Background of the Invention

Replacement of defective or severely injured tissues and organs has been a medical objective as long as medi- cine has been practiced. Grafts from an individual to himself almost invariably succeed, and are especially important in the treatment of burn patients. Likewise, grafts between two genetically identical individuals almost invariably succeed. However, grafts between two genetically dissimilar individuals would not succeed without immunosuppressive drug therapies. The major reason for their failure is a T cell mediated immune response to cell-surface antigens that distinguish donor from host. Immunosuppressive agents are also indicated in the treatment of autoimmune diseases such as rheumatoid ar¬ thritis or type I diabetes mellitus. One particular condition worth mentioning here is psoriasis. This dis¬ ease is characterized by erythematous patches of skin accompanied by discomfort and itching. Hyperplasia of the epidermis involving proliferation of keratinocytes is also a hallmark feature of psoriasis. An inflammatory compo¬ nent is suggested by: (i) the finding of lymphocytic infiltration of epidermis, and (ii) the fact that immuno- suppressive agents such as cyclosporin and corticosteroids have beneficial effect on the disease.

A number of drugs are currently being used or inves¬ tigated for their immunosuppressive properties. Among these drugs, the most commonly used immunosuppressant is cyclosporin A. However, usage of cyclosporin has numerous side effects such as nephrotoxicity, hepatotoxicity and other central nervous system disorders. Thus, there is presently a need to investigate new immunosuppressive

agents that are less toxic but equally as effective as those currently available.

Summary of the Invention

This invention relates to ruthenium complexes and their use as immunosuppressive agents to prevent or sig¬ nificantly reduce graft rejection in organ and bone marrow transplantation. The ruthenium complexes can also be used as an immunosuppressant drug for T lymphocyte mediated autoimmune diseases, such as diabetes, rheumatoid arthri- tis, multiple sclerosis, lupus erythematosus and steroid resistant asthma.

In another aspect, other diseases with suspected inflammatory components, such as psoriasis, contact derma¬ titis and hyperplasia of the epidermis, can be treated with a ruthenium complex of this invention to alleviate symptoms associated with these disease states.

It has also been demonstrated that the ruthenium complexes have antiproliferative properties and in partic¬ ular can inhibit cardiac smooth muscle cells. Based upon this, the ruthenium complexes can be used for the treat¬ ment of hyperproliferative vascular disorders, such as restenosis and atherosclerosis.

Detailed Description of the Invention

This invention is based upon the discovery that ruthenium complexes can inhibit antigen specific T lympho¬ cyte proliferation in vitro . The data suggest that ruthe¬ nium complexes have potential use as immunosuppressants to reduce undesirable immune responses in humans. Ruthenium complexes can be used to facilitate organ transplantation, and to treat human autoimmune disorders where the specific activation of T cells is responsible for, or contributes to the pathology and progression of the diseases, such as

diabetes, rheumatoid arthritis, multiple sclerosis, lupus erythematosus and steroid resistant asthma.

This invention pertains to ruthenium complexes that have immunosuppressive properties of the general formula:

[RuM-^TVP- _. Z

and physiologically acceptable salts thereof; wherein Ru is ruthenium having an oxidation state of 2 , 3 or 4 ; wherein M is a monodentate ligand selected from the group consisting of nitrogen containing ligands, phospho¬ rus containing ligands, sulfur containing ligands, carbon containing ligands, oxygen containing ligands and halide (e.g., F, Br, Cl, I); wherein m is 0, 1, 2, 3, 4 or 6; wherein b is 0, 1, 2 or 3; wherein t is 0, 1 or 2 ; wherein p is 0 or 1; wherein B is a bidentate ligand selected from the group consisting of aliphatic amines, heterocyclic aromat- ic amines, sulfur containing ligands, carbon containing ligands, oxygen containing ligands and phosphorus contain¬ ing ligands; wherein T is a tridentate ligand selected from the group consisting of nitrogen containing ligands, sulfur containing ligands, carbon containing ligands, oxygen containing ligands and phosphorus containing ligands; wherein P is a polydentate ligand selected from the group consisting of nitrogen containing ligands, carbon containing ligands, oxygen containing ligands, sulfur containing ligands and phosphorus containing ligands; wherein when the complex is charged, then Z is at least one counterion of appropriate charge to render the

overall charge of the complex neutral, for example a counterion selected from the group consisting of F ^~ , Cl ^~ , Br ^" , I ^" , NO, ^" , NH ₄ ⁺ , NR' ₄ ⁺ , PF ₆ ^~ , BP ₄ -, S0 ₄ ^"2 , S _% ² , S ₂ 0 ₇ ^"2 , RuCl ₄ ^"2 , K ⁺ , Na ⁺ , Li ⁺ , C10 ₄ ^_ and R'lmH ⁺ , where Im is imidaz- ole; and wherein R ¹ is a linear or branched alkyl (e.g., 1 to 8 carbon atoms) or aryl.

In a preferred embodiment, novel ruthenium complexes are represented by the general formula:

[RuM ₆ ]Z

and physiologically acceptable salts thereof; wherein M is the same or different and is indepen¬ dently a heterocyclic aromatic amine, provided that for novel complexes of this formula the ligands cannot be 6- membered aromatic rings containing one or more nitrogens, such as pyridine, pyrazine, pyridazine or pyrimidine, or derivatives of these.

In another embodiment, novel ruthenium complexes have the general formula:

[Ru(NH,) ₃ M _m B _b T _l ]Z

and physiologically acceptable salts thereof; wherein Ru is ruthenium having an oxidation state of 2 or 3; wherein M, B and T are ligands (i.e., monodentate, bidentate and tridentate, respectively) that are heterocy¬ clic aromatic amines (e.g., substituted or unsubstituted imidazole as defined below) coordinated to the ruthenium through aromatic nitrogens; provided that the ligand is not pyridine; wherein m is 0, 1 or 3; wherein b is 0 or 1; and

wherein t is 0 or 1.

Examples of complexes which are covered by this formula are:

[Ru(NH ₃ ) ₃ (Im) ₃ ]Cl ₃ [Ru(NH ₃ ) ₃ (l- eIm) ₃ ]Cl ₃ .

The coordination sphere of the metal center may contain all six ligands (referred to as monodentate) to be equivalent or a mixture of different ligands. The mixture of ligands can consist of different monodentate ligands; a mixture of bidentate/monodentate in a ratio of 1:4 or 2:2; three bidentate ligands; a mixture of bidentate/ tridentate/monodentate in a ratio of 1:1:1; two tridentate ligands; or tridentate/monodentate in a 1:3 ratio; or a mixture of polydentate and bidentate in a ratio of 1:1; or a mixture of polydentate/monodentate in a 1:1 or 1:2 ratio depending on the nature of the polydentate ligand.

For the purposes of this application, the terms "monodentate", "bidentate" and "tridentate" will have their generally accepted meaning in the art. That is, a monodentate ligand is defined as a chemical moiety or group which has one potential coordinating atom. More than one potential coordinating atom is termed a multi- dentate ligand where the number of potential coordinating atoms is indicated by the terms bidentate, tridentate, etc. Ligands that are protonated are well within the scope of the invention.

Ruthenium complexes of this invention can contain a ruthenium metal center of different oxidation states, e.g., Ru(II) , Ru(III) or Ru(IV) . Depending upon the ligands, the complex can inherently be neutral, i.e. , it will not require counterion(s) to neutralize the overall charge of the complex. Alternatively, the complex can

contain counterion(s) of appropriate charge to render the overall charge of the complex neutral and optionally to enhance solubility of the complex in a physiological environment. The number of counterions (e.g., 1 to 5 counterions that are the same or different from each other) will be that which is required to render the over¬ all charge of the complex. Counterions which result in physiologically acceptable salts of the complexes, includ¬ ing protonated salts thereof, are within the scope of this invention and include but are not limited to salts derived from inorganic cations such as sodium (Na ⁺ ) , potassium (K ⁺ ) , lithium (Li ⁺ ) , and the like; organic bases such as mono-, di- and trialkyl amines of 1-8 carbon atoms, per alkyl group and mono-, di and trihydroxyalkyl amines of 1- 8 carbon atoms per alkyl group, and the like; and organic and inorganic acids such as acetic, lactic, citric, tar- taric, succinic, maleic, malonic, gluconic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesul¬ fonic, and similarly known acceptable acids. Some specif- ic examples are listed above under the definition for Z.

In one embodiment, the ruthenium complex can comprise six monodentate ligands which can contain nitrogen (e.g., heterocyclic aromatic amines, aliphatic amines), sulfur, phosphorus or oxygen groups. Examples of suitable ligands include but are not limited to imidazole, pyridine, ammo¬ nia, triazole, picoline, pyrazole, quinoline, pyrazine, pyridazine, pyrimidine, quinoxaline, quinazoline, isoquin- azoline, tetrahydroquinoline, tetrahydroquinazoline, tetrahydroquinoxaline, piperidine, phosphine, phosphite, thiolate, sulfoxide, alkoxide, phenolate and carboxylate. Derivatives of these ligands can also be incorporated into the complex in various combinations with the non-substi¬ tuted ligands. A derivative is a ligand in which one or more of the hydrogen atoms has been substituted with a

moiety, such as C1-C8 alkyl, C2-C8 alkenyl, hydroxy, nitro, amino, carboxyl, ester, di-Cl-C8 alkyl amine, phenyl, benzyl, imidazole and combinations of these.

Preferred ligands are imidazole derivatives having the general formula:

where R ² and R ³ are independently selected from the group consisting of aryl, heteroaryl, linear and branched (e.g., 1 to 8 carbons) alkyl, -C(0)H, -(CH ₂ )„COOR', -(CH ₂ ) _n SH, -(CH ₂ ) _n OH, -(CH ₂ ) _n NH ₂ , -(CH ₂ ) _n OS0 ₃ , -(CH ₂ ) _n COH, -(CH ₂ ) _n COR', -(CH ₂ ) _n CONR', -(CH ₂ ) _n COOH, - (CH ₂ ) _n CH(X) (Y) , H, Cl, Br, F, I and N0 ₂ ; where R ¹ is a linear or branched alkyl (e.g., 1 to 8 carbon atoms) or aryl group; X is NH ₂ ; Y is COOH and n is 0 to 8. Ligands which are protonated are also con¬ templated herein. Preferred ligands also include pyridine derivatives having the following general formula:

wherein R ¹ is selected from the substituents defined above for R ₂ and R ₃ .

Examples of preferred ruthenium complexes having monodentate ligands are listed below.

[Ru(Im) ₆ ]Cl ₂ where Im = imidazole

[Ru(l-MeIm) ₆ ]Cl ₂ where l-Melm = 1-methyl imidazole

[Ru(l-Melm) ₆ ] (PF ₆ ) ₃

[Ru ( l-MeIm) ₆ ] Cl ₃

[Ru(Im) ₆ ]Cl ₃ trans- [Ru(NH ₃ ) ₄ (Im) (py) ]C1 ₃ where py = pyridine cis- [ Ru (NH ₃ ) ₄ ( im) ₂ ] Cl ₃ trans- [Ru (NH ₃ ) ₄ (Im) ₂ ] Cl ₃ [Ru(NH ₃ ) ₅ (L-his) ]C1 ₃ where his = histidine

[Ru(NH ₃ ) ₅ (py)]Cl(RuCl ₄ ) cis-[Ru(NH ₃ ) ₄ (py) ₂ ]Cl ₃

[Ru(NH ₃ ) ₅ (4-pic) ]C1 ₃ where pic = picoline cis-[Ru(NH ₃ ) ₄ (l-MeIm) ₂ ]Cl ₃

In another embodiment, a ruthenium complex can be made having multidentate ligands, in combination with other multidentate ligands and/or monodentate ligands. Suitable bidentate ligands (B) will include, but are not limited to, aliphatic amines (e.g., ethylene diamine, propylene diamine, 1, 2-cyclohexane diamine and the corre¬ sponding alkylated amines thereof) ; heterocyclic aromatic amines (e.g., 2, 2'-bipyridine, 1, 10-phenanthroline) ; pyridine based ligands (e.g., 2-aminopicoline) ; pyrazole based ligands (e.g., potassium-bis-pyrazolyl borate, bis- pyrazolyl methane); carboxylates; and bis-phosphines

(e.g., 1, 2-bis(dimethylphosphino) ethane) . Preferred are imidazole based ligands having the general formula:

where R ⁴ to R ⁹ are the same or different and are indepen¬ dently selected from the substituents defined above for R ² and R ³ .

The ligand can be a tridentate ligand (T) such as aromatic heterocyclic amines (e.g., 2, 2' , 6" , 2"-terpyri- dine, bis- (2-pyridylmethyl) amine) ; imidazole based ligands

(e.g., bis- (2-imidazolylmethyl) amine) ; pyrazole based ligands (e.g., potassium tris pyrazolyl borate) ; macro- cyclic amines (e.g., 1,4 , 7-triazacyclononane) ; macrocyclic sulfur based ligands (e.g., 1,4 ,7-trithiacyclononane and 2-(arylazophenyl)thio ether); and macrocyclic oxygen containing ligands Na{ (C ₅ H _S ) Co[P(O)R ₂ ] ₃ } •

The ligand can be a polydentate ligand (P) such as nitrogen containing ligands (e.g., 1,4 ,7, 10-tetraazacyclo- dodecane; 1, 4 , 8, 11-tetraazacyclotetradecane; 1,3,5,7- tetrakis-(2-(4-sec-butylpyridyl) imino) benzodipyrrole; 3, 6, 10, 13, 16, 19-hexaazabicyclo[6.6.6]eicosane; and, 1,4,8, 11-tetrakis- (2-pyridylmethyl) -1,4,8, 11-tetraaza¬ cyclotetradecane) ; sulfur containing ligands (e.g., 1 , , 7 , 10-tetrathiacyclotridecane and 1, 4 , 8, 11-tetrathia- cyclotetradecane) ; and phosphorus containing ligands

(e.g. , , '-bis- (bis- (2-biphenylphosphino) ethyl) amino) - ethane and α, a '-bis-(bis-(2-diphenylphosphino)m-xylene) .

The invention also pertains to di ers and trimers of the ruthenium complexes described above. The coordination sphere of the metal center contain monodentate ligands

(that are the same or different from each other) or it can contains a mixture of monodentate, bidentate and/or tri¬ dentate ligands. The counterions are the same as those described above. Dimers will have the general formula:

[(RuM _m B _h T _t )-0-(RuM' _m .B' _h .TV)]Z

wherein the variables are described above and further wherein m and m' are independently 0, 1, 2, 3 or 5; b and b' are independently 0, 1 or 2; and t and t' are independently 0 or 1.

Trimers will have the general formula:

[(RuM _m B _b T _l )-0-(RuM" _m ,.B" _h ..T" _t ^„ )-0-(RuM' _m ^, B' _b .T' _t .)]Z

wherein the variables are described above and further wherein m and m' are independently 0, 1, 2, 3 or 5; " is 0, 1, 2 or 4; b, b' and b" are independently 0, 1 or 2; and t, t' and t" are independently 0 or 1. General procedures for making monomeric ruthenium complexes include: Vogt, Jr. et a_l. , Inorq. Chem. , 4 _. : 1157 (1965) ; Ford et aL. , J. Am. Chem. Soc.. 9_0:1187 (1968) ; Marchant et a_l. , Inorq. Chem. , 16:2160 (1977); Sullivan et al.. Inorq. Chem. , 12:3334 (1978) ; Klassen et al. , Inorq. Chem. , 12:1977 (1980) ; Klassen et al. , Inorq. Chem., JL4 _. :2733 (1975) ; Leising et _ ., Inorq. Chem. , 22=4569, (1990) ; Bessel et a_l. , J. Chem. Soc.. Dalton trans. , pp. 1563 (1993) ; Bernhard and Sargeson, J. Chem. Soc. Chem. Co mun. , pp. 1516 (1985) ; Poon and Che, J__ Chem. Soc, Dalton trans. , pp. 491 (1981) ; Walker and Taube, Inorq. Chem.. 12: 828 (1981) ; Mazzetto et al. , Polyhedron. 12:971 (1993) ; Khan et al. , Inorq. Chi . Acta, 189: 165 (1991) ; Keppler et al. , Inorq. Chem. , 26:844 (1987) ; and Kraus, Inorq. Chim. Acta.. 2 209 (1977) . General procedures for making dimeric and trimeric ruthe¬ nium complexes include: Dopplet and Meyer, Inorq. Chem.. 2 _. 6:2027 (1987) ; Geselowitz et al. , Inorq. Chem.. 25:2015 (1986); Neubold et al. , Inorq. Chem.. 2 459 (1989); Sasaki et aj . , J. Am. Chem. Soc.. 110: 6251 (1988) ; Smith et a_l. , Inorq. Chem. , 10:1943 (1971) ; Sudha et a_l. , J. Am. Chem. Soc.. 2 380 (1993) ; Weaver et a!., J. Am. Che . Soc.. 22:3039 (1975) and Emerson et a_l. , J. Am. Chem. Soc.. 115:11799 (1993) .

-li¬ lt has now been discovered that the ruthenium com¬ plexes of this invention possess immunosuppressive activi¬ ty as confirmed through a drug screen. Specific T cell proliferation was measured in response to antigen exposure in the presence or absence of ruthenium complexes. It was found that ruthenium complexes inhibited T cell prolifera¬ tion by 50% (IC ₅ „) at a concentration of about 1 to 100 nM. This compares favorably with cyclosporin A, which has an IC ₅₀ at 20nM (Table 1) . Ruthenium complexes can be administered orally, parenterally (e.g. intramuscularly, intravenously, subcu- taneously) , topically, nasally or via slow releasing microcarriers in dosage formulations (e.g. , therapeutical- ly effective amount) containing a physiologically accept- able vehicle and optional adjuvants and preservatives. Suitable physiologically acceptable vehicles include saline, sterile water, creams, ointments, solutions, gels, pastes, emulsions, lotions, oils, solid carriers and aerosols. Ruthenium complexes can be applied topically as a cream or ointment to locally deliver immunosuppressive concentrations of the drug without significant systemic exposure. Topical application may be the ideal way to deliver the compound in psoriasis and other inflammatory skin diseases, such as contact dermatitis and pemphigus vulgariε.

The specific dosage level of active ingredient will depend upon a number of factors, ■ including biological activity of the ruthenium complexes, age, body weight, sex, general health, severity of the particular disease to be treated and the degree of immune suppression desired, as well as appropriate pharmacokinetic properties. It should be understood that ruthenium complexes can be

administered to mammals other than humans for immunosup- pression of mammalian autoimmune diseases.

Ruthenium complexes can be administered in combina¬ tion with other drugs to boost the immunosuppressive effect. Compounds that can be coadministered include steroids (e.g. methyl prednisolone acetate) , NSAIDS and other known immunosuppressants such as azathioprine, 15- deoxyspergualin, cyclosporin, mizoribine, mycophenolate mofetil, brequinar sodium, leflunomide, FK-506, rapamcyin and related molecules. Dosages of these drugs will also vary depending upon the condition and individual to be treated.

The assay used to measure T cell growth inhibition was a human peripheral blood lymphocyte (PBL) prolifera- tion assay using standard procedures known in the art.

PBL's were chosen due to their known ability to prolifer¬ ate in the presence of antigens derived from herpes sim¬ plex virus (HSV) , Rubella or tetanus toxoid (TT) . PBL growth inhibition was measured in terms of ruthenium complexes's ability to interfere with antigen induced lymphocyte proliferation.

Ruthenium complexes can be used to produce antibodies (e.g., polyclonal and monoclonal) against the complexes. Methods for making antibodies are well known. The anti- bodies can be used as a diagnostic tool for monitoring the amount of ruthenium complex in patient blood levels. The ability to closely monitor the amount of ruthenium complex provides a suitable means for controlling drug delivery to patients in both preclinical and clinical settings. It has also been demonstrated that the ruthenium complexes have antiproliferative properties and in partic¬ ular can inhibit cardiac smooth muscle cells. Based upon this, the ruthenium complexes can be used for the treat¬ ment of hyperproliferative vascular disorders, such as restenosis and atherosclerosis. See Table 2.

The invention will be further illustrated by the following non-limiting Exemplification:

Examples

Example 1 - Preparation of [Ru(l-Melm) ₆ C1-, RuCl ₃ (1.871 g, 9.04 mmol) was added slowly to l-Melm (lO L, 125 mmol, 14 eq.) . The mixture was placed in a preheated oil bath (230°C) , and the mixture was refluxed for 2 hours. The mixture was cooled down to room tempera¬ ture and acetone (50-70 mL) was added to the mixture. The mixture was filtered and the solid washed with acetone (3 X 10 mL) . The product was dried under vacuum.

The product was dissolved in MeOH (30 mL) , and fil¬ tered over celite. The product was obtained as a light yellow crystalline (3.27 g, 55%) solid after triple crys- tallization from MeOH/ether.

[Ru(l-MeIm) ₆ ]Cl ₂ was characterized by X-ray crystal¬ lography, 1H NMR, UV/Vis and elemental analysis.

Example 2 - Preparation of fRu(l-Melm) ₆ ]Cl ₃

[Ru(l-MeIm) ₆ ]Cl ₂ (0.405 g, .609 mmol) was dissolved in HCl (0.25 M, 30 mL) and H ₂ 0 ₂ was added slowly until the starting material had disappeared (reaction followed by UV/Vis spectroscopy) . The solvent was removed to dryness and the product was purified by recrystallization from MeOH/ether. The product was characterized by UV/Vis.

Example 3 - Preparation of fRu(NHQ ,(l-Melm) ₃ ]C1 _?

Zn/Hg was added to a previously degassed suspension of [Ru(NH ₃ ) ₃ Cl,] (69.1 mg, 0.267 mmol) and l-Melm (200 μL, 2.51 mmol) in HCl (0.1M, 15 mL) . The mixture was stirred at room temperature (under Ar) for 20 hours. It was filtered over celite and H ₂ 0 ₂ (30%, 2 drops) and HCl (IM, 1

L) were added. The color of the solution turned from light yellow to orange. The mixture was stirred at room temperature for 1 hour and the solvent was removed to dryness. The mixture was redissolved in water (1 mL) and ethanol (400 mL) added, and then it was placed in the refrigerator overnight. The mixture was filtered, washed with ethanol (2 X 10 L) and then dried under vacuum. The product was crystallized from water/ethanol/acetone. The product was obtained as an orange solid (53.7 mg, 37% yield) . It was characterized by UV/Vis and elemental analysis.

Example 4 - PBL Antigen Specific Proliferation Assay

The lymphocytes were prepared by first separating them from the blood samples of several donors by Ficoll gradient separation as described by standard procedure known in the art. The isolated lymphocytes were then grown in RPMI 1640 medium containing 5% human AB serum, glutamine (2mM) , penicillin/streptomycin, 100 U/ml/100 μg/ml sodium pyruvate

(ImM) and HEPES buffer (lOmM) . For assay purposes, PBL's were incubated at a density of 10 ^s per 200μl of medium per well of a 96-well plate.

Tetanus toxoid (TT; Connaught Labs, Willow Dale, ON) was used as a stimulating antigen at a concentration of 5

LF/ml. The test wells containing PBL's, were exposed to antigen, along with various dilutions of the ruthenium complexes solutions, as shown in Table 1.

Subsequently, TT antigen/ruthenium complexes exposed

PBL's were pulsed with 1 μCi/well of ³ H-thymidine on day 5 using a standard procedure known in the art. The cells were then harvested 16 hours later onto a glass fiber filter using a TOMTEC cell harvester. Thymidine incorporation was

measured by liquid scintillation counting using a Beta plate counter (Pharmacia, Inc., Piscataway, N.J.).

The results of the assay are shown in Table 1.

Table 1

Compound # Structure (itq/mL)

PRO 1305 [Ru ₃ O ₂ (en) ₂ (NH0 ₁₍₁ ]Cl ₆ 0.06

PIC 1097 [Ru ₂ (μ-0) (NH,)„C1,]C1, 0.13

PIC 1099 [Ru ₂ (μ-0) (NH,) _H (HC0 ₂ ) ₂ ]C1, 0.10

PIC 1101 [Ru,(μ-0) (NH,) _H (H-0),] (C10 ₄ ) _S 0.12

PRO 1261 [Ru ₂ 0(OAc) ₂ (py) (PF _ft ) ₂ >100

PRO 1306 [Ru ₂ 0(OAc) ₂ (Bipy) ₂ (py) ₂ ] (PF ₆ ) ₂ >100

PIC 1497 [Ru ₂ 0(Bipy) ₄ (H ₂ 0) ₂ ] (C10 ₄ ) ₄ 4.5

PIC 1095 [RuCl(NH,)j]Cl ₃ ¹⁵

PIC 1096 [Ru(NH ₃ ) _s (4-MeIm) ]Cl _λ 0.45

PIC 1098 cis-[RuCl ₂ (NH ₃ ) ₄ ]Cl >10

PIC 1100 trans- [Ru(S0 ₄ ) (py) (NH,) ₄ ]C1 >10

PRO 1422 cis-Ru(DMSO) ₄ Cl ₂ >100

PRO 1423 [Ru(l-MeIm) ₆ ]Cl ₂

PRO 1424 [Ru(l-Melm)„] (PF„) ₃

PRO 1492 [Ru(l-MeIm) _ή ]Cl,

PIC 1548 trans-[Ru(Im) (py) (NH ) ₄ ]C1,

PIC 1549 cis-[Ru(Im) ₂ (NH ₁ ) ₄ ]Cl ₃

Table 1

Compound # Structure (μq/mL)

PIC 1550 trans- [Ru (Im) ₂ (NH ₃ ) ₄ ]Cl ₃ 0.0033 PIC 1551 trans-[Ru(Im)Cl(NH ₃ ) ₄ ]Cl ₂ >50 PIC 1552 ImH [ trans-Ru ( Im) ₂ C1 ₄ ] 35 PIC 1553 trans- [RuCl ₂ (cyclam) ]C1 >10

PIC 1554 trans- [Ru(S0 ) (Im) (NH,) ]C1 30

PIC 1555 K ₂ [Ru(H ₂ 0)Cl _s ] 40 PRO 1556 [Ru(Im) ₆ ]Cl ₂ 0.0067

PRO 1696 trans- [ Ru ( l-Melm) C1,] Cl 22

PIC 1746 2-MeImH [ trans-Ru ( 2-MeIm) ,C1 ₄ ]

PIC 1747 4 -MelmH [ trans-Ru ( 4 -Melm) ₂ C1 ₄ ]

PRO 1949 [Ru(4-MeIm) _ή ]Cl ₂

PRO 1952 [Ru(Im) _ft ]Cl ₃

PRO 1986 [Ru(NH,) _s (BzIm) ]C1 ₃

PRO 1987 _Ru(NH ₃ ) ₅ (Im) ]C1,

PRO 1988 [Ru(NH ₃ ) _s (py) ] (PF ₂

PRO 2032 [Ru(NH ₃ ) _s (py) ]Cl(RuCl ₄ )

PIC 2447 [Ru(NH ₃ ) ₆ ]Cl ₃

PRO 2449 [Ru(NH,) _s (L-his) ]C1,

PRO 2450 [Ru(NH ₃ ) _s (4-MeIm-5-CH0) ]C1 ₃

PRO 2453 tranε-[Ru(NH,) ₄ (py) ₂ ] (PF _ft ) ₂

Table 1

Compound # Structure (ug/ L)

PRO 2503 trans-[Ru(NH ₃ ) ₄ (py) ₂ ]Cl ₃ 0.026

PRO 2841 cis-[Ru(NH ₃ ) ₄ (L-his) ₂ ]Cl 0.027

PRO 2842 cis-[Ru(NH ₃ ) ₄ (py) ₂ ]Cl ₃ 0.004

PRO 2843 cis-[Ru(NH ₃ ) ₄ (PPh,) ₂ ]Cl ₃ 0.57

PRO 2844 [Ru(NH,) _s (4-pic) ]Cl(RuCl ) 0.0006

PRO 2844B [ Ru (NH ₃ ) ₅ ( 4-pic) ] Cl 0.0012

PRO 2846 cis-[Ru(NH ₃ ) ₄ (l-MeIm) ₂ ]Cl, 0.004

PRO 3006 [Ru(en) ₃ ]Cl, 0.13

PRO 3428 [Ru(NH ₃ ) ₅ (2-NH ₂ -5-Me-py) ]C1 ₃ 0.04

PRO 3429 [Ru(NH ₃ ) ₅ (4-NH ₂ -py) ]C1 0.0016

PRO 4322 cis-[Ru(NH,) ₄ (4-pic),]Cl ₃ 0.012

PRO 4325 [Ru(NH,) _s (PhCCH) ]C1 ₂ 0.05

PRO 4514 [Ru(NH ₃ ) (4-CH ₂ CO,H-py) ]Cl, 0.0045

PRO 4758 [Ru(NH ₃ ) _s (3-/3-py-ala-OH) ]C1, 0.015

PRO 5024 [Ru(NH ₃ ),(Im) ₃ ]Cl, 0.0011

PRO 5237 [Ru(NH ) (l-MeIm) ₃ ]Cl, 0.0035

PRO 6201 [Ru(NH ₃ ),(l-MeIm) ]C1 ₃ 0.015

PRO 6338 {Ru(NH ),[4-(CH ₂ CH ₂ NH ₃ ⁺ )-Im] }C1 ₄ 0.037

Im = Imidazole DMSO = dimethylsulfoxide

PY ⁼ pyridine en = ethylenediamine bipy = 2 , 2 '-bipyridine Bzlm = benzimidazole his = histidine pic = picoline phen = 1, 10-phenanthroline ala = alanine cyclam = 1, 4 , 8 , 11-tetraazacyclotetradecane

Melm = methylimidazole

Ph = phenyl PPh = phenyl phosphine

Example 5 - Inhibition of Human Coronary Aortic Smooth Muscle Cell Proliferation

The assay used to determine inhibition of human coronary aortic smooth muscle cell proliferation was reported by Morris, Heart Lung Transplant ll(pt2) :197 (1992). The results are shown in Table 2 below.

Table 2

Compound # IC ,

PRO 2844B 137 nM PRO 2846 4300 nM

PRO 1549 750 nM

PRO 1556 5 nM

PRO 2449 3000 nM

PRO 6201 3000 nM

Equivalents

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims: