Synthesis of glycoporphyrin derivatives and their antiviral activity against herpes simplex virus types 1 and 2

Article abstract of DOI:10.1016/j.bmc.2005.04.015

Studies on the synthesis, structural elucidation, and antiviral evaluation of several carbohydrate-substituted meso-tetraarylporphyrins against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are described. The potential of those photosensitizers, and of their precursors, on the photoinactivation of HSV-1 and HSV-2 was examined in Vero cells. Their virucidal and viral replication effects were assessed under white light, at their maximum noncytotoxic concentrations. The highest inhibitory effects on viral replication, for both viruses, were obtained with the glycoporphyrins where the sugar moiety bears unprotected hydroxyl groups. Strong inhibition of virus yield was observed even at concentrations much lower than their maximum noncytotoxic concentrations. These compounds can be postulated to be useful as potential drugs for the treatment of herpes simplex viruses infections.

Full text of DOI:10.1016/j.bmc.2005.04.015

Bioorganic & Medicinal Chemistry 13 (2005) 3878–3888
Synthesis of glycoporphyrin derivatives and their antiviral
activity against herpes simplex virus types 1 and 2
a
Joa˜o P. C. Tome, Maria G. P. M. S. Neves, Augusto C. Tome, Jose A. S. Cavaleiro,
a
a
a,
*
´
Ana F. Mendonc¸a, Ines N. Pegado, Ricardo Duarte and Maria L. Valdeira
´
´
b
b
b
b
ˆ
aDepartment of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
bCenter of Genetics and Molecular Biology, Faculty of Pharmacy, University of Lisbon, 1649-019 Lisbon, Portugal
Received 8 February 2005; accepted 8 April 2005
Available online 4 May 2005
Abstract—Studies on the synthesis, structural elucidation, and antiviral evaluation of several carbohydrate-substituted meso-tetra-
arylporphyrins against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are described. The potential of those photosensi-
tizers, and of their precursors, on the photoinactivation of HSV-1 and HSV-2 was examined in Vero cells. Their virucidal and viral
replication effects were assessed under white light, at their maximum noncytotoxic concentrations. The highest inhibitory effects on
viral replication, for both viruses, were obtained with the glycoporphyrins where the sugar moiety bears unprotected hydroxyl
groups. Strong inhibition of virus yield was observed even at concentrations much lower than their maximum noncytotoxic concen-
trations. These compounds can be postulated to be useful as potential drugs for the treatment of herpes simplex viruses infections.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
odologies to inactivate viruses. Photodynamic therapy
(PDT) can become a promising method. It is being used
in the treatment of cancer, psoriasis, age-related macular
degeneration, and also in the inactivation of microor-
ganisms and viruses.8–14 This type of therapy requires
a combination of a photosensitizer, oxygen, and light.
The mechanism by which the cell destruction occurs is
thought to involve mainly the disruption of the cellular,
mitochondrial, or nuclear membranes by cytotoxic
agents such as singlet oxygen.15 The main advantages
associated with the use of PDT over conventional che-
motherapy are: (a) the action of the pharmacophore
can be ꢀturned on or offꢁ, since no reaction occurs in
the absence of light; (b) the duration time of the action
can be controlled, and (c) both the photosensitizer and
light can be selectively directed to the locations to be
treated.15
Herpesviruses are responsible for a wide range of human
diseases, of special concern in immunocompromised
individuals, namely AIDS, hematological malignancies,
after organ bone marrow transplantation, fulminant
hepatitis, and infections in transplant recipients.1–4 They
have also been implicated in the development of several
malignancies of epithelial or lymphatic origin.5
The transmission of pathogenic viruses by blood trans-
fusion is still a subject of concern, since absolute safety
has not yet been achieved. Sterilization of blood and its
components seems to be the best way to obtain safe
products. However, the sterilization of cellular blood
components presents a unique challenge, since cell struc-
ture and function are easily disrupted.6 The photochem-
ical sterilization of such products using effective
photosensitizers might be a solution to this problem.7
Amongst the various types of photosensitizers used in
PDT, the porphyrins are the most extensively studied
and are the ones approved so far for clinical use.9,16–19
Several groups are involved in structural modification
of porphyrins in order to generate compounds with
the photophysical and hydrophobic/hydrophilic proper-
ties required for an ideal photosensitizer. Porphyrins
with carbohydrate moieties have been described as effi-
cient photosensitizers to be used in PDT.18,20–28 That
The fact that few antiviral drugs of proven effectiveness
exist has prompted the search for new drugs and meth-
Keywords: Porphyrins; Sugars; Antiviral compounds; HSV-1; HSV-2.
*
Corresponding author. Tel.: +351 234 370 712; fax: +351 234 370
084; e-mail: jcavaleiro@dq.ua.pt
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.015

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3879
is mainly due to the specific affinity of several carbohy-
drates for cancer cells.29,30 Following our interest on the
development of porphyrins with potential use in medi-
cine,31–33 we have set up a work program on the synthe-
sis of carbohydrate-substituted porphyrins and the
study of their efficiency for the photo-inactivation of
HSV-1 and HSV-2. Our interest in finding new drugs
to inactivate herpes simplex virus is related to the fact
that some viral strains are very often resistant to drugs
commonly employed in the treatment of herpetic infec-
tions.34–37 Acyclovir38 and foscarnet36 are often em-
ployed to treat infections such as herpetic encephalitis.
However, these antiviral agents are not able to inhibit
resistant viruses and can lead to hemotoxicity.38
hexafluorobenzene, in DMF, in the presence of sodium
hydride.
The monosaccharide derivatives selected for this work
were 1-bromo-2,3,4,6-tetra-O-acetyl-a-D-glucopyranose
and 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose.
These are commercially available starting materials,
with protecting groups which can be easily and selec-
tively removed in the presence of other functional
groups, leading to the desired glycoporphyrins with
the sugar moiety bearing free hydroxyl groups. The
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose
was
also used to prepare 6-iodo-1,2:3,4-di-O-isopropylid-
ene-a-D-galactopyranose,40,41 a useful compound for
the synthesis of other glycoporphyrins. Two series of
porphyrins were obtained by coupling these sugars with
meso-tetraphenylporphyrins containing an hydroxyl
group (1) or a carboxylic group (4); the other two series
were obtained by direct substitution of a fluorine atom
on meso-tetrakis(pentafluorophenyl)porphyrin (3) or
on a meso-tetraphenylporphyrin containing a pentaflu-
orobenzene spacer (2).
In this article, we describe the synthesis of four series of
porphyrins containing carbohydrate moieties with pro-
tected and unprotected hydroxyl groups. The antiviral
activity of these compounds, and of the corresponding
porphyrin precursors, was examined against HSV-1
and HSV-2 in different situations of cell culture infec-
tion. At the same time, the influence of the amphiphilic
character of the porphyrins on the inhibitory capacity
was evaluated. We have found that under the weak
white light exposure of the laminar flow cabinet
(3 mW/cm2during 15 min), some of the synthesized gly-
coporphyrins show similar efficiency to the currently
used drugs acyclovir and foscarnet.
Glycoporphyrin 1a was obtained from the reaction of
porphyrin 1 with 1-bromo-2,3,4,6-tetra-O-acetyl-a-D-
glucopyranose (Scheme 2). The reaction was carried
out at room temperature in the presence of freshly pre-
pared silver carbonate. After the work up, the reaction
mixture was separated by column chromatography
affording a mixture of the desired porphyrin 1a with
the corresponding silver complex 1a0 (Scheme 2). Treat-
ment of this mixture with trifluoroacetic acid (TFA)
gave, after purification, pure porphyrin 1a in 66% yield.
The carbohydrate protection groups were removed by
2. Results and discussion
2.1. Chemistry
Porphyrins 1, 3, and 4 (Scheme 1), key compounds in
the synthesis of the glycoporphyrins, were obtained
from Rothemund and crossed-Rothemund reactions
using the appropriate benzaldehydes and pyrrole in
refluxing acetic acid and nitrobenzene.39 Porphyrin 2
was obtained in 85% yield by coupling porphyrin 1 with
Ph
OAc
O
NH
N
N
AcO
AcO
Ph
+
HO
AcO
HN
Br
O
1
Ph
R1
Ag2CO3
Ph
NH
N
N
OAc
R1
R2
N
N
N
AcO
AcO
Ph
O
M
HN
N
AcO
R1
Ph
1a, M = 2H
1a', M = Ag
R1 =
R1 =
R2 =
OH
TFA
,
1,
2,
NaOMe
F
F
F
F
R2 =
F
O
F
,
Ph
F
OH
NH
N
N
R1 = R2
=
O
OH
3,
4,
F
HO
HO
Ph
O
HN
F
,
F
R2 =
CO H
2
1
R =
1b
Ph
Scheme 1. Key compounds for the synthesis of the glycoporphyrins.
Scheme 2.

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3880
treating 1a with sodium methoxide in dry methanol; the
glycoporphyrin 1b was obtained in 80% yield.
TLC afforded the a and b anomers in 43% and 40%
yields, respectively.
Glycoporphyrins 2a and 3a were obtained, respectively,
from the reaction of porphyrins 2 and 3 with 1,2:3,4-di-
O-isopropylidene-a-D-galactopyranose (Scheme 3). Both
compounds were formed by nucleophilic substitution of
a fluorine atom by the alkoxide generated from the reac-
tion of the carbohydrate with sodium hydride. The
fluorobenzene bridge was introduced in order to evaluate
any effect on the separation of the carbohydrate unit
from the porphyrin macrocycle.
The structures of all porphyrins were confirmed by
NMR, UV–vis, mass spectrometry, and HRMS or ele-
mental analysis. The H NMR spectra of porphyrins
1
2a, 3a, and 4a show two distinct regions: the signals at
lower field (between 7 and 9 ppm) are due to the reso-
nances of the protons of the porphyrinic moiety and
the ones at higher field (between 1 and 6 ppm) are due
to the resonances of the protons of the carbohydrate
unit. Each spectrum shows four singlets, between 1.3
and 1.6 ppm, due to the isopropylidene protons; the reso-
nances of the other protons of the carbohydrate unit
appear between 4 and 6 ppm. The resonances of the
anomeric protons of the three compounds appear as
doublets at 5.54, 5.70, and 5.66 ppm, respectively. For
each one of these derivatives, the multiplet centered at
ca. 8.9 ppm was assigned to the b-pyrrolic protons; the
resonances of the protons of the nonsubstituted phenyl
groups appear at ca. 8.2 ppm (ortho protons) and at
7.7 ppm (meta and para protons). The resonances of
the protons of the para-substituted phenyl groups ap-
pear as two doublets at 7.35 and 8.16 ppm (for 2a)
and at 8.30 and 8.45 ppm (for 4a). The 19F NMR spec-
trum of 2a confirmed the substitution of the para-fluo-
rine atom by the sugar unit. The resonances of the
ortho- and meta-fluorine atoms appear as two doublets
(J = 22.6 Hz) at ꢀ151.91 and ꢀ152.40 ppm, respec-
tively. The 1H NMR spectra of 2b, 3b, and 4b confirmed
Attempts to couple 1,2:3,4-di-O-isopropylidene-a-D-
galactopyranose with porphyrin 4, in the presence of
N,N-dicyclohexylcarbodiimide (DCC) and 4-pyrrolo-
pyridine, did not lead to the expected compound 4a
but to the corresponding N-acylurea derivative.42 How-
ever, we were able to obtain 4a in 50% yield from the
reaction of 4 with 6-iodo-1,2:3,4-di-O-isopropylidene-
a-D-galactopyranose in the presence of potassium car-
bonate (Scheme 4). The removal of the carbohydrate
protection groups in compounds 2a, 3a, and 4a to the
corresponding porphyrins 2b, 3b, and 4b was performed
with aqueous TFA at room temperature.
The methyl galactopyranosides a-4c and b-4c were
prepared by bubbling gaseous hydrogen chloride
through a solution of 4b in dry methanol and chloro-
form at room temperature; purification by preparative
OH
O
O
O
O
3, NaH
F
2, NaH
O
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
NH
NH
N
N
O
O
O
HN
N
HN
N
F
O
O
F
O
O
O
F
F
F
F
O
2a
O
O
3a
F
F
O
O
TFA/H2O (9:1)
TFA/H2O (9:1)
F
F
F
F
F
F
F
F
NH
N
O
O
F
F
F
F
F
F
HN
N
NH
N
HO
O
N
HN
O
F
F
F
HO
HO
2b
HO
OH
O
F
F
F
F
OH
OH
3b
OH
F
Scheme 3.

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3881
I
O
NH
N
O
O
N
K2CO3
O
4
+
O
HN
O
O
O
O
4a
TFA/H2O (9:1)
O
O
O
O
O
O
O
NH
N
NH
N
N
HN
MeOH
N
HN
HCl (g)
HO
HO
O
O
α- 4c
β-4c
HO
4b
HO
OH
OMe
OH
OH
Scheme 4.
the deprotection of the carbohydrate moieties with the
disappearance of the four singlets due to the isopropyl-
idene protons. The spectra show several multiplets be-
tween 3 and 6 ppm due to the resonances of the
protons of the sugar units. As expected, there are no sig-
nificant differences between the lower field regions of the
1H NMR spectra of porphyrins 2b, 3b, and 4b and that
72 h in the presence of different concentrations of the
drugs, and then the cell viability was determined. These
studies indicated that the cytotoxic effects of the porphy-
rin derivatives, as well as acyclovir and foscarnet (antivi-
ral controls), are concentration dependent. The values of
the MNCSC for each compound are listed in Table 1.
1
of the corresponding precursors. Each H NMR spec-
The antiviral activity of the porphyrin derivatives was
determined by the observed virucidal effect inhibition
and the decrease on the virus yield (Table 1). Porphyrins
with the sugar moiety bearing unprotected hydroxyl
trum of porphyrins a-4c and b-4c shows a singlet due
to the methoxy group at 3.47 and 3.59 ppm, respec-
tively. The resonance of the anomeric proton appears
as a singlet at 5.02 ppm for the a anomer and as a dou-
blet at 4.94 ppm (J = 4.8 Hz) for the b anomer.
Table 1. Effect of the synthesized compounds on HSV-1 and HSV-2
infectivity
The UV–vis spectra of the glycoporphyrin derivatives
show, as expected, spectroscopic features analogous to
those of the starting porphyrins. The linking of carbo-
hydrate units to the meso-aryl groups of the porphyrins
does not change the macrocycle conjugation in order to
affect the absorption properties of the new derivatives.
Compound MNCSCa
Virucidal effect
inhibition
Replication cycle
inhibition
(lg/mL)
(% control)b
(% control)b
Vero cells HSV-1 HSV-2 HSV-1 HSV-2
1
1a
30
30
0
0
100
40
40
20
100
33
14
86
50
80
99
25
50
100
97
98
99
99
0
0
1b
15
50
ND
ND
ND
57
ND
ND
ND
25
100
42
2
2a
3. Biological evaluation
50
50
50
80
2b
3.1. Results and discussion
3
3a
40
50
ND
ND
60
ND
ND
50
30
60
All the described porphyrin derivatives, at their maxi-
mum noncytotoxic studied concentration (MNCSC),
were tested against HSV-1 and HSV-2 in Vero cells.
Studies were carried out at room temperature under
the white light of a laminar flow cabinet type B, corre-
sponding to a fluence rate of 3 mW/cm2, for 15 min.
Under these conditions, the intensity of the Soret band
of each porphyrin did not show any decrease, indicating
that the compounds are photostable under such condi-
tions. To evaluate the cytotoxic effect of the porphyrin
derivatives, monolayers of Vero cells were cultured for
3b
15
90
30
4
4a
5
50
85
30
60
50
35
4b
a-4c
10
50
70
15
82
26
100
100
99
b-4c
25
20
45
31
0
Acyclovir
Foscarnet
100
1000
99
99
20
0
ND—not done.
a Maximum noncytotoxic studied concentration.
b Each value represents the average of three independent experiments.

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3882
groups (1b, 2b, 3b, and 4b) and the methyl galactopyr-
anosides a-4c and b-4c showed virus yield inhibitions
comparable to the ones obtained with the control com-
pounds (Fig. 1a). The virus yield inhibition, quite similar
for both viruses, must be specific of the viral cycle,
because no significant virucidal effect was observed with
the same porphyrins (Fig. 1b).
From such results, it can be stated that the glycoporphy-
rins 1b, 2b, 3b, 4b, a-4c, and b-4c are more effective than
their precursors in the inactivation of both viruses. This
structure–activity relationship indicates that the sugar
moiety plays an important role in the interaction of
the photosensitizer with the biological target.
The antiviral activity of the most severe inhibitors was
also determined at concentrations lower than the
MNCSC. The results show that all these compounds
continue to be highly effective against both viruses even
at concentrations much lower than the MNCSC (Fig. 2).
Porphyrins 1b, 3b, and 4b show antiviral effects higher
than 40% even at 0.01 lg/mL.
Inhibition (% control)
20 40 60 80 100 120
(a)
(b)
Inhibition (% control)
20 40 60 80 100 120
0
0
1
1a
1b
1
1a
1b
Methyl glycosides a-4c and b-4c show similar inhibitions
as porphyrin 4b (Fig. 1). These results indicate that it is
not required to have a reducing carbohydrate moiety for
a good antiviral activity. This is also confirmed with the
results from porphyrin 1b.
2
2a
2b
2
2a
2b
3
3a
3b
3
3a
3b
Adsorption and post-adsorption assays for HSV-1 were
carried out with compound 1b at concentrations of 15
and 3 lg/mL. Despite no inhibition being observed in
the adsorption assay using compound 1b at 15 lg/mL,
a total inhibition (100%) in the post-adsorption test
was reached even when a lower concentration (3 lg/
mL) of the drug was used. These results are consistent
with the hypothesis that the viral inhibition occurs in a
late step of the viral cycle.
HSV-1
HSV-2
4
4a
4b
4
4a
4b
alfa-4c
beta-4c
alfa-4c
beta-4c
HSV-1
HSV-2
Acyclovir
Foscarnet
Acyclovir
Foscarnet
Figure 1. (a) Viral replication effect and (b) virucidal effect of the HSV-
1 and HSV-2 incubated with the compounds at their MNCSC, and
exposed for 15 min to 3 mW/cm2 of white light. The inhibition values
(in percentage) were calculated in relation to the control. Data are
reported as the means of three independent assays, each run in
duplicate. Error bars represent standard deviations.
When the effect on the viral replication was studied with
1b, at the MNCSC, under careful exclusion of light, the
virus titration showed a much lower inhibition (40%)
than the one obtained under the currently used condi-
tions, that is, with exposure to the light of the laminar
120
100
80
60
40
20
0
120
100
80
60
40
20
0
15 10
5
2.5
1
0.5 0.1 0.01
50 25 15 10
5
2.5
1
Porphyrin 2b (µg/mL)
Porphyrin 1b (µg/mL)
120
100
80
120
100
80
HSV11
HSV--22
60
60
40
20
40
20
0
0
10
5
2.5
1
0.5 0.1 0.01
15 10
5
2.5
1
0.5 0.1 0.01
Porphyrin 4b (µg/mL)
Porphyrin 3b (µg/mL)
Figure 2. Viral replication effect of the HSV-1 and HSV-2 incubated with the compounds at concentrations lower than MNCSC and exposed for
15 min to 3 mW/cm2 of white light. The inhibition values (in percentage) were calculated in relation to the control. Data are reported as the means of
three independent assays, each run in duplicate. Error bars represent standard deviations.

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3883
flow cabinet. Although several reports show that certain
porphyrins under dark conditions can have cytotoxic
effects in cancer cells43 and on HIV-1,44 our results
suggest that the antiviral activity of our compounds is
significantly enhanced by light.
mixture of chloroform–petroleum ether (1:1) as eluent.
The first fraction was identified by TLC as 5,10,15,20-
tetraphenylporphyrin (TPP). The desired porphyrin 1
was then eluted with chloroform. Evaporation of the
solvent and recrystallization from chloroform/petroleum
ether gave purple crystals (300 mg, 7% yield).
1
Mp >300 ꢁC. H NMR (CDCl3): d ꢀ2.78 (s, 2H, NH),
4. Conclusion
7.15 (d, J = 8.5 Hz, 2H, 5-Ar-m-H), 7.72–7.78 (m, 9H,
10,15,20-Ar-m- and p-H), 8.06 (d, J = 8.5 Hz, 2H, 5-
Ar-o-H), 8.20–8.23 (m, 6H, 10,15,20-Ar-o-H), 8.84–
8.88 (m, 8H, b-H). 13C NMR (CDCl3): d 113.6, 119.8,
120.1, 126.7, 127.7, 131.0, 131.1, 134.5, 134.7, 135.7,
142.2, 155.4. Anal. Calcd for C44H30N4OÆ5/2H2O: C,
78.20; H, 5.22; N, 8.29. Found: C, 78.41; H, 4.97; N,
8.41. UV–vis (CHCl3): kmax (log e): 419 (5.65), 515
(4.24), 551 (3.92), 591 (3.73), 647 (3.67) nm. MS
(FAB+) m/z: 631 (M+H)+.
We have demonstrated that glycoporphyrins can signifi-
cantly inhibit the infection of Vero cells by HSV-1 and
HSV-2. Our results show that glycoporphyrins 1b and
4b (with a sugar moiety bearing unprotected hydroxyl
groups) have a similar inhibitory capacity in the forma-
tion of infectious virions as acyclovir or foscarnet. The
glycoporphyrins are active against both viruses; only
marginal differences were observed, in all cases, for the
inhibition of HSV-1 and HSV-2. Post-adsorption studies
with glycoporphyrin 1b showed a reduction on the rep-
lication cycle by 6 log in relation with the control with-
out the drug (data not shown). This might mean that a
specific inhibitory action occurs during the formation of
new viral particles. Some of the tested compounds
showed strong virus yield inhibition, even at concentra-
tions much lower than the MNCSC, and so they may be
useful for the treatment of herpesvirus infections.
5.2.2. 5-[4-(Pentafluorophenyloxy)phenyl]-10,15,20-triphen-
ylporphyrin (2). To a suspension of porphyrin 1 (50.0
mg, 80.9 lmol) in dimethylformamide (2 mL) was
added an excess of sodium hydride (ꢁ25 mg) and the
mixture was heated at 70 ꢁC during approximately
10 min. Hexafluorobenzene (1 mL, 8.7 mmol, 107 equiv)
was then added and the mixture was maintained at that
temperature for 4 h in the dark and under nitrogen.
After cooling to room temperature, the reaction mixture
was neutralized with a saturated aqueous citric acid
solution, and the resulting porphyrin was extracted with
chloroform. The organic layer was washed with water
(2 · 100 mL), dried (Na2SO4), and evaporated to dry-
ness. The residue was purified by column chromatogra-
phy on silica using chloroform–petroleum ether (70:30)
as eluent. The major fraction afforded product 2
(55 mg, 85% yield) after recrystallization from chloro-
5. Experimental
5.1. General
1H, 13C, and 19F solution NMR spectra were recorded
on a Bruker AMX 300 spectrometer at 300.13, 75.47,
and 282.38 MHz, respectively. Tetramethylsilane was
used as internal reference. Mass spectra and HRMS
were recorded on VG AutoSpec Q and M mass spec-
trometers using chloroform as solvent and NBA as ma-
trix. Elemental analyses were performed with a Leco 932
CHNS analyzer. The UV–vis spectra were recorded on
an Uvikon spectrophotometer using dichloromethane
or chloroform as solvent. Melting points were measured
on a Reichert Thermovar apparatus fitted with a micro-
scope and are uncorrected. Column chromatography
was carried out in silica gel (Merck, 35–70 mesh). Pre-
parative thin-layer chromatography was carried out on
20 · 20 cm glass plates coated with silica gel (1 mm
thick, Merck). Analytical TLC was carried out on pre-
coated sheets with silica gel (0.2 mm thick, Merck).
1
form/methanol. Mp 295–296 ꢁC. H NMR (CDCl3): d
ꢀ2.80 (s, 2H, NH), 7.33 (d, J = 8.6 Hz, 2H, 5-Ar-m-
H), 7.73–7.78 (m, 9H, 10,15,20-Ar-m- and p-H), 8.17
(d, J = 8.6 Hz, 2H, 5-Ar-o-H), 8.20–8.23 (m, 6H,
10,15,20-Ar-o-H), 8.82–8.87 (m, 8H, b-H). 13C NMR
(CDCl3): d 113.7, 118.6, 120.25, 120.3, 126.7, 127.7,
131.2, 134.6, 135.7, 138.0, 142.1, 157.0. 19F NMR
(CDCl3): d ꢀ158.10 (t, J = 21.2 Hz, 2F, Ar-m-F),
ꢀ155.88 (t, J = 21.2 Hz, 1F, Ar-p-F), ꢀ149.91 (d, J =
16.9 Hz, 2F, Ar-o-F). Anal. Calcd for C50H29F5N4OÆ
3H2O: C, 70.58; H, 4.15; N, 6.58. Found: C, 70.36; H,
3.91; N, 6.24. UV–vis (CHCl3) kmax (log e): 420 (5.71),
516 (4.30), 551 (3.92), 591 (3.76), 646 (3.63) nm. MS
(FAB+) m/z: 797 (M+H)+.
5.2. Synthesis
5.2.3. meso-Tetrakis(pentafluorophenyl)porphyrin (3).
Pentafluorobenzaldehyde (4 mL, 32 mmol) was added
to a refluxing mixture of glacial acetic acid (200 mL)
and nitrobenzene (150 mL). Pyrrole (2.5 mL, 36 mmol)
was then added dropwise over 15 min and the mixture
was refluxed for a further 1 h. After cooling to room
temperature, the acetic acid and nitrobenzene were dis-
tilled under reduced pressure to dryness. The crude
material was taken into chloroform and submitted to
column chromatography (silica gel) using a mixture of
chloroform–petroleum ether (1:1) as eluent. After evapo-
ration of the solvent, the desired porphyrin 3 was
recrystallized from dichloromethane/petroleum ether
5.2.1. 5-(4-Hydroxyphenyl)-10,15,20-triphenylporphyrin (1).
4-Hydroxybenzaldehyde (1.07 g, 8.8 mmol, 1.2 equiv)
and benzaldehyde (2.20 mL, 21.7 mmol, 3 equiv) were
added to a refluxing mixture of glacial acetic acid
(200 mL) and nitrobenzene (150 mL). Pyrrole (2.00
mL, 28.9 mmol, 4 equiv) was then added dropwise over
15 min and the mixture was refluxed for a further 1 h.
After cooling to room temperature, the acetic acid,
and nitrobenzene were distilled under reduced pressure.
The crude material was taken into chloroform and sub-
mitted to column chromatography (silica gel) using a

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3884
1
(15% yield). H NMR (CDCl3): d ꢀ2.93 (s, 2H, NH),
8.92 (s, 8H, b-H). Anal. Calcd for C44H10F20N4Æ
3/2H2O: C, 52.76; H, 1.31; N, 5.59. Found: C, 52.74;
H, 0.90; N, 5.98. UV–vis (CHCl3) kmax (log e): 411
(5.36), 504 (4.28), 579 (3.83), 632 (2.96) nm. MS
(FAB+) m/z: 976 (M+2H)+.
the organic phase was washed with water (2 · 50 mL).
The organic extracts were dried (Na2SO4) and evapo-
rated under reduced pressure. The resulting residue
was taken into chloroform and purified by column chro-
matography (silica gel) using the same solvent as eluent.
The first fraction was identified as the silver complex of
the starting porphyrin 1 and the second one as a mixture
of porphyrin 1a and its silver complex 1a0. After evapo-
ration of the eluent, the second fraction was stirred with
TFA (5 mL) for 10 min at room temperature. Chloro-
form (5 mL) was then added and after 5 min the solution
was neutralized with saturated aqueous sodium carbon-
ate. The organic phase was dried (Na2SO4) and the sol-
vent removed under vacuum. The residue was purified
by column chromatography (silica gel) using chloroform
as eluent. The desired porphyrin was then crystallized
from chloroform/petroleum ether (101 mg, 66% yield).
5.2.4. 5-(4-Carboxyphenyl)-10,15,20-triphenylporphyrin
(4). Methyl 4-formylbenzoate (1.44 g, 8.77 mmol,
1.2 equiv) and benzaldehyde (2.20 mL, 21.7 mmol,
3 equiv) were added to a refluxing mixture of glacial
acetic acid (200 mL) and nitrobenzene (150 mL). After
complete dissolution of methyl 4-formylbenzoate,
pyrrole (2.00 mL, 28.9 mmol, 4 equiv) was then added
dropwise and the mixture was refluxed for 1 h. The
solvents were then distilled under reduced pressure and
the crude material was taken into chloroform and
submitted to column chromatography (silica gel) using
a mixture of chloroform–petroleum ether (1:1) as eluent.
The first fraction was identified as 5,10,15,20-tetra-
phenylporphyrin (TPP) and the second one as the
methyl ester of porphyrin 4 (evaporation of the solvent
and recrystallization from chloroform/methanol
gave the pure compound in 13% yield). Mp 268–
1
Mp >300 ꢁC. H NMR (CDCl3): d ꢀ2.79 (s, 2H, NH),
2.10, 2.11, 2.12, 2.22 (4s, 12H, 4 · acetyl), 4.06 (ddd,
J = 9.6, 5.4, 2.4 Hz, 1H, Glc-H5), 4.30 (dd, J = 12.3,
2.4 Hz, 1H, Glc-H6), 4.42 (dd, J = 12.3, 5.4 Hz, 1H,
Glc-H6), 5.30 (t, J = 9.6 Hz, 1H, Glc-H4), 5.42–5.50
(m, 3H, Glc-H1, H2, H3), 7.38 (d, J = 8.7 Hz, 2H, 5-
Ar-m-H), 7.73–7.79 (m, 9H, 10,15,20-Ar-m- and p-H),
8.14 (d, J = 8.7 Hz, 2H, 5-Ar-o-H), 8.20–8.23 (m, 6H,
10,15,20-Ar-o-H), 8.85 (s, 8H, b-H). 13C NMR: d 20.6
(CH3CO2), 20.7 (CH3CO2), 20.8 (2 · CH3CO2), 62.1
(Glc-C6), 68.4, 71.4, 72.3, 72.9 (Glc-C2, 3, 4, 5), 99.2
(Glc-C1), 115.0, 119.2, 120.2, 126.7, 127.7, 131.2,
134.5, 135.6, 137.3, 142.1, 156.6, 169.5 (2 · CH3CO2),
170.3 (CH3CO2), 170.6 (CH3CO2). Anal. Calcd for
C58H48N4O10Æ1/2H2O: C, 71.81; H, 5.09; N, 5.78.
Found: C, 71.74; H, 5.21; N, 5.73. UV–vis (CHCl3) kmax
(log e): 418 (5.68), 515 (4.28), 550 (3.93), 590 (3.75), 645
(3.62) nm. MS (FAB+) m/z: 961 (M+H)+.
1
270 ꢁC. H NMR (CDCl3): d ꢀ2.79 (s, 2H, NH), 4.11
(s, 3H, CO2CH3), 7.72–7.81 (m, 9H, 10,15,20-Ar-m-
and p-H), 8.20–8.22 (m, 6H, 10,15,20-Ar-o-H), 8.31 (d,
J = 8.9 Hz, 2H, 5-Ar-o-H), 8.44 (d, J = 8.9 Hz, 2H, 5-
Ar-m-H), 8.78–8.87 (m, 8H, b-H). HRMS (FAB+): m/z
calcd for C46H32N4O2 (M+H)+: 673.2604. Found:
673.2594.
The previous porphyrin (162 mg, 0.241 mmol) was then
dissolved in tetrahydrofuran (4 mL) and pyridine
(0.8 mL). KOH (4.00 g, 71.3 mmol) dissolved in metha-
nol (40 mL) was added to this solution and the mixture
was refluxed for 38 h. After cooling to room tempera-
ture, the mixture was neutralized with a saturated aque-
ous citric acid solution and the resulting suspension
extracted with chloroform–methanol (85:15). The or-
ganic layer was washed with water (2 · 100 mL), dried
(Na2SO4), and evaporated to dryness. Porphyrin 4
(151 mg, 95% yield) was obtained after recrystallization
in chloroform–methanol (85:15)/petroleum ether. Mp >
300 ꢁC. 1H NMR (CDCl3/CD3OD): d 7.73–7.80 (m, 9H,
10,15,20-Ar-m- and p-H), 8.21–8.24 (m, 6H, 10,15,20-
Ar-o-H), 8.32 (d, J = 8.2 Hz, 2H, 5-Ar-o-H), 8.46 (d,
J = 8.2 Hz, 2H, 5-Ar-m-H), 8.87 (br s, 8H, b-H). UV–
vis (CHCl3): kmax (log e): 418 (5.78), 513 (4.40), 549
(4.07), 588 (3.94), 649 (3.81) nm. Anal. Calcd for
C45H30N4O2: C, 82.05; H, 4.59; N, 8.51. Found: C,
82.22; H, 4.77; N, 8.25.
5.2.6. 5-(4-b-D-Glucopyranosyloxyphenyl)-10,15,20-triphen-
ylporphyrin (1b).45 To porphyrin 1a (24.9 mg, 25.9
lmol) in dichloromethane (6 mL) and methanol
(6 mL) was added a catalytic amount of sodium methox-
ide in methanol (0.1 mol dmꢀ3) (57.6 lL, 5.76 lmol,
0.22 equiv). The reaction mixture was stirred in the dark
at room temperature under nitrogen for 6 h. Water was
then added and the mixture was extracted with dichloro-
methane. The organic extracts were washed again with
water and dried over Na2SO4. The solvent was evapo-
rated under reduced pressure to dryness and the product
was crystallized from chloroform/petroleum ether
1
(16.4 mg, 80% yield). Mp >300 ꢁC. H NMR (DMSO-
d6): d ꢀ2.92 (s, 2H, NH), 3.52–3.57 (m, 4H, Glc-OH),
3.59–3.61 (m, 2H, Glc-H), 4.74 (br s, 1H, Glc-H), 5.14
(d, J = 6.0 Hz, 1H, Glc-H), 5.23 (br s, 2H, Glc-H),
5.55 (s, 1H, Glc-H), 7.48 (d, J = 8.3 Hz, 2H, 5-Ar-m-
H), 7.84 (m, 9H, 10,15,20-Ar-m- and p-H), 8.14 (d,
J = 8.3 Hz, 2H, 5-Ar-o-H), 8.22 (m, 6H, 10,15,20-Ar-o-
H), 8.83–8.88 (m, 8H, b-H). 13C NMR (DMSO-d6): d
60.8 (Glc-C6), 69.8, 73.5, 76.7, 77.2 (Glc-C2, 3, 4, 5),
100.5 (Glc-C1), 114.6, 119.9, 120.0, 127.0, 128.1, 131.4,
134.2, 134.6, 135.3, 141.2, 157.5. Anal. Calcd for
C50H40N4O6Æ4H2O: C, 69.43; H, 5.59; N, 6.48. Found:
C, 69.41; H, 5.42; N, 6.46. UV–vis (CHCl3) kmax (log e):
418 (5.64), 515 (4.23), 550 (3.88), 590 (3.70), 645 (3.57)
nm. MS (FAB+) m/z: 793 (M+H)+.
5.2.5. 5-[4-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyloxy)-
phenyl]-10,15,20-triphenylporphyrin (1a).45 To a solu-
tion of porphyrin
dichloromethane (150 mL), sodium sulfate (1.84 g, 13.0
mmol, 81.2 equiv), freshly prepared silver carbonate
(0.95 g, 3.4 mmol, 21.2 equiv), and 1-bromo-2,3,4,6-
tetra-O-acetyl-a-D-glucopyranose (1.00 g, 2.43 mmol,
15 equiv) were added. The reaction mixture was stirred
for 24 h at room temperature, in the dark and under
nitrogen. The reaction mixture was then filtered and
1 (101 mg, 160 lmol) in dry

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3885
5.2.7. 5-{4-[4-(1,2:3,4-Di-O-isopropylidene-a-D-galactopyr-
anosyl-6-oxy)tetrafluorophenyloxy]phenyl}-10,15,20-tri-
phenylporphyrin (2a). A mixture of 1,2:3,4-di-O-iso-
propylidene-a-D-galactopyranose (4.5 mg, 17.3 lmol)
and porphyrin 2 (20.0 mg, 25.1 lmol, 1.45 equiv) in
DMF (2.5 mL) was heated at 70 ꢁC until total dissolu-
tion of reagents (approximately 10 min). An excess of
sodium hydride (ꢁ25 mg) was then added and the
resulting mixture was maintained at that temperature
for 1.5 h in the dark and under nitrogen. After cooling
to room temperature, the mixture was neutralized with
a saturated aqueous citric acid solution, and extracted
with chloroform. The organic layer was washed with
water (2 · 100 mL), dried (Na2SO4), and the solvent re-
moved. The residue was purified by column chromato-
graphy (silica gel) using chloroform–petroleum ether (1:1)
as eluent. The major fraction afforded, after recrystalli-
zation from chloroform/petroleum, the desired product
(12.5 mg, 70% yield). Mp 131–133 ꢁC. 1H NMR
(CDCl3): d ꢀ2.80 (s, 2H, NH), 1.33, 1.35, 1.47, 1.55
[4s, 12H, C(CH3)2], 4.20 (dt, J = 5.7, 2.0 Hz, 1H, Gal-
H5), 4.34 (dd, J = 5.2, 2.6 Hz, 1H, Gal-H2), 4.37 (dd,
J = 8.0, 2.0 Hz, 1H, Gal-H4), 4.47 (br d, J = 5.7 Hz,
2H, Gal-H6), 4.66 (dd, J = 8.0, 2.6 Hz, 1H, Gal-H3),
5.54 (d, J = 5.2 Hz, 1H, Gal-H1), 7.35 (d, J = 8.6 Hz,
2H, 5-Ar-m-H), 7.73–7.80 (m, 9H, 10,15,20-Ar-m- and
p-H), 8.16 (d, J = 8.6 Hz, 2H, 5-Ar-o-H), 8.20–8.23 (m,
6H, 10,15,20-Ar-o-H), 8.84–8.86 (m, 8H, b-H). 13C
NMR (CDCl3): d 24.4, 24.9, 25.9, 25.9 [4 · C(CH3)2],
67.0 (Gal-C6), 70.4, 70.7, 71.0 (Gal-C2, 3, 4, 5), 96.2
(Gal-C1), 108.8, 109.6 [2 · C(CH3)2], 113.6, 118.9,
120.2, 126.7, 127.7, 134.6, 135.6, 137.5, 142.1, 157.3.
19F NMR (CDCl3): d ꢀ152.40 (d, J = 22.6 Hz, 2F, Ar-
m-F), ꢀ151.91 (d, J = 22.6 Hz, 2F, Ar-o-F). Anal. Calcd
for C62H48F4N4O7: C, 71.81; H, 4.67; N, 5.40. Found:
C, 71.82; H, 5.01; N, 5.07; UV–vis (CHCl3): kmax (log e):
420 (5.67), 516 (4.25), 551 (3.89), 590 (3.73), 646 (3.56)
nm. MS (FAB+) m/z: 1037 (M+H)+.
and meso-tetrakis(pentafluorophenyl)porphyrin (30.0
mg, 30.8 lmol, 1.46 equiv), in dry toluene (2.5 mL),
was added an excess of sodium hydride (ꢁ25 mg). The
reaction mixture was refluxed for 24 h in the dark and
under nitrogen. After cooling at room temperature,
the solution was neutralized with saturated aqueous cit-
ric acid and the resulting porphyrin was extracted with
chloroform. The organic layer was washed with water
(2 · 100 mL), dried (Na2SO4), and the solvent removed.
The residue was purified by column chromatography
(silica gel) using chloroform–petroleum ether (1:1) as
eluent. The major fraction afforded, after crystallization
from chloroform/petroleum ether, porphyrin 3a
(10.1 mg, 40% yield). Mp 171–174 ꢁC. 1H NMR
(CDCl3): d ꢀ2.92 (s, 2H, NH), 1.44, 1.45, 1.56, 1.67
[4s, 12H, C(CH3)2], 4.40–4.53 (m, 3H, Gal-H), 4.75–
4.78 (m, 3H, Gal-H), 5.70 (d, J = 3.0 Hz, 1H, Gal-H1),
8.89–9.02 (m, 8H, b-H). 19F NMR (CDCl3): d ꢀ157.95
to ꢀ157.75 (m, 6F, 10,15,20-Ar-m-F), ꢀ152.62 (dd,
J = 22.6, 8.5 Hz, 2F, 5-Ar-m-F), ꢀ147.90 to ꢀ147.73
(m, 3F, 10,15,20-Ar-p-F), ꢀ135.80 to ꢀ135.68 (m, 2F,
5-Ar-o-F), ꢀ133.01 to ꢀ132.90 (m, 6F, 10,15,20-Ar-o-
F). UV–vis (CHCl3) kmax (log e): 414 (5.54), 507 (4.39),
585 (3.89) nm. HRMS (FAB+) m/z: Calcd for
C56H30F19N4O6 (M+H)+: 1215.1862. Found: 1215.1866.
5.2.10. 5-[4-(a/b-D-Galactopyranosyl-6-oxy)tetrafluorophen-
yl]-10,15,20-tris(pentafluorophenyl)porphyrin (3b).
A
mixture of TFA–water (9:1, 4 mL) was added to por-
phyrin 3a (20.0 mg, 16.5 lmol). The resulting mixture
was stirred in the dark at room temperature for
30 min. Chloroform (20 mL) and water (20 mL) were
then added and the mixture was neutralized with aque-
ous sodium carbonate. The organic layer was separated,
washed with water (100 mL) and dried over Na2SO4.
The solvent was evaporated under reduced pressure to
dryness and the residue was crystallized from chloro-
form/petroleum
ether
(16.8 mg,
90%
yield).
1
Mp >300 ꢁC. H NMR (CDCl3): d ꢀ3.04, ꢀ3.01 (2s,
2H, NH), 3.05–3.55, 4.23–4.53, 4.79–5.10 (3m, 10H,
Gal-H and OH), 5.70 (s, 1H, Gal-H1), 8.73–8.89 (m,
8H, b-H). 19F NMR (CDCl3): d ꢀ158.41 to ꢀ157.82
(m, 6F, 10,15,20-Ar-m-F), ꢀ153.58 to ꢀ153.21 (m, 2F,
5-Ar-m-F), ꢀ148.37 to ꢀ147.78 (m, 3F, 10,15,20-Ar-p-
F), ꢀ135.00 to ꢀ134.91 (m, 2F, 5-Ar-o-F), ꢀ133.44 to
ꢀ133.00 (m, 6F, 10,15,20-Ar-o-F). Anal. Calcd for
C50H21F19N4O6: C, 52.92; H, 1.87; N, 4.94. Found: C,
52.91; H, 2.40; N, 4.57. UV–vis (CHCl3) kmax (log e):
414 (5.56), 507 (4.40), 585 (3.89) nm. MS (FAB+) m/z:
1135 (M+H)+.
5.2.8. 5-{4-[4-(a/b-D-Galactopyranosyl-6-oxy)tetrafluoro-
phenyloxy]phenyl}-10,15,20-triphenylporphyrin (2b). Por-
phyrin 2a (15.0 mg, 14.5 lmol) was dissolved in TFA–
water (9:1) (3 mL); the mixture was stirred in the dark
at room temperature for 30 min. Then, chloroform
(10 mL) and water (20 mL) were added and the mixture
was neutralized with aqueous sodium carbonate. The
organic layer was separated, washed with water (100
mL), and dried over Na2SO4. The solvent was evapo-
rated under reduced pressure to dryness and the residue
was crystallized from chloroform/petroleum ether (12.9
1
mg, 93% yield). Mp 215–216 ꢁC. H NMR (CDCl3): d
ꢀ2.94 (br s, 2H, NH), 3.63–5.42 (m, 11H, Gal-H and
OH), 6.90–8.03 (m, 19H, 5,10,15,20-Ar-H), 8.52–8.66
(m, 8H, b-H). 19F NMR (CDCl3): d ꢀ153.38 (br s, 2F,
Ar-m-F), ꢀ151.26 (br s, 2F, Ar-o-F). UV–vis (CHCl3)
kmax (log e): 420 (5.66), 518 (4.24), 551 (3.98), 591
(3.76), 646 (3.55) nm. HRMS (FAB+) m/z: Calcd for
C56H41F4N4O7 (M+H)+: 957.2911. Found: 957.2915.
5.2.11. 5-[4-(1,2:3,4-Di-O-isopropylidene-a-D-galactopyr-
anosyl-6-oxycarbonyl)phenyl]-10,15,20-triphenylporphy-
rin (4a). To a solution of porphyrin 4 (50 mg, 76 lmol)
and 6-iodo-1,2:3,4-di-O-isopropylidene-a-D-galactopyr-
anose (176 mg, 475 lmol, 6.2 equiv) in dry dimethyl-
formamide (10 mL) was added potassium carbonate
(125 mg, 0.9 mmol, 11.9 equiv). The reaction mixture
was stirred for 30 h at 100 ꢁC in the dark and under
nitrogen. After cooling to room temperature, the solu-
tion was neutralized with aqueous saturated citric acid,
and the resulting porphyrin extracted with chloroform.
The organic phase was washed with water
5.2.9. 5-[4-(1,2:3,4-Di-O-isopropylidene-a-D-galactopyrano-
syl-6-oxy)tetrafluorophenyl]-10,15,20-tris(pentafluorophen-
yl)porphyrin (3a). To
a mixture of 1,2:3,4-di-O-
isopropylidene-a-D-galactopyranose (5.5 mg, 21.1 lmol)

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3886
(2 · 100 mL), dried (Na2SO4), and the solvent removed.
The residue was purified by column chromatography
(silica gel) using chloroform as eluent. The major frac-
tion afforded, after crystallization from chloroform/
methanol, porphyrin 4a (34 mg, 50% yield).
1H, Gal-OH), 4.07, 4.17, and 4.32 (2d and 1s, J = 9.9,
11.2 Hz, 3H, Gal-H2,3,4), 4.39–4.41 (m, 1H, Gal-H5),
4.46 (dd, J = 11.7, 3.7 Hz, 1H, Gal-H6), 4.78 (dd,
J = 11.7, 7.9 Hz, 1H, Gal-H6), 5.02 (s, 1H, Gal-H1),
7.73–7.79 (m, 9H, 10,15,20-Ar-m-H and p-H), 8.20–
8.23 (m, 6H, 10,15,20-Ar-o-H), 8.33 (d, J = 8.1 Hz,
2H, 5-Ar-o-H), 8.46 (d, J = 8.1 Hz, 2H, 5-Ar-m-H),
8.77–8.88 (m, 8H, b-H). HRMS (FAB+) m/z: Calcd for
C52H43N4O7 (M+H)+: 835.3132. Found: 835.3099.
UV–vis (CHCl3) kmax (log e): 418 (5.68), 514 (4.28),
550 (3.92), 589 (3.76), 645 (3.60) nm.
1
Mp >300 ꢁC. H NMR (CDCl3): d ꢀ2.79 (s, 2H, NH),
1.38, 1.42, 1.55, 1.63 [4s, 12H, C(CH3)2], 4.35–4.49,
4.61–4.75 (2m, 6H, Gal-H2, H3, H4, H5, H6), 5.66 (d,
J = 5.1 Hz, 1H, Gal-H1), 7.73–7.79 (m, 9H, 10,15,20-
Ar-m- and p-H), 8.20–8.23 (m, 6H, 10,15,20-Ar-o-H),
8.30 (d, J = 8.4 Hz, 2H, 5-Ar-o-H), 8.45 (d, J = 8.4 Hz,
2H, 5-Ar-m-H), 8.78–8.88 (m, 8H, b-H). 13C NMR
(CDCl3): d 24.6, 25.0, 26.0, 26.1 [4 · C(CH3)2], 64.2
(Gal-C6), 66.2 (Gal-C5), 70.6, 70.8 (Gal-C3, 4), 71.2
(Gal-C2), 96.4 (Gal-C1), 108.9, 109.8 [2 · C(CH3)2],
118.5, 120.4, 120.5, 126.7, 127.8, 128.0, 129.4, 134.5,
142.0, 147.1, 166.7 (CO). UV–vis (CHCl3) kmax (log e):
420 (5.73), 516 (4.30), 551 (3.92), 591 (3.74), 646 (3.60)
nm. HRMS (FAB+) m/z: Calcd for C57H49N4O7
(M+H)+: 901.3601. Found: 901.3590.
Anomer b-4c: 9 mg (40% yield), mp 210–212 ꢁC. 1H
NMR (CDCl3): d ꢀ2.79 (s, 2H, NH), 2.48 (d,
J = 9.9 Hz, 1H, Gal-OH), 2.61 (s, 1H, Gal-OH), 2.77
(d, J = 6.8 Hz, 1H, Gal-OH), 3.59 (s, 3H, Gal-OMe),
4.15–4.21 (m, 3H, Gal-H2, H3, H4), 4.35–4.38 (m, 1H,
Gal-H5), 4.59 (dd, J = 11.5, 4.8 Hz, 1H, Gal-H6), 4.67
(dd, J = 11.5, 6.1 Hz, 1H, Gal-H6), 4.94 (d, J = 4.8 Hz,
1H, Gal-H1), 7.73–7.79 (m, 9H, 10,15,20-Ar-m-H and
p-H), 8.20–8.23 (m, 6H, 10,15,20-Ar-o-H), 8.31 (d,
J = 8.2 Hz, 2H, 5-Ar-o-H), 8.46 (d, J = 8.2 Hz, 2H, 5-
Ar-m-H), 8.77–8.87 (m, 8H, b-H). HRMS (FAB+) m/z:
Calcd for C52H43N4O7 (M+H)+: 835.3132. Found:
835.3152. UV–vis (CHCl3) kmax (log e): 418 (5.62), 515
(4.22), 550 (3.89), 589 (3.71), 645 (3.53) nm.
5.2.12. 5-[4-(a/b-D-Galactopyranosyl-6-oxycarbonyl)phen-
yl]-10,15,20-triphenylporphyrin (4b). Porphyrin 4a (15.0
mg, 16.5 lmol) in TFA–water (9:1) (3 mL) was
stirred in the dark at room temperature for 30 min.
Chloroform (20 mL) and water (20 mL) were then
added and the mixture was neutralized with aqueous so-
dium carbonate. The mixture was extracted with chloro-
form–methanol (85:15), and then the organic phase was
washed with water (100 mL) and dried over Na2SO4.
The solvent was evaporated under reduced pressure to
dryness and the residue was crystallized from chloro-
5.2.14. Photosensitizers. Each compound stock solution
was prepared at a concentration of 8 mg/mL in dimethyl
sulfoxide (DMSO) and diluted to the final concentration
in Dulbeccoꢁs modified Eagleꢁs medium (DMEM) or in
phosphate buffer solution (PBS).
1
form/methanol (12.9 mg, 94% yield). Mp >300 ꢁC. H
NMR (CDCl3/CD3OD): d 3.52–3.70, 3.90–4.15, 4.50–
4.68, 4.68–4.75, and 5.32 (4m and 1d, J = 3.2 Hz, 11H,
Gal-H and OH), 7.74–7.81 (m, 9H, 10,15,20-Ar-m-H
and p-H), 8.21–8.23 (m, 6H, 10,15,20-Ar-o-H), 8.33 (d,
J = 8.1 Hz, 2H, 5-Ar-o-H), 8.47 (d, J = 8.1 Hz, 2H, 5-
Ar-m-H), 8.88 (br s, 8H, b-H). UV–vis [CHCl3–MeOH
(85:15)] kmax (log e): 420 (5.52), 518 (4.12), 551 (3.75),
590 (3.60), 645 (3.41) nm. HRMS (FAB+) m/z: Calcd
for C51H41N4O7 (M+H)+: 821.2975. Found: 821.2988.
5.2.15. Photostability of the compounds. The photostabi-
lity of the photosensitizers was determined by keeping
1 lM solutions of the various porphyrin samples under
the white light of the laminar flow cabinet (3 mW/cm2)
at room temperature, with magnetic stirring, for
30 min. At fixed intervals of time, the intensity of the
Soret band of each porphyrin was monitored by visible
absorption spectrophotometry.
5.2.16. Cells and virus. African green monkey kidney
(Vero) cells were grown as monolayers in DMEM sup-
plemented with 10% fetal calf serum (FCS) and genta-
mycin (50 lg/mL), both purchased from Gibco,
Scotland, and incubated at 37 ꢁC in a humidified atmo-
sphere containing 5% CO2. HSV-1 strain SC16 and
HSV-2 strain HD from ATCC (USA), adapted to grow
in Vero cells, were produced in the same conditions of
cells.46
5.2.13. 5-[4-(a-Methyl-D-galactopyranosyl-6-oxycarbonyl)-
phenyl]-10,15,20-triphenylporphyrin (a-4c) and 5-[4-(b-
methyl-D-galactopyranosyl-6-oxycarbonyl)phenyl]-10,15,20-
triphenylporphyrin (b-4c). Porphyrin 4b (22 mg, 26.8
lmol) in anhydrous methanol (10 mL) and chloroform
(4 mL) was saturated with gaseous hydrogen chloride.
The mixture was stirred for 4 h in the dark and at room
temperature. It was then neutralized with aqueous
sodium carbonate. The organic layer was separated,
washed with water (100 mL), and dried over Na2SO4.
The two anomers a-4c and b-4c were separated by pre-
parative TLC using CHCl3–MeOH (95:5) as eluent; the
one with higher Rf (0.39) was identified by NMR as the
a-anomer. The two methyl glycosides were crystallized
from chloroform/petroleum ether.
5.2.17. Photocytotoxicity. Confluent monolayer Vero
cells in 96-well plates with 100 lL of solutions with
decreasing concentrations of the compounds (prepared
in DMEM from the stock solutions) were kept under
the white light of the laminar flow cabinet for 15 min
at room temperature, and then incubated for 72 h at
37 ꢁC in a humidified atmosphere containing 5% CO2.
The dye uptake assay47,48 was then performed to deter-
mine the percentage of living cells related to the control
(cells incubated without drug and kept under the same
conditions). The maximum noncytotoxic studied
1
Anomer a-4c: 9.5 mg (43% yield), mp 241–243 ꢁC. H
NMR (CDCl3): d ꢀ2.79 (s, 2H, NH), 2.93 (d,
J = 11.3 Hz, 1H, Gal-OH), 3.47 (s, 3H, Gal-OMe),
3.56 (d, J = 3.7 Hz, 1H, Gal-OH), 3.98 (d, J = 10.2 Hz,

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3887
concentration (MNCSC) is the highest concentration of
the drug, which gives identical results as the control
after 24, 48, and 72 h.
to FCT for a post-Doc grant. Thanks are also due to
Prof. Edgar Cruz e Silva and Dr. Adelaide Almeida,
Department of Biology, University of Aveiro, for
helpful discussions.
5.2.18. Virucidal effect. HSV-1 and HSV-2 (107 PFU/
mL) were incubated with the compounds, at their
MNCSC in PBS, for 1 h at 37 ꢁC and then kept for
15 min under the laminar flow cabinet light. The drugs
were then removed by ultracentrifugation (44,000g).
Controls were similarly processed. Virucidal effect was
determined by plaque titration: confluent Vero cells in
a 24-well plate were infected with 100 lL of serial dilu-
tions of the viruses in PBS. After 2 h of adsorption at
37 ꢁC the inoculum was removed, the cells were washed
and covered with 2% of Sephadex in DMEM with 2%
FCS. On the fourth day post-infection, the cells were
fixed with 10% formaldehyde in ethanol and stained
with a solution of 0.1% crystal violet. Viral plaques were
counted under the microscope. The inhibitory activity of
each compound was calculated in relation to the control.
References and notes
1. Pinna, A. D.; Rakela, J.; Demetris, A. J.; Fung, J. J.
Digest. Dis. Sci. 2002, 47, 750.
2. Kleymann, G.; Fischer, R.; Betz, U. A. K.; Hendrix, M.;
Bender, W.; Schneider, U.; Handke, G.; Eckenberg, P.;
Hewlett, G.; Pevzner, V.; Baumeister, J.; Weber, O.;
Henninger, K.; Keldenich, J.; Jensen, A.; Kolb, J.; Bach,
U.; Popp, A.; Maben, J.; Frappa, I.; Haebich, D.;
Lockhoff, O.; Rubsamen-Waigmann, H. Nat. Med. 2002,
8, 392.
3. Rand, K. H.; Rasmussen, L. E.; Pollard, R. B.; Arvin, A.;
Merigen, T. C. N. Eng. J. Med. 1997, 296, 1372.
4. Kleymann, G. Expert Opin. Invest. Drugs 2003, 12, 165.
5. Henry, S.; Sacaze, C.; Berrajah, L.; Karray, H.; Drira, M.;
Hammami, A.; Icart, J.; Mariame, B. Int. J. Cancer 2001,
91, 698.
5.2.19. Viral replication effect. To evaluate the effect of
compounds on the viral replication cycle, Vero cell mono-
layers were incubated for 30 min at 37 ꢁC with the
compounds at their MNCSC and, when specified, at
lower concentrations. Then, the compounds were re-
moved and the cells were washed with PBS or DMEM
with 2% FCS at the same temperature. Treated and un-
treated (control) cell monolayers were then infected with
HSV-1 or HSV-2, at a multiplicity of infection (MOI) of
approximately 1 PFU per cell. After adsorption of the
viruses for 2 h at 37 ꢁC, the unbound viruses were re-
moved by washing the cells with PBS. DMEM 2% with
or without drug was then added and the plates were kept
for 15 min under the laminar flow cabinet light. Such
plates were incubated at 37 ꢁC until an extensive cyto-
pathic effect was observed in the control infected cells,
normally 24 h post-infection. The cells were collected
and the extracellular infectious virions were evaluated
by standard plaque assay on Vero cells.
6. Ben-Hur, E.; Margolis-Nunno, H.; Rywkin, S.; Gottlieb,
P.; Lustigman, S.; Kenney, M. E.; Horowitz, B. In
Photodiagnostic and Phototerapeutic Techniques in Medi-
cine; Jori, G., Perria, C., Eds.; Documento Editoriale:
Milan, 1995, pp 135–143.
7. Alter, H. J.; Morel, P. A.; Dorman, B. P.; Smith, G. C.;
Creagan, R. P.; Wiesehahn, G. P.; Corash, L.; Popper, H.;
Eichberg, J. W. The Lancet 1988, 31, 1446.
8. The Porphyrin Handbook—Applications: Past, Present and
Future; Kadish, K. M., Smith, K. M., Guilard, R., Eds.;
Academic: New York, 2000; Vol. 6.
9. Bonnett, R. In Chemical Aspects of Photodynamic Ther-
apy; Gordon and Breach Science: London, 2000.
10. Photosensitizing Compounds: Their Chemistry, Biology and
Clinical Use; Bock, G., Harnett, S., Eds.; Wiley: Chich-
ester, 1989.
11. Sternberg, E. D.; Dolphin, D.; Bruckner, C. Tetrahedron
1998, 54, 4151.
12. Bonnett, R. Rev. Contemp. Pharmaco. 1999, 10, 1.
13. Lim, D.-S.; Ko, S.-H.; Kim, S.-J.; Park, Y.-J.; Park, J.-H.;
Lee, W.-Y. J. Photochem. Photobiol. B: Biol. 2002, 67, 149.
14. Vzorov, A. N.; Bixon, D. W.; Trommel, J. S.; Marzilli, L.
G.; Compans, R. W. Antimicrob. Agents Chemother. 2002,
12, 3917.
15. North, J.; Neyndorff, H.; Levy, J. G. J. Photochem.
Photobiol. B: Biol. 1993, 17, 99.
16. Stojiljkovic, I.; Evavold, B. D.; Kumar, V. Expert Opin.
Invest. Drugs 2001, 10, 309.
5.2.20. Effect of glycoporphyrin 1b on viral adsorption and
post-adsorption. Confluent Vero cells in 35 mm diameter
dishes were cooled to 4 ꢁC for 15 min and then infected
with an ice-cold suspension of HSV-1 or HSV-2
(107 PFU/mL), at a MOI of approximately 2 PFU per
cell, with 15 lg/mL of compound 1b. The dishes were
then gently shaken for 2 h at 4 ꢁC. The free viruses were
then removed and the cell monolayers were washed
twice with cold PBS. New medium without drug was
added. The virus yield was determined 24 h post-infec-
tion, when extensive cytopathic effect was observed in
the control cells. The viral post-adsorption assay was
carried out in the same way, but during the virus adsorp-
tion the drug was not present.
17. Polo, L.; Segalla, A.; Bertoloni, G.; Jori, G.; Schaffner, K.;
Reddi, E. J. Photochem. Photobiol. B: Biol. 2000, 59, 152.
´
18. Carre, V.; Gaud, O.; Sylvain, I.; Bourdon, O.; Spiro, M.;
Blais, J.; Granet, R.; Krausz, P.; Guilloton, M. J.
Photochem. Photobiol. B: Biol. 1999, 48, 57.
19. Soukos, N. S.; Ximenez-Fyvie, L. A.; Hamblin, M. R.;
Socransky, S. S.; Hasan, T. Antimicrob. Agents Chemo-
ther. 1998, 42, 2595.
20. Gaud, O.; Granet, R.; Kaouadji, M.; Krausz, P.; Blais, J.
C.; Bolbach, G. Can. J. Chem. 1996, 74, 481.
21. Hombrecher, H. K.; Ohm, S.; Koll, D. Tetrahedron 1996,
52, 5441.
Acknowledgements
22. Driaf, K.; Granet, R.; Krausz, P.; Kaouadji, M.; Thom-
asson, F.; Chulia, A. J.; Verneuil, B.; Spiro, M.; Blais, J.
C.; Bolbach, G. Can. J. Chem. 1996, 74, 1550.
23. Maillard, P.; Hery, C.; Momenteau, M. Tetrahedron Lett.
1997, 38, 3731.
ˆ
Thanks are due to FCT—Fundac¸a˜o para a Ciencia e a
Tecnologia (projects POCTI/38750/FCB/2001 and POC-
TI/32851/QUI/1999) and the University of Aveiro for
funding this work. One of us (J.P.C.T.) is also grateful

´
J. P. C. Tome et al. / Bioorg. Med. Chem. 13 (2005) 3878–3888
3888
24. Mikata, Y.; Onchi, Y.; Tabata, K.; Ogura, S.; Okura, I.;
Ono, H.; Yano, S. Tetrahedron Lett. 1998, 39, 4505.
36. Safrin, S.; Kemmerly, S.; Plotkin, B.; Smith, T.; Weiss-
bach, N.; De Varanez, D.; Phan, L. D.; Cohn, D. J. Infect.
Dis. 1994, 169, 193.
37. De Clercq, E. Nat. Rev. 2002, 1, 13.
38. Ernst, M. E.; Franey, R. J. Ann. Pharmaco. 1998, 32,
111.
39. Gonsalves, A. M. A. R.; Vareja˜o, J. M. T. B.; Pereira, M.
M. J. Heterocycl. Chem. 1991, 28, 635.
40. Classon, B.; Liu, Z. J. Org. Chem. 1988, 53, 6126.
41. Garegg, P. J.; Samuelsson, B. J. Chem. Soc. Perkin Trans.
1 1980, 2866.
42. Lloyd-Williams, P.; Albericio, F.; Giralt, E. In Chemical
Approaches to the Synthesis of Peptides and Proteins; CRC:
Boca Raton, 1997, p 50.
43. Guo, C.-C.; Tong, R.-B.; Li, K.-L. Bioorg. Med. Chem.
2004, 12, 2469.
44. Dixon, D. W.; Kim, M. S.; Kumar, V.; Obara, G.;
Marzilli, L. G.; Schinazi, R. F. Antiviral Chem. Chemo-
ther. 1992, 3, 279.
´
25. Sol, V.; Blais, J. C.; Carre, V.; Granet, R.; Guilloton, M.;
Spiro, M.; Krausz, P. J. Org. Chem. 1999, 64, 4431.
´
26. Kaldapa, C.; Blais, J. C.; Carre, V.; Granet, R.; Sol, V.;
Guilloton, M.; Spiro, M.; Krausz, P. Tetrahedron Lett.
2000, 41, 331.
27. Sylvain, I.; Zerrouki, R.; Granet, R.; Huang, Y. M.;
Lagorce, J.-F.; Guilloton, M.; Blais, J.-C.; Krausz, P.
Bioorg. Med. Chem. 2002, 10, 57.
28. Laville, I.; Figueiredo, T.; Loock, B.; Pigaglio, S.; Mail-
lard, Ph.; Grierson, D. S.; Carrez, D.; Croisy, D.; Blais, J.
Bioorg. Med. Chem. 2003, 11, 1643.
29. Sharon, N.; Lis, H. Science 1989, 246, 227.
30. Dwek, R. A. Chem. Rev. 1996, 96, 683.
31. Faustino, M. A. F.; Neves, M. G. P. M. S.; Cavaleiro, J.
A. S.; Neumann, M.; Brauer, H.-D.; Jori, G. Photochem.
Photobiol. 2000, 72, 217.
´
32. Tome, J. P. C.; Mendonc¸a, A. F.; Neves, M. G. P. M. S.;
´
Tome, A. C.; Valdeira, M. L.; Cavaleiro, J. A. S. PT
Patent 102,572, 2001.
45. Oulmi, D.; Maillard, P.; Guerquin-Kern, J.-L.; Huel, C.;
Momenteau, M. J. Org. Chem. 1995, 60, 1554.
46. Valdeira, M. L.; Duque-Magalha˜es, M. C.; Geraldes, A.
Arch. Virol. 1990, 113, 125.
47. Finter, N. B. J. Gen. Virol. 1969, 5, 419.
48. Gong, Y.; Matthews, B.; Cheung, D.; Tam, T.; Gadawski,
I.; Leung, D.; Holan, G.; Raff, J.; Sacks, S. Antiviral Res.
2002, 55, 319.
´
´
33. Tome, J. P. C.; Neves, M. G. P. M. S.; Tome, A. C.;
Cavaleiro, J. A. S.; Soncin, M.; Magaraggia, M.; Ferro, S.;
Jori, G. J. Med. Chem. 2004, 47, 6649.
34. Coen, D. M. Ann. N.Y. Acad. Sci. 1990, 616, 224.
35. Morfin, F.; Thouvenot, D.; Najioullah, F.; Aymard, M.;
Lina, B. Virologie 1999, 3, 57.