Novel porphyrin-psoralen conjugates: Synthesis, DNA interaction and cytotoxicity studies

Article abstract of DOI:10.1039/c3ob41224e

A Cu(i)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) has been utilized to prepare novel triazole-linked cationic porphyrin-psoralen conjugates that exhibited significant photocytotoxicity against A549 cancer cells (IC ₅₀ = 84 nM).

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Novel porphyrin-psoralen conjugates: synthesis, DNA interaction and
cytotoxicity studies
Dalip Kumar,*a Bhupendra A Mishra,a K. P. Chandra Shekar,a Anil Kumar,a Kanako Akamatsu,b
Ryohsuke Kurihara,b and Takeo Ito*b
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
it was found to be less effective than the existing anticancer
drugs and radiation therapy in terms of killing carcinoma
cells. Affinity ligands based on antibodies, protein cage,
50 polypeptide, nanoparticles, and bacteriophage have been
successfully reported to direct lethal photosensitizers to
cancer cells.14 For example, conjugates porphyrin–platin and
porphyrin–paclitaxel exhibited better efficacies and selectivity
towards cancerous cells over normal cells as compared to
55 parent drugs.15aꢀc These studies showed that “dual” approach
has higher therapeutic effect and improved cellular uptake
than single drug therapy.15cꢀe
To attain highly efficient DNA photocleavge activity we
designed novel waterꢀsoluble cationic porphyrin–psoralen
60 conjugates. Porphyrin, a commonly used photosensitizer due
to its clinical significance in both PDT and photodynamic
diagnosis (PDD), was chosen as a high affinity DNA binding
agent to design psoralen conjugate 11. Also, motivated with
the encouraging results of cationic porphyrin conjugates at our
65 hands16 and effectiveness of psoralen scaffolds against
cancers, we prepared waterꢀsoluble novel cationic porphyrin–
psoralen conjugates using click chemistry approach. Click
chemistry is the new concept developed by Sharpless and coꢀ
workers that has attracted significant interest due to its
70 versatility, high yield, simple reaction conditions and their
tolerance of water and oxygen.17 Click reaction involves the
copperꢀmediated cycloaddition of organic azides and alkynes
leading to 1,2,3–triazoles. It is a powerful linking reaction
which has quickly found numerous applications in chemistry,
75 biology, and materials science.17
A
Cu(I)-catalyzed azide-alkyne cycloaddition reaction
(CuAAC) has been utilized to prepare novel triazole–linked
cationic porphyrin–psoralen conjugates that exhibited
10 significant photocytotoxicity against A549 cancer cells (IC50
84 nM).
=
Since many years photodynamic therapy (PDT) has emerged as
an effective modality for the selective treatment of tumors and
neoplastic lesions.1 The mechanism of the tumor destruction is
15 interconnected through several biological responses and still
needs to be fully understood. However, a key step in PDT
requires the accumulation and retention of a photosensitizing
agent in the neoplastic tissue to generate cytotoxic species upon
light exposure.2 Number of strategies have been examined to
20 improve selectivity and specificity of photodynamic sensitizers
towards tumor cells.3 The use of porphyrin hybrids has emerged
as one of the attractive methods.3a,c,d Coupling of porphyrin with
carbohydrates,4 monoclonal antibodies,5 oligoꢀnucleotides,6
peptides,7 has been reported in the recent past. Linking of
25 porphyrin with synthetic compounds or natural products with
established biological significance is found to give better
efficacy, selectivity, and stability as compared with porphyrin
alone.8,9 Moreover, the structural diversity that can be introduced
on porphyrin is limited, whereas any number of hybrids as
30 combinations of parts of different biologically potent motifs can
be prepared. The idea of synthetic porphyrin conjugates as a
treatment modality is still emerging and its potential remains to
be explored.
The photosensitizing 8ꢀmethoxypsoralen (8ꢀMOP), 5ꢀ
35 methoxypsoralen (5ꢀMOP), and congeners are widely used in
psoralen plus ultravioletꢀA radiation (PUVA) therapy as an
effective treatment for various skin diseases and, recently, for
Tꢀcell lymphoma by means of photochemotherapy. The
biological activity of these derivatives has been attributed to
40 their ability to photoreact with DNA.10,11 The photoadducts
were found to be effective towards the inhibition of DNA
synthesis through photoinduced mutagenicity.12 In spite of
this, PUVA therapy has some adverse effects such as
photosensitive skin, formation of cutaneous tumors and
45 induced genotoxicity.13 Some studies revealed that PUVA
induced apoptosis in cultured lymphoma cell lines, however,
Scheme 1 (i) AlCl3, CH2Cl2, rt, 5 h; (ii) 1,2ꢀdibromoethane, K2CO3,
DMF, rt, 6 h; (iii) NaN3, DMF: H2O (3: 1 v/v), 40 °C, 8 h.
We envisaged the synthesis of porphyrin conjugate 11 from
80 psoralen azide 4 and (propargyloxyphenyl)porphyrin 7. The
psoralen azide 4 was prepared as described in Scheme 1.
Demethylation of the 8ꢀmethoxy psoralen 1 with anhydrous
aluminium chloride at room temperature produced exclusively
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DOI: 10.1039/C3OB41224E
hydroxypsoralen 2.18 The alkylation of 2 with dibromoethane led
to 3, which upon treatment with sodium azide in DMF afforded
psoralen azide 4 in good yield (70%).
displayed a peak at m/z 1004 for [M]+ ion that corresponds to the
molecular formula C57H36N10O5Zn.
Porphyrin–psoralen conjugate 10 was demetallated using
We next prepared the porphyrin 5 by using Adler–Longo 45 aqueous HCl in chloroform and treated with excess of methyl
5
protocol.19 Hydrolysis of 5 with potassium hydroxide produced
5–(4′–hydroxylphenyl)–10,15,20–tri(4'–pyridyl)porphyrin in
iodide to afford water–soluble cationic porphyrin–triazole 11.
The structure of conjugate 11 was elucidated by H NMR and
1
6
excellent yield (85%). Treatment of 6 with propargyl bromide
(1.2 equiv.) in the presence of fused K2CO3 in dry DMF afforded
MALDI–TOF spectral data. The 1H NMR spectrum of 11 showed
a characteristic singlet at δ 4.73 for –N+CH3 protons. The β–
50 pyrrolic and N–methylpyridinium protons (H–2' & H–6') in 11
were shifted downfield when compared to porphyrin conjugate
10.20 The HRMS (MALDI–TOF) spectrum of 11 displayed a
peak at m/z 987.2841 that is in agreement with calculated mass
987.2814 for C60H47N10O5 (M+–3I−). The purity of the isolated
5–(4′–propargyloxyphenyl)–10,15,20–tri(4'–pyridyl)porphyrin
7
1
10 in 53% yield (Scheme 2). The H NMR spectrum of 7 showed
two characteristic singlets at δ 5.10 (OCH2) and δ 2.88 (C≡CH)
along with other protons. The coupling reaction of 7 with
psoralen azide 4 (3.0 equiv.) in the presence of CuSO4.5H2O (1.0
equiv.) and sodium ascorbate (2.0 equiv.) in DMF: H2O (1: 1 v/v) 55 porphyrin conjugate 11 was greater than 99% as evaluated by
15 at 80 °C for 36 h afforded metallated porphyrin–psoralen
conjugate 9. We attempted demetallation of compound 9 in 10%
TFA/H2SO4 but the reaction afforded (4′ꢀhydroxypheny)ꢀ
10,15,20–tri(4'–pyridyl)porphyrin 6 as the only isolable product
with the cleavage of propargyloxy group. So to avoid metallation
20 of porphyrin core during the CuAAC reaction we converted the
free base porphyrin 7 to zinc metallated porphyrin 8 by treatment
with Zn(OAc)2 in chloroform–methanol. Cycloaddition reaction
of 8 with 4 (3 equiv.) using CuSO4.5H2O (4 equiv.) and sodium
ascorbate (8 equiv.) at 80 °C in DMF: H2O for 168 h gave
25 porphyrin–psoralen conjugate 10 (Scheme 2). With lesser
equivalents of CuSO4.5H2O and sodium ascorbate, and at lower
temperatures, the reaction was very sluggish which led to
decomposition of psoralen azide 4 and poor yield of the desired
product.
HPLC analysis.20
The interactions of cationic porphyrin conjugate 11 and
5,10,15,20ꢀtetrakis(Nꢀmethylpyridiniumꢀ4ꢀyl)porphyrin (TMPyP)
with calf thymus DNA (ctDNA) were studied using UVꢀvis
60 spectroscopy (Fig. 1). The absorption spectra of cationic
porphyrin conjugate 11 (2 ꢁM) was recorded with increasing
0.4
ctDNA conc.
0
ꢀ
M
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
0.2
0.4
0.6
0.8
1.0
2.0
3.0
4.0
5.0
M
M
M
M
M
M
M
M
M
0.2
0.0
30
400
500
Wavelength (nm)
600
700
Fig. 1 Absorption spectra of conjugate 11 (2 ꢁM) with increasing ctDNA
concentrations 5 mM TrisꢀHCl, 0.1 M NaCl, pH 8.0, 25 °C).
(
65 amounts of ctDNA (0ꢀ55 ꢁM). Absorption spectra of 11 showed
a red shift of 12 nm and hypochromicity of 27% indicating
intercalative binding towards ctDNA. Whereas, TMPyP showed a
redshift of 16 nm and hypochromicity of 41%. Further, we
carried out competitive binding study for the conjugate 11 using
70 the emission intensity of DNA intercalator ethidium bromide
(EB).21 EB bound to ctDNA exhibits several fold enhancement in
its fluorescence intensity which can be quenched by the addition
of another DNA binding molecule. As shown in Fig. 2 with the
increasing amounts of conjugate 11 an appreciable reduction in
75 the fluorescence intensity of the ctDNAꢀbound EB was observed.
The quenching plot of I0/I vs [Por 11]/[DNA] was found to be in
good agreement with the linear Stern–Volmer equation with a
slope of 3.31 for 11 (ESI, Fig. S1).22 The decrease in fluorescence
intensity could be attributed to the appreciable replacement of the
80 EB bound to ctDNA by the intercalation of conjugate 11.
Scheme 2 (i) KOH, MeOH: H2O, reflux, 5 h; (ii) propargyl bromide (1.2
equiv), K2CO3 (1.2 equiv), DMF, 0–27 °C, 24 h; (iii) Zn(OAc)2, CHCl3:
MeOH, reflux, 2 h; (iv) CuSO4.5H2O, sodium ascorbate, DMF: H2O (1: 1
35 v/v), 80 °C, 32–168 h; (v) 10, CHCl3, aq HCl (25%), 1 h; (vi) MeI (120
equiv), DMF, rt, 36 h.
The proton NMR spectrum of porphyrin–psoralen conjugate
10 showed a singlet at δ 5.77 (–C6H4OCH2–N), two triplets at δ
5.00 (triazolylꢀNCH2) and δ 3.76 (triazolylꢀNCH2CH2Oꢀ) along
40 with two doublets at δ 8.17 and 6.82 for C3′–H and C4′–H protons
of psoralen, respectively. Mass spectrum of conjugate 10
The DNA cleavage activities of the porphyrin conjugate 11
were examined by its effectiveness in converting circular
2
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DOI: 10.1039/C3OB41224E
30
25
20
15
10
5
[Por 11]
0.0
ꢀM
0.23
0.35
0.46
0.59
0.75
0.94
1.17
1.41
1.88
ꢀ
M
M
M
M
M
M
M
M
M
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
11 (UV)
11 (Vis)
9 (UV)
9 (Vis)
45 Fig. 4 Cell viablility data for porphyrin conjugates (9 and 11).
0
600
Wavelength (nm)
700
Table 1 Phototoxicity results of 2, 9, 11 and TMPyP towards A549.
a
IC50
Fig. 2 Emission spectra of EB (5 ꢂM) bound to DNA (12 ꢂM) in 5 mM
TrisꢀHCl, 0.1 M NaCl, (pH 8.0) at λex = 510 nm in the absence and
presence of varying concentrations of 11.
Porphyrin
Dark
Visible
84 nM
UVꢀA
146 nM
3.18 ꢁM
659 nM
>100 ꢁM
11
>100 ꢁM
>100 ꢁM
>100 ꢁM
>100 ꢁM
9
>10 ꢁM
171 nM
>100 ꢁM
5
TMPyP
supercoiled DNA (form I) to circular relaxed (form II) DNA (Fig.
3). The conjugate 11 (1ꢀ25 ꢁM) and plasmid ΦX174 DNA (0.5
ꢁg) were incubated in TrisꢀHCl (20 mM) buffer solution (pH 7.2)
at ambient temperature for 30 min, then exposed to UV–A (310–
10 390 nm, 4 mW) or visible (400–800 nm, 2 mW) light. As shown
in Fig. 3, significant single strand cleavage was observed for the
2
a The results are the mean values of at least 3 independent experiments.
This enhanced photocytotoxicity relative to the dark control is
50 an essential property for a photochemotherapeutic agent. As
envisioned the cationic conjugate 11 exhibited higher
phototoxicity than TMPyP (IC50 171–659 nM), which has been
extensively studied as a tumor–localizing, potent photosensitizing
drug.24
To visualize nuclear changes and apoptotic body formation that
are characteristic of apoptosis, A549 cells were treated with 11
(0.1 ꢀM) for 24 h, washed with phosphate buffered saline, and
15
55
Fig. 3 Photoinduced DNA cleavage by 11. ΦX174 supercoiled DNA (0.5
20 ꢀg) was incubated with 11 (0ꢀ25 ꢀM) in Tris–HCl (20 mM, pH 7.2)
containing NaCl (20 mM), DMSO (2.5 vol%) at ambient temperature in
the dark for 30 min, and then exposed to (lanes 1–4) UV–A or (lanes 5–8)
visible light for 10 min.
25 UV and visible–light exposed 11. It should be noted that high
concentrations of 11 caused mobility change and darkening of the
DNA bands, probably due to intercalation of 11 into plasmid
DNA. It has been reported that psoralen intercalates into DNA
duplex and forms interstrand crossꢀlinks in DNA upon UVꢀ
30 irradiation.23 We performed restriction enzyme assay for the
photoꢀirradiated plasmid DNA samples and confirmed that
counjgate 11 could induce interstand crosslinking (See ESI,
Fig S5,6). It is thus likely that the crossꢀlink formation
between 11 and DNA lowered mobility of the products.
35
The photocytotoxicities of 9 and 11 towards A549, a human
epithelial cell line derived from a lung carcinoma, were
determined by WST assay. The cell viabilities were ploted
against the concentrations of conjugates (Fig. 4) and the results
are listed in Table 1, in which cytotoxicities are expressed as the
60 Fig. 5 Fluorescence microscopic images of Hoechst 33342ꢀstained A549
cells. Normal appearance of nuclei in control cells (a); Cells were
incubated in the presence of 11 (0.1 ꢀM) in the dark (b); Cells were
exposed to UVꢀlight (4 mW, 10 min) (c, d) or visibleꢀlight (2 mW, 10
min) (e, f) in the presence (c, e) or absence (d, f) of 11 (0.1 ꢀM).
40 concentration of the drugs that inhibit 50% of cell proliferation
(IC50). Interestingly, 9 and 11 showed potent toxicities with IC50
values of 3.18 ꢁM and 84 nM, respectively after UV–A or visible
light exposure; whereas both conjugates are nontoxic (IC50 >100
ꢁM) in the dark.
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51, 2915; (c) J. Kralova, Z. Kejık, T. Briza, P. Pouckova, A. Kral, P.
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then were exposed to UV or visible light for 10 min. After further
incubation for 24 h, cells were stained with Hoechst 33342 (Fig.
5). The nonꢀirradiated cells (Fig. 5d & 5f) showed no remarkable
changes as compared to the control (Fig. 5a), while after the cells
were exposed to UV or visible light (Fig. 5c & 5e), a number of
bright spots appeared in the cells. Such morphological changes of
nuclei are often observed in apoptotic cells, and thus we
preliminarily conclude that the cationic porphyrin conjugate 11
may induce cell death in A549 cells through an apoptotic
8
9
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In summary, we have prepared a novel water soluble cationic
porphyrin–psoralen conjugate which exhibited high
photocytotoxicity towards A549 cancer cells when compared to a
tumor–localizing and potent photosensitizing agent, TMPyP. This
15 work demonstrates that porphyrin–psoralen conjugate 11 could
be a potential candidate for PDT. Further mechanistic and
structureꢀactivity relationship studies of porphyrin–psoralen
conjugates are underway in our laboratory and will be reported in
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20 Acknowledgements
The authors acknowledge DST, New Delhi (SR/S1/OC 77/2006)
and UGC(SAP), New Delhi for the financial support. BM is
grateful to CSIR, New Delhi for award of SRF (F. No.
09/719(0042) /2011.EMRꢀI). We thank AIRF, JNU and SAIF,
25 Panjab University for spectral analysis.
95
Notes and references
aDepartment of Chemistry, Birla Institute of Technology & Science,
Pilaniꢀ 333 031 India Fax: +91–1596–244183; Tel: +91–1596–245073–
5238; E–mail: dalipk@pilani.bits–pilani.ac.in
30 bDepartment of Energy and Hydrocarbon Chemistry, Graduate School of
100
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Engineering,
Kyoto
University,
Kyoto,
615–8510,
Japan;
E–mail: takeoit@scl.kyoto–u.ac.jp
†Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/b000000x/
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C. Casas, B. S. Jalmes, C. Loup, J. Lacey and B. Meunier, J. Org.
Chem., 1993, 58, 2913.
20 Preparation of cationic porphyrin–psoralen conjugate 11: To a
stirred solution of metallated porphyrin 8 (0.12 g, 0.163 mmol) in
DMF:H2O (1:1; 70 mL) was added CuSO4.5H2O (0.163 g, 0.653
mmol), sodium ascorbate (0.259 g, 1.306 mmol) followed by psoralen
azide 4 (0.132 g, 0.49 mmol). The reaction mixture was allowed to
stir at 80 °C for 168 h. After completion of the reaction, the contents
were diluted with chloroform (4 × 80 mL) and ammonia solution
(20%, 30 mL). The resulting solution filtered over a thin celite bed
and washed thoroughly with chloroform. The combined organic phase
was washed with water, dried over anhydrous sodium sulfate and the
solvents were evaporated in vacuo. The residue thus obtained was
purified by column chromatography using chloroform/methanol (8:2)
as eluent to afford pure porphyrin conjugate 10 (0.082 g, yield 52%)
as a red solid. To a cooled solution (5ꢀ10 °C) of Zn(II) conjugate 10
(0.091 g) in chloroform (80 mL) was added aq. HCl (25%, 20 mL)
and stirred for 1 h at 27 °C. After complete demetallation, the
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temperature was lowered to 0–2 °C and the reaction mixture was
basified with ammonia solution, the organic layer was separated and
dried over anhydrous sodium sulfate. The solvents were distilled off
in vacuo to afford demetallated porphyrin triazole (0.085 g), which
was dissolved in dichloromethane:methanol (9:1), precipitated by
adding hexane, filtered, dried and used as such for further reaction. To
a solution of demetallated porphyrin triazole (0.085 g, 0.091 mmol) in
dry N,N'ꢀdimethylformamide (20 mL) was added methyl iodide (0.75
mL, 11.73 mmol) and allowed to stir at room temperature until
starting material was consumed completely (36 h). The solvent was
removed in vacuo and methanol (5 mL) was added. The compound
was precipitated by adding diethyl ether (15 mL), and the resulting
solid was filtered. This procedure was repeated four times to afford
compound 11 (0.071 g, yield 65%) as brown solid. m.p. > 300 °C; 1H
NMR (500 MHz, DMSO–d6) δ 9.48–9.47 (m, 6H, 2′,6′ Me–+N–
pyridiniumyl H), 9.32–9.01 (m, 14H, 3′,5′ Me–+N–pyridiniumyl H &
β–pyrrole H), 8.25 (d, J = 9.9 Hz, 1H, psoralen C3′H), 8.20 (d, J = 8.9
Hz, 1H, psoralen C7′H), 7.88 (s, 1H, triazolyl C5′′H), 7.48 (d, J = 8.2
Hz, 2H, 2′,6′ mesoꢀphenyl H), 7.33 (s, 1H, psoralen C5′H), 7.24–7.23
(m, 3H, 3′,5′ meso–phenyl H & psoralen C6′H), 6.75 (d, J = 9.9 Hz,
1H, psoralen C4′H) 5.83 (s, 2H, –C6H4OCH2–triazol), 5.22 (t, J = 7.5
Hz, 2H, triazolyl–CH2CH2O–), 4.73 (s, 9H, 3 × –N+CH3 ), 3.99 (t, J =
7.4 Hz, 2H, triazolyl–CH2CH2O–), −3.01 (s, 2H, pyrrole NH); 13C
NMR (126 MHz, DMSO–d6) δ 161.10 (O–C=O–, psoralen) 159.05,
151.66, 151.00, 148.58, 148.08, 147.10, 144.28, 143.27, 142.75,
140.44,138.45, 133.18, 132.13, 131.89, 131.18, 127.35, 126.94,
124.18, 122.49, 118.81, 116.11, 114.98. 114.88, 107.19, 67.21
(OCH2–triazol), 56.97 (–OCH2CH2–), 47.30 (–N+CH3); MALDI–
TOF: m/z calcd for C60H47N10O5 [M+–3Iꢀ]: calcd: 987.2814; found:
987.2841; HPLC purity: 99.24%.
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21 J. ꢀB. Le Pecq, P. Yot and C. C. Paoletti, R. Acad. Sci., 1964,
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22 W. –J. Mei, J. Liu, H. Chao and L. –N. Ji, Transit. Met. Chem., 2003,
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3523 (a) D. W. Ussery, R. W. Hoepfner and R. R. Sinden, Method
Enzymol., 1992, 212, 242; (b) D. Bethea, B. Fullmer, S. Syed, G.
Seltzer, J. Tiano, C. Rischko, L. Gillespie, D. Brown and F. P.
Gasparro, J. Dermatol. Sci., 1999, 19, 78.
24 A. Rück, T. Köllner, A. Dietrich, W. Strauss and H.
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Schneckenburger, J. Photochem. Photobiol. B: Biol., 1992, 12, 403.
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