Benzamide derivatives and their constrained analogs as histamine H3 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2015.12.005

A series of 4-(1-substituted piperidin-4-yloxy) benzamides and 6-(1-substituted piperidin-4-yloxy)-3,4-dihydro-2H-isoquinolin-1-one derivatives have been synthesized and tested for their binding affinity towards H₃ receptor. Most of these synth

Full text of DOI:10.1016/j.ejmech.2015.12.005

Accepted Manuscript
Benzamide derivatives and their constrained analogs as histamine H receptor
3
antagonists
Ramakrishna Nirogi, Anil Shinde, Vinaykumar Tiriveedhi, Laxman Kota, Sangram
Keshari Saraf, Rajesh Kumar Badange, Abdul Rasheed Mohammed, Ramkumar
Subramanian, Nageshwararao Muddana, Gopinadh Bhyrapuneni, Renny Abraham
PII:
S0223-5234(15)30392-5
10.1016/j.ejmech.2015.12.005
EJMECH 8245
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 July 2015
Revised Date: 26 October 2015
Accepted Date: 2 December 2015
Please cite this article as: R. Nirogi, A. Shinde, V. Tiriveedhi, L. Kota, S.K. Saraf, R.K. Badange, A.R.
Mohammed, R. Subramanian, N. Muddana, G. Bhyrapuneni, R. Abraham, Benzamide derivatives
and their constrained analogs as histamine H receptor antagonists, European Journal of Medicinal
3
Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.005.
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Graphical Abstract
R2
N
R2
N
n
n
O
O
R1
R
R1
R
N
N
N
N
H
O
O
Compounds 2
2a, R = H, R1 = R2 = iPr,
Compounds 3
3d, R = H, R1 = R2 = cyclobutyl
hH3 Ki (nM) = 5.7, hH3 IC50 (nM) = 323.8
hH3 Ki (nM) = 0.8, hH3 IC50 (nM) = 4.8
Bringing conformational constraint in Compounds 2 resulted in Compounds 3 having improved brain
penetration.

ACCEPTED MANUSCRIPT
Benzamide derivatives and their constrained analogs as
histamine H3 receptor antagonists
Ramakrishna Nirogi*, Anil Shinde, Vinaykumar Tiriveedhi, Laxman Kota, Sangram Keshari
Saraf, Rajesh Kumar Badange, Abdul Rasheed Mohammed, Ramkumar Subramanian,
Nageshwararao Muddana, Gopinadh Bhyrapuneni and Renny Abraham
Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road-5, Avenue-7, Banjara
Hills, Hyderabad - 500 034, India
Keywords: Histamine H3 receptor, SAR, pharmacokinetic profile, receptor occupancy
ABSTRACT: A series of 4-(1-substituted piperidin-4-yloxy) benzamides and 6-(1-substituted
piperidin-4-yloxy)-3,4-dihydro-2H-isoquinolin-1-one derivatives have been synthesized and
tested for their binding affinity towards H3 receptor. Most of these synthesized compounds have
displayed potent binding affinity for H3 receptor when tested in in vitro binding assay.
Preliminary SAR studies, functional activity, pharmacokinetic profile and efficacy profile
constitute the subject matter of this communication.
1. Introduction
The histamine 3 receptor (H3R) is a G protein-coupled receptor (GPCR), mainly expressed in
presynaptic terminals of central nervous system (CNS) neurons.[1] H3Rs function as both
autoreceptors by modulating histamine release [2] and as inhibitory hetero receptors by
regulating the release of neurotransmitters including histamine, serotonin, acetylcholine,
norepinephrine and dopamine.[3-6] All these neurotransmitters play an important role in
1

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modulating cognition, mood and sensory gating. Preliminary literature reports suggest that H3R
antagonists have shown reversal effect on cognitive deficits in several preclinical models of
cognition.[7] Thus, H3R antagonists may have promising utility for the treatment of various CNS
disorders including Alzheimer’s disease (AD), schizophrenia (SZ), attention-deficit hyperactivity
disorder (ADHD), epilepsy, narcolepsy, neuropathic pain and metabolic disorders.[8] H3R
antagonists also play an important role in wakefulness by way of regulation of sleep-wake cycle
and circadian rhythm.[9] Plancher reviewed the several discovery challenges for the
advancement of H3 antagonists that include species differences, CYP-450 liability, human ether-
a-go-go-related gene (hERG) interactions, pharmacokinetic issues and phospholipidosis.[10]
Over the past several years, a number of histamine H3 antagonist chemotypes were reported in
the literature. Imidazole based compounds were not progressed further because of CYP liabilities
and brain penetration issues.[11] Early stage phenoxypropylamine based compounds [12] which
suffer from phospolipidosis were optimized by several research groups to modulate ADME
properties, selectivity and physical properties.[13] However the class which found frequent
occurrences in literature is aryl and heteroarylamide based compounds (Figure 1) as reported by
JnJ, GSK and Merck.[14] Zhou et al [15] has also reported their work on constraining the
benzamide to dihydroisoquinoline as H3R antagonists. With the information gained from the
above literature precedence, we set our project goal to identify a compound series with high
potency, no hERG liability, clean CYP-450 profile and no phospholipidosis (PL) liabilities while
maintaining the duration of action appropriate for a compound that promotes wakefulness.
2

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Fig. 1. Structures of reported H3R antagonists
O
O
N
N
N
N
N
O
N
O
Bavisant
hH3R Ki = 5.4 nM
GSK239512
hH3R pKi = 10 nM
N
N
N
N
N
CF3
O
O
N
CF3
O
O
MK-3134
hH3R Ki Not reported
MK-0249
hH3R Ki = 6.8 nM
In vitro screening of benzamide based analog series previously prepared in our laboratories
resulted in advanced hit compound 1 (unpublished work, Fig. 2) with moderate binding affinity
(Ki of 103 nM) at human H3R. In order to improve upon the in vitro potency of this arylamide
compound, we began our exploration of the western domain by way of replacement of
tetrahydropyran moiety with introduction of a basic amine center in the form of piperidine ring
(Compounds 2) followed by the rigidization of amidic group with central core on the eastern
domain (Compounds 3). Details of structure activity relationship (SAR), pharmacokinetic
properties and in vivo assessment in animal models were presented over here.
3

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Fig. 2. Design of ligands
Eastern domain
O
O
CH3
O
R2
N
n
n = 1, 2
R1
R2
N
R1
N
n
N
N
O
N
N
N
CH3
H
H
O
O
O
n = 1, 2
R
R
Compound 1
Compounds 3
Compounds 2
Western domain
2. Results and discussion
2.1. Chemistry
The synthetic steps for the targeted compounds were described in schemes 1 and 2. 4-
hydroxymethyl benzoate derivatives (4a-4f) were reacted with commercially procured N-
substituted-4-piperidinols (Fig. 3) under Mitsunobu reaction conditions to obtain intermediates 5a-
5i. The latter compounds, thus obtained, were upon potassium hydroxide mediated hydrolysis gave
intermediates 6a-6i respectively. The reaction of intermediates 6a-6i with either N-Boc-3-
aminopyrrolidine or N-Boc-4-aminopiperidine under acid-amine coupling conditions afforded
intermediates 7a-7l. Trifluoroacetic acid mediated Boc deprotection of intermediates 7a-7l gave
intermediates 8a-8l respectively. The reaction of intermediates 8a-8l with various carbonyl
compounds under reductive amination conditions yielded targeted series of Compounds 2 (a-q)
(Scheme 1).
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Scheme 1. Synthesis of targeted Compounds 2
O
O
R
R
R1
b
a
OCH3
N
OCH3
45 - 50 %
75 - 80 %
HO
O
4
5
5a (R = H, R1 = isopropyl), 5b (R = H, R1 = cyclobutyl),
4a (R = H), 4b (R = 3-F), 4c (R
2-F), 4d (R 2-Cl),
4e (R = 2-CH3), 4f (R = 3-CH3)
5c (R = H, R1 = cyclopentyl), 5d (R = 3-F, R1
=
=
=
isopropyl), 5e (R = 2-F, R1 = isopropyl), 5f (R = 2-Cl,
R1 = isopropyl), 5g (R = 2-Cl, R1 = isobutyl), 5h (R = 2-
CH3, R1 = isopropyl), 5i (R = 3-CH3, R1 = isopropyl)
O
O
R
R1
N
n
R1
R
boc
N
OH
c
d
N
N
H
O
57 - 62 %
80 - 85 %
O
6
7
7a (R = H, R1 = isopropyl, n =1), 7b (R = H, R1
cyclobutyl, n = 1), 7c (R = H, R1 = cyclopentyl, n =1),
7d (R = H, R1 = isopropyl, n =2), 7e (R = H, R1
cyclobutyl, n = 2), 7f (R = 3-F, R1 = isopropyl, n = 2),
7g (R = 2-F, R1 = isopropyl, n = 2), 7h (R = 2-Cl, R1 =
isopropyl, n = 1), 7i (R = 2-Cl, R1 = isobutyl, n = 2), 7j
=
6a (R = H, R1 = isopropyl), 6b (R = H, R1 = cyclobutyl),
6c (R = H, R1 = cyclopentyl), 6d (R = 3-F, R1
isopropyl), 6e (R = 2-F, R1 = isopropyl), 6f (R = 2-Cl,
R1 = isopropyl), 6g (R = 2-Cl, R1 = isobutyl), 6h (R = 2-
CH3, R1 = isopropyl), 6i (R = 3-CH3, R1 = isopropyl)
=
=
(R = 2-F, R1 = isopropyl, n = 1), 7k (R = 2-CH3, R1
=
isopropyl, n = 1), 7l (R = 3-CH3, R1 = isopropyl, n = 1)
O
R2
O
R
N
n
R1
e
68 - 73 %
R
NH
n
R1
N
N
N
N
H
H
O
O
2
8
2a (R = H, R1 = isopropyl, R2 = isopropyl, n =1), 2b (R = H, R1 = cyclobutyl,
R2 = isopropyl, n = 1), 2c (R = H, R1 = cyclopentyl, R2 = isopropyl, n =1), 2d
(R = H, R1 = isopropyl, R2 = cyclobutyl, n = 1), 2e (R = H, R1 = isopropyl,
R2 = isobutyl, n = 1), 2f (R = H, R1 = isopropyl, R2 = isopropyl, n = 2), 2g
(R = H, R1 = cyclobutyl, R2 = isopropyl, n = 2), 2h (R = 3-F, R1 = isopropyl,
R2 = cyclobutyl, n = 1), 2i (R = 2-F, R1 = isopropyl, R2 = cyclobutyl, n = 2),
8a (R = H, R1 = isopropyl, n =1), 8b (R = H, R1
cyclobutyl, n = 1), 8c (R = H, R1 = cyclopentyl, n =1),
8d (R = H, R1 = isopropyl, n =2), 8e (R = H, R1
cyclobutyl, n = 2), 8f (R = 3-F, R1 = isopropyl, n = 2),
8g (R = 2-F, R1 = isopropyl, n = 2), 8h (R = 2-Cl, R1 =
isopropyl, n = 1), 8i (R = 2-Cl, R1 = isobutyl, n = 2), 8j
(R = 2-F, R1 = isopropyl, n = 1), 8k (R = 2-CH3, R1 =
isopropyl, n = 1), 8l (R = 3-CH3, R1 = isopropyl, n = 1)
=
=
2j (R = 2-F, R1 = isopropyl, R2 = isopropyl, n = 2), 2k (R = 2-Cl, R1
=
isopropyl, R2 = cyclobutyl, n = 1), 2l (R = 2-Cl, R1 = cyclobutyl, R2 =
isopropyl, n = 2), 2m (R = 2-Cl, R1 = cyclobutyl, R2 = cyclobutyl, n = 2), 2n
(R = 2-F, R1 = isopropyl, R2 = cyclobutyl, n = 1), 2o (R = 2-CH3, R1
=
isopropyl, R2 = cyclobutyl, n = 1), 2p (R = 3-CH3, R1 = isopropyl, R2 =
isopropyl, n = 1), 2q (R = 3-CH3, R1 = isopropyl, R2 = cyclobutyl, n = 1)
Reagents and conditions:a) 1-substituted-4-piperidinol, DEAD, PPh3, THF, rt, 16 h. b) KOH, Ethanol: Water,
reflux, 4 h. c) PyBOP, Et3N, appropriate Boc protected amine, DCM, rt, 16 h. d) CF3CO2H, DCM, rt, 4 h. e)
Carbonyl compound, NaBH(OAc)3, DCE, rt, 2 h.
5

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OH
N
OH
N
OH
N
OH
N
(I)
(IV)
(III)
(II)
Fig. 3. N-Substituted-4-piperidinols
2-(3-Methoxyphenyl) ethylamine (9) on reaction with either N-benzyl-3-pyrrolidinone or N-
benzyl-4-piperidone under reductive amination conditions gave intermediates 10a-10b, which on
further reaction with ethyl chloroformate yielded intermediates 11a-11b respectively. Reaction of
intermediates 11a-11b with a mixture of POCl3 and P2O5 at 110 °C gave the cyclized
intermediates 12a-12b, which on O-demethylation with BBr3 at RT afforded intermediates 13a-
13b. Intermediates 13a-13b on reaction with N-substituted-4-bromo piperidine in presence of
base gave the ether intermediates 14a-14b that on Pd/C assisted debenzylation yielded
pyrrolidine derivatives 15a-15b. The intermediates 15a-15b on reductive amination with various
carbonyl compounds yielded the targeted compounds 3 (a-d) (Scheme 2).
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Scheme 2. Synthesis of targeted Compounds 3
R
R
b
a
N
n
H3CO
NH2
H3CO
N
68 - 73 %
60 - 65 %
H
10
9
10a (R = H, n = 1),
10b (R = H, n = 2),
(R = H)
O
R
N
R
N
d
n
c
N
H3CO
48 - 53 %
40 - 45 %
H3CO
N
n
COOEt
12
11
12a (R = H, n = 1),
12b (R = H, n = 2),
11a (R = H, n = 1),
11b (R = H, n = 2),
O
O
R
N
R1
e
R
f
N
N
N
n
N
n
65 - 70 %
55 - 60 %
O
HO
13
14
14a (R = H, R1 = isopropyl, n = 1),
14b (R = H, R1 = cyclobutyl, n = 2)
13a (R = H, n = 1),
13b (R = H, n = 2),
O
O
R
R
R2
NH
n
N
R1
N
R1
g
N
N
N
n
60 - 65 %
O
O
15
3
15a (R = H, R1 = isopropyl, n= 1),
15b (R = H, R1 = cyclobutyl, n = 2)
3a (R = H, R1 = isopropyl, R2 = isopropyl, n = 1),
3b (R = H, R1 = isopropyl, R2 = cyclobutyl, n = 1),
3c (R = H, R1 = isopropyl, R2 = cyclobutyl, n = 2),
3d (R = H, R1 = cyclobutyl, R2 = cyclobutyl, n = 2),
Reagents and conditions:a) N-benzyl-3-pyrrolidinone or N-benzyl-4-piperidone, NaBH(OAc)3, DCE, rt, 2 h. b)
Ethyl chloroformate, Et3N, THF, rt, 2 h. c) POCl3, P2O5, 110 °C, 2 h. d) BBr3, rt, 16 h e) N-substituted-4-bromo
piperidine, K2CO3, DMF, 145 °C, 2 h. f) 10 % Pd/C, methanol, H2(↑), 16 h.g) Carbonyl compound, NaBH(OAc)3,
DCE, rt, 2 h.
7

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2.2. Structure-activity relationships
Several of the synthesized compounds depicted in Table 1 showed improved in vitro affinity
indicating the importance of introduction of second basic center in western domain of Compound
1. In Compounds 2, the diverse substitutions on piperidine nitrogen like isopropyl (2a, Ki = 5.7
nM), cyclobutyl (2b, Ki = 26.9 nM) and cyclopentyl (2c, Ki = 8.6 nM) maintained the in vitro
affinity with 2a being the most potent. By keeping the isopropyl substitution of 2a constant, two
additional analogs were synthesized wherein R2 position was substituted with cyclobutyl (2d, Ki
= 2.5 nM) and isobutyl (2e, Ki = 1.0 nM) groups. These two compounds have shown better in
vitro affinity as compared to that of 2a. Replacing the five membered pyrrolidine moiety in
eastern domain of Compounds 2 with six membered piperidine resulted in compounds 2f (Ki =
8.3 nM) and 2g (Ki = 1.6 nM) that maintained potent in vitro binding affinity towards H3R.
Compound 2f exhibited equipotent binding affinity compared to its parent compound 2a (Ki =
5.7 nM) whereas compound 2g was 16 fold more potent compared to its parent compound 2b.
From these results, it could be concluded that insertion of a piperidine ring in the eastern domain
of Compound 1 is beneficial as it enhances the binding affinities towards H3R. In order to
expand the SAR, and also to explore the effect of introduction of small substituents such as
chloro, fluoro and methyl groups on central aromatic ring of Compounds 2, new analogs with
small substituents on central aromatic ring were synthesized and checked their in vitro affinity
towards H3R. Insertion of fluoro, chloro at ortho and meta position on the central aromatic ring
with respect to amide linkage retained the affinity as can be seen from the Ki values of
compounds 2h (Ki = 7.3 nM), 2i (Ki = 10.5 nM), 2j (Ki = 11.4 nM), 2k (Ki = 18.4 nM), 2l (Ki =
7.0 nM), 2m (Ki = 6.7 nM) and 2n (Ki = 5.7 nM). From the Ki values, it is evident that though the
insertion of halogen group has produced potent compounds both in pyrrolidine and piperidine
8

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bearing analogs, however these compounds are less potent in terms of in vitro binding affinity as
compared to compounds having no substitution on central aromatic ring. Insertion of a methyl
group at ortho and meta position of central aromatic ring led to compounds 2o (Ki = 9.2 nM), 2p
(Ki= 1357 nM) and 2q (Ki = 384.3 nM). As can be seen from in vitro results of these
compounds, introduction of methyl group at ortho position (2o) is a favorable modification,
whereas methyl substitution at meta position (2p, 2q) found to be detrimental to affinity. In
summary, the substitution of alkyl groups (isopropyl and isobutyl) or cycloalkyl groups
(cyclobutyl, cyclopentyl) on both the basic nitrogen atoms of alicyclic rings is well tolerated.
9

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Table 1: hH3R bindinga and functional potenciesb of Compounds 2 (a-q) & 3 (a-d)
R2
n
N
O
O
R
6
R2
N
n
R1
R1
R
5
N
N
N
N
1
H
O
2
O
4
3
Compounds 2
Compounds 3
hH3
R
hH3R
hH3R
n
Comp.
R
R1
R2
IC50
(nM)
Kb
Ki (nM)
(nM)
1
103*
2a
H
H
H
H
H
H
isopropyl
isopropyl
isopropyl
1
1
1
1
1
2
5.7*± 0.3 0.9
26.9*±
323.8
273.9
2b
2c
2d
2e
2f
cyclobutyl
3.9
8.6*± 0.9
cyclopentyl isopropyl
1
2.5*±
0.23
isopropyl
isopropyl
isopropyl
cyclobutyl
isobutyl
1.0*± 0.1 1.20 458.7
8.3 ±
0.73
isopropyl
1.6 ±
0.18
7.3*±
2g
2h
2i
H
cyclobutyl
isopropyl
isopropyl
isopropyl
isopropyl
cyclobutyl
cyclobutyl
isopropyl
2
1
2
2
3-fluoro
2-fluoro
2-fluoro
0.65
10.5 ±
1.3
11.4 ±
1.75
2j
18.4*±
1.4
2k
2l
2-chloro
2-chloro
2-chloro
isopropyl
cyclobutyl
cyclobutyl
cyclobutyl
isopropyl
cyclobutyl
1
2
2
7 ± 1.1
6.7 ±
0.71
2m
5.7*±
0.41
9.2*± 1.3
1357*±
2n
2o
2p
2-fluoro
2-methyl
3-methyl
isopropyl
isopropyl
isopropyl
cyclobutyl
cyclobutyl
isopropyl
1
1
1
29
384.3*±
17
2q
3-methyl
isopropyl
cyclobutyl
1
2.1* ±
0.31
3a
3b
H
H
isopropyl
isopropyl
isopropyl
1
1
0.1
14.3
1.2*±
cyclobutyl
10

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0.15
1.8 ±
0.21
3c
3d
H
H
isopropyl
cyclobutyl
cyclobutyl
2
2
cyclobutyl
0.8 ± 0.1 0.03 4.8
All compounds were characterized by 1H NMR, ESI-MS and purity was assessed using HPLC.
aThe affinity of the test compounds were determined using in-vitro radioligand binding assay using recombinant
human H3R membrane and [3H] R-α-Methyl histamine radioligand and are mean of two independent
experiments.[16]
bHuman H3 functional assay measuring cAMP utilizing a luciferase reporter geneassay in CHO-K1 cells stably
expressing in human H3R.
*Values are generated on racemic compounds and are mean of three independent experiments.
2.3. Discovery profiling, pharmacokinetics and in vivo activity
The two potent compounds (2a and 2e) were further profiled for their hERG (human Ether-a-go-
go-Related Gene) inhibition and human H3 functional activity. Both 2a and 2e compounds, have
shown mild hERG inhibition of negligible 21 % and 35 % respectively when tested at 1 &10 µM
concentration respectively, indicating cardiovascular safety concerns. Compounds 2a and 2e
found to be antagonists when tested in luciferase reporter gene based functional assay on human
H3R with IC50 values of 323.8 and 458.7 nM respectively (Table 1).
The compounds 2ª and 2e were evaluated for their CYP affinity and metabolic stability. The IC50
values were found to be >45 µM for compound 2a and >30 µM for compound 2b at
metabolizing enezymes CYP2D6 and CYP3A4, indicating minimal drug-drug interaction
potential. The compound 2a was found to be 4% metabolized in rat and 12% metabolized in
human liver microsomes whereas compound 2e was found to be 8% and 18% metabolized in rat
and human liver microsomes respectively, confirming good metabolic stability of these
compounds. Both the compounds 2a and 2e were further profiled for their pharmacokinetic
properties in male Wistar rats at 5 mg/kg p.o. dose. Compound 2a displayed good oral exposure
with AUC 444 ± 29 ng*h/mL and Cmax 122 ± 85 ng/mL, with longer i.v. half-life (t1/2 = 11.67 h),
and 75 ± 18 % mean oral bioavailability whereas compound 2e displayed low oral exposure with
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AUC 281 ± 38 ng*h/mL and Cmax18 ± 1 ng/mL with longer half life of 17.51 h and 81 ± 11 %
oral bioavailability (Table 2). The high oral bioavailability of compounds 2a and 2e in rats
correlated well with theirhigh metabolic stability in rat liver microsomes. However, both
compounds 2a and 2e showed lowbrain penetration at10 mg/kg p.o., when assessed in male non
fasted Wistar rats.
Poor brain penetration coupled with longer rodent half-lives of lead Compounds 2a and 2e
warranted further modification around this scaffold. As a next step of SAR, conformationally
restrained analogs of Compounds 2 with subsequent reduction in hydrogen bond count [17,18]
were undertaken as next targets. The amidic nitrogen in Compounds 2 was connected with
central core to obtain Compounds 3 as summarized in Fig. 1. All the compounds (3a-3d)
synthesized in this series retained the high potency towards histamine H3R (Table 1). Among all
the synthesized derivatives, compound 3d was most potent with a Ki= 0.8 ± 0.1 nM whereas the
binding affinity values for compounds 3a, 3b and 3c were found to be Ki= 2.1 ± 0.31, Ki= 1.2 ±
0.15, and 1.8 ± 0.21 respectively. Representative analogs 3a and 3d were antagonists when tested
in functional assay with IC50 values 14.3 nM and 4.8 nM respectively (Table 1). The most potent
compound 3d was further evaluated for its metabolic stability and hERG inhibition potential.
Compound 3d was metabolically stable in rat (6% metabolized) and human (10% metabolized)
liver microsomes. The hERG inhibition was minimal as it showed 23 % and 26 % inhibition at 1
µM and 10 µM concentrations respectively, a similar metabolic stability profile as compared to
compound 2a. The pharmacokinetic profile of 3d was assessed in male Wistar rats at 3 mg/kg,
p.o. dose. Compound 3d has shown high oral exposure with AUC 1429 ± 626 ng*h/mL anda
Cmax of 80 ± 35 ng/mL withimprovedbrain penetration (Cbrain/Cblood = 0.54 ± 0.16 at 10 mg/kg
12

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p.o). However it showed longer i.v. half-life (t1/2) of 23.49 h and moderate bioavailability (Table
2).
Table 2: Pharmacokinetic profile of compounds 2a, 2e and 3d in male Wistar ratsa
Compound
p.o.
2a
2e
3d
dose
Cmax (ng/mL)
AUCt (ng.h/mL)
F (%)
5 mg/kg
122 ± 85
444 ± 29
75 ± 18
5 mg/kg
18 ± 1
281 ± 38
81 ± 11
3 mg/kg
80 ± 35
1429 ± 626
27 ± 4
i.v.
dose
t1/2 (h)
5 mg/kg
11.67
5 mg/kg
17.51
3 mg/kg
23.49
Vz (mL/kg)
CL (mL/h/kg)
119 ± 32
90 ± 8
101 ± 10
97 ± 11
13 ± 5
2 ± 1
aFasted male Wistar rats, Vehicle used: water for injection for both oral and i.v. routes.
Dosing Volumes: 10 mL/kg for p.o and 2 mL/kg for i.v.
In order to assess the translation of in vitro affinity to in vivo target engagement, cortical
histamine H3 receptor occupancy of compound 3d was determined [19] using GSK-189254 as
non-radio lablelled tracer. At doses, of 3 mg/kg, 10mg/kg and 30 mg/kg, compound 3d showed
dose dependent receptor occupancy with an ED50 = 15.54 mg/kg for histamine H3 receptors (Fig.
4), confirming the target engagement.The brain exposures associated with receptor occupancy of
compound 3d depicted in Table 3. Having achieved the brain penetration and receptor
occupancy, our focus shifted on to shorten the half life so as to have required window of efficacy
with reduced side effect profile. The results of our focussed SAR to have appropriate half lives
would be published in due course of time.
Table 3: Brain and plasma exposures of compound 3d in rat tested at 3, 10 and 30 mg/kg oral
doses
Compound 3d
Dose; mg/kg, p.o.
Brain (ng/g)
Avg SEM
Plasma (ng/mL)
Avg
3.7
SEM
0.4
3
10
30
28.5
175.3
765.4
6.0
33.5
102.4
15.7
76.3
3.6
6.5
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Fig 4. H3 receptor occupancy data of compound 3d
100
75
50
25
0
3
10
30
Compound 3d (mg/kg, p.o.)
3. Conclusion
In summary, a new series of Compounds 2, synthesized by introducing the second basic center
have shown improvement in binding affinities towards hH3R, with good oral bioavailability. To
overcome the issues of longer half-life and low brain penetration with these compounds, the
constrained analogs of Compounds 2 were designed and synthesized which resulted in
Compounds 3. The Compounds 3 found to be potent at H3R, showed adequate brain penetration,
showed good RO and exhibited acceptable pharmacokinetic properties. Further efforts are in
progress on this scaffold and also on chemically different scaffolds (non-chemotypes), to
optimize the overall biological properties, details of which will be published in due course.
4. Experimental section
4.1. Biological testing
The affinity of the test compounds were determined using in-vitro radioligand binding assay
using recombinant human H3R membrane and [3H] R-α-Methyl histamine radioligand and are
mean of two independent experiments. The metabolic stability of compounds was tested in
pooled rat and human liver microsomes, results were expressed as % metabolized at 30 min post
incubation. CYP3A4 and 2D6 inhibitory potential was evaluated by incubating the test
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compound (0.1 – 45 µM) in duplicates with pooled human liver microsomes in presence of
isoform-specific substrate (midazolam-3A4 or dextromethorphan-2D6) and metabolites were
measured by LC-MS/MS, and IC50 was determined. Oral bioavailability and brain penetrations
study was assessed in male Wistar rats (225 ± 25 gm). Oral bioavailability was assessed
following 3 mg/kg, p.o. (overnight fasted) and intravenous administration, blood samples were
collected from jugular vein cannulated rats up to 24 hours post dose. Brain penetration was
determined in discrete manner in rats; compounds were formulated in water and administered
orally at 10 mg/kg. Blood samples were removed via cardiac puncture by using isoflurane
anesthesia at one hour post dose. The concentrations of the compound in plasma and brain were
determined using LC-MS/MS method. Pharmacokinetic parameters were calculated by non
compartmental model by using WinNonLin 5.0.1 version Software package. Extent of brain-
plasma ratio was calculated (Cb/Cp).
4.2. General chemistry methods
All the reagents and chemicals used were of “reagent grade”. The derivatives of 4-
hydroxymethyl benzoate, 2-(3-methoxyphenyl) ethylamine and 1-substituted-4-piperidinols were
commercially procured and used as such without further purification. Thin layer chromatography
(TLC) was performed on Merck silica gel 60 F254 plates, and spots were visualized in UV light
(254 nm) and/or iodine. Purification of compounds was done by column chromatography
performed using 60-120 mesh silica. Purity of the final compounds (>95%) was established
1
using Agilent 1100 high-performance liquid chromatography (HPLC) system. H NMR spectra
were obtained on a Bruker NMR spectrometer (Fallanden, Switzerland) at 400 MHz and 13C
spectra at 100 MHz. Tetramethylsilane (TMS) was used as an internal standard, chemical shift
values are expressed in parts per million (δ) and coupling constants are expressed in Hz.
15

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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’. Electrospray ionization mass spectra were recorded on a API 2000 triple quadrupole
instrument (MDS-SCIEX, Concord, Ontario, Canada).
4.2.1. Methyl 4-(1-isopropyl piperidin-4-yloxy) benzoate (5a)
A solution of methyl 4-hydroxy benzoate 4a (9.0 g, 59.21 mmol), 1-isopropyl-4-piperidinol (I)
(11.00 g, 76.97 mmol) and triphenylphosphine (23.26 g, 88.81 mmol) in tetrahydrofuran(90 mL)
was treated with diethyl azodicarboxylate (13.9 mL, 88.81 mmol) dropwise at 0 °C. The mixture
was left at 0 °C for 15 min and stirred overnight at room temperature. After completion of the
reaction, the reaction mixture was poured onto cold water (300 mL), the resulting solution was
extracted with dichloromethane (3 x 100 mL), washed with brine (50 mL), dried over Na2SO4,
concentrated in vacuo and purified by flash chromatography (methanol: chloroform, 1:9) to yield
8.03 g (49.0 %) of compound 5a. 1H NMR (400 MHz, DMSO – d6) δ: 7.87 (2H, d, J = 8.72 Hz),
7.03 (2H, d, J = 8.74 Hz), 4.42 – 4.46 (1H, m), 3.79 (3H, s), 2.70 – 2.78 (3H, m), 2.33 – 2.38
(2H, m), 1.94 – 1.99 (2H, m), 1.58 – 1.68 (2H, m), 0.96 (6H, d, J = 6.30 Hz); ESI mass (m/z):
278.4 (M+H)+.
4.2.2. 4-(1-Isopropyl piperidin-4-yloxy) benzoic acid (6a)
Potassium hydroxide (4.85 g, 86.64 mmol) was added to a mixture of methyl 4-(1-isopropyl
piperidin-4-yloxy) benzoate 5 (8.00 g, 28.88 mmol), ethanol (40 mL) and water (20 mL) at room
temperature.The mixture was stirred at 80 °C for 3 hours. After completion of the reaction, the
reaction mass was concentrated under reduced pressure and quenched onto chilled water (200
mL). The resulting mass was acidified with 2N HCl and extracted with dichloromethane (3 x 100
mL). The organic layer was washed with brine (50 mL), dried over Na2SO4 and concentrated in
16

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1
vacuo to yield 5.70 g (58.0 %) of compound 6a. H NMR (400 MHz, DMSO – d6) δ: 10.95 –
11.01 (1H m) , 7.86 – 7.89 (2H, m), 7.05 – 7.12 (2H, m), 4.69 – 4.88 (1H, m), 3.23 – 3.26 (1H,
m), 3.27 – 3.41 (2H, m), 3.01 – 3.14 (2H, m), 2.30 – 2.37 (1H, m), 2.19 – 2.22 (1H, m), 2.03 –
2.15 (2H, m), 1.27 (6H, d, J = 6.68 Hz); ESI mass (m/z): 264.3 (M+H)+.
4.2.3. N-(1-tert butyloxycarbonyl pyrrolidin-3-yl)-4-(1-isopropyl piperidin-4-yloxy) benzamide
(7a)
4-(1-Isopropyl piperidin-4-yl oxy) benzoic acid 6 (6.00 g, 28.81 mmol), PyBOP (17.79 g, 34.22
mmol), Et3N (4.67 mL, 34.22 mmol) and tert butyl 3-amino pyrrolidine-1-carboxylate (5.09 g,
27.37 mmol) in dichloromethane (40 mL) was stirred overnight at room temperature. After
completion of the reaction, the mass was diluted with dichloromethane (200 mL), washed with
water (100 mL), dried over Na2SO4, concentrated in vacuo and purified by flash chromatography
1
(methanol: chloroform, 0.5:9.5) to yield 5.70 g (58.0 %) of compound 7a. H NMR (400 MHz,
DMSO – d6) δ: 8.34 (1H, d, J = 6.32 Hz), 7.79 (2H, d, J = 8.71 Hz), 6.98 (2H, d, J = 8.75 Hz),
4.35 – 4.45 (2H, m), 3.38 – 3.53 (2H, m), 3.13 – 3.18 (1H, m), 2.74 (3H, bs), 2.39 (1H, bs), 1.85
– 2.07 (5H, m), 1.59 – 1.61 (2H, m), 1.38 (9H, s), 0.98 (6H, d, J = 6.40 Hz); ESI mass (m/z):
432.5 (M+H)+.
4.2.4. N-(Pyrrolidin-3-yl)-4-(1-isopropyl piperidin-4-yloxy) benzamide (8a)
A solution of N-(1-tert butyloxycarbonyl pyrrolidin-3-yl)-4-(1-isopropyl piperidin-4-yloxy)
benzamide 7 (5.70 g, 13.22 mmol) in dichloromethane (50 mL) was treated with trifluoroacetic
acid (10.12 mL, 132.25 mmol) dropwise at 10 °C. The mixture was left at 10 °C for 15 min and
further stirred for 4 hours at room temperature. After completion of the reaction, mass was
quenched onto cold water (200 mL) and basified with lye solution. The layers were separated
and the aqueous layer was extracted with dichloromethane (2 x 100 mL), washed with brine (50
17

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1
mL), dried over Na2SO4 and concentrated in vacuo to yield 3.67 g (84.0 %) of compound 8a. H
NMR (400 MHz, DMSO – d6) δ: 8.12 (1H, d, J = 7.06 Hz ), 7.77 (2H, d, J = 8.67 Hz), 6.96 (2H,
d, J = 8.69 Hz), 4.38 – 4.43 (1H, m), 4.25 – 4.27 (1H, m), 2.87 – 2.93 (2H, m), 2.59 – 2.71 (5H,
m), 2.28 – 2.34 (2H, m), 1.92 – 1.95 (3H, m), 1.55 – 1.62 (3H, m), 1.21 – 1.32 (1H, m), 0.96
(6H, d, J = 6.54 Hz); ESI mass (m/z): 332.3 (M+H)+.
4.2.5.N-(1-Isopropyl pyrrolidin-3-yl)-4-(1-isopropyl piperidin-4-yloxy)benzamide (2a)
A solution of N-(pyrrolidin-3-yl)-4-(1-isopropyl piperidin-4-yl oxy) benzamide 8 (3.60 g, 10.87
mmol) and acetone (0.87 mL, 11.96 mmol) in ethylene dichloride (30 mL) was treated with
sodium triacetoxyborohydride (4.61 g, 21.75 mmol) portion wise and the mixture was stirred at
room temperature for 2 hours. After completion of the reaction, the mass was quenched onto cold
water (100 mL), basified with lye solution and layers separated. Aqueous layer was extracted
with dichloromethane (3 x 50 mL) and the combined organic layer was washed with brine (50
mL), dried over Na2SO4, concentrated in vacuo and purified by flash chromatography (methanol:
1
chloroform, 1:9) to yield 2.92 g (72.0 %) of compound 2a. H NMR (400 MHz, DMSO – d6) δ:
8.24 (1H, d, J = 6.91 Hz ), 7.79 (1H, d, J = 8.63 Hz), 6.95 (1H, d, J = 8.66 Hz), 4.38 – 4.43 (1H,
m), 4.31 – 4.34 (1H, m), 2.77 – 2.82 (1H, m), 2.62 – 2.70 (4H, m), 2.44 – 2.58 (1H, m), 2.24 –
2.42 (4H, m), 2.01 – 2.12 (1H, m), 1.79 – 1.99 (2H, m), 1.64 – 1.78 (1H, m), 1.49 – 1.62 (2H,
m), 0.9 (6H, d, J = 6.15 Hz), 0.96 (6H, d, J = 6.51 Hz); HPLC: 96.03%; ESI mass (m/z): 374.4
(M+H)+. Anal. (C22H35N3O2) C, H, N.: C, 70.76; H, 9.42; N, 11.29
4.2.6. N-(1-Benzyl piperidin-4-yl)-2-(3-methoxy phenyl) ethanamine (10b)
A solution of 2-(3-methoxyphenyl) ethylamine 9 (6.0 g, 39.7 mmol) and 1-benzyl-4-piperidone
(8.26 g, 43.7 mmol) in ethylene dichloride (150 mL) was treated with sodium
triacetoxyborohydride (16.84 g, 79.47 mmol) portion wise and the mixture was stirred at room
18

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temperature for 2 hours. The reaction mixture was quenched onto cold water (200 mL) and
basified with lye solution. The layers were separated and the aqueous layer was extracted with
dichloromethane (2 x 100 mL). The combined organic layer was washed with brine (50 mL),
1
dried over Na2SO4 and concentrated in vacuo to yield 8.04 g (62.5 %) of compound 10b. H
NMR (400 MHz, CDCl3) δ: 7.30 – 7.36 (4H, m), 7.19 – 7.27 (2H, m), 6.75 – 6.81 (3H, m), 3.80
(3H, s), 3.49 (2H, s), 2.88 – 2.92 (2H, m), 2.74 – 2.86 (4H, m), 2.44 – 2.50 (1H, m), 1.98 – 2.05
(2H, m), 1.78 – 1.86 (2H, m), 1.61 – 1.72 (3H, m); ESI mass (m/z): 325.3 (M+H)+.
4.2.7. N-(1-Benzyl piperidin-4-yl) N-(ethoxycarbonyl) 2-(3-methoxy phenyl) ethanamine (11b):
N-(1-Benzyl piperidin-4-yl)-2-(3-methoxy phenyl) ethanamine 10b (8.0 g, 24.69 mmol) was
dissolved in tetrahydrofuran (30 mL) and added triethylamine (10.1 mL, 74.07 mmol) at room
temperature. To the resulting mass, ethyl chloroformate (2.86 mL, 37.03 mmol) was added drop
wise at 0 °C. The mixture was stirred at room temperature for 2 hours and the reaction mixture
was quenched onto cold water (200 mL). The resulting mass was extracted with dichloromethane
(2 x 100 mL), washed with brine (50 mL), dried over Na2SO4 and concentrated in vacuo to yield
1
7.03 g (72.0 %) of compound 11b. H NMR (400 MHz, CDCl3) δ: 7.32 – 7.39 (4H, m), 7.19 –
7.28 (2H, m), 6.75 – 6.81 (3H, m), 4.15 – 4.20 (2H, m), 3.81 (3H, s), 3.52 (2H, s), 3.38 – 3.44
(2H, m), 2.92 – 2.98 (2H, m), 2.72 – 2.86 (2H, m), 2.02 – 2.18 (2H, m), 1.52 – 1.86 (5H, m),
1.25 – 1.32 (3H, m); ESI mass (m/z): 397.3 (M+H)+.
4.2.8. 2-(1-Benzyl piperidin-4-yl)-6-methoxy-3,4-dihydro-2H-isoquinolin-1-one (12b)
Phosphorus pentoxide (7.42 g, 53.03 mmol) was added to a mixture of N-(1-Benzyl piperidin-4-
yl) N-(ethoxycarbonyl) 2-(3-methoxy phenyl) ethanamine 11b (7.00 g, 17.67 mmol) and
phosphorus oxychloride (40 mL) at room temperature under nitrogen atmosphere. The mixture
was stirred at 110 °C for 2 hours. After completion of the reaction, mass was concentrated under
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reduced pressure and quenched onto chilled water (500 mL). The resulting mass was basified
with lye solution (pH ~ 13) and extracted with dichloromethane (3 x 100 mL). The combined
organic layer was washed with brine (100 mL), dried over Na2SO4, concentrated in vacuo and
purified by flash chromatography (ethyl acetate: hexane, 1:1) to yield 2.72 g (44.0 %) of
1
compound 12b. H NMR (400 MHz, CDCl3) δ: 8.02 (1H, d, J = 8.63 Hz), 7.26 – 7.34 (5H, m),
6.82 – 6.85 (1H, dd, J = 2.38, 8.62 Hz), 6.65 (1H, d, J = 2.15 Hz), 4.68 – 4.74 (1H, m), 3.83
(3H, s), 3.55 (2H, s), 3.44 – 3.48 (2H, t, J = 6.40 Hz ), 2.96 – 3.00 (2H, m), 2.87 – 2.90 (2H, t, J
= 6.48 Hz ), 2.16 – 2.22 (2H, m), 1.66 – 1.84 (4H, m);ESI mass (m/z): 351.4 (M+H)+.
4.2.9. 2-(1-Benzyl piperidin-4-yl)-6-hydroxy-3,4-dihydro-2H-isoquinolin-1-one (13b)
Borontribromide (16.2 mL, 6.0 vol) was added to 2-(1-benzyl piperidin-4-yl)-6-methoxy-3,4-
dihydro-2H-isoquinolin-1-one 12b (2.70 g, 7.71 mmol) at 0 °C. The reaction mass was stirred
overnight at room temperature. After completion of the reaction, the mass was quenched onto
cold water (200 mL) and basified with aqueous ammonia. The resulting mass was extracted with
dichloromethane (2 x 100 mL), washed with brine (50 mL), dried over Na2SO4 and concentrated
1
in vacuo to yield 1.30 g (50.2 %) of compound 13b. H NMR (400 MHz, DMSO – d6) δ: 9.96
(1H, s), 7.54 – 7.68 (3H, m), 7.24 – 7.30 (3H, m), 6.66 – 6.68 (1H, dd, J = 2.24, 8.48 Hz), 6.59
(1H, d, J = 1.88 Hz), 4.32 – 4.44 (1H, m), 3.47 (2H, s), 3.23 – 3.36 (2H, m), 2.85 – 2.92 (2H, m),
2.77 – 2.80 (2H, m), 1.92 – 2.06 (2H, m), 1.71 – 1.80 (2H, m) 1.42 – 1.52 (2H, m); ESI mass
(m/z): 337.3 (M+H)+.
4.2.10.
2-(1-Benzyl
piperidin-4-yl)-6-(1-cyclobutyl
piperidin-4-yloxy)-3,4-dihydro-2H-
isoquinolin-1-one (14b)
A solution of 2-(1-benzyl piperidin-4-yl)-6-hydroxy-3,4-dihydro-2H-isoquinolin-1-one 13b (1.3
g, 3.86 mmol), potassium carbonate (1.6 g, 11.60 mmol) and 4-bromo-1-cyclobutyl-piperidine
20

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(1.01 g, 4.64 mmol) in N, N - dimethylformamide (10 mL) were stirred for 2 hours at 145 °C.
After completion of the reaction, the mass was cooled to room temperature, quenched onto cold
water (50 mL). The mass was extracted with dichloromethane (3 x 50 mL), washed with brine
(50 mL), dried over Na2SO4, concentrated in vacuo and purified by flash chromatography
1
(methanol: chloroform, 1:9) to yield 1.06 g (58.0 %) of compound 14b. H NMR (400 MHz,
CDCl3) δ: 8.01 (1H, d, J = 8.64 Hz), 7.25 – 7.35 (5H, m), 6.82 – 6.85 (1H, dd, J = 2.47, 8.65
Hz), 6.66 (1H, d, J = 2.36 Hz), 4.68 – 4.75 (1H, m), 4.34 – 4.44 (1H, m) 3.55 (2H, s), 3.45 –
3.51 (2H, m), 2.98 – 3.04 (2H, m), 2.87 – 2.90 (2H, t, J = 6.46 Hz), 2.78 – 2.82 (1H, m), 2.56 –
2.66 (2H, m) 1.98 – 2.33 (8H, m), 1.81 – 1.96 (6H, m), 1.53 – 1.78 (4H, m); ESI mass (m/z):
474.3 (M+H)+.
4.2.11. 6-(1-Cyclobutyl piperidin-4-yloxy)-2-(piperidin-4-yl)-3,4-dihydro-2H-isoquinolin-1-one
(15b)
Hydrogen gas was bubbled through a solution of 2-(1-benzyl piperidin-4-yl)-6-(1-cyclobutyl
piperidin-4-yloxy)-3,4-dihydro-2H-isoquinolin-1-one 14b (1.0 g, 2.11 mmol) over 10 % Pd/C in
methanol (50 mL) overnight at room temperature. The mixture was filtered through a pad of
1
celite and the filtrate was concentrated in vacuo to yield 0.53 g (66.4 %) of compound 15b. H
NMR (400 MHz, DMSO – d6) δ: 7.76 (1H, d, J = 8.54 Hz), 6.83 – 6.88 (2H, m), 3.28 – 3.40
(3H, m), 3.04 – 3.12 (2H m), 2.82 – 2.86 (2H, m), 2.62 – 2.69 (3H, m), 2.50 – 2.58 (2H, m), 2.02
– 2.08 (2H, m), 1.77 – 1.96 (8H, m), 1.54 – 1.63 (8H, m); ESI mass (m/z): 384.4 (M+H)+.
4.2.12. 2-(1-Cyclobutyl piperidin-4-yl)-6-(1-cyclobutyl piperidin-4-yloxy)-3,4-dihydro-2H-
isoquinolin-1-one (3d)
A solution of 6-(1-cyclobutyl piperidin-4-yloxy)-2-piperidin-4-yl-3,4-dihydro-2H-isoquinolin-1-
one 15b (0.53 g, 1.38 mmol) and cyclobutanone (0.10 g, 1.52 mmol) in ethylene dichloride (15
21

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mL) was treated with sodium triacetoxyborohydride (0.58 g, 2.76 mmol) portionwise and the
mixture was stirred at room temperature for 2 hours. After completion of the reaction, the mass
was quenched onto cold water (50 mL) and basified with lye solution (pH ~ 13). Aqueous layer
was extracted with dichloromethane (3 x 30 mL), washed with brine (30 mL), dried over
Na2SO4, concentrated in vacuo and purified by flash chromatography (methanol: chloroform,
1
0.5:9.5) to yield 0.38 g (64.0 %) of compound 3d. H NMR (400 MHz, CDCl3) δ: 7.99 – 8.02
(1H, dd, J = 3.32, 8.63 Hz), 6.83 – 6.85 (1H, dd, J = 2.44, 8.65 Hz), 6.66 (1H, d, J = 2.32 Hz),
4.68 – 4.76 (1H, m), 4.34 – 4.48 (1H, m), 3.47 (2H, d, J = 6.29 Hz), 2.99 – 3.24 (2H, m), 2.87 –
2.90 (2H, t, J = 6.46 Hz), 2.72 – 2.82 (2H, m), 2.63 (2H, bs), 2.19 (2H, bs), 2.03 – 2.07 (6H, m),
13
1.88 – 1.94 (6H, m), 1.80 – 1.86 (4H, m), 1.35 – 1.74 (6H, m); C NMR (100 MHz, CDCl3) δ:
164.11, 160.16, 139.96, 130.48, 122.65, 113.87, 113.62, 60.32, 60.27, 50.17, 49.25, 46.62, 40.07,
30.34, 28.84, 28.72, 27.49, 27.43, 14.16, 14.05; HPLC: 98.65%; ESI mass (m/z): 438.5 (M+H)+.
Anal. (C27H39N3O2) C, H, N.: C, 74.15; H, 8.95; N, 9.59
Animal care & ethics
Animal care and all the experiments were conducted according to protocols approved by the
Institutional Animal Ethics Committee of Suven Life Science Ltd, constituted according to the
directions of the Committee for the Purpose of Control and Supervision of Experiments on
Animals, India.
ASSOCIATED CONTENT
Supplementary Information. Characterization data for target compounds 2b-q and 3a-c and 1H
NMR data for compound 2a associated with this article can be found at free of charge via
Internet at
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AUTHOR INFORMATION
Ramakrishna Nirogi* Tel: +91-40-23556038/ 23541142, E-mail: nvsrk@suven.com
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. All the authors contributed equally.
ACKNOWLEDGEMENTS
The support received from discovery analytical department and Mr. Venkateswarlu Jasti, CEO,
Suven Life Sciences Ltd., Hyderabad, India is gratefully acknowledged. Authors further wish to
thank Dr. K. R. Sastry for critically reviewing the manuscript.
ABBREVIATIONS
H3R, histamine 3 receptor; AD, Alzheimer’s Disease; GPCR, G protein-coupled receptor; CNS,
Central Nervous System; SZ, schizophrenia; ADHD, attention-deficit hyperactivity disorder;
CYP-450, Cytochrome p450; hERG, human ether-a-go-go-related gene; ADME, absorption,
distribution, metabolism, and excretion; PL, phospholipidosis; SAR, Structure-Activity
Relationships; p.o., oral dosing; LC-MS/MS, Liquid Chromatography-Mass Spectrometry; CHO
cells, Chinese hamster ovary cells.
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ACCEPTED MANUSCRIPT
Highlights:
ꢀ About twenty novel Benzamide derivatives were synthesized and evaluated for their
affinity at H3R
ꢀ Most of the compounds showed good in vitro potency
ꢀ Selected compounds showed acceptable ADME properties
ꢀ Compound 3d showed good exposures and adequate brain penetration in rat
ꢀ Compound 3d also showed dose dependent receptor occupancy (RO) at H3R confirming
the target engagement
ꢀ Compound 3d had minimal hERG liability and showed minimal inhibition of tested
metabolizing enzymes CYP3A4 and CYP2D6 there by showing reduced drug-drug
interaction potential