Investigation of pyrazolo-sulfonamides as putative small molecule oxytocin receptor agonists

Article abstract of DOI:10.1016/j.ejmech.2017.05.013

The neuropeptide oxytocin has been implicated in multiple central nervous system functions in mammalian species. Increased levels have been reported to improve trust, alleviate symptoms related to autism and social phobias, and reduce social anxiety. Hoffman-La Roche published a patent claiming to have found potent small molecule oxytocin receptor agonists, smaller than the first non-peptide oxytocin agonist reported, WAY 267,464. We selected two of the more potent compounds from the patent and, in addition, created WAY 267,464 hybrid structures and determined their oxytocin and vasopressin receptor activity. Human embryonic kidney and Chinese hamster ovary cells were used for the expression of oxytocin or vasopressin 1a receptors and activity assessed via IP1 accumulation assays and calcium FLIPR assays. The results concluded that the reported compounds in the patent and the hybrid structures have no activity at the oxytocin or vasopressin 1a receptors.

Full text of DOI:10.1016/j.ejmech.2017.05.013

Accepted Manuscript
Investigation of pyrazolo-sulfonamides as putative small molecule oxytocin receptor
agonists
Timothy A. Katte, Tristan A. Reekie, Eryn L. Werry, William T. Jorgensen, Rochelle
Boyd, Erick C.N. Wong, Damien W. Gulliver, Mark Connor, Michael Kassiou
PII:
S0223-5234(17)30374-4
10.1016/j.ejmech.2017.05.013
EJMECH 9443
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 December 2016
Revised Date: 2 May 2017
Accepted Date: 3 May 2017
Please cite this article as: T.A. Katte, T.A. Reekie, E.L. Werry, W.T. Jorgensen, R. Boyd, E.C.N.
Wong, D.W. Gulliver, M. Connor, M. Kassiou, Investigation of pyrazolo-sulfonamides as putative small
molecule oxytocin receptor agonists, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.013.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Investigation of Pyrazolo-Sulfonamides as Putative Small Molecule
Oxytocin Receptor Agonists
Timothy A. Katte,a Tristan A. Reekie,a Eryn L. Werry,b William T. Jorgensen,a Rochelle
Boyd,c Erick C.N. Wong,d Damien W. Gulliver,d Mark Connor,c Michael Kassiou*a
a School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
b Faculty of Health Sciences, The University of Sydney, Sydney, NSW 2006, Australia
c Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie
University, NSW 2109, Australia
d School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, Australia
KEYWORDS Oxytocin agonists, sulfonamide, small molecule
Abstract
The neuropeptide oxytocin has been implicated in multiple central nervous system functions
in mammalian species. Increased levels have been reported to improve trust, alleviate
symptoms related to autism and social phobias, and reduce social anxiety. Hoffman-La
Roche published a patent claiming to have found potent small molecule oxytocin receptor
agonists, smaller than the first non-peptide oxytocin agonist reported, WAY 267,464. We
selected two of the more potent compounds from the patent and, in addition, created WAY
267,464 hybrid structures and determined their oxytocin and vasopressin receptor activity.
Human embryonic kidney and Chinese hamster ovary cells were used for the expression of
oxytocin or vasopressin 1a receptors and activity assessed via IP1 accumulation assays and
calcium FLIPR assays. The results concluded that the reported compounds in the patent and
the hybrid structures have no activity at the oxytocin or vasopressin 1a receptors.
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Introduction
Oxytocin (OT) is a nine amino acid cyclic peptide with well-known neuromodulatory
activity in complex social behaviour. Increased levels have been reported to improve trust,
alleviate symptoms related to autism and social phobias, and reduce social anxiety.[1]
Problems with the use of OT as a therapeutic are related to is peptidic nature, including low
oral bioavailability, short half-life, and poor blood brain barrier penetration. In addition, it
displays cross-talk with arginine vasopressin (AVP) receptors.[2]
Interest in the oxytocin receptor (OTR) as a potential therapeutic is reinforced by the recent
patent by Hoffman-La-Roche.[3] This patent outlined a series of non-peptidic compounds
based on a 5-sulfonamidopyrazole scaffold. The patent contained 26 compounds with
heteroaromatic variations on the N1 position of the pyrazole along with various
functionalized phenyl rings on the sulfonamide moiety. The exemplary drug like properties of
the pyrazolo sulphonamides, coupled with their high OTR potency validates their therapeutic
potential.
We chose to synthesize and explore the activity of two of the more potent molecules from
this patent, 2a and 2b (Figure 1). EC50 values were reported to be 19 nM for 2a and 8 nM for
2b at the human oxytocin receptor (hOTR) as determined by Fluorometric Imaging Plate
Reader (FLIPR) measurement of calcium influx. Such was the interest in this report that it
was detailed in a patent highlight.[4] Considering the remarkable activity achieved for these
compounds, and favorable physicochemical properties, we sought to investigate them further
and compare them to observations made with the reported non-peptidic agonist, WAY
267,464 (1). After detailing improved methods of synthesis of WAY 267,464 [5-7] further
studies showed that rather than being a selective OTR agonist, it was an AVP receptor
antagonist, particularly at the vasopressin 1a receptor (V1aR), (Ki of 27 nM and 230 nM for
V1aR and OTR respectively)[8] and that this antagonism was predominately responsible for
the observed in vivo effects.[9-11] Further structure-activity relationship and truncation
studies have shown the importance of the rigid aromatic linker and the resorcinol moiety for
selectivity.[8] Comparing compound 1 with sulfonamides 2 shows that in addition to the
acyclic vs. cyclic structure, both contain the 5-aminopyrazole moiety that differ in N1-
substitution, (pyridyl vs. methyl), and 5-amino substitution, (sulfonamide vs. benzyl). We
aimed to synthesize lead compounds 2a-b in addition to the hybrid structures 3a-b and
evaluate their activity at the hOTR and hV1aR.
Figure 1. Lead compounds for OTR binding and targeted analogues.
Synthesis
The synthesis of target compounds 2a-b and 3a-b began with pyrazole formation between
acrylate 4 and either 2-hydrazinopyridine or methylhydrazine to form compounds 5 and 6
respectively. Saponification and decarboxylation gave the 5-aminopyrazoles 7 and 8.
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Sulfonamide formation with the requisite sulfonyl chloride afforded lead compounds 2a-b
and their methyl analogues 3a-b which were then subjected to biological testing.
Scheme 1: Synthesis of 2a-b and 3a-ba
aConditions: a) methylhydrazine (1.1 equiv.) or 2-hydrazinopyridine (1.1 equiv.), ethanol,
reflux, 2 h, 68-73%; b) 1) 3 M aqueous NaOH (1.3 equiv.), ethanol, reflux, 2 h; 2) 185 °C,
85-87%; c) appropriate sulfonyl chloride (1.0 equiv.), pyridine, rt, 6 h, 23-34%.
Results and Discussion
We evaluated these compounds using HEK cells transfected with the hOTR for binding
and, both agonism (Figure 2) and antagonism (Table 1). An inositol monophosphate (IP1)
accumulation assay was used, which has been successfully employed previously for OTR
functional studies.[8]
Competitive binding assays run on compounds 2a–b and 3a–b revealed a lack of binding,
with an inhibition constant (Ki) of greater than 10 µM for all compounds at both the hOTR
and the hV1aR. The IP1 values obtained for compounds 2a–b and 3a–b at concentrations of
100 nM and 10 µM showed no difference to those obtained from the negative control (basal)
(Figure 2). Due to the patent compounds being evaluated on CHO cells, we sought to verify
that the HEK cells result in an accurate measurement of OT activity. It was found that OT
effectively stimulated IP1 release in transfected cells in a manner that was able to be inhibited
by the OTR antagonist atosiban (IC50: 372 ± 97 nM), and OT did not elicit IP1 release in wild
type HEK cells, suggesting its effect was specifically through action on the hOTR (Figure 2).
Figure 2. IP1 values obtained from treatment of HEK cells transfected with the hOTR with
2a–b, 3a–b (100 nM, 10 µM), vehicle (basal) and OT (10 nM (~EC50)), and values obtained
from treatment of non-transfected HEK cells (WT) with vehicle and OT (10 nM). Values are
mean ± standard error of the mean (S.E.M.) ***p<0.001
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As these compounds did not stimulate IP1 release on their own, we investigated whether
they could act as positive allosteric modulators by increasing the IP1 levels stimulated by an
~EC10 (3 nM) concentration of OT. They did not increase the response of the cells to OT,
and in fact, at a high concentration (10 µM) a number of these compounds significantly
decreased the response of OT (Figure 3). The concentration of OT used in Figure 2 is too low
to allow conclusion about whether these compounds act as antagonists, so for this reason, we
investigated whether they acted as antagonists by examining their effect on a concentration
usually used in antagonist studies (EC70). Data obtained for all compounds clearly showed
no antagonism at the hOTR (Table 1).
Figure 3. IP1 values obtained from treatment of HEK cells transfected with the hOTR with an
~EC10 concentration of OT (3 nM) with vehicle (0.1% DMSO) or 2a–b (10 µM) and 3a–b
(10 µM). Values are mean ± S.E.M. *p<0.05, **p<0.01 compared to OT + vehicle.
Table 1. Antagonism in hOTR-transfected HEK cells
2a
2b
3a
3b
Mean % at 100 nM compounda 89.5 ± 4.2 70.6 ± 6.2 82.5 ± 2.8 98.3 ± 3.3
Mean % at 10 µM compounda
78.1 ± 4.1 65.5 ± 1.5 72.5 ± 7.6 90.4 ± 2.7
pIC50
<5
<5
<5
<5
a
HEK cells transfected with the hOTR were exposed to an ~EC70 concentration of OT (30
nM) with compounds. Measured levels of IP1 were expressed as a percentage of the levels
stimulated by 30 nM OT with a DMSO (0.1%) control. Experiments were repeated in
triplicate with the mean values ± standard S.E.M;
We have previously shown that the reported hOTR agonist activity attributed to 1 was in
fact antagonism of the V1aR [9-11] so we sought to investigate that possibility here.
Additionally, we were concerned by the statement in the patent asserting, “The
compounds…were also functionally tested on cell lines expressing the human vasopressin 1a
and the human vasopressin 2 receptor to measure potential agonist activity and were found to
be selective over the human oxytocin receptor.” This statement seemed to be at odds with the
title and theme of the patent, namely that the compounds were OTR agonists, however no
clear description was provided as to which receptor, hOTR, hV1aR, or hV2R, functional data
provided was for. To clarify the position the same functional experiments were conducted at
the hV1aR (Figure 4 and Table 2). Results clearly show that these compounds display no
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agonism at the hV1aR (Figure 4). Results also conclusively showed no antagonism at the
hV1aR (Table 2).
Figure 4. IP1 values obtained from treatment of HEK cells transfected with the hV1aR with
2a–b, 3a–b (100 nM, 10 µM), basal levels and AVP (10 nM). Values are mean ± S.E.M.
***p<0.001
Table 2. Antagonism in hV1aR-transfected HEK cells
2a
2b
3a
3b
Mean % at 100 85.9 ± 9.3
nM compounda
109.1 ± 4.9
112.0 ± 5.7
84.7 ± 1.2
Mean % at 10 80.2 ± 0.8
104.0 ± 1.9
105.2 ± 6.2
77.9 ± 2.8
µM compounda
pIC50
<5
<5
<5
<5
a
HEK cells transfected with the hV1aR were exposed to an ~EC70 concentration of AVP (25
nM) with compounds. Measured levels of IP1 were expressed as a percentage of the levels
stimulated by 25 nM AVP with a DMSO (0.1%) control. Experiments were repeated in
triplicate with the mean values ± S.E.M;
The discrepancy in our results and those reported by the patent for the same compounds
were of concern and so we attempted to determine their source. The most notable difference
in experimental procedure was the use of an IP1 assay as opposed to a Fluorometric Imaging
Place Reader (FLIPR) Ca2+ flux assay. Therefore, we tested compounds 2a-b and 3a-b using
the same assay. None of the compounds at 100 nM or 10 µM produced an increase in
intracellular Ca2+ ([Ca]i), in HEK cells expressing hOTR or hV1aR. Pre-incubation of cells
with 2a-b and 3a-b (10 µM) for 5 minutes did not significantly affect the potency of OT or
AVP to elevate [Ca]i, in cells expressing their cognate receptor, nor was the maximum effect
of either OT or AVP changed (Table 3).
Table 3. Effects on [Ca]i
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hOTR
hV1aR
Max
pEC50
Max
pEC50
in-vehicle
9.9 ± 0.1
10.1 ± 0.1
10 ± 0.1
4.5 ± 0.1
4.5 ± 0.2
4.4 ± 0.2
4.0 ± 0.2
4.4 ± 0.2
9.8 ± 0.1
9.9 ± 0.1
9.8 ± 0.05
9.9 ± 0.1
9.8 ± 0.1
5.2 ± 0.2
5.3 ± 0.1
5.3 ± 0.1
5.0 ± 0.2
5.1 ± 0.2
2a
2b
3a
3b
10.1 ± 0.1
10.0 ± 0.1
HEK cells expressing hOTR were exposed to OT and cells expressing hV1aR were exposed to
AVP after 5-minute pre-incubation with compounds (10 µM) or vehicle (0.1 % DMSO).
Peak changes in Ca5 fluorescence, reflecting increases in [Ca]i, were calculated as a fold-
change compared to baseline, and a 4-parameter concentration response relationship fitted to
the data to obtain a pEC50 in each condition. Max is a fold change in fluorescence over the
basal measurement of calcium ions in the cell. Experiments were performed in duplicate with
5-6 independent replicates. Values represent the mean ± S.E.M.
A further point of difference between our methods and those used in the patent related
to the cell line in which the OTR and V1aR were expressed. Our experiments utilized
transfected HEK cells, while those in the patent were performed on transfected Chinese
hamster ovary (CHO) cells. To eliminate this as a source of difference, we established stably
transfected CHO cells expressing the hOTR. None of the compounds at 100 nM or 10 µM
produced an increase in intracellular Ca2+ ([Ca]i), and no significant agonist or antagonist
activity at the OTR was seen in the IP1 assay performed on these cells (Figure 5, Table 4).
Figure 5. IP1 values obtained from treatment of CHO cells transfected with the hOTR with
2a–b, 3a–b (100 nM, 10 µM), vehicle (basal) and OT (10 nM). Values are mean ± S.E.M.
***p<0.001
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Table 4. Antagonism in hOTR-transfected CHO cells
2a
2b
3a
3b
Mean % at 100 nM compounda 83.9 ± 9.9 83.2 ± 6.4 90.5 ± 8.9 85.5 ± 4.0
Mean % at 10 µM compounda
71.1 ± 3.6 82.1 ± 4.2 94.7 ± 6.7 79.5 ± 6.4
pIC50
<5
<5
<5
<5
a
CHO cells transfected with the hOTR were exposed to an ~EC70 concentration of OT (30
nM) with compounds. Measured levels of IP1 were expressed as a percentage of the levels
stimulated by 30 nM OT with a DMSO (0.1%) control. Experiments were repeated in
triplicate with the mean values ± standard error of the mean (S.E.M).
To also examine the contribution of cell background to the differing reports of activity
of these compounds at the V1aR, we established stably transfected CHO cells expressing the
hV1aR. None of the compounds at 100 nM or 10 µM produced an increase in intracellular
Ca2+ ([Ca]i), and the compounds did not demonstrate agonist or antagonist activity at the
hV1aR in the IP1 assay performed on these cells (Figure 6, Table 5). These results suggest
that differences in cell line between the current study and the results reported in the patent
literature are unlikely to be the cause of the loss of function at the hOTR and hV1aR in this
study.
Figure 6. IP1 values obtained from treatment of CHO cells transfected with the hOTR with
2a–b, 3a–b (100 nM, 10 µM), vehicle (basal) and AVP (10 nM). Values are mean ± S.E.M.
***p<0.001
Table 5. Antagonism in hV1aR-transfected CHO cells
2a
2b
3a
3b
Mean % at 100 nM compounda 103.2 ± 2.5 84.8 ± 4.3 100.1 ± 3.8 86.5 ± 1.6
Mean % at 10 µM compounda
116.1 ± 6.7 83.1 ± 4.0 105.2 ± 1.7 79.3 ± 0.7
pIC50
<5
<5
<5
<5
a CHO cells transfected with the hV1aR were exposed to an ~EC70 concentration of AVP (25
nM) with compounds. Measured levels of IP1 were expressed as a percentage of the levels
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stimulated by 30 nM OT with a DMSO (0.1%) control. Experiments were repeated in
triplicate with the mean values ± standard error of the mean (S.E.M).
Conclusion
The results from this work have shown that the previously reported compounds (2a
and b) and their corresponding hybrid structures (3a and b) have no notable activity at the
hOTR. Furthermore, their activity at the hV1aR was assessed and analogous results were
obtained. These results have been obtained in both CHO and HEK cells, and from both IP1
and FLIPR Ca2+ assays to determine functional activity. The FLIPR Ca2+ assays on CHO
cells recapitulate the conditions in which the patent results were generated, whilst the results
on HEK cells, and in the IP1 assay, acts to compliment the FLIPR assay to ensure activity of
these compounds is accurately measured. Therefore, these compounds are not functionally
active at the hOTR or hV1aR.
ASSOCIATED CONTENT
Supporting Information
1
Experimental procedures, H and 13C NMR spectra, HPLC purity analysis and additional
biological data (PDF).
AUTHOR INFORMATION
Corresponding Author
* michael.kassiou@sydney.edu.au
Funding Sources
Work performed was supported by the NHMRC.
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Highlights
•
•
•
•
Patent compounds and new hybrids were synthesized
Showed no binding or activity at the oxytocin receptor contradicting patent
Further investigated in vasopressin which also lacked activity
Concluded that these are not viable oxytocin agonists