U.S. patent application number 13/387407 was filed with the patent office on 2012-05-31 for phthalocyanine derivative consisting of a mixture of 4 isomers.
Invention is credited to Giacomo Chiti, Donata Dei, Daniele Nistri, Gabrio Roncucci.
Application Number | 20120135972 13/387407 |
Document ID | / |
Family ID | 41822411 |
Filed Date | 2012-05-31 |
United States Patent
Application |
20120135972 |
Kind Code |
A1 |
Roncucci; Gabrio ; et
al. |
May 31, 2012 |
PHTHALOCYANINE DERIVATIVE CONSISTING OF A MIXTURE OF 4 ISOMERS
Abstract
There is described a phthalocyanine derivative of formula (I)
consisting of 4 isomers wherein the relative isomer B content is
less than or equal to 1% by weight. ##STR00001##
Inventors: |
Roncucci; Gabrio;
(Poggibonsi, IT) ; Dei; Donata; (San Gimignano,
IT) ; Chiti; Giacomo; (Prato, IT) ; Nistri;
Daniele; (Prato, IT) |
Family ID: |
41822411 |
Appl. No.: |
13/387407 |
Filed: |
July 30, 2010 |
PCT Filed: |
July 30, 2010 |
PCT NO: |
PCT/EP10/61112 |
371 Date: |
January 27, 2012 |
Current U.S.
Class: |
514/185 ;
540/140 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 31/409 20130101; A61P 17/12 20180101; A61P 35/00 20180101;
A61P 17/02 20180101; A61P 17/00 20180101; C07D 487/22 20130101;
C07F 3/06 20130101; A61P 31/02 20180101; A61P 17/06 20180101 |
Class at
Publication: |
514/185 ;
540/140 |
International
Class: |
A61K 31/555 20060101
A61K031/555; A61P 31/00 20060101 A61P031/00; A61P 17/06 20060101
A61P017/06; A61P 17/12 20060101 A61P017/12; C07F 3/06 20060101
C07F003/06; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 30, 2009 |
IT |
FI2009A000168 |
Claims
1. Phthalocyanine derivative of formula (I) ##STR00002## consisting
of 4 isomers: A, B, C, D identified according to the symmetry
classes D.sub.2h, C.sub.4h, C.sub.s, C.sub.2v respectively, wherein
the isomer B is .ltoreq.1% by weight.
2. Phthalocyanine derivative of formula (I) according to claim 1
wherein the isomer B is .ltoreq.1% by weight and isomer A is
.ltoreq.12.5% by weight.
3. Phthalocyanine derivative of formula (I) according to claim 1
wherein the isomer B is .ltoreq.0.2% by weight.
4. Phthalocyanine derivative of formula (I) according to claim 1
wherein the isomer B is .ltoreq.0.2% by weight and isomer A is
.ltoreq.12.5% by weight.
5. Phthalocyanine derivative of formula (I) according to claim 4
wherein the isomers A/C/D are present in the following relative
contents: 5.5-7.0%/56.0-63.5%/30.0-38.0%.
6. Process for the preparation of a derivative of formula (I)
according to claim 1 comprising a chromatographic purification of
compound of formula (II) ##STR00003## to give the amine
intermediate of formula (III).
7. Process according to claim 6 wherein said chromatographic
purification is carried out using a chromatographic column wherein
the stationary phase is silica gel and the mobile phase is a
constant or variable mixture of organic solvents.
8. Process according to claim 7 wherein the silica gel is the
stationary phase made of irregularly shaped or spherical shape
silica gel particles, with particle size in the range 5-75 .mu.m
and pore size in the range 60-150 Angstrom.
9. Process according to claim 7 wherein the stationary phase is
used for more than one chromatographic run
10. Process according to claim 7 wherein the mobile phase is
constituted by at least two organic solvents comprising among
dichloromethane, chloroform, ethyl acetate, acetone,
tetrahydrofurane, N,N-dimethylformamide, methanol, ethanol,
acetonitrile, diethyl ether, pentanes, hexanes, heptanes, petroleum
ether, alkylamines.
11. Process according to claim 7 wherein said mobile phase consists
of two phases X and Y mixed in constant or variable manner along
the time.
12. Process according to claim 11 wherein X is constituted from at
least 90% of chlorinated solvents and Y is constituted by pentanes,
hexanes, heptanes, or petroleum ethers.
13. Process according to claim 11 wherein X consists of a mixture
of dichloromethane or chloroform with 0.1-10% of an agent or a
mixture of agents with stronger elution power, comprising among
tetrahydrofuran, ethyl acetate, methanol, ethanol, acetone,
alkylamines and Y is constituted by pentanes, hexanes, heptanes, or
petroleum ethers,
14. Process according to claim 11 wherein X consists of a mixture
94/5/1 dichloromethane/tetrahydrofuran/methanol and Y consists of
n-hexane or n-heptane.
15. Compounds of formula (III) ##STR00004## and (IV) ##STR00005##
as intermediates in the process according to claim 6.
16. Pharmaceutical compositions comprising as active principle a
phthalocyanine derivative of formula (I) according to claim 1 in
combination with pharmaceutically acceptable excipients and/or
diluents, possibly in combination with chelating agents.
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. A method for the treatment of hypderproliferative diseases,
psoriasis, actinic keratosis and pre-tumour or tumour pathologies
with a pharmaceutical composition comprising as active principle a
compound according to claim 1.
25. A method for the treatment by PDT of hyperproliferative
diseases, psoriasis, actinic keratosis and pre-tumour or tumour
pathologies with a pharmaceutical composition comprising as active
principle a compound according to claim 1.
26. A method for the preventive or curative treatment of microbial
infections with a pharmaceutical composition comprising as active
principle a compound according to claim 1.
27. A method for the preventive or curative photodynamic treatment
of microbial infections with a pharmaceutical composition
comprising as active principle a compound according to claim 1.
28. A method for the incorporation of a pharmaceutical composition
comprising as active principle a compound according to claim 1 as a
component of medical devices.
29. A method according to claim 28 wherein said medical devices are
for the disinfection of wounds, as a sterilization agent, also ex
vivo, and as in vivo diagnostic agent.
30. A method for the preventive or curative treatment of microbial
infections in animals with a pharmaceutical composition comprising
as active principle a compound according to claim 1.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the field of photosensitising
compounds for therapeutic use.
BACKGROUND ART
[0002] It is known that molecules containing the chromo/fluorophore
phthalocyanine macrocycle produce reactive species of oxygen, such
as radicals or singlet oxygen, by interaction with visible
light.
[0003] For these properties phthalocyanine compounds have proposed
in photodynamic therapy (hereinafter indicated with the initials
"PDT") both for therapeutic treatment and for diagnostic purposes
prior treatment.
[0004] Examples of these compounds are described by Ogura et al.
Journal of Porphyrins and Phthalocyanines 2006, 10, 1116-1124.
[0005] Photosensitizing agents useful in PDT are Zinc
phthalocyanine compounds and their conjugates described in the
patent EP 906 758, by the Applicant.
[0006] Moreover, EP 1 444 236 and in EP 1 883 640 (both by the
Applicant) respectively describe a process for the separation of
regioisomers mixtures and a process for the preparation of
chlorides of phthalocyanine compounds.
[0007] The compounds described and obtained according to the
processes indicated in the above documents have proven to be
effective photosensitising agents in PDT treatment, both for
tumours and for microbial infections; in particular the product
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate tetraiodide, corresponding to
example 53 of EP 906 758, has proved to be particularly active. It
is known that tetra-substituted phthalocyanine derivatives, such as
the compound
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate tetraiodide, corresponding to
example 53 of EP 906 758, are obtained as a mixture of four
positional isomers (see FIG. 1), identified according to the
symmetry classes (D.sub.2h, C.sub.4h, C.sub.s, C.sub.2v), and
hereinafter respectively named with the letters of the alphabet (A,
B, C, D). The aforesaid isomers are formed during the synthesis of
the phthalocyanine macrocycle in relative percentages which differ
from the theoretical (defined by statistics and respectively equal
to 12.5%, 12.5%, 50% and 25%) as a function of the synthesis method
used, the nature of the phthalonitrile substituents employed for
preparation and, finally, of the central metal inserted, but which
are constant, given the same compound and method (Phthalocyanines:
Properties and Applications, Volume 4, Chapter 1 C. C. Leznoff and
A. B. P. Lever (York University, Canada). VCH: New York, 1996).
[0008] As a result, a particular compound, prepared through a
specific synthesis method, such as example 53 of EP 906 758, will
have a typical and reproducible isomers distribution.
[0009] Also using the synthesis process described in the patent EP
1 883 640, through which the compound
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate is obtained as tetrachloride
(example 4), the new salt is in any case present as a mixture of
four isomers, according to the isomers distribution expected and
already obtained with the process indicated in EP 906 758.
[0010] The exact isomers distribution of a particular
tetra-substituted phthalocyanine derivative can be known and
controlled using the separation process described in the patent EP
1 444 236.
[0011] During characterization studies of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate (tetraiodide or tetrachloride), the
Applicant has discovered that the compound identified above,
obtained by following known synthesis processes, contains related
substances in a percentages greater than 0.1%, whose
reduction/elimination is difficult to achieve. Even using the
synthesis process described in the European Patent EP 1 883 640,
and therefore preparing said compound as chloride (example 4),
although obtaining an improvement in terms of solubility and
product purity, it is not possible to reduce said impurities in the
final product below 0.1% by weight (threshold above which
structural identification and toxicological qualification is
required). Moreover the related substances described above have
considerable structural similarities compared to the active
substance, are able to interact with the light radiation used in
PDT and, unlike all other impurities, cannot be eliminated from
batches of the ammonium derivative
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate (tetraiodide or tetrachloride).
Therefore, there is still the need of improving the purity of the
compound [1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate tetrachloride, in view of its use as
a drug substance.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 shows the chemical structure of the four isomers of
the compound
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate (tetrachloride).
[0013] FIG. 2 shows the synthetic way to be used for the obtainment
of compound of formula (I) with low isomer B content in comparison
with the pathway used to obtain compound (VI), having isomers
distribution by statistics.
[0014] FIG. 3 a and b respectively show the HPLC chromatograms of
compound (II) and of compound (III).
[0015] FIG. 4 shows the superimposed HPLC chromatograms for
compound (II) after solubilisation in DMF and in THF/MeOH mixture
(1:1).
[0016] FIG. 5 a and b respectively show the chromatograms of the
product of formula (VI) and of the product of formula (I).
SUMMARY OF THE INVENTION
[0017] The present invention relates to a phthalocyanine compound
of formula (I) as indicated below, consisting of 4 isomers wherein
the isomer B is present in a quantity equal to or less than 1% by
weight.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The Applicant has now surprisingly found that the unwanted
related substances found in the product
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate (both in the form of iodide (V) and
chloride (VI)) derive from impurities present that are formed
during the synthetic process of the amino intermediate (II).
[0019] The Applicant has also surprisingly found that these
impurities can be removed through a chromatography process of the
intermediate, characterized by simultaneous significant elimination
of the isomer B, one of the four positional isomers of the active
substance. The Applicant, on the basis of the chromatographic
affinities existing between the isomer B and the unwanted related
substances, has also defined the maximum quantity of isomer B that
must be present in compound (I) to have a simultaneous quantity of
unwanted impurities 0.1% by weight. The value of the percentage of
B has been found to correspond to 1% by weight.
[0020] Therefore, the subject matter of the present invention is
the phthalocyanine derivative of formula (I), consisting of a
mixture of 4 isomers, identified as A/B/C/D, wherein the percentage
of isomer B present is .ltoreq.1% by weight. The state of the art
closest to the subject matter of the present invention is
represented by the compound described in example 53 of EP 906 758.
Example 53 has the same substituents on the phthalocyanine
macrocycle, but nonetheless differs from (I) for two principal and
mutually related characteristics:
[0021] 1) the four positional isomers are present according to the
distribution expected from the synthesis of the phthalocyanine
nucleus, in fact after their formation, none of the obtained
isomers is partially or totally removed;
[0022] the unwanted impurities are present in a quantity
.gtoreq.0.1% by weight, which makes the example 53 of EP 906 758
less adequate to be used as a drug substance. Conversely the
aforesaid impurities are instead below 0.1% in the batches of
product (I), obtained according to a preparation process that
includes the following steps (see also FIG. 2, where the synthesis
that leads to obtainment of (I) is compared with the process
described in the patents EP 906 758 and EP 1 883 640, in which the
chromatography step ii) is not performed):
[0023] i) Base-catalyzed tetramerisation of 3-dimethylamino phenoxy
phthalonitrile and preliminary purification of the obtained amino
phthalocyanine intermediate (II);
[0024] ii) Chromatographic purification of compound (II) to give
the amino intermediate "with low isomer B content" (III);
[0025] iii) Methylation of the amino intermediate (III) with a
suitable methylating agent to give the ammonium salt intermediate
"with low isomer B content" (IV);
[0026] iv) Ion exchange of the ammonium salt intermediate (IV) to
give the ammonium salt "with low isomer B content" in the form of
chloride (I).
[0027] For the preparation of the desired product (I), the
Applicant has discovered that the impurities of the intermediate
(II), which are those that make compound (VI) unsuitable for the
desired purposes, cannot be eliminated other than using further
chromatography purification of this intermediate (II).
[0028] The Applicant has also surprisingly discovered that the
chromatography process which allows effective separation of the
unwanted impurities from the amino intermediate (II) involves a
coelution with the aforesaid impurities of one of the four
positional isomers (isomer B), which is thus mainly removed from
the rest of the isomers mixture. The amino intermediate (III)
obtained from this chromatography purification has a different
isomers distribution, and is therefore a new compound with respect
to compound (II), besides being efficiently purified from the
impurities initially present.
[0029] With reference to the synthetic process described in FIG. 2,
the ammonium salt (IV) obtained by methylation of the intermediate
(III) has a different isomers composition from (V) and is therefore
a new compound with improved purity. Likewise, the final chloride
(I) obtained through ion exchange of (IV) has a different isomers
distribution and is therefore a new compound with respect to (VI).
Finally, compound (I) is purer than (VI).
[0030] The Applicant has also surprisingly and unexpectedly
discovered that the new isomers distribution of product (I) "with
low isomer B content" with respect to those indicated in patents EP
906 758 and EP 1 883 640, respectively compounds (V) and (VI), does
not imply any difference in terms of pharmacological and
toxicological activity.
[0031] In other words, the product thus obtained, in which the
relative percentage of one of the four isomers (B) is substantially
decreased, maintains the therapeutic properties of the original
mixture and has improved purity, both fundamental requirements for
the use of (I) as pharmaceutical active substance and therefore for
the practical application of the claimed therapeutic properties.
Moreover, the almost total elimination of isomer B, which occurs as
a consequence of chromatographic purification, leads to a series of
further advantages of compound (I), both at the level of final
product and of the process intermediates, in light of the
preparation and use of (I) as an active substance, advantages that
are presented below, The poor solubility of the isomer B of the
amino intermediate (II), due to its symmetrical structure, makes
the solutions of (II) variable in terms of general isomers
distribution, because incomplete dissolution and/or precipitation
can occur. This takes place both during the synthesis and in the
course of analytical controls, with consequent decrease of the
reproducibility of the synthesis process and difficulty of
validation and routine use of analytical methods, problems that are
however totally inexistent for the intermediate (III).
[0032] Moreover, the low content of isomer B facilitates the
characterization, by means of HPLC chromatography methods, of the
product (I) (and, for the same reasons, of the last process
intermediate (IV)) which results in a better control of the
reproducibility of the synthesis batches.
[0033] In a preferred condition of the present invention, the
content of isomer A in compound (I) is lower than 12.5% by
weight.
[0034] Chromatographic purification of the amino intermediate (II),
which allows removal of the unwanted impurities and the majority t
of the isomer B, leading to the intermediate (III), is carried out
using silica gel as stationary phase and a constant or variable
mixture of organic solvents as mobile phase (elution in isocratic
or gradient conditions).
[0035] For the aforesaid purification every one of the following
methods can be used: Flash Chromatography (FC), Medium Pressure
Liquid Chromatography (MPLC) and preparative High Pressure Liquid
Chromatography (prep HPLC)
[0036] In a preferred condition, the purification is performed in
FC or MPLC.
[0037] According to the selected purification method, the
stationary phase can be made of irregularly shaped or spherical
shape silica gel particles, with particle size in the range 5-75
.mu.m and pore size in the range 60-150 Angstrom. The silica gel
can be packed before the chromatography run or used as pre-packed
cartridges/columns.
[0038] In a preferred condition, the stationary phase is made of
irregularly shaped silica gel particles, with particle size in the
range 20-75 .mu.m and pore size of 60 Angstrom.
[0039] In a particular preferred condition the stationary phase can
be used for several chromatographic runs.
[0040] In a preferred condition the mobile phase is constituted by
at least two organic solvents such as dichloromethane, chloroform,
ethyl acetate, acetone, tetrahydrofurane, N,N-dimethylformamide,
methanol, ethanol, acetonitrile, diethyl ether, pentanes, hexanes,
heptanes, petroleum ether, alkylamines.
[0041] In a particularly preferred condition a constant or variable
mixture of two phases X and Y is used as mobile phase.
[0042] In a particularly preferred condition X prevalently contains
chlorinated solvents and Y consists of pentanes, hexanes, heptanes
or petroleum ether.
[0043] In a particularly preferred condition X consists of a
mixture of dichloromethane or chloroform with 0.1-10% of an agent
or a mixture of agents with stronger elution power, such as
tetrahydrofuran, ethyl acetate, methanol, ethanol, acetone,
alkylamines.
[0044] In a particularly preferred condition X consists of a
mixture 94/5/1 dichloromethane/tetrahydrofuran/methanol and Y
consists of n-hexane or n-heptane.
[0045] In a preferred condition the elution is performed eluting
with X/Y 3/1 in isocratic conditions.
[0046] In another preferred condition the elution is performed
initially eluting with X/Y 3/1 (until elution of the isomers C and
D starts) and then with X/Y 4/1 until complete elution of the
isomer A.
[0047] The described chromatographic process can be applied to
samples of compound (II) having different initial isomers
distribution (depending both on the synthetic method and on the
pre-chromatographic treatments carried out), in any case allowing
the total removal of undesired related substances and lowering of
isomer B content, with minimal impact on the percentages of the
other three isomers.
[0048] The following examples are provided by way of non-limiting
illustration of the present invention.
Example 1
Synthetic Preparation of the Compound of Formula (I)
i) Synthesis and Preliminary Purification of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-Dimethylamino henoxy)]zinc
phthalocyaninate (compound II)
[0049] In nitrogen atmosphere, 55 g of 3-(3-N,N-dimethylamino
phenoxy)phthalonitrile (0.21 moles) are solubilised in 300 ml of
DMF. Subsequently 18.3 g of Zn(AcO).sub.2 (0.11 moles) and 150 ml
of DBU (1 mole) are added and the reaction mixture is then heated
to 130.degree. C. and maintained at this temperature, in the dark,
in nitrogen atmosphere and under vigorous stirring for 12 hours.
The reaction mixture is then cooled to 0.degree. C., treated with
1.81 of deionised H.sub.2O, and maintained under stirring at
0.degree. C. for half an hour, the suspension is then filtered,
washing the solid with H.sub.2O in portions (1.3 l total) and MeOH
(1.times.750 ml+1.times.180 ml).
[0050] The product is then subjected to chromatographic
purification on silica gel (mobile phase: CH.sub.2Cl.sub.2/DMF
50/1), followed by treatment of the purified solid with ethyl ether
(200 ml) for 1 hour, filtration and washing of the solid with ethyl
ether (2.times.25 ml).
[0051] The product thus purified is solubilised in 0.5 l of
CH.sub.2Cl.sub.2 and reprecipitated by adding 4 l of n-hexane.
After filtration, washing with n-hexane (2.times.1 l) and drying,
60.1 g of compound (II) are obtained.
[0052] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta.=9.22-8.69 (m,
4H), 8.17-7.89 (m, 4H), 7.75-7.37 (m, 4H), 7.20-6.40 (m, 16H),
3.02-2.80 (m, 24H) ppm.
[0053] ESI-MS: m/z 1117.4 [(M+H).sup.+] in positive mode
[0054] HPLC data (see FIG. 3a): [0055] isomers distribution:
B=13.9%; A=5.7%; C+D=80.4% [0056] related substances=0.8%.
ii) Chromatographic Purification of Compound (II) to Give
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylamino phenoxy)]zinc
phthalocyaninate "with Low Isomer B Content" (Compound III)
[0057] 25 g of [1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylamino
phenoxy)]zinc phthalocyaninate (II) are solubilised in 200 ml of an
eluent mixture X/Y 3/1 where X=DCM/THF/MeOH 94/5/1 and Y=n-hexane.
The solution is loaded on a chromatography column (diameter 100 mm,
height 460 mm) in silica gel preconditioned with X/Y 3/1. The
elution is performed at a flow of 130 ml/min by using as mobile
phase X/Y 3/1, during elution of the isomer B and until initial
elution of the isomers C/D, then with X/Y 4/1 during elution of the
isomers C/D and A. The fractions are controlled by TLC (SiO.sub.2,
X/Y 4/1): all the fractions in which the isomers C+D and/or the
isomer A are present are combined and concentrated to give 17.5 g
of [1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylamino
phenoxy)]zinc phthalocyaninate "with low isomer B content"
(III).
[0058] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta.=9.19-8.68 (m,
4H), 8.16-7.90 (m, 4H), 7.74-7.40 (m, 4H), 7.20-6.49 (m, 16H),
3.02-2.80 (m, 24H) ppm.
[0059] ESI-MS: m/z 1117.4 [(M+H).sup.+] in positive mode
[0060] HPLC Data (see FIG. 3b): [0061] isomers distribution:
B.ltoreq.0.1%; A=6.1%; C+D=93.9% [0062] related substances
0.1%.
iii) Synthesis of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate tetraiodide "with Low Isomer B
Content" (Compound IV)
[0063] 34 g (0.03 moles) of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylamino phenoxy)]zinc
phthalocyaninate obtained as described above at point ii) are
solubilised in 850 ml of NMP, to the solution 85 ml (1.4 moles) of
MeI are then added and the reaction mixture is maintained in the
dark, under stirring, in nitrogen atmosphere, at ambient
temperature for 96 hours.
[0064] The reaction mixture is diluted with 1.7 l of MeOH, then
treated with 6.8 l of ethyl ether, obtaining a suspension that is
left under stirring for half an hour and to rest for 1 hour, then
filtered; the solid is washed with ethyl ether (2.times.0.5 l),
obtaining 60 g of damp product (incalculable yield) which was found
to be [1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate tetraiodide "with low isomer B
content" (IV), characterized by mass spectrometry and NMR.
[0065] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta.=9.49-7.20 (m,
28H), 3.79-3.58 (m, 36H) ppm.
[0066] ESI-MS: m/z 294.1 [(M-4I).sup.4+] in positive mode.
iv) Treatment With Ion Exchange Resin that Leads to the Obtainment
of [1,8(11),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammonium
phenoxy)]zinc phthalocyaninate tetrachloride "with Low Isomer B
Content" (Compound I)
[0067] 60 g of compound (IV) are solubilised in 5.5 l of MeOH, and
the solution is passed through a chromatographic column, having as
stationary phase 500 g of Amberlite.RTM. IRA 400 (CI) resin,
previously washed with HCl 0.5 M acidic aqueous solution and
conditioned with MeOH. To the eluate (approximately 6 l),
maintained under stirring, ethyl ether (24 l) is slowly added, the
suspension obtained is left to rest for an hour and then filtered.
The solid is washed with ethyl ether (2.times.250 ml) and dried on
the filter for about one hour to give 36 g of compound (I)
.sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta.=9.45-7.23 (m, 28H),
3.77-3.58 (m, 36H) ppm.
[0068] .sup.13CNMR (75 MHz, DMSO-d.sub.6): .delta.=160.5, 160.4,
160.3, 157.9, 154.4, 154.1, 153.9, 153.5, 153.2, 153.1, 153.0,
152.7, 152.5, 151.9, 150.6, 150.5, 150.2, 149.6, 149.5, 149.1,
142.0, 141.9, 141.8, 132.1, 131.3, 129.4, 129.3, 129.1, 127.7,
127.5, 123.0, 122.8, 122.7, 120.7, 120.4, 118.6, 118.2, 118.0,
117.6, 116.5, 114.7, 113.4, 111.0, 57.3, 57.2 ppm.
[0069] UV-vis (DMF) .lamda..sub.max %: 690(100), 621(16), 391(17),
327(17).
[0070] UV-vis (H.sub.2O) .lamda..sub.max %: 691(100), 643(49),
331(45).
[0071] ESI-MS: m/z 294.1 [(M-4Cl).sup.4+] in positive mode
[0072] HRMS: m/z 294.1114 [(M-4Cl).sup.4+] in positive mode.
.DELTA.=1.08 ppm from the theoretical mass of
C.sub.68H.sub.64N.sub.12O.sub.4Zn
[0073] Isomers distribution: A=6.5%, B<0.2%, C=62.0%,
D=31.5%.
Example 2
[0074] Starting from a Batch of Compound (II) with the Following
Isomers Distribution:
[0075] A=8.6%, B=4.0% C+D=87.4%;
[0076] Performing the Chromatography Purification (Step ii)) as
Described:
[0077] 28.6 g of [1,8(11),15(18),22(25)-tetrakis-(3-N,N-di
methylaminophenoxy)]zinc phthalocyaninate (II) are dissolved in 192
ml of an eluent mixture X/Y 3/1 in which X=DCM/THF/MeOH 94/5/1 e
Y=n-hexane. The sample is loaded on a chromatographic column (d=100
mm, h=460 mm) packed with silica gel Davisil 60 (40-63 .mu.m)
conditioned with X/Y 3/1. The elution (Flow: 130 ml/min), is
performed with X/Y 3/1 until all isomer B is eluted, then ratio of
the mixture X/Y is carried to 4/1 and so kept for the elution of
isomers C, D and A. The selected fractions are collected and dried
to give 23.2 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=7.8%, B=0.3%
C+D=91.9%;
[0078] Performing Steps iii) and iv) on this Batch of Compound
(III):
[0079] a batch of compound (I) with the following isomers
distribution is obtained: A=8.2%, B=0.4%, C=65.5%, D=25.9%.
Example 3
[0080] Starting from a Batch of Compound (II) with the Following
Isomers Distribution:
[0081] A=7.9%, B=9.9% C+D=82.2%;
[0082] Performing the Chromatography Purification (Step ii)) as
Described:
[0083] 40.0 g of [1,8(11),15(18),22(25)-tetrakis-(3-N,N-di
methylaminophenoxy)]zinc phthalocyaninate (II) are dissolved in 192
ml of an eluent mixture X/Y 3/1 in which X=DCM/THF/MeOH 94/5/1 e
Y=n-hexane. The sample is loaded on a chromatographic column (d=100
mm, h=460 mm) packed with silica gel Davisil 60 (20-45 .mu.m)
conditioned with X/Y 3/1. The elution (Flow: 130 ml/min) is
performed with X/Y 3/1 until all isomer B is eluted, then ratio of
the mixture X/Y is carried to 4/1 and so kept for the elution of
isomers C, D and A. The selected fractions are collected and dried
to give 30.1 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=7.5%, B=0.3%
C+D=92.2%;
[0084] Performing Steps iii) and iv) on this Batch of Compound
(III):
[0085] a batch of compound (I) with the following isomers
distribution is obtained: A=7.8%, B=0.3%, C=64.9%, D=27.0%.
Example 4
[0086] Starting from a Batch of Compound (II) with the Following
Isomers Distribution:
[0087] A=7.7%, B=4.4% C+D=87.9%;
[0088] Performing the Chromatography Purification (Step ii)) as
Described:
[0089] 49.6 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate (II) are dissolved in 235 ml of an eluent mixture
X/Y 3/1 in which X=DCM/THF/MeOH 94/5/1 e Y=n-hexane. The sample is
loaded on a chromatographic column (d=100 mm, h=460 mm) packed with
silica gel Davisil 60 (20-45 .mu.m) conditioned with X/Y 3/1. The
elution (Flow: 130 ml/min) is performed with X/Y 3/1 until all
isomer B is eluted, then ratio of the mixture X/Y is carried to 4/1
and so kept for the elution of isomers C, D and A. The selected
fractions are collected and dried to give 38.2 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=7.6%, B=0.9%
C+D=91.5%;
[0090] Performing Steps iii) and iv) on this Batch of Compound
(III):
[0091] a batch of compound (I) with the following isomers
distribution is obtained: A=5.6%, B=0.9%, C=61.4%, D=32.1%.
Example 5
[0092] Starting from a Batch of Compound (II) with the Following
Isomers Distribution:
[0093] A=7.0%, B=4.6% C+D=88.4%;
[0094] Performing the Chromatography Purification (Step ii)) as
Described:
[0095] 32.0 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate (II) are dissolved in 288 ml of dichloromethane.
The sample is loaded on a chromatographic column (d=100 mm, h=460
mm) packed with silica gel Davisil 60 (40-63 .mu.m) and conditioned
with X/Y 3/1. The elution (Flow: 130 ml/min) is performed in
isocratic mode with mobile phase X/Y 3/1 The selected fractions are
collected and dried to give 30.1 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=6.2%, B<0.1%
C+D=93.8%;
[0096] Performing Steps iii) and iv) on this Batch of Compound
(III):
[0097] a batch of compound (I) with the following isomers
distribution is obtained: A=6.4%, B=0.2%, C=55.8%, D=37.6%.
Example 6
[0098] Starting from a Batch of Compound (II) with the Following
Isomers Distribution:
[0099] A=6.9%, B=7.8% C+D=85.3%;
[0100] Performing Three Chromatography Purifications (Step ii) a,
b, c) on the Same Stationary Phase, as Described:
[0101] a) 17.6 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate (II) are dissolved in 256 ml of dichloromethane.
The sample is loaded on a chromatographic column (d=100 mm, h=460
mm) packed with silica gel Davisil 60 (40-63 .mu.m) conditioned
with X/Y 3/1. The elution (Flow: 130 ml/min) is performed in
isocratic mode with mobile phase X/Y 3/1 The selected fractions are
collected and dried to give 13.1 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=5.1%, B<0.2%
C+D=94.9%;
[0102] b) After a washing of the column with 16 l of mobile phase
X/Y 3/1, the second run is performed: 14.4 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate (II) are dissolved in 256 ml of dichloromethane.
The sample is loaded on a chromatographic column (d=100 mm, h=460
mm) packed with silica gel Davisil 60 (40-63 .mu.m) and conditioned
with X/Y 3/1. The elution (Flow: 130 ml/min) is performed in
isocratic mode with mobile phase X/Y 3/1 The selected fractions are
collected and dried to give 10.2 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=3.5%, B=0.5%
C+D=96.0%;
[0103] c) After a washing of the column with 16 l of mobile phase
X/Y 3/1, the third run is performed: 11.2 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate (II) are dissolved in 256 ml of dichloromethane.
The sample is loaded on a chromatographic column (d=100 mm, h=460
mm) packed with silica gel Davisil 60 (40-63 .mu.m) and conditioned
with X/Y 3/1. The elution (Flow: 130 ml/min) is performed in
isocratic mode with mobile phase X/Y 3/1 The selected fractions are
collected and dried to give 7.9 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=3.5%, B=0.5%
C+D=96.0%;
[0104] Performing Steps iii) and iv) on the Batches of Compound
(iii) Obtained from Step ii a, b, c
[0105] three batches compound (I) with the following isomers
distribution are obtained:
[0106] a) A=4.8%, B<0.2% C=56.9%, D=38.2%;
[0107] b) A=3.2%, B=0.4%, C=57.2%, D=39.2%;
[0108] c) A=3.3%, B=0.5%, C=56.8%, D=39.4%.
Example 7
[0109] Starting from a Batch of Compound (II) with the Following
Isomers Distribution:
[0110] A=6.7%, B=6.5%, C+D=86.8%;
[0111] Performing the Chromatography Purification (Step ii)) as
Described:
[0112] 1.8 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate (II) are dissolved in 12 ml of an eluent mixture
X/Y 3/1 in which X=DCM/THF/MeOH 94/5/1 e Y=n-hexane. The sample is
loaded on a chromatographic pre-packed column (Biotage SNAP KP-Sil
Cartridge, silica gel 100 g, mesh 40-63 .mu.m) pre-conditioned with
X/Y 3/1. The elution (Flow: 40 ml/min) is performed with X/Y 3/1
until all isomer B is eluted, then ratio of the mixture X/Y is
carried to 4/1 and so kept for the elution of isomers C, D and A.
The selected fractions are collected and dried to give 1.4 g of
[1,8(11),15(18),22(25)-tetrakis-(3-N,N-dimethylaminophenoxy)]zinc
phthalocyaninate "with low isomer B content" (III) with the
following isomers distribution are obtained: A=7.4%, B=0.4%,
C+D=92.2%;
[0113] Performing Steps iii) and iv) on this Batch of Compound
(III):
[0114] a batch of compound (I) with the following isomers
distribution is obtained: A=7.6%, B=0.5%, C=62.6%, D=29.3%.
Example 8
Experimental Proof of the Difficulty of Preparing Solutions of
(II), Due to the Low Solubility of the Isomer B
[0115] Two samples of the same batch of intermediate (II) are
solubilised (2 mg/ml) in, DMF and THF/MeOH 1/1. The solutions are
appropriately diluted and analysed in HPLC. As shown in Table 1,
and in FIG. 4, the two samples have a different percentage of
isomer B, which is much lower in the sample coming from the
solution in THF/MeOH 1/1, This is due to the precipitation of
isomer B from the THF/MeOH 1/1 solution, as a consequence of the
lower solubility of the isomer B in comparison to the other
isomers. This behaviour can lead to the preparation of samples
having variable composition and, ultimately, to the production of
not reliable analytical data including the ones concerning the
other isomers
TABLE-US-00001 TABLE 1 HPLC analysis of the isomer distribution of
two samples of the same batch of compound (II) Compound (II)
Compound (II) from stock from stock solution solution 2 mg/ml in
DMF 2 mg/ml in THF/MeOH (1:1) % isomers An. N.. 0315/09 An. N.
0315/09 % A A: 5.9% A: 6.7% % C + D C + D: 79.2% C + D: 86.7% % B
B: 14.9% B: 6.6%
Example 9
Demonstration of Non Resolution Between the HPLC Peaks of t Isomers
B and C of Compound (VI)
[0116] FIG. 5 compares the HPLC chromatograms relative to the
isomers distribution of a sample of compound (VI) (5a) and of a
sample of compound (I) (5b). It is evident that the resolution
between the peaks relative to the isomers B and C is not complete;
when the presence of isomer B is beyond 1%, a non-negligible error
is introduced in the quantification of the isomers abundances,
making the method described unsuitable for the characterization of
batches of a drug substance. Conversely the problem is non-existent
if a batch of compound (I) is analysed, as the very small quantity
of isomer B generates a negligible estimation error of the isomers
A, C and D ratio.
[0117] The compound of formula (I), prepared as described, can be
used as a drug substance in the preparation of pharmaceutical
compositions, included those intended to be used for photodynamic
therapy, comprising, in combination with the aforesaid drug
substance, pharmaceutically acceptable excipients and/or diluents.
These pharmaceutical compositions can be intended to be used for
both systemic and topical administration to humans and animals and
can be in solution, suspension, cream, ointment, gel or spray form.
Controlled release administration of the composition is also
possible. These pharmaceutical compositions can also contain a
metal chelating agent, with preference for metal chelating agents
having specificity for the ions Ca.sup.2+ and Mg.sup.2+.
[0118] In a particularly preferred condition, said metal chelating
agent is selected in the group consisting of
ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA),
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) and
diethylene triamine pentaacetic acid (DTPA).
[0119] Due to the non specific (multi-target) mechanism of action,
the aforesaid pharmaceutical compositions, containing the
phthalocyanine derivative of formula (I) can be intended to be used
for photodynamic treatment of various pathologies (microbial
infections, hyperproliferative diseases, psoriasis, actinic
keratosis and pre-tumour or tumour pathologies). In a particularly
preferred condition, the pharmaceutical compositions containing the
phthalocyanine derivative of formula (I) are intended to be used
for the preventive or curative photodynamic treatment of microbial
infections.
[0120] Finally, the derivative of formula (I) can also be used as
component of medical devices, in particular in the disinfection of
wounds, as sterilisation agent, also ex vivo, and as in vivo
diagnostic agent.
* * * * *