Novel bioconjugates of aminolevulinic acid with vitamins

Article abstract of DOI:10.1021/ol801496j

(Chemical Equation Presented) 5-Aminolevulinic acid (ALA) and derivatives thereof have been successfully used in photodynamic cancer therapy (PDT). The synthesis of novel bioconjugates combining ALA with two lipophilic and one hydrophilic vitamins is repo

Full text of DOI:10.1021/ol801496j

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4453-4455
Novel Bioconjugates of Aminolevulinic
Acid with Vitamins
Ramakrishnan Vallinayagam, Joanne Weber, and Reinhard Neier*
Institut de Chimie, UniVersite´ de Neuchaˆtel, Rue Emile-Argand,
11, PO 158, Neuchatel, Switzerland. CH-2009
reinhard.neier@unine.ch
Received July 2, 2008
ABSTRACT
5-Aminolevulinic acid (ALA) and derivatives thereof have been successfully used in photodynamic cancer therapy (PDT). The synthesis of
novel bioconjugates combining ALA with two lipophilic and one hydrophilic vitamins is reported. The new bioconjugates allow studying the
potential synergies between the two components in PDT. The synthetic methodology is robust giving the bioconjugates in good to satisfactory
yield.
Photodynamic therapy (PDT) is a minimally invasive treat-
ment for the cure of malignant diseases.1-3 As clearing of
the photosensitizer is often a limiting factor in PDT, the study
of 5-aminolevulinic acid (ALA), the natural precursor of the
endogenous photosensitizer protoporphyrin IX (PpIX), has
been receiving increasing attention.4,5 ALA is unstable at
neutral or slightly basic pH6 and lacks selectivity toward
malignant tissues.7 Esters of ALA have been successfully
applied to circumvent the problem of the insufficient lipo-
philicity.8 Bioconjugates of ALA with amino acids and
simple peptides have been synthesized and studied to target
tumor cells (Figure 1).9 We recently synthesized a new class
of ALA bioconjugates, using monosaccharides.10 On the
(1) (a) Hopper, C. Lancet Oncol. 2000, 1, 212. (b) Dolmans, D. E. J. G.
J.; Fukumura, D.; Jain, R. K. Nat. ReV. Cancer 2003, 3, 380. (c) Alison,
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J. L.; Flotte, T. J.; Deutsch, T. F.; Anderson, R. R. Arch. Dermatol. Res.
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E. H.; MacRobert, A.; Riley, P. A. Br. J. Cancer 1999, 80, 1525. (c) Di
Venosa Gabriela, M.; Casas, A. G.; Battah, S.; Dobbin, P.; Fukuda, H.;
Macrobert, A. J.; Batlle, A. Int. J. Biochem. Cell Biol. 2006, 38, 82. (d)
Godal, A.; Nilsen, N. O.; Klaveness, J.; Braenden, J. E.; Nesland, J. M.;
Peng, Q. J. EnViron. Pathol. Toxicol. Oncol. 2006, 25, 109.
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37, 272. (b) Lopez, R. F. V.; Lange, N.; Guy, R.; Bentley, M. V. L. B.
AdV. Drug DeliVery ReV. 2004, 56, 77.
(5) (a) Kloek, J.; Beijersbergen van, H. Photochem. Photobiol. 1996,
64, 994. (b) Peng, Q.; Moan, J.; Warloe, T.; Iani, V.; Steen, H. B.; Bjorseth,
A.; Nesland, J. M. J. Photochem. Photobiol., B 1996, 34, 95.
(6) (a) Gadmar, O. B.; Moan, J.; Scheie, E.; Ma, L.-W.; Peng, Q. J.
Photochem. Photobiol., B 2002, 67, 187. (b) Bunke, A.; Zerbe, O.; Schmid,
H.; Burmeister, G.; Merkle, H. P.; Gander, B. J. Pharm. Sci. 2000, 89,
1335. (c) Elfsson, B.; Walin, I.; Eksborg, S.; Rudaeus, K.; Ros, A. M.;
Ehrsson, H. Eur. J. Pharm. Sci. 1999, 7, 87. (d) Butler, A. R.; Georger, S.
Tetrahedron 1992, 48, 7879.
(9) (a) Berger, Y.; Greppi, A.; Siri, O.; Neier, R.; Juillerat-Jeanneret,
L. J. Med. Chem. 2000, 43, 4738. (b) Berger, Y.; Ingrassia, L.; Neier, R.;
Juillerat-Jeanneret, L. Bioorg. Med. Chem. 2003, 11, 1343. (c) Rogers,
L. M. A.; McGivern, P. G.; Butler, A. R.; MacRobert, A. J.; Eggleston,
I. M. Tetrahedron 2005, 61, 6951.
(10) (a) Vallinayagam, R.; Schmitt, F.; Barge, J.; Wagnieres, G.; Wenger,
V.; Neier, R.; Juillerat-Jeannerat, L. Bioconjugate Chem. 2008, 19, 821.
(b) Vallinayagam, R. Synthesis of Novel Prodrugs for Targeted Photody-
namic Therapy, Ph.D. thesis, University of Neuchatel, 2007.
10.1021/ol801496j CCC: $40.75
Published on Web 09/18/2008
2008 American Chemical Society

Scheme 1. Synthesis of Tocopherol-ALA Esters Using the
Boc-Protected ALA 9 or the Azido-Protected ALA 11
Figure 1. Examples of ALA pseudopeptides synthesized and tested
in PDT as reported in refs 9a and b.
basis of our synthetic methodology, we have undertaken
studies investigating a variety of bioconjugates of ALA. We
were interested to expand our approach to novel classes of
bioconjugates coupling ALA with lipophilic and hydrophilic
vitamins. Herein, we report the first successful synthesis of
these new bioconjugates. Our approach is robust and can
therefore be used for the synthesis of many other derivatives
of ALA.
In the past, 5-ALA-hydrochloride (ALA-HCl) has been
used as the starting material of choice. ALA-HCl is
hygroscopic and easily forms side products. In our hands,
the Boc-protected form of ALA 9 (Boc-ALA), the activated
oxime ester of Boc-ALA 10, or the 5-azidolevulinic acid 11
proved to be reliable precursors for the synthesis of novel
ALA bioconjugates11 avoiding most of the problems associ-
ated with the use of 5-ALA-HCl.
Bioconjugates between vitamins and ALA were attractive
choices to show the applicability of our methodology. We
choose vitamin E (12) and vitamin D3 (16) as representatives
for the lipophilic vitamins and biotin (19) as an example of
hydrophilic vitamins. R-Tocopherol was chosen because of
its ease of incorporation into natural membranes, which might
facilitate the transport of ALA into the interior of cancer
cells.12 The lipophilic character of cholecalciferol (16) should
facilitate the incorporation of the bioconjugate into cell
membranes. It has been proposed that the observed anticancer
effects of vitamin D are mediated through the vitamin D
receptors (VDR).13 In contrast to the lipophilic vitamins
mentioned above, biotin (vitamin H) is water soluble, and
no reports on activity related to cancer have been reported.
We decided to test the application of our strategy to this
vitamin to find out if our methodology is compatible with
water-soluble components.
the presence of DMAP allowed us to achieve the efficient
synthesis of the bioconjugates between 9 and vitamin E.
Deprotection of the Boc group under acidic conditions
provided the vitamin E ALA bioconjugate 14 in good yield
and high purity. The coupling of 9 with vitamin D3 (16) was
realized successfully under standard conditions. Trials to
deprotect the Boc group of 17 using trifluoroacidic acid led
to degradation (Scheme 2), probably due to the sensitivity
of the vitamin part.
We tested an alternative approach to these two bioconju-
gates using the azido-protected ALA 11 as a precursor (see
Schemes 1 and 2). We could couple 11 with the two vitamins
under standard coupling conditions in good yields. Treating
the mixture of 11 with R-tocopherol (12) in equimolar
amounts with DCC as the coupling reagent and DMAP in
catalytic quantities gave an excellent yield of the ester 15 of
83% in high purity. The azido derivative is stable and can
be stored without degradation. The conversion of the azido
function in the bioconjugate with tocopherol 15 could be
achieved by catalytic hydrogenation using palladium on
charcoal in the presence of trifluoroacetic acid to trap the
amino group. The deprotected bioconjugate 14 could be
isolated in 90% yield and in excellent purity. The biocon-
jugate 18 between azido-protected ALA 11 and cholecal-
ciferol (16) could be obtained in moderate yield (Scheme
2). We were not able to transform the azido function into
the protonated amine under the conditions mentioned above.
To form the bioconjugate between biotin and the amine
of ALA via an amide linkage, we synthesized the pentafluoro
phenyl ester of biotin first, which was treated with methyl
5-azidolevulinate (21) under catalytic hydrogenation condi-
tions (Scheme 3).
We synthesized the direct conjugate between vitamin E
(12) and ALA using the Boc-protected precursors of ALA
9 (Scheme 1). Standard coupling conditions using DCC in
(11) Vallinayagam, R.; Bertschy, H.; Berger, Y.; Wenger, V.; Neier, R.
Synthesis 2007, 3731.
(12) (a) Goh, S. H.; Hew, N. F.; Norhanom, A. W.; Yadav, M. Int. J.
Cancer 1994, 57, 529. (b) Sigounas, G.; Anagnostou, A.; Steiner, M. Nutr.
Cancer 1997, 28, 30.
(13) (a) Gewirtz, D. A.; Gupta, M. S.; Sundaram, S. Curr. Med. Chem.:
Anti-Cancer Agents 2002, 2, 683. (b) Evans, T. R. J.; Anthoney, D. A.;
DeBono, J. S.; O’Neill, V. J.; Morrison, R.; McInnes, A. Curr. Top. Steroid
Res. 2000, 3, 89.
The methyl azidolevulinate (21) is reduced under these
conditions in situ to methyl aminolevulinate. The newly
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Org. Lett., Vol. 10, No. 20, 2008

Scheme 2
.
Synthesis of Cholecalciferol-ALA Esters Using the
Scheme 3. Synthesis of the Biotin-ALA Amide
Boc-Protected ALA 9 and the Azido-Protected ALA 11
The concomitant delivery of both partners of the bioconjugate
to the cancer cells might create synergies. The synthetic
methodologies reported for the synthesis of bioconjugates
are robust and general. The most delicate step is the final
deprotection, which has not been successful in the case of
the bioconjugate with the acid-sensitive cholecalciferol.
Acknowledgment. This research was supported by the
Swiss National Science Foundation (Funding number 3152A0-
102184) and the University of Neuchatel. NMR and mass
spectra were measured on instruments at the Universities of
Neuchatel and Fribourg acquired with the assistance of grants
from the Swiss National Science Foundation. We would like
to thank Dr. Lucienne Juillerat-Jeanneret, Fre´de´ric Schmitt
from CHUV Lausanne, for discussions on biological and
clinical aspects of this research.
formed amine attacks the activated pentafluoro phenyl ester
of biotin. The activated pentafluoro phenyl ester of biotin
was added in excess to prevent the formation of the pyrazine-
type impurity by self-condensation of two methyl aminole-
vulinate molecules. Under these optimized conditions, a
moderate, but reproducible, yield of the bioconjugate 22
could isolated.
We report the synthesis of bioconjugates combining ALA
with lipophilic and hydrophilic vitamins. These novel
compounds open the opportunity to target the delivery of
ALA to tumor cells and to enhance the efficiency of PDT.
Supporting Information Available: Detailed procedures
for the preparation and characterization of all compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL801496J
Org. Lett., Vol. 10, No. 20, 2008
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