Bicyclic α-Iminophosphonates as High Affinity Imidazoline I2 Receptor Ligands for Alzheimer's Disease

Article abstract of DOI:10.1021/acs.jmedchem.9b02080

Imidazoline I₂ receptors (I₂-IR), widely distributed in the CNS and altered in patients that suffer from neurodegenerative disorders, are orphans from a structural point of view, and new I₂-IR ligands are urgently required

Full text of DOI:10.1021/acs.jmedchem.9b02080

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Article
Bicyclic #-iminophosphonates as highly affinity
imidazoline I2 receptor ligands for Alzheimer’s Disease
Carmen Escolano, Sònia Abás, Sergio Rodríguez-Arévalo, Andrea Bagán, Christian Griñán-Ferré,
Foteini Vasilopoulou, Iria Brocos-Mosquera, CAROLINA MUGURUZA, Belén Pérez, Elies Molins,
F. Javier Luque, Pilar Pérez-Lozano, Steven De Jonghe, Dirk Daelemans, Lieve Naesens, José
M. Brea, María Isabel Loza, Elena Hernández-Hernández, Jesús A. García-Sevilla, M. Julia Dr
García-Fuster, Milica Radan, Teodora Djikic, Katarina Nikolic, Mercè Pallàs, and Luis F. Callado
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02080 • Publication Date (Web): 09 Mar 2020
Downloaded from pubs.acs.org on March 10, 2020
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Pharmacology
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Bicyclic -iminophosphonates as highly affinity
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imidazoline I receptor ligands for Alzheimer’s
2
Disease
Sònia Abás,†,∇ Sergio Rodríguez-Arévalo,†,∇ Andrea Bagán, Christian Griñán-Ferré,
†
‡
‡
§
§
‖
Foteini Vasilopoulou, Iria Brocos-Mosquera, Carolina Muguruza, Belén Pérez, Elies

#
○
Δ
Δ
Molins, F. Javier Luque, Pilar Pérez-Lozano, Steven de Jonghe, Dirk Daelemans,
Δ
⧫
⧫
ǂ
Lieve Naesens, José Brea, M. Isabel Loza, Elena Hernández-Hernández, Jesús A.
ǂ
ǂ
#
#
García-Sevilla, M. Julia García-Fuster, Milica Radan, Teodora Djikic, Katarina
#
‡
§
†,
Nikolic, Mercè Pallàs, Luis F. Callado, Carmen Escolano *
†
Laboratory of Medicinal Chemistry (Associated Unit to CSIC), Department of Pharmacology,
Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, and Institute of
Biomedicine (IBUB), University of Barcelona, Av. Joan XXIII, 27-31, E-08028 Barcelona,
Spain.
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‡
Pharmacology Section, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food
Sciences, and Institut de Neurociències, University of Barcelona, Av. Joan XXIII, 27-31, E-
0
8028 Barcelona, Spain.
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§
Department of Pharmacology, University of the Basque Country, UPV/EHU, E-48940 Leioa,
Bizkaia, and Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain.
‖
Department of Pharmacology, Therapeutic and Toxicology. Autonomous University of
Barcelona, E-08193 Barcelona, Spain.

Institut de Ciència de Materials de Barcelona (CSIC), Campus UAB, E-08193 Cerdanyola,
Spain.
#
Department of Nutrition, Food Sciences and Gastronomy, School of Pharmacy and
Food Sciences, Institute of Biomedicine (IBUB), and Institute of Theoretical and
Computational Chemistry (IQTCUB), University of Barcelona, E-08921, Santa Coloma
de Gramanet, Spain.
○
Unit of Pharmaceutical Technology, Pharmacy and Pharmaceutical Technology, and
Physical Chemistry Department, Faculty of Pharmacy and Food Sciences, University of
Barcelona, Av. Joan XXIII, 27-31, E-08028 Barcelona, Spain.
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Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven,
Belgium.
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Innopharma screening platform. BioFarma research group. Centro de Investigación en
Medicina Molecular y Enfermedades Crónicas (CIMUS). Universidad de Santiago de
Compostela, Santiago de Compostela, Spain.
#
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of
Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia.
ǂ
IUNICS University of the Balearic Islands (UIB), and IdISBa, Cra. Valldemossa km 7.5, E-
0
7122 Palma de Mallorca, Spain.
KEYWORDS. Alzheimer’s disease, bicyclic -iminophosphonates, imidazoline I2
receptors, imidazoline I ligands, neuroprotection, 3D-QSAR, 5xFAD.
2
ABSTRACT.
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Imidazoline I receptors (I -IR), widely distributed in the CNS and altered in patients that
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suffered from neurodegenerative disorders, are orphan from the structural point of view
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and new I -IR ligands are urgently required for improving their pharmacological
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characterization. We report the synthesis and 3D-QSAR studies of a new family of
bicyclic -iminophosphonates endowed with relevant affinities for human brain I -IR.
2
Acute treatment in mice with a selected compound significantly decreased the FADD
protein in the hippocampus, a key marker in neuroprotective actions. Additionally, in
vivo studies in the familial Alzheimer’s disease 5xFAD murine model revealed beneficial
effects in behavior and cognition. These results are supported by changes in molecular
pathways related to cognitive decline and Alzheimer’s disease. Therefore bicyclic -
iminophosphonates are tools that may open new therapeutic avenues for I -IR,
2
particularly for unmet neurodegenerative conditions.
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INTRODUCTION
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The imidazoline receptors (IR) (non-adrenergic receptors for imidazolines) have
attracted the attention of the scientific community during decades building a body of
knowledge that place them as relevant biological targets.2,3 IR are classified in I , I and
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I3-types depending on the specific radio-labelled ligands that recognize their binding
sites. These receptors are situated in different locations and are involved in different
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physiological functions. I , I and I -IR have been unequally studied. Pharmacologically,
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I1-IR are well characterized and understood, leading to the clinically approved
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antihypertensive drugs moxonodine and rilmenidine. The most-unknown are I -IR,
3
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identified in pancreatic -cells and involved in insulin secretion. Regarding I -IR,
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although structurally undescribed, a considerable understanding has been achieved on
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these heterogeneous receptors by using well-characterized I -IR ligands. I -IR are
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2
widely distributed in the brain and, at the molecular level, are located in the outer
membrane of mitochondria. Selective I -IR ligands have proven that I -IR are involved in
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2
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analgesia, inflammation, and a plethora of human brain disorders. Dysregulations in
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2,13
the levels of I -IR are a hallmark in illnesses such as glial tumors,
Huntington’s
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16,17
disease, Parkinson’s disease, and depression
amongst others. In particular, I -IR
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are reported to be increased in the brain of patients that suffered from Alzheimer’s
Disease (AD).18,19 Recently, two I -IR ligands, CR4056 1 and [ C]BU99008 2, have
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been progressed to clinical trials. CR4056 1,20,21 described as the first-in-class I -IR
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ligand embodying analgesic properties, is in clinical phase II studies for osteoarthritis
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and postoperative dental pain, and [ C]BU99008 2
is in early clinical phase I for
PET diagnosis for patients that suffer from AD.
The implication of I -IR in many physiological and pathological processes emphasizes
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their pharmacological relevance and deserves in-depth studies. Since the structural
data for I -IR remains unknown, the discovery of better and more selective I -IR ligands
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2
is crucial to build a comprehensive understanding of the pharmacological implications of
I2-IR.
Although there are a few exceptions, LSL60101 3 and most notably the clinical
candidate CR4056 1, the vast majority of known I -IR ligands (idazoxan, 4; tracizoline 5,
2
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and 2-BFI, 6) are 2-substituted-2-imidazolines without further decoration in the 1-, 4-
and 5-positions (Figure 1).24
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H
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CR4056, 1
BU99008, 2
LSL60101, 3
HCl
H
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N
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idazoxan, 4
tracizoline, 5
2-BFI, 6
Figure 1. Representative I -IR ligands.
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In order to explore new imidazoline-based I -IR ligands moving out of the comfort zone
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offered by the rather structurally homogeneous I -IR ligands reported so far (Figure 1),
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we have recently disclosed a family of (2-imidazolin-4-yl)phosphonates.25,26 The
putative therapeutic relevance of a member of this new family of I -IR ligands, MCR5 7,
2
was validated in a murine model of neurodegeneration, the senescence accelerated
mouse-prone 8 (SAMP8).27 An improvement in the cognitive decline and related
biomarkers was found when MCR5 7 was orally administered to the animals. This study
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was the first in vivo evidence that reinforced I -IR as a promising target for the treatment
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of cognitive impairment, associated to multiple neurodegenerative diseases.27
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Separately, we had reported that the diastereoselective [3+2] cycloaddition of diethyl
isocyanomethylphosphonate with ten diversely substituted maleimides in acetonitrile under
AgOAc catalysis furnished a series of bicycles of general structure Ia (Scheme 1a).28 The
presence, within this series of compounds of an -iminophosphonate unit, also featured in the
abovementioned (2-imidazolin-4-yl)phosphonates, prompted us to evaluate whether these
bicycloderivatives would also behave as I -IR ligands. We indeed found that two of these
2
ten already reported compounds, 8a and 8c (Scheme 1b), displayed an affinity for the I -IR
2
similar to that of idazoxan 4 (see below). These promising results encouraged us to resume our
research with this family of bicyclic -iminophosphonates with the two-fold aim of further
exploring the scope of the aforementioned [3+2] cycloaddition reaction and of establishing their
structure-activity relationships (SAR) as I -IR ligands.
2
Scheme 1. a) General structure of bicyclic -iminophosphonates Ia (previously
a
reported) and Ib (reported herein) and reaction conditions; b) Chemical structures of 8a
and 8c; and c) Chemical structure of MCR5 7.
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R'
N
a)
O
O
H
O
H
(EtO) (O)P
NC
AgOAc
N R'
2
+
Acetonitrile
R
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(EtO) (O)P
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R
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Ia, R = H, previous synthetic work, 8
Ib, R = Ph, this work, 9
Me
Ph

-iminophosphonate moiety
O
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N
O
H
O
H
O
H
N
b)
c)
(
Cl
N
(
EtO) (O)P
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N
(EtO) (O)P
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N
EtO) (O)P
F
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N
H
H
Ph
MCR5 7
8a
8c
aReagents and conditions, (a) N-substituted maleimide derivative (1.5 mmol), PhosMic
(
1 mmol), AgOAc (0.06 mmol), acetonitrile, room temperature, overnight.
Herein, we explore the synthetic scope of the [3+2] cycloaddition reaction of -
substituted PhosMic derivatives and diversely substituted maleimides. Particular attention
was given to derivatives including a phenyl substituent in the -position of the phosphonate
leading to general structures 9 depicted as Ib in the Scheme 1a, in order to resemble
the structure of MCR5 7 (Scheme 1c). We also assessed the pharmacological profile
and selectivity of a wide range of bicyclic -iminophosphonates through competition
3
binding studies against the selective I -IR radioligand [ H]-2-[(2-benzofuranyl)-2-
2
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imidazoline] (2-BFI).29 Selectivity versus two related targets, the I -IR and the  -
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2
adrenergic receptor ( -AR) was evaluated through competition studies using the
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selective radioligands [3H]clonidine and [3H]RX821002 (2-methoxyidazoxan),
respectively. Complementary, we performed 3D-QSAR studies. Compound 9d,
endowed with outstanding I -IR affinity and excellent selectivity index regarding I -IR
2
1
and  -AR, was selected for further studies. We first compared the affinity for the
2
human I -IR of 9d with those of the standards shown in Figure 1. Additionally, the
2
affinity for I -IR from different species was considered for idazoxan 4 and 9d. Next, we
2
performed preliminary DMPK studies for 9d, including chemical stability, PAMPA-BBB
permeability assay, solubility, cytotoxicity, microsomal stability, cytochromes inhibition,
and safety. Finally, we characterized its in vivo neuroprotective effects in the 5xFAD
murine model of AD.
RESULTS AND DISCUSSION
Chemistry
Synthesis and structural characterization
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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
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Considering the previously described compounds 8a and 8c as promising starting points for
designing potent I -IR ligands, we resolved to prepare bicyclic compounds functionally close to
2
MCR5 7 by including a -phenyliminophosphonate moiety in their structure. To this end, we
decided to increase the scope of the original [3+2] cycloaddition by using diversely -substituted
PhosMic derivatives (Figure 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
10a, R = phenyl
10b, R = 4-fluorophenyl
(
EtO) (O)P
NC
2
(PhO) (O)P
NC
2
10c, R = 4-methoxyphenyl
R
10d, R = benzyl
Ph
10e, R = 4-fluorobenzyl
11
Figure 2. -Substituted PhosMic derivatives used in this work.
The preparation of the -substituted PhosMic derivatives was performed adapting previously
described procedures (for references and experimental procedures, see Supporting Information).
Briefly, the four phenylisocyanomethylphosphonates 10a, 10b, 10c and 11 were prepared by
conversion of the required (-aminophenyl)phosphonate derivative to the corresponding
formamide followed by dehydration with phosphorus oxychloride. While diethyl (-
aminophenyl)phosphonate is a commercially available compound, the other three precursors
were synthesized according to published procedures. A different approach was followed for the

-benzylisocyano derivatives 10d and 10e. Alkylation of commercially available PhosMic with
either benzylbromide or 4-fluorobenzylbromide, using potassium tert-butoxide furnished diethyl
benzylisocyanomethylphosphonate 10d and diethyl 4-fluorobenzylisocyanomethylphosphonate
1
0e, respectively.
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The maleimides used in the cycloaddition reaction were commercially available or were prepared
following previously described procedures.
Gratifyingly, although the targeted compounds feature increased steric hindrance in the -
phosphonate position, our previously optimized set of conditions for the [3+2] cycloaddition
reaction of maleimides with PhosMic also worked for the current set of -substituted PhosMic
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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2
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0
1
2
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4
5
6
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8
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0
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4
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8
9
0
1
2
3
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7
8
9
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1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
8
derivatives. In this way, 36 new bicycloderivatives (Schemes 2 and 3) having a quaternary
stereocenter, were synthesized in medium to high yields (experimental section). The products
were purified by column chromatography and, when solids, analytical samples were obtained by
recrystallization. For the sake of clarity in the section I -IR binding activity and structure-activity
2
relationships the new -substituted bicycles, depicted in Schemes 2 and 3, were ordered and
numbered attending to the SAR discussion.
Analogously to our previous work,28 all the [3+2] cycloaddition reactions occurred in a
diastereoselective manner and only one of the two possible diastereoisomers was formed. The
relative configuration of the three stereocenters in the new compounds was unambiguously
confirmed by X-ray crystallographic analysis for five examples and the stereochemistry of the
1
13
other compounds was assigned by comparison of their H and C-NMR spectra (Tables S12 and
S13).
Scheme 2. General procedure for the synthesis of bicyclic -iminophosphonates.a
2
8
Compounds prepared in previous work (R = H) and compounds prepared in this work (R
=
Ph, 4FPh, 4-MeOPh, PhCH , 4FPhCH ).
2
2
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1
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2
2
2
2
2
2
3
3
3
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R'
N
O
O
H
O
H
(
EtO) (O)P
NC
a
2
N R'
O
+
R
(EtO)2(O)P
N
10a, 10b, 10c, 10d, 10e
R
0
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2
3
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0
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0
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8
9
0
R- = H-
Ph-
4-FPh- 4-MeOPh- PhCH - 4FPhCH -
2
2
R'-
Me
cyclohexyl
Ph
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
12c
12d
1
13d
3c
14c
15c
3
-Cl,4-FPh
4-MeOPh
aReagents and conditions, (a) N-substituted maleimide derivative (1.5 mmol), -
substituted PhosMic (10a, 10b, 10c, 10d, 10e, 1 mmol), AgOAc (0.06 mmol),
acetonitrile, room temperature, overnight.
Scheme 3. Second-round of compounds synthesized, featuring modified N-maleimide
substituents inspired by compounds 9a and 9b, R’ = alkyl and 9c and 9d, R’ =aryl.a
R'
O
O
H
O
H
N
(EtO) (O)P
NC
a
2
N R'
+
Ph
O
(EtO)2(O)P
N
1
0a
Ph
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9
R'- = alkyl, 9a 9b
9f, ethyl
R'- = aryl, 9c 9d
n, 4-CF Ph-
9
3
9o, 3-CF Ph-
9
9
9
g, propyl
h, t-butyl
i, (1-adamantyl)methyl-
3
9p, 4-FPh-
9q,
4-ClPh-
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
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5
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0
1
2
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7
8
9
0
1
2
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5
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7
8
9
0
1
2
3
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9r, 2-ClPh-
9j, PhCH2-
9
9
9
s, 3-ClPh-
t, 4-BrPh-
u, 3,5-diClPh-
9k, PhCH CH -
2
2
9l, 4-FPhCH CH -
2 2
9
m, Ph(CH ) CH-
2
2
9v, 3,4-diClPh-
9
9
w, 2,4,6-triClPh-
x, 3-NO Ph-
2
9y, 3-NO ,6-CH Ph-
2
3
9z, 4-PhPh-
9
aa, 4-CH Ph-
3
9ab, 4-PhOPh-
9
9
ac, 1-naphthyl
ad, 2-Cl,3-pyridyl
aReagents and conditions, (a) N-alkyl or aryl substituted maleimide derivative (1.5
mmol), -PhenylPhosMic (10a, 1 mmol), AgOAc (0.06 mmol), acetonitrile, room
temperature, overnight.
As previously noted the [3+2] cycloaddition reaction between -substituted PhosMic derivatives
and diversely substituted maleimides was completely diastereoselective, only one of the two
possible diastereoisomers was observed. Iminophosphonates 9b, 9c, 9d, 9v and 9ab were
recrystallized as monocrystals from ethyl acetate. Their relative configuration was
unambiguously confirmed by X-ray crystallographic analysis, indicating a trans relationship
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2
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3
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5
6
between the hydrogen atoms on the bridged positions and the substituent at the α phosphonate
carbon atom (Figure 3).
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3
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8
9
0
9
b
9
c
9d
9v
9ab
Figure 3. X-Ray structures of 9b, 9c, 9d, 9v and 9ab .
Finally, the origin of the diastereoselective [3+2] cycloaddition was investigated by
quantum mechanical (QM) calculations that were performed for the addition of N-
methylmaleimide to -phenylPhosMic (in this latter case the ethyl groups were replaced
by methyl in order to reduce the cost of QM computations). In addition, a silver cation
bound to acetonitrile was introduced to account for the catalytic effect on the chemical
reaction. Reactants, transition states and products for the cis and trans [3+2]
cycloadditions were determined from geometry optimizations at the B3LYP/6-31+G(d)
(LANL2DZ for silver) level, and the nature of the stationary points was verified from the
analysis of the vibrational frequencies. The geometries of the transition states point out
that the cycloaddition occurs via an asynchronous concerted process as the length of the
bond that is formed by carbon atom 3a is shorter than the bond formed by carbon atom
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a by 0.51 and 0.23 Å in the cis and trans addition, respectively (Figure 4). Moreover, a
significant deviation from linearity is observed in the isocyano group, as the C-N-C angle
is close to 144 degrees in the two transition states. The results also point out that the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
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0
1
2
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4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
−
1
transition state leading to the trans addition was more stable by 2.3 kcal mol relative to
the cis cycloaddition (Table S1), presumably due to the destabilizing electrostatic
interactions between the oxygen atoms of the phosphonate and maleimide moieties. The
preferred stability of the trans transition state was further checked by geometry
optimizations performed the cis and trans cycloadditions with the MN15L density
functional, leading to a free energy difference of 1.2 kcal mol−1 favoring the trans
cycloaddition. The contribution due to the solvation effects in acetonitrile was determined
by means of continuum solvation calculations (see Methods). The results (Table S1)
reveal that solvation leads to a slight destabilization of the transition state relative to the
reactants. Nevertheless, this effect cancels out for the cis and trans addition, which can
be understood from the similar structural features of the two transition states. Overall,
these results justify the preferential formation of the diastereoselective compound
originated from the trans cycloaddition (Figure 4).
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Representation of the transition states for the cis and trans [3+2] cycloaddition
between N-methylmaleimide and -phenylPhosMic (ethyl groups substituted by methyl) located
.
..
from B3LYP calculations (C C distances in a; C-N-C angle in degrees).
I2-IR binding activity and Structure-Activity Relationships
The pharmacological activity of the compounds depicted in Schemes 2 and 3 was evaluated
3
through competition binding studies against the selective I -IR radioligand [ H]-2-BFI and the
2
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selective  -AR radioligand [ H]RX821002. The studies were performed in membranes from
2
post-mortem human frontal cortex, a brain area that shows an important density of I -IR and  -
2
2
30
AR. Idazoxan 4, a compound with well-established affinity for I -IR (pKi = 7.27 ± 0.07) and
2
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
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4
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5
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5
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0
1
2
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4
5
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7
8
9
0
1
2
3
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8
9
0
1
2
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9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
2
-AR (pKi = 7.51 ± 0.07) was used as reference. The inhibition constant (K ) for each
i
compound was obtained and is expressed as the corresponding pKi (Table 1). The selectivity for
these two receptors was expressed by the I / index, calculated as the antilogarithm of the ratio
2
2
between pKi values for I -IR and pKi values for  -AR (Table 1). Competition experiments
2
2
3
against [ H]2-BFI were monophasic for most of the compounds (for a few exceptions, see
below).
Among the set of ten bicycles of general structure Ia (Scheme 1a) already reported28 five
representative compounds, 8a, 8b, 8c, 8d and 8e, were selected for evaluation as potential I -IR
2
ligands considering the substitution in the N-maleimide by an alkyl (8a), cycloalkyl (8b),
unsubstituted phenyl (8c), electron withdrawing-disubstituted phenyl (8d) and electron donating-
substituted phenyl (8e) groups.
Pleasantly, 8a and 8c displayed pKi I affinity of 6.79 and 7.73, respectively, in the range of that
2
of idazoxan 4 (7.41). However, no promising results were found for 8b, 8d and 8e (Table 1). As
a first structural approximation, we turned our attention to compounds bearing a quaternary
center in the -position by including a phenyl group. In this manner, the new compounds would
resemble the -phenyliminophosphonate moiety of MCR5 7 (in pink color in Scheme 1a and
1c). In order to maintain the homology with the first series of evaluated compounds (Scheme 2,
R = H), analogous maleimide derivatives were considered to give access to compounds 9a, 9b,
9
c, 9d, and 9e (Scheme 2, R = phenyl). Indeed, this change was highly positive for the whole
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1
1
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2
2
2
2
2
2
2
2
2
3
3
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3
3
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3
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4
4
4
4
4
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5
5
5
5
6
series, increasing the pKi I affinity for all the phenyl-substituted derivatives compared to their
2
unsubstituted congeners, with the added benefit, in three cases (9a, 9d and 9e), of an enhanced
I / selective ratio up to 195. A remarkably benefit in the I -IR affinity, pKi I 9.74 (Ki = 18
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
nM) was observed in N-cyclohexyl derived 9b, 4-fold compared with analogous 8b with an I /
2
2
selectivity of 5. The rise in the affinity was also conserved in compounds bearing an N-arylimide
substitution. In particular, the presence of an N-phenyl group led to 9c, with an outstanding
activity binding pKi I 10.28 (Ki = 63.0 pM), but not I / selectivity. Gratifyingly, introduction
2
2
2
of halogen atoms (3-chloro-4-fluoro) in the N-phenyl ring of 9c led to congener 9d that kept a
nice affinity, with a pKi I 8.56. Of note, 9d fitted significantly better to a two-sites binding
2
model, with a high pKi I 8.61 (Ki = 2.45 nM) and a low pKi I 4.29 (Ki = 51.2 µM), with the
2
H
2
L
3
high-affinity site representing a calculated 37% of the specific binding of [ H]2-BFI at 2 nM
concentration.
The enhancement, both in terms of affinity and of selectivity, observed when moving from the -
unsubstituted to the -substituted phosphonates prompted us to briefly consider additional
variations. The introduction in the -phosphonate position of p-fluorophenyl (12c) or p-
methoxyphenyl (13c), benzyl (14c), and p-fluorobenzyl (15c) groups was highly deleterious for
the affinity (pKi I = 6.59 for 14c and pKi I < 3 up to 5.35 for 12c, 13c and 15c, respectively).
2
2
However, for the p-substituted phenyl derivatives, the further introduction of halogen atoms (3-
chloro-4-fluoro) in the N-phenyl ring (compounds 12d and 13d), nicely restored the affinity (pKi
I 7.55 for 12d and pKi I 7.87 for 13d). Additionally, due to the lack of binding of 12d and 13d
2
2
to  -AR, their I / selectivity was outstanding, 14791 and 74131, respectively.
2
2
2
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Taking into account the aforementioned results, for a second round of compounds the general
structure depicted in Scheme 3 was conserved, featuring the unsubstituted phenyl group in the -
position of the phosphonate, and modifying the substituents in the maleimide. New compounds
were classified in two groups taking into consideration whether an alkyl or an aryl substituent
was introduced in the N-maleimide.
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
Inspired by 9a and 9b, compounds bearing an alkyl substituents with different length, 9f and 9g,
ramified alkyl, 9h, and polycycloalkane, 9i, were prepared. From 9a, the elongation of the N-
alkyl chain, from methyl to ethyl, led to 9f, with an increase in the affinity to pKi I = 8.37 (Ki =
2
4
.3 nM) and I / selectivity to 331, while the n-propyl derivative, 9g, was much less affine pKi
2
2
I = 4.02. For 9f, the best fit was a two-site model of binding with a high pKi I = 8.95 and a low
2
2
pK I = 5.86, high affinity site occupancy is 62%. Further increase of the size of the N-alkyl
i
2
substituent to a tert-butyl, 9h, or an adamantylmethyl, 9i, did not improve the affinity. Taking
together the affinity values for 9a, 9b, 9f, 9g, 9h and 9i, it seems that small and large substituents
are compatible with good affinity values, but that conformational freedom, as in 9g, is
deleterious.
Compounds 9j, 9k, 9l and 9m, with N-benzyl, N-phenethyl, N-4-fluorophenethyl, and N-
phenylpropyl substituents, respectively, were accessed to increase the examples in the SAR
study. However, their affinities revealed a remarkable decrease in the biological properties,
leading to pKi I = 5.26, 6.35 <3 and 3.84 values, respectively.
2
Taking into account that 9c displayed an outstanding affinity for I -IR, but lacked selectivity
2
over  -AR, further R’= aryl derivatives were explored. As we knew that 9d (pKi I = 8.56, I /
2
2
2
2
=
195) was endowed with excellent affinity and remarkable selectivity, we mainly focused on
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6
electron withdrawing groups (9n, 9o, 9p, 9q, 9r, 9s, 9t, 9u, 9v, 9w, 9x and 9y), although a few
electron donating substituents were briefly examined (9z, 9aa, and 9ab). Overall, neither these
new phenyl derivatives nor the N-naphthyl derivative 9ac, outperformed the excellent affinity of
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
9
c (Table 1), although 9z (pK I = 7.90) had an improved I / ratio of 602. Finally, 9ad with an
i
2
2
2
N-(2-chloro-3-pyridyl) substituent gave a pKi I = 7.96, in the range of standard idazoxan 4, but
2
it offered as an outstanding advantage a null affinity upon  -AR, leading to an I / selectivity
2
2
2
of 91201.
Table 1. I -IR and  -AR Binding Affinities (pKi) of five previously reported compounds
2
2
828
and new compounds.
pKi
Compound
R-
R’-
[3H]-2-BFI,
I2 one site
[3H]-
RX821002,
2
aSelectivity
I2/2
General
structure
a[3H]-2-
BFI, I two
2
R'
sites
O
N
O
H/L; High
affinity site
%
(EtO) (O)P
2
N
R
Idazoxan
7.41  0.63 8.35  0.16
6.79  0.51 9.49  0.18
5.74  0.51 5.02  0.58
7.73  0.19 8.49  0.36
-
5
-
8
a
H
H
H
H
H
Me
8
b
cyclohexyl
Ph
8
c
-
8
d
3-Cl,4-FPh
4-MeOPh
<3
10.27  0.32
-
8e
5.11  0.13 6.14  0.85
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7
8
9
9
a
Ph
Ph
Ph
Me
cyclohexyl
Ph
7.97  0.55 5.93  0.41
9.74  0.29 9.01  0.51
10.28  10.38  0.22
110
5
9
b
9
c
1
0
.37
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
9
d
Ph
3-Cl,4-FPh
8.56  0.32 6.27  0.56
195
2
19
8
.61  0.28/
4.29  0.20;
3
7  4
6.65  1.27 4.59  0.22
<3
6.77  0.64
9
e
Ph
4-MeOPh
Ph
115
1
2c
2d
3c
4-FPh
4-FPh
-
14791
-
1
3-Cl,4-FPh
Ph
7.55  0.32 3.38  0.33
3.39  0.62 3.85  0.31
1
4-
MeOPh
1
3d
4-
MeOPh
3-Cl,4-FPh
7.87  0.40
<3
74131
1
1
4c
5c
CH Ph
Ph
Ph
6.59  0.77 3.94  0.16
5.35  0.35 7.20  1.02
447
-
2
4-
FCH Ph
2
9f
Ph
Et
8.37  0.27 5.85  0.53
331
1259
8
5
.95  0.36/
.86  0.66;
6
2  11
9
g
Ph
Ph
Ph
propyl
4.02  0.41

-
3
9
h
t-butyl
7.35  0.43 6.77  0.66
7.01  0.76 4.31  0.29
9
i
(1-
501
adamantyl)methyl
9
j
Ph
Ph
Ph
Ph
PhCH2
5.26  0.22 8.11  0.28
-
380
-
9
k
PhCH CH
6.350.38
3.77  0.09
5.65  0.39
2
2
9l
9m
4-FPhCH CH
<3
2
2
Ph(CH ) CH
2
3.84  0.31 3.44  0.28
2
2
2
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7
8
9
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
6
3
.87  0.81/
.20  0.99;
22  2
2691
9n
9o
9p
9q
Ph
Ph
Ph
Ph
Ph
4-CF Ph
<3
<3
<3
<3
4.73  0.60
-
-
3
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
3-CF Ph
3

5.34  0.35

4-FPh
4-ClPh
2-ClPh
-
-
9
r
5.09  0.16 6.15  0.44
-
24
7
4
.53  0.66/
.74  0.23;
2
5  7
9
s
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
3-ClPh
4-BrPh
<3


-
9
t
<3
-
9
u
3,5-diClPh
3,4-diClPh
2,4,6-triClPh
5.81  0.37 6.22  0.26
-
9v
9w
9x
9y
<3
<3

-
5.16  0.19
-
3-NO Ph
6.81  0.27 10.18  0.41
<3
-
2
3-NO 6-CH Ph

-
602
-
2
,
3
9
z
4-PhPh
7.90  0.46 5.12  0.14
5.44  0.16
6.96  0.30 5.43  0.21
9
aa
ab
ac
ad
4-CH Ph

3
9
4-PhOPh
1-naphthyl
34
9
3.11  0.7
<3
<3
-
9
2-Cl,3-pyridyl
7.96  0.41
91201
a
b
Selectivity I -IR/ -AR expressed as the antilog (pKi I -IR-pKi  -AR). The best fit of
2
2
2
2
the data for 9d, 9f, 9m and 9r was to a two-site binding model of binding with high pKi
(
pKi ) and low pKi (pKi ) affinities for both binding sites respectively.
H
L
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2
3
4
5
6
7
8
9
Selectivity I -IR versus I -IR
2
1
After evaluating the affinity of the indicated compounds for  -AR, we assessed the affinity of
2
some representative compounds for I -IR. To this end, I -IR binding site assays were conducted
1
1
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
in membranes obtained from the rat kidney using moxonidine, a known I -IR selective
1
compound, as reference. The results are summarized in Table 2 and only 8e deserves a mention
with a pKi I 8.09. Gratifyingly, the values for the rest of the assessed compounds led to the
1
conclusion that there was not a significant interaction with I -IR highlighting the I -IR selective
1
2
behavior of this family of ligands.
Table 2. I -IR potencies (pIC ) of representative compounds
1
50
pIC50
Compound
[3H]-Clonidine
8.45 ± 0.85
5.13  0.44
5.14  0.54
5.47  0.31
<3
Moxonidine
8
a
8
b
8
c
8
d
8
e
8.09  0.34
6.19  0.27
7.54  0.79
6.74  0.74
3.04  0.45
3.22  0.67
5.12  0.85
9
a
9b
9c
9d
9e
1
4c
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8
9
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
9
j
5.87  0.19
9
k
7.98  0.31
5.26  0.43
7.19  0.33
9
x
9
z
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
Overall, considering their excellent I -IR affinity (Ki = 2.8 nM) and the remarkable selectivity
2
versus  -AR (Ki = 53 µM) and I -IR (Ki = 91 mM), we identified 9d as the most promising
2
1
compound for performing further studies.
Comparison of I -IR human receptor binding affinities (pKi) of 9d and other ligands, and
2
across species
A problem typically encountered when working with I -IR ligands is that the binding
2
experiments reported in the bibliography have been performed in a variety of non-human species
and using tissues from different anatomical parts (e.g., kidney, whole brain, cortex). Another
factor of potential discrepancies is that different radioligands have been used. Overall, this makes
difficult the comparison amongst studies. For this reason, and in order to better place 9d as a new
3
31
I -IR ligand, unprecedented experiments of displacement of [ H]2-BFI in samples from post-
2
mortem human brains were performed with clinical candidates BU99008 232 and CR4056 133
and the widely used I -IR ligands tracizoline, LSL60101 3 and 2-BFI 6 (Table 3).
2
As previously observed with 9d, the affinity data found for BU99008 2 and CR4056 1 fitted best
to a two-site model of binding. In particular, BU99008 2 showed a pK I = 6.89 (K = 128 nM)
iH
2
iH
and pKi I = 3.82 (Ki = 15.1 mM), and a good I / selectivity ratio of 331. CR4056 1 showed
L
2
H
2
2
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5
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7
8
9
a pKi I = 7.72 (Ki = 19.0 nM) and pKi I = 5.45 (Ki = 3.5 µM) with an excellent I /
H
2
H
L
2
H
2
2
selectivity of 117490. The percentage of occupancy for the high affinity site was different for
BU99008 2 (51%) compared with CR4056 1 (29%). Other well-stablished I -IR ligands,
2
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
tracizoline 5, LSL60101 3 and 2-BFI 6 also resulted in clearly biphasic curves. Tracizoline 5
displayed a pKi I = 8.48 (Ki = 3.3 nM) and pKi I = 6.48 with an excellent I / selectivity of
H
2
H
L
2
2
2
1
4125. 2-BFI 6 had a pKi I = 9.87 (Ki = 0.13 nM) and pKi I = 7.94, with a good I /
H
2
H
L
2
2
2
selectivity of 1698 and LSL60101 3 a pKi I = 9.03 (Ki = 0.9 nM) and pKi I = 5.25 (Ki = 5.6
H
2
H
L
2
L
µM), with a good I / selectivity of 7244. The high-affinity site represented 38, 21 and 49%
2
2
occupancy for tracizoline 5, 2-BFI 6 and LSL60101 3, respectively (Table 3). Previous studies
3
34
35,36
31
have reported [ H]2-BFI identifying two binding sites in rabbit, rat
and human brain. It
remains unclear whether these two sites observed represent distinct receptors or interconvertible
conformational states of the I -IR. For tracizoline 5 a single binding site of pKi I = 8.72, similar
2
2
37
to the affinity described for human tissues, was described in the rabbit kidney membranes. In
the rat cerebral cortex, LSL60101 3 is less affine than in human tissues, with a Ki = 350 nM and
H
38
Ki = 116 µM.
L
Therefore, compounds BU99008 2, tracizoline 5 and 2-BFI 6, that have a non-substituted 2-
imidazolin-2-yl) group, CR4056 1 and LSL60101 3, that feature an imidazole ring, and the
structurally dissimilar 9d, have similar affinity profiles upon I -IR in human brain.
(
2
Table 3. I -IR and  -AR binding affinities (pKi) of BU99008 2, CR4056 1, tracizoline 5
2
2
and LSL60101 3 and 9d in postmortem human brain cortical membranes.
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1
2
3
4
5
6
7
8
9
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
Compound
[3H]-2-BFI
High-
affinity site
%
[3H]-RX821002 Selectivity I /
2
2
3
I2
2
for [ H]-2-BFI
(high-affinity
site)
pKi two sites
pKi
BU99008, 2
CR4056, 1
Tracizoline, 5 8.48 ± 0.51
-BFI, 6
LSL60101, 3
9d
6.89 ± 0.21
7.72 ± 0.31
3.82 ± 0.30
5.45 ± 0.15
6.48 ± 0.32
7.94 ± 0.11
5.25 ± 0.24
4.29 ± 0.20
51 ± 6
29 ± 6
38 ± 13
21 ± 5
49 ± 4
37 ± 4
4.37 ± 0.17
2.65 ± 1.24
4.33 ± 0.22
6.64 ± 0.38
5.17 ± 1.32
6.27 ± 0.56
331
117490
14125
1698
7244
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
9.87 ± 0.33
9.03 ± 0.21
8.61 ± 0.28
219
3
Of note, the ability of BU99008 2 to displace [ H]2-BFI from I -IR in rat brain was described to
2
fit to a two-site model of binding, with a Ki = 1.4 ± 0.6 nM and Ki = 238.6 ± 63.3 nM, and
H
L
with a percentage % fraction of high occupancy of 58 ± 7. That is, an enhanced affinity by 100
times in rat brain, compared with human brain, being the % of occupancy similar in the high site.
Regarding selectivity, a good I / ratio of 909 was reported in rat, 4.5 times higher than that
2
2
found in human. Of note, the opposite trend was found for CR4056 1, the inhibition recorded in
3
rat whole-brain for [ H]2-BFI binding was IC of 596 ± 76 nM, with an improved affinity to 19
5
0
nM showed in human brain.39 Therefore, significant differences between species occur within
the two I -IR ligands in clinical trials, BU99008 2 and CR4056 1.
2
In an attempt to incorporate additional data regarding the differences in I -IR binding affinities
2
between species, idazoxan 4 and 9d were investigated (Table 4). In our hands, idazoxan 4 gave
similar results in human frontal cortex, pKi 7.74, as compared to rat brain cortex, pKi 7.17, and
was considerably less affine in mouse brain cortical membranes, pKi 5.68. Importantly,
differences for 9d were not only found among species but also in its binding
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9
characteristics. As previously mentioned, the binding to I -IR in human frontal cortex
2
displayed a biphasic curve, whereas a monophasic one was observed in rat and mouse
brain cortex with affinity values of pKi 6.92 and 6.41, respectively.
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
Table 4. I -IR binding affinities (pKi) of idazoxan 4 and 9d in the brain cortex of different
2
species.
Human
Rat
Mice
Idazoxan, 4
7.74 ± 0.10
7.17 ± 0.11
6.92 ± 0.35
5.68 ± 0.31
6.41 ± 0.39
9d
8.61 ±
.28
4.29 ±
0.20
0
Finally, in order to verify if the high affinity site observed for 9d in competition experiments
3
against [ H]2-BFI corresponded to the I -IR, we performed additional experiments in the
2
presence of MCR5 7, a high-affinity I -IR selective compound previously reported by our
2
2
5
-5
group. Interestingly, in the presence of MCR5 7 (10 M) the 9d competition curve against
[3H]2-BFI became monophasic (pKi = 6.96 ± 0.46), and the high-affinity site recognized by 9d
was completely blocked. These results confirm that the high affinity site bound by 9d is the I2-
IR.
3
D-QSAR study
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