Hydroxyphthalocyanines as potential photodynamic agents for cancer therapy

Article abstract of DOI:10.1021/jm970336s

A series of benzyl-substituted phthalonitriles, substituted at the 3-, 4-, and 4,5-positions, underwent varied condensations with phthalonitrile to give a series of protected (monohydroxy- and polyhydroxyphthalocyaninato)zinc(II) derivatives which were readily cleaved to give several hydroxyphthalocyanines (ZnPc) (phthalocyanine phenol analogues). Their efficacy as sensitizers for the photodynamic therapy (PDT) of cancer was evaluated on the EMT-6 mammary tumor cell line. In vitro, the 2-hydroxy ZnPc (32) was the most active, followed by the 2,3- and 2,9- dihydroxy ZnPc (39 and 45), with the 2,9,16-trihydroxy ZnPc (33) exhibiting the least activity. In vivo, the monohydroxy derivative 32 and the 2,3- dihydroxy derivative 39 were both efficient in inducing tumor necrosis at 1 μmol kg^-1, but complete tumor regression was poor, even at 2 μmol/kg. In contrast, the 2,9-dihydroxy isomer 45, at 2 μmol kg^-1, induced tumor necrosis in all animals treated, with 75% complete regression. These results underline the importance of the position of the substituents on the Pc macrocycle to optimize tumor response and confirm the PDT potential of the unsymmetrical Pcs bearing functional groups on adjacent benzene rings.

Full text of DOI:10.1021/jm970336s

J . Med. Chem. 1998, 41, 1789-1802
1789
Hyd r oxyp h th a locya n in es a s P oten tia l P h otod yn a m ic Agen ts for Ca n cer
Th er a p y
Mougang Hu,† Nicole Brasseur,‡ S. Zeki Yildiz,†,§ J ohan E. van Lier,*,‡ and Clifford C. Leznoff*,†
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada and MRC Group in the Radiation Sciences,
Faculty of Medicine, Universite´ de Sherbrooke, Que´bec J 1H 5N4, Canada
Received May 21, 1997
A series of benzyl-substituted phthalonitriles, substituted at the 3-, 4-, and 4,5-positions,
underwent varied condensations with phthalonitrile to give a series of protected (monohydroxy-
and polyhydroxyphthalocyaninato)zinc(II) derivatives which were readily cleaved to give several
hydroxyphthalocyanines (ZnPc) (phthalocyanine phenol analogues). Their efficacy as sensitizers
for the photodynamic therapy (PDT) of cancer was evaluated on the EMT-6 mammary tumor
cell line. In vitro, the 2-hydroxy ZnPc (32) was the most active, followed by the 2,3- and 2,9-
dihydroxy ZnPc (39 and 45), with the 2,9,16-trihydroxy ZnPc (33) exhibiting the least activity.
In vivo, the monohydroxy derivative 32 and the 2,3-dihydroxy derivative 39 were both efficient
in inducing tumor necrosis at 1 µmol kg-1, but complete tumor regression was poor, even at 2
µmol/kg. In contrast, the 2,9-dihydroxy isomer 45, at 2 µmol kg-1, induced tumor necrosis in
all animals treated, with 75% complete regression. These results underline the importance of
the position of the substituents on the Pc macrocycle to optimize tumor response and confirm
the PDT potential of the unsymmetrical Pcs bearing functional groups on adjacent benzene
rings.
In tr od u ction
rivatives, as porphyrin analogues, have attracted much
interest because of their many advantages over Photo-
frin such as existing as single substances, having large
extinction coefficients and absorption in the convenient
650-800-nm range, and having no dark toxicity. Pcs
and naphthalocyanines (Ncs) form stable chelates with
metal cations, and the photosensitizing properties of
aluminum, gallium, zinc, and silicon Pcs6 and Ncs7 have
been documented.
Photodynamic therapy (PDT) has been proposed as
an alternative treatment modality to complement con-
ventional protocols in the management of malignant
tumors. Thousands of patients have been treated by
PDT worldwide, and a large majority of them have
favorably responded to treatment.1 PDT using a mixture
of hematoporphyrin derivatives (Photofrin) as the pho-
tosensitizer is in an advanced experimental phase in
several centers, especially for the endoscopic irradiation
of lung, bronchial, and esophageal tumors and for the
photosterilization of the tumor bed after surgical resec-
tion of large neoplasm,2 and is approved as a sensitizer
for photodynamic treatment of esophageal and early
lung cancer in the United States, France, The Nether-
lands, and J apan. It is well-known that Photofrin is
far from being the ideal drug for PDT3 mainly because
of its complex, variable, and unstable composition,4 its
low extinction coefficient at 630 nm, and the onset of
long-term toxic effects, since some components of Photo-
frin are retained in amounts as large as several micro-
grams per gram of tissue by liver and spleen.4d For
these reasons it appears essential to develop new tumor
photosensitizers in order to overcome the present limi-
tations of Photofrin.
A very important property of a photosensitizing drug
is its hydrophobic and hydrophilic properties. Both the
porphyrin and Pc skeletons are essentially hydrophobic.
Tumor localization can be improved by introducing polar
substituents to confer amphiphilicity and improve se-
lectivity. Sulfonic acid, carboxylic acid, phosphonic acid,
hydroxyl, and quaternary ammonium salts are among
some of the common functional groups which can be
used for this purpose. The relationship between the
chemical structure and the tumor-localizing activity of
photosensitizers has been investigated in detail using
cultured cells and different experimental tumors.8 It
appears that optimal cell uptake efficiency is imparted
to the porphyrin-type macrocycles by the presence of two
substituents in adjacent rings, yielding an amphiphilic
molecule which retains a hydrophobic matrix.9 It has
been shown that water-soluble disulfonated Pcs, par-
ticularly the “adjacent” disulfonated Pc isomers,9 can
diffuse readily through a cellular membrane and dem-
onstrated that such disulfonated Pcs are particularly
efficient drugs for inducing direct cell kill during in vivo
PDT.10 Comparison of cellular uptake of disulfonated
tetraphenylporphyrin with sulfonate groups on adjacent
opposite phenyl rings also showed the former as being
more efficient.11 Some 2,9,16,23-tetrahydroxy Pcs12
have shown good photodynamic potential under in vitro
and in vivo assays. Unfortunately, both the sulfonated
Many synthetic porphyrin derivatives have been
tested for toxicity and phototoxicity in PDT studies, and
polyhydroxyporphyrins, especially meso-tetra(hydroxy-
phenyl)porphyrin, meso-tetra(sodium sulfonated)por-
phyrins, and (3,4-dihydroxyphenyl)porphyrin, are among
the most active compounds.5 Phthalocyanine (Pc) de-
† York University.
‡ Universite´ de Sherbrooke.
§ Current address: Department of Chemistry, Karadeniz Technical
University, 61080 Trabzon, Turkey.
S0022-2623(97)00336-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/28/1998

1790 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Hu et al.
Sch em e 1
phthalocyanines (including sulfonated porphyrins) and
tetrahydroxyphthalocyanines were only prepared as a
mixture of different isomers. Furthermore, the mono-
or the “adjacent” disulfonated Pcs were only available
in small amounts by tedious separation by HPLC, and
these Pcs still existed as a mixture of sodium 3- and
4-monosulfonated Pcs or a mixture of “adjacent” and
“opposite” Pc isomers.13 In this paper, we report new
directed specific synthetic methods to prepare pure
mono-, “adjacent” di-, tri-, and tetrahydroxy Pcs. Their
photobiological potentials were evaluated on EMT-6
mammary tumor cells growing on monolayer or im-
planted intradermally in BALB/c mice.
Resu lts a n d Discu ssion
Ch em istr y. Because the self-condensation of hy-
droxyphthalonitrile cannot give the desired hydroxy-
phthalocyanines,12a the desired polyhydroxyphthalocya-
nines can only be obtained from the cleavage of the
corresponding substituted phthalocyanines. Some pub-
lished methods for the preparation of unsymmetrical
Pcs include functionalization of preformed phthalocya-
nines and the mixed condensation of two different
precursors.14 Partial functional substitution of Pcs can
be used to prepare unsymmetrical Pcs and to introduce
hydrophilic groups. An example of this is the partial
sulfonation of Pcs13 which gives a very complex isomeric
mixture. Time-consuming and tedious chromatographic
separation procedures have to be used to isolate sul-
fonated Pcs from the complex mixture. Mixed conden-
sation methods using two different precursors can also
be used to prepare unsymmetrical Pcs and have similar
disadvantages. These mixtures are very difficult to
isolate by common chromatography due to the Pcs
tendency toward aggregation.15 There are some meth-
ods developed specifically for the preparation of unsym-
metrical Pcs such as the solid-phase synthetic method16,17
and the “sub-phthalocyanine” method,18 but each needs
several reaction steps and gives low yields. Thus, to
prepare unsymmetrical Pcs a key aspect is the develop-
ment of a suitable separation method to isolate these
mixtures of Pcs.
Although flash chromatography does not efficiently
separate these complex mixtures of Pcs, size exclusion
chromatography is useful in the separation of these
mixtures if there is a sufficient difference in size among
these Pcs. The strategy of synthesis was to choose a
suitable protecting group to prepare protected hydroxy-
phthalonitriles. The protecting group should be bulky
enough to result in a large size difference among these
substituted Pcs, so that it is possible to isolate these
Pcs using gel permeation chromatography (size exclu-
sive chromatography) (GPC). The protecting group
should also be a good leaving group, because these
unsymmetrical Pcs would be cleaved in the final step
to give the designed polyhydroxyphthalocyanines.
1,2-bis(benzyloxy)benzene (4) and 1,2-bis(4-tert-butyl-
benzyloxy)benzene (5) in 92% and 90% yield, respec-
tively (Scheme 1). Compound 4 could be easily bromi-
nated in dichloromethane to give 4,5-dibromo-1,2-
bis(dibenzyloxy)benzene (6) in 83% yield, but unfor-
tunately, when compound 5 reacted with bromine, the
hydrogen bromide released from the bromination hy-
drolyzed the ether bond, which resulted in a complex
mixture which included bromocatechol and 3,4,5-tri-
bromo-1,2-bis(4-tert-butylbenzyloxy)benzene. An alter-
native route, including bromination followed by protec-
tion, was used to prepare substituted phthalonitriles.
Treatment of catechol (1) with bromine in acetic acid
gave 4,5-dibromocatechol (7)19 in 85% yield. Mass
spectroscopy of the crude products showed 7 as the main
product but contaminated with 3,4,5- and 3,4,6-tribro-
mocatechols which were very difficult to remove from
the crude products. Flash column chromatography on
silica gel, while very slowly eluting with benzene,
yielded pure 7. The reactions between 7 and benzyl
chloride (2), 3, and 2,5-dimethylbenzyl bromide (8) gave
compound 6, 4,5-dibromo-1,2-bis(4-tert-butylbenzyloxy)-
benzene (9), and 4,5-dibromo-1,2-bis(2,5-dimethylben-
zyloxy)benzene (10) in 90%, 85%, and 91% yield, re-
spectively (Scheme 1). Treatment of 6, 9, or 10 with
cuprous cyanide in refluxing DMF gave 4,5-bis(benzyl-
oxy)phthalonitrile (11), 4,5-bis(4-tert-butylbenzyloxy)-
phthalonitrile (12), or 4,5-bis(2,5-dimethylbenzyloxy)-
phthalonitrile (13) in 60%, 45%, or 21% yield, respec-
tively (Scheme 1).
Treatment of 4-nitrophthalonitrile (14) with diphen-
ylmethanol (15) in DMSO gave 4-(diphenylmethoxy)-
phthalonitrile (16) in 92% yield (Scheme 2).20 The
reactions between 3-nitrophthalonitrile (17), potassium
carbonate, and p-n-butylbenzyl alcohol (18), benzyl
alcohol (19), propargyl alcohol (20), or neopentyl alcohol
(21) in DMSO gave 3-substituted phthalonitrile 22, 23,
24, or 25 in 90%, 95%, 88%, or 90% yield, respectively
(Scheme 2).20 Treatment of 17 with sodium methoxide
and methanol (26) in DMSO gave the sterically less
Syn th esis of Su bstitu ted P h th a lon itr iles. Since
benzyl and substituted benzyl groups can be readily
cleaved under a variety of conditions and are sufficiently
bulky to provide one-isomer Pcs, separated by GPC, they
were chosen to prepare the protected hydroxy-
phthalonitriles in this research. Thus, catechol (1) was
treated in N,N-dimethylformamide (DMF) with benzyl
chloride (2) and 4-tert-butylbenzyl bromide (3) to give

Hydroxyphthalocyanines as Agents for Cancer Therapy
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1791
chromatography20 was used to purify all products (see
Experimental Section).
Sch em e 2
Syn th esis of “Ad ja cen tly” Su bstitu ted P h th a lo-
cya n in es. To prepare a desired polyhydroxy Pc with
amphiphilic properties for PDT study, a pure “adja-
cently” substituted Pc has to be synthesized first. A
mixed condensation of an unsubstituted phthalonitrile
and a substituted phthalonitrile could only give a
mixture of the “adjacent” and “opposite” substituted Pcs
which could not be separated by gel permeation chro-
matography.
A phthalonitrile dimer intermediate (“half Pc”) has
been shown to be an intermediate in Pc formation.22a
To prepare the “adjacently” substituted Pcs a “half-Pc”
has to be prepared. The “half-Pc” can undergo further
condensation with another phthalonitrile in a large
excess to give the desired “adjacent” Pcs without the
“oppositely” substituted Pc.22b
The first attempt to prepare the “adjacent” Pc was to
treat 4,5-disubstituted phthalonitriles with lithium
methoxide in methanol, and then commercially avail-
able phthalonitrile (28) was added in a large excess.
Unfortunately, this route gave 2,3-disubstituted Pcs as
the main product because the two benzyloxy groups
inactivated both cyano groups so that monomer inter-
mediates were the main product instead of the desired
“half Pc”, when a disubstituted phthalonitrile such as
4,5-bis(benzyloxy)phthalonitrile (11) was treated with
lithium methoxide.
Thus, 28 was first treated with lithium methoxide in
methanol for a few hours to give a mixture of intermedi-
ates with the desired unsubstituted “half-Pc” as the
main product (Scheme 6). After evaporation of metha-
nol under reduced pressure, the mixture was directly
used to undergo a further mixed condensation with 11,
16, or 4,5-dimethoxyphthalonitrile (40) in a large excess
in 1-octanol to give a mixture containing the “adjacently”
substituted Pc but without the “oppositely” substituted
Pc (Scheme 6). The isolation of this mixture could give
a pure “adjacent” Pc. In this way the “adjacent” alkoxy-
substituted Pcs 41-43 were isolated. Pcs 42 and 43
were cleaved with TFA/TMB as before to yield the
2,3,9,10-tetrahydroxyphthalocyanine (44) and 2,9-dihy-
droxyphthalocyanine (45) (see Experimental Section).
demanding 3-methoxyphthalonitrile (27) in 90% yield
(Scheme 2). These phthalonitriles were fully character-
ized by MS, IR, and different NMR techniques such as
1H NMR, 13C NMR, J MOD 13C NMR, and proton-
carbon correlation spectroscopy in attempts to assign
different signals (Table 1).
Syn t h esis a n d Sep a r a t ion of Un sym m et r ica l
P h th a locya n in es. Mixed condensation methods were
used to prepare unsymmetrical Pcs using two precur-
sors. Since both phthalonitrile (28) and protected
hydroxyphthalonitriles are hydrophobic, optimal reac-
tion conditions are readily selected for these mixed
condensations. Although a mixed condensation of two
precursors in a 1:1 ratio gives a mixture of all Pc
products, the use of one of the precursors in a large
excess, such as a ratio of 10:1, promotes the possibility
that one of these isomers becomes the main product.
Thus, treatment of 4-(diphenylmethoxy)phthalonitrile
(16) and phthalonitrile (28) in a ratio of 1:10 with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in 1-butanol at
100 °C for 1 h and followed by the addition of excess
zinc acetate gave a mixture of [2-(diphenylmethoxy)-
phthalocyaninato]zinc(II) (29) (50%) and zinc Pc with
trace amounts of disubstituted Pcs (Scheme 3).
The same procedure was used to prepare [2,9,16-tris-
(diphenylmethoxy)phthalocyaninato]zinc(II) (30) in 48%
yield from 16 and 28 in a ratio of 10:1 (Scheme 3).
Treatment of 3-(p-n-butylbenzyloxy)phthalonitrile (22)
with 28 in a ratio of 1:10 using lithium in 1-octanol
followed by addition of zinc acetate gave [1-(p-n-butyl-
benzyloxy)phthalocyaninato]zinc(II) compound (31) in
62% yield (Scheme 4). The cleavage of 29-31 by
trifluoroacetic acid (TFA) and 1,2,4,5-tetramethylben-
zene (TMB)21 gave the corresponding (2-hydroxyphtha-
locyaninato)zinc(II) (32), (2,9,16-trihydroxyphthalocya-
ninato)zinc(II) (33), or (1-hydroxyphthalocyaninato)-
zinc(II) (34) in 90%, 77%, or 85% yield, respectively
(Schemes 3 and 4). The 1H NMR spectrum of 32 showed
a typical ABX splitting pattern with a doublet at 9.18
ppm (J ) 8.0 Hz), a small doublet at 8.70 ppm (J ) 1.6
Hz), and a doublet-doublet at 7.67 ppm (J ) 8.0, 1.6
Hz) for the substituted benzene ring. There is a singlet
at 10.70 ppm (OH), which disappeared after D2O
exchange.
The 1H NMR spectrum of a pure “adjacent” Pc,
(2,3,9,10-tetramethoxyphthalocyaninato)zinc(II) (41),
showed two doublets at 9.79 ppm (J ) 7.4 Hz, 2H) and
9.71 ppm (J ) 7.4 Hz, 2H) which were assigned to two
hydrogens at the 15- and 25-positions and two hydro-
gens at the 18- and 22-positions of the Pc macrocycle,
respectively. Two singlet signals at 9.24 and 9.19 ppm
were assigned to the two hydrogens at the 1- and 11-
positions and the 4- and 8-positions of the Pc molecule,
respectively. Four hydrogens at the 15-, 16-, 22-, and
23-positions of the Pc gave multiplet signals. There
were two singlet signals at 4.31 and 4.11 ppm which
were assigned to two groups of hydrogens from the four
methyl groups. 1H NMR spectrum showed that the
compound 41 is a pure adjacent Pc without contamina-
tion with the opposite Pc.
4,5-Bis(benzyloxy)phthalonitrile (11) was first used
to determine the possibility for this precursor to form
Pcs. Treatment of 11, DBU and zinc(II) in 1-butanol
did give [2,3,9,10,16,17,23,24-octakis(benzyloxy)phtha-
locyaninato]zinc(II) (35) as a blue solid in 34% yield
(Scheme 5). Similary, 11-13 with excess 28 gave 2,3-
disubstituted Pcs 36-38 in 38%, 24%, and 18% yield,
respectively (Scheme 5). The cleavage of 37-39 by TFA
and TMB gave (2,3-dihydroxyphthalocyaninato)zinc(II)
(39) in 58%, 84%, and 62% yield, respectively. Exten-
sive flash chromatography followed by size exclusion
Syn th esis of Sin gle-Isom er 1,8,15,22-Tetr a su b-
stitu ted P h th a locya n in es. In one of our previous
publications, an example in which 1,8,15,22-tetrakis(p-
n-butylbenzyloxy)phthalocyanine was prepared from

1792 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Hu et al.
Ta ble 1. 13C NMR Chemical Shifts (ppm, δ) of Substituted Phthalonitriles
compd
C1
C2
C3
C4
C5
C6
C7
C8
16
22
23
24
27
107.57
117.22
117.22
117.43
117.50
117.47
105.52
105.53
105.80
105.78
120.09
161.00
161.14
159.83
159.88
161.78
117.22
117.62
117.61
117.64
119.80
134.46
134.44
134.34
134.30
135.29
125.40
125.30
126.03
125.98
115.29
115.26
115.32
115.15
115.10
115.69
112.95
113.02
112.68
112.67
Sch em e 3
Sch em e 4
substituent can skew the reaction to give exclusively a
Pc as a pure single isomer, it is believed that if the
reaction temperature increases the intermediate will
have enough energy to overcome steric interactions to
form different isomers. To prove the supposition, the
self-condensation of 3-(neopentyloxy)phthalonitrile was
performed at different temperatures. As expected, when
the reaction temperature is higher than 150 °C, Pc 54
was produced as a mixture of all possible isomers; when
the reaction temperature was 60-80 °C, the Pc was
produced as a pure single isomer (53); and when the
reaction temperature was around 120 °C, a nonstatis-
tical mixture of isomers was obtained. Self-condensa-
tions of other phthalonitriles gave similar results. 1H
NMR of compound 53 exhibited the desired doublet,
triplet, and doublet generated by the hydrogens of the
Pc macrocycle of the highly symmetrical molecule, and
the 13C NMR spectrum of 53 gave the 11 singlet signals
which represent 11 groups of carbons in this sym-
metrical molecule. Compared to the spectra of 53, the
1H and 13C NMR spectra of 54 gave multiplet signals.
3-(p-n-butylbenzyloxy)phthalonitrile as a pure single
isomer20 was reported. In this paper more 1,8,15,22-
tetrasubstituted Pcs were synthesized as pure single
isomers. The effects of reaction temperature and size
of substituents on the isomer distribution were also
studied.
Treatment of phthalonitriles 22-25 and 27 with
lithium in 1-octanol at different temperatures gave the
corresponding 1,8,15,22-tetrakis(p-n-butylbenzyloxy)
Pcs (46, 47), 1,8,15,22-tetrakis(benzyloxy) Pcs (48,
49, 50), (tetrapropargyloxyphthalocyaninato)zinc(II) (51),
1,8,15,22-tetrakis(neopentyloxy) Pcs (52, 53, 54), and
(1,8,15,22-tetramethoxyphthalocyaninato)zinc(II) (55)
(Scheme 7). Cleavage of 47 in TFA/TMB as before gave
1,8,15,22-tetrahydroxyphthalocyanine (56) as a single
isomer. The reaction temperature was believed to play
an important role in the formation of single-isomer Pcs.
Although a phthalonitrile intermediate with a bulky
The distribution of isomers depended on the bulky
properties of substituents. Self-condensation of a 3-sub-

Hydroxyphthalocyanines as Agents for Cancer Therapy
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1793
Sch em e 5
Sch em e 6
important role in the condensation because there should
exist enough space in a Pc macrocycle for two methoxy
groups to exist in the “face-to-face” positions. Thus, to
a solution of lithium in 1-octanol was added a solution
of 27 in THF at 40-50 °C, and the solution was kept
stirring overnight. Zinc acetate was added, and the
reaction mixture was stirred for another 1-2 days. The
reaction did give a blue zinc Pc in 50% yield. 1H and
13C NMR spectra of the Pc showed that the Pc, 1,8,15,-
22-tetramethoxy zinc Pc (55), was a pure single isomer.
Because the electron-donating effects of the methoxy
group can affect ortho- and para-positions, it is obvious
that the alkoxy anion RO- favors attack at the cyano
at the meta-position. The reactivities of the phthaloni-
trile will decrease as the reaction temperature de-
creases. When the temperature is low enough (40 °C)
as used in this research, the cyano group at the
2-position will be totally inactive. Thus, at this tem-
perature the alkoxy anion attacks only one of the two
cyano groups and results in only one phthalonitrile
monomer which undergoes further condensation to a Pc
macrocycle.
stituted phthalonitrile with a bulky substituent such as
a p-n-butylbenzyloxy group gave the corresponding Pc
as a pure single isomer even when the reaction was
performed at 120-130 °C, but phthalonitriles with less
bulky substituents such as neopentoxy or propargyloxy
gave the Pc as a mixture of isomers at the same
temperature. The results could be also used to explain
why a high-temperature reaction gave a much lower
yield. Instead of cyclizing to a Pc macrocycle, the strong
steric interactions between two substituents may cause
different intermediates to form linear polymers. FAB-
MS spectra of the crude products show signals of linear
polymers which possess higher molecular weights than
the corresponding Pc.
Electronic effects are also considered to play a role in
the reaction. It is believed that the electron-donating
effects of the substituent could partially control the
initial nuclear attack of alkoxy anion RO- to only one
of the two cyano groups to lead to only one intermediate
which underwent further condensation to a Pc. 3-Meth-
oxyphthalonitrile (27), the smallest hindered alkoxy-
substituted phthalonitrile, was prepared for this study.
It was believed that if the self-condensation of a 3-meth-
oxyphthalonitrile could exclusively give one single iso-
mer, it could be that the electronic effects played an
In summary, both electronic effects and steric interac-
tions were considered to affect the isomer distribution.
When the reaction temperature is low (40 °C) electronic
effects control the initial nucleophilic attack of RO- to

1794 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Hu et al.
Ta ble 2. EMT-6 Cell Photoinactivation and Dye Mobility of
Sch em e 7
Different ZnPcs
a
b
LD90 (J cm-2
)
LC90 (µM)
compound
e
(substituents)
1 h
24 h
1 h
24 h
Rf
ZnPc
(pyridinium salt)
32
0.14c
0.40
2.40
7.50
19.00
NA
NA
NA
0.0
(2-hydroxy)
39
(2,3-dihydroxy)
45
(2,9-dihydroxy)
33
(2,9,16-trihydroxy)
ZnPc(OH)4
0.15
0.25
0.40
0.48
NA
0.25
1.00
3.20
4.50
0.12 0.62
0.30 0.0f
0.58 0.55
0.80 0.47
(2,9,16,23-tetrahydroxy) NA
>100d NA
NA
a
LD90, light dose (λ > 590 nm, fluence rate 10 mW cm-2
)
required to kill 90% of cells after 1- or 24-h incubation with a fixed
b
drug concentration of 1 µM (SEM < 20%). LC90, drug dose
required to kill 90% of cells after 1- or 24-h incubation with a fixed
fluence of 2.4 J cm-2 (SEM < 20%). c Datum taken from ref 23.
d
As determined by colony formation assay with Chinese hamster
V-79 fibroblasts.12b e Dye mobilities relative to the solvent front
(Rf) on silica gel TLC in ethyl acetate/methanol (50:50) and 3%
triethylamine. f Compound 39 did not migrate, possibly due to air
oxidation to the quinone.
After 1-h incubation with 0.1 µM drug, the monohydroxy
derivative 32 gave a LD90 of 0.25 J cm-2 whereas 33,
39, and 45 exhibited LD90 > 2.4 J cm-2. Increasing the
dye concentration to 1 µM augmented the differences
in phototoxicities among the dyes with LD90 varying
between 0.4 and 19 J cm-2
. The relative activities
correlate to some extent with differences in hydropho-
bicities. The latter is known to affect the rate of cell
uptake, explaining in part variations in phototoxicity,
although differences in intracellular localization also
constitute an important parameter.9b Relative mobili-
ties (Rf) on silica gel TLC in ethyl acetate/methanol (50:
50) and 3% triethylamine are included in Table 2 as a
measure of relative hydrophobicity. The 2,3-dihydroxy
ZnPc (39) did not migrate in our TLC system, possibly
due to its facile conversion to a 2,3-quinoid species.
Decreasing hydrophobicity of the unsubstituted parent
ZnPc via the addition of hydroxyl groups onto the
macrocycle, i.e., 2-hydroxy (32), and 2,9-dihydroxy (45),
2,9,16-trihydroxy (33), 2,9,16,23-tetrahydroxy ZnPc,12b
parallels the observed pattern of decreasing phototox-
icity, with LC90 (Table 2: 2.4 J cm-2 after 1 h) from 0.25,
3.2, 4.5, to >100 µM, respectively. The ZnPc(OH)4 was
completely void of cellular photoactivity, even after 1-h
incubation at 150 µM followed by exposure to a 2.4 J
cm-2 light dose.12b The highly insoluble unsubstituted
ZnPc is active as a pyridinium salt solubilized in
Cremophor,23 exhibiting a photocytotoxicity comparable
to that of the monohydroxy derivative 32. Both isomeric
dihydroxy ZnPc 39 and 45, as well as 32, appear to
remain monomeric upon dilution in serum from either
THF solutions or Cremophor emulsions, as judged by
the absence of a blue shift of the Q-band. In fact, their
absorption maxima in serum are slightly red-shifted
(data not shown). Quantum yields of singlet oxygen of
monomeric metallo phthalocyanines are around 0.5;
however, the actual in vivo yields of this cytotoxic
species may vary substantially due to differences be-
tween the various dyes in tendencies to aggregate in
the aqueous environment.6d
one of two cyano groups and give only one intermediate
which undergoes further condensation to a pure single-
isomer Pc. This observation matches the experimental
results that self-condensation of all 3-substituted
phthalonitriles described at 40-60 °C or lower have
given Pcs as single isomers. When the reaction tem-
perature increases to 120-130 °C, the nucleophilic
attack of RO- can result in two intermediates. Now the
steric interaction controls the isomer distribution, and
only phthalonitriles with a very bulky substituent such
as p-n-butylbenzyloxy can give the corresponding Pc as
a pure isomer. Those phthalonitriles with less bulky
substituents such as propargyloxy and neopentyloxy
give Pcs as mixtures of isomers. When the reaction is
performed at a very high temperature such as 170 °C,
different intermediates can be obtained, and they can
overcome the activation barrier of steric interactions to
form different Pc isomers. Thus, all Pcs are obtained
as mixtures of isomers, but only a much lower yield can
be obtained because of the increase of byproducts such
as linear polymers.
P h otobiologica l P r op er ties. 1. Cell P h otoin a c-
tiva tion . Cell survival was estimated by means of the
colorimetric MTT assay. The light dose (LD90) at 1 µM
dye, or drug concentration (LC90) at a constant fluence
of 2.4 J cm-2, required to inactivate 90% of the cells is
given in Table 2. No dark toxicity was observed with
any of the dyes at the concentrations under study. The
four compounds tested (32, 33, 39, 45) exhibited good
photoactivities, especially after 24-h incubation with 1
µM drug, with LD90 between 0.15 and 0.48 J cm-2
.
2. P h otod yn a m ic Th er a p y. The EMT-6 tumor

Hydroxyphthalocyanines as Agents for Cancer Therapy
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1795
Ta ble 3. EMT-6 Tumor Response Induced in BALB/c Mice after ZnPc (650-700 nm)- and Photofrin (600-650 nm)-Mediated PDT
(200 mW cm-2, 400 J cm-2
)
tumor response (%)
compound
(substituents)
concn
(µmol kg-1
)
n
necrosisa
regressionb
edemac
NA
ZnPc24
2
NA
NA
100
(pyridinium salt)
32
1
2
1
2
1
2
10
8
4
8
4
3
8
9
100
25
62
25
0
12
25
25
0
0
75
0
++++
++++
+++
+++
+
(2-hydroxy)
39
(2,3-dihydroxy)
45
(2,9-dihydroxy)
100
NA
++
12b
Zn-Pc(OH)4
NA
(2,9,16,23-tetrahydroxy)
Photofrin5c
5 (mg kg-1
)
10
100
70
+++
a
b
Necrosis, percent of animals showing flat and necrotic tumors within 2-3 days after PDT. Regression, percent of animals showing
complete tumor regression 3 weeks after PDT. c Edema is determined 24 h after PDT: light (+), medium (++), important (+++), extensive
(++++).
vials. Unless otherwise noted, Matheson high-purity argon
was used to maintain inert atmosphere conditions, and
magnetic stirring methods were utilized during distillation and
reaction processes. Thin-layer chromatography (TLC) was
performed using silica gel G as the adsorbent. Flash chroma-
tography was performed using silica gel of particle size 20-
45 µm. Gel permeation chromatography was performed using
Bio-beads, SX-2, 200-400 mesh. Melting points (mp) were
determined using a Kofler hot stage melting point apparatus
and are uncorrected. Infrared (IR) spectra were recorded on
a Pye Unicam SP3-200 or a Perkin-Elmer infrared spectro-
photometer, and FT-IR was performed on a Unicam Mattson
3000 FT-IR spectrometer using KBr disks. The ultraviolet-
visible spectra (UV-vis) were recorded on a Hewlett-Packard
HP8451A diode array spectrophotometer. Mass spectra (MS)
were performed by Dr. B. Khouw (York University, Toronto,
Ontario, Canada) and recorded at 70 eV on a VG Micromass
16F or a Kratos MS-50 triple analyzer mass spectrometer in
the EI mode. The FAB-MS spectra were obtained with a
Kratos MS-50 triple analyzer mass spectrometer equipped
with a FAB ion source of standard Kratos design and Ion Tech
atom gun. Microanalyses were performed by Guelph Chemical
Laboratories Ltd., Guelph, Ontario (only analyses above (0.4%
calculated values are given as Pcs are notoriously difficult to
combust). Nuclear magnetic resonance (NMR) spectra for
protons and carbons were recorded on either a Bruker AM300
NMR spectrometer or a Bruker ARX400 high-field Fourier
transform instrument. The positions of signals are reported
in δ units (parts per million relative to tetramethylsilane
(TMS)). The splitting patterns of the signals of proton
resonances are described as singlets (s), doublets (d), triplets
(t), quartets (q), doublets of doublets (dd), multiplets (m), or
broad signals (br). 13C NMR resonances are reported as the
proton-decoupled chemical shifts, and in most cases the J MOD
or/and DEPT 13C NMR technique was used to differentiate
carbons.
response induced in mice after photosensitization with
the monohydroxy (32), the two isomeric dihydroxy (39,
45), and the trihydroxy (33) ZnPc are presented in Table
3. For comparison, earlier reported data for the unsub-
stituted ZnPc, the ZnPc(OH)4, and Photofrin are also
included. The monohydroxy derivative 32 and the 2,3-
dihydroxy isomer 39 induce substantial tumor necrosis
at 1 µmol kg-1. However, this effect was accompanied
by extensive edema and resulted in low tumor cure
rates. Surprisingly, increasing the dye dose to 2 µmol
kg-1 decreased the PDT efficiency, most likely as a
result of the extensive inflammatory response which
obscures tumor necrosis. In contrast, the 2,9-dihydroxy
isomer 45, while being totally inactive at 1 µmol kg-1
,
induced tumor necrosis and cure in 100% and 75% of
animals, respectively, at 2 µmol kg-1 (1.2 mg kg-1).
Furthermore, edema was less as observed with com-
pounds 32 and 39 at 1 µmol kg-1. To obtain a similar
tumor response with Photofrin requires a 5 mg kg-1
drug dose.5c The highly insoluble, unsubstituted ZnPc
shows good tumor response at 2 µM kg-1 if administered
as a pyridinium salt formulated in a Cremophor emul-
sion.24 For comparison, the ZnPc(OH)4 failed to induce
tumor cure under the same light conditions, even at 10
µmol kg-1 12b
This lack of activity of the tetrahydroxy
.
Pc is in strong contrast with the high photodynamic
activities of tetrahydroxyphenyl porphyrins and chlor-
ins. These differences likely reflect the conformational
flexibility of the latter molecules. Several reports12b,5a
indicate that biologically active conformations of sub-
stituted porphyrins and phthalocyanines require a
critical distance between oxygen atoms, a requirement
which cannot be fulfilled with the rigidly locked hy-
droxyls of the ZnPc(OH)4. In our hydroxy Pc series it
thus appears that the presence of hydroxyl groups on
two adjacent benzene rings, e.g., as in 45, optimizes the
in vivo photoactivity of the ZnPc molecule. Such
structural arrangements augment amphiphilicity of the
dye, a property which promotes uptake by tumor cells
and intracellular localization at photosensitive sites.9b,c
1,2-Bis(ben zyloxy)ben zen e (4). From a solution of 22 g
(0.20 mol) of catechol (1) in 150 mL of DMF, dissolved oxygen
was removed by repeated evacuation followed by the admission
of argon. Benzyl chloride (2) (53.13 g, 0.42 mol) was added
and the system again deoxygenized. Hydrated potassium
hydroxide (84% KOH) (26.5 g, 0.4 mol) and 1 mL of Aliquate
336 (phase-transfer agent)25 were then added, and the mixture
was heated at 120 °C under argon for 4 h with vigorous
stirring. Water (300 mL) and 200 mL of methylene chloride
were added. The organic layer was separated, and the aqueous
layer was extracted with two 50-mL portions of methylene
chloride. The combined organic extracts were washed with
150 mL of water and dried over MgSO4. After evaporation of
the solvent, the residue was purified by column chromatog-
raphy on silica gel, eluting with toluene. Evaporation of the
solvent gave a colorless solid. Recrystallization from methyl-
ene chloride gave 4 as white crystals: yield 90%; mp 60-61
°C; IR (KBr, cm-1) ν 3030, 2941, 1260 and 1020 (Ar-O-C),
Exp er im en ta l Section
Ch em istr y. All organic solvents were dried by appropriate
methods and distilled before use. All reagents were freshly
distilled or were recrystallized and then dried under reduced
pressure before use. Anhydrous potassium carbonate (K2CO3)
and zinc acetate (Zn(AcO)2‚2H2O) were finely ground, dried
at 110 °C under vacuum for 36 h, and then stored in sealed

1796 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Hu et al.
770, 741; 1H NMR (CDCl3) δ 7.45 (d, J ) 8 Hz, 4H), 7.39-
7.30 (m, 6H), 6.97-6.86 (m, 4H), 5.16 (s, 4H); 13C NMR (CDCl3)
δ 149.10, 137.43, 128.47, 127.76, 127.32, 121.67, 115.34, 71.35;
EI-MS m/z (rel intensity) 291 [(M + 1)+, 42], 290 (M+, 53), 199
[(M - PhCH2)+, 50], 91 (PhCH2+, 100). Anal. (C20H18O2) C,
H.
- (CH3)2PhCH2]+, 15, 30, 14}, 266, 268, 270 {[M - 2(CH3)2-
PhCH2]+, [M + 2 - 2(CH3)2PhCH2]+, [M + 4 - 2(CH3)2-
PhCH2]+, 18, 37, 17}, 119 {(CH3)2PhCH2+, 100}. Anal. Calcd
(C24H24Br2O2): C, 57.14; H, 4.76. Found: C, 57.69; H, 4.32.
4,5-Dibr om o-1,2-bis(p-ter t-bu tylben zyloxy)ben zen e (10).
Method II via 7 was used to prepare 0.47 g of 10 (from 0.20 g
of 7 and 0.45 g of 3) as white crystals in 85% yield: mp 83.5-
84.5 °C; IR (KBr, cm -1) ν 3018, 2920, 1376, 1250 and 1080
1,2-Bis(4-ter t-bu tylben zyloxy)ben zen e (5). A mixture
of 5.5 g (0.05 mol) of 1, 25 g (0.11 mol) of 4-tert-butylbenzyl
bromide (3), 5.6 g of hydrated potassium hydroxide (84%
KOH), and 1 mL of Aliquate 336 was stirred at 120 °C for 4 h
under argon. To this mixture were added 50 mL of water and
50 mL of methylene chloride, and the water layer was
extracted twice with 30 mL of methylene chloride. The organic
layer was washed with 50 mL of water and dried over MgSO4
overnight. Evaporation of the solvent gave a brown oil. The
residue was purified by silica gel chromatography, eluting with
ethyl acetate and hexane (3:97). Recrystallization of the crude
product from acetone gave 5 as white crystalline needles: yield
88%; mp 65-66 °C; IR (KBr, cm -1) ν 3080, 3040, 2970, 1260
and 1015 (Ar-O-C), 756, 700; 1H NMR (CDCl3) δ 7.46 (m,
8H), 6.98-6.94 (m, 2H), 6.91-6.87 (m, 2H), 5.14 (s, 4H), 1.33
(s, 18H); 13C NMR (CDCl3) δ 150.75, 149.34, 134.51, 127.29,
125.42, 121.57, 115.51, 71.34, 34.61, 31.51; EI-MS m/z (rel
intensity) 402 (M+, 30), 255 {[M - (CH3)3CPhCH2)]+, 28}, 147
[(CH3)3CPhCH2)+, 100]. Anal. (C28H34O2) C, H.
4,5-Dibr om oca tech ol (7).19 Column chromatography on
silica gel was used to purify 7 by removing tribromocatechols:
yield 85%; IR (KBr, cm-1) ν 3600-3250 (OH); 1H NMR
(acetone-d6) δ 8.60 (s, br, 2H), 7.14 (s, 2H); 13C NMR (acetone-
d6) δ 146.94, 120.72, 113.58; EI-MS for C6H4Br2O2 m/z (rel
intensity) 266, 268, 270 {(M+, (M + 2)+, (M + 4)+, 25, 51, 26}.
1,2-Bis(ben zyloxy)-4,5-d ibr om oben zen e (6). Meth od I:
A solution of 9.48 g (0.06 mol) of bromine in 15 mL of
methylene chloride was slowly added to an ice-salt-cooled
solution of 8.7 g (0.03 mol) of 4 in 15 mL of methylene chloride.
The mixture was stirred for 2 h. The temperature was then
raised to room temperature, and the mixture stirred for
another 1 h. Excess bromine was neutralized with 10 mL of
a 10% solution of sodium bisulfide. The solution was then
washed with a 10% solution of sodium bicarbonate and water.
The extract was dried overnight using MgSO4. The residue
was purified by silica gel chromatography, eluting with toluene
and hexane (1:3). The crude product was then recrystallized
from methylene chloride to give 6 as white crystals: yield 92%.
1
(Ar-O-C); H NMR (CDCl3) δ 7.41 (d, J ) 8.2 Hz, 4H), 7.36
(d, J ) 8.2 Hz, 2H), 7.19 (s, 2H), 5.09 (s, 4H), 1.30 (s, 18H);
EI-MS m/z (rel intensity) 558, 560, 562 {M+, (M + 2)+, (M +
4)+, 30, 59, 32}, 411, 413, 415 (9, 18, 8), 266, 268, 270 (13, 24,
12), 147 (100). Anal. (C28H32Br2O2) C, H.
4,5-Bis(ben zyloxy)p h th a lon itr ile (11). A mixture of 3.2
g (7.17 mmol) of compound 6 and 1.98 g (22.0 mmol) of CuCN
in 60 mL of DMF was stirred at 150 °C for 8 h. The reaction
mixture was cooled and then poured into 300 mL of concen-
trated ammonia solution. The mixture was stirred overnight
and then filtered. The solid was washed with 10% aqueous
ammonia and water and then extracted with 200 mL of
acetone. The residue was then purified by column chroma-
tography on silica gel, eluting with benzene. Recrystallization
of the crude product from benzene gave white crystalline
needles: yield 60%; mp 183.5-184.5 °C; IR (KBr, cm-1) ν 3120,
3070, 2950, 2900, 2242 (CN), 1595, 1530, 1370, 1300, 1240 and
1
1095 (Ar-O-C), 898, 765, 745; H NMR (CDCl3) δ 7.45 (m,
10H), 7.17 (s, 2H), 5.24 (s, 4H); EI-MS m/z (rel intensity) 340
(M+, 40), 249 (100), 158 (22), 91 (92). Anal. (C22H16N2O2) C,
H, N.
4,5-Bis(4-ter t-bu tylben zyloxy)ph th alon itr ile (12). Simi-
larly, 9 was used to prepare 0.20 g of compound 12 (from 0.56
g of 9) as white crystals in 45% yield: mp 181-182 °C; IR
(KBr, cm-1) ν 3022, 2241 (CN), 1255 and 1094 (Ar-O-C), 770,
750; 1H NMR (CDCl3) δ 7.40 (d, J ) 8.4 Hz, 4H), 7.30 (d, J )
8.4 Hz, 4H), 7.15 (s, 2H), 5.17 (s, 4H), 1.31 (s, 18H); EI-MS
m/z (rel intensity) 453 [(M + 1)+, 15], 305 [(M - t-BuPhCH2)+,
20], 147 (t-BuPhCH2+, 100). Anal. (C30H32N2O2) C, H, N.
4,5-Bis(2,5-dim eth ylben zyloxy)ph th alon itr ile (13). Simi-
larly, 10 was used to prepare 0.083 g of compound 13 (from
0.5 g of 10) as white crystals: yield 21%; mp 188.5-190 °C;
IR (KBr, cm-1) ν 3020, 2240 (CN), 1260 and 1092 (Ar-O-C),
768, 748; 1H NMR (CDCl3) δ 7.23 (s, 2H), 7.15 (s, 2H), 7.10 (d,
J ) 7.0 Hz, 2H), 7.06 (d, J ) 7.0 Hz, 2H), 5.09 (s, 4H), 2.28 (s,
6H), 2.27 (s, 6H); EI-MS m/z (rel intensity) 397 {(M + 1)+, 18},
277 {(M - 2,5-dimethylPhCH2)+, 18}, 119 (2,5-dimeth-
ylPhCH2+, 70). Anal. Calcd (C26H24N2O2): C, 78.70; H, 6.10;
N, 7.06. Found: N, 6.42.
4-(Dip h en ylm eth oxy)p h th a lon itr ile (16). A previously
described procedure20 was used to prepare 16 (0.86 g) (from
14 (0.52 g) and diphenylmethanol (15) (0.55 g)) in 92% yield
as white crystals: mp 156-157 °C; IR (KBr, cm-1) ν 3080,
3020, 2880, 2230 (CN), 2220 (CN), 1575, 1468, 1295, 1245 and
1080 (Ar-O-C), 1040, 820, 790; 1H NMR (CDCl3) δ 7.67 (d, J
) 9 Hz, 1H), 7.42-7.32 (m, 11H), 7.25 (dd, J ) 9, 2.4 Hz, 1H),
6.28 (s, 1H); 13C NMR (CDCl3) δ 161.09, 139.00, 135.15, 129.05,
128.64, 126.74, 121.14, 120.68, 117.44, 115.54, 115.17, 107.71,
83.16; EI-MS m/z (rel intensity) 310 (M+, 6), 284 (48), 237 (32),
167 (Ph2CH+, 100), 152 (58), 115 (54), 89 (50), 82 (73), 63 (32),
51 (16). Anal. (C21H14N2O) C, H, N.
3-(p-n -Bu tylben zyloxy)p h th a lon itr ile (22).20 To a solu-
tion of 0.52 g of 3-nitrophthalonitrile (17) and 1.18 g of p-n-
butylbenzyl alcohol (18) (7.2 mmol) in 10 mL of DMSO was
added 0.44 g of potassium carbonate. The solution was stirred
for 24 h at room temperature. TLC showed three spots (first,
18; second, the product 22; third, 17). Potassium carbonate
(0.44 g) and 17 (0.507 g) were added to this solution which
was stirred for 48 h. Again, to the solution was added 0.3 g of
potassium carbonate, and the solution was stirred for another
48 h. TLC did not show the presence of any remaining 17.
The reaction mixture was poured into 150 g of ice-water; the
yellow precipitate was filtered, washed with cold water, and
dried. The crude product was purified by flash column
chromatography on silica gel, eluting with THF and hexane
(8:92, v/v) to remove a small amount of 18 and then with THF
Meth od II: A mixture of 2.0 g (0.01 mol) of compound 7,
4.0 g (0.032 mol) of benzyl chloride (2), 2.0 g of hydrated
potassium hydroxide (84% KOH), and 0.5 mL of Aliquate 336
was heated to 120 °C for 3 h while stirring under argon. The
mixture was cooled and then poured into 300 mL of ice-water.
The mixture was extracted with three 100-mL portions of
ether. The ether layer was dried with MgSO4 overnight. The
reaction residue was purified by column chromatography on
silica gel, eluting with toluene and hexane (1:3). Recrystal-
lization of the crude product from methylene chloride gave 6
as white crystals: yield 90%; mp 144.5-155.5 °C; IR (KBr,
cm-1) ν 3070, 3050, 2940, 2880, 1250 (Ar-O-C), 1200, 1050,
765, 710; 1H NMR (acetone-d6) δ 7.49 (d, J ) 7 Hz, 4H), 7.41-
1
7.32 (m, 8H), 5.20 (s, 4H); H NMR (CDCl3) δ 7.42-7.32 (m,
10H), 7.15 (s, 2H), 5.10 (s, 4H); 13C NMR (CDCl3) δ 148.83,
136.23, 128.61, 128.14, 127.35, 119.41, 115.49, 71.59; EI-MS
m/z (rel intensity) 446, 448, 450 {M+, (M + 2)+, (M + 4)+, 38,
70, 35}, 357 {(M - PhCH2)+, 19}, 91 (PhCH2+, 100). Anal.
(C20H16Br2O2) C, H.
4,5-Dibr om o-1,2-bis(2,5-dim eth ylben zyloxy)ben zen e (9).
Method II via 7 was used to prepare 0.46 g of compound 9
from 0.2 g of 7 and 0.40 g of 2,5-dimethylbenzyl bromide (8):
yield 91%; mp 114-115.5 °C; IR (KBr, cm-1) ν 3020, 2920,
1
1375, 1250 and 1080 (Ar-O-C); H NMR (CDCl3) δ 7.21 (s,
2H), 7.20 (s, 2H), 7.09 (d, J ) 8 Hz, 2H), 7.05 (d, J ) 8 Hz,
2H), 5.03 (s, 4H), 2.33 (s, 6H), 2.31 (s, 6H); 13C NMR (CDCl3)
δ 151.01, 149.18, 136.01, 135.18, 130.28, 130.13, 129.22,
128.38, 119.21, 70.51, 20.96, 18.35; EI-MS m/z (rel intensity)
502, 504, 506 {(M+, (M + 2)+, (M + 4)+, 22, 45, 21}, 383, 385,
387 {[M - (CH3)2PhCH2]+, [M + 2 - (CH3)2PhCH2]+, [M + 4

Hydroxyphthalocyanines as Agents for Cancer Therapy
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1797
and hexane (25:75, v/v) to collect 22. Evaporation of the
solvent gave a white solid which recrystallized from benzene
as white crystals: yield 95%; mp 70-71 °C; IR (KBr, cm-1) ν
3080, 3020, 2950, 2920, 2850, 2250 (CN), 2220 (CN), 1575,
1470, 1295, 1280, 1250 (Ar-O-C), 1180, 1080, 1040, 840, 790;
1H NMR (CDCl3) δ 7.59 (t, J ) 7.6 Hz, 1H), 7.54-7.16 (m,
6H), 5.24 (s, 2H), 2.63 (t, J ) 7.7 Hz, 2H), 1.62-1.57 (m, 2H),
1.39-1.31 (m, 2H), 0.93 (t, J ) 7.3 Hz, 2H); 13C NMR (CDCl3)
δ 161.14, 143.68, 134.44, 131.75, 128.98, 127.34, 125.30,
117.62, 117.22, 115.32, 113.02, 105.53, 71.57, 35.38, 33.51,
22.34, 13.91; EI-MS m/z (rel intensity) 291 {(M + 1)+, 18}, 247
(20), 147 (C4H9PhCH2+, 100), 127 (22), 116 (33), 104 (44), 91
(40), 78 (27), 64 (23), 51 (20). Anal. (C19H18N2O) C, H, N.
3-(Ben zyloxy)p h th a lon itr ile (23). Similarly 19 was used
to prepare 23 (0.63 g) in 90% yield (from 0.52 g of 17 and 0.32
g of benzyl alcohol (19)) as shining white crystals: mp 168-
169 °C; IR (KBr, cm-1) ν 3075, 3020, 2950, 2920, 2850, 2250
(CN), 2225 (CN), 1575, 1470, 1290, 1280, 1250 (Ar-O-C),
1185, 1080, 1040, 840, 790; 1H NMR (CDCl3) δ 7.62 (t, J ) 7.9
Hz, 1H), 7.58-7.34 (m, 6H), 7.31 (d, J ) 8.8 Hz, 1H), 5.23 (s,
2H); 13C NMR (CDCl3) δ 161.00, 134.57, 134.46, 128.94, 128.69,
127.01, 125.40, 117.53, 117.22, 115.26, 112.95, 105.52, 71.48;
EI-MS m/z (rel intensity) 235 {(M + 1)+, 19}, 91 (PhCH2+, 100).
Anal. (C15H10N2O) C, H, N.
ninato]zinc(II), [2,9,16-tris(diphenylmethoxy)phthalocyaninato]-
zinc(II) (30), or 2,9,16,23-tetrakis(diphenylmethoxy) zinc Pc.
An alternative isolation was performed only by size exclu-
sion chromatography columns on Bio-beads gel (SX-2). The
crude product (20 mg) was put onto a column and eluted with
THF. A few different blue-colored bands could be seen on the
column. These bands were collected and purified by another
size exclusion column chromatography, respectively. In gen-
eral, three or four columns were needed to obtain a pure Pc.
The ideal isolation method was achieved by a combination
of flash chromatography and gel permeation chromatography.
The crude product was put onto a silica gel column and first
eluted with benzene to collect a mixture of tetrasubstituted
zinc Pc and 30 (with small amounts of disubstituted zinc Pcs).
Further elution with benzene and ethyl acetate (1:1) gave a
mixture enriched in disubstituted zinc Pcs together with small
amounts of 29 and zinc Pc. The last eluted with ethyl acetate
gave 29 with small amounts of zinc Pc and disubstituted zinc
Pcs. The three fractions were purified separately by size
exclusion column chromatography on Bio-beads gel (SX-2) and
eluted with THF to give 29 (6%), disubstituted zinc Pcs (13%),
30 (12%), and zinc Pc (17%) in a total yield of 48% (calculation
based on 16). Because a fast silica gel column could give
several fractions and each of them enriched in one of the
products, and another gel permeation column could give an
almost pure Pc, the method is convenient, time-saving, and
used to separate other mixtures of Pcs. FAB-MS: for tetrakis-
(diphenylmethoxy) zinc Pc, C84H56N8O4Zn m/z (rel intensity)
1304, 1305 {M+, (M + 1)+, 88, 95}, 1137 {(M - Ph2CH)+, 60},
970 {(M - 2Ph2CH)+, 40}, 803 {(M - 3Ph2CH)+, 32}, 636 {(M
- 4Ph2CH)+, 20}; for 30, C71H46N8O3Zn m/z (rel intensity) 1122
(M+, 100), 955 {(M - Ph2CH)+, 65}, 788 {(M - 2Ph2CH)+, 38},
621 {(M - 3Ph2CH)+, 32}; for bis(diphenylmethoxy) zinc Pcs,
3-(P r op a r gyloxy)p h th a lon itr ile (24). Similarly, 20 was
used to prepare 0.48 g of 24 from 0.52 g of 17 and 0.18 g of
propargyl alcohol (20) as white crystals in 88% yield: mp 149-
151 °C; IR (KBr, cm-1) ν 3130, 3045, 2910, 2920, 2840, 2245
(CN), 2220 (CN), 1572, 1460, 1445, 1275, 1175, 1035, 1015;
1H NMR (CDCl3) δ 7.69 (t, J ) 7.6 Hz, 1H), 7.45-7.41 (m,
2H), 4.91 (d, J ) 2.1 Hz, 2H), 2.62 (t, J ) 2.1 Hz, 1H); 13C
NMR (CDCl3) δ 159.83, 134.34, 126.03, 117.61, 117.43, 115.15,
112.68, 105.80, 76.57, 75.94, 57.20; EI-MS m/z (rel intensity)
183 [(M + 1)+, 25], 39 (100). Anal. (C11H6N2O) C, H, N.
C
58H36N8O2Zn m/z (rel intensity) 940 (M+, 85), 773 {(M -
Ph2CH)+, 80}, 606 {(M - 2Ph2CH)+, 100}; for 29, C45H26N8-
3-(Neop en tyloxy)p h th a lon itr ile (25).26 Compound 25
was prepared by following the literature,26 from the corre-
sponding neopentyl alcohol and 17.
OZn m/z (rel intensity) 758 (M+, 78), 591 {(M - Ph2CH)+, 60}.
[2-(Dip h en ylm eth oxy)p h th a locya n in a to]zin c(II) (29).
A mixture of 0.24 g (0.78 mmol) of 16, 1.00 g (7.8 mmol) of 28,
and 1 mL of DBU in 20 mL of 1-butanol was heated to 100 °C
for 1 h. To the solution was added zinc acetate, and the
solution was stirred for 20 h. The mixture was cooled to room
temperature, and 50 mL of methanol and water (1:1) was
added. The precipitate was collected by centrifugation and
purified to give 0.25 g of 29 as a blue solid in 42% yield: mp
>320 °C; UV-vis (THF) λmax (log ꢀ) 672 (5.20), 640 (4.38), 606
(4.45), 344 (4.78); IR (KBr, cm-1) ν 3010, 1250 (Ar-O-C), 1120,
3-Meth oxyp h th a lon itr ile (27).27 To 1 mL of freshly
distilled methanol (26) was added 0.02 g of sodium. To the
solution was added 5 mL of DMSO and 0.8 g (5 mmol) of 17,
and the solution was stirred for 3 h. TLC analysis of the
reaction mixture showed that some 17 remained; 1 mL of
methanol and 0.4 g of potassium carbonate were added, and
the solution was stirred until all of 17 reacted. The mixture
was poured into 100 mL of ice-water to give a yellow
precipitate, which was filtered, washed with cold water, and
dried. After chromatography on silica gel, eluting with
benzene, 0.80 g of 27 (90%) was obtained. Recrystallization
of 27 from benzene gave shining crystals: mp 185.5-186.5
°C (lit.27 mp 183-184 °C); IR (KBr, cm-1) ν 3020, 2920, 2840,
2245 (CN), 2225 (CN), 1572, 1460, 1445, 1255 and 1075 (Ar-
O-C), 1035, 1015; 1H NMR (CDCl3) δ 7.68 (t, J ) 8.2 Hz, 1H),
7.38 (d, J ) 8.1 Hz, 1H), 7.27 (d, J ) 8.1 Hz, 1H), 4.03 (s, 3H);
13C NMR (CDCl3) δ 159.88, 134.30, 125.98, 117.64, 117.50,
115.10, 112.67, 105.78, 56.30; EI-MS m/z (rel intensity) 158
(M+, 100], 143 [(M - CH3)+, 25].
Gen er a l P r ep a r a tion a n d Sep a r a tion of Un sym m etr i-
ca l Su bstitu ted P cs. A mixture of 0.5 g (3.9 mmol) of 28
and 1.21 g (3.9 mmol) of 16 in 15 mL of n-butanol was heated
to 100 °C and stirred until the phthalonitriles dissolved. To
the solution was added 1.5 mL of DBU, and the solution was
stirred for 1 h. Zinc acetate (0.1 g) was added, and the solution
was stirred at 100-110 °C overnight. The reaction mixture
was cooled to 60 °C, and 100 mL of methanol and water (1:1)
was added to give a blue precipitate. The precipitate was
collected by centrifugation, washed with methanol and hexane,
and dried. TLC analysis of the crude products showed several
colored spots. Half of the crude products were dissolved in 10
mL of THF, preabsorbed on 10 g of flash silica gel, and put
onto a flash silica gel column. Elution with THF and hexane
(from 3:97 to 20:80, v/v) gave different fractions which were
separately purified by another flash column chromatography
on silica gel to give [2-(diphenylmethoxy)phthalocyaninato]-
zinc(II) (29), [2,9- and 2,16-bis(diphenylmethoxy)phthalocya-
1
1085 (Ar-O-C); H NMR (pyridine-d5) δ 9.70-9.68 (m, 6H),
9.55 (d, J ) 8.2 Hz, 1H), 9.52 (s, 1H), 8.21-8.16 (m, 6H), 8.11
(dd, J ) 8.2, 2.0 Hz, 1H), 7.98 (d, J ) 8 Hz, 4H), 7.48 (t, J )
7.8 Hz, 4H), 7.32 (t, J ) 6.3 Hz, 2H), 6.98 (s, 1H); FAB-MS
m/z (rel intensity) 758 (M+, 78), 591 {(M - Ph2CH)+, 60}. Anal.
Calcd (C45H26N8OZn): C, 71.10; H, 3.45; N, 14.75. Found: N,
15.30.
[2,9,16-Tr is(d ip h en ylm eth oxy)p h th a locya n in a to]zin c-
(II) (30). The procedure described for the preparation of 29
was repeated with a different ratio to prepare 0.12 g of 30 as
a blue solid (from 0.03 g of 28 and 0.24 g of 16 in 1:10 ratio)
in 40% yield: mp >320 °C; UV-vis (THF) λmax (log ꢀ) 676
(5.12), 608 (4.22), 348 (4.70); IR (KBr, cm-1) ν 3030, 2862, 1250
1
and 1085 (Ar-O-C); H NMR (pyridine-d5) δ 9.74-9.71 (m,
2H), 9.57-9.51 (m, 6H), 8.20-8.17 (m, 2H), 8.13-8.06
(m, 3H), 7.96 (d, J ) 8 Hz, 12H), 7.50-7.20 (m, 21H). Anal.
(C71H46N8O3Zn) C, H, N.
[1-(p-n -Bu tylben zyloxy)ph th a locya n in a to]zin c(II) (31).
Lithium (0.2 g) was dissolved in 20 mL of 1-octanol. To the
solution was added a solution of 0.5 g (3.9 mmol) of 28 and
0.11 g (0.39 mmol) of 22 in 4 mL of THF. The solution was
stirred at 100 °C for 5 h. To the solution was added zinc
acetate (0.15 g), and the solution was stirred overnight. The
mixture was cooled to room temperature and quenched with
methanol and water. The crude product was purified by gel
permeation chromatography to give 0.18 g of 31 as a blue solid
in 62% yield: mp >320 °C; UV-vis (THF) λmax (log ꢀ) 676
(5.18), 646 (4.24), 610 (4.45), 342 (4.73); IR (KBr, cm-1) ν 3010,

1798 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Hu et al.
2920, 2862, 1255 and 1085 (Ar-O-C); 1H NMR (pyridine-d5)
δ 9.73-9.69 (m, 6H), 9.46 (d, J ) 7.8 Hz, 1H), 8.32 (d, J ) 8.0
Hz, 2H), 8.25-8.18 (m, 7H), 7.86 (d, J ) 8.0 Hz, 1H), 7.63 (d,
J ) 8.0 Hz, 2H), 5.92 (s, 2H), 2.83 (t, J ) 7.5 Hz, 2H), 1.77-
1.73 (m, 2H), 1.45-1.41 (m, 2H), 0.96 (t, J ) 6.9 Hz, 3H); FAB-
MS m/z (rel intensity) 738, 739 {M+, (M + 1)+, 91, 100}, 591
{(M - R)+, 60}. Anal. (C43H30N8OZn) C, H, N.
(2-Hyd r oxyp h th a locya n in a to)zin c(II) (32). A previ-
ously described procedure20 was used to prepare 32 by the
cleavage of compound 29 (76 mg, 0.1 mmol) with trifluoroacetic
acid (TFA) and 1,2,4,5-tetramethylbenzene (TMB). This reac-
tion gave 32 (53 mg, 0.09 mmol) as a blue solid in 90% yield:
UV-vis (THF) λmax (log ꢀ) 672 (5.05), 604 (4.42), 346 (4.72);
IR (KBr, cm- 1) ν 3650-3300 (OH); 1H NMR (DMSO-d6) δ 10.70
(s, 1H), 9.36-9.35 (m, 6H), 9.18 (d, J ) 8 Hz, 1H), 8.70 (d, J
) 1.6 Hz, 1H), 8.24-8.21 (m, 6H), 7.67 (dd, J ) 9.5, 1.6 Hz,
1H); FAB-MS m/z (rel intensity) 592 (M+, 100), 593 (94), 594
FAB-MS m/z (rel intensity) 788 (M+, 100), 697 (M - PhCH2
,
+
35), 606 (M - 2PhCH2+, 28). Anal. (C46H28N8O2Zn) C, H, N.
[2,3-Bis(p-ter t-bu tylben zyloxy)p h th a locya n in a to]zin c-
(II) (37). Similarily, 12 gave 37 (21 mg) (from 45 mg of 12
and 128 mg of 7) in 24% yield: mp >320 °C; UV-vis (THF)
λ
max (log ꢀ) 666 (5.05), 638 (4.38), 604 (4.40), 342 (4.65); IR (KBr,
1
cm-1) 3020, 1080 (Ar-O-C); H NMR (pyridine-d5) δ 9.79-
9.76 (m, 4H), 9.64 (d, J ) 7.8 Hz, 2H), 9.50 (s, 2H), 8.26-8.16
(m, 6H), 7.89 (d, J ) 8 Hz, 4H), 7.66 (d, J ) 8 Hz, 4H), 5.73 (s,
4H), 1.34 (s, 18H); FAB-MS m/z (rel intensity) 900 (M+, 100),
753 (55), 606 (32). Anal. (C54H44N8O2Zn) C, H, N.
[2,3-Bis(2,5-dim eth ylben zyloxy)ph th alocyan in ato]zin c-
(II) (38). Similarily, 13 gave 38 (30 mg) (from 79 mg of 13
and 256 mg of 7) in 18% yield: mp >320 °C; UV-vis (THF)
λ
max (log ꢀ) 664 (5.00), 638 (4.39), 602 (4.38), 338 (4.74); IR (KBr,
cm-1) ν 3022, 1080 (Ar-O-C); 1H NMR (pyridine-d5) δ 9.77-
9.74 (m, 4H), 9.67 (d, J ) 7.3 Hz, 2H), 9.56 (s, 2H), 8.48-8.14
(m, 6H), 7.67 (s, 2H), 7.16 (d, J ) 7.5 Hz, 2H), 7.11 (d, J ) 7.5
Hz, 2H), 5.75 (s, 4H), 2.56 (s, 6H), 2.30 (s, 6H); FAB-MS m/z
(rel intensity) 844 (M+, 100), 725 (42), 606 (36). Anal.
(C50H36N8O2Zn) Calcd: C, 70.97; H, 4.23; N, 13.24. Found:
N, 13.88.
(90), 595 (88); HRMS (FAB) m/z calcd for
C32H16N8OZn
592.0738, found 592.0710.
(2,9,16-Tr ih ydr oxyph th alocyan in ato)zin c(II) (33). Sim-
ilarily, the cleavage of 30 (112 mg, 0.1 mmol) gave 33 (48 mg,
0.077 mmol) in 77% yield: UV-vis (THF) λmax (log ꢀ) 678 (4.95),
1
610 (4.32), 348 (4.69); IR (KBr, cm-1) ν 3650-3300 (OH); H
(2,3-Dih yd r oxyp h th a locya n in a to)zin c(II) (39). A mix-
ture of 40 mg of compound 36 and 200 mg of TMB in 10 mL
of TFA was refluxed for 24 h under argon. The solvent was
then evaporated under vacuum, and the residue was washed
with hexane. The blue solid was dissolved in 4 mL of THF,
and the solution was applied to a gel permeation column (SX-
2), eluting with THF to give 14 mg of 39 as a blue solid in
58% yield. Cleavage of 37 and 38 gave 39 in 84% and 62%
yield, respectively: UV-vis (THF) λmax (log ꢀ) 672 (5.06), 608
(4.30), 346 (4.67); IR (KBr, cm-1) ν 3650-3300 (OH); 1H NMR
(DMSO-d6) δ 10.34 (s, br, 2H), 9.47-9.37 (m, 6H), 8.76 (s, 2H),
8.30-8.20 (m, 6H); FAB-MS m/z (rel intensity) 608 (M+, 100),
609 (92), 610 (90), 611 (78), 612 (76), 613 (53). Anal.
(C32H16N8O2Zn) Calcd: C, 63.01; H, 2.64; N, 18.38. Found:
N, 17.90.
NMR (DMSO-d6) δ 10.69 (s, br, 3H), 9.39-9.37 (m, 2H), 9.21-
9.16 (m, 3H), 8.72-8.69 (m, 3H), 8.23-8.21 (m, 2H), 7.66-
7.64 (m, 3H); FAB-MS m/z (rel intensity) 624, 625, 626 {M+,
(M + 1)+, (M + 2)+, 88, 93, 100}. Anal. (C32H16N8O3Zn) C, H,
N.
(1-Hyd r oxyp h th a locya n in a to)zin c(II) (34). Cleavage of
31 (74 mg, 0.1 mmol) with TFA as above gave 34 (50 mg, 0.085
mmol) as a blue solid in 85% yield: mp >320 °C; UV-vis
(THF) λmax (log ꢀ) 686 (5.11), 622 (4.48), 338 (4.75); IR (KBr,
1
cm-1) ν 3650-3300 (OH); H NMR (DMSO-d6) δ 10.70 (s, br,
1H), 9.53 (br, 1H), 9.38-9.27 (m, 6H), 8.95 (br, 1H), 8.25-
8.21 (m, 6H), 8.09 (br, 1H); FAB-MS m/z (rel intensity) 592
(M+, 100), 593 (90), 594 (94), 595 (87). Anal. (C32H16N8OZn)
C, H, N.
[2,3,9,10,16,17,23,24-Octa k is(ben zyloxy)p h th a locya n i-
n a to]zin c(II) (35). A mixture of 100 mg of compound 11 and
0.5 mL of DBU in 10 mL of 1-butanol was heated to 100 °C
under argon for 1 h. To the mixture was added zinc acetate
(0.1 g), and the solution was stirred overnight at 120 °C under
argon. The mixture was cooled to room temperature, and 20
mL of methanol and water (1:1) was added. The blue
precipitate was collected by centrifugation and was washed
with methanol and then hexane. The blue solid was purified
by flash column chromatography on silica gel, eluting with
benzene and THF (3:1) to give pure 0.035 g of 35 in 34%
yield: mp >320 °C; UV-vis (THF) λmax (log ꢀ) 672 (5.10), 608
(4.35), 356 (4.78); IR (KBr, cm-1) ν 3020, 1250 and 1082 (Ar-
O-C); 1H NMR (pyridine-d5) δ 9.34 (s, 8H), 7.86 (d, J ) 8 Hz,
16H), 7.47 (t, J ) 7.5 Hz, 16H), 7.39 (t, J ) 7.5 Hz, 8H), 5.58
(s, 16H); FAB-MS m/z (rel intensity) 1424 (M+, 38), 1333,
1242, 1151, 1060 {(M - 4PhCH2)+, 100}, 969, 878. Anal.
(C88H64N8O8Zn) C, H, N.
(2,3,9,10-Tetr a m eth oxyp h th a locya n in a to)zin c(II), a n
“Ad ja cen t” P c (41). To 20 mL of freshly distilled methanol
were added 0.07 g of lithium (10 mmol) and 2.56 g (20 mmol)
of 7. The solution was refluxed for 2 h under argon. To 2 mL
of the dark-green solution was added 1.84 g (10 mmol) of 4,5-
dimethoxyphthalonitrile (40)28 in 10 mL of 1-octanol. The
mixture was heated to 100 °C and stirred overnight. Zinc
acetate was added to the mixture, which was stirred for
another 8 h. The mixture was poured into 50 mL of methanol
and water (1:1) to give a blue precipitate which was collected
by centrifugation. The residue was passed through a silica
gel column to remove some impurities by eluting with THF
and hexane (1:1). Further purification was performed by size
exclusion column chromatography on Bio-beads gel (SX-2), and
two main fractions, 41 and (2,3-dimethoxyphthalocyaninato)-
zinc(II), were collected in 28% and 22% yield, respectively.
Compound 41 is a blue solid: mp >320 °C; UV-vis (THF) λmax
(log ꢀ) 668 (5.11), 640 (4.32), 604 (4.34), 354 (4.68); IR (KBr,
cm-1) ν 3010, 2960, 2862, 1375, 1085 (Ar-C-O); 1H NMR
(pyridine-d5) δ 9.79 (d, J ) 7.4 Hz, 2H), 9.71 (d, J ) 7.4 Hz,
2H), 9.24 (s, 2H), 9.19 (s, 2H), 8.25-8.18 (m, 4H), 4.31 (s, 6H),
4.11 (s, 6H); FAB-MS m/z (rel intensity) 696 (M+, 95), 697 (75),
698 (84), 699 (100), 700 (65). Anal. (C36H24N8O4Zn) C, H, N.
(2,3-Dimethoxyphthalocyaninato)zinc(II) is also a blue solid:
mp >320 °C; 1H NMR (pyridine-d5) δ 9.78-9.73 (m, 8H), 8.24-
8.22 (m, 6H), 4.31 (s, 6H); FAB-MS for C34H20N8O2Zn m/z (rel
intensity) 636 (M+, 100), 637 (90), 638 (96).
[2,3-Bis(ben zyloxy)p h th a locya n in a to]zin c(II) (36). A
mixture of 60 mg (0.18 mmol) of compound 11, 276 mg (2.16
mmol) of 28, and 1.0 mL of DBU in 50 mL of 1-butanol was
heated to 100 °C for 1 h under argon. To the mixture was
added zinc acetate (0.15 g), and the solution was stirred at
100-110 °C overnight. The mixture was then cooled to room
temperature, and 100 mL of methanol and water (1:1) was
added to this mixture to give a blue precipitate. The precipi-
tate was collected by centrifugation, washed with methanol
and hexane, and extracted with ethyl acetate for 24 h in a
Soxhlet extractor. The reaction residue was purified by size
exclusion chromatography (SX-2), eluting with THF. Further
purification was performed on a short flash silica gel column,
eluting with hexane and THF (3:1): yield 38%; mp >320 °C;
UV-vis (THF) λmax (log ꢀ) 668 (5.20), 638 (4.44), 604 (4.49),
[2,3,9,10-Tet r a k is(b en zyloxy)p h t h a locya n in a t o]zin c-
(II), a n “Ad ja cen t” P c (42). To 30 mL of freshly distilled
methanol were added 0.07 g of lithium (10 mmol) and 2.56 g
(20 mmol) of 7. The solution was refluxed for 2 h under argon.
To 1 mL of the solution was added 1.13 g (3.33 mmol) of 11 in
10 mL of 1-octanol. The mixture was heated to 80 °C and
stirred overnight. Zinc acetate (0.35 g) was added to the
mixture, which was stirred for another 8 h. The mixture was
poured into 50 mL of methanol and water (1:1) to give a blue
precipitate which was collected by centrifugation. Further
1
348 (4.75); IR (KBr, cm-1) ν 3020, 2840, 1080 (Ar-O-C); H
NMR (pyridine-d5) δ 9.73-9.70 (m, 4H), 9.62 (d, J ) 7.2 Hz,
2H), 9.36 (s, 2H), 8.23-8.16 (m, 6H), 7.92 (d, J ) 6.5 Hz, 4H),
7.50 (t, J ) 7.4 Hz, 4H), 7.39 (t, J ) 7.2 Hz, 2H), 5.77 (s, 4H);

Hydroxyphthalocyanines as Agents for Cancer Therapy
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1799
purification was performed by size exclusion column chroma-
tography on Bio-beads gel (SX-2), and two main fractions, 42
and 36, were collected in 18% and 12% yield, respectively.
Compound 42 is a blue solid: mp >320 °C; UV-vis (THF) λmax
(log ꢀ) 672 (5.03), 638 (4.20), 608 (4.26), 354 (4.67); IR (KBr,
for 12 h at 100 °C. The temperature was raised to 120 °C,
and the solution was stirred for another 2 h. The mixture was
cooled to room temperature and quenched with methanol and
water (1:1) to form a shining blue precipitate. The precipitate
was collected by centrifugation, washed with water, methanol
and hexane, successively, and dried. The blue solid was
examined under a microscope and exhibited crystalline needles.
Further recrystallization from THF gave 0.44 g (80%) of
shining, blue, square crystals, 47.
Compound 47 can also be prepared from 46: A mixture of
80 mg (0.069 mmol) of 46 and 50 mg of zinc acetate in 5 mL
of DMF and toluene (1:1) was refluxed for 24 h. To the mixture
was added 10 mL of water, and the solution was centrifuged
to separate a shining, blue solid. The solid was washed with
water, methanol, and hexane and dried to give 76 mg (90%)
of shining, blue 47 as crystalline needles or square crystals:
mp >320 °C; UV-vis (THF) λmax (log ꢀ) 696 (5.34), 665 (4.37),
626 (4.46), 369 (4.45); IR (KBr, cm-1) ν 3010, 2940, 2920, 2840,
1580, 1485, 1335, 1260, 1235, 1130, 1090, 1060, 1040, 1020,
800, 760, 735; 1H NMR (pyridine-d5) δ 9.43 (d, J ) 7.5 Hz,
4H), 8.33 (d, J ) 6.6 Hz, 8H), 8.22 (t, J ) 7.6 Hz, 4H), 7.87 (d,
J ) 8.0 Hz, 4H), 7.65 (d, J ) 7.6 Hz, 8H), 5.91 (s, 8H), 2.83 (t,
J ) 7.6 Hz, 8H), 1.83-1.73 (m, 8H), 1.51-1.39 (m, 8H), 0.98
(t, J ) 7.3 Hz, 12H); J MOD 13C NMR (pyridine-d5) δ 156.85,
154.18, 154.07, 143.01, 142.25, 135.20, 130.91, 129.20, 128.62,
126.41, 116.79, 113.68, 71.40, 35.84, 34.20, 22.72, 14.25; FAB-
MS m/z (rel intensity) 1226 {(M + 2)+, 100}, 1079 {[(M + 2) -
R]+, 88}, 932 {[(M + 2) - 2R]+, 96}, 785 {[(M + 2) - 3R]+,
84}, 638 {[(M + 2) - 4R]+, 68}. Anal. (C76H72N8O4Zn) C, H,
N.
1
cm-1) ν 3020, 2862, 1850 (Ar-C-O); H NMR (pyridine-d5) δ
9.83-9.72 (m, 4H), 9.52 (s, 2H), 9.50 (s, 2H), 8.25-8.15 (m,
6H), 7.92-7.88 (m, 8H), 7.35-7.20 (m, 12H), 5.93 (s, 4H), 5.88
(s, 4H); FAB-MS m/z (rel intensity) 1000 (M+, 90), 909 {(M -
PhCH2)+, 80}, 818 {(M - 2PhCH2)+, 61}, 727 {(M - 3PhCH2)+,
50}, 636 {(M - 4PhCH2)+, 32}. Anal. (C60H40N8O4Zn) C, H,
N.
[2,9-Bis(d ip h en ylm eth oxy)p h th a locya n in a to)zin c(II)
(43). The procedure previously described for the preparation
of 41 was used to prepare 43 from 16 and 28: yield 30%; mp
>320 °C; UV-vis (THF) λmax (log ꢀ) 676 (5.20), 606 (4.40), 346
(4.79); IR (KBr, cm-1) ν 3010, 1250, 1120, 1085 (Ar-O-C); 1H
NMR (pyridine-d5) δ 9.74-9.70 (m, 4H), 9.62-9.59 (m, 4H),
8.23-8.20 (m, 4H), 8.16-8.8.13 (m, 2H), 7.97-7.95 (m, 8H),
7.53-7.30 (m, 12H), 7.05-6.98 (m, 2H); FAB-MS m/z (rel
intensity) 940 (M+, 100), 773 {(M - Ph2CH)+, 66}, 606 {(M -
2Ph2CH)+, 52}. Anal. (C58H36N8O2Zn) Calcd: C, 73.93; H,
3.85; N, 11.89. Found: C, 72.95.
(2,3,9,10-Tetr a h yd r oxyp h th a locya n in a to)zin c(II) (44).
Cleavage of 42 (100 mg, 0.1 mmol), as for 32, gave 44 (27 mg,
0.042 mmol) as a blue solid in 42% yield: mp >320 °C; UV-
vis (THF) λmax (log ꢀ) 676 (5.06), 610 (4.32), 344 (4.67); IR (KBr,
1
cm-1) ν 3650-3300 (OH); H NMR (DMSO-d6) δ 10.64 (s, br,
2H), 10.57 (s, br, 2H), 9.57-9.40 (m, 4H), 8.68 (s, 1H), 8.58 (s,
1H), 8.30-8.20 (m, 4H); FAB-MS m/z (rel intensity) 640 (M+,
100). Anal. (C32H16N8O4Zn) C, H, N.
1,8,15,22-Tetr a k is(ben zyloxy)p h th a locya n in e (48). As
for 46, compound 23 (0.94 g) was used to prepare 0.39 g of 48
at 100 °C as a blue solid in 42% yield: UV-vis (THF) λmax
(log ꢀ) 726 (5.01), 692 (5.05), 662 (4.12), 632 (3.18), 352 (2.75),
318 (2.99); IR (KBr, cm-1) ν 3310 (N-H), 3010, 2960, 2920,
2860, 1665, 1530, 1440, 1385, 1240, 1175, 1145, 1110, 1010,
925, 850; 1H NMR (pyridine-d5) δ 9.41 (d, J ) 7.5 Hz, 4H),
8.43 (d, J ) 8 Hz, 8H), 8.18 (t, J ) 7.5 Hz, 4H), 7.99-7.90 (m,
12H), 7.55 (t, J ) 7.5 Hz, 4H), 5.73 (s, 8H), -2.2 (br, 2H); FAB-
MS m/z (rel intensity) 940 {(M + 2)+, 92}, 849 {[(M + 2) -
R]+, 70}, 758 {[(M + 2) - 2R]+, 65}, 667 {[(M + 2) - 3R]+,
80}. Anal. (C60H42N8O4) C, H, N.
(2,9-Dih yd r oxyp h th a locya n in a to)zin c(II) (45). Cleav-
age of 43 (94 mg, 0.1 mmol) by TFA gave 45 (45 mg, 0.088
mmol) as a blue solid in 88% yield: mp >320 °C; UV-vis
(THF) λmax (log ꢀ) 676 (4.99), 610 (4.25), 344 (4.60); IR (KBr,
cm-1) ν 3650 (br, OH); 1H NMR (DMSO-d6) δ 10.74 (s, br, 2H),
9.40-9.35 (m, 2H), 9.20-9.17 (m, 2H), 8.76-9.71 (m, 2H),
8.25-8.08 (m, 6H), 7.80-7.75 (m, 2H); FAB-MS m/z (rel
intensity) 608 (M+, 100). Anal. (C32H16N8O2Zn) Calcd: C,
63.01; H, 2.64; N, 18.38. Found: C, 63.60.
1,8,15,22-Te t r a k is(p -n -b u t ylb e n zyloxy)p h t h a locya -
n in e (46). Lithium (0.30 g) was suspended in 30 mL of
1-octanol. The suspension was heated to 170 °C and stirred
for 4 h. To the homogeneous solution, cooled to 40 °C, was
added 0.35 g (1.2 mmol) of 22 in 4 mL of dried THF. The
temperature was raised to 60 °C, and the solution was stirred
for 2 h. Then, the temperature was raised to 100 °C, and the
mixture stirred for 12 h. The temperature was further raised
to 120 °C, and the solution was stirred for another 2 h. The
mixture was cooled to room temperature, and the reaction was
quenched with methanol and water (1:1) to form a blue
precipitate. The precipitate was collected by centrifugation,
washed successively with water, methanol, and hexane, and
dried to give 126.7 mg (40%) of a dark-green solid ,46: mp
>320 °C; UV-vis (THF) λmax (log ꢀ) 724 (4.70), 690 (4.71), 658
(4.02), 627 (2.95), 395 (3.46), 351 (3.96); IR (KBr, cm-1) ν 3310
(N-H), 3010, 2960, 2920, 2860, 1665, 1530, 1440, 1385, 1240
[1,8,15,22-Tetr a k is(ben zyloxy)p h th a locya n in a to]zin c-
(II) (a s a sin gle isom er ) (49). Using the procedure previ-
ously described for the preparation of 47 (method I), 23 (0.13
g) was used to prepare 49 at 100 °C as cubic crystals in 68%
yield: mp >320 °C; UV-vis (THF) λmax (log ꢀ) 696 (5.24), 664
(4.30), 624 (4.26), 368 (4.12); IR (KBr, cm-1) ν 3010, 2920, 2840,
2820, 1575, 1480, 1335, 1262, 1230, 1130, 1090, 1060, 1040,
1
1020, 800, 760, 735; H NMR (pyridine-d5) δ 9.35 (d, J ) 7.4
Hz, 4H), 8.40 (d, J ) 7.2 Hz, 8H), 8.15 (t, J ) 7.6 Hz, 4H),
7.85-7.78 (m, 12H), 7.67 (t, J ) 7.4 Hz, 4H), 5.90 (s, 8H);
J MOD 13C NMR (pyridine-d5) δ 156.05, 154.08, 154.00, 142.15,
138.51, 135.02, 130.61, 129.10, 128.40, 126.21, 116.49, 113.47,
71.27; FAB-MS m/z (rel intensity) 1002 {(M + 2)+, 100}, 911
{[(M + 2) -R]+, 65}, 820 {[(M + 2) - 2R]+, 64}, 729 {[(M + 2)
- 3R]+, 45}, 638 {[(M + 2) - 4R]+, 40}. Anal. (C60H40N8O4-
Zn) C, H, N.
1
(Ar-O-C), 1175, 1145, 1110, 1010, 925, 850; H NMR (pyri-
dine-d5) δ 9.47 (d, J ) 7.5 Hz, 4H), 8.33 (d, J ) 7.8 Hz, 8H),
8.25 (t, J ) 7.5 Hz, 4H), 7.90 (d, J ) 8.0 Hz, 4H), 7.65 (d, J )
7.7 Hz, 8H), 5.93 (s, 8H), 2.83 (t, J ) 7.5 Hz, 8H), 1.83-1.73
(m, 8H), 1.51-1.42 (m, 8H), 0.99 (t, J ) 7.5 Hz, 12H), -4.2 (s,
br, 2H); FAB-MS m/z (rel intensity) 1164 {(M + 2)+, 68}, 1017
{[(M + 2) - R]+, 56}, 870 {[(M + 2) - 2R]+, 23}, 723 {[(M +
2) - 3R]+, 100}, 578 {[(M + 2) -4R]+, 91}. Anal. (C76H74N8O4)
C, H, N.
[1,8,15,22-Tetr a k is(p-n -bu tylben zyloxy)p h th a locya n i-
n a to]zin c(II) (47). Lithium (0.60 g) was suspended in 60 mL
of 1-octanol. The mixture was heated to 170 °C and stirred
for 4 h. To the homogeneous solution, cooled to 40 °C, was
added 0.52 g (1.79 mmol) of 22 in 5 mL of THF. The solution
was slowly heated to 100 °C and underwent a color change
from colorless to green. To the green solution was added 326
mg (1.79 mmol) of zinc acetate, and the solution was stirred
[1,8,15,22-Tetr a k is(ben zyloxy)p h th a locya n in a to)zin c-
(II) (a s a m ixtu r e of isom er s) (50). The above-described
procedure for the preparation of 49 was repeated at 160 °C,
and compound 50 was obtained in 8% yield: 1H NMR (pyri-
dine-d5) δ 9.48-9.28 (m, 4H), 8.48-8.36 (m, 8H), 8.22-8.08
(m, 4H), 7.89-7.62 (m, 16H), 5.88 (s, br, 8H). Compound 50
exhibited the same FAB-MS spectrum as 49.
[1,8,15,22-Tetr a k is(p r op a r gyloxy)p h th a locya n in a to]-
zin c(II) (a m ixtu r e of isom er s) (51). As for 47 (method I),
24 (0.15 g) was used to prepare 51 at 120 °C as a blue solid in
26% yield: mp >320 °C; UV-vis (THF) λmax (log ꢀ) 702 (5.14),
666 (4.20), 620 (4.06), 360 (4.12); IR (KBr, cm-1) ν 3010, 2920,
2840, 2820, 1575, 1480, 1335, 1262, 1230, 1130, 1090, 1060,
1040, 1020, 800, 760, 735; 1H NMR (pyridine-d5) δ 9.70-9.41
(m, 4H), 8.34-8.11 (m, 4H), 7.91-7.73 (m, 4H), 5.75-5.58 (m,
8H), 2.80-2.73 (m, 4H); FAB-MS m/z (rel intensity) 792 (M+,

1800 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Hu et al.
30). Anal. (C44H24N8O4Zn) Calcd: C, 66.61; H, 3.05; N, 14.12.
Found: N, 15.00.
(Gibco, Canada), according to an established protocol.29 The
photocytotoxicity test was conducted by means of the colori-
metric MTT assay.30 Briefly, 15 × 103 EMT-6 cells/well were
inoculated in 100 µL of Waymouth’s growth medium in 96-
multiwell plates and incubated overnight at 37 °C in an
atmosphere containing 5% CO2. The cells were rinsed twice
with PBS and incubated for 1 or 24 h at 37 °C with 100 µL of
a 1 µM dye solution prepared by diluting the Cremophor
emulsion in Waymouth 1% FBS. After incubation, the cells
were rinsed twice with PBS, refed with 100 µL of Waymouth
15% FBS, and exposed to red light. The light source consisted
of two 500-W tungsten/halogen lamps (GTE Sylvania, Canada)
fitted with a circulating, refrigerated, aqueous rhodamine
filter. The fluence rate calculated over the absorbance peaks
of the dyes (660-700 nm) was 10 mW cm-2 for a total fluence
of 0.1-20 J cm-2. The cells were incubated at 37 °C overnight
before assessing cell viability. Fifty microliters of a 5-fold
diluted MTT stock solution (5 mg mL-1 PBS) in Waymouth
15% FBS was added to each well. After 3 h, 100 µL of sodium
dodecyl sulfate (SDS; 10% in 0.01 N HCl) was added in the
wells. Plates were incubated overnight at 37 °C, whereafter
the absorbance was read at 595 nm by means of a microplate
reader (BioRad, Mississauga, Ontario, Canada). The average
absorbance of the control wells without cells was subtracted.
The average absorbance of the control cells which were
incubated with dye-free Waymouth 1% FBS was taken as
100% cell survival. The light doses (LD90) required to inacti-
vate 90% of the cells incubated with 1 µM dye were extrapo-
lated from the survival curves. Eightfold replicates were run
per light dose, and the experiment was repeated at least three
times.
1,8,15,22-Tetr a k is(n eop en tyloxy)p h th a locya n in e (a s a
sin gle isom er ) (52). Using the procedure previously de-
scribed for the preparation for 47, the self-condensation of 0.88
g of 3-neopentoxyphthalonitrile (25)26 gave 0.26 g of 52 at 60-
70 °C as a blue solid in 30% yield: mp >320 C; UV-vis (THF)
λ
max (log ꢀ) 726, 698, 664, 628, 354, 320; IR (KBr, cm-1) ν 3285
(N-H), 2945, 2840, 1575, 1480, 1335, 1255 (Ar-O-C), 1230,
1
1130, 1090, 1060, 1020, 735; H NMR (pyridine-d5) δ 9.74 (d,
J ) 7.8 Hz, 4H), 8.41 (t, J ) 7.7 Hz, 4H), 7.66 (d, J ) 7.8 Hz,
4H), 4.57 (s, 8H), 1.70 (s, 36H), -2.80 (s, br, 2H); FAB-MS
m/z (rel intensity) 858 (M+, 100). Anal. (C52H58N8O4) C, H,
N.
[1,8,15,22-Tetr a k is(n eop en tyloxy)p h th a locya n in a to]-
zin c(II) (a s a sin gle isom er ) (53). As for 52 (method I), 25
was used to prepare 53 at 60-70 °C in 65% yield as crystalline
needles: mp >320 °C; UV-vis (THF) λmax (log ꢀ) 698 (5.24),
662 (4.30), 622 (4.26), 365 (4.12); IR (KBr, cm-1) ν 3010, 2920,
2840, 2820, 1575, 1480, 1335, 1255 (Ar-O-C), 1230, 1130,
1
1090, 1060, 1040, 1020, 800, 760, 735; H NMR (pyridine-d5)
δ 9.71 (d, J ) 7.4 Hz, 4H), 8.33 (t, J ) 7.7 Hz, 4H), 7.76 (d, J
) 7.4 Hz, 4H), 4.37 (s, 8H), 1.72 (s, 36H); J MOD 13C NMR
(pyridine-d5) δ 158.10, 154.97, 154.73, 142.85, 132.00, 126.47,
116.90, 113.25, 79.70, 33.32, 27.35; FAB-MS m/z (rel intensity)
920 (M+, 100). Anal. (C52H56N8O4Zn) Calcd: C, 67.71; H, 6.12;
N, 12.14. Found: N, 11.47.
[1,8,15,22-Tetr a k is(n eop en tyloxy)p h th a locya n in a to)-
zin c(II) (m ixtu r e of isom er s) (54). As for 47 (method I),
27 was repeated at >130 °C, and 54 was obtained as a blue
solid in 24% yield. Compound 54 exhibited the same FAB-
MS as 53 but gave multiple absorption peaks in its 1H NMR
spectrum: 1H NMR (pyridine-d5) δ 9.74-9.51 (m, 4H), 8.41-
8.21 (m, 4H), 8.00-7.75 (m, 4H), 4.40-4.32 (m, 8H), 1.70-
1.35 (m, 36H).
(1,8,15,22-Tetr am eth oxyph th alocyan in ato)zin c(II) (55).
As for 47 (method I), 27 was used to prepare 0.35 g of 55 (from
0.63 g of 27) at 50 °C in 50% yield as blue crystals: mp >320
°C; UV-vis (THF) λmax (log ꢀ) 694 (5.24), 662 (4.37), 624 (4.43),
368 (4.37); IR (KBr, cm-1) ν 3010, 2920, 2840, 2820, 1575, 1480,
1335, 1262, 1230, 1130, 1090, 1060, 1040, 1020, 800, 760, 735;
1H NMR (pyridine-d5) δ 9.55 (d, J ) 8.0 Hz, 4H), 8.18 (d, J )
8.0 Hz, 4H), 7.69 (t, J ) 8.0 Hz, 4H), 4.58 (s, 12H); J MOD 13C
NMR (pyridine-d5) δ 156.85, 155.07, 154.89, 142.93, 132.03,
127.04, 117.04, 113.81, 56.88; FAB-MS m/z (rel intensity) 696
(M+, 65), 697 {(M + 1)+, 100}, 698 {(M + 2)+, 90}, 699 {(M +
3)+, 80}. Anal. (C36H24N8O4Zn) C, H, N.
(1,8,15,22-Tetr a h yd r oxyph th a locya n in a to)zin c(II) (56).
To 109 mg (0.086 mmol) of 55 in 5 mL of TFA was added 46
mg (0.35 mmol) of TMB. The mixture was refluxed for 15 h.
The TFA was evaporated, and the residue was washed with
hexane a few times. The blue solid was dissolved in THF, and
the solution was put onto a gel permeation column, eluting
with THF. Evaporation of the solvent gave 46.8 mg (85%) of
56 as a blue solid: UV-vis (THF) λmax (log ꢀ) 719 (4.85), 686
(4.12), 646 (4.13), 344 (4.06); IR (KBr, cm-1) ν 3500-3100 (br
signal, phenolic group), 1650, 1520, 1430, 1360, 1275, 1215,
1060, 925, 840; 1H NMR (DMSO-d6) δ 10.56 (s, 4H), 8.57 (d, J
) 7.5 Hz, 4H), 8.25 (t, J ) 7.5 Hz, 4H), 7.82 (d, J ) 7.6 Hz,
4H); FAB-MS m/z (rel intensity) 640 (M+, 100). Anal.
(C32H16N8O4Zn) Calcd: C, 59.88; H, 2.51; N, 17.46. Found:
N, 16.89.
3. P h otod yn a m ic Th er a p y. All experiments were per-
formed on male BALB/c mice (18-22 g) (Charles River
Breeding Laboratories, Montreal, Canada) following a protocol
approved by the Canadian Council on Animal Care and an
in-house ethics committee. The animals were allowed free
access to water and food throughout the course of the experi-
ments. Before tumor implantation, hair on the hind legs and
back of the mice was removed by shaving and chemical
depilating (Nair, Whitehall, Mississauga, Canada). A tumor
was implanted on each hind thigh by intradermal injection of
2 × 105 EMT-6 cells suspended in 0.05 mL of Waymouth
growth medium; 6-8 days after tumor cell inoculation (tumor
size: 3-5-mm diameter, 2-3-mm thickness), animals were
given an intravenous injection of 1 or 2 µmol kg-1 dye prepared
as a Cremophor emulsion (0.2 mL/20 g) and treated with red
light 24 h later. One tumor served as a control, while the other
was illuminated with 650-700-nm light generated by a
1000-W xenon lamp, equipped with a 10-cm circulating water
filter to eliminate possible hyperthermic effects and two glass
filters (Corion LL-650 and LS-700). The fluence rate over a
8-mm diameter beam was 200 mW cm-2 for a total fluence of
400 J cm-2
. Under these conditions an increase of tumor
temperature of approximately 2 °C was recorded, as monitored
by means of a microthermocouple inserted at the base of the
tumor. A positive tumor response was assigned to tumors
which appeared macroscopically as flat and necrotic tissues
within a few days after PDT. A complete tumor regression is
defined as the absence of a palpable tumor at 3 weeks after
PDT.
Ack n ow led gm en t. The authors wish to thank the
Natural Science and Engineering Research Council of
Canada for support of this research and Carole La
Madeleine for expert technical assistance. S. Zeki Yildiz
is grateful to The Scientific and Technical Research
Council of Turkey for a NATO Science Fellowship.
Biologica l Stu d ies. 1. Dye F or m u la tion . ZnPcs were
first diluted in a minimal volume of THF; Cremophor EL
(polyethoxylated castor oil; BASF, Toronto, Canada; 10% final)
and 1,2-propanediol (3% final) were added under sonication,
whereafter THF was evaporated under vacuum. The solution
was then diluted with phosphate-buffered saline (PBS; pH 7.4)
and filtered (0.2 µm). Pc concentration of stock solutions was
determined spectroscopically upon dilution in THF by measur-
ing the absorbance at the λmax around 670-680 nm.
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