Water soluble anionic bacteriochlorophyll derivatives and their uses

Abstract

The invention provides anionic water-soluble tetracyclic and pentacyclic bacteriochlorophyll derivatives (Bchls) containing at least one, preferably two or three, negatively charged groups and/or acidic groups that are converted to negatively charged groups at the physiological pH, preferably Bchls having a group COO<−>, COS<−>, SO3<−>, PO3<2−>, COOH, COSH, SO3H, and/or PO3H2 bound through an ester or amide bond to one or more of the positions 17<3>, 13<3>, and 3<2> of the tetracyclic or pentacyclic Bchl molecule, for photodynamic therapy and diagnosis.

1 . A bacteriochlorophyll derivative containing at least one, preferably two or three, negatively charged groups and/or acidic groups that are converted to negatively charged groups at the physiological pH, excluding pentacyclic bacteriochlorophyll derivatives having a free CH 2 CH 2 COOH or a CH 2 CH 2 COO −  group at position 17, and tetracyclic bacteriochlorophyll derivatives devoid of a central metal atom and having a —CH 2 CH 2 COOH group at position 17, a —CH 2 COOH or —COOH group at position 15, a —COOH group at position 13, methyl groups at the positions 2, 7, 12, 18, and ethyl groups at the positions 3 and 8. 
     
     
         2 . A bacteriochlorophyll derivative according to  claim 1  containing two negatively charged groups. 
     
     
         3 . A bacteriochlorophyll derivative according to  claim 1  containing three negatively charged groups. 
     
     
         4 . A bacteriochlorophyll derivative according to any one of  claims 1  to  3  wherein said negatively charged groups are selected from the group consisting of COO − , COS − , SO 3   − , and/or PO 3   2− . 
     
     
         5 . A bacteriochlorophyll derivative according to  claim 1  wherein said acidic groups that are converted to negatively charged groups at the physiological pH are selected from the group consisting of COOH, COSH, SO 3 H, and/or PO 3 H 2 . 
     
     
         6 . A bacteriochlorophyll derivative according to any one of  claims 1  to  5  derived from a natural or synthetic derivative of bacteriochlorophyll, including compounds in which the central Mg atom has been deleted or replaced by other metal atoms. 
     
     
         7 . A bacteriochlorophyll derivative according to  claim 1  of the formula I or II: 
       
         
           
           
               
               
           
         
       
       wherein
 M represents 2H or a metal atom selected from divalent Pd, Pt, Co, Sn, Ni, Cu, Zn and Mn, and trivalent Fe, Mn and Cr; 
 R 1 , R 2 , and R 4  each independently is Y—R 5 ; 
 Y is O, S or NR 5 R 6 ; 
 R 3  is selected from —CH═CH 2 , —C(═O)—CH 3 , —C(═O)—H, —CH═NR 7 , —C(CH 3 )═NR 7 , —CH 2 —OR 7 , —CH 2 —SR 7 , —CH(CH 3 )—OR 7 , —CH(CH 3 )—SR 7 , —CH(CH 3 )—NR 7 R′ 7 , —CH(CH 3 )Hal, —CH 2 -Hal, —CH═CR 7 R′ 7 , —C(CH 3 )═CR 7 R′ 7 , —CH═CR 7 Hal, —C(CH 3 )=CR 7 Hal, and —C≡CR 7 ; 
 R 5 , R 6 , R 7  and R′ 7  each independently is H or selected from the group consisting of: 
 (a) C 1 -C 25  hydrocarbyl optionally containing one or more heteroatoms, carbocyclic or heterocyclic moieties, and/or optionally substituted by one or more functional groups selected from the group consisting of halogen, oxo, OH, SH, CHO, NH 2 , CONH 2 , a negatively charged group, and an acidic group that is converted to a negatively charged group at the physiological pH; 
 (b) a residue of an amino acid, a peptide or of a protein; and 
 (c) when Y is O or S, R 5  may further be R 8   + ; 
 m is 0 or 1; and 
 R 8   +  is H +  or a cation; 
 provided that: 
 (i) at least one, preferably two, of R 5 , R 6 , R 7  and R′ 7  is a hydrocarbon chain as defined in (a) above substituted by a negatively charged group or by an acidic group that is converted to a negatively charged group at the physiological pH; or 
 (ii) at least one, preferably two, of R 1 , R 2 , and R 4  is OH, SH, O − R 8   +  or S − R 8   + ; or 
 (iii) at least one of R 1 , R 2 , and R 4  is OH, SH, O − R 8   +  or S − R 8   +  and at least one of R 5 , R 6 , R 7  and R′ 7  is a hydrocarbon chain substituted by a negatively charged group or by an acidic group that is converted to a negatively charged group at the physiological pH; or 
 (iv) at least one of R 1 , R 2 , and R 4  is OH, SH, O − R 8   +  or S − R 8   +  and at least one of R 5 , R 6 , R 7  and R′ 7  is a residue of an amino acid, a peptide or of a protein; or 
 (v) at least one of R 5 , R 6 , R 7  and R′ 7  is a hydrocarbon chain substituted by a negatively charged group or by an acidic group that is converted to a negatively charged group at the physiological pH and at least one of R 5 , R 6 , R 7  and R′ 7  is a residue of an amino acid, a peptide or of a protein; 
 but excluding the compounds of formula I wherein M is as defined, R 3  is —C(═O)CH 3 , R 1  is OH or OR 8   +  and R 2  is —OCH 3 , and the compound of formula II wherein M is 2H, R 3  is —C(═O)CH 3 , R 1 , R 2  and R 4  are OH, and m is 0 or 1. 
 
     
     
         8 . A bacteriochlorophyll derivative of the formula I or II according to  claim 7  wherein said negatively charged groups are selected from the group consisting of COO − , cos − , SO 3   − , and/or PO 3   2− . 
     
     
         9 . A bacteriochlorophyll derivative of the formula I or II according to  claim 7  wherein said acidic groups that are converted to negatively charged groups at the physiological pH are selected from the group consisting of COOH, COSH, SO 3 H, and/or PO 3 H 2 . 
     
     
         10 . A bacteriochlorophyll derivative of the formula I or II according to  claim 7  wherein R 1  is Y—R 5 ; Y is O, S or NH; and R 5  is a hydrocarbon chain substituted by functional groups selected from OH, SH, SO 3 H, NH 2 , CONH 2 , COOH, COSH, PO 3 H 2 . 
     
     
         11 . A bacteriochlorophyll derivative of the formula I or II according to  claim 7  wherein R 5  is the residue of an amino acid, a peptide or a protein. 
     
     
         12 . A bacteriochlorophyll derivative of the formula I or II according to  claim 1  containing a central Pd metal atom. 
     
     
         13 . A bacteriochlorophyll derivative of the formula I according to  claim 7  wherein:
 M is Pd; 
 R 1  is —NH—(CH 2 ) n —SO 3   − R 8   + , —NH—(CH 2 ) n —COO − R 8   + ; —NH—(CH 2 ) n —PO 3   2− (R 8   + ) 2 ; 
 R 2  is methoxy; 
 R 3  is —C(═O)—CH 3 ; 
 R 8   +  is a monovalent cation such as K + , Na + , Li + , NH 4   + ; and 
 n is an integer from 1 to 10, preferably 2 or 3. 
 
     
     
         14 . A bacteriochlorophyll derivative of the formula II according to  claim 7  wherein:
 M represents 2H, divalent Pd, Cu, or Zn or trivalent Mn; 
 R 1  is —O − R 8   + , —NH—(CH 2 ) n —SO 3   − R 8   + , —NH—(CH 2 ) n —COOR 8   + ; —NH—(CH 2 ) n —PO 3   2− (R 8   + ) 2 ; or Y—R 5  wherein Y is O, S or NH and R 5  is the residue of an amino acid, a peptide or a protein; 
 R 2  is C 1 -C 6  alkoxy such as methoxy, ethoxy, propoxy, butoxy, more preferably methoxy; 
 R 3  is —C(═O)—CH 3 , —CH═N—(CH 2 ) n —SO 3   −R   8   + ; —CH═N—(CH 2 ) n —COO −   8   + ; —CH═N—(CH 2 ) n —PO 3   2− (R 8   + ) 2 ; —CH 2 —NH—(CH 2 ) n —SO 3   − R 8   + ; —NH—(CH 2 ) n —COO − R 8   + ; or —NH—(CH 2 ) n —PO 3   2− (R 8   + ) 2 ; 
 R 4  is —NH—(CH 2 ) n —SO 3   − R 8   + ; —NH—(CH 2 ) n —COO − R 8   + ; —NH—(CH 2 ) n —PO 3   2− (R 8   + ) 2 ; 
 R 8   +  is a monovalent cation such as K + , Na + , Li + , NH 4   + , more preferably K + ; and 
 m is 1, and n is an integer from 1 to 10, preferably 2 or 3. 
 
     
     
         15 . A bacteriochlorophyll derivative of formula II in  claim 7  wherein:
 M is divalent Pd; 
 R 1  is —O − R 8   + , —NH—(CH 2 ) n —SO 3   − R 8   + , or Y—R 5  wherein Y is O, S or NH and R 5  is the residue of an amino acid, a peptide or a protein; 
 R 2  is C 1 -C 6  alkoxy, preferably methoxy; 
 R 3  is —C(═O)—CH 3 , —CH═N—(CH 2 ) n —SO 3   − R 8   + ; or —CH 2 —NH—(CH 2 ) n —SO 3   − R 8   + ; 
 R 4  is —NH—(CH 2 ) n —SO 3   −  R 8   + ; NH—(CH 2 ) n —COO − R 8   + ; NH—(CH 2 ) n —PO 3   2− (R 8   + ) 2 ; 
 R 8   +  is a monovalent cation, preferably K + ; 
 m is 1, and n is 2 or 3. 
 
     
     
         16 . A bacteriochlorophyll derivative of the formula I according to  claim 13 , consisting of the compound Palladium bacteriopheophorbide a 17 3 -(3-sulfopropyl)amide potassium salt. 
     
     
         17 . A bacteriochlorophyll derivative of the formula II according to  claim 15 , consisting of the compounds:
 Palladium 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt;   3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt;   Palladium 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 ,17 3 -di(3-sulfopropyl)amide dipotassium salt;   Palladium 3 1 -(3-sulfopropylimino)-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 ,17 3 -di(3-sulfopropyl)amide tripotassium salt;   Copper(II) 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt;   Zinc 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt;   Manganese(III) 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl)amide dipotassium salt;   Palladium 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide, 17 3 -(N-immunoglobulin G) amide potassium salt;   Palladium 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-carboxy-ethyl)amide dipotassium salt;   Palladium 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(3-phosphopropyl)amide tripotassium salt; and   Palladium 3 1 -(3-sulfopropylamino)-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 ,17 3 -di(3-sulfopropyl)amide tripotassium salt.   
     
     
         18 . Palladium 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt. 
     
     
         19 . A pharmaceutical composition comprising a bacteriochlorophyll derivative according to any one of  claims 1  to  18 , and a pharmaceutically acceptable carrier. 
     
     
         20 . The pharmaceutical composition according to  claim 19  for photodynamic therapy. 
     
     
         21 . The pharmaceutical composition according to  claim 20  for vascular-targeting photodynamic therapy. 
     
     
         22 . The pharmaceutical composition according to  claim 20  or  21  for photodynamic therapy of tumors, including metastatic tumors. 
     
     
         23 . The pharmaceutical composition according to  claim 22  for photodynamic therapy of melanoma, colon, breast, lung, or prostate cancer. 
     
     
         24 . The pharmaceutical composition according to  claim 20  or  21  for photodynamic therapy of age-related macular degeneration. 
     
     
         25 . The pharmaceutical composition according to  claim 20  or  21  for photodynamic therapy of benign prostate hypertrophy. 
     
     
         26 . The pharmaceutical composition according to  claim 19  for tumor diagnosis. 
     
     
         27 . A pharmaceutical composition according to  claim 19  for killing cells or infectious agents comprising bacteria and viruses. 
     
     
         28 . The pharmaceutical composition according to  claim 27  for in vitro killing of cells or infectious agents comprising bacteria and viruses in a biological product upon illumination of said product. 
     
     
         29 . The pharmaceutical composition according to  claim 28  wherein said biological product is blood. 
     
     
         30 . Use of a compound according to any one of  claims 1  to  18  for the manufacture of a pharmaceutical composition for use in photodynamic therapy. 
     
     
         31 . The use according to  claim 30  for photodynamic therapy of tumors, including metastatic tumors. 
     
     
         32 . The use according to  claim 31  for photodynamic therapy of melanoma, colon, breast, lung, or prostate cancer. 
     
     
         33 . The use according to  claim 30  for photodynamic therapy of age-related macular degeneration. 
     
     
         34 . Use of a compound according to any one of  claims 1  to  18  for the manufacture of a pharmaceutical composition for diagnosis of tumors. 
     
     
         35 . Use of a compound according to any one of  claims 1  to  18  for the manufacture of a pharmaceutical composition for killing cells or infectious agents comprising bacteria and viruses. 
     
     
         36 . A method for tumor photodynamic therapy which comprises:
 (a) administering to an individual in need a compound according to any one of  claims 1  to  18 ; and   (b) irradiating the local of the tumor.   
     
     
         37 . A method for photodynamic therapy of age-related macular degeneration which comprises: (a) administering to an individual in need a compound according to any one of  claims 1  to  18 ; and (b) irradiating the local of the macular degeneration. 
     
     
         38 . A method for tumor diagnosis which comprises:
 (a) administering to a subject suspected of having a tumor, a compound according to any one of  claims 1  to  18 ; and   (b) irradiating the subject by standard procedures and measuring the fluorescence of the suspected area, wherein a higher fluorescence indicates tumor sites.   
     
     
         39 . In a method for photodynamic therapy using a photosensitizer, the improvement wherein said photosensitizer is a bacteriochlorophyll derivative according to any one of  claims 1  to  18 . 
     
     
         40 . In a method for diagnosis of tumors using a photosensitizer, the improvement wherein said photosensitizer is a bacteriochlorophyll derivative according to any one of  claims 1  to  18 . 
     
     
         41 . In an in vitro method for killing of cells or infectious agents comprising bacteria and viruses, using a photosensitizer, the improvement wherein said photosensitizer is a bacteriochlorophyll derivative according to any one of  claims 1  to  18 . 
     
     
         42 . The compound Palladium bacteriopheophorbide a 17 3 -(3-sulfo-1-oxysuccinimide) ester sodium salt, as an intermediate. 
     
     
         43 . A method for the preparation of compounds of formula II In  claim 7  wherein R 1  is —O − R 8   + ; R 2  is OCH 3 ; R 3  is acetyl; R 4  is a group —NH—(CH 2 ) n —SO 3   − R 8   + ; R 8   +  is a monovalent cation; m is 1 and n is 1 to 10, which comprises:
 (i) reacting the corresponding M-bacteriopheophorbide of formula I wherein R 1  is OH with an aminosulfonic acid of the formula H 2 N—(CH 2 ) n —SO 3 H in a R 8   + -buffer; and 
 (ii) isolating the desired compound of formula II. 
 
     
     
         44 . The method according to  claim 43  for preparation of palladium 3 1 -oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 1 -(2-sulfoethyl) amide dipotassium salt which comprises: (i) reacting Pd-bacteriopheophorbide a with taurine of the formula H 2 N—(CH 2 ) 2 —SO 3 H in a K + -buffer; and (ii) isolating the title compound. 
     
     
         45 . A method for the preparation of compounds of formula II In  claim 7  wherein R 1  is —O − R 8   + ; R 2  is —OCH 3 ; R 3  is acetyl; R 4  is a group —NH—(CH 1   2 ) n -COO −  R 8   + ; R 8   +  is a monovalent cation; m is 1 and n is 1 to 10, which comprises:
 (i) reacting the corresponding M-bacteriopheophorbide of formula I wherein R 1  is OH with an aminocarboxylic acid of the formula H 2 N—(CH 2 ) n —COOH in a R 8   + -buffer; and 
 (ii) isolating the desired compound of formula II. 
 
     
     
         46 . A method for the preparation of compounds of formula II in  claim 7  wherein R 1  is —O − R 8   + ; R 2  is —OCH 3 ; R 3  is acetyl; R 4  is a group —NH—(CH 2 ) n —PO 3   2− (R 8   + ) 2 ; R 8   +  is a monovalent cation; m is 1 and n is 1 to 10, which comprises:
 (i) reacting the corresponding M-bacteriopheophorbide of formula I wherein R 1  is OH with an aminophosphonic acid of the formula H 2 N—(CH 2 ) n —PO 3 H 2  in a R 8 -buffer; and 
 (ii) isolating the desired compound of formula II. 
 
     
     
         47 . A method for the preparation of compounds of formula II in  claim 7  wherein R 1  and R 4  contain the same negatively charged group, which comprises:
 (i) reacting the corresponding M-bacteriopheophorbide with an excess of the aminosulfonic, aminocarboxylic or aminophosphonic acid in a R 8   + -buffer; and 
 (ii) isolating the desired 13,17-disubstituted derivative of formula II. 
 
     
     
         48 . A method for the preparation of compounds of formula II in  claim 7  wherein R 1  and R 4  are each a group —NH—(CH 2 ) n —SO 3   − R 8   + ; R 2  is —OCH 3 ; R 3  is acetyl; R 8   +  is a monovalent cation; m is 1 and n is 1 to 10, which comprises:
 (i) coupling the corresponding M-bacteriopheophorbide of formula I wherein R 1  is OH with N-hydroxy-sulfosuccinimide (sulfa NHS) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC); 
 (ii) reacting the resulting M-bacteriopheophorbide-17 3 -N-hydroxy-sulfosuccinimide ester with an excess of an aminosulfonic acid of the formula H 2 N—(CH 2 ) n —SO 3 H in a R 8   + -buffer, thus obtaining a compound of formula I having a sole negatively charged group at position 17; 
 (iii) reacting the product of step (ii) with an excess of H 2 N—(CH 2 ) n —SO 3 H in a R 8   + -buffer; and 
 (iv) isolating the desired compound of formula II. 
 
     
     
         49 . A method for the preparation of compounds of formula II in  claim 7  wherein R 1  and R 4  are each a group —NH—(CH 2 ) n —COO − R 8   + ; R 2  is —OCH 3 ; R 3  is acetyl; R 8   +  is a monovalent cation; m is 1 and n is 1 to 10, which comprises:
 (i) coupling the corresponding M-bacteriopheophorbide of formula I wherein R 1  is OH with N-hydroxy-sulfosuccinimide (sulfo NHS) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC); 
 (ii) reacting the resulting M-bacteriopheophorbide-17 3 -N-hydroxy-sulfosuccinimide ester with an excess of an aminocarboxylic acid of the formula H 2 N—(CH 2 ) n —COOH in a R 8   + -buffer, thus obtaining a compound of formula I having a sole negatively charged group at position 17; 
 (iii) reacting the product of step (ii) with an excess of H 2 N—(CH 2 ) n —COOH in a R 8   + -buffer; and (iv) isolating the desired compound of formula U. 
 
     
     
         50 . A method for the preparation of compounds of formula II in  claim 7  wherein R 1  and R 4  are each a group —NH—(CH 2 ) n —PO 3   2− R 8   + ; R 2  is —OCH 3 ; R 3  is acetyl; R 8   +  is a monovalent cation; m is 1 and n is 1 to 10, which comprises:
 (i) coupling the corresponding M-bacteriopheophorbide of formula I wherein R 1  is OH with N-hydroxy-sulfosuccinimide (sulfo NHS) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC); 
 (ii) reacting the resulting M-bacteriopheophorbide-17 3 -N-hydroxy-sulfosuccinimide ester with an excess of an aminophosphonic acid of the formula H 2 N—(CH 2 ) n -PO 3 H 2  in a R 8   + -buffer, thus obtaining a compound of formula I having a sole negatively charged group at position 17; 
 (iii) reacting the product of step (ii) with an excess of H 2 N—(CH 2 ) n —PO 3 H 2  in a R 8   + -buffer; and (iv) isolating the desired compound of formula II.