Polyamine conjugates of meso-tritolylporphyrin and protoporphyrin IX: Potential agents for photodynamic therapy of cancers

Article abstract of DOI:10.1016/j.bmc.2005.09.071

An efficient five-step synthesis method was developed to obtain tritolylporphyrin and protoporphyrin IX polyamine conjugates. These compounds were composed of either one polyamine unit (spermidine or spermine) covalently tethered to monocarboxyphenyl trit

Full text of DOI:10.1016/j.bmc.2005.09.071

Bioorganic & Medicinal Chemistry 14 (2006) 1364–1377
Polyamine conjugates of meso-tritolylporphyrin and protoporphyrin
IX: Potential agents for photodynamic therapy of cancers
a
a
a,
a
Vincent Sol, Fran c¸ ois Lamarche, Michaela Enache, Guillaume Garcia,
a
a
b
a,
*
Robert Granet, Michel Guilloton, J. C. Blais and Pierre Krausz
a
Universit e´ de Limoges, Facult e´ des Sciences et Techniques, Laboratoire de Chimie des Substances Naturelles (LCSN),
23, Avenue Albert Thomas, F-87060 Limoges, France
Universit e´ Pierre et Marie Curie, Synth e` se, Structure et Fonction de Mol e´ cules Bioactives, C.N.R.S,
place Jussieu F-75252, Paris Cedex 05, France
1
b
4
Received 5 April 2005; revised 23 September 2005; accepted 27 September 2005
Available online 2 November 2005
Abstract—An efficient five-step synthesis method was developed to obtain tritolylporphyrin and protoporphyrin IX polyamine con-
jugates. These compounds were composed of either one polyamine unit (spermidine or spermine) covalently tethered to monocarb-
oxyphenyl tritolylporphyrin or two molecules of polyamines borne by protoporphyrin IX. In each compound, an aliphatic spacer
4
arm is linked to the N polyamine position. Photocytotoxicity of these new compounds was evaluated against K562 human chronic
myelogenous leukemia cells and compared to Photofrin II ; protoporphyrin IX polyamine conjugates exhibited much stronger
ꢀ
ꢀ
photocytocicity than Photofrin II and were shown to readily induce necrosis in treated cells.
ꢁ
2005 Elsevier Ltd. All rights reserved.
ꢀ
1
. Introduction
chlorin (m-THPC), also known by trade name Foscan ,
received regulatory approval, in 2002 in the European
3
Photodynamic therapy (PDT) is an established modality
for treatment of neoplasic diseases. PDT involves selec-
tive accumulation of photosensitizer by cancer cells and
in situ photoactivation of the photosensitizer by visible
Union for palliative treatment of head and neck cancer.
Benzoporphyrin derivative monoacid ring A (BPD-MA)
trade named Visudyne (Verteporfin for injection) is used
ꢀ
in Phase III clinical trial for basal cell carcinoma (BCC)
and Mono-L-aspartylchlorin e6 (MACE) is being devel-
oped in Japan, under the acronym NPe6, for the treat-
1
light, leading to the destruction of treated cells. Two
types of photoreaction mechanisms are invoked to ex-
plain photosensitizer action: light-activated photosensi-
tizers in its triplet state can generate free radicals by
4
ment of endobronchial lung tumours.
electron or proton transfer (type I photochemical reac-
tions) or singlet oxygen ( O ) is produced by energy
Vectored or targeted drugs, which have enhanced affin-
ity for cancer cells, would be an important advance in
cancer therapy. To this end, we have devised a synthetic
route to obtain porphyrin derivatives designed for selec-
tive uptake by tumor cells and for binding DNA. Poly-
amines such as spermine, spermidine or putrescine,
required for optimal growth and replication of various
cell types, are present in high concentrations in rapidly
1
2
transfer (type II reactions). Singlet oxygen seems to be
2
the major mediator of photochemical cell damage, yet
the mechanism of action is not well understood. Photofrin
II is an efficient first-generation photosensitizer ap-
ꢀ
provedfor PDTbut it suffers from several drawbacks such
as a lack of selectivity toward tumor cells and a persistent
photosensitization of the skin. At present, considerable
efforts are being devoted to the development of new
PDT agents. Among them, meta-tetrahydroxyphenyl-
5
proliferating cells. Cancer cells, whose polyamine
requirements exceed biosynthetic capabilities, use a
polyamine transport system (PAT) to fulfill their needs.
This system displays at the same time a strong affinity
and a low specificity for polyamines. Therefore, poly-
amine transport system can afford selective accumula-
tion of polyamine analogs in neoplastic tissues and
presents a very attractive anticancer chemotherapeutic
Keywords: Polyamines; Photodynamic therapy; Porphyrins.
*
Corresponding author. Tel.: 33(0)555457490; fax: +33(0)5 55 45 72
2; e-mail: krausz@ unilim.fr
Present e-mail address: Mihaela.Enache@univ-lille1.fr
0
6
,7
strategy. Literature study has shown that one can
0
968-0896/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.071

V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
1365
utilize polyamine conjugation to convey cytotoxic drugs
acridine-carborane, chloroambucil, nitroimidazole)
fluoroacetic acid (TFA). Thus, spermidine 1 and
spermine 2 (Scheme 1) have been protected using 2-
(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (Boc-
(
8
into rapidly growing cells. On the other hand, poly-
amines are known to interact with the phosphate moiety
of nucleic acids by charge interaction and by hydrogen
bonding. So, in their cationic form, these compounds
constitute an interesting class of DNA binding
1
1c
ON) in THF as described in our preliminary work,
to obtain in one step, respectively, spermidineBoc 3
2
1
4
(83%) and spermineBoc 4 (72%). Then, compounds
3
3 and 4 were monoalkylated with N-(4-bromobu-
tyl)phthalimide in acetonitrile in the presence of potas-
sium carbonate and the yields obtained for resulting
compounds 5 and 6 were 96% and 95%, respectively.
In each case, spermidine and spermine were conjugated
7
a,9,10
molecules.
In connection with our research program on porphyrin
and their use in PDT, it occurred to us that porphy-
rin-based sensitizers in combination with an intracellu-
lar recognition element might acquire Ôdual actionÕ
4
at the N position via an aliphatic carbon tether to the
phtalimide nucleus. This central attachment was pre-
1
1,12
13
capabilities.
To this end, we thought to generate a
dicted by the findings by Poter in 1982, wherein sper-
midine could be derivatized at the central N position
4
suitable hybrid porphyrin with polyamine moieties. In
this paper, we report the synthesis of porphyrin deriva-
tives 16–19 bearing spermine and spermidine linked by
spacer arm on the macrocycle. As porphyrins moiety,
we used either a synthetic porphyrin as tritolylporphyrin
which bore one polyamine unit (spermidine or spermine)
1
5
and could be taken up by the polyamine transporter.
The N alkylation step was designed to maintain the
4
4
basicity of N nitrogen, which was also shown to be
critical for uptake. Hydrazinolysis of compounds 5
and 6 gave the respective amines 7 (95%) and 8 (86%).
1
6, 17 or protoporphyrin IX bearing two units of poly-
amines 18, 19. In all cases, spermine and spermidine
analogs 7, 8 have been attached to the porphyrin core
by amide linkages.
meso-Monohydroxyphenyltritolylporphyrins
(Scheme 2) were synthesized by LittleÕs standard method
(Adler modified method), condensation of pyrrole
9a,b
(4 equiv) with para-tolylaldehyde (3 equiv) and para-or
ortho-hydroxybenzaldehyde (1 equiv) in propionic acid
giving porphyrins para 9a in 6% yield and porphyrins
ortho 9b in 5% yield. These compounds were converted
into the derivatives 10a,b by treatment with ethyl 4-
bromobutyrate (10 equiv) with K CO (20 equiv) in
dry DMF at room temperature for 18 h. Purification
of the resulting products on TLC gave 90% and 85%
yields for 10a and b, respectively. The carboxy-function-
2
. Results
.1. Synthesis of porphyrins
Different approaches to the selective protection of
2
2
3
1
3
polyamines have been reported. We chose Boc-pro-
tective group, which can be selectively removed by tri-
NHR
H N
N
2
H
1
2
: R = H : spermidine
: R = -(CH ) -NH : spermine
2
3
2
i
NHR1
3 : R = Boc
1
BocNH
N
4 : R = -(CH ) -NHBoc
1
2 3
H
ii
iii
NHR2
NHR2
BocNH
N
BocNH
N
NH2
O
N
O
7
8
: R = Boc
2
5
6
: R = Boc
2
: R = -(CH ) -NHBoc
2
2 3
: R = -(CH ) -NHBoc
2
2 3
Scheme 1. Reagents and conditions: (i) with 1, Boc-ON (2 equiv), THF, 0 ꢂC, 18 h, 83% ; with 2, Boc-ON (3 equiv), THF, 0 ꢂC, 18 h, 72%; (ii)
N-(4-bromobutyl)phthalimide (1.25 equiv), K CO (5 equiv), CH CN, rt,18 h, 96% 5, 95% 6; (iii) hydrazine monohydrate (50 equiv), THF/EtOH
0:20, 90 ꢂC 5 h then 50 ꢂC 18 h, 95% 7, 86% 8.
2
3
3
8

1366
V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
H C
3
ii
12a,b
13a,b
i
iii
9
a,b
10a,b
11a,b
R1
N
NH HN
N
iv
12a,b
16a,b
7a,b
13a,b
1
Porphyrins
9a,b
R1
H C
CH3
3
OH
1
0a,b
1a,b
O(CH ) CO Et
2 3 2
H N
N
Spd =
2
NH2
1
O(CH ) COOH
2
3
Spm = H N
N
2
N
NH2
12a,b
3a,b
6a,b
7a,b
O(CH ) CONH(CH ) Spd(Boc)
2 3 2 4 2
H
1
O(CH ) CONH(CH ) Spm(Boc)
2 3 2 4 3
1
O(CH ) CONH(CH ) Spd
2 3 2 4
1
O(CH ) CONH(CH ) Spm
2
3
2 4
a and b refer to para and ortho position of R group respectively
1
Scheme 2. Reaction conditions: (i) Br(CH
(1.1 equiv), HOBt (1.1 equiv), DMF, rt, 72 h 80% 12a,b, 85%, 13a,b; (iv) CF
2
)
3
CO Et/K
2
2
CO
3
/DMF, 18 h, rt; (ii)KOH/EtOH/DMF 2 h, reflux; (iii) polyamine 7 or 8 (1.1 equiv), DCC
COOH/CH Cl (1:1), rt, 2 h, quantitatively yields 16a,b and 17a,b.
3
2
2
alized porphyrins 11a,b were obtained in excellent yields
by saponification of compounds 10a and b with KOH/
EtOH (1 M in DMF) at reflux for 2 h.
room temperature (2 h). Finally, the attempted porphy-
rins were obtained in a nearly quantitative yield.
Under the same conditions, protoporphyrin IX (Scheme
3) reacted with N -(4-aminobutyl)-N , N -bis-tert-
4
1
8
The synthesis of polyamine porphyrins 12a,b and 13a,b
was carried out by reaction of carboxy-porphyrins 11a,b
with N -(4-aminobutyl)-N ,N -bis-tert-butoxycarbonyl-
spermidine or N -(4-aminobutyl)-N ,N ,N -tris-tert-
butoxycarbonylspermine in the presence of dicyclo-
hexylcarbodiimide (DCC) and 1-hydroxybenzotriazole
4
butoxycarbonylspermidine or N -(4-aminobutyl)-N ,
1
4
1
8
8
12
N ,N -tris-tert-butoxycarbonylspermine in the presence
of dicyclohexylcarbodiimide (DCC) and 1-hydrox-
ybenzotriazole (HOBt) in DMF. After purification by
TLC (eluent: CH Cl /EtOH 80:20 + 1% Et N), protect-
4
1
8
12
2
2
3
(
HOBt) in dimethylformamide. Finally, the protecting
ed polyamine porphyrin conjugates 14 and 15 were ob-
tained in 78% and 84% yields, respectively. After
cleavage of protecting groups (Boc), the expected com-
pounds 18 and 19 were obtained in quantitative yields.
groups (Boc) were removed by standard methods in high
yields with trifluoroacetic acid in dichloromethane at
N
H
N
H
N
H
i
ii
N
N
N
N
N
N
H
H
N
H
N
N
COOH
COR
COR'
COOH
COR
COR'
Protoporphyrin IX
14 : R=NH-(CH
5 : R=NH-(CH
2
)
4
-spermidineBoc
-spermineBoc
2
18 : R’=NH-(CH
19 : R’=NH-(CH
2
)
)
4
4
-spermidine
-spermine
1
2
)
4
3
2
Scheme 3. Reagents and conditions: (i) Protoporphyrin IX (1 equiv), polyamine 7 or 8 (2.2 equiv), DCC (2.2 equiv), HOBt (2.2 equiv), DMF, rt, 72 h
0% 14, 85%, 15; (ii) CF COOH/CH Cl (1:1), rt, 2 h, quantitatively yields 18 and 19.
8
3
2
2

V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
1367
2
.2. Mass characterization
10b, 11b, 12b, and 13b), displayed an obvious change
1
1b
in the chemical shift of the phenyl ring. Furthermore,
compounds (12b, 13b) which are characterized by ortho
spermidine and spermine substitutions, are subjected to
Mass spectrometry of all porphyrin polyamine deriva-
tives was performed using the matrix-assisted laser
desorption ionization-time-of-flight (MALDI-TOF)
technique. Most of the studied compounds gave one
1
considerable changes in the H NMR chemical shift
and/or in figure relative to most of the nuclei. Thus,
for example, all the protons of spacer arm and poly-
amine (spermine or spermidine) experience a strong
+
main peak (protonated molecule MH , no fragments).
Nevertheless, compounds bearing polyamine units with
protective groups (Boc) gave additional signals. The
MALDI-TOF mass spectrum of compound 13a
shielding from ꢀ0.12 ppm (CH Boc) to ꢀ1.43 ppm
3
(H , spacer arm). These nuclei are obviously located well
c
(
Fig. 1) clearly indicated the presence of the protonated
within the range of the shielding current above the por-
phyrin macrocycle.
+
porphyrin derivatives (M+H ) along with metastable
fragment ions that corresponded to the loss of one,
two, and three protective groups, respectively.
1
H NMR spectra of protoporphyrin IX polyamine con-
jugates were realized in CDCl for 14–15 and in CD OD
3
3
1
.3. H NMR characterization
2
for 18–19. We have observed all chemical shifts and fig-
ures relative to most of the nuclei of protoporphyrin IX
and polyamine units.
1
H NMR spectra recorded at 400.13 MHz were used for
characterization of 9–13 in CDCl or CDCl /CD OD
3
3
3
(
9/1) for 16–17. The detailed resonance assignments
2.4. UV–vis absorption
are based on integration and selective homonuclear
decoupling and 2D homonuclear COSY experiments.
The spectra of these compounds are governed by the
symmetry properties of the molecule and by the orienta-
tion of the spacer arm in 10–11, polyamines bearing Boc
units in 12–13 (Fig. 2), and polyamines with free amino
functions in 14–15. Porphyrin derivatives that possess
one meso phenyl group with ortho substitution (9b,
The meso-phenylporphyrin conjugates 9–11 synthesized
in this work show typical electronic spectra, with a Soret
band near 420 nm and four less intense Q bands with an
etio outline. Polyamine- protected mesoporphyrin conju-
gates 12–13 were characterized by basically identical etio
spectra with very little difference in extinction coeffi-
cients. On the other hand, the unprotected counterparts
Counts
2
500
000
2
MH+1314.60
1
500
000
Metastable fragments
1
M+Na+
500
0
600
800
1000
1200
1400
1600
Mass (m/z)
Figure 1. Example of MALDI mass spectra for compound 13a.
H-β pyrrole
CH
3
CH Boc
3
tolyl
H spacer arm
and spermine
H aryl
NH pyrrole
NH Boc
1
0
ppm
8
6
4
2
0
-2
-4
1
3
Figure 2. H NMR spectrum in CDCl of compound 13a.

1368
V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
ꢀ3
ꢀ1
ꢀ1
Table 1. UV–vis spectra [knm (e · 10 , cm mol L)] of protoporphyrin-polyamine conjugates in organic solvents (CH
2
Cl
2
, MeOH) and water
Derivatives
Soret
Q IV
Q III
Q II
Q I
a
1
1
1
1
1
1
4
5
8
9
8
9
407 (113.9)
406 (133.5)
403 (27.0)
402 (29.7)
399 (59.9)
402 (95.3)
504 (8.8)
504 (10.5)
503 (2.5)
503 (2.6)
505 (5.5)
505 (7.8)
541 (8.4)
540 (9.3)
538 (2.1)
538 (2.1)
540 (4.5)
541 (6.8)
575 (5.3)
574 (6.6)
575 (1.3)
575 (1.4)
569 (2.8)
571 (4.4)
629 (3.2)
629 (3.7)
629 (0.9)
629 (0.9)
626 (1.0)
624 (2.5)
a
b
b
c
c
The solvents used are as follows:
a
2 2
CH Cl .
b
c
MeOH.
Water.
1
6-17 in methanol solution displayed a difference in
acetate epidioxide with nearly quantitative yields. In
the same experimental conditions, our porphyrins had
Soret band height with respect to substitution positions
Experimental): the extinction coefficients of para
1
the same efficiency for O production than HP.
(
2
substituted compounds 16a, 17a were lower than the
extinction coefficients of ortho substituted compounds
2.7. Biological tests
16b, 17b although electronic configuration should not
be modified.
The results presented in Figure 3 show that porphyrin
ꢀ6
polyamine conjugates 18 and 19 at 2 · 10 M display
a very strong photocytotoxicity, even for the shortest
irradiation time. These effects were much stronger than
With regard to protoporphyrin-polyamine conjugates
1
4, 15 and 18, 19, their UV–vis spectra are typical of
ꢀ
protoporphyrin IX, consisting of the intense Soret band
around 408 nm and weaker Q bands in the 500–650 nm
interval. Nevertheless, in water, their molar absorption
coefficients decrease as shown in Table 1 and the Soret
band is blueshifted and broadened. These features
strongly suggest an association of protoporphyrin
with Photofrin II used at an equivalent ponderal con-
centration. Lowering porphyrin conjugate concentra-
ꢀ
7
tions to 2 · 10 M resulted in a drop in toxicity so
that their effects were quite similar to those of Photofrin
ꢀ
II whatever the irradiation time, if the dead cells were
counted at once after irradiation.
1
6
polyamine conjugates in aqueous solutions.
ꢀ
Although irradiation in the presence of Photofrin
resulted in an increase in dead cells after an additional
2
.5. Partition coefficients
2
4 h incubation in the dark (Fig. 3E, solid bars), such
In medicinal chemistry, lipophilicity has proven to be an
important molecular descriptor that often is well corre-
lated with the bioactivity of drugs. Lipophilicity is indi-
cated, for example, by the logarithm of a partition
coefficient, logP, which reflects the equilibrium parti-
tioning of a molecule between a nonpolar and a polar
phase, such as the 1-octanol/water system. In this work,
we have determined logP of tritolylporphyrin-poly-
amine 16, 17 and protoporphyrin-polyamine derivatives
an effect was not seen when polyamine conjugates 18
or 19 were used (Figs. 3C and D, solid bars). This dis-
crepancy could indicate a difference in the death path-
ways induced by either Photofrin or polyamine
conjugates.
ꢀ
Biological assays were also conducted with trit-
olylporphyrin monopolyamine conjugates 16, 17 at
ꢀ6
2 · 10 M (Fig. 4). We have observed a less important
photocytotoxicity, especially for short irradiation times;
it is worth noticing that photoactivity displayed by ortho
compounds 16b, 17b (Figs. 4B and D) was always signif-
icantly stronger than their para counterparts 16a, 17a
1
8, 19 as log([Porphyrin]1-octanol/[Porphyrin]water) which
indicates that compounds 16, 17 are more lipophilic
than 18, 19 (Table 2). Determinations were repeated
three times.
(
Figs. 4A and C).
2
.6. Singlet oxygen production
Annexin V-FITC/propidium iodide (PI) fluorescent cell
staining was used to check cell death induced by poly-
amine porphyrins 18 and 19. Results presented in
Fig. 5 show that the massive death recorded after
In order to determine the photosensitizing properties of
porphyrins 16, 17, 18, and 19, trapping reactions of O
with ergosterol acetate were carried out. Reference
experiments with eosin, rose bengal or hematoporphyrin
1
2
1
7
ꢀ
6
30 min irradiation in the presence of 2 · 10 M porphy-
rin polyamine 19 was probably the consequence of a
rapid necrotic death: more than 80% of the treated cells
became nonviable (PI permeable cells) although they did
not display the strong FITC fluorescence characteristic
of apoptotic cells (Fig. 5A). Irradiation in the presence
(
HP), known singlet oxygen producers, gave ergosterol
Table 2. Partition coefficient of tritolylporphyrin and protoporphyrin-
polyamine conjugates
ꢀ
7
of the same compounds at 2 · 10 M resulted in a
much lower nonviable cell count, again without any
consistent increase in FITC fluorescence (Fig. 5B).
Experiments conducted with porphyrin polyamine 18
Derivatives
16a
16b
17a
17b
18
19
a
a
>3
a
>3
a
LogP
>3
>3
ꢀ0.12
ꢀ 0.51
a
Nearly insoluble in water.

V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
1369
A 100
B 100
80
8
6
4
2
0
0
0
0
0
60
40
20
0
0
30
60
90
120
0
30
60
90
120
Irradiation time (min)
Irradiation time (min)
C 100
D 100
80
8
6
4
2
0
0
0
0
0
60
40
20
0
0
30
60
90
120
0
30
60
90
120
Irradiation time (min)
Irradiation time (min)
E 100
8
6
4
2
0
0
0
0
0
0
30
60
90
120
Irradiation time (min)
Figure 3. Cell irradiation and analysis were conducted as described in Experimental. Cells were incubated with porphyrin polyamine conjugates 18
(A) 2 · 10 M, (C) 2 · 10 M) or 19 ((B) 2 · 10 M, (D) 2 · 10 M), or 1.25 lg/mL Photofrin IIꢀ (E). Dead cell count was estimated by flow
ꢀ
6
ꢀ7
ꢀ6
ꢀ7
(
cytometry just after irradiation (void bars) or after a further 24 h incubation in the dark (solid bars). Error bars are based upon standard deviations.
1
8
led to virtually identical results (not shown). The same
to increase transport and uptake in cells. The six
drugs examined do not have the same solubility in
aqueous medium. So, only protoporphyrin IX poly-
amine derivatives 18, 19 are soluble in water with
logP near 0 which seems to be characteristic of an
amphiphilic molecule. The other compounds 16a,b
and 17a,b were shown to have a relatively high par-
tition coefficient (logP > 3) which indicates their very
low water solubility. Based on the results of the loss
of cell viability, we observed that none of the com-
pounds studies exhibited cytotoxicity in the dark.
Moreover, after photoactivation with white light,
polyamine porphyrin conjugates 18 and 19 at
ꢀ1
ꢀ
irradiation in the presence of 1.25 lg mL Photofrin
Fig. 5D) resulted in a slight increase in FITC-stained
(
viable cell population. These results are consistent with
photocytotoxicity tests presented in Figure 3, since irra-
diation in the presence of Photofrin is followed by an
ꢀ
increase in dead cell count within the next 24 h, contrary
to irradiation in presence of porphyrin polyamines 18
and 19.
3. Discussion
ꢀ
6
In this study, we have investigated the synthesis of
six new polyamine-photosensitizers which differ by
the nature, number, and position on macrocycle of
polyamine units. We have tried to correlate these dif-
ferent structures with cell killing efficiency. The choice
of the number and position of polyamine units of the
porphyrin rings was didacted by the necessity to
modulate the hydrophilic/lipophilic balance of the
molecule which seems to be a very important factor
2 · 10 M displayed a very strong photocytotoxicity,
higher than that of Photofrin , even for the shortest
ꢀ
irradiation time (95% of death cells after 30 min of
irradiation). Thus, the efficacy of the photoactivity
is influenced by the hydrophophilic/lipophilic charac-
ter of the compounds; the presence of two polyamine
units on the same side of the macrocycle (18–19)
increases in parallel amphiphilic character and photo-
toxicity of these molecules.

1370
V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
A 100
B 100
80
8
6
4
2
0
0
0
0
0
60
40
20
0
0
30
60
90
120
0
30
60
90
120
Irradiation time (min)
Irradiation time (min)
C 100
D 100
80
8
6
4
2
0
0
0
0
0
60
40
20
0
0
30
60
90
120
0
30
60
90
120
Irradiation time (min)
Irradiation time (min)
ꢀ6
Figure 4. Cell irradiation and analysis were conducted as described in Experimental. Cells were incubated with tritolylporphyrin (2 · 10 M) with
monopolyamine substituent in ortho position 16b (A), 17b (B) and in para position 16a, 17a (C and D). Dead cell count was estimated by flow
cytometry just after irradiation (void bars) or after a further 24 h incubation in the dark (solid bars). Error bars are based upon standard deviations.
A
B
50.3 %
33.1 %
20.1 %
14.1 %
<
0.1 %
8.2 %
0.5 %
C
D
5.9 %
1.9 %
24.3 %
2.8 %
5.7 %
Figure 5. AnnexinV-FITC/propidium iodide assays were performed on cells irradiated for 30 min in the presence of porphyrin polyamine conjugate
ꢀ7
9 at 2 · 10 M (A), 2 · 10 M (B) or 1.25 lg/mL Photofrin IIꢀ (D); (C) control cells without photosensitizer.
ꢀ
6
1
In order to evaluate the influence of polyamine units on
the cell viability, biological assay with porphyrin conju-
gates devoid of free amino functions (compounds 12, 13
with Boc-protective groups) or without spermine, sper-
midine (Protoporphyrin IX dimethylester) was realized
(data not shown). These compounds induced 20% and

V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
1371
6
0% of cell death, respectively, after 120 min; this photo-
spectrophotometer using 10 or 50 mm quartz cells.
Infra-Red spectra were recorded on a Perkin-Elmer
spectrum 1000 with KBr pellets. Mass spectrometry
(ESI) was performed by the Centre R e´ gional de Mesures
Physiques de lÕOuest, CRMPO, Rennes. MALDI-TOF
mass spectra were recorded with a Voyager Elite (Fra-
mingham MA, USA) time-of-flight mass spectrometer
equipped with a 337 nm nitrogen laser (VSL 337ND).
It was operated in the reflectron delayed extraction
mode at an acceleration voltage of 20 kV. Internal stan-
dards (peptides) were used to calibrate the mass scale
with the two- point calibration Software version 3.07.1
from PerSeptive Biosystems. One microliter of an ace-
tone solution of matrix (a-cyano-4-hydroxycinnamic
acid) and compounds at concentrations of 0.1 M and
activity is significantly reduced if compared with 18, 19
So, the presence of polyamine with free amino functions
on the macrocycle was essential to induce massive cell
death. For tritolylporphyrins 16–17, the position on
the phenyl ring of one polyamine unit was different (para
to ortho). Within the later series, photoactivity displayed
by the ortho compounds was always significantly stron-
ger than their para counterparts. It should be noted that
these results are reminiscent of those previously ob-
served with mono ortho and para b-glycosyl porphy-
1
9
rins. On the other hand, the nature of polyamine
units does not seem to be important; indeed, porphyrin
conjugates bearing spermine or spermidine units showed
the same activity.
0.01 mM, respectively, was deposited onto the stainless
Then and with the aim of correlating cell death mecha-
steel sample slide and, dried in air. Elemental analyses
were carried by the ÔService R e´ gional de Microanalyse
de lÕUniversit e´ Pierre et Marie Curie, Paris.Õ
2
0
nism with the structure of photosensitizers, we have
used Annexin V-FITC/PI staining and flow cytometry
to characterize the cell death pathway, that is, the pres-
2
1
ence of apoptotic or necrotic cells. Our experiments
realized with amphiphilic compounds 18, 19 showed a
massive death probably corresponding to a rapid
necrosis.
5.2. Synthesis
1 8
N ,N -Bis-tert-butoxycarbonylspermidine
5.2.1.
3.
Spermidine (2.16 mL, 13.8 mmol, 1 equiv) was dissolved
in 30 mL of anhydrous THF. A solution of 2-(tert-butyl-
oxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON,
6.80 g, 27.6 mmol, 2 equiv) dissolved in 30 mL of anhy-
drous THF was added dropwise with constant stirring
and then 5.77 mL of triethylamine (41.4 mmol, 3 equiv)
was added. The reaction was carried out under an argon
atmosphere and stirred during 16 h at 0 ꢂC. The disap-
pearance of spermidine 1 was monitored by TLC
4. Conclusion
A series of new polyamine porphyrin conjugates have
been designed, synthesized, and characterized. Prelimin-
ary in vitro tests confirm previous observations suggest-
ing the requirement of amphiphilicity for efficient
photodynamic activity. The binding of two polyamine
units is obviously a good means to bring a balance be-
tween hydrophilicity and hydrophobicity. Massive cell
death induced by these compounds after 30 min irradia-
tion is likely the consequence of rapid necrotic death.
Further in vitro experiments are presently conducted
for evaluating other parameters as cellular uptake, sub-
cellular binding of sensitizers (lysosomes, mitochondria,
and/or cell membranes), and cell death pathway.
(R = 0.1). After evaporation under reduced pressure,
f
residue was redissolved in methylene chloride (50 mL)
and washed with 5% aqueous solution of sodium
hydroxyde (2 · 50 mL) and water (3 · 50 mL). The
organic layer was separated and dried over anhydrous
magnesium sulfate, filtered, concentrated, and subjected
to flash chromatography (CHCl /MeOH, 100:0 to
3
50:50) to give compound 3, which was recrystallized in
petroleum ether (3.76 g, 76%). R = 0.56 (ethyl acetate/
f
1
4
acetone/acetic acid/water, 5:3:1:1). T = 80 ꢂC (lit.
F
T = 79–80 ꢂC). IR (KBr) 3373, 2977, 2929, 1685,
F
ꢀ
1 1
5. Experimental
1165 cm ; H NMR (CDCl , 400.13 MHz) d 1,44 (s,
3
1
8H), 1.57 (m, 2H), 1.68 (m, 2H), 1.85 (m, 2H), 2.80
(t, JH,H = 6.56 Hz, 2H), 2.84 (t, JH,H = 6.56 Hz, 2H),
.13 (m, 2H), 3.25 (m, 2H), 4.90 (br s, 1H), 5.26 (br s,
5
.1. General
3
1
3
All solvents and reagents were purchased from Aldrich,
Prolabo or Acros. Pyrrole was distilled over CaH under
1H). RMN C (CDCl ) d, 156.1, 156.0, 78.9, 49.4,
3
47.7, 40.4, 39.2, 29.9, 28.4, 27.8, 27.4.
2
reduced pressure immediately before use. Dimethyl-
formamide was distilled over CaH under reduced pres-
1
8
12
5.2.2. N ,N ,N -Tris-tert-butoxycarbonylspermine 4.
Spermine (500 mg, 2.47 mmol, 1 equiv) was dissolved
in 20 mL of anhydrous THF. A solution of 2-(tert-butyl-
oxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON,
1.83 g, 7.41 mmol, 3 equiv) dissolved in 25 mL of anhy-
drous THF was added dropwise with constant stirring
and then 1.38 mL of triethylamine (9.88 mmol, 4 equiv)
was added. The reaction was carried out under an argon
atmosphere and stirred for 20 h at 0 ꢂC. The disappear-
2
sure and stored under argon. Methylene chloride was
distilled over P O and then CaH . Protoporphyrin IX
2
5
2
was purchased from Aldrich. Analytical thin-layer chro-
matography (TLC) was performed on silica gel Merck
6
0F254. Column chromatography was carried out with
C
1
13
silica gel (60 ACC; 15–40 lm, Merck). H and
NMR spectroscopy was performed with a Brucker
DPX-400 spectrometer. Chemical shifts are reported as
d ppm, downfield from internal TMS, and are listed
according to the standard numbering of meso-arylpor-
phyrins and protoporphyrin IX. UV–vis spectra were
recorded on a Perkin-Elmer Lambda 25 double-beam
ance of spermine 2 was monitored by TLC (R = 0.1).
f
After evaporation under reduced pressure, oil was redis-
solved in methylene chloride (50 mL) and washed with
5% aqueous solution of sodium hydroxyde (2 · 50 mL)

1372
V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
4
1
8
and water (3 · 50 mL). The organic layer was separated
and dried over anhydrous magnesium sulfate, filtered,
concentrated, and subjected to flash chromatography
5.2.5. N -(4-Aminobutyl)-N ,N -bis-tert-butoxycarbonyl-
spermidine 7. Compound 5 (1.070 g, 1.96 mmol, 1 equiv)
and an excess of hydrazine monohydrate (4.76 g,
98 mmol, 50 equiv) were dissolved in THF/MeOH (80/
20) (12 mL). The mixture was stirred 5 h at 90 ꢂC and
then 18 h at 50 ꢂC. After evaporation under vacuum,
the residue was redissolved in methylene chloride
(50 mL) and washed with 5% sodium hydroxide solution
(2 · 50 mL) and water (3 · 50 mL). The organic phase
was separated, dried over anhydrous magnesium sulfate,
filtered, concentrated, and subjected to flash chromatog-
raphy using 3% NH OH/CH OH to give the desired
(
(
CHCl /MeOH, 100:0 to 50:50) to give compound 4
3
897 mg, 72%). R = 0.48 (ethyl acetate/acetone/acetic
f
acid/water, 5:3:1:1). IR (KBr) 3347, 2976, 2932, 1694,
ꢀ
1 1
1
2
172 cm ; H NMR (400.13 MHz, CDCl ) d 1.44 (s,
3
7 H), 1.46 (m, 2H), 1.55 (m, 2H), 1.65 (m, 2H), 2.60
(
(
(
t, JH,H = 7.0 Hz, 2H), 2.66 (t, JH,H = 6.8 Hz, 2H), 3.17
m, 8H), 4.79 (br s, 1H), 5.15 (br s, 1H). RMN
1
3
C
CDCl ) d 156.1, 156.8, 155.4, 79.7, 79.5, 78.9, 49.6,
3
4
7.7, 46.8, 43.9, 39.2, 37.5, 30.0, 29.2, 28.5, 27.4, 26.5.
4
3
amine
NH OH). IR (KBr) 3347, 2974, 2933, 1693,
7
(776 mg: 95%). R = 0.35 (MeOH + 5%
f
4
1
8
.2.3. N -(4-Phthalimidobutyl)-N ,N -bis-tert-butoxycar-
5
bonylspermidine 5. Compound 3 (875 mg, 2.48 mmol,
4
ꢀ
1 1
1173 cm ; H NMR (400.13 MHz, CDCl ) d 1.44 (m,
3
1 equiv) and N-(4-bromobutyl)phthalimide (875 mg,
3.10 mmol, 1.25 equiv) were dissolved in dry acetonitrile
26H), 1.60 (m, 2H), 2.40 (m, 6H), 2.70 (t, J = 6.4 Hz,
2H), 3.14 (m, 4H), 4.87 (br s, 1H), 5.53 (br s, 1H).
1
3
C
(
20 mL) with an excess of K CO (5 equiv). The mixture
3
RMN (CDCl ) d , 156.1, 79.5, 78.8, 53.9, 53.7, 52.1,
2
3
was stirred for 18 h under reflux. After completion of the
reaction, acetonitrile was evaporated under vacuum and
the crude product was dissolved in dichloromethane.
The organic layer was washed with NaHCO3
42.2, 40.5, 40.1, 29.6, 28.5 , 28.0, 26.7, 24.4, 24.3. HRMS
(ESI): calcd for C H N O : m/z 416.3433. Found: m/z
2
1
44
4
4
+
417.3440 [M+H] .
4
1
8
12
(
2 · 50 mL) and water (2 · 50 mL), dried (MgSO ),
5.2.6. N -(4-Aminobutyl)-N ,N ,N -tris-tert-butoxycar-
bonylspermine 8. Compound 6 (1.225 g, 1.78 mmol,
1 equiv) and an excess of hydrazine monohydrate
(4.33 g, 89 mmol, 50 equiv) were dissolved in THF/
MeOH (80/20) (15 mL). The mixture was stirred for 5 h
at 90 ꢂC and then for 18 h at 50 ꢂC. After evaporation un-
der vacuum, the residue was redissolved in methylene
chloride (50 mL) and washed with 5% sodium hydroxide
solution (2 · 50 mL) and water (3 · 50 mL). The organic
phase was separated, dried over anhydrous magnesium
sulfate, filtered, concentrated, and subjected to flash chro-
matography using 3% NH OH/CH OH to give the de-
4
and then evaporated to afford, after purification by col-
umn chromatography (chloroform/ethanol, 100:0 to
8
EtOH, 90:10).IR (KBr) 3356, 2974, 2935, 1771, 1711,
0:20), 1.3 g of compound 5 (96%). R = 0.44 (CH Cl /
f 2 2
ꢀ
1 1
1
1
171 cm ; H NMR (400.13 MHz, CDCl ) d 1.44 (s,
3
8H), 1.48 (m, 6H), 1.65 (m, 4H), 2.44 (m,6H), 3.14
(
5
m, 4H), 3.70 (t, JH,H = 7.2 Hz, 2H), 4.91 (br s, 1H),
.30 (br s, 1H), 7.71 (dd, JH,H = 3.1 Hz and
JH,H = 5.4 Hz, 2H), 7.8513 (dd, JH,H = 3.1 Hz and
JH,H = 5.4 Hz, 2H). RMN C (CDCl ) d 168.4, 156.1,
3
1
3
33.4, 132.1, 123.7, 79.5, 78.7, 53.6, 53.3, 52.3, 40.5,
9.9, 37.8, 29.7, 28.5, 26.9, 26.5, 24.5, 24.0. HRMS
4
3
sired amine 8 (780 mg: 86%). R = 0.40 (MeOH + 5%
f
ꢀ
NH OH). IR (KBr) 3353, 2974, 2932, 1693, 1172 cm ;
4
1
(
m/z 547.3496 [M+H] .
ESI): calcd for C H N O : m/z 546.3416. Found:
47
2
9
+
4
6
1
H NMR (400.13 MHz, CDCl ) d 1.43 (m, 35H), 1.60
3
(
m, 4H), 2.40 (m, 6H), 2.70 (t, J = 6.4 Hz, 2H), 3.17 (m,
4
.2.4. N -(4-Phthalimidobutyl)-N ,N ,N -tris-tert-but-
1
8
12
13
5
8H), 4.88 (br s, 1H), 5.36 (br s, 1H). C RMN (CDCl3)
d 156.1, 79.5, 78.8, 77. 3, 54.0, 53.7, 52.7, 46.9, 43.7,
42.1, 40.0 , 37.5, 31.6, 29.7, 28.5, 26.9, 24.5, 24.3. HRMS
(ESI): calcd for C H N O : m/z 573.4536. Found: m/z
oxycarbonylspermine 6. Compound
.54 mmol, 1 equiv) and N-(4-bromobutyl)phthalimide
896 mg, 3.17 mmol, 1.25 equiv) were dissolved in dry
acetonitrile (20 mL) with an excess of K CO (5 equiv).
4
(1.277 mg,
2
(
2
9
59
5
6
+
574.4544 [M+H] .
2
3
The mixture was stirred for 18 h under reflux. The disap-
pearance of 4 was monitored by TLC (CH Cl /EtOH,
Monohydroxyphenylporphyrins 9a,b were synthesized
1d
according to the literature.
2
2
1
9
0:10). Acetonitrile was evaporated under vacuum and
the crude product was dissolved in dichloromethane.
The organic layer was washed with NaHCO3
5.2.7. General procedure for the synthesis of monocarb-
oxypropyloxyphenylporphyrins. Porphyrins 9a,b
(
2 · 50 mL) and water (2 · 50 mL), dried (MgSO ),
4
and then evaporated to afford, after purification by col-
umn chromatography (chloroform/ethanol, 100:0 to
(1 equiv) were dissolved in dry DMF (10 mL) with a
large excess of K CO (20 equiv). The mixture was stir-
2
3
9
EtOH, 90:10). IR (KBr) 3354, 2975, 2932, 1772, 1713,
0:10), 1.7 g of compound 6 (95%). R = 0.49 (CH Cl /
red for 15 min at room temperature. Ethyl 4-bromobu-
tyrate (5 equiv) was added and then the solution was
stirred at room temperature overnight in the dark. After
reaction, DMF was evaporated under vacuum and the
crude product was dissolved in methylene chloride.
The organic layer was washed several times with water,
dried (MgSO ), and then evaporated to afford, after
4
purification by thin-layer chromatography, the pure
porphyrins 10a,b.
f
2
2
ꢀ
1 1
1
2
170 cm ; H NMR (CDCl , 400.13 MHz) d 1.44 (m,
7H), 1.46 (m, 8H), 1.65 (m, 4H), 2.42 (m, 6H), 3.14
3
(
7
m, 8H), 3.70 (m, 2H), 4.91 (br s, 1H), 5.34 (br s, 1H),
.71 (dd, JH,H = 3.14 Hz and JH,H = 5.08 Hz, 2H), 7.84
dd, J = 3.13 Hz and JH,H = 5.08 Hz, 2H). RMN
(
H,H
1
3
C (CDCl ) d, 168.4, 156.0, 133.9, 132.1, 123.2, 79.5,
3
7
2
8.7, 77.2, 53.7, 53.4, 52.6, 46.9, 44.1, 43.9, 39.9, 37.8,
8.5, 28.4, 28.3, 27.0, 26.5, 26.0, 24.3. HRMS (ESI):
calcd for C H N O : m/z 703.4518. Found: m/z
5
7
Tritolyl derivatives 10a or b were dissolved in DMF
(8 mL) and KOH (2 mL, 1 M in ethanol) was added.
3
7
62
8
+
04.4592 [M+H] .

V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
1373
The mixture was stirred under reflux for 2 h. After cool-
ing, solvent was evaporated under vacuum and the res-
idue was dissolved in methylene chloride. The solution
was neutralized by addition of HCl (1 M) washed with
water and dried over magnesium sulfate. Column chro-
matography performed with CHCl3 and increasing
amounts of ethanol (0–10%) allowed purification of por-
phyrins 11a,b.
5.2.11. 5-(2-[3-Carboxypropyloxy]phenyl)-10,15,20-tris
(4-methylphenyl) porphyrin (11b). The saponification of
10b carried out on 120 mg gave porphyrin 11b in 97%
yield. R = 0.49 (CHCl /EtOH, 95:5); UV–vis (CH Cl ):
f
3
2
2
ꢀ
1
ꢀ1
3
kmax, nm (e, L cm mol · 10 ) 420 (388.6), 517 (15.7),
553 (9.8), 592 (4.9), 648 (4.2); H NMR (400.13 MHz,
1
CDCl , 25 ꢂC): d ꢀ2,76 (br s, 2H, NH pyr), 2.68 (s,
3
9H, CH tolyl), 7.23 (m, 2H, H Ar), 7.52 (br d, 6H,
3
3,5
JH,H = 7.8 Hz, H3,5 tolyl), 8.07 (d, 2H,JH,H = 7.7 Hz
H2,6 Ar), 8.09 (d, 6H, JH,H = 7.6 Hz, H2,6 tolyl), 8.83+
b
monoisotopic calcd: 758.3).
5
2
.2.8. 5-(4-[3-Ethoxycarbonylpropyloxy]phenyl)-10,15,
0-tris(4-methylphenyl) porphyrin (10a). Porphyrin 9a
(br s, 8H, H pyr). MS (MALDI) m/z 759.8 ([M+H]
(
(
150 mg, 0.22 mmol, 1 equiv), ethyl 4-bromobutyrate
159 mL, 1.1 mmol, 5 equiv), and K CO3 (615 mg,
2
4
.4 mmol, 20 equiv) afforded pure product 10a, 167 mg,
5.2.12. General procedure for the synthesis of porphyrins
bearing spermine or spermidine units. N -(4-Aminobutyl)-
4
(
(
(
90%). R = 0.56 (CHCl ); UV–vis (CH Cl ): kmax, nm
f
ꢀ
3
2
2
1
ꢀ1
3
1
8
N ,N -bis-tert-butoxycarbonylspermidine 7 (1.1 equiv),
e, L cm mol · 10 ): 420 (354.0), 516 (14.3), 552
1
7.2), 592 (4.7), 648 (4.2); H NMR (400.13 MHz, CDCl ,
4
1
8
12
or N -(4-aminobutyl)-N , N ,N -tris-tert-butoxycar-
bonylspermine 8 (1.1 equiv) was dissolved in DMF. A
solution of carboxy-porphyrins 11a,b (1 equiv) or proto-
3
2
3
2
4
5 ꢂC):
H,J
H,H
d
= 7.2 Hz, CH ethyl), 2.31 (m, 2H, H –CH –),
ꢀ2,71 (br s, 2H, NH pyr),1.36 (t,
3
b
2
0
.66 (m, 2H, H –CH –C@O), 2.69 (s, 9H, CH tolyl),
porphyrin IX (0.5 equiv) and N,N -dicyclohexylcarbodii-
b
2
3
.25 (q, 2H, J
= 7.1 Hz, CH ethyl), 4.30 (t, 2H,
mide (DCC) (1.1 equiv) in dry DMF (1 mL) was added.
After addition of 1-hydroxybenzotriazole (HOBt)
(1.1 equiv), the mixture was kept at room temperature,
in the dark, under argon, for 42 or 72 h (protoporphyrin
IX). DMF was evaporated under vacuum and the crude
product was dissolved in dichloromethane. The organic
H,H
2
JH,H = 6.0 Hz, H –O–CH –), 7.26 (d, 2H, J = 8.6 Hz,
H,H
a
2
H3,5 Ar), 7.56 (d, 6H, JH,H = 7.8 Hz, H3,5 tolyl), 8.11 (d,
H, JH,H = 7.7 Hz H2,6 Ar), 8.12 (d, 6H, JH,H = 7.7 Hz,
H2,6 tolyl), 8.88 (s, 8H, H pyr.). MS (MALDI) m/z
2
b
+
7
87.9 ([M+H] monoisotopic calcd: 786.4).
layer was washed with water (2 · 50 mL), dried MgSO ,
4
5
2
.2.9. 5-(2-[3-Ethoxycarbonylpropyloxy]phenyl)-10,15,
0-tris(4-methylphenyl) porphyrin (10b). Porphyrin 9b
and then evaporated to afford, after purification by
thin-layer chromatography, the pure product.
(
(
4
1
150 mg, 0.22 mmol, 1 equiv), ethyl 4-bromobutyrate
159 mL, 1.1 mmol, 5 equiv), and K CO3 (615 mg,
4
1
8
5.2.13. 5-(4-[N -(4-Amidobutyl)-N , N -bis-tert-butoxy-
carbonylspermidine-3-amidopropyloxy]phenyl)-10,15,20-
tris(4-methylphenyl) porphyrin (12a). Porphyrin 11a
(52 mg, 68.6 lmol, 1 equiv) and compound 7 (31 mg,
72.5 lmol, 1.1 equiv) reacted with DCC (16 mg,
68.6 lmol, 1.1 equiv) and 1-hydroxybenzotriazole
(HOBt) (10 mg, 68.6 lmol, 1.1 equiv) to afford pure
product 12a, 65 mg (81%). R = 0.63 (CH Cl /EtOH,
2
.4 mmol, 20 equiv) afforded pure product 10b,
58 mg, (85%). R = 0.66 (CHCl ); UV–vis (CH Cl ):
f
3
3
2
2
ꢀ1
ꢀ1
kmax, nm (e, L cm mol · 10 ) 420 (391.0), 516
(
(
pyr), 0.73 (t, 3H,J
4
2
1
13.4), 552 (8.1), 592 (4.6), 648 (2.8); H NMR
400.13 MHz, CDCl , 25 ꢂC): d ꢀ2.72 (s, 2H, NH
3
= 7.2 Hz, CH ethyl), 1.30 (m,
3
H,H
H, –CH -CH -C@O), 2.69 (s, 9H, CH tolyl), 3.59 (q,
2
2
3
f
2
2
ꢀ
1
H, J
= 7.2 Hz, CH ethyl), 3.91 (br t, 2H,
70:30). UV–vis (CH Cl ): kmax
,
nm (e, L cm
H,H
2
2
2
ꢀ
1
3
JH,H = 5.2 Hz, H –CH –), 7.30 (br d, 1H, JH,H = 8.0 Hz,
2
H Ar), 7.34 (br t, 1H, J
4
mol · 10 ) 420 (420.3), 516 (14.3), 553 (12.2), 592
1
(9.4), 648 (8.2). H NMR (400.13 MHz, CDCl ,
= 7.2 Hz, H Ar), 7.53 (d,
6
H,H
3
6
H, JH,H = 7.6 Hz, H3,5 tolyl), 7.73 (dt, 1H,
JH,H = 8.4–1.6 Hz H5 Ar), 8.02 (dd, 1H, JH,H = 7.6–
.6 Hz, H Ar), 8.10 (br d, 6H, JH,H = 8.0 Hz, H2,6 tol-
25 ꢂC): d ꢀ2,76 (br s, 2H, NH pyr), 1.43 (m, 18H,
CH Boc), 1.49 (m, 4H, N-CH -(CH ) -CH -NHBoc),
2 2
3
2
2
1
1.57 (quint, 2H, JH,H = 6.0 Hz, N-CH -CH -CH -
2 2 2
3
yl), 8.77 (d, 2H, JH,H = 4.8 Hz, H pyr), 8.83 (d, 2H,
b
JH,H = 4.7 Hz, H pyr), 8.84 (s, 4H, H pyr). MS (MAL-
NHBoc), 1.65 (m, 4H, CO-NH-CH -(CH ) -CH -N),
2 2 2 2
2.29 (quint, 2H, Porph-O-CH -CH –), 2.53 (t, 2H,
2 2
JH,H = 7.4 Hz, CO-NH-CH -(CH ) -N),2.65-2.69 (m,
2 2 3
b
DI) m/z 787.9 ([M+H] monoisotopic calcd: 786.4).
b
+
6
H,
NHBoc and N-CH -(CH ) -NHBoc), 2.70 (s, 9H, CH
3
Porph-O-(CH ) -CH -C@O,
N-CH -(CH ) -
2
2
2
2 2 2
5
(
1
.2.10. 5-(4-[3-Carboxypropyloxy]phenyl)-10,15,20-tris
4-methylphenyl) porphyrin (11a). The saponification of
0a carried out on 100 mg gave porphyrin 11a in 98%
yield. R = 0.49 (CHCl /EtOH, 95:5); UV–vis (CH Cl ):
2
2 3
tolyl), 3.09 (m, 2H, N-(CH ) -CH -NHBoc), 3.16 (m,
2 3 2
2H, N-(CH ) -CH -NHBoc), 3.31 (m, 2H, CO-NH-
2 2 2
(CH ) -CH -N), 4.27 (t, 2H, J = 6.0 Hz, Porph-O-
H,H
f
3
2
2
2 3
2
ꢀ1
ꢀ1
3
kmax, nm (e, L cm mol · 10 ) 420 (355.7), 517 (13.4),
5
CH –), 4.74 (br s, 1H, NHBoc), 5.30 (br s, 1H, NHBoc),
2
1
53 (7.2), 592 (4.0), 648 (3.8); H NMR (400.13 MHz,
7.25 (d, 2H, JH,H = 8.4 Hz, H3,5 Ar), 7.53 (d, 6H,
JH,H = 7.8 Hz, H3,5 tolyl), 8.08 (d, 6H, JH,H = 7.8 Hz,
H2,6 tolyl), 8.10 (d, 2H,JH,H = 8.4 Hz H2,6 Ar), 8.85 (s,
8H, H pyr). MS (MALDI) m/z 1157.5 ([M+H] mono-
b
isotopic calcd: 1156.7).
CDCl , 25 ꢂC): d ꢀ2,76 (br s, 2H, NH pyr), 2.35 (m,
3
2
H, H –CH –), 2.68 (s, 9H, CH tolyl), 2.77 (t, 2H,
b
2
3
+
JH,H = 7.1 Hz, Hc –CH -C@O), 4.31 (t, 2H,
2
JH,H = 6.1 Hz, H O-CH -), 4.30 (t, 2H, J = 6.0 Hz,
H,H
a
H –O–CH –), 7.23 (m, 2H, H Ar), 7.52 (br d, 6H,
2
a
JH,H = 7.8 Hz, H3,5 tolyl), 8.07 (d, 2H, JH,H = 7.7 Hz
2
3,5
4
1
8
5.2.14. 5-(2-[N -(4-Amidobutyl)-N , N -bis-tert-butoxy-
carbonylspermidine-3-amidopropyloxy]phenyl)-10,15,20-
tris(4-methylphenyl) porphyrin (12b). Porphyrin 11b
(41 mg, 54.1 lmol, 1 equiv) and compound 7 (25 mg,
H2,6 Ar), 8.09 (d, 6H, JH,H = 7.6 Hz, H2,6 tolyl), 8.83
+
(br s, 8H, H pyr). MS (MALDI) m/z 759.9 ([M+H]
b
monoisotopic calcd: 758.3).

1374
V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
5
5
9.5 lmol, 1.1 equiv) reacted with DCC (12 mg,
9.5 lmol, 1.1 equiv) and 1-hydroxybenzotriazole
NMR (400.13 MHz, CDCl , 25 ꢂC): d ꢀ2.74 (br s, 2H,
3
NH pyr), 1.08–1.25 (m, 12H, Porph-O-CH -(CH ) -
2 2
2
(
uct 12b, 57 mg (77%). R = 0.41 (CH Cl /EtOH, 85:15).
HOBt) (8 mg, 59.5 lmol, 1.1 equiv) to afford pure prod-
CONH-CH -(CH ) -CH -N and N-CH -(CH ) -CH -
2 2 2 2 2 2 2 2
NH-(CH ) -NHBoc). 1.32 (m, 18H, CH Boc), 1.43
3
f
2
2
2 4
ꢀ
1
ꢀ1
3
UV–vis (CH Cl ): kmax, nm (e, L cm mol · 10 ) 419
(m, 4H, N-CH -CH -CH -NHBoc and –(CH ) NH-
2 2 2 2 4
CH -CH -CH -NHBoc), 1.91–1.93 (m, 4H, CONH-
2 2 2
2
2
1
487.9), 516 (16.0), 553 (10.5), 593 (6.1), 648 (4.3). H
(
NMR (400.13 MHz, CDCl , 25 ꢂC): d ꢀ2,75 (br s, 2H,
CH -(CH ) -N and N-CH -(CH ) -NHBoc), 2.70 (s,
2 2 3 2 2 3
3
NH pyr), 1.13–1.25 (m, 12H, Porph-O-CH -(CH ) -
2
9H, CH tolyl), 3.83 (t, 2H, J
3
= 5.4 Hz, Porph-O-
H,H
2 2
CONH-CH -(CH ) -CH -N and N-CH -(CH ) -CH -
2
CH -(CH ) -CONH-), 2.62 (m, 2H, CONH-(CH ) -
2 2 2 2 3
2
2 2
2
2
2 2
NHBoc). 1.34 (m, 18H, CH Boc), 1.42 (m, 2H, N-CH -
3
CH -N), 2.86 (m, 2H,-(CH ) NH-(CH ) -CH -NHBoc)
2 2 4 2 2 2
2
CH -CH -NHBoc), 1.91–2.00 (m, 6H, CONH-CH -
2
3.11–3.16 (m, 6H, CO-NH-(CH ) -CH -NH-CH -
2 3 2 2
2
2
(
NHBoc), 2.64–2.87(m, 6H, N-CH -CH -CH -NHBoc,
CH ) -N, N-CH -(CH ) -NHBoc and N-CH -(CH ) -
(CH ) -NHBoc and N-(CH ) -CH -NHBoc) 4.74 (br s,
2 2 2 2 2
2
3
2
2 3
2
2 2
1H, NHBoc), 5.31 (br s, 1H, NHBoc), 7.29 (br d, 1H,
JH,H = 8.3 Hz, H Ar), 7.39 (br t, 1H, J = 7.3 Hz,
2
2
2
N-(CH ) -CH -NHBoc and CONH-(CH ) -CH -N),
2
2
3
2
2 3
3
H,H
2
ph-O-CH -(CH ) -CONH–), 4.73 (br s, 1H, NHBoc),
.70 (s, 9H, CH tolyl), 3.82 (t, 2H, J
= 5.5 Hz, Por-
H Ar) 7.55 (d, 6H, J
5
t, 1H, JH,H = 8.2–1.6 Hz, H Ar), 8.06 (m, 6H, H tol-
4 2,6
= 7.5 Hz, H3,5 tolyl), 7.76 (br
H,H
3
H,H
2
2 2
5
H Ar), 7.40 (br t, 1H, J = 7.3 Hz, H Ar) 7.55 (m,
.29 (br s, 1H, NHBoc), 7.29 (br d, 1H, JH,H = 8.2 Hz,
yl), 8.13 (m, 1H, H Ar), 8.86 (m, 8H, H pyr). MS
6 b
(MALDI) m/z 1314.7 ([M+H] monoisotopic calcd:
+
3
H,H
5
6
8
8
H, H3,5 tolyl), 7.75 (dt, 1H, JH,H = 8.3–1.5 Hz, H Ar),
4
1313.8).
.06 (m, 6H, H2,6 tolyl), 8.13 (m, 1H, H Ar), 8.87 (m,
6
+
H, H pyr). MS (MALDI) m/z 1157.6 ([M+H] monoi-
4
1
8
5.2.17. 13,17-di-[3-Amidoethyl-N -(4-aminobutyl)-N ,N -
bis-tert-butoxycarbonylspermidine]-2,7,12,18-tetramethyl-
b
sotopic calcd: 1156.7).
8
,13-divinyl-porphyne (14). Protoporphyrin IX (70 mg,
4
1
8
12
.2.15. 5-(4-[N -(4-Aminobutyl)-N ,N ,N -bis-tert-but-
5
oxycarbonylspermine-3-amidopropyloxy]phenyl)-10,15,20-
0.12 mmol, 0.5 equiv) and compound 7 (108.3 mg,
0.26 mmol, 1.1 equiv) reacted with DCC (53.6 mg,
0.26 mmol, 1.1 equiv) and 1-hydroxybenzotriazole
(HOBt) (35.1 mg, 0.26 mmol, 1.1 equiv) in DMF
(20 mL). After 72 h of reaction and treatment, 132 mg
(80%) of compound 14 was obtained after thin-layer
chromatography (CH Cl /EtOH, 80:20 + 1% Et N).
R = 0.45 (ethyl acetate/acetone/acetic acid/water,
f
5:3:1:1). UV–vis (CH Cl ): kmax, nm (e, L cm
mol · 10 ): 407(113.9); 504(8.8); 541 (8.4); 575 (5.3);
629(3.2). H NMR (CDCl , 400.13 MHz): d (ppm) =
tris(4-methylphenyl) porphyrin (13a). Porphyrin 11a
(
106 mg, 0.14 mmol, 1 equiv) and compound 8 (86 mg,
0
0
.15 mmol, 1.1 equiv) reacted with DCC (31 mg,
.15 mmol, 1.1 equiv) and 1-hydroxybenzotriazole
(HOBt) (20 mg, 0.15 mmol, 1.1 equiv) to afford pure
product 13a, 65 mg (74%). R = 0.48 (CH Cl /EtOH,
2
2
3
f
2
2
ꢀ
1
ꢀ1
8
0:20). UV–vis (CH Cl ): kmax
,
nm (e, L cm
2
2
2
2
ꢀ
1
3
ꢀ1
3
mol · 10 ): 422 (460.6), 517 (18.7), 553 (14.6), 595
1
6.5), 648 (4.4). H NMR (400.13 MHz, CDCl ,
1
(
3
3
2
CH Boc), 1.51 (m, 4H, N-CH -(CH ) -CH -NH-
5 ꢂC): d ꢀ2,76 (br s, 2H, NH pyr), 1.44 (m, 18H,
ꢀ3.69 (s, 2H, NH pyrrole), 1.36 (s, 36H, CH Boc),
3
1.43–1.20 (m, 20H, –CONHCH (CH ) CH N(CH ) ,
2
3
2
2 2
2
2
2 2
2 2
(
CH -CH -CH -NHBoc), 1.65 (m, 4H, CO-NH-CH -
CH ) -NHBoc), 1.57 (m, 4H, N-CH -CH -CH -NH-
(CH ) NCH CH CH CH NHBoc and –(CH ) NCH
2 2 2 2 2 2 2 2 2
CH CH NHBoc), 3.1–2.2 (m, 24H, CH -N, CH -NH-),
2 2 2 2
2
3
2
2
2
2
2
2
2
(
CH ) -CH -N), 2.30 (quint, 2H, J
= 6.2 Hz, Por-
ph-O-CH -CH –), 2.53(m, 6H, CO-NH-CH -(CH ) -
3.50 (m, 16H, Proto-CH -CH -CO and CH b pyrrole),
4.26 (m, 4H, Proto-CH -CH -CO), 4.79 (s, 2H, –NHCO),
2 2
2
2
2
H,H
2 2 3
2
2
2
2 3
N, N-CH -(CH ) NH-CH -(CH ) -NHBoc), 2.67 (m,
2 2
5.27 (s, 4H, NHBoc), 6.21 (bd, 2H, J = 10.7 Hz, CH2
vinyl), 6.34 (d, 2H, J = 17.8 Hz, CH vinyl), 8.14 (dd,
2
2 3
2
2
yl), 3.08 (m, 2H, N-(CH ) -CH -NH-(CH ) -NHBoc),
H, Porph-O-(CH ) -CH -C@O), 2.69 (s, 9H, CH tol-
2
2
2
3
2
2H, J = 11.6–17.7 Hz, CH vinyl), 9.76, 9.69, 9.54 (s, 4H,
H-meso). Anal. calcd for C H N O : C, 67.12; H,
2
3
2
2 3
3
CH -CH -CH -NHBoc), 3.31 (m, 2H, CO-NH-(CH ) -
.17 (m, 6H, N-(CH ) -CH -NHBoc and -(CH ) -NH-
2
2
2
2 4
76 118 12 10
8.75; N, 12.36. Found: C, 67.01; H, 8.63; N, 12.25. MS
(MALDI) m/z 1360.4 ([M+H] monoisotopic calcd:
1359.8).
2
2
2
2 3
+
CH -N), 4.28 (t, 2H, J
2
= 5.9 Hz, Porph-O-CH –),
2
H,H
4
.74 (br s, 1H, NHBoc), 5.30 (br s, 1H, NHBoc), 7.25
d, 2H, JH,H = 8.2 Hz, H3,5 Ar), 7.54 (d, 6H,
(
JH,H = 7.7 Hz, H3,5 tolyl), 8.08 (d, 6H, JH,H = 7.7 Hz,
H2,6 tolyl), 8.10 (d, 2H,JH,H = 8.2 Hz H2,6 Ar), 8.85
(s, 8H, Hb pyr). MS (MALDI) m/z 1314.6 ([M+H]
monoisotopic calcd: 1313.8).
4
1
5.2.18. 13,17-di-[3-Amidoethyl-N -(4-aminobutyl)-N ,N ,
8
1
2
N -bis-tert-butoxycarbonylspermine]-2,7,12,18-tetrameth-
yl-8,13-divinyl- porphyne (15). Protoporphyrin IX
+
(55 mg, 0.97 mmol, 0.5 equiv) and compound
(
8
108.3 mg, 0.21 mmol, 1.1 equiv) reacted with DCC
4
1
8
.2.16. 5-(4-[N -(4-Aminobutyl)-N ,N ,N -bis-tert-but-
12
5
oxycarbonylspermine-3-amidopropyloxy]phenyl)-10,15,20-
(44 mg, 0.21 mmol, 1.1 equiv) and 1-hydroxybenzotriaz-
ole (HOBt) (29 mg, 0.21 mmol, 1.1 equiv) in DMF
(20 mL). After 72 h of reaction and treatment, 139 mg
(85%) of compound 15 was obtained after
thin-layer chromatography (CH Cl /EtOH, 80:20 + 1%
tris(4-methylphenyl) porphyrin (13b). Porphyrin 11b
(
35 mg, 46.15 lmol, 1 equiv) and compound 8 (29 mg,
5
5
0.77 lmol, 1.1 equiv) reacted with DCC (10 mg,
0.8 lmol, 1.1 equiv) and 1-hydroxybenzotriazole
2
2
Et N). R = 0.62 (ethyl acetate/acetone/acetic acid/
3
f
(
uct 13b, 46 mg (76%). R = 0.48 (CH Cl /EtOH, 80:20).
HOBt) (7 mg, 50.8 lmol, 1.1 equiv) to afford pure prod-
water, 5:3:1:1). UV–vis (CH Cl ): kmax, nm (e,
2 2
1 3
ꢀ
ꢀ1
L cm mol · 10 ): 406 (133.5); 504 (10.5); 540 (9.3);
f
2
2
ꢀ
1
ꢀ1
3
1
UV–vis (CH Cl ): kmax, nm (e, L cm mol · 10 ) 420
574 (6.6); 629 (3.7). H NMR (CDCl , 400.13 MHz): d
2
2
3
1
446.4), 516 (14.4), 554 (9.0), 594 (5.6), 645 (3.7). H
(
(ppm) = ꢀ3.71(s,
2H,
NH-pyrrole),
1.45–1.26

V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
1375
(
m, 74H, CH3 Boc, CONH-CH (CH ) CH N(CH ) ,
2
N, 11.71. Found: C, 77.51; H, 7.01; N, 11.52. MS
(MALDI) m/z 957.6 ([M+H ] monoisotopic calcd:
956.5).
2 2
2
2 2
+
(
CH -NHBoc and (CH ) NCH CH CH N(CH )), 3.22–
CH ) NCH CH CH CH NHBoc, –(CH ) NCH CH
2
2
2
2
2
2
2
2 2
2
2
2 2
2
2
2
2
2
3
.88 (m, 32H, CH -N, –(CH ) N-CO and CH NHCO),
2 2 2 2
4
5.2.22. 5-(4-[N -(4-Aminobutyl)spermine-3-amidopropyl-
.71–3.46 (m, 16H, CH3b pyrrole and Proto-CH -CH -
2
2
CO), 4.33 (m, 4H, Proto-CH -CH -CO), 4.75 (bs, 2H,
2
oxy]phenyl)-10, 15,20-tris(4-methylphenyl) porphyrin
(17a). R = 0.60 (CH CN/H O 7/3 + 1% TFA). UV–vis
2
-
J = 11.4 Hz, CH vinyl), 6.36 (d, 2H, J = 17.8 Hz, CH
NHCO), 5.25 (bs, 4H, NHBoc), 6.21 (d, 2H,
f
3
2
ꢀ
1
ꢀ1
3
(MeOH): kmax, nm (e, L cm mol · 10 ): 415(258.4);
514(12.7); 549(6.9); 590 (4.6); 646(3.7). H NMR (400
2
2
1
vinyl), 8.19 (dd, 2H, J = 11.6–17.7 Hz, CH vinyl), 9.94,
.90, 9.88, 9.75 (s, 4H, H-meso). Anal. calcd for
C H N O : C, 66.00; H, 8.91; N, 11.71. Found: C,
9
MHz, CDCl /CD OD 9/1, 25 ꢂC): d 1.67(m, 6H, N-
3
3
CH -(CH ) -CH -NH-CH -CH -CH -NH ), 1.84 (m,
6H, CO-NH-CH -(CH ) -CH -N and N-CH -CH -
2 2 2 2 2 2
9
2
148 14 14
2
2 2
2
2
2
2
2
6
(
5.83; H, 8.78; N, 11.59. MS (MALDI) m/z 1674.7
[M+H] monoisotopic calcd: 1374.2).
+
CH -NH ), 2.30 (quint, 2H, J
H,H
= 6.7 Hz, Porph-O-
= 7.1 Hz, CO-NH-
= 6.6 Hz, Porph-O-
2
2
CH -CH -CH ), 2.60 (t, 2H, J
2
2
2
H,H
CH -(CH ) -N), 2.67 (t, 2H, J
2 2 3 H,H
5
.2.19. General procedure for the removal of Boc-protec-
tive groups. The protecting groups (Boc) were removed
with standard method in high yields with TFA in
CH Cl at room temperature (2 h).
(CH ) -CH -C@O), 2.71 (s, 9H, CH tolyl), 2.79 (m,
2
2
2
3
2H, N-CH -(CH ) -NH-), 2.89 (t, 2H, J = 6.4 Hz,
H,H
2
2 2
N-CH -(CH ) -NH ), 3.10 (m, 6H, N-(CH ) -CH -NH-
2
2
2
2 2
2
2 3
2
(
CH ) -CH -NH and N-(CH ) -CH -NH ), 3.24 (t,
2 2 2 2 2 2 2 2
4
5
.2.20. 5-(4-[N -(4-Amidobutyl)spermidine-3-amidopro-
pyloxy]phenyl)-10,15,20-tris(4-methylphenyl) porphyrin
16a). R = 0.58 (CH CN/H O, 7:3 + 1% TFA). UV–
2H, JH,H = 6.0 Hz, N-(CH ) -NH-CH -(CH ) -NH )
2 4 2 2 2 2
3.35 (m, 2H, CO-NH-(CH ) -CH -N), 4.32 (t, 2H,
2
JH,H = 6.0 Hz, Porph-O-CH -), 7.32 (d, 2H, J
2
8.3 Hz, H3,5 Ar), 7.58 (d, 6H, JH,H = 7.7 Hz, H3,5 tolyl),
8.07 (d, 6H, H2,6 tolyl, JH,H = 7.7Hz), 8.12 (d, 2H, H2,6
Ar, JH,H = 8.3 Hz), 8.87 (br s, 8H, H pyr). Anal. calcd
for C H N O : C, 77.01; H, 7.46; N, 12.44. Found: C,
9
76.84; H, 7.21; N, 12.09. MS (MALDI) m/z 1014.5
([M+H ] monoisotopic calcd: 1013.60).
3
2
(
=
H,H
f
3
2
ꢀ
1
ꢀ1
3
vis (MeOH): kmax, nm (e, L cm mol · 10 ): 416
1
234.1); 515 (7.9); 547 (5.1); 590 (4.1); 650 (3.1). H
(
NMR (400 MHz, CDCl /CD OD, 9:1, 25 ꢂC): d 1.58
3
3
b
(
NH-CH -(CH ) -CH -N), 1.99 (quint, 2H, J = 6.6 Hz,
m, 4H, N-CH -(CH ) -CH -NH ), 1.78(m, 4H, CO-
2
2 2
2
2
65 75
2
2
2 2
2
+
N-CH -CH -CH -NH ) 2.29 (quint, 2H, J = 6.7 Hz,
2
2
2
2
Porph-O-CH -CH -CH ), 2,60(t, 2H, J = 6.6 Hz,
H,H
2
2
2
4
5.2.23. 5-(2-[N -(4-Aminobutyl)spermine-3-amidopropyl-
N-CH -(CH ) -NH ), 2.66 (m, 4H, Porph-O-(CH ) -
2 2
2
2 3
2
CH -CO-NH-CH -(CH ) -N), 2.70 (s, 9H, CH tolyl),
2
oxy]phenyl)-10, 15,20-tris(4-methylphenyl) porphyrin
(17b). R = 0.64 (CH CN/H O 7/3 + 1% TFA). UV–vis
2
2 3
3
2
.84 (t, 2H, JH,H = 6.6 Hz, N-CH -(CH ) -NH ), 2.97
2 2
2
2
f
3
2
ꢀ
1
ꢀ1
3
(
t, 2H, JH,H = 6.1 Hz, N-(CH ) -CH -NH ), 3.11 (t,
2 3
(MeOH): kmax, nm (e, L cm mol · 10 ): 415(143.1);
515(10.7); 550 (7.7); 591 (5.9); 648(5.3). H NMR
2
2
1
2
JH,H = 6.5 Hz, CO-NH-(CH ) -CH -N), 4.30 (t, 2H,
H, JH,H = 6.4 Hz, N-(CH ) -CH -NH ) 3.30 (t, 2H,
2 2
2
2
(400 MHz, CDCl /CD OD 9/1, 25 ꢂC): d 1.18-1.26 (m,
2 3
2
3
3
JH,H = 6.1 Hz,
Porph-O-CH –),
2
7.27
(d,
2H,
12H,
Porph-O-CH -CH -CH -CO-NH-CH -(CH ) -
2 2 2 2 2 2
JH,H = 8.4 Hz, H3,5 Ar), 7.55 (d, 6H, JH,H = 7.8 Hz,
H3,5 tolyl), 8.08 (d, 6H, H2,6 tolyl, JH,H = 7.8 Hz),
CH -N and N-CH -(CH ) -CH -NH-), 1.37 (m, 4H, N-
2 2 2 2 2
(CH ) -NH-CH -CH -CH -NH
2
and N-CH -CH -
2 2
2 4
2
2
2
8
pyr). Anal. calcd for C H N O : C, 77.85; H, 7.17;
.10(d, 2H, H2,6 Ar, JH,H = 8.4 Hz), 8.86 (br s, 8H, Hb
CH -NH ), 2.15 (m, 6H, N-CH -(CH ) NH-, CO-NH-
2 2 2 2 3
CH - and N-CH -(CH ) -NH ), 2.65 (m, 2H, CO-NH-
2
6
2
68
8
2
2
2 2
2
N, 11.71. Found: C, 77.69; H, 7.08; N, 11.59. MS
(
(CH ) -CH -N), 2.71 (s, 9H, CH tolyl), 2.78 (m, 6H,
2 3 2 3
N-(CH ) -CH -NH-, N-(CH ) -CH -NH and –(CH ) -
2 3 2 2 2 2 2 2 4
+
MALDI) m/z 957.6 ([M+H ] monoisotopic calcd:
9
56.5).
NH-(CH ) -CH -NH ), 2.90 (m, 2H, -(CH ) -NH-CH -
2 2 2 2 2 4 2
(CH ) -NH ), 3.97 (t, 2H, J
CH –), 7.43 (d, 1H, J
= 5.4 Hz, Porph-O-
H,H
= 8.4 Hz, H Ar), 7.40 (m, 1H,
H,H 3
2
2
2
4
.2.21. 5-(2-[N -(4-Amidobutyl)spermidine-3-amidopro-
5
pyloxy]phenyl)-10,15,20-tris(4-methylphenyl) Porphyrin
(
(
(
2
H5 Ar), 7.61 (m, 6H, H3,5 tolyl), 7.81 (dt, 1H,
JH,H = 8.4–1.4 Hz, H Ar), 8.04 (dd, 1H, J = 7.7–
16b). R = 0.45 (CH CN/H O 7/3 + 1% TFA). UV–vis
f
3
2
4
H,H
ꢀ
1
ꢀ1
3
MeOH): kmax
,
nm (e, L.cm mol · 10 ): 4115
162.7); 515 (9.6); 550 (6.3); 591 (3.9); 648(3.4).
1.4 Hz, H Ar), 8.09 (m, 6H, H tolyl), 8.87 (br s, 8H,
6 2,6
H
H pyr) Anal. calcd for C H N O : C, 77.01; H, 7.46;
b 65 75 9 2
N, 12.44. Found: C, 76.89; H, 7.38; N, 12.11. MS (MAL-
NMR (400 MHz, CDCl /CD OD 9/1, 25 ꢂC): d 1.13
3
3
+
(
Porph-O-CH -CH -CH -CO-NH-CH -(CH ) -CH -N
m, 4H, N-CH -(CH ) -CH -NH ), 1.20-1.37 (m, 10H,
DI) m/z 1014.5 ([M+H ] monoisotopic calcd: 1013.6).
2
2 2
2
2
2
2
2
2
2 2
2
4
5.2.24. 13,17-di-[3-Amidoethyl-N -(4-aminobutyl)spermi-
and N-CH -CH -CH -NH ), 2.03-2.10 (m, 6H, N-CH -
2
2
2
2
2
(
(
2
CH ) -NH and CO-NH-CH -(CH ) -N and N-CH -
2
dine]-2,7,12,18-tetramethyl-8, 13-divinyl-porphine (18).
R = 0.58 (CH CN/H O 7:3 + 1% TFA). UV–vis
2
3
2
2
2 3
CH ) -NH ), 2.66 (m, 2H, CO-NH-(CH ) -CH -N),
2
2
2
2
2 3
f
3
2
ꢀ1
ꢀ1
3
.71 (s, 9H, CH3 tolyl), 2.75 (t, 2H, JH,H = 7.0 Hz,
= 7.0 Hz, N-
(MeOH): kmax, nm (e, L cm mol · 10 ): 403 (27.0);
503 (2.5); 538 (2.1); 575 (1.3); 629 (0.9). H NMR
1
N-(CH ) -CH -NH ), 2.81 (t, 2H, J
H,H
2
3
2
2
(
O-CH -), 7.43 (d, 1H, J
CH ) -CH -NH ), 3.96 (t, 2H, J
= 5.4 Hz, Porph-
= 8.4 Hz, H Ar), 7.39 (m,
(CD OD, 400.13 MHz): d (ppm) = 1.55–1.16 (m, 20H,
2
2
2
2
H,H
3
–CONHCH (CH ) CH N(CH ) ,
(CH ) NCH CH
2
H,H
3
2
2 2
2
2 2
2 2 2 2
1
JH,H = 8.2–1.4 Hz, H Ar), 8.04 (dd, 1H, J
H, H Ar), 7.59 (m, 6H, H3,5 tolyl), 7.80 (dt, 1H,
= 7.6-1.4
Hz, H Ar), 8.07 (m, 6H, H tolyl), 8.87 (m, 8H, Hb
CH CH NH and –(CH ) NCH CH CH NH ), 3.01–
2 2 2 2 2 2 2 2 2
2.64 (m, 16H, CH -N, CH -NH–), 3.34 (s, 4H, Proto-
2 2
CH -CH -CO), 3.50 (m, 12H, CH pyrrole), 4.29 (m,
2 2 3b
5
4
H,H
6
2,6
pyr). Anal. Calcd.for C H N O : C, 77.85; H, 7.17;
8
4H, Proto-CH -CH -CO), 6.40–6.30 (m, 4H, CH
2 2 2
6
2
68
2

1376
V. Sol et al. / Bioorg. Med. Chem. 14 (2006) 1364–1377
vinyl), 8.09 (dd, 1H, J = 12.0–17.1 Hz, CH vinyl), 8.17
dd, 1H, J = 11.6–17.4 Hz, CH vinyl), 9.46 (br s, 3H, H-
meso), 9.34 (br s, 1H, H-meso). Anal. calcd for
(PI)atonceafterilluminationandafterafurther24 hincu-
2
0,21
(
bation in the dark.
All the compounds tested did not
presentanycytotoxicityinthedark.DatashowninFigures
3 and 4 are always means of three independent experi-
ments. Annexin V/Propidium iodide co-staining assays
(Roche Annexin-V-FLUOS) were conducted according
to the manufacturerÕs protocol.
C H N O : C, 70.15; H, 9.04; N, 20.45. Found: C,
5
6
86 12
2
6
9.88; H, 8.91; N, 20.14. MS (MALDI) m/z 959.7 ([
M+H ] monoisotopic calcd: 958.7).
+
4
.2.25. 13,17-di-[3-Amidoethyl-N -(4-aminobutyl)sperm-
5
ine]-2,7,12,18-tetramethyl-8,13-divinyl-porphyne
(19).
R = 0.60(CH CN/H O 7:3 + 1%TFA).UV–vis(MeOH):
Acknowledgments
f
3
2
ꢀ
1
ꢀ1
3
kmax, nm (e, L cm mol · 10 ): 402 (29.7); 503 (2.6); 538
2.1); 575 (1.4); 629 (0.9).
00.13 MHz): d (ppm) = 1.45–1.26 (m, 24H, CONHCH2
CH ) CH N(CH ) , (CH ) NCH CH CH CH NH-
1
H
(
4
(
NMR (CD OD,
We thank ÔConseil R e´ gional du LimousinÕ for financial
support and ÔAssociation de Recherche sur le CancerÕ
(ARC) for a fellowship attributed to one of us (F.L.).
3
2
2
2
2 2
2 2
2
2
2
2
CH CH CH NH and (CH ) NCH CH CH NH2),
2
2
2
2
2
2 2
2
2
3
.22-2.88 (m, 36H, (CH ) N, –(CH ) NH, CONHCH -,
2 3 2 2 2
CH NH and Proto-CH -CH -CO), 3.50 (m, 12H, CH
3
2
2
2
2
References and notes
b pyrrole), 4.33 (m, 4H, Proto-CH -CH -CO), 6.36 (m,
2
2
4
H, CH vinyl), 8.33-8.17 (m, 2H, CH vinyl), 9.52–9.41
2
1
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