Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M

doi:10.1016/S0968-0896(99)00107-8

Bioorganic & Medicinal Chemistry

Volume 7, Issue 8, August 1999, Pages 1539-1547

https://doi.org/10.1016/S0968-0896(99)00107-8 Get rights and content

Abstract

Two subseries of nonquaternized Figure 1, Figure 2, Scheme 2

and quaternized derivatives Figure 1, Figure 2, Scheme 2 related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M_l rabbit vas deferens, M₂ guinea pig left atrium and M₃ guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative Figure 1, Figure 2, Scheme 2, which behaved as a mixed M₃-agonist/M_l-antagonist (pD₂=5.85; pA₂=4.76, respectively). In in vivo tests nonquaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

Introduction

The muscarinic receptor population represents a class of cholinergic receptors abundant in the parasympathetic and in the central nervous systems, where they mediate both excitatory and inhibitory effects.¹ At present, the muscarinic receptor pharmacological classification distinguishes among M₁, M₂, M₃, and M₄ subtypes. A fifth subtype, termed m₅, has been predicted by molecular cloning studies, even though its physiological role is at present missing.² The characterization of these multiple muscarinic receptors came from the availability of ligands, primarily antagonists, able to selectively interact with one of the subtypes.3, 4, 5, 6 In the last decade a number of potent and selective muscarinic antagonists has been successfully designed.7, 8, 9, 10, 11 However, current interest is also addressed to the research of agonists, since the development of new highly potent and subtype-selective muscarinic receptor agonists could provide both improved experimental tools and novel therapeutic agents, useful, for example, in the treatment of pain and Alzheimer's disease.12, 13

In the course of previous studies on the structure–activity relationships of muscarinic ligands structurally related to natural muscarine 1,14, 15, 16, 17, 18 we synthesized the highly effective agonist azamuscarone 2 (Fig. 1), which proved to be as potent as the parent compound at guinea pig, atrial and ileal muscarinic sites.14, 15 More recently, we prepared derivatives Figure 1, Scheme 2 (Fig. 1) by incorporating the isoxazolidin-3-one moiety of 2 into the skeleton of the potent yet nonselective muscarinic agonists oxotremorine (3) and oxotremorine-M (4).¹⁹ Compounds Figure 1, Scheme 2 displayed different binding affinities at brain and heart muscarinic subtypes.¹⁹

Based on these preliminary results, a larger set of oxotremorine/oxotremorine-M-like derivatives has been designed by modifying further both the nature of the heterocyclic ring and the attachment point of the acetylenic side chain. Accordingly, in addition to the isoxazolidin-3-one derivatives Figure 1, Scheme 2, we prepared and tested the regioisomeric Δ²-isoxazolinyl-ethers Figure 1, Scheme 2 and the related isoxazolyl-ethers Figure 1, Figure 2, Scheme 2 (Fig. 1).

This paper deals with the synthesis of derivatives Figure 1, Figure 2, Scheme 2 and the evaluation of their pharmacological profile by classical in vitro functional assays, in order to estimate their agonist potency at muscarinic M_l, M₂, and M₃ receptor subtypes. In addition, we investigated the ability of the compounds under study to produce tremor in mice after ip administration. Such a symptom can be taken as a measure of the activity of the compounds at central muscarinic receptors.²⁰

Section snippets

Chemistry

Target compounds were synthesized along the reaction sequences reported in Scheme 1, Scheme 2. Isoxazolidin-3-one 13 was conveniently prepared by converting known 3-nitro-Δ²-isoxazoline 11²¹ into the corresponding 3-benzyloxy derivative 12, followed by catalytic hydrogenolysis (Scheme 1). Electrophilic addition of 1,4-dichloro-2-butyne to 13, carried out in refluxing acetone in the presence of K₂CO₃, produced a 4:1 mixture of regioisomers Scheme 1, Scheme 2 (Scheme 1). The N- and O-alkynyl

Results and Discussion

The pharmacological evaluation of the agonist potency and selectivity of compounds Figure 1, Figure 2, Scheme 2 towards M_l, M₂, and M₃ muscarinic receptors was performed in vitro on three suitable preparations. Rabbit electrically-stimulated vas deferens, guinea pig electrically-driven left atrium and guinea pig ileum were used, respectively, since in each preparation the functional response is primarily due to the activation of a single receptor population subtype.24, 25, 26 Oxotremorine and

Conclusion

The overall data of the present investigation evidenced that the new oxotremorine/oxotremorine-M-like analogues retained variable muscarinic agonist activity with respect to the parent agonists. The most interesting results, in terms of potency or selectivity, were achieved when the pyrrolidin-2-one ring was replaced either by the Δ²-isoxazoline or the isoxazole moieties, with a concomitant shift of the acetylenic side chain. The first modification produced trimethylammonium derivative 8b,

Material and methods

¹H NMR spectra were recorded with a Bruker AC-E 200 (200 mHz) spectrometer in CDCl₃ solutions (unless otherwise indicated); chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hertz. TLC analyses were performed on commercial silica gel 60 F₂₅₄ aluminum sheets; spots were further evidenced by spraying with a dilute alkaline potassium permanganate solution. Melting points were determined with a Büchi Mod.B 540 apparatus and are uncorrected. Liquid compounds were characterized by

Acknowledgements

We thank G. Domenichini for his excellent technical assistance. Financial support from MURST (Ministero della Ricerca Scientifica e Tecnologica), Rome is gratefully acknowledged.

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However, selectivity of oxotremorine and gallamine for the M2 receptor at 20 µM and 100 µM, respectively, seems rather unlikely. Indeed, low selectivity for oxotremorine between M1, M2, and M3 receptors has been observed earlier in classical in vitro functional assays (Dallanoce et al., 1999) while in cells expressing the 5 mAChR subtypes gallamine has a pKi of 7.4 for M2 receptors, displaying 30–360 fold selectivity over other muscarinic receptor subtypes (Dong et al., 1995). There are no truly selective antagonists for M3 receptors, in particular over M4-M5 receptors.
Acetylcholine can modulate hippocampal network function through activation of both nicotinic and muscarinic acetylcholine receptors (mAChRs). All five mAChR subtypes have been identified in the hippocampus. Besides by their involvement in excitability of hippocampal cells, synaptic plasticity and memory, a large body of research has demonstrated the involvement of presynaptic mAChRs in the inhibition of glutamatergic transmission in the hippocampus. Over the years, however, pharmacological and molecular genetic studies have yielded quite contradictory results regarding the mAChR subtype(s) involved. In this study, multi-electrode array technology was used for the pharmacological elucidation of the subtype of mAChR mediating the depression of excitatory synaptic transmission at the SC-CA1 synapse. Using selective antagonists (VU0255035, MT7, tripinamide, MT3) and allosteric potentiators (VU 10010, VU 0238429) the involvement of M₁, M₂, M₄, and M₅ subtypes was ruled out thereby implying a major modulatory role for M₃ receptors in the inhibition of excitatory synaptic transmission in area CA1 of rat hippocampus.
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Since M1 and M3 receptors use the same intracellular signaling pathway, the activation of all of these receptors might not be needed to produce a full response. ACh and OxoM are known to be non-selective full muscarinic agonists (Dallanoce et al., 1999). In the presents study, these two agonists induced inward currents with similar affinity and potency.
Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of −60 mV. Removal of extracellular Ca²⁺ markedly increased the OxoM-induced current (I_OxoM). The inward I_OxoM in the absence of extracellular Ca²⁺ was fully inhibited by removal of extracellular Na⁺, indicating the involvement of non-selective cation channels. The I_OxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The I_OxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M₁ receptor, produced partial inhibition of the I_OxoM. In the presence of MT-7, concentration–inhibition curve of the M₃-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M₁ and M₃ receptors to the OxoM response. The I_OxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP₃ receptor blocker xestospongin C also inhibited the I_OxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the I_OxoM, while KN-62, a blocker of Ca²⁺/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca²⁺ from intracellular Ca²⁺ stores and calmodulin.
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¹: Second corresponding author. Tel.: +39-0521-905090; fax: +39-0521-905091; e-mail: [email protected]

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Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M

Abstract

Introduction

Section snippets

Chemistry

Results and Discussion

Conclusion

Material and methods

Acknowledgements

Pharmacol. Therap.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Life Sci.

Life Sci.

Life Sci.

Eur. J. Med. Chem.

Bioorg. Med. Chem. Lett.

Tetrahedron

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Pharmacol. Toxicol.

Br. J. Pharmacol.

Nature

Trends Pharmacol. Sci.

Clin. Pharmacol. Therap.