Ancistrobrevines E-J and related naphthylisoquinoline alkaloids from the West African liana Ancistrocladus abbreviatus with inhibitory activities against Plasmodium falciparum and PANC-1 human pancreatic cancer cells

Article abstract of DOI:10.1016/j.fitote.2018.11.006

From the roots of the West African liana Ancistrocladus abbreviatus (Ancistrocladaceae), ten new naphthylisoquinoline alkaloids (7a, 7b, 8a, 8b, and 9–14), displaying three different coupling types (5,1′, 5,8′, and 7,8′), were isolated, among them a series of five 5,1′-linked representatives and four metabolites belonging to the rare group of 7,8′-coupled alkaloids. Two of the alkaloids, the ancistrobrevines I (13) and J (14), are only the fourth and fifth examples of 7,8′-linked naphthyldihydroisoquinolines ever found in nature. The stereostructures of the new plant metabolites were determined by spectroscopic, chemical (oxidative degradation), and chiroptical (electronic circular dichroism) methods. For the assignment of the axial configuration of 13 and 14 relative to the stereocenter at C-3, which is too far away for significant NOE long-range interactions, these 7,8′-coupled naphthyldihydroisoquinolines were stereoselectively converted into the respective cis-configured tetrahydroisoquinoline analogs. The newly generated ‘auxiliary’ stereocenter at C-1 permitted decisive NOE interactions between the isoquinoline and the naphthalene parts, and thus a reliable attribution of the axial configuration of 13 and 14. In addition, five known compounds (3, 5, 16, 17, and 20), previously discovered in related African and Asian Ancistrocladus species, have now for the first time been identified in A. abbreviatus. All of these alkaloids are S-configured at C-3 and bear an oxygen function at C-6, and are, thus, typical Ancistrocladaceae-type compounds. Some of the alkaloids of A. abbreviatus exhibited promising activities against the malaria parasite Plasmodium falciparum and PANC-1 human pancreatic cancer cells.

Full text of DOI:10.1016/j.fitote.2018.11.006

Fitoterapia131(2018)245–259
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Ancistrobrevines E-J and related naphthylisoquinoline alkaloids from the
West African liana Ancistrocladus abbreviatus with inhibitory activities
against Plasmodium falciparum and PANC-1 human pancreatic cancer cells
Shaimaa Fayeza, Doris Feineisa, Laurent Aké Assib,1, Marcel Kaiserc,d, Reto Brunc,d
,
Suresh Awalee, Gerhard Bringmanna,⁎
a Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
b Centre National de Floristique (Conservatoire et Jardin Botaniques), Université d’ Abidjan, Abidjan 08, Ivory Coast
c Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
d University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
e Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
From the roots of the West African liana Ancistrocladus abbreviatus (Ancistrocladaceae), ten new naphthyliso-
quinoline alkaloids (7a, 7b, 8a, 8b, and 9–14), displaying three different coupling types (5,1′, 5,8′, and 7,8′),
were isolated, among them a series of five 5,1′-linked representatives and four metabolites belonging to the rare
group of 7,8′-coupled alkaloids. Two of the alkaloids, the ancistrobrevines I (13) and J (14), are only the fourth
and fifth examples of 7,8′-linked naphthyldihydroisoquinolines ever found in nature. The stereostructures of the
new plant metabolites were determined by spectroscopic, chemical (oxidative degradation), and chiroptical
(electronic circular dichroism) methods. For the assignment of the axial configuration of 13 and 14 relative to
the stereocenter at C-3, which is too far away for significant NOE long-range interactions, these 7,8′-coupled
naphthyldihydroisoquinolines were stereoselectively converted into the respective cis-configured tetra-
hydroisoquinoline analogs. The newly generated ‘auxiliary’ stereocenter at C-1 permitted decisive NOE inter-
actions between the isoquinoline and the naphthalene parts, and thus a reliable attribution of the axial con-
figuration of 13 and 14. In addition, five known compounds (3, 5, 16, 17, and 20), previously discovered in
related African and Asian Ancistrocladus species, have now for the first time been identified in A. abbreviatus. All
of these alkaloids are S-configured at C-3 and bear an oxygen function at C-6, and are, thus, typical
Ancistrocladaceae-type compounds. Some of the alkaloids of A. abbreviatus exhibited promising activities against
the malaria parasite Plasmodium falciparum and PANC-1 human pancreatic cancer cells.
Ancistrocladus abbreviatus,Ancistrocladaceae
Naphthylisoquinoline alkaloids
Ancistrobrevines
Antiplasmodial agents
Anticancer agents
1. Introduction
investigated representatives are A. korupensis from Cameroon [6–10],
A. likoko [11–13], and A. ealaensis [14–16] from the Democratic Re-
Ancistrocladus Wall. is the only genus of the Ancistrocladaceae plant
family. Presently, it comprises 18 accepted species (twelve from Africa
and six from Asia) and is characterized by a disjunct distribution in the
palaeotropics with two areas of speciation, one in tropical West,
Central, and East Africa, and one in Southeast Asia [1–5]. The name
Ancistrocladus comes from the Greek words ankistron (= hook) and
klados (= shoot) and reflects the botanical pecularity of the Ancis-
trocladaceae. They are scandent shrubs or woody lianas with tendril-
like modified shoots provided with circinate woody hooks as climbing
devices [1,2]. Most prominent and phytochemically thoroughly
public of the Congo, the Indian liana A. heyneanus [6,17–21], and A.
tectorius from Southeast Asia [6,22–28]. All these plants are rich sources
of naphthylisoquinoline alkaloids. Only one further, even smaller plant
family is known to produce these structurally, biosynthetically, and
pharmacologically unusual biaryl natural products, the Dioncophylla-
ceae [29], which are endemic to West Africa [6,30–32]. Some naph-
thylisoquinoline alkaloids exhibit potent antiplasmodial activities like
e.g., 5-epi-ancistectorine A2 (3) [22], while other representatives of this
unprecedented class of polyketide-derived isoquinoline alkaloids
[6,31,33] display strong cytotoxic effects against multiple myeloma and
⁎ Corresponding author.
E-mail address: bringman@chemie.uni-wuerzburg.de (G. Bringmann).
1 Deceased on January 14, 2014.
https://doi.org/10.1016/j.fitote.2018.11.006
Received 27 September 2018; Accepted 7 November 2018
Availableonline09November2018
0367-326X/©2018ElsevierB.V.Allrightsreserved.

S. Fayez et al.
Fitoterapia131(2018)245–259
Fig. 1. Secondary metabolites isolated from the roots of the West African liana A. abbreviatus, among them ten new naphthylisoquinoline alkaloids: ancistrobrevine E
(7a), 5-epi-ancistrobrevine E (7b), ancistrobrevine F (8a), 5-epi-ancistrobrevine F (8b), ancistrobrevine G (9), 5′-O-demethylancistrobrevine B (10), ancistrobrevine H
(11), and its 6-O-demethyl analog 12, the ancistrobrevines I (13) and J (14), as well as six previously known [6,17,18,22,30] compounds: the tetralone cis-
isoshinanolone (5), ancistrocladine (6a), hamatine (6b), the antileukemic dioncophylline A (1) and its N-methyl analog 2, and the antiplasmodial 5-epi-ancistectorine
A2 (3); ancistroyafungine B (4) [35] is an example of a naphthylisoquinoline with a high preferential cytotoxicity against pancreatic cancer cells, but it is not
produced by the investigated plant.
leukemia cell lines such as dioncophylline A (1) [34] or against pan-
creatic cancer cells like e.g., ancistroyafungine B (4) [35] (Fig. 1).
Ancistrocladus abbreviatus AIRY SHAW (Ancistrocladaceae) [1,36,37] is
widely distributed within lowland wet evergreen and swamp forests, on
coastal bluffs, and along rivers extending from Guinea to Cameroon.
Previous phytochemical work revealed that this West African liana
mainly produces 5,1′- and 7,1′-coupled naphthylisoquinoline alkaloids
[6,38–42]. Most of the representatives that have a 5,1′-linkage are ty-
pical Ancistrocladaceae-type compounds like e.g., ancistrocladine (6a)
and its atropo-diastereomer hamatine (6b) (Fig. 1), with S-configura-
tion at C-3 and an oxygen function at C-6 [6,38,42]. The majority of the
7,1′-coupled compounds, by contrast, belong to the subclass of Dion-
cophyllaceae-type alkaloids, being devoid of an oxygen function at C-6
and possessing an R-configuration at C-3, like e.g., dioncophylline A (1)
and its N-methyl analog 2 [6,38,41,42]. The latter two alkaloids are
also the main metabolites of the related Dioncophyllaceae liana Tri-
phyophyllum peltatum [6,30], which shares a common habitat with A.
abbreviatus. While A. abbreviatus obviously uses the ‘synthetic re-
pertoires' of both plant families, T. peltatum was found to contain
Dioncophyllaceae-type naphthylisoquinoline alkaloids exclusively
[6,30,31,38,41].
one
constitutionally
and
configurationally
identical
tetra-
hydroisoquinoline portion. The sixth new metabolite, 5’-O-demethy-
lancistrobrevine B (10), is a 5,8′-linked naphthylisoquinoline. This
finding is quite remarkable with respect to the fact that it is only the
second representative of this coupling type detected in A. abbreviatus
and that such 5,8′-linked alkaloids have so far been detected in West
African Ancistrocladus species only rarely [38,43], whereas the meta-
bolite profiles of most of the Central and East African taxa are domi-
nated by the presence of a high number of mono- and dimeric 5,8′-
linked alkaloids [6–9,11–16]. Four of the new compounds isolated from
the roots of A. abbreviatus, ancistrobrevine H (11), its 6-O-demethyl
analog 12, and the ancistrobrevines I (13) and J (14), belong to the
small group of 7,8′-coupled naphthylisoquinoline alkaloids. So far, only
eleven such compounds have been discovered in nature
[10,19,22,34,42,44,45], including three naphthyldihydroisoquinolines
[10,19] such as 13 and 14. Attempts to elucidate the absolute axial
configuration of 13 and 14 by NMR measurements were not successful,
since decisive long-range NOE interactions were not observable. To
overcome this problem, an ‘auxiliary’ stereocenter at C-1 was generated
by conversion of 13 and 14 into their corresponding cis-configured
tetrahydroisoquinoline analogs using NaBH4 as the reducing agent. In
conjunction with the absolute stereoarrays in the tetrahydroisoquino-
line portions, the now spatially differentiated spin systems finally per-
mitted diagnostically valuable long-range couplings across the biaryl
axis and, thus, reliable attribution of the relative and absolute axial
configurations of 13 and 14.
Here, we describe the isolation and structural elucidation of ten new
naphthylisoquinoline alkaloids (Fig. 1) from the roots of A. abbreviatus,
exhibiting three different coupling types, viz., 5,1′, 5,8′, and 7,8′.
Among them are five new 5,1′-coupled compounds, namely ancistro-
brevines E (7a) and F (8a), their atropo-diastereomers 7b and 8b, re-
spectively, and ancistrobrevine G (9). Interestingly, all of them possess
In addition, four known naphthylisoquinolines (3, 16, 17, and 20)
246

S. Fayez et al.
Fitoterapia131(2018)245–259
Fig. 2. Known alkaloids isolated from the roots of A. abbreviatus now subjected to biotesting: 6-O-methylhamatine (15), 6-O-methyl-4′-O-demethylhamatine (16), 6-
O-methylhamatinine (17), ancistrobrevines B (18) and D (19), ancistrocladisine B (20), ancistrobrevine A (21) and its 6-O-demethyl analog 22; the compounds 16,
17, and 20 have now been identified for the first time in this West African liana.
previously discovered in related Ancistrocladus species, have now been
identified for the first time in A. abbreviatus. These alkaloids (Fig. 1 and
Fig. 2) have been isolated along with the naphthalene-related tetralone
cis-isoshinanolone (5) (Fig. 1), which has widely been detected in An-
cistrocladaceae and Dioncophyllaceae plants [6,12,30,31,46,47]. All of
the naphthylisoquinoline alkaloids of A. abbreviatus presented in this
paper are S-configured at C-3 and oxygenated at C-6, thus being typical
Ancistrocladaceae-type compounds.
The pronounced antiparasitic activities of some of the naphthyli-
soquinoline alkaloids against pathogens belonging to the genera
Plasmodium, Leishmania, and Trypanosoma [14–16,22,24,34,48,49] en-
couraged us to screen not only the new compounds (Fig. 1), but also
some of the known, yet uninvestigated constituents of A. abbreviatus
presented in Fig. 2. Furthermore, the metabolites of this West African
liana were tested for their antiproliferative potential against PANC-1
human pancreatic cancer cells under nutrient-deprived conditions.
Plus diode array detector). Resolution of the crude extracts was per-
formed on a Waters Symmetry RP18 column (19 × 300 mm, 7 μm,
Waters) at a flow rate of 10 mL min−1; mobile phases: (A) 90% H2O
with 10% MeCN (0.05% trifluoroacetic acid) and (B) 90% MeCN with
10% H2O (0.05% trifluoroacetic acid). For further purification of al-
kaloid-enriched fractions
a Waters XSelect™ HSS PFP column
(4.6 × 250 mm, 4 μm) was applied at a flow rate of 4 mL min−1; mobile
phases: (A′) 90% H2O with 10% MeOH (0.05% trifluoroacetic acid) and
(B′) 90% MeOH with 10% H2O (0.05% trifluoroacetic acid). Solvents
were either purchased from commercial suppliers or purified by stan-
dard techniques. Ultra-pure water was obtained from an Elga Purelab
Classic system. (S)-MTPA was purchased from Sigma-Aldrich (Stein-
heim, Germany).
2.2. Plant material
Roots of Ancistrocladus abbreviatus AIRY SHAW (Ancistrocladaceae)
were collected by late Prof. L. Aké Assi (Centre National de Floristique,
Université Abidjan) in May 1996, in the Parc National de Taï, in the
South-Western region of the Ivory Coast. A voucher specimen (No. 3)
has been deposited at the Herbarium Bringmann, Institute of Organic
Chemistry, University of Würzburg.
2. Experimental
2.1. General experimental procedures
IR spectra were taken on a Jasco FT/IR-410 spectrometer, UV
spectra on a Shimadzu UV-1800 spectrophotometer, ECD spectra on a
Jasco J-715 spectropolarimeter, and optical rotations on a Jasco P-1020
polarimeter operating with a sodium light source (λ = 589 nm). The
ECD data are reported in Δε values (cm 2 mol−1) at the given wave-
length λ (nm) and are processed using SpecDis [50,51]. 1D and 2D
NMR spectra were monitored on Bruker AMX 400 and DMX 600 in-
struments, using methanol‑d4 (δ 3.31 and 49.15 ppm) as the solvent,
with the 1H or 13C signals of methanol (1H, δ 3.31 ppm, 13C, δ
49.0 ppm) in the deuterated solvent as the internal reference. Chemical
shifts (δ) are reported in parts per million (ppm), and coupling con-
stants (J) are given in Hertz (Hz). NMR signal multiplicities are denoted
as singlet (s), doublet (d), doublet of doublets (dd), quartet (q), pseu-
dotriplet (pt), or multiplet (m). Spectra were acquired and processed
using an ACD/NMR processor (version 12.01) and the Topspin 3.2
(Bruker Daltonics) or the MestReNova NMR (Mestrelab Research)
software. LC-MS measurements were performed on an Agilent 1100
Series System, equipped with a binary high-pressure mixing pump, with
a degasser module, an autosampler, a 1100 series photodiode array
(PDA) detector (Agilent Technology, Germany), and an Esquire 3000
Plus ion-trap mass spectrometer with an electrospray ionization inter-
face (Bruker Daltonics, Bremen, Germany). HRESIMS analyses were
done on a Bruker micrOTOF-focus mass instrument. GC-MSD measur-
ementes were conducted on a GCMS-QP 2010SE System (Shimadzu).
Preparative HPLC was performed on a Jasco System (PU-1580 Plus) in
combination with UV/Vis detection at 195–650 nm (Jasco MD-2010
2.3. Extraction and isolation
Air-dried, powdered root bark material (ca. 420 g) of A. abbreviatus
was extracted with MeOH for 1 h, assisted by ultrasonication. The ex-
tract was filtered and then concentrated in vacuo. The solid residue was
dissolved in MeOH/H2O (9:1) and repeatedly washed with n-hexane to
remove the non-polar impurities. The aqueous methanolic extract
(17.5 g) was directly subjected to silica gel column chromatography
(CC) using a linear solvent system of MeOH in CH2Cl2 (0 → 90%), fi-
nally giving rise to five metabolite-enriched fractions (F1-F5). The
fractionation was guided by LC-MS, searching for masses hinting at the
presence of naphthylisoquinoline alkaloids.
Preparative HPLC of fraction F1 (2.1 g) on a SymmetryPrep™ C18
column, using a linear gradient (0 min: 20% B, 15 min: 30% B, 35 min:
40% B, 45 min, 49% B, 50 min, 60% B) furnished some alkaloid-con-
taining subfractions, which were further purified on an XSelect HSS PFP
HPLC column using a gradient system consisting of the solvents A′ and
B′ (0 min: 50% of B, 28min: 100% of B), finally affording 0.9 mg of cis-
isoshinanolone (5) (retention time 13.9 min), 2.0 mg of ancistrobrevine
A (21) (retention time 16.2 min), 10.5 mg of 6-O-methylhamatine (15)
(retention time 18.4 min), and 1.0 mg of 6-O-methyl-4′-O-demethylha-
matine (16) (retention time 26.0 min).
Fraction F2 (3.7 g) was resolved on a SymmetryPrep™ C18 column
using a linear gradient (0 min: 22% B, 35 min: 34% B, 40 min: 50% B).
247

S. Fayez et al.
Fitoterapia131(2018)245–259
The collected (still impure) compounds were further purified on an
XSelect HSS PFP HPLC column using an isocratic solvent system of A′
and B′ (40:60, v/v), finally yielding five pure compounds, namely
1.8 mg of ancistrobrevine B (18) (retention time 15.0 min), 0.8 mg of 5′-
O-demethylancistrobrevine B (10) (retention time 16.5 min), 6.0 mg of
ancistrobrevine D (19) (retention time 17.5 min), 1.0 mg of ancistro-
brevine H (11), (retention time 21.0 min), and 1.9 mg of 6-O-methyl-
hamatinine (17) (retention time 24.7 min).
Table 2
13C NMR data of ancistrobrevine E (7a), 5-epi-ancistrobrevine E (7b), ancis-
trobrevine F (8a), 5-epi-ancistrobrevine F (8b), and ancistrobrevine G (9) in
methanol-d4 (δ in ppm).
7a
7b
8a
8b
9
no.
δC, type
δC, type
δC, type
δC, type
δC, type
1
49.5, CH
44.9, CH
32.9, CH2
117.7, C
156.4, C
102.0, CH
155.4, C
113.1, C
132.4, C
124.9, C
137.4, C
110.3, CH
157.7, C
158.6, C
106.9, CH
127.8, CH
118.5, CH
137.7, C
117.7, C
18.3, CH3
19.1, CH3
20.2, CH3
56.6, CH3
56.8, CH3
48.8, CH
44.2, CH
32.4, CH2
117.7, C
156.4, C
101.1, CH
155.7, C
112.9, C
132.3, C
125.7, C
137.3, C
110.0, CH
157.6, C
158.6, C
106.5, CH
127.4, CH
118.5, CH
137.7, C
117.7, C
18.5, CH3
19.1, CH3
20.7, CH3
56.7, CH3
56.8, CH3
49.2, CH
44.8, CH
33.0, CH2
117.6, C
156.7, C
102.0, CH
155.4, C
113.1, C
132.4, C
123.4, C
138.5, C
113.6, CH
155.2, C
157.9, C
104.6, CH
127.3, CH
119.4, CH
137.1, C
114.9, C
18.3, CH3
19.1, CH3
20.0, CH3
49.0, CH
45.2, CH
32.8, CH2
117.5, C
156.6, C
102.0, CH
155.5, C
113.0, C
132.5, C
123.5, C
138.3, C
113.6, CH
155.2, C
158.0, C
104.0, CH
127.2, CH
119.6, CH
137.2, C
115.0, C
18.4, CH3
19.1, CH3
20.3, CH3
49.5, CH
45.1, CH
33.0, CH2
117.5, C
156.6, C
102.1, CH
155.6, C
113.1, C
132.4, C
126.1, C
137.1, C
108.0, CH
157.1, C
156.1, C
110.6, CH
128.9, CH
116.7, CH
137.3, C
115.1, C
18.4, CH3
19.2, CH3
20.3, CH3
56.6, CH3
From fraction F3 (2.9 g) six naphthylisoquinoline alkaloids were
isolated by preparative HPLC on a SymmetryPrep™ C18 column under
linear-gradient conditions (0 min: 20% B, 25min: 47% B). The com-
pounds were again further purified on an XSelect HSS PFP column
applying linear gradients conditions (0 min: 60% B′, 55 min: 70% B′) or
using isocratic solvent systems of A′ and B′ (v/v), containing 50, 54, or
56% of B′, thus providing the following metabolites in a pure form:
1.3 mg of ancistrobrevine E (7a) (retention time 14.8 min), 1.3 mg of 5-
epi-ancistrobrevine E (7b) (retention time 15.0 min), 1.7 mg of 5-epi-
ancistrobrevine F (8b) (retention time 16.3 min), 1.5 mg of 6-O-de-
methylancistrobrevine A (22) (retention time 19.2 min), 4.7 mg of 5-
epi-ancistectorine A2 (3) (retention time 20.3 min), 1.0 mg of ancistro-
3
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
6′
7′
8′
brevine
F (8a) (retention time 24.7 min), and 0.9 mg of ancis-
trocladisine B (20) (retention time 32.0 min).
9′
10′
Resolution of fraction F4 (1.2 g) on a SymmetryPrep™ C18 column
using a linear gradient (0 min: 20% B, 35min: 38% B), followed by
further purification steps on an XSelect HSS PFP column applying the
conditions given above, afforded five naphthylisoquinoline alkaloids:
0.92 mg of ancistrobrevine G (9) (retention time 14.8 min), 1.1 mg of 6-
O-demethylancistrobrevine H (12) (retention time 15.1 min), 1.0 mg of
hamatine (6b) (retention time 16.0 min), 0.9 mg of ancistrobrevine I
(13) (retention time 21.1 min), and 1.3 mg of ancistrobrevine J (14)
(retention time 27.3 min).
1-CH3
3-CH3
2′-CH3
4′-OCH3
5′-OCH3
56.7, CH3
56.6, CH3
The spectra were recorded at 100 MHz.
(ATR) νmax: 3314, 1670, 1596, 1258, 1198, 1125 cm−1
.
1H and 13C
NMR data are given in Table 1 and Table 2. HR-ESI-MS: m/z 394.20140
[M + H]+ (calcd for C24H28NO4: 394.20128).
2.3.1. Ancistrobrevine E (7a)
Yellow amorphous solid: [α]D20–30.3° (c 0.05, MeOH). UV/Vis
(MeOH): λmax (logε): 228 (3.5), 292 (2.8) nm. ECD (c 0.1, MeOH): λmax
(Δε): 200 (+2.1), 217 (−0.9), 284 (−0.3) nm. IR (ATR) νmax: 3357,
2.3.3. Ancistrobrevine F (8a)
Yellow amorphous solid: [α]D20-25.2° (c 0.02, MeOH). UV/Vis
(MeOH): λmax (logε): 229 (3.6) nm. ECD (c 0.1, MeOH): λmax (Δε): 212
(−2.5), 221 (−2.4), 240 (+0.5), 261 (+0.1), 284 (−0.4), 335
(+0.06) nm. IR (ATR) νmax: 2977, 2889, 2159, 1671, 1384, 1252, 1140,
2973, 1675, 1584, 1462, 1376, 1130, 949 cm−1 1H and 13C NMR data
.
are given in Table 1 and Table 2. HR-ESI-MS: m/z 394.20198 [M + H]+
(calcd for C24H28NO4: 394.20128).
1073, 954 cm−1 1H and 13C NMR data are given in Table 1 and
.
2.3.2. 5-epi-Ancistrobrevine E (7b)
Table 2. HR-ESI-MS: m/z 380.18599 [M + H]+ (calcd for C23H26NO4:
Yellow amorphous solid: [α]D20+21.8° (c 0.10, MeOH). UV/Vis
(MeOH): λmax (logε): 230 (3.9) nm. ECD (c 0.1, MeOH): λmax (Δε): 210
(+20.3), 224 (+19.2), 240 (−12.2), 241 (−6.9), 296 (−1.3) nm. IR
380.18563).
Table 1
1H NMR data of ancistrobrevine E (7a), 5-epi-ancistrobrevine E (7b), ancistrobrevine F (8a), 5-epi-ancistrobrevine F (8b), and ancistrobrevine G (9) in methanol-d4 (δ
in ppm).
7a
7b
8a
8b
9
no.
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
1
4.75, q
4.75, q
4.74, q
4.74, q
4.74, q
3
4ax
4eq
7
3.60, m
3.66, m
3.60, m
3.65, m
3.60, m
2.11, dd (10.6, 18.0)
2.22, dd (5.0, 17.8)
6.46, s
2.03, dd (12.0, 17.9)
2.33, dd (4.8, 17.9)
6.46, s
2.10, dd (11.3, 17.09)
2.22, dd (5.0, 17.7)
6.46, s
2.01, dd (11.7, 18.0)
2.32, dd (4.7, 17.9)
6.46, s
2.10a
2.24, dd (5.1, 17.3)
6.46, s
3′
6.90, s
6.91, s
6.79, s
6.79, s
6.90, s
6′
7′
6.86, d (7.7)
7.21, pt
6.85, d (7.0)
7.18, pt
6.86, d (7.7)
7.20, pt
6.85, d (7.1)
7.16, pt
6.70, d (8.6)
7.16, pt
8′
6.87, dd (0.9, 8.5)
1.67, d (6.7)
1.19, d (6.3)
2.10, s
3.95, s
3.91, s
6.80, dd (0.9, 8.4)
1.65, d (6.7)
1.17, d (6.3)
2.13, s
3.95, s
3.91, s
6.87, dd (0.85, 8.6)
1.66, d (6.7)
1.20, d (6.4)
2.05, s
6.80, dd (0.8, 8.5)
1.64, d (6.7)
1.17, d (6.3)
2.05, s
6.73, d (8.4)
1.66, d (6.7)
1.19, d (6.3)
2.11, s
1-CH3
3-CH3
2′-CH3
4′-OCH3
5′-OCH3
4.10, s
4.07, s
4.07, s
The spectra were recorded at 400 MHz.
a
Signal overlapped with that of 2′-CH3.
248

S. Fayez et al.
Fitoterapia131(2018)245–259
2.3.4. 5-epi-Ancistrobrevine F (8b)
Table 4
1H NMR data of 5′-O-demethylancistrobrevine B (10), ancistrobrevine H (11),
6-O-demethylancistrobrevine H (12), and ancistrobrevines I (13) and J (14) in
methanol-d4 (δ in ppm).
Yellow amorphous solid: [α]D20+9.2° (c 0.10, MeOH). UV/Vis
(MeOH): λmax (logε): 232 (3.1) nm. ECD (c 0.1, MeOH): λmax (Δε): 212
(+31.1), 222 (+28.7), 240 (−12.2), 281 (+1.1), 301 (−1.8) nm. IR
(ATR) νmax: 3369, 1670, 1608, 1182, 1135 cm−1. 1H and 13C NMR data
are given in Table 1 and Table 2. HR-ESI-MS: m/z 380.18590 [M + H]+
(calcd for C23H26NO4: 380.18563).
10b
11a
12a
13a
14a
no.
δC, type
δC, type
δC, type
δC, type
δC, type
1
3
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
1-CH3
3-CH3
6-OCH3
8-OCH3
2′-CH3
4′-OCH3
5′-OCH3
49.1, CH
44.9, CH
32.8, CH2
119.9, C
157.3, C
98.5, CH
157.5, C
113.9, C
133.2, C
118.7, CH
137.4, C
107.4, CH
158.0, C
155.8, C
110.4, CH
131.0, CH
124.4, C
137.1, C
114.8, C
18.6, CH3
19.1, CH3
52.4, CH
51.0, CH
35.3, CH2
107.4, CH
159.6, C
123.6, C
158.1, C
119.8, C
134.9, C
118.9, CH
137.4, C
109.5, CH
158.5, C
158.4, C
106.4, CH
130.5, CH
124.6, C
137.5, C
117.1, C
20.5, CH3
18.8, CH3
56.2, CH3
60.3, CH3
22.0, CH3
56.9, CH3
56.7, CH3
52.4, CH
51.0, CH
35.1, CH2
111.4, CH
157.3, C
122.3, C
158.3, C
118.5, C
134.6, C
119.1, CH
137.4, C
109.7, CH
158.5, C
158.5, C
106.5, CH
130.9, CH
124.4, C
137.4, C
117.3, C
20.5, CH3
18.8, CH3
176.2, C
49.8, CH
35.1, CH2
112.3, CH
167.1, C
122.9, C
165.3, C
112.6, C
142.3, C
118.5, CH
138.1, C
109.8, CH
159.1, C
158.7, C
106.3, CH
131.1, CH
122.8, C
137.4, C
117.3, C
24.1, CH3
18.1, CH3
176.5, C
49.7, CH
35.4, CH2
105.1, CH
167.2, C
117.6, C
167.2, C
109.9, C
142.6, C
117.7, CH
138.0, C
109.8, CH
158.6, C
159.2, C
106.5, CH
131.4, CH
121.0, C
137.7, C
117.6, C
24.6, CH3
18.1, CH3
56.8, CH3
2.3.5. Ancistrobrevine G (9)
Yellow amorphous solid: [α]D20-36.8° (c 0.05, MeOH). UV/Vis
(MeOH): λmax (logε): 197 (4.1), 229 (3.6) nm. ECD (c 0.1, MeOH): λmax
(Δε): 214 (−1.7), 223 (−2.1), 241 (+0.4), 283 (−0.2), 335 (+0.05)
nm. IR (ATR) νmax: 3370, 1671, 1609, 1200, 1138, 837, 721 cm−1 1H
.
and 13C NMR data are given in Table 1 and Table 2. HR-ESI-MS: m/z
380.18536 [M + H]+ (calcd for C23H26NO4: 380.18563).
2.3.6. 5′-O-Demethylancistrobrevine B (10)
Yellow amorphous solid: [α]D20-27.8° (c 0.05, MeOH). UV/Vis
(MeOH): λmax (logε): 236 (3.7), 308 (3.0) nm. ECD (c 0.1, MeOH): λmax
(Δε): 208 (+10.8), 214 (+9.0), 225 (+17.7), 239 (−13.0), 309 (−1.0)
nm. IR (ATR) νmax: 3370, 1670, 1589, 1202, 1129, 1111 cm−1 1H and
.
13C NMR data are given in Table 3 and Table 4. HR-ESI-MS: m/z
394.20129 [M + H]+ (calcd for C24H28NO4: 394.20128).
56.0, CH3
22.1, CH3
56.7, CH3
60.1, CH3
22.0, CH3
56.9, CH3
56.7, CH3
61.5, CH3
22.0, CH3
56.9, CH3
56.6, CH3
2.3.7. Ancistrobrevine H (11)
21.8, CH3
56.8, CH3
56.5, CH3
Yellow amorphous solid: [α]D20-32.9° (c 0.05, MeOH). UV/Vis
(MeOH): λmax (logε): 236 (3.8), 308 (3.1) nm. ECD (c 0.1, MeOH): λmax
(Δε): 222 (+17.1), 238 (−11.2), 261 (+0.3), 280 (−2.4), 306 (+1.2)
The spectra were recorded at 100a or 150b MHz.
nm. IR (ATR) νmax: 3372, 1671, 1579, 1464, 1195, 1090 cm−1 1H and
.
13C NMR data are given in Table 3 and Table 4. HR-ESI-MS: m/z
2.3.9. Ancistrobrevine I (13)
422.23260 [M + H]+ (calcd for C26H32NO4: 422.23258).
Yellow amorphous solid: [α]D20-52.8° (c 0.05, MeOH). UV/Vis
(MeOH): λmax (logε): 199 (4.2), 232 (3.9), 322 (3.6) nm. ECD (c 0.1,
MeOH): λmax (Δε): 212 (−5.4), 231 (+3.1), 245 (−2.2), 269 (+0.5),
303 (+0.3), 335 (−0.5) nm. IR (ATR) νmax: 3395, 2928, 1671, 1585,
2.3.8. 6-O-Demethylancistrobrevine H (12)
Yellow amorphous solid: [α]D20+183.6° (c 0.05, MeOH). UV/Vis
(MeOH): λmax (logε): 231 (3.9), 307 (3.3), 319 (3.2), 334 (3.1) nm. ECD
(c 0.1, MeOH): λmax (Δε): 222 (+0.7), 232 (+0.1), 239 (+0.2), 256
(+0.4), 282 (−0.4), 309 (+0.2) nm. IR (ATR) νmax: 3419, 1671, 1585,
1321, 1273, 1194, 1134 cm−1 1H and 13C NMR data are given in
.
Table 3 and Table 4. HR-ESI-MS: m/z 406.20233 [M + H]+ (calcd for
25H28NO4: 406.20128).
C
1280, 1136, 1093, 1068 cm−1 1H and 13C NMR data are given in
.
Table 3 and Table 4. HR-ESI-MS: m/z 408.21767 [M + H]+ (calcd for
25H30NO4: 408.21693).
2.3.10. Ancistrobrevine J (14)
C
Yellow amorphous solid: [α]D20-77.5° (c 0.05, MeOH). UV/Vis
Table 3
1H NMR data of 5′-O-demethylancistrobrevine B (10), ancistrobrevine H (11), 6-O-demethylancistrobrevine H (12), and ancistrobrevines I (13) and J (14) in
methanol‑d4 (δ in ppm).
10b
11a
12a
13a
14a
no.
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
1
4.75, q
4.70, q
4.67, q
3
3.67, m
3.55, m
3.53, m
4.05, m
4.05, m
4ax
4eq
5
2.06, dd (11.8, 17.5)
2.65, dd (4.8, 18.0)
3.06, overlapped
3.06, overlapped
6.72, s
2.95, overlapped
2.95, overlapped
6.56, s
2.92, dd (12.4, 16.8)
3.12, dd (4.9, 17.0)
6.73, s
2.97, dd (11.5, 15.9)
3.22, dd (5.6, 16.0)
6.79, s
7
6.56, s
1′
6.64, s
6.72, s
6.83, s
6.78, s
6.66, s
3′
6.78, s
6.75, s
6.77, s
6.79, s
6.79, s
6′
7′
6.80, d (7.8)
7.09, d (7.8)
1.61, d (6.7)
1.19, d (6.4)
6.90, d (8.0)
7.14, d (8.0)
1.73, d (6.6)
1.52, d (6.4)
3.60, s
3.05, s
2.29, s
3.91, s
3.94, s
6.93, d (8.1)
7.19, d (8.0)
1.72, d (6.0)
1.50, d (6.5)
6.95, d (8.1)
7.24, d (8.0)
2.75, d (1.5)
1.50, d (6.7)
6.94, d (8.1)
7.17, d (7.9)
2.78, d (1.5)
1.51, d (6.7)
3.74, s
1-CH3
3-CH3
6-OCH3
8-OCH3
2′-CH3
4′-OCH3
5′-OCH3
3.91, s
2.29, s
4.08, s
3.04, s
2.31, s
3.92,s
3.95, s
3.17, s
2.33, s
3.93, s
3.96, s
2.30, s
3.92, s
3.96, s
The spectra were recorded at 400a or 600b MHz.
249

S. Fayez et al.
Fitoterapia131(2018)245–259
Table 5
Antiparasitic activities of naphthylisoquinoline alkaloids (7a, 7b, 8a, 8b, 9–11, 15, 18, 19, 21, and 22) isolated from the roots of A. abbreviatus, and the previously
reported [22] data of 5-epi-ancistectorine A2 (3) against Plasmodium falciparum (NF54 strain), Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania
donovani, and cytotoxicities against rat skeletal myoblast (L6) cells.
IC50 [μM]a
Compound
P. falciparum
T. cruzi
T. brucei rhodesiense
L. donovani
L6 cells (cytotoxicity)
Selectivity indexb
b
Standard
0.065c
0.0300 (K1)h
0.0074 (NF54)
0.213
0.663
0.928
0.846
0.134
6.62
2.39
5.59
3.68
2.05
2.447d
135.4h
—
114
130
0.007e
21.1h
—
1.357f
> 220h
—
> 100
> 100
235
> 100
141
112
> 100
> 100
142
> 100
105
0.0096g
100h
94.1
129
145
162
—
3h
3340h
12,720
604
225
175
7a
7b
8a
8b
9
15
10
18
11
19
21
22
16.2
16.8
16.2
16.4
4.22
8.35
34.1
14.7
4.55
38.7
13.1
9.54
129
124
117
138
183
54.8
38.6
136
70.9
42.1
115
24.5
95.9
150
80.5
40.2
109
14.5
62.8
14.4
10.9
53.4
9.70
62.7
6.59
1.45
76.4
76.7
63.9
90.9
> 100
a
The IC50 values are the means of two independent assays; the individual values vary by a factor of < 2.
The selectivity index is calculated as the ratio of the IC50 values concerning L6 cells to the IC50 data relative to P. falciparum.
Chloroquine.
Benznidazole.
Melarsoprol.
Miltefosine.
Podophyllotoxin.
Values reported earlier (see ref. [22]).
b
c
d
e
f
g
h
(MeOH): λmax (logε): 197 (4.1), 231 (3.6), 321 (3.2) nm. ECD (c 0.1,
MeOH): λmax (Δε): 214 (−6.1), 231 (+2.9), 242 (−2.6), 259 (+0.3),
319 (−0.7) nm. IR (ATR) νmax: 3323, 1630, 1457, 1381, 1305, 1273,
1196, 1131, 1015 cm−1. 1H and 13C NMR data are given in Table 3 and
Table 4. HR-ESI-MS: m/z 406.20145 [M + H]+ (calcd for C25H28NO4:
406.20128).
13C NMR (methanol‑d4, 100 MHz): δ 18.7 (Me-3), 19.7 (Me-1), 21.9
(Me-2′), 35.4 (C-4), 51.1 (C-3), 52.4 (C-1), 56.0 (OMe-6), 56.7 (OMe-
5′), 56.9 (OMe-4′), 103.5 (C-5), 106.7 (C-6′), 109.8 (C-3′), 114.4 (C-9),
116.7 (C-7), 117.7 (C-10′), 118.5 (C-1′), 123.0 (C-8′), 131.1 (C-7′),
134.7 (C-10), 137.5 (C-2′), 138.1 (C-9′), 154.1 (C-8), 158.5 (C-5′),
158.7 (C-4′), 159.3 (C-6) ppm. HR-ESI-MS: m/z 408.2169 [M + H]+
(calcd for C25H30NO4: 408.2169).
2.3.11. Stereoselective reduction of ancistrobrevine I (13)
To a cooled solution of 13 (1.50 mg, 3.70 μmol) in dry MeOH
(5 mL), an excess of NaBH4 (5 mg, 0.132 mmol) was added under a
nitrogen atmosphere at 0 °C. The mixture was stirred at 0 °C and
monitored by HPLC until complete reduction was achieved after 2 h.
The mixture was then filtered over Celite® and purified by HPLC on an
XSelect HSS PFP HPLC column using a linear gradient solvent system of
A′ and B′ (0 min: 50% B′ → 30 min: 70% B′), yielding the cis-configured
compound 12 as a yellow amorphous solid (1.1 mg, 2.71 μmol, 73%):
All physical and spectroscopic data of 12 (see Supporting Information)
were found to be fully in accordance with those of the natural product
6-O-demethylancistrobrevine H (12) obtained by isolation from the
roots of A. abbreviatus (see paragraph 2.3.8).
2.3.13. Known alkaloids isolated: 3, 5, 6a, 6b, and 15–22
Likewise isolated were the known naphthylisoquinoline alkaloids 5-
epi-ancistectorine A2 (3) (Fig. 1), 6-O-methyl-4′-O-demethylhamatine
(16), 6-O-methylhamatinine (17), and ancistrocladisine B (20) (Fig. 2),
and the tetralone cis-isoshinanoline (5) (Fig. 1), now discovered for the
first time in the roots of A. abbreviatus. They were found to be identical
in their spectroscopic, physical, and chromatographic behavior with the
data reported previously [22,26,34,47,52]. Furthermore, hamatine
(6b) (Fig. 1), 6-O-methylhamatine (15), the ancistrobrevines B (18) and
D (19), ancistrobrevine A (21), and its 6-O-demethyl analog 22 (Fig. 2),
which are well-known metabolites of A. abbreviatus [6,38,39,42], were
isolated for biotesting.
2.3.12. Stereoselective reduction of ancistrobrevine J (14)
As described in the last paragraph, a cooled solution of 14 (1.30 mg,
3.20 μmol) in dry MeOH (8 mL) was treated with an excess of NaBH4
(5 mg, 0.132 mmol) under a nitrogen atmosphere at 0 °C until complete
reduction was achieved after 30 min, followed by purification of the
crude product by HPLC, gving rise to the cis-configured compound 23
as a yellow amorphous solid (1.20 mg, 2.96 μmol, 92%): [α]D20 +83.5°
(c 0.10, MeOH). UV/Vis (MeOH): λmax (logε): 230 (3.9), 304 (3.2), 319
(3.1) nm. ECD (c 0.1, MeOH): λmax (Δε): 210 (−1.6), 230 (+0.9), 276
(+0.06), 333 (−0.05) nm. IR (ATR) νmax: 2973, 1713, 1677, 1578,
2.4. Oxidative degradation
Following a procedure described earlier [53], ca. 0.5 mg of the
corresponding alkaloid (7a, 7b, 8a, 8b, or 9–14) were subjected to a
ruthenium(VIII)-catalyzed periodate oxidation, followed by conversion
of the resulting chiral amino acids with MeOH/SOCl2 to the respective
methyl esters and Mosher-type derivatization with (R)-α-methoxy-α-
trifluoromethylphenylacetyl chloride [(R)-MTPA-Cl, prepared from (S)-
MTPA]. The absolute configurations of 7a, 7b, 8a, 8b, and 9–14 at C-3
(and, in some cases, at C-1) were assigned by gas chromatography
coupled to mass-selective detection (GC-MSD) and comparison with the
corresponding derivatives of the authentic amino acids of known con-
figuration.
1457, 1375, 1280, 1228, 1199, 767 cm−1 1H NMR (methanol‑d4,
.
400 MHz): δ 1.51 (3H, d, J = 6.4 Hz, Me-3), 1.74 (3H, d, J = 6.5 Hz,
Me-1), 2.26 (3H, s, Me-2′), 3.00 (2H, overlapped, H2–4), 3.54 (1H, m,
H-3), 3.57 (3H, s, OMe-6), 3.90 (3H, s, OMe-4′), 3.94 (3H, s, OMe-5′),
4.67 (1H, q, H-1), 6.49 (1H, s, H-5), 6.64 (1H, s, H-1′), 6.75 (1H, s, H-
3′), 6.92 (1H, d, J = 8.0 Hz, H-6′), 7.15 (1H, d, J = 7.90 Hz, H-7′) ppm.
250

S. Fayez et al.
Fitoterapia131(2018)245–259
Table 6
3. Results and discussion
Preferential cytotoxicity of naphthylisoquinoline alkaloids isolated from the
roots of A. abbreviatus (3, 6b, 7a, 7b, 8b, 12, 16, 17, 19, 21, and 22) against
human PANC-1 pancreatic cancer cells under nutrient-deprived conditions.
3.1. Isolation and structural elucidation of naphthylisoquinoline alkaloids
Powdered material of air-dried roots of A. abbreviatus was ma-
cerated with MeOH, assisted by ultrasonication, and further partitioned
between water and CH2Cl2 to extract the secondary metabolites.
Fractionation of the organic layer by column chromatgraphy (CC) on
silica gel and further resolution and purification by preparative re-
versed-phase HPLC afforded ten new naphthylisoquinoline alkaloids,
along with five known [22,26,34,47,52] compounds, which were for
the first time detected in A. abbreviatus, viz., 5-epi-ancistectorine A2 (3)
(Fig. 1), 6-O-methyl-4′-O-demethylhamatine (16), 6-O-methylhamati-
nine (17), ancistrocladisine B (20) (Fig. 2), and the tetralone cis-iso-
shinanolone (5) (Fig. 1).
Compound
PC50 (in μM)a
Compound
PC50 (in μM)a
3
11.5
35.6
35.3
45.0
32.2
32.2
42.0
16
17
19
21
7.50
18.8
45.5
19.0
41.1
41.1
0.8
6b
7a
7b
8b
12
15
22
22
arctigeninb
a
Concentration at which 50% of the cells were killed preferentially in nu-
trient-deprived medium (NDM).
b
Used as the reference compound.
The three 5,1′-coupled alkaloids 5-epi-ancistectorine A2 (3) [22], 6-
O-methyl-4′-O-demethylhamatine (16) [26], and 6-O-methylhamati-
nine (17) [25] had earlier been isolated from the Southeast Asian liana
A. tectorius. Compound 17 is also a well-known constituent of A. co-
chinchinensis from Vietnam [52] and has likewise been detected in an as
yet unidentified Congolese Ancistrocladus species [56]. The 7,1′-linked
ancistrocladisine B (20) has only recently been discovered as a minor
metabolite in A. ileboensis endemic to the South-Central Congo Basin
[34]. With their S-configuration at C-3 and an oxygen function at C-6,
the alkaloids 3, 16, 17, and 20 belong to the subclass of Ancis-
trocladaceae-type [6,31] naphthylisoquinolines.
2.5. Antiprotozoal assay
The in vitro antiparasitic activities (Table 5) of the ancistrobrevines
E (7a) and F (8a), their atropo-diastereomers 7b and 8b, ancistro-
brevine G (9), 5′-O-demethylancistrobrevine B (10), and ancistro-
brevine H (11) against the pathogens Plasmodium falciparum (NF54
strain), Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leish-
mania donovani, and the cytotoxicity against mammalian host cells (rat
skeletal myoblast L6 cells) were determined as described previously
[54]. Likewise tested were the known [6,22,38,39,42] alkaloids 5-epi-
ancistectorine A2 (3), 6-O-methylhamatine (15), ancistrobrevines A
(21), B (18), and D (19), and 6-O-demethylancistrobrevine A (22).
The first new metabolite was obtained as a yellow amorphous solid.
It had a molecular formula of C24H27NO4, as evidenced from HRESIMS
(m/z 394.20198 [M + H]+) and from the number of signals in the 13C
NMR spectrum (Table 2). The compound showed 1H NMR data
(Table 1) typical of a naphthyl-1,3-dimethyltetrahydroisoquinoline al-
kaloid equipped with two methoxy functions (δH 3.91 and 3.95), three
C-methyl groups (δH 1.19, 1.67, and 2.10), two aliphatic methines, H-1
(δH 4.75) and H-3 (δH 3.60), two diastereotopic protons, Hax-4 (δH 2.11)
and Heq-4 (δH 2.22), and five aromatic ones (Fig. 3A). The coupling
pattern of the latter displayed two singlets, H-7 (δH 6.46) and H-3′ (δH
6.90), and a spin system of three contiguous protons in the naphthalene
portion, viz., H-6′ (δH 6.86), H-7′ (pseudotriplet at δH 7.21), and H-8′
(δH 6.87), thus, establishing the coupling site of the biaryl axis in the
naphthalene portion to be at C-1′. This assignment was further corro-
borated by the highfield-shifted resonance of the Me-2′ signal (δH 2.10),
by two NOESY correlation sequences in the series {Me-2′ ↔ H-3′ ↔
OMe-4′} and {OMe-5′ ↔ H-6′ ↔ H-7′ ↔ H-8′}, and by HMBC interac-
tions from H-8′, Me-2′, and H-3′ to the quaternary carbon atom C-1′ (δC
124.9). The positions of the two methoxy functions at C-4′ (δH 3.95)
and C-5′ (δH 3.91) in the naphthalene subunit were deduced from
NOESY interactions to H-3′ and H-6′, respectively, and were in ac-
cordance with HMBC cross peaks to C-4′ (δC 157.7) and C-5′ (δC 158.6)
(Fig. 3B). The remaining two oxygen functions thus had to be free
hydroxy groups at C-6 and C-8 in the tetrahydroisoquinoline portion.
From 3J HMBC interactions of H-4ax (δH 2.11) and H-7 (δH 6.46), both
to the quaternary carbon atom C-5 (δC 117.7), the coupling position of
the biaryl linkage in the tetrahydroisoquinoline moiety was deduced to
be C-5 (Fig. 3B). Thus, the isolated alkaloid was 5,1′-coupled, which
was also in accordance with the close proximity of the C-4 position to
the axis, as evidenced by the high-field shifts of the signals of H-4ax (δH
2.11) and H-4eq (δH 2.22). Thus, the new metabolite was attributed the
constitution 7 as outlined in Fig. 3.
2.6. Preferential cytotoxicity against PANC-1 cells
The naphthylisoquinoline alkaloids 3, 6b, 7a, 7b, 8b, 15–17 (5,1′-
coupled), 19 (7,1′-coupled), and 12, 21, and 22 (7,8′-coupled) were
evaluated for their preferential cytotoxicity against PANC-1 human
pancreatic cancer cells (RBRC-RCB2095) (Table 6). The cell line was
purchased from the Riken BRC cell bank and maintained in standard
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
serum (FBS) supplement and stored at 37 °C under a humidified at-
mosphere of 5% CO2 and 95% air. Briefly, the cancer cells were seeded
in 96-well plates (1.5 × 104/well), incubated in fresh DMEM, and wa-
shed twice with PBS. The medium was changed to serially diluted test
samples in both, nutrient-rich medium (DMEM) and nutrient-deprived
medium (NDM) [55], with a control and blank in each test plate. The
composition of the NDM was as follows: 265 mg L−1 CaCl2 (2 H2O),
0.1 mg L−1 Fe(NO3)3 (9 H2O), 400 mg L−1 KCl, 200 mg L−1 MgSO4 (7
H2O), 6400 mg L−1 NaCl, 700 mg L−1 NaHCO3, 125 mg L−1 NaH2PO4,
15 mg L−1 phenol red, 25 mM L−1 HEPES buffer (pH 7.4), and MEM
vitamin solution (Life Technologies, Inc., Rockville, MD, USA); the final
pH was adjusted to 7.4 with 10% NaHCO3. Arctigenin, the positive
control in this study, was isolated from the seeds of Arctium lappa [55].
After 24 h of incubation with each of the compounds in DMEM and
NDM, the cells were washed twice with PBS and replaced by 100 μL of
DMEM containing 10% WST-8 cell counting kit solution. After 3 h of
incubation, the absorbance at 450 nm was measured on an EnSpire
Multimode plate reader (PerkinElmer, Inc., Waltham, MA, USA). Cell
viability was calculated from the mean values of three wells using the
following equation:
The absolute configuration at C-3 in the tetrahydroisoquinoline
portion was established by ruthenium-mediated oxidative degradation
according to a procedure developed by us earlier [53]. The formation of
(S)-3-aminobutyric acid proved the new alkaloid to be S-configured at
C-3. The relative configuration at C-1 versus C-3 was deduced to be
trans from a NOESY interaction between Me-1 (δH 1.67) and H-3 (δH
3.60), which, given the absolute S-configuration at C-3, determined C-1
to be S-configured, too. On the basis of this absolute 1S,3S-stereoarray
Cell viability (%) = [Abs(test sample)–Abs(blank) /Abs(control)–Abs(blank)
× 100%.
]
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Fitoterapia131(2018)245–259
Fig. 3. (A) Selected 1H and 13C NMR spectroscopic data (in methanol‑d4, δ in ppm) of ancistrobrevine E (7a); in {}: the values of 5-epi-ancistrobrevine E (7b) that are
different from those of 7a. All other values of 7a and 7b are nearly identical ( 0.02 ppm in 1H NMR and 0.5 ppm in 13C NMR). (B) Key HMBC (green single
arrows) and NOESY (red double arrows) interactions evidencing the constitution of 7a and 7b. (C) NOESY correlations proving the relative configurations of 7a and
7b at the biaryl axis and at the stereogenic centers C-1 and C-3 in the tetrahydroisoquinoline subunit. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
in the tetrahydroisoquinoline portion, the configuration at the biaryl
axis was assigned to be P due to long-range NOESY correlations be-
tween Me-2′ and H-4ax and between H-8′ and H-4eq (Fig. 3C, top). This
assignment was corroborated by the ECD spectrum of the new alkaloid,
which displayed an almost mirror-image like ECD curve compared to
that of the known [22] – and co-occurring – likewise 5,1′-coupled, but
M-configured alkaloid 5-epi-ancistectorine A2 (3) (see Supporting In-
formation). The new metabolite thus had the full absolute stereo-
structure 7a as presented in Fig. 1. It was henceforth named ancistro-
brevine E, in continuation of the series of alkaloids previously isolated
from A. abbreviatus. Alternatively, 7a might also be addressed as the 8-
O-demethyl analog of the co-occurring ancistrocladine (6a), a well-
known major metabolite of various African and Asian Ancistrocladus
species [6,17,31,32,57].
stereostructure 7b and was hence the atropo-diastereomer of 7a. Thus,
the new alkaloid was named 5-epi-ancistrobrevine E. It can likewise be
addressed as the 8-O-demethyl analog of the well-known and widely
occurring [6,31,32,58] alkaloid hamatine (6b).
Three further new alkaloids were isolated from the roots of A. ab-
breviatus, all of them with a molecular weight of 14 u below that of 7a
and 7b, which is typical of the loss of CH2 due to O-demethylation.
HRESIMS indicated a molecular formula of C23H26NO4, suggesting that
these metabolites might be O-demethyl analogs of 7a or 7b. In agree-
ment with this assumption, their 1D and 2D NMR spectra were, apart
from that missing methoxy function and little variations of the chemical
shifts (see Table 1 and Table 2), very similar to those of 7a and 7b, thus
suggesting the presence of likewise 5,1′-coupled naphthylisoquinoline
alkaloids, but, in contrast to 7a and 7b, equipped with only one
methoxy group.
HRESIMS analysis of the second new compound isolated from the
root extract of A. abbreviatus gave the same molecular formula of
In the case of the third and fourth new alkaloids, this methoxy group
(δH 4.07/4.07) showed a NOESY interaction to H-6′ (δH 6.85/6.86) and
an HMBC correlation to C-5′ (δC 158.0/157.9). Thus, the only methoxy
function of the compounds was attributed to be located at C-5′, which
was in agreement with the lacking NOESY correlation of H-3′ (δH 6.79/
6.79) to a possible methoxy group at C-4′ (δC 155.2/155.2) (see
Supporting Information). The relative configuration at C-1 versus C-3
was assigned to be trans in both compounds. And, likewise similar to
the results with 7a and 7b, oxidative degradation again established the
absolute configuration in the isoquinoline portions of the two new
metabolites to be 1S,3S. This, in conjunction with specific NOESY in-
teractions between Heq-4 (δH 2.22) and H-8′ (δH 6.87), and between
C
24H27NO4 as that of ancistrobrevine E (7a). The 1H (Table 1) and 13C
NMR data (Table 2) were almost identical to those of 7a, with just
slightly different chemical shifts, thus evidencing that the new isolated
compound possessed the same constitution as 7a. Significant differ-
ences between 7a and the new metabolite were only observed for the
chemical shifts of the diastereotopic protons Hax-4 (δH 2.03) and Heq-4
(δH 2.33) (see Fig. 3A), which are located next to the biaryl axis, thus
suggesting that these two metabolites were diastereomers, probably
atropo-diastereomers.
NOESY cross peaks of the protons of Me-1 (δH 1.65) to H-3 (δH 3.66)
indicated the methyl groups at C-1 and C-3 to be trans to each other
(Fig. 3C, bottom). The oxidative degradation procedure again delivered
only (S)-3-aminobutyric acid, thus, together with the relative trans-
configuration, the absolute configuration at C-1 had to be S, too. Given
the identical constitution and same configurations at the two stereo-
centers in the isoquinoline subunit, the differences between the new
metabolite and 7a thus had to be due to an opposite configuration at
the biaryl linkage. This assumption was confirmed by the long-range
NOESY correlation between Hax-4 (δH 2.03) and H-8′ (δH 6.80) and
between Heq-4 (δH 2.33) and Me-2′ (δH 2.13) (Fig. 3C, bottom), which
attributed an M-configuration at the biaryl axis. This was further cor-
roborated by the fact that the ECD spectrum of the new alkaloid was
virtually identical to that of the likewise M-configured 5-epi-ancis-
tectorine A2 (3) (Fig. 4A).
H
ax-4 (δH 2.10) and Me-2′ (δH 2.05) demonstrated the biaryl axis of the
third compound, 8a, to be P-configured, while the fourth new alkaloid,
8b, was M-configured, as obvious from complementary NOESY corre-
lations between Hax-4 (δH 2.01) and H-8′ (δH 6.80) and between Heq-4
(δH 2.32) and Me-2′ (δH 2.05) (see Supporting Information). Thus, the
new metabolites 8a and 8b, as outlined in Fig. 1, constituted a pair of
atropo-diastereomers. This finding was also in agreement with the ECD
spectra of 8a and 8b, which were fully opposite to each other (Fig. 4B).
In continuation of the series of alkaloids discovered in A. abbreviatus,
the two new compounds were named ancistrobrevine F (8a) and 5-epi-
ancistrobrevine F (8b). These as yet unknown metabolites might also be
addressed as the 8,4′-O-didemethyl analogs of their well-known
[17,18,57,58] parent compounds ancistrocladine (6a) and hamatine
(6b).
The new naphthyltetrahydroisoquinoline thus possessed the
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Fitoterapia131(2018)245–259
Fig. 4. Assignment of the absolute axial configuration of the new 5,1′-coupled ancistrobrevines by ECD spectroscopy: (A) comparison of the ECD spectrum of 5-epi-
ancistrobrevine E (7b) with that of the known [22], structurally related and likewise M-configured alkaloid 5-epi-ancistectorine A2 (3); (B) mirror-image ECD curves
of ancistrobrevine F (8a) and its atropo-diastereomer 8b; and (C) comparison of the ECD spectrum of ancistrobrevine G (9) with that of 3; for the structure of 3, see
Fig. 1.
NMR measurements revealed that the fifth new 5,1′-coupled alka-
loid, different from 8a and 8b, had to be equipped with a methoxy
group (δH 4.10) at C-4′. Again, similar to 7a/7b and 8a/8b, the two
methyl groups at C-1 and C-3 were trans to each other, and both C-1 and
C-3 were S-configured. Like for 7a and 8a, long-range NOE interactions
evidenced the biaryl axis to be P-configured (see Supporting
Information). As shown in Fig. 4C, this finding was confirmed by ECD
spectroscopy. The new alkaloid thus had the stereostructure 9 as pre-
sented in Fig. 1. It was hence the 8,5′-O-didemethyl analog of ancis-
trocladine (6a) and was named ancistrobrevine G.
The sixth new alkaloid was isolated as a trace metabolite from the
roots of A. abbreviatus. It had a molecular formula of C24H27NO4, as
determined by HRESIMS, and again exhibited 1H NMR signals typical of
an Ancistrocladaceae-type naphthyltetrahydroisoquinoline with four
oxygen functions (Table 1). Its proton pattern in the aromatic region
was different from those of the five 5,1′-coupled ancistrobrevines 7a/b,
8a/b, and 9 presented above. Given the spin system of three singlets (δH
6.56, 6.64, and 6.78) and two doublets (δH 6.80 and 7.09) (Fig. 5A),
thus evidencing the presence of a pair of adjacent aromatic protons in
the naphthalene part, this substitution pattern hinted either at a 6′- or
an 8′-coupling site of the biaryl axis. This was corroborated by the
‘normal’ chemical shift of the 2′-Me group (δH 2.29), which would
otherwise, with the axis neighboring the 1′- or the 3′-position, be high-
field shifted, as in 7, 8, and 9. NOESY interactions in the series {H-1′ ↔
Fig. 5. (A) Selected 1H and 13C NMR data (in methanol-d4, δ in ppm), NOESY
(red double arrows) and HMBC (green single arrows) interactions of 5′-O-de-
methylancistrobrevine B (10); (B) NOESY interactions indicative of the con-
figuration at the biaryl axis of 10 relative to the stereogenic centers in the
tetrahydroisoquinoline subunit. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
Me-2′ ↔ H-3′ ↔ OMe-4′} and a cross-peak between H-6′ (δH 6.80) and
H-7′ (δH 7.09), in conjunction with HMBC correlations from H-1′ (δH
6.64) and H-6′ to the quaternary carbon atom C-8′ (δC 124.4) (Table 2),
established the isoquinoline portion to be located at C-8′ (Fig. 5A). The
naphthalene subunit was deduced to be coupled to C-5 (δC 119.9) due
to HMBC interactions between H-7′, H-7 (δH 6.56), and H-4eq (δH 2.65)
with the quaternary atom C-5 (δC 119.9). The new 5,8′-coupled alkaloid
possessed two methoxy groups with NMR resonances at δH 3.91 and
4.08. From NOESY interactions between OMe-4′ and H-6′, and between
OMe-8 and H-7, their positions were determined to be at C-4′ in the
naphthalene part and at C-8 in the isoquinoline half, which was in
agreement with HMBC correlations from OMe-4′ to C-4′, and from
OMe-8 to C-8 (Fig. 5A).
The relative configuration of the two stereogenic centers at C-1 and
C-3 was deduced to be trans from a NOESY interaction between Me-1
(δH 1.61) and H-3 (δH 3.67) (Fig. 5B). This assignment was confirmed
by oxidative degradation [53], giving rise to (S)-3-aminobutyric acid
and L-alanine, thus clearly establishing the new metabolite to be 1S,3S-
configured. Long-range NOESY cross-peaks between H-7′ and Heq-4,
and between H-1′ and Hax-4 (Fig. 5B), in combination with the absolute
1S,3S-stereoarray in the tetrahydroisoquinoline portion, assigned the
biaryl axis to be M-configured. This result was in accordance with the
fact that the ECD spectrum of the new minor constituent (see Sup-
porting Information) was nearly identical to that of the known [38],
likewise 5,8′-coupled alkaloid ancistrobrevine B (18), which is a main
constituent of A. abbreviatus. The new metabolite thus had the full
stereostructure 10 as presented in Fig. 1. Owing to its close structural
relationship to 18, it was named 5′-O-demethylancistrobrevine B. With
its constitution identical to that of ancistroguineine A, a major con-
stituent of the Nigerian species A. guineënsis [43], and with the same
1S,3S-configuration, but differing in its absolute axial configuration,
the new alkaloid 10 might also be addressed as 5-epi-ancistroguineine
A.
According to HRESIMS, the molecular formula of the seventh new
metabolite
Comprehensive analysis of the 1H (Table 3) and 13C NMR data (Table 4)
revealed the presence of naphthyl-1,3-
was
C
26H31NO4
(m/z
422.23260,
[M + H]+).
a
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Fitoterapia131(2018)245–259
across the biaryl axis between OMe-8 (δH 3.05) and H-7′ (Fig. 6B).
The relative configuration of the two methyl groups at C-1 and C-3
was deduced from a NOESY interaction between H-1 (δH 4.70) and H-3
(δH 3.55) (Fig. 6B) revealing that these two protons – and, thus, also the
two respective methyl groups, Me-1 (δH 1.73) and Me-3 (δH 1.52) –
were cis to each other. The absolute configurations at C-1 and C-3 were
established to be 1R,3S by ruthenium-catalyzed oxidation affording D-
alanine and (S)-3-aminobutyric acid as the main products, which was in
agreement with the cis-configuration assigned by NMR.
The configuration at the biaryl axis relative to the stereogenic
centers was determined from a long-range NOESY interaction between
H-7′ and Me-1, indicating that these spin systems were on the same side
of the tetrahydroisoquinoline plane, so that, with Me-1 below (as evi-
dent from the degradation), the biaryl axis had to be P-configured, as
illustrated in Fig. 6B. Consequently, the new compound had the full
absolute stereostructure 11 as presented in Fig. 1, and was named an-
cistrobrevine H. Its axial P-configuration was further supported by the
ECD spectrum (Fig. 6C, left), which was virtually identical to that of the
closely related known [6,42] – and co-occurring – alkaloid ancistro-
brevine A (21) (Fig. 1), the latter differing from 11 only by an addi-
tional N-methyl group.
The eighth metabolite, isolated from the roots of A. abbreviatus,
showed 1H (Table 3) and 13C NMR (Table 4) data hinting at the pre-
sence of a further 7,8′-coupled naphthyltetrahydroisoquinoline alka-
loid. Its constitution strongly resembled that of the above presented
ancistrobrevine H (11), yet with a lower degree of methylation, as
obvious from its molecular formula of C25H29NO4, suggesting that this
alkaloid might be an O-demethyl analog of 11. Indeed, NMR in-
vestigations revealed that the new compound possessed a free hydroxy
function at C-6 instead of a methoxy substituent as found in 11
(Fig. 6A). Again,
a
relative cis-configuration in the tetra-
hydroisosquinoline part was evident from a NOESY interaction between
H-1 (δH 4.67) and H-3 (δH 3.53) and, similar to 11, the absolute con-
figuration at C-1 and C-3, as determined by oxidative degradation, was
established to be 1R,3S. Furthermore, the isolated minor alkaloid had
the same axial P-configuration as 11, which was again deduced from a
long-range NOE interaction between Me-1 (δH 1.72) and H-7′ (δH 7.19)
(Fig. 6B), and from the ECD spectrum of the new compound. As pre-
sented in Fig. 6C (right), the ECD curve was virtually identical to that of
the structurally closely related alkaloid 6-O-demethylancistrobrevine A
(22) (Fig. 2), which is well known from previous isolation work on A.
abbreviatus [6,42]. Hence, the new alkaloid possessed the stereo-
structure 12 (Fig. 1). Since it was the 6-O-demethyl analog of 11, it was
named 6-O-demethylancistrobrevine H.
The molecular formula of the ninth new alkaloid, which occurred as
a minor constituent in the root extract, was C25H27NO4, as deduced
from HRESIMS. The compound again exhibited NMR signals (Table 3
and Table 4) typical of a 7,8′-coupled naphthylisoquinoline alkaloid,
but in contrast to the ancistrobrevines 11 and 12, it was equipped with
a dihydroisoquinoline subunit, as evident from the significant lack of
the H-1 quartet around δ 4.60–4.80, the downfield shifted Me-1 signal
at δ 2.75, and the 13C NMR resonance of C-1 (δC 176.2) (Fig. 7A). Three
of the four oxygen functions were found to be methoxy groups, re-
sonating at δH 3.96, 3.93, and 3.17. Of these, the latter displayed an
NOE interaction with Me-1, thus revealing this O-methyl group to be
located at C-8. Its high-field shift indicated the proximity of the naph-
thalene portion, hence confirming C-7 to be the coupling position in the
dihydroisoquinoline moiety. Additional evidence of a 7,8′-linked biaryl
was obtained from a NOESY correlation between H-7′ (δH 7.24) and
MeO-8 (δH 3.17) (Fig. 7A).
Fig. 6. (A) Selected 1H NMR data (in methanol-d4, δ in ppm), NOESY (red
double arrows) and HMBC (green single arrows) interactions of ancistrobrevine
H (11) and its 6-O-demethyl analog 12; (B) NOESY interactions indicative of
the relative configurations of 11 and 12 at C-1 and C-3 in the tetra-
hydroisoquinoline halves and at the biaryl axes; (C) confirmation of the abso-
lute axial configuration of 11 and 12 by comparison of their ECD spectra with
those of the structurally closely related known [6,42] – and likewise P-config-
ured – alkaloids ancistrobrevine A (21) and 6-O-demethylancistrobrevine A
(22); for their structures, see Fig. 2. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this
article.)
dimethyltetrahydroisoquinoline alkaloid possessing four methoxy
groups (δH 3.05, 3.60, 3.91, and 3.94) (Fig. 6A). The normal, not high-
field shifted signal of Me-2′ (δH 2.29) and the two NOESY correlations
sequences in the series {H-1′ ↔ Me-2′ ↔ H-3′ ↔ MeO-4′} and {MeO-5′
↔ H-6′ ↔ H-7′} evidenced the naphthalene portion to be coupled via C-
8′. This assignment was further proven by HMBC interactions from H-1′
(δH 6.72) and H-6′ (δH 6.90) to the quaternary carbon atom C-8′ (δC
124.6). The NOESY correlation sequence {MeO-6 ↔ H-5 ↔ Heq-4} and
HMBC interactions from H-5 (δH 6.72) to C-4 (δC 35.3) and to C-7 (δC
123.6), and from H-7′ (δH 7.14) to C-7 showed that the biaryl axis was
not linked to C-5 (δC 107.4), but to C-7 in the isoquinoline part
(Fig. 6A). In conclusion, the new alkaloid was established to be 7,8′-
coupled, which was additionally confirmed by the NOESY correlation
Oxidative degradation established the absolute configuration at C-3
to be S. A reliable assignment of the absolute axial configuration of the
new alkaloid, however, proved to be demanding: The experimental ECD
spectrum of the new alkaloid did not fit perfectly with that of the
known [10,59] and likewise 7,8′-coupled naphthyldihydroisoquinoline
yaoundamine A, which differed from the new alkaloid by possessing an
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S. Fayez et al.
Fitoterapia131(2018)245–259
is too far away from Me-2′ and H-7′ in the naphthalene subunit.
To overcome these difficulties, a new, less remote ‘auxiliary’ ste-
reocenter at C-1 was introduced by subjecting the isolated metabolite to
a cis-selective reduction using NaBH4 [62] to give the corresponding
naphthyltetrahydroisoquinoline 12 (Fig. 7B). The cis-configuration at
the two stereogenic centers C-1 versus C-3 of the semi-synthetic product
12 was established by a NOESY interaction between H-1 and H-3. The
position of the previously ‘flat’ Me-1, now below the plane, enabled a
long-range NOESY cross-peak of its protons with H-7′, which, thus, also
had to be below the plane, too. This, in conjunction with the absolute
1R,3S-stereoarray in the tetrahydroisoquinoline portion, un-
ambiguously established the biaryl axis of the semi-synthetic product
12 to be P-configured (Fig. 7B), and, simultaneously, evidenced that the
new metabolite had the stereostructure 13 (Fig. 1).
This stereochemical assignment was firmly corroborated by the fact
that in this case, the semi-synthetic reduction product of 13 happened
to be the – co-occurring (see above) – natural product 6-O-demethy-
lancistrobrevine H (12) (Fig. 6), with which it turned out to be fully
identical, also proven by perfectly matching ECD spectra (Fig. 7C) and
co-chromatography on HPLC (see Supporting Information). In con-
tinuation of the series of alkaloids previously isolated from A. ab-
breviatus, the new alkaloid 13 was named ancistrobrevine I. It is only
the fourth representative of this rare coupling type possessing a dihy-
droisoquinoline subunit [10,19].
According to HRESIMS and NMR analysis, the tenth new alkaloid
isolated from the roots of A. abbreviatus again was a 7,8′-coupled
naphthyldihydroisoquinoline with the same molecular formula of
C25H27NO4 as the above-described ancistrobrevine
I
(13).
Constitutionally, it differed from 13 only by its OH/OMe substitution
pattern in the dihydroisoquinoline portion, possessing a methoxy group
(δH 3.74) at C-6 and a free phenolic hydroxy function at C-8. The ru-
thenium-mediated oxidative degradation procedure [53] again afforded
(S)-3-aminobutyric acid, thus assigning the absolute configuration at C-
3 to be S. As in the case of 13, no significant long-range NOESY in-
teractions indicative of the axial configuration were detected. ECD
measurements revealed the new alkaloid and ancistrobrevine I (13) to
display nearly perfectly matching spectra (Fig. 8, top), thus indicating
that these two naphthyldihydroisoquinoline alkaloids had the same
axial configurations (for formal reasons, however, 13 and the new al-
kaloid have opposite descriptors according to the Cahn-Ingold-Prelog
formalism).
For an unambiguous assignment of the full stereostructure of the
new metabolite, the above-established methodology was applied: It was
reacted with NaBH4 to give the corresponding cis-configured tetra-
hydroisoquinoline 23 with a high stereoselectivity (see Supporting
Information). The axis of the (as yet) merely semi-synthetic compound
23 was established to be M-configured by a long-range NOESY cross-
peak between H-7′ and Me-1 as part of the newly generated additional
stereocenter. This assignment was corroborated by the virtually oppo-
site ECD spectrum of 23 compared to that of the structurally closely
related 7,8′-coupled, but P-configured alkaloid ancistrogriffine C (24)
Fig. 7. (A) Selected 1H and 13C NMR data (in methanol‑d4, δ in ppm), NOESY
(red double arrows) and HMBC (green single arrows) interactions of ancistro-
brevine I (13); (B) diastereoselective reduction of 13 using NaBH4, to give the
cis-configured naphthyl-1,3-dimethyltetrahydroisoquinoline 12 (the diag-
nostically valuable new stereocenter is underlaid in yellow), and assignment of
the absolute axial configuration of 13 as P by the decisive now possible long-
range NOE interactions observed for the semi-synthetic 12; (C) confirmation of
the absolute configuration at the biaryl axis by comparison of the ECD spectrum
of the semi-synthetic 12 with that of the authentic natural product 6-O-de-
methylancistrobrevine H (12) isolated from A. abbreviatus. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
[44] (Fig. 8, bottom), which likewise has
a 6-methoxy-8-hydro-
R-configuration at C-3 and a hydroxy group at C-5′, thus only giving a
first hint that the isolated metabolite might be P-configured at the
biaryl axis (see Supporting Information).
xytetrahydroisoquinoline portion, thus displaying a similar substitution
pattern next to the biaryl axis as in 23. Accordingly, the new alkaloid
possessed the stereostructure 14. It was named ancistrobrevine J.
Even more difficult seemed an assignment of the axial configuration
of the new alkaloid by NMR, since (different from the new alkaloids
described above) no significant long-range NOESY interactions, i.e. no
cross-peaks between H-7′ (δH 7.24) or H-1′ (δH 6.78) with any ‘out-of-
plane’ protons in the dihydroisoquinoline part, were observed. Such
complications regarding a reliable attribution of the axial configuration
of 7,8′-coupled naphthyldihydroisoquinoline alkaloids by NMR are
known in the literature [10,60,61]. They are caused by fact that, due to
the sp2 character of C-1, the C1-methyl group is located more or less in
the isoquinoline plane and the only stereogenic center in the dihy-
droisoquinoline part, C-3, with its ‘out-of-plane’ protons, H-3 and Me-3,
3.2. Antiprotozoal activities of ancistrobrevines and related
naphthylisoquinoline alkaloids
In view of the strong anti-infective activities of some of the naph-
thylisoquinoline alkaloids [14–16,22,24,34,48,49] previously isolated
from related African or Asian Ancistrocladus species, the new com-
pounds 7a, 7b, 8a, 8b, and 9–11 were investigated for their inhibitory
effects against the protozoan parasites causing malaria, Chagas disease,
African sleeping sickness, and leishmaniasis (Table 5). Likewise as-
sessed were five of the known, but so far not yet tested metabolites of A.
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ancistrobrevines 7a, 7b, 8a, 8b, and 9 indicate that a free hydroxy
group at C-8 may favor the antiplasmodial activities of 5,1′-linked
naphthylisoquinolines, since the parent compounds of this series of
alkaloids, ancistrocladine (6a) and its atropo-diastereomer hamatine
(6b), which bear a methoxy function at C-8, but also the fully O-me-
thylated 6-O-methylhamatine (15), displayed distinctly lower anti-
plasmodial activities, with IC50 values of 45.0 μM (for 6a) [64], 8.19 μM
(for 6b) [64], and 6.62 μM (for 15) (Table 5).
Against Trypanosoma cruzi and Leishmania donovani only very weak
or virtually no activities were determined for any of the evaluated
naphthylisoquinoline alkaloids, while for Trypanosoma brucei rhode-
siense at least some low activities could be recorded (Table 5). This
finding confirms the distinct structure dependency – and thus high
specificity – of the antiplasmodial activities of the naphthylisoquinoline
alkaloids presented in Table 5. This makes further biological studies on
the most active compound, 5-epi-ancistectorine A2 (3), a rewarding
task.
3.3. Preferential cytotoxicity of ancistrobrevines and related alkaloids
against PANC-1 cells
The antiausterity strategy [54,65–68] focusses on the discovery of
new anticancer agents that inhibit the tolerance of cancer cells to nu-
trient deprivation. Human pancreatic PANC-1 cancer cells aggressively
proliferate even under extremely hypovascular (austerity) and hypoxic
conditions, i.e., in the complete absence of essential nutrients such as
glucose, amino acids, and serum [69,70]. Following this approach, we
have most recently identified naphthylisoquinoline alkaloids to exert
strong antiproliferative effects towards human PANC-1 pancreatic
cancer cells in nutrient-deprived medium (NDM) [12,35,71,72]. This
preferential cytotoxicity, also referred to as antiausterity activity, is
represented by PC50 values, i.e., the concentration at which 50% of the
pancreatic cancer cells are killed in NDM without displaying toxicity in
normal, nutrient-rich Dulbecco's modified Eagle's medium (DMEM).
All of the metabolites of A. abbreviatus tested exhibited moderate to
strong preferential cytotoxicities against the PANC-1 cell line, with PC50
values ranging from 7.50 to 45.5 μM (Table 6). Within this series of
naphthylisoquinoline alkaloids, the two 5,1′-coupled alkaloids 6-O-
methyl-4′-O-demethylhamatine (16) and 5-epi-ancistectorine A2 (3)
were the most potent compounds, showing pronounced antiausterity
activities in NDM in a concentration-dependent manner, with PC50
values of 7.50 and 11.5 μM, respectively (Fig. 9).
Fig. 8. Assignment of the absolute axial configuration of ancistrobrevine J (14)
by comparison of the ECD spectrum of 14 with that of ancistrobrevine I (13),
and by NMR after transformation of 14 into the (as yet merely semi-synthetic)
naphthyl-1,3-dimethyltetrahydroisoquinoline 23 by cis-selective reduction
using NaBH4, corroborating the biaryl axis of 14 to be M-configured due to the
detection of a decisive long-range NOESY cross-peak between Me-1 and H-7′ in
23 (the diagnostically valuable new stereocenter is underlaid in yellow), and
due to the ECD spectrum of 23, which was virtually opposite to that of the
known [44], likewise 7,8′-coupled, but P-configured alkaloid ancistrogriffine C
(24). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Interestingly, within the small library of 5,1′-coupled alkaloids
presented here (cmpds. 3, 6b, 7a, 7b, 8b, and 15–17), all of the com-
pounds possessing a 6,8-dihydroxy substituted tetrahydroisoquinoline
half such as ancistrobrevine E (7a) (PC50, 35.3 μM) and its atropo-
diastereomer 7b (PC50, 45.0 μM) displayed only weak to moderate
preferential cytotoxic effects. This finding complements results pre-
viously obtained for 5,8′-linked naphthylisoquinoline alkaloids
[12,35,72], among them korupensamine A and its 5-epimer kor-
upensamine B (PC50, 110.1 and 94.9 μM, respectively) [12], yet both
with R-configuration at C-1 and C-3. Thus, the oxygenation patterns of
the isoquinoline portions seem to have a distinct impact on the cyto-
toxic potential of naphthylisoquinoline alkaloids, more than e.g., the
configuration at C-3 in the isoquinoline part. This influence was clearly
recognizable from the strong preferential activity of 5-epi-ancistectorine
A2 (3) (PC50, 11.5 μM) with its mixed 6-OMe/8-OH substituted tetra-
hydroisoquinoline part as compared to the strongly (by a factor of al-
abbreviatus, viz., the alkaloids 15, 18, 19, 21, and 22. All of these
naphthylisoquinoline alkaloids displayed good to moderate activities
against the NF54 strain of Plasmodium falciparum (sensitive to all known
drugs), the most potent compounds were the 5,1′-coupled alkaloids
ancistrobrevines E (7a) and F (8a), their atropo-diastereomers 7b and
8b, and ancistrobrevine G (9), exhibiting half-maximum inhibitory
concentrations (IC50) of 0.213 μM (for 7a), 0.663 μM (for 7b), 0.928 μM
(for 8a), 0.846 μM (for 8b), and 0.134 μM (for 9). Their inhibitory
potentials, however, were less efficient than that of 5-epi-ancistectorine
A2 (3) (Table 5), which had already attracted attention as a promising
lead compound according to the TDR/WHO guidelines [63], due to its
excellent activity against the chloroquine-resistant K1 strain of P. fal-
ciparum (IC50, 0.03 μM) and a likewise high selectivity index of > 3000
[22]. That alkaloid 3, earlier discovered in the Chinese liana A. tectorius
[22], and here isolated from the roots of A. abbreviatus, too, was now
tested for its activity against the NF54 strain of P. falciparum: It ex-
hibited a high inhibitory potential in vitro (IC50, 0.0074 μM) and was
found to be even about 8.5 times more active than the standard
chloroquine (IC50, 0.065 μM). Moreover, 5-epi-ancistectorine A2 (3)
showed an excellent selectivity index of ca. 12,700, displaying only
extremely weak cytotoxic effects against L6 cells (Table 5).
most 4) reduced cytotoxicity of 5-epi-ancistrobrevine E (7b) (PC50
,
45.0 μM), which is equipped with two free hydroxy groups at C-8 and C-
6. The free hydroxy function at C-8 obviously plays a crucial role for the
cytotoxic potential of 5,1′-coupled naphthylisoquinoline alkaloids,
since hamatine (6b) (PC50, 35.6 μM), with its 6-OH/8-OMe substitution
pattern, was found to be about three times less active than compound 3.
A similar effect was observed for the 7,8′-linked ancistrobrevine A (21)
(PC50, 19.0 μM), which showed a more than two times stronger
The results presented for 5-epi-ancistectorine A2 (3) and the
256

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Fitoterapia131(2018)245–259
otherwise rare 7,8′-coupled naphthylisoquinoline alkaloids (six com-
pounds). Four new representatives of this coupling type have now been
discovered, among them the only fourth and fifth examples of 7,8′-
linked naphthyldihydroisoquinolines at all, viz., ancistrobrevines I (13)
and J (14).
Simultaneously, A. abbreviatus is a rewarding source for the search
of new potent bioactive compounds. Some of the metabolites displayed
promising activities against the malaria parasite Plasmodium falciparum,
while other constituents were found to exhibit pronounced preferential
cytotoxicities against PANC-1 human pancreatic cancer cells. The rapid
development of resistance against conventional drugs is a major ob-
stacle both in the management and global control of malaria and in the
fight against cancer. The promising antiplasmodial activities and the
strong preferential cytotoxic effects against tumor cells exerted by some
of the naphthylisoquinoline alkaloids warrant further investigations on
this intriguing class of compounds in general. This work is currently in
progress.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(SFB 630 “Agents against Infectious Diseases”, project A2), and by a
grant of the German Academic Exchange Service (Deutscher
Akademischer Austauschdienst, DAAD) to SF. The assessment of the
antiausterity activities was supported by a grant from the Japanese
Society for the Promotion of Science (Japan, Kakenhi, 16K08319) to
SA. We thank Dr. D. Tshitenge Tshitenge for fruitful discussions and for
experimental assistance (ECD spectroscopy). Furthermore, we ac-
knowledge experimental assistance from Dr. M. Büchner and Mrs. J.
Adelmann (MS), and from Dr. M. Grüne and Mrs. P. Altenberger (NMR).
Fig. 9. Preferential cytotoxic activity of (A) 5-epi-ancistectorine A2 (3) and (B)
6-O-methyl-4′-O-demethylhamatine (16) against the PANC-1 human pancreatic
cancer cell line in nutrient-deprived medium (NDM) and Dulbecco's modified
Eagle medium (DMEM).
cytotoxicity as compared to its 6-O-demethyl analog 22 (PC50
,
41.1 μM).
The test results likewise indicated that the degree of O-methylation
in the naphthalene subunit might be of importance for the anti-
proliferative effect of naphthylisoquinoline alkaloids, too: 6-O-
Methylhamatine (15) (PC50, 42.0 μM), with two methoxy functions at
C-5′ and C-4′, was about six times less active than its 4′-O-demethyl
analog 16 (PC50, 7.5 μM) (see Table 6). The nearly two times higher
cytotoxicity of 6-O-demethylancistrobrevine H (12) (PC50, 19.0 μM)
compared to that of its N-methylated analog 6-O-demethylancistro-
brevine A (22) (PC50, 41.1 μM) suggested that N-methylation of the
alkaloids might also have a negative impact on the cytotoxic potential
of naphthylisoquinoline alkaloids.
In conclusion, by this and previous phytochemical work [6,38–42],
A. abbreviatus has been demonstrated to produce a broad spectrum of
naphthylisoquinoline alkaloids. So far, about 30 such biaryl natural
products have been isolated from the roots and stem bark of this plant.
Among the main metabolites are five Dioncophyllaceae-type com-
pounds [6,38,41] related to dioncophylline A (1) (Fig. 1), lacking an
oxygen function at C-6 and with R-configuration at C-3, and, in parti-
cular, representatives of the Ancistrocladaceae type (22 compounds)
[6,38,39,42], i.e., 3S-configured alkaloids with a methoxy or hydroxy
group at C-6. Thus, from the chemotaxonomic point of view, this West
African liana significantly differs from many Central African Ancis-
trocladus species, which are characterized by the presence of a high
number of naphthylisoquinoline alkaloids belonging to the subclass of
hybrid-type alkaloids (i.e., oxygenated at C-6 and R-configured at C-3),
and by the fact that 5,8′-coupled compounds are the most common ones
[6–9,11–16,31,32]. On the contrary, as presented in this paper, a
striking feature of the metabolite pattern of A. abbreviatus is the pre-
dominant occurrence of 5,1′-coupled Ancistrocladaceae-type naphthy-
lisoquinoline alkaloids (twelve alkaloids), among them most prominent
representatives such as ancistrocladine (6a) and its atropo-diastereomer
hamatine (6b) [6,38], and, as described here for the first time, a series
of five different alkaloids related to 6a and 6b, yet all of them equipped
with the same 6,8-dihydroxy substituted tetrahydroisoquinoline por-
tion. Likewise remarkable in A. abbreviatus is the occurrence of
Appendix A. Supplementary data
Supplementary data to this article comprising NMR (1H, 13C, DEPT,
1H,1H-COSY, HSQC, HMBC, and NOESY) and HRESIMS and ECD
spectra of 7a, 7b, 8a, 8b, and 9-14, including GC-MSD chromatograms
after oxidative degradation of the alkaloids and Mosher derivatization
can be found online at https://doi.org/10.1016/j.fitote.2018.11.006.
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