Synthesis and evaluation of the anti-proliferative and NF-κB activities of a library of simplified tylophorine analogs

Article abstract of DOI:10.1016/j.bmc.2012.07.044

Tylophorine and many related phenanthropiperidine alkaloids are extraordinarily potent anti-proliferative agents. Despite their impressive anti-cancer activity, clinical development of these alkaloids has been hampered by their poor solubility and neurological side effects. Although it has been suggested that developing polar phenanthropiperidines will mitigate these undesired properties, the lack of practical methods for the synthesis of such analogues has limited this effort. Here, we present a concise synthetic approach to N-substituted phenanthropiperidines, which enabled a systematic investigation of structure-activity relationships at an underexplored region of the tylophorine scaffold. This work suggests that ring E of tylophorine is essential for the anti-proliferative activity of the 6,7,10,11-tetramethoxy-1,2, 3,4-tetrahydrodibenzo[f,h]isoquinoline core scaffold.

Full text of DOI:10.1016/j.bmc.2012.07.044

Bioorganic & Medicinal Chemistry 20 (2012) 5893–5900
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of the anti-proliferative and NF-
of a library of simplified tylophorine analogs
j
B activities
⇑
Micah J. Niphakis , Bryant C. Gay , Kwon Ho Hong, Nicholas P. Bleeker, Gunda I. Georg
Department of Medicinal Chemistry and the Institute for Therapeutics Discovery & Development, University of Minnesota, 717 Delaware Street SE, Minneapolis, MN 55414, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tylophorine and many related phenanthropiperidine alkaloids are extraordinarily potent anti-prolifera-
tive agents. Despite their impressive anti-cancer activity, clinical development of these alkaloids has been
hampered by their poor solubility and neurological side effects. Although it has been suggested that
developing polar phenanthropiperidines will mitigate these undesired properties, the lack of practical
methods for the synthesis of such analogues has limited this effort. Here, we present a concise synthetic
approach to N-substituted phenanthropiperidines, which enabled a systematic investigation of structure-
activity relationships at an underexplored region of the tylophorine scaffold. This work suggests that ring
E of tylophorine is essential for the anti-proliferative activity of the 6,7,10,11-tetramethoxy-1,2,3,4-tetra-
hydrodibenzo[f,h]isoquinoline core scaffold.
Received 3 June 2012
Revised 14 July 2012
Accepted 24 July 2012
Available online 3 August 2012
Keywords:
Phenanthropiperidine
Tylophorine
Synthesis
Simplified analogs
Cytotoxicity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
activity in mouse models of cancer,4,8 lupus9 and arthritis6 without
overt signs of toxicity. These intriguing pharmacological properties
Phenanthroindolizidine and phenanthroquinolizidine natural
products (hereon referred to as phenanthropiperidines) have been
extensively studied for over 50 years due to their extraordinary
biological activity.1–3 The NCI (NCI 60 tumor cell lines) and other
research groups have shown that members of this class exhibit
extremely potent anti-proliferative activity against normal and
multi-drug resistant cancer cell lines and possess anti-inflamma-
tory properties.1,3–6 These findings have inspired efforts to develop
novel chemotherapeutic agents based on the phenanthropiperi-
dine scaffold. Although the molecular target(s) of these natural
products have not been unambiguously identified, and may even
differ between members of this class of compounds, recent studies
have shown that these alkaloids exert their growth inhibitory
activity in a manner that is distinct from known anti-cancer
agents.4,7,8
Tylophorine and its hydroxylated analogue, DCB-3503, have
been subjected to the most extensive mechanistic studies (Fig. 1).
DCB-3503, for instance, suppresses protein biosynthesis resulting
in a down-regulation of multiple pro-oncogenic and pro-survival
proteins such as cyclin D1, survivin, b-catenin, p53 and p21.4,8
DCB-3503 specifically interferes with the elongation phase of
translation during protein biosynthesis.4 When regimented cor-
rectly, DCB-3503 has been shown to possess promising therapeutic
suggest that the phenanthropiperidines could enhance our under-
standing of cancer and inflammatory diseases and provide a means
to treat them.
Synthetic approaches to the phenanthropiperidines have
greatly improved over the past few decades and compound
availability is no longer limited by natural abundance.10–15 Despite
these advanced synthetic approaches, a systematic investigation of
phenanthropiperidine structure–activity relationships (SAR) has
remained challenging, because many of the current synthetic
strategies are highly linear, making library synthesis impractical.
A better understanding of phenanthropiperidine SARs would prove
highly beneficial for the further development of phenanthropiperi-
dine chemotherapeutics. There are at least two important reasons
why the parent alkaloids require further development. First, phen-
anthropiperidines are notoriously insoluble. This is most likely
attributable to their high lipophilicity and planarity.16 Secondly,
many members of this class have physicochemical properties con-
ducive to BBB-penetration.17 This is a concern because the one
member of this class to have been studied in humans, tylocrebrine
(Fig. 1), exhibited neurological side effects.18 Although the design
of polar analogs may mitigate CNS-exposure and improve solubil-
ity, attempts to incorporate polar functional groups in a pharmaco-
logically silent manner have been largely unsuccessful. Indeed,
new synthetic approaches that enable the synthesis of informative
libraries19 to systematically identify the essential and nonessential
structural features for bioactivity would be invaluable for this
endeavor.
⇑
Corresponding author.
E-mail address: georg@umn.edu (G.I. Georg).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.044

5894
M. J. Niphakis et al. / Bioorg. Med. Chem. 20 (2012) 5893–5900
OMe
stereogenic center and dramatically simplifying the synthesis.
OMe
MeO
MeO
Importantly, this simplification enables diversification of the
secondary amine through reductive alkylation reactions, which
can be used to access each library member in a single step from
a common intermediate.
MeO
MeO
OH
A
B
C
H
H
N
E
D
N
2. Results and discussion
OMe
OMe
(R)-tylophorine
DCB-3503
2.1. Synthesis of N-substituted phenanthropiperidines
OMe
In order to obtain sufficient quantities of the library precursor,
we first devised a concise and high-yielding sequence that allowed
us to prepare gram quantities of the piperidine 6 (Scheme 1).
4-Bromoveratrole (1) was first dimerized using a PIFA-mediated
oxidative biaryl coupling reaction.24 The resultant dibromoverat-
role dimer 2 was desymmetrized though a lithium-halogen ex-
change using a single equivalent of n-butyllithium. Notably,
these two steps could be carried out on large scale (>5 g) without
relying on chromatography for purification. Following another
lithium-halogen exchange with aryl bromide 3, the biaryl moiety
was added to piperidinone 4 which spontaneously dehydrated to
afford tetrahydropyridine 5. Using our recently developed aryl-
alkene coupling conditions,13 the phenanthrene system could be
constructed in excellent yields. Subsequent deprotection of
the Boc group with TFA provided the desired phenanthropiperidine
6. This sequence provided gram quantities of the key intermediate
6 in 5 steps and 61% overall yield.
Previous SAR studies have shown that amide analogues of the
phenanthropiperidines are substantially less active than the natu-
rally occurring amines.25,26 As such, we chose to prepare a library
of N-alkylated phenanthropiperidines from intermediate 6. We
decided that the reductive amination reaction would be most prac-
tical for this endeavor based on the commercial availability of a di-
verse collection of aldehydes and the amenability of this reaction
for parallel synthesis. It should be noted that phenanthropiperidine
MeO
H
MeO
MeO
N
(R)-tylocrebrine
Figure 1. Structures of phenanthroindolizidine alkaloids tylophorine, DCB-3503
and tylocrebrine.
Herein, we report the synthesis and biological evaluation of a li-
brary of novel, simplified tylophorine analogues in order to gain a
better understanding of phenanthropiperidine SAR. Tylophorine
was used as a representative of the phenanthropiperidines, be-
cause it is one of the most well-studied members of its class. We
chose to focus on the aliphatic portion of the scaffold since SARs
in this region are underexplored. Previous studies have shown that
disconnections within ring D result in a significant loss of activity
or change in their mode of action.20–22 Moreover, ring E was found
to be highly sensitive to heteroatom incorporation.19 Surprisingly,
whether or not ring E is required for activity at all remains
elusive.23 In this regard, we chose to prepare a library of tylopho-
rine analogues lacking ring E, consequently eliminating the sole
OMe
MeO
OMe
Br
PIFA, BF3•Et2O
n-BuLi (1.0 equiv)
MeO
Br
CH2Cl2
–78 ºC
THF; then MeOH
80% (2 steps)
Br
OMe
OMe
1
2
OMe
OMe
MeO
MeO
MeO
t-BuLi, THF
–78 ºC;
Br
N
Boc
then
Boc
O
MeO
N
5
4
OMe
OMe
84%
3
OMe
OMe
MeO
i) VOF3, TFAA, TFA,
EtOAc, CH2Cl2, –78 ºC
NH
ii) TFA:CH2Cl2 (1:3)
91% (2 steps)
MeO
6
Scheme 1. Synthesis of phenanthropiperidine core 6.

M. J. Niphakis et al. / Bioorg. Med. Chem. 20 (2012) 5893–5900
5895
Benzyl
7a (R = H), 74%
OMe
OMe
OMe
MeO
7b (R = 4-NO2), 71%
7c (R = 4-OMe), 81%
7d (R = 3-OMe), 85%
7e (R = 2-OMe), 56%
7f (R = 4-NMe2), 73%
7g (R = 2-OH, 3-OMe), 67%
7h (R = 4-t-Bu), 81%
7i (R = 2-OH), 65%
MeO
MeO
CHO
Na(OAc)3BH
DCE
R
+
NH
N
R
MeO
7a–k
6
OMe
7j (R = 3,5-Cl2), 81%
7k (R = 2,6-(OMe)2, 55%
Na(OAc)3BH
DCE
R
CHO
Heteroaryl
Alkyl
Me
O
7l (R = H), 71%
N
N
OMe
O
R =
O
N
7m (R = CH2Ph), 76%
7n (R = CH2NHBoc), 95%
MeO
7p, 55%
7r, 72%
7q, 91%
N
N
R
HCl
OH
N
Me
O
MeO
7l–t
7o (R = CH2NH2•HCl) 84%
OMe
7s, 72%
7t, 62%
Scheme 2. Synthesis of N-substituted phenanthropiperidine library using reductive amination reactions.
6 is poorly soluble in most organic solvents, limiting the chemistry
that can be used to derivatize this intermediate. Fortunately,
halogenated solvents such as CH2Cl2 and DCE could sufficiently
solubilize this amine, albeit at low concentrations (0.02 M), to en-
able efficient N-alkylations with a diverse collection of commer-
cially available aldehydes. In general, these reactions proceeded
in excellent yield (55–95%) providing the desired benzyl-(7a–k),
alkyl-(7l–o) and heteroaryl-(7p–t) derivatives of tylophorine to
aid our understanding of phenanthropiperidine SAR (Scheme 2).
numerous studies have established SARs for the phenanthrene
and piperidine systems, the necessity of the pyrrolidine (ring E)
system of tylophorine for activity remains underexplored. In order
to assess the potential of these compounds for further develop-
ment, we first tested the anti-proliferative activity of each com-
pound at 5
We were pleased to find that many library members exhibited
greater than 50% inhibition of cell growth at 5 M. Most com-
lM in MCF-7 cells (Fig. 2).
l
pounds, however, were significantly less active than tylophorine.
Notably, the unsubstituted phenanthropiperidine core 6 and N-
methyl analogue 7l, the simplest library members, inhibited cell
proliferation by 50% suggesting that the intact indolizidine and
quinolizidine moieties found in phenanthropiperidine natural
products contribute significantly to their activity. Benzylation of
the nitrogen (7a) resulted in near complete loss of activity;
however, a clear phenyl-substituent effect was observed for these
benzyl analogues. 4-Substituted benzyl groups (7b and 7c)
displayed low activity, whereas 2-substituted benzyl analogues
7e, 7g and 7i were the most potent compounds in the series. The
2.2. Anti-proliferative activity of phenanthropiperidines
The anti-proliferative activity of phenanthropiperidines has
been investigated in many cell lines.1 Interestingly, the most well
studied alkaloids show little discrimination between these cell
lines and even retain their activity in those resistant to the most
commonly used anti-cancer drugs. The NCI 60-tumor cell line
screen of several members of this class bears witness to the
potent and broad-spectrum activity of these alkaloids. Although
Figure 2. Anti-proliferative activity of N-substituted phenanthropiperidines at 5.0 lM in MCF-7 cells.

5896
M. J. Niphakis et al. / Bioorg. Med. Chem. 20 (2012) 5893–5900
Table 1
Anti-proliferative and NF-jB activity of compounds 7e and 7g
Compound
MCF-7
GI50
(l
M)a
NF-j (lM)
B inhibitionbIC50
DU-145
A549
( )-Tylophorinec
7e
7g
0.042 0.001
0.6 0.2
0.071 0.004
1.5 0.1
2.1 0.1
0.045 0.003
1.6 0.1
2.1 0.1
0.030 0.01
15
18.9 0.7
4
3
1
GI50 and IC50 values are reported as the mean SEM from three independent experiments.
a
MCF-7 = human breast carcinoma; DU-145 = human prostate carcinoma; A549/NF-
j
B-luc = human lung carcinoma.
B inhibition was measured by monitoring the luciferase activity from A549/NF- B-luc cells upon treatment with the indicated compound.
GI50 MCF-7: (R)-tylophorine: 0.032 M; (S)-tylophorine 0.042
M.13
b
c
NF-j
j
l
l
2-methoxybenzyl analogue 7e was particularly active showing
ꢀ90% growth inhibition at 5 M. However, incorporation of an
4. Materials and methods
4.1. Materials
l
additional 2-methoxy substituent significantly reduced activity
(7k). N-Alkyl substituted analogues 7l–7n were equipotent to the
unsubstituted analog 6. Boc-deprotection of 7n significantly re-
duced its anti-proliferative activity (7o). We also tested several
heterocyclic analogues, which we hoped would improve the solu-
bility of these highly lipophilic molecules. Unfortunately, only a
slight improvement of activity was observed for the most active
heterocyclic compounds, isooxazole 7r and furan 7s.
All commercially available reagents and solvents were used
without further purification. Flash column chromatography was
carried out on silica gel. TLC was conducted on silica gel 250 mi-
cron, F254 plates. 1H NMR spectra were recorded on a 400 MHz
NMR instrument. Chemical shifts are reported in ppm with TMS
or CHCl3 as an internal standard (TMS: 0.0 ppm, CHCl3/7.26). Data
are reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), inte-
gration and coupling constants (Hz). 13C NMR spectra were re-
corded on a 100 MHz NMR spectrometer with complete proton
decoupling. Chemical shifts are reported in ppm with the solvent
as internal standard (CDCl3: 77.2 ppm).
After obtaining these preliminary results, we analyzed the
most active compounds in more detail. Two compounds, 7e and
7g, were assayed to determine their GI50 values against MCF-7,
DU-145 and A549 cell proliferation. The most active of these
compounds was the 2-OMe analogue 7e, which displayed a GI50
of 600 nM in MCF-7 and 1.5 and 1.6 lM in DU-145 and A549 cells,
respectively (Table 1). Despite this respectable anti-proliferative
activity, this analogue was greater than 10-fold less potent than
the parent compound, tylophorine. Motivated by previous reports
4.2. Synthesis of the phenanthropiperidine core
that tylophorine inhibits NF-
examined the effect of 7e and 7g on NF-
cells stably expressing the luciferase reporter gene downstream
of NF- B. Consistent with previous findings, tylophorine potently
inhibited NF- B activity. In contrast, analogues 7e and 7g only
inhibited this pathway at concentrations of greater than 10 M.
j
B-mediated transcription,8 we
B activity using A549
4.2.1. 2-Bromo-30,4,40,5-tetramethoxy-1,10-biphenyl (3)
j
Dimerization: 4-Bromoveratrole (20.0 g, 92.1 mmol, 1.00 equiv)
was dissolved in CH2Cl2 (1000 mL) in a 2 L round-bottomed flask
and cooled to ꢁ78 °C. To this solution was added dropwise (over
1 h) a pre-mixed solution of PIFA (21.8 g, 50.7 mmol, 0.55 equiv)
and BF3ꢂEt2O (12.7 mL, 101 mmol, 1.10 equiv) in CH2Cl2 (500 mL).
The reaction mixture was stirred until the starting material was
completely consumed (ꢀ1 h). The reaction was quenched by
adding 10% NaOH (500 mL) and the reaction mixture was vigor-
ously stirred for 1 h. The product was extracted with CH2Cl2
(3ꢃ). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo and recrystallized from EtOH. The veratrole
dimer was obtained as a white crystalline solid (18.8 g, 95%).
Analytical data was in agreement with that reported.24
Dehalogenation: Veratrole dimer (10.0 g, 23.0 mmol, 1.00 equiv)
was dissolved in anhydrous THF (400 mL) and cooled to ꢁ78 °C. To
this solution was added n-BuLi (9.30 mL, 23.0 mmol, 1.00 equiv,
2.5 M in hexanes) dropwise via syringe pump (0.62 mL/min). Upon
complete addition of n-BuLi the reaction was stirred for another
20 min before 2.0 mL of MeOH was added as a proton source.
The quenched reaction mixture was stirred for 5 min at ꢁ78 °C
and then poured into a separatory funnel with 100 mL of H2O.
The product was extracted with EtOAc (3ꢃ). The combined organic
layers were dried over Na2SO4 and concentrated in vacuo and
recrystallized from MeOH. The title compound was obtained as
an off-white crystalline solid (6.79 g, 84%). Analytical data was in
agreement with that reported.28
j
j
l
Considering the structurally sensitive nature of the alkaloids’
mechanism of action,19,27 it is tempting to speculate that these
simplified tylophorine analogs could also have a different mecha-
nism of action.
3. Conclusion
We have prepared a library of novel tylophorine analogues,
which was used to explore the effects of N-substitution at the
phenanthropiperidine core. The synthetic route developed here en-
abled a highly efficient synthesis of the phenanthropiperidine scaf-
fold from which each library member could be prepared through
reductive alkylation reactions. Although the library members, in
general, had reduced activity relative to tylophorine, we noted a
significant improvement of anti-proliferative activity for 2-substi-
tuted N-benzyl analogues, the best of which, 7e, had a GI50 value
of 600 nM against MCF-7 cell proliferation. The lack of NF-jB
inhibitory activity, however, suggests that these structural analogs
may exert their anti-proliferative activity through a mechanism
distinct from tylophorine.
Given the synthetic accessibility of these analogues and the
suitability of this route for library development, this work opens
new avenues to explore the stripped-down phenanthropiperidine
core. Importantly, this work has also shed light on the operative
pharmacophore and establishes the necessity of ring E for potent
anti-proliferative activity of analogues of the 6,7,10,11-tetrameth-
oxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline core structure 6.
4.2.2. tert-Butyl 4-(30,4,40,5-tetramethoxy-[1,10-biphenyl]-2-yl)-
5,6-dihydropyridine-1(2H)-carboxylate (5)
Biaryl monobromide (6.74 g, 19.1 mmol, 1.1 equiv) was dis-
solved in anhydrous THF and cooled to ꢁ78 °C. To this solution
was added t-BuLi (23.5 mL, 39.9 mmol, 2.3 equiv, 1.7 M in

M. J. Niphakis et al. / Bioorg. Med. Chem. 20 (2012) 5893–5900
5897
hexanes) dropwise via a syringe pump. Upon complete addition of
t-BuLi the reaction was stirred for 30 min before N-Boc-piperidone
was added (3.46 g, 17.3 mmol, 1.0 equiv) dropwise in 30 mL THF.
That solution was allowed to warm to room temperature while
stirring slowly. Upon completion (ꢀ3 h) of the reaction, silica gel
was added, stirred for 1 h, concentrated, loaded directly on to a
column, and purified via column chromatography (25% EtOAc/
Hex) to yield 6.64 g (84%) of the title compound as an orange oil.
1H NMR (400 MHz, CDCl3) d 6.94–6.89 (m, 2H), 6.86 (d, J = 8.8 Hz,
1H), 6.82 (s, 1H), 6.75 (s, 1H), 5.87–5.52 (m, 1H), 4.03–3.94 (m,
2H), 3.91 (s, 3H) 3.89 (s, 3H), 3.89 (s, 3H) 3.84 (s, 3H), 3.29 (s,
2H), 2.00–1.79 (m, 2H), 1.44 (s, 9H); 13C NMR (100 MHz, CDCl3) d
154.96, 148.54, 148.10, 148.07, 147.88, 138.73, 134.47, 133.85,
131.96, 123.23, 121.09, 113.35, 112.83, 112.36, 111.02, 79.63,
56.14, 56.08, 56.06, 55.96, 55.94, 29.49, 28.54. IR (neat) 2933,
2836, 1694, 1603, 1505, 1464, 1417, 1248, 1211, 1173, 1151,
1248, 1214, 1156, 1044, 1013, 843, 799, 764 cmꢁ1; HRMS (ESI+)
m/e calcd for [M+H]+ C21H24NO4+: 354.1700, found 354.1698.
4.3. Synthesis of the N-alkylated phenanthropiperidine library
4.3.1. General procedure for reductive amination reactions
Phenanthropiperidine 6 (1.00 equiv, 0.05–0.08 mmol) and alde-
hyde (3.00 equiv) were dissolved in anhydrous DCE (0.02 M) and
allowed to stir for 5 min at rt. Na(OAc)3BH (5.00 equiv) was added
in one portion (as a solid) and the reaction was allowed to stir at
room temperature until completion (6–24 h, monitored via TLC).
The reaction was quenched with 10% NaOH and extracted with
CH2Cl2 (3ꢃ). The combined organic layers were dried over anhy-
drous MgSO4, concentrated, and purified via prep TLC (2.5–5%
MeOH (2.0 M NH3 in MeOH)/CH2Cl2).
1028, 864,764 cmꢁ1
;
HRMS (ESI+) m/e calcd for [M+H]+
4.3.2. 2-Benzyl-6,7,10,11-tetramethoxy-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7a)
C26H34NO6+: 456.2386, found 456.2884.
The title compound was isolated as an off-white solid (74%): 1H
NMR (400 MHz, CDCl3) d 7.81 (s, 2H), 7.53 (d, J = 3.9 Hz, 2H), 7.38
(d, J = 7.1 Hz, 3H), 7.25 (s, 1H), 6.99 (s, 1H), 4.34 (s, 2H), 4.16 (s,
2H), 4.11 (d, J = 3.3 Hz, 6H), 4.03 (s, 3H), 3.97 (s, 3H), 3.30 (s,
4H); 13C NMR (100 MHz, CDCl3) d 149.08, 148.92, 133.04, 130.38,
130.23, 130.20, 128.92, 128.62, 128.40, 125.10, 124.66, 124.62,
123.99, 123.84, 103.95, 103.65, 103.48, 102.87, 59.98, 56.21,
56.19, 56.09, 56.03, 51.77, 48.13, 29.83; IR (neat) 2928, 1514,
1467, 1247, 1147, 1047, 841 cmꢁ1; HRMS (ESI+) m/e calcd for
[M+H]+ C28H30NO4+: 444.2169, found 444.2165.
4.2.3. tert-Butyl 6,7,10,11-tetramethoxy-3,4-dihydrodibenzo
[f,h]-isoquinoline-2(1H)-carboxylate (5b)
Biphenyl 5 (6.53 g, 12.7 mmol, 1.0 equiv) was dissolved in
anhydrous CH2Cl2 (40 mL) and cooled to ꢁ78 °C. Two drops of
TFAA were then added to this solution. In a separate flask contain-
ing VOF3 (3.45 g, 27.9 mmol, 2.2 equiv) was added anhydrous
CH2Cl2 (40 mL), anhydrous EtOAc (20 mL), TFA (1.5 mL) and TFAA
(2 drops). The VOF3 solution was then added over 20 min to the
biphenyl 5 solution. The reaction mixture was stirred until the
starting material had been consumed (ꢀ1 h) and quenched with
10% NaOH (50 mL). The biphasic mixture was vigorously stirred
for 1 h at room temperature. The aqueous layer was extracted
(3ꢃ) with CH2Cl2. The combined organic layers were washed with
brine and dried over anhydrous Na2SO4. Filtration and concentra-
tion in vacuo gave the crude product. Purification by flash chroma-
tography (silica gel, 50% EtOAc/hexanes) yielded 6.18 g (95%) of 5b
as a white solid. 1H NMR (400 MHz, CDCl3) d 7.81 (s, 2H), 7.28 (s,
1H), 7.17 (s, 1H), 4.96 (s, 2H), 4.12 (d, J = 3.5 Hz, 6H), 4.03 (d,
J = 5.2 Hz, 6H), 3.87 (t, J = 5.7 Hz, 2H), 3.15 (t, J = 5.6 Hz, 2H), 1.54
(s, 9H); 13C NMR (100 MHz, CDCl3) d 155.06, 149.02, 148.93,
148.81, 148.76, 125.52, 123.96, 123.71, 123.55, 103.97, 103.53,
103.46, 102.88, 80.11, 56.18, 56.03, 55.98, 28.64, 28.55 IR (neat)
2972, 2834, 1690, 1620, 1514, 1471, 1424, 1248, 1169, 1150,
1047, 1021, 838, 767 cmꢁ1; HRMS (ESI+) m/e calcd for [M+H]+
C26H32NO6+: 454.2230, found 454.2209.
4.3.3. 6,7,10,11-Tetramethoxy-2-(4-nitrobenzyl)-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7b)
The title compound was isolated as an off-white solid (71%): 1H
NMR (400 MHz, CDCl3) d 8.21 (d, J = 8.7 Hz, 2H), 7.82 (s, 1H), 7.81
(s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.27 (s, 1H), 7.05 (s, 1H), 4.11 (s,
3H), 4.11 (s, 3H), 4.07 (s, 2H), 4.03 (s, 3H), 3.99 (s, 3H), 3.96 (s,
2H), 3.19 (t, J = 5.6 Hz, 2H), 2.91 (t, J = 5.8 Hz, 2H); 13C NMR
(100 MHz, CDCl3)
d 148.74, 148.71, 148.58, 148.47, 147.21,
146.64, 129.31, 125.75, 125.48, 125.21, 124.04, 123.61, 123.50,
123.42, 103.81, 103.46, 103.26, 102.82, 61.82, 55.99, 55.98, 55.90,
55.80, 54.42, 49.79, 27.14; IR (neat) 2917, 1516, 1344, 1248,
1146, 1046, 842 cmꢁ1
;
HRMS (ESI+) m/e calcd for [M+H]+
C28H29N2O6+: 489.2026, found 489.2030.
4.3.4. 6,7,10,11-tetramethoxy-2-(4-methoxybenzyl)-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7c)
The title compound was isolated as an off-white solid (81%): 1H
NMR (400 MHz, CDCl3) d 7.82 (d, J = 2.6 Hz, 2H), 7.39 (d, J = 8.6 Hz,
2H), 7.27 (s, 1H), 7.10 (s, 1H), 6.91 (d, J = 8.6 Hz, 2H), 4.12 (dd,
J = 6.3, 1.2 Hz, 6H), 4.03 (dd, J = 12.0, 5.1 Hz, 8H), 3.84–3.80 (m,
5H), 3.17 (t, J = 5.5 Hz, 2H), 2.90 (t, J = 5.7 Hz, 2H); 13C NMR
4.2.4. 6,7,10,11-Tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]
isoquinoline (6)
Piperidine 5b (6.01 g, 13.26 mmol, 1.00 equiv) was dissolved in
anhydrous CH2Cl2 (40 mL). TFA (13 mL) was added to the solution.
The reaction mixture immediately turned dark purple and slowly
turned a light brown color. After the reaction was stirred for 1 h
the solvent was removed by passing N2 over it. To the remaining
residue was added CH2Cl2 (50 mL) and NH3 (10 mL, 2.0 M in
MeOH). This solution was stirred for 30 min and then poured into
a separatory funnel containing 10% NaOH (aq.). The product was
extracted with CH2Cl2 (3ꢃ) and combined organic layers were
dried over anhydrous Na2SO4. Filtration and concentration in va-
cuo furnished 4.64 g (96%) the crude product (yellow solid) which
was used without further purification. 1H NMR (400 MHz, CDCl3) d
7.78 (s, 2H), 7.23 (s, 1H), 7.05 (s, 1H), 4.32 (s, 2H), 4.11 (s, 3H), 4.10
(s, 3H), 4.02 (s, 3H), 3.99 (s, 3H), 3.31 (t, J = 5.9 Hz, 2H), 3.02 (t,
J = 5.8 Hz, 2H), 2.17 (s, 1H); 13C NMR (100 MHz, CDCl3) d 148.81,
148.79, 148.60, 148.53, 126.96, 126.16, 125.98, 124.26, 123.54,
123.31, 103.71, 103.49, 103.41, 102.91, 56.15, 56.13, 55.95, 46.56,
43.67, 26.89; IR (neat) 3390, 2956, 2835, 1618, 1515, 1462, 1425,
(100 MHz, CDCl3)
d 158.96, 148.79, 148.76, 148.58, 148.48,
130.60, 130.45, 128.76, 126.17, 125.83, 124.44, 123.59, 123.51,
114.05, 113.85, 104.01, 103.53, 103.37, 103.11, 65.18, 62.28,
56.15, 56.03, 55.96, 55.44, 54.50, 49.54, 27.33; IR (neat) 2928,
1514, 1467, 1247, 1147, 1047, 841 cmꢁ1; HRMS (ESI+) m/e calcd
for [M+H]+ C29H32NO5+: 474.2280, found 474.2285.
4.3.5. 6,7,10,11-Tetramethoxy-2-(3-methoxybenzyl)-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7d)
The title compound was isolated as an off-white solid (85%): 1H
NMR (400 MHz, CDCl3) d 7.80 (d, J = 1.6 Hz, 2H), 7.25 (d, J = 6.1 Hz,
2H), 7.08 (s, 1H), 7.04 (d, J = 6.7 Hz, 2H), 6.88–6.79 (m, 1H), 4.09 (d,
J = 1.0 Hz, 6H), 4.04 (s, 2H), 4.00 (d, J = 5.7 Hz, 6H), 3.84 (s, 2H), 3.80
(s, 3H), 3.15 (d, J = 5.0 Hz, 2H), 2.89 (t, J = 5.6 Hz, 2H).; 13C NMR
(101 MHz, CDCl3)
d 159.94, 148.84, 148.81, 148.63, 148.53,
140.32, 129.43, 126.15, 125.85, 125.82, 124.43, 123.63, 123.54,

5898
M. J. Niphakis et al. / Bioorg. Med. Chem. 20 (2012) 5893–5900
121.55, 114.49, 112.95, 104.06, 103.60, 103.45, 103.14, 62.81,
56.17, 56.16, 56.04, 55.97, 55.38, 54.59, 49.68, 27.32; IR (neat)
2928, 1514, 1467, 1247, 1147, 1047, 841 cmꢁ1; m/e calcd for
[M+H]+ C32H32NO4+: 474.2280, found 474.2269.
7.25–7.19 (m, 1H), 7.10 (d, J = 7.3 Hz, 1H), 7.05 (s, 1H), 6.89–6.82
(m, J = 12.2, 4.7 Hz, 2H), 4.16 (br s, 2H), 4.12 (s, 3H), 4.12 (s, 3H),
4.09 (br s, 2H), 4.02 (s, 3H), 4.02 (s, 3H), 3.23 (t, J = 5.2 Hz, 2H),
3.03 (br s, 2H); 13C NMR (101 MHz, CDCl3) d 158.15, 148.97,
148.90, 148.77, 129.10, 128.94, 125.61, 125.38, 124.37, 123.99,
123.77, 123.66, 121.28, 119.33, 116.48, 103.97, 103.66, 103.45,
102.96, 61.16, 56.20, 56.19, 56.13, 55.97, 53.85, 48.95, 26.73; IR
(neat) 1619, 1514, 1249, 1143, 1045, 845 cmꢁ1; HRMS (ESI+) m/e
calcd for [M+H]+ C28H30NO5+: 460.2124, found 460.2137.
4.3.6. 6,7,10,11-Tetramethoxy-2-(2-methoxybenzyl)-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7e)
The title compound was isolated as an off-white solid (56%): 1H
NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 7.81 (s, 1H), 7.53 (d,
J = 7.8 Hz, 1H), 7.32–7.27 (m, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 7.01–
6.87 (m, 2H), 4.16 (br s, 2H), 4.11 (s, 3H), 4.11 (s, 3H), 4.03 (s,
3H), 4.01 (s, 3H), 4.01 (br s, 2H), 3.87 (s, 3H), 3.22 (t, J = 5.1 Hz,
2H), 3.06 (br s, 2H). 13C NMR (100 MHz, CDCl3) d 157.86, 148.65,
148.61, 148.42, 148.31, 130.45, 128.18, 126.33, 125.96, 125.73,
124.35, 123.43, 123.34, 120.47, 110.38, 103.92, 103.43, 103.30,
102.99, 56.01, 56.01, 55.88, 55.83, 55.81, 55.42, 54.14, 49.83,
4.3.11. 2-(3,5-Dichlorobenzyl)-6,7,10,11-tetramethoxy-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7j)
The title compound was isolated as an off-white solid (81%): 1H
NMR (400 MHz, CDCl3) d 7.80 (s, 2H), 7.36 (s, 2H), 7.28 (s, 1H), 7.17
(d, J = 18.9 Hz, 1H), 7.04 (s, 1H), 4.10 (s, 6H), 4.01 (d, J = 6.7 Hz, 8H),
3.80 (s, 2H), 3.17 (s, 2H), 2.89 (d, J = 5.2 Hz, 2H); 13C NMR
27.16; IR (neat) 2928, 1514, 1467, 1247, 1147, 1047, 841 cmꢁ1
;
(101 MHz, CDCl3)
d 148.88, 148.86, 148.71, 148.59, 142.52,
HRMS (ESI+) m/e calcd for [M+H]+ C29H32NO5+: 474.2280, found
135.09, 127.52, 127.34, 125.95, 125.68, 125.40, 124.93, 124.24,
123.66, 123.56, 104.00, 103.62, 103.43, 102.99, 61.79, 56.16,
56.05, 55.97, 54.47, 49.89, 27.30; IR (neat) 2932, 1619, 1569,
1514, 1425, 1249, 1147, 846 cmꢁ1; HRMS (ESI+) m/e calcd for
[M+H]+ C28H28NO4Cl2+: 512.1395, found 512.1400.
474.2269.
4.3.7. N,N-Dimethyl-4-((6,7,10,11-tetramethoxy-3,4-
dihydrodibenzo[f,h]isoquinolin-2(1H)-yl)methyl)aniline (7f)
The title compound was isolated as an off-white solid (73%): 1H
NMR (400 MHz, CD2Cl2) d 7.81 (d, J = 1.3 Hz, 2H), 7.29 (d, J = 8.6 Hz,
2H), 7.26 (s, 1H), 7.08 (s, 1H), 6.73 (d, J = 8.7 Hz, 2H), 4.04 (d,
J = 1.2 Hz, 6H), 4.01 (s, 2H), 3.95 (d, J = 5.3 Hz, 6H), 3.77 (s, 2H),
3.13 (t, J = 5.6 Hz, 2H), 2.92 (s, 6H), 2.90–2.85 (m, 2H); 13C NMR
4.3.12. 2-(2,6-Dimethoxybenzyl)-6,7,10,11-tetramethoxy-
1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (7k)
The title compound was isolated as an off-white solid (55%): 1H
NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 7.80 (s, 1H), 7.27 (s, 1H), 7.24
(d, J = 8.3 Hz, 1H), 7.15 (s, 1H), 6.61 (d, J = 8.4 Hz, 2H), 4.10 (t,
J = 2.3 Hz, 8H), 4.04 (s, 5H), 4.02 (s, 3H), 3.86 (s, 6H), 3.16 (d,
J = 5.4 Hz, 2H), 3.08 (t, J = 5.6 Hz, 2H); 13C NMR (100 MHz, CDCl3)
d 159.65, 148.72, 148.60, 148.42, 148.29, 128.98, 126.50, 125.96,
125.92, 124.65, 123.47, 123.42, 114.20, 104.07, 103.89, 103.55,
103.44, 103.15, 56.14, 55.93, 55.91, 55.82, 53.25, 49.94, 48.91,
(101 MHz, CD2Cl2)
d 150.77, 149.49, 149.45, 149.30, 149.20,
130.73, 128.97, 126.47, 126.09, 124.72, 123.93, 123.85, 112.95,
112.64, 104.68, 104.18, 104.04, 103.78, 65.56, 62.44, 56.47, 56.45,
56.29, 56.23, 49.76, 41.03, 40.94, 30.25, 27.49; IR (neat) 2919,
1615, 1515, 1470, 1248, 1146, 1045, 840 cmꢁ1; HRMS (ESI+) m/e
calcd for [M+H]+ C30H35N2O4+: 487.2597, found 487.2604.
27.05; IR (neat) 2928, 1514, 1467, 1247, 1147, 1047, 841 cmꢁ1
;
4.3.8. 2-Methoxy-6-((6,7,10,11-tetramethoxy-3,4-
HRMS (ESI+) m/e calcd for [M+H]+ C30H34NO6+: 504.2386, found
dihydrodibenzo[f,h]isoquinolin-2(1H)-yl)methyl)phenol (7g)
The title compound was isolated as an off-white solid (67%): 1H
NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 7.81 (s, 1H), 7.24 (s, 1H), 7.05
(s, 1H), 6.92–6.84 (m, 1H), 6.81 (t, J = 7.7 Hz, 1H), 6.75 (d, J = 7.1 Hz,
1H), 4.20 (s, 2H), 4.11 (d, J = 1.4 Hz, 8H), 4.02 (s, 3H), 4.01 (s, 3H),
3.87 (s, 3H), 3.25 (d, J = 4.7 Hz, 2H), 3.06 (s, 2H); 13C NMR
504.2380.
4.3.13. 6,7,10,11-Tetramethoxy-2-methyl-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7l)
The title compound was isolated as an off-white solid (71%): 1H
NMR (400 MHz, CDCl3) d 7.83 (s, 1H), 7.83 (s, 1H), 7.30 (s, 1H), 7.14
(s, 1H), 4.12 (s, 6H), 4.05 (s, 3H), 4.05 (s, 3H), 3.95 (s, 2H), 3.22 (t,
J = 5.8 Hz, 2H), 2.90 (t, J = 5.9 Hz, 2H), 2.66 (s, 3H); 13C NMR
(101 MHz, CDCl3)
d 148.97, 148.91, 148.76, 148.18, 147.41,
125.57, 125.37, 124.12, 123.99, 123.77, 123.67, 121.22, 121.12,
119.03, 111.35, 103.96, 103.67, 103.45, 102.98, 60.48, 56.19,
56.14, 56.03, 55.96, 53.66, 48.85, 26.48; IR (neat) 2933, 1619,
1514, 1474, 1249, 1144, 1044, 842 cmꢁ1; HRMS (ESI+) m/e calcd
for [M+H]+ C29H32NO6+: 490.2230, found 490.2230.
(100 MHz, CDCl3)
d 148.88, 148.66, 148.55, 125.79, 125.75,
125.64, 124.32, 123.63, 123.54, 104.08, 103.63, 103.49, 103.10,
56.19, 56.13, 56.07, 56.01, 52.51, 46.58, 27.51; IR (neat) 3373,
2975, 1515, 1425, 1366, 1249, 1150, 844 cmꢁ1; HRMS (ESI+) m/e
calcd for [M+H]+ C22H26NO4+: 368.1862, found 368.1869.
4.3.9. 2-(4-(tert-Butyl)benzyl)-6,7,10,11-tetramethoxy-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7h)
The title compound was isolated as an off-white solid (81%): 1H
NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 7.81 (s, 1H), 7.40 (s, 4H), 7.26
(s, 1H), 7.08 (s, 1H), 4.11 (s, 3H), 4.11 (s, 3H), 4.08 (s, 2H), 4.02 (s,
3H), 4.00 (s, 3H), 3.88 (s, 2H), 3.18 (t, J = 5.4 Hz, 2H), 2.95 (t,
J = 5.3 Hz, 2H), 1.34 (s, 9H); 13C NMR (101 MHz, CDCl3) d 150.45,
148.82, 148.80, 148.64, 148.53, 138.12, 129.14, 126.99, 125.72,
125.59, 125.43, 124.35, 123.65, 123.55, 104.01, 103.58, 103.43,
103.11, 65.27, 56.15, 56.01, 55.93, 54.29, 49.37, 34.65, 31.53,
4.3.14. 6,7,10,11-Tetramethoxy-2-phenethyl-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (7m)
The title compound was isolated as an off-white solid (76%): 1H
NMR (400 MHz, CD2Cl2) d 7.83 (s, 1H), 7.82 (s, 1H), 7.32 (dd, J = 5.0,
1.5 Hz, 4H), 7.29 (s, 1H), 7.24–7.19 (m, 1H), 7.09 (s, 1H), 4.06 (d,
J = 1.5 Hz, 8H), 3.99 (d, J = 8.9 Hz, 6H), 3.21 (t, J = 5.6 Hz, 2H),
3.09–2.98 (m, 6H); 1H NMR (400 MHz, CD2Cl2) d 7.83 (s, 1H),
7.82 (s, 1H), 7.32 (dd, J = 5.0, 1.5 Hz, 4H), 7.29 (s, 1H), 7.24–7.19
(m, 1H), 7.09 (s, 1H), 4.06 (d, J = 1.5 Hz, 8H), 3.99 (d, J = 8.9 Hz,
6H), 3.21 (t, J = 5.6 Hz, 2H), 3.09–2.98 (m, 6H); 13C NMR
26.96; IR (neat) 2959, 1515, 1479, 1248, 1147, 1046, 839 cmꢁ1
;
HRMS (ESI+) m/e calcd for [M+H]+ C32H38NO4+: 500.2801, found
(100 MHz, CD2Cl2)
d 149.52, 149.50, 149.34, 149.21, 141.23,
500.2789.
129.32, 128.93, 126.58, 126.40, 126.03, 124.67, 123.94, 123.85,
104.67, 104.20, 104.04, 103.66, 60.45, 56.47, 56.46, 56.36, 56.26,
4.3.10. 2-((6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo
[f,h]isoquinolin-2(1H)-yl)methyl)phenol (7i)
50.59, 34.25, 27.54; IR (neat) 1615, 1513, 1422, 1248, 1149,
1042, 840 cmꢁ1; HRMS (ESI+) m/e calcd for [M+H]+ C29H32NO4
:
+
The title compound was isolated as an off-white solid (65%): 1H
NMR (400 MHz, CDCl3) d 7.83 (s, 1H), 7.82 (s, 1H), 7.25 (s, 1H),
458.2331, found 458.2329.

M. J. Niphakis et al. / Bioorg. Med. Chem. 20 (2012) 5893–5900
5899
4.3.15. tert-Butyl (2-(6,7,10,11-Tetramethoxy-3,4-
4.3.20. (5-((6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo
dihydrodibenzo[f,h]isoquinolin-2(1H)-yl)ethyl)carbamate (7n)
The title compound was isolated as an off-white solid (95%): 1H
NMR (400 MHz, CDCl3) d 7.80 (s, 1H), 7.80 (s, 1H), 7.27 (s, 1H), 7.10
(s, 1H), 4.10 (s, 6H), 4.03 (s, 6H), 4.00 (s, 2H), 3.45 (d, J = 5.0 Hz, 2H),
3.18 (d, J = 5.1 Hz, 2H), 2.95 (t, J = 5.6 Hz, 2H), 2.84 (t, J = 5.7 Hz,
2H), 1.48–1.43 (s, 9H); 13C NMR (101 MHz, CDCl3) d 156.24,
148.88, 148.71, 148.59, 125.94, 125.59, 125.35, 124.23, 123.64,
123.52, 103.97, 103.59, 103.43, 102.93, 79.30, 57.15, 56.15, 56.07,
55.98, 54.08, 49.98, 37.64, 29.81, 28.55, 28.49, 28.45, 28.10,
[f,h]isoquinolin-2(1H)-yl)methyl)furan-2-yl)methanol (7s)
The title compound was isolated as an off-white solid (72%): 1H
NMR (400 MHz, CDCl3) d 7.80 (s, 1H), 7.79 (s, 1H), 7.24 (s, 1H), 7.07
(s, 1H), 6.30 (d, J = 3.1 Hz, 1H), 6.26 (d, J = 3.1 Hz, 1H), 4.60 (s, 2H),
4.10 (s, 3H), 4.10 (s, 3H), 4.04 (br s, 2H), 4.02 (s, 3H), 4.01 (s, 3H),
3.88 (s, 2H), 3.18 (t, J = 5.4 Hz, 2H), 2.98 (t, J = 5.8 Hz, 2H); 13C
NMR (101 MHz, CDCl3) d 154.04, 151.75, 148.70, 148.67, 148.51,
148.40, 125.65, 125.51, 125.13, 124.14, 123.48, 123.40, 109.79,
108.38, 103.85, 103.46, 103.29, 102.90, 57.53, 56.02, 55.91, 55.83,
54.64, 53.64, 49.59, 26.82; IR (neat) 3393, 1619, 1514, 1425,
27.23; IR (neat) 3373, 2975, 1701, 1515, 1425, 1366, 1249, 1150,
844 cmꢁ1
;
HRMS (ESI+) m/e calcd for [M+H]+ C28H27N2O6
:
1248, 1146, 1012 cmꢁ1 HRMS (ESI+) m/e calcd for [M+H]+
;
+
497.2652, found 497.2665.
C27H30NO6+: 464.2073, found 464.2055.
4.3.16. 2-(6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo
[f,h]isoquinolin-2(1H)-yl)ethanamine (7o)
4.3.21. 6,7,10,11-Tetramethoxy-2-((1-methyl-1H-imidazol-2-yl)
methyl)-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (7t)
The title compound was isolated as an off-white solid (84%): 1H
NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 7.80 (s, 1H), 7.26 (s, 1H), 7.07
(s, 1H), 4.10 (t, J = 5.6 Hz, 10H), 4.05 (s, 6H), 3.69 (d, J = 5.1 Hz, 2H),
3.22 (s, 2H), 3.03 (d, J = 21.2 Hz, 4H); 13C NMR (100 MHz, CDCl3) d
149.08, 149.07, 148.99, 148.83, 125.29, 123.92, 123.80, 123.67,
103.92, 103.67, 103.46, 102.77, 56.22, 56.19, 56.13, 56.03, 55.39,
53.84, 49.72, 36.61, 29.84.; IR (neat) 3373, 2975, 1701, 1515,
1425, 1366, 1249, 1150, 844 cmꢁ1; HRMS (ESI+) m/e calcd for
[M+H]+ C23H29N2O4+: 397.2122, found 397.2135.
The title compound was isolated as an off-white solid (62%): 1H
NMR (400 MHz, CDCl3) d 7.83 (s, 1H), 7.82 (s, 1H), 7.28 (s, 1H), 7.11
(s, 1H), 6.99 (d, J = 1.3 Hz, 1H), 6.89 (d, J = 1.3 Hz, 1H), 4.12 (s, 3H),
4.11 (s, 3H), 4.03 (s, 7H), 3.98 (s, 2H), 3.75 (s, 3H), 3.17 (t, J = 5.8 Hz,
2H), 2.94 (t, J = 5.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 148.88,
148.83, 148.66, 148.56, 145.05, 127.24, 126.04, 125.68, 125.65,
124.30, 123.61, 123.44, 121.97, 103.97, 103.51, 103.38, 103.04,
56.17, 56.09, 55.97, 55.14, 54.35, 49.83, 33.19, 27.44; IR (neat)
2917, 1620, 1514, 1425, 1248, 1147, 1044 cmꢁ1; HRMS (ESI+) m/e
calcd for [M+H]+ C26H30N3O4+: 448.2236, found 448.2223.
4.3.17. 4-((6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo[f,h]
isoquinolin-2(1H)-yl)methyl)pyridine 1-Oxide (7p)
4.4. Biological testing of the phenanthropiperidine library
The title compound was isolated as an off-white solid (55%): 1H
NMR (400 MHz, CD2Cl2) d 8.13 (d, J = 6.3 Hz, 2H), 7.83 (s, 2H), 7.40
(d, J = 6.4 Hz, 2H), 7.29 (s, 1H), 7.06 (s, 1H), 4.13–3.92 (m, 14H),
3.83 (s, 2H), 3.19 (d, J = 5.9 Hz, 2H), 2.91 (t, J = 5.9 Hz, 2H); 13C
NMR (101 MHz, CD2Cl2) d 149.57, 149.53, 149.40, 149.29, 139.41,
138.80, 126.51, 126.40, 126.00, 125.85, 124.56, 123.97, 123.88,
104.61, 104.22, 104.05, 103.63, 60.95, 56.47, 56.45, 56.35, 56.25,
54.75, 50.48, 27.77; IR (neat) 2918, 1620, 1514, 1474, 1249,
4.4.1. Anti-proliferative activity
To improve solubility, each phenanthroindolizidine alkaloid
was converted to its HCl salt by dissolving the free amine in CH2Cl2,
adding a large excess (>10 equiv) of HCl (2.0 M in diethylether) and
removing the solvent in vacuo. Stock solutions (10 mM) of the each
compound were prepared in DMSO.
MCF-7, DU-145 and A549/NF-jB-luc cancer cells were har-
1146, 1043, 843 cmꢁ1
;
HRMS (ESI+) m/e calcd for [M+H]+
vested (125 G centrifuge for 5 min) from an exponential-phase
maintenance culture. The cells were re-suspended in new culture
media (RPMI1640 (GIBCO11875) with 10% FBS, EMEM (ATCC P/N
30-2003) with 10% FBS, and DMEM (ATCC P/N 30-2002) with
10% FBS, penicillin, streptomycin and hygromycin) and the cell
density was adjusted to 105 cells/mL and dispensed in 96-well cul-
C27H29N2O5+: 461.2076, found 461.2077.
4.3.18. 4-((6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo[f,h]
isoquinolin-2(1H)-yl)methyl)oxazole (7q)
The title compound was isolated as an off-white solid (91%): 1H
NMR (400 MHz, CD2Cl2) d 7.91 (s, 1H), 7.82 (d, J = 2.1 Hz, 2H), 7.73
(s, 1H), 7.28 (s, 1H), 7.12 (s, 1H), 4.06 (d, J = 1.5 Hz, 8H), 3.99 (s, 3H),
3.97 (s, 3H), 3.85 (s, 2H), 3.17 (t, J = 5.9 Hz, 2H), 2.99 (t, J = 5.8 Hz,
ture plates at a density of 5000 cells per well (50
were incubated overnight to allow cells to adhere to the wells.
Fresh culture medium (50 L) containing varying concentrations
of the test and control compounds was added to each well. The cul-
tures were grown for an additional 72 h and alamarBlueÒ (10
L)
lL). The cells
l
2H); 13C NMR (101 MHz, CD2Cl2)
d 151.67, 149.49, 149.47,
l
149.30, 149.20, 138.17, 137.20, 126.42, 126.14, 126.09, 124.70,
123.91, 123.83, 104.67, 104.19, 104.05, 103.75, 56.47, 56.45,
56.33, 56.24, 50.48, 27.77; IR (neat) 2935, 1620, 1514, 1425,
1248, 1146, 1045, 843 cmꢁ1; HRMS (ESI+) m/e calcd for [M+H]+
C25H27N2O5+: 435.1920, found 435.1911; 91%.
was added. After 1.5 h, the fluorescence excitation (530 nm) and
emission (590 nm) of each well were measured to determine the
optical density. Each compound was tested in triplicate with less
than 5% variation. Compound 7h was tested in duplicate.
4.4.2. NF-
jB activity assay
4.3.19. 5-Methyl-3-((6,7,10,11-tetramethoxy-3,4-
A549/NF-
jB-luc cells were cultured using complete growth
dihydrodibenzo[f,h]isoquinolin-2(1H)-yl)methyl)isoxazole (7r)
The title compound was isolated as an off-white solid (72%): 1H
NMR (400 MHz, CD2Cl2) d 7.82 (s, 1H), 7.82 (s, 1H), 7.27 (s, 1H),
7.10 (s, 1H), 6.12 (d, J = 1.1 Hz, 1H), 4.08–4.03 (m, 8H), 3.98 (s,
3H), 3.97 (s, 3H), 3.89 (s, 2H), 3.25–3.10 (m, 2H), 2.94 (t,
J = 5.8 Hz, 2H), 2.41 (d, J = 0.9 Hz, 3H); 13C NMR (100 MHz, CD2Cl2)
d 170.20, 162.48, 149.51, 149.34, 149.24, 126.36, 126.04, 125.99,
124.63, 123.94, 123.84, 104.64, 104.19, 104.04, 103.72, 102.22,
56.46, 56.45, 56.37, 56.24, 54.60, 50.44, 27.77, 12.62; IR (neat)
medium (ATCC P/N 30-2002, 10% FBS, penicillin and streptomycin)
containing hygromycin at 37 °C with 5% CO2. After trypsinizing and
centrifuging the cells (150 ꢃ G for 5 min), the cell density was ad-
justed to 105 cells/mL and dispensed in 96-well culture plates at a
density 5000 cells per well (50
24 h, allowing cells to attach. The appropriate compound in the
complete growth medium (50 L) was added to each well and
the cells were incubated for another 30 min. TNF- (5 L of
TNF- in 1X PBS))
solution in 1 ꢃ PBS (1:667 dilution of TNF-
was then added to each well. After incubating for another 7 h,
luciferin reagent (100
L; Bright-GloTM from Promega) was
added to each well. After 2 min, luciferase activity was measured
lL). The cells were incubated for
l
a
l
a
a
2918, 1607, 1514, 1424, 1248, 1145, 1046, 841 cmꢁ1; HRMS
(ESI+) m/e calcd for [M+H]+ C26H29N2O5
: 449.2076, found
+
l
449.2062.

5900
M. J. Niphakis et al. / Bioorg. Med. Chem. 20 (2012) 5893–5900
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This work was supported by the University of Minnesota
through the Vince and McKnight Endowed Chairs (to GIG) and an
ACS Division of Medicinal Chemistry Predoctoral Fellowship (to
M. J. N).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.044.
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