Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γ-Aminobutyric Acid Type A Receptor (GABAAR) α6 Subtype with Improved Metabolic Stability and Enhanced Bioavailability

Article abstract of DOI:10.1021/acs.jmedchem.7b01664

Recent reports indicate that α6β2/3γ2 GABA_AR selective ligands may be important for the treatment of trigeminal activation-related pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6β2/3γ2 GABA_AR selective pyrazoloquinolinones, 42 novel analogs were synthesized, and their in vitro metabolic stability and cytotoxicity as well as their in vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion were investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased their duration of action via improved metabolic stability and bioavailability, while their selectivity for the GABA_AR α6 subtype was retained. 8b was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6β2/3γ2 GABA_ARs and were functionally silent at diazepam sensitive α1β2/3γ2 GABA_ARs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by GABA_AR α6β2/3γ2 subtypes.

Full text of DOI:10.1021/acs.jmedchem.7b01664

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Article
Design and Synthesis of Novel Deuterated Ligands Functionally
A
Selective for the #-Aminobutyric Acid Type A Receptor (GABAR) #6
Subtype with Improved Metabolic Stability and Enhanced Bioavailability
Daniel E. Knutson, Revathi Kodali, Branka Divovic, Marco Treven, Michael Rajesh
Stephen, Nicolas M. Zahn, Vladimir Dobri#i#, Alec T. Huber, Matheus A. Meirelles, Ranjit
S. Verma, Laurin Wimmer, Christopher Witzigmann, Leggy A. Arnold, Lih-Chu Chiou,
Margot Ernst, Marko D. Mihovilovic, Miroslav M Savic, Werner Sieghart, and James M. Cook
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01664 • Publication Date (Web): 26 Feb 2018
Downloaded from http://pubs.acs.org on February 27, 2018
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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of their duties.

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&
Biochemistry, Milwaukee Institute of Drug Discovery
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Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γꢀ
Aminobutyric Acid Type A Receptor (GABA R) α6 Subtype with Improved Metabolic
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Stability and Enhanced Bioavailability
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a
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b
e
a
Daniel E. Knutson , Revathi Kodali , Branka Divović , Marco Treven , Michael R. Stephen ,
a
c
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a
Nicolas M. Zahn , Vladimir Dobričić , Alec T. Huber , Matheus A. Meirelles , Ranjit S. Verma ,
d
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f,g,h
Laurin Wimmer , Christopher Witzigmann , Leggy A. Arnold , LihꢀChu Chiou , Margot
e
d
b
e
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Ernst , Marko D. Mihovilovic , Miroslav M. Savić , Werner Sieghart , and James M. Cook *
a
Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery,
University of WisconsinꢀMilwaukee, 3210 N. Cramer St., Milwaukee, Wisconsin 53211, USA.
b
Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe
450, 11221 Belgrade, Serbia.
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade,
Vojvode Stepe 450, 11221 Belgrade, Serbia.
d
TU Wien—Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163, Aꢀ1060 Vienna,
Austria.
e
Department of Molecular Neurosciences, Center for Brain Research, Medical University of
Vienna, Spitalgasse 4, Aꢀ1090 Vienna, Austria.
f
g
Department of Pharmacology, Graduate Institute of Brain and Mind Sciences, College of
h
Medicine, National Taiwan University, Taipei 10051, Taiwan, Graduate Institute of
Acupuncture Science, China Medical University, Taichung 40402, Taiwan.
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Abstract:
Recent reports indicate that α6β2/3γ2 GABA R selective ligands may be important for the
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treatment of trigeminal orofacial pain and neuropsychiatric disorders with sensoriꢀmotor gating
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deficits. Based on 3 functionally α6β2/3γ2 GABA R selective pyrazoloquinolinones, 42 novel
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analogs were synthesized and their in vitro metabolic stability and cytotoxicity as well as their in
vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion was
investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased
their duration of action via improved metabolic stability and bioavailability, while their
selectivity for the GABA R α6 subtype was retained. 8b was identified as the lead compound
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with a substantially improved pharmacokinetic profile. The ligands allosterically modulated
diazepam insensitive α6β2/3γ2 GABA Rs and were functionally silent at diazepam
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sensitive α1β2/3γ2 GABA Rs, thus no sedation was detected. In addition, these analogs were not
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cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by
GABA R α6β2/3γ2 subtypes.
A
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Introduction
GABA receptors (GABA Rs) are the major inhibitory neurotransmitter receptors in the
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mammalian brain and the target of many clinically important drugs, which act at GABA R
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binding sites. GABA Rs are ligandꢀgated pentameric chloride channels mainly comprised of
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combinations of 19 different subunits (α , β , γ , δ, ε, π, θ, ρ ). Classical GABA Rs
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1–3 1–3
1ꢀ3
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, 3
consist of two α, two β and one “tertiary” subunit (γ, δ, ε, θ, or π).
Due to the vast array of
subunit combinations and their diverse regional, cellular and subcellular distribution, a wideꢀ
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, 4, 5
range of pharmacological properties are potentially mediated by these ion channels.
GABAergic drugs including benzodiazepines (Bzs) and barbiturates modulate GABA Rs via
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allosteric binding sites. Many GABAergic drugs on the market today offer little alpha subtype
selectivity and thus exhibit undesired side effects (sedation, ataxia, amnesia, tolerance, and
dependence), in addition to their therapeutic benefits. Recently, a renewed search for GABA R
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subtype selective ligands began with the discovery of a new GABA R α +β ꢀ ligand binding
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y
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site. The identification of new ligands which specifically interact with this interface might lead
9ꢀ11
to novel therapeutic entities and provided the initial interest in this area of research.
GABA Rs containing the α6 subunit (α6βγ2, Figure 1, or α6βδ) were found primarily expressed
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in the granule cells of the cerebellum, where they are located synaptically or extrasynaptically,
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respectively, as well as in the olfactory bulb and the cochlear nucleus. Lower expression of
α6ꢀGABA Rs has recently been reported for the hippocampus where they might play a role in
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depressive behaviors. Recent reports also suggest that α6ꢀcontaining receptors may play a role
in neuropsychiatric disorders with sensoriꢀmotor gating deficits (such as tic disorders, certain
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5, 16
symptoms of schizophrenia, obsessive compulsive disorder and attention deficit disorders),
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as well as in migraine, and trigeminal orofacial pain. However, our understanding of the
function of α6ꢀcontaining receptors in the CNS is still in its infancy, and currently it is not
possible to indicate with certainty whether α6βγ2, or α6βδ, or both of these receptors, play a
major role in the above mentioned disorders. In any case, the synthesis of ligands selective for α6
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subunitꢀcontaining GABA Rs is important to help determine which physiological processes
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might be mediated by these particular ion channels and if they are a valid target for drug
development.
The CibaꢀGeigy Corporation reported pyrazoloquinolinones (PQs), CGS 8216 and CGS 9896, as
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9, 20
the first nonbenzodiazepine ligands to interact with the Bz site of GABA Rs.
These
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compounds exhibited Bz site agonist (positive allosteric modulatory) and antagonist (null
modulatory) properties at GABA Rs, but their receptor binding selectivity between diazepam
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sensitive (DS, αβγ GABA Rs containing α1, α2, α3, or α5 subunits) and diazepam insensitive
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(
DI, αβγ GABA Rs containing α4 or α6 subunits) GABA R subtypes was rather poor.
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Incorporation of a 7ꢀmethoxy substituent on the “Aꢀring” (see Figure 2) of PQs by the Skolnick
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group resulted in improved DI affinity. Deconstruction of the CGSꢀrelated compounds via
application of the pharmacophore/receptor model for GABA R subtypes was used to incorporate
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structural modifications of the “Aꢀring” and “Dꢀring” (Figure 2) of PQs.
These
modifications resulted in the synthesis of PZꢀIIꢀ029 (8a) and several other ligands which bound
25
tightly to the Bz/GABA R α6 subtype with a similar affinity as imidazobenzodiazepines. The
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size and position of substituents on the “Dꢀring” affected the affinity towards the Bz/GABA R
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α6 subtype.
More recently, structural analogues including 8a were found not only to bind to
α6β2/3γ2 GABA Rs, but also to potentiate these receptors, representing the first functionally
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selective α6β3γ2 subtype ligands reported to date. Prototypes of these ligands mediated their
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physiological effects at the recently discovered α6+β3ꢀ interface of GABA Rs as positive
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allosteric modulators, while in addition acting as antagonists (null modulators) at the Bz site
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(α+γ2ꢀ interface) of α1ꢀ6βγ2 receptors.
In a parallel study, the Ernst group synthesized very
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similar analogs LAU 159 (8i) and LAU 463 (8n). These compounds exhibited a functional
selectivity at α6β2/3γ2 GABA Rs comparable to 8a, within the series of α1ꢀ6β2/3γ2 receptors.
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Compounds 8a, 8i and 8n thus represent the three lead compounds described in this study (see
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Figure 2). Since all these PQs exert their positive allosteric modulation via the α6+β3ꢀ
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interface of GABA Rs, they also are able to modulate α1ꢀ6β2/3δ receptors or any receptors
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containing an α1ꢀ6β2/3 interface. But the modulation of α1β3δ, α4β3δ, or α6β3δ receptors by
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all three lead compounds (8a, 8i, 8n) is much weaker than that of α6β3γ2 receptors, and was
thus not investigated in the present study.
Due to the possibility of Oꢀdemethylation in vivo in this series, as well as their limited solubility
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in water, the bioavailability and halfꢀlife of these α6β3γ2 GABA R selective ligands were of
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concern. One way to enhance the bioavailability of compounds is via selective replacement of
hydrogen atoms with deuterium at key metabolic positions on the ligand (see Figure 3).
Deuteration has been shown to positively affect the metabolic stability while retaining the
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pharmacologic profile of active compounds. Consequently, the primary deuterium kinetic
33
isotope (D vs H) effect (DIE) was employed here to enhance metabolic stability. The kinetic
DIE results from a difference in the zeroꢀpoint energy of CꢀD and CꢀH bonds due to the lower
vibrational frequency of CꢀD bonds. Consequently, CꢀD bond cleavage requires a higher
activation energy, followed by a concomitant slower metabolic rate than a CꢀH bond. Since the
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lead compounds contained methoxy groups, it was assumed that those would be primary
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metabolic sites for Oꢀdemethylation by cytochrome P450 enzymes. Thus, one could take
advantage of the stability of the CꢀD bond to reduce Oꢀdemethylation of deuterated analogs.
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Herein, the first synthesis of novel deuterated GABA R α6 subtype selective ligands is reported,
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as well as the concomitant improved metabolic profile with respect to their parent OCH3
containing counterparts. Moreover, it was found that their improved metabolic stability resulted
in improved bioavailability in vivo and ultimately the achievement of higher brain concentrations
via both oral and intraperitoneal (IP) administration, a circumstance that is very important in
regard to drug development.
Another approach to improve ligand bioavailability of CNSꢀacting drugs is to improve the
solubility and lipophilicity since these modifications can impact membrane permeability and
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enhance their bioavailability and ability to reach the desired biological target.
nitrogen atoms were incorporated into the “Aꢀring” or “Dꢀring” of the lead ligands (8a, 8n and
i) to improve the water solubility (see Figure 3), while still maintaining a reasonable cLogP.
Therefore,
8
In addition to their metabolic stability, GABA Rꢀα6 subtype selective efficacy and the
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pharmacokinetic profile of these Nꢀheteroꢀmodified ligands were also studied.
To expand the structureꢀactivity relationship (SAR) library of the GABA Rꢀα6 subtype selective
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ligands, a series of 42 structural analogs related to 8a, 8n and 8i were synthesized. GABA Rꢀα6
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subtype efficacy of some of these novel ligands was measured to analyze the effect of
deuteration and structural modifications. Furthermore, a binding affinity screen over a panel of
4
6 receptors (including the hERG) was conducted to determine any possible offꢀtarget
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interactions of these novel compounds. In addition, the effects of these compounds on the
rotarod test and on HEPG2 (liver) and HEK293 (kidney) cell lines were examined to screen
motor activity and hepato and renal toxicities, respectively. Finally, basic in vivo behavioral
tests on the elevated plus maze, grip strength and cued waterꢀmaze training were run on these
ligands to assess the occurrence of anxiolysis, muscle strength and basic sensorimotor activity,
respectively.
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Results
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Chemistry. The synthesis of 8a and 8i, but not that of 8n has been reported before. Since we
improved the route of their synthesis as well as their purification, we also report this here. The
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synthesis of analogs of the α6β2/3γ2ꢀsubtype selective ligand 8a, which contained deuterium
on the methoxy groups of the “Aꢀring” and/or “Dꢀring” of the quinolinone nucleus was
undertaken. Analogs of 8i and 8n, which also contained deuterium on the methoxy groups of the
“Dꢀring” of the PQ nucleus, were also prepared. To permit an extension of the SARs of possible
α6β2/3γ2ꢀselective PQ compounds to be performed, additional deuterated compounds were
synthesized, in which the OCD group in the “Dꢀring” was moved to alternative positions, or in
3
which the 8ꢀCl of 8i or 8k in the “Aꢀring” was replaced by 8ꢀOCD (Table 1). The compounds
3
(8aꢀt) in Table 1 were generated in a convergent fashion following the steps outlined in Scheme
1
. First, the deuterated methoxy phenyl hydrazines (4aꢀc) were synthesized starting from the
appropriate hydroxyanilines (1aꢀc). Amide protection of the amine functionality was achieved
using acetic anhydride. NꢀAcetylation was accomplished over Oꢀacetylation via strict
stoichiometric (1.05 eq.) control of the amount of acetic anhydride. This was followed by
installation of the OCD groups via alkylation of the hydroxyphenylacetamides (2aꢀc) using
3
iodomethaneꢀd and base to yield the deuterated methoxy phenyl acetamides (3aꢀc) in high yield.
3
Aryl hydrazines (4aꢀc) were obtained via a oneꢀpot procedure, which involved the deprotection
of the amine with hydrochloric acid, diazonium salt formation with sodium nitrite and finally
3
8
reduction of the diazonium salts to yield the desired hydrazines using tin (II) chloride. As
39
o
reported by Frahn and Illman, control of the temperature (ꢀ20 C) of the diazotization and
reduction steps was key to avoid elimination of nitrogen from the decomposition of the
3
9
diazonium salt, especially in the cases of the para- and orthoꢀmethoxy substituted series.
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The hydrolysis of methoxyphenylacetamides (3a and 3b) yielded deuterated anisidines (5a and
5b) in excellent yields. The 4ꢀhydroxyquinoline derivatives (6aꢀe) were then prepared in a
4
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modified “one pot” procedure following previous reports (6c and 6e ) which involved
condensation of the substituted anilines (5aꢀe) with diethyl ethoxymethylenemalonate and this
4
2
was followed by cycloꢀacylation to the quinolone core using the GouldꢀJacobs reaction. The
application of para-substituted anilines typically resulted in higher yields than metaꢀsubstituted
anilines since the cycloꢀacylation with para-anilines resulted in only one 4ꢀhydroxyquinoline
regioisomer with the substituent in the 6ꢀposition of the final compound. The cycloꢀacylation
with metaꢀanilines resulted in the formation of the 5ꢀsubstituted 4ꢀhydroxyquinolines in addition
to the desired 7ꢀsubstituted 4ꢀhydroxyquinolines in a 1:5 ratio. The undesired 5ꢀsubstituted 4ꢀ
hydroxyquinolines were easily removed during the isolation process by using ethanol to
selectively dissolve the undesired isomer. The 4ꢀchloroquinolines (7aꢀe) were obtained by
chlorination of the respective 4ꢀhydroxyquinolines (6aꢀe) by generation of the catalytic Vilsmeier
43
reagent in situ using oxalyl chloride and a catalytic amount of N,Nꢀdimethylformamide in high
yields (80ꢀ90%). Typically, the hydrochloride salts of the 4ꢀchloroquinolines were formed in the
chlorination reactions and the free bases were generated by quenching the reaction mixtures into
aqueous potassium carbonate solution during the isolation of 4ꢀchloroquinolines (7aꢀe).
Utilization of the catalytic Vilsmeier reagent simplified the purification and improved the yield
4
1
and safety of this chlorination reaction over the reported phosphorus oxychloride (7c) and
40
thionyl chloride (7e) methods.
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Finally, PQs (8aꢀt) were obtained by the convergent coupling of phenylhydrazines (4aꢀe) with
appropriate chloroquinolines (7aꢀe) by simple modification of the previous work of Cibaꢀ
1
9, 20
o
Geigy
in mixed xylenes at 135ꢀ140 C. A similar procedure was reported in the
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deconstruction approach to PQs by He et al. In all cases, excess triethylamine (1.2 eq.) was
used to scavenge the hydrogen chloride formed from the nucleophilic aromatic substitution
(SNAr) of chlorine by the primary amino group of the respective phenylhydrazines. Following
the S Ar reaction, thermal cyclization via the secondary amine of the phenylhydrazine occurred
N
with subsequent elimination of ethanol to form the PQ core. Upon completion of the cyclization,
the reaction mixtures were quenched into ethanol and filtered to yield the pure PQs (8aꢀt) in 40 ꢀ
60% yields. This improved procedure required no further purification in contrast to the
40, 44
previously reported methods.
In cases where phenylhydrazine hydrochloride salts were
employed, the amount of triethylamine was adjusted to accommodate for the additional hydrogen
chloride.
To increase the solubility and lipophilicity of the lead compounds, the “Aꢀring” and “Dꢀring” Nꢀ
hetero PQs (13aꢀi) in Table 2 were synthesized in a convergent fashion following the steps
depicted in Scheme 2 and Scheme 3. Initially, to install the “Nꢀhetero” methoxypyridyl groups
into the “Dꢀring” of PQs (13aꢀh), pyridylhydrazines (12aꢀb) were synthesized. The 2ꢀmethoxyꢀ
d3ꢀ5ꢀnitropyridine (10) was generated in 87% yield by reacting 2ꢀchloroꢀ5ꢀnitropyridine (9) in
deuterated methanol (CD OD) as the solvent in the presence of potassium t-butoxide.
3
Reduction of the nitro group with iron metal in aqueous hydrogen chloride yielded 5ꢀaminoꢀ2ꢀ
methoxyꢀd ꢀpyridine (11a) in 95% yield. The aminopyridines (11aꢀb) were converted into their
3
respective pyridylhydrazines (12aꢀb) using the same procedure via diazonium salt formation and
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tin reduction, as described above for aryl hydrazines (4aꢀc). A similar procedure for the
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synthesis of 12b has been reported. Finally, reaction of the pyridylhydrazines (12aꢀb) with
chloroquinolines (7aꢀe) yielded PQs (13aꢀh) in moderate yields (40ꢀ75%) using the same
condensation process as described above.
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The “Aꢀring” substituted Nꢀhetero PQ (13i) provided a unique challenge (Scheme 3). The poor
electron density of the amino pyridine ring (11c) disfavored the GouldꢀJacobs cyclization as
compared to the previous syntheses that were described. The lower yields (15%) to form 4ꢀ
hydroxynaphthyridine (6f) was not desirable. In addition, chlorination of 4ꢀ
hydroxynaphthyridine (6f) using the catalytic Vilsmeier reagent was also not as selective as in
the 4ꢀhydroxyquinoline series. Cleavage of the pyridyl methoxy group under the acidic reaction
conditions to a pyridone appeared to be the problem, which resulted in degradation. Despite,
degradation of the product which resulted in a low overall yield (40%) of 4ꢀchloronapthridine
(7f), as mentioned, the pure product was isolated. Lastly, the reaction of 4ꢀ
methoxyphenylhydrazine hydrochloride (4d) with 4ꢀchloronapthyridine (7f) to form PQ (13i)
proceeded in 69% yield.
The synthesis of 14 additional structural analogs (14aꢀn) of the parent compounds were also
pursued to further expand the SAR library of GABA Rꢀα6 subtype selective ligands (see (S)
A
Table S4 and Scheme S1). These analogs incorporated substituent modifications of the “Aꢀring”
and “Dꢀring” of the PQ core which included: bromoꢀ, chloroꢀ, fluoroꢀ, methoxyꢀ, methylꢀ, nitroꢀ,
trifluoromethylꢀ, and trifluoromethoxyꢀ groups (see (S) Experimental).
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Metabolic Stability with Human Liver (HLM) and Mouse Liver (MLM) Microsomes.
Since it was assumed that the α6β3γ2 selectivity of deuterated ligands would not be significantly
different from that of nonꢀdeuterated ligands, and since the generation of compounds with an
increased metabolic stability was the main aim of this study, the compounds synthesized were
first investigated for their metabolic stability. The deuterated analogs displayed improved
metabolic stability in both HLM and MLM, as compared to their respective nondeuterated parent
compounds in all cases (Table 3). The nonꢀdeuterated parent ligands 8a and 8i both exhibited
similar HLMꢀmediated metabolic halfꢀlives of 3.6 and 3.4 hours, respectively, while 8n
displayed the most rapid metabolic rate of 1.7 hours in the presence of HLM. In MLM, 8a
exhibited a halfꢀlife of 3.2 hours and 8n displayed a halfꢀlife of 1.9 hours, while 8i displayed the
most rapid metabolic rate in MLM with a halfꢀlife of 1.5 hours. Among the 8a series,
deuteration of the “Aꢀring” methoxy group (8c) exhibited a threeꢀfold increase in stability in the
presence of HLM and a fourꢀfold increase of stability in MLM with respect to the parent ligand.
Deuteration of the “Dꢀring” methoxy group (8b) exhibited a 2.5ꢀfold increased halfꢀlife in HLM
and a threeꢀfold increase in MLM, as compared to 8a. These results indicated a slightly faster
metabolism of the “Aꢀring” methoxy group over the “Dꢀring” methoxy group in the 8a parent
series within experimental error. The comparison of metabolic stability in the presence of HLM
of “Dꢀring” metaꢀOCD isomers between “Aꢀring” OCD ligand 8e and “Aꢀring” OCH ligand
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g further supports the evidence of faster metabolism of the “Aꢀring” versus the “Dꢀring”
methoxy group. Additionally, a similar trend was observed for the “Dꢀring” orthoꢀOCD isomers
3
between “Aꢀring” OCD ligand 8f and “Aꢀring” OCH ligand 8h. The % percent remaining after
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3
1
hour in the presence of HLM and MLM for each of the three deuterated ligands (8b, 8c, and
8d) was virtually identical between 91.5 and 93.1%, within experimental error. In the case of 8d,
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deuteration of both methoxy groups did not have an additive effect on the metabolic stability in
comparison with the monoꢀOCD analogs 8b and 8c. Thus, from a cost and ease of synthesis
3
point of view the monoꢀOCD substituted analogs were pursued versus the diꢀOCD substituted
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analog 8d for further studies. In the 8i series, the deuterated analog 8j exhibited a threeꢀfold
increase in HLMꢀmediated halfꢀlife and a twoꢀfold increase in MLMꢀmediated halfꢀlife.
Similarly, the deuterated analog 8o of the parent 8n exhibited a twoꢀfold increase in HLMꢀ
mediated halfꢀlife and a 20 % increase in MLMꢀmediated halfꢀlife. In summary, deuteration of
the methoxy substituent(s) resulted in the desired improvement in metabolic stability in the
presence of both HLM and MLM for the compounds.
Interestingly, the position of the methoxy substituent on the “Dꢀring” also seemed to have an
impact on the metabolic stability of the PQs, and the effects observed differed in HLM and
MLM, as indicated by a comparison of ortho-OCD ligands with their paraꢀOCD analogs (8e vs
3
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8
d; 8h vs 8b; 8m vs 8l; 8q vs 8o). A similar mixed halfꢀlife improvement in the presence of
HLM and MLM was observed when metaꢀOCD ꢀisomers were compared with the para-OCD3
3
series (8c vs 8d; 8g vs 8b; 8j vs 8l; 8p vs 8o). The introduction of ortho-OCD substituents
3
appeared to have the largest effect on metabolic stability in the 8i and 8n series (8m and 8q,
respectively), but notably this effect was not the case in the 8a series (8f and 8h). Meta-OCD3
substituted ligands generally had the least improvement on metabolic stability in all three series
(8e, 8g, 8j, and 8p). Clearly, the observed mixed effect on metabolic stability governed by the
position of the OCD group on the “Dꢀring” was in line with the activity of CYP450 enzymes in
3
HLM versus MLM. Therefore, it is not possible, at present, to predict trends in the metabolic
stability between the para- versus ortho- versus meta-methoxy substituted analogs as the HLM
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and MLM enzymes appeared nonꢀdiscriminate in regards to the position of the methoxy group
on the “Dꢀring” during the microsomal studies.
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The Nꢀheterocyclic ligands also exhibited better metabolic stability than the parent compounds.
The Nꢀhetero ligands are more polar (see cLogP data in Table 4) than their respective parent
ligands and therefore should not undergo metabolism as quickly as the more lipophilic parent
ligands, since the CYP450 enzymes primarily consist of lipophilic membraneꢀassociated
46
proteins. Here, the Nꢀhetero ligand 13a when compared to the parent ligand 8a exhibited a
fourꢀfold increase in halfꢀlife in the presence of HLM, yet a slightly decreased halfꢀlife was
observed in the presence of MLM. Interestingly, the “Aꢀring” Nꢀhetero ligand 13i displayed a
fourꢀfold increase in the presence of both HLM and MLM. Similar improvement was seen when
1
3e was compared to its parent compound 8k where a nearly fourꢀfold increased halfꢀlife (HLM)
and a twoꢀfold increased halfꢀlife (MLM) was observed. In the case of 8n, the Nꢀhetero ligand
3g exhibited a sevenꢀfold increase in halfꢀlife in both HLM and MLM which is significant.
1
Notably, the effect of incorporation of both the “Dꢀring” Nꢀhetero (pyridineꢀlike) substituents
into and the deuterated methoxy groups onto the lead ligands, exhibited even further
improvement in the metabolic stability over either case individually. Here, the OCD substituted
3
Nꢀhetero ligand 13f when compared to the OCH substituted Nꢀhetero ligand 13e exhibited a
3
40% increased halfꢀlife (HLM) and no change in respect to halfꢀlife in the presence of MLM.
Additionally, the OCD substituted Nꢀhetero ligand 13h exhibited a threeꢀfold increased stability
3
(HLM) and sixꢀfold increased stability (MLM) with respect to their halfꢀlives when compared to
the OCH substituted Nꢀhetero ligand 13g. Of special note, the OCD substituted Nꢀheterocyclic
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compound 13h displayed the best metabolic stability of all the analogs in the series with halfꢀ
lives of 38.6 hours (HLM) and 70.8 hours (MLM), respectively.
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The effect of deuteration of the “Aꢀring” and / or “Dꢀring” methoxy groups was also examined in
the case of the 13a related analogs (13b, 13c and 13d). A definitive metabolic rate difference
between the “Aꢀring” methoxy group and the “Dꢀring” methoxy group (see 13c, "Aꢀring" OCD3
group and 13b, "Dꢀring" OCD group) as compared to 13a was not evident; the effect was much
3
more subtle than the metabolic stability observed in the α6 selective 8a series above and again
was different in the presence of HLM or MLM. In the case of diꢀOCD analog 13d, deuteration
3
of both methoxy groups did not have an additive effect on the metabolic stability in comparison
to the monoꢀOCD analogs 13c and 13b. Obviously, the concurrent effect on the metabolic
3
stability between Nꢀhetero and OCD substitution is too complex to draw clear conclusions on
3
the impact of deuteration of the “Aꢀring” versus “Dꢀring” methoxy groups in the case of 13a
related analogs. It is very possible that retarding the rate of the Oꢀdemethylation reaction in both
the “Aꢀring” and “Dꢀring” has directed the metabolism rapidly towards another metabolic
pathway.
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Metabolite Study. Analysis of the metabolites from the HLM and MLM study confirmed that
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4
the primary metabolic function of CYP450 enzymes with respect to
methoxyarylpyrazoloquinolinones proceeded by Oꢀdemethylation of the methoxy groups (OCH3)
in either the “Aꢀring” or “Dꢀring” to form hydroxyarylpyrazoloquinolinones. This is in
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35
33
agreement with Nelson et al. Furthermore, this study confirmed the kinetic DIE and
illustrated that k > k for compounds with both a OCD and a OCH group in either the “Aꢀring”
H
D
3
3
or “Dꢀring” (Scheme 4) was in operation. Qualitative assessment by mass spectroscopy
confirmed for “Dꢀring” OCD substituted ligand 8b (Scheme 4A), that primary metabolite I (in
3
bold) existed at a much higher concentration than metabolite II and the diꢀdesmethoxy
metabolite III in both (HLM and MLM) metabolism studies after sixty minutes. Similarly, for
“
Aꢀring” OCD substituted ligand 8c (Scheme 4B), primary metabolite IV (in bold) was present
3
at a higher concentration than either metabolite V or VI. Examples A and B in Scheme 4,
clearly indicated that the kinetic DIE predominated in the metabolic pathway (k > k ) of the Oꢀ
H
D
demethylation (OCD versus OCH ) of these ligands over which ring (“Aꢀring” or “Dꢀring”) the
3
3
deuterated versus nonꢀdeuterated methoxy groups was placed upon.
For the case of Nꢀhetero substituted ligand 13b (Scheme 4A), primary metabolite VII (in bold)
existed at a much higher concentration than metabolite VIII and the diꢀdesmethoxy metabolite
IX in both the HLM and MLM metabolism study after 60 minutes. Similarly, for “Aꢀring”
OCD substituted ligand 13c (Scheme 4B), primary metabolite X (in bold) was present at a
3
higher concentration than either metabolite XI or XII. Examination of examples A and B in
Scheme 4 not only indicated that substitution of OCD for OCH predominated during
3
3
metabolism over the effect of which ring (“Aꢀring” versus “Dꢀring)” the deuterated methoxy
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group was placed on, but also showed that the kinetic DIE prevailed over the effect of Nꢀhetero
substitution onto the “Dꢀring” of the ligands on the rate of metabolism.
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In the cases of 8o and 8j, it was also confirmed (see Scheme 4C and 4D) that the primary
metabolic pathway occurred via Oꢀdemethylation of the methoxy groups since primary
metabolite XIII was found for 8o and primary metabolite XIV was found for 8j. In summary,
this metabolite study confirmed the importance of deuteration of the methoxy groups to
positively impact the metabolic stability of this series of GABA R α6 subtype selective ligands.
A
In addition, the metabolic rate of these ligands was governed predominately by the kinetic DIE
over the position of the deuterated versus nonꢀdeuterated methoxy group on the “Aꢀring” versus
“Dꢀring” or the impact of a “Dꢀring” Nꢀhetero substituent, as mentioned above.
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Study of GABA R α6β3γ2 Subtype Efficacy and Selectivity. The three lead ligands (8a, 8n
A
1
0, 29
and 8i) all displayed a pronounced functional selectivity for the GABA R α6β3γ2 subtype,
A
and modulated the EC ꢀGABA induced currents strongly with efficacies at 30 µM ranging from
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~400% (8i) to ~ 900% (8a and 8n) potentiation (Figure 4). Electrophysiological experiments
confirmed that within experimental error, replacement of OCH by OCD groups did not
3
3
significantly change the efficacy of five different deuterated ligands for modulating GABAꢀ
induced currents at the α6β3γ2 GABA R subtype (Figure 5), as compared to the three lead
A
compounds. It thus could be assumed that this also would hold true for the efficacy of these
ligands for modulating GABAꢀinduced currents at the other receptor subtypes that was zero or
close to zero for the three lead compounds up to a concentration of 1 µM (Figure 4), and that
these compounds would exhibit a comparable α6β3γ2 GABA R selectivity. Specifically, the
A
deuterated analogs of 8a (8b, 8c, 8d) and 8n (8o) exhibited the best efficacy and selectivity.
2
5
In a different series of compounds based on PZꢀIIꢀ028 (8k) which exhibited higher efficacy but
10
poor α6 selectivity, as compared with 8a, the methoxy group on the “Dꢀring” was moved from
para to meta (8i) and to ortho (8m) positions. These modifications dramatically changed the
properties of the compounds. As a result the meta-OCH substituted compound, LAU 159 (8i),
3
in contrast to its nonꢀselective parent compound 8k was a selective modulator of the α6β3γ2
28
subtype, while the orthoꢀOCD analogue 8m was completely inactive (Figure 6). The same
3
2
8
turned out to be true for the nonꢀdeuterated analogue of 8m (LAU 165). Since LAU 165 was
2
8
not able to inhibit the effects of 8i, it might not be able to bind to the α6+β3ꢀ interface. Since
deuteration does not change the activity of compounds at the receptor, this conclusion also holds
true for 8m. Consequently, 8m can be further investigated in in vivo studies as a negative
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control compound from the same chemotype to establish that an observed biological effect was
connected with the modulation of the α6β3γ2 subtype.
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In an additional study, ligands with heteroatoms in the core scaffold with substituents at
positions analogous to the parent ligands (8a, 8n and 8k) were also investigated. Incorporation
of the Nꢀhetero atoms into the “Aꢀring” or “Dꢀring” of the PQ core was accompanied by a drop
in efficacy at the α6β3γ2 subtype relative to parent compounds 8a and 8k, but not relative to the
parent compound 8n, as revealed in Figure 7. Four compounds retained some efficacy (13b,
1
3c, 13h, and 13i), while one Nꢀhetero ligand 13f displayed much poorer efficacy. The
deuterated Nꢀhetero analog 13c was then characterized in the whole panel of six receptors
Figure 8). While the maximum efficacy of 13c was weaker than that for the lead ligands, the
(
α6 subtype selectivity was very good and if its bioavailability is as good or better, this compound
may be a good alternative to 8a for use in in vivo studies.
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PDSP Receptor Study. In addition to this GABA R α6β3γ2 subtype selectivity, a screening of
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the ligands against a panel of 46 receptors, transporters and ion channels including the hERG
channel confirmed selective binding of these ligands towards the Bz/GABA receptors ((S)
A
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Table S1). The 8i related analogs only displaced the radiolabeled probe [ H]flunitrazepam from
the benzodiazepine (BZP) rat brain site of GABA Rs, except for 8l, that also weakly interacted
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3
with DOR ([ H]DADLE). No affinity of these analogs was observed for the other 45 receptors
((S) Table S1). The BZP rat brain site is a homogenized mixture of rat brain cells and
membranes consisting of all GABA R subtypes (α1ꢀα6) where the binding affinity reflects
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affinity at the α+γꢀ site. The 8a related structures showed no activity in all assays except the BZP
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rat brain site of GABA Rs, serotonin (5ꢀHT , [ H]LSD), and µꢀopioid (MOR, [ H]DAMGO)
7
A
receptors. However, individual ligands such as 8a, also showed activity at Dopamine D5
3
3
(
[ H]SCH233930), or 13a at Muscarinic M ([ H]QNB) receptors. The 8n related structures
4
again only showed activity at the BZP rat brain site of GABA Rs, but individual compounds
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3
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weakly interacted also with Serotonin 5ꢀHT ([ H]LSD), 5ꢀHT ([ H]Mesulergine) and 5ꢀHT
7
2
B
2C
3
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(
[ H]LSD) receptors, or µꢀopioid (MOR, [ H]DAMGO) receptors. Most of these individual
additional interactions resulted in about 50% inhibition of binding of the respective ligand at a 10
µM drug concentration, suggesting an IC of about 10 µM. Secondary binding studies ((S)
5
0
Table S2) were then conducted to determine the binding affinities for any ligand that displaced
50% radioligand in the primary (PDSP) assay, which demonstrated that offꢀtarget binding for
most ligands was 1,000 times less potent compared to the primary target. The ortho isomers of
b and 8o (8h and 8q, respectively) exhibited significantly less binding to the BZP rat brain site,
while the ortho-OCD isomer of 8i (8m) still displayed a reasonable binding affinity to the BZP
>
8
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rat brain site, but less as compared to its para-OCD isomer 8l. Thus, the ortho isomer 8m was
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identified as a control compound because of its lack of efficacy exhibited at the GABA R
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α6β3γ2 subtype, as described above. Overall, the library of potential α6 ligands do have high
specificity for binding at the BZP rat brain site and importantly do not bind at the hERG channel
nor any of the other 44 receptors, transporters and channels examined in a significant fashion.
However, it cannot be ruled out from the PDSP assays that these compounds do not interact with
binding sites at these receptors for which no radiolabeled ligands are available or at other
receptors, transporters and channels that were not investigated in this study. Thus, although it is
proposed that these α6 subtype selective ligands bind at both the α6+β3ꢀ (PQ) site as positive
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allosteric modulators and at the α6+γ2ꢀ (DIꢀBz) site as null modulators of GABA Rs,
there
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is no way to assess their binding affinity at the α6+β3ꢀ (PQ) site at present. Deuterated and Nꢀ
hetero analogs of the parent compounds did not display a meaningful change in binding behavior
in the panel of radioligand binding assays explored (B. Roth et al., NIMH Psychoactive Drug
Screening Program, UNC, available at http://pdsp.med.unc.edu) when compared to the parent
OCH lead compounds.
3
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Study of Solubility and cLogP. The introduction of Nꢀhetero atoms into either the “Aꢀring”
(13i) or “Dꢀring” (13a) of α6 ligands such as 8a increased the water solubility, as compared to
the parent ligands in both neutral and acidic media only significantly in the case of the lead
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ligand 8a (see Table 4). In the case of 8k, introduction of a pyridineꢀlike nitrogen atom into the
“Dꢀring” (13e) actually decreased the water solubility by almost 10ꢀfold in both neutral and
acidic aqueous media. Additionally, Nꢀhetero introduction into the “Dꢀring” of 8n resulted in
only a minimal impact on the neutral and acidic solubility in the case of 13g. Finally, 8i had
similar solubility as 8k and 8n. Ultimately, the aqueous solubility for 8a was the best over the
other nonꢀNꢀhetero substituted ligands; however, Nꢀhetero ligands 13i and 13a displayed
significant improvement in water solubility. The acidic water solubility in 0.01 M HCl (pH = 2)
of the ligands did not improve over the water solubility (pH = 7) substantially in any of the cases
explored, presumably because the nitrogen of the Nꢀhetero substituent lacks sufficient basicity to
positively impact hydrogen chloride salt formation. Because the “Aꢀring” analog 13i was
difficult to synthesize, only the deuterated analogs of 8a and 13a were carried forward for
37
pharmacokinetic studies. Of note, the calculated cLogP (octanolꢀwater partition coefficient )
values (Table 4) also dropped in all cases for the Nꢀhetero analogs as expected; however, this did
have an important positive effect on metabolic stability, as indicated by a comparison of 8a and
1
3a, 8k and 13e, or 8n and 13g. Interestingly, however, Nꢀhetero substitution together with
deuteration resulted in an 11ꢀfold increase in metabolic stability in HLM, and a 30ꢀfold increase
in stability in MLM in the 8n series (compare 8o and 13h in Table 3).
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Study of Potential Cytotoxicity in HEK293 (Kidney) cells and HEPG2 (Liver) cells. An
examination of the cell viability, indicated that these ligands are nonꢀtoxic even at 400 µM in the
presence of HEK293 as well as HEPG2 cell lines (see (S) Table S3 and Figure S1 for details).
In both cell lines, substitution of OCD for OCH in either the “Aꢀring” or “Dꢀring” of the parent
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ligands did not have a negative impact on cell viability. Additionally, variation of the
substitution pattern of the methoxy groups on the “Dꢀring” to the meta position (8e and 8j) or
ortho position (8f and 8m) had no impact on cytotoxicity. Finally, Nꢀhetero atom insertion into
the “Dꢀring” to provide 13c, 13e and 13g also had no impact of cell viability. All of the potential
α6β3γ2 subtype selective ligands tested here were nontoxic.
Study of the Lack of Rotarod Motor Impairment. Analysis of the data from the rotarod
studies indicated that none of the GABA Rꢀα6 subtype selective PQ analogs were incapacitating
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in mice on the rotarod when dosed by oral gavage at 40mg/kg. Deuteration of the methoxy
groups of the parent ligands ((S) Figure S2), and introduction of Nꢀhetero substituents into the
“Dꢀring”, as well as deuteration of the Nꢀhetero analogs ((S) Figure S2) resulted in no further
change of the physiological response in contrast to the effect on motor impairment of diazepam
at 5mg/kg.
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Pharmacokinetic Study. In vivo plasma and brain kinetic profiles of six ligands (8a, 8b, 8c, 8d,
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3c, and 8m) suspended in a standard vehicle and dosed by IP injection at 10 mg/kg in male
Wistar rats, are illustrated in Figure 9. The timeꢀconcentration curves of all ligands tested
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indicated they were rapidly absorbed and distributed in the brain. The nonꢀdeuterated molecule
8
a demonstrated a low brainꢀtoꢀplasma partition coefficient (Kp = AUC , brain/AUC , plasma)
0ꢀt 0ꢀt
of 0.08 ± 0.01. The three deuterated analogs 8b, 8c and 8d had increased Kp values, equal to
.13 ± 0.01, 0.16 ± 0.02, and 0.17 ± 0.02, respectively, together with longer halfꢀlives, both in
plasma and brain. The brain AUC0ꢀ12 value of the Nꢀhetero ligand 13c was only 45%, 36% and
5% of that obtained with 8b, 8c and 8d, respectively. The orthoꢀOCD substituted ligand 8m
0
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exhibited a reasonable Kp value of 0.15 ± 0.01 which additionally illustrated its usefulness in
further in vivo studies as a control compound with a favorable pharmacokinetic profile.
Because the three deuterated analogs of 8a had generally similar values of pharmacokinetic
parameters, it was decided to pursue the study of 8b and 8d. The key ligands 8b and 8d were
employed for calculation of the estimated free concentrations available for binding to receptors
in brain tissue and free fractions were found to be 0.194 and 0.105, respectively. These values
gave rise to more than twoꢀfold higher AUC values of estimated free brain concentrations of 8b
ꢀ1
ꢀ1
as compared to 8d (651.72 ± 217.70 ng h g and 288.78 ± 61.07 ng h g , respectively). Having
in mind the electrophysiological activity presented in Figure 5A, the free (unbound) brain
concentrations of 8b in the range close to 100 nM were sufficiently high to mediate α6β3γ2
modulation in vivo. Hence, the “Dꢀring” substituted OCD ligand 8b was chosen to be tested in a
3
battery of basic behavioral paradigms. The “Aꢀring” OCD ligand 8c was not pursued further
3
because it requires more steps to synthesize than ligand 8b.
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The concentration–time profiles of 8a and 8b in rat plasma and brain after oral (gavage) as well
as after intravenous (IV) administration, with the calculated pharmacokinetic parameters, are
presented in Figure 10. The values of absolute oral bioavailability of 8a and 8b from
nanoemulsion formulations, calculated on the basis of the plasma AUC values obtained
following two routes of administration, were 66.9% and 100.0%, respectively. These data reveal
that deuteration of 8a resulted in optimization of pharmacokinetic behavior after oral
administration, which was further supported by respective values of brain bioavailability of 8a
and 8b (82.6% vs. 89.5%).
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In Vivo behavioral studies on 8b versus the control compound 8m. Since the ligand 8b
exhibited a longer duration of action, it was subjected to in vivo studies after IP administration at
a 10 mg/kg dose. In addition, the control ligand 8m of the same chemotype, was also studied in
parallel. Behavioral assays were run on these ligands to determine the occurrence of anxiolysis,
assess muscle strength and basic sensorimotor activity on the elevated plus maze, grip strength
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and cued waterꢀmaze training, respectively (Table 5). The nanoemulsion formulation
employed for the behavioral studies, which permits a more reliable dosing, did not grossly affect
the pharmacokinetic profile of 8b obtained with the suspension formulation (data not shown).
Moreover, the corresponding placebo (nanoemulsion alone) was not behaviorally active when
compared to saline (data not shown). The changes in behavioral parameters analyzed above,
47
which are generally observed with typical Bzs or Bz site ligands, were not elicited by either of
the two ligands. In addition, motor coordination and balance on the rotarod were retained after IP
doses as high as 55 mg/kg 8b and 60 mg/kg 8m. Taken together, these results indicate that
selective in vivo potentiation of GABA Rs containing the α6 subtype did not mimic any of the
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behavioral effects of classical Bzs supporting the role of the GABA R α6 (DI) receptor in the
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following in vivo data.
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In Vivo hyperlocomotor response studies on 8b versus the control compound 8m. In rats,
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b, but not 8m at an IP dose of 10 mg/kg induced a hyperlocomotor response (Figure 11A); in
the repeated experiment, the effect of 10 mg/kg 8b did not reach statistical significance (Figure
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11C), while in the third experiment it was present at both the dose of 3 and 10 mg/kg (Figure
11E). In mice, both 8b and 8a induced a hyperlocomotor action in a wide range of IP doses (3,
10 and 30 mg/kg, Figure 11B and 11D, respectively). The hyperlocomotor effect of 10 mg/kg 8b
was present even in mice that received a sedative IP dose of diazepam (3 mg/kg) in combination
Figure 11F), suggesting that 8b is able to antagonize the effects of diazepam at Bz binding sites
(
and additionally elicit locomotor stimulation presumably exerted through GABA Rs containing
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Discussion and Conclusions
GABA Rs containing the α6 subunit are primarily located in the granule cells of cerebellum,
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, 12
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but are also expressed in other brain regions.
So far, not much is known about the function
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of these receptors in the brain, but recent evidence indicates that they might play a role in a
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variety of diseases. These include disorders of the trigeminal system, neuropsychiatric
disorders with sensoriꢀmotor gating deficits (such as tic disorders, certain symptoms of
1
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schizophrenia, obsessive compulsive disorder and attention deficit disorders),
and
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depression, as well as migraine. Recently, some PQs were identified as the very first
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compounds that are able to modulate α6ꢀGABA Rs with high selectivity.
Based on
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three highly α6ꢀGABA Rꢀselective PQ lead compounds (8a, 8n and 8i), a total of 42 PQs were
A
31
synthesized in the present study with the aim to increase their limited water solubility, as well
as their metabolic stability. Exchanging deuterium for hydrogen has been shown previously to
positively affect the metabolic stability of drugs while retaining the pharmacologic profile of
32
active compounds. Despite these successes, deuteration sometimes has also been observed to
4
9,50
have no effect on metabolic stability.
In the present study, the substitution of OCD groups
3
for OCH groups in the lead ligands (8a, 8n and 8i) resulted in the deuterated α6β3γ2 GABA R
3
A
subtype selective ligands (8b, 8c, 8d, 8j and 8o) with an improved metabolic stability in both
HLM and MLM assays (Table 3) and improved pharmacokinetic profile (Figure 9). However,
the halfꢀlife of compounds not only increased but also decreased on deuteration depending on the
structure of the compound investigated and the position of the incorporated deuterium and was
different in HLM and MLM (Table 3). The changes in halfꢀlife observed on deuteration were
thus not predictable. The same was true for the Nꢀheterocyclic compounds (13a, 13e, 13g and
13i) and their deuterated derivatives (13b, 13c, 13d, 13f and 13h) (Table 3). The Nꢀheterocyclic
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