Synthesis and biological evaluation of novel isoellipticine derivatives and salts

Article abstract of DOI:10.1039/c2ob26181b

Synthesis of novel 7-substituted isoellipticines and isoellipticinium salts is described, with optimisation of routes, representing a new class of anti-cancer agent. Initial assessment of biological activity using a topoisomerase II decatenation assay and NCI screening highlighted strong anti-cancer activity, further developed in a panel of isoellipticinium salts. Interestingly, low correlation between results of the topoisomerase II decatenation assay and NCI screen throughout the panel suggest that topo II is not the most important biological target with respect to anti-cancer activity in this new class of compounds. Results also suggest that solubility is not the limiting factor in activity of the isoellipticinium salts. Overall, 20 novel ellipticine analogues were prepared and full anti-cancer profiling was completed for 13 isoellipticine derivatives and salts. Two compounds display significant specificity towards CNS cancer cell lines and are lead compounds for future development.

Full text of DOI:10.1039/c2ob26181b

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PAPER
Synthesis and biological evaluation of novel isoellipticine derivatives and
salts†
Charlotte M. Miller,a Elaine C. O’Sullivan,a Ken J. Devineb and Florence O. McCarthy*a
Received 20th June 2012, Accepted 24th August 2012
DOI: 10.1039/c2ob26181b
Synthesis of novel 7-substituted isoellipticines and isoellipticinium salts is described, with optimisation of
routes, representing a new class of anti-cancer agent. Initial assessment of biological activity using a
topoisomerase II decatenation assay and NCI screening highlighted strong anti-cancer activity, further
developed in a panel of isoellipticinium salts. Interestingly, low correlation between results of the
topoisomerase II decatenation assay and NCI screen throughout the panel suggest that topo II is not the
most important biological target with respect to anti-cancer activity in this new class of compounds.
Results also suggest that solubility is not the limiting factor in activity of the isoellipticinium salts.
Overall, 20 novel ellipticine analogues were prepared and full anti-cancer profiling was completed for
13 isoellipticine derivatives and salts. Two compounds display significant specificity towards CNS cancer
cell lines and are lead compounds for future development.
Introduction
The tetracyclic natural product ellipticine 1 (5,11-dimethyl-6H-
pyrido[4,3-b]carbazole, Fig. 1) was isolated from the leaf
material of Ochrosia elliptica Labill by Goodwin et al. in 1959.1
This small tropical evergreen tree belonging to the Apocynaceae
family also contained several other alkaloids, including 9-meth-
oxyellipticine 2. Ellipticine has since been isolated from several
other plants of the Apocynaceae family (Ochrosia vieillardii,
Ochrosia acuminate and Ochrosia moorei)2–4 and from Strychnos
dinkagei of the Loganiaceae family.5,6
Fig. 1 Ellipticine derivatives.
Ellipticine and its derivatives were found to exhibit potent
anticancer activity and have been subject to much study over the
last fifty years. In particular, Celiptium (9-hydroxy-N-methyl-
ellipticinium acetate) 3 and 9-hydroxyellipticine 4 were early
lead compounds which progressed to phase II clinical trials.7–9
Several key mechanisms of action have been found to contrib-
ute to ellipticine anticancer activity. These include DNA inter-
calation and topoisomerase II inhibition, two closely related
mechanisms which have been well established in the
literature.10–12 Biooxidation and adduct formation have also been
proposed to play a role in ellipticine cytotoxicity, and have been
subject to much study in the last decade.13–15 More recent
investigations have shown that ellipticines induce multifaceted
biological responses, including interaction with kinases such as
c-Kit and AKT and also p53 tumour suppressor.16–23
In 1994, collation of NCI screening results for a large panel of
ellipticinium salts revealed significant CNS cancer cell line
selectivity.24,25 A range of N-methylellipticinium salts with
varying counterions (I, OAc, Cl, OSO2Me) and C-9 substituents
(H, OMe, Me, Cl) displayed selectivity for the six CNS cancer
cell lines over the other 54 lines in the panel. Interestingly,
neither the parent ellipticine compounds nor the lead elliptici-
nium salt, Celiptium 3, displayed this selectivity. In general, a
decrease in selectivity for CNS cell lines was observed with
increasing size of the C-9 substituent.25
Isoellipticine 5 (Fig. 2) is a non-natural isomer of ellipticine,
first synthesised in 1967, it possesses similar anticancer proper-
ties to ellipticine but has not been extensively investigated.26
Our aim was to further investigate the biological activity of the
isoellipticine family of compounds by synthesis of novel 7-sub-
stituted isoellipticines and isoellipticinium salts. In particular, if
the isoellipticinium salts proved to be as potent and selective as
their ellipticine analogues, we proposed further investigation of
aDepartment of Chemistry, Analytical and Biological Chemistry
Research Facility, University College Cork, Western Road, Cork,
Ireland. E-mail: f.mccarthy@ucc.ie; Tel: +353 21 4901695
bSchool of Pharmacy, University College Cork, Western Road, Cork,
Ireland
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26181b
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the side chain functionally at the N-2 position. The appeal of
this strategy lay in the possibility of increasing both solubility
and activity through salt formation.
v/v solution in methanol resulted in full dissolution of 7-formyl-
isoellipticine and an increased yield of 71%. These reaction con-
ditions were repeated several times and gave consistently high
yields between 60 and 70%.
Fujishiro and Mitamura investigated the Baeyer–Villiger reac-
tion of polycyclic aromatic aldehydes and found that use of
formic acid as both acid and reaction solvent gave excellent
yields and purity.28 These conditions were applied to the prep-
aration of 7-hydroxyisoellipticine 7, however only starting
material was recovered after work-up. Finally, a solid phase
Dakin reaction was investigated on the basis of work published
by Da Silva et al.29 The reaction carried out by carefully grind-
ing 7-formylisoellipticine 6 and m-CPBA with a pestle and
mortar (for safety precautions, see Experimental section) to give
7-hydroxyisoellipticine 7 in 12% yield. Overall, the best results
for the Baeyer–Villiger reaction were obtained with hydrogen
peroxide and a 5% v/v solution of sulfuric acid in methanol.
In an alternative preparation of 7-hydroxyisoellipticine 7,
7-methoxyisoellipticine 8 (synthesised via the Gribble route
from 5-methoxyindole) was demethylated with pyridine hydro-
chloride in a yield of 82%.
Results and discussion
Synthesis of isoellipticine and deazaellipticine derivatives
Isoellipticine 5 was prepared via Sauliner and Gribble’s versatile
route.27 The novel derivatives 7-formylisoellipticine 6 and
7-hydroxyisoellipticine 7 were synthesised using Plug’s con-
ditions for the Duff and Baeyer–Villiger reactions.7 The highly
regioselective Duff reaction of isoellipticine 5 with hexamethy-
lenetetramine in trifluoroacetic acid gave 7-formylisoellipticine
6 in 77% yield (Scheme 1).
The Baeyer–Villiger reaction was initially carried out under
the conditions reported by Plug et al. (Scheme 1, conditions (i)),
however this procedure gave low yields (15%–34%), so a more
reliable preparation was sought. First, the issue of solubility was
considered, increasing the amount of sulfuric acid to a 5%
As an additional comparison with ellipticine and isoellipticine
derivatives, the novel 2-hydroxydeazaellipticine 9 (6,11-dimethyl-
5H-benzo[b]carbazol-2-ol), was synthesized in 18% yield over
six steps from 5-methoxy-1-(phenylsulfonyl)-1H-indole 10
(Scheme 2). Lithiation at C-2 of the indole, followed by coupling
with phthalic anhydride gave 2-(5-methoxy-1-(phenylsulfonyl)-1H-
Fig. 2 Isoellipticine 5.
Scheme 1 Synthesis of 7-substituted isoellipticines. Conditions of Baeyer–Villiger reaction: (i) H2O2, H2SO4, MeOH; (ii) H2O2, H2SO4 in MeOH
(5% v/v); (iii) H2O2, formic acid; (iv) m-CPBA, solid phase.
Scheme 2 Synthesis of 2-hydroxydeazaellipticine 9.
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Synthesis and biological evaluation of isoellipticinium salts
indole-2-carbonyl)benzoic acid 11 in 94% yield. Deprotection to
12 proceeded smoothly (78%), followed by cyclisation to
2-methoxyindolo[1,2-b]isoquinoline-6,11-dione 13 in 66%
yield. 2-Methoxydeazaellipticine 14 was prepared by alkylation
with methyllithium followed by cyclisation with sodium boro-
hydride and was subsequently demethylated with pyridine
hydrochloride to give the novel 2-hydroxydeazaellipticine 9 in
57% yield.
These favourable initial results prompted investigation of novel
salts of 7-formylisoellipticine 6 and 7-hydroxyisoellipticine 7. A
large panel of salts was prepared by heating isoellipticine 5,
7-formylisoellipticine 6 or 7-hydroxyisoellipticine 7 with an
alkyl halide in dimethylformamide and triturating with diethyl
ether to give the novel isoellipticinium salts in excellent to
modest yields (Scheme 3 and Table 2). The salts of 7-hydroxy-
isoellipticine were highly hydroscopic, resulting in lower yields.
Given the anti-cancer activity of the N-methylellipticinium
salts, N-methylisoellipticinium iodide 16 was predicted to show
some activity and indeed it displayed moderate topoisomerase II
inhibition and cytotoxicity in the NCI screen. In the single dose
screen (10 μM concentration), 16 displayed 65.75% mean
growth over 58 cell lines and also displayed the CNS cell line
selectivity that had previously been observed in the N-methyl-
ellipticinium salts, with growth reduced to between 11 and 36%
across all CNS lines.24 This equates to selectivity of 3.36 (mean
of 52 non-CNS lines/mean of 6 CNS lines; where 1 = no selec-
tivity). N-Methylisoellipticinium iodide 16 also gave excellent
activity against the breast cancer cell line MDA-MB-468 with
−31.95% growth.
Next, the optimum length of the side chain was investigated
using nitrile halides (17, 18, 19), of these the six carbon chain
give moderate topo II inhibition and cytotoxicity. Investigation
of acid, amide and sulphonamide functionality resulted in
decreased cytotoxicity, while maintaining some topo II inhi-
bition, with the amide, 2-(6′-carboxamidohexyl)isoellipticinium
bromide 21 displaying inhibition at 10 μM.
7-Formylisoellipticine 6 had performed well in initial testing
and it was therefore surprising that none of its salt derivatives
were cytotoxic in the NCI screen, however, 2-(5′-cyanopentyl)-
7-formylisoellipticinium bromide 24 showed the highest level of
topo II activity in the screen, with inhibition at 10 μM and
partial inhibition at 1 μM. Surprisingly, given the activity of
Celiptium, none of the 7-hydroxyisoellipticinium salts showed
significant topo II inhibition or general cytotoxicity.
Biological activity of isoellipticines versus ellipticines
Inhibition of topoisomerase II is considered to be a key mecha-
nism of action for the ellipticine family of compounds and was
evaluated using a decatenation assay. In addition to this, the
overall cytotoxicity of the novel derivatives was assessed using
the NCI 60-cell line screen. Initially, the effect of A and D-ring
variation across ellipticine, isoellipticine and deazaellipticine
analogues was investigated (Table 1).
All compounds showed inhibition of topoisomerase II decate-
nation at 100 μM concentration except 2–hydroxydeazaellipti-
cine 9, indicating that the pyridine nitrogen is essential for
activity. The novel 7-substituted isoellipticines are at least as
active as their ellipticine counterparts and, in addition to this,
9-hydroxyellipticine 4 and 7-formylisoellipticine 6 are both
active at 10 μM.
In the NCI 60-cell line screen, 7-formylisoellipticine 6 and
7-hydroxyisoellipticine 7 both displayed moderate activity, with
mean growth values across the cell line panel of 51.47% and
53.50% respectively at 10 μM concentration. 7-Formylisoellipti-
cine 6 showed selectivity for leukaemia cell lines, with mean
growth in most cell lines below 10%. Highest activity was seen
against the SR cell line at −28.84% growth after 48 hours. Other
lines that responded well to 7-formylisoellipticine (10.80%) and
MCF7 (6.80%). 7-Hydroxyisoellipticine 7 also displayed selec-
tivity against leukaemia cell lines, with most lines below 30%
growth. It also showed good activity against two non-small cell
lung cancer lines and several colon cancer lines. These early
results established the promise of the isoellipticine class of com-
pounds as anti-cancer therapeutics.
Overall, three novel isoellipticines displayed strong inhibition
of topo II at 10 μM, two of which are isoellipticinium salts. It
appears that both long and short chain salts are tolerated in the
DNA–topoisomerase II-drug complex, however a six carbon
chain with either nitrile or amide functionality was optimum for
topo II inhibition (21, 24). Interestingly, none of the salts with a
carboxylic acid side chain were active (20, 23 and 27). A-ring
substitution is also favourable for inhibition (Table 1), however
the combination of an A-ring substituent and quaternary iso-
ellipticinium salt did not dramatically increase activity, and in
fact several such derivatives were less active.
Table 1 Comparison of topoisomerase II activity in ellipticine
analogues
Compound number
R1
Y
Z
Topo II inhibition
In the NCI screen, the carboxylic acid, nitrile, amide and sul-
phonamide isoellipticinium salts displayed rather disappointing
5
1
7
4
9
6
15
H
H
OH
OH
OH
CHO
CHO
CH
N
CH
N
CH
CH
N
N
CH
N
CH
CH
N
+
+
+
++
−
++
+
CH
+ Inhibition observed at 100 μM; ++ inhibition at 10 μM; +++ inhibition
at 1 μM; − no activity observed.
Scheme 3 Salt formation.
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Table 2 Yields and biological activity of novel isoellipticinium salts
Compound number
R1
R2
X
Yield %
Topo II inhibition
NCI mean growth %
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
H
H
H
H
CHO
CHO
CHO
CHO
OH
OH
OH
OH
CH3
C3H6CN
C4H8CN
C5H10CN
C5H10COOH
C5H10CONH2
C5H10CONHSO2CH3
C5H10COOH
C5H10CN
C5H10CONH2
C5H10CONHSO2CH3
C5H10COOH
C5H10CN
I
47
79
88
82
77
84
76
49
63
60
54
32
31
46
52
+
65.75
—
—
75.68
99.78
91.68
—
95.87
92.33
92.99
95.43
—
89.52
95.78
84.58
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
−
−
+
−
++
+
−
++
−
−
−
+
C5H10CONH2
C5H10CONHSO2CH3
+
−
+ Inhibition observed at 100 μM; ++ inhibition at 10 μM; +++ inhibition at 1 μM; − no activity observed.
cytotoxicity overall. The best mean growth was recorded for
2-(5′-cyanopentyl)isoellipticinium bromide 19 (75.68%) and this
was also the only other salt to display the CNS selectivity
observed in the N-methylisoellipticinium salt. CNS cell line
growth ranged between 25.19 and 69.30% for this compound
and with lower selectivity than N-methylisoellipticinium iodide
16 at 1.67 (mean of 52 non-CNS lines/mean of 6 CNS lines;
where 1 = no selectivity). Growth of the melanoma cell line,
SK-MEL-5 was also significantly inhibited at 14.31%, along
with the renal line, SN12C (19.33%).
Table 3 Selected five dose data for 7-formylisoellipticine 6
Cancer type
Leukaemia
Breast
Cell line
GI50 (μM)
LC50 (μM)
CCRF-CEM
SR
MCF7
HS 578T
LOX IMIV
0.894
0.487
0.879
0.620
0.861
>100
>100
41.3
78.2
56.0
Melanoma
The biological importance of C-9 substituents in the ellipticine
series is well established, and a similar effect was anticipated in
the 7-substituted isoellipticine series, however this was not the
case, 2-(5′-cyanopentyl)-7-formylisoellipticinium bromide 24
and 2-(5′-cyanopentyl)-7-hydroxyisoellipticinium bromide 28
gave mean growth values of 92.33% and 89.52%. This trend was
common to all the 7-formyl and 7-hydroxyisoellipticinium salts.
Fig. 3 Dose response curves for 7-formylisoellipticine 6.
NCI five-dose data for isoellipticine derivatives
Of the novel isoellipticine and isoellipticinium derivatives, four
were selected by the NCI for five dose screening, these include
7-formylisoellipticine 6, 7-hydroxyisoellipticine 7, N-methyl-
isoellipticinium iodide 16 and 2-(5′-cyanopentyl)isoellipticinium
bromide 19.
7-Formylisoellipticine 6 exhibited good GI50 values (concen-
tration at which growth is limited to 50%) in leukaemia and
breast cancer cell lines, particularly in CCRF-CEM, SR, MCF7,
HS578T and also the melanoma line, LOX IMIV (Table 3 and
Fig. 3).
These results correlate well with the single dose data discussed
above. While the dose response curve for leukaemia cell lines
levels off to give high LC50 values i.e. a cytostatic rather than
cytotoxic effect, that of breast cancer and the melanoma line
LOX IMIV display moderate LC50 values (concentration at
which <50% of cells survive after 48 hours).
Each of the compounds selected for five-dose testing was also
analysed using the COMPARE program to identify agents with
similar activity patterns across the NCI-60 panel. The correlation
was carried out using COMPARE analysis of GI50 values of
each isoellipticine derivative with those of the NCI synthetic
compounds database. Surprisingly, in the case of 7-formyliso-
ellipticine 6, no significant correlation was found with any ellip-
ticine or isoellipticine analogue or indeed with any of the NCI
standard agents. This unusual result suggests that 7-formyliso-
ellipticine 6 does not exert its effects via the currently known
mechanisms of NCI agents and further elucidation of its cellular
effects is currently underway.
7-Hydroxyisoellipticine 7 displayed a mean growth of 53.50%
in the single dose testing, however five dose testing highlights a
decline in activity at lower concentrations, particularly in leukae-
mia cell lines. Thus, the sub-micromolar GI50 values are found
in one leukaemia cell line and two NSCLC lines (Table 4 and
Fig. 4).
COMPARE analysis of 7-hydroxyisoellipticine 7 give a corre-
lation of 0.705 with another ellipticine isomer, 5,11-dimethyl-
10H-pyrido[2,3-b]carbazol-7-ol 31, although 7 is the more
active of the two (Fig. 5). An additional correlation of 0.785
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Table 4 Selected five dose data for 7-hydroxyisoellipticine 7
Cancer type
Cell line
GI50 (μM)
LC50 (μM)
Leukaemia
NSCLC
MOLT-4
A549/ATCC
NCI-H460
0.517
0.888
0.569
>100
34.3
30.6
Fig. 6 Dose response curves for N–methylisoellipticinium iodide 16.
Table 6 Selected five dose data for 2-(5′-cyanopentyl)isoellipticinium
bromide 19
Cancer type
CNS
Cell line
GI50 (μM)
LC50 (μM)
SF-268
SF-295
SF-539
SNB-19
U251
SN12C
MDA-MB-468
4.95
2.47
4.64
2.40
2.05
1.50
1.22
>100
>100
>100
>100
>100
>100
51.6
Fig. 4 Dose response curves for 7-hydroxyisoellipticine 7.
Renal
Breast
Fig. 5 Compounds with COMPARE analysis correlation to 7-hydroxy-
isoellipticine 7.
Fig. 7 Dose response curves for 2-(5′-cyanopentyl)isoellipticinium
bromide 19.
Table 5 Selected five dose data for N-methylisoellipticinium iodide 16
Cancer type
CNS
Cell line
GI50 (μM)
LC50 (μM)
SF-268
SNB-19
U251
A498
SN12C
0.676
0.607
0.276
0.683
0.526
0.442
40.2
>100
40.2
>100
>100
6.51
Renal
Breast
MDA-MB-468
Fig. 8 Berberine 33.
was found with the tricyclic compound 5-((3-(dimethylamino)-
propyl)amino)-6-oxo-6H-[1,2,3]triazolo[4,5,1-de]acridin-8-yl
propionate 32, however the mechanism of action of this drug is
unknown.
Significant selectivity for CNS cancer cell lines was displayed
in the single dose data for N-methylisoellipticinium iodide 16
and this translated to excellent GI50 values in the five dose assay.
In addition, strong activity was seen in two renal cancer and one
breast cancer line (Table 5 and Fig. 6).
As expected, COMPARE analysis of N-methylisoellipticinium
iodide 16 gave a strong correlation to the corresponding ellipti-
cine, N-methylellipticinium iodide. The correlation value was
0.775, with the ellipticine isomer displaying marginally stronger
activity.
The nitrile salt, 2-(5′-cyanopentyl)isoellipticinium bromide 19
also displayed CNS specificity in the single dose mean graph,
however with a mean growth of 75.68%, this compound was not
expected to be particularly active in the five dose assay. Indeed,
no sub-micromolar GI50 values were recorded for 19 (Table 6
and Fig. 7), although the renal line SN12C and the breast cancer
line MDA-MB-468 displayed GI50 values of 1.50 μM and
1.22 μM respectively.
2-(5′-Cyanopentyl)isoellipticinium bromide 19 gave a corre-
lation of 0.681 to 2-methylisoellipticinium iodide 16 and a stron-
ger correlation of 0.789 to the natural product berberine 33
(Fig. 8). Interestingly, berberine is known to interact with multi-
drug resistance protein MDR1 or P-glycoprotein, a mechanism
also attributed to the ellipticines.30
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spectrometer. 1H (600 MHz) and 13C (150.9 MHz) NMR spectra
were recorded on a Bruker Avance III 600 MHz NMR spec-
trometer equipped with a dual CH cryoprobe. All spectra were
recorded at room temperature (∼20 °C) in deuterated chloroform
(CDCl3) with tetramethylsilane (TMS) as an internal standard, or
deuterated dimethylsulfoxide (DMSO-d6). 1H NMR spectra
recorded in deuterated dimethylsulfoxide (DMSO-d6) were
assigned using the DMSO-d6 peak as the reference peak. Chemi-
cal shifts (δH and δC) are expressed in parts per million (ppm)
relative to the reference peak. Coupling constants (J) are
Conclusions
The 7-substituted isoellipticines represent a new class of anti-
cancer agent with a unique NCI activity profile. Interestingly,
low correlation between results of the topoisomerase II decate-
nation assay and NCI screen throughout the panel suggests that
topo II is not the most important biological target with respect to
anti-cancer activity in this new class of compounds.
In particular, the CNS specific agents N-methylisoelliptici-
nium iodide 16 and 2-(5′-cyanopentyl)isoellipticinium bromide
19 are of significant interest in treatment of a disease with very
poor prognosis and a lower age profile than most other cancers.
Preliminary results show that the longer chain isoellipticinium
salts are less active than the simple methyl derivative and further
investigation of the structure activity relationship is required.
These results also suggest that solubility is not the limiting
factor in activity of the isoellipticinium salts, given that all
the longer chain salts had improved aqueous solubility over the
predecessor 16.
The mechanism of action of these new compounds is largely
unknown and comparison with current data on NCI cell line
characteristics has not yielded any significant correlations.31 In
particular, studies which characterised the NCI cell line
expression of p53 tumour suppressor, reductase enzyme and
topoisomerase I gave no correlation with isoellipticines 6, 7, 16
and 19.32–34 On COMPARE analysis, compounds 7, 16 and 19
appear to correlate to some extent with ellipticine congeners,
however 7-formylisoellipticine 6 has a unique mode of action
and may be a significant lead for future work especially given its
poor solubility. Further investigation of the mechanism of action
of all four compounds will be pursued.
Overall, 20 novel ellipticine analogues were prepared and full
anti-cancer profiling was completed for 13 isoellipticine deriva-
tives and salts. Significant SAR studies will now follow in order
to determine where potency and solubility can be improved in
this new class of anti-cancer compounds.
1
expressed in Hertz (Hz). Splitting patterns in H NMR spectra
are designated as s (singlet), br s (broad singlet), d (doublet),
br d (broad doublet), t (triplet), q (quartet), dd (doublet of
doublets), dt (doublet of triplets), ddd (doublet of doublet of
doublets), ddt (doublet of doublet of triplets) and m (multiplet).
Low resolution mass spectra were recorded on a Waters
Quattro Micro triple quadrupole spectrometer (QAA1202) in
electrospray ionisation (ESI) mode using 50% acetonitrile–water
containing 0.1% formic acid as eluent. High resolution mass
spectra (HRMS) were recorded on a Waters LCT Premier Time
of Flight spectrometer in electrospray ionisation (ESI) mode
using 50% acetonitrile–water containing 0.1% formic acid as
eluent.
Elemental analyses were preformed by the Microanalysis
Laboratory, National University of Ireland, Cork, using Perkin
Elmer 240 and Exeter Analytical CE440 elemental analysers.
Melting points were measured in a uni-melt Thomas Hoover
capillary melting point apparatus and are uncorrected. Thin layer
chromatography (TLC) was carried out on precoated silica gel
plates (Merck 60 PF254) or aluminium oxide TLC plates
(Sigma). Visualisation was achieved by UV light detection
(254 nm).
Synthesis of 7-substituted isoellipticines
5,11-Dimethyl-10H-pyrido[3,4-b]carbazole-7-carbaldehyde 6. A
solution of 5,11-dimethyl-10H-pyrido[3,4-b]carbazole 5 (2.433 g,
9.88 mmol) in trifluoroacetic acid (160 mL), was treated with
hexamethylenetetramine (13.847 g, 98.78 mmol, 10 eq.) portion-
wise over 5 minutes. The resulting deep red solution was heated
to reflux for 30 minutes. On cooling, the reaction mixture was
concentrated to approx. one quarter volume and water (250 mL)
was added. The solution was cooled to 0 °C and neutralized with
solid sodium bicarbonate while stirring vigorously. Dichloro-
methane (300 mL) was added and the mixture was stirred for
2 hours. The aqueous layer was extracted with dichloromethane–
methanol (90 : 10, 5 × 200 mL). Combined organic extracts were
dried and concentrated under reduced pressure to give the
product as an orange solid (1.013 g). A precipitate in the
aqueous layer, which did not extract into the organic phase, was
filtered and dried thoroughly under vacuum (0.1 mm Hg) to give
additional pure product (1.064 g, combined yield 2.077 g,
76.7%). m.p. 270–271 °C; vmax/cm−1 (KBr): 3153 (NH), 1672
(CvO), 1599 (CvC arom.), 1469 (CvC arom.), 1381, 1285,
1216, 1113, 1014, 810; δH (600 MHz, DMSO-d6): 2.86 [3H, s,
C(11)CH3], 3.05 [3H, s, C(5)CH3], 7.61 [1H, d, J 8.3, C(9)H],
8.01 [1H, d, J 8.5, C(8)H], 8.07 [1H, d, J 5.9, C(3)H], 8.42
[1H, d, J 5.9, C(4)H], 8.74 [1H, s, C(6)H], 9.52 [1H, s, C(1)H],
Experimental
General procedures
Solvents were distilled prior to use as follows: dichloromethane
was distilled from phosphorous pentoxide; ethyl acetate was
distilled from potassium carbonate; ethanol and methanol were
distilled from magnesium in the presence of iodine; toluene was
distilled from sodium and benzophenone; hexane was distilled
prior to use; tetrahydrofuran was freshly distilled from sodium
and benzophenone. Diethyl ether was obtained pure from
Riedel-de Haën. Organic phases were dried using anhydrous
magnesium sulphate. All commercial reagents were used without
further purification unless otherwise stated. 5-Methoxy-1-(phenyl-
sulfonyl)-1H-indole 10 was prepared from 5-methoxyindole in
94% yield.
Infrared spectra were recorded as a thin film on sodium chlor-
ide plates for liquids or potassium bromide (KBr) disc for solids
on a Perkin Elmer Spectrum 100 FT-IR spectrometer.
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded
on a Bruker Avance 300 NMR spectrometer. 1H (400 MHz)
NMR spectra were recorded on a Bruker Avance 400 NMR
This journal is © The Royal Society of Chemistry 2012
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10.04 [1H, s, C(7)CHO], 11.83 [1H, s, N(10)H]; δC
(150.9 MHz, DMSO-d6): 11.7 [CH3, C(11)CH3]; 14.4 [CH3, C
(5)CH3]; 110.9 (CH, aromatic CH), 111.9 (C, aromatic C),
116.9 (CH, aromatic CH), 122.5 (C, aromatic C), 125.1 (C, aro-
matic C), 125.4 (C, aromatic C), 125.6 (C, aromatic C), 127.7
(CH, aromatic CH), 128.2 (C, aromatic C), 128.4 (CH, aromatic
CH), 128.8 (C, aromatic C), 138.4 (C, aromatic C), 138.7 (CH,
aromatic CH), 146.9 (C, aromatic C), 148.7 (CH, aromatic CH),
191.8 (C, CHO); m/z (ESI+): 275 [(M + H)+ 30%], 115 (100%),
105 (70%); HRMS (ESI+): Exact mass calculated for
C18H15N2O+ 275.1184. Found 275.1179. Anal. calculated for
C18H14N2O: C, 78.81; H, 5.14; N, 10.21. Found: C, 78.96;
H, 5.42; N, 9.91.
product was obtained after concentrating under reduced pressure.
During extraction an precipitate formed in the aqueous layer, this
was filtered to give a brown solid, which was found to be
unreacted starting material (63 mg, 48.8% recovery).
(c) Solid phase Baeyer–Villiger reaction with m-CPBA. 5,11-
Dimethyl-10H-pyrido[3,4-b]carbazole-7-carbaldehyde
6
(413 mg, 1.51 mmol) was ground with a pestle and mortar to
give a fine orange powder. m-Chloroperbenzoic acid (519 mg,
2.32 mmol, 77%) was added and gently ground.‡ After
5 minutes the mixture melted and bubbled slightly and the reac-
tion was continued for a further 15 minutes. Sodium hydroxide
(10% soln., 5 mL) was added and stirred for 10 minutes before
transferring to a conical flask and neutralising with 20% aqueous
HCl. The solution was concentrated under reduced pressure and
purified by column chromatography with dichloromethane–
methanol (95 : 5–90 : 10) containing 10 drops of triethylamine
per 500 mL of eluent. However further impurity remained and
additional column chromatography was required to give the
desired product as a red solid (47 mg, 11.9%).
(d) Demethylation of 7-methoxyisoellipticine. 7-Methoxy-5,11-
dimethyl-10H-pyrido[3,4-b]carbazole 8 (69 mg, 0.250 mmol)
and pyridine hydrochloride (1.155 g, 10.0 mmol) were heated
with a heat gun for 3 minutes until melted and bubbling slightly
and then on a sand bath at ∼220 °C for 40 minutes. The reaction
was cooled to 0 °C, brine (10 mL) was added and stirred for
30 minutes. The mixture was filtered, washed with water
(20 mL) and dried. Column chromatography on basic alumina
with dichloromethane–methanol (95 : 5) gave the product as a
red solid (53 mg, 81.5%). Analysis was identical to that quoted
in method A above.
5,11-Dimethyl-10H-pyrido[3,4-b]carbazol-7-ol 7
(a) Baeyer–Villiger reaction with hydrogen peroxide and sulfu-
ric acid. 5,11-Dimethyl-10H-pyrido[3,4-b]carbazole-7-carbalde-
hyde 6 (201 mg, 0.733 mmol) was added to a solution of
sulfuric acid in methanol (150 mL, 5% v/v). The deep red solu-
tion was treated with hydrogen peroxide (1 mL, 30% w/v soln.)
and heated to reflux for 6 hours. The mixture was cooled to 0 °C
and stirred vigorously while adjusting to pH 12 with solid
sodium bicarbonate. After 30 minutes stirring at room temp-
erature the solution was neutralised to pH 7 with 20% aqueous
HCl and stirred overnight with dichloromethane (70 mL). The
layers were separated and the aqueous layer was extracted with
dichloromethane–methanol (90 : 10, 6 × 30 mL) while sequen-
tially adjusting the pH (7, 5, 10, 7) with 20% aqueous HCl and
10% aqueous NaOH. Combined organic extracts were dried and
concentrated under reduced pressure to give a bright orange
solid (137 mg, 71.3%). m.p. >300 °C (without melting);
v
max/cm−1 (KBr): 3388 (OH), 3241 (NH), 1658 (CvC arom.),
Synthesis of 2-hydroxydeazaellipticine
1635 (CvC arom.), 1451, 1410, 1220, 1116 (C–O stretch);
δH (300 MHz, DMSO-d6): 2.85 [3H, s, C(11)CH3], 3.02 [3H, s,
C(5)CH3], 7.00 [1H, dd, J 8.6, 2.3, C(8)H], 7.33 [1H, d, J 8.6,
C(9)H], 7.72 [1H, d, J 2.3, C(6)H], 8.01 [1H, dd, J 6.0, 0.2,
C(3)H], 8.30 [1H, d, J 5.8, C(4)H], 9.05 [1H, s, C(7)OH,
assigned from D2O exchange experiment], 9.48 [1H, s, C(1)H],
10.96 [1H, s, N(10)H]; δC (75.5 MHz, DMSO-d6): 11.8 [CH3,
C(11)CH3], 14.3 [CH3, C(5)CH3], 109.3 [CH, C(6)H], 110.3
(C, aromatic C), 111.0 [CH, C(9)H], 116.6 [CH, C(8)H], 116.8
[CH, C(3)H], 123.2 (C, aromatic C), 124.6 (C, aromatic C),
125.1 (C, aromatic C), 125.8 (C, aromatic C), 127.6 (C, aromatic
C), 136.9 (C, aromatic C), 138.1 [CH, C(4)H], 139.1 (C, aro-
matic C), 148.7 [CH, C(1)H], 150.5 (C, aromatic C); m/z (ESI+):
263 [(M + H)+ 100%]; HRMS (ESI): Exact mass calculated for
C17H15N2O+ 263.1184. Found 263.1181.
2-(5-Methoxy-1-(phenylsulfonyl)-1H-indole-2-carbonyl)benzoic
acid 11. A solution of diisopropylamine (14.7 mL, 0.108 mol) in
THF (150 mL) at −78 °C, under N2, was treated with n-BuLi
(39.6 mL, 0.099 mol, 2.5 M soln.) via cannula and stirred for
20 minutes. 5-Methoxy-1-(phenylsulfonyl)-1H-indole 10
(25.850 g, 0.090 mol) in THF (150 mL) was added over
15 minutes, maintaining the temperature below −78 °C. The
mixture was allowed to warm to 15 °C over 3 hours. The
mixture was recooled to −78 °C and treated with phthalic anhy-
dride (19.991 g, 0.135 mol) in THF (100 mL), maintaining the
temperature below −78 °C. The reaction mixture was allowed to
warm to room temperature overnight. The solvent was evapor-
ated under reduced pressure and the residue was dissolved in
water (600 mL). The solution was cooled to 0 °C and acidified
to pH 2 with 20% aqueous HCl. The resulting precipitate was
collected by vacuum filtration and washed with water (200 mL)
to give a cream solid. The crude product was recrystallised from
acetone and collected in two crops as a cream solid (36.634 g,
93.5%). m.p. 205–207 °C (acetone); vmax/cm−1 (KBr): 2982
(OH, broad) 1720 (CvO, acid), 1682 (CvO, ketone), 1584
(b) Baeyer–Villiger reaction with hydrogen peroxide and formic
acid. A stirred solution of 5,11-dimethyl-10H-pyrido[3,4-b]car-
bazole-7-carbaldehyde 6 (129 mg, 0.470 mmol) in formic acid
(10 mL) was treated with hydrogen peroxide (0.06 mL, 30% w/v
soln., 1.2 eq.) and stirred at room temperature for 24 hours. TLC
analysis showed no reaction, so additional hydrogen peroxide
(0.06 mL) was added, and the reaction was heated to 40 °C for
6 hours. On cooling, water (10 mL) was added and the solution
was basified with 10% aqueous NaOH to pH 12. After stirring
for 15 minutes, the solution was neutralised with 20% aqueous
HCl and extraction was attempted with dichloromethane–metha-
nol (90 : 10, 3 × 20 mL), however this was unsuccessful and no
‡Note: Due to the explosion and fire hazard of grinding m-CPBA,
several precautions were taken. These included the use of a blast
shield for the duration of the reaction and heavy-duty gloves worn over
disposable gloves to protect hands and arms. A sand–gravel mixture was
kept in the fumehood in case of fire.
7918 | Org. Biomol. Chem., 2012, 10, 7912–7921
This journal is © The Royal Society of Chemistry 2012

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(CvC arom.), 1444, 1371, 1351 (SO2 asymm. stretch), 1243
(C–O), 1144 (SO2 symm. stretch); δH (300 MHz, DMSO-d6):
3.75 [3H, s, C(5)OCH3], 6.99 [1H, s, C(3)H], 7.13 [1H, dd,
J 9.1, 2.7, C(6)H], 7.21 [1H, d, J 2.3, C(4)H], 7.57–7.59
[1H, m, C(3′)H], 7.64–7.79 [5H, m, C(4′)H, C(5′)H, SO2Ph-
C(3)H, SO2Ph-C(4)H, SO2Ph-C(5)H], 7.87–7.89 [1H, m, C(6′)
H], 8.06 [1H, d, J 9.1, C(7)H], 8.13–8.16 [2H, m, SO2Ph-C(2)
H, SO2Ph-C(6)H], 13.30 (1H, br s, COOH); δC (75.5 MHz,
DMSO-d6): 55.4 [CH3, C(5)OCH3], 104.8 [CH, C(4)H], 116.1
[CH, C(7)H], 117.8 [CH, C(6)H], 121.1 [CH, C(3)H], 127.1
[CH, two overlapping aromatic CH, SO2Ph-C(2)H, SO2Ph-C(6)
H], 128.4 (C, aromatic C), 129.2 [CH, C(3′)H], 129.4 [CH, three
overlapping aromatic CH, C(6′)H, SO2Ph-C(3)H, SO2Ph-C(5)
H], 131.29 (CH, aromatic CH), 131.32 (CH, aromatic CH),
132.4 (C, aromatic C), 133.3 (C, aromatic C), 134.3 (CH, aro-
matic CH), 138.6 (C, aromatic C), 138.8 (C, aromatic C), 139.3
(C, aromatic C), 156.3 (C, aromatic C), 167.8 [C, C(2′)COOH],
185.2 [C, C(2)CvO]; m/z (ESI+): 436 [(M + H)+ 100%]; HRMS
(ESI+): Exact mass calculated for C23H18NO6S+ 436.0855.
Found 436.0862.
with water (500 mL) and dried at 0.1 mbar to give the product as
gold needles (2.932 g). The filtrate was completely evaporated
under reduced pressure and recrystallised from acetone in two
crops to give additional product (0.702 g, combined yield
3.634 g, 66.1%). m.p. 223–225 °C (acetone); vmax/cm−1 (KBr):
3070 (CH), 2974 (CH3 asymm. stretch), 2833 (CH3 symm.
stretch), 1693 (CvO, ketone), 1659 (CvO, lactam), 1599
(CvC arom.), 1547 (CvC arom.), 1477, 1377, 1255 (C–O),
1152; δH (300 MHz, DMSO-d6): 3.84 [3H, s, C(5)OCH3], 7.24
[1H, dd, J 9.1, 2.6, C(3)H], 7.36 [1H, d, J 2.5, C(1)H], 7.65
[1H, s, C(12)H], 7.89–7.98 [2H, m, C(8)H, C(9)H], 8.15–8.18
[1H, m, C(7)H or C(10)H], 8.30–8.33 [1H, m, C(7)H or C(10)
H], 8.38 [1H, d, J 9.1, C(4)H]; δC (75.5 MHz, DMSO-d6): 55.5
[CH3, C(2)OCH3], 105.3 (CH, aromatic CH), 115.2 (CH, aro-
matic CH), 117.2 (CH, aromatic CH), 119.0 (CH, aromatic CH),
126.3 (CH, aromatic CH), 128.7 (CH, aromatic CH), 129.5 (C,
aromatic C), 130.8 (C, aromatic C), 131.2 (C, aromatic C), 133.0
(C, aromatic C), 134.0 (C, aromatic C), 134.3 (CH, aromatic
CH), 134.7 (CH, aromatic CH), 156.9 (C, aromatic C), 158.6
[C, C(6)vO], 175.0 [C, C(11)vO]; m/z (ESI+): 278 [(M + H)+
100%]; HRMS (ESI+): Exact mass calculated for C17H12NO3
+
2-(5-Methoxy-1H-indole-2-carbonyl)benzoic acid 12. A solu-
tion of 2-(5-methoxy-1-(phenylsulfonyl)-1H-indole-2-carbonyl)-
benzoic acid 11 (36.635 g, 0.084 mol) and potassium carbonate
(46.438 g, 0.336 mol) in methanol (900 mL) and water
(300 mL) was heated to reflux for 5 hours. The mixture was
allowed to cool and the methanol was evaporated under reduced
pressure. The residue was treated with water (500 mL), cooled to
0 °C and acidified to pH 2 with 20% aqueous HCl, with vigor-
ous stirring. The aqueous layer was extracted with ethyl acetate
(3 × 200 mL) and the combined organic extracts were washed
with water (1 × 200 mL) and brine (2 × 200 mL), dried and con-
centrated under reduced pressure to give an orange solid. This
was recrystallised from acetone to give a yellow solid in several
crops (19.361 g, 78.1%). m.p. 219–221 °C (acetone); vmax/cm−1
(KBr): 3212 (OH, broad), 1703 (CvO × 2, broad), 1590 (CvC
arom.), 1524 (CvC arom.), 1455, 1397, 1266 (C–O), 1134,
1034; δH (400 MHz, DMSO-d6): 3.73 [3H, s, C(5)OCH3], 6.49
[1H, dd, J 2.2, 0.7, C(3)H], 6.96 [1H, dd, J 9.0, 2.5, C(6)H],
7.07 [1H, d, J 2.4, C(4)H], 7.38 [1H, d, J 9.0, C(7)H], 7.57 [1H,
dd, J 7.4, 1.3, C(3′)H], 7.67 [1H, td, J 7.5, 1.3, C(5′)H], 7.72
[1H, td, J 7.4, 1.4, C(4′)H], 7.97 [1H, dd, J 7.6, 1.3, C(6′)H],
11.88 [1H, s, N(1)H], 13.12 (1H, br s, COOH); δC (75.5 MHz,
DMSO-d6): 55.7 [CH3, C(5)OCH3], 102.4 (CH, aromatic CH),
110.5 (CH, aromatic CH), 113.6 (CH, aromatic CH), 117.3 (CH,
aromatic CH), 127.1 (C, aromatic C), 127.9 (CH, aromatic CH),
129.6 (CH, aromatic CH), 129.9 (CH, aromatic CH), 130.8 (C,
aromatic C), 131.8 (CH, aromatic CH), 133.5 (C, aromatic C),
136.2 (C, aromatic C), 140.8 (C, aromatic C), 153.9 (C, aromatic
C), 167.3 [C, C(2′)COOH], 187.7 [C, C(2)CvO]; m/z (ESI−):
294 [(M − H)− 100%]; HRMS (ESI+): Exact mass calculated for
C17H14NO4+ 296.0923. Found 296.0933.
278.0817. Found 278.0806.
2-Methoxy-6,11-dimethyl-5H-benzo[b]carbazole 14. A solu-
tion of 2-methoxyindolo[1,2-b]isoquinoline-6,11-dione 13
(0.401 g, 1.45 mmol) in THF (100 mL) at −100 °C under N2
was treated drop-wise with methyllithium (1.9 mL, 3.05 mmol,
1.6 M soln.). The resulting mixture was maintained below
−100 °C for 1 hour and then allowed to warm to room temp-
erature overnight. The reaction was quenched with water (5 mL)
and solvent was removed under reduced pressure. The residue
was dissolved in absolute ethanol (130 mL), treated with sodium
borohydride (0.546 g, 14.4 mmol) and heated to reflux. The
reaction was refluxed for a total of 48 hours with a second
portion of sodium borohydride (0.546 g, 14.4 mmol) added after
6 hours and a third portion (0.546 g, 14.4 mmol) after 26 hours.
On cooling, the solvent was evaporated under reduced pressure
and water (120 mL) was added. The aqueous layer was extracted
with dichloromethane (3 × 100 mL), adjusted to pH 7 with 20%
aqueous HCl and extracted again with dichloromethane (2 ×
50 mL). Combined organic extracts were washed with water (1 ×
100 mL) and brine (1 × 100 mL), dried and concentrated under
reduced pressure. Purification by column chromatography,
eluting with hexane–ethyl acetate (70 : 30), gave 14 (0.245 g,
61.4%). m.p. 184–186 °C (Lit. 188 °C);17 vmax/cm−1 (KBr):
3414 (NH), 2930 (CH3 asymm. stretch), 2849 (CH3 symm.
stretch), 1624 (CvC arom.), 1582 (CvC arom.), 1485, 1223
(C–O), 1132; δH (300 MHz, DMSO-d6): 2.81 [3H, s, C(6)CH3],
3.17 [3H, s, C(11)CH3], 3.90 [3H, s, C(2)OCH3], 7.15 [1H, dd,
J 8.7, 2.3, C(3)H], 7.39–7.43 [1H, m, C(9)H], overlapping with
7.45 [1H, d, J 8.6, C(4)H], 7.48–7.54 [1H, m, C(8)H], 7.87 [1H,
d, J 2.4, C(1)H], 8.10 [1H, d, J 8.5, C(7)H], 8.32 [1H, d, J 8.5,
C(10)H], 10.84 [1H, s, N(5)H]; δC (75.5 MHz, DMSO-d6): 12.6
[CH3, C(6)CH3], 15.0 [CH3, C(11)CH3], 55.8 [CH3, C(2)
OCH3], 107.7 [CH, C(1)H], 108.8 (C, aromatic C), 110.7 [CH,
C(4)H], 114.6 [CH, C(3)H], 121.5 [CH, C(9)H], 122.7 (C, aro-
matic C), 123.1 [CH, C(7)H], 123.7 (C, aromatic C), 124.3 [CH,
C(8)H], 124.5 [CH, C(10)H], 125.8 (C, aromatic C), 126.4 (C,
aromatic C), 130.6 (C, aromatic C), 137.5 (C, aromatic C), 138.9
2-Methoxyindolo[1,2-b]isoquinoline-6,11-dione 13. 2-(5-
Methoxy-1H-indole-2-carbonyl)benzoic acid 12 (5.857 g,
19.8 mmol) in acetic anhydride (500 mL) was heated to 90 °C
under N2 for 24 hours. The reaction mixture was concentrated to
approximately half volume and stored at 4 °C overnight. The
resulting precipitate was collected by vacuum filtration, washed
This journal is © The Royal Society of Chemistry 2012
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(C, aromatic C), 152.6 (C, aromatic C); m/z (ESI+): 276
[(M + H)+ 100%]; HRMS (ESI+): Exact mass calculated for
C19H18NO+ 276.1388. Found 276.1388.
128.0 (C, aromatic C), 129.7 (CH, aromatic CH), 129.8 (CH,
aromatic CH), 130.0 (C, aromatic C), 139.8 (C, aromatic C),
143.7 (C, aromatic C), 146.5 (CH, aromatic CH); m/z (ESI+):
261 [(M)+ 100%]; HRMS (ESI+): Exact mass calculated for
C18H17N2+ 261.1392. Found 261.1385.
6,11-Dimethyl-5H-benzo[b]carbazol-2-ol 9. 2-Methoxy-6,11-
dimethyl-5H-benzo[b]carbazole 14 (145 mg, 0.527 mmol) and
pyridine hydrochloride (1.217 g, 10.5 mmol) were heated to
220 °C for 2 hours. The reaction was cooled to 0 °C, water
(10 mL) was added and extracted with dichloromethane (2 ×
10 mL) and dichloromethane–methanol (90 : 10, 2 × 10 mL).
Combined organic extracts were washed with 1 M HCl (2 ×
10 mL) and brine (1 × 10 mL), dried and concentrated under
reduced pressure. Purification by column chromatography,
eluting with hexane–ethyl acetate (85 : 15), gave the product as a
yellow solid (78 mg, 56.5%). m.p. 215–217 °C; vmax/cm−1
(KBr): 3343 (OH), 2917 (CH3 asymm. stretch), 2849 (CH3
symm. stretch), 1622 (CvC arom.), 1565 (CvC arom.), 1477,
1222 (C–O stretch), 1185, 1131; δH (300 MHz, DMSO-d6): 2.79
[3H, s, C(6)CH3], 3.12 [3H, s, C(11)CH3], 6.98 [1H, dd, J 8.5,
2.3, C(3)H], 7.33 [1H, d, J 8.5, C(4)H], 7.40 [1H, overlapping
ddd, J 8.3, 6.7, 1.4, C(9)H], 7.50 [1H, overlapping ddd, J 8.3,
6.7, 1.4, C(8)H], 7.75 [1H, d, J 2.3, C(1)H], 8.09 [1H, d, J 8.1,
C(7)H], 8.29 [1H, d, J 7.9, C(10)H], 8.95 [1H, s, C(2)OH],
10.68 [1H, s, N(5)H]; δC (75.5 MHz, DMSO-d6): 12.5 [CH3,
C(6)CH3], 15.0 [CH3, C(11)CH3], 107.7 (C, aromatic C), 108.8
(CH, aromatic CH), 110.7 (CH, aromatic CH), 114.5 (CH, aro-
matic CH), 121.5 (CH, aromatic CH), 122.7 (C, aromatic C),
123.0 (CH, aromatic CH), 123.7 (C, aromatic C), 124.2 (CH,
aromatic CH), 124.5 (CH, aromatic CH), 125.8 (C, aromatic C),
126.3 (C, aromatic C), 130.6 (C, aromatic C), 137.5 (C, aromatic
C), 138.9 (C, aromatic C), 152.6 (C, aromatic C); m/z (ESI+):
262 [(M + H)+ 40%], 261 (100%); HRMS (ESI+): Exact mass
calculated for C18H16NO+ 262.1232. Found 262.1226.
2-(3′-Cyanopropyl)isoellipticinium chloride 17. 5,11-Dimethyl-
10H-pyrido[3,4-b]carbazole 5 (198 mg, 0.80 mmol) and 4-
chlorobutryonitrile (0.091 mL, 0.96 mmol) in dimethylforma-
mide (5 mL) were heated to 120 °C for 48 hours. The dark pink
solution was cooled to 0 °C and excess cold ether added. The
resulting dark pink precipitate was filtered and washed with cold
ether to yield product as a red powder (222 mg, 79%).
m.p. 236–240 °C; vmax/cm−1 (KBr): 3384 (NH), 3153 (CH, aro-
matic), 3091 (CH3), 2246 (CN, nitrile), 1634 (CvC, aromatic),
1619 (CvC, aromatic), 1417, 1321; δH (300 MHz, DMSO-d6):
2.28–2.41 [2H, m, C(2′)H2], 2.69 [2H, t, J 7.3, C(3′)CH2], 2.91
[3H, s, C(11)CH3], 2.98 [3H, s, C(5)CH3]. 4.80 [2H, t, J 7.0,
C(1′)H2], 7.18–7.25 [1H, m, C(7)H], 7.53–7.59 [2H, m, C(8)H,
C(9)H], 8.25 [1H, d, J 8.1, C(6)H], 8.44 [1H, d, J 7.1, C(3)H],
8.57 [1H, d, J 7.1, C(4)H], 9.97 [1H, s, C(1)H], 12.13 (1H, s,
NH); δC (75.5 MHz; DMSO-d6): 12.55 [CH3, C(11) CH3],
13.64 (CH2), 14.64 [CH3, C(5)CH3], 26.44 (CH2), 58.57 [CH2,
C(1′)H2], 111.38 (CH, aromatic), 115.61 (C, aromatic C), 119.68
(C, aromatic C), 119.81 (CH, aromatic), 121.35 (C, CN), 122.32
(CH, aromatic), 123.58 (C, aromatic C), 124.82 (CH, aromatic),
126.07 (C, aromatic C), 128.06 (C, aromatic C), 128.56 (CH,
aromatic), 129.47 (CH, aromatic), 129.89 (C, aromatic C),
139.65 (C, aromatic C), 143.71 (C, aromatic C), 146.21 (CH,
aromatic); m/z (ESI+): 314 [(M+), 100%]; HRMS (ESI): Exact
+
mass calculated for C21H20N3 : 314.1657. Found: 314.1664.
Topoisomerase II decatenation assay
The decatenation assay kit was obtained from Inspiralis,
Norwich Bioincubator, Norwich Research Park, Colney,
Norwich, UK. The kit comprised of the following: assay buffer
(supplied as 10× stock) containing 50 mM Tris-HCl (pH 7.5),
125 mM NaCl, 10 mM MgCl2, 5 mM DTT and 100 μg mL−1
albumin; dilution buffer containing 50 mM Tris-HCl (pH 7.5),
100 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 50% (v/v) glycerol,
50 μg mL−1 albumin; ATP 30 mM; kDNA (100 ng μL−1);
10 U μL−1 human topoisomerase II in dilution buffer; 5× stop
buffer containing 2.5% SDS, 15% Ficoll-400, 0.05% bromo-
phenol blue, 0.05% xylene cyanol and 25 mM EDTA. Tris-
acetate-EDTA buffer (supplied as 10× buffer) and agarose were
obtained from Sigma Life Sciences (Dublin, Ireland) and Safe
View Stain was supplied by NBS Biologicals, Cambridgeshire,
England.
The topo II decatenation assay protocol involved initial incu-
bation of each inhibitor candidate (100 μM) along with a stock
solution containing water, ATP, assay buffer, kDNA obtained
from the mitochondrial DNA of Crithidia fasciculate, and topo
II, at 37 °C for 1 hour. Following addition of stop buffer, agarose
DNA gel electrophoresis was run at 50 V for 2 hours using a
Consort EV243 power pack, to determine the relative amounts of
decatenated DNA bands obtained in each compound lane. Posi-
tive (water), as well as negative controls (ellipticine) were incor-
porated in order to validate the results of each run. The resulting
Representative syntheses of isoellipticinium salts
Full experimental data for salts 18–30 may be found in the ESI.†
2-Methylisoellipticinium iodide 16. To a stirred suspension of
5,11-dimethyl-10H-pyrido[3,4-b]carbazole 5 (72 mg, 0.292 mmol)
in dimethylformamide (4 mL), was added iodomethane
(0.02 mL, 0.321 mmol, 1.1 eq.). The reaction mixture was stirred
at room temperature for 3 hours. The mixture was cooled to 0 °C
and cold diethyl ether (3 mL) was added. The resulting precipi-
tate was collected by vacuum filtration and washed with hexane
(3 mL) and cold diethyl ether (5 mL) to give the product as an
orange solid (53 mg, 46.7%). m.p. >350 °C without melting;
v
max/cm−1 (KBr): 3400 (NH), 3195 (CH), 3004 (CH), 2862
(symm. CH3 stretch), 1636 (CvC arom.), 1619 (CvC arom.),
1418, 1316, 1193; δH (600 MHz, DMSO-d6): 2.94 [3H, s, C(11)
CH3], 3.13 [3H, s, C(5)CH3], 4.45 [3H, s, N(2)CH3], 7.31 [1H,
overlapping ddd appearing as a td, J 6.9, 0.7, C(7)H], 7.61–7.64
[2H, m, C(8)H, C(9)H], 8.37 [1H, d, J 6.9, C(3)H], 8.40 [1H, d,
J 8.0, C(6)H], 8.69 [1H, d, J 7.0, C(4)H], 9.89 [1H, s, C(1)H],
11.90 [1H, s, N(10)H]; δC (150.9 MHz, DMSO-d6): 12.3 [CH3,
C(11)CH3], 14.8 [CH3, C(5)CH3], 47.2 [CH3, N(2)CH3], 111.3
(CH, aromatic CH), 115.2 (C, aromatic C), 120.1 (CH, aromatic
CH), 121.6 (C, aromatic C), 122.1 (CH, aromatic CH), 123.6 (C,
aromatic C), 125.1 (CH, aromatic CH), 126.4 (C, aromatic C),
7920 | Org. Biomol. Chem., 2012, 10, 7912–7921
This journal is © The Royal Society of Chemistry 2012

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NCI-60 anti-cancer screening
Tested compounds were initially solubilised in DMSO, diluted
into RPMI 1640 and 5% fetal bovine serum/L-glutamine, and
added to 96-well plates containing cell lines previously cultured
for 24 hours. After 48-hour incubation, the media were removed,
and the cells were fixed and stained with sulforhodamine B to
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Acknowledgements
The authors would like to acknowledge the Higher Education
Authority of Ireland under the auspices of PRTLI4 for funding
this research and also the National Cancer Institute (NCI) screen-
ing program for 60-cell line testing.
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This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7912–7921 | 7921