Targeting Serotonin 2A and Adrenergic α1 Receptors for Ocular Antihypertensive Agents: Discovery of 3,4-Dihydropyrazino[1,2-b]indazol-1(2H)-one Derivatives

Article abstract of DOI:10.1002/cmdc.201800199

Glaucoma affects millions of people worldwide and causes optic nerve damage and blindness. The elevation of the intraocular pressure (IOP) is the main risk factor associated with this pathology, and decreasing IOP is the key therapeutic target of current pharmacological treatments. As potential ocular hypotensive agents, we studied compounds that act on two receptors (serotonin 2A and adrenergic α₁) linked to the regulation of aqueous humour dynamics. Herein we describe the design, synthesis, and pharmacological profiling of a series of novel bicyclic and tricyclic N2-alkyl-indazole-amide derivatives. This study identified a 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative with potent serotonin 2A receptor antagonism, >100-fold selectivity over other serotonin subtype receptors, and high affinity for the α₁ receptor. Moreover, upon local administration, this compound showed superior ocular hypotensive action in vivo relative to the clinically used reference compound timolol.

Full text of DOI:10.1002/cmdc.201800199

Accepted Article
Title: Targeting Serotonin 2A and Adrenergic α1 Receptors for Ocular
Antihypertensive Agents: Discovery of 3,4-Dihydropyrazino[1,2-
b]indazol-1(2H)-one Derivatives
Authors: Guido Furlotti, Maria Alessandra Alisi, Nicola Cazzolla,
Beatrice Garrone, Gabriele Magarò, Giorgina Mangano,
Marco Vitiello, Francesca Ceccacci, Tecla Gasperi, Angela La
Bella, Francesca Leonelli, Maria Antonietta Loreto, Emanuela
Masini, Rinaldo Marini Bettolo, Martina Miceli, and Luisa
Maria Migneco
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201800199
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800199
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Targeting Serotonin 2A and Adrenergic α1 Receptors for Ocular
Antihypertensive Agents: Discovery of 3,4-Dihydropyrazino[1,2-
b]indazol-1(2H)-one Derivatives
Guido Furlotti,*[a] Maria Alessandra Alisi,[a] Nicola Cazzolla,[a] Francesca Ceccacci,[b] Beatrice
Garrone,[a] Tecla Gasperi,[c] Angela La Bella,[b] Francesca Leonelli,[b,§] Maria Antonietta Loreto,[b]
Gabriele Magarò,[a] Giorgina Mangano,[a] Rinaldo Marini Bettolo,[b] Emanuela Masini,[d] Martina Miceli,[c]
Luisa Maria Migneco,[b] Marco Vitiello.[a]
[a]
Dr M.A. Alisi, Dr. N. Cazzolla, Dr. B. Garrone, Dr. G. Magarò, Dr. G. Mangano, Dr. M. Vitellio and Dr. G. Furlotti.
Angelini RR&D (Research, Regulatory & Development), Angelini S.p.A., Piazzale della stazione snc, S.Palomba-Pomezia (Rome), 00071, Italy
E-mail: g.furlotti@angelini.it
[b]
[c]
[d]
Dr. F. Ceccacci, Dr. A. La Bella, Prof. Dr. F. Leonelli, Prof. Dr. M.A. Loreto, Prof. Dr. R. Marini Bettolo, Dr. L.M. Migneco.
Chemistry Department “S. Cannizzaro” and § Dipartimento di Biologia Ambientale, University of Rome “La Sapienza”, P.le Aldo Moro 5, I-00185 Rome, Italy
Dr. T. Gasperi, Dr. Martina Miceli.
Department of Science – Section of Nanoscience and Nanotechnology, University of Roma Tre, via della Vasca Navale 79, I-00146 Rome, Italy
Dr. E. Masini
Departments of NEUROFARBA, Section of Pharmacology, University of Florence, Viale Pieraccini 6, I – 50139 Florence, Italy
Supporting information for this article is given via a link at the end of the document.
Abstract: Glaucoma affects millions of people worldwide, causing
optic nerve damage and blindness. The elevation of the intraocular
pressure (IOP) is the main risk factor associated with this pathology,
and nowadays, the key therapeutic target of the existing
pharmacological treatments. We studied, as potential ocular
hypotensive agents, compounds acting on two receptors (serotonin
2A, 5-HT2A, and adrenergic α1) linked to the regulation of the humour
aqueous dynamic. In this article we describe the design, synthesis,
and pharmacological profiling of a series of novel bicyclic and tricyclic
2N-alkyl-indazole-amide derivatives. The work brought to the
identification of compound 28, a 3,4-dihydropyrazino[1,2-b]indazol-
1(2H)-one derivative, with potent 5-HT2A antagonism, >100 fold
selectivity over other serotonin subtype receptors and high affinity for
the α1 receptor. Moreover, when administered locally, 28 showed
superior ocular hypotensive action in vivo, compared to the clinically
used reference compound timolol.
F2α analogues such as latanoprost and travoprost, that increase
the uveoscleral outflow pathway, meanwhile the latter mode of
action is typical of the adrenergic beta blockers and carbonic
anhydrase inhibitors.[6]
Unfortunately these treatments are associated with a number of
side effects and a significant percentage of non-responders, with
the overall result that more than 50% of glaucoma patients fail to
control the disease progression.[2] Consequently, substantial
unmet medical needs still stimulate researchers to find novel
biological targets and mechanisms of action for the
pharmacological reduction of the IOP level. [7]
In this context, we envisaged the dual modulation of the 5-HT2A
and adrenergic alpha1 (1) receptors as promising and
efficacious pharmacological approach for the reduction of IOP.
Both receptors are phospholipase C-coupled proteins, which
promote, when activated, the production of the intracellular
second messenger inositol triphosphate, and subsequent
modification of the cellular calcium ion level.[8] The two receptor
systems are intimately involved in aqueous humor dynamics
being expressed in human ocular tissues, in particular in iris,
ciliary body and trabecular meshwork.[9] Several studies provide
evidence that the specific antagonism of the 5-HT2A and α1
receptors can influence the IOP level, increasing capacity of the
two eye’s outflow systems: respectively the trabecular meshwork
and the uveoscleral.[10] However, the use of topical selective 5-
HT2A or α1 antagonists, such as BVT.28949 and bunazosin, while
showing significant decrease in IOP level do not reach the
potency of the reference ocular hypotensive agent timolol.[24,25]
With these evidences, we may assume that the serotoninergic
and the adrenergic mechanism of action, effecting on different
points of the aqueous humor pathway, could act synergistically to
produce a substantial IOP reduction increasing the drug’s
therapeutic window. This hypothesis has already been indirectly
substantiated, by some previous studies demonstrating that the
topical administration of non-selective 5-HT2A receptor
Introduction
Glaucoma is an ocular disease that produces a progressive optic
nerve damage and visual field loss. The primary open-angle
glaucoma (POAG) is the most common form of this disease,
affecting about 2.2 million of people in the USA, and causing
about 90,000 blindness each year.[1] The prolonged elevation of
the intraocular pressure (IOP) is considered one of the main risk
factors associated with POAG, in addition to a family history of
glaucoma, myopia and hypertension. The strong relationship
between glaucomatous damage and high IOP level dictates the
current pharmacological and surgical treatments of open-angle
glaucoma, which are essentially aimed at reducing the IOP
level.[2],[3] The ocular hypotensive effect can be obtained
pharmacologically, either by improving the aqueous humor
outflow or by a reduction of the ocular liquid production.[4,5] The
former dynamic action is ascribed, for example, to prostaglandin
1
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antagonists ketanserin (1), trazodone (2) and sarpogrelate (3)
(Figure 1), (which possess residual adrenergic α1 binding affinity;
(see S.I. Table 7.4 for α1 and 5-HT2A receptors binding affinity and
functional activity data), had excellent ocular hypotensive effects
in animal models and patients, without showing evident adverse
effects.[10g,11,12]
The dual-activity approach for IOP-lowering compounds, while
already studied for few mechanism of actions,[13,14] for 5-HT2A and
α1 antagonists has not been rationally pursued.
According to these observations, we decided to search novel,
potent serotonin subtype-2A and adrenergic 1 receptors
antagonists, to be used as topical ocular hypotensive agents.
Herein we described the design, synthesis, and pharmacological
profiling of a series of novel bicyclic and tricyclic 2N-alkyl-
indazole-amide derivatives (A, Figure 1) that, among the
serotoninergic receptor families, selectively inhibit only the 5-HT2A
subtype, while maintaining good potency also for the adrenergic
1 receptors. The intraocular hypotensive effect of the most
promising compound was assayed in a carbomer-induced albino
rabbit model of ocular hypertension.[15]
In light of the very scarce SAR information published about this
class of molecules, and the unpredictable structure-activity
relationship response showed, we decided to focus our synthetic
efforts on close derivatives of compound 5, assaying primarily the
binding affinity for the human recombinant 5-HT2A receptor,
subsequently, the selectivity over the human recombinant 5-HT2B,
5-HT2C and 5-HT4 receptor subtypes together with the affinity for
the adrenergic α1 receptor. This approach was chosen to
maximize the probability to obtain selective 5-HT2A antagonist,
with the option, in case of lack of residual adrenergic potency for
the most interesting compounds, to dedicate the late part of the
program to fine tune the α1 affinity.
Table 1. Binding results of 1N-methyl (4) and 2N-methyl (5) derivatives for rat
5-HT2A and 5-HT4 receptor subtypes.
pKi[a]
5-HT2A
pKi[a]
5-HT4
Entry
X
Y
4
5
NCH3
N
N
7.42
7.49
8.5
-
8.34
5.83
-
NCH3
methysergide
SB207266A (piboserod)
9.68
[a] Displacement of [3H]-ketanserin (5-HT2A receptor) and [3H]-GR113808 (5-
HT4 receptor) both from rat cortex homogenate. pKi = log 1/Ki. (n = 2) (see
experimental section for further details). Values of 95% confidence interval
(CI95%) were always less than or equal to the 5% of Ki values. – not tested.
Keeping unaltered the [1-(2-phenylethyl)piperidin-4-yl]methyl}-
indazole-3-carboxamide core structure of 5, we started to
evaluate the modulation of the human recombinant 5-HT2A
receptor affinity as a function of the substitution pattern of the
phenyl ring, the amide nitrogen and the 2N indazole nitrogen. The
results are summarized in Table 2.
Based on the SAR results obtained by other research groups,[18]
focus of aromatic ring substitution was on F, CF3, OCH3 and OH
substitutions.
Figure 1. Structure of reference compounds 1 - 3 and general formula of 2-
alkyl-indazole-amide derivatives A.
The introduction of a fluorine on the phenyl ring showed an
increase in binding affinity, in comparison with the un-substituted
parent compound (5), with the ortho position (8, pKi = 9.00)
preferred to the para (7, pKi= 8.34). No further improvement in
binding affinity was found with the o,p-difluoro substitution
(derivatives 8 and 11). Among the evaluated para-substitutions,
two log unit loss of binding affinity was observed with the
trifluoromethyl group (9) and with the methoxy group (10), while
the hydroxyl group (6) retained binding potency comparable to the
un-substituted compound 5.
Increasing the steric hindrance on indazole ring by different 2N-
alkyl groups reduced 5-HT2A receptor binding affinity by more than
one log unit on moving from methyl (7) to ethyl (12) or to
methoxyethyl (13) group. N-methylation of the indazole-3-
carboxamide nitrogen (14) also markedly lowered receptor
binding affinity by more than 2 log units in comparison to the un-
substituted compound 5. An indazole ring 5-methoxy substituent
(16), selected because negatively relevant for other serotonin
Results and Discussion
Structure-Activity Relationship
In this project, the design approach was mainly driven by our
knowledge of the SAR for the two G protein-coupled receptors
from several classes of compounds.[16] The real starting point of
the project were some binding results obtained during a hit to lead
program aiming to obtain selective 5-HT4 receptor antagonist, and
in particular, those obtained for the two indazole-amide isomers
depicted in Table 1.[17] In the competition binding assay, the shift
of the methyl position from the 1-nitrogen (4) to the 2-nitrogen of
the indazole ring (5) results in a 300-fold drop in 5-HT4 receptor
affinity while maintaining affinity for the 5-HT2A receptor. This
sharp change in the receptor affinity profile, due to a subtle
structural modification, opened a very promising scenario, making
us select 2N-substituted indazole-3-carboxamide scaffold as
starting point for our program.
2
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subtype receptors,[19] gave a slight drop in affinity (about half log
Table 3. SAR: Effect of ring A and linker modification on affinity at the 5-HT2A
unit) in comparison with the parent compound 6.
receptor.
Table 2. SAR: Effect of aromatic ring substitution on N-{[1-(2-
phenylethyl)piperidin-4-yl]methyl}-indazole-3-carboxamide scaffold on 5-HT2A
receptor affinity.
pKi[a]
5-HT2A
Entry
7
A
n
1
1
1
1
R1
F
R2
H
H
F
8.34
pKi[a]
5-HT2A
Entry
R1
R2
R3
R4
R5
17
F
6.46
6.69
<5
5
6
CH3
CH3
H
H
H
OH
F
H
H
H
F
H
H
7.98
8.00
8.34
9.00
6.10
6.11
8.96
7.65
7.08
6.12
5.41
7.59
9.44
18
H
F
19
H
7
CH3
H
H
8
CH3
H
H
H
20
21
22
1
0
2
F
H
F
H
H
H
6.77
4.61
6.63
9
CH3
H
CF3
CH3O
F
H
H
F
H
10
11
12
13
14
15
16
CH3
H
H
CH3
H
H
[a] Displacement of [3H]-ketanserin from human recombinant 5-HT2A receptor,
expressed in CHO-K1 cell line. pKi = log 1/Ki. (n = 2) (see experimental section
for further details). Values of 95% confidence interval (CI95%) always lower or
equal to the 5% of Ki values.
CH3CH2
CH3O(CH2)2
CH3
H
F
H
H
H
H
H
H
H
F
H
H
H
CH3
CH3
H
CH3
H
CH3O
OH
Table 4. Modification of the central backbone.
CH3
CH3O
pKi[a]
5-HT2A
Structure
Entry
R
ketanserin
[a] Displacement of [3H]-ketanserin from human recombinant 5-HT2A receptor,
expressed in CHO-K1 cell line. pKi = log 1/Ki. (n = 2) (see experimental section
for further details). Values of 95% confidence interval (CI95%) always lower or
equal to the 5% of Ki values.
23
24
H
7.98
CH3CH2
8.00
In a further approach to improve binding affinity for the 5-HT2A
receptor modification of the central 4-aminomethylpiperidine
scaffold, varying the relative distances between the indazole-3-
carboxamide group, the central basic nitrogen and the terminal
phenyl ring were investigated. Unfortunately, all the modification
made on the central part of the molecules (Table 3 and 4), gave
only medium or low affinity ligands.
pKi[a]
5-HT2A
Structure
Entry
n
25
26
1
2
6.74
7.63
Among the combinations of central ring and linkers tested, none
gave better results with respect to the parent compound 7, with
pKi values peaking at 7.89 and 7.63 respectively for the [1-(2-
aminoethyl)piperidin-4-yl](phenyl)methanone derivative 27 and
the 3-(4-phenylpiperidin-1-yl)propanamine derivative 26. The
most potent 5-HT2A ligands, found in the 2N-alkyl-indazolamide
series, were evaluated for serotonin sub-type receptors selectivity
(human recombinant 5-HT4, 5-HT2C and 5-HT2B) and for
adrenergic α1 receptor binding inhibition (Table 5).
27
-
7.89
[a] Displacement of [3H]-ketanserin from human recombinant 5-HT2A receptor,
expressed in CHO-K1 cell line. pKi = log 1/Ki. (n = 2) (see experimental section
for further details). Values of 95% confidence interval (CI95%) always lower or
equal to the 5% of pKi values.
As initially noted for reference compound 5, all the assayed 2N-
substituted compounds continued to show optimum selectivity
towards the 5-HT4 and 5-HT2B receptors but lacking selectivity
over the 5-HT2C receptor subtype. Interestingly, among the more
potent compounds, the best selectivity over the 5-HT2C receptor
3
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was obtained with the 2N-ethyl indazole derivative 12, with about
[a] pKi = log 1/Ki, human recombinant serotonin receptors (n = 2) (see
experimental section for further details). Values of 95% confidence interval
(CI95%) always lower or equal to the 5% of Ki values. [b] Percent of inhibition
compound @ 1 µM (pKi) of binding of [3H]-prazosin from rat cerebral cortex,
non-selective α1 receptor (n = 3) (see experimental section for further details).
– not tested.
1.7 log unit (50 fold) selectivity for 5-HT2A over 5-HT2C
.
Table 5. Serotonin and adrenergic 1 selectivity of selected compounds.
pKi[a]
% inhib.[b]
For the adrenergic receptor, compounds tested at concentration
of 1 M showed generally low to moderate percent inhibition of
binding, with the highest values seen for compounds 26 and 27,
83% and 95% respectively. Increase in α1 receptor potency
probable ascribing respectively to the 3-(4-phenylpiperidin-1-
Entry
1 n.s..
5-HT2A
7.98
8.00
8.34
9.00
8.96
7.65
7.59
7.63
7.89
-
5-HT4
5-HT2C
7.05
6.83
7.27
7.70
7.37
5.97
-
5-HT2B
5
5.83
5.84
(6.17)
33
55
29
61
56
n.t.
83
95
-
6
5.59
5.82
yl)propanamine
and
[1-(2-aminoethyl)piperidin-4-
yl](phenyl)methanone skeletons, structural features already
proven on Ondansetron and Ketanserin analogues with optimal
affinity results.[22,23]
7
5.99
5.82
8
11
-
-
In the attempt to pursuit the promising results obtained with
compound 12, both in the serotoninergic selectivity and in the
adrenergic α1 binding potency, we decided to link the indazole-2-
nitrogen atom with the nearby 3-carboxamide group, forming 3,4-
dihydropyrazino[1,2-b]indazol-1(2H)-one and 2,3,4,5-tetrahydro-
1H-[1,4]diazepino[1,2-b]indazol-1-one tricyclic derivatives. This
modification was aimed at mimicking the steric hindrance of the
indazole 2-N-ethyl group of 12, constraining the conformational
freedom of amide bond freezing it in the anti orientation . The
effect of this approach was already described by other authors,
applied to indazole amide derivatives. It was helpful to obtain
-
-
12
-
-
16
6.30
-
26
-
5.93
6.51
-
-
27
-
-
SB207266A
Prazosin
SB242084
Ketanserin
9.68
-
-
-
-
-
-
-
-
(9.80)
-
potent, non-selective, dopaminergic and serotonin ligands.[26,27]
A
-
9.28
-
focused series of tricyclic 1-[1-(2-phenylethyl)piperidin-4-
yl]methanamide derivatives were synthesized and tested. The
most relevant results are reported in Table 6.
9.44
6.74
-
Table 6. SAR: Substitution on pyrazino[1,2-b]indazol-1(2H)-one and 1H-[1,4]diazepino[1,2-b]indazol-1-one derivatives.
pKi[a]
% inhib.[b]
1 n.s.
Entry
n
R1
R2
X
5-HT2A
8.50
8.74
8.14
7.68
7.78
7.53
<5
5-HT4
5-HT2C
5.77
5.74
4.74
<5
5-HT2B
28
29
30
31
32
33
34
35
1
1
2
2
1
1
1
2
H
F
F
H
F
CH2
CH2
CH2
CH2
CH2
CH2
CO
<6.0
5.93
83
43
72
40
35
66
8
<6.0
<6.0
H
H
H
H
H
H
<6.0
<6.0
H
H
F
-
-
-
5.5
5.13
<5
-
-
-
H
H
-
-
CO
<5
-
<5
-
-5
Ketanserin
SB207266A
SB242084
Prazosin
9.44
-
-
-
6.74
9.68
-
-
-
-
-
-
-
9.28
-
-
(9.80)
4
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[a] pKi = log 1/Ki, human recombinant serotonin receptors (n = 2) (see experimental section for further details). Values of 95% confidence interval (CI95%) always
lower or equal to the 5% of Ki values. [b]Percent of inhibition, compound @ 1 µM (pKi), of binding of [3H]-prazosin from rat cerebral cortex, non-selective α1 receptor
(n = 3) (see experimental section for further details). – not tested.
The shift from bicyclic to tricyclic system produced a slight effect
on 5-HT2A receptor affinity e.g. compare 29 with 8 or 28 and 7).
However, this modification had a substantial impact on 5-HT2C
receptor binding affinity. Selectivity for the 5-HT2A receptor of up
to 3 log units (1000 fold) was seen but without effect on either the
selectivity over the other serotonin sub-type receptors assayed
(5-HT4 and 5-HT2B) or the residual affinity for the adrenergic α1
receptor (in the case of 28, α1 binding inhibition peaks at 83% at
1 µM).
The enlargement of the new formed ring system, from 6 to 7
membered did not increase the 5-HT2A binding affinity (compare
the two p-fluoro-phenyl derivatives 30 and 28 or the un-
substituted derivatives 31 and 32). The partial reduction of the
indazole ring system, to obtain the tetra-hydro derivative 33,
produced a ten-fold drop in 5-HT2A receptor affinity. Conversely,
the complete loss of binding affinity of the two non-basic
compounds 34 and 35 pointed out the importance of a basic
center for this series of compounds.
41 showed some decrease in 5-HT2A receptor affinity (about half
log unit in comparison with their parent compound 32), with a
marked increase of adrenergic α1 binding inhibition (about 80%
at 1M compared with 35% for the parent compound 32) (Table
7).
It is hard, and also off topic, for this article, try to explain, in
function of the different receptors subtype features, the
serotoninergic and adrenergic pharmacological profile showed by
the two classes of compounds studied. This difficulty could be the
reason for the fact that the dual activity on 5-HT2A and α1
receptors, although shown in some poly-pharmacological drugs
or reported in studies to identify selective agents, has not been
rationally pursued and rationalized in the scientific literature, so
far.[28,29]
The most active and selective 5-HT2A receptor ligands were
selected to investigate their adrenergic α1 sub-type receptors (1A,
1B and 1D) affinity (Table 8).
Also in the tricycloindazol-3-carboxamide system we investigated
the 3-methylenepiperidine as central basic moiety (compounds
36-39, Table 7) but found a sharp drop of 5-HT2A receptor affinity
(pKi < 6.6).
Table 8. Adrenergic 1A, 1B and 1D Selectivity of Selected Compounds
pKi[a]
Entry
1A
6.80
7.16
6.64
7.32
6.80
6.64
6.64
9.96
-
1B
6.66
7.62
6.85
7.96
7.33
7.38
7.14
-
1D
5.96
<5
Table 7 SAR: Modification of the central backbone of tricyclic systems.
6
7
8
28
6.28
6.97
6.52
6.21
6.42
-
29
pKi[a]
[5-HT2A
Entry]
n
R1
R2
31
]
32
36
37
38
39
1
1
2
2
H
F
H
F
F
H
F
5.94
WB4101
Prazosin
6.16
10.00
9.64
6.56
H
6.59
[a] Displacement of [3H]-prazosin from human recombinant α1 subtype
receptors expressed in stable CHO cell line. pKi = log 1/Ki. (n = 2) (see
experimental section for further details). Values of 95% confidence interval
(CI95%) always lower or equal to the 5% of Ki values. – Not tested
The multi-concentration response curves for the α1 subtype
receptors were in good agreement with the initial single
concentration results obtained from the 1 non-selective assay
used in the initial SAR studies. Among the tested compounds,
derivative 28 gave the most potent α1 receptors ligand, with pKi
values ranging between 7.96 (α1B) and 6.97 (α1D).
pKi[a]
[5-HT2A
pKi[a]
[5-HT2C
% inhib.[b]
1 n.s.
Entry
n
]
]
40
41
1
2
7.13
7.34
<5
<5
74
88
Pharmacology profiling studies
We selected compound 28 for a full in vitro pharmacology profile
in a standard test panel (Eurofins), including more than 60
molecular targets (see the Supplementary Information for the full
list of receptors, ion channels, transporters and enzymes
assayed). In the single concentration binding assays, only human
α2B and α2C receptors, and Ca2+ (type L) ion channel showed a
percent of inhibition > 50%, the cut-off limit for a significant affinity.
[a] pKi = log 1/Ki, human recombinant serotonin receptors (n = 2) (see
experimental section for further details). Values of 95% confidence interval
(CI95%) always lower or equal to the 5% of pKi values. [b] Percent of inhibition
(compound @ 1 µM) of binding of [3H]-prazosin from rat cerebral cortex, non-
selective α1 receptor (n = 3) (see experimental section for further details).
Similarly, to the results obtained for the bicyclic 2-alkylindazol-3-
amide 26, the two 1-propyl-4-phenylpiperidine derivatives 40 and
5
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10.1002/cmdc.201800199
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For this reason, we selected these targets for the pKi
measurement with the concentration response curves assays.
Compound 28 showed low residual target affinity (pKi > 6.0) only
for α2B and α2C receptors with pKi of 6.18 and 6.46, respectively.
Was interesting to note that compound 28 displays almost full
selectivity for other classes of G protein–coupled receptors
(GPCRs), such as dopamine and histamine. This uncommon
property could be due to the specific, rigid nature of the 3,4-
dihydropyrazino[1,2-b]indazol-1(2H)-one moiety, absent in other
more flexible unselective ligands.[30,31]
Moreover, the functional activity of compound 28 was assessed
on 5-HT2A serotonin and selected adrenergic receptor subtypes.
Compound 28, for all the receptor system assayed, showed
antagonist effect, in particular, a good antagonist activity could be
observed for 5-HT2A (IC50 = 18 nM) and alpha1 receptor subtypes
(IC50s ranging from 220 nM to 1.5 µM). Inhibitory activity was
measured also for alpha2 receptors. In this case antagonistic
activity is exerted al higher concentrations, in line with affinity data
(Table 9).
the IOP measured for 240 min following treatment. As reported in
figure 2, compound 28, administered the day of the experiment,
reduced IOP levels in carbomer-induced glaucoma model, with an
IOP decrease of 6.5 mmHg (ΔIOP about 22%) at 120 min post-
administration.
5 0
4 0
3 0
2 0
1 0
vehicle
Table 9. Functional activity of compound 28 on Serotonin 2A, Adrenergic 1A
1B, 1D, 2B and 2C
,
c o m p o u n d 28 0 .5 %
0
Compound 28
Receptor
IC50[b] (nM)
Kb (nM)
Functional activity[a]
b as al
0
30 60
120
180
240
tim e (m in )
5-HT2A
α1A
α1B
18
4
Antagonist
1500
190
Antagonist
Figure 2. IOP (mmHg) versus time (min), in carbomer-induced ocular
hypertension in rabbit eyes treated twice with one drop of compound 28 at 0.5%
and vehicle. Errors were within 10-15% of the reported IOP values (from three
different measurements for each of the six animals in the study group) and were
statistically significant (p < 0.05 by the Student’s t test).
220
24
Antagonist
α1D
910
260
Antagonist
α2B
67000
36000
8800
1700
Antagonist
α2C
Antagonist
[a] Functional inhibitory activity vs a reference agonist was measured in stable
recombinant cell lines expressing human selected receptors. [b] Results are
expressed as IC50 and Kb (apparent dissociation constant) (n = 2). See
experimental section for further details.
5 0
4 0
3 0
2 0
In the perspective of using this compound as a topical ophthalmic
substance, we also evaluated its apparent water solubility
(g/100ml in phosphate buffer, pH = 5.5). We selected a slightly
acid buffer to enable solubility of the basic compound without
moving away from the tolerated pH range of ophthalmic
formulation.[20] Fortunately, derivative 28 showed a very good
solubility (1.9% w/v), that was ideal for further in vivo investigation.
An ocular tolerability test was performed for 28 in order to select
the concentrations range to be used for efficacy assessment
studies. New Zealand white male rabbits were used and the
ocular effects of the compound were scored by means of the
Draize scale.[21] The ocular tolerability of 28 (0.1 ml of solution in
phosphate buffer pH = 5.5) was assessed at concentrations of 1.6
% and 0.8 %. At the highest dose 28 showed slight hyperemia of
conjunctivae, moderate lacrimation and blinking up to 2h after
treatment, so we selected 0.8% as the tolerated dose and we
used it as maximum dose for the subsequent efficacy assessment
tests.
1 0
tim olol 0.5%
c o m p o u n d 28 0 .5 %
0
b as al
0
30 60
120
180
240
tim e (m in )
Figure 3. IOP (mmHg) versus time (min), in carbomer-induced ocular
hypertension in rabbit eyes treated twice with one drop of compound 28 at 0.5%
and timolol 0.5%. Errors were within 10-15% of the reported IOP values (from
three different measurements for each of the six animals in the study group) and
were statistically significant (p < 0.05 by Student’s t test).
In hypertensive rabbits (eye pressure of around 28-46 mmHg,
generated as reported in reference 27), compound 28 (solution,
0.5%) was investigated in an acute administration schedule, and
6
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EtO2C
R
a, b
d
MsO
Interestingly in a different experiment, compound 28, showed
stronger IOP lowering activity along all the course of the
experiment (ΔIOP about 30%) in comparison of the reference
compound timolol (ΔIOP about 23%) (Figure 3).
NBoc
NH
OR
NBoc
50
54
51: R = CO2H
c
52: R = CH2OH
O
O
O
Chemistry
OCH3
NH
f
g,h
A linear synthetic pathway was devised to prepare the initially
tested bicyclic 2N-alkyl-indazole-amide derivatives as, for
example, depicted in Scheme 1 for compound 8. Specifically, after
protecting the primary amine moiety as a Schiff’s base 44, the
piperidinyl core was easily decorated with the commercially
available 1-(2-bromoethyl)-2-fluorobenzene (45) furnishing the
intermediate 46, which was straightforwardly transformed in the
corresponding diamine 47 upon acid hydrolysis. Subsequently,
the obtained advanced intermediate 47 was used as nucleophilic
counterpart in the final amide formation with the freshly prepared
acyl chloride 49 to afford the target product 8 (Scheme 1).
N
N
N
N
N
NHBoc
N
H
55: R = H
58
59
e
56: R = CH3
NR
N
O
O
i
N
j
N
F
N
N
N
N
28
60: R = Boc
61: R = H
g
a
b
H2N
Ph
N
Scheme 2. Reagents and conditions: (a) 2M NaOH, Δ; (b) (Boc)2O, Et3N, 25 °C
(70% over two steps); (c) 1M BH3THF, 25 °C (85%); (d) MsCl (53), Et3N, DCM,
NH
NH
25 °C (73%); (e) H2SO4 conc., MeOH,
Δ (82%); (f) tert-butyl (2-
42
44
bromoethyl)carbamate (57), Cs2CO3, DMF, 25 °C, (50%); (g) HCl (1.25M in
EtOH), MeOH, 25 °C; (h) Cs2CO3, MeOH (91%, over two steps); (i) NaH,
THF/DMF, 0 °C, than 54, Δ (85%); (j) 1-(2-bromoethyl)-2-fluorobenzene (62),
Cs2CO3, DMF (55%, over two steps).
Ph
N
F
H2N
F
c, d
N
N
Conclusions
47
46
At the beginning of our project, we depicted the dual-receptor (5-
HT2A and α1) interaction approach as a promising method to treat
topically one of the main risk factor of the open angle Glaucoma,
high intraocular pressure. With this goal we discovered a novel
3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one 5-HT2A antagonist,
compound 28, with >100-fold selectivity over other serotonin sub-
type receptors and high affinity for α1 receptors. We obtained the
selective, nanomolar range potency compound 28 during a hit-to-
lead program starting from the initial hit 5. Compound 28, besides
its in vitro potency, showed a favorable water solubility and ocular
tolerability, that permitted its use in an ocular hypertensive-rabbit
model. In this carbomer-induced glaucoma model, 28,
administered locally, showed a clear ocular hypotensive action,
superior in magnitude for the whole course of the experiment, to
the reference compound timolol. These results support the initial
findings related to the promising potential of a multi-receptors (5-
HT2A and α1) action as a new pharmacological mechanism for the
treatment of Glaucoma. To fully understand the actual effect of 28
on the aqueous humor dynamic, further pharmacological
characterization is in progress.
F
O
N
X
f
O
NH
N
N
N
8
N
48: X = OH
49: X = Cl
e
Scheme 1. Preparation of Target Compound 8. Reagents and conditions: (a)
benzaldehyde (43), toluene, 25°C; (b) 1-(2-bromoethyl)-2-fluorobenzene (45),
K2CO3, abs. EtOH, 25°C; (c) 3N HCl, 25 °C; (d) 6N NaOH, 25 °C (72%, over
four steps); (e) SOCl2, toluene, Δ; (f) 47, toluene, 25 °C (27% over two steps).
Similarly, as an exemplar of the tricyclic analogues, the
retrosynthetic analysis of the target structure 28 suggested as
suitable main building blocks (i) an indazole head, herein fused
with either a six or a seven-membered ring, (ii) a central body with
a nitrogen six-membered ring, and (iii) a short aromatic tail.
Specifically, as outlined in Scheme 2, the preparation of the
piperidine central building block 54 was readily accomplished: (i)
the N-protection and the subsequent basic hydrolysis of the amino
ester 50 to the corresponding acid derivative 51, (ii) the
straightforward reduction of 51 to the primary alcohol 52 and (iii)
the conversion of the hydroxyl moiety to a better leaving group.
The tricyclic initial scaffold 59 was obtained through the de-
protection and the ring closure of the intermediate 58, which, in
turn, was prepared starting from the simple indazole-3-carboxylic
acid 55 upon esterification and subsequent N-alkylation (Scheme
2).
Experimental Section
Chemistry (General). Unless otherwise indicated, all materials were
obtained from commercial suppliers (Sigma-Aldrich) and used as received
without further purification. All solvents were analytical grade. Anhydrous
solvents were obtained from Sigma-Aldrich. Yields were not optimized. All
reactions involving air- or moisture-sensitive reagents were performed
under argon atmosphere. All evaporations were carried out under reduced
pressure. Reaction progress was monitored by TLC using Merck silica gel
60 F254 (0.04-0.063 mm) with detection by UV (214 or 254 nm). Merck
silica gel (200-400 mesh) or aluminum oxide 90 (active neutral) were used
7
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10.1002/cmdc.201800199
ChemMedChem
FULL PAPER
for flash column chromatography. Melting points (uncorrected) were
determined in open Pyrex capillary tubes using a Buchi 510 melting point
apparatus. Final compounds purity, always >95%, was determined both
by high pressure liquid chromatography (HPLC), and Ultra Performance
Liquid Chromatography (UPLC), using the area percentage of all peaks
detected. HPLC analyses were carried out with a pump/autosampler
Waters (2695 – Alliance model), a UV photo diode array detector Waters
(2996 model) and a system data management Waters (Empower 2). The
2-Methyl-2H-indazole-3-carbonyl chloride (49). To a suspension of the 2-
alkyl-2H-indazole-3-carboxylic acid 48 (3.9 mmol) in toluene (20 mL)
SOCl2 (7.8 mmol) was added cautiously drop-wise. The whole was
refluxed for 10 hours. After cooling toluene was evaporated under reduced
pressure and the residue was taken-up 3 times with toluene, which was
every time evaporated, to give the crude 2-alkyl-2H-indazole-3-carbonyl
chloride 49, which was used in the next reaction without any further
purification. Yield 95%; 1H NMR (300 MHz, DMSO-d6) δ 8.15–8.00 (m,
1H), 7.85–7.70 (m, 1H), 7.65–7.50 (m, 1H), 7.45–7.30 (m, 1H), 3.97 (s,
3H) ppm.
column used was generally Suplex pkb-100 (250x4.6 mm,
5 m).
UPLC/QToF Cromatography (exact mass data) were obtained by means
of a SYNAPT MS - ACQUITY UPLC system, Waters. The system was
operated in positive ion mode in the “V-Optics” configuration. Leucine-
enkephalin (200 pg/µl) was employed as the lock mass in order to provide
authenticated exact mass measurement in MS and MS/MS modes within
5 ppm RMS mass accuracy. The column was an Acquity BEH C18 (2.1x50
mm, 1.7m). Nuclear Magnetic Resonance Spectroscopy (1H-NMR and
13C-NMR) were obtained using a both a Varian Gemini and a Bruker
Avance system, operating respectively at 200 or 300.13 MHZ for 1H-NMR
and 75.48 and 50 MHz for 13C-NMR. All resonance bands referenced to
tetramethylsilane (internal standard). For 1H-NMR spectroscopy: (s) =
singlet; (d) = doublet; (t) = triplet; (br) = broad; (dd) = double doublet; (dt)
= double triplet; (ddd) = double double doublet ;(dtd) = double triple
doublet; (m) = multiplet; J = coupling constant; δ = chemical shift (in ppm).
Preparative HPLC/MS system consisted of a Waters 2767 Sample
manager, a Waters 2478 dual  absorbance detector and a Waters
Micromass ZQ single quadrupole mass spectrometer with an electrospray
N-({1-[2-(2-Fluorophenyl)ethyl]piperidin-4-yl}methyl)-2-methyl-2H-
indazole-3-carboxamide (8). The monoalkylated diamine 47 (2.7 mmol),
dissolved in anhydrous toluene (20 mL), was allowed to react for 12 hours
at r.t. with the 2-alkyl-2H-indazole-3-carbonyl chloride 49 (2.7 mmol). The
reaction mixture was then filtered under reduced pressure and the residue,
after having been washed with toluene, was taken-up with CH2Cl2. The
resulting solution was washed with 1M NaOH, dried over anhydrous
Na2SO4, and evaporated to dryness. The crude product obtained was
purified by flash chromatography on silica gel (eluent CHCl3/MeOH ratio
from 100/0 to 95/5) affording the N-({1-[2-(2-fluorophenyl)ethyl]piperidin-4-
yl}methyl)-2-methyl-2H-indazole-3-carboxamide 8. Yield: 27%. 1H NMR
(200 MHz, CDCl3)  7.84–7.58 (m, 2H), 7.42–6.91 (m, 6H), 6.29 (t, J = 5.2
Hz, 1H), 4.46 (s, 3H), 3.45 (t, J = 6.2 Hz, 2H), 3.14–2.97 (m, 2H), 2.94–
2.77 (m, 2H), 2.68–2.50 (m, 2H), 2.07 (td, J = 11.6 Hz, J =2.0 Hz, 2H),
1.96–1.62 (m, 3H), 1.57–1.30 (m, 2 H) ppm; 13C NMR (50 MHz, CDCl3) 
161.1 (d, J = 244.9 Hz), 160.4, 147.2, 130.9 (d, J = 5.1 Hz), 127.8 (d, J =
8.1 Hz), 127.6, 127.1 (d, J = 16.3 Hz), 125.9, 124.3, 124.0 (d, J = 3.5 Hz),
120.2, 118.6, 118.2, 115.2 (d, J = 22.1 Hz), 59.0, 53.2, 45.4, 40.7, 36.3,
30.2, 26.9 ppm; Elemental Analysis for C23H27N4OF calcd. 70.03%C,
6.90%H, 14.20%N, found 70.21%C, 6.79%H, 14.39%N.
ionization (ESI) source. The column used was a X-Bridge Prep C18 5
m
with 19x10mm (Waters) pre-column. Fraction collection was available from
the system software MassLynxTM v. 4.1. Detection wavelength was set to
230 nm and temperature to 25°C. Elemental Analysis was conducted by
means of a CHNS-O EA1108 elemental analyser, Carlo Erba Instruments,
and the results were within + 0.3 % of the theoretical values, unless
otherwise noted. Final compounds, described in this article, were purified
with one of the following techniques: flash chromatography on silica gel
(Grace Reveleris flash chromatography system with 40 μM silica cartridge
Flow = 60 ml/min; a proper gradient of the mixture CHCl3 and CH3OH as
eluents); preparative HPLC/MS system (sample was dissolved (50 mg/mL)
in DMSO/CH3CN in 1:1 ratio; a proper gradient of the two phases CH3CN
+ 0.1% formic acid and H2O + 0.1% formic acid; flow = 40 mL/min).
Chlorine ion determination was performed by precipitation titrations on
automatic titration instrument including stirring plate and automatic burette
(Metrohm Titrando 809). Silver nitrate 0.05 M was used as titrant and data
were registered adopting an Ag-Titrode electrode with Ag2S coating
(6.0430.100).
2-({1-[2-(4-Fluorophenyl)ethyl]piperidin-4-yl}methyl)-3,4-
dihydropyrazino[1,2-b]indazol-1(2H)-one hydrochloride (28).
1-(tert-Butoxycarbonyl)piperidine-4-carboxylic acid (51). In a two-necked
flask equipped with a condenser, the ethyl isonipecotate (50, 20 mmol)
was placed with NaOH aq. (2N, 40.9 mmol). The mixture was heated under
reflux and stirred for 3 hours. Afterwards, HCl 6N was added until pH 5
and the volume of the resulting mixture was reduced under vacuum. The
obtained crude was diluted with a solution of H2O/dioxane (1/1 in volume)
and placed in a two-necked round bottomed flask equipped with a
magnetic stirring bar. Boc-anhydride (25.5 mmol) and trimethylamine (20.5
mmol) were cautiously added and the reaction mixture was kept under
stirring for 7 hours. Subsequently, the mixture was directly washed with
water (2 x 50 mL), dried over anhydrous Na2SO4, and concentrated to
furnish the desired 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid 51
(13.8 mmol), which was directly used in the subsequent reaction without
N-({1-[2-(2-Fluorophenyl)ethyl]piperidin-4-yl}methyl)-2-methyl-2H-
indazole-3-carboxamide (8)
1
any further purification. Yield: 70% (over two steps); H NMR (200 MHz,
1-(2-Fluorophenethyl)piperidin-4-yl)methanamine (47). Benzaldehyde
CDCl3)  4.02 (brd, J = 13.2, 2H), 2.85 (dt, J = 13.2 Hz, J = 3.0 Hz, 2H),
2.49 (tt, J = 10.9 Hz, J = 3.90 Hz, 1H), 1.99–1.90 (m, 2H), 1.78–1.50 (m,
2H), 1.45 (s, 9H) ppm.
(43; 264 mol) was added, dropwise, to
a
solution of 4-
aminomethylpiperidine 42 (264 mol) in toluene (100 mL). The solution thus
obtained was kept stirred at room temperature. After 3 hours, the solvent
was removed by evaporation at reduced pressure and the residue was
taken up twice in toluene. The crude N-hexahydro-4-pyridinylmethyl-N-
phenylmethylidenamine 44 (about 260 mmol) was dissolved in absolute
ethanol (500 mL) containing anhydrous potassium carbonate (528 mmol)
and 1-(2-bromoethyl)-2-fluorobenzene (45, 290 mmol). The reaction
mixture thus obtained was boiled under reflux for 24 hours. After cooling
to room temperature, the suspension was filtered and the solvent was
evaporated at reduced pressure. The residue thus obtained was
suspended in 3N HCl (200 mL) and stirred at room temperature for 2
hours. The acidic aqueous phase was washed 4 times with ethyl acetate
and then alkalized to pH approx. 13 with 6N NaOH and extracted with
dichloromethane. The combined organic phase was dried over anhydrous
Na2SO4 and the solvent was removed by evaporation at reduced pressure.
The residue was subjected to flash chromatography on silica gel to afford
(1-(2-fluorophenethyl)piperidin-4-yl)methanamine (47) in 74% yield. 1H
NMR (300 MHz, CDCl3) δ 7.20–6.80 (m, 4H), 3.00–2.40 (m, 10H), 2.10–
1.50 (m, 5H), 1.40–1.00 (m, 2H) ppm.
tert-Butyl 4-(hydroxymethyl)piperidine-1-carboxylate (52). In a two round
bottomed flask, the carboxylic acid 51 (3.0 mmol) was placed under stirring
in anhydrous THF. To the resulting solution, BH3•THF (1M, 4.5 mmol) was
slowly added at room temperature over 20 min taking care of the possible
bubbling. The obtained mixture was kept at room temperature for
additional
2 hours (until completion of reaction detected by TLC,
hexane/AcOEt = 6/4). Afterwards the crude was slowly poured in a
saturated solution of NaHCO3 and the organic layer was extracted several
times with Et2O. The combined organic phases were washed with brine,
dried over anhydrous Na2SO4 and concentrated at reduced pressure to
furnish the desired product 52 (2.5 mmol), which was employed without
any further purification. Yield: 85%; 1H NMR (200 MHz, CDCl3)  4.12 (brd,
J = 12.2 Hz, 2H), 3.50 (t, J = 5.7 Hz, 2H), 2.70 (brt, J = 12.7 Hz, 2H), 1.78–
1.60 (m, 3H), 1.45 (s, 9H), 1.27–1.01(m, 2H) ppm.
tert-Butyl 4-((methylsulfonyloxy)methyl)piperidine-1-carboxylate (54). In a
two-necked round bottomed flask equipped with a magnetic stirring bar,
the N-protected alcohol 52 (2.0 mmol) and trimethylamine (2.0 mmol) were
placed in CH2Cl2 (4.5 mL) To the mixture, CH3SO3Cl (53, 2.0 mmol) was
8
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10.1002/cmdc.201800199
ChemMedChem
FULL PAPER
added slowly over 20 min taking care of the exothermic reaction using an
ice-bath. The system was kept at room temperature for 12 h (the substrate
disappearance was monitored by TLC, hexane/AcOEt = 3/7). Afterwards
the reaction wasquenched adding H2O and extracting the aqueous phase
with CH2Cl2 several times. The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, filtered and concentrated under
(chloroform/methanol = 9/1 -> 7/3) affording the advanced intermediate 60.
Yield: 85%; 1H NMR (200 MHz, CDCl3):  8.16 (dt, J = 7.3 Hz, J = 1.2 Hz,
1H), 7.76 (dt, J = 8.4 Hz, J = 1.1 Hz, 1H), 7.45–7.22 (m, 2H), 4.70 (t, J =
6.2 Hz, 2H), 4.14 (brd, J = 11.7 Hz, 2H), 3.89 (t, J = 6.2 Hz, 2H), 3.51 (bs,
2H), 2.71 (brt, J = 12.6 Hz, 2H), 2.01–1.81 (m, 1H), 1.80–1.64 (m, 2H),
1.45 (s, 9H), 1.39–1.11 (m, 2H) ppm.
vacuum
to
furnish
the
desired
tert-butyl
4-
2-({1-[2-(4-Fluorophenyl)ethyl]piperidin-4-yl}methyl)-3,4-
((methylsulfonyloxy)methyl)piperidine-1-carboxylate (54, 1.5 mmol) that
was used without any further purification in the subsequent step. Yield:
73%; 1H NMR (200 MHz, CDCl3)  4.24-4.03 (m, 2H), 4.06 (d, J = 6.4 Hz,
2H), 3.01 (s, 3H), 2.71 (brt, J = 12.9 Hz, 2H), 2.02–1.81 (m, 1H), 1.81–1.66
(m, 2H), 1.45 (s, 9H), 1.35–1.04 (m, 2H) ppm.
Methyl 1H-indazole-3-carboxylate (56). To indazole-3-carboxylic acid (55,
100 mmol) suspended in methanol (150 mL) was slowly added
concentrated H2SO4. The mixture was vigorously stirred and refluxed at
80°C following the reaction by TLC (AcOEt/i-PrOH = 7/3). After 6 h,
methanol was distilled off and the remained pale yellow solid was treated
with water, crushed to fine powder and collected by filtration. The crude
product was purified by crystallization with AcOEt affording indazole-3-
carboxylic acid methyl ester (56) as a pale yellow solid. Yield: 82%; 1H
NMR (200 MHz, CDCl3)  8.19 (t, J = 8.1 Hz, 1H); 7.74 (dt, 1H, J = 8.1 Hz,
J = 0.9 Hz, 1H), 7.53–7.29 (m, 2H), 4.08 (s, 3H) ppm.
Methyl 2-(2-(tert-butoxycarbonylamino)ethyl)-2H-indazole-3-carboxylate
(58). To a solution of the substrate 56 (2.0 mmol) and Boc-aminoalkyl
bromide (57, 4.0 mmol) in DMF (3 mL) was added solid Cs2CO3 (6.0
mmol). The mixture was vigorously stirred until the reaction completion
(TLC, Hexane/Ethyl Acetate = 7/3). Water and AcOEt were added and the
resulting solution was acidified to pH 6-6.5 using 1M HCl. The layers were
separated and the organic phase was washed with water and brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified by chromatography on silica gel (hexane/AcOEt=6/4)
affording the methyl 2-(2-(tert-butoxycarbonylamino)ethyl)-2H-indazole-3-
carboxylate 58 as whitish solid. Yield: 45%; 1H NMR (200 MHz, CDCl3) 
8.02 (dt, J = 7.5 Hz, J = 1.6 Hz, 1H), 7.79 (dt, J = 7.5 Hz, J = 1.6 Hz,1 H),
7.42–7.23 (m, 2H), 5.03 (t, J = 5.5 Hz, 2H), 5.07–4.89 (brs, 1H), 4.03 (s,
3H, OCH3), 3.74 (q, 2H, J = 5.5 Hz), 1.36 (s, 9H) ppm.
dihydropyrazino[1,2-b]indazol-1(2H)-one hydrochloride (28). To a
solution of the substrate (60, 2.0 mmol) in CH2Cl2 (18 mL) was added TFA
(9 mL) and the reaction mixture was stirred at room temperature for 3
hours. The solvents were removed under reduced pressure and the crude
product was re-dissolved in ethyl acetate. The organic layer was washed
with saturated aqueous NaHCO3, brine and dried over anhydrous Na2SO4.
Filtration and removal of solvent under reduced pressure gave the product
61 that was directly used in the subsequent reaction without any further
purification. To a solution of the amino derivative 61 (2.0 mmol) and
alkylating agent 62 (4.0 mmol) in dry DMF (3 mL) was added solid Cs2CO3
(6.0 mmol). The mixture was vigorously stirred for 8h (reaction completion
followed by TLC, chloroform/methanol = 7/3). Water and AcOEt were
added and the resulting solution was extracted several times with AcOEt.
The combined organic layers were dried (anhydrous Na2SO4), filtered and
evaporated under reduced pressure. The residue was purified by
chromatography on silica gel eluted with chloroform/methanol 9/1. Yield:
1
68%. H NMR (300 MHz, DMSO-d6):  10.51 (brs, 1H), 8.01 (dt, J = 8.3
Hz, J = 1.2 Hz, 1H), 7.76 (dt, J = 8.6 Hz, J = 1.0 Hz, 1H), 7.42–7.11(m,
6H), 4.75 (t, J = 6.1 Hz, 2H), 3.93 (t, J = 6.1 Hz, 2H), 3.67–3.51 (m, 2H),
3.47 (d, J = 6.6 Hz, 2H), 3.40–3.17 (m, 2H), 3.12–3.00 (m, 2H), 2.99–2.76
(m, 2H), 2.10–1.81 (m, 3H), 1.78–1.49 (m, 2H) ppm. 13C NMR (75 MHz,
DMSO-d6)  162.1 (d, J = 251.8 Hz), 148.9, 144.9, 134.9, 129.8, 129.1(d,
J = 8.0 Hz, 2C), 128.1, 122.8, 122.6, 119.6, 115.6 (d, J = 19.5 Hz, 2C),
114.3, 57.3, 53.1, (2C), 50.7, 47.2, 45.7, 34.3, 33.9, 28.8 (2C) ppm. HRMS
m/z 465.1830 [M+Na]+ (465.1833 calcd for C24H28ClFN4NaO).
Radioligand binding assays
Rat and human 5-HT2A receptor binding assay
3,4-Dihydropyrazino[1,2-b]indazol-1(2H)-one (59). To a stirred solution of
the substrate (58, 2.0 mmol) in ethyl acetate was added HCl 1.25 M in
ethanol (6.0 mmol). The reaction was kept under stirring at room
temperature and monitored by TLC (hexane/AcOEt = 7/3) until its
completion. After 6 hours, solvents were removed under reduced pressure
and the residue was taken up with ethyl acetate and filtered to give the
product which was directly used in the subsequent procedure without any
further purification. Subsequently, a solution of the obtained hydrochloride
(2.0 mmol) in methanol was treated with Cs2CO3 and stirred at room
temperature for 16 hours. The solution was evaporated to dryness and
taken up with water. Afterwards the solution was extracted several times
with AcOEt. The combined organic layers were dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure affording the
Membranes from rat cerebral cortex were prepared using standard
procedure. The binding of 3H-ketanserin (1nM) was performed using
standard filtration methods. Non-specific binding was evaluated in the
presence of 10µM of methysergide; 100nM prazosin and 100nM
pyrilamine were added to prevent the binding of 3H-Ketanserin to
α1adrenergic and H1 histamine receptors, respectively. Alternatively,
membrane extracts containing human 5-HT2A receptor (stable
recombinant CHO-K1 cell line expressing the 5-HT2A receptor) were used
(Euroscreen) in order to study binding at human receptor. Displacement
studies were performed using 1 nM of 3H-ketanserin. Non-specific
binding was measured in the presence of 20 µM of mianserin. For each
compound a displacement curve was performed, at least in duplicate,
using eight concentrations in a Log scale ranging from 10-12 to 10-5 M
and the affinity constant (pKi), confidence interval (±CI95%) and Hill slope
(nH) values were obtained. Compounds were dissolved in DMSO at 10mM
concentration, then 1:10 serial dilutions were performed in incubation
buffer and subsequently plated on 96 well plates (final DMSO
concentration 0.1%). Ketanserin was used as reference compound.
Assays were conducted in a 96 well plate with 60 minutes of incubation at
27°C. Membranes were then harvested onto glass fiber (GF/B) filters
(presoaked in 0.5% polyethylenimine) using filtermate cell harvester
(PerkinElmer). Subsequently the filters were washed ten times dried for 30
min in an oven at 45°C. 35 µl of Microscint 20 were added to each well. At
least 16 hours later the radioactivity was measured using a TopCount
(Packard) for 3 min/well.
pure desired 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one 59 as
a
glittering white solid. Yield: 91% (over two steps); 1H NMR (200 MHz,
CDCl3)  8.16 (dt, J = 8.4 Hz, J = 1.4 Hz, 1H), 7.78 (dt, J = 8.4 Hz, J = 1.4
Hz, 1H), 7.46-7.27 (m, 2H), 6.35 (brs, 1H), 4.72 (t, J = 6.7 Hz, 2H), 3.93
(dt, 2H, J = 6.7 Hz, J = 1.7 Hz, 2H) ppm.
tert-Butyl
4-((1-oxo-3,4-dihydropyrazino[1,2-b]indazol-2(1H)-yl)methyl)
piperidine-1-carboxylate (60). In a three-necked bottomed flask fitted with
a magnetic stirring bar, a reflux condenser and a septum, NaH (55% in
mineral oil, 2.4 mmol) was placed in anhydrous THF (2.4 mL) under argon
atmosphere at 0°C. After the H2 gas evolution stopped, the suspension
was heated at room temperature for 15 min. At the same temperature, a
solution of the substrate (59, 2.0 mmol) in 4 mL THF/DMF (1:1) was added
dropwise to the mixture that was kept under stirring for an additional hour.
Finally, the alkylating agent 54 (3.0 mmol) in 6 mL THF/DMF (1:1) was
slowly added to the solution and the resulting mixture was heated under
reflux until the reaction completion (TLC using as eluent either
hexane/AcOEt, or chloroform/methanol). After removing the external
heating, the reaction was quenched by a slow addition of AcOEt and
filtered over a silica pad. The solvents were removed under reduced
pressure and the crude product was purified by flash chromatography
Human 5-HT2B receptor binding assay
Binding assay on human 5-HT2B receptor was performed using standard
radioligand methods. Human recombinant CHO-K1 cells were used as a
source of the human receptor. 1.2 nM [3H] Lysergic acid diethylamide
(LSD) was used as radioligand. Non-specific binding was measured in the
presence of 10 µM of serotonin. Compounds were dissolved in DMSO.
9
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10.1002/cmdc.201800199
ChemMedChem
FULL PAPER
They were assayed in duplicate at five concentration dose response
curves (in a Log scale ranging from 10-9 to 10-5 M). DMSO final
concentration was 1%. Ketanserin was used as reference compound.
Assays were conducted in a 96 well plate with 60 minutes of incubation at
37°C, then membranes were harvested and scintillation counting was
performed.
Data Analysis
Results obtained at single concentration were expressed as a percent of
inhibition of control specific binding obtained in the presence of the test
compounds. The IC50 values (concentration causing a half-maximal
inhibition of control specific binding) were determined by computer
assisted non-linear regression analysis of concentration response curves.
The inhibition constants pKi (defined as the negative Log of the Ki) were
calculated from the Cheng Prusoff equation (Ki = IC50/(1+(L/KD)), where
L = concentration of radioligand in the assay, and KD = affinity of the
radioligand for the receptor. Regarding functional assays, Kb (apparent
dissociation constant) was calculated using a modified Cheng Prusoff
equation (Kb = IC50/ 1+(A/EC50A) where A = concentration of reference
agonist in the assay, and EC50A = EC50 value of the reference agonist.
Human 5-HT4(b) receptor binding assay
HEK293 (Human Embryonal Kidney) cell line transfected with the
serotoninergic 5-HT4 was used. Cell homogenate was incubated at 27°C
for 120 minutes in 96 well microtiter plates. Displacement studies were
performed using 0.2 nM of 3H-GR113808. Non- specific binding was
evaluated using 10µM SB207266A. Compounds were dissolved in DMSO.
They were assayed in duplicate at eight concentration dose response
curves (in a Log scale ranging from 10-12 to 10-5 M). DMSO final
concentration was 0.1%. SB207266A was used as reference compound.
After incubation, the reaction was terminated by rapid filtration through pre-
soaked (0.1 % polyethylenimine) glass fiber (GF/B) filters (Unifilter,
Packard) using Filtermate cell harvester (Packard). Subsequently the
filters were washed dried for 30 min in an oven at 45°C. 30-35µl of
Microscint 20 (Packard) were added to each well. At least 16 hours later
the radioactivity was measured using a TopCount (Packard) for 1 min.
Alternatively, binding at serotoninergic receptor 5-HT4 was measured on
Guinea pig striatum homogenates in the same experimental conditions,
using 0.70 nM [3H]-GR113808 as radioligand.
IOP-lowering studies in carbomer-induced IOP raise in rabbit
Male New Zeland albino rabbits weighing 1500−2000 g were used in these
studies. Glaucoma was induced by an injection of 0.1 ml 0.25% carbomer
(Siccafluid, FarMila-THEA Pharmaceuticals) into anterior eye-chamber
bilaterally in animals anesthetized with tiletamine and zolazepam (Zoletil
100, 0.05 mg/Kg, b.w.) plus xilazine (Xilor 2%, 0.05 ml/kg b.w.) i.m.1. IOP
was measured before carbomer injection and after 1, 2 and 4 h on the first
day, then thrice daily until stabilization, and every 24 h thereafter. All
rabbits treated with carbomer presented a net increase in IOP. One drop
of 0.2% oxybuprocaine hydrochloride (Novesine, Sandoz) diluted 1:1 with
sterile saline was instilled in each eye immediately before each set of
pressure measurements. IOP was measured using a Tono-Pen XL
tonometer (Medtronic Solan, USA). As soon as a stable IOP increase was
achieved, the treatment was applied in six animals in one eye, and
compared to the contralateral eye, treated with vehicle alone (pH 5.5) or
timolol 0.5% (reference drug). The drug efficacy at lowering IOP was
evaluated after drug administration throughout a four-hour period: before
the administration and then 30, 60, 120, 180 and 240 min after drug
administration. All animal-use procedures conformed to the guidelines of
the European Community’s Council for Animal Experiments.
Rat and human α1 adrenergic receptor binding assay
α1 non-selective binding was measured in rat cerebral cortex
homogenates. Compounds were assayed at single 1 µM concentration in
duplicate. Incubation was performed for 60 minutes at 22 °C using 0.25
nM [3H]prazosin as radioligand. Non- specific binding was evaluated using
0.5 µM of prazosin. Prazosin was also used as reference compound. After
membrane filtration radioactivity was measured by scintillation counting.
Human α1 adrenergic receptor subtypes binding was evaluated using
homogenates from human recombinant CHO cells transfected
respectively with human 1A, 1B or 1D receptors. Compounds were
dissolved in DMSO (final concentration 1%). They were assayed in
duplicate at five serially diluted concentrations (in a Log scale ranging from
10-9 to 10-5 M). [3H]prazosin (0.1, 0.15 and 0.2 nM for 1A, 1B or 1D
respectively) was used as radioligand. For 1A non-specific binding was
evaluated using 0.1 mM epinephrine and WB4101 was used as reference
compound. For the other two subtypes non- specific binding was evaluated
using 10 µM phentolamine and prazosin was used as reference
compound. Incubation was performed for 60 minutes at 22°C, then after
membrane filtration radioactivity was detected by scintillation counting.
Acknowledgements
The authors would like to thank Prof. Giovanni Zappia and Dr. Rod Porter
for the excellent support provided during the preparation of the manuscript.
GF would also thank the Angelini’s Physical Chemistry Laboratory for the
analytical supports provided for the characterization of the compounds.
Keywords: Serotonin 2A receptor, 5-HT2A, α1 receptor,
antagonists, intraocular pressure, antihypertensive compounds
Human 5-HT2A and alpha adrenergic receptors functional assays.
Compound antagonistic activity was measured on 5-HT2A human receptor
expressed in recombinant HEK293 cells.100 nM serotonin was used as
reference agonist, cells were incubated for 30 minutes at 37°C and IP1
was measured by HTRF. Regarding alpha receptors, antagonism on
References:
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Entry for the Table of Contents
Graphic for Table of Contents
Text for Table of Contents
The elevation of the intraocular pressure (IOP) is the main risk factor associated with Glaucoma. We studied, as potential ocular hypotensive
agents, compounds acting on two receptors: serotonin 2A (5-HT2A) and adrenergic α1, both linked to the regulation of the humour aqueous
dynamic. We identified the 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivative 28, as effective “dual” 5-HT2A and α1 antagonist. 28 showed
potent ocular hypotensive action in vivo.
12
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