Elsevier

Steroids

Volume 104, December 2015, Pages 284-293
Steroids

Synthesis of novel 13α-18-norandrostane–ferrocene conjugates via homogeneous catalytic methods and their investigation on TRPV1 receptor activation

https://doi.org/10.1016/j.steroids.2015.10.016Get rights and content

Highlights

  • New 13α-18-norandrostane–ferrocene conjugates were synthesized.

  • Ferrocene containing triazoles were obtained via azide–alkyne cycloaddition.

  • Pd-catalyzed aminocarbonylation was investigated with ferrocenylmethylamine.

  • X-ray structures of three compounds were determined.

  • An effect on the activation of TRPV1 receptor on TRG neurons was observed.

Abstract

13α-Steroid–ferrocene derivatives were synthesized via two reaction pathways starting from an unnatural 16-keto-18-nor-13α-steroid. The unnatural steroid was converted to ferrocene derivatives via copper-catalyzed azide–alkyne cycloaddition or palladium-catalyzed aminocarbonylation. 16-Azido- and 16-N-(prop-2-ynyl)-carboxamido-steroids were synthesized as starting materials for azide–alkyne cycloaddition with the appropriate ferrocene derivatives. Based on our earlier work, aminocarbonylation of 16-iodo-16-ene and 16-iodo-15-ene derivatives was studied with ferrocenylmethylamine. The new products were obtained in moderate to good yields and were characterized by 1H and 13C NMR, IR and MS. The solid state structure of the starting material 13α-18-norandrostan-16-one and two carboxamide products were determined by X-ray crystallography. Evidences were provided that the N-propargyl-carboxamide compound as well as its ferrocenylmethyltriazole derivative are able to decrease the activation of TRPV1 receptor on TRG neurons.

Introduction

The ferrocenyl derivatives of steroids could be interesting because of their potential application in materials science, analytical chemistry and medicine.

The functionalization of cholesterol with ferrocene led to liquid–crystalline materials and redox-controlled aggregation of these compounds was studied [1]. A cholesteryl glycinate ferrocenoylamide was prepared as a low-molecular-mass gelator. The chemical oxidation of the ferrocenyl unit led to a phase transition from gel state to solution state [2].

A labeling method with ferrocene has been developed for the detection enhancement of steroids after HPLC separation using electrochemical or UV detectors [3], [4]. On the other hand, the derivatization could be an alternative method to enhance the electrospray-mass spectrometry performance. Earlier studies proved that the detection was specific and the derivatization enhanced the ionization efficiencies of steroids [5].

Relative binding affinities of 17α-(ferrocenylethynyl)estradiol were determined for natural estrogen receptors. It was found that the modified hormone retained its affinity for the two subtypes of the estrogen receptor [6]. Some 16α- and 16β-(ferrocenylmethyl)amino-estratrienes showed broad antimicrobial activity [7]. Other steroid–ferrocene derivatives exhibited antiproliferative effect [8], [9].

Although there are a lot of examples about the derivatization of natural steroids, to the best of our knowledge, there has been no report on labeling unnatural steroids with ferrocene.

Unnatural 13α-steroids have different shape compared to the natural steroids because of the cis junction between rings C and D [10]. 16,17-Substituted 13α-steroids with 17α-substituents were investigated by X-ray and NMR measurements. It was found that ring C possessed chair conformation while ring D with restricted conformation was forced above the plane of the steroid [11]. These structural characteristics could change possible interactions with the receptors and consequently, could modify biological activity. For example, for optimal GABAA receptor modulation, the hydrogen bond-accepting substituent should be above the plane of the steroid rings [12]. Therefore, 13α-steroids bearing such substituents in ring D seems to be ideal candidates for such applications.

During the present work, two different synthetic strategies were developed to introduce nitrogen-containing groups together with a ferrocene label to a 18-nor-13α-steroid. The first route involved a copper-catalyzed azide–alkyne cycloaddition that had been used efficiently for the synthesis of natural steroid–ferrocene conjugates in our research group before [13].

The other methodology, the palladium-catalyzed aminocarbonylation had been applied before for the introduction of carboxamido groups to C-17 position of 13α-steroids [14]. In our recent work, a 16-keto-18-nor-13α-steroid was transformed to alkenyl-iodides to serve as starting materials in aminocarbonylation reaction with simple amines [15]. In this paper, investigations concerning the conversion of the same substrates to ferrocene-containing carboxamides are presented.

Several endogenous steroids are able to bind and inhibit the Transient Receptor Potential Vanilloid 1 (TRPV1) cation channel. TRPV1 is important regulator of nociceptive and inflammatory processing. Besides the two classical vanilloid type agonists, capsaicin and resiniferatoxin, TRPV1 is activated by several highly lipophilic compounds as endogenous arachidonic acid or other fatty acid metabolites. Protons (pH < 6.0) and noxious heat (>43 °C) are also able to open this channel [16], [17]. Dehydroepiandrosterone (DHEA), a major blood steroid, can inhibit capsaicin-evoked currents in dorsal root ganglion neurons [18]. It is not clear if steroids bind directly to the capsaicin-binding domain of the TRPV1 receptor or if they are allosteric modulators of TRPV1. Since the relation between some steroid-type compounds and TRPV1 receptor inhibition, we investigated the effect of our steroid compounds on TRPV1 receptor activation in in vitro cell culture of trigeminal ganglion (TRG) neurons.

Section snippets

General methods

1H and 13C NMR spectra were recorded in CDCl3 on a Varian Inova 400 spectrometer at 400.13 MHz and 100.62 MHz, respectively. Chemical shifts (δ) are reported in ppm relative to CHCl3 (7.26 and 77.00 ppm for 1H and 13C, respectively). GC–MS of compounds 3a, 3b and 4 was recorded on a HP-5971A MSD connected to a HP-5890/II gas chromatograph, while those of steroids 10a and 10b were measured on a Shimadzu GCMS-QP2010 SE instrument. Samples 2a, 7a, 12, 14a and 14b were analyzed using triple quadruple

Synthesis of steroid–ferrocene conjugates via azide–alkyne cycloaddition

The azide–alkyne cycloaddition has been proved to be a versatile methodology to connect ferrocene to molecules with diverse structures. It was also used efficiently for the functionalization of steroids by us and others [13], [26], [27], [28], [29], [30], [31], [32]. The steroid component may play the role of either of the two reaction partners. To clarify the efficiency of both methodologies, reaction routes towards azido- and alkynyl derivatives of a 13α-18-nor steroid were developed.

A

Conclusions

Ferrocene labelling of the 13α-18-nor-androstane skeleton was attempted by an azide–alkyne cycloaddition. The synthetic route led to inseparable mixtures of 16α and 16β epimers in each step. At the same time, the ferrocene label can easily be introduced into position 16 of 13α-15-enes with the help of carbonylation reactions either via an aminocarbonylation–azide–alkyne cycloaddition sequence or via aminocarbonylation of the iodo derivative with aminomethylferrocene. The two methods result in

Acknowledgments

The support of the Hungarian National Science Foundation (OTKA K105632, K113177) and project TÁMOP-4.2.2.B-15/1/KONV-2015-0004, realized with the support of the Hungarian Government and the European Union, with the co-funding of the European Social Fund, is acknowledged. This work was sponsored by Hungarian Brain Research Program (grant KTIA_NAP_13-2-2014-0022 MTA-PTE NAP B Chronic Pain Research Group). É. Sághy was supported by Richter Tálentum Alapítvány. Mrs. B. Norberg is acknowledged for

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