Non-peptide oxytocin agonists

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Abstract

A library of compounds targeted to the vasopressin/oxytocin family of receptors was screened for activity at a cloned human oxytocin receptor using a reporter gene assay. Potency and selectivity were optimised to afford compound 39, EC50 = 33 nM. This series of compounds represents the first disclosed, non-peptide, low molecular weight agonists of the hormone oxytocin (OT).

Graphical

The first non-peptide, low molecular weight agonists of the hormone oxytocin (OT) are reported. The most potent compound, 39, showed an EC50 = 33 nM and was selective for the OT receptor.

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Introduction

Oxytocin (OT) is a cyclic nonapeptide that acts at the OT receptor, a member of the super-family of G-protein coupled receptors (GPCRs). OT and the closely related hormone arginine vasopressin (AVP) are neurohypophyseal hormones secreted by the posterior pituitary gland.1 AVP acts at the vasopressin receptor of which three subtypes, V1a, V1b and V2 have so far been identified. The OT receptor is approximately 10-fold selective for OT over AVP whereas the vasopressin receptor subtypes show stronger selectivity, up to 400-fold, in favour of AVP.[2], [3] Within the periphery the OT receptor is localised in a number of different organs including the uterus and mammary glands. In the uterus it is involved in the onset and progress of labour. As such it has been shown that OT antagonists have utility in the treatment of pre-term labour and the peptide OT antagonist atosiban (Tractocile) is marketed as a treatment for this indication.4 The OT receptor has also been localised in central tissues such as the paraventricular nucleus where it is involved in regulation of both male and female sexual response.[1], [5] It is clear that OT has a range of physiological roles that have not been fully elaborated. Thus there is a need for new, potent, selective and efficacious OT receptor agonists that may be used as pharmacological tools and as potential drugs. Furthermore, the peptide vasopressin and oxytocin analogues such as carbetocin (Duratocin) are poorly absorbed due to their high hydrophilicities.6 Thus there is a need to develop non-peptide compounds that may have potential to be orally bioavailable. These may have utility as drugs where OT function is compromised, in particular in the treatment of various sexual disorders including male erectile dysfunction. Also in promoting labour, controlling post-partum bleeding and increasing milk let-down.

A number of non-peptide antagonists of OT have been discovered.[7], [8], [9] However, it is generally perceived to be more difficult to identify non-peptide agonists, principally because of the much reduced molecular size compared to the parent peptide hormone.[10], [11] However, non-peptide agonists of the V2 receptor have already been reported by us (compound 1), as well as by Wyeth-Ayerst (WAY-VNA-932, 2) and Otsuka (OPC-51803, 3), the structures of which are shown in Figure 1.[12], [13], [14] Given the close relationship between OT and AVP, we hypothesised that a library designed around V2 agonists would yield OT agonists. To test this hypothesis these V2 agonists were assayed for their OT activity in a functional reporter gene assay, vide infra. Compounds 2 and 3 showed <10% response at 3 μM. However, 1 gave a response relative to OT of 33% at 3 μM. Therefore, a library of 50,000 structures related to 1 was screened and the resulting OT agonists are described in this paper.

Section snippets

Chemistry

The compounds related to 1 were prepared through the synthesis of ‘building blocks’, which were coupled together at the benzoyl-amide and at the urea as shown schematically in Scheme 1.

The synthesis of the top groups is shown in Scheme 2, Scheme 3. The building block 5,6,7,8-tetrahydro-4H-thieno[3,2-b] azepine, 6, present in compounds 2838 was prepared via a DIBAL mediated Beckmann rearrangement from commercially available 4-keto-4,5,6,7-tetrahydrothianaphthene 4.15

The cyclisation of 9 that

Biology

The human OT and V2 receptors were cloned from ovary total RNA and kidney total RNA (BD Clontech) using the reverse transcriptase polymerase chain reaction (RT-PCR). The receptors were subcloned into the KpnI and EcoRI sites of pcDNA3. The cAMP response element (CRE) and the nuclear factor of activated T-cells (NFAT) element was subcloned into the BamHI and BglII sites of pGL3 promoter (Promega), which contains the Firefly Luciferase gene. Plasmids were amplified in Novablue E. coli (Merck

Results and discussion

Screening of our own in-house assembled vasopressin targeted library for OT activity identified two hits, 27 and 28 (Fig. 2), which were related to 1 (Fig. 1). Common to all three compounds was the substituted benzoyl linker and a bicyclic or tricyclic fused azepine as a top group. A urea functionality attached to an aryl ring or an amino acid amide formed the bottom group. The relative responses compared to OT of these hit compounds, determined at a concentration of 3 μM, were 41% for 27 and

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