Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2011 Sep 17;2(11):866–869. doi: 10.1021/ml200183z

Identification of a Potent and Selective Cannabinoid CB1 Receptor Antagonist from Auxarthron reticulatum

Mahmoud Fahmi Elsebai †,∥, Viktor Rempel §, Gregor Schnakenburg ‡, Stefan Kehraus †, Christa E Müller §,*, Gabriele M König †,*
PMCID: PMC4017988  PMID: 24900275

Abstract

graphic file with name ml-2011-00183z_0001.jpg

The fungus Auxarthron reticulatum derived from the marine sponge Ircinia variabilis produced the diketopiperazine alkaloid amauromine (1) and the quinolinone methyl-penicinoline (2). Compound 2 is identical to the reported methyl-marinamide, whose structure is herewith revised. In radioligand binding studies at human cannabinoid CB1 and CB2 receptors recombinantly expressed in Chinese hamster ovary (CHO) cells, amauromine (1) was found to exhibit high affinity and selectivity for the CB1 receptor (Ki = 178 nM). The compound was shown to be a neutral CB1 antagonist with a Kb value of 66.6 nM determined in cAMP assays. Compound 2 exhibited only weak or no effects at CB receptors. To the best of our knowledge, compound 1 is the first fungal natural product that shows affinity for cannabinoid CB1 receptors. Because of its high antagonistic potency and selectivity, it may have potential for use as a drug and/or serve as a lead structure for drug development.

Keywords: Auxarthron reticulatum, amauromine, cannabinoid receptors, CB1, CB2, natural products

Cannabinoid receptors are located in the cell membrane and belong to the G protein-coupled receptor (GPCR) superfamily.1 They are divided into two distinct subtypes designated CB1 and CB2, both of which are Gi protein-coupled mediating inhibition of adenylate cyclase, and thus, their activation results in reduced intracellular cAMP levels.2 The CB1 receptor is expressed in the central nervous system (CNS) in high density but also is present in peripheral tissues, including the lungs, liver, kidneys, and adipocytes.3 CB1 activation mediates analgesia, stimulation of appetite, and euphoria, among other effects. CB1 antagonists show appetite-suppressing and antischizopathic properties.3 The CB2 receptor is mainly present in organs and cells of the immune system including spleen, tonsils, and thymus, and its activation results in analgesic and antiinflammatory effects.3,4

Regarding the origin of the ligands of cannabinoid receptors, they can be classified into three groups: (1) endocannabinoids, such as N-arachidonoylethanolamine, which are found in the mammalian nervous and immune system;5 (2) phytocannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC), which are produced by the plant Cannabis sativa;6 and (3) synthetic cannabinoids such as the agonist nabilone, a synthetic analogue of Δ9-THC, and the antagonist rimonabant, which is synthetically produced. Nabilone (Cesamet) is therapeutically used for the suppression of chemotherapy-induced nausea and vomiting,7 for the treatment of neuropathic pain, and for the therapy of anorexia in patients with AIDS.3 Further indications include chronic pain, fibromyalgia, and multiple sclerosis.3 CB1 receptor antagonists are potential drugs for the treatment of schizophrenia, especially for treating the negative symptoms of the disease, and for the therapy of drug and alcohol addiction.3 Another indication for CB1 receptor antagonists is the treatment of obesity, due to an appetite-suppressing effect mediated via central CB1 receptors in the hypothalamus. Besides weight loss, a reduction in HbA1c and triglyceride levels and an increase in high-density lipoprotein levels can be observed. Investigations have shown that these metabolic improvements are based on peripheral CB1 receptor blockade. Thus, peripherally acting CB1 receptor antagonists without CNS penetration would be promising drugs for the treatment of metabolic disorders associated with abdominal obesity, as they would be devoid of side effects caused by central CB1 receptor activation, for example, depression and anxiety.3,4,8 CB2 receptor inverse agonists/antagonists are potential drugs for the treatment of osteoporosis and were also shown to have beneficial effects in animal models of dermatitis.3,7 In the present study, we isolated compounds 1 and 2 from the marine sponge-derived fungus Auxarthron reticulatum and investigated the natural products for their interaction with CB receptors.

The fungal metabolite 1 is a dipeptide composed of two modified tryptophan units, which are cyclized forming a central diketopiperazine ring. Compound 1 is further characterized by the presence of two prenyl moieties, which are attached to the core structure through “reversed prenylation” (Figure 1). The molecular formula of compound 1 was deduced by accurate mass measurement [high-resolution electrospray ionization mass spectrometry (HRESIMS), m/z = 509.2911 [M + H]+] to be C32H36N4O2. The NMR spectroscopic data showed only resonances for C16H18, and thus, it was concluded that a homodimeric structure was present and that each resonance signal in the 1H and 13C NMR spectra represented at least two magnetically equivalent nuclei. Indeed, compound 1 is characterized by the presence of a C2-axis of symmetry (Supporting Information, Figure S2 and Table S3).

Figure 1.

Figure 1

Open in a new tab

Structures of amauromine (1) and methyl-penicinoline (2).

Because several stereoisomers of compound 1 are possible, it is difficult to delineate the absolute configuration of the molecule. Thus, for the determination of the absolute configuration, acid hydrolysis was performed followed by chiral HPLC chromatography (Supporting Information, Figure S14). This resulted in the detection of l-tryptophan indicating the S-configuration of the α-carbon atom of compound 1 (C-11/11′). Our data therefore showed that compound 1 is the alkaloid amauromine9−11 (=nigrifortine12). Compound 1 was previously investigated for its pharmacological effects and reported to be a vasodilator acting as a calcium entry blocker.9−11

HRESIMS analysis of compound 2 (Figure 1) yielded the molecular formula C15H12N2O3 with 11 degrees of unsaturation. This molecular formula was supported by 1H and 13C NMR spectra (Supporting Information, Figure S4 and Table S5), which showed resonance signals for two carbonyl groups (δc 173.8 for C-14 and 167.8 for C-16), five unsaturated quaternary carbon atoms (δc 123.0 for C-5, 140.9 for C-6, 139.7 for C-8, 124.3 for C-13, and 113.6 for C-15), seven unsaturated methine carbon atoms (δH/C 6.47/112.1 for CH-1, 6.25/110.1 for CH-2, 7.13/122.8 for CH-3, 7.66/118.7 for CH-9, 7.70/132.6 for CH-10, 7.36/123.9 for CH-11, and 8.07/124.9 for CH-12), and one methoxyl group (δH/c 3.67/52.1 for CH3-17). In addition, in the 1H and 13C NMR spectra, there are two downfield shifted resonances at δH 11.63 and 11.70, which were determined to arise from NH-4 and NH-7, respectively. The downfield shift may be explained by intermolecular hydrogen bonds of the NH groups with the carbonyl group at C-14 as seen in the X-ray derived structure (Supporting Information, Figure S1).

The methine carbons CH-9 to CH-12 are connected due to the mutual cross-peak correlations in both 1H–1H correlation spectroscopy (COSY) and 1H–13C heteronuclear multiple bond correlation (HMBC) spectra (Supporting Information, Figure S6). They are also bound to the quaternary aromatic carbons C-13 and C-8, as confirmed by 1H–13C HMBC correlations from H-9 and from H-11 to C-13 and from H-10 and H-12 to C-8. Compound 2 is thus an ortho-disubstituted benzene derivative. C-13 is attached to the carbonyl carbon C-14 due to the 1H–13C HMBC correlation from H-12 to C-14, and C-8 is attached to NH-7 due to 1H–13C HMBC correlations from NH-7 to both C-9 and C-13. Also, in the 1H–13C HMBC spectrum, NH-7 exhibits correlations to C-6 and C-15, which results in a γ-pyridone ring including carbons C-8, C-13, and C-14. Such an arrangement forms a 4-quinolinone ring, which is substituted at C-15 and C-6. The substitution at C-15 is a methyl carboxylate residue due to the 1H–13C HMBC cross-peak correlations from the methoxyl group CH3-17 to both C-15 and C-16, whereas the substitution at C-6 is an α-pyrrolyl moiety. The latter was proven from the data acquired in 1H–1H COSY and 1H–13C HMBC experiments. Thus, H-1 to H-3 are forming a spin system, which together with the key HMBC correlations between them and the carbons C-1 to C-5 implies the presence of a pyrrol ring. The nuclear Overhauser effect spectroscopy correlation of NH-4 to H-3 confirmed this conclusion. The pyrrol ring is linked to the quinolinone nucleus at C-6 due to the 1H–13C HMBC cross-peak correlation from NH-7 to C-5. X-ray diffraction analysis allowed the structure of 2 to be unambiguously established. It is a structurally unique 4-quinolinone, linked to a pyrrole ring on one side and a methyl carboxylic acid ester moiety at the other side.

A similar compound, that is, methyl-marinamide,13 which was described as an isoquinolinone analogue of 2, was already described in the literature (Supporting Information, Figure S13). Surprisingly, this compound has the same NMR chemical shifts as compound 2, and thus, we assume that the structures of methyl-marinamide and also of the related marinamide are wrong. This is further confirmed by the recently reported compound penicinoline,14 which shows the same structural features as compound 2, except that it is not methylated (i.e., CH3-17). Penicinoline, according to our analysis, is identical to marinamide.

Because of the structural similarity between amauromine and the CB1 antagonist rimonabant (Figure 2), we investigated the isolated natural products 1 and 2 in radioligand binding studies15 at human cannabinoid CB1 and CB2 receptors recombinantly expressed in Chinese hamster ovary (CHO-K1) cells obtained by retroviral transfection (Hinz, S.; Karcz, T.; Müller, C. E. Unpublished results). Compound 1 showed very high affinity for CB1 receptors exhibiting a Ki value of 178 nM (Figure 3), while it did not show any affinity at a high concentration of 10 μM at CB2 receptors (Figure S7, Supporting Information). In functional assays measuring forskolin-induced cAMP accumulation in CHO cells,16 expressing the human CB1 receptor amauromine (1) did not show agonistic effects (Figure S8, Supporting Information). However, compound 1 (300 nM) led to a significant rightward shift of the concentration–response curve for the potent CB receptor agonist CP55,940 in inhibiting forskolin-induced cAMP accumulation at the Gi protein-coupled CB1 receptor (Figure 4). A Kb value of 66.6 nM was determined for 1. To the best of our knowledge, compound 1 is the first compound of fungal origin to show affinity toward cannabinoid receptors. Compound 2 exhibited only weak affinity for CB1 and CB2 receptors (Figure S7, Supporting Information).

Figure 2.

Figure 2

Open in a new tab

Comparison of the structure of amauromine (1) with that of the CB1 antagonist/inverse agonist rimonabant. Essential structural features (carbonyl group, lipophilic, and aromatic residues) of rimonabant and corresponding features in amauromine are circled by dashed lines.

Figure 3.

Figure 3

Open in a new tab

Concentration-dependent inhibition of specific [3H]CP55,940 binding by amauromine at membrane preparations of CHO cells recombinantly expressing human CB1 receptors. Data points represent means ± SEMs of three independent experiments, performed in duplicate.

Figure 4.

Figure 4

Open in a new tab

Concentration-dependent inhibition of forskolin (10 μM)-induced cAMP accumulation by the CB agonist CP55,940 in CHO cells recombinantly expressing the human CB1 receptor in the absence and in the presence of amauromine (300 nM). Data points are means ± SEMs of three independent experiments performed in duplicate. A KB value of 66.6 ± 57.0 nM was determined for amauromine (1).

In light of our results, it is interesting to note that many synthetic indole derivatives have already been examined with regard to their affinity for CB receptors.7,17−19 In a number of studies, indole derivatives were identified that exhibit potent agonistic, cannabimimetic effects on CB2, but only weak or no affinity for CB1 receptors. In contrast to those previous results, compound 1 functions as a selective antagonist at the CB1 receptor. Only very recently, the first natural alkaloids with CB1-antagonistic activity isolated from the plant Voacanga africana were described.19

Functional studies indicate that compound 1 appears to be a neutral competitive antagonist, as no increased cAMP levels could be detected in cAMP accumulation assays. There is evidence that neutral antagonists may be preferable over inverse agonists when used as drugs, because they do not alter basic activity arising from spontaneous receptor signaling and, thus, may cause less side effects. For example, it has been reported that the neutral CB1 receptor antagonist AM4113 had effects comparable to that of the CB1 inverse agonist AM251 regarding food intake and food-reinforcement behavior in rats, but in contrast to AM251, it failed to induce nausea.7,20 Furthermore, neutral antagonists are useful experimental tools as they show no effect on their own and, thus, will facilitate the investigation of the physiological role of the endocannabinoid system.7,21

Recent studies have emphasized important roles for the endocannabinoid system, that is, receptors and endogenous ligands, in many pathophysiological processes. Therefore, CB1 and CB2 receptors are regarded as important drug targets for a number of common diseases for which new drugs are urgently needed, including Parkinson's and Alzheimer's disease, major depression, inflammation, neuropathic pain, and metabolic syndrome.22 The identification of the structurally novel cannabinoid receptor ligand amauromine (compound 1) provides an opportunity for the development of a new chemical class of therapeutic agents for the treatment of a number of disorders involving CB1 cannabinoid receptors.

Dedication

This paper is dedicated to Professors Hassan Elrady A. Saad, Sahar Gedara, and Ahmed Gohar.

Financial support was provided by the Egyptian Government (M.F.E.) and the BMBF (G.M.K., project no. 03F0415A). Funding by the BMBF (project no. 01EW0911) in the frame of ERA-NET NEURON is gratefully acknowledged (C.E.M.).

Supporting Information Available

Full experimental procedures, complete crystallographic data for compound 2, compound characterization for compounds 1 and 2, 1H and 13C NMR spectra for compounds 1 and 2, purity data of compound 1, diagrams of affinities of compounds 1 and 2 toward CB receptors, and results for compound 1 in cAMP accumulation assays at CB1 receptor. This material is available free of charge via the Internet at http://pubs.acs.org.

Supplementary Material

ml200183z_si_001.pdf (411.8KB, pdf)

References

  1. Graham E. S.; Ashton J. C.; Glass M. Cannabinoid receptors: A brief history and “what’s hot”. Front. Biosci. 2009, 14, 944–957. [DOI] [PubMed] [Google Scholar]
  2. Matsuda L. A.; Lolait S. J.; Brownstein M. J.; Young A. C.; Bonner T. I. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990, 346, 561–564. [DOI] [PubMed] [Google Scholar]
  3. Pacher P.; Batkai S.; Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol. Rev. 2006, 58, 389–462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Racz I.; Nadal X.; Alferink J.; Banos J. E.; Rehnelt J.; Martin M.; Pintado B.; Gutierrez-Adan A.; Sanguino E.; Manzanares J.; Zimmer A.; Maldonado R. Crucial role of CB(2) cannabinoid receptor in the regulation of central immune responses during neuropathic pain. J. Neurosci. 2008, 28, 12125–12135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Devane W. A.; Hanus L.; Breuer A.; Pertwee R. G.; Stevenson L. A.; Griffin G.; Gibson D.; Mandelbaum A.; Etinger A.; Mechoulam R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992, 258, 1946–1949. [DOI] [PubMed] [Google Scholar]
  6. Fusar-Poli P.; Crippa J. A.; Bhattacharyya S.; Borgwardt S. J.; Allen P.; Martin-Santos R.; Seal M.; Surguladze S. A.; O’Carrol C.; Atakan Z.; Zuardi A. W.; McGuire P. K. Distinct effects of {delta}9-tetrahydrocannabinol and cannabidiol on neural activation during emotional processing. Arch. Gen. Psychiatry 2009, 66, 95–105. [DOI] [PubMed] [Google Scholar]
  7. Pertwee R. G. Receptors and channels targeted by synthetic cannabinoid receptor agonists and antagonists. Curr. Med. Chem. 2010, 17, 1360–1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Scheen A. J.; Paquot N. Use of cannabinoid CB1 receptor antagonists for the treatment of metabolic disorders. Best. Pract. Res. Clin. Endocrinol. Metab. 2009, 23, 103–116. [DOI] [PubMed] [Google Scholar]
  9. Takase S.; Kawai Y.; Uchida I.; Tanaka H.; Aoki H. Structure of amauromine, a new alkaloid with vasodilating activity produced by Amauroascus sp. Tetrahedron Lett. 1984, 25, 4673–4676. [Google Scholar]
  10. Takase S.; Iwami M.; Ando T.; Okamoto M.; Yoshida K.; Horiai H.; Kohsaka M.; Aoki H.; Imanaka H. Amauromine, a new vasodilator. Taxonomy, isolation and characterization. J. Antibiot. 1984, 37, 1320–1323. [DOI] [PubMed] [Google Scholar]
  11. Takase S.; Kawai Y.; Uchida I.; Tanaka H.; Aoki H. Structure of amauromine, a new hypotensive vasodilator produced by Amauroascus sp. Tetrahedron 1985, 41, 3037–3048. [Google Scholar]
  12. Laws I.; Mantle P. G. Nigrifortine, a diketopiperazine metabolite of Penicillium nigricans. Phytochemistry 1985, 24, 1395–1397. [Google Scholar]
  13. Zhu F.; Lin Y. Marinamide, a novel alkaloid and its methyl ester produced by the application of mixed fermentation technique to two mangrove endophytic fungi from the South China Sea. Chin. Sci. Bull. 2006, 51, 1426–1430. [Google Scholar]
  14. Shao C.-L.; Wang C.-Y.; Gu Y.-C.; Wei M.-Y.; Pan J.-H.; Deng D.-S.; She Z.-G.; Lin Y.-C. Penicinoline, a new pyrrolyl 4-quinolinone alkaloid with an unprecedented ring system from an endophytic fungus Penicillium sp. Bioorg. Med. Chem. Lett. 2010, 20, 3284–3286. [DOI] [PubMed] [Google Scholar]
  15. Behrenswerth A.; Volz N.; Torang J.; Hinz S.; Bräse S.; Müller C. E. Synthesis and pharmacological evaluation of coumarin derivatives as cannabinoid receptor antagonists and inverse agonists. Bioorg. Med. Chem. 2009, 17, 2842–2851. [DOI] [PubMed] [Google Scholar]
  16. Drabczynska A.; Yuzlenko O.; Köse M.; Paskaleva M.; Schiedel A. C.; Karolak-Wojciechowska J.; Handzlik J.; Karcz T.; Kuder K.; Müller C. E.; Kiec-Kononowicz K. Synthesis and biological activity of tricyclic cycloalkylimidazo-, pyrimido- and diazepinopurinediones. Eur. J. Med. Chem. 2011, 46, 3590–3607. [DOI] [PubMed] [Google Scholar]
  17. Poso A.; Huffman J. W. Targeting the cannabinoid CB2 receptor: modelling and structural determinants of CB2 selective ligands. Br. J. Pharmacol. 2008, 153, 335–346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huffman J. W.; Zengin G.; Wu M.-J.; Lu J.; Hynd G.; Bushell K.; Thompson A. L. S.; Bushell S.; Tartal C.; Hurst D. P.; Reggio P. H.; Selley D. E.; Cassidy M. P.; Wiley J. L.; Martin B. R. Structure-activity relationships for 1-alkyl-3-(l-naphthoyl)indoles at the cannabinoid CB1 and CB2 receptors: steric and electronic effects of naphthoyl substituents. New highly selective CB2 receptor agonists. Bioorg. Med. Chem. 2005, 13, 89–112. [DOI] [PubMed] [Google Scholar]
  19. Kitajima M.; Iwai M.; Kikura-Hanajiri R.; Goda Y.; Iida M.; Yabushita H.; Takayama H. Discovery of indole alkaloids with cannabinoid CB1 receptor antagonistic activity. Bioorg. Med. Chem. Lett. 2011, 21, 1962–1964. [DOI] [PubMed] [Google Scholar]
  20. Sink K. S.; McLaughlin P. J.; Wood J. A.; Brown C.; Fan P.; Vemuri V. K.; Peng Y.; Olszewska T.; Thakur G. A.; Makriyannis A.; Parker L. A.; Salamone J. D. The novel cannabinoid CB1 receptor neutral antagonist AM4113 suppresses food intake and food-reinforced behavior but does not induce signs of nausea in rats. Neuropsychopharmacology 2008, 33, 946–955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wiley J. L.; Breivogel C. S.; Mahadevan A.; Pertwee R. G.; Cascio M. G.; Bolognini D.; Huffman J. W.; Walentiny D. M.; Vann R. E.; Razdan R. K.; Martin B. R. Structural and pharmacological analysis of O-2050, a putative neutral cannabinoid CB(1) receptor antagonist. Eur. J. Pharmacol. 2010, 651, 96–105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heimann A. S.; Gomes I.; Dale C. S.; Pagano R. L.; Gupta A.; de Souza L. L.; Luchessi A. D.; Castro L. M.; Giorgi R.; Rioli V.; Ferro E. S.; Devi L. A. Hemopressin is an inverse agonist of CB1 cannabinoid receptors. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 20588–20593. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml200183z_si_001.pdf (411.8KB, pdf)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES