Synthesis and biological evaluation of a D-ring-contracted analogue of lamellarin D

Article abstract of DOI:10.1016/j.bmc.2017.02.005

A D-ring contracted analogue of the strongly cytotoxic marine pyrrole alkaloid lamellarin D was synthesized and investigated for its antiproliferative action towards a wild type and a multidrug resistant (MDR) cancer cell line. The compound was found to inhibit tumor cell growth at submicromolar concentrations and showed a lower relative resistance in the MDR cell line than the antitumor drug camptothecin to which lamellarin D shows cross resistance and with which lamellarin D shares the same binding site.

Full text of DOI:10.1016/j.bmc.2017.02.005

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a D-ring-contracted analogue
of lamellarin D
Vanessa Colligs a, Steven Peter Hansen a, Dennis Imbri a, Ean-Jeong Seo b, Onat Kadioglu b,
Thomas Efferth b, , Till Opatz a,
⇑
⇑
a Institute of Organic Chemistry, Johannes Gutenberg-University, Duesbergweg 10–14, 55128 Mainz, Germany
b Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University, Staudinger Weg 5, 55128 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A D-ring contracted analogue of the strongly cytotoxic marine pyrrole alkaloid lamellarin D was synthe-
sized and investigated for its antiproliferative action towards a wild type and a multidrug resistant (MDR)
cancer cell line. The compound was found to inhibit tumor cell growth at submicromolar concentrations
and showed a lower relative resistance in the MDR cell line than the antitumor drug camptothecin to
which lamellarin D shows cross resistance and with which lamellarin D shares the same binding site.
Ó 2017 Elsevier Ltd. All rights reserved.
Received 1 November 2016
Revised 15 January 2017
Accepted 2 February 2017
Available online xxxx
Keywords:
Alkaloid synthesis
Transition metal catalysis
Natural product mimetics
Antitumor activity
Molecular docking
1. Introduction
between Lam-D and the natural product camptothecin (CPT, II),
another well-known topoisomerase-I poison, suggests Lam-D to
The lamellarins are a class of polycylic pyrrole alkaloids of mar-
ine origin. More than 50 representatives have been isolated and
characterized to date and strong biological activities have been
encountered. In particular, strong antiproliferative activities have
been observed in a subclass of the lamellarins, characterized by a
common 6H-chromeno[40,30:4,5]pyrrolo[2,1-a]isoquinolin-6-one
core. The most potent member of this subclass, the so-called
type-Ib lamellarins, is lamellarin D (Lam-D, I, Fig. 1) which was first
isolated from a prosobranch mollusk of the genus Lamellaria by
Faulkner and Clardy et al. in 1985.1
Lam-D was intensively investigated because of its strong cyto-
toxicity against various tumor cell lines and it was found to be a
potent inhibitor of human topoisomerase I (topo I).2 Recently, the
inhibition of mitochondrial topoisomerase (Top1mt) by Lam-D
was postulated to be responsible for the observed direct action
of the alkaloid on mitochondria.3,4 Several analogues of Lam-D
have been synthesized in order to improve its biological activity
or its poor water solubility.5,6 Although no crystal structure of
the Lam-D-topoisomerase I complex exists, the cross-resistance
address the same binding site. Bailly, Gago, and coworkers have
proposed a binding mode for Lam-D to topo I based on (a) struc-
tural similarities between Lam-D and CPT and (b) the known
crystal structure of the CPT-complex with topo I and DNA.7 We
became interested in the synthesis of structural analogues of
Lam-D as well as in molecular docking approaches to simulate
the binding behavior of these analogues to their biological target
structure. Herein, the synthesis and biological evaluation of a D-
ring contracted analogue of lamellarin D is presented along with
the in silico analysis of its potential binding mode to human
topoisomerase I.
2. Synthesis of a ring-contracted lamellarin D analogue
a
-Aminonitriles are interesting compounds which provide a
variety of different reactivities and can serve as versatile
starting materials for the synthesis of diverse nitrogen-contain-
ing products.8–10 If an aromatic, heteroaromatic or olefinic
a-substituent is present, they can be deprotonated quantita-
tively in
a-position even though one or two free NH-protons
are present.11 This methodology has successfully been applied
to the synthesis of the lamellarin core in an extended von
Miller-Plöchl-type pyrrole synthesis.12–15 The reaction of
⇑
Corresponding authors.
E-mail addresses: efferth@uni-mainz.de (T. Efferth), opatz@uni-mainz.de
(T. Opatz).
http://dx.doi.org/10.1016/j.bmc.2017.02.005
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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2
V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
O
O
HO
BnBr, K2CO3, MeOH,
HO
BnO
A
B
O
96 h, reflux
N
C
N
N
O
O
3
MeO
23
10
MeO
MeO
87%
O
D
1
2
11
1
17
MeO
F
19
16
E
CH3NO2, NH4OAc,
AcOH, reflux
69%
HO
HO
O
OH
MeO
LiAlH4, THF, Et2O,
2 h, reflux
II
BnO
I
BnO
NO2
camptothecin
lamellarin D
NH2
91%
MeO
MeO
4
3
Fig. 1. Structure of lamellarin D and camptothecin.
EtOCOH,
24 h, reflux
quant.
BnO
BnO
MeO
PCl5, DCM, 0.5 h, rt
HN
O
R1
R1
KHMDS
N
73%
MeO
NH
NH
CN
5
6
H
BnO
NC
H
IV
KCN, MeOH,
H2O, 16 h, rt
III
CHO
NH
CN
MeO
69%
7
R2
R3
V
Scheme 2. Synthesis of the northern fragment 7.
R1
R1
N
N
− H2O
− HCN
NC
OH
R2
R2
O
O
R3
R3
VII
VI
BnBr, K2CO3, MeOH,
48 h, reflux
MeO
BnO
MeO
HO
Scheme 1. Synthesis of the pyrrolo[2,1-a]isoquinoline core using deprotonated a-
97%
aminonitriles.
9
8
BnBr, K2CO3, acetone,
4 h, reflux
MeO
BnO
MeO
deprotonated 1,2,3,4-tetrahydoisoquinoline-1-carbonitriles IV
with -arylcinnamaldehydes V furnishes pyrrolo[2,1-a]isoquino-
lines VII in a sequence of vinylogous addition, cyclodehydration
CN
CN
a
quant.
HO
11
10
OMe
and dehydrocyanation (Scheme 1).
If R3 is an ortho-halo aryl moiety, a five-membered ring ketone
can be formed through a cyclocarbonylation reaction on the
unsubstituted position on the pyrrole ring. This reaction proved
to be a synthetic dead end in the attempted synthesis of lamellar-
ins in a Bayer-Villiger ring expansion which did not provide the
desired lactone structure.16 However, the resulting 8H-indeno
[10,20:4,5]pyrrolo[2,1-a]isoquinolin-8-one system has a slightly
lower molecular curvature than the lamellarin core making it more
similar to the CPT core. Although the basicity of the carbonyl
oxygen in 8H-indeno[10,20:4,5]pyrrolo[2,1-a]isoquinolin-8-ones
should be lower compared to Lam-D in which the sp3-hydridized
ring oxygen acts as an additional electron donor, the compound
can provide a similar H-bond donor/acceptor pattern as its natural
analogue. Moreover, 8H-indeno[10,20:4,5]pyrrolo[2,1-a]isoquino-
lin-8-ones are also flat which has been recognized as a prerequisite
for the biological activity of the type-Ib lamellarins.17 They could
thus also act as topo-I inhibitors. To investigate this issue, suitable
precursors were synthesized and an ortho-iodo substituent
BnO
NIS, TFA, MeCN,
16 h, rt
94%
9
, NaOMe, EtOH,
7 h reflux
MeO
X
CN
I
74%
I
BnO
12
DIBAL-H, DCM,
1.) 0.5 h, −78 °C,
2.) 2.3 h, rt
13: X = CN
14: X = CHO
MeO
OBn
75%
Scheme 3. Synthesis of the southern fragment 14.
reduced with DIBAL-H to aldehyde 14 which was obtained in
52% yield over four linear steps (Scheme 3).
The building blocks 7 and 14 were joined in an extended von
Miller-Plöchl-type pyrrole synthesis earlier developed by us to fur-
nish pyrrole 15 in 44% yield. Cyclocarbonylation was effected
according to a protocol by Larock18 using Pd(dba)2, PCy3ꢀHBF4
and caesium pivalate with CO at ambient pressure in 96% yield
(Scheme 4).
The dehydrogenation of the B-ring proved to be cumbersome
and required considerable optimization. Tetrachloro-o-benzo-
quinone (TBQ) turned out to be the only oxidant which permitted
the aromatization of compound 16 in appreciable yield while the
more frequently used quinone-type reagents such as DDQ or chlo-
ranil failed. To obtain Lam-D analogue 18, the resulting 8H-indeno
[10,20:4,5]pyrrolo[2,1-a]isoquinolin-8-one 17 was debenzylated
with boron trichloride. The compound was subjected to biological
evaluation and its binding mode to topoisomerase I was analyzed
in silico.
was
introduced
to
facilitate
the
palladium-catalyzed
cyclocarbonylation.
The synthesis of the AB-ring fragment started from isovanillin
(1) which was O-benzylated and converted to 2-(3-benzyloxy-4-
methoxy)phenylethylamine (4) through condensation with
nitromethane and LiAlH4-reduction. N-Formylation, Bischler-
Napieralski-cyclization, and HCN-addition to the resulting 3,4-
dihydroisoquinoline furnished bicyclic aminonitrile 7 in 28% yield
over six synthetic steps (Scheme 2).
The enal component 14 was synthesized starting from commer-
cial (4-hydroxy-3-methoxy)phenylacetonitrile (10) by O-benzyla-
tion and iodination using the NIS/TFA-system. Condensation with
O-benzylvanillin (9) leads to 2-arylcinnamonitrile 13 which was
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V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Table 1
BnO
MeO
1. KHMDS, THF,
3 min, 78 °C
IC50 values (mean SD) of camptothecin and compound 18 for CCRF-CEM and CEM/
ADR5000 cells as assayed by the resazurin reduction assay.
BnO
MeO
−
N
NH
CN
Compound
Camptothecin 0.003 0.0004 mM 0.011 0.003 mM 3.7
18 0.113 0.04 mM 0.172 0.01 mM 1.5
CCRF-CEM
CEM/ADR5000
Relative resistance
2. 14, 0.5 h, rt
3. AcOH, EtOH,
2 h, reflux
I
MeO
BnO
7
44%
OBn
15 MeO
CsOPiv, Cy3PHBF4 (20 mol%),
Pd(dba)2 (10 mol%), CO (1 bar),
DMF, 24 h, 110 °C
4. Cell cycle analysis
96 %
In order to investigate the effect of camptothecin and com-
pound 18 on the cell cycle, CCRF-CEM cells were treated with sol-
vent (DMSO) or the two drugs at various concentrations. Upon
camptothecin treatment, cells initially accumulated in the S phase
after 6 h. Subsequently, the percentage of G2/M phase increased in
a dose-dependent manner after 24 h. Finally, we observed apopto-
sis induction after 72 h camptothecin treatment (Fig. 3). Upon
treatment with compound 18, cells did not display S phase or
G2/M arrests, unlike camptothecin. However, apoptosis was
induced after 24 h and 72 h and the percentage of sub-G1 cells
increased after 72 h treatment with compound 18 (Fig. 4).
TBQ,
BnO
BnO
MeO
MeO
Pyridine,
3 h, µW
O
N
O
N
MeO
(150 °C,
150 W)
MeO
BnO
53%
OBn
OBn
BnO
17
OMe
OMe
16
HO
BCl3,DCM
1. 0.5 h, −78 °C
2. 3 h, 0 °C
N
O
MeO
94%
5. Discussion
MeO
HO
OH
The well-known DNA topoisomerase I inhibitor camptothecin
was used as control drug in the cytotoxicity assays. Multidrug-
resistant tumor cells are known to exert cross-resistance to camp-
tothecin and other DNA topoisomerase I inhibitors.21–23 We mea-
18
OMe
Scheme 4. Synthesis of lamellarin D-analogue 18.
sured
a 3.7-fold cross-resistance of CEM/ADR5000 cells to
3. Cytotoxic effect of camptothecin and compound 18 on CCRF-
CEM and CEM/ADR5000 cells
camptothecin. P-glycoprotein expressing CEM/ADR5000 exhibit
high degrees of resistance to anthracyclines, Vinca alkaloids, tax-
anes and other anticancer drugs.24 The low degree of resistance
to camptothecin indicates that this compound may be only a weak
substrate of P-glycoprotein and that other mechanisms may also
be operative in CEM/ADR5000 cells. Indeed, these multidrug-resis-
tant cells reveal down-regulated DNA topoisomerase I compared to
parental, drug-sensitive CCRF-CEM cells (unpublished data). Com-
pound 18 did not or only very weakly provoke cross-resistance in
CEM/ADR5000 cells (1.5-fold), indicating that it may be a poor or
no substrate of both P-glycoprotein and DNA topoisomerase I. This
result is comparable to a previous report on the parent compound
lamellarin D and further supports the similarity of Lam-D and its
ring-contracted analogue 18.25
The effects of CPT and lamellarin D analogue 18 towards sensi-
tive CCRF-CEM and multidrug-resistant (MDR) CEM/ADR5000
cells, which overexpress the MDR-conferring drug transporter P-
glycoprotein/MDR1,19,20 were evaluated. Camptothecin showed
IC50 values of 0.003 mM and 0.011 mM against CCRF-CEM and
CEM/ADR5000 cells, respectively. Compound 18 inhibited prolifer-
ation by 50% in CCRF-CEM and CEM/ADR5000 at concentrations of
0.113 mM and 0.172 mM, respectively (Fig. 2, Table 1). When com-
pared to CCRF-CEM cells, CEM/ADR5000 cells showed higher resis-
tance to camptothecin than to compound 18 (3.7- and 1.5-fold,
respectively) (Table 1).
Fig. 2. Cytotoxic effect of camptothecin (a) and compound 18 (b) against CCRF-CEM and CEM/ADR5000 cells. Resazurin reduction assays were performed to determine dose
response curve. Viability of CCRF-CEM and CEM/ADR5000 cells is represented by mean values SD of three independent experiments with each six parallel measurements
values are expressed as percentage survival of control.
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V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 3. Cell cycle analysis of CCRF-CEM cells after treatment with camptothecin. (a) Representative DNA content histograms of CCRF-CEM cells treated with increasing
concentrations of camptothecin for various incubation times. (b) Statistical analysis of cell cycle distribution of CCRF-CEM cells after treatment with different concentrations
of camptothecin for 6 h, 24 h and 72 h. The bar diagrams represent mean values of three independent experiments. The SD values which cannot be represented in this kind of
bar diagrams were in a range from 0.2 to 6.2% over all concentrations and incubation times analyzed.
Camptothecin arrested the cell cycle of CCRF-CEM cells in the S-
phase and G2/M-phase. This compares to results reported in the
literature.26 In contrast, compound 18 did not cause cell cycle
arrest, while both compounds induced apoptosis. The missing cell
cycle arrest is another hint that compound 18 may inhibit DNA
topoisomerase I to a weaker extent than camptothecin. In line with
this hypothesis are reports that lamellarins are low affinity DNA-
binding agents2 that interfere with several DNA-manipulating
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V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 4. Cell cycle analysis of CCRF-CEM cells after treatment with 18. (a) Representative DNA content histograms of CCRF-CEM cells treated with increasing concentrations of
18 for various incubation times. (b) Statistical analysis of cell cycle distribution of CCRF-CEM cells after treatment with different concentrations of 18 for 6 h, 24 h and 72 h.
The bar diagrams represent mean values of three independent experiments. The SD values which cannot be represented in this kind of bar diagrams were in a range from 0.3
to 9.8% over all concentrations and incubation times analyzed.
enzymes.27 Hence, other mechanisms in addition to topo-I inhibi-
tion may be taken into account to explain the cytotoxicity of com-
pound 18 towards tumor cells.
residue is located in the known topotecan binding site and is
hyphotesized to cause campthothecin resistance.28 Lamellarin D
and compound 18 addressed the same binding site and showed
predicted binding energies comparable to that of camptothecin.
Interestingly, the mutation did not affect the docking pose of both
lamellarin D and compound 18. However, the docking pose of
camptothecin was shifted when it was docked on the mutant pro-
tein instead of the wild type. This is in agreement with the hypoth-
esis that the mutation is related to camptothecin resistance. It can
be speculated that the missing asparagine side chain at position
5.1. Molecular docking
To analyze the binding properties of compound 18 to the DNA-
topoisomerase I complex in silico, two rounds of molecular docking
were performed on wild type and a mutant human topoisomerase I
(asparagine in position 722 mutated to serine). The mutated
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V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 5. Molecular docking analyses of lamellarin D (red) and camptothecin (blue) on wild type (violet) and mutant (orange) human topoisomerase I. (A) Blind docking result
for the wild type protein. (B) Blind docking result for the mutant protein. (C) Defined docking result for the wild type protein. (D) Defined docking result for the mutant
protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2
Molecular docking results of lamellarin D, its derivative and camptothecin on wild type and mutant (N722S) human topoisomerase I.
Wild type blind docking
LBE (kcal/mol)
pKi (nM)
interacting residues
residues forming H-bonds
camptothecin
lamellarin D
18
mutant blind docking
camptothecin
lamellarin D
18
–9.237 0.006
–9.263 0.006
–9.027 0.035
LBE (kcal/mol)
–9.497 0.006
–9.143 0.012
–9.333 0.015
168.757 1.607
161.907 0.868
TGP11, DC112, DA113, Asp533, Thr718
DT10, TGP11, DA113, Ala351, Asn352, Glu356, Arg364, Asn722
–
Asn352, Arg364, Asn722
242.257 14.209 DT10, TGP11, DC112, DA113, Aal351, Asn352, Glu356, Arg364, Asn722 Asn352, Arg364, Asn722
pKi (nM)
108.927 1.188
199.087 4.743
144.707 3.687
pKi (nM)
interacting residues
residues forming H-bonds
Asp533
Ser722
DA113, Ser722
residues forming H-bonds
Asp533
DT10, DG12, DC112, DA113, Arg364, Asp533, Thr718
DT10, DG11, DC112, DA113, Arg352, Arg364, Ser722, Lys751
DT10, DG11, DC112, DA113, Arg352, Arg364, Ser722, Lys751
interacting residues
wild type defined docking LBE (kcal/mol)
camptothecin
–
28.633 0.674
DT10, TGP11, DC112, DA113, Arg364, Asp533, Thr718
10.293 0.015
–9.960 0.026
–9.940 0.030
LBE (kcal/mol)
–9.937 0.031
–9.733 0.103
–9.713 0.025
lamellarin D
18
mutant defined docking
camptothecin
lamellarin D
18
50.140 2.102
51.723 2.383
pKi (nM)
52.303 2.826
74.003 13.263
75.957 3.532
DT10, TGP11, DC112, DA113, Asn352, Glu356, Arg364, Asn722
DT10, TGP11, DC112, DA113, Glu356, Arg364, Thr718, Asn722
interacting residues
DA15, DA16, DA17, DT18, DT19, DT107, DT108, DT109
DT10, DG11, DA113, Asn352, Glu356, Arg364
Asn352, Glu356, Asn722
Glu356
residues forming H-bonds
DA16
Arg352, Glu356, Arg364
DA113, Asn352, Ser722
DG11, DC112, DA113, Asn352, Arg364, Leu721, Ser722, Lys751
Fig. 6. Molecular docking analyses of compound 18 (red) and camptothecin (blue) on wild type (violet) and mutant (orange) human topoisomerase I. (A) Blind docking result
for the wild type protein. (B) Blind docking result for the mutant protein. (C) Defined docking result for the wild type protein. (D) Defined docking result for the mutant
protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
residue might be critical for its binding. Lamellarin D and its ana-
logue 18 were not affected by the mutation and can still bind with
similar strength at the topotecan binding site. Camptothecin is pre-
dicted to bind slightly stronger (ꢁ10.293 0.015 kcal/mol) than
chemical shifts are references to the (residual) solvent signal and
are expressed in ppm downfield from tetramethylsilane. CDCl3
(1H: d = 7.26 ppm; 13C: d = 77.16 ppm); DMSO-d6 (1H:
d = 2.50 ppm; 13C: d = 39.52 ppm). NMR
HPLC-MS-and ESI-MS spectra were recorded on an Agilent 1200
series HPLC connected to a Bruker LC/MSD-Trap spectrometer.
Sample concentrations amounted to 0.1 mg/mL in H2O/MeCN
1:1, the mass range was set to 150–900 u. An Ascentis Express
lamellarin
D
(ꢁ9.960 0.026 kcal/mol) and its derivative
(ꢁ9.940 0.030 kcal/mol) for the docking on wild type topoiso-
merase I. Regarding the mutant topoisomerase I docking run, pre-
dicted binding affinity of camptothecin (ꢁ9.937 0.031 kcal/mol)
was lower than that of wild type topoisomerase I docking run.
C18 column (30 ꢂ 2 mm, Supelco) with 2.7
lm particle size was
Lamellarin
(ꢁ9.713 0.025 kcal/mol)
D
(ꢁ9.733 0.103 kcal/mol) and its derivative
used. The dry temperature was set to 350 °C, the nebulizer pres-
sure to 60 psi and the dry gas flow to 11 L/min. The capillary exit:
voltage was set to 137.5 V. HR-ESI-MS analyses were performed on
a Water Q-TOF Ultima-III instrument equipped with a LockSpray
interface and a suitable external calibrant. For the calibration,
exact masses of electroneutral species were used, the exact masses
(calculated and recorded) of the actual cationic species are
5.5 ꢀ 10ꢁ4 Da lower. Melting point ranges were recorded on a Krüss
Optronic electrothermal apparatus, IR spectra were recorded on a
Bruker Tensor 27-FT-IR spectrometer using a diamond ATR unit.
Reactions accelerated by microwave heating were performed in
a Discover monomode apparatus from CEM in glass vials sealed
with septa under monitoring of temperature (IR sensor) and
pressure.
bound slightly weaker than
campthothecin which is in agreement with the literature data. It
should however be noted that these values are only indications
and an exact prediction of binding affinities is still impossible.
Docking results for lamellarin D are summarized in Fig. 5 and
Table 2 while docking results for compound 18 are summarized
in Fig. 6 and Table 2.
6. Conclusion
The D-ring contracted analogue of lamellarin D, compound 18,
was synthesized and evaluated for its antiproliferative activity in
a system of a susceptible (CCRF-CEM) and a resistant tumor cell
line (CEM/ADR5000). While the absolute potency was lower than
that of the antitumor drug camptothecin, compound 18 was less
susceptible to the drug resistance mechanism of the CEM/
ADR5000 cell line. Molecular docking suggests that compound 18
occupies the same binding site as camptothecin on human topoiso-
merase I and in contrast to the latter, it should be less influenced
by the N722S mutation of this enzyme.
7.1.1. Cell culture
Human CCRF-CEM leukemia and multi-drug-resistant CEM/
ADR5000 cells were kindly provided by Prof. Axel Sauerbrey
(University of Jena, Jena, Germany). The original source of these
cell lines is the ATCC. Cells were maintained in RPMI 1640 contain-
ing 100 units/mL penicillin and 100 mg/L streptomycin and sup-
plemented with heat-inactivated 10% fetal bovine serum (FBS), in
a humidified environment at 37 °C with 5% CO2. To maintain the
multidrug-resistance phenotypes, the MDR1-expressing CEM/
ADR5000 cells were treated with 5000 ng/mL doxorubicin.
7. Experimental section
7.1. Materials and methods
7.1.2. Resazurin reduction assay
All air and moisture sensitive reactions were performed in
flame-dried glassware under an argon atmosphere. THF was dried
over potassium/benzophenone and distilled under argon, ethanol
was dried with the aid of sodium and diethyl phthalate. Dichloro-
methane was distilled from CaH2 under argon. DMF and pyridine
were purchased in predried quality (over molecular sieves) from
Acros. Removal of solvents in vacuo was performed using a rotary
evaporator (40 °C bath temperature) and a membrane pump. CDCl3
was stored over basic alumina. All other chemicals were obtained
from commercial suppliers and were used without further
purifications.
Resazurin reduction assay was used to investigate cytotoxicity
of camptothecin and compound 18 towards CCRF-CEM and CEM/
ADR5000 cells.29 The assay is based on reduction of the indicator
dye, resazurin, to the highly fluorescent resazurin by viable cells.
Non-viable cells rapidly lose the metabolic capacity to reduce resa-
zurin and thus do not produce a fluorescent signal. The investiga-
tion of cytotoxic compounds using the resazurin assay and CCRF-
CEM and CEM/ADR5000 cells has been described by us.30,31 Briefly,
10,000 cells were placed in each well of a 96-well cell culture plate
(Thermo Scientific, Dreieich, Germany) in a total volume of 100
Cells were attached overnight and then were treated with different
concentrations of drugs. After 72 h incubation, 20 L resazurin
lL.
For analytical TLC, silica-coated aluminium plates (E. Merck, sil-
ica 60 F254) were used. Substance bands were visualized by UV-
light or with the following reagents: DNPH reagent for detection
of aldehydes: 2,4-Dinitrophenylhydrazine (1.0 g) in ethanol
(25 mL), water (8 mL) and sulfuric acid (5 mL). Ninhydrin reagent
for the detection of amines: Ninhydrin (0.3 g) in acetic acid
(3 mL) and 1-butanol (100 mL). Ehrlich reagent for pyrroles: Pre-
pared by freshly mixing equal volumes of a 2% ethanolic solution
of 4-(dimethylamino)benzaldehyde and 20% hydrochloric acid.
For flash chromatography, silica with 35–70 mm particle size
from Acros was used. The eluents were purchased in technical
quality and were purified by distillation prior to use. Alternatively,
compounds were purified with the aid of a semiautomatic Biotage
Isolera Flashmaster with integrated UV detector and silica
cartridges.
l
(Sigma-Aldrich, Taufkirchen, Germany) 0.01% w/v in ddH2O was
added to each well and the plates were incubated at 37 °C for
4 h. Fluorescence was measured by an Infinite M2000 Proplate
reader (Tecan, Crailsheim, Germany) using an excitation wave-
length of 544 nm and an emission wavelength of 590 nm. Each
experiment was performed three times, with six replicates each.
The viability was analyzed based on a comparison with untreated
cells. IC50 values indicate the drug concentrations required to inhi-
bit 50% of cell proliferation and were calculated from a calibration
curve by linear regression using Microsoft Excel.
7.2. Cell cycle analysis by flow cytometry
For cell cycle analysis, CCRF-CEM cells were treated with vari-
ous concentrations of camptothecin and compound 18 for 6 h,
24 h, and 72 h. Afterwards, cells were washed by PBS and fixed
in ice-cold 95% ethanol (Sigma-Aldrich). Fixed cells were washed
with PBS and were incubate with 50 mg/mL propidium iodide (PI,
NMR spectra were recorded on a Bruker AC300 (300 MHz for 1H
NMR and 75.5 MHz for 13C NMR) or on a Bruker Avance-II 400
(400 MHz for 1H NMR and 100.6 MHz for 13C NMR, including 2D
NMR) using 5 mm probes and standard pulse sequences. The
Please cite this article in press as: Colligs V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.005

8
V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Carl-Roth, Karlsruhe, Germany) in PBS for 1 h in the dark. Cells
were measured by means of a BD FACS Calibur flow cytometer
(Becton-Dickinson, Heidelberg, Germany). 1 ꢂ 104 cells were
counted for each sample. PI was measured with 488 nm excitation
(100 mW) and detected using a 610/20 nm band pass filter. Cyto-
graphs were analyzed using FlowJo software (Celeza, Olten,
Switzerland). All experiments were performed three times.
with DCM (3 ꢂ 10 mL). The combined organic layers were washed
with water (2 ꢂ 10 mL), brine (2 ꢂ 10 mL) and dried over Na2SO4.
The solvent was removed in vacuo, the residue was recrystallized
from ethanol. The product was obtained as bright yellow needles
(813 mg, 2.85 mmol, 69%).
Rf = 0.53 (cyclohexane/ethyl acetate = 3:2).
Mp: 127–128 °C.
1H NMR (300 MHz, CDCl3): d = 7.91 (d, 3J = 13.6 Hz, 1H,
CH = CH-NO2), 7.49–7.37 (m, 5H, C6H5), 7.37–7.31 (m, 1H,
CH = CH-NO2), 7.18 (dd, 3J = 8.4 Hz, 4J = 2.0 Hz, 6-H), 7.03 (d,
4J = 2.0 Hz, 1H, 2-H), 6.93 (d, 3J = 8.4 Hz, 1H, 5-H), 5.18 (s, 2H, O-
CH2-C6H5), 3.95 (s, 3H, –OCH3) ppm.
7.2.1. Molecular docking
The protocol for molecular docking was previously reported by
us.32 An X-ray crystallography-based structure of wild-type human
topoisomerase I (PDB ID: 1K4T) and its N722S mutant (PDB ID:
1RRJ) were obtained from Protein Data Bank (http://www.rcsb.
org/pdb). This mutation was hypothesized to cause camptothecin
resistance.33 Bound topotecan was deleted from the protein struc-
tures. Both structures carry phosphorylated tyrosine at the posi-
tion of 723. The selected residues of human topoisomerase I for
the defined docking were obtained from a topotecan docking pose
on human topoisomerase I and the interacting residues34; DA5,
DG6, DA7, DC8, DT9, DC117, DT118, DT119, Arg364, Lys532,
Asp533, Asn722, Tyr723. A grid box was then constructed to define
docking spaces in each protein according to the selected residues
for the defined docking and whole protein surface was covered
for the blind docking. Docking parameters were set to 250 runs
and 2,500,000 energy evaluations for defined docking, 25,000,000
for blind docking. Docking was performed three times indepen-
dently by Autodock4 and with AutodockTools-1.5.7rc135 using
the Lamarckian algorithm. The corresponding lowest binding ener-
gies and predicted inhibition constants were obtained from the
docking log files (dlg). Mean SD of binding energies were calcu-
lated from three independent docking. Visual Molecular Dynamics
(VMD) was used to depict the docking poses of camptothecin,
lamellarin D and compound 18.
13C NMR (101 MHz, CDCl3): d = 153.6 (CAr), 148.7 (CAr), 139.4,
136.4 (C-10), 135.2, 128.8 (C-30, C-50), 128.3 (C-40), 127.4 (C-20, C-60),
125.0, 122.8, 113.3, 111.9, 71.3 (CH2-O), 56.2 (-OCH3) ppm.
The spectroscopic data matched those reported in the
literature.38
7.3.3. 2-[3-(Benzyloxy)-4-methoxyphenyl]ethylamine (4)
The title compound was prepared according to a modified pro-
cedure of Bermejo et al.37 To a suspension of LiAlH4 (5.65 g,
149 mmol, 3.8 eq.) in diethyl ether (226 mL), a solution of nitros-
tyrene 3 (11.24 g, 39.4 mmol) in dry THF (157 mL) was added
dropwise, under vigorous stirring. The solution was heated to
reflux for 2 h. Once it was cooled to 0 °C and diluted with water
(5.7 mL). After the addition of 15% NaOH-solution (5.7 mL) and
water (17.1 mL), the solution was stirred for another hour at room
temperature. The mixture was filtered under suction. The remain-
ing colourless solid was washed with DCM. The filtrate was
obtained as colourless liquid which was washed with saturated
NaHCO3-solution (1 ꢂ 100 mL), water (2 ꢂ 100 mL) and brine
(2 ꢂ 100 mL). The solution was dried over Na2SO4 and the solvent
was removed in vacuo. After complete evaporation the product
remained as a light brown oil (9.27 g, 36.0 mmol, 91%)
7.3. Synthetic procedures and compound characterization
Rf = 0.24 (DCM/MeOH = 20:1, 1% of NEt3)
1H NMR (300 MHz, CDCl3): d = 7.49–7.28 (m, 5H, C6H5), 6.87–
6.77 (m, 3H, HAr), 5.17 (s, 2H, O-CH2-C6H5), 3.88 (s, 3H, -OCH3),
2.89 (t, 3J = 6.8 Hz, 2H, CH2-CH2-NH2), 2.65 (t, 3J = 6.8 Hz, 2H,
CH2-CH2-NH2), 1.32 (br s, 2H, NH2) ppm.
7.3.1. O-Benzylisovanillin (2)
The title compound was prepared according a procedure of
Zhang et al.36 To a solution of isovanillin (1, 30.0 g, 197 mmol) in
methanol (238 mL) was added benzyl bromide (23.4 mL, 33.3 g,
197 mmol) and K2CO3 (32.9 g, 238 mmol). The mixture was heated
to reflux for 96 h and was filtered while still hot. The filtrate was
concentrated in vacuo and taken up in DCM (100 mL). The solution
was washed with dilute NaOH solution (1 M, 50 mL), water
(50 mL), and brine (50 mL), dried over Na2SO4 and the solvent
was removed in vacuo to yield the title compound as a light yellow
microcrystalline solid (41.4 g, 171 mmol, 87%).
13C NMR (101 MHz, CDCl3): d = 148.2 (Cq-Ar), 148.0 (Cq-Ar),
137.2 (C-10), 132.3 (C-1), 128.5 (C- 30, C-50), 127.8 (C-40), 127.4
(C-20, C-60), 121.5 (CAr), 115.1 (CAr), 112.0 (CAr), 71.1 (CH2-O-),
56.1 (-OCH3), 43.5 (CH2-NH2), 39.3 (CH2-CH2-NH2) ppm.
The spectroscopic data matched those reported in the
literature.38
7.3.4. 6-(Benzyloxy)-7-methoxy-3,4-dihydroisoquinoline (6)
Amine 4 (2.75 g, 10.8 mmol) and ethyl formate (2.86 mL, 2.63 g,
35.5 mmol, 3.3 eq.) were heated to reflux for 24 h. The remaining
ethyl formate was removed in vacuo. Formamide 5 was obtained
as a brown oil (3.08 g, 10.8 mmol, quant.) which was used in the
next step without further purification. The title compound was
prepared according to a modified procedure of J.C. Rohloff et al.39
PCl5 (4.49 g, 21.6 mmol, 1.2 eq.) was suspended in dry DCM
(4.3 mL) under vigorous stirring. A solution of formamide 5
(5.12 g, 18.0 mmol) in dry DCM (12 mL) was added with the
required speed to keep the solution boiling. The reaction mixture
was allowed to cool to room temperature and stirred for another
30 min. It was quenched with an ice/hexane-mixture (11 g in
4.5 mL) and the organic layer was separated and extracted with
HCl (0.01 N, 5 ꢂ 20 mL). To the combined aqueous layers, solid
KOH was added until pH 12 was reached. The solution was
extracted with DCM (5 ꢂ 20 mL). The solution was dried over Na2-
SO4 and evaporated to furnish the title compound as a yellow solid
(3.51 g, 13.1 mmol, 73%)
Rf = 0.47 (cyclohexane/ethyl acetate, 3:2).
Mp: 56–58 °C.
1H NMR (300 MHz, CDCl3): d = 9.82 (s, 1H, CHO), 7.49–7.45 (m,
5H, C6H5), 7.40–7.32 (m, 2H, 2-H, 6-H), 7.01–6.98 (d, 1H, 3J = 9 Hz),
5.19 (s, 2H, O-CH2-C6H5), 3.96 (s, 3H, -OCH3) ppm.
13C NMR (75.5 MHz, CDCl3): d = 191.0 (CHO), 155.2 (C-4), 148.8
(C-3), 136.4 (C-10), 130.1 (C-1), 128.8 (C-30, C-50), 128.3 (C-40), 127.6
(C-20, C-60), 127.0 (C-6), 111.5 (CAr), 110.9 (CAr), 71.0 (-O-CH2-),
56.3 (-OCH3) ppm.
The spectroscopic data matched those reported in the
literature.36
7.3.2. 3-Benzyloxy-4-methoxy-b-nitrostyrene (3)
The title compound was prepared according to a modified pro-
cedure of Bermejo et al.37 To a solution of O-benzylisovanillin (2,
1.00 g, 4.13 mmol) in 12.5 mL acetic acid, nitromethane (0.7 mL,
0.80 g, 13.1 mmol, 3.2 eq) and NH4OAc (0.80 g, 10.4 mmol, 2.5 eq)
were added. The solution was heated to reflux for 3 h. After cool-
ing, the solution was diluted with water (10 mL) and extracted
Mp: 102–103 °C
Please cite this article in press as: Colligs V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.005

V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
IR (ATR):
1117 cmꢁ1
m
= 3032, 3003, 2938, 2838, 1574, 1516, 1323, 1277,
127.3 (CH-2, CH-6, Ph), 126.7, 112.6, 109.5, 71.0 (OCH2Ph), 56.2
(OCH3) ppm.
.
1H NMR (400 MHz, CDCl3): d = 8.22 (t, J = 2.0 Hz, 1H, 1-H), 7.46–
7.30 (m, 5H, C5H6), 6.83 (s, 1H, 5-H), 6.69 (s, 1H, 8-H), 5.18 (s, 2H,
O-CH2-C6H5), 3.90 (s, 3H, -OCH3), 3.73–3.66 (pseudo–dt, 3Jd = 7.8,
Jt = 2.1 Hz, 2H, 3-H), 2.65–2.58 (pseudo-t, 3J = 7.8 Hz, 2H, 4-H) ppm.
13C NMR (101 MHz, CDCl3) d = 159.8 (-C=N), 150.5 (Cq-Ar),
148.5 (Cq-Ar), 136.7 (C-10), 129.8 (Cq-Ar), 128.8 (C-30, C-50), 128.2
(C-40), 127.3 (C-20, C-60), 122.1 (Cq-Ar), 112.8 (C-Ar), 111.1 (C-Ar),
71.1 (CH2-O-), 56.4 (-OCH3), 47.5 (CH2-N=C-), 24.8 (CH2-CH2-
N=C-) ppm.
The spectroscopic data matched those reported in the
literature.41
7.3.7. 4-(Benzyloxy)-3-methoxyphenylacetonitrile (11)
The title compound was prepared according to a modified pro-
cedure of Imbri et al.12 To
a solution of 4-hydroxy-3-
methoxyphenylacetonitrile (10, 1.08 g, 6.60 mmol, 1.0 eq.) in ace-
tone (14 mL), K2CO3 (1.10 g, 7.92 mmol, 1.2 eq.) was added. The
suspension was stirred for 10 min. Subsequently, benzyl bromide
(1.24 g, 7.26 mmol, 1.1 eq.) was added dropwise with a syringe.
The reaction mixture was heated to reflux for 4 h. The suspension
was filtered with suction the precipitated solid was washed with
acetone (10 mL). The filtrate was concentrated under reduced pres-
sure, the remaining solution was diluted with DCM (50 mL). The
organic layers were washed with 2 N NaOH (30 mL), brine
(30 mL) and dried over Na2SO4. The solvent was removed in vacuo
to furnish the title compound as a yellow solid (1.67 g, 6.59 mmol,
quant.).
ESI-MS: m/z (%) = 268.1 (100) [M + H]+.
ESI-HRMS: calculated for [C17H17NO2 + H]: m/z = 268.1338,
observed: 268.1333.
7.3.5. 6-(Benzyloxy)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-1-
carbonitrile (7)
The title compound was prepared according to a modified pro-
cedure of Werner et al.40 To 3,4-dihydroisoquinoline 6 (5.67 g,
21.2 mmol) dissolved in methanol (8 mL), a solution of KCN
(4.96 g, 76.2 mmol, 3.6 eq.) in water (24 mL) was added. The solu-
tion was cooled to 0 °C at this temperature aqueous HCl (1 M,
3 mL) was added with a syringe. The reaction was stirred for
another hour at this temperature. It was allowed to warm to room
temperature and stirred for another 16 h. Saturated NaHCO3-solu-
tion was added until pH 8 was reached. The aqueous layers were
extracted with DCM (5 ꢂ 10 mL). The solvent was removed in
vacuo to furnish the title compound as a brown oil (6.07 g
(20.6 mmol, 97%). In contrast to its 6,7-dimethoxy-analogue, com-
pound 7 undergoes spontaneous dehydrocyanation in solution (see
NMR spectra in the Supporting Information) and must be freshly
prepared.
Yield: 1.67 mg (6.59 mmol, 100 % Lit.).
Rf = 0.43 (cyclohexane/ethyl acetate, 2:1).
Mp: 65–66 °C.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 7.44–7.29 (m, 5H,
Ph), 6.86 (d, 3J = 8.2 Hz, 1H, 5-H), 6.84 (d, 4J = 2.1 Hz, 1H, 2-H), 6.78
(dd, 3J = 8.2 Hz, 4J = 2.1 Hz, 1H, 6-H), 5.15 (s, 2H, PhCH2O), 3.90 (s,
3H, OCH3), 3.68 (s, 2H, ArCH2CN) ppm.
13C NMR (100.6 MHz, CDCl3): d = 150.2 (Cq-3), 148.0 (Cq-4),
136.9 (Cq-1, Ph), 128.7 (CH-3, CH-5, Ph), 128.0 (CH-4, Ph), 127.3
(CH-2, CH-6, Ph), 122.8 (Cq-1), 120.3 (CH-6), 118.2 (CN), 114.4
(CH-2), 111.6 (CH-5), 71.2 (PhCH2O), 56.2 (OCH3), 23.3 (ArCH2CN)
ppm.
IR (ATR):
1216, 1192, 1101, 700.
m
= 3350, 2932, 2877, 2859, 2833, 1575, 1257, 1228,
The spectroscopic data matched those reported in the
literature.42
1H NMR (400 MHz, CDCl3): d = 7.43–7.31 (m, 5H, C6H5), 6.69 (s,
1H, 5-H), 6.63 (s, 1H,8-H), 5.13 (s, 2H, O-CH2-C6H5), 4.95 (s, 1H, 1-
H), 3.87 (s, 3H, -OCH3), 3.26–3.18 (m, 2H, H-3), 2.79 (pseudo-dt,
J = 9.8, 4.8 Hz, 1H, H-4), 2.64–2.58 (m, 1H, H-4) ppm.
13C NMR (75 MHz, CDCl3) d = 148.6 (Cq-Ar), 136.9 (C-10), 128.7
(C-30, C-50), 128.1 (C-40), 127.4 (C-20, C-60) 127.0 (Cq-Ar), 121.7 (Cq-
Ar), 120.4 (Cq-Ar), 114.7 (C-Ar), 110.1 (C-Ar), 71.1 (-CH2-O-), 56.3
(-OCH3), 48.2 (CH2-NH-), 40.9 (C-1), 27.9 (CH2-CH2-NH-) ppm.
ESI-MS: m/z (%) = 295 (20) [M + H]+, 268 (100) [M - HCN]+.
ESI-HRMS: calculated for [C18H18N2O2 + H]: m/z = 295.1447,
observed: 295.1451.
7.3.8. 4-(Benzyloxy)-2-iodo-5-methoxyphenylacetonitrile (12)
The title compound was prepared according to a modified pro-
cedure of Imbri et al.12
A solution of nitrile 11 (1.67 g, 6.60 mmol, 1.0 eq.), N-iodosuc-
cinimide (1.49 g, 6.60 mmol, 1.0 eq.) and trifluoroacetic acid
(1 mL, 1.5 eq.) in acetonitrile (25 mL) was stirred for 16 h at room
temperature. The solution was concentrated under reduced pres-
sure and poured on ice. The precipitated solid was filtered with
suction and washed with water (50 mL) and dried under fine vac-
uum. The product was obtained as
6.17 mmol, 94 %).
a yellow solid (2.34 g,
7.3.6. 4-(Benzyloxy)-3-methoxybenzaldehyde (9)
Rf = 0.49 (cyclohexane/ethyl acetate, 3:2).
The title compound was prepared according to a procedure of
Shen and Sun.41 To a solution of vanillin (8, 10.0 g, 65.8 mmol,
1.0 eq.) in methanol (40 mL), K2CO3 (10.9 g, 79.0 mmol, 1.2 eq.)
was added and the resulting suspension was stirred for 10 min at
room temperature. Benzyl bromide (9.38 mL, 79.0 mmol, 1.2 eq.)
was added dropwise and the reaction mixture was heated to reflux
for 48 h. The suspension was filtered with suction the filtrate was
concentrated under reduced pressure and diluted with DCM
(80 mL). The solution was washed with (2 ꢂ 30 mL) and dried over
Na2SO4. The solvent was removed under reduced pressure. The
remaining solid was dried in vacuo. The product was obtained as
a colourless solid (15.3 g, 63.6 mmol, 97%).
Mp: 118–119 °C.
IR (ATR):
m = 3031, 2857, 1594, 1507, 1378, 1257, 1218, 1020,
865, 738, 658 cmꢁ1
.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 7.44–7.32 (m, 5H,
Ph), 7.30 (s, 1H, 3-H), 7.02 (s, 1H, 6-H), 5.10 (s, 2H, OCH2Ph), 3.90 (s,
3H, OCH3), 3.76 (ArCH2CN) ppm.
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3): d = 150.6 (Cq-5),
148.6 (Cq-4), 136.2 (Cq-1, Ph), 128.8 (CH-3, CH-5, Ph), 128.4 (Cq-
4, Ph), 127.6 (CH-2, CH-6, Ph), 126.0 (Cq-1), 124.4 (CH-3), 117.7
(CN), 112.4 (CH-6), 86.9 (Cq-2), 71.5 (OCH2Ph), 56.3 (OCH3), 29.6
(ArCH2CN) ppm.
ESI-MS: m/z (%) = 402.0 (100) [M + Na]+.
Rf = 0.45 (cyclohexane/ethyl acetate, 3:2).
ESI-HRMS: Calculated for [C16H14INO2 + Na]: m/z = 401.9967,
observed: 401.9955.
Mp: 60–61 °C.
1H NMR (300 MHz, CDCl3): d = 9.84 (s, 1H, CHO), 7.45–7.31 (m,
7H, Ar), 6.99 (d, 3J = 8.2 Hz, 1H, 5-H), 5.25 (s, 2H, OCH2Ph), 3.95 (s,
3H, OCH3) ppm.
7.3.9. (2Z)-2-[4-(benzyloxy)-2-iodo-5-methoxyphenyl]-3-[4-
(benzyloxy)-3-methoxyphenyl]prop-2-enenitrile (13)
13C NMR (75.5 MHz, CDCl3): d = 191.0 (CHO), 153.7, 150.2, 136.2
(Cq-1, Ph), 130.5 (Cq-1), 128.9 (CH-3, CH-5, Ph), 128.4 (CH-4, Ph),
The title compound was prepared according to a protocol by
Waddell and Rapoport.43 (4-Benzyloxy)-3-methoxybenzaldehyde
Please cite this article in press as: Colligs V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.005

10
V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
(1.61 g, 6.70 mmol, 1.1 eq.) and nitrile 12 (2.31 g, 6.09 mmol,
1.0 eq.) were suspended in dry ethanol (70 mL) under argon atmo-
sphere. A suspension of sodium methoxide in methanol (5.4 M,
1.24 mL, 6.70 mmol, 1.1 eq.) was added. The resulting mixture
was heated to reflux for 7 h. The solution was cooled to room tem-
perature and the solvent was removed under reduced pressure. To
remove excess aldehyde from the reaction mixture, it was diluted
with methanol (50 mL). Acetic acid (6.5 mL), Girard reagent T
(511 mg, 3.05 mmol, 0.5 eq.) were added and heated to reflux for
2 h. The solution was washed with saturated NaHCO3-solution
(30 mL) and diluted with water (50 mL). Once the aqueous layers
were extracted with DCM (3 ꢂ 50 mL), the combined organic lay-
ers were dried over Na2SO4. The solvent was removed in vacuo.
The product was purified by automatic flash chromatography
(cyclohexane/ethyl acetate 4:1, Isolera Flashmaster, gradient: 10–
80% ethyl acetate). The product was obtained as colourless solid
(2.71 g, 4.49 mmol, 74%).
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3): d = 192.2 (CHO),
150.9 (Cq-50), 150.7 (Cq-400), 150.4 (CH-3), 149.3 (Cq-300), 148.6
(Cq-40), 141.9 (Cq-2), 136.4 (2C, Cq, Ph), 132.7 (Cq-10), 128.8 (4C,
C-3 C-5, Ph), 128.3 (C-4, Ph), 128.2 (C4, Ph), 127.5 (2C, C-2 C-6,
Ph), 127.3 (2C, C-2 C-6, Ph), 127.0 (Cq-100), 126.0 (CH-600), 124.2
(CH-30), 113.2 (CH-60), 112.9 (CH-500), 112.7 (Cq-200), 87.4 (Cq-20),
71.3 (40-OCH2Ph), 70.8 (400-OCH2Ph), 56.2 (50-OCH3), 55.5 (300-
OCH3) ppm.
ESI-MS: m/z (%) = 629.1 (100) [M + Na]+.
ESI-HRMS: Calculated for [C31H27IO5 + Na]: m/z = 629.0801,
observed: 629.0801.
7.3.11. 8-Benzyloxy-2-(4-benzyloxy-2-iodo-5-methoxyphenyl)-1-[4-
(benzyloxy)-3-methoxyphenyl]-9-methoxy-5,6-dihydropyrrolo[2,1-a]
isoquinoline (15)
The title compound was prepared according to a procedure of
Meyer at al.14 In an oven-dried Schlenk-flask, the freshly prepared
Rf = 0.22 (cyclohexane/ethyl acetate, 4:1).
a-aminonitrile 7 (863 mg, 2.93 mmol, 1.3 eq.) was dissolved in dry
Mp: 158–160 °C.
THF (33 mL) at room temperature. The solution was cooled to
ꢁ78 °C under argon atmosphere KHMDS (540 mg, 2.71 mmol,
1.2 eq.) in dry THF (3.5 mL) was added. After 3 min, a solution of
enal 14 (1.37 g, 2.26 mmol, 1.0 eq.) in dry THF (3 mL) was added
dropwise. The resulting mixture was stirred for another 30 min
at this temperature. The mixture was allowed to warm to room
temperature and quenched by addition of ethanol (11 mL). The
solution was acidified with acetic acid (1.0 mL) and heated to
reflux for 3 h. It was diluted with ethyl acetate (150 mL) and
washed with 2 N HCl (60 mL), saturated NaHCO3-solution (2 x
60 mL) and brine (60 mL). The solution was dried over Na2SO4
and the solvent was removed under reduced pressure. The crude
product was purified by automatic flash chromatography (SiO2,
toluene/ethyl acetate 20:1, Isolera Flashmaster, gradient: 10–80%
IR (ATR):
m = 3037, 2878, 2197, 1598, 1509, 1375, 1144, 997,
850, 746, 696 cmꢁ1
.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 7.74 (d,
4J = 2.1 Hz, 1H, 200-H), 7.46–7.30 (m, 10H, Ph), 7.37 (s, 1H, 30-H),
7.25 (dd, 3J = 8.5 Hz, J = 2.1 Hz, 1H, 600-H), 7.03 (s, 1H, 3-H), 6.93
4
(d, 3J = 8.5 Hz, 1H, 500-H), 6.91 (s, 1H, 60-H), 5.24 (s, 2H, 400-OCH2Ph),
5.13 (s, 2H, 40-OCH2Ph), 3.98 (s, 3H, 300-OCH3), 3.88 (s, 3H, 50-OCH3)
ppm.
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3): d = 150.7 (Cq-400),
150.3 (Cq-50), 149.8 (Cq-300), 149.1 (Cq-40), 148.0 (CH-3), 136.5
(Cq, Ph), 136.2 (Cq, Ph), 133.2 (Cq-10), 128.8 (4C, C-3 C-5, Ph),
128.4 (C-4, Ph), 128.2 (C-4, Ph), 127.6 (2C, C-2 C-6, Ph), 127.3
(2C, C-2 C-6, Ph), 126.5 (Cq-100), 124.4 (CH-600), 124.4 (CH-30),
118.2 (CN), 113.4 (CH-60), 113.3 (CH-500), 111.1 (CH-200), 110.4
(Cq-2), 86.4 (Cq-20), 71.4 (40-OCH2Ph), 70.9 (400-OCH2Ph), 56.3 (50-
OCH3), 56.2 (300-OCH3) ppm.
ethyl acetate). The product was obtained as
(833 mg, 0.97 mmol, 44 %).
a green solid
Rf = 0.33 (toluol/ethyl acetate, 20:1).
Mp: 127.6–130.9 °C.
ESI-MS: m/z (%) = 626.1 (100) [M + Na]+.
ESI-HRMS: Calculated for [C31H26INO4 + Na]: m/z = 626.0804,
observed: 626.0829.
IR (ATR):
m = 3032, 3010, 2933, 1464, 1453, 1325, 1239, 1120,
996, 802, 773, 697 cmꢁ1
.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 7.44–7.29 (m,
15H, Ph), 7.32 (s, 1H, 300-H), 6.82 (dd, 3J = 8.2 Hz, 4J = 1.8 Hz, 1H,
60-H), 6.82 (s, 1H, 10-H), 6.79 (s, 1H, 3-H), 6.78 (d, 3J = 8.2 Hz, 1H,
7.3.10. (2E)-2-(4-Benzyloxy-2-iodo-5-methoxyphenyl)-3-[4-
(benzyloxy)-3-methoxyphenyl]prop-2-enal (14)
4
The title compound was prepared according to a procedure by
Chuang et al.44 In an oven-dried Schlenk-flask, prop-2-enenitrile
13 (2.33 g, 3.86 mmol, 1.0 eq.) was dissolved in DCM (30 mL) under
argon atmosphere. The solution was cooled to ꢁ78 °C, and a solu-
tion of DIBAL-H in hexane (1 M, 5.50 mL, 5.41 mmol, 1.4 eq.) was
added, the resulting mixture was stirred 30 min at ꢁ78 °C. The
cooling bath was removed and the mixture was allowed to warm
to room temperature over a period of 3 h. Hydrochloric acid (10
%, 22 mL) was added, the solution was stirred another 30 min at
room temperature. The aqueous solution was extracted with
DCM (3 ꢂ 60 mL). The combined organic layers were washed with
water (2 ꢂ 50 mL) and brine (1 ꢂ 50 mL) and dried over Na2SO4.
The solvent was removed in vacuo and the residue purified by
automatic flash chromatography (cyclohexane/ethyl acetate 2:1,
Isolera Flashmaster, gradient: 10–80% ethyl acetate). The title com-
pound was obtained as a yellow oil (1.74 g, 2.83 mmol, 75%).
Rf = 0.27 (cyclohexane/ethyl acetate, 2:1).
50-H), 6.75 (d, J = 1.8 Hz, 1H, 20-H), 6.72 (s, 1H, 7-H), 6.59 (s, 1H,
600-H), 5.15 (s, 2H, 40-OCH2Ph), 5.12 (s, 2H, 8-OCH2Ph), 5.08 (s,
2H, 400-OCH2Ph), 4.07 (pseudo-t, 3J = 6.5 Hz, 2H, 5-H), 3.65 (s, 3H,
30-OCH3), 3.53 (s, 3H, 500-OCH3), 3.31 (s, 3H, 9-OCH3), 3.00
(pseudo–t, 3J = 6.5 Hz, 2H, 6-H) ppm.
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3): d = 149.3 (Cq-30),
149.2 (Cq-500), 148.2 (Cq-9), 147.2 (Cq-400), 146.4 (Cq-40), 146.1
(Cq-8), 137.4 (Cq, Ph), 137.3 (Cq, Ph), 136.7 (Cq, Ph), 134.0 (Cq-
100), 129.5 (Cq-10), 128.7 (4C, C-3 C-5, Ph), 128.6 (2C, C-3 C-5, Ph),
128.1 (C-4, Ph), 127.9 (2C, C-4, Ph), 127.6 (2C, C-2 C-6, Ph), 127.4
(2C, C-2 C-6, Ph), 127.2 (2C, C-2 C-6, Ph), 126.7 (Cq-2), 125.1 (Cq-
10b), 123.9 (Cq-6a), 123.7 (CH-300), 123.1 (CH-60), 123.0 (Cq-10a),
119.9 (Cq-1), 119.8 (CH-3), 115.3 (CH-600), 114.8 (CH-20), 114.1
(CH-7), 113.8 (CH-50), 107.9 (CH-10), 89.4 (Cq-200), 71.3 (400-OCH2-
Ph), 71.3 (8-OCH2Ph), 70.9 (40-OCH2Ph), 56.0 (30-OCH3), 55.8 (500-
OCH3), 55.4 (9-OCH3), 44.9 (CH2-5), 29.5 (CH2-6) ppm.
ESI-MS: m/z (%) = 856.3 (100) [M + H]+.
IR (ATR):
m
= 3032, 2934, 2831, 1673, 1616, 1500, 1268, 1244,
ESI-HRMS: Calculated for [C48H42INO6 + H]: m/z = 856.2135,
observed: 856.2148.
1170, 907, 726 cmꢁ1
.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 9.70 (s, 1H, CHO),
7.47–7.30 (m, 10H, Ph), 7.37 (s, 1H, 3-H), 7.43 (s, 1H, 30-H), 6.87 (dd,
3J = 8.5 Hz, 4J = 2.0 Hz, 1H, 600-H), 6.81 (d, 3J = 8.5 Hz, 1H, 500-H), 6.62
(s, 1H, 60-H), 6.62 (d, 4J = 2.0 Hz, 1H, 200-H), 5.16 (s, 2H, 400-OCH2Ph),
7.3.12. 3,10-Bis(benzyloxy)-13-[4-(benzyloxy)-3-methoxyphenyl]-
2,11-dimethoxy-5,6-dihydro-8H-indeno[10,20:4,5]pyrrolo[2,1-a]
isoquinolin-8-one (16)
2
5.15 (d, J = 12.1 Hz, 2H, 40-OCH2Ph), 3.78 (s, 3H, 50-OCH3), 3.44 (s,
The title compound was prepared according to a procedure of
3H, 300-OCH3) ppm.
Campo and Larock.18 In an oven-dried Schlenk-flask under argon-
Please cite this article in press as: Colligs V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.005

V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
atmosphere, Pd(dba)2 (50.4 mg, 87.6 mmol, 10 mol%), PCy3HBF4
(64.6 mg, 175 mmol, 20 mol%), CsOPiv (410 mg, 1.75 mmol,
2.0 eq.) and iodide 15 (750 mg, 876 mmol, 1.0 eq.) were dissolved
in dry DMF (24 mL) and stirred for 5 min at room temperature.
The flask was filled with CO (1 atm) and stirred for 24 h at
110 °C. The mixture was cooled to room temperature, the solvent
was removed in vacuo. The residue was diluted with toluene
(100 mL). To remove excess dba from the mixture, it was filtered
over a short pad of silica, washed with toluene and a toluene-ethyl
acetate-mixture (10:1). The eluate was washed with saturated
NaHCO3-solution (2 ꢂ 50 ml) and brine (50 mL) and dried over
Na2SO4. The solvent was removed under reduced pressure to yield
the title compound as cherry-red crystals (635 mg, 0.84 mmol,
96%).
10-OCH2Ph), 3.92 (s, 3H, 30-OCH3), 3.68 (s, 3H, 11-OCH3), 3.37 (s,
3H, 2-OCH3) ppm.
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3): d = 178.6 (Cq-8),
152.6 (Cq-11), 150.1 (Cq-2), 149.8 (Cq-30), 148.9 (Cq-3), 147.4
(Cq-10), 147.4 (Cq-40), 142.2 (Cq-12b), 137.1 (Cq, Ph), 136.9 (Cq,
Ph), 136.4 (Cq, Ph), 135.5 (Cq-13a), 133.1 (Cq-8a), 132.9 (Cq-12a),
128.8 (2C, C-3 C-5, Ph), 128.7 (4C, C-3 C-5, Ph), 128.3 (C, C-4, Ph),
128.1 (2C, C-4, Ph), 128.0 (Cq-10), 127.6 (2C, C-2 C-6, Ph), 127.4
(2C, C-2 C-6, Ph), 127.2 (2C, C-2 C-6, Ph), 124.1 (Cq-4a), 123.5
(Cq-7a), 122.8 (CH-60), 122.4 (CH-6), 120.9 (Cq-13b), 114.1 (CH-
9), 114.1 (CH-5), 114.0 (Cq-13), 113.9 (CH-20), 111.0 (CH-50),
109.7 (CH-4), 105.9 (CH-12), 104.7 (CH-1), 71.4 (10-OCH2Ph),
70.9 (40-OCH2Ph), 70.9 (3-OCH2Ph), 56.4 (11-OCH3), 56.3 (30-
OCH3), 55.5 (2-OCH3) ppm.
Rf = 0.19 (toluol/ethyl acetate, 30:1).
ESI-MS: m/z (%) = 754.4 (100) [M + H]+.
Mp: 179–180 °C.
ESI-HRMS: Calculated for [C49H39NO7 + Na]: m/z = 776.2624,
observed: 776.2629.
IR (ATR):
m = 3062, 3003, 1678, 1601, 1540, 1382, 1272, 1225,
1212, 1013, 852, 813, 734, 696 cmꢁ1
.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 7.47–7.29 (m,
7.3.14. 3,10-Dihydroxy-13-(4-hydroxy-3-methoxyphenyl)-2,11-
dimethoxy-8H-indeno[10,20:4,5]pyrrolo[2,1-a]isoquinolin-8-one (18)
In an oven-dried Schlenk-tube, compound 17 (15.7 mg,
20.8 mmol, 1.0 eq.) was dissolved in DCM (1.5 mL) under argon-
atmosphere and stirred at ꢁ78 °C. A solution of BCl3 in n-hexane
(1.0 M, 0.19 mL, 0.19 mmol, 9.0 eq.) was added dropwise. After
30 min, the solution was warmed to 0 °C and stirred another 3 h.
The reaction mixture was diluted with NaHCO3 (5 mL) and
extracted with DCM (3 ꢂ 30 mL). The combined organic layers
were washed with brine (30 mL) and dried over Na2SO4. The sol-
vent was removed in vacuo the remaining residue was washed
with methanol (2 mL) and dried in vacuo. The product was
obtained as black crystals (9.40 mg, 19.4 mmol, 94%).
15H, Ph), 7.06 (d, J = 1.8 Hz, 1H, 20-H), 7.00 (dd, 3J = 8.2 Hz,
4
4J = 1.8 Hz, 1H, 60-H), 6.99 (s, 1H, 9-H), 6.95 (d, J = 8.2 Hz, 1H, 50-
3
H), 6.75 (s, 1H, 1-H), 6.72 (s, 1H, 4-H), 6.46 (s, 1H, 12-H), 5.25 (s.
2H, 40-OCH2Ph), 5.13 (s, 2H, 3-OCH2Ph), 5.08 (s, 2H, 10-OCH2Ph),
3
4.17 (pseudo-t, J = 6.7 Hz, 2H, 6-H), 3.88 (s, 3H, 30-OCH3), 3.70 (s,
3H, 11-OCH3), 3.32 (s, 3H, 2-OCH3), 2.95 (pseudo-t, 3J = 6.7 Hz,
2H, 5-H) ppm.
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3): d = 180.5 (Cq-8),
153.0 (Cq-11), 149.8 (Cq-30), 148.1 (Cq-2), 147.7 (Cq-3), 147.3
(Cq-40), 146.5 (Cq-10), 140.2 (Cq-12b), 137.1 (Cq, Ph), 137.0 (Cq,
Ph), 136.9 (Cq, Ph), 135.6 (Cq-13a), 134.2 (Cq-12a), 131.5 (Cq-8a),
130.0 (Cq-7a), 128.8 (2C, C-3 C-5, Ph), 128.7 (2C, C-3 C-5, Ph),
128.6 (2C, C-3 C-5, Ph), 128.1 (2C, C-4, Ph), 128.0 (C-4, Ph), 127.9
(Cq-10), 127.6 (2C, C-2 C-6, Ph), 127.4 (2C, C-2 C-6, Ph), 127.3 (2C,
C-2 C-6, Ph), 125.2 (Cq-4a), 122.2 (CH-60), 121.3 (Cq-13b), 117.2
(Cq-13), 114.2 (CH-50), 113.7 (CH-4), 113.4 (CH-20), 111.6 (CH-9),
108.7 (CH-1), 105.1 (CH-12), 71.6 (10-OCH2Ph), 71.1 (3-OCH2Ph),
70.9 (40-OCH2Ph), 56.4 (30-OCH3), 56.3 (11-OCH3), 55.6 (2-OCH3),
42.1 (CH2-6), 28.6 (CH2-5) ppm.
Rf = 0.31 (DCM/ethyl acetate, 1:3).
Mp: 227–228 °C.
IR (ATR):
m = 3049, 2924, 2853, 2606, 1636, 1430, 1264, 1217,
1057, 808, 754 cmꢁ1
.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 9.88 (s, 1H, 3-OH),
9.25 (s, 1H, 40-OH), 9.21 (s, 1H, 10-OH), 8.10 (d, 3J = 7.1 Hz, 1H, 6-H),
7.23 (s, 1H, 1-H), 7.14 (d, J = 1.9 Hz, 1H, 20–H), 7.11 (d, 3J = 7.1 Hz,
4
ESI-MS: m/z (%) = 756.4 (100) [M + H]+
ESI-HRMS: Calculated for [C49H41NO7 + Na]: m/z = 778.2781,
observed: 778.2805.
1H, 5-H), 7.10 (s, 1H,4-H), 7.04 (dd, 3J = 8.1 Hz, 4J = 1.9 Hz, 1H, 60-H),
3
7.00 (d, J = 8.1 Hz, 1H, 50-H), 6.74 (s, 1H, 9-H), 6.43 (s, 1H, 12-H),
3.80 (s, 3H, 30-OCH3), 3.64 (s, 3H, 11-OCH3), 3.43 (s, 3H, 2-OCH3)
ppm.
13C NMR, HSQC, HMBC (100.6 MHz, CDCl3): d = 177.2 (Cq-8),
149.8 (Cq-11), 148.9 (Cq-2), 147.9 (Cq-3), 147.8 (Cq-30), 146.2
(Cq-40), 145.9 (Cq-10), 141.8 (Cq-12b), 135.1 (Cq-13a), 132.8 (Cq-
8a), 129.8 (Cq-12a), 124.8 (Cq-10), 124.3 (Cq-4a), 122.8 (CH-60),
122.0 (Cq-7a), 121.5 (CH-6), 119.0 (Cq-13b), 115.8 (CH-50), 114.2
(CH-5), 114.0 (CH-20) 113.6 (Cq-13), 112.0 (CH-9), 111.6 (CH-4),
106.0 (CH-12), 104.3 (CH-1), 55.8 (30-OCH3), 55.8 (11-OCH3), 54.8
(2-OCH3) ppm.
7.3.13. 3,10-Bis(benzyloxy)-13-[4-(benzyloxy)-3-methoxyphenyl]-
2,11-dimethoxy-8H-indeno[10,20:4,5]pyrrolo[2,1-a]isoquinolin-8-one
(17)
A mixture of compound 16 (30.0 mg, 39.7 mmol, 1.0 eq.) and
TBQ (9.76 mg, 39.7 mmol, 1.0 eq.) in pyridine (2.5 mL) were trans-
ferred to a microwave reaction vessel (15 mL). The mixture was
radiated at 130 °C for one hour (maximum temperature 150 °C,
maximum pressure 10 bar, power: 150 W). Afterwards, another
portion of TBQ (9.76 mg, 39.7 mmol, 1.0 eq.) was added and the
mixture was irradiated again. This procedure was repeated another
time. In total, 3.0 eq of TBQ were added. The solvent was removed
in vacuo, the residue was diluted with toluene (10 mL), filtered
over silica, washed with toluene and a toluene-ethyl acetate
(10:1). The solvent was removed under reduced pressure to furnish
the title compound as a dark red oil (15.7 mg, 20.8 mmol, 53 %).
Rf = 0.19 (toluene/ethyl acetate, 30:1).
ESI-MS: m/z (%) = 484.2 (100) [M + H]+.
ESI-HRMS: Calculated for [C28H21NO7 + H]: m/z = 484.1396,
observed: 484.1401.
Acknowledgements
This work was supported by the Carl Zeiss Foundation (project
ChemBioMed). We thank Dr. Johannes C. Liermann (Mainz) for
NMR spectroscopy and Dr. N. Hanold (Mainz) for mass
spectrometry.
IR (ATR):
m = 3062, 3032 2935, 1714, 1506, 1438, 1265, 1222,
1064, 856, 809 cmꢁ1
.
1H NMR, COSY, NOESY (400 MHz, CDCl3): d = 8.15 (d,
3J = 7.1 Hz, 1H, 6-H), 7.49–7.29 (m, 15H, Ph), 7.19 (s, 1H, 1-H),
4
7.14 (d, J = 1.9 Hz, 1H, 20-H), 7.09 (dd, 3J = 8.1 Hz, 4J = 1.9 Hz, 1H,
A. Supplementary material
3
60-H), 7.03 (d, J = 8.1 Hz, 1H, 50-H), 7.02 (s, 1H, 9-H), 6.95 (s, 1H,
4-H), 6.81 (d, 3J = 7.1 Hz, 1H, 5-H), 6.43 (s, 1H, 12-H), 5.29 (d,
2J = 12.6 Hz, 2H, 40-OCH2Ph), 5.19 (s, 2H, 3-OCH2Ph), 5.10 (s, 2H,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.005.
Please cite this article in press as: Colligs V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.005

12
V. Colligs et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
24. Efferth T, Konkimalla VB, Wang Y-F, et al. Clin Cancer Res. 2008;14:2405–2412.
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Please cite this article in press as: Colligs V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.005