Synthesis, Structure-Activity Relationships, and Preclinical Evaluation of Heteroaromatic Amides and 1,3,4-Oxadiazole Derivatives as 5-HT4 Receptor Partial Agonists

Article abstract of DOI:10.1021/acs.jmedchem.8b00457

Alzheimer's disease (AD) is a neurodegenerative disorder that has a higher prevalence and incidence in people older than 60 years. The need for improved AD therapies is unmet as the current therapies are symptomatic with modest efficacy. Partial agonists of the 5-HT₄ receptor (5-HT₄R) offer both symptomatic and disease-modifying treatments as they shift amyloid-precursor-protein (APP) processing from the amyloidogenic pathway to the nonamyloidogenic pathway by activating the α-secretase enzyme. In addition, they also offer symptomatic treatment by increasing levels of the neurotransmitter acetylcholine in the brain. Because of this fascinating dual mechanism of action, several chemical scaffolds having 5-HT₄R pharmacophores were designed and evaluated. Most of the synthesized compounds showed potent in vitro affinities and in vivo efficacies. Upon analysis of focused structure-activity relationships, compound 4o was identified as a potent 5-HT₄R partial agonist with favorable ADME properties and good in vivo efficacy. GR-125487, a selective 5-HT₄R antagonist, attenuated the activity of compound 4o in the novel-object-recognition-test cognition model.

Full text of DOI:10.1021/acs.jmedchem.8b00457

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Article
Synthesis, Structure Activity Relationships and Preclinical
evaluation of Heteroaromatic Amides and 1,3,4-
Oxadiazole Derivatives as 5-HT4 Receptor Partial Agonists
Ramakrishna Nirogi, Abdul Rasheed Mohammed, Anil K. Shinde, Shankar Reddy Gagginapally,
Durga Malleshwari Kancharla, Vanaja Reddy Middekadi, Narsimha Bogaraju, Srinivasa
Rao Ravella, Pooja Singh, Sumit Raosaheb Birangal, Ramkumar Subramanian, Raghava
Choudary Palacharla, Vijay Benade, Nageswararao Muddana, and Pradeep Jayarajan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00457 • Publication Date (Web): 15 May 2018
Downloaded from http://pubs.acs.org on May 15, 2018
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Synthesis, Structure Activity Relationships and
Preclinical evaluation of Heteroaromatic Amides
and 1,3,4ꢀOxadiazole Derivatives as 5ꢀHT4
Receptor Partial Agonists
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Ramakrishna Nirogi,* Abdul Rasheed Mohammed, Anil K. Shinde, Shankar Reddy
Gagginapally, Durga Malleshwari Kancharla, Vanaja Reddy Middekadi, Narsimha Bogaraju,
Srinivasa Rao Ravella, Pooja Singh, Sumit Raosaheb Birangal, Ramkumar Subramanian,
Raghava Choudary Palacharla, Vijay Benade, Nageswararao Muddana, Pradeep Jayarajan.
Suven Life Sciences Limited, Discovery Research, Serene Chambers, Roadꢀ5, Avenueꢀ7,
Banjara Hills, Hyderabad ꢀ 500 034, India
KEYWORDS. 5ꢀHT receptor, Structure Activity Relationships, Pharmacokinetic profile,
4
Receptor occupancy, Cognitive deficits, Alzheimer’s disease, GRꢀ125487
ABSTRACT. Alzheimer's disease (AD) is a neurodegenerative disorder which has a higher
prevalence and incidence in people older than 60 years. The need for improved AD therapies is
unmet as the current therapies are symptomatic with modest efficacy. The 5ꢀHT receptor (5ꢀ
4
HT R) partial agonists offer both symptomatic and disease modifying treatment as they shift
4
amyloid precursor protein (APP) processing from amyloidogenic to nonꢀamyloidogenic pathway
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by activating αꢀsecretase enzyme. In addition, they also offer symptomatic treatment by
increasing neurotransmitter acetylcholine levels in the brain. Fascinated by dual mechanism of
action, several chemical scaffolds having 5ꢀHT R pharmacophore were designed and evaluated.
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Most of the synthesized compounds showed potent in vitro affinities and in vivo efficacies. Upon
focused structure activity relationships, compound 4o was identified as potent 5ꢀHT R partial
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agonist with favorable ADME properties and good in vivo efficacy. GRꢀ125487, a selective 5ꢀ
HT R antagonist attenuated the activity of compound 4o in cognition model NORT.
4
INTRODUCTION
AD is a progressively debilitating neurodegenerative disorder, affecting mostly the aging
population. Although the underlying causative factors are yet to be identified, symptoms include
confusion, memory loss, and dementia, ultimately leading to death. The current pharmacotherapy
provides only symptomatic relief through augmentation of cholinergic function with undesired
side effects such as nausea, vomiting and bradycardia. Moreover, no disease modifying therapy
is available to the patients till date. Thus, there is a need of new therapeutic targets for the
treatment of this disorder and 5ꢀHT R is one such receptor which has attracted lot of attention.
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The 5ꢀHT R belongs to the superfamily of seven transꢀmembrane Gꢀprotein coupled receptors
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(
GPCRs) and is coupled to G protein containing Gαs subunit. The 5ꢀHT R is reported to have
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potential role in many central nervous system (CNS) such as AD and peripherally mediated
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disorders such as irritable bowel syndrome and gastroparesis. The 5ꢀHT receptors are highly
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expressed in brain regions like hippocampus, amygdala and cerebral cortex suggesting the
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involvement of receptor in cognitive processes. The 5ꢀHT R agonists modulate amyloid
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precursor protein (APP) derived peptides, amyloid beta (Aβ) and soluble amyloid precursor
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protein alpha (sAPPα).
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The sAPPα is nonꢀamyloidogenic protein and it is reported to have potent neuroprotective role
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against neurotoxic effects of glutamate and βꢀamyloid. Human 5ꢀHT R isoforms are positively
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coupled to adenylyl cyclase production. Thus, activation of this Gꢀprotein coupled receptor
activates adenylyl cyclase consequently increasing the production of cAMP. The increased
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cAMP in turn increases sAPPα release. The stimulatory effect of 5ꢀHT R receptor on sAPPα
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release is mimicked by forskolin, a direct activator of adenylyl cyclase, and 8ꢀbromoꢀcAMP
(membrane permeant cAMP analogue) suggesting the involvement of adenylyl cyclase and
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cAMP in modulating sAPPα by 5ꢀHT R. The 5ꢀHT R also increases the neuronal acetylcholine
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(
ACh) levels in the brain. Thus, 5ꢀHT R partial agonists may be of use for both diseaseꢀ
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modifying and symptomatic treatment of cognitive disorders. The 5ꢀHT R owing to its dual
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mechanism of action to treat AD and other cognition related diseases became an attractive target
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ꢀ13
for drug discovery. Several structurally diverse heteroaromatic derivatives
as 5ꢀHT R
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agonists/partial agonists have been explored for both CNS and peripheral indications. The 5ꢀ
HT R partial agonist PRXꢀ03140 showed significant improvement of ADASꢀcog score in Phaseꢀ
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a study for AD.
Apart from this compound, several other compounds were in development
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such as BIMUꢀ1, RSꢀ67333, prucalopride and PFꢀ04995274 among others (Figure 1). In our
continuous efforts in discovering novel small molecules to treat AD with diverse mechanism of
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actions,
we shifted our attention on 5ꢀHT R agonists partly due to their dual mechanism of
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action and partly due to availability of proof of concept in the form of PRXꢀ03140 phaseꢀ2
positive results. Our internally discovered clinical candidate SUVNꢀD4010, a 5ꢀHT R partial
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agonist has completed Phaseꢀ1 clinical trials in healthy subjects and also completed Phaseꢀ2
enabling long term safety studies. The structure of this compound has not been disclosed so far.
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Most of the reported 5ꢀHT R ligands, which have been explored for both CNS and peripheral
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indications belong to either benzamide or imidazole carboxamide scaffold. Previous work from
our group disclosed a series of 3ꢀisopropylimidazo[1,5ꢀa]pyridine carboxamide derivatives (5a,
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Seriesꢀ1) as 5ꢀHT R partial agonists which displayed cognition enhancing properties in animal
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models. However, the brain penetration of these compounds was poor (Cbrain/Cplasma = <0.3). As
we are targeting the CNS receptor, low brain penetration in rat makes this scaffold, unsuitable
for further development. Our goal was to identify a potent 5ꢀHT R agonist with adequate brain
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penetration, acceptable ADME and preclinical efficacy as a possible drug candidate for the
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treatment of AD. The Seriesꢀ1 compounds were modified by keeping the basic pharmacophore
and the isopropyl substituent unchanged (Figure 2) to obtain several diverse chemical scaffolds.
The 5ꢀHT R pharmacophore consists of an aromatic moiety, a coplanar carbonyl group or its
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bioisostere, and a voluminous substituent in the basic amino framework of the molecule. ,
RESULTS AND DISCUSSION
Successive modifications in the position of the nitrogen atom, position of isopropyl group,
position of carboxamide group in heteroaromatic ring resulted in novel 1ꢀisopropylimidazo[1,5ꢀ
a]pyridine carboxamide derivatives (Seriesꢀ2), 2ꢀisopropylimidazo[1,2ꢀa]pyridine carboxamide
derivatives (Seriesꢀ3), 2ꢀisopropylpyrazolo[1,5ꢀa]pyridine carboxamide derivatives (Seriesꢀ4).
The replacement of carboxamide group in each of the series with amide bioisostere resulted in
1
,3,4ꢀoxadiazole derivatives. The results of these modifications covering in vitro binding
affinity, pharmacokinetic, hERG affinity, receptor occupancy and in vivo efficacy profiling
constitute the subject matter of this manuscript.
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Chemistry: The synthesis of heteroaromatic carboxylic acid A1 commenced from
commercially available 2ꢀbromopyridine 1 which was treated with nꢀBuLi followed by
isobutyraldehyde to obtain compound 2. The hydroxyl group in compound 2 was converted into
a leaving group by mesylation and then was displaced with sodium azide to obtain azido
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compound 3. The azide group in compound 3 was reduced with triphenylphosphine/H O
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combination (Staudinger reaction) which afforded amino compound 4. The amino compound 4
was treated with ethyl chlorooxoacetate to obtain intermediate 5 which on cyclization with
POCl in 1,2ꢀdichloroethane afforded imidazo[1,5ꢀa]pyridine ethyl ester 6. The ester 6 was
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hydrolyzed with NaOH and thus obtained sodium salt upon acidification yielded 1ꢀisopropylꢀ
imidazo[1,5ꢀa]pyridineꢀ3ꢀcarboxylic acid A (Scheme 1). The 2ꢀisopropylimidazo[1,2ꢀa]pyridine
1
carboxylic acid A was synthesized starting from commercially available ethyl 2ꢀaminonicotinate
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7, which on refluxing with 1ꢀchloroꢀ3ꢀmethylꢀbutanꢀ2ꢀone in ethanol yielded imidazo[1,2ꢀ
a]pyridine ester derivative 8 which after hydrolysis afforded the corresponding acid A (Scheme
2
2
2
). Synthesis of 2ꢀisopropylpyrazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylic acid A started from reaction of
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ꢀpicoline 9 with isobutyryl chloride which afforded intermediate 10. The intermediate 10 on
cyclization with Oꢀmesitylenesulfonylhydroxylamine, afforded 2ꢀisopropylpyrazolo[1,5ꢀ
a]pyridine 11 which on reaction with nꢀBuLi followed by carboxylation with carbon dioxide (dry
ice) and acidification yielded acid A (Scheme 3).
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The ethyl isonipecotate 12 was alkylated, either by reacting with alkyl halides or by reductive
amination with aldehydes to obtain compounds 13a, 13b and 13c. Compounds 13a, 13b and 13c
on ester hydrolysis with lithium hydroxide afforded lithium carboxylates 14a, 14b and 14c
respectively (Scheme 4). The acid hydrazides 15, 16 and 18 were synthesized from
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corresponding esters 6, 8 and 17. The ester 17 was obtained from acid A upon treatment with
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dry HCl in methanol (Scheme 5).
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The acids A A or A were reacted with earlier reported NꢀBoc substituted piperidine B or
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ꢀazaꢀbicyclo[3.1.0]hexane amines B respectively in presence of TBTU or HATU to obtain the
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Boc protected carboxamide derivatives 19a, 19b, 21a, 21b, 23a, 23b which on Boc deprotection
with dry HCl in isopropanol afforded compounds 20a, 20b, 22a, 22b, 24a, 24b respectively.
These compounds upon Nꢀalkylation, either by reacting with alkyl halides or reductive amination
with aldehydes afforded 1ꢀisopropylimidazo[1,5ꢀa]pyridine carboxamides 2aꢀ2g or 2ꢀ
isopropylimidazo[1,2ꢀa]pyridine carboxamides 3aꢀ3q or 2ꢀisopropylpyrazolo[1,5ꢀa]pyridineꢀ7ꢀ
carboxamides 4aꢀ4l respectively (Scheme 6). The 1,3,4ꢀoxadiazole derivatives 2hꢀ2j, 3rꢀ3s, 4mꢀ
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o were synthesized by reacting hydrazides 15, 16 or 18 with substituted lithium carboxylates
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4a, 14b or 14c in presence of POCl (Scheme 7).
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The isopropylimidazo[1,5ꢀa]pyridine carboxamide derivatives 2aꢀ2e with different
substitutions on piperidine (B ) nitrogen showed in vitro potency EC <100 nM towards 5ꢀHT R
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with partial agonist activity (Emax<100%, Table 1). Compounds with bulkier substitutions such as
methylpiperidine (2c) and methylpyran (2d) on piperidine nitrogen showed increased potency
(
EC <10 nM) as compared to simple substitution bearing compounds such as isopropyl (2a)
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and methoxypropyl (2b). Similarly, compounds with Nꢀsubstituted 3ꢀazaꢀbicyclo[3.1.0]hexane
ring (B ) showed increased potency at 5ꢀHT R as compared to simple piperidine (B )
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counterparts. Compounds with methoxypropyl substitution (2f) and pyran substitution (2g) on
nitrogen of 3ꢀazaꢀbicyclo[3.1.0]hexane showed increased potency as compared to the piperidine
counterparts 2b and 2e respectively.
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The development and application of bioisosteres have been adopted as a fundamental tactical
approach useful to address a number of aspects such as improving potency, enhancing
selectivity, altering physical property, reducing or redirecting metabolism, eliminating or
modifying toxicophores or acquiring novel intellectual property associated with drug
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candidates.
The 1,3,4ꢀoxadiazoles derivatives 2hꢀ2j as carboxamide bioisosteres were
synthesized and evaluated. However, these compounds showed weak in vitro potency at 5ꢀHT R.
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In brief, the carboxamide derivatives showed moderate in vitro potency and 1,3,4ꢀoxadiazole
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derivatives showed weak potency towards 5ꢀHT R. Compounds from this series were evaluated
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for their metabolic stability in rat and human microsomes. These compounds showed moderate
to good microsomal metabolic stability. The piperidine analogues having methoxypropyl group
2
b, tetrahydropyran group 2e and 3ꢀazaꢀbicyclo[3.1.0]hexane analogue having methoxypropyl
group 2f, pyran group 2g showed higher metabolic stability. Compounds 2b and 2g, which
showed good metabolic stability, were evaluated in rat pharmacokinetics (PK) studies. Both the
compounds showed poor oral bioavailability suggesting lack of correlation between microsomal
stability and oral exposures in rats. The brain penetration (C /C ) however had substantially
b
p
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improved (1.8 to 2) as against earlier reported Series 1 compound 5a (0.31). The 1,3,4ꢀ
oxadiazole derivatives were not evaluated in PK experiments owing to their poor in vitro
potency.
Given the improvement in brain penetration in Series 2 compounds, the scope of SAR was
further expanded by synthesizing and evaluating a series of isopropylimidazo[1,2ꢀa]pyridine
derivatives (Seriesꢀ3 , Table 2). Most of the piperidine ring (B ) compounds (3aꢀ3h) showed
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potent in vitro potency at 5ꢀHT R (EC <10 nM). Compounds with Nꢀsubstituted 3ꢀazaꢀ
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bicyclo[3.1.0]hexane rings (3iꢀ3m) however were 2ꢀ10 fold less potent (Compounds 3a vs 3i; 3b
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vs 3j). During the course of this research work, a publication from Pfizer reported the
formation of a cyclized oxazolidine metabolite of PFꢀ04995274 which was reported as unique to
human and was not observed in animal studies. Given the partial structural similarity i.e., tertꢀ
hydroxyl group on piperidine Nꢀsubstituents of compounds 3f, 3g and 3h to PFꢀ04995274,
theoretically these compounds may also have potential to form such unique metabolites. Though
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these compounds showed potent in vitro activity at 5ꢀHT R, no additional explorations were
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carried out. The rat pharmacokinetic studies of compounds 3b and 3c showed high clearance,
poor exposures and poor bioavailability, an observation akin to Series 2 compounds. Compound
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c based on its high in vitro potency and high brain penetration, was evaluated in the longꢀterm
memory deficit assay the Novel Object Recognition Test (NORT) to see the translation of in
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vitro activity into in vivo efficacy. The NORT
has been regarded as a spontaneous Delayedꢀ
NonꢀMatchingꢀtoꢀSample (DNMS) test. The test is based on a spontaneous behavior. The main
assumption at the base of this test is that access to novelty (object or environment) can elicit
approach behaviors in animals. NORT doesn’t involve reference memory components, thus it
can be considered a “pure” recognition memory test and a valid task to assess working memory.
Also the test doesn’t involve positive or negative reinforces (food or electric shocks) and this
makes NORT comparable to memory tests currently used in humans. These advantages make
NORT a quick and simple to be implemented and, therefore, it has been widely used for
assessing mild cognitive impairment in preꢀclinical research. This apparent ‘unconditioned
preference’ for novelty has been used in the NORT in order to study memory functions,
assessing the ability of animals to recognize a novel object in a familiar environment. Although
this model has face and predictive validity for memory deficits, the percentage of translational
success from rat to human is poor till date. In this experiment, the rats treated with 3c at 0.3 and
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mg/kg, p.o. spent significantly more time exploring the novel object than the familiar object in
the test phase, i.e., the rats remembered the familiar object even after 24 h time gap. However,
the vehicle treated rats spent almost equal time in exploring the novel object as well as the
familiar object forgetting the familiar object after 24 h time gap (Figure 3). Encouraged by these
results, further modification of the chemical scaffold was done with the aim to obtain compounds
with good oral bioavailability. Chloro substitution was brought in the 3ꢀposition of imidazo[1,2ꢀ
a]pyridine to obtain compounds 3n and 3o and carbon (Cꢀ3) was replaced with nitrogen atom to
obtain triazolo compounds 3p and 3q. However, these modifications resulted in very weak
potency (3q) or complete loss of potency (3nꢀ3p). However, the microsomal metabolic stability
of these compounds has increased substantially. We hypothesized that this improvement in
microsomal metabolic stability is probably due to blocking of the high electron density 3ꢀ
position of imidazo[1,2ꢀa]pyridine. However, the 1,3,4ꢀoxadiazole derivatives, 3r and 3s
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showed weak affinity towards 5ꢀHT R with favorable metabolic stability.
4
As attempts to improve the metabolic stability by structural modifications resulted in less
potent or inactive compounds, further SAR was carried out by synthesizing pyrazolo[1,5ꢀ
a]pyridine derivatives (Series 4, Table 3). As it was evident from the results shown in Table 3,
the compounds having piperidine (B ) with Nꢀsubstitutions like methoxypropyl (4a),
1
pyranylmethyl (4b) groups were more potent as compared to isopropyl group (4c). The
corresponding Nꢀsubstituted 3ꢀazaꢀbicyclo[3.1.0]hexane derivatives (4dꢀ4f) were less potent,
similar to Series 2 and Series 3 compounds. Most of the evaluated compounds were extensively
metabolized both in rat and human liver microsomes. Based on the availability of resources,
compounds 4b and 4c have been explored in pharmacokinetics (PK) studies. Both the
compounds showed poor oral bioavailability while 4c showed poor brain penetration as well. In
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an attempt to increase the microsomal metabolic stability, 3ꢀchloroꢀpyrazolopyridine derivatives
(4gꢀ4i) and triazolopyridine derivatives (4jꢀ4l) were synthesized. However, these compounds did
not show any activity towards 5ꢀHT R. In line with earlier observations, the metabolic stability
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of 3ꢀchloroꢀpyrazolopyridine derivatives 4gꢀ4i was moderately improved whereas the metabolic
stability of triazolopyridine derivatives 4kꢀ4l was increased substantially. The 1,3,4ꢀoxadiazole
derivatives (4mꢀ4o) however, showed potent in vitro activity and also showed improved
metabolic stability. Compounds 4m, 4n and 4o showed good oral bioavailability; Compounds 4n
and 4o showed improved brain penetration. However, the Nꢀisopropyl substituted derivative 4m
showed poor brain penetration (Table 3). Compounds 4n and 4o were evaluated in efficacy
models of cognition. In NORT, compound 4o showed robust efficacy at 3 and 10 mg p.o.
whereas, compound 4n showed efficacy at 1 and 10 mg p.o. (Figure 3). The reference
1
7
compound RSꢀ67333 a 5ꢀHT R partial agonist showed efficacy at 0.3 and 1.0 mg/kg i.p. In a
4
separate experiment, the observed activity of 4o in NORT was blocked by administering the
31,32
selective 5ꢀHT R antagonist GRꢀ125487
giving ample evidence that the efficacy observed
4
may be arising from its agonistic activity at 5ꢀHT R (Figure 4).
4
In order to further substantiate the finding that the observed efficacy is arising from 5ꢀHT R
4
3
3
agonism, compounds 4n and 4o were evaluated for their receptor occupancy (R.O.). Both the
compounds showed significant R.O. showing absolute ED values of 8.47 and 6.15 mg/kg, p.o.,
5
0
respectively (Figure 5) suggesting the in vivo efficacy observed for these compounds may be
mediated through 5ꢀHT R. The partial agonist from Pfizer, PFꢀ04995274 showed dose dependent
4
R.O. with ED value of 0.017 mg/kg, s.c. which is comparable with the reported value (0.008
5
0
3
4
mg/kg, s.c.).
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The protein binding experiment was done in rat, dog and human liver microsomes to ascertain
the free fraction available for 4n and 4o. Compound 4n showed 26% free fraction in rat plasma
and 14% in brain homogenate whereas compound 4o showed 17% free fraction in rat plasma and
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4% in brain homogenate. The percentage free fraction for both compounds in plasma was
similar in other species like dog and human. Both 4n and 4o showed minimal inhibition of
cytochrome P450 3A4 and 2D6 enzymes and showed absolute IC50 values of >10 µM.
Compound 4o based on its favorable in vitro affinity, higher oral exposures, dose dependent
R.O., robust efficacy in NORT and significant free fraction in brain was selected for further
evaluation in microdialysis studies. It is known in the literature that 5ꢀHT R agonists improve
4
memory formation by enhancing the synaptic release of acetylcholine (the neurotransmitter
responsible for cognition process) in the brain which would in turn enhance the cholinergic
8
transmission. The microdialysis experiments showed that compound 4o increased acetylcholine
levels doseꢀdependently in frontal cortex upon oral dosing at 3 and 10 mg/kg (Figure 6). The 5ꢀ
HT R partial agonist PRXꢀ03140 was used as a positive control. At 1 mg/kg, i.p. PRXꢀ03140
4
produced about 254% increase from preꢀdose levels. The increase in acetylcholine levels by
3
5
PRXꢀ03140 was in agreement with the literature reports.
The 5ꢀHT R agonists have the ability to shift the equilibrium of amyloid precursor protein
4
(APP) processing from amyloidogenic to nonꢀamyloidogenic form showing disease modifying
3
6
potential. The APP processing shifts equilibrium from amyloidogenic to nonꢀamyloidogenic
7
,37,38
form by generating sAPPα which is reported to provide neuroprotective effect.
In this
experiment, 5ꢀHT R agonist prucalopride was used as a positive control. Treatment with
4
prucalopride (10 mg/kg, s.c.) produced 65% increase above control levels. These results are in
3
7
agreement with the literature. Increase in sAPPα levels produced by compound 4o (1ꢀ10
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mg/kg, s.c.) were higher than the effects showed by prucalopride (10 mg/kg, s.c.). The increase
in cortical sAPPα levels in mice demonstrated that compound 4o may have disease modifying
potential (Figure 7).
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Compounds 4n and 4o showed comparable potencies at 5ꢀHT R isoforms (5ꢀHT D, 5ꢀHT E
4
4
4
and 5ꢀHT L) with EC values ranging from 6 to 62 nM. Compounds 4n and 4o showed weak
4
50
propensity to inhibit hERG channel in electrophysiology patch clamp assay and the IC was
5
0
found to be 2.3 µM with 76% Imax and 1.9 µM with 100% Imax at 10 µM concentration
respectively. Compounds 4n and 4o showed excellent selectivity against 5ꢀHT receptors (5ꢀ
HT , 5ꢀHT , 5ꢀHT , 5ꢀHT ), Adrenergic receptors (Adα , Adα ), Adenosine receptor A ,
1
A
2A
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1B
2C
2A
Cannabinoid receptor (CB and CB ), Dopamine (D , D , D and D ), Histamine (H and H ),
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2
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2S
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5
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Muscarinic receptors (M , M , M , M and M ) and serotonin transporter. Both the compounds
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4
5
did not show any significant binding at highest tested concentration of 10 µM towards GPCRs,
ion channels and transporters (See Supporting Information). Compound 4o showed partial
39
agonist activity at 5ꢀHT B receptor when evaluated in rat fundus assay (See Supporting
2
Information). Based on the panel of selectivity assessment involving majority of GPCRs which
are responsible for cognition, we hypothesized that the compound 4o selectively modulates 5ꢀ
HT R. Further evaluation of compound 4o in additional efficacy models, receptor mapping and
4
extended safety will be done in due course of time.
CONCLUSIONS
In summary, starting from a poorly brain penetrant 5a, effecting successive modifications in
the position of nitrogen atom, position of isopropyl group, position of carboxamide group in
heteroaromatic ring resulted in identification of a series of pyrazolo[1,5ꢀa]pyridine derivatives
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represented by compound 4o. The lead compound 4o is a potent and selective 5ꢀHT R partial
4
agonist with high oral exposures and good brain penetration. In vivo R.O. studies demonstrated a
clear relationship between the in vitro affinity, free brain exposure, in vivo efficacy and R.O. .
We have also demonstrated in this report that the compounds 3c, 4n and 4o improved longꢀterm
memory deficits in NORT, a finding that is consistent with previous reports. The attenuation of
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compound 4o efficacy in NORT by selective 5ꢀHT R antagonist GRꢀ125487 and dose dependent
4
5
ꢀHT R.O. provides ample evidence that the cognitive effects of compound 4o was mediated
4
through 5ꢀHT R. Compound 4o increased neurotransmitter ACh release in prefrontal cortex, a
4
finding that is consistent with previous reports. Compound 4o increased neuroprotective sAPPα
levels thus offering disease modifying therapy. A safety assessment of this compound is in
progress to ascertain the therapeutic margins between efficacious concentrations and safety
findings.
EXPERIMENTAL SECTION
Unless stated otherwise, all reagents and solvents were purchased from common commercial
1
suppliers and were used without further purification. HꢀNMR spectra were recorded at 400 MHz
1
3
and CꢀNMR at 100 MHz on a Bruker NMR spectrometer instrument (Fallanden, Switzerland).
1
All H NMR shifts are reported in δ units (ppm) relative to the signals for chloroform (7.27
ppm), DMSO (2.50 ppm), and MeOH (3.31 ppm). All coupling constants (J values) are reported
in hertz (Hz). NMR abbreviations are as follows: bs, broadened singlet; s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet and dd, doublet of doublets. Thin layer chromatography (TLC)
was performed on Merck silica gel 60 F254 plates. Electrospray ionization mass spectra were
recorded on API 4000 triple quadrupole instrument (MDSꢀSCIEX, Concord, Ontario, Canada).
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Infrared spectra were recorded on KBr disc and in solid state using PerkinꢀElmer model 1600
FTꢀIR spectrophotometer (PerkinꢀElmer, Norwalk, CT, USA). Elemental analyses were carried
out in an ‘Elementar’ GmbH, Vario micro cube instrument and the ‘analyses indicated by the
symbols of the elements were within ± 0.4 % of the theoretical values’. Column chromatography
was performed using 100ꢀ200 mesh silica gel. HPLC purity of the final compounds was done
using Agilant systems (Modelꢀ1100 series). DSC was recorded on Waters DSC Q100 instrument.
Melting points of synthesized compounds were determined using Electro Derman open capillary
apparatus and are uncorrected. The reference standards PRXꢀ03140, RSꢀ67333, PFꢀ04995274,
GRꢀ125487 prucalopride and others were synthesized and characterized inꢀhouse using
respective reported procedures. All final compounds were determined to have a purity of >95%
by one of the aforementioned methods unless stated otherwise.
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Synthesis of 2ꢀMethylꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ1ꢀpropanol 2: To a stirred solution of 2ꢀbromopyridine,
1 (13 g, 82.3 mmol) in dry THF (150 mL) cooled at −78 °C, nꢀBuLi (45.0 mL, 1.6 M in hexane)
was added dropꢀwise over a period of 30 minutes. Isobutyraldehyde (15.0 mL, 153.0 mmol) was
added over a period of 15 minutes and after stirring the reaction mixture for 15 minutes at this
temperature, it was gradually warmed to r.t. and was stirred for 2 h. The reaction mixture was
then cooled to 0 °C and was quenched by adding ice water followed by addition of EtOAc. The
two layers were separated and the aqueous layer was extracted again with EtOAc. The combined
organic layer was dried over anhydrous Na SO and the solvent was removed under reduced
2
4
pressure to afford crude product which was purified by silica gel column chromatography to
1
obtain 6.1 g of the above titled compound 2 in 47% yield. H ꢀ NMR (400 MHz, CDCl ): δ 8.55
3
(d, J = 4.7 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.19 (d, J = 2.2 Hz, 2H), 4.19 (d, J = 5.8 Hz, 1H),
+
2
.15ꢀ2.05 (m, 1H), 1.02 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.4 Hz, 3H); Mass (m/z): 152.1 (M+H) .
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Synthesis of 2ꢀMethylꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ1ꢀazidopropane 3: To a stirred solution of compound 2
(5.5 g, 36.3 mmol), triethylamine (10.0 mL, 72.6 mmol) in dry DCM (150.0 mL) cooled at 0 °C
was added methanesulfonyl chloride (3.0 mL, 36.3 mmol) over a period of 15 minutes. After
stirring the reaction mixture for another 30 minutes, it was diluted with water (50.0 mL), DCM
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(50 mL) and the two layers were separated. The aqueous layer was further extracted with DCM
(2x50 mL). The combined organic layer was washed with water (150 mL), brine (150 mL), dried
over anhydrous Na SO and the solvent was removed under reduced pressure to obtain the
2
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mesylated intermediate which was dissolved in DMF (66.0 mL) and K CO (10.0 g, 72.6 mmol)
2
3
followed by NaN (3.5 g, 57.3 mmol) were added. The reaction temperature was gradually raised
3
to 80 °C and the reaction mass was maintained at this temperature for 2 h before bringing it to r.t.
The reaction mixture was diluted with water (100 mL) and EtOAc (150 mL) and the two layers
were separated. The organic layer was further washed with water followed by brine solution,
dried over anhydrous Na SO and the solvent was removed under reduced pressure to obtain the
2
4
1
titled azido 2ꢀmethylꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ1ꢀazidopropane 3 (6.0 g) in 99% yield. H ꢀ NMR (400
MHz, CDCl ): δ 8.60 (d, J = 4.4 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.24
3
(dd, J = 4.4, 7.8 Hz, 1H), 4.26 (d, J = 7.3 Hz, 1H), 2.30ꢀ2.10 (m, 1H), 1.0 (d, J = 6.6 Hz, 3H),
+
0
.88 (d, J = 6.7 Hz, 3H); Mass (m/z): 177.2 (M+H) .
Synthesis of 2ꢀMethylꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ1ꢀaminopropane 4: To a stirred solution of compound
3 (6.0 g, 36.3 mmol) in THF (68.0 mL) at r.t., triphenylphosphine (11.0 g, 44.6 mmol) followed
by water (3.1 mL, 185.0 mmol) was added. After 16 hours stirring at r.t., the volatiles were
removed under reduced pressure and the crude reaction mixture was purified by silica gel
1
column chromatography to obtain compound 4 (3.83 g) in 70% yield. H ꢀ NMR (400 MHz,
CDCl ): δ 8.55 (d, J = 2.4 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.26 (d, J = 1.8 Hz, 2H), 7.13 (t, J =
3
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.0 Hz, 1H), 3.69 (d, J = 1.2 Hz, 1H), 2.07ꢀ2.01 (m, 1H), 0.95 (d, J = 1.6 Hz, 3H), 0.79 (d, J =
+
.5 Hz, 3H); Mass (m/z): 151.1 (M+H) .
Synthesis of Ethyl Nꢀ[2ꢀmethylꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀpropyl]ꢀoxalamate 5: To a stirred solution of
compound 4 (3.8 g, 25.3 mmol) and triethylamine (10.0 mL, 50.6 mmol) in solvent DCM (50
mL) cooled at 0 °C, ethyl chlorooxoacetate (3.0 mL, 25.3 mmol) was added over a period of 10
minutes. After stirring the reaction mixture at this temperature for 1h, it was diluted with DCM
(50 mL), washed with water (50 mL), dried over anhydrous Na SO and the solvent was
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removed under reduced pressure to obtain compound 5 (5.52 g) in 87% yield. H ꢀ NMR (400
MHz, CDCl ): δ 8.57 (d, J = 3.9 Hz, 1H), 8.28 (d, J = 7.2 Hz, 1H), 7.68 (t, J = 12.2 Hz, 1H),
3
7
0
.19 (d, J = 7.4 Hz, 2H), 4.88 (t, J = 7.2 Hz, 1H), 4.35 (q, 2H), 2.23ꢀ2.15 (m, 1H), 1.37 (t, 3H),
+
.95 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H); Mass (m/z): 251.1 (M+H) .
Synthesis of Ethyl 1ꢀisopropyl imidazo[1,5ꢀa]pyridineꢀ3ꢀcarboxylate 6: To a stirred solution
of compound 5 (5.5 g, 22.0 mmol) in 1,2ꢀdichloroethane (50.0 mL) at r.t., POCl (20.0 mL) was
3
added dropꢀwise over a period of 15 minutes. The reaction mixture was then heated to 130 °C for
7
2 h. The volatiles were removed under reduced pressure to obtain a crude mass which was
neutralized with NaHCO solution and extracted with EtOAc. The combined organic layer was
3
dried over anhydrous Na SO and the solvent was removed under reduced pressure. The crude
2
4
product was purified by silica gel column chromatography to obtain compound 6 (3.58 g) in 70%
1
yield. H ꢀ NMR (400 MHz, CDCl ): δ 9.32 (d, J = 7.1 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.00
3
(t, J = 6.6 Hz, 1H), 6.88 (t, J = 6.7 Hz, 1H), 4.58ꢀ4.50 (m, 2H), 3.47ꢀ3.39 (m, 1H), 1.43 (d, J =
+
6
.6 Hz, 6H), 1.32 (t, 3H); Mass (m/z): 233.1 (M+H) .
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Synthesis of Ethyl 2ꢀisopropylꢀimidazo[1,2ꢀa]pyridineꢀ8ꢀcarboxylate 8: To a stirred solution
of ethyl 2ꢀaminonicotinate 7 (0.8 g, 3.44 mmol) in ethanol (4.72 mL), at r.t., 1ꢀchloroꢀ3ꢀ
methylbutanꢀ2ꢀone (6.9 g, 41.2 mmol) was added. The reaction mixture was stirred at 95 °C for
1
1
1
1
1
1
1
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1
1
2
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2
2
2
2
2
2
2
2
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4
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5
5
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5
6
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
h. The solvent was removed under reduced pressure and the crude reaction mixture was
1
purified by silica gel column chromatography to obtain compound 8 (0.45 g) in 58% yield. H ꢀ
NMR (400 MHz, CDCl ): δ 8.22 (d, J = 6.6 Hz, 1H), 7.87 (d, J = 7.1 Hz, 1H), 7.40 (s, 1H), 6.79
3
(t, J = 6.9 Hz, 1H), 4.50 – 4.45 (q, 2H), 3.26–3.19 (m, 1H), 1.39 (d, J = 6.8 Hz, 6H); Mass (m/z):
+
2
33.2 (M+H) .
Synthesis of 3ꢀMethylꢀ1ꢀpyridinꢀ2ꢀylꢀ2ꢀbutanone 10: To a stirred solution of 2ꢀpicoline 9 (20.0
g, 214.7 mmol) in dry THF (860.0 mL) cooled at −78 °C, nꢀBuLi (1.6 M in hexanes, 134.2 mL,
2
14.7 mmol) was added over a period of 15 minutes. After the reaction mixture was stirred for 1
hour at this temperature, isobutyryl chloride (25.2 g, 236.2 mmol) was added at once and the
reaction mass was stirred for another 30 minutes. The reaction mass was quenched by adding
saturated ammonium chloride solution. The two layers were separated and the aqueous layer was
extracted with EtOAc. The combined organic layer was dried over anhydrous Na SO and the
2
4
volatiles were removed under reduced pressure to obtain a crude mass which was purified by
1
silica gel column chromatography to obtain the titled compound 10 (5.4 g) in 13% yield. H ꢀ
NMR (400 MHz, CDCl ): δ 8.54 (d, J = 4.3 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.23 (t, J = 7.7 Hz,
3
1
H), 7.16 (d, J = 6.7 Hz, 1H), 3.96 (s, 2H), 2.86ꢀ2.77 (m, 1H), 1.13 (d, J = 6.4 Hz, 6H); Mass
+
(
m/z): 164.33 (M+H) .
Synthesis of 2ꢀIsopropyl pyrazolo[1,5ꢀa]pyridine 11: To a stirred solution of compound 10
(3.3 g, 3.44 mmol) in CHCl (67.0 mL), cooled at 0 °C, Oꢀmesitylenesulfonylhydroxylamine
3
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2
2
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6
(
4.35 g, 20.2 mmol) was added. The reaction mass stirred at r.t. for 1 hour, upon which TLC
revealed completion of the reaction, the volatiles were removed and the crude mass was purified
by silica gel column chromatography to obtain above titled compound 11 (1.0 g) in 31% yield.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
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9
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2
3
4
5
6
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9
0
1
2
3
4
5
6
7
8
9
0
H ꢀ NMR (400 MHz, CDCl ): δ 8.38 (d, J = 8.8 Hz, 1H), 7.4 (d, J = 8.8 Hz, 1H), 7.04 (t, J = 8.2
3
Hz, 1H), 6.66 (t, J = 6.4 Hz, 1H), 6.3 (s, 1H), 3.21 (m, 1H), 1.37 (d, J = 6.8 Hz, 6H); Mass (m/z):
+
161.1 (M+H) .
Synthesis of compounds 13a, 13b and 13c by Nꢀalkylation with alkyl halides: Followed the
procedure as reported in our own patent publication WO2013042135 with minor modifications.
To a stirred solution of ethyl isonipecotate 12 (1.0 mmol) in acetonitrile (2.0 mL), at r.t., was
added Cs CO (2.2 mmol) and alkyl halide (1.1 mmol). The reaction mass was gradually heated
2
3
to reflux temperature and stirred at this temperature for 4ꢀ12 h until all starting material gets
consumed. The reaction mixture was cooled to r.t., and was filtered through a small pad of celite.
The filtrate was evaporated under reduced pressure which afforded the Nꢀalkylated ethyl
isonipecotates 13a, 13b and 13c respectively in quantitative yields.
Synthesis of compounds 14a, 14b and 14c: Followed the procedure as reported in our own
patent publication WO2013042135 with minor modifications. To a stirred solution of Nꢀalkylated
ethyl isonipecotate (13a, 13b or 13c, 1.0 mmol) in THF (2.0 mL), and water (2.0 mL) cooled at 0
°
C was added LiOH.H O (1.1 mmol). After stirring for 30 minutes at this temperature, the
2
reaction mass was brought to r.t., and stirred for 6ꢀ12 h until all ester gets hydrolyzed. Upon
completion of the reaction, the reaction mass was diluted with EtOAc and the two layers were
separated. The aqueous layer was further extracted with EtOAc and the resultant aqueous layer
evaporated under reduced pressure which afforded Nꢀalkylated lithium carboxylates 14a, 14b or
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1
4c in quantitative yields respectively which were used in the next step without further
purification.
Synthesis of 1ꢀIsopropylꢀimidazo[1,5ꢀa]pyridineꢀ3ꢀcarboxylic acid hydrazide 15: To a stirred
solution of 6 (0.23 g, 1.0 mmol) in EtOH (10.0 mL) at r.t., hydrazine hydrate (2.0 mL) was
added. The reaction mixture was heated to reflux for 8 h until all ester got consumed. The
volatiles were removed under reduced pressure to obtain a crude mass which was triturated with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
hexane followed by ether which afforded hydrazide 15 as brown color solid (0.21 g) in 90%
1
yield. H ꢀ NMR (400 MHz, CDCl ): δ 9.36 (s, 1H), 9.28 (d, J = 7.0 Hz, 1H), 7.78 (d, J = 8.9 Hz,
3
1
H), 6.99ꢀ6.94 (m, 2H), 4.47 (s, 2H), 3.39ꢀ3.30 (m, 1H), 1.32 (d, J = 6.9 Hz, 6H); Mass (m/z):
+
219.2 (M+H) .
Synthesis of 2ꢀIsopropylꢀimidazo[1,2ꢀa]pyridineꢀ8ꢀcarboxylic acid hydrazide 16: Following
the procedure followed for preparing compound 15, compound 16 was obtained as dark brown
1
color solid in 85% yield. H ꢀ NMR (400 MHz, CDCl ): δ 11.39 (bs, 1H), 8.17 (d, J = 6.6 Hz,
3
1
3
H), 8.13 (d, J = 7.0 Hz, 1H), 7.38 (s, 1H), 6.90 (t, J = 6.9 Hz, 1H), 4.29 (d, J = 3.7 Hz, 2H),
+
.14 (m, 1H), 1.38 (d, J = 6.8 Hz, 6H); Mass (m/z): 219.2 (M+H) .
Synthesis of methyl 2ꢀisopropylpyrazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylate 17: To a stirred
solution of acid A (0.23 g, 1.1 mmol) in MeOH (5.0 mL), cooled at 0 °C, dropꢀwise addition of
3
thionyl chloride (0.2 mL). The reaction temperature was gradually raised to 65 °C, and the
reaction mixture was stirred at this temperature for 4 h. The volatiles were removed under
reduced pressure and the crude mass was diluted with DCM and aqueous NaHCO solution. The
3
two layers were separated, the organic layer was dried over anhydrous Na SO and the solvent
2
4
1
was removed under reduced pressure to obtain ester 17 (0.25 g) in 100% yield. H ꢀ NMR (400
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9
1
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1
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1
1
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2
2
2
2
2
2
2
2
2
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3
3
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3
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4
4
4
4
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6
MHz, CDCl ): δ 7.63 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 6.8 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1H), 6.46
3
+
(
s, 1H), 4.0 (s, 3H), 3.34 (m, 1H), 1.38 (d, J = 6.7 Hz, 6H); Mass (m/z): 219.2 (M+H) .
Synthesis of 2ꢀIsopropylpyrazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylic acid hydrazide 18: Following
the procedure used for preparing compound 15, compound 18 was obtained as dark brown color
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
solid in 95% yield. H ꢀ NMR (400 MHz, DMSO): δ 11.4 (bs, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.69
(d, J = 6.9 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 6.68 (s, 1H), 4.95 (bs, 2H), 3.28ꢀ3.20 (m, 1H), 1.35
+
(
d, J = 6.8 Hz, 6H); Mass (m/z): 219.2 (M+H) .
Synthesis of Nꢀ[Nꢀtertꢀbutyloxycarbonylꢀ3ꢀazabicyclo[3.1.0]hexaneꢀ6ꢀyl methyl]1ꢀisopropyl
imidazo[1,5ꢀa]pyridineꢀ3ꢀcarboxamide 19b: To the stirred solution of acid A (600.0 mg, 2.94
1
mmol) in DCM (11.8 mL) cooled at 0 °C, diisopropylethyl amine (0.77 mL, 4.41 mmol), tertꢀ
butyl 6ꢀaminomethylꢀ3ꢀazaꢀbicyclo[3.1.0]hexaneꢀ3ꢀcarboxylate B (624.0 mg, 2.94 mmol) and
2
TBTU (944.0 mg, 2.94 mmol) were sequentially added. The reaction temperature was gradually
brought to r.t. and the reaction mixture was stirred for 16 h before being diluted with DCM and
water. The two layers were separated, the aqueous layer was extracted once with DCM and the
combined organic layer was washed with brine solution. The resultant organic layer was dried
over anhydrous Na SO and the solvent was evaporated under reduced pressure. The crude mass
2
4
was purified by silica gel column chromatography to obtain above titled compound 19b (1.03 g)
1
in 85% yield. H ꢀ NMR (400 MHz, CDCl ): δ 9.38 (d, J = 7.1 Hz, 1H), 7.56 (d, J = 9.9 Hz, 1H),
3
7.44 (t, J = 5.2 Hz, 1H), 6.86 (t, J = 9.9 Hz, 1H), 6.75 (t, J = 6.8 Hz, 1H), 3.67 (d, J = 6.7 Hz,
2
1
H), 3.39 ꢀ 3.36 (m, 4H), 3.09 – 3.07 (m, 1H), 1.42 (s, 9H), 1.39 ꢀ 1.37 (m, 8H), 1.03ꢀ0.97 (m,
+
H); Mass (m/z): 399.3 (M+H) .
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Synthesis of Nꢀ[Nꢀ3ꢀazabicyclo[3.1.0]hexaneꢀ6ꢀyl methyl]1ꢀisopropyl imidazo[1,5ꢀ
a]pyridineꢀ3ꢀcarboxamide hydrochloride 20b: To the stirred solution of Boc protected amide
derivative 19b (1.0 g, 2.51 mmol) in isopropanol (5.0 mL) cooled at 0 °C, a solution of dry HCl
in IPA (5.0 mL) was added. The contents were stirred for 16 h at r.t. before the volatiles were
removed under reduced pressure. The HCl salt thus obtained was triturated with several portions
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of ether to obtain the above titled compound 20b (630.0 mg) as white solid in 98% yield. m.p.
1
1
72ꢀ174 °C. H ꢀ NMR (400 MHz, DMSO): δ 9.55 (bs, 1H), 9.35 (d, J = 7.0 Hz, 1H), 8.95 (bs,
1
H), 8.59 (t, J = 5.6 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.01 (t, J = 8.4 Hz, 1H), 6.95 (t, J = 6.8
Hz, 1H), 3.43 – 3.41 (m, 1H), 3.27 – 3.19 (m, 6H), 1.77 (s, 2H), 1.35 (d, J = 6.9 Hz, 7H); Mass
+
(
m/z): 299.2 (M+H) .
Synthesis of 1ꢀIsopropyl imidazo[1,5ꢀa]pyridineꢀ3ꢀcarboxylic acid A : To a stirred solution of
1
compound 6 (1.56 g, 6.7 mmol) in a mixture of ethanol and water (1:1, 20 mL), NaOH (0.3 g,
7.38 mmol) was added and the reaction mixture was heated to reflux for 2 hours. The volatiles
were removed under reduced pressure to one third of its volume, diluted with water, extracted
with solvent ether. The aqueous layer was acidified with con. HCl to pH 4ꢀ5 and extracted the
acidified aqueous layer with mixture of methanol and DCM (1:9). The combined organic layer
was dried on anhydrous Na SO and the solvent was removed under reduced pressure to obtain
2
4
1
acid A (1.33 g) in 97% yield. H ꢀ NMR (400 MHz, CDCl ): δ 9.20 (d, J = 7.0 Hz, 1H), 7.86 (d,
1
3
J = 8.9 Hz, 1H), 7.09 (t, J = 6.6 Hz, 1H), 7.02 (t, J = 6.7 Hz, 1H), 3.45ꢀ3.36 (m, 1H), 1.30 (d, J =
+
6
.5 Hz, 6H); Mass (m/z): 205.2 (M+H) .
Synthesis of 2ꢀIsopropylꢀimidazo[1,2ꢀa]pyridineꢀ8ꢀcarboxylic acid A : To a stirred solution of
2
compound 8 (0.44 g, 2.0 mmol) in a mixture of THF and water (1:1, 7.2 mL), cooled at 0 °C,
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
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5
6
NaOH (83.4 mg, 2.2 mmol) was added. The reaction mixture temperature was slowly raised to
r.t. and after 3 hours of stirring, the TLC revealed complete consumption of ester. The reaction
mass was diluted with ether and the two layers were separated. The aqueous layer was acidified
with 1N HCl to pH 4ꢀ5and was extracted with DCM. The combined organic layer was dried over
anhydrous Na SO and the solvent was removed under reduced pressure to obtain acid A (380.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
2
1
mg) in 98% yield. H ꢀ NMR (400 MHz, DMSO): δ 8.76 (d, J = 6.5 Hz, 1H), 7.96 (d, J = 6.8 Hz,
1
H), 7.94 (s, 1H), 7.13 (t, J = 6.2 Hz, 1H), 2.95 – 2.93 (m, 1H), 1.28 (d, J = 6.4 Hz, 6H); Mass
+
(
m/z): 205.3 (M+H) .
Synthesis of 2ꢀIsopropylpyrazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylic acid A : To a stirred solution of
3
compound 11 (0.5 g, 3.1 mmol) in dry THF (12.0 mL), cooled at −78 °C, nꢀBuLi (1.6 M in
hexanes, 2.0 mL, 3.2 mmol) was added. The reaction mass was stirred at the same temperature
for another hour before being quenched by dry ice (solid CO ). The reaction mixture was diluted
2
with water and EtOAc and the two layers were separated. The aqueous layer was acidified with
1
N HCl to pH 2ꢀ3 and extracted with chloroform. The combined organic layer was dried over
anhydrous Na SO and the solvent was removed under reduced pressure to obtain acid A (35.0
2
4
3
1
mg) in 55% yield. H ꢀ NMR (400 MHz, CDCl ): δ 15.2 (s, 1H), 7.88 (d, J = 7.0 Hz, 1H), 7.73
3
(d, J = 8.7 Hz, 1H), 7.31 (t, J = 7.4 Hz, 1H), 6.51 (s, 1H), 3.29ꢀ3.22 (m, 1H), 1.40 (d, J = 6.8 Hz,
+
6
H); Mass (m/z): 205.2 (M+H) .
Synthesis of Nꢀ[Nꢀ3ꢀmethoxypropylꢀ3ꢀazabicyclo[3.1.0]hexaneꢀ6ꢀyl methyl]1ꢀisopropyl
imidazo[1,5ꢀa]pyridineꢀ3ꢀcarboxamide 2f. To a stirred solution of deprotected amide
derivative 20b (630.0 mg, 1.88 mmol) in acetonitrile (7.5 mL), K CO (390.0 mg, 2.82 mmol)
2
3
and 1ꢀbromoꢀ3ꢀmethoxypropane (380.0 mg, 2.5 mmol) were added. The reaction mass was
heated to reflux for 7 h. The volatiles were removed under reduced pressure. The crude product
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was purified by silica gel column chromatography to obtain the desired compound 2f (620.0 mg)
1
as offꢀwhite color gummy liquid in 67% yield. H ꢀ NMR (400 MHz, CDCl ): δ 9.40 (d, J = 7.2
3
Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.37 (bs, 1H), 6.83 (t, J = 8.0 Hz, 1H), 6.72 (t, J = 4.0 Hz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
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3
4
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8
9
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1
H), 3.39 (t, J = 8.0 Hz, 2H), 3.25 (m, 1H), 3.27 (s, 3H), 3.30 (t, J = 4.0 Hz, 2H), 3.06 (d, J = 8.0
Hz, 2H), 2.45 (t, J = 8.0 Hz, 2H), 2.30 (d, J = 8.0 Hz, 2H), 1.97ꢀ1.90 (m, 1H), 1.72ꢀ1.65 (m, 2H),
+
1.49ꢀ1.43 (m, 1H), 1.47ꢀ1.43 (m,1H), 1.39 (d, J = 8.0 Hz, 6H); Mass (m/z); 371.3 (M+H) . δ
1
60.2, 139.2, 129.1, 127.2, 124.6, 120.3, 117.4, 114.7, 69.1, 57.5, 55.8, 54.4, 42.3, 39.7, 26.6,
5.7, 21.7, 11.7. HPLC. 99.32%. C,H,N for C H N O , calc. C, 68.08, H, 8.16, N, 15.12;
2
21
30
4
2
found, C, 68.32, H, 8.51, N, 15.32.
Synthesis of Nꢀ[Nꢀ(tetrahydropyranꢀ4ꢀylꢀmethyl) piperidinꢀ4ꢀyl methyl]ꢀ2ꢀisopropylꢀ
imidazo[1,2ꢀa]pyridineꢀ8ꢀcarboxamide 3c: Compund 3c was synthesized following the
1
procuedres as used in synthesis of compound 2f, in 49% overall yield. H ꢀ NMR (400 MHz,
DMSO): δ 10.428 (t, J = 5.5 Hz, 1H), 8.67 (d, J = 6.5 Hz, 1H), 7.95 (d, J = 7.0 Hz, 1H), 7.85 (s,
1
H), 7.04 (t, J = 7.0 Hz, 1H), 3.87ꢀ3.79 (m, 2H), 3.39 (s, 4H), 3.34ꢀ3.29 (m, 1H), 2.8 (s, 4H),
1
.99 (s, 1H), 1.87ꢀ1.80 (m, 2H), 1.67ꢀ1.58 (m, 4H), 1.30 (d, J = 6.6 Hz, 6H); Mass (m/z): 399.5
+
(M+H) . HPLC. 98.12%. C,H,N for C H N O , calc. C, 61.46, H, 7.43, N, 11.47; found, C,
25
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4
6
6
1.55, H, 7.89, N, 11.62.
Synthesis of 2ꢀIsopropylꢀ7ꢀ{5ꢀ[1ꢀ(3ꢀmethoxypropyl)ꢀpiperidinꢀ4ꢀyl]ꢀ[1,3,4]oxadiazolꢀ2ꢀyl}ꢀ
pyrazolo[1,5ꢀa]pyridine 4n:
To a stirred solution of lithium 1ꢀ(3ꢀmethoxypropyl)ꢀpiperidineꢀ4ꢀcarboxylate 14b (250.0 mg,
1
.2 mmol) in POCl (2.0 mL) at r.t., hydrazide 18 (220.0 mg, 1.0 mmol) was added. The reaction
3
mixture temperature was raised to 80 °Cꢀ85 °C and was stirred for 10 h. The reaction mixture
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was cooled to iceꢀbath temperature and was quenched by adding 1N NaOH solution. The
reaction mass was extracted with CHCl . The combined organic layer was dried over anhydrous
3
Na SO and the volatiles were removed under reduced pressure to obtain a crude mass which
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was purified by silica gel column chromatography to obtain compound 4n (138.0 mg) in 36%
1
yield. H ꢀ NMR (400 MHz,CDCl ): δ 7.64 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.14 (t,
3
J = 7.2 Hz, 1H), 6.49 (s, 1H) 3.44 (t, J = 6.4 Hz, 2H), 3.34 (s, 3H), 3.29ꢀ3.24 (m, 1H), 3.16ꢀ3.11
(m, 1H), 3.02 (t, 2H), 2.49 (t, 2H), 2.20 (t, 2H), 2.12 (t, J = 12.0 Hz, 2H), 1.83ꢀ1.80 (m, 2H),
+
1
.64ꢀ1.54 (m, 2H), 1.39 (d, J = 8.0 Hz, 6H); Mass (m/z); 384.2 (M+H) . C,H,N for C H N O ,
2
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5
2
calc. C, 65.77, H, 7.62, N, 18.26; found, C, 65.49, H, 7.37, N, 17.98.
Oxalate salt formation: To the stirred solution of above obtained 1,3,4ꢀoxadiazole 4n (96.0 mg,
.25 mmol) in isopropanol (4.0 mL) cooled at 0 °C, oxalic acid (23.0 mg, 0.25 mmol) was added
0
and the reaction mass was stirred for 1 h. The volatiles were removed under reduced pressure
and the crude salt thus obtained was triturated with several portions of solvent ether to obtain
1
oxalate salt of 4n in quantitative yield. H ꢀ NMR (400 MHz, DMSOꢀd ): δ 7.90 (d, J = 8.8 Hz,
6
1
H), 7.52 (d, J = 6.8 Hz, 1H), 7.34 (dd, J = 7.5, 8.4 Hz, 1H), 6.68 (s, 1H) 3.50ꢀ3.36 (m, 5H),
3.24 (s, 3H), 3.20ꢀ3.10 (m, 1H), 3.10ꢀ2.95 (m, 4H), 2.40ꢀ2.30 (m, 2H), 2.20ꢀ2.05 (m, 2H), 1.95ꢀ
+
13
1
.85 (m, 2H), 1.32 (t, J = 6.8 Hz, 6H); Mass (m/z); 384.3 (M+H) . C NMR (75 MHz, DMSOꢀ
d6): δ 168.3, 164.4, 161.6, 159.2, 150.8, 141.3, 123.4, 122.4, 121.0, 115.3, 95.0, 69.0, 57.8, 53.6,
0.2, 30.0, 27.6, 25.9, 23.8, 22.6, 22.5. M.R. 143.8 °Cꢀ149.9 °C. HPLC. 99.08%
5
Synthesis of 2ꢀIsopropylꢀ7ꢀ{5ꢀ[1ꢀ(tetrahydropyranꢀ4ꢀylmethyl)ꢀpiperidinꢀ4ꢀyl]ꢀ
1,3,4]oxadiazolꢀ2ꢀyl}ꢀpyrazolo[1,5ꢀa]pyridine 4o: Following the procedure used in the
[
synthesis of compound 4n as depicted above, compound 4o was prepared (180.0 mg) in 44%
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yield using lithium 1ꢀ(tetrahydropyranꢀ4ꢀylmethyl)ꢀpiperidineꢀ4ꢀcarboxylate 14c (280.0 mg, 1.2
1
mmol) and hydrazide 18 (220.0 mg, 1.0 mmol). H ꢀ NMR (400 MHz, CDCl3): δ 8.10 (s, 1H),
8.0 (d, J = 7.0 Hz, 1H), 7.86 (d, J = 8.32Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 5.03–4.98 (m, 1H),
1
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.99 (d, J = 3.0 Hz, 1H), 3.96 (d, J = 3.2 Hz, 1H), 3.42 (t, J = 11.2 Hz, 2H), 3.14ꢀ3.06 (m, 1H),
.95 (d, J = 10.1 Hz, 2H), 2.22 (d, J = 6.9 Hz, 2H), 2.13 – 1.99 (m, 6H), 1.77 (m, 1H), 1.69 (d, J
+
= 6.6 Hz, 6H), 1.32ꢀ1.22 (m, 2H); Mass (m/z); 410.3 (M+H) . C,H,N for C H N O , calc. C,
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31
5
2
6
7.46, H, 7.63, N, 17.10; found, C, 67.23, H, 7.54, N, 17.47.
Fumarate salt formation: To the stirred solution of above obtained 1,3,4ꢀoxadiazole 4o (103.0
mg, 0.25 mmol) in isopropanol (4.0 mL) cooled at 0 °C, fumaric acid (30.0 mg, 0.25 mmol) was
added and the reaction mass was stirred for 1 h. The volatiles were removed under reduced
pressure and the crude salt thus obtained was triturated with several portions of solvent ether to
1
obtain fumarate salt of 4o in quantitative yield. H ꢀ NMR (400 MHz, DMSOꢀd + D O): δ 8.58
6
2
(s, 1H), 8.01 (d, J = 8.2, Hz, 1H), 7.96 (d, J = 6.9 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 6.46 (s, 2H),
4
2
.94 – 4.88 (m, 1H), 3.84ꢀ3.80 (m, 2H), 3.50ꢀ3.25 (m, 5H), 3.0ꢀ2.90 (m, 2H), 2.88ꢀ2.80 (m, 2H),
.35ꢀ2.25 (m, 2H), 2.10ꢀ1.97 (m, 3H), 1.65ꢀ1.60 (m, 2H), 1.58 (d, J = 6.6 Hz, 6H), 1.20 – 1.10
+
13
(
m, 2H); Mass (m/z); 410.0 (M+H) . C NMR (75 MHz, DMSOꢀd ): δ 169.5, 166.3, 161.6,
6
1
59.0, 141.3, 134.2, 123.5, 122.4, 120.9, 115.2, 95.0, 66.8, 63.6, 52.1, 32.1, 31.6, 28.4, 27.5,
22.6. M.R. 205.2 °Cꢀ209.6 °C. HPLC. 98.38%.
Animals and ethics
All animal care and experiments were carried out according to protocols approved by the
Institutional Animal Ethics Committee (IAEC) of Suven Life Science Ltd constituted according
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to the directions of the Committee for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), India.
Determination of EC values for 5ꢀHT R
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The compounds were screened in the cell based reporter gene assay an end point luminescence
assay which measures the levels of cAMP inside cells upon activation or inhibition of the
receptor, using the luciferase reporter. Serotonin (5ꢀHT) and Luciferin was purchased from
Sigma Aldrich (St. Louis, Missouri, USA). T4 DNA Ligase and high fidelity Taq polymerase
were procured from Roche (Basel, Switzerland). Superscript Reverse Transcriptase and
mammalian expression vector pcDNA3.1 were purchased from Invitrogen (Carlsbad, California,
USA). CREꢀLuc reporter gene construct was supplied by Stratagene (La Jolla, California, USA).
Cell culture media and sera were procured from Invitrogen (Carlsbad, California, USA). All
other DNA restriction and modification enzymes were from New England Biolabs (Ipswich,
Massachusetts, USA). All other reagents and common chemicals were purchased from Sigma
Aldrich (St. Louis, Missouri, USA). Human 5ꢀHT R cDNA clone was purchased from Origene
4
(Rockville, Maryland, USA). The coding sequence was amplified by PCR using gene specific
primers. Amplified DNA was cloned in to mammalian expression vector pcDNA 3.1. The
authenticity of the cloned genes was determined by restriction analysis and nucleotide
sequencing. CHO cell line was purchased from American Type Culture Collection, Manassas,
Virginia, USA. The cells were routinely cultured in Hams F12 medium supplemented with 10%
fetal bovine serum at 37 °C in 5% CO . The cells were transfected with 5ꢀHT isoforms along
2
4
with threeꢀfold excess of CREꢀLuc reporter gene using Lipofectamine as recommended by the
supplier. Cells were further cultured in the same medium supplemented with 800 ꢀg/ ml G418.
Individual colonies were picked up after two weeks of selection and screened for serotonin
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induced luciferase activity. Colonies exhibiting maximum serotonin induced luciferase activity
were selected and amplified for further investigations. The recombinant CHO cells were plated
in 96 well clear bottom white plates (Corning) and cultured overnight as described above. Cells
were grown overnight in serum free medium before incubation with compounds to prevent
receptor desensitization due to presence of endogenous ligands in the serum.
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The reference endogenous agonist serotonin and test compounds in OptiꢀMEM medium at 11
different test concentrations starting from 10 µM until 0.1 nM in threeꢀfold serial dilutions were
incubated with the cells separately in individual wells for 4 hours at 37 ºC in 5 % CO2.
Compounds whose potency is less than 1 nM were tested in dose response study whose test
concentrations were Starting from 1 µM until 0.01 nM. After incubation, the medium was
removed; cells were washed with phosphate buffered saline and lysed in lysis buffer (Trisꢀ10mM
(pHꢀ8.0), NaClꢀ50mM, DTTꢀ1mM, Protease inhibitor cocktailꢀ1X, NP40ꢀ0.1%). The luciferase
activity was measured in individual wells using luciferin substrate in Victor Light Luminometer
Perkin Elmer.
The maximum response produced by each drug was normalized to the 5ꢀHT induced maximum
response. Data was analyzed using Graphpad prism software to derive EC50 values that
correspond to the concentration of agonists required to obtain halfꢀmaximal stimulation of
adenylyl cyclase. Values were expressed as the geometric mean of two independent experiments
9
performed in triplicates. Reference compound PFꢀ04995274 a reported partial agonist showed
partial agonist activity in our 5ꢀHT cell based reporter gene assay with an EC value of 1.3 nM
4
e
50
and Emax 18% which is very well in agreement with published data of EC / Emax : 0.26 nM /
50
7
%. Some of other reported compounds EC values were also determined using our assay and
50
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compared with the published data. The EC values obtained versus reported values tabulated in
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the supporting information of this manuscript.
Microsomal Metabolic Stability
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Compounds were incubated with microsomes at 0.5 mg protein /mL in 100 mM
phosphate buffer pH 7.4 at 37 °C. Reactions were initiated by addition of NADPH. All
incubations were carried out for 30 min. At 30 min, an aliquot of the sample was removed and
transferred into 300 µL of acetonitrile. The samples were stored at –20 °C until analysis.
Samples were analyzed using LCꢀMS/MS. Results were expressed as % metabolized at 30 min
post incubation.
CYP 3A4 and 2D6 inhibition
CYP 3A4 and 2D6 inhibitory potential of test compounds was studied using human liver
microsomes. Inhibitory activity was evaluated by incubating the test compound (0.1 – 45 µM) in
duplicates for 2 (3A4) and 12 minutes (2D6) with human liver microsomes in presence of
isoformꢀspecific substrate (midazolamꢀ3A4 or dextromethorphanꢀ2D6) and NADPH
regeneration system. Incubations were terminated by adding iceꢀcold acetonitrile containing an
internal standard and metabolites were quantified using LCꢀMS/MS. Peak area ratios of analyte
versus internal standards were used for calculating IC .
5
0
Pharmacokinetic Study in Rats
Male Wistar rats (225 ± 25 gm) were used as experimental animals. Three animals were housed
in each cage. Two days prior to dosing day, male Wistar rats were anesthetized with isoflurane
for surgical placement of jugular vein catheter. Animals were fasted over night before oral
dosing (p.o.) and food pellets were allowed 2 hours post dosing, whereas during intravenous
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dosing food and water were provided ad libitum. Three rats were dosed with compounds orally
(3 mg/kg) and intravenously (1 mg/kg). Dose formulation was prepared by using water as a
vehicle (10 mL/kg for oral and 2 mL/kg for intravenous dosing). At each time point blood was
collected through jugular vein and immediately replenished with an equivalent volume of normal
saline from freely moving rats. Collected blood was transferred into a labeled eppendorf
containing 10 µL of heparin as anticoagulant. The time points for blood samples collection was;
pre dose, 0.08 (only i.v.), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post dose (n=3). The collected
blood was centrifuged at 4000 rpm for 10 minutes. Plasma was prepared and stored at −70 °C
until analysis. The test compound concentrations were extracted from plasma samples by protein
precipitation using four volumes of acetonitrile (v/v) containing predetermined internal
standard. The precipitants are centrifuged (at 11292xg and 10 °C for 10 min) and supernatant is
transferred and injected in LCꢀMS/MS. The analytes of interest are quantified using suitable
multiple reaction monitoring mode in the range of 2ꢀ2000 ng/mL or ng/g against set of
calibration standards and quality control samples. Study samples were analyzed using calibration
samples in the batch and quality control samples spread across the batch. Pharmacokinetic
parameters were determined by using standard nonꢀcompartmental analysis (Phoenix
WinNonLin® 6.2 version or higher software) using linear trapezoidal with linear interpolation
method.
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Rodent Brain Penetration Study
Male Wistar rats (225 ± 25 gm) were used as experimental animals. Three animals were housed
in each cage. Animals were given water and food ad libitum throughout the experiment and
maintained on a 12 h light/dark cycle. Brain penetration was determined in discrete manner in
rats. The compounds were formulated in water and administered orally at 10 mg/kg (free base
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