Photoinduced cytotoxicity and thioadduct formation by a prodigiosin analogue

Article abstract of DOI:10.1021/ol061998r

(Chemical Equation Presented) The prodigiosin alkaloid 1 and the synthetic analogue 2 show photoinduced cytotoxicity against HL-60 cancer cells. Photoirradiation of 1 and 2 causes photofading, photooxidation, and thioadduct formation. These results provide a model for the redox properties of prodigiosins that play a role in their biological activity and provide a new way to functionalize their pyrromethene entity with water-soluble thiol groups.

Full text of DOI:10.1021/ol061998r

ORGANIC
LETTERS
2006
Vol. 8, No. 21
4951-4954
Photoinduced Cytotoxicity and
Thioadduct Formation by a Prodigiosin
Analogue
John T. Tomlinson,† Gyungse Park,† Jacob A. Misenheimer,† Gregory L. Kucera,‡
Kevin Hesp,§ and Richard A. Manderville*,§
Department of Chemistry, Wake Forest UniVersity, Winston-Salem, North Carolina
27109-7486, Section of Hematology-Oncology, Wake Forest UniVersity School of
Medicine, Winston-Salem, North Carolina 27157, Department of Chemistry, UniVersity
of Guelph, Guelph, Ontario, Canada N1G 2W1
rmanderV@uoguelph.ca
Received August 11, 2006
ABSTRACT
The prodigiosin alkaloid 1 and the synthetic analogue 2 show photoinduced cytotoxicity against HL-60 cancer cells. Photoirradiation of 1 and
2 causes photofading, photooxidation, and thioadduct formation. These results provide a model for the redox properties of prodigiosins that
play a role in their biological activity and provide a new way to functionalize their pyrromethene entity with water-soluble thiol groups.
The prodigiosins are a family of red pigments produced by
certain strains of Serratia marcesens. These alkaloids contain
a characteristic pyrrolylpyrromethene skeleton with a B-ring
methoxy group.1 Prodigiosin (1, Figure 1), the parent member
in dozens of human cancer cells with little effect on
nonmalignant cells.3
The therapeutic potential of prodigiosins has stimulated
research into their mechanism of action. Structure-activity
relationships establish that a nitrogen-containing heterocyclic
A-ring4 and a methoxy group5 at the 3-position of the B-ring
are important for cytotoxic potency. In addition to proposed
(1) For reviews, see: (a) Manderville, R. A. Curr. Med. Chem.: Anti-
Cancer Agents 2001, 1, 195-218. (b) Fu¨rstner, A. Angew. Chem., Int. Ed.
2003, 42, 3582-3603.
(2) (a) Magae, J.; Yamashita, M.; Nagai, K. Ann. N.Y. Acad. Sci. 1993,
685, 339-340. (b) Mortellaro, A.; Songia, S.; Gnocchi, P.; Ferrari, M.;
Fornasiero, C.; D’Alessio, R.; Isetta, A.; Colotta, F.; Golay, J. J. Immunol.
1999, 162, 7102-7109. (c) Stepkowski, S. M.; Erwin-Cohen, R. A.; Behbod,
F.; Wang, M. E.; Qu, X.; Tejpal, N.; Nagy, Z. S.; Kahan, B. D.; Kirken, R.
A. Blood 2002, 99, 680-689.
Figure 1. Prodigiosin (1) and synthetic analogue 2.
(3) (a) Yamamoto, C.; Takemoto, H.; Kuno, K.; Yamamoto, D.; Tsubura,
A.; Kamata, K.; Hirata, H.; Yamamoto, K.; Kano, H.; Seki, T.; Inoue, K.
Hepatology 1999, 30, 894-902. (b) Baldino, C. M.; Parr, J.; Wilson, C. J.;
Ng, S.-C.; Yohannes, D.; Wasserman, H. H. Bioorg. Med. Chem. Lett. 2006,
16, 701-704.
of this family, has been noted for immunosuppressive
activity2 and has been identified as a mediator of apoptosis
(4) D’Alessio, R.; Bargiotti, A.; Carlini, O.; Colotta, F.; Ferrari, M.;
Gnocchi, P.; Isetta, A.; Mongelli, N.; Motta, P.; Rossi, A.; Rossi, M.; Tibolla,
M.; Vanotti, E. J. Med. Chem. 2000, 43, 2557-2565.
† Wake Forest University.
‡ Wake Forest University School of Medicine.
§ University of Guelph.
(5) Boger, D. L.; Patel, M. J. Org. Chem. 1988, 53, 1405-1415.
10.1021/ol061998r CCC: $33.50
© 2006 American Chemical Society
Published on Web 09/15/2006

H+/Cl- symport activities,6,7 prodigiosins also bind DNA
effectively8 and facilitate oxidative DNA cleavage in the
presence of copper2+ (Cu(II)).9 The nuclease activity is
thought to occur via π-radical cation formation at the
electron-rich pyrrolylpyrromethene entity. This event could
be stimulated by the interaction of Cu(II) to yield Cu(I),
which is known to foster reductive activation of molecular
oxygen (O2) leading to the formation of a superoxide radical
anion (O2•-) and hence hydrogen peroxide (H2O2). The
interaction of H2O2 with a Cu-bound prodigiosin species10
is thought to initiate DNA cleavage. Structure-activity
relationships demonstrate that replacement of the individual
metal-coordinating pyrrole rings by other weaker Cu(II)-
ligating arenes results in marked loss of nuclease activity
and cytotoxicity.11
Scheme 1. Synthesis of Prodigiosin Analogue 2
Although coordination of 1 to a redox-active metal cation
represents one way of triggering oxidation of the natural
alkaloid, Roth noted that prodigiosins also possess photo-
sensitizing activity.12 Here, exposure of colorless mutant
Sarcina lutea cells to prodigiosin and visible light led to cell
death in an O2-dependent process. Thus, we speculated that
1 would undergo photooxidation to reductively activate O2
and yield prodigiosin-derived π-radical cations that may act
to facilitate tumor destruction. Presently, we report on our
initial findings regarding photoinduced cytotoxicity of pro-
digiosin (1) and the synthetic analogue 2 (Figure 1) against
HL-60 leukemia cancer cells. Our results provide new
insights into the redox properties of prodigiosin analogues
and afford design ideas for their development as photoac-
tivatable anticancer agents.
Although prodigiosin (1) exhibited an increase in cyto-
toxicity when exposed to visible light, the pigment was too
active in the absence of light. Our initial strategy to inhibit
dark cytotoxicity was to replace the A-pyrrole of 1 with an
alternative, nonmetal-coordinating arene.4,12 Scheme 1 shows
the synthesis of the phenyl analogue 2 that was prepared
using the strategies outlined by D’Alessio and Rossi13 (see
Supporting Information for details).
using human promyelocytic leukemia (HL-60) cells. These
cells have been utilized previously by our research group to
determine structure-activity relationships for the prodigio-
sins.10,12b Prodigiosin (1) exhibited an IC50 value of ∼6.6
µM following 4 h of drug exposure. Under analogous
conditions, the synthetic derivative 2 failed to inhibit colony
formation at 25 µM, which was the maximum drug concen-
tration examined (IC50 (dark), see Table 1). The photocyto-
Table 1. Inhibition of HL-60 Cell Growth by Prodigiosinsa
compound
IC50 (dark)
IC50 (light)c
1
2
6.6 ( 0.1
>25b
2.5 ( 0.2
16.2 ( 0.2
a Inhibition of colony formation was assessed using the soft agar
clonogenic survival assay as described in the Supporting Information. Values
in micromolar are expressed as the mean of three determinations. b No
inhibition at 25 µM drug. c Inhibition following 4 h exposure to the drug
in the presence of 30 min exposure to visible light (>495 nm).
The ability of 1 and 2 to inhibit cancer cell growth in the
absence and presence of visible light was then determined
toxicity of 1 and 2 was then determined by exposing HL-60
cells to drug and visible light (λ > 495 nm) for 30 min
followed by incubation for an additional 3.5 h in the absence
of light. Cells exposed to 30 min of light in the absence of
drug showed no inhibition of colony formation. As shown
in Table 1, prodigiosin 1 was more active with irradiation
(IC50 (dark) vs IC50 (light)), as was the derivative 2 (IC50
(light) ) 16.2 µM). These results demonstrated that prodi-
giosin-based pigments can serve as photoactivatable anti-
cancer agents.
To determine how 1 and 2 may stimulate photocytotox-
icity, their photoreactions in buffered water were studied
using absorption spectroscopy and LC-MS for product
analysis. Figure 2 shows the absorption spectra of 1 (7 µM)
and 2 (18 µM) before and after different irradiation times.
For 1 (Figure 2A), the absorption maxima at 538 nm
(6) Sessler, J. L.; Eller, L. R.; Cho, W.-S.; Nicolaou, S.; Aguilar, A.;
Lee, J. T.; Lynch, V. M.; Magda, D. J. Angew. Chem., Int. Ed. 2005, 44,
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Wasserman, H.; Ohkuma, S. J. Biol. Chem. 1998, 273, 21455-21462. (b)
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Smith, B. D. Chem. Commun. 2005, 3773-3775.
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A. J. Org. Chem. 1999, 64, 6861-6869. (b) Montaner, B.; Castillo-AÄ vila,
W.; Martinell, M.; O¨ llinger, R.; Aymami, J.; Giralt, E.; Pe´rez-Toma´s, R.
Toxicol. Sci. 2005, 85, 870-879.
(9) (a) Melvin, M. S.; Tomlinson, J. T.; Saluta, G. R.; Kucera, G. L.;
Lindquist, N.; Manderville, R. A. J. Am. Chem. Soc. 2000, 122, 6333-
6334. (b) Melvin, M. S.; Wooton, K E.; Rich, C. C.; Saluta, G. R.; Kucera,
G. L.; Lindquist, N.; Manderville, R. A. J. Inorg. Biochem. 2001, 87, 129-
135.
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S.; Manderville, R. A. Org. Lett. 2003, 5, 113-116.
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R.; Kucera, G. L.; Manderville, R. A. Chem. Res. Toxicol. 2002, 15, 734-
741.
decreased together with the shoulder at 487 nm (ꢀ487/ꢀ538
)
(12) Roth, M. M. Photochem. Photobiol. 1967, 6, 923-926.
(13) D’Alessio, R.; Rossi, A. Synlett 1996, 513-514.
0.74) by 38% following 30 min. For 2 (Figure 2B), the
4952
Org. Lett., Vol. 8, No. 21, 2006

analogue 2 did not show photoproducts following 30 min
of irradiation (not shown). Replacement of the A-pyrrole ring
of 1 with a phenyl group raises the electrochemical oxidation
potential by ∼240 mV,17 and so 2 would be more resistant
to photooxidation and hence photofading14 (Supporting
Information, Figure 2B).
Further experiments with 2 were carried out to determine
whether irradiation triggers its attachment to a thiol nucleo-
phile. For these studies, we utilized thioglycolic acid (TGA)
that has been employed to trap indole electrophiles.18 The
synthetic derivative 2 is also more water-soluble than 1 and
was available in our laboratory in sufficient quantities to carry
out such a product analysis. For these experiments, 4.8 mM
2 in the presence of 48 mM TGA was irradiated in buffered
water (0.5 M MES, pH 6.0, 0.1 M NaCl, total volume of 5
mL) for 30 min and then the mixture was incubated for 2 h
at 37 °C. Reaction products were analyzed by ES+-MS and
isolated using semipreparative HPLC.
Figure 3 shows an overlay of the 1H NMR spectra
Figure 2. Absorption spectra of 1 (7 µM) (A) and 2 (18 µM) (B)
in buffered water (MOPS, 10 mM, pH 7, 0.1 M NaCl) after different
irradiation times: 0, 5, 10, 15, 20, 25, and 30 min.
absorption maxima at 490 nm also decreased, but only by
20% following 30 min of irradiation. These results demon-
strated that 1 is more photoreactive than 2, which is
consistent with its superior photocytotoxicity (Table 1).
Certain dyes produce reactive oxygen species (ROS) that,
in turn, attack the dye and result in photofading.14 Pyr-
romethene (BODIPY) dyes15 and pyrroles16 are known to
undergo photooxidation in the presence of O2. Laser flash
photolysis studies on BODIPY dyes show radical cation
formation by photoinduced electron-transfer processes.15
These dyes possess electrochemical oxidation potentials ∼
0.9 V (vs SCE),15 which are 400 mV higher than the
oxidation potential of 1,17 suggesting that 1 would be even
more prone to photooxidation. Thus, the irradiated sample
of 1 was examined by ES+-MS to determine whether
photoproducts were produced. As shown in Figure S1
(Supporting Information), two photoproducts (a and b) were
generated with m/z 340, which is 16 mass units heavier than
the parent 1 ([M + H]+ ) 324). Collision-induced dissocia-
tion of b yielded a species with m/z 323 for loss of 17 mass
units (OH). Photoproduct a generated species with m/z 325
and 308 for losses of 15 (CH3) and 17 (OH) mass units,
respectively (Supporting Information, Figure S1); prodigiosin
1 shows a loss of 15 mass units (CH3) from the B-ring
methoxy group. These results suggested photooxidation of
1 leading to OH attachment, which correlates with our
previous findings that Cu(II) complexation by 1 yields a
complex with an OH group attached to the C-ring.10 The
1
Figure 3. Aromatic region of the 500 MHz H NMR spectra of
(A) 2; (B) 6; (C) 7; and (D) 8, recorded in DMSO-d6.
(aromatic region) of 2 and three TGA conjugates that were
isolated from the photoreaction. The conjugates formed in a
sequential fashion with the monoconjugate 6 forming first,
followed by the (TGA)2 conjugate 7 and finally the (TGA)3
conjugate 8 with [6] > [7] > [8].19 The conjugate 6 had an
[M + H]+ peak at m/z 355 and a UV-vis absorbance at
(14) Kanony, C.; Akerman, B.; Tuite, E. J. Am. Chem. Soc. 2001, 123,
7985-7995.
(15) Jones, G., II; Kumar, S.; Klueva, O.; Pacheco, D. J. Phys. Chem. A
2003, 107, 8429-8434.
(16) Lightner, D. A.; Crandall, D. C. Tetrahedron Lett. 1973, 1799-
1803.
(17) Melvin, M. S.; Calcutt, M. W.; Noftle, R. E.; Manderville, R. A.
Chem. Res. Toxicol. 2002, 15, 742-748.
(18) Skordos, K. W.; Laycock, J. D.; Yost, G. S. Chem. Res. Toxicol.
1998, 11, 1326-1331.
Org. Lett., Vol. 8, No. 21, 2006
4953

romethene moiety. We propose the mechanism outlined in
Scheme 2 for the production of 6. The first step involves
Scheme 2. Proposed Mechanism for Production of 6
π-radical cation formation with reduction of O2 producing
O2•-, as proposed for BODIPY dyes.15 Attachment of TGA
to the π-radical cation would yield a radical intermediate
that requires H-atom abstraction, possibly by O2•-, to yield
6. The conjugate 6, which is more electron rich than the
parent 2, undergoes further reactions to generate 7 and 8.
These results highlight the photoinduced redox properties
of prodigiosins that may provide a model for biological
activity. For example, Fu¨rstner and co-workers reported that
prodigiosins act as tyrosine phosphatase inhibitors and
speculated that prodigiosin-derived electrophiles may target
active site cysteine residues.20 Our results give credence to
this hypothesis and provide a new means of functionalizing
the pyrromethene chromophore with water-soluble thiol
groups. Compared to the parent pigments, such thioadducts
may display superior biological properties.
Figure 4. NOESY spectrum of 6 in DMSO-d6 showing the B3′-
B4, C4′-C3, and C2′-B4 cross-peaks.
λ
max 510 nm (diode array detector) that is red shifted by 20
1
nm compared to 2. Its H NMR spectrum in Figure 3B
showed three singlets on the pyrromethene ring system
suggesting TGA attachment to the C-ring of 2. Unambiguous
structural assignment of 6 was afforded by the NOESY
spectrum shown in Figure 4; the B3′-B4, C4′-C3, and C2′-
C3 cross-peaks confirmed C4 attachment by the first TGA.
The (TGA)2 conjugate 7 had an [M + H]+ peak at m/z 445
and a UV-vis absorbance at λmax 526 nm that is red-shifted
by 16 nm compared to 6 and by 36 nm compared to 2. For
7, assignment of the B4 proton was achieved from observa-
tion of o-B4 and B3′-B4 cross-peaks in the NOESY
spectrum (Supporting Information, Figure S2). The C3 proton
assignment followed from HMQC and HMBC spectra
(Supporting Information, Figure S3) in which the C2′ methyl
protons at 2.3 ppm showed a long-range correlation (HMBC
spectrum) with the C3 carbon at 113 ppm. The final (TGA)3
conjugate 8 with the single proton on the pyrromethene
moiety (Figure 3D) had an [M + H]+ peak at m/z 535 and
a UV-vis absorbance at λmax 536 nm; its assignment was
confirmed by its NOESY spectrum shown in Supporting
Information Figure S4 that displayed o-B4 and B3′-B4
cross-peaks.
In conclusion, we have demonstrated the photocytotoxicity
of prodigiosins and have shown that the photoreaction
generates thioadducts for the synthetic analogue 2. The
development of more potent prodigiosin-based photocyto-
toxic agents appears possible.
Acknowledgment. Acknowledgment is made to the Drug
Synthesis & Chemistry Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis of
the National Cancer Institute for the sample of prodigiosin.
Supporting Information Available: Experimental details
and Figures S1-S4 described in the text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Photoinduced thioadduct formation by 2 demonstrates the
existence of a reactive intermediate centered on the pyr-
OL061998R
(19) Thioadducts 6-8 are depicted in the R-form, with an E double bond
connecting the B and C rings, for convenience. Our NMR data do not
differentiate between the R-form and the â-form used to depict 1 and 2 in
Figure 1.
(20) Fu¨rstner, A.; Reinecke, K.; Prinz, H.; Waldmann, H. ChemBioChem
2004, 5, 1575-1579.
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