Boehmeriasin A as new lead compound for the inhibition of topoisomerases and SIRT2

Article abstract of DOI:10.1016/j.ejmech.2015.01.038

Two synthetic approaches to boehmeriasin A are described. A gram scale racemic preparation is accompanied by an efficient preparation of both the pure enantiomers using the conformationally stable 2-piperidin-2-yl acetaldehyde as starting material. The an

Full text of DOI:10.1016/j.ejmech.2015.01.038

Accepted Manuscript
Boehmeriasin A as new lead compound for the inhibition of Topoisomerases and
SIRT2
Michael S. Christodoulou, Francesco Calogero, Marcus Baumann, Aída Nelly García-
Argáez, Stefano Pieraccini, Maurizio Sironi, Federico Dapiaggi, Raffaella Bucci,
Gianluigi Broggini, Silvia Gazzola, Sandra Liekens, Alessandra Silvani, Maija Lahtela-
Kakkonen, Nadine Martinet, Alfons Nonel-Canals, Eduardo Santamaría-Navarro, Ian
R. Baxendale, Lisa Dalla Via, Daniele Passarella
PII:
S0223-5234(15)00058-6
10.1016/j.ejmech.2015.01.038
EJMECH 7654
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 October 2014
Revised Date: 15 January 2015
Accepted Date: 19 January 2015
Please cite this article as: M.S. Christodoulou, F. Calogero, M. Baumann, A.N. García-Argáez, S.
Pieraccini, M. Sironi, F. Dapiaggi, R. Bucci, G. Broggini, S. Gazzola, S. Liekens, A. Silvani, M. Lahtela-
Kakkonen, N. Martinet, A. Nonel-Canals, E. Santamaría-Navarro, I.R. Baxendale, L. Dalla Via, D.
Passarella, Boehmeriasin A as new lead compound for the inhibition of Topoisomerases and SIRT2,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.01.038.
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ACCEPTED MANUSCRIPT
Boehmeriasin A as new lead compound for the inhibition of
Topoisomerases and SIRT2
a¥
a¥
b
c
Michael S. Christodoulou , Francesco Calogero , Marcus Baumann, Aída Nelly García-Argáez,
a
a
a
a
d
g
Stefano Pieraccini, Maurizio Sironi, Federico Dapiaggi, Raffaella Bucci, Gianluigi Broggini,
Silvia Gazzola, Sandra Liekens, Alessandra Silvani, Maija Lahtela-Kakkonen, Nadine Martinet,
Alfons Nonel-Canals, Eduardo Santamaría-Navarro, Ian R. Baxendale, Lisa Dalla Via and
d
e
a
f
h
h
b
c
a,
Daniele Passarella *
Graphical Abstract

ACCEPTED MANUSCRIPT
Boehmeriasin A as new lead compound for the inhibition of
Topoisomerases and SIRT2
a¥
a¥
b
c
Michael S. Christodoulou , Francesco Calogero , Marcus Baumann, Aída Nelly García-Argáez,
a
a
a
a
d
g
Stefano Pieraccini, Maurizio Sironi, Federico Dapiaggi, Raffaella Bucci, Gianluigi Broggini,
Silvia Gazzola, Sandra Liekens, Alessandra Silvani, Maija Lahtela-Kakkonen, Nadine Martinet,
Alfons Nonel-Canals, Eduardo Santamaría-Navarro, Ian R. Baxendale, Lisa Dalla Via and
d
e
a
f
h
h
b
c
a,
Daniele Passarella *
a
Dipartimento di Chimica - Università degli Studi di Milano - Via Golgi 19, 20133 Milano - Italy
Department of Chemistry - University of Durham - South Road, DH1 3LE, Durham – United
b
Kingdom
c
Dipartimento di Scienze del Farmaco - Università degli Studi di Padova - Via F. Marzolo 5,
3
5131 Padova - Italy
d
Dipartimento di Scienza e Alta Tecnologia - Università degli Studi dell’Insubria - Via Valleggio
1, 22100 Como - Italy
Rega Institute for Medical Research - Minderbroedersstraat 10, B-3000 Leuven – Belgium
School of Pharmacy - University of Eastern Finland - P. O. Box 1627, 70211 Kuopio – Finland
Institute of Chemistry - UMR CNRS 7272 - Parc Valrose, Nice 06108, Cedex 2 - France
Mind the Byte, S.L. - Parc Científic de Barcelona - C/Baldiri Reixac, 4-8, 08028 Barcelona –
1
e
f
g
h
Spain
¥
The authors contributed equally to this work
ABSTRACT
Two synthetic approaches to boehmeriasin A are described. A gram scale racemic preparation is
accompanied by an efficient preparation of both the pure enantiomers using the conformationally
stable 2-piperidin-2-yl acetaldehyde as starting material. The anti-proliferative activity in three
cancer cell lines (CEM, HeLa and L1210) and two endothelial cell lines (HMEC-1, BAEC)
indicates promising activity at the nanomolar range. Topoisomerases and SIRT2 are identified as
biological targets and the experimental data has been supported by docking studies.

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1
. INTRODUCTION
Boehmeriasin A (Scheme 1), a phenanthroquinolizidine alkaloid, was first isolated from the
aqueous ethanolic extract of Boehmeria siamensis Craib (Urticaceae) by bioassay-guided
1
fractionation. In vitro tests showed that boehmeriasin A possesses a strong cytotoxic activity, more
potent than taxol, against 12 cell lines from 6 panels of cancer including breast, kidney, prostate,
colon, lung cancer and leukemia, with GI values between 0.2 and 100 ng/mL. Furthermore, this
5
0
alkaloid restrains the expression of a series of genes related to cell proliferation and cell cycle
2
regulation. This results in G1 phase arrest of the cell cycle and differentiation in the breast cancer
3
cell line MDA-MB-232. Consequently, we were attracted by its biological activity, the uncertainty
of its biological mechanism and by the features of its structure that show boehmeriasins as possible
building blocks for further structural modification. In this context, we planned a convenient
synthesis to both enantiomers of boehmeriasin A with the potential extension of this methodology
to create a library of analogues. Several synthetic methodologies have been developed for the
elaboration of some boehmeriasin A congeners (e.g. cryptopleurine and its hydroxy derivatives) in
4
-15
both racemic and optically active forms.
Herein, we report two synthetic approaches to obtain the racemic mixture and the pure enantiomers
of boehmeriasin A. Biological evaluation showed high anti-proliferative activity on cancer and
endothelial cells, with topoisomerases and SIRT2 as involved targets. Virtual screening (Hurakan
1
6
tool) and docking studies support the experimental results.
2
. RESULTS AND DISCUSSION
2
.1 Chemistry
The retrosynthetic plan (Scheme 1) indicates the possibility to first create the phenantrene nucleus
and then conduct a further cyclization to form the quinolizidine ring (path A) or (path B). This
would permit the generation of the nitrogen containing bicyclic system in a stereospecific way by
an intramolecular aldol type reaction followed by an intamolecular arylation reaction.
Driven by a desire to access both enantiomers of boehmeriasin A, we initiated our synthetic efforts
first through a racemic preparation of the target compound. To this end, we pursued the synthesis
outlined in Scheme 2 which through its simplicity and robustness would allow gram quantities of
the desired natural product to be obtained.

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Scheme 1. Retrosynthetic Plan
The synthesis commences with a Perkin reaction between commercially available 4-
methoxybenzaldehyde (3) and 3,4-dimethoxyphenylacetic acid (4). This reaction can be easily
performed at >100 mmol scale. Next, a microwave-assisted Fischer esterification delivered the
corresponding ester 5 in quantitative yield through rapid flash heating. A ferrous chloride mediated
oxidative biaryl coupling was then performed generating the quinolizidine ring. Again, this
transformation can be performed on multigram scale in an efficient manner; however, the
generation of stoichiometric amounts of insoluble inorganic by-products requires close reaction
monitoring and specific work-up strategies in order to achieve a reproducible reaction outcome (see
Experimental Section). Reduction of the ester functionality of intermediate 6 using LiAlH and
4
chlorodehydroxylation of the resulting benzylic alcohol 7 with concentrated HCl cleanly affords the
benzylic chloride 8. This material is subjected to nucleophilic substitution, with the potassium salt
of racemic pipecolic acid, furnishing the desired adduct 9 without recourse to any protecting group
chemistries. In order to prepare the pentacyclic scaffold of boehmeriasin A, a Friedel-Crafts
acylation in neat polyphosphoric acid was used. This transformation was found to reliably furnish
the desired ketone 10 in good yield, as long as the instability of this material under basic conditions
is addressed by maintaining the pH during the aqueous work-up below 8. Finally, removal of the

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ketone functionality was accomplished in a two-step fashion firstly using LiAlH followed by
4
dehydroxylation under TFA/triethylsilane conditions. In summary, this sequence allowed the
preparation of racemic boehmeriasin A in a short 7 step sequence and in 22% overall yield. In
addition, the feasibility of separating the racemic natural product by means of chiral HPLC was
evaluated. Pleasingly, it was quickly established that for analytical purposes a short chiral HPLC
column (AD, 5 cm) can be used in order to achieve resolution of the racemic boehmeriasin A (see
insert in Scheme 2). Consequently, we feel confident that separation of the racemate on a
preparative scale using a larger AD column can be accomplished; however, before doing so we
elected to carry out an asymmetric synthesis of both (S)- and (R)-boehmeriasin A.
a
Scheme 2. Racemic synthesis of boehmeriasin A and its resolution via chiral HPLC (path A)
a
Conditions: : (a) i) Et N, Ac O, 120 °C, ii) MeOH, H SO , MW, 125 °C; (b) DCM, anhydr. FeCl , r.t.; (c) LiAlH ,
3
2
2
4
3
4
THF; (d) conc. HCl, DCM, r.t.; (e) pipecolic acid, NaOH, iPrOH, 40 °C; f) PPA, 90 °C; g) i) LiAlH , THF, r.t. ii)
4
Et SiH, TFA, DCM, r.t..
3
In this second approach we developed an enantioselective synthesis using enantiopure
1
7
piperidinethanol 2 as the starting material, which can be produced at gram scale and used for
1
7-21
22
target-
and diversity-oriented synthesis. The aldehyde 11, obtained from oxidation of the
alcohol 2, was converted to the secondary alcohol 12 by Grignard addition using (4-
methoxyphenyl)magnesium bromide in high yield (90-92%, Scheme 3). DMP (Dess-Martin
periodinane) oxidation of alcohol 12, followed by deprotection of the Boc group (13 → 14) and N-
2
3
acylation of the amine 14 with the easily prepared 2-bromo-4,5-dimethoxyphenylacetic acid
yielded compound 15 in 63% (3 steps) (Scheme 3, S-enantiomer shown). An intramolecular aldol-

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type condensation in the presence of KOH furnished the desirable intermediates 16 as mixture of
atropoisomers (85%, 1:1 ratio).
a
Scheme 3. Enantioselective synthesis of boehmeriasin A (path B)
a
Conditions: (a) (4-methoxyphenyl)magnesium bromide, THF, -78 ºC to r.t.; (b) DMP, r.t., 1 h; (c) TMSCl, MeOH, 0
ºC to r.t.; (d) 2-bromo-4,5-dimethoxyphenylacetic acid, DIPEA, HATU, THF, r.t., 1 h; (e) KOH, EtOH, reflux, 2 h; (f)
Pd(OAc) , K CO , 2′-(diphenylphosphino)-N,N′-dimethyl-(1,1′-biphenyl)-2-amine, DMA, reflux, 5 h; (g) LiAlH4, THF,
2
2
3
reflux, 2 h.
To obtain the phenanthroquinolizidine skeleton a palladium catalyzed intramolecular coupling was
investigated (see Supp. Inf.). The use of Pd(OAc) in dimethylacetamide (125 °C for 5 h), in the
2
presence of K CO3 and 2′-(diphenylphosphino)-N,N′-dimethyl-(1,1′-biphenyl)-2-amine as the
2
ligand furnished 17 in 60% yield. A final reduction with LiAlH , completed the synthesis of
4
boehmeriasin A. HPLC analysis confirmed the purity of both enantiomers was in accordance with
the e.e. of the starting materials (2).
2
.2 Biological Evaluation
.2.1 Anti-proliferative activity. Both enantiomers of boehmeriasin A, as well as the racemic
mixture were evaluated for their anti-proliferative activity (Table 1) in three cancer cell lines
human lymphoblastic leukemia (CEM), human cervical carcinoma (HeLa) and mouse lymphocytic
leukemia (L1210) cells] and two endothelial cell lines [human microvascular endothelial cells
2
[

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(HMEC-1) and bovine aortic endothelial cells (BAEC)]. As a reference compound, we used the
vascular-targeting agent combretastatin A4P (CA-4P), which inhibits the proliferation of the tumor
cells, and endothelial cells with IC values around 80 nM and 3 nM, respectively. In accordance
5
0
1
-3, 14
with other results reported in the literature
both enantiomers showed potent cytostatic activity
against the different tumor cells, with IC50 values in the nanomolar range. Interestingly, (R)-1
proved to be significantly more potent than the (S)-enantiomer against HeLa and L1210 cells. Both
compounds also inhibited the proliferation of endothelial cells, with IC values of 23 nM in BAEC
5
0
and 7 and 82 nM for the (R)- and (S)-enantiomers in HMEC-1, respectively. Together, these data
confirm the inhibitory activity of (R)- and (S)-boehmeriasin A against both tumor and endothelial
cells, the (R)-enantiomer being up to 11-fold more potent than the (S)-enantiomer in selected cell
lines. The anti-proliferative activity of the racemic mixture was comparable to the activity of the
(R)-enantiomer, being equally active in HeLa cells, about 2-fold more active in CEM cells and
BAEC and 3-4-fold less active in L1210 cells and HMEC-1.
Table 1. Anti-proliferative activity of (R)- and (S)-boehmeriasin A in comparison with combretastatin A4P.
Tumor cell lines
Endothelial cell lines
Compound
[a]
[a]
(
IC50 [nM])
(IC50 [nM])
HeLa
66 ± 56
182 ± 164
76 ± 52
79 ± 3
CEM
L1210
19 ± 10
111 ± 11
71 ± 23
82 ± 12
HMEC-1
7.4 ± 1.1
82 ± 66
29 ± 8
BAEC
23 ± 13
23 ± 6
(
R)-1
S)-1
185 ± 156
201 ± 127
119 ± 71
95 ± 6
(
rac.-1
9.0 ± 2.1
3.9 ± 0.1
CA-4P
2.9 ± 0
[a]
5
0% inhibitory concentration. CA-4P: combretastatin A4P. CEM: human lymphoblastic
leukemia. HeLa: human cervical carcinoma. L1210: mouse lymphocytic leukemia cells. HMEC-
: human microvascular endothelial cells. BAEC: bovine aortic endothelial cells.
1
We were attracted by the possibility to identify the biological targets that could justify the anti-
proliferative activity on different cell lines. We wanted to avoid any structural change that could be
useful for the application of interesting approaches such as surface plasmon resonance, but has the
drawback to require SAR information to identify the proper alterable region of the molecule.
1
6
2.2.2 Virtual screening. We planned to use virtual screening with the help of Hurakan software
that compares the input molecule with the structures present in the reference database using
CoMSIA fields on a 3D grid. Hurakan uses ChemblDB as a reference database because it contains
molecules, targets and biological activities. In this way, Hurakan predicts the biological profile of
an input molecule and, in our case, it predicted 13 proteins for the R-enantiomer and 15 proteins for

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the S-enantiomer (see Supporting Information). We were driven by the fact that topoisomerases
were identified as a possible target for the (S)-enantiomer.
2
5-29
2.2.3 Topoisomerase inhibition. Based on our previous efforts in this field,
both (R)- and (S)-
boehmeriasin A were tested for their capacity to affect the activity of topoisomerases.
Figure 1 shows the effect of (R)- and (S)-boehmeriasin A on the relaxation of supercoiled plasmid
pBR322 DNA, mediated by topoisomerase II. The enzyme relaxes supercoiled DNA giving rise to a
series of topoisomers, representing differently relaxed forms (Topo II). The test compounds were
assayed at 10, 25 and 50 µM concentration, while m-AMSA, taken as reference drug, was used at
10 µM. Both enantiomers of boehmeriasin A exhibit a comparable and dose-dependent effect on
enzymatic activity. Indeed, at 10 µM both (R)- and (S)-enantiomers are unable to exert a significant
inhibitory activity, while the topoisomerase-mediated relaxation is completely inhibited at the
higher concentration taken into account (50 µM).
Figure 1. Effect of (R)- and (S)-boehmeriasin A on relaxation of supercoiled pBR322 DNA by human recombinant
topoisomerase II. m-AMSA was taken as reference.
Similar experiments were performed to assay the effect on topoisomerase I relaxation activity, and
the results indicate the occurrence of a higher inhibitory effect compared to that obtained on
topoisomerase II. Indeed, at 10 µΜ concentration both (R)- and (S)-boehmeriasin A completely
inhibit the relaxation mediated by topoisomerase I (Figure 2).
Figure 2. Effect of (R)- and (S)-boehmeriasin A on relaxation of supercoiled pBR322 DNA by human recombinant
topoisomerase I
.

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Some anti-proliferative agents, called topoisomerase poisons, interfere with topoisomerase activity
by stabilizing a biological intermediate, the cleavable complex, into a lethal agent. The occurrence
of the cleavable complex can be demonstrated experimentally by the enzyme-dependent formation
of linear (topoisomerase II) or nicked (topoisomerase I) DNA from supercoiled DNA. Figure 3
shows a cleavable complex assay performed on topoisomerase II in the presence of 100 µM of (R)-
and (S)-boehmeriasin A and 10 µM m-AMSA used as reference. The results show that both
derivatives do not stabilize the formation of the cleavage complex, although tested at a significantly
higher concentration compared to that of the reference drug. Otherwise, as expected, m-AMSA,
which is a well-known topoisomerase II poison, induces the formation of a detectable amount of
linear DNA (Figure 3).
Figure 3. Effect of (R)- and (S)-boehmeriasin A on the stabilization of covalent DNA-topoisomerase II complex. m-
AMSA was taken as reference.
With respect to topoisomerase I, the results reported in Figure 4 also show the inability of both (R)-
and (S)-boehmeriasin A to act as a poison at the considered concentration (0.5 µM). On the other
hand, the topoisomerase I poison camptothecin (CPT), taken as reference, induces in the same
experimental results that of occurrence of the band corresponding to nicked DNA (Figure 4).

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Figure 4. Effect of (R)- and (S)-boehmeriasin A on the stabilization of covalent DNA-topoisomerase I complex.
Camptothecin (CPT) was taken as reference.
Finally, the ability of (R)- and (S)-boehmeriasin A to interfere with topoisomerase-mediated
relaxation activity, along with the lack of poisoning effects, suggest that the effect on the enzymatic
activity could come from the capacity of the compounds to form a molecular complex with DNA.
2.2.4 Sirtuins inhibition. We were driven to study boehmeriasin A against sirtuins due to its
3
0
structural similarity with known sirtuin inhibitors. Both enantiomers were screened in vitro
against SIRT1 and SIRT2 at 200 µM concentration (Table 2). For SIRT1 (R)- and (S)-boehmeriasin
A showed no inhibition. Interestingly, for SIRT2 (R)-boehmeriasin A gave ~65 % inhibition,
whereas (S)-boehmeriasin A gave only 41% inhibition.
Table 2. Evaluation of (R)- and (S)-boehmeriasin A against SIRT1 and SIRT2.
a
a
Compound
R)-1
S)-1
SIRT1
SIRT2
(
12 ± 1.4
10 ± 4.2
65 ± 1.6
(
41 ± 0.56
a
Inhibition % at 200 µM inhibitor concentration.
2.2.5 Docking Studies. To gain better insight into the interactions between boehmeriasin A the
topoisomerases and sirtuins, molecular dockings studies were carried out. Docking simulations on
topoisomerase I showed that both boehmerasin enantiomers display good binding affinity to the
target. Cluster analysis showed two major clusters, comprising 50% and 36% of the docked
structures of the S enantiomer (Figure 5a), and two minor clusters. Cluster analysis of the docking
results on the topoisomerase II showed only one cluster for the S enantiomer, with the phenantrene
moiety stacked between the DNA bases (Figure 5b). A slight preference toward topoisomerase II

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can, on the other hand, be inferred on the basis of the docking results. It is worth noting that in all of
the docked conformations both S and R enantiomers of boehmeriasin A do not display any
significant contact with either one of the topoisomerase isoforms that have been tested, but seem to
mainly interact with the DNA strand through stacking interactions (see Supp. Inf.).
Figure 5. a) Docked structure of (S)-boehmeriasin A. The best fit structure of the principal cluster is depicted in orange,
while the best fit structure of secondary cluster is green. Boehmerasin is intercalated between DNA bases of the nucleic
acid double helix complexed with topoisomerase I. b) Docked structure of (S)-boehmeriasin A (orange molecule). The
phenantrene moiety is intercalated between base pairs of the DNA in complex with topoisomerase II; c) (R)-
Boehmeriasin A (green) in the putative binding site of SIRT2. NAD+ (black) and Ex-527(purple) is also presented
based on their position in SIRT1.
a)
b)
c)
In SIRT1, both enantiomers of boehmeriasin A were docked into the place of the nicotinamide-
moiety (so called C-pocket in sirtuins) showing π-interactions with His363 or Phe273 but they
could not adopt a good orientation in the binding pocket (Figure 5c). As for SIRT2, (R)-
boehmeriasin A was docked into the C-pocket having an interaction of the phenyl-ring with Ile169
and showed good complementarity with the binding site. Whereas (S)-boehmeriasin A had no
interactions with SIRT2, but showed also complementary binding. Based on the modeling there was
no clear difference in the putative binding modes of the enantiomers (see Supp. Inf.).
3
. CONCLUSION
We described two different approaches for the synthesis of boehmeriasin A which in spite of being
strategically based on previous reported preparations present some novelties that result in increased
efficacy and simplicity which is relevant for large-scale synthesis and analogue preparation. The
high anti-proliferative activity in three endothelial and two cancer cell lines has been described. The

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biological evaluation accompanied by virtual screening and docking studies permitted to identify
the interaction with DNA and SIRT2 as biological mechanisms that justify their activity. These
results offer new suggestions for the design and practical synthesis of new topoisomerase and SIRT-
2
inhibitors based on the boehmeriasin A scaffold.
4
. EXPERIMENTAL SECTION
4
.1. Chemistry General procedures.
All reactions were carried out in oven-dried glassware and dry solvents under nitrogen
atmosphere. Unless otherwise stated, all solvents were purchased from Fisher Scientific and Sigma
Aldrich and used without further purification. Substrates and reagents were purchased from Alfa
Aesar or Sigma Aldrich and used as received. Thin layer chromatography (TLC) was performed on
Merck precoated 60F254 plates. Reactions were monitored by TLC on silica gel, with detection by
UV light (254 nm) or by a solution of KMnO with heating. Flash chromatography was performed
4
using silica gel (240-400 mesh, Merck). All tested compounds possessed a purity of > 98%
confirmed via elemental analyses (CHN) in Perkin Elmer 2400 instrument. Optical rotation was
1
measured on a JAS.CO P-1030 instrument. H-NMR spectra were recorded on either Bruker DRX-
400 or Bruker Avance-400 or Varian VNMRS-700 instruments and are reported relative to residual
1
3
CHCl (δ 7.26 ppm) or DMSO (δ 2.50 ppm). C-NMR spectra were recorded on the same
3
instruments (100 MHz or 175 MHz) and are reported relative to CDCl (δ 77.16 ppm) or d -DMSO
3
6
(δ 39.52 ppm). Chemical shifts (δ) for proton and carbon resonances are quoted in parts per million
(ppm) relative to tetramethylsilane (TMS), which was used as an internal standard. Low and high
resolution mass spectrometry was performed using the indicated techniques on either Waters LCT
Premier XE or Waters TQD instruments equipped with Acquity UPLC and a lock-mass
electrospray ion source. EI mass spectra were recorded at an ionizing voltage of 6Kev on a VG 70-
70 EQ. Melting ranges were recorded on an Optimelt automated melting point system and are
uncorrected operating at a heating rate of 1 °C/min.

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4.1.1.
(E)-Methyl
2-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)acrylate
(5).
4-
Methoxybenzaldehyde (3) (13.6 g, 100 mmol) and 3,4-dimethoxyphenylacetic acid (4) (19.6 g, 100
mmol) were dissolved in a mixture of triethylamine (10 mL) and acetic anhydride (20 mL) and
heated at 120 °C for 30 h. After the reaction mixture was cooled to room temperature ethyl acetate
(150 mL) was added leading to precipitation of a yellow solid. After filtration and drying this
material was charged into 20 mL microwave vessels (~4 g each), filled with 7 mL methanol and 0.1
mL conc. sulfuric acid and sealed with the appropriate cap. Each of these samples was heated in a
Biotage Microwave Synthesizer for 90 min at 125 °C. Upon cooling to rt the desired ester product
precipitated as pale yellow solid and was isolated by filtration. Yield (over 2 steps) 70-75 % (23-
1
2
5g); mp 106.7-107.9 °C (MeOH). H NMR (CDCl , 400 MHz) δ: 7.77 (1H, s), 7.02 (2H, d, J = 8.8
3
Hz), 6.89 (1H, d, J = 8.0 Hz), 6.78 (1H, dd, J = 8.0 Hz J = 1.6 Hz), 6.73 (1H, d, J = 1.6 Hz), 6.69
1
3
(
2H, d, J = 8.8 Hz), 3.92 (3H, s), 3.80 (3H, s), 3.78 (3H, s), 3.75 (3H, s). C NMR (CDCl , 100
3
MHz) δ: 168.7, 160.3, 149.1, 148.6, 140.2, 132.4, 129.6, 128.6, 127.3, 122.1, 113.7, 112.8, 111.4,
+
5
5.88, 55.81, 55.22, 52.29. ESI-MS: 351.8 [M+Na] . HR-MS: calculated for C H O 329.1389,
1
9
21
5
found 329.1393.
4.1.2. Methyl 3,6,7-trimethoxyphenanthrene-9-carboxylate (6). (E)-methyl 2-(3,4-
dimethoxyphenyl)-3-(4’’-methoxyphenyl)acrylate (5) (4 g, 12.2 mmol) was dissolved in DCM (40
mL) at rt. To this solution anhydrous FeCl (5 g, 31 mmol) was added portionwise. The resulting
3
reaction mixture was stirred at ambient temperature for 3 h after which more anhydrous FeCl (1 g,
3
6
mmol) was added. After a total reaction time of 5 h complete consumption of substrate was
1
achieved (monitored by H NMR). Methanol (~30 mL) was added to this crude mixture resulting in
a homogeneous red-brownish solution, which was subsequently extracted with DCM/water giving
the desired phenanthrene product as dark brown foam after evaporation of the solvents. Filtration of
the dried organic layer over a plug of silica gel (10 g) prior to solvent removal yields the desired

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product as light brown foam. At this scale the isolated yield commonly varied from 60-75% (>90%
1
purity). H NMR (CDCl , 400 MHz) δ: 8.64 (1H, s), 8.43 (1H, s), 7.85 (1H, s), 7.84 (1H, d, J = 8.8
3
Hz), 7.79 (1H, d, J = 2.4 Hz), 7.20 (1H, dd, J = 8.8 Hz J = 2.4 Hz), 4.10 (3H, s), 4.08 (3H, s), 4.02
1
3
(
3H, s), 4.01 (3H, s). C NMR (CDCl , 100 MHz) δ: 168.2, 160.2, 149.9, 148.9, 133.4, 131.8,
3
1
3
31.3, 125.2, 125.0, 124.2, 121.6, 116.0, 106.9, 103.7, 103.2, 55.90, 55.87, 55.53, 52.03. ESI-MS:
+
27.0 [M+H] . HR-MS: calculated for C H O 327.1232, found 327.1240.
1
9
19
5
4.1.3.
(3,6,7)-Trimethoxyphenanthren-9-yl)-methanol
(7).
Methyl
3,6,7-
trimethoxyphenanthrene-9-carboxylate (6) (3.26 g, 10.0 mmol) was dissolved in THF (20 mL) and
cooled to 0 °C. To this vigorously stirred solution, LiAlH (880 mg, 25 mmol) was added in small
4
portions over a period of 10 min. After 30 min the ice bath was removed allowing the reaction
mixture to warm to rt where it was maintained for 2 h. Upon quenching of the reaction mixture with
sat. NH Cl (2 mL) a greyish slurry was obtained which was filtered over a plug of silica (eluent
4
DCM). The desired reduction product was obtained after removal of the volatiles as a pale yellow
1
amorphous solid. Yield 95% (2.82 g). H NMR (CDCl , 700 MHz) δ: 7.79 (1H, s), 7.73 (1H, d, J =
3
2
5
.1 Hz), 7.69 (1H, d, J = 9.1 Hz), 7.51 (1H, s), 7.45 (1H, s), 7.15 (1H, dd, J = 9.1 Hz J = 1.2 Hz),
1
3
.01 (2H, s), 4.06 (3H, s), 4.01 (3H, s), 3.99 (3H, s), 1.92 (1H, br. s). C NMR (CDCl , 175 MHz)
3
δ: 158.3, 149.3, 148.7, 131.3, 131.1, 130.1, 125.7, 125.5, 124.8, 124.4, 115.4, 104.8, 103.9, 103.7,
+
6
2
4.59, 55.91, 55.86, 55.47. ESI-MS: 281.0 [M-OH] . HR-MS: calculated for C H O (M-OH)
1
8
17
3
81.1178, found 281.1186.
4.1.4. 10-(Chloromethyl)-2,3,6-trimethoxyphenanthrene (8). To a vigorously stirred solution of
(3,6,7)-trimethoxyphenanthren-9-yl)-methanol (7) (3.0 g, 10 mmol, in DCM) was added conc. HCl
(37 %, 5 mL) changing the color of the initial pale yellow solution to dark brown. After 2 h at rt the
reaction is directly extracted (DCM/H O) giving the title compound 8 as brown solid after removal
2
1
of the solvent. Yield 98% (3.1 g). H NMR (CDCl , 400 MHz) δ: 7.76 (1H, s), 7.70 (1H, d, J = 2.4
3

ACCEPTED MANUSCRIPT
Hz), 7.66 (1H, d, J = 8.4 Hz), 7.51 (1H, s), 7.40 (1H, s), 7.14 (1H, dd, J = 8.4 Hz J = 2.4 Hz), 4.92
1
3
(
2H, s), 4.05 (3H, s), 4.04 (3H, s), 3.96 (3H, s). C NMR (CDCl , 100 MHz) δ: 158.8, 149.4, 148.9,
3
131.7, 130.4, 127.9, 126.8, 125.2, 125.1, 125.0, 115.6, 104.7, 103.8, 55.96, 55.91, 55.47, 46.11.
ESI-MS (ASAP): 316.1 [radical cation]. HR-MS: calculated for C H O Cl 317.0944, found
1
8
18
3
317.0931.
4.1.5. Potassium 1-[(3,6,7-trimethoxyphenanthren-9-yl)methyl]piperidine-2-carboxylate (9). A
suspension containing rac-pipecolic acid (750 mg, 5.8 mmol) and potassium hydroxide (1.0 g, 17.9
mmol) in isopropanol (6 mL) is stirred at r.t. for 30 min. To this mixture 10-(chloromethyl)-2,3,6-
trimethoxyphenanthrene (8) (1.6 g, 5 mmol) is added portionwise over 30 min creating a beige
slurry. This mixture is stirred at 40 °C for 14 h before cooling to rt. Filtration and washing of this
crude material, with a minimal amount of cold isopropanol (3 mL), yields the title compound 9 as
beige solid which is used in the subsequent step without further purification. Yield 87% (1.9 g > 90
1
%
pure). H NMR (d -DMSO, 700 MHz) δ: 8.81 (1H, s), 7.94 (2H, br. s), 7.73 (1H, d, J = 8.4 Hz),
6
7.39 (1H, s), 7.11 (1H, dd, J = 8.4 Hz J = 2.8 Hz), 4.58 (1H, d, J = 12.6 Hz), 3.97 (3H, s), 3.96 (3H,
s), 3.92 (3H, s), 3.03 (1H, d, J = 12.6 Hz), 2.54 (1H, m), 2.49 (1H, m), 1.68-1.73 (2H, m), 1.50-1.58
1
3
(
2H, m), 1.25-1.30 (1H, m), 1.10-1.18 (2H, m). C NMR (d -DMSO, 175 MHz) δ: 177.7, 158.1,
6
1
5
49.2, 149.0, 131.3, 131.0, 130.0, 127.8, 126.0, 125.5, 124.5, 115.9, 109.6, 104.2, 103.8, 71.8, 60.5,
6.68, 56.11, 55.88, 51.41, 30.87, 25.97, 24.63.
4.1.6.
3,6,7-Trimethoxy-12,13,14,14a-tetrahydro-9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-
15(11H)-one
(10).
Potassium
1-((3,6,7-trimethoxyphenanthren-9-yl)methyl)piperidine-2-
o
carboxylate (9) (900 mg, 2.0 mmol) is added to polyphosphoric acid (~3 g) and stirred at 90 C for
4-5 h. Within 30 min a thick black solution is obtained which is maintained at this temperature until
full conversion of the substrate is obtained (monitored by LC-MS). The reaction mixture is then
cooled to rt and carefully quenched by addition of methanol not allowing the temperature to rise

ACCEPTED MANUSCRIPT
above ~40 °C (ice-bath). The resulting solution is then neutralized by careful addition of a saturated
solution of potassium carbonate. Extractive work-up of this material with DCM/H O gives a crude
2
product which can be purified further by column chromatography (15 % EtOAc/Hex) providing the
1
title compound 10 as a pale yellow solid. Yield 70%. H NMR (CDCl , 700 MHz) δ: 9.26 (1H, d, J
3
=
9.8 Hz), 7.24 (1H, s), 7.72 (1H, d, J = 1.4 Hz), 7.22 (1H, dd, J = 9.8 Hz J = 1.4 Hz), 7.11 (1H, s),
.36 (1H, d, J = 15.4 Hz), 4.07 (3H, s), 4.01 (3H, s), 3.97 (3H, s), 3.65 (1H, d, J = 15.4 Hz), 3.19
1H, d, J = 10.5 Hz), 2.77 (1H, d, J = 10.5 Hz), 2.47 (1H, d, J = 13.3 Hz), 2.40 (1H, t, J = 13.3 Hz),
.96 (1H, d, J = 13.3 Hz), 1.70-1.77 (1H, m), 1.66 (1H, tq, J = 13.3 Hz J = 2.8 Hz), 1.60 (1H, q, J =
4
(
1
1
1
5
1
3
3.3 Hz), 1.45 (1H, tq, J = 13.3 Hz J = 2.8 Hz). C NMR (CDCl , 175 MHz) δ: 197.3, 157.8,
3
51.0, 149.5, 139.3, 130.9, 129.3, 127.3, 123.1, 123.0, 122.7, 115.6, 104.3, 104.3, 103.7, 68.92,
+
5.99, 55.96, 55.89, 55.34, 54.85, 27.38, 24.98, 23.97. ESI-MS: 392.0 [M+H] . HR-MS: calculated
for C H O N 392.1861, found 392.1855.
2
4
26
4
4.1.7. 1-(4-Methoxyphenyl)-2-(1-tert-butoxycarbonylpiperidin-2-yl)ethanol (12). To a solution
of aldehyde 11 (0.34g, 1.5 mmol) in THF (13 mL) at −78 ºC, (4-methoxyphenyl)magnesium
bromide (6 mL, 3 mmol) was added, and the new solution was stirred for 10 min at −78 ºC and then
overnight at rt. After the completion of the reaction, the solvent was evaporated in vacuum, sat.
NH Cl was added and the aqueous layer was extracted 3 times with EtOAc. The combined organic
4
layers were washed with water and brine, dried with Na SO , filtered and evaporated. The residue
2
4
was purified by flash column chromatography (Hex/EtOAc 7:3) to provide the two diastereomer
alcohols 12 as oils. 12a (R-COH) and 12b (S-COH): Yield 92-95%; R = 0.39 (Hex/EtOAc, 7:3);
f
1
2a (R-COH):
found 336.2156; 12b (S-COH):
NMR (CDCl , 400 MHz): δ = 7.32 (2H, d, J = 8.4 Hz), 6.90 (2H, d, J = 8.4 Hz), 4.61 (1H, m), 4.41
= + 35.9 (c = 0.90 in CHCl ), HR-MS: calculated for C H NO 336.2175,
3 19 30 4
1
= − 36.6 (c = 0.92 in CHCl ), HR-MS found 336.2179. H
3
3
(1H, m), 4.04 (1H, m), 3.82 (3H, s), 2.80 (1H, t, J = 11.6 Hz), 2.20 (1H, td, J = 14.0 Hz J = 2.0 Hz),
13
1
.78 – 1.47 (8H, m), 1.52 (9H, s). C NMR (CDCl , 100 MHz): δ = 159.0, 155.6, 136.9, 127.5,
3

ACCEPTED MANUSCRIPT
114.4, 81.08, 70.16, 55.99, 47.36, 40.88, 40.25, 29.97, 29.13, 26.20, 19.87. 12a (S-COH) and 12b
(
R-COH): oils; yields 92-95 %; R = 0.17 (Hex/EtOAc, 7:3); 12a (S-COH):
= + 71.9 (c = 1.06
f
in CHCl ), HR-MS: found 336.2160; 12b (R-COH):
= − 73.3 (c = 0.98 in CHCl ), HR-MS:
3
3
1
found 336.2158. H NMR (CDCl , 400 MHz): δ = 7.32 (2H, d, J = 8.4 Hz), 6.89 (2H, d, J = 8.4
3
Hz), 4.71 (1H, m), 4.38 (1H, m), 3.94 (1H, m), 3.82 (3H, s), 2.82 (1H, td, J = 13.2 Hz J = 1.6 Hz),
2
1
1
.12 (1H, dt, J = 14.4 Hz J = 6.8 Hz), 1.85 (1H, dt, J = 14.4 Hz J = 5.6 Hz), 1.62 – 1.51 (6H, m),
13
.48 (9H, s), 1.43 – 1.40 (1H, m). C NMR (CDCl , 100 MHz): δ = 159.6, 156.2, 137.6, 127.7,
3
14.4, 80.41, 73.03, 55.98, 49.32, 40.86, 40.19, 30.39, 29.17, 25.96, 19.80.
4.1.8. 1-(4-Methoxyphenyl)-2-(1-tert-butoxycarbonylpiperidin-2-yl)ethanone (13). To a solution
of alcohol 12 (0.22 g, 0.65 mmol) in DCM (8.4 mL), Dess-Martin periodinane (0.34 g, 0.78 mmol)
was added and the new mixture was stirred for 1 h at rt. After the completion of the reaction, the
solvent was evaporated in vacuum, 10% K CO was added and the aqueous layer was extracted 3
2
3
times with EtOAc. The combined organic layers were washed with water and brine, dried with
Na SO , filtered and evaporated. The residue was purified by flash column chromatography
2
4
(
Hex/EtOAc 7:3) to provide ketone 13 as oil. Yield 83%; R = 0.38 (Hex/EtOAc, 7:3); 13a:
=
f
−
34.2 (c = 0.95 in CHCl ), HR-MS: calculated for C H NO 334.2018, found 334.2009; 13b:
3 19 28 4
1
=
+ 35.6 (c = 1.00 in CHCl ), HR-MS: found 334.2006. H NMR (CDCl , 400 MHz): δ =
3 3
8.00 (2H, d, J = 8.8 Hz), 6.96 (2H, d, J = 8.8 Hz), 4.82 (1H, m), 4.05 (1H, m), 3.89 (3H, s), 3.18
(1H, dd, J = 14.0 Hz J = 6.4 Hz), 3.12 (1H, dd, J = 14.0 Hz J = 6.4 Hz), 2.90 (1H, td, J = 13.0 Hz J
1
3
=
2.3 Hz), 1.64 (5H, m), 1.41 (10H, s). C NMR (CDCl , 100 MHz): δ = 197.0, 163.6, 154.8,
3
130.6, 130.1, 113.8, 79.57, 55.49, 48.44, 39.44, 38.99, 28.38, 28.13, 25.34, 18.90.
4.1.9. 1-(4-Methoxyphenyl)-2-(piperidin-2-yl)ethanone hydrochloride (14). To a cooled solution
at 0 ºC of ketone 13 (0.31 g, 0.94 mmol) in MeOH (7.5 mL), TMSCl (0.59 mL, 5.7 mmol) was

ACCEPTED MANUSCRIPT
added and the new solution was stirred overnight at rt. After the completion of the reaction, the
solvent was evaporated in vacuum, to provide ketone 14 as oil. Yield 95%; 14a:
= + 17.5 (c =
1
1
.14 in CHCl ); 14b:
= − 18.5 (c = 1.25 in CHCl ) for (S)-14. H NMR (CDCl , 400 MHz): δ
3
3
3
=
9.73 (1H, s), 9.30 (1H, s), 7.92 (2H, d, J = 8.0 Hz), 6.86 (2H, d, J = 8.0 Hz), 3.84 (3H, s), 3.78 –
3
1
5
.72 (1H, m), 3.70 – 3.64 (1H, m), 3.55 – 3.47 (2H, m), 2.97 – 2.91 (1H, m), 2.00 – 1.80 (5H, m),
1
3
.58 – 1.50 (1H, m). C NMR (CDCl , 100 MHz): δ = 195.7, 164.7, 131.4, 129.7, 114.6, 56.21,
3
4.67, 45.82, 41.62, 29.12, 23.08, 22.82.
4.1.10. 2-(2-Bromo-4,5-dimethoxyphenyl)-1-(2-(2-(4-methoxyphenyl)-2-oxoethyl)piperidin-1-
yl)ethanone (15). To a solution of 2-bromo-4,5-dimethoxyphenylacetic acid (0.25 g, 0.90 mmol) in
THF (23 mL), HATU (0.39 g, 0.99 mmol) and DIPEA (0.31 mL, 1.8 mmol) were added and the
new mixture was stirred for 30 min at rt. Then, the solution was cooled at 0 ºC and a solution of
compound 14 (0.24 g, 0.90 mmol) in THF (12 mL) and DIPEA (200 µL) was added and the new
solution was stirred for 1 h at rt. After the completion of the reaction, the solvent was evaporated in
vacuum, sat. NH Cl was added and the aqueous layer was extracted 3 times with DCM. The
4
combined organic layers were washed with water and brine, dried with Na SO , filtered and
2
4
evaporated. The residue was purified by flash column chromatography (Hex/EtOAc 3:7) to provide
compound 15 as oil. Yield 80%; R = 0.25 (Hex/EtOAc, 3:7); 15a: = − 1.2 (c = 0.82 in
= + 1.3 (c =
f
CHCl ), HR-MS: calculated for C H BrNO 490.1229, found 490.1215; 15b:
3
24 29
5
1
0
.86 in CHCl ), HR-MS: found 490.1210. H NMR (CDCl , 400 MHz, amide rotamers 1:1):
3
3
rotamer a δ = 8.05 (2H, d, J = 8.8 Hz), 7.03 (1H, s), 6.97 (2H, d, J = 8.8 Hz), 6.83 (1H, s), 5.33 –
.28 (1H, m), 4.00 (1H, d, J = 16.0 Hz), 3.90 (3H, s), 3.87 (3H, s), 3.85 (3H, s), 3.78 – 3.72 (2H,
5
m), 3.26 (2H, d, J = 6.8 Hz), 3.23 – 3.20 (1H, m), 1.78 – 1.56 (5H, m), 1.46 – 1.30 (1H, m) and
rotamer b δ = 7.93 (2H, d, J = 8.8 Hz), 6.96 (1H, s), 6.95 (2H, d, J = 8.8 Hz), 6.83 (1H, s), 4.78 –
4.74 (1H, m), 4.67 – 4.64 (1H, m), 3.87 (3H, s), 3.85 (3H, s), 3.81 (3H, s), 3.78 – 3.72 (2H, m), 3.18

ACCEPTED MANUSCRIPT
1
3
–
3.09 (2H, m), 2.70 (1H, td, J = 13.0 Hz J = 2.4 Hz), 1.78 – 1.56 (5H, m), 1.46 – 1.30 (1H, m). C
NMR (CDCl , 100 MHz): rotamer a δ = 197.5, 170.4, 164.5, 149.2, 149.0, 131.5, 130.5, 128.1,
3
116.0, 115.3, 114.6, 114.0, 56.74, 56.21, 47.40, 42.72, 41.55, 39.65, 29.99, 26.29, 20.00 and
rotamer b δ = 196.4, 170.1, 164.3, 149.2, 149.0, 131.0, 130.3, 127.7, 116.0, 115.0, 114.6, 113.5,
56.74, 56.14, 50.36, 41.13, 39.25, 38.32, 27.96, 26.16, 19.35.
4.1.11.
3-(2-Bromo-4,5-dimethoxyphenyl)-2-(4-methoxyphenyl)-1,6,7,8,9,9a-
hexahydroquinolizin-4-one (16). A solution of compound 15 (0.37 g, 0.76 mmol) in 5% ethanolic
KOH (15.8 mL) was refluxed for 2 h. After the completion of the reaction, the solvent was
evaporated in vacuum, sat. NH Cl was added and the aqueous layer was extracted 3 times with
4
DCM. The combined organic layers were washed with water and brine, dried with Na SO , filtered
2
4
and evaporated. The residue was purified by flash column chromatography (Hex/EtOAc 4:6) to
provide atropisomer compounds 16 as oils. Yield 85%; 16a (mode 1): R = 0.35 (Hex/EtOAc, 4:6);
f
=
+ 57.6 (c = 0.89 in CHCl ); HR-MS: calculated for C H BrNO 472.1123, found
3 24 27 4
1
472.1110. 16b (mode 1):
= − 58.4 (c = 0.62 in CHCl ); HR-MS: found 472.1109. H NMR
3
(
CDCl , 400 MHz): δ = 7.01 (2H, d, J = 8.4 Hz), 7.00 (1H, s), 6.70 (2H, d, J = 8.4 Hz), 6.46 (1H, s),
3
4.56 (1H, br. d, J = 13.6 Hz), 3.86 (3H, s), 3.76 (3H, s), 3.68 (1H, m), 3.65 (3H, s), 2.86 – 2.69 (3H,
1
3
m), 1.95 – 1.84 (3H, m), 1.58 – 1.44 (3H, m). C NMR (CDCl , 100 MHz): δ = 166.8, 159.8,
3
149.3, 148.7, 146.3, 132.2, 131.3, 131.2, 129.6, 116.1, 115.6, 115.5, 114.0, 56.74, 56.58, 55.82,
5
4.11, 43.46, 38.30, 34.06, 25.37, 23.97. EI-MS 392: [M-Br]. 16a (mode 2): R = 0.23 (Hex/EtOAc,
f
4:6);
= − 39.2 (c = 1.09 in CHCl ); HR-MS: found 472.1113. 16b (mode 2):
= + 40.4
3
1
(
c = 1.10 in CHCl ); HR-MS: found 472.1111. H NMR (CDCl , 400 MHz): δ = 7.04 (2H, d, J =
3
3
8.8 Hz), 6.99 (1H, s), 6.71 (2H, d, J = 8.8 Hz), 6.50 (1H, s), 4.62 (1H, br. d, J = 13.6 Hz), 3.85 (3H,
s), 3.76 (3H, s), 3.70 (3H, s), 3.67 – 3.60 (1H, m), 3.09 (1H, dd, J = 17.2 Hz J = 6.4 Hz), 2.69 –
.63 (2H, m), 1.97 – 1.92 (1H, m), 1.89 – 1.82 (1H, m), 1.76 – 1.73 (2H, m), 1.70 – 1.59 (2H, m).
2

ACCEPTED MANUSCRIPT
1
3
C NMR (CDCl , 100 MHz): δ = 166.1, 159.2, 148.6, 148.0, 145.7, 131.9, 130.5, 130.4, 128.9,
3
115.8, 115.2, 115.0, 113.4, 56.08, 55.95, 55.15, 53.46, 44.64, 36.09, 32.54, 25.10, 24.42.
4.1.12. 3,6,7-Trimethoxy-11,12,13,14,14a,15-hexahydro-9H-phenanthro[9,10-b]quinolizin-9-
one (17). To a solution of compound 16 (0.12 g, 0.24 mmol) in DMA (5.3 mL), Pd(OAc) (13 mg,
2
0.058 mmol), 2′-(diphenylphosphino)-N,N′-dimethyl-(1,1′-biphenyl)-2-amine (28 mg, 0.072 mmol)
and K CO (0.066 g, 0.48 mmol) were added and the new mixture was heated at 125 ºC for 5 h.
2
3
After the completion of the reaction, H O was added and the aqueous layer was extracted 3 times
2
with EtOAc. The combined organic layers were washed with water and brine, dried with Na SO ,
2
4
filtered and evaporated. The residue was purified by flash column chromatography (Hex/EtOAc
4
:6) to provide compound 17 as oil. Yield 60%; R = 0.46 (Hex/EtOAc, 3:7); 17a:
= − 108.4
c = 0.44 in CHCl ), HR-MS calculated for C H NO 392.1861, found 392.1850; 17b: = +
3 24 26 4
f
(
1
1
8
4
10.2 (c = 0.65 in CHCl ), HR-MS found 392.1847. H NMR (CDCl , 400 MHz): δ = 9.41 (1H, s),
3 3
.01 (1H, d, J = 8.8 Hz), 7.88 (1H, d, J = 3.2 Hz), 7.87 (1H, s), 7.23 (1H, dd, J = 9.2 Hz J = 2.4 Hz),
.74 (1H, br. d, J = 11.6 Hz), 4.12 (6H, s), 4.05 (3H, s), 3.64 – 3.56 (1H, m), 3.52 (1H, dd, J = 16.4
Hz J = 4.8 Hz), 3.05 (1H, dd, J = 16.0 Hz J = 5.2 Hz), 2.90 (1H, td, J = 13.2 Hz J = 3.2 Hz), 2.01
1
3
(
1H, br. d, J = 10.0 Hz), 1.93 – 1.91 (2H, m), 1.67 – 1.48 (3H, m). C NMR (CDCl , 100 MHz): δ
3
=
168.2, 160.2, 150.2, 149.0, 135.1, 133.9, 127.2, 126.4, 125.2, 123.4, 120.3, 116.1, 109.5, 105.1,
103.7, 56.63, 56.52, 56.19, 53.54, 43.48, 36.65, 33.37, 25.39, 23.64. EI-MS: 391 [M].
4.1.13.
3,6,7-Trimethoxy-11,12,13,14,14a,15-hexahydro-9H-dibenzo[f,h]pyrido[1,2-
b]isoquinoline, Rac-boehmeriasin A (1, path A). To a solution of 3,6,7-trimethoxy-12,13,14,14a-
tetrahydro-9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-15(11H)-one (10) (391 mg, 1.0 mmol) in THF
(
5 mL, cooled to 0 °C) LiAlH (100 mg, 2.6 mmol) was added portionwise. After 10 min the
4
mixture was allowed to warm to rt where it was stirred for 2 h, prior to careful quenching by
addition of aqueous NH Cl solution. After aqueous extraction with DCM the combined organic
4

ACCEPTED MANUSCRIPT
layers were dried over anhydrous Na SO and evaporated under reduced pressure to give the
2
4
intermediate aminoalcohol product as yellow oil which was not purified further. This crude material
was redissolved in DCM (2 mL) and combined with TFA (0.3 mL) and Et SiH (0.3 mL). After
3
o
stirring for 4 h at 40 C the reaction had reached completion (monitored by LC-MS) and was
quenched by addition of aqueous NaHCO solution. The crude product was isolated after aqueous
3
extraction as yellow foam. Final purification was accomplished by flash column chromatography
furnishing racemic boehmeriasin A (1) as solid (308 mg, 82%) after evaporation of the solvents. IR
(neat) ν 2931.1 (m), 1610.4 (m), 1511.5 (s), 1467.9 (s), 1253.9 (s), 1201.9 (s), 1138.6 (s), 1038.7
(s), 837.8 (m), 783.1 (m), 728.9 (s) cm-1. LR-MS (ESI): 377.9 (M+H). HR-MS: calculated for
C H O N 378.2069, found 378.2055 (ꢀ -3.7 ppm).
24
28
3
HPLC: (AD 5 cm, EtOH: Hexane 1:9, 1 ml/min, 22 °C) can be used in order to achieve resolution
of racemic boehmeriasin A (see Supporting Information).
4.1.14.
3,6,7-Trimethoxy-11,12,13,14,14a,15-hexahydro-9H-dibenzo[f,h]pyrido[1,2-
b]isoquinoline, boehmeriasin A (1, path B). To a cooled at 0 ºC suspension of LiAlH (0.020 g,
4
0.49 mmol) in THF (5 mL), a solution of compound 17 (48 mg, 0.12 mmol) in THF (2.5 mL) was
added dropwise and the new mixture was refluxed for 2 h. After the completion of the reaction, the
reaction mixture was cooled at 0 ºC, and carefully quenched by addition of 10% NaOH aqueous
solution and after the THF was evaporated in vacuum. Then, the aqueous layer was extracted 3
times with EtOAc. The combined organic layers were washed with water and brine, dried with
Na SO , filtered and evaporated. The residue was purified by flash column chromatography
2
4
(
DCM/MeOH 9.6:0.4) to provide boehmeriasin A (1) as solid. Yield 80%; R = 0.24 (DCM/MeOH,
f
9.6:0.4); 1a:
= – 79.2 (c = 0.12 in MeOH), HR-MS: calculated for C H O N 378.2069,
24 28 3
found 378.2057. Anal. Calcd for C H NO : C, 76.36; H, 7.21; N, 3.71. Found: C, 75.58; H, 7.02;
2
4
27
3
N, 3.56; 1b:
= + 80.6 (c = 0.10 in MeOH), HR-MS: found 378.2058; Found: C, 75.53; H,
1
7
.04; N, 3.53. H NMR (CDCl , 400 MHz): δ = 7.92 – 7.90 (3H, m), 7.22 (1H, dd, J = 8.8 Hz J =
3

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2.4 Hz), 7.14 (1H, s), 4.64 (1H, d, J = 15.2 Hz), 4.12 (3H, s), 4.07 (3H, s), 4.03 (3H, s), 3.58 (1H, d,
J = 15.2 Hz), 3.30 (1H, d, J = 11.2 Hz), 3.18 (1H, dd, J = 16.4 Hz J = 2.8 Hz), 2.94 (1H, dd, J =
1
1
1
2
6.4 Hz J = 6.0 Hz), 2.41 – 2.29 (2H, m), 2.03 (1H, d, J = 13.2 Hz), 1.92 – 1.78 (3H, m), 1.56 –
13
.45 (2H, m). C NMR (CDCl , 100 MHz): δ = 158.2, 150.1, 148.9, 130.9, 126.6, 125.9, 125.7,
3
24.9, 123.9, 115.4, 105.3, 104.7, 103.8, 58.19, 57.05, 56.84, 56.66, 56.19, 35.35, 34.44, 26.67,
5.05.
4.2. Virtual Screening
(R)- and (S)-boehmeriasin A were used as input structures for Hurakan running the jobs on default
parameters.
4
4
.3. Biological Evaluation.
.3.1. Cell proliferation: Endothelial cells. Bovine aortic endothelial cells (BAEC) and human
dermal microvascular endothelial cells (HMEC-1) were seeded in 48-well plates at 10,000
cells/well and 20,000 cells/well, respectively. After 24 h, 5-fold dilutions of the compounds were
added. The cells were allowed to proliferate 3 days (or 4 days for HMEC-1) in the presence of the
compounds, trypsinized, and counted by means of a Coulter counter (Analis, Belgium). Tumor
cells. Human cervical carcinoma (HeLa) cells were seeded in 96-well plates at 15,000 cells/well in
the presence of different concentrations of the compounds. After 4 days of incubation, the cells
were trypsinized and counted in a Coulter counter. Suspension cells (Mouse leukemia L1210 and
human lymphoid Cem cells) were seeded in 96-well plates at 60,000 cells/well in the presence of
different concentrations of the compounds. L1210 and Cem cells were allowed to proliferate for 48
h or 96 h, respectively and then counted in a Coulter counter. The 50% inhibitory concentration
(
IC ) was defined as the compound concentration required to reduce cell proliferation by 50%.
50
Combretastatin A-4 phosphate was added as reference compound.

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4.3.2. Analysis of in vitro sirtuin inhibition. SIRT1 and SIRT2 in Vitro Assay. The compounds
were studied using the Fluor de Lys fluorescence assays which are described in the BioMol product
sheet (Enzo Life Sciences). In assays the BioMol KI177 substrate was used for SIRT1 and the
KI179 substrate for SIRT2. The determined Km value of SIRT1 for KI177 was 58 µM and the Km
3
1
of SIRT2 for KI179 was 198 µM. The Km values of SIRT1 and SIRT2 were 558 µM and 547µM
for NAD+ reported by BioMol, respectively. Briefly, assays were carried out using the Fluor de Lys
acetylated peptide substrate at 0.7 Km and NAD+ (Sigma N6522 or BioMol KI282) at 0.9 Km,
recombinant GST-SIRT1/2-enzyme and SIRT assay buffer (KI286). GST-SIRT1 and GST-SIRT2
3
2, 33
were produced as described previously.
The buffer together with Fluor de Lys acetylated
peptide substrate, NAD+ and DMSO/compounds in DMSO (2.5 µL in 50 µL total reaction volume;
DMSO from Sigma, D2650) were preincubated for 5 min at room temperature. Then enzyme was
added to start the reaction. The reaction mixture was incubated for one hour at 37 °C and after that,
Fluor de Lys developer (KI176) and 2 mM nicotinamide (KI283) in SIRT assay buffer (total
volume 50 µL) were added. The incubation was continued for 45 min at 37 °C. Finally,
fluorescence readings were obtained using EnVision 2104 Multilabel Reader (PerkinElmer) with
excitation wavelength 370 nm and emission 460 nm. The Fluor de Lys fluorescence assays of
sirtuins are regularly performed with compounds from our own collections to calibrate data between
assay runs.
4.3.3. Topoisomerase-Mediated DNA Relaxation. Supercoiled pBR322 plasmid DNA (0.25 µg,
Fermentas Life Sciences) was incubated with 1U topoisomerase II (human recombinant
topoisomerase II α, USB Corporation) or 2U topoisomerase I (human recombinant topoisomerase I,
TopoGen) and the test compounds as indicated, for 60 min at 37 °C in 20 µL reaction buffer.
Reactions were stopped by adding 4 µL stop buffer (5% sodium dodecyl sulfate (SDS), 0.125%
bromophenol blue, and 25% glycerol), 50 µg/mL proteinase K (Sigma) and incubating for a further

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30 min at 37 °C. The samples were separated by electrophoresis on a 1% agarose gel at room
temperature. The gels were stained with ethidium bromide 1 µg/mL in TAE buffer (0.04 M Tris-
acetate and 0.001 M EDTA), transilluminated by UV light, and fluorescence emission was
visualized by a CCD camera coupled to a Bio-Rad Gel Doc XR apparatus.
4.3.4. Topoisomerase II-mediated DNA cleavage. Reaction mixtures (20 µL) containing 10 mM
Tris-HCl (pH=7.9), 50 mM NaCl, 50 mM KCl, 5 mM MgCl , 0.1 mM EDTA, 15 µg/mL bovine
2
serum albumin, 1 mM ATP, 0.25 µg pBR322 plasmid DNA (Fermentas Life Sciences), 10 U
topoisomerase II (human recombinant topoisomerase II α, USB Corporation) and test compounds
were incubated for 60 min at 37 °C. Reactions were stopped by adding 4 µL stop buffer (5% SDS,
0.125% bromophenol blue and 25% glycerol), 50 µg/mL proteinase K (Sigma) and incubating for a
further 30 min at 37 °C. The samples were separated by electrophoresis on a 1% agarose gel
containing ethidium bromide 0.5 µg/mL at room temperature in TBE buffer (0.09 M Tris-borate
and 0.002 M EDTA), transilluminated by UV light, and fluorescence emission was visualized by a
CCD camera coupled to a Bio-Rad Gel Doc XR apparatus.
4.4.5. Topoisomerase I-mediated DNA cleavage. Reaction mixtures (20 ꢁL) containing 35 mM
Tris-HCl (pH=8.0), 72 mM KCl, 5 mM MgCl , 5 mM DTT, 5 mM spermidine, 0.01% bovine
2
serum albumin , 20 ng pBR322 plasmid DNA (Fermentas Life Sciences), 5 U topoisomerase I
(human recombinant topoisomerase I, TopoGen) and test compounds were incubated for 60 min at
37°C. Reactions were stopped by adding 4 ꢁL of stop buffer (5% SDS, 0.125% bromophenol blue
and 25% glycerol), 50 µg/mL proteinase K (Sigma) and incubating for a further 30 min at 37°C.
The samples were separated by electrophoresis on a 1% agarose gel containing ethidium bromide
0.5 ꢁg/mL (Sigma) at room temperature in TBE buffer (0.09 M Tris-borate and 0.002 M EDTA),

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transilluminated by UV light, and fluorescence emission was visualized by a CCD camera coupled
to a Bio-Rad Gel Doc XR apparatus.
4.5. Docking Studies.
Topoisomerase: (R)- and (S)-Boehmeriasin A were docked in the enzyme mediated DNA cleavage
site in the crystal structure of the topoisomerase I and of the topoisomerase II-beta, both in complex
with DNA using AutoDock 4.2 software. Sirtuins: (R)- and (S)-Boehmeriasin A were docked in the
crystal structure of SIRT1 complex with Ex-527(PDB id 4I5I) and the homology model of SIRT2
using Schrödinger’s Glide software.
5
. ACKNOWLEDGEMENTS
This research has been developed under the umbrella of CM1106 COST Action “Chemical
Approaches to Targeting Drug Resistance in Cancer Stem Cells”. D. Passarella and G. Broggini
thank PRIN 2012 “Identification, sustainable synthesis and study of molecular drugs efficacy in
brain tumors treatment.” The authors express their gratitude to Ms. Ioana Stupariu for the revision
of the manuscript.
6
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Captions
Figure 1. Effect of (R)- and (S)-boehmeriasin A on relaxation of supercoiled pBR322 DNA by human
recombinant topoisomerase II. m-AMSA was taken as reference.
Figure 2. Effect of (R)- and (S)-boehmeriasin A on relaxation of supercoiled pBR322 DNA by human
recombinant topoisomerase I.
Figure 3. Effect of (R)- and (S)-boehmeriasin A on the stabilization of covalent DNA-topoisomerase II
complex. m-AMSA was taken as reference.
Figure 4. Effect of (R)- and (S)-boehmeriasin A on the stabilization of covalent DNA-topoisomerase I
complex. Camptothecin (CPT) was taken as reference.
Figure 5. a) Docked structure of (S)-boehmeriasin A. The best fit structure of the principal cluster is
depicted in orange, while the best fit structure of secondary cluster is green. Boehmerasin is intercalated
between DNA bases of the nucleic acid double helix complexed with topoisomerase I. b) Docked structure of
(
S)-boehmeriasin A (orange molecule). The phenantrene moiety is intercalated between base pairs of the
DNA in complex with topoisomerase II; c) (R)-Boehmeriasin A (green) in the putative binding site of
SIRT2. NAD+ (black) and Ex-527(purple) is also presented based on their position in SIRT1.
Scheme 1. Retrosynthetic Plan
a
Scheme 2. Racemic synthesis of boehmeriasin A and its resolution via chiral HPLC (path A)
a
Scheme 3. Enantioselective synthesis of boehmeriasin A (path B)
Table 1. Anti-proliferative activity of (R)- and (S)-boehmeriasin A in comparison with combretastatin A4P.
Table 2. Evaluation of (R)- and (S)-boehmeriasin A against SIRT1 and SIRT2.