New synthesis and tritium labeling of a selective ligand for studying high-affinity γ-hydroxybutyrate (GHB) binding sites

Article abstract of DOI:10.1021/jm4011719

3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [³H]-1 is described. The radiosynthesis employs in

Full text of DOI:10.1021/jm4011719

Brief Article
pubs.acs.org/jmc
New Synthesis and Tritium Labeling of a Selective Ligand for
Studying High-Affinity γ‑Hydroxybutyrate (GHB) Binding Sites
Stine B. Vogensen,*,†,⊥ Ales Marek,‡,§,⊥ Tina Bay,† Petrine Wellendorph,† Jan Kehler,∥
̌
Christoffer Bundgaard,∥ Bente Frølund,† Martin H. F. Pedersen,*,‡ and Rasmus P. Clausen*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100
Copenhagen, Denmark
‡The Hevesy Laboratory, DTU Nutech, Technical University of Denmark, 4000 Roskilde, Denmark
§Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 6 Prague, Czech Republic
∥DiscoveryChemistry and DMPK, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
S
* Supporting Information
ABSTRACT: 3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites
in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis
employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high
selectivity, and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the
high-affinity GHB binding sites.
of GHB with that of the GABAB agonist baclofen (Figure 1) in,
e.g., c-Fos expression studies5 and behavioral studies,6 it is clear
that the two compounds have different pharmacological
profiles. Thus, GHB effects may also be mediated by non-
GABAB receptors. The fact that GHB high-affinity binding sites
are preserved in brains of GABAB knockout mice4 make them
likely receptor candidates and has prompted the uncovering of
their identity. Using photoaffinity labeling combined with
proteomic analysis and molecular pharmacology studies, we
recently reported that α4β1−3δ ionotropic GABAA receptors
correlate with the GHB high-affinity sites.7 To acquire a more
complete understanding of the physiological relevance of GHB
high-affinity binding sites in the CNS, and particularly the
nanomolar effects mediated by GHB at the α4β1δ receptors,
better tool compounds and further pharmacological inves-
tigations are needed.
We have previously described 3-hydroxycyclopent-1-enecar-
boxylic acid (HOCPCA, 1, Figure 1) as a selective ligand for
the high-affinity GHB binding site (27 times higher affinity than
GHB itself) and, importantly, with no affinity for the GABAB
receptor. Given the close structural relationship to GHB and its
relatively small size, 1 is a highly attractive compound to
investigate GHB-like pharmacology. We therefore decided to
explore synthetic routes for incorporation of hydrogen and
carbon isotopes into 1 and at the same time provide a more
feasible and preparative synthesis of 1, which is needed to
facilitate in vivo studies. At present, [3H](E,RS)-(6,7,8,9-
tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid
([3H]-NCS-382) is the standard radioligand in binding
experiments for the high-affinity GHB binding site.8 NCS-382
(Figure 1) is a putative antagonist8 at the high-affinity GHB
INTRODUCTION
γ-Hydroxybutyrate (GHB, Figure 1) is an endogenous
substance that is present in micromolar concentrations in the
■
Figure 1. Chemical structures of the two neuroactive substances GHB
and GABA. HOCPCA (1) and NCS-382 bind selectively to the high-
affinity GHB binding site. Baclofen is a selective GABAB agonist.
mammalian brain. It is a metabolite of the major inhibitory
neurotransmitter γ-aminobutyric acid (GABA) (Figure 1) but is
also believed to be a neuromodulator on its own1 although the
specific function of GHB remains to be elucidated. GHB is
administered orally for the treatment of narcolepsy (sodium
oxybate)2 and alcoholism.3 GHB is tasteless and odorless, and
combined with its central nervous system (CNS) effects (mild
euphoria, sedation, respiratory depression, and eventually coma
in high doses) it is a hazardous drug of abuse (fantasy). In the
CNS, GHB binds to both low-affinity and high-affinity binding
sites.1 The low-affinity site is believed to be identical to the
GABA orthosteric site at the metabotropic GABAB receptor, as
high dose effects of GHB (micromolar in the brain) are
prevented by preincubating with a GABAB receptor antagonist3
and several prominent GHB-induced effects are absent in
GABAB knockout mice.4 However, when comparing the effect
Received: August 1, 2013
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
binding site with a Ki 14 times lower than that of GHB.9 The
higher affinity of 1 compared to NCS-3829 and closer structural
relationship to GHB underscores the relevance of developing a
better preparative and radiolabeling procedure. This will
provide a better alternative to the only available radiolabeled
agonist [3H]GHB, which has several caveats, both in terms of
selectivity due to its affinity for the GABAB receptor and in
terms of its susceptibility to metabolism and uptake.10
Here, we describe a highly improved preparative synthesis of
1 and the synthesis of tritium labeled 1. Furthermore, the
stability of 1 and [3H]-1 are evaluated, binding competition
studies using [3H]-1 are presented, and the selectivity profile of
1 is investigated. Finally, the brain penetration of GHB, 1, and
NCS-382 is investigated in vivo.
amine.11 It was crucial for a high yield to use freshly distilled
triethylamine dried over LiAlH4, whereas sublimation (110 °C,
0.2 mbar) of the commercial brownish 1.3-cyclopentanedione
to white crystals did not improve the yield. Luche reduction
with NaBH4 in methanol gave the alcohol that was immediately
TBS-protected to give 6 in 86% overall yield.
Metal−halogen exchange of 6 using t-BuLi followed by
addition of CO2(g) afforded the carboxylic acid 7 in 78% yield.
Finally, deprotection of the TBS-protected alcohol using an
aqueous solution of H2SiF6 as described by Pilcher and
DeShong12 gave 1 as white crystals in 75% yield upon
recrystallization. The product was stable as the free acid as well
as the sodium salt. This new synthetic route is high yielding
(56% overall), includes more simple purifications, and produces
a nonhygroscopic stable product without the use of chromic
acid. The route is also applicable for late-stage isotopic carbon
labeling of 1 because labeled CO2(g) can be employed when
incorporating the carboxylic acid group. Alternatively, the
bromide 6 can be transformed to the corresponding pinacol
boronic ester and treated with [11C]CO2(g) in the presence of
CuI/crypt-222/KF as recently described by Riss et al.13 On the
other hand, the route is not stereoselective and provide both
enantiomers.
RESULTS AND DISCUSSION
■
Improved Synthesis of 1. The previously9 reported
synthesis of 1 is outlined in Scheme 1. Reduction of the
starting material (2) followed by mesylation and elimination
gave α,β-unsaturated ethyl ester (3a) in 53% yield.
Scheme 1. Previous Synthesis Route to 1 and Its Carbonyl
a
Precursor 9
Tritium Labeling. The previously reported synthesis of 1
outlined in Scheme 1 was a good starting point for a route to
tritium labeled 1 as reduction of the α,β-unsaturated ketone (4)
enabled introduction of tritium late in the synthesis. Still, some
optimization of this route was feasible. 3b is commercially
available, but volatility of both 3b and 4b compromised the
yield in the first step and 3b was therefore transesterified to the
propylester 3c in 70% yield. Attempts to replace the excess
amount of chromic acid with KMnO414 or TBHP/Pd(OH)C15
were unsuccessful. The use of catalytic amounts of Mn(OAc)2·
4H2O16 was at first glance successful (75% yield), but
unfortunately an unidentified impurity not visible by NMR
affected the course of the following reduction negatively and
the oxidation was therefore performed with chromic acid to
afford 4c. The Luche reduction (1 equiv of NaBH4 in MeOH in
the presence of CeCl3) gave the alcohol 5b in 98% yield
(Supporting Information (SI), Table S1). To prepare [3H]-1
with a maximal specific activity, we decided to screen the
reaction conditions using borodeuterides prepared in situ from
deuterium gas instead of using commercial deuterides. In the
literature, a broad spectrum of in situ prepared boro-
deuterides17,18 and tritides,19,20 respectively, are described. To
suppress the risk of reduction of the double bond and ester
group, we were especially interested in less reactive
borodeuterides. As demonstrated by Zippi et al.,19 LiB-
a
Reagents and conditions: (a) (i) NaBH4, EtOH, 0 °C − rt, 2.5 h, (ii)
CH3SO2Cl, Py, rt, 3 h, (iii) Et3N, CHCl3, reflux, 23 h; (b) CrO3, Ac2O,
AcOH, rt, 30 min; (c) NaBH4, CeCl3, MeOH, 0 °C, 30 min; (d)
Na2CO3, 50 °C, 16 h; (e) 1-propanol, H2SO4, reflux, 1 h.
Oxidation of 3a with 3 equiv of CrO3 gave 4a in varying
yields below 39%. Successful Luche reduction gave 5a, and final
deprotection with sodium carbonate gave the product 1 as
hygroscopic crystals. The overall yield of the six-step synthesis
was 18%. However, this route posed several problems as a
preparative synthesis, i.e., varying yields, tedious purification in
particular in the oxidation of 3a, and a hygroscopic end product
that is unstable during storage.
In light of the potential of 1 as a pharmacological tool for
future in vivo and ex vivo studies, we wanted to develop a more
suitable synthesis of 1 that could circumvent these obstacles
and generate large quantities of 1. The new synthetic route to 1
is outlined in Scheme 2. First, 1,3-cyclopentanedione was
brominated with dibromo-triphenylphosphine and triethyl-
(OMe)3 H (8) can be readily generated from Li2H by addition
2
of trimethoxyborate (Scheme 3). Lithium deuteride is formed
by the treatment of a n-BuLi-TMEDA solution under gaseous
2H2 atmosphere17 (Scheme 3).
a
Scheme 2. New Synthesis Route to 1
Scheme 3. Synthesis of the Lithiumboronic Reducing
a
Reagent
a
Reagents and conditions: (a) (i) Br2PPh3, benzene, Et3N, rt, 18 h, (ii)
NaBH4, CeCl3, MeOH, 0 °C − rt, 2.5 h, (iii) TBS-Cl, DMAP,
imidazole, DCM, rt, 16 h; (b) t-BuLi, THF, −78 to −50 °C, 2 h, −78
°C, CO2(g), 20 min; (c) H2SiF6 (aq 23%), CH3CN, rt, 1 h.
a
Reagents and conditions: (a) TMEDA, 2 h, room temperature; (b)
B(OMe)3, 30 min, room temperature.
B
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Journal of Medicinal Chemistry
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The yield of in situ generated borodeuteride (8) was
determined indirectly using 4-anisaldehyde as a trapping agent
in independent reactions. The reaction was carried out under
various ratios (1:1, 2:1, and 1:2) of (8) (theoretically generated
amounts based on n-BuLi) and anisaldehyde, and the yield of 8
found to be a solution of 1 mCi/mL in ethanol or in an
aqueous solution of gentisic acid (50 mM).
Binding Studies. Binding of [3H]-1 to rat cortical
membranes was investigated using assay conditions previously
described for [3H]NCS-3829 with GHB (1 mM) for
determining nonspecific binding (Figure 2A). IC50 [pIC50
1
was determined by HPLC and H NMR to be in the range of
40−47% in all three cases. That is in accordance with the yield
of Li3H (35%) reported by Zippi et al.19 For radiation safety
reasons during the 3H-labeling of 1, it was desirable to perform
the reduction with borotritide and the ester hydrolysis of 4c in
one pot. Therefore, upon lyophilization of the solvent, aq
NaOH was added and hydrolysis of the propylester gave [2H]-1
(Scheme 4). The lyophilization was crucial as the yield dropped
a
Scheme 4. Deuterium and Tritium Labeling of 1
a
Reagents and conditions: (a) (i) 8 or 9, THF, room temperature, 2 h,
room temperature, lyophilized, (ii) 1 M NaOH, 1 h, room
temperature, (iii) 1 M HCl.
from 88% to 15% if omitted. Next the reaction conditions for
the reduction of 4c (Scheme 4) was investigated (see SI Table
S1).
The protic solvent MeOH of the original experiment could
not be exchanged with EtOH in the presence of CeCl3.
Actually, the presence of CeCl3 apparently resulted in an
increased amount of hydrolyzed 4c and could successfully be
omitted. In the absence of CeCl3, both DMF and even the
easily lyophilizable solvent THF could be used.
Lowering of the reaction temperature to 0 or −78 °C did not
improve the deuterated yield nor suppressed the hydrolysis of
the ketone. For comparison, in situ synthesized Li2H and
LiB2H4 (see SI) were also attempted but resulted in lower
yields and more complex reaction mixtures.
Figure 2. (A) Concentration-dependent inhibition of [3H]-1 binding
(10 nM) to rat cerebrocortical membranes by GHB and 1. Results are
expressed as mean (SD of a single representative experiment
performed in triplicate). Three additional experiments gave similar
results. (B) Displacement of 10 nM [3H]-1 binding to rat
cerebrocortical membranes by 1 mM GHB, 100 μM NCS-382, 1
mM 1, 1 mM GABA, 100 μM gabazine, and 100 μM baclofen. Results
are expressed as mean CPM (SD of a single representative experiment
performed in triplicate) and were repeated at least twice with similar
results.
2
Finally, the best conditions (LiB(OMe)3 H, THF, room
temperature) were used to prepare [3H]-1 (Scheme 4). To
achieve full conversion of 4c, excess amounts (1.8 equiv) of
3
3
LiB(OMe)3 H were used. Reduction of 4c gave the H-labeled
propylester in 95% yield according to HPLC (245 nm).
Subsequent hydrolysis gave the desired product [3H]-1 (97%,
245 nm). We determined the amounts of prepared [3H]-1 to
637 mCi with a specific activity of 28.9 Ci/mmol (determined
by MS and HPLC/LSC, which accounts for close to 1 tritium/
molecule) and a radio chemical purity (RCP) and chemical
purity >99% after purification (HPLC, SI Figure S11).
SEM] values for 1, sodium salt of 1, and GHB were,
respectively, 0.073 μM [7.14
0.02], 0.116 μM [6.93
0.01], and 1.14 μM [5.94 0.06]. This is in relative accordance
with values obtained in the comparable [3H]NCS-382 binding
assay (0.16 and 4.3 μM, respectively).9 As expected, [3H]-1
binding was inhibited by the GHB-selective ligand NCS-382
and the GABAA antagonist gabazine7,21 but notably not by
baclofen and GABA at high concentrations.7,9 These data
suggest that [3H]-1 selectively labels the high-affinity GHB
binding site. The selectivity was further explored in The
National Institute of Mental Health’s Psychoactive Drug
Screening Program (NIMH-PDSP) (SI Table S2), where 1
was found to be more than 100 times selective for the [3H]-1
Stability. Both 1 and [3H]-1 was subjected to stability
testing by HPLC (area%, 245 nm). The rate of decomposition
of 1 was tested at both room temperature and −30 2 °C in
Milli-Q water (2 mg/mL). pH of this solution was 3.5. At −30
°C, compound 1 proved completely stable (1 year and 4
months after formulation) and at room temperature only a
minor decomposition of 1 was observed (100% at day 0 to 96%
at day 35 and 88% at day 99). The stability of [3H]-1 was
investigated in four different formulations for the protection
against radiolysis (see SI). The best stability of [3H]-1 was
C
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Journal of Medicinal Chemistry
Brief Article
3-Hydroxycyclopent-1-enecarboxylic Acid (1). 3-((tert-
Butyldimethylsilyl)oxy)cyclopent-1-enecarboxylic acid (3.17 g, 13.08
mol) was dissolved in acetonitrile (130 mL) in a polypropylene vial,
and H2SiF6 (aq, 20−25% wt, 2.3 mL, 3.9 mmol) was added. The
solution was stirred for 1 h at room temperature. Satrated Na2CO3
(100 mL) and water (50 mL) were added, and the aqueous phase was
washed with diethyl ether (2 × 20 mL). The aqueous phase was made
acidic by the addition of HCl (aq, 4M) and extracted with ethyl acetate
(4 × 150 mL). The combined organic phase was dried (MgSO4) and
the solvent removed in vacuo to give a white solid. CC (2:1 heptane/
ethyl acetate + 2% AcOH) gave a white solid. Recrystallization
(acetonitrile) gave the product as white crystals (1.26 g, 75%); mp
binding site over 45 different receptors and transporters. The
high selectivity of 1 combined with a ligand efficiency22 of 1.05
kcal/mol establish 1 as a highly interesting compound for
further studying the high-affinity GHB binding site.
Brain Penetration Studies. The brain to plasma
distribution ratio of GHB, 1, and NCS-382 was investigated
acutely in mice 30 min after oral administration of 10 mg/kg.
All three compounds were found to be brain penetrant,
reflected by their brain to plasma ratios of 0.20, 0.37, and 0.38
for GHB, 1, and NCS-382, respectively. Considering the
carboxylic acid group shared by all three compounds, often
associated with restricted passive brain penetration, these
results may suggest an involvement of active transport across
the blood−brain barrier. This hypothesis was supported by
assessment of the in vitro permeability using MDCK-MDR1
cells23 which showed that GHB, 1, and NCS-382 all exhibited
very low passive permeability. Thus, after spiking of test
compounds on either the apical or basal side of the cell
monolayers, no compound could be detected at the respective
receiver sides.
1
134−136 °C. H NMR (CD3OD): δ 1.71 (m, 1H), 2.30−2.48 (m,
2H), 2.62−2.70 (m, 1H), 4.86−4.91 (m, 1H), 6.63−6.65 (m, 1H). 13C
NMR (CD3OD): δ 30.81, 34.23, 77.65, 139.93, 144.72, 168.62. Anal.
Calcd for C6H8O3: C, 56.24; H, 6.29. Found: C, 56.34; H, 6.17. LC-
MS (system B): (M − H2O)H+: 111.1.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental data for compounds (3c, 4c, 6, 7, [2H]-5b, [2H]-
1), description of stability studies, in vitro pharmacological
methods, and NIMH-PDSP screening results. This material is
available free of charge via the Internet at http://pubs.acs.org.
CONCLUSION
■
In conclusion, we have developed a new preparative synthetic
route to 1 that can be scaled up with a high and reproducible
overall yield. The new synthesis can readily be developed
further for carbon labeling. Compound 1 is stable during
storage and has a favorable selectivity profile when tested
against other relevant targets. Furthermore, compound 1 was
successfully tritium labeled using in situ generated lithium
trimethoxyborotritide. The incorporation of one tritium atom
proceeded in high radioactive yield, with close to theoretical
specific activity and with high radiochemical purity. Binding of
[3H]-1 could be displaced by GHB and 1 in a concentration-
dependent manner and not by the GABAB receptor agonist
baclofen. This new radioligand will be useful for further
understanding the molecular mechanism of GHB at its high-
affinity binding site and α4βδ GABAA receptors and for
elucidating the pharmacology of the GHB system in the
mammalian CNS, given the brain permeability of 1, for instance
through ex vivo or in vivo labeling of the binding sites with
positron-emitting or other isotopes.
AUTHOR INFORMATION
Corresponding Authors
■
*S.B.V.: phone, +45 35 33 66 80; E-mail, sbv@sund.ku.dk.
*M.H.F.P. (radiochemistry): phone, +45 31 12 27 64; E-mail,
martin@friborg.org.
*R.P.C.: phone, +45 35 33 65 66; E-mail, rac@sund.ku.dk.
Author Contributions
⊥S.B.V. and A.M. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Lundbeck Foundation
(S.B.V.), the Danish Medical Research Council (T.B.), and
the Drug Research Academy (T.B.). A.M.’s fellowship at the
Hevesy laboratory was founded by The International Atomic
Energy Agency (IAEA) (C6/CZR/11001). This work was
supported by the Academy of Sciences of the Czech Republic
(RVO: 61388963) We gratefully acknowledge the Prague
institute for access to tritium NMR. Dr. Steve White is kindly
acknowledged for generously organizing the NIMH-PDSP
compound screen (contract no. HHSN-271-2008-00025-C).
The NIMH PDSP is directed by Bryan L. Roth MD, Ph.D. at
the University of North Carolina at Chapel Hill and Project
Officer Jamie Driscol at NIMH, Bethesda, MD, USA.
EXPERIMENTAL SECTION
■
[3H]-3-Hydroxycyclopent-1-enecarboxylic Acid ([3H]-1). Ca-
reer free 3H2 (13.8 Ci) trapped on a uranium bed (as uranium tritide)
was released by heating (500 °C) and directed in a tritium manifold
into a dried reaction vial (1 mL two-necked round-bottom flask).
N,N,N′,N′-Tetramethylethylenediamine (50 μL) was added. n-Butyl
lithium (100 μL, 160 μmol, 1.6 M in hexane) was added dropwise, and
within 10 min a white precipitate of Li3H was formed. After 2 h of
vigorous stirring, trimethyl borate (18 μL, 160 μmoles) was added and
ABBREVIATIONS USED
■
3
the reaction was stirred for 30 min to generate LiB(OMe)3 H. A
GHB, γ-hydroxybutyrate; HOCPCA, 3-hydroxycyclopent-1-
enecarboxylic acid; NCS-382, (2E)-(5-hydroxy-5,7,8,9-tetrahy-
dro-6H-benzo[a][7]annulen)-6-ylidene ethanoic acid
solution of propyl 3-oxo-cyclopent-1-en carboxylate (4c) (15 mg, 89
μmol) in THF (300 μL) was added dropwise, and the reaction was
stirred for 2 h. Solvents were lyophilized. Water (500 μL) was added,
and a sample showed predominantly [3H]-5b by HPLC analysis (95%,
245 nm). Aqueous NaOH (2 M, 100 μL) was added, and the reaction
was stirred for 1 h. The reaction mixture was neutralized with 1N HCl
to reach pH 6. Water was lyophilized and the solid residue dissolved in
accurate amounts of water. Then 1/10 of the crude mixture was used
for qualitative and quantitative analysis (see SI). Preparative HPLC
gave [3H]-1 ((97%, 245 nm), 637 mCi, 28.9 Ci/mmol) as white solid.
3H NMR (320 MHz, DMSO-d6/H2O 10:1): δ 4.71 (s). MS (ESI):
128.9 (100, M − 1).
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