Synthesis and evaluation of novel ellipticines as potential anti-cancer agents

Article abstract of DOI:10.1039/c2ob27186a

Drugs that inhibit DNA topoisomerase I and DNA topoisomerase II have been widely used in cancer chemotherapy. We report herein the results of a focused medicinal chemistry effort around novel ellipticinium salts which target topoisomerase I and II enzymes with improved solubility. The salts were prepared by reaction of ellipticine with the required alkyl halide and evaluated for DNA intercalation, topoisomerase inhibition and growth inhibition against 12 cancer cell lines. Results from the topoisomerase I relaxation assay indicated that all novel ellipticine derivatives behaved as intercalating agents. At a concentration of 100 μM, specific topoisomerase I inhibition was not observed. Two of the derivatives under investigation were found to fully inhibit the DNA decatenation reaction at a concentration of 100 μM, indicative of topoisomerase II inhibition. N-Alkylation of ellipticine was found to enhance the observed growth inhibition across all cell lines and induce growth inhibition comparable to that of Irinotecan (CPT-11; GI₅₀ 1-18 μM) and in some cell lines better than Etoposide (VP-16; GI₅₀ = 0.04-5.2 μM). 6-Methylellipticine was the most potent growth inhibitory compound assessed (GI₅₀ = 0.47-0.9 μM). N-Alkylation of 6-methylellipticine was found to reduce this response with GI₅₀ values in the range of 1.3-28 μM.

Full text of DOI:10.1039/c2ob27186a

Organic &
Biomolecular Chemistry
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Synthesis and evaluation of novel ellipticines as
potential anti-cancer agents†
Cite this: Org. Biomol. Chem., 2013, 11,
1334
Fiona M. Deane,a Elaine C. O’Sullivan,a Anita R. Maguire,b Jayne Gilbert,c
Jennette A. Sakoff,c,d Adam McCluskey*d and Florence O. McCarthy*a
Drugs that inhibit DNA topoisomerase I and DNA topoisomerase II have been widely used in cancer
chemotherapy. We report herein the results of a focused medicinal chemistry effort around novel
ellipticinium salts which target topoisomerase I and II enzymes with improved solubility. The salts were
prepared by reaction of ellipticine with the required alkyl halide and evaluated for DNA intercalation,
topoisomerase inhibition and growth inhibition against 12 cancer cell lines. Results from the topoiso-
merase I relaxation assay indicated that all novel ellipticine derivatives behaved as intercalating agents. At
a concentration of 100 μM, specific topoisomerase I inhibition was not observed. Two of the derivatives
under investigation were found to fully inhibit the DNA decatenation reaction at a concentration of
100 μM, indicative of topoisomerase II inhibition. N-Alkylation of ellipticine was found to enhance
the observed growth inhibition across all cell lines and induce growth inhibition comparable to that of
Received 9th November 2012,
Accepted 3rd January 2013
Irinotecan (CPT-11; GI50 1–18 μM) and in some cell lines better than Etoposide (VP-16; GI50
0.04–5.2 μM). 6-Methylellipticine was the most potent growth inhibitory compound assessed (GI50
=
=
DOI: 10.1039/c2ob27186a
0.47–0.9 μM). N-Alkylation of 6-methylellipticine was found to reduce this response with GI50 values in
the range of 1.3–28 μM.
www.rsc.org/obc
group attached to the scissile strand on the phosphotyrosine
linkage to rejoin the DNA strand and eliminate the topo
Introduction
In order to manage the compact and complex supercoiled enzyme. Those enzymes that cleave only one strand of DNA are
nature of DNA, cells have enzymes known as topoisomerases defined as topo I enzymes whereas topos that cleave both
(topos). By introducing transient strand breaks in the DNA, strands to generate staggered double-strand breaks are defined
these ubiquitous enzymes alleviate topological problems gen- as topo II enzymes.1–4 Regarding both topo I and topo II
erated during fundamental cellular processes such as replica- enzymes, the end result is similar, the reaction has produced a
tion, transcription, recombination and repair. DNA strand DNA molecule which is chemically unchanged and covalently
breaks are created when an activated tyrosine residue attacks a closed, but in a different topology.
phosphodiester group on the backbone of DNA to form a new
Given that the inherent functions of topo enzymes are
phosphotyrosine linkage. DNA topology is modified during the crucial for ensuing genomic integrity, the ability to interfere
lifetime of this covalent intermediate and subsequent DNA with these enzymes or generate enzyme-mediated DNA
religation occurs by nucleophilic attack of the free hydroxyl damage is an effective target for cancer therapy.5 In this
respect, DNA topo I and topo II inhibitors represent a class of
anti-cancer agents forming the basis of many chemotherapy
combinations widely used in a broad spectrum of tumours.6,7
Topo inhibitors can be classified as either poisons or suppres-
sors based on the manner in which they interfere with these
enzymes. Poisons stabilize the transient phosphotyrosine
covalent bond between the enzyme and DNA thus converting it
into a permanent feature. Suppressors prevent the formation
of such a complex.3,4
aDepartment of Chemistry and Analytical and Biological Chemistry Research Facility,
University College Cork, Cork, Ireland. E-mail: f.mccarthy@ucc.ie;
Fax: +353 214901656; Tel: +353 214901695
bDepartment of Chemistry and School of Pharmacy, Analytical and Biological
Chemistry Research Facility, University College Cork, Cork, Ireland
cDepartment of Medical Oncology, Calvary Mater Newcastle Hospital, Edith Street,
Waratah, NSW 2298, Australia
dCentre for Chemical Biology, Chemistry, School of Environmental and Life Science,
The University of Newcastle, University Drive, Callaghan NSW 2308, Australia.
E-mail: Adam.McCluskey@newcastle.edu.au; Fax: +61 249215472;
Tel: +61 249216486
The natural product ellipticine (5,11-dimethyl-6H-pyrido-
[4,3-b]carbazole, 1) has engendered interest due to the
reported anticancer activity mediated by its ability to inhibit
topo II. Like many anti-cancer agents, ellipticine (1) exhibits a
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c2ob27186a
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Fig. 1 Chemical structures of ellipticine (1), Celiptium 2 and 6-methylellipticine
3.
multi-modal mechanism of action with DNA intercalation and
topo II inhibition.8 Bio-oxidation, adduct formation, mito-
chondrial membrane modulation and inhibition of p53
tumour suppressor have been the subject of much research in
the past decade and in addition ellipticines have been uncov-
ered as kinase inhibitors against c-Kit and AKT kinase.9 To
date, the efficacy of ellipticines as potential therapeutic agents
has been hindered by poor aqueous solubility. Endeavours to
address this limitation identified the formation of an elliptici-
nium salt (by quaternisation at N-2) as a modification that led
to improved aqueous solubility and increased anti-tumour
activity. The most clinically successful ellipticinium salt to
date is 9-hydroxy-N-methylellipticinium acetate (Celiptium, 2),
a drug for the treatment of metastatic breast cancer, myelo-
blastic leukemia and some solid tumors (Fig. 1).10,11
Multi-target inhibition is an attractive target in cancer
chemotherapy as it blocks >1 biological targets crucial to cell
cycle integrity. This leads to the possibility of a more effica-
cious treatment allied with the possibility of synergistic effects
for the treatment of cancer. The present study describes the
synthesis and DNA binding affinity, topo I and II inhibition
and cytotoxicity against a panel of cancer cell lines: HT29 and
SW480 (colon), MCF-7 (breast), A2780 (ovarian), H460 (lung),
A431 (skin), DU145 (prostate), BE2-C (neuroblastoma), SJ-G2,
SMA and U87 (glioblastoma), MIA (pancreas) of a series of
ellipticinium and 6-methylellipticinium salts.
Scheme 1 Reagents and conditions. (i) R2-Br, DMF, 120 °C for 4 h, r.t. for 12 h.
Fig. 2 Topo I unwinding assay. Influence of ellipticine (1), 6-methylellipticine 3
and ellipticinium salts 12–23 on the relaxation of plasmid DNA by topoisome-
rase (topo) I. Supercoiled DNA (SC DNA) was incubated without or with human
topo I in the absence and presence of the test compounds which were all ana-
lysed at a final concentration of 100 μM. Control inhibitor camptothecin (CPT)
was used at a final concentration of 200 μM. DNA samples were separated on
agarose gel without ethidium bromide. Lanes 1, 12–17 and 3, 18–23 contain
selected compounds 1, 12–17 and 3, 18–23 respectively, SC DNA and topo I. A
= SC DNA; B = relaxed DNA; C = SC DNA and topo I; D = SC DNA, topo I and
CPT.
Four of the required alkyl bromides: 3-bromopropionic
acid, 5-bromopentanoic acid, 6-bromohexanoic acid and
6-bromohexanenitrile were commercially available. 6-Bromo-
hexanamide (24) and 6-bromo-N-(methylsulfonyl)hexanamide
(25) were synthesized via 6-bromohexanoyl chloride which was
accessed from the corresponding carboxylic acid, 6-bromo-
hexanoic acid, on reaction with oxalyl chloride. Dropwise
addition of the acid chloride to stirring concentrated aqueous
ammonia afforded 6-bromohexanamide (24). 6-Bromo-N-
(methylsulfonyl)hexanamide (25) was generated by reaction of
the aforementioned acid chloride with methanesulfonamide at
90 °C for 1 hour. Coupling of these alkyl bromides as shown in
Scheme 1 with ellipticine (1) and 6-methylellipticine 3 gave
rise to two focused compound libraries of ellipticinium salts
12–17 and 6-methylellipticinium salts 18–23.
Results and discussion
The synthesis of ellipticine (1) and 6-methylellipticine 3 was
accomplished in either seven or eight steps with overall yields
of 11–14% using a modified Cranwell Saxton procedure (see
ESI† for details).12–14
With sufficient quantities of 6-methylellipticine 3 and
ellipticine (1) in hand, we next turned our attention to two
focused libraries of ellipticinium salts. The ellipticinium salts
were prepared by heating ellipticine (1) or 6-methylellipticine 3
with an excess of alkyl bromide in DMF (Scheme 1).
DNA intercalation
The alkyl bromides were selected for reaction with parent
ellipticines based on a number of important merits; (i) the We first examined DNA intercalation status of the ellipticinium
polar terminal functional groups would serve to alleviate solu- salts based on 1 (Scheme 1, 12–17) for their ability to affect
bility issues known with ellipticines; and (ii) the ability of the topo I activity in a topo I unwinding assay (Fig. 2). Negatively
functional group at N-2 to probe the drug DNA topoisomerase supercoiled plasmid DNA (SC DNA) was incubated with topo I
ternary complex.
in the presence or absence of the selected compounds. The
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DNA samples were treated with SDS and proteinase K to
remove any covalently bound protein and were resolved in a
1% agarose gel without ethidium bromide. The topo I unwind-
ing assay primarily investigates the DNA binding affinities of
the drug in question. Briefly, intercalators will cause re-
supercoiling of plasmid DNA after initial relaxation by topo I
and subsequent removal of the compound and enzyme. Re-
supercoiling is due to the change in the linking number that
accompanies relaxation by the enzyme. Conversely minor
groove binders should not induce DNA re-supercoiling due to
negligible DNA unwinding and binding.15–17 As detailed in
Fig. 2, SC DNA (Lane A) migrates faster than the relaxed DNA
(Lane B). The disappearance of SC DNA is indicative of the
complete conversion of SC DNA to relaxed DNA by the action
of topo I (Lane C), however in this case the topoisomers of
relaxed DNA have not fully resolved. While camptothecin
(CPT) is a known topo I inhibitor it is not an intercalating
molecule and therefore failed to show re-supercoiling in this
assay (Lane D).18
As anticipated, ellipticine (1) behaved as a typical intercala-
tor and displayed non-specific inhibition (suppression) of topo
I giving rise to a band consistent with SC DNA.19 Ellipticine
salts 12, 13, 14 and 17 gave rise to the same DNA band as 1
suggesting that they all intercalated with DNA and altered the
linking number leading to re-SC DNA. A different pattern was
observed for ellipticine salts 15 and 16, as the DNA bands
appeared in between the supercoiled DNA band and the
relaxed DNA band. This would suggest that these compounds
are again intercalators of DNA but the re-supercoiling of the
DNA (after the intercalator has locally unwound DNA) has
been followed by a change in plasmid mobility (Fig. 2).
On examination of the data obtained for 6-methylellipticine
3 and 6-methylellipticinium salts 18–23, it was clear that the
ellipticinium salts behaved in a similar manner to the parent
3. All of the analogues were intercalators and caused re-
supercoiling of plasmid DNA after initial relaxation by topo I
and subsequent removal of compound and enzyme. However,
compounds 21 and 22 caused a significant shift in plasmid
mobility after re-supercoiling of the DNA plasmid. This behav-
iour mirrors that observed with ellipticinium salts 15 and 16,
which may suggest an increased level of DNA binding for these
analogues. All four analogues display enhanced DNA binding
most probably due to the terminal amide and nitrile moiety
favourably interacting with DNA. This may have occurred via
interactions within the DNA minor or major groove.
Fig. 3 Topo I cleavage assay. Influence of ellipticine (1), ellipticinium salts
12–17; 6-methylellipticine 3, and 6-methylellipticinium salts 18–23 on the relax-
ation of plasmid DNA by topoisomerase (topo) I. Conditions were identical to
the topo I unwinding assay (Fig. 2) with the exception that DNA samples were
separated on a gel containing ethidium bromide (0.5 μg mL−1). Supercoiled
DNA (SC DNA) was incubated without or with human topo I in the absence and
presence of the test compounds which were all analysed at a final concentration
of 100 μM. Control inhibitor camptothecin (CPT) was used at a final concen-
tration of 200 μM. Gel lanes are identified by the compound number at the top
of each gel. Gel flow was from top to bottom. A = SC DNA; B = relaxed DNA; C
= SC DNA and topo I; D = SC DNA, topo I and CPT.
actively consumes the enzyme leading to stoichiometric com-
plexes between topo I and DNA.20 From Fig. 3, it can be seen
that, while CPT has relaxed DNA, there is also a significant
amount of nicked DNA present (Lane D). The same pattern
would be observed if any of the compounds being examined
possessed the ability to poison topo I. In order to better ident-
ify cleaved DNA from relaxed or SC DNA the gels have been
prestained with ethidium bromide. The ethidium bromide
induced-DNA unwinding effects also causes relaxed DNA to co-
migrate or migrate faster than SC DNA.
The effects of the ellipticinium and 6-methylellipticinium
libraries on the cleavage of plasmid DNA by topo I was exami-
ned. Elution of the DNA bands, as before, highlighted a clear
plasmid mobility shift, which reflected the DNA binding prop-
erties of the analogues evaluated (Fig. 3). The observed electro-
phoretic mobility shift was attributed to a decrease in the
plasmid DNA linking number as a result of intercalation. Ana-
logue 22 was observed to create a strong shift in the mobility
of the DNA band, which was subsequently observed to occur
in the same area as nicked DNA. This is most likely coinciden-
tal as a typical topo I poison would not cause complete conver-
sion of SC DNA to nicked DNA. Interestingly, ellipticinium salt
22 was also detected from the topo I unwinding assay to
strongly interact with DNA (Fig. 2). In order to fully assess the
ability of these compounds to bind to DNA, similar experi-
ments should be carried out in the absence of the enzyme.
None of the analogues examined (at 100 μM drug concen-
tration) elicited the formation of a second DNA band that
corresponded to nicked DNA. This is consistent with the ellip-
ticines acting as non-specific inhibitors rather than topo I
poisons. These analogues bind DNA strongly but display no
obvious specific topo I inhibition at the concentrations evalu-
ated. However, the nitrile and amide salts merit further investi-
gation to identify the specific nature of interaction with DNA.
Topoisomerase I cleavage assay
To establish whether 1, 3 and 12–23 act as topo poisons and
specifically inhibit topo I, a topo I cleavage assay was utilized
(Fig. 3). Topo I relaxes SC DNA by introducing transient nicks
in the DNA substrate. Topo I poisons such as CPT have the
ability to stabilize the intermediate and lead to an increase in
the nicked DNA product. The cleavage reaction is usually a low
Topoisomerase II decatenation assay
yielding event and for this reason the cleavage product will The inhibitory effects of these compounds against human
generally only be weakly detected, even with well established topo II catalytic activity was measured by a kinetoplast DNA
poisons such as CPT. This is because the cleavage event (kDNA) decatenation assay (Fig. 4). Kinetoplast DNA, obtained
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to 50% that observed with an untreated control). The out-
comes from our antiproliferative screening protocol are pre-
sented in Table 1. The clinically approved topo I and II
inhibitors Irinotecan (CPT-11, an analogue of CPT) and
Etoposide (VP-16) respectively, were also included.
From the ellipticinium and 6-methylellipticinium libraries
two ellipticinium analogues, 13 and 16 and four 6-methyl-
ellipticinium analogues 19, 21–23 proceeded to full dose
response evaluation (Table 1). Ellipticine (1) and 6-methylellip-
ticine 3 together with CPT-11 and VP-16 were screened, and
Fig. 4 Topo II decatenation assay. Influence of ellipticine (1), ellipticinium salts
12–17; 6-methylellipticine 3, and 6-methylellipticinium salts 18–23 on DNA dec-
atenation. All of the compounds were tested at a 100 μM final concentration.
ATP was added to all reactions containing topo II and DNA samples were separ-
ated on a 1% agarose gel containing ethidium bromide (0.5 μg mL−1). Gel lanes the data presented for comparative purposes. As can be seen
are identified by the compound number at the top of each gel. Gel flow was
from the data presented in Table 1, ellipticine (1) did not
proceed to full dose response evaluation. Both 13 and 16 dis-
played considerably enhanced broad spectrum cytotoxicity
from top to bottom. A = catenated kDNA (Cat DNA); B = decatenated kDNA
(Decat DNA); C = linearized kDNA (Lin DNA); D = Cat DNA, ATP and topo II; E =
Cat DNA, ATP, topo II and ellipticine; F = Cat DNA, ATP, topo II and VP-16.
compared with 1 with average GI50 values of 2.0 and ≥9.7 μM
respectively. Ellipticinium salt 13 displayed modest selectivity
from the mitochondrial DNA of Crithidia fasciculate, is a
network consisting of thousands of interlocked closed circular
DNA molecules called ‘minicircles’. Because of its overall size,
catenated DNA cannot enter a typical agarose gel during elec-
trophoresis unless minicircles are previously released (Lane A).
A transient double strand break is the only way to take a mini-
circle out of the network and preserve its circular nature which
is accomplished through the decatenation activity of topo II
(Lane D).21 The compounds under evaluation were compared
with that of the standard topo II inhibitors ellipticine (1, Lane
E) and etoposide (VP-16), a highly specific non-intercalative
poison that stabilizes the cleavage complex (Lane F). As the
cleavage step is indeed reversible, etoposide does not greatly
affect the overall reaction.22 On the other hand, ellipticine (1),
while it modestly promotes the formation of more cleavage
intermediates, can strongly inhibit the topo II decatenation
reaction at the same time that it intercalates into the DNA
molecule.23 Compared with ellipticine (1), all the compounds
examined were found to retain potency and, at the very least,
partially inhibit the decatenation reaction. At a concentration
of 100 μM, compounds 21 and 22 were found to fully inhibit
the decatenation reaction. These salts were also found to
strongly bind to DNA in the topo I relaxation assay (Fig. 2).
towards the murine glioblastoma SMA cell line (GI50
=
0.67 0.12 μM) while ellipticinium 16 was inactive against
the neuroblastoma BE2-C cell line (GI50 > 50 μM). N-Alkylation
of ellipticine (1) in addition to enhancing aqueous solubility
also enhanced the observed cytotoxicity.
The 6-methylellipticinium salts were uniformly less potent
than their ellipticinium counterparts returning average GI50
values of 8.6, 12.1, 5.1 and 13.7 μM for analogues 19 and
21–23 respectively. These growth inhibition values compare
poorly with 3 (average GI50 = 0.60 μM). As was noted with 16,
these 6-methylellipticinium salts displayed reduced cyto-
toxicity against the neuroblastoma BE2-C cell line with GI50
values of 33, 16 and 14 μM for analogues 21–23. With the
6-methylellipticinium salts additional N-alkylation reduced
cytotoxicity. In terms of the average GI50 value across all cell
lines only 13, 3 and 22 showed enhanced growth inhibition
compared with the clinically approved topo I inhibitor CPT-11
(GI50 = 5.95 μM). Enhanced inhibition was also observed for
16, 19, 21 but only in selected cell lines. In comparison with
the topo II inhibitor VP-16, with an average growth inhibition
of 1.55 μM, only 3 showed enhanced antiproliferative qualities,
although 13, 16, and 22 did show slight enhancement in some
cell lines.
Antiproliferative activity
Having established that the ellipticinium and 6-methylelliptici-
nium libraries retained the DNA intercalating properties of the
Conclusion
parent ellipticine we next sought to determine the extent, if The library of compounds synthesized during this research
any, of their cytotoxicity towards a panel of cancer cell lines. In consists of novel ellipticinium salts with side chains at the N-2
our other anticancer drug development programs we have position to probe the drug DNA topoisomerase ternary
established a broad array of cancer cell lines that we routinely complex. Carboxylic acid functional groups of different chain
screen potential cytotoxic agents against viz: HT29, SW480 lengths were examined in order to determine which length
(colon); MCF-7 (breast); A2780 (ovarian); H460 (lung); A431 was most effective. The preliminary results obtained during
(skin); DU145 (prostate); BE2-C (neuroblastoma); SJ-G2, U87 biological testing did not exclusively show any preference
(glioblastoma); MIA (pancreas) and SMA (murine glioblas- between a propyl, butyl or pentyl group with respect to activity.
toma). Analogues were screened at a 25 μM initial drug testing Ellipticinium salts with side chains containing amides and
and those that pass our internal filter (growth inhibition nitriles, and sulphonacetamides, were also analyzed. It was
≥90% over 12 cell lines; unless unusual specificity was ident- observed that ellipticinium salts with terminal nitrile or amide
ified) progress to full dose response screening and GI50 deter- groups such as 15, 16, 21 and 22 were seen to display much
mination (the concentration of drug that inhibits cell growth stronger binding affinities for DNA than their carboxylic acid
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counterparts and this is somewhat correlated in the cyto-
toxicity data with 16, 21 and 22 progressing to full dose
evaluation.
The topoisomerase I inhibitory properties of the com-
pounds were initially examined using a DNA unwinding
assay. Like their parent ellipticines (1 and 3), all of the com-
pounds evaluated were found to bind to DNA via inter-
calation. Due to the high degree of binding observed
concerning many of the ellipticinium salts investigated it is
plausible that these compounds might function as topo I
suppressors. Topo I suppressors bind to DNA and prevent
the topo I enzyme from recognizing the site at which it acts
therefore suppressing the action of the enzyme. Ethoxidine
is one such example of a topo I suppressor and was shown
by Clark et al. to suppress the activities of topo I by inter-
calating with DNA.24
To establish whether these compounds functioned as
specific topo I inhibitors (topo I poisons), a topo I cleavage
assay was carried out. At a concentration of 100 μM, no sign
of topo I poisoning was observed. This however, does not
mean that the ellipticinium salts are inactive as topo I
poisons, but at a concentration of 100 μM, no specific
inhibition was observed. It is not uncommon to observe a
drug acting as a topo I suppressor at high concentrations
and a topo I poison at lower concentrations.25,26
On examination of the influence of selected compounds
1, 3 and 12–23, it could be seen that, at a concentration of
100 μM, all compounds partially inhibited the ability of
topo II to decatenate kDNA. Ellipticinium salts 21 and 22
were seen to completely inhibit the decatenation reaction.
Remarkably, there is good correlation of activity with a
recently reported series of isoellipticinium salts incorporat-
ing an isomeric pyridine nitrogen.27 This suggests
that there is some flexibility in the binding site and will
explored in detail in future work.
Results from the cytotoxicity screen across 12 different
cancer cell lines highlighted 6-methylellipticine 3 to be the
most potent with an average value of GI50 = 0.66 μM. Inter-
estingly, even though compounds 21 and 22 were found to
bind to DNA strongly via intercalation and completely
inhibit the decatenation reaction, they were less active than
6-methylellipticine 3 scoring average GI50 values of 12.0
and 5.1 μM respectively across cell lines. The fact that no
clear relationship existed between cytotoxicity and DNA
binding/topo II inhibition could be due to the involvements
of targets and mechanisms different from DNA binding
and topo II inhibition.
Experimental
Chemistry
General. Melting points were measured on a Uni-Melt
Thomas Hoover Capillary Melting Point apparatus and are
uncorrected. Low resolution mass spectra were recorded
on a Waters Micromass Quattro Micro mass spectrometer
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(Instrument number QAA1202) in electrospray ionization (ESI) (pH 7.9), 10 mM EDTA, 1.5 M NaCl, 1% bovine serum
positive and negative modes. High resolution mass spectra albumin, 1 mM spermidine and 50% glycerol. To the solution
(HRMS) were recorded on a Waters Micromass LCT Premier was added the drug to be tested (3 μL, 100 μM) and the reac-
mass spectrometer (Instrument number KD 160) in electro- tion was initiated by addition of recombinant human topo I
spray ionization (ESI) positive or negative modes. Infrared (IR) (8 U) (Inspiralis, Norwich UK). Immediately after the enzyme
spectra were recorded as thin films on sodium chloride plates, was added, the reactions were incubated at 37 °C for
as potassium bromide (KBr) discs for solids or as solution 30 minutes and terminated by the addition of 5× stop buffer
spectra in deuterated chloroform on a Perkin-Elmer FT-IR containing 5% sarkosyl, 0.125% bromophenol blue, 25%
Paragon 1000 or a Spectrum One FT-IR spectrophotometer. glycerol. Proteinase K was added to a concentration of 50 μg
1H (300 MHz) and 13C NMR (75 MHz) were recorded on a mL−1 and the reactions were incubated at 37 °C for another
Bruker Avance 300 NMR spectrometer, All spectra were 30 minutes. The reactions were subsequently vortexed and
recorded at 20 °C in deuterated dimethylsulphoxide (DMSO) microcentrifuged briefly and the blue aqueous layer was
with tetramethylsilane (TMS) as an internal standard unless loaded onto a 1% agarose gel containing either 1× TBE buffer
otherwise specified. Chemical shifts (δH and δC) are reported or 1× TAE buffer. Gels were electrophoresed at 2 V cm−1 for
in parts per million (ppm), relative to TMS and coupling con- 2.5 hours, stained with ethidium bromide (0.5 μg mL−1) for
stants are expressed in Hertz (Hz). Splitting patterns in 30 minutes at room temperature, destained with distilled
1H NMR spectra are designated as s (singlet), br s (broad water for 10 minutes and visualized using a UV transillumina-
singlet), d (doublet), dd (doublet of doublets), dt (doublet of tor (DNR bio-imaging system).
triplets), ddt (doublet of doublets of triplets), t (triplet), tt
Topoisomerase I cleavage assay. Reactions were assembled
(triplet of triplets), q (quartet) and m (multiplet). The Micro- in microcentrifuge tubes on ice and were made up to a 30 μL
analysis Laboratory, University College Cork, preformed final volume. Supercoiled pBR322 DNA (0.25 μg) (TogoGEN) in
elemental analysis using a Perkin-Elmer 240 and Exeter TE buffer [TE: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA] was
Analytical CE440 elemental analysers. Column chromato- added to a solution containing water (variable volume) and
graphy was carried out using Kieselgel 60, 0.040–0.063 nm 10× cleavage buffer (3 μL) containing 10 mM Tris-HCl (pH
(Merck).
7.9), 1 mM EDTA, 0.15 M NaCl, 0.1% bovine serum albumin,
The synthetic routes to ellipticine (1) and 6-methylellipti- 0.1 mM spermidine and 5% glycerol. To the solution was
cine 3 have been described previously.14
added the drug to be tested (3 μL, 100 μM) and the reaction
was initiated by addition of recombinant human topo I (8 U)
(Inspiralis, Norwich UK). Immediately after the enzyme was
Biology
Drugs and chemicals. Ellipticine, ellipticine derivatives and added, the reactions were incubated at 37 °C for 30 minutes
ellipticinium salts were dissolved in DMSO and water (1 : 1) at and terminated by the addition of sodium dodecyl sulphate
2 mM and then further diluted with water. The final concen- (10%, 3 μL). Proteinase K was added to a concentration of
tration of DMSO never exceeded 3% (v/v) in reactions. Under 50 μg mL−1 and the reactions were incubated at 37 °C for
these conditions DMSO was also used in the controls and was another 30 minutes. Gel loading buffer (10×) (3 μL, 0.25%
not seen to affect topoisomerase I or topoisomerase II activity. bromophenol blue, 50% glycerol) was added to the reaction
In the case of 6-methylellipticine 3, the compound was dis- mixture along with CIA (30 μL, chloroform : isoamyl alcohol
solved initially in a solution containing DMSO (20%), metha- 24 : 1). The reactions were vortexed and microcentrifuged
nol (20%), acetonitrile (20%) and water (40%) at a 2 mM briefly and the blue aqueous layer was loaded onto a 1%
concentration. The final concentration of the combination of agarose gel containing 1× TAE buffer and ethidium bromide
DMSO, methanol and acetonitrile in reactions did not exceed (0.5 μg mL−1). Gels were electrophoresed at 3 V cm−1 for
0.9% (each solvent contributing 0.3%). Under these conditions 2 hours and visualized using a UV transilluminator (DNR bio-
controls containing this solvent mixture were not seen to affect imaging system).
topo I or topo II. Ellipticine Stock solutions of 2 mM and
Topoisomerase II decatenation assay. Reactions were
1 mM concentrations were stored at room temperature in the assembled in microcentrifuge tubes on ice and were made up
dark for up to 2 months without any loss of activity (although to a 30 μL final volume. To a solution containing water and
trace precipitation was seen with 3. Control drugs used in the buffer (10× stock contains: 50 mM Tris-HCl at a pH of 7.5,
experiments such as camptothecin (CPT), irinotecan (CPT-11) 125 mM NaCl, 10 mM MgCl2, 5 mM DTT, 100 μg mL−1
and etoposide (VP-16) were purchased from Sigma (Dorset, albumin) was added 0.2 μg Kinetoplast DNA (topoGEN). ATP
UK). Stock solutions of each were dissolved in DMSO and was added to a final concentration of 1 mM along with tested
stored at −20 °C.
the drug to be tested (3 μL, 100 μM) and the reactions were
Topoisomerase I relaxation assay. Reactions were assembled initiated by addition of 3 U topo II (inspiralis). Immediately
in microcentrifuge tubes on ice and were made up to a 30 μL after the enzyme was added, the reactions were incubated at
final volume. Supercoiled pBR322 DNA (0.25 μg) (TogoGEN) in 37 °C for 30 minutes and terminated by the addition of 5×
TE buffer [TE: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA] was stop loading dye (5% sarkosyl, 0.125% bromophenol blue,
added to a solution containing water (variable volume) and 25% glycerol). Proteinase K was added to give a final concen-
10× topo I assay buffer (3 μL) containing 100 mM Tris-HCl tration of 50 μg mL−1 and the reactions were incubated at
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37 °C for another 30 minutes. The reactions were loaded onto
a 1% agarose gel containing 1× TAE buffer and ethidium 1400;
bromide (0.5 μg mL−1). Gels were electrophoresed at 4 V cm−1
νmax/cm−1 (KBr) 3429, 3154, 2963, 2912, 1728, 1602, 1464,
δH (300 MHz, d6-DMSO) 2.78 [3H, s, C(5)CH3], 3.18 [2H, t,
for 3 hours and visualized using a UV transilluminator (DNR J 7.0, C(2′)H2], 3.24 [3H, s, C(11)CH3], 4.93 [2H, t, J 6.9,
bio-imaging system). C(1′)H2], 7.35–7.45 [1H, m, C(9)H], 7.62–7.73 [2H, m, C(7)H,
Growth inhibition assay. All cell lines were maintained in C(8)H], 8.45–8.52 [2H, m, C(4)H, C(10)H], 8.57 [1H, d, J 7.3,
Dulbecco’s Modified Eagle’s media (DMEM) supplemented C(3)H], 10.14 [1H, s, C(1)H], 12.10 (1H, s, OH), 15.01 (1H, s,
with 10% foetal bovine serum and 5% penicillin/streptomycin, NH);
at 37 °C in a humidified 5% CO2 atmosphere. Growth inhi-
δC (75 MHz, d6-DMSO) 12.0 [CH3, C(5)CH3], 14.9 [CH3,
bition was determined by plating cells in duplicate in medium C(11)CH3], 34.9 [CH2, C(2′)H2], 55.1 [CH2, C(1′)H2], 110.2
(100 μL) at a density of 2500–4000 cells per well in a 96 well (C, aromatic C), 111.5 (CH, aromatic CH), 120.1 (C, aromatic
plate. On day 0 (24 h after plating), when the cells were in log- C), 120.6 (CH, aromatic CH), 120.7 (CH, aromatic CH), 122.0
arithmic growth, medium (100 μL) with or without the test (C, aromatic C), 124.3 (CH, aromatic CH), 125.7 (C, aromatic
agent was added to each well. After 72 h of drug exposure, C), 128.5 (CH, aromatic CH), 130.9 (CH, aromatic CH), 132.4
growth inhibitory effects were evaluated using the MTT (3-(4,5- (C, aromatic C), 133.2 (C, aromatic C), 142.5 (C, aromatic C),
dimethyltiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay 144.3 (C, aromatic C), 147.0 (CH, aromatic CH), 171.7 (C,
and their absorbance was read at 540 nm. Percentage growth CvO);
inhibition was determined at a fixed drug concentration of
25 μM. A value of 100% is indicative of total cell growth inhi-
m/z (ES+) 319 (M)+, (100%), 247 (95), 152 (4);
HRMS (ES+) Exact mass calculated for C20H19N2O2 (M)+,
bition. Those agents inducing appreciable percentage growth 319.1447. Found 319.1436.
inhibition underwent further dose response analysis to enable
the calculation of GI50 values. The GI50 value is defined as the
drug concentration at which cell growth is inhibited by 50%
N2-(4′-Carboxybutyl)-ellipticinium bromide (13)
based on the difference between the optical density values on Employing the procedure described, ellipticine (1, 155 mg,
day 0 and those at the end of drug exposure.
0.628 mmol) and 5-bromovaleric acid (227 mg, 1.260 mmol)
* Caution – Ethidium Bromide is a potent mutagen and were reacted in DMF. The isolated brown solid was seen to
was handled with necessary care during the course of this convert into a gummy oil on the filter paper and was sub-
research.
sequently dissolved in methanol–water (1 : 1, 30 mL) and evap-
** MiniBIS Pro in conjunction with GelCapture image orated under reduced pressure to afford a green/brown oil.
acquisition software, both from DNR Bio-Imaging Systems Ltd. Chloroform (20 mL) was added to the oil resulting in the pre-
cipitation of a solid which was determined by TLC analysis to
be still contaminated with starting material 1. The brown
residue was washed repeatedly with chloroform and analysed
by TLC until no starting material 1 was found to remain
(45.4 mg, 17%): m.p. > 300 °C.
General procedure for the synthesis of ellipticinium and
6-methylellipticinium salts
A stirring solution of the appropriate ellipticine (1 mmol,
1 eq.) and the required alkyl bromide (2 mmol, 2 eq.) in DMF
(5 mL) was heated at 120 °C for 4 hours, followed by stirring at
room temperature overnight. Cold anhydrous diethyl ether
(10 mL) was added dropwise to the reaction mixture resulting
in the precipitation of a residue which was isolated by
filtration to afford a solid.
νmax/cm−1 (KBr) 3435, 3154, 2913, 2841, 1698, 1599, 1419,
1241;
δH (300 MHz, d6-DMSO) 1.51–1.63 [2H, m, C(3′)H2],
2.00–2.07 [2H, m, C(2′)H2], 2.34 [2H, t, J 7.3, C(4′)H2], 2.82 [3H,
s, C(5)CH3], 3.27 [3H, s, C(11)CH3], 4.74 [2H, t, J 7.1, C(1′)H2],
7.33–7.42 [1H, m, C(9)H], 7.61–7.68 [2H, m, C(7)H, C(8)H],
8.36–8.47 {2H, m, including 8.40 [1H, d, J 8.0, C(10)H], C(4)H},
8.53 [1H, d, J 7.3, C(3)H], 9.92 (1H, br s, OH), 10.07 [1H, s,
N2-(2′-Carboxyethyl)-ellipticinium bromide (12)
Employing the procedure described above, ellipticine (1, C(1)H], 12.08 (1H, s, NH);
150 mg, 0.609 mmol) and 3–bromopropionic acid (186 mg,
δC (75 MHz, d6-DMSO) 12.4 [CH3, C(5)CH3], 15.3 [CH3,
1.220 mmol) were reacted in DMF. Following isolation by fil- C(11)CH3], 21.5 [CH2, C(3′)H2], 30.7 [CH2, C(4′)H2], 33.3 [CH2,
tration, TLC and LCMS analysis determined that the product C(2′)H2], 59.4 [CH2, C(1′)H2], 110.0 (C, aromatic C), 111.5 (CH,
was contaminated with unreacted starting material 1 (starting aromatic CH), 120.1 (C, aromatic C), 120.7 (CH, aromatic CH),
material 1–product 12, 0.6 : 1.0). The solid was transferred to a 120.8 (CH, aromatic CH), 122.1 (C, aromatic C), 124.4 (CH,
conical flask containing a mixture of ether and dichloro- aromatic CH), 125.6 (C, aromatic C), 128.5 (CH, aromatic CH),
methane (1 : 1, 100 mL) and the solution was heated gently 130.8 (CH, aromatic CH), 133.2 (C, aromatic C), 133.8 (C,
while stirring. A mixture of dichloromethane–methanol (4 : 1, aromatic C), 142.5 (C, aromatic C), 144.1 (C, aromatic C), 146.4
1 mL) was also added and the suspension was filtered to yield (CH, aromatic CH), 174.5 (C, CvO);
a mixture of ellipticine 1 and N2-(2′-carboxyethyl)-ellipticinium
bromide 12 as bright yellow solid (starting material
m/z (ES+) 347 (M)+, (100%), 247 (20);
HRMS (ES+) Exact mass calculated for C22H23N2O2 (M)+,
347.1760. Found 347.1767.
a
1–product 12, 0.11 : 1.0), (116 mg):
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N2-(5′-Carboxypentyl)-ellipticinium bromide (14)
m/z (ES+) 360 (M)+, (100%), 193 (2), 146 (2);
HRMS (ES+) Exact mass calculated for C23H26N3O (M)+,
360.2076. Found 360.2071.
Employing the procedure described, ellipticine (1, 50 mg,
0.203 mmol) and 6-bromohexanoic acid (75 mg, 0.380 mmol)
were reacted in DMF and the resulting yellow precipitate was
filtered to afford N2-(5′-carboxypentyl)-ellipticinium bromide
14 as a yellow solid (54 mg, 60%): m.p. >300 °C.
N2-(5′-Cyanopentyl)-ellipticinium bromide (16)
Using the procedure described, compound 16 was obtained
from 1 (150 mg, 0.610 mmol) and 6-bromohexanenitrile
(215 mg, 1.220 mmol) to afford a lime green solid (151 mg,
59%): m.p. 290–291 °C.
νmax/cm−1 (KBr) 3436, 3056, 2942, 2864, 1695, 1602, 1464,
1422, 1403;
δH (300 MHz, d6-DMSO) 1.30–1.43 [2H, m, C(3′)H2],
1.54–1.65 [2H, m, C(4′)H2], 1.99–2.09 [2H, m, C(2′)H2], 2.25
[2H, t, J 7.3, C(5′)H2], 2.87 [3H, s, C(5)CH3], 3.34 [3H, s,
C(11)CH3], 4.72 [2H, t, J 7.4, C(1′)H2], 7.41 [1H, ddd, J 8.1, 6.2,
2.1, C(9)H], 7.63–7.72 [2H, m, C(7)H, C(8)H], 8.48 [1H, d, J 8.0
C(10)H], 8.49 [1H, d, J 7.2, C(4)H], 8.57 [1H, d, J 7.3, C(3)H],
10.01 [1H, s, C(1)H], 12.01 (1H, br s, OH or NH), 12.19 (1H, s,
OH or NH);
νmax/cm−1 (KBr) 3413, 3088, 2944, 2863, 2240, 1602, 1422;
δH (300 MHz, d6-DMSO) 1.41–1.54 [2H, m, C(3′)H2],
1.60–1.73 [2H, m, C(4′)H2], 1.99–2.14 [2H, m, C(2′)H2], 2.55
[2H, t, J 7.0, C(5′)H2], 2.82 [3H, s, C(5)CH3], 3.32 [3H, s, C(11)-
CH3], 4.74 [2H, t, J 7.4, C(1′)H2], 7.40 [1H, ddd, J 7.9, 6.0, 2.3,
C(9)H], 7.62–7.72 [2H, m, C(7)H, C(8)H,], 8.46 [1H, d, J 7.9,
C(10)H], 8.48 [1H, d, J 7.2, C(4)H], 8.56 [1H, d, J 7.3, C(3)H],
10.11 [1H, s, C(1)H], 12.18 (1H, s, NH);
δC (75 MHz, d6-DMSO) 12.1 [CH3, C(5)CH3], 15.1 [CH3,
C(11)CH3], 23.9 [CH2, C(3′)H2], 25.2 [CH2, C(4′)H2], 30.6 [CH2,
C(2′)H2], 33.4 [CH2, C(5′)H2], 59.3 [CH2, C(1′)H2], 110.4 (C, aro-
matic C), 111.6 (CH, aromatic CH), 120.2 (C, aromatic C),
120.5 (CH, aromatic CH), 120.8 (CH, aromatic CH), 122.2
(C, aromatic C), 124.5 (CH, aromatic CH), 126.0 (C, aromatic
C), 128.7 (CH, aromatic CH), 130.8 (CH, aromatic CH), 132.5
(C, aromatic C), 133.4 (C, aromatic C), 142.6 (C, aromatic C),
144.4 (C, aromatic C), 146.6 (CH, aromatic CH), 174.3
(C, CvO);
δC (75 MHz, d6-DMSO) 12.0 [CH3, C(5)CH3], 15.1 [CH3,
C(11)CH3], 16.0 [CH2, C(5′)H2], 24.2 [CH2, C(4′)H2], 24.7 [CH2,
C(3′)H2], 30.0 [CH2, C(2′)H2], 59.0 [CH2, C(1′)H2], 110.4 (C,
aromatic C), 111.6 (CH, aromatic CH), 120.2 (C, aromatic C),
120.4 (CH, aromatic CH), 120.6 (C, CN nitrile), 120.7 (CH,
aromatic CH), 122.2 (C, aromatic C), 124.4 (CH, aromatic CH),
126.0 (C, aromatic C), 128.7 (CH, aromatic CH), 130.8 (CH,
aromatic CH), 132.5 (C, aromatic C), 133.3 (C, aromatic C),
142.6 (C, aromatic C), 144.3 (C, aromatic C), 146.5 (CH,
aromatic CH);
m/z (ES+) 361 (M)+ (100%), 247 (5%).
HRMS (ES+) Exact mass calculated for C23H25N2O2 (M)+,
361.1916. Found 361.1899.
m/z (ES+) 342 (M)+, (100%), 146 (10), 105 (90);
HRMS (ESI) Exact mass calculated for C23H24N3 (M)+,
342.1970. Found 342.1980.
Found C, 64.18; H, 5.62; N, 9.64; C23H24BrN3·12H2O requires
C, 64.04; H, 5.84; N, 9.74%.
N2-(5′-Carboxyaminopentyl)-ellipticinium bromide (15)
Using the procedure described, compound 15 was obtained
as a mustard yellow solid from reaction of 1 (152 mg,
0.618 mmol) and 6-bromohexanamide (24, 239 mg,
1.230 mmol) (180 mg, 66%): m.p. 281–282 °C.
N2-(5′-Hexanoylmethylsulphonamide)-ellipticinium
bromide (17)
νmax/cm−1 (KBr) 3327, 3140, 3055, 2929, 2859, 1682, 1598, Using the procedure described, compound 17 was obtained
1597, 1423;
from 1 (33 mg, 0.134 mmol) and N-methanesulphonyl-
δH (300 MHz, d6-DMSO) 1.27–1.43 [2H, m, C(3′)H2], 6-bromohexanamide (25, 73 mg, 0.268 mmol). Upon filtration,
1.52–1.65 [2H, m, C(4′)H2], 1.95–2.13 {4H, m, including 2.08 the isolated solid was seen to convert into a gummy black
[2H, t, J 7.2, C(5′)H2], C(2′)H2}, 2.83 [3H, s, C(5)CH3], 3.30 residue which was subsequently dissolved in a mixture of
[3H, s, C(11)CH3], 4.72 [2H, t, J 7.4, C(1′)H2], 6.71 [1H, br s, dichloromethane–methanol (1 : 1, 10 mL) and evaporated
one of CONH2], 7.26 [1H, br s, one of CONH2], 7.38 [1H, ddd, under reduced pressure to afford a dark yellow oil. Acetone
J 7.9, 5.8, 2.5, C(9)H], 7.60–7.69 [2H, m, C(7)H, C(8)H], 8.42 (10 mL) was added resulting in the precipitation of a solid,
[1H, d, J 7.8, C(10)H], 8.44 [1H, d, J 7.0, C(4)H], 8.54 [1H, d, which was stored in a freezer (−20 °C) for 4 hours and then
J 7.2 C(3)H], 10.08 [1H, s, C(1)H], 12.15 (1H, s, NH);
isolated by suction filtration to afford 17 (36 mg, 52%) as
δC (75 MHz, d6–DMSO) 12.0 [CH3, C(5)CH3], 15.1 [CH3, yellow crystals (starting material 1–product 17, 0.1 : 1.0): m.
C(11)CH3], 24.4 [CH2, C(3′)H2], 25.3 [CH2, C(4′)H2], 30.7 [CH2, p. 235–236 °C.
C(2′)H2], 34.7 [CH2, C(5′)H2], 59.2 [CH2, C(1′)H2], 110.4
(C, aromatic C), 111.6 (CH, aromatic CH), 120.2 (C, aromatic 1325, 1125;
νmax/cm−1 (KBr) 3425, 3154, 3066, 2923, 2857, 1706, 1599,
C), 120.5 (CH, aromatic CH), 120.8 (CH, aromatic CH), 122.9
δH (300 MHz, d6-DMSO) 1.28–1.43 [2H, m, C(3′)H2],
(C, aromatic C), 124.5 (CH, aromatic CH), 126.0 (C, aromatic 1.54–1.69 [2H, m, C(4′)H2], 1.95–2.15 [2H, m, C(2′)H2], 2.29
C), 128.7 (CH, aromatic CH), 130.8 (CH, aromatic CH), 132.5 [2H, t, J 7.2, C(5′)H2], 2.86 [3H, s, C(5)CH3], 3.21 [3H, s, C(11)-
(C, aromatic C), 133.4 (C, aromatic C), 142.6 (C, aromatic C), CH3], 3.33 (3H, s, SO2CH3), 4.73 [2H, t, J 7.1, C(1′)H2],
144.3 (C, aromatic C), 146.5 (CH, aromatic CH), 174.1 7.34–7.45 [1H, m, C(9)H], 7.62–7.74 [2H, m, C(7)H, C(8)H,],
(C, CvO)
8.47 [1H, d, J 7.6, C(10)H], 8.48 [1H, d, J 7.0, C(4)H], 8.56
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[1H, d, J 7.1, C(3)H], 10.11 [1H, s, C(1)H], 12.09 (1H, br s,
νmax/cm−1 (KBr) 3294, 2978, 2929, 1720, 1638, 1396;
CONHSO2), 12.19 (1H, s, NH);
δH (300 MHz, d6-DMSO) 1.51–1.65 [2H, m, C(3′)H2],
δC (75 MHz, d6-DMSO 12.1 [CH3, C(5)CH3], 15.1 [CH3, 1.99–2.12 [2H, m, C(2′)H2], 2.34 [2H, t, J 7.3, C(4′)H2], 3.12 [3H,
C(11)CH3], 23.5 [CH2, C(4′)H2], 24.0 [CH2, C(3′)H2], 30.6 [CH2, s, C(5)CH3], 3.30 [3H, s, C(11)CH3], 4.26 [3H, s, N(6)CH3], 4.74
C(2′)H2], 35.1 [CH2, C(5′)H2], 41.0 (CH3, SO2CH3), 59.2 [CH2, [2H, t, J 7.2, C(1′)H2], 7.45 [1H, overlapping ddd, J 7.9, 7.0, 0.9,
C(1′)H2], 110.4 (C, aromatic C), 111.6 (CH, aromatic CH), 120.2 C(9)H], 7.74 [1H, overlapping ddd, J 8.2, 7.1, 0.8, C(8)H], 7.81
(C, aromatic C), 120.5 (CH, aromatic CH), 120.8 (CH, aromatic [1H, d, J 8.1, C(7)H], 8.48 [1H, d, J 8.0, C(10)H], 8.56 [1H, dd,
CH), 122.2 (C, aromatic C), 124.5 (CH, aromatic CH), 125.5 J 7.4, 0.8, C(3)H], 8.61 [1H, d, J 7.3, C(4)H], 10.12 [1H, br s,
(C, aromatic C), 128.7 (CH, aromatic CH), 130.8 (CH, aromatic C(1)H], 12.10 (1H, s, OH);
CH), 132.5 (C, aromatic C), 133.3 (C, aromatic C), 142.6 (C, aro-
δC (75 MHz, d6-DMSO) 13.6 [CH3, C(5)CH3], 15.2 [CH3,
matic C), 144.3 (C, aromatic C), 146.5 (CH, aromatic CH), C(11)CH3], 21.1 [CH2, C(3′)H2], 30.3 [CH2, C(4′)H2], 32.9 [CH2,
172.4 (C, CvO);
C(2′)H2], 33.6 [CH3, N(6)CH3], 58.9 [CH2, C(1′)H2], 109.9 (CH,
aromatic CH), 110.9 (C, aromatic C), 120.2 (C, aromatic C),
m/z (ES+) 438 (M)+, (100%), 361 (20), 247 (2);
HRMS (ES+) Exact mass calculated for C24H28N3O3S (M)+, 120.6 (CH, aromatic CH), 121.1 (CH, aromatic CH), 121.2
438.1851. Found 438.1840.
(C, aromatic C), 124.2 (CH, aromatic CH), 126.2 (C, aromatic
C), 128.6 (CH, aromatic CH), 131.0 (CH, aromatic CH), 133.0
(C, aromatic C), 133.5 (C, aromatic C), 144.1 (C, aromatic C),
N2-(2′-Carboxyethyl)-6-methylellipticinium bromide (18)
Using the procedure described, compound 18 was obtained 144.3 (C, aromatic C), 146.0 (CH, aromatic CH), 174.1
from 3 (150 mg, 0.577 mmol) and 3-bromopropionic acid (C, CvO);
(176 mg, 1.15 mmol) as an orange solid (137 mg) however
LCMS analysis found the solid to be contaminated with trace
starting material 3.*
m/z (ES+) 361 (M)+, (100%), 261 (6), 221 (6);
HRMS (ES+) Exact mass calculated for C23H25N2O2 (M)+,
361.1916. Found 361.1914.
νmax/cm−1 (KBr) 3414, 2920, 1717, 1637, 1470, 1384;
δH (300 MHz, d6-DMSO) 2.89 [3H, s, C(5)CH3], 3.05 [3H, s,
N2-(5′-Carboxypentyl)-6-methylellipticinium bromide (20)
C(11)CH3], 3.19 [2H, t, J 6.9, C(2′)H2], 4.02 [3H, s, N(6)CH3], Using the procedure described, the reaction of 3 (100 mg,
4.91 [2H, t, J 6.9, C(1′)H2], 7.28–7.37 [1H, m, C(9)H], 7.59–7.65 0.380 mmol) and 6–bromohexanoic acid (150 mg,
[2H, m, C(7)H, C(8)H], 8.22 [1H, d, J 8.0, C(10)H], 8.41 [1H, d, 0.740 mmol) afforded 20 as a yellow orange solid (128 mg,
J 7.3, C(4)H], 8.50 [1H, d, J 7.4, C(3)H], 9.77 (1H, s, OH), 10.00 74%): m.p. 117–119 °C.
[1H, s, C(1)H];
νmax/cm−1 (KBr) 3370, 3033, 2937, 2846, 1698, 1451, 1400,
δC (75 MHz, d6-DMSO) 13.5 [CH3, C(5)CH3], 15.0 [CH3, 748;
C(11)CH3], 33.5 [CH3, N(6)CH3], 34.9 [CH2, C(2′)H2], 55.0 [CH2,
δH (300 MHz, d6-DMSO) 1.31–1.44 [2H, m, C(3′)H2],
C(1′)H2], 109.8 (CH, aromatic CH), 110.7 (C, aromatic C), 119.8 1.52–1.66 [2H, m, C(4′)H2], 1.98–2.10 [2H, m, C(2′)H2], 2.25
(C, aromatic C), 120.2 (CH, aromatic CH), 121.0 (CH, aromatic [2H, t, J 7.2, C(5′)H2], 3.15 [3H, s, C(5)CH3], 3.33 [3H, s, C(11)
CH), 121.1 (C, aromatic C), 124.1 (CH, aromatic CH), 126.0 (C, CH3], 4.28 [3H, s, N(6)CH3], 4.73 [2H, t, J 7.4, C(1′)H2], 7.46
aromatic C), 128.5 (CH, aromatic CH), 131.1 (CH, aromatic [1H, overlapping ddd, J 7.9, 7.1, 0.9, C(9)H], 7.74 [1H, overlap-
CH), 132.9 (C, aromatic C), 133.4 (C, aromatic C), 143.9 (C, ping ddd, J 8.2, 7.1, 0.8, C(8)H], 7.83 [1H, d, J 8.2, C(7)H], 8.51
aromatic C), 144.1 (C, aromatic C), 146.6 (CH, aromatic CH), [1H, d, J 7.9, C(10)H], 8.58 [1H, dd, J 7.4, 0.9, C(3)H], 8.62 [1H,
171.7 (C, CvO);
d, J 7.3, C(4)H], 10.13 [1H, br s, C(1)H], 12.04 (1H, br s, OH);
δC (75 MHz, d6-DMSO) 13.6 [CH3, C(5)CH3], 15.2 [CH3,
m/z (ES+) 333 (M)+, (100%), 261 (80), 146 (6);
HRMS (ES+) Exact mass calculated for C21H21N2O2 (M)+, C(11)CH3], 23.9 [CH2, C(3′)H2], 25.1 [CH2, C(4′)H2], 30.6 [CH2,
333.1603. Found 333.1611.
C(2′)H2], 33.4 [CH2, C(5′)H2], 33.6 [CH3, N(6)CH3], 59.1 [CH2,
* It was not possible to tell the exact ratio of starting C(1′)H2], 110.0 (CH, aromatic CH), 110.9 (C, aromatic C), 120.2
material 3 present due to the fact that 3 is only sparingly (C, aromatic C), 120.6 (CH, aromatic CH), 121.1 (CH, aromatic
soluble in d6-DMSO. It was not possible to elucidate the ratio CH), 121.3 (C, aromatic C), 124.3 (CH, aromatic CH), 126.3
from LCMS analysis due to co-elution of peaks.
(C, aromatic C), 128.7 (CH, aromatic CH), 131.0 (CH, aromatic
CH), 133.1 (C, aromatic C), 133.6 (C, aromatic C), 144.2 (C, aro-
matic C), 144.3 (C, aromatic C), 146.1 (CH, aromatic CH),
N2-(4′-Carboxybutyl)-6-methylellipticinium bromide (19)
Employing the procedure described, 6-methylellipticine 3 174.3 (C, CvO);
(207 mg, 0.794 mmol) and 5–bromovaleric acid (290 mg,
1.60 mmol) were reacted in DMF and the resulting yellow pre-
m/z (ES+) 375 (M)+ (100%), 261 (10), 489 (8);
HRMS (ES+) Exact mass calculated for C24H27N2O2 (M)+,
cipitate was filtered to afford a yellow/green solid. The product 375.2073. Found 375.2065.
was found by LCMS analysis to be contaminated with starting
material. A mixture of chloroform–ether (1 : 1, 10 mL) was
added and the resulting suspension was heated until boiling
N2-(5′-Carboxyaminopentyl)-6-methylellipticinium
bromide (21)
and subsequently filtered to afford pure 19 as a yellow solid Using the procedure described, the reaction of 3 (150 mg,
(263 mg, 75%): m.p. 254–255 °C.
0.577 mmol) and 6–bromohexanamide (24, 224 mg,
1342 | Org. Biomol. Chem., 2013, 11, 1334–1344
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1.150 mmol) afforded 21 as a mustard yellow solid (203 mg,
77%): m.p. 222–223 °C.
Found C, 63.21; H, 5.97; N, 9.32; C24H26BrN3·12H2O requires
C, 63.44; H, 6.21; N, 9.25%.
νmax/cm−1 (KBr) 3283, 3066, 2924, 2852, 1664, 1612, 1587,
1396;
N2-(5′-Hexanoylmethylsulphonamide)-6-methylellipticinium
δH (300 MHz, d6-DMSO) 1.28–1.42 [2H, m, C(3′)H2],
1.52–1.65 [2H, m, C(4′)H2], 1.95–2.07 [2H, m, C(2′)H2], 2.09
[2H, t, J 7.2, C(5′)H2], 2.99 [3H, s, C(5)CH3], 3.14 [3H, s,
C(11)CH3], 4.12 [3H, s, N(6)CH3], 4.70 [2H, t, J 7.4, C(1′)H2],
6.71 [1H, br s, one of CONH2], 7.29 [1H, br s, one of CONH2],
7.37 [1H, ddd, J 8.0, 5.9, 2.3, C(9)H], 7.63–7.72 [2H, m, C(8)H,
C(7)H], 8.31 [1H, d, J 7.9, C(10)H], 8.48 [1H, d, J 7.3, C(4)H],
8.52 [1H, d, J 7.4, C(3)H], 10.02 [1H, s, C(1)H];
δC (75 MHz, d6-DMSO) 13.6 [CH3, C(5)CH3], 15.1 [CH3,
C(11)CH3], 24.4 [CH2, C(3′)H2], 25.3 [CH2, C(4′)H2], 30.7 [CH2,
C(2′)H2], 33.5 [CH3, N(6)CH3], 34.7 [CH2, C(5′)H2], 59.1 [CH2,
C(1′)H2], 109.9 (CH, aromatic CH), 110.8 (C, aromatic C), 120.1
(C, aromatic C), 120.6 (CH, aromatic CH), 121.1 (CH, aromatic
CH), 121.2 (C, aromatic C), 124.2 (CH, aromatic CH), 126.2
(C, aromatic C), 128.6 (CH, aromatic CH), 130.9 (CH, aromatic
CH), 133.0 (C, aromatic C), 133.4 (C, aromatic C), 144.0 (C, aro-
matic C), 144.2 (C, aromatic C), 146.0 (CH, aromatic CH),
174.2 (C, CvO);
bromide (23)
Compound 23 was obtained as an orange solid (0.208 g, 64%)
from the reaction of 3 (159 mg, 0.612 mmol) and N-methane-
sulphonyl-6-bromohexanamide (25, 331 mg, 1.220 mmol)
using the procedure described: m.p. 235–236 °C.
νmax/cm−1 (KBr) 3414, 2934, 2857, 2785, 1701, 1585, 1302,
1150;
δH (300 MHz, d6-DMSO) 1.30–1.44 [2H, m, C(3′)H2],
1.55–1.69 [2H, m, C(4′)H2], 1.95–2.09 [2H, m, C(2′)H2], 2.34
[2H, t, J 7.1, C(5′)H2], 3.01 [3H, s, C(5)CH3], 3.17 [3H, s, C(11)-
CH3], 3.21 [3H, s, SO2CH3], 4.14 [3H, s, N(6)CH3], 4.72 [2H, t,
J 7.2, C(1′)H2], 7.38 [1H, overlapping ddd, J 7.8, 6.2, 1.5, C(9)H],
7.64–7.74 [2H, m, C(7)H, C(8)H], 8.34 [1H, d, J 7.9, C(10)H],
8.50 [1H, d, J 7.4, C(4)H], 8.53 [1H, d, J 7.5, C(3)H], 10.05 [1H,
s, C(1)H], 11.68 (1H, br s, CONHSO2);
δC (75 MHz, d6-DMSO) 13.6 [CH3, C(5)CH3], 15.2 [CH3,
C(11)CH3], 23.5 [CH2, C(3′)H2], 25.0 [CH2, C(4′)H2], 30.6 [CH2,
C(2′)H2], 33.6 [CH3, N(6)CH3], 35.1 [CH2, C(5′)H2], 41.0 (CH3,
SO2CH3), 59.0 [CH2, C(1′)H2], 109.9 (CH, aromatic CH), 110.9
(C, aromatic C), 120.2 (C, aromatic C), 120.6 (CH, aromatic
CH), 121.1 (CH, aromatic CH), 121.3 (C, aromatic C), 124.2
(CH, aromatic CH), 126.2 (C, aromatic C), 128.6 (CH, aromatic
CH), 131.0 (CH, aromatic CH), 133.1 (C, aromatic C), 133.5
(C, aromatic C), 144.1 (C, aromatic C), 144.3 (C, aromatic C),
146.1 (CH, aromatic CH), 172.5 (C, CvO);
m/z (ES+) 374 (100%), 105 (20);
HRMS (ESI) Exact mass calculated for C24H28N3O (M)+,
374.2232. Found 374.2239.
Found C, 60.89; H, 6.69; N, 8.89; C24H28BrN3O·12H2O
requires C, 61.02; H, 6.40; N, 8.89%.
N2-(5′-Cyanopentyl)-6-methylellipticinium bromide (22)
m/z (ES+) 452 (M)+, (100%), 375 (20), 261 (2);
Using the procedure described, the reaction of 3 (150 mg,
0.577 mmol) and 6-bromohexanitrile (203 mg, 1.154 mmol)
afforded 22 as a mustard yellow solid (169 mg, 62%): m.p.
100–102 °C.
HRMS (ES+) Exact mass calculated for C25H30N3O3S (M)+,
452.2008. Found 452.1996.
Found C, 55.49; H, 5.63; N, 7.80; C25H30BrN3O3S·12H2O
requires C, 55.45; H, 5.77; N, 7.76%.
νmax/cm−1 (KBr) 2989, 2931, 2857, 2241, 1637, 1445;
δH (300 MHz, d6-DMSO) 1.34–1.53 [2H, m, C(3′)H2],
1.60–1.73 [2H, m, C(4′)H2], 2.00–2.13 [2H, m, C(2′)H2], 2.55
[2H, t, J 7.0, C(5′)H2], 3.12 [3H, s, C(5)CH3], 3.30 [3H, s, C(11)
6-Bromohexanamide (24)
Preparation of 6-bromohexanoyl chloride. To a stirring solu-
CH3], 4.25 [3H, s, N(6)CH3], 4.74 [2H, t, J 7.4, C(1′)H2], 7.44 tion containing 6-bromohexanoic acid (5.000 g, 0.026 mol) in
[1H, overlapping ddd, J 7.9, 7.0, 0.9, C(9)H], 7.73 [1H, overlap- anhydrous diethyl ether (20 mL) at 0 °C was added oxalyl
ping ddd, J 8.2, 7.1, 0.8, C(8)H], 7.80 [1H, d, J 8.0, C(7)H], 8.47 chloride (2.5 mL, 0.029 mol) and the reaction mixture was left
[1H, d, J 7.9, C(10)H], 8.57 [1H, d, J 7.5, C(3)H], 8.61 [1H, d, to warm to room temperature while stirring overnight. After
J 7.3, C(4)H], 10.12 [1H, s, C(1)H];
this time the reaction mixture was evaporated under reduced
δC (75 MHz, d6-DMSO) 13.7 [CH3, C(5)CH3], 15.3 [CH3, pressure to form a clear oil. IR analysis determined that the
C(11)CH3], 16.0 [CH2, C(5′)H2], 24.2 [CH2, C(4′)H2], 24.7 [CH2, reaction had gone to completion to afford 6-bromohexanoyl
C(3′)H2], 30.0 [CH2, C(2′)H2], 33.7 [CH3, N(6)CH3], 59.0 [CH2, chloride (5.383 g, 97%) which was used without further
C(1′)H2], 110.1 (CH, aromatic CH), 111.1 (C, aromatic C), 120.3 purification.
(C, aromatic C), 120.6 (C, CN nitrile), 120.8 (CH, aromatic CH),
Preparation of 6-bromohexanamide. To a stirring solution
121.2 (CH, aromatic CH), 121.4 (C, aromatic C), 124.4 (CH, aro- of concentrated ammonia (50 mL, 35% aq., v/v) at −10 °C was
matic CH), 126.5 (C, aromatic C), 128.8 (CH, aromatic CH), added 6-bromohexanoyl chloride (5.383 g, 0.025 mol), drop-
131.1 (CH, aromatic CH), 133.2 (C, aromatic C), 133.7 (C, aro- wise, over the course of 10 minutes resulting in the immediate
matic C), 144.4 (C, aromatic C), 144.5 (C, aromatic C), 146.3 formation of a white precipitate. The reaction mixture was
(CH, aromatic CH);
stirred at 0 °C for a further 20 minutes after which time the
precipitate was isolated by filtration to afford 6-bromohexan-
m/z (ES+) 356 (M)+, (100%), 357 (40);
HRMS (ESI) Exact mass calculated for C24H26N3 (M)+, amide (24) as a pure solid which was used without further
356.2123. Found 356.2127.
purification (4.757 g, 94%): m.p. 107 °C.
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νmax/cm−1 (KBr) 3363, 3195, 2947, 2900, 2872, 2849, 2798,
Organic & Biomolecular Chemistry
7 Y. Pommier, DNA, Nat. Rev. Cancer, 2006, 6, 789–802.
8 K. R. Hande, Topoisomerase II inhibitors, Update Cancer
Ther., 2008, 3, 13–26.
9 (a) N. C. Garbett and D. E. Graves, Curr. Med. Chem.: Anti-
cancer Agents, 2004, 4, 149–172; (b) C. M. Miller and
F. O. McCarthy, RSC Adv., 2012, 2(24), 8883–8918.
1661, 1629, 513;
δH (300 MHz, d6-DMSO) 1.29–1.42 [2H, m, C(4)H2],
1.44–1.52 [2H, m, C(3)H2], 1.80 [2H, overlapping tt, J 7.3, 6.8,
C(5)H2], 2.04 [2H, t, J 7.3, C(2)H2], 3.52 [2H, t, J 6.7, C(6)H2],
6.68 [1H, br s, one of CONH2], 7.23 [1H, br s, one of CONH2];
m/z (ES+) 196 (C6H13NO81Br) (M
+
H)+ (56%), 194 10 J. Rouëssé, M. Spielmann, F. Turpin, T. Le Chevalier,
M. Azab and J. M. Mondésir, Eur. J. Cancer, 1993, 29(6),
856–859.
(C6H13NO79Br) (M + H)+ (58), 105 (18), 100 (12).
N-Methanesulphonyl-6-bromohexanamide (25)
11 A. U. Buzdar, G. N. Hortobagyi, L. T. Esparza, F. A. Holmes,
J. S. Ro, G. Fraschini and B. Lichtiger, Oncology, 1990, 47,
101–104.
12 Y. Murakami, Y. Yokoyama and N. Okuyama, Tetrahedron
Lett., 1983, 24, 2189–2192.
To a round bottomed flask containing 6-bromohexanoyl
chloride (0.027 mol, 5.602 g), prepared using the procedure
above, was added methanesulphonamide (2.600 g, 0.027 mol)
and the reaction mixture was heated at 90 °C for 1 hour. Once
the solution had cooled, ethyl acetate (20 mL) was added
and the solution was washed with water (2 × 20 mL) and brine
(2 × 20 mL). The reaction mixture was then cooled to 0 °C for
30 minutes and the resulting white precipitate was collected by
filtration to afford N-methanesulphonyl-6-bromohexanamide
(25) as a white solid which was used without further purifi-
cation (6.312 g, 86%): m.p. 75–76 °C.
13 Y. Yokoyama, N. Okuyama, S. Iwadate, T. Momoi and
Y. Murakami, Synthetic studies of indoles and related com-
pounds. Part 22. The Vilsmeier–Haack reaction of N-benzyl-
1,2,3,4-tetrahydrocarbazoles and its synthetic application
to olivacine and ellipticine, J. Chem. Soc., Perkin Trans. 1,
1990, 1319–1329.
14 F. M. Deane, C. M. Miller, A. R. Maguire and
F. O. McCarthy, J. Heterocycl. Chem., 2011, 48(4), 814–823.
15 Y. Pommier, J. M. Covey, D. Kerrigan, J. Markovits and
R. Pham, Nucleic Acids Res., 1987, 15, 6713–6731.
16 R. E. McKnight, A. B. Gleason, J. A. Keyes and S. Sahabi,
Bioorg. Med. Chem. Lett., 2007, 17, 1013–1017.
17 R. E. McKnight, B. Onogul, S. R. Polasani, M. K. Gannon
and M. R. Detty, Bioorg. Med. Chem., 2008, 16, 10221–
10227.
νmax/cm−1 (KBr) 3220, 2944, 2908, 2850, 1726, 1340, 1130,
511;
δH (300 MHz) 1.45–1.52 [2H, m, C(4)H2], 1.64–1.77 [2H, m,
C(3)H2], 1.83–1.94 [2H, m, C(5)H2], 2.36 [2H, t, J 7.4, C(2)H2],
3.31 (3H, s, SO2CH3) 3.42 [2H, t, J 6.6, C(6)H2];
δC (75 MHz) 23.4 [CH2], 27.4 [CH2], 32.2 [CH2], 33.5 [CH2,
C(6)H2], 36.1 [CH2, C(2)H2], 41.6 (CH3, SO2CH3), 172.1 (CvO);
m/z (ES−) 272 (C7H13NO3S81Br) (M − H)− (100%), 270
(C7H13NO3S79Br) (M − H)− (96), 195 (4), 127 (6), 125 (5);
HRMS (ES−) Exact mass calculated for C7H13NO3S79Br
(M − H)−, 269.9800. Found 269.9808.
18 Y. Hsiang, R. Hertzberg, S. Hecht and L. F. Liu, J. Biol.
Chem., 1985, 260, 14873–14878.
19 M. Hranjec, I. Piantanida, M. Kralj, L. Šuman, K. Pavelić
and G. Karminski-Zamola, J. Med. Chem., 2008, 51, 4899–
4910.
Found C, 31.05; H, 5.10; N, 4.92; S, 11.47. C7H14NSO3Br
requires C, 30.89; H, 5.18; N, 5.15; S, 11.47%.
20 C. Bailly, L. Dassonneville, P. Colson, C. Houssier,
K. Fukasawa, S. Nishimura and T. Yoshinari, Cancer Res.,
1999, 59, 2853–2860.
Acknowledgements
21 M. T. Muller, K. Helal, S. Soisson and J. R. Spitzner, Nucleic
Acids Res., 1989, 17, 9499–9505.
22 N. Osheroff, Biochemistry, 1989, 28, 6157–6160.
23 S. D. Cline, T. L. Macdonald and N. Osheroff, Biochemistry,
1997, 36, 13095–13101.
24 R. L. Clark, F. M. Deane, N. G. Anthony, B. F. Johnston,
F. O. McCarthy and S. P. Mackay, Bioorg. Med. Chem., 2007,
15, 4741–4752.
25 F. Fleury, A. Sukhanova, A. Ianoul, J. Devy, I. Kudelina,
O. Duval, A. P. J. Alix, J. C. Jardillier and I. Nabiev, J. Biol.
Chem., 2000, 275, 3501–3509.
We are grateful to Waters, Enterprise Ireland, Cancer Research
Ireland and Cork County council higher education grant for
their financial support.
References
1 K. Chikamori, A. G. Grozav, T. Kozuki, D. Grabowski,
R. Ganapathi and M. K. Ganapathi, Curr. Cancer Drug
Targets, 2010, 10, 758–771.
2 J. C. Wang, Nat. Rev. Mol. Cell Biol., 2002, 3, 430–440.
3 J. C. Wang, Annu. Rev. Biochem., 1996, 65, 635–692.
4 J. J. Champoux, Annu. Rev. Biochem., 2001, 70, 369–413.
5 J. A. Holden, Curr. Med. Chem.: Anti-Cancer Agents, 2001, 1,
1–25.
26 K. Wassermann, J. Markovits, C. Jaxel, C. Capranico,
K. W. Kohn and Y. Pommier, Mol. Pharmacol., 1990, 38,
38–45.
27 C. M. Miller, E. C. O’Sullivan, K. J. Devine and
F. O. McCarthy, Org. Biomol. Chem., 2012, 10(39), 7912–
7921.
6 S. Salerno, F. Da Settimo, S. Taliani, F. Simorini, C. La
Motta, G. Fornaciari and G. A. M. Marini, Curr. Med.
Chem., 2010, 17, 4270–4290.
1344 | Org. Biomol. Chem., 2013, 11, 1334–1344
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