Synthesis of β-functionalized Temoporfin derivatives for an application in photodynamic therapy

Article abstract of DOI:10.1016/j.bmcl.2011.07.113

The synthesis of novel β-functionalized derivatives of the clinically used photosensitizer Temoporfin has been achieved by nucleophilic addition reactions to a corresponding diketo chlorin. The β-substituted dihydroxychlorin products exhibit a strong abso

Full text of DOI:10.1016/j.bmcl.2011.07.113

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5808–5811
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of b-functionalized Temoporfin derivatives for an application
in photodynamic therapy
Daniel Aicher a,b, Susanna Gräfe c, Christian B.W. Stark a, , Arno Wiehe c,
⇑
⇑
a Universität Leipzig, Institut für Organische Chemie, Johannisallee 29, 04103 Leipzig, Germany
b Freie Universität Berlin, Institut für Chemie und Biochemie, Takustr. 3, 14195 Berlin, Germany
c biolitec AG, Otto-Schott-Str. 15, 07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of novel b-functionalized derivatives of the clinically used photosensitizer Temoporfin has
been achieved by nucleophilic addition reactions to a corresponding diketo chlorin. The b-substituted
dihydroxychlorin products exhibit a strong absorption in the red spectral region, a high singlet oxygen
quantum yield, and were found to be highly effective in in vitro assays against HT-29 tumor cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 27 July 2010
Revised 27 July 2011
Accepted 29 July 2011
Available online 8 August 2011
Keywords:
Photodynamic therapy (PDT)
Photosensitizer
Photocytotoxicity
Temoporfin
Chlorins
Photodynamic therapy (PDT) is a well established modality for
selective destruction of malignant cells. After administration of
the photosensitizer it is locally activated with laser light. Thus,
cytotoxic singlet oxygen is produced resulting in cell damage or
cell death.1 The first generation sensitizers were based on a por-
phyrin core system but their efficacy is limited by the weak absor-
bance in the red spectral region. However, chlorins and other
second generation sensitizers possess significantly stronger
absorptions at longer wavelengths, thus increasing both the effi-
cacy and the depth of effect.2
has been established as the standard oxidizing agent for the synthe-
sis of such b-dihydroxy-substituted chlorins.5 Other reagents
proved insufficient. We here report the ‘osmium free’ synthesis of
novel b-substituted Temoporfin derivatives6 by nucleophilic addi-
tion reactions to a corresponding diketo chlorin. In addition,
in vitro studies were carried out to preliminarily assess the PDT effi-
cacy of these new chlorins against HT-29 tumor cells.
Our synthetic strategy is related to a route developed by Cross-
ley and co-workers.7 In the present case, this approach involved
preparation of the common dicarbonyl precursor 5. Its synthesis
was accomplished in four steps starting from known porphyrin
Chlorins can principally be obtained either by reduction or oxi-
dation of one of the b-pyrrolic double bonds of porphyrins. For in-
stance, simple b-unsubstituted chlorins are synthesized by
reduction with in situ formed diimide.3 However, the resulting
chlorins are oxidation-susceptible and are often not easily
1
8 (Scheme 1). First, selective mononitration and copper complex-
separable from the starting porphyrin and by-products.4
A
b-unsubstituted chlorin is Temoporfin (FoscanÒ) which carries
meta-hydroxyphenyl groups in meso-positions (Fig. 1). It is a clini-
cally approved photosensitizer in Europe for the palliative treat-
ment of head and neck cancer. An example for the oxidative
functionalization of the porphyrin double bond is the cis-dihydr-
oxylation. Although expensive and very toxic, osmium tetroxide
⇑
Corresponding authors. Address: Universität Hamburg, Institut für Organische
Chemie, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany. Tel.: +49 40 42 838
9014; fax: +49 40 42 838 4325 (C.B.W.S.); tel.: +49 3641 519530 (A.W.).
E-mail addresses: stark@chemie.uni-hamburg.de (C.B.W. Stark), arno.wiehe@-
biolitec.com (A. Wiehe).
Figure 1. Structure of Temoporfin and its novel derivatives.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.113

D. Aicher et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5808–5811
5809
Scheme 1. Synthesis of precursor-chlorin 5.
ation of porphyrin 1 was achieved in one step using Cu(NO3)2 in a
mixture of acetic anhydride and acetic acid.9 The nitroporphyrin
copper(II)-complex 2 was obtained in 87% yield and subsequently
converted to the 2-hydroxyporphyrin derivative 3 by treatment
with benzaldoxime in a solution of sodium methyl-sulfinylmetha-
nide.10 The following decomplexation of copper(II)-porphyrin 3
using concentrated sulfuric acid in dichloromethane failed11 and
led to sulfonated products. Instead, a 10:1 mixture of trifluoroace-
tic acid and sulfuric acid afforded the copper-free meta-methoxy-
phenyl-substituted porphyrin derivative 4 in 79% yield over two
steps. Finally, oxidation using Dess-Martin periodinane (DMP)11
yielded the diketo chlorin 5 in 67% yield. Thus, porphyrin 1 was
efficiently converted to the diketo chlorin 5 in four steps with an
overall yield of 46% (Scheme 1).
With common precursor 5 in hand, the addition of nucleophilic
agents was investigated. In a first experiment chlorin 5 was reacted
with hexyl magnesium bromide affording the dialkylated chlorin 6
in 53% yield (Scheme 2). No mono-addition product was detected.
Pleasingly, the desired double addition product was formed as a
single diastereoisomer. Selective formation of the presumed
trans-diol is believed to be favored due to steric reasons.12
We next focused on fluoro-substituted dihydroxy-chlorins for
mainly two reasons: (i) Generally, fluorinated porphyrins exhibit
higher triplet quantum yields13 and singlet oxygen quantum
yields, respectively, and (ii) the substitution with electron-with-
drawing groups in b-position stabilizes the vicinal diol against
pinacol–pinacolone rearrangement.14 We therefore treated dike-
tone 5 with 3,5-bis(trifluoromethyl)phenyl magnesium bromide
and surprisingly only the two-fold addition product 7 was ob-
tained. However, the use of the electron-deficient and very bulky
Grignard reagent led to a reduced yield of only 44% (Scheme 2).
In order to minimize the steric stress we inserted trifluoromethyl
groups directly without any spacer by using the Ruppert–Prakash
reagent CF3SiMe3/TBAF (Scheme 2).15 This reaction afforded a mix-
ture of the dihydroxychlorin and the corresponding trimethylsilyl
ether. After treatment with additional TBAF the clean diol product
8 was isolated in 78% yield.16
Finally, chlorins 6, 7, and 8 were subjected to boron tribromide
mediated cleavage of the phenolic methyl ethers. The correspond-
ing meta-hydroxyphenyl-substituted products 9, 10, and 11 were
obtained in yields of 57–87%.17,18 These b-substituted chlorins
were found to consist of a mixture of atropisomers because the
Scheme 2. Synthesis of Temoporfin derivatives. Reagents and conditions: (a) For the synthesis of product 6: n-HexMgBr, THF, À45 °C, 3 h, 53%; for the synthesis of product 7:
(CF3)2PhMgBr, THF, À45 °C, 3 h, 44%; for the synthesis of product 8: Me3SiCF3/TBAF, THF, À40 °C, 8 h, 78%. (b) 9, 10, 11 BBr3, CH2Cl2, À50 °C, 16 h, 57–87%.

5810
D. Aicher et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5808–5811
Table 1
as compared to Temoporfin. Chlorin 10 displayed a lower activity
and was only effective at a concentration of 10 mol. None of the
tested sensitizers showed dark toxicity.
Absorption data and 1O2 yield of chlorins 9, 10, and 11a
l
b
Sensitizer
kmax
e
kmax
e
kmax
e
kmax
e
kmax
e
1O2
In conclusion, an ‘osmium free’ strategy for the synthesis of b-
functionalized Temoporfin derivatives is presented. The approach
via a common diketo chlorin intermediate gives broad access to
novel b-substituted dihydroxy-chlorins.20 Alkyl-, aryl-, and trifluo-
romethyl groups could be used as nucleophiles and a clean double
addition reaction was observed. Compared to Temoporfin, the b-
substituted sensitizers possess a significantly increased chemical
stability. They exhibit a comparatively strong absorption in the
red spectral region, a high singlet oxygen quantum yield, and were
highly effective in in vitro assays against HT-29 tumor cells.
9
418
518
544
600
653
21900
644
25000
653
27600
0.97
1.56
1.10
116800
412
10800
514
7400
543
4600
592
10
11
213300
407
18300
518
17800
547
9000
599
166900
15200
15600
7700
a
Absorption spectra in acetone [kmax in nm,
1O2 yields (in EtOH) are given relative to 1O2 quantum yield of Temoporfin.
e
in dm3 molÀ1 cmÀ1].
b
meta-substituted aryl groups next to the oxidized pyrrolic subunit
are hindered in their rotation and the substituents can point inde-
pendently in the same or opposite directions.
Acknowledgments
The support of this research by the Arbeitsgemeinschaft indus-
trieller Forschungsvereinigungen ‘Otto-von-Guericke’ e. V. (AiF)
(Project: KF0579901UL7 and KF0249303UL7) is gratefully
acknowledged.
We next investigated the photophysical properties of the syn-
thesized dihydroxychlorins. In Table 1 the absorption data and
the singlet oxygen yields (relative to 1O2 quantum yield of Temo-
porfin) of the novel chlorins 9, 10, and 11 are summarized. Chlorins
9 and 11 possess a high extinction coefficient at 653 nm whereas
the corresponding Q band of chlorin 10 is shifted to 644 nm. The
singlet oxygen yields for compounds 9 and 11 are quite similar
to that of Temoporfin (Table 1). For the trifluoromethyl-substituted
chlorin 10 an increased relative quantum yield of 1.56 was ob-
served (Table 1). Finally, the photocytotoxicity of sensitizers 9,
10, and 11 was evaluated in cell assays against human colon ade-
nocarcinoma cells HT-29 (Fig. 2).19 The assays were carried out
after incubation for 24 h in 10% FCS containing medium and both
the dark and the phototoxicity were determined at two different
References and notes
1. (a) MacDonald, I. J.; Dougherty, T. J. J. Porphyrins Phthalocyanines 2001, 5, 105;
(b) Allison, R. R.; Downie, G. H.; Cuenca, R.; Hu, X.-H.; Childs, C. J. H.; Sibata, C.
H. Photodiagn. Photodyn. Ther. 2004, 1, 27.
2. Hopper, C. Lancet Oncol. 2000, 1, 212.
3. (a) Whitlock, H. W., Jr.; Hanauer, R.; Oester, M. Y.; Bower, B. K. J. Am. Chem. Soc.
1969, 91, 7485; (b) Bonnett, R.; White, R. D.; Winfield, U.-J.; Berenbaum, M. C.
Biochem. J. 1989, 261, 277.
4. Laville, I.; Figueiredo, T.; Loock, B.; Pigaglio, S.; Maillard, P.; Grierson, D. S.;
Carrez, D.; Croisy, A.; Blais, J. Bioorg. Med. Chem. 2003, 11, 1643.
5. (a) Fischer, H.; Eckoldt, H. Liebigs Ann. Chem. 1940, 543, 138; (b) Brückner, C.;
Dolphin, D. Tetrahedron Lett. 1995, 36, 3295; (c) Sutton, J. M.; Fernandez, N.;
Boyle, R. W. J. Porphyrins Phthalocyanines 2000, 4, 655; (d) Macalpine, J. K.;
Boch, R.; Dolphin, D. J. Porphyrins Phthalocyanines 2002, 6, 146; (e) Pandey, S. K.;
Gryshuk, A. L.; Graham, A.; Ohkubo, K.; Fukuzumi, S.; Dobhal, M. P.; Zheng, G.;
Ou, Z.; Zhan, R.; Kadish, K. M.; Oseroff, A.; Ramaprasad, S.; Pandey, R. K.
Tetrahedron 2003, 59, 10059; (f) Rancan, F.; Wiehe, A.; Nöbel, M.; Senge, M. O.;
Omari, S. A.; Böhm, F.; John, M.; Röder, B. Photochem. Photobiol. 2005, 78, 17; (g)
McCarthy, J. R.; Bhaumik, J.; Merbouh, N.; Weissleder, R. Org. Biomol. Chem.
2009, 7, 3430.
sensitizer concentrations (2 and 10 lmol). A laser with a wave-
length of 652 nm at a dose rate of 50 J/cm2 was used as the light
source. The photodynamic activity was compared to the approved
sensitizer Temoporfin. Chlorins 9 and 11 showed phototoxicity at
both concentrations and exhibited a very similar level of activity
6. For the use of Temoporfin derivatives in PDT see also: (a) Wiehe, A.; Shaker, Y.
M.; Brandt, J. C.; Mebs, S.; Senge, M. O. Tetrahedron 2005, 61, 5535; (b) Gravier,
J.; Schneider, R.; Frochot, C.; Bastogne, T.; Schmitt, F.; Didelon, J.; Guillemin, F.;
Barberi-Heyob, M. J. Med. Chem. 2008, 51, 3867.
control
7. Crossley, M. J.; Burn, P. L.; Langford, S. J.; Pyke, S. M.; Stark, A. G. J. Chem. Soc.,
Chem. Commun. 1991, 1557. see also Ref. 10.
2
10
8. Lindsey, J. S.; Schreimann, I. C.; Hsu, H. C.; Kearney, P. C.; Marguerettaz, A. M. J.
Org. Chem. 1987, 52, 827.
9. Giraudeau, A.; Callot, H. J.; Jordan, J.; Ezhar, I.; Gross, M. J. Am. Chem. Soc. 1979,
101, 3857.
Temoporfin
2
10
10. Crossley, M. J.; King, L. G.; Pyke, S. J. Tetrahedron 1987, 43, 4569.
11. Beavington, R.; Rees, P. A.; Burn, P. L. J. Chem. Soc., Perkin Trans. 1 1998, 2847.
12. Moriconi, E. J.; O’Connor, W. F.; Kuhn, L. P.; Keneally, E. A.; Wallenberger, F. T. J.
Am. Chem. Soc. 1959, 81, 6472.
13. Yang, S. I.; Seth, J.; Strachan, J.-P.; Gentemann, S.; Kim, D.; Holten, D.; Lindsey, J.
S.; Bocian, D. F. J. Porphyrins Phthalocyanines 1999, 3, 117.
2
10
2
9
dark
10
14. Chen, Y.; Medforth, C. J.; Smith, K. M.; Alderfer, J.; Dougherty, T. J.; Pandey, R. K.
J. Org. Chem. 2001, 66, 3930.
toxicity
15. (a) Ruppert, I.; Schlich, K.; Volbach, W. Tetrahedron Lett. 1984, 25, 2195; (b)
Prakash, G. K. S.; Krishnamurti, R.; Olah, G. H. J. Am. Chem. Soc. 1989, 111, 393;
(c) Li, G.; Chen, Y.; Missert, J. R.; Rungta, A.; Dougherty, T. J.; Grossman, Z. D.;
Pandey, R. K. J. Chem. Soc., Perkin Trans. 1 1999, 1785.
photo
2
10
2
toxicity
10
16. Typical procedure for the nucleophilic addition:
A solution of 7,8-dioxo-
10
5,10,15,20-tetrakis-(3-methoxyphenyl)-7,8-chlorin 5 (100 mg, 0.13 mmol) in
dry tetrahydrofuran (7 mL) was cooled under an argon atmosphere to À40 °C.
Trifluoromethyltrimethylsilane (350
lL, 2.66 mmol) and TBAFÁ3 H2O (10 mg,
2
10
2
0.03 mmol) were added and the mixture was stirred for 8 h. In order to cleave
the trimethylsilyl ether additional TBAFÁ3 H2O (100 mg, 0.3 mmol) was added
and the reaction mixture was stirred until TLC analysis showed complete
conversion. Then, water (40 mL) and CH2Cl2 (50 mL) were added, the organic
layer was separated, washed with water (40 mL), dried over anhydrous sodium
sulfate, and the solvent was removed under reduced pressure. The residue was
purified by flash chromatography with CH2Cl2/EtOAc 99:1 as eluent. The
11
10
0
50
cell viability
Figure 2. HT-29 cell assays.
100
compound
7,8-dihydroxy-5,10,15,20-tetrakis-(3-methoxyphenyl)-7,8-bis-
(trifluoromethyl)-7,8-chlorin
CH2Cl2/MeOH. (92 mg, 78%).
8 was obtained after recrystallization from
Compound 8: mp 177 °C; 1H NMR (500 MHz, CDCl3) d 8.63-8.61 (m, 2H), 8.47

D. Aicher et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5808–5811
5811
(s, 2H), 8.03–8.00 (m, 2H), 7.89–7.77 (m, 4H), 7.68–7.43 (m, 6H), 7.35–7.26 (m,
6H), 4.13–4.11 (m, 1H), 4.02–4.01 (m, 1H), 4.00–3.98 (br m, 3H), 3.98-3.97 (m,
3H), 3.91–3.89 (br m, 3H) 3.87–3.85 (m, 3H), À1.48–(À1.52) (br m, 2H); 13C
(126 MHz, CDCl3) d 159.02, 158.20, 153.63, 149.54, 149.17, 142.55, 141.55,
138.98, 138.82, 136.03, 133.24, 128.97, 128.48, 128.38, 127.80, 127.46, 126.91,
125.87, 125.09, 124.53, 124.46, 120.63, 119.81, 119.29, 115.40, 113.84, 111.57,
111.45, 55.61, 55.50; 19F (376 MHz, CDCl3) d À73.92, À73.94, À74.12, À74.15;
128.02, 127.96, 127.90, 127.52, 127.45, 126.21, 126.00, 125.53, 125.48, 125.43,
125.38, 125.33, 125.28, 125.23, 124.37, 124.30, 121.95, 121.39, 121.35, 121.16,
115.58, 115.52, 115.47, 115.40, 115.14, 112.52, 112.44, 112.28, 112.22, 90.4–
89.8; 19F (471 MHz, CD3OD
d
À75.02, À75.05; HRMS (ESI) m/z calcd for
+
C
46H31F6N4O6 (M+H)+ 849.2142. Found 849.2163.
18. It has recently been demonstrated that meta-hydroxy groups in related
porphyrins can efficiently be glycosylated to increase solubility and
amphiphilicity: Aicher, D.; Wiehe, A.; Stark, C. B. W. Synlett 2010, 395.
HRMS (ESI) m/z calcd for C50H39F6N4O6 (M+H)+ 905.2768. Found 905.2774.
+
17. Procedure for the deprotection of methyl ethers: A solution of 7,8-dihydroxy-
5,10,15,20-tetrakis-(3-methoxyphenyl)-7,8-bis-(trifluoromethyl)-7,8-chlorin 8
(80 mg, 0.09 mmol) in dry CH2Cl2 (30 mL) was cooled under an argon
atmosphere to À50 °C. A boron tribromide solution in CH2Cl2 (1 M, 1.6 mL)
was added dropwise over a period of 10 min. The reaction mixture was allowed
to warm up slowly to room temperature and stirred for 18 h. Then water
(100 mL) and ethyl acetate (100 mL) were added as well as sodium hydroxide
solution 30% until neutral. The organic layer was separated, washed with water
(2 Â 100 mL), dried over anhydrous sodium sulfate, and the solvent was
removed under reduced pressure. The residue was purified by flash
chromatography with CH2Cl2/MeOH 95:5 as eluent. Further purification was
achieved by column chromatography with C18 reversed phase silica gel using
MeOH/H2O 95:5 as eluent. The title compound 7,8-dihydroxy-5,10,15,20-
19. Typical procedure for in vitro biological studies: HT-29 cells were grown in
DMEM (cc-pro GmbH) supplemented with 10% heat-inactivated fetal calf
serum (FCS, cc-pro GmbH), 1% penicillin (10000 IU) and streptomycin (10
000 lg/ml, cc-pro GmbH).
A photosensitizer stock solution (2 mM) was prepared in DMSO and was kept
in the dark at 4 °C. Further dilution was performed in RPMI 1640 medium
(without phenol red) supplemented with 10% FCS to reach
a final
photosensitizer concentration of 2 or 10 lM, respectively.
2 Â 104 cells/well were seeded in micro plates and incubated for 24 h with
fresh medium (RPMI without phenol red) containing 10% FCS with 2 or 10 lM
of the photosensitizer. For photosensitization, cells were irridiated at room
temperature with a 652 nm diode laser (Ceralas PDT 652, biolitec AG) at a fixed
fluence rate of 100 mW/cm2 (50 J/cm2).
tetrakis-(3-hydroxyphenyl)-7,8-bis-(trifluoromethyl)-7,8-chlorin
11
was
The cell viability was assessed using the XTT assay and the absorbance of the
samples was measured with a spectrophotometer (Bio-Kinetics Reader EL312
e; Bio-Tek Instruments Inc.) at a wavelength of 490 nm. In order to measure
reference absorbance (to measure non-specific readings) a wavelength of 630–
690 nm was used.
obtained after recrystallization from CH2Cl2/aq. MeOH. (65 mg, 87%).
Compound 11: mp >300 °C; 1H NMR (700 MHz, (CD3)2CO) d 8.82–8.66 (m, 6H),
8.50 (s, 2H), 8.14–8.12 (m, 2H), 7.81–7.11 (m, 16H), 5.75–5.57 (m, 2H), À1.38
to À1.42 (m, 2H); 13C NMR (176 MHz, (CD3)2CO) d 156.35, 156.30, 156.05,
155.99, 155.46, 155.41, 153.44, 150.47, 150.39, 150.28, 150.19, 142.42, 142.39,
141.68, 141.61, 141.58, 141.51, 139.91, 139.87, 135.75, 132.86, 128.15, 128.12,
20. Aicher, D. Ph.D. Thesis, Freie Universität Berlin, May 2010.