WO1996040614A1

WO1996040614A1 - Protein kinase c modulators .x.

Info

Publication number: WO1996040614A1
Application number: PCT/US1996/009710
Authority: WO
Inventors: Paul E. Driedger; James Quick
Original assignee: Procyon Pharmaceuticals, Inc.
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1996-12-19

Abstract

Compounds having anti-inflammatory and other activities are disclosed. The compounds are derived from diterpene and benzolactan phorboids.

Description

PROTEIN KINASE C MODULATORS. X.

Background

Protein kinase C (also known as "calcium/phospholipid-dependent protein kinase", "PKC" or "C-kinase") is a family of closely related enzymes; one or more members of the protein kinase C family are found in nearly all animal tissues and animal cells that have been examined. The identity of protein kinase C is generally established by its ability to phosphorylate certain proteins when adenosine triphosphate and phospholipid cofactors are present, with greatly reduced activity when these cofactors are absent. Protein kinase C is believed to phosphorylate only serine and/or threonine residues in the proteins that are substrates for protein kinase C. Additionally, some forms of protein kinase C require the presence of calcium ions for maximal activity.

Protein kinase C comprises a family of eleven or more closely related protein molecules [Parker, P.J. et al., Mol. Cell. Endocrin. 65: 1-11 (1989)]. Because of their high degree of relatedness they are referred to as "isozymes", "isotypes" or "isoforms".

Occasionally the term "subtypes" is used, but this term is usually reserved to designate, as a subdivision, two or more variants of a single isotype.

The currently known isotypes of protein kinase C have all been characterized in molecular detail by numerous laboratories, and are subdivided into several subfamilies: α, β₁, β₂ and γ (the "A-group"); δ, ε, ε' [Ono, Y. et al., J. Biol. Chem. 263: 6927-6932 (1988)], η [also known as protein kinase C-L; Osada, S. et al., J. Biol. Chem. 265: 22434- 22440 (1990) and Bacher, N. et al., Mol. Cell. Biol. 11: 126-133 (1991), respectively], and θ [Osada, S.-I. et al., Mol. Cell. Biol. 12: 3930-3938 (1992) (the "B-group"); ζ [Ono, Y. et al., J. Biol. Chem. 263: 6927-6932 (1988)]and i [Selbie, L.A. et al., J. Biol. Chem. 268: 24296-24302 (1993), the latter also being known as PKCλ [Akimoto, K. et al., J. Biol. Chem. 269: 12677-12683 (1994)] (the "C-group"); and μ [Johannes, F.-J. et al., J. Biol. Chem. 269: 6140-6148 (1994)], also known as PKD [Valverde, A.M. et al., Proc. Natl. Acad. Sci. USA 91: 8572-8576 (1994) (the "D-group"). Members of the A-group require calcium ions for maximal activation, whereas the B-, C- and D-group members are thought to be largely calcium-independent for activation. The genes for each of the isotypes above have been cloned from one or more animal and yeast species and the clones have been sequenced; the relatedness of the genes and their product polypeptides is thus well established. Beyond the ability of phospholipid and, for some isotypes, calcium to stimulate protein kinase C activity, members of the A-, B- and D-groups of the protein kinase C family are 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 the diacylglycerol binding site, and it is located on the amino-terminal portion of protein kinase C, the so-called "regulatory domain". The carboxy-terminal portion of protein kinase C carries the site at which protein phosphorylation is effected, and this portion is therefore called the "kinase domain" or the "catalytic domain".

Thus, the rate at which various protein kinase C family members carry out their enzymatic phosphorylation of certain substrates can be markedly enhanced by the presence of the cofactors such as phospholipids, diacylglycerols (except for the C-group) and, for some protein kinase C family members, calcium ions. This stimulation of protein kinase C activity is referred to as protein kinase C "activation", and the activation of protein kinase C by the binding of diacylglycerols to the regulatory domain of protein kinase C is of particular importance in the normal and pathological functions of protein kinase C.

In contrast to the activation of protein kinase C, some chemical compounds have been shown, when added to protein kinase C enzyme assays, to reduce the rate at which protein kinase C phosphorylates its substrates; such compounds are referred to as protein kinase C "inhibitors" or, in some cases, "antagonists". In some circumstances, protein kinase C inhibitors are capable of inhibiting various cellular or tissue phenomena which are thought to be mediated by protein kinase C.

Activation of protein kinase C by diacylglycerols has been shown to be an important physiological event that mediates the actions of a wide variety of hormones,

neurotransmitters, and other biological control factors such as histamine, vasopressin, α- adrenergic agonists, dopamine agonists, muscarinic cholinergic agonists, platelet activating factor, cytokines, growth factors and many others [see Nishizuka, Y., Nature 308: 693-698 (1984) and Science 225: 1365-1370 (1984) for reviews].

The biological role of protein kinase C 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 at present six other known classes of compounds that bind to this site: diterpenes such as the phorbol esters; indole alkaloids (indolactams) such as the teleocidins, lyngbyatoxin, and indolactam V; polyacetates such as the aplysiatoxins and oscillatoxins; certain derivatives of diaminobenzyl alcohol; macrocyclic lactones of the bryostatin class; and, benzolactams such as (-)-BL-V8-310; these seven classes of compounds are collectively referred to herein as "phorboids".

The chemical structural aspect of the term "benzolactams" shall be herein defined as a class of chemical compounds having at least two fused rings (the "primary rings") wherein the two vertices of fusion comprise adjacent carbon atoms. One ring of this fused bicyclic structure contains six atoms, including the atoms common to the two primary rings, is aromatic in character and contains either six carbon atoms or, optionally, may have one or two nitrogen atoms in any position except the vertices at which the two primary rings are fused. The other primary ring may be comprised of eight or nine atoms, including the atoms common to the two primary rings, is not aromatic, and, in the case of the parent

benzolactams from which the benzolactam portion of the present invention is derived, carries a hydroxymethyl or 1-hydroxyethyl group. The definition of benzolactam as used herein is also conditioned by functional constraints (see below under "Detailed Description of the Invention").

The phorbol esters have long been known as powerful tumor promoters, the teleocidins, aplysiatoxins, diacylglycerols and, though weak, the bryostatins are now known to have this activity, and it appears likely that additional classes of compounds will be found to have the toxic and tumor promoting activities associated with the capability to bind to the diacylglycerol site of protein kinase C and thus activate the enzyme. Other toxicities of these agents when administered to animals include lung injury and profound changes in blood elements, such as leukopenia and neutropenia, among many others.

Representative examples of these seven classes of previously known protein kinase

C-activating compounds, collectively referred to herein as "phorboids", are depicted below:

lt can be seen that the phorboids depicted have diverse, dissimilar structural elements of both hydrophilic and hydrophobic nature, with one prominent exception, namely that each class of phorboid contains a hydroxymethyl or 1-hydroxyethyl group (indicated by the dashed-line boxes in each structure). In each case the phorboids depicted are among the most potent of their respective structural class, and among the seven classes the diterpenes, indolactams, polyacetates, bryostatins and benzolactams have members of especially high potency, in the range of low nanomolar affinities for protein kinase C.

In addition to potent tumor promoting activity, these seven classes of compounds display a vast range of biological activities, as would be expected from the widespread distribution of their target enzyme. Many of these activities indicate the involvement of protein kinase C in important normal or pathological processes in animals, as shown by experiments utilizing both genetic and pharmacological approaches. Thus, the phorboids are potent skin inflammatory agents, cause smooth muscle contraction in several tissues, alter immune system function and can be used to cause or mimic a wide variety of other normal or pathological biological responses. Published evidence indicates that disease states such as the development of cancer, the onset and/or maintenance of inflammatory disease, the role of vasoconstriction in hypertension, the role of bronchoconstriction in asthma, the life cycles of many pathogenic human viruses, and the role of certain classes of cholinergic, adrenergic, serotoninergic and dopaminergic synapses in diseases of the central/peripheral nervous systems, may be mediated in vivo by the stimulation of protein kinase C or other diacylglycerol binding site-bearing entities by diacylglycerols, the latter being generated in the cell by either normal physiological or by pathological agents or conditions.

In analyzing the activity of a pharmaceutical or other bioactive compound, it is useful to consider two properties: the efficacy of the compound, defined as the capability to elicit a full or partial biological result, such as complete displacement of a Iigand from its receptor site or the complete inhibition of inflammation or edema caused by a standard stimulus; and the potency, defined as that amount or concentration of drug that causes 50% of the full response (often abbreviated as the ED₅₀). It is frequently the case within a given class of pharmaceutical agents that individual members of the class all have equal efficacy, i.e. they each can generate a full biological effect, but they show differing potencies. Thus, the structural modifications within such a class generally affect only the amount necessary to achieve a given result (i.e. the potency), and the modified compounds otherwise generally retain the same central biological characteristic (i.e. efficacy as a pharmaceutical or in a given biological assay or test). There may also be differences between members of such a class as regards properties other than the central biological characteristic; for example, members of the class might differ in side effects or susceptibility to metabolism by an organism.

Well-known pharmaceuticals that have been in extensive use for years or decades show a wide range of optimal therapeutic potencies. Aspirin, for example, is often taken in multi-gram amounts per day for treatment of inflammation or arthritis, and detailed analyses of its mechanism of action in vitro show that a concentration in the millimolar range is required for certain therapeutic effects of aspirin. In contrast, steroid-based topical anti- inflammatory compounds such as fluocinolone acetonide are many thousand-fold more potent, and, beyond this, some oral contraceptive agents are prescribed in daily doses in the microgram range. Thus, although high potency is generally advantageous for a

pharmaceutical, it is not an absolute requirement.

The concepts of potency and efficacy provide a useful basis for understanding the pharmacological properties of the nearly one thousand analogs of the typically skin- inflammatory and tumor-promoting phorboids that have been reported in the literature, including numerous examples on which major or minor chemical modifications have been made [see Evans and Soper, Lloydia 41: 193-233 (1978) and references cited therein]. When the structures of these phorboids are compared, and their activities for inflammation, tumor promotion and protein kinase C modulation are analyzed from the perspective of efficacy and potency, a remarkable unity is observed. To a degree that is nearly unique among known ligand-receptor phenomena, the structures of the different classes of phorboids vary quite markedly from one to the other class, yet widespread testing of their biological activities has shown that these classes have essentially at least one common general target site, namely the diacylglycerol binding site on protein kinase C, and generally have very similar biological properties. In particular, the numerous known phorboids of the diterpene, indolactam, diacylglycerol, polyacetate, bryostatin and benzolactam classes appear to have, with very minor exceptions, virtually identical efficacies as skin irritants and tumor promoters [Sugimura, T., Gann 73: 499-507 (1982)]. Typical exceptions involve: (i) a few compounds that have a short duration of irritant activity and/or manifest diminished tumor promoting activity [Hergenhahn, M. et al., Experentia 30: 1438-1440 (1974)], perhaps due to slightly different protein kinase C isotype selectivity, toxicity or secondary parameters such as differing metabolic destruction rates; (ii) the bryostatins, which, though having weak but detectable inflammatory and tumor-promoting activity, show other, non- correlating pharmacological properties [Sako, T. et al., Cancer Res. 47: 5445-5450 (1987)]; or (iii) certain short-chain diesters of phorbol which are tumor promotion inhibitors at low doses but fully efficacious tumor promoters when tested alone at higher

concentrations [Schmidt and Hecker, E., Carcinogenesis, Vol. 7, ed. by E. Hecker et al., Raven Press, New York, 1982, pp. 57-63] .

In contrast to the essentially equal efficacies among the vast majority of phorboids, their relative potencies cover a wide range, as measured in inflammation and promotion tests and as measured in numerous other in vivo and in vitro systems. Example compounds can be found in the diterpene, indolactam, polyacetate and benzolactam classes that have nearly equal, very high potencies. At the same time there are compounds in each of these classes which embody significant structural changes that do not diminish efficacy but do result in potency decreases of 10-fold to 100,000-fold or more [see, for example, Driedger, P.E. and Blumberg, P., Cancer Res. 37: 3257-3265 (1977); idem, Cancer Res. 39: 714-719 (1979)]. Thus, all these compounds appear to be capable of achieving generally the same biological results, and merely differ in the amount which must be used to obtain a given result.

In vitro measurements of biochemical properties provide an even more sensitive method for comparing the properties of the various phorboids. For example, using a radioactively labeled phorboid such as [³H]phorbol 12,13-dibutyrate or [³H]lyngbyatoxin, one can measure the potency of a test compound as a competitive Iigand for the

diacylglycerol binding site, which is also referred to herein as the "phorboid binding site" on protein kinase C or on other biological molecules which have phorboid binding sites (see below). Alternatively, one can measure the ability of a given phorboid to stimulate the protein kinase C-mediated incorporation of radioactive phosphate from [³²P]adenosine triphosphate into a standard acceptor substrate such as histone H1. These tests reveal a difference in potency between given phorboid agonists of as much as 10,000,000-fold or more [Dunn and Blumberg, Cancer Res. 43: 4632-4637 (1983), Table 1].

These basic data regarding the phorboid agonists are an important consideration because they underscore the concept that the structural differences among the phorboids known prior to the instant invention [with the exception of previous discoveries in this related to the present invention (vide infra)], especially the widely studied diterpenes, indolactams, diacylglycerols and polyacetates, generally do not affect their efficacies as toxic agonists, and indeed a remarkably wide variety of structural changes are tolerated in this regard. Such changes generally alter potency only and do not provide agents with therapeutic utility, since the resulting compounds retain their toxicity.

In the case of parent diterpenes of the bryostatin class, the presence of the unmodified 1-hydroxyethyl group in typical bryostatins such as bryostatin 1 or bryostatin 2 is associated with substantially less skin-inflammatory and tumor-promoting activity than for the other classes of parent phorboids such as diterpenes, etc. However, the inflammatory and tumor-promoting activity of bryostatin 1 and 2 are nonetheless still detectable, indicating an incomplete separation between these toxicities and other properties of the bryostatins.

Some minor changes in phorboid structure are known to result in generally inactive compounds, such as a stereochemical change from 4-β to 4-α in the phorbol series, and indeed some of the diterpene skeleton structures carry hydroxy groups that must be esterified in order for inflammatory activity to be observed. However, these inactive compounds are quite few in number among the known phorboids, and no therapeutic utility has been demonstrated for them.

The phorbol esters, indolactams, polyacetates, diaminobenzyl alcohols, bryostatins and benzolactams are generally found in plants, molds, and algae, or are synthetic in origin. Although they are found in many parts of the world, normal human contact with these classes of phorboids is thought to be low and of negligible medical significance. In contrast, the diacylglycerols are part of the functioning of virtually every type of animal cell, and the undesirable activation (and, alternatively as discussed below, the cessation of desirable activation) of protein kinase C by the diacylglycerols is thought to have a very widespread role in human diseases. Thus, compounds capable of blocking the activation of, or inhibiting, protein kinase C by acting as specific pharmacological antagonists of the diacylglycerols at the

diacylglycerol binding site on protein kinase C, would be valuable agents in the prevention and treatment of a wide variety of diseases in animals and humans. For example, the need for, and potential utility of, protein kinase C inhibitors/antagonists as agents for the treatment of cancer has received much attention [Corda, D. et al., Trends in

Pharmacological Sciences 11: 471-473 (1990); Powis, G., Trends in Pharmacological Sciences 12: 188-194 (1991); Gandy, S. and Greengard, P., Trends in Pharmacological Sciences 13: 108-113 (1992); Henderson, B. and Blake, S., Trends in Pharmacological Sciences 13: 145-152 (1992)].

It is possible that the different protein kinase C isozymes have different biological roles, and published evidence supports this idea [Homan, E., Jensen, D. and Sando, J., J. Biol. Chem. 266: 5676-5681 (1991); Gusovsky, F. and Gutkind, S., Mol. Pharm. 39: 124- 129 (1991); Borner, C., "The role of protein kinase C in growth control", Sixth

International Symposium on Cellular Endocrinology, W. Alton Jones Cell Science Center, Lake Placid, NY, August 12-15, 1990; Naor, Z. et al., Proc. Natl. Acad. Sci. USA 86: 4501-4504 (1989); Godson, C., Weiss, B. and Insel, P., J. Biol. Chem. 265: 8369-8372 (1990); Melloni, E. et al., Proc. Natl. Acad. Sci. USA 87: 4417-4420 (1990); Koretzky, G. et al., J. Immunology 143: 1692-1695 (1989)]. For example, the stimulation of one protein kinase C isotype or a limited subset of protein kinase C isotypes might lead to undesirable results such as the development of inflammation [Ohuchi, K. et al., Biochim. Biophys. Acta 925: 156-163 (1987)], the promotion of tumor formation [Slaga, T., Envir. Health Perspec. 50: 3-14 (1983)] 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) [Harada, S. et al., Virology 154: 249-258 (1986)].

On the other hand, other protein kinase C isozymes might be responsible for the many beneficial effects observed when protein kinase C is stimulated by known protein kinase C activators in a variety of biological settings; such beneficial effects include the cessation of division of leukemic cells [Rovera, G., O'Brien, T. and Diamond, L., Science 204: 868-870 (1979)], multiplication of colonies of lymphocytes [Rosenstreich, D. and Mizel, S., J. Immunol. 123: 1749-1754 (1979)] and leucocytes [Skinnider, L. and McAskill, J., Exp. Hematol 8: 477-483 (1980)] or the secretion of useful bioregulatory factors such as interferon-γ [Braude, I., U.S. Patent #4,376,822] and interleukin-2 [Gillis, S., U.S.

Patent #4,401,756].

Recent publications indicate that diacylglycerol binding sites (as exemplified by the capability to bind phorbol esters) exist on newly-described proteins which lack the kinase domain, and, thus, lack the kinase activity, of protein kinase C. One such protein family is the n-chimaerins, found in human brain [Ahmed et al., Biochem. J. 272: 767-773 (1990)]. Another family is the unc-13 gene product of the nematode Caenorhabditis elegans,

[Maruyama, I. and Brenner, S., Proc. Natl. Acad. Sci. USA 88: 5729-5733 (1991)] and the homologous human counterparts to the unc-13 gene, known as munc13-1, -2 and -3

[Brose, N. et al., J. Biol. Chem. 270: 25273-25280 (1995)]. The presence of the diacylglycerol binding sites on these two classes of protein was demonstrated by standard binding experiments with [³H]phorbol 12,13-dibutyrate. These new proteins may have other enzymatic or biological activities which can be modulated by compounds which bind to their diacylglycerol binding sites. Thus, diacylglycerol binding site-directed compounds may have utility on non-protein kinase C biological targets.

Given that there are now numerous distinct biological entities bearing diacylglycerol binding sites, it would be highly desirable to obtain chemical compounds which could specifically and selectively target one or another type of diacylglycerol binding site, thus permitting one to selectively activate or inhibit one such site without affecting the others. Such compounds would be valuable experimental tools for studying the role of individual types of proteins bearing diacylglycerol binding sites as well as providing novel means for treating diseases in which protein kinase C or other non-protein kinase C-type

diacylglycerol binding site-bearing proteins are involved.

There are several published reports describing chemical compounds capable of selectively distinguishing several diacylglycerol/phorboid-type binding sites in mouse skin [Dunn and Blumberg, op. cit.] and in purified preparations of protein kinase C isotypes [Ryves, W.J., et al., FEBS Letters 288: 5-9 (1991)]. However, in these studies, even the compounds showing the clearest differences in affinity for these distinct classes, namely phorbol 12,13-dibutyrate, 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-phenylacetate and thymeleatoxin, are only selective by a factor of 10-1000 in

dissociation constant among the different binding sites. Furthermore, these compounds have potent skin inflammatory activity and are not desirable in human or animal medicine because of this toxicity.

Thus, to briefly recapitulate, two kinds of new compounds relating to diacylglycerol binding sites would be highly desirable. The first type would be capable of selectively activating one or a few useful, but not other, deleterious, diacylglycerol binding site-bearing targets. The second type would be capable of inhibiting, or antagonizing the stimulation of, one or more diacylglycerol binding site-bearing targets without blocking activity and/or activation of phorboid-activated target entities whose activation is physiologically harmless or desirable. These kinds of compounds would be valuable agents for the study of diacylglycerol binding site-bearing entities and for the prevention or treatment of a wide range of human and animal diseases thought to involve protein kinase C or other entities bearing diacylglycerol binding sites.

The physiology of protein kinase C includes, in certain cases, a phenomenon known as "down-regulation", manifested as the ability of protein kinase C activators of the phorboid class to initially stimulate protein kinase C at or shortly after the time of application of the phorboid, followed by a net, phorboid-induced metabolic lowering of total protein kinase C levels. [See e.g. Cooper, D.R. et al., Biochem. Biophys. Res. Comm. 161: 327-334 (1989); Isakov, N. et al., J. Biol. Chem. 265: 2091-2097 (1990); Strulovici, B. et al., J. Biol. Chem. 266: 168-173 (1991); and Gschwendt, M. et al., FEBS Letters 307: 151-155 (1992). Several of these studies illustrate the selective down-regulation of one but not another protein kinase C isotype via extended exposure to standard protein kinase C-activating phorboids such as phorbol esters.] Thus, at short times the phorboids generally act as protein kinase C stimulants, but at longer times, for certain protein kinase C isotypes in certain biological settings, the net loss of one or more protein kinase C isotypes in response to the (initially stimulatory) phorboid results in substantial or complete loss of the protein kinase C. This important effect is therefore functionally equivalent to inhibition of protein kinase C, even though the effect is achieved by nominal activators of this enzyme family, and it is clear that, on a long-term time scale, net inhibition of protein kinase C can be achieved either with inhibitors or, in many cases, activators of protein kinase C. A given compound might therefore show quite complex properties on protein kinase C; for example, a non-toxic agonist might fail to stimulate the protein kinase C isotype(s) responsible for inflammation while at the same time activating many other isotypes on a short time scale and inhibiting a subset of the latter isotypes on a long time scale.

On the other hand, down-regulation of one or more protein kinase C isotypes in response to treatment with a protein kinase C activator would be undesirable in situations where protein kinase C activation per se provides a direct therapeutic benefit. In such situations a persistent protein kinase C activation is desired, rather than activation terminated by subsequent down-regulation. It is an object of this invention to provide novel non-toxic protein kinase C activators capable of eliciting persistent activation of protein kinase C.

With the exception of clinical tests of bryostatin 1 itself [Prendiville, J. et al., Brit. J. Cancer 68: 418-424 (1993)], of certain modifications to the hydroxymethyl/1-hydroxyethyl group present in all previously known phorboids [Driedger, P.E. and Quick, J., U.S. Patent 5,145,842 (September 8, 1992) and related patents and patent applications including PCT WO 87/07599 (published December 17, 1987) and PCT WO 92/02484 (published February 20, 1992)], and diterpenes having polar substituents at position 13 as exemplified by phorbol 12-esters [Driedger, P.E., European Patent 0310622 (April 15, 1992) and related patents and applications], efforts to make medical use of the previously known phorboids themselves or to modify the structures of these known phorboids in medically useful ways, have generally not been successful in producing useful compounds with toxicity low enough for use in humans.

For example, it has been known for some time that several of the toxic,

inflammatory and tumor-promoting compounds such as phorbol 12-tigliate 13-decanoate, mezerein, lyngbyatoxin and aplysiatoxin have anti-leukemic activity in mouse model tests [Sugimura, T., op cit.; Kupchan, S.M. and Baxter, R.L., Science 187: 652-653 (1975); Kupchan, S.M. et al., Science 191: 571-572 (1976); Territo, M.C. and Koeffler, HP., Br. J. Haematol 47, 479-483 (1981)].

As another example, the toxic and inflammatory diterpenes of the phorbol ester type have been reported to cause human cells to decrease their production of the 40-43 amino acid peptide family known as Aβ, amyloid peptide or BAP, which is thought to be a primary pathological cause of Alzheimer's disease [Buxbaum, J.D. et al., Proc. Acad. Sci. USA 87: 6003-6006 (1990); Demaerschalk, I. et al., Biochim. Biophys. Acta 1181: 214-218 (1993); Buxbaum, J.D. et al., Proc. Acad. Sci. USA 90: 9195-9198 (1993); Slack, B.E. et al., J. Biol. Chem. 268: 21097-21101 (1993); Hung, AY. et al., J. Biol. Chem. 268:

22959-22962 (1993); Jacobsen, J.S. et al., J. Biol. Chem. 269: 8376-8382 (1994);

Felsenstein, K.M. et al., Neurosci. Lett. 174: 173-176 (1994); Citron, M. et al., Proc. Acad. Sci. USA 91: 11993-11997 (1994)]. These reports show reduction of Aβ production during brief treatments of an hour or two. Such toxic phorbol esters cannot be used advantageously in human medicine because of their toxicity at appropriate doses.

Ganong, et al. [Proc. Nat. Acad. Sci. USA 83: 1184-1188 (1986)] tested a series of diacylglycerols and found no antagonistic activity in that series against the standard agonist, 1,2-dioctanoylglycerol. Compounds tested in this work were modified in the

hydroxymethyl or other portions of the diacylglycerol molecule, and these modifications produced only a loss of activity or a weakened activity that was not distinguishable from the agonist activity of 1,2-dioctanoylglycerol itself, a compound which is toxic to mouse skin [Smart, R. et al., Carcinogenesis 7: 1865-1870 (1986); Verma, A, Cancer Res. 48: 2168- 2173 (1988)]. These modified diacylglycerols were not antagonists in these tests and no utility was found.

Schmidt and Hecker ("Simple phorbol esters as inhibitors of tumor promotion by TPA in mouse skin". Carcinogenesis, Vol. 7, ed. by E. Hecker et al., Raven Press, New York, 1982, pp. 57-63) studied the abilities of a series of diterpene phorboids to inhibit tumor promotion by the standard phorboid agonist tumor promoter phorbol 12-myristate 13-acetate (PMA, also known as TPA). They found that, at low doses, some short-chain ester derivatives of phorbol, differing from PMA only in the chain length of the 12- and 13- ester substituents, were able to block the tumor promotion by PMA However, all of the compounds that were active as antagonists at low doses are also known to be very efficacious skin irritants themselves at slightly higher doses and most of them are also known to have tumor promoting activity. Thus, these short-chain esters still have toxic inflammatory and tumor promoting activity at doses only slightly different from those which would be needed to exhibit a therapeutic effect in mice. In the same study (Schmidt and Hecker, op.cit.) phorbol 12-myristate (abbreviated "TP" in Schmidt and Hecker, op. cit.), which differs from PMA only in the lack of a substituent on the 13-hydroxy group, was tested as an inhibitor of tumor promotion and was found to be inactive.

It is an object of this invention to provide non-inflammatory compounds that are not cancer suspect agents and have anti-leukemia or anti-cancer activity or are able to reduce human cellular or organismal production of Aβ. It is also an object of this invention to provide compounds able to maintain a reduced production of Aβ for periods of time much longer than a 1- or 2-hour exposure. It is a further object of this invention to provide compounds capable of inhibiting the infection cycle of the human immunodeficiency virus (HIV; vide infra). It is also an object of this invention to provide compounds having anti- inflammatory activity. Moreover, it is an object of this invention to provide phorboid diterpenes, in the class having a polar group at position 13, with improved properties over previously reported members of this class, and to provide, in a class comprising

benzolactam phorboids, improved compounds of the phorboid class having altered hydroxymethyl/l-hydroxyethyl groups. Finally, it is an object of this invention to provide medical uses for benzolactam phorboids having modified hydroxymethyl/1-hydroxyethyl groups and to provide new medical uses for diterpenoid phorboids having polar groups at position 13.

Summary of the Invention

The primary focus of the diterpenoid portion of this invention comprises novel compounds of the diterpenoid class of phorboids, designated herein as "13-polar diterpenoids". Such 13-polar diterpenoids have the following defining characteristics (in addition to the basic definition of diterpene phorboids): (i) the usual hydroxymethyl or 1- hydroxyethyl group attached to carbon 6 [using common phorbol numbering] is intact, (ii) at least one substituent other than hydrogen or hydroxy is present at carbon 12, and (iii) at least one generally polar group, as exemplified without limitation by hydroxy, amino, thiol, hydroxymethyl, mercaptomethyl, aminomethyl, 2-hydroxyethyl, carboxy, unsubstituted carboxamido, unsubstituted aminocarbonyloxy or unsubstituted aminothiocarbonyloxy in the α or β configuration on carbon 13 or an oxo or thiono group is present at carbon 13. The initial discovery of the 13-polar diterpenoids [Driedger, European Patent 0310622] was in contrast to the previous belief, based on published experimental results [Thielmann, H.-W. and Hecker, E., "Beziehungen zwischen der Struktur von Phorbolderivaten und ihren entzundlichen und tumorpromovierenden Eigenschaften." Fortschr. Krebsforsch. 7: 171- 179 (1969)], that diterpenes such as phorbol 12-decanoate, phorbol 12-dodecanoate and phorbol 12-myristate are biologically inactive, and it is also in contrast to the usual association of an intact hydroxymethyl or 1-hydroxyethyl groups in previously known phorboids with proinflammatory activity.

In the instant extension of previous discoveries [Driedger, P.E., European Patent 0 310 622] it has now been found that specific new classes of such compounds similarly lack the inflammatory activity of other members of the diterpene class of phorboids and also have useful activities as anti-inflammatory, anti-cancer, anti-HTV, anti-psoriatic agents, as agents to treat numerous other diseases involving protein kinase C and as modulators of protein kinase C. Moreover, in the instant invention various changes in the chemical structure of 13 -polar diterpenoids at certain locations other than the hydroxymethyl leads to substantial and useful improvements in chemical and biological properties over previously disclosed [Driedger, European Patent 0310622] 13-polar diterpenoids. The compounds of this portion of the invention are distinguished, in part, by improvements in their chemical and/or biochemical stability.

The previously disclosed 13-polar diterpenoid compounds embodied at least one ester group and an intact cyclopropyl ring, making the compounds susceptible to degradation under esterolytic/basic and mildly acidic conditions, respectively. The present invention provides, variously, compounds lacking one or another or both of the sensitive ester and cyclopropyl groups, with consequent improved stability in the presence of esterolytic conditions such as biological fluids, acidic conditions such as gastric fluids and chemical synthesis procedures, and/or basic conditions such as are routinely encountered in chemical synthesis. Thus, acidic conditions mimicking gastric fluids degrade phorbol 12-esters, whereas compounds of the present invention comprising 12-esters or other 12-derivatives of compounds such as bisdehydrophorbol, which lack the cyclopropyl group found in many diterpenes, are completely resistant to destruction under the conditions employed for this test. Also, whereas phorbol 12-esters are susceptible to hydrolysis by esterases, the present invention provi-ies novel compounds which lack such ester groups yet retain comparable or improved potencies in medical uses such as treatment of inflammation.

Compounds of the present invention show novel profiles of differential affinity for the known classes of protein kinase C isotypes and other classes of proteins bearing diacylglycerol-type binding sites. The present invention also provides new medical uses for phorbol 12-esters and other 13-polar diterpenoids, such as anti-cancer, anti-leukemia, anti-HIV and anti- Alzheimer's activity.

The structural features associated with the non-toxicity and the diacylglycerol binding site-modulating properties of the compounds of the benzolactam portion of the present invention relate primarily to the hydroxymethyl and 1-hydroxyethyl feature common to all classes of toxic phorboids. Previous discoveries [Driedger, P.E. and Quick, J., U.S. Patent 5,145,842 and related patents and patent applications including PCT WO 87/07599 and PCT WO 92/02484] showed that specific modifications of the latter chemical groupings yields non-skin inflammatory compounds that show anti-inflammatory activity in several test systems, whereas, except as noted above, any of a very wide variety of changes in other parts of the then-known parent phorboid structures, including but not limited to diterpenes, indole alkaloids, polyacetates, diaminobenzyl alcohol derivatives, aplysiatoxins and bryostatinoids, have very markedly less effect on the overall biological properties of the derivatives, other than changes in potency.

The benzolactam portion of this invention is based on the finding that the hydroxymethyl and 1-hydroxyethyl groups, which previously were thought to be required for biological activity of phorboids containing these groups, not only can be replaced by other substituents of very diverse nature, even though these new substituents are very substantially larger and of more diverse structure and heteroatom composition than the original hydroxymethyl/ 1-hydroxyethyl group, but also that the resulting compounds have improved chemical and biological properties over the previously discovered six classes of hydroxymethyl/1-hydroxyethyl-modified phorboids [Driedger, P.E. and Quick, J., U.S. Patent 5,145,842 and related patents and patent applications including PCT WO 87/07599 and PCT WO 92/02484].

Compounds of the benzolactam portion of the present invention show several unexpected advantages over previously disclosed hydroxymethyl/1-hydroxyethyl-modified phorboids. For example, compounds of the present invention show novel profiles of differential affinity for the known classes of protein kinase C isotypes and other classes of proteins bearing diacylglycerol-type binding sites. Compounds of the present invention have improved potencies over hydroxymethyl/l-hydroxyethyl derivatives of other phorboid parent classes such as diacylglycerols and diaminobenzyl alcohols. The hydroxymethyl/1- hydroxyethyl-modified benzolactam compounds of the present invention also provide novel properties in terms of biodistribution. This invention provides compounds with a range of protein kinase C-modulatory properties. In particular, this invention provides, among others, compounds generally able to stimulate many members of the protein kinase C family but which lack the inflammatory toxicity of previously known protein kinase C activators. Because protein kinase C activators can, with prolonged exposure, result in the down-regulation of certain protein kinase C isotypes in certain cells, tissues and organs, this invention also provides a means for blocking certain protein kinase C activities. This invention also provides new

compounds that discriminate in novel ways among phorboid receptor-type targets, whether embodied in protein kinase C or others such as the n-chimaerins or unc-13 gene and munc- 13 gene proteins, having useful differences in relative binding affinities for members of the family of phorboid receptor-types.

This invention pertains to novel phorboid derivatives which variously block the toxic effects of the hydroxymethyl-containing phorboids, lack the toxic properties of previously available phorboids and show activity for applications as therapeutics. The phorboid derivatives of the present invention embody very diverse structures and have utility as anti- inflammatory agents, as cancer cell and leukemic cell inhibitory agents, anti-asthmatic and anti-hypertensive agents, as modulators of human immune cell function, as anti-viral agents, as stimulators of the production of lymphokines such as interferon and the interleukins and as central nervous system pharmaceuticals for several pathological conditions. Detailed Description of the Invention

The phorboid derivatives of the diterpenoid portion of the invention are generally represented by the formula:

I_o-D

wherein I₀ represents a radical, formally derived from a phorbol- or daphnane-type diterpenoid parent compound, which compound:

a. binds reversibly or irreversibly to a diacylglycerol-type receptor; and/or b. activates any form of the enzyme protein kinase C; and

c. contains an hydroxymethyl or 1-hydroxyethyl group bonded to carbon 6; and d. contains at least one substituent other than hydrogen or hydroxy at carbon 12; and

wherein D is a polar group attached to carbon 13. The group, D, may be further exemplified, without limitation, by hydroxy, amino, thioL hydroxymethyl, mercaptomethyl, aminomethyl, 2-hydroxyethyl, carboxy, unsubstituted carboxamido, unsubstituted aminocarbonyloxy, unsubstituted aminothiocarbonyloxy, ketonic or thionic groups.

Requirements for a polar group at position 13 may also be met by an amino group carrying 1-2 substituents or by guanidino or other such polar nitrogen containing groups. However, the invention does not include 12-O-methylphorbol, 12-O-ethylphorbol or compounds of the exact phorbol structure with acyl groups at the 12-hydroxy group, also known as "phorbol 12-esters."

More specifically the diterpene portion of the invention provides compounds of the general formula (I):

in the form of an individual isomer, an isomer mixture, a racemate or optical antipode, or a pharmaceutically acceptable salt thereof; wherein A¹ and A² may be individually selected from hydrogen and a straight chain or branched chain, cyclic or acyclic, saturated, unsaturated and/or aromatic carbon- and/or heteroatom-containing substituent having not more than 34 carbon atoms, not more than 24 halogen atoms and not more than 6 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus, boron and sulfur or wherein A¹ and A² taken together complete a 5- or 6-membered carbocyclic or heterocyclic ring, optionally substituted by, respectively, up to six or up to eight straight chain or branched chain, cyclic or acyclic, saturated, unsaturated, and/or aromatic carbon- and/or heteroatom-containing groups, which groups may optionally form one or two additional rings by connection among themselves and/or to J¹ or A⁴ and which taken together contain a total of not more than 30 carbon atoms, not more than 24 halogen atoms and not more than 9 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus, boron and sulfur; wherein A³ is a three atom chain which completes a 7-membered carbocyclic ring optionally substituted by up to six straight chain or branched chain, cyclic or acyclic, saturated, unsaturated, and/or aromatic carbon- and/or heteroatom-containing groups, which groups taken together, excluding S, contain not more than 12 carbon atoms, not more than 8 halogen atoms, and not more than 5 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur; provided that, excluding S, the middle carbon atom of A³ is not substituted by hydroxymethyl or 1-hydroxyethyl; wherein A⁴ completes a 6-membered carbocyclic ring connected in the β configuration to carbon atom 9, optionally substituted by up to eight straight chain or branched chain cyclic or acyclic, saturated, unsaturated and/or aromatic carbon- and/or heteroatom-containing groups linked to A⁴ by single or double bonds, the group or groups optionally completing one or two additional rings by themselves and/or one or two additional rings when taken together with A¹, A², a ring formed by A¹ and A² together, and/or a bond to carbon atom 9, which groups taken together include not more than 50 carbon atoms, not more than 24 halogen atoms, and not more than 15 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus, boron and sulfur, and wherein A⁴ carries at least one substituent other than hydrogen or hydroxyl at carbon 12 and a hydroxy, unsubstituted amino, thiol, hydroxymethyl, mercaptomethyl, aminomethyl, 2-hydroxyethyl, carboxy, unsubstituted carboxamido, unsubstituted aminocarbonyloxy or unsubstituted aminothiocarbonyloxy group in the α or β configuration on carbon 13 or an oxo or thiono group on carbon 13; wherein carbon 9 may be optionally bound to hydrogen or to a substituent selected from hydroxy, acyloxy, orthoesteroxy, ether or silyl ether; wherein J¹ is selected from hydrogen, fluoro, chloro, hydroxy, amino, mono- or di(lower-alkyl)amino, methyl, ethyl, vinyl, ethynyl, propargyl, cyano, methoxy, ethoxy, trifluoromethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, acetoxy,

propanoyloxy, acetyl, propanoyl, hydroxyacetyl, 2-hydroxypropanoyloxy,

3-hydroxypropanoyl, acetamido, propanamido, hydroxyacetamido, 2-hydroxypropanamido, or 3-hydroxypropanamido, any of which must be situated in the β configuration, or wherein J¹ taken together with A¹, A², or a ring formed by A¹ together with A² completes a 3- to 7- membered substituted or unsubstituted carbocyclic or heterocyclic ring; wherein J² is selected from hydrogen, methyl, ethyl, hydroxymethyl, hydroxyethyl, vinyl, ethynyl, allyl, propargyl, n-propyl and iso-propyl; and wherein S is bound to carbon 5, 6, or 7 and is selected from hydroxymethyl, 1-hydroxyethyl or 2-hydroxy-2-propyl or from esters or ethers of hydroxymethyl, 1-hydroxyethyl or 2-hydroxy-2-propyl.

The compounds of this invention are further illustrated by the formula I_R:

wherein J¹, J² and S are as defined above; and wherein carbons (1 and 2) or (2 and 3) may optionally be joined by a double bond; carbons (5 and 6) or (6 and 7) may optionally be joined by a double bond; S may be bonded to carbon 5, 6 or 7; R_A1 represents not more than 6 identical or different substituents bonded independently via single and/or double bonds to carbons 1, 2 and/or 3, which substituents may optionally form one or two additional rings by connection among themselves and/or to J¹ or the substituents on the 6- membered ring and which may independently be halogen(s) and/or other groups, which halogens and groups taken together contain a total of not more than 30 carbon atoms, not more than 24 halogen atoms and not more than 9 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur; R_A3 represents not more than 6 identical or different substituents bonded independently via single and/or double bonds to carbons 5, 6 and/or 7, which substituents may independently be halogen(s) and/or other groups, which halogens and groups taken together contain not more than 12 carbon atoms, not more than 8 halogen atoms, and not more than 5 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur; and R_A4 represents not more than 8 identical or different substituents bonded independently via single or double bonds to carbons 11, 12, 13 and/or 14, which substituents may independently be halogen(s) and/or other groups, said group or groups optionally completing 1-3 additional rings through bonds among themselves and/or 1-5 additional rings when taken together with the 5-membered ring and/or its substituent(s) R_A1 and/or J⁸, and which halogen(s) and groups taken together for R_A4 include not more than 50 carbon atoms, not more than 24 halogen atoms, and not more than 15 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur; J⁵ is a hydroxy, amino, thiol, hydroxymethyl, mercaptomethyl, aminomethyl, 2-hydroxyethyl, carboxy, unsubstituted carboxamido or unsubstituted aminocarbonyloxy group in the α or β configuration on carbon 13 or is an oxo or thiono group doubly bonded to carbon 13; the substituents on carbon 12, J⁶ and J⁷, may not both be hydrogen; if either J⁶ or J⁷ is hydroxyl, the other may not be hydrogen or hydroxyl; J⁸ is selected from hydrogen, hydroxy, acyloxy,

orthoesteroxy, ether, silyl ether or a bond with J⁴ to form a ring.

Illustrative examples of compounds of the I_R type include, without limitation, derivatives of crotophorbolone, bisdehydrophorbol and 4,9,20-trihydroxy-15,16,17- trinortigliadien-(1,6)-dione-(3,13),

such as 12-decyl-15,17-dihydrocrotophorbolone, 12-benzylidene-15,17- dihydrocrotophorbolone, bisdehydrophorbol 12-myristate, 13-deoxo-13-beta- hydroxybisdehydrophorbol 12-octyldimethylsilyl ether, 13-deoxo-13-beta- carboxybisdehydrophorbol 12-[3',5'-bis(trifluoromethyl)phenyl]carbamate and 12-decyl- 4,9,20-trihydroxy-15,16,17-trinortigliadien-(1,6)-dione-(3,13).

It will also be appreciated by those skilled in the pharmaceutical arts that various compounds of the present invention will have different preferred structural features, depending on the intended use of the compounds in question. For example, it has generally been found that adding substituents of moderate hydrophobicity, e.g. substituents carrying hydrocarbon moieties totaling from about 4 to about 25 carbon atoms, to the A-ring and/or C-ring of diterpene phorboids increases the biological potency of the resulting compounds. Such highly potent compounds might be desired in some therapeutic settings to have a long biological half-life in the patient, while in other therapeutic settings a short half-life might be desired.

To achieve the metabolic stability needed for long biological half-life, for example, substituents may be bound to the A-ring or C-ring via heteroatom-free carbon linkages or, depending on the organism and tissue involved, the metabolism-resistant heteroatom linkages such as ether, dialkyl- or alkylarylphosphinate and derivatives, carbonate, carbamate, amides of certain types, sterically hindered ester, sulfur-for-oxygen analogs of the foregoing heteroatom linkages, and linkages comprising silyl-carbon bonds, silyl ethers, diradylsulfoxides, diradylsulfones, or amines at the secondary level of alkyl substitution or greater.

On the other hand, selection of A-ring or C-ring substituents with metabolically labile linkages to the A- or C-rings would be preferred when compounds short in vivo half- lives are desired. Examples of such linkages, without limitation, include unhindered esters and phosphodiesters.

Other preferred embodiments of I_R are illustrated by, but not limited to, structures carrying a substituted or unsubstituted cyclopropyl ring, forming I_p

wherein J¹, J², J⁵, J⁶, J⁷, J⁸, R_A1, R_A3 and S are as defined for I_R above, except that in I_p J⁵ is necessarily in the α configuration and may not be oxo or thiono; and wherein the R_A5 and R_A6 radicals may independently be hydrogen, halogen and/or other groups, which halogens and groups taken together contain a total of not more than 30 carbon atoms, not more than 24 halogen atoms and not more than 9 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur.

In preferred compounds further illustrating I_p, carbon 2 carries a methyl group, J¹ is hydroxy, carbon 9 carries a hydroxy group in the α configuration, carbons 10 and 14 carry hydrogens in the α configuration, carbon 11 carries a methyl group in the α configuration and R_A5 and R_A6 are both methyl, forming I_PP:

wherein J⁵, J⁶, J⁷, J⁸ , R_A1, R_A3 and S are as defined above for I_p; and wherein either J⁶ or J⁷ is linked to carbon 12 via a carbon atom and the other of J⁶ or J⁷ is linked to carbon 12 via an oxygen atom.

The primary focus of the benzolactam portion of this invention are benzolactam phorboid derivatives generally represented by the formula:

P - G

The formula depicts a radical, P, derived from a benzolactam parent compound, which compound:

c. contains an hydroxymethyl or 1-hydroxyethyl group bonded to a carbon atom, and

wherein G is any group of 55 or fewer atoms selected from carbon, hydrogen, oxygen, nitrogen, halogen, sulfur, phosphorus, silicon, arsenic, boron and selenium either: i) singly or doubly bonded to the carbon atom of the parent compound in place of the hydroxymethyl or 1-hydroxyethyl group; or ii) singly or doubly bonded to a carbon atom immediately adjacent to the carbon atom to which the hydroxymethyl or 1-hydroxyethyl group is bound in the parent compound; and wherein the hydroxymethyl or 1-hydroxyethyl group of the parent compound is absent or has been replaced by G. Excluded from the present invention are: (S)-1,2,3,4,5,7-hexahydro-3-(1-methylethyl)-2-[(4-methylphenyl)sulfonyl]-4-oxo-6H- 2,5-benzodiazonine-6,6-dicarboxylic acid diethyl ester, [S-(R*,R*)]-1,2,3,5,6,7-hexahydro- 3-(1-methylethyl)-2-[(4-methylphenyl)sulfonyl]-4-oxo-4H-2,5-benzodiazonine-6-carboxylic acid, [S-(R*,S*)]-1,2,3,5,6,7-hexahydro-3-(1-methylethyl)-2-[(4-methylphenyl)sulfonyl]-4- oxo-4H-2,5-benzodiazonine-6-carboxylic acid, (S)-1,2,3,5,6,7-hexahydro-6,6- bis[(acetyloxy)methyl]-3-(l-methylethyl)-2-[(4-methylphenyl)sulfonyl]-4H-2,5- benzodiazonin-4-one and (S)-1,2,3,5,6,7-hexahydro-6,6-bis(hydroxymethyl)-3-(1- methylethyl)-4H-2,5-benzodiazonin-4-one.

More specifically, the phorboid derivatives of the benzolactam portion of this invention are represented by the formula:

P₀ - S₀ - E₀

The formula depicts a radical P₀, formally derived from a parent hydroxymethyl-containing phorboid compound, bonded to an S₀-E₀ substituent.

P₀ represents a radical, formally derived from a compound which contains an hydroxymethyl (or the equivalent 1-hydroxyethyl) group rather than S₀E₀, and which binds reversibly or irreversibly to a diacylglycerol-type receptor and/or activates any form of the enzyme protein kinase C. P₀ may be formally derived from a protein kinase C activator of the benzolactam class. These parent benzolactam phorboids contain an hydroxymethyl or

1-hydroxyethyl group which is replaced by S₀E₀ in the present invention and is shown in the present invention to be associated with the toxic biological activity of the parent benzolactam phorboids, such as skin inflammatory activity measured on the mouse ear.

S₀-E₀ represents a substituent which is either:

i) singly or doubly bonded to the carbon atom of the parent compound in place of the hydroxymethyl or 1-hydroxyethyl group; or

ii) singly or doubly bonded to a carbon immediately adjacent to the carbon atom to which the hydroxymethyl or 1-hydroxyethyl group is bound in the parent compound.

In this S₀-E₀ substituent, S₀ 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 separates P₀ and E₀ by a linear count of not more than 12 atoms and contains and/or carries not more than 9 heteroatoms selected from oxygen, nitrogen, silicon, sulfur, phosphorus, arsenic, boron and selenium, and not more than 16 halogen atoms; provided that the total number of atoms does not exceed 35; and in some cases S₀ may be a single or double bond; and E₀ can be hydrogen, halogen or a saturated or singly or multiply unsaturated group containing up to 15 carbon atoms and optionally containing 1 to 12 halogen atoms and/or 1 to 6 heteroatoms selected from oxygen, nitrogen, silicon, sulfur, phosphorus, arsenic, boron and selenium. S₀E₀ taken together may also be a hydrogen, halogen, thionic sulfur atom or ketonic oxygen atom or a hydroxy, amino, or thiol group singly or doubly bonded to the carbon atom of the parent compound P₀ in place of the hydroxymethyl or 1-hydroxyethyl group.

In a preferred embodiment, the benzolactam portion of the present invention is represented as follows:

P_x - S_x - E₁

wherein P_x is a benzolactam-type parent structure as defined below, wherein S_x is selected from seven different structural types as defined below and E₁ is as defined below.

Thus, the benzolactam-type parent structure, P₇, is a radical of the formula:

in the form of an individual isomer, an isomer mixture, a racemate or optical antipode, or a pharmaceutically acceptable salt thereof, wherein up to two of B⁷-B¹⁰ may optionally be nitrogen; represents 1-4 identical or different substituents located independently at

carbons in any of positions 7, 8, 9 and/or 10, which substituents may independently be hydrogen, halogen and/or other groups which, taken together, contain not more than 9 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur, the groups being optionally connected to one another and/or to

to form 1-2 additional carbocyclic or heterocyclic rings;

is selected from oxygen, sulfur

wherein is hydrogen, hydroxy, methyl, ethyl, fluoro, n-propyl, allyl, or propargyl and is hydrogen, methyl, ethyl, halogen, trifluoromethyl or cyano;

may be the same or may differ and each may independently be hydrogen, halogen, a substituent group, or may complete an additional ring connecting

or connecting either

such that

taken together contain not more than 18 carbon atoms, not more than 12 halogen atoms, and not more than 8 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur;

₇ is selected from oxygen, sulfur, sulfoxide, sulfone or

wherein

is hydrogen or a group containing not more than 30 carbon atoms, not more than 24 halogen atoms, and not more than 8 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur, and is hydrogen, methyl, ethyl,

n-propyl, hydroxy, halogen, allyl, propargyl, cyano, or trifluoromethyl; y may either be 0 or 1; and, Q_L is hydrogens or is Q as defined below; provided that if S_xE₁ is hydroxymethyl or 1-hydroxyethyl then S_xE₁ may not be bound to carbon 5.

S_x may represent any of a broad range of connecting chains or groups of atoms, designated S_B, S₁, S₂, S₃, S₄, S₅ and S₆. Surprisingly, these organic functional groups may be hydrophobic in nature, with few if any polar or heteroatoms present, may be extensively halogen-substituted, or may contain one or several polar atoms such as oxygen, nitrogen, silicon, phosphorus, arsenic, boron, selenium and/or sulfur in any of numerous chemical groupings. Such functional groupings may even bear positive or negative charges at physiologic pH, and the values which are permissible for S_x also may include combinations of hydrophobic, halogenated, hydrophilic and/or charged functional groups. The resultant compounds in any case generally display, variously, the protein kinase C-modulatory, nontoxic agonist, and/or antagonistic properties, selectivities and therapeutic and other utilities described in this invention.

S_B-S₆ may comprise the following values.

S_B is a single or double bond.

S₁ is a chain of atoms of the formula:

wherein a, b, d, e, and g may independently be from 0 to 3; c and f may independently be 0 or 1; the sum of (a + b + c + d + e + f + g) is at least 1 but not more than 12; and if c and f are both 1, then the sum of (d + e) must be at least 1; and X and X' are as defined below.

S₂ is a chain of atoms of the formula:

wherein h, i, k, m, p, and q may be independently be from 0 to 3; j and n may independently be 0 or 1; if j and n are both 1 and 1 is 0, then the sum of (k + m) must be at least 1; if n is 1 and o is 0, then the sum of (p + q) must be at least 1; the sum of (1 + o) is 1-3; and the sum of (h + i + j + k + 21 + m + n + 2o + p + q) is at least 1 but not more than 12; and X, X', Y and Y¹ are as defined below.

S₃ is a chain of atoms of the formula:

wherein r, s, u, y, a', and b' may independently be from 0 to 3; the sum of (t + z) is 0 or 1; the sum of (v + w + x) is 1; the sum of (y + z + a' + b') is at least 1; and the sum of (r + s + 2t + u + 2v + 3w + 4x + y + 2z + a' + b') is at least 1 but not more than 12; and Y, Y', Z¹, Z², and Z³ are as defined below.

S₄ is a chain of atoms defined by:

wherein c', d', e', h', and i' may independently be from 0 to 3; the sum of (f' + g') must be 1 or 2; f' and g' may independently be 0 or 1; and the sum of (c' + d' + e' + f' + g' + h' + i') is at least 1 but not more than 12; and M, M', and R^Q are as defined below.

S₅ is a chain of atoms defined by:

wherein j', k', m', q', and s' may independently be from 0 to 3; l' and r' may each be 0 or 1, but the sum of (l' + r') must be 1 or 2; n' and p' may each be 0 or 1, but the sum of (n' + p') must be 0 or 1; the value of o' may be 0-2; if the sum of (n' + p') is l and l' is 0, then q' must be at least 1; if the sum of (n' + p') is 1 and r' is 0, then m' must be at least 1; and the sum of (j' + k' + l' + m' + n' + o' + p' + q' + r' + s') is at least 1 but not more than 12; and Q, Q', X, X', and Y are as defined below.

S₆ is a chain of atoms defined by:

wherein u', v', w', x', y', z', and m" may each be 0 or 1; t' and a" may each independently be 0-6; the sum of (t' + u' + v' + 2w' + x' + 2y' + z' + a") must be 0-8; b", d", e", f", h", j", k" and n" may each independently be 0 or 1; c", g", i", and l" may each independently be 0-3; if d" and j" are both 1, then the sum of (g" + i") must be at least 1; if either j" or k" is 1, then l" must be at least 1; if b" is 1, then the sum of (c" + g" + h" + i" + l") must be at least 1; if d" is 1, then the sum of (g" + h" + i" + l") must be at least 1; and the sum of (t' + u' + v' + 2w' + x' + 2y' + z' + a" + b" + c" + d" + e" + 2f" + g" + 2h" + i" + j" + k" + l") must be 0-14; if m" is zero,

may optionally comprise an additional bond to G² as defined below, thus completing an unsaturated linkage; if n" and b" are zero,

| | may optionally comprise an additional bond to G², thus completing an unsaturated linkage; one of the substituents

and/or one of the substituents

may optionally comprise the same or different values of G¹, as defined below; and M, M', R^Q, R^Q', R^Q", R^Q'", X, X', X", Y, Y', Z⁴, Z^4', Z⁵ and Z^5' are as defined below.

For S₁-S₆,

through

may be the same or different and each may be hydrogen, halogen or an acyclic substituent containing not more than 20 carbon atoms, not more than 16 halogen atoms, and not more than 6 heteroatoms selected from oxygen, nitrogen, sulfur, silicon, boron, arsenic, phosphorus and selenium; one substituent selected from

may optionally comprise an additional bond completing an unsaturated linkage to P_x; one or two of the substituents

may optionally comprise the same or different values of G¹, as defined below; one substituent selected from

may optionally comprise an additional bond to E₁, thereby completing an unsaturated linkage; one of the substituents

may be linked to either the atom in P_x that carries the S_x chain or to an atom in P_x adjacent thereto, to form a saturated, unsaturated or aromatic, carbocyclic or heterocyclic 4-8 membered ring optionally containing 1-4 identical or different ring hetero members selected from X and =N-, the ring being optionally substituted by 1-8 identical or different substituents, preferably selected from halogen, hydroxy, methoxy, ethoxy, methyl, ethyl, cyano, azide, nitro, hydroxymethyl, 1-hydroxyethyl, 2-hydroxy-2-propyl, CF₃, OCF₃, SH, SCH₃, SOCH₃, SCF₃, COOH, COOCH₃, COCH₃, CH=O, acetoxy, amino, mono- or dialkylamino totaling 1-4 carbon atoms inclusive, acetamido, N-methylacetamido, carboxamido, N-alkylated carboxamido containing 1-4 carbon atoms inclusive, hydroxyacetyl and hydroxyacetoxy.

The foregoing description of S₁-S₆ is subject to the restriction that, for any given S₁, S₂, S₃, S₄, S₅ or S₆, but excluding P_x and E₁: the total of carbon atoms is 25 or less; the total of halogen atoms is 16 or less; the total of oxygen atoms is 6 or less; the total of nitrogen atoms is 4 or less; the sulfur, silicon, boron and phosphorus atoms each total 3 or less; the arsenic and selenium atoms each total 1 or less; and the total of oxygen, nitrogen, silicon, boron, arsenic, phosphorus, selenium and sulfur atoms together is 8 or less.

In a preferred mode of this invention, the oxygen, nitrogen, sulfur, silicon and/or phosphorus atoms in

may be situated in a variety of functional groups such as hydroxy, amino, hydroxylamine, tertiary amine oxide, Schiffs base, hydrazine, thiol, nitro, nitroso, oxime, azide, ether, acetal, ketal, thioether, aldehyde, keto, hydrazone, carboxy, mercaptocarbonyl, mercaptothionocarbonyl, sulfonate, sulfonyl, sulfoxide, phosphate, phosphonate, phosphate ester, phosphonate ester, phosphine, phosphine oxide,

thionophosphine, phosphite, phosphonium, phosphorothioate, thionophosphate ester, thiophosphonate, thionophosphonate ester, silane, silanol, silanediol, fluorinated silane, ester, amide, cyano, hydrazide, carbonate, carbamate, urea, isourea, carboxamidine, imidate, guanidine, thioester, thioamide, thiocarbonate, dithiocarbonate, thiocarbamate,

dithiocarbamate, thiourea, isothiourea, thioimidate, nitroguanidine, cyanoguanidine and xanthate. Preferably, for any given S_x, the total of - OH groups is 3 or less, the total of -NH₂ groups is 2 or less, the total of -SH groups is 2 or less, and the total of -OH, -SH and -NH₂ groups is 4 or less.

X, X', X" may be the same or different and are selected from:

or from - Se- ,

wherein

may independently be hydrogen; through may be the same or different and each may be

an acyclic substituent containing 1-20 carbon atoms, not more than 16 halogen atoms, and not more than 6 heteroatoms selected from oxygen, nitrogen, and sulfur, such that for any substituent the oxygen atoms total 4 or less, the nitrogen atoms total 4 or less, and the sulfur atoms total 2 or less;

may independently be hydroxy; Q and Q' are as defined below;

may optionally represent an additional bond to P_x, thus completing an unsaturated linkage; and, one to four of the substituents may optionally

comprise the same or different values of G¹, as defined below.

Y and Y' may be the same or different and are selected from:

wherein

each pair being cis or trans relative to one another, may be the same or different and each may be hydrogen or an acyclic substituent containing not more than 20 carbon atoms, not more than 16 halogen atoms, and not more than 6 heteroatoms selected from oxygen, nitrogen, and sulfur, such that for any substituent the oxygen atoms total 4 of less, the nitrogen atoms total 4 or less, and the sulfur atoms total 2 or less;

y _γ may also independently be halogen; one or two of the substituents

may optionally comprise the same or different values of G¹, as defined below; and one of the substituents

may be linked to either the atom in P_x that carries the chain containing X, X', and/or X" or to an atom in P_x adjacent thereto, to form a saturated, unsaturated or aromatic, carbocyclic or heterocyclic 4-8 membered ring defined as for the analogous

-containing ring above.

In a preferred embodiment, the substituents

are selected from hydroxy, amino, thiol, nitro, azide, ether, thioether, aldehyde, keto, carboxy,

mercaptocarbonyl, mercaptothionocarbonyl, sulfonate, sulfonyl, sulfoxide, ester, amide, cyano, carbonate, carbamate, urea, isourea, carboxamidine, guanidine, thioester, thioamide, thiourea, nitroguanidine, cyanoguanidine and xatthate.

wherein the substituents

are generally selected from hydrogen, halogen in cases where a chemically stable structure results, and a radical containing about 1-12 carbon atoms and optionally containing 0-12 halogens and 0-6 heteroatoms selected from oxygen, nitrogen and sulfur.

In a preferred embodiment of the invention, the substituents

comprise a range of saturated or unsaturated substituents as described below, wherein the terms alkyl, halogenated alkyl and acyl are taken to include alkenyl, alkynyl, alkenoyl and alkynoyl and their halogenated forms.

Thus:

be any of the values specified for

above

individually may be -O-, -S-, or

wherein

may be hydrogen, C_1-4 alkyl, 2-hydroxyethyl, 2-hydroxy-n-propyl, 2-acetoxyethyl, or 2-acetoxy-n-propyl; and

l individually may be hydrogen or a substituent selected from C_1-4 alkyl; C_1-4 alkoxy; C_1- ₄ alkylthio; phenoxy or thiophenoxy optionally substituted by methyl, hydroxy,

hydroxymethyl, thiol, carboxy, carboxymethyl, amino, methoxy, halogen, and/or nitro; or amino optionally mono- or disubstituted by C_1-4 alkyl or monosubstituted by cyano, nitro or phenyl optionally substituted by halogen, hydroxy, hydroxymethyl, thiol, carboxy, carboxymethyl, amino, and/or nitro may be hydrogen, a

saturated or unsaturated substituent selected from C_1-6 alkyl, C_1-6 halogenated alkyl, and cyclohexyl, or may be phenyl or benzyl, each optionally substituted by methyl, ethyl, hydroxy, hydroxymethyl, thiol, carboxy, carboxymethyl, amino, methoxy, nitro, cyano, trifluoromethyl, and/or halogen; may also be

independently selected from C_1-6 acyl, C_1-6 halogenated acyl, C_2-6 monohydroxyacyl, and C_2-6 hydroxyalkyl; | | may also be independently selected from C_1-6 hydroxyalkyl,

C_1-6 alkoxy, C_1-6 hydroxyalkoxy; and

| | | may also independently be C_2-6 hydroxyalkyl; one of the substituents f may be linked to

either the atom in P_x that carries the chain containing Z¹ or to an atom in P_x adjacent thereto, to form a saturated, unsaturated, or aromatic, carbocyclic or heterocyclic 4-8 membered ring optionally containing 1-4 other identical or different hetero ring members selected from O, S, =N-, and NH, the ring being optionally substituted on its carbon and/or NH members by 1-8 identical or different substituents selected from halogen, hydroxy, methoxy, ethoxy, methyl, ethyl, cyano, azide, nitro, hydroxymethyl, 1 -hydroxyethyl, 2-hydroxy-2-propyl, CF₃, OCF₃, SH, SCH₃, SOCH₃, SCF₃, COOH, COOCH₃, COCH₃, CH=O, acetoxy, amino, mono- or dialkylamino totaling 1-4 carbon atoms inclusive, acetamido, N-methylacetamido, carboxamido, N-alkylated carboxamido containing 1-4 carbon atoms inclusive, hydroxyacetyl and hydroxyacetoxy;

may comprise G¹ as defined below; an optional ring may be formed between

and P_x as described above for may comprise an additional bond to P_x, thus completing

an unsaturated linkage; and only one of the substituents

may be substituted or unsubstituted phenyl or benzyl.

M and M' independently may be:

k wherein

may be the same or different and each may be hydrogen or a saturated or singly or multiply unsaturated, straight or branched, acyclic substituent containing 1-20 carbon atoms, not more than 16 halogen atoms, and not more than 6 heteroatoms selected from oxygen, nitrogen, and sulfur, in which the oxygen atoms total 4 or less, the nitrogen atoms total 4 or less, and the sulfur atoms total 2 or less, the heteroatoms being preferably situated in functional groups selected from hydroxy, amino, thiol, nitro, azide, ether, thioether, aldehyde, keto, carboxy, ester, amide, cyano, nitroguanidine, and cyanoguanidine; may optionally comprise an additional bond to P_x group, thus completing an

unsaturated linkage;

may optionally comprise the same or different values of G¹, as defined below;

may be linked to either the atom in P_x that carries the chain containing M and/or M' or to an atom in P_x adjacent thereto, to form a saturated, unsaturated or aromatic, carbocyclic or heterocyclic 4-8 membered ring defined as for the analogous

-containing ring above.

Q-Q'" independently may be: wherein R

may be the same or different and each may have the values specified above for

Q Q may optionally comprise the same or different values of G¹, as defined below;

may be linked to either the atom in P_x that carries the chain containing Q and/or Q' or to an atom in P_x adjacent thereto, to form a saturated, unsaturated or aromatic, carbocyclic or heterocyclic 4-8 membered ring defined as for the analogous

p ^-containing ring above.

R^Q-R^Q'" are independently selected from:

wherein R may be the same or different and each may be selected from halogen

and the values specified above for

may optionally comprise the same or different values of G¹, as defined below; Q and Q' are as defined above; one of

_Q and may be linked to either the atom in P_x bonded to the chain that carries R^Q or to an atom in P_x adjacent thereto, to form a saturated, unsaturated or aromatic, carbocyclic or heterocyclic 4-8 membered ring defined as for the analogous

-containing ring above.

G¹ and G² independently comprise a group containing 1-3 fused or separate, saturated, unsaturated or aromatic, carbocyclic or heterocyclic 4-8 membered rings, each ring optionally containing 1-4 identical or different hetero ring members selected from X and =N-, each ring being optionally substituted on its carbon and/or NH members by 1-8 identical or different substituents, preferably selected from halogen, hydroxy, methoxy, ethoxy, methyl, ethyl, cyano, azide, nitro, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-propyl, CF₃, OCF₃, SH, SCH₃, SOCH₃, SCF₃, COOH, COOCH₃, COCH₃, CH=O, acetoxy, amino, mono- or dialkylamino totaling 1-4 carbon atoms inclusive, acetamido, N-methylacetamido, carboxamido, N-alkylated carboxamido containing 1-4 carbon atoms inclusive, hydroxyacetyl and hydroxyacetoxy; wherein for G¹ the optional second and third rings may be fused to the first ring and/or to one another or may be separate rings connected to one another and/or to the atom bearing G¹ by a single or double bond or by an intervening substituted or unsubstituted, linear or branched, saturated or unsaturated chain containing not more than 8 carbon atoms, not more than 8 halogens, and not more than 4 heteroatoms selected from oxygen, nitrogen, silicon, boron, arsenic, phosphorus, selenium and sulfur; and wherein for G² the first ring is singly or doubly bonded to P_x or to a component atom of the S₆ chain connecting P_x and G², and the optional second and third rings may be fused to the first ring or to one another or may be separate rings connected to one another and/or to the first ring by single or double bonds or by an intervening substituted or unsubstituted, linear or branched, saturated or unsaturated chain containing not more than 8 carbon atoms, not more than 8 halogens, and not more than 4 heteroatoms selected from oxygen, nitrogen, silicon, boron, arsenic, phosphorus, selenium and sulfur.

The capping group E₀ that terminates the connecting chain also may be selected from any of a surprisingly broad array of chemical groupings, and these chemical groupings can be composed of a far larger number of atoms than is found in the hydroxymethyl or 1- hydroxyethyl group. These chemical groupings may include, without limitation, hydrophobic entities such as alkyl, hydrogen, and halogenated alkyl, or may include, without limitation, quite hydrophilic organic functional groups, such as hydroxy, thiol, carboxy and carboxy esters, amines, etc. It is well-known in the art that organic functional groups spanning a wide range of properties, from ionized and very hydrophilic to very hydrophobic, can be formed from multi-atom groupings of elements selected from carbon, hydrogen, halogen, oxygen, nitrogen, silicon, phosphorus, arsenic, boron and selenium. Indeed, for this invention the single restriction appears to be that S₀E₀ taken together should not be hydroxymethyl or 1-hydroxyethyl bonded in the usual position in the parent compounds, since such compounds correspond to the skin-inflammatory and often tumor- promoting parent natural products.

Thus, E₀ may comprise E₁, wherein E₁ is selected from =O, =S, =NH,

wherein

is hydrogen or a C₁-C₈ normal or branched alkyl radical, =N-NH₂, hydrogen, halogen, -OH, -SH, -NH₂, -NH-NH₂, -N₃, -CN, -NO, -NO₂, -NHOH, -ONH₂, or is selected from:

f

wherein T¹ is selected from -O -, -S-, and -NH-; T² is selected from =O, =S, and

| in which

| may be hydrogen, hydroxy, cyano, or nitro; T^2' is selected from =O and =S; T³, T⁴ and T^4' are independently selected from -OH, -NH₂, -SH, -N₃, -NH-NH₂, and

in which

may be hydrogen, C_1-3 alkyl or C_1-3 acyl; T³ may also be hydrogen or halogen; T⁵-T^5" are independently selected from hydrogen and hydroxy; T⁵ may also be halogen;

is selected from hydrogen, halogen, hydroxy, nitro, nitroso, cyano, azide, -NH₂, -NH-OH, -SH, -O-NH₂, -NH-NH₂,

-T¹-C(=T²)-T³, -C(=T²) -T³, - SiT⁵T^5'T^5", - T¹-S(=O)(=T²)-T⁴,

-S(=O)(=T²)-T⁴, -T¹-P(-T⁴)-T^4', -P(-T⁴)-T^4', -T¹-P(=T^2')(-T³)-T⁴, and -P(=T^2')(-T³)-T⁴; are individually selected from hydrogen,

-C(=T²)-T³, cyano, nitro, aade, halogen and a C₁-C₁₅ straight or branched chain, saturated, unsaturated and/or aromatic substituent optionally containing not more than 10 halogen atoms and not more than 4 heteroatoms selected from oxygen, nitrogen and sulfur.

Finally, if

is cyano or -C(=T²)-T³, then R

| | may optionally be selected from - SiT⁵T^5'T^5", -T¹-P(=T^2')(-T³)-T⁴, and -P(=T^2')(-T³)-T⁴; and

are individually selected from hydrogen, halogen, cyano, nitro, -C(=T²)-T³,

-T¹-C(=T²)-T³, -SiT⁵T^5'T^5", -S(=O)(=T²) -T⁴, and

-P(=T^2')(-T³)-T⁴.

Among the phorboids of the P₇ class of benzolactams, the novel compounds of this invention may embody the structure P_7B,

, wherein

are as defined above.

In preferred compounds further illustrating P_7B, is hydrogen, is methyl and y

is 0, forming P_7BL

wherein

represents 1-2 identical or different substituents attached independently to carbons 8 and/or 9, which substituents may independently be hydrogen, halogen(s) and/or other groups which taken together, contain not more than 40 carbon atoms, not more than 24 halogen atoms and not more than 9 heteroatoms selected from oxygen, nitrogen, silicon, phosphorus and sulfur, the groups being optionally connected to one another to form 1-2 additional carbocyclic or heterocyclic rings. In a preferred embodiment is a C₁-C₁₄ saturated or unsaturated alkyl group.

It will be appreciated that the many different permissible changes to the

hydroxymethyl or 1-hydroxyethyl groups of the parent phorboids lead to diverse compounds with diverse biological properties, and different embodiments will be preferred for different utilities. If different protein kinase C isotypes and other proteins bearing phorboid-type binding sites have different biological functions, as has been extensively hypothesized and to some extent demonstrated in biological experiments, then the novel compounds of this invention with differing activity on different protein kinase C isotypes will obviously display a wide range of differing utilities.

For example, a preferred set of compounds for anti-inflammatory activity is generated by replacing the hydroxymethyl or 1-hydroxyethyl groups of parent benzolactam phorboids with the following organic functional groups: (i) dihalomethyl, trihalomethyl, -N₃, -NH₂, -CN, -COOH, -CH=CHCOOH, -C≡CCOOH, -CSSH, -COSH, -SO₂H, -SO₃H, -PO₃H₂, -

|

-CH=NOH, -CH=NOCH₃, -CH=N(-O)CH₃, -C(CH₃)=NOH,

| - Si(CH₃)₂OH, -Si(OH)₂CH₃, -Si(CH₃)₂F,

or

| in which

is hydrogen or C_1-12 linear or branched, saturated, unsaturated and/or aromatic hydrocarbon optionally substituted by not more than 16 halogens; or (ii) -CH₂- or -CH(CH₃)- , to either of which is bonded -

| -F, -N₃, -NH₂, -CN, -COOH, -COSH, -CSSH, -Si(CH₃)₂OH, -Si(OH)₂CH₃, -Si(CH₃)₂F, -SO₂H, -SO₃H, -PO₃H₂,

-PO₃H₂,

the o-, m- or p-isomer of -M-C₆H₄CH₂-T³, the o-, m- or p-isomer of -C₆H₄CH₂-T³,

-SCH₂CH₂OH, -S(=O)-CH₂CH₂OH, -S(CH₂)₃OH, -S(CH₂)₄OH, -SCH₂CH₂SH,

(except -OC(=O)NH₂), -OCH₂C(=O)CH₃, -OCH₂C(=NOH)CH₃, the o-, m- or p-isomer of

- M-C(=T²)- M'-C₆H₄-T³, the o-, m- or p-isomer of

-M-C(=T²)- M'-C₆H₄CH₂-T³, or - imidazol-2-yl.

Preferred compounds for anti-inflammatory use, for example, incorporate parent benzolactam radicals selected from P₇, P_7B and P_7BL bearing functionally diverse S_xE₁ groups selected from (i) dihalomethyl, trihalomethyl, -N₃, -NH₂, -CN, -COOH, -CH=CHCOOH, -C≡CCOOH, -CSSH, -COSH, -SO₂H, -SO₃H, -PO₃H₂, -CH=NOH, -Si(CH₃)₂OH, -Si(CH₃)₂F,

-Si(OH)₂CH₃, or (ii) -CH₂- or -CH(CH₃)-, to either of which is bonded

| -F, -N₃, -NH₂, -CN, -COOH, -COSH, -CSSH, -Si(CH₃)₂OH, -Si(OH)₂CH₃, -SO₃H, -PO₃H₂, -

| H, the o-, m- or p-isomer of -M-C₆H₄CH₂-T³, the o-, m- or p-isomer of -C₆H₄CH₂-T³, -SCH₂CH₂OH, -S(=O)-CH₂CH₂OH,

-S(CH₂)₃OH, -S(CH₂)₄OH, -SCH₂CH₂SH, the o-, m- or p-isomer of

-M-C(=T²)-M'-C₆H₄-T³, or the o-, m- or p-isomer of

-M-C(=T²)-M'-C₆H₄CH₂-T³, in which

is hydrogen or C_1-12 linear or branched, saturated, unsaturated and/or aromatic hydrocarbon optionally substituted by not more than 16 halogens.

The compounds of this invention have been found to possess valuable

pharmacological properties for human and veterinary medicine. For therapeutic use in humans or animals the compounds of this invention are dispensed in unit dosage form comprising 0.001 to 1000 mg per unit dosage in a pharmaceutically acceptable carrier. In particular, unit dosages in the range of 0.1 to 100 mg are preferred. The compounds of this invention may also be incorporated in topical formulations in concentrations of about 0.001 to 10 weight percent, with concentrations of 0.01 to 10 weight percent being preferred.

The compounds of this invention have been found to possess valuable

pharmacological properties for human and veterinary medicine. For therapeutic use in humans or animals the compounds of this invention are dispensed in unit dosage form comprising from about 0.001 to 3000 mg per unit dosage in a pharmaceutically acceptable carrier. In particular, unit dosages in the range of 0.1 to 100 mg are preferred. The compounds of this invention may also be incorporated in topical formulations in

concentrations of about 0.001 to 20 weight percent, with concentrations of 0.01 to 10 weight percent being preferred.

It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, and the particular sites and organism being treated. Compounds of this invention having higher potencies should be used in generally smaller amounts, and compounds with lower potencies should be used in generally larger amounts. Dosages for a given host, whether a small animal such as a cat or a human patient, can be determined using conventional considerations, e.g., the host's weight or body surface area. In general, the compounds of the present invention are administered in unit doses of about 0.000015 to about 50 mg/kg of body weight, and quantities of about 0.01 to about 15 mg/kg of body weight are preferred.

As specific examples, the compounds of this invention variously block inflammation; show cytostatic and/or cytotoxic activity against diverse types of human cancer cells representative of several human cancers such as leukemia, carcinoma and melanoma; inhibit the ability of HIV to infect human cells; and induce production of thrombolytic activity. These effects are demonstrated by (i) inhibition in the standard topical in vivo mouse ear inflammation model for anti-inflammatory and anti-psoriatic agents wherein inflammation and/or edema by established agonists such as PMA and the ionophore A23187 are blocked; (ii) by the inhibition of proliferation of human leukemic cells in culture via induction of differentiation; (iii) by assays of cytotoxicity against human carcinoma cancer cells; (iv) by assays of inhibition of growth of human melanoma cells; (v) by assays of inhibitory activity against HIV viral replication in human lymphocyte cells; and (vi) by measurement of stimulation of fibrinolytic activity in cultured cells. These demonstrations of efficacy are achieved at doses per ear, per kg of body weight or per kg of bodily fluid equivalent, using representative compounds of the present invention, as follows: (i) about 0.01-1000 nanogram/ear, corresponding to about 0.0001-10 mg per square meter of body surface being treated; (ii) about 0.1-100 mg/kg fluids; (iii) about 0.1-100 mg/kg fluids; (iv) about 1- 100 mg per kg of fluids; (v) about 0.1-100 mg/kg fluids; and (vi) about 0.1-100 mg/kg fluids, respectively.

The activities of representative compounds of this invention against the three diverse types of human cancer cells described above are particularly noteworthy. Cancer is in fact a broad classification containing at least 110 clinically different types of neoplastic diseases. The activities of compounds of this invention against three very different types of cancer cells demonstrate not only their utilities against specific human cancer cells but also the significant breadth for the anti-cancer effects of this invention beyond a single class of cancer disease, indicative of additional anti-cancer utilities.

The anti-viral activities of compounds of this invention are also of great importance.

For example, the tests demonstrating the anti-HIV properties of these compounds [see Example 23] were carried out in widely validated and accepted cellular assays of HIV infectivity in human cells that are indicative of in vivo activity. Thus the anti-HIV properties of the compounds of this invention relate directly to the in vivo activities of standard anti-HIV reverse transcription inhibitors such as azidothymidine, dideoxyinosine, dideoxycytidine and non-nucleoside reverse transcription inhibitors, HIV-protease inhibitors and inhibitors of tat-gene function, which are fully active in the anti-HIV assay by which the compounds of this invention were tested. This is in contrast to many in vitro HIV-related assays that are unable to provide predictive information about the anti-HIV effects of test compounds in living cells, such as assays of inhibition against isolated HIV enzymes. For example, the inadequacy of isolated enzyme assays was indicated by the finding that, of two compounds able to inhibit the HIV protease when assayed on the purified protease enzyme, only one of the compounds was able to inhibit HIV infectivity in the whole-cell assay in human lymphocytes [Antonucci, T.K. et al., "Characterizations of HIV-1 protease inhibitors" in Innovations in therapy of human viral diseases: a Wellcome Symposium, December 6-9, 1992, Book of Abstracts, Page 2.]

The compounds of this invention also show selective effects as antagonists for protein kinase C in some cases, as noninflammatory agonists for protein kinase C in other cases, and as selective ligands for protein kinase C and/or for phorboid receptors.

Thus, compounds of the present invention can be used as agents for the abrogation of pathophysiological conditions and disease states such as inflammation, psoriasis, cancer, ulcer, hypertension, asthma, arthritis, autoimmune, nociperception, secretory disease, parasitic infections, amoebic infections, viral disease including HIV disease, Alzheimer's disease, multiple sclerosis, in prophylaxis against infection by any HIV form, and any other application in which pathological activity or pathological absence of activity of protein kinase C and/or other proteins bearing diacylglycerol-type binding sites is found. The compounds of the present invention may also be used as agents for the induction of cardiac pre-conditioning effects.

As an example of the numerous diseases for which extensive literature data are available to demonstrate a role of PKC as a therapeutic target, the connection between PKC and anti-viral indications provides a general model of such analysis. Evidence for involvement of protein kinase C in the physiology of many human and animal pathogenic viruses has long been known, particularly from experiments in which a standard protein kinase C activator such as a phorbol ester greatly stimulates viral production for many different kinds of viruses [see, for example: zur Hausen, H. et al., "Persisting oncogenic herpesvirus induced by the tumour promoter TPA", Nature 272: 373-375 (1978); zur Hausen, H. et al., "Tumor initiators and promoters in the induction of Epstein-Barr virus", Proc. Natl. Acad Sci. USA 76: 782-785 (1979); Ablashi, D.V. et al., "Increased infectivity of oncogenic herpes viruses of primates with tumor promoter 12-O-tetradecanoylphorbol- 13-acetate", Proc. Soc. Exp. Biol. Med.164: 485-490 (1980); Colletta, G. et al.,

"Enhancement of viral gene expression in Friend erythroleukemia cells by 12-O- tetradecanoylphorbol-13-acetate", Cancer Research 40: 3369-3373 (1980); Arya, S.K., "Phorbol ester-mediated stimulation of the synthesis of mouse mammary tumour virus", Nature 284: 71-72 (1980); Hellman, K.B. and Hellman, A., "Induction of type-C retrovirus by the tumor promoter TPA", Int. J. Cancer 27: 95-99 (1981); Amtmann,E. and Sauer, G., "Activation of non-expressed bovine papilloma virus genomes by tumour promoters", Nature 296: 675-677 (1982); Kucera, L.S. et al., "12-O-Tetradecanoyl-phorbol-13-acetate enhancement of the tumorigenic potential of herpes simplex virus type 2 transformed cells", Oncology 40: 357-362 (1983); and Wunderlich, V. et al., "Enhancement of primate retrovirus synthesis of tumor promoters", Symposium on role of cocarcinogens and promoters in human and experimental carcinogenesis, 16-18 May, 1983, Budapest, Hungary, Book of Abstracts, p. 88].

Similar experiments indicate involvement of protein kinase C in the life cycle of HIV, and initial molecular genetics studies helped illuminate the mechanisms by which cellular protein kinase C can influence HIV [see, for example: Harada, S. et al., "Tumor promoter, TPA, enhances replication of HTLV-III/LAV", Virology 154: 249-258 (1986); Dinter, H. et al., "In vitro activation of the HIV-1 enhancer in extracts from cells treated with a phorbol ester tumor promoter", EMBO Journal 6: 4067-4071 (1987); Kaufman, J.D. et al., "Phorbol ester enhances human immunodeficiency virus-promoted gene expression and acts on a repeated 10-base-pair functional enhancer element", Mol. Cell. Biol. 7: 3759- 3766 (1987); and Siekevitz, M. et al., "Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I", Science 238: 1575-1578 (1987)]. These and later molecular genetics-based virological investigations provided clear mechanistic explanations for the effects, observed much earlier, of protein kinase C modulators on the life cycles of numerous human and animal viruses. Such studies showed that many viruses contain genetic control elements, called enhancers, whose functions in controlling viral expression involve the protein kinase C of the host cell. Of particular importance for the role of protein kinase C in virus-cell interactions are the enhancers known as AP-1 and NF-κB [see, for example: Marich, J.E. et al., "The phylogenetic relationship and complete nucleotide sequence of human papillomavirus Type 35", Virology 186: 770-776 (1992); Smith, R.L. et al., "Activation of second-messenger pathways rectivates latent Herpes Simplex virus in neuronal cultures", Virology 188: 311-318 (1992); Gdovin, S.L. and Clements, J.E. "Molecular mechanisms of visna virus tat: identification of the targets for transcriptional activation and evidence for a post-transcriptional effect", Virology 188: 438-450 (1992); Shih, D.S. et al., "Involvement of FOS and JUN in the activation of visna virus gene expression in macrophages through an AP-1 site in the viral LTR", Virology 190: 84-91 (1992); Mirza, A., "Stimulation of adenovirus early gene expression by phorbol ester: its possible mechanism", Virology 190: 645-653 (1992); Wade, E.J. et al., "An AP-1 binding site is the predominant cis-acting regulatory element in the 1.2-kilobase early RNA promoter of human cytomegalovirus", J. Virology 66: 2407- 2417 (1992); Stubenrauch, F. et al., "Late promoter of human papillomavirus Type 8 and its regulation", J. Virology 66: 3485-3493 (1992); Thierry, F. et al., "Two AP-1 sites binding JunB are essential for human papillomavirus Type 18 transcription in

keratinocytes", J. Virology 66: 3740-3748 (1992); Liu, J. et al., "Specific NF-κB subunits act in concert with tat to stimulate human immunodeficiency virus Type 1 transcription", J. Virology 66: 3883-3887 (1992); Jensen, W.A. et al., "Inhibition of protein kinase C results in decreased expression of bovine leukemia virus", J. Virology 66: 4427-4433 (1992); Shiroki, K. et al., "Adenovirus El A proteins stimulate inositol phospholipid metabolism in PC12 cells", J. Virology 66: 6093-6098 (1992); Cross, J.C. et al., "Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine-threonine kinases", Proc. Natl. Acad. Sci. USA 90: 8078-8082 (1993); and Kekule, A.S. et al., "Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway", Nature 361: 742-745 (1993)].

The compounds of this invention can also be used in combination with other therapeutic agents, for example for use in the treatment of viral infections. Thus, a compound of this invention can be used in combination with a nucleoside analog such as azidothymidine or dideoxyinosine, a tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)- one derivative, other HIV reverse transcriptase inhibitors, HIV protease inhibitors, or HIV tat-gene function inhibitors for the prophylaxis against or treatment of HIV infections. A method for treating a mammal infected with a virus comprises administering to a mammal in need of such treatment an antivirally effective quantity of a composition comprising an acceptable pharmaceutical carrier and an antivirally active compound or compounds or a pharmaceutically acceptable salt thereof.

In a further aspect, the present invention provides the use of a compound, novel or known, of the formulae I₀-D, I, I_R, I_P, I_PP, P-G, P₀-S₀-E₀, P₇-S_x-E₁, P_7B-S_x-E₁ or

P_7BL-S_x-E₁ for the manufacture of a medicament for medical use, e.g., treating

inflammatory conditions, viral infections and central nervous system disorders such as Alzheimer's disease.

Also provided is a pharmaceutical composition containing a compound of the formulae I₀-D, I, I_R, I_P, I_PP, P-G, P₀-S₀-E₀, P₇-S_x-E₁, P_7B-S_x-E₁ or P_7BL-S_x-E₁ as defined and a pharmaceutically acceptable carrier.

Furthermore, the compounds of this invention may be used to achieve desired physiological results such as interferon release, interleukin induction, tumor necrosis factor production, immune system stimulation and/or reconstitution, insulin secretion,

insulinomimetic activity, acceleration of wound healing, improvement in central nervous system functions such as memory and learning and abrogation of the symptoms or progress of Alzheimer's disease, and any other application for which desirable actions of protein kinase C are found.

As phorboid receptor subtype- and/or protein kinase C subtype-selective ligands, the compounds of this invention also have very valuable application as experimental agents for research into the role of protein kinase C and/or phorboid receptors in important biological processes and in human and veterinary diseases. Thus, their value extends to their use as pharmacological tools for in vitro and in vivo research, in a manner similar to the important roles that selective agonists and antagonists have played in the studies of the mechanism of action of adrenergic, dopaminergic, opiate, benzodiazepine, cholinergic, and serotoninergic receptor systems, among others.

In addition, the compounds can be used in in vitro diagnostics (e.g., in an assay for protein kinase C). They are also useful as intermediates in the production of other drugs, e.g., as described in the present invention and/or related inventions.

The compounds of this invention are generally administered to animals, including but not limited to fish, avians, and mammals including humans.

The pharmacologically active compounds of this invention can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans.

The compounds of this invention can be employed in admixture with conventional excipients and carriers, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcoholics, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., enzyme inhibitors, to reduce metabolic degradation.

For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. Ampoules are convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules. A syrup, elixir, or the like can be used wherein a sweetened vehicle is employed.

A preferred method of administration comprises oral dosing, with tablets, dragees, 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.

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.

Another preferred route of administration comprises topical application, for which are employed nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity compatible with topical application, preferably greater than water. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a freon.

The compounds of this invention, admixed with appropriate carriers, may also be delivered to subjects by means of an externally connected or internally implanted pumping device to give a controlled and/or sustained release of the therapeutic mixture, or by means of a patch of natural or synthetic fabric and/or polymer impregnated with the compounds in a suitable carrier and affixed to the skin to achieve transdermal release and absorption of the active compounds.

The compounds of this invention may also be modified by covalent attachment of metabolically modifiable groups, to form "prodrugs" which are released by cleavage in vivo of the metabolically removable groups. For example, amine, hydroxy and/or thiol groups present in many compounds of this invention may 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, may be converted to prodrugs by formation of their esters or amides by covalent attachment of alcohols, amines, amino acids and the like. Compounds of this invention may also incorporate N-alkyldihydropyridine functional groups, which become localized to the central nervous system after administration to the subject and subsequent metabolic modification of the N-alkyldihydropyridine group in the central nervous system.

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 acceptable steric hindrance, to permit access of metabolizing enzymes, other bioreactants or water.

It will be appreciated that starting materials for obtaining compounds of the diterpene portion of this invention from natural sources or from total or partial synthesis may be altered in very diverse ways, consistent with this invention, to obtain compounds with novel and diverse primary biological/medicinal activities resulting from, and controlled by, the polar group attached to carbon 13; such properties include, for example, loss of skin inflammatory activity and appearance or retention of anti-inflammatory, anti-HIV, anti- Alzheimer's disease, anti-leukemic and cytokine-induction activities. The introduction of an extremely wide variety of changes into the diterpene parent structure and its substituents in the present invention, especially the substituent at carbon 12, to obtain new entities with improved secondary properties, such as, variously, hydrophobicity, water solubility, high potency, oral availability, metabolic and chemical stability or lability as desired, reduced therapeutic side effects, and so on, is routinely accomplished using strategies and techniques widely recognized and used by workers of ordinary skill in the art of medicinal chemistry and pharmacology.

Starting materials for the synthesis of the compounds of this portion of the invention may be obtained from any of a wide variety of natural sources and by total synthesis.

Notable among the abundantly available natural starting materials is phorbol which has long been known in the literature and art of phorboid chemistry [see e.g. Hecker, E. and

Schmidt, R., Fortschritte der Chemie Organischer Naturstoffe 31: 377-467 (1974) and Naturally Occurring Phorbol Esters, ed. F.J. Evans, CRC Press, Boca Raton (1986), chapters 7, 8 and 9 and references cited therein]. Other starting materials may be readily prepared from the naturally occurring materials by well known synthetic steps. Among these materials, bisdehydrophorbol, crotophorbolone and 4,9,20-trihydroxy-15,16,17- trinortigliadien-(1,6)-dione-(3,13), among others, are readily prepared from phorbol

[Thielmann, H.-W. and Hecker, E., LiebigsAnn. Chem. 728: 158-183 (1969); Gschwendt, M. and Hecker, E., Z. Naturforsch. 24b: 80-91 (1969)].

Furthermore, the diterpene phorboids of this invention are available by total synthesis from common organic chemical starting materials. These syntheses provide a variety of approaches and associated flexibility in arriving at widely diverse functionalities on the parent nucleus [see Paquette, L. et al., J. Am. Chem. Soc. 106: 1446-1454 (1984); Rigby, J. and Moore, T., J. Org. Chem. 55: 2959-2962 (1990); Wender, P. et al., J. Am. Chem. Soc. 111: 8954-8957 (1989); Wender, P. et al., J. Am. Chem. Soc. 111: 8957-8958 (1989); and Wender, P. and McDonald, F., J. Am. Chem. Soc. 112: 4956-4958 (1990)].

The means for modifying the diterpenoid phorboid starting materials to produce the compounds of this invention will be obvious to workers with ordinary skill in synthetic organic chemistry. Using naturally occurring parent compounds, routine synthetic routes, transformations and procedures common in the art of synthetic organic chemistry and extensively exemplified in, for example, Hecker and Schmidt, op.cit. and in Thielmann and Hecker, op.cit, are sufficient for highly varied and extensive practice of this invention. Compounds of this invention may be obtained by semisynthetic procedures, starting from any of a variety of compounds from naturally occurring sources, using very routine synthetic strategies to protect sensitive hydroxy and keto groups at, for example, the 3, 4, 9 and/or 20 positions of the diterpenes (phorbol numbering) during subsequent steps aimed at modifying the C-ring.

Frequently one or more oxygen atoms must be blocked before some types of chemical modifications may be accomplished on the other portions of the diterpene parents available easily from natural sources. Many widely used and thoroughly characterized protecting groups for the oxygen atoms present as hydroxy groups are acyl, benzyl, trialkylsilane, benzyloxycarbonyl, 4'-methoxyphenyldiphenylmethyl and

trimethylsilylyethoxycarbonyl, which are variously stable to or removed under acidic, basic or reducing conditions or with fluoride ion reagents. Carbonyl functions may be protected by conversion to acetals or ketals, or by reduction to the alcohol level followed by protection with standard protecting groups for the hydroxy group.

C-ring modifications in suitably protected crotophorbolone and bisdehydrophorbol compounds, for example, can include a wide range of very routine manipulations of keto and hydroxy groups, including oxidation, reduction, conversion to a thiono or amino group, alkylation, esterification, etherification, formation of carbamates, carbonates, iminoesters, thioureas, etc.

Standard techniques for removal of protecting groups then provide any of a very diverse selection of agents having the particular features taught in this invention, particularly with respect to the substitution patterns on carbons 12 and 13. The use and removal of such groups is obvious and accessible to any worker with modest skill in the art of synthetic organic chemistry without undue experimentation.

Protection of reactive functional groups such as hydroxy and ketone moieties in regions other than the 12 and 13 positions in the diterpene series is accomplished using routine procedures in protecting group chemistry. The 20-hydroxyl is protected with silyl, trityl, methoxytrityl groups, etc. The 4-hydroxyl is protected with small silyl or acyl groups, or, in combination with a 3β-hydroxy, ketal-type protecting groups such as an acetonide, phenyl boronate, etc. The latter method also protects a 3-keto group, which, after synthetic manipulations in the 12 and 13 positions, can be regenerated by routine oxidation following acetonide hydrolysis, all under mild conditions. The 13-hydroxyl group of standard diterpenes can be protected with silyl, acyl or acetal-type groups. Such protected intermediates as described above are then derivatized at 12- and/or 13- hydroxyls by standard chemical methods, such as reaction with isocyanates, isothiocyanates, chloro- or fluoroformates and the like. A protected phorbol starting material with a free 12-hydroxyl and protected 13-hydroxyl may be oxidized to a 12-ketone, followed by further

functionalization or reduction to a 12-α-hydroxyl group, thus providing entry into 12-α diterpenoids. Suitably protected diterpenoid starting materials of the

crotophorbolone/bisdehydrophorbol class are similarly derivatized at hydroxyl in the 12/13 region. Such starting materials are also converted to novel compounds via manipulation of e.g. a ketonic oxygen group at position 13, by such routine synthetic procedures as reduction to hydroxy, conversion to a thione group, reductive amination, alkylation of enols and enolates, etc.

As an illustration, treatment of 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol 13-methoxyacetate, in which the 13- and 20-hydroxyls are selectively protected, with octadecylisocyanate in the presence of 4-dimethylaminopyridine and dibutyltin dilaurate affords 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol 12-(N-octadecyl)carbamate 13-methoxyacetate. The protecting groups at 13 and 20 may be sequentially removed by treatment with sodium carbonate or sodium methoxide followed by treatment with alcoholic trifluoroacetic acid to afford phorbol 12-(N-octadecyι)carbamate. The partially deprotected intermediate, 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol

12-(N-octadecyl)carbamate, may be treated with other reagents such as

trimethylsilylisocyanate to afford, after removal of the protecting group at position 20, another compound of the present invention, phorbol 12-(N-octadecyl)carbamate 13- carbamate.

As a further illustration, 20-O-[diphenyl(4'-methoxyphenyl)methyl]-

13-O-isopropyldimethylsilylphorbol may be reduced by treatment with sodium borohydride in the presence of cerium(III) chloride and the resulting alcohol treated with

dimethoxypropane to afford, 3-deoxo-3-hydroxy-20-O-[diphenyl(4'-methoxyphenyl)- methyl]-13-O-isopropyldimethylsilylphorbol-3,4-acetonide. Treatment of that compound with one equivalent of a base, such as sodium hydride, and an alkylating agent, such as decyl iodide or decyl nonaflate, affords, after deprotection with alcoholic trifluoroacetic acid, 12-O-decyl-3-deoxo-3-hydroxyphorbol, a compound of the present invention.

Reprotection of the 13- and 20-hydroxyls and oxidation of the 3-hydroxyl by any of several well known methods affords 12-O-decylphorbol. In an alternative route, 20-O-[diphenyl- (4'-methoxyphenyl)methyl]-13-O-isopropyldimethylsilylphorbol, may be acylated with methoxyacetic anhydride to afford 20-O-[diphenyl(4'-methoxyphenyl)methyl]- 13-O-isopropyldimethylsilylphorbol 12-methoxyacetate. Silylation of this material with trifluoromethyltriflate in the presence of base and deprotection with a trace of methanolic sodium methoxide affords 4-O-trimethylsilyl-20-O-[diphenyl(4'-methoxyphenyl)methyl]- 13-O-isopropyldimethylsilylphorbol. Treatment of the last compound with one equivalent of a base, such as sodium hydride, and an alkylating agent, such as decyl iodide or decyl nonaflate, affords, after deprotection with tetra-n-butylammonium fluoride followed by alcoholic trifluoroacetic acid, 12-O-decylphorbol.

The use of the methods of total synthesis as described in the literature cited above permits specific modifications of the parent structures of the diterpenoids. By established techniques in the art of organic synthesis modified parent structures may be obtained which embody alterations at any of the numerous permitted substituent locations and which have useful biological activity as taught by the present invention. This wide variety of modified diterpenoid structures may result from the use of modified starting materials, from modifications of one or more synthetic steps or from a combination of both, as applied to the examples in the synthetic organic and natural product literature cited above by workers of ordinary skill in synthetic organic chemistry.

It will be appreciated that starting materials for obtaining compounds of the benzolactam portion of this invention from synthesis may be altered in very diverse ways, consistent with this invention, to obtain compounds with novel and diverse primary biological/medicinal activities resulting from, and controlled by, the replacements for the hydroxymethyl/1-hydroxyethyl; such properties include, for example, loss of skin inflammatory activity and appearance or retention of anti-inflammatory, anti-HIV, anti- Alzheimer's disease, anti-leukemic and cytokine-induction activities. The introduction of an extremely wide variety of changes into the benzolactam parent structure and its substituents permitted in the present invention to obtain new entities with improved secondary properties, such as, variously, hydrophobicity, water solubility, high potency, oral availability, metabolic and chemical stability or lability as desired, reduced therapeutic side effects, and so on, is routinely accomplished using strategies and techniques widely recognized and used by workers of ordinary skill in the art of medicinal chemistry and pharmacology.

Compounds of this invention from the benzolactam (P₇) class of phorboids are readily available by total synthesis from common organic chemical starting materials. These syntheses provide flexibility in arriving at diverse functionalities on the parent nucleus and on the S₀E₀, or S_xE₁ side chain. Modifications of the parent structure may also be made by the application of well known organic synthetic procedures to the benzolactam as well as on precursor molecules. [See Kozikowski, A.P. et al. "Synthesis, molecular modeling, 2-D NMR, and biological evaluation of ILV mimics as potential modulators of protein kinase C", J. Am. Chem. Soc. 115: 3957-3965 (1993) and Ohno, M. et al. "Designed molecules. Reproducing the two conformations of teleocidins", Tetrahedron Lett. 34: 8119-8122 (1993).]

Benzolactams having one or two nitrogens incorporated into the benzenoid portion of the basic fused bicyclic system are easily derived via total syntheses using starting benzenoid compounds having one or two nitrogen atoms already present in the aromatic nucleus. Chemical manipulations of this type have been extensively developed in the routine practice of heterocyclic chemistry, for example in the field of nucleoside and nucleoside analog synthesis, and permit a wide variety of nitrogen heterocycles to be synthesized by routes of inherently high predictability. The ready adaptation of such approaches are obvious to synthetic chemical practitioners of ordinary skill in the pharmaceutical industry.

The means for modifying the hydroxymethyl group of benzolactam phorboids to produce the compounds of this invention will be obvious to workers with ordinary skill in synthetic organic chemistry.

The limited reactivity of the other portions of the simplest benzolactam parents enhances the ability to achieve selective modification of the hydroxymethyl group with or without the need for protecting groups. For example, the hydroxymethyl group may be conveniently capped under very mild conditions by the treatment with a substituted or unsubstituted alkyl, aryl, or aralkyl isocyanate, optionally containing silicon or phosphorus atoms in a variety of functional groups, in the presence of a catalyst such as dibutyltin dilaurate or activated and displaced with a wide variety of nucleophiles. The resulting compounds lack the toxic inflammatory activity of the phorboid from which they were derived, and have themselves anti-inflammatory utility. As an illustration, treatment of (-)- BL-V8-310 with methylisocyanate in the presence of dibutyltin dilaurate and

4-dimethylaminopyridine in tetrahydrofuran affords 11-O-(N-methyl)carbamoyl-(2S,5S)- BL-V8-310.

Conversion of the hydroxy group to a halogen or pseudohalogen not only in itself provides active and useful compounds, but also permits the displacement of the resultant electrophile from the protected or in some cases, unprotected, parent nucleus by a very wide range of nucleophiles. Persons with ordinary skill in the art of organic synthesis will recognize that such nucleophiles may include, without limitation, reagents having carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, arsenic, boron, and/or selenium atoms in their structures. Particular examples would be reaction with ammonia,

methylamine, the sodium salt of dimethylphosphine, trimethylphosphite, triphenylphosphine, potassium sulfite, trimethylsilylmethyl-metal salts, lithium trimethylsilylacetylide, sodium cyanide, N-methyl-2-hydroxyethyl amine, 1[H]-tetrazole, or with the sodium salt of 2-mercaptoethanol, 3-mercaptoethanol, or of hydroxymethylphenol. Many variations may be executed as described in standard textbooks of synthetic organic chemistry, such as J. March, Advanced Organic Chemistry, Third Edition, Wiley-Interscience, New York, 1985. As an illustration, 11-O-methanesulfonyl-(2S,5S)-BL-V8-310 yields 11-deoxy-(2S,5S)-BL- V8-310 11-sulfonic acid upon treatment with sodium sulfite. As a further illustration, treatment of 11-O-methansulfbnyl-(2R,5S)-BL-V8-310 with the sodium salt of 4-t- butyldimethylsilyloxyphenol followed by treatment of the product with tetrabutylammonium fluoride in tetrahydrofuran affords 11-O-(4'-hydroxyphenyl)-(2R,2S)-BL-V8-310.

The hydroxy group of the hydroxymethyl of benzolactams may be replaced by a metal and then reacted with an electrophile, effectively replacing the hydroxy with a group derived from the electrophile. The techniques for this replacement are obvious to workers with ordinary skill in organic synthesis, in that replacement of the hydroxymethyl hydroxy group by halogen in a suitably protected or modified benzolactam permits strong and/or hard nucleophiles to be generated by the use of metals or strong bases, and persons with ordinary skill in the art of organic chemistry will recognize that such nucleophiles can be contacted with a very diverse range of electrophilic reagents having carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, arsenic, boron and/or selenium atoms in their structures to obtain hydroxymethyl-modified benzolactams. As one approach among many, the 14-O-methanesulfonyl derivative of an appropriate benzolactam may be converted to an iodo compound with sodium iodide in acetone. The resulting iodide may be modified by halogen-metal exchange using appropriately active metals or metal-containing reagents such as magnesium, zinc, alkali metals, metal alkyl reagents and so on, to obtain carbanionic character at the carbon atom previously bearing the hydroxy group, as shown: ^*

wherein Met is the metal ion, and PG₇ may be unnecessary (i.e. may be hydrogen or an anionic charge), may be benzyl (removable by hydrogenolysis) or may be a stable group, such as methyl or ethyl, intended to remain in the final bioactive synthetic product. The simultaneous presence or subsequent addition to the reaction of an electrophile such as, without limitation, an aldehyde, ketone, epoxide or oxetane then provides, after reaction and workup, compounds having one or more methylenes inserted between the original hydroxy group and the methylene to which it was attached. This methodology is particularly advantageous for replacement of the hydroxy with a group containing silicon, phosphorus or other atoms by contacting the anionic metal derivative of the parent nucleus with electrophilic reagents having halogen or pseudohalogen groups, in addition to other functions chosen for biological specificity, bonded directly to the silicon or phosphorus atoms, affording compounds with useful biological activity.

Alternatively, the hydroxy group on the hydroxymethyl of benzolactams may be oxidized to an aldehyde and then reacted via condensation or addition chemistries to provide a very wide variety of modified benzolactams. Examples, without limitation, would be reactions with Wittig reagents, hydroxylamines, or Grignard reagents. Many variations may be executed as described in standard textbooks of synthetic organic chemistry, such as J. March, op cit. As an illustration, the aldehyde, 11-deoxy-11-oxo-epi-BL-V9-310, may be prepared by oxidation of the parent hydroxymethyl compound with periodinane among other oxidizing agents. This aldehyde may be treated with methyl

triphenylphosphoranylideneacetate in toluene to afford 5-deshydroxymethyl-5-[2'- (methoxycarbonyl)-(E)-vinyl]-epi-BL-V9-310.

These aldehydes may also be obtained by reduction of appropriate carboxylic acid derivatives by application of well-known techniques. These carboxylic acid derivatives may be prepared by modification of the published synthetic routes, which modifications are well understood by those with skill in the art of organic synthesis. The carboxylic acid derivatives of the benzolactam phorboids may also be prepared by oxidation of the hydroxymethyl group of suitable benzolactam phorboids or of aldehydic derivatives of benzolactams by methods well-known in the art.

Besides being useful examples of this invention themselves, these acids permit the preparation of further modified benzolactams. For example, this carboxylic group may be activated for condensation reactions by any of a number of ordinary and well-known methods, e.g. by conversion to an acyl halide or to an active ester such as the

N-succinimidyl ester. The resultant activated carboxyl may then be easily converted to simple or multifunctional ester, amide, or thioester derivatives by reaction with alcohols, amines, or thiols respectively, alone or in the presence of condensation catalysts.

The total syntheses described in the literature cited above are also amenable to extensive adaptations so as to provide a wide variety of modifications in the parent structures of the benzolactam group. By established techniques in the art of organic synthesis modified parent structures may be obtained which embody alterations at the hydroxymethyl group and which have useful biological activity. This extremely wide variety of modified benzolactam structures may result from the use of modified starting materials, from modifications of one or more synthetic steps or from a combination of both.

Beyond the extensive changes in the hydroxymethyl group of benzolactam and related P₇-type parent phorboids described above, the present invention also discloses broad and diverse alterations which are accommodated in the non-hydroxymethyl regions of the parent P₇-type phorboids. Such modifications of the benzolactam parents can be carried out before, after or in alternating fashion with respect to construction of the hydroxymethyl modifications, depending on obvious considerations of chemical stability of the various functional groups in intermediates being subjected to chemical modifications.

It is obvious to one skilled in the art that many modified benzolactam parents may be obtained by carrying a modification through the de novo synthesis as described in the literature cited above. Specifically modified parents of the benzolactam class may also be further modified at positions other than the hydroxymethyl group either before or after the modifications of hydroxymethyl group to produce embodiments of this invention. The means for accomplishing these modifications are obvious to workers with ordinary skill in organic synthesis.

This invention is illustrated further by the following examples.

EXAMPLE 1

Phorbol 12-[4'-(9",10"-Dihydrophenanthrene-2")butyrate]

A solution of 2.95 g of 4-(9',10'-dihydrophenanthrene-2')butyric acid and 1.13 g of dicyclohexylcarbodiimide in 20 mL of tetrahydrofuran was warmed briefly with a hot air gun and then stirred for 5 min at room temperature. The mixture was then filtered and concentrated in vacuo to obtain 2.79 g of the 4-(9',10'-dihydrophenanthrene-2')butyric anhydride. This anhydride was mixed with 1.6 g of 20-O-[diphenyl(4'-methoxyphenyl)- methyl]-13-O-isopropyldimethylsilylphorbol and then 15 mL of pyridine was added. After 18 h of stirring under nitrogen 100 mg of 4-dimethylaminopyridine was added followed 7 h later with 400 mg of dicyclohexylcarbodiimide. After another 22 h at room temperature approximately 10 mL of methanol was added followed by the concentration of the mixture in vacuo. The residue was partitioned between ethyl acetate and phosphate buffer (pH 2) and the organic layer washed with phosphate buffer (pH 8), filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and again concentrated in vacuo. Purification by preparative liquid chromatography [silica; hexane/ethyl acetate (75:25)] afforded slightly impure 20-O-[diphenyl(4'-methoxyphenyl)methyl]- 13-O-isopropyldimethylsilylphorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate].

The entire sample of the above compound was dissolved in 35 mL of t-butylmethyl ether and treated with 150 mL of 90% acetic acid in water. After 70 min the mixture was concentrated in vacuo and partitioned between ethyl acetate and phosphate buffer (pH 8) followed by concentration of the organic layer in vacuo. Preparative liquid chromatography [silica; methylene chloride/methanol (97:3)] afforded 890 mg of phorbol 12-[4'-(9",10"- dihydrophenanthrene-2")butyrate]. EXAMPLE 2

20-O-[Diphenyl(4'-methoxyphenyl)methyl]phorbol 12-[3'.5'-Bis(trifluoromethyl)phenylcarbamate] 13-Methoxyacetate To a solution of 396 mg of 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol

13-methoxyacetate, 70 mg of 4-dimethylaminopyridine and 72 mg of dibutyltin dilaurate in 5 mL of tetrahydrofuran was added 220 mg of 3,5-bis(trifluoromethyl)phenyl isocyanate in 1 mL tetrahydrofuran. After 7 h another 100 mg of the isocyanate was added. After stirring for another 12 h at room temperature the reaction mixture was partitioned between ethyl acetate/hexane and water. The organic layer was filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo to afford 872 mg of crude 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol

12-[3',5'-bis(trifluoromethyl)phenylcarbamate] 13-methoxyacetate.

EXAMPLE 3

In a manner similar to the methods of Example 2 the following compounds are prepared:

(i) 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol 12-n-octadecylcarbamate

13-methoxyacetate; and

(ii) 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol

12-(pentafluorophenyl)carbamate 13-methoxyacetate.

EXAMPLE 4

Phorbol 12-[3',5'-Bis(trifluoromethyl)phenylcarbamate]

To a solution of 174 mg of 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol 12-[3',5'- bis(trifluoromethyι)phenylcarbamate] 13-methoxyacetate in 5 mL of methanol was added approximately 20 mg of sodium carbonate. After 4.5 h the mixture was partitioned between water and ethyl acetate. The organic layer was filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo. The resulting residue was dissolved in 2.7 mL dry methylene chloride and 0.7 mL of absolute ethanol and treated with o.05 mL of trifluoroacetic acid. After 1.5 h the mixture was partitioned between ethyl acetate and phosphate buffer (pH 8). The organic layer was filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo. Preparative liquid chromatography [silica; hexane/ethyl acetate (50:50)] afforded 27 mg of phorbol 12-[3',5'-bis(trifluoromethyl)phenylcarbamate]. The structure was confirmed by NMR and high resolution mass spectral analysis.

EXAMPLE 5

In a manner similar to the methods of Examples 2-4 the following compounds are prepared:

(i) phorbol 12-(N-octadecyl)carbamate;

(ii) phorbol 12-[N-(4'-propylphenyl)]carbamate;

(iii) phorbol 12-[N-(3'-phenylpropyl)]carbamate;

(iv) phorbol 12-[N-(cyclopropylmethyl)]carbamate;

(v) phorbol 12-(N-pentafluorophenyl)carbamate; and

(vi) phorbol 12-[N-(3',5'-dimethoxybenzyl)]carbamate.

EXAMPLE 6

20-O-[Diphenyl(4'-methoxyphenyl)methyl]phorbol 12-[(1'-Adamantyl)carbonate]

13-Methoxyacetate

To a solution of 750 mg of 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol

13-methoxyacetate, a few mg of 4-dimethylaminopyridine in about 5 mL of 2,6-lutidine was added 570 mg of 1-adamantyl fluoroformate. After about 12 hours at room temperature the reaction mixture was treated with methanol, diluted with ethyl acetate and washed with phosphate buffer (pH 2) until the wash was acidic. The organic layer was washed with phosphate buffer (pH 8), filtered through a sintered glass funnel containing silica, sodium suifate and sodium chloride and concentrated in vacuo. Preparative liquid chromatography [silica; hexane/ethyl acetate (75:25)] afforded 847 mg of 20-O-[diphenyl(4'- methoxyphenyl)methyl]phorbol 12-[(1'-adamantyl)carbonate] 13-methoxyacetate.

EXAMPLE 7

Phorbol 12-[(1'-Adamantyl)carbonate]

To a solution of 748 mg of 20-O-[diphenyl(4'-methoxyphenyl)methyl]phorbol

12-[(1'-adamantyl)carbonate] 13-methoxyacetate in 20 mL of methanol was added 1-5 mg of sodium methoxide in 10 μL of methanol. After 2 h the mixture was treated with 2 mL of trifluoroacetic acid in 5 mL of methylene chloride and 5 mL of methanol. After 1 h the mixture was partitioned between ethyl acetate and phosphate buffer (pH 8). The organic layer was washed with water, filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo. Preparative liquid chromatography [silica; hexane/ethyl acetate (10:90)] afforded 340 mg of phorbol

12-[(1'-adamantyl)carbonate].

EXAMPLE 8

In a manner similar to the methods of Examples 6 and 7 the following compounds are prepared:

(i) phorbol 12-n-octadecylcarbonate;

(ii) phorbol 12-(3'-sec-butylphenyι)carbonate;

(iii) phorbol 12-(4'-butoxyphenyι)carbonate;

(iv) phorbol 12-phenylcarbonate;

(v) phorbol 12-n-tetradecylcarbonate;

(vi) phorbol 12-(cyclohexylmethyl)carbonate;

(vii) phorbol 12-(2'-phenylethyl)carbonate; and

(viii) phorbol 12-(4'-phenoxypropyl)carbonate.

EXAMPLE 9

Bisdehydrophorbol 12-[3'.5'-Bis(trifluoromethyl)phenylcarbamate]

To a solution of 500 mg of 20-O-[diphenyl(4'-methoxyphenyl)methyl]bisdehydrophorbol, 98 mg of 4-dimethylaminopyridine and 106 mg of dibutyltin dilaurate in 6 mL of tetrahydrofuran was added 520 mg of 3,5-bis(trifluoromethyl)phenyl isocyanate in 1 mL tetrahydrofuran. After stirring for 2 h at room temperature the reaction mixture was partitioned between ethyl acetate/hexane and water. The organic layer was filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and

concentrated in vacuo to afford crude 20-O-[diphenyl(4'- methoxyphenyl)methyl]bisdehydrophorbol 12-[3',5'-bis(trifluoromethyl)phenylcarbamate]. To a solution of crude 20-O-[diphenyl(4'-methoxyphenyl)methyl]bis dehydrophorbol 12-[3',5'-bis(trifluoromethyl)phenylcarbamate] in 12 mL of methanol was added 2 mL of trifluoroacetic acid in 5 mL methylene chloride and 5 mL of methanol. After 1 h the mixture was partitioned between ethyl acetate and phosphate buffer (pH 8). The organic layer was filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo. Preparative liquid chromatography [silica; hexane/ethyl acetate (35:65)] afforded 168 mg of bisdehydrophorbol

12-[3',5'-bis(trifluoromethyl)phenylcarbamate].

EXAMPLE 10

In a manner similar to the methods of Example 9 the following compounds are prepared:

(i) bisdehydrophorbol 12-n-octadecylcarbamate;

(ii) bisdehydrophorbol 12-(pentafluorophenyl)carbamate; and

(iii) bisdehydrophorbol 12-(3',5'-dimethoxybenzyl)carbamate.

EXAMPLE 11

Bisdehydrophorbol 12-Myristate

To a solution of 500 mg of 20-O-[diphenyl(4'-methoxyphenyl)methyl]bisdehydrophorbol, about 20 mg of 4-dimethylaminopyridine and 3 mL of pyridine was added 1.5 mL of myristoyl fluoride. After 3 hours 330 mg of myristic anhydride was added. After another 16 hours at room temperature the reaction mixture was partitioned between ethyl acetate and phosphate buffer (pH 2). The organic layer was washed with phosphate buffer (pH 8), filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo to afford crude 20-O-[diphenyl(4'- methoxyphenyl)methyl]bisdehydrophorbol 12-myristate.

To a solution of crude 20-O-[diphenyl(4'-methoxyphenyl)methyl]bisdehydrophorbol 12-myristate in 10 mL of methanol containing a few mLs of methylene chloride was added 2 mL of trifluoroacetic acid in 5 mL of methylene chloride and 5 mL of methanol. After 1 h the mbcture was partitioned between ethyl acetate and phosphate buffer (pH 8). The organic layer was washed with water, filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo. Preparative liquid chromatography [silica; hexane/ethyl acetate (45:55)] afforded 293 mg of

bisdehydrophorbol 12-myristate.

EXAMPLE 12

In a manner similar to the methods of Example 11 the following compounds are prepared:

(i) bisdehydrophorbol 12-n-octylcarbonate;

(ii) bisdehydrophorbol 12-phenylcarbonate; and

(iii) bisdehydorphorbol 12-(1'-adamantyl)carbonate.

EXAMPLE 13

Phorbol 12-[4'-(9",10"-Dihydrophenanthrene-2")buryratel 3-Oxime

A mbcture of 50 mg of phorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate] and 153 mg of hydroxylamine hydrochloride in 2 mL of pyridine was heated at 55°C for 2.5 h. After cooling to room temperature the mbcture was partitioned between ethyl acetate and phosphate buffer (pH 2). The organic layer was washed with phosphate buffer (pH 8), filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo. Preparative liquid chromatography [silica; methylene chloride/ispopropyl alcohol (92:8)] afforded 41 mg of phorbol 12-[4'-(9",10"- dihydrophenanthrene-2")butyrate] 3-oxime.

EXAMPLE 14

3-Deoxo-3-hydroxyphorbol 12-[4'-(9",10"-Dihydrophenanthrene-2")butyrate]

To a mixture of 50 mg of phorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate] and 32 mg of cerium(III) chloride heptahydrate in 1.1 mL of methanol was added 6 mg of sodium borohydride. After about 6 minutes the mixture was partitioned between ethyl acetate and phosphate buffer (pH 2). The organic layer was washed with phosphate buffer (pH 8), dried over sodium sulfate and sodium chloride and concentrated in vacuo. Preparative liquid chromatography [silica; methylene chloride/isopropyl alcohol (95:5)] afforded 29 mg of 3-deoxo-3-hydroxyphorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate]. EXAMPLE 15

In a manner similar to the methods of Examples 1-14 the following compounds are prepared:

(i) phorbol 12-(N-octadecyl)thiocarbamate;

(ii) bisdehydrophorbol 12-(N-octadecyl)tbiocarbamate;

(iii) phorbol 12-[N-(pentafluorophenyl)]thiocarbamate;

(iv) bisdehydrophorbol 12-[N-(pentafluorophenyl)]thiocarbamate;

(v) phorbol 12-(N-decyl)carboxamidine;

(vi) phorbol 12-(N-phenyl)carboxamidine;

(vii) bisdehydrophorbol 12-(N-decyl)carboxamidine; and

(viii) bisdehydrophorbol 12-(N-phenyl)carboxamidine.

EXAMPLE 16

11-O-(N-Methyl)carbamoyl-(2S,5S)-BL-V8-310

To a solution of the 5 mg benzolactam (-)-BL-V8-310 [obtained by the method of Y. Endo et al., Bioorg. Med. Chem. Lett. 4: 491-494 (1994)], 1 mg of 4-dimethylaminopyridne and 1 mg of dibutyltin dilaurate in 100 μL of anhydrous butyl methyl ether was added 10-20 molar excess of methyl isocyanate. After several hours at room temperature the mbcture was partitioned between ethyl acetate and phosphate buffer (pH 2). The organic layer was washed with phosphate buffer (pH 8), dried over sodium sulfate and concentrated in vacuo. The residue was purified by thin layer chromatography [silica; methylene chloride/acetone (80:20)] affording 2.8 mg of 11-O-(N-methyl)carbamoyl-(2S,5S)-BL-V8-310.

EXAMPLE 17

11-Deoxy-(2S,5S)-BL-V8-310 11-Sulfonic Acid

A solution of (-)-BL-V8-310 and pyridine in anhydrous methylene chloride is cooled in a cold bath under a nitrogen atmosphere and is treated with methanesulfonyl chloride. After a period of time the mixture is warmed to room temperature and washed with brine, pH 2 phosphate buffer and brine sequentially. The organic layer is then dried and concentrated in vacuo to afford 11-O-methanesulfonyl-(2S,5S)-BL-V8-310. A mixture of 11-O-methanesulfonyl-(2S,5S)-BL-V8-310, sodium sulfite and sodium iodide in a 1:1 mixture of ethanol/water containing a small amount of acetic acid is stirred for a period of time at an elevated temperature. After that time the mixture is concentrated in vacuo and partitioned between ethyl acetate and brine to afford a residue which is purified by liquid chromatography to yield 11-deoxy-(2S,5S)-BL-V8-310 11-sulfonic acid.

EXAMPLE 18

In a manner similar to the methods of Examples 16-17 the following compounds are prepared:

(i) 11-O-(4'-hydroxyphenyl)-(2R,2S)-BL-V8-310;

(ii) 11-deoxy-11-(2'-hydroxyethylthio)-(2R,2S)-BL-V9-310;

(iii) 11-deoxy-11-(2'-hydroxyethylthio)-(2S,2S)-BL-V8-310;

(iv) 11-deoxy-11-(2'-carboxyethylthio)-(2S,2S)-BL-V8-310;

(v) 11-O-(N-ethyl)carbamoyl-(2S,5S)-BL-V8-310

(vi) 11-deoxy-11-cyano-(2S,5S)-BL-V8-310; and

(vii) 11-deoxy-11-azido-(2S,5S)-BL-V8-310.

EXAMPLE 19

11-Deoxy-11-oxo-BL-V8-310

To a solution of 35 mg of BL-V8-310 in 2 mL of methylene chloride was added a 10-fold molar excess of periodinane reagent [Dess, D.B. and Martin, J.C., J. Org. Chem. 48: 4155- 4156 (1983)]. After 3 h the mbcture was quenched with phosphate buffer (pH 8) and aqueous sodium thiosulfate (20%) and extracted with ethyl acetate. The organic layer was washed sequentially with phosphate buffer (pH 2) and phosphate buffer (pH 8), filtered through a sintered glass funnel containing silica, sodium sulfate and sodium chloride and concentrated in vacuo. The residue was purified by thin layer chromatography [silica; hexane/ethyl acetate] to afford 11-deoxy-11-oxo-BL-V8-310.

EXAMPLE 20

5-Des(hydroxymethyl)-5-carboxy-BL-V8-310

To a solution of 11-deoxy-11-oxo-BL-V8-310 in a mixture of methylene chloride, t-butyl alcohol and 2-methyl-2-butene is added a 10% (wt/vol) of sodium chlorite in a dihydrogen phosphate buffered solution. After a period of time 20% aqueous sodium thiosulfate is added and the mbcture is partially concentrated in vacuo. The residue is partitioned between ethyl acetate and phosphate buffer (pH 8). The organic layer is washed sequentially with pH 2 and pH 8 phosphate buffers and dried over sodium sulfate. After concentration in vacuo and purification by preparative liquid chromatography,

5-des(hydroxymethyl)-5-carboxy-BL-V8-310 is obtained.

EXAMPLE 21

11-Deoxy-11-[3'-(hydroxymethyl)phenylamino]-BL-V8-310 To a solution of 11-deoxy-11-oxo-BL-V8-310 in acetonitrile containing a little acetic acid is added a molar excess of 3-aminobenzyl alcohol. After a period of time sodium cyanoborohydride is added to this mixture. The mixture is partitioned between ethyl acetate and phosphate buffer (pH 2). The organic layer is washed with phosphate buffer (pH 8), dried over sodium sulfate and concentrated in vacuo. Chromatographic purification of the residue affords 11-doxy-11-[3'-(hydroxymethyl)phenylamino]-BL-V8-310.

EXAMPLE 22

Demonstration of Anti-inflammatory Activity

A stock solution of 300 pmoles of the standard inflammatory compound phorbol 12- myristate 13-acetate (PMA) per 5 μL acetone was prepared. This solution was used to prepare four-fold dilutions of the agent to be tested, covering concentrations of the latter typically selected from a range of about 4.0 to about 1,200,000 pmoles per 5 μL. These solutions containing both PMA and the test agent were used to demonstrate the anti- inflammatory activity of the test compound by application of 5 μL to the insides of the ears of mice (one ear per mouse), followed by the observation of ear inflammation/erythema at intervals from 1 to 48 hours after application.

In this manner, the anti-inflammatory activities of the following compounds are

demonstrated; lower doses produce shorter periods of inflammation/erythema and higher doses produce complete, inhibition during the entire assay period. (i) phorbol 12-[3',5'-bis(trifluoromethyl)phenylcarbamate];

(ii) phorbol 12-n-octadecylcarbamate;

(iii) phorbol 12-[(1'-adamantyl)carbonate];

(iv) bisdehydrophorbol 12-myristate;

(v) bisdehydrophorbol 12-[3',5'-bis(trifluoromethyl)phenylcarbamate];

(vi) 11-deoxy-(2S,5S)-BL-V8-310 11-sulfonic acid;

(vii) 11-O-(N-methyl)carbamoyl-(2S,5S)-BL-V8-310;

(viii) 11-deoxy-11-(2'-hydroxyethylthio)-(2S,2S)-BL-V8-310;

(ix) 11-deoxy-11-(2'-carboxyethylthio)-(2S,2S)-BL-V8-310;

(x) 11-deoxy-11-cyano-(2S,5S)-BL-V8-310;

(xi) 5-des(hydroxymethyl)-5-carboxy-BL- V8-310; and

(xϋ) 11-deoxy-11-[3'-(hydroxymethyl)phenylamino]-BL-V8-310.

EXAMPLE 23

Demonstration of Anti-HIV Activity

Human peripheral blood lymphocytes were isolated from the buffy coat fiactions of blood donations. The lymphocytes were then stimulated with 5 micrograms/ml of

phytohemagglutinin for 48 hours. Prior to infection with HIV, the lymphocytes were washed and resuspended in mitogen-free medium. On day 0 the cells were infected with HIV and were cultured for four days in the presence or absence of graded concentrations of the test agent. On days 3 and 4 the supernatant levels of total viral RNA and viral core protein p24 were determined at each drug concentration and dose-response curves were used to determine the concentration of drug giving 50% inhibition of production of viral RNA and of p24 core protein.

Using this method, the anti-HIV ED₅₀ value for RNA [drug concentration is in brackets] for the following compound was determined from dose-response curves or by estimation from one or more experimental drug concentrations:

(i) phorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate] [450 nM];

(ii) phorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate] 3-oxime [p24:

9.3% and 13.4% inhibition at 3 and 4 days, respectively; HIV-RNA: 17% and 9% inhibition at 3 and 4 days, respectively; both at 10 μM];

(iii) 3-deoxy-3-β-hydroxyphorbol 12-[4'-(9",10"-dihydrophenanthrene-

2")butyrate] [HIV-RNA: 17% inhibition at day 4, 10 μM]; and (iv) phorbol 12-[bis(3',5'-trifluoromethyl)phenyl]carbamate [HIV-RNA: 21%

inhibition; p24: 7.9% inhibition; both at day 4, 10 μM].

EXAMPLE 24

Demonstration of Anti-melanoma Activity

Human RPMI-7272 melanoma cells were grown in the standard culture medium under normal incubation conditions. On day 1 the cells were cultured in the absence (control) or presence of graded concentrations of the test agent in separate tubes. On day 4 after 72 h of exposure the number of cells in each tube was measured and the number of cell doublings determined. The drug treated tubes were compared to the control tube to arrive at the ID₅₀ (the concentration of drug required to inhibit cell doublings by 50%) for the test agent.

By this procedure the anti-melanoma activity of phorbol 12-[4'-(9",10"- dihydrophenanthrene-2")butyrate] was determined, showing an ID₅₀ of 12 μM.

EXAMPLE 25

Demonstration of Anti-leukemic Activity

HL-60 promyelocytic leukemia cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells (7,500) were seeded into 96-well microtiter plates and incubated overnight. Serial dilutions of the test agent (dissolved in DMSO and then diluted with culture medium) were added to the wells on day 1. The plates were incubated for 8 days to allow the control cultures to undergo at least 3 cell divisions. The cell growth was monitored by using the calorimetric MTT (tetrazolium) assay [Mosmann, T., J. Immunol. Meth. 65: 55-63 (1983)]. After the incubation period, the cells were washed with phosphate-buffered saline in the microtiter plate. DMSO was then added to each well and the dish was put on a shaker for 20 min. The optical density was measured at 540 nm and compared using the formula: (OD Test - OD Start)/(OD Control - OD Start) x 100. The IC₅₀ was defined as the dmg concentration which leads to 50% of cells per well compared to control cultures (100%) at the end of the incubation period.

By this method the anti-leukemic activities of the following other compounds were demonstrated:

(i) phorbol 12-(pentafluorophenyl)acetate (IC₅₀= 50.8 μM); and

(ii) phorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate] (IC₅₀= 1.8 μM).

EXAMPLE 26

Demonstration of Anti-cancer Activity

T-24 human bladder carcinoma cells were cultured in Eagle's minimal essential medium supplemented with 5% fetal bovine serum. Cells (1,000) were seeded into 96-well microtiter plates and incubated overnight. Serial dilutions of the test agent (dissolved in DMSO and then diluted with culture medium) were added to the wells on day 1. The plates were incubated for 5-6 days to allow the control cultures to undergo at least 3 cell divisions. After the incubation period, the cells were fixed with glutaraldehyde, washed with water and stained with 0.05% methylene blue. After washing the dye was eluted with 3% HCl. The optical density per well was measured at 665 nm and compared using the formula: (OD Test - OD Start)/(OD Control - OD Start) x 100. The IC₅₀ was defined as the drug concentration which leads to 50% of cells per well compared to control cultures (100%) at the end of the incubation period.

By this method the anti-cancer activity of the following compound was demonstrated:

(i) phorbol 12-[4'-(9",10"-dihydrophenanthrene-2")butyrate] (IC₅₀= 4.0 μM).

EXAMPLE 27

Topical Gel

An illustrative composition for a topical gel is the following:

bisdehydrophorbol 12-myristate 20 mg

hydroxypropylcellulose 60 mg

ethyl alcohol 920 mg

The materials are mixed, homogenized and filled into containers each holding 1 gram of gel.

Claims

CLAIMS What is claimed is:

1. A compound of the formula: I₀ - D, wherein I₀ represents a radical, formally derived from a phorbol- or daphnane-type diterpenoid parent compound, which compound:

a. binds reversibly or irreversibly to a diacylglycerol-type receptor; and/or

b. activates any form of the enzyme protein kinase C; and

c. contains an hydroxymethyl or 1-hydroxyethyl group bonded to carbon 6; and

d. contains at least one substituent other than hydrogen or hydroxy at carbon 12; and

wherein D is a polar group attached to carbon 13; and provided that I₀-D may not be 12-O-methylphorbol, 12-O-ethylphorbol or compounds of the exact phorbol structure with acyl groups at the 12-hydroxy group.

2. A compound according to Claim 1 selected from:

(i) phorbol 12-[3',5'-bis(trifluoromethyl)phenylcarbamate];

(ii) phorbol 12-n-octadecylcarbamate;

(iii) phorbol 12-[(1'-adamantyl)carbonate];

(iv) bisdehydrophorbol 12-myristate; and

(v) bisdehydrophorbol 12-[3',5'-bis(trifluoromethyl)phenylcarbamate].

3. A compound of the formula: P - G, wherein P is a radical derived from a

benzolactam parent compound, which compound:

a. binds reversibly or irreversibly to a diacylglycerol-type receptor; and/or

b. activates any form of the enzyme protein kinase C; and

c. contains an hydroxymethyl or 1-hydroxyethyl group bonded to a carbon atom; and

wherein G is any group of 55 or fewer atoms selected from carbon, hydrogen, oxygen, nitrogen, halogen, sulfur, phosphorus, silicon, arsenic, boron and selenium either: i) singly or doubly bonded to the carbon atom of the parent compound in place of the hydroxymethyl or 1-hydroxyethyl group; or ii) singly or doubly bonded to a carbon atom immediately adjacent to the carbon atom to which the

hydroxymethyl or 1-hydroxyethyl group is bound in the parent compound; and wherein the hydroxymethyl or 1 -hydroxyethyl group of the parent compound has been replaced by G; provided that P-G may not be: (S)-1,2,3,4,5,7-hexahydro-3-(1- methylethyl)-2-[(4-methylphenyl)sulfonyl]-4-oxo-6H-2,5-benzodiazonine-6,6- dicarboxylic acid diethyl ester, [S-(R*, R*j]-1,2,3,5,6,7-hexahydro-3-(1- methylethyl)-2-[(4-methylphenyl)sulfonyl]-4-oxo-4H-2,5-benzodiazonine-6- carboxylic acid, [S-( R*,S*;]-1,2,3,5,6,7-hexahydro-3-(1-methylethyl)-2-[(4- methylphenyl)suιfonyl]-4-oxo-4H-2,5-benzodiazonine-6-carboxylic acid, (S)- 1,2,3,5,6,7-hexahydro-6,6-bis[(acetyloxy)methyl]-3-(1-methylethyl)-2-[(4- methylphenyl)sulfonyl]-4H-2,5-benzodiazonin-4-one and (S)-1,2,3,5,6,7-hexahydro- 6,6-bis(hydroxymethyl)-3-(1-methylethyl)-4H-2,5-benzodiazonin-4-one.

4. A compound according to Claim 3 selected from:

(i) 11-deoxy-(2S,5S)-BL-V8-310 11-sulfonic acid;

(ϋ) 11-O-(N-methyl)caιbamoyl-(2S,5S)-BL-V8-310;

(iii) 11-deoxy-11-(2'-hydroxyethylthio)-(2S,2S)-BL-V8-310;

(iv) 11-deoxy-11-(2'-carboxyethylthio)-(2S,2S)-BL-V8-310;

(v) 11-deoxy-11-cyano-(2S,5S)-BL-V8-310;

(vi) 5-des(hydroxymethyl)-5-carboxy-BL-V8-310; and

(vii) 11-deoxy-11-[3'-(hydroxymethyl)phenylamino]-BL-V8-310.