Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2002-05-20
2004-08-17
Berch, Mark L (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C540S145000, C514S410000
Reexamination Certificate
active
06777402
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the chemistry of biologically active compounds, namely, to a new method to prepare water-soluble porphyrin derivatives, particularly chlorin, bacteriochlorin, pheophorbide and bacteriopheophorbide derivatives of types 1 and 2. The compounds of the present invention can be used as photosensitizers for the photodynamic therapy of cancer, infections and other diseases as well as for light irradiation treatments in other cases.
Wherein B is a ring having the structure:
Wherein:
R
1
═—CH═CH
2
, —CH(OAlk)CH
3
, —CHO, —C(O)CH
3
, —CH
2
CH
3
, —CH(Alk)CH(COAlk)
2
, —CH
2
CH(COAlk)
2
, —CH(Alk)CH
2
COAlk, —CH(Alk)CH
2
CH(OH)CH
3
, and —CH
2
CH
2
CH(OH)CH
3
R
2
═—CH
3
, —CHO, —CH(OH)Alk, —CH═CHAlk, CH
2
OH, and CH
2
OAlk;
R
3
═—OH, —OAlk, —NH-Alk, NH—X—COO
−
(HG)
+
, —NH—Y—NR
8
R
9
, -and NH—Y—OH;
R
4
═—OAlk, —NH-Alk, and NH—X—COO
−
(HG)
+
;
R
5
═—OAlk, —NH-Alk, and NH—X—COO
−
(HG)
+
;
R
6
═H and —COOAlk;
R
7
═—O
−
(HG)
+
, —OAlk, —NH-Alk, and —NH—X—COO
−
(HG)
+
;
R
8
═H and Alk
R
9
═H and Alk
Wherein:
—NH—X—COO
−
=the residue of organic amino acid;
X=alkylidene, peptides, oligopeptides and —(CH
2
CH
2
O)
n
CH
2
CH
2
—, wherein n=1-30;
Y=alkylidene and —(CH
2
CH
2
O)
n
CH
2
CH
2
—, wherein n=1-30;
G=a hydrophilic organic amine (f.ex. N-methyl-D-glucamine and other amino-group containing carbohydrate derivatives, TRIS, amino acids, oligopeptides); and
Alk=an alkyl substituent.
BACKGROUND OF THE INVENTION
Photodynamic therapy (PDT) is one of the most promising new techniques now being explored for use in a variety of medical applications (Photodynamic therapy, basic principles and clinical applications. Eds. B. W. Henderson, Th. J. Dougherty, Marcel Dekker, 1992, New York), and particularly is a well-recognized treatment for the destruction of tumors (Photodynamic tumor therapy. 2
nd
and 3
rd
generation photosensitizers. Ed. J. G. Moser, Harwood Academic Publishers, 1998, Amsterdam). Porphyrins are compounds widely used in PDT. A major problem in the pharmaceutical application of porphyrins is their low solubility in physiological solutions. This renders it nearly impossible to prepare effective pharmaceutical grade injectable solutions for PDT and other applications.
Methods to prepare water soluble porphyrin derivatives for PDT are known in the art. U.S. Pat. No. 5,330,741 by Smith et al discloses a method to prepare trisodium lysyl-chlorin p
6
involving the reaction between purpurin 18 methyl ester, resulting from methyl pheophorbide a transformation, and aqueous lysine in methylene chloride in the presence of pyridine. The mixture is stirred at room temperature for 12 hours, followed by the removal of the solvents in a high vacuum. The so prepared crude product is purified by reversed-phase HPLC and subsequently lyophilized. To prepare an injectable solution for the PDT of cancer, the preparation is first dissolved in phosphate buffer solution and then 0.1 N sodium hydroxide is added The pH value of the solution is adjusted to pH 7.35 using 0.1 N HCl followed by sterility filtration through a microporous filter.
Drawbacks of the above mentioned method include a lack of reproducibility and difficulty in the work-up and utilization of toxic reagents, which make it hardly appropriate for pharmaceutical manufacturing. Additionally, the prepared water soluble product of interest is stable in an aqueous solution for only 24 hours at 4° C. in the dark, and in solid form for up to 4 months at 4° C. in the dark [M. W. Leach, R. J. Higgins, J. E. Boggan, S.-J. Lee, S. Autry, K. M. Smith, Effectiveness of a Lysylchlorin p
6
/Chlorin p
6
mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat. Cancer Res., 1992, 52, 1235-1239; U.S. Pat. No. 5,330,741].
There is a method to prepare a water-soluble sodium salt of pheophorbide a (3), described in U.S. Pat. No. 5,378,835 by Nakazato. According to this invention, pheophorbide a (4) is dissolved in diethyl ether, and a very diluted solution of alkali in n-propanol, iso-propanol or in their mixture is added dropwise and very slowly to the solution. The reaction is maintained up to the complete precipitation of pheophorbide a salt, which is separated by centrifugation and dried in vacuo. Then the product is dissolved in water resulting in a solution with concentration 0.5% and pH 9.2-9.5 that is then diluted with a phosphate buffer with pH 7.4-7.8.
The drawback of the method described by Nakazato is the fact that a concentrated (>1%) injectable pheophorbide a solution in water can not be generated by this technique. Additionally, the authors of the present invention demonstrated the chemical instability of such salts when stored dry, and their incomplete ability to dissolve in water after having been stored in the dry state.
The closest analogue to the present invention is the method disclosed in Russian Patent No. RU2144538 by G. V. Ponomarev et al to prepare water-soluble complexes of chlorin e
6
(7) with spacious organic amines including N-methyl-D-glucosamine by a multi-step straightforward sequence of chemical reactions including preparation of chlorophyll a from
Spirulina Platensis
cyanobacteria biomass, further conversion into chlorin e
6
according to standard procedures [S. Lötjönen, P. H. Hynninen, An improved method for the preparation of (10R)- and (10S)-pheophytins a and b.
Synthesis.
1983, 705-708; P. H. Hynninen, S. Lötjönen, Preparation of phorbin derivatives from chlorophyll mixture utilizing the principle of selective hydrolysis.
Synthesis.
1980, 539-541; S. Lötjönen, P. H. Hynninen, A convenient method for the preparation of wet chlorin e
6
and rhodin g
7
trimethyl esters.
Synthesis,
1980, 541-543] with an overall yield exceeding 50% after precipitation of chlorin e
6
by way of stepwise addition of water to its acetone solution, followed by separation by centrifugation and 3-fold washing with water and subsequent treatment of wet chlorin e
6
with water solution of 2 g-eq. spacious organic amine.
The key disadvantages of this method, which cause critical difficulties for preparative syntheses of water-soluble chlorins and particularly for industrial syntheses and drug manufacturing, are the following:
1. Chlorin e
6
as an intermediate product is obtained as a wet mass with unknown definite content of chlorin e
6
. This instability of the amount of chlorin e6 obtained creates uncertainties that undermine the ability to standardize further resultant solutions.
2. The key intermediate in the synthetic sequence is pheophorbide a (4) which is difficult to handle for purification and standardization due to its acidic properties. Separation of pheophorbide a (4) via repeatable precipitations (as used by Ponomarev) is not quantitative and thus not convenient for large scale preparations.
3. Pheophorbide a (4) obtained by the indicated method contains impurities that are difficult to separate. This disadvantage causes uncertainty in the quantification of pheophorbide a (4) and disturbs the chemical opening of cyclopentanon ring in the course of transformation of pheophorbide a (4) into chlorins.
4. It should be noted that the samples of water soluble salts of chlorin e
6
being prepared according to Ponomarev contain a variety of impurities of non-porphyrin and porphyrin types which could not be separated from the target chlorin e
6
product with the use of the procedures described therein. Particularly, among the porphyrin impurities one could note by using TLC and HPLC methods are pheophorbide a (4), purpurin 18 (8), chlorin p6 (9) and some other concomitants.
It could be noted that the compounds of types (4), (8) and (9) as salts with hydrophilic amines of the above invention are characterized by remarkably lower water solubility as compared with their respective chlorin e
6
salts. Nevertheless in the presence of chlorin e
6
salts the compounds of types (4), (8) and (9
Nifantiev Nikolay E.
Yashunsky Dmitri V.
Berch Mark L
BJ Associates
CeramOptec Industries Inc.
Habte Kahsay
Ryan Thomas J.
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