WO2004078118A2

WO2004078118A2 - Protein kinase c inhibitor, related composition, and method of use

Info

Publication number: WO2004078118A2
Application number: PCT/US2004/005855
Authority: WO
Inventors: Shaomeng Wang; Peter M. Blumberg; Nancy Lewin
Original assignee: The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services; Georgetown University; The Regents Of The University Of Michigan
Priority date: 2003-02-28
Filing date: 2004-02-26
Publication date: 2004-09-16
Also published as: WO2004078118A3

Abstract

Compounds of the formula (1), (2), (3), (4), (5) or (6) or a pharmaceutically acceptable salt thereof, wherein A is a DAG receptor-binding moiety, L is a linker moiety, and B is a zinc finger reactive moiety, and, a pharmaceutical composition comprising an above compound, and methods of use.

Description

PROTEIN KTNASE C INHIBITOR, RELATED COMPOSITION, AND METHOD OF USE

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0001] This patent application claims priority to U.S. Provisional Patent Application No. 60/451,214, filed February 28, 2003, which is herein incorporated by reference.

FIELD OF THE INVENTION [0002] The present invention relates to a method of inhibiting protein kinase C function and to compounds and compositions suitable for use in such method.

BACKGROUND OF THE INVENTION [0003] Protein kinase C (PKC) consists of a family of ten or more closely related enzymes. Because of their high degree of relatedness they are referred to as "isotypes," "isozymes" or "isoforms." The known isotypes of PKC are divided into three subfamilies: conventional (cPKC), novel (nPKC). and atypical (aPKC). The known conventional isotypes include α, βi, β₂, and γ. The known novel isotypes include δ, ε, ε', η, and θ. The known atypical isotypes include ξ and i. Two recently described isotypes are μ and v. The identity of PKC is generally established by its ability to phosphorylate certain proteins in the presence of adenosine triphosphate and phospholipid cofactors. PKC is believed to phosphorylate only serine and/or threonine residues in the proteins that are substrates for PKC. Some forms of PKC, such as members of the cPKC group, require the presence of calcium ions for maximal activity, whereas members of the nPKC and aPKC groups are thought to be calcium independent.

[0004] PKC is also substantially stimulated by certain 1,2-sn-diacylglycerols that bind specifically and stoichiometrically to a recognition site or sites on the enzyme. This site is called diacylglycerol (DAG) binding site, and it is located on the amino-terminal portion of PKC, the so-called "regulatory domain." The carboxy-terminal portion of PKC carries the site at which protein phosphorylation is effected, and thus, this portion is called the "kinase domain."

[0005] This stimulation of PKC activity is referred to as PKC "activation." The activation of PKC by the binding of diacylglycerols to the regulatory domain of PKC is of particular importance in the normal and pathological functions of PKC. [0006] Some chemical compounds have been shown to reduce the rate at which PKC phosphorylates its substrates; such compounds are referred to as PKC "inhibitors" or, in some cases, "antagonists." In some circumstances, PKC inhibitors are capable of inhibiting various cellular or tissue phenomena, which are thought to be mediated by PKC. [0007] The biological role of PKC is also of great interest because of the discovery that certain very powerful tumor-promoting chemicals activate this enzyme by binding specifically, and with very high affinity to, the diacylglycerol-binding site on the enzyme. In addition to diacylglycerols, there are several other known classes of compounds that bind to this site, including diterpenes such as phorbol, ingenol, and daphnane derivatives; indole alkaloids (indolactams) such as the teleocidins, lyngbyatoxin, and indolactam V; certain derivatives of diaminobenzyl alcohol; polyacetates such as the aplysiatoxins and oscillatoxins; macrocyclic lactones of the bryostatin class; and benzolactams such as (-)-BL- V8-310. Representative st ctures of these classes of known PKC-activating compounds, collectively referred to herein as "DAG receptor-binding moieties," are depicted in Figure 1.

[0008] Thus, compounds capable of blocking the activation of, or inhibiting, PKC by acting as specific pharmacological antagonists of the diacylglycerols at the diacylglycerol binding site on PKC would be valuable agents in the prevention and treatment of a wide variety of diseases in mammals, such as humans. In particular, the need for, and potential utility of, PKC inhibitors/antagonists as agents for the treatment of cancer has been a focus of active scientific research (see, e.g., Hofinann, Rev. Physiol. Biochem. Pharmacol, 142, 1-96 (2001); and O'Brian et al., Cancer Metastasis Rev., 20, 95-100 (2001)). [0009] Recent studies suggest that the different PKC isotypes have different biological roles. For example, the stimulation of one PKC isotype or a limited subset of PKC isotypes might lead to undesirable results, such as the development of inflammation, the promotion of tumor formation, or an increased rate of viral replication in cells (i.e., de novo infection of cells and/or expression, assembly and release of new viral particles). On the other hand, other PKC isotypes might be responsible for the many beneficial effects observed when PKC is stimulated by known PKC activators in a variety of biological settings; such beneficial effects include the cessation of division of leukemic cells, multiplication of colonies of lymphocytes, and leucocytes, or the secretion of useful bioregulatory factors such as interferon-c and interleukin-2.

[0010] Although compounds that demonstrate specificity to PKC over other proteins have been discovered, very little is known regarding isotype selectivity. For example, staurosporine shows little isotype selectivity, with the exception of poor inhibition of the ζ isotype relative to the other isotypes. Studies of the PKC-selective compound, 3-[l-(3- dimethylaminopropyl)-indol-3-yl-4-(lH-indol-3-yl)-lH-pyrrole-2,5-dione₅ suggest a slight selectivity for the calcium-dependent isotypes. Subsequent studies of this compound observed no difference, or possibly slight selectivity, for α over βj_. and β₂ isotypes. Therefore, despite years of research and the identification of classes of compounds that inhibit PKC versus other protein kinases, there remains a need for therapeutically effective isotype-selective PKC inhibitors. Such inhibitors would be expected to be more efficacious and less toxic, due to their specificity.

[0011] The present invention provides a new class of PKC inhibitor compounds, which are selective to PKC over other kinases and show potential for isotype-selectivity. As selective inhibitors, the compounds are useful in treating conditions associated with diabetes mellitus and its complications, ischemia, inflammation, central nervous system disorders, cardiovascular disease, dermatological disease, and cancer. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention provides a new class of compounds for inhibiting PKC.

The compounds include compounds of the formula:

or a pharmaceutically acceptable salt thereof, wherein A is a DAG receptor-binding moiety selected from the group consisting of diacylglycerols, diterpenes, indolactams, polyacetates. diaminobenzyl alcohol derivatives, bryostatins, and benzolactams, wherein L is a linker moiety, which can be a substituted or unsubstituted, saturated, unsaturated and/or aromatic, straight or branched, acyclic, ring-containing and/or ring- carrying chain of atoms, which has a linear count of at least one but not more than 20 atoms, and optionally contains one or more heteroatoms selected from oxygen, nitrogen, silicon, sulfur, phosphorus, arsenic, boron, and selenium; wherein L is optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁.₁₅)alkyl, (C₂.₁₅)alkenyl, (C₂-i₅)alkynyl, (C₃-₈)cycloalkyl, (C₃-₈)heterocycloalkyl, (C₃-₈)cycloalkyl(C₁-₁₅)alkyl, (C₃.₈)cycloalkyl(C₂-₁₅)alkenyl, (C_3-8)cycloalkyl(C_2-15)alkynyl, (C₁-₁₅)alkoxy, (C_1-15)alkanoyl, (C_1-15)alkanoyloxy, (C_6-14)aryl, (C₆-₁₄)heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(=O)OR^a, C(=O)NR R^c, C(=O)NHNR R^c, C(=O)NH=CHR^d, OC(=O)NR R^c, NR^eR^f, SH, SO₂R^d, and SO₂NR^eR^f, wherein each R^a is independently hydrogen or (Cι-₆)alkyl; each of R and R^c is independently hydrogen or (C₁-₁o)alkyl; or R and R^c, together with the nitrogen to which they are attached, are a 5-6 membered heterocyclic ring; each R^d is independently hydrogen, (Cι.₆)alkyl, (C₆-₁₄)aryl, or (C₆-ι₄)heteroaryl; each of R^e and R^f is independently hydrogen, (C₁-₁o)alkyl, (C₆-₁₄)aryl, or (C₆-ι₄)heteroaryl; and wherein B is a zinc finger reactive moiety selected from the group consisting of aryl disulfides, aliphatic disulfides, heterocyclic disulfides, xanthic disulfides, amidine disulfides, benzothiazole disulfides, thiuram disulfides, dithianes, disulfoxides, maleimides, benzoisothiazolin-3-ones, sulfonic acid thioesters, aryl nitroso compounds, quinones, and azodicarboxylic acids. The invention also includes a salt of any of the above compounds. [0013] In another aspect, the invention includes a pharmaceutical composition comprising a compound as described above and a pharmaceutically acceptable carrier. [0014] In yet another aspect of the invention, the invention includes a method of inhibiting PKC activity in a mammal by administering to the mammal a PKC-inhibiting effective amount of the pharmaceutical composition, whereupon PKC in the mammal is inhibited.

[0015] In another aspect, the invention includes a method of selectively inhibiting the δ- isoform of PKC in a mammal in need thereof by administering to the mammal the pharmaceutical composition in an amount effective to inhibit the δ-isoform of PKC, whereupon the δ-isoform of PKC in the mammal is selectively inhibited. [0016] In yet another aspect, the invention includes a method of inhibiting the proliferation of cancerous cells in a mammal, said method comprising administering to the mammal the pharmaceutical composition in an amount to inhibit the proliferation of cancerous cells, whereupon the proliferation of the cancerous cells in the mammal is inhibited.

[0017] In still another aspect of the invention, the invention includes a method of inhibiting PKC activity in a mammal by administering to the mammal a PKC-inhibiting effective amount of an aryl disulfide compound, whereupon PKC in the mammal is inhibited.

[0018] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS [0019] Figure 1 illustrates DAG receptor binders suitable for use as "DAG receptor- binding moieties" (A) in a compound of the invention.

[0020] Figure 2 illustrates zinc finger reactive compounds suitable for use as "zinc finger reactive moieties" (B) in a compound of the invention.

[0021] Figure 3 illustrates aryl disulfide compounds suitable for use as "zinc finger reactive moieties" (B) in a compound of the invention.

DETAILED DESCRIPTION OF THE INVENTION [0022] PKC is involved in regulatory signaling related to cell proliferation and apoptosis, making it a viable target for cancer therapies. Applicants have discovered that changing the structure of the zinc finger of PKC can inhibit the activity of PKC (e.g., by ejecting zinc). Such changes in zinc finger structure are induced by contacting the zinc finger with a compound that reacts or interacts with a zinc finger. [0023] Many compounds are known in the art as effective for interacting with zinc fingers of other proteins, including those described in Rice et al., J. Med. Chem., 39:3606- 3616 (1996); Otsuka et al., J. Med. Chem., 37:4267-4269 (1994); Otsuka et al., J. Med. Chem., 38:3264-3270 (1995); Fujita et al, J Med. Chem., 39:503-507 (1996); Loo et al., J Med. Chem., 39:4313-4320 (1996); Jaffe et al., J. Biol. Chem., 259:5032-5036 (1984); and Louie et al., Proc. Natl. Acad. Sci. USA, 95:6663-6668 (1998). Such zinc finger reactive moieties include those selected from the group consisting of aryl disulfides, aliphatic disulfides, heterocyclic disulfides, xanthic disulfides, amidine disulfides, benzothiazole disulfides, thiuram disulfides, dithianes, disulfoxides, maleimides, benzoisothiazolin-3- ones, sulfonic acid thioesters, aryl nitroso compounds, quinones, and azodicarboxylic acids. Examples of these compounds are shown in Figure 2.

[0024] Applicants have discovered that, in particular, aryl disulfide compounds of the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen, halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁-₁s)alkyl, (C_2-15)alkenyl, (C2_-15)alkynyl, (C₃.₈)cycloalkyl, (C₃.₈)heterocycloalkyl, (C_3-8)cycloalkyl(C_1-15)alkyl, (C_3-8)cycloalkyl(C_2-15)alkenyl, (C_3-8)cycloalkyl(C_2-15)alkynyl, (C_1-15)alkoxy, (C_1-15)alkanoyl, (C_1-15)alkanoyloxy, (C_6-14)aryl, (C_6-1 )heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(=O)OR^a, C(=O)NR^bR°, C(=O)NHNR^bR^c, C(=O)NH=CHR^d, OC(=O)NR^bR^c, NR^eR^f, SH, SO₂R^d, or SO₂NR^eR^f; wherein each R^a is independently hydrogen or (C_1-6)alkyl; each of R^b and R^c is independently hydrogen or (C_1-1o)alkyl; or R^b and R^c, together with the nitrogen to which they are attached, are a 5-6 membered heterocyclic ring; each R^d is independently hydrogen, (Cι.₆)alkyl, (C₆.₁₄)aryl, or (C₆-₁₄)heteroaryl; each of R^e and R is independently hydrogen, (C^i^alkyl, (C₆-₁ )aryl, or (C₆-₁₄)heteroaryl; and wherein any of the above R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^a, R^b, R^c, R^d, R^e, f and R optionally further comprises one or more substituents selected from the group consisting of halo, hydroxy, nitro, cyano, phospho, sulfo, sulfonamide, amino, amide, trifluoromethyl, trifluoromethoxy, (C₁-₁₅)alkyl, and combinations thereof, are effective at inhibiting the activity of PKC. Moreover, such compounds have been found to selectively inhibit the activity of the δ-isoform of PKC. [0025] To increase the specificity of zinc finger-reactive moieties for PKC over other zinc finger containing proteins, any of the zinc finger-reactive moieties described herein can be linked, e.g., covalently linked, to a compound known to bind to the DAG receptor site of the PKC. Specificity or potency of the compounds of the invention can be increased by bringing a zinc finger-reactive moiety into close proximity to the zinc finger via an active site-binding component.

[0026] Accordingly, the compounds of the invention typically contain three components: a DAG receptor site-binding moiety, a zinc finger-reactive moiety, and a linker, which joins the DAG receptor site-binding moiety and the zinc finger-reactive moiety together. Specifically, the compounds are represented by the general formula:

or a pharmaceutically acceptable salt thereof, wherein A is a DAG receptor-binding moiety, L is a linker moiety, and B is a zinc finger reactive moiety.

[0027] The DAG receptor-binding moiety (A) can be any suitable DAG receptor- binding moiety. The DAG receptor-binding moiety desirably has a high affinity for binding to the zinc finger region of PKC. Suitable DAG receptor-binding moieties include those selected from the group consisting of diacylglycerols, diterpenes, indolactams, polyacetates, diaminobenzyl alcohol derivatives, bryostatins, and benzolactams (see, e.g., Fig. 1). The diacylglycerols include, for example, 1,2-dioctanoyl-sn-glycerol, l-oleoyl-2-acetyl-sn- glycerol, l-stearoyl-2-arachidonyl-sn-glycerol, 2-deoxy-L-ribonolactone derivatives, 4- hydroxymethyl-4-tetradecanoyloxymethyl-4-butanolide, and 3-hydroxymethyl-l ,6-dioxo- 2,5-dioxacyclocosane. The diterpenes include, for example, phorbol esters such as phorbol 12-myristate 13-acetate, phorbol 12-retinoate 13-acetate, and des-(ring A)-phorbol 12- myristate 13-acetate, daphnane derivatives such as daphnopsis factor R₆, ingenane derivatives such as ingenol 3-tetradecanoate, synaptolepis factor Kl, pimelea factor P₂, pimelea factor S₂, mezerein, simplexin, and gnidimacrin. The indolactams include, for example, teleocidin B-4, indolactam V, lyngbyatoxin A, and blastmycetin A. The polyacetates include, for example, aplysiatoxins and oscillatoxins. The diaminobenzyl alcohol derivatives include for example, 3-acetylamino-5-(N-decyl-N-methylamino)benzyl alcohol and 6-(N-decylamino)-4-hydroxymethylindole. The bryostatins include, for example, bryostatin 1 and bryostatin 2. The benzolactams include, for example, (-)-BL-V8- 310, (+)-epi-BL-V8-310, and epi-BL-V9-310. Desirably, the DAG receptor-binding moiety is selected from indolactams, benzolactams, diacylglycerols, and polyacetates. Preferably, the DAG receptor-binding moiety is an indolactam or benzolactam. [0028] The DAG receptor-binding moiety is substituted by one or two linker moieties, L. The linker moiety can be bound to the DAG receptor-binding moiety at any suitable position. The point of connection of the linker to the DAG receptor-binding moiety will depend on the identity of the DAG receptor-binding moiety. For example, when the DAG receptor-binding moiety is a benzolactam or indolactam, the linker desirably is connected to the aromatic benzene. When the DAG receptor-binding moiety is a phorbol derivative, the linker desirably is connected at the 12- or 13-position. The linker moiety also can be bound at the hydroxymethyl or 2-hydroxyethyl site of the DAG receptor-binding moiety, or to various other ester, amino, ether, or amido sites that may be present on the DAG receptor- binding moiety. Desirably, the DAG receptor-binding moiety is connected to the linker moiety via a direct bond or an ether, ester, amide, thioether, or amine linkage. [0029] Optionally, the DAG receptor-binding moiety contains one or more substituents in addition to the linker moiety L, and at any position around the basic DAG receptor binder structure. Suitable substituents include (C_1-15)alkyl, (C_2-15)alkenyl, (C₂-₁₅)alkynyl, (C _-8)cycloalkyl, (C₃.₈)heterocycloalkyl, (C₃.₈)cycloalkyl(C₁-₁₅)alkyl, (C .₈)cycloalkyl(C₂-ι₅)alkenyl, (C₃.₈)cycloalkyl(C .₁₅)alkynyl, (C₁.₁₅)alkoxy, (C₁-₁₅)alkanoyl, (Cι.₁₅)alkanoyloxy, (C₆-₁₄)aryl, (C₆-₁₄)heteroaryl, and combinations thereof. [0030] The linker moiety (L) can be any suitable linker. Desirably, the linker moiety introduces a spatial separation between the the DAG receptor-binding moiety (A) and the zinc finger reactive moiety (B). Suitable linker moieties include substituted or unsubstituted, saturated, unsaturated and/or aromatic, straight or branched, acyclic, ring- containing and/or ring-carrying chain of atoms. Optionally, the linker moiety contains one or more heteroatoms selected from oxygen, nitrogen, silicon, sulfur, phosphorus, arsenic, boron, and selenium. The linker moiety is optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁-₁₅)alkyl, (C_2-i₅)alkenyl, (C₂-₁₅)alkynyl, (C₃-₈)cycloalkyl, (C₃.₈)heterocycloalkyl, (C₃-₈)cycloalkyl(C₁-₁₅)alkyl, (C₃-₈)cycloaU yl(C₂.₁₅)alkenyl, (C₃-₈)cycloalkyl(C₂-₁₅)alkynyl, (Cι-ι₅)alkoxy, (C₁-₁₅)alkanoyl, (Cι-₁₅)alkanoyloxy, (C₆-₁₄)aryl, (C₆-ι₄)heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(=O)OR^a, C(=O)NR R^c, C(=O)NHNR^bR^c, C(=O)NH=CHR^d, OC(=O)NR^bR^c, NR^eR^f, SH, SO₂R^d, and SO₂NR^eR^f, wherein each R^a is independently hydrogen or (C₁-₆)alkyl; each of R^b and R^c is independently hydrogen or (C₁-₁₀)alkyl; or R^b and R^c, together with the nitrogen to which they are attached, are a 5-6 membered heterocyclic ring; each R^d is independently hydrogen,

(C₆.₁₄)aryl, or (C₆-₁₄)heteroaryl; each of R^e and R^f is independently hydrogen, (Cι_ιo)alkyl, (C₆.₁ )aryl, or (C₆-ι₄)heteroaryl; and wherein any of the above R^a, R^b, R°, R^d, R^e, and R^f, optionally further comprises one or more substituents selected from the group consisting of halo, hydroxy, nitro, cyano, phospho, sulfo, sulfonamide, amino, amide, trifluoromethyl, trifluoromethoxy, (C₁-ι₅)alkyl, and combinations thereof.

[0031] The linker can be as short as a direct bond or as long as a C₁₈ alkylene chain. When the linker is an alkylene chain, it can optionally contain ether, thioether, amine, ester, thioester, or amide. For example, the alkylene chain can containing 1 to 5 amine groups as in -(CH₂)₂-NH-(CH₂) -NH-(CH2)2-- The linker can also be a branched alkylene chain, for example, -CH(-(CH₂)₃-)-O-(CH₂)₃-O-(CH₂)₂-. The ether, thioether, amine, ester, thioester, or amide group can also be present at the ends of the linker, thus joining the other two moieties to the linker. Desirably, L is a direct bond, a Cι-ι₂ alkylene chain optionally containing 1 to 5 heteroatoms selected from O, S, NR, C(O)O, C(S)O, and C(O)NR, wherein R is hydrogen or C_1-3 alkyl, a Cι-₁₂ alkylene chain optionally containing one or more unsaturated bonds, or a more rigid chain containing one or more aryl (e.g., 1,4- phenylene) groups. Preferably, L is a Cι-₁₂ alkylene chain optionally containing 1 to 5 heteroatoms selected from O, S, NR, C(O)O, C(S)O, and C(O)NR. [0032] Advantageously, the linker moiety separates the DAG receptor-binding moiety (A) from the zinc finger reactive moiety (B) by a linear count of about 1 atom (e.g., about 2 atoms, about 3 atoms, or about 4 atoms) to about 20 atoms (e.g., about 18 atoms, about 16 atoms, or about 14 atoms). Preferably, the linker is a saturated or unsaturated straight chain linker. Desirably, the linker moiety introduces a spatial separation between the DAG receptor-binding moiety and the zinc finger reactive moiety in the range of about 2 A to about 50 A (e.g., about 4 A to about 40 A, or about 5 A to about 30 A). [0033] The zinc finger reactive moiety (B) can be any suitable moiety that can interact with a cysteine and/or histidine residue of a zinc finger. Typically, the zinc finger reactive moiety is selected from the group consisting of aryl disulfides, aliphatic disulfides, heterocyclic disulfides, xanthic disulfides, amidine disulfides, benzothiazole disulfides, thiuram disulfides, dithianes, disulfoxides, maleimides, benzoisothiazolin-3-ones, sulfonic acid thioesters, aryl nitroso compounds, quinones, and azodicarboxylic acids, as described above (see, e.g., Fig. 2). In some embodiments, the zinc finger reactive moiety desirably is a disulfide selected from aryl disulfides, aliphatic disulfides, heterocyclic disulfides, xanthic disulfides, amidine disulfides, benzothiazole disulfides, thiuram disulfides, and dithianes, preferably an aryl disulfide. In other embodiments, the zinc finger reactive moiety desirably is a compound selected from disulfoxides, maleimides, benzoisothiazolin-3-ones, sulfonic acid thioesters, aryl nitroso compounds, quinones, and azodicarboxylic acids. [0034] The zinc finger reactive moiety (B) is substituted by one or two linker moieties (L), which are in turn bound to a DAG receptor-binding moiety (A). Desirably, the linker moiety is connected to the zinc finger reactive moiety via a direct bond, or an ether, ester, amide, thioether, or amine linkage. The linker can be attached to the zinc finger reactive moiety at any suitable position.

[0035] In one preferred embodiment, the zinc finger reactive moiety is an aryl disulfide of the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently -L-A of any of the formulae described above, hydrogen, halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁-₁₅)alkyl, (C_2-ι₅)alkenyl, (C₂-ι₅)alkynyl, (C₃-₈)cycloalkyl, (C₃-₈)heterocycloalkyl, (C_3-8)cycloalkyl(Cι-i₅)alkyl, (C₃-₈)cycloalkyl(C_2-15)alkenyl, (C₃-₈)cycloa-lcyl(C_2-ι₅)alkynyl, (C_1-15)allcoxy, (C₁-ι₅)allcanoyl, (Cι-₁₅)alkanoyloxy, (C₆-₁₄)aryl, (C₆-ι₄)heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR R°, OC(=O)OR^a, C(=O)NR R^c, C(=O)NHNR^bR^c, C(=O)NH=CHR^d, OC(=O)NR R^c, NR^eR^f, SH, SO₂R^d, or SO₂NR^eR^f.

[0036] The aryl disulfide can be modified from an aryl disulfide compound well known in the art. Preferably, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are selected from the group consisting of-L-A of any of the formulae described above, hydrogen, halo, sulfo, (Ci- ₁₅)alkyl, (C₂-ι₅)alkenyl, (C₁-ι₅)alkoxy, (C₆-ι₄)aryl, (C₆-ι₄)heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(=O)OR^a, C(=O)NR R°, C(=O)NHNR R^c, C(=O)NH=CHR^d, OC(=O)NR^bR^c, NR^eR^f, SO₂R^d, SO₂NR^eR^f; and combinations thereof. More preferably, R¹ and R⁶ are selected from NR^eR^f, C(=O)NR R^c, C(=O)OR^a, and SO₂R^d, and R⁵ and R¹⁰ are hydrogen. The aryl disulfide can be symmetrically substituted or asymmetrically substituted. Examples of preferred aryl disulfide moieties are shown in Figure 3. Such compounds have been found to eject or otherwise interact with the zinc ion from the zinc finger of PKC by either forming bonds (e.g., covalent, ionic, or hydrogen bonds) with the ejected zinc ion directly or with the amino acid residues (e.g., cysteine or histidine residues), which coordinate with the zinc ion. Most preferably, the aryl disulfide moiety is a 2,2'-diamino-4,4'-disulfonamido phenyl disulfide derivative of NSC 83217. [0037] Only one or two of the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are -L-A. If two or more linkers are attached to the aryl disulfide, the linkers can be bound to the same aryl ring or to different aryl rings. Preferably, the linkers are bound to different aryl rings. Desirably, the linker L is bound the aryl disulfide group at the meta-position relative to the disulfide group. [0038] In a particularly preferred embodiment, the compound is of the formula:

A— I,— B, or a pharmaceutically acceptable salt thereof, wherein L is a linker moiety as defined above, A is an indolactam, B is aryl disulfide moiety of the formula:

wherein R¹ and R⁶ are NH₂, R³ and R⁸ are SO₂NH₂, and R², R⁴, R⁵, R⁷, R⁹, and R¹⁰ are as defined above; and with the proviso that only one of R², R⁴, R⁷, and R⁹ is -L-A of the formula A— L— B. [0039] In this embodiment, L is advantageously a C_1-12 alkylene, optionally containing 1 to 5 heteroatoms selected from O, S, NR, C(O)O, C(S)O, and C(O)NR. Preferably, L introduces a spatial separation between the aryl disulfide and the indolactam of about 10 A to about 25 A.

[0040] There exist many different routes for the preparation of the compounds of the invention. Generally, the compounds of the invention are prepared by coupling a DAG receptor-binding moiety (A) to a linker moiety (L), and subsequently coupling the linker moiety to a zinc finger reactive moiety (B). Typically, the substituents of the DAG receptor-binding moiety are modified to form a linker moiety containing a functional group at its terminus for coupling to a zinc finger reactive moiety. Suitable linker terminal functional groups include typical leaving groups for substitution reactions, e.g., halides, amine groups for forming amide linkages with activated carboxylic acid derivatives (e.g., acid halides), or thio groups for forming disulfide linkages with other thio-containing compounds.

[0041] The following is an example of a suitable method for preparing compounds of the invention, in particular a compound of the formula:

[0042] Commercially available 4-bromo-5-chloro-phenol is reacted with sulfonyl chloride followed by reaction with PC1₅ and ammonia to produce the sulfonamide derivative, 3. The phenol group is protected, for example, by reaction with methoxymethylchloride (MOMCl) to produce compound 4. Intermediate 4 is then coupled with ethanolamine via a copper-catalyzed coupling (e.g., as described in Gabriel et al, Org. Lett. 2002, 4, 3703-6) and subsequently reacted with lead acetate to produce the amino- functionalized derivative, 5, in high yields. Treatment of compound 5 with H₂S under the conditions described in Pappalardo, Boll. Sci. Chim. Ind., 1959, 17, 23-7, followed by oxidization produces an aryl disulfide derivative, 7. The amino groups are then protected by reaction with tert-butyloxycarbonyl ether ((Boc)₂O). Removal of the MOM hydroxyl protecting groups produces the aryl disulfide zinc finger reactive moiety, 9.

PTSA

9

[0043] The DAG receptor-binding indolactam moiety can be prepared from indolactam, 10, which can be prepared following known methods. Suitable methods include those described in, e.g., Nakagawa et al., Bioscience, Biotechnology, and Biochemistry, (1998), 62(8), 1568-1573 and Irie et al. Tetrahedron, (1995), 51(22), 6255-66. Indolactam, 10, is coupled to a hydroxy-substituted alkyne using a palladium catalyst to produce compound 12, which is subsequently reduced to produce compound 13. The hydroxyl group of the indolactam is activated upon reaction with mesityl chloride to yield compound 14. A coupling reaction of the zinc finger reactive aryl disulfide moiety 9 (from the previous scheme) with compound 14 under basic conditions produces compound 15. Removal of the amine protecting groups yields the target compound of the invention.

[0044] Alternately, the indolactam starting compound 10 used in the above scheme can be converted via Pd-catalyzed carboxylation reaction to produce compound 16, which can then be coupled with a substituted amine (e.g., an amine bearing a linker moiety) to yield the amide intermediate 17. After standard deprotection and protection steps, the indolactam-linker intermediate 19 is obtained. Similar compounds of the invention can be prepared following the same procedure, but using different DAG receptor binders, different linkers, and/or different zinc finger reactive moieties.

17 18

19

[0045] Pharmaceutical compositions of the present invention comprise, as an active ingredient, at least one of the compounds described above, together with a pharmaceutically acceptable carrier. The active compound can be present as a solid, crystal, granule, or a mixture thereof. The active ingredient is present in the composition in an amount sufficient to produce the desired effect (e.g., to inhibit PKC activity in mammals, or to inhibit cell proliferation in mammals). Preferably, the pharmaceutical composition of the invention includes the active ingredient in a quantity selected from 0.01 μg to about 10 g (e.g., from about 0.1 μg to about 1 g, or about 1 μg to about 500 mg), advantageously, about 5 μg to about 100 mg (e.g., about 10 μg to about 75 mg), per dosage unit, depending on the specific derivative and route of administration. The dosage administered to a mammal, particularly a human, should be sufficient to affect a prophylactic or therapeutic response in the mammal over a reasonable time frame. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the strength of the particular compound employed, the condition of the mammal, the body weight of the mammal, as well as the severity and stage of the disease state being treated. The size of the dosage will also be determined by the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular compound. The preferred dosage is the amount that results in maximum inhibition of PKC activity, without significant side effects. The compounds of the present invention can be used alone or in appropriate association, and also can be used in combination with other pharmaceutically active compounds. [0046] The compounds and compositions of the invention can be administered by parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal, vaginal, topical, or transdermal administration. The compositions of the invention also can be administered by the use of surgical implants, which release the compounds of the invention.

[0047] The pharmaceutical composition of the invention can be present in dosage unit form. For example, the composition can take the form of a tablet, capsule, granules, powder, inhalant, syrup, lozenge, emulsion (e.g., lipid emulsion), gel, ointment, cream, lotion, paste, foam, transdermal patch, suppository, sterile injectable liquid, as well as a liquid suspension or solution (e.g., aqueous or nonaqueous). The pharmaceutical composition of the present invention can be prepared by conventional techniques. [0048] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, coπistarch, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, potato starch, macrocrystalline cellulose, alkyl cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, solubilizers (e.g., nonionic surfactants), and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0049] The compounds of the present invention can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer.

[0050] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0051] Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier; as well as creams, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

[0052] Additionally, formulations suitable for rectal administration can be presented as suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. [0053] A preferred method of administration comprises oral dosing, with tablets, liquids, drops, or capsules. For the oral route of administration, either compounds of this invention lacking functional groups destroyed by acid, or tablets or capsules, which protect the active compound from upper gastrointestinal acidity, are preferred. [0054] Sustained or directed release compositions can be formulated, e.g., in liposomes or in compositions wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, absorption onto charcoal, entrapment in human serum albumin microspheres, etc. It is also possible to freeze-dry the new compounds and use the lyophilizates obtained, for example, for the preparation of products for injection.

[0055] The compounds of this invention also can be modified by covalent attachment of metabohcally modifiable groups to form "prodrugs," which are released by cleavage in vivo of the metabohcally removable groups. For example, amine, hydroxy, and/or thiol groups present in many compounds of this invention can be converted to prodrugs by covalent attachment of acyl or aminoacyl organic functional groups. Likewise, compounds of this invention containing carboxylic, sulfonic, phosphoric, phosphonic or related free acids, including those in which one or more oxygen atoms are replaced by sulfur, can be converted to prodrugs by formation of their esters or amides by covalent attachment of alcohols, amines, amino acids and the like. It will be recognized by persons with ordinary skill in medicinal chemistry that conversion of alcohol-, amine-, thiol- or acid-containing compounds of this invention to prodrugs is preferably done by derivatization of such groups located in regions of the molecule having minimal steric hindrance, to permit access of metabolizing enzymes, other bioreactants or water. Such alcohol-, amine-, thiol- or acid- containing groups can be located in any of the parent, side-chain or capping-group organic functional groups described above.

[0056] A typical daily dose will contain from about 0.01 mg/kg to about 500 mg/kg of the active compound of this invention. Preferred daily doses will be about 0.05 mg/kg to about 100 mg/kg, ideally about 0.1 mg/kg to about 50 mg/kg. However, for topical administration a typical dosage is about 1 to about 500 μg compound per cm of an affected tissue. Preferably, the applied amount of compound will range from about 30 to about 300 μg/cm², more preferably, from about 50 to about 200 μg/cm², and, most preferably, from about 60 to about 100 μg/cm². Appropriate concentrations and dosage unit sizes can be readily determined by one of ordinary skill in the art.

[0057] The compounds of this invention possess valuable pharmacological properties for human and veterinary medicine. In particular, the compounds of this invention show selective effects as antagonists for PKC and as selective ligands for PKC and/or for DAG receptors. Thus, these compounds can be used as agents for the abrogation of pathophysiological conditions and disease states in applications such as anti-inflammatory, anti-psoriatic, anti-cancer, anti-ulcer, anti-hypertensive, anti-asthma, anti-arthritic, anti- autoimmune, anti-nociceptive, anti-secretory, anti-parasitic, anti-amoebic, anti-viral including anti-HIV replication, in prophylaxis against infection by any hepatitis E virus form, and any other application in which pathological involvement of PKC is found. [0058] As regards these applications, a method for inhibiting PKC activity in a mammal comprises administering to the mammal a PKC-inhibiting effective amount of a pharmaceutical composition of the invention, whereupon PKC in the mammal is inhibited. Desirably, the method of inhibiting PKC activity selectively inhibits the δ-isoform of PKC over the other PKC isoforms. The δ-isoform of PKC is implicated in cellular proliferation in mammals, whereas other PKC isoforms are implicated in cellular apoptosis. Thus, in the treatment of disease states such as cancer, the activity of PKC isoforms can have opposing effects on treatment. Thus, it is desirable to identify compounds, which are capable of selectively acting upon one isoform over all others. Compounds that can selectively inhibit PKC-δ without inhibiting cell apoptosis are particularly desirable. Thus, the compounds of the invention desirably are used in a method of selectively inhibiting the δ-isoform of PKC in a mammal in need thereof. The method comprises administering to the mammal a pharmaceutical composition comprising a compound of the invention in an amount effective to inhibit the δ-isoform of PKC, whereupon the δ-isoform of PKC in the mammal is selectively inhibited.

[0059] In a particularly preferred embodiment, the method of administration is useful for inhibiting the proliferation of cancerous cells in a mammal. Such a method comprises administering to the mammal a pharmaceutical composition comprising a compound of the invention in an amount sufficient to inhibit the proliferation of cancerous cells, whereupon the proliferation of the cancerous cells in the mammal is inhibited.

[0060] The following example further illustrates the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES [0061] The following studies illustrate the ability of aryl disulfide compounds to inhibit the activity of PKC when administered in the presence of DAG receptor binders. The studies further show that the aryl disulfide compounds can selectively inhibiting the δ- isoform of PKC.

[0062] Binding Assay: ΣD₅₀ values of the experimental ligands shown in Figure 1, i.e., NSC 83217, 4493, 4492, 58950, 121513, 38069, 666724, 210281, 633232, 405662, and 633231 (Developmental Therapeutics Program, NCI), were determined by inhibition of [³H]ρhorbol 12,13-dibutyrate ([³H]PDBu) (21 Ci/mmol) (Perkin Elmer, Boston, MA) binding to PKC α, PKC δ, or the Clb domain of PKC δ. The intact PKCs were partially purified as described Kazanietz et al. Mol. Pharmacol. 44, 298-307 (1993). The Clb domain of PKC δ was expressed as a GST fusion construct and purified as described in Kazanietz et al, J Biol. Chem., 270, 21852-21859 (1995). [³H]PDBu binding to PKC α, PKC δ, or the Clb domain of PKC δ was measured using the polyethylene glycol precipitation method as described in Lewin et al., Protein Kinase C Protocols, Newton, A., Humana Press, Totowa, N.J. (2002), with some modifications to allow pre-incubation of the ligands with the protein. The ligands dissolved in dimethylsulfoxide (DMSO) (Pierce, Rockford, IL) were diluted in 50 mM Tris-Cl, pH 7.5, containing 1 mg/ml γ-globulins (Cohn Fraction 11,111) (Sigma, St. Louis, MO), and were added in increasing concentrations to 1 mg/ml γ-globulins, 50 mM Tris-Cl, pH 7.5, 0.1 mM Ca^or 1 mM ethyleneglycol- bis(β-aminoethylether)-N,N,N',N',-tetraacetic acid (EGTA), and PKC α, PKC δ or PKC δ Clb in a total volume of 200 μl. The final concentration of dithiothreitol (DTT) from the protein stock solutions was less than 0.25 μM. The tubes were pre-incubated at 37° C for 1 hr., unless otherwise specified. After pre-incubation, the tubes were placed on ice and 0.1 mg/ml phosphatidylserine (PS) (Avanti Polar Lipids, Alabaster, AL), 2.5 nM ([³H]PDBu) in γ-globulins, and, for the controls to determine non-specific binding, non-radioactive phorbol 12,13-dibutyrate (PDBu) (LC Laboratories, Woburn, MA) was added to give a final assay volume of 250 μl and a final concentration of 1.8 mg/ml γ-globulins. The tubes were incubated for 5 min at 37°C. After incubation, the samples were chilled at 0°C for 7 min., 200 μl of 35% polyethylene glycol 6000 (EM Science, Gibbstown, NJ) was added to the samples to produce a final concentration of 15.5%, and the assay tubes were handled as described.

[0063] For assay of the protection by phosphatidylserine of PKC α and PKC δ from inhibition by NSC 83217, the usual assay was modified as follows. The 1 hr pre-incubation at 37°C was carried out in a 200 μl volume containing 1.8 mg/ml γ-globulins, 50 mM Tris- Cl, pH 7.5, 0.1 mM Ca⁺⁺, (0 to 0.125 mg/ml) PS, 2.5 nM ([³H]PDBu), (0 or 1 μM) NSC 83217 and PKC α or PKC δ. After pre-incubation, the tubes were placed on ice and PS was added to yield a final concentration of 0.1 mg/ml in 50 mM Tris-Cl, pH 7.5. The tubes were incubated at 37°C for 5 minutes and the assay completed as described above. [0064] Kinase Assay: Activation of PKC was determined by measuring the incorporation of ³³P from [γ-³³P 3000 Ci/mmol] ATP (ICN, Costa Mesa, CA) into PKC α pseudosubstrate peptide [Ser²⁵]PKC (19-31) (hivitrogen Life Technologies, Rockville, MD) in the presence of PKC . The ligand NSC 83217 and the protein were pre-incubated for 1 hr at 37°C in a volume of 40 μl in the presence of 20 mM Tris-Cl, pH 7.5, 0.25 mg/ml bovine serum album (BSA) (fraction V), (Sigma, ST. Louis, MO), O.lmM Ca⁺⁺, and PKC α. After pre-incubation, the assay tubes were placed on ice and the reagents for the kinase assay were added to yield a final assay volume of 50 μl containing: 20 mM Tris-Cl, pH 7.5, 0.25 mg/ml BSA, 7.5 mM magnesium acetate, 100 μg/ml phospholipid (phosphatidylserine : phosphatidylcholine, 1:10, w/w) (Avanti Polar Lipids, Alabaster, AL), 300 nM PKC pseudosubstrate peptide, and ATP (50 μM final concentration, 1 μCi). Included was an assay point without NSC 83217 but with 1 μM phorbol 12-myristate 13- acetate (PMA) (LC Laboratories, Woburn, MA) as a control for activation. The assay tubes were incubated for 10 min at 30°C. The reaction was stopped by chilling on ice. A 25 μl aliquot was spotted onto DE81 ion exchange chromatography paper (Whatman Ltd., Maidstone, England); the paper was washed three times in 0.5% phosphoric acid, and the bound radioactivity was measured in a scintillation counter. In each experiment, each ligand concentration was assayed in triplicate. The dose response curve was fitted to the relationship y=[(Aι-A₂)/(l+X/X)^p) ]+A₂ where Ai represents basal activity, A₂ represents maximal stimulated activity, and X represents the half-maximally effective dose. [0065] For assay of inhibition of PMA activation of PKC α by NSC 83217, the assay was performed as described, except that 1 μM PMA was included in the kinase reaction mixture. Because the inhibition by NSC 83217 was irreversible (vide infra), the concentrations indicated are those during the pre-incubation before the final assay of phorbol ester binding or enzymatic activity. Of the eleven compounds tested for inhibition of PMA activation, NSC 83217 showed the greatest potency (μM) and was selected for further evaluation.

[0066] Characteristics of [³H]Phorbol 12,13-Dibutyrate [³H]PDBu binding inhibition by NSC 83217: The inhibition of [³H]PDBu binding was time-dependent, blocked by the presence of competing thiols, and required access of the inhibitor to the CI domain. Depending on concentration, composition, and surface charge of the phospho lipid surface, the CI domains of PKC bind to, and insert into, the lipid bilayer. The presence of the phorbol ester enhances this association and insertion. To assess whether or not the phospholipid and phorbol ester might shield the CI domain from inhibition, the inhibitory activity of NSC 83217 that was pre-incubated with PKC α for 60 min at 37 °C was compared with its inhibitory activity if all components of the binding reaction were included throughout the incubation. The omission of phosphatidylserine and [³H]PDBu during the initial exposure to NSC 83217 led to a 30-fold enhancement of inhibitory activity, yielding a -D₅o of260 ± 37 nM.

[0067] To confirm the time-dependence of the inhibition, PKC α was pre-incubated with varying concentrations of the aryl disulfide, NSC 83217, for 5, 30 or 60 min in the absence of phosphatidylserine and [³H]PDBu. These components were then added and, after a further incubation of 5 min, the level of remaining binding activity was evaluated. Binding decreased with first-order kinetics as a function of time of pre-incubation. [0068] The possible interference of reducing agents, commonly included in preparations of PKC, with the aryl disulfide compounds, was evaluated by determining the dose- dependence for the protection of PKC from inhibition by NSC 83217 in the presence of DTT. At a concentration of NSC 83217 of 500 nM, which in the absence of DTT yielded 65 % inhibition of [³H]PDBu binding to PKC α, DTT protected by approximately 90%. Its ED₅o for protection was 1.0 μM.

[0069] Structural comparison of Cys-reactive compounds: Based on the above experiments, the 11 Cys-reactive aryl disulfide compounds were compared for their ability to inhibit binding of [³H]PDBu to PKC α, using for the assays a pre-incubation for 60 min at 37 °C in the absence of phosphatidylserine and [³H]PDBu and then evaluating residual binding upon inclusion of all constituents for a final 5 min incubation (Table 1). ID₅o values displayed a 400-fold range, from 17011M for NSC 4493 to 73,000 nM for NSC 633231. Table 1:

[0070] Site(s) of action of the Cys-reactive compounds: The inhibition of [³H]PDBu by NSC 83217 was evaluated using the isolated Clb domain of PKC δ. The inhibition of [³H]PDBu binding to the Clb domain yielded an ID₅₀ of 340 nM (Table 2). This value for inhibition was similar to that of the intact PKC δ as well as that of intact PKC . Based on these results, it was concluded that interaction with the Cys residues of the CI domain suffices to account for the inhibition of ligand binding.

Table 2:

[0071] Although [³H]PDBu binding provides a direct measure of CI domain function, the evaluation was extended to the enzymatic activity of the intact PKC. Two different measures of function were examined.

[0072] NSC 83217 activation of enzymatic activity was assessed in the absence of the co-activators of PKC, namely phosphatidylserine and PMA. PKC α was pre-incubated with NSC 83217, then assayed for kinase activity using the α-pseudosubstrate peptide as a substrate. Negligible stimulation was observed, with inhibition of basal activity at a concentration of NSC 83217 of 3000 nM (Figure 5A). NSC 83217 activation of enzymatic activity was then assessed in the presence of the co-activators PMA and phosphatidylserine. The absolute level of enzymatic activity was considerably greater under these conditions, as expected, and inhibition was again observed at 3000 nM (Figure 5B). [0073] Taken together, the results indicated that NSC 83217 alone does not activate PKC. Rather, NSC 83217 inhibits the kinase activity of PKC in the presence of a PKC activator, such as PMA.

[0074] Isoform selectivity of inhibition: As described above, NSC 83217 inhibited PKC α and PKC δ with similar potencies (Table 2). A potential problem for comparison was that PKC α requires Ca²⁺ as a co-activator, whereas PKC δ is independent of Ca²⁺. However, inhibition of PKC δ by NSC 83217 was confirmed to be similar in the presence or absence of Ca²⁺ (Table 2). Different PKC isoforms have been shown to insert differently into the lipid bilayer and to have different requirements for lipid binding. Thus, it was concluded that this difference in interaction might provide a potential isotype-specific characteristic that could contribute isoform selectivity for inhibition. To test this hypothesis, the ability of phosphatidylserine to protect PKC α from inactivation by 1 μM NSC 83217 was compared to its ability to protect PKC δ from inactivation under the same conditions, hi contrast to the 90% protection of PKC α at 100 μg/ml of phosphatidylserine, PKC δ was only protected by 20% under these conditions. Indeed, at 1 μg/ml of phosphatidylserine, no protection of PKC δ was observed, whereas PKC α was protected to a maximal degree. Reciprocally, the potency of NSC 83217 to inhibit [³H]PDBu binding to PKC α and PKC δ in either of the presence or absence of 1.25 μg/ml of phosphatidylserine was also determined. In contrast to the similar potencies of NSC 83217 for PKC α and PKC δ in the absence of phosphatidylserine, in the presence of phosphatidylserine, NSC 83217 displayed 20-fold selectivity for PKC δ. The results described above demonstrate that aryl disulfide compounds can selectively inhibit PKC δ over other PKC isoforms in the presence of DAG receptor binders.

[0075] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0076] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0077] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein A is a DAG receptor-binding moiety selected from the group consisting of diacylglycerols, diterpenes, indolactams, polyacetates, diaminobenzyl alcohol derivatives, bryostatins, and benzolactams, wherein L is a linker moiety, which can be a substituted or unsubstituted, saturated, unsaturated and/or aromatic, straight or branched, acyclic, ring-containing and/or ring- carrying chain of atoms, which has a linear count of at least one but not more than 20 atoms and optionally contains one or more heteroatoms selected from oxygen, nitrogen, silicon, sulfur, phosphorus, arsenic, boron, and selenium; wherein L is optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁.₁₅)alkyl, (C₂-₁₅)alkenyl, (C₂-₁₅)alkynyl, (C₃.₈)cycloalkyl, (C₃-₈)heterocycloalkyl, (C₃-₈)cycloalkyl(C n ₅)alkyl, (C₃.₈)cycloalkyl(C₂.₁₅)alkenyl, (C_3-8)cycloalkyl(C₂-₁₅)alkynyl, (C_1-15)alkoxy, (C_1-15)alkanoyl, (Cι-₁₅)alkanoyloxy, (C₆-ι₄)aryl, (C₆.₁₄)heteroaryl, C(=O)R^a, C(=0)OR^a, C(=0)NR^bR^c, OC(=O)OR^a ₅ C(=O)NR^bR°, C(=O)NHNR^bR^c, C(=O)NH=CHR^d, OC(=O)NR^bR^c, NR^eR^f, SH, SO₂R^d, and SO₂NR^eR^f, wherein each R^a is independently hydrogen or (C_1-6)alkyl; each of R and R^c is independently hydrogen or (C_1-1o)alkyl; or R^b and R^c, together with the nitrogen to which they are attached, are a 5-6 membered heterocyclic ring; each R is independently hydrogen, (C_1-6)alkyl, (C₆-₁₄)aryl, or (C_6-14)heteroaryl; each of R^e and R^f is independently hydrogen, (Cι.ιo)alkyl, (C₆.₁₄)aryl, or (C₆.₁₄)heteroaryl; and wherein B is a zinc finger reactive moiety selected from the group consisting of aryl disulfides, aliphatic disulfides, heterocyclic disulfides, xanthic disulfides, amidine disulfides, benzothiazole disulfides, thiuram disulfides, dithianes, disulfoxides, maleimides, benzoisothiazolin-3-ones, sulfonic acid thioesters, aryl nitroso compounds, quinones, and azodicarboxylic acids.

2. The compound of claim 1, wherein the compound is of the formula A— L— B.

3. The compound of claim 1, wherein the compound is of the formula:

B— L— A— L— B.

4. The compound of claim 1 , wherein the compound is of the formula:

A— L— B— L— A.

5. The compound of claim 1, wherein A is an indolactam or benzolactam.

6. The compound of claim 1, wherein B is an aryl disulfide of the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently -L-A of any of the formulae of claim 1, hydrogen, halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁.₁₅)alkyl, (C₂-₁₅)alkenyl, (C₂-₁₅)alkynyl, (C₃- ₈)cycloalkyl, (C₃.₈)heterocycloalkyl, (C₃.₈)cycloalkyl(Cι-₁₅)alkyl, (C_3-8)cycloalkyl(C_2- ₁₅)alkenyl, (C₃-₈)cycloalkyl(C₂-i₅)alkynyl, (Cι-ι₅)alkoxy, (C_1-i₅)alkanoyl, (C_1-ι₅)alkanoyloxy, (C₆-i4)aryl, (C_6-14)heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(=O)OR^a, C(=O)NR^bR^c, C(=O)NHNR R^c, C(=O)NH=CHR^d, OC(=O)NR^bR^c, NR^eR^f, SH, SO₂R^d, or SO₂NR^eR^f; wherein any of the above R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^a, R^b, R°, R^d, R^e, and R optionally further comprises one or more substituents selected from the group consisting of halo, hydroxy, nitro, cyano, phospho, sulfo, sulfonamide, amino, amide, trifluoromethyl, trifluoromethoxy, (Cι-ι₅)alkyl, and combinations thereof; with the proviso that only one or two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ is -L-A of any of the formulae of claim 1.

7. The compound of claim 1, wherein L is a direct bond, a C_MS alkylene chain optionally comprising ether, thioether, amine, ester, thioester, or amide, a C π alkylene chain optionally containing one or more unsaturated bonds, or a chain containing one or more 1,4-phenylene groups.

8. The compound of claim 7, wherein L is a C_MS alkylene chain optionally comprising ether, thioether, amine, ester, thioester, or amide.

9. The compound of claim 1, wherein L is connected to A and/or B via a direct bond, or an ether, ester, amide, thioether, or amine linkage.

10. A pharmaceutical composition comprising a compound of any of claims 1 -9 and a pharmaceutically acceptable carrier.

11. A method of inhibiting protein kinase C (PKC) activity in a mammal, said method comprising administering to the mammal a PKC-inhibiting effective amount of the pharmaceutical composition of claim 10, whereupon PKC in the mammal is inhibited.

12. A method of selectively inhibiting the δ-isoform of PKC in a mammal in need thereof, said method comprising administering to the mammal the pharmaceutical composition of claim 10 in an amount effective to inhibit the δ-isoform of PKC, whereupon the δ-isoform of PKC in the mammal is selectively inhibited.

13. A method of inhibiting the proliferation of cancerous cells in a mammal, said method comprising administering to the mammal the pharmaceutical composition of claim 10 in an amount to inhibit the proliferation of cancerous cells, whereupon the proliferation of the cancerous cells in the mammal is inhibited.

14. A compound of the formula:

A—l_^-B, or a pharmaceutically acceptable salt thereof, wherein L is a linker moiety, which can be a substituted or unsubstituted, saturated, unsaturated and/or aromatic, straight or branched, acyclic, ring-containing and/or ring- carrying chain of atoms, which has a linear count of at least one but not more than 20 atoms and optionally contains one or more heteroatoms selected from oxygen, nitrogen, silicon, sulfur, phosphorus, arsenic, boron, and selenium; wherein L is optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁.₁₅)alkyl, (C₂.₁₅)alkenyl, (C₂-₁₅)alkynyl, (C₃-₈)cycloalkyl, (C .₈)heterocycloalkyl, (C₃-₈)cycloalkyl(C₁-₁₅)alkyl, (C₃-₈)cycloalkyl(C₂-₁₅)alkenyl, (C₃-s)cycloalkyl(C₂-₁₅)alkynyl, (Cι-i₅)alkoxy, (C₁-₁₅)alkanoyl, (Cι-₁₅)alkanoyloxy, (C₆-ι₄)aryl, (C₆-ι₄)heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(=O)OR^a, C(=O)NR R°, C(=O)NHNR^bR^c, C(=O)NH=CHR^d, OC(=O)NR^bR^c, NR^eR^f, SH, SO₂R^d, and SO₂NR^eR^f, wherein each R^a is independently hydrogen or (C_1-6)alkyl; each of R and R^c is independently hydrogen or (C₁-₁₀)alkyl; or R^b and R^c, together with the nitrogen to which they are attached, are a 5-6 membered heterocyclic ring; each R is independently hydrogen, (C_1-6)alkyl, (C₆-₁ )aryl, or (C₆-₁₄)heteroaryl; each of R^e and R^f is independently hydrogen, (Cι-ι₀)alkyl, (C₆-₁₄)aryl, or (C₆-ι₄)heteroaryl; and wherein A is an indolactam, wherein B is aryl disulfide moiety of the formula:

wherein R¹ and R^δ are NH₂, R³ and R⁸ are SO₂NH₂, and R², R⁴, R⁵, R⁷, R⁹, and R¹⁰ are each independently -L-A of the formula A — L — B, hydrogen, halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (Cι-₁₅)alkyl, (C₂-ι₅)alkenyl, (C₂- ₁₅)alkynyl, (C₃-₈)cycloalkyl, (C₃-₈)heterocycloalkyl, (C₃-₈)cycloalkyl(C₁-₁₅)allcyl, (C₃-₈)cycloalkyl(C₂-ι₅)alkenyl, (C₃-₈)cycloalkyl(C₂-i₅)alkynyl, (Cι-₁₅)alkoxy, (Ci-^alkanoyl, (C_M5)alkanoyloxy, (C_6-ι₄)aryl, (C_6-14)heteroaryl, C(-O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(-O)OR^a, C(=O)NR R^c, C(=O)NHNR^bR°, C(=O)NH=CHR^d, OC(=O)NR R^c, NR^eR^f, SH, SO₂R^d, or SO₂NR^eR^f; and wherein any of the above R², R⁴, R⁵, R⁷, R⁹, R¹⁰, R^a, R^b, R°, R^d, R^e, and R^f optionally further comprises one or more substituents selected from the group consisting of halo, hydroxy, nitro, cyano, phospho, sulfo, sulfonamide, amino, amide, trifluoromethyl, trifluoromethoxy, (Cι-i₅)alkyl, and combinations thereof; with the proviso that only one of R², R⁴, R⁷, and R⁹ is -L-A of the formula A— L— B.

15. The compound of claim 14, wherein L is a direct bond, a C_1-18 alkylene chain optionally comprising ether, thioether, amine, ester, thioester, or amide, a C_1-12 alkylene chain optionally containing one or more unsaturated bonds, or a chain containing one or more 1,4-phenylene groups.

16. A pharmaceutical composition comprising a compound of claim 14 or 15 and a pharmaceutically acceptable carrier.

17. A method of inhibiting protein kinase C (PKC) activity in a mammal, said method comprising administering to the mammal a PKC-inhibiting effective amount of the pharmaceutical composition of claim 16, whereupon PKC in the mammal is inhibited.

18. A method of selectively inhibiting the δ-isoform of PKC in a mammal in need thereof, said method comprising administering to the mammal the pharmaceutical composition of claim 16 in an amount effective to inhibit the δ-isoform of PKC, whereupon the δ-isoform of PKC in the mammal is selectively inhibited.

19. A method of inhibiting the proliferation of cancerous cells in a mammal, said method comprising administering to the mammal the pharmaceutical composition of claim 16 in an amount to inhibit the proliferation of cancerous cells, whereupon the proliferation of the cancerous cells in the mammal is inhibited.

20. A method of inhibiting protein kinase C (PKC) activity in a mammal, said method comprising administering to the mammal a PKC-inhibiting effective amount of an aryl disulfide compound of the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen, halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C₁.₁s)alkyl, (C₂-₁₅)alkenyl, (C₂-ι₅)alkynyl, (C_3-8)cycloalkyl, (C₃-₈)heterocycloalkyl, (C_3-8)cycloalkyl(C_1- ₁₅)alkyl, (C₃.₈)cycloalkyl(C₂-₁₅)alkenyl, (C_3-8)cycloaU (C_2-15)alkynyl, (C₁-₁₅)alkoxy, (Ci- ₁₅)alkanoyl, (Cι.ι₅)alkanoyloxy, (C₆-₁₄)aryl, (C_6-14)heteroaryl, C(=O)R^a, C(=O)OR^a, C(=O)NR^bR^c, OC(=O)OR^a, C(=O)NR^bR^c, C(=O)NHNR^bR^c, C(=O)NH=CHR^d, OC(=O)NR^bR^c, NR^eR^f, SH, SO₂R^d, or SO₂NR^eR^f; wherein each R^a is independently hydrogen or (C_1-6)alkyl; each of R^b and R^c is independently hydrogen or (C₁.₁₀)alkyl; or R and R^c, together with the nitrogen to which they are attached, are a 5-6 membered heterocyclic ring; each R is independently hydrogen, (Cι.₆)alkyl, (C₆.ι₄)aryl, or (C₆.₁₄)heteroaryl; each of R^e and R^f is independently hydrogen, (Cι-ιo)alkyl, (C₆-₁₄)aryl, or (C₆-₁₄)heteroaryl; and wherein any of the above R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^a, R^b, R^c, R^d, R^e, and R^f optionally further comprises one or more substituents selected from the group consisting of halo, hydroxy, nitro, cyano, phospho, sulfo, sulfonamide, amino, amide, trifluoromethyl, trifluoromethoxy, (C_1-15)alkyl, and combinations thereof; or a pharmaceutically acceptable salt thereof, whereupon PKC in the mammal is inhibited.

21. The method of claim 20, wherein the δ-isofonn of PKC is selectively inhibited.

22. The method of claim 20, wherein the proliferation of cancerous cells in a mammal is inhibited.