CA2188029C - Porphocyanine and cnc-expanded porphyrins - Google Patents

Abstract

Compounds of formula (1) and the metalated forms and salts thereof; wherein n is an integer of 1-4; and wherein each Pi is independently a pyrrole residue of formula (II), wherein each Ria and Rib is independently a noninterfering substituent, and wherein each Zi is independently a covalent bond; or is a meso bridging group of formula (III); or is an N-meso bridging group of formula (IV); or is a CC linkage of formula (V) or is a CNCCNC linkage of formula (VI), wherein each Ric, Rid, Rie, Rif and Rig is independently a noninterfering substituent; or is a CNC linkage of formula (VII), wherein at least one Zi is said CNC linkage, are disclosed. These compounds are useful in photodynamic therapy and diagnosis. The metalated forms when the metal is paramagnetic are useful as MRI contrast agents.

Description

=

PORPHOCYANINE AND CNC-EXPANDED PORPHYRINS

Field of the Invention The invention relates to CNC-expanded porphyrins, such as the porphocyanines, to methods for their preparation and the use of these compounds to mediate the detection or destruction of target cells or tissues by light irradiation. In addition, the invention relates to the use of the compounds of the invention in radioimaging and magnetic resonance imaging methods.

Background Art Although considerable effort has been devoted to the synthesis and study of porphyrins and other tetrapyrrolic macrocycles, much less is known about the larger aromatic pyrrole-containing systems, the so-called "expanded porphyrins."
Such systems, by virtue of containing a greater number of 7 electrons, additional coordinating heteroatoms and a larger central binding core, may offer advantages over the porphyrins.
Many of the expanded porphyrin systems contain more than 4 pyrrole rings. The synthesis of saphyrin from tripyrrane dicarboxylic acid and bipyrroledicarboxaldehyde to yield a pentapyrrole compound was reported several decades ago by Woodward, R.B., Aromaticity Conference, Scheffield, England, 1966. See also, Broadhurst et al., J Chem Soc Perkins Trans (1972) 1:2111 and Bauer et al. J Am Chem Soc (1983) jQ5:6429).
The synthesis of smaragdyrin from bipyrroledicarboxaldehyde and pyrroledipyrromethane dicarboxylic acid was reported in 1970 by M.M. King. (Ph.D. Dissertation, Harvard University, Cambridge, MA) The uranyl complex of superphthalocyanine is another pentapyrrolic macrocyclic compound of historical importance.

'WO 95/29180 PCT/CA95/00242 218802 =

..This compound was prepared by direct template condensation of dicyanobenzene with uranyl dichloride, however, thefree base is -unstable (Day et al. J Am Chem Soc..(1975) ,97:4519).
Demetalation resulted in contraction of the ring to form phthalocyanine (Marks, T.J. and D.R. Stojakovic J Am Chem Soc (1978) 100:1695).
Gossauer synthesized the first hexaphyrin by condensing a bis-a-tripyrrane with_a tripyrrane dialdehyde, followed by oxidation (Bull Soc Chim Bela (1983) 21:793). Of the six methene bridges present in the hexaphyrin, two have E
configuration (Id.). Charriere reported that hexaphyrin forms bimetallic complexes with several transition metals (1987, Thesis, University de Fribourg, Suisse). Another hexapyrrolic system, rubyrin, has been recently synthesized and structurally characterized (Sessler et al. Angew Chem Int Ed Enal (1991a) 977) .
Vinylogous porphyrins or platyrins are another important class of pyrrole-containing macrocycles first described by R.A. Berger and E.LeGoff (Tetra Lett (1978) 4~:4225; see also, LeGoff, E. and O.G. Weaver J Org Chem (1977) .za:711; and Franck et al. Proc SPIE Int Soc OAt Eng, Ser 5 (1988) 997:107). These compounds are generally synthesized by reacting a dipyrromethane with an acrylaldehyde-substituted dipyrromethane (Beckman et al. Anaew Chem Int Ed Enal (1990) 22:1395). Bisvinylogous expanded porphyrins were further expanded to tetravinylogous porphyrins in which all four of the meso bridges are enlarged. Tetravinylogous porphyrins are made by an acid-catalyzed self-condensation of the N-protected, pyrrole-substituted allyl alcohol. Tetravinylogous porphyrins have a very intense Soret-like band shift of more than 150 nm from that of the normal porphyrins (Gosmann, M. and B. Franck Angew Chem Int Ed Enal (1986) 2.1L:1100; Kntilbel, G. and B. Franck Angew Chem Int Ed Enal (1988) 2.Z:1170). -In addition, the synthesis ofbisvinylogous porphycene has recently been reported (Jux et al. Angew Chem Int Ed Enal (1990) 2a:1385; Vogel et al.
Anaew Chem Int Ed Enal (1990) 212:1387).
Schiff-base compounds represented by the texaphyrins comprise another c].ass of pyrrole containing macrocycles WO 95/29180 PCTlCA95/00242 = ~ YS

(Sessler et al. J Ora Chem (1987) U:4394; Sessler et al. J A
Chem Soc (1988) },LQ:5586); Sessler, J.L. et al. Acc Chem Res (1994) 27:43-50._ Texaphyrins are synthesized by acid-catalyzed condensation of tripyrrane dialdehyde with o-phenylenediamine.
Several texaphyrins have been prepared using similar strategies (Sessler et al. (1991) Abstract of the 201st Natl Soc Mtg, Inorganic Division; Sessler et al. Inorcr Chem (1992) 2&:529).
The use of porphyrins, combined with irradiation, for the detection and treatment of malignant cells has,.by this time, some considerable history. (See, e.g., Porphyrin Photosensitization (Kessel, D. et al., eds. Plenum Press, 1983).
Certain porphyrins seem "naturally" capable of localizing malignant cells. When irradiated, porphyrins have two properties which make them useful. First, when irradiated with ultraviolet or visible light, they may fluoresce, and thus be useful in diagnostic methods related to detection of malignancy (see, for example, Kessel et al., supra; Gregory, H.B. Jr. et al., Ann Surg (1968) ~&7:827-829).
In addition, when irradiated with light, certain porphyrins exhibit a cytotoxic effect on the cells in which they are localized (see, for example, Diamond, I. et al., Lancet (1972) 2:1175-1177; Dougherty, T.J. et al., Cancer Research (1978) U:2628-2635; Dougherty, T.J. et al., The Science of Photo Medicine 625-638 (J.D. Regan & J.A. Parrish, eds., 1982);
Dougherty, T.J. et al., Cancer: Principles and Practice of Oncolocav 1836-1844 (V.T. DeVita Jr. et al., eds., 1982).
Certain of the expanded porphyrins such as sapphyrin, texaphyrin and vinylogous porphyrins possess unique long-wavelength and singlet oxygen producing properties which also make them attractive as potential photosensitizers for use in tumor phototherapy (Maiya et al. J Phys Chem (1990) ~4:3597; Sessler et al. SPIE Soc (1991) 1426:318; Franck et al., supra).
Porphyrins, such as hematoporphyrin, monohydrobenzoporphyrin derivatives (BPDs) and porfimer sodium have been conjugated to immunoglobulins specific for targeted cells to refine their ability to home to the desired cells or tissue.

' WO 95/29180 PCT/CA95/00242 21880~9 One problem ancillary to the practice of photodynamic therapy is that the wavelength for_irradiation required to activate certain porphyrins is in the range of 630 nm, which wavelength is also readily absorbed by other porphyrins and.
natural chromophores normally present in the blood and other tissues. Therefore, the depth of the effective treatment has been limited to a few millimeters because of blocking effects of light-absorbing natural chromophores such as hemoglobin. When greater penetration is desired it is desirable to administer compounds to mediate the effects of irradiationwhich can be excited at longer wavelengths such_as the BPDs thus avoiding the blocking effects of natural chromophores present throughout the subject organism. - - -In addition to phototherapy, expanded porphyrins are useful in magnetic resonance imaging (MRI). MRI is a noninvasive, nonionizing method that allows normal and abnormal tissue to be observed and recognized at the early stages of development. At this time MRI has a significant drawback, in that the degree of signal enhancement for diseased versus normal tissues is often insufficient to allow this method to be used in many clinical situations. To overcome this problem, contrast reagents for MRI have been developed. Paramagnetic metal complexes, such as those derived from gadolinium(III) (Gd) have recently proven particularly efficient in clinical trials.
To date, the coordination of gadolinium in MRI
contrast agents has been achieved using carboxylate-type ligands including porphyrins, but more successfully with texaphrins.
(See, for example, Lauffer, R.B. Chem Rev (1987)_a2:901;
Kornguth et al. J Neurosurg (1987) EJ;898; Koenig et al. Invest Radiol (1986) 21:697; Chacheris et al. Inora Chem (1987) JJ;958;
Loncin et al. Inora Chem (1986) J.~:2646; Chang, C.A. and V.C.
Sekhar Inora Chem (1987) ,2i5:1981)..-.Sessler et al. reported that texaphyrin forms an extremely stable Gd(III) complex in vitro (Sessler et al. inora Chem (1989) 2&:3390). Gd(III) does no-t form stable complexes with normal porphyrins. In addition to Gd(III), texaphyrin has been reported to form complexes with a variety of transition metals such as Cd and-Eu (Sessler, J.L.
and A.K. Burrell Top Cur Chem (1992) 161:177). _ Disclosure of the Invention The invention provides novel light-absorbing compounds suitable for use in detecting and/or treating target tissues, cells and pathogens. The compounds of the invention can be utilized in photodynamic therapy and in diagnosis in a manner analogous to that in which the porphyrins, phthalocyanines, BPDs, and related compounds can be used. If desired, the invention compounds may be administered in relatively low dosage due to their ability to absorb radiation in an energy range outside of that normally absorbed by the components present in high concentration in blood or other tissues, in particular the porphyrin residues normally associated with hemoglobin and myoglobin. This is advantageous when penetration of tissues by light is required. In some instances, where superficial treatment is sufficient, shorter wavelengths can be used.
These compounds are preferentially retained in target tissues and cells as compared to nontarget tissues and cells, though this property may not be necessary for effective treatment. See, e.g., WO 94/06424 and WO 94/12239.

Thus, in one aspect, the invention is directed to CNC-expanded porphyrins of the formula P;-Z;- (P;-Z;) õ-Pi-Zj I I (1) wherein n=1-4;
wherein each P; is independently a pyrrole residue of the formula R~a R b P ia ib N/
H

WO 95129180 PCTlCA95/00242 wherein each Rr, and R. is independently a noninterfering substituent, and wherein each Z; is independently a covalent bond; or is a meso bridging group of the formula Ic I I c_ -, ,- ' -, -C- 4- -C= or is an N-meso bridging group of the formula -N- H -N=; or is a CC linkage of the formula iid re id i e -C= C- ~-~ =,C- C.=
or is a CNCCNC linkage of the formula Rir R;9 Rir R;9 I I I I
- C H=N-C= C-N=CH-.--- =C H-N=C- C=N-CH=
wherein each _R;., Rid, R;,, R;f and R;. is independently a noninterfering substituent; or is a CNC linkage of the formula -CH=N-CH ~-~ CH-NCH
wherein at least one Z; is said CNC linkage.
In the compounds of the invention at least one Z; is said CNC linkage. The compounds ofthe invention also include ~ ~e,[f241U~e1 the metalated forms and the salts of the compound of formula (1) .
The prototype of this group of "CNC-expanded porphyrins" is porphocyanine, as described below.
In other aspects, the invention is directed to pharmaceutical and diagnostic compositions containing the compounds of the invention, to conjugates wherein the invention compounds are covalently linked with specific targeting agents, such as immunoglobulins, and to the use of these compounds or conjugates in photodynamic therapy and diagnosis. In another aspect, the invention is directed to forms of the invention compounds suitable for use as radioimaging and magnetic resonance imaging contrast agents, as well as methods of using these. Another aspect relates to synthesis of the invention compounds.

Brief Description of the Drawings Figure 1 shows the emission spectrum of octaethylporphocyanine in the free-base form.
Figure 2 shows the emission spectrum of octaethylporphocyanine in the acid-addition-salt form.
Figure 3 shows some preferred embodiments of the invention compounds.

Modes of Carrying Out the Invention The compounds of the invention, designated herein "CNC-expanded porphyrins", contain at least 3 pyrrole nuclei linked through covalent bonds, or through optionally substituted conventional meso linkages found natively in porphyrins, or through N-meso linkages, or through optionally substituted CC
linkages as found in the phorphacenes or through optionally substituted CNCCNC linkages analogous to the o-phenylene diamine linkages found in the texaphins, or by CNC linkages, but wherein at least one linkage is a CNC linkageof the formula -CH=N-CH= ~ - =CH-N=CH-The members of one specific class belonging to this group are designated porphocyanines. These are represented by the formula -Rlb R2a Rla I - N R2b N HN =
R4c R2c 1 ( a ) NH N_ Rib / , N R3a R4n P3b The various R substituents are those which do not interfere with the basic porphocyanine nucleus in its ability to absorb light of appropriate wavelengths and to achieve at least one desired effect, such as mediating the destruction of target tissues and-cells in a photodynamic therapy context, serving as an MRI contrast agent, and the like. The nature of-suitable R
substituents which will not interfere is further discussed below. However, it can be stated that a wide variety of substituents may be employed without interfering with the usefulness of these compounds. The appropriate choice of these noninterfering substituents will depend on the specific intended use. For example, if intended for in vivo use, the substituents must be nontoxic. When employed in vitro, especially if the possibility for removal of the compounds of the invention from ,the appropriately treated material is-available, this may not be of concern. Those or ordinary skill will understand the parameters that constrain the choice of each R;.
In addition to the compounds of formula (la), other CNC-expanded porphyrins areincluded in the scope of the invention. In one preferred embodiment, where n=2, formula (1) can be represented by P1 -Zi Pz wherein each P; and Z;
is as above defined.

Illustrative of these -CNC- compounds where n=2 are the compounds P

of formulas (1b)-(1e):

R b R2a Rla N R2b NH N-' N N 1(b) HN
R 1b \ / N~ 1135 R4e R3b Rlb R2e Rqa N' R2b -N HN
R4c \ 1(c) NH HN
R4b N R3a R4a R3b Rlb R2e Rta N' \ \ Rzb NH N
R4C R2c 1(d) NH HN
Rqb R3a R4a R3b ,. i 2188029 _io R1b Rza -Rla / ~ ~ N \ \ Rzb N HN
R4~ 1 \ zc: e)--or, generically, NH N_ -/ ~ ~ ~ ~ ~3a R,p Rv R4b Ru ~ N~Ra R4a R3b NH R3t Z Zi 1(f) R. ~JN N1~ Ry Rõ Z Re As seen from these illustrative formulas, at least one bridge between the four pyrrole nuclei must be a CNC linkage.
Otherwise, the linkage may be a covalent bond or the conventional meso single-atom methine linkage optionally substituted by Rk, or the other linkages described above. The resultant arrangement of a-bonds in the CNC porphyrins of the invention will be dependent on the choice of linkage, as is understood by those of ordinary skill in organic chemistry. For example, replacing an odd number of meso linkages with CNC
converts an aromatic system to a nonaromatic, albeit stable one;
replacement of an even number retains aromaticity.
Additional embodiments include those wherein n=1:
R ib R ia Ria ",~ , N Rib NH N 1C5) Z Z~
~ N

Rib Ria W O 95/29180 PCTlCA95100242 or wherein n=3:

Rib Ria Ria 1 \ Inl \ Rib NH N-Z Z 1(h) N
Rib ~ Z Z- o Rm \i/ N\/ i Ra '(~/ Rib Rm/}~(\Rla or wherein n=4:

Rib Ra R,a \ N Rib NH N-ZI Z;
Rw~l I Rm ~ ( )N NQ 1 ( J ) R'//n~~Z. Z rRib Riy CJN Nl~ Ru Z, Rin Rie Particularly preferred are those embodiments shown in Figure 3.
With respect to the substituents R;,, R;b and R;,, R;d, R;c, R;f and R;Y in general, each R; is independently H, halo, nitro, cyano, NR'21 SR', OR', SOR', SOzR', COOR', CONR'2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or optionally substituted arylalkyl. The substituent R' denotes hydrogen or alkyl (1-6C). Amino, sulfhydryl and hydroxyl substituents may also be acylated (1-6C). Suitable substituents for said optional substitutions include those named above, i.e., halo, nitro, cyano, NR'2, SR', OR', and the like. Of course, aromatic nuclei may also be substituted by alkyl, alkenyl or alkynyl 15, groups.
As used herein, alkyl, alkenyl and alkynyl are defined conventionally as hydrocarbyl substituents containing 1-6C, are either saturated or unsaturated, and are straight-chain, branched or cyclic moieties. Thus, alkyl would include methyl, tertiary butyl, cyclohexyl, n-hexyl and the like; alkenyl would include these carbon backbones with one or more double bonds;
and alkynyl would include similar carbon frameworks with one or more triple bonds. Arylalkyl (5-18C) refers to an aryl substituent linked to the CNC-expanded porphyrin nucleus through an alkylene group, where alkylene is defined in a manner corresponding to the definition of alkyl (1-6C).
By "aryl" is meant an aromatic moiety of 4-12C, optionally containing one or more heteroatoms. Thus, suitable aryl groups include phenyl, naphthyl, pyridyl, pyrimidyl, quinolyl, and the like.
Where the substituents contain carboxylic acid or amino substitutions, the relevant salts are also included within the scope of the invention compounds.
Salts of -COOH can be derived from inorganic or organic bases, including pharmaceutically acceptable nontoxic inorganic and organic bases. Suitable inorganic bases include sodium, potassium, lithium, ammonium, calcium, and magnesium, hydroxides, and the like. Particularly preferred are the potassium and sodium salts. Pharmaceutically acceptable organic nontoxic bases include primary, secondary, tertiary and quaternary amines including cyclic amines, and basic ion-exchange resins. Examples include isopropylamine, trimethylamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, glucosamine, theobromine, purines, piperazine, polyamine resins, and the like.
Any amino groups contained in the substituents may also be present in the form of their acid-addition salts. In addition, the CNC-expanded porphyrin nucleus itself may be present in the form of an acid-addition salt. These salts are formed from inorganic acids such as hydrochloric, sulfuric and phosphoric acid or with organic acids such as acetic, oxalic, benzoic acid and the like.
Conversion of the invention compounds from free acid or free base to the corresponding salt forms and vice versa are conducted by methods-well understood in the art. --Preferred embodiments of R;, and R;b include hydrogen, optionally substituted alkyl, optionally substituted aryl and optionally substituted arylalkyl. A particularly preferred substituent is carboxyl. Most preferred are hydrogen and alkyl.
In addition, while each R;, and Rib is independently determined, symmetries in the choice of these substituents facilitate the preparation of the compounds since the number of components in the resulting product mixture is reduced. While chromatographic separation techniques are adequate to permit an arbitrary number of components to be separated from the product mixture, yields are increased when the number of possible products is reduced.
Thus, particularly preferred are compounds of the invention wherein all R;, are the same and all R;y are the same, especially where all R. and R;y are the same. Another preferred embodiment is that wherein in the compound of formula 1(a), R1õ Rlb, Ra, and R4b are the same and wherein R2,, R2b, R3i and R3b are the same as each other although different from Rlõ Rib, R4õ and R46.
Particularly preferred embodiments of R;,-R4 include H
and optionally substituted aromatic (aryl) substituents.
Particularly preferred embodiments of R;, are H and optionally substituted phenyl.
Illustrative preferred embodiments of the invention compounds wherein n=1 are:
1. Zl is CNC, Z2 is meso wherein Rk is H, Z3 is a covalent bond, Rla and RZy are H, and Rlb, R2õ R3a and R3b are methyl;
2. Zt is CNC, Z2 is meso wherein R2. is phenyl, Z3 is a covalent bond, Rla, R2õ R3a and R3b are (CH2) 2COOH, Rlb is methyl and R2, is H;
3. Zl is CNC, Z2 is CNCCNC wherein R2f and RZ8 are H, Z3 is CC wherein R3d and R3, are H, and wherein Rl, and R2b are H, and Rly, RZõ R3a and R3b are methyl;
4. Z, is CNC, Z2 is CNCCNC wherein RZf and RZY are phenyl, Z3 is CC wherein R3d and R3e are phenyl, Rla, R2õ R3a and R3b are (CH2)ZCOOH, Rly is methyl and R2b is H;
5. Zt is CNC, Z2 is CNCCNC wherein R2, is phenyl and RZ$ is H, and Z3 a CC linkage wherein R3d is phenyl and R3e is H and wherein R!õ Rza and R3, are phenyl and Rly, R2b and R3b are H.

~~QtOC~~!~ . _ =
~. .

Preferred embodiments wherein n=2 are:
1. Z1 is CNC, ZZ is N-meso, Z3 is meso wherein R3c is methyl, and Z4 is CNC and wherein Ri, and R2b are H and Rib, Ra,, R3a R3b, Ra: and R4b are methyl;
2. Zl is CNC, Z2 is N-meso, Z3 is meso wherein R3. is phenyl and Z4 is CNC and wherein Riõ R2õ R3a R3b and Rq, are (CH2) 2COOH and Rlb, R2, and R4b are H;
3. Z1 is CNC, ZZ is N-meso, Z3 is meso wherein R3c is H, and Z4 is CNC and wherein R,õ R2, R3a and R4, are phenyl and Rlb, R2y, R3b and R4b are ethyl;
4. Zl ia CNC, ZZ ia CNCCNC wherein R2f and RZ. are both phenyl, Z3 is a CC linkage wherein R3d and R3e are H, and Z4 is a covalent bond and wherein Rl, and R2y are H and Rtb, R21, R3õ R3b, P41 and R4b are methyl;
5. Z1 is CNC, Z2 is CNCCNC wherein R2f and R2. are H, or Z3 is a CC linkage wherein R3d ia ethyl and R3e is H, and Z4 is meso wherein Rk is ethyl and wherein Rlõ R2õ R3õ R3b and R4a are (CH2)2COOH and Rib, R2y and R4b are H;

6. Zl is CNC, Z2 is CNCCNC wherein R2f is ethyl and RZj is H, Z3 is meso wherein R3, is phenyl and Z4 is a CC linkage wherein R3d and R3e are methyl and wherein Rlõ RZõ R3a and R4, are phenyl and Rlb, R2b, R3b and R4b are ethyl.
In instances where n=3, preferred compounds are:
1. ZI is CNC, Z2 is meso wherein R2. is methyl, Z3 is meso wherein R3c is methyl, Z4 is a covalent bond and ZS is CNC
and wherein Rlõ Raõ R3a R4n and R5a are methyl and Rib, R2b, R3b+ R4b and R5b are H;
2. Zl is CNC, Z2 is N-meso, Z3 is meso wherein R3. is phenyl, Z4 is CNC and ZS is CC wherein Rsd and R5e are phenyl, and wherein Rlõ Rlb, R3õ R3b, Rs, and R5b are (CHa)2COOH and R7õ R2b, R4a and R4b are methyl;
3. ZI is CNC, Z2 is N-meso, Z3 is meso wherein R3c is H, Z4 is N-meso, and ZS is CNC and wherein Rlõ R3a and R5, are phenyl, Rlb, R3b and R5b are H and R2õ R2b, Ra, and R4b are methyl;
4. Z1 is CNC, Z2 is meso wherein Rk is methyl, Z3 is CNCCNC wherein R3f and R3y are phenyl, Z4 is N-meso, Zs is a CC
linkage wherein R3d and R3e are H, and wherein-Rl, and R2b are H and Rib, Rv. R3a, R3b. Rc, and R4b are ethyl; - - - --WO 95/29180 FCTlCA95100242 Gi~FA(Z.~(,el - 15 -5. Zl is CNC, Z2 is CNCCNC wherein R2f and R2y are H, Z3 is a CC linkage wherein R3d is ethyl and R3c is H, Z4 is meso .wherein R. is ethyl and Z5 is a covalent bond, and wherein RIa, R?õ R3a R3b and R4, are (CH2)ZCOOH and Rib, R2bõ R46, R5, and R5b are H;
6. ZI is CNC, Z. is CNCCNC wherein R2f is ethyl and RZg is H, Z. is meso wherein R3, is phenyl, Z4 is a CC linkage wherein R4d and Rk are methyl and Z5 is CNC, and wherein R1a R29 R3a, R4, and R5i are methyl and Rlb, R2b. R3b' R4b and R5b are ethyl;

7. Z1 is CNC, Z2 is a CC linkage wherein R2d and R2.
are phenyl, Z3 is CNCCNC wherein R3f and R3g are H, Z4 is N-meso and Z5 is meso wherein-Rs, is methyl and wherein Rlõ Rlb, R3a, R3b, Rs, and Rs, are (CH2)2COOH and R2õ R2b, R4a and R4b are methyl;

8. Z1 is CNC, Z2 is a covalent bond, Z3 is meso wherein R3. is phenyl, Z4 is CNC and Z5 is a CC linkage wherein R5d and RS, are methyl and wherein Rlõ R3a and Rs, are phenyl, Rlb, R3b and Rsb are H and Rza R2b, R4n and R4b are methyl.
For compounds wherein n=4, preferred embodiments include: --1. Z1 and Z6 are CNC, Z2 and ZS are CC linkages where all of R2d, RZ,, Rsd and R5, are phenyl, and Z3 and Z4 are meso where R3. and Rk are ethyl, and wherein RIõ Rtõ R3a, R4õ Rs, and R& are i-propyl and Rlbl R2b, R3b' Rab. R5, and Rs, are (CH2)2COOH;
2. ZI is CNC, Z2 and Z4 are N-meso, Z3 and Z5 are covalent bonds and Z6 is meso wherein R& is H and Rlõ R2õ R3aI R4n1 Rs, and Rf, are phenyl and Rlb, R2b' R3b' RebI Rsb and R6b are methyl.
The possibility of obtaining CNC-expanded porphyrins with a multiplicity of substituents has the advantage that the properties of the_core CNC-expanded porphyrin nucleus can be modified by the choice of such substituents. For example, solubility of the compounds can be enhanced by employing substituents with polar groups. By altering the symmetry of the arrangement of these groups, the compounds can be made ampiphilic, i.e., the compound will have a patterned distribution of + and - charges. This property is helpful in solving problems of biodistribution -- membrane transition and the like. In addition, the light-absorbing qualities of the core nucleus can be modified to some extent by conjugated n-bonds in the substituents giving the usual auxochromic shift.

16 - ~
Metalation/Addition of Radio Isotopes The CNC-expanded porphyrin compounds of the present invention include those where the CNC-expanded porphyrin itself is coupled with a radioisotope for radioimaging (scintigraphic imaging) or with certain metals for use as ions a magnetic resonance.image contrast agent, or simply as a convenient form of the compound. Examples of radioisotopes which would be_ useful labels include Iodine-123, Iodine-131, Technetium-99m, Indium-111 and gallium-67. Examples of metals which would be appropriate as MRI contrast agents include paramagnetic ions of elements such as Gd, Mn, Eu, Dy, Pr, Pa, Cr, Co, Fe, Cu, Ni, Ti, and V, preferably Gd and-Mn.

Coniu4ates of the Invention ComAounds For use in some contexts, it is helpful to link the compounds of the invention to a target-specific moiety such as an immunoglobulin or a ligand for a receptor. The ability of the invention compounds to home todiseased tissues and cells such as tumors, if desired, can be enhanced by coupling the compound to a moiety that specifically binds epitopes or receptors located on the surface of such target tissues or cells.
Thus, target-specific moieties within the present invention include ligands such as steroids, such asestrogen and testosterone and derivatives thereof, peptides comprising ligands for T cell receptors, saccharides, such as mannose for which monocytes and macrophages have receptors, and H2 agonists.
Target-specific moieties may also be immunospecific components. The TSM may be derived from polyclonal or monoclonal antibody preparations and may contain whole -antibodies or immunologically reactive fraginents of these antibodies such as (F(ab')2, Fab, or Fab' fragments. Use of such immunologically reactive fragments.as substitutes for whole antibodies is well known in the art. See, for example, Spiegelberg, H.L., Immunoassavs in the Clinical Laboratorv (1978) 1:1-23.
Polyclonal antisera are prepared in conventional ways by injecting a suitable mammal with antigen to which antibody is WO 95/29180 PCTlCA95l00242 ~ 2188029 _ 17 _ desired, assaying the antibody level in serum against the antigen, and preparing antisera when the titers are high.
Monoclonal antibody preparations may also be prepared conventionally such as by the method of Koehler and Milstein using peripheral blood lymphocytes or-spleen cells from immunized animals and immortalizing these cells either by viral infection, by fusion with myelomas, or_by other conventional procedures, and screening for production of the desired antibodies by isolated colonies. Formation of the fragments from either monoclonal or polyclonal preparations is effected by conventional means as described by Spiegelberg, H.L., supra.
Particularly useful antibodies exemplified herein include the monoclonal antibody preparation CAMAL-1 which can be prepared as described by Malcolm et al. (Ex Hematol (1984) la:539-547); polyclonal or monoclonal preparations of anti-M1 antibody as described by Mew et al. J Immunol (1983) 13Q:1473-1477 and B16G antibody which is prepared as described by Maier et al. J Immunol (1983) 111:1843 and Steele et aI. Cell Immunol (1984) 2Q:303. -The foregoing list is exemplary and not limiting; once the target tissue is known, antibody specific for this tissue may be prepared by conventional means. Therefore, the invention is applicable to effecting toxicity against any desired target.
Coupling of the target-specific moiety to a CNC-expanded porphyrin of the present invention can be effected by any convenient means known in the art, depending on the nature of the substituents on the CNC-expanded porphyrin moieties. For example, if at least one R; contains a carboxylic group, a covalent bond to an amino-containing TSM may be effected using a dehydrating agent such as a carbodiimide. A particularly preferred method of covalently binding a CNC-expanded porphyrin to the target-specific moiety is treatment with i-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) in the presence of a reaction medium consisting essentially of dimethylsulfoxide (DMSO). A preparation using this preferred procedure is illustrated below. Other dehydrating agents such as dicyclohexylcarbodiimide or diethylcarbodiimide could also be ' F; ..

used as well as conventional aqueous and partially aqueous media.
The active moieties of the conjugate may also be coupled through linker compounds which are bifunctional, and are capable of covalently binding each of the two active components.
A large variety of these linkers is commercially available, and a typical listwouldinclude those found, for example, in the catalog of the Pierce Chemical Co., Rockford, IL. These linkers are either homo- or heterobifunctional moieties and include functionalities capable of forming disulfides, amides, hydrazones, and a wide variety of other linkages. Other linkers include polymers such as polyamines, polyethers, polyamine alcohols, derivatized to the components by means of ketones, acids, aldehydes, isocyanates, or a variety of other groups.
The techniques employed in coupling the active moieties of the conjugate include any conventional means and the method for coupling does not form part of the invention.

Use of the Invention ComAounds in PDT and Other Applicatio*+A
The invention compounds can be used in photodynamic therapy protocols analogous to those known for porfimer sodium and the BPDs. Thus, the compounds can be used to destroy or impair the functioning of unwanted--cells or tissues or to inactivate pathogens such as bacteria and viruses.
Examples of target cells=and tissues for which PDT is useful include, but are not limited to, tumors, including blood tumors, malignant bone marrow, virally-infected blood cells or bone marrow, dysplastic cells or tissues, sites of inflammation or infection, hyperproliferative tissue such aspsoriatic plaque or papillomavirus lesions (warts) or neointimal hyperplasia lesions, hypervascularization such-as portwine stains and hemangiomas, atherosclerotic plaque, hair follicles, free viruses, bacteria, protozoa or other pathogenic-parasites.
Pathogens that may be adversely affected by PDT
include certain viruses, bacteria, protozoa and other pathogenic parasites. Viruses, for example, include enveloped viruses such as human cytomegaloviruses, Epstein-Barr virus, Marek's disease herpes virus, human herpes simplex-viruses, varicella-zoster WO 95/29180 PCTlCA95100242 virus, members of the family Poxviridae, members of the family Hepadnaviridae such as human hepatitis A virus (HAV), human hepatitis B virus (HBV) and non-A, non-B hepatitis viruses, including human hepatitis C virus, members of the family Orthomyxoviridae such as influenza virus types A, B and C, members of the family Retroviridae such as human T cell leukemia viruses, human immunodeficiency viruses, and members of the family Flaviviridae such as tick-borne encephalitis virus or yellow fever virus.
Illustrative parasites include Plasmodium malariae, P.
falciparum, P. ovale, P. vivax and Trypanosoma cruzi. Bacteria include Bacillus subtilis, Streptococcus faecalis, Pseudomonas spp õ Mycobacterium spp. and other opportunistic organisms treatable by photodynamic activation.
The manner of use of-the invention compounds in these contexts follows procedures that are established in the art.
The choice of wavelength absorption for the CNC-expanded porphyrin used in a particular technique may be influenced by the-nature of the use. For example, where the target tissue is in a context with a number of interfering materials such as those found in the blood, it is advantageous to use CNC-expanded porphyrins which have wavelength absorptions not shared with these contaminants. On the other hand, for treatment of superficial target tissues, this consideration is not as relevant. Thus, the presence or absence of alternative compounds capable of absorbing light in the environment to be irradiated with light is taken into consideration in choosing an appropriate member of the class of CNC-expanded porphyrins of the invention and in the choice of wavelength.
In addition to their use in photodynamic therapy, the compounds of the invention may be used for diagnosis in a manner analogous to that for porphyrins per se. The CNC-expanded porphyrins, like the porphyrins themselves, are capable of fluorescence when excited by light of an appropriate wavelength.
As the CNC-expanded porphyrins, like the porphyrins themselves, will home to certain target tissues, such as atherosclerotic plaques, tumors, and the like. If appropriate, the invention compounds can be provided with target-specific moieties to =

assure this homing". The compounds of the invention may thus be allowed to accumulate in such target tissues or cells if present, and these tissues or cells may be detected or localized .
by detecting the fluorescence.
In addition, the compounds of the invention may include radioisotopes either as covalently bonded substituents or in the metalated complexes so that the invention compounds can be located by radio- or scintigraphic imaging.
Certain_metal ions also provide the compounds of the invention with the ability to serve as MRI contrast agents.
These contrast agents are used conventionally in obtaining MRI
scans with enhanced readability.

Preparation of the Invention Comzaounds A number of strategies may be employed to effectthe CNC linkage of the invention.- A single CNC linkage may be formed, for example, by reacting a pyrrole substituted in the 2-position with a carboxylic acid moiety with a pyrrole substituted in the 2-position with aminomethyl. This general method for forming the CNC linkage is shown in Reaction Scheme 1.

SNH H HN
Hz0 \ ~ N ~ \
NH N- - -- -Reaction Scheme 1_ WO 95/29150 PCTlCA95100242 ~ 2188029 As shown, conventional amide formation is followed by dehydration to form the CNC linkage of the invention. The CNC
linkage is stabilized by its inclusion in the resonance system of the expanded porphyrin nucleus.
An additional approach is illustrated by Reaction Scheme 2 whicheffects the linkage by condensation of two cyano groups, each linked to the 2-position of an adjoining pyrrole.

R. R, R; R; Li. AIHq Ri P.
R;
R, tHN/
NHZCH2 /H' /H GHzNHz NC (2) CN
(3) z Ri Ri Ri N Ri N HN
NH N

R N Ri Ri (1) Ri Reaction Scheme 2 As shown in Reaction Scheme 2, the cyano-substituted dipyrrole in this case is first reduced with lithium aluminum hydride and then treated with oxygen to form the resultant linkage. The reduced compound (3) is partly ionized and the ionized form combines with the compound of formula (3) in the presence of 02 to give the product (1).
An alternative to Reaction Scheme 2 is shown in Reaction Scheme 3. Instead of treating the lithium aluminum hydride-reduced dicyano compound with oxygen; DDQ in methylene chloride and TI-IF are used.

u. : .

R R R R -L iaiH, Ri R
NHzCHz /N /N\ C-H2NHZ NC N1 N CN
H (3) H H (2) H
Ri . . RI

Ri N R~

N HN
NH N
Ri N/ R

Ri Ri Reaction Scheme 3 It will be apparent from Reaction Scheme 3 that rather than starting with the bisnitrile, the starting materials could also comprise the bisaminomethyl-derived dipyrromethane shown as formula (3). This starting material is especially advantageously employed in obtaining asymmetric forms of the.
porphocyanines of formula (la). This is illustrated in Reaction Scheme 3' which illustrates the condensation of two different "halves" of the porphocyanine nucleus to obtain the desired asymmetric product. Of course, the two symmetric products are also formed, but these are readily separated from the desired asymmetric product using standard chromatographic techniques.

WO 95/29180 PCTlCA95100242 R, (X)~ N Ri RRi Ri Ri N HN \ -= I 1 DDOICHZCIZITHF
H2N N N NHt t ~\ -il -H H H2NH2C NH HN CHZNH2 (X), NH N_ N
Ri Reaction Scheme 3' In another variant of Reaction Scheme 3', rather than condensing two diaminomethyldipyrromethanes as shown, the bisaldehyde (which can be obtained as an intermediate in Reaction Scheme 5 below) is added. This facilitates the production of asymmetric forms of the porphocyanines in which the nature of Rtõ Rtb, R4a and R4b (as well as Ri,) can be different from Ry, R2õ R3a R3b and R3c. Thus, the substituents on the compound of formula (3) shown in Reaction Scheme 3 can be different from those on the dialdehyde and a single asymmetric product is obtained.
In the alternative, a dialdehyde form of the dipyrrole intermediate can be condensed directly using ammonia in ethanol and water as shown in Reaction Scheme 4.
R
R[it N R
RI Ri RI --R N HN
H H NH31Et0 (6) - NH N_ RN R R

Reaction Scheme 4 The dipyrrole moiety containing two cyano groups for condensation in Reaction Schemes 2 or 3, or the dialdehyde for condensation in Reaction Scheme 4 can be obtained as shown in Reaction Scheme 5.

R.~N~ R ) - -- ,- ---8z0 -- ~ CHzOAc (6) 1(e' Ri Ri- R, Ri R~ R~ R R;
1. 1H
Bz0 N OB 0 H H' 0 0 HH 0 o 1. H2NOH, NaOAc + CH20 2.AcZo R R R R
NC N N CN
H (2) H
Reaction Scheme 5 As shown, the dipyrrole is formed by the condensation of two molecules of 2-benzoyl carbonyl-4-acetoxylmethyl pyrrole.-The resulting dipyrrole is reducedto the corresponding dialdehyde and converted to the cyano derivative using standard -reagents. -Finally, Reaction Scheme 6 shows an additional 1D alternative for obtaining the dicyano intermediate typified by formula (2). The dicyano compound can then be converted to the appropriate porphocyanine using the approaches set forth above in Reaction Schemes 2 or 3.

WO 95/29180 PCTfCA95100242 X X
Ri / NH Y Y Y Y
2 - ~ ~ ---- I
Z Z Z Z
CHO R R R
R
NH HN

Xi Y Y' ~ \
Z Z~
R~ R~
R \ / R
\ NH HN / ~
Reaction Scheme 6 NC CN
In the initial step of Reaction Scheme 6, benz-aldehyde optionally substituted with 1-5 independently selected substituents is used to form the meso bridge between the two condensed pyrrole nuclei. However, other aldehydes can also be used, including formaldehyde and simple alkyl aldehydes such as acetaldehyde, butyraldehyde, hexylaldehyde, and the like. In addition, other aryl aldehydes, such as 1-naphthylaldehyde, can also be used. In the second step, chlorosulfonyl isocyanate (C1S02N=C=O; CSI) in a polar aprotic solvent, such as DMF, derivatizes the pyrrole nuclei so as to contain the desired cyano groups in the 5-positions. The specific components shown in Reaction Scheme 6 are intended to be illustrative, not limiting.
Concerning more details related to Reaction Scheme 5, the tetraethyl-substituted dicyanodipyrrole is used as an illustration as follows: The starting material, 2-benzyloxycarbonyl-3,4-diethyl-5-methyl pyrrole (not shown) in glacial acetic acid is treated with lead tetraacetate. Ethylene glycol is added to reduce any remaining Pb(IV). Water is added and the 5-acetoxymethyl-2-benzyloxycarbonyl-3,4-diethylpyrrole, 26 - ~

shown as the starting material in Reaction Scheme 5, is collected by filtration and washed with additional water.
The 5-acetoxymethyl-2-benzyloxycarbonyl-3,4-diethylpyrrole is added to acetic acid in water and heated. The 5 solid product, is precipitated as large chunks when the foregoing solution cools to room temperature. Water is added and the product is collected by filtration and then-washed with additional water_ The filtrate is extracted with CHZC12 and then evaporated to produce a solid product. The solid products are combined, and then recrystallized from a solution of CH2C12 and hexanes to obtain 5,5'-bis(benzyloxycarbonyl)-3,3'4,4'-tetraethyl-2,2'-dipyrromethane. -This product is dissolved in tetrahydrofuran (THF) and stirred under hydrogen in the presence of Pd/C and triethylamine. After the catalyst is filtered through celite, the filtrate is evaporated to dryness, dissolved in N,N-dimethylformamide and heated to boiling under argon. The solution is chilled and an excess of chilled benzoyl chloride is added. The reaction mixture is stirred and the solid product collected by filtration. The solid product is added to water and basified using NaHCO3 and heated to 60 C. The pale yellow product, 3,3',4,4'-tetraethyl-5,5'-diformyl-2,2'-dipyrromethane, crystallizes from the-solution and_is filtered and washed with water.
This product is dissolved in ethanol and bubbled with argon; hydroxylamine hydrogen chloride and sodium acetate are added. This mixture is heated under argon and the solvent is removed and the product dried overnight in vacuo. The resulting bisoxime is dissolved in acetic anhydride and saturated with argon to generate the crude bisnitrile product, which is obtained as a black solid after removal of acetic anhydride and dried under vacuum. The product shown as the end product of Reaction Scheme 5 is purified by silica gel column with 0.5%
methanol in CH2C12, followed by an alumina column with 10-20%
EtOAc. Evaporation of the solvent yields the 3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane aspale pink crystals.
The product obtained above can then be converted to the desired_corresponding porphocyanine as illustrated in -~ ~~SSQ29 Reaction Scheme 2. The 3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane in anhydrous THF is added to a THF
suspension of LiAlH4 under nitrogen at 0 C. The mixture is stirred and water is added to quench the reaction and the precipitate is filtered off. The golden colored solution is transferred to a two-neck flask containing equimolar portions of Pb(SCN)2 and anhydrous sodium sulphate. Anhydrous methanol is added and the mixture is brought to reflux. The color gradually changes from purple to dark green. The reaction is stopped and air is bubbled slowly through the solution. The crude product was dissolved in methylene chloride and the solid was filtered off. The volume of the green solution is reduced to approximately 5 ml and then charged on an alumina column and eluted with ethylacetate in CH2Cla. The bright green eluent containing the porphocyanine is collected and evaporated to dryness.
If Reaction Scheme 3 is to be followed in order to obtain the desired porphocyanine, the conduct of this sequence of reactions may be illustrated, again using the tetraethyl-substituted dipyrromethane as an example, as follows:
The 3,3'4,4'-tetraethyl-5,51-cyanodipyrromethane in anhydrous THF is added to a THF suspension of LiAlH4 under nitrogen at 0 C. The mixture is stirred and water is added to quench the reaction and the precipitate is filtered off. A ten-fold excess of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) 'in a THF/CH2C12 suspension under nitrogen at 0 C is added to the 3,314,4'-tetraethyl-5,5'-bisaminomethyl-2,2'-dipyrromethane in anhydrous THF/CH2C12. The solution turns dark green immediately.
The crude product is dissolved in methylene chloride and the solid filtered off. The volume of the green solution is reduced to approximately 5 ml and then charged on an alumina column and eluted with ethylacetate in CHZC12. The bright green eluent containing the porphocyanine is collected and evaporated to dryness. A significant increase in yield is obtained from this synthetic process as compared to the yield obtained via air oxidation.
To prepare the product from the bisaldehyde and the bisnitrile, the 3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane, 2igS429 - 28 - ~

for example, is dissolved in THF and added to a TEF suspension of LiAlH4. The 'rqsi,tl'tii3g mixture is stirred and water is added.
A solid product forms which is filtered off. The bisamine product is obtained after evaporation of the solvent by drying under vacuum. The bisamine is dissolved in anhydrous methanol and bisaldehyde is added. The solution is bubbled and brought to reflux with nitrogen. Lead thiocyanate (Pb(SCNa)) is added and the solution is refluxed. Oxygen gas is bubbled through the solution at room temperature. After evaporation of the solvent, the crude porphocyanine_productisdried undervacuum. The-product is purified by A1203 column with ethyl acetate in CHzC12.
The green eluent is collected and concentrated. Crystals of the product porphocyanine are obtained after evaporation of the solvent.
I5 The conduct of still another alternative, Reaction-Scheme 4, is illustrated as follows. An illustrative dialdehyde (6) 3,3'4,4'-tetraethyl-5,5'-diformyl-2,2'-dipyrromethane is suspended in dry EtOH. The resulting ethanol solution is chilled at 0 C and ammonia gas is bubbled through it. Theflask is then placed in an oil bathat 60 C for 72 hours. The reaction is stopped and-cooled at 0 C. Ethanol is removed on the roto-vap, the residue is chromatographed on neutral Alumina with dichloromethane. This greatly simplified synthetic process produces high yields of-the porphocyanine.
In order to prepare the alternative embodiments of the CNC-expanded porphyrins of the invention, appropriate modifications of the Reaction Schemes leading to the porphocyanine compounds per se are employed as would generally be understood in the art. For example, to obtain the CNC-expanded porphyrin of formula (lb), the condensation reactions set forth in Reaction Schemes 2 or 3 are employed using as starting material 2,5-dicyanopyrrole. To obtain the embodiments of formula (lc), the-condensation of the diaminomethyl derivative with a dialdehyde is_preferably employed. The dialdehyde of the dipyrrole methane along with the diaminomethylated pyrrolyl pyrrole.are reacted to form the desired product. For production of the compounds of formula (id), and (le), the CNC bonds are formed as described above from ~ ~~18802~

the relevant tetrapyrrole starting material. Such modifications are well understood by ordinary practitioners and conventional means to synthesize these alternatives may be employed.
Administration and Use The invention compounds or conjugates thereof are formulated into pharmaceutical compositions for administration to the subject using techniques known in the art generally. A
summary of such pharmaceutical compositions may be found, for example, in Reminaton's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pennsylvania, latest edition).
The invention compounds and their conjugates are administered systemically for some indications, preferably by injection. Injection may be intravenous, subcutaneous, intramuscular, or even intraperitoneal. injectables can be prepared in conventional forms, either as liquid solutions or -suspensions, solid form suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol and the 1ike. Of course, these compositions may also contain minor amounts of nontoxic, auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
Systemic administration can also be implemented through implantation of a slow release or sustained release system, by suppository, or, if properly formulated, orally.
Formulations for these modes of administration are well known in the art, and a summary of such methods may be found, for example, in Remin4ton's Pharmaceutical Sciences (supra).
If the treatment is to be localized, such as for the treatment of superficial tumors or skin disorders, the compounds may be topically administered using standard topical compositions involving lotions, suspensions, pastes, or creams.
The quantity of compound to be administered depends on the,choice of active ingredient, the condition to be treated, the mode of administration, the individual subject, and the judgment of the practitioner. Depending on the specificity of the preparation, smaller or larger doses may be needed. Dosages in the range of about 0.05-10 mg/kg are suggested for systemic 'W095/29180 P CT/CA 95/00242 r-'~'_ administration. Dosages in the range of about 0.01-20%
concentration of active i.ngredient, preferably 1-5%, are suggestedfor topical administration. A total daily dosage in the range of about 10-300 mg are suggested for oral_--_ administration. The foregoing ranges are_merely suggestive,as the number of variables in regard to an individual treatment regime is large and considerable excursions from these recommended values are expected.
The radioimaging (scintigraphic imaging) method of the -present invention is practiced by injecting an individual parenterally with an effective amount of the porphocyanine=-radioimaging agent. By parenterally is meant, e.g., -intravenously, intraarterially, intrathecally, interstitially or intracavitarily. It is contemplatedthat an individual will receive a dosage of from about 1 mCi to 50 mCi of the radioimaging agent, the amount being a function of the particular radioisotope and mode of administration. For intravenous injection, the amount are normally: about 10-40 mCi, preferably about 20 mCi of Tc-99m; about 2-5 mCi, preferably about 4 mCi of In-ill or Ga-67.
The radioimaging agent is conveniently provided as an injectable preparation, preferably a sterile injectable preparation for human use, for targeting the agent to diseased tissue or cells, preferably comprising: a sterile injectable solution containing an effective amount of.the radiolabeled agent in a pharmaceutically acceptable sterile injection vehicle, preferably phosphate buffered saline (PBS)at physiological pH and concentration. Other pharmaceutically acceptable vehicles may be utilized as required for_the site of parenteral administration.
A representative preparation to be parenterally administered in accordance with this invention will normally contain about 0.1 to 20 mg, preferably about 2 mg, of radiolabeled agent in a sterile solution.
Once enough isotope has been deposited at the target site, scanning is effected with either a conventional planar and/or SPECT gamma camera, or by use of a hand held gamma probe used externally or internally to localize the inflammation or the lesion. The scintigram is normally taken by a gamma imaging camera having one or more windows for detection of energies in the 50-500 KeV range.
Administration of the contrast agents for magnetic resonance imaging (MRI) is effected in an analogous method to administration for radioimaging except that the compounds of the invention will be in the metalated forms employing paramagnetic ions. Normally the signal generated is correlated with the relaxation times of the magnetic moments of protonsin the nuclei of the hydrogen atoms of water molecules in the region to be imaged. The magnetic resonance image contrast agent acts by increasing the rate of relaxation, thereby increasing the contrast between water molecules in the region where the imaging agent accretes and water molecules elsewhere in the body.
However, the effect of the agent is to increase both Tt and T2, the former resulting in greater contrast, while the latter results in lesser contrast. Accordingly, the phenomenon is concentration dependent, and there is normally anoptimum concentration of a paramagnetic species for maximum efficiency.
The optimum concentration will vary with the particular agent used, the locus of imaging, the mode of imaging, i.e., spin-echo, saturation-recovery, inversion-recovery and for various other strongly T1 dependent or T2 dependent imaging techniques, and the composition of the medium in which the agent is dissolved or suspended. These factors, and their relative importance are known in the art. See, e.g., Pykett, Scientific American (1982) 2JJ:78, and Runge et al., Am J Radiol (1987) ,]J1:1209.
The MRI method of the invention is practiced by injecting an individual parenterally with an effective amount of the MRI contrast agent of the invention which includes a metallic element such as gadolinium. It is contemplated that an individual will receive a dosage of contrast agent sufficient to enhance the MRI signal at the targeted site by at least about 20t, preferably 50-500g, the amount being a function of the particular paramagnetic species and the mode of administration.
A contrast agent within the present invention is conveniently provided as an injectable preparation for use, ; ,: ; . ~:: =
~~~~t329 preferably a sterile injectable preparation for human use, for targeting an MRI agent to diseased tissues or cells, preferably comprising: a sterile injectable solution containing an effective amount of the contrast agent in a pharmaceutically acceptable sterile injection vehicle, preferably phosphate-buffered saline. Other conventional pharmaceutically acceptable vehicles for parenteral administration may be utilized as required for the site of parenteral administration.--A representative preparation to be parenterally administered in accordance with this invention will normally contain about 0.1 to 20 mg, preferably about 2 mg of contrast agent, in a sterile solution.

Examnles The following examples are intended to illustrate_the invention but not to limit its scope.

Example 1 Synthesis nf an Octaethvl Porphocvanine RPactinn Scheme 5 followedbV
RPrlõctinn and Imine Formation_.
This-example describes the conduct of Reaction Scheme 5 to prepare both the bisaldehyde and the bisnitrile dipyrrole intermediates. It also illustrates the-condensatian of the bisnitrile with the bisaldehyde to obtain the CNC linkages of formula (la).
Lead tetraacetate (18.2 g, 0.041 mole) was added-to a stirred solution of -2-benzyloxycarbonyl-3,4-diethyl-5-methyl pyrrole (10.1 g, 0.037 mole) in 60 ml glacial acetic acid. The mixture was warmed briefly to 60 C. Ten ml of ethylene glycol was added to reduce any remaining_Pb(IV). Twenty ml of water was added and the 5-acetoxymethyl-2-benzyloxycarbonyl-3,4-diethylpyrrole (8) was collected by filtration and washed with additional water. The 5-acetoxymethyl-2-benzyloxycarbonyl-3,4-diethylpyrrole was added to 80% acetic acid in 100 ml water and heated at 100 C for one hour. The solid product precipitated as large chunks when the foregoing solution cooled to xoom temperature. One hundred ml of water was added and the product WO 95/29180 PCTlCA95100242 = ~~,t2Q~I~~

was collected by filtration and then washed with additional water. The filtrate was extracted with CHaClZ and then evaporated to produce a solid product. The solid products were combined, and then recrystallized from a solution of CHaClZ and hexanes to obtain 5,5'-bis(benzyloxycarbonyl)-3,3'4,4'-tetraethyl-2,2'-dipyrromethane (7).
Yield: 6.6 g, 67.4&
Mol. Wt. Calcd. for C33H38O4N2: 526.2831 High resolution MS: 526.2835 'H NMR in CDC13: 1.05 (t, 6H), 1.12 (t, 6H), 2.43 (q, 4H), 2.75 (q, 4H), 3.85 (s, 2H), 5.25 (s, 4H), 7.28-7.40 (m, 10H), 8.70 (br s, 2H).
The above product (8.1 g, 0.015 mole) in 200 ml tetrahydrofuran (THF) was stirred under hydrogen at atmospheric pressure and room temperature overnight in the presence of 0.4 g 10g Pd/C and 5 drops of triethylamine. After the catalyst was filtered through celite, the filtrate was evaporated to dryness on a roto-vap resulting in dicarboxylic acid. The dicarboxylic acid was dissolved in 100 ml N,N-dimethylformamide and heated to boiling under argon for one and one-half hours. The solution was then chilled on ice and an excess of chilled benzoyl chloride (7.2 ml) was added drop-wise and the reaction mixture was stirred for 2 hours at 5 C and the solid product collected by filtration. The solid product was added to 50 ml water and basified using NaHCO3. The solution was heated and held at 60 C
for one hour. The pale yellow product which crystallized from the solution was filtered and then washed with water to obtain 3,3',-4,4'-tetraethyl-5,5'-diformyl-2,2'-dipyrromethane (6).
Yield: 3.4 g, 70.0t Mol. Wt. Calcd. for C19H26NZOZ: 314.1994 High resolution MS: 314.1994 1H NMR in CDC13: 1.10 (t, 6H), 1.25 (t, 6H), 2.50 (q,4H), 2.70 (q, 4H), 4.00 (s, 2H), 9.55 (s, 2H), 10.90 (s, 2H).
This product (1.03 g, 0.0033 mole) in 300 ml ethanol was bubbled with argon for 20minutes. Hydroxylamine hydrogen chloride (0.51 g, 0.0073 mole) and sodium acetate (1.20 g, 0.015 mole) were added. This mixture was heated at 60 C under argon for two and one-half hours. The solvent was removed on a roto-WO 95/29180 PCTlCA95100342 21&8029 vap and the product dried overnight in vacuo. The bisoxime was dissolved in 5 ml acetic anhydride and saturated with argorn==for 30 minutes. The crude bisnitrileproduct (2) was obtained as a , black solid after removal ofacetic anhydride and dried und-er vacuum overnight. The product was purified-by silica gel column (40 g) with 0.5% methanol in CHaC12, followedby an alumina column (40 g) with 10-201 EtOAc. Evaporation of the solvent yielded pale pink crystals of 3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane (2).
Yield: 0.43 g, 42t Mol. Wt. Calcd. for C19H24N4: 308.2001 High resolution MS: 308.2002.
'H NMR in CDC13: 1.10 (t, 6H), 1.25 (t, 6H), 2.45 (q, 4H), 2.60 (q, 4H), 3.90 (s, 2H), 8.45 (s, 2H).
This product (2) (0.053 g, 1.7 X 104 mole) was dissolved in 10 ml anhydrous THF and added dropwise to a THF
suspension of LiAlH4 (0.050 g, 1.5 X 10"; mole) under N. at 0 C.
The resulting mixture was stirred for 30 minutes and two drops of water was added. A solid product formed which was filtered off. The bisamine product was obtained after evaporation of the solvent by drying under vacuum overnight. The bisamine (3) was dissolved in 50 ml anhydrous methanol and bisaldehyde (6) (0.050 g, 1.6 X 104 was added. The solution was bubbled with N2 for 20 minutes and brought to reflux under N2. Lead thiocyanate (Pb(SCNa)) (0.055 g, 1.7 X 10'4 mole) was added and the solution 'refluxed for-:4 hours. Oxygen gas was bubbled through the solution at room temperature overnight. After evaporation of the solvent, the crude porphocyanine product was dried unde-r vacuum overnight. The product was purified by A1203 column (4%
water added) with 10% ethyl acetate in CH2C12. The green e1-uent was collected and concentrated on the roto-vap. Crystals of 2,3,9,10,14,15,21,22-octaethyl porphocyanine (1) were obtained -after evaporation of the solvent.
Yield: 19.3 mg, 19.1k macrocycle -- Mol. Wt. Calcd. C38H4aN6: 588.3941 _ High resolution MS: 588.3933 'H NMR (300 MHz, CDC13), -4.50 (bs, 2H), 2.05 (t, 12H), 2.13 (t, 12H), 4.28 (q, 8H), 4.40 (q, 8H), 10.50 (s, 2H), 13.0 (s, 4H).
IIV/VIS in CH2C12 455, 592, 800 nm.
Examole 2 Synthesis of an Octaethvl Porphocvanine (Reaction Scheme 2) This example illustrates the formation of a porphocyanine of formula (la) using the condensation procedure shown in Reaction Scheme 2.
3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane was prepared according to the methodology of Example 1. 0.102 g, 3.3 X 10'4 mole of 3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane in 10 ml anhydrous THF was slowly added to a 20 ml THF
suspension of LiA1H4 under N2 at OOC. The mixture was stirred for 30 minutes and two drops of water were added to quench the reaction. The precipitate was filtered off. The golden colored solution was transferred to a two-neck flask containing equimolar portions of Pb(SCN)z and anhydrous sodium sulphate. Fifteen ml of anhydrous methanol were added and the mixture was brought to reflux. The color of the solution gradually changed from purple to dark green. The reaction was stopped after four and one-half hours and air was bubbled slowly through the solution overnight. The crude product was dissolvedin methylene chloride and the solid was filtered off. The volume of the green solution was reduced to approximately 5 ml and then charged on an alumina column (120 g, 4k water added) and eluted with 10%- ethylacetate in CH2C12. Two liters of the bright green eluent containing the porphocyanine was collected and evaporated to dryness.
Yield: 23.4 mg, 24.11 The spectroscopic data of this compound are identical to the compound prepared in Example 1 above.

ExamAle 3 Synthesis of an Octaethvl Porphocyanine (Reaction Scheme 3) This example illustrates Reaction Scheme 3. 3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane (2) was prepared according to the methodology of Example 1. A ten-fold excessof 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ) was added to 69 mg of 3,3'4,4'-tetraethyl-5,5'-cyanodipyrromethane in 10 ml anhydrous THF. The resulting solution turned dark green immediately. Theresidue was chromatographed according to the methodology of Example 2. A significant increase in yield was obtained when compared to air oxidation_ Yield: 32 mg, 48!k The spectroscopic data of this compound are identical to the compound prepared in Example 1 above.-Example 4 Synthesis of Porphocvanine - -(Reaction Scheme 4) This example illustrates Reaction Scheme 4. The dialdehyde was prepared according to the methodology of Example 1. 25 mg of 3,3'4,4'-tetraethyl-5,5'-diformyl-2,2'-dipyrromethane (6) was suspended in 30 ml dry EtOH_The -resulting ethanol solution was chilled at 0 C and ammonia gas was bubbled through it for 30 minutes. The gas inlet was then 'removed and the flask placed in an oil bath at 60 C for 72 hours. The reaction was stopped and cooled at 0 C. Ethanol was removed on the roto-vap and the residue was chromatographed on neutral Alumina (4!kHZO added) with dichloromethane. -Yield: 4.6 mg, 20%
The spectroscopic data of this compound are identical to the compound prepared in Example 1 above. -Another 1.5 mg of product was obtained-from the oxidation of the more polar component by DDQ. -Example 5 Synthesis of 2.2'-bis cyano meso arvl dipyrromethanea This example illustrates Reaction Scheme 6.
General procedure: 250 ml round bottomed flask is charged with methanol (115 ml), pyrrole (10 ml; 105 mmol) and p-toluene sulfonic acid (2.15g; 11.5 mmol). To this stirred solution-is added a solution of the aromatic aldehyde (11.25 mmol) in methanol (30 ml) over 100 min. After this time the reaction is poured into water (350 ml) and extracted with dichloromethane (3 x 150 ml). The organic phase is washed with brine (3 x 200 ml) and dried over anhydrous potassium carbonate.
Evaporation of solvent in vacuo followed by flash chromatography (silica gel; 100g) of the residue gives the meso aryl dipyrromethane.
The meso aryl dipyrromethane (0.32 mmol) is dissolved in N,N-dimethylformamide (5 ml), this solution is then diluted with acetonitrile (5 ml) and the stirred mixture is cooled to -78 C. To the cooled mixture, under an atmosphere of N2 is added dropwise a solution of chlorosulfonyl isocyanate (1.42 mmol) in acetonitrile (3 ml). The mixture is stirred at -78 C for 1 hour then at -40 C for a further hour before being allowed to rise to room temperature. The reaction mixture is poured into a mixture of 5t aqueous sodium hydrogen carbonate (15 ml), aqueous potassium hydroxide (3M; 2.7 ml) and ice then diluted with brine (200 ml) and extracted with dichloromethane (3 x 50 ml). The organic phase is dried over anhydrous potassium carbonate, the solvent evaporated in vacuo and the residue chromatographed on silica gel to give the 2,2'-bis cyano meso aryl dipyrromethane.

Example 6 Condensation of 2.2'-bis cvano meso arvl diAVrromethanes to Prepare Symmetric Pornhocvanines This illustrates the method of Reaction Scheme 3 where at least one Rk is aryl.
General method: A solution of 2,2'-bis cyano meso aryl dipyrromethane (0.124 mmol) in tetrahydrofuran (5 ml) is added dropwise to a stirred suspension of lithium aluminum hydride (1.3 mmol) in tetrahydrofuran (10 ml) under an 2188029 . . .. r atmosphere of N2 at 0 C. The reduction is followed by thin-layer chromatography (silica gel), eluting with 10t ethyl acetate in dichloromethane until complete, then excess LiAlH4 is quenched with water, dichloromethane (15 ml) is added, and the mixture is filtered. The filtrate is dried over anhydrous sodium sulfate and the solvent evaporated in vacuo. The residue is redissolved in dry dichloromethane (100 ml) and to the stirred solution is -added, dropwise, a solution of dichlorodicyanobenzoquinone (5 equivalents) in toluene (5 ml). The oxidation is followed by UV-vis spectroscopy and when no further porphocyanine formation is detectedthe reaction mixture is filtered through a plug of neutral alumina to remove excess DDQ, and the solvent is evaporated in vacuo. The crude diphenyl porphocyanine is purified by chromatography on alumina.

- ExamAle 7 Application of the Methods of Examples 5 and 6 In the examples below which describe various porphocyanines, the following numbering system will-be used:

2~ 6 10 1 - N, H N11 23 N H 2 N/,3 22 ~ 20\\ i N ~ 16 ~ 14 Using the Method of Examples 5 and 6, the following 20 were prepared.
1. 12.24-DiDhenvl porphocyanine: ig benzaldehyde gives 732 mg (35k) meso phenyl dipyrromethane after chromatography (silica gel; 10% hexane in dichloromethane). 500 mg meso phenyl dipyrromethane gives 195 mg (32%) 2,2'-bis cyano 25 rneso phenyl dipyrromethane after chromatography (silica gel; 5%
acetonitrile in dichloromethane). 100 mg 2,2'-bis-cyano meso ~ 2188029 phenyl dipyrromethane gives 28 mg (29t 1,11-diphenyl porphocyanine after chromatography (60-325 mesh neutral alumina;
101 ethyl acetate in dichloromethane).
Spectroscopic data: 'H NMR (CDC13) E7.92 (m, 6H), 8.44 (m, 4H), 9.38 (d, j=5.7Hz, 4H), 9.8 (d, J=5.7Hz, 4H), 12.95 (s, 4H) ; UV-vis: X452,598,640,814nm in CHaCla; HRMS (EI) for C34H24N6(M*) calcd. 516.2062, found 516.2058.
2. 12,24-Di(3',4',5'-trimethoxvwhenvl) porohocvanine: 2.21g 3,4,5-trimethoxybenzaldehyde gives 1.147g (33k) meso (3,4,5-trimethoxyphenyl) dipyrromethane after chromatography (silica gel; 3k acetone in dichloromethane). lg meso (3,4,5-trimethoxyphenyl) dipyrromethane gives 460 mg (40!k) w,w'-bis cyano meso (3,4,5-trimethoxyphenyl) dipyrromethane after chromatography (silica gel; 10& ethyl acetate in dichioromethane). 100 mg 2,2'-bis cyano meso (3,4,5-trimethoxyphenyl) dipyrromethane gives 31 mg (32k 1,11-di(3',4',5'-trimethoxyphenyl) porphocyanine after chromatography (60-325 mesh neutral alumina; ethyl acetate/bichloromethane (1:1)), Spectroscopic data: 'H NMR (CDC13) 54.1 (s, 12H), 4.25 (s, 6H), 7.67 (s, 4H) 9.44 (d, J=4.8Hz, 4H) 9.8 (d, J=4.8Hz, 4H), 12.95 (s, 4H) ; UV-vis: X460,602,644,818nm in CHZCla; HRMS
(EI) for C40H36V6O6 (M*) calcd. 696.2696, found 696.2690.
3. 12.24-Di(oentafluorophenvl) porphocvanine:
2.22g pentaflubrobenzaldehyde gives 1.48g (42%-) meso (pentafluorophenyl) dipyrromethane after--chromatography (silica gel; 25W hexane in dichloromethane). 200 mg meso (pentafluorophenyl) dipyrromethane gives 89 mg (38% 2,2'-bis cyano meso (pentafluorophenyl) dipyrromethane after chromatography (silica gel; 5% ethyl acetate in dichloromethane). 89 mg 2,2'-bis cyano meso (pentafluorophenyl) dipyrromethane gives 23 mg (28%-) 1,11-di(pentafluorophenyl) porphocyanine after chromatography (60-325 mesh neutral alumina;
5% acetone in dichloromethane).
Spectroscopic data: 'H NMR ((D3C)2C0) 59.55 (dd, J,=15.22Hz, J2=4.15Hz, 4H), 10.1 (d, J=4.lSHz, 4H), 13.24 (s, 4H); '9F NMR ((D3C)ZCO) 60.53, 60.92, 61.45 referenced to F3CCOOH;

~~~~~~g =

UV-vis: 442,582,624,814nm in acetone; HRMS (EI) for C3aH1aNeFlo (M*) calcd. 696.1120, found 696.112.4.

Example 8 Qgndensation to Obtain Asymmetric Porohocvanines This example employs the method of Reaction Scheme 3.
General procedure: Two differently substituted 2,2'-bis cyano dipyrromethanes (0.1 mmol) are dissolvedin dry tetrahydrofuran (5 ml) and the two solutions are mixed. The mixed solution is added dropwise to a suspension ofLiAlH4 in dry THF (10 ml) under an atmosphere of N2 at 0 C. The reduction is followed by TLC and, when complete, excess LiAlH4 is_quenched with water and dichloromethane (5 ml) is added. The resulting slurry is gravity filtered and thefiltrate is dried over -anhydrous sodium sulfate. Evaporation of solvent in vacuo, followed by redissolution in dichloromethane (20 ml) gave a golden solution. To this solution_is added, dropwise, a solution of DDQ (5 equivalents) in toluene (5ml). The oxidation is followed by UV-vis spectroscopy and, when no further porphocyanine formation is-seen, the reaction mixture is filtered through a plug of neutral_alumina. Evaporation of the solvent from the filtrate gives the crude mixture of porphocyanines which are resolved by chromatography.

Example 9 AoAlication of the Method of Example 8__ Using the method of Example 8, the following was prepared: -12-Phenvl-2.3.21,22-tetraethvl Aorohocvanine: 51 mg 2,2'-bis cyanomeso phenyl dipyrromethane + 58 mg 2,2'-bis cyano 3,3'4,4'-tetraethyl dipyrromethane gives 48 mg of crude mixed porphocyanine. Afterchromatography (C18 reversed phase; 20%
0.1% aqueous trifluoroacetic acid in 0.1% trifluoroacetic acid/acetonitrile) 21_7 mg (21%) of the title compound is isolated. --Spectroscopic data: 'H NMR (CDC13) 'H NMR (CDC13) 2.05 (t, J=7.5Hz, 3H), 2.07 (t, J=7.5Hz; 3fI), 4.21 (q, J=7.5Hz, 2H), --4.32 (q, J=7.5Hz, 2H), 7.88 (m, 3H), 8.41 (m, 2H), 9.31 (d, WO 95/29180 PCTICA95l00242 ~

J=4.5Hz, 2H), 9.7 (d, J=4.SHz, 2H), 10.3 (s, 1H), 12.72 (s, 2H), 12.96 (s, 2H) ; W-vis: X456,592,632,804nm in CH2C172; HRMS (EI) for C36H36N6 (M+) calcd. 552.3001, found 552,2996.

ExamAle 10 12.24-DiAhenvl li-alkvl porphocvanine This general procedure, encompassing part of Reaction Scheme 5 and Reaction Scheme 2, begins with the bis carboxy dipyrromethane: 2,2'-bis carboxy 3,3'4,4'-tetraalkyl meso phenyl dipyrromethane (0.25 mmol) or 2,2' bis carboxy 3,3'-dialkyl meso phenyl dipyrromethane (0.25 mmol) are dissolved in N,N-dimethylformamide (10 ml) and the solution is heated to reflux under a stream of N2. the reaction is monitored by TLC
and when decarboxylation is complete the mixture is diluted with acetonitrile (10 ml) and cooled to -78 C. A solution of chlorosulfonyl isocyanate (2.9 mmol) in acetonitrile (1 ml) is added dropwise to the stirred solution under an atmosphere of N2.
The mixture is stirred for 1 hour at -78 C and then for a further hour at -40 C before being allowed to rise to room temperature. The reaction mixture is poured into a mixture of 5k aqueous sodium hydrogen carbonate (20 ml), aqueous potassium hydroxide (4 ml) and ice, then diluted with brine (200 ml) and extracted with dichloromethane (3 x 50 ml). The organic phase is washed with brine (5 x 100 ml), dried over anhydrous potassium carbonate, and thesolvent is evaporated in vacuo.
Chromatography of the residue gives 2,2'-bis cyano meso phenyl-3,3'4,4'-tetraalkyl dipyrromethane or 2,2'-bis cyano meso phenyl-3,3'-dialkyl dipyrromethane.
The bis cyano compound (0.11 mmol) is dissolved in dry THF (10 ml) and this solution is added dropwise to a stirred suspension of LiAlH4 (1.3 mmol) in dry THF (10 ml) under an atmosphere of N2 at 0 C. The reduction is monitored by TLC and when complete the excess LiA1H4 is quenched with water, dichloromethane (15 ml) is added and the resulting slurry is gravity filtered. The filtrate is dried over anhydrous sodium sulfate and solvent is evaporated in vacuo. The residue is redissolved in dichloromethane (100 ml) and to the stirred solution is added dropwise a solution of DDQ (5 equivalents) in toluene (5 ml). The oxidation is followed by UV-vis spectroscopy and, when no further porphocyanine formation is observed, the reaction mixture is filtered through a plug of alumina. The filtrate is evaporated in vacuo and the residue is chromatographed to give 1,11-diphenyl 0-octaethyl porphocyanine or 1,11-diphenyl 0-tetraalkyl porphocyanine.

Example 11 Application of the Method of Example 10 _ Using the method of Example 10, the following was prepared:
12.24-Diphenvl-3.9,15,21-tetraethvl-2,10.14.22-tetramethvl Aorohocvanine: 100 mg_2,2'-bis carboxy 3,3'-diethyl-4,4'-dimethyl meso phenyl dipyrromethane gives 40 mg (44~;) bis cyano 3,3'-diethyl-4,4'-dimethyl meso phenyl dipyrromethane. 40 mg bis cyano 3,3'-diethyl-4,4'-dimethyl meso phenyl dipyrromethane gives 90 g (0.23%) of the title compound after chromatography (60-325 mesh neutral alumina; 10%- ethyl acetate in dichloromethane).
Spectroscopic data: W-vis: 462,604,642,802nm in CH2C12; HRMS (EI) for C4J1,N6 (M+) calcd. 684.394A, found 684.3932.
Examnle 12 rn Vitro Toxicity of Pornhocvanine Cells are washed three times in serum-free medium (DME), counted and made up to a concentration of 10' cells per ml.
For the "affinity" assay, in the dark, 100 l of the cell suspension and 100 l of the test or control compoundare mixed. "Labeling" is allowed-to continue for one hour at 4 C, and labeled cells are washed in-the dark three times with 3 ml medium each time and resuspended in fresh medium. The resuspended cells are then subjected to light exposure at 300-850 nm for 30 minutes.
In a "direct" assay the cells are irradiated immediately upon addition of the test or control compound. The -effect of irsadiation is estimated=using inethods appropriate to the target cells. _ when human erythrocytes (RECs) are used as test cells, the hemolysis caused by irradiation of control (hematoporphyrin, ,Hp)-labeled and porphocyanine (formula (1))-labeled cells is estimated visually.
When the murine mastocytoma cell line P815 is used, the results are determined as follows:
The cells are labeled as above using concentrations of 10-50 ng/ml of Hp as control and the porphocyanine of formula (la) as the test substance. The resuspended cells are treated with 300-850 nm light for 30 minutes and the viability resulting is estimated by,direct counting using eosin-Y exclusion, a standard procedure for differentiating living from dead cells.
In other determinations conducted as above, the cells recovered from light exposure are assayed for viability by incubating them for 18 hours in 10 fcCi/ml tritium-labeled thymidine according to the standard procedure whereby thymidine incorporation is equated with viability. The cells are harvested and radioactivity uptake is measured by a scintillation counter.

Example 13 Selective Binding of Porohocvanine P815 cells are incubated as described in Example 12 using 1-200 ng/ml Hp or the porphocyanine of formula (1a). The cells are labeled in the dark for 30 minutes, washed free of unabsorbed porphyrins, resuspended, and then exposed to 300-850 nm light for another 30 minutes. Viability of the cells is established by tritiated thymidine incorporation after labeling with 30 Ci/ml tritiated thymidine and incubating at 370C for 18 hours.

Example 14 Preparation of Immunoconiuaates This example describes methods of preparation for immunoconjugates of four different antibody preparations with either hematoporphyrin (Hp) or a porphocyanine (Pc) of formula (la). The antibodies employed are CAMAL-1, anti-M1 antibody, and B16G antibody, all prepared as described hereinabove, and 2188029 ~-affinity purified rabbit anti-mouse Ig (RcMIg). In addition, a purified irrelevant monoclonal preparation (C-MAb) is used where a control is desired_ One preparation of the conjugates is performed basically as described by Mew et al. J Immunol (1983) ],3Q:1_473).
Briefly, to 220 mg HpØ2 HC1-(Sigma Chemical Co., St. Louis, MO) in 25 ml water and 0.8 ml N,N-dimethylformamide is added 20 mg 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HC1. (EDCI) in 0.6 ml water. After 30 minutes, this solution is mixed with 15 mg of the antibody protein dissolved in 5 ml distilled water and incubated for 5 hours. During this period, the pH of the solution is monitored and adjusted to between 6 and 7. Then 50 l of monoethanolamine is added, ahd the solution is allowed to stand overnight at room temperature. The solution is dialyzed against 0.001 M phosphate buffer pH 7.4 for four days with three changes per day and overnight against PBS. The conjugate of porphocyanine is analogously prepared.
in a preferred protocol, 2 ml of a dispersion in DMSO
containing 5 mg each of the Hp or Pc and the dehydrating agent is prepared and stirred for 30 minutes at room temperature-under nitrogen. To this is added a dispersion containing 2 mg of the appropriate immunoglobulin in 2 ml of DMSO, and theresulting mixture stirred for another 10 minutes. This mixture is then worked up by dilution in phosphate-buffered.saline, pH 7.4 (PBS) by adding 5 times the volume of PBS containing 50 l monoethanolamine, and is then dialyzed against PBS using three changed of wash. - -Alternatively, 2 ml of a dispersion containing 5.mg each of Hp or Pc, a linking agent, and a dehydrating agent-is prepared and stirred for approximately 15 minutes at room temperature-under nitrogen. To this is then added a dispersion containing about 2 mg of the immunospecific protein in 2 ml of tetrahydrofuran and the resulting-mixture stirred for another 10 minutes. The mixture is then worked up as described above.
The foregoing procadures are appropriate for CAMAL-1 and for the remaining antibody preparations listed above.
in addition, the following preparations are made specifically with-B16G and RaMIg:

~~ -Eleven mg of hematoporphyrin plus 11 mg of EDCI in 4 ml spectral grade DMSO are stirred for 30 minutes under nitrogen at room temperature before the addition of 20 mg lyophilized B16G antibodies, prepared as described by Maier et al. J Immunol (1983) 121:1843, in 2 ml DMSO. The resulting mixture is stirred for 40 second at room temperature and working up as described above. The resulting product contains approximately 375 g Hp/mg B16G. A similar procedure is used substituting Pc for Hp.
RgMIg Four hundred g of EDCI and 400 g hematoporphyrin in 1 ml DMSO are stirred for 30 minutes under nitrogen at room temperature as above before the addition of 800 g lyophilized RaMIg antibodies, prepared as described by Mew et al. J Immunol (1983) J,~-O-:1473) in 1 ml DMSO. The resulting mixture is stirred for 30 seconds and worked up as described above to obtain a product containing 200 ag Hp/mg RaMIg. A similar procedure is used substituting Pc for Hp.

Examvle 15 Specificity of Immunoconiucrates In Vitro The TSM-Hp and TSM-Pc conjugates wherein the TSM is comprised of an immunoglobulin are tested against cells in vitro by mixing the conjugates with the appropriate cell types, along with suitable controls, and then exposing the labeled cells to irradiation. Procedures for carrying out this assay are described in detail in Mew et al., Cancer Research (1985) for CAMAL-1, and by Mew et al., J. Immunol. (1983) for Anti-Mi, both references cited hereinabove are incorporated herein by reference. - -Briefly, for CAMAL-1, three cell lines, WC4, WC6 and WC2 (WC4 and WC6 produce the CAMAL antigen, but WC2 does not), are labeled with the appropriate TSM-Hp or TSM-Pc preparation as described above in Example 14. The labeled cell preparations containing 106 cells each are introduced to Rose chambers and exposed to light activation with a laser at 630 nm. The results for various preparations are then compiled_ ~~E20Q~~ ~

For the anti-Mi conjugate, Ml tumor cells-are used as target cells and treated with the TSM-Hp, TSM-Pc conjugates or drug or antibody alone or the combination of antibody and drug, but uncoupled, by incubating them in 6t Co2 humidified incubator at 37 C for two hours. The cells are washed three times in PBS
and then plated and exposed to fluorescent light overnight. The cells are assessed for viability by tritiated thymidine uptake as above. -For the B16G conjugates, A10, P815, and L1210 cells are used as target cells. (A10 cells are a T cell hybridoma which secretes a B16G-reactive T-suppressor factor; P815 cells are also reactive with B16G.) The invitro study is done using a direct method employing the B16G=Hp or B16G-Pc conjugate or indirectly using unlabeled B16G antibodies and labeled RaMIg-Hp or RaMIg-Pc.
In a direct method, 5 x 105 cells are suspended in 1 ml DME/Hepes containing the appropriate TSM-drug conjugate as test or control at Hp or Pc concentrations of 320, 160, 80, 40 and 20 ng drug/ml. The cells are incubated in-the dark at 370C for 1 hour, then washed 3 times in 5 ml DME/Hepes, and then resuspended in 1 ml of the same buffer. Three 100 l test portions of the labeled preparations are dispensed into flat bottom microtiter wells and the remainder of the cell suspensions (700 l) are exposed to incandescent light (22.5 mW/cm7) at a distance of 20 cm for-1 hour. Then three additional 100 fcl aliquots are removed to microtiter wells. Tritium-labeled thymidine diluted in-DME/Hepes containing 20t FCS is then added to all microtiter wells in 100 l aliquots so that 2 {cCi of labeled thymidine is added to each well. Cultures are incubated for 18 hours at 37 C and humidified 10t CO2 and then harvested on a MASH harvester. Thymidine incorporation is measured with an Hp scintillation counter (Tri-Carb Model 4550).
In an indirect-assay, the A10suspended cells, prepared as described above, are exposed to 50- g/ml of either B16G or a control antibody C-MAb at 4 C for 30 minutes, washed in DME/Hepes, and then exposed for an additional 30 minutes at 4 C
in the dark to varying concentrations of RaMIg-Hp or RaMIg-Pc between 2 g/ml and 15 ng/ml of Hp or Pc. The cells are WO 95129180 pCTlCA95/00242 ~ 2188029 assessed for viability using labeled thymidine uptake as described above.

Examole 16 In Vivo Cvtotoxicitv of the Porohocyanine and ConiucTates Thereof The in vivo efficacy of porphocyanine (Pc) and conjugates thereof is also assessed. For the CAMAL-1 and anti-M1 conjugates, the procedures are as described in the two Mew et al. papers referenced above in Example 15. For the B16G-Hp and B16G-Pc conjugates and for the Pc (formula (1)) alone, the in vivo studies are conducted as follows:
The in vivo test relies on the indirect effect of a population of T-suppressor cells on tumors, which then serve as means to assess the effectiveness of the irradiation treatment.
P815 mastocytoma cells grown in syngeneic DBA/2 mice stimulate T-suppressor cells specific for the tumor. These T-suppressor cells impede the development of specific T-killer cells which would otherwise aid in the regression of the tumor. The T cell hybridoma designated A10 above secretes a T-suppressor factor which is associated with these T-suppressor cells. Thus, selective killing of these T-suppressor cell populations through reaction with conjugates in which the TSM is an antibody specific for the T-suppressor factor on the surface of the cells (namely B16G) should result in tumor regression in mice bearing the P815 tumors.
Therefore, in this assay, DBA/2 mice are injected in the right flank subcutaneously with 10 P815 cells to incorporate the tumor. On day eight, when the tumors are palpable (approx.
25-42 sq. mm), the mice are randomly sorted into groups of eight and injected IV with 150 l PBS containing nothing, Hp or Pc, B16G-Hp or B15G-Pc, B16G plus either drug, B16G alone or C-MAb-Hp or C-MAb-Pc. The levels of Hp are 50 g per animal in all cases and B16G 310 g in all cases (where appropriate).
The animals are maintained in the dark for two hours and then exposed to strong light at 300-850 nm and 22.5 mW/cm2.
The animals are then treated normally and monitored on a daily basis. -ExamAle 17 Aiacmostic imaaina -A 32-year old female patient developsfever and abdominal pain. The patient is maintained on antibiotic therapy for a period of one week without effect. A CAT scan fails to demonstrate any abnormal mass. Radioimaging studies are performed using Tc-99m-labeled porphocyanine. Ar injection of 20 mCi of the radiolabeled porphocyanine is used andthe patient is scanned with a gamma camera in SPECT mode. The scan of the patient's abdomen demonstrates a focus of accumulation of -Tc-99m. Surgery is performed and an abscess is found at the site of the Tc-99m activity.

Claims (33)

Claims

1. A compound of the formula:

and the metalated forms and salts thereof; wherein n is an integer of 1-4; and wherein each P i is independently a pyrrole residue of the formula wherein each R ia and R ib is independently a noninterfering substituent, and wherein each Z i is independently a covalent bond; or is a meso bridging group of the formula or is an N-meso bridging group of the formula =N- ~ -N=; or is a CC linkage of the formula or is a CNCCNC linkage of the formula wherein each R ic, R id, R ie, R if and R ig is independently a noninterfering substituent; or is a CNC linkage of the formula wherein at least one Z i is said CNC linkage, and with the proviso that, when Z1 and Z3 are both - CNC - and Z2 and Z4 are both meso bridging groups of =CR ic-, then n cannot be 2.

2. The compound of claim 1 wherein n=2.

3. The compound of claim 2 wherein Z1 and Z3 are CNC
linkages and Z2 and Z4 are meso linkages.

4. The compound of claim 2 wherein Z, is a CNC
linkage and Z2, Z3 and Z4 are meso linkages.

5. The compound of claim 2 wherein Z1 is a CNC
linkage, Z2 and Z4 are covalent bonds, and Z3 is a CC linkage.

6. The compound of claim 2 wherein Z1 and Z3 are CNC
linkages, Z2 is a covalent bond and Z4 is a meso linkage.

7. The compound of claim 2 wherein Z1 and Z3 are CNC
linkages, Z2 is a meso linkage, and Z4 is a CNCCNC linkage.

8. The compound of claim 2 wherein Z1 and Z3 are CNC
linkages, Z2 is an N-meso linkage, and Z4 is a meso linkage.

9. The compound of claim 2 wherein all of Z1, Z2, Z3 and Z4 are CNC linkages.

10. The compound of claim 1 wherein n=1.

11. The compound of claim 10 wherein Z1 is a CNC
linkage, and Z2 and Z3 are meso linkages.

12. The compound of claim 1 wherein n=3.

13. The compound of claim 12 wherein Z1 and Z2 are CNC
linkages, and Z3, Z4 and Z5 are meso linkages.

14. The compound of claim 1 wherein n = 4.

15. The compound of claim 14 wherein Z1 and Z4 are CNC
linkages and Z2, Z3, Z5 and Z6 are meso linkages.

16. The compound of claim 1 wherein each R ia, R ib, R ic, R id, R ie, R if and R ig is independently H, halo, nitro, cyano, NR'2, SR', OR', SOR', S02R', COOR', CONR'2, wherein each R' is independently H or alkyl (1-6C), and wherein amino, sulfhydryl and hydroxyl substituents may optionally be acylated (1-6C), optionally substituted alkyl (1-6C), optionally substituted alkenyl (1-6C), optionally substituted alkynyl (1-6C), optionally substituted aryl (4-12C), or optionally substituted arylalkyl (5-18C).

17. The compound of claim 1 wherein each R ic is independently optionally substituted aryl, optionally substituted alkyl, optionally substituted arylalkyl or is H.

18. The compound of claim 17 wherein each R ic is independently optionally substituted aryl or H.

19. The compound of claim 3 wherein one R ic is substituted aryl and the other R ic is H.

20. The compound of claim 1 wherein all R ia and all R ib are identical.

21. The compound of claim 3 wherein R1a, R1b, R4a and R4b are identical to each other and wherein R2a, R2b, R3a and R3b are identical to each other but different from R1a, R1b, R4a and R4b.

22. The compound of claim 21 wherein each of R ia and R ib is independently H or optionally substituted alkyl.

23. The compound of claim 2 which is selected from the group consisting of porphocyanine; 2,3,9,10,14,15,21,22-octaethylporphocyanine, 12,24-diphenylporphocyanine; 12,24-di(3',4',5'-trimethoxyphenyl) porphocyanine; 12,24-di(pentafluorophenyl) porphocyanine; 12-phenyl-2,3,21,22-tetraethyl porphocyanine, 12,24-diphenyl 2,3,9,10,14,15,21,22-octaethyl porphocyanine and 12,24-diphenyl 3,9,15,21-tetraethyl-2,10,14,22-tetramethyl porphocyanine.

24. A composition of matter which is the compound of formula (1) covalently linked to a target-specific moiety (TSM).

25. The composition of claim 24 wherein said covalent linking includes a linking group.

26. The composition of claim 24 wherein the TSM is an immunoglobulin or an immunoglobulin fragment, a steroid, or a saccharide.

27. A pharmaceutical composition which is cytotoxic to specific cells or tissues when irradiated with light, which comprises an effective amount of the compound of claim 1 in admixture with at least one pharmaceutically acceptable excipient.

28. A pharmaceutical composition which is cytotoxic to specific cells or tissues when irradiated with light, which comprises an effective amount of the composition of claim 24 in admixture with at least one pharmaceutically acceptable excipient.

29. The compound of claim 1 which is in metalated form.

30. The compound of claim 20 wherein the metal is a paramagnetic ion.

31. The compound of claim 30 wherein the paramagnetic ion element is gadolinium(III) or manganese(II).

32. A magnetic resonance imaging (MRI) contrast agent comprising the compound of claim 1 in metalated form with a paramagnetic ion.

33. The compound of claim 1 which includes a radioisotope selected from the group consisting of technetium, indium and gallium.