Novel Tetrahydroquinazolinamines as Selective Histamine 3 Receptor Antagonists for the Treatment of Obesity

Article abstract of DOI:10.1021/acs.jmedchem.9b00241

The histamine 3 receptor (H3R) is a presynaptic receptor, which modulates several neurotransmitters including histamine and various essential physiological processes, such as feeding, arousal, cognition, and pain. The H3R is considered as a drug target for the treatment of several central nervous system disorders. We have synthesized and identified a novel series of 4-aryl-6-methyl-5,6,7,8-tetrahydroquinazolinamines that act as selective H3R antagonists. Among all the synthesized compounds, in vitro and docking studies suggested that the 4-methoxy-phenyl-substituted tetrahydroquinazolinamine compound 4c has potent and selective H3R antagonist activity (IC₅₀ < 0.04 μM). Compound 4c did not exhibit any activity on the hERG ion channel and pan-assay interference compounds liability. Pharmacokinetic studies showed that 4c crosses the blood brain barrier, and in vivo studies demonstrated that 4c induces anorexia and weight loss in obese, but not in lean mice. These data reveal the therapeutic potential of 4c as an anti-obesity candidate drug via antagonizing the H3R.

Full text of DOI:10.1021/acs.jmedchem.9b00241

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Article
Novel tetrahydroquinazolinamines as Selective Histamine
Receptor Antagonists for the Treatment of Obesity
3
Ajeet Kumar, Venkata Reddy Pasam, Ravi Kumar Thakur, Maninder Singh, Kartikey Singh,
Mahendra Shukla, Anubhav yadav, Shalini Dogra, Chandan Sona, Deepmala Umrao, Swati
Jaiswal, Hafsa Ahmad, Mamunur Rashid, Sandeep singh, Muhammad Wahajuddin, Anil Kumar
Dwivedi, Mohammad Imran Siddiqi, Jawahar Lal, Rama Pati Tripathi, and Prem N Yadav
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00241 • Publication Date (Web): 18 Apr 2019
Downloaded from http://pubs.acs.org on April 18, 2019
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Novel tetrahydroquinazolinamines as Selective Histamine 3 Receptor Antagonists for the
Treatment of Obesity
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1,†
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Ajeet Kumar , Venkata Reddy Pasam ,Ravi Kumar Thakur , Maninder Singh , Kartikey
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Singh , Mahendra Shukla , Anubhav Yadav , Shalini Dogra , Chandan Sona , Deepmala
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Umrao , Swati Jaiswal , Hafsa Ahmad , Mamunur Rashid , Sandeep K Singh , Muhammad
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Wahajuddin Anil Kumar Dwivedi , Mohammad Imran Siddiqi , Jawahar Lal , Rama Pati
Tripathi2,5,6,* and Prem N Yadav1,5*
1Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow-226031, India
2Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-
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26031, India. Molecular and Structural Biology Division, CSIR-Central Drug Research
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Institute, Lucknow- 226031, India. Pharmaceutics and Pharmacokinetics Division, CSIR-
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Central Drug Research Institute, Lucknow- 226031, India . Academy of Scientific and
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Innovative Research (AcSIR), New Delhi- 110001, India. Dean, National Institute of
Pharmaceutical Education and Research Raebareli, New Transit Campus, Bijnor Road, Sarojani
Nagar, Near CRPF Base Camp, Lucknow 226002, U.P., India
†These authors contributed equally
*Correspondence:rpt.cdri@gmail.com, pn.yadav@cdri.res.in
Key words: Histamine 3 receptor (H3R), GPCR, obesity, GloSensor, Quinazolinamines
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The histamine 3 receptor (H3R) is a presynaptic receptor, which modulates several
neurotransmitters including histamine and various essential physiological processes, such as
feeding, arousal, cognition and pain. The H3R is considered as a drug target for the treatment of
several central nervous system disorders. We have synthesized and identified a novel series of 4-
Aryl-6-methyl-5,6,7,8-tetrahydroquinazolinamines that act as selective H3R antagonist. Among
all the synthesized compounds, in vitro and docking studies suggested that 4-methoxy-phenyl
substituted tetrahydroquinazolinamine compound 4c has potent and selective H3R antagonist
activity (IC <0.04 μM). Compound 4c did not exhibit any activity on hERG ion channel and
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Pan-assay interference compounds (PAINS) liability. Pharmacokinetic studies showed that 4c
crosses the blood brain barrier and in vivo studies demonstrated that 4c induces anorexia, and
weight loss in obese, but not in lean mice. These data reveal the therapeutic potential of 4c as an
anti-obesity candidate drug via antagonizing the H3R.
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Introduction
The histamine 3 receptor (H3R) is an inhibitory G-protein coupled receptor (GPCR), which
is abundantly expressed in various brain regions, but modestly expressed in periphery.1
Activation of presynaptic H3Rs expressed on histaminergic neurons (autoreceptor) inhibits the
synthesis and release of histamine, while presynaptic hetero H3Rs (expressed on other neurons)
negatively regulates several other neurotransmitters such as dopamine, serotonin, and
norepinephrine.1, 2 Several rodent studies suggest that antagonists or inverse agonists of the H3R
increase the levels of various neurotransmitters, including histamine, dopamine, serotonin,
acetylcholine and norepinephrine.3-5 Multiple lines of evidence indicate that histaminergic
neurons originate from the tuberomammilary nucleus of hypothalamus and project to major brain
area including the satiety centre of the hypothalamus that play a crucial role in eating behavior
and energy homeostasis.2,3, 6-8 Indeed, several preclinical studies showed that H3R antagonists
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(
i.e. NNC38-1049, NNC38-1202, JNJ-5207852, GT-2394, A-423579 and A-331440) reduced the
food intake, body weight and blood glucose level in obese animals.7-13 On the basis of several
such preclinical observations two H3R antagonists SCH-497079 (NCT00642993,
NCT00673465) and HPP-404 (NCT01540864) were evaluated in clinical trials for the treatment
of obesity and diabetes, but both of these molecules were stopped from further development due
14
to the lack of desired level of efficacy. A recent study also suggests that Pitolisant (a selective
inverse agonist of the H3R) treatment reduces body weight in obese mice.15 Moreover,
Betahistine, a partial inverse H3R agonist and H1R partial agonist, induced modest weight loss
with minimal adverse events in women under 50 years of age and significant weight loss in
animals that developed obesity after olanzapine treatment.16-18 A recent study mentioned several
melanin-concentrating hormone receptor 1 (MCHR1) antagonist with anti-obesity effects that
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also exhibited H3R antagonist activity and suggested a polypharmacology approach for better
therapeutics for obesity.19
Besides metabolic conditions, H3R antagonists have also been evaluated for neuropathic
pain, sleeping disorder, cognitive impairments, Tourette syndrome and attention deficit
hyperactivity disorder (ADHD).14, 20-22 For example, GSK239512 (NCT01009255), MK-0249
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(NCT00420420), ABT-288 (NCT01018875) and SAR110894 (NCT01266525) were evaluated
in Phase 2 clinical trials for the treatment of Alzheimer’s disease, but none of them were
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approved due to lack of efficacy or side effects. Nevertheless, recently one H3R antagonist-
Pitolisant has been approved by the European Medicines Agency for the treatment of
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narcolepsy. Furthermore, the positive effect of betahistine (H3R antagonist and modest H1R
agonist) on weight gain induced by atypical antipshycotics, 18 but not robust and consistent effect
in obese subjects, suggest that variations in basal histaminergic tone in the brains of obese
populations might be responsible for inconsistencies between the preclinical and clinical effects
of H3R antagonist. Therefore, despite several setbacks in the development of H3R antagonists
for the treatment of obesity, more potent and selective H3R antagonist might still be useful to
treat obesity, especially when this disease has become a global epidemic with severe CNS related
co-morbidities.11, 24
Pyrimidines are considered a versatile scaffold from a medicinal chemistry point of view
because they display a broad spectrum of biological activities, including antihistaminic
activity.25-29 Examples of biologically active pyrimidine derivatives include 4-(4'-chlorophenyl)-
30
6
-(4''-methylphenyl)-2-hydrazinepyrimidine that show antidepressant properties
and
2
6
dihydropyrimidine containing calcium channel blockers
Aminopyrimidine moieties are also
found to show a wide variety of biological activity.31-34 Further, aminoquinazolines and
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tetrahydro-aminoquinazolines are also considered as aminopyrimidine derivatives, which have
been shown to exhibit analgesic, narcotic, sedative and hypoglycemic activities.35, 36 We have
also shown that 2-aminopyrimidine derivatives exhibit antitubercular and antidiabetic
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activities.37, 38
Figure 1: Designed prototype based on known tetrahydroquinazolinamine derived GPCRs
ligands.
Interestingly, Savall et al. have described a tricyclic aminopyrimidine derivative as a
histamine 4 receptor (H4R) antagonists (JNJ 40279486).39 Previous reports also suggest
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aminopyrimidine moiety is active at other GPCRs including H3R.40-44 Other previously reported
pyrimidine compounds include the potent anti-obesity compound 6 agonist of human
cholecystokinin-1 receptor (CCK-1R), compound 7 antagonist of MCHR1, H3R antagonists
compounds 4 and 5, and H4R antagonists compounds 1-3 (Figure 1). With these structures in
mind we designed and synthesized a novel series of tetrahydroquinazolinamine derivatives and
evaluated them for agonist as well as antagonist actions at histamine receptors.39, 40, 42, 43, 45-47
Interestingly, we discovered a very selective and potent H3R antagonist that induced anorexia
and weight loss in rodent models of obesity.
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Results and discussion
Chemistry
Synthesis of 4-aryl-tetrahydroquinazolin-2-amines. This study was mainly focused on the
synthesis of various substituted tetrahydroquinazolinamines and their biological evaluations at
histamine receptors in vitro followed by in vivo efficacy in mouse models of obesity. We first
synthesized various arylidenylcyclohexanones 3 as the important precursors, following our
earlier protocol 48 by employing the Claisen-Schmidt condensation reaction of cyclohexanone
(1a-1d) and hexanone (1e) with different aromatic aldehydes 2 in the presence of 10% aqueous
KOH in ethanol at ambient temperature (Scheme 1). The oily crude products thus obtained were
purified with column chromatography to give the required arylidenylcyclohexanone derivatives
in good to moderate yields (55-68%). These molecules were characterized on the basis of their
spectroscopic data and are supported by previous literature.49-57 The appropriately substituted
tetrahydroquinazolinamines
4
were
accessed
by
condensation
of
respective
arylidenylcyclohexanones 3 with guanidine hydrochloride in the presence of NaH in DMF as the
solvent following earlier reported protocols 37, 38 (Scheme 2). The reaction involves conjugate 1,
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addition of guanidine to the olefinic bond of the enone followed by cyclization and
condensation at the carbonyl end, tautomerization and aromatization by aerial oxidation resulting
into the formation of 2-aminopyrimidine nucleus. The targeted compounds were thus obtained in
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moderate yield and their characterization was done by H, C NMR and mass spectrometry.
Furthermore, purity of all compounds (4a-4y, 4a'-4c'and 4d') were evaluated by general HPLC
and found to be ≥95% pure.
O
O
R1
R2
R1
R2
KOH, EtOH
Ar
Ar CHO

2
N HCl, 30 C, 4h
1
1
1
a
b
c
3a-3w (55-65%)
3x (60%)
R3
2a-2y
R3
a-3z
3y (56%)
3
1a-1d
1d 3z (68%)
R1 = CH3, R2 = R3 =H (1a)
R1 = R2 = CH3, R3 =H (1b) Comp. Ar
R1 = R2 = H, R3 =CH3 (1c)
Comp.
3j
Ar
Comp.
3s
Ar
R1 = R2 = R3 = H (1d)
3a
phenyl
4-isopropyl-phenyl
3-bromo-phenyl
4-CN-phenyl
3
3
3
3
3
3
3
b
c
d
e
f
4-Br-phenyl
4-methoxy-phenyl
4-methyl-phenyl
4-Cl-phenyl
3k
3t
3-benzyloxy-phenyl
4-nitro-phenyl
3l
4-methylthio-phenyl
3u
3m
3n
3,4,5-trimethoxy-phenyl 3v
4-ethoxy-phenyl
cyclohexyl
3,4,-dimethoxy-phenyl
3-methoxy-phenyl
4-CF3-phenyl
3w
3x
3y
3z
4-F-phenyl
3o
4-methoxy-phenyl
4-methoxy-phenyl
4-methoxy-phenyl
g
h
naphthyl
3p
4-benzyloxy-phenyl 3q
pyridinyl
3i
4-N(CH3)2-phenyl
3r
4-OCF3-phenyl
O
O
KOH, EtOH
Ar CHO
2c

2
N HCl, 30 C, 4h
Ar
3a'
1e
(Ar = 4-methoxy-phenyl)
(Ar = 4-methoxy-phenyl)
Scheme 1: Synthesis of different substituted 2-arylidene-cyclohexanones (3a-3z) & 2-
arylheptenones (3a')
Two of the final compounds 4h and 4t, having benzyloxy substitution at 4 and 3 position of
phenyl ring were subjected to debenzylation using 10% Palladium over charcoal and hydrogen
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gas atmosphere, to get the debenzylated analogues 4x and 4y, respectively (Scheme 2).
NH2
O
N
N
NH.HCl
NaH, DMF
R1
R2
R1
R2
0
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Ar
Ar


H N
2
NH2
0 C - 100 C
24h
Guanidine
hydrochloride
R3
R3
3
a-3z
4a-4w & 4a'-4c'
R = CH , R = R =H (3a-3w)
1
3
2
3
(19-28%)
R = R = CH , R =H (3x)
1
2
3
3
3a-3w 4a-4w (19-26%) 4h; Ar = 4-benzyloxy-phenyl
R = R = H, R =CH (3y)
1
2
3
3
4t ; Ar = 3-benzyloxy-phenyl
3x
3y
3z
4a' (28%)
4b' (24%)
4c' (22%)
R = R = R = H (3z)
1
2
3
10% Pd/C, H2
Debenzylation
MeOH, rt, 8h
4
4
x; Ar = 4-hydroxy-phenyl
y ; Ar = 3-hydroxy-phenyl
NH2
O
NH.HCl
NaH, DMF
N
N
Ar


H N
NH2
0 C - 100 C
4h
2
3a'
Ar
2
(
Ar = 4-methoxy-phenyl)
Guanidine
hydrochloride
4
d' (30%)
Ar = 4-methoxy-phenyl)
(
Scheme 2: Synthesis of various substituted 4-aryl-tetrahydroquinazolin-2-amines (4a-4y, 4a'-
c') and 4-aryl-2-aminopyrimidine (4d')
4
Biology
In vitro activity of compounds at human histamine receptors. The newly synthesized 25
tetrahydroquinazolinamines 4a-4y (Scheme 2) were evaluated for their agonist and antagonist
activity at the human H3R by the GloSensor assay in which we measured cAMP production in
live cells. First, all compounds were tested at 10µM concentration in triplicate for agonist or
antagonist activity, and if any compound exhibited a minimum of 50% inhibition or stimulation
in comparison to control (for agonist activity- forskolin 10 μM and for antagonist activity-
histamine 100 nM + forskolin 10 μM), then that particular compound was further evaluated for
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concentration-dependent (10 μM to 10 pM) response to determine the EC or the IC . After the
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primary screening, we found 14 compounds active as antagonist at H3R, and therefore further
tested in a concentration dependent manner to determine IC value at H3R (Table 1 and figure
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). All 25 compounds were also evaluated for activity at the other histamine receptors H1R, H2R
and H4R. None of them showed any activity at H1R and H4R, while 8 compounds exhibited
agonist activity at H2R (Table 1 and Fig S1).
Figure 2. Representative concentration-response curve of H3R antagonist mediated
blockade of HA response (cAMP): Concentration response curve (A) GSK334429 (known
H3R antagonist) and compounds 4c, (B) 4a, 4b, 4d, 4e and 4f, (C) 4g, 4k, 4o and 4q and (D) 4s,
4
u, 4y and 4w at human H3R for antagonist activity in transiently transfected HEK293T cells.
Data are expressed as mean ± S.E.M. of normalized result of two individual experiments in
triplicates.
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Structure-activity relationship (SAR). Among all of these, compound 4c having 4-methoxy-
phenyl substituted tetrahydroquinazolinamine, exhibited very good antagonist activity at H3R
with IC50 0.04 µM (Fig 2A and Table 1), while no activity at any other HA receptors.
Compounds 4o having 3-methoxy-phenyl substitution and 4k having 3-bromo-phenyl
0
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substitution also exhibited antagonist activity at H3R with IC values of 0.07 µM and 0.09 µM,
50
respectively (Fig 2C and Table 1), almost similar to compound 4c but both compounds also
displayed moderate agonist activity at human H2R with EC50 value 0.25 µM and 0.84 µM
(Table 1 and Fig S1), respectively. Compound 4w having cyclohexyl substituted at 4-position of
tetrahydroquinazolinamine showed modest H3R antagonist activity (IC =0.24 µM, Fig 2D) and
5
0
also have similar agonist activity at H2R with an EC of 0.36 µM (Fig S1). On the other hand,
5
0
compound 4a with an unsubstituted phenyl ring and compounds 4e and 4f having 4-chloro and
4-fluoro substituted phenyl ring, exhibited almost similar antagonist action with IC50 1.30 µM,
0
.90 µM and 0.56 µM, respectively (Fig 2B and Table 1). Compounds 4a, 4e and 4f also
showed better agonist activity at H2R with EC 0.10 µM, 0.08 µM and 0.08 µM, respectively
5
0
(Table 1 and Fig S1).
Table 1: Summary of hit compounds screened at human histamine receptor:
S.No
Compounds
Structure
hH2R
hH3R
Code
LogEC50
EC50
LogIC50
IC50
(µM)
(µM)
NH2
N
1
4a
-7.02 ± 0.21
0.10
-5.89 ± 0.13
1.30
N
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NH2
N
2
4b
NA
-
-5.51 ± 0.13
3.1
N
Br
NH2
N
3
4
5
4c
4d
4e
NA
-
-7.35 ± 0.12
-5.53 ± 0.18
-6.04 ± 0.07
0.04
2.93
0.90
N
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
OMe
NH2
-6.05 ± 0.20
-7.11 ± 0.23
0.88
0.08
N
N
NH2
N
N
Cl
NH2
N
6
4f
-7.12 ± 0.21
0.08
-6.26 ± 0.08
0.56
N
F
NH2
N
7
8
9
4g
4k
4o
NA
-
-5.57 ± 0.14
-7.07 ± 0.10
-7.18 ± 0.07
2.71
0.09
0.07
N
NH2
N
-6.50 ± 0.22
-6.61±0.06
0.84
0.25
N
Br
NH2
N
N
OMe
NH2
10
4q
NA
-
-5.17 ± 0.16
-5.26 ± 0.15
6.80
5.50
N
N
N
NH2
1
1
1
2
4s
NA
-
N
N
CN
NH2
4u
-5.27 ± 0.32
5.29
-4.91 ± 0.21 12.30
N
N
NO2
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NH2
N
1
1
3
4
4y
NA
-
-5.34 ± 0.17
-6.61 ± 0.80
4.61
0.24
N
OH
NH2
4w
-6.44±0.05
0.36
N
N
0
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NH2
N
1
5
4b'
-5.67 ± 0.13
2.12
-5.33 ± 0.10
4.68
N
OMe
NH2
N
1
1
6
7
4c'
4d'
-5.89 ± 0.28
-5.28 ± 0.25
1.39
5.18
-5.56 ± 0.09
-5.35 ± 0.07
2.75
4.45
N
OMe
NH2
N
N
OMe
NA= Not Active
The H3R antagonist potential of compounds 4d (IC = 2.93 µM), 4b (IC = 3.1 µM), 4s
5
0
50
(IC = 5.50 µM) and 4u (IC = 12.30 µM) were reduced H3R activity with corresponding -CH ,
5
0
50
3
Br, –CN and -NO groups substitutions at para-position of phenyl ring. While compound 4d and
2
4
u also showed H2R agonist activity with EC value 0.88 µM and 5.29 µM (Table 1 and Fig
50
S1). The effect of -OH group at both meta- and para- position of phenyl ring was also checked,
compound 4y with meta-position substitution showed antagonism at H3R (IC =4.61 µM), while
5
0
4
x with para-position substitution showed no activity at H3R. Substitution of the aryl group with
a naphthyl (4g, IC = 2.71 µM) and pyridinyl (4q, IC = 6.80 µM) ring also reduced antagonist
5
0
50
activity at H3R. The other compounds with substitutions at para position of phenyl ring such as -
OCH2Ph, -dimethylamino, -isopropyl, -methylthio, -CF3 and -OCF3 did not exhibit any
antagonistic potential at H3R. Homology modelling showed that some of the active compounds
(4e, 4b, and 4b') show similar binding orientation and comparable docking score (4e= -7.25, 4b=
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-
7.25, 4b'= -7.68) like the most potent compound 4c having methoxy group in the phenyl ring,
but no TYR115 hydrogen bonding with oxygen atom of methoxy group, suggesting the critical
nature of this molecular interaction for high affinity binding. Furthermore, the compound 4v
having ethoxy group docks in different orientation, resulting in the loss of hydrogen bond
interaction with TYR115 and did not show any antagonistic activity at H3R at all, again
underlining the importance of hydrogen bond interaction at TYR115 for desired activity. We also
determined the role of methyl group at tetrahydroquinazolin ring compounds with 8,8-dimethyl
substitution (4a') and found that this modification lead to loss of activity at all HA receptors,
including H3R. However, compounds with 6-methyl substitution (4b'), without methyl
substitution (4c'), and an open chain 2-aminopyrimidine derivative (4d') showed reduced
antagonist activity at H3R as compared to 4c having same 4-methoxy-phenyl group (Table 1,
Fig S1 A). Compounds 4b', 4c' and 4d' also showed modest agonist activity at H2R with EC50
2.12 µM, 1.39 µM and 5.18 µM, respectively (Table 1, Fig S1 D). This signifies the importance
of monosubstituted 8-methyl group and para-methoxy group in the most potent compound 4c to
act as an efficient antagonist at H3R and negligible activity at other histamine receptors.
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Selectivity profiling of 4c. In vitro activity of 4c, 4k and 4o show approximately similar IC as
50
H3R antagonist but compounds 4k and 4o have agonist activity at H2R, while 4c has no activity
towards H2R. On the basis of histamine receptor functional activity data we considered 4c as a
lead compound with potent antagonist activity against human H3R, while lacking any noticeable
activity in the functional assays to other subtypes of histamine receptors- H1R, H2R, and H4R.
Furthermore, pre-treatment with compound 4c (10 μM) significantly blocks the H3R agonists
(i.e. histamine or imitet; 1 µM) induced inhibition of cAMP response, which suggest that 4c
selectively work through H3R and did not show receptor independent activity (Fig. 3).
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Figure 3. Compound 4c selectively inhibits H3R agonist response. Bar graph showing cAMP
dependent relative luminescence unit (RLU) induced by forskolin (10 µM) treatment and pre-
treatment with compound 4c or reference ligand GSK (GSK334429, a known H3R antagonist) at
1
0 μM concentration significantly block the H3R agonists (i.e. histamine or imitet; 1 µM)
induced inhibition of cAMP response. $$$p<0.001 denote forskolin treatment significantly
increased RLU compare to control cells, ***p<0.001 denote histamine or imitet (IMT)
significantly decreased the cAMP compared to forskolin alone, ###p<0.001 and δδδp<0.001 denote
significant blockade of cAMP by pretreatment with 4c or GSK compared to histamine and
imitet, respectively.
Next, we evaluated the off target activity of lead compound 4c at other families of GPCRs such
as dopamine receptors (D1-D5), muscarinic receptors (M1 and M5), G-protein-coupled bile acid
receptor 1, G-protein coupled receptor 40, serotonin receptor (5-HT ) and kappa opioid receptor
2
C
(KOR). Lead compound 4c did not exhibit noticeable activity at GPCRs of other families (Table
). These in vitro results demonstrate that compound 4c exhibits selective antagonist activity at
2
H3R, over the panel of GPCRs tested.
Since we were interested to discover and develop a selective H3R antagonist with
desirable safety profile for the treatment of obesity, we preferred to evaluate compound 4c for
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the binding at the protein of the Human Ether-a-go-go-related Gene (hERG), a potassium ion
channel in heart responsible for long QT syndrome using a predictor hERG assay kit (Invitrogen
Corp, USA). We observed that test compound 4c did not bind to the hERG up to 10 µM , while
reference compound E-4031 (well known hERG ligand) showed activity with IC < 0.30 µM
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3
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9
0
5
0
(Table 2 and Fig S2). We also found that all 25 compounds including 4c (Table 1) have no Pan-
assay interference compounds (PAINS) liability as determined by PAINS- Remover online web
5
8
server. Although evaluation at CEREP panel targets would have yielded better clarity on the
safety profile of 4c, our data for 15 targets indicate a reasonable selectivity and specificity for
this compound.
Table 2. Off-targets activity of 4c
S. No
Targets
Activity
Agonist
Antagonist
1
2
3
4
5
6
7
8
9
10
Dopamine D1 receptor (D1R)
Dopamine D2 receptor (D2R)
Dopamine D3 receptor (D3R)
Dopamine D4 receptor (D4R)
Dopamine D5 receptor (D5R)
Histamine H1 receptor (H1R)
Histamine H2 receptor (H2R)
Histamine H4 receptor (H4R)
Muscarinic M1 receptor (M1R)
Muscarinic M5 receptor (M5R)
Opioid κ receptor (KOR)
NA
NA
NA
NA
NA
NA
NA
NA
> 10 μM NA
NA NA
> 10 μM NA
NA
NA
NA
NA
NA
NA
NA
1
1
>10 μM
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3
Serotonin 5-HT2 receptor (5-HT2 R)
NA
NA
NA
NA
NA
C
C
G-protein-coupled receptor40 (GPR40)
14
G-protein-coupled bile acid receptor 1 (GPBAR1) NA
0
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6
hERG liability
PAINS liability
No
No
Characterization of 4c enantiomers activity at H3R. First we evaluated activity of both (R and
S) enantiomer of 4c at H3R by in silico docking experiment with homology modelling. We first
generated 50 models, and the top 10 models were selected on the basis of DOPE score and
validated with PROCHECK and QMEAN. Models having more than 90% residues in the most
favoured regions of the Ramachandran plot were considered good quality models.59, 60. Models
having QMEAN score close to 1 indicate a better model quality.61 Visual inspection was done
for the correct orientation of side chains of conserved residues favourable for binding of
antagonists and stabilization of receptor in the inactive state.62,63, 64
Figure 4 Ramachandran Plot of human H3R model protein and Predicted binding mode of
R- and S- isomer of the compound 4c: (A) 96.9% residues in most favoured region and 0.0%
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residues in disallowed region. (B) Binding pose of R-isomer (blue) and (C) S-isomer (dark
green) within the H3R model constructed from 4U15. Amino acid residue interacted with both
the isomers of the compound 4c via hydrogen bond showing in green.
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Previous studies showed that GLU206, TYR115 and TYR374 lining the orthosteric site
are the gripping points for agonists, including histamine. Binding of more complex ligands were
described as extending to the allosteric site residues, TYR189, ALA190, ARG381, PHE193,
TYR194 and TYR394.62, 65-68 A Ramachandran plot was generated using a PROCHECK server
and the best model showed 96.9% residues in the maximum allowed region. Further, the
calculated QMEAN was 0.600 (Fig 4A). Both R- and S- isomers showed relatively similar
molecular interactions with the surrounding binding site residues of the protein with highest
docking score of -7.642 and -7.518, respectively (Fig 4B and 4C). Interactions involved in the
binding of R- and S- isomer of the compound 4c were shown in figure 5A and 5B and
summarized in Table 3. Aliphatic ring I formed -alkyl interactions with TYR394. In the
extracellular loop 2 (ECL2) on the top edge of the binding site interacted with R-isomer,
PHE193 and TYR194 interacted with the R-isomer by forming two conventional hydrogen
bonds with amine group IV of the R-isomer. Aromatic ring II participated in a -lone pair
interaction with TYR394 and a π-alkyl interaction with ALA190.
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Figure 5. Interaction of R and S isomer of compound 4c with the human H3R model
protein. (A) Amino acid residues of H3R involved in interaction with R-isomer (blue) and (B)
S-isomer (dark green). Amino acid residues involved in hydrogen bonding showed in green. (C)
Interacting groups of compound 4c with the human H3R.
The methoxy oxygen atom of interacting group III forms hydrogen bond with TYR115.
In addition, charged residues ASP114, ARG381 and ASP391 formed ionic interaction with the
R-isomer. Moreover, residues LEU111, TRP110, PHE398, TYR91, TYR194, TRP196 and
LEU199 formed hydrophobic interactions with the R-isomer. The S-isomer formed relatively
similar molecular interactions with binding site residues that were observed for the R-isomer
except interaction between TYR194 and ASP114. TYR194 was observed to form both hydrogen
bonding and hydrophobic interactions with the R-isomer, whereas TYR194 formed only
hydrophobic interactions with the S-isomer. These direct and indirect interactions stabilized the
R-and S-isomers of compound 4c in the H3R model. Docking experiments suggest that both R
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and S enantiomer exhibits equal activity at H3R with almost similar docking score and
interaction.
Table 3 Summary of interactions between R- and S-isomers of the 4c and the human H3R
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
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3
4
4
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4
4
4
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4
4
4
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5
5
5
5
5
5
5
6
0
1
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3
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9
0
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3
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9
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Molecule feature
Interacting residue
TYR394 (R,S)
TYR394 (R,S)
ALA190 (R,S)
TYR115 (R,S)
PHE193 (R,S)
TYR194 (R)
Interaction type
π-Alkyl
I
II
π-Lone Pair
π-Alkyl
III
IV
Hydrogen Bond
Hydrogen Bond
Hydrogen Bond
π-Alkyl
V
HIS387 (R,S)
Furthermore, enantiomers of compound 4c were separated as pure enantiomers E1 and
E2 by chiral HPLC using polysaccharide based chiral column known as Chiralpak IA
(Supporting information: Chiral separation section ). After separation, both enantiomers were
evaluated for their in vitro antagonist activity at H3R. We found that E1 and E2 have almost
similar activity at H3R with IC 0.008 and 0.003 µM, slightly more than racemate (4c) IC (Fig
5
0
50
S2). These data suggest that IC of enantiomers (E1 and E2) and racemate (4c) have almost the
5
0
same in vitro activity. Therefore, we did not perform further studies with the pure enantiomers of
c (Fig S2).
4
Pharmacokinetic profile of compound 4c. In mice, levels of compound 4c and its metabolite in
brain and blood serum were determined using LC-MS/MS. Following a 20 mg/kg oral dose, 4c
showed a peak serum concentration (Cmax, 3356.7 ± 343.8 ng/mL) at 0.25 h indicating rapid
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absorption (Fig 6). Its demethylated metabolite (4x) showed Cmax (3333.3 ± 249.4 ng/mL) at 0.5
h.
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Figure 6. Mean serum concentration-time profile of 4c and its metabolite after single 20 mg/kg
oral dose in mice. Bar represents SEM (n=4)
The concentration-time profile was analyzed using both a non-compartmental and
compartmental approach. The pharmacokinetic models were compared according to the maximal
correlation between the observed and predicted concentrations, the minimal sum of squared
residuals, Akaike’s Information Criterion and Schwarz Bayesian Criterion.69,
70
The
concentration-time profile was best described by a two-compartmental open model and the
pharmacokinetic parameters are shown in Table 4. The volume of distribution (28.5 L/kg) was
larger than the total blood volume of mice (0.085 L/kg) 71 and systemic clearance (10.3 L/h/kg)
71
was higher than the total hepatic blood flow in mice (5.4 L/h/kg) indicating extra-vascular
distribution along with the extra hepatic elimination of the compound. Following oral
administration of test compound (4c; 20 mg/kg), both 4c and its metabolite crossed the blood
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brain barrier but the availability was lower than that in serum (Fig 6). Compound 4c showed a
peak brain concentration (Cmax, 322.8 ± 80.5 ng/mL) at 0.25 h, while concentration of metabolite
(Cmax, 101.4 ± 31.7ng/mL) was lower than the parent compound (Fig 6 and Table 4).
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Furthermore, plasma protein binding (PPB) and unbound fraction in brain (F ) of compound 4c
u
was estimated in plasma and brain tissue homogenate by an equilibrium dialysis method. We
found high PPB in plasma (95.76±0.94%) of compound 4c, whereas F was 28.41±3.51% of 4c
u
in mouse brain tissue. Thus, freely available 4c to antagonize H3R in brain (unbound fraction,
Fu) at Cmax would be around 90 ng/ml (equivalent to 334.7 nM, 8.3 times of IC of 4c), and level
50
of 4c at 4h in brain would be 5.6 ng/ml (equivalent to 20.8 nM). Thus, we believe that this brain
concentration of 4c might be enough to antagonize the H3R in brain and produce CNS specific
beneficial effects mediated by H3R.
Table 4. Pharmacokinetic parameters of 4c and its metabolite (4x) in C57BL/6J mice
Parameters
4c (20mg/kg, PO)
Metabolite (4x)
Serum Brain
3333.3 ± 249.4 101.4 ± 31.7
Serum
Brain
Cmax (ng/mL)
tmax (h)
3356.7 ± 343.8
0.25
322.8 ± 80.5
0.5
310.4
1.1
0.25
0.5
100.7
1.2
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AUC(ng h/mL)
T (h)
1919.3
1.9
1646.9
2.1
1
/2
Cl/F(L/h/kg)
10.3
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-
-
V /F (L/kg)
d
28.5
28.3
-
AUC /AUC (%)
85.8
16.2
32.4
6.1
M
P
AUC /AUC (%)
B
S
Each value represents the average of five mice. Concentrations are mean ± SEM from five mice.
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Abbreviations: AUC = area under the serum concentration-time curve up to last observation,
AUC /AUC = metabolite to 4c AUC ratio, AUC /AUC = brain to serum AUC ratio, Cl/F =
M
P
B
S
clearance, Cmax = peak concentration, tmax = time to Cmax, V /F = volume of distribution, T =
d
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elimination half-life.
Acute treatment with 4c reduced food intake via H3R in obese mice. Considering the
encouraging pharmacokinetic profile, the selectivity profile and the IC50 of 4c at H3R, we
investigated the acute anorexic (suppression of food intake) effect of 4c in a High Fat diet (HFD)
and the db/db mouse model of obesity. We observed that oral administration of 4c and lorcaserin
significantly reduced cumulative food intake during the four hour night phase (9:00 pm to 1:00
am) (Fig 7). Compound 4c at 20 mg/kg oral dose reduced food intake by 28% compared to
vehicle treated HFD fed mice (0.9693 ± 0.04339 g, N=15 vs 0.6967 ± 0.03972 g, N=12), while
an oral dose of 10 mg/kg reduced food intake by approximately 26% in comparison to the
**
vehicle group (0.9693 ± 0.04339 g, N=15 to 0.7150 ± 0.07869 g, N=6 (Fig 7 A, p<0.01,
***p<0.001, One Way ANOVA). We used lorcaserin (10 mg/kg) as a standard anti-obesity drug
for comparison, which showed robust anorexic effect (40% decrease) better than 4c treated
(
0.9693 ± 0.04339 g N=15 to 0.5830 ± 0.06178 g N=11) (Fig 7 A, *** p<0.001, One Way
ANOVA). These data suggest that temporary blockade of H3R by 4c in brain might lead to
increase histamine secretion like the previously reported antagonist and concomitant activation
of various downstream effectors that induced anorexia.7, 11, 72-74
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Figure 7. Compound 4c produced an anorexic effect in Diet-induced and db/db obese mice,
via H3R inhibition: Representative bar graph shown 4h cumulative food intake 30 minutes after
oral administration, of test compound 4c (20 mg/kg and 10 mg/kg body weight) or LRC
(lorcaserin,-reference drug, 10 mg/kg body weight) or vehicle in diet-induced obese C57BL/6J
mice (A), db/db obese mice (B), lean C57BL/6J mice treated with compound 4c (20 mg/kg, PO)
or vehicle (C) and pre-treatment with Imitet (H3R selective agonist, 5mg/kg, i.p. 30 min before
4
c) or HDC inhibitor (α-MDLH, 20 mg/kg, i.p. 2 h before 4c) significantly blocked the anorexic
effect (i.e. reduced food intake) of 4c (20 mg/kg, oral) in db/db mice (D). p<0.01, ***p<0.001
*
*
denote significant decrease in food intake after the treatment with 4c and #p<0.05 and ###p<0.001
denote significant blocked anorexic effect of 4c (20 mg/kg, PO) by pre-treatment with imitet
(5mg/kg, i.p. 30 min before 4c) or α-MDLH (20 mg/kg, i.p. 2 h before 4c), p value calculated by
One Way ANOVA followed by Newman-Keuls multiple comparison test.
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We next investigated the anorexic effect of 4c in a genetically obese db/db mice model to
explore if the mode of satiety is a leptin dependent or a leptin independent mechanism. The
db/db obese mice lack the leptin system due to a point mutation in lpr gene.75 Oral
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administration of compound 4c (20 mg/kg) in 12 hour fasted db/db mice significantly reduced
cumulative food intake (33%) in comparison to vehicle treated mice (1.956 ± 0.1431g, N=10 vs
1
.315 ± 0.1183 g, N=13), while we did not find any change at a dose of 10 mg/kg dose (Figure
B, ** p<0.01, n=8-12, One Way ANOVA followed by Newman-Keuls multiple comparison
7
test). The anorexic effect of 4c in db/db mice suggests that H3R antagonists exhibit anorexia in a
leptin independent manner. Next, we examined the anorexic activity of compound 4c in db/db
mice is H3R dependent or not. Pre-treatment with an H3R selective agonist (Imitet, 5mg/kg, i.p.
3
0 min before 4c) or the irreversible inhibitor of histidine decarboxylase (histamine synthesizing
enzyme) α-Methyl-DL-histidine dihydrochloride (α-MDLH, 20 mg/kg, i.p. 2 h before 4c)
significantly reversed the anorexic effect of compound 4c (20 mg/kg, oral) and alone treatment
with imitet and α-MDLH did not exhibits any effects (Fig 7D, p<0.05 and ###p<0.001, n=4-7,
One Way ANOVA followed by Newman-Keuls multiple comparison test). These results suggest
that compound 4c probably increased the brain histamine level via inhibition of H3R and this
increased histamine level produced the anorexic effect observed in the obese db/db mice. We
also determined the food intake in normal healthy lean mice after 12 hours of food deprivation.
Healthy lean mice acutely treated with vehicle or 4c at 20 mg/kg dose, did not display any effect
on food intake (Fig 7C), suggesting no adverse effects in normal healthy mice after treatment
with 4c.
#
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Acute administration of 4c induce c-FOS expression in hypothalamic nuclei of db/db mice.
Since 4c enters in to the brain where H3Rs are expressed and induces the suppression of food
intake, we sought to determine the activation of c-FOS (a surrogate marker of neuronal
activation) in the hypothalamus. We observed that administration of 4c (20 mg/kg; i.p.)
significantly increased c-Fos expression compared to vehicle treated db/db mice in the para-
ventricular region of the hypothalamus (PVN), which is considered a satiety centre (Fig 8). This
result is similar to results reported for other H3R antagonists.74
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Figure 8. H3R antagonist 4c activates c-FOS in the PVN. Representative immuno-stained
fluorescent image showing c-Fos induction in the para-ventricular nucleus (PVN) of
hypothalamus 90 minutes after intraperitoneal administration of the test compound 4c (20
mg/kg) or vehicle in db/db mice. After treatment with 4c, the number of c-Fos positive neurons
increased in the PVN as compare to vehicle treated mice (Scale bar 50µm)
Compound 4c ameliorates body weight gain in HFD fed mice. Several previous reports
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clearly suggest that treatment with H3R antagonist shows anti-obesity effect by reducing body
weight in rodent model of obesity.7, 9, 10 Figure 9A shows the percent body change of normal
chow fed mice and high-fat diet fed mice treated with vehicle or 4c or lorcaserin in a time
dependent manner. Chronic oral administration of 4c at a dose of 20 mg/kg resulted in a
significant reduction in percent body weight gain evident from day 20, compared to vehicle
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*
**
positive control, the known anti-obesity drug, Lorcaserin, also shows significant reduction in
body weight gain at day 20 (N=5-6, p<0.05, p<0.01 and ***p<0.001; two-way ANOVA). This
anti-obesity effect of test compound 4c consistently increased during the course of treatment and
reached an effect equal to that of lorcaserin by day 27. Although 4c at dose 10 mg/kg also
modestly reduced the percent body weight gain, this effect was not statistically significant in
comparison to vehicle treated HFD mice.
*
**
Figure 9. H3R antagonist 4c act as anti-obesity agent in diet-induced obese mice: Line graph
showing % change in body weight relative to the treatment days (A) and bar graph showing net
body weight change after six weeks of treatment (B) with 4c (10 mg/kg and 20 mg/kg body
weight), or lorcaserin (10 mg/ kg body weight), or vehicle. p<0.05, p<0.01 and ###p<0.001;
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n=5-6 Denotes significant body weight increase of HFD mice compared to chow mice and
*
**
***
*
p<0.05, p<0.01 and p<0.001; n=5-6, denote significant body weight decrease treated with
4c or lorcaserin mice compare to HFD vehicle mice. p values were determined by two-way
ANOVA or by one-way ANOVA followed by Newman-Keuls Multiple Comparison Test.
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Thus, six weeks of chronic administration of compounds 4c significantly reduced the net
body weight gain compared to vehicle treated HFD fed mice (3.86± 0.99g vs 0.80 ± 0.70g, Fig
*
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B, p<0.05; one way ANOVA), while 4c at a dose of 10 mg/kg did not show significant body
weight reduction (3.86 ± 0.99g vs 2.6 ± 0.06g). As expected, the positive control drug lorcaserin
*
significantly reduced the body weight gain (3.867 ± 0.99g vs 0.95 ± 0.34g; Fig 10B, p<0.05;
one way ANOVA). Furthermore, we also investigated the chronic effect of 4c in healthy lean
C57BL/6J mice. We found that chronic administration of 4c did not show any significant effect
on body weight in normal lean mice (Fig S4). We next analyzed the body composition of mice
after the six weeks of treatment with 4c by EchoMRI live scanning of mice. EchoMRI data
suggested that high fat diet fed mice have significantly higher amount (63.2%) of fat mass
compared to normal chow fed mice (13.8%) and did not have any significant difference in lean
mass weight (Fig 10). Chronic administration of compounds 4c at 20 mg/kg dose significantly
reduced the body fat mass (27.1%) compared to HFD mice treated with vehicle (Fig 10 A and C,
*
N=5-6; p<0.05, one way ANOVA). This effect was similar to the anti-obesity drug lorcaserin,
which reduced the fat mass approximately 22.4% compared to HFD mice (Fig 10C, N=5-6;
*p<0.05, one way ANOVA). However, compound 4c at dose 10 mg/kg showed a very minimal
effect on fat mass. Compound 4c treatment did not alter lean mass of HFD mice compared to
vehicle treated mice (Fig 10B). We did not find any change in blood glucose and lipid
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parameters (Fig S5 and S6A) after the chronic treatment with 4c (dosage) in HFD fed mice.
These observations confirm that 4c abrogated body weight gain via suppressing food intake and
consequently reducing body fat mass in obese mice.
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Figure 10. H3R antagonist 4c treatment reduced body fat composition in diet-induced
obese mice: Representative bar graph shows body fat mass (A), lean mass (B) and % body fat
mass (C) of normal chow fed mice and HFD fed mice treated with 4c (10 mg kg and 20 mg/kg
body weight) or lorcaserin (10 mg/kg body weight) or vehicle. ###p<0.001 (n=5-6, chow vs
*
HFD), p<0.05 (n=5-6) denote significant decrease of body fat after treatment with 4c or
lorcaserin compared to vehicle treated HFD fed mice. p values were determined by one-way
ANOVA followed by Newman-Keuls Multiple Comparison Test.
Compound 4c reduces the body weight and blood glucose in db/db mice. Since previous
studies reported that concentration of hypothalamic histamine is significantly lower in leptin
signaling disrupted rodents such as ob/ob, db/db and Zucker fatty (fa/fa) rats 76 and chronic i.c.v.
infusion of histamine decreases food intake and body weight in leptin signaling disrupted obese
7
3
mice , we also evaluated the effect of chronic treatment with 4c (20 mg/kg body weight, oral)
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on body weight loss in db/db mice. As expected, we observed that four weeks of chronic oral
administration of 4c significantly reduced the body weight of db/db mice compared to vehicle
*
treated db/db mice (Fig 11A, p<0.05; N=4, t-test). Food intake and chronic studies data
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suggested that compounds 4c shows anti-obesity effect in db/db mice via leptin independent
signalling (Fig 7 and 11). These results suggest that 4c shows anti-obesity effect in db/db mice
by increasing histaminergic signaling in satiety neurons.
Curiously, we also measured the fasting blood glucose levels and glucose tolerance
which is highly impaired in db/db mice, and found that chronic administration of 4c significantly
*
reduced fasting glucose (Fig S7, N= 4-5; p<0.05; t-test) and improve glucose homeostasis,
*
observed in an intra peritoneal glucose tolerance test (ipGTT; Fig11 B and C, N= 4-5; p<0.05
one way ANOVA and t-test). However, we did not found any change in blood lipid parameters
after the chronic treatment with 4c in db/db mice (Fig S6B).
Figure 11. H3R antagonist 4c acts as anti-obesity agent and improves glucose homeostasis
in db/db obese mice: (A) Net body weight change after four weeks treatment with 4c (20mg/kg)
in db/db mice, (B) Line graph shows glucose level relative to time after glucose administration (1
g/kg body weight) in 16 hour fasted db/db mice and (C) Change in blood glucose AUC analysis
*
of ipGTT results. p<0.05 (n=4), p value were determined by two-way ANOVA or student’s t
test.
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