Metformin, an Anthropogenic Contaminant of Seidlitzia rosmarinus

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Metformin, an Anthropogenic Contaminant of Seidlitzia rosmarinus Collected in a Desert Region near the Gulf of Aqaba, Sinai Peninsula Ahmed R. Hassan,†,∥ Salah M. El-Kousy,‡ Sayed A. El-Toumy,§ Karla Frydenvang,† Truong Thanh Tung,† Jesper Olsen,⊥ John Nielsen,*,† and Søren Brøgger Christensen*,† †

Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark Chemistry of Tannins Department, National Research Centre, Dokki 12622, Cairo, Egypt § Chemistry Department, Menoufia University, Shebin El-Kom 32861, El-Menoufia, Egypt ⊥ Department of Physics and Astronomy, Aarhus University, DK-8000, Aarhus C, Denmark ∥ Medicinal and Aromatic Plants Department, Desert Research Center, El-Matariya 11753, Cairo, Egypt ‡

S Supporting Information *

ABSTRACT: A phytochemical investigation of Seidlitzia rosmarinus collected along the shoreline of the Gulf of Aqaba in the remote southern desert region of the Sinai peninsula has revealed the presence of the registered drug metformin (4). However, analysis of the 14C content revealed the drug to be an anthropogenic contaminant. Consequently, natural product researchers should be aware that compounds isolated from plants might originate from environmental contamination rather than biosynthesis. The new natural product N-(4-hydroxyphenylethyl)-α-chloroferuloylamide was isolated as a mixture of the E and Z isomers along with a number of other well-established secondary metabolites.

M

Tramadol (2), a synthetic painkiller designed based on the structure of morphine (3), was found in roots of Nauclea latifolia6 but was later found to be an anthropogenic contaminant.7 The discovery of the true origin of tramadol in this tree should be very instructive to natural products researchers. The increased use of pharmaceuticals in humans and in domesticated animals results in an increased excretion of their metabolites or in the unchanged form. Agriculture, aquaculture, and increased use of pharmaceuticals in humans consequently afford the potential for contamination of the environment inclusive of landfill and wastewater treatment plants.8 The increasing amounts of unmodified pharmaceuticals and their metabolites in the environment cause an increased risk for the anthropogenic contamination of local plants and animals. The origin of compounds found in plants also relates to an ongoing discussion as to whether some metabolites are produced by the plant or associated microorganisms such as its endophytes.9 The development of metformin (4) was based on the traditional use of the leaves from Galega off icinalis (Fabaceae) for the treatment of polyuria and halitosis, most likely symptoms associated with type 2 diabetes.10 A debate is still ongoing as to whether this effect is caused by galegine [Ndimethylallylguanidine (5)]11 or guanidines.12 Metformin was first described in 1922.13 Even though the drug has been used successfully for the treatment of type 2 diabetes in the U.K. and

any examples of natural products used as drugs can be found in textbooks of pharmacognosy1 and natural products chemistry2 or in major reviews.3 Prominent examples include the anticancer drugs paclitaxel, vincristine, and vinblastine. Moreover, natural products are highly important as lead compounds for the development of new drugs. Newman and Cragg have determined that a third of all drugs developed in the period 1981 to 2010 originated from living organism sources, but only 6% were unmodified structurally, whereas 28% were modified natural products.3 Among the drugs prepared by total synthesis, 20% were compounds inspired by natural products molecules.3 In contrast, there appears to be only one example of a drug developed synthetically that subsequently turned out to be an authentic natural product, namely, 5-fluorouracil (1). 5-Fluorouracil is a chemotherapeutic agent that was established as a constituent of a marine sponge long after the drug had been synthesized and registered.4

Figure 1. Structures of 5-fluorouracil (1), tramadol (2), and morphine (3). Whereas morphine always is used as the enantiomer (3), tramadol is used as a (1R,2R) and (1S,2S) racemic mixture since this is superior clinically to the pure enantiomers.5 © 2017 American Chemical Society and American Society of Pharmacognosy

Received: February 21, 2017 Published: September 20, 2017 2830

DOI: 10.1021/acs.jnatprod.7b00106 J. Nat. Prod. 2017, 80, 2830−2834

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the 14C content by high-precision accelerator mass spectrometry (AMS). The carbon source for metabolites formed in plants almost exclusively is atmospheric CO2, and consequently the 14C content must equal the content of the contemporary atmosphere. In contrast, the carbon source of chemically synthesized metformin is fossil coal, in which the 14C atoms have decayed.7b Hence, by analyzing the 14C of the isolated metformin in the plant it is possible to distinguish sources of carbon for the metformin synthesis. If the carbon source for metformin synthesis is coal, the expected fraction modern ratio, F14C, would be much lower than 1.03, the value for the contemporary atmosphere. However, if the contemporary sources of carbon are in equilibrium with the atmosphere via photosynthetic uptake of CO2, contemporary sources of carbon will result in an F14C value equal to the 14C content of the contemporary atmosphere. This analysis revealed that the fraction modern F14C ratio was 0.0056 ± 0.0004 in the isolated metformin hydrochloride. This number is very close to zero, indicating an age of more than 50 000 years of the carbon source of this compound. This finding was substantiated by determining the fraction modern F14C ratio of metformin isolated from a Sandoz metformin tablet to be F14C = 0.0013 ± 0.0003, very similar to that isolated from S. rosmarinus. In contrast the ratio of N-E-feruloyltyramine (compound 7) also isolated from the plant was F14C = 1.0308 ± 0.0039, equal to the content of the contemporary atmosphere, proving that the carbon source is atmospheric CO2. Consequently, compound 7 is a product formed in the plant. The finding of metformin as an anthropogenic contaminant in a plant growing in the desert region on the remote shoreline of the Gulf of Aqaba, South Sinai Peninsula, might be related to the extensive use of metformin as an oral diabetes medicine in Egypt. Recent studies have revealed that 16% of the population in Egypt, corresponding to 10 million persons, suffer from type 2 diabetes.20 Since metformin is the drug of first choice with a daily dose up to 2.5 g and with more than 90% of the drug being secreted unconverted with the urine,10,21 a substantial amount of the drug must occur in Egyptian soil, rivers, and possibly the seas around Egypt. This could explain the presence of synthesized metformin in substantial amounts in plants since it has previously been established that metformin may accumulate in plants.22 A review on contamination of rivers, lakes, and even oceans in or around the U.S.A. has revealed that pharmaceuticals are emerging contaminants since they are observed in substantial amounts in animals living in the surface water.23 In particular, metformin has been detected as a contaminant in soil24 and in German rivers and as far away as 200 km from the coast of Germany.8,25 In contrast, no systematic search for pharmaceuticals as xenobiotics in plants has been performed. Besides metformin, three N-phenethylferuloylamide derivatives (6−8) were isolated from fractions of the extract along with salicylic acid,26 rutin,27 isorhamnetin-3-O-robinbioside,28 isorhamnetin-3-O-rutinoside (narcissin),28 isorhamnetin-3-O-β28 D-glucopyranoside, quercetin, 3,4-dihydroxybenzoic acid,29 30 and ferulic acid. The spectroscopic data for these wellestablished compounds were in agreement with those published. Likewise NMR spectra of 7 and 8 were in accordance with those reported.31 The 1H NMR spectrum (Figure S8, Supporting Information) of one of the isomers of 6 was very similar to that of 7, except for the signals of the vinylic protons. In the spectrum of 6, only one singlet at 7.73 ppm was found in this region. Inspection of the MS (Figure S14,

Canada since 1958 and since 1972 in the U.S.A., it was not until 2012 that it was established as a drug of first choice.10 A daily dosage can reach up to 2.5 g. The half-life of the drug in the human body is about 20 h, and the drug leaves the body through the urine without metabolism. Its pharmacological mechanism of action is still not understood in detail.14

Figure 2. Structures of metformin (4) and galegine (5).

As a part of a search for useful natural products in plants growing in the Egyptian desert, the aerial parts of Seidlitzia rosmarinus Bunge ex Boiss. were investigated. Some screening procedures for different biological activities of the aerial parts have been performed previously, but no in-depth phytochemical investigation has occurred.15 The plant grows in saline marshes along the shoreline of the Gulf of Aqaba on the south Sinai peninsula and is not found anywhere else in Egypt.16 Extraction with EtOH−H2O afforded an extract, which was concentrated and then fractionated by chromatography. One of the fractions was concentrated to give a crystalline compound. The 1H and 13C NMR data (Figures S1−S5, Supporting Information) were in agreement with those published for metformin17 except for the presence of signals that were assigned for an acetate ion. Since the presence of many nitrogen atoms prevented a valid establishment of the connectivity, a crystal was investigated by X-ray analysis, proving the suggested structure (Figure 3). Previously, a similar structure of metformin has been published.18

Figure 3. Perspective drawing (ORTEP-3)19 of the acetate of metformin (4). Displacement ellipsoids of the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are represented by spheres of arbitrary size. Oxygen atoms are colored red and nitrogen atoms blue.

To ensure that no contamination had occurred during fractionation, the NMR spectrum of the extract obtained after defatting of the crude plant extract was recorded. A singlet at 3.04 ppm originating from the two methyl groups in metformin was observed (Figure S7, Supporting Information). Addition of authentic metformin to the sample analyzed by NMR resulted in an increase of intensity of only this peak and no other changes in the spectrum, which would be consistent with the presence of metformin in the crude extract. Realizing that no biguanidines have previously been reported as natural products, we decided to establish if the compound was a genuine metabolite or an anthropogenic contaminant. An unequivocal procedure to resolve this question is to measure 2831

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distilled H2O, subsequently adding MeOH until 100% MeOH to afford four fractions, A−D. Fraction B (8.6 g) was further fractionated by passage over a Sephadex LH-20 column (MeOH−H2O, 20% to 100%, v/v) to provide three subfractions, B1−B3. The subfraction B1 (2.1 g) was rechromatographed by passage over a Sephadex LH-20 column using n-butanol (n-BuOH) saturated with H2O as eluent to obtain two subfractions, B1a and B1b. The first subfraction, B1a (1.3 g), was further subjected to a Sephadex LH-20 column and eluted with EtOH−H2O (7:3) to yield 3,4-dihydroxybenzoic acid (10 mg) and 110 mg of a mixture of two isomers (isorhamnetin-3-O-robinbioside, isorhamnetin-3-O-rutinoside) in the approximate ratio 1:2. The second subfraction, B1b (0.3 g), was also loaded onto a Sephadex LH-20 column using the same eluent to afford rutin (27 mg). Subfraction B2 (0.4 g) was further subjected to silica gel column chromatography and eluted with CH2Cl2 as the initial eluent, gradually increasing the polarity by addition of MeOH until 20% of MeOH in CH2Cl2 to afford two fractions, B2a (35 mg) and B2b (20 mg). Subfraction B2a was separated on a Biotage SP1 flash chromatography purification system by passage over a Biotage Snap Ultra 10 g cartridge, by eluting with CH2Cl2−EtOAc (1:1) to afford two fractions, each consisting of two isomers. The first fraction consisted of a mixture of the two isomers 6E and 6Z (4.8 mg) in an approximately 1:2 proportion based on 1H NMR spectra. The two isomers can isomerize under UV light, suggesting E/Z photoisomerization as a possible mechanism during sample extraction and/or chromatography. The second fraction consisted of a mixture of the two isomers 7E and 7Z (8.9 mg). Subfraction B2b was separated in the same way to afford a mixture of 7E and 7Z (2.7 mg) and a mixture of the two isomers 8E and 8Z (4.7 mg). The subfraction B3 (0.1 g) was purified by passage over a Sephadex LH-20 column using n-BuOH saturated with H2O as eluent to yield isorhamnetin-3-O-β-Dglucopyranoside (15 mg). Fraction C (4.2 g) was further fractionated by passage over a Sephadex LH-20 column (MeOH−H2O, 20% to 100%, v/v) to afford two subfractions, C1 and C2. Fraction C1 (0.96 g) was purified on a Sephadex LH-20 column using n-butanol saturated with H2O as eluent to yield compound 4 (240 mg). Fraction C2 (0.15 g) was separated on a Sephadex LH-20 column using as eluent nBuOH saturated with H2O to afford ferulic acid (3.2 mg) and salicylic acid (15.7 mg). Fraction D (0.9 g) was separated on a Sephadex LH20 column using n-BuOH saturated with H2O as eluent and further purified by passage over a Sephadex LH-20 column, by eluting with MeOH−H2O (20% to 100%, v/v) to yield quercetin (19 mg). 2-Chloro-N-Z-feruloyltyramine (6Z): 1H NMR (600 MHz, CD3OD) δ 7.73 (1H, s, H-3), 7.49 (1H, d, J = 1.9 Hz, H-5), 6.85 (1H, d, J = 8.3 Hz, H-8), 7.33 (1H, dd, J = 8.4, 1.9 Hz, H-9), 3.49 (2H, t, J = 7.6 Hz, H-1′), 2.77 (2H, t, J = 7.6 Hz, H-2′), 7.06 (2H, d, J = 8.5 Hz, H-4′/H-8′), 6.72 (2H, d, J = 8.5 Hz, H-5′/H-7′), 3.88 (3H, s, OCH3-6); 13C NMR (150 MHz, CD3OD) δ 165.36 (C-1), 121.56 (C2), 134.54 (C-3), 126.23 (C-4), 114.71 (C-5), 148.79 (C-6), 148.93 (C-7), 116.28 (C-8), 126.27 (C-9), 43.30 (C-1′), 35.59 (C-2′), 131.10 (C-3′), 130.80 (C-4′/C-8′), 116.30 (C-5′/C-7′), 156.97 (C-6′), 56.42 (OCH3-6); HRESIMS m/z 346.0848 [M − H]− (calcd for C18H17ClNO4 346.0846). 2-Chloro-N-E-feruloyltyramine (6E): 1H NMR (600 MHz, CD3OD) δ 6.83 (1H, s, H-3), 6.96 (1H, d, J = 1.8 Hz, H-5), 6.74 (1H, d, J = 8.2 Hz, H-8), 6.80 (1H, dd, J = 8.2, 1.5 Hz, H-9), 3.37 (2H, t, J = 7.6 Hz, H-1′), 2.66 (2H, t, J = 7.6 Hz, H-2′), 6.95 (2H, d, J = 8.5 Hz, H-4′/H-8′), 6.67 (2H, d, J = 8.5 Hz, H-5′/H-7′), 3.80 (3H, s, OCH3-6); 13C NMR (150 MHz, CD3OD) δ 167.52 (C-1), 123.94 (C2), 132.60 (C-3), 126.80 (C-4), 112.60 (C-5), 148.88 (C-6), 148.63 (C-7), 116.26 (C-8), 123.25 (C-9), 42.77 (C-1′), 35.14 (C-2′), 130.87 (C-3′), 130.70 (C-4′/C-8′), 116.29 (C-5′/C-7′), 156.93 (C-6′), 56.37 (OCH3-6); HRESIMS m/z 346.0849 [M − H]− (calcd for C18H17ClNO4 346.0846). Accelerator Mass Spectrometry Analysis.33 Metformin HCl and compound 7 were combusted to CO2 at 900 °C with CuO in sealed evacuated tubes. The CO2 was reduced by hydrogen to produce graphite at 550 °C using Fe as a catalyst. The mixed Fe and graphite powder was pressed into targets, which were analyzed with AMS. The radiocarbon content of the sample was calculated as the measured

Supporting Information) clearly indicated the presence at a chlorine atom (a pseudomolecular peak at m/z 348.0826 with one-third of the intensity of the pseudomolecular peak at m/z 346.0848). The shift values of the vinylic proton revealed that it was located in the β-position. Compound 6 was obtained as a mixture of the E and Z isomers. Compound 6, consequently, is an additional example of a halogenated compound isolated from a terrestrial plant.32

Figure 4. Structures of N-phenethylferuloylamides isolated from S. rosmarinus.

In conclusion, a phytochemical investigations on the halophyte S. rosmarinus has revealed that N,N-dimethylguanidine (metformin) is an anthropogenic contaminant. The absence of 14C in this compound unequivocally proves that the compound is synthesized from starting materials originating from fossil materials. In addition, S. rosmarinus forms chlorinecontaining feruloylphenethylamides. The increased use of pharmaceuticals for treatment of patients and animals in agriculture and aquaculture is an increasing load of our environment.8,23−25 It might lead to finding synthetic compounds in organisms found even at remote locations. Thus, natural product researchers should be careful not to confuse an anthropogenic contaminant with a secondary metabolite.

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EXPERIMENTAL SECTION

General Experimental Procedures. The NMR spectra were recorded on Bruker Advance 400 and 600 MHz spectrometers. Standard Bruker software was used for the 2D NMR spectra. HRESIMS were recorded on a Bruker micrOTOF-Q instrument using electrospray ionization, in the negative mode. Accelerator mass spectrometry was performed on the HVE 1MV Tandetron accelerator at Department of Physics and Astronomy, Aarhus University. Column chromatography was performed over polyamide 6S or Sephadex LH20. Plant Material. The aerial parts of Seidlitzia rosmarinus were collected in November 2012 from the Red Sea coast, South Sinai, Egypt, near the shoreline of the Gulf of Aqaba (Nuweiba−Taba) road, approximately 12 km from Nuweiba (geographic position coordinates N: 29°15′34.97″; E: 34°43′33.69″) and identified by Dr. Attia Mohamed, Desert Research Center, Cairo, Egypt. A voucher specimen of the plant has been deposited at the Herbarium of Desert Research Center, Ministry of Agriculture, Cairo, Egypt (S. rosmarinus 30-112012-S). Extraction and Isolation. The aerial parts of S. rosmarinus plant were air-dried in the shade and ground to a fine powder. The powder (1.9 kg) was submitted to exhaustive maceration five times utilizing 70% ethanol in a water bath at 40 °C for 48 h. The combined ethanol solutions were concentrated in a rotary evaporator under reduced pressure until dryness, affording a residue of 120 g. The residue was dissolved in 500 mL of distilled H2O acidified by 5% acetic acid and defatted by partitioning the aqueous suspension with petroleum ether. The aqueous phase was concentrated to 140 mL. Then, a small amount of polyamide 6S was added and the solvent was removed. The residue was applied on a polyamide column, which was eluted with 2832

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ed.; Elsevier: Edinburgh, 2009. (c) Bruneton, J. Pharmacognosy, 2nd ed.; Intercept Ltd: Hampshire, UK, 1999. (2) Dewick, P. M. Medicinal Natural Products, 3rd ed.; John Wiley and Sons Ltd.: Chicester, UK, 2009. (3) (a) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335. (b) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629−661. (4) Xu, X.-H.; Yao, G.-M.; Li, Y.-M.; Lu, J.-H.; Lin, C.-J.; Wang, X.; Kong, C.-H. J. Nat. Prod. 2003, 66, 285−288. (5) Buschmann, H. In Analogue-Based Drug Discovery III; Ganellin, C. R.; Rotella, D. P., Eds.; Wiley-VCH Verlag GmbH & Co.: Weinheim, Germany, 2013; pp 295−318. (6) (a) Boumendjel, A.; Sotoing Taiwe, G.; Ngo Bum, E.; Chabrol, T.; Beney, C.; Sinniger, V.; Haudecoeur, R.; Marcourt, L.; Challal, S.; Ferreira Queiroz, E.; Souard, F.; Le Borgne, M.; Lomberget, T.; Depaulis, A.; Lavaud, C.; Robins, R.; Wolfender, J.-L.; Bonaz, B.; De Waard, M. Angew. Chem., Int. Ed. 2013, 52, 11780−11784. (b) LecerfSchmidt, F.; Haudecoeur, R.; Peres, B.; Ferreira Queiroz, M. M.; Marcourt, L.; Challal, S.; Ferreira Queiroz, E.; Sotoing Taiwe, G.; Lomberget, T.; Le Borgne, M.; Wolfender, J.-L.; De Waard, M.; Robins, R. J.; Boumendjel, A. Chem. Commun. 2015, 51, 14451− 14453. (7) (a) Kusari, S.; Tatsimo, S. J. N.; Zuehlke, S.; Talontsi, F. M.; Kouam, S. F.; Spiteller, M. Angew. Chem., Int. Ed. 2014, 53, 12073− 12076. (b) Kusari, S.; Tatsimo, S. J. N.; Zuehlke, S.; Spiteller, M. Angew. Chem., Int. Ed. 2016, 55, 240−243. (8) Gaw, S.; Thomas, K.; Hutchinson, T. H. Issues Environ. Sci. Technol. 2016, 41, 70−71. (9) (a) Kusari, S.; Lamshoeft, M.; Kusari, P.; Gottfried, S.; Zuehlke, S.; Louven, K.; Hentschel, U.; Kayser, O.; Spiteller, M. J. Nat. Prod. 2014, 77, 2577−2584. (b) Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009, 109, 3012−3043. (10) Alusik, S.; Paluch, Z. Minerva Med. 2015, 106, 233−238. (11) Bailey, C. J.; Day, C. Practical Diabetes Int. 2004, 21, 115−117. (12) Cragg, G. M.; Newman, D. J. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 3670−3695. (13) Werner, E. A.; Bell, J. J. Chem. Soc., Trans. 1922, 121, 1790− 1795. (14) An, H.; He, L. J. Endocrinol. 2016, 228, R97−R106. (15) (a) Aynehchi, Y.; Salehi Sormaghi, M. H.; Amin, G.; Khoshkhow, M.; Shabani, A. Int. J. Crude Drug Res. 1985, 23, 33− 41. (b) Sabahi, M.; Mansouri, S. H.; Ramezanian, M.; GholamHoseinian, A. Int. J. Crude Drug Res. 1987, 25, 72−76. (c) Al-Shamma, A.; Mitscher, L. A. J. Nat. Prod. 1979, 42, 633−642. (d) Al-Yahya, M. A.; Mossa, J. S.; Al-meshal, I. A.; Antoun, M. D.; McCloud, T. G.; Cassady, J. M.; Jacobsen, L. B.; McLaughlin, J. L. Int. J. Crude Drug Res. 1985, 23, 45−66. (16) Boulos, L. Flora of Egypt; Al-Hadara Publishing: Cairo, 1999; Vol. 1, p 123. (17) Gadape, H. H.; Parikh, K. S. E-J. Chem. 2011, 8, 767−781. (18) Olar, R.; Badea, M.; Marinescu, D.; Chifiriuc, C.-M.; Bleotu, C.; Grecu, M. N.; Iorgulescu, E. E.; Bucur, M.; Lazar, V.; Finaru, A. Eur. J. Med. Chem. 2010, 45, 2868−2875. (19) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565. (20) Hegazi, R.; El-Gamal, M.; Abdel-Hady, N.; Hamdy, O. Ann. Glob. Health 2015, 81, 814−820. (21) Alkhalaf, F.; Soliman, A. T.; De Sanctis, V. J. Diabetes Metab. 2014, 05, 5−12. (22) Eggen, T.; Lillo, C. J. Agric. Food Chem. 2012, 60, 6929−6935. (23) Deo, R. P. Curr. Environ. Health Rep. 2014, 1, 113−122. (24) Mrozik, W.; Stefanska, J. Chemosphere 2014, 95, 281−288. (25) Trautwein, C.; Berset, J.-D.; Wolschke, H.; Kuemmerer, K. Environ. Int. 2014, 70, 203−212. (26) Takac, M. J.-M.; Topić, D. V. Acta Pharm. (Zagreb, Croatia) 2004, 54, 177−191. (27) Markham, K. R.; Ternai, B.; Stanley, R.; Geiger, H.; Mabry, T. J. Tetrahedron 1978, 34, 1389−1397. (28) Yeskaliyeva, B.; Mesaik, M. A.; Abbaskhan, A.; Kulsoom, A.; Burasheva, G. S.; Abilov, Z. A.; Choudhary, M. I.; Atta-ur-Rahman. Phytochemistry 2006, 67, 2392−2397.

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C/12C ratio of the sample relative to the 14C/12C ratio of a standard (OX-II) representing the atmospheric 14C activity of 1950 A.D. All samples were normalized to a δ13C value of −25‰ to account for natural fractionation.34 The results were provided as a fraction modern carbon, F14C, where an F14C value of 0 indicates an infinitive age, an F14C value of 1 indicates 1950 A.D., and an F14C values above 1 indicates the age after 1950 A.D. X-ray Crystallographic Analysis of the Acetate of Metformin (4). Single crystals suitable for X-ray diffraction studies were grown from a solution in MeOH. A single crystal was mounted and immersed in a stream of nitrogen gas [T = 123(1) K]. Data were collected, using graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å), on a Bruker D8 Venture diffractometer. Crystal data: Colorless crystal, prism, size: 0.23 × 0.17 × 0.11 mm, C6H15N5O2, MW 189.23, monoclinic system, space group P21/n, unit cell a = 9.8260(6) Å, b = 8.8711(7) Å, c = 10.6841(7) Å, β = 90.834(3)°, V = 931.21(11) Å3, Z = 4, dcalc = 1.350 g/cm3, μ(Mo Kα) = 0.104 mm−1; 10 196 reflections were measured, resulting in 3164 unique reflections (Rint = 0.047, Rsigma = 0.057), which were used for all calculations. The structure was solved using direct methods (SHELXS97),29 and full-matrix leastsquares refinements were performed using SHELXL97.29 Refinement (139 parameters) converged at RF = 0.0491, wRF2 = 0.1082 [2312 unique reflections with Fo > 4σ(Fo)]. Crystallographic data for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC 1525740).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00106. Copies of 1H, 13C, HCOSY, HSQC, HMBC, and ROESY NMR spectra of the isolated compounds; detailed description of the data for the X-ray structure (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +45 3533 6253. E-mail: [email protected] (S. B. Christensen). *Tel: +45 3533 6706. E-mail: [email protected] (J. Nielsen). ORCID

Ahmed R. Hassan: 0000-0002-1060-2326 Truong Thanh Tung: 0000-0002-5263-203X John Nielsen: 0000-0002-2854-8188 Søren Brøgger Christensen: 0000-0002-5773-6874 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The technical assistance of Mr. N. Vissing Holst, Department of Chemistry, University of Copenhagen, with collecting X-ray data is gratefully acknowledged. This work was supported by the Danish Agency for Higher Education and a Danish Government Scholarship under the Cultural Agreement to A.R.H.

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REFERENCES

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