6
652 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Brief Articles
compounds studied were able to photoinactivate directly
both strains of S. aureus.
The photoinactivation of E. coli occurred only in the
presence of the cationic derivatives 3, 6, and 7. The
highest efficiency was obtained with conjugates 6 and
porphyrinic activated ester 4 (80.0 mg, 67.6 µmol), dissolved
in dry DMSO (14 mL), was added dropwise at room temper-
ature. The mixture was stirred in the dark and under nitrogen
for 24 h at 40 °C. The progress of the reaction was monitored
for TLC using acetic acid/methanol/water(5:2:1) as eluent.
After the workup (as described for the preparation of conjugate
5), the conjugate 6‚nHCl (68.0 mg) was obtained as a green
flocculated solid.
7
; thus, positive charges are required to photoinactivate
Gram (-) cells and that polylysine significantly in-
creases the activity of the photosensitizers. However,
the extraordinary efficiency of 6 after 5 min of irradia-
tion when compared with 7 shows that the conversion
of the free amino groups into quaternary ammonium
ones in polylysine induces a smaller efficiency. From
these data we can conclude that the use of conjugate 6
represents the most convenient choice in order to
achieve the most efficient photosensitized inactivation
of both Gram (+) and Gram (-) bacteria.
Porphyrin-Polylysine Conjugate 7. The activated ester
2
(21.0 mg, 27.7 µmol) dissolved in dry DMSO (10 mL) was
added to a solution of poly-S-lysine hydrobromide (26 mg) in
dry DMSO (16 mL) and triethylamine (0.5 mL). After 24 h at
4
0 °C, the mixture was cooled to room temperature, and
methyl iodide (3 mL) was added. The mixture was stirred for
h at 40 °C. After the workup (as described for conjugates 5),
7
the mixture was dialyzed against (1%) hydrochloric acid
aqueous solutions (3 × 300 mL). The conjugate 7‚nHCl (21.5
mg) was obtained as a green flocculated solid. The degree of
homogeneity of the sample was assayed by HPLC analysis (see
Supporting Information).
These results are also promising for the progress of
any research work on the synthesis of novel porphyrin
derivatives and their potential application for the
inactivation of highly antibiotic-resistant Gram (+) and
Gram (-) bacteria. Potentially, this opens the possibility
to obtain a drug with a large spectrum of antimicrobial
activity.
Acknowledgment. Thanks are due to FCT - Fun-
da c¸ a˜ o para a Ci eˆ ncia e a Tecnologia - FEDER (projects
POCTI/38750/FCB/2001 and POCTI/32851/QUI/1999)
and to Universities of Aveiro and Padova, for funding
this work. J. P. C. Tom e´ is also grateful to FCT for a
Post-Doc grant.
Experimental Section
Supporting Information Available: Detailed informa-
tion on the synthesis of porphyrin derivatives 2 and 4,
analytical and spectroscopic data, photostability studies, and
biological results (graphics) of all compounds. This material
is available free of charge via the Internet at http://
pubs.acs.org.
5
-(4-Carboxyphenyl)-10,15,20-tris(4-pyridyl)porphy-
rin (1). 4-Formylbenzoic acid (1.27 g, 8.46 mmol, 1.2 equiv)
and 4-pyridinecarbaldehyde (2.00 mL, 20.9 mmol, 2.9 equiv)
were added to a refluxing mixture of glacial acetic acid (200
mL) and nitrobenzene (150 mL). After the dissolution of the
4-formylbenzoic acid, pyrrole (2.00 mL, 28.9 mmol, 4 equiv)
was added dropwise (ca. 4 min) to the mixture. The reaction
mixture was then refluxed for 1 h. The solvents were distilled
under reduced pressure, and the crude material was taken
into chloroform/methanol (85:15) and directly chromato-
graphed on a silica column using a mixture of chloroform/
methanol (85:15) as eluent. The first fraction was identified
by TLC as 5,10,15,20-tetrakis(4-pyridyl)porphyrin (93 mg, 3%).
The second fraction gave porphyrin 1 (257 mg, 6% yield) after
evaporation ofthe solvent and recrystallization from chloroform/
methanol(85:15)/acetone.
References
(
1) (a) The Porphyrin Handbook - Applications: Past, Present and
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Press: New York, 2000; Vol. 6. (b) Bonnett, R. Chemical Aspects
of Photodynamic Therapy; Gordon and Breach Science Publish-
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Editore: P a´ dua, 1985. (d) Stojiljkovic, I.; Evavold, B. D.; Kumar,
V. Expert Opin. Inv. Drug. 2001, 10, 309-320. (e) Koning, K.;
Teschke, M.; Sigusch, B.; Glockmann, E.; Eick, S.; Pfister, W.
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2) Photosensitizing Compounds: Their Chemistry, Biology and
Clinical Use; Bock, G., Harnett, S., Eds.; John Wiley: Chichester,
General Procedure for the Methylation of Porphyrins
and 2. To a stirred solution (or suspension) of porphyrins 1
1
1
989.
and 2 in dry DMF (5 mL for 25 mg of porphyrin) was added a
large excess of methyl iodide, and the reaction mixture was
heated at 40 °C in one flask equipped with a condenser for
(
3) Bonnett, R.; Buckley, D. G.; Burrow, T.; Galia, A. B. B.; Saville,
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3-4 h. The progress was monitored by TLC using a mixture
2
002, 75, 462-470. (b) Polo, L.; Segalla, A.; Bertoloni, G.; Jori,
of acetic acid/methanol/water (5:2:1) as eluent. When the
reactions were complete, the cationic porphyrins 3 or 4 were
precipitated with diethyl ether, filtered, and washed several
times with the same solvent. The solids were dissolved in
acetone/water (1:1) and reprecipitated with acetone. The
products were dried under reduced pressure.
G.; Schaffner, K.; Reddi, E. J. Photochem. Photobiol. B: Biol.
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Porphyrin-Polylysine Conjugate 5. Poly-S-lysine hy-
drobromide (29 mg) was dissolved in dry DMSO (19 mL), and
triethylamine (0.5 mL) was added to this solution. The
porphyrinic activated ester 2 (24.0 mg, 31.7 µmol), in dry
DMSO (10 mL), was then added dropwise at room tempera-
ture. The reaction mixture was stirred in the dark and under
nitrogen for 24 h at 40 °C. The coupling reaction was monitored
by TLC using chloroform or acetic acid/methanol/water(5:2:1)
as eluents. The reaction mixture was then dialyzed using
membranes of 1000 MW cutoff for 48 h against a 1:1 solutions
of aqueous hydrochloric acid (1%) and methanol (3 × 300 mL).
The retentate solution was concentrated to a few milliliters
under reduced pressure at a maximum temperature of 35 °C.
The resulting solution was frozen in liquid nitrogen and lyo-
philized to give 5‚nHCl (25.5 mg) as a green flocculated solid.
Porphyrin-Polylysine Conjugate 6. Poly-S-lysine hy-
drobromide (64 mg) was dissolved in dry DMSO (50 mL), and
triethylamine (1.5 mL) was added to this solution. The
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(8) The Porphyrin Handbook - Synthesis and Organic Chemistry;
Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press:
New York, 2000; Vol. 1.
(
9) The data are available at Supporting Information.
(
10) Wainwright, M. J. Antimicrob. Chemother. 1998, 42, 13-28.
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(
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JM040802V