A SARS-coronovirus 3CL protease inhibitor isolated from the marine sponge Axinella cf. corrugata: structure elucidation and synthesis

SHORT REPORT

A SARS-coronovirus 3CL protease inhibitor isolated from the marine sponge Axinella cf. corrugata: structure elucidation and synthesis

Simone P. de Lira^I; Mirna H. R. Seleghim^I; David E. Williams^II; Frederic Marion^II; Pamela Hamill^III; François Jean^III; Raymond J. Andersen^II; Eduardo Hajdu^IV; Roberto G. S. Berlinck^{*, I}

^IInstituto de Química de São Carlos, Universidade de São Paulo, CP 780, 13560-970 São Carlos-SP, Brazil

^IIDepartment of Chemistry and Department of Earth and Ocean Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z1 Canada

^IIIDepartment of Microbiology and Immunology, Life Sciences Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada

^IVMuseu Nacional, Universidade Federal do Rio de Janeiro, Quinta da Boa Vista, s/n, 20940-040 Rio de Janeiro-RJ, Brazil

ABSTRACT

Two coumarin derivatives, esculetin-4-carboxylic acid methyl ester (1) and esculetin-4-carboxylic acid ethyl ester (2), have been isolated from the marine sponge Axinella cf. corrugata. Structure determination included analysis of spectroscopic data and total synthesis of compound 2. These are the first coumarin derivatives isolated from a marine sponge. The ethyl ester 2 was found to be an in vitro inhibitor of SARS 3CL-protease and an effective inhibitor of SARS-CoV replication in Vero cells at non-cytotoxic concentrations.

Keywords: esculetin, marine sponge, Axinella cf. corrugata, SARS, anti-viral

RESUMO

Dois derivados cumarínicos, o ester metílico do ácido 4-esculetinocarboxílico (1) e o éster etílico do ácido 4-esculetinocarboxílico (2), foram isolados da esponja marinha Axinella cf. corrugata. A determinação estrutural dos compostos isolados foi realizada pela análise de seus dados espectroscópicos e pela síntese do composto 2. Estes são os primeiros derivados cumarínicos isolados de uma esponja marinha. O éster etílico 2 apresentou atividade in vitro contra a SARS 3Cl-protease e de inibição de células Vero infectadas com o SARS coronavírus, em concentrações que não provocaram citotoxicidade.

Introduction

In 2002 and 2003, several cases of a life-threatening respiratory disease that was ultimately named "severe acute respiratory syndrome" (SARS) were reported from Guangdong Province in mainland China, Hong Kong, Canada, and Vietnam.¹ The etiological agent responsible for SARS was quickly found to be a novel coronavirus (SARS-CoV). Among the viral gene products are several large polyproteins that must be proteolytically processed to generate the individual proteins required for viral replication to occur. Two viral proteases, PL2^pro and 3CL^pro, are involved in degrading the large polyproteins.² The viral protease 3CL^pro is considered the most important of the two because it is responsible for the release of the key replicative proteins of the vírus, including the viral RNA polymerase and helicase proteins. The central role of 3CL^pro in SARS-CoV replication has made it a high profile target for developing antiviral drugs to treat SARS.² Recently, a novel fluorescence resonance energy transfer (FRET)-based assay to screen for 3CL^pro inhibitors has been developed in one of our laboratories.³ As part of a screening of crude marine sponge extracts and pure compounds isolated from the marine sponges using this assay, we have found that the new natural product esculetin-4-carboxylic acid ethyl ester (2) isolated from Axinella cf. corrugata collected in Brazil is a an effective inhibitor of 3CL^proin vitro.

In our current search for new biologically active marine natural products,^4,5 we decided to investigate the MeOH crude extract of the sponge Axinella cf. corrugata, which displayed cytotoxic activity against breast MCF-7 and colon B16 cancer cell lines. Although the cytotoxic activity was lost during the crude extract fractionation, photodiode array detection-HPLC analysis of the chromatographic fractions obtained indicated the presence of metabolites with UV absorptions at l_max 207, 239, 271 and 375 nm, an absorption profile not found in MARINLIT database. Further separation and purification yielded the new metabolites esculetin-4-carboxylic acid methyl ester (1) and esculetin-4-carboxylic acid ethyl ester (2), probably as artifacts of isolation of the herein reported new coumarin derivative esculetin-4-carboxylic acid with MeOH and EtOH, used in the extraction procedure. The pure compounds 1 and 2 were subsequently screened in a panel of biological assays resulting in the discovery that compound 2 was an inhibitor of 3CL^pro.³ Herein we present details of the isolation, structure determination of 1 and 2, along with the total synthesis of compound 2 in order to confirm the structure proposal.

Results and Discussion

Esculetin-4-carboxylic acid methyl ester (1) displayed a molecular ion peak corresponding to its dimerized sodium adduct at m/z 495 (TOF/ESI-MS). A high resolution mass measurement at m/z 495.0544 (Calc. 495.0539) indicated the formula C₂₂H₁₆O₁₂Na. Considering the ¹³C NMR spectrum of 1 which displayed only eleven carbons, the molecular weight measurement indicated that either the compound presented internal symmetry or it was detected as a dimerized molecular ion. The last assumption has proven to be true after the total synthesis of compound 2 (see below) and recording the mass spectrum of the synthetic product, which displayed the same dimerized molecular ion. Seven carbons of esculetin-4-carboxylic acid methyl ester (1) were shown to be quaternary sp² by DEPT ¹³C NMR (d 164.4, 160.0, 150.9, 148.8, 143.0, 142.5, 107.0). Three of the remaining carbons were methine sp² (d 113.5, 110.3, 102.9) and one methyl group (d 53.0). The ¹H NMR spectrum of 1 displayed six singlets. The HSQC experiment indicated that two of these singlets could be assigned to phenol hydrogens (d 10.44 and 9.60), three to vinylic and/or aromatic hydrogens at d 7.46, 6.80 and 6.60 attached to the corresponding sp² carbons at d 110.3, 102.9 and 113.5, respectively, and one methoxyl group at d 3.91 (d 53.0). The ¹H-¹H COSY spectrum of 1 indicated a very weak long-range ¹H-¹H coupling between the singlets at d 7.46 and 6.80, suggesting a 1,4-aromatic relationship between these hydrogens. Since the HMBC spectrum displayed correlations of both hydrogens at d 7.46 and 6.80 with carbons at d 150.9 and 148.8, which were also coupled with both phenolic hydrogens at d 10.44 and 9.60, the ¹H-¹H and ¹H-¹³C correlation data enabled us to define the aromatic portion of esculetin-4-carboxylic acid methyl ester (1) as a 1,2,4,5-tetrasubstituted benzene ring, with two hydroxyl groups at vicinal positions. The remaining vinylic hydrogen at d 6.60 displayed long range correlations with two carbonyl groups at d 164.4 and 160.0. Since the methyl group at d 3.91 (s) also presented a long range correlation with carbonyl at d 164.4, it was assigned to a methyl ester group. Considering the number of eight unsaturations established by half of the molecular formula as indicated above and the number of sp² carbons observed in the ¹³C NMR spectrum, the remaining carbonyl group at d 160.0 was assigned to a lactone moiety of a coumarin-like skeleton. The positions of the hydroxyl groups were assigned to carbons 6 and 7, as in esculetin (3). The methyl ester group might be placed at either C-3 or C-4, giving rise to two possible isomeric structures, 1 or 4, which could not be distinguished by the long range correlations observed for the hydrogen at d 6.60 with both carbonyl groups, since in both cases these are correlations through two or three bonds. However, the HMBC spectrum clearly indicated that hydrogens at d 6.60 and 6.80 presented a long-range correlation with the carbon at d 107.0. Therefore, structure 1 was favoured, in which the carbon with chemical shift at d 107.0 was assigned to C-4, which was ³J coupled with the aromatic hydrogen at d 6.80 and ²J coupled to the vinylic hydrogen at d 6.60. Additionally, a hydrogen at C-4 position would present a higher chemical shift, since it would be at a b-position of an a,b-conjugated double bond with two carbonyl groups. The two remaining quaternary sp² carbons at d 142.5 and 143.0 were assigned to C-9 and C-10, both assignments being interchangeable.

Esculetin-4-carboxylic acid ethyl ester (2) displayed a molecular ion peak for its dimerized sodium adduct at m/z 523. Since the high resolution mass measurement at m/z 523.0843 (Calc. 523.0852) indicated the formula C₂₄H₂₀O₁₂Na, and its ¹H, ¹³C, and HMBC NMR spectra indicated the presence of an ethoxy group (d ¹H 3.37, q, 7 Hz, 2H; d ¹³C 62.0; d ¹H 1.35, t, 7 Hz, 3H; d ¹³C 13.7) coupled to the C-11 ester carbonyl as the only difference to 1, we have assigned the structure of the corresponding ethyl ester to 2.

In order to confirm our structure proposal, we have performed the total synthesis of compound 2 in three steps from 3-hydroxy-4-methoxybenzaldehyde (5) following the procedure described for the synthesis of scopoletin (Scheme 1).⁶ The intermediate scopoletin-4-carboxylic acid ethyl ester (7) was deprotected with using boron tribromide providing esculetin-4-carboxylic acid ethyl ester (2) in 27% overall yield.

The isolation of esculetin-4-carboxylic acid as its corresponding methyl and ethyl esters is probably due to the extraction of Axinella cf. corrugata with MeOH and EtOH. Esculetin-4-carboxylic acid is not known as a natural product, but it has been synthesized during a survey for inhibitors of DOPA decarboxylase.⁷ Considering the small amount isolated of both esculetin-4-carboxylic acid methyl ester and ethyl ester, it is reasonable to suggest a microbial origin for such compounds, although the occurrence of coumarin derivatives in fungi and bacteria has not been frequently observed.⁸ Metabolites bearing a coumarin moiety have been isolated from the marine fungus Trichoderma virens⁹ and from the marine ascomycete Leptosphaeria oraemaris.¹⁰ The isolation of plant-related metabolites from sponges is rare. Only two examples are found in the literature: resorcinol derivatives from Haliclona sp.¹¹ and triptophol, an auxin derivative, from Ircinia spinulosa.¹²

Esculetin-4-carboxylic acid ethyl ester (2) was found to inhibit recombinant 3CL^pro in vitro with an ID₅₀ value of 46 mmol L^-1.³ It has also been shown that 2 is an effective inhibitor of SARS-CoV replication in Vero cells (EC₅₀ = 112 mmol L^-1) and that it mediates these effects at non-cytotoxic concentrations (2: Vero cell cytotoxicity IC₅₀ > 800 µmol L^-1).^4b Further studies on the antiviral properties of 2 are ongoing and the results will be reported elsewhere.

Supplementary Information

Experimental details and spectra are given as supplementary information at http://jbcs.sbq.org.br, as PDF file.

Acknowledgments

The authors thank Professor Maria da Glória B. S. Moreira, past principal of the Centro de Biologia Marinha, for providing facilities for the sponge collection and also Prof. Gil V. J. Silva and Virginia H. B. Glass (Depertamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo) in obtaining the NMR spectra of the natural products. Financial support was provided by FAPESP (01/03095-5 to RGSB) and the Natural Sciences and Engineering Research Council of Canada (RJA). SPL thanks FAPESP for the award of a scholarship and FM was supported by a Fellowship from Pierre Fabre.

References

1. Drosten, C.; Gunther, S.; Preiser, W.; van der Werf, S.; Brodt, H. R.; Becker, S.; Rabenau, H.; Panning, M.; Kolesnikova, L.; Fouchier, R. A. M.; Berger, A.; Burguiere, A. M.; Cinatl, J.; Eickmann, M.; Escriou, N.; Grywna, K.; Kramme, S.; Manuguerra, J. C.; Muller, S.; Rickerts, V.; Sturmer, M.; Vieth, S.; Klenk, H. D.; Osterhaus, A. D. M. E.; Schmitz, H.; Doerr, H. W.; N. Engl. J. Med. 2003, 348, 1967.

2. Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R.; Science 2003, 300, 1763.

3. Hamill, P.; Hudson, D.; Kao, R. Y.; Chow, P.; Raj, M.; Xu, H.; Richer, M. J.; Jean, F.; Biol. Chem. 2006, 387, 1063.

4. Oliveira, J. H. H. L.; Seleghim, M. H. R.; Timm, C.; Grube, A.; Köck, M.; Nascimento, G. G. F.; Martins, A. C. T.; Silva, E. G. O.; Souza, A. O.; Galetti, F. C. S.; Minarini, P. R. R.; Silva, C. L. L.; Hajdu, E.; Berlinck, R. G. S.; Mar. Drugs 2006, 4, 1; Oliveira, M. F.; Oliveira, J. H. H. L.; Galetti, F. C. S.; Souza, A. O.; Silva, C. L.; Hajdu, E.; Peixinho, S.; Berlinck, R. G. S.; Planta Med. 2006, 72, 437.

5. Williams, D. E.; Austin, P.; Diaz-Marrero, A. R.; Van Soest, R.; Matainaho, T.; Roskelley, C. D.; Roberge, M.; Andersen, R. J.; Org. Lett. 2005, 7, 4173; Williams, D. E.; Patrick, B. O.; Behrisch, H. W.; Van Soest, R.; Roberge, M.; Andersen, R. J.; J. Nat. Prod. 2005, 68, 327.

6. Croby, D. G.; J. Org. Chem. 1961, 26, 1215.

7. Hartman, W. J.; Akawie, R. I.; Clark, W. G.; J. Biol. Chem. 1955, 216, 507.

8. Literature searches in SciFinder database provide only a limited number of records related to the occurrence of coumarin derivatives in bacteria and fungi. See Chen, H.; Walsh, C. T.; Chem. Biol. 2001, 8, 301; Pedras, M. S. C.; Chumala, P. B.; Phytochemistry 2005, 66, 81.

9. Garo, E., Starks, C. M.; Jensen, P. R.; Fenical, W.; Lobkovsky, E.; Clardy, J.; J. Nat. Prod. 2003, 66, 423.

10. Guerriero, A.; D'Ambrosio, M.; Cuomo, V.; Pietra, F.; Helv. Chim. Acta 1991, 74, 1445.

11. Barrow, R. A.; Capon, R. J.; Aust. J. Chem. 1991, 44, 1393.

12. Erdogan, I.; Sener, B.; Higa, T.; Biochem. Syst. Ecol. 2000, 28, 793.

Received: August 21, 2006

Web Release Date: March 16, 2007

FAPESP helped in meeting the publication costs of this article.

Supplementary Information

Experimental

General experimental procedures

UV spectra were recorded on a Hitachi U-3210 spectrophotometer. IR spectra (film on Si plate) were recorded on a FT-IR Bomem MB102 infrared spectrometer. NMR spectra were recorded either on a Bruker ARX 9.4 T instrument, operating at 400.35 MHz for ¹H and 100.10 MHz for ¹³C channels, respectively, or on a Bruker Avance 300 Spectrometer with Bruker QNP 5 mm probe 300.00 MHz for ¹H and 75.0 MHz for ¹³C, respectively. All NMR spectra were obtained at 25 ºC using TMS as internal reference. Low and high resolution ESI-QIT-MS were recorded on a Bruker-Hewlett Packard 1100 Esquire-LC system mass spectrometer. Solvents used for extraction and flash chromatography were glass distilled prior to use. HPLC-grade solvents were utilized without further purification in HPLC separations. TLC analyses were performed with plastic-backed Si gel TLC sheets, eluting with different mixtures of MeOH and CH₂Cl₂. Plates were visualised under UV and also using phosphomolybdic acid as spray reagent, followed by heating at 100 ºC. HPLC separations were performed either with a Waters quaternary pump 600, double beam UV detector 2487, and data module 746, or with a Waters autosampler 717, Waters 600 pump, Waters 2996 photodiode array detector monitored by Waters Millenium 32.

Animal Material

The sponge Axinella cf. corrugata sp. was collected in several sites in the São Sebastião Channel, São Paulo, Brazil during the summer of 1995 and immediately stored in EtOH at –20 ºC until processed. A voucher specimen was deposited at the Museu Nacional of the Universidade Federal do Rio de Janeiro (MNRJ 1749).

Isolation of compounds 1 and 2 from the sponge Axinella cf. corrugata

The sponge (ca. 2.0 kg wet weight) was separated from the EtOH (3 L) in which the sponge samples were stored, triturated in MeOH (3 L) in a waring blender and left overnight. After filtration of the MeOH extract, the solid material was re-extracted with MeOH (3 L). Both EtOH (3 L) and MeOH (6 L) extracts were pooled and evaporated until 500 mL of an aqueous suspension was obtained. The H₂O phase was partitioned with EtOAc. The EtOAc extract (0.574 g) was subjected to a chromatography on Sephadex LH-20 (MeOH), to give eight fractions. Fraction EtOAc-4 was subjected to a chromatography on a Waters silica gel Sep Pak column (10 g), eluted with a gradient of 1:1 EtOAc-MeOH in CH₂Cl₂, to give six fractions (EtOAc-4A to -4F). Fractions EtOAc-4C and EtOAc-4E were further purified by HPLC, using a Waters mBondapak Phenyl column (125 Å, 7.8 × 300 mm), using 2:3 MeOH/H₂O (containing 0.1% TFA) as the eluent, to give 3.9 mg of esculetin-4-carboxylic acid methyl ester (1, 0.20 10^-3%) and 4.5 mg of esculetin-4-carboxylic acid ethyl ester (2, 0.22 10^-3%).

Esculetin-4-carboxylic acid methyl ester (1)

Green amorph solid. UV (MeOH) l_max/ nm: 207, 239, 271 and 375; IR (film) n_max/ cm^-1: 3400-3000 (broad), 1709, 1619, 1566, 1447, 1384, 1283, 1202, 1142; ¹H NMR (DMSO-d₆, 400 MHz) d 10.44 (1H, s, OH-6), 9.60 (1H, s, OH-7), 7.46 (1H, s, H-8), 6.80 (1H, s, H-5), 6.60 (1H, s, H-3), 3.91 (3H, s, CH₃); ¹³C NMR (DMSO-d₆, 100 MHz) d 164.4 (C-11), 160.0 (C-2), 150.9 (C-6), 148.8 (C-7), 143.0 (C-9), 142.5 (C-10), 113.5 (C-3), 110.3 (C-8), 107.0 (C-4), 102.9 (C-5), 53.0 (C-12); Positive HRTOF/ESI-MS m/z 495.0544 [2×M+Na]⁺ (Calc. for C₂₂H₁₆O₁₂Na 495.0539).

Esculetin-4-carboxylic acid ethyl ester (2)

Green amorph solid; UV (MeOH) l_max/ nm: 207, 239, 271 and 375; IR (film on a Si plate) n_max/ cm^-1: 3400-3000 (broad), 1709, 1619, 1566, 1447, 1384, 1283, 1202, 1142; ¹H NMR (DMSO-d₆, 400 MHz) d 10.43 (1H, s, OH-6), 9.62 (1H, s, OH-7), 7.46 (1H, s, H-8), 6.80 (1H, s, H-5), 6.58 (1H, s, H-3), 4.37 (2H, q, 7 Hz, CH₂-12), 1.33 (3H, t, 7Hz, CH₃-13); ¹³C NMR (DMSO-d₆, 100 MHz) d 163.9 (C-11), 160.0 (C-2), 150.9 (C-6), 148.8 (C-7), 143.0 (C-9), 142.8 (C-10), 113.3 (C-3), 110.2 (C-8), 107.0 (C-4), 102.9 (C-5), 62.0 (C-12), 13.7 (C-13); HRESIMS m/z 523.0843 [2×M+Na]⁺ (Calc. for C₂₄H₂₀O₁₂Na 523.0852).

Synthesis of esculetin-4-carboxylic acid ethyl ester (2). Preparation of 3-hydroxy-4-methoxyphenyl formate (6)

A mixture of isovanillin (5) (760 mg, 5.0 mmol) and m-CPBA (1.5 equiv.) and NaHCO₃ (1.5 equiv.) in CH₂Cl₂ (25 mL) was stirred at rt for 2 h. The solids were removed by filtration and washed with CH₂Cl₂. The filtrate was extracted with an aqueous solution of NaHCO₃ and brine. The organic phase was dried over MgSO₄, filtered and evaporated under reduced pressure. The crude was chromatographed on silica gel (20% EtOAc in hexanes) to provide formate 6 (583 mg) in 70% yield.

3-Hydroxy-4-methoxyphenyl formate (6)

¹H NMR (CDCl₃, 400 MHz) d 8.23 (1H, s), 6.79 (1H, d, J 8.7 Hz), 6.70 (1H, d, J 2.6 Hz), 6.59 (1H, dd, J 8.7, 2.6 Hz), 5.84 (1H, bs), 3.84 (3H, s); ¹³C NMR (CDCl₃, 100 MHz) d 159.7, 146.2, 144.8, 143.7, 111.9, 110.7, 108.0, 56.1; Positive HRESIMS [M+H]⁺m/z 169.0505 (C₈H₉O₄, Calc. 169.0501).

Preparation of compound 7

A mixture of 6 (1.32 g, 7.8 mmol), sodium diethyloxalacetate (SDO, 2.0 g, 9.36 mmol) and H₃PO₄ (85%, 5 mL) was heated at 100 ºC for 2 h. The mixture was then poured into ice and the solids collected by filtration and washed with H₂O. This crude product was purified by silica gel column chromatography (5% MeOH in CH₂Cl₂) affording 947 mg of 7 (3.6 mmol, 46% yield). ¹H NMR (CDCl₃, 300 MHz) d 7.82 (1H, s), 6.91 (1H, s), 6.83 (1H, s), 6.18 (1H, s), 4.42 (2H, q, J 7.0 Hz), 3.95 (3H, s), 1.41 (3H, t, J 7.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) d 164.1, 160.8, 150.6, 149.9, 144.0, 141.6, 119.1, 108.5, 106.5, 103.0, 62.2, 56.2, 13.9; Positive HRESIMS [M+Na]⁺m/z 287.0527 (C₁₃H₁₂O₆Na Calc. 287.0732).

Preparation of esculetin-4-carboxylic acid ethyl ester (2)

To a solution of 7 (25 mg, 0.095) in CH₂Cl₂ was added at –78 ºC a solution of BBr₃ (3.0 equiv., 1 mol L^-1 in CH₂Cl₂). The mixture was allowed to warm to rt and silica gel was then added. Concentration under reduced pressure and chromatography on silica gel (5% MeOH in CH₂Cl₂) afforded 2 (20 mg) in 84% yield. HRESIMS m/z 523.0850 [2xM+Na]⁺ (Calc. for C₂₄H₂₀O₁₂Na 523.0852), [M+Na]⁺m/z 273.0353 (C₁₂H₁₀O₆Na, Calc. 273.0375).

Publication Dates

• Publication in this collection
  13 June 2007
• Date of issue
  Apr 2007

History

• Received
  21 Aug 2006
• Accepted
  16 Mar 2007