Phytochemistry Letters 10 (2014) 287–290

Leonurosides A–D: Steroid N-acetylglucosaminides from the fruitsof Leonurus japonicus

Miao Ye a,b,c, Guang-Lei Ma a, Jing-Jing Su c, Juan Xiong a, Jin-Feng Hu a,c,*a Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR Chinab School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, PR Chinac Department of Chemistry and Institutes for Advanced Interdisciplinary Research, East China Normal University (ECNU), Shanghai 200062, PR China

A R T I C L E I N F O

Article history:

Received 29 May 2014

Received in revised form 10 October 2014

Accepted 14 October 2014

Available online 25 October 2014

Keywords:

Leonurus japonicus

Lamiaceae

Steroid N-acetylglucosaminides

Leonurosides

A B S T R A C T

Four new naturally occurring steroidal glycosides (leonurosides A–D, 1–4) were isolated from the dried

fruits of Leonurus japonicus (motherwort fruit). Their structures were established by spectroscopic and

chemical methods. The monosaccharide unit was identified to be a 2-acetamido-2-deoxy-b-D-glucose

and the aglycone is either a common 3b-stigmasterol or 3b-ergosterol derivative. These compounds are

the first representatives of steroid N-acetylglucosaminides from vascular plants.

� 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Phytochemistry Letters

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate /p hyt ol

1. Introduction

Naturally occurring steroid N-acetylglucosaminides are interest-ing secondary metabolites that generally have a 2-acetamido-2-deoxy-b-D-glucose moiety bound to a steroid nucleus. They havebeen often found from the excretion of some animals [e.g., the urineor serum of mammals (Marschall et al., 1989; Meng et al., 1996,1997; Yamaga and Kohara, 1994) and the defense secretion of fishes(Tachibana et al., 1984, 1985)], and only a few examples werepreviously reported from marine organisms (Gulavita et al., 1994;Lin et al., 2010). However, none of this class of steroidal glycosideshas been so far isolated from any vascular plants. During ourcontinuing research toward the discovery of novel bioactive sterol-type derivatives from terrestrial higher plants (Wu et al., 2009; Yeet al., 2013) and fungi (Zang et al., 2013), four (1–4) new steroid N-acetylglucosaminides (Fig. 1) were identified and characterizedfrom the air-dried ripe fruits of the herbaceous plant Leonurus

japonicus Houtt (family Lamiaceae). In this study, compounds 3 and4 were obtained as a mixture of C-24 epimers of 3-b-O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-ergosta-5-ene. We herein

* Corresponding author at: Department of Natural Products Chemistry, School of

Pharmacy, Fudan University, Shanghai 201203, PR China. Tel.: +86 21 51980172;

fax: +86 21 51980172.

E-mail address: jfhu@fudan.edu.cn (J.-F. Hu).

http://dx.doi.org/10.1016/j.phytol.2014.10.016

1874-3900/� 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rig

describe the isolation and structural elucidation of the newcompounds.

2. Results and discussion

Compound 1 was obtained as a white, amorphous powder. Itsmolecular formula was established as C37H63NO6 based on apseudomolecular ion peak [M+Na]+ at m/z 640.4556 (calcd forC37H63NO6Na, 640.4548) in its HRESIMS. In addition to eightcharacteristic signals attributed to an N-acetylglucosamine moiety(d 100.5, 57.8, 76.1, 72.2, 78.2, 62.5; N-Ac: d 170.3, 23.2) (Iida et al.,2001), the 13C and DEPT NMR (Table 1) spectra of 1 exhibited29 carbon signals. Acid hydrolysis of 1 (see Section 3) gave theaglycone 1a and the sugar moiety (existed as glucosaminehydrochloride). 1a was ascertained to be b-sitosterol by comparingits NMR data (Ahmad et al., 2010; Wright et al., 1978) and opticalrotation (Gutierrez-Lugo et al., 2004) with those published. Theaglycone was also confirmed by a direct TLC analysis with anauthentic specimen. Meanwhile, the HMBC correlations betweenthe acetyl carbonyl (d 170.3) and d 8.91 (NH)/d 2.15 (3H, s)indicated the presence of an acetamido group in the sugar unit.Furthermore, the 2-acetamido-2-deoxysugar moiety was con-firmed by the HMBC correlation from NH (d 8.91, D2O exchange-able) to C-20 (d 57.8) and the COSY correlations between NH (d8.91) and H-20 (d 4.50), especially the long-range ‘‘W’’ couplingbetween NH (d 8.91) and H-10 (d 5.28) (see Supplementary data).

hts reserved.

Fig. 1. Chemical structures of steroid N-acetylglucosaminides 1–4.

Fig. 2. Observed key HMBC correlations of 1.

M. Ye et al. / Phytochemistry Letters 10 (2014) 287–290288

Moreover, a 3J HMBC correlation was observed from theanomeric proton at d 5.28 (1H, d, J = 7.3 Hz) to C-3 at d 78.2,demonstrating C-3 as the glycosidic linkage position (Fig. 2), whilethe b-glycosidic linkage could be easily determined based on theobserved coupling constant (7.3 Hz) of the anomeric proton. Themeasured optical rotation value of the glucosamine hydrochlo-ride, [a]D

20 + 67.4 (c 0.05, H2O), was in good accordance with thatreported in the literature {[a]D

25 + 71.8 (after 20 h, c 4, H2O)}(Armarego and Chai, 2003). Finally, the structure of 1 waselucidated to be 3-b-O-(2-acetamido-2-deoxy-b-D-glucopyrano-syl)-b-sitosterol. Actually, compound 1 has been previously semi-synthesized by coupling of b-sitosterol with glucosaminehydrochloride in 2009 (Grover et al., 2009). Herein, it wasreported as a new naturally occurring steroid N-acetylglucosa-minide, and its proton and carbon NMR data were completelyassigned for the first time.

Table 113C NMR data of compounds 1–4 (125 MHz, C5D5N).

No. 1 2 3 4

1 37.1 37.1 36.9

2 29.9 29.9 31.5

3 78.2 78.2 78.0

4 39.2 39.2 38.9

5 140.6 140.9 140.6

6 121.6 122.7 121.3

7 31.8 31.8 31.6

8 31.7 31.7 31.6

9 50.0 50.0 49.7

10 36.5 36.6 36.3

11 20.9 20.9 20.7

12 39.6 39.6 39.3

13 42.1 42.2 41.9

14 56.5 56.5 56.2

15 24.2 24.1 23.9

16 29.6 29.6 27.8

17 55.9 55.8 55.7 55.6

18 11.6 11.6 11.4

19 19.1 19.1 19.7

20 36.0 35.6 35.5 35.8

21 18.7 18.5 18.8 18.6

22 33.9 34.7 33.4

23 26.1 30.9 29.6 30.0

24 45.7 156.4 38.5 38.7

25 29.1 33.7 32.0 31.5

26 18.9 21.8 20.1 17.7

27 19.6 21.6 18.3 20.7

28 23.0 106.3 14.9 15.0

29 11.8

OAc 23.2 23.3 23.0

170.3 170.3 170.0

10 100.5 100.5 100.2

20 57.8 57.8 57.7

30 76.1 76.1 75.8

40 72.2 72.2 71.9

50 78.2 78.3 78.2

60 62.5 62.6 62.2

Compound 2 was deduced to be a C-28 steroid N-acetylglu-cosaminide by comparison of its 1H and 13C NMR data (Tables 1 and2) with those of compound 1. The obvious difference between 2and 1 was that the terminal methyl at d 0.89 (3H, t, J = 7.5 Hz, H3-29) in 1 was replaced by an exomethylene group [d 4.87 and 4.85(each 1H, br s), d 106.3 (CH2), 156.4 (qC)] in 2, which was supportedby the molecular formula (C36H59NO6.) of 2. The D24(28)

exomethylene unit in the side chain of the aglycone in 2 wasconfirmed by the HMBC correlations from H3-26 (d 1.05)/H3-27 (d1.06) to C-24 (d 156.4). Thus, compound 2 was elucidated to be 3-b-O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-ergosta-5,24(28)-diene.

Compounds 3 and 4 were obtained as a mixture, and both havethe same molecular formula (C36H61NO6) based on their HRESIMSdata. The 1H and 13C NMR spectroscopic data of 3 and 4(Tables 1 and 2) were quite similar to 1, indicating that 3 and 4were also steroid N-acetylglucosaminide derivatives. 3 and 4possessed the same aglycone of ergosta-5-en-3b-ol by comparisonof its NMR spectroscopic data with those of 1. In the 13C NMRspectrum of the mixture, two sets of carbon signals assignable to C-17–C-24 (Table 1) were clearly observed, implying 3 and 4 were apair of C-24 epimers. As summarized by Wright et al. (Wright et al.,1978), for some diastereomeric C-24 alkyl sterols, slight differ-ences in the chemical shifts of side-chain carbons permitted thedetermination of the absolute configuration at C-24. As for 3 (24R),C-26 (d 20.1) and C-27 (d 18.3) have a chemical shift difference ofonly 1.8 ppm in contrast to a bigger difference of 3.0 ppm (C-26:17.7; C-27: 20.7) in 4 (24S). Consequently, 3 and 4 were deduced as(24R)-3-b-O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-ergosta-5-ene and (24S)-3-b-O-(2-acetamido-2-deoxy-b-D-gluco-pyranosyl)-ergosta-5-ene, respectively.

To the best of our knowledge, none steroidal glycoside with a 2-acetamido-2-deoxy-b-D-glucose moiety has been so far isolatedfrom vascular plants. Steroid N-acetylglucosaminides 1–4 weretested for their cytotoxic effects against five human cancer celllines (BGC-823 and KE-97 gastric carcinoma, Huh-7 hepatocarci-noma, Jurkat T cell lymphoblasts, and MCF-7 breast adenocarci-noma) using the CellTiter GloTM luminescent cell viability assay (Yeet al., 2013, 2014; Zang et al., 2013), but none of them was active(IC50s > 10 mM).

3. Experimental

3.1. General procedures

Optical rotations were measured on a Perkin-Elmer 341 polar-imeter (Perkin-Elmer, Waltham, MA, USA). IR spectra weremeasured on an Avatar 360 FT-IR spectrophotometer (ThermoScientific, Waltham, MA, USA). NMR spectra were recorded on aBruker Avance DRX-500 spectrometer (Bruker Daltonics, Boston,

Table 21H NMR data of compounds 1–4 (500 MHz, J in Hz, C5D5N).

No. 1 2 3/4

1 1.68, 1H, m

0.95, 1H, m

1.64, 1H, m

0.91, 1H, m

1.69, 1H, m

0.83–0.80, 1H, m

2 2.08, 1H, m

1.73, 1H, m

2.06, 1H, m

1.73, 1H, m

2.08, 1H, m

1.74, 1H, m

3 3.87, 1H, dddd, 10.6, 9.4, 4.5, 3.9 3.88, 1H, dddd, 10.5, 10.2, 4.5, 3.9 3.87, 1H, dddd, 10.6, 9.4, 4.5, 3.9

4 2.67, 1H, m

2.50, 1H, m

2.67, 1H, m

2.42, 1H, m

2.66, 1H, m

2.50, 1H, m

6 5.33, 1H, br s 5.32, 1H, br s 5.32, 1H, br s

7 1.91, 1H, m

1.51, 1H, m

1.91, 1H, m

1.52, 1H, m

1.91, 1H, m

1.51, 1H, m

8 1.39, 1H, m 1.37, 1H, m 1.51–1.40, 1H, m

9 0.88, 1H, m 0.86, 1H, m 0.84, 1H, m

11 1.41, 2H, m 1.38, 2H, m 1.51, 2H, m

12 1.99, 1H, m

1.09, 1H, m

1.89, 1H, m

1.03, 1H, m

1.94–1.98, 1H, m

1.08, 1H, m

14 0.93, 1H, m 0.91, 1H, m 0.89, 1H, m

15 1.55, 1H, m

1.03, 1H, m

1.55, 1H, m

1.03, 1H, m

1.55, 1H, m

1.03, 1H, m

16 1.30, 2H, m 1.47, 1H, m

1.29, 1H, m

1.36–1.25, 2H, m

17 1.09, 1H, m 1.08, 1H, m 1.08, 1H, m

18 0.66, 3H, s 0.64, 3H, s 0.65, 3H, s

19 0.94, 3H, s 0.94, 3H, s 3: 0.85, 3H, s

4: 0.87, 3H, s

20 1.39, 1H, m 1.41, 1H, m 1.51–1.40, 1H, m

21 0.99, 3H, d, 6.4 0.98, 3H, d, 5.5 3: 0.94, 3H, d, 6.8

4: 0.96, 3H, d, 6.8

22 1.39, 1H, m

1.08, 1H, m

1.62, 1H, m

1.23, 1H, m

1.51–1.40, 1H, m

1.08, 1H, m

23 1.24, 2H, m 2.19, 1H, m

1.99, 1H, m

1.36–1.25, 2H, m

24 0.99, 1H, m 0.96, 1H, m

25 1.72, 1H, m 2.26, 1H, m 1.69, 1H, m

26 0.86, 3H, d, 6.9 1.05, 3H, d, 6.6 3: 0.83, 3H, overlapped

4: 0.84, 3H, overlapped

27 0.87, 3H, d, 5.4 1.06, 3H, d, 6.6 3: 0.82, 3H, overlapped

4: 0.81, 3H, overlapped

28 1.26, 2H, m 4.87, 1H, br s

4.85, 1H, br s

3: 0.81, 3H, overlapped

4: 0.82, 3H, overlapped

29 0.89, 3H, t, 7.5

COCH3 2.15, 3H, s 2.15, 3H, s 2.15, 3H, s

NH 8.91, 1H, d, 5.8 8.91, 1H, d, 7.3 8.91, 1H, d, 6.7

10 5.28, 1H, d, 7.3 5.28, 1H, d, 7.3 5.27, 1H, d, 7.5

20 4.50, 1H, overlapped 4.50, 1H, overlapped 4.50, 1H, overlapped

30 4.50, 1H, overlapped 4.50, 1H, overlapped 4.50, 1H, overlapped

40 4.23, 1H, dd, 9.0, 7.1 4.23, 1H, dd, 9.4, 7.7 4.24, 1H, dd, 9.0, 7.4

50 3.98, 1H, m 3.98, 1H, m 3.98, 1H, m

60 4.56, 1H, br d, 11.6

4.38, 1H, dd, 11.7, 5.3

4.56, 1H, br d, 11.4

4.38, 1H, dd, 11.4, 5.5

4.56, 1H, br d, 11.6

4.39, 1H, dd, 11.8, 5.3

M. Ye et al. / Phytochemistry Letters 10 (2014) 287–290 289

MA, USA). Chemical shifts are expressed in d (ppm) and arereferenced to the residual solvent signals. ESIMS were recorded on aBruker Daltonics micrOTOF-QII mass spectrometer (Bruker Dal-tonics, Boston, MA, USA). EIMS were recorded on an Agilent 5975CMSD mass spectrometer (Agilent Technologies, Santa Clara, CA,USA). Semi-preparative HPLC was performed on a Beckman Systemconsisting of a Beckman Coulter System Gold 508 autosampler, Gold126 gradient HPLC pumps with a Beckman System Gold 168 diodearray detector (DAD) and a Sedex 80 (SEDERE, France) evaporativelight-scattering detector (ELSD) and a YMC-Pack ODS-A column(250 � 10 mm, dp 5 mm). Column chromatography (CC) was carriedout on silica gel (200–300 mesh, Yantai Kang-Bi-Nuo SilysiaChemical Ltd., Yantai, China), and TLC on silica gel (GF254,0.25 mm, Yantai Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, China).Spots were visualized using UV light (254 nm) and 15% H2SO4–EtOH.

3.2. Plant material

The dried fruits of L. japonicus were purchased from ShanghaiJiu-Zhou-Tong Medicine Co. Ltd., and were originally collected in

August 2009, from Bozhou City, Anhui Province of China. The plantwas identified by Prof. Bao-Kang Huang (Department of Pharma-cognosy, Second Military Medical University of China). A voucherspecimen (No. 100126) was deposited at the Herbarium of theDepartment of Natural Products Chemistry, School of Pharmacy,Fudan University.

3.3. Extraction and isolation

Dried and powdered fruits of L. japonicus (20.0 kg) wereextracted with MeOH (15 L � 4) under reflux for 4 h. The solventwas removed at reduced pressure to give a dark brown residue(ca. 832.6 g), which was then suspended in H2O (2 L) andextracted with EtOAc three times (3� 2 L). The entire EtOAc-soluble extract (ca. 340.2 g) was then subjected to a flash CC oversilica gel eluting with gradients of petroleum ether (PE)–EtOAc–MeOH (2:1:0 to 0:0:1, v/v/v) to yield ten fractions (Fr. 1–Fr. 10).In an earlier work (Ye et al., 2014), Fr. 3 (PE-EtOAc, 1:1, v/v),Fr. 4 (neat EtOAc), and Fr. 5 (EtOAc–MeOH, 20:1, v/v) werefound to yield a number of 28-nor-oleanane-type spirocyclic

M. Ye et al. / Phytochemistry Letters 10 (2014) 287–290290

triterpenoids with interesting cytotoxic activity (Ye et al., 2014).Only Fr. 8 (EtOAc–MeOH, 10:1, v/v, 28.2 g) was positive toLiebermann–Burchard and Molish tests. The rest fractions werenot interesting and were discarded. Fr. 8 was later subjected tosilica gel CC and eluted with EtOAc–EtOH–H2O (7:1:0.12, v/v/v)and then separated by semi-preparative HPLC [96% MeOH in H2O(with 0.1% TFA), flow rate: 3.0 mL/min] to afford compounds 1(15.3 mg, tR = 37.1 min), 2 (2.2 mg, tR = 27.5 min), and a mixtureof 3/4 (10.7 mg, tR = 35.8 min).

3.3.1. Leonuroside A (1)

White amorphous powder; [a]D22 – 31.1 (c 0.22, MeOH); IR

(KBr) nmax: 3419, 3005, 2898, 2360, 1668, 1643, 1545, 1386, 1051,673 cm�1; 1H and 13C NMR data (in C5D5N) see Tables 1 and 2; (+)-ESIMS: m/z 640 [M+Na]+; (+)-HRESIMS: m/z 640.4556 [M+Na]+

(calcd for C37H63NNaO6, 640.4548).

3.3.2. Leonuroside B (2)

White amorphous powder; [a]D22 – 68.2 (c 0.15, MeOH); IR

(KBr) nmax: 3420, 3012, 2975, 2360, 1680, 1669, 1351, 673 cm�1;1H and 13C NMR data (in C5D5N) see Tables 1 and 2; (+) ESIMS: m/z624 [M+Na]+; (+)-HRESIMS: m/z 624.4236 [M+Na]+ (calcd forC36H59NNaO6, 624.4235).

3.3.3. Leonurosides C (3) and D (4)

White amorphous powder; [a]D22 – 18.1 (c 0.08, MeOH); IR

(KBr) nmax: 3419, 3001, 2898, 2360, 1686, 1515, 1386, 1051,612 cm�1; 1H and 13C NMR data (in C5D5N) see Tables 1 and 2; (+)ESIMS: m/z 626 [M+Na]+; (+)-HRESIMS: m/z 626.4436 [M+Na]+

(calcd for C36H61NNaO6, 626.4391).

3.4. Hydrolysis of compound 1

The sugar moiety of 1 was identified by the strategy applied byShiono et al. (2011). Briefly, a solution of 1 (10.3 mg) in MeOH(1.5 mL) was treated with 1.4 N HCl (5.0 mL), and refluxed at 85 8Cfor 8 h. After neutralized with 0.7 M Na2CO3 (5.0 mL), the solutionwas extracted with EtOAc five times (15.0 mL � 5). The organicphase was combined and then evaporated under vacuum to givean oil residue, which was purified by silica gel CC (PE/Acetone,20:1) to furnish the aglycone (1a, 5.3 mg). b-Sitosterol (1a):[a]D

20 – 32.6 (c 0.2, CHCl3) {lit. (Gutierrez-Lugo et al., 2004):[a]D

20 – 28.4 (c 0.1, CHCl3)}; 1H and 13C NMR data (in CDCl3) werein full agreement with those reported in the literature (Ahmadet al., 2010; Wright et al., 1978). EIMS m/z (rel. int.): 414 (M+, 26),396 (9), 381 (6), 273 (5), 232 (2), 231 (3), 213 (7), 43 (100).Meanwhile, the aqueous layer was concentrated under reducedpressure and then worked up as usual to give D-glucosaminehydrochloride {[a]D

20 + 67.4 (c 0.05, H2O), lit. (Armarego and Chai,2003): [a]D

25 + 71.8 (c 4, H2O)}.

Supporting information

Supporting information (The 1D, 2D NMR and HR-ESIMSspectra of compounds 1–4) of this article can be found online.

Acknowledgements

This work was supported by NSFC grants (Nos. 81273401,81202420, 21472021), grants from the Ph.D. Programs Foundationof Ministry of Education (MOE) of China (Nos. 20120071110049,20120071120049).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytol.2014.10.016.

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