Formation of nudicaulins in vivo and in vitro and the biomimetic synthesis and bioactivity of O-methylated nudicaulin derivatives

Article abstract of DOI:10.3390/molecules23123357

Nudicaulins are yellow flower pigments accounting for the color of the petals of Papaver nudicaule (Papaveraceae). These glucosidic compounds belong to the small group of indole/flavonoid hybrid alkaloids. Here we describe in vivo and in vitro experiments which substantiate the strongly pH-dependent conversion of pelargonidin glucosides to nudicaulins as the final biosynthetic step of these alkaloids. Furthermore, we report the first synthesis of nudicaulin aglycon derivatives, starting with quercetin and ending up at the biomimetic fusion of a permethylated anthocyanidin with indole. A small library of nudicaulin derivatives with differently substituted indole units was prepared, and the antimicrobial, antiproliferative and cell toxicity data of the new compounds were determined. The synthetic procedure is considered suitable for preparing nudicaulin derivatives which are structurally modified in the indole and/or the polyphenolic part of the molecule and may have optimized pharmacological activities.

Full text of DOI:10.3390/molecules23123357

molecules
Article
Formation of Nudicaulins In Vivo and In Vitro and
the Biomimetic Synthesis and Bioactivity of
O-Methylated Nudicaulin Derivatives
Bettina Dudek 1,†, Florian Schnurrer 1,†, Hans-Martin Dahse 2, Christian Paetz 1
,
Anne-Christin Warskulat 1, Christiane Weigel 2, Kerstin Voigt 2 and Bernd Schneider 1,
*
1
Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany;
bdudek@ice.mpg.de (B.D.); fschnurrer@ice.mpg.de (F.S.); cpaetz@ice.mpg.de (C.P.);
anne.warskulat@yahoo.de (A.-C.W.)
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI),
2
Adolf-Reichwein-Straße 23, D-07745 Jena, Germany; hans-martin.dahse@hki-jena.de (H.-M.D.);
christiane.weigel@hki-jena.de (C.W.); kerstin.voigt@hki-jena.de (K.V.)
*
†
Correspondence: schneider@ice.mpg.de; Tel.: +49-3641-5549682
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 9 November 2018; Accepted: 15 December 2018; Published: 18 December 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Nudicaulins are yellow flower pigments accounting for the color of the petals of
Papaver nudicaule (Papaveraceae). These glucosidic compounds belong to the small group of
indole/flavonoid hybrid alkaloids. Here we describe in vivo and in vitro experiments which
substantiate the strongly pH-dependent conversion of pelargonidin glucosides to nudicaulins
as the final biosynthetic step of these alkaloids. Furthermore, we report the first synthesis of
nudicaulin aglycon derivatives, starting with quercetin and ending up at the biomimetic fusion of a
permethylated anthocyanidin with indole. A small library of nudicaulin derivatives with differently
substituted indole units was prepared, and the antimicrobial, antiproliferative and cell toxicity data of
the new compounds were determined. The synthetic procedure is considered suitable for preparing
nudicaulin derivatives which are structurally modified in the indole and/or the polyphenolic part of
the molecule and may have optimized pharmacological activities.
Keywords: nudicaulins; flower pigments; indole alkaloids; biomimetic synthesis; bioassays
1. Introduction
The yellow pigment of Papaver nudicaule L. (“Iceland poppy”) petals was first reported in 1939
and named nudicaulin [
with two stereoisomeric aglycons were identified from petals of the yellow blooming cultivar of this
plant. An indole/flavonoid hybrid structure was proposed for these alkaloids in 2006 [ ], and the
revised constitution and the absolute configuration (Figure 1) were reported in 2013 [ ]. The relative
1]. In 2006, eight differently substituted compounds, also named nudicaulins,
2
3
and absolute configurations of nudicaulins (Figure 1) were assigned by NOESY spectroscopy and by
comparison of the experimental CD spectra with calculated spectra, respectively [3].
Biosynthetic studies [
precursors as well as the building blocks of these unique polyphenolic molecules (Scheme S1).
A mechanism for the formation of the nudicaulins was proposed [ ], but the conditions or enzymes
4,5] revealed pelargonidin glucosides and indole as probable biosynthetic
5
necessary to enable the reaction in the plant remained unknown. In the first part of the present study,
we therefore aimed at shedding light on the final step of the nudicaulin biosynthesis. Our in vivo and
in vitro experiments provide evidence that the proposed precursors are indeed used by P. nudicaule
and that their fusion is possible without enzymatic catalysis.
Molecules 2018, 23, 3357; doi:10.3390/molecules23123357
www.mdpi.com/journal/molecules

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Nudicaulins can be considered flavonoidal indole alkaloids. Compared to the large class of
monoterpenoidal indole alkaloids, the flavonoidal indole alkaloids are a small group with only a few
known compounds, such as lotthanongine [
immense pharmacological importance of some indole alkaloids, e.g., the anticancer drugs vincristine
and vinblastine [ 10], we aimed at the development of a synthesis of nudicaulin aglycon derivatives
and investigated their bioactivity.
6] and yuremamine [7,8]. Nevertheless, because of the
9
,
Figure 1. Structures of nudicaulin I (A) and II (B), flower pigments of Papaver nudicaule L.
2. Results
2.1. The Final Step of the Nudicaulin Biosynthesis
An in vivo experiment using a petal of the orange P. nudicaule cultivar was carried out to prove
that the free indole is incorporated into the nudicaulin molecule. In orange petals, a mixture of red
pelargonidin glucosides and yellow nudicaulins is responsible for the color. Under natural conditions,
the low level of indole [11] probably confines the conversion of an excess of pelargonidin glucosides
into nudicaulins. Therefore, drops of an aqueous indole solution were applied to the surface of orange
petals. After approximately 30 min, the area covered by the indole solution had turned yellow while
the untreated petal surface remained orange (Figure 2A). Treating a whole petal with indole solution
for 20 min, followed by extraction, HPLC analysis and comparison with the extract of an untreated
petal, clearly showed the disappearance of pelargonidin glucosides and the formation of nudicaulins
(Figure 2B). This result showed that free indole and pelargonidin glucosides are indeed the final
precursors of nudicaulins.
Additionally, we aimed to perform in vitro experiments. Preliminary investigations indicated
that the color of petal extracts depended on their pH value. The UV/Vis absorption of cell sap from
yellow P. nudicaule petals at pH values 4 and 3 differed completely. At a pH value higher than or equal
to 4, spectra show a strong maximum at 348 nm and a weak maximum at 510 nm. If the pH value
is lower than or equal to 3, the maxima are around 450 and 337 nm. In comparison, the absorption
spectrum of a petal measured under in vivo conditions has a broad maximum around 422 nm and two
shoulders at 331 and 360 nm (Figure 2C). The shoulders originate mainly from kaempferol glucosides,
which are also present in high amounts in the petals [2]. The absorption in the blue region is much
more intense than the absorptions at shorter wavelengths. In the in vitro experiment, this difference
holds true only for a pH of 2, suggesting that the vacuoles where the pigments are stored possess an
unusually acidic pH value [12], probably equal to or lower than pH 2.

Molecules 2018, 23, 3357
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Figure 2.
applied to a petal of the orange P. nudicaule cultivar. After 30 min, the color of the treated petal area
changed to yellow. ( ) HPLC elution profile (460 nm, HPLC 1, method 1) of a petal extract of the
(
A)
In vivo formation of nudicaulins: Drops of aqueous indole solution (1 mg
mL−1) were
·
B
orange P. nudicaule cultivar (dark green line) compared with the elution profile of an extract of a petal
which had been immersed in indole solution for 20 min (gray line). Pelargonidin glucosides were
transformed into nudicaulins during treatment with indole solution. (C) UV/Vis absorption spectra
of the cell sap of yellow P. nudicaule petals at different pH values (solid lines) in comparison with the
spectra of intact petals (dotted lines). The typical absorption in the spectral region around 450 nm
which accounts for the yellow color is present only in strongly acidic cell sap.
To mimic the nudicaulin formation in vitro, an aqueous petal extract of the orange P. nudicaule
cultivar was mixed with indole solution and analyzed by HPLC. Within 70 min, only a marginal
conversion of pelargonidin glucosides to nudicaulins occurred. In light of the presumed acidic
conditions in the flower, hydrochloric acid was added to another aliquot of the petal extract, decreasing
the pH from 5.8 to 1.3. Lowering the pH initiated a structural change of the pelargonidin glucosides
to their flavylium cation which was accompanied by an immediate color change from pale to bright
orange. Ten min after the addition of indole solution, the HPLC peaks of the pelargonidin glucosides
were reduced to about one-third of their original intensity. After 70 min, the peaks of the pelargonidin
glucosides had been almost entirely replaced by the peaks of the nudicaulins (Figure 3A). The color
change of the extracts illustrates this conversion (Figure 3B). These results demonstrate that the
production of nudicaulins in vitro is possible but requires an acidic pH value and a relatively long
reaction time.

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Figure 3. In vitro formation of nudicaulins in an extract of orange P. nudicaule petals supplemented
with indole solution. ( ) HPLC elution profile (460 nm, HPLC 1, method 2) of an extract without the
A
addition of HCl (blue dotted line: 10 min; gray line: 70 min after indole addition) and after adding HCl
(dark green line: 10 min; light green line: 70 min after indole addition), indicating the conversion of
pelargonidin glucosides into nudicaulins. (B) Petal extracts without HCl: the color stays pale orange,
indicating a mixture of pelargonidin glucosides and nudicaulins. Petal extracts with HCl: the color
changes from bright orange (color of the pelargonidin flavylium cations and nudicaulins in mixture) to
bright yellow, the color of nudicaulins.
2.2. Synthesis of O-Methylated Nudicaulin Derivatives
According to the “rule of five” for drug-likeness [13], the molecular mass of glucosidic nudicaulins,
e.g., nudicaulins I and II (Figure 1, molar mass 871) far exceeds the optimum defined for most
pharmacologically active compounds. An in silico drug-likeness prediction using MolCart [14] resulted
in positive scores for both nudicaulin glycosides and aglycons. Hence, bioactivity does not necessarily
require glycosylation. For these reasons, and for structural simplification, we aimed to synthesize
nudicaulin aglycons. However, as shown by hydrolytic experiments, completely deglucosylated
nudicaulins are unstable, due to their free hemiacetal structural moiety, and decompose without
forming any isolatable compounds [3]. Hence, we describe another structural condition for the
nudicaulin derivatives to be synthesized: a small substituent (e.g., methyl) in position 11 of the
non-glucosidic structure.
Due to the finding that nudicaulins are biosynthetically formed from pelargonidin glucosides
and indole [5], and the presented new results on the spontaneous formation of nudicaulins in vitro,
a biomimetic approach following Scheme 1 seemed reasonable. The reductive synthesis from
commercially available flavonols was considered the fastest way to prepare the anthocyanidin part.
We started with quercetin (3), the flavonol analogue of cyanidin, instead of the more expensive
kaempferol. Cyanidin possesses an additional hydroxyl group in ring B compared to pelargonidin,
the actual precursor of the nudicaulins in P. nudicaule (Scheme S1). Precursor-directed biosynthetic
(PDB) experiments had already shown that cyanidin can be incorporated into 30-hydroxynudicaulin.
But since the PDB experiments afforded nudicaulin derivatives only in a non-preparative scale [
method was unsuitable for the chemical synthesis.
5], this

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Scheme 1. Retrosynthetic analysis of nudicaulin derivatives. R = carbohydrate.
In the first step, quercetin (
acetone/water [15]. Reduction of 3,5,7,3 4 -penta-O-methylquercetin (
3,5,7,3040-penta-O-methylcyanidin (
) [16]. In the final step, compound
yielded a racemic mixture of permethylated nudicaulin diastereomers . The CD spectrum (Figure S43)
confirmed the presence of a racemate. The reaction required acidic conditions in order to facilitate
3) was permethylated with Me2SO4 under alkaline conditions in
0
0
4) with LiAlH4 in THF afforded
5
5
reacted with indole and
6
formation of a C–C bond between the electrophilic C-2 of
(Scheme 2).
5 and the nucleophilic C-3 of indole
Scheme 2. Synthesis of permethylated nudicaulin derivatives including the biomimetic reaction of
0
0
3,5,7,3 4 -penta-O-methylcyanidin (5) with different indoles.
0
0
Using 3,5,7,3 4 -penta-O-methylcyanidin (5) as an educt for the polyphenolic part of the nudicaulin
molecule and a series of substituted indoles (5-methylindole, 6-methylindole, 7-methylindole,
6-fluoroindole, 5-hydroxyindole) as educts for the nitrogen-containing part, compounds 7 to 11 were
obtained. According to the 30,40-dihydroxyphenyl ring of starting compound
3, the skeleton of all
synthetic compounds differs from that of the native nudicaulins by an additional hydroxyl group
0
(3 -OH). The structures were confirmed by the characteristic absorption between 460 and 480 nm, mass
spectrometry, and 1D and 2D NMR spectroscopic data.
2.3. Bioactivity
Compounds
and K-562 (Figure S45) cells and also the strongest cell toxicity against HeLa (Figure S46) cells.
The antiproliferative activity (GI50) values for compound were 1.3 mol
−1 against HUVEC and
6, 10 and 11 showed the highest antiproliferative effects against HUVEC (Figure S44)
6
µ
·
L

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1.1
and the values for compound 11 were 2.0
varied between 3.4 mol
−1 (compound
exhibited lower antiproliferative activity against HUVEC and K-562 cells, and lower cytotoxicity
µmol
·
L−1 against K-562 cells, the values for compound 10 were 2.2
mol mol
L−1 and 1.0 −1, respectively. Cytotoxicity
) and 5.7 mol and
−1 (compound 10). Compounds
µmol
·
L−1 and 1.0
µ
mol
·
L−1
µ
·
µ
·
L
µ
·
L
6
µ
·
L
7, 8
9
against HeLa cells. The antiproliferative and cytotoxicity data from all tested compounds are shown in
Table S1, with corresponding dose-response curves in Figure S44–46.
No antimicrobial activity against the different bacterial and fungal strains was found for
compounds
6
,
7
and
9
–
11 despite the application of high concentrations of 1 mg
·
mL−1. Only compound
8
exhibited marginal activity against Bacillus subtilis, Staphylococcus aureus (MRSA and non-MRSA
variant) as well as Mycobacterium vaccae. Detailed results are shown in Table S2.
3. Discussion
3.1. The Final Step of the Nudicaulin Biosynthesis
Nudicaulins are indole/flavonoid hybrid alkaloids that until now have only been known as
natural products from the petals of some Papaveraceous plants such as P. nudicaule and Meconopsis
cambrica [
1
,
17]. Their precursors have been proposed to originate from the phenylpropanoid
], but the conditions for the final biosynthetic step
and the indole biosynthetic pathways [4,5
have remained unknown. Our experiments aimed at clarifying the final step of the nudicaulin
formation. We confirmed that pelargonidin glucosides and free indole are the precursors of
nudicaulins and demonstrated their biosynthetic fusion by treating intact orange petals with an
indole solution. Additionally, the in vitro production of nudicaulins in an aqueous petal extract, which
was supplemented with indole, is reported here for the first time. The in vitro reaction was largely pH-
and time-dependent. While a slightly acidic pH yielded only a marginal amount of nudicaulins after
70 min, a strongly acidic pH resulted in conversion of approximately 67% of pelargonidins after 10
min and full conversion after 70 min. This result suggests that the pelargonidin glucosides, whose
structure also depends on pH value [18], have to be present in their flavylium cation form to react
with indole. As the different anthocyanin forms are in equilibrium with each other, a small portion of
flavylium cations is also present at pH 6, which explains the small amounts of nudicaulins produced
at this pH. Additionally, our investigation of the UV/Vis absorption spectra of P. nudicaule petal cell
sap revealed that the structure of the nudicaulins might also depend on pH as the spectra varied
substantially as pH changed. A spectrum comparable to the one of the living flower was seen only
around pH 2. This finding suggests that the vacuoles of P. nudicaule petals exhibit a strongly acidic pH
value. Even though a spontaneous last step of the nudicaulin biosynthesis seems to be possible under
these conditions, subcellular compartmentation studies need to be carried out, because both precursors
are present simultaneously during petal development without any nudicaulins being produced [5].
Furthermore, other factors such as transporters for indole, a proton pump to adjust the pH value in the
vacuoles, or the stabilization of anthocyanins and possibly nudicaulins by co-pigmentation or stacking
may play a role [19,20].
3.2. Synthesis of O-Methylated Nudicaulin Derivatives
In view of the activity of other indole alkaloids, the synthesis of nudicaulins appeared a promising
way to obtain new compounds with interesting properties, e.g., potential antiproliferative activity.
To synthesize nudicaulin derivatives, we chose a biomimetic approach, taking advantage of the
spontaneous fusion of anthocyanins with the indole moiety that occurs under strongly acidic conditions.
The synthetic route mimics the final step of nudicaulin biosynthesis as it occurs in the petals of the
yellow and orange P. nudicaule cultivars. Since the glucose moieties of native nudicaulins were
considered nonessential for potential bioactivity, we aimed to synthesize aglycon derivatives instead of
glucosides. Furthermore, to avoid the instability of the molecules caused by the hemiacetal structure,
the hydroxyl group in position 11 had to be protected by methylation, as were the other hydroxyl

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groups of the cyanidin part of the molecule. Thus, we were successful in the first synthesis of
non-glycosylated nudicaulins.
Optimizing the reaction conditions (reaction time and temperature, adjustment of pH, etc.) is
assumed to result in improved yield for the final step of the reaction. In general, our work provides
easy synthetic access to flavonoidal indole alkaloids.
3.3. Bioactivity
The synthetic nudicaulin derivatives
6
–11 showed high antiproliferative activity against HUVEC
and K-562 cells and cytotoxic activity against HeLa cells. Compounds
6
–11 possessed an activity
against K-562 cells similar to doxorubicin, an established chemotherapeutic agent. The antiproliferative
activity against HUVEC cells and cytotoxicity against HeLa cells was slightly lower than against K-562
cells (Table S1). However, as the activities varied only little within the same order of magnitude,
further investigations are necessary to evaluate the structure–activity relationship of the nudicaulin
derivatives in-depth. Our study is suggesting the nudicaulin skeleton as a possible new core structure
for further research, aiming at the enhancement of the antiproliferative selectivity.
4. Materials and Methods
4.1. General Experimental Procedures
Solvents and reagents were purchased from Sigma-Aldrich, Deisenhofen, Germany and used
without further purification. All reactions were monitored by thin-layer chromatography (TLC) carried
out on 0.25 mm Merck silica gel sheets 60-F254 (Merck, Darmstadt, Germany) and visualized by UV
light (254 nm).
HPLC 1: The analytical Agilent 1100 HPLC system consisted of a degasser G1322A, binary pump
G1312A, autosampler G1313A, and photodiode array detector (PDA) G1315B (Agilent, Waldbronn,
Germany) equipped with an EC250/4 Nucleodur C18 HTec column (5 µm; Macherey-Nagel, Düren,
Germany). Solvent A was acidified water (0.1% trifluoroacetic acid (TFA) (v/v)); solvent B, methanol
(MeOH); and solvent C, acidified MeOH (0.1% TFA (v/v)). The column temperature was 25 ◦C, the
flow rate was 1 mL
min−1, and the detection wavelengths were 211, 254, 281, 351 and 460 nm. Method
·
1 included isocratic elution with 30% B/70% A for 20 min followed by a 35-min gradient from 30% to
65% of B with a subsequent washing step (65% to 100% solvent B in 10 min/100% solvent B for 10 min)
and equilibration to starting conditions (100% to 30% solvent B in 5 min/30% solvent B for 10 min).
Method 2 consisted of a linear binary gradient from 0 to 76% of solvent B in solvent A in 38 min with a
subsequent washing step (76% to 100% solvent B in 2 min/ 100% solvent B for 5 min) and equilibration
to starting conditions (100% to 0% solvent B in 5 min/0% solvent B for 10 min). Method 3 included a
linear binary gradient from 50 to 100% of solvent C in solvent A in 35 min with a subsequent washing
step (100% solvent C for 5 min) and equilibration to starting conditions (100% to 50% solvent B in 5
min/50% solvent B for 10 min).
HPLC 2: Preparative HPLC separations were carried out using a Shimadzu Prominence HPLC
system (Shimadzu, Duisburg, Germany), controlled by the Shimadzu LCSolution ver. 1.21, and
consisting of a DGU-20A5 degasser, LC-20AT gradient pump, SIL-10AP autosampler, CTO-20A
column oven, SPD-20A UV detector, FRC-10A fraction collector and CBM-20A system controller.
A Nucleodur VP250/10 Nucleodur C18 HTec column (5
µ
m; Macherey-Nagel, Düren, Germany) was
◦
used at 25 C with a flow rate of 3.5 mL
·
min−1. The binary solvent system consisted of acidified water
(0.1% formic acid (FA) (v/v), solvent A) and acidified MeOH (0.1% FA (v/v), solvent B). Method 4
included a linear binary gradient from 70–100% B in 15 min with a subsequent washing step (100%
solvent B) and fraction collection using intensity and time.
LC 3: Flash chromatography was carried out by a Biotage Isolera Spectra One (Biotage, Uppsala,
Sweden) equipped with Biotage Snap-Ultra cartridges (Biotage HP-Sphere, 25 g) and a PDA detector in
λ-al detection mode. The binary solvent system consisted of acidified dichloromethane (0.5% TFA (v/v);

Molecules 2018, 23, 3357
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solvent A) and acidified methanol (0.5% TFA (v/v); solvent B). The flow rate was 30 mL
·
min−1. Method
5 consisted of a linear binary gradient from 0–10% B in 34 min, followed by a linear binary gradient
from 10–100% in 2 min with a subsequent washing step (100% solvent B) and fraction collection
using intensity.
NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer (operating at
1
400.13 MHz for H and 100.61 MHz for 13C) equipped with 5 mm BBFO probe (Bruker Biospin,
Rheinstetten, Germany) or a Bruker Avance III HD 500 MHz spectrometer (operating at 500.13 MHz
for 1H and 125.75 MHz for 13C) equipped with a 5 mm TCI cryoprobe. Structures were assigned with
the aid of 1H NMR, 13C NMR, APT, 1H,1H-COSY, HSQC, 1H,13C-HMBC, and 1H,15N-HMBC spectra.
CDCl3 (for compounds
4 and 5) or MeOH-d4/1% TFA (for nudicaulin derivatives 6–11) were used
as solvents. Chemical shifts were referenced to the residual solvent peaks at δH 7.26 and δC 77.0 for
CDCl3 and δH 3.31 and δC 49.15 for MeOH-d4. Data acquisition and processing were accomplished
using TopSpin 3.2 (Bruker Biospin, Rheinstetten, Germany). Standard Bruker pulse sequences as
implemented in Bruker TopSpin were used for data acquisition.
For HPLC-HRESIMS measurements, an Agilent Infinity 1260 HPLC was coupled to a Bruker
Compact OTOF mass spectrometer (Bruker Daltonics, Bremen, Germany), controlled by the Bruker
Compass control suite (version 1.9, Bruker Daltonics, Bremen, Germany) together with Bruker
OTOFControl version 4.0 (Bruker Daltonics, Bremen, Germany). The samples were analyzed in
the positive ionization mode in the mass range m/z 50 to 1300 using 30,000 m/Vm resolving power.
UV/Vis absorption spectra were recorded with a Jasco V-550 spectrophotometer (Jasco, Pfungstadt,
Germany) between 200 and 700 nm. Blank sample measurements were used for baseline correction.
CD spectra were recorded on a Jasco J-810 spectropolarimeter (Jasco, Pfungstadt, Germany).
All compounds were measured in MeOH using a quartz cuvette of 1 mm width. Spectra were recorded
between 200 and 600 nm and were accumulated 5 times. Baseline correction was performed on the
basis of a MeOH blank sample.
4.2. Plant Material
The Papaver nudicaule L. plants were raised from seeds of ‘Summer Breeze Yellow’ and ‘Summer
Breeze Orange’ (Jelitto Staudensamen, Schwarmstedt, Germany) in soil (clay substratum and TS1
substratum, Klasmann-Deilmann, Geeste, Germany) in the glasshouse facilities of the Max Planck
Institute for Chemical Ecology, Jena, Germany. The glasshouse chamber was equipped with Phillips
Sun-T Agro 400 Na lights to support an illumination period of 14 h. Temperatures ranged from
19–21 ◦C in the night and from 21–23 ◦C during the day with a relative air humidity of 50% to 60%.
Plants were irrigated for 6 min every day.
4.3. Final Step of Nudicaulin Biosynthesis
In vivo: One petal of the orange P. nudicaule cultivar was placed in an aqueous indole solution
(1 mg
·
mL−1, Merck, Darmstadt, Germany) for 20 min until its color changed to yellow. Afterwards, the
petal and an untreated control petal were extracted with methanol–water mixture 1:1 (20
µL solvent
per mg tissue, 30 min ultrasound) and centrifuged (13,200 rpm) for 20 min, after which the supernatant
was used for HPLC analysis (HPLC 1, method 1).
In vitro: Petals from the orange cultivar were homogenized in deionized water (10 µ
L mg−1
tissue) by Bertin Minilys Homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France; 120 s,
0.3 g of 1.4 mm ceramic (zirconium oxide) beads, 5000 rpm). After 10 min of ultrasound and 10 min of
centrifugation (13,200 rpm), 100
(1 mg
mL−1), vortexed and measured by HPLC 1 after 10 min and 70 min (method 2). Additionally,
100 L of the extract was mixed with 1 L of hydrochloric acid (37%), intensifying the color. The pH
value decreased from 5.8 to 1.2 (measured by a pH-electrode HI1093B, Hanna Instruments, Vöhringen,
Germany). Afterwards, 10 L of aq. indole solution (1 mg
mL−1) was added, and after vortexing and
10 or 70 min of reaction time, the samples were measured by HPLC 1 (method 2).
µL of the supernatant was mixed with 10 µL of an aq. indole solution
·
µ
µ
µ
·

Molecules 2018, 23, 3357
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pH dependence of nudicaulin UV/Vis absorption: The cell sap was pressed from the petals of the
yellow P. nudicaule cultivar and diluted with deionized water (1:2). UV/Vis absorption spectra were
recorded in a quartz cell with a thickness of 10 mm. The pH of the sample as well as the blank were
adjusted with hydrochloric acid and sodium hydroxide solution (1 mol L
−1) using the pH-electrode
·
HI1093B (Hanna Instruments, Vöhringen, Germany). To obtain a spectrum of a petal, the tissue
was cut in pieces and its transparency was increased by vacuum infiltration in deionized water [21].
Afterwards, the moistened petal was placed between two slides and measured using the UV/Vis
spectrophotometer. The spectra were normalized to a maximal intensity of 1 mAU.
4.4. Synthetic Procedures and Analytical Data
4.4.1. 3,5,7,3040-Penta-O-Methylcyanidin (5)
3,5,7,3040-Penta-O-methylquercetin (
Me2SO4 in acetone/water 2:1 according to a reported procedure [22]. Compound
4
) was obtained from quercetin (
3
) by methylation with
4
was purified by
recrystallization from CH2Cl2/EtOAc (1:6). Reduction of compound
under argon atmosphere as described by Kimura et al. [16], afforded 3,5,7,3 4 -penta-O-methylcyanidin
(5). Analytical data of compounds 4 and 5 matched those reported in the literature [16,22].
4
,0u0sing LiALH4 in dry THF
4.4.2. General Procedure for the Preparation of Nudicaulin Derivatives (Racemic Mixtures)
0
0
3,5,7,3 ,4 -Penta-O-methylcyanidin (1 eq.) was dissolved in a 1:1 solution of MeOH/water at room
temperature. The pH of the resulting solution was set to 2.5 by adding 0.1 M HCl aq., followed by a
methanolic indole solution (3.0–5.5 eq.). The reaction mixture was stirred for 2.5 h and was stopped
by the evaporation of MeOH. The aq. residue was spiked with FA (4 mL) and extracted with DCM
(
4 × 25 mL). The combined extracts were dried over MgSO4, and the solvent was evaporated in vacuo
until dryness. Purification was done by LC 3, method 5. Final purification was done by preparative
HPLC 2, method 4. Evaporation of the solvent by a N2 stream and drying in vacuo yielded the desired
nudicaulin. Purity was verified by HPLC 1, method 3.
4.4.3. 5,7,11,30,40-Penta-O-Methylnudicaulin (6)
3,5,7,30,40-Penta-O-methylcyanidin (17.8 mg, 0.038 mmol, 1 eq.) dissolved in MeOH/water
mixture (1:1, 100 mL) and indole (22.25 mg, 0.19 mmol, 5 eq.) reacted according to the general
procedure. The title compound was obtained as a yellow solid (yield: 3.1 mg, 0.007 mmol, 17%). MS:
(M + H)calc: 472.1755 m/z, (M + H)exp: 472.1772 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 210 nm, 472 nm.
4.4.4. 16-Methyl-5,7,11,30,40-Penta-O-Methylnudicaulin (7)
3,5,7,30,40-Penta-O-methylcyanidin (44.8 mg, 0.095 mmol, 1 eq.) dissolved in MeOH/water
mixture (1:1, 350 mL) and 5-methylindole (53.7 mg, 0.41 mmol, 4 eq.) reacted according to the general
procedure. The title compound was obtained as a yellow solid (yield: 3 mg, 0.006 mmol, 6.4%). MS:
(M + H)calc: 486.1911 m/z, (M + H)exp: 486.1913 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 209 nm, 469 nm.
4.4.5. 17-Methyl-5,7,11,30,40-Penta-O-Methylnudicaulin (8)
3,5,7,30,40-Penta-O-methylcyanidin (17.9 mg, 0.038 mmol, 1 eq.) dissolved in MeOH/water
mixture (1:1, 100 mL) and 6-methylindole (14.6 mg, 0.11 mmol, 3 eq.) reacted according to the general
procedure. The title compound was obtained as a yellow solid (yield: 2.4 mg, 0.005 mmol, 12%). MS:
(M + H)calc: 486.1911 m/z, (M + H)exp: 486.1929 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 206 nm, 473 nm.
4.4.6. 18-Methyl-5,7,11,30,40-Penta-O-Methylnudicaulin (9)
3,5,7,30,40-Penta-O-methylcyanidin (44.8 mg, 0.095 mmol, 1 eq.) dissolved in MeOH/water
mixture (1:1, 350 mL) and 7-methylindole (68.5 mg, 0.52 mmol, 5.5 eq.) reacted according to the general

Molecules 2018, 23, 3357
10 of 14
procedure. The title compound was obtained as a yellow solid (yield: 4.5 mg, 0.009 mmol, 10%). MS:
(M + H)calc: 486.1911 m/z, (M + H)exp: 486.1925 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 211 nm, 478 nm.
4.4.7. 17-Fluoro-5,7,11,30,40-Penta-O-Methylnudicaulin (10)
3,5,7,30,40-Penta-O-methylcyanidin (50 mg, 0.1 mmol; 1 eq.) dissolved in MeOH/water mixture
(1:1, 350 mL) and 6-fluoroindole (64.6 mg, 0.5 mmol, 5 eq.) reacted according to the general procedure.
The title compound was obtained as a yellow solid (yield: 7.7 mg, 0.016 mmol, 16%). MS: (M + H)calc
:
490.1660 m/z, (M + H)exp: 490.1668 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 212 nm, 395 nm, 478 nm.
4.4.8. 16-Hydroxy-5,7,11,30,40-Penta-O-Methylnudicaulin (11)
0
0
3,5,7,3 ,4 -Penta-O-methylcyanidin (37.8 mg, 0.08 mmol, 1 eq.) dissolved in MeOH/water mixture
(1:1, 350 mL) and 5-hydroxyindole (53.7 mg, 0.40 mmol, 5 eq) reacted according to the general procedure.
The title compound was obtained as a yellow solid (yield: 2.6 mg, 0.005 mmol, 7%). MS: (M + H)calc
:
488.1704 m/z, (M + H)exp: 488.1708 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 203 nm, 465 nm.
For 1H-NMR data of compounds to 11, see Table 1, and for 13C-NMR data, see Table 2. NMR,
6
MS, and UV/Vis spectra are available in the Supplementary Materials (Figures S1–30, S31–36 and
S37–42).

Molecules 2018, 23, 3357
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1
Table 1. H-NMR data of nudicaulin derivatives 6 to 11 (500 MHz, MeOH-d4/1% TFA).
6
7
8
9
10
11
Position
δ, mult., J [Hz]
δ, mult., J [Hz]
δ, mult., J [Hz]
δ, mult., J [Hz]
δ, mult., J [Hz]
δ, mult., J [Hz]
3
6
8
5.39, s
6.30, d, 1.9
6.25, d, 1.9
8.34, d, 7.8
7.60, dd, 7.6, 7.8
7.68, dd, 7.6, 8.0
7.75, d, 8.0
7.97, d, 2.0
7.37, d, 8.7
8.26, dd, 2.0, 8.7
4.01, s
5.36, s
5.36, s
5.33, s
6.29, d, 1.9
6.25, d, 1.9
8.15, dd, 2.1, 6.7
7.47, overlap
7.48, overlap
5.36, s
6.30, d, 1.8
6.24, d, 1.8
5.31, s
6.30, d, 1.9
6.25, d, 1.9
8.15, brs
6.30, d, 1.8
6.25, d, 1.8
8.20, d, 8.2
7.42, brd, 8.2
6.29, d, 1.9
6.25, d, 1.9
7.71, d, 2.3
15
16
17
18
8.29, dd, 8.7, 4JH-15/F-17 = 4.9
7.32, ddd, 8.7, 2.3, 3JH-16/F-17 = 8.7
7.50, brd, 8.3
7.62, d, 8.3
7.96, d, 2.1
7.38, d, 8.7
8.25, dd, 2.1, 8.7
4.00, s
7.07, dd, 2.3, 8.8
7.53, d, 8.8
7.90, d, 2.1
7.34, d, 8.7
8.18, dd, 2.1, 8.7
3.99, s
7.55, brs
7.95, d, 2.0
7.35, d, 8.7
8.23, dd, 2.0, 8.7
4.00, s
7.49, dd, 2.3, 3JH-18/F-17 = 8.4
0
2
5
6
7.94, d, 2.0
7.34, d, 8.6
8.23, dd, 2.0, 8.6
3.99, s
7.92, d, 2.0
7.34, d, 8.7
8.20, dd, 2.0, 8.7
3.99, s
0
0
5-OMe
7-OMe
11-OMe
3 -OMe
4 -OMe
3.76, s
3.66, s
3.99, s
4.08, s
3.76, s
3.65, s
3.99, s
4.08, s
3.76, s
3.65, s
3.99, s
4.07, s
3.75, s
3.68, s
3.98, s
4.07, s
3.75, s
3.65, s
3.98, s
4.06, s
3.76, s
3.64, s
3.99, s
4.06, s
0
0
16-Me
17-Me
18-Me
2.52, s
2.54, s
2.64, s

Molecules 2018, 23, 3357
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Table 2. 13C-NMR data of nudicaulin derivatives 6 to 11 (125 MHz, MeOH-d4/1% TFA).
Position
6
7
8
9
10
11
δ
δ
δ
δ
δ, J13C-19F [Hz]
δ
2
3
178.2
46.0
177.7
45.9
176.9
46.0
178.4
46.0
179.8
46.2
176.2
45.8
4
5
6
100.6
159.0
94.3
100.7
159.0
94.2
100.3
158.8
94.2
100.9
158.8
94.2
100.7
158.7
94.2
100.8
158.7
94.2
7
8
166.0
90.1
166.0
90.1
166.0
90.1
165.9
90.1
166.0
90.1
166.0
90.1
9
161.7
127.8
169.7
132.5
123.1
125.8
129.0
132.1
117.7
148.9
124.5
116.4
151.3
158.9
113.3
132.0
56.6
161.7
127.7
169.2
132.5
123.2
126.2
139.7
133.0
117.3
146.9
124.4
116.3
151.2
158.0
113.5
131.8
56.9
161.7
127.8
168.6
132.5
120.4
125.6
129.9
143.8
118.0
149.3
124.5
116.3
151.2
158.6
113.4
131.7
56.9
161.6
127.8
169.1
132.9
123.1
123.3
128.9
133.7
128.4
147.8
124.4
116.2
151.2
158.7
113.4
131.7
56.9
161.7
127.6
168.8
132.0
161.6
127.8
168.6
132.9
124.5
112.3
159.5
119.2
118.4
141.5
124.4
116.0
151.2
158.3
113.3
131.1
56.9
11
12
13
14
15
16
17
18
19
119.7, d, 2.5
127.2, d, 10.4
115.9, d, 24.0
165.2, d, 251.0
105.7, d, 27.4
151.2
0
1
2
3
4
5
6
124.5
116.3
151.3
158.9
113.5
131.9
56.6
56.4
0
0
0
0
0
5-OMe
7-OMe
11-OMe
3 -OMe
4 -OMe
56.5
53.7
56.9
57.3
56.5
53.7
56.6
57.3
56.5
53.6
56.6
57.3
56.4
53.7
56.5
57.3
56.4
53.3
56.6
57.2
53.7
56.9
57.3
0
0
16-Me
17-Me
18-Me
21.7
22.0
17.1
4.5. Cell Toxicity
To determine cell toxicity and antiproliferative activity, nudicaulin derivatives
6–11 were used
in a dimethylsulfoxide solution (conc. 10 mg
·
mL−1). Five replicates were investigated for each
assay. Cell toxicity assays were conducted using a HeLa cell line, and antiproliferative activity was
assayed using HUVEC and K-562 cell lines. All cell lines are from the Leibniz Institute for Natural
Product Research and Infection Biology, Hans Knöll Institute (HKI), Adolf-Reichwein-Straße 23,
07745 Jena, Germany. The test substances were dissolved in dimethylsulfoxide before being diluted
in the respective medium of the cells to concentrations between 0.1 and 100
µ
mol L
−1. The adherent
·
cells were harvested at the logarithmic growth phase after soft trypsinization using 0.25% trypsin in
phosphate-buffered saline (PBS) containing 0.02% ethylenediaminetetraacetic acid (EDTA). For each
experiment, approximately 10,000 cells were seeded with 0.1 mL culture medium per well of the 96-well
microplates. HeLa cells were pre-incubated for 48 h prior to the addition of the test compounds, which
were carefully diluted on the subconfluent monolayers. Incubation was then conducted in a humidified
◦
atmosphere at 37 C and 5% CO2. In the case of K-562 cells, the number of viable cells in every well was
determined using the CellTiter-Blue1 assay [23]. The adherent HUVEC and HeLa cells were fixed by
glutaraldehyde and stained with a 0.05% solution of methylene blue for 10 min. After gentle washing,
the stain was eluted with 0.2 mL of 0.33 n HCl in the wells. The optical densities were measured at
660 nm in a SUNRISE microplate reader (Tecan Trading AG, Männedorf, Switzerland).

Molecules 2018, 23, 3357
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4.6. Antimicrobial Activity
To determine their antimicrobial activity, nudicaulin derivatives
6
–
11 were used in a methanolic
solution (concentration 1 mg
mL−1). Antibacterial bioassays were performed in accordance with Krieg
·
et al., 2017 [24] using strains of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, Enterococcus faecalis and Mycobacterium vaccae. The bacteria were cultivated on standard
I nutrient agar (NA I) in Petri dishes at 37 ◦C. Antifungal bioassays were conducted at 30 ◦C using
strains of Sporobolomyces salmonicolor and Penicillium notatum, which were cultivated on malt agar
(MA), and Candida albicans, which was cultivated on yeast morphology agar (YMA). After inoculation,
a disc (9 mm in diameter) was removed from the center of the Petri dish and 50
was added to the cavity. After 18 h of incubation at the respective temperatures, the inhibition areolas
were measured. Ciprofloxacin (5
mL−1 in deionized water) and amphotericin B (10 mL−1 in
µL of the test solution
µ
g
·
µ
g
·
DMSO/MeOH 1:1) were used as reference compounds against bacterial and fungal strains, respectively.
Supplementary Materials: The following are available online. Scheme S1: Formation of nudicaulin derivatives,
Figures S1–30: NMR spectra of compounds
UV/Vis spectra of compounds
relationship for antiproliferative effect and cytotoxicity of compounds
S1: Antiproliferative and cytotoxicity data of compounds 11, Table S2: Antimicrobial data of compounds
6
–
11, Figures S31–36: HR-MS of compounds
11, Figure S43: CD spectra of compound , Figure S44–46: Dose-response
11 on HUVEC, K-562 and HeLa cells, Table
11.
6–11, Figures S37–42:
6
–
6
6
–
6–
6
–
Author Contributions: Conceptualization, B.D., A.-C.W. and B.S.; Data curation, B.D. and B.S.; Formal analysis,
B.D., F.S. and B.S.; Investigation, B.D., F.S., H.-M.D., C.W. and K.V.; Methodology, B.D., F.S., C.P., A.-C.W. and B.S.;
Project administration, B.S.; Supervision, B.S.; Visualization, B.D., F.S., H.-M.D. and K.V.; Writing–original draft,
B.D. and B.S.; Writing–review & editing, B.D., F.S. and B.S.
Funding: This research received no external funding.
Acknowledgments: The authors thank Emily Wheeler for editorial assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 6–11 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).