1,3-Benzodioxole-Modified Noscapine Analogues: Synthesis, Antiproliferative Activity, and Tubulin-Bound Structure

Article abstract of DOI:10.1002/cmdc.202100363

Since the revelation of noscapine's weak anti-mitotic activity, extensive research has been conducted over the past two decades, with the goal of discovering noscapine derivatives with improved potency. To date, noscapine has been explored at the 1, 7, 6′, and 9′-positions, though the 1,3-benzodioxole motif in the noscapine scaffold that remains unexplored. The present investigation describes the design, synthesis and pharmacological evaluation of noscapine analogues consisting of modifications to the 1,3-benzodioxole moiety. This includes expansion of the dioxolane ring and inclusion of metabolically robust deuterium and fluorine atoms. Favourable structural modifications were subsequently incorporated into multi-functionalised noscapine derivatives that also possessed modifications previously shown to promote anti-proliferative activity in the 1-, 6′- and 9′-positions. Our research efforts afforded the deuterated noscapine derivative 14 e and the dioxino-containing analogue 20 as potent cytotoxic agents with EC₅₀ values of 1.50 and 0.73 μM, respectively, against breast cancer (MCF-7) cells. Compound 20 also exhibited EC₅₀ values of <2 μM against melanoma, non-small cell lung carcinoma, and cancers of the brain, kidney and breast in an NCI screen. Furthermore, compounds 14 e and 20 inhibit tubulin polymerisation and are not vulnerable to the overexpression of resistance conferring P-gp efflux pumps in drug-resistant breast cancer cells (NCI^ADR/RES). We also conducted X-ray crystallography studies that yielded the high-resolution structure of 14 e bound to tubulin. Our structural analysis revealed the key interactions between this noscapinoid and tubulin and will assist with the future design of noscapine derivatives with improved properties.

Full text of DOI:10.1002/cmdc.202100363

Full Papers
doi.org/10.1002/cmdc.202100363
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1
,3-Benzodioxole-Modified Noscapine Analogues:
Synthesis, Antiproliferative Activity, and Tubulin-Bound
Structure
[a]
+ [a]
+ [b]
[a]
Cassandra Yong, Shane M. Devine , Anne-Catherine Abel , Stefan D. Tomlins,
Divya Muthiah, Xuexin Gao, Richard Callaghan, Michel O. Steinmetz,
Andrea E. Prota,* Ben Capuano,* and Peter J. Scammells*
[c]
[c]
[c]
[b, d]
[b]
[a]
[a]
Since the revelation of noscapine’s weak anti-mitotic activity,
extensive research has been conducted over the past two
decades, with the goal of discovering noscapine derivatives
with improved potency. To date, noscapine has been explored
at the 1, 7, 6’, and 9’-positions, though the 1,3-benzodioxole
motif in the noscapine scaffold that remains unexplored. The
present investigation describes the design, synthesis and
pharmacological evaluation of noscapine analogues consisting
of modifications to the 1,3-benzodioxole moiety. This includes
expansion of the dioxolane ring and inclusion of metabolically
robust deuterium and fluorine atoms. Favourable structural
modifications were subsequently incorporated into multi-
functionalised noscapine derivatives that also possessed mod-
ifications previously shown to promote anti-proliferative activity
in the 1-, 6’- and 9’-positions. Our research efforts afforded the
deuterated noscapine derivative 14e and the dioxino-contain-
ing analogue 20 as potent cytotoxic agents with EC50 values of
1.50 and 0.73 μM, respectively, against breast cancer (MCF-7)
cells. Compound 20 also exhibited EC50 values of <2 μM against
melanoma, non-small cell lung carcinoma, and cancers of the
brain, kidney and breast in an NCI screen. Furthermore,
compounds 14e and 20 inhibit tubulin polymerisation and are
not vulnerable to the overexpression of resistance conferring P-
ADR/RES
gp efflux pumps in drug-resistant breast cancer cells (NCI
).
We also conducted X-ray crystallography studies that yielded
the high-resolution structure of 14e bound to tubulin. Our
structural analysis revealed the key interactions between this
noscapinoid and tubulin and will assist with the future design
of noscapine derivatives with improved properties.
Introduction
with MT dynamics, subsequently inducing cancer cell arrest at
[1]
different phases of the cell division cycle. Nature has been one
of the greatest sources of chemotherapeutics currently in
The clinical success of tubulin-binding agents over the past few
decades has made microtubules (MT) one of the most attractive
molecular targets for the treatment of cancer. The potent anti-
proliferative effects of these drugs are due to their interference
[2,3]
clinical use. A large majority of tubulin-targeting agents are
either directly derived from natural sources or generated
through manifestation of nature-derived structures. These
clinical agents either inhibit tubulin assembly (e.g., vinca
alkaloids, colchicine) or inhibit MT disassembly (e.g., paclitaxel,
+
[a] Dr. C. Yong, Dr. S. M. Devine, Dr. S. D. Tomlins, Prof. B. Capuano,
Prof. P. J. Scammells
[4]
epothilones). The development of chemoresistance has led to
[
5]
Medicinal Chemistry
Monash Institute of Pharmaceutical Sciences
Monash University
the downfall of these tubulin-targeted anti-cancer agents and
many of these agents are also associated with several toxicity
[4]
issues, including neuropathy. Thus, there is undoubtedly a
Parkville, VIC 3052 (Australia)
E-mail: peter.scammells@monash.edu
ben.capuano@monash.edu
b] A.-C. Abel, Prof. M. O. Steinmetz, Dr. A. E. Prota
Laboratory of Biomolecular Research,
Division of Biology and Chemistry
Paul Scherrer Institut
demand for novel tubulin-binding agents that are devoid of
these side effects.
+
[
Noscapine (1, Figure 1), a naturally-occurring phthalideiso-
quinoline alkaloid constituting 1–10% of alkaloid content in the
opium poppy, Papaver somniferum, was found to inhibit tubulin
5232 Villigen PSI (Switzerland)
[6]
polymerisation. Noscapine exhibited cytotoxic activities of 25,
E-mail: andrea.prota@psi.ch
c] Dr. D. Muthiah, X. Gao, Prof. R. Callaghan
Research School of Biology
Australian National University
Canberra, ACT 2061 (Australia)
d] Prof. M. O. Steinmetz
[
[
39 and 42 μM against cervical (HeLa), bladder (Renal 1983) and
breast (MCF-7) cancers, respectively. Further evaluation studies
showed that noscapine significantly inhibits the progression of
tumour growth with low toxicity in surrounding healthy
[7]
Biozentrum
University of Basel
tissues. Noscapine was previously used as a clinical antitussive
agent with an oral bioavailability of 30% and half-life of 4.5 h,
thereby demonstrating an excellent safety and pharmacokinetic
4056 Basel (Switzerland)
+
[
] These authors contributed equally to this work.
[8,9]
profile.
Our research group has recently shown that the
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100363
efficacy of noscapine was not reduced by the multidrug
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noscapine would only bind to tubulin after metabolic oxidation
and demethylation at the 7-position.
As part of our SAR investigation at the 6’-position of
noscapine, we have previously reported the synthesis and
pharmacological evaluation of N-substituted cyclic ether nosca-
pine derivatives (6a–e) against breast (MCF-7), colon (Caco-2)
[26]
and prostate (PC3) cancer cells (Figure 2). Several compounds
from this focussed library showed potencies of �10 μM. The
replacement of N-methyl for longer N-alkyl chains (6a) was
found to be detrimental to activity. N-Acyl (6b) and N-
alkoxycarbonyl analogues (6c) with phenyl and benzyl sub-
stitution resulted in moderate mitotic arrest activity. Most
notably, N-carbamoyl (6d) and N-thiocarbamoyl (6e) derivatives
with short side chains displayed promising anti-mitotic activity.
The cyclic ether N-ethylcarbamoyl noscapine analogue 7 was
found to be most active within this library, with EC50 values of
[
14–24]
Figure 1. Noscapine (1) and noscapinoids 2–5.
[
26]
3
.6 μM and 6.7 μM against MCF-7 and PC3 cells, respectively.
[10–12]
resistant phenotype.
Further investigation revealed nosca-
The inclusion of a 9-chloro group gave a further boost in
pine’s inhibitory effects on P-glycoprotein (P-gp) through direct
interaction with the transporter.
potency with compound 8 exhibiting IC50 values of 1.7 μM and
1.5 μM against MCF-7 and PC3 cells, respectively. We have
[13]
[10]
In the past two decades, many researchers have explored
modifications at the 1, 7, 6’ and 9’-positions of noscapine, in
search of more potent noscapine analogues. The manipulation
at the C9’-position was first explored with halo-substitution
also recently disclosed the replacement of N-methyl with an (N-
methylimidazol-4-yl)sulfonyl moiety (9) at the N6’-position
resulted in a 64-fold potency increase relative to noscapine
[12]
(1). Noscapinoid 9 possesses sub-micromolar activity against
a range of cell lines (EC50 values: 560 nM against MCF-7 cells;
980 nM against PANC-1 cells; 271 nM against MDA-MB-435 cells;
443 nM against SK-MEL-5 cells). Unlike many clinical anti-cancer
agents, this potency is also maintained against drug-resistant
(Figure 1), where the analogues exhibited tubulin-binding
activities greater than noscapine (1) in the following order of
magnitude: 9’-iodo (2a) >9’-chloro (2b) >9’-bromo (2c) >9’-
[14,15]
fluoronoscapine (2d).
with several nitrogen-containing functional groups such as
NO , -NH and -N , where 9’-nitronoscapine (2e) demonstrated
This position was further explored
RES
NCI/Adr cells, evidence of circumvention of common efflux
-
mechanisms. Furthermore, 9 demonstrated a strong MT
2
2
3
anti-cancer activity against ovarian cancer cells (1A9) and drug-
destabilising profile, where the rate of tubulin polymerisation
was significantly reduced by 8-fold.
Though noscapine has been the subject of extensive
research, a region of the molecule that has remained unex-
plored is the 1,3-benzodioxole functionality. This motif is a
common functionality found in natural products such as the
resistant ovarian cancer cells (1A9/PTX22) of 30.1 and 27.5 μM,
[16–18]
respectively.
A novel series of 9’-aryl noscapinoids were
assessed and 9’-(4-vinylphenyl)noscapine (2f) was shown to
inhibit triple-negative breast cancer cells (MDA-MB-231) with an
[19]
IC50 of 6 μM.
The development of structure-activity relationships (SAR) at
the southern isobenzofuranone ring have also been inves-
tigated by various research groups. This work was first
conducted by Anderson et al., where a regioselective O-
[20]
demethylation was conducted at the C7-position. Following
the discovery of 7-hydroxy noscapine derivative 3a, a number
of functional groups were explored at the C7-postion (Figure 1).
This includes O-acyl, O-benzyl, O-aryl, S-benzyl and 7-amino-
[21–23]
noscapine derivatives.
The 7-hydroxy and 7-aminonosca-
pine derivatives (3a and 5, respectively) caused the arrest of
cells at the G /M phase at approximately 500 times increased
2
potency in contrast with noscapine (1). Compound 5 displayed
a potency (IC ) to inhibit proliferation of 94.3 and 237.4 nM
5
0
[24]
against SNU398 and PLC/PRF/5 cells, respectively. Moreover,
5
was found to inhibit tubulin assembly with a tubulin-binding
5
À 1
affinity of 1.1×10 M at 25°C. Despite its outstanding anti-
mitotic profile, microsomal and S9 stability data suggests that 5
has high in vivo clearance. More recently, a new pro-drug
hypothesis was introduced based on a high-resolution crystal
[25]
structure of 7-aminonoscapine (5),
which proposed that
Figure 2. N-Substituted noscapine and multi-functionalised derivatives 6a-e
[
12, 26]
and 7–9.
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[
27]
terpenes, safrole, benzylisoquinoline and aporphine alkaloids.
tubulin of one of these potent multi-functionalised noscapi-
noids to high resolution by X-ray crystallography. This crystal
structure illuminated the observed SAR profile, most notably
providing an explanation for poor activity of the noscapine
derivatives with an unmodified lactone moiety relative to the
series in which the lactone had been reduced to the desired
cyclic ether.
The 1,3-benzodioxole moiety can also be found in clinical
agents such as stiripentol and paroxetine for the treatment of
[28,29]
epilepsy and depression, respectively.
The dioxolane ring is
a substrate for the cytochrome P450 system, and is commonly
metabolised to an acetal moiety, which subsequently under-
goes hydrolytic or oxidative cleavage to give the catechol or
[30,31]
carbene species, respectively.
The resultant carbene species
is known to form an irreversible coordination to cytochrome
[30,32]
enzyme CYP2D6, leading to the inactivation of this enzyme.
Results and Discussion
As CYP2D6 is an enzyme that is involved in the metabolism of
numerous drugs which includes antihypertensive agents, β-
blockers, antiarrhythmic drugs, and antidepressants, inhibition
of CYP2D6 produces an unwanted reduction in clearance of
Chemistry
The first steps in the synthesis of the targeted noscapine
derivatives involved the preparation of N-ethylcarbamoyl
noscapine (11) via N-demethylation of noscapine using an iron
salt-mediated Polonovski reaction followed by reaction of the
resultant secondary amine with ethyl isocyanate (Scheme 1). In
order to introduce the proposed modifications to the 1,3-
benzodioxole moiety of 11 the 1,3-dioxolane ring was sub-
sequently cleaved to generate the corresponding catechol 12.
[33]
these clinical agents.
In this study, we have investigated a range of bioisosteres
of the 1,3-benzodioxole motif present in noscapine in order to
map the structure-activity relationships associated with this
region of the molecule, identify more potent derivatives and
potentially improve metabolic stability. The modifications
include the replacement of the methylenedioxy group with
dialkoxy substituents (including deuterium and fluorine con-
taining alkyloxy groups), the expansion of the dioxolane ring
and the replacement of the hydrogens of the dioxolane ring
with deuterium atoms. Herein, we report the synthesis and
biological evaluation of a series of novel noscapine derivatives
with a range of aforementioned modifications to the 1,3-
benzodioxole functionality. These structural modifications were
also combined with the modifications to the 1-, 6’- and 9’-
positions that were previously shown to promote anti-prolifer-
ative activity. Finally, we determined the binding pose to
The ring was cleaved via a BCl -mediated reaction, where the
resultant complex (RÀ O-BCl ) was hydrolysed in an acidic
3
2
environment to give 12 in 95% yield. The subsequent alkylation
reactions were achieved by treating the catechol 12 with the
appropriate alkyl halide or dihalide and potassium carbonate.
The only exception was the difluoromethoxy analogue 13b
which was prepared by treating 12 with sodium chlorodifluor-
oacetate and potassium carbonate. In the final step, the lactone
moiety of compounds 13a–h was reduced using NaBH /BF Et O
4
3·
2
Scheme 1. Reagents and conditions: (a) (i) m-CPBA, CHCl
3
, 0°C, 2. HCl, 1 h, 95%, (ii) FeSO4·7H
2
O, MeOH, À 5°C, 1 h, 78%; (b) EtNCO, MeCN, 25°C, 2 h, 84%; (c)
BCl
1
3
, DCM, 25°C, 16 h, 95%; (d) 13a: MeI, K
2
CO
3
, DMF, 81% 13b: NaOOCF
2
Cl, K
2
CO
/BF3·Et
3
, DMF, 15%, 13c: CD
O, THF, 25°C, 16 h, 6–75%.
3
I, K
2
CO
3
, DMF, 35%, 13d: (CH
3
)
2
CHI, K
2
CO
3
, DMF, 63%
3e: CD
2
I
2
, K
2
CO
3
, DMF 42%, 13f-h: Br(CH
2
)
n
Br, K
2
CO
3
, DMF (14-60%); (e) NaBH
4
2
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to yield the corresponding cyclic ethers 14a–h in yields ranging
from 6 to 75%.
Biology
We have previously reported the benefits of installing a 9’-
chloro group in combination with modifications to the 6’-
position and reduction of the lactone of the
The impact of modification to the 1,3-dioxolane of noscapine
on cell viability in cancer cells was determined by screening the
noscapine analogues against breast (MCF-7) and pancreatic
(PANC-1) cancer cells. Potent noscapinoids identified from the
preliminary cell viability assay were then subjected to further
assessment in tubulin polymerisation and resistance studies.
[10]
isobenzofuranone.
Accordingly we targeted the 9-chloro
derivatives of the 1,4-dioxinotetrahydroisoquinoline 14f as well
as the corresponding analogue bearing a (N-methylimidazol-4-
yl)sulfonyl moiety in the 6’-position. The synthesis of these
compounds is shown in Scheme 2. Briefly, noscapine (1) was
first converted to N-nornoscapine (10) via a non-classical
Cell viability assays
[34,35]
Polonovski reaction.
The secondary amine was then reacted
with ethyl isocyanate or N-methylimidazolesulfonyl chloride to
give 11 and 15, respectively. Following previously described
To assess the anti-proliferative impact of these noscapine
derivatives, the compounds were subjected to cell viability
studies against MCF-7 and PANC-1 cell lines. The MCF-7 and
PANC-1 cells were treated with a broad range of concentrations
(0.12–250 μM) of the noscapine analogues, and cell viability was
BCl -mediated conditions, the 1,3-dioxolane ring was then
3
cleaved to give the catechols 12 and 16, respectively.
Installation of the 6-membered dioxane was conducted with
K CO and 1,2-dibromoethane, and the resultant products (13f
determined at the conclusion of treatment using a resazurin-
2
3
®
and 17) were chlorinated at the 9’-position using SO Cl to
based assay (CellTiter-Blue , Promega). EC values were deter-
2
2
50
afford 18 and 19. Lastly, the lactone was reduced to the cyclic
ether with a NaBH /BF Et O system affording 20 and 21 in
mined from the mean concentration-viability curves, in tripli-
cate (n=3), using GraphPad Prism v7.
4
3·
2
yields of 47% and 52%, respectively.
A vast majority of these analogues, which includes the
catechols 12 and 16 and the lactones 13a–h and 17, displayed
low cytotoxic activities of >250 μM (Table 1), with the
exception for 13e, which displayed a potency of 94.4ꢀ2.4 μM.
Scheme 2. Reagents and conditions: (a) N-methylimidazol-4-yl-SO
2
Cl, Et
3
N, DCM, 25°C, 16 h, 82%; (b) BCl
3
, DCM, 25°C, 16 h, 82–95%; (c) Br(CH
2 2 2 3
) Br, K CO ,
DMF, 25°C or 80°C, 16 h, 20–60%; (d) SO
Cl
, CHCl
, 25°C, 2 h, 31–75%; (e) NaBH
4 2
/BF3·Et
O, THF, 25°C, 16 h, 47–52%.
2
2
3
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ylimidazol-4-ylsulfonyl, was instead met with a reduction of
Table 1. Cell viability of noscapine derivatives.
activity from 9 (EC : 17.3ꢀ1.6 μM against MCF-7 and 3.8ꢀ
5
0
0
.1 μM against PANC-1).
The noscapine derivative 20 was subjected to a further
screening process against a panel of 60-cancer cell lines. This
screening was performed by the Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis, National
Cancer Institute (http://dtp.cancer.gov). Compound 20 was
found to inhibit carcinoma cell growth with activities of >2 μM
against melanoma (MDA-MB-435), non-small cell lung
[
a]
EC50 [μM]
MCF-7
Cpd No.
R
R’
Y
X
PANC-1
1
2
1
1
1
1
1
1
1
1
1
7
1
1
1
1
1
1
1
1
2
À OCH
2
3
OÀ
À CH
3
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
35.9ꢀ0.1 19.3ꢀ0.1
(
NCIÀ H522), CNS (SF-295) and renal (RXF) cancer lines (Figur-
3a
3b
3c
3d
3e
3f
À OCH
À C(O)NHEt
À C(O)NHEt
À C(O)NHEt
À C(O)NHEt
À C(O)NHEt
>250
>250
>250
>250
>250
>250
>250
>250
es S1 and S2). With a GI value of 1.16 μM, 20 can be
À OCHF
2
50
À OCD
3
acknowledged as a potent growth inhibitor for MDA-MB-435.
Compound 20 also displayed an exemplary cytotoxic potency,
with an LC50 of 79.8 μM against melanoma (SK-MEL-5) cells.
À O-iPr
À OCD
À O(CH
À O(CH
À O(CH
2
O-
94.4ꢀ2.4 >250
2
)
2
)
2
)
2
3
4
O- À C(O)NHEt
O- À C(O)NHEt
O- À C(O)NHEt
>250
>250
>250
3.6ꢀ0.4
>250
>250
>250
nd
3g
3h
30
À OCH
À OCH
2
OÀ
À C(O)NHEt
À C(O)NHEt
À C(O)NHEt
À C(O)NHEt
À C(O)NHEt
À C(O)NHEt
2
2
2
2
2
2
2
2
2
2
4a
4b
4c
4d
4e
4f
4g
4h
0
3
28.9ꢀ1.5 72.7ꢀ1.9
73.1ꢀ1.4 65.3ꢀ1.5
49.3ꢀ1.9 2.5ꢀ0.2
Cellular antiproliferative studies against sensitive- and
resistant-cancer cells
À OCHF
2
À OCD
3
À O-iPr
>250
1.5ꢀ0.1
>250
4.1ꢀ0.2
The development of resistance towards chemotherapeutic
agents is currently one of the major causes of treatment failure.
A key mechanism of acquired resistance is the increased
expression of P-gp, subsequently increasing the efflux of
À OCD
À O(CH
À O(CH
À O(CH
À O(CH
2
OÀ
2
2
2
2
)
)
)
)
2
3
4
2
OÀ À C(O)NHEt
OÀ À C(O)NHEt
OÀ À C(O)NHEt
OÀ À C(O)NHEt
12.5ꢀ1.9 7.9ꢀ1.4
28.5ꢀ1.4 28.2ꢀ1.6
34.3ꢀ1.4 28.2ꢀ1.6
Cl 0.73ꢀ0.1 1.1ꢀ0.1
[36]
cytotoxic agents from the cytoplasm of the cell. To determine
2
1
À O(CH
2
)
2
OÀ
CH
2
Cl 17.3ꢀ1.6 3.8ꢀ0.1
if the promising anti-proliferative activity of compounds 14e
and 20 is affected by the overexpression of P-gp, the ability of
[
a] Cell viability was determined using standard resazurin reduction
WT
2
0 to inhibit proliferation in the drug-sensitive MCF-7 breast
®
method with CellTiter-Blue assay . EC50 values shown represent the mean
SEM for at least 3 independent observations.
ADR/RES
cancer cell line and its drug-resistant variant, the NCI
cancer cell line (high P-gp expression levels) was assessed. The
clinical anti-mitotic agent, vinblastine, displayed an EC50 of
ꢀ
WT
ADR/RES
0
.24ꢀ0.04 nM in MCF-7 cells and 33.1ꢀ7.3 nM in NCI
In contrast, the reduction of these lactones to N-ethylcarbamoyl
cyclic ether noscapine analogues (14a–h) led to significant
improvements in potency (Table 1).
cells (Table 2).
The statistically significant (p <0.05, n=8) 138-fold differ-
ence in potency between the drug-sensitive and resistant cells
The observed cytotoxic activity of N-ethylcarbamoylnosca-
pine (11) was improved with the modification of 1,3-dioxolane
to the dimethoxy (14a), bis-difluoromethoxy (14b), bis-
trideuteromethoxy (14c), 7-membered (14g) and 8-membered
confirmed that vinblastine was affected by the MDR phenotype
ADR/RES
exhibited by the NCI
cells. Conversely, noscapine (1)
demonstrated anti-proliferative activities of 13.7ꢀ3.0 μM and
WT
ADR/RES
24.7ꢀ3.5 μM against MCF-7 and NCI
cells, respectively.
(
14h) dioxacyclic N-ethylcarbamoyl noscapine analogues (EC50
Although there was a statistically significant difference in
noscapine potency between the two cell lines (p<0.05), the
degree of resistance was a modest 1.8-fold, which is consistent
values: 28.5–73.1 μM). However, no cytotoxic activity was
detected following bis-isopropyl replacements in 14d. The
results furnished that 14e, whereby the naturally occurring
methylene of the dioxolane ring in noscapine has been
deuterated, displayed anti-cancer activities of 1.50ꢀ0.1 μM and
Table 2. Cell proliferation of Vinblastine, Noscapine (1), 14e and 20 in
4
.1ꢀ0.2 μM against MCF-7 and PANC-1 cells, respectively.
WT
ADR/RES
drug-sensitive (MCF-7 ) and -resistant (NCI
) breast cancer cell lines
Expansion to the 6-membered dioxane ring (14f) enhanced the
anti-proliferative activity, with EC50 values of 12.5ꢀ1.9 μM in
MCF-7 cell line and 7.9ꢀ1.4 μM in PANC-1 cell line.
[
a]
EC50 [μM]
MCF-7
WT
ADR/RES
Cpd No.
NCI
Fold Ratio
[
b]
Vinblastine
0.24ꢀ0.04
13.7ꢀ3.0
1.6ꢀ0.1
33.1ꢀ7.3
24.7ꢀ3.5
1.5ꢀ0.1
138
1.8
0.94
1.2
Introduction of a chloro substituent to 14f at the 9’-position
Noscapine (1)
14e
(
20) further promoted cytotoxic activity (EC : 0.73ꢀ0.1 μM
5
0
20
0.65ꢀ0.17
0.81ꢀ0.21
against MCF-7 and 1.1ꢀ0.1 μM against PANC-1), with a
considerable 52-fold improvement in activity when contrasted
with noscapine (1). This positive result further confirms the
benefits with multi-functionalisation that was previously deter-
[a] Cells were treated with varying concentrations of test compounds, and
cell viability was determined using a standard MTT assay. Potency was
determined by nonlinear least-squares regression of the sigmoidal doseÀ
response equation. The data represent the means ꢀ SEM for at least eight
independent observations. [b] The EC50 values for vinblastine are in
nanomolar (nM).
[10]
mined by DeBono et al. The anticipated potency gain with
1, where N-ethylcarbamoyl in 14 was replaced with N-meth-
2
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with previous findings and markedly lower than observed for
noscapine (1), the anti-proliferative effect observed against
MCF-7 cells is likely due to interference with MT dynamics.
Apart from the significant 13-fold reduction in Vmax, 20
remained in the growth phase of the polymerisation curve over
the course of the 60-minute experiment. Given that the other
tubulin ligands, paclitaxel, noscapine (1) and 14e, reached a
steady state at t=25 min onwards, this result indicates that 20
possesses a strong inhibitory effect on tubulin polymerisation.
[10–12]
vinblastine.
the drug-sensitive and resistant cell lines, with EC50 values of
.6ꢀ0.1 μM and 1.5ꢀ0.1 μM, respectively. A similar outcome
was observed for 20, with EC values of 0.65ꢀ0.17 μM and
Compound 14e was found to be equipotent in
1
5
0
0
.81ꢀ0.21 μM against drug-sensitive and the resistant cells,
respectively. This indicates that the overexpression of P-gp in
the drug-resistant NCI
ADR/RES
does not alter the efficacies of
noscapine (1), 14e or 20. Therefore, the noscapine derivatives
4e and 20 are not susceptible to the common MDR
1
phenotype that many clinical anti-mitotic agents are vulnerable
to, including vinblastine.
X-ray crystallography
Crystal structure of the tubulin-compound 14e complex. To
support the SAR of 14e and to define its interaction mode with
tubulin, we sought to determine the crystal structure of the
compound in complex with tubulin by X-ray crystallography. To
this end, we soaked 14e in a crystal formed by a protein
complex composed of two αβ-tubulin heterodimers, the
Inhibitory effects on tubulin polymerisation
The effectiveness in tubulin-targeting agents currently used in
cancer treatments are due to their ability to disrupt MT
dynamics through binding to tubulin, subsequently triggering
apoptotic mechanisms of the affected carcinoma. As a MT-
destabilising agent, noscapine (1) was previously found to
reduce the maximum initial velocity (Vmax) of tubulin polymer-
stathmin-like protein RB3 and tubulin tyrosine ligase (T R-
2
[1]
[38,39]
TTL).
We solved the crystal structure of T R-TTL-14e at a
2
resolution of 2.2 Å (Table S1).
Compound 14e binds to the colchicine site of tubulin,
which is formed by residues of the αT5 loop of α-tubulin
together with residues of strands βS8, βS9, βS10, helices βH7
and βH8 and loop βT7 of β-tubulin (PDB ID: 7AU5) (Figure 4).
We determined the occupancy of compound 14e at 75%
[6]
isation in a concentration-dependant manner. This inhibitory
action has also been consistently observed across the years by
[11,12,24,37]
various noscapine research groups.
The key compounds
of this series, 14e and 20, were evaluated for their capacity to
inhibit tubulin polymerisation.
within the colchicine site of αβ-tubulin (chains A and B of T R-
2
Tubulin polymerisation was induced with GTP, followed by
addition of ligands at 10 μM. Fluorescence intensities were
measured at 1-minute intervals across a period of 60 minutes,
with the temperature maintained at 37°C throughout the
TTL) (Figure S2). In addition, the observed binding mode is in
accord with the one recently published for the closely related 7-
[25]
aminonoscapine. Superimposition of the tubulin structure in
[38]
the absence of any ligand with the T R-TTL-14e structure
2
experiment. The control (V =24.5ꢀ1.2 mOD/min) represents
suggests that binding of compound 14e has no impact on the
max
a classic polymerisation curve in the absence of tubulin ligands
overall T R-TTL structure (PDB ID: 4I55, overall rmsd
of
T2R-TTL-17e
2
(Figure 3). Paclitaxel produced a conventional MT-stabilising
0.30 Å over 2072 C -atoms; chain B: rmsd
of 0.18 Å over
α
T2R-TTL-17e
polymerisation curve, whereby a three-fold increase in Vmax was
observed (Vmax =70.7ꢀ6.2 mOD/min). In contrast, noscapine (1)
instigated a reduction of Vmax to a polymerisation rate of 18.1ꢀ
378 C -atoms).
α
The main interactions between tubulin and compound 14e
are via hydrogen bonds and electrostatic interactions. The
tetrahydro[1,3]dioxolo[4,5-g]isoquinoline ring (Figure 4B) of 14e
is stacked between helices βH7 and βH8, specifically between
the residues βCys342 and βLeu255 (Figure 4B,C). Furthermore,
the residues βTyr202, βVal238, βLeu242, βLeu255, βIle318,
βIle378 form a pocket for coordination of the 1,3-dioxolo
moiety containing the deuterated methylene group. The oxy-
gen of the 4-methoxy group forms a hydrogen bond to the
main chain amide of βAla250, whereas the methyl group
engages in a hydrophobic contact to βLeu242.
1
.0 mOD/min. The novel noscapine analogues, 14e and 20,
inhibited tubulin polymerisation to Vmax rates of 12.0ꢀ0.8
mOD/min and 1.9ꢀ0.03 mOD/min, respectively. These results
confirmed that 14e and 20 both bind to tubulin, and like
The 4,5-dimethoxy-1,3-dihydroisobenzofuran ring is posi-
tioned at the αβ-tubulin heterodimer interface and rests
between the helix βH8 and the αT5 loop. The residue βMet259
(βH8 helix), the amino-terminal end of the strand βS9 and the
side chain of αVal181 (αT5-loop) together form a cavity in
which the 6-methoxy group is coordinated. The oxygen of the
7
-methoxy group is involved in a weak polar interaction (4.3 Å
distance) with the main chain amide of αVal181. Lastly, the N-
ethylcarbamoyl group at the N6’-position is oriented towards
the nucleotide with the carbonyl facing the βS9 strand. The
Figure 3. Polymerisation of porcine brain derived tubulin dimers in the
absence (!) and presence of ligands (paclitaxel,
; 20, &).
◆
; noscapine (1), *; 14e,
~
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Figure 4. Tubulin-14e complex structure (PDB ID: 7AU5) and superposition with the tubulin-7-aminonoscapine structure; (A) Overview of compound 14e
bound to the colchicine site in αβ-tubulin (chains A and B of T R-TTL). Ribbon representations of the α- and β-tubulin chains are coloured in dark and light
2
grey, respectively. Compound 14e (light blue) and the GTP molecule (light orange) are displayed in stick and surface representation. All carbon atoms are
coloured according to their chain assignment; oxygen atoms and nitrogen atoms are coloured red and blue, respectively. (B), (C) Two close-up views of the
compound 14e (cyan) binding mode. Interacting tubulin residues are labelled and shown in stick representation; α-tubulin residues are specifically labelled as
such. Secondary structural elements are labelled in blue. Water molecules are shown as red spheres and hydrogen bonds are indicated by black-dashed lines.
The GTP molecule (orange) is shown in stick representation and the disordered segment of the β-tubulin T7 loop is displayed as a dashed line. (D)
2 2
Superposition of the T R-TTL-7-aminonoscapine structure (PDB ID: 6Y6D, yellow) and the T R-TTL-14e structure (tubulin in grey, 14e in cyan). Both ligands as
well as interacting tubulin residues are displayed in stick representation. The shown tubulin residues are labelled and α-tubulin residues are specifically
labelled as such. Oxygen and nitrogen atoms are coloured red and blue, respectively. Secondary structural elements are labelled in blue; hydrogen-bonds are
displayed as black dashed lines. A dashed ellipse is centred around the α-tubulin αT5 loop to highlight the two observed conformations.
contained ethyl group displaces residue βLeu249, thereby
causing disorder of this segment of the βT7 loop.
tetrahydro[1,3]dioxolo[4,5-g]isoquinoline moiety of both mole-
cules is identical, except for the deuterated bridging methylene
moiety in 14e, coordination of this ring system occurs in the
same manner in both complexes.
Comparison between 14e and 7-aminonoscapine (5). To date,
only one high-resolution crystal structure for noscapine deriva-
[25]
tives has been published, introducing the hypothesis that the
parental noscapine is a pro-drug. Here, we show that com-
pound 14e is able to bind to tubulin, despite containing the
critical 7-methoxy moiety. Hence, we superimposed our crystal
Interestingly, in the T R-TTL-7-aminonoscapine structure the
2
αT5 loop is flipped at αThr179 compared to the apo-T R-TTL
2
structure. This reorganisation takes place to enable water
mediated hydrogen bonds with the carbonyl group of 7-
aminonoscapine. As this carbonyl group is reduced to a
methylene in compound 14e, the flip of the αT5 loop does not
structure to the T R-TTL-7-aminonoscapine one (Figure 4D) to
2
investigate the differences in the compound binding modes.
Superimposition of the T R-TTL-7-aminonoscapine structure
occur in the T R-TTL-14e structure. Furthermore, this allows the
2
2
with the T R-TTL-14e structure shows only very small deviations
at an overall structural level (PDB ID: 6Y6D, overall:
whole dihydroisobenzofuran moiety of 14e to rest closer to the
dioxolo-tetrahydroisoquinoline group at the centre of the
colchicine site compared to its position in the tubulin-7-
aminonoscapine complex. This repositioning prevents clashes
2
rmsdT2R-TTL-7-aminonoscapine 0.23 Å over 1989 Cα atoms chain B:
,
rmsdT2R-TTL-7-aminonoscapine 0.20 over 370 Cα atoms). Since the
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of the 7-methoxy group with the αT5 loop that hinder binding
of the parental compound noscapine and thus enables binding
of 14e despite the presence of the same 7-methoxy group.
The N-ethylcarbamoyl group is oriented as the N6’-methyl
group in the 7-aminonoscapine structure towards the nucleo-
tide and the βT7 loop. In the 7-aminonoscapine structure, a flip
in the βT7 loop, compared to the apo position is sufficient to
accommodate the N6’-methyl group. However, this flip is not
sufficient to accommodate the elongated N-ethylcarbamoyl
moiety in 14e. As a consequence, it displaces both the residues
βAsn249 and βLeu249, and causes them to remain unresolved
in the structure.
that these noscapine derivatives are not susceptible to the
common MDR phenotype.
Furthermore, structural studies yielded a crystal structure of
14e bound to tubulin, which revealed that it binds to the
colchicine site of tubulin. This structural study pinpointed the
key interactions between 14e and tubulin as well as its binding
mode relative to the only other reported high-resolution crystal
structure of a noscapine derivative (7-aminonoscapine) bound
to tubulin. The structural studies of noscapine derivatives in
complex with tubulin may eventually enable the rational design
of this class of anti-tubulin compounds with the aim to produce
further variants with superior pharmacological profiles.
Both 14e and 7-aminonoscapine adopt a similar pose
within the colchicine site, suggesting a common mechanism to
perturb MTs. The higher impact of 14e on the overall Experimental Section
organization of the binding site could explain the higher
potency of the ligand compared to noscapine. The reduction of
the 1-carbonyl group in compound 14e enables the αT5 loop
to coordinate the compound closer to the centre of the binding
site and thereby enables accommodation of the 7-methoxy
Chemistry
1
19
13
NMR spectra ( H, F and C) were recorded on a Bruker Avance
Nanobay III 400 MHz Ultrashield Plus spectrometer at 400.13,
376.85 and 100.62 MHz, respectively coupled to a BACS 60
automatic sample changer at 25°C. Chemical shifts (δ) are recorded
[
25]
group, which is predicted to hinder noscapine binding.
in parts per million (ppm) by correction with reference to the
chemical shift of the solvent. 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). LCÀ MS were
run on an Agilent 6120 series Single Quad coupled to an Agilent
1260 series HPLC. The following buffers were used: buffer A, 0.1%
Conclusion
A series of 1,3-benzodioxole modified noscapine analogues
were synthesised to allow for the novel SAR exploration at this
region of the noscapine scaffold. The tubulin inhibitory profile
and anti-cancer activities of these compounds were evaluated
in comparison to noscapine (1), with the natural 1,3-benzodiox-
ole group in place. The lack of anti-proliferative activity against
MCF-7 and PANC-1 cells in lactone analogues 13a–h were
recovered following reduction to its respective cyclic ether N-
ethylcarbamoyl noscapine derivatives 14a–h. The isosteric
replacement of the hydrogens of the methylenedioxy bridge
with deuterium (14e) resulted in a 25-fold increase in potency
formic acid in H O; buffer B, 0.1% formic acid in MeCN. The
following gradient was used with a Phenomenex Luna 3 μM C8(2)
2
1
5 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–
min 0% buffer A and 100% buffer B, 7–12 min 95% buffer A and
7
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 214 nm and 254 nm. All screening compounds were
of >95% purity.
Analytical HPLC was performed using an Agilent 1260 Infinity
Analytical HPLC with a Zorbax Eclipse Plus C18 Rapid Resolution
(
EC : 1.5ꢀ0.1 μM) against MCF-7 cells, relative to that of
50
4
.6×100 mm, 3.5 μM column. Buffer A: 0.1% TFA in H O and buffer
2
noscapine (1). Compound 14e was also found to inhibit tubulin
polymerisation to a rate of 12.0ꢀ0.8 mOD/min. This preference
for the deuterium over hydrogen indicates that substitution at
the 2’-methylene position is tolerated within this region of the
tubulin-binding site. Our research efforts to form the second
generation of multi-functionalised noscapine derivatives af-
B: 0.1% TFA in MeCN were used. Samples were run at a gradient of
0% buffer B/ buffer A (0–5 min) to 100% buffer B (5–6 min) at a
3
flow rate of 1 mL/min. Unless otherwise indicated, all compounds
were >95% by HPLC prior to biological evaluation. 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 μm, 70–230 mesh).
forded 20 as a potent tubulin polymerisation inhibitor (Vmax
:
1
.9ꢀ0.04 mOD/min). Compound 20 instigated a 13-fold
General Procedure A: 1,3-Dioxolane Ring Cleavage. To a stirring
solution of 11 or 15 (1.0 eq) in DCM (50 mL), was added BCl (1 M
reduction in tubulin polymerisation rate relative to the DMSO
control. Furthermore, 20 exhibited pronounced cytotoxic
activity of 0.73ꢀ0.1 μM and 1.1ꢀ0.1 μM against MCF-7 and
PANC-1 cells, respectively. Screening against a panel of 60-
cancer cell lines conducted by the NCI revealed that 20 was
also active against MDA-MB-435, NCIÀ H522, SF-295 and RXF
cells, with activities of >2 μM. Compound 20 was particularly
active against melanoma cells, with GI50 of 1.16 μM and LC50 of
3
in DCM, 2.0 eq) and the mixture stirred at 25°C for 16 h. The
reaction was quenched with MeOH (50 mL) and the solvent was
removed in vacuo. The resultant solid was then stirred in 1 M HCl
(
20 mL) at 90 C for 30 min. The mixture was basified to pH 8 with
°
sat. NaHCO and precipitation was observed. The suspension was
filtered to afford the desired catechol (12 or 16).
3
General Procedure B: Catechol Alkylation. To a solution of 12 or 16
(1.0 eq) in DMF (2 mL) was added K CO or NaOH, and the mixture
2
3
was left to stir at 25
°
C for 5 min. The appropriate alkylating agent
7
9.8 μM against MDA-MB-435 and SK-MEL-5, respectively. More
was added and the reaction was stirred at the described conditions.
The resultant mixture was then pipetted over ice and precipitation
was observed. The suspension was filtered to yield the crude
importantly, both compounds 14e and 20 maintain their
potency in the drug-resistant NCI
ADR/RES
cell line, an indication
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product. The crude residue was purified by flash chromatography
1:1; EtOAc:pet. spirits) to afford the title products.
line-2(1H)-carboxamide (13b). To a stirring solution of 12 (254 mg,
(
0.555 mmol), K2CO3 (307 mg, 2.22 mmol) and H O (40μL,
2
2
.22 mmol) in DMF (2 mL) was added sodium chlorodifluoroacetate
General Procedure C: Lactone Reduction. To a stirring solution of
(
338 mg, 2.22 mmol). The mixture was heated to 80°C for 16 h,
NaBH (5.0 eq) in THF at À 5°C, was added 13a–h, 18 or 19 (1.0 eq)
4
then cooled to 25°C and diluted with H O (10 mL). The aqueous
2
dissolved in BF ·Et O (39 eq) dropwise. The mixture was warmed to
3
2
phase was extracted with EtOAc (3×15 mL) and the combined
2
5°C and stirred for 16 h. The reaction was quenched with 10% aq.
organic fractions were washed with H O (3×10 mL), dried with
2
HCl and stirred for 1 h. The solution was extracted with CHCl (3 ×).
The combined organic layers were washed with 10% aq. NaOH,
3
MgSO , filtered, and concentrated in vacuo. The crude material
4
purified by flash chromatography (1:1; EtOAc:pet. spirits) give the
dried with MgSO , filtered and the solvent removed in vacuo. The
1
4
title product as a colourless oil (47 mg, 15%). H NMR (CDCl ): δ
3
crude product was then purified by flash chromatography (EtOAc)
to yield the desired cyclic ether (14a–h, 20 and 21).
7
.11 (d, J=8.3 Hz, 1H), 6.84 (d, J=8.2 Hz, 1H), 6.80 (s, 1H), 6.52 (dd,
J=73.2, 73.2 Hz, 1H), 6.27 (dd, J=75.3, 73.8 Hz, 1H), 5.94 (d, J=
3
3
.1 Hz, 1H), 5.81 (d, J=3.1 Hz, 1H), 4.86 (t, J=5.3 Hz, 1H), 3.97 (s,
H), 3.86 (s, 3H), 3.77 (s, 3H), 3.59–3.50 (m, 1H), 3.38–3.25 (m, 2H),
.10–2.99 (m, 1H), 2.90–2.81 (m, 1H), 2.80–2.70 (m, 1H), 1.19 (t, J=
(
R)-5-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
ethyl-4-methoxy-7,8-dihydro[1,3]dioxolo[4,5-g]isoquinoline-
6
0
1
(5H)-carboxamide (11). To a cooled solution (-5°C) of 10 (353 mg,
3
1
9
7
.2 Hz, 3H); F NMR (CDCl ): δ À 81.05 (d, J=164.0 Hz), À 81.29 (d,
.886 mmol) in CH CN (15 mL) was added EtNCO (105μL,
3
3
1
3
.33 mmol). The reaction was warmed to 25°C and stirred for 2 h.
J=162.6 Hz), À 81.68 (d, J=164.0 Hz), À 82.35 (d, J=162.6 Hz);
C
NMR (CDCl ): δ 167.2, 158.5, 152.9, 150.7, 148.2, 144.0 (app t, J=
The reaction was then concentrated in vacuo and H O (15 mL) was
3
2
2
1
8
.7 Hz), 139.6, 136.5, 133.4 (app t, J=3.3 Hz), 122.6, 119.1, 118.5,
18.3, 116.7 (app t, J=263.0 Hz), 115.7 (app t, J=263.0 Hz), 115.6,
1.9, 62.4, 61.3, 57.0, 53.5, 39.5, 36.1, 28.2, 15.7. HR-ESMS calcd. for
added to the resulting brown solid. The mixture was extracted with
DCM (3×15 mL), washed with sat. NaCl (20 mL), with the organic
layer dried over anhydrous MgSO , filtered and concentrated in
4
+
C H F N O [M+H] 559.1698, found 559.1709.
vacuo to yield the crude product as a yellow foam. The crude
25 27
4
2
8
(
R)-1-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
1
ethyl-8-methoxy-6,7-bis(methoxy-d )-3,4-dihydroisoquinoline-
3
2
(1H)-carboxamide (13c). Compound 13c was synthesised from 12
(
(
496 mg, 1.42 mmol) according to General Procedure B, with K CO3
747 mg, 5.40 mmol) and iodomethane-d (168μL, 2.70 mmol). The
2
3
1
product was obtained as a white foam (188 mg, 35%). H NMR
(CDCl ): δ 6.99 (d, J=8.3 Hz, 1H), 6.53 (d, J=8.2 Hz, 1H), 6.36 (s, 1H),
3
5
3
2
.94 (d, J=3.1 Hz, 1H), 5.58 (d, J=3.3 Hz, 1H), 5.05 (t, J=5.4 Hz, 1H),
.92 (s, 3H), 3.77 (s, 3H), 3.76 (s, 3H), 3.63–3.54 (m, 1H), 3.31–3.11 (m,
13
H), 2.74–2.53 (m, 2H), 2.42–2.30 (m, 1H), 1.11 (t, J=7.2 Hz, 3H);
C
NMR (CDCl ): δ 167.1, 158.8, 153.0, 152.5, 149.9, 147.9, 139.9, 139.7,
(
R)-1-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
3
1
3
4
32.3, 118.7, 118.3, 118.3, 115.8, 106.9, 81.4, 62.2, 60.6, 56.7, 53.6,
ethyl-6,7-dihydroxy-8-methoxy-3,4-dihydroisoquinoline-2(1H)-
carboxamide (12). Compound 12 was synthesised from 11
+
8.9, 35.8, 27.9, 15.5. HR-ESMS calcd. for C H D N O [M+H]
25 25
6
2
8
93.2452, found 493.2432.
(
(
251 mg, 0.534 mmol) according to General Procedure A, with BCl3
1 M in DCM, 1.07 mL, 1.07 mmol). The product was obtained as a
(
R)-1-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
1
white foam (232 mg, 95%). H NMR (DMSO-d ): δ 9.27 (s, 1H), 8.40
6
ethyl-6,7-diisopropoxy-8-methoxy-3,4-dihydroisoquinoline-2(1H)-
carboxamide (13d). Compound 13d was synthesised from 12
(
(
s, 1H), 7.32 (d, J=8.4 Hz, 1H), 6.57–6.44 (m, 2H), 6.38 (s, 1H), 5.76
d, J=5.1 Hz, 1H), 5.73 (d, J=5.0 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H),
(
207 mg, 0.452 mmol) according to General Procedure B, with
3
.68 (s, 3H), 3.47–3.37 (m, 2H), 3.05 (s, 2H), 2.53–2.49 (m, 2H), 1.01 (t,
K CO (313 mg, 2.26 mmol) and 2-iodopropane (136μL, 1.36 mmol).
13
2
3
J=7.1 Hz, 3H); C NMR (CDCl ): δ 167.4, 157.9, 152.6, 147.3, 146.6,
1
3
The product was obtained as a white solid (154 mg, 63%). H NMR
DMSO-d ): δ 7.36 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.3 Hz, 1H), 6.62 (s,
1
45.7, 141.0, 136.5, 126.6, 119.9, 118.7, 118.6, 116.2, 110.9, 80.8,
2.0, 60.2+, 57.0, 51.3, 35.5, 27.2, 16.1. HR-ESMS calcd. for
(
6
6
1
1
3
H), 6.54 (t, J=5.4 Hz, 1H), 5.78 (d, J=4.5 Hz, 1H), 5.72 (d, J=4.4 Hz,
H), 4.62–4.50 (m, 1H), 4.17–4.04 (m, 1H), 3.81 (s, 3H), 3.80 (s, 3H),
.65 (s, 3H), 3.45–3.35 (m, 1H), 3.14–3.03 (m, 2H), 2.97–2.87 (m, 1H),
.86–2.76 (m, 1H), 2.70–2.60 (m, 1H), 1.25 (d, J=6.0 Hz, 6H), 1.13 (d,
C H N O [M+H] 459.1762, found 459.1769.
2
3
27
2
8
(
R)-1-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
2
ethyl-6,7,8-trimethoxy-3,4-dihydroisoquinoline-2(1H)-carboxa-
mide (13a). Compound 13a was synthesised from 12 (1.30 g,
1
3
J=6.1 Hz, 3H), 1.08 (d, J=6.2 Hz, 3H), 1.03 (t, J=7.1 Hz, 3H);
C
NMR (DMSO-d ): δ 166.9, 157.4, 152.2, 151.1, 150.8, 146.8, 140.4,
2
8
.84 mmol) according to General Procedure B, with NaOH (340 mg,
.52 mmol) and iodomethane (583μL, 9.36 mmol). The aqueous
6
1
6
38.1, 131.6, 119.5, 118.5, 118.0, 117.2, 109.9, 81.2, 74.4, 70.0, 61.5,
0.2, 56.6, 51.3, 35.1 (×2), 27.4, 22.3, 22.1, 22.0, 21.9, 15.7. HR-ESMS
layer was extracted with EtOAc (3 × 25 mL) and solvent removed in
vacuo to yield the crude product. The product was purified with
flash column chromatography (1:1; EtOAc;1 pet. spirits) and
+
calcd. for C H N O [M+H] 543.2701, found 543.2711.
29 39
2
8
(
R)-5-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
obtained as a pale yellow foam (1.12 g, 81%). H NMR (CD OD): δ
3
ethyl-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline-2,2-
d2-6(5H)-carboxamide (13e). Compound 13e was synthesised from
7
.29 (d, J=8.3 Hz, 1H), 6.78 (d, J=8.1 Hz, 1H), 6.61 (s, 1H), 5.88 (q,
J=3.9 Hz, 2H), 3.90 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 3.68
1
2 (240 mg, 0.522 mmol) according to General Procedure B, with
(217 mg, 1.57 mmol) and diiodomethane-d2 (64μL,
.784 mmol). The product was obtained as a pale yellow oil
(s, 3H), 3.50–3.40 (m, 1H), 3.29–3.20 (m, 2H), 2.87 (dt, J=19.3,
K2CO
3
1
3
9
.2 Hz, 2H), 2.77–2.64 (m, 1H), 1.16 (t, J=7.2 Hz, 3H); C NMR
0
(CD OD): δ 160.4, 154.7, 154.2, 151.5, 148.9, 141.6, 141.2, 141.2,
1
3
(103 mg, 42%). H NMR (CDCl ): δ 7.06 (d, J=8.3 Hz, 1H), 6.70 (d,
3
1
5
+
33.8, 120.7, 120.0, 119.5, 117.6, 108.3, 83.3, 62.4, 61.2, 57.3, 56.4,
J=8.2 Hz, 1H), 6.33 (s, 1H), 5.94 (dd, J=3.2, 0.6 Hz, 1H), 5.68 (d, J=
+
3.6, 40.8, 36.9, 36.7, 29.0, 15.9. HR-ESMS calcd. for C H N O [M
25
31
2
8
3
3
.1 Hz, 1H), 4.94 (br s, 1H), 4.01 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H),
.62–3.51 (m, 1H), 3.39–3.18 (m, 2H), 2.82–2.58 (m, 2H), 2.55–2.44
H] 487.2075, found 487.2087.
1
3
(m, 1H), 1.18 (t, J=7.2 Hz, 3H); C NMR (CDCl ): δ 167.3, 158.9,
(
R)-6,7-Bis(difluoromethoxy)-1-((S)-4,5-dimethoxy-3-oxo-1,3-dihy-
3
152.6, 149.0, 148.1, 140.1, 139.8, 134.0, 131.4, 118.8, 118.6, 118.5,
droisobenzofuran-1-yl)-N-ethyl-8-methoxy-3,4-dihydroisoquino-
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1
15.1, 102.8, 81.7, 62.5, 59.2, 56.9, 53.7, 39.2, 36.0, 28.2, 15.7. HR-
61.2, 60.5, 56.7, 56.4, 36.7, 36.6, 29.1, 16.0. HR-ESMS calcd. for
+
+
ESMS calcd. for C H D N O [M+H] 473.1887, found 473.1880.
C H N O [M + H] 473.2282, found 473.2294.
24
25
2
2
8
25 33
2
7
(
R)-6-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
(R)-6,7-Bis(difluoromethoxy)-1-((S)-4,5-dimethoxy-1,3-dihydroiso-
benzofuran-1-yl)-N-ethyl-8-methoxy-3,4-dihydroisoquinoline-
2(1H)-carboxamide (14b). Compound 14b was synthesised from
13b (130 mg, 0.233 mmol) according to General Procedure C, with
NaBH (44 mg, 1.17 mmol) and BF ·Et O (1.12 mL, 9.10 mmol). The
ethyl-5-methoxy-2,3,8,9-tetrahydro-[1,4]dioxino[2,3-g]
isoquinoline-7(6H)-carboxamide (13f). Compound 13f was syn-
thesised from 12 (209 mg, 0.456 mmol) according to General
Procedure B, with K CO3 (190 mg, 1.38 mmol) and 1,2-dibromo-
ethane (59μL, 0.684 mmol). No precipitation was observed follow-
2
4
3
2
1
product was obtained as a colourless oil (42 mg, 33%). H NMR
ing addition to ice. The aqueous layer was extracted with EtOAc
(CDCl ): δ 6.78 (s, 1H), 6.63 (d, J=8.2 Hz, 1H), 6.75–6.37 (m, 1H), 6.49
3
(3 × 10 mL) and solvent removed in vacuo to yield the crude
(dd, J=75.4, 73.8 Hz, 1H), 5.96 (d, J=8.0 Hz, 1H), 5.87–5.80 (m, 2H),
5.36 (d, J=3.5 Hz, 1H), 5.11 (s, 2H), 3.97 (s, 3H), 3.92–3.84 (m, 1H),
3.81 (s, 3H), 3.80 (s, 3H), 3.40–3.12 (m, 2H), 2.72–2.61 (m, 1H), 2.50–
product. The product was purified with flash column chromatog-
raphy (EtOAc) and obtained as a pale yellow foam (131 mg, 60%).
1
19
H NMR (DMSO-d ): δ 7.37 (d, J=8.4 Hz, 1H), 6.72 (d, J=8.3 Hz, 1H),
2.35 (m, 1H), 2.28–2.15 (m, 1H), 1.18 (t, J=7.2 Hz, 3H); F NMR
6
6
4
1
2
1
1
2
4
.56–6.48 (m, 2H), 5.77 (d, J=5.4 Hz, 1H), 5.66 (d, J=5.2 Hz, 1H),
.31–4.17 (m, 4H), 3.85 (s, 3H), 3.82 (s, 3H), 3.67 (s, 3H), 3.40–3.34 (m,
H), 3.09–3.00 (m, 2H), 2.91–2.81 (m, 1H), 2.76–2.67 (m, 1H), 2.66–
(CDCl ): δ À 80.98 (d, J=163.9 Hz), À 81.10 (d, J=163.1 Hz), À 81.62
3
1
3
(d, J=163.8 Hz), À 82.04 (d, J=163.1 Hz); C NMR (CDCl ): δ 159.7,
3
151.9, 150.6, 143.5 (app t, J=2.8 Hz), 143.4, 136.4, 133.6 (app t, J=
3.2 Hz), 132.1, 131.4, 123.9, 117.1, 116.9 (app t, J=262.6 Hz), 116.1,
13
.56 (m, 1H), 1.00 (t, J=7.1 Hz, 3H); C NMR (DMSO-d ): δ 167.3,
6
57.8, 152.7, 147.3, 145.6, 144.1, 141.0, 135.3, 128.7, 120.1, 118.8,
18.5, 118.3, 111.7, 81.0, 64.5, 64.4, 61.9, 60.7, 57.1, 55.4, 51.2, 35.5,
115.8 (app t, J=262.8 Hz), 112.4, 86.1, 72.0, 61.5, 60.2, 56.5, 56.4,
+
37.8, 35.8, 28.1, 15.8. HR-ESMS calcd. for C H F N O
[M+H]
2
5
29
4
2
7
+
7.1, 16.1. HR-ESMS calcd. for C H N O [M+H] 485.1918, found
545.1905, found 545.1918.
25
29
2
8
85.1927.
(
R)-1-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-N-ethyl-
(
R)-7-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
8-methoxy-6,7-bis(methoxy-d )-3,4-dihydroisoquinoline-2(1H)-
3
ethyl-6-methoxy-3,4,9,10-tetrahydro-2H-[1,4]dioxepino[2,3-g]
isoquinoline-8(7H)-carboxamide (13g). Compound 13g was syn-
thesised from 12 (203 mg, 0.443 mmol) according to General
Procedure B, with K CO (306 mg, 2.22 mmol) and 1,3-dibromopro-
carboxamide (14c). Compound 14c was synthesised from 13c
(167 mg, 0.338 mmol) according to General Procedure C, with
NaBH (64 mg, 1.69 mmol) and BF ·Et O (1.63 mL, 13.2 mmol). The
4
3
2
1
product was obtained as a colourless oil (74 mg, 46%). H NMR
2
3
(CDCl ): δ 6.58 (d, J=8.2 Hz, 1H), 6.38 (s, 1H), 6.00 (s, 1H), 5.93–5.83
3
1
(m, 2H), 5.27 (d, J=3.5 Hz, 1H), 5.20–5.07 (m, 2H), 3.97 (s, 3H), 3.95–
3.87 (m, 1H), 3.81 (s, 3H), 3.78 (s, 3H), 3.40–3.14 (m, 2H), 2.68–2.55
(m, 1H), 2.32–2.18 (m, 1H), 2.12–1.99 (m, 1H), 1.18 (t, J=7.2 Hz, 3H);
1
3
C NMR (CDCl ): δ 160.0, 152.7, 151.7, 150.1, 143.3, 140.0, 132.2,
3
132.2, 131.9, 117.4, 117.3, 112.2, 107.2, 86.2, 71.9, 60.9, 60.2, 56.6,
+
56.3, 37.8, 35.7, 28.2, 15.8. HR-ESMS calcd. for C H D N O [M+H]
25 27
6
2
7
1
18.7, 118.5, 118.3, 118.2, 116.2, 81.0, 71.4, 70.9, 62.5, 61.2, 56.8,
479.2659, found 479.2663.
+
54.0, 38.6, 36.0, 32.0, 27.3, 15.6. HR-ESMS calcd. for C H N O [M
26
31
2
8
(
R)-1-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-N-ethyl-
,7-diisopropoxy-8-methoxy-3,4-dihydroisoquinoline-2(1H)-car-
+
H] 499.2075, found 499.2082.
6
(
R)-8-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-N-
boxamide (14d). Compound 14d was synthesised from 13d
(588 mg, 1.08 mmol) according to General Procedure C, with NaBH4
(205 mg, 5.42 mmol) and BF ·Et O (5.22 mL, 42.3 mmol). The
ethyl-7-methoxy-2,3,4,5,10,11-hexahydro-[1,4]dioxocino[2,3-g]
isoquinoline-9(8H)-carboxamide (13h). Compound 13h was syn-
thesised from 12 (201 mg, 0.438 mmol) according to General
Procedure B, with K CO (302 mg, 2.19 mmol) and 1,4-dibromobu-
tane (56μL, 0.482 mmol). The product was obtained as a white
foam (31 mg, 14%). H NMR (CDCl ): δ 7.03 (d, J=8.2 Hz, 1H), 6.54
3
2
1
product was obtained as a yellow oil (43 mg, 8%). H NMR (CDCl ):
3
δ 6.57 (d, J=8.2 Hz, 1H), 6.39 (s, 1H), 6.01 (s, 1H), 5.94–5.81 (m, 2H),
5.28 (d, J=3.6 Hz, 1H), 5.22–5.07 (m, 2H), 4.62–4.48 (m, 1H), 4.43–
4.32 (m, 1H), 3.98 (s, 3H), 3.96–3.90 (m, 1H), 3.81 (s, 3H), 3.79 (s, 3H),
3.39–3.14 (m, 2H), 2.67–2.54 (m, 1H), 2.29–2.16 (m, 1H), 2.12–1.99
(m, 1H), 1.36 (app t, J=6.1 Hz, 6H), 1.30 (dd, J=6.2, 3.0 Hz, 6H), 1.19
2
3
1
3
(s, 1H), 6.41 (d, J=8.2 Hz, 1H), 6.02 (d, J=3.4 Hz, 1H), 5.62 (d, J=
3
3
2
.4 Hz, 1H), 4.96 (s, 1H), 4.44–4.34 (m, 2H), 4.31–4.10 (m, 2H), 4.02 (s,
1
3
H), 3.85 (s, 6H), 3.72–3.61 (m, 1H), 3.54–3.41 (m, 1H), 3.37–3.21 (m,
(t, J=7.2 Hz, 3H); C NMR (CDCl ): δ 160.2, 151.7, 151.5, 151.2,
3
1
3
H), 2.68–2.61 (m, 2H), 2.12–1.79 (m, 4H), 1.20 (d, J=7.2 Hz, 3H);
C
143.4, 139.1, 132.2, 132.0, 131.8, 117.4, 117.2, 112.2, 110.4, 86.2,
75.5, 71.9, 70.8, 60.9, 60.2, 56.7, 56.4, 37.9, 35.8, 28.2, 22.6, 22.6, 22.3,
NMR (CDCl ): δ 167.1, 159.0, 152.6, 151.5, 149.8, 148.2, 140.5, 140.0,
3
+
1
31.9, 118.8, 118.5, 118.4, 118.1, 116.4, 81.4, 73.1, 72.9, 62.5, 61.1,
22.1, 15.9. HR-ESMS calcd. for C H N O [M+H] 528.2830, found
2
9
41
2
7
5
6.9, 54.0, 39.0, 36.0, 27.9, 27.5, 26.7, 15.6. HR-ESMS calcd. for
528.2772.
+
C H N O [M+H] 513.2231, found 513.2240.
2
7
33
2
8
(
R)-5-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-N-ethyl-
(
6
(
0
R)-1-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-N-ethyl-
,7,8-trimethoxy-3,4-dihydroisoquinoline-2(1H)-carboxamide
14a). Compound 14a was synthesised from 13a (130 mg,
4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinoline-2,2-d2-
6(5H)-carboxamide (14e). Compound 14e was synthesised from
13e (94 mg, 0.199 mmol) according to General Procedure C, with
NaBH (38 mg, 0.994 mmol) and BF ·Et O (957μL, 7.75 mmol). The
.267 mmol) according to General Procedure C, with NaBH (30 mg,
4
4
3
2
1
0
.801 mmol) and BF ·Et O (428 μL, 3.47 mmol). The product was
product was obtained as a colourless oil (14 mg, 16%). H NMR
3
2
1
obtained as an off-white solid (95 mg, 75%). H NMR (CD OD): δ
(CDCl ): δ 6.62 (d, J=8.2 Hz, 1H), 6.32 (s, 1H), 6.02 (d, J=8.1 Hz, 1H),
3
3
6
.78 (d, J=8.2 Hz, 1H), 6.58 (s, 1H), 6.19 (s, 1H), 5.72 (s, 1H), 5.50 (d,
J=5.6 Hz, 1H), 5.02 (d, J=12.3 Hz, 1H), 4.92 (d, J=5.6 Hz, 1H), 3.85
s, 6H), 3.79 (s, 3H), 3.77 (s, 3H), 3.76 (s, 3H), 3.74–3.70 (m, 1H) 3.64–
5.97 (s, 1H), 5.80 (t, J=3.0 Hz, 1H), 5.32 (d, J=3.5 Hz, 1H), 5.20–5.07
(m, 2H), 4.04 (s, 3H), 3.87–3.83 (m, 1H), 3.82 (s, 3H), 3.81 (s, 3H),
3.41–3.15 (m, 2H), 2.63–2.51 (m, 1H), 2.31–2.11 (m, 2H), 1.19 (t, J=
(
1
3
3
3
1
.56 (m, 1H), 3.29–3.15 (m, 2H), 2.67–2.57 (m, 2H), 1.16 (t, J=7.2 Hz,
7.2 Hz, 3H); C NMR (CDCl ): δ 160.0, 151.7, 148.6, 143.3, 139.9,
3
13
H); C NMR (CD OD): δ 154.3, 153.2, 151.5, 149.2, 142.0, 141.3,
134.3, 132.2, 131.9, 131.1, 117.7, 116.6, 112.2, 103.0, 86.2, 71.9, 60.3,
59.6, 56.6, +56.4, 38.1, 35.8, 28.3, 15.8. HR-ESMS calcd. for
C H D N O [M+H] 459.2095, found 459.2102.
3
33.6, 133.4, 120.1, 118.9, 118.9, 113.7, 108.4, 87.6, 72.4, 72.3, 61.3,
2
4
27
2
2
7
ChemMedChem 2021, 16, 1–14
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ChemMedChem
(
R)-6-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-N-ethyl-
(S)-3-((R)-6,7-Dihydroxy-8-methoxy-2-((1-methyl-1H-imidazol-4-yl)
sulfonyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)-6,7-dimeth-
oxyisobenzofuran-1(3H)-one (16). Compound 16 was synthesised
from 15 (771 mg, 1.42 mmol) according to General Procedure A,
5
7
1
-methoxy-2,3,8,9-tetrahydro[1,4]dioxino[2,3-g]isoquinoline-
(6H)-carboxamide (14f). Compound 14f was synthesised from
3f (220 mg, 0.453 mmol) according to General Procedure C, with
NaBH (223 mg, 5.89 mmol) and BF ·Et O (2.18 mL, 17.7 mmol). The
product was obtained as a colourless oil (50 mg, 25%). H NMR
with BCl3 (1 M in DCM, 2.84 mL, 2.84 mmol). The product was
4
3
2
1
1
obtained as a pale brown foam (614 mg, 82%). H NMR (CDCl ): δ
3
(
CDCl ): δ 6.59 (d, J=8.2 Hz, 1H), 6.42 (s, 1H), 6.10 (s, 1H), 5.91 (d,
7.41 (d, J=1.0 Hz, 1H), 7.38–7.34 (m, 1H), 7.02 (d, J=8.4 Hz, 1H),
6.48 (d, J=8.3 Hz, 1H), 6.34 (s, 1H), 5.80 (d, J=4.3 Hz, 1H), 5.68 (d,
J=4.5 Hz, 1H), 3.95 (s, 3H), 3.81 (s, 3H), 3.76 (s, 3H), 3.68 (s, 3H), 3.55
3
J=8.2 Hz, 1H), 5.87 (t, J=3.2 Hz, 1H), 5.29 (d, J=3.7 Hz, 1H), 5.23–
.08 (m, 2H), 4.40–4.27 (m, 4H), 3.94 (s, 3H), 3.90–3.84 (m, 1H), 3.83
s, 3H), 3.80 (s, 3H), 3.43–3.15 (m, 2H), 2.64–2.47 (m, 1H), 2.26–2.06
5
1
3
(
(
(dd, J=6.7, 3.9 Hz, 1H), 2.63–2.50 (m, 2H), 2.48–2.36 (m, 1H);
C
13
m, 2H), 1.19 (t, J=7.2 Hz, 3H); C NMR (CDCl ): δ 160.3, 151.8,
NMR (CDCl ): δ 167.9, 152.7, 147.9, 145.8, 144.6, 139.7, 139.4, 139.2,
3
3
1
1
1
4
45.2, 143.9, 143.3, 135.2, 132.2, 131.7, 129.2, 117.7, 117.5, 112.2,
135.7, 126.7, 124.5, 123.8, 118.9, 118.8, 118.6, 114.7, 110.9, 80.6,
+
12.1, 86.4, 71.9, 64.5, 64.5, 60.9, 60.3, 56.7, 56.4, 38.3, 35.9, 27.6,
62.4, 60.5, 56.8, 42.7, 40.9, 34.2. HR-ESMS calcd. for C H N O S [M
2
4
26
3
9
+
5.9. HR-ESMS calcd. for C H N O
[M+H] 471.2126, found
+H] 532.1384, found 532.1394.
25
31
2
7
71.2124.
(
S)-6,7-Dimethoxy-3-((R)-5-methoxy-7-((1-methyl-1H-imidazol-4-
(
6
R)-7-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-N-ethyl-
-methoxy-3,4,9,10-tetrahydro-2H-[1,4]dioxepino[2,3-g]
yl)sulfonyl)-2,3,6,7,8,9-hexahydro[1,4]dioxino[2,3-g]isoquinolin-6-
yl)isobenzofuran-1(3H)-one (17). Compound 17 was synthesised
from 16 (220 mg, 0.413 mmol) according to General Procedure B,
with K CO (171 mg, 1.24 mmol) and 1,2-dibromoethane (54μL,
isoquinoline-8(7H)-carboxamide (14g). Compound 14g was syn-
thesised from 13g (495 mg, 0.992 mmol) according to General
Procedure C, with NaBH4 (188 mg, 4.96 mmol) and BF ·Et O
2
3
0.619 mmol). The product was obtained as a clear oil (45 mg, 20%).
3
2
1
(
(
(
4.77 mL, 38.7 mmol). The product was obtained as a pale yellow oil
58 mg, 12%). H NMR (CDCl ): δ 6.59–6.52 (m, 1H), 6.50 (s, 1H), 6.03
s, 1H), 5.89–5.82 (m, 2H), 5.26 (d, J=3.7 Hz, 1H), 5.20–5.04 (m, 2H),
H NMR (CDCl ): δ 7.47 (s, 1H), 7.36 (s, 1H), 7.01 (d, J=8.3 Hz, 1H),
3
1
6.48 (d, J=8.2 Hz, 1H), 6.41 (s, 1H), 5.88 (d, J=4.2 Hz, 1H), 5.80 (d,
J=4.2 Hz, 1H), 4.26 (s, 4H), 3.98 (s, 3H), 3.84 (s, 6H), 3.75 (s, 3H),
3
1
3
4
3
2
3
1
6
.39–4.25 (m, 2H), 4.16–4.04 (m, 2H), 3.94 (s, 3H), 3.88–3.81 (m, 1H),
.79 (s, 3H), 3.77 (s, 3H), 3.40–3.12 (m, 2H), 2.59–2.46 (m, 1H), 2.31–
.22 (m, 1H), 2.21–2.11 (m, 2H), 2.10–2.01 (m, 1H), 1.16 (t, J=7.2 Hz,
3.71–3.64 (m, 1H), 2.88–2.71 (m, 1H), 2.68–2.49 (m, 2H); C NMR
(CDCl ): δ 167.3, 152.7, 148.1, 145.4, 144.3, 140.3, 139.8, 138.7, 135.3,
3
128.2, 124.0, 118.8, 118.6, 118.5, 116.2, 112.3, 80.8, 64.4, 64.4, 62.5,
13
+
H); C NMR (CDCl ): δ 160.1, 151.9, 151.6, 148.6, 143.2, 143.0,
61.0, 56.8, 53.4, 41.1, 34.1, 27.4. HR-ESMS calcd. for C H N O S [M
3
26 28
3
9
32.1, 131.7, 131.3, 119.6, 117.5, 116.1, 112.1, 86.1, 71.8, 71.4, 70.9,
+H] 558.1541, found 558.1543.
1.2, 60.1, 56.7, 56.2, 37.8, 35.7, 32.1, 27.5, 15.8. HR-ESMS calcd. for
+
(R)-10-Chloro-6-((S)-4,5-dimethoxy-3-oxo-1,3-dihydroisobenzofur-
an-1-yl)-N-ethyl-5-methoxy-2,3,8,9-tetrahydro[1,4]dioxino[2,3-g]
isoquinoline-7(6H)-carboxamide (18). To a cooled (-5°C) solution
C H N O [M+H] 485.2282, found 485.2293.
2
6
33
2
7
(
7
R)-8-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-N-ethyl-
-methoxy-2,3,4,5,10,11-hexahydro[1,4]dioxocino[2,3-g]
of 13f (174 mg, 0.359 mmol) in CHCl (9 mL) was added a solution
3
isoquinoline-9(8H)-carboxamide (14h). Compound 14h was syn-
thesised from 13h (251 mg, 0.489 mmol) according to General
Procedure C, with NaBH (93 mg, 2.44 mmol) and BF ·Et O (2.35 mL,
1
6
of SO Cl (87μL, 1.08 mmol) diluted in CHCl (9 mL) dropwise. The
2
2
3
reaction was warmed to 25°C and stirred for 2 h. The reaction was
then pipetted over ice (~20 g) and neutralised with sat. aq.
NaHCO . The mixture was extracted with CHCl (3×50 mL), with the
4
3
2
9.1 mmol). The product was obtained as a pale yellow oil (14 mg,
3
3
1
%). H NMR (CDCl ): δ 6.59 (d, J=8.2 Hz, 1H), 6.51 (s, 1H), 6.01 (br
organic layer dried over anhydrous MgSO , filtered and concen-
3
4
s, 1H), 5.92–5.83 (m, 2H), 5.30 (d, J=3.7 Hz, 1H), 5.22–5.08 (m, 2H),
trated in vacuo to yield the crude product as a pale yellow foam.
The crude product was then purified by flash column chromatog-
4
3
2
.43–4.22 (m, 4H), 3.98 (s, 3H), 3.92–3.84 (m, 1H), 3.82 (s, 3H), 3.79 (s,
H), 3.43–3.14 (m, 2H), 2.66–2.47 (m, 1H), 2.28–2.16 (m, 1H), 2.15–
raphy (EtOAc) to give the title product as a white foam (140 mg,
1
3
1
.02 (m, 1H), 1.97–1.86 (m, 4H), 1.18 (t, J=7.2 Hz, 3H); C NMR
75%). H NMR (CDCl ) d6.99 (d, J=8.3 Hz, 1H), 6.47 (d, J=8.3 Hz,
3
(
1
CDCl ): δ 160.1, 151.7, 150.9, 149.7, 143.3, 140.8, 132.2, 131.9, 131.8,
1H), 5.92 (d, J=3.5 Hz, 1H), 5.58 (d, J=3.6 Hz, 1H), 5.08 (s, 1H), 4.39–
4.29 (m, 2H), 4.29–4.20 (m, 2H), 3.96 (s, 3H), 3.79 (s, 3H), 3.77 (s, 3H),
3.72–3.63 (m, 1H), 3.31–3.09 (m, 2H), 2.68–2.56 (m, 2H), 2.26–2.12
3
19.5, 117.5, 116.6, 112.2, 86.3, 73.0, 71.9, 61.2, 60.2, 56.8, 56.3, 38.0,
+
3
5.8 (×2), 27.9, 27.7, 27.0, 15.9. HR-ESMS calcd. for C H N O [M+
27
35
2
7
1
3
H] 499.2439, found 499.2444.
(m, 1H), 1.10 (t, J=7.2 Hz, 3H); C NMR (CDCl ) d167.1, 159.0, 152.8,
3
1
1
48.5, 143.9, 140.7, 139.7, 135.9, 127.2, 118.9, 118.6, 118.1, 117.2,
(
S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-((1-methyl-1H-imidazol-4-
16.5, 80.9, 65.0, 64.1, 62.6, 61.0, 56.9, 53.6, 38.2, 36.1, 24.8, 15.7.
yl)sulfonyl)-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinolin-5-yl)
isobenzofuran-1(3H)-one (15). To a solution of 10 (299 mg,
0
+
HR-ESMS calcd. for C H ClN O
[M+H] 519.1529, found
2
5
28
2
8
519.1540.
.749 mmol) dissolved in DCM (2 mL) was added Et N (313μL,
3
2
.25 mmol) and the mixture was left to stir at 25°C for 15 min. 1-
Methyl-1H-imidazole-4-sulfonyl chloride (154 mg, 0.786 mmol) was
then added and the reaction was stirred at 25°C for 16 h. Following
the addition of H O (5 mL) to the reaction, the mixture was
2
extracted with DCM (3×20 mL) and the organic layer was dried
over MgSO , filtered and the solvent was removed in vacuo. The
4
crude product was purified by flash column chromatography
1
(
EtOAc) to yield the title product as a white foam (342 mg, 82%). H
NMR (CDCl ): δ 7.60–7.56 (m, 2H), 7.05 (d, J=8.3 Hz, 1H), 6.42 (dd,
J=8.3, 0.5 Hz, 1H), 6.33 (s, 1H), 5.93 (s, 2H), 5.78 (dd, J=4.5, 0.7 Hz,
CHCl3 (3×50 mL), with the organic layer dried over anhydrous
3
MgSO , filtered and concentrated in vacuo to yield the crude
4
1
H), 5.63 (d, J=4.5 Hz, 1H), 4.02 (s, 3H), 3.94 (s, 3H), 3.86 (s, 3H), 3.76
product as a pale yellow foam. The crude product was then purified
1
3
(
s, 3H), 3.60–3.51 (m, 1H), 2.83–2.70 (m, 1H), 2.61–2.47 (m, 2H);
NMR (CDCl ): δ 167.3, 152.9, 149.5, 148.4, 142.8, 139.8, 139.6, 137.4,
C
by flash column chromatography (EtOAc) to give the title product
1
as a white foam (40 mg, 31%). H NMR (CDCl ): δ 7.45 (d, J=1.2 Hz,
3
3
34.2, 129.6, 127.3, 118.9, 118.6, 118.2, 114.4, 103.2, 101.2, 79.4,
1H), 7.33 (d, J=0.8 Hz, 1H), 7.03 (d, J=8.3 Hz, 1H), 6.54 (d, J=
8.3 Hz, 1H), 5.83 (d, J=4.2 Hz, 1H), 5.78 (d, J=4.2 Hz, 1H), 4.41–4.34
(m, 2H), 4.33–4.24 (m, 2H), 3.99 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H),
+
2
5
26
3
9
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11
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3
.81–3.75 (m, 1H), 3.73 (s, 3H), 2.85–2.72 (m, 1H), 2.68–2.54 (m, 2H);
plotted against log concentration of the inhibitor, and EC50 values
were calculated using GraphPad Prism statistical software (v7).
1
3
C NMR (CDCl ): δ 167.2, 152.8, 148.2, 143.9, 140.7, 140.1, 139.7,
3
1
38.8, 135.9, 126.0, 124.0, 118.8, 118.6, 118.2, 117.1, 116.4, 80.3,
RES
Cell cytotoxicity assays: MCF-7 and NCI/Adr
cells were both
6
4.9, 64.1, 62.5, 61.0, 56.8, 53.2, 40.2, 34.1, 24.8. HR-ESMS calcd. for
+
cultured in high-glucose (25 mM) DMEM supplemented with 10%
FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. Every
C H ClN O S [M+H] 592.1151, found 592.1165.
2
6
27
3
9
RES
(
R)-10-Chloro-6-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-
third passage, the NCI/Adr
cells were treated with 3 μM
yl)-N-ethyl-5-methoxy-2,3,8,9-tetrahydro[1,4]dioxino[2,3-g]
isoquinoline-7(6H)-carboxamide (20). Compound 20 was synthes-
ised from 18 (139 mg, 0.267 mmol) according to General Procedure
C, with NaBH4 (30 mg, 0.801 mmol) and BF ·Et O (428μL,
doxorubicin to maintain selection pressure for the resistance
phenotype. Cells were seeded at a density of 3×10 cells per well
in 96-well plates and recovered in standard culture condition for
48 hours prior to drug treatment. Following the addition of
compounds, cells were incubated for a further 96 h before
measuring the number of live cells using a standard MTT assay.
3
3
2
3
4
1
2
3
2
.47 mmol). The product was obtained as a white foam (64 mg,
1
7%). H NMR (CDCl ) d 6.59 (d, J=8.2 Hz, 1H), 5.93 (d, J=8.2 Hz,
3
H), 5.85 (m, 1H), 5.27 (d, J=3.7 Hz, 1H), 5.15 (m, 2H), 4.42–4.37 (m,
H), 4.37–4.28 (m, 2H), 3.98–3.93 (m, 1H), 3.92 (s, 3H), 3.81 (s, 3H),
.78 (s, 3H), 3.38–3.26 (m, 1H), 3.25–3.12 (m, 1H), 2.59–2.47 (m, 1H),
The percentage of live cells was plotted as a function of drug
concentration and fitted with the general dose response relation-
ship. The extent of cell death and the drug potency EC50 were
estimated from the non-linear regression using GraphPad Prism
1
3
.45–2.35 (m, 1H), 2.10–1.97 (m, 1H), 1.17 (t, J=7.2 Hz, 3H); C NMR
(CDCl ) d159.8, 151.7, 143.7, 143.3, 140.2, 135.8, 132.0, 131.5, 127.1,
3
(v5). The EC50 values of noscapine and the derivatives were
1
3
5
18.3, 117.3, 116.3, 112.2, 86.1, 71.9, 64.9, 64.1, 61.0, 60.2, 56.3, 56.2,
+
compared using One-way ANOVA with the Bonferroni post-hoc
test. A value of p <0.05 was considered statistically significant.
7.3, 35.7, 25.1, 15.8. HR-ESMS calcd. for C H ClN O
7
[M+H]
25
30
2
05.1736, found 505.1745.
Tubulin polymerisation assay: Tubulin polymerisation was recorded
turbidimetrically at 340 nm in PerkinElmer EnVision2101 Multilabel
Reader equipped with temperature controllers. Compounds of
interest (2 mM in sterile-filtered DMSO) were dissolved in General
(
R)-10-Chloro-6-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-
yl)-5-methoxy-7-((1-methyl-1H-imidazol-4-yl)sulfonyl)-2,3,6,7,8,9-
hexahydro[1,4]dioxino[2,3-g]isoquinoline (21). Compound 21 was
synthesised from 19 (120 mg, 0.202 mmol) according to General
Procedure C, with NaBH (38 mg, 1.01 mmol) and BF ·Et O (973μL,
Tubulin Buffer (80 mM PIPES pH 6.9, 2 mM MgCl , 0.5 mM EGTA) to
2
4
3
2
a final concentration of 10 μM and were added to 96-half area well
7
.89 mmol). The product was obtained as a yellow oil (61 mg, 52%).
1
plate and kept at 37°C. Tubulin was dissolved to
a final
H NMR (CDCl ): δ 7.33 (s, 2H), 6.67 (d, J=8.2 Hz, 1H), 6.28 (d, J=
3
concentration of 3 mg/mL in 80 mM PIPES pH 6.9, 0.5 mM MgCl ,
2
8
.2 Hz, 1H), 5.59 (d, J=2.4 Hz, 1H), 5.47 (d, J=4.5 Hz, 1H), 5.11 (dd,
0
.5 mM EGTA, 1 mM GTP, 10.2% glycerol and was kept at 4°C prior
J=12.4, 2.4 Hz, 1H), 5.03 (d, J=12.3 Hz, 1H), 4.40–4.33 (m, 2H),
to addition to plate. Kinetic absorbance reading started immedi-
ately following addition of tubulin to the plate, and was recorded
at 37°C over a period of 60 min. The tubulin polymerisation assay
4
1
1
1
3
5
.33–4.22 (m, 2H), 3.80 (s, 6H), 3.77 (s, 4H), 3.64 (s, 3H), 2.85–2.71 (m,
13
H), 2.69–2.57 (m, 1H), 2.55–2.43 (m, 1H); C NMR (CDCl ): δ 151.6,
3
44.0, 143.1, 140.3, 140.0, 138.8, 135.7, 132.6, 132.3, 125.7, 123.9,
18.7, 117.5, 115.9, 112.1, 85.0, 71.9, 64.9, 64.1, 60.8, 60.0, 56.3, 55.4,
kit (BK006P) used to conduct this study was supplied by
Cytoskeleton Inc.
+
9.9, 34.0, 24.1. HR-ESMS calcd. for C H ClN O S [M+H]
26
29
3
8
78.1358, found 578.1371.
Structural studies
Biology
Crystallisation, data collection and structure determination: T R-TTL
2
[
38,39]
À 2
crystals were grown as previously described by Prota et al.
via
Compound preparation and storage: A 10 M stock solution was
prepared for each compound by dissolving the compound in
sterile-filtered DMSO (Sigma D2650), and the solution was stored at
the vapor diffusion method. Crystals grew over more than 2 days in
reservoir solution containing 4% PEG 4 K, 7% glycerol, 30 mM
MgCl , 30 mM CaCl , 5 mM tyrosine and 100 mM MES/Imidazole
2
2
–
20°C until use.
pH 6.5. Compound 14e was soaked into crystals overnight at 20°C
at a final concentration of 5 mM in reservoir solution. Subsequently,
the crystals were transferred into two cryoprotectant solutions (i)
10% PEG 4 K, 16% glycerol, 30 mM MgCl , 30 mM CaCl , 5 mM
Cell culture and reagents: Human breast cancer cell line MCF-7 (HTB-
2
2) and pancreatic cancer cell line PANC-1 (CRL-1469) were
purchased directly from American Type Culture Collection (ATCC)
2
2
tyrosine, 100 mM MES/Imidazole pH 6.5 and (ii) 10% PEG 4 K, 22%
glycerol, 30 mM MgCl , 30 mM CaCl , 5 mM tyrosine, 100 mM MES/
2
2
Imidazole pH 6.5 and then flash-cooled in liquid nitrogen. X-Ray
diffraction data were collected at 100 K at the beamline X06DA of
the Swiss Light Source (Paul Scherrer Institut, Villigen PSI, Switzer-
[41]
land). The obtained data were processed and merged using XDS
atmosphere containing 5% CO at 37°C. Before use, cell lines were
to 2.2 Å resolution. The T R-TTL-14e structure was determined in
2
2
[42]
tested for the presence of mycoplasma by using a MycoAlert
Mycoplasma detection kit as per manufacturer’s instructions; cells
employed in the assays tested negative for mycoplasma.
Phenix by the difference Fourier method using a T R-TTL complex
2
template model in the absence of ligands and solvent molecules
(PDB ID 5LXT). Difference density delineating the shape of a
compound was observed after three cycles of rigid body and
3
Cell viability assays: MCF-7 and PANC-1 cells were seeded at 5×10
[42]
restrained refinement using Phenix. After correction of geometry,
cells per well in 96-well plates 16 h before treatment. Cells were
then treated with various concentrations of noscapine analogues.
After 72 h of treatment, cell viability was determined following
incubation with resazurin dye (CellTiter-Blue assay–Promega
G8080). The emitted fluorescence was detected using Envision
microplate reader (PerkinElmer) at 560 nm excitation/590 nm
emission wavelengths. The obtained fluorescence values were
Ramachandran and rotamer outliers and the addition of solvent
[43]
molecules, compound 14e was inserted into the density. Refmac
was used to concomitantly refine the β-T7 loop and compound
1
4e in order to estimate the occupancy level of the ligand. The
quality of[44]the T2R-TTL-14e structure was assessed using
MolProbity. The final model was refined to 2.20 Å resolution with
crystallographic R values of 17.8% (Rwork) and 21.3% (Rfree). Small
ChemMedChem 2021, 16, 1–14
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12
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ChemMedChem
root-mean-square deviations (rmsd) from ideal values for bond
lengths and bond angles show the good geometry of the refined
model. PyMOL (the PyMOL Molecular Graphics System, Version
[15] R. Aneja, S. N. Vangapandu, M. Lopus, V. G. Viswesarappa, N. Dhiman, A.
Verma, R. Chandra, D. Panda, H. C. Joshi, Biochem. Pharmacol. 2006, 72,
4
15–426.
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2
been deposited at the Protein Data Bank (www.rcsb.org) under
accession number PDB: 7AU5. Authors will release the atomic
coordinates and experimental data upon article publication.
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their support through an Australian Postgraduate Award to CY.
The authors also thank the National Cancer Institute (NCI) for
screening selected compounds in their NCI-60 Human Tumour Cell
Lines Screen, and the staff of the Swiss Light Source responsible
for beamline X06DA, for their support during X-ray data collection.
This work was supported by the H2020-MSCA-ITN-2019 (860070
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Manuscript received: May 24, 2021
Accepted manuscript online: June 22, 2021
Version of record online: ■■■, ■■■■
ChemMedChem 2021, 16, 1–14
www.chemmedchem.org
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FULL PAPERS
Key interactions: We describe the
design, synthesis and pharmacologi-
cal evaluation of noscapine deriva-
tives with modifications to the 1,3-
benzodioxole moiety. These efforts
afforded the deuterated noscapinoid
Dr. C. Yong, Dr. S. M. Devine, A.-C.
Abel, Dr. S. D. Tomlins, Dr. D. Muthiah,
X. Gao, Prof. R. Callaghan, Prof. M. O.
Steinmetz, Dr. A. E. Prota*, Prof. B.
Capuano*, Prof. P. J. Scammells*
1
– 14
1
4e and the dioxino-containing
analogue 20 as potent cytotoxic
agents with EC50 values of <2 μM
against breast cancer (MCF-7) cells.
These compounds were shown to
inhibit tubulin polymerisation and
were not vulnerable to the overex-
pression of resistance conferring P-
gp efflux pumps in drug-resistant
MCF-7 cells. We also conducted X-ray
crystallography studies that yielded a
high-resolution structure of 14e
bound to tubulin.
1,3-Benzodioxole-Modified
Noscapine Analogues: Synthesis,
Antiproliferative Activity, and
Tubulin-Bound Structure