(+)-Meyeniins A-C, Novel Hexahydroimidazo[1,5-c]thiazole Derivatives from the Tubers of Lepidium meyenii: Complete Structural Elucidation by Biomimetic Synthesis and Racemic Crystallization

Article abstract of DOI:10.1021/acs.jafc.6b05805

(+)-Meyeniins A-C (1-3), a novel class of sulfur-containing hexahydroimidazo[1,5-c]thiazole derivatives, were isolated from the tubers of Lepidium meyenii (maca) cultivated in Lijiang, Yunnan province, China. Guided by their biosynthetic hypothesis, a ste

Full text of DOI:10.1021/acs.jafc.6b05805

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Article
(+)-Meyeniins A–C, Novel Hexahydroimidazo[1,5-c]thiazole
Derivatives from the Tubers of Lepidium meyenii: Complete Structural
Elucidation by Biomimetic Synthesis and Racemic Crystallization
Min Zhou, Hang-Ying Ma, Zhi-Hua Liu, Guang-Yu Yang,
Gang Du, Yan-Qing Ye, Gan-Peng Li, and Qiu-Fen Hu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05805 • Publication Date (Web): 17 Feb 2017
Downloaded from http://pubs.acs.org on February 23, 2017
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(+)-Meyeniins A–C, Novel Hexahydroimidazo[1,5-c]thiazole
Derivatives from the Tubers of Lepidium meyenii: Complete Structural
Elucidation by Biomimetic Synthesis and Racemic Crystallization
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Min Zhou†,‡,*, Hang-Ying Ma†, Zhi-Hua Liu‡, Guang-Yu Yang‡, Gang Du†, Yan-Qing
Ye†, Gan-Peng Li†,*, and Qiu-Fen Hu†,*
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†Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and
Ministry of Education, Yunnan Minzu University, Kunming 650031, Yunnan, People’s Republic of
China;
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‡Key Laboratory of Tobacco Chemistry of Yunnan Province, China Tobacco Yunnan Industrial Co.,
Ltd, Kunming 650231, Yunnan, People’s Republic of China.
*Corresponding
Author:
E-mail:
zhouminynun@163.com;
ganpeng_li@sina.com.cn;
ygy1110@163.com. Tel: +86 871 65910017.
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ABSTRACT: (+)-Meyeniins A–C (1–3), a novel class of sulfur-containing
hexahydroimidazo[1,5-c]thiazole derivatives, were isolated from the tubers of Lepidium
meyenii cultivated in Lijiang, Yunnan Province, China. Guided by their biosynthetic
hypothesis, a stereocontrolled biomimetic synthesis of meyeniins A–C and their
individual enantiomers was efficiently accomplished by a combination of a
condensation reaction and Edman degradation. The formation of high-quality crystals
for X-ray crystallography occurred much more readily from a racemic mixture of
(±)-meyeniin A than with the single enantiomer alone in this case. These extensive
strategies, combined with circular dichroism (CD) spectra, allowed the complete
structural assignments of (+)-meyeniins A–C. Among them, (+)-meyeniin A showed
moderate selective cytotoxicities against the HL-60, A549 and MCF-7 human cell lines
with IC50 values of 14.41, 32.22, and 33.14 μM, respectively. To some extent, these
findings support traditional applications of maca as healthy nutritional supplements or
functional foods for cancer prevention.
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KEYWORDS: (+)-meyeniins A–C, sulfur-containing hexahydroimidazo[1,5-c]thiazole,
Lepidium meyenii, biomimetic synthesis, racemic crystallization, cytotoxicity
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INTRODUCTION
Lepidium meyenii Walp. (Brassicaceae), commonly known as ‘maca’, is a perennial
herbaceous plant grown on Andean plateau in Peru under conditions of low temperature
and high altitude (over 3500 m).1 Maca tubers were consumed by the indigenous
population as both a nutritional supplements and fertility medicine since pre-Columbian
times.1 Recently, pharmacological research on maca extracts provided evidence to
support its diverse health-promoting properties, such as fertility-enhancing effect,2
antioxidative activity,3 immunomodulatory activity,4 anticancer,5,6 memory impairment,7
regulation of hormonal secretion,8 and effectiveness for decreasing serum lipid and
blood sugar.9 And, some of these activities have been closely linked to the lipidic
constituents, especially the characteristic glucosinolates, macamides, and
macaenes.5,10-13 Due to these putative properties, maca often referred as “Peruvian
ginseng” or “ginseng of the Andes”, has gained worldwide attention as dietary
supplements or functional foods with claims of anabolic effects since the 1990s.1 In
China, more than two hundred thousand tons of raw materials and a wide array of
commercial products have been produced by the largest maca cultivation base located in
northwest Yunnan province every year, which possesses a high diurnal temperature
range and relatively low altitude (2800–3500 m) comparable to the Andean region.14
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Considering that the bioactive principles of this important resource plant would
plausibly be influenced by the ecological environment, we carefully examined the
chemical components of the maca tubers collected from Lijiang, Yunnan. Interestingly,
except for previously reported glucosinolates, macamides, and macaenes, a new class of
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sulfur-containing hexahydroimidazo[1,5-c]thiazole derivatives, (+)-meyeniins A–C, 1–3
(Figure 1) were isolated as one of the major constituents from the lipidic fraction of the
title plant. Their structures including absolute configurations were determined by
extensive spectroscopic data, bioinspired synthesis combined with racemic
crystallization and X-ray crystallographic analysis, as well as comparison of circular
dichroism (CD) spectra. Herein, the complete structural assignments and biological
properties of meyeniins A–C are described in detail.
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MATERIALS AND METHODS
General Experimental Procedures. Optical rotations were obtained on a JASCO
DIP-370 digital polarimeter (Jasco, Tokyo, Japan). Circular dichroism spectra were
measured on a Chirascan instrument (Applied Photophysics Limited, Leatherhead,
England). Melting points were determined on a Yuhua melting point apparatus (Yuhua
Instrument co., Gongyi, China). UV data were measured with a Shimadzu UV-2401A
spectrophotometer (Shimadzu, Kyoto, Japan). A BioRad FtS-135 spectrophotometer
(Bio-Rad, Richmond, CA, USA) was used for scanning IR spectroscopy with KBr
pellets. 1D and 2D NMR spectra were realized on AV-400, DRX-500 NMR or Avance
III 600 spectrometers (Bruker, Karlsruhe, Germany) with TMS as an internal standard.
Mass spectra were recorded on a VG-Auto-Spec-3000 spectrometer (VG, Manchester,
England). X-ray diffraction was realized on a Bruker SMART APEX II CCD
crystallography system (Bruker, Karlsruhe, Germany). 5,5'-Dithio-bis-(2-nitrobenzoic)
acid (DTNB, Ellman’s reagent), S-butyrylthiocholine iodide, S-acetylthiocholine iodide,
acetylcholinesterase
derived
from
human
erythrocytes,
and
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased
from Sigma Chemical Co. (St. Louis, MO, USA). HL-60 (human promyelocytic
leukemia cells), A549 (human lung adenocarcinoma epithelial cells), SMMC-7721
(human hepatocarcinoma cells), MCF7 (human breast adenocarcinoma cells), and
SW480 (human colon cancer cells) tumor cells were purchased from the American Type
Culture Collection (ATCC, Manassas, VA, USA). Column chromatography was
performed with silica gel (100-200 mesh) (Qingdao Marine Chemical, Inc., Qingdao,
China). Preparative HPLC was performed using a Shimadzu LC-8A preparative liquid
chromatography (Shimadzu, Kyoto, Japan) with a Shimadzu PRC-ODS column.
Semi-preparative HPLC was performed on an Agilent 1100 or 1200 semi-preparative
liquid chromatography (Agilent Technologies, Foster City, CA, USA) with Zorbax
SB-C18 (10 μm, 9.4 mm × 25 cm), Welch Ultimate XB-Phenyl (10 μm, 4.6 mm × 25
cm), or Welch Ultimate Cellu-D (5 μm, 4.6 mm × 25 cm) columns. Fractions were
monitored by TLC (Qingdao Marine Chemical, Inc., Qingdao, China), and spots were
detected by spraying with 8% H2SO4 in EtOH, followed by heating silica gel plates. All
solvents were distilled prior to use.
Plant Materials. The tubers of Lepidium meyenii were collected from Lijiang,
Yunnan Province, China, in September 2014, and identified by Prof. Ning Yuan. A
voucher specimen (YNNI-14-09-25) has been deposited in the Key Laboratory of
Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and
Ministry of Education, Yunnan Minzu University.
Extraction and Isolation. The air-dried tubers of L. meyenii (25 kg) were powdered
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and extracted with 80% aqueous acetone (3 × 60 L, 4 days each) at room temperature.
After removal of the solvent by evaporation, the crude extract (5.8 kg) was suspended in
H2O, and then extracted successively with CH2Cl2 and EtOAc. The merged CH2Cl2 and
EtOAc-soluble parts (280 g) were purified by CC (column chromatography on SiO2
with CH2Cl2-Me2CO gradient system 1:0, 9:1, 8:2, 7:3, 6:4 and 1:1) to yield six main
fractions A-F. Fr. B (CH2Cl2/acetone 9:1, 35.0 g) eluted with PE/CH2Cl2 (10:1, 8:1 5:1,
2:1, and 0:1) and yielding subfractions B1-B5. Subfraction B4 (12.0 g, PE/ CH2Cl2 2:1)
was fractionated on RP-18 with gradient elution with MeOH/H2O (40:60 to 1:0) to yield
fractions B41-B47, Subsequently fraction B45 (5.5 g) was further purified by
preparative HPLC (20 mL/min, detector UV λmax 202 and 254, MeOH/H2O 75:15) and
followed by semipreparative HPLC (3 mL/min, detector UV λmax 202, 220, 254, and
280 nm, MeOH/H2O 85:15) with an Ultimate XB-Phenyl (10 μm, 4.6 mm × 25 cm)
column to yield 1 (155 mg, Rf = 12.5 min), 2 (189 mg, Rf = 17.2 min) and 3 (120 mg, Rf
= 16.5 min), respectively.
(+)-Meyeniin A, 1, C14H14N2O3S2, obtained as colorless powder; mp 51 C; [α]D25.2
o
+235.8 (c 0.20, MeOH); UV (MeOH) λmax (log ε): 276 (0.246), 241 (0.190), 203 (0.548)
nm; CD (c 0.005, MeOH) Δε205 +13.0, Δε235 +11.8, Δε250 -4.81; IR (KBr) νmax 3682,
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3430, 3068, 2969, 2924, 2855, 2779, 2046, 1852, 1748, 1444, 1037 cm-1; H and C
NMR (400 and 100 MHz, in CDCl3) (Table 1); ESIMS (positive ion mode) m/z 323 [M
+ H]+; HRESIMS (positive ion mode) m/z 323.0520 [M + H]+ (calcd 323.0519 for
C14H15N2O3S2).
(+)-Meyeniin B, 2, C13H14N2OS2, obtained as colorless powder; mp 31 C; [α]D23.3
o
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+258.8 (c 0.20, MeOH); UV (MeOH) λmax (log ε): 274 (0.451), 246 (0.315), 204 (0.458)
nm; CD (c 0.009, MeOH) Δε202 +19.8, Δε237 +21.4, Δε252 -10.1; IR (KBr) νmax 3427,
3108, 3062, 2974, 2926, 2582, 1956, 1856, 1751, 1425, 938, 701 cm-1; 1H and 13C NMR
(400 and 100 MHz, in CDCl3) (Table 2); ESIMS (positive ion mode) m/z 279 [M + H]+;
HRESIMS (positive ion mode) m/z 279.0627 [M + H]+ (calcd 279.0620 for
C13H14N2OS2).
(+)-Meyeniin C, 3, C14H16N2O2S2, obtained as colorless powder; mp 33 C; [α]D25.1
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+175.7 (c 0.20, MeOH); UV (MeOH) λmax (log ε): 201 (0.319), 216 (0.186), 245 (0.128)
nm; CD (c 0.004, MeOH) Δε204 +11.3, Δε237 +9.60, Δε253 -4.60; IR (KBr) νmax 3679,
1
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3427, 3054, 2925, 2854, 1749, 1604, 1424, 1341, 1267, 1158, 1042 cm-1; H and C
NMR (400 and 100 MHz, in CDCl3) (Table 2); ESIMS (positive ion mode) m/z 309 [M
+ H]+; HRESIMS (positive ion mode) m/z 309.0729 [M + H]+ (calcd 309.0726 for
C14H16N2O2S2).
Synthesis Procedures.
Synthesis of (±)-Meyeniin A: a suspension of L-cysteine, 5 (1.21 g, 10.0 mmol) and
KOAc (1.18 g, 12.0 mmol) was dissolved in aqueous methanol (1:1, 40 mL), and after
10 min, acetaldehyde (0.66 g, 15.0 mmol) was added. The reaction was performed by
stirring the mixture under nitrogen at room temperature for 3h, and the solution then
was reduced under a vacuum. The white precipitate was isolated by filtration, then,
washed with methanol and dried under high vacuum overnight to yield 6 (1.35 g, 9.2
mmol, 92%) (Figure 2). The diastereomeric mixture of (2R,4R)-6 and (2S,4R)-6 (147
mg, 1.0 mmol) and 3,4-(methylenedioxy)benzyl isothiocyanate (232 mg, 1.2 mmol)
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were further dissolved in dry pyridine (10 mL) under ambient conditions. The reaction
mixture was stirred at room temperature for 12 h. The resulting solution was acidified
with 1 M HCl, exhaustively extracted with EtOAc (3 × 20 mL), dried over sodium
sulfate, and concentrated in vacuo. The resulting mixture was directly subjected to
preparative HPLC to afford a white solid (264 mg, 0.82 mmol, 82%) (Figure 2).
Analysis HPLC with an Ultimate Cellu-D chiral column combined with optical rotation
measurements showed that the synthetic meyeniin A was a 1:1 enantiomers mixture.
Synthesis of (+)-Meyeniin A: the diastereomeric mixture of (2R,4R)-6 and (2S,4R)-6
(147 mg, 1.0 mmol) and 3,4-(methylenedioxy)benzyl isothiocyanate (232 mg, 1.2 mmol)
were dissolved in dry EtOH (10 mL) and placed in a sealed tube under ambient
conditions. The tube was placed into a 70 oC oil bath for 2 h. The resulting solution was
exhaustively reduced under a vacuum. Concentration in vacuo gave meyeniin A (274
mg, 0.85 mmol, 78% over two steps) (Figure 3) as a white solid, which was further
analysed by HPLC with chiral column. The result showed that the synthetic meyeniin A
under this condition is an optically pure compound with positive absorption in optical
rotations. The similar synthesis procedures led to the synthesis of (-)-meyeniin A (82%),
(+)-meyeniin B (80%), (-)-meyeniin B (80%), (+)-meyeniin C (70%), and (-)-meyeniin
C (74%), respectively.
X-ray Crystal Structure Analysis. The intensity data for a racemic mixture of
synthetic or purified (+)-meyeniin A and synthetic (-)-meyeniin A was measured on a
Bruker diffractometer equipped with an APEX II CCD, using graphite-monochromated
Mo Kα radiation (Figure 4). The crystal structure was solved by direct methods using
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SHELXS97, expanded by difference Fourier techniques, and refined by full-matrix
least-squares calculations and the program NOMCSDP. The non-hydrogen atoms were
refined using anisotropic displacement parameters, and hydrogen atoms were placed in
calculated positions. The CIF files of crystallographic data for the structures of
(±)-meyeniins A have been deposited in the Cambridge Crystallographic Data Centre
database (deposition number CCDC 1518102). Copies of the data can be obtained free
of charge on application to the CCDC, 12 Union Road, Cambridge, CB2 1EZ, U.K. (fax
+ 44 1223 336033; e-mail: deposit@cced.cam.ac.uk).
X-ray crystallographic analysis of (±)-meyeniins A: C14H14N2O3S2, M = 322.39, a =
13.0549(13) Å, b = 6.8643(7) Å, c = 15.8088(15) Å, α = 90°, β = 97.519(2)°, γ = 90°, V
= 1404.5(2) Å3, T = 100(2) K, space group P2/c, Z = 4, μ(MoKα) = 0.390 mm-1. The
total number of reflections measured was 15102, of which 4170 were observed, R1 =
0.0299 (I > 2σ(I)), wR(F2) = 0.0800 (I > 2σ(I)), R1 = 0.0347 (all data), wR(F2) = 0.0835
(all data), F2 = 1.085.
Acetylcholinesterase Inhibitory Activity. Acetylcholinesterase (AChE) inhibitory
activity of the isolated (+)-meyeniins A–C was assayed by the spectrophotometric
method in 96-well microplates developed by Ellman et al.15 with slightly modification.
Briefly, the reaction mixture (total 200 μL) contained test compound (50 μM),
phosphate buffer (PB, pH 8.0), and acetyl cholinesterase solution (0.02 U/mL), was
o
incubated at 37 C for 20 min. Then, 40 μL of solution containing DTNB (0.625 mM)
was added and the reaction was started by adding acetylthiocholine iodide (0.625 mM).
The hydrolysis of acetylthiocholine was monitored by a 96-well plate reader at 405 nm
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every 30 s for 1 h. Tacrine served as positive control (0.333 μM). All the reactions were
measured three times. The percentage inhibition was calculated as follows: inhibition
(%) = 1-S/E × 100 (S is the absorbance of the test compound and E is the absorbance of
the blank).
Cytotoxicity Assay. The cytotoxicity of the isolated (+)-meyeniins A–C was tested
using the MTT method, as reported previously.16 All human tumor cells were cultured
in RPMI-1640 or DMEM medium (Hyclone, Logan, UT), supplemented with 10%
o
fetal bovine serum (Hyclone, Logan, UT) at 37 C in 5% CO2. Cell viability was
assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay. Cells were seeded into 96-well plates 24 h before treatment and
exposed to the test compound at different concentrations in triplicate for 72 h with
DDP as positive control. The IC50 value of each compound was calculated by Reed
and Muench's method.17
RESULTS AND DISCUSSION
Structure Elucidation. Compound 1 was obtained as colorless powder. Its molecular
formula C14H14N2O3S2 was established from HRESIMS and 13C NMR spectra,
possessing an index of hydrogen deficiency of nine. The IR spectrum showed
absorption bands due to carbonyl group (1748 cm–1). In the 1D NMR spectra (Table 1),
a set of characteristic signals of a methylene group (δH 4.89 and 4.82, d, J = 14.4 Hz,
H2-7'; δC 45.0, t, C-7'), a methylenedioxy group (δH 5.92, s, H2-1''; δC 101.1, t, C-1'') and
a 1,3,4-trisubstituted benzene ring (δH 6.97, br s, H-2'; 6.73, d, J = 7.9 Hz, H-5'; 6.94, d,
J = 7.9 Hz, H-6'; δC 129.2, s, C-1'; 109.5, d, C-2'; 147.4, s, C-3'; 147.7, s, C-4'; 108.2, d,
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C-5'; 122.8, d, C-6') indicated the appearance of a 3,4-(methylenedioxy)benzyl
substituent (ring C, partial molecular formula: C8H7O2), which was further confirmed
by the 1H-1H COSY cross peak of H-5'/H-6' (fragment c) and by the HMBC correlations
observed between H2-7' and C-1'/C-2'/C-6', between H-5' and C-1'/C-3'/C-4'/C-6', and
between H2-1'' and C-3'/C-4' (Figure 5).
The remaining six carbon resonances belonged to the nucleus, comprising of one
methyl at δC 23.8 (C-8), one methylene at δC 31.5 (C-1), two heteroatom-bearing
methines at δC 60.4 and 65.7 (C-2 and C-7a), one amide carbonyl at δC 170.8 (C-7), and
one amide thiocarbonyl at δC 183.7 (C-5), attributable to a multi-heteroatom-containing
bicyclic ring system (rings A and B, partial molecular formula: C6H7N2OS2). Since
nitrogen (3.04) is more electronegative than sulfur (2.58),18 the two downfield methines
at δC 60.4 and 65.7 might be connected by a nitrogen atom, respectively, while the
upfield methylene at δC 31.5 should be linked with a sulfur atom. Analysis of the 1H-1H
COSY spectrum belong to the nucleus of 1 exhibited two structural fragments [a:
-CH2(1)-CH(7a)-; b: -CH(3)-CH3(8)] (Figure 5). Then, the HMBC spectrum was
applied to assemble the two subunits with an amide carbonyl (C-7), an amide
thiocarbonyl (C-5), two nitrogen atoms (N-4 and N-6), and a sulfur atom (S-2). Firstly,
the HMBC correlations from protons of H2-1 in fragment a to the methine at δC 60.4 (d,
C-3) in fragment b allowed the connection between C-1 and C-3 by a sulfur atom (S-2).
Meanwhile, the correlations from H-3 to the methine at δC 65.7 (d, C-7a) indicated that
C-7a was linked with C-3 through a nitrogen atom (N-4). These data revealed that C-1,
S-2, C-3, N-4, C-7a, and C-8 constructed a 2-methylthiazolidine moiety (ring A).
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Secondly, the HMBC correlations from H-3 and H-7a to the amide thiocarbonyl C-5,
and from H2-1' and H-7a to the amide carbonyl C-7, led to the N-4 and C-5 linkage and
the C-7 and C-7a linkage, respectively. This structural assignment also revealed that N-4,
C-5, C-7, C-7a, together with the remaining nitrogen atom (N-6) formed a
2-thioxoimidazolidin-4-one moiety (ring B), which is coupled to 2-methylthiazolidine
moiety (ring A) at N-4 and C-7a. Finally, the direct linkage between the
hexahydroimidazo[1,5-c]thiazole
nucleus
(rings
A
and
B)
and
3,4-methylenedioxy-benzyl substituent through the nitrogen atom N-6 could be readily
established by the key HMBC correlations observed between H2-7' and C-5/C-7 (Figure
5). The above evidence led to the planar structural assignment of 1 as depicted in Figure
1. Unfortunately, no reliable NOESY correlation could be observed to determine the
relative or absolute stereochemistry at C-3 and C-7a.
The 1H and 13C NMR data of 2 (Table 2) were similar to those of 1, with the exception
that the 3,4-(methylenedioxy)benzyl substituent in 1 was replaced by a benzyl group in
2. This inference was further proved by the HMBC correlations observed between H2-7'
and C-1'/C-2'/C-6', between H-2' or H-6' and C-1'/C-3'/C-4'/C-5', and between H-3' or
H-5' and C-1'/C-2'/C-4'/C-6' in 2. Similarly, a direct comparison of the NMR data of 1
and 3 (Tables 1 and 2) suggested that 3,4-(methylenedioxy)benzyl moiety in 1 were
replaced by 3-methoxybenzyl moiety in 4. Such assignment was further supported by
the HMBC correlations observed from H2-7' to C-1', C-2', and C-6', and from H-2' to
C-1', C-3', C-4', C-6' and C-7', and from H3-1'' to C-3'.
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The relative configuration at C-3 and C-7a in 2 and 3 was determined to be identical
to that of 1 based on the same 1H and 13C chemical shifts of the
hexahydroimidazo[1,5-c]thiazole nucleus. However, their relative stereochemistry could
not be assigned by spectroscopic data alone, and all the efforts to obtain single crystals
for X-ray crystallography analysis also failed. Finally, the absolute configuration of
natural compounds 1−3 was determined as (3S,7aR) by biosynthetic prediction,
biomimetic synthesis combined with racemic crystallization and X-ray cry-stallographic
analysis (Figure 4), as well as comparison of circular dichroism (CD) spectra. Thus,
compounds 1−3 were elucidated as shown (Figure 1) and they were given trivial names
of (+)-meyeniins A−C, respectively.
Hypothetical Biogenetic Pathway of (+)-meyeniins A−C. Though several synthetic
counterparts have been reported previously,19 and 20 meyeniins A−C represent the first
examples of naturally occurring hexahydroimidazo[1,5-c]thiazole derivatives. This is an
interesting case that the basic skeleton of the synthetic analogues were discovered from
natural origins. Our postulated biosynthetic pathway of (+)-meyeniins A−C involves the
construction of the 2-methylthiazolidine ring by condensation reaction between
acetaldehyde, 4, and L-cysteine, 5, followed by the formation of the
2-thioxoimidazolidin-4-one moiety via the Edman degradation reaction between a
diastereomeric mixture of 2-methylthiazolidine-4-carboxylic acid, 6, and related
isothiocyanate, 7 (Figure 6).19 and 20 Especially, in the second key step, the intermediate
E must undergo a ring opening and subsequent epimerization at C-2, followed by ring
re-closure. This process may lead exclusively to the formation of (+)-meyeniins A−C
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possessing the less strained ring system with trans-configuration between C-3 and
C-7a.21
Biomimetic Synthesis and Racemic Crystallization for Complete Structural
Elucidation of (+)-meyeniins A−C. Guided by this biosynthetic consideration, a
two-step stereocontrolled synthesis of (+)-meyeniins A–C and their individual
enantiomers was efficiently accomplished. Briefly, treatment of L-cysteine (5) and
acetaldehyde (4) with KOAc in carboxylic acid, 6, as a diastereomeric mixture (Figure
2).19-21
Surprisingly,
the
diastereoisomers
further
condensed
with
3,4-(methylenedioxy)benzyl isothiocyanate in pyridine to form a racemic mixture of
(±)-meyeniin A in 75% overall yield,21 which was determined by HPLC analysis with a
chiral column and optical rotation measurement (Figure 3). We propose a mechanism
involving a racemization at C-4 catalyzed by pyridine and an epimerization at C-2 by
Edman degradation reaction as stated previously (Figure 6).
With this in mind, the same reaction materials were dissolved in anhydrous ethanol
and placed in a sealed tube under ambient conditions, which led to optically pure
(+)-meyeniin A with a large positive optical rotation. Similarly, (+)-meyeniin B,
(+)-meyeniin C and (-)-meyeniins A−C were easily prepared under the same reaction
conditions (Figure 6). The NMR, optical rotation, and circular dichroism data of the
synthetic (+)-meyeniins A−C were identical in all respects to those of the natural
counterparts, indicating that the cysteine residues in the isolated structures are the
L-(+)-form. Although the synthetic meyeniins A−C were readily available, we still
failed to obtain their single crystals for confirming their absolute configuration at C-3
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even after extensive trials.
Research has suggested that the racemic crystallization of a 1:1 enantiomeric mixture
from solution occurred much more readily than the formation of homochiral crystals
from a solution of the single enantiomer in some cases (an approximate 9:1 ratio).22 and 23
This propensity might be attributed to the new possibilities for favorable packing
arrangements available in racemic space groups.21 Accordingly, diffraction quality
crystals appeared rapidly from a solution of acetone/methanol (1:4) containing a
racemic mixture of synthetic or purified (+)-1 and synthetic (-)-1 in only ten minutes.
X-ray crystallographic analysis was realized, which clarified not only the planar
structure but also the absolute configuration to be 3S,7aR (Figure 7). In the CD spectra
of compounds 1−3, the Cotton effects were all negative at 205 and 235 nm and positive
at 250 nm, suggesting the same absolute configuration for these three compounds.
Acetylcholinesterase Inhibitory Activity and Cytotoxicity Assay. Considering the
medicinal applications of the plant species,5-7 compounds 1–3 were tested for
acetylcholine esterase inhibitory activity using the Ellman method.15 No significant
activity of these compounds was detected at concentrations up to 50 μM. Compounds
1–3 were also evaluated for cytotoxic activity against a panel of human cancer cell lines
using the MTT method with DDP as positive control.16 (+)-Meyeniin A showed
selective cytotoxicities against the HL-60, A549 and MCF-7 human cell lines with IC50
values of 14.41, 32.22, and 33.14 μM, respectively. To some extent, these findings
support maca’s traditional applications as healthy nutritional supplements or functional
foods for cancer prevention.
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ACKNOWLEDGMENT
This project was supported by the National Natural Science Foundation of China (No.
31600282), China Tobacco Yunnan Industrial Co., Ltd (2016539200340107), and the
Innovative Research Team in University of Yunnan Province (IRTSTYN 2014-11).
SUPPORTING INFORMATION
1D and 2D NMR, and CD data of natural (+)-meyeniins A–C, 1D NMR and CD data of
synthetic (±)-meyeniins A–C, and X-ray structure of synthetic racemic meyeniin A are
supplied as supporting information, which is available free of charge via the Internet at
http: //pubs.acs.org.
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REFERENCES
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multi-pharmacological functions. Food Res. Int. 2007, 40, 783–792.
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Simánek, V. The influence of maca (Lepidium meyenii) on antioxidant status, lipid and
glucose metabolism in rat. Plant Foods Hum. Nutr. 2007, 62, 59−63.
(10) Piacente, S.; Carbone, V.; Plaza, A.; Zampelli. A.; Pizza. C. Investigation of the
tuber constituents of maca (Lepidium meyenii Walp.). J. Agric. Food Chem. 2002, 50,
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(11) Muhammad, I.; Zhao, J.; Dunbar, D. C.; Khan, I. A. Constituents of Lepidium
meyenii 'maca'. Phytochemistry 2002, 59, 105−110.
(12) Zhao, J.; Muhammad, I.; Dunbar, D. C.; Mustafa, J.; Khan, I. New alkamides from
maca (Lepidium meyenii). J. Agric. Food Chem. 2005, 53, 690−693.
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(21) Miskolczi, I.; Zékány, A.; Rantal, F.; Linden, A.; Kövér, K. E.; Györgydeák, Z.
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(22) Gavezzotti, A.; Rizzato, S. Are racemic crystals favored over homochiral crystals
by higher stability or by kinetics? Insights from comparative studies of crystalline
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Am. Chem. Soc. 1991, 113, 9811–9820.
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FIGURE CAPTIONS
Figure 1. The structures of isolated (+)-meyeniins A–C (1–3).
Figure 2. Synthesis of a racemic mixture of meyeniin A in pyridine.
Figure 3. Synthesis of (+)/(-)-meyeniins A−C in ethanol.
Figure 4. Key 1H-1H COSY and selected HMBC correlations of compound 1.
Figure 5. ORTEP diagram of compound 1.
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Figure 6. Hypothetical biogenetic pathway of (+)-1–3.
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Table 1. NMR Spectroscopic Data of Natural and Synthetic (+)-1 in CDCl3 (δ ppm)
1 (natural)
1 (synthetic)
no.
δC
δH
HMBC
3, 7a, 7
δC
δH
1β
1α
3α
5
31.5 t
2.91, t (11.0)
31.6 t
2.91, t (11.0)
3.27, dd (11.0, 6.8)
3.27, dd (11.0, 6.8)
60.4 d
5.63, q (6.3)
1, 7a, 5
60.5 d
5.63, q (6.3)
183.7 s
170.8 s
65.7 d
-
-
183.8 s
170.9 s
65.8 d
-
7
-
-
-
7aβ
8β
1'
4.56, dd (11.0, 6.8)
1.63, d (6.3)
-
1, 5, 7
4.56, dd (11.0, 6.8)
1.63, d (6.3)
-
23.8 q
3
23.9 q
129.2 s
109.5 d
147.4 s
147.7 s
108.2 d
122.8 d
45.0 t
-
129.2 s
109.6 d
147.5 s
147.8 s
108.4 d
122.9 d
45.1 t
2'
6.97, br s
-
1', 3', 4', 6', 7'
6.97, br s
-
3'
-
4'
-
-
-
5'
6.73, d (7.9)
6.94, d (7.9)
4.89, d (14.4)
4.82, d (14.4)
5.92, s
1', 3', 4', 6'
1', 2', 4', 5', 6', 7'
5, 7, 1', 2', 6'
6.73, d (7.9)
6.94, d (7.9)
4.89, d (14.4)
4.82, d (14.4)
5.92, s
6'
7'
101.1 t
3', 4'
101.2 t
1''
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Table 2. NMR Spectroscopic Data of Compounds 2 and 3 in CDCl3 (δ ppm)
2 (natural)
3 (natural)
no.
δC
δH
HMBC
δC
δH
HMBC
1β 31.7 t
1α
2.92, dd (11.0, 9.9) 3, 7a, 7
3.28, dd (11.0, 6.8)
31.7 t
2.93, dd (11.0, 9.9) 3, 7a, 7
3.28, dd (11.0, 6.8)
3α 60.5 d
5.65, q (6.3)
1, 7a, 5
60.5 d
5.65, q (6.3)
1, 7a, 5
5
7
183.9 s
170.9 s
-
-
-
-
183.9 s
170.9 s
65.7 d
-
-
-
-
7aβ 65.8 d
8β 23.9 q
4.58, dd (9.9, 6.8) 1, 5, 7
4.58, dd (9.9, 6.8) 1, 5, 7
1.64, d (6.3)
-
3
23.9 q
1.64, d (6.3)
3
1'
2'
3'
4'
5'
6'
7'
-
-
-
135.5 s
128.9 d
128.7 d
128.2 d
136.9 s
114.5 d
159.7 s
113.6 d
7.24, m
7.11, m
7.11, m
1', 3', 4', 7'
1', 2', 4', 6'
2', 3', 5', 6'
1', 2', 4', 6'
1', 3', 4', 7'
7.00, d (1.2)
-
1', 3', 4', 7'
-
6.82, dd (8.0, 1.2) 2', 3', 5', 6'
128.7 d 7.11, m
128.9 d 7.24, m
129.7 d 7.23, t (8.0)
121.1 d 7.01, d (8.0)
1', 2', 4', 6'
1', 3', 4', 7'
5, 7, 1', 2', 6'
45.3 t
5.01, d (14.5)
5, 7, 1', 2', 6' 45.2 t
4.97, d (14.5)
4.88, d (14.5)
4.91, d (14.5)
-
1''
3'
-
-
55.3 q
3.79, s
407
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Figure 1. The structures of isolated (+)-meyeniins A–C (1–3).
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Figure 2. Synthesis of a racemic mixture of meyeniin A in pyridinea.
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414
aReagents and conditions: (i) L-cysteine (5) (1 equiv), acetaldehyde (4) (1.5 equiv),
KOAc (1.2 equiv), rt, H2O:MeOH = 1:1, 3 h; (ii) 2-methyl-thiazolidine-4-carboxylic
acid (6) (1 equiv), 3,4-(methylenedioxy)benzyl isothiocyanate (1.2 equiv), rt, pyridine,
12 h, 75% for (+)-1:(-)-1 = 1:1 over two steps.
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Figure 3. Synthesis of (+)/(-)-meyeniins A−C in ethanola.
aReagents and conditions: (i) 2-methylthiazolidine-4-carboxylic acid (6) (1 equiv),
related isothiocyanates (1.2 equiv), rt, ethanol, 2 h, 78% for (+)-1, 82% for (-)-1, 80%
for (+)-2, 80% for (-)-2, 70% for (+)-3, and 74% for (-)-3 over two steps.
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422
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Figure 4. Key 1H-1H COSY and selected HMBC correlations of compound 1.
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Figure 5. ORTEP diagram of compound 1.
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Figure 6. Hypothetical biogenetic pathway of (+)-1–3.
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