Synthesis and SAR of Imidazo[1,5-a[pyridine derivatives as 5-HT4 receptor partial agonists for the treatment of cognitive disorders associated with Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2015.08.051

Alzheimer's disease (AD) is a neurodegenerative disease which has a higher prevalence and incidence in older people. The need for improved AD therapies is unmet. The 5-hydroxytryptamine₄ receptor (5-HT₄R) partial agonists may be of benefit for both the symptomatic and disease-modifying treatment of cognitive disorders associated with AD. Herein, we report the design, synthesis and SAR of imidazo[1,5-a] pyridine derivatives as 5-HT₄R partial agonists. The focused SAR, optimization of ADME properties resulted the discovery of compound 5a as potent, selective, brain penetrant 5-HT4 partial agonist as a lead compound with good ADME properties and efficacy in both symptomatic and disease modifying animal models of cognition.

Full text of DOI:10.1016/j.ejmech.2015.08.051

European Journal of Medicinal Chemistry 103 (2015) 289e301
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and SAR of Imidazo[1,5-a]pyridine derivatives as 5-HT4
receptor partial agonists for the treatment of cognitive disorders
associated with Alzheimer's disease
*
Ramakrishna Nirogi , Abdul Rasheed Mohammed, Anil K. Shinde, Narsimha Bogaraju,
Shankar Reddy Gagginapalli, Srinivasa Rao Ravella, Laxman Kota, Gopinadh Bhyrapuneni,
Nageswara Rao Muddana, Vijay Benade, Raghava Chowdary Palacharla, Pradeep Jayarajan,
Ramkumar Subramanian, Vinod Kumar Goyal
Discovery Research, Suven Life Sciences Ltd., Serene Chambers, Road-5, Avenue-7, Banjara Hills, Hyderabad, 500 034, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2015
Received in revised form
Alzheimer's disease (AD) is a neurodegenerative disease which has a higher prevalence and incidence in
older people. The need for improved AD therapies is unmet. The 5-hydroxytryptamine
HT R) partial agonists may be of benefit for both the symptomatic and disease-modifying treatment
of cognitive disorders associated with AD. Herein, we report the design, synthesis and SAR of imidazo
1,5-a] pyridine derivatives as 5-HT R partial agonists. The focused SAR, optimization of ADME properties
4
receptor (5-
4
25 August 2015
Accepted 26 August 2015
Available online 1 September 2015
[
4
resulted the discovery of compound 5a as potent, selective, brain penetrant 5-HT4 partial agonist as a
lead compound with good ADME properties and efficacy in both symptomatic and disease modifying
animal models of cognition.
Keywords:
4
5-HT receptor
©
2015 Elsevier Masson SAS. All rights reserved.
Imidazo[1,5-a]pyridines
Cognitive impairment
sAPPa
Disease modifying
Alzheimer's disease
1
. Introduction
tangles in brain provide the best differentiator of AD from other
forms of dementia. Current therapies treat only the disease
symptoms and are associated with modest efficacy, offset by dose-
Dementia is a prevalent disease with estimated 36 million
people currently affected. Alzheimer's disease (AD) is the most
common disease representing such patient pool. AD is a progres-
sive neurodegenerative disorder with ageing as the major risk
factor. Extracellular beta amyloid deposits and intracellular tau
4
limiting side effects [1]. The 5-HT R belonged to serotonin (5-HT)
receptor superfamily and is positively coupled to adenylate cyclase
thereby increases the cAMP production. These receptors are widely
expressed throughout the body, and the highest density is observed
in the cortex and hippocampus regions of brain associated with
learning [2]. In brain microdialysis experiment, an increase in
acetylcholine level was observed in the rat frontal cortex and hip-
Abbreviations: AD, Alzheimer's disease; SAR, structureeactivity relationships;
CNS, central nervous system; 5-HT, serotonin; cAMP, cyclic adenosine mono-
phosphate; ADAS-Cog, Alzheimer's disease assessment scale e cognitive subscale;
ADME, absorption, distribution, metabolism, and excretion; PK study, pharmaco-
pocampus upon intracerebroventricular injection of 5-HT
nists [3]. Behavioral studies in animal models of learning and
memory supports the role of 5-HT R agonists in cognition [4,5]. 5-
HT R also regulates the production of the neurotoxic amyloid
peptide (A ), which is one of the major pathogenic pathways in AD
6,7]. The 5-HT agonists can also stimulate the non-
amyloidogenic pathway leading to the release of soluble form of
amyloid precursor protein (sAPP ) in the experimental animal
studies, which has putative neurotropic and neuroprotective
properties [8e10]. 5-HT receptors therefore, attracts as a potential
4
R ago-
4
kinetic study; IBS, irritable bowel syndrome; 5-HT R, 5-hydroxytryptamine 4 Re-
4
ceptor; RO, receptor occupancy; NORT, novel object recognition task; RAM, radial
arm maze; IND, investigational new drug; DAST, diethylaminosulfurtrifluoride; TLC,
thin layer chromatography; min, minutes; LC-MS/MS, liquid chromatography-mass
spectrometry p.o., oral dosing; s.c., subcutaneous dosing; min, minutes; mm,
millimeter; rpm, rounds per minute; cps, counts per second; hERG, human ether-
aꢀ -go-go-related gene; CHO cells, Chinese hamster ovary cells; WFI, water for
4
b-
b
[
4
R
a
injection.
*
Corresponding author.
4
E-mail address: nvsrk@suven.com (R. Nirogi).
http://dx.doi.org/10.1016/j.ejmech.2015.08.051
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

290
R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
target for the treatment of AD with both symptomatic and disease
modifying potential. Besides neuroprotective properties, 5-HT
CH
3
N
4
R
also modulate mood which could be exploited for the treatment of
depression [11]. One of the non-CNS (peripheral) applications of 5-
HT R agonists is in the treatment of gastrointestinal disorders,
4
especially those associated with reduced esophageal, gastric
motility disorders, functional dyspepsia; conditions associated
with constipation, irritable bowel syndrome (IBS) and esophagitis
O
N
H
N
O
N
R
[
12,13]. Thus, 5-HT
4
R agonists represent a viable target for the
R = H DAU 6215
R = Et BIMU 1
R = iPr BIMU 8
treatment of multiple disorders like AD, schizophrenia, depression
and gastrointestinal disorders. Published literature [14e19] anal-
ysis shows that several structurally diverse 5-HT
been explored for both CNS and peripheral indications. Pfizer has
published [17] about a 5-HT R partial agonist PF-04995274 (Fig. 1)
for the treatment of AD. A poster from Raqualia pertaining to the
efficacy of RQ-00000009, a selective 5-HT partial agonist in ro-
dents was presented [20] in Alzheimer's conference. While the
chemical structure of RQ-00000009 remains undisclosed, a patent
publication [21] specifically covering the CNS indication was pub-
lished by Raqualia, with detailed efficacy data around Raqualia's
4
R agonists have
4
Fig. 2. Representative examples showing 5-HT R agonists structural requirements.
4
R1
N
4
O
H
O
N
N
NH
R1
O
R2
N
N
N
N
N
O
compound as depicted (Fig. 1). Another 5-HT
3140 from Epix showed positive phase IIa data with significant
improvement in ADAS-cog score [22,23]. Suven's internally
discovered 5-HT partial agonist SUVN-D4010 (structure not dis-
4
partial agonist, PRX-
H
Compounds 3o - 3r
O
Compounds 4a - 4b
N
0
O
O
H
N
N
N
H
N
4
R1
N
closed) has successfully completed IND enabling preclinical efficacy
and safety studies [24].
N
N
H
R1
N
N
Compounds 3a - 3n
R1
N
The 5-HT
4
pharmacophore model consists of an aromatic moi-
Example E1 of WO1993/08187
reported as 5-HT4 antagonist
N
Compounds 4c - 4f
ety, a coplanar carbonyl group, and a voluminous substituent in the
basic amino framework of the molecule as exemplified by com-
pounds shown in Fig. 2. Suitable aromatic systems [25] include 4-
amino-5-chloro-2-methoxy benzoic acid, N-alkyl quinolones and
N-alkyl indazoles among others. Several basic amines with volu-
minous substituents such as tropane [26], quinuclidine [27],
piperizines [28], and piperidines [17,25] have been used. Literature
evidence suggests that the potency and the pharmacological profile
of benzimidazolone scaffold (Fig. 2) is dependent on the alkyl
substitution on the nitrogen atom of the benzimidazole ring [15].
N
H
O
R1
N
R1
N
R2
O
N
N
NH
H
O
NH
N
N
Compounds 2a - 2e
N
N
Compounds 5a - 5p
Compounds 1a - 1j
Fig. 3. Genesis of 5-HT R partial agonists.
The reported 5-HT
4
R ligand DAU-6215 with no alkyl substituent on
R ligands BIMU-1 and
4
the nitrogen was inactive whereas 5-HT
4
BIMU-8 with ethyl and isopropyl substitutions respectively were
potent agonists [15,29].
4
from treatment with 5-HT R full agonists. The GPCR receptor full
On examining the different scaffolds which were reported as
agonists in general, on prolonged exposure could desensitize the
receptor thereby limit their therapeutic utility. The desensitization
of GPCR receptors by agonists is often dependent on the degree of
their efficacy (Emax) and the partial agonists minimize the potential
for desensitization. Based on this information we intend to develop
active at 5-HT
to be developed further [30e32]. One such Imidazo[1,5-a]pyridine
30,31] scaffold represented by N-(1-butyl) piperidin-4-yl methyl]
-ethyl imidazo[1,5-a]pyridine-1-carboxylate (Example E1) [31]
has attracted our attention. The compounds from this scaffold
were reported as dual antagonists at 5-HT R and 5-HT R (Fig. 3).
Using the afore mentioned knowledge that the alkyl sub-
stituents influence the potency of the molecules towards 5-HT R,
4
R, we felt that, some of the scaffolds have potential
[
3
4
5-HT R partial agonists with appropriate efficacy. We focused our
structure activity relationships (SAR) around imidazo[1,5-a]pyri-
dine carboxamide derivatives rather ester derivatives as the pres-
ence of the ester function in the pharmacologically-active
molecules could be a drawback for their use as drugs for their
putative short half-lives [33]. The isopropyl group was introduced
in the third position of imidazo[1,5-a]pyridine ring as it was
observed that, presence of isopropyl group in this position in-
3
4
4
we started our work by modifying the alkyl substituents both on
imidazo[1,5-a]pyridine ring and piperidine ring, anticipating to
4
obtain the desired 5-HT R partial agonists. It is proposed [17] that
the 5-HT R partial agonists would be beneficial in CNS related
4
disorders as it is preferable to reduce or avoid undesired increase in
gastric motility and other peripheral side effects which may result
creases the efficacy of the compounds towards 5-HT
imidazo[1,5-a]pyridine carboxamide derivatives were synthesized
4
R. Several
O
O
OH
N
O
O
O
N
N
N
N
O
N
N
HO
H
H
O
N
N
O
S
O
OH
Raqualia's compound
O
PF-04995274
PRX-03140
Fig. 1. Structures of 5-HT
4
agonists PF-04995274, Raqualia's compound, and PRX-03140 reported for CNS indication.

R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
291
as part of structure activity relationships (SAR) to improve upon the
in vitro affinity, efficacy and ADME properties. The 5-HT R partial
agonists with potent in vitro affinity and acceptable ADME profile
were achieved by substantial modification both on eastern and
western domain of the scaffold. These efforts culminated into novel
imidazo[1,5-a]pyridine carboxamide derivatives as 5-HT R partial
4
agonists (compounds 1 through 5) as depicted in Fig. 3. Among
these compounds, compound 5a showed high in vitro affinity and
O
O
OEt
OEt
OH
NH2
N3
4
H C
3
H C
3
H
3
C
H C
3
a
b
c
d
N
N
N
N
N
Boc
Boc
Boc
Boc
Boc
17
18
19
20
22b
O
N
O
N
OH
desired partial agonistic activity towards 5-HT
4
R with drug-like
NH
2
e
f
properties such as adequate brain penetration, good oral bioavail-
ability and efficacy in animal models of cognition.
N
Boc
Boc
Boc
2
. Results and discussion
21
2
2c
Scheme 2. Synthesis of 4-substituted piperidinylmethyl amines. Reaction conditions:
2.1. Synthetic aspects
ꢁ
ꢁ
(a) LDA, MeI, THF, ꢀ78 C (b) LiAlH
4
, THF, 0 C (c) i. Et
3
N, MsCl, DCM, r.t., ii. NaN
3
, DMF,
in
ꢁ
r.t. d) Pd/C, H
2
, MeOH, r.t. (e) NaH, Trimethylsulfoxonium iodide, THF, 10 C (f) NH
3
The 3-alkyl substituted imidazo[1,5-a]pyridine carboxylic acids
MeOH, r.t.
were synthesized starting from ethyl-2-pyridylacetate 6 as per re-
ported procedures [34] as depicted in Scheme 1. The compound 6
on treatment with sodium nitrite in glacial acetic acid afforded
compound 7 which was on hydrogenation in the presence of Pd/C
afforded aminoester 8. The intermediate 9 was obtained when
aminoester 8 reacted with butyryl chloride. This intermediate on
cyclization in presence of thionyl chloride yielded imidazo[1,5-a]
pyridine ester 10. The ester 10 was hydrolyzed to get acid 11. The
hydrazide 12 was obtained when the ester 10 was refluxed with
hydrazine hydrate in ethanol.
The Boc protected piperidinylmethyl amine 22a (Scheme 4)
with which compounds 1a-1d have been synthesized was prepared
by using literature reported procedures [35]. The 4-substituted
piperidine amines 22b and 22c used in the preparation of com-
pounds 1g-1j were prepared as shown in Scheme 2.
The N-alkyl substituted isonipecotic acid derivatives 25 which
are part of compounds 2a to 2e were prepared as per the in-house
developed procedures [36]. The N-Boc-4-aminopiperidine 22d a
building block for compounds 3ae3n is commercially available. The
Boc protected morpholinemethyl amines 22e [37] representing the
compounds 3oe3p, the Boc protected tropane amine 22f [38]
representing the compounds 4ae4b and the Boc protected bicy-
clic piperidine amine 22g [39] representing the compounds 4ce4d
were synthesized as reported in the literature. The Boc protected
bicyclic piperidinylmethyl amine 22h which forms the starting
material for the compounds 5ae5p was synthesized from 6-
hydroxymethyl-3-aza-bicyclo[3.1.0]hexane 13 [40] by a series of
simple high yielding transformations as shown in Scheme 3.
The N-Boc protected imidazo[1,5-a]pyridine carboxamide de-
rivatives (23ae23h) were prepared by converting imidazo[1,5-a]
pyridine carboxylic acid 11 into its acid chloride followed by
coupling with different N-Boc protected alicyclic amines (22ae22h)
or the carboxylic acid was coupled with amines using coupling
reagents TBTU, HATU etc as shown in Scheme 4. The 4-
fluoropiperidinecarboxamide derivative 23i was synthesized from
4-hydroxypiperidinecarboxamide derivative 23c by reacting it with
diethylaminosulfur trifluoride (DAST). The Boc group was depro-
tected in compounds (23ae23i) to obtain compounds 24ae24i. The
Boc deprotected analogues were N-alkylated to obtain compounds
1, 3, 4 & 5 either by reaction with alkyl halides in presence of
inorganic bases or by reductive amination with aldehydes or ke-
tones. The oxadiazole analogues (2ae2e) were synthesized from
imidazopyridine hydrazide 12 and N-alkylated isonipecotic acids
25.
2.2. Assessment of in vitro activity
Diverse imidazo[1,5-a]pyridine derivatives were designed and
synthesized using combination of classical medicinal chemistry
and fragment based drug discovery approach. The fully character-
ized compounds were evaluated for their affinity towards 5-HT
4
R
using cell based in vitro reporter gene assay [41e43]. 5-HT R partial
4
O
a
O
b
O
agonists program was started with the synthesis of compounds
with isopropyl group at C3 carbon of imidazo[1,5-a]pyridine scaf-
fold (Western domain). Several modifications were done on the
basic amine framework in the eastern domain. These modifications
resulted in the novel imidazo[1,5-a]pyridine analogues (1ae1d)
with potent in vitro affinities when tested in CHO stable cell line
expressing human 5-HT4eR. However, these compounds turned out
to be very weak partial agonists (Emax 20e30%, Table 1).
N
OEt
N
OEt
N
OEt
OH
6
N
7
8
NH2
O
HO
O
OEt
c
O
d
f
N
N
N
OEt
N
N
HN
O
9
10
11
O
NH2
NHNH2
OH
H
OH
H
OMs
H
N3
H
e
10
N
H
H
H
H
a
H
b
c
H
d
N
N
N
N
N
N
Boc
12
H
Boc
Boc
Boc
13
14
15
16
22h
Scheme 1. Synthesis of 3-isopropylimidazo[1,5-a]pyridine carboxylic acid derivatives.
ꢁ
Reaction conditions: (a) NaNO
2
, ACOH, ꢀ10 C-r.t. (b) H
2
, Pd/C, MeOH, r.t. (c) Isobutyryl
eNH $H O, reflux (f) NaOH,
Scheme 3. Synthesis of N-tert-butyloxycarbonyl-3-azabicyclo[3.1.0]hexane-6-yl-
ꢁ
ꢁ
ꢁ
3
chloride, Et N, DCM, 0 C-r.t. (d) POCl
3
, DCE, 90 C (e) NH
2
2
2
methyl amine. Reaction conditions: (a) Boc
2
O, Et
3
N, DCM, r.t. (b) MsCl, Et
3
N, DCM, 0 C-
ꢁ
MeOH, reflux.
r.t. (c) NaN
3
, DMF, 90 C (d) Pd/C, H
2
, EtOAc, r.t.

292
R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
O
O
O
H
O
H
O
H
NH
2
N
N
N
OH
a
Cl
b
c
d
N
N
+
N
N
N
N
N
N
N
N
NBoc
NBoc
NHCl
NR
1
1
1
22
23a-23i
24a-24i
Compounds 1, 3, 4 and 5
NH
2
NH2
NH2
NH2
NH2
NH
N
NH2
NH
2
2
H
3
C
HO
H
H
H
H
O
=
NBoc
NBoc H
2
N
NBoc
N
N
N
N
N
Boc
2a
Boc
Boc
Boc
Boc
Boc
22h
2
22b
22c
22d
22e
22f
22g
R
1
N
N
O
N
O
OLi
O
NHNH
2
e
+
N
N
N
N
N
R
1
1
2
25
Compounds 2a-2e
, DCE, reflux (b) iPr
ꢁ
ꢁ
Scheme 4. Synthesis of imidazo[1,5-a]pyridine derivatives. Reaction conditions: (a) SOCl
methoxypropane, K CO , CAN, reflux (e) POCl , DCE, reflux.
2
2
EtN, DCM, 0 C to r.t. (c) Dry HCl in isopropanol, 0 C (d) 1-bromo-3-
2
3
3
Introduction of small substitutions such as fluoro, methyl or hy-
droxy group on C-4 carbon of the piperidine ring resulted in the
compounds 1ee1j which showed decreased in vitro affinity; how-
ever a trend towards increase in efficacy was observed.
The increase in efficacy for C-4 substituted piperidine analogues
prompted us to design the compounds with steric hindrance or
conformational constrain near the primary binding amidic site. The
amide group was replaced with its stable 1,3,4-oxadiazole bio-
isostere [44]. Gratifyingly, the efficacy of the compounds (2ae2e)
was increased; however, the in vitro affinity remained on the lower
side (Table 2).
The next set of SAR iteration was carried out to understand the
role of methylene linker connecting the amide group with the
piperidine moiety. The methylene linker was removed in com-
pounds 3a, 3b, 3f, 3i, 3l and 3n. On positive note, these compounds
showed potent in vitro affinity and adequate efficacy (Table 3).
2.3. ADME properties
Some of the representative compounds from each series based
on their cLogP, efficacy, intrinsic activity and ease of synthesis, were
profiled further for ADME properties like metabolic stability, CYP
liability, rat pharmacokinetic and brain penetration studies
(Table 6). Compounds such as 1a, 1b, 3a, and 3f, 5a, 5d and 5f were
tested for their metabolic stability in both rat and human liver
microsomes. Compounds 5d and 5f completely metabolized in the
tested conditions whereas compounds 1a, 1b, 3f, 3g, 3h and 5a
turned out to be metabolically stable. Compounds 1a, 1b, 3f, 3g, 3h
and 5a showed good oral exposures when evaluated for their
pharmacokinetic parameters in male Wistar rats. The brain pene-
tration was adequate (C
The inhibitory potential of 3f, 3g, 3h and 5a on CYP 2D6 and CYP
3A4 was low with absolute IC50 values > 30 M for both metabolic
b p
/C ~ 0.3) for most of these compounds.
m
Apart
from
the
above
set
of
compounds,
2-
enzymes indicating less likely chances for drugedrug interactions
in the clinic.
These compounds were further profiled in animal models of
cognition to evaluate the in vitro to in vivo translatability.
aminomethylmorpholine derivatives (3oe3p, Table 3) which
maintain almost equidistance between amide group and the
pipe0072idine nitrogen were on evaluation, turned out to be
inactive.
Additional conformational constraint was brought in the mol-
ecules by replacing piperidine ring with a bicyclic tropane ring
system. The compounds from this series retained the efficacy (Emax
2
.4. In vivo efficacy of selected compounds
Based on bio-availability and oral exposures (Table 6), com-
~90%) but the in vitro potency was reduced substantially (com-
pounds 1a, 3f, 3g and 5a have been evaluated for their cognition
enhancement ability in in vivo efficacy model, the novel object
recognition task (NORT) [45]. The compounds 3f and 3g found to be
inactive at the tested doses (0.3, 1.0, 3.0 & 10.0 mg/kg, p.o) while
compound 1a showed moderate activity at highest tested dose
pounds 4a & 4b in Table 4). Another set of conformational con-
strained derivatives comprising bicyclic 3-azabicyclo hexylamine
have been synthesized and evaluated for their in vitro activity;
however, these compounds (compounds 4c and 4d in Table 4)
found to be inactive.
Reasoning severe conformational strain in bicyclic 3-azabicyclo
hexylamine ring near the amide bond for the inactivity of these
compounds, a series of compounds having methylene linker be-
tween the amide group and the bicyclic ring system were synthe-
sized. Gratifyingly, this modification led to potent compounds
(10 mg/kg, p.o.). The compound 5a reversed the time induced
memory deficit in NORT assay at tested doses of 1, 3 & 10 mg/kg, p.o.
and the effect was significant at doses 1 & 3 whereas inverted-U
phenomenon was observed at 10 mg dose (Fig. 4).
As the compound 5a showed acceptable ADME properties
(Table 6) and good precognitive effects in NORT, it was further
(
compounds 5a-5l, Table 5) which showed good in vitro affinity and
evaluated in various in vitro, ADME, cognition and safety assays. The
compound 5a had rapid absorption and good oral bio-availability in
efficacy (Emax ~50%).

R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
293
Table 1
Table 2
Structure and in vitro potencies of 4-aminomethylpiperidine series compounds.
Structure and in vitro potencies of 1, 3, 4-oxadiazole series compounds.
a
Compound
R
1
R
2
5HT
4
R EC50 (nM) /Emax (%)
a
Compound
R
1
4
5HT R EC50 (nM) /Emax (%)
PRX-03140/VRX-3011
N.A.
N.A.
H
58/48
Lit. 17e52/30-61% [22]
10/18
PRX-03140/VRX-3011
N.A
58/48
Lit. 17e52/30-61% [22]
840/55
1a
2a
2
b
520/63
1
1
b
c
H
H
4.8/22
11/29
2c
857/73
500/65
2d
1
d
H
9.8/20
2e
736/67
1
1
e
f
F
F
633/82
a
In vitro assay. Values are the geometric mean of three to six determinations. CHO
1358/36
stable cell line expressing human 5-HT4eR and reporter gene was used.
1
1
g
OH
OH
315.7/41
56.7/34
The compound 5a also showed potential disease modifying ef-
fect as it increased the neuroprotective soluble alpha-amyloid
h
precursor proteins (sAPP
was antagonized by 5-HT
cating the observed effect was mediated through activation of 5-
HT R. The in vitro sAPP increase was complemented by in vivo in-
crease in sAPP levels when compound 5a was tested in Balb/c57
mouse brain cortex (Fig. 7). Prucalopride a 5-HT R agonist was used
as the positive control. Statistically significant increase of sAPP
a
) in in vitro testing [9,48]. The effect
4
R selective antagonist GR113808 indi-
1
1
i
j
OH
72.3/41
53.0/56
4
a
CH
3
4
a
was observed at 3 mg/kg s.c. dosing with inverted U-shaped dose
response at 10 mg/kg s.c.
N.A. Not applicable.
a
In vitro assay. Values are the geometric mean of three to six determinations. CHO
stable cell line expressing human 5-HT4eR and reporter gene was used.
Compound 5a showed dose dependent in vivo 5-HT
4
R occu-
pancy [49] (RO, Fig. 8) indicating the in vivo efficacy observed for
compound 5a may be mediated through 5-HT
value found to be 4.11 mg/kg, p.o.
4
R. The absolute ED50
In-house selectivity profiling against 5-HT1A, 5-HT2A, 5-HT2C, 5-
, SERT, DAT, 5-HT , 5-HT , CB , CB , Muscarinic M , M , M , M
Histaminergic H , H , Dopaminergic D , D , D , Adrenergic receptors
at the highest tested concentration of 10 M showed <50% inhi-
bition in functional response assay both in agonist and antagonist
mode indicating the selectivity of 5a over the above tested re-
ceptors. The IC50 for hERG channel in electrophysiology patch
Beagle dogs. A comparative assessment of compound 5a oral ex-
posures in both rat and dog is given in Table 7.
HT
3
6
7
1
2
1
2
3
5
,
I
3
1
3
5
4
The compound 5a activity across 5-HT R isoforms (5-HT4A, 5-
m
HT4E, 5-HT4D and 5-HT4L) found to be similar with EC50 values
ranging from 20 to 40 nM having 50e100% Emax values. In Caco-2
permeability assay, compound 5a showed good permeability
ꢀ
6
(
P
app 34.8 10 cm/s) and it is not a P-gp substrate (B-A/A-B ¼ 1.6).
clamp assay found to be 5.1 mM with 51% Imax at 10 mM concen-
In protein binding studies across tested rat, dog and human species,
an appreciable amount of free fraction was found for compound 5a
with no significant species difference (Table 8).
tration. Compound 5a showed very weak agonist like activity to-
wards 5HT2b receptor in rat fundus assay [50e52] (Fig. 9) and
owing to its weak activity, EC50 calculation could not be
ascertained.
In another cognition model, the radial arm maze (RAM) assay,
compound 5a at 3 mg/kg, p.o. significantly reversed the scopol-
amine induced working memory deficits (Fig. 5) by increasing the
choice accuracy and decreasing the total error in comparison with
the scopolamine treated groups.
Further detailed profiling of compound 5a is currently ongoing.
3. Conclusion
4
Activation of 5-HT R under preclinical conditions is demon-
strated to improve neurotransmission and enhance the release of
acetylcholine resulting in the memory formation [3]. Effect of
compound 5a on acetylcholine modulation was tested in rat
microdialysis experiment [46,47]. Per oral administration of com-
pound 5a produced significant increase in cortical acetylcholine
levels in frontal cortex (Fig. 6).
The chemical optimization of a class of imidazo[1,5-a]pyridine
derivatives covering optimization of alicyclic ring, chain length,
alkyl substitutions on the nitrogen has led to diverse set of in vitro
potent compounds. Modification in the structure of compounds so
as to have better metabolic stability, low hERG potential, minimum
cytochrome
P 450 enzyme's (CYP450) liability, acceptable

294
R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
Table 3
4. Experimental section
Structures and in vitro potencies of 4-aminopiperidine & morpholine series com-
pounds.
4.1. General synthesis
Unless stated otherwise, all reagents and solvents were pur-
chased from common commercial suppliers and were used
1
without further purification. H NMR spectra were recorded at
13
4
00 MHz and C NMR at 100 MHz on a Bruker NMR spec-
trometer instrument (Fallanden, Switzerland). Deuterated re-
agents were used as solvents and were commercially procured.
Tetramethylsilane (TMS) was used as an internal standard.
Chemical shift values are expressed in parts per million (d) and
a
coupling constants are expressed in Hz. Thin layer chromatog-
raphy (TLC) was performed on Merck silica gel 60 F254 plates.
Electrospray ionization mass spectra were recorded on API 4000
triple quadrupole instrument (MDS-SCIEX, Concord, Ontario,
Canada). Infrared spectra were recorded on KBr disc and in solid
state using PerkineElmer model 1600 FT-IR spectrophotometer
Compound
R
1
5HT
4
R EC50 (nM) /Emax (%)
PRX-03140/VRX-3011
N.A.
58/48
Lit. 17e52/30-61% [22]
3
3
a
3.4/50
b
15.5/50
(
PerkineElmer, 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 100e200 mesh silica gel. Analytical HPLC
was done using Agilant systems (Model-1100 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. 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). CRE-Luc reporter
gene construct was supplied by Stratagene (La Jolla, California,
USA). Cell culture media and sera were procured from either
Invitrogen (Carlsbad, California, USA). All other DNA restriction
and modification enzymes were from New England Biolabs
(Ipswich, Massachusetts, USA). Perkin Elmer Victor light Model
3
3
c
50/100
49/46
d
3
3
e
f
144/72
40/80
3
3
g
49/46
h
144/72
3
3
3
i
40/80
j
132/92
196/71
k
1420 was used to measure the luminescence counts.
3
3
l
3.9/86
47/74
4
4
3
.2. Chemistry
m
.2.1. Ethyl 2-hydroxyimino-2-(pyridin-2-yl) acetate (7)
To a stirred solution of ethyl 2-pyridylacetate 6 (50.0 g,
02.6 mmol s) in water (120 mL) cooled at 0 C, glacial acetic acid
3
n
25/81
ꢁ
(
120 mL) was added. After stirring the reaction mixture for 15 min,
an aqueous solution of sodium nitrite (40.5 g, 605.3 mmol s in
0 mL of water) was added over a period of 15 min. The reaction
3
3
o
p
2247
6
467/35
mixture was then stirred at r.t. for 24 h before it was diluted with
water (200 mL). The solids thus precipitated were filtered, washed
ꢁ
with water and dried under reduced pressure at 50 C to obtain the
N.A. Not applicable.
a
In vitro assay. Values are the geometric mean of three to six determinations. CHO
stable cell line expressing human 5-HT4eR and reporter gene was used.
above titled compound 7 (53.8 g) as light brown gummy liquid in
1
91% yield. H NMR (CDCl
3
):
d
17.49 (bs, 1H), 8.56 (d, J ¼ 4.5 Hz, 1H),
8
1
.14 (d, J ¼ 8.2 Hz,1H), 7.97 (t, J ¼ 9.3 Hz,1H), 7.53 (dd, J ¼ 5.2, 7.2 Hz,
pharmacokinetic properties, adequate brain penetration and good
in vivo efficacy resulted in diverse series of in vitro potent com-
pounds which in turn led to the discovery of compound 5a, a potent
H), 4.43 (q, J ¼ 7.1 Hz, 2H), 1.43 (t, J ¼ 7.1 Hz, 3H). Mass (m/z): 195.2
þ
(M þ H) . Anal. (C
9 10 2 3
H N O ) C, H, N.
4
and selective 5-HT R partial agonist which showed potent in vitro
4
.2.2. Ethyl 2-amino-2-(pyridin-2-yl) acetate (8)
To a stirred solution of compound 7 (52.0 G, 267.7 mmol s) in
affinity with acceptable ADME profile. Compound 5a showed effi-
cacy in tested models of cognition such as NORT, RAM, and
increased acetylcholine levels in frontal cortex. Compound 5a also
methanol (1.5 L), Pd/C (10.0 g) was added. Hydrogen pressure was
applied by fixing the hydrogen filled balloon on the reaction vessel
and the reaction mixture was stirred at r.t. for 48 h. The reaction
mass was filtered through celite and the filtrate was concentrated
under reduced pressure to obtain compound 8 (48.4 g) as brown
gummy mass in 99% yield. H NMR (CDCl
showed disease modifying effect by increasing the sAPP
a levels
both in vitro and in vivo. Compound 5a showed target engagement
as it showed dose dependent receptor occupancy. Compound 5a
showed good selectivity over tested GPCR & ion channels apart
1
3
):
d
8.58 (d, J ¼ 4.3 Hz,
3
from closely related 5-HT receptor.
1
H), 7.69 (t, J ¼ 7.6 Hz, 1H), 7.42 (d, J ¼ 7.7 Hz, 1H), 7.24 (dd, J ¼ 5.0,

R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
295
Table 4
Structure and in vitro potencies of bicyclic aminopiperidine series compounds.
a
Compound
Cy
R
1
4
5HT R EC50 (nM) /Emax (%)
PRX-03140/VRX-3011
N.A.
N.A.
58/48
Lit. 17e52/30-61% [22]
4a
88/89
4b
39/87
4
4
4
c
>10,000
>10,000
1061/19
d
e
N.A. Not applicable.
a
In vitro assay. Values are the geometric mean of three to six determinations. CHO stable cell line expressing human 5-HT4eR and reporter gene was used.
7
.1 Hz, 1H), 4.71 (s, 1H), 4.19 (q, J ¼ 7.1 Hz, 2H), 2.14 (bs, 2H), 1.21 (t,
2 4
layer was dried over anhydrous Na SO and the solvent was
removed under reduced pressure to obtain a crude solid which was
þ
J ¼ 7.1 Hz, 3H). Mass (m/z): 181.4 (M þ H) . Anal. (C
9
H
12
N
2
O
2
) C, H,
N.
triturated with hexanes to obtain compound 11 (21.0 g) as off-white
ꢁ
1
solid in 95% yield. m.p. 134e137 C. H NMR (CDCl
3
): d 8.41 (d,
4
.2.3. Ethyl 3-isopropylimidazo[1,5-a]pyridine-1-carboxylate (10)
J ¼ 7.0 Hz, 1H), 8.0 (d, J ¼ 9.1 Hz, 1H), 7.17 (t, J ¼ 6.5 Hz, 1H), 6.88 (t,
13
To a stirred solution of compound 8 (48.4 g, 268.6 mmol s) in
J ¼ 6.6 Hz, 1H), 3.58e3.44 (m, 1H), 1.32 (d, J ¼ 6.8 Hz, 6H). C NMR
ꢁ
toluene (500 mL) cooled at 0 C isobutyryl chloride (30.0 mL,
68.6 mmol s) was added over a period of 15 min. After stirring the
(DMSO-d
6
):
d
164.5, 144.6, 134.3, 124.3, 123.4, 119.6, 119.1, 113.7,
þ
2
25.3, 20.6. Mass (m/z): 205.1 (M þ H) . Anal. (C11
12 2 2
H N O ) C, H, N.
reaction mass for additional 15 min, the volatiles were removed
under reduced pressure and the intermediate 9, thus obtained was
4.3. Synthesis of tert-butyl 6-aminomethyl-3-azabicyclo[3.1.0]
diluted with dichloroethane (500 mL). After cooling the reaction
hexane-3-carboxylate (22h)
ꢁ
mass to 0 C, POCl
of 15 min. The reaction mixture was then gradually heated to 90 C
3
(60.0 mL, 618.0 mmol) was added over a period
ꢁ
4.3.1. tert-Butyl 6-hydroxymethyl-3-azabicyclo[3.1.0]hexane-3-
carboxylate (14)
ꢁ
and was stirred for 24 h. The reaction mass was cooled to 0 C and
then quenched by adding saturated aqueous NaHCO
reaction mass then extracted with ethylacetate (2 ꢂ 300 mL). The
organic layer was dried over anhydrous Na SO and the volatiles
3
solution. The
Di-tert-butyl dicarbonate (16.96 g, 77.0 mmol s) was added to a
solution of 6-hydroxymethyl-3-azabicyclo[3.1.0]hexane 13 (8.0 g,
70.0 mmol s) and triethylamine (15.7 mL, 112.0 mmol s) in DCM
(150 mL) at 10 C. The reaction mass was stirred for 2 h at 10 C,
while monitoring the progress of the reaction by TLC. The reaction
mass was washed with chilled water (50 mL), brine solution
2
4
ꢁ
ꢁ
were removed under reduced pressure. The crude mass was puri-
fied by silica gel column chromatography to obtain compound 10
1
(
40.0 g) as light yellow gummy liquid in 65% yield. H NMR (CDCl
3
):
d
1
1
8.17 (d, J ¼ 9.3 Hz, 1H), 7.92 (d, J ¼ 6.9 Hz, 1H), 7.06 (t, J ¼ 6.8 Hz,
2 4
(50 mL) and dried over Na SO . The organic phase was concen-
trated under vacuum to obtain a crude residue, which was further
purified by flash chromatography using EtOAc: n-hexane (50:50) to
H), 6.77 (t, J ¼ 6.8 Hz, 1H), 4.47 (q, J ¼ 7.2 Hz, 2H), 3.43e3.30 (m,
13
H), 1.49 (d, J ¼ 6.9 Hz, 6H), 1.44 (t, J ¼ 7.2 Hz, 3H). C NMR
OD): 163.7, 163.4, 145.4, 134.4, 124.3, 122.4, 118.8, 113.8, 59.8,
(
CD
3
d
afford the title compound 14 as light brown gummy liquid (7.84 g)
ꢀ
1
1
2
5.5, 19.2, 13.5. IR (cm ): 2963, 2871, 1693, 1634, 1538, 1408, 1324,
in 52% yield. H NMR (CDCl
3
):
d
3.68e3.42 (m, 4H), 3.40e3.30 (m,
þ
1
210, 1058, 928, 784, 753, 698. Mass (m/z): 233.2 (M þ H) . Anal.
2H), 1.69 (s, 1H), 1.43 (s, 11H), 0.98e0.92 (m, 1H). Mass (m/z): 214.3
þ
(
C
13
16
H N
2
O
2
) C, H, N.
(M þ H) . Anal. (C11
3
H19NO ) C, H, N.
4
.2.4. 3-Isopropylimidazo[1,5-a]pyridine-1-carboxylic acid (11)
To a stirred solution of compound 10 (25.0 g, 107.6 mmol s) in a
4.3.2. tert-Butyl 6-methanesulfonyloxymethyl-3-azabicyclo[3.1.0]
hexane-3-carboxylate (15)
mixture of ethanol (400 mL) and water (150 mL) at r.t, sodium-
hydroxide (5.28 g,129.2 mmol s) was added over a period of 15 min.
The reaction mass was gradually heated to reflux and stirred at this
temperature for 6 h. The reaction mixture was concentrated to its
one third volume under reduced pressure and the residue was
acidified with acetic acid (8.1 mL, 135.6 mmol s) and extracted with
A solution of methanesulfonylchloride (4.42 g, 38.0 mmol s) in
DCM (25.0 mL) was added to a solution of compound 14 (7.8 g,
36.0 mmol s) and triethylamine (7.70 mL, 55.0 mmol s) in DCM
(100.0 mL) at 0 C. The reaction mass was stirred over night at r.t,
while monitoring the progress of the reaction by TLC. The reaction
mass was washed with chilled water (50 mL), brine solution
ꢁ
5
% methanol in dichloromethane solution. The combined organic
2 4
(50 mL) and dried over Na SO . The organic phase was

296
R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
Table 5
Structure and in vitro potencies of bicyclic aminomethylpiperidine series compounds.
Compound
R
1
5HT
4
R EC50 (nM)/Emax (%)a
PRX-03140/VRX-3011
N.A.
58/48
Lit. 17e52/30-61% [22]
22.0/50
5a
5b
13.2/50
5
5
5
c
18/59
d
e
18.3/50
20.7/43
5
5
f
31/53
9.7/42
g
5h
8.7/35
5
5
i
j
5.7/40
18/31
5
5
k
l
19.8/53
34/28
N.A. Not applicable.
a
In vitro assay. Values are the geometric mean of three to six determinations. CHO stable cell line expressing human 5-HT4eR and
reporter gene was used.
concentrated under vacuum to afford the title compound 15 (9.3 g)
NMR (CDCl
3
): d 3.73e3.55 (m, 2H), 3.45e3.32 (m, 2H), 3.30e3.20
1
as light brown gummy liquid in 87% yield. H NMR (CDCl
3
):
(m, 1H), 3.18e3.08 (m, 1H), 1.48 (s, 2H), 1.44 (s, 9H), 1.04e0.95 (m,
13
d
4.20e4.05 (m, 2H), 3.70e3.55 (m, 2H), 3.42e3.32 (m, 2H), 3.02 (s,
1H). C NMR (CDCl
3
):
d
154.7, 79.3, 52.6, 47.8, 47.5, 28.3, 21.8, 21.3,
þ
3
H), 1.59 (bs, 2H), 1.42 (s, 9H), 1.16e1.06 (m, 1H). Mass (m/z): 292.3
21.2. Mass (m/z): 239.3 (M þ H) . Anal. (C11
18 4 2
H N O ) C, H, N.
þ
(
M þ H) .
4.3.4. tert-Butyl 6-aminomethyl-3-azabicyclo[3.1.0]hexane-3-
4.3.3. tert-Butyl 6-azidomethyl-3-azabicyclo[3.1.0]hexane-3-
carboxylate (22h)
carboxylate (16)
A solution of tert-butyl 6-azidomethyl-3-azabicyclo[3.1.0]hex-
ane-3-carboxylate 16 (1.50 g, 6.0 mmol s) in THF (30.0 mL) and
water (3.0 mL) mixture was treated with triphenylphosphine (2.1 g,
8.0 mmol s). The reaction mass was stirred for 36 h at r.t., and
concentrated under vacuum to obtain a crude residue, which was
further purified by flash chromatography using a mixture of TEA:
Sodium azide (7.30 g, 112.0 mmol s) was added to a solution of
compound 15 (9.30 g, 39.0 mmol s) and potassium carbonate
ꢁ
(
11.0 g, 79.0 mmol s) in DMF (100.0 mL) at 10 C. Then the reaction
mass was stirred over night at r.t., and poured onto chilled water
200 mL). The product was extracted with EtOAc (3 ꢂ 150 mL) and
the combined organic phase was washed with chilled water
150 mL), brine solution (150 mL) and dried over Na SO . The
(
MeOH: DCM (2:8:9) to afford the title compound 22h (1.20 g) as
1
(
2
4
light brown gummy liquid in 90% yield. H NMR (CDCl
3
):
organic phase was concentrated under vacuum to afford the title
compound 16 (7.0 g) as light brown gummy liquid in 90% yield. H
d 3.66e3.46 (m, 2H), 3.40e3.30 (m, 2H), 2.80e2.60 (m, 2H),
2.60e2.40 (m, 4H),1.43 (s, 9H), 0.95e0.80 (m,1H). C NMR (CDCl ):
3
1
13

R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
297
Table 6
Summary of in vitro potency, in vitro intrinsic clearance, CYP inhibition and in vivo pharmacokinetic parameters in rat for select compounds.
Compound
CYP inhibition
IC50 M)
RLM clint
scaled
HLM clint
scaled
In-vivo rat pharmacokinetic profile
(
m
(mL/min/kg)
(mL/min/kg)
2D6
3A4
Clearance (mL/min/kg)
Vdss (L/kg)
t
1/2 (h)
C
brain/Cplasma
C
max (ng/mL)
% F
1
1
3
3
3
3
3
3
5
5
5
5
5
5
a
b
a
d
f
g
h
I
a
d
f
h
j
7.5
48.6
50.4
140.1
36.1
ND
25.5
27.2
0.2
20.0
16.3
ND
ND
27.3
0.0
7.5
25.5
27.2
0.2
20.0
16.3
ND
ND
27.3
0.0
64.2 ± 4.7
74.8 ± 5.1
115 ± 3.1
125 ± 28
98 ± 2.8
83 ± 2
43 ± 6
88 ± 11
43 ± 5
119 ± 13
60 ± 2
50 ± 2
54 ± 12
60 ± 6
4.0 ± 0.6
5.2 ± 0.7
4.7 ± 0.2
5.3 ± 1.1
4.9 ± 0.3
4.6 ± 0.1
4.7 ± 2.1
2.9 ± 0.9
3.7 ± 0.2
7.6 ± 0.7
10.4 ± 0.9
3.3 ± 0.3
4.9 ± 0.2
3.8 ± 0.5
0.80 ± 0.2
1.69 ± 0.4
0.6 ± 0.0
0.6 ± 0.1
1.2 ± 0.0
0.9 ± 0.1
1.5 ± 2
0.5 ± 0.1
1.0 ± 0.1
NA
0.31
0.22
0
232 ± 44
109 ± 20
166 ± 28
74 ± 33
45 ± 5.2
30 ± 2.3
10 ± 1.9
3.7 ± 1.2
44 ± 1.1
98 ± 10
63 ± 4
11 ± 1.9
26 ± 6
5 ± 3
48.6
50.4
140.1
36.1
ND
ND
0.31
0.31
0.38
1.46
0.31
0.14
0.60
0.69
0.38
0.31
848 ± 49
1761 ± 116
434 ± 127
99 ± 33
188 ± 35
80 ± 41
120 ± 13
150 ± 142
8.1 ± 2.9
277 ± 251
ND
ND
93.5
21.9
ND
90.2
88.5
734.3
43.7
93.5
21.9
76.2
90.2
88.5
734.3
43.7
ND
4.9
41.4
54.3
44.7
18.9
41.4
54.3
44.7
18.9
1.6 ± 0.2
1.3 ± 0.2
2.2 ± 0.6
0.9 ± 0.1
17 ± 1.7
12 ± 6.9
1.5 ± 0.2
34 ± 15
I
Incubation of test compound (0.1e45
m
M) in duplicates for 2 (3A4) and 12 min (2D6) with human liver microsomes.
ꢁ
Compounds were incubated with liver microsomes at 0.5 mg protein/mL in 100 mM phosphate buffer pH 7.4 at 37 C for 30 min.
Three male Wistar rats were used for each PK study. Values were calculated from at least eight data points.
For oral dosing, 10 mg/kg and for IV dosing, 5 mg/kg compound was used.
ND. Not done.
***
2
0
0
***
*
1
0
Vehicle, WFI
ml/kg, p.o.
5a, 1 mg/kg, p.o.
5a, 3 mg/kg, p.o.
5a, 10 mg/kg, p.o.
2
Data represents Mean ± SEM of Exploration Time (Students paired ‘t’ test) N=8-11 (*P<0.05, ***P<0.001)
WFI: Water for injection
White bars: Time spent by rats to explore the known object
Shaded bars: Time spent by rats to explore the novel object
Fig. 4. Compound 5a reverses time induced memory deficit in NORT.
Table 7
Summary of compound 5a non-clinical pharmacokinetic parameters in rat and dog.
Species
Route/Dosea (mg/kg)
AUC0-t (ng*hr/mL)
CLplasma (mL/min/kg)
Vdz (L/kg)
t1/2 (hr)
Rat
1 (i.v.)
389 ± 42
43 ± 5
13 ± 4
3.7 ± 0.2
2.1 ± 0.3
Tmax (hr)
0.25e0.50
0.5
1.0 ± 0.1
2.7 ± 1.3
% F
26 ± 6
94 ± 13
Dog
Species
Rat
Dog
Rat
1.0 (i.v., 5 min infusion)
Route/Dose (mg/kg)
3 (p.o.)
1311 ± 469
AUC0-t (ng*hr/mL)
301 ± 69
Cmax (ng/mL)
188 ± 35
412 ± 18
1 (p.o.)
1217 ± 466
C
brain/Cplasma ¼ 0.31 ± 0.12
a
Values were calculated from at least eight data points.
d
154.7, 79.0, 48.1, 47.8, 43.8, 28.3, 25.9, 21.6, 20.8. Mass (m/z): 213.2
þ
(M þ H) . Anal. (C11
20 2 2
H N O ) C, H, N.
Table 8
Summary of compound 5a unbound fractions in plasma, brain homogenate and
microsomes.
4
.4. Synthesis of N-[N-(3-methoxy propyl)-3-azabicyclo[3.1.0]
Species
Fraction unbound, fu, mean ± SEM
hexane-6-yl methyl] 3-isopropyl imidazo[1,5-a]pyridine-1-
carboxamide oxalate (5a)
Plasma
Brain
Microsomes
Rat
Dog
Human
0.314 ± 0.015
0.242 ± 0.013
0.289 ± 0.018
0.247 ± 0.007
NA
NA
0.636 ± 0.032
0.661 ± 0.027
0.677 ± 0.015
4.4.1. N-[N-tert-butyloxycarbonyl-3-azabicyclo[3.1.0]hexane-6-yl
methyl] 3-isopropyl imidazo[1,5-a]pyridine-1-carboxamide (23h)
To the imidazo[1,5-a]pyridine carboxylic acid 11 (2.0 g,
NA-Not applicable.

298
R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
1
5
0
5
0
8
0
*
*
70
*
*
60
1
50
**
*
*
*
*
40
30
*
*
20
10
0
Treatment
Vehicle 1 mL/kg, p.o. + Vehicle 1 mL/kg, i.p.
Vehicle 1 mL/kg, p.o. + Scopolamine 0.8 mg/kg, i.p.
Treatment
5a, 1 mg/kg/kg, p.o. + Scopolamine 0.8 mg/kg, i.p.
5a, 3 mg/kg/kg, p.o. + Scopolamine 0.8 mg/kg, i.p.
5a, 10 mg/kg, p.o. + Scopolamine 0.8 mg/kg, i.p.
*
*P<0.01 Scopolamine vs. 5a, 1, 3 & 10 mg/kg n=5-6 /group, Vehicle (WFI) one way ANOVA followed by
Dunnett’s test
Fig. 5. Compound 5a reduces working memory deficits in RAM.
8
6
4
00
00
00
*
*
*
400
300
200
100
0
Vehicle (n=7)
Compound 5a, 3.0 mg/kg, p.o. (n=7)
Compound 5a, 10.0 mg/kg, p.o. (n=8)
200
0
Vehicle
n=7)
3.0 mg/kg
(n=7)
10.0 mg/kg
(n=8)
(
*
**
**
*
**
Compound 5a
*
***
**
*
*
-
80 -60 -40 -20
0
20 40 60 80 100 120 140 160 180 200 220 240
Time (min)
Data represents mean ± SEM of percent change in acetylcholine levels. Arrow indicates the point of treatment.
p<0.05, **p<0.01, ***p<0.001 Vs Vehicle (Bonferroni post test). Inset graph shows the area under the curve
*
(AUC) value calculated for the increase in acetylcholine levels. *p<0.05, **p<0.01 Vs Vehicle (Dunnett’s post test)
Fig. 6. Compound 5a increases cortical acetylcholine levels in male Wistar rats.
ꢁ
9
.8 mmol s) cooled at 0 C, thionyl chloride (20.0 mL) was added
the solvent was removed under reduced pressure. The crude
and the reaction mass was stirred at this temperature for 2 h. Excess
thionyl chloride was distilled off and the crude acid chloride thus
obtained (2.2 g) was dissolved in DCM (40.0 mL) and transferred
through addition funnel to the RB flask containing a mixture of Boc
protected bicyclic piperidine amine 22h (2.0 g, 8.13 mmol s) and
product was purified by silica gel column chromatography to obtain
the desired carboxamide derivative 23h (2.5 g) as light brown
1
gummy liquid in 71% yield. H NMR (CDCl
3
):
d
8.27 (d, J ¼ 9.0 Hz,
1H), 7.83 (d, J ¼ 6.6 Hz, 1H), 7.26 (bs,1H), 6.95 (t, J ¼ 7.4 Hz,1H), 6.69
(t, J ¼ 6.2 Hz,1H), 3.70e3.55 (m, 2H), 3.50e3.26 (m, 5H),1.67 (s, 2H),
13
triethylamine (3.4 mL, 24.5 mmol s) in DCM (16.0 mL) cooled at
1.45 (d, J ¼ 6.6 Hz, 6H), 1.42 (s, 9H), 1.06e0.95 (m, 1H). C NMR
ꢁ
0
C. The reaction mass was gradually warmed to r.t and stirred for
3
(CDCl ): d 163.7, 154.7, 142.8, 132.0, 122.4, 121.9, 120.9, 120.3, 113.3,
1
6 h before diluting it with DCM and water. The two layers were
79.1, 40.2, 38.4, 28.3, 25.9, 22.7, 21.8, 21.2, 20.2. Mass (m/z): 399.4
þ
separated; the organic layer was dried over anhydrous Na SO and
2
4
30 4 3
(M þ H) . Anal. (C22H N O ) C, H, N.

R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
. In vitro sAPPα assay
2. sAPPα in Balb/c57 mouse brain cortex
299
1
200
200
*
*
*
150
100
100
50
0
0
Vehicle
1
3
10
Prucalopride
Compound 5a dose (mg/kg, s.c.)
Data are Mean ± SD of sAPPα levels expressed as percentage of control values (mice which received water for
injection). Values were compared between the different groups by using unpaired t test. The significance level was
set at *p<0.05; **p<0.01; ***p<0.001.
Fig. 7. Compound 5a promotes non-amyloidogenic processing of sAPPa: 1. In vitro increase of sAPPa in transiently transfected Tsa201 cell line with APP695 & HTR4E plasmids with
respect to 5-HT and 5-HT R antagonist GR113808 2. sAPP increase in Balb/c57 mice at 60 min after compound 5a dosing.
4
a
4
.4.2. N-(N-azabicyclo[3.1.0]hexane-6-yl methyl) 3-isopropyl
J ¼ 7.1 Hz, 2H), 2.30 (d, J ¼ 8.3 Hz, 2H), 1.82e1.68 (m, 2H), 1.45 (d,
J ¼ 6.8 Hz, 6H), 1.50e1.40 (m, 1H), 1.39 (s, 2H). Mass (m/z): 371.3
imidazo[1,5-a]pyridine-1-carboxamide hydrochloride (24h)
þ
To the stirred solution of Boc protected amide derivative 23h
(M þ H) .
(
154.8 mg, 0.39 mmol) in isopropanol (0.5 mL) at r.t., a solution of
dry HCl in IPA (1.5 mL) was added. The contents were stirred for
6 h before the volatiles were removed under reduced pressure.
4
.4.4. N-[N-(3-methoxy propyl)-3-azabicyclo[3.1.0]hexane-6-yl
methyl] 3-isopropyl imidazo[1,5-a]pyridine-1-carboxamide oxalate
5a)
To the stirred solution of methoxypropyl imidazo[1,5-a]pyridine
1
The HCl salt thus obtained was triturated with several portions of
ether to obtain the above titled compound 24h (129.0 mg) as white
(
ꢁ
1
solid in 99% yield. m.p. 165e167 C. H NMR (DMSO-d
H), 8.91 (bs, 1H), 8.40 (d, J ¼ 7.0 Hz, 1H), 8.18 (bs, 1H), 8.11 (d,
J ¼ 9.0 Hz, 1H), 7.10 (dd, J ¼ 6.6, 8.7 Hz, 1H), 6.87 (t, J ¼ 6.6 Hz, 1H),
.65e3.52 (m, 1H), 3.30e3.12 (m, 6H), 1.75 (s, 2H), 1.38 (d,
6
): d 9.52 (bs,
amide derivative (358.0 mg, 0.96 mmol) in isopropanol (10.0 mL),
oxalic acid (90.0 mg, 1.0 mmol) was added. The reaction mass was
stirred for 1 h before the volatiles were evaporated under reduced
pressure. The crude salt was triturated with plenty of solvent ether
1
3
13
J ¼ 6.7 Hz, 6H), 1.35e1.25 (m, 1H). C NMR (DMSO-d
6
): d 160.1,
and ethyl acetate to obtain the cream color crystalline powder as an
143.4, 131.5, 125.7, 123.7, 119.4, 117.9, 116.0, 46.7, 38.5, 25.1, 21.3,
1
þ
oxalate salt 5a (413.0 mg) in 93% yield. H NMR (DMSO-d
6
):
d
8.36
2
0.4, 20.1. Mass (m/z): 299.2 (M þ H) . Anal. (C17
4
H23ClN O) C, H, N.
(
(
d, J ¼ 7.1 Hz, 1H), 8.07 (d, J ¼ 9.1 Hz, 1H), 7.99 (t, J ¼ 6.0 Hz, 1H), 7.06
dd, J ¼ 6.5, 9.0 Hz, 1H), 6.82 (t, J ¼ 6.7 Hz, 1H), 3.58e3.38 (m, 3H),
4.4.3. N-[N-(3-methoxy propyl)-3-azabicyclo[3.1.0]hexane-6-yl
3.30 (t, J ¼ 5.8 Hz, 2H), 3.19 (s, 3H), 3.15 (t, J ¼ 6.4 Hz, 2H), 3.05e2.93
(m, 2H), 1.83e1.70 (m, 4H), 1.47e1.40 (m, 1H), 1.35 (d, J ¼ 6.8 Hz,
methyl] 3-isopropyl imidazo[1,5-a]pyridine-1-carboxamide (5a free
base)
1
3
6H). C NMR (DMSO-d ):
6
d 165.1, 163.4, 143.5, 131.7, 123.2, 123.1,
To a stirred solution of deprotected amide derivative 24h
122.5, 119.4, 113.6, 69.3, 58.2, 55.2, 52.4, 25.9, 25.4, 21.1, 20.8. IR
(
559.0 mg, 1.87 mmol s) in acetonitrile (8.0 mL), K
2
3
CO (387.0 mg,
2
2
7
.8 mmol s) and 1-bromo-3-methoxypropane (380.0 mg,
.5 mmol s) were added. The reaction mass was heated to reflux for
h. The volatiles were removed under reduced pressure. The crude
product was purified by silica gel column chromatography to obtain
the desired methoxypropyl imidazo[1,5-a]pyridine amide deriva-
1
tive 5a (358.0 mg) as off-white color semi solid in 54% yield. H
NMR (CDCl
bs, 1H), 6.94 (dd, J ¼ 6.5, 9.0 Hz 1H), 6.68 (t, J ¼ 7.0 Hz,1H), 3.38 (t,
J ¼ 6.5 Hz 2H), 3.38e3.26 (m, 6H), 3.06 (d, J ¼ 8.6 Hz 2H), 2.46 (t,
3
):
d
8.28 (d, J ¼ 9.1 Hz, 1H), 7.82 (d, J ¼ 7.1 Hz, 1H), 7.24
(
100
75
50
25
0
GR113808
0.3
1
3
10
Compound 5a, dose (mg/kg, p.o.)
R occupancy in male Wistar rats in non-fasted condition.
30
6
mg/kg, i.v.
Fig. 8. Compound 5a 5-HT
4
Fig. 9. Compound 5a activity towards 5-HT2bR in rat fundus functional assay.

300
R. Nirogi et al. / European Journal of Medicinal Chemistry 103 (2015) 289e301
ꢀ
1
(
1
cm ): 3377, 2996, 2928, 2908, 1713, 1631, 1558, 1513, 1262, 1220,
Appendix A. Supplementary data
þ
ꢁ
111, 840, 732, 710. Mass (m/z): 371.3 (M þ H) . DSC: 174.54 C.
Oxalate content: 20.26%. HPLC purity: 99.59%. Anal. (C23
H, N.
H
32
N
4
O
6
) C,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.08.051.
4
2
.5. Synthesis of 1-[5-(N-cyclobutylpiperidin-4-yl)-1,3,4-oxadiazol-
-yl]-3-isopropyl-imidazo[1,5-a]pyridine (2a)
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re-
1
gummy liquid in 83% yield. H NMR (CDCl
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d
8.25 (d, J ¼ 9.2 Hz,
(
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[
4
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