Phytochemical meanings of tetrahydro-β-carboline moiety in strictosidine derivatives

Article abstract of DOI:10.1016/j.bmc.2015.12.028

Synthesis of 13 different tetrahydro-β-carbolines (THBC) was accomplished by applying the Pictet-Spengler reaction with seven aldehydes, which have been coupled with tryptamine (6) and l-tryptophan methyl ester (7), respectively. The resulting products represent analogues of strictosidine (1) and carboxystrictosidine (5). They were investigated with respect to possible effects on herbivores in feeding bioassays upon the generalist Spodoptera littoralis. Maximum inhibition averages were 42% after four and 46% after six days for the most effective product (19) at 1000 ppm. Additionally, the frass of this particular bioassay was investigated via HPLC-UV for THBC digestion. All synthesized THBCs were also tested for their radical scavenger activity by monitoring their interaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds 16-20, 24 and 25 exhibited radical scavenging activity, ranging from 50% to 74% compared to that of α-tocopherol. All results were discussed with respect to possible contributions of tetrahydro-β-carboline moieties in bioactivities of strictosidine (1) and its biodegradation products.

Full text of DOI:10.1016/j.bmc.2015.12.028

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Phytochemical meanings of tetrahydro-b-carboline moiety in
strictosidine derivatives
a
b
a,
⇑
ˇ
´
Nicole Sudzukovic , Johann Schinnerl , Lothar Brecker
a Institute of Organic Chemistry, University of Vienna, Währingerstrasse 38, A-1090 Wien, Austria
b Chemodiversity Research Group, Division of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, A-1030 Wien, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of 13 different tetrahydro-b-carbolines (THBC) was accomplished by applying the Pictet–
Received 29 August 2015
Revised 14 December 2015
Accepted 15 December 2015
Available online xxxx
Spengler reaction with seven aldehydes, which have been coupled with tryptamine (6) and L-tryptophan
methyl ester (7), respectively. The resulting products represent analogues of strictosidine (1) and car-
boxystrictosidine (5). They were investigated with respect to possible effects on herbivores in feeding
bioassays upon the generalist Spodoptera littoralis. Maximum inhibition averages were 42% after four
and 46% after six days for the most effective product (19) at 1000 ppm. Additionally, the frass of this par-
ticular bioassay was investigated via HPLC-UV for THBC digestion. All synthesized THBCs were also tested
for their radical scavenger activity by monitoring their interaction with 2,2-diphenyl-1-picrylhydrazyl
(DPPH). Compounds 16–20, 24 and 25 exhibited radical scavenging activity, ranging from 50% to 74%
Keywords:
Strictosidine
Pictet–Spengler
Psychotria
Radical scavenging activity
Spodoptera littoralis
Tetrahydro-b-carbolines (THBC)
compared to that of
a-tocopherol. All results were discussed with respect to possible contributions of
tetrahydro-b-carboline moieties in bioactivities of strictosidine (1) and its biodegradation products.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
condensation of tryptamine (6) and secologanin.5 Strictosidine
(1) is a highly functionalised key intermediate in the biosynthesis
The huge group of monoterpene indole alkaloids (MIAs) con-
tains more than 2500 different substances, which have mostly
been isolated from the families Loganiaceae, Apocynaceae and
Rubiaceae.1 Many of these natural products show high biological
activities, which are often revealing their function in planta.1 MIAs
are hence interesting candidates for pharmacological purposes.2
Most popular examples are vinblastine and vincristine as well as
ajmalicine, all isolated from Catharanthus roseus (L.) G. Don, which
are potent anti-cancer drugs and used against hypertension. Aja-
maline, another MIA from Rauvolfia sepentina (L.) Benth. ex Kurz
(both Apocynaceae) is applied in cases of cardiac arrhythmia.3
A wide variety of MIAs have been reported in different species
of the genus Psychotria L., a member of the Rubiaceae family.4
Plenty of those MIAs belong to the group of tryptamine-iridoid
alkaloids bearing a tetrahydro-b-carboline moiety (THBC, 2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole). Its most widespread repre-
sentative is strictosidine (1, Scheme 1), generated during the stric-
tosidine synthase (STR, E.C. 4.3.3.2) catalysed Pictet–Spengler
of several other tryptamine-iridoid alkaloids.6 Moreover, even
strictosidine (1) itself shows antifungal activity7 and its decompo-
sition and biodegradation products are assumed to be involved in
plant defence mechanisms.7 At an herbivore attack, where cell
compartments are destroyed, the strictosidine (1) stock from the
vacuoles uncontrollably meets the strictosidine-b-D-glucosidase
(SGD, E.C. 3.2.1.105) from the nucleus. The hence catalysed
deglycosylation leads to strictosidine aglycon (2) which very
rapidly converts to a dialdehyde (3), as shown in Scheme 1.
Multiple tests show this reactive intermediate and follow up
products to possess various biological potentials, like antifungal
and protein cross-linking activities.7,8 Many efforts have been
made identifying involved compounds and enzymes in the
compartments of the plants.9
Oleuropein (4), isolated from leaves of Ligustrum obtusifolium
Siebold & Zucc. (Oleaceae), also comprises a secologanin-moiety
and is a described subject to comparable biodegradation (Fig. 1).
The resulting products show similar protein-crosslinking activity
and have an important contribution to the defence mechanism of
the plant.10 Strictosidine (1), however, contains the tetrahydro-b-
carboline moiety, which differs from oleuropein (4) and its pheno-
lic moiety. Consequently, the phytochemical meaning of this THBC
moiety has not yet been described in detail.
Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; MIA, monoterpene indole
alkaloid; THBC, tetrahydro-b-carboline.
⇑
Corresponding author. Tel.: +43 1 4277 52131; fax: +43 1 4277 852131.
E-mail address: lothar.brecker@univie.ac.at (L. Brecker).
http://dx.doi.org/10.1016/j.bmc.2015.12.028
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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2
Hence we synthesized a small library of THBC derivatives using
the Pictet–Spengler reaction. Resulting compounds (15–27) mim-
icked this part of the strictosidine aglycon (2), but did not carry
reactive aldehyde groups. Their bioactivity was tested towards
possible growth-retarding effects on the herbivore Spodoptera lit-
toralis, as well as their potential ability as a radical scavenger.
These two investigated bioactivities represent the most probable
tasks of strictosidine (1) itself in its natural environment. The
results provided several details about the meaning of the THBC
moiety in strictosidine (1) and its decomposition products.
2. Results and discussion
First goal was the synthesis of strictosidine analogues, being
tetrahydro-b-carboline (THBC) derivatives suitable for the bioac-
tivity tests. Apart from the tricyclic THBC structure present in all
compounds, different moieties replace the skeleton of the dialde-
hyde (3) which derived from the deglycosylated secologanin. These
residues should not possess any significant own bioactivity and
remain chemically stable. Hence mainly aliphatic and aromatic
moieties were used (Scheme 2), but additionally one heterocyclic
pyran skeleton was introduced. Although such structural range
was rather narrow, it allowed determining the influence of these
residues on bioactivities of the THBC groups. In an additional group
of compounds a methoxycarbonyl group (–COOCH3) was intro-
duced at position 5. These latter compounds mimic carboxystricto-
sidine (5) compounds (Fig. 1), which also occur in species of the
genus Psychotria.4a,11
2.1. Synthetic approach
To obtain the desired THBC derivatives the Pictet–Spengler
reaction was utilised.12 In the applied procedure the amine was
suspended in dichloromethane and afterwards the respective alde-
hyde and trifluoracetic acid were added.13 The reaction was per-
formed with tryptamine (6) or
gain the strictosidine (1) or carboxystrictosidine (5) analogues,
respectively. -Tryptophan methyl ester (7) was gained by esterifi-
cation of
L-tryptophan methyl ester (7), to
L
L
-tryptophan in high yields without significant N-methy-
lation.14 The seven aldehydes introducing the different side chains
in position 3 were commercially available and led to the formation
of 13 THBC derivatives, as shown in Scheme 2. All products were
purified by column chromatography.
Scheme 1. Strictosidine (1) biodegradation induced by strictosidine b-D-glucosi-
dase (SGD, E.C. 3.2.1.105), which catalyses deglycosylation. The intermittently
occurring strictosidine aglycon (2) further converts to a dialdehyde (3), which is a
chemical reactive agent in plant defence mechanism. The THBC moiety is not
involved in this reaction cascade.
The achieved yields performing the Pictet–Spengler reaction
ranged distinctly between 11% and 83%, mostly dependent on the
applied aldehyde (Table S1). Yield differences between the use of
Figure 1. Oleuropein (4) from Ligustrum obtusifolium as a representative substance for being essentially involved in the defence mechanism of the plant.10 The similarity to
strictosidine (1) and carboxystrictosidine (5) lies in the secologanin moiety, which is therefore assumed to be responsible for possible biological activities within the
Psychotria genus.
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N. Sudzukovic et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Scheme 2. Pictet–Spengler reaction of tryptamine (6) and L-tryptophan methyl ester (7) with the respective aldehydes (8–14), to gain THBCs (15–27). Numbering of atoms in
NMR data are in accordance with the numbering of strictosidine (1) to allow easier comparison between the compounds and the natural products.4b,c The numbering is hence
not necessarily according to IUPAC name of the compounds.
tryptamine (6) and
L
-tryptophan methyl ester (7) were rather small
After 4 d and 6 d the average mass of the larvae in each experiment
was measured and compared to those of the larvae in the control
group. Most of the substances did not show a significant impact
on the growth of the larvae; in some cases the larvae even gained
more weight compared to the control. However, the substances 15,
18, 19 and 20 showed inhibitory activity in the range 22–34% of
the gained mass. All those compounds were tryptamine (6) based
and mimicked the tetrahydro-b-carboline moiety of strictosidine
and varied mostly in the reaction with heptanal (13) (21% and
47%). The lowest yield was achieved when performing the reaction
with tryptamine (6) and (tetrahydro-2H-pyran-4-yl)-acetaldehyde
(14). Nevertheless only three reactions delivered a yield less than
50% and the gained material was in all cases sufficient for structure
determination, bioassays and redox activity experiments. Gener-
ally all compounds were characterized by one- and two-dimen-
sional NMR measurements, as well as ESI-MS analysis.
Exemplarily some substances also underwent elemental analysis
to validate their purity concerning possible inorganic residues.
The Pictet–Spengler reaction was not stereoselective and led to
enantiomeric mixtures, with respect to the newly generated chiral
centre in the annulated tetrahydropyridine ring when using trypta-
(1). The carboxystrictosidine analogues which derived from L-tryp-
tophan methyl ester (7), however, did not show any significant
inhibitory effect. Most effective inhibition of 34% after 4 d and
6 d was gained by 20 in a concentration of 1000 ppm. The further
three substances 15, 18 and 20 were most efficient at 500 ppm
after 6 d, with inhibitory effects of 22%, 28% and 26%, respectively.
Summed inhibition rates are listed in Tables 1 and S2.
mine (6) as reagent. In cases of utilising L-tryptophan methyl ester
(7), the expected diastereomers were formed and detected in the
The four most active compounds were all tryptamine-derived
and possessed different substituents, namely a linear hexyl, a
branched iso-butyl, a phenyl and a benzyl group (15, 18, 19 and
20, Scheme 2). All these moieties were rather bulky and had a non-
polar aliphatic or aromatic character. The lipophilic compounds
with an unsubstituted THBC moiety had a moderate but signifi-
cantly larger influence on growth compared to the more hydrophi-
lic carboxystrictosidine analogues. This implied a gentle effect on
the growth rate of the herbivore, likely caused by the structural
features, for example, by protein binding. The bioactivity of
NMR spectra. Racemisation of the chiral centre, which was intro-
duced from L
-tryptophan, was not investigated in these cases.15
For all compounds the stereoisomers were not separated and the
bioactivity as well as radical scavenging tests were performed with
the mixtures of the stereoisomers.
2.2. Bioassays on herbivores Spodoptera littoralis
The herbivores chosen for the different feeding bioassays were
neonate larvae of S. littoralis (Lepidoptera, Noctuidae). This noctuid
moth is a large, polyphagous insect which can ruin crops of more
than 100 other different plant species from various families by
feeding from leaves.16 Nevertheless, it is also utilised as a model
organism for bioassays, especially for its features of the larvae
being very polyphagous and for the insensitivity towards multiple
pesticides.17 An additional advantage is the rather uncomplicated
way to culture a laboratory colony of S. littoralis.18 We used these
insects to investigate the phytochemical potential of the synthe-
sized THBCs. In particular, their growth inhibitory effects against
neonate larvae were tested.
Table 1
Selected activities of tetrahydro-b-carbolines on neonate larvae of S. littoralis. Growth
inhibition is indicated in [% m/m] against growth of a control group
Compound
Concentration [ppm]
4 d [% m/m]
6 d [% m/m]
15
1000
500
125
12
15
4
16
22
10
18
19
20
1000
500
125
12
8
4
6
28
ꢀ20
16
26
5
1000
500
125
17
20
1
2.2.1. Short term growth tests
All 13 substances (15–27) were screened in feeding bioassays
over a period of 6 d to study their possible activity against the
growth of the neonate larvae. Three different concentrations
(250, 500 and 1000 ppm) were added to the food and in parallel
a control group was fed without addition of the THBC derivatives.
1000
500
34
13
34
13
125
ꢀ9
ꢀ10
Negative values mean that the larvae gained more mass compared to the control.
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Table 2
The three THBCs 18–20 with the highest inhibitory effect were
further investigated by triple-fold repetition of the 6 d lasting feed-
ing experiments using ten concentrations between 50 ppm and
1000 ppm. The results were slightly different, but principally in
good agreement with those of the screening experiment. Here,
compound 19 showed the highest inhibition on the S. littoralis lar-
vae growths, with an effect of 42% after 4 d and 46% after 6 d
(1000 ppm), while 18 and 20 caused a slightly smaller inhibition
of growth. The averaged results summarised in Table 2 showed a
correlation between the concentration of the THBC derivatives
and the reduced gain in weight. However, compounds 18 and 20
showed higher effect after 4 d, compared to those after 6 d, which
implied a recovery of the larvae on habituation after a short time
period. This could be explained due to optimised digestion and
improved nitrogen incorporation.
Further investigation of the three most active substances (18–20) after the screening
(S1), to examine the biological activity in closer detail
Compound
Concentration [ppm]
4 d [% m/m]
6 d [% m/m]
1000
800
600
500
400
300
250
200
100
50
15
28
18
4
13
9
12
14
10
ꢀ6
6
2
10
0
18
4
ꢀ1
0
7
ꢀ3
1
1000
800
600
500
400
300
250
200
100
50
42
30
37
39
19
12
17
16
11
11
46
38
38
33
21
16
18
8
19
2.2.2. Growth experiment until metamorphosis
A long term investigation, lasting from hatching of the larvae
until the beginning of metamorphosis, was carried out with com-
pound 15 applying 125 and 250 ppm concentrations. During the
first week the higher concentrated feed led to an inhibitory
impact of ca. 39% mass decrease compared to the control group.
However, the caterpillars were afterwards able to recover suc-
cessfully, showing no negative long term effects on growth rate
after 14 d. At that time most of the larvae had already pupated
and the other were on their way to metamorphosis. Afterwards,
hatching of the nymphs was also observed, indicating no signifi-
cant long term effects on their development. The investigated
strictosidine analogue hence influenced the growth of S. littoralis
in an early growth phase, but the moths were able to accustom
themselves to the exposition and continue their lifecycle. The
moderate effect on the growth rate of the herbivore caused by
the structural features was hence not sufficient to sustainably
affect this herbivore.
7
3
1000
800
600
500
400
300
250
200
100
50
35
20
9
28
22
6
16
10
4
30
18
0
24
13
6
10
ꢀ2
ꢀ4
ꢀ7
20
4
Negative values mean that the larvae gained more mass compared to the control.
strictosidine (1) against herbivores is hence not only based on the
reactivity of the dialdehyde (3) follow up product shown in
Scheme 1, but in a minor amount caused by the THBC moiety.
Figure 2. HPLC of frass collected from neonate larvae of S. littoralis. Panel A shows the chromatogram of an extract from frass collected after larvae were fed with diet not
containing any synthesized THBC. In panel B a chromatogram of pure compound 19 is shown. The significant peak is indicated as dashed section. Inlet D shows the UV
spectrum recorded from this peak. Panel C presents a chromatogram of frass collected from larvae fed with diet containing compound 19. The peak of 19 is also indicated as
dashed section and inlet E shows the UV spectrum recorded of this peak. Concurrent retention times and UV absorption maxima indicate that 19 has not been digested by the
larvae.
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Table 3
Radical scavenging activity of the synthesized tetrahydro-b-carboline derivatives
3. Conclusion
Compound
5 min
30 min
Tetrahydro-b-carboline moieties are present in strictosidine (1)
as well as in related MIAs and biodegradation products. Model
compounds bearing this structural building block were easily syn-
thesized in moderate to good yields by Pictet–Spengler reactions.
The resulting products mimicked the THBC moiety and were used
for bioassays to investigate its phytochemical meaning in the nat-
ural products. Only moderate growth-retarding effects were deter-
mined during the feeding experiments with neonate larvae of S.
littoralis. The THBC skeleton was hence supposed to contribute
only moderately to strictosidine (1) derived input in plant defence
mechanisms against herbivores. As opposed to this, the substances
with THBC moiety showed a good ability as radical scavengers in
investigations using the DPPH method. This indicates a significant
contribution to antioxidant activity in THBC bearing MIAs.
*
*
EC50
2.36
%
EC50
2.31
1.64
1.97
2.00
1.47
1.36
3.96
15.37
31.95
5.89
23.81
2.54
4.93
1.00
%
15
16
17
18
19
20
21
22
23
24
25
26
27
43
32
26
29
11
14
21
3
44
62
51
50
68
74
25
7
3.17
3.86
3.48
9.17
7.33
4.69
35.13
57.89
1.83
1.82
5.28
2.97
1.00
2
3
55
55
19
34
100
17
4
40
20
100
a-Tocopherol
These findings indicated that the functions of strictosidine (1)
and related natural products of genus Psychotria are not limited
to their known contribution in plant defence or to act as a key
intermediate in biosynthesis for further tryptamine-iridoid alka-
loids.5,8 In particular the quite distinct radical scavenging activity
of the THBC skeleton was a not yet thoroughly investigated feature,
with respect to its phytochemical meaning of this strictosidine
moiety. It is hence of interest to further study the importance
and functions of strictosidine derivatives in Psychotria with respect
to its successful pantropic distribution.
Listed are EC50 values and relative activity compared to those of
a
-tocopherol.
*
Relative antioxidant activity compared to those of
a-tocopherol.
2.2.3. THBC derivatives in the frass
During the more detailed feeding experiments of compounds
15, 18, 19 and 20 the frass of the larvae was collected, extracted
with methanol, and examined to study possible decomposition
products after digestion. The investigated faeces of compounds
15, 18 and 19 showed only one additional significant peak during
the HPLC analysis, which appeared at the same retention time as
the synthesized substance fed to the caterpillars. Also the UV spec-
tra of the originally fed compounds and the recovered substances
from faeces did match in all cases. Figure 2 shows the congruence
exemplary for compound 19. In case of compound 20, significant
line broadening was observed in the chromatogram, indicating
their incomplete solution. Therefore no distinct conclusion can be
made for this compound. However, the compliances in the other
three cases allow the assumption, that the tetrahydro-b-carboline
derivatives were passing the insects without being metabolized
by digestion. The moderate effects on growth rates can hence be
ascribed to structural moieties of the originally fed THBC stricto-
sidine analogues.
4. Experimental
4.1. General
All substances and solvents were purchased from Sigma Aldrich
or Alfa Aesar and used without further purification. For column
chromatography between 30 and 50 g of silica gel (Silicagel 60 M
(40–63 lm) from Macherey-Nagel was used with different ratios
of DCM/MeOH as mobile phases. TLC was performed on silica gel
plates (0.25 mm), Macherey-Nagel (ALUGRAMÒ Xtra SIL G/UV
254) developed in different organic solvents.
4.2. Spectroscopic an analytical techniques
2.3. Antioxidant activity of THBCs
The 1H, 13C and 2D NMR-spectra were recorded on Bruker DRX-
400 (400.13 MHz) or on Bruker AVIII-600 (600.13 MHz), using Top-
spin 1.3 and Topspin 3.1 software, respectively. 1D spectra were
recorded by acquisition of 32 k data points. 13C NMR have been
recorded using the SEFT (Spin-Echo Fourier Transform) technique.
After zero filling to 64 k data points Fourier transformation spectra
were performed with a range of 14 ppm (1H) and 240 ppm (13C),
respectively. To determine the 2D COSY, TOCSY, NOESY, HMQC
and HMBC spectra 128 experiments with 2048 data points each
were recorded and Fourier transformed to 2D-spectra with a range
of 10 ppm and up to 220 ppm for 1H and 13C, respectively. Mea-
surement temperature was 298 0.05 K. To perform the measure-
ments, each compound was dissolved in CDCl3 (ꢁ5.0 mg in 0.7 mL)
and transferred into 5 mm high precision NMR sample tubes. All
NMR spectral data were expressed as d values and the residual
CDCl3 was used as an internal standard for 1H (dH 7.26) and for
13C (dC 77.02) measurements.
Mass spectra were recorded by direct infusion electrospray ion-
isation (ESI) in positive ionisation mode (mass accuracy 5 ppm).
The analyser was a high resolution time-of-flight (TOF) mass spec-
trometer (maXis, Bruker Daltonics). TOF measurements have been
performed within the selected mass range of m/z 100–2500. ESI
was achieved by applying a capillary voltage of 4 kV to uphold a
(capillary) current between 30 and 50 nA. The flow rate was
To evaluate the possible radical scavenging activity of the syn-
thesized tetrahydro-b-carboline derivatives (15–27) the DPPH
method (2,2-diphenyl-1-picrylhydrazyl) was applied.19 Data col-
lected after 5 min and 30 min were used to calculate the accord-
ing EC50 values for each tested substance in comparison to those
of the additionally tested
dant of the vitamin
a
-tocopherol, the most active antioxi-
E
family.1a,20 Seven investigated THBC
derivatives (16–20, 24 and 25) exhibited rather high radical
scavenging activities, ranging from 50% to 74% compared to
those of
a-tocopherol. Results of all compounds are summarised
in Table 3.
Most of the investigated substances showed highest radical
scavenging activities after ca. 30 min when reaching their equilib-
rium. At that point in time the activities of the strictosidine ana-
logues were averagely higher compared to those of the
carboxystrictosidine analogues, independent from the structure
of the modified alkyl or aryl moieties. The activity after 5 min,
however, was highest for carboxystrictosidine analogues 24 and
25, both carrying small alkyl chain residues, one linear and the
other branched. These two compounds had about half the effi-
ciency of
a-tocopherol after 5 min and showed a distinctly lower
activity after 30 min. Compound (27) showed similar behaviour,
but not as differentiated and with lower activity.
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6
adjusted to 4.0 mL/min at a nitrogen temperature of 180 °C,
whereas the nebulizer gas pressure of N2 was 0.3 bar.
139.3 ppm (C-10) (Fig. S2); ESI-MS (C17H16N2): calculated m/z
249.1386 [M+H]+, observed m/z 249.1387 [M+H]+.
Elemental analyses were performed at the Microanalytical Lab-
oratory of the University of Vienna with a Eurovector EA3000 ele-
mental analyser and are within 0.4% of the calculated values,
confirming their P95% purity.
4.4.2. 1-Cyclohexyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
(16)
Yield 81% (128 mg, 0.50 mmol), mobile phase ratio 8:2 (DCM/
1
The HPLC measurements were performed on Agilent 1100 ser-
ies with using a Hypersil BDS-C18 column (250 mm ꢂ 4.6 mm,
MeOH), H NMR (600 MHz, CDCl3, d): 1.14 (m, 1H, H-20), 1.19 (m,
2H, H-30/40), 1.32 (m, 1H, H-50), 1.42 (m, 1H, H-60), 1.54 (m, 1H,
H-20), 1.71 (m, 1H, H-40), 1.75 (m, 1H, H-30), 1.77 (m, 1H, H-60),
1.81 (m, 1H, H-10), 1.84 (m, 1H, H-50), 2.69–2.79 (m, 2H, H-6),
3.00 (m, 1H, H-5), 3.38 (m, 1H, H-5), 3.99 (m, 1H, H-3), 7.10 (m,
1H, H-11), 7.15 (m, 1H, H-10), 7.32 (d, J = 8.0 Hz, 1H, H-12),
7.49 ppm (d, J = 7.7 Hz, 1H, H-9); 13C NMR (150 MHz, CDCl3, d):
22.5 (C-6), 26.4 (C-40), 26.5 (C-30), 26.8 (C-50), 27.5 (C-20), 30.2 (C-
60), 42.2 (C-10), 42.9 (C-5), 57.7 (C-3), 109.8 (C-7), 110.6 (C-12),
117.9 (C-9), 119.2 (C-11), 121.4 (C-10), 127.5 (C-13), 135.0 (C-2),
135.6 ppm (C-8) (Fig. S3); ESI-MS (C17H22N2): calculated m/z
255.1857 [M+H]+, observed m/z 255.1855 [M+H]+.
5 lm particle size) with UV diode array detection at 230 nm. The
eluent contained MeOH (B) and an aqueous buffer containing
15 mM ortho-phosphoric acid and 1.5 mM tetrabutylammonium
hydroxide (A). The method applied a linear MeOH gradient 0–
15 min:15–60% (B) followed by 5 min 60–100% and (B) hold for
5 min. The flow rate was 0.8 mL/min and the injection volume
10
lL.
An incubator (WTB, Binder) was used for rearing the larvae. The
antioxidant activity was measured by employing a Tecan Sunrise-
Basic plate reader.
4.3. Esterification of
A suspension of
(5.0 mL) was cooled to 0 °C before thionyl chloride (350 mg,
12.94 mmol) was added slowly. The solution was stirred for 3 h
at rt. MeOH was evaporated, water (3.0 mL) added to the residue
and the pH adjusted to 9–10 with aq NaOH (10%). The solution
was extracted with ethyl acetate and the organic phase was
washed with brine and dried over MgSO4. After removal of the
solvent column chromatography was performed using DCM/
L
-tryptophan
4.4.3. 1-Ethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (17)
Yield 50% (62 mg, 0.31 mmol), mobile phase ratio 8:2 (DCM/
1
L
-tryptophan (200 mg, 0.98 mmol) in MeOH
MeOH), H NMR (400 MHz, CDCl3, d): 1.07 (t, J = 7.3 Hz, 3H, H-20),
1.72 (m, 1H, H-10), 1.94 (m, 1H, H-10), 2.75 (m, 2H, H-6), 3.04 (m,
1H, H-5), 3.38 (m, 1H, H-5), 4.02 ppm (m, 1H, H-3), 7.10 (m, 1H,
H-11), 7.15 (m, 1H, H-10), 7.31 (d, J = 7.5 Hz, 1H, H-12), 7.49 ppm
13
(d, J = 7.5 Hz, 1H, H-9); C NMR (100 MHz, CDCl3, d): 10.2 (C-20),
22.7 (C-6), 27.7 (C-10), 42.6 (C-5), 53.9 (C-3), 109.2 (C-7), 110.7
(C-12), 118.0 (C-9), 119.3 (C-11), 121.5 (C-10), 127.6 (C-13),
135.6 (C-2), 136.1 ppm (C-8) (Fig. S4); ESI-MS (C13H16N2): calcu-
lated m/z 201.1386 [M+H]+, observed m/z 201.1385 [M+H]+.
MeOH (9:1). Yield: 60% of L-tryptophan methyl ester (7, 129 mg,
0.59 mmol). 1H NMR (400 MHz, CDCl3, d): 2.03 (s, 2H, H-12),
3.05 (dd, J = 14.4, 7.8 Hz, 1H, H-10), 3.29 (dd, J = 14.4, 4.8 Hz,
1H, H-10), 3.71 (s, 3H, H-14), 3.83 (dd, J = 7.8, 4.8 Hz, 1H, H-11),
6.96 (s, 1H, H-2), 7.12 (dd, J = 7.4, 7.8 Hz, 1H, H-7), 7.19 (dd,
J = 7.4, 7.8 Hz, 1H, H-6), 7.31 (d, J = 7.8 Hz, 1H, H-8), 7.60 ppm
(d, J = 7.8 Hz, 1H, H-5), 8.85 (s, H-1); 13C NMR (100 MHz, CDCl3,
d): 30.5 (C-10), 51.9 (C-14), 54.7 (C-11), 110.3 (C-3), 111.2
(C-8), 118.4 (C-5), 119.2 (C-7), 121.8 (C-6), 123.1 (C-2), 127.2
(C-9), 136.2 (C-4), 175.5 ppm (C-13) (Fig. S1); ESI-MS
(C12H14O2N2): calculated m/z 219.1128 [M+H]+, observed m/z
219.1120 [M+H]+.
4.4.4. 1-(2-Methyl-propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole (18)
Yield 67% (95 mg, 0.42 mmol), mobile phase ratio 8:2 (DCM/
1
MeOH), H NMR (400 MHz, CDCl3, d): 1.01 ppm (m, 3H, H-30),
1.04 (m, 3H, H-40), 1.63 (m, 2H, H-10), 1.98 (m, 1H, H-20), 2.75 (m,
2H, H-6), 3.03 (m, 1H, H-5), 3.35 (m, 1H, H-5), 4.11 (m, 1H, H-3),
7.10 (m, 1H, H-11), 7.15 (m, 1H, H-10), 7.30 (d, J = 7.6, 1H, H-12),
7.49 ppm (d, J = 7.6, 1H, H-9); 13C NMR (400 MHz, CDCl3, d): 21.7
(C-40), 22.7 (C-6), 23.8 (C-30), 24.6 (C-20), 42.4 (C-5), 44.4 (C-10),
50.5 (C-3), 108.7 (C-7), 110.6 (C-12), 118.0 (C-9), 119.3 (C-11),
121.4 (C-10), 127.6 (C-13), 135.6 (C-8), 136.6 ppm (C-2) (Fig. S5);
ESI-MS (C15H20N2): calculated m/z 229.1699 [M+H]+, observed m/
z 229.1707 [M+H]+.
4.4. General procedure of the Pictet–Spengler reaction
To
a
cooled solution (0 °C) of tryptamine (6, 100 mg,
-tryptophan methyl ester (7, 100 mg, 0.46 mmol) in
0.62 mmol)/
L
4.4.5. 1-Benzyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (19)
Yield 58% (94 mg, 0.36 mmol), mobile phase ratio 8:2 (DCM/
MeOH), 1H NMR (400 MHz, CDCl3, d): 2.69–2.83 (m, 2H, H-6),
3.02 (m, 1H, H-5), 3.09 (d, J = 7.1 Hz, 2H, H-10), 3.33 (m, 1H, H-5),
4.37 (m, 1H, H-3), 7.09 (m, 1H, H-11), 7.13 (m, 1H, H-10), 7.21
(d, J = 7.3 Hz, 1H, H-12), 7.28 (m, 2H, H-40/60), 7.31 (m, 1H, H-50),
7.35 (m, 2H, H-30/70), 7.49 ppm (d, J = 7.3 Hz, 1H, H-9); 13C NMR
(100 MHz, CDCl3, d): 22.5 (C-6), 41.5 (C-10), 42.3 (C-5), 53.9 (C-3),
109.2 (C-7), 110.7 (C-12), 118.1 (C-9), 119.3 (C-11), 121.5 (C-10),
126.9 (C-50), 127.1 (C-13), 128.8 (C-30/70), 129.3 (C-40/60), 135.3
(C-8), 135.5 (C-2), 138.0 ppm (C-20) (Fig. S6); ESI-MS (C18H18N2):
calculated m/z 263.1542 [M+H]+, observed m/z 263.1541 [M+H]+.
DCM (1.5 mL) the respective aldehyde (8–14, 1.2 mol equiv) and
the trifluoracetic acid (1.5 mol equiv) were added slowly. The reac-
tion was stirred for 24 h at room temperature and then the solvent
was evaporated. The residue was triturated with aq K2CO3 (5%),
extracted with DCM and dried over MgSO4. After removal of the
solvent column chromatography was performed using variable
DCM/MeOH mixtures.
Numbering of atoms in NMR data are in accordance with the
numbering of strictosidine (1) to allow easier comparison.4b,c The
numbering is shown in Schemes 1 and 2.
4.4.1. 1-Phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (15)
Yield 83% (128 mg, 0.52 mmol), mobile phase ratio 9:1 (DCM/
MeOH), 1H NMR (400 MHz, CDCl3, d): 2.87 (m, 1H, H-6), 2.94 (m,
1H, H-6), 3.12 (m, 1H, H-5), 3.31 (m, 1H, H-5), 5.39 (s, 1H, H-3),
7.13 (m, 1H, H-11), 7.16 (m, 1H, H-10), 7.22 (m, 1H, H-12), 7.30
(m, 5H, H-20-60), 7.52 ppm (m, 1H, H-9); 13C NMR (100 MHz, CDCl3,
d): 21.3 (C-6), 41.7 (C-5), 57.4 (C-3), 109.9 (C-7), 111.0 (C-12),
118.3 (C-9), 119.6 (C-11), 122.1 (C-10), 127.0 (C-13), 128.7 (C-40),
128.9 (C-30/50), 129.0 (C-20/60), 132.3 (C-2), 136.1 (C-8),
4.4.6. 1-Hexyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (20)
Yield 21% (33 mg, 0.13 mmol), mobile phase ratio 9:1 (DCM/
1
MeOH), H NMR (600 MHz, CDCl3, d): 0.88 (t, J = 6.8 Hz, 3H, H-60),
1.29 (m, 4H, H-40/50), 1.35 (m, 2H, H-30), 1.45 (m, 1H, H-20), 1.52
(m, 1H, H-20), 1.71 (m, 1H, H-10), 1.89 (m, 1H, H-10), 2.77 (m, 2H,
H-6), 3.06 (m, 1H, H-5), 3.37 (m, 1H, H-5), 4.12 (m, 1H, H-3),
7.08 (dd, J = 7.0, 7.4 Hz, 1H, H-11), 7.13 (dd, J = 7.0, 7.4 Hz, 1H, H-
10), 7.32 (d, J = 7.4 Hz, 1H, H-12), 7.50 ppm (d, J = 7.4 Hz, 1H, H-
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N. Sudzukovic et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9); 13C NMR (150 MHz, CDCl3, d): 14.0 (C-60), 22.2 (C-6), 22.6 (C-50),
25.8 (C-20), 29.5 (C-30), 31.7 (C-40), 34.8 (C-10), 42.3 (C-5), 52.7 (C-
3), 108.6 (C-7), 110.8 (C-12), 118.0 (C-9), 119.3 (C-11), 121.5 (C-
10), 127.4 (C-13), 135.6 (C-2), 135.7 ppm (C-8) (Fig. S7); ESI-MS
(C17H24N2): calculated m/z 257.2012 [M+H]+, observed m/z
257.2020 [M+H]+.
5), 106.9/108.2 (C-7), 110.8 (C-12), 118.0 (C-9), 119.4/119.5 (C-
11), 121.7 (C-10), 127.0/127.2 (C-13), 135.0/135.3 (C-2),
135.9/136.0 (C-8), 173.6/173.9 ppm (C-14) (Fig. S11); ESI-MS
(C15H18N2O2): calculated m/z 259.1441 [M+H]+, observed m/z
259.1433 [M+H]+. Elemental analysis: C15H18N2O2 calcd C, 69.74;
H, 7.02; N, 10.84; found C, 64.24; H, 6.61; N, 9.52.
4.4.7. 1-[(Tetrahydro-2H-pyran-4-yl)methyl]-2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole (21)
4.4.11. Methyl-1-(2-methyl-propyl)-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole-3-carboxylate (25)
Yield 11% (18 mg, 0.07 mmol), mobile phase ratio 85:15 (DCM/
MeOH), 1H NMR (400 MHz, CDCl3, d): 1.34 ppm (m, 1H, H-30), 1.42
(m, 1H, H-60), 1.60–1.64 (m, 1H, H-60), 1.69 (m, 2H, H-10), 1.85 (m,
1H, H-30), 1.96 (m, 1H, H-20), 2.75 (m, 2H, H-6), 3.03 (m, 1H, H-5),
3.35 (m, 1H, H-5), 3.45 (m, 2H, H-50), 3.99 (m, 2H, H-40), 4.16 (m,
1H, H-3), 7.10 (m, 1H, H-11), 7.15 (m, 1H, H-10), 7.32 (d,
J = 7.8 Hz, 1H, H-12), 7.48 ppm (d, J = 7.6 Hz, 1H, H-9); 13C NMR
(100 MHz, CDCl3, d): 22.8 (C-6), 31.3 (C-20), 32.5 (C-30), 34.2 (C-
60), 42.3 (C-5), 42.4 (C-10), 49.4 (C-3), 67.9 (C-40), 68.0 (C-50),
109.1 (C-7), 110.7 (C-12), 118.0 (C-9), 119.5 (C-11), 121.6 (C-10),
127.6 (C-13), 135.6 (C-2), 136.3 ppm (C-8) (Fig. S8); ESI-MS
(C17H22N2O): calculated m/z 271.1804 [M+H]+, observed m/z
271.1805 [M+H]+.
Yield 58% (76 mg, 0.27 mmol), mobile phase ratio 95:5 (DCM/
MeOH), diastereomeric mixture, 1H NMR (400 MHz, CDCl3, d):
1.01 (d, J = 6.8 Hz, 3H, H-30), 1.04 (d, J = 6.8 Hz, 3H, H-40),
1.52/1.68 (m, 2H, H-10), 2.03 (m, 1H, H-20), 2.83/2.99 (m, 1H, H-
6), 3.12/3.14 (m, 1H, H-6), 3.76/3.83 (s, 3H, H-15), 3.80/3.98 (m,
1H, H-5), 4.23/4.30 (m, 1H, H-3), 7.11 (m, 1H, H-11), 7.16 (m, 1H,
H-10), 7.31 (d, J = 7.6 Hz, 1H, H-12), 7.48 ppm (d, J = 7.6 Hz, 1H,
13
H-9); C NMR (100 MHz, CDCl3, d): 21.6/21.7 (C-40), 23.6/23.8
(C-30), 24.3/24.6 (C-20), 25.0/26.0 (C-6), 44.3/44.5 (C-10), 48.1/50.6
(C-3), 52.1 (C-15), 52.4/56.5 (C-5), 107.8 (C-7), 110.7 (C-12),
117.9 (C-9), 119.4/119.5 (C-11), 121.6 (C-10), 127.2 (C-13), 135.9
(C-2), 136.0 (C-8), 173.8/174.3 ppm (C-14) (Fig. S12); ESI-MS
(C17H22N2O2): calculated m/z 287.1754 [M+H]+, observed m/z
287.1756 [M+H]+.
4.4.8. Methyl-1-phenyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylate (22)
4.4.12. Methyl-1-benzyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylate (26)
Yield 81% (115 mg, 0.38 mmol), mobile phase ratio 98:2 (DCM/
MeOH), diastereomeric mixture, 1H NMR (400 MHz, CDCl3, d): 3.02
(m, 1H, H-6), 3.24 (m, 1H, H-6), 3.81/4.04 (s, 3H, H-15), 3.98 (dd,
J = 4.3, 11.1 Hz, 1H, H-5), 5.23 (s, 1H, H-3), 7.09–7.13 (m, 1H, H-
11), 7.13–7.17 (m, 1H, H-10), 7.18–7.21 (m, 1H, H-12), 7.34–7.41
(m, 5H, H-20-60), 7.51–7.56 ppm (m, 1H, H-9); 13C NMR
(100 MHz, CDCl3, d): 25.6 (C-6), 52.2/52.6 (C-15), 56.9 (C-5), 58.6
(C-3), 108.8 (C-7), 110.9 (C-12), 118.2 (C-9), 119.6 (C-11), 121.9
(C-10), 127.1 (C-13), 128.6 (C-20/60), 128.9 (C-30-50), 134.5 (C-2),
136.1 (C-8), 140.6 (C-10), 173.1 ppm (C-14) (Fig. S9); ESI-MS
(C19H18N2O2): calculated m/z 307.1441 [M+H]+, observed m/z
307.1438 [M+H]+.
Yield 51% (75 mg, 0.24 mmol), mobile phase ratio 95:5 (DCM/
MeOH), diastereomeric mixture, 1H NMR (400 MHz, CDCl3, d):
2.86/3.04 (m, 1H, H-6), 3.05/3.09 (m, 1H, H-10), 3.14/3.19 (m, 1H,
H-6), 3.15/3.22 (m, 1H, H-10), 3.74/3.81 (s, 3H, H-15), 3.79/4.53
(m, 1H, H-5), 4.07/4.64 (m, 1H, H-3), 7.09 (m, 1H, H-11), 7.13 (m,
1H, H-10), 7.18/7.20 (m, 1H, H-12), 7.25 (m, 1H, H-40), 7.27 (m,
1H, H-60), 7.34 (m, 2H, H-30/50), 7.37 (m, 1H, H-70), 7.48 ppm (m,
1H, H-9); 13C NMR (100 MHz, CDCl3, d): 24.7/25.8 (C-6),
41.5/41.9 (C-10), 52.1/52.7 (C-3), 52.2/52.6 (C-15), 54.1/56.4 (C-5),
107.0/108.3 (C-7), 110.8 (C-12), 118.0/118.1 (C-9), 119.4/119.5
(C-11), 121.8 (C-10), 126.6/126.7 (C-13), 127.0/127.2 (C-50),
128.8/129.0 (C-30/70), 129.3/129.4 (C-40/60), 134.0/134.8 (C-20),
135.7/135.8 (C-2), 137.5/137.7 (C-8), 173.2/173.4 ppm (C-14)
(Fig. S13); ESI-MS (C20H20N2O2): calculated m/z 321.1604 [M+H]+,
observed m/z 321.1606 [M+H]+.
4.4.9. Methyl-1-cyclohexyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylate (23)
Yield 70% (101 mg, 0.32 mmol), mobile phase ratio 98:2 (DCM/
MeOH), diastereomeric mixture, 1H NMR (600 MHz, CDCl3, d):
1.10–1.36 (m, 2H, H-20/60), 1.42 (m, 2H, H-20/60), 1.59–1.95 (m,
6H, H-30/40/50), 1.99 (m, 1H, H-10), 2.81 (m, 1H, H-6), 3.12 (m, 1H,
H-6), 3.72/3.81 (s, 3H, H-15), 3.77 (m, 1H, H-5), 4.23 (m, 1H, H-
3), 7.09/7.32 (m, 1H, H-11), 7.15/7.56 (m, 1H, H-10), 7.34/7.61
(m, 1H, H-12), 7.46/8.75 ppm (m, 1H, H-9); 13C NMR (150 MHz,
CDCl3, d): 25.6/25.8 (C-6), 26.2–31.0 (C-20/30/4050/60), 42.2/43.2 (C-
10), 52.3/52.6 (C-15), 56.3 (C-5), 57.7 (C-3), 108.8 (C-7),
110.9/112.0 (C-12), 116.2/117.8 (C-9), 119.5/120.7 (C-11),
121.7/128.5 (C-10), 127.0 (C-13), 134.0 (C-2), 136.1 (C-8),
173.4 ppm (C-14) (Fig. S10); ESI-MS (C19H24N2O2): calculated m/z
313.1910 [M+H]+, observed m/z 313.1905 [M+H]+. Elemental anal-
ysis: C19H24N2O2 calcd C, 73.05; H, 7.74; N, 8.97; found C, 72.13; H,
7.68; N, 8.84.
4.4.13. Methyl-1-hexyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylate (27)
Yield 47% (68 mg, 0.22 mmol), mobile phase 95:5 (DCM/MeOH),
diastereomeric mixture, 1H NMR (600 MHz, CDCl3, d): 0.89 (m, 3H,
H-60), 1.29 (m, 2H, H-50), 1.30 (m, 2H, H-40), 1.35 (m, 2H, H-30),
1.46/1.50 (m, 2H, H-20), 1.69/1.77 (m, 1H, H-10), 1.74/1.94 (m, 1H,
H-10), 2.82/3.00 (m, 1H, H-6), 3.13 (m, 1H, H-6), 3.75/3.82 (s, 3H,
H-15), 3.79/4.01 (m, 1H, H-5), 4.18/4.24 (m, 1H, H-3), 7.09/7.11
(m, 1H, H-11), 7.15 (m, 1H, H-10), 7.29/7.31 (m, 1H, H-12),
13
7.48 ppm (m, 1H, H-9); C NMR (150 MHz, CDCl3, d): 14.0 (C-60),
22.5/22.6 (C-40), 24.8/25.9 (C-6), 25.2/26.1 (C-20), 29.3/29.5 (C-30),
31.6/31.7 (C-50), 34.7/35.5 (C-10), 50.6/52.7 (C-3), 52.1/52.2 (C-
15), 52.5/56.4 (C-5), 106.6/107.9 (C-7), 110.7/110.8 (C-12), 117.9
(C-9), 119.3/119.4 (C-11), 121.6 (C-10), 127.0/127.1 (C-13),
135.3/135.9 (C-2), 135.6/135.9 (C-8), 173.7/174.0 ppm (C-14)
(Fig. S14); ESI-MS (C19H26N2O2): calculated m/z 315.2067 [M+H]+,
observed m/z 315.2069 [M+H]+.
4.4.10. Methyl-1-ethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylate (24)
Yield 40% (48 mg, 0.19 mmol), mobile phase ratio 9:1 (DCM/
MeOH), diastereomeric mixture, 1H NMR (400 MHz, CDCl3, d):
1.07 (m, 3H, H-20), 1.78 (m, 1H, H-10), 2.02 (m, 1H, H-10), 2.83 (m,
1H, H-6), 3.14 (m, 1H, H-6), 3.75/3.83 (s, 3H, H-15), 3.80/4.02 (m,
1H, H-5), 4.17/4.21 (m, 1H, H-3), 7.11 (m, 1H, H-11), 7.16 (m, 1H,
H-10), 7.30/7.32 (d, J = 7.8 Hz, 1H, H-12), 7.48 ppm (d, J = 7.6 Hz,
4.5. Preparation of the artificial diet
Each tested compound (15–27) was dissolved in acetone or
MeOH, according to the desired final concentration (50–
1000 ppm), and added to 367 mg freeze-dried feed powder.16a,21
Afterwards the content was overlaid with the previously used
13
1H, H-9); C NMR (100 MHz, CDCl3, d): 9.5/10.5 (C-20), 24.8/25.9
(C-6), 27.3/28.3 (C-10), 51.8/53.7 (C-3), 52.2 (C-15), 52.6/56.4 (C-
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8
solvent (acetone or MeOH) and shaken for 15 min. The same rou-
tine was applied to the control. The mixture was dried at rt for
3 h (acetone) and for 14 h (MeOH) and then an aq chloramphenicol
Acknowledgements
ˇ ´
The authors thank Stefan Mikulicic (Chemodiversity Research
Group, University of Vienna) for insect breeding and support with
the feeding bioassays. Susanne Felsinger and Peter Unteregger
(both Institute of Organic Chemistry, University of Vienna) are
acknowledged for performing the NMR and MS measurements,
respectively. We are also grateful to the Microanalytical Laboratory
of the University of Vienna for the elemental analyses.
solution (725 lL; 52 mg/mL) was added to each sample. An aq
agar-solution (70 mg/mL) was shortly boiled and 1.1 mL was
quickly transferred to each sample. After cooling to rt each feeding
sample was placed in a labelled petri dish.16a,22
4.6. Feeding bioassays
Supplementary data
4.6.1. Short term tests
Feeding bioassays were conducted on neonate larvae of S. lit-
toralis from a laboratory colony. Ten larvae were transferred on
top of the artificial diet in each petri dish, and held in an incubator
with >90% humidity at 26 °C under exclusion of light. After 4 d and
6 d the number of living insects was counted and their average
mass was measured. Results were compared with those of a con-
trol group not being exposed to the investigated compounds. The
frass was collected during several experiments for HPLC analysis
of possible digestion products.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.12.028.
References and notes
1. (a) Michal, G.; Schomburg, D. Biochemical Pathways: An Atlas of Biochemistry and
Molecular Biology, 2nd ed.; Wiley: Oxford, 2013; (b) do Nascimento, N. C.;
Fragoso, V.; Moura, D. J.; e Silva, A. C.; Fett-Neto, A. G.; Saffi, J. J. Environ. Mol.
Mutagen 2007, 48, 728.
2. Panjikar, S.; Stoeckigt, J.; O’Connor, S.; Warzecha, H. Nat. Prod. Rep. 2012, 29,
1176.
3. O’Connor, S. E.; Maresh, J. J. Nat. Prod. Rep. 2006, 23, 532.
4. (a) Faßhuber, H.K. Master Thesis, University of Vienna, Vienna, 2011.; (b)
Berger, A.; Fasshuber, H.; Schinnerl, J.; Brecker, L.; Greger, H. Phytochem. Lett.
2012, 5, 558; (c) Plagg, M. Master Thesis, University of Vienna, Vienna, 2012.
5. Stoeckigt, J.; Antonchick, A. P.; Wu, F.-R.; Waldmann, H. Angew. Chem., Int. Ed.
2011, 50, 8538.
6. (a) Schinnerl, J.; Orlowska, E. A.; Lorbeer, E.; Berger, A.; Brecker, L. Phytochem.
Lett. 2012, 5, 586; (b) Bernhardt, P.; O’Connor, S. E. Tetrahedron Lett. 2009, 50,
7118.
7. Verpoorte, R.; Van der Heijden, R.; Moreno, P. R. H. In Chemistry and
Pharmacology; Cordell, G. A., Ed.; Elsevier Science, 1997; pp 221–299.
8. Guirimand, G.; Courdavault, V.; Lanoue, A.; Mahroug, S.; Guihur, A.; Blanc, N.;
Giglioli-Guivarc’h, N.; St-Pierre, B.; Burlat, V. BMC Plant Biol. 2010, 10, 182.
9. Guirimand, G.; Guihur, A.; Ginis, O.; Poutrain, P.; Hericourt, F.; Oudin, A.;
Lanoue, A.; St-Pierre, B.; Burlat, V.; Courdavault, V. FEBS J. 2011, 278, 749.
10. Konno, K.; Hirayama, C.; Yasui, H.; Nakamura, M. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 9159.
4.6.2. Long term tests
Substance 15 was used for the long term feeding bioassay with
concentrations of 125 and 250 ppm, respectively. Again ten larvae
were transferred on top of the artificial diet in each petri dish and
held in an incubator with >90% humidity at 26 °C under exclusion
of light. After 4, 6, 10, 12 and 14 d number of living insects was
counted and their mass was measured. The insects were provided
with a sufficient amount of freshly prepared artificial diet after
each evaluation. The frass was collected for HPLC analysis of possi-
ble digestion products.
4.7. Antioxidant activity test
11. Paul, J. H. A.; Maxwell, A. R.; Reynolds, W. F. J. Nat. Prod. 2003, 66, 752.
12. Pictet, A.; Spengler, T. Ber. Dtsch. Chem. Ges. 1911, 44, 2030.
13. Hadjaz, F.; Yous, S.; Lebegue, N.; Berthelot, P.; Carato, P. Tetrahedron 2008, 64,
10004.
14. Tang, J.; Wang, Y.; Wang, R.; Dong, Z.; Yang, L.; Zheng, Y.; Liu, J. Chem. Biodivers.
2008, 5, 447.
Freshly formulated methanolic DPPH solution (0.4 mM) and
methanolic solutions of the substances 15–27 were used to pre-
pare mixtures of multiple concentrations on Microtiter plates.
The THBC concentration varied form 6.15 mg/mL to 1.52 lg/
15. Bailey, P. D.; Hollinshead, S. P.; McLay, N. R.; Morgan, K.; Palmer, S. J.; Prince, S.
N.; Reynolds, C. D.; Wood, S. D. J. Chem. Soc., Perkin Trans. 1 1993, 431.
16. (a) Brem, B.; Seger, C.; Pacher, T.; Hofer, O.; Vajrodaya, S.; Greger, H. J. Agric.
Food Chem. 2002, 50, 6383; (b) Gelbic, I.; Adel, M.; Hussein, H. Cent. Eur. J. Biol.
2011, 6, 861; (c) EFSA PLH Panel (EFSA Panel on Plant Health) EFSA J. 2015, 13,
3987.
17. Burow, M.; Müller, R.; Gershenzon, J.; Wittstock, U. J. Chem. Ecol. 2006, 32,
2333.
18. Meissle, M.; Vojtech, E.; Poppy, G. M. IOBC WPRS Bull. 2004, 27, 117.
19. Blois, M. S. Nature 1958, 181, 1199.
mL, while an equal quantity of a DPPH (0.08 mg/mL) was sup-
plied in each sample. 5 min and 30 min after adding the DPPH
to the dilution series the UV extinctions of all samples were
measured at 550 nm (free radical DPPH) and 700 nm (reference),
respectively and compared to those of sole, freshly prepared
DPPH solution. The average extinction was used to calculate
EC50 values, which was performed with Sigma Plot (Systat
Software Inc.) version 12.5. The calculated antioxidant activity
20. Finney, D. J. J. Am. Pharm. Assoc. 1952, 41, 627.
values were compared to the results of
the investigated concentrations ranged from 4.11 mg/mL to
0.02
g/mL.19
a-tocopherol, for which
21. Srivastava, R. P.; Proksch, P. Entomol. Gen. 1990, 15, 265.
22. Kaltenegger, E.; Brem, B.; Mereiter, K.; Kalchhauser, H.; Kählig, H.; Hofer, O.;
Vajrodaya, S.; Greger, H. Phytochemistry 2003, 63, 803.
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Please cite this article in press as: Sudzukovic, N.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.12.028