binding
modes of the different aliphatic heterocyclic groups. The docking
poses and the XP docking summary [50] reveal that proposed
ligand 14, containing the (1S,4S)-2,5-diazabicyclo[2.2.1]heptane
core of agonist 9, is predicted to be a much more potent binder at a7
receptors than SEN12333 (8) as indicated by its more negative
GScore (Table 1).
All newly synthesized compounds and SEN12333 (8) were
subjected to competition binding measurements against racemic
[3H]epibatidine. The binding assays were performed using mem-
brane homogenates of stably transfected HEK293 cells expressing
The docking studies highlight the putative key interactions of
compound 14 in a homology model for the human
a7 receptor
(Fig. 6). The basic amine in the 5-position of the (1S,4S)-2,5-
diazabicyclo[2.2.1]heptane core of compound 14 is engaged in a
hydrogen bond interaction with the indole nitrogen of Trp171, a
rat
a
7,
a4
b2, or
a3
b
4 nAChR subtype [53e55]. All compounds were
selective for the
a
7 subtype over the 2 and 4 subtypes and a
a
4
b
a3b
known key interaction of the cationic centre of
a7 agonists [51]. The
range of affinities were obtained. The results are summarized in
Tables 2 and 3. The first series of compounds (Table 2), reveal
replacement of the morpholine ring of SEN12333 (8) with a
piperidine functionality as in analogue 10 to result in an increase in
Table 1
G-scores of SEN12333 (8) and compound 14.
Compound
Ligand
GScore
potency at
a7 nAChRs; evident in the direct comparison of
SEN12333 (8) and 10 (Ki: 670 nM vs 220 nM). Conversion to the
biphenyl analogue of 10, gave a compound (11) with similar po-
tency (Table 2, entry 3). The use of the 1-methylpiperazine func-
tionality (12) led to a dramatic loss of potency, this was partially
restored in the corresponding biphenyl analogue 13. The results of
compound 10 and 12 are in agreement with work previously
described by Haydaar and co-workers, however, these compounds
were included in the present work for completeness of the study
[41]. Interestingly incorporation of the (1S,4S)-2,5-diazabicyclo
[2.2.1]heptane functionality as in compound 14 corresponded
with a complete loss of potency at a7 nAChRs, again, this was
SEN12333 (8)
ꢀ5.14
14
ꢀ13.14

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
281
Fig. 6. Compound 14 docked into the active site of the AChBP.
Scheme 1. Reagents and conditions: (a) (COCl)2, rt, 45 min; (b) 4-bromoaniline, Et3N, CH2Cl2, e78 ꢁC to rt, 4 h, 91% yield over 2 steps; (c) appropriate amine, NaI, Et3N, DMF, reflux,
16 h, 51e93%; (d) Pd(PPh3)4, appropriate boronic acid, MeCN/sat. aq. Na2CO3 (1:1), reflux, 18 h, 70e90%; * when amine 25 was used additional deprotection and methylation steps
were performed as follows; HCl (4 M in 1,4-dioxane), MeOH, rt, 2 h; (d) 37% aq. HCOH, NaBH(OAc)3, ClCH2CH2Cl, rt, 18 h, 48e53% over two steps.
Scheme 2. Reagents and conditions: (a) appropriate amine, NaI, Et3N, THF, reflux, 16 h, 74e91%; (b) NH2NH2$H2O, EtOH, reflux, 1 h; (c) 4-bromobenzoyl chloride, Et3N, CH2CH2,
e78 ꢁC to rt, 2 h, 35e66% over two steps; (d) Pd(PPh3)4, appropriate boronic acid, MeCN/sat. aq. Na2CO3 (1:1), reflux, 18 h, 55e91%; * when amine 25 was used additional
deprotection and methylation steps were performed as follows; HCl (4 M in 1,4-dioxane), MeOH, rt, 2 h; (d) 37% aq. HCOH, NaBH(OAc)3, ClCH2CH2Cl, rt, 18 h, 58e62% over two steps.
somewhat restored in the analogous biphenyl compound 15. This is
in direct contrast to our molecular modelling results, and suggests
larger 2-oxa-6-azaspiro[3.3]heptane functionality (16 and 17),
similarly resulted in a loss of potency at 7 receptors. The physi-
a
the
a
7 homology model is not predictive of binding affinity at
a
7
cochemical properties, calculated 1eoctanol-water partition coef-
ficient (cLogP), polar surface area (PSA) and ligand lipophilicity
nAChRs for this series of compounds. Incorporation of the slightly

282
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
Table 2
Binding affinities of basic centre exploration of SEN12333 (8).
Ligand
Binding affinity Ki (nM)a,b
Physicochemical properties
Compound
Basic centre
X
N
a
7
a
4
b
2
a
3
b
4
LogPc
PSA
LLEd
3.92
SEN12333 (8)
670 119
>10,000
>10,000
2.25
53.93
10
11
N
CH
220 30
190 30
>10,000
>10,000
>10,000
2600 200
3.46
4.79
44.7
32.34
3.20
1.93
12
13
N
CH
9330
1620
DNC
DNC
313,000
88,100
1.32
2.65
47.94
35.58
3.71
3.14
14
15
N
CH
>10,000
3200 100
>10,000
>10,000
>100,000
>10,000
1.07
2.41
47.94
35.58
ND
3.08
16
17
N
CH
>10,000
>10,000
>10,000
>10,000
>50,000
>50,000
2.40
3.73
53.93
41.57
2.32
1.04
a
Competition binding assays were carried out in membrane homogenates of stably transfected cells or rat forebrain tissue as described previously. The nAChRs were
labelled with [3H]-epibatidine. The Kd values (nM) for [3H]-epibatidine used for calculating Ki values were 0.3 for a3b4, 0.04 for a4b2 and 1.8 for a7. Ki values are the mean SEM
of three to six independent measurements. For low binding affinity compounds, estimated Ki value ranges were provided.
b
DNC ¼ does not converge.
c
Calculated using ChemDraw12.0.
d
ND ¼ not determined.
Table 3
Binding affinities of basic centre exploration in reverse amide series.
Ligand
Binding affinity Ki (nM)a,b
Physicochemical properties
Compound
Basic centre
X
a
7
a
4
b
2
a
3
b4
LogPc
PSA
LLEd
37
38
N
CH
1400 200
410 80
>10,000
>10,000
>50,000
>10,000
3.15
4.49
44.70
32.34
2.70
1.90
39
40
N
CH
>10,000
5400 700
>10,000
>10,000
>10,000
>100,000
1.00
2.35
47.94
35.58
ND
2.92
41
42
N
CH
>10,000
6100 500
>10,000
>10,000
>10,000
>10,000
0.76
2.11
47.94
35.58
3.38
3.10
a
Competition binding assays were carried out in membrane homogenates of stably transfected cells or rat forebrain tissue as described previously. The nAChRs were
labelled with [3H]-epibatidine. The Kd values (nM) for [3H]-epibatidine used for calculating Ki values were 0.3 for a3b4, 0.04 for a4b2 and 1.8 for a7. Ki values are the mean SEM
of three to six independent measurements. For low binding affinity compounds, estimated Ki value ranges were provided.
b
DNC ¼ does not converge.
c
Calculated using ChemDraw12.0.
d
ND ¼ not determined.
efficiency (LLE) of all newly synthesized compounds are also shown
in Table 2. Drug lipophilicity is reportedly changing less over time in
comparison to other physical properties and suggests it to be a key
drug-like property [56,57]. The success of a lead in drug develop-
ment is therefore largely affected by its lipophilicity and accord-
ingly, the role of LogP in influencing drug potency,
pharmacokinetics and toxicity has been recognized for numerous
years [57]. All compounds in the current study display predicted
values clogP < 5, defined by Lipinski to be a requirement for
desirable drug-like and physicochemical properties, all compounds
also exhibited acceptable PSA values [58]. The recent development
of a novel index known as ligand lipophilicity efficient (LLE) has
served as an important guide in the process of lead optimization,
and also as a flag for selection of preclinical candidates [59]. LLE is

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
283
defined as:
biaryl motif of SEN12333 (8), in order to potentially enhance the
hydrogen bonding capacity of the biaryl system within the a7 active
site [44].
LLE ¼ pIC50ðor pKiÞ ꢀ cLogP
The synthesis of compound 43 required a revised synthesis in
order to successfully incorporate the 2-pyridine moiety (Scheme 3).
Following the procedure of Baran and co-workers [61], boronic acid
51 was reacted with pyridine to afford the 2-substituted pyridine
52 in 27% yield. Subsequent hydrogenation afforded aniline 53
(87%), which, in a manner similar to that described above, could be
reacted with the acid chloride formed from acid 18, to afford amide
54 (85% from acid over two steps). Final iodide assisted displace-
ment of the bromide contained within compound 54 with mor-
pholine afforded target compound 43 (74%).
Compounds 44e46 were synthesized in an analogous manner
to that described in Scheme 1 from bromide 20 using the appro-
priate boronic acid (57e72%). Pyridazine analogue 47 could be
synthesized from starting material 55, which underwent mono-
substitution to afford aniline 56 (66%) followed by amide forma-
tion in a manner similar to that described above and thus providing
compound 57 (20% over two steps, Scheme 4). Iodide assisted
morpholine substitution (62%) provided amine 58 which, followed
by Suzuki cross-coupling with phenylboronic acid, afforded the
target pyridazine containing analogue 47 (63%).
A typical oral drug with cLogP of 2.5 and potency within the
range of 1e10 nM suggests a target LLE of 5e7 or greater. The ad-
vantages of high in vivo potency are low total doses in humans and
decreased adventitious compound related toxicity [59]. In essence,
the target objective for molecule optimization is to enhance po-
tency without simultaneously elevating lipophilicity. Whilst all
compounds 8 and 10e17 display acceptable cLogP values the
calculation of their LLE values confers some interesting results.
Piperidine containing analogues 10 and 11 respectively display a 3
and 3.5 fold increase in affinity relative to SEN12333 (8), however,
their LLE values are much lower, 3.2 and 1.9 respectively. This is a
direct result of their increased cLogP values (3.46 and 4.79
respectively vs 2.25 of 8) and is particularly evident in the more
lipophilic 11. Interestingly, while compound 12 is a much weaker
binder at
a
7 receptors (Ki ¼ 9330 nM), its low cLogP value results in
an LLE value similar to that of SEN12333 (8) (3.70 vs 3.92 respec-
tively). Though compound 12 is unsuitable for further development
due to its low binding affinity, the importance of lipophilicity is
highlighted. The importance of binding affinity upon LLE values and
drug-likeness is evident in compounds 14 and 15. Whilst these
compounds display acceptable cLogP values, their extremely low
binding affinities diminish their LLE values and renders them un-
suitable for further development.
Pyrazole containing analogues 48e50 were synthesized as
depicted in Scheme 5 [62]. Methyl benzoate (59) was treated with
the anion derived from acetonitrile to afford compound 60 (63%),
which could then be reacted with hydrazine to form pyrazole 61
(80%). The amine contained within pyrazole 61 could then be used
to form amide 60 (74%) and finally amine displacement as utilized
earlier afforded analogues 48e50 (56e62%)
The binding affinities of the reverse amide hybrid series as
summarized in Table 3 were generally less potent than their
counterparts in Table 2, thus indicating the original amide bond
orientation of SEN12333 (8) to be favourable for
a
7 binding. This is
In the competition binding assay, compounds 43e50 showed a
evident in the direct comparison of data obtained for compounds
37 with 10 and 38 with 11, despite the similar cLogP values between
amide and reverse amide hybrids. A similar trend was seen with
piperidine being the favoured aliphatic heterocycle and biphenyl
compounds were consistently more potent than their phenyl-
pyridyl counterparts. In terms of binding affinity, the results indi-
cate piperidine to be the optimal functionality for the basic centre
portion of SEN12333 (8) and suggest that structural features of the
basic centre of lead compound 9 cannot be transposed onto
SEN12333 (8). It was postulated that substitution with a slightly
branched, larger heterocycle and the introduction of a second po-
tential site of protonation on the heterocycle leads to this decrease
in potency. Consistent with our results from Table 2 the PSA and LLE
values of compounds 37e42 were included. Again, the importance
of lipophilicity is highlighted with the most potent compound of
the series 38 returning an LLE value of only 1.90 as a result of its
increased lipophilicity. Despite the low binding affinity of 42, its
extremely low cLogP value results in its LLE being an acceptable
3.38, even though it is unsuitable for further development.
range of affinities and all compounds were selective for the
a7
subtype over the 2 and 4 subtypes (Table 4). Compounds 43
a
4
b
a3b
and 44 containing the 2- and 4-position pyridine nitrogen both
display a 2-fold increased potency over the lead SEN12333 (8) and
importantly an increase in their LLE values. This indicates that
currently the pyridine nitrogen of 8 is in the least favourable po-
sition within the terminal aromatic ring and suggests the potential
of further development of compounds 43 and 44. Upon conserving
the 3-position of the pyridine nitrogen and incorporating the
electronegative 2-fluorine substituent as in compound 45, a dra-
matic increase in potency and LLE is seen as compared to SEN12333
(8) (85 nM vs 670 nM respectively). Introduction of the 5-indolyl
moiety (46) was well tolerated and led to increased potency rela-
tive to compound 8, suggesting the additional H-bonding possi-
bilities of indole 46 are favourable and that the biaryl moiety can be
extended without sacrificing potency. However, the increased lip-
ophilicity of compound 46 results in a somewhat diminished LLE
value. Hybrid compound 47 containing the biaryl ring system of
compound 9 exhibited a complete loss of potency at a7 receptors,
We then turned our attention to the SAR exploration of the
biaryl moiety of SEN12333 (8). Compounds 43 and 44 examine the
effect of the position of the pyridine nitrogen within the terminal
aromatic ring. Compounds 45 and 46 respectively explore the
incorporation of a fluorine atom, a known bioisostere of hydrogen
[60], onto the terminal aromatic ring of SEN12333 (8) and the
replacement of pyridine with indole, previously described to
exhibit excellent human liver microsome stability in similar com-
pounds (>97% remaining after 30 min incubation) [46]. We sought
to potentially improve the biological properties of SEN12333 (8)
and also to enhance the hydrogen bonding capacity of the terminal
aromatic ring through incorporation of these motifs. Hybrid com-
pound 47 explores the direct transposition of the biaryl moiety of
lead compound 9 onto SEN12333 (8). Compounds 48e50 involve
the incorporation of a phenyl-substituted-pyrazole ring as the
indicating that the structural features of compound 9 cannot be
incorporated into SEN12333 (8). The development of such hybrids
is catastrophic for
nent 5-phenyl-1H-pyrazole moiety resulted in a severe loss of po-
tency at 7 receptors evident in direct comparison of compounds
a7 binding affinity. Incorporation of the promi-
a
48 and SEN12333 (8). This drop in potency was restored by incor-
poration of the more favourable aliphatic heterocycle piperidine
(49). Consistent with our SAR results from the basic centre explo-
ration of SEN12333 (8), incorporation of the 1-methylpiperazine
functionality was unfavourable and resulted in a loss of potency
at a7 receptors (50). While compound 49 displays a similar binding
affinity to SEN12333 (8), its increased lipophilicity lowers its LLE
value as compared to 8 (2.29 vs 3.92 respectively). Additionally, the
comparison of compound 49 with 38 suggests that the phenyl to
pyrazolyl swap has minimal potency penalty but offers
a

284
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
Scheme 3. Synthesis of compound 43. Reagents and conditions: (a) pyridine, trifluoroacetic acid, AgNO3, K2S2O8, CH2Cl2, H2O, rt, 24 h, 27%; (b) H2 (1 atm), Pd/C, MeOH, 18 h, rt, 87%;
(c) (i) 5-bromovaleric acid, (COCl)2, rt, 1 h (ii) 53, Et3N, CH2Cl2, e78 ꢁC to rt, 10 h, 85%; (d) morpholine, NaI, Et3N, THF, reflux, 16 h, 74%.
Scheme 4. Synthesis of compound 47. Reagents and conditions: (a) NH4OH, m
wave, 120 ꢁC, 300 W, 30 min, 66%; (b) (i) 5-bromovaleric acid, (COCl)2, rt, 1 h (ii) 56, Et3N, CH2Cl2,
e78 ꢁC to rt, 10 h, 20%; (c) morpholine, NaI, Et3N, DMF, reflux, 16 h, 62%; (d) Pd(PPh3)4, phenylboronic acid, MeCN/sat. aq. Na2CO3 (1:1), reflux, 18 h, 63%.
Scheme 5. Synthesis of pyrazole containing analogues 48e50. Reagents and conditions: (a) NaH, MeCN, PhMe, reflux, 18 h, 63%; (b) NH2NH2$H2O, CH3COOH, EtOH, reflux, 18 h, 80%;
(c) (i) 5-bromovaleric acid, (COCl)2, rt, 1 h (ii) 60, Et3N, CH2Cl2, e78 ꢁC to rt, 10 h, 74%; (d) appropriate amine, NaI, Et3N, THF, reflux, 16 h, 56e62%.
lipophilicity benefit.
altering the heterocycle comprising the basic centre of the mole-
cules while still retaining the more truncated structure that dis-
tinguishes these two lead compounds. Similar to the previous work
with SEN12333 (8), (1S,4S)-2,5-diazabicyclo[2.2.1]heptane was
replaced by small azacycles morpholine, piperidine and 1-
methylpiperazine. These azacycles were selected as again they
represent the stepwise regression from the (1S,4S)-2,5-diazabicyclo
[2.2.1]heptane heterocycle contained within compound 9 to the
The structures described thus far have focused on the devel-
opment of hybrids of SEN12333 (8) by incorporating elements of
compound 9 onto the SEN12333 (8) scaffold. This has resulted in a
more comprehensive SAR study of SEN12333 (8) being developed.
We then turned our attention to synthesizing hybrids of compound
9; to expand its SAR and better compare binding data with that
obtained for SEN12333 (8) analogues. This was to be achieved by

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
285
Table 4
Binding affinities of biaryl moiety exploration analogues of SEN12333 (8).
Ligand
Binding affinity Ki (nM)a,b
Physicochemical properties
Compound
Biaryl moiety
a
7
a
4
b2
a
3
b
4
LogPc
PSA
LLEd
3.92
SEN12333 (8)
670 119
330 80
350 90
85 10
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2.25
53.93
43
44
45
46
2.46
2.25
2.48
3.57
53.93
53.93
53.93
53.6
4.02
4.21
4.59
2.94
310 30
47
48
>10,000
>10,000
>10,000
>10,000
>10,000
2.39
2.96
66.29
65.96
1.65
2.63
2600 300
49
50
660 70
>50,000
>10,000
>10,000
>10,000
3.89
1.74
56.73
59.97
2.29
2.85
>10,000
a
Competition binding assays were carried out in membrane homogenates of stably transfected cells or rat forebrain tissue as described previously. The nAChRs were
4, 0.04 for 2 and 1.8 for 7. Ki values are the
labelled with [3H]-epibatidine. The Kd values (nM) for [3H]-epibatidine used for calculating Ki values were 0.3 for
a
3
b
a
4
b
a
mean SEM of three to six independent measurements. For low binding affinity compounds, estimated Ki value ranges were provided.
b
DNC ¼ does not converge.
c
Calculated using ChemDraw12.0.
d
ND ¼ not determined.
morpholine ring contained within SEN12333 (8). The synthesis of
compound 9 and analogues 63e65 is shown in Scheme 6 and be-
gins with mono-chloride substitution of pyridazine 55 with various
amines to afford compounds 67e70 (37e83%). Suzuki cross-
coupling of the remaining chloride with phenylboronic acid affor-
ded analogues 63e65 (59e78%). 5-Indolyl analogue 66 was syn-
thesized analogously so as to draw comparisons with previously
tested SEN12333 (8) hybrid 46.
Competition binding assays of the newly synthesized hybrids of
compound 9 are summarized in Table 5. It was immediately evident
that the potency trend for the basic centre of compound 9 was in
contradiction to that of SEN12333, (8). Aliphatic azacycles mor-
pholine and piperidine which corresponded to potency and selec-
tivity for compounds of similar structure to SEN12333 (8) were the
less active in the series of truncated hybrids (63 and 64). Incorpo-
ration of 1-methylpiperazine (65) marginally restored potency at

286
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
Scheme 6. Reagents and conditions: (a) appropriate amine, DIPEA, DMF, reflux, 17 h, 37e83%; (b) Pd(PPh3)4, phenylboronic acid, MeCN/sat. aq. Na2CO3 (1:1), reflux, 18 h, 59e78%; *
when amine 25 was used an additional deprotection and methylation steps were performed as follows; HCl (4 M in 1,4-dioxane), MeOH, rt, 2 h; (d) 37% aq. HCOH, NaBH(OAc)3,
ClCH2CH2Cl, rt, 18 h, 66% over two steps.
a
7 receptors; however, compound 65 interestingly was the only
the dramatic loss of potency of compounds 72e75. Similar to the
results of Table 5, introduction of the 1-methylpiperazine func-
tionality (76 and 77) marginally restored potency. The most potent
compounds of this series were 78 and 79 containing the (1S,4S)-
2,5-diazabicyclo[2.2.1]heptane functionality. Interestingly, in
accordance with the SEN12333-hybrids (Tables 2 and 3), the
biphenyl compounds were of enhanced potency as compared to
their corresponding phenyl-pyridyl counterparts. While compound
79 was considerably more potent than compound 78, the high
cLogP value of compound 79 has resulted in its LLE value being
below that of compound 78. For compounds containing the general
compound to show any potency for the 2 receptors. The most
a4b
potent compound of the series was compound 9 thus suggesting
that the (1S,4S)-2,5-diazabicyclo[2.2.1]heptane functionality is the
optimal heterocycle for the binding and selectivity of the synthe-
sized compounds which possess this truncated structure. This is
further highlighted by the excellent LLE value of compound 9.
Incorporation of the 5-indolyl moiety into the truncated scaffold
resulted in hybrid 66, previously shown to improve the potency of
SEN12333 hybrid e 46 (Table 4), here resulted in a complete loss of
potency at a7 receptors.
The final set of hybrids synthesized investigated the necessity of
the pyridazine moiety within compound 9 (general structure 2) and
the consequence of incorporating the biphenyl or phenyl-pyridyl
motif originally associated with SEN12333 (8) (general structure
structure 2, it can be concluded that potency at a7 receptors is
largely dependent on the aliphatic heterocyclic ring, alteration of
this to a smaller, unsubstituted heterocycle results in a striking loss
of potency at a7 receptors irrespective of the biaryl ring system.
1). The synthesis of analogues 72e79 began with
a
Buch-
Substitution of the biaryl ring system of compound 9 (general
structure 2) with that of SEN12333 (general structure 1) was well
tolerated and suggests that these ring systems can be interchanged
on these smaller truncated hybrids.
waldeHartwig amination between an appropriate amine and 1,4-
dibromobenzene (80) or, in the case of using piperidine as the
amine, a Cu(II) mediated coupling to afford compounds 81e84
(47e82%) (Scheme 7). A subsequent Suzuki cross-coupling with the
appropriate boronic acid led to the target compounds 72e79
(70e90%).
2.3. Functional activity of selected compounds
In consistency with our results of the previous truncated series
(Table 5), small unsubstituted azacycles morpholine and piperidine
are not tolerated for compounds bearing the scaffold of general
structure 2, the results are summarized in Table 6. This is evident in
Several of the
a7 receptor ligands described were selected,
based upon their binding profiles and LLE values, for evaluation in
functional activity studies. The aim of these studies was to deter-
mine the agonist activity of the selected synthesized ligands at the
Table 5
Binding affinities of basic centre exploration of truncated analogues.
Ligand
Binding affinity Ki (nM)a,b
Physicochemical properties
Compound
Basic centre
a
7
a
4
b
2
a
3
b
4
cLogPc
PSA
LLEd
1.67
63
>10,000
>10,000
4070
>100,000
>10,000
417
>1,000,000
>10,000
>10,000
>50,000
>10,000
1.78
37.19
64
65
9
3.18
2.36
2.50
1.79
27.96
31.20
31.20
49.22
ND
ND
5.73
ND
5.9 0.6
>10,000
>30,000
>10,000
66
a
Competition binding assays were carried out in membrane homogenates of stably transfected cells or rat forebrain tissue as described previously. The nAChRs were
labelled with [3H]-epibatidine. The Kd values (nM) for [3H]-epibatidine used for calculating Ki values were 0.3 for a3b4, 0.04 for a4b2 and 1.8 for a7. Ki values are the mean SEM
of three to six independent measurements. For low binding affinity compounds, estimated Ki value ranges were provided.
b
DNC ¼ does not converge.
c
Calculated using ChemDraw12.0.
d
ND ¼ not determined.

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
287
Scheme 7. Reagents and conditions (a) appropriate amine, Pd2(dba)3, -BINAP, toluene, reflux, 15 min then 80, KOtBu, reflux, 16 h; (b) Cu(OAc)2$H2O, 4-bromophenylboronic acid,
piperidine, 4 Å MS, CH2Cl2, 20 h, rt, 58% (c) Pd(PPh3)4, appropriate boronic acid, MeCN/sat. aq. Na2CO3 (1:1), reflux, 18 h, 70e90%; * when amine 25 was used an additional
deprotection and methylation steps were performed as follows; HCl (4 M in 1,4-dioxane), MeOH, rt, 2 h; (d) 37% aq. HCOH, NaBH(OAc)3, ClCH2CH2Cl, rt, 18 h, 54e61%% over two
steps.
Table 6
Binding affinities of SEN12333 (8)-hybrid-truncated analogues.
Ligand
Binding affinity Ki (nM)a,b
Physicochemical properties
Compound
Basic centre
X
a
7
a
4
b2
a
3
b
4
LogPc
PSA
LLEd
72
73
N
CH
>10,000
>10,000
DNC
>10,000
>10,000
>10,000
2.10
3.49
24.83
12.47
1.50
ND
74
75
N
CH
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
3.48
4.87
15.6
3.24
ND
ND
76
77
N
CH
9400 1800
7700 800
>10,000
>10,000
>10,000
>10,000
2.66
4.05
18.84
6.48
2.37
1.06
78
79
N
CH
140 40
34 10
>10,000
>10,000
>10,000
>10,000
2.80
4.19
18.84
6.48
4.05
3.28
a
Competition binding assays were carried out in membrane homogenates of stably transfected cells or rat forebrain tissue as described previously. The nAChRs were
labelled with [3H]-epibatidine. The Kd values (nM) for [3H]-epibatidine used for calculating Ki values were 0.3 for a3b4, 0.04 for a4b2 and 1.8 for a7. Ki values are the mean SEM
of three to six independent measurements. For low binding affinity compounds, estimated Ki value ranges were provided.
b
DNC ¼ does not converge.
c
Calculated using ChemDraw12.0.
d
ND ¼ not determined.
a
7 receptor. The ligands included in the functional activity studies
terminal aromatic ring, previously displayed a 2-fold increase in
binding affinity compared to SEN12333 (8) (Ki ¼ 350 vs 670 nM). In
the functional assay, compound 44 exhibited comparable potency
(EC50 ¼ 522 nM) to SEN12333 (8), indicating that the 4-position of
the pyridine nitrogen within the terminal aromatic ring is favour-
able for both binding affinity and agonist potency. Compound 45,
incorporating the electronegative fluorine atom, previously dis-
played an 8-fold increase in binding affinity compared to SEN12333
(8) (Ki ¼ 85 nM vs 670 nM). Compound 45 additionally demon-
strated an EC50 of 145 nM, a dramatic improvement in functional
activity compared to SEN12333 (8), which may result from atten-
uation of pyridine basicity, the addition of another hydrogen-bond
acceptor or by increasing the torsional angle between the hetero-
aromatics. Considering the increased potency of 45, this region of
the molecule could be targeted for further examination. Compound
46 containing the 5-indolyl motif, previously demonstrated a 2-fold
improvement in binding affinity compared to SEN12333 (8)
(Ki ¼ 310 nM vs 670 nM). However, in studies of agonist potency,
compared to SEN12333 (8), compound 46 displayed comparable
were compounds 44, 45, 46, hybrid compounds 78 and 79.
SEN12333 (8) and lead agonist 9 were also included as references to
directly correlate SAR as a result of modifying these leads. To
determine their agonistic activity at
compounds were evaluated in a fluorescent calcium assay using a
GH4C1 cell line expressing rat
marized in Table 7.
The EC50 of SEN12333 (8) was determined to be 687 nM, cor-
responding to its binding affinity (Ki ¼ 670 nM). Compound 44
which contains the pyridine nitrogen at the 4-position within the
a7 receptors, the selected
a7 receptors. The results are sum-
Table 7
The functional activities of selected compounds.
Compound
EC50 (nM)a
SEN12333 (8)
687
522
145
997
126
253
25
44
45
46
78
79
9
agonist activity at the a7 receptor, with an EC50 of 997 nM, sug-
gesting the extension of the indolyl motif into the hydrophobic
pocket is similarly tolerated for agonist activity. Lead agonist 9
(Ki ¼ 5.9 nM) was the most potent compound of the series with an
EC50 of 25 nM. Analogues 78 and 79 examined the incorporation of
the biaryl motif initially associated with SEN12333 (8). These
a
Compounds were tested for agonist activity on the
a
7nAChR in a fluorescent calcium assay using a GH4C1
cells stably expressing rat
values, all compounds were tested at 0.03, 0.1, 0.3, 1, 3
and 10
M (n ¼ 3) except for compound 9 which was
M. Replicates have been
a7 nAChR. To determine EC50
compounds maintained binding affinity at the
and 34 nM respectively), however, this was diminished compared
a
7 receptor (Ki ¼ 140
m
to the lead compound 9. These compounds additionally maintained
agonist activity at the a7 receptor, albeit below that of lead agonist
tested (n ¼ 3) at 0.001e0.3
m
factored in each datapoint, the error is contained in the
datapoints.

288
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
9. Compound 78, containing the phenyl-pyridyl motif, displayed an
EC50 of 126 nM. Although this is diminished compared to the lead 9,
this is still a 5-fold increase in agonist activity compared to
SEN12333 (8) (EC50 687 nM). Interestingly, biphenyl containing
compound 79, displayed higher binding affinity (Ki ¼ 34 nM) than
the related 78, but exhibited somewhat diminished potency
(EC50 ¼ 253 nM).
2.4. Electrophysiology
Two compounds, SEN12333 (8) and compound 45, were tested
for agonist activity on the
nAChR) in manual patch-clamp electrophysiology using GH4C1
cells stably expressing rat 7 nAChR. The results are summarized in
a7 nicotinic acetylcholine receptor (a7
a
Table 8 and Fig. 7. Both SEN12333 and 45 are full agonists and more
potent than the endogenous agonist, acetylcholine (ACh). The po-
tency in electrophysiology is much lower for these agonists than in
the binding and fluorescence assays (300- to 600-fold less potent),
which is a well-established phenomenon with reported agonists.
For example, compound (9) and its analogues showed 200- to
1000-fold drop-offs from binding to electrophysiology [45].
Consistent with the binding and fluorescence data, compound 45
was at least as potent as SEN12333 by electrophysiology.
Fig. 7. Electrophysiology studies of SEN12333 (8), compound 45 and ACh.
nitrogen or argon, unless otherwise stated. Temperatures quoted as
0 ꢁC and ꢀ78 ꢁC were achieved with cooling baths of ice-water and
dry ice-acetone, respectively. Acetonitrile, dichloromethane, 1,2-
dichloroethane, methanol, and triethylamine were distilled from
calcium hydride. Tetrahydrofuran and diethyl ether were dried over
sodium wire and distilled from sodium benzophenone ketyl.
Ethanol, n-propanol and pyridine were distilled from calcium hy-
dride and stored over activated 3 Å molecular sieves. Crude tosyl
chloride (~10 g) was dissolved in chloroform (25 mL), filtered and
diluted with hexanes (125 mL) to precipitate impurities. The solu-
tion was then filtered, clarified with activated charcoal and
concentrated under reduced pressure to give analytically pure
material (~7 g). All reagents were purchased from either Sigma
Aldrich Chemical Company, Matrix Scientific, Boron Molecular, or
Tokyo Chemical Industry Co and used without further purification.
Melting point determinations were measured on either a Gallen-
kamp melting point apparatus or Optimelt Automated Melting
Point System and are recorded uncorrected. Infrared absorption
spectra were run as Nujol (KBr) mulls for solids using a BIO-RAD FT-
IR spectrophotometer/DRIFTS A 4000e400 cmꢀ1 or as thin films on
sodium chloride (NaCl) plates for oils using either Shimadzu FTIR-
8400S Bruker Alpha-E FT-IR spectrometers. 1H and 13C NMR
spectra were acquired at 300 1 K using either a Bruker Avance DP
282 (300 MHz), DP X400 (400 MHz) or DPX500 (500 MHz). 1H
chemical shifts are reported relative to residual non-deuterated
solvent resonance or tetramethylsilane. Data are reported as
chemical shifts (dH ppm), relative integral, multiplicity (s ¼ singlet;
br s ¼ broad singlet; app. br s ¼ apparent broad singlet;
d ¼ doublet; dd ¼ doublet of doublets; t ¼ triplet; dt ¼ doublet of
triplets; q ¼ quartet; quin ¼ quintet; m ¼ multiplet range), coupling
constants (J, Hz) and assignments are made where unambiguous.
13C chemical shifts are reported relative to the internal perdeu-
terated solvent resonance and are specified as quaternary (C),
methine (aryl or CH), methylene (CH2) or methyl (CH3). Residual
acid in deuterated chloroform was removed prior to use by filtering
through a pad of basic alumina. Low-resolution mass spectra were
recorded on a Finnigan LCQ mass spectrometer. High-resolution
mass spectra were recorded by the Mass Spectrometry Unit of the
School of Chemistry, University of Sydney. Positive electrospray
ionisation (ESI) and atmospheric pressure chemical ionisation
(APCI) utilized methanol and/or dichloromethane. The purity of all
new compounds was ꢂ95% based on C, H and N analyses conducted
at the Department of Chemistry and Biomolecular Sciences,
3. Conclusion
In conclusion, the synthesis and SAR analysis within two
structurally distinct series of a7 agonists (represented by SEN12333
and compound 9) have been described. Hybrids of both lead
compounds have been synthesized and the functional groups
interchanged to gain greater insight into the requirements of these
two agonists for a7 receptor binding. We have identified two an-
alogues of SEN12333, compound 44 and 45, which are respectively
of a 2- and 8-fold increase in affinity relative to SEN12333. Both
compounds exhibit acceptable predicted logP values and display
the potential for further development of the series. After investi-
gating whether compound 9 represents a truncated version of
SEN12333 (8), it was concluded that the functional groups of the
two structures cannot be interchanged and maintain potency at a7
receptors, particularly in relation to the aliphatic heterocycle. The
divergent SAR between the series suggests there may be a different
binding mode for the basic heterocycles. Biaryl ring substitution of
compound 9 with those present in SEN12333 (8) was the only
functional group exchange tolerated as evident in compounds 78
and 79.
4. Experimental section
4.1. General synthetic details
Reactions were conducted under a positive pressure of dry
Table 8
Electrophysiology studies of SEN12333 (8), compound 43 and ACh.
Compound
EC50
(m
M)
nH
Efficacy (%)
ACh
SEN12333 (8)
45
127.6
42
27.3
1.76
1.83
1.28
100
104.3
136.5
SEN12333 (8) and compound 45, were tested for agonist activity on the
a7 nAChR in
manual patch-clamp electrophysiology using GH4C1 cells stably expressing rat
a
7
nAChR. The manual patch-clamp recordings used the fast application add-on
Dynaflow® system. To determine EC50 values, the compounds were tested at
3
m
M, 10
m
M, 30
m
M, 100
m
M, 300
m
M, 1 mM and 3 mM (n ¼ 5). Peak currents were
normalized to saturating acetylcholine. Replicates have been factored in each
datapoint, the error is therefore contained in the datapoints.

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
289
Macquarie University, Sydney, Australia. Analytical thin layer
chromatography was carried out using aluminium backed silica gel
60 F254 (0.2 mm) Merck KGaA sheets and plates were visualized
(1H, br s), 7.38 (4H, m), 2.36e2.27 (8H, m), 1.69 (2H, m), 1.58e1.51
(6H, m), 1.41 (2H, m); dC (75 MHz, CDCl3) 171.8, 137.2, 131.9, 121.8,
116.8, 58.8, 54.7, 37.3, 26.2, 25.9, 24.3, 23.7; m/z (þESI) 339
([MþH]þ, 80); Found: C, 56.67; H, 7.04; N, 8.22. Calc for
under a UV lamp at
l
¼ 254 nm. Plates were routinely stained with
phosphomolybdic acid (5% solution in absolute ethanol). Flash
column chromatography employed Merck Kieselgel 60
(230e400 mesh) silica gel. Solvents for flash column chromatog-
raphy were distilled prior to use and are quoted as volume/volume
mixtures where applicable.
C16H23BrN2O: C, 56.64, H, 6.83; N, 8.26%
4.1.2.3. N-(4-Bromophenyl)-5-(4-methylpiperazin-1-yl)pentanamide
(22) [41]. Subjecting compound 19 to general procedure 2 using
THF as the solvent and N-methyl piperazine as the amine source,
the crude amide was obtained and purified by flash chromatog-
raphy on silica eluting with CH2Cl2-methanol-aqueous ammonia
(90:9:1) to give compound 22 (1.84 g, 51%) as a colourless solid; mp
4.1.1. General procedure 1 for the synthesis of
bromophenyl)alkanamides [43]
u-bromo-N-(4-
99e100 ꢁC; max/cmꢀ1 2933, 1738, 1665, 1587, 1529, 1499, 1387,
n
The appropriate u-bromoalkanoic acid (1 mmol) was suspended
in oxalyl chloride (ca. 2 mL), stirred at ambient temperature for
45 min, and the excess oxalyl chloride evaporated under reduced
pressure. The residue was dissolved in CH2Cl2 (10 mL), cooled
to ꢀ78 ꢁC, and treated dropwise with a solution of 4-bromoaniline
(1.1 mmol) and triethylamine (1.1 mmol) in CH2Cl2 (11 mL). The
mixture was warmed to ambient temperature, stirred for 4 h, and
CH2Cl2 (50 mL) was added. The solution was washed with H2O
(50 mL), hydrochloric acid (1 M; 50 mL), saturated aqueous sodium
hydrogencarbonate (50 mL), brine (50 mL), dried (MgSO4), filtered,
and the solvent evaporated under reduced pressure to give the
crude amides.
824; dH (200 MHz, CDCl3) 7.56 (1H, br s), 7.41 (4H, br m), 2.49e2.33
(12H, m), 2.29 (3H, s), 1.80e1.57 (4H, m); dC (125 MHz, CDCl3) 171.4,
137.2, 132.0, 121.6, 116.8, 57.0, 54.9, 53.1, 46.0, 37.4, 26.2, 23.5; m/z
(þESI) 355 ([MþH]þ, 100).
4.1.2.4. 4-Bromo-N-((1S,4S)-tert-butyl5-(2,5-diazabicyclo[2.2.1]hep-
tane-2-carboxylate)butyl)benzamide (23). Subjecting compound 19
to general procedure 2 using THF as the solvent and 25 as the amine
source, the crude amide was obtained and purified by flash chro-
matography on silica eluting with CH2Cl2-methanol-aqueous
ammonia (90:9:1) to give compound 23 (0.19 g, 84%) as a tan oil;
½aꢄD25ꢀ19.4 (0.7, CHCl3); nmax/cmꢀ1 2931, 1666, 1590, 1531, 1488,
1393, 1364, 1328, 1246, 1100, 1007, 826; dH (500 MHz, CDCl3) 8.06
(1H, br s), 7.42 (2H, d, J 8.5 Hz), 7.37 (2H, d, J 8.5 Hz), 4.29 (0.5H, br s),
4.19 (0.5H, br s), 3.74e3.40 (2H, m), 3.10 (1H, d, J 9.0 Hz), 2.88 (0.5H,
d, J 9.5 Hz), 2.81 (0.5H, d, J 9.0 Hz), 2.57e2.43 (3H, m), 2.33 (2H, t, J
7.5 Hz), 1.78e1.61 (4H, m), 1.50e1.43 (11H, m) (mixture of rotam-
ers); dC (125 MHz, CDCl3) 171.6, 154.5, 154.3, 137.4, 131.9, 121.5,
116.6, 79.6, 79.5, 61.4, 60.8, 60.3, 60.1, 57.8, 56.8, 54.2, 53.6, 49.8,
48.9, 37.3, 36.2, 35.5, 28.79, 28.73, 28.6, 23.4 (mixture of rotamers);
m/z (APCI) 396 ([MꢀC4H9]þ, 100) 254 ([MꢀC10H17N2O2]þ, 80) 452
([MþH]þ, 100); Found: MHþ 452.15389 C21H36BrN3O3 þ H requires
452.15488.
4.1.1.1. 5-Bromo-N-(4-bromophenyl)pentanamide
(19)
[43].
Subjecting 5-bromopentanoic acid (18) to general procedure 1
gave, after purification by flash chromatography on silica eluting
with hexane-ethyl acetate (70:30), amide 19 (0.91 mmol, 91%) as a
colourless solid; mp 165e167 ꢁC;
n
max/cmꢀ1 3303, 1660, 1591,
1522, 1394, 818; dH (300 MHz, CDCl3) 7.41 (4H, m), 7.22 (1H, br s),
3.43 (2H, t, J 9.2 Hz), 2.38 (2H, t, J 9.2 Hz), 1.91e1.84 (4H, m); dC
(75 MHz, CDCl3) 171.2,137.5,132.1,121.3,116.4, 58.2, 37.0, 25.4, 23.2;
m/z (APCI) 255 ([MꢀBr]ꢀ, 100).
4.1.2. General procedure 2 for the synthesis of N-(4-bromophenyl)-
u
-morpholinoalkanamides [43]
A solution of amide 19 (1.0 mmol) in DMF (10 mL) was treated
4.1.2.5. N-(4-Bromophenyl)-5-(2-oxa-6-azaspiro[3.3]heptan-6-yl)
pentanamide (24). Subjecting compound 19 to general procedure 2,
using 2-oxa-6-azaspiro[3.3]heptane as the amine source, gave the
crude amide. Purification by flash chromatography on silica eluting
with CH2Cl2-methanol-aqueous ammonia (92:8.5:0.5) yielded
with sodium iodide (1.0 mmol), triethylamine (1.0 mmol) and the
appropriate amine (1.1 mmol), and heated at reflux for 16 h. After
cooling to ambient temperature, the solution was partitioned be-
tween H2O (100 mL) and ethyl acetate (100 mL), the layers sepa-
rated, and the aqueous layer extracted with ethyl acetate
(2 ꢃ 100 mL). The combined organic extracts were washed with
H2O (100 mL), brine (100 mL), dried (MgSO4), filtered, and the
solvent evaporated under reduced pressure to give crude amides.
amide 24 (0.63 g, 74%) as a colourless solid; mp 125e127 ꢁC; nmax
/
cmꢀ1 2937, 1734, 1690, 1524, 1502, 1475, 1248, 1106, 892; dH
(500 MHz, CDCl3) 7.48 (1H, br s), 7.43e7.39 (4H, m), 4.72 (4H, s),
3.32 (4H, s), 2.39 (2H, t, J 7.5 Hz), 2.34 (2H, t, J 7.5 Hz), 1.72 (2H, quin,
J 7.5 Hz), 1.4 (2H, quin, J 7.5 Hz); dC (125 MHz, CDCl3) 171.2, 137.2,
132.1, 121.5, 116.9, 81.5, 64.0, 59.2, 39.2, 37.5, 27.2, 23.2; m/z (ESI)
353, 355 ([MþH]þ, 100); Found: C, 54.11; H, 5.55; N, 7.80. Calc for
4.1.2.1. N-(4-Bromophenyl)-5-morpholinopentanamide (20) [43].
Subjecting compound 19 to general procedure 2, using morpholine
as the amine source, gave, after purification by flash chromatog-
raphy on silica eluting with ethyl acetate, compound 20
C16H21BrN2O2: C, 54.40, H, 5.99; N, 7.93%
(0.85 mmol, 85%) as a colourless solid; mp 147e148 ꢁC; max/cmꢀ1
n
2939,1665, 1590,1531,1488, 1395,1302,1246,1114,1070,1007, 825;
dH (300 MHz, CDCl3) 7.54 (1H, br s), 7.42 (4H, m), 3.74 (4H, t, J
4.4 Hz), 2.51e2.36 (8H, m), 1.77 (2H, m), 1.61 (2H, m); dC (75 MHz,
CDCl3) 171.4, 137.2, 132.0, 121.5, 116.7, 66.7, 58.4, 53.7, 37.4, 25.8,
23.3; m/z (þESI) 341 ([MþH]þ, 100).
4.1.3. General procedure 3 for the synthesis of morpholino-N-(4-
(pyridin-3-yl)phenyl)alkanamides [43]
A suspension of the appropriate amide (1.0 mmol) and pyridine-
3-boronic acid (1.1 mmol) were suspended in acetonitrile-aqueous
sodium carbonate (1:1, 0.4 M, 10 mL) and deoxygenated with
bubbling argon at ambient temperature. A catalytic amount of
Pd(PPh3)4 (0.05 mmol, 5 mol%) was added, and the mixture heated
at reflux for 18 h. The reaction was partitioned between ethyl ac-
etate (30 mL) and saturated aqueous sodium carbonate (30 mL), the
layers separated, and the aqueous layer extracted with ethyl acetate
(2 ꢃ 30 mL). The combined organic extracts were washed with
brine (40 mL), dried (MgSO4), filtered, and the solvent evaporated
under reduced pressure to give the crude product.
4.1.2.2. N-(4-Bromophenyl)-5-(piperidin-1-yl)pentanamide
(21).
Subjecting amide 19 to general procedure 2 using THF as the sol-
vent and piperdine as the amine source, the crude amide was ob-
tained and purified by flash chromatography on silica eluting with
CH2Cl2-methanol-aqueous ammonia (90:9:1) to give compound 21
(1.0 g, 82%) as a colourless solid; mp 123e124 ꢁC; max/cmꢀ1 2934,
n
1739, 1665, 1592, 1535, 1489, 1394, 824; dH (300 MHz, CDCl3) 8.09

290
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
4.1.3.1. 5-Morpholino-N-(4-(pyridin-3-yl)phenyl)pentanamide
(8,
m); dC (125 MHz, CDCl3) 171.4, 140.6, 137.4, 137.2, 128.9, 127.7, 127.2,
127.0, 120.4, 58.1, 55.1, 53.3, 46.1, 37.6, 26.4, 23.7; m/z (þESI) 352
([MþH]þ, 100); Found: C, 75.10; H, 8.37; N, 11.86. Calc for
SEN12333) [43]. Subjecting compound 20 to general procedure 3
gave, after purification by flash chromatography on silica eluting
with CH2Cl2-methanol-aqueous ammonia (90:9:1), compound 8
C
22H29N3O: C, 75.18, H, 8.32; N, 11.96%
(0.83 mmol, 83%) as an off-white solid; mp 112e114 ꢁC; max/cmꢀ1
n
2954, 1669, 1599, 1532, 1474, 1289, 1184, 1115, 805, 711; dH
(300 MHz, CDCl3) 8.79 (1H, s), 8.54 (1H, d, J 4.6 Hz), 8.20 (1H, br s),
7.83 (1H, d, J 7.9 Hz), 7.65 (2H, d, J 8.5 Hz), 7.50 (2H, d, J 8.5 Hz), 7.33
(1H, m), 3.69 (4H, t, J 6.0 Hz), 2.45e2.35 (8H, m), 1.76 (2H, m), 1.58
(2H, m); dC (75 MHz, CDCl3) 171.6, 148.2, 148.2, 138.5, 136.2, 134.2,
133.3,127.7, 123.8, 120.5, 66.8, 58.5, 53.7, 37.4, 25.9, 23.5; m/z (þESI)
340 ([MþH]þ, 100).
4.1.3.6. 5-((1S,4S)-tert-Butyl5-(2,5-diazabicyclo[2.2.1]heptane-2-
carboxylate)-N-(4-(pyridin-3-yl)phenyl)pentanamide (26).
Subjecting compound 23 to general procedure 3 the crude residue
was obtained and purified by flash chromatography on silica
eluting with CH2Cl2-methanol-aqueous ammonia (90:9.5:0.5) to
give compound 26 (0.17 g, 93%) as a colourless oil; ½aꢄ2D5ꢀ25.1 (1.0,
CHCl3); n
max/cmꢀ1 2974, 1687, 1598, 1529, 1474, 1397, 1328, 1250,
1103; dH (400 MHz, CDCl3) 8.81 (1H, br s), 8.56 (1H, d, J 4.8 Hz), 7.84
(1H, d, J 7.6 Hz), 7.65 (2H, d, J 8.4 Hz), 7.53 (2H, d, J 8.4 Hz), 7.34 (1H,
dd, J 4.8, 7.6 Hz), 4.34 (0.5H, s), 4.22 (0.5H, s), 3.54e3.44 (2H, m),
3.14 (1H, d, J 6.8 Hz), 2.95 (0.5H, d, J 9.2 Hz), 2.88 (0.5H, d, J 9.2 Hz),
2.63e2.56 (2H, m), 2.42 (2H, t, J 7.6 Hz), 1.83e1.76 (4H, m), 1.68 (1H,
m), 1.54 (2H, quin, J 7.6 Hz), 1.45 (9H, s) (mixture of rotamers); dC
(100 MHz, CDCl3) 171.5,154.3,148.4,148.2,138.4,136.2,134.2,133.5,
127.8, 123.7, 120.5, 79.6, 79.5, 61.6, 60.9, 60.4, 60.3, 57.8, 56.8, 54.2,
53.6, 49.7, 48.7, 37.6, 36.4, 35.6, 28.7, 23.5 (mixture of rotamers); m/
z (ESI) 451 ([MþH]þ, 100), 253 ([MꢀC10H17N2O2]þ, 63), 395
([MꢀC4H9]þ, 34); Found: MHþ 451.27040 C26H34N4O3 þ H requires
451.27092.
4.1.3.2. 5-(Piperidin-1-yl)-N-(4-(pyridin-3-yl)phenyl)pentanamide
(10). Subjecting compound 21 to general procedure 3, the crude
amide was obtained and purified by flash chromatography on silica
eluting with CH2Cl2-methanol-aqueous ammonia (93:6.5:0.5) to
give compound 10 (0.12 g, 71%) as
a colourless solid; mp
117e118 ꢁC; max/cmꢀ1 2932, 1668, 1599, 1530, 1473, 1375, 1116; dH
n
(500 MHz, CDCl3) 8.82 (1H, d, J 2.0 Hz), 8.56 (1H, dd, J 1.0, 4.5 Hz),
7.88 (1H, br s), 7.84 (1H, dt, J 1.5, 8.0 Hz), 7.64 (2H, d, J 8.5 Hz), 7.5
(2H, d, J 8.5 Hz), 7.34 (1H, dd, J 5.0, 8.0 Hz), 2.43e2.32 (8H, m), 1.76
(2H, m), 1.62e1.55 (6H, m), 1.42 (2H, m); dC (125 MHz, CDCl3) 171.7,
148.3, 148.2, 138.4, 136.2, 134.2, 133.6, 127.8, 123.7, 120.7, 59.0, 54.8,
37.6, 26.5, 26.1, 24.5, 23.9; m/z (þESI) 169 ([MꢀC11H9N2], 100);
(APCI) 253 ([MꢀC5H10N]þ, 100); Found: C, 74.66; H, 8.24; N, 12.29.
Calc for C21H27N3O: C, 74.74, H, 8.06; N, 12.45%
4.1.3.7. 5-((1S,4S)-tert-Butyl5-(2,5-diazabicyclo[2.2.1]heptane-2-
carboxylate)-N-[1,1'-biphenyl]-4-carboxamide
(27). Subjecting
compound 23 to general procedure 3 using phenylboronic acid the
crude residue was obtained and purified by flash chromatography
on silica eluting with CH2Cl2-methanol-aqueous ammonia
(92:7.5:0.5) to give compound 27 (0.1 g, 59%) as a colourless oil;
4.1.3.3. N-([1,10-Biphenyl]-4-yl)-5-(piperidin-1-yl)pentanamide (11).
Subjecting compound 21 to general procedure 3 using phenyl-
boronic acid, the crude amide was obtained and purified by flash
chromatography on silica eluting with CH2Cl2-methanol-aqueous
ammonia (90:9:1) to give compound 11 (0.15 g, 52%) as a colourless
½aꢄ2D5ꢀ22.3 (1.0, CHCl3);
n
max/cmꢀ1 2916, 1668, 1598, 1529, 1486,
1405, 1165; dH (500 MHz, CDCl3) 7.60e7.54 (6H, m), 7.42 (2H, d, J
7.5 Hz), 7.32 (1H, d, J 7.5 Hz), 4.34 (0.5H, s), 4.21 (0.5H, s), 3.54 (0.5H,
d, J 10.0 Hz), 3.48e3.44 (1.5H, m), 3.14 (1H, t, J 9.0 Hz), 2.94 (0.5H, d,
J 9.5 Hz), 2.89 (0.5H, d, J 9.5 Hz), 2.62e2.55 (1.5H, m), 2.47 (0.5H, d, J
9.5 Hz), 2.40 (2H, t, J 7.5 Hz), 1.83e1.76 (4H, m), 1.70 (0.5H, d, J
9.0 Hz), 1.54 (0.5H, d, J 9.5 Hz), 1.54 (2H, quin, J 7.5 Hz), 1.45 (9H, s),
NH proton not observed (mixture of rotamers); dC (100 MHz, CDCl3)
171.4, 154.5, 154.4, 140.7, 137.4, 137.2, 128.9, 127.7, 127.2, 127.0, 120.3,
79.6, 79.5, 61.5, 60.9, 60.4, 60.2, 57.9, 56.8, 54.2, 53.6, 49.7, 48.7, 37.6,
36.4, 35.6, 28.8, 28.7, 23.5 (mixture of rotamers); m/z (ESI) 472
([MþNa]þ, 100), 252 ([MꢀC10H17N2O2]þ, 85), 394 ([MꢀC4H9]þ, 34);
Found: MNaþ 472.25675 C27H35N3O3 þ Na requires 472.25761.
solid; mp 109e111 ꢁC; max/cmꢀ1 2933, 1654, 1596, 1532, 1488,
n
1395, 826; dH (500 MHz, CDCl3) 7.58e7.53 (4H, m), 7.42e7.39 (4H,
m), 7.31 (1H, m), 2.44e2.37 (8H, m), 1.78 (2H, m), 1.65e1.59 (6H, m),
1.45 (2H, m) NH proton not observed; dC (125 MHz, CDCl3) 171.5,
140.9, 137.5, 132.1, 128.9, 127.7, 127.2, 127.0, 120.7, 58.9, 54.8, 37.5,
26.4, 26.0, 24.5, 23.9; m/z (APCI) 252 ([MꢀC5H10N], 100), 168
([MꢀC11H9N2]þ, 95), 337 ([MþH], 80); Found: C, 78.74; H, 8.01; N,
8.23. Calc for C22H28N2O: C, 78.53, H, 8.39; N, 8.33%
4.1.3.4. 5-(4-Methylpiperazin-1-yl)-N-(4-(pyridin-3-yl)phenyl)pen-
tanamide (12) [41]. Subjecting compound 22 to the general pro-
cedure 3 gave, after purification by flash chromatography on silica
eluting with CH2Cl2-methanol-aqueous ammonia (90:9:1) com-
4.1.4. General procedure 5 for N-methylation of ((1S,4S)-5-methyl-
2,5-diazabicyclo[2.2.1]heptan-2-yl)pentanamides
pound 12 (0.28 g, 57%) as a colourless solid; mp 144e146 ꢁC;
nmax/
cmꢀ1 3551, 2945, 2803, 1740, 1653, 1532, 1425, 1289, 843; dH
(500 MHz, CDCl3) 8.82 (1H, d, J 1.5 Hz), 8.56 (1H, dd, J 1.5, 4.5 Hz),
7.84 (1H, dt, J 2.0, 8.0 Hz), 7.63 (2H, d, J 8.5 Hz), 7.54 (2H, d, J 8.5 Hz),
7.35 (1H, dd, J 5.0, 8.0 Hz), 2.43e2.34 (12H, m), 2.27 (3H, s),
1.80e1.74 (2H, m), 1.62e1.56 (2H, m) NH proton not observed; dC
(125 MHz, CDCl3) 171.3, 148.3, 148.0, 138.1, 136.0, 134.0, 132.1, 127.7,
123.6, 120.4, 58.1, 55.1, 53.3, 46.1, 37.5, 26.4, 23.6; m/z (þESI) 353
([MþH]þ, 100).
A solution of the appropriate N-Boc protected pentanamide
(1.0 mmol) in methanol (0.1 M) was treated with hydrogen chloride
gas in dioxane (5.0 mmol) and stirred at room temperature for 2 h.
The solvent was then removed under a steam of nitrogen gas and
the residue partitioned between hydrochloric acid (1 M, 20 mL) and
CH2Cl2 (50 mL), the aqueous layer was further extracted with
CH2Cl2 (20 mL) and then basified to pH 14 with addition of aqueous
sodium hydroxide (3 M, ca 10 mL). The aqueous layer was then
extracted with CH2Cl2 (3 ꢃ 50 mL) and the combined organic ex-
tracts dried (MgSO4), filtered and the solvent removed under
reduced pressure. The free amine was then suspended in
ClCH2CH2Cl (0.1 M) and treated with aqueous formaldehyde (37%,
1.5 mmol) and NaBH(OAc)3 (5 mmol) and the resulting mixture
stirred at ambient temperature for 17e20 h. The reaction was
quenched with addition of aqueous sodium hydroxide (3 M, 5 mL)
and the layers separated. The aqueous layer was extracted with
CH2Cl2 (3 ꢃ 30 mL) and the combined organic layers were washed
with brine (30 mL), dried (MgSO4), filtered and the solvent
4.1.3.5. N-([1,10-Biphenyl]-4-yl)-5-(4-methylpiperazin-1-yl)pentana-
mide (13). Subjecting 22 to the general procedure 3 using phe-
nylboronic acid gave, after purification by flash chromatography on
silica
(85:13.5:1.5) 13 (0.34 mmol, 68%) as a colourless solid; mp
147.5e148.9 ꢁC; max/cmꢀ1 3552, 2942, 2805, 1738, 1656, 1596,
eluting
with
CH2Cl2-methanol-aqueous
ammonia
n
1532, 1372, 1216, 834, 762; dH (500 MHz, CDCl3) 7.59 (2H, d, J
8.5 Hz), 7.56e7.53 (4H, m), 7.42 (2H, t, J 6.0 Hz), 7.32 (1H, t, J 6.0 Hz),
2.47e2.38 (12H, m), 2.28 (3H, s), 1.78e1.74 (2H, m), 1.63e1.58 (2H,

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
291
evaporated to give the crude residue.
C22H26N2O2: C, 75.40; H, 7.48; N, 7.99%
4.1.4.1. 5-((1S,4S)-5-Methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-N-
(4-(pyridin-3-yl)phenyl)pentanamide (14). Subjecting compound
26 to general procedure 5 the crude residue was obtained and
purified by flash chromatography on silica eluting with CH2Cl2-
methanol-aqueous ammonia (88:11.5:0.5) to give methylated
amine 14 (0.05 g, 48% over two steps) as a colourless oil; ½aꢄ2D5ꢀ21.5
(1.0, CHCl3); nmax/cmꢀ1 2929, 1668, 1597, 1525, 1473, 1425, 1288,
1250, 1182, 1001, 845; dH (500 MHz, CDCl3) 8.81 (1H, d, J 1.5 Hz),
8.55 (1H, dd, J 1.5, 4.5 Hz), 7.99 (1H, br s), 7.84 (1H, dt, J 2.0, 8.0 Hz),
7.66 (2H, d, J 8.5 Hz), 7.52 (2H, d, J 8.5 Hz), 7.34 (1H, dd, J 5.0, 8.0 Hz),
3.23 (1H, s), 3.17 (1H, s), 2.82 (1H, d J 10.0 Hz), 2.71 (1H, dd, J 2.5,
10.0 Hz), 2.66e2.60 (2H, m), 2.51 (2H, m), 2.42 (2H, t, J 7.5 Hz), 2.36
(3H, s), 1.79 (2H, quin, J 7.5 Hz), 1.69 (2H, m), 1.54 (2H, quin, J 7.5H);
dC (125 MHz, CDCl3) 171.7, 148.4, 148.2, 138.4, 136.2, 134.2, 133.5,
127.8, 123.7, 120.5, 63.7, 62.4, 57.1, 56.6, 53.5, 41.6, 37.5, 33.6, 28.5,
23.6; m/z (APCI) 253 ([MꢀC6H11N2]þ, 100), 169 ([MꢀC11H19N2O]þ,
68), 365 ([MþH]þ, 24); Found: MHþ 365.23377C22H28N4O þ H re-
quires 365.23414.
4.1.4.5. 2-(4-(Piperidin-1-yl)butyl)isoindoline-1,3-dione
(29).
N-(4-Bromobutyl)phthalimide (28) (2.0 g, 7.09 mmol) was sus-
pended in THF (64 mL) and treated with sodium iodide (1.06 g,
7.09 mmol), triethylamine (0.99 mL, 7.09 mmol) and piperidine
(0.77 mL, 7.79 mmol). The mixture was then heated at reflux for
16 h. After cooling to ambient temperature, the solvent was
removed in vacuo and the residue partitioned between H2O
(100 mL) and CHCl3 (100 mL). The aqueous layer was further
extracted with CHCl3 (100 mL) and the organic extracts combined,
dried (MgSO4), filtered, and the solvent removed under reduced
pressure. The crude product was purified by flash chromatography
on silica eluting with ethyl acetate to give phthalimide 29 (1.52 g,
74%) as a colourless solid; mp 73e74 ꢁC; max/cmꢀ1 2933, 2765,
n
1711,1395,1367,1215,1040; dH (300 MHz, CDCl3) 7.82e7.80 (2H, m),
7.70e7.67 (2H, m), 3.68 (2H, t, J 6.9 Hz), 2.32e2.26 (6H, m), 1.66 (2H,
m), 1.56e1.46 (6H, m), 1.39 (2H, m); dC (75 MHz, CDCl3) 168.5, 133.9,
132.2, 123.2, 59.0, 54.7, 38.0, 26.8, 26.1, 24.5, 24.4; m/z (APCI) 287
([MþH]þ, 83); Found: C, 71.42; H, 8.06; N, 9.78. Calc for C17H22N2O:
C, 71.30, H, 7.74; N, 9.78%
4.1.4.2. N-([1,10-Biphenyl]-4-yl)-5-((1S,4S)-5-methyl-2,5-
diazabicyclo[2.2.1]heptan-2-yl)pentanamide (15). Subjecting com-
pound 27 to general procedure 5 the crude residue was obtained
and purified by flash chromatography on silica eluting with CH2Cl2-
methanol-aqueous ammonia (85:14:1) to give methylated amine
15 (0.06 g, 50% over two steps) as a colourless oil; ½aꢄ2D5ꢀ24.7 (1.0,
CHCl3); nmax/cmꢀ1 2910, 1670, 1587, 1529, 1480, 1428, 1165, 809; dH
(500 MHz, CDCl3) 8.32 (1H, br s), 7.62 (2H, d, J 8.5 Hz), 7.54 (4H, t, J
9.0 Hz), 7.40 (2H, t, J 7.5 Hz), 7.3 (1H, t, J 7.5 Hz), 3.32 (1H, s), 3.18
(1H, s), 2.92 (1H, d, J 10.0 Hz), 2.72e2.60 (3H, m), 2.49 (2H, m), 2.40
(2H, t, J 7.5 Hz), 2.36 (3H, s), 1.79e1.69 (4H, m), 1.54 (2H, m); dC
(125 MHz, CDCl3) 171.9, 140.7, 137.8, 136.9, 128.9, 127.6, 127.1, 126.9,
120.3, 63.6, 62.1, 57.0, 56.7, 53.4, 41.8, 37.3, 33.1, 28.2, 23.6; m/z (ESI)
364 ([MþH]þ, 100); Found: MHþ 364.23859 C23H29N3O þ H re-
quires 364.23889.
4.1.4.6. 2-(4-(4-Methylpiperazin-1-yl)butyl)isoindoline-1,3-dione
(30). N-(4-Bromobutyl)phthalimide (28) (2.0 g, 7.09 mmol) was
suspended in THF (64 mL) and treated with sodium iodide (1.06 g,
7.09 mmol), triethylamine (0.99 mL, 7.09 mmol) and N-methyl
piperazine (0.86 mL, 7.09 mmol). The mixture was then heated at
reflux for 16 h. After cooling to ambient temperature, the solvent
was removed in vacuo and the residue partitioned between H2O
(100 mL) and CHCl3 (100 mL). The aqueous layer was further
extracted with CHCl3 (100 mL) and the organic extracts combined,
dried (MgSO4), filtered and the solvent removed under reduced
pressure. The crude product was purified by flash chromatography
on silica eluting with ethyl acetate to give phthalimide 30 (1.94 g,
91%) as a colourless solid; mp 71e72 ꢁC; max/cmꢀ1 2794, 1707,
n
1394, 1164, 1041; dH (300 MHz, CDCl3) 7.83e7.81 (2H, m), 7.72e7.69
(2H, m), 3.70 (2H, t, J 6.4 Hz), 2.43e2.33 (10H, m), 2.26 (3H, s), 1.69
(2H, m), 1.52 (2H, s); dC (75 MHz, CDCl3) 168.6, 134.0, 132.2, 123.3,
58.1, 55.2, 53.3, 46.2, 38.0, 26.7, 24.4; m/z (APCI) 302 ([MþH]þ, 100);
Found: C, 67.82; H, 7.64; N, 13.84. Calc for C17H23N3O2: C, 67.75, H,
7.69; N, 13.94%
4.1.4.3. N-(4-(Pyridin-3-yl)phenyl)-5-(2-oxa-6-azaspiro[3.3]heptan-
6-yl)pentanamide (16). Subjecting amide 24 to the general proce-
dure 3 gave the crude residue which was purified by flash chro-
matography on silica eluting with CH2Cl2-methanol-aqueous
ammonia (90:9:1) to give pentanamide 16 (0.29 g, 70%) as a yellow
oil;
n
max/cmꢀ1 3673, 2925, 1738, 1598, 1535, 1365, 1215; dH
4.1.4.7. (1S,4S)-tert-Butyl-5-(4-(1,3-dioxoisoindolin-2-yl)butyl)-2,5-
diazabicyclo[2.2.1]heptane-2-carboxylate (31).
N-(4-Bromobutyl)phthalimide (28) (0.32 g, 1.15 mmol) was sus-
pended in THF (11 mL) and treated with sodium iodide (0.17 g,
(400 MHz, CDCl3) 8.78 (1H, d, J 1.6 Hz), 8.53 (1H, dd, J 1.6, 4.8 Hz),
8.39 (1H, br s), 7.81 (1H, m), 7.62 (2H, d, J 8.4 Hz), 7.49 (2H, d, J
8.4 Hz), 7.32 (1H, dd, J 4.8, 7.6 Hz), 4.68 (4H, s), 3.27 (4H, s),
2.37e2.33 (4H, m), 1.70 (2H, quin, J 7.6 Hz), 1.36 (2H, quin, J 7.6 Hz);
dC (100 MHz, CDCl3) 171.6, 148.1, 147.9, 138.6, 136.2, 134.2, 133.3,
127.6, 123.7, 120.5, 81.4, 63.9, 59.2, 39.1, 37.3, 27.2, 23.3; m/z (ESI)
352 ([MþH]þ, 100), 725 ([2MþNa]þ, 43); Found: MHþ 352.01969.
1.15 mmol), triethylamine (160
mL, 1.15 mmol) and 157 (0.25 g,
1.26 mmol). The mixture was then heated at reflux for 16 h. After
cooling to ambient temperature, the solvent was removed in vacuo
and the residue partitioned between H2O (100 mL) and CHCl3
(100 mL). The aqueous layer was further extracted with CHCl3
(100 mL) and the organic extracts combined, dried (MgSO4),
filtered and the solvent removed under reduced pressure. The
crude product was purified by flash chromatography on silica
eluting with ethyl acetate to give phthalimide 31 (0.35 g, 76%) as an
C
21H25N3O þ H requires 352.20250.
4.1.4.4. N-([1,10-Biphenyl]-4-yl)-5-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)pentanamide (17). Subjecting amide 24 to the general procedure
3 using phenylboronic acid gave the crude residue which was pu-
rified by flash chromatography on silica eluting with CH2Cl2-
methanol-aqueous ammonia (92:7.5:0.5) to give pentanamide 17
off-white solid; ½aꢄ2D5ꢀ22.8 (0.9, CHCl3);
n
max/cmꢀ1 3129, 1689,
1419, 1320, 890; dH (500 MHz, CDCl3) 7.83e7.82 (2H, m), 7.71e7.69
(2H, m), 4.31 (0.5H, s), 4.19 (0.5H, s), 3.69 (2H, t, J 7.0 Hz), 3.50 (0.5H,
d, J 10.0 Hz), 3.45e3.41 (1.5H, m), 3.41 (1H, t, J 10.0 Hz), 2.92 (0.5H,
d, J 9.5 Hz), 2.85 (0.5H, d, J 9.5 Hz), 2.58e2.52 (2.5H, m), 2.43 (0.5H,
d, J 9.5 Hz), 1.78e1.63 (4H, m), 1.51e1.41 (11H, m) (mixture of
rotamers); dC (125 MHz, CDCl3) 168.6, 154.4, 154.3, 134.0, 132.3,
123.3, 79.4, 79.3, 61.4, 60.9, 60.21, 60.16, 57.9, 56.9, 54.0, 53.4, 49.8,
48.7, 38.0, 36.5, 35.6, 28.7, 26.7, 26.6 (mixture of rotamers); m/z
(ESI) 400 ([MþH]þ, 100); Found: MHþ 400.22329 C22H29N3O4 þ H
(0.088 g, 90%) as a colourless solid; mp 1089e111; n
max/cmꢀ1 3729,
2927, 1740, 1662, 1597, 1531, 1408, 1355, 1331, 1237; dH (500 MHz,
CDCl3) 7.75 (1H, br s), 7.58e7.53 (4H, m), 7.43e7.37 (4H, m), 7.32
(1H, t, J 7.5 Hz), 4.70 (4H, s), 3.38 (4H, s), 2.42 (2H, t, J 7.5 Hz), 2.35
(2H, t, J 7.5 Hz), 1.72 (2H, quin, J 7.5 Hz), 1.45 (2H, quin, J 7.5 Hz); dC
(125 MHz, CDCl3) 171.3, 140.6, 137.4, 137.2, 128.9, 127.7, 127.2, 126.9,
120.3, 81.4, 63.7, 59.0, 38.8, 37.4, 26.8, 23.3; m/z (ESI) 351 ([MþH]þ,
100), 723 ([2MþNa]þ, 33); Found: C, 75.52; H, 7.39 N, 7.88. Calc for

292
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
requires 400.22363.
n
max/cmꢀ1 2940, 2794, 1636, 1590, 1544, 1482, 1284, 1164, 1011; dH
(300 MHz, CDCl3) 7.61 (2H, d, J 8.1 Hz), 7.54 (2H, d, J 8.1 Hz), 3.43
(2H, t, J 6.0 Hz), 2.39e2.35 (10H, m), 2.42 (3H, s), 1.65e1.59 (4H, m),
NH proton not observed; dC (75 MHz, CDCl3) 166.8, 134.0, 131.8,
128.7, 125.9, 58.0, 55.1, 53.3, 46.1, 40.2, 27.5, 24.6; m/z (APCI) 354
([MþH]þ, 100); Found: C, 54.18; H, 7.06; N, 11.56. Calc for
4.1.4.8. 4-Bromo-N-(4-(piperidin-1-yl)butyl)benzamide
(32).
Phthalimide 29 (0.85 g, 2.97 mmol) was suspended in anhydrous
ethanol (30 mL) and treated with hydrazine monohydrate (0.72 mL,
14.85 mmol). The mixture was heated under reflux for 2 h and then
cooled to room temperature, filtered, and the filtrate evaporated
under reduced pressure. The remaining residue was suspended in
hydrochloric acid (1 M; 50 mL) and heated under reflux for 1 h.
After cooling to room temperature, the mixture was filtered and the
filtrate washed with CH2Cl2 (50 mL). The aqueous layer was basified
to pH 14 with addition of aqueous sodium hydroxide (3 M), and
extracted with CH2Cl2 (3 ꢃ 50 mL). The combined organic layers
were dried (MgSO4), filtered, and the solvent removed in vacuo to
give crude amine which was used directly in the next step without
further purification or characterization due to its volatile nature.
4-Bromobenzoyl chloride (0.52 g, 2.39 mmol) was suspended in
CH2Cl2 (15 mL) and cooled to 0 ꢁC. A solution of crude amine
(2.97 mmol) and triethylamine (0.33 mL, 2.39 mmol) in CH2Cl2
(15 mL) was added dropwise and stirring continued at 0 ꢁC for 0.5 h.
The mixture was then allowed to reach room temperature and
stirring continued for a further 2 h. CH2Cl2 (50 mL) was added and
the solution washed with saturated aqueous sodium carbonate
(50 mL), saturated aqueous ammonium chloride (50 mL), H2O
(50 mL) then brine (50 mL). The organic layer was then dried
(MgSO4), filtered, and the solvent removed under reduced pressure
to give the crude residue. Purification by flash chromatography on
silica eluting with CH2Cl2-methanol-aqueous ammonia (90:9:1),
gave amide 32 (0.99 g, 50% over two steps) as a colourless solid; mp
C16H24BrN3O: C, 54.24, H, 6.83; N, 11.86%
4.1.4.10. (1S,4S)-tert-Butyl 5-(4-(4-bromobenzamido)butyl)-2,5-
diazabicyclo[2.2.1]heptane-2-carboxylate (34). Phthalimide 31
(0.40 g, 1.12 mmol) was suspended in anhydrous ethanol (12 mL)
and treated with hydrazine monohydrate (273 L, 5.62 mmol). The
m
mixture was heated under reflux for 2 h and then cooled to room
temperature, filtered, and the filtrate evaporated under reduced
pressure. The remaining residue was suspended in hydrochloric
acid (1 M; 50 mL) and heated under reflux for 1 h. After cooling to
room temperature, the mixture was filtered and the filtrate washed
with CH2Cl2 (50 mL). The aqueous layer was basified to pH 14 with
addition of aqueous sodium hydroxide (3 M), and extracted with
CH2Cl2 (3 ꢃ 50 mL). The combined organic layers were dried
(MgSO4), filtered and the solvent removed in vacuo to give the
crude amine which was used directly in the next step without
further purification or characterization.
4-Bromobenzoyl chloride (0.25 g, 1.12 mmol) was suspended in
CH2Cl2 (10 mL) and cooled to 0 ꢁC. A solution of the crude amine
(1.12 mmol) and triethylamine (156 mL, 0.8 mmol) in CH2Cl2 (10 mL)
was added dropwise and stirring continued at 0 ꢁC for 0.5 h. The
mixture was then allowed to reach room temperature and stirring
continued for a further 2 h. CH2Cl2 (50 mL) was added and the
solution washed with saturated aqueous sodium carbonate
(50 mL), saturated aqueous ammonium chloride (50 mL), H2O
(50 mL) and brine (50 mL). The organic layer was then dried
(MgSO4), filtered and the solvent removed under reduced pressure
to give the crude residue. Purification by flash chromatography on
112e113 ꢁC; max/cmꢀ1 3316, 2932, 1742, 1632, 1536; dH (300 MHz,
n
CDCl3) 7.62 (2H, d, J 8.4 Hz), 7.53 (2H, d, J 8.4 Hz), 3.43 (2H, q, J
5.4 Hz), 2.34e2.30 (6H, m), 1.66e1.50 (8H, m), 1.40 (2H, m); dC
(75 MHz, CDCl3) 166.9, 134.1, 131.8, 128.8, 125.9, 58.7, 54.7, 40.2,
27.5, 25.9, 24.7, 24.5; m/z (APCI) 185 ([MꢀC9H19N2]þ, 100), 339
([MþH], 42); Found: C, 56.69; H, 6.97; N, 8.25. Calc for
silica
eluting
with
CH2Cl2-methanol-aqueous
ammonia
C
16H23BrN2O: C, 56.64, H, 6.83; N, 8.26%
(93:6.5:0.5), gave amide 34 (0.33 g, 66% over two steps) as a tan oil;
½aꢄ2D5ꢀ22.6 (1.0, CHCl3);
n
max/cmꢀ1 2938, 1678, 1591, 1531, 1492,
4.1.4.9. 4-Bromo-N-(4-(4-methylpiperazin-1-yl)butyl)benzamide
(33). Phthalimide 30 (1.20 g, 3.98 mmol) was suspended in anhy-
drous ethanol (40 mL) and treated with hydrazine monohydrate
(0.97 mL, 19.9 mmol). The mixture was heated under reflux for 2 h
and then cooled to room temperature, filtered, and the filtrate
evaporated under reduced pressure. The resulting residue was
suspended in hydrochloric acid (1 M; 50 mL) and heated under
reflux for 1 h. After cooling to room temperature, the mixture was
filtered and the filtrate washed with CH2Cl2 (50 mL). The aqueous
layer was basified to pH 14 with addition of aqueous sodium hy-
droxide (3 M), and extracted with CH2Cl2 (3 ꢃ 50 mL). The com-
bined organic layers were dried (MgSO4), filtered and the solvent
removed in vacuo to give the crude amine which was used directly
in the next step without further purification or characterization due
to its volatile nature.
4-Bromobenzoyl chloride (0.18 g, 0.8 mmol) was suspended in
CH2Cl2 (10 mL) and cooled to 0 ꢁC. A solution of crude the amine
(0.87 mmol) and triethylamine (0.11 mL, 0.8 mmol) in CH2Cl2
(10 mL) was added dropwise and stirring continued at 0 ꢁC for 0.5 h.
The mixture was then allowed to reach room temperature and
stirring continued for a further 2 h. CH2Cl2 (50 mL) was added and
the solution washed with saturated aqueous sodium carbonate
(50 mL), saturated aqueous ammonium chloride (50 mL), H2O
(50 mL) then brine (50 mL). The organic layer was then dried
(MgSO4), filtered and the solvent removed under reduced pressure
to give the crude residue. Purification by flash chromatography on
silica eluting with CH2Cl2-methanol-aqueous ammonia (90:9:1),
gave amide 33 (0.3 g, 35%) as a colourless solid; mp 121e123 ꢁC;
1387, 1330, 1245, 1105, 1010, 826; dH (500 MHz, CDCl3) 7.61 (2H, d, J
8.5 Hz), 7.53 (2H, d, J 8.5 Hz), 4.31 (0.5H, s), 4.19 (0.5H, s), 3.5 (0.5H,
d, J 10.5 Hz), 3.44e3.40 (3.5H, m), 3.12 (1H, t, J 8.0 Hz), 2.88 (0.5H, d,
J 9.5 Hz), 2.83 (0.5H, d, J 9.5 Hz), 2.60e2.52 (2.5H, m), 2.43 (0.5H, d, J
9.5 Hz), 1.71 (1H, d, J 9.5 Hz), 1.68e1.64 (3H, m), 1.53 (2H, m), 1.44
(9H, s), NH peak not observed (mixture of rotamers); dC (125 MHz,
CDCl3) 166.8, 154.4, 154.3, 134.0, 131.8, 128.7, 126.0, 61.3, 60.7, 60.2,
60.1, 57.7, 56.7, 53.8, 53.3, 49.6, 48.6, 40.1, 36.4, 35.6, 28.7, 27.5, 27.4,
26.9 (mixture of rotamers); m/z (ESI) 454, 452 ([MþH]þ, 100);
Found: MHþ 452.15429 C21H30BrN3O3 þ H requires 452.15429.
4.1.4.11. N-(4-(Piperidin-1-yl)butyl)-4-(pyridin-3-yl)benzamide (37).
Subjecting compound 32 to general procedure 3 gave, after puri-
fication by flash chromatography on silica eluting with CH2Cl2-
methanol-aqueous ammonia (95:4.5:0.5) compound 37 (0.126 g,
63%) as a colourless solid; mp 115e117 ꢁC; max/cmꢀ1 3315, 2932,
n
1739, 1639, 1546, 1366, 1305, 1216; dH (300 MHz, CDCl3) 8.87 (1H, d,
J 2.1 Hz), 8.62 (1H, dd, J 1.5, 4.8 Hz), 7.91 (1H, t, J 2.1 Hz), 7.87 (2H, d, J
8.4 Hz), 7.64 (2H, d, J 8.4 Hz), 7.39 (1H, dd, J 4.8, 8.78 Hz), 6.95 (1H,
br s), 3.50 (2H, q, J 6.0 Hz), 2.37e2.32 (6H, m), 1.69e1.51 (8H, m),
1.42 (2H, m); dC (75 MHz, CDCl3) 167.3, 149.2, 148.5, 140.7, 135.8,
134.8,134.5,128.0,127.3, 123.8, 58.8, 54.7, 40.1, 27.6, 25.9, 24.7, 24.5;
m/z (APCI) 182 ([MꢀC11H8N]þ, 100), 253 ([MꢀC5H10N]þ, 35);
Found: C, 74.38; H, 8.37; N, 12.41. Calc for C21H27N3O: C, 74.74, H,
8.06; N, 12.45%
4.1.4.12. N-(4-(Piperidin-1-yl)butyl)-[1,10-biphenyl]-4-carboxamide
(38). Subjecting compound 32 to general procedure 3 using

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
293
phenylboronic acid gave, after purification by flash chromatog-
raphy on silica eluting with CH2Cl2-methanol-aqueous ammonia
(95:4.5:0.5) compound 38 (0.19 g, 67%) as a colourless solid; mp
phenylboronic acid the crude amide was obtained and purified by
flash chromatography on silica eluting with CH2Cl2-methanol-
aqueous ammonia (95:4.5:0.5) to give the desired amide 36 (0.11 g,
80%) as a colourless oil; ½aꢄ2D5ꢀ17.5 (1.0, CHCl3); nmax/cmꢀ1 3279,
2914, 1689, 1546, 1486, 1408, 1327, 1105, 858; dH (500 MHz, CDCl3)
7.82 (2H, d, J 8.5 Hz), 7.63 (2H, d, J 8.5 Hz), 7.59 (2H, d, J 7.5 Hz), 7.44
(2H, t, J 7.5 Hz), 7.37 (1H, t, J 7.5 Hz), 6.86 (1H, br s), 4.32 (0.5H, s),
4.19 (0.5H, s), 3.53e3.43 (4H, m), 3.12 (1H, t, J 8.5 Hz), 2.91 (0.5H, d, J
9.5 Hz), 2.85 (0.5H, d, J 9.5 Hz), 2.62e2.54 (2.5H, m), 2.45 (0.5H, d, J
9.5 Hz), 1.75 (1H, d, J 9.5 Hz), 1.70e1.62 (3H, m), 1.56 (2H, br s), 1.44
(9H, s) (mixture of rotamers); dC (125 MHz, CDCl3) 167.5, 154.4,
154.3, 144.2, 140.2, 133.7, 129.0, 128.1, 127.5, 127.28, 127.25, 79.6,
79.4, 61.2, 60.7, 60.2, 60.1, 57.8, 56.7, 53.8, 53.3, 49.6, 48.6, 40.1, 36.4,
35.6, 28.6, 27.5, 26.9 (mixture of rotamers); m/z (ESI) 921
([2MþNa]þ, 100); Found: MHþ 450.27531 C27H35N3O3 þ Na re-
quires 450.27567.
129e131 ꢁC;
n
max/cmꢀ1 3327, 2932, 1632, 1541, 1304, 852; dH
(400 MHz, CDCl3) 7.83 (2H, d, J 8.8 Hz), 7.64 (2H, d, J 8.8 Hz), 7.61
(2H, d, J 7.2 Hz), 7.46 (2H, t, J 7.2 Hz), 7.36 (1H, m), 3.49 (2H, q, J
6.4 Hz), 2.37e2.34 (6H, m), 1.70e1.62 (4H, m), 1.58e1.53 (4H, m),
1.41 (2H, m), NH proton not observed; dC (100 MHz, CDCl3) 167.5,
144.1, 140.3, 133.8, 129.0, 128.0, 127.6, 127.3, 127.2, 58.7, 54.7, 40.0,
27.6, 25.8, 24.5, 24.4; m/z (APCI) 181 ([MꢀC12H9]þ, 100), 252
([MꢀC5H10N]þ, 38); Found: C, 78.01; H, 8.68; N, 8.32. Calc for
C
22H28N2O: C, 78.53, H, 8.39; N, 8.33%
4.1.4.13. N-(4-(4-Methylpiperazin-1-yl)butyl)-4-(pyridin-3-yl)benza-
mide (39). Subjecting compound 33 to the general procedure 3
gave, after purification by flash chromatography on silica eluting
with CH2Cl2-methanol-aqueous ammonia (95:4.5:0.5) compound
39 (0.05 mmol, 55%) as a colourless solid; mp 113e115 ꢁC;
nmax/
4.1.4.17. N-(4-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)
butyl)-4-(pyridin-3-yl)benzamide (41). Subjecting compound 35 to
general procedure 5 the crude residue was obtained and purified by
flash chromatography on silica eluting with CH2Cl2-methanol-
aqueous ammonia (88:11:1) to give methylated amine 41 (0.08 g,
cmꢀ1 3295, 2939, 2798, 1737, 1638, 1545, 1458, 1371, 1301, 1216,
1164, 1012; dH (500 MHz, CDCl3) 8.82 (1H, d, J 2.0 Hz), 8.60 (1H, dd, J
1.5, 5.0 Hz), 7.87e7.83 (3H, m), 7.60 (2H, d, J 8.0 Hz), 7.36 (1H, dd, J
5.0, 8.0 Hz), 6.89 (1H, br s), 3.46 (2H, q J 6.5 Hz), 2.38e2.35 (10H, m),
2.22 (3H, s), 1.68e1.58 (4H, m); dC (125 MHz, CDCl3) 167.3, 149.2,
148.4, 140.8, 135.7, 134.7, 134.5, 127.9, 127.3, 123.8, 58.1, 55.1, 53.3,
46.2, 40.2, 27.6, 24.7; m/z (APCI) 182 ([MꢀC9H20N3]þ, 100), 253
([MꢀC5H11N2]þ, 37); Found: C, 71.49; H, 8.31; N, 15.81. Calc for
58% over two steps) as a colourless oil; ½aꢄ2D5ꢀ17.6 (1.0, CHCl3); nmax
/
cmꢀ1 2930, 1665, 1582, 1525, 1425, 1289, 1250, 1184, 1005, 845; dH
(500 MHz, CDCl3) 8.85 (1H, d, J 2.0 Hz), 8.61 (1H, dd, J 1.5, 5.0 Hz),
7.98 (2H, d, J 8.5 Hz), 7.89 (1H, dt, J 1.5, 8.0 Hz), 7.64 (2H, d, J 8.5 Hz),
7.38 (1H, dd, J 4.0, 8.0 Hz), 3.63 (1H, s), 3.51 (2H, m), 3.47 (1H, s),
3.22 (1H, d, J 11.0 Hz), 3.08 (1H, d, J 11.0 Hz), 2.96 (1H, dd, J 2.5,
11.0 Hz), 2.89 (2H, m), 2.76e2.70 (3H, m), 2.51 (3H, s), 1.71 (4H, m),
NH proton not observed; dC (125 MHz, CDCl3) 167.3, 149.2, 148.2,
140.8, 135.8, 134.6, 134.3, 128.1, 127.3, 123.8, 63.6, 62.5, 56.5, 55.8,
52.7, 41.1, 39.3, 33.6, 26.9, 25.0; m/z (ESI) 365 ([MþH]þ,100); Found:
MHþ 365.23366 C22H28N4O þ H requires 365.23414.
C
21H28N4O: C, 71.56, H, 8.01; N, 15.9%
4.1.4.14. N-(4-(4-Methylpiperazin-1-yl)butyl)-[1,10-biphenyl]-4-
carboxamide (40). Subjecting compound 33 to the general proce-
dure 3 using phenylboronic acid gave, after purification by flash
chromatography on silica eluting with CH2Cl2-methanol-aqueous
ammonia (95:4.5:0.5) compound 40 (0.057 mmol, 71%) as a col-
ourless solid; mp 132e133 ꢁC; max/cmꢀ1 3320, 2932, 2791, 1741,
n
1632, 1543, 1485, 1372, 1166; dH (500 MHz, CDCl3) 7.82 (2H, d, J
8.0 Hz), 7.62 (2H, d, J 8.0 Hz), 7.58 (2H, d, J 7.5 Hz), 7.44 (2H, t, J
7.5 Hz), 7.37 (1H, t, J 7.5 Hz), 6.79 (1H, br s), 3.47 (2H, q, J 6.5 Hz),
2.60e2.36 (10H, m), 2.25 (3H, s), 1.68e1.59 (4H, m); dC (125 MHz,
CDCl3) 167.6, 144.2, 140.2, 133.8, 129.0, 128.1, 127.6, 127.30, 127.29,
58.1, 55.1, 53.2, 46.0, 40.1, 27.6, 24.6; m/z (APCI) 181 ([MꢀC9H20N3]þ,
100), 252 ([MꢀC5H11N2]þ, 37); Found: C, 75.17; H, 8.36; N, 11.87.
Calc for C22H29N3O: C, 75.18, H, 8.32; N, 11.95%
4.1.4.18. N-(4-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)
butyl)-[1,1'-biphenyl]-4-carboxamide (42). Subjecting compound 36
to general procedure 5 the crude residue was obtained and purified
by flash chromatography on silica eluting with CH2Cl2-methanol-
aqueous ammonia (88:11:1) to give methylated amine 42 (0.075 g,
62% over two steps) as a colourless oil; ½aꢄ2D5ꢀ25.8 (1.0, CHCl3); nmax
/
cmꢀ1 3306, 2910, 1710, 1509, 1462, 1408, 1318, 1106, 923; dH
(500 MHz, CDCl3) 7.84 (2H, d, J 8.5 Hz), 7.64 (2H, d, J 8.5 Hz), 7.60
(2H, d, J 7.5 Hz), 7.45 (2H, t, J 7.5 Hz), 7.37 (1H, t, J 7.5 Hz), 7.05 (1H, br
s), 3.48 (2H, m), 3.33 (1H, s), 3.20 (1H, s), 2.88 (1H, d, J 10.0 Hz),
2.76e2.64 (3H, m), 2.57e2.52 (2H, m), 2.37 (3H, s), 1.74e1.66 (4H,
m), 1.59 (2H, m); dC (125 MHz, CDCl3) 167.6, 144.2, 140.2, 133.8,
129.0, 128.1, 127.6, 127.31, 127.25, 63.6, 62.2, 56.7, 56.2, 53.1, 41.4,
40.0, 33.7, 27.5, 26.7; m/z (ESI) 364 ([MþH]þ, 100); Found: MHþ
364.23840 C23H29N3O þ H requires 364.23889.
4.1.4.15. (1S,4S)-tert-Butyl-5-(4-(4-(pyridin-3-yl)benzamido)butyl)-
2,5-diazabicyclo[2.2.1]
heptane-2-carboxylate
(35). Subjecting
compound 34 to general procedure 3 the crude residue was ob-
tained and purified by flash chromatography on silica eluting with
CH2Cl2-methanol-aqueous ammonia (95:4.5:0.5) to give the
desired amide 35 (0.12 g, 89%) as a colourless oil; ½aꢄ2D5ꢀ20.3 (1.0,
CHCl3); nmax/cmꢀ1 2935, 1688, 1547, 1409, 1103, 859; dH (500 MHz,
CDCl3) 8.84 (1H, d, J 2.0 Hz), 8.6 (1H, dd, J 1.5, 4.5 Hz), 7.89 (1H, t, J
2.0 Hz), 7.89 (2H, d, J 8.5 Hz), 7.86 (2H, d, J 8.5 Hz), 7.37 (1H, dd, J 4.5,
8.0 Hz), 6.93 (1H, br s), 4.31 (0.5H, s), 4.19 (0.5H, s), 3.49e3.45 (4H,
m), 3.13 (1H, m), 2.91 (0.5H, d, J 9.0 Hz), 2.84 (0.5H, d, J 9.0 Hz),
2.62e2.54 (2.5H, m), 2.45 (0.5H, d, J 0.5H), 1.74e1.68 (4H, m), 1.56
(2H, m), 1.43 (9H, s) (mixture of rotamers); dC (125 MHz, CDCl3)
167.2, 154.4, 154.3, 149.2, 148.4, 140.8, 135.7, 134.64, 134.55, 127.9,
127.3, 123.77, 79.6, 79.5, 61.3, 60.7, 60.19, 60.10, 57.8, 56.7, 53.8, 53.4,
49.7, 48.6, 40.1, 36.4, 35.6, 28.6, 27.5, 26.9 (mixture of rotamers); m/
4.1.4.19. 2-(4-Nitrophenyl)-pyridine (52) [63]. To a solution of pyr-
idine (323
mL, 4.0 mmol) in dichloromethane (20 mL) was added
trifluoroacetic acid (308
mL, 4.0 mmol) followed by 4-
nitrophenylboronic acid (51) (2.0 g, 12.0 mmol). Water (12 mL)
was then added, followed by silver(I) nitrate (0.14 g, 0.8 mmol) in
water (8 mL). Potassium persulfate (3.2 g, 12.0 mmol) was then
added and the solution was stirred vigorously at room temperature
and monitored by thin-layer chromatography analysis of the
organic layer. After 3 h, a second addition of solid silver(I) nitrate
(0.14 g, 0.8 mmol) and potassium persulfate (3.2 g, 12.0 mmol) was
added. After stirring for 24 h, the reaction was diluted with
dichloromethane (70 mL) and washed with aqueous sodium hy-
droxide (2 M; 50 mL). The layers were separated, and the aqueous
layer was extracted with dichloromethane (2 ꢃ 50 mL), the organic
extracts were combined, dried (MgSO4), filtered, and evaporated in
z
C
(ESI) 923 ([2MþNa]þ, 100); Found: MHþ 451.27035
26H34N4O3 þ Na requires 451.27092.
4.1.4.16. (1S,4S)-tert-butyl-5-(4-([1,10-biphenyl]-4-ylcarboxamido)
butyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate
(36).
Subjecting compound 34 to general procedure
3
using

294
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
vacuo. Purification by flash chromatography on silica eluting with
hexane-ethyl acetate (1:1) gave compound 52 (0.22 g, 27%) as a
yellow solid; mp 131e133 ꢁC (lit mp 131e132 ꢁC); nmax/cmꢀ1 1583,
1516, 1435, 1347, 856; dH (500 MHz, CDCl3) 8.75 (1H, d, J 4.5 Hz),
8.32 (2H, d, J 8.5 Hz), 8.18 (2H, d, J 8.5 Hz), 7.84e7.81 (2H, m), 7.34
(1H, m); dC (125 MHz, CDCl3) 155.0, 150.3, 148.3, 145.4, 137.3, 127.8,
124.3, 123.7, 121.4; m/z (ESI) 154 ([MꢀNO2]þ, 100).
dC (125 MHz, CDCl3) 171.3, 156.9, 149.7, 138.9, 136.9, 135.3, 127.7,
122.0, 120.3, 119.9, 67.1, 58.7, 53.9, 37.6, 26.2, 23.5; m/z (ESI) 340
([MþH]þ, 100); Found: C, 70.74; H, 7.37; N, 12.37. Calc for
C20H25N3O2: C, 70.77, H, 7.42; N, 12.38%
4.1.4.23. 5-Morpholino-N-(4-(pyridin-4-yl)phenyl)pentanamide
(44). Subjecting compound 20 to general procedure 3 using 4-
pyridineboronic acid, the crude residue was obtained and purified
by flash chromatography on silica eluting with CH2Cl2-methanol-
aqueous ammonia (92:7.5:0.5) to give pentanamide 44 (0.067 g,
57%) as a colourless solid; mp 103e104 ꢁC; nmax/cmꢀ1 2949, 1689,
1596, 1525, 1292, 1116, 814; dH (400 MHz, CDCl3) 8.64 (1H, br s),
7.67e7.59 (6H, m), 7.49 (2H, d, J 4.4 Hz), 3.71 (4H, t, J 4.4 Hz),
2.44e2.36 (8H, m), 1.79 (2H, quin, J 7.6 Hz), 1.60 (2H, quin, J 7.6 Hz);
dC (100 MHz, CDCl3) 171.4, 150.3, 147.7, 139.1, 133.8, 127.8, 121.4,
120.3, 67.1, 58.6, 53.9, 37.6, 26.1, 23.5; m/z (ESI) 340 ([MþH]þ, 100);
Found: C, 63.74; H, 7.87; N, 11.39. Calc for C20H25N3O2 þ 2H2O: C,
63.98, H, 7.79; N, 11.19%
4.1.4.20. 4-(Pyridin-2-yl)aniline (53) [64,65]. A solution of com-
pound 52 (0.2 g, 1.03 mmol) in methanol (10 mL) was treated with
10% palladium on carbon (20 mg) and subjected to a hydrogen at-
mosphere. The mixture was stirred for a further 18 h and then
filtered through a pad of Celite®. The pad was washed with meth-
anol (50 mL) and the combined filtrates evaporated in vacuo to give
the crude residue which was purified by flash chromatography on
silica eluting with CH2Cl2-methanol-aqueous ammonia (95:4.5:0.5)
to give amine 53 (0.15 g, 87%) as a brown solid; mp 89e92 ꢁC (lit mp
94e95 ꢁC); nmax/cmꢀ1 3333, 3210, 1606, 1586, 1519, 1294, 1181; dH
(500 MHz, CDCl3) 8.62 (1H, dt, J 1.0, 5.0 Hz), 7.83 (2H, d, J 8.5 Hz),
7.68e7.61 (2H, m), 7.11 (1H, m) 6.75 (2H, d, J 8.5 Hz), 3.83 (2H, br s);
dC (125 MHz, CDCl3) 157.6, 149.5, 147.5, 136.6, 129.8, 128.1, 121.0,
119.4, 115.2; m/z (ESI) 171 ([MþH]þ, 100).
4 . 1. 4 . 2 4 . N - ( 4 - ( 2 - fl u o r o p y r i d i n - 3 - y l ) p h e n y l ) - 5 -
morpholinopentanamide (45). Subjecting amide 20 to general pro-
cedure 3 using 2-fluoro-3-pyridineboronic acid, the crude residue
was obtained and purified by flash chromatography on silica
eluting with CH2Cl2-methanol-aqueous ammonia (92:7.5:0.5) to
give pentanamide 45 (0.12 g, 72%) as a colourless solid; mp
129e130 ꢁC; nmax/cmꢀ1 2953, 1664, 1599, 1527, 1437, 1397, 1249,
1116; dH (500 MHz, CDCl3) 8.17 (1H, d, J 5.0 Hz), 7.85 (1H, m), 7.63
(2H, d, J 8.5 Hz), 7.54 (2H, d, J 8.5 Hz), 7.41 (1H, br s), 7.26 (1H, m),
3.71 (4H, t, J 4.5 Hz), 2.44e2.37 (8H, m),1.77 (2H, quin, J 8.0 Hz),1.61
4.1.4.21. 5-Bromo-N-(4-(pyridin-2-yl)phenyl)pentanamide
(54).
5-bromovaleric acid (0.14 g, 0.75 mmol) was suspended in oxalyl
chloride (ca. 2 mL), stirred at ambient temperature for 45 min, and
the excess oxalyl chloride evaporated under reduced pressure. The
residue was dissolved in CH2Cl2 (10 mL), cooled to ꢀ78 ꢁC, and
treated dropwise with a solution of amine 53 (0.14 g, 0.82 mmol)
1
and triethylamine (115
mL, 0.82 mmol) in CH2Cl2 (10 mL). The
(2H, quin, J 8.0 Hz); dC (125 MHz, CDCl3) 171.3, 160.5 (d, JC-F
mixture was warmed to ambient temperature, stirred for 10 h, and
CH2Cl2 (70 mL) was added. The solution was washed with H2O
(50 mL), hydrochloric acid (1 M; 50 mL), saturated aqueous sodium
hydrogencarbonate (50 mL), brine (50 mL), dried (MgSO4), filtered,
and the solvent evaporated under reduced pressure to give the
crude amide which was purified by flash chromatography on silica
eluting with CH2Cl2-methanol-aqueous ammonia (98:1.5:0.5) to
give amide 54 (0.21 g, 85%) as a colourless solid; mp 97e98 ꢁC;
nmax/cmꢀ1 3230, 1718, 1543, 1418, 1375, 1214, 907, 814; dH
(500 MHz, CDCl3) 8.67 (1H, d, J 5.0 Hz), 7.98 (2H, d, J 8.5 Hz),
7.76e7.70 (2H, m), 7.64 (2H, d, J 8.5 Hz), 7.21 (1H, t, J 5.5 Hz), 3.46
(2H, t, J 7.0 Hz), 2.43 (2H, t, J 7.0 Hz), 2.01e1.87 (4H, m), NH proton
not observed; dC (125 MHz, CDCl3) 170.6, 156.9, 149.8, 138.7, 137.7,
127.6, 120.1, 120.3, 119.9, 115.3, 36.8, 33.3, 32.2, 24.1; m/z (ESI) 333,
335 ([MþH]þ, 100); Found: C, 57.74; H, 5.29; N, 8.43. Calc for
238.8 Hz), 146.2 (d, 3JC-F 13.8 Hz), 140.5 (d, 3JC-F 5 Hz), 138.3, 129.63,
2
129.60, 123.4 (d, JC-F 28.8 Hz), 122.0, 120.0, 67.1, 58.7, 53.9, 37.6,
26.2, 23.5; m/z (ESI) 358 ([MþH]þ, 100); Found: C, 67.27; H, 6.82; N,
11.58. Calc for C20H24FN3O2: C, 67.21, H, 6.77; N, 11.76%
4.1.4.25. N-(4-(1H-indol-5-yl)phenyl)-5-morpholinopentanamide
(46). Subjecting amide 20 to general procedure
3 using 5-
indolylboronic acid, the crude residue was obtained and purified
by flash chromatography on silica eluting with CH2Cl2-methanol-
aqueous ammonia (90:9:1) to give pentanamide 46 (0.14 g, 81%) as
a colourless solid; mp 205e206 ꢁC; nmax/cmꢀ1 3290, 1663, 1597,
1470, 1318, 1114, 807, 734; dH (500 MHz, DMSO-d6) 11.09 (1H, br s),
9.88 (1H, s), 7.76 (1H, s), 7.65 (2H, d, J 9.0 Hz), 7.58 (2H, d, J 9.0 Hz),
7.44 (1H, d, J 8.5 Hz), 7.37e7.35 (2H, m), 6.46 (1H, t, J 2.0 Hz), 3.56
(4H, t, J 4.5 Hz), 2.32e2.27 (8H, m), 1.62 (2H, quin, J 7.5 Hz), 1.47 (2H,
quin, J 7.5 Hz); dC (125 MHz, DMSO-d6) 171.1, 137.7, 136.5, 135.2,
131.0, 128.2, 126.7, 125.9, 120.1, 119.4, 117.6, 111.7, 101.4, 66.2, 58.0,
53.4, 36.2, 25.5, 23.0; m/z (ESI) 378 ([MþH]þ, 100); Found: C, 73.22;
H, 7.21; N, 11.13. Calc for C23H27N3O2: C, 73.18, H, 7.21; N, 11.13%
C
16H17BrN2O: C, 57.67, H, 5.14; N, 8.41%
4.1.4.22. 5-Morpholino-N-(4-(pyridin-2-yl)phenyl)pentanamide
(43). A solution of amide 54 (0.1 g, 0.3 mmol) in THF (5 mL) was
treated with sodium iodide (0.046 g, 0.3 mmol), triethylamine
(42
m
L, 0.3 mmol) and morpholine (64
m
L, 0.34 mmol), and heated
4.1.4.26. 6-Chloropyridazin-3-amine (56) [66]. To a thick-wall bo-
rosilicate glass vial was added 3,6-dichloropyridazine (55) (1.5 g,
10.1 mmol) and ammonium hydroxide solution (5 mL; NH3 con-
tent: 28e30%). The vial was sealed and placed in the microwave
reactor for 30 min at 120 ꢁC (power: 300 W). After cooling, the
precipitate that deposited was filtered off, washed with ethyl
acetate-hexane (3:7) and dried to give amine 56 (0.86 g, 66%) as a
yellow solid; mp decomposition >206 ꢁC (lit mp 229e232 ꢁC);
nmax/cmꢀ1 3147, 1643, 1596, 1455, 1055, 838; dH (200 MHz, DMSO)
7.36 (1H, d, J 10.0 Hz), 6.84 (1H, d, J 10.0 Hz), 6.61 (2H, br s); dC
(75 MHz, DMSO) 160.2, 145.0, 128.9, 117.5; m/z (ESI) 130 ([MþH]þ,
100).
at reflux for 16 h. After cooling to ambient temperature, the solvent
was removed under reduced pressure and the residue partitioned
between H2O (50 mL) and ethyl acetate (70 mL). The layers were
separated, and the aqueous layer extracted with ethyl acetate
(2 ꢃ 50 mL). The combined organic extracts were washed with
brine (100 mL), dried (MgSO4), filtered, and the solvent evaporated
under reduced pressure to give the crude residue which was pu-
rified by flash chromatography on silica eluting with CH2Cl2-
methanol-aqueous ammonia (90:9:1) to give amide 43 (0.073 g,
74%) as a colourless solid; mp 155e156.0 ꢁC; nmax/cmꢀ1 3302, 2915,
1658, 1598, 1466, 1118, 782; dH (500 MHz, CDCl3) 8.65 (1H, d, J
5.0 Hz), 7.96 (2H, d, J 8.5 Hz), 7.74e7.68 (2H, m), 7.62 (2H, d, J
8.5 Hz), 7.58 (1H, br s), 7.19 (1H, t, J 6.5 Hz), 3.69 (4H, t, J 4.5 Hz),
2.42e2.34 (8H, m), 1.77 (2H, quin, J 7.5 Hz), 1.59 (2H, quin, J 7.5 Hz);
4.1.4.27. 5-Bromo-N-(6-chloropyridazin-3-yl)pentanamide
(57).
5-bromovaleric acid (18) (0.46 g, 2.6 mmol) was suspended in

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
295
oxalyl chloride (ca. 2 mL), stirred at ambient temperature for
45 min, and the excess oxalyl chloride evaporated under reduced
pressure. The residue was dissolved in CH2Cl2 (20 mL), cooled
to ꢀ78 ꢁC, and treated dropwise with a solution of amine 56 (0.3 g,
layer was further extracted with ethyl acetate (2 ꢃ 70 mL) and the
combined organic extracts washed with brine (150 mL), dried
(MgSO4), filtered and the solvent removed under reduced pressure.
The crude residue was then purified by flash chromatography on
silica eluting with hexane-ethyl acetate (1:1) to give nitrile 60
(4.7 g, 63%) as a colourless solid; mp 79e81 ꢁC (lit mp 81e82 ꢁC);
nmax/cmꢀ1 2950, 2837, 1690, 1427, 1365, 1210, 847; dH (500 MHz,
CDCl3) 7.92 (2H, d, J 8.0 Hz), 7.66 (1H, t, J 7.5H), 7.52 (2H, t, J 8.0 Hz),
4.10 (2H, s); dC (125 MHz, CDCl3) 187.3, 134.9, 134.4, 129.3, 128.6,
114.0, 29.5.
2.3 mmol) and triethylamine (355 mL, 2.6 mmol) in CH2Cl2 (20 mL).
The mixture was warmed to ambient temperature, stirred for 10 h,
and CH2Cl2 (100 mL) was added. The solution was washed with H2O
(70 mL), saturated aqueous ammonium chloride (70 mL), saturated
aqueous sodium hydrogencarbonate (70 mL), brine (70 mL), dried
(MgSO4), filtered, and the solvent evaporated under reduced
pressure to give the crude amide. The orange residue was sus-
pended in methanol and the resulting solid filtered off and dried to
give amide 57 (0.13 g, 20%) as a colourless solid; mp decomposition
>169 ꢁC; nmax/cmꢀ1 3191, 1690, 1582, 1515, 1409, 1141, 1088, 735; dH
(300 MHz, CDCl3) 10.5 (1H, br s), 8.66 (1H, d, J 9.0 Hz), 7.58 (1H, d, J
9.0 Hz), 3.48 (2H, t, J 6.0 Hz), 2.71 (2H, t, J 6.0 Hz), 2.01e1.92 (4H, m);
dC (75 MHz, CDCl3) 173.1, 155.0, 152.0, 130.9, 122.2, 36.7, 33.1, 32.1,
23.9; m/z (ESI) 292 ([MþH]þ, 100); Found: C, 36.97; H, 3.68; N,
14.06. Calc for C9H11BrClN3O: C, 36.95, H, 3.79; N, 14.36%
4.1.4.31. 3-Phenyl-1H-pyrazol-5-amine (61) [68]. A solution of
compound 60 (3.5 g, 24.1 mmol) in absolute ethanol (50 mL) was
treated with hydrazine monohydrate (2.5 mL, 48.3 mmol) and
glacial acetic acid (2.4 mL, 38.6 mmol) and heated at reflux for 18 h.
The mixture was allowed to cool to ambient temperature and the
solvent removed under reduced pressure. The residue was sus-
pended between CH2Cl2 (100 mL) and hydrochloric acid (1M;
100 mL), the aqueous layer was further extracted with CH2Cl2
(50 mL) and then basified to pH 12 with addition of aqueous so-
dium hydroxide (3 M; ca 40 mL). The aqueous layer was then
extracted with CH2Cl2 (3 ꢃ 100 mL) and the basified organic ex-
tracts combined, dried (MgSO4), filtered and the solvent removed in
vacuo to give the crude residue which was purified by flash chro-
matography on silica eluting with CH2Cl2-methanol-aqueous
ammonia (90:9.5:0.5) to give amine 61 (3.06 g, 80%) as an off-white
solid; mp 144e145 ꢁC (lit mp 145e146 ꢁC); nmax/cmꢀ1 3205, 1571,
1510, 758, 693; dH (500 MHz, CDCl3) 7.55 (2H, d, J 7.0 Hz), 7.36 (2H, t,
J 7.0 Hz), 7.30 (1H, t, J 7.0 Hz), 5.90 (1H, s), 3NH protons not
observed; dC (125 MHz, CDCl3) 154.5, 145.9, 130.5, 129.0, 128.4,
125.6, 90.5; m/z (ESI) 160 ([MþH]þ, 100).
4.1.4.28. N-(6-chloropyridazin-3-yl)-5-morpholinopentanamide
(58). A solution of amide 57 (0.1 g, 0.34 mmol) in DMF (8 mL) was
treated with sodium iodide (0.051 g, 0.34 mmol), triethylamine
(50 mL, 0.34 mmol) and morpholine (33 mL, 0.38 mmol), and heated
at reflux for 19 h. After cooling to ambient temperature, the solu-
tion was partitioned between H2O (50 mL) and ethyl acetate
(50 mL), the layers separated, and the aqueous layer extracted with
ethyl acetate (2 ꢃ 50 mL). The combined organic extracts were
washed with H2O (100 mL), brine (50 mL), dried (MgSO4), filtered,
and the solvent evaporated under pressure to give the crude res-
idue which was purified by flash chromatography on silica eluting
with CH2Cl2-methanol-aqueous ammonia (90:9:1) to give amide
58 (0.061 g, 62%) as a colourless solid; mp decomposition >152 ꢁC;
nmax/cmꢀ1 2937, 1686, 1588, 1518, 1406, 1136, 1116, 866; dH
(300 MHz, CDCl3) 10.47 (1H, br s), 8.61 (1H, d, J 9.0 Hz), 7.52 (1H, d, J
9.0 Hz), 3.71 (4H, t, J 6.0 Hz), 2.70 (2H, t, J 6.0 Hz), 2.47e2.41 (6H, m),
1.78 (2H, m), 1.65 (2H, m); dC (75 MHz, CDCl3) 173.5, 155.3, 151.7,
130.3,121.8, 66.9, 58.5, 53.8, 37.4, 25.8, 23.3; m/z (ESI) 299 ([MþH]þ,
100); Found: C, 52.55; H, 6.46; N, 18.69. Calc for C13H19ClN4O2: C,
52.26, H, 6.41; N, 18.75%
4.1.4.32. 5-Bromo-N-(5-phenyl-1H-pyrazol-3-yl)pentanamide (62).
5-bromovaleric acid (1.24 g, 6.8 mmol) was suspended in oxalyl
chloride (ca. 5 mL), stirred at ambient temperature for 45 min, and
the excess oxalyl chloride evaporated under reduced pressure. The
residue was dissolved in CH2Cl2 (50 mL), cooled to ꢀ78 ꢁC, and
treated dropwise with a solution of compound 61 (1.2 g, 7.5 mmol)
and triethylamine (1.05 mL, 7.5 mmol) in CH2Cl2 (20 mL). The
mixture was warmed to ambient temperature, stirred for 10 h, and
CH2Cl2 (150 mL) was added. The solution was washed with H2O
(100 mL), hydrochloric acid (1 M; 100 mL), saturated aqueous so-
dium hydrogencarbonate (100 mL), brine (100 mL), dried (MgSO4),
filtered, and the solvent evaporated under reduced pressure to give
the crude amide which was purified by flash chromatography on
silica eluting with CH2Cl2-methanol-aqueous ammonia (95:4.5:0.5)
to give amide 62 (1.6 g, 74%) as an off white solid. Due to the un-
stable nature of amide 62 it was used directly in the next step
without further characterisation.
4.1.4.29. 5-Morpholino-N-(6-phenylpyridazin-3-yl)pentanamide
(47). Subjecting amide 58 to general procedure 3 using phenyl-
boronic acid gave, after purification by flash chromatography on
silica eluting with CH2Cl2-methanol-aqueous ammonia (90:9:1)
compound 47 (0.027 g, 63%) as a colourless solid; mp decomposi-
tion >220 ꢁC; nmax/cmꢀ1 2942, 1679, 1590, 1519, 1406, 1218, 1147,
1120, 866; dH (500 MHz, CDCl3) 10.46 (1H, br s), 8.65 (1H, d, J
9.0 Hz), 8.02 (2H, d, J 7.0 Hz), 7.91 (1H, d, J 9.0 Hz), 7.54e7.48 (3H,
m), 3.67 (4H, t, J 4.5 Hz), 2.79 (2H, t, J 7.5 Hz), 2.37e2.34 (6H, m),1.82
(2H, quin, J 7.5 Hz), 1.61 (2H, quin, J 7.5 Hz); dC (125 MHz, CDCl3)
173.4, 156.4, 154.9, 136.2, 129.9, 129.2, 126.8, 126.3, 119.7, 67.1, 58.7,
53.9, 37.6, 26.1, 23.5; m/z (ESI) 363 ([MþNa]þ, 100); Found: C, 67.10;
H, 7.24; N, 16.53. Calc for C19H24N4O2: C, 67.04, H, 7.11; N, 16.46%
4.1.4.33. 5-Morpholino-N-(5-phenyl-1H-pyrazol-3-yl)pentanamide
(48). Subjecting compound 62 to general procedure 2 using THF as
the solvent, the crude amide was obtained and purified by flash
chromatography on silica eluting with CH2Cl2-methanol-aqueous
ammonia (92:8.5:1.5) to give compound 48 (0.12 g, 59%) as a col-
ourless solid; mp 158e160 ꢁC; nmax/cmꢀ1 3218, 2949, 1664, 1578,
1547, 1492, 1115, 1008; dH (500 MHz, CDCl3) 8.66 (1H, s), 7.62 (2H, d,
J 7.5 Hz), 7.41 (2H, t, J 7.5 Hz), 7.35 (1H, t, J 7.5 Hz), 6.83 (1H, br s),
3.70 (4H, t, J 4.5 Hz), 2.41e2.38 (6H, m), 2.35 (2H, t, J 7.5 Hz), 1.75
(2H, quin, J 7.5 Hz), 1.56 (2H, quin, J 7.5 Hz), one NH peak not
observed; dC (100 MHz, CDCl3) 171.0, 129.2, 128.8, 125.6, 93.6, 67.0,
58.6, 53.9, 36.8, 26.0, 23.4, pyrazole peaks not observed; m/z (ESI)
329 ([MþH]þ, 100); Found: C, 65.89; H, 7.32; N, 16.92. Calc for
4.1.4.30. 3-Oxo-3-phenylpropanenitrile (60) [67]. A cooled (0 ꢁC)
solution of methyl benzoate (59) (7.0 g, 51.4 mmol) in toluene
(60 mL) was treated portionwise with sodium hydride (60%
dispersion in mineral oil, 4.08 g, 102.8 mmol). The mixture was
allowed to warm to room temperature and treated dropwise with a
solution of acetonitrile (13.5 mL, 257.0 mmol) in toluene (15 mL).
The mixture was heated at reflux for 18 h during which time a thick
precipitate formed. The mixture was cooled (0 ꢁC), quenched by
addition of hydrochloric acid (3 M; 30 mL) and then partitioned
between ethyl acetate (150 mL) and H2O (100 mL). The aqueous
C18H24N4O2: C, 65.83; H, 7.37; N, 17.06%

296
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
4.1.4.34. N-(5-Phenyl-1H-pyrazol-3-yl)-5-(piperidin-1-yl)pentana-
mide (49). Subjecting compound 62 to general procedure 2 using
THF as the solvent and piperidine as the amine source, the crude
amide was obtained and purified by flash chromatography on silica
eluting with CH2Cl2-methanol-aqueous ammonia (92:8.5:1.5) to
146.0, 128.5, 115.2, 46.3, 25.3, 24.4; m/z (APCI) 198 ([MþH]þ, 100).
4.1.4.38. 3-Chloro-6-(4-methylpiperazin-1-yl)pyridazine (69) [69].
To a solution of 3,6-dichloropyridazine (55) (1.0 g, 6.7 mmol) in
DMF (67 mL) was added N,N-diisopropylethylamine (1.29 mL,
give compound 49 (0.11 g, 56%) as
a
colourless solid; mp
7.38 mmol) and 1-methylpiperazine (744 mL, 6.7 mmol). The
163e164 ꢁC; nmax/cmꢀ1 3239, 2932, 1658, 1577, 1550, 1492, 959; dH
(500 MHz, CDCl3) 9.20 (1H, br s), 7.65 (2H, d, J 7.5 Hz), 7.41 (2H, t, J
7.5 Hz), 7.33 (1H, t, J 7.5 Hz), 6.83 (1H, br s), 2.40e2.36 (8H, m), 1.71
(2H, quin, J 7.5 Hz),1.60e1.54 (6H, m),1.42 (2H, m), one NH peak not
observed; dC (125 MHz, CDCl3) 171.5,146.4,130.5,129.1,128.5,125.7,
93.5, 58.7, 54.7, 36.8, 26.3, 25.8, 24.4, 23.7, one pyrazole peak not
observed; m/z (ESI) 327 ([MþH]þ, 100); Found: C, 66.20; H, 8.47; N,
16.24. Calc for C19H26N4O þ H2O: C, 66.25; H, 8.19; N, 16.27%
mixture was heated at reflux for 17 h and after cooling to room
temperature, partitioned between ethyl acetate (200 mL) and H2O
(70 mL). The aqueous layer was further extracted with ethyl acetate
(2 ꢃ 50 mL) and the combined organic layers then further washed
with H2O (100 mL), brine (100 mL) then dried (MgSO4), filtered and
the solvent removed under reduced pressure to give the crude
product. Purification by flash chromatograph on silica eluting with
CH2Cl2-methanol-aqueous ammonia (95:4.5:0.5) yielded the
desired pyridazine 69 (0.88 g, 62%) as a pale yellow solid; mp
108e109 ꢁC (lit mp 106e107 ꢁC); nmax/cmꢀ1 3780, 2953, 2927, 1443,
1258,1168,1143, 613; dH (500 MHz, CDCl3) 7.12 (1H, d, J 9.5 Hz), 6.89
(1H, d, J 9.5 Hz), 3.64 (4H, t, J 5.0 Hz), 2.51 (4H, t, J 5.0 Hz), 2.34 (3H,
s); dC (125 MHz, CDCl3) 159.2, 147.0, 128.8, 115.3, 54.7, 46.3, 45.2; m/
z (APCI) 213 ([MþH], 100).
4.1.4.35. 5-(4-Methylpiperazin-1-yl)-N-(5-phenyl-1H-pyrazol-3-yl)
pentanamide (50). Subjecting compound 62 to general procedure 2
using THF as the solvent and 1-methylpiperazine as the amine
source, the crude amide was obtained and purified by flash chro-
matography on silica eluting with CH2Cl2-methanol-aqueous
ammonia (92:8.5:1.5) to give compound 50 (0.14 g, 62%) as a col-
ourless solid; mp 162e164 ꢁC; nmax/cmꢀ1 3222, 3198, 2942, 2813,
1661, 1579, 1543, 1457, 1109; dH (500 MHz, CDCl3) 8.9 (1H, br s), 7.63
(2H, d, J 7.5 Hz), 7.40 (2H, t, J 7.5 Hz), 7.33 (1H, t, J 7.5 Hz), 6.84 (1H, br
s), 2.45e2.34 (12H, m), 2.56 (3H, s), 1.72 (2H, quin, J 7.5 Hz), 1.56
(2H, quin, J 7.5 Hz), one NH peak not observed; dC (125 MHz, CDCl3)
171.2,145.3,130.4,129.1,128.7,125.7, 93.7, 58.0, 55.0, 53.2, 46.1, 36.7,
26.2, 23.5, one pyrazole peak not observed; m/z (ESI) 705
([2MþNa]þ, 100), 342 ([MþH]þ, 100); Found: C, 63.72; H, 8.52; N,
19.54. Calc for C19H26N4O þ H2O: C, 63.48; H, 8.13; N, 19.48%
4.1.4.39. (1S, 4S)-tert-Butyl 5-(6-chloropyridazin-3-yl)-2,5-
diazabicyclo[2.2.1]heptane-2-carboxylate (70) [46]. To a solution of
3,6-dichloropyridazine (0.11 g, 0.76 mmol) in DMF (7.5 mL) was
added N,N-diisopropylethylamine (145 mL, 0.83 mmol) and amine
25 (0.15 g, 0.76 mmol). The mixture was heated at reflux for 14 h
and after cooling to room temperature, partitioned between ethyl
acetate (200 mL) and H2O (70 mL). The aqueous layer was further
extracted with ethyl acetate (2 ꢃ 50 mL) and the combined organic
layers then further washed with H2O (100 mL), brine (100 mL) then
dried (MgSO4), filtered and the solvent removed under reduced
pressure to give the crude product. Purification by flash chro-
matograph on silica eluting with hexane-ethyl acetate (1:1) yielded
compound 70 (0.09 g, 37%) as a colourless solid; ½aꢄ2D5ꢀ50.9 (1.0,
4.1.4.36. 4-(6-chloropyridazin-3-yl)morpholine (283) [69]. To a so-
lution of 3,6-dichloropyridazine (67) (1.0 g, 6.7 mmol) in DMF
(67 mL) was added N,N-diisopropylethylamine (1.29 mL,
7.38 mmol) and morpholine (0.58 mL, 6.7 mmol). The mixture was
heated at reflux for 17 h and after cooling to room temperature,
partitioned between ethyl acetate (200 mL) and H2O (100 mL). The
aqueous layer was further extracted with ethyl acetate (2 ꢃ 80 mL)
and the combined organic layers then further washed with H2O
(200 mL), brine (200 mL) then dried (MgSO4), filtered and the
solvent removed under reduced pressure to give the crude product.
Purification by flash chromatograph on silica eluting with ethyl
acetate yielded the desired pyridazine 67 (1.1 g, 83%) as a yellow
solid; mp 132e135 ꢁC (lit mp 132.5e134.5 ꢁC); nmax/cmꢀ1 2985,
2924, 1582, 1527, 1454,1266,1164, 1123, 927, 821, 765; dH (300 MHz,
CDCl3) 7.22 (1H, d, J 9.0 Hz), 6.89 (1H, d, J 9.0 Hz), 3.82 (4H, t, J
6.0 Hz), 3.58 (4H, t, J 6.0 Hz); dC (75 MHz, CDCl3) 159.3, 147.4, 128.9,
115.3, 66.4, 45.4; m/z (APCI) 200 ([MþH]þ, 100).
CHCl3); mp dec >195 ꢁC; max/cmꢀ1 3029, 2914, 1507, 1423, 1289,
n
1258, 1172, 1103, 842; dH (400 MHz, CDCl3) 7.20 (1H, d, J 9.2 Hz),
6.59 (1H, d, J 9.2 Hz), 5.08 (0.6H, s), 4.93 (0.4H, s), 4.70 (0.6H, s), 4.57
(0.4H, s), 3.54 (1H, m), 3.45e3.37 (3H, m), 1.97 (2H, m), 1.44 (9H, m)
(mixture of rotamers); dC (75 MHz, CDCl3) 157.1, 154.3, 146.4, 129.1,
115.1, 80.1, 57.2, 56.8, 56.4, 55.9, 55.6, 52.9, 52.5, 37.7, 37.1, 28.5
(mixture of rotamers); m/z (APCI) 254 ([MꢀC4H9]þ, 100), 311
([MþH]þ, 20).
4.1.4.40. 4-(6-Phenylpyridazin-3-yl)morpholine
(63)
[69].
Subjecting 67 to general procedure 3 using phenyl boronic acid, the
crude residue was obtained and purified by flash chromatography
on silica eluting with ethyl acetate-hexane (2:1) to give pyridazine
63 (0.14 g, 61%) as a colourless solid; mp 153e154 ꢁC (lit mp
153e155 ꢁC); max/cmꢀ1 2854, 1594, 1451, 1432, 1378, 1263, 1238,
n
4.1.4.37. 3-Chloro-6-(piperidin-1-yl)pyridazine (68) [69]. To a solu-
tion of 3,6-dichloropyridazine (55) (0.3 g, 2.0 mmol) in DMF
1120, 930, 831, 784, 746, 697; dH (500 MHz, CDCl3) 8.0 (2H, d, J
7.5 Hz), 7.67 (1H, d, J 9.5 Hz), 7.47 (2H, t, J 7.5 Hz), 7.4 (1H, m), 6.97
(1H, d, J 9.5 Hz), 3.87 (4H, t, J 5.0 Hz), 3.67 (4H, t, J 5.0 Hz); dC
(125 MHz, CDCl3) 159.1, 151.7, 136.6, 128.84, 128.75, 126.0, 125.5,
112.8, 66.6, 45.4; m/z (APCI) 242 ([MþH]þ, 100).
(20 mL) was added N,N-diisopropylethylamine (377
mL, 2.2 mmol)
and piperidine (200 L, 2.0 mmol). The mixture was heated at
m
reflux for 17 h and after cooling to room temperature, partitioned
between ethyl acetate (100 mL) and H2O (100 mL). The aqueous
layer was further extracted with ethyl acetate (2 ꢃ 80 mL) and the
combined organic layers then further washed with H2O (200 mL),
brine (200 mL) then dried (MgSO4), filtered and the solvent
removed under reduced pressure to give the crude product. Puri-
fication by flash chromatograph on silica eluting with hexane-ethyl
acetate (7:2) yielded the desired pyridazine 68 (0.29 g, 72%) as an
off white solid; mp 75e77 ꢁC (lit mp 74e76 ꢁC); nmax/cmꢀ1 3050,
2928, 2854,1587,1529,1479,1438,1263,1249,1213,1171,1004, 923,
830; dH (500 MHz, CDCl3) 7.14 (1H, d, J 9.5 Hz), 6.88 (1H, d, J 9.5 Hz),
3.60 (4H, t, J 5.5 Hz), 1.69e1.65 (6H, m); dC (125 MHz, CDCl3) 159.1,
4.1.4.41. 3-Phenyl-6-(piperidin-1-yl)pyridazine
(64). Subjecting
compound 68 to general procedure 3 using phenyl boronic acid, the
crude residue was obtained and purified by flash chromatography
on silica eluting with ethyl acetate-hexane (1:1) to give pyridazine
64 (0.16 g, 68%) as a colourless solid; mp 120e121 ꢁC; max/cmꢀ1
n
2934, 2851, 1592, 1541, 1430, 1248, 1128, 923, 745, 694; dH
(500 MHz, CDCl3) 8.0 (2H, d, J 7.5 Hz), 7.62 (1H, d, J 9.5 Hz), 7.46 (2H,
t, J 7.5 Hz), 7.39 (1H, t, J 7.5 Hz), 6.98 (1H, d, J 9.5 Hz), 3.71 (4H, m),
1.71 (6H, br s, signals overlapping); dc (125 MHz, CDCl3) 159.1, 150.5,
137.1, 128.9, 128.6, 125.9, 125.2, 113.0, 46.4, 25.6, 24.8; m/z (APCI)

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
297
240 ([MþH]þ, 100); Found: C, 75.07; H, 7.13; N, 17.90. Calc for
mixture was allowed to cool to room temperature before potassium
tert-butoxide (0.53 g, 4.73 mmol), 1,4-dibromobenzene (80) (0.6 g,
C
15H17N3: C, 75.28, H, 7.16; N, 17.56%
2.54 mmol) and morpholine (332 mL, 3.82 mmol) were added. The
4.1.4.42. 3-(4-Methylpiperazin-1-yl)-6-phenylpyridazine (65) [70].
Subjecting compound 69 to general procedure 3 using phenyl
boronic acid, the crude residue was obtained and purified by flash
chromatography on silica eluting with ethyl acetate-hexane (1:1) to
resulting mixture was heated at reflux for 16 h, cooled to room
temperature and filtered through a pad of Celite®, the pad was
further washed with toluene (30 mL) and the combined filtrates
evaporated under reduced pressure to give the crude product. Pu-
rification by flash chromatography on silica eluting with hexane-
ethyl acetate (6:1) gave the desired amine 81 (0.19 g, 47%) as a
give pyridazine 65 (0.14 g, 59%) as
a colourless solid; mp
120e122 ꢁC (lit mp 121e122 ꢁC); max/cmꢀ1 2927, 1592, 1443,
n
1236,1117, 985; dH (300 MHz, CDCl3) 8.0 (2H, d, J 6.9 Hz), 7.65 (1H, d,
J 9.6 Hz), 7.49e7.39 (3H, m), 6.98 (1H, d, J 9.6 Hz), 3.74 (4H, t, J
5.1 Hz), 2.56 (4H, t, J 5.1 Hz), 2.36 (3H, s); dc (75 MHz, CDCl3) 159.1,
151.3, 136.9, 128.9, 128.8, 126.0, 125.3, 113.0, 54.8, 46.4, 45.2; m/z
(APCI) 255 ([MþH]þ, 100).
colourless solid; mp 110e111 ꢁC; max/cmꢀ1 2965, 2857, 2831,
n
1589, 1495, 1259, 1236, 1120, 923, 818; dH (300 MHz, CDCl3) 7.35
(2H, d, J 9.0 Hz), 6.78 (2H, d, J 9.0 Hz), 3.84 (4H, t, J 6.0 Hz), 3.11 (4H,
t, J 6.0 Hz); dC (75 MHz, CDCl3) 150.3, 132.0, 117.4, 112.3, 66.8, 49.3;
m/z (APCI) 242/244 ([MþH]þ, 68/100).
4.1.4.43. (1S, 4S)-tert-butyl 5-(6-phenylpyridazin-3-yl)-2,5-
4.1.4.47. 1-(4-Bromophenyl)piperidine (82) [71,72]. A suspension of
4-bromophenylboronic acid (0.3 g, 1.5 mmol), copper(II) acetate
monohydrate (0.03 g, 0.15 mmol) and powdered 4 Å molecular
sieves (0.56 g) in CH2Cl2 (10 mL) was stirred at room temperature
diazabicyclo[2.2.1]heptane-2-carboxylate
(71)
[46]. Subjecting
compound 70 to general procedure 3 using phenyl boronic acid, the
crude residue was obtained and purified by flash chromatography
on silica eluting with ethyl acetate-hexane (1:1) to give pyridazine
71 (0.18 g, 78%) as a colourless solid; mp 208e210 ꢁC; ½aꢄD25ꢀ67.4
(1.0, CHCl3); nmax/cmꢀ1 1650, 1489, 1476, 1452, 1209, 1186, 809, 627;
dH (500 MHz, CDCl3) 7.98 (2H, d, J 7.0 Hz), 7.63 (1H, t, J 7.5 Hz), 7.45
(2H, t, J 7.5 Hz), 7.38 (1H, t, J 7.5 Hz), 6.67 (1H, d, J 9.0 Hz), 5.16 (0.6H,
s), 5.01 (0.4H, s), 4.71 (0.6H, s), 4.57 (0.4H, s), 3.61 (1H, m),
3.53e3.42 (3H, m), 1.99 (2H, m), 1.43 (9H, m) (mixture of rotamers);
dc (125 MHz, CDCl3) 156.9, 156.7, 154.3, 150.7, 136.9, 128.9, 128.6,
125.8, 125.4, 112.8, 112.7, 80.0, 79.9, 57.3, 57.1, 56.6, 56.4, 55.7, 55.4,
53.0, 52.6, 37.6, 37.1, 28.6, 28.5 (mixture of rotamers); m/z (ESI) 353
([MþH]þ, 100).
for 5 min. Piperidine (74 mL, 0.74 mmol) was then added and the
resulting mixture sealed and stirred under an atmosphere of air at
room temperature for 20 h. The reaction mixture was filtered
through a pad of Celite® and the solvent removed under reduced
pressure to give the crude residue. Purification by flash chroma-
tography on silica eluting with hexane-ethyl acetate (7:2) yielded
amine 82 (0.1 g, 58%) as a colourless solid; mp 73e74 ꢁC (lit mp
72e73 ꢁC); max/cmꢀ1 2937, 2816,1588,1496,1246,1221,1127, 807,
n
537; dH (500 MHz, CDCl3) 7.31 (2H, d, J 9.0 Hz), 6.79 (2H, d, J 9.0 Hz),
3.12 (4H, t, J 5.5 Hz), 1.69 (4H, quin, J 5.5 Hz), 1.58 (2H, m); dC
(125 MHz, CDCl3) 151.3, 131.9, 118.1, 111.2, 50.6, 25.8, 24.3; m/z
(APCI) 240, 242 ([MþH]þ, 100).
4.1.4.44. (1S, 4S)-2-Methyl-5-(6-phenylpyridazin-3-yl)-2,5-
diazabicyclo[2.2.1]heptane (9) [46]. Subjecting compound 71 to
general procedure 5 the crude residue was obtained and purified by
flash chromatography on silica eluting with CH2Cl2-methanol-
aqueous ammonia (92:7.5:0.5) to give methylated amine 9 (0.03 g,
66% over two steps) as a colourless solid; mp 137e138 ꢁC;
½aꢄD25ꢀ49.7 (1.0, CHCl3); nmax/cmꢀ1 2853, 1709, 1594, 1545, 1460,
1435, 1360, 1219, 1028, 834, 746, 695; dH (500 MHz, CDCl3) 7.97 (2H,
d, J 7.0 Hz), 7.6 (1H, d, J 9.0 Hz), 7.44 (2H, t, J 7.0 Hz), 7.36 (1H, t, J
7.0 Hz), 6.66 (1H, d, J 9.0 Hz), 3.62 (1H, d, J 9.5 Hz), 3.61 (1H, s), 3.44
(1H, dd, J 2.0, 9.5 Hz), 2.97 (1H, dd, J 2.0, 10.0 Hz), 2.73 (1H, d, J
10.0 Hz), 2.41 (3H, s), 2.24 (1H, s), 2.03 (1H, d, J 9.5 Hz), 1.87 (1H, d, J
10.0 Hz); dC (125 MHz, CDCl3) 156.8, 150.3, 137.1, 128.9, 128.5, 125.8,
125.2, 112.7, 62.9, 60.4, 57.8, 51.1, 41.1, 35.9; m/z (ESI) 267 ([MþH]þ,
100).
4.1.4.48. 1-(4-Bromophenyl)-4-methylpiperazine (83) [73]. A solu-
tion of tris(dibenzylideneacetone)dipalladium(0) (0.04 g,
0.084 mmol) and ( )-BINAP (0.10 g, 0.16 mmol) in toluene (10 mL)
was degassed with argon for 10 min and then heated at 110 ꢁC for
15 min. The reaction mixture was allowed to cool to room tem-
perature before potassium tert-butoxide (0.44 g, 3.94 mmol), 1.4-
dibromobenzene (80) (0.5 g, 2.12 mmol) and 1-methylpiperazine
(260 mL, 2.34 mmol) were added. The resulting mixture was heat-
ed at reflux for 16 h, cooled to room temperature and filtered
through a pad of Celite®, the pad was further washed with toluene
(30 mL) and the combined filtrates evaporated under reduced
pressure to give the crude product. Purification by flash chroma-
tography on silica eluting with hexane-ethyl acetate (3:1) gave the
desired amine 83 (0.16 g, 50%) as a colourless solid; mp 81e83 ꢁC;
n
max/cmꢀ1 2940, 2814, 1568, 1456, 1245, 1220, 1107, 930; dH
4.1.4.45. 4-(6-(1H-indol-6-yl)pyridazin-3-yl)morpholine
(66).
(500 MHz, CDCl3) 7.33 (2H, d, J 9.0 Hz), 6.79 (2H, d, J 9.0 Hz), 3.19
(4H, t, J 5.0 Hz), 2.57 (4H, t, J 5.0 Hz), 2.35 (3H, s); dC (125 MHz,
CDCl3) 150.4, 132.0, 117.8, 111.9, 55.1, 49.1, 46.3; m/z (ESI) 255/257
([MþH]þ, 70/100).
Subjecting 67 to general procedure 3 using 5-indolylboronic acid,
the crude residue was obtained and purified by flash chromatog-
raphy on silica eluting with ethyl acetate-hexane (1:1) to give
pyridazine 66 (0.11 g, 74%) as a colourless solid; mp decomposition
>160 ꢁC;
n
max/cmꢀ1 3305, 1541, 1340, 1262, 1241, 1175, 1107, 1071,
4.1.4.49. (1S, 4S)-tert-Butyl 5-(4-bromophenyl)-2,5-diazabicyclo
[2.2.1]heptane-2-carboxylate (84). A solution of tris(dibenzylide-
neacetone)dipalladium(0) (0.034 g, 0.037 mmol) and ( )-BINAP
(0.046 g, 0.074 mmol) in toluene (10 mL) was degassed with argon
for 10 min and then heated at 110 ꢁC for 15 min. The reaction
mixture was allowed to cool to room temperature before potassium
tert-butoxide (0.19 g, 1.71 mmol), 1,4-dibromobenzene (80) (0.22 g,
0.92 mmol) and 157 (0.2 g, 1.01 mmol) were added. The resulting
mixture was heated at reflux for 16 h, cooled to room temperature
and filtered through a pad of Celite®, the pad was further washed
with toluene (30 mL) and the combined filtrates evaporated under
reduced pressure to give the crude product. Purification by flash
chromatography on silica eluting with hexane-ethyl acetate (3:1)
936, 810, 727, 540; dH (500 MHz, DMSO-d6) 11.2 (1H, s), 8.18 (1H, s),
7.97 (1H, d, J 9.5 Hz), 7.84 (1H, dd, J 2.0, 8.5 Hz), 7.48 (1H, d, J 8.5 Hz),
7.38 (1H, t, J 3.0 Hz), 7.33 (1H, d, J 9.5 Hz), 6.51 (1H, t, J 4.0 Hz); 3.76
(4H, t, J 4.5 Hz), 3.58 (4H, t, J 4.5 Hz); dC (125 MHz, DMSO-d6) 158.8,
152.3, 136.2, 131.5, 128.7, 126.2, 125.1, 119.3, 117.7, 113.7, 111.7, 101.8;
65.9, 45.1; m/z (ESI) 281 ([MþH]þ, 100); Found: C, 68.56; H, 5.71; N,
19.77. Calc for C16H16N4O: C, 68.55, H, 5.75; N, 19.99%
4.1.4.46. 4-(4-Bromophenyl)morpholine (81). A solution of tris(di-
benzylideneacetone)dipalladium(0) (0.093 g, 0.1 mmol) and
( )-BINAP (0.13 g, 0.21 mmol) in toluene (7 mL) was degassed with
argon for 10 min and then heated at 110 ꢁC for 15 min. The reaction

298
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
gave the desired amine 84 (0.27 g, 82%) as a colourless solid; mp
(0.048 g, 63%) as a colourless solid; mp 110e111 ꢁC; max/cmꢀ1
n
210e211 ꢁC; ½aꢄD25ꢀ22.0 (0.5, CHCl3);
n
max/cmꢀ1 3543, 2965, 1693,
2939, 2843, 2799, 1609, 1525, 1477, 1450, 1291, 1243, 1161, 1147,
1006, 919, 826; dH (400 MHz, CDCl3) 8.82 (1H, dd, J 0.8, 2.4 Hz), 8.51
(1H, dd, J 1.6, 4.8 Hz), 7.82 (1H, ddd, J 1.6, 2.4, 7.9 Hz), 7.50 (2H, d, J
9.2 Hz), 7.31 (1H, dd, J 4.8, 5.6 Hz), 7.01 (2H, d, J 9.2 Hz), 3.28 (4H, t, J
5.2 Hz), 2.59 (4H, t, J 5.2 Hz), 2.36 (3H, s); dC (100 MHz, CDCl3) 151.2,
148.0, 147.7, 136.5, 133.7, 128.6, 127.9, 123.6, 116.2, 55.2, 48.8, 46.3;
m/z (ESI) 254 ([MþH]þ, 100); Found MHþ 254.16540 C16H19N3 þ H
requires 254.16572.
1592, 1496, 1406, 1365,1167,1105; dH (400 MHz, CDCl3) 7.29 (2H, d, J
8.4 Hz), 6.42 (2H, d, J 8.4 Hz), 4.62 (0.6H, s), 4.48 (0.4H, s), 4.33 (1H,
s), 3.54 (1H, d, J 8.8 Hz), 3.45 (0.5H, d, J 9.6 Hz), 3.39e3.36 (1.5H, m),
3.16 (0.6H, d, J 8.8 Hz), 3.07 (0.4H, d, J 8.4 Hz), 1.99e1.87 (2H, m),
1.43 (9H, m) (mixture of rotamers); dC (100 MHz, CDCl3) 154.2,
145.9, 132.1, 114.1, 108.6, 108.4, 57.4, 57.3, 57.0, 56.96, 56.47, 51.4,
51.1, 37.9, 37.4, 28.7, 28.6 (mixture of rotamers); m/z (ESI) 353/355
([MþH],
16H21BrN2O2 þ H requires 353.08647, 355.08647; Found: C, 54.49;
H, 6.00; N, 7.55. Calc for C16H21BrN2O2: C, 54.40, H, 5.99; N, 7.93%
61/100);
Found
MHþ
353.08620,
355.08412
C
4.1.4.55. 1-([1,10-Biphenyl]-4-yl)-4-methylpiperazine
(77).
Subjecting compound 83 to general procedure 3 using phenyl-
boronic acid, the crude residue was obtained and purified by flash
chromatography on silica eluting with hexane-ethyl acetate (2:1) to
give piperazine 77 (0.055 g, 74%) as a colourless solid; mp
4.1.4.50. 4-(4-(Pyridin-3-yl)phenyl)morpholine
(72). Subjecting
compound 81 to general procedure 3, the crude residue was ob-
tained and purified by flash chromatography on silica eluting with
ethyl acetate-hexane (2:1) to give amine 72 (0.085 g, 56%) as a
113e114 ꢁC; max/cmꢀ1 2940, 2846, 2837, 2791, 1735, 1607, 1527,
n
1491, 1292, 1243, 1163, 1008, 921, 821; dH (400 MHz, CDCl3) 7.48
(2H, d, J 8.8 Hz), 7.44 (2H, d, J 7.2 Hz), 7.32 (2H, t, J 7.2 Hz), 7.20 (1H,
d, J 7.2 Hz), 6.92 (2H, d, J 8.8 Hz), 3.20 (4H, t, J 5.2 Hz), 2.53 (4H, t, J
5.2 Hz), 2.29 (3H, s); dC (100 MHz, CDCl3) 150.7, 141.1, 132.4, 128.8,
127.9, 126.7, 126.6, 116.2, 55.3, 49.0, 46.3; m/z (ESI) 253 ([MþH]þ,
100); Found MHþ 253.17017 C17H20N2 þ H requires 253.17047.
colourless solid; mp 162e163 ꢁC; max/cmꢀ1 2970, 2846, 1609,
n
1350, 1259, 921, 823; dH (300 MHz, CDCl3) 8.82 (1H, s), 8.52 (1H, d, J
6.0 Hz), 7.84 (1H, m), 7.52 (2H, d, J 9.0 Hz), 7.34 (1H, dd, J 6.0, 9.0 Hz),
7.00 (2H, d, J 9.0 Hz), 3.88 (4H, t, J 6.0 Hz), 3.22 (4H, t, J 6.0 Hz); dC
(75 MHz, CDCl3) 151.2, 147.6, 147.4, 136.4, 133.9, 128.8, 127.9, 123.7,
115.9, 66.9, 48.9; m/z (APCI) 241 ([MþH]þ, 100); Found: C, 74.86; H,
6.81; N, 11.29. Calc for C15H16N2O: C, 74.97, H, 6.71; N, 11.66%
4.1.4.56. (1S, 4S)-tert-Butyl 5-(4-(pyridin-3-yl)phenyl)-2,5-
diazabicyclo[2.2.1]heptane-2-carboxylate (85). Subjecting com-
pound 84 to general procedure 3 gave, after purification by flash
chromatography on silica eluting with hexane-ethylacetate (1:1)
compound 85 (0.053 g, 52%) as a colourless solid; mp 179e180 ꢁC;
4.1.4.51. 4-([1,10-biphenyl]-4-yl)morpholine
(73)
[74].
Subjecting compound 81 to general procedure 3 using phenyl-
boronic acid, the crude residue was obtained and purified by flash
chromatography on silica eluting with ethyl acetate-hexane (6:1) to
give amine 73 (0.093 g, 72%) as a colourless solid; mp 179e180 ꢁC;
½aꢄ2D5ꢀ25.0 (1.0, CHCl3);
n
max/cmꢀ1 2918, 1692, 1609, 1525, 1474,
1402, 1322, 1184, 1166, 1102, 813; dH (500 MHz, CDCl3) 8.81 (1H, s),
8.49 (1H, d, J 4.5 Hz), 7.81 (1H, d, J 7.5 Hz), 7.47 (2H, t, J 9.0 Hz), 7.30
(1H, dd, J 4.5, 8.0 Hz), 6.65 (2H, d, J 8.0 Hz), 4.67 (0.6H, s), 4.52 (0.4H,
s), 4.44 (1H, s), 3.60 (1H, m), 3.52 (0.5H, d, J 10.0 Hz), 3.45e3.39
(1.5H, m), 3.27 (0.6H, d, J 9.0 Hz), 3.18 (0.4H, d, J 8.5 Hz), 2.01e1.92
(2H, m), 1.43 (9H, m) (mixture of rotamers); dC (125 MHz, CDCl3)
154.2, 147.8, 147.4, 146.8, 136.6, 133.3, 128.2, 16.0, 125.81, 123.6,
113.0, 79.9, 79.8, 57.4, 57.3, 57.2, 57.0, 56.8, 56.5, 51.7, 51.3, 37.9, 37.5,
28.7, 28.6 (mixture of rotamers); m/z (ESI) 352 ([MþH]þ, 100);
Found MHþ 352.20195 C21H25N3O2 þ H requires 352.20250.
n
max/cmꢀ1 2837, 1607, 1490, 1259, 1234, 1121, 927, 820, 761, 692; dH
(500 MHz, CDCl3) 7.57e7.53 (4H, m), 7.41 (2H, t, J 8.0 Hz), 7.29 (1H, t,
J 8.0 Hz), 6.99 (2H, d, J 7.0 Hz), 3.89 (4H, t, J 5.0 Hz), 3.21 (4H, t, J
5.0 Hz); dC (125 MHz, CDCl3) 150.7, 141.0, 132.9, 128.9, 128.0, 126.69,
126.67, 115.9, 67.1, 49.4; m/z (APCI) 240 ([MþH]þ, 100).
4.1.4.52. 3-(4-(Piperidin-1-yl)phenyl)pyridine
(74). Subjecting
compound 82 to general procedure 3, the crude residue was ob-
tained and purified by flash chromatography on silica eluting with
hexane-ethyl acetate (2:1) to give amine 74 (0.05 g, 72%) as a col-
ourless solid; mp 134e136 ꢁC;
n
max/cmꢀ1 2934, 1606, 1244, 1130,
4.1.4.57. (1S, 4S)-tert-Butyl 5-([1,10-biphenyl]-4-yl)-2,5-diazabicyclo
[2.2.1]heptane-2-carboxylate (86). Subjecting compound 84 to
general procedure 3 using phenylboronic acid gave, after purifica-
tion by flash chromatography on silica eluting with hexane-
ethylacetate (3:1) compound 86 (0.059 g, 58%) as a colourless
802, 711; dH (500 MHz, CDCl3) 8.82 (1H, d, J 2.0 Hz), 8.50 (1H, dd, J
2.0, 5.0 Hz), 7.83 (1H, dt, J 2.0, 8.5 Hz), 7.49 (2H, d, J 8.5 Hz), 7.31 (1H,
m), 7.02 (2H, d, J 8.5 Hz), 3.24 (4H, t, J 5.5 Hz), 1.73 (4H, quin, J
5.5 Hz), 1.61 (2H, m); dC (125 MHz, CDCl3) 152.0, 147.9, 147.5, 136.6,
133.7, 127.88, 127.82, 123.6, 116.5, 50.3, 25.8, 24.5; m/z (ESI) 239
([MþH]þ, 100); Found: C, 80.53; H, 7.71; N, 11.63. Calc for C16H18N2:
C, 80.63, H, 7.61; N, 11.75%
solid; mp 156e157 ꢁC; ½aꢄD25ꢀ20.0 (1.0, CHCl3);
n
max/cmꢀ1 2950,
1695, 1491, 1405, 1365, 1186, 1167, 1103; dH (400 MHz, CDCl3) 7.57
(2H, d, J 8.0 Hz), 7.51 (2H, t, J 8.4 Hz), 7.42 (2H, t, J 8.0 Hz), 7.28 (1H,
m), 6.66 (2H, d, J 8.0 Hz), 4.69 (0.6H, s), 4.53 (0.4H, s), 4.46 (1H, s),
3.64 (1H, d, J 8.4 Hz), 3.56 (0.4H, d, J 9.6 Hz), 3.50e3.40 (1.6H, m),
3.84 (0.6H, d, J 8.4 Hz), 3.20 (0.4H, d, J 8.8 Hz), 2.04e1.93 (2H, m),
1.46 (9H, m) (mixture of rotamers); dC (100 MHz, CDCl3) 154.3,
146.3, 129.7, 129.5, 128.8, 128.2, 126.4, 126.2, 112.9, 112.8, 79.8, 79.7,
57.5, 57.4, 57.2, 57.0, 56.9, 56.5, 51.6, 51.2, 37.9, 37.5, 28.7, 28.6; m/z
(ESI) 373 ([MþNa]þ,100); Found MNaþ 373.18865 C22H26N2O2 þ Na
requires 373.18920.
4.1.4.53. 1-([1,10-Biphenyl]-4-yl)piperidine (75) [75]. Subjecting
compound 82 to general procedure 3 using phenylboronic acid, the
crude residue was obtained and purified by flash chromatography
on silica eluting with hexane-ethyl acetate (95:5) to give amine 75
(0.05 g, 71%) as a colourless solid; mp 122e123 ꢁC; max/cmꢀ1
n
2937, 2850, 1607, 1490, 1241, 820, 760, 692; dH (500 MHz, CDCl3)
7.56 (2H, d, J 7.5 Hz), 7.5 (2H, d, J 9.0 Hz), 7.39 (2H, t, J 7.5 Hz), 7.26
(1H, t, J 7.5 Hz), 7.0 (2H, d, J 9.0 Hz), 3.21 (4H, t, J 5.5 Hz), 1.73 (4H,
quin, J 5.5 Hz), 1.60 (2H, m); dC (125 MHz, CDCl3) 151.6, 141.2, 131.8,
128.8, 127.8, 126.6, 126.4, 116.6, 50.6, 25.9, 24.5; m/z (ESI) 238
([MþH]þ, 100).
4.1.4.58. (1S, 4S)-2-Methyl-5-(4-(pyridin-3-yl)phenyl)-2,5-
diazabicyclo[2.2.1]heptane (78). Subjecting compound 85 to gen-
eral procedure 5, the crude residue was obtained and purified by
flash chromatography on silica eluting with CH2Cl2-methanol-
aqueous ammonia (90:9.5:0.5) to give methylated amine 78 (0.03 g,
4.1.4.54. 1-Methyl-4-(4-(pyridin-3-yl)phenyl)piperazine
(76).
Subjecting compound 83 to general procedure 3, the crude residue
was obtained and purified by flash chromatography on silica
eluting with hexane-ethyl acetate (2:1) to give piperazine 76
61% over two steps) as a colourless oil; ½aꢄ2D5ꢀ14.6 (1.0, CHCl3); nmax
/
cmꢀ1 2877, 1608, 1525, 1476, 1370; dH (400 MHz, CDCl3) 8.81 (1H, d,
J 1.6 Hz), 8.47 (1H, dd, J 1.2, 4.4 Hz), 7.81 (1H, dt, J 8.0 Hz), 7.46 (2H, d,

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
299
J 8.8 Hz), 7.29 (1H, dd, J 4.8, 7.6 Hz), 6.65 (2H, d, J 8.8 Hz), 4.26 (1H,
s), 3.51 (1H, s), 3.41e3.37 (2H, m), 2.98 (1H, dd, J 2.0, 9.6 Hz), 2.66
(1H, d, J 9.2 Hz), 2.39 (3H, s), 2.01 (1H, d, J 9.2 Hz), 1.91 (1H, d, J
9.6 Hz); dC (100 MHz, CDCl3) 147.7, 147.2, 147.1, 136.8, 133.3, 128.0,
125.4, 123.6, 113.0, 63.1, 59.0, 58.2, 52.0, 40.7, 36.6; m/z (ESI) 266
([MþH]þ, 100); Found: MHþ 266.16517 C17H19N3 þ H requires
266.16572.
preparation procedures and binding assays were described previ-
ously [53e55,76]. Briefly, cultured cells at >80% confluence were
removed from their flasks (80 cm2) with a disposable cell scraper
and placed in 10 mL of 50 mM Tris.HCl buffer (pH 7.4, 4 ꢁC). The cell
suspension was centrifuged at 10,000 ꢃ g for 5 min and the pellet
was collected. The cell pellet was then homogenised in 10 mL buffer
with a polytron homogenizer and centrifuged at 36,000 g for
10 min at 4 ꢁC. The membrane pellet was resuspended in fresh
buffer, and aliquots of the membrane preparation were used for
binding assays. The concentrations of [3H]EB used in competition
binding assays were ~500 pM for a3b4 and a4b2 nAChRs and ~2 nM
for a7 nAChRs. Nonspecific binding was assessed in parallel in-
4.1.4.59. 2-([1,10-Biphenyl]-4-yl)-5-methyl-2,5-diazabicyclo[2.2.1]
heptane (79). Subjecting compound 86 to general procedure 5, the
crude residue was obtained and purified by flash chromatography
on silica eluting with CH2Cl2-methanol-aqueous ammonia
(90:9.5:0.5) to give methylated amine 79 (0.02 g, 54% over two
steps) as a colourless oil; ½aꢄ2D5ꢀ16.5 (1.0, CHCl3); nmax/cmꢀ1 2914,
1609, 1527, 1487, 1365, 823; dH (500 MHz, CDCl3) 7.54 (2H, d, J
7.5 Hz), 7.47 (2H, d, J 8.5 Hz), 7.38 (2H, t, J 8.5 Hz), 7.24 (1H, t, J
9.0 Hz), 6.63 (2H, d, J 8.5 Hz), 4.25 (1H, s), 3.51 (1H, s), 3.42e3.37
(2H, m), 2.98 (1H, dd, J 2.0, 9.8 Hz), 2.67 (1H, d, J 9.5 Hz), 2.39 (3H, s),
2.0 (1H, d, J 9.5 Hz), 1.91 (1H, d, J 9.5 Hz); dC (125 MHz, CDCl3) 146.5,
141.4, 129.1, 128.8, 128.0, 126.3, 126.0, 112.9, 63.1, 58.9, 58.2, 51.9,
40.5, 36.7; m/z (ESI) 265 ([MþH], 100); Found: MHþ
265.16993C18H20N2 þ H requires 265.17047.
cubations in the presence of 300 mM nicotine. Bound and free li-
gands were separated by vacuum filtration through Whatman GF/C
filters treated with 0.5% polyethylenimine. The filter-retained
radioactivity was measured by liquid scintillation counting. Spe-
cific binding was defined as the difference between total binding
and nonspecific binding. Data from competition binding assays
were analysed using Prism 5.
4.4. Functional activity studies of selected a7 ligands
All compounds were tested for agonist activity on the
in a fluorescent calcium assay using a GH4C1 cells stably expressing
rat 7 nAChR. To determine EC50 values, all compounds were tested
at 0.03, 0.1, 0.3, 1, 3 and 10
M (n ¼ 3) except for compound 9 which
was tested (n ¼ 3) at 0.001e0.3 M Fluo4 Direct Dye from Molec-
a7 nAChR
4.2. Docking studies (Table 9)
a
m
Table 9
m
Summary of XP [50] docking results for SEN12333 (8) and 14.
ular Probes was used and the assay was performed on a Perkin
Elmer CellLux high throughput fluorescent plate reader. A baseline
read was recorded prior to addition of compound. Peak/Base (P/B)
ratios were calculated. Values >1.00 indicate agonist activity
(shown in orange). GraphPad Prism, Version 4.03 was used to
produce dose response curves for each compound and calculate the
XP terma
Compound
SEN12333 (8)
14
GScore
LipophilicEvdW
PhobEn
PhobEnHB
HBond
Electro
Sitemap
PiCat
LowMW
Penalties
Rot Penalties
ꢀ5.14
ꢀ4.48
ꢀ0.9
0
ꢀ1.14
ꢀ0.57
ꢀ0.25
0
ꢀ13.14
ꢀ4.61
ꢀ2.7
EC50 for agonist activity at rat
a7 nAChR. Replicates have been
0
ꢀ1.11
ꢀ1.19
ꢀ0.27
ꢀ4.12
ꢀ0.28
1.64
factored in each datapoint, the error is contained in the datapoints,
as opposed to calculating each replicate as a dose response and
getting the error on the EC50.
ꢀ0.37
4.69
0.49
4.5. Electrophysiology studies of SEN12333 (8) and compound 45
Two compounds, SEN12333 (8) and compound 45, were tested
0.49
a
Description of XP terms. GScore: total Glide score; LipophilicEvdW: ChemScore
lipophilic pair term and fraction of the total protein-ligand vdW energy; PhobEn:
Hydrophobic enclosure reward; HBond: ChemScore H-bond pair term; Electro:
Electrostatic rewards; Sitemap: Sitemap ligand/receptor non-H-bonding polar/hy-
drophobic and hydrophobic/hydrophilic complementarity terms; PiCat: Reward for
for agonist activity on the
nAChR) in manual patch-clamp electrophysiology using GH4C1
cells stably expressing rat 7 nAChR. The manual patch-clamp re-
cordings used the fast application add-on Dynaflow® system. To
determine EC50 values, the compounds were tested at 3 M, 10 M,
30 M, 100 M, 300
a7 nicotinic acetylcholine receptor (a7
a
cat-
p interactions; LowMW: Reward for ligands with low molecular weight; Pen-
alties: Polar atom burial and desolvation penalties, and penalty for intra-ligand
contacts; RotPenal: Rotatable bond penalty.
m
m
m
m
m
M, 1 mM and 3 mM (n ¼ 5). Peak currents
were normalized to saturating acetylcholine. Replicates have been
factored in each datapoint, the error is contained in the datapoints,
as opposed to calculating each replicate as a dose response and
getting the error on the EC50.
4.3. Binding affinity studies of a7 ligands
Tissue culture medium and antibiotics were obtained from
Invitrogen Corp (Carlsbad, California). Foetal bovine serum was
purchased from Gemini Bio-products (West Sacramento, Califor-
nia). [3H] EB was obtained from PerkinElmer Life and Analytical
Sciences (Boston, Massachusetts). Three cell lines, KXa3b4R2,
KXa4b2R2 and KXa7R1, which express defined rat nAChR subtypes,
were established previously by stably transfecting HEK293 cells
with rat nAChR subunit genes [53e55,76].
Acknowledgements
C.B. acknowledges receipt of an Australian Postgraduate Award
and a John A. Lamberton Research Scholarship. The authors grate-
fully acknowledge Dr Munikumar Reddy Doddareddy for his
assistance with the molecular modelling studies.
These cell lines were maintained in minimum essential medium
(MEM) supplemented with 10% foetal bovine serum, 100 U/mL
penicillin G, 100 mg/mL streptomycin and selective antibiotics at
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.025.
37 ꢁC with 5% CO2 in
a humidified incubator. Membrane

300
C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
[26] X.-L. Qi, A. Nordberg, J. Xiu, Z.-Z. Guan, The consequences of reducing
expression of the 7 nicotinic receptor by RNA interference and of stimulating
its activity with an 7 agonist in SH-SY5Y cells indicate that this receptor
plays a neuroprotective role in connection with the pathogenesis of Alz-
heimer's disease, Neurochem. Int. 51 (2007) 377e383.
[27] A. Mazurov, T. Hauser, C.H. Miller, Selective [alpha]7 nicotinic acetylcholine
receptor ligands, Curr. Med. Chem. 13 (2006) 1567e1584.
[28] C.A. Briggs, D.J. Anderson, J.D. Brioni, J.J. Buccafusco, M.J. Buckley,
J.E. Campbell, M.W. Decker, D. DonnellyeRoberts, R.L. Elliott,
M. Gopalakrishnan, M.W. Holladay, Y.-H. Hui, W.J. Jackson, D.J.B. Kim,
K.C. Marsh, A. O'Neill, M.A. Prendergast, K.B. Ryther, J.P. Sullivan, S.P. Arneric,
Functional characterization of the novel neuronal nicotinic acetylcholine re-
ceptor ligand GTS-21 in vitro and in vivo, Pharmacol. Biochem. Behav. 57
(1997) 231e241.
[29] W. Kem, F. Soti, K. Wildeboer, S. LeFrancois, K. MacDougall, D.-Q. Wei, K.-
C. Chou, H.R. Arias, The nemertine toxin anabaseine and its derivative DMXBA
(GTS-21): chemical and pharmacological properties, Mar. Drugs 4 (2006)
255e273.
References
a
a
[1] J.A. Dani, D. Bertrand, Nicotinic acetylcholine receptors and nicotinic cholin-
ergic mechanisms of the Central nervous system, Annu. Rev. Pharmacol.
Toxicol. 47 (2007) 699e729.
[2] D. D'Hoedt, D. Bertrand, Nicotinic acetylcholine receptors: an overview on
drug discovery, Expert Opin. Ther. Targets 13 (2009) 395e411.
[3] M. Bencherif, S.T. Narla, M.S. Stachowiak, Alpha7 neuronal nicotinic receptor:
a pluripotent target for diseases of the central nervous system, CNS Neurol.
Disord. Drug Targets 13 (2014) 836e845.
[4] T.L. Wallace, D. Bertrand, Alpha7 neuronal nicotinic receptors as a drug target
in schizophrenia, Expert Opin. Ther. Targets 17 (2013) 139e155.
[5] P. Seguela, J. Wadiche, K. Dineley-Miller, J.A. Dani, J.W. Patrick, Molecular
cloning, functional properties, and distribution of rat brain
a7: a nicotinic
cation channel highly permeable to calcium, J. Neurosci. 13 (1993) 596e604.
[6] R. Hurst, H. Rollema, D. Bertrand, Nicotinic acetylcholine receptors: from basic
science to therapeutics, Pharmacol. Ther. 137 (2013) 22e54.
[7] A. Taly, P.-J. Corringer, D. Guedin, P. Lestage, J.-P. Changeux, Nicotinic re-
ceptors: allosteric transitions and therapeutic targets in the nervous system,
Nat. Rev. Drug Discov. 8 (2009) 733e750.
[30] A. Olincy, J.G. Harris, L.L. Johnson, et al., Proof-of-concept trial of an a7 nico-
tinic agonist in schizophrenia, Arch. Gen. Psychiatry 63 (2006) 630e638.
[31] G. Mullen, J. Napier, M. Balestra, T. DeCory, G. Hale, J. Macor, R. Mack, J. Loch,
E. Wu, A. Kover, P. Verhoest, A. Sampognaro, E. Phillips, Y. Zhu, R. Murray,
R. Griffith, J. Blosser, D. Gurley, A. Machulskis, J. Zongrone, A. Rosen, J. Gordon,
(ꢀ)-spiro[1-azabicyclo[2.2.2]octane-3,50-oxazolidin-20-one], a conformation-
ally restricted analogue of acetylcholine, is a highly selective full agonist at the
ꢀ
ꢀ
ꢀ
[8] B. Lendvai, F. Kassai, A. Szajli, Z. Nemethy, a7 nicotinic acetylcholine receptors
and their role in cognition, Brain Res. Bull. 93 (2013) 86e96.
[9] P. Lippiello, M. Bencherif, T. Hauser, K. Jordan, S. Letchworth, A. Mazurov,
Nicotinic receptors as targets for therapeutic discovery, Expert Opin. Drug
Discov. 2 (2007) 1185e1203.
a
7 nicotinic acetylcholine receptor, J. Med. Chem. 43 (2000) 4045e4050.
[10] W.R. Kem, The brain a7 nicotinic receptor may be an important therapeutic
[32] M. Kampen, K. Selbach, R. Schneider, E. Schiegel, F. Boess, R. Schreiber, AR-R
target for the treatment of Alzheimer's disease: studies with DMXBA (GTS-
21), Behav. Brain Res. 113 (2000) 169e181.
[11] B. Lendvai, F. Kassai, A. Szajli, Z. Nemethy, alpha7 nicotinic acetylcholine re-
ceptors and their role in cognition, Brain Res. Bull. 93 (2013) 86e96.
[12] A.H.C. Wong, H.H.M. Van Tol, Schizophrenia: from phenomenology to
neurobiology, Neurosci. Behav. Rev. 27 (2003) 269e306.
17779 improves social recognition in rats by activation of nicotinic
a7 re-
ceptors, Psychopharmacology 172 (2004) 375e383.
[33] E.D. Levin, C. Bettegowda, J. Blosser, J. Gordon, AR-R 17779, an alpha7 nicotinic
agonist, improves learning and memory in rats, Behav. Pharmacol. 10 (1999)
675e680.
[34] P. Pichat, O.E. Bergis, J.-P. Terranova, A. Urani, C. Duarte, V. Santucci,
C. Gueudet, C. Voltz, R. Steinberg, J. Stemmelin, F. Oury-Donat, P. Avenet,
[13] A. Marutle, X. Zhang, J. Court, M. Piggott, M. Johnson, R. Perry, E. Perry,
A. Nordberg, Laminar distribution of nicotinic receptor subtypes in cortical
regions in schizophrenia, J. Chem. Neuroanat. 22 (2001) 115e126.
[14] C.M. Martin-Ruiz, V.H. Haroutunian, P. Long, A.H. Young, K.L. Davis, E.K. Perry,
J.A. Court, Dementia rating and nicotinic receptor expression in the prefrontal
cortex in schizophrenia, Biol. Psychiatry 54 (2003) 1222e1233.
[15] Z.-Z. Guan, X. Zhang, K. Blennow, A. Nordberg, Decreased protein level of
nicotinic receptor [alpha]7 subunit in the frontal cortex from schizophrenic
brain, Neuroreport 10 (1999) 1779e1782.
[16] R. Freedman, H. Coon, M. Myles-Worsley, A. Orr-Urtreger, A. Olincy, A. Davis,
M. Polymeropoulos, J. Holik, J. Hopkins, M. Hoff, J. Rosenthal, M.C. Waldo,
F. Reimherr, P. Wender, J. Yaw, D.A. Young, C.R. Breese, C. Adams, D. Patterson,
L.E. Adler, L. Kruglyak, S. Leonard, W. Byerley, Linkage of a neurophysiological
deficit in schizophrenia to a chromosome 15 locus, Proc. Natl. Acad. Sci. U. S. A.
94 (1997) 587e592.
[17] V. De Luca, A.H.C. Wong, D.J. Muller, G.W.H. Wong, R.F. Tyndale, J.L. Kennedy,
Evidence of association between smoking and [alpha]7 nicotinic receptor
subunit gene in schizophrenia patients, Neuropsychopharm 29 (2004)
1522e1526.
[18] S. Leonard, C. Breese, C. Adams, K. Benhammou, J. Gault, K. Stevens, M. Lee,
L. Adler, A. Olincy, R. Ross, R. Freedman, Smoking and schizophrenia:
abnormal nicotinic receptor expression, Eur. J. Pharmacol. 393 (2000)
237e242.
[19] R. Freedman, A. Olincy, R.W. Buchanan, J.G. Harris, J.M. Gold, L.L. Johnson,
D. Allensworth, B.A.A. Guzman-Bonilla, B. Clement, M.S.M. Ball, J. Kutnick,
V. Pender, L.F. Martin, K. Stevens, B. Wagner, G.O. Zerbe, F. Soti, W.R. Kem,
Initial phase 2 trial of a nicotinic agonist in schizophrenia, Am. J. Psychiatry
165 (2008) 1040e1047.
[20] B.A. Acker, E.J. Jacobsen, B.N. Rogers, D.G. Wishka, S.C. Reitz, D.W. Piotrowski,
J.K. Myers, M.L. Wolfe, V.E. Groppi, B.A. Thornburgh, P.M. Tinholt, R.R. Walters,
B.A. Olson, L. Fitzgerald, B.A. Staton, T.J. Raub, M. Krause, K.S. Li,
W.E. Hoffmann, M. Hajos, R.S. Hurst, D.P. Walker, Discovery of N-[(3R,5R)-1-
azabicyclo[3.2.1]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide as an agonist of
G. Griebel, B. Scatton, SSR180711, a novel selective
a7 nicotinic receptor
partial agonist: (II) efficacy in experimental models predictive of activity
against cognitive symptoms of schizophrenia, Neuropsychopharmacology 32
(2007) 17e34.
[35] B. Biton, O.E. Bergis, F. Galli, A. Nedelec, A.W. Lochead, S. Jegham, D. Godet,
C. Lanneau, R. Santamaria, F. Chesney, J. Leonardon, P. Granger, M.W. Debono,
G.A. Bohme, F. Sgard, F. Besnard, D. Graham, A. Coste, A. Oblin, O. Curet,
X. Vige, C. Voltz, L. Rouquier, J. Souilhac, V. Santucci, C. Gueudet, D. Francon,
R. Steinberg, G. Griebel, F. Oury-Donat, P. George, P. Avenet, B. Scatton,
SSR180711, a novel selective a7 nicotinic receptor partial agonist: (1) binding
and functional profile, Neuropsychopharmacology 32 (2007) 1e16.
[36] S. Barak, M. Arad, A. De Levie, M.D. Black, G. Griebel, I. Weiner, Pro-Cognitive,
Antipsychotic, Efficacy of the [alpha]7 nicotinic partial agonist SSR180711 in
pharmacological and neurodevelopmental latent inhibition models of
schizophrenia, Neuropsychopharmacology 34 (2009) 1753e1763.
[37] K. Hashimoto, T. Ishima, Y. Fujita, M. Matsuo, T. Kobashi, M. Takahagi,
H. Tsukada, M. Iyo, Phencyclidine-induced cognitive deficits in mice are
improved by subsequent subchronic administration of the novel selective a7
nicotinic receptor agonist SSR180711, Biol. Psychiatry 63 (2008) 92e97.
[38] J. Malysz, D.J. Anderson, J.H. Gronlien, J. Ji, W.H. Bunnelle, M. Hakerud,
K. Thorin-Hagene, H. Ween, R. Helfrich, M. Hu, E. Gubbins, S. Gopalakrishnan,
P.S. Puttfarcken, C.A. Briggs, J. Li, M.D. Meyer, T. Dyhring, P.K. Ahring,
E.O. Nielsen, D. Peters, D.B. Timmermann, M. Gopalakrishnan, In vitro phar-
macological characterization of
a novel selective a7 neuronal nicotinic
acetylcholine receptor agonist ABT-107, J. Pharmacol. Exp. Ther. 334 (2010)
863e874.
[39] J. Toyohara, K. Hashimoto,
a7 nicotinic receptor agonists: potential thera-
peutic drugs for treatment of cognitive impairments in schizophrenia and
Alzheimer's disease, Open Med. Chem. J. 4 (2010) 37e56.
[40] A.H. Rezvani, E. Kholdebarin, F.H. Brucato, P.M. Callahan, D.A. Lowe, E.D. Levin,
Effect of R3487/MEM3454, a novel nicotinic a7 receptor partial agonist and 5-
HT3 antagonist on sustained attention in rats, Prog. Neuro-Psychopharmacol.
Biol. Psychiatry 33 (2009) 269e275.
the a7 nicotinic acetylcholine receptor: in vitro and in vivo activity, Bioorg.
Med. Chem. Lett. 18 (2008) 3611e3615.
[21] Z.-Z. Guan, X. Zhang, R. Ravid, A. Nordberg, Decreased protein levels of
nicotinic receptor subunits in the hippocampus and temporal cortex of pa-
tients with Alzheimer's disease, J. Neurochem. 74 (2000) 237e243.
[41] S.N. Haydar, C. Ghiron, L. Bettinetti, H. Bothmann, T.A. Comery, J. Dunlop, S. La
Rosa, I. Micco, M. Pollastrini, J. Quinn, R. Roncarati, C. Scali, M. Valacchi,
M. Varrone, R. Zanaletti, SAR and biological evaluation of SEN12333/WAY-
317538: novel alpha 7 nicotinic acetylcholine receptor agonist, Bioorg. Med.
Chem. 17 (2009) 5247e5258.
€
[22] M. Mousavi, E. Hellstrom-Lindahl, Z.Z. Guan, K.R. Shan, R. Ravid, A. Nordberg,
Protein and mRNA levels of nicotinic receptors in brain of tobacco using
controls and patients with Alzheimer's disease, Neuroscience 122 (2003)
515e520.
[42] R. Roncarati, C. Scali, T.A. Comery, S.M. Grauer, S. Aschmi, H. Bothmann, B. Jow,
D. Kowal, M. Gianfriddo, C. Kelley, U. Zanelli, C. Ghiron, S. Haydar, J. Dunlop,
G.C. Terstappen, Procognitive and neuroprotective activity of a novel
a7
[23] H.-Y. Wang, D.H.S. Lee, C.B. Davis, R.P. Shank, Amyloid peptide A
b1-42 binds
nicotinic acetylcholine receptor agonist for treatment of neurodegenerative
and cognitive disorders, J. Pharmacol. Exp. Ther. 329 (2009) 459e468.
selectively and with picomolar affinity to
J. Neurochem. 75 (2000) 1155e1161.
[24] H.-Y. Wang, D.H.S. Lee, M.R. D'Andrea, P.A. Peterson, R.P. Shank, A.B. Reitz,
a7 nicotinic acetylcholine receptors,
[43] C. Beinat, S.D. Banister, S. van Prehn, M.R. Doddareddy, D. Hibbs, M. Sako,
M. Chebib, T. Tran, N. Al-Muhtasib, Y. Xiao, M. Kassiou, Consequences of linker
length alteration of the a7 nicotinic acetylcholine receptor (nAChR) agonist,
SEN12333, Bioorg. Med. Chem. Lett. 22 (2012) 2380e2384.
[44] R. Zanaletti, L. Bettinetti, C. Castaldo, G. Cocconcelli, T. Comery, J. Dunlop,
G. Gaviraghi, C. Ghiron, S.N. Haydar, F. Jow, L. Maccari, I. Micco, A. Nencini,
b-
amyloid1e42 binds to 7 nicotinic acetylcholine receptor with high affinity:
implications for Alzheimer's disease pathology, J. Biol. Chem. 275 (2000)
5626e5632.
a
[25] T. Kihara, S. Shimohama, H. Sawada, J. Kimura, T. Kume, H. Kochiyama,
T. Maeda, A. Akaike, Nicotinic receptor stimulation protects neurons against b-
amyloid toxicity, Ann. Neurol. 42 (1997) 159e163.
C. Scali, E. Turlizzi, M. Valacchi, Discovery of
a novel alpha-7 nicotinic
acetylcholine receptor agonist series and characterization of the potent,

C. Beinat et al. / European Journal of Medicinal Chemistry 95 (2015) 277e301
301
selective, and orally efficacious agonist 5-(4-Acetyl[1,4]diazepan-1-yl)penta-
noic acid [5-(4-Methoxyphenyl)-1H-pyrazol-3-yl] amide (SEN15924, WAY-
361789), J. Med. Chem. 55 (2012) 4806e4823.
[60] W.K. Hagmann, The Many roles for fluorine in medicinal chemistry, J. Med.
Chem. 51 (2008) 4359e4369.
[61] I.B. Seiple, S. Su, R.A. Rodriguez, R. Gianatassio, Y. Fujiwara, A.L. Sobel,
P.S. Baran, Direct CꢀH arylation of electron-deficient heterocycles with aryl-
boronic acids, J. Am. Chem. Soc. 132 (2010) 13194e13196.
[45] C. Ghiron, S.N. Haydar, S. Aschmies, H. Bothmann, C. Castaldo, G. Cocconcelli,
T.A. Comery, L. Di, J. Dunlop, T. Lock, A. Kramer, D. Kowal, F. Jow, S. Grauer,
B. Harrison, S. La Rosa, L. Maccari, K.L. Marquis, I. Micco, A. Nencini, J. Quinn,
A.J. Robichaud, R. Roncarati, C. Scali, G.C. Terstappen, E. Turlizzi, M. Valacchi,
M. Varrone, R. Zanaletti, U. Zanelli, Novel Alpha-7 nicotinic acetylcholine re-
ceptor agonists containing a urea Moiety: Identification and characterization
of the potent, selective, and orally efficacious agonist 1-[6-(4-fluorophenyl)
[62] S.D. Banister, S.M. Wilkinson, R. Hanani, A.J. Reynolds, D.E. Hibbs, M. Kassiou,
A
practical, multigram synthesis of the 2-(2-(4-alkoxyphenyl)-5,7-
dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (DPA) class of high affin-
ity translocator protein (TSPO) ligands, Tetrahedron Lett. 53 (2012)
3780e3783.
pyridin-3-yl]-3-(4-piperidin-1-ylbutyl)
J. Med. Chem. 53 (2010) 4379e4389.
[46] T. Li, W.H. Bunnelle, K.B. Ryther, D.J. Anderson, J. Malysz, R. Helfrich,
J.H. Gronlien, M. Hakerud, D. Peters, M.R. Schrimpf, M. Gopalakrishnan, J. Ji,
Syntheses and structure-activity relationship (SAR) studies of 2,5-diazabicyclo
urea
(SEN34625/WYE-103914),
[63] A.R. Katritzky, A. Chermprapai, R.C. Patel, A. Tarraga-Tomas, Pyridinium ylides
derived from pyryliums and amines and a novel rearrangement of 1-vinyl-
1,2-dihydropyridines, J. Org. Chem. 47 (1982) 492e497.
[64] S.-H. Kim, R.D. Rieke, 2-Pyridyl and 3-pyridylzinc bromides: direct prepara-
tion and coupling reaction, Tetrahedron 66 (2010) 3135e3146.
[65] S.-H. Kim, R.D. Rieke, Coupling reactions with haloaromatic amines and al-
[2.2.1]heptanes as novel
a7 neuronal nicotinic receptor (NNR) ligands, Bioorg.
Med. Chem. Lett. 20 (2010) 3636e3639.
cohols for
a practical synthetic route to 2-substituted aminophenyl and
€
[47] G. Wuitschik, M. Rogers-Evans, A. Buckl, M. Bernasconi, M. Marki, T. Godel,
H. Fischer, B. Wagner, I. Parrilla, F. Schuler, J. Schneider, A. Alker,
W.B. Schweizer, K. Müller, E.M. Carreira, Spirocyclic oxetanes: synthesis and
properties, Angew. Chem. Int. Ed. 47 (2008) 4512e4515.
hydroxyphenyl pyridines, Tetrahedron Lett. 50 (2009) 6985e6988.
[66] N. Gavande, G.A.R. Johnston, J.R. Hanrahan, M. Chebib, Microwave-enhanced
synthesis of 2,3,6-trisubstituted pyridazines: application to four-step syn-
thesis of gabazine (SR-95531), Org. Biomol. Chem. 8 (2010) 4131e4136.
[48] K. Brejc, W.J. van Dijk, R.V. Klaassen, M. Schuurmans, J. van der Oost, A.B. Smit,
T.K. Sixma, Crystal structure of an ACh-binding protein reveals the ligand-
binding domain of nicotinic receptors, Nature 411 (2001) 269e276.
[67] C.R. Hauser, C.J. Eby, The conversion of b-ketonitriles to b-ketoamides by
boron fluoride in aqueous acetic acid and by polyphosphoric acid, J. Am.
Chem. Soc. 79 (1957) 725e727.
ꢁ
[49] P.-J. Corringer, N.L. Novere, J.-P. Changeux, Nicotinic receptors at the amino
[68] V.V.V.N.S.R. Rao, B.P.V. Lingaiah, G. Ezikiel, R. Yadla, P.S. Rao, Facile synthesis of
fluorinated 2-aryl-5,7-bisalkylpyrazolopyrimidines from aryl-alkyne nitriles,
Heterocycl. Commun. 12 (2006) 275e280.
acid level, Annu. Rev. Pharmacol. Toxicol. 40 (2000) 431e458.
[50] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood,
T.A. Halgren, P.C. Sanschagrin, D.T. Mainz, Extra precision glide: docking and
scoring incorporating a model of hydrophobic enclosure for proteinꢀligand
complexes, J. Med. Chem. 49 (2006) 6177e6196.
[51] P.T.F. Williamson, A. Verhoeven, K.W. Miller, B.H. Meier, A. Watts, The
conformation of acetylcholine at its target site in the membrane-embedded
nicotinic acetylcholine receptor, Proc. Natl. Acad. Sci. U. S. A. 104 (2007)
18031e18036.
ꢀ
[69] S. Sengmany, A. Vitu-Thiebaud, E. Le Gall, S. Condon, E. Leonel, C. Thobie-
Gautier, M. Pipelier, J. Lebreton, D. Dubreuil, An electrochemical nickel-
catalyzed arylation of 3-amino-6-chloropyridazines, J. Org. Chem. 78 (2012)
370e379.
[70] Y. Rival, R. Hoffmann, B. Didier, V. Rybaltchenko, J.-J. Bourguignon,
C.G. Wermuth, 5-HT3 antagonists derived from aminopyridazine-type
muscarinic M1 agonists, J. Med. Chem. 41 (1998) 311e317.
[52] C. Beinat, S.D. Banister, C.S.P. McErlean, M. Kassiou, A practical synthesis of
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane, Tetrahedron Lett. 54 (2013)
5345e5347.
[71] T.D. Quach, R.A. Batey, Ligand- and base-free copper(II)-catalyzed CꢀN bond
formation: cross-coupling reactions of organoboron compounds with
aliphatic amines and anilines, Org. Lett. 5 (2003) 4397e4400.
[53] Y. Xiao, K.J. Kellar, The comparative pharmacology and up-regulation of rat
neuronal nicotinic receptor subtype binding sites stably expressed in trans-
fected mammalian cells, J. Pharmacol. Exp. Ther. 310 (2004) 98e107.
[54] Y. Xiao, M. Baydyuk, H. Wang, H.E. Davis, K.J. Kellar, Pharmacology of the
agonist binding sites of rat neuronal nicotinic receptor subtypes expressed in
HEK 293 cells, Bioorg. Med. Chem. Lett. 14 (2004) 1845e1848.
[55] Y. Xiao, G.R. Abdrakhmanova, M. Baydyuk, S. Hernandez, K.J. Kellar, Rat
neuronal nicotinic acetylcholine receptors containing [alpha]7 subunit:
pharmacological properties of ligand binding and function, Acta Pharmacol.
Sin. 30 (2009) 842e850.
[56] P.D. Leeson, B. Springthorpe, The influence of drug-like concepts on decision-
making in medicinal chemistry, Nat. Rev. Drug Discov. 6 (2007) 881e890.
[57] H. van de Waterbeemd, D.A. Smith, K. Beaumont, D.K. Walker, Property-based
design: optimization of drug absorption and pharmacokinetics, J. Med. Chem.
44 (2001) 1313e1333.
[72] X.-Z. Shu, X.-F. Xia, Y.-F. Yang, K.-G. Ji, X.-Y. Liu, Y.-M. Liang, Selective func-
tionalization of sp3 CꢀH bonds adjacent to nitrogen using (Diacetoxyiodo)
benzene (DIB), J. Org. Chem. 74 (2009) 7464e7469.
[73] J. Renaud, S.F. Bischoff, T. Buhl, P. Floersheim, B. Fournier, M. Geiser,
C. Halleux, J. Kallen, H. Keller, P. Ramage, Selective estrogen receptor modu-
lators with conformationally restricted side chains. Synthesis and structur-
eꢀactivity relationship of ER
a-selective tetrahydroisoquinoline ligands,
J. Med. Chem. 48 (2004) 364e379.
[74] L. Ackermann, R. Born, J.H. Spatz, D. Meyer, Efficient Aryle(Hetero)Aryl
coupling by activation of C-Cl and C-F bonds using nickel complexes of air-
stable phosphine oxides, Angew. Chem. Int. Ed. 44 (2005) 7216e7219.
[75] N. Marion, O. Navarro, J. Mei, E.D. Stevens, N.M. Scott, S.P. Nolan, Modified
(NHC)Pd(allyl)Cl (NHC¼N-Heterocyclic carbene) complexes for room-
temperature SuzukiꢀMiyaura and BuchwaldꢀHartwig reactions, J. Am.
Chem. Soc. 128 (2006) 4101e4111.
[58] C.A. Lipinski, Lead- and drug-like compounds: the rule-of-five revolution,
Drug Discov. Today Technol. 1 (2004) 337e341.
[76] Y. Xiao, E.L. Meyer, J.M. Thompson, A. Surin, J. Wroblewski, K.J. Kellar, Rat
a3/
b4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a
[59] A.R. Leach, M.M. Hann, J.N. Burrows, E.J. Griffen, Fragment screening: an
introduction, Mol. Biosyst. 2 (2006) 429e446.
transfected cell line: pharmacology of ligand binding and function, Mol.
Pharmacol. 54 (1998) 322e333.