Small molecule piperazinyl-benzimidazole antagonists of the gonadotropin-releasing hormone (GnRH) receptor

Article abstract of DOI:10.1039/c7md00320j

In this communication, we report the synthesis and characterization of a library of small molecule antagonists of the human gonadotropin releasing hormone receptor based upon the 2-(4-tert-butylphenyl)-4-piperazinyl-benzimidazole scaffold via Cu-catalysed

Full text of DOI:10.1039/c7md00320j

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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Small molecule piperazinyl-benzimidazole antagonists of the gonadotropin-
releasing hormone (GnRH) receptor
Richard Fjellaksel,a,b,c Marc Boomgaren,c Rune Sundset,a,d Ira H. Haraldsen,e Jørn H. Hansenc and Patrick J.
Risse,f,g
aMedical Imaging Group, Department of Clinical Medicine, UiT The Arctic
University of Norway, 9037 Tromsø, Norway.
bDrug Transport and Delivery Group, Department of Pharmacy, UiT The
Arctic University of Norway, 9037 Tromsø, Norway.
cOrganic Chemistry Group, Department of Chemistry, UiT The Arctic
University of Norway, 9037 Tromsø, Norway.
dPET imaging center, division of diagnostics, UNN – University Hospital of
North-Norway, 9038 Tromsø, Norway.
eDepartment of neuropsychiatry and psychosomatic medicine, Oslo
University Hospital, Oslo, Norway.
fRealomics SFI, Department of Chemistry, University of Oslo, PO BOX 1033,
Oslo 0371, Norway.
gNorsk Medisinsk Syklotronsenter AS, Postboks 4950ꢀ Nydalen, ꢀ0424 Oslo
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In this communication, we report the synthesis and
We were interested in the development of a GnRH antagonist
characterization of a library of small molecule antagonists of the of moderate potency to address cognitive and behavioral correlates
human gonadotropin releasing hormone receptor based upon the of GnRH-R attenuation. In our reasoning, a reversibly binding
2-(4-tert-butylphenyl)-4-piperazinyl-benzimidazole scaffold via Cu- antagonist would reduce gonadal overstimulation by limiting the
availability of binding sites by competition with the agonist.
catalysed azide alkyne cycloaddition. Our main purpose was to
Thereby, the central effects of testosterone and estrogen would be
buffered, while hormone homeostasis would be preserved. Small
molecule antagonists of GnRHR are particularly interesting due to
their direct, dose dependent inhibition of GnRH activity, lack of
stimulatory side effects on the receptor and superior passive
permeability into tissues relative to peptide agonists.
find a more soluble compound based on the WAY207024 lead with
nanomolar potency to inhibit the GnRH receptor. A late stage
diversification by the use of click chemistry was, furthermore
developed to allow for expansion of the library in future
optimisations. All compounds were tested in a functional assay to
determine the individual potency of inhibiting stimulation of the
receptor by the endogenous agonist GnRH. In conclusion, we
found that compound 8a showed improved solubility compared to
WAY207024 and nanomolar affinity to GnRH receptor.
Several classes of small molecule antagonists have been
developed previously. Pelletier and co-workers reported a 2-(4-
tertbutylphenyl)-4-piperazinyl-benzimidazole-scaffold as
molecule antagonist of the GnRHR with nanomolar inhibition
Gonadotropin-releasing hormone (GnRH) is a peptide hormone potency, albeit low solubility was cited as a shortcoming by the
a small
secreted from the hypothalamus into the hypophysial portal authors.9,
Further optimization lead to the discovery of the
10
slightly more soluble WAY 207024 compound as shown in figure
1.11
bloodstream. Once in circulation, the peptide acts as an endocrine
signalling hormone mediating release of follicle stimulating
hormone (FSH) and luteinizing hormone (LH) via gonadotropin- Based on these results, we have designed a novel library of
releasing hormone receptor (GnRHR) activation in the pituitary potential antagonists addressing the following criteria:
a) A late stage diversification approach based on the 1,3-triazole
motive to facilitate straightforward structural modification.
gland. LH and FSH directly regulate gender-specific production of
androgens, estrogens, progesterone and inhibin in the gonads. The
regulatory circuit is closed by the permeability of the blood-brain b) A moderate potency permitting competition of the ligand in
circulation with endogenous GnRH for GnRHR binding sites,
which would make GnRH activation subject to both GnRH
concentration and the concentration of the inhibitor in tissue.
barrier to steroid hormones such as estrogen and testosterone,
which form negative feedback loops. GnRH, LH and FSH are unlikely
to permeate the blood-brain barrier, thus impeding any regulatory
feedback on hormone balance, which substantiates the impact of c) Improved solubility in aqueous media to improve
bioavailability and pharmacokinetic profile of the lead.
testosterone and estrogen on neurochemical correlates of their
respective effects on social behaviour, decision-making and ageing.
The hypothalamic-pituitary-gonadal (HPG) signalling circuit is key to
gender hormone homeostasis, which can lead to substantial
implications in cognitive and behavioural traits.1-4
GnRH agonists are well-established pharmaceuticals used at low
dose to stimulate hypofunctional GnRH in hypogonadism. At high
dose, GnRH agonists deplete GnRH receptor function in the
pituitary, limiting LH and FSH secretion and thereby the levels of
androgens and estrogens in circulation and suppressing hormone
production completely. GnRH receptor agonists play an important
role in clinical care, e.g. in the treatment of hormone responsive
cancer, reproductive diseases and for behavioral modification of
sexual offenders via these mechanisms. However, GnRH agonists
cannot address mild GnRH overfunction, which would require
attenuation of the signaling circuit, rather than stimulation or
depletion. To address this shortcoming, GnRH antagonists have
attracted considerable attention in recent years specifically to treat
diseases, which require some reduction of GnRH stimulation.5-8
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suggests a Cannizarro-like side reaction.16 The route is shown in
Scheme 1.
Figure 1. Key properties and molecular structure of WAY 207024,
the lead compound for GnRH antagonist development. A (blue oval)
depicts the scaffold body; B (green oval) depicts the moiety of
interest for further structural optimization. IC50 – concentration to
inhibit GnRH binding to the receptor by 50%. hGnRH – human GnRH
receptor. rGnRH – rat GnRH receptor. PSA – Polar surface area in
Å2. CLog P – calculated octanol-water partitioning coefficient.
Figure 1 shows the title scaffold and some relevant properties
discussed herein. As apparent from the high partitioning coefficient
log P, the compound is highly lipophilic, which may hamper
solubility in aqueous formulation as well as non-specific binding of
the compound in vivo. In previous studies, it was found that the
body of the scaffold (blue oval, A) is central to binding selectively
the GnRH receptor, whereas structural modifications in the upper
section of the lead (green oval, B) with planar, hydrogen bonding
functional groups were found to be beneficial for modulating
antagonist potency.
Scheme 1. Synthetic route to alkyne intermediate 7.
A further modification was made relative to literature to
facilitate cleavage of the BOC protecting group. Substitution of the
TFA used originally with HCl in dioxane gave a colourless solid
instead of an oily product, which benefits the scalability of this
route. Notably, compound 4 is fully converted into heterocyclic
products, although the competing formation of 6b in about 8% yield
limits the overall yield of 6a to 75% over two steps. Intermediate 7
We surmised that the 1,3-functionalised triazole moiety would
resemble well the planar geometry required for successful
structural modification and simultaneously allow for introducing a
broad spectrum of structural variations in the last synthetic stage
using one robust reaction. Since the 1,3-functionalised triazole is
obtained via the Cu-catalysed azide-alkyne cycloaddition reaction, was synthesized by alkylation with propargyl bromide in dry DMF
using potassium carbonate as base in 51% yield to furnish the
desired substrate 7 for azide alkyne cycloaddition.
we opted for working with a building block based on A to obtain an
alkyne substrate for the reaction.
The overall design strategy in this communication was to
generate triazoles from different functionalized azides that
would be beneficial for the potency to inhibit the GnRH receptor
while improving the aqueous solubility. We incorporated a
variety of azides via click chemistry, including sugar moieties,
which have been thoroughly demonstrated to effect favourable
solubility characteristics and are discussed extensively in the
literature.12-15 Furthermore, the reduced solubility of the alkyl
and aromatic azides would also counteract our intention to
improve aqueous solubility through reducing clogP (and hence,
the bioavailability and pharmacokinetically desirable profile).
The desired intermediate (7) was synthesised in 19% overall
With a robust and high yielding route to gram amounts of 7 in
hand, further diversification using the copper-catalysed azide-
alkyne cycloaddition (CuAAC) reaction with a range of commercially
available azide substrates became possible. See Scheme 2 for an
overview.
yield over six steps on
a multigram scale. In brief, 2,5-
difluoronitrobenzene was converted to phenylene diamine 4 over
three steps as described previously.9 Contrary to the published
method, we found that the oxidative conversion of 4 into imidazole
6 proceeded smoothly in absence of a transition metal catalyst with
air as the oxidant. The observation of 6b as a minor by-product
Scheme 2. Diversification of the scaffold body using CuAAC.
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Since products 8a-8j were obtained in more than sufficient yields
optimise the reaction were made at this point.
for biological testing following a published protocol, no attempts to
Figure 2. Investigated molecular entities, isolated yields, inhibition potency, inhibition constant and properties computed for evaluation.
For comparison, test results of lead 1 (green box) and positive controls 8k and 8l should be considered. aReference value from literature.17,
18 IC50 values are given as the geometric mean of two experiments, see ESI for details. Ki was calculated from mean value using the Cheng-
Prusoff equation (Ki = IC50/(1+[L]/Kd)).19 cReplicated twice with broader range of concentrations. dBased on single experiment.
Following synthesis and characterisation, inhibition of receptor (8k) and AG04557 (8l) as independent, positive controls and the
activation by LHRH/GnRH was measured in division arrested cell metabolically stable peptide agonist buserelin as negative control.
lines from Multispan Inc. stably expressing functional hGnRH The lead compound WAY207024 (1) was included as a reference in
receptors. As described in the literature, the solubility of 1 and the cell assay. All IC50 values obtained for each test compound in
some of its derivatives is rather poor, which lead to a cut-off at the concentration dependent screening were converted into Ki
5x103 nM for the maximum concentration in the cell culture assay. values using the equation of Cheng and Prusoff.15
Compounds insoluble in DMSO at 10 mM were dropped from
As illustrated in Figure 2, compounds derived from 1 using Cu-
testing. Nonetheless, concentration dependent competition studies AAC were found to exhibit competitive antagonism against the
were performed in vitro in presence of 5 nM LnRH antagonist with endogeneous agonist GnRH. While alkyl piperazines 7a and 7b show
the remainder of derivatives. Compounds showing a pIC50 > 5 were only moderate potency, 21 and 43-fold lower than 1, respectively,
tested again over a wide range of concentrations (logarithmic, 0.1- their Clog P values are lower than that of 1, which results in higher
103 nM). A one-site competitive inhibition model was found to work solubility (>100 nM). In line with our working hypothesis, the
best for fitting of the data curves. To evaluate the obtained introduction of a planar triazole-core has a positive effect on
inhibition potency values and to validate the assay, we included the potency. The 2-fluoroethyl triazole 8a is equally potent as 1,
well-described, commercially available GnRH antagonists T98475 Interestingly the thermodynamic solubility assay showed that 8a is
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‡ Supplementary information available.
1.5 times more soluble than 1, 3 µg/ml at pH 7.4 (Phosphate
buffered saline) and 2.098 mg/mL at pH 1.2 (Simulated Gastric
Fluid) which was indicated by the lower Clog P-value. The
introduction of an N-BOC-Ala functionalised triazole 8b further
improves polarity, albeit at the cost of a drop of one order of
magnitude in potency. While these results may indicate that the
triazole moiety is a useful pharmacophore, its vicinity was found to
be much less tolerant to the introduction of pyranose moieties (8c-
f), which was attempted to improve solubility. While these sugar
derivatives lead to a major improvement of polarity as indicated by
Clog P values between 2 and 5, their potency to inhibit GnRH
binding dropped out of the desired range. Masking the polyol as a
tetraacetate (8c) helped with retaining potency, which does not
bode well for making use of the solubility improvement by
glycosylation. However, when varying the azide component to a
synthetic glycoside to obtain 8g, a viable compromise between
solubility and inhibition potency was obtained. Derivative 8g was
measured to be soluble at pH 1.2, 1.2 mg/ml and <1 µg/ml at pH
7.4. With a potency of 38 nM, barely threefold lower than that of 1,
this hit may create an opening for further exploration using a library
of diverse analogues of 8g. Products 8h-j, obtained from aromatic
and heteroaromatic azides and 7, did not lead to increased
solubility or affinities compared to WAY207024.
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ACKNOWLEDGEMENTS
RF and RS acknowledge the Northern Norway Regional Health Authority for
funding [project number: SFP1196-14]. PJR gratefully acknowledges the
research council of Norway and the University of Oslo (realomics SFI). JHH
gratefully acknowledges the Department of Chemistry at UiT, The Arctic
University of Norway, for funding parts of this research project.
17.
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KEYWORDS
GnRH Receptor, Binding affinities, Alzheimer`s disease
Conflict of Interest
The author declare no competing interests.
Notes and references
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