Synthesis and Pharmacological Evaluation of Noscapine-Inspired 5-Substituted Tetrahydroisoquinolines as Cytotoxic Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b00986

A series of 5-substituted tetrahydroisoquinolines was synthesized via a 10-step linear synthesis to assess whether replacement of noscapine's southern isobenzofuranone with other moieties resulted in retained cytotoxic activity. One such molecule, 18g, bearing a para-methoxybenzyl functionality with N-ethylcarbamoyl substitution, produced cell-cycle arrest at the G2/M phase with an EC₅₀ of 2.7 μM in the MCF-7 breast-cancer cell line, a 7-fold increase compared with that of noscapine (5). This molecule had similar activity (EC₅₀ of 2.5 μM) against the resistant NCI/Adr^RES cell line, demonstrating its potential to overcome or avert known resistance mechanisms, unlike current cytotoxic agents. Compound 18g was found to modify the drug-efflux activity of P-gp and, in combination studies, potentiate the antiproliferative activity of vinblastine. These results provide insights into structural modifications to noscapine that will guide future development toward more potent cytotoxic agents that are active against resistant cancer cells.

Full text of DOI:10.1021/acs.jmedchem.8b00986

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Article
Synthesis and Pharmacological Evaluation of Noscapine Inspired
5-Substituted Tetrahydroisoquinolines as Cytotoxic Agents
Shane M. Devine, Cassandra Yong, Dzifa Amenuvegbe, Luigi Aurelio, Divya
muthiah, Colin W. Pouton, Richard Callaghan, Ben Capuano, and Peter J. Scammells
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00986 • Publication Date (Web): 29 Aug 2018
Downloaded from http://pubs.acs.org on August 30, 2018
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Synthesis and Pharmacological Evaluation of Noscapine-
Inspired 5-Substituted Tetrahydroisoquinolines as Cytotoxic
Agents
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Shane M. Devine,† Cassandra Yong,† Dzifa Amenuvegbe,‡ Luigi Aurelio,† Divya
Muthiah,‡ Colin Pouton,§ Richard Callaghan,‡ Ben Capuano,† Peter J. Scammells†,*
§
† Medicinal Chemistry and
Drug Delivery, Disposition and Dynamics, Monash Institute of
Pharmaceutical Sciences; Monash University, Parkville, VIC 3052, Australia
‡ Research School of Biology, Australian National University, Canberra, ACT 0200, Australia
ABSTRACT: A series of 5ꢀsubstituted tetrahydroisoquinolines was synthesised via a 10ꢀstep linear
synthesis to assess whether replacement of noscapine’s southern isobenzofuranone with other
moieties retained cytotoxic activity. One such molecule, 18g bearing a paraꢀmethoxybenzyl
functionality with Nꢀethylcarbamoyl substitution produced cell cycle arrest at the G2/M phase with
an EC50 of 2.7 ꢁM in the MCFꢀ7 breast cancer cell line; a 7ꢀfold increase compared with noscapine
(5). This molecule had similar activity (EC50 of 2.5 ꢁM) against the resistant NCI/AdrRES cell line,
demonstrating its potential to overcome or avert known resistance mechanisms, unlike current
cytotoxic agents. Compound 18g was found to modify the drug efflux activity of Pꢀgp and in
combination studies, potentiate the antiproliferative activity of vinblastine. These results provide
insights into the structural modifications to noscapine that will guide future development towards
more potent cytotoxic agents that are active at resistant cancer cells.
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ꢀ INTRODUCTION
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Tubulin targeting compounds have been, and continue to be, of major importance in cancer
chemotherapy. Compounds acting on tubulin disrupt microtubule dynamics; these microtubules are
cytoskeletal fibres crucial throughout the cell cycle, especially in the mitotic phase. Tubulinꢀ
disrupting agents fall into two classes; namely tubulinꢀpolymerisation inhibitors (TPIs) and tubulinꢀ
depolymerisation inhibitors (TDIs). There are many classes of molecules that act via these two
mechanisms and these have historically been derived from natural products. Examples of TPIs
include vinca alkaloids, such as vincristine (1) and colchicine (2), whereas taxanes, such as paclitaxel
(3) and epothilones, including epothilone B (4) act as TDIs (Figure 1). Although these agents have
found great utility as anticancer agents and significantly decreased the burden of cancer on human
society, their efficacy is limited in vivo due to numerous toxic side effects and multiple resistance
mechanisms. Another example of a TPI is that of noscapine (5), the motivation of this current work.
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Figure 1. Examples of tubulin disrupting agents 1–5.
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Noscapine is a phthalideisoquinoline alkaloid isolated from the opium poppy, Papaver
somniferum constituting approximately 1ꢀ10% of the alkaloid content of the poppy. Noscapine’s
initial indication was as an antitussive agent since the discovery of its cough suppressing abilities.1,2
It is orally administered with a measured absolute oral bioavailability of 30%,3 and a halfꢀlife of 4.5
hours.4 It has been used clinically since the 1950s, demonstrating its good safety and
pharmacokinetic profile. Unlike morphine and codeine, noscapine does not exhibit addictive,
analgesic and sedative traits indicating action at a distinctly separate receptor to that of the opiate
receptors.2 In 1998, Ye et al. demonstrated that noscapine could arrest cells at mitosis, induce
apoptosis, act on tubulin to impair microtubule dynamics and shrink tumours in vivo.5 Noscapine’s
cytotoxic activity was relatively weak measured against cervical (HeLa), breast (MCFꢀ7) and bladder
(Renal 1983) cancer cell lines with IC50 values of 25 ꢁM, 42 ꢁM and 39 ꢁM, respectively. The
structures of noscapine and colchicine are seemingly similar, consisting of polyꢀoxygenated ring
systems with electronꢀdonating methoxy groups and bridged by a saturated ring. However,
competition binding experiments with [3H]colchicine/noscapine indicate that noscapine acts at a
different binding site on tubulin, compared with colchicine.5 Experimental and computational
insights revealed noscapine to bind tubulin at either a partially overlapping site to colchicine or a
distinctly nonꢀoverlapping sight that does not interfere with colchicine binding to tubulin.6ꢀ10
Notably, there is no evidence to suggest that noscapine can bind the vinca alkaloid site. Acting as a
TPI, noscapine arrests cells at the G2/M phase, whereby the cell remains in a
prometaphase/metaphaseꢀlike state, which subsequently elicits apoptosis. Many groups have
demonstrated noscapine’s potential as an anticancer agent both in vitro and in vivo over the last two
decades.11ꢀ18 Our work aims to better define the structural changes that can be incorporated to
enhance this anticancer activity.
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Modification of the noscapine scaffold has been of considerable interest in the intervening years,
with changes occurring primarily at the 1, 7, 6′ꢀ and 9′ꢀpositions.16,19 The deployment of halogens at
the 9′ꢀposition gave better activity in the order of 2ꢀfold magnitude.20 It was also demonstrated that
the potentially deleterious lactone moiety at the 1ꢀposition could be replaced with a cyclic ether, as
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exemplified by 9′ꢀchloronoscapine derivative 6 (Figure 2), which retained activity.20 Anderson et al.
established a method to selectively demethylate at the 7ꢀposition using MeMgBr and BnOH to afford
the corresponding phenol (7).21 This approach afforded Oꢀsubstituted products, which exhibited an
Sꢀphase arresting phenotype, as well as 7ꢀamino containing moieties. The 7ꢀhydroxy and 7ꢀamino
containing noscapinoids were shown to be G2/M phase arresting molecules that exhibit
approximately 500ꢀfold increased potency compared to noscapine (5).22 These compounds also
demonstrated good oral bioavailability in mice. In particular, the aniline 8 exhibited an IC50 of 94
nM in SNU398 cells. Further work detailed tubulinꢀbinding experiments by STD NMR
demonstrating the aniline to have a similar binding site to that of colchicine. Unfortunately though,
this molecule suffered from high in vivo clearance in rats.23 Our group has strived to further these
contributions to enhance the activity of noscapine’s cytotoxic activity and generate a more global
picture of the structureꢀactivity relationship (SAR). The combination of Nꢀsubstitution reactions,
coupled with lactone removal resulted in a series of 6′ꢀsubstituted noscapinoids of which the 6′ꢀ
ethylcarbonyl containing analogue 9 was the most promising.24 This analogue exhibited EC50 values
of 6.7 ꢁM and 3.6 ꢁM for the prostate (PC3) and breast (MCFꢀ7) cancer cell lines, respectively. This
was enhanced with the added benefits of 9′ꢀhalogen insertion and 7ꢀdemethylation, with the 9′ꢀ
chloro–cyclic ether analogue 10 displaying EC50 values of 1.5, 1.7 and 0.9 ꢁM for the prostate (PC3),
breast (MCFꢀ7) and pancreatic (PANCꢀ1) cell lines, respectively.25
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Figure 2. Key modifications made to noscapine to map structureꢀactivity relationships.
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Importantly, this molecule and similar noscapinoids demonstrate comparable efficacy against
sensitive and resistant breast cancer cell lines, indicating their inability to act as substrates for efflux
transporter pumps ABCB1 and ABCG2.25 This is contrasted with known potent cytotoxic agents
such as mitoxantrone and vinblastine exhibiting marked differences between these cell lines, with
reductions of 17ꢀfold and 260ꢀfold differences, respectively. Clearly, the promise of a more potent
noscapine analogue with these features will be of broad significance to the field as a novel cytotoxic
agent with lower susceptibility to resistance mediators.
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One aspect of noscapine’s structure that could be deleterious in vivo is that of the lactone on the
southern heterocycle. Zimmermann and coꢀworkers showed that removal of the southern
isobenzofuranone and replacement with various alkynes could retain activity similar to that of
noscapine. This series of novel molecules was made possible by BischlerꢀNapieralski cyclization of
readily accessible carbamates to the corresponding 3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀones generating the
appropriate tetrahydroisoquinoline (THIQ) scaffold. Many compounds were inactive, however one
featuring a (mꢀmethoxyphenyl)ethynyl group (11) exhibited greater inhibition of mitosis and a higher
antiproliferative activity (Figure 3).26 Crucially, it exhibited similar activity against multidrugꢀ
resistant HeLa KBꢀV1 cell line, which expresses the multidrug efflux pump Pꢀglycoprotein (Pꢀgp).
Figure 3. Southern heterocycle replacement retains activity.26
With these historical changes in mind, we sought to exploit modifications to the southern
heterocycle to enhance activity via a linear route. These changes would incorporate aromatic rings
containing electronꢀdonating methoxy groups with monoꢀ, diꢀ or triꢀsubstitution patterns. These
modifications would demonstrate the smallest deviation from noscapine’s structure, whilst hopefully
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maintaining activity. Our Nꢀethylcarbamoyl group was also incorporated for anticipated higher
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ꢀ RESULTS AND DISCUSSION
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Chemistry. To generate our target 5ꢀsubstituted noscapinoid molecules we envisaged a linear
route which included a BischlerꢀNapieralski cyclisation27 to generate the tetrahydroisoquinoline
scaffold. Firstly, selective bromination of vanillin (12) afforded 5ꢀbromovanillin in excellent yield
(Scheme S1, Supporting Information). This was then subjected to a copperꢀmediated hydroxylation
to form a catechol. Selective bromination once again was employed and further cyclisation with
diiodomethane generated the precursor dioxole. The incorporation of the bromine atom was
important for two reasons. Firstly, halogens in this position of noscapine engender better activity.
Secondly, under the following dehydrative BischlerꢀNapieralski conditions with this position
unoccupied, inseparable mixtures of tricyclic products ensued (unpublished work). A nitroꢀaldol
reaction with nitromethane formed the nitroꢀalkene and subsequent reduction with borane produced
the key intermediate, namely arylethanamine (13). The borane was necessary as the reducing agent
as other standard methods also removed the bromine atom. Unfortunately, a significant amount of
hydroxylamine byꢀproduct was also formed, similar to that described by Guy et al.28 Pleasingly,
sufficient material was obtained to complete the synthesis. Reaction with an appropriate benzoic
acid via EDCI/HOBt amide coupling conditions generated the diversity in our series, the amides (14)
(Scheme 1). Installation of various benzoic and phenylacetic acids containing mono, di or
trimethoxy groups furnished the coverage of potential SAR assessment to address the suitability of
the southern heterocycles removal. The choice of electronꢀdonating methoxy groups was selected to
give comparable information to that of previously identified noscapinoid and THIQ compounds.
BischlerꢀNapieralski cyclisation with POCl3 generated the dihydroisoquinolines (15), which were
directly reduced with NaBH4 to the tetrahydroisoquinolines (16). Finally, Nꢀsubstitution was
undertaken with a methylating agent (MeI or CH2O) or ethyl isocyanate to give the N6ꢀsubstituted
compounds 17 and 18, respectively. MeI was initially used for the alkylation, however this reaction
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suffered from poor yields (12–37%) due to the fact that unwanted quaternized material formed
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preferentially.
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Scheme 1. Synthesis of 5-substituted noscapinoidsa
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O
Br
Br
(a)
O
O
O
O
6 steps
NH2
HN
O
HO
OMe
12
OMe
OMe
R
13
14a-j
(b)
Br
Br
Br
O
O
O
O
(d)
(c)
N
NH
N
O
O
Me
OMe R
OMe R
OMe R
16a-j
15a-j
17a-j
(e)
a R = Ph
b R = 4-OMePh
c R = 2,3-diOMePh
d R = 3,4-diOMePh
e R = 3,4,5-triOMePh
f R = Bn
Br
O
O
N
NHEt
g R = 4-OMeBn
h R = 2,3-diOMeBn
i R = 3,4-diOMeBn
j R = 3,4,5-triOMeBn
OMe R
18a-j
O
aReagents and conditions: (a) RꢀCO2H, HOBt, EDCI, DIPEA, 25 °C, 16 h, 87–98%; (b) POCl3,
PhMe, 120 °C, 2 h, 86–99%; (c) NaBH4, MeOH, ꢀ78 °C, 30 min, 47–94%; (d) i) MeI, K2CO3, DMF,
25 °C, 3 h, for 17a, c, f, g, h, i, j, 12–37%; ii) HCOOH, CH2O, 80 °C, 2 h, for (17b, d, e), 83–85%;
(e) EtNCO, MeCN, 0 °C, 1 h, 62–93%.
Compounds 17b, 17d and 17e were synthesised by an alternative method using CH2O in formic
acid as the methyl source. Under these conditions, when 2ꢀ(2,3ꢀdimethoxyphenyl)acetic acid was
installed as for 16h, an unexpected result ensued. The 1H NMR spectrum of the isolated compound
lacked the Nꢀmethyl signal (usually present at ~2.3 ppm), the disappearance of an aromatic proton
and the presence of an additional methylene signal, corresponding to the berberineꢀlike structure 19
(Scheme 2). This was confirmed by 2D NMR spectroscopy. Ring cyclisation affording berberineꢀ
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like entities in the presence of formic acid and formaldehyde have been previously described by Sun
et al.29 We speculate that the formation of this berberineꢀlike compound takes place through
formaldehyde insertion, an iminium intermediate and subsequent cyclisation.
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Scheme 2. Synthesis of the berberine analogue 19a
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aReagents and conditions: (a) HCOOH, CH2O, 80 °C, 2 h, 47%.
Pharmacology. Cell cycle arrest assays. Cell cycle arrest assays provided preliminary
evaluation to assess the ability of novel 5ꢀsubstituted THIQs to cause advanced G2/M phase arrest
through disruption of microtubule assembly (Table 1). Cells were stained with Hoechst 33342
following 18 h treatment with positive controls, vincristine (100 nM), noscapine (5) (10 ꢁM) or test
compounds 17a–j, 18a–j and 19 (10 ꢁM) and analysed with FACSCanto II fluorescenceꢀactivated
cell sorting (FACS) analyser. The percentage of cells present at three stages of the cell proliferation
cycle (G1, S and G2/M) was obtained following data processing with FlowJo (v10). The percentage
increase in G2/M arrest was calculated with reference to the vehicle control (0.1% DMSO).
The standout compounds based on the cell cycle arrest data were the 5ꢀ(4ꢀmethoxybenzyl)ꢀ
substituted THIQs 17g and 18g, both of which produced significantly greater increases in G2/M
arrest than noscapine (5) in MCFꢀ7 and PANCꢀ1 cells. For example, 18g produced a 208% increase
in G2/M arrest in MCFꢀ7 cells and a 175% increase in PANCꢀ1 cells, relative to the 2% and 16%
increases observed for 5. The 4ꢀmethoxy group was clearly a key contributor to the target binding
interactions as the compounds which lacked this moiety / possessed an unsubstituted benzyl in the 5ꢀ
position exhibited a minimal percentage increase in G2/M arrest (0–13%). Furthermore, the
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presence of more than one methoxy group in compounds 17h–j and 18h–j also led to a reduction in
activity relative to 17g and 18g, with moderate G2/M arrest of 0ꢀ32% across the two cell lines. The
berberineꢀlike THIQ derivative 19 showed 28% and 9% increase in G2/M arrest with reference to the
control, in MCFꢀ7 and PANCꢀ1 cells, respectively.
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Table 1. Percentage increase in arrested cells trapped in the G2/M phase for THIQs 17a-j, 18a-
j and 19.
Structural Modifications
% Increase in G2/M Arrest[a]
Cpd
R
ꢀ
R1
MCF-7
283 ± 15
2 ± 0.4
18 ± 4.4
23 ± 7.6
29 ± 7.7
20 ± 6.7
48 ± 24
9 ± 3.4
180 ± 23
32 ± 5.8
19 ± 4.0
ꢀ8 ± 1.7
6 ± 1.9
18 ± 2.7
19 ± 12
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3 ± 0.3
PANC-1
265 ± 12
16 ± 4.0
ꢀ7 ± 7.5
26 ± 6.9
16 ± 7.6
8 ± 14
12 ± 5.0
13 ± 12
226 ± 44
8 ± 12
Vincristine[b]
5
ꢀ
ꢀ
ꢀ
Me
Ph
17a
17b
17c
17d
17e
17f
17g
17h
17i
Me
Me
Me
Me
4ꢀOMePh
2,3ꢀdiOMePh
3,4ꢀdiOMePh
3,4,5ꢀtriOMePh
Bn
Me
Me
Me
Me
Me
4ꢀOMeBn
2,3ꢀdiOMeBn
3,4ꢀdiOMeBn
3,4,5ꢀtriOMeBn
Ph
4ꢀOMePh
2,3ꢀdiOMePh
3,4ꢀdiOMePh
3,4,5ꢀtriOMePh
Bn
0 ± 8.6
ꢀ1 ± 7.5
ꢀ2 ± 2.8
21 ± 5.6
14 ± 1.4
11 ± 6.5
2 ± 2.8
17j
C(O)NHEt
C(O)NHEt
C(O)NHEt
C(O)NHEt
C(O)NHEt
C(O)NHEt
C(O)NHEt
C(O)NHEt
C(O)NHEt
C(O)NHEt
18a
18b
18c
18d
18e
18f
18g
18h
18i
1 ± 3.0
ꢀ8 ± 8.8
175 ± 37
28 ± 12
ꢀ10 ± 9.6
ꢀ4 ± 1.2
9 ± 9
4ꢀOMeBn
2,3ꢀdiOMeBn
3,4ꢀdiOMeBn
3,4,5ꢀtriOMeBn
208 ± 7.2
11 ± 10
12 ± 1.4
5 ± 6.9
18j
19
28 ± 2.6
[a]
[b]
The percentage increase in arrested cells was calculated with reference to the vehicle control. The data
represents the mean ± SEM observed in three independent experiments.
Vincristine, as a positive control, was tested at a concentration of 100 nM. All other compounds were
tested at 10 ꢁM.
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Figure 4. FACS analysis of PANCꢀ1 cells treated for 18 h with (A) 0.1% DMSO, (B) vincristine
(100 nM), (C) noscapine (5) (10 ꢁM), (D) 18g (10 ꢁM). FACS data was processed with FlowJo
(v10) and % cells were obtained at the G1 (blue), S (yellow) and G2/M (green) phases of the cell
proliferation cycle.
Tubulin polymerisation assay. Cytotoxic agents that interfere with MT dynamics are often found
to alter tubulin polymerisation through direct interaction with tubulin. For example, paclitaxel (3), a
microtubuleꢀstabilising agent, significantly enhances maximal initial velocity (Vmax) in the growth
phase of tubulin polymerisation, in comparison to control (tubulin with DMSO).30 Conversely, the
Vmax of tubulin polymerisation was reduced in the presence of noscapine (5) (in a concentrationꢀ
dependent manner).5 This characteristic inhibition of microtubule assembly has also previously been
observed with noscapinoids.23 To ascertain that the antiꢀproliferative effect of 18g against breast
cancer cells are due to interactions with tubulin, 18g was subjected to tubulin polymerisation
experiments (Fig. 5). The control (DMSO + tubulin) represents normal rate of polymerisation in the
absence of ligands (Vmax = 27 ± 2 mOD/min). At 10 ꢁM, paclitaxel (3) enhances polymerisation with
a Vmax of 76 ± 6 mOD/min. Noscapinoid 18g was found to behave in a similar manner to parent
noscapine (5), decreasing the initial polymerisation rate to 20 ± 2 mOD/min at 10 ꢁM.
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Figure 5. Tubulin polymerisation assay in the absence (in blue), and presence of ligand (paclitaxel,
in green; 18g, in red)
Compound 18g demonstrates efficacy in drug resistant cancer cells expressing P-gp.
Noscapinoid 18g demonstrates antiꢀproliferative activity by arrest of cells at the G2/M stage of the
cell cycle. The mechanism of action involves tubulin perturbation; however, several tubulin
disrupting drugs including paclitaxel and vinblastine are affected by the presence of Pꢀgp. This
efflux pump mediates their active extrusion from resistant cells and its influence on noscapine (5)
and 18g was investigated in a drug resistant variant (NCI/AdrRES) of MCFꢀ7WT breast cancer cells.
The antiꢀproliferative efficacy was measured in both cell lines using a MTT assay and data
shown in Table 2. The anticancer drug vinblastine caused potent inhibition of MCFꢀ7WT cell growth
with an IC50 value of 0.36 ± 0.01 nM. The potency was reduced by ~267ꢀfold in the drug resistant
cells expressing high levels of Pꢀgp. In contrast, the antiꢀproliferative effects of noscapine and 18g
did not differ between the two cell lines (Table 2). This demonstrates that the expression of Pꢀgp
does not alter the efficacy of these two compounds, in contrast to the TPI vinblastine.
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Table 2. Cell proliferation, calcein transport and ATP hydrolysis profile of noscapine (5), 18g,
nicardipine and vinblastine, in drug-sensitive (MCF-7WT) and -resistant (NCI/AdrRES) breast
cancer cell lines.[a]
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Drug
Cell Proliferation
Potency IC50( M)
CalceinꢀAM
Transport
Maximal rate Maximal rate
ATP Hydrolysis
Potency
IC50( M)
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ꢀ
(RFU/min)
(fold-basal)
ꢀ
MCFꢀ7
18.8 ± 6.4
2.7 ± 1.0
NCI/AdrRES
17.4 ± 4.5
2.5 ± 1.3
Noscapine (5)
26.7 ± 4.2
78 ± 20
17.6 ± 2.8
6.2 ± 1.1
69.7 ± 8.9
2.4 ± 0.6
18g
ꢀꢀ
ꢀꢀ
Nicardipine
Vinblastine[b]
107 ± 15
13.6 ± 2.3
4.7 ± 0.5
ꢀꢀ
ꢀꢀ
ꢀꢀ
0.36 ± 0.01
96 ± 15
[a]
Cells were treated with varying concentrations of test compounds and cell viability was
determined using a standard MTT assay. Potency was determined by nonꢀlinear least squares
regression of the sigmoidal doseꢀresponse equation. The data represented the mean ± SEM for at
least 8 independent observations. [b] The IC50 values for vinblastine are in nM.
The above observation may offer the potential of synergy to generate growth arrest in cancer
cells between noscapine derivatives and cytotoxic anticancer drugs. This would be of particular
importance in resistant cells where Pꢀgp generates an accumulation deficit. Therefore, the ability of
noscapine (5) and 18g to alter Pꢀgp mediated transport was assessed using the fluorescent substrate
calceinꢀAM. The calceinꢀAM rapidly accumulates in MCFꢀ7 cells where it is cleaved to the highly
fluorescent calcein, a charged species that is trapped in the cytoplasm.31 Addition of 5, 18g or
nicardipine failed to alter the rate of calcein accumulation in MCFꢀ7WT cells (data not shown). In
contrast, the NCI/AdrRES cells displayed negligible fluorescence over a 10 minute incubation period.
The addition of the Pꢀgp modulator nicardipine caused a rapid increase in fluorescent signal to a
maximal rate of calcein accumulation in rate of 107 ± 15 RFU/min (Table 2). Noscapine (5) also
elicited an increase in calcein accumulation, although the maximal rate was 4ꢀfold lower than
observed for nicardipine. Similarly, 18g increased the fluorescent signal to 78 ± 20 RFU/min, which
was 3ꢀfold greater than the effect produced by noscapine (5). This data suggests that noscapine (5)
and 18g are able to modulate the transport activity of Pꢀgp.
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To ascertain whether the altered transport rate was due to direct interaction with Pꢀgp, a more
direct assay was used. Pꢀgp was purified and reconstituted into lipid bilayers as previously
described,32,33 and drug dependent stimulation of ATP hydrolysis was measured. The basal rate of
ATP hydrolysis by purified Pꢀgp was 190 ± 40 nmol Pi/min/mg protein and this was stimulated to
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1266 ± 197 nmol Pi/min/mg protein in the presence of 3
ꢀM nicardipine. Full doseꢀresponse
analysis of the effect of 5, 18g and nicardipine on ATP hydrolysis are summarised in Table 2. All
three compounds produced significant stimulation of ATP hydrolysis by Pꢀgp. The degree of
stimulation by 18g was less than that observed for noscapine (5), but its potency was significantly
greater. This indicates that 18g interacts directly with Pꢀgp to modulate its function.
In summary, the antiꢀproliferative effects of noscapine and 18g were unaffected by the presence
of the resistance conferring efflux pump Pꢀgp. Moreover, both compounds were able to directly
modulate the function of Pꢀgp. This offers the potential for coꢀadministration of noscapine
derivatives with anticancer drugs to achieve synergistic growth inhibitory effects, even in drug
resistant cells. This activity could be potentially higher due to the fact this was not an
enantioselective synthesis and presumably only one enantiomer would confer activity based on
previous work.26 Obviously, the activity of noscapine and 5ꢀsubstituted THIQs is moderate and
further work is continuing to increase the potency of these molecules.
Synergistic Effects. Both vinblastine and 18g cause growth inhibitory effects by disruption of
tubulin dynamics, although they bind to distinct sites on the tubulin monomer. Consequently, the
two compounds may interact in a synergistic manner. To investigate this possibility, the effects of
vinblastine on cell proliferation were measured in the presence of fixed concentrations of 18g (Figure
6). In both cell lines the doseꢀresponse curves were shifted (by varying degrees) to the left, thus
indicating a higher apparent potency (IC50) for vinblastine in the presence of 18g. The range of
vinblastine concentrations used in the two cell lines differs and the shift in the curves was not
equivalent. In addition, the upper level of the doseꢀresponse curve was reduced for increasing
concentrations of 18g, which reflects the inherent growth inhibitory effects of the noscapine
derivatives.
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Figure 6. The effect of 18g on the cytotoxicity of vinblastine in the drugꢀsensitive MCFꢀ7WT and the
drugꢀresistant NCI/AdrRES cells. The (a) MCFꢀ7WT and (b) NCI/AdrRES cells were treated with
varying concentrations of vinblastine alone (ꢀ) or in combination with 0.1 (ꢁ), 0.3 (ꢁ), 1.0 (ꢀ), 3.0
(ꢂ) or 10 ꢀM (ꢃ) of 18g and incubated for 6 d. The cell viability was determined using a MTT
assay and data fitted with the general doseꢀresponse relationship. All values represent mean ± SEM
obtained from at least three independent observations.
Combination Index analysis provides a quantitative measure of the changes in doseꢀresponse
curves and reveals whether the drug interactions are additive or synergistic.34 Table 3 shows the
combination indices (CI) for the combination of vinblastine and 18g in both cell lines. In the MCFꢀ
7WT cell line, the CI values did not differ significantly from 1.0 at any concentration of 18g. This
demonstrates an additive interaction between 18g and vinblastine in MCFꢀ7WT cells. In contrast, the
CI values obtained using 18g concentrations of 0.3 and 0.1 ꢀM were significantly different from 1.0
in the resistant cells. The values were <1.0 thereby demonstrating a synergistic interaction. The CIꢀ
value at a concentration of 1.0 ꢀM of 18g was not different to 1.0 and at higher concentrations of
18g, the “downward shift” in the doseꢀresponse curve precluded accurate assignment of a CIꢀvalue.
Thus, compound 18g provides a novel route to restoration of the efficacy of chemotherapy in
resistant cancer cells. It modulates the drug efflux activity of Pꢀgp and the combined effects on
tubulin result in a synergistic antiproliferative effect with vinblastine.
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Table 3. Combination Indices for co-administration of 18g and vinblastine in MCF-7 and
NCI/AdrRES cells.
18g (ꢀM)
MCF-7WT
1.50 ± 0.17
1.02 ± 0.19
1.09 ± 0.26
NCI/AdrRES
0.95 ± 0.18
0.57 ± 0.11
0.60 ± 0.17
1
9
0.3
0.1
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Vinblastine and 18g were coꢀadministered to drug sensitive (MCFꢀ7) and drug resistant
(NCI/AdrRES) cancer cells. Combination indices (CI) were calculated at 50% cell viability using
Lowe’s Additivity Model as described in the Experimental section. The CIs are represented as the
mean ± SEM of at least 4 independent observations.
ꢀ
CONCLUSIONS
We have synthesised a series of 5ꢀsubstituted THIQs 17a-j and 18a-j via a 10ꢀstep synthesis and two
of these compounds (17g and 18g) exhibited marked improvement in their ability to halt mitosis at
the G2/M phase compared with noscapine (5). The 5ꢀ(4ꢀmethoxybenzyl) substituted analogue 18g
featuring a Nꢀethylcarbamoyl moiety produced a 208% increase in G2/M arrest in MCFꢀ7 cells and a
175% increase in PANCꢀ1 cells in a cell cycle arrest assay. Compound 18g was assessed in a tubulin
polymerisation assay and found to reduce the maximal initial velocity of tubulin polymerisation
(Vmax) relative to the control. This compound displayed an EC50 of 2.7 ꢁM against a sensitive breast
cancer line (MCFꢀ7WT), a 7ꢀfold increase in potency relative to noscapine. This potency was
maintained against a resistant breast cancer line (NCI/AdrRES), which demonstrates the potential to
limit resistance effects seen for most current cytotoxic agents. Furthermore, noscapine and 18g were
shown to interact directly with Pꢀgp to modulate its function in an ATP hydrolysis assay. This
highlights the potential for coꢀadministration of noscapine and derivatives with anticancer drugs to
achieve synergistic growth inhibitory effects.
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ꢀ
EXPERIMENTAL SECTION
3
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Chemistry. H and 13C NMR spectra were recorded on a Bruker Avance Nanobay III 400 MHz
5
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Ultrashield Plus spectrometer at 400.13 and 100.62 MHz, respectively coupled to a BACS 60
automatic sample changer at 25 °C. Chemical shifts (δ) are recorded in parts per million (ppm) by
correction with reference to the chemical shift of the solvent, according to the procedure described
by Gottlieb et al.35 Coupling constants (J) are recorded in Hz, and the significant multiplicities
described by singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), multiplet (m), doublet of
doublets (dd), and doublet of triplets (dt). LCꢀMS were run to verify reaction outcome and purity
using an Agilent 6100 series Single Quad coupled to an Agilent 1200 series HPLC. The following
buffers were used: buffer A, 0.1% formic acid in H2O; buffer B, 0.1% formic acid in MeCN. The
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following gradient was used with a Phenomenex Luna 3 ꢁM C8(2) 15 mm × 4.6 mm column, and a
flow rate of 0.5 mL/min and total run time of 12 min; 0–4 min 95% buffer A and 5% buffer B, 4–7
min 0% buffer A and 100% buffer B, 7–12 min 95% buffer A and 5% buffer B. Mass spectra were
acquired in positive and negative ion mode with a scan range of 0–1000 m/z at 5 V. UV detection
was carried out at 254 nm. All compounds were of >95% purity. Thin layer chromatography was
conducted on 0.2 mm plates using Merck silica gel 60 F254. Column chromatography was achieved
using Merck silica gel 60 (particle size 0.063–0.200 ꢀm, 70–230 mesh). Calculated partition coꢀ
efficient values (cLogP) were calculated using ChemAxon’s Instant JChem program. Instant JChem
was used for structure database management, search and prediction (Instant JChem 5.9.4, 2013,
ChemAxon; http://www.chemaxon.com).]
General Procedure for the Formation of Amides 14a-j. To a stirred solution of 2ꢀ(4ꢀbromoꢀ
7ꢀmethoxybenzo[d][1,3]dioxolꢀ5ꢀyl)ethanꢀ1ꢀamine (13) (typically 300 mg, 1.0 eq), HOBt (1.2 eq),
EDCI.HCl (1.2 eq) and the appropriate phenylacetic or benzoic acid (1.01 eq) in DMF (3 mL) was
added DIPEA (2.5 eq) and the solution stirred for 16 h at 25 °C. The solution was added dropwise to
ice/water (~ 50 mL) and the resultant precipitate filtered, washed with water and dried thoroughly to
give the corresponding amide 14.
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-(2-(4-Bromo-7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)benzamide (14a). Compound 14a
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2
N
3
4
5
6
7
8
9
1
was formed in 92% as a colourless solid. H NMR (CDCl3) δ 7.76–7.71 (m, 2H), 7.52–7.47 (m, 1H),
7.40–7.45 (m, 2H), 6.47 (s, 1H), 6.20 (br s, 1H), 6.05 (s, 2H), 3.81 (s, 3H), 3.70 (t, J = 6.8 Hz, 2H),
3.02 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3) δ 167.7, 147.3, 143.0, 134.6, 134.4, 132.1, 131.7, 128.7
(× 2), 127.0 (× 2), 109.9, 102.0, 95.3, 56.8, 40.2, 34.8.
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N
-(2-(4-Bromo-7-methoxybenzo[
d
][1,3]dioxol-5-yl)ethyl)-4-methoxybenzamide
(14b).
1
Compound 14b was formed in 92% as a colourless solid. H NMR (CDCl3) δ 7.69 (d, J = 8.9 Hz,
2H), 6.91 (d, J = 8.9 Hz, 2H), 6.47 (s, 1H), 6.12 (br s, 1H), 6.04 (s, 2H), 3.84 (s, 3H), 3.81 (s, 3H),
3.67 (dd, J = 12.9, 6.8 Hz, 2H), 3.00 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3) δ 167.2, 162.4, 147.3,
143.0, 134.4, 132.2, 128.8 (× 2), 126.9, 113.9 (× 2), 109.9, 102.0, 95.4, 56.9, 55.6, 40.2, 34.9.
N
-(2-(4-Bromo-7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)-2,3-dimethoxybenzamide (14c).
Compound 14c was formed in 97% as a colourless solid. 1H NMR (CDCl3) δ 8.05 (t, J = 5.0 Hz,
1H), 7.71 (dd, J = 8.0, 1.6 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 7.03 (dd, J = 8.0, 1.6 Hz, 1H), 6.50 (s,
1H), 6.03 (s, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 3.71 (dd, J = 13.0, 7.0 Hz, 2H), 3.01 (t, J =
7.0 Hz, 2H). 13C NMR (CDCl3) δ 165.4, 152.7, 147.6, 147.2, 142.9, 134.3, 132.2, 126.7, 124.5,
122.9, 115.5, 109.8, 101.9, 95.4, 61.3, 56.9, 56.2, 39.8, 34.9.
N
-(2-(4-Bromo-7-methoxybenzo[
d
][1,3]dioxol-5-yl)ethyl)-4-methoxybenzamide
(14d).
1
Compound 14d was formed in 94% as a colourless solid. H NMR (CDCl3) δ 7.39 (d, J = 2.0 Hz,
1H), 7.21 (dd, J = 8.3, 2.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.45 (s, 1H), 6.24 (t, J = 5.6 Hz, 1H),
6.02 (s, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 3.80 (s, 3H), 3.66 (dd, J = 12.9, 6.8 Hz, 2H), 3.00 (t, J = 6.9
Hz, 2H). 13C NMR (CDCl3) δ 167.3, 151.8, 149.1, 147.2, 143.0, 134.4, 132.2, 127.2, 119.3, 110.6,
1l10.4, 109.9, 102.0, 95.3, 56.8, 56.1, 40.3, 34.8.
N
-(2-(4-Bromo-7-methoxybenzo[
d
][1,3]dioxol-5-yl)ethyl)-3,4,5-trimethoxybenzamide (14e).
1
Compound 14b was formed in 98% as a colourless solid. H NMR (CDCl3) δ 6.95 (s, 2H), 6.48 (s,
1H), 6.12 (t, J = 5.4 Hz, 1H), 6.04 (s, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 3.84 (s, 3H), 3.68 (dd, J = 12.8,
6.8 Hz, 2H), 3.02 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3) δ 167.5, 153.4 (× 2), 147.3, 143.1, 141.1,
134.5, 132.2, 130.1, 110.2, 104.4, 102.0 (× 2), 95.5, 61.1, 57.0, 56.5 (× 2), 40.5, 34.7.
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N
-(2-(4-Bromo-7-methoxybenzo[
d
][1,3]dioxol-5-yl)ethyl)-2-phenylacetamide
(14f).
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6
7
8
9
Compound 14f was formed in 93% as a colourless solid. 1H NMR (CDCl3) δ 7.36–7.27 (m, 3H),
7.24–7.18 (m, 2H), 6.33 (s, 1H), 6.02 (s, 2H), 5.45 (br s, 1H), 3.83 (s, 3H), 3.56 (s, 2H), 3.45 (dd, J =
13.1, 6.8 Hz, 2H), 2.82 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3) δ 171.2, 147.2, 142.9, 134.8, 134.3,
131.9, 129.6 (× 2), 129.1 (× 2), 127.5, 109.9, 101.9, 95.3, 57.0, 44.0, 39.7, 34.7.
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N
-(2-(4-Bromo-7-methoxybenzo[
d
][1,3]dioxol-5-yl)ethyl)-2-(4-methoxyphenyl)acetamide
1
(14g). Compound 14g was formed in 94% as a colourless solid. H NMR (CDCl3) δ 7.13–7.09 (m,
2H), 6.88–6.83 (m, 2H), 6.32 (s, 1H), 6.03 (s, 2H), 5.39 (br s, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.49 (s,
2H), 3.44 (dd, J = 12.9, 6.8 Hz, 2H), 2.81 (t, J = 6.8 Hz, 2H). 13C NMR (CDCl3) δ 171.6, 159.0,
147.2, 142.9, 134.4, 131.9, 130.7 (× 2), 126.7, 114.6 (× 2), 109.9, 101.9, 95.3, 57.0, 55.4, 43.1, 39.7,
34.7.
N
-(2-(4-Bromo-7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)-2-(2,3-
1
dimethoxyphenyl)acetamide (14h). Compound 14h was formed in 93% as a colourless solid. H
NMR (CDCl3) δ 7.04–6.98 (m, 1H), 6.88–6.80 (m, 2H), 6.36 (s, 1H), 6.01 (s, 2H), 5.92 (br s, 1H),
3.87 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H), 3.54 (s, 2H), 3.41 (dd, J = 13.1, 6.9 Hz, 2H), 2.81 (t, J = 7.0
Hz, 2H). 13C NMR (CDCl3) δ 171.3, 153.0, 147.1, 147.0, 142.9, 134.3, 132.1, 129.2, 124.6, 122.9,
111.9, 109.8, 101.9, 95.4, 60.7, 56.9, 55.8, 39.7, 38.8, 34.9.
N
-(2-(4-Bromo-7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)-2-(3,4-
dimethoxyphenyl)acetamide (14i). Compound 14i was formed in 94% as a colourless solid. 1H
NMR (CDCl3) δ 6.81 (d, J = 8.0 Hz, 1H), 6.74–6.69 (m, 2H), 6.33 (s, 1H), 6.03 (s, 2H), 5.43 (t, J =
4.7 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.49 (s, 2H), 3.45 (dd, J = 12.9, 6.9 Hz, 2H),
2.82 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3) δ 171.5, 149.4, 148.5, 147.2, 142.9, 134.3, 131.8, 127.2,
121.7, 112.5, 111.6, 109.8, 101.9, 95.3, 56.9, 56.0, 55.9, 43.6, 39.8, 34.5.
N
-(2-(4-Bromo-7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)-2-(3,4,5-
1
trimethoxyphenyl)acetamide (14j). Compound 14j was formed in 87% as a colourless solid. H
NMR (CDCl3) δ 6.41 (s, 2H), 6.36 (s, 1H), 6.03 (s, 2H), 5.51 (t, J = 5.5 Hz, 1H), 3.85 (s, 3H), 3.84
(s, 3H), 3.83 (s, 6H), 3.50–3.43 (m, 4H), 2.85 (t, J = 6.9 Hz, 2H). 13C NMR (CDCl3) δ 171.1, 153.7
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(× 2), 147.2, 142.9, 137.3, 134.3, 131.9, 130.3, 109.8, 106.5 (× 2), 101.9, 95.4, 61.0, 57.0, 56.2 (× 2),
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44.3, 40.0, 34.4.
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General Procedure for the Bischler-Napieralski cyclisation (compounds 15a-j). To a
mixture of the amide 14 (typically 300 mg, 1.0 eq) in toluene (3 mL) in a teflonꢀcapped sealed tube
was added POCl3 (5.0 eq) dropwise. The fluffy white mixture was heated at 120 °C for 2 h. The
yellow solution, which formed, was cooled to room temperature and added to DCM (30 mL).
Saturated NaHCO3 (20 mL) and 5 M KOH (0.5 mL) were carefully added and stirred for 30 min.
The organic phase was separated, washed with sat. NaHCO3 (20 mL) and 5 M KOH (0.5 mL), then
sat. NaCl, dried with MgSO4, filtered and the filtrate evaporated under reduced pressure to give the
cyclised imine 15. This was immediately taken forward in the reduction step.
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9-Bromo-4-methoxy-5-phenyl-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline
(15a).
1
Compound 15a was formed in 95% as a yellow gum. H NMR (CDCl3) δ 7.47–7.42 (m, 2H), 7.40–
7.37 (m, 3H), 6.06 (s, 2H), 3.74–3.66 (m, 2H), 3.49 (s, 3H), 2.79–2.72 (m, 2H).
9-Bromo-4-methoxy-5-(4-methoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline
(15b). Compound 15b was formed in 94% as a yellow gum. 1H NMR (CDCl3) δ 7.45 (d, J = 8.8
Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 6.07 (s, 2H), 3.83 (s, 3H), 3.72 – 3.64 (m, 2H), 3.57 (s, 3H), 2.82–
2.72 (m, 2H).
9-Bromo-5-(2,3-dimethoxyphenyl)-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline
1
(15c). Compound 15c was formed in 86% as a yellow gum. H NMR (CDCl3) δ 7.08 (t, J = 7.9 Hz,
1H), 6.96 (dd, J = 7.7, 1.6 Hz, 1H), 6.90 (dd, J = 8.1, 1.5 Hz, 1H), 6.00 (s, 2H), 4.05–3.95 (m, 1H),
3.86 (s, 3H), 3.57 (s, 3H), 3.44 (s, 3H), 2.94–2.82 (m, 2H), 2.67–2.55 (m, 1H).
9-Bromo-5-(3,4-dimethoxyphenyl)-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline
1
(15d). Compound 15d was formed in 99% as a yellow oil. H NMR (CDCl3) δ 7.37 (d, J = 1.5 Hz,
1H), 7.16 (d, J = 6.7 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 6.19 (s, 2H), 4.02 (s, 3H), 3.94 (s, 3H), 3.90 –
3.83 (m, 2H), 3.70 (s, 3H), 3.09–3.01 (m, 2H).
9-Bromo-4-methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline (15e). Compound 15e was formed in 98% as a yellow gum. 1H NMR (CDCl3) δ
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6.75 (s, 2H), 6.10 (s, 2H), 3.88 (s, 6H), 3.87 (s, 3H), 3.75–3.69 (m, 2H), 3.60 (s, 3H), 2.84–2.78 (m,
3
4
2H).
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5-Benzyl-9-bromo-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline (15f). Compound
7
8
15f was formed in 98% as an orange gum. 1H NMR (CDCl3) δ 7.32–7.25 (m, 2H), 7.24–7.19 (m,
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3H), 6.05 (s, 2H), 4.41 (s, 2H), 3.93 (s, 3H), 3.68 – 3.62 (m, 2H), 2.80–2.72 (m, 2H).
9-Bromo-4-methoxy-5-(4-methoxybenzyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline
1
(15g). Compound 15g was formed in 95% as an orange gum. H NMR (CDCl3) δ 7.08 (d, J = 8.7
Hz, 2H), 6.77 (d, J = 8.7 Hz, 2H), 6.00 (s, 2H), 4.20 (br s, 2H), 3.90 (s, 3H), 3.75 (s, 3H), 3.58–3.52
(m, 2H), 2.68–2.61 (m, 2H).
9-Bromo-5-(2,3-dimethoxybenzyl)-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline
1
(15h). Compound 15h was formed in 99% as a yellow gum. H NMR (CDCl3) δ 6.91 (t, J = 7.9 Hz,
1H), 6.76 (dd, J = 8.2, 1.4 Hz, 1H), 6.66–6.57 (m, 1H), 5.98 (s, 2H), 4.21 (s, 2H), 3.84 (s, 3H), 3.82
(s, 3H), 3.78 (s, 3H), 3.53 (dd, J = 7.7, 6.6 Hz, 2H), 2.64 (dd, J = 7.9, 6.5 Hz, 2H).
9-Bromo-5-(3,4-dimethoxybenzyl)-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline
1
(15i). Compound 15i was formed in 96% as a yellow gum. H NMR (CDCl3) δ 6.74–6.68 (m, 3H),
5.99 (s, 2H), 4.15 (s, 2H), 3.90 (s, 3H), 3.82 (s, 3H), 3.80 (s, 3H), 3.56–3.49 (m, 2H), 2.63–2.58 (m,
2H).
9-Bromo-4-methoxy-5-(3,4,5-trimethoxybenzyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline
1
(15j). Compound 15j was formed in 94% as a yellow gum. H NMR (CDCl3) δ 6.40 (s, 2Η), 6.00
(s, 2Η), 4.14 (s, 2Η), 3.92 (s, 3Η), 3.79 (s, 3Η), 3.78 (s, 6Η), 3.56–3.51 (m, 2Η), 2.64–2.59 (m, 2Η).
General Procedure for Imine Reduction (compounds 16a-j). The imine 15 (typically 275
mg, 1.0 eq) was taken up in MeOH (3 mL), cooled to ꢀ78 °C and NaBH4 (3.0 eq) added portionwise.
The reaction was stirred at this temperature for 30 min, then allowed to slowly warm up to room
temperature. Water (5 mL) and sat. NaCl (10 mL) were added and extracted with DCM (2 × 25 mL),
dried with MgSO4, filtered and the filtrate evaporated under reduced pressure to give the reduced
tetrahydroisoquinoline 16.
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9-Bromo-4-methoxy-5-phenyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
g
]isoquinoline
(16a).
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Compound 16a was formed in 94% as a yellow solid. H NMR (CDCl3) δ 7.31–7.20 (m, 3H), 7.15–
7.11 (m, 2H), 5.99 (d, J = 1.4 Hz, 1H), 5.97 (d, J = 1.4 Hz, 1H), 5.25 (s, 1H), 3.52 (s, 3H), 2.99–2.87
(m, 2H), 2.77–2.65 (m, 2H).
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9-Bromo-4-methoxy-5-(4-methoxyphenyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5
-
g
]isoquinoline (16b). Compound 16b was formed in 85% as a mauve solid. 1H NMR (CDCl3) δ
6.98 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.6 Hz, 2H), 5.91 (d, J = 1.2 Hz, 1H), 5.90 (d, J = 1.2 Hz, 1H),
5.16 (s, 1H), 3.71 (s, 3H), 3.48 (s, 3H), 2.91–2.79 (m, 2H), 2.71–2.56 (m, 2H).
9-Bromo-5-(2,3-dimethoxyphenyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
g
]isoquinoline (16c). Compound 16c was formed in 90% as a yellow solid. 1H NMR (CDCl3) δ
6.90 – 6.81 (m, 2H), 6.26 (dd, J = 7.4, 1.7 Hz, 1H), 5.98 (dd, J = 4.0, 1.4 Hz, 2H), 5.60 (s, 1H), 3.95
(s, 3H), 3.89 (s, 3H), 3.60 (s, 3H), 2.93–2.79 (m, 2H), 2.74–2.70 (m, 2H).
9-Bromo-5-(3,4-dimethoxyphenyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5
-
g
]isoquinoline (16d). Compound 16d was formed in 47% as a mauve solid. 1H NMR (CDCl3) δ
6.86 (d, J = 1.7 Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 6.51 (dd, J = 8.2, 1.9 Hz, 1H), 5.99 (s, 2H), 5.27
(s, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.58 (s, 3H), 3.03 – 2.90 (m, 2H), 2.79–2.69 (m, 2H).
9-Bromo-4-methoxy-5-(3,4,5-trimethoxyphenyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5
-
g
]isoquinoline (16e). Compound 16e was formed in 80% as a yellow solid. 1H NMR (CDCl3) δ
6.38 (s, 2H), 6.01 (d, J = 1.3 Hz, 1H), 6.00 (d, J = 1.3 Hz, 1H), 5.25 (s, 1H), 3.83 (s, 3H), 3.81 (s,
6H), 3.61 (s, 3H), 3.03 – 2.94 (m, 2H), 2.83 – 2.69 (m, 2H).
5-Benzyl-9-bromo-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinoline
(16f).
1
Compound 16f was formed in 92% as a yellow solid. H NMR (CDCl3) δ 7.38–7.31 (m, 2H), 7.30–
7.21 (m, 3H), 6.00 (d, J = 1.4 Hz, 1H), 5.97 (d, J = 1.4 Hz, 1H), 4.25 (dd, J = 10.5, 2.8 Hz, 1H), 4.07
(s, 3H), 3.24 (ddd, J = 12.3, 10.8, 5.2 Hz, 1H), 3.03 (ddd, J = 12.4, 5.5, 2.7 Hz, 2H), 2.84 (dd, J =
13.7, 10.5 Hz, 1H), 2.77–2.59 (m, 2H).
9-Bromo-4-methoxy-5-(4-methoxybenzyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
g
]isoquinoline (16g). Compound 16g was formed in 71% as a yellow solid. 1H NMR (CDCl3) δ
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7.19 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.6 Hz, 2H), 5.98 (dd, J = 8.7, 1.4 Hz, 2H), 4.19 (dd, J = 10.5,
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2.8 Hz, 1H), 4.07 (s, 3H), 3.81 (s, 3H), 3.28–3.17 (m, 1H), 3.06–2.92 (m, 2H), 2.82–2.58 (m, 3H).
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9-Bromo-5-(2,3-dimethoxybenzyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5
-
7
8
1
g
]isoquinoline (16h). Compound 16h was formed in 54% as a colourless solid. H NMR (CDCl3) δ
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7.04 (t, J = 7.9 Hz, 1H), 6.87 (dd, J = 7.7, 1.4 Hz, 1H), 6.82 (dd, J = 8.1, 1.3 Hz, 1H), 5.98 (d, J = 1.3
Hz, 1H), 5.96 (d, J = 1.3 Hz, 1H), 4.32 (t, J = 6.8 Hz, 1H), 4.05 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H),
3.33–3.23 (m, 1H), 3.04–2.94 (m, 3H), 2.75–2.55 (m, 2H).
9-Bromo-5-(3,4-dimethoxybenzyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
1
g
]isoquinoline (16i). Compound 16i was formed in 81% as a yellow solid. H NMR (CDCl3) δ 6.87
– 6.77 (m, 3H), 5.99 (dd, J = 9.1, 1.4 Hz, 2H), 4.23 (dd, J = 10.3, 2.8 Hz, 1H), 4.07 (s, 3H), 3.90 (s,
3H), 3.88 (s, 3H), 3.21 (ddd, J = 12.3, 10.3, 5.4 Hz, 1H), 3.05–2.94 (m, 2H), 2.78 (dd, J = 13.8, 10.4
Hz, 1H), 2.73–2.58 (m, 2H).
9-Bromo-4-methoxy-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
g
]isoquinoline (16j). Compound 16j was formed in 78% as a yellow solid. 1H NMR (CDCl3) δ
6.48 (s, 2H), 5.99 (dd, J = 9.6, 1.4 Hz, 2H), 4.27 (dd, J = 10.3, 2.8 Hz, 1H), 4.07 (s, 3H), 3.88 (s,
6H), 3.85 (s, 3H), 3.21 (ddd, J = 12.4, 10.3, 5.4 Hz, 1H), 3.07–2.94 (m, 2H), 2.81–2.59 (m, 3H).
General Procedure for N-Methylation (compounds 17a-j). Method A. To a stirred mixture
of THIQ (16) (typically 100 mg, 1.0 eq) and K2CO3 (2.5 eq) in DMF (1 mL) cooled on ice, was
added MeI (1.1 eq) dropwise and the reaction stirred at 25 °C for 3 h. Water (15 mL) was added and
extracted with EtOAc (30 mL). The aqueous fraction was reꢀextracted with EtOAc (10 mL). The
combined organic fractions were washed with water (3 × 10 mL), sat. NaCl, dried with MgSO4,
filtered and the filtrate evaporated under reduced pressure to give the reduced Nꢀmethyl product 17.
Method B.36 To a stirred solution of THIQ (16) (typically 100 mg, 1.0 eq) in HCOOH (1 mL)
was added CH2O solution (37% wt. in water) (7.0 eq) dropwise. The reaction was heated at 80 °C
for 2 h and the resultant brown solution was cooled to room temperature. The mixture was then
basified with 5 M NaOH to pH 9, extracted with EtOAc (3 × 15 mL), dried with MgSO4, filtered and
the filtrate evaporated under reduced pressure to give the reduced Nꢀmethyl product 17.
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g]isoquinoline
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9-Bromo-4-methoxy-6-methyl-5-phenyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
3
4
5
6
7
8
9
(17a). Compound 17a was formed via Method A in 14% as a colourless oil. 1H NMR (CDCl3) δ
7.31–7.19 (m, 3H), 7.12–7.07 (m, 2H), 5.96 (dd, J = 9.7, 1.3 Hz, 2H), 4.77 (s, 1H), 3.38 (s, 3H),
2.93–2.73 (m, 3H), 2.67–2.61 (m, 1H), 2.35 (s, 3H). 13C NMR (CDCl3) δ 146.2, 141.6, 139.9, 135.5,
129.5 (× 2), 127.9, 127.7 (× 2), 127.0, 125.2, 101.2, 96.4, 63.6, 59.0, 46.2, 42.6, 27.6. HRꢀESMS
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+
calcd. for C18H19BrNO3 [M + H] 376.0543, found 376.0548.
9-Bromo-4-methoxy-5-(4-methoxyphenyl)-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5
-
1
g
]isoquinoline (17b). Compound 17b was formed via Method B in 83% as a yellow oil. H NMR
(CDCl3) δ 7.03–6.97 (m, 2H), 6.82–6.74 (m, 2H), 5.95 (d, J = 1.2 Hz, 1H), 5.93 (d, J = 1.3 Hz, 1H),
4.77 (s, 1H), 3.76 (s, 3H), 3.43 (s, 3H), 2.90–2.71 (m, 3H), 2.62 (dt, J = 9.0, 3.5 Hz, 1H), 2.33 (s,
3H). 13C NMR (CDCl3) δ 158.6, 146.1, 139.8, 135.4, 133.3, 130.5 (× 2), 127.6, 125.3, 113.0 (× 2),
+
101.1, 96.3, 62.6, 59.1, 55.3, 45.8, 42.4, 27.5. HRꢀESMS calcd. for C19H21BrNO4 [M + H]
406.0648, found 406.0640.
9-Bromo-5-(2,3-dimethoxyphenyl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
g
]isoquinoline (17c). Compound 17c was formed via Method A in 26% as a colourless solid. 1H
NMR (CDCl3) δ 6.88 (t, J = 7.9 Hz, 1H), 6.80 (dd, J = 8.2, 1.5 Hz, 1H), 6.31 (dd, J = 7.7, 1.6 Hz,
1H), 5.94 (dd, J = 10.0, 1.4 Hz, 2H), 5.31 (s, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 3.46 (s, 3H), 2.93–2.72
(m, 3H), 2.65–2.56 (m, 1H), 2.35 (s, 3H). 13C NMR (CDCl3) δ 152.6, 147.7, 146.1, 139.6, 135.3,
134.9, 128.2, 125.6, 122.9, 122.0, 111.2, 101.1, 96.3, 60.3, 58.8, 55.9 (× 2), 46.1, 42.2, 27.8. HRꢀ
+
ESMS calcd. for C20H23BrNO5 [M + H] 436.0754, found 436.0765.
9-Bromo-5-(3,4-dimethoxyphenyl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
g
]isoquinoline (17d). Compound 17d was formed via Method B in 83% as a colourless oil. 1H
NMR (CDCl3) δ 6.79–6.68 (m, 2H), 6.51 (dd, J = 8.2, 1.9 Hz, 1H), 5.95 (d, J = 0.9 Hz, 1H), 5.94 (d,
J = 1.0 Hz, 1H), 4.75 (s, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.45 (s, 3H), 2.93–2.56 (m, 4H), 2.35 (s,
3H). 13C NMR (CDCl3) δ 148.4, 148.1, 146.2, 139.9, 135.4, 134.1, 127.6, 125.0, 121.7, 113.1,
+
110.2, 101.2, 96.3, 63.0, 59.2, 56.1, 55.9, 45.7, 42.5, 27.1. HRꢀESMS calcd. for C20H23BrNO5 [M +
H] 436.0754, found 436.0758.
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3
4
[1,3]dioxolo[4,5-g]isoquinoline (17e). Compound 17e was formed via Method B in 85% as a
5
6
7
8
1
yellow oil. H NMR (CDCl3) δ 6.32 (s, 2H), 5.97 (d, J = 1.3 Hz, 1H), 5.97 (d, J = 1.3 Hz, 1H), 4.72
(s, 1H), 3.81 (s, 3H), 3.79 (s, 6H), 3.49 (s, 3H), 2.99–2.90 (m, 1H), 2.89–2.79 (m, 1H), 2.77–2.62 (m,
2H), 2.38 (s, 3H). 13C NMR (CDCl3) δ 152.6 (× 2), 146.3, 140.0, 137.5, 137.2, 135.4, 127.7, 124.6,
106.9 (× 2), 101.2, 96.3, 63.7, 60.9, 59.1, 56.4 (× 2), 46.1, 42.6, 27.0. HRꢀESMS calcd. for
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+
C21H25BrNO6 [M + H] 466.0860, found 466.0869.
5-Benzyl-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinoline
(17f). Compound 17f was formed via Method A in 18% as a colourless oil. 1H NMR (CDCl3) δ
7.32–7.25 (m, 4H), 7.22–7.17 (m, 1H), 5.97 (dd, J = 5.5, 1.4 Hz, 2H), 4.01 (dd, J = 7.9, 4.7 Hz, 1H),
3.98 (s, 3H), 3.32 (ddd, J = 13.6, 11.3, 5.5 Hz, 1H), 2.91–2.78 (m, 3H), 2.70 (ddd, J = 17.7, 11.3, 6.8
Hz, 1H), 2.45 (ddd, J = 17.5, 5.5, 1.7 Hz, 1H), 2.29 (s, 3H). 13C NMR (CDCl3) δ 146.8, 141.3,
140.0, 134.4, 129.2 (× 2), 128.2 (× 2), 127.1, 125.9, 125.2, 100.9, 96.2, 60.3, 59.3, 44.0, 42.3, 40.7,
+
23.2. HRꢀESMS calcd. for C19H21BrNO3 [M + H] 390.0699, found 390.0707.
9-Bromo-4-methoxy-5-(4-methoxybenzyl)-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5
-
g
]isoquinoline (17g). Compound 17g was formed via Method A in 36% as a colourless oil. 1H
NMR (CDCl3) δ 7.17 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 5.97 (d, J = 1.4 Hz, 1H), 5.96 (d,
J = 1.4 Hz, 1H), 3.97 (s, 3H), 3.79 (s, 3H), 3.36–3.25 (m, 1H), 2.87 (dd, J = 13.6, 6.2 Hz, 1H), 2.79
(d, J = 6.1 Hz, 2H), 2.76–2.64 (m, 2H), 2.45 (ddd, J = 7.1, 5.3, 1.3 Hz, 1H), 2.31 (s, 3H). 13C NMR
(CDCl3) δ 157.9, 145.9, 140.0, 134.4, 133.0, 130.1 (× 2), 126.9, 113.7 (× 2), 101.2, 100.9, 96.1, 60.5,
+
59.4, 55.3, 44.0, 42.2, 39.7, 23.2. HRꢀESMS calcd. for C20H23BrNO4 [M + H] 420.0805, found
420.0810.
9-Bromo-5-(2,3-dimethoxybenzyl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5
-
1
g
]isoquinoline (17h). Compound 17h was formed via Method A in 12% as a yellow oil. H NMR
(CDCl3) δ 7.00 (t, J = 7.9 Hz, 1H), 6.88 (dd, J = 7.7, 1.4 Hz, 1H), 6.78 (dd, J = 8.1, 1.4 Hz, 1H), 5.97
(d, J = 1.4 Hz, 1H), 5.95 (d, J = 1.4 Hz, 1H), 4.14 (dd, J = 9.8, 4.0 Hz, 1H), 3.94 (s, 3H), 3.86 (s,
3H), 3.84 (s, 3H), 3.38 (ddd, J = 13.6, 11.5, 5.8 Hz, 1H), 2.97 (dd, J = 14.1, 9.8 Hz, 1H), 2.89–2.78
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(m, 2H), 2.70 (ddd, J = 18.2, 11.4, 7.1 Hz, 1H), 2.49 (ddd, J = 17.5, 5.6, 1.3 Hz, 1H), 2.28 (s, 3H).
13C NMR (CDCl3) δ 152.8, 147.6, 145.7, 140.2, 134.8, 134.5, 127.1, 125.6, 123.7, 122.5, 110.2,
100.8, 96.3, 60.7, 59.4, 59.2, 55.8, 43.6, 42.1, 33.9, 23.2. HRꢀESMS calcd. for C21H25BrNO5+ [M +
H] 450.0911, found 450.0928.
9
10
11
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9-Bromo-5-(3,4-dimethoxybenzyl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-
g
]isoquinoline (17i). Compound 17i was formed via Method A in 37% as a pale yellow oil. 1H
NMR (CDCl3) δ 6.82–6.78 (m, 3H), 5.96 (dd, J = 7.3, 1.4 Hz, 2H), 4.00–3.94 (m, 4H), 3.87 (s, 3H),
3.86 (s, 3H), 3.27 (ddd, J = 13.5, 11.2, 5.5 Hz, 1H), 2.89–2.81 (m, 1H), 2.78 (d, J = 6.2 Hz, 2H), 2.68
(ddd, J = 17.7, 11.1, 6.7 Hz, 1H), 2.43 (ddd, J = 17.4, 5.4, 1.8 Hz, 1H), 2.31 (s, 3H). 13C NMR
(CDCl3) δ 148.6, 147.3, 145.7, 140.0, 134.4, 134.0, 127.2, 125.1, 121.1, 112.5, 111.1, 100.8, 96.1,
+
60.3, 59.4, 56.0, 55.9, 44.1, 42.3, 40.2, 23.3. HRꢀESMS calcd. for C21H25BrNO5 [M + H] 450.0911,
found 450.0921.
9-Bromo-4-methoxy-6-methyl-5-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-
[1,3]dioxolo[4,5-g]isoquinoline (17j). Compound 17j was formed via Method A in 14% as a
1
colourless oil. H NMR (CDCl3) δ 6.49 (s, 2H), 5.96 (dd, J = 9.0, 1.4 Hz, 2H), 4.02 (dd, J = 7.7, 4.5
Hz, 1H), 3.98 (s, 3H), 3.85 (s, 6H), 3.83 (s, 3H), 3.26 (ddd, J = 13.6, 11.2, 5.5 Hz, 1H), 2.91–2.76
(m, 3H), 2.70 (ddd, J = 17.7, 11.1, 5.5 Hz, 1H), 2.45 (ddd, J = 17.4, 5.4, 1.8 Hz, 1H), 2.35 (s, 3H).
13C NMR (CDCl3) δ 153.0 (× 2), 145.8, 140.0, 137.1, 136.0, 134.2, 127.2, 125.0, 106.1 (× 2), 100.9,
+
96.2, 61.0, 60.1, 59.4, 56.2 (× 2), 44.2, 42.4, 41.0, 23.3. HRꢀESMS calcd. for C22H27BrNO6 [M +
H] 480.1016, found 480.0994.
General Procedure for N-Ethyl Urea Formation (compounds 18a-j). To a stirred solution of
THIQ (16) (typically 150 mg, 1.0 eq) in MeCN (1 mL) cooled on ice, was added ethyl isocyanate
(1.05 eq) and the reaction stirred for 1 h. The reaction mixture was evaporated at reduced pressure,
and purified by column chromatography to give Nꢀethyl urea product 18.
9-Bromo-
N
-ethyl-4-methoxy-5-phenyl-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline-6(5H)-
1
carboxamide (18a). Compound 18a was formed in 93% as a colourless foam. H NMR (CDCl3) δ
7.35–7.09 (m, 5H), 6.58 (s, 1H), 5.99 (s, 2H), 4.60 (t, J = 5.2 Hz, 1H), 3.81 (s, 3H), 3.59–3.51 (m,
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1H), 3.35–3.26 (m, 3H), 2.86 (ddd, J = 15.3, 9.1, 6.0 Hz, 1H), 2.62 (dt, J = 16.5, 5.1 Hz, 1H), 1.14 (t,
J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ 157.3, 146.6, 142.2, 139.6, 135.0, 128.4, 128.3 (× 2), 127.5 (×
5
6
+
2), 127.2, 124.0, 101.3, 95.8, 59.6, 52.4, 38.8, 36.0, 27.5, 15.7. HRꢀESMS calcd. for C20H22BrN2O4
7
8
[M + H] 433.0757, found 433.0764.
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10
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18
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60
9-Bromo-N-ethyl-4-methoxy-5-(4-methoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline-6-(5H)-carboxamide (18b). Compound 18b was formed in 71% as a colourless
1
foam. H NMR (CDCl3) δ 7.10 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.49 (s, 1H), 6.00 (s,
2H), 3.82 (s, 3H), 3.77 (s, 3H), 3.59–3.49 (m, 1H), 3.36–3.26 (m, 3H), 2.86 (ddd, J = 15.6, 9.3, 6.1
Hz, 1H), 2.66 (dt, J = 16.5, 5.0 Hz, 1H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ 158.8, 157.3,
146.6, 139.6, 135.1, 134.4, 128.7 (× 2), 128.3, 124.5, 113.7 (× 2), 101.3, 95.9, 59.7, 55.4, 52.1, 38.6,
+
36.0, 27.6, 15.8. HRꢀESMS calcd. for C21H24BrN2O5 [M + H] 463.0863, found 463.0858.
9-Bromo-5-(2,3-dimethoxyphenyl)-
N
-ethyl-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline-6-(5H)-carboxamide (18c). Compound 18c was formed in 88% as a colourless
1
solid. H NMR (CDCl3) δ 6.95–6.89 (m, 1H), 6.85 (dd, J = 8.2, 1.6 Hz, 1H), 6.45 (dd, J = 7.6, 1.6
Hz, 1H), 6.30 (t, J = 5.0 Hz, 1H), 6.15 (s, 1H), 5.95 (dd, J = 5.2, 1.3 Hz, 2H), 4.29 (ddd, J = 12.3,
4.9, 3.0 Hz, 1H), 4.02 (s, 3H), 3.89 (s, 3H), 3.51 (s, 3H), 3.23 (qd, J = 7.3, 5.1 Hz, 2H), 2.95 (ddd, J
= 13.5, 11.4, 5.2 Hz, 1H), 2.85–2.70 (m, 2H), 1.18 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3) δ 158.0,
152.6, 146.7, 145.8, 138.8, 135.9, 135.0, 129.1, 123.8, 123.2, 120.9, 111.9, 101.3, 96.7, 60.4, 58.8,
+
55.9, 50.0, 35.8, 35.2, 27.6, 15.3. HRꢀESMS calcd. for C22H26BrN2O6 [M + H] 493.0969, found
493.0967.
9-Bromo-5-(3,4-dimethoxyphenyl)-N-ethyl-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline-6-(5H)-carboxamide (18d). Compound 18d was formed in 80% as a colourless
foam. 1H NMR (CDCl3) δ 6.89 (d, J = 2.0 Hz, 1H), 6.69 (d, J = 8.3 Hz, 1H), 6.56–6.45 (m, 2H),
5.98 (dd, J = 3.4, 1.4 Hz, 2H), 4.62 (t, J = 5.3 Hz, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.52
(dt, J = 12.5, 5.2 Hz, 1H), 3.41–3.18 (m, 3H), 2.83 (ddd, J = 15.9, 9.5, 6.1 Hz, 1H), 2.64 (dt, J =
16.6, 4.8 Hz, 1H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ 157.3, 148.9, 148.2, 146.5, 139.6,
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135.0, 134.9, 128.2, 124.0, 119.5, 111.2, 110.3, 101.3, 95.9, 59.6, 56.0, 55.9, 52.1, 38.5, 35.9, 27.6,
3
4
+
15.7. HRꢀESMS calcd for C22H26BrN2O6 [M + H] 495.0948, found 495.0955.
5
6
7
8
9-Bromo-
N
-ethyl-4-methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-
)-carboxamide (18e). Compound 18e was formed in 65% as a colourless foam.
g
]isoquinoline-6(5
H
9
1H NMR (CDCl3) δ 6.51 (s, 1H), 6.42 (s, 2H), 6.01 (dd, J = 7.9, 1.2 Hz, 2H), 3.86 (s, 3H), 3.80 (s,
3H), 3.77 (s, 6H), 3.50 (dd, J = 12.4, 6.3 Hz, 1H), 3.39 (td, J = 8.6, 4.5 Hz, 1H), 3.31 (q, J = 7.2 Hz,
2H), 2.88 (ddd, J = 15.0, 8.8, 6.0 Hz, 1H), 2.68 (dt, J = 16.5, 5.2 Hz, 1H), 1.15 (t, J = 7.2 Hz, 3H).
13C NMR (CDCl3) δ 157.3, 153.1 (× 2), 146.7, 139.6, 138.1, 136.3, 135.0, 128.4, 123.8, 104.9 (× 2),
10
11
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14
15
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19
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55
56
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58
59
60
+
101.4, 95.9, 60.9, 59.7, 56.4 (× 2), 52.6, 39.2, 36.0, 27.5, 15.8. HRꢀESMS calcd. for C23H28BrN2O7
[M + H] 523.1074, found 523.1089.
5-Benzyl-9-bromo-
N
-ethyl-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline-6(5H)-
1
carboxamide (18f). Compound 18f was formed in 62% as a pale yellow gum. H NMR (CDCl3) δ
7.34–7.20 (m, 5H), 5.97 (dd, J = 4.0, 1.2 Hz, 2H), 4.91 (d, J = 7.7 Hz, 1H), 4.34–4.23 (m, 1H), 4.08
(s, 3H), 3.42 (t, J = 5.2 Hz, 1H), 3.24 (ddd, J = 13.3, 10.4, 5.2 Hz, 1H), 3.10 (dd, J = 13.4, 2.9 Hz,
1H), 2.93–2.75 (m, 3H), 2.67 (ddd, J = 16.8, 12.8, 9.0 Hz, 2H), 0.73 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3) δ 157.7, 146.3, 139.7, 138.8, 134.4, 129.3 (× 2), 128.9 (× 2), 128.2, 126.9, 124.0, 101.1,
+
96.0, 59.5, 54.5, 40.4, 36.2, 35.5, 27.8, 15.1. HRꢀESMS calcd. for C21H24BrN2O4 [M + H]
447.0914, found 447.0923.
9-Bromo-N-ethyl-4-methoxy-5-(4-methoxybenzyl)-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline-6-(5H)-carboxamide (18g). Compound 18g was formed in 74% as a colourless
1
gum. H NMR (CDCl3) δ 7.13 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 5.98 (dd, J = 3.8, 1.4
Hz, 2H), 4.85 (d, J = 7.6 Hz, 1H), 4.35–4.25 (m, 1H), 4.09 (s, 3H), 3.78 (s, 3H), 3.42 (t, J = 4.9 Hz,
1H), 3.22 (ddd, J = 13.2, 10.4, 5.2 Hz, 1H), 3.05 (dd, J = 13.6, 2.9 Hz, 1H), 2.98–2.60 (m, 5H), 0.76
(t, J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ 158.7, 157.7, 146.4, 138.9, 134.4, 131.7, 130.3 (× 2), 128.2,
124.0, 114.3 (× 2), 101.1, 96.0, 59.6, 55.4, 54.7, 39.5, 36.2, 35.6, 27.9, 15.2. HRꢀESMS calcd. for
+
C22H26BrN2O5 [M + H] 477.1020, found 477.1018.
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9-Bromo-5-(2,3-dimethoxybenzyl)-N-ethyl-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-
3
4
g
]isoquinoline-6-(5H)-carboxamide (18h). Compound 18h was formed in 65% as a colourless
5
6
7
8
1
foam. H NMR (CDCl3) δ 6.96 (t, J = 7.9 Hz, 1H), 6.80 (dd, J = 8.2, 1.3 Hz, 1H), 6.67 (dd, J = 7.6,
1.3 Hz, 1H), 5.96 (d, J = 1.4 Hz, 1H), 5.96 (d, J = 1.4 Hz, 1H), 5.15 (dd, J = 9.5, 4.6 Hz, 1H), 4.43 (t,
J = 5.3 Hz, 1H), 4.22–4.05 (m, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 3.85 (s, 3H), 3.31 (ddd, J = 13.4, 9.6,
5.6 Hz, 1H), 3.12 (dd, J = 13.4, 4.7 Hz, 1H), 3.02 (ddd, J = 13.0, 7.2, 5.8 Hz, 1H), 2.96–2.86 (m,
2H), 2.82–2.66 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ 157.6, 152.9, 147.4, 146.3,
139.1, 134.5, 132.6, 128.4, 124.5, 124.4, 123.1, 111.2, 101.1, 96.2, 60.7, 59.4, 55.8, 51.9, 36.4, 35.6,
9
10
11
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13
14
15
16
17
18
19
20
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23
24
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45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
35.5, 27.9, 15.1. HRꢀESMS calcd. for C23H28BrN2O6 [M + H] 507.1125, found 507.1141.
9-Bromo-5-(3,4-dimethoxybenzyl)-
N-ethyl-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-
g
]isoquinoline-6(5 )-carboxamide (18i). Compound 18i was formed in 66% as a pale yellow gum.
H
1H NMR (CDCl3) δ 6.81 (d, J = 8.1 Hz, 1H), 6.76–6.71 (m, 2H), 5.97 (dd, J = 4.3, 1.4 Hz, 2H), 4.93
(d, J = 6.8 Hz, 1H), 4.27–4.24 (m, 1H), 4.07 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.54 (t, J = 4.8 Hz,
1H), 3.22 (ddd, J = 13.1, 10.4, 5.0 Hz, 1H), 3.03 (dd, J = 13.6, 3.1 Hz, 1H), 2.96 (qd, J = 7.2, 3.6 Hz,
1H), 2.92–2.83 (m, 1H), 2.81–2.67 (m, 2H), 2.62 (ddd, J = 16.8, 4.8, 3.5 Hz, 1H), 0.78 (t, J = 7.2 Hz,
3H). 13C NMR (CDCl3) δ 157.7, 149.3, 148.1, 146.4, 138.9, 134.4, 132.2, 128.2, 124.0, 121.4,
112.4, 111.6, 101.1, 96.0, 59.6, 56.1 (× 2), 54.4, 40.1, 36.5, 35.6, 27.8, 15.2. HRꢀESMS calcd. for
+
C23H28BrN2O6 [M + H] 507.1125, found 507.1133.
9-Bromo-
N
-ethyl-4-methoxy-5-(3,4,5-trimethoxybenzyl)-7,8-dihydro-[1,3]dioxolo[4,5-
)-carboxamide (18j). Compound 18j was formed in 74% as a colourless gum.
g
]isoquinoline-6(5
H
1H NMR (CDCl3) δ 6.41 (s, 2H), 5.96 (dd, J = 5.5, 1.4 Hz, 2H), 5.00 (d, J = 7.0 Hz, 1H), 4.19 (dd, J
= 7.6, 4.6 Hz, 1H), 4.05 (s, 3H), 3.83 (s, 6H), 3.80 (s, 3H), 3.68 (t, J = 4.6 Hz, 1H), 3.22 (ddd, J =
13.0, 10.2, 4.9 Hz, 1H), 3.05–2.86 (m, 3H), 2.81–2.68 (m, 2H), 2.61 (dt, J = 8.5, 4.6 Hz, 1H), 0.83 (t,
J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ 157.7, 153.5 (× 2), 146.4, 138.8, 136.9, 135.4, 134.4, 128.2,
123.9, 106.1 (× 2), 101.2, 95.9, 60.9, 59.5, 56.3 (× 2), 54.0, 41.0, 36.7, 35.6, 27.8, 15.2. HRꢀESMS
+
calcd. for C24H30BrN2O7 [M + H] 537.1231, found 537.1230.
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4-Bromo-11,12,14-trimethoxy-5,8,13,13a-tetrahydro-6
H
-[1,3]dioxolo[4,5-
3
4
g
]isoquinolino[3,2-a]isoquinoline (19). Compound 19 was formed from 16h via Method B in 47%
5
6
7
8
1
as a brown oil. H NMR (CDCl3) δ 6.85 – 6.71 (m, 2H), 6.00 (d, J = 1.4 Hz, 1H), 5.98 (d, J = 1.4
Hz, 1H), 4.17–4.10 (m, 1H), 4.04 (dd, J = 11.1, 4.3 Hz, 1H), 4.00 (s, 3H), 3.89 (d, J = 15.6 Hz, 1H),
3.84 (s, 3H), 3.78 (s, 3H), 3.42 (dd, J = 17.4, 3.6 Hz, 1H), 3.06 (dt, J = 11.6, 6.0 Hz, 1H), 2.87–2.79
(m, 2H), 2.66 (dt, J = 11.3, 5.6 Hz, 1H), 2.52 (dd, J = 17.4, 11.1 Hz, 1H). 13C NMR (CDCl3) δ
151.0, 146.6, 145.9, 139.8, 135.0, 129.1, 127.9, 127.0, 126.3, 121.8, 110.7, 101.2, 96.5, 60.1, 59.6,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
57.0, 56.0, 54.3, 46.5, 30.1, 26.6. HRꢀESMS calcd. for C21H23BrNO5 [M + H] 448.0754, found
448.0759.
Pharmacology. Compound preparation and storage. A 10ꢀ2 M stock solution was prepared for
each compound by dissolving the compound in sterileꢀfiltered DMSO (Sigma D2650), and the
solution was stored at –20 °C until use.
Cell culture and reagents. The human breast cancer cell line MCFꢀ7WT (HTBꢀ22) and pancreatic
cancer cell line PANCꢀ1 (CRLꢀ1469) were purchased directly from American Type Culture
Collection (ATCC) and cultured in minimum essential medium alpha (MEMα) (Invitrogen 32561ꢀ
037) and Dulbecco’s modified eagle medium (DMEM) (Invitrogen 10566ꢀ016), respectively. In
addition, each culture medium was supplemented with 10% foetal bovine serum (FBS) (Invitrogen
10100ꢀ147). The doxorubicinꢀselected Pꢀgp expressing variant NCI/AdrRes cell line was obtained
from Dr K Cowan (National Cancer Institute, Bethesda, MD).37 The NCI/AdrRES cell line was
cultured in DMEM supplemented with 10% foetal calf serum (FCS) and penicillin/streptomycin,
with every third passage supplemented with 3 ꢁM doxorubicin to maintain selection pressure. Cells
were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Before use, cell lines
were tested for the presence of mycoplasma by using a MycoAlert Mycoplasma detection kit (Lonza
LT07ꢀ318) according to the manufacturer’s instructions; cells employed in the assays tested negative
for mycoplasma.
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