OA10198A

OA10198A - Stable copper(i) complexes and methods related thereto

Info

Publication number: OA10198A
Application number: OA60750A
Authority: OA
Inventors: Alexander J Pallenberg; Andrew Branca; Thomas M Marschner; Leonard M Patt
Original assignee: Procyte Corp
Priority date: 1993-06-02
Filing date: 1995-12-01
Publication date: 1996-12-18
Also published as: AU7051794A; JPH08511006A; EP0701439A1; TW239077B; CA2163640A1; WO1994027594A2; WO1994027594A3; ZA943857B

Abstract

There is disclosed stable copper(I) complexes and methods relating thereto. The stable copper(I) complexes comprise a copper(I) ion complexed by a multi-dentate ligand which favors the +1 oxidation state for copper. Methods of this invention include the use of the stable copper(I) complexes as wound healing agents, anti-oxidative agents, anti-inflammatory agents, lipid modulating agents, signal transduction modulating agents, hair growth agents, and anti-viral agents. Exemplary stable copper(I) complexes include neocuproine copper(I) and bathocuproine disulfonic acid copper(I).

Description

010198

Description

STABLE COPPER(I) COMPLEXESAND METHODS RELATED THERETO

Technical Field

This invention is generally directed to a copper(I)complex and methods relating to the use thereof and, morespecifically, to copper(I) complexed by a multi-dentateligand such that the +1 oxidation State for copper isfavored in the resulting complex. aackgrpund of tte Intention

Copper is found in both plants and animais, and anumber of copper-containing proteins, including enzymes,hâve been isolated. Copper may exist in a variety ofoxidation States, including the 0, +1, +2 and +3 oxidationStates (i.e., copper(0), copper(I), copper(II) andcopper(III), respectively), with copper(I) and copper(II)the most common. The relative stabilities of copper(I)and copper (II) in açrueous solution dépend on the nature ofthe anions or other ligands présent in the solution.Moreover, only low equilibrium concentrations of copper(I)in açrueous solutions (i.e., < 10-2M) can exist. Thisinstability is due, in part, to the tendency of copper(I)to disproportionate to copper(II) and copper(0). Mostcopper(I) compounds readily oxidize to copper(II)compounds, although further oxidation to copper(III) isdifficult (ses, generally, A.F. Cotton and G. Wilkinson,Advanced Inorganic Chemistry. 5th ed., John Wiley & Sons,New York, pp. 903-922, 19ΘΘ).

Due to the relatively well-defined aqueous chemistryof ccpper(II), a large number of copper(IÏ) salts andcomplexes are known. For example, a great deal ofresearch has been directed to the biological activity ofpeptide/copper(II) complexes, and such copper(II) 2 010198 complexes hâve been shown to possess utility for a varietyof therapeutic and cosmetic purposes. In particular, thenaturally occurring glycyl-histidyl-lysine:copper(II) complex ("GHK-Cu(II)") has been shown to be an effectiveagent in the enhancement of wound healing in warm-bloodedwell as generally serving as an anti-agent (see U.S. Patent No. 4,760,051).

Various dérivatives of GHK-Cu(II) possess similar activity(see U.S. Patent Nos. 4,665,054 and 4,877,770). GHK-Cu (II) and other peptide-copper(II) complexes hâve alsobeen shown to be effective for stimulating hair growth5,177,061 and 5,120,831), for inducing coverings in wounds (U.S. Patent No. for preventing ulcers (U.S. Patent Nos. animais, asinflammatory (U.S. Patent Nosbiological4,810,693) ,4,767,753, 5,023,237, 5,145,838), ror cosmetic applications (U.S. Patent No. 5,135,913), and for healingbone (U.S. Patent No. 5,509,588). Moreover, anti-oxidative and anti-inflammatory activity of métal(II)-peptide complexes has been disclosed (U.S. Patent No.5,118,665), as well as the use of copper {II)-containingcompounds to accelerate wound healing (U.S. Patent No.5,164,367) .

Although great strides hâve been made in the study ofcopper(II) complexes, and particularly peptide/copper(II)complexes, there is still a need in the art for additionalcopper complexes which posses biological activity. Theprésent invention fulfills this need, and provides furtherrelated advantages.

Summary of the Invention

This invention is generally directed to stablecopper(I) complexes and methods relating thereto. Morespecifically, the stable copper(I) complexes of theprésent invention comprise copper(I) complexed by a multi-dentate ligand such that the +1 oxidation State for copperis favored. 3 010198

The stable copper(I) complexes hâve utility forenhancing wound healing in warm-blooded animais, forenhancing or restoring the résistance of warm-bloodedanimais to oxidative or inflammatory damage associatedwith reactive oxygen species and/or lipid mediators, forstimulating the growth of hair in warm-blooded animais,for modulating lipid metabolism, for modulating signaltransduction in cells by inhibiting protein kinases, andfor inhibiting viral activity, including (but not limitedto) HIV réplication in an HIV-infected animal. Methods ofthe présent invention comprise administering an effectiveamount of a stable copper(I) complex to the animal.

Other aspects of this invention will become évidentupon reference to the attached figures and followingdetailed description. Ail references identified in thedetailed description,, including the examples, are herebyincorporated by reference in their entirety

Description of the Figures

Figure 1 illustrâtes the activity of a représentativecopper(I) complex of this invention (i.e., bathocuproinedisulfonic acid ("SCDS") copper(D) to accelerate woundhealing.

Figure 2 illustrâtes the ability of a représentativecopper(I) complex of the présent invention, BCDScopper(I) , to inhibit viral (i.e., HIV) réplication.

Figure 3 illustrâtes synthesis pathways forprostaglandins and leukotrienes, as well as certain keyenzymes associated therewith.

Figure 4 illustrâtes a synthesis pathway forcholestérol formation, including the intermediates acetylCoA and HMG-CoA and the enzymes acetyl CoA synthetase andEMG-CoA reductase.

Figure 5 illustrâtes the action of Protein Kinase C (PKC) and protein tyrosine kinase in signal transduction (PI - phosphatidyl inositol, IP3 = inositol triphosphate, 010198 PG = phosphatyl glycerol, P-Protein = phosphorylatedprotein, CDR PK = calmoduln-regulated protein kinase,PKA = Protein Kinase A, Protein Kinase = Protein TyrosineKinase (cytoplasmic), and EGF-R Protein Kinase = Epidermal 5 growth factor receptor protein tyrosine kinase).

Detail ad Descrip-Lion

This invention is generally directed to copper(I)complexes and' methods relating to the use thereof, and 10 more specifically, to copper(I) complexed by a multi-dentate ligand to form a stable copper(I) complex. Asused herein, a "stable copper(I) complex" is copper(I)chelated by at least one multi-dentate ligand such thatthe resulting complex favors the +1 oxidation State of 15 copper. The most common States of copper(I) are associated with four coordination sites, and are generallyof a tetrahedral configuration. In general, chelatingagents are coordination compounds in which a single ligandoccupies more than one coordination position of a métal •20 ion. If the ligand occupies two coordination positions,it is considered a bi-dentate ligand; if more than twocoordination positions are occupied by the ligand, it isconsidered a poly-dentate ligand (such as a tri-dentateligand or a tetra-dentate ligand). As used herein, a .25 "multi-dentate ligand" is a bi-, tri- or tetra-dentateligand which occupies two, three or four coordinationsites, respectively, of copper (I).

The stable copper(I) complexes of this inventioninclude ail complexes of copper(I) chelated by at least -30 one multi-dentate ligand which structurally favors the +1oxidation State of copper. Copper(I) complexes may beformed by reacting a multi-dentate ligand with a source ofcopper(I) (such as CuCl, Cu2O or CuCN) in aqueous solution.The resulting copper(I) complex may then be observed by 35 suitable analytical techniques, such as ESR, NMR and/or ÜV-VIS, to détermine the oxidation State of the copper in 5 010198 the complex (see Munakata et al., Copper CoordinationChemistry: Biochemical and Inorganlc Perspectives. Karlinand Zubieta editors, Adenine Press, Guilderland, N.Y., pp.473-495, 1983). For example, copper(I) complexes can beidentified by their characteristic absence of an ESRsignal, while copper(II) complexes will generally possessan ESR signal. Furthermore, copper(II) complexes exhibitbroadening of proton NMR signais, and copper(I) complexesexhibit relatively sharp proton NMR signais. Followingidentification of the copper(I) complex, its stability canbe evaluated by determining its susceptibility tooxidation by, for example, exposing the copper(I) complexto air. As used herein, a "stable" copper(I) complex hasa half-life of at least 5 minutes, preferably of at leastone hour, and more preferably of 24 hours or more (i.e.,half of the copper (I) complex remains in the +1 oxidationState) upon exposure to air, at room température (23°C) andatmospheric pressure. In other words, stable copper(I)complexes of this invention resist oxidation, while non-stable copper(I) complexes are readily oxidized to yieldcopper(II) complexes upon exposure to air.

As mentioned above, any multi-dentate ligand whichchelates copper(I) to yield a stable copper(I) complex issuitable in the practice of this invention. However, in apreferred embodiment, the multi-dentate ligands of thisinvention are selected from the following general,structures I through VII: 010198

10 wherein A and B represent heteroatoms which may occupycoordination sites of copper(I), and are preferablyselected from nitrogen, oxygen, sulfur and phosphorous.

The rings of structures I through VII may bearomatic, non-aromatic or a mixture of both aromatic andnon-aromatic rings. For example, the following structuresare représentative of such combinations: .15

Via \

VZIa 20

7 010198

Représentative examples of multi-dentate ligands ofthis invention having structures I through VII are setforth in Table 1. Specifically, Table 1 identifies thestructure of the représentative multi-dentate ligand, 5 lists the corresponding Chemical name, identifies theChemical Abstracts Registration Number ("CA Reg. No."),and provides a corresponding reference (if available)describing the synthesis and/or chemistry of theidentified multi-dentate ligand. 10

Table 1

Structura Name Heu. Reference fCb s S benzo (2,l-b:3,4- b) dithicphene 211-53-0 Sturaro et al. , Hets’Ocvcl. Chem, 22:1867, 1990 0 0 benzo (2,l-b:3,4- b) difuran 211-47-2 Rene et al., Sur. J. Med, Chem.-Chim. Ther. 13:435. 1978 O thieno (3,2-g) 438-31-9 Cagniant and 0=0 0 s benzofuran Kirsch, Hebd. Séances Acad. Sci. C. 232:465, 1976 OO— 2H-furo(3,2-g) indole 103671-62-1 Lawrence Jr. , Sur. Pat. Appl. S? 173,520, 1986 rS~ N N H H 2H-benzo (2,1- b:3,4-b>) dipyrrole 112149-08-3 3erlin et al. , ü. Chem. Soc. Chem. Commun. (15):1176, 1987 8 010198 ^NZ H 1H-cyclopenta (2,l-b:3,4-b') bipyridine 42262-29-3 1,10- phenanthroline 66-71-7 furo (3,2-h) quinoline 234-28-6 2,2'-bipyridyl 366-18-7

In structures I through VII above, further ringsubstitutions with heteroatoms are permitted. Preferably,such heteroatoms are selected from nitrogen, oxygen, 5 sulfur, and phosphorus. For example, the compounds listedin Table 2 illustrate further représentative multi-dentateligands of the présent invention having additional ringsubstitutions. As with Table 1, Table 2 identifies thestructure of the représentative multi-dentate ligands, 10 lists the corresponding Chemical name, identifies the CAReg. No., and provides a corresponding reference (if - available) describing the synthesis and/or chemistry ofthe identified multi-dentate ligand. 9 010198

Tabla 2.

Structure Name ΩΔ. Reg- Raxarencft 0¾ 0 0 furano (3,2-g) benzoxazole 25885-39-6 cCh N 0 H furano (2,3-e) benzoxazole 66037-80-1 Turin et al., Fr. Demande 2,338,041, 1977 0¾ 0 s thieno (3,2-g) benzoxazole 58188-85-5 Iddon et al., iL Chem. ..Soc, Perkin Trans. I 11:1686, 1975 thieno (3,2-g) benzothiazole 72121-58-5 cQi N S thieno (2,3-e) benzothiazole 211-36-9 rCb 0 CT benzo (l,2-d:3,4- d’) bis (1,3) dioxide 211-50-7 Dallacker and Weiner. Justus Liebias Ann. ChfiBU 121:99, 1969 N=Q=N ^N Ν'" benzo (l,2-d:3,4- d1) diimidazole 211-10-9 cCb N N pyrrolo(2,3-e) benzimidazole 53068-46-5 Chetverikov et al., U.S.S.R. 425,906, 1974 °O~° "'S S"" benzo (2,l-d:3,4- d’) bis (1,3) oxathiole 211-54-1 10 010198 s<yN H 2H-imidazo (4,5- e) benzothiazole 42341-40-2 Q “V 2H-imidazo (4,5- g) benzothiazole 211-23-4 rQj 0 N 1,3-dioxolo (4,5- e) benzothiazole 77482-58-7 Foerster et al., Ger. Offen. 2,903,966, 1980 rQi S N benzo (l,2-d:3,4- d1) bisthiazole 211-37-0 rCb N N benzo (2,l-d:3,4- d’) bisthiazole 23147-19-5 N<yN benzo (l,2-d:4,3- d') bisthiazole 10558-80-2 Grandolini et al., Ann. Chim. 5À:91, 1968 rCb ''S N" thiazolo(5,4-e) benzoxazole 211-35-8 . nCb thiazolo (5,4-g) benzoxazole 51273-21-3 -- ’7=\ - · rv-rj "N thiazolo (4,5-e) benzoxazole 315-47-9 rvn '"0 s" thiazolo (4,5-f) benzoxazole 67239-73-0 Fridman et al., Ikr, Khim. Zh. 44:399,1978 /=\ ?Λ-Γ° ^'n' benzo (2,l-d:3,4- d‘) bisoxazole 211-19-8 /=Ά r^4~° xo N'' benzo (l,2-d:3,4- d') bisoxazole 211-20-1 010198 rQj benzo (l,2-d:4,3- d') bisôxazole 54935-19-2 Barker et al., J. Chem. R»s. Synop. (9):328, 1986 ,N=\ ç-y furo (2,3-d) thieno (3,2-b) pyridine 110655-19-5 n-o N SX lH-imidazo {4,5- d) thieno (3,2- b)-pyridine 111163-54-3 Takada et al., Eur. Pat. Appl. EP 223,420, 1987 N ·ΣΓ\ iri^Cÿ. xs s dithieno (3,2- b:2‘,3’-d) pyridine 40826-38-8 Yang et al,, Synthssis. 2:130, 1989; Heeres et al., Syn.. Commun.. 2:365, 1972 N —\ N S 5H-oxazolo (4,5- e) thiazolo (3,2- c) pyrimidine 211-46-1 N-N 0-0xs s dithieno (3,2- C:2',3'-e) pyridazine 51974-92-6 Nonciaux et al., Bull. Soc. Chim. Fr. 12 Pt 2, 3318, 1973 Ν-λ N"\ V-N 1 N—y V' XN N' H 1H-(1,2,4) triazolo (5,1-b) purine 387-96-2 n-N N-n < 4 v - XN N bis (1,2,4) triazolo (1,5- d:5’,l'-c) pyrazine 55366-22-8 Vercek et al., Iatrahe,dzcr. (51/52) :4539, 1974 H λ°> WoO -V benzo (2,1-b:3,4- b') dipyran 231-29-8 Menassch 80:743 . 1949 12 010198 benzo (l,2-b:4,3- b') bis'(1,4)- oxathiin 231-34-5 Λ benzo (l,2-e:3,4- e') dipyrazine N=\ /= N benzo (l,2-d:3,4- d*) diimidazole 211-10-9 CO)· '—N N=/ pyrazino (2,3-f) quinoxaline 231-23-2 Shim et al., Svnrhesis 2:116, 1980; Nasielski- Hinkins et al., J. Chem. Soc. Perkin Trans. 1:1229, 1975 0¾ N N bis (1,2,4) oxadiazolo (2,3- d:3',2’-c) pyrazine 743Θ2-83-5 /=\ N Ν'" (1,2,4) - oxadiazolo (3,2- i) purine 56248-95-4 Miura et al., Chem. Pharm. Bull. 22:464, 1975 Ν^Λ Sn t. N-N J- '"N N< - bis (1,2,4) triazolo (1,5- b:5’,l'-f) pyridazine 51519-32-5 Polanc et al. , J. Oro. Chem. 22:2143, 1974 N'\ Λ^Ν '< XN N bis (1,2,4) triazolo (1,5- d:l',5'-c) pyrimidine 76044-62-7 Brown and Shir.ozuka, Aust, J. Chem. 22:11-47, 1980 13 - General structures I through VII identified above maypossess further Chemical moieties covalently attached tothe structural backbone, as illustrated below:

Illb IVb

wherein Rj_ through Rg are the same or different, and are selected from the following Chemical moieties: -H, -OH, -X, -0X, -COOH, -COOX, -CHO, -CXO, -F, -Cl, -Br, -I, -CN, 14 010198 -nh2, -nhx, -nx2, -PX2, -SO3H, -SO3Na, - -SO3K, -SO3X,-PO3H, -OPO3H, -PO3X, -OPO3X and -NO2. As used herein,"X" représente and an alkyl moiety or an aryl moiety. An"alkyl moiety" is a straight chain or branched, cyclic ornoncyclic, saturated or unsaturated, substituted orunsubstituted carbon chain containing from 1-20 carbonatoms; and an "aryl moiety" is a straight chain orbranched, cyclic or noncyclic, saturated or unsaturated,substituted or unsubstituted carbon chain containing atleast one substituted or unsubstituted aromatic moiety andcontaining from S-20 carbon atoms. Such Chemical moietiesmay also be covalently attached to the ring fusion atoms.Représentative examples of the Chemical moieties of thisinvention include, but are not limited to, the moietiesidentified in Table 3 beiow.

Table 3 -H -ch3 -CH2Br -CH2OH -ch2ci -CBr3 -ch2c6h5 -c6h5 "(CH2)i-i2CH3 -Cl -CHO -COOH -COOMe -CH=NOH -ch2nh2 -ch2och -ch=ch2 -P(C6H5)2 -CH2CH(CO2H) 2 -CON(CH2COOH)2 -CH2N(CH2COOH)2 _ch2 ' CH2OH ch3 _n-Ch-ch-%h5ch3 oh -CH2N ch3 -Ph-SO3Na

Représentative examples of the multi-dentate ligandspossessing further Chemical moieties covalently attachedto the structural backbor.e of structures I through VII arepresented in Table 4. In particular, Table 4 identifiesthe structure of the représentative multi-dentate ligands,lists the corresponding Chemical name, identifies the CA 15 010198

Reg. No., and provides a corresponding reference (ifavailable) describing the ·synthesis and/or chemistry ofthe multi-dentate ligand.

Tabla.4

Structura Name CA Reg.. Rsfsrenca CO H CO H I 2 1 2 N N 2,2'- bipyridine- 4,4'- dicarboxylic acid 6813-38-3 .,Λ’ΚΓ' 3 1 ? œ3 - H H 2,2'-bis (4.5- dimethyl imidazole) 69286-06-2 J. Orçanomet. Chenu À£L2:39, 1986 2,3-bis (2- pyridyl) pyrazine 25005-96-3 (Aldrich: 28,164-16} h3<<xsv ,-sας 5,5'-dimethyl- 2,2'- bitbiophene 16303-58-5 k ·' k " 6,6'-dimethyl- 2,2'-dipyridine 4411-80-7 Kauffmann et 109:3864. 1976

The Chemical moieties covalently attached to thestructural backbone may be joined to yield an aromatic or 10 nonaromatic cyclic Chemical moiety. Représentative examples of such cyclic Chemical moieties are set forth in

Table 5, which identifies the structure of the représentative multi-dentate ligands, lists the 16 010198 corresponding Chemical name, identifies the CA Reg. No.,and provides a corresponding reference (if available)describing the synthesis and/or chemistry of the multi-dentate ligand.

Table 5

Structure Name CA Reg. No. Reference h3c Γ -A CH \ / 3 6,7-dihydro- 5298-71-5 N □ 5,8-dimethyl dibenzo — (b) (1,10) phenanthroline H H 5 bibenzimidazole 123057-51-6 .N 2,2'- 119-91-5 (Aldrich: 1 bisquinoline 33,540-7)

The synthesis of représentative examples of the10 multi-dentate ligands of this invention are disclosed inTable 6 and Table 7 below. Specifically, in these tablesthe structure of the multi-dentate ligands are identifiedalong with their CA Reg. No. and one or more references disclosing their synthesis and/or chemistry. •15 17 010198

Table 6.

Synthesis of Représentative Copperfl) Complexes

Having the Structurer

{R2 through R7 » hydrogen, unless indicated)

Kl ES. CA-Sag-t-No..· Ee£ersnce -ch3 -ch3 484-11-7 0'Reilly et al.,Aust. J. Chem,. 11:145, I960 -CH2Br -CH2Br 78831-37-5 Weijen et al. , J .Qzg-K-ChSffl. 12:7258,1992; Jukkalaet al., Kelv. Chim.Acta. 21:1621, 1992;Chandler et al., J. Estsrocycl. Chem. 11:599, 1981 -CH2Br -ch2oh 142470-16-4 Weijen et al., J.Org. Chem. 12:7258,1992 -CBr3 -CBr3 Chandler et al., JL.Heterocycl. Chem. 11:599, 1981 -CH2C1 -CH2C1 Newkome et al., J.Qrg^. Chan. il: 3807,1985; Newcorae etal., J. Org. Chem.£1:5112, 1983 -CCI3 -CCI3 Chandler et al., J.Sfitesocycl..Chem. 11:599, 1991;Newcome et al., J.Orc. Chem. 48:5112. 1983 -CN -CN 57709-63-4 Chandler et al., J.Heterocvcl. fhem. 11:599, 1931;Sjoegren et al.,Otsancmscallies 11:3954, 1992 18 010198 -ch2c6h5 -ch2c6h5 223-20-1 Sjoegren et al.,Oroanometallics 11:3954, 1992 - (CH2)hCH3 -(CH2)11CH3 Menger et al., J, Am. Chem. Soc. 111:4017, 1991 -(CH2)3CH3 -{CH2)3CH3 85575-93-5P Sugihara et al., JP 02096578 A2, Jpn .Kokai Tokkvo Koho 111(15):132159v (R3=Rg=H, Ph -(CH2)3CH3 -(CH2)3CH3 Delton et al., EP339973 Al. Eur. Pat. appI^ 112(21):19835p, 1989 (r4=r5=-ch3) -Cl -Cl 29176-55-4 Sjoegren et al.,Qrgar.cme tailles 11:3954, 1992;Delton et al., EP339973 Al. Eur. Pat. Appl- 112(21):19835p, 1989 -CH2OH -CH2OH 78831-36-4 Chandler et al., J. Heterflcycl., Chenu11:599, 1981; Deltonet al., EP 339973 Al, Eur. Pat. Appl 112(21):19835p, 1989; Newcome etal., J. Qro. Chem.41:5112, 1983 -CHO -CHO 57709-62-3 Ziessel. Tetrahedron Lett. 2Q:463, 1989;Toner, E? 288256 A2,Eur. Pat. Appl. 111(15):130322c; - , Bell et al., J.Inclus ion.. Pasnonu 2:149, 1987 -COOH -COOH Chandler et al., iL Eeterocvcl. Chem. 11:599, 1981 -COOMe -COOMe Chandler et al., jL. Heterccycl. Chem. 11:599, 1981;Newcome et al., iL.Or?. Ch£tl. £1:5112,1933 19 010198 -CH=NOH -CH=NOH Chandler et al., J.Eeterocycl. Chem. 15:599, 1981 -ch2nh2 -CH2NH2 Chandler et al., J, Heterocvc1 . Chem. 11:599, 1981 -CHO -H 33795-37-8 Toner, E? 28825S A2, Eur. Pat. AppI. 111(15):130322c -COOH -H 1891-17-4 Toner, EP 288256 A2,Eur. Pat. AppI. 111(15):130322c -CH2CsCH -CH2CsCH - Sjoegren et al.,Organometallies 11:3954, 1992 -c6h5 -c6h5 Dietrich-Buchecker et al., TetrahedronLett. 21:5291, 1982 -Cl -ch3 Newcome et al., J.Orç. Chem. 51:1766,1989 -ch=ch2 -ch=ch2 Newkome étal., J.Orç. Chem. 55:3807,1985 -p(c6h5)3 -P(C6H5)3 Ziessel, TetrahedronLâH 12.:463, 1989 CH2CH(CO2H)2 -CH2CH(CO2H)2 Newcome et al.,Inorg. Chem. 21:811,1985 -ch2n(ch2)11ch ch3 -CH2 N .'Œ20H Weijen et al., J.Qrc. Chem. 52:7258,1992 _ch2 „ CH9OH.N., 2 } · _ch2 « CH 9 OH 2 L>H Weijen et al., J.Orcr. Chem. 57:7258. 1992 -ch2oh _ch2 N ,'Œ2°H Γ Weijen et al., J.Orc. Chem. 57:7253. 1992 -CH2N(CK2)1;1CH *ck3 ch3 -N-CH-CH-CgK5CK3 OH Weijen et al., J.Orc. Chem. 57 :7258. 2S92 -CH2N(CH2COOH)2 -CH2N(CK2COOH)2 Mukkala et al.,Helv. Chim, Acta 25:1521, 1992;Toner, ΞΡ 288256 A2,EUX_Pat. Appl. 111(15):130322c 20 01Q198 -CON(CH2COOH)2 -CON(CH2COOH)2 Toner, EP 288256 A2,Sur. Pat. AddI. 111(15):130322c -ch3 (R3=Rg= -Ph-SO3Na -ch3 52698-84-7 Blair et al, Talanta 2:163, 1961

Table 7

Synthesis of Représentative Copperil) Complexes

Having the Structure;

(R2 through R7 = hydrogen, unless indicated) RI 23 CA Reg, No, Referencs -CN -CN 4411-83-0 Sjoegren et al.,Qrsanpmat allies 11:3954, 1992 -ch2ci -CK2C1 74065-64-8 Bell et al., J. Inclusion Phenom. 5.:149, 1987 -CHO -CHO Newkome et al.. J. Orc. Chem. 55:3807, 1985 -ch=ch2 -CH=CH2 Newkome et al.. J. Orc. Chem. 55:3807, 1985 (R^ and R2 = benzomoiety) (R7 andRg = benzo moiety) 119-91-5 (Aldrich: 33,540-7)

In one embodiment of this invention, the multi-dentate ligands are selected from the followingstructures : 10 21 010198

wherein Rj through Rg are the same or different, and areselected from hydrogen, an alkyl moiety and an aryl 5 moiety.

In a preferred embodiment, the multi-dentate ligandis 6,6'-dimethyl-2,2'-dipyridine having structure Id:

In a further preferred embodiment, the multi-dentateligand is neocuproine (2,9-dimethyl-l,10-phenanthroline)having structure Ild, or is bathocuproine disulfonic acid("BCDS") having one of the isomeric structures Ile, Ile' 15 or Ile' ' :

Ild 22 010198 SO3Na

Ile SO3Na SO3Na

O3Na

Ile'

NaO·

Ile ' 1

Unless otherwise indicated, BCDS refers to a physicalmixture of the above isomers (i.e., Ile, Ile' and Ile'').Typically, the ratio of the various isomers (i.e.,Ile : Ile': Ile' ') vary depending upon the commercial sourceof BCDS as follows: Aldrich Chemical Co., Inc. (Milwaukee,Wisconsin) 9.1:38.6:41.2; Spectrum Chemical ManufacturingCorp. (Gardena, California) 8.5:39.7:45.2; GFS Chemicals(Columbus, Ohio) 8.4:38.5:45.3; Janssen Pharmaceutica(subsidiary of Johnson & Johnson) (Beerse, Belgium)4.6-8.7:36.4-39.4:44.4-55.9.

As discussed above, stable copper(I) complexes ofthis invention may be made by contacting a multi-dentateligand with a copper(I) source. The multi-dentate ligandsmay be obtained from commercial sources, or may besynthesized by known organic synthesis techniques fromcommercially available reagents. Preferably, water soluble multi-dentate ligands are complexed with the copper(I) in aqueous solution, employing CuCl, CU2O or

CuCN as the copper(I) source. The resuiting copper(I) 23 010198 complex may then be recovered by évaporation of solvent toyield the copper(I) complex: Alternatively, if the multi-dentate ligand is not readily soluble in water, copper(I)complexes may be formed by the above procedure employing asuitable non-aqueous (e.g., organic) solvent.

In the practice of this invention, the ratio of themulti-dentate ligand to copper(I) may be any ratio whichresults in a stable copper(I) complex. Preferably, theligand to copper ratio is at least 1:1. In a morepreferred embodiment, the ligand to copper ratio rangesfrom 1:1 to 3:1 (including 2:1). Such copper(I) complexesmay be made by the procedures identified in the precedingparagraph by reacting the appropriate molar ratios of themulti-dentate ligand and the copper(I) ion source.

Although not intending to be limited by the followingtheory, it is believed that copper (I) has enhancedbiological activity over copper(II) in certain biologicalevents. For example, it is believed that copper(I) may bean important intermediate for copper metabolism, includingcopper uptake and/or transfer, as well as cellulardelivery. Thus, the réduction of copper(II) to copper(I)is bypassed by direct delivery of copper(I). Furthermore,the stable copper(I) complexes of this invention aresuitable for systemic delivery to warm blooded animais,and may provide a sustained release of copper to theanimal.

The stable copper(I) complexes of this inventionpossess utility as therapeutic substances, includingutility as anti-oxidative and anti-inflammatory agentsgenerally and, more specifically, as wound healing agents.The copper(I) complexes of this invention also possessactivity as hair growth agents, lipid modulation agents,signal transduction modulating agents, and anti-viralagents. For purpose of clarity, the various biologicalactivities of the stable copper(I) complexes of thisinvention are acdressed individually below. 24 010198

Highly reactive oxygen species such as the superoxideanion (03*, hydrogen peroxide (H2O2), hydroxyl radical(HO·) , and lipid peroxides (LOOH) are involved in a numberof human diseases. For example, such oxygen species hâve 5 been implicated in autoimmune diseases, arthritis, tissuedamage caused by environmental pollutants, cigarette smokeand drugs, tissue injury during, for example, surgery andtransplantation, as well as a variety of other conditions(see, e.g., Halliwell, B., Fed, Amer. Soc, Exp, 3iol. 10 1:358-364, 1987). Reactive oxygen species are also generated during the response to injury by phagocyticcells. One of the early events in the wound healingresponse is the cleansing and sterilization of the woundby neutrophils and macrophages. A mechanism for this 15 sterilization is the génération of the superoxide anionand hydrogen peroxide, and generally results in an - . inflammatory response. Moreover, superoxide anion andhydrogen peroxide will, in the presence of iron or other .. . . redox active transition métal complexes, generate the 20 hydroxyl radical. The hydroxyl radical is a potent oxidant which initiâtes the free radical oxidation of fatty acids, as well as the oxidative dégradation cf otherbiomolecules. For example, an important area in which . reactive oxygen species cause tissue damage is in post-..-.:25 . injury damage to the brain and spinal chord, and in reperfusion injury to ischémie tissue following surgery·and transplantation (such as heart surgery and/ortransplantation). A sudden inrush of oxygenated blood andactivated phagocytic cells leads to superoxide anion and '3 0 hydrogen peroxide formation. These species do directdamage to tissue, and also react with iron (as discussedabove) to generated the very reactive hydroxyl radical.

The stable copper(I) complexes of this inventiongenerally serve as anti-oxidative agents which prevent or 35 limit the oxidative damage caused by reactive oxygenspecies, and further serve as anti-inflammatory agents by 25 010198 reducing the inflammatory response associated with suchreactive oxygen species. More specifically, the copper(I)complexes of the présent invention are useful in theenhancement and/or restoration of the defense of warm-blooded animais to oxidative or inflammatory damage causedby the highly reactive oxygen species, and may be used inpharmaceutical préparations to inhibit oxidative andinflammatory processes which lead to tissue damage.Moreover, the stable copper(I) complexes of this inventionaccelerate the wound healing process by "detoxifying"tissue damage by the highly reactive oxygen species.

In addition to highly reactive oxygen species,macrophages and neutrophils induce or continue aninflammatory response through the génération of certainlipid mediators of inflammation {e.g., leukotrienes andprostaglandins). The involvement of such mediators ininflammatory bowel disease (I3D) and related chronicinflammatory conditions, such as arthritis, is evidencedby a strong corrélation between disease progression andthe levels and presence of leukotrienes and prostaglandinsin the circulation and effected tissue. Prostaglandinsenhance vasodilation and edema formation, whileleukotrienes are potent chemoattractive agents forleukocytes, especially neutrophils, and stimulatedegranulation and the release of damaging lysosomalenzymes and superoxide production.

The distribution of the two major pathways leadingeither to prostaglandins or to leukotrienes variesaccording to cell type. While most cells possess thecyclooxygenase pathway, the 5-lipoxygenase pathway leadingto the leukotrienes is less widely distributed and isprominent in inflammatory cells, such as neutrophils,macrophages, monocytes and mast cells. The general schemefor lipid mediator synthesis is illustrated in Figure 3.

The stable copper(I) complexes of this invention inhibit the formation of prostaglandins and/or 26 010198 leukotrienes by inhibiting the enzymes involved in theirformation. Referring to Figure 3, the stable copper(I)complexes are effective inhibitors of both cyclooxygenase-1 and cyclooxygenase-2, thereby inhibiting the formation 5 of prostaglandins. Similarly, the stable copper(I) complexes are effective inhibitors of 5-lipoxygenase andleukotriene C4 (LCT4) synthetase, thereby inhibiting theformation of leukotrienes.

In addition, proteolysis of various cellular targets10 by elastase (a neutrophil-released serine protease) at thesite of inflammation has been implicated in a number ofpathologie conditions, including emphysema, rheumatoidarthritis, and psoriasis. Thus, inhibitors of elastasemay be used to treat, prevent or limit the breakdown of 15 normal tissue at the site of inflammation, and the stablecopper(I) complexes of this invention are effectiveinhibitors of elastase.

The stable copper(I) complexes of this invention mayalso be used in the régulation and/or modulation of lipid 20 metabolism in general. For example, hypercholesterolemiaand hyperlipidemia are common and serious health problemswhich are treatable with the stable copper(I) complexes ofthis invention.

Hypercholesterolemia has been observed in marginal 25 and severely copper-def icient rats, as well as otheranimais, including humans (Lei, "Plasma CholestérolResponse in Copper Deficiency," Rôle of Copper in LipidMstabolism, ed. Lei, CRC Press, pages 1-24, 1990) .

Elévation in sérum cholestérol level has been linked to 30 increases in the activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase,E.C.1.1.1.34) and glutathione levels (3unce,

"Hypercholesterolemia of Copper Deficiency is Linked toGlutathione Metabolism and Régulation of HMG CoA 35 Reductase," Nutr. Rev. 51: 305-307, 1993; Kim et al., "Inhibition of Elevated Hepatic Glutathione Abolishes 010198 27

Copper Deficiency Cholesterolemia," FASEB J. 2467-2471, 1992) .

Similar increases in the synthesis and level of otherhepatic lipids (fatty acids, triacylglycerols andphospholipids) hâve been observed in copper déficient rats(al-Othman et al., "Copper Deficiency Increases In VivoHepatic Synthesis of Fatty Acids, Triacylglycerols, andPhospholipids," Proc. Soc. Exp, Biol. Med. 204 fl): 97-103, 1993) and treatment with a copper(II) complex has been shown to lower the activity of liver enzymes involved inlipid metabolism, including acetyl CoA synthetase in vivo(Hall et al., "Hypolipidémie Activity of Tetetrakis-mu-(trimethylamine-boranecarboxylato)-bis(trimethylamine-carboxylborane)-dicopper(II) in Rodents and its Effect onLipid Metabolism," J, Phartn. Sci. 73 (7) : 973-977, 1984).

Conversely, it has been reported that treatment byinjection of copper(II) increased sérum cholestérolconcentrations in rats, possibly by increasing theactivity of the HMG CoA reductase (Tanaka et al., "Effectof Cupric Ions on Sérum and Liver Cholestérol Metabolism,"Lipids 22: 1016-1019, 1987) . Accordingly, it is believed that copper may be an important factor in the régulationof lipid levels.

Acetyl CoA synthetase catalyzes the formation ofacetyl CoA from acetate. As illustrated in Figure 4,acetyl CoA can be further metabolized along many differentpathways leading primarily to the formation of cholestéroland fatty acids or energy production. Agents whichinhibit this enzyme influence the biosynthesis of variouslipids. KMG-CoA reductase (3-hydroxy-3-methylglutarylcoenzyme A reductase) is located biochemically later inthe lipid synthesis scheme and converts HMG-CoA tomevalonic acid, and is the rate limiting reaction incholestérol biosynthesis (see Figure 4). Stable copper(I)compounds of this invention inhibit certain key enzymesinvolved in the formation of lipids, and thus serve as 28 010198 lipid modulating or regulatïng agents. (The ability ofstable copper(I) complexes to inhibit enzymes in theformation of lipids is disclosed in further detail inExamples 12-13.) 5 The stable copper(I) complexes of this invention may also serve as modulating agents of signal transduction incells. Most intracellular signaling processes are regulated by réversible phosphorylation of spécifie proteins by kinases. Breakdown of phosphatidylinositol 10 leads to the formation of diacylglycérol and inositoltriphosphate, the former acting synergistically withcalcium to activate Protein Kinase C (PKC), resulting intranslocation of the enzyme from cytosol to the membrane.Phosphorylation of proteins by PKC has been implicated as 15 a pivotai regulatory element in signal transduction,cellular régulation and tumor promotion. Inhibitors ofPKC, as well as other protein kinases, hâve the potentialto block proliférative signaling in tumor induction,atherosclerosis and immune modulation. 20 Examples of factors which stimulate the G-protein linked phospholipase C breakdown of phosphatidylinositolinclude angiotensin II, bradykinin, endothelin, f-Met-Leu-Phe, and vasopressin. These protein kinase C enzymes arealso directly activated by tumor promoters such as phorbol . 25. esters. Examples of Receptor linked tyrosine kinasesinclude Epidermal Growth Factor, Nerve Growth Factor, and ; Platelet Derived Growth Factor. Examples of cytoplasmictyrosine kinase activators include cytokines such asInterleukin 2, Interleukin 3, and Interleukin 5. These 30 factors bind to spécifie lymphocyte receptors whichactivate the cytoplasmic tyrosine kinase.

The action of PKC and protein tyrosine kinase actionis illustrated in Figure 5. The stable copper(I) complexesof this invention serve as signal transduction modulating . .35 agents by inhibiting or.e cr more enzymes involved in 010198 29 intracellular signal transduction, including PKC andprotein tyrosine kinases.

When administered to an animal to treat theconditions discussed above, the stable copper(I) complexesmay first be combined with one or more suitable carriersor diluents to yield a pharmaceutical préparation suitablefor topical, oral or parentéral application. Such diluents or carriers, however, should not interact withthe stable copper(I) complex to significantly reduce theeffectiveness thereof, or oxidize copper(I). Effectiveadministration will preferably deliver a dosage ofapproximately 0.01 to 100 mg of the stable copper(I)complex per kg of body weight.

Methods for encapsulating compositions (such as in acoating of hard gelatin) for oral administration are wellknown in the art (see, e.g., Baker, Richard, ControlledRelease of Biological Active Agents, John Wiley and Sons,1986)(incorporated herein by reference). Suitable carriers for parentéral application (such as intravenous,subcutaneous or intramuscular injection) include stérilewater, physiological saline, bacteriostatic saline (salinecontaining 0.9 mg/ml benzyl alcohol) and phosphate-buffered saline. The stable copper(I) complexes may betopically applied in the form of liquids, containingpharmaceutically acceptable diluents (such as saline andstérile water) or may be applied as lotions, creams orgels, containing additional ingrédients to impart thedesired texture, consistency, viscosity and appearance.Such additional ingrédients are familiar to those skilledin the art and include emulsifying agents such as non-ionic ethoxylated and nonethoxylated surfactants, fattyalcohols, fatty acids, organic or inorganic bases,preserving agents, wax esters, steroid alcohols,triglycéride esters, pnospholipids such as lecithin andcephalin, polyhydric alcohol esters, fatty alcohol esters,hydrcphilic lanolin dérivatives, hydrophilic beeswax 30 010198 dérivatives, hydrocarbon oils such as palm oil, coconutoil, minerai oil, cocoa butter waxes, Silicon oils, dHbalancers and cellulose dérivatives.

Topical administration may by accomplished byapplying an amount of the préparation, directly to thedesired area, such as a wound or an inflamed area. Therequired dosage will vary according to the particularcondition to be treated, the severity of the condition,and the duration of the treatment. Preferably, when thestable copper(I) complex is topically applied in the formof a lotion, cream or gel, the préparation may containabout 1% to about 20% of a pénétration enhancing agent.Sxamples of pénétration enhancing agents includedimethylsulfoxide (DMSO), urea and eucalyptol. In thecase of a liquid préparations for topical application, theconcentration of pénétration enhancing agent (such asDMSO) may comprise about 3 0% to about 80% of thepréparation.

In addition to the activity discussed above, thestable copper(I) complexes of this invention also possessutility as hair growth agents. Hair loss is a commonaffliction of humans, the most common being "alopecia"where males lose scalp hair as they get older (also called"male pattern baldness"). Other hair loss afflicationsinclude alopecia areata (AA), female pattern baldness andsecondary alopecia (e.g., hair loss associated withchemotherapy and/or radiation treatment). The stablecopper(I) complexes of this invention are particularlyuseful in stimulating hair growth associated with any hairloss afflication, including the spécifie afflicationsidentified above.

Hair is normally divided inco two types, "terminal"and "vellus" ’nairs. Terminal hair is ccarse, pigmentedhair which arises frem follicles which are developed deepwithin the dermis. Vellus hairs are typically thin, non-pigmented hairs which grew from hair follicles which are 31 010198 smaller and located superficially in the dermis. Asalopecia progresses, there is a change from terminal tovellus type hair. Other changes that contribute to alopecia are alterations in the growth cycle of hair.Hair typically progresses through three cycles, anagen(active hair growth), catagen (transition phase), andtelogen (resting phase during which the hair shaft is shedprior to new growth). As baldness progresses, there is ashift in the percentages of hair follicles in each phase,with the majority shifting from anagen to telogen. Thesize of hair follicles is also known to decrease while thetotal number remains relatively constant.

As mentioned above, the stable copper(I) complexes ofthis invention hâve utility as stimulating agents for thegrowth of hair in warm-blooded animais. In one embodimentof the présent invention, the copper(I) complex may beadministered intradermally in the area to be treated,along with a suitable vehicle, at a concentration ofapproximately 100-500 micrograms of copper(I) complex per0.1 ml of vehicle. Suitable vehicles in this regardinclude saline, stérile water, and the like.

In another embodiment, the stable copper(I) complexmay be topically applied in the form of a liquid, lotion,cream or gel by applying an effective amount of thetopical préparation directly to the scalp. Any guantitysufficient to stimulate the rate of hair growth iseffective, and treatment may be repeated as often as theprogress of hair growth indicates. Preferably, suitabletopical hair growth préparations contain from about 0.1%to about 20% by weight of the stable copper(I) complex(based on the total weight of the préparation).

Topical hair growth préparations of the présentinvention may contain about 0.5% to about 10% of anemulsifving cr surface active agent. Non-icnic surfaceactive agents and ionic surface active agents may be usedfer the purposes of the présent invention. Examples of 32 010198 suitable non-ionic surface active agents arenonylphenoxypolyethoxy éthanol (Nonoxynol-9) , polyoxyethylene oleyl ether (Srij-97), variouspolyoxyethylene ethers (Tritons), and block copolymers of 5 ethylene oxide and propylene oxide of various molecularweights (Pluronic 68, for exemple). Acceptable préparations may also contain about 1% to about 10% ofcertain ionic surface active agents. These ionic surfaceactive agents may be used in addition to or in place of, 10 the non-ionic surface active agents. Examples of ionicsurface active agents are sodium lauryl sulfate andsimilar compounds.

In addition to, or in place of, the emulsifying orsurface active agent, topical hair growth préparations of 15 this invention may contain about 1% to about 20% of apénétration enhancing agent. Examples of penetratingenhancing agents are DMSO and Urea. In the case of aliquid préparation to be applied topically, theconcentration of a penetrating enhancing agent, such as 20 DMSO, may comprise about 3 0% to about 80% of the topicalpréparation. The balance of the topical hair growthpréparation may comprise an inert, physiologicallyacceptable carrier. Suitable carriers include, but arenot limited to, water, physiological saline, 25 bacteriostatic saline (saline containing 0.9 mg/ml benzylalcohol), petrolatum based creams (e.g., USP hydrophilicointments and similar creams, Unibase, Parke-Davis), -- :.i.. . various types of pharmaceutically acceptable gels, andshort chain alcohols and glycols (e.g., ethyl alcohol and ' 3 0 propylene glycol) .

The following are examples of suitable hair growthpréparations within the context of the présent invention:

Préparation A :

Copper(I) Complex 10.0% (w/w)

Hydroxy Ethyl Cellulose 3.0% - 35 33 ίο 15 20 25

Propylene Glycol · .··· 20.0%

Nonoxynol-9 3.0%

Sodium Lauryl Sulfate 2.0%

Benzyl Alcohol 2.0% 0.2N Phosphate Buffer 60.0%

Préparation B:

Copper(I) Complex 10.0%

Nonoxynol-9 3.0%

Ethyl Alcohol 87.0%

Préparation C:

Copper(I) Complex 5.0%

Ethyl Alcohol 47.5%

Isopropyl Alcohol 4.0%

Propylene Glycol 20.0%

Laoneth-4 1.0%

Water 22.5%

Préparation D:

Copper(I) Complex 5.0%

Water 95.0%

Préparation E:

Copper(I) Complex 5.0%

Hydroxypropyl Cellulose 2.0%20.0%3.0% • . 70.0% (w/w) (w/v) (w/v) (w/v) 010198

Glycerin

Nonoxynol-9

Water

Préparation F:

Copper(I) ComplexNonoxynol-9 ..Unibase Cream

Préparation G:

Copper(I) ComplexNonoxynol-9Propylene GlycolEthanol

Water 1. 5. 94 . 2 .3 . 50 .30.15. 0% 0% 0% 0% 0% 0% 0% 0% (w/w) (w/w) 30 35 34 010198

The copper(I) complexes of the présent invention alsoposses utility as anti-viral agents, and are particularlyeffective in the inhibition of the AIDS virus. Humanacquired immunodeficiency syndrome or "AIDS" is a fataldisease for which there is presently no cure. The diseaseis believed to be caused by a virus known as the humanimmunodeficiency virus, commonly referred to as "HIV."The virus is transmitted by HIV-infected individualsthrough the exchange of bodily fluids. HIV infectionresults most commonly from sexual contact with an infectedpartner and the sharing among intravenous drug users ofhypodermic syringes previously used by an infectedindividual. A prégnant HIV-infected mother may infect herunborn child by trans-placental transmission, and HIV-contaminated blood is a possible source of infection forindividuals subject to blood transfusion. HIV infection causes a suppression of the immuneSystem. The immune suppression renders the infectedindividual vulnérable to a variety of opportunisticinfections and conditions that are otherwise kept inbalance by a healthy immune System. Fatalities resuitfrom HIV infection due to the inability of AIDS patientsto respond to treatment of the opportunistic infectionsand conditions as a conséquence of their compromisedimmune Systems. Because the virus may .often remaindormant, the manifestation of AIDS from HIV infection maytake as long as ten years.

One approach to the treatment of AIDS has targetedthe opportunistic infections or conditions which resuitfrom HIV infection. The treatment of such infections orconditions, however, is ultimately ineffective and, whileprolongir.g the life of the infected individual, does nottreat the underlying HIV infection. A second approach tothe treatment of AIDS targets the cause of the diseaseitself. 3ecause AIDS results from viral infection, it isbelieved that viral inactivation may ultimately provide a 35 0tθ198 subséquent prévention cure. Materials which are capable of viral inactivationor inhibition are referred to herein as "antiviralagents."

To understand the mode of action of antiviral agentsin the treatment of AIDS, an understanding of the processof HIV infection is necessary. HIV chronically infectsspécifie immune cells known as T-helper cells, which arerequired for normal immune response. The HIV infected T-helper cells serve as hosts to the virus and facilitatethe reproduction of the virus (the process of viralreproduction is commonly referred to as "réplication").After HIV infection, the infected host cell eventuallydies, the replicated HIV virus is released, and theinfection spreads to additional cells. This cycle continues unabated, depleting the population of T-helpercells and, in time, weakens the immune System to the onsetof AIDS symptems. Because T-helper cells are continucuslyproduced by the body, the population of these cells may bereestablished in the absence of further HIV infection.Therefore, the progression of HIV infection (and theonset of AIDS) may be arrested byor inhibition of viral réplication, antiviral agents capable of inhibiting or preventing theréplication of HIV should be effective in the treatment ofAIDS . the and

At the genetic level, HIV réplication requires theinsertion of viral deoxyribonucleic acid ("DNA") into thegenome of the 'host cell. The genome of the host cellconsists of the cell's own DNA, and is responsible for thesynthesis of materials essential to the cell's ownfunction and prolifération. Once the viral DNA isinserted into the host genome, the host facilitâtesréplication of HIV. The inserted viral DNA is anenzymatic product derived from viral ribonucleic acid("RNA") and the action of an enzyme known as HIV reversetranscriptase. Inhibition of HIV reverse transcriptase 36 010198 precludes the formation of viral DNA required forinsertion into the genome of the host. Viral réplicationis prevented by the absence of viral DNA in the host cellgenome. Antiviral agents which inhibit HIV reversetranscriptase are thus potential therapeutic drugs fortreatment of AIDS.

Accordingly, in yet another embodiment of the présentinvention, antiviral agents are disclosed for inhibitingHIV réplication, as well as methods relating to theadministration thereof to an HIV-infected patient. Theantiviral agents of this invention are the stablecopper(I) complexes discloses above, and the methodsinclude administration of a therapeutically effectiveamount of a composition which includes a stable copper(I)complex in combination with a pharmaceutically acceptablecarrier or diluent. Although not limited by the followingtheory, it is believed that the copper(I) complexes ofthis invention enhance transport of copper(I) into HIVinfected cells which, in turn, inhibits or inactivâtes HIVprotease and thus inhibits the réplication of HIV. Asused herein, the term "HIV" includes the various strainsof the virus such as HIV-1 and HIV-2.

Administration of the stable copper(I) complexes ofthe présent invention may be accomplished in any mannerwhich will resuit in a systemic dose of a therapeuticallyeffective amount of the copper(I) complex to an HIV-infected animal cr patient (including human patients) .For example, such administration may be by injection(intramuscular, intravenous, subcutaneous or intradermal),oral, nasal, or suppository applications. Typically,préparations of the présent invention include stablecopper(I) complexes in solution for various forms ofinjection, or in préparations which are formulated for thesustained release of the stable copper(I) complexes fororal, nasal, or suppository dosage application andgenerally include one cr more inert, physiological 37 010198 acceptable carriers. As used herein, the term "effectiveamount" means an amount of the stable copper(I) complexwhich inhibits HIV réplication in the patient. Suitabledosages may range from approximately 0.01 to 100 mg of 5 stable copper(I) complex per kg body weight.

The stable copper(I) complexes of this invention may be screened for their ability to inhibit HIV réplicationusing known techniques. For example, HIV virus réplication may be monitored using the Cytopathic Effect 10 (CPE) assay disclosed by Bergeron et al. (J. Virol.66 :5777-5787. 1992). In this assay, the degree of infection is monitored by the appearance of fused cellularmembranes ("syncitium"). Altematively, assays directedto activity of HIV protease may be employed. For example, 15 the assays and techniques disclosed in the followingreferences may be employed: Ashorn et al., Proc. Natl .

Acad. Sci. U.S.A. £2:7472-7476, 1990; Schramm et al., BÏQChenï__Biophys. Res. Commun. 122:847-851, 1991; Sham et al., BiQchern.„3iQphyau..„Zes.fc,Commun. 125:914-919, 1991; and 20 Roberts et al., Science 248:358-361. 1990. Moreover, theability of the stable copper(I) complexes of thisinvention to inhibit HIV réplication may be determined bythe assay disclosed in Example 5 herein below.

The stable copper(I) complexes of this invention, in• 25 addition to inhibiting HIV réplication, may also inhibitréplication of other viruses, including human T-cellleukemia (HTLV) I ar.d/or II, human herpes virus, cytomegalo virus (CMV), encephalomyocarditis virus (EMCV),Epstein Barr virus (E3V), human hepatitis virus, Varicella — 30 Zoster virus, Rhinovirus, and rubella virus. One skilledin the art could readilv assay the stable copper(I)complexes of this invention for their inhibitory activitywich regard to these viruses. For example, Example 15illustrâtes the inhibitory affect of stable copper(I) 3 5 complexes of this invention on both encephalomyocarditis virus (EMCV) and cytomegalo virus (CMV). 38 010198

In addition to the biological activity of the stablecopper(I) complexes of the présent invention, the multi-dentate ligands of this invention also possess biologicalactivity when administered alone as the "free" multi-dentate ligand (i.e., without copper(I)). Such biologicalactivity includes the activities identified above,including anti-viral activity, as well as a preventativeagent against gastric tissue damage. Although notintending to be limited to the following theory, when themulti-dentate ligands of this invention are administeredas the free ligand, it is believed that they function, atleast in part, by scavenging copper(I) to yield the stablecopper(I) complex in vivo.

The following examples are offered by way ofillustration, and not by way of limitation.

EXEMPLES

The examples which follow illustrate the préparationand utility of certain exemplary embodiments of the stablecopper(I) complexes of the présent invention. To summarize the examples that follow: Example 1 illustrâtesthe synthesis of neocuproine copper(I) at a molar ratio of1:1 and 2:1; Example 2 illustrâtes the superoxideactivity of représentativethis invention (employing a copper(II)-peptide complex as a positive control); Example3 illustrâtes the wound healing activity of areprésentative copper(I) complex of hair growthcomDlex of dismutasecopper(I) (SOD)-mimeticcomDlexes of this invention;activity of athis invention;

Example 4 illustrâtesreprésentative copper(I)

Example 5 illustrâtes inhibition of HIV réplication by areoresentative copper(I) complex of this invention;Example δ illustrâtes the activity of a représentative"free” multi-dentate ligand of this invention for bothwound healing and protection against éthanol-inducedgastric mucosal damage; Examples 7 and 8 illustrâtes the 39 010198 inhibition of cyclooxygenase-1 and cyclooxygenase-2,respectively, by représentative stable copper(I)complexes; Example 9 illustrâtes the inhibition of 5-lipoxygenase by représentative stable copper(I) complexes;Example 10 illustrâtes the inhibition of leukotriene C4synthetase by représentative stable copper(I) complexes;Example 11 illustrâtes the inhibition of elastase by areprésentative stable copper(I) complex; Example 12illustrâtes the inhibition of acetyl coenzyme A synthetaseby représentative stable copper(I) complexes; Example 13illustrâtes the inhibition of HMG-CoA reductase byreprésentative stable copper(I) complexes; Example 14illustrâtes the inhibition of HIV-1 activity by variousisomers of a représentative stable copper(I) complex;Example 15 illustrâtes the anti-viral activity ofreprésentative stable copper(I) complexes and areprésentative free mult'i-dentate ligand; Example 16illustrâtes inhibition of HIV-1 and HIV-2 proteases byreprésentative stable copper(I) complexes; Example 17illustrâtes the inhibition of HIV reverse transcriptase byreprésentative stable copper(I) complexes; and Examples 18and 19 illustrate the inhibition of Protein Kinase C andvarious tyrosine kinases, respectively, by représentativestable copper(I) complexes.

Example 1

Synthesis.. of Copper ( I ) -Neocuproine

Neocuproine hydrate was used as received from AldrichChemical Company, naving the following properties: mplSl-163°C; S-H NMR (500MHz, DMSO-ds) δ 8.32 (2H, d, J = 8.2), 7.85 (2H, s), 7.60 (2H, d, J = 8.1), 2.79 (6H, s); 13C MMR(125MHz, DMSO-ds) δ 153.0, 144.6, 136.1, 126.4, 125.3,123.1, 24.9. 40 010198 A. Neocuproine CopperdJ—(Ixll

Cuprous chloride (1.98g, 20.0mmol) was added to a stirred, vacuum-degassed solution of neocuproine hydrate(4.53g, 20.0mmol) iii acetonitrile (l50mL) . This solution was stirred for 2 hours. The resulting suspension waswarmed to boiling and filtered. The filtrate was boiled toa volume of about lOOmL. This solution was allowed tocool slowly to give dark red needles: mp280-284°C(decomp., lit. 310-320°C)(Healy et al., iL_Chem,_S&CL_Dalton Trans . 2531, 1985); 1H NMR (500MHz, DMSO-d6) 6 8.74 (2H, d, J =8.2), 8.21 (2H, s), 7.95 (2H, d, J = 8.2), 2.38 (6H, s) ;13C NMR (125MH?, DMSO-ds) δ 157.6, 142.2, 137.4, 127.1, 125.9, 125.6, 25.1; Anal. calcd. for Ci4H12ClCuN2 : C, 54.73; H, 3.94; N, 9.12; Cl, 11.54. Found: C, 54.67; H,3.89; N, 9.04; Cl, 11.40. B. Neocuproine Copper(I)_(2j_1). A vacuum degassed solution of neocuproine hydrate(4.53g, 20.0mmol) in absolute éthanol (150mL) was added tocuprous chloride (990mg, lO.Ommol) via cannula under anatmosphère of nitrogen. The resulting bright red solutionwas stirred at room température for 2 hours. This mixturewas filtered, to remove a small amount of insolublematter, and evaporated to give 5.64g (100%) of bright redsolid. Recrystallization from aqueous methanol gave veryfine needles: mp231-233°C; UV-vis (95% éthanol) 207nm (ε = 63, 750M"1cm-1) , 226nm (ε = 76,250), 272nm (ε = 60,000),454nm (ε = 6,750), NMR (500MHz, DMSO-d6) δ 8.75 (2H, brs), 8.22 (2H, s), 7.96 (2H, br s), 2.40 (6H, s); 13C NMR(125MHz, OMSO-dg) 6 157.6, 142.2, 137.3, 127.1, 125.8,125.6, 25.0; Anal, calcd. for C2gH24ClCuN4 : C, 65.24; K,4.69; N, 10.87; Cl, 6.83; Cu, 12.33. Found: C, 65.01; H,4.73; N, 10.75; Cl, 6.34; Cu, 12.70. 41 010198

Example 2

Superoxide Dismutase Mime tic_ Activity of Copper(I) Complex

As used herein, compounds which possess activity in asuperoxide dismutase (SOD) assay are termed "SODmimetics." In this example, représentative copper(I)complexes of this invention were evaluated for SOD mimeticactivity as measured by the Xanthine Oxidase/NBT method (see Oberly and Spitz, HandbOQk_ûf_Methods for Oxygen

Radical Research. R. Greenwald (ed.), pp. 217-220, 1985;

Auclair and Voisin, Handbook of Methods for Oxygen RadicalResearch, R. Greenwald (ed.), pp. 123-132, 1985). The reactions contained the following: 100 uM Xanthine, 56 uMNBT (Nitro Blue Tétrazolium), 1 . unit of Catalase, 50 mMPotassium Phosphate .Buffer, pH 7.8. The reaction wasinitiated by the addition of Xanthine Oxidase insufficient quantity to obtain an increase in absorbance at560 nm of approximately 0.025/min. in a total volume of1.7 ml. The Xanthine Oxidase was prepared fresh daily andstored on ice until used. Ail the components of thereaction are added except the Xanthine Oxidase and thespectrophotometer was adjusted to zéro at 560 nm. Thereaction was initiated by the addition of the XanthineOxidase. Ail reagents were obtained from Sigma ChemicalCo.

Measurements of the Absorbance at 560 nm were takenat 1-2 minute intervals for at least 16 minutes. Thecontrol consisted of reactions containing zéro copper(I)complex. The copper(I) complexes tested in this examplewere as follows: bathocuproine disulfonate copper(I) ("3CDS;Cu(I)"); neocuproine copper(I) (”NC:Cu(I)"); and2,2'-biquinoline copper(I) ("BQ:Cu(I)"). As a positivecontrol, reactions containing a peptide-copper(II) complex(i.e., glycyl-L-histidyl-L-lysine:copper(II) or "GHX:Cu"),which is a known SOD mimetic (see U.S. Patent No. 42 010198 4,760,051) , were also employed. One unit of SOD activity was taken as that amount of î □ample in micromoles which inhibits the control reaction with the NBT by 50¾. The relative activity is then obtained by comparing the micromoles of copper(I) complex necessary to product a 50¾ inhibition of the control reactions. The lower the value, the more i active the compound is as an SOD mimetic. The results of this experiment are presented in Table 8 below. Tabla... a. SOD-Mimetic Activitv of Copper(I )Complexes .Copper Activity Relative Exp. No. Compound Ratia (urool per Activitv to Q icand:Cu) _Inhiba , ) Control 1 GHK:Cu(II) 2:1 0.055 BCDS:Cu(I) 2:1 0.034 1.6 2 GHK:Cu(II) 2:1 0.0503 — BCDS:Cu(I) 2:1 0.0278 1.8 BCDS:Cu(I) 2 :1 0.0018 28 • NC :Cu(I) 2 :1 0.0014 36 3 GHK:Cu(II) 2:1 0.0479 — NC :Cu(I ) 1:1 0.0018 27 BQ :Cu(I) 2 :1 0.0028 17 Exampla 3 Wound Healing Activitv of Copper ( I ).. Complexes The subcutaneous implantation of stainless steel wound chambers in rats provides a model for the healing ofcpen cavity wounds. Implantation of these chamberstriggers a sériés of responses which reflect the sériés of 43 010198 phases involved in wound healing - fibrin clôt formation,infiltration of white cells, collagen synthesis, and newblood vessel formation.

This assay involves the implantation of a stainless5 Steel chamber (1 X 2.5 cm cylindrical 312 SS, 20 mesh,with Teflon end caps) on the dorsal mid-line of rats.After one week to allow for encapsulation of the chamber,the chamber on each rat was injected with a 0.2 ml salinesolution containing 2.7 gmol of the copper(I) complex 10 (i.e., BCDS copper(I) 1:1 or 2:1), or with the same volume of saline (0.2 ml) without the copper(I) complex (i.e.,control) . Injections were made on days 5, 7, 9, 12, 14, 16 and 19. The chambers were then removed on day 21.

The chambers were lyophilized and the interior 15 contents removed for biochemical analysis. The biochemical parameters examined included the total dryweight, protein content, collagen content (i.e.,hydroxyproline content after acid hydrolysis) andglycosaminoglycan content or "GAG" (i.e., uronic acid 20 content after acid hydrolysis).

The protein was determined by the method of Lowry ët al. (J. Biol, Chem. 193: 265-275, 1951) using Bovine Sérum Albumin (BSA) as a standard. The collagen contentwas determined by acid hydrolysis and a colorimétrie assay 25 for hydroxyproline (Bergman et al., Clin. Chim, Acta 22.:347-349, 1970), an amino acid spécifie for collagen..

Glycosaminoglycan content was determined by quantitationof the amount of uronic acid (UA) . Aliquots of thehomogenate were dissolved in 0.5M NaOH, precipitated and '30 washed with éthanol, and uronic acid was determined by acolorimétrie assay using 2-phenylphenol as a reagent(Vilim V., Biomed. 3lochem. Acta. 44(ll/12s):1717-1720,1985). Glycosaminoglycan content was expressed as μσ ofuronic acid per chamber. 35 The results of this experiment are illustrated in

Figure 1. Specifically, BCDS copper(I) at botn the 1:1 44 0)0)98 and 2:1 ratio significantly stimulated theglycosaminoglycan content of the injected chamber.Moreover, BCDS copper(I) at both ratios stimulated thecollagen content of the injected chambers. Collagen andglycosaminoglycans are two of the critical extracellularmatrix components important for tissue associated with wound healing. régénération 10

ExampLe ,.A

Stimulation of Haïr Growth bv CopperfT) Complexes

The following example illustrâtes the stimulation ofhair growth in warm-blooded animais after intradermalinjection of a copper(I) complex of this invention. 15 The backs of C3H mice (60 days old, telogen hair growth phase) were closely clipped on day 1 using anelectric clipper. A stérile saline solution containingthe .indicated copper complex was then injectedintradermally (i.e., infiltrated under the skin) at two 20 locations within the clipped areas of the mice. Injectionat two locations provided two test locations within theclipped area of each mouse. Each injection (0.1 ml)contained the indicated amount of the copper(I) complex(i.e., BCDS copper(I) (1:1) complex at 0.14 pmol and 1.4 ,25__μιπο1) within a stérile saline solution. A group of salineinjected mice (0.1 ml) served as Controls. Followinginjection of the copper(I) complex, indications of hairgrowth were seén within 10 days. The first Visual signswere a darkening of the skin in a circular région 30 surrounding the injection site. The size of this régionis generally dose dépendent, increasing with an increasein dose. The 0.1 ml injections used in this experimentproduced a circle of hair growth measuring approximately0.5 cm2 to 5 cm2 in diameter. Active hair growth cccurred 35 between 14-20 days following injection, with a maximum effect seen by day 29. Both the number of mice growing 45 010198 hair at the injection site and the diameter of the hairgrowth région were determined at day 21. A positiveresponse was expressed as the number of mice exhibitinghair growth at the injection sites compared to the totalnumber of mice injected in the study. The résulte of thisexperiment are presented in Table 9 below'.

Table 3

Hair Growth Activity of-BCDS Copper(I) Complex

Amount Injected (μτηοΐ) Growth Area (cm2) 0.0 (control) 0.0 0.14 1.35 (Std. Dev. 0.42) 1.4 3.06 (Std. Dev. 0.47)

Example 5

Inhibition of HIV Réplication pf -.Copper ί I ). .Complex

In this experiment, the inhibitory effect ofbathocuproine disulfonic acid (BCDS) copper(I) (2:1)complex on phytohemagglutinin (PHA) stimulated peripheralblood mononuclear cells is demonstrated. PHA stimulated peripheral blood mononuclear cells(PSMC) were infected by HIVIIIS in the presence of thecopper(I) complex identified above and cultured in thepresence of the copper(I) complex for two weeks. Theextent of HIV réplication was assayed at 1 and 2 weeks bya p24 antigen capture ELISA assay. More specifically,PBMC was stimulated with PHA for 24 to 72 hours in basalmedium, containing RPMI-1640, 10V fêtai bovine sérum, and

50 gg/mL gentamicin, and then cultured overnight in thepresence of 250 units/ml IL-2. Treated PBMC were pelletedby centrifugation and resuspended to 0.75 x 10s/mL in basalmedium with appropriate dilutions of the copper(I) complexor with no copper(I) complex added (i.e., control). Toeach 0.5 mL aliquot of cells, 0.5 mL of appropriate HIV 46 010198 .. dilution was added. The virus-cell mixture was incubatedfor 2 hours at 37°C in a · 5% CO2 humidified atmosphère.Following the incubation period, the PBMC were washedtwice in phosphate-buffered saline. Cells were 5 resuspended in 5 mL to 7 x 104 cells/mL in basal mediumwith (or without) the copper(I) complex. Each cellaliquot was dispensed into four replicate wells of a 48well tissue culture plate. Cells were fed twice a weekwith appropriâte medium. 10 At one week and two week culture timepoints the extent of HIV réplication was assayed by a p24 antigencapture assay kit (Coulter Corp., Hialeah, Florida). PBMCwere treated with buffered detergent to release viralproteins. The cell extract was absorbed to immunoassay 15 titer plates and p24 was detected by binding of amonoclonal anti-p24 ar.tibody coupled to an enzyme.Following the addition of a chromogenic substrate, theamount of p24 was guantified spectrophotometrically.

The results of this experiment are presented in 20 Figure 2. In particular, a 50 uM concentration of theBCDS copper(I) (2:1) complex completely inhibited HIVréplication at both week 1 and week 2 at the identifiedvirus dilutions. Furthermore, the 5 uM concentration of •BCDS copper(I) (2:1) complex completely inhibited HIV-•25 -réplication at week 2 at the 10“^ virus dilution.

Examp 1

Activity of "Free" Multi-Dentate Ligand

This example illustrâtes the activity of the freemulti-dentate ligands of this invention. As used herein,the free ligand is not complexed to the copper(I) ionprior to administration. ' 30 47 010198 A. Inhibition of Ethanol-Tnduced Gastric Mucosal Damage

Juvénile Sprague-Dawley rats were used in this example. After fasting for 24 hours, the rats weretreated by oral gavage with bathocuproine disulfonic acid(BCDS) as the copper(I)-free ligand at various dosages(i.e., 0, 7.6 and 37.6 mg/kg body weight). One hour afterBCDS treatment, the animais were challenged with 1 ml of95% éthanol by oral gavage to cause érosion of the gastricmucosa. As shown in Table 10, BCDS pre-treatment led to adose-dépendent protection against the mucosal damageobserved in the control animais.

Table.. 10 t ,ρς aCDa.. on., atnanoi-inou Gastric Mucosal Damace Cefl Dosage Mucosal Damage mc/kc body we-îcht % of total area Mean 0.0 45.48 6.94 7.6 32.95 7.49 37.6 23.45 8.18 B. Wound Healing Activity

The BCDS ligand was also examined in the rat woundchamber model as disclosed above in Example 3. Theresults of this experiment are presented in Table 11.

Table, 11

Effect of_3CDS on Wound Healing rns/injection ug uronic acid/mg protein 0.0 (control) 1.5 7.5 28.3 (Std. Dev. ± 8.7)57.6 (Std. Dev. ± 9.1)79.2 (Std. Dev. ± 10.8) 48 010198

These results indicate that glycosaminoglycan synthesis isstimulated by administration of the free BCDS ligand.

Example 7

Inhibition of Cyclooxygenase-1 byNeocuproine and BCDS CoEoeriI)__Complexes (2:D

Cyclooxygenase is involved in the formation ofprostaglandins and thromboxanes by the oxidativemetabolism of arachidonic acid (see Figure 3).

In this experiment, cyclooxygenase-1 from ram séminalvesicles was incubated with arachidonic acid {100 uM) for2 minutes at 37° C in the presence or absence ofneocuproine ccpper(I) (2:1) or BCDS copper(I) (2:1) atincreasing concentrations of neocuproine copper(I) or BCDScopper(I) from 0.3 to 300 μΜ. The assay was terminated bythe addition of trichloroacetic acid (TCA), andcyclooxygenase-1 activity was determined by reading theabsorbance at 530 nm (Evans et al., "Actions of CannabisConstituents on Enzymes of Arachidonate Metabolism:Anti-inflammatory Potential," Biochem, Pharmacol, Ifi.: 2035-2037, 1987; Boopathy and Balasubramanian, "Purificationand Characterization of Sheep Platelet cyclooxygenase,"Biochem J. 222.' 371-377, 1988) .

Neocuproine copper(I) (2:1) was found to inhibitcyclooxygenase-1 with an IC50 of 23μΜ (see Table 12) .3CDS copper(I) (2:1) ccmplex produced approximately 44%inhibition at a concentration of 300 μΜ. These resultsdemonstrate that the stable copper(I) complexes cf thisinvention are potent inhibitors of prostaglandin synthesisthrough inhibition of cyclooxygenase-1. 49

Table 12

Inhibition of Cyclooxygenase-1 by

Stable Copper(I) Complexes 010198

Compound Conc. (μΜ) Percent Inhibition (Mean ± SEM) BCDS Copper(I) (2:1) 300 43.5 ± 1.5 Neocuproine Copper(I) (2:1) 300 77.3 ± 1.5 30 54.5 ± 0.5 3.0 15.5 4- 2.5 0.3 6.5 0.5

Example ...8

Inhibition of Cyclooxygenase-2 by

Neocuproine and BCDS Copper(I) Complexes (2:1)

Cyclooxygenase-2,synthase-2, catalyzesDrecursors to form also known as prostaglandin Hthe oxygénation of unesterifiedcyclic endoperoxide dérivatives,including prostaglandin H (see Figure 3).

In this experiment, cyclooxygenase-2 frotn sheepplacenta, 80 units/tube, was pre-incubated with 1 mMglutathione (GSH), 1 mM hydroquinone, 2.5 μΜ hemoglobin, and either neocuproine copper(I) (2:1) or BCDS copper(I)(2:1) at increasing concentrations of neocuproinecopper(I) or BCDS copper(I) from 0.3 to 300 μΜ for 1minute at 25°C. The reaction was initiated by the additionof arachidonic acid (100 μΜ) , and terminated after 20minutes at 37° C by the addition of TCA. centrifugal séparation of the precipitated thiobarbiturate was added and cyclooxygenase activity wasdetermined by absorbance at 53 0 nm (see Evans et al.,supra ,· Boopathy and Balasubramanian, supra : 0'Sullivan etal., "Lipopolysaccharide Induces Prostaglandin H Synthese-2 in Alveolar Macrophages," Biochem, Biophys. Res. Commun.1^2:1123-1127, 1992).

Following protein, 50 010198

Neocuproine copper(I) (2:1) was found to inhibitcyclooxygenase-2 at an estimated IC50 of 25μΜ (see Table13) , which is similar to the results of Example 7 withcyclooxygenase-1. BCDS copper(I) (2:1) produced 5 approximately 34% inhibition at the screeningconcentration of 300 μΜ. These results show that stablecopper(I) complexes of this invention are also notentinhibitors of prostaglandin synthesis through inhibitionof cyclooxygenase-2. 0

Table-13.

Inhibition of.Cyclooxvgenase-2 by

Stable Copper(I) Complexes

Compound Cc>ng. Percent. .Inhib.i.tig.n (μΜ) (Mean + SEM) BCDS Copper(I) (2:1) 300 34.0 + 1.0 Neocuproine Copper(I) (2:1) 300 63.8 + 0.5 30 54.0 + 1.0 3.0 7.0 + 1.0 0.3 6.5 + 2.5 25

Exemple .2.

Inhibition of 5-Lipoxygenase by

Neocuproine and 5CDS Copper(I) Complexes (2:1)

The 5-lipoxygenase is the principal lipoxygenase inbasophils, polymorphonuclear (PMN) leukocytes, macrophages, mast cells, and any organ undergoing aninflammatory response. As illustrated in Figure 3, theaction of 5-lipoxygenase leads to the formation of 5-HPETEand 5-ΗΕΤΞ, which are precursors to the leuokotriene LTB4and LTC4.

In this experiment, 5-lipoxygenase assays were run using a crude enzyme préparation prepared from rat 51 010198 basophilie leukemia cells (RBL-1). Neocuproine copper(I)(2:1) or BCDS copper(I) (2:1) at increasing concentrationsfrom 0.3 to 300 μΜ were pre-incubated with the 5-lipoxygenase for 5 minutes at room température, and thereaction was initiated by addition of arachidonic acidsubstrate. After incubation at room température for 8minutes, the reaction was terminated by the addition ofcitric acid. The levels of 5-HETE were determined by aspécifie 5-HETE RIA (Shimuzu et al., "Enzyme with DualLipoxygenase Activities Catalyzes Leukotriene A4 Synthesisfrom Arachidonic Acid," Proc. Natl. Acad. Sci, U,S,A, 689-693, 1984; Egan and Gale, "Inhibition of Mammalian 5-Lipoxygenase by Aromatic Disulfides," J. Biol. Chem.2ÆÛ:11554-11559, 1985).

Both BCDS copper(I) (2:1) and neocuproine copper (I)(2:1) were found to be inhibitors of 5-lipoxygenase withestimated ICSO's of less than 10 μΜ (see Table 14). Theseresults show that stable copper(I) complexes of thisinvention are potent inhibitors of neutrophil 5-lipoxygenase, thus preventing the accumulation ofinflammatory lipid mediators at the sites of inflammation.

Tabla 14

Inhibition .of ScLipoxygenase ,by

Stable Copper.il) Complexes

Compound BCDS Copper(I) (2:1)

Neocuproine Copper(I) (2:1)

Conc. Percent Inhibi t >'on (μΜ) (Mean ± SEM) 30 71.3 ± 2.5 3.0 29.0 ± 5.0 0.3 5.5 i 3.5 0.03 4.0 ± 1.0 30 99.0 ± 0.5 3.0 51.0 ± S.C 0.3 15.5 ± 2.5 0.03 7.0 ± 0.0 52 010198

Example 1Q

Inhibition of Leukotriene Synthetase bv

Neocuproine and BCDS Copper(I) Complexes (2:1)

Leukotriene C4 (LTC4) Synthetase is involved in theformation of LTC4 from LTA4, as illustrated in Figure 3,by the addition of a reduced glutathione at the CS site.

In this example, LTC4 Synthetase was prepared as acrude fraction from rat basophilie leukemia cells (RBL-1).The crude enzyme fraction was incubated with testcompounds, LTA4 methyl ester, albumin (to stabilize theproduct), and serine borate (to prevent conversion of LTC4to LTD4) for 15 minutes at 37° C. The reaction wasterminated by the addition of ice cold methanol, and LTC4concentration was determined by a spécifie RIA (Bach etal., "Inhibition by Sulfasalazine of LTC4 Synthetase andof Rat Liver Glutathione S-Transferases," Biochem.Pharmacol. JA:2695-2704, 1985; Fitzpatrick et- al.,"Albumin Stabilizes Leukotriene A4," J. Biol, Chem,257:4680-4683 . 1982).

Both BCDS copper(I) (2:1) and neocuproine copper(I)(2:1) were found to be inhibitors of LTC4 Synthetase withestimated IC50's of 87 and 285 μΜ, respectively (see Table15). These results show that stable copper(I) complexes,are potent inhibitors of neutrophil LTC4 Synthetase, thuspreventing the accumulation of inflammatory lipidmediators at the sites of inflammation. t 010198 53

. . Table -1S

Inhibition of Leukotriene fLTC/Π Svnthetase by

Stable Copper ( II, ..Complexes compound Cqqg. Percent-, Inhibit i en (μΜ) (Mean ± SEM) BCDS Copper(I) (2:1) 1000 77.8 1.9 100 51.0 + 4.0 10 26.5 + 1.5 1 11.0 + 2.0 Neocuproine Copper(I) (2:1) 1000 71.0 + 1.9 100 32.5 0.5 10 15.0 4. 1.0 1 9.0 4. 1.0 E2samiila_ll Inhibition of Elastase by BCDS Copper(I) (2:1) Proteolysis of various cellular 1 targets by elastase has been implicated in a number of pathologie conditions, including emphysema, rheumatoid arthritis, and psoriasis.

In this experiment, human neutrophil was the source 15 of the elastase. In particular, human neutrophil elastasewas prepared in crude form from fresh blood followingdextran sédimentation, leukocyte isolation, cell lysis andhomogenization of sub-cellular granules containing theelastase. BCDS copper(I) (2:1) was incubated with the 20 enzyme . and substrate (methoxysuccinly-alanyl-alanyl-propyl-valine-4-nitroanalide) for Θ minutes at 25°C. Thereaction is terminated by immersing the test tubes inboiling water for 5 minutes. Spectrophotometric analysisof the proteolytic product is measured at 410 nm (Baugh 25 and Travis, "Human Leukocyte Granule Elastase, Rapid 010198 54

Isolation and Characterization," Biochemistry 13:836-841,1976) . BCDS copper(I) (2:1) was found to inhibit human neutrophil elastase with an estimated IC50 of 12 μΜ (seeTable 16). These results show that stable copper(I)complexes of this invention are potent inhibitors ofneutrophil elastase, thus preventing or limiting thebreakdown of normal tissue at the sites of inflammation.

Table. LS Inhibition of Human Neutrophil Elastase by Stabl' e Coooer(I) Complexes CompQuad Conc. Percent Inh ibition (μΜ) (Mean ± SEM) BCDS Copper(I) (2:1) 30 65.8 i 3.1 3.0 25.0 + 5.0 0.3 18.5 + 0.5 0.03 5.5 + 0.5

Inhibition of Acetyl Coenzyme A . (CoA). Synthetase by

Neocuproine and BCDS Copper(I)_(2j1)

In this experiment, the ability of two stablecopper(I) complexes, neocuproine copper(I) (2:1) and BCDScopper (I) (2:1), to inhibit certain key enzymes involved in the formation of lipids is demonstrated.

CoA synthetase (yeast) activity was monitored byutilization of a labeled substrate, sodium [3H]acetate(Grayson and WestKaemper, "Stable Analogs of AcylAdenylaes, Inhibition of Acetyl and Acyl (acyl-CoA) CoASynthetase by Adenosine 5'-alkylphosphates, " Life Sci. 43.:437-444, 1988). A reaction buffer including 0.1 M glycine-NaOH (pK 9.0), AT?, and the substrate was pre- 55 010198 incubated for 5 minutes at 27°C, followed by addition of 2nM coenzyme A for an additional 5 minute incubation at 27°C. The reaction was terminated by addition of HCl, andthe remaining substrate determined by scintillationcounting.

The results of this experiment are presented in Table17. Both BCDS copper(I) (2:1) and neocuproine copper(I)(2:1) were found to inhibit acetyl CoA synthetaseactivity.

Table 17

Inhibition of Acetyl CoA Synthetase by

Stable Copper (II.., Complexes

Cg.mp.Qiind -^M). BCDS Copper(I) (2:1) 29 Neocuproine Copper(I) (2:1) 47 Reference compounds: Ethyl-5-AMP 60 Lovastatin >100 Orotic Acid >100

Both stable copper(I) complexes tested were found toinhibit acetyl CoA synthetase with estimated ICsq's of 30-50 μΜ. These results indicate that the stable copper(I)complexes of this invention may serve as lipid modulating(e.g., lipid lowering) agents.

Exampls ,.13

Inhibition of HMG-CoA Reductase by

Neocuproine and BCDS Copper(I) (2:1)

In this experiment, HMG-CoA reductase was isolated from rat liver and incubated with [14C] HMG-CoA and either neocuproine copper(I) (2:1) or BCDS copper(I) (2:1) for 15 56 010198 minutes at 37°C. The reaction is terminated by addition ofHCl, and [14C] MVA is separated from the intact substrateby column filtration (Kubo and Strott, "DifferentialActivity of 3-hydroxy-3-methylglutaryl Coenzyme AReductase in Zones of the Adrenal Cortex," Endocrinology120; 214-221, 1987; Heller and Gould, "Solubilization and

Partial Purification of Hepatic 3-hydroxy-3-methylglutaryl

Coenzyme A Reductase," Biochém._BÏQphys __Ses,_Comm. 5Ώ.: 859-865, 1973) .

Testing at 3 0 μΜ indicated that both neocuproinecopper(I) (2:1) and BCDS copper(I) (2:1) inhibited théHMG-CoA reductase enzyme. The results of this experimentare presented in Table 18.

Tablé 18

Inhibition of HMG-CoA Reductase by

Stable Copper(I) Complexes

Compound BCDS Copper(I) (2:1) >30 μΜ, <50 μΜ Neocuproine Copper(I) (2:1) >30 μΜ, <50 μΜ Reference compound: Lovastatin . >12 ηΜ

Both stable copper(I) complexes tested were found to.· .inhibit HMG-CoA reductase with ICsq's estimated at greaterthan 30 μΜ. These results indicate that the stablecopper(I) complexes of this invention may serve as lipid modulating (e.g., lipid lowering) agents. 57 010198

Examp la. IA

Inhibition of HIV-1 Activitv bv BCDS Copper(I) (2:1) Isomers

The experiments presented in this example demonstratethe effect on anti-HIV activity of different isomers ofBCDS copper(I) (2:1). Two experiments utilized p24antigen capture as a marker for viral réplication, whiletwo further experiments utilized reverse transcriptaseactivity to monitor the course of infection. Theinfection in ail three experiments was performed incultures of human peripheral blood mononuclear cells(PBMC) treated with HIV-1.

The positional isomers of BCDS copper(I) employed inthis experiment are identified above as structures Ile,Ile' and Ile'', and are set forth below:

Ile' '

Structure Ile is referred to herein as the para-para ("P?") BCDS isomer since both disulfonic acid/sodium sait 58 010198 moieties are located in the para position. Similarly,structure Ile' and Ile'1 are referred to herein as themeta-para ("MP”) and meta-meta ("MM") BCDS isomers,respectively. In addition, a mixture of the PP, MP and MMBCDS isomers was also tested (referred tç herein simply as"BCDS”), having a ratio of PP:MP:MM of approximately5:39:56.

In the first experiment, the anti-HIV activity ofBCDS, MP-BCDS and MM-BCDS copper(I) (2:1) at twoconcentrations (i.e., 10 and 25 μΜ) was compared. Theseconcentrations had been previously determined to bepartially and completely effective, respectively, forinhibition of HIV réplication by BCDS copper(I).

The same methodology as described above in Example 5for evaluating inhibition of HIV réplication was employedin the experiment. The results of this experiment areprésent in Table 19.

Table 13,

Inhibition of HIV Réplication byBCDS, MP-BCDS and MM-BCDS Copper(I) (2 ; 1) . as._Measured..±y p24 Antigen Capture (@1:1000 viral dilution) tëe.ek .1

Compound (SEM) % Inhibition Control (infected cells) 30910.00 3770.00 BCDS Copper(I) (10μΜ) 1959.00 317.16 93.66 BCDS Copper(I) (25μΜ) 0.25 0.25 99.99 MP-3CDS Copper(I) (10μΜ) 404.50 124.66 98.69 MP-BCDS Copper(I) (25μΜ) 0.50 0.50 99.99 MM-BCDS Copper(I) (10μΜ) 346.50 106.27 98.88 MM-3CDS Copper(I) (25μΜ) 0.00 0.00 100.00 59 010198

Week 2

CompQ.unà p2£ (SEM) 1. Inhibition Control (infected cells) 10483.80 1109.73 -- BCDS Copper(I) (ΙΟμΜ) 3286.00 242.36 68.66 BCDS Copper(I) (25μΜ) 0.00 •o.oo 100.00 MP-BCDS Copper(I) (10μΜ) 901.75 277.26 91.40 MP-BCDS Copper(I) (25μΜ) 0.00 0.00 100.00 MM-BCDS Copper(I) (10μΜ) 549.50 176.25 94.76 MM-BCDS Copper(I) (25μΜ) 0.00 0.00 100.00 In a second experiment, the activity of BCDS copper(I) and PP-BCDS copper(I) was compared in the mannerdescribed above. The results of this experiment are setforth in Table 20. In this experiment the p24 concentrations were lower than in the above experiment.This is due to a different ELISA technique used in thisexperiment. The standard curve for p24 détectionmaximizes at 300 pg/ml. Any values over 300 require akinetic extrapolation to estimate the p24 concentration.Such extrapolation gives a substantial underestimation ofthe actual p24 concentration. . To obtain a more accurateestimate, a sériés of dilutions of the sample was made toarrive at a reading that is in the middle of the standardcurve, and the dilution factor applied to the reading togive the p24 concentrations. This method (which was usedin the first experiment, see Table 19 above) while moreaccurate, yields an overestimate due to the errors’ ofdilution. Nevertheless, the comparisons frcm one sampleto the next in each experiment reflect the inhibitoryeffects of stable copper(I) complexes tested. 60 010198

Table. 22

Inhibition ,Qf...HIV Réplication by BCDS and PP-BCDS Copper(I)_(2:1) as Measured by p24 Antlgen Capture ((31:1000 viral dilution)

Week—I

Compaund p21 .(SEML ^-..Inhibition Control (infected cells) 1649.75 29.32 BCDS Copper(I) (10μΜ) 474.25 41.22 71.25 BCDS Copper(I) (25μΜ) 39.50 6.06 97.61 PP-BCDS Copper(I) (10μΜ) 480.00 49.65 70.90 PP-BCDS Copper(I) (25μΜ) 34.50 4.57 97.91 Hfigk-2 Compound p24 (SEM) % Inhibition Control (infected cells) 2256.50 45.93 BCDS Copper(I) (10μΜ) 1785.00 49.03 20.90 BCDS Copper(I) (25μΜ) 22.00 6.38 99.02 PP-BCDS Copper(I) (10μΜ) 1915.75 69.75 15.10 PM-BCDS Copper(I) (25μΜ) 33.25 6.60 98.53 In a third experiment , the anti-HIV activity of BCDS,. PP-3CDS, MP-BCDS and MM-BCDS copper(I) (2:1) was determined by monitoring the same type of culture (i.e.,HIV-1, PBMC) by measuring the reverse transcriptaseactivity as an infection marker. The -PBMC cultureconditions for this experiment are described above inExample 5. Following 6 days of incubation, the activityof HIV-1 reverse transcriptase in cellular extracts wasdetermined as a marker for the réplication of the virus inculture. The measurement of HIV-1 reverse transcriptasein P3MC cultures may be performed by known techniques(Chattopadhyay et al., "Purification and Characterizationof Heterodimeric Human Immuncdeficiency Virus Type 1(HIV-1) Reverse Transcriptase Produced by an In VitroProcessing of p66 with Recombinant HIV-1 Protease," 61 010198

Biol. Chem, 252:14227-14232, 1992). The results of this experiment are presented in. Table 21.

Tabla 21 5 Inhibition of HIV Réplication by BCDS. PP-BCDS. MP-BCDS and MM-BCDS Copper(I) (2:1) as Measured by Reverse Transcriptase Activity

Reverse Transcriptase Activity

Compound Conc. (μΜ) Inhibition None(control) 0 29283 NA BCDS Copper(I) 0.001 23963 18.17 0.01 19585 33.12 0.1 17340 40.78 1 17623 39.82 10 4974 83.01 100 585 93.00 PP-BCDS Copper(I) 0.001 26934 8.02 0.01 28097 4.05 0.1 12742 56.49 • 1 12247 58.18 10 1846 93.70 100 566 98.07 MP-BCDS Copper(I) 0.001 19966 31.82 0.01 15040 48.64 0.1 12369 57.76 1 9880 ..... 66.26 10 1408 95.19 100 540 93.16 MM-BCDS Copper(I) 0.001 22679 22.55 0.01 18212 37.81 0.1 13464 36.95 1 2085 92.88 10 583 98.01 52 010198

In a fourth experiment, inhibition of HIV-i, HIV-2and SIV, as compared to AZT, was determined for BCDScopper(I), PP-BCDS copper(I), MP-BCDS copper(I) and MM-BCDS copper(I). The experimental conditions describedabove where employed utilizing Reverse Transcription assayto monitor infection. The results of this experiment arepresented in Table 22. It should be noted that the datapresented in Table 22 are report in a different formatfrom that of Table 21. In particular, the data of Table22 represent the calculated EC50 values. The EC5Q isdetermined by non-linear régression from inhibition data(such as that presented in Table 21), and extrapolated forthe concentration of the test compound required toaccomplish a 50% inhibition of reverse transcriptaseactivity.

Table .22 Inhibition of HIV-1. HIV-2 and SIV Réplication bv BCDS, PP-BCDS, MP-BCDS and MM-BCDS Copper(I) (2:1) as Measured by Reverse Transcriptase Activity EC50 (μΜ) Compound HIV-1 HIV-2 SIV BCDS Copper(I) 1.7 17.6 4.6 PP-BCDS Copper(I) 0.25 1.2 6.4 MP-BCDS Copper(I) 0.04 12.1 4.3 MM-BCDS Copper(I) 0.13 0.62 6.1 AZT 0.004-0.009 0.0004 0.00€

Example .15.

Anti-Viral Activity of Stable Copperil)_Complexes

This example illustrâtes that the stable copper (I) compounds cf this invention, as well as the free ligands, 63 010198 hâve general anti-viral activity. In this experiment,BCDS copper(I) and BCDS alone (i.e., the free ligand) wereassayed for the ability to inhibit the murine virusencephalomyocarditis (EMCV) and the cytomegalo virus(CMV).

Inhibition o£, EMCV

Cultures of A549 cells (human lung) were infectedwith EMCV for 24-48 hours in the presence of either BCDScopper(I) or BCDS alone. The cells were cultured in DMEM(10% FBS) for 3-4 days prior to use. The medium was thenremoved, and the cells incubated with sufficient EMCV insérum free DMEM to kill between 30-90% of the cells in theculture. After 2-3 hours of incubation of the cells withEMCV in their presence (or absence) of the test compounds,complété medium (DMEM + 10% FBS) was added and the cellsallowed to incubât e for 1-2 days in the presence orabsence of the test compounds at concentrations rangingfrom 0.0001-0.0005 M.

The viability of the cultures was then measured bymitochondrial function test (Mossman, J, Immunol, Meth.£5.:55-63,1983). The ability of the test compounds toprotect the cells from the lethality of the EMCV infectionwas calculated as a percent protection compared to themitochondrial activity of parallel, uninfected cells. Theresults of this experiment are presented in Table 23.

Table 23

Inhibition of EMCV byStable .Copper (I) Complex and Free Ligand % Protection (uM) BCDS CopDerm (2:1) BCDS Ucand 100 30.2 10.7 200 67.2 3.2 400 97.7 22.2 500 153.2 84.2 64 010198

Inhibition of CMV

Normal Diploid Human Fibroblasts were isolated andcultured with Minimal Essential Medium (MEM) containingEarles balanced salts and supplemented with 10% FêtaiBovine Sérum (FBS). Cytomegalo virus (CMV) was added tothe cultures in the presence or absence of BCDS and BCDScopper(I) (2:1). Five cultures were employed in each testgroup, with the exception of the uninfected cell groupswhich utilized 8 cultures. The uninfected cell groupswere used to ensure that antiviral activity was achievedin the absence of any direct cytotoxic effect of the testcompounds.

After one week of incubation, cellular viability(i.e., mitochondrial functicn) was determined, and theability of the test compounds to prevent the cytopathiceffect (CPE) of the virus was calculated as percentprotection by the following formula: % Protection = (Vt-Vv)/Vu-Vv) x 100 where Vt represents viability of the test culture, Vvrepresents the viability of culture with virus alone, andVu represents the viability of uninfected cells.

The results of this experiment are presented in Table 24. No cytotoxic effects were observed on the uninfectedcompounds treated with the test compounds.

Table 24

Inhibition of CMV by

Stable Coppsrd) Complex and Free Ligand % Protection (SEM) Conc. (uM) BCDS Copper(I) (2:1' BCDS Licand 25 13.1 (7.7) 34.2 (8.1) 100 117.3 (13.3) 35.4 (6.2) 250 92.9 (5.2) 23.6 (8.3) 65 010198

Exampls 16

Inhibition of HIV-land HIV-2. Proteases by5 Stable Copper(I) Complexes

This example illustrâtes the ability of stablecopper(I) complexes of this invention to inhibit HIV-l andHIV-2 proteases. 10 HIV-l Protease 125t-spa Assay

In this experiment, SPA beads (ScintillationProximity Assay) were coupled with a peptide substrate toassay for HIV-l protease. The substrate was a 12 residue 15 peptide with the following sequence:

AcN-Tyr-Arg-Ala-Arg-Val-Phe-Phe-Val-Arg-Ala-Ala-Lys-COOH

The peptide was monoiodinated on the terminal tyrosine20 residue, biotinylated through the ε-amino group on theterminal lysine, and linked to the SPA bead via a streptavidin link. HIV-l protease cleaves the peptide substrate at thePhe-Phe bond, releasing the 125I-fragment from the bead. 25 Once the peptide is cleaved, it can no longer stimulatethe scintillant in the SPA bead and the signal is reduced.The rate of réduction is proportional to the activity ofthe HIV-l protease. Recombinant HIV-l protease, affinitypurified for kinetic and assay studies, was used in this 30 experiment.

Two types of Controls were conducted with this assay, one without enzyme to test for possible scintillation quenching by the test compound (2:1)), and another positive 35' pepstatin. At concentrations 10 (i.e., 3CDS copper(I)control with acetyltimes that used in the 66 010198 assay, there was no quench detected in the presence ofBCDS copper(I) (2:1).

The results of this experiment are presented in Table 25. The data presented is the mean ± SD of the percent 5 inhibition relative to a no enzyme control reaction. Asdiscussed above, the IC5Q was estimated from the point atwhich the dose inhibition line crossed the 50% inhibitionline. The estimated IC50 with this HIV-1 protease assaywas ΙΙμΜ. 10

Tabla 25

Inhibition of HIV-1 Protease by

Stable Copper(I) Complexes ,Compound BCDS Copper(I) (2:1)

ReferenceAcetyl

Compound:Pepstatin

Conc. Percent Inhibitii (μΜ) (Mean ± SEM) 25 86.7 + 2.1 10 45.2 ± 2.3 5 17.6 -U 2.3 2 12.2 ± 1.8 1 8.5 ± 4.6 0.5 1.8 ± 0.6 0.1 0.0 4. 1.9 0.5 67.4 + 1.1 0.25 50.1 + 0.4 0.1 28.4 7.7 0.05 16.6 4- 0.5 0.025 10.2 ± 2.6 0.01 2.4 ± 3.5 15 HIV-2 Protease 125.1-sPA AssayAs in the above experiment, SPA beads were coupled with a peptide substrate to assay for HIV-2 protease. The20 substrate was the 12 residue peptide identified above and 010198 67 monoiodinated on the terminal tyrosine residue,biotinylated through the ε-amino group on the terminallysine, and linked to the SPA bead via a streptavidinlink. 5 HIV-2 protease cleaves the peptide substrate at the

Phe-Phe bond, releasing the 125I-fragment from the bead.Once the peptide is cleaved, it can no longer stimulatethe scintillant in the SPA bead and the signal is reduced.The rate of réduction is proportional to the activity of 10 the HIV-2 protease. Recombinant HIV-2 protease, affinitypurified for kinetic and assay studies, was used in thisexperiment. HIV-2 protease has about 50% seguencehomology with HIV-1 protease, and is similar to simianimmunodeficiency virus (SIV) protease. 15 Two types of control assays were again run, one without enzyme and the other using acetyl pepstatin as apositive control.

The results of this experiment are presented in Table 26. The data presented is the mean ± SD of the percent 20 inhibition relative to a no enzyme control reaction. TheIC50 was estimated from the point at which the doseinhibition line crossed the 50% inhibition line. Theestimated IC5Q with this HIV-2 protease assay was ΙΟμΜ. 68 10 010198

Table 2,6,

Inhibition of HIV-2 Protease bv

Stable Copperil).Complexes

Compound BCDS Copper(I) (2:1)

Reference Compound:Acetyl Pepstatin COUP. Percent Inhibition (μΜ) (Mean ± SEM) 25 10 5 2 1 0.5 0.1 5.02.51.00.50.250.1 51.9 ± 5.5 49.6 ± 2.9 32.2 ± 2.8 14.1 ± 1.1 5.3 + 0.9 0.8 ± 5.3 2.5 ± 0.6 88.8 ± 0.570.6 ± 2.345.5 ± 1.837.2 ± 0.3 19.9 ± 5.64.3 ± 12.3

Example 17

Inhibition of HIV Reverse Transcriptase bv

Stable Popper(I) Complexes 15

This example illustrâtes the ability of a stablecopper(I) complex of this invention, BCDS copper(I) (2:1),to inhibit HIV reverse transcriptase activity.

As in Example 16 above, SPA (Scintillation ProximityAssay) beads were used to assay for the reversetranscriptase activity. The reverse transcriptase (10 uL)was incubated with the 3H-deoxyribonucleotides (10 uL) ,the DNA primer linked to biotin (10 uL) , and the RNAtemplate. After incubation at 37° for 20 minutes, thereaction was stopped and the labeled product was recovered 20 69 010198 by addition of the SPA beads coupled to streptavidin whichbinds to the biotin linked DNA primer.

The extent of the reaction was determined byscintillation counting. Increasing concentrations of BCDScopper(I) (2:1) were added and the extent of the reactiondetermined by the method described above.

The results of this experiment are presented in Table 27. The data show the mean ± SD of the percent inhibitionrelative to a no test compound control reaction. The IC5Qis estimated from the point at which the dose inhibitionlike crosses the 50% inhibition line. The estimated IC50was ΙΙμΜ.

Table 27

Inhibition of HIV Reverse Transcriptase by

Stable Cgppgr.il) Complexes

Compound

Conc.(μΜ)

Percent Inhibition (Mean) BCDS Copper(I) (2:1) 25 63.2 10 35.4 5 26.8 2 27.0 1 9.8 ' ........ 0.5 0.2 0.1 1.8

Sxample 16,

Inhibition of Protein Kinase C by

Stable Coppexil) Complexes

This example illustrâtes the ability of thereprésentative stable copper(I) complexes, BCDS copper(I)(2:1) and neocuproine copper(I) (2:1), to inhibit enzymesinvolved in intracellular signal transduction. The 70 010198 enzymes tested in this experiment were various proteinkinase C isozymes [and protein tyrosine kinases spécifiefor growth factors and cytokines]. 5 Protein Kinase C (non-selective) Assay

In this experiment, the reaction mixture included 20

mM Tris-HCl, pH 7.4, [32P]-ATP, phosphatidylserine,partially purified PKC from rat brain, and one of the testcompounds (Hunnun, et al. "Activation of Protein Kinase C 10 by Triton X-100 Mixed Micelles Containing Diacylglycéroland Phosphatidylserine," J. Biol. Chem. 260:10039-10043,1985; Jeng, et al., "Purification of Stable Protein KinaseC from Mouse 3rain Cytosol by Spécifie Ligand ElutionUsing Fast Protein Liquid Chromatography," Cancer. Res. 15 46:1966-1971, 1986). Following a 10 minute incubation, 25 ul aliçruots are removed, spotted on phosphocellulosepaper, washed three times in cold phosphoric acid, dried,and counted to détermine phosphorylated product. Theresults of this experiment are presented in Table 28. 20

Table. 29

Inhibition of Protein Kinase C (non-selective) by

Stable CopperiT) Complexes

Compound BCDS Copper(I) (2:1)

Neccuproine Ccpper(I) (2:1)

Conc. Percent Inhibition (μΜ) (Mean ± SEM) 300 87.5 ± 2.7 30 9.5 ± 4.5 3.0 7.5 ± 4.5 0.3 2.5 ± 4.5 300 62.0 ± 1.9 30 22.0 ± 7.0 3.0 6.0 ± 4.0 0.3 -12.0 - 2.0 71 010)98

Protein Kinase Ca Assav

Protein Kinase Ca is one of the major protein kinaseC isoforms. Protein kinase C is a family of serine/threonine protein kinases that médiate the actionsof a wide variety of growth factor, hormone, and neurotransmitter action.

In this experiment, protein Kinase Ca was purified tohomogeneity from rat brain using a modification of a thepublished procedure(3). The purity of the isolated PKCawas confirmed by SDS/polyacrylamide gel electrophoresisand isoform-specific antibodies. The enzyme was pre-incubated with the test compounds, and its activity ismeasured by the ability of the enzyme to phosphorylatehistone H1 in the absence and presence of calcium,phosphatidylserine, diolein and [32P]ATP. Following a 5minute incubation, the reaction was terminated by theaddition of acetic acid, 50 ul aliguots are removed,spotted on phosphocellulose paper, washed three times inwater, dried, and counted to détermine phosphorylatedproduct. The data presented in Table 29 show that theaddition of the stable copper(I) complexes inhibit theactivity of Protein K'inase Ca.

Table .22,

Inhibition of Protein Kinase Ca by

Stable copper(I) Complexes

Compound 3CDS Copper(I) (2:1)

Conc. Percent Inhibitio: (μΜ) (Mean ± SEM) 100 88.3 ± 0.5 10 19.0 ± 2.0 1.0 0.0 ± 3.0 0.1 -4.5 ± 5.5 72 010198

Neocuproine Copper(I) (2:1) 100 10 1.0 0.1 87.5 23.6 -1.5 -5.0 + 4- ± 1.8 4.5 3.5 1.0 Protein Kinase CB Assav Protein Kinase Οβ is another major protein kinase C isoforms. Protein kinase C is a family of serine/threonine protein kinases that médiate the actionsof a wide variety of growth factor, hormone, andneurotransmitter action.

In this experiment, Protein Kinase Οβ (which includes βΐ and βΐΐ forms) was purified to homogeneity from ratbrain using a modification of a published protocol(Woodgett and Hunter, "Isolation and Characterization ofTwo Distinct Forms of Protein Kinase C, " J. Biol, Chem.252:4836-4848. 1987). The purity of the isolated PKCa wasconfirmed by SDS/polyacrylamide gel electrophoresis andisoform-specific antibodies. The enzyme was pre-incubatedwith test compounds, and its activity is measured by theability of the enzyme to phosphorylate histone Kl in theabsence and presence of calcium, phosphatidylserine,diolein and [32P]ATP. Following a 5 minute incubation,the reaction was terminated by the addition of aceticacid, 50 ul aliguots are removed, spotted onphosphocellulose paper, washed three times in water,dried, and counted to détermine phosphorylated product.

The data presented in Table 30 show that the additionof the stable copper(I) complexes inhibit the activity ofProtein Kinase Οβ. 73 010198

Tahla..3fl inhibition of ..Erotein Kinaae-Cfi. bv

Stable Coooer(I) Complexes

ComDOund Conc. Percent Inhibition (μΜ) (Me an SEM) BCDS Copper(I) (2: 1) 100 96.8 + 2.0 10 20.0 2.0 1.0 3.5 + 6.5 0.1 6.5 + 4.5 Neocuproine Copper (I) (2:1) 100 84.5 1.9 10 25.5 U. 1.5 1.0 4.0 + 7.0 0.1 3.5 4. 6.5 Protein Kinase Cy Assav

Protein Kinase Cy is another major protein kinase Cisoform. Protein kinase C is a family of serine/threonineprotein kinases that médiate the actions of a wide varietyof growth factor, hormone, and neurotransmitter action.

In this experiment, Protein Kinase Cy was purifiedfrom insect cells expressing a baculovirus recombinantrabbit brain protein kinase Cy isoform. The enzyme waspre-incubated with the test compounds, and its activitywas measured by the ability of the enzyme to phosphorylatehistone H1 in the absence and presence of calcium,phosphatidylserine, diolein and [32P]ATP. Following a 5minute incubation, the reaction was terminated by theaddition of acetic acid, 50 ul aliquots were removed,spotted on phospnocellulose paper, washed three times inwater, dried, and counted to détermine phosphorylatedproduct.

The data presented in Table 31 show that the additionof the stable copper(I) complexes inhibit the activity ofProtein Kinase Cy. 74 010198

Table 31

Inhibition of Protein Kinase Cy by

Stable Copper(I) Complexes

Compound Conc (μΜ) Percent Inhibition BCDS Copper(I) (2:1) 100 99 10 51 1.0 21 0.1 5 Neocuproine Copper(I) (2 :1) 100 97 10 40 1.0 28 0.1 17 The data in Tables 23-31 was used to détermine the 50% inhibitory dose (IC50) of each enzyme. This data ispresented below in Table 32. These results show that thestable copper(I) complexes of this invention are potent 10 inhibitors of Protein Kinase C.

Table 32

Inhibition of Protein Kinase C Isoforms by

Stable Coppjar.il) Complexes

Protein Kinase C I£sq (μΜ)

Isoform Copper(T) (2:1) Neocuproine Coooer(I) (2:1) Non-sélective 97 145 Ca 28 25 cp 25 25 cy 8.8 15 75 010198

Ex ample, 13.

Inhibition of Protein Tyrosine Kinases by

Stable Cooper(I) Complexes 5 This example illustrâtes the ability of the représentative stable copper(I) complexes, BCDS copper(I)(2:1) and neocuproine copper(I) (2:1), to inhibit enzymesinvolved in intracellular signal transduction. Theenzymes tested in this experiment were protein tyrosine 10 kinases spécifie for growth factors and cytokines.

Epidermal Growth Factor (EGF)

Receptor Tyrosine Kinase (human recombinant) Assay

The binding of EGF or TGF-α (Transforming Growth15 Factor a) to the EGF receptor results in activation of thetyrosine kinase portion of the receptor. This kinasephosphorylâtes several cytosolic proteins which lead toinduction of intracellular signaling pathways eventuallyleading to cell mitogenesis and in some cases cellular 20 transformation. Inhibition of the EGF tyrosine kinase isuseful for chemotherapy for malignant cells.

In this experiment, a recombinant form of the humanEpidermal Growth Factor Tyrosine Kinase domain was assayed(Geissler et al., "Thiazolidine-Diones:Biochemical and 25 Biological Activity of a Novel Class of Tyrosine ProteinKinase Inhibitors," J. Biol. Chem. 155:22255-22261, 1990;

Wedegartner and Gill, "Activation of the Purified ProteinKinase Domain of the Epidermal Growth Factor Receptor, " J.3lol. Chem. 254:11346-11353. 1989; Yaish et al., "31ocking 30 of EGF-dependent Cell Prolifération by EGF-Receptor KinaseInhibitors," Science 242 : 933-935. 1988).

TÎie kinase assay measures the activity of the 6 9kD kinase domain by employing an immobilized svnthetic polypeptide as a substrate. Following a 10 minute 35 réaction, phosphorylated tyrosine residues were detected by incubation with a monoclonal anti-phosphotyrosine 76 010198 antibody. Bound anti-phosphotyrosine antibody was quantitated by incubation with a biotin-linked anti-mouseIgG, followed by streptavidin linked β-galactosidaseenzyme. Fluorescence resulting from conversion of fluoroscein-di-p-galactoside to fluorescein was measured’The results of this experiment are presented in Table 33.

Table 33 . Inhibition of Epidermal__GrQWth Factor (EGF) Receptor

Tyrosine Kinase ihuman recombinant) by

Stable Copper(I) Complexes

Compound 3CDS Copper(I) (2:1)

Neocuproine Copper(I) (2:1)

Conc. Percent Inhibition (μΜ) (Mean ± SEM) 10 102.3 ± 2.3 1 40.0 ± 3.6 0.1 11.7 ± 2.7 0.01 0.3 ± 2.4 10 96.7 ± 1.0 1 43.7 ± 5.5 0.1 12.0 ± 4.0 0.01 -5.7 ± 0.9 p56lck Tyrosine Kinase AssayThe lck tyrosine kinase is a member of the src family of cytoplasmic tyrosine kinases. It is expressed only inT-lymphocytes and NK cells. The p56^-c^ Tyrosine Kinase isa 56 kD protein that is found associated with thecytoplasmic side of the plasma membrane of these cells.It is responsable of transmission of the »IL-2 signalleading to T-lymphocyte activation. The binding of IL-2to spécifie IL-2 receptors leads to activation of the p56tyrosine kinase. In addition, the p56^c^ Tyrosine Kinasehas been found to function in signal transduction forantigen activated CD4 and CD8 T-cell receptors. 77 010198

In this experiment, the p56^-ck Tyrosine Kinase waspurified from bovine thymus. The kinase assay measuresthe activity of the 69kD kinase domain by employing animmobilized synthetic polypeptide as a substrate. Thetest compounds were pre-incubated with the enzyme of 15minutes. Following a 10 minute reaction with 100 uM ATP,phosphorylated tyrosine residues are detected byincubation with a monoclonal anti-phosphotyrosineantibody. Bound anti-phosphotyrosine antibody wasquantitated by incubation with a biotin-linked anti-mouseIgG, followed by streptavidin linked β-galactosidaseenzyme. Fluorescence resulting from conversion offluoroscein-di-P-galactoside to fluorescein was measured(Hatekeyama et al., "Interaction of the IL-2 Receptor withthe src-Family Kinase p56^c^: Identification of NovelIntermolecular Association," Science 252:1523-1528, 1991;Caron et al., "Structural · Requirements for Enhancement ofT-cell Responsiveness by the Lymphocyte Spécifie TyrosineProtein Kinase p5Slck," Mol, Cell Biol. 12:2720-2729,1992; Cheng et al., "A Synthetic Peptide Derived fromp34cdc2 is a Spécifie and Efficient Substrate of src-Family Tyrosine Kinases." J. Biol. Chem, 2£2-· 9248-925S,1992) .

Both the BCDS copper(I) and neocuproine copper(I)complexes were found to be potent inhibitors of the kinaseactivity. The results of this experiment are presented inTable 34.

Table 34 Inhibition of p56^·^ Tvrosine Kinase Acf’v-’tv by Stable Copper(I) Compl exes Compound Conc. Percent înh’bit’on (μΜ) (Mean ± SEM) BCDS Copper(I) (2:1) 10 97.5 ± 1.7 1 19.5 ± 1.5 78 010198

Neocuproine

Copper(I) (2:1) 0.1 -3.5 + 0.5 0.01 2.5 ± 7.5 10 90.0 + 2.3 1 19.5 + 0.5 0.1 -8.0 + 2.0 0.01 -1.0 + 6.0 p59-£yn Tyrosine Kinase Assay

The fyn tyrosine kinase is also a member of the srcfamily of non-receptor linked cytoplasmic tyrosinekinases. The ροθ^ϊ71 Tyrosine Kinase is responsible formediating signal transduction through the T-cell receptor(TCR) . This receptor is responsible for a signal cascadeleading to lymphokine sécrétion and cell prolifération.The pSg^Y71 Tyrosine Kinase is also one of several kinasesassociated with the B-cell receptor.

In this experiment, the ρδ^Υ11 Tyrosine Kinase waspurified from bovine thymus. The kinase assay measures-the activity of. the 69kD kinase domain by employing animmobilized synthetic polypeptide as a substrate. Thetest compounds are preincubated with the enzyme of 15minutes. Following a 10 minute reaction with 100 uM AT?,phosphorylated tyrosine residues are detected byincubation with a monoclonal anti-phosphotyrosineantibody. Bound anti-phosphotyrosine antibody isquantitated by incubation with a biotin-linked anti-mouseIgG, followed by streptavidin linked β-galactosidaseenzyme. Fluorescence resulting from conversion offluoroscein-di-P-galactoside to fluorescein is measured(Cooke et al., "Régulation of T-cell Receptor Sicnaling bya src Family Protein Tyrosine Kinase pSS1^, " Cell65:281-291, 1991; Grassman et al, "Protein Tyrosine Kinasep59fyn is Associated with the T-cell Receptor CD3 Complexin Functional Human Lymphocytes," Fur, J. Immunol. 22:233-286, 1992; Appleby et al., "Defective T-cell Receptor 13 010198

Signaling in Mice Lacking the Thymie Isoform of ρδθ^Υ11, "Cell 2û:751-763, 1992). Both the BCDS copper(I) and neocuproine copper(I) complexes were found to be potentinhibitors of the kinase activity. The results of this 5 experiment are presented in Table 35. 10

Compound BCDS Copper)(I) (2:1)

Neocuproine Copper(I) (2:1)

Table 3.5

Inhibition of p59-£gl Tyrosine Kinase Activity byStable Copper(I) Complexes

Conc. Percent Inh ibition (μΜ) (Mean + SEM) 10 99.0 ± 2.7 1 38.0 5.0 0.1 20.5 + 0.5 0.01 2.0 + 8.0 10 91.0 3.0 1 25.5 ± 1.5 0.1 1.0 6.0 0.01 2.5 ± 4.5

The data in Tables 33-35 were used to détermine the50% inhibitory dose (IC5Q) of each protein tyrosine kinasetested. This data is shown in Table 36. These resultsshow that the stable copper(I) complexes of this inventionare potent inhibitors of this class of tyrosine kinase. 15 ΘΟ 010198

Table.1&

Inhibition of Protein Tyrosine Kinases.by

Stable Copper(I) Complexes

Protein Tyrosine Kinase

BCDS

Copper(I) EGF Receptor Tyrosine Kinase 1.3 p5glck Tyrosine Kinase 2.4 pSgfyn Tyrosine Kinase 1.5

Neocuproinp(2:1). Casser(I) (2:1) 1.42.7 2.4

From the foregoing, it will be appreciated that,although spécifie embodiments of the invention hâve beendescribed herein for purposes of illustration, various 10 modifications may be made without deviating from thespirit and scope of the invention. Accordingly, theinvention is not to be limited except as by the appendedclaims.

Claims

81 010108 Claime :

1. Use of a stable copper(I) complex as an activetherapeutic substance.
2. A composition comprising a stable copper(I) complex incombination with a pharmaceutically acceptable carrier or 5 diluent.
3. The composition of claim 2 wherein the stable copper(I) complex is (2,9-dimethyl-4,7-diphenyl-l,10-phenanthroline disulfonic acid disodium sait) copper(I) (2:1).
4. The composition of claim 3, wherein the stable10 copper(I) complex is a single isomer of (2,9-dimethyl-4,7- diphenyl-1,10-phenanthroline disulfonic acid disodium sait)copper(I) (2:1).
5. The composition of claim 2 wherein the stable copper(I)complex is (2,9-dimethyl-l,10-phenyl-l,10-phenanthroline) 15 copper(I) (2:1).
6. A stable copper(I) complex for use in a method forenhancing wound healing in a warm blooded animal, comprisingadministering to the animal an effective amount of the complex.
7. A stable copper(I) complex for use in a method for 20 enhancing or restoring the résistance of a warm-blooded animal to oxidative or inflammatory damage associated with reactiveoxygen species, comprising administering to the animal aneffective amount of the complex.
8. A stable copper(I) complex for use in a method for 2^ treating inflammation in a warm-blooded animal, comprising administering to the animal an effective amount of the complex. 82 010198
9. A stable copper(I) complex for use in a method formodulating lipid metabolism in a warm-blooded animal, comprisingadministering to the animal an effective amount of the complex.
10. A stable copper(I) complex for use in a method forstimulating the growth of haïr in a warm-blooded animal,comprising administering to the animal an effective amount of thecomplex.
11. A stable copper(I) complex for use in a method formodulating signal transduction in a warm-blooded animal,comprising administering to the animal an effective amount of thecomplex.
12. A stable copper(I) complex for use in a method forinhibiting Protein Kinase C in a warm-blooded animal, comprisingadministering to the animal an effective amount of the complex.
13. A stable copper(I) complex for use in a method forinhibiting a protein tyrosine kinase in a warm-blooded animal,comprising administering to the animal an effective amount of thecomplex.
14. A stable copper(I) complex for use in a method forinhibiting viral réplication in a warm-blooded animal, comprisingadministering to the animal an effective amount of the complex.
15. The complex of claim 14, wherein the virus is selectedfrom the group consisting of human T-cell leukemia I and/or II,human herpes virus, cytomegalo virus, encephalomyocarditis virus,Epstein Barr virus, human hepatitis virus, Varicella Zostervirus, Rhinovirus, and rubella virus.
16. The complex of claim 14, wherein the virus is human immunodeficiency virus.