5,10b-Ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel bicyclic ring systems with activity against drug resistant cancer cells

Article abstract of DOI:10.1016/j.ejmech.2016.05.004

Plants of the Amaryllidaceae family produce a large variety of alkaloids and non-basic secondary metabolites, many of which are investigated for their promising anticancer activities. Of these, crinine-type alkaloids based on the 5,10b-ethanophenanthridin

Full text of DOI:10.1016/j.ejmech.2016.05.004

Accepted Manuscript
5,10b-Ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel
bicyclic ring systems with activity against drug resistant cancer cells
Sean Henry, Ria Kidner, Mary R. Reisenauer, Igor V. Magedov, Ria Quintana, Robert
Kiss, Véronique Mathieu, Florence Lefranc, Ramesh Dasari, Antonio Evidente,
Xiaojie Yu, Xiuye Ma, Alexander Pertsemlidis, Regina Cencic, Jerry Pelletier, David
A. Cavazos, Andrew J. Brenner, Alexander V. Aksenov, Snezna Rogelj, Alexander
Kornienko, Liliya V. Frolova
PII:
S0223-5234(16)30370-1
10.1016/j.ejmech.2016.05.004
EJMECH 8591
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 January 2016
Revised Date: 1 May 2016
Accepted Date: 2 May 2016
Please cite this article as: S. Henry, R. Kidner, M.R. Reisenauer, I.V. Magedov, R. Quintana, R.
Kiss, V. Mathieu, F. Lefranc, R. Dasari, A. Evidente, X. Yu, X. Ma, A. Pertsemlidis, R. Cencic, J.
Pelletier, D.A. Cavazos, A.J. Brenner, A.V. Aksenov, S. Rogelj, A. Kornienko, L.V. Frolova, 5,10b-
Ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel bicyclic ring systems
with activity against drug resistant cancer cells, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.05.004.
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ACCEPTED MANUSCRIPT
5
,10b-Ethanophenanthridine Amaryllidaceae Alkaloids Inspire the
Discovery of Novel Bicyclic Ring Systems with Activity Against Drug
Resistant Cancer Cells†
a
a
a
a
a
Sean Henry, Ria Kidner, Mary R. Reisenauer, Igor V. Magedov, Ria Quintana,
b
b
c
d
Robert Kiss, Véronique Mathieu, Florence Lefranc, Ramesh Dasari, Antonio
e
f
f
f
g
Evidente, Xiaojie Yu, Xiuye Ma, Alexander Pertsemlidis, Regina Cencic, Jerry
g
h
h
i
Pelletier, David A. Cavazos, Andrew J. Brenner, Alexander V. Aksenov, Snezna
a
d,*
a,*
Rogelj, Alexander Kornienko, and Liliya V. Frolova
a
Departments of Chemistry and Biology, New Mexico Institute of Mining and Technology, Socorro,
NM 87801, USA
b
Laboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université
Libre de Bruxelles (ULB), Campus de la Plaine, CP205/1, Boulevard du Triomphe, Brussels, Belgium
c
Service de Neurochirurgie, Hôpital Erasme, ULB, 808 route de Lennik, 1070 Brussels, Belgium
d
Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666, USA
e
Dipartimento di Scienze Chimiche, Universita’ di Napoli Federico II, Complesso Universitario
Monte Sant’Angelo, Via Cintia 4, 80126 Napoli, Italy
1

ACCEPTED MANUSCRIPT
f
Greehey Children’s Cancer Research Institute, UT Health Science Center at San Antonio, 8403 Floyd
Curl Drive, San Antonio, TX 78229, USA
g
Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada
h
Cancer Therapy and Research Center, The University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA
i
Department of Chemistry, North Caucasus University, 1a Pushkin St., Stavropol 355009, Russian
Federation
*
Corresponding authors. Email addresses: a_k76@txstate.edu (A. Kornienko); lfrolova@nmt.edu
(
L. Frolova).
th
†
This manuscript is dedicated to Prof. Yuri I. Smushkevich on the occasion of his 80 birthday.
Abstract
Plants of the Amaryllidaceae family produce a large variety of alkaloids and non-basic secondary
metabolites, many of which are investigated for their promising anticancer activities. Of these, crinine-
type alkaloids based on the 5,10b-ethanophenanthridine ring system were recently shown to be
effective at inhibiting proliferation of cancer cells resistant to various pro-apoptotic stimuli and
representing tumors with dismal prognoses refractory to current chemotherapy, such as glioma,
melanoma, non-small-cell lung, esophageal, head and neck cancers, among others. Using this
discovery as a starting point and taking advantage of a concise biomimetic route to the crinine
skeleton, a collection of crinine analogues were synthetically prepared and evaluated against cancer
2

ACCEPTED MANUSCRIPT
cells. The compounds exhibited single-digit micromolar activities and retained this activity in a variety
of drug-resistant cancer cell cultures. This investigation resulted in the discovery of new bicyclic ring
systems with significant potential in the development of effective clinical cancer drugs capable of
overcoming cancer chemotherapy resistance.
Keywords
Glioblastoma; apoptosis resistance; multidrug resistance; translation inhibition; lycorine;
haemanthamine
Abbreviations
ATCC, American Type Culture Collection; DAPI, 4’,6-diamidino-2-phenylindole; DMEM,
Dulbecco’s modified Eagle’s medium; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DSMZ,
Deutsche Sammlung von Mikroorganismen and Zellkulturen; ECACC, European Collection of Cell
Culture; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; GBM, glioblastoma
6
multiforme; HRMS, high resolution mass spectrometry; MGMT, O -methylguanine-DNA-
methyltransferase;
MDR,
multidrug
resistance;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; NSCLC, non-small cell lung cancer; PAO, phenyl arsine oxide; P-gp, P-
glycoprotein; PODO, podophyllotoxin; SAR, structure-activity relationship; PIFA, phenyliodine (III)
bis(trifluoroacetate); TMZ, temozolomide; TMS, tetramethylsilane; TLC, thin layer chromatography;
SD, standard deviation.
3

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1
. Introduction
Apoptosis resistance is a hallmark of cancer, because defects in apoptosis regulators invariably
accompany tumorigenesis and sustain malignant progression. Because most standard chemotherapeutic
1
,2
agents work by inducing apoptosis in cancer cells, its disruption during tumor evolution can promote
intrinsic drug resistance and result in therapy failure. For example, one type of cancer with such
intrinsic resistance to apoptosis induction is glioblastoma multiforme (GBM). GBM cells are generally
highly resistant to many different apoptotic stimuli resulting in low effectiveness of classical pro-
3
,4
apoptotic therapeutic approaches. Indeed, GBM is characterized by a deregulated tumor genome
containing opportunistic deletions of tumor suppressor genes as well as amplification or mutational
5
hyper-activation of receptor tyrosine kinase receptors. These genetic changes result in enhanced
5
survival pathways and systematic defects in the apoptotic machinery. As a result, GBM is associated
4
-8
with dismal prognoses and GBM patients have a median survival expectancy of less than 14 months
when treated with a standard protocol of surgical resection, radiotherapy and chemotherapy with
9
,10
temozolomide (TMZ).
Other apoptosis-resistant cancers include the tumors of the lung, liver,
stomach, esophagus, pancreas as well as melanomas, and they are all associated with dismal
1
1
prognoses.
In addition to intrinsic resistance to chemotherapeutic agents, tumors often develop acquired
resistance, in which they are initially susceptible to treatment and patients respond to chemotherapy
but then become refractory to a broad spectrum of structurally and mechanistically diverse antitumor
1
2,13
agents. This phenomenon, referred to as multidrug resistance (MDR),
usually results from
upregulation of protein pumps, such as P-glycoprotein (P-gp), leading to the reduction of drug
concentration in cancer cells. MDR is one of the major causes of chemotherapy failure, for example
1
4
15
with such widely used anticancer drugs as the vinca alkaloids or the taxanes.
4

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In search of medicinal leads capable of combating drug-resistant cancers, we have been
investigating small molecule constituents of the Amaryllidaceae plants, whose medicinal value has
been recognized for a long time. The use of Amaryllidaceae plants dates back to at least the fourth
century BC, when Hippocrates of Cos used oil from the daffodil Narciclasus poeticus L. for the
1
6
treatment of cancer. In more recent times, a large number of structurally diverse alkaloids, along with
non-basic amidic constituents (isocarbostyrils), possessing a wide spectrum of biological activities
1
7-20
have been isolated from the Amaryllidaceae species.
A number of these natural products, such as
phenanthridines narciclasine, lycorine and so-called crinine-type alkaloids containing a C11,C12
ethylene bridge on the phenanthridine skeleton, such as haemanthamine, haemanthidine and
2
1
bulbispermine (Figure 1), are of interest due to their promising anticancer properties. Thus,
narciclasine and lycorine have been actively investigated for their potent anti-tumor effects, both in
2
2-30
vitro and in vivo, in various pre-clinical models of human cancers by many research groups,
3
1-37
including their evaluation as cytostatic agents active against apoptosis-resistant cancers by us.
The
antiproliferative activity of the crinine-type alkaloids has also been reported by natural product
3
8-44
chemists on numerous occasions.
However, despite some dated work reporting the inhibition of
4
5
46-48
protein biosynthesis by crinine-type alkaloids, the paucity of in-depth biological studies
of the
anticancer effects of these compounds has led to insufficient attention to these agents from the
medicinal chemistry community. We recently showed, however, that these 5,10b-ethanophenanthridine
alkaloids, including haemanthamine, haemanthidine and bulbispermine, appear to be as promising in
3
2,48
the treatment of apoptosis-resistant cancers as their congeners, narciclasine and lycorine.
We
demonstrated that at antiproliferative GI50 doses, these crinine-type alkaloids exhibit non-apoptotic
cytostatic effects, while apoptosis induction (in apoptosis-sensitive cell lines) require 5-10 times higher
3
2
doses. Furthermore, using bulbispermine as a model crinine-type alkaloid, we demonstrated that
4
8
these cytostatic effects occur through impairing the actin cytoskeleton organization.
5

ACCEPTED MANUSCRIPT
This discovery makes the crinine-type alkaloids an excellent starting point for the generation of
compounds able to combat cancers, which are naturally resistant to chemotherapy, such as
glioblastoma, melanoma, non-small-cell lung cancer (NSCLC), among others. The investigation
reported herein describes the generation of a collection of synthetic compounds based on the crinine-
type 5,10b-ethanophenanthridine skeleton with the aim of obtaining new structural leads with activity
against drug-resistant cancer cells.
Figure 1. Structures of selected Amaryllidaceae anticancer constituents
2
. Results and Discussion
2
.1 Chemistry
Our synthetic route is based on the use of a biomimetic synthesis of the crinine skeleton. The results
of radiotracer experiments revealed that the biosynthesis of the crinine skeleton involves an
intramolecular para-para oxidative coupling of O-methylnorbelladine with the subsequent
2
1
intramolecular Michael cyclization to afford noroxomaritidine (Scheme 1). This oxidative phenolic
coupling has been successfully performed in laboratory and the first biomimetic synthesis of the
crinine skeleton was achieved using a vanadium oxytrichloride-promoted reaction reported in 1970 by
4
9
Schwartz and Holton. More recent modifications of this method involved the reactions utilizing
hypervalent iodine to achieve the desired phenolic coupling. Such efforts led to the total syntheses of
6

ACCEPTED MANUSCRIPT
4
9-51
52
52
52
53
54
maritidine,
siculine, oxocrinine, epicrinine, powelline, and 14,15-dideoxycripowellin.
Analysis of all published total syntheses of the crinine-type alkaloids reveals that the biomimetic
strategy provides the shortest route to these natural products. However, prior to our investigation, it
had not been applied to the generation of synthetic analogues with the crinine-like 5,10b-
ethanophenanthridine ring system.
O
HO
MeO
MeO
H
N
HO
HO
NH
O-methylnorbelladine
O
MeO
N
HO
noroxomaritidine
Scheme 1. Biosynthesis of the crinine skeleton
5
2
Thus, to utilize this biomimetic approach, various aromatic aldehydes 1a-l were reacted with
tyramine to form imines, which without isolation were reduced with sodium borohydride to the
corresponding amines 2a-l (Scheme 2). These were protected as trifluoroacetamides 3a-l and then
converted to spirodienones 4a-l upon the oxidative cyclization promoted with phenyliodine (III)
bis(trifluoroacetate) (PIFA) in 2,2,2-trifluoroethanol. Removal of the trifluoroacetamide protection led
to an intramolecular Michael addition to generate the 5,10b-ethanophenanthridine ring system 5a-l.
The structures of the synthesized compounds and reaction yields are given in Table 1. As can be seen,
in all shown examples the key oxidative cyclization of 3→4 was highly regioselective with the
exception of substrates 3h and 3j, where mixtures of isomeric spirodienones 4h/i and 4j/k were
obtained. These subsequently gave pure regioisomeric crinine analogues 5h and 5i as well as 5j and
5
k, respectively (Table 1).
7

ACCEPTED MANUSCRIPT
HO
HO
OH
1
. H N
2
, MeOH, 0.5 h
. NaBH , MeOH, 24 h
TFAA
CHO
a-l
0 oC, py
2
4
1
N
N
O
H
CF3
2
a-l
3a-l
O
O
PIFA
CF CH OH, -25 C
KOH, H O/MeOH
2
o
3
2
rt
N
O
N
CF3
5a-l
4a-l
Scheme 2. General route toward synthetic analogues of the crinine-like alkaloids
Table 1. Compound structures and synthetic yields
Compounds 1
Compounds 2
Compounds 3
Compounds 4
Compounds 5
HO
HO
O
O
O
a
O
O
O
O
O
O
O
O
O
CHO
N
O
N
N
H
N
O
CF3
CF3
7
5%
9
0%
9
7
4%
HO
3
4
3%
HO
O
MeO
MeO
O
MeO
MeO
b
MeO
MeO
MeO
MeO
MeO
MeO
CHO
N
O
N
O
N
N
CF
3
CF3
H
8
9
2%
7%
9%
66%
66%
HO
HO
O
O
O
O
O
O
c
O
O
O
O
O
O
CHO
N
O
N
O
N
N
H
CF3
CF3
5%
8
2%
3
O
6%
HO
HO
O
d
CHO
O
O
N
N
O
N
O
N
O
H
CF3
O
O
O
CF
3
O
O
O
O
9
5%
5
3%
2
6%
8
0%
8

ACCEPTED MANUSCRIPT
HO
HO
O
MeO
BnO
O
O
O
O
MeO
BnO
e
MeO
BnO
MeO
BnO
MeO
BnO
CHO
N
O
N
O
N
H
CF
N
N
N
N
3
CF
3
8
9
9%
9
8
5%
5
O
5%
7
3
4%
0%
HO
HO
BnO
MeO
BnO
MeO
f
BnO
MeO
BnO
MeO
BnO
MeO
CHO
N
O
N
O
N
H
CF
3
CF
3
7%
1%
4
O
1%
HO
HO
BnO
BnO
BnO
BnO
g
h
i
BnO
BnO
BnO
BnO
BnO
BnO
CHO
N
O
N
O
N
H
CF3
CF3
used as crude
6
8
4%
2
O
0%
44%
40%
HO
HO
BnO
CHO
BnO
N
O
BnO
N
O
BnO
N
CF3
H
CF3
BnO
9
5%
8%
2
1%
O
O
BnO
OBn
N
N
CF3
O
4
5%
1
1%
HO
HO
O
S
O
j
S
S
S
S
CHO
N
N
O
N
O
N
H
CF3
CF3
4
0% (from 4k+4j)
6
0%
isolated as inseparable
7
7%
mixture with 4k
O
O
k
S
S
N
N
CF3
O
4
5% (from 4k+4j)
isolated as inseparable
mixture with 4j
9

ACCEPTED MANUSCRIPT
HO
HO
O
S
O
l
S
S
S
S
CHO
N
O
N
N
N
O
H
CF3
CF3
8
9%
4
1%
9
5%
74%
To obtain additional 5,10b-ethanophenanthridines inaccessible by the direct oxidative coupling
approach, crinine alkaloid noroxomaritidine (5m), containing a free phenolic hydroxyl, was prepared
utilizing a published procedure involving debenzylation of spirodienone 4e to phenol 4m followed by
intramolecular cyclization upon treatment with potassium hydroxide (Scheme 3). This synthetically
prepared alkaloid was derivatized at the phenolic hydroxyl to obtain esters 5n, 5o, 5p and converted to
oxime 5q.
O
O
BCl , CH Cl
MeO
BnO
3
2
2
MeO
HO
5
3%
N
O
N
O
CF3
CF3
4e
4m
KOH
H O/MeOH 71%
2
O
rt
CO H
2
O
O
MeO
O
DCC, DMAP, DMF, rt
MeO
BzO
BzCl, py
rt, 52%
MeO
N
76%
N
N
5
o
HO
O
5
n
5m
2
Ac O, py
3 2 4
(NH OH) SO
0 oC, 62%
py/EtOH, rt, 70%
O
NOH
MeO
AcO
MeO
HO
N
N
5
p
5q
Scheme 3. Synthesis and derivatization of noroxomaritidine (5m)
2
2
.2 Pharmacology
.2.1 Structure-Activity Relationship
1
0

ACCEPTED MANUSCRIPT
Because spirodienones 4 have not to our knowledge been evaluated for anticancer activities before,
these compounds, together with the desired 5,10b-ethanophenanthridines 5, were subjected to in vitro
5
5
growth inhibition MTT assay against two cell lines, human HeLa cervical and MCF-7 breast
adenocarcinomas (Table 2). Depending on the type of the aromatic ring A, the antiproliferative GI50
values for 5,10b-ethanophenanthridines 5a-5p, containing the C3-carbonyl moiety, ranged from single
digit to >100 micromolar. High potency appeared to be associated with compounds containing large
hydrophobic ether substituents in the ring A portion of the molecule (e.g., 5e-5i and 5l). The most
noteworthy are compounds 5g and 5l, incorporating two benzyl substituents and a novel
benzothiophene-based bicyclic ring system, respectively, possessing single digit micromolar GI50
values. In contrast, the compounds, whose ring A is substituted with large ester moieties (5n and 5o)
were only weakly active, demonstrating the importance of maintaining the electron-rich character of
ring A. Conversion of the C3-carbonyl to the oxime functionality also resulted in a loss of activity (see
5
q). Most unexpectedly, spirodienones 4a-4m produced a range of activities that paralleled that of the
,10b-ethanophenanthridines 5a-5m, to which they are converted upon hydrolytic removal of the
5
trifluoracetamide protection. For example, this is clearly pronounced in the potent 4l/5l, inactive
m/5m or moderately active 4a/5a pairs. This raises the question of whether compounds 4 undergo
4
5
6
intracellular hydrolysis by non-specific amidases or even esterases followed by an intramolecular
cyclization to release the active compounds 5. The hydrolytic lability of the trifluoroacetamide group
5
7
has been observed to be even higher than that of a methyl ester and, although it appears there are no
examples of this functionality in clinically approved drugs, ester-containing prodrugs that undergo
5
8
hydrolysis to release the active drug are common.
Table 2. Antiproliferative activities of spirodienones 4 and 5,10b-ethanophenanthridines 5
1
1

ACCEPTED MANUSCRIPT
compound
a
compound
a
compound
a
cell viability
cell viability
cell viability
GI50, µM
GI50, µM
GI50, µM
HeLa
24 ± 2
25 ± 3
83 ± 5
64 ± 3
21 ± 1
13 ± 2
>100
MCF-7
30 ± 2
24 ± 1
>100
HeLa
34 ± 4
17 ± 2
17 ± 1
3 ± 1
MCF-7
34 ± 2
17 ± 0
25 ± 2
4 ± 0
HeLa
NI
MCF-7
NI
4
5
4
5
a
a
b
b
4f
5f
4k
5k
4l
25 ± 5
10 ± 0
8 ± 1
>100
>100
35 ± 1
43 ± 2
>100
>100
44 ± 2
7 ± 1
5 ± 0
>100
>100
45 ± 1
4g
5g
4h
5h
4i
>100
5l
4
5
c
c
34 ± 12
19 ± 1
>100
16 ± 0
8 ± 0
40 ± 1
14 ± 1
14 ± 2
11 ± 1
NI
4m
5m
5n
5o
5p
5q
4
5
d
d
11 ± 2
7 ± 1
61 ± 5
38 ± 2
12 ± 1
73 ± 3
48 ± 3
15 ± 1
5i
53 ± 1
b
4
5
e
e
4j
5j
NI
>100
>100
8 ± 1
12 ± 0
a
Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative to a
b
DMSO control ± SD from two independent experiments, each performed in 4 replicates, as determined by the MTT assay. NI = not
isolated in pure form
2
.2.2 Activity against cells exhibiting intrinsic resistance to apoptosis, MDR phenotype, stem-like
cell properties and other clinically relevant types of drug resistance
4
8
Based on our previous studies with the alkaloid bulbispermine, found to retain activity against
apoptosis-resistant glioblastoma cells, the synthesized compounds were evaluated for in vitro growth
5
9,60
61
61
59
inhibition against a panel of additional cancer cell lines containing U373,
U251, U87, T98G
6
2
human GBM and A549 NSCLC, all exhibiting various degrees and mechanisms of apoptosis
5
9
resistance, as well as an apoptosis-sensitive tumor model, Hs683 anaplastic oligodendroglioma, used
as reference. The obtained GI50 values associated with spirodienone 4l, 5,10b-ethanophenanthridines 5l
and 5g and bulbispermine control are shown in Table 3. The data reveal that these compounds retain
the single-digit micromolar antiproliferatve GI values in this challenging cancer cell panel making
5
0
1
2

ACCEPTED MANUSCRIPT
them more potent than our previously studied lead, alkaloid bulbispermine, displaying double-digit
micromolar potencies.
Table 3. Antiproliferative activities of spirodienone 4l and crinine analogues 5g and 5l against
cancer cells displaying various types of drug resistance
a
GI50 in vitro values (µM)
glioma
U87
lung cancer
H1993
uterine sarcoma
GBM
GBM
031810
MES-
SA/Dx5
compound
Hs683
U373
U251
T98G
A549
H2073
50
MES-SA
0
90909
b
4
5
l
l
5
3
6
7
2
4
1
6
2
15
7
7
8
--
12
3
5
5
5
5
--
--
1
--
9
4
11
7
7
5
g
7
5
5
7
--
--
2
bulbisper
mine
2
7
53
--
--
41
--
--
38
--
--
99
--
--
--
--
--
--
--
--
--
--
--
10
TMZ
--
50
--
>1000
--
--
--
--
paclitaxel
--
0.004
2.4
0.007
a
Average concentration required to reduce the viability of cells by 50% after a 48-96 h treatment with the indicated compounds in
b
varied numbers of replicates, as determined by the MTT assay. -- = not tested
.
It is often observed that cells resistant to various pro-apoptotic stimuli contain a sensitive population
that quickly responds to pro-apoptotic agents, but a significant portion of the cells in the culture resists
60
the effects of the pro-apoptotic agents even at concentrations 100- or 1000-fold of their GI values.
5
0
1
3

ACCEPTED MANUSCRIPT
Figures 2A-C show the absence of such resistant populations in five GBM cell lines treated with
spirodienone 4l and 5,10b-ethanophenanthridines 5g and 5l. Indeed, as can be seen on the example of
apoptosis-resistant A549 NSCLC cells (Figure 2D), crinine analogues 5g and 5l are equally effective
against most of the cells in the A549 NSCLC cultures and, with increasing concentration, rapidly reach
antiproliferative levels of a non-discriminate cytotoxic agent phenyl arsine oxide (PAO). In contrast,
common proapoptotic agents paclitaxel and podophyllotoxin have no effect on proliferation of ca. 50%
of cells in these cultures at concentrations up to 100 µM.
Figure 2. Activity of spirodienone 4l and crinine analogues 5g and 5l against cell populations resistant
to proapoptotic agents determined with the MTT assay. A-C: growth curves of five GBM cell cultures
treated with 4l, 5l and 5g, respectively (one experiment performed in six replicates). D: growth curves
of A549 NSCLC cell cultures treated with 5l, 5g, paclitaxel, phenyl arsine oxide (PAO) and
podophyllotoxin (PODO, two independent experiments, each performed in 4 replicates).
1
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It has been shown that glioma cells grown in neurosphere conditions exhibit stem-like properties
through the ability of self-renewal and differentiation into multiple neural lineages. Furthermore, such
cells recapitulate human gliomas on both histological and genetic levels more faithfully than serum
6
3-66
cultured glioma cell lines when injected into the brains of mice
and are generally resistant to
It is thus noteworthy that spirodienone 4l and 5,10b-
ethanophenanthridine 5l retained similar levels of activity in neurosphere cultures 090909 and 031810
Table 3). The former line, 090909, was derived from a patient who initially responded to TMZ,
6
7-70
radiation and chemotherapy.
(
whereas the latter was derived from the same patient after progressing on temozolomide due to high
6
O -methylguanine-DNA-methyltransferase (MGMT) expression. MGMT is a protein capable of
repairing mutagenic lesions produced by TMZ, therefore elevated MGMT expression confers
resistance to TMZ even at concentrations as high as 1000 micromolar (Table 2). Importantly,
methylation arrays show that almost 50% of patients with GBM have tumors that are unmethylated at
7
1
the MGMT promoter site, leading to temozolomide resistance and poor outcome. To date, no
71
alternative treatment exists for this group of patients.
Further, spirodienone 4l and 5,10b-ethanophenanthridine 5l were evaluated against patient-derived
NSCLC cell line H1993 and its drug-resistant variant H2073. The former was obtained from a lymph
node metastasis isolated prior to chemotherapy, whereas the latter was derived from a regrown tumor
7
2
at the primary site after the patient relapsed several months later. Drug resistance of the H2073 cells
is exemplified by the loss of responsiveness toward paclitaxel by several orders of magnitude relative
to their H1993 counterparts (Table 3). Although the potencies of 4l and 5l also decrease against the
H2073 cells, the drop is significantly less pronounced than that observed with paclitaxel (Table 3).
Furthermore, it was found that both 4l and 5l synergize with paclitaxel in their antiproliferative action
against the H1993 cell line (Figure 3), indicating a potential use of these compounds as adjuvants to
conventional chemotherapy for drug-resistant cancers.
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5

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Figure 3. Combination treatment of paclitaxel with 4l (A and B) and 5l (C and D) illustrating
synergism against H1993 cells. H1993 is an NSCLC cell line and H2073 is its drug-resistant
counterpart.
Finally, to assess whether our compounds are substrates for the MDR cancer efflux systems, 4l, 5l
and 5g were evaluated against the MDR uterine sarcoma cell line MES-SA/Dx5. This cell line was
established from the parent uterine sarcoma MES-SA, grown in the presence of increasing
7
3
concentrations of doxorubicin and is known to be resistant to a number of P-gp substrates. Both
paclitaxel (Table 3) and vinblastine (not shown) displayed more than a thousand fold drop in potency
when tested for antiproliferative activity against the MDR cell line as compared with the parent one. In
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6

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contrast, there was only a small variation in the activities of our compounds, indicating their potential
to overcome clinical multi-drug resistance (Table 3).
2
.2.3 Inhibition of translation
Because the earlier work revealed the translation inhibitory properties associated with the
4
5
74
crinine-type alkaloids it was important to test benzothiophene 5l in quantitative assays assessing
this biological activity (Figure 4). As such, we performed in vitro translation assays in both Krebs-2
and rabbit reticulocyte lysates programmed with the bicistronic FF/HCV/Ren mRNA (Figure 4A).
Compared to previously characterized inhibitors of translation initiation (Hipp; hippuristanol) and
elongation (Aniso; anisomycin, Lyco; lycorine, and Haeman; haemanthamine), 5l (CR-10) showed
modest activity at inhibiting translation from both the FF (firefly) and Ren (renilla) cistrons (~25%
inhibition). We then assessed the potency of 5l in vivo by monitoring polysome profiles (Figure 4B).
The polysome distribution revealed that cells treated with lycorine and haemanthamine retained intact
polysomes as previously reported for amaryllidaceae alkaloids which “freeze” polysomes and do not
4
5
allow run-off or disaggregation of polysomes. In contrast, 5l (CR-10) led to a complete
disaggregation of polysomes (Figure 4B). We then probed extracts from HeLa cells exposed to
lycorine, haemanthamine, or 5l and found that 5l (CR-10) caused profound eIF2α phosphorylation,
compared to vehicle, lycorine or haemanthamine (Figure 4C). These results indicate that 5l is not an
effective inhibitor of translation in vitro, but blocks translation in vivo by a mechanism that appears
distinct from lycorine and haemanthamine involving eIF2α phosphorylation.
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8

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Figure 4. 5l (CR-10) Blocks Translation Initiation. A. Effect of CR-10 on cap- and HCV- mediated in
vitro translation in Krebs-2 extract and rabbit reticulocyte lysate (RRL). Upper panel: Schematic
diagram of bicistronic mRNA used for in vitro translation assays. Lower panels: Krebs-2 translation
extracts (left) or RRL (right) were programmed with FF/HCV/Ren mRNA and incubated with vehicle
(0.5% DMSO) or the indicated compounds. The relative luciferase activity compared to the DMSO
control from 2 independent translation reactions along with the error of the mean is presented.
Reactions contained 1 µM hippuristanol (Hipp), 10 µM anisomycin (aniso), 50 µM lycorine (Lyco), 50
µM haemanthamine (Haeman), or 50 µM 5l (CR-10). B. Polysome profiling analyses of HeLa S3 cells
exposed to 50 µM compound for 1 h. Polysomes for Lyco and 5l (CR-10) are from the same
experiment so the DMSO control profile is the same in the two plots shown. C. 5l (CR-10) induces
phosphorylation of eIF2� . HeLa S3 cells were treated with 50 µM compound for 1 h. Extracts were
prepared, fractioned by SDS-PAGE, and transferred to a Immobilon-P PVDF membrane. Western
blots were performed with the antibodies directed to the indicated proteins.
3
. Conclusion
Drug resistance is one of the main causes for the failure of cancer chemotherapy. It can be intrinsic,
involving apoptosis-resistant cancers such as glioma, melanoma or NSCLC, among others, or acquired,
in which tumors innately respond to chemotherapy but eventually become refractory to a broad
spectrum of structurally and mechanistically diverse antitumor agents. The results presented herein
demonstrate the potential of a synthetic collection of compounds inspired by the crinine group of the
Amaryllidaceae alkaloids for the treatment of drug-resistant cancers, regardless of whether the latter
harbor intrinsic or acquired resistance mechanisms. The structural scaffold associated with the
benzothiophene ring system 5l represents a new chemotype, whose further investigation is warranted
by the described findings and should be facilitated by the straightforward biomimetic synthetic
1
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methodology utilized in this study. The spirodienone ring system, such as in 4l, also represents an
interesting structural type, which may serve as a prodrug of 5l, undergoing enzymatic removal of the
trifluoroacetamide group with the subsequent intramolecular cyclization to produce 5l in the cell.
Although direct proof of this hypothesis requires further studies, the similarity of the biological effects
between structurally different 4l and 5l certainly strongly supports this argument. In addition to these
experiments, the ongoing work includes optimization of potency, further elucidation of mechanisms
responsible for cytostatic/cytotoxic effects in this series of compounds as well as studies involving
animal models of drug-resistant human cancer.
4
. Experimental Section
4
.1 General Experimental.
All reagents, solvents and catalysts were purchased from commercial sources (Acros Organics and
Sigma-Aldrich) and used without purification. All reactions were performed in oven-dried flasks open
to the atmosphere or under nitrogen or argon and monitored by thin layer chromatography (TLC) on
TLC precoated (250 ꢀm) silica gel 60 F254 glass-backed plates (EMD Chemicals Inc.). Visualization
was accomplished with UV light. Flash column chromatography was performed on silica gel (32-63
1
13
ꢀ
m, 60 Å pore size). H and C NMR spectra were recorded on a Bruker 400 spectrometer. Chemical
shifts (δ) are reported in ppm relative to the TMS internal standard. Abbreviations are as follows: s
singlet), d (doublet), t (triplet), q (quartet), m (multiplet). The synthesized compounds are at least 95%
pure according to an HPLC/MS analysis.
(
4
.2 General procedure for the synthesis of compounds 2a-h, 2j and 2l
o
To a flask containing 4.1 mmol of a selected aromatic aldehyde in 10.2 mL of methanol at 0 C were
added 0.7452 g (5.4 mmol) of tyramine. The solution was allowed to stir for 45 min and 0.1808 g (4.8
2
0

ACCEPTED MANUSCRIPT
mmol) of sodium borohydride was added. The solution was allowed to stir for 4.5 h, at which point
0
.1679 g (4.4 mmol) of additional sodium borohydride was added. This mixture was stirred overnight
and the formed precipitate was collected via vacuum filtration. No further purification was required at
5
1
50
50
this stage. The NMR spectral data for known compounds (2a, 2b, and 2e ) matched those in the
literature. The characterization of all new compounds is given below.
4
9
5
.3 Compound 2c
5%, white solid, mp: 91-93 C (decomp.); H NMR (CDCl
H), 4.22 (m, 4H), 3.69 (broad s, 2H), 2.84 (t, J=7.1 Hz, 2H), 2.72 (t, J=7.1 Hz, 2H); C NMR
o
1
3
): δ 6.96 (d, J=8.0 Hz, 2H), 6.78-6.66 (m,
1
3
(
CDCl ): δ 154.6 (C), 143.4 (C), 142.6 (C), 132.9 (C), 131.1 (C), 129.8 (CHx2), 121.3 (CH), 117.2
3
(
CHx2), 117.1 (CH), 115.6 (CH), 64.3(CH x2), 53.1 (CH ), 50.2 (CH ), 35.0 (CH ); HRMS calc’d for
2
2
2
2
+
C H NO (M+H) : 286.1438, found 286.1441.
1
7
20
3
4
9
5
.4 Compound 2d
5%, white solid, mp: 89-91 C (decomp.); H NMR (CDCl
H), 5.81 (s, 2H), 5.46 (broad s, 1H), 3.79 (s, 2H), 2.85 (t, J=7.1 Hz, 2H), 2.74 (t, J=7.1 Hz, 2H). C
o
1
3
): δ 6.95 (d, J=8.4 Hz, 2H), 6.89-6.65 (m,
13
NMR (CDCl ): δ 156.1 (C), 147.3 (C), 145.5 (C), 129.9 (CHx2), 122.3 (C), 121.6 (C), 120.6 (CH),
3
1
16.3(CHx2), 116.1 (CH), 107.8 (CH), 100.8 (CH ), 50.0 (CH ), 47.8 (CH ), 34.7 (CH ). HRMS
2 2 2 2
+
calc’d for C H NO (M+H) : 272.1281, found 272.1286.
1
6
18
3
4
.5 Compound 2f
3
7%, white solid, mp: 65-67 C (decomp.); H NMR (CDCl ): 7.40 (d, J=7.2 Hz, 2H), 7.33 (t, J=7.6
o
1
9
Hz, 2H), 7.28 (d, J=7.2 Hz, 1H), 7.01 (d, J=8.4 Hz, 2H), 6.82 (d, J=3.6 Hz, 1H), 6.78 (d, J=8.2 Hz,
H), 6.70-6.68 (m, 3H), 5.11 (s, 2H), 3.82 (s, 3H), 3.70 (s, 2H), 2.84 (t, J=6.5 Hz, 2H), 2.73 (t, J=7.2
1
2
1

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1
3
Hz, 2H). C NMR (CDCl ): δ 154.4 (C), 149.8 (C), 147.2 (C), 137.4 (C), 133.2 (CHx2), 131.5 (C),
3
1
7
3
29.8 (C), 128.5 (C), 127.8 (CH), 127.3 (CHx2), 120.3 (CH), 115.4 (CHx2), 114.0 (CH), 112.0 (CH),
+
1.2 (CH ), 56.0 (CH ), 53.6 (CH ), 50.7 (CH ), 35.2 (CH ). HRMS calc’d for C H NO (M+H) :
2
3
2
2
2
23 26
64.1907, found 364.1912.
4
9
1
.6 Compound 2h
o
1
5%, white solid, mp: 83-85 C (decomp.); H NMR (CDCl ): δ 7.39-7.29 (m, 5H), 7.18 (t, J=7.8 Hz,
3
H), 6.97 (d, J=8.4 Hz, 2H), 6.89 (s, 1H), 6.83 (d, J=8.4 Hz, 1H), 6.82 (d, J=6.4 Hz, 1H), 6.65 (d,
1
3
J=8.4 Hz, 2H), 4.94 (s, 2H), 3.75 (s, 2H), 2.85 (t, J=6.6 Hz, 2H), 2.73 (t, J=7.0 Hz, 2H). C NMR
(
CDCl ): δ 159.0 (C), 154.5 (C), 141.4 (C), 137.1 (CHx2), 131.4 (CHx2), 129.8 (CHx2), 129.5 (CH),
3
1
28.6 (C), 127.9 (C), 127.5 (CH), 120.8 (C), 115.5 (CHx2), 114.5 (CH), 113.6 (CH), 69.9 (CH ), 53.7
2
+
(
CH
2
), 50.4 (CH
2
), 35.1 (CH
2
). HRMS calc’d for C22
H24NO
2
(M+H) : 334.1802 found 334.1807.
4
6
7
3
1
3
.7 Compound 2j
o
1
0%, white solid, mp: 103-105 C (decomp.); H NMR (Acetone-d ): δ 7.32 (dd, J=3.0, J=2.5 Hz, 1H),
6
.17-7.16 (m, 1H), 7.02 (dd, J=1.0, J=2.5 Hz, 1H), 7.00 (d, J=8.4 Hz, 2H), 6.70 (d, J=8.4 Hz, 2H),
1
3
.75 (s, 2H), 2.75 (t, J=6.9 Hz, 2H), 2.65 (t, J=6.9 Hz, 2H). C NMR (CDCl ): δ 155.6 (C), 142.5 (C),
3
31.2 (CHx2), 129.5 (C), 127.7 (CH), 125.2 (CH), 120.9 (CH), 115.0 (CHx2), 51.0 (CH ), 48.4 (CH ),
2
2
+
5.4 (CH
2
). HRMS calc’d for C13
H
16NOS (M+H) : 234.0947, found 234.0952.
4
8
1
2
.8 Compound 2l
o
1
9%, white solid, mp: 99-101 C (decomp.); H NMR (CDCl ): δ 7.83-7.81 (m, 1H), 7.70-7.67 (m,
3
H), 7.35-7.29 (m, 2H), 7.00 (d, J=8.3 Hz, 2H), 6.67 (d, J=8.4 Hz, 2H), 4.03 (s, 2H), 2.94 (t, J=7.0 Hz,
1
3
H), 2.76 (t, J=7.0 Hz, 2H). C NMR (CDCl ): δ 155.6 (C), 140.6 (C), 138.7 (C), 136.1 (C), 131.2
3
2
2

ACCEPTED MANUSCRIPT
(CHx2), 129.6 (C), 124.2 (CH), 123.7 (CH), 122.6 (CH), 122.6 (CH), 122.3 (CH), 115.0 (CHx2), 51.3
+
(
CH ), 47.3 (CH ), 35.4 (CH ). HRMS calc’d for C H NOS (M+H) : 284.1104, found 285.1108.
2
2
2
17 18
4
.9 General procedure for the synthesis of compounds 3a-h, 3j and 3l
To a dry flask containing 2.7 mmol of a selected amine 2a-l in 9.6 mL pyridine under argon were
o
added 0.75 mL (5.2 mmol) of trifluoroacetic anhydride. The reaction was stirred at 0 C for 1 day and
quenched with 3 mL of methanol. The solution was concentrated in vacuo, dissolved in ethyl acetate,
and washed twice with water and twice with brine. The solution was dried with sodium sulfate,
filtered, concentrated in vacuo, and the desired product 3a-h, 3j and 3l was purified by column
7
5
chromatography (hexanes/ethyl acetate, 1.5:1). The NMR spectral data for known compounds (3a,
5
0
50
3
b and 3e ) matched those in the literature. The characterization of all new compounds is given
below.
4
8
4
.10 Compound 3c
1
2%, oil; H NMR (CDCl ): δ 6.99 (d, J=11.2 Hz, 2H), 6.95-6.58 (m, 5H), 5.46 (s, 1H), 4.51 (s, 1H),
3
1
3
.26-4.22 (m, 5H), 3.45-3.41 (m, 2H), 2.78 (t, J=7.1 Hz, 1H), 2.72 (t, J=7.1 Hz, 1H). C NMR
(
CDCl ): δ 157.1 (q, J=34.9 Hz, C), 154.8 (C), 154.6 (C), 143.9 (C), 143.8 (C), 143.6 (C), 143.5 (C),
3
1
1
30.1(C), 129.9 (C), 129.8 (C), 129.2 (C), 128.5 (CHx2), 127.8 (CHx2), 121.3 (CH), 120.6 (CH),
17.7 (CF ), 117.7(CHx2), 117.1(CHx2), 116.5 (CH), 115.8 (CH), 115.7 (CH), 115.6 (CH), 64.4
x2), 51.0 (CH
1.9 (CH ). HRMS calc’d for C19
3
(CH
2
2
), 50.9 (CH
2
), 49.1 (CH
2
2 2 2 2
), 48.4 (CH ), 48.3 (CH ), 48.3, 34.3 (CH ), 34.1 (CH ),
+
3
2
H
18
F
3
NNaO
4
(M+Na) : 404.1080, found 404.1085.
4
.11 Compound 3d
2
3

ACCEPTED MANUSCRIPT
1
8
1
0%, oil; H NMR (CDCl ): δ 6.99-6.96 (m, 2H), 6.81-6.72 (m, 5H), 5.94 (d, J=7.0 Hz, 2H), 5.23 (s,
3
1
3
H), 4.65 (s, 1H), 4.43 (s, 1H), 3.49 (m, 2H), 2.83 (t, J=7.8 Hz, 1H), 2.74 (t, J=7.8 Hz, 1H). C NMR
(
CDCl ): δ157.3(q, J=25.7 Hz, C), 155.0 (C), 154.8 (C), 147.6 (C), 147.4 (C), 146.0 (C), 145.8 (C),
3
1
29.9 (C), 129.8 (CH
18.0 (CH), 116.7 (CH), 116.1 (CF
), 108.7 (CH), 108.6 (CH), 101.2 (CH
CH ), 44.2 (CH ), 34.2 (CH ), 31.9 (CH ). HRMS calc’d for C H F NNaO (M+Na) : 390.0924,
2
), 129.7 (CH
2
), 129.0 (C), 122.3 (CH), 122.2 (CH), 121.0 (C), 118.1 (CH),
), 115.8 (CF ), 115.7 (CF ), 115.6 (CF ), 115.3 (CH ), 115.1
), 101.1 (CH ), 48.9 (CH ), , 48.7 (CH ), 46.0 (CH ), 45.9
1
3
3
3
3
2
(CH
2
2
2
2
2
2
+
(
2
2
2
2
18 16
3
4
found, 390.0927.
4
.12 Compound 3f
1
8
1%, oil; H NMR (CDCl ): δ 7.43 (d, J=7.8 Hz, 2H), 7.38-7.34 (m, 2H), 7.32-7.28 (m, 1H), 6.98-6.95
3
(m, 2H), 6.85 (dd, J=6.6, J=4.0 Hz, 1H), 6.79-6.70 (m, 3H), 6.65-6.61 (m, 1H), 5.15 (d, J=3.8 Hz, 2H),
4
.58 (s, 1H), 4.33 (s, 1H), 3.85 (d, J=1.6 Hz, 3H), 3.49-3.43 (m, 2H), 2.80 (t, J=7.4 Hz, 1H), 2.73 (t,
1
3
J=7.1 Hz, 2H), C NMR (CDCl ): δ 157.3 (q, J=86.7 Hz, C)155.1 (C), 154.9 (C), 150.1 (C), 148.2
3
(C), 148.1 (C), 136.9 (C), 136.8 (C), 129.9 (CHx2), 129.7 (CHx2), 128.9 (C), 1, 128.6 (CHx2), 128.4
(
CHx2), 128.0 (CH), , 127.5 (CHx2), 127.4 (CHx2), 120.8 (CH), 120.2 (CF ), 115.8 (CHx2), 114.1
3
(
CH), 114.0 (CH), 111.9 (CH), 111.1 (CH), 71.1 (CH ), 56.1 (CH ), 56.0 (CH ), , 49.6 (CH ), 48.4
2
3
3
2
+
(
CH ), 34.3 (CH ), 32.0 (CH ). HRMS calc’d for C H F NNaO (M+Na) : 482.1550, found
2
2
2
25 24
3
4
4
82.1555.
4
.13 Compound 3g
4%, white solid, mp: 45-47 C; H NMR (CDCl ): δ 7.42-7.26 (m, 10H), 7.03 (d, J=6.6 Hz, 1H), 6.90
o
1
6
3
(
dd, J=8.4, J=1.8 Hz, 2H), 6.86 (dd, J=8.2, J=3.4 Hz, 1H), 6.79-6.77 (m, 1H), 6.74-6.69 (m, 2H), 5.14
d, J=2.2 Hz, 2H), 5.12 (s, 2H), 4.49 (s, 1H), 4.21 (s, 1H), 3.36-3.28 (m, 2H), 2.67 (t, J=7.8 Hz, 1H),
(
2
4

ACCEPTED MANUSCRIPT
1
3
2
1
1
.61 (t, J=7.1 Hz, 1H). C NMR (CDCl ): δ 154.5 (C), 154.3 (C), 148.4 (q, J=21.9 Hz, C) , 137.1 (C),
3
37.0 (C), 136.9 (C), 130.4 (CH ), 129.9 (C), 129.8 (C), 129.5 (C), 128.6 (CHx2), 128.5 (CHx2),
2
28.0 (CH), 127.9 (CH), 127.7 (C), 127.4 (CHx2), 127.3 (CHx2), 121.4 (CH), 120.8 (CF ), 115.8
3
(CHx2), 115.6 (CHx2), 115.5 (CH), 115.1 (CH), 114.9 (CH), 114.5 (CH), 71.4 (CH
2
2
), 71.3 (CH ), 71.2
(
CH
2
), 49.4 (CH
2
), 48.2 (CH
NNaO
2
), 41.2 (CH
2
), 34.3 (CH
2
), 34.1 (CH
2
), 31.9 (CH
2
), 29.7 (CH ). HRMS
2
+
calc’d for C31
H
28
F
3
4
(M+Na) : 558.1863, found 558.1863.
4
.14 Compound 3h
1
8
8%, oil; H NMR (CDCl ): δ 7.43-7.23 (m, 5H), 6.96-6.91 (m, 3H), 6.83-6.71 (m, 4H), 5.93 (d, J=8.3
3
13
Hz, 1H), 5.04 (d, J=4.2 Hz, 2H), 4.61 (s, 1H), 4.35 (s, 1H), 3.48-3.42 (m, 2H), 2.79-2.70 (m, 2H). C
NMR (CDCl ): δ 159.3 (C), 157.4 (C) (q, J=33.9 Hz, C), 155.0 (C), 154.8 (C), 136.8 (C), 136.7 (C),
3
1
1
1
36.6 (C), 136.3 (C), 130.2 (C), 130.1 (C), 129.9 (CHx2), 129.8 (CHx2), 129.0 (CH), 128.7 (CH),
28.2 (CHx2), 128.1 (CHx2), 127.5 (CHx2), 120.6 (CH), 120.0 (CF ), 115.8 (CHx2), 115.7 (CHx2),
14.7 (CHx2), 114.6 (CHx2), 114.2 (CH), 70.2 (CH ), 70.1 (CH ), 51.52 (CH ), 51.49 (CH ), 49.8
3
2
2
2
2
+
(
CH ), 48.7 (CH ), 34.3 (CH ), 31.9 (CH ). HRMS calc’d for C H F NNaO (M+Na) : 452.1444,
2 2 2 2 24 22 3 3
found 452.1448.
4
7
2
.15 Compound 3j
1
7%, oil; H NMR (CDCl
3
): δ 7.33-7.28 (m, 1H), 7.15-7.07 (m, 1H), 6.98-6.9 (m, 2H), 6.78-6.74 (m,
1
3
H), 4.60 (s, 1H), 4.37 (s, 1H), 3.51-3.45 (m, 2H), 2.79 (t, J=7.8 Hz, 1H), 2.70 (t, J=7.2 Hz, 1H). C
): δ 157.0 (C) (q, J=38.1 Hz, C), 154.8 (C), 154.6 (C), 135.9 (C), 135.7 (C), 130.0
CHx2), 129.9 (CHx2), 129.8 (C), 129.2 (C), 127.5 (CH), 127.2 (CH), 126.9 (CH), 126.7 (CH), 123.8
NMR (CDCl
3
(
(
CH), 123.6 (CF ), 115.7 (CHx2), 115.6 (CHx2), 48.6 (CH ),, 47.1 (CH ), 47.0 (CH ), 45.2 (CH ),
3
2
2
2
2
+
3
4.4 (CH ), 32.0 (CH ). HRMS calc’d for C H F NNaO S (M+Na) : 352.0590, found 352.0595.
2 2 15 14 3 2
2
5

ACCEPTED MANUSCRIPT
4
9
6
1
.16 Compound 3l
1
5%, oil; H NMR (CDCl ): δ 7.88-7.84 (m, 1H), 7.72-7.70 (m, 1H), 7.41-7.36 (m, 2H), 7.30 (s, 1H),
3
.95-6.92 (m, 2H), 6.77-6.73 (m, 2H), 5.29 (broad s, 1H), 4.89 (s, 1H), 4.60 (s, 1H), 3.52 (t, J=7.1 Hz,
1
3
H), 3.46 (t, J=7.1 Hz, 1H), 2.80-2.73 (m, 2H). C NMR (CDCl
C), 154.7 (C), 140.7 (C), 140.5 (C), 137.6 (C), 137.3 (C), 130.0 (CHx2), 129.9 (CHx2), 129.8 (C),
29.6 (C), 129.1 (C), 126.0 (CH), 125.0 (CH), 124.7 (CH), 124.6 (CH), 123.2 (CH), 123.1 (CH), 121.6
CH), 120.9 (CH), 118.1 (CH), 115.8 (CHx2), 115.6 (CHx2), 49.0 (CH ), , 48.2 (CH ), 46.3 (CH ),
3
): δ 156.9 (q, J=85.6 Hz, C) 154.8
(
1
(
2
2
2
+
4
4
6.2 (CH ), 43.6 (CH ), 34.5 (CH ), 32.2 (CH ). HRMS calc’d for C H F NNaO S (M+Na) :
2 2 2 2 19 16 3 2
02.0746, found 402.0752.
4
.17 General procedure for the synthesis of compounds 4a-l
To a dry flask containing 1.2 mmol of a selected coupling precursor 3a-l in 6 mL of 2,2,2-
trifluoroethanol under argon were added 0.5956 g (1.4 mmol) phenyliodine bis(trifluoroacetate) in
o
dissolved in 6 mL of 2,2,2-trifluoroethanol. The reaction was stirred at -40 C for 30 min and the
solution was concentrated in vacuo. Purification by column chromatography (hexanes/ethyl acetate,
5
0
50
2
:1) yielded pure spirodienone 4a-l. The NMR spectral data for known compounds (4a, 4b and
5
0
4
e ) matched those in the literature. The characterization of all new compounds is given below.
4
.18 Compound 4c
o
1
3
3
6%, white solid, mp: 71-73 C (decomp.); H NMR (CDCl ): δ 7.00 (d, J=10.1 Hz, 1H), 6.88 (d,
J=10.1 Hz, 1H), 6.81 (s, 1H), 6.65 (s, 1H), 6.55 (s, 1H), 6.24 (d, J=9.9 Hz, 1H), 6.22 (d, J=9.9 Hz,
1
H), 4.69 (d, J=6.6 Hz, 2H), 4.19-4.14 (m, 4H), 3.92-3.86 (m, 2H), 2.31 (tt, J=17.5 Hz, 6.2 Hz, 2H).
1
3
C NMR (CDCl ): δ 185.3 (C), 185.2 (C), 156.4 (q, J=44.7 Hz, C), 152.8 (CHx2), 152.3 (CHx2),
3
2
6

ACCEPTED MANUSCRIPT
1
43.4 (C), 143.3 (C), 142.8 (C), 129.0 (C), 128.9 (C), 128.5 (CH ), 128.4 (CH ), 127.2 (CF ), 127.0
2
2
3
(
CF ), 120.2 (CH), 118.9 (CH), 118.6 (CH), 64.4 (CH ), 64.3 (CH ), 48.7 (CH ), 48.5 (CH ), 48.2 (C),
3 2 2 2 2
4
7.9 (CH ), 45.6 (CH ), 45.5 (CH ), 44.3 (CH ), 36.1 (CH ), 33.9 (CH ). HRMS calc’d for
2
2
2
2
2
2
+
C
19
H
16
F
3
4
NNaO (M+Na) : 402.0924, found 402.0926.
4
.19 Compound 4d
6%, white solid, mp: 147-149 C (decomp.): H NMR (CDCl ): δ 6.99 (d, J=10.3 Hz, 1H), 6.86 (d,
o
1
2
3
J=10.3 Hz, 1H), 6.65-6.54 (m, 2H), 6.26-6.22 (m, 2H), 5.98 (d, J=14.1 Hz, 2H), 4.85 (d, J=15.4 Hz,
1
3
2
H), 3.95-3.91 (m, 2H), 2.37-2.31 (m, 2H). C NMR (CDCl ): δ 185.3 (C), 185.1 (C), 156.5 (q,
3
J=104.5 Hz, C), 152.5 (CHx2), 152.2 (CHx2), 146.9 (C), 146.7 (C), 146.5 (C), 146.0 (C), 129.9 (C),
1
1
29.8 (C), 127.1 (CHx2), 126.9 (CHx2), 123.5 (C), 123.1 (CH), 117.8 (CF ), 117.3 (CF ), 108.3 (CH),
3
3
08.1 (CH), 101.7 (CH
2
), 101.6 (CH
2
), 48.4 (C), 48.0 (C), 44.6 (CH
2
), 41.0 (CH
2
2
), 36.2 (CH ), 34.2
+
(
CH
2
). HRMS calc’d for C18
H
14
F
3
NNaO
4
(M+Na) : 388.0767, found 388.0773.
4
.20 Compound 4f
1%, white solid, mp: 109-111 C (decomp.): H NMR (CDCl ): δ 7.32-7.24 (m, 5H), 6.85 (d, J=10.0
o
1
4
3
Hz, 1H), 6.76 (d, J=13.4 Hz, 1H), 6.70 (d, J=35.9 Hz, 1H), 6.47 (d, J=2.6 Hz, 1H), 6.17 (dd, J=10.1,
1
3
J=3.1 Hz, 2H), 5.00 (s, 2H), 4.73 (d, J=14.6 Hz, 2H), 3.88 (s, 3H), 3.86 (s, 2H), 2.33-2.27 (m, 2H). C
NMR (CDCl ): δ 185.1 (C),185.0 (C),156.4 (q, J=36.6 Hz, C), 152.7 (CHx2), 152.3 (CHx2), 148.8
C), 147.6 (C), 147.5 (C), 136.6 (C), 136.5 (C), 128.7 (CHx2), 128.6 (CHx2), 128.3 (C), 128.1 (C),
3
(
1
3
28.0 (CHx2), 127.8 (CHx2), 127.6 (CH), 127.4 (C), 127.3 (CF ), 127.2 (CHx2), 127.1 (CHx2), 116.1
(
CH), 114.5 (CH), 113.5 (CH), 71.3 (CH ), 71.2 (CH ), 56.2 (CH ), 56.1 (CH ), 48.3 (C), 48.2 (C),
2 2 2 2
+
4
8.0 (C), 44.2 (CH ), 35.5 (CH ), 33.6 (CH ). HRMS calc’d for C H F NNaO (M+Na) : 480.1393,
3 2 2 25 22 3 4
found 480.1398.
2
7

ACCEPTED MANUSCRIPT
4
.21 Compound 4g
0%, white solid, mp: 79-79 C; H NMR (CDCl ): δ 7.43-7.24 (m, 10H), 6.87 (d, J=9.4 Hz, 1H), 6.76
o
1
2
3
(d, J=10.0 Hz, 1H), 6.63 (s, 1H), 6.50 (d, J=1.6 Hz, 1H), 6.19 (dd, J=9.9 Hz, J=6.7 2H), 5.19 (s, 2H),
1
3
5
.01 (d, J=2.4 Hz, 2H), 4.67 (d, J=14.2 Hz, 2H), 3.89-3.84 (m, 2H), 2.32-2.26 (m, 2H). C NMR
): δ 185.2 (C), 185.1 (C), 152.7 (CHx2), 152.3 (CHx2), 148.3 (C), 148.1 (q, J=44.7 Hz, C),
36.7 (Cx2), 136.6 (Cx2), 128.6 (CHx4), 128.5 (CHx4), 128.4 (C), 128.3 (C), 128.2 (CHx2), 128.1
(CDCl
3
1
(
CHx2), 128.0 (C), 127.4 (CHx2), 127.3 (CF ), 127.1 (CHx4), 117.1 (CH), 117.0 (CH), 116.8 (CH),
3
1
16.6 (CH), 71.4 (CH ), 71.4 (CH ), 71.3 (CH ), 71.2 (CH ), 48.3 (CH ), 48.2 (CH ), 48.0 (C), 44.2
2 2 2 2 2 2
+
(
CH ), 35.5 (CH ), 33.6 (CH ), 29.7 (CH ). HRMS calc’d for C H F NNaO (M+Na) : 556.1712,
2 2 2 2 31 26 3 4
found 556.1715.
4
.22 Compound 4h
1%, white solid, mp: 58-58.5 C; H NMR (CDCl ): δ 7.39-7.30 (m, 5H), 7.02-6.95 (m, 3H), 6.89 (d,
o
1
2
3
J=10.2 Hz, 1H), 6.82-6.77 (m, 1H), 6.29-6.25 (m, 2H), 5.02 (s, 2H), 4.78 (d, J=8.0 Hz, 2H), 3.96-3.90
1
3
(
m, 2H), 2.38-2.31 (m, 2H). C NMR (CDCl ): δ 185.3 (C), 185.2 (C), 158.2 (C), 158.1 (C), 156.5 (q,
3
J=77.8 Hz, C), 152.8 (CHx2), 152.4 (CHx2), 136.7 (C), 136.5 (C), 136.4 (C), 131.7 (C), 131.2 (CH),
1
1
4
28.7 (CHx2), 128.2 (CH), 128.1 (CH), 127.5 (CHx2), 127.4 (CHx2), 127.3 (CHx2), 127.1 (CHx2),
17.8 (CF ), 117.0 (CH), 114.8 (CH), 114.7 (CH), 70.2 (CH ), 70.1 (CH ), 49.3 (CH ),49.1 (CH ),
), 36.1 (CH ), 34.0 (CH ). HRMS calc’d for
3
2
2
2
2
8.2 (C), 47.9 (C), 45.6 (CH
2
), 45.5 (CH
2
), 44.5 (CH
2
2
2
+
C
24
H
20
F
3
NNaO
3
(M+Na) : 450.1287, found 450.1292.
4
.23 Compound 4i
2
8

ACCEPTED MANUSCRIPT
o
1
1
1%, white solid, mp: 74-76 C; H NMR (CDCl ): δ 7.31-7.29 (m, 3H), 7.24-7.15 (m, 3H), 6.95-6.77
3
1
3
(
m, 4H), 6.08 (d, J=10.0 Hz, 2H), 4.86 (s, 2H), 4.84 (s, 2H), 3.88-3.81 (m, 2H), 2.34-2.31 (m, 2H). C
NMR (CDCl ): δ 185.0 (C), 158.4 (C), 156.4 (q, J=92.9 Hz, C), 153.5 (CHx2), 153.3 (CHx2), 137.4
3
(
(
(
C), 137.3 (C), 135.4 (C), 135.3 (C), 129.1 (CH), 128.6 (CHx2), 128.3 (CH),127.9 (CHx2), 127.4
CHx2), 125.3 (C), 125.0 (C), 123.8 (CH), 122.9 (CF ), 112.6 (CH), 112.5 (CH), 70.9 (CH ), 48.8
), 45.1 (CH ), 44.2 (CH ), 38.4 (C), 37.0 (C).
3
2
CH
2
), 47.8 (CH
2
), 47.4 (CH
2
), 47.1 (CH
2
), 45.2 (CH
2
2
2
4
.24 Compound 4l
o
1
4
1%, light yellow solid, mp: 89-92 C (decomp.); H NMR (CDCl ): δ 7.84 (d, J=8.1, 1H), 7.71 (t,
3
J=7.8 Hz, 1H), 7.63 (d, J=8.1 Hz, 1H), 7.43-7.31 (m. 2H), 7.13-7.11 (m, 1H), 7.02-6.99 (m, 1H), 6.36-
1
3
6
1
1
1
4
4
.32 (m, 2H), 5.05 (s, 1H), 4.98 (s, 1H), 4.11-4.08 (m, 2H), 2.45-2.39 (m, 2H). C NMR (CDCl ): δ
3
84.7 (C), 156.5 (q, J=58.0 Hz, C), 150.0 (CHx2), 149.5 (CHx2), 140.2 (C), 139.5 (C), 138.8 (C),
38.3 (C), 137.8 (C), 130.3 (C), 129.8 (C), 128.6 (CHx2), 128.4 (CHx2), 125.3 (CH), 125.0 (CH),
24.8 (CH), 122.6 (CH), 122.4 (CH), 121.4 (CH), 121.0 (CF ), 46.0 (CH ), 45.7 (CH ), 45.6 (CH ),
3
2
2
2
+
5.0 (CH ), 42.8 (CH ), 41.9 (CH ), 37.5 (C), 35.7 (C). HRMS calc’d for C H F NNaO S (M+Na) :
2
2
2
23 26
3
2
00.0590, found 400.0593.
4
.25 General procedure for the synthesis of compounds 5a-l
To a flask containing 0.15 mmol of a selected spirodienone 4a-m in 0.78 mL of methanol were added
.8 mL of 10% aqueous potassium hydroxide at room temperature. The reaction was stirred for 30 min
0
and the solution was extracted several times with chloroform. The organic layers were collected,
filtered, and concentrated in vacuo. Purification by column chromatography (chloroform/methanol,
1
4:1) yielded the desired 5,10b-ethanophenanthridine 5a-5l. The NMR spectral data for known
2
9