CA2227808A1 - Photoactivatable compounds for the prevention of intimal hyperplasia and other diseases - Google Patents

Abstract

A broad class of photosensitive compounds having enhanced in vivo target tissue selectivity and versatility in photodynamic therapy. Many furocoumarin compounds, such as psoralens, exhibit cytostatic activity when photoactivated but exhibit little in vivo specificity for selectively accumulating in any particular target tissue such as atheromatous plaques. Reactive Oxygen Producing Photosensitizers ("ROPPs") are photoactivatable compounds having an affinity for hyperproliferating cells (such as atheromatous plaque cells), which when photoactivated, produce cytotoxic reaction products. The photoactivity of a ROPP, such as a porphyrin, may be reduced by metalating the porphyrin while the selective affinity of the metalized ROPP for hyperproliferating tissue remains substantially unchanged. By linking a furocoumarin compound to a ROPP to form a F-ROPP, the cytostatic properties of the furocoumarin portion of the F-ROPP can be exploited while the selective affinity of the ROPP portion of the compound for hyperproliferating cells such as atheromatous plaque provides enhanced tissue selectivity without cytotoxicity. In vivo, certain F-ROPPs may be forced to selectively accumulate in a target tissue by illuminating only the target tissue with light having a wavelength operable for photoactivating the F portion of the F-ROPP thereby causing the F-ROPP to either form a monoadduct with or cross-link the cellular DNA in the target tissue. Light of a second wavelength can then be delivered to the target tissue to photoactivate the ROPP portion causing further interference with cellular activity.

Description

PHOTOACTrVATABLE COMPO~NDSFOR T~EPREVENTION OFLNTDMk~L
HYPERrLASLAAiND OT~ER DT~F.A.~

BACKGROUND OF T~E INVENTION

2 1. Field of the Invention 3 This invention relates to photoactivatable compounds and to methods for using 4 the compounds for diagnosing and treating medical conriitior 2. Prior Art 6 Photodynamic Therapy (PDT) is used for treating various ~ e~cPs in~ in~
7 cancer, psoriasis, vascular disease, non-cancerous hyperplastic disease such as benign 8 prostatic hyperplasia, macular degeneration, glaucoma, and certain viral infections.
9 PDT requires conce~ ing a photos~nciti7~or drug in a target tissue then 10 photoactivating the compound with a device which in~ des a light source providing 11 light at a particular wavelength and power level. The drugs ~mini~t~red for PDT are 12 commonly known as photos.?n.citi7~rs (PS) due to their inherent ability to absorb 13 photons of light and transfer that energy to oxygen which then converts to a cytotoxic 14 or cytostatic species. Table 1 presents a list of classes of photosenc~iti7~r compounds 15 cornmonly employed in PDT, which PS's are referred to hereinafter in the alttom~tive 16 as "ROPPs" (Reactive Oxygen Producing PhotosPnciti7~or molecules) and "LEPs"
17 (Light F.mitting Photosensitive molecules). While not ~xh~llctive, the list of PDT
18 photosenc if i7~r drugs presented in Table 1 is exemplary of the variety of ROPPs and 19 LEPs currently used in the art.

The photoactivating device employed for PDT usually comprises a 21 monochromatic light source such as a laser, the light output of which may be coupled to an invasive light delivery catheter for conduction and delivery to a remote target 2 tissue. Such interventional light delivery catheters are well known in the art and are 3 described, for example, in U.S. Patents 5,169,395; 5,196,005; and 5,231,684. Other 4 devices which are frequently used in conjunction with a light source and light delivery catheter include drug delivery devices and/or a balloon pPrfilcion catheter (IJ.S. Patent 6 5,213,576) and/or various medicament-dispensing stents for the slow localized release 7 ofthe photos~ ;7P~~. PDT is presently an approved procedure in Canada, Japan, and 8 The Netherlands for the treatment of various cancers.
9 In addition to cancer therapy, PDT is being tested for the ll e~ l of psoriasis. Extra-corporal PDT of blood is being evaluated for the prevention of intimal 11 hyperplasia following transplant surgery. PDT is also being evaluated for the 12 treatment of vascular disease; most commonly the prevention of intimal hyperplasia 13 following angioplasty. ROPPs are presently in clinical trials for the L~ .ll of 14 cutaneous cancers such as basal cell carcinoma, basal cell nevus syndrome, squamous cell carcinoma, and AIDS related Kaposi's sarcoma. ROPPs are also being 16 investig~ted for the tre~tment of a cancer precursor, Barrett's esophagus. In addition, 17 ROPPs may have utility for treating invasive cancers, cancer precursors, psoriasis, non-18 cancerous urological disorders, viral inactivation, macular degeneration, glaucoma and 19 various vascular diseases.

W O 97/OS127 PCT~US96/12128 Table 1: ROPPs and LEPs ~ Pyrrole-derived macrocyclic compounds T~i~hylills and de~;vdliv~s thereof (11) Naturally occl.nnn~J or synthetic porphyrins phPnt~Y~7inP dyes and derivatives and d~l;vdliv~s thereof (l)* thereof (12) Naturally oc~ . . . ;. .~ or synthetic chlorins Pl .~ ,; . .. c and d~,.ivdliv~ thereof (13) and de~ivdlivtis thereof (2) Ch~lcool~duld~y.yliu.. dyes and d~,liv Naturally occ--rrinlJ or synthetic bacterio- thereof (14) chlorins and derivatives thereof (3) T ia-y~ .. Pc and dt;-iv~Livt;, thereof (15) Synthetic icob~ctPnnrhk~nnc and ~hnd ~ s and de.;vdli~,.,, thereof (16)d~;vdli~ thereof (4) Fluu~sc~les and d~.;v~.L~,~;. thereof (17) Phthalocyanines and derivatives thereof (5) A~d~Jol~Jhyliuls and de~ivdLivti~ thereof (18) N~phth~lr~;y~u~illes and d~l;vdliv~;s RPn7orh1rJrinc and dt;-ivdliv~s thereof (19) thereof (6) Pu~u~hls and d~ivdLivc;:, thereof (20) Pu~l~hy-;~--es and derivatives thereof (7) Chlorophylls and de~;vdliv~s thereof (21) Poll.l.y~;ydl~i--es and de~ivdLives thereof (8) Verdins and derivatives thereof (22) P- IlLd~JLylill and delivdLivt;s thereof (9) Sd~ Lyli ls and dcrivatives thereof (lû) 5 * (m) refers to the compound having molecular structure in-lic~ted at (m) in the 6 specification where m is an integer between 1 and 22.

ROPPs and LEPs such as those in~ic~ted in Table 1, and as illustrated in 2 Figures 1-23, have been shown to selectively ~z~4cc-lmll1z4t~o, both in vifro and in vivo, in 3 catheter in~ ced atheromatous plaques in rabbit and swine models as evidenced by 4 laser in-1uced fluorescence and chemical extraction (HL Narciso, et al, Retention of tin ethyl etiopurpurin (SnET2) by atheromatous plaques: Studies in vitro & in vivo 6 rabbits, Proceedings of 4SPIE: Diagnostic and T*erapeufic Cardiovascular 7 Interve7?tio7ls IY, 1994, 2130:30-41). In vitro studies utili~ing human cadaver aortas 8 demonstrate the passive accumlll~tion of photosPn~iti7~rs such as ROPPs and LEPs 9 into naturally occurring atheromatous plaques. Certain ROPPs and LEPs have the ability to penetrate the nuclear membrane within a cell and to intercalate into the 11 nuclear DNA, particularly ROPPs bearing a positive charge (cationic).
12 Psoralen-type compounds have also been investig~ted for their ability to 13 prevent intimal hyperplasia. Psoralens and other furocoumarins (furane fused to 14 coul~lalill and derivatives thereof) are also photosensitive compounds which have been used in the tre~tment of psoriasis for over 40 years. Such psoralen-based phototherapy 16 is alternatively referred to herein as PWA; Psoralen activated with UltraViolet A
17 light. An exemplary list of some furocourmarin compounds is presented in Table 2.
18 Systemically ~mini~tered psoralen-type compounds penetrate the nuclear membrane 19 of cells and may intercalate with the nuclear DNA in target tissue cells. Following intercalation with the target tissue' s nuclear DNA, the psoralen compound is 21 photoactivated with ultraviolet light or short wavelength visible light (see, for example, 22 FP Gasparro, et al, The excitation of 8-Methoxvpsoralen with visible li~ht: Reversed 23 phase HPLC quantitation of monoadducts and cross-links. P*otoc*em. PhotobioL, 24 1993, ~7(6):1007-1010.), which UV light is preferably delivered only to the target W O 97/05127 PCT~US96/l~I28 tissue by a light delivery catheter or similar delivery device, to cause DNA crosclinking 2 and ~lltim~tely a mutagenic effect in the cells comprising the target tissue. (KL March, 3 et al, 8-Methoxypsoralen and lon~wave ultraviolet irradiation are a novel 4 antiproliferative combination for vascular smooth muscle, Circulation, 1993, 87:184-91; BE Sumpio, et al, Control of smooth muscle cell proliferation by psoralen 6 photochemotherapy~ J. vasG Surg, 1993, 17: 1010-1018; KW Gregory, et al, 7 Photochemotherapy of intimal hyperplasia usin~ psoralen activated by ultraviolet li~ht 8 in a porcine model, Lasers i~t Surg Med., 1994, (Suppl 6):12 Abstract).
9 Furocoumarins are photochemical agents showing potential for both diagnostic and therapeutic applications in mcdir~ine. The DNA cross-linking by furocoumarins 11 such as described above proceeds by a two step process. Following injection of the 12 furocoumarin into the body of an animal, the (planar) furocoumarin molecllle first 13 intercalates within the double helix of intracP~ r DNA or RNA. Following 14 intercalation, the covalent addition of the furocoumarin to the polynucleic acid is achieved through the addition of light energy within the absorption band of the specific 16 furocoumarin. Either furocoumarin -RNA or -DNA mono~c~d~lcts or cross-links may 17 be created upon illllmin~tion of the intercalated species. By forming covalent cross-18 links with base-pair structures, furocoumarins can alter the metabolic activity of a cell 19 and induce cytostasis (GD Cimino, HB Gamper, ST Isaacs, JE Hearst, Psoralens as photoactive probes of nucleic acid structures and function: Or~anic chemi~try. and 21 biochemistry. Ann. Rev. Biochem., 1985, 54: 1154-93).

WO 97/05127 PCT~US96/12128 1 Table2: Furocoumarins~
2 Compounds co.,l~ g Furocoumarin sub-components (23)*
3 Psoralens and dG~;v~L;v~ thereof (24) 4 Isopsoralens (~n~lirinc) and d~;vdl;v~ thereof (25) S Pse~opsor~ll nc and d~;vaL;~ , thereof (26) 6 Pse.lf~ cu~.s.,, ~ nc and d~l;vill;vt;:, thereof (27) 7 ~llopsor~l.?ne and d~:l;val;v~s thereof (28) 8 Pse~ lops~ nc and d~l;vaL;v~s thereof (29) 21 * (m) refers to the compound having the structure indicated at Figure m in the 22 appended figures where m is an integer 23 ~ m < 29.
23 ~ The furocoumarins may be either naturally occurring or synthetic.

Coronary artery disease is thought to be inifi~ted by a disruption of fatty 2 streaks which form early in life on the vessel wall which disruption, in turn, plulnotes 3 ll~c,r,lbus formation. Over time the thrombus becomes o,~,al~ized and provides 4 structure for the ~ccl-m~ tion of fatty lipids, foam cells, chol~st~rol, c~ -m, fibrin, S and collagen. A fibrous cap forms over this collection of lipid-rich ms~tl~ri~l 6 Periodically this fibrous cap ruptures; rtole~in~ some of the lipid-rich m5-tçri~1 and 7 exposing the len~ lg plaque materials to the circulating blood. Growth factors 8 within the blood initiate the migration of smooth muscle cells (SMCs), from the media 9 to the intima where proliferation of the SMCs begins. The ulcerated plaque induces 10 the deposition of platelets and thrombus fonn~tion in a "response to injury" mode.

11 This cycle recurs until eventually the plaque lu~lules, the distal coLunaly artery is 12 occluded by an thrombus and a heart attack occurs (V. Fuster, et al, Clinical-13 Patholo~ical Correlations of Coronarv Disease Pro~ression and Re~ression, 14 Supplement to Circulatio~, Vol. 86, No. 6, 1992:III-1-III-11 and JJ Rar~imon 15 Coronary Atherosclerosis, A Multifactorial Disease, Sl~pplement to Circula~ion, Vol.

16 87, No. 3, 1993 :II-3-II-16).

17 Restenosis occurs when coronary disease is treated with an interventional 18 therapy such as Percutaneous Tr~n~ min~l Coronary Angioplasty, PTCA, or 19 atherectomy, or laser angioplasty, or stenting~ or a myriad of newer terhnologies 20 Restenosis refers to the over- aggressive autogenous repair of an injury to a blood 21 vessel by the body. Intimal hyperplasia or the hyperproliferation of medial (and 22 possibly adventitial) smooth muscle cells (SMCs,) is a major contributing factor to 23 restenosis. Hyperproliferating SMCs form a neo-intima which can reduce the bore of 24 the arterial lumen and thus the capacity of the artery to deliver oxygen rich blood. This WO 97/05127 PCT~US96/12128 reduction in cross-sectional luminal area can be more severe than the original 2 constricted area which was treated. The foregoing problems are l~plese,lLa~iv~ of 3 some medical conditions which the compounds of the present invention may have 4 particular application.
DNA cross-linking by furocoumarins results in the re~uction of smooth muscle 6 cell (SMC) proliferation and, since their DNA cross-linking activity is cytostatic, 7 furocoumarins may have certain advantages over cytotoxic photosenchi7~rs (ROPPs 8 and LEPs) in the prevention of intimal hyperplasia as described by March, et al, U.S.
9 Patent 5,116,864 and Deckelbaum, et al, U.S. Patent 5,354,774 the t~chin~c of which patents are incorporated herein by reference thereto. The cytotoxicity of 11 ROPPs and LEPs currently used in PDT results in the extravasation of intr~c~ r 12 organelles, cytoplasm, and cytokines which, in turn, elicits an i~ hllllll~toly response.
13 The infl~mm~tory response elicited by extravasation of cellular conLel,ls is14 hypotheci7~d as a key contributing factor to restçnocic The disadvantage of 1~ employing psoralens to prevent rçsten~ siC (when con-pa,ed to photos~n.citi7Prs such as 16 ROPPs and LEPs) is that psoralens do not exhibit a selective affinity for atheromatous 17 plaques over normal intimal tissue.

It is a primary object of the present invention to provide a photoactivatable 21 compound which can be used to treat a variety of diseases.
22 It is an object of the present invention to provide a photoactivatable therapeutic 23 compound which causes cytostasis but not cytolysis when bound to a cell and activated 24 with light.
-WO 97/05127 PCT~US96/12128 It is another object of the present invention to provide a photoactivatable 2 compound which has a selective affinity for rapidly proliferating cells.
3 It is still a further object of the present invention to provide a photoactivatable 4 compound which will reduce the incidence of restçnocic following phototherapy of atheromatous plaque.
6 It is a further object of the present invention to provide a photoactivatable 7 compound which can cause cytostasis when activated by a specific wavelength of light.
8 It is still a filrther object of the present invention to provide a photoactivatable 9 compound which can cause cytostasis when activated by one particular wavelength of light and cause cytolysis when activated with light having a dirr~ .L wavelength.
11 It is yet a further object of the present invention to provide a method for 12 treating such di.ce~ces as atherosclerosis, restenosis, cancer, cancer precursors, 13 nonc~ncerous hyperproliferative ~lice~cec~ psoriasis, macular degeneration, gl~coln~
14 and viruses employing photoactivatable compounds.
It is a further object of the present invention to provide a method for employing 16 such photoactivatable compounds for diagnosing such ~lice-~cçs as atherosclerosis, 17 restenosis, cancer, cancer precursors, noncancerous hyperproliferative ~ice~cec 18 psoriasis, m~ r degeneration, glaucoma and viruses.
19 The features of the invention believed to be novel are set forth with particularity in the appended claims. However, the invention itself, both as to 21 composition and manner of use, together with further advantages of these compounds 22 may best be understood by reference to the following description of plerelled 23 embo~limentc.

BRIEF DESCRIPTION OF THE FIGURES
2 Figures 1-22 present the chemical structures of various photosensitive pyrrole-3 derived macrocyclic compounds which exhibit as follows:
4 Figure 1 illustrates the chemical structure of photoactivatable compositions compricing a porphyrin core.
6 Figure 2 shows clorin compounds.
7 Figure 3 shows bacterioclorin-derived compounds.
8 Figure 4 illustrates isobacteriochlorin compounds 9 Figure 5 shows phthalocyanines.
Figure 6 shows naphthalocyanine compounds.
11 Figure 7 illustrates porphycene-cont~inin~ compounds.
12 Figure 8 is porpl,y~;y~nille compounds.
13 Figure 9 is pentaphyrin derivatives.
14 Figure 10 shows sapphryin and derivatives thereof.
Figure 11 illustrates texaphyrin and derivatives thereo~
16 Figure 12 shows the chemical structures of phenoxazine dyes and derivatives 17 thereof.
18 Figure 13 is phenothiazine and derivatives thereof.
19 Figure 14 illustrates chalcoorganapyrylium dyes.
Figure 15 shows triarylmethane derivatives.
21 Figure 16 gives the structure of rhodamine and derivatives thereo~
22 Figure 17 is fluorescene derivatives.
23 Figure 18 shows azaporphyrin and derivatives thereof.
24 Figure 19 shows benzochlorin and derivatives thereof.

W O 97~05127 PCT~US96/12~28 Figure 20 illustrates the structure of purpurin and derivatives thereof.
2 Figure 21 shows chlorophyll and derivatives thereof.
3 Figure 22 is verdin and derivatives thereof.
4 Figure 23 shows the chemical structure of compounds co~ ;";n~ furoc-)ulllalin sub-components.
6 Figure 24 illustrates psoralens and derivatives thereo~
7 Figure 25 shows the structure of isopsoralens (angelicins) and de~;v~lives 8 thereof.
9 Figure 26 is the chemical structure of pseudopsoralens and derivatives thereo~
Figure 27 illustrates the chemical structure of pseudoisopsoralen compounds.
11 Figure 28 shows allopsoralen and derivatives thereo~
12 Figure 29 is pseudoallQpsoralen and derivatives thereof.

14 DESCRIPTION OF T~IE PREFERRED EMBODIMENTS
A problem encountered when using conventional cytotoxic photosç~ el 16 compounds such as those listed in Table 1 for PDT is the post-~minictration 17 infl~mm~tory sequella such as restenosis of a blood vessel. While photose~
18 such as ROPPs and LEPs exhibit çnh~nced selectivity and avidity for rapidly19 proliferating cells in comparison with normal, more q~ sc~nt cells, the cytotoxic and cytolytic activity of such compounds may be undesirable.
21 A problem encountered when using PWA for the tre~tmt~nt of 22 hyperproliferative conditions is that furocoumarins exhibit little, if any, specificity and 23 avidity for hyperproliferative cells over normal cells. Notwith~t~n-ling the folegoing, 24 furocourmarins have the advantage that upon photoactivation with light they may either form a monoadduct to DNA or crosslink the nuclear DNA, thereby rendering 2 the cell quiescent. Such cytostatic activity does not produce ~ orl to the same 3 extent as PDT employing ROPPs and LEPs. A novel class of photos~
4 compounds exhibiting the enh~nced specificity of ROPPs and LEPs for hyperproliferating cells and the photocytostatic activity offurocourmarin compounds is 6 described.
7 The compounds of the present invention form a super-class of compounds 8 characterized by a furocoumarin compound or component thereof, ~ ;vely 9 referred to hereinafter as "F", conjugated with one or more of the followingphotosensitive molecules: (a) a ROPP (Reactive Oxygen Producing Photos~ *~) or 11 a component thereof; or (b) a LEP (Light Fmitting Photosçnciti7Pr) or a component 12 thereof to forrn a F-ROPP or F-LEP. The individual compounds within this super-13 class of compounds are useful for the diagnosis and tre~tm~?nt of a myriad of ~ice~ce 14 as previously described. F-ROPPs contained within this super-class of compounds are classes of compounds cont~ininSJ all possible combinations of any of the compounds 16 set forth in Table 1 conjugated to compounds listed in Table 2. Additional compounds 17 not explicitly listed in Tables 1 and 2 which exhibit the photosensi~ive and/or tissue 18 specificity properties exemplified by ROPPs or LEPs conjugated to furocoumarins (F-19 ROPPs) should be construed as inc~ ded within, and part of, this super-class of compounds. Each class of compound contains a plethora of specific compounds 21 differing only in the particular functional groups ~tt~t~hçd to the basic structure.
22 For example, furocoumarins and derivatives thereof can be conjugated with 23 porphyrins, chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, 24 naphthalocyanines, porphycenes, porphycyanines, pentaphyrin, sapphyrins, W O 97/05127 PCTAUS96/lZlZ8 texaphyrins, phenoxazine dyes, phenothi~7inec, chaloolganapyrylium dyes, 2 rhodamines, fluorescenes, azoporphyrins, benzochlorins, purpurins, chlorophylls, 3 verdins and triarylmethanes and derivatives thereof, thereby creating 23 new classes of 4 compounds. Compounds within each class are conveniently rer~ d by first specifying the particular furocoumarin followed by the particular ROPP or LEPP For example,6 isopsoralen conjugated with chlorin would be isopsor~t hl~ rin 7 As a further example, furocou~l&lins such as naturally occurring or synthetic 8 psoralens, as well as derivatives thereof, can be conjugated with one of the following 9 photosensitive compounds from Table 1: porphyrins, chlorins, bacteriochlorins, synthetic isobacteriochlorins, phthalocyanines, naphthalocyanines, porphycenes, 11 porphycyanines, pentaphyrin, sapphyrins, texaphyrins, phPnox~7ine dyes, 12 phenothi~7inec, chaloorganapyrylium dyes, rho-l~minec, fluorescenes, a7oporphyrins, 13 benzochlorins, purpurins, chlorophylls, verdins and triarylmeth~nPc, as well as 14 derivatives of such photos~ e~ The foregoing conjugates form new classes of compounds which may conveniently be referred to, for example, as: Psoraporphyrins, 16 Psorachlorins, Psora-bacteriochlorins, Psoraisobacteriochlorins, Psor~phth~lncyanines, 17 Psoranaphthalocyanines, Psoraporphycenes, Psorapo,l,hy~;y~nines, PsorapeLaphy.i.l 18 Psorasapphyrins, Psoratexaphyrins, Psoraphenoxa~ine dyes, Psoraphenothi~7in.oc, 19 Psorachaloorgana-pyrylium dyes, Psorarhod~min~?c Psorafluoresc.?ne~s, Psoraa_aporphyrins, Psoraben_o-chlorins, Psorapurpurins, Psorachlorophylls, 21 Psoraverdins, and Psoratriarylmeth~nes, and derivatives thereof, respectively 22 The following examples pr~s~ntins~ the synthesis of particular photos.onciti7~r 23 compounds in accordance with the present invention are lel)~esenlaLi~e of the variety W O 97/0~127 PCTAUS96/12128 of photoactive furocourmain-photoscnciti7çr conjugates which can be made and the 2 conditions therefor.

4 Example 1.
Pvropheophorbide linked 8-MOP. (8-MOP PPhe) 6 Pyropheophorbide (300mg) was dissolved in dry tetrahydrofuran (lOOmL) and 7 1,3-dicyclohexylcarbodiimide (lOOmg) and dimethylaminopyridine (lOOmg) were 8 added. After stirring at room temperature for 15 min., a solution of 5-~minomethyl_8_ 9 methoxypsoralen (250mg) in dry tetrahydrofuran (60mL) was added. The solution was 10 stirred at room temperature overnight. The solvent was removed by rotary 11 evaporation, and the residual solid dissolved in dichlornmefh~n~, washed with dilute 12 HCI then sodium carbonate solution. The organic layer was collected, dried over 13 sodium sulfate, filtered and evaporated to dryness on a rotary evaporator. The crude 14 residue was chromatographed on silica using m.oth~nol / dichlorometh~ne (2%) and the 15 major green band collected and evaporated. The residue, 8 16 Methoxypsorapyropheophoribide (Structure I below), was cryst~lli7ecl from 17 dichloromethane / methanol.

19 Example 2.
Meso-Pyropheophorbide linked 8-MOP. (8-MOP MPPhe) 21 Meso-Pyropheophorbide (300mg) was dissolved in dry tetrahydrofuran 22 (lOOmL) and 1,3-dicyclohexylcarbodiimide (lOOmg) and dimethylaminopyridine 23 (lOOmg) were added. A~Ler stirring at room temperature for 15 min., a solution of S-24 arninomethyl-8-methoxypsoralen (250mg) in dry tetrahydrofuran (60mL) was added.
-W O 97/05127 PCT~US96/12128 The solution was stirred at room temperature overnight. The solvent was removed by 2 rotary evaporation, and the residual solid dissolved in dichlorometh~ne, washed with 3 dilute HCl then sodium carbonate solution. The organic layer was collected, dried over 4 sodium sulfate, filtered and evaporated to dryness on a rotary evaporator. The crude 5 residue was chromatographed on silica using m~th~nol / dichlorometh~ne (2%) and the 6 major green band collected and evaporated. The residue, 8-7 Methoxymesopyropeophoribide (Structure II below), was cryst~lli7ed from 8 dichloromethane / meth~nol.

Example 3.
11 2-(1-Hexyloxyethyl)pyropheophorbidelinked8-MOP. (8-MOPHPPhe) 12 2-(1-Hexyloxyethyl) pyropheophorbide (200mg) was dissolved in dry 13 tetrahydrofuran (100mL) and 1,3-dicyclohexylcarbodiimide (lOOmg) and 14 dimethylaminopyridine (lOOmg) were added. After stirring at room t~ pe-~Lul~ for 15 min., a solution of 5-aminomethyl-8-methoxypsoralen (170mg) in dry tetrahydrofuran 16 (60rnL) was added. The solution was stirred at room temperature overnight. The 17 solvent was removed by rotary evaporation, and the residual solid dissolved in 18 dichloromethane, washed with dilute HCl then sodium carbonate solution. The organic 19 layer was collected, dried over sodium sulfate, filtered and evaporated to dryness on a 20 rotary evaporator. The crude residue was chromatographed on silica using m~th~nol /
21 dichloromethane (2%) and the major green band collected and evaporated. The 22 residue, 8-MOP HPPhe (Structure III), was cryst~lli7ed from dichlorometh~nP /
23 meth~nol.

W O 97/OS127 PCT~US96/12128 1 ExamPle4.
2 Octaethvlbenzochlorin linked 8-MOP. (8-MOP OEBCS) 3 To a stirred solution of octaethylbenzochlorin sulfonylchloride (300mg) in dry 4 dichloromethane (SOmL), was added 5-aminomethyl-8-methoxypsoralen (170mg) indry dichlorometh~ne (20ml) and dry triethylamine (0.1mL). The resulting solution was 6 stirred at room temperature for 1 hr and the solvent removed by rotary evaporation.
7 The crude residue was columned on silica using dichlorom~th~ne and the major grey 8 band collected and recryst~lli7ed from dichlorometh~ne / m~th~nol to give the title 9 compound (Structure IV below).
11 Example 5.
12 Zinc octaethvlbenzochlorin linked 8-MOP. (8-MOP ZnOEBCS) 13 To a stirred solution of octaethylbenzochlorin sulfonylchloride (300mg) in 14 dichlorometh~ne (50mL), was added 5-aminomethyl-8-methoxypsoralen (150mg) indichlorometh~ne (20ml) and dry triethylamine (0.1mL). The resulting solution was16 stirred at room temperature for 1 hr. Zinc acetate (200mg) dissolved in m~th~nol 17 (lOmL) was added to the reaction solution and the solution was warmed on a hot 18 water bath until metallation of the benzochlorin was complete by Uv / vis spectroscopy 19 (as seen by a band I absorption at 673nm). The solvent was then removed by rotary evaporation and the crude residue redissolved in dichloromPth~ne (5mL) and 21 chromatographed on silica using dichloromethane. The major green band collected and 22 recrystallized from dichloromethane / methanol to give the title compound (Structure 23 V below).

Example 6.
2 Cu iminium octaethylbenzochlorin linked 8-MOP. (8-MOP Cu Im OEBCS) 3 To copper octaethylbenzochlorin sulfonic acid (300mg) dissolved in 4 dichloromethane (100rnL) was added (chloromethylene) dimethylammonium chloride S (500mg) and the solution stirred overnight at room temperature, protected ~om 6 moisture. The solution was poured into ice cold water quickly, the organic layer 7 washed with water rapidly, separated and dried over sodium sulfate. The solution was 8 filtered to remove sodium sulfate and S-aminomethyl-8-methoxypsoralen (200mg) in 9 dichloromethane (20mL) was added. The solution was stirred for 20 minutes at room temperature, then poured into water. The organic layer was washed with dilute HC1 11 and dried over sodium sulfate. The solution was filtered and evaporated to dryness.
12 The resulting reside was chlorl.alographed on silica using 2% m~th~n~
13 dichloromethane and the major green band collected and evaporated. The title14 compound (Structure VI below) was obtained as a green powder by precipitation from lS dichloromethane / hexane.

17 Example 7.
18 Indium texaphyrin linked 8-MOP. (8-MOP InT) 19 To a solution of Indium texaphyrin-16-carboxylic acid (200mg) was dissolved in dry terahydrofuran (50mL) and 1,3-dicyclohexylcarbodiimide (SOmg) and 21 dimethylaminopyridine (50mg) added. After stirring at room temperature for 15 min., a 22 solution of S-aminomethyl-8-methoxypsoralen (lOOmg) in dry terahydrofuran (20mL) 23 was added. The solution was stirred under argon at room temperature overnight. The 24 solvent was removed by rotary evaporation, and the residual solid dissolved in dichloromethane and washed with dilute HCl and finally with water. The organic phase 2 was separated, dried over sodium sulfate, revaporated under reduced pLes~ule and 3 chromatographed on silica using methanol / dichlorometh~n~ (2%). The major green 4 band was collected and evaporated. The residue, 8-MOP InT (Structure VIII below), 5 was cryst~lli7ed from dichlorometh~ne / hexane.

7 Example 8.
8 Protoporphvrin linked 8-MOP. (8-MOP PP~
9 Protoporphyrin (200mg) was dissolved in oxalyl chloride (3mL) and the solution warmed at 40~C for Ihr, while being protected from moisture. The excess 11 oxalyl chloride was removed under high vacuum and dry dichlorometh~ne (5mL) was 12 added. This was also removed under high vacuum, to give a purple residue that was 13 protected from moisture via a drying tube. Dry dichlorometh~ne (10mL) and dry 14 triethylamine (lmL) were added to the residue, followed by a solution of 5-aminomethyl-8-methoxypsoralen (160mg) in dry dichlorometh~ne (20mL). The 16 solution was stirred overnight, protected from moisture via a drying tube. The solution 17 was then poured into water and the organic phase washed well with water, collected 18 and dried over sodium sulfate. After filtration and evaporation to dryness, the resulting 19 residue was columned on silica using 2% acetone / dichlorometh~ne as eluent. The major red band was collected and recryst~lli7ed from dichlorometh~n~ / m~th~nol to 21 yield the title compound VIII.

WO 97/0~127 PCTAUS96/12I~8 Example 9.
2 Tetraphenvlporphyrin linked 8-MOP. (8-MOP TPP) 3 Meso-terakis-(4'-carboxyphenyl) porphyrin (200mg) was dissolved in oxalyl 4 chloride (SrnL) and the solution warmed at 40~C for Ihr, while being protected from moisture. The excess oxalyl chloride was removed under high vacuum and dry 6 dichlorometh~ne (5mL) was added. This was also removed under high vacuum, to7 give a green residue that was protected from moisture via a drying tube. Dry8 dichloromethane (lOmL) and dry triethylamine (ImL) were added to the residue and a 9 solution of 5-aminomethyl-8-methoxypsoralen (400mg) in dry dichloromPth~n~
(20mL) was added. The solution was stirred overnight, protected from moisture via a 11 drying tube. The solution was then poured into water and the organic phase washed 12 well with water, collected and dried over sodium sulfate. A~er filtration and 13 evaporation to dryness, the resulting residue was columned on silica using 2% acetone 14 / dichlorometh~ne as eluent. The major red band comprised 8-MOP TPP (Structure IX) and was collected and recryst~lli7ed from dichlorom~h~ne / m-oth~nol.

17 Example 10.
18 2.8,12,18-Tetraethvl-3.7,13,17-tetramethyl-5,15-bist2'-fi~ran~ porphyrin. (5.15-19 DFP!.
4,4'-Diethyl-3,3'-dimethyl-2,2'-dil~yllyllllethane (4.0g) and 2-furaldehyde 21 (1.67g) were dissolved in meth~nt~l (lOOmL) and the solution deaerated by bubbling 22 with argon for 15min. 4-Toluenesulfonic acid (0.9Sg) was added and the solution 23 stirred for 2hrs in the dark, then refrigerated overnight. The precipitated porphyrinogen 24 was collected, washed with ice cold methanol (20mL) and resuspended in m~th~nol W O 97/05127 PCT~US96/12128 (lOOmL). o-Chloranil (6.0g) was added and the solution stirred in the dark for 2hrs.
2 Triethylamine (2mL) was added and the precipitated porphyrin was collected by 3 filtration, washed well with methanol and dried under high vacuum. The porphyrin was 4 recryst~lli7ed from dichloromethane / meth~nQI to yield the title compound (X).

s 6 ExamPle 11.
7 Texas red linked 8-MOP. (8-MOP TR) 8 Sulforhodamine 101 acid chloride (200mg) was dissolved in dry 9 tetrahydrofuran (lOOmL) and 5-aminomethyl-8-metho~y~so~len (lOOmg) added, followed by triethylamine (O. lmL). The solution was left overnight at room 11 telllpe~ re. The following day the solution was evaporated to dryness, redissolved in 12 dichlorometh~ne and columned on silica using 2% meth~nnl / dichloromçth~ne as 13 eluent. The major fluorescent red fraction was collected and evaporated to dryness.
14 The residue, comprising 8-MOP TR (Structure XI) was recryst~lli7ed from ~ dichloromethane / hexane.

17 Example 12.
18 Rhodamine B linked 8-MOP. (8-MOP RB) 19 Sulforhodamine B acid chloride (200mg) was dissolved in dry tetrahydrofuran 20 (lOOmL) and 5-aminomethyl-8-methoxypsoralen (lOOmg) added, followed by dry 21 triethylamine (O.lml). The solution was left overnight at room temperature. The 22 following day the solution was evaporated to dryness, redissolved in dichloromçth~ne 23 and columned on silica using 2% methanol / dichloromethane as eluent. The major WO 97/05127 PCT~US96/12128 fluorescent red fraction was collected and evaporated to' dryness. The residue 2 (Structure XII) was recryst~lli7~d from dichloromethane / hexane.
4 Example 13.
S Porphocyanine linked 8-MOP. (8-MOP Pocy!
6 2,3,21,22-tetraethyl-12-(4'-carboxyphenyl) polphocyanine (200mg) was 7 dissolved in dry tetrahydrofilran (lOOmL) and 1,3-dicyclohexylcarbodiimide (lOOmg) 8 and dimethylaminopyridine (lOOmg) were added. After stirring at room te~ Gl~lult;
9 for 15 min., a solution of 5-aminomethyl-8-methoxypsoralen (300mg) in dry tetrahydrofuran (60mL) was added. The solution was stirred at room telllpel~luie11 overnight. The solvent was removed by rotary evaporation, and the residual solid 12 dissolved in dichlorometh~nç, washed with dilute HCl then sodium carbonate sQl-ltion 13 The organic layer was collected, dried over sodium sulfate, filtered and evaporated to 14 dryness on a rotary evaporator. The crude residue was ch,ulll~ographed on silica using methanol / dichloromethane (2%) and the major green band collected and 16 evaporated. The residue (Structure XIII) was cryst~lli7Pd from dichloloe~h~ne /
17 mPth~n~

19 Example 14.
Phthalocvanine linked 8-MOP. (8-MOPPth) 21 Phthalocyanine tetra sulfonate (200mg) was dissolved in phosphorus 22 oxychloride (20mL) and the solution refluxed for 2 hrs. The excess phosphorus 23 oxychloride was removed by rotary evaporation and the residue dissolved in dry, cold 24 pyridine (lOmL). A solution of S-aminomethyl-8-methoxypsoralen (300mg) in dry .

WO 97/05127 PCT~US96/12128 pyridine (60mL) was added. The solution was stirred at room temperature overnight.
2 The solvent was removed by rotary evaporation, and the residual solid dissolved in 3 dichloromethane, washed with dilute HCl then sodium carbonate solution. The organic 4 layer was collected, dried over sodium sulfate, filtered and evaporated to dryness on a S rotary evaporator. The crude residue was chromatographed on silica using meth~nol /
6 dichlorom~th~ne (5%) and the major green band collected and evaporated. The residue 7 (Structure XIV) was crystallized from dichlorom~th~ne / m~th~nn =

WO 97/05127 PCTAUS96/lZlZ8 ~

N N - ~\ N N - ~\

CONH ~ CONH

lo ~u~

(8-MOP PPhe) 8-MOP HPPhe III

CO~NH

'~J
~ 8-MOP InT
8-MOP PPhe VII
II

W O 97/05127 PCT~US96/12128 DN~

5O2NH ~ SOzNH /

8-M OP ZnOEBCS
8-M OP CuIm OEBCS

\r~S03 ~~

SOzNH ~ ~ 50zNH

IV ~1 o/

~I

CA 02227808 l998-0l-26 W O 97/05127 PCT~US96/12128 - ~,~ , ~ CO2NH/~

~ CO2NH

VIII ~/

~o/

o Co.~

~o\

IX

5, 15-DFP
X

~/HNOzS~ ~/

o N N

--\HNO S/~ J~502NH
~ 0/
8-MOP Pth o XlV

_<~ j/~'N ~ ~ ~ ~

~H ~ ~)~ \~O/

8-MOP Pocy ~II

W O 97/~5127 PCT~US96/1212 The preceding super-class of photos~nci~i7ing compounds may be characterized 2 by: a) a fi~rocoumarin ~tt~ched to a Reactive Oxygen Producing Photose~ l type 3 compound, F-ROPP; b) a furocoumarin sub-component ~tt~ch~ to a ROPP, FS-4 ROPP; c) a cationic filrocoumarin attached to an ROPP (neutral or cationic), to produce either CF-ROPP or CFS-ROPP; d) a cationic ROPP att~çhPd to a 6 furocoumarin (neutral or cationic); e) any one of the above compounds wherein the 7 ROPP is met~li7e~1; and f) a furocoumarin conjugated ~,vith a light ~ g 8 photos~n.citi7~r, F-LEP.
9 The foregoing super-class of conjugated compounds can be used to treat a variety of cii~ es such as atherosclerosis, restenosis, cancer, cancer precursors, non-11 cancerous hyperproliferative t~ es, psoriasis, macular degeneration, glaucoma, and 12 certain viruses. These compounds are light activatable drugs which may or may not be 13 photodynamically active (i.e. produce singlet oxygen and/or oxygen radicals to mediate 14 cytotoxicity), but will be photoactive (i.e. exhibit photorh~mic~l cross-linking with DNA or RNA or the production of monoadducts of the compound therewith) to 16 modulate the metabolic activity of cells. More specifically, these novel photoactive 17 compounds will retain the ability of the ROPP or LEP to localize to a greater extent in 18 the target tissue and the ability of the furocoumarin (such as psoralen) to intercalate 19 into target tissue DNA and forrn cross-linked and/or monoadducts adducts upon the addition of light energy.
21 Previous studies indicate that utilizing a cationic ROPP or LEP to synthe~i7e a 22 CF-ROPP or CF-LEP facilitates the intercalation of the compound into target cell 23 DNA. Once the F-ROPP or CF-ROPP is localized in target cells, light activation can 24 be used therapeutically and/or diagnostically. The use of these novel compounds for WO 97/05127 PCT~US96/12128 the detection and/or treatment and the prevention of restenosis and intimal hyperplasia 2 following cardiac transplantation surgery (or AV shunt procedures such as dialysis) is 3 an exemplary application which is ~ c~ssed in particular detail to teach and illustrate a 4 use for the invention, but it should be kept in mind that such an application is illustrative and should not be construed as a lirnitation of this invention.
6 For example, another application for the photos~ r compounds described 7 herein is the light activated tre~tmçnt of a target tissue which does not selectively 8 concentrate either ROPPs or furocoumarins. An F-ROPP, selected as described below 9 from the super-class of compounds described above, can be atlminict~red systPmic~lly 10 to a biological organism, which organism could be an animal, a plant or even a single 11 cell or a polynucleic acid fragment. Following systemic a~mini~tration of the F-ROPP, 12 and while the F-ROPP is present in the animal's serum, a light source operating at a 13 strong absorption wavelength of the furocoumarin co~ onellL of the F-ROPP, is 14 directed toward the volume of target tissue in which high concentrations of the F-15 ROPP are desired. The selection of the particular furocoumarin used in the F-ROPP is 16 preferably a species which creates mono-adducts with polynucleic acids when activated 17 with W or short wavelength visible light. By ~lmini~t~rinp the activating light to the 18 target tissue, mono-adducts of F-ROPPs with DNA and RNA are formed. Increasing 19 the intensity of the activating light delivered to the target tissue increases the DNA-bound F-ROPP therein. When the F-ROPP reaches the desired conce~ Lion in the 21 target tissue, a longer wavelength of light which activates the ROPP portion of the F-22 ROPP may be used to photoactivate the cell bound F-ROPP in the target tissues to 23 selectively destroy or modify the target tissue. In effect, the F-ROPP creates a light-24 induced selectivity of the F-ROPP for binding to the target tissue because only the W O 97/05127 PCTtUS96tl2128 targe~ tissue is illllmin~ted with the shorter wavelength of light thereby causing 2 covalent bonding of F-ROPP only in the DNA/RNA of the target tissue.
3 While particular embodiments of the present invention have been illustrated and 4 described, it would be obvious to those skilled in the art that various other changes and S modifications can be made without departing from the spirit and scope of the 6 invention. It is therefore intended to cover in the impending claims all such changes 7 and modifications that are within the scope of this invention.

Claims (10)

1. Photoactive compounds comprising a functional furocoumarin conjugated with a photosensitive benzochlorin compound.

2. The photoactive compounds of Claim 1 wherein the photosensitive benzochlorin compound is cationic.

3. The photoactive compound of Claim 1 wherein said photosensitive compound comprises a metal coordinated to a benzochlorin molecule.

4. The photoactive compound of Claim 3 wherein the metal is selected from the group consisting of copper, aluminum, tin, zinc, gadolinium, manganese, magnesium or iron.

5. The photoactive compounds of Claim 1 wherein said functional furocoumarin comprises a psoralen.

6. A photoactive compound having the structure R-R1 wherein R comprises a furocoumarin and wherein R1 is a reactive oxygen-producing benzochlorin compound.

7. A photoactive compound in accordance Claim 9 wherein R1 is a light-emitting benzochlorin compound.

8. A photoactive compound in accordance Claim 9 wherein said furocoumarin is selected from the group consisting of compounds comprising isopsoralen, pseudopsoralen, pseudoisopsoralen, allopsoralen and pseudoallopsoralen; or derivatives thereof.

9. A photoactive composition for treating diseased target tissue cells within an organism, said photoactive composition having the form R-R' wherein R is a photoactivatable furocoumarin compound which covalently bonds to target tissue cells only when the furocoumarin compound is photoactivated with light having a first wavelength, and R' is a photosensitive benzochlorin compound which interferes with normal cellular activity within the diseased target tissue only when photoactivated with light having a second wavelength, which second wavelength is different from said first wavelength.

10. Photoactive compounds comprising a functional furocoumarin conjugated with a photosensitive pyrrole-derived macrocyclic compound.