Investigations regarding the utility of prodigiosenes to treat leukemia

Article abstract of DOI:10.1039/c2ob26535d

Prodigiosenes, possessing a 4-methoxypyrrolyldipyrrin skeleton, are known for their anti-cancer activity. Structural modification of the C-ring resulted in a series of prodigiosenes that displayed promising activity against leukemia cell lines during in v

Full text of DOI:10.1039/c2ob26535d

Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Volume 11 | Number 1 | 7 January 2013 | Pages 1–180
ISSN 1477-0520
PAPER
Alison Thompson et al.
Investigations regarding the utility of prodigiosenes to treat leukemia

Organic &
Biomolecular
Chemistry
View Article Online
View Journal | View Issue
PAPER
Investigations regarding the utility of prodigiosenes to
treat leukemia†
Cite this: Org. Biomol. Chem., 2013, 11, 62
Deborah A. Smithen,a A. Michael Forrester,b,c Dale P. Corkery,d Graham Dellaire,d,e
Julie Colpitts,f Sherri A. McFarland,f Jason N. Bermanb,c,g and Alison Thompson*a
Prodigiosenes, possessing a 4-methoxypyrrolyldipyrrin skeleton, are known for their anti-cancer activity.
Structural modification of the C-ring resulted in a series of prodigiosenes that displayed promising activity
against leukemia cell lines during in vitro analysis against the NCI 60 cancer cell line panel. Further in vivo
studies of these compounds using the zebrafish model showed persistence of anti-leukemia properties in
human K562 chronic myelogenous leukemia cells.
Received 3rd August 2012,
Accepted 2nd October 2012
DOI: 10.1039/c2ob26535d
www.rsc.org/obc
Selective cytotoxicity of prodigiosin has been noted against
various human cancer cell lines, for example haematopoietic,13
Introduction
Prodigiosin (1) is the parent member of a family of tripyrrolic colon14 and gastric15 cancer cell lines. Although there are brief
natural products, isolated from several Serratia, Streptomyces descriptions in the patent literature,19–21 the use of such tripyr-
and Bacillus strains of bacteria. Prodigiosin (1) possesses rolic compounds for the treatment of leukemia was not pub-
potent immunosuppressive, antimicrobial and cytotoxic lished in the journal literature until 1988,16 whereby in vitro
properties.1–3 Coley’s Toxin,4 consisting of a mixture of bac- studies showed prodigiosin to be exceptionally potent against
terial extracts including Serratia Marcescens, contains prodigio- P388 leukemia, alongside significant activity against other
sin (1) as well as lipopolysaccharides, and was successfully cancerous cell lines. Omission of the methoxy (R1) substituent
used (USA 1893–1963) as an anti-cancer treatment before its in ring B, as in prodigiosene 2,16,17 resulted in greatly diminished
withdrawal by the FDA due to systemic toxicity.5 The anti- cytotoxicity and removal of all substituents (3) led to equal or
cancer activity displayed by prodigiosin is thought to be the further reduced activity, relative to prodigiosene 2, in the
result of several mechanistic modes of action: for example, it is majority of cancer cell lines examined.16
known to transport H+/Cl− over phospholipid membranes1,6–8
This trend in activity was studied further by D’Alessio and
and to induce copper-mediated double-strand DNA co-workers,18–20 who found that substitution of the
cleavage.6–12
C-6 methoxy group with alkoxy substituents of increasing size
lead to a step-wise reduction in activity and cytotoxicity. Other
studies pertaining to the treatment of leukemia focused on the
elucidation of signalling pathways and modes of action
involved in prodigiosin-induced apoptosis. Cell lines examined
include Jurkat-T cells,27–29 HL-60 leukemia cells9 and B-cell
chronic lymphocytic leukemia (B-CLL) cells.21 In the latter case
it was found that prodigiosin induced apoptosis of B-CLL cells
through caspase activation and was the first report showing
that prodigiosin induces apoptosis in human primary cancer
cells.21
Myeloid leukemias are a heterogeneous group of diseases
in need of novel therapeutic agents.22,23 Acute myeloid leuke-
mia (AML) remains fatal for 40% of patients,23 both due to
refractory disease and toxicity from traditional therapeutic
agents. Some experts believe that AML treatment has a reached
a “plateau” in efficacy.24 AML is the most common acute leuke-
mia in adults and accounts for nearly 20% of childhood leuke-
mias. While pediatric clinical trials over the past decades have
resulted in significant improvements to overall survival of
aDepartment of Chemistry, Dalhousie University, Halifax, Nova Scotia B4P 2R6,
Canada. E-mail: alison.thompson@dal.ca
bDepartment of Microbiology and Immunology, Dalhousie University, Halifax, Nova
Scotia B4P 2R6, Canada
cIWK Health Centre, Halifax, Nova Scotia B3K 6R8, Canada
dDepartment of Biochemistry and Molecular Biology, Dalhousie University, Halifax,
Nova Scotia B4P 2R6, Canada
eDepartment of Pathology, Dalhousie University, Halifax, Nova Scotia B4P 2R6,
Canada
fDepartment of Chemistry, Acadia University, Wolfville, Nova Scotia B4P 2R6,
Canada
gDepartment of Pediatrics, Dalhousie University, P.O. Box 15000, Halifax, Nova
Scotia B3H 4R2, Canada
†Electronic supplementary information (ESI) available: General Experimental;
Zebrafish Husbandry, Embryo Collection and Embryo Staging; Xenotransplanta-
tion of Human Leukemia Cells Into Zebrafish Embryos, Dissociation, and Immu-
nofluorescence; Experimental Procedures and Data; NMR spectra for all novel
compounds; and HPLC traces for all prodigiosenes. See DOI: 10.1039/
c2ob26535d
62 | Org. Biomol. Chem., 2013, 11, 62–68
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
patients, cure rates for AML have peaked in the 60% range,
which is significantly inferior to the 90% childhood cure rates
for acute lymphoblastic leukemia (ALL).25 The need for new
anti-leukemia agents is thus pressing, particularly ones that
avoid overlapping toxicities with current chemotherapies, such
as AraC and anthracyclines.35,36
With this in mind, we synthesized C-ring modified prodi-
giosenes and saw activity against leukemia cell lines in vitro,
including K562 erythroleukemia cells. K562 cells harbour the
t(9;22)(q34;q11) translocation termed the Philadelphia chromo-
some, which results in the pathognomonic BCR-ABL fusion
gene found in chronic myelogenous leukemia (CML).26 Since
the resulting BCR-ABL oncoprotein can be successfully tar-
geted by tyrosine kinase inhibitors, including the prototypic
imatinib mesylate (Gleevec),27 this cell line provided a positive
control with which to evaluate efficacy of our prodigiosene
compounds. To validate our in vitro studies, we investigated
the efficacy of our novel prodigiosenes against leukemia cell
survival in vivo using a novel zebrafish xenotransplantation
platform that we have recently pioneered for the study of anti-
leukemia agents in a live animal model.
Scheme 1 Synthesis of prodigiosenes 8a and 8b.
was less successful for C-ring pyrroles lacking a conjugated
carbonyl moiety, prompting the development of a modified
protocol to include the formation of bromo-substituted dipyr-
rins, as a more robust alternative to the triflate analogue.33
With the exception of this modification, the syntheses followed
the same pathway to that used originally,32 with the first steps
being hydrogenolysis and decarboxylation of Knorr-type pyr-
roles 4a and 4b (Scheme 1) followed by formylation of the
resulting α-free pyrroles using trimethylorthoformate
(TMOF).34
Condensation of the 2-formyl pyrroles (5a and 5b) with 4-
methoxy-3-pyrrolin-2-one,35–37 under basic conditions, fol-
lowed by re-esterification of the resulting carboxylic acids in
acidic methanol, afforded dipyrrinones 6a and 6b, respectively,
in good yields. Reaction of 6a and 6b with phosphorous oxy-
bromide proceeded over 5 days: the extended reaction time
gave optimum conversion to the desired 2-bromodipyrrins (7a
and 7b), as did the use of excess POBr3 (4 equiv.) added in two
portions during the course of the reaction. Subsequent Suzuki
coupling was successful for the bromide derivatives 7a and 7b,
giving the target prodigiosenes 8a and 8b in yields of 55 and
65%, respectively.
Results and discussion
Most structure–activity relationship (SAR) studies concerning
prodigiosenes have focused on the A-ring (3),28–31 with some
success in retaining the anti-proliferative behaviour of the
parent natural product. Our work concerns structural modifi-
cation of the C-ring within the prodigiosene skeleton (see 3,
Fig. 1), an area that was previously under-developed. We
hereby report the use of pendant alkyl ester groups i.e., those
lacking a carbonyl group directly adjacent to the pyrrole ring.
In addition, we varied the position of the functional group on
the C-ring and examined the effect of di-ester functionality.
Synthesis of C-ring modified prodigiosenes generally
begins with a Knorr-type pyrrole (4, Scheme 1) bearing the
desired functionality in the 4-position. It is this heterocycle
that becomes the C-ring (3) of the final product. Our initial
synthetic route32 involved Suzuki cross-coupling of triflate-acti-
vated dipyrrins with N-Boc pyrrole-2-boronic acid.24,25 This
strategy facilitated the introduction of various C-ring pyrroles,
most notably those stabilized by a deactivating carbonyl group
directly adjacent to the pyrrole ring.32 However, this method
Synthesis of 8a and 8b demonstrated the significance of the
improved methodology and allowed us to design more challen-
ging target compounds that also featured alkanoate substitu-
ents. Variation of the position of the C-ring substituent and
incorporation of multiple substituents were of interest to
further probe the SAR.
Pyrroles 4c and 4d (Scheme 2) were both derived from
methyl-4,6-dioxoheptanoate (12, Scheme 2),34 itself syn-
thesised from tert-butyl acetoacetate (9). Indeed, reaction of
tert-butyl acetoacetate with magnesium methoxide afforded
the Grignard reagent 10,38 and subsequent reaction of 10 with
3-methoxycarbonyl propionyl chloride, followed by thermolysis
of the resulting Boc-protected product (11) in the presence of
catalytic p-tosic acid gave 12 in a 39% yield over the three
steps. From here, the 5-methyl and 5-ethyl ethanoate diketones
13a and 13b were synthesized by reaction of 12 with methyl
iodide or ethyl bromoacetate, respectively.34 The two diketones
were subsequently employed in separate Knorr-type reactions
Fig. 1 Prodigiosin (1), analogue 2 and prodigiosene core structure 3.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 62–68 | 63

View Article Online
Paper
Organic & Biomolecular Chemistry
Table 1 Mean in vitro activity of prodigiosin (1) and prodigiosenes 8a–d over
60 cancer cell lines; average of two repeat screens (http://dtp.cancer.gov)
Log10 mean
GI50
Log10 mean
TGI
Log10 mean
LC50
Entry
Compound
1
2
3
4
5
1
−7.85
−6.24
−6.70
−6.27
−5.45
−5.68
−5.44
−5.71
−5.27
−4.82
−6.65
−4.59
−4.81
−4.52
−4.29
8a
8b
8c
8d
calculated based on its fit to a non-linear sigmoidal dose–
response curve. Significant DNA cleavage was observed from
8a (EC50 = 14.1 μM), indicating that C-ring modified prodigio-
senes maintain the cleavage ability of prodigiosin (1, EC50
=
Scheme 2 Synthesis of 3,4-disubstituted pyrroles.
9.2 μM).
All four novel prodigiosenes 8a–d were selected for screen-
ing by the National Cancer Institute (NCI) against their stan-
dard panel of 60 human cell lines, derived from nine cancer
cell types. The averaged results of these in vitro screens are
shown in Table 1, alongside those previously determined for
prodigiosin (1).
With the exception of 8d (entry 5) all compounds showed
sufficient anti-cancer activity to warrant further study. Prodi-
giosene 8b (entry 3) was selected by the NCI for additional toxi-
city studies and it was found that this compound caused no
deaths in mice at 50 mg kg−1 versus acute system toxicity at
4 mg kg−1 for prodigiosin (1). Of further interest, during the
original 60-line screen prodigiosenes 8a–d displayed enhanced
activity against leukemia cell lines compared to lines repre-
senting the other eight types of cancer included in the NCI
panel (Fig. 2). Indeed, compounds 8a–c uniformly effected cell
kill against the leukemia cell lines: note the negative growth
percentages across the leukemia cell lines versus both negative
and positive growth of cells across all of the other cancer
types. This preferential activity was not observed for any of the
previous prodigiosenes that have been synthesized in our
group and evaluated in the NCI 60-line screen.
We explored this new-found activity against leukemia cells
in vivo and decided to employ the zebrafish model, an emmer-
ging robust model for vertebrate blood development and leu-
kemia.39 We chose to use mutant casper fish40 which lack all
pigmentation and are thus ideal for tracking fluorescently-
labelled cells in vivo during real-time analysis. Micro-injec-
tion41 (Fig. 3) of CM-DiI-labelled K562 human leukemia cells
(∼50 cells) directly into the yolk sac of casper embryos at
48 hours of life, allowed successful xenotransplantation to be
determined via the presence of a fluorescent mass at the injec-
tion site 24 hours post injection (hpi).
Scheme 3 Synthesis of 8c and 8d.
to generate the pyrroles 4c and 4d. Hydrogenolysis of the
benzyl esters followed by decarboxylation and formylation gave
the 2-formyl pyrroles 5c and 5d (Scheme 3).
Condensation of 5c and 5d with 4-methoxy-3-pyrrolin-2-
one,35–37 followed by re-establishment of the esters gave 6c
and 6d (Scheme 3). In the latter case (6d), this resulted in two
terminal methyl ester groups, as opposed to the precursor
(5d), which possessed both ethyl and methyl ester groups. Bro-
mination of 6c and 6d followed by palladium-catalyzed cross-
coupling of the resulting bromodipyrrins 7c and 7d with N-Boc
pyrrole-2-boronic acid completed the syntheses of 8c and 8d,
respectively (Scheme 3).
We were interested to learn whether the prodigiosenes 8a–d
had the ability to induce copper-mediated DNA cleavage, akin
to prodigiosin (1). Compound 8a was selected as representative
of the group and subjected to agarose gel electrophoresis with
supercoiled plasmid DNA in the presence of Cu(OAc)2.11 Fol-
lowing incubation at 37 °C for two hours (8a : Cu 1 : 1), the con-
centration required to effect 50% DNA cleavage (EC50) was
We then sought to determine whether the prodigiosenes
8a–d could inhibit growth of the leukemia cells: injected
embryos were divided into six groups; two controls and one
each with addition of a prodigiosene (8) to the water. Prodigio-
senes were resuspended in DMSO and the optimal dose (50%
of maximum tolerated dose, MTD) of each compound was
determined by conducting toxicity curves using 48-hour
64 | Org. Biomol. Chem., 2013, 11, 62–68
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 2 In vitro activity of prodigiosenes 8a–d, at 10 μM concentration, against 60 human cancer cell lines representing 9 different cancer types; negative growth %
represents cell death; http://dtp.cancer.gov.
Embryos treated with 0.3% DMSO served as a negative
control, showing abundant K562 cell proliferation and entry of
cells into circulation. Embryos treated with 20 μM imatinib
mesylate (IM), a targeted inhibitor of the BCR-ABL1 oncopro-
tein in K562 cells, served as a positive control, demonstrating
no cell proliferation or migration.41 By comparison, prodigio-
senes (8a–d) inhibited K562 cell activity to a similar or greater
degree than IM, preventing proliferation of the leukemia cells
(Fig. 5). The effects of prodigiosenes appeared to be graduated:
8a (n = 7/13) was predominantly cytostatic, 8b (n = 6/14) and
8d (n = 5/14) were moderately cytotoxic. Activity of compound
8c (n = 8/11) was of particular note, being strongly cytotoxic.
Migration of K562 cells was sometimes observed in embryos
treated with prodigiosenes, independent of apparent cytostatic
activity of the compound. This indicates that prodigiosenes are
not likely to affect cell migration. Migration of K562 cells in
fish with clear cytotoxic effects and tumour regression were
rarely seen.
Fig. 3 Baseline proliferation (24 hpi) of xenotransplanted K562 leukemia cells
in zebrafish embryo. Brightfield (left) and 555 nm fluorescent (right) images of
casper zebrafish embryos following successful injection of K562 human leuke-
mia cells at 48 hours of life. Scale bar = 1 mm, intervals = 250 μm. (Abbrevi-
ations: hpi = hours post-injection.)
zebrafish embryos treated for 72 hours. Prodigiosenes 8c
(2 μM) and 8d (3 μM) were better tolerated than 8a (0.2 μM)
and 8b (0.2 μM). When the MTD was exceeded with 8a and 8b,
the fish died; with 8c the embryos exhibited reduced activity,
but were still alive; and with 8d, the embryos seemed
unaffected by increasing concentrations, even above the MTD
value that was selected as higher concentrations exhibited
incomplete dissolution and started to undesirably stain the
embryos a brown-red colour. At the MTD concentrations, all
prodigiosenes were water-soluble: no precipitation was
observed for any compound. Engrafted embryos were followed
using live cell microscopy and living embryos were imaged
every 24 hours to monitor cell numbers and migration (Fig. 4).
We observed that K562 cells engrafted in vehicle and drug-
treated fish, and either exhibited proliferation and extensive
migration throughout the fish (i.e. proliferative), a single
stable mass of cells (i.e. cytostatic), or visible regression of
tumour mass (i.e. cytotoxic) over time. We classified the
embryos into these three categories, via scoring.
Conclusions
Four previously inaccessible prodigiosenes bearing pendant
alkyl ester groups on the C-ring were synthesised. These com-
pounds showed particular activity for leukemia cells in vitro
that was explored in vivo using the zebrafish model. Initial
results indicate that all compounds demonstrate anti-prolifer-
ation and, in some cases, selective induction of cell-death in
K562 leukemia cells. Our work also extends the use of zebra-
fish xenografts to determine the in vivo sensitivity of human
cancer cells to novel drugs and suggests that tailored prodigio-
senes may represent novel therapeutic agents for leukemia
with improved anti-cancer potency and reduced toxicity.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 62–68 | 65

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 5 Bar graph summarizing in vivo activity of prodigiosenes 8a–d against
human K562 leukemia cell proliferation in zebrafish. The effects of treatments
on K562 activity were scored relative to DMSO control, then normalized to a
percentage value of the total number of embryos of that treatment group.
White bar = “proliferative”; grey bar = “cytostatic”; black bar = “cytotoxic”; n =
number of embryos used for analysis.
ethyl acetate. The aqueous phase was then acidified to pH 4
(pH paper) with 5% citric acid and the product was extracted
with ethyl acetate (3 × 80 mL). The organic extracts were com-
bined and washed with brine, dried over magnesium sulfate,
filtered and concentrated to give the 2-carboxy pyrrole product,
which did not require further purification. (b) TFA (44 mmol,
11 eq.) was added drop-wise, over 15 minutes, to a solution of
2-carboxy pyrrole (4 mmol, 1 eq.) in freshly distilled dichloro-
methane (10 mL), with stirring at 0 °C under N2 for
15 minutes. Trimethylorthoformate (20 mmol, 5 eq.) was then
added drop-wise, over 15 minutes, and the reaction mixture
was stirred under N2 for a further 20 minutes, warming to r.t.,
before being poured into ice-water (50 mL) followed by slow
addition of 6 M aq. sodium hydroxide (32 mmol, 8 eq.) and
extraction with chloroform (3 × 50 mL). The combined organic
extracts were carefully washed with 5% aq. NaHCO3 solution
(2 × 80 mL), water (80 mL) and brine (80 mL), before being
dried over anhydrous sodium sulfate, filtered and concentrated
in vacuo to give the crude product, which was purified using
column chromatography on silica, eluting with 40–60% ethyl
acetate in hexanes, to give the product (5).
Fig. 4 Anti-leukemia activity of prodigiosenes 8a–d in vivo against
K562 human leukemia cells injected into casper zebrafish. Embryos injected at
48 hours of life with K562 human leukaemia cells. K562 cell numbers and
migration were monitored through live cell microscopy every 24 hours up to
96 hpi. Brightfield (left panels in each pair) and 555 nm fluorescent (right
panels in each pair) images of casper zebrafish embryo at indicated time-points.
Embryo displayed in side profile, anterior to the left. (A–F) Embryos were treated
with (A) 0.3% DMSO (negative control); (B) 20 μM IM (positive control);41 (C)
0.2 μM 8a; (D) 0.2 μM 8b; (E) 2 μM 8c; and (F) 3 μM 8d. Drugs were added
directly to water at 50% maximum tolerated dose [MTD]. Treatment was
initiated when embryos were 24 hpi, and incubation in chemical continued until
end of assessment. Asterisks indicate autofluoresence of prodigiosenes in the
zebrafish liver [*] and swim bladder [**]; white arrows indicate leukaemia cells;
IM = imatinib mesylate; hpi = hours post-injection.
General procedure for the synthesis of dipyrrinones (6)
Experimental section
Aqueous potassium hydroxide (4 M, 20 mmol, 2 equiv.) was
added drop-wise to a solution of 4-methoxy-3-pyrrolin-2-one
(15.5 mmol, 1.55 equiv.) in THF (100 mL) and the resulting
solution was purged with nitrogen for 15 minutes before
warming to 60 °C, with stirring under nitrogen for 1 hour. The
reaction mixture was then cooled slightly and the 2-formyl
pyrrole (5, 10 mmol, 1 equiv.) was added slowly before re-
heating to 60 °C with stirring under nitrogen for 48 hours.
After this time the reaction mixture was concentrated in vacuo,
the residue was dissolved in methanol and conc. HCl (12 M,
30 mmol, 3 equiv.) was added drop-wise. The solution was
General procedure for the synthesis of 2-formyl pyrroles (5)
(a) Triethylamine (∼10 drops) was added to a solution of the
2-benzyl ester pyrrole (4, 4 mmol, 1 eq.) in ethanol (95%) or
THF (65 mL) and the solution was degassed with nitrogen for
10 minutes. 10% Pd/C (∼10% by weight of pyrrole 4) was then
added and the reaction was placed under a hydrogen atmos-
phere, with use of a hydrogen-filled balloon, with stirring at
r.t. overnight. The reaction mixture was then filtered through
Celite, concentrated to dryness and the residue was dissolved
in 5% aq. ammonium hydroxide (60 mL) and washed with
66 | Org. Biomol. Chem., 2013, 11, 62–68
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
then left to cool in the freezer, whereby a precipitate formed cell line. A.M.F. was supported by graduate Awards from the
and was collected by filtration, and washed with hexane to give Canadian Institutes of Health Research (CIHR) and The Cana-
the saponified product as a yellow solid. Re-esterification was dian Cancer Society, in conjunction with The Beatrice Hunter
achieved by heating a solution of the preceding carboxylic acid Cancer Research Institute. D.C. was supported by a trainee
(10 mmol, 1 equiv.) and sulfuric acid (9 mmol, 0.9 equiv.) in award from the Beatrice Hunter Cancer Research Institute
methanol (750 mL) to reflux temperature for 3 hours, with stir- (BHCRI) with funds provided by The Canadian Breast Cancer
ring under nitrogen. The reaction mixture was then concen- Foundation (CBCF) – Atlantic Region. G.D. is a Canadian Insti-
trated in vacuo and the residue was dissolved in tutes of Health New Investigator (CIHR) and both G.D. and
dichloromethane (80 mL), washed with water (50 mL) and J.N.B. are Senior Scientists of the BHCRI. This work was
brine (50 mL), dried over anhydrous sodium sulfate, filtered funded by research grants to: A.T. from CIHR and the Nova
and concentrated in vacuo to give the product (6).
Scotia Health Research Foundation; G.D. from the CBCF-Atlan-
tic; J.N.B. from the Dalhousie Clinical Research Scholar
Program and S.A.M. from NSERC. Human cell-line screening
was performed by the Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer
Institute (http://dtp.cancer.gov).
General procedure for the synthesis of 2-bromodipyrrins (7)
Phosphoryl bromide (4 mmol, 2 eq.) was added to a solution
of dipyrrinone (6, 2 mmol, 1 eq.) in anhydrous dichloro-
methane (60 mL) under N2 and the reaction mixture was
heated to reflux temperature with stirring for 24 hours. The
reaction mixture was then cooled slightly before further phos-
phoryl bromide (4 mmol, 2 eq.) was added. Reflux temperature
was then re-established, with continued stirring under nitro-
gen for 72 hours or until consumption of starting material was
observed using TLC. The reaction mixture was then cooled to
0 °C and saturated aq. NaHCO3 (40 mL) was added slowly with
stirring. When gas evolution had ceased, the reaction mixture
was diluted with water (40 mL) and thoroughly extracted with
dichloromethane (4 × 50 mL). The organic fractions were com-
bined and dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo to give the crude product, which was dis-
solved in dichloromethane and filtered through a short pad of
neutral alumina, washing with dichloromethane, to give the
product (7).
Notes and references
1 A. Fürstner, Angew. Chem., Int. Ed., 2003, 42, 3582–3603.
2 R. A. Manderville, Curr. Med. Chem.: Anti-Cancer Agents,
2001, 1, 195–218.
3 B. Montaner and R. Pérez-Tomás, Curr. Cancer Drug
Targets, 2003, 3, 57–65.
4 G. Rook, Nature, 1992, 357, 545.
5 R. Péréz-Tomas, B. Montaner, E. Llagostera and V. Soto-
Cerrato, Biochem. Pharmacol., 2003, 66, 1447–1452.
6 A. Fürstner and E. J. Grabowski, ChemBioChem, 2001, 2,
706–709.
7 M. S. Melvin, M. W. Calcutt, R. E. Noftlet and
R. A. Manderville, Chem. Res. Toxicol., 2002, 15, 742–748.
8 M. S. Melvin, D. C. Ferguson, N. Lindquist and
R. A. Manderville, J. Org. Chem., 1999, 64, 6861–6869.
9 M. S. Melvin, J. T. Tomlinson, G. Park, C. S. Day,
G. R. Saluta, G. L. Kucera and R. A. Manderville, Chem. Res.
Toxicol., 2002, 15, 734–741.
10 M. S. Melvin, J. T. Tomlinson, G. R. Saluta, G. L. Kucera,
N. Lindquist and R. A. Manderville, J. Am. Chem. Soc., 2000,
122, 6333–6334.
11 M. S. Melvin, K. E. Wooton, C. C. Rich, G. R. Saluta,
G. L. Kucera, N. Lindquist and R. A. Manderville, J. Inorg.
Biochem., 2001, 87, 129–135.
12 G. Park, J. T. Tomlinson, M. S. Melvin, M. W. Wright,
C. S. Day and R. A. Manderville, Org. Lett., 2003, 5, 113–116.
13 B. Montaner, S. Navarro, M. Pique, M. Vilaseca,
M. Martinell, E. Giralt, J. Gil and R. Perez-Tomas,
Br. J. Pharmacol., 2000, 131, 585–593.
14 B. Montaner and R. Perez-Tomas, Life Sci., 2001, 68, 2025–
2036.
15 C. Diaz-Ruiz, B. Montaner and R. Perez-Tomas, Histol. His-
topathol., 2001, 16, 415–421.
General procedure for the synthesis of prodigiosenes (8)
N-Boc-pyrrole-2-boronic acid (0.36 mmol, 1.2 equiv.), lithium
chloride (0.9 mmol, 3 equiv.) and tetrakis(triphenylphosphine)
palladium (0.03 mmol, 0.1 equiv.) were added to a solution of
the preceding 2-bromodipyrrin (7, 0.3 mmol, 1 equiv.) in 1,2-
dimethoxyethane (12 mL, 0.025 M) and the resulting mixture
was purged with nitrogen for 15 minutes. Aqueous sodium car-
bonate (2 M, 1.2 mmol, 4 equiv.), previously bubbled with
nitrogen, was then added drop-wise and the reaction mixture
was heated to 85 °C, with stirring under nitrogen for 24 hours.
After cooling to room temperature, the reaction mixture was
diluted with water (30 mL) and thoroughly extracted with ethyl
acetate (4 × 30 mL). The organic extracts were combined and
washed with water (60 mL) and brine (60 mL), then dried over
anhydrous magnesium sulfate before filtering and concentrat-
ing in vacuo to give the crude product, which was purified
using column chromatography over neutral alumina, eluting
with 2–40% ethyl acetate in hexanes, to give the product (8).
16 D. L. Boger and M. Patel, J. Org. Chem., 1988, 53, 1405–
1415.
Acknowledgements
The authors thank Drs. Donald Small and Robert Arceci 17 W. R. Hearn, M. K. Elson, R. H. Williams and J. Medina-
(Johns Hopkins School of Medicine) for provision of the K562
Castro, J. Org. Chem., 1970, 35, 142–146.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 62–68 | 67

View Article Online
Paper
Organic & Biomolecular Chemistry
18 R. D’Alessio, A. Bargiotti, O. Carlini, F. Colotta, M. Ferrari, 29 K. Daïri, S. Tripathy, G. Attardo and J.-F. Lavallée, Tetrahe-
P. Gnocchi, A. M. Isetta, N. Mongelli, P. Motta, A. Rossi, dron Lett., 2006, 47, 2605–2606.
M. Rossi, M. Tibolla and E. Vanotti, J. Med. Chem., 2000, 30 H. H. Wasserman, A. K. Petersen, M. Xia and J. Wang,
43, 2557–2565.
Tetrahedron Lett., 1999, 40, 7587–7589.
31 H. H. Wasserman, M. Xia, J. Wang, A. K. Petersen,
M. Jorgensen, P. Power and P. Parr, Tetrahedron, 2004, 60,
7419–7425.
19 R. D’Alessio and A. Rossi, Synlett, 1996, 513–514.
20 V. Rizzo, A. Morelli, V. Pinciroli, D. Sciangula and
R. D’Alessio, J. Pharm. Sci., 1999, 88, 73–78.
21 C. Campas, M. Dalmau, B. Montaner, M. Barragan, 32 J. Regourd, A. Al-Sheikh Ali and A. Thompson, J. Med.
B. Bellosillo, D. Colomer, G. Pons, R. Perez-Tomas and
J. Gil, Leukemia, 2003, 17, 746–750.
22 G. Marcucci, T. Haferlach and H. Doehner, J. Clin. Oncol.,
2011, 29, 475–486.
23 G. J. Roboz, Hematology Am. Soc. Hematol. Educ. Program,
2011, 2011, 43–50.
24 B. Lowenberg, T. Pabst, E. Vellenga, P. W. van,
Chem., 2007, 50, 1528–1536.
33 M. I. Uddin, S. Thirumalairajan, S. M. Crawford,
T. S. Cameron and A. Thompson, Synlett, 2010, 2561–2564.
34 T. D. Lash, U. N. Mani, E. A. Lyons, P. Thientanavanich and
M. A. Jones, J. Org. Chem., 1999, 64, 478–487.
35 L. Duc, J. F. McGarrity, T. Meul and A. Warm, Synthesis,
1992, 391–394.
H. C. Schouten, C. Graux, A. Ferrant, P. Sonneveld, 36 R. C. F. Jones and A. D. Bates, Tetrahedron Lett., 1986, 27,
B. J. Biemond, A. Gratwohl, G. G. E. de, L. F. Verdonck, 5285–5288.
M. R. Schaafsma, M. Gregor, M. Theobald, U. Schanz, 37 K. S. Kochhar, H. J. Carson, K. A. Clouser,
J. Maertens and G. J. Ossenkoppele, N. Engl. J. Med., 2011,
364, 1027–1036.
25 W. G. Woods, Pediatr. Blood Cancer, 2006, 46, 565–569.
J. W. Elling, L. A. Gramens, J. L. Parry, H. L. Sherman,
K. Braat and H. W. Pinnick, Tetrahedron Lett., 1984, 25,
1871–1874.
26 R. P. Gale and E. Canaani, Proc. Natl. Acad. Sci. U. S. A., 38 A. R. Battersby, C. J. R. Fookes, M. J. Meegan, E. McDonald
1984, 81, 5648–5652.
27 T. P. Hughes, A. Hochhaus, S. Branford, M. C. Muller,
and H. K. W. Wurziger, J. Chem. Soc., Perkin Trans. 1, 1981,
2786–2799.
J. S. Kaeda, L. Foroni, B. J. Druker, F. Guilhot, R. A. Larson, 39 J. F. Amatruda and L. I. Zon, Dev. Biol., 1999, 216,
S. G. O’Brien, M. S. Rudoltz, M. Mone, E. Wehrle, 1–15.
V. Modur, J. M. Goldman and J. P. Radich, Blood, 2010, 40 R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc,
116, 3758–3765.
C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns
28 C. M. Baldino, J. Parr, C. J. Wilson, S.-C. Ng, D. Yohannes
and L. I. Zon, Cell Stem Cell, 2008, 2, 183–189.
and H. H. Wasserman, Bioorg. Med. Chem. Lett., 2006, 16, 41 D. P. Corkery, G. Dellaire and J. N. Berman,
701–704.
Br. J. Haematol., 2011, 153, 786–789.
68 | Org. Biomol. Chem., 2013, 11, 62–68
This journal is © The Royal Society of Chemistry 2013