Chemistry of hermidin: Insights from extraction experiments with the main Alkaloid of mercurialis perennis L. Tracked by GC/MS and LC/MSn

Article abstract of DOI:10.1002/hlca.201400046

Hermidin (1), a piperidine-2,3-dione alkaloid, has been previously detected as a lipophilic constituent in Mercurialis perennis L. and other Mercurialis species. Because of strong electronwithdrawing effects of its carbonyl groups, an acidic H-atom is easily subtracted from 1, whereas the latter shows high reactivity towards oxidation reactions or the attack of C-nucleophilic agents. To obtain a better understanding of possible chemical pathways upon extraction of root parts of M. perennis, the products obtained with different solvents from 1 were investigated. Extraction of M. perennis with aqueous MeOH or EtOH yielded a mixture of hermidin quinone (3), 5-hydroxy-4-methoxy-5- (alkoxycarbonyl)-1-methyl-3-pyrrolin-2-ones, 7 and 8, and d,l- And meso-isochrysohermidins, 5 and 6, all of them being investigated by GC/MS and LC/MSn. The latter compounds were supposedly formed by free-radical reactions, followed by spontaneous benzilic acid rearrangement and esterification. Furthermore, extraction of M. perennis with aqueous Me₂CO produced an aldol condensation product, the known alkaloid speranskatine A (9a), which was identified by NMR after chromatographic purification. In a similar manner, a CH₂ homolog of speranskatine A (10a) was obtained as a novel compound when ethyl methyl ketone (=butan-2-one; EtCOMe) instead of Me₂CO was used for extraction. Consequently, 1 easily undergoes artefact formation upon extraction of plant material with polar or slightly polar solvents.

Full text of DOI:10.1002/hlca.201400046

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Chemistry of Hermidin: Insights from Extraction Experiments with the Main
Alkaloid of Mercurialis perennis L. Tracked by GC/MS and LC/MSn
by Peter Lorenz*a), Jꢀrgen Conradb), Sarina Ducksteina), Dietmar R. Kammerera), and
Florian C. Stintzinga)
a) WALA Heilmittel GmbH, Department of Analytical Development & Research, Section
Phytochemical Research, Dorfstrasse 1, DE-73087 Bad Boll/Eckwꢀlden
(phone: þ 4971649306661; fax: þ 497164930227; e-mail: peter.lorenz@wala.de)
b) Hohenheim University, Institute of Chemistry, Bioorganic Chemistry, Garbenstraße 30,
DE-70599 Stuttgart
Hermidin (1), a piperidine-2,3-dione alkaloid, has been previously detected as a lipophilic
constituent in Mercurialis perennis L. and other Mercurialis species. Because of strong electron-
withdrawing effects of its carbonyl groups, an acidic H-atom is easily subtracted from 1, whereas the latter
shows high reactivity towards oxidation reactions or the attack of C-nucleophilic agents. To obtain a
better understanding of possible chemical pathways upon extraction of root parts of M. perennis, the
products obtained with different solvents from 1 were investigated. Extraction of M. perennis with
aqueous MeOH or EtOH yielded a mixture of hermidin quinone (3), 5-hydroxy-4-methoxy-5-
(alkoxycarbonyl)-1-methyl-3-pyrrolin-2-ones, 7 and 8, and d,l- and meso-isochrysohermidins, 5 and 6, all
of them being investigated by GC/MS and LC/MSn. The latter compounds were supposedly formed by
free-radical reactions, followed by spontaneous benzilic acid rearrangement and esterification.
Furthermore, extraction of M. perennis with aqueous Me2CO produced an aldol condensation product,
the known alkaloid speranskatine A (9a), which was identified by NMR after chromatographic
purification. In a similar manner, a CH2 homolog of speranskatine A (10a) was obtained as a novel
compound when ethyl methyl ketone (¼ butan-2-one; EtCOMe) instead of Me2CO was used for
extraction. Consequently, 1 easily undergoes artefact formation upon extraction of plant material with
polar or slightly polar solvents.
Introduction. – Extraction solvents play a crucial role in the isolation of natural
products from plants and in bioanalytical chemistry [1], because side reactions may
occur with the respective compounds. This has been reported in the literature for
alkaloids [2][3], anthocyanins [4], or several other compounds [1][5][6]. The artefacts
formed differ structurally from the genuine metabolites biosynthesized by the
organisms. These novel constituents may exert biological or pharmacological proper-
ties different from those of the primary natural products. At the same time, extracts can
reveal activity losses due to degradation of their active principles, or toxic compounds
might be formed [1]. In the course of phytochemical investigations on different herbal
plants, we identified dogꢁs mercury (Mercurialis perennis L.) as a rich source of various
natural constituents, such as terpenes, lipids, nitrogenous compounds, low-molecular
phenolics, n-alkylresorcinols, depsides, flavonoids, etc. [7 – 10]. M. perennis is an old
medicinal plant, belonging to the Euphorbiaceae family. Besides its use in ancient
phytomedicine, it nowadays becomes increasingly important in complementary
ꢂ 2014 Verlag Helvetica Chimica Acta AG, Zꢃrich

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medicine for the treatment of inflammations [11]. Comprehensive metabolomic studies
of M. perennis [7][9][10] revealed a closer insight on the alkaloid content of this
species. Hermidin (¼ 5-hydroxy-4-methoxy-1-methylpyridine-2,6(1H,3H)-dione, 1),
the main alkaloid of M. perennis, is an O2-sensitive compound, entirely stable under
reducing physiological conditions [12]. After extraction from the plant matrix, 1 is
easily oxidized in solution, forming a semistable blue anionic radical, the so-called
cyanohermidin (2), in a first step (Scheme 1) [7]. Compound 2 is stabilized via
mesomeric effects and possibly by interaction with cations, and has been early
characterized by redox titration [12] and ESR studies [13]. Downstream oxidation of 2
affords hermidin quinone (¼4-methoxy-1-methylpyridine-2,3,6(1H)-trione; 3) or the
dimeric quinone 4a via free-radical dimerization (Scheme 1). The latter is in a redox-
equilibrium with 4b, a dimeric analog of 1 [7][14]. We have recently shown that 1 and
its oxidation products undergo metabolic conversion during fermentation, by
Lactobacteria [15]. In the same line, the high reactivity of 1 towards C-nucleophilic
agents such as MeCHO was demonstrated for the first time [15].
Scheme 1. Proposed Pathways for the Reaction of Hermidin (1) under the Influence of Aqueous Organic
Solvents during Extraction of M. perennis
a) Deprotonation and oxidation (ꢀ2 Hþ; ꢀ eꢀ). b) Oxidation (ꢀeꢀ). c) Dimerization and oxidation
(þ2 H; ꢀ 2eꢀ). d) Reduction (þ2 H). e) ROH (R ¼ Me, Et)/H2O, benzilic acid rearrangement and
esterification. f) MeCOR (R ¼ Me, Et)/H2O, aldol condensation.

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With the aim to achieve a standardization, we investigated the impact of different
solvents on the secondary metabolite profile of the resulting extracts. In particular,
potential artefact formation from 1, which may occur upon the extraction of M.
perennis, was considered in the present study. Two common solvent classes, alcohols
and ketones, were used for extraction, and the resulting metabolite profiles were
extensively analyzed by chromatographic and MS techniques.
Results and Discussion. – Hermidin (1) and its oxidation products are prone to
benzilic acid rearrangements in the presence of aqueous alcohols. To investigate the
solvent influence on the constituent profile, root parts of M. perennis were first
extracted with aqueous MeOH. The obtained extract was evaporated to remove
MeOH, and the remaining H2O portion (pH 6.8) was extracted with AcOEt. GC/MS
Analyses of this extract showed a complex compound pattern (Fig. 1,a). By alignment
of mass spectra with the NIST database [16], several known compounds were assigned,
such as 3,4-dimethoxyphenol (tR 16.6 min), and simple fatty acids (palmitic, linoleic,
and linolenic acids), as well as the corresponding methyl esters. Furthermore,
nitrogenous compounds with odd-numbered molecular ion, Mþ peaks, were detected,
like the formerly identified hermidin (1) and hermidin quinone (3) at tR 16.5 and
20.3 min, respectively [7]. However, one N-containing compound at tR 20.2 min
(Fig. 1,a) could not readily be assigned. The Mþ peak at m/z 201, and the release of
COOMeþ and COþ fragments in EI-MS experiments (Table 1) revealed this compound
to be an N-heterocyclic methyl ester. Comparison with literature MS data [17]
disclosed the coincidence of this compound with methyl oxopyrrole carboxylate 7.
Compound 7 has been previously obtained by 1O2-mediated oxidative decarboxylation
of a pyrroledicarboxylate [17]. Furthermore, two peaks (tR 43.8 and 44.5 min) with
identical Mþ ions at m/z 400 and fragmentation patterns similar to those of 7 were
found (Fig. 1,a and Table 1). Based on literature MS data [18] and comparison with
synthesized reference material, these peaks could be assigned to a diastereoisomeric
mixture of isochrysohermidin (d,l/meso-5 1.2 : 1.0 (w/w)). Interestingly, compound 5
was first isolated from the Asian Mercurialis leiocarpa [19], but obviously as an
artefact, since the authors used MeOH for extraction. Later, a multistep total synthesis
of d,l- and meso-5 was accomplished by Boger and Baldino, and Wasserman et al.
[17][18][20][21], and it has been even shown earlier that 5 could be obtained from
chrysohermidin (4a) in one step via benzilic acid rearrangement in MeOH, catalyzed by
alkoxides or tertiary amines [22][23] (see Scheme 1). In this context, it appears
interesting that, in the current investigation, the reactivities of 3 and 4a were high
enough for a benzilic acid rearrangement, and that esterification already proceeded in
MeOH/H2O at pH 6.8. This finding was supported when the extraction of the plant
material was performed with EtOH/H2O. The GC/MS analysis of the resulting AcOEt
extract exhibited a similar but more complex compound pattern (Fig. 1,b) as compared
to the MeOH/H2O extract. Among others, an unknown peak at tR 21.5 min with an Mþ
peak at m/z 215 was striking. Because of structural resemblance to rac-7 (Table 1), the
novel compound, 8, could be readily assigned as a CH2 homolog of 7. Moreover, rac-8
was independently synthesized from 3 by reaction with EtOH/EtONa, and its structure
was determined by NMR. Furthermore, in the tR range between 38 and 47 min of the
EtOH/H2O extracts, several peaks of dimeric nitrogenous compounds with even-

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Fig. 1. Sections of GC/MS profiles (EI) showing metabolites of AcOEt fractions from M. perennis root
extracts obtained with different solvents. a) MeOH/H2O. b) EtOH/H2O. c) Me2CO/H2O. d) EtCOMe/
H2O. Assignment: 3,4-DMP, 3,4-dimethoxyphenol; C16 :O, palmitic acid; C18 :2, linoleic acid; C18 :3,
a-linolenic acid; and their corresponding methyl and ethyl esters (ME and EE, resp.)
numbered Mþ ions were analyzed (Fig. 1,b). Besides chrysohermidin (4a; tR 26.7 min),
two peaks of the diastereoisomers d,l- and meso-6 (Scheme 1) were detected at tR 44.3
and 46.0 min, respectively (Fig. 1,b). The characterization of d,l- and meso-6 was based
again on comparison with literature MS data [23] and with a reference compound,
obtained by reaction of 4a with EtOH/Et3N, as described in [23]. Interestingly, an
unknown compound with a peak at tR 45.3 min in GC/MS analyses attracted our
attention. On the basis of an Mþ peak at m/z 382 and peaks of fragment-ions
[COOEt]þ, [CO]þ, and [NMe]þ in the EI mass spectrum, this compound was
tentatively assigned as the half-benzilic acid rearrangement product 11 (see Scheme 2).
However, complete structure elucidation only based on MS data was impossible. To
support the proposed structure, a synthesis of 11 was attempted by treating 4a with
EtOH in the presence of low amounts of Et3N. It was possible to trap the intermediate,
and chromatographic purification on SiO2 yielded 11 as a yellow solid, the structure of

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Scheme 2. Possible Pathways of the Reaction of Chrysohermidin (4a) with EtOH/H2O
a) Benzilic acid rearrangement and esterification. b) Oxidative/reductive N-demethylation.
1
which was confirmed by comprehensive NMR investigations. The H-NMR spectrum
of the unknown compound (Table 2) exhibited a double set of seven different 1H spin
systems corresponding to one OH group (s), a CH2 unit (2dq) coupled with a Me group
(t), two MeO groups (2s), and two MeN groups (2s) (Table 2). A CH-edited gHSQC
spectrum revealed the CH2 group and enabled the assignment of all other protonated
C-atoms (data not shown). Further NMR experiments involving 13C and gHMBC
allowed complete assignment of the NMR signals of 11, but left the question regarding
its configuration unanswered, because all H- and C-signals were doubled (Table 2).
Since the synthesis of 11 was expected to yield a racemic mixture with respect to C(13),
indistinguishable by NMR, the observation of a double signal set indicating, e.g.,
diastereoisomers could be explained by the presence of a second chiral element in 11.
Obviously, free rotation of the C(3)ꢀC(11) is hindered by steric strain energy barrier of
neighboring substituents (MeO and C¼O). Thus, the axial chirality produces
atropisomers [24] which, in combination with the stereogenic center C(13), enables
the detection of diastereoisomers by NMR; however, unfortunately, they are not
separable by GC.
In addition to products resulting from the benzilic acid rearrangement, in the
EtOH/H2O extract, two further compounds were detected by GC/MS, at tR 39.5 and
41.3 min, which were tentatively assigned to the N-demethylation products 12 and 13,
respectively (Scheme 2), based on their Mþ peaks and fragmentation behavior
(Table 1). N-Demethylation of tertiary amines like alkaloids occurs in nature by
means of oxidative enzymes, e.g., cytochrome P 450, but it has been also achieved under
oxidative/deoxidative laboratory conditions [25][26]. Since 1 and 3 form part of a
common redox system, it is feasible that the latter may catalyze the N-demethylation of
4a and 11 to yield 12 and 13, respectively (Scheme 2). Remarkably, in a commercial
EtOH/H2O extract of M. perennis (tincture) the constituents d,l- and meso-6, and 13
were also detected as predominant N-containing artefacts by GC/MS (data not shown),
thus confirming the aforementioned results.

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Table 2. 1H- and 13C-NMR Data of the Novel Constituent 11/11’ (diastereoisomeric atropisomer
mixture)a). In CDCl3; d in ppm, J in Hz.
Position
d(H)
d(C)
HMBC
1
2
3
4
5
6
7
8
9
–
–
–
–
–
–
–
–
–
–
–
–
–
162.63/162.71 (C¼O)
119.47/119.85 (C)
157.00/157.16 (C)
171.80/171.91 (C¼O)
155.43/155.58 (C¼O)
27.65/27.73 (MeN)
60.81/61.05 (MeO)
–
3.32 (s)/3.33 (s)
4.10 (s)/4.13 (s)
–
C(6), C(2)
C(4)
–
10
11
12
13
14
15
16
17
–
–
–
–
168.34/168.49 (C¼O)
–
–
–
–
98.62/98.79 (C)
167.81/167.88 (C)
86.92/86.94 (C)
24.12/24.20 (MeN)
58.52/59.03 (MeO)
168.51/168.94 (C¼O)
63.91/64.31 (CH2)
2.78 (s)/2.79 (s)
3.84 (s)/3.85 (s)
–
4.38, 4.40 (2dq, J ¼ 7.1, 10.8)/4.34, 4.35
(2dq, J ¼ 7.1, 11.0)
1.31 (t, J ¼ 7.1)/1.35 (t, J ¼ 7.1)
4.58/4.63
C(13), C(10)
C(12)
–
C(18), C(16)
18
OH
14.08/14.09 (Me)
–
C(17)
C(13), C(16)
a) Relative configurations of the diastereoisomers 11 and 11’, together with arbitrary atom numbering.
To provide another analytical method to detect the assigned alkaloid reaction
products, these were also independently investigated for the first time by HPLC-DAD/
MSn (Table 3). Sections of a total ion current (TIC) and UV chromatogram of an
EtOH/H2O extract are shown in Fig. 2,a and b, respectively. After HPLC separation,
the compounds were analyzed in the ESIþ mode, which provides optimum results for
ionization of low-polar N-Me heterocycles, characterized in collision-induced dissoci-
ation (CID) experiments. All alkaloid homologs assigned by GC/MS (except 12) could
also be detected by LC/MS. As expected, differences in the EI-MS and ESIþ-MS
fragmentation patterns were observed (Table 3 and Scheme 3). Thus, MSn spectra of
the pyrrolidones 11 and d,l-6 are shown, as examples, in Fig. 2,c and d, respectively. An
initial H2O loss from the pseudo-molecular ion [M þ H]þ was observed for both
constituents resulting in fragment-ion peaks at m/z 365 and 411, respectively. While the
peaks for the [365 ꢀ CO]þ, [337 ꢀ CO2]þ, and [293 ꢀ CO]þ ions were detected for 11

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Fig. 2. HPLC(DAD)/MS Sections of an AcOEt fraction obtained from an EtOH/H2O extract of M.
perennis roots. a) Total ion chromatogram (TIC) recorded in the positive-ion mode (ESIþ). b) UV
Chromatogram recorded at 200 nm. c) MSn data of compound 11 (C16H18N2O6; Mr 382.32). d) MSn data
of compound d,l-6 (C18H24N2O10; Mr 428.39).

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Scheme 3. Proposed MS Fragmentation of d,l-/meso-6. a) Electron-impact mode (EI-MS; 70 eV). b)
Positive-ionization mode (ESIþ).
(Fig. 2,c) upon CID, the 2,2’-dioxo-3,3’-bipyrrole-dicarboxylate 6 displayed [411 ꢀ
MeOH]þ and [379 ꢀ MeN¼C¼O]þ fragment-ion peaks (Fig. 2,d, and Scheme 3,b).
The latter neutral loss of MeN¼C¼O (57 Da; see Fig. 2,d) has been formerly reported
for the fragmentation of N-Me carbamates, analyzed in the ESIþ mode [27 – 29], and
appears to be a key step in the fragmentation of bipyrrole-dicarboxylates. Hence, by
applying a combination of GC/MS and LC/MS techniques, it was possible to obtain a
clear picture of the respective chemical structures.
Formation of Aldol Condensation Products upon Extraction with Aqueous Ketones.
Besides alcohols also ketones are occasionally used for the extraction of medicinal
plants, even though ketones are capable of artefact formation [2][4]. The extraction of
M. perennis roots with a Me2CO/H2O mixture yielded an extract which was further
purified by AcOEt extraction as mentioned before. While TLC analysis (SiO2; AcOEt/
hexane 5 :1) of the latter gave an orange spot with anisaldehyde/H2SO4 spraying
reagent, GC/MS chromatograms exhibited an intense peak at tR 26.7 min (Fig. 1,c).
MS Experiments revealed an Mþ ion peak at m/z 227, and peaks of [M ꢀ CO]þ and
[M ꢀ CO ꢀ NMe]þ fragment ions (Table 1). A pure crystalline compound was isolated
from the crude AcOEt extract via preparative TLC (yield, 0.1% of the plant material).
By comparison of its 1H- and 13C-NMR, UV/VIS, and MS data, as well as melting point,
with literature data, the known pyridine-2,6-(1H,3H)-dione alkaloid speranskatine A
(9a) was identified. Compound (þ)-(R)-9a has previously been isolated from the Asian

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plant Speranskia tuberculata (Euphorbiaceae) [30][31]. Recently, a stereoselective
synthesis of (þ)-(R)-9a from 3 and Me2CO in the presence of the chiral amine l-
leucinol has been achieved [32]. However, in the present investigation no optical
rotation ([a]2D2 ¼ꢁ 0.00; c ¼ 0.25, MeOH) could be observed. Consequently, the
isolated compound was a racemate (rac-9a). Surprisingly, aldol condensation products
of the dimer 4a were not detected.
The formation of 9a by condensation of Me2CO with 1 or 3 via an aldol-type
oxidative addition reaction appears plausible. An aldol reaction is generally defined as
the nucleophilic addition of a carbonyl compound, in form of its enol or enolate, to
another C¼O compound acting as an electrophile; the initial enolization is promoted by
an acid or a base. However, aldol condensations proceed also in neutral media, e.g.,
under phase-transfer conditions [33], catalyzed by amino acids in salt-buffered
solutions [34], enzymatically [35], or spontaneously, such as the condensation of
Me2CO with the alkaloid berberine [2]. Hence, when 1 was stirred under N2 in Me2CO/
H2O (phosphate-buffered saline (PBS); pH 7.4) for 24 h, the GC/MS analysis revealed
a complete conversion to 9a (data not shown). However, intermediate formation of 3
from 1 cannot be ruled out, since AcOEt extraction and further sample preparation was
performed in the presence of O2.
Interestingly, low amounts of 9a were likewise detected in AcOEt fractions of
fermented aqueous extracts of M. perennis [15] when investigated by GC/MS. This
example also demonstrates that 9a is readily formed from 1 or 3 in the presence of
Me2CO, with the latter being presumably produced as a microbial side fermentation
product (data not shown).
Moreover, when the extraction of M. perennis was performed with ethyl methyl
ketone (EtCOMe)/H2O, and subsequent partitioning was conducted with AcOEt, an
indigo-blue spot on the TLC of this extract was observed, when sprayed with
anisaldehyde/H2SO4. Chromatographic purification of the crude extract yielded a
yellowish semicrystalline compound (0.15% of the plant material). EI-MS Fragmen-
tation (GC/MS; Fig. 1,d, and Scheme 4,a) of the compound was similar to that of 9a,
but the Mþ ion peak indicated elongation by a CH2 unit (m/z 241). Fragmentation of
the unknown compound in the EI-MS mode exhibited the formation of [M ꢀ Et]þ,
[M ꢀ Et ꢀ H2O]þ, and [M ꢀ Et ꢀ CO]þ ions (Table 1 and Scheme 4,a). In contrast,
collision-induced dissociation (CID) in LC/MS experiments showed the predominance
of the following fragments: [M þ H ꢀ H2O]þ, [M þ H ꢀ H2O ꢀ CO]þ, [M þ H ꢀ H2Oꢀ
CO ꢀ MeOH]þ (Table 3 and Scheme 4,b). Moreover, in ESIþ-MS experiments the
release of the MeN¼C¼O fragment (57 Da) was observed, similar to the fragmentation
of bipyrrole-dicarboxylates 5 and 6 (Scheme 3,b). Based on 1D- and 2D-NMR
experiments, i.e., 1H- and 13C-NMR, gHSQC, gHMBC (see Table 4), the novel
compound was identified as the CH2 homolog, rac-10, of speranskatine A (rac-9a).
Interestingly, GC/MS and LC/MS investigations revealed also N-demethylated
side-products of 9a and 10a, the piperidine-2,3-diones 9b and 10b, respectively (Fig. 1,c
and d, and Tables 1 and 3). Fig. 3,a, displays the fragmentation pattern of 10b, obtained
by LC/MSn. The peak at m/z 210 indicated an initial loss of a H2O moiety [M þ Hꢀ
H2O]þ from the pseudo-molecular ion [M þ H]þ. However, in the MS2 experiment, 10b
exhibited fragment-ion peaks at m/z 210 and 211, evidencing both a homolytic, as well
as a heterolytic cleavage of the C(3)ꢀO bond. Thus, upon CID [36], [M ꢀ OH]þ. and

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Scheme 4. Proposed MS Fragmentation of 10a. a) Electron-impact mode (EI-MS; 70 eV). b) Positive-
ionization mode (ESIþ).
Table 4. 1H- and 13C-NMR Data of the Novel Compound rac-10aa). In (D6)DMSO, d in ppm, J in Hz.
Position
d(H)
d(C)
HMBC
2
3
4
5
6
7
8
9
10
11
–
–
–
172.38
69.22
169.87
93.53
164.80
25.97
56.70
48.16
208.49
34.94
5.47 (s)
–
3.05 (s)
3.68 (s)
C(3), C(4)
–
C(2), C(6)
C(4)
C(2), C(3), C(4), C(10)
–
C(10), C(12)
3.35 (d, J ¼ 17.4, Ha), 3.24 (d, J ¼ 17.4, Hb)
–
2.43 (dq, J ¼ 7.3, 17.9, Ha),
2.36 (dq, J ¼ 7.2, 17.9, Hb)
0.84 (t, J ¼ 7.3)
12
OH
7.30
–
C(11), C(10)
C(9)
6.64 (s)
a) Arbitrary atom numbering as shown below.

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Fig. 3. a) MS, MS2, and MS3 data of 10b as obtained in the ESIþ mode. b) Section of MS2 data showing
fragment ions at m/z 210 and 211, suggesting homo- and heterolytic dissociation of HO. and/or H2O from
the pseudo-molecular ion ([M þ H]þ).
[M ꢀ H2O]þ ions are produced (Fig. 3,b), depending on the localization of the positive
charge. Moreover, a neutral loss of a H2O moiety appears possible. Furthermore,
fragment ions such as COþ, Etþ, and C3H4Oþ confirm the proposed structure 10b.
Conclusions. – The results of the present study have revealed the high sensitivity of
M. perennis constituents towards extraction solvents. Hermidin (1) and its oxidation
products 3 and 4a react with aqueous alcohols to yield mono- and bipyrrole-
carboxylates via benzilic acid rearrangement and esterification. Furthermore, the
extraction of the plant material with aqueous ketones was shown to yield highly
selective aldol condensation products via oxidative condensation. The spontaneous
formation of these products under neutral conditions is unusual, since such reactions
are generally proceed under strongly basic or acidic conditions. The current
investigations form a basis for the development of standardized Mercurialis extracts
with defined compound signatures. This approach of pinpointing artefact chemistry
may be applied to other plant secondary metabolites. In this way, it appears worthwhile
to study extraction chemistry by using specific solvents to obtain tailor-made extracts.

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Experimental Part
General. Fluorescence indicator green, 254 nm, for chromatographic purifications with a Chroma-
totronꢄ, was purchased from SigmaꢀAldrich (Saint Louis, MO, USA). 4-Methoxy-1-methylpyridine-
2,6(1H,3H)-dione (14; m.p. 111 – 1128) was synthesized as described in [7]. 3-(Hydroxyimino)-4-
methoxy-1-methylpyridine-2,6-(1H,3H)-dione (15; m.p. 195 – 1968) and hermidin (1) were obtained as
described by Swan [7][14].
GC/MS Analyses. GC/MS Analyses were performed with a PerkinElmer Clarus 500 gas chromato-
graph by split injection (split ratio, 30 :1; injection volume, 1.0 ml) coupled to a mass detector. The column
used was a Zebron ZB-5ms cap. column (60 m ꢂ 0.25 mm i.d. ꢂ 0.25 mm film thickness, 5% phenyl-
polysiloxane, and 95% dimethylpolysiloxane coating; Phenomenex, Torrance, USA). Carrier gas, He at a
flow rate of 1 ml/min. The injector used was a PSS (programmed-temp. split/splitless injector; temp.,
2508). The temp. program for the column oven was 100 to 3208 with a linear gradient of 48/min and a final
hold time of 30 min. The mass spectrometer was run in electron ionization (EI) mode (70 eV).
RP-HPLC-ESI-MSn Analyses. Chromatographic analyses were carried out on an Agilent 1200 HPLC
system (Agilent Technologies Inc., Palo Alto, USA), equipped with a binary pump, a micro-vacuum
degasser, an autosampler, a thermostatic column compartment, and a UV/VIS diode array detector. A
Sunfireꢄ C18-reversed phase (RP) column (5 mm particle size, 150 ꢂ 2.1 mm i.d.; Waters Corporation,
Milford, MA, USA) was used for chromatographic separation at 258 and a flow rate of 0.21 ml/min. The
UV detection of heterocyclic compounds (Fig. 2,a and Table 3) was performed at 200 nm. The mobile
phase consisted of HCOOH/H2O 0.2 :99.8 (mobile phase A) and MeCN (100%; mobile phase B).
Starting with 0% B for 10 min, a linear gradient was followed to 10% B at 10 min, then increasing to 23%
B at 60 min, further increasing to 100% B at 65 min, continuing for 5 min, before re-equilibration to
starting conditions. The injection volume of each sample was 10 ml. The LC system was coupled to an
HCTultra ion trap (Bruker Daltonik GmbH, D-Bremen) with an ESI source operating in the positive-
ionization mode by applying the following parameters: HV voltage, ꢀ 4000 V; dry gas, N2; 9.00 l/min with
a dry gas temp. of 3658; nebulizer, 35 psi. Full-scan mass spectra (mass range, m/z 50 – 2000) of the HPLC
eluates were recorded during chromatographic separation to yield [M þ H]þ ions. To obtain further
structural information, CID experiments were performed. MSn data were acquired in the auto MS/MS
mode. The instruments were controlled by Agilent Chemstation and EsquireControl Software (V6.1).
NMR Spectroscopy. NMR Spectra were recorded in (D6)DMSO or CDCl3 at 500 (1H) and 125 MHz
(13C), resp., with a Varian Unity Inova NMR spectrometer (D-Darmstadt); d in ppm rel. to residual
solvent signals of (D6)DMSO (1H: d(H) 2.50; 13C: d(C) 39.51) and CHCl3 (1H: d(H) 7.27; 13C: d(H)
77.00) as internal standard; J in Hz. 13C-NMR Signal assignments of the novel compounds 8, 10a, and 11
were based on 2D-heteronuclear NMR experiments (gHMBC and gHSQC). For evaluation of NMR
spectra, the program SpinWorks 3.1.7. (Copyrightꢄ 2010, K. Marat, University of Manitoba, USA) was
used.
Plant Material. Extraction (General Procedure (GP)) and Sampling. Root parts of M. perennis were
collected in September 2008 and 2013 in the forest close to Bad Boll/Eckwaelden (Germany), cleaned by
rinsing with tap H2O, and stored at ꢀ 808 until investigation. Voucher specimens of M. perennis were
deposited with the herbarium of the Department of Botany, Hohenheim University (Germany), and the
plant material was identified by Prof. O. Spring (voucher Nos. HOH-011281, HOH-011282, and HOH-
011283). For extraction, root parts of M. perennis (60.0 g) were immersed in an organic solvent/H2O
mixture (600 ml) and bubbled with N2 (15 min). Subsequently, the plant material was minced for 3 min
using an Ultra-turraxꢄ (15.000 rpm; IKA-Werke GmbH & Co. KG, D-Staufen), and the slurry was
allowed to stand for 24 h at 48. The sediment was recovered by vacuum suction over Celite, and the filter
cake was re-extracted in the same manner. The org. solvent was removed from the combined filtrates by
vacuum roto-evaporation, and the remaining aq. layer was saturated with NaCl and extracted with
AcOEt (200 ml and 2 ꢂ 100 ml). After drying (Na2SO4), the solvent was evaporated in vacuo to yield a
crude extract. For GC/MS analysis, the crude extract was re-dissolved in AcOEt (10 ml), and 1 ml injected
into the GC/MS. For LC/MS investigations, samples (20 mg each) were dissolved in MeCN/H2O 1:9 and
centrifuged for 5 min (14.000 rpm) before injection. The reaction products of 1 were isolated from the
crude extracts prior to compound characterization (see below).

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Helvetica Chimica Acta – Vol. 97 (2014)
Isolation of rac-Speranskatine A ( ¼ 3-Hydroxy-4-methoxy-1-methyl-3-(2-oxopropyl)pyridine-
2,6(1H,3H)-dione; 9a). Compound 9a was isolated from the crude AcOEt extract (0.16 g), obtained
by Me2CO/H2O extraction of the roots (see GP) using a Chromatotronꢄ (2-mm layer, SiO2/gypsum/
fluorescence indicator 254 nm 45 :18 :1.2 (w/w/w); preconditioned with hexane). Elution of 9a was
performed with hexane/AcOEt 100 :0 to 80 :20, and the corresponding compound bands were monitored
by fluorescence extinction (lmax 254 nm). The fraction containing 9a was evaporated to dryness using a
vacuum rotoevaporator. Yield: 0.062 g (0.1% of the plant material). Colorless crystals. TLC (SiO2;
AcOEt/hexane 5 :1): Rf 0.22; GC/MS purity: > 99% at tR 26.6 min. The m.p. (158 – 1608), UV/VIS, and
MS data were in agreement with those reported in [30]. The NMR data of 9a (recorded in (D6)DMSO);
corresponded to those recorded in CDCl3 [30]. [a]2D2 ¼ꢁ 0.00 (c ¼ 0.25, MeOH). 1H-NMR ((D6)DMSO,
500 MHz): 6.63 (br., OH); 5.47 (s, HꢀC(5)); 3.03 (s, HꢀC(7)); 3.68 (s, HꢀC(8); 3.36 (d, J ¼ 17.5,
HaꢀC(9); 3.25 (d, J ¼ 17.5, HbꢀC(9)); 2.05 (s, HꢀC(11)).13C-NMR ((D6)DMSO, 125 MHz): 206.04
(C(10)); 172.34 (C(2)); 169.88 (C(4)); 164.80 (C(6)); 93.51 (C(5)); 69.13 (C(3)); 56.71 (C(8)); 49.30
(C(9)); 29.68 (C(11)); 25.95 (C(7)).
Detection of 9a in Fermented Aq. Extracts. Fermented aq. extracts of M. perennis were obtained
according to an official procedure (German Homoeopathic Pharmacopoeia (GHP), 2008) [37] described
in [15]. Samples of three batches were taken after 2 years of storage. Aliquots of 4 ml were extracted with
AcOEt (2 ꢂ 10 ml), dried (Na2SO4), and the solvent was removed by vacuum rotoevaporation. The
residues were re-dissolved in AcOEt (1 ml) and analyzed by GC/MS. Isolated 9a (1 mg/10 ml AcOEt)
was used as a reference.
Isolation of rac-3-Hydroxy-4-methoxy-1-methyl-3-(2-oxobutyl)pyridine-2,6(1H,3H)-dione (10a).
After extraction of M. perennis roots with EtCOMe/H2O and partitioning of the corresponding H2O
phase with AcOEt (see GP), the obtained extract (0.37 g) was subjected to a Chromatotronꢄ separation
(for conditions, see above) to afford 10a. Yield: 0.093 g (0.15% of the plant material). Yellowish
semicrystalline solid; purity: > 95% (GC/MS). For NMR characterization, the material was further
purified via a second Chromatotronꢄ separation. TLC (SiO2; AcOEt/hexane 5 :1): Rf 0.28. UV/VIS
(MeCN): 219 (4.12), 260 (3.73). GC/MS (70 eV) purity: 98% (at tR 29.0 min). For 1H- and 13C-NMR: see
Table 4.
Hermidin Quinone ( ¼ 4-Methoxy-1-methylpyridine-2,3,6(1H)-trione; 3). Compound 15 (1.00 g,
5.43 mmol) was dissolved in a mixture of SnCl2 · 2 H2O (2.00 g, 8.86 mmol) and HCl (37% (w/w),
100 ml). After stirring (22 h), H2O (100 ml) was added, the mixture was neutralized (pH 7.0) by addition
of NaOH (16% in H2O (w/w)), and extracted with CHCl3 (3 ꢂ 100 ml). After drying (Na2SO4), the
solvent was evaporated in vacuo to yield 3 (0.23 g; GC/MS purity: 87% at tR 20.3 min). Yield: 22% of the
theory. A Chromatotronꢄ purification yielded 3 in higher purity (> 97%). UV/VIS (MeCN): 274 (3.96),
326 (3.16). MS (GC/MS, 70 eV; at tR 20.2 min): data in agreement with those reported in [7].
rac-Methyl 2,5-Dihydro-2-hydroxy-3-methoxy-1-methyl-5-oxo-1H-pyrrole-2-carboxylate (7). Com-
pound 3 (0.23 g; GC/MS purity: 87%; 1.183 mmol) was dissolved in MeOH (65 ml), and MeONa (3.2 ml;
30% in MeOH (w/w)) was added under N2 to yield a blue soln. After stirring (19 h), the reaction was
quenched by adding sat. NH4Cl/H2O soln. (25% (w/w); 240 g), and the mixture was extracted with CHCl3
(3 ꢂ 100 ml). Subsequently, the CHCl3 extract was dried (Na2SO4), and the solvent was evaporated in
vacuo. Compound 7 was isolated from the crude residue (0.185 g) by applying a Chromatotronꢄ
separation (2-mm layer; SiO2/gypsum/fluorescence indicator 254 nm 45 :18 :1.2 (w/w/w); preconditioned
with hexane). Elution of 7 was performed with hexane/AcOEt 70 :30 to 0 :100. Two fractions containing
7 (at 100% AcOEt) were separated, and the solvent was removed in vacuo to yield white crystals (0.084
and 0.046 g; GC/MS (at tR 20.2 min) purity: 86 and > 97%, resp.; total yield: 49% of the theory). M.p.
132 – 1338. TLC (SiO2; CHCl3/MeOH 4 :1): Rf 0.59. UV/VIS (MeCN): 207 (4.10), 250 (sh). The MS, 1H-
and 13C-NMR data were in agreement with those reported in [17].
rac-Ethyl 2-Hydroxy-3-methoxy-1-methyl-5-oxo-2,5-dihydro-1H-pyrrole-2-carboxylate (8). Synthe-
sis and purification of 8 were performed as described for 7, by treatment of 3 (0.14 g; GC/MS purity:
85%, 7.036 mmol) with EtOH (100 vol-%, 50 ml) and EtONa (1.0 ml, 20% in EtOH (w/w) under N2.
After stirring (23 h), 8 was isolated from the mixture as described above. By Chromatotronꢄ purification
(for conditions, see above), three fractions (46, 18, and 20 mg) were obtained from the crude extract
(0.165 g); GC/MS purity (at tR 21.5 min): 91, 98 and 94%, resp. Overall yield: 49% of the theory. TLC

Helvetica Chimica Acta – Vol. 97 (2014)
1621
(SiO2; CHCl3/MeOH 4 :1); Rf 0.61. UV/VIS (MeCN): 207 (4.14), 250 (sh). 1H-NMR (CDCl3,
500 MHz): 5.08 (s, HꢀC(3)); 4.60 (s, OH); 4.33 (dq, J ¼ 7.1, 10.7, HaꢀC(9); 4.27 (dq, J ¼ 7.1, 10.7,
HbꢀC(9)); 3.81 (s, HꢀC(7)); 2.73 (s, HꢀC(6)); 1.27 (t, J ¼ 7.1, HꢀC(10)). 13C-NMR (CDCl3, 125 MHz):
171.80 (C(8)); 170.59 (C(2)); 168.97 (C(4)); 94.11 (C(3)); 87.09 (C(5)); 63.83 (C(9)); 58.71 (C(7)); 23.46
(C(6)); 14.04 (C(10)). MS: see Tables 1 and 3.
Mixture of Chrysohermidin ( ¼ 4,4’-Dimethoxy-1,1’-dimethyl-3,3’-bipyridine-2,2’,5,5’,6,6’(1H,1’H)-
hexone; 4a) and 3. Compound 14 (1.00 g, 6.45 mmol) was dissolved in CHCl3 (50 ml) containing 1 drop of
HCl (37% (w/w)). Subsequently, SeO2 (1.20 g, 10.82 mmol) was added, and the mixture was stirred 24 h
at r.t. Then, precipitated Se was filtered off by vacuum suction over Celite and washed with CHCl3 (3 ꢂ
25 ml). The solvent was removed in vacuo to yield a crude product (2.71 g) containing high amounts of
Se. Purification was achieved by vacuum liquid chromatography (VLC) on silica 60 G (60 g),
preconditioned with hexane. Elution was performed with hexane/AcOEt 100 :0 to 20 :80. The
corresponding fractions containing 4a (analyzed by GC/MS) were combined, and the solvent was
distilled off in vacuo to yield an orange residue (0.21 g). GC/MS revealed a 3/4a ratio of 54/46% (w/w;
corrected by Mr; at tR 20.2 and 43.3 min, resp.). This mixture was used without further purification.
Isochrysohermidin ( ¼ d,l- and meso-Dimethyl 2,2’,5,5’-Tetrahydro-5,5’-dihydroxy-4,4’-dimethoxy-
1,1’-dimethyl-2,2’-dioxo-1H,1’H-3,3’-bipyrrole-5,5’-dicarboxylate; d,l-5 and meso-5, resp.). Compound 5
was synthesized as described by Abe et al. in [22]. In brief, 3/4a (0.25 g; see above) was dissolved in
MeOH (75 ml) and treated under N2 with a MeONa soln. (3.6 ml; 30% in MeOH (w/w)). After stirring
(19 h), the reaction was quenched with NH4Cl soln. (240 g, 25% in H2O (w/w)), and the mixture was
extracted with CHCl3 (3 ꢂ 100 ml). Then, the extract was dried (Na2SO4), and the solvent was
evaporated in vacuo to yield a crude product (0.21 g). The mixture d,l-5/meso-5 1.2 : 1.0 (w/w) and small
amounts of 7 were isolated from this crude material by using Chromatotronꢄ (2 mm layer; SiO2/gypsum/
fluorescence indicator 254 nm 45 :18 :1.2 (w/w/w); preconditioned with hexane). The elution of 7 was
performed with hexane/AcOEt 100 :0 to 0 :100. Yield: 0.045 g (GC/MS purity: 96% (at tR 20.2 min); 2%
of the theory. Subsequently, d,l-5 and meso-5 were eluted with CHCl3/MeOH 90 :30. Yield: 0.108 g (2.5%
of the theory; calc. on 14); GC/MS purity: 94% (at tR 43.8 and 44.5 min, resp.). Further purification of 5
1
was achieved by applying a second Chromatotronꢄ. The MS, and H- and 13C-NMR data of d,l-5 and
meso-5 were in agreement with those in [17].
d,l- and meso-Diethyl 2,2’,5,5’-Tetrahydro-5,5’-dihydroxy-4,4’-dimethoxy-1,1’-dimethyl-2,2’-dioxo-
1H,1’H-3,3’-bipyrrole-5,5’-dicarboxylate (d,l-6 and meso-6, resp.). Compound 6 was synthesized
according to a modified procedure [23]. In brief, the mixture 3/4a (0.21 g; see above) was dissolved in
EtOH (50 ml, 96 vol-%) under N2, treated with Et3N (1 ml), and stirred 20 h at r.t. The reaction was
quenched with a NH4Cl/H2O soln. (see above), and the mixture was extracted with CHCl3 (3 ꢂ 100 ml).
After drying (Na2SO4), the solvent was distilled off in vacuo to yield a black residue (0.336 g).
Purification was performed by using Chromatotronꢄ (2-mm layer; SiO2/gypsum/fluorescence indicator
254 nm 45 :18 :1.2 (w/w/w); preconditioned with CHCl3). Elution of 6 was performed with CHCl3/MeCN
100 :0 to 60 :40. A fraction at a CHCl3/MeCN ratio of 60 :40 yielded d,l-6/meso-6 as 1:1 (white solid,
0.059 g; yield: 2.1% calculated on 14; GC/MS purity: > 99%, at tR and 44.3 and 46.0 min, resp.). The
1H-NMR data of the product were in accordance with those reported in [23]. 13C-NMR (CDCl3,
125 MHz): 172.77 (C(8,8’), d,l-6/meso-6, overlapped); 169.65 (C(2,2’), d,l-6/meso-6, ov); 166.63 (C(4,4’),
d,l-6/meso-6, ov); 97.11 (C(3,3’), d,l-6); 95.96 (C(3,3’), meso-6); 87.46 (C(5,5’), d,l-6); 86.86 (C(5,5’),
meso-6); 62.62 (C(9,9’), d,l-6/meso-6, ov); 59.08 (C(7,7’), meso-6); 58.87 (C(7,7’), d,l-6); 24.58 (C(6,6’),
d,l-6); 24.15 (C(6,6’), meso-6); 14.09 (C(10,10’), d,l-6); 14.07 (C(10,10’), meso-6).
Ethyl 2-Hydroxy-3-methoxy-4-(4-methoxy-1-methyl-2,5,6-trioxo-1,2,5,6-tetrahydropyridin-3-yl)-1-
methyl-5-oxo-2,5-dihydro-1H-pyrrole-2-carboxylate (11). The mixture 3/4a (0.30 g) was dissolved in
EtOH (100 ml, 96 vol-%) and treated under N2 with Et3N (4 drops). After stirring (6 h), the reaction was
quenched by addition of HCl (37% (w/w); 5 drops), and EtOH was removed by vacuum rotoevapora-
tion. Subsequently, the residue was dissolved in AcOEt (50 ml), ammonium salts were filtered off by
vacuum suction, washed with AcOEt (50 ml), and the solvent was removed again in vacuo. The novel
compound 11 (0.017 g) was isolated from the crude (0.35 g) as a yellow solid by Chromatotronꢄ
separation (for conditions, see above); GC/MS purity: > 95%, at tR 45.3 min. NMR Data: see Table 2.

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Helvetica Chimica Acta – Vol. 97 (2014)
The authors wish to acknowledge Prof. O. Spring (Institute of Botany, Hohenheim University, DE-
Stuttgart) for identification of the plant specimens.
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Received February 11, 2014