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Sulfadiazine Masquerading as a Natural Product from madeirensis () Luke P. Robertson,* Lindon W. K. Moodie, Darren C. Holland, K. Charlotte Jander,́ and Ulf Göransson

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ABSTRACT: The structure of 2,4-(4′-aminobenzenamine)- pyrimidine (1), a pyrimidine alkaloid previously isolated from the of (, synonym Autonoë madeirensis), has been revised. These conclusions were met via comparison of reported NMR and EIMS data with those obtained from synthetic standards. The corrected structure is the antibiotic sulfadiazine (2), which has likely been isolated as a contaminant α from the site of collection. The reported bioactivity of 1 as an 1- adrenoceptor antagonist should instead be ascribed to sulfadiazine. Our findings appear to show another example of an anthropogenic contaminant being identified as a natural product and emphasize the importance of considering the biosynthetic origins of isolated compounds within a phylogenetic context.

he reported isolation of the analgesic tramadol from the via comparison of the reported spectroscopic data with those T roots of the African medicinal Nauclea latifolia Sm. obtained from synthetic standards. (Rubiaceae) in 20131 garnered substantial attention from the The Scilloideae (Asparagaceae) is a subfamily of bulbous scientific community in what would later be called “The containing approximately 900 across 70 genera. Tramadol Wars” by medicinal chemist and columnist Derek The chemistry of the Scilloideae has been recently reviewed, Lowe.2 The authors were met with skepticism by their revealing it to be particularly rich in homoisoflavones, conclusion that tramadol was being produced naturally,3 and, bufadienolides, cardenolides, and steroidal glycosides. In 4 even after a convincing biosynthetic proposal, it was terms of alkaloids, pyrrolizidines, pyrrolidines, and piperidines ultimately determined that the tramadol was an anthropogenic are particularly common.9 However, the structure of 2,4-(4′- ff Downloaded via UPPSALA UNIV on July 2, 2020 at 07:33:39 (UTC). contaminant arising from its o -label administration to aminobenzenamine)pyrimidine (1), an alkaloid purportedly 5 livestock in the region. The tramadol story has since served isolated from the bulbs of S. madeirensis,10 appeared to us as as a warning to natural products chemists to be wary of the slightly unusual, with no structurally similar compounds potential isolation of synthetic contaminants from natural previously reported from the Scilloideae. sources. This lesson is especially important in an age where Upon further investigation, we were unable to corroborate See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. environmental drug contamination, most notably from those 6 7 the proposed structure of 1 with the reported NMR used in the agricultural sector, is very high. spectroscopic data (Figure 1; Table 1). Of particular note is The lessons learned from the tramadol incident were ′ ′ δ the deshielded chemical shift of C-2 /C-6 ( C 130.2) considered by Hassan et al. in 2017 after their isolation of ′ ′ δ compared to C-3 /C-5 ( C 112.5), which is not consistent the antidiabetes drug metformin from a specimen of Seidlitzia 11 ′ δ 8 with a p-phenylenediamine moiety. Moreover, C-4 ( C rosmarinus (Amaranthaceae). The authors quickly determined 153.4) is too deshielded to be an amine-bound aromatic that the metformin was an anthropogenic contaminant by carbon para to an amino group.12 One explanation for the analyzing its 14C content, the same method used to prove the ′ ′ ′ 5 highly uneven chemical environments of C-2 /C-6 and C-3 / synthetic origin of tramadol from N. latifolia in 2016. The C-5′ is a sulfonamide group linking C-1′ and C-2. This also examples of tramadol and metformin notwithstanding, to the best of our knowledge, no other cases involving the attribution of synthetic drug contaminants as natural products have been Received: February 13, 2020 reported. Herein, we report what appears to be another Published: March 25, 2020 example of the isolation of an anthropogenic environmental contaminant (sulfadiazine) and its mistaken identity as a plant natural product from Scilla madeirensis (Asparagaceae, synonym Autonoëmadeirensis). These conclusions were met

© 2020 American Chemical Society and American Society of Pharmacognosy https://dx.doi.org/10.1021/acs.jnatprod.0c00163 1305 J. Nat. Prod. 2020, 83, 1305−1308 Journal of Natural Products pubs.acs.org/jnp Note

Figure 2. Proposed ions of sulfadiazine (2) formed after the loss of 13 SO2 (m/z 186) and HSO2 (m/z 185)

eV) of synthetic 1 show the authors’ predicted base [M − H]+ peak at m/z 185, while the molecular ion is observed at m/z Figure 1. Proposed (1) and revised structure of sulfadiazine (2). 186 (rel int 96%) (Supporting Information). Although we were unable to obtain any of the original justifies the deshielded chemical shift of C-4′.12 The presence isolated compound or plant material, the site of the plant of a sulfonamide group at this position would identify 1 as the collection, the Botanical Garden of Funchal, Madeira, Portugal, antibiotic sulfadiazine (2). supplied two freshly collected samples of S. madeirensis bulbs. NMR analyses of commercially acquired sulfadiazine LC-MS analyses of their MeOH and CH3CN/H2O (1:4) demonstrate that it shares identical spectroscopic data to extracts did not show any molecular ions associated with either those originally reported for 1 (Table 1).10 As attempts to 1, 2, or the main mammalian metabolites of sulfadiazine (4- obtain samples from the original publication were unsuccessful, hydroxysulfadiazine, N-acetylsulfadiazine).15 The original we synthetically prepared a standard of 1. The acquired NMR collection appears to have been performed approximately 20 data underscore the clear spectroscopic differences between years ago,10,16 and thus there are a litany of potential reasons synthetic 1 and sulfadiazine (Table 1). The most prominent (e.g., decomposition over time, new specimens being planted, differences in the 13C NMR data are the chemical shifts of C- changes in horticultural practices) that sulfadiazine was not 2′/C-6′ and C-4′, which are shielded by 8.5 and 10.3 ppm, observed in the current study. However, given that sulfadiazine respectively, in comparison to sulfadiazine. has never previously been reported as a natural product, we We then sought to determine whether the EIMS data were led to the hypothesis that this is another example of an support our structural revision. The original authors reported a anthropogenic contaminant being mistakenly identified as a base peak observed in EIMS (70 eV) at m/z 185, which was natural product. ascribed as the [M − H]+ ion of 1, while the molecular ion was Sulfadiazine is a known environmental contaminant due to proposed to be at m/z 186 (rel int 61%).10 Previously its widespread use in veterinary medicine.6 Its low bioavail- published EIMS data of sulfadiazine report that its molecular ability leads to excretion in animal feces and urine, which can ion peak occurs at m/z 250 (rel int 0.3%), while the base peak then be spread throughout the environment as fertilizer.17 − + 18 is observed at m/z 185 [M HSO2] . A prominent peak also Studies on willow (Salix fragilis L.), maize (Zea mays L.), and − + 13,14 19 occurs at m/z 186 [M SO2] (Figure 2). The low wheat (Triticum aestivum L.) growing in sulfadiazine-spiked intensity of the molecular ion peak of sulfadiazine appears to soil have demonstrated that sulfadiazine may accumulate in haveunderstandablycomplicated the assignment of its plant roots and even be translocated to stems and leaves. molecular formula. Our acquired EIMS data of sulfadiazine Natural products containing a sulfonamide moiety are also (Supporting Information) match with both previously quite rare. They are predominantly produced by bacteria20,21 published data for sulfadiazine13,14 and the originally reported but have also been isolated from marine invertebrates.22,23 To EIMS data of 1.10 Interestingly, our acquired EIMS data (70 the best of our knowledge, no sulfonamide-containing natural

Table 1. Comparison of the Previously Reported NMR Data of 2,4-(4′-Aminobenzenamine)pyrimidine (1)10 with Those of d Synthetic 1 and Commercially Acquired Sulfadiazine (2) (All Data Are Reported in DMSO- 6) 2,4-(4′-aminobenzenamine)-pyrimidine (1) 2,4-(4′-aminobenzenamine)-pyrimidine (1) (reported by Dias et al.)10 (synthetic) sulfadiazine (2) (commercially acquired) δ a δ b δ c δ d δ e δ f position C, type H (J in Hz) C, type H (J in Hz) C, type H (J in Hz) 1NNN 2 157.6, C 160.4, C 157.2, C 2-NH NH 11.26, brs NH 9.07, brs NH 11.24, brs 3NNN 4 158.6, CH 8.47, d (4.8) 157.8, CH 8.34, d (4.6) 158.2, CH 8.47, d (4.8) 5 115.8, CH 7.00, t (4.8) 111.1, CH 6.67, t (4.6) 115.5, CH 6.99, t (4.8) 6 158.6, CH 8.47, d (4.8) 157.8, CH 8.34, d (4.6) 158.2, CH 8.47, d (4.8) 1′ 125.2, C 130.0, C 124.9, C 2′ 130.2, CH 7.61, d (8.7) 121.3, CH 7.32, d (8.4) 129.8, CH 7.62, d (8.8) 3′ 112.5, CH 6.56, d (8.7) 114.3, CH 6.54, d (8.4) 112.1, CH 6.57, d (8.8) 4′ 153.4, C 142.7, C 153.0, C ′ 4 -NH2 NH2 6.00, s NH2 5.18, brs NH2 5.98, s 5′ 112.5, CH 6.56, d (8.7) 114.3, CH 6.54, d (8.4) 112.1, CH 6.57, d (8.8) 6′ 130.2, CH 7.61, d (8.7) 121.3, CH 7.32, d (8.4) 129.8, CH 7.62, d (8.8) a100 MHz. b400 MHz. c200 MHz. d600 MHz. e125 MHz. f500 MHz.

1306 https://dx.doi.org/10.1021/acs.jnatprod.0c00163 J. Nat. Prod. 2020, 83, 1305−1308 Journal of Natural Products pubs.acs.org/jnp Note products have been isolated from or observed in terrestrial of 5 mg/mL and subject to LC-MS analysis. The mobile phase used plants. Given this and the known capacity for sulfadiazine to was a linear gradient from 1% to 90% CH3CN (0.1% FA) over 50 min at a flow rate of 0.3 μL/min. Data were analyzed using MZmine 2.26 bioaccumulate in plants, it is most likely that its isolation from 2,4-(4′-Aminobenzenamine)pyrimidine (1). S. madeirensis is a consequence of anthropogenic contami- Synthesis of 1 was performed using a method patented by Jautelat et al. (2008).27 A nation at the site of collection. This is supported by our failure suspension of 4-nitroaniline (28 mg, 0.20 mmol) and 2-chloropyr- to redetect it from fresh plant samples. 14 imidine (23 mg, 0.20 mmol) in acetonitrile (2 mL) was treated with Analysis of radiocarbon ( C) levels provides an exper- HCl (4 M in dioxane, 50 μL) and water (50 μL). The reaction imental platform to distinguish between carbon atoms recently mixture was then refluxed for 16 h before cooling to room incorporated into a molecule of interest via photosynthesis and temperature. Triethylamine (34 μL, 0.24 mmol) was added, and the those derived from petroleum-based precursors, where 14C resulting solid washed with acetonitrile and water and then dried nuclides have long since decayed.5,8 Unfortunately, the lack of under vacuum overnight. The solid was then suspended in ethyl fl access to the originally isolated sample or plant material acetate and ethanol (1:1, 4 mL), and the vessel ushed with nitrogen. precludes this analysis, and we cannot definitively rule out the Pd/C 10% (5 mg) was added, and the reaction placed under a hydrogen atmosphere. After stirring for 20 h, the reaction mixture was possibility that sulfadiazine was biosynthesized by S. filtered through Celite, and the filtrate concentrated to afford the title madeirensis or an associated microorganism. α compound (26 mg, 69%). The original authors also report that 1 is active as an 1- adrenoceptor antagonist.10 This activity should instead be ■ ASSOCIATED CONTENT attributed to sulfadiazine. To the best of our knowledge, *sı Supporting Information sulfadiazine has not been previously reported as an α-blocker. α The Supporting Information is available free of charge at Several clinically used -blockers (e.g., prazosin, terazosin, https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00163. alfuzosin, doxazosin) contain a core 2,4-diaminoquinazoline ′ functionality, and sulfadiazine shares some structural similar- NMR and EIMS spectra for synthetic 2,4-(4 - ities with these drugs.24 aminobenzenamine)pyrimidine (1) and commercially These findings illustrate the importance of considering the acquired sulfadiazine (2)(PDF) biosynthetic origins of newly discovered natural products within a phylogenetic context. Our attention was drawn to 1 ■ AUTHOR INFORMATION due to its structural dissimilarity to any other alkaloids isolated Corresponding Author from the Scilloideae, which is typically known to produce Luke P. Robertson − Plant Ecology and Evolution, Department alkaloids of the pyrrolizidine, pyrrolidine, and piperidine of Ecology and Genetics, Evolutionary Biology Centre and types.9 These alkaloids are biosynthesized by a limited set of Pharmacognosy, Department of Medicinal Chemistry, precursors (L-ornithine in the case of pyrrolizidine and Biomedical Centre, Uppsala University, 75236 Uppsala, 25 pyrrolidine alkaloids; L-lysine for piperidines), and it is Sweden; orcid.org/0000-0001-5987-2426; challenging to propose a biosynthetic pathway for 1 within this Email: [email protected] framework. While the structures of new natural products should always be thoroughly validated, particular scrutiny must Authors Lindon W. K. Moodie − be paid to proposed structures that deviate considerably from Drug Design and Discovery, those known to occur within a taxonomic group. Department of Medicinal Chemistry, Biomedical Centre and Uppsala Antibiotic Centre, Uppsala University, 75123 Uppsala, EXPERIMENTAL SECTION Sweden ■ Darren C. Holland − Environmental Futures Research Institute, General Experimental Procedures. NMR spectra were acquired Griffith University, 4222 Gold Coast, Australia; orcid.org/ 13 15 1 at 298 K on a Bruker 500 MHz (TXO CRPHe TR- C/ N/ H5 0000-0002-2498-6084 mm-Z CryoProbe) and a Bruker 600 MHz (TCI CRPHe TR-1H and K. Charlotte Jandeŕ− Plant Ecology and Evolution, 19F/13C/15N 5 mm-EZ CryoProbe) spectrometer. Chemical shifts δ δ Department of Ecology and Genetics, Evolutionary Biology were referenced to the solvent peak for (CD3)2SO at H 2.50 and C Centre, Uppsala University, 75236 Uppsala, Sweden 39.52. GC-MS analyses were performed on an Agilent Technologies Ulf Goransson̈ − 7890A GC system equipped with an Agilent Technologies 7693 Pharmacognosy, Department of Medicinal autosampler and an Agilent Technologies 5975 Series MSD. A linear Chemistry, Biomedical Centre, Uppsala University, 75123 gradient from 70 to 300 °C was employed, and EI was set at 70 eV. Uppsala, Sweden × The column used was a CP-SIL 8 CB Low Bleed (30 m 0.25 mm) Complete contact information is available at: capillary column. High-resolution mass spectrometric measurements https://pubs.acs.org/10.1021/acs.jnatprod.0c00163 were acquired using a UPLC-QToF nanospray MS (Waters nanoAcquity, QToF Micro). The UPLC column used was a Waters μ Notes ACQUITY UPLC M-Class Peptide BEH C18 column (1.7 m, 130 Å, μ × The authors declare no competing financial interest. 75 m 150 mm). DMSO-d6 (99.8%) was from Teknolab Sorbent. Sulfadiazine (≥99.0%) was purchased from Sigma-Aldrich. Plant Material. S. madeirensis material was collected and identified ■ ACKNOWLEDGMENTS in September 2019 at the Botanical Garden of Funchal, Madeira, L.P.R. and K.C.J. acknowledge the Wenner-Gren Foundations Portugal, by Francisco Manuel Fernandes. Voucher specimens for scholarship provision. The authors thank L. Odell and B. (MADJ 14346 and MADJ 14347) are housed at the Botanical Skillinghaug for assistance with GC-MS analysis. L.W.K.M. Garden of Funchal, Madeira, Portugal. would like to acknowledge the Uppsala Antibiotic Centre. The Extraction and LC-MS Analysis. Small portions (1 g) of oven- − dried (40 °C) and ground S. madeirensis bulbs (100 g) were NMR Uppsala infrastructure, Department of Chemistry BMC exhaustively extracted via sonication (20 min) in MeOH (20 mL) and and Disciplinary Domain of Medicine and Pharmacy, Uppsala 15 then 1:4 CH3CN/H2O (20 mL). The samples were evaporated, University, is acknowledged. The authors also thank Francisco filtered (0.45 μm), and redissolved in 20% MeOH at a concentration Manuel Fernandes and the Botanical Garden of Funchal,

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Madeira, Portugal, for provision of samples. We credit Tim Entwisle as the owner of the image in the graphical abstract. ■ REFERENCES (1) Boumendjel, A.; Sotoing Taïwe, G.; Ngo Bum, E.; Chabrol, T.; Beney, C.; Sinniger, V.; Haudecoeur, R.; Marcourt, L.; Challal, S.; Ferreira Queiroz, E. Angew. Chem., Int. Ed. 2013, 52, 11780−11784. (2) Lowe, D. The Tramadol Wars. https://blogs.sciencemag.org/ pipeline/archives/2015/06/25/the_tramadol_wars (accessed Feb 6, 2020). (3) Kusari, S.; Tatsimo, S. J. N.; Zühlke, S.; Talontsi, F. M.; Kouam, S. F.; Spiteller, M. Angew. Chem. 2014, 126, 12269−12272. (4) Romek, K. M.; Nun, P.; Remaud, G. S.; Silvestre, V.; Taïwe, G. S.; Lecerf-Schmidt, F.; Boumendjel, A.; De Waard, M.; Robins, R. J. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 8296−8301. (5) Kusari, S.; Tatsimo, S. J. N.; Zühlke, S.; Spiteller, M. Angew. Chem. 2016, 128, 248−251. (6) Bartíková ,́ H.; Podlipna,́ R.; Skalová ,L.́ Chemosphere 2016, 144, 2290−2301. (7) Zuccato, E.; Calamari, D.; Natangelo, M.; Fanelli, R. Lancet 2000, 355, 1789−1790. (8) Hassan, A. R.; El-Kousy, S. M.; El-Toumy, S. A.; Frydenvang, K.; Tung, T. T.; Olsen, J.; Nielsen, J.; Christensen, S. B. J. Nat. Prod. 2017, 80, 2830−2834. (9) Mulholland, D. A.; Schwikkard, S. L.; Crouch, N. R. Nat. Prod. Rep. 2013, 30, 1165−1210. (10) Dias, C.; Paulo, A.; Nascimento, J.; Houghton, P.; Hawkes, J. E.; Goncalves,̧ M. L. Planta Med. 2003, 69, 1060−1062. (11) Rodriguez, F.; Rozas, I.; Ortega, J. E.; Erdozain, A. M.; Meana, J. J.; Callado, L. F. J. Med. Chem. 2009, 52, 601−609. (12) Pretsch, E.; Bühlmann, P.; Badertscher, M. Structure Determination of Organic Compounds: Tables of Spectral Data; Springer: Berlin, 2009. (13) Spiteller, G.; Kaschnitz, R. Monatsh. Chem. 1963, 94, 964−980. (14) Bulk, A.; Plug, C. M. Silver Sulfadiazine. In Analytical Profiles of Drug Substances, Vol. 13; Florey, K., Ed.; Academic Press: Orlando, 1984; pp 553−571. (15) Förster, M.; Laabs, V.; Lamshöft, M.; Pütz, T.; Amelung, W. Anal. Bioanal. Chem. 2008, 391, 1029−1038. (16) Paulo, A.; Dias, C.; Jimeno, M. L.; Borges, C.; Nascimento, J. Fitoterapia 2005, 76, 765−767. (17) Schauss, K.; Focks, A.; Heuer, H.; Kotzerke, A.; Schmitt, H.; Thiele-Bruhn, S.; Smalla, K.; Wilke, B.-M.; Matthies, M.; Amelung, W.; Klasmeier, J.; Schloter, M. TrAC, Trends Anal. Chem. 2009, 28, 612−618. (18) Michelini, L.; Reichel, R.; Werner, W.; Ghisi, R.; Thiele-Bruhn, S. Water, Air, Soil Pollut. 2012, 223, 5243−5257. (19) Grote, M.; Schwake-Anduschus, C.; Michel, R.; Stevens, H.; Heyser, W.; Langenkamper, G.; Betsche, T.; Freitag, M. Land- bauforschung Volkenrode 2007, 57, 25. (20) Takahashi, A.; Kurasawa, S.; Ikeda, D.; Okami, Y.; Takeuchi, T. J. Antibiot. 1989, 42, 1556−1561. (21) Kim, K. K.; Kang, J. G.; Moon, S. S.; Kang, K. Y. J. Antibiot. 2000, 53, 131−136. (22) Peters, L.; König, G. M.; Terlau, H.; Wright, A. D. J. Nat. Prod. 2002, 65, 1633−1637. (23) Exposito, M.; Lopez,́ B.; Fernandez,́ R.; Vazquez, M.; Debitus, C.; Iglesias, T.; Jimenez,́ C.; Quinoa, E.; Riguera, R. Tetrahedron 1998, 54, 7539−7550. (24) Narayan, P.; Tewari, A. Urology 1998, 51,38−45. (25) Aniszewski, T. Alkaloids - Secrets of Life; Elsevier: Netherlands, 2007; pp 62−63. (26) Pluskal, T.; Castillo, S.; Villar-Briones, A.; Oresič,M.̌ BMC Bioinf. 2010, 11, 395. (27) Jautelat, R.; Siemeister, G.; Luecking, U. WIPO Patent WOWO/2008/074515, 2008.

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