WO1997043268A1 - 8-hydrocarbyl substituted benzodizocine derivatives, their preparation and their use as protein kinase c (=pkc) modulators - Google Patents

Abstract

Compounds of formula (I) wherein R1 is H, (C1-C5)alkyl, OR?a, SRa, N(Ra)(Rb¿), halo, NO¿2?, NHC(O)[(C1-C4)alkyl] or NHOH; R2 is a (C5-C22) hydrocarbyl group, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof, or (C6-C12)aryl(C2-C10)alkyl, wherein the alkyl moiety optionally comprises 1-2 double bonds, 1-2 triple bonds or a mixture thereof; wherein said (C5-C22) hydrocarbyl group or said (C6-C12)aryl(C2-C10)alkyl may optionally be substituted with 1 or 2 substituents independently selected from the group consisting of halo, hydroxy, cyano, nitro, (C1-C5)alkyl, (C1-C5)alkoxy, trifluoromethyl, trifluoromethoxy, -C(=O)O(C1-C5)alkyl, and N(R?e)(Rf); R¿1 and R2 together are -CH(Rc)-CH2-C(O)-N(Rd)-, -C(Rc)=CH-C(O)N(Rd)-, -C(Rc)=CH-N(Rd)- or -C(Rc)=CH-O-; R¿3? is H, OH or halo; R?a and Rb¿ are independently H or (C¿1?-C5)alkyl; R?c¿ is a (C¿5?-C22) hydrocarbyl group; R?d¿ is H or (C¿1?-C5)alkyl; R?e and Rf¿ are independently hydrogen, (C¿1?-C5)alkyl, or (C1-C5)alkanoyl, or together with the nitrogen to which they are attached are pyrrolidino, piperidino or morpholino; Z is H or (C1-C5)alkyl, and Y is H or (C1-C5)alkyl; and their pharmaceutically acceptable salts, are PKC modulators and are useful for treating i.e. cancer mammals. Also disclosed are pharmaceutical compositions comprising compounds of formula (I), processes for preparing compounds of formula (I), and intermediates useful for preparing compounds of formula (I).

Description

8-HYDROCARBYL SUBSTITUTED BEN20DEOCINE DERIVATIVES. THEIR PREPARAΗON AND THEIR USE AS PROTEIN KINASE C (-PKC) MODULATORS

5 Priority of Invention

This application claims priority from U.S. Provisional Application Number 60/017,532, filed May 10, 1996.

Background ofthe Invention 0 Protein kinases serve a regulatory function which is crucial for all aspects of cellular development, differentiation and transformation. One ofthe largest gene families of non-receptor serine-threonine protein kinases is protein kinase C (PKC). Since the discovery of PKC more than a decade ago by Nishizuka and coworkers (Kikkawa et al., J. Biol. Chem.. 257. 13341 (1982)), 5 and its identification as a major receptor for phorbol esters (Ashendel et al., Cancer Res.. 43, 4333 (1983)), a multitude of physiological signaling mechanisms have been ascribed to this enzyme. The intense interest in PKC stems from its unique ability to be activated in vitro by diacylglycerol (and its phorbol ester mimetics), an effector whose formation is coupled to phospholipid 0 turnover by the action of growth and differentiation factors.

The PKC gene family consists presently of 1 1 genes which are divided into four subgroups: 1) classical PKCα, β,, β₂ (β, and β₂ are alternately spliced forms ofthe same gene) and γ, 2) novel PKCδ, e, η, and θ, 3) atypical PKCζ, λ, η and i and 4) PKCμ. PKCμ resembles the novel PKC isoforms but 5 differs by having a putative transmembrane domain (reviewed in Blobe et al., Cancer Metast. Rev.. 13, 41 1 (1994)); Hug et al., Biochem J.. 291. 329 (1993); Kikkawa et al., Ann. Rev. Biochem. 5.8, 31 (1989)) (Figure 1). The α, β„ β₂ and γ isoforms are Ca²⁺, phospholipid- and diacylglycerol-dependent and represent the classical isoforms of PKC, whereas the other isoforms are activated by 0 phospholipid and diacylglycerol but are not dependent on Ca²'. All isoforms encompass 5 variable (VI -V5) regions and the a, β and γ isoforms contain four (C1-C4) structural domains which are highly conserved. All isoforms except PKCα, β, and γ lack the C2 domain, and the λ, η and i isoforms also lack one of two cysteine-rich zinc finger domains in Cl to which diacylglycerol binds. The Cl domain also contains the pseudo substrate sequence which is highly conserved among all isoforms, and which serves an autoregulatory function by blocking the substrate-binding site to produce an inactive conformation ofthe enzyme (House et al. Science. 238. 1726 ( 1987)).

Because of these structural features, diverse PKC isoforms are thought to have highly specialized roles in signal transduction in response to physiological stimuli (Nishizuka, Cancer. 10. 1892 (1989)), as well as in neoplastic transformation and differentiation (Glazer, Protein Kinase C. J. F. Kuo, ed., Oxford U. Press (1994) at pages 171-198).

From a pharmacological perspective, PKC has served as a focal point for the design of anticancer drugs (Gescher, Brit. J. Cancer. 66. 10 (1992)). Antisense expression of either the PKCα cDNA (Ahmad et al., Neurosurgery. 35- 904 (1994)) or a phosphorothioate oligodeoxynucleotide (S- oligo) for PKCα has shown the efficacy of targeting PKC to inhibit the proliferation of A549 lung carcinoma cells (Dean et al., J. Biol. Chem.. 269. 16416 (1994)) and U-87 glioblastoma cells. Similar studies have not been conducted with breast tumors, but historical and preliminary data suggest that PKC is a logical molecular target by which to inhibit tumor growth and/or induce apoptosis. However, it is not clear which isoforms are most crucial for tumor proliferation and what role different PKC isoforms play in such critical cellular processes as cell proliferation and apoptosis. Nonetheless, it is reasonable to conclude that isoform selective, non-tumor promoting modulators of PKC that cause downregulation may find use in cancer treatment through the initiation of cancer cell death through apoptosis. Selective cancer cell killing may be achieved either through the targeting of those isoforms found to be overexpressed in the cancer cells, or through the synergistic interaction of a cytotoxic drug like 1-β-D-arabinofuranosylcytosine with an appropriate PKC- based signaling interceptor. Teleocidin was first isolated from the mycelia of Streptomyces mediocidicus as a mixture of highly toxic compounds by Takahashi et al., Bull. Agr. Chem. Soc. Japan. 24. 647 (1960). The structure of one of these metabolites was assigned by Hirata as shown by Figure 2, formula 1. The lyngbyatoxin series can be obtained together with the teleocidin B group from Streptomyces mediocidicus as disclosed by S. Sakai et al., Tetrahedron Lett.. 27. 5219 (1986). Therefore, as depicted in Figure 2, they were named as teleocidin A-l (2a) and A-2 (2b) by Sakai. Indolactam V (3, ILV), which contains the basic ring structure ofthe teleocidins, is the simplest member ofthe family, and is produced in large quantities by actinomycetes strain NA34- 17 (Figure 2).

Investigations with 12-O-tetradecanoylphorbol-13-acetate (TPA) have provided considerable information on tumor promotion. In the two stage model of skin carcinogenesis, it is believed that initiators bind to DNA and that tumor promoters such as TPA bind non-covalently to membrane-associated high affinity receptors, most likely protein kinase C. Thus, TPA, the teleocidins, and the lyngbyatoxins as well as aplysiatoxin serve as diacylglycerol mimics, binding to the diacylglycerol site of protein kinase C, thus activating the kinase. Indeed, computer assisted molecular modeling studies of these tumor promoters have revealed a commonality of their hydrophobic regions and certain heteroatoms. On the basis of both solution NMR studies and molecular mechanics calculations, it was additionally reported that the indolactam portion (indolactam V, 3) ofthe teleocidins and lyngbyatoxins can exist in two conformational states, the sofa or twist-like conformations. At equilibrium, the ratio of twist/sofa was 2.8; the twist form of ILV represents the biologically active conformation.

Compounds related to the teleocidins are disclosed in Kozikowski, A. et al. Journal of the American Chemical Society. 1993, 1 15.

3957-3965; in PCT Application WO/95-09,160 (1995); in Endo Y. et al. Journal ofthe American Chemical Society. 1996, 118, 1841-1855; and in Endo Y. et al. Chem. Pharm. Bull. 1997, 45, 424-426. However, a continuing need exists for novel compounds which can selectively modulate PKC so as to effect the selective killing of cancer cells. Summary ofthe Invention The present invention provides certain benzolactam PKC modulators, which exhibit PKC isoform selectivity. The compounds are of general formula (I):

wherein

R, is H, (C,-C₅)alkyl, OR", SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C,-C₄)alkyl] or NHOH;

R₂ is a (C₅-C₂₂) hydrocarbyl group, optionally comprising 1 -3 double bonds, 1-2 triple bonds or a mixture thereof, or (C₆-C₁₂)aryl(C₂-C₁₀)alkyl, wherein the alkyl moiety optionally comprises 1-2 double bonds, 1-2 triple bonds or a mixture thereof; wherein said (C₅-C₂₂) hydrocarbyl group or said (C₆- C₁₂)aryl(C₂-C₁₀)alkyl may optionally be substituted with 1 or 2 substituents independently selected from the group consisting of halo, hydroxy, cyano, nitro, (C,-C₅)alkyl, (C,-C₅)alkoxy, trifluoromethyl, trifluoromethoxy, -C(-O)O(C,-

C₅)alkyl, and N(R^e)(R^*);

R, and R₂ together are -CH(R^c)-CH₂-C(O)-N(R^d)-, -C(R^C)=CH- C(O)N(R^d)-, -C(R^c)=CH-N(R^d)- or -C(R^c)=CH-O-;

R₃ is H, OH or halo; R^a and R^b are independently H or (C,-C₅)alkyl;

R^c is a (C₅-C₂₂) hydrocarbyl group; R^d is H or (C,-C₅)alkyl;

R^c and R^f are independently hydrogen, (C,-C₅)alkyl, or (C,-C₅)alkanoyl, or together with the nitrogen to which they are attached are pyrrolidino, piperidino or morpholino; Z is H or (C,-C₅)alkyl; and

Y is H or (C_rC₅)alkyl; or a pharmaceutically acceptable salt thereof.

Pharmaceutical compositions comprising an amount of one or more compounds of formula (I) effective to treat mammalian conditions associated with pathological cellular proliferation, particularly human cancers, such as solid tumors and leukemias, are also an embodiment ofthe invention. The present invention also provides a method to inhibit the pathological proliferation of mammalian cells, such as cancer cells, by administering to a mammal afflicted with such a condition, an effective inhibitory amount of one or more ofthe compounds of formula I, preferably formulated as said pharmaceutical composition, i.e., in unit dosage form. Novel intermediates and processes to prepare compounds of formula (I), as depicted in Figures 6-12 are also embodiments ofthe invention.

The discovery of these new modulators of PKC that exhibit isotype selectivity will permit elucidation ofthe functional importance ofthe different PKC isoforms in the regulation of cell function, and can provide PKC based therapeutics that may find use not only in the treatment of cancer, but potentially autoimmune diseases, and inflammation.

Brief Description ofthe Figures

FIG. 1 shows the structural organization ofthe PKC gene family.

FIG. 2 shows the structures of telocidin B-4, A-l , A-2, and indolactam V.

FIG. 3 shows compounds ofthe invention.

FIG. 4 shows an electrophile useful for preparing compounds ofthe invention. FIG. 5 shows an electrophile useful for preparing compounds ofthe invention.

FIG. 6 shows a scheme for preparing compounds of the invention.

FIG. 7 shows a scheme for preparing compounds ofthe invention. FIG. 8 shows a scheme for preparing compounds ofthe invention.

FIG. 9 shows a scheme for preparing compounds ofthe invention.

FIG. 10 shows a scheme for preparing compounds ofthe invention.

FIG. 11 shows a scheme for preparing compounds ofthe invention.

FIG. 12 shows a scheme for preparing compounds ofthe invention. FIG. 13 shows the cytotoxicity of compound 17 and ILV in MCF-7 and

MDA-MB-231 breast carcinoma cells.

FIG. 14 shows the Western blot of PKC isoform levels 24 hours after treatment with compound 17.

FIG. 15 shows the antitumor activity of compound 17 against the MDA-MB-231 xenograft in nude mice.

Detailed Description ofthe Invention In the following description ofthe preferred embodiments, reference is made to the accompanying figures which form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

The following definitions are used, unless otherwise described. Halo is fluoro, chloro, bromo, or iodo. The term "alkyl" encompasses branched or unbranched alkyl, cycloalkyl or (cycloalkyl)alkyl, but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to. Aryl comprises a phenyl radical, an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic, as well as simple (C,-C₄)_n alkylaryl wherein n is 1-3. It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymoφhism. It is to be understood that the present invention encompasses any racemic, optically- active, polymoφhic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution ofthe racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

Specific values listed below for radicals, substituents, and ranges, are for illustration only and they do not exclude other defined values or other values within defined ranges for the radicals and substituents

Specifically, (C,-C₅)alkyl is methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, or cyclopentyl; and aryl is phenyl, methylphenyl, ethylphenyl, propylphenyl, dimethylphenyl, diethylphenyl, indenyl, methylindenyl, dimethylindenyl, naphthyl, methylnaphthyl, or dimethylnaphthyl.

A specific value for R, is OR^a, SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C,-C₄)alkyl] or NHOH; for R₂ is 1-decynyl or decyl; for R₃ is H; for R^c is (C₅-C₁₅)alkyl; for Y is H; and for Z is methyl.

A more specific value for R, is OR^a.

A specific group of compounds are compounds of formula I wherein R, and R₂ together are -CH(R^c)-CH₂-C(O)-N(R^d)-, -C(R^C)=CH- C(O)N(R^d)-, -C(R^c)=CH-N(R^d)- or -C(R^c)-=CH-0-H; or a pharmaceutically acceptable salt thereof.

Another specific group of compounds are compounds of formula I wherein Z is CH₃; Y is H; R, is OR^a, SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C,- C₄)alkyl] or NHOH; R₂ is (C₅-C,₅)alkyl, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof; and R₃ is H; or a pharmaceutically acceptable salt thereof. Another specific group of compounds are compounds of formula I wherein: R, is OR\ SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C,-C₄)aIkyl] or NHOH; and R₂ is a hydrophobic (C₅-C₂₂) hydrocarbyl group, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof, or (C₆- C|₂)aryl(C₂-C₁₀)alkyl, wherein the alkyl moiety optionally comprises 1 -2 double bonds, 1 -2 triple bonds or a mixture thereof; or R, and R₂ together are -CH(R^C)- CH₂-C(O)-N(R^d)-, -C(R^c)=CH-C(O)N(R^d)-, -C(R^c)=CH-N(R^d)- or -C(R^c)=CH-O- H; or a pharmaceutically acceptable salt thereof.

Another specific group of compounds are compounds of formula I wherein: R₂ is (C₅-C₁₅)alkyl, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof; or a pharmaceutically acceptable salt thereof. Another specific group of compounds are compounds of formula I wherein: R₂ is (C₆-C_]2)aryl(C₂-C₁₀)alkyl, wherein the alkyl moiety optionally comprises 1 -2 double bonds, 1 -2 triple bonds or a mixture thereof; or a pharmaceutically acceptable salt thereof.

A preferred group of compounds are compounds of formula I wherein Z is CH₃; Y is H; R₂ is (C₅-C,₅)alkyl, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof; and R₃ is H; or a pharmaceutically acceptable salt thereof. Another preferred group of compounds are compounds of formula

I wherein R₂ is 1-decynyl; or a pharmaceutically acceptable salt thereof.

Processes for preparing compounds of formula I are provided as further embodiments ofthe invention and are illustrated by the following procedures in which the meanings ofthe generic radicals are as given above unless otherwise qualified.

Compounds of formula I wherein R, is 1 -alkynyl can be prepared from a corresponding compound wherein R, is iodide by coupling with the requisite alkyne using a suitable catalyst, such as for example palladium. Suitable conditions for such a coupling reaction are illustrated in Example 1. Compounds of formula I wherein R, is 1 -alkyl can be prepared from a corresponding compound wherein R, is 1 -alkynyl by hydrogenation of the alkyne bond using a suitable catalyst, such as for example palladium on carbon. Suitable conditions for such a hydrogenation are illustrated in Example 2.

Compounds of formula I can generally be prepared by the reaction of 2,6-disubstituted arylmetal compounds as nucleophiles with enantiomerically pure three carbon electrophiles incoφorating the necessary amino and hydroxyl groups in protected form.

Electrophiles. Two readily available electrophiles (Figures 4 and 5) are suitable for preparing compounds ofthe invention, the protected aziridinemethanol 21, and the protected serine aldehyde 23 ("Enantiospecific Synthesis of D-α,ω-Diaminoalkanoic Acids" Beaulieu, P. L.; Schiller, P. W. Tetrahedron Lett. 1988, 29, 2019-2022). To obtain compound 21, the known L-serine-derived methyl or benzyl esters 20 can be reduced to the aziridinemethanol, e. g., with NaBH₄ and the hydroxyl group silylated [(a) "Construction of Optically Pure Tryptophans from Serine Derived Aziridine-2-carboxylates" Sato, K.; Kozikowski, A. P. Tetrahedron Lett. 1989, 31, 4073-4076. (b) "One-Step Synthesis of Optically Active Benzyl N-Trityl-Z,-Aziridine-2-Carboxylic Esters" Kyul-Yeheskiely, E.; et al. Tetrahedron Lett. 1992, 33, 3013-3016] ("Derivatives of heterocyclic α-iminocarboxylic acids. 4. Reduction of N-alkoxycarbonyl derivatives of α-iminocarboxylic acids" Nurdinov, R.; et al. Khim. Geterotsikl. Soedin. 1993, 1567-1573; Chem. Abstr. 1995, 123, 83337x). Since cuprates exhibit enhanced reactivity towards aziridines compared with organolithium reagents, stoichiometric or catalytic amounts of copper(I) salts may be included in reaction mixtures involving 21.

Nucleophiles. The most convenient nitrogen substituent on the aromatic ring would be a free amino group (NH₂). While, N-alkylarylamines have been successfully ortho-metalated by N-lithiation, reacted with CO₂ to form the lithium carbamate, and further treated with tert-butyllithium, this procedure failed in the case of aniline ("Carbon Dioxide: A Reagent for the Simultaneous Protection of Nucleophilic Centers and the Activation of Alternative Locations to Electrophilic Attack. 17. Substitution of N-Methyl-1- and

N-Methyl-2-naphthylamine and Side-Chain Functionalization of o-Toluidine" Katritzky, A. R.; et al. J. Org. Chem. 1991, 56, 5045-5048). N-(methoxycarbonyl)-O-(methoxymethyl)-w-aminophenol has been reported to undergo directed metalation mainly in the 2-position, whereas the corresponding Ν-Boc derivative reacted on the opposite side ofthe nitrogen in position 4 ( "Biosynthesis of Sarubicin A. Synthesis and Incoφoration of 6-Hydroxy[¹³CO^,5ΝH₂]anthranilamide" Gould, S. J.; Eisenberg, R. L. J. Org. Chem. 1991, 56, 6666-6671). Since the nitrogen may need to be deprotected in the presence of protecting groups such as N-Cbz and O-TBDMS, the N-(allyloxycarbonyl) derivative 24 (Figure 6) is a convenient starting material for preparing compounds ofthe invention. While the allyloxycarbonyl group is not bulky, it is readily removed by various nucleophiles or hydride donors in the presence of a Pd catalyst.

An intermediate of formula 28 is particularly useful for preparing compounds of formula I. An intermediate of formula 28 can be prepared as shown in Figure 6 by reaction of a nucleophile of formula 24 and an aziridine (electrophile) of formula 21 followed by deprotection ofthe aniline nitrogen. The resulting aniline 25 can be alkylated to give a compound of formula 26. Hydrogenation of 26 followed by lactam formation yields an intermediate of formula 28.

The methoxymethoxy substituent in intermediate 28 provides access not only to 7-hydroxy- and 7-alkoxybenzolactams but, as discussed below, to a variety of other compounds ofthe invention via aryl triflate chemistry. Benzolactams containing 7-halo or 7-CF₃ substituents can conveniently be synthesized from the requsite N-(allyloxycarbonyl)-w- substituted anilines using a procedure similar to the one described above. A variety of potentially suitable substrates have been reported which differ in their thermal stability as well as in the ease or difficulty with which the second substituent can be transformed into NH₂. 3-Chlorobenzonitrile and N-tert-butyl-3-chlorobenzamide undergo directed lithiation in position 2 at -70°C, and the resulting organolithiums can be trapped with an electrophile ("Heteroatom-Facilitated Lithiations" Gschwend, H. W.; Rodriguez, H. R. Org. React. 1979, 26, 1-360 (unpublished results by Rodriguez, H. R.)). 3-Fluoro- and 3-chIorophenyloxazolines have been metalated in position 2 ("A New Route to 3-Hydroxyphthalides: Application to the Synthesis of Racemic f5-^,3C]

Daunomycinone" Becker, A. M.; et al. Tetrahedron Lett. 1986, 27, 3431 -3434.) and ("The Oxazoline-Benzyne Route to 1,2,3-Trisubstituted Benzenes. Tandem Addition of Organolithiums and α-Lithionitriles to Benzynes" Pansegrau, P. D.; et al. J. Am. Chem. Soc. 1988, 110, 7178-7184). Additionally, 3-Fluorobenzaldehyde dimethyl acetal undergoes metalation and subsequent carboxylation in high yield ("Synthesis of Functionalized Hydroxyphthalides and Their Conversion to 3-Cyano-l(3H)-isobenzofuranones. The Diels-Alder Reaction of Methyl 4,4-Diethoxybutynoate and Cyclohexadienes" Freskos, J. N.; Morrow, G. W.; Swenton, J. S. J Org. Chem. 1985, 50, 805-810). The 7-halo-6-hydroxybenzolactams ofthe invention can conveniently be prepared as illustrated in Figure 7. Lithiated 3-fluoro- and 3-chlorobenzaldehyde dimethyl acetal 35a, b can be reacted with aldehyde 23. Separation ofthe resulting stereoisomers followed by acid hydrolysis of the aryllithium addition product 36, yields an aldehyde, which forms a hemiacetal with the benzylic hydroxyl group (37). Benzylic alcohol 37 can be selectively oxidized to a lactone using, for example, MnO₂. Acetonide protection can then be restored. The lactam can be ammonolyzed to yield 39, and the liberated benzylic hydroxyl group protected by silylation. Hofmann degradation gives an aniline of formula 40, which can be converted to the bis-tert-butyldimethylsilyl ether 41. Using a sequence similar to that illustrated in Figure 6, a compound of formula 41 can be converted to a 7-halo-6-hydroxybenzolactam of formula 42. 97/43268 P /US97/08141

12

Compounds ofthe invention wherein R₃ is fluoro (such as for example a compound of formula 43 can be prepared by treatment of a corresponding compound wherein R₃ is hydroxy (such as for example a compound of formula 42) with (diethylaminosulfur)trifluoride (DAST) as shown in Figure 7.

A compound of formula I wherein R₃ is hydrogen can be prepared from a corresponding compound of formula I wherein R₃ is hydroxy, such as for example a compound of formula 42 , by formation of a cyclic thionocarbonate of formula 44 followed by Barton deoxygenation ("Synthesis of Deoxysugars and Deoxynucleosides from Diol Thiocarbonates" Barton, D. H. R.; Subramanian, R. J. Chem. Soc., Chem. Commun. 1976, 867-868).

As illustrated in Figure 8, the intermediate aryl triflate 46, can be used to prepare compounds of formula I having a variety of 7-substituents. Methoxycarbonylation of 46 ( "Palladium Catalysed Alkoxycarbonylation of Phenols to Benzoate Esters" Dolle, R. E.; et al. J. Chem. Soc., Chem. Commun. 1987, 904-905) gives the ester 47 which can be transformed into the derivatives 48 and subsequently compounds of formula 49, by selective reduction of the ester moiety, followed by cuprate alkylation of the derived triflate.

The ester function of a compound of formula 47 can also be used to introduce a nitrogen atom into position 7 by means of a Curtius degradation. The product 50 can be alkylated in position 8 as previously illustrated in Figure 6 to obtain compound 51. This intermediate can be used to prepare 7-nitro- and 7-(hydroxyamino)benzolactams 53, 54 by peracid oxidation and reduction with zinc. Diazonium chemistry can be applied to 51 to prepare the 7-iodo- and 7-mercapto derivatives 55, 56. The corresponding chlorides and bromides may be obtained from 51 by action of tert-butyl nitrite and the anhydrous copper(II) halides ("Alkyl Nitrite-Metal Halide Deamination Reactions. 2. Substitutive Deamination of Arylamines by Alkyl Nitrites and Copper(II) Halides. A Direct and Remarkably Efficient Conversion of Arylamines to Aryl Halides" Doyle, M. P.; et al. J. Org. Chem. 1977, 42, 2426-2431). The corresponding fluoride may be obtained from 51 using a procedure similar to that described in "A Mild and Efficient Method of Aromatic Fluorination" Rosenfeld, M. N.; Widdowson, D. A. J Chem. Soc, Chem. Commun. 1979, 914-916. Compounds of formula I wherein R, and R₂ together are -

CH(R^c)-CH₂-C(O)-N(R^d)-, -C(R^c)=CH-C(O)N(R^d)-, -C(R^c)=CH-N(R^d)- or - C(R^c)=CH-O- can be prepared using procedures similar to those illustrated in in Figure 9. Iodophenol 58 and the iodoaniline 59 can be alkylated with the allylic halide 60, and the resulting intermediates cyclized under Pd catalysis to obtain the benzofuran 63 ("Synthesis of Benzofurans, Tetrahydrobenzopyrans, and Related Cyclic Ethers via Cyclic Carbopalladation" Negishi, E.; et al. Heterocycles 1989, 28, 55-58) and the indole 64 ("Conversion of 2-Halo-N-allylanilines to Indoles via Palladium(O) Oxidative Addition-Insertion Reactions" Odle, R.; et al. J. Org. Chem. 1980, 45, 2709-2710). An intermolecular Heck reaction between 59 and dimethyl maleate gives quinolinone 65 ( "Palladium-Catalyzed Synthesis of 2-Quinolone Derivatives from 2-Iodoanilines" Cortese, N. A.; et al. J. Org. Chem. 1978, 43, 2952-2958). Elaboration ofthe methoxycarbonyl side chain in quinolinone 65 to an alkyl group, using standard conditions, yields the alkylated compound 66. Hydrogenation ofthe unsaturated heterocyclic ring of compound 66 gives the lactam stereoisomers 67.

An intermediate of formula 28 can alternatively be prepared as illustrated in Figure 10. 1 ,3-Cyclohexanedione 69 can be alkylated with aziridine 68 obtained by coupling the serine and valine building blocks. Removal ofthe N-protective group yields a compound which can close to the eight-membered lactam ring under conditions of enamine formation (high dilution), to give enaminone 73. Aromatization of enaminone 73, using standard conditions, gives compound 75, which can be protected to give an intermediate of formula 28. Compounds of formula I can also be prepared using a sequence similar to that described in the previous paragraph. As shown in Figure 10, alkylation ofthe dianion of 69 gives the substituted cyclohexanedione 70.

Subsequent alkylation with the aziridine 68, followed by N-deprotection gives the cyclic enaminone 74, which can be elaborated to compounds of formula 76 and 33. Compounds of formula I wherein R, is H can generally be prepared using procedures similar to those described in Examples 1 and 2, as illustrated in Figure 1 1.

Compounds of formula I wherein R, is OR^a can generally be prepared using procedures similar to that described in Example 3, as illustrated in Figure 12.

It is noted that many ofthe starting materials employed in the synthetic methods described above are commercially available or are reported in the scientific literature.

In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes. Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incoφorated directly with the food ofthe patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% ofthe weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials ofthe above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form ofthe solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incoφorated into sustained-release preparations and devices. The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions ofthe active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absoφtion ofthe injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incoφorating the active compound in the required amount in the appropriate solvent with various ofthe other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration ofthe compound(s) of formula I in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt- %. Single dosages for injection, infusion or ingestion will generally vary between 50-1500 mg, and may be administered, i.e., 1-3 times daily, to yield levels of about 0.5 - 50 mg/kg, for adults.

Accordingly, the invention includes a pharmaceutical composition comprising a compound of formula I as described hereinabove; or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent or carrier.

The ability of compounds ofthe invention to modulate PKC can be demonstrated using standard models which are well known in the art, or can be demonstrated using the tests described hereinbelow.

Isozyme Studies.

The isozyme selectivity of representative compounds of the invention and ILV (3) was determined by investigating their ability to displace [³H]PDBU binding to recombinant PKC isozymes expressed in the baculovirus system, as described by Kazanietz, M. G.; et al. Characterization of ligand and substrate specificity for the calcium-dependent and calcium-independent PKC isozymes. Mol. Pharmacol. 1993, 44, 298-307. As shown in Table 1, the compound of Example 1 (17) showed a higher affinity for the α and β isozymes, in comparison to γ, δ, and ε, with approximately a ten-fold difference in affinity between PKC α and ε. It is apparent that the ectronic effect of the acetylenic group influences isozyme selectivity, since the saturated alkyl compound of Example 2 (18) shows only a four-fold difference in the K₍ for α versus ε.

Table 1- K; values ± SEM for the inhibition of [Η]PBDU binding by the compounds tested.

α β Y δ ε

Compound

Acetylene- 14.7 ± 1.3 17.4 ± 2.2 40.7 ± 8.9 122 ± 22 142 ± 3 Benzolactam

17

Saturated- 46.6 ± 8.0 58.2 ± 12.6 145 ± 25 185 ± 30 187 ± 22

Benzolactam

18

ILV* 11.0 6.1 19.4 8.2 21.9

*Data taken from Mol. Pharmacol. 1993, 44, 298-307.

Cell proliferation assay and PKC Downregulation.

Representative compounds of the invention were also tested for antiproliferafive activity against breast carcinoma cell lines MCF-7 and MDA- MB-231 (Yu, G.; et al. Transfection with protein kinase Cα confers increased multidrug resistance to MCF-7 cells expressing P-glycoprotein. Cancer Commun. 1991, 3, 181-189). Exposure of MCF-7 and MDA-MB-231 cells to the compound of Example 1 (17) for four days resulted in IC₅₀ values of 20 and 30 μM, respectively, whereas ILV (3) was inactive (Figure 13).

PKC isozyme levels were determined in MCF-7 and MDA-MB- 231 cells exposed to the compound of Example 1 (17) for 24 hours (Figure 14). In MCF-7 cells, PKCβ and PKCε were virtually eliminated, PKCδ was reduced to a lesser extent, and PKCζ was unchanged. MDA-MB-231 cells exhibited a similar reduction in PKCβ, whereas PKCδ and ζ were slightly reduced, while PKCα and PKCε remained unchanged. These results indicate that the compound of Example 1 (17), while not completely selective, preferentially downregulates PKCβ in both cell lines. The varying degree of selectivity ofthe compound of Example 1 for other PKC isozymes was to some degree cell type specific. This result was not completely unexpected, since different tumors would be expected to exhibit different PKC isozyme patterns as well as different pathways governing their stability and turnover.

Using a procedure similar to that described in: Price, J.E., et al. Cancer Res. 50:717-721 , 1990, the antitumor activity of compound 17 was evaluated in vivo in MDA-MB-231 human breast carcinoma xenographs at the maximum dose evaluated thus far for toxicity, but not necessarily the maximum tolerated dose (Figure 15). Daily i.p. administration ofthe compound for three consecutive weeks resulted in 65% inhibition of tumor growth three weeks after treatment was initiated. No overt general cytotoxic effects were observed. Compounds ofthe invention have been shown to be downregulators of PKC, and are therefore useful to treat conditions ameliorated by reduction of PKC activity. Such conditions include but are not limited to cancer, autoimmune diseases, and inflammation. Accordingly, the invention includes a method for modulating PKC in a mammal comprising administering to said mammal a pharmaceutically effective dose of a compound of formula I; or a pharmaceutically acceptable salt thereof. The invention also includes a method for the treatment of cancer in a mammal comprising administering to said mammal a pharmaceutically effective dose of a compound of formula I; or a pharmaceutically acceptable salt thereof.

Compounds of formula (I) are generally effective to treat mammalian conditions associated with pathological cellular proliferation. In addition to the utilities described above, they may also be useful to treat conditions which include restenosis, atherosclerosis, coronary heart disease, thrombosis, myocardial infarction, stroke, uterine fibroid or fibroma, and obliterative disease of vascular grafts and transplanted organs. The invention will now be illustrated by way ofthe following non-limiting Examples.

Examples Example 1. (25',55)-8-(l-Decynyl)benzolactam (Figure 11, compound 17).

To a mixture of 16 (Figure 11) (1 16 mg, 0.27 mmol), Et₂NH (2 mL), and PdCl₂(PPh₃)₂ (26 mg, 0.018 mmol) were added 1-decyne (0.13 mL, 0.74 mmol) and Cul (4 mg, 0.01 mmol). The resulting solution was stirred at room temperature for 24 hours. The solvent was evaporated, the residue was dissolved in 1 mL of 20% aqueous NaOH and 4 mL of MeOH, and the solution was stirred at room temperature for 0.5 hours. The mixture was partitioned between 10 mL of water and 100 mL of EtOAc. The organic layer was separated, washed with brine, and dried over NajSO₄. Evaporation and chromatography on silica gel (2/1 ethyl acetate/petroleum ether as eluent) afforded 87 mg (81%) of the title compound (17): [α]²°_D -303.3° (c 0.5, ethanol); 'H NMR (300 MHz,

CDC1₃) δ 7.18 (d, J= 7.8 Hz, IH), 7.09 (s, IH), 6.85 (d, J= 7.8 Hz, IH), 6.55 (s, IH), 3.82 (m, IH), 3.58 (m, IH), 3.48 (m, 2H), 3.12 (dd, J = 16.3, 8.1 Hz, I H), 2.80 (s, 3H), 2.73 (d, J= 16.2 Hz, IH), 2.33 (m, I H), 2.30 (t, J= 7.8 Hz, 2H), 1.68-1.15 (m, 12H), 1.05 (d, J= 7.2 Hz, 3H), 0.88 (t, J= 7.4 Hz, 3H), 0.85 (d, J = 7.2 Hz, 3H); MS m/z 398 (M⁺), 312, 91; HRMS calc. for C_2SH„N₂0₃ 398.293, found 398.294. Anal, calcd. for C„H_3»N₂O₂: C, 75.34; H, 9.61 ; N, 7.03. Found: C, 74.98, H, 9.86; N, 6.71.

The intermediate 16 was prepared as follows (Figure 1 1). a. (5)-0-Acetyl-2-[(ethoxycarbonyl)amino]-3-phenyl-l-propanol (Figure

11 , compound 78). To a suspension of (5)-2-amino-3-phenyl-l-propanol 77 (25.0 g, 0.165 mol) and Na,CO₃ (20.1 g, 0.19 mol) in 80 mL of water was added EtOCOCl (26.0 mL, 0.27 mol) dropwise at room temperature. The solution was stirred at room temperature for 4 hours and extracted with CH₂C1₂ (4 x 250 mL). The organic layer was dried over Na^O, and evaporated to give an oil which was dissolved in Et,N (100 mL, 0.72 mol) and Ac₂O (60 mL, 0.58 mol). The W

21 mixture was stirred overnight at room temperature and partitioned between 400 mL of EtOAc and 150 mL of water. The organic layer was washed with 50 mL of brine and dried over NajSO₄. Evaporation and chromatography (1/2 ethyl acetate-petroleum ether as eluent) afforded 40.2 g (91%) ofthe di-acetate: [α]^2ϋ _D - 17.9° (c 0.9, CHC1₃); IR (KBr) 2980, 1700, 1500, 1200 cm ¹; 'H NMR (300 MHz, CDC1₃) δ 7.31-7.15 (m, 4H), 4.72 (br s, IH), 4.09 (q, J = 7.3 Hz, 2H), 4.07-4.02 (m, 3H), 2.80 (m, 2H), 2.04 (s, 3H), 1.18 (t, J= 7.3 Hz, 3H); MS m/z 265 (M⁺), 165, 91. Anal. Calcd for C₁₄H„NO₄: C, 63.38; II, 7.22; N, 5.28. Found: C, 63.36; H, 7.30; N, 5.06.

b. (LS)-0-Acetyl-3-(2-aminophenyl)-2-[(ethoxycarbonyl)amino]- 1 -propanol (Figure 1 1, compound 9). To a solution of (S)-O-Acetyl-2- [(ethoxycarbonyl)amino]-3-phenyl-l -propanol (25.0 g, 0.094 mol) in Ac,O (25 mL) was added nitric acid (10 mL, 0.24 mol) dropwise at 0 °C. The mixture was stirred at room temperature for 24 hours, poured into 200 mL of ice- water, and extracted with EtOAc (3 x 250 mL). The organic layers were washed with brine, dried over Na₂SO₄, and evaporated. The residue was dissolved in 200 mL of EtOAc, and 500 mg of Pd/C was added. The mixture was stirred under 1 atm of H₂ at room temperature for 24 hours. Filtration from the catalyst, evaporation, and chromatography (2/3 ethyl acetate/petroleum ether as eluent) provided 9

(6.02 g, 23%) and its isomer 10 (13.2 g, 50%). Compound 9: [α]²⁰ _D +4.6° (c 0.5, CHC1,); IR (KBr) 3360, 1720, 1700, 1500, 1200, 730 cm^*'; Η NMR (300 MHz, CDCL) δ 7.06 (t, J= 7.5 Hz, IH), 6.93 (d, J= 7.6 Hz, I H), 6.67 (m, 2H), 5.26 (br s, IH), 4.14 (q, J= 7.3 Hz, 2H), 4.10-4.05 (m, 3H), 4.01 (br s, 2H), 2.94 and 2.61 (ABq, 2 H, J= 13.6 Hz), 2.10 (s, 3H), 1.25 (t, J= 7.3 Hz, 3H); MS m/z 280 (M⁺), 191, 132, 106. Anal. Calcd for C_I4H₂₀N₂O₄: C, 59.98; H, 7.19; N, 9.99. Found: C, 59.65; H, 7.19; N, 9.89.

c. (2S,2'S)-N-[3'-Acetoxy-2^r-((ethoxycarbonyl)amino)phenyl]valine benzyl ester (Figure 11, compound 12). A mixture of (Λ)-benzyl α-[[(trifluoromethyl)- sulfonyl]oxy]isovalerate (11, 5.03 g, 18.1 mmol), 9 (5.02 g, 18.1 mmol), and 22

2,6-lutidine (2.4 mL, 20 mmol) in 40 mL of 1 ,2-dichloroethane was stirred at 70 °C for 10 hours. Evaporation and chromatography on silica gel (1/5 ethyl acetate/petroleum as eluent) gave compound 12 (6.05g, 70%) as a colorless oil: [α]^M _D -20.6° (c 0.07, CHC1₃); IR (KBr) 3300, 1725, 1710, 1520 cm ¹; 'H NMR (300 MHz, CDC1₃) δ 7.29 (m, 5H), 7.05 (t, J- 7.5 Hz, IH), 6.95 (d, J= 7.6 Hz, IH), 6.67 (t, J= 7.5 Hz, IH), 6.68 (d, J= 7.6 Hz, IH), 5.14 (m, 3H), 4.12 (m, 5H), 3.91 (d, J= 6.2 Hz, IH), 2.98 (m, IH), 2.67 (m, IH), 2.23 (m, IH), 2.02 (s, 3H), 1.16 (t, J= 7.2 Hz, 3H), 1.03 (d, J= 7.0 Hz, 3H), 0.84 (d, J= 7.0 Hz, 3H); MS m/z 471 (M + H'), 335, 275, 186, 91 ; HRMS calcd for C₂₆H,₄N₂O₆ 470.241 ; found 470.241.

d. (2S, 5S)-Benzolactam (Figure 1 1, compound 15). A mixture of 12 (6.0 g,

12.8 mmol) and KOH (5.0 g, 89 mmol) in 40 mL of MeOH/H₂O (1 :1) was stirred at 30 °C for 3 days. After neutralization to pH approx. 7 with cone. HCl, 5 di-t_v7-butyl dicarbonate (2.7 g, 12.5 mmol) and NaHCO₃ (1.5 g, 18 mmol) were added. The mixture was stirred at room temperature for 24 hours, washed with petroleum ether, and adjusted to approximately pH 2. Extraction with EtOAc (4 x 150 mL), drying over Na^O,, and evaporation gave 4.1 g of crude 13. This product (1.61g, 4.37 mmol) and N-hydroxysuccinimide (0.52 g, 4.41 mmol) 0 were dissolved in 20 mL of CH₃CΝ. Dicyclohexylcarbodiimide (DCC) (1.21 g, 5.81 mmol) in 20 mL of CH₃CN was added dropwise at 0 °C, and the mixture was stirred at room temperature overnight. Filtration from the precipitate, evaporation, and chromatography on silica gel (1/1 CH₂Cl₂/EtOAc as eluent) provided 1.79 g of 14. Without further purification, this product was directly 5 dissolved in 10 mL of dried CH₂C1₂ and the solution was cooled to 0 °C before 10 mL of CF₃COOH was added. The mixture was stirred at 0 °C for 2 hours, and the volatiles were removed in vacuo below 30 °C. The residue was dissolved in 100 mL of EtOAc, and 5 mL of saturated aqueous NaHCO, solution was added. The mixture was heated at 80 °C for 24 hours with vigorous stirring. After 0 cooling to room temperature, 40 mL of water was added. The organic layer was separated, and the aqueous layer was extracted with EtOAc (4 x 20 mL). Drying over NajSO, and evaporation produced a yellow oil, which was dissolved in 10 mL of CH₃CN. This solution was cooled to 0 °C, and 4.0 mL (40 mmol) of formalin, 1.0 g (16 mmol) of NaBH₃CN, and 0.27 mL of AcOH were added. The resulting mixture was stirred at 0 °C for 2 hours before quenching with phosphate buffer (pH = 2). The solvent was evaporated, and the residue was dissolved in EtOAc, washed with water, saturated NaHCO₃, and brine, and dried over NajSO₄. Evaporation and chromatography on silica gel (1/10 MeOH/CH,Cl₂ as eluent) gave 15 (480 mg, 44% overall from 12): [α]²⁰ _D -271 ° (c 0.08, CHC1,); 'H NMR (300 MHz, CDC1,) δ 6.80-7.20 (m, 4H), 6.72 (br s, III), 4.10 (m, IH), 3.73 (m, IH), 3.61 (m, IFI), 3.46 (d, J= 8.1 Hz, IH), 3.11 (dd, J = 15.8, 8.1 Hz, I H), 2.85 (dd, J= 15.8, 3.2 Hz, IH), 2.84 (s, 3H), 2.48 (m, IH), 1.15 (d, J= 6.4 Hz, 3H), 0.94 (d, J= 6.6 Hz, 3H); MS m/z 262 (M⁺), 245, 1 17, 91.

e. (25',55)-8-Iodobenzolactam (Figure 1 1 , compound 16). A mixture of 15 (850 mg, 3.2 mmol), Et,N (15 mL), and Ac₂O (5 mL) was stirred at room temperature for 24 hours. The mixture was poured into 100 mL of water, extracted with EtOAc (4 x 100 mL), washed with brine, and dried over Na₂SO₄. After evaporation, the residue was dissolved in 10 mL of 1 ,4-dioxane and 2 mL of pyridine. To this solution 1.70 g (6.7 mmol) of I₂ was added, and the deep brown solution was stirred at room temperature for 3 days. The mixture was partitioned between EtOAc and water, and the organic layer was washed with 10 mL of 10% aqueous NaHSO, and dried over Na₂SO₄. Chromatography on silica gel provided 598 mg of 16 together with 338 mg of unreacted starting material (83% of 16 based on conversion): [α]²⁰ _D -279° (c 0.21 , CHCI₃); IR (KBr) 3200, 1740, 1680, 1260, 1240, 1065 cm ¹; Η NMR (300 MHz, CDC1₃) δ 7.18-7.00 (m, 3H), 6.05 (s, IH), 4.54 (m, IH), 4.13 and 3.98 (ABq, 2 H, J= 1 1.1 Hz, both parts d with J= 8.2 Hz), 3.45 (d, J= 8.2 Hz, IH), 3.01-2.87 (m, 2H), 2.76 (s, 3H), 2.46 (m, IH), 2.10 (s, 3H), 1.08 (d, J= 7.2 Hz, 3H), 0.92 (d, J= 7.2 Hz, 3H); MS m/z 430 (M0, 304, 261, 233, 158, 132; HRMS calcd for C₁₇H_MNAι: 430.076, found: 430.075. Example 2. (2S,5S)-8-Decylbenzolactam (Figure 1 1, compound 18).

A mixture ofthe compound of Example 1 (17) (20 mg, 0.05 mmol), Pd/C (10 mg), and EtOAc (5 mL) was hydrogenated under 10 atm of H₂ at room temperature for 2 hours. Filtration from the catalyst, evaporation, and chromatography gave 18 mg (90%) ofthe title compound 18: [α]²⁰ _D -247.5° (c 1.0, ethanol); Η NMR (300 MHz, CDC1₃) δ 6.93 (m, 2H), 6.87 (s, IH), 6.45 (s, IH), 4.14 (m, IH), 3.78 and 3.51 (ABq, 2H, J= 15.8 Hz, both parts d, J= 12.4 and 8.6 Hz, resp.), 3.39 (d, J= 8.3 Hz, IH), 2.96 (dd, J= 16.2, 10.6 Hz, IH), 2.83 (d, J = 16.2 Hz, 1 H), 2.70 (s, 3H), 2.36 (t, J = 7.6 Hz, 2H), 2.30 (m, 1 H), 1.60-1.06 (m, 16H), 1.01 (d, J= 7.6 FIz, 3FI), 0.82 (d, J = 7.6 Hz, 3H). 0.79 (t, J = 7.4 Hz, 3H); MS m/z 402 (M⁺), 359, 331 , 316; HRMS calc. for C₂,H₄,N₂O. 402.324, found 402.325. Anal, calcd. for C₂₅H₄₂N₂O₂: C, 74.58; H, 10.51 ; N, 6.96. Found: C, 74.26, H, 10.82; N, 6.71.

Example 3. (2S,5S)-7-Methoxy-8-(l-decynyl) benzolactam (Figure 12, compound 8).

To a suspension solution of 7(Figure 12) (25 mg, 0.085 mmol) and FIgCl₂ (23 mg, 0.085 mmol) in 2 mL of methylene chloride was added I₂ (22 mg, 0.085 mmol). The mixture was stirred at room temperature overnight and filtered. The filtrate was washed with aqueous 0.1 M sodium thiosulfate, and a saturated aqueous solution of potassium iodide. The organic layer was dried and concentrated by rotary evaporation. To a mixture ofthe above iodide, 2 mL of diethylamine and

PdCl₂(Ph₃P)₂ (13 mg. 0.009 mmol) was added 1-decyne (0.065 mL, 0.37 mmol) and Cul (4 mg, 0.01 mmol). The resulting solution was stirred at room temperature for 24 hours. The solvent was removed under reduced pressure, and the residual oil was purified by chromatography (silica gel, 2/1 ethyl acetate/petroleum ether as eluent) to afford 27 mg (74% yield) ofthe title compound 8. [α]²⁰ _D = -302° (c 0.63, CHC1₃); 'H NMR (300 MHz, CDC1₃) δ 7.18 (d, J = 8.3 Hz, IH), 6.70 (br s, IH), 6.62 (d, J = 8.3 Hz, IH), 3.89 (s, 3H), 3.72- 3.53 (m, 3H), 3.46 (d, J = 9.2 Hz, IH), 3. 26 and 2.78 (AB q, d, J = 17.3 Hz, 2H), 2.79 (s, 3H), 2.48 (t, J = 7.5 FIz, 2H), 2.46 (m, IH), 1.53 (m, 2H), 1.46(m, 2H), 1.37 (m, 8H), 1.02 (d, J - 7.4 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H), 0.78 (d, J = 7.4 Hz, 3H): MS m/z 428 (M+), 329, 291, 249, 221, 104, 77. HRMS calcd for C₂₆H₄₀N₂O₃: 428.304, found: 428.302.

a) 3-Hydroxy-2-(hydroxymethyl)nitrobenzene (Figure 12, compound 80). A mixture of 15.0 g of 5-nitro-l ,3-benzodioxane79 (Ando, M.; Emoto, S., Bull. Chem. Soc. Jpn. 46, 2093, 1973) and 800 mL of 1 N HCl was allowed to reflux for 24 hours. The cooled suspension was extracted with ethyl acetate (400 mL x 3). The combined organic layers were washed with water and brine, and dried over MgSO₄. After removal of solvent, the residual oil was purified by chromatography to afford 9.7 g ofthe diol. Η NMR (300 MHz CDC1₃) δ 7.41 (d, J = 7.2 Hz, IH), 7.28 (dd, J = 7.2 Hz, IH), 7.13 d, J = 7.2 Hz), 5.09 (s, 2H); MS M/z 169 (M⁺), 151, 133, 121, 105, 93, 77.

b) 2-(Hydroymethyl)-3-(methoxy)nitrobenzene (Figure 12, compound 81). To a solution of 9.7 g of 80 (57 mmol) in 300 mL of dry acetone was added 81 g of idomethane (57 mmol) and 11.8 g of potassium carbonate (85 mmol). The mixture was allowed to reflux overnight, and the cooled solution was partitioned between 300 mL of ethyl acetate and 100 mL of water. The organic layer was washed with water and brine, and dried over Na₂SO₄. After removal of solvent, the residual oil was passed a short column (silica gel, 1/1 ethyl acetate/petroleum ether as eluent) to afford 10.3 g (98%) ofthe methoxy alcohol; Η NMR (300 MHz, CDCLJ δ 7.38 (d, J = 7.2 Hz, III), 7.19 (dd, J = 7.2 Hz, IH), 7.09 (2, J - 7.2 Hz), 5.10 (s, 2H), 3.81 (s, 3H); MS m/z 183 (M+), 166, 150, 108, 92, 77; HRMS calcd for C₈H₉NO₄: 183.053, found: 183.052.

c) 2-[2-(tert-Butoxycarbonylamino)-2-(ethoxycarbonyl)ethyl]-3-(methoxy) nitrobenzene (Figure 12, compound 4). To a solution of 81 (4.0 g, 22 mmol) and triethylamine (3.3 g, 33 mmol) in 160 mL of THF was added mesyl chloride (3.8 g, 33 mmol), dropwise with cooling at -20 °C. The solution was warmed to room temperature slowly, and the THF was removed by rotary evaporation. The residue was partitioned between 200 mL of ethyl acetate and 70 mL of water. The organic layer was washed with water and brine, and dried over Na₂SO₄. After removal of solvent, the residue was dried in vacuo and then this residue was dissolved in 50 mL of methylene chloride. To the resulting solution was added Bu₄NBr (6.16 g, 19.2 mmol), (5.0 g, 19.2 mmol) and 50 mL of 10% NaOH. The mixture was stirred for 36 hours at room temperature. The organic layer was separated and aqueous layer was extracted with ether. The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated by rotary evaporation. The residue was dissolved in 50 mL of THF, and 50 mL of 5% HCl was added. The resulting solution was stirred overnight and then saturated aqueous NaHCO₃ was added to adjust pH to 1 1. After removal of the THF by rotary evaporation, the residue was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine, dried over Na₂SO₄ and concentrated. The residual oil was dissolved in 60 mL of acetonitrile, and then 3.75 g of di-tert-butyl-dicarbonate (17.1 mmol) was added. After stirring overnight, the solution was concentrated and the residue was chromatographed to afford 3.80 g (49% yield from 3) ofthe ester. 'II NMR (300 MHz, CDC13) δ 7.41 (d, J = 7.2 Hz, IH), 7.33 (dd, J = 7.2 Hz), 5.22 (br s, IH), 4.36-4.10 (m, 3H), 3.92 (s, 3H), 3.04 (M, 2H), 1.01 (s, 9H), 0.99 (t, J + 7.2 Hz, 3H); MS m/z 368 (M+), 271, 150, 104, 77: FIRMS calcd for C₁₇H₂₄N₂O₇; 368.159, found: 358.156.

d) 2-[2-(tert-Butoxycarbonylamino)-3-hydroxypropyI]-3-(methoxy)aniline (Figure 12, compound 5). A mixture of 4 (5.0 g, 14.2 mmol), NaBH₄ (1.62g, 42.6 mmol) in 350 mL of dry ethanol was heated at reflux for 2 hours. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was partitioned between 400 mL of ethyl acetate and 100 mL of water. The organic layer was separated, washed with water and brine, and dried over Na₂SO₄. After removal ofthe solvent, the residual oil was chromatographed to afford the nitro-alcohol.

A suspension ofthe nitro-alcohol and 100 mg of 10% Pd/C in 100 mL of methanol was exposed to hydrogen under atmospheric pressure with vigorous stirring. After no more hydrogen was taken up, the Pd/C was filtered off, and the filtrate was concentrated. Chromatography ofthe residual oil afforded 3.65 g (92% yield) of 5. 'H NMR (300 MHz, CDC1₃) 66.98 (dd, J = 7.2 Hz, IH), 6.25 (d, J = 7.3 Hz, IH), 6.20 (d, J + 7.2 Hz, IH), 5.03 (br s, IH), 4.88 (br, s, IH), 4.18 (m, IH), 3.79 (s, 3H), 3.29 and 3.18 (AB q, d, J = 7.8 Hz, 2H), 2.85 and 2.74 (AB q, d, J = 15.4 Hz, 2H), 1.44 (s, 9H); MS m/z 296 (M+), 104, 77: HRMS calcd for C₁₅H₂₄N₂)(4: 296.174 found: 296.171.

e) N-[(S)- 1 -Ethoxycarbonyl-2-methylbuty l]-2-[2-(tert- butoxycarbonylamino)-3-hydroxypropyl]-3-(methoxy)aniline (Figure 12, compound 6). To a solution of (R)-ethyl-α-[[(trifluoromethyl)sulfonyl]oxy] isovalerate (2.9 g, 10.5 mmol), 5 (2.8 g, 10 mmol in 35 mL of 1 ,2-dichloroethane was added 2,6-lutidine (1.87 g, 1 1 mmol). The resulting solution was heated at 70 °C for 40 hours, and the cooled solution was directly chromatographed to afford 2.46 g (67%) of 6. "H NMR (300 MHz, CDC1₃) δ 7.03 (dd, J = 7.2 Hz, IH), 6.30 (d, J = 7.2 Hz, IH), 6.23 (d, J = 7.2 Hz) 5.29 ()br, s, IH), 4.1 1 (m, 3H), 3.84-3.44 (m, 2H), 3.79 (s, 3H), 2.98-2.72 (m, 2H), 1.86 (m, IH), 1.43 (s, 9H), 1.34-0.88 (m, 9H).

f) (2S,5S)-7-Methoxybenzolactam (Figure 12, compound 7). A mixture of 6 (2.1 g, 5.15 mmol), 20 mL of 0.5 N aqueous NaOH, and 20 mL of ethanol was stirred for 10 hours at room temperature. The resulting solution was neutralized to a pH of ~5 with concentrated hydrochloride acid and extracted with ether to afford 1.52 g of crude acid.

This acid (1.5 g, 4.0 mmol) and N-hydroxysuccinimide (1.2 g, 10.0 mmol) were dissolved in 20 mL of acetonitrile. To this solution DCC (1.5 g, 7.5 mmol) in 20 mL of acetonitrile was added dropwise at 0 °C. The mixture was stirred at room temperature overnight. After filtering off the resulting solid, the solution was evaporated under reduced pressure, and the residue was chromatographed on silica gel (1/1 methylene chloride/ethyl acetate as eluent) to provide 1.34 g ofthe activated ester. Without further purification, this product was directly dissolved in 10 mL of dried methylene chloride, and the resulting solution was cooled at 0 °C prior to the addition of 10 mL of trifluoroacetic acid. The reaction mixture was stirred at 0 °C for 2 hours, and the trifluoroactic acid was removed in vacuo at below 30 °C. The residue was dissolved in 100 mL of ethyl acetate and 10 mL of a saturated aqueous NaHCO₃ solution. The mixture was heated at 80 °C for 24 hours with vigorous stirring. After cooling to room temperature, 40 mL of water was added to the mixture, and the mixture was extracted with ethyl acetate (4 x 100 mL) and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was dissolved in 10 mL of acetonitrile, and 4 mL (40 mmol) of formalin, 1.0 g (16 mmol) of sodium cyanoborohydride, and 0.27 mL of acetic acid were added sequentially at 0 °C. The resulting solution was stirred at 0 °C for 2 hours. After quenching with phosphate buffer (pH = 2), the solvent was removed by evaporation. The residue was dissolved in ethyl acetate, and the solution was washed with water, saturated sodium bicarbonate, and brine, and dried over Na₂SO₄. After concentration, the residue was purified by chromatography on silica gel (9/1 ethyl acetate/methylene chloride as eluent) to give 7 (280 mg, 20% overall yield from 6). [α]²⁰ _D = -252° (c 1.3, CHC1₃); H NMR (300 MHz, CDC1₃) δ 7.15 (dd, J = 8.1 Hz, IH), 6.83 (br s, IH), 6..67 (d, J = 8.1 Hz, IH), 6.51 (d, J = 8.1 Hz, IH), 3.92 (m, IH), 3.82 (s, 3H). 3.75-3.18 (m, 4H), 2.78 (s, 3H), 2.70 (m, IH), 2.38 (m, IH), 1.08 (d, J = 7.2 Hz, 3H), (d, J = 7.2 Hz, 3H); MS m/z 292 (W), 261, 249, 221, 174, 162, 137, 1 14; HRMS calcd for C_l6H₂₄N₂O₃: 292.179, found: 292.173. All patents, patent applications, and publications cited herein, and specifically, U.S. Provisional Patent Application Number 60/017,532, are incoφorated by reference herein as though fully set forth.

Claims

CLAIMSWhat is claimed is:

1. A compound of formula I:

wherein

R, is H, (C,-C₅)alkyl, OR", SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C,-C₄)alkyl] or NHOH; R₂ is a (C₅-C₂₂) hydrocarbyl group, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof, or (C₆-C_I2)aryl(C₂-C₁₀)alkyl, wherein the alkyl moiety optionally comprises 1-2 double bonds, 1-2 triple bonds or a mixture thereof; wherein said (C₅-C₂₂) hydrocarbyl group or said (C₆- C_I2)aryl(C₂-C₁₀)alkyl may optionally be substituted with 1 or 2 substituents independently selected from the group consisting of halo, hydroxy, cyano, nitro, (C,-C₅)alkyl, (C,-C₅)alkoxy, trifluoromethyl, trifluoromethoxy, -C(=O)O(C,- C₅)alkyl, and N(R^e)(R^*);

R, and R₂ together are -CH(R^c)-CH₂-C(O)-N(R^d)-, -C(R^C)=CH- C(O)N(R^d)-, -C(R^c)=CH-N(R^d)- or -C(R^c)=CH-O-; R₃ is H, OH or halo;

R^a and R^b are independently H or (C_rC₅)alkyl;

R^c is a (C₅-C₂₂) hydrocarbyl group;

R^d is H or (C,-C₅)alkyl

R^e and R^f are independently hydrogen, (C,-C₅)alkyl, or (C,-C₅)alkanoyl, or together with the nitrogen to which they are attached are pyrrolidino, piperidino or moφholino; Z is H or (C_rC₅)alkyl; and

Y is H or (C,-C₅)alkyl; or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 wherein:

R, is OR\ SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C,-C₄)alkyI] or NHOH; and

R₂ is a (C₅-C₂₂) hydrocarbyl group, optionally comprising 1 -3 double bonds, 1-2 triple bonds or a mixture thereof, or (C₆-C₁₂)aryl(C₂-C_l0)alkyl, wherein the alkyl moiety optionally comprises 1 -2 double bonds, 1 -2 triple bonds or a mixture thereof; or

R, and R₂ together arc -CH(R^c)-CH₂-C(O)-N(R^d)-, -C(R^C)=CH- C(O)N(R^d)-, -C(R^c)=CH-N(R^d)- or -C(R^c)=CH-O-.

3. A compound of claim 1 wherein (C,-C₅)alkyl is methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, or cyclopentyl; and aryl is phenyl, methylphenyl, ethylphenyl, propylphenyl, dimethylphenyl, diethylphenyl, indenyl, methylindenyl, dimethylindenyl, naphthyl, methylnaphthyl, or dimethylnaphthyl.

4. A compound of claim 1 wherein R, is OR\ SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C,-C₄)alkyl] or NHOH.

5. A compound of claim 1 wherein R, is OR^a.

6. A compound of claim 1 or 2 wherein R₂ is 1 -decyl or 1-decynyl.

7. A compound of claim 1 or 2 wherein R₃ is H.

8. A compound of claim 1 or 2 wherein R^c is (C₅-C|₅)alkyl.

9. A compound of claim 1 or 2 wherein Y is H.

10. A compound of claim 1 or 2 wherein Z is methyl.

11. A compound of claim 1 wherein R, and R₂ together are -CFI(R^C)-

CH₂-C(O)-N(R^d)-, -C(R^c)=CH-C(O)N(R^d)-, -C(R^c)=CH-N(R^d)- or -C(R^c)=CH-O-.

12. A compound of claim 1 wherein Z is CH₃; Y is H; R, is OR^a, SR^a, N(R^a)(R^b), halo, NO₂, NHC(O)[(C_rC₄)aIkyl] or NHOH; R₂ is (C₅-C,₅)alkyl, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof; and R₃ is H; or a pharmaceutically acceptable salt thereof.

13. A compound of claim 1 or 2 wherein Z is CH₃; Y is H; R₂ is (C₅- C_I5)alkyl, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof; and R₃ is H; or a pharmaceutically acceptable salt thereof.

14. A compound of claim 1 or 2 wherein R₂ is 1-decynyl.

15. A compound of claim 1 or 2 wherein R₂ is (C₅-Cι₅)alkyl, optionally comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof.

16. A compound of claim 1 or 2 wherein R₂ is (C₆-C_I2)aryl(C₂- C_l0)alkyl, wherein the alkyl moiety optionally comprises 1-2 double bonds, 1 -2 triple bonds or a mixture thereof.

17. A compound of claim 1 which is (2S,5S)-8-(] -decynyl)- benzolactam 17; or a pharmaceutically acceptable salt thereof.

18. A compound of claim 1 which is (25',5S)-8-decylbenzolactam 18; or a pharmaceutically acceptable salt thereof.

19. A compound of claim 1 which is (2S,5S)-7-methoxy-8-(l- decynyl) benzolactam 8; or a pharmaceutically acceptable salt thereof.

20. A pharmaceutical composition comprising an amount ofthe compound of claim 1 effective to inhibit pathological proliferation of mammalian cells, in combination with a pharmaceutically acceptable carrier.

21. A therapeutic method to treat a condition characterized by the pathological proliferation of mammalian cells comprising administering to a mammal afflicted with such a condition, an effective amount of a compound of claim 1.

22. The method of claim 21 wherein the condition is a cancer.

23. A method for modulating PKC in a mammal comprising administering to said mammal a pharmaceutically effective dose of a compound of formula I; or a pharmaceutically acceptable salt thereof.