COMPOUNDS THAT INHIBIT COMPLEMENT AND/OR SUPPRESS IMMUNE ACTIVITY

Abstract: The present invention is directed to compounds which suppress immune responses and/or selectively inhibit complement. These compounds contain an aromatic ring and are substituted dihydrobenzofurans, spirobenzofuran-2(3H)-cycloalkanes, and their open chain intermediates. The compounds of the present invention, and the pharmaceutically acceptable salts thereof, interrupt the proteolytic processing of C5 to bioactive components, exhibit immunosuppressive activities, and have therapeutic utility in the amelioration of disease and disorders mediated by complement and/or immune activity.

Description: COMPOUNDS THAT INHIBIT COMPLEMENT AND/OR SUPPRESS IMMUNE ACTIVITY 1. FIELD OF THE INVENTION The present invention relates to compounds which . . . inhibit complement and/or possess immunosuppressive activ¬ ity. In particular, the compounds of the present inven¬ tion, and the pharmaceutically acceptable salts thereof, selectively inhibit complement at the C5 step of comple¬ ment activation. The compounds of the invention are substituted dihydrobenzofurans, spιrobenzofuran-2(3H)- cycloalkanes, and their open chain intermediates that exhibit such inhibitory activates. Compounds of the invention also include 6,7-disubstituted spirobenzofuran- 2(3H)-cycloalkanes and 4-substituted spirobenzofuran- 2(3H)-cycloalkanes. The invention relates to the use of these compounds for therapy of immune and/or inflammatory disorders. 2. BACKGROUND OF THE INVENTION 2.1. THE COMPLEMENT SYSTEM The complement system is a group of proteins that constitutes about 10 percent of the globulins in the normal serum of humans (Hood, L.E. et al. 1984, Immunolo- -TY' --- Edition, The Benjamin/Cummings Publishing Co. , Menlo Park, California, p. 339) . Complement (C) plays an important role in the mediation of immune and allergic reactions (Rapp, H.J. and Borsos, T. , 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts (Meredith) , New York) . The activation of C components leads to the generation of a group of factors, including chemotactic peptides that mediate the inflammation associ¬ ated with complement-dependent diseases. The sequential activation of the complement cascade may occur via the classical pathway involving antigen-antibody complexes, or by an alternative pathway which involves the recognition of certain cell wall polysaccharides. The activities mediated by activated complement proteins include lysis of target cells, chemotaxis, opsonization, stimulation of vascular and other smooth muscle cells, degranulation of mast cells, increased permeability of small blood vessels, directed migration of leukocytes, and activation of B lymphocytes, macrophages and neutrophils (Eisen, H.N., 1974, Immunology, Harper & Row, Publishers, Inc., Hagerstown, Maryland, p. 512). During proteolytic cascade steps, biologically active peptide fragments, the anaphylatoxins C3a, C4a, and C5a (See WHO Scientific Group, WHO Tech. Rep. Ser. 1977, 606, 5 and re erences cited therein) , are released from the third (C3) , fourth (C4) , and fifth (C5) native complement components (Hugli, T.E. CRC Crit. Rev. Immunol. 1981, 1, 321; Bult, H. and Herman, A.G. Agents Actions 1983, 13, 405) . The C5a fragment, a cationic peptide derived from the first 74 amino acids of the amino-terminus of the C5 alpha subunit (Tack, B.F. et al. Biochemistry 1979, 18, 1490) , is of particular pathological relevance. Regula¬ tion of C5a activity is by the endogenous plasma enzyme carboxypeptidase N (E.C. 3.4.12.7), which rapidly removes the carboxy-terminal arginine from C5a, producing the less potent but still active C5a des Arg. Reported effects of C3a and C5a upon specific immune responses are listed in Table I. TABLE I EFFECTS OF COMPLEMENT COMPONENTS C3a AND C5a ON SPECIFIC IMMUNE RESPONSES Immune Response C3a C5a/C5a des Arσ Specific antibody production in response to sheep red blood cells Suppression Enhancement Polyclonal antibody production in response to Fc antibody fragment Suppression Enhancement T cell proliferation in response to tetanous toxoid Suppression Enhancement T cell proliferation in mixed lymphocyte reaction No effect Enhancement T cell-mediated cytotoxicity Suppression Enhancement Among the wide variety of biological activities exhibited by C5a are contraction of smooth muscle (Wissler, J.H. Eur. J. Immunol. 1972, 2, 73), degranulation of mast cells (Johnson, A.R. et al., Immunol. 1975, 28, 1067), secretion of azurophilic granular enzymes from polymorphonuclear neutrophils (PMN) (Webster, R.O. et al., Immunopharmacol. 1980, 2, 201), and the chemotaxis of PMN (Wissler, J.H. Eur. J. Immunol. 1972, 2, 73; Becker, E.L. Trends Pharmacol. Sci. 1983, 4, 223) (Table II). TABLE II BIOLOGICAL EFFECTS OF C5a I. Stimulation of neutrophil functions involved in inflammation A. chemotaxis B. chemokinesis C. aggregation D. lysosomal enzyme release E. generation of toxic oxygen products II. Smooth muscle effects A. stomach smooth muscle contraction B. vasodilation II. Promotion of histamine release A. mast cells B. basophils IV. Immunoregulatory effects The active chemotactic factor in vivo is considered to be C5a des Arg (Becker, E.L. Trends Pharmacol. Sci. 1983, 4, 223) . The C5a or C5a des Arg fragments have been implicated in the infiltration of PMN (the chemotactic effect) in rheumatoid arthritis, certain forms of glomerulonephritis, experimental vasculitides such as the Arthus reaction, the acute pneumonitis produced by the instillation of chemo¬ tactic factors into the lungs of experimental animals with resulting release of leukotrienes C-4 and D-4 (LTC 4 and LTD 4 ) , etc. In addition, the interactions between C5a and neutrophils have been considered to underlie tissue damage in several clinical situations. For instance, there exists a growing body of evidence for the role of ' oxygen-derived free radicals in mediating myocardial tissue injury during myocardial ischemia and, in particu¬ lar, during the phase of myocardial reoxygenation and reperfusion. Among a number of possible sources of these radicals, the polymorphonuclear neutrophil has been the focus of primary attention. Studies have documented that neutrophil depletion or suppression of neutrophil function results in a significant salvage of myocardial tissue that is subjected to a period of regional ischemia followed by reperfusion (Simpson, P.J. and Lucchesi, B.R. J. Lab. Clin. Med. 1987, 110(1), 13-30). Neutrophil depletion in dogs resulted in significantly smaller myocardial infarcts after 90 minute occlusion with 24 hour reperfusion (Jolly, S.R. et al. Am. Heart J. 1986, 112, 682-690). One study documented the activation of complement and generation of oxygen-derived free radicals during cardio- pulmonary bypass. The administration of protamine during cardiopulmonary bypass further activated complement (Cavarocchi, N.C. et al., Circulation 1986, 74, 130-133; Kirklen, J.K. et al. J. Thorac. Cardiovasc. Surg. 1983, 86, 845-857) . Also, recombinant tissue plasminogen acti¬ vator (r-TPA) , which in recent clinical trials has been found to be an effective thrombolytic agent in patients with acute myocardial infarction, was shown to activate complement. A striking increase in the level of C4a, C3a, and C5a was found in patients receiving r-TPA as compared to the level of these complement peptides before adminis¬ tration of the drug (Bennett, W.R. et al. J. Am. Coll. Cardiol. 1987, 10(3), 627-632). Schafer and co-workers were able to positively identify the deposition of termi¬ nal C5b-9 complement complex in myocardial cells located within zones of infarction in human tissue (J. Immunol. 1986, 137(6), 1945-1949). Likewise, the selective accumu¬ lation of the first component of complement and leukocytes in ischemic canine heart muscle has been found (Rosen, R.D. et al. Circ. Research. 1985, 57, 119-230) . In one study, the depletion of complement was found to increase the blood flow in ischemic canine myocardium. This in¬ creased blood flow was found, in turn, to increase the 5supply and utilization of oxygen in complement depleted animals versus control animals (Grover, G.J. and Weiss, H.R. Basic Res. Cardio. 1987, 82(1) , 57-65) . Complement activation is also believed to initiate adult respiratory distress syndrome (ARDS) . This syndrome, also known as 10 adult respiratory failure, shock lung, diffuse alveolar damage, or traumatic wet lungs, is characterized clinical¬ ly by the rapid onset of severe life-threatening respira¬ tory insufficiency that is refractory to oxygen therapy. (Miescher, P. . and Muller-Eberhard, H.J., eds., 1976, 15 Text Book of Immunopathology, 2d Ed., Vols. I and II, Grune and Stratton, New York; Sandberg, A.L., 1981, in Cellular Functions in Immunity and Inflammation, Oppenheim, J.J. et al. , eds. Elsevier/North Holland, New York, p. 373; Conrow, R.B. et al. J. Med. Chem 1980, 23. 20242; Regal, J.F.; and Pickering, R.H. Int. J. Immunopharmacol. 1983, 104. 617). Some of the clinical implications of C5a release are listed in Table III. 5 0 5 TABLE III CLINICAL IMPLICATIONS OF C5a RELEASE Rheumatoid Arthritis Acute Gouty Arthritis Acute Immunological Arthritis Pulmonary Disorders Adult Respiratory Distress Syndrome Pulmonary Dysfunction - Hemodialysis Chronic Progressive Pulmonary Dis-Cystic Fibrosis Byssinosis Asbestos-Induced Inflammation Inflammation of Systemic Lupus Erythematosus Inflammation of Glomerulonephritis Purtscher ■ s Retinopathy Hemorrhagic Pancreatitis Renal Cortical Necrosis Primary Biliary Cirrhosis Inflammation Nephropathology Cranial Nerve Damage in Meningitis Tumor Cell Metastasis Extended Tissue Destruction in Myocardial Infarction Extended Tissue Destruction in Burns 2.2. CELL-MEDIATED IMMUNE RESPONSES A variety of immune responses independent of the complement system are known to be mediated by specifically reactive lymphocytes. These responses may give rise to autoimmune diseases, hypersensitivity, or simply allergic reactions. Some examples of these responses include delayed-type hypersensitivity, allograft rejection, graft versus host disease, drug allergies, or resistance to infection. Autoimmune disorders may include atrophic gastrititis, thyroiditis, allergic encephalomyelitis, gastric mucosa, thyrotoxicosis, autoimmune hemolytic anemia, and sympathetic ophthalmia (Eisen, H.N., 1979, Immunology, Harper and Row, Hagerstown, Maryland, pp. 557- 595) . 2.3. ORGANIC COMPOUNDS WHICH INHIBIT COMPLEMENT, AMELIORATE INFLAMMATION, AND/OR POSSESS IMMUNOSUPPRESSIVE ACTIVITY Many chemicals have been reported to diminish comple¬ ment-mediated activity. Such compounds include: amino acids (Takada, Y. et al. Immunology 1978, 34, 509); phosphonate esters (Becker, L. Biochem. Biophy. Acta 1967, 147, 289); polyanionic substances (Conrow, R.B. et al. J. Med. Chem. 1980, 23, 242); sulfonyl fluorides (Hansch, C.; Yoshimoto, M. J. Med. Chem. 1974, 17, 1160, and references cited therein) ; polynucleotides (DeClercq, P.F. et al. Biochem. Biophys. Res. Commun. 1975, 67, 255); pimaric acids (Glovsky, M.M. et al. J. Immunol. 1969, 102, 1); porphines (Lapidus, M. and Tomasco, J. Immunopharmacol. 1981, 3, 137); several antiinflammatories (Burge, J.J. et al. J. Immunol. 1978, 120, 1625); phenols (Muller-Eberhard, H.J. 1978, i.n Molecular Basi.s of Bi.olog¬ ical Degradative Processes, Berlin, R.D. et al., eds. Academic Press, New York, p. 65) ; and benzamidines (Vogt, W. et al Immunology 1979, 36, 138). Some of these agents express their activity by general inhibition of proteases and esterases. Others are not speci.fi.c to any parti.cular intermediate step in the complement pathway, but, rather, inhibit more than one step of complement activation. Examples of the latter compounds include the benzamidines, which block Cl, C4 and C5 utilization (Vogt, W. et al. Immunol. 1979, 36, 138). 2.4. SPIROBENZOFURAN-2.3H.-CYCLOALKANES AND K-76 K-76 is a fungal metabolite from Stachybotrys complementi nov. sp. K-76. Metabolite K-76 has a drimane skeleton combined with a benzene ring attached through a spirofuran, and has been determined as 6,7-diformyl- 3• ,4• ,4a' ,5' ,6• ,7' ,8' ,8a » -octahydro-4,6• ,7•-trihydroxy- 2 % ,5' ,5',8a'-tetramethyl spiro [1' (2'H)-naphthalene-2(3H)- gbenzofuran] (Kaise, H. et al. J. Chem. Soc. Chem. Commun. 1979, 726). The monocarboxylic acid derivative, K-76 COOH, is obtained when K-76 is selectively oxidized by silver oxide (Corey, E.J. and Das, J. J. Amer. Chem. Soc. 1982, 104, 5551). Both K-76 and K-76 COOH have been shown to inhibit complement mainly at the C5 step (Hong, K. et al. J. Immunol. 1979, 122, 2418; Miyazaki, W. et al. Microbiol. Immunol. 1980, 24, 1091). In a classical hemolytic reac¬ tion system, hemolysis of sensitized sheep erythrocytes by guinea pig serum was reduced 50% by K-76 at 7.45 x 10' 5 M, or K-76 COONa at 3.41 x 10- 4 M (Hong, K. et al. J. Immunol. 1979, 122, 2418; Miyazaki, W. et al. Microbiol. Immunol. 1980, 24, 1091). Similar results were observed in a hemolytic reaction system via the alternative pathway of complement activation. Both K-76 and K-76 COOH prevented the generation of a chemotactic factor from normal human complement (Bumpers, H. and Baum, J. J. Lab. Clinc. Med. 1983, 102, 421). K-76 has been shown to reduce the amount of protein excreted in urine of rats with nephrotoxic glomerulonephritis (Iida, H., et al., Clin. Exp. Immunol. 1987, 67, 130-134), and is reported to greatly increase the survival of mice with a spontaneous systemic lupus erythematosis-like disease and to suppress Forssman shock in guinea pigs and mice (Miyazaki, W. et al. Microbiol. Immunol. 1980. 24. 1091) . At high concentrations of K-76 or K-76 COOH, some inhibi¬ tion of the reactions of C2, C3, C6, C7, and C9 with their respective preceding intermediaries is exhibited. Howev¬ er, both compounds' inhibitory action is mainly the gener- . . ation in vitro of EAC1, 4b,2a,3b,5b (sensitized sheep erythrocytes carrying the indicated complement components) from C5 and EACl,4b,2a,3b; the acceleration of the decay of any EACl,4b,2a,3b,5b present; and blocking generation of the chemotactic peptides (Hong, K. et al. J. Immunol. 5 1981, 127, 109; Ramm, L.E., et al. Mol. Immunol. 1983, 20, 155) . K-76 COOH is also reported to be an anti-hepatitic agent (West German Patent Application, Publication No. 3,031,788, published March 12, 1981, by Shinohara, M. et 10al.), and possesses the ability to inhibit antibody-depen¬ dent cell-mediated cytotoxicity and natural killer lytic activity (Hudig, D. et al. J. Immunol. 1984, 133, 408-413) . K-76 or K-76 COOH has also been reported to inhibit the C3b inactivator system of complement (Hong, K. 15et al. J. Immunol. 1981, 127, 104-108). Semi-synthetic derivatives of K-76 have been patented as anti-allergy, anti-tumor, and anti-nephritic agents (Belgium Patent No. 867,095, published November 16, 1978, by Shinohara, M. et al.). The isolation of K-76, its uses in the treatment of 20 autoimmune diseases, and the preparation of its deriva¬ tives have been described in a number of patents (See Japanese Patent Applications (Kokai) , Publication Nos. 54 092680 (published July 23, 1979), 54 106458 (published August 21, 1979), 57 083281 (published May 25, 1982), by 25 shinohara, M. et al.; Japanese Patent (Kokoku) No. 85 030289 (published March 20, 1979)). A number of additional compounds which contain the substructure of a spirobenzofuran-2(3H)-cycloalkane are known. These compounds include griseofulvin (Weinberg, 30 E.D., 1981, in Principles of Medicinal Chemistry, 2d Ed., Foye, W.O., ed. , Lea & Febiger, Philadelphia, PA., p. 813), isopannarin (Djura, P. and Sargent, M.V. Aust. J. Chem. 1983, 36, 1057), and metabolites of Siphonodictyon coral1i-phaqum (Sullivan, B., et al. Tetrahedron 1981, 37, 35 979). The general synthetic methodology utilized for the synthesis of the compounds of the present invention in¬ volves regioselective aromatic lithiation reactions. Numerous oxygen-containing heterocycles have been made 5using regioselective aromatic lithiation reactions, but few preparations of dihydrobenzo[b]furans have been de¬ scribed (Narasimhan, N.S. and Mali, R.S. Synthesis 1983, 957) . Three reported syntheses of K-76 itself (Corey, E.J. and Das, J.J. J. Am. Chem. Soc. 1982, 104, 5551; loMcMurray, J.E. et al. Am. Chem. Soc. 1985, 107, 2712 Mori, K. et al. Ann. Chem. 1988, 107-119) utilize metalation techniques in the coupling of the terpenoid portion to the aromatic moiety, but neither provide sufficient flexibili¬ ty to allow for analog preparations via subsequent elabo- 15ration of the aromatic ring. 3. SUMMARY OF THE INVENTION The present invention is directed to compounds which suppress immune responses and/or selectively inhibit 0 complement. In a specific embodiment, such compounds interrupt the proteolytic processing of C5 to bioactive components, blocking the release of C5a. The compounds of the present invention also exhibit immunosuppressive activities, such as for example, the ability to inhibit 5 natural killer activity, lymphocyte proliferations, and T cell activation. The compounds of the present invention have therapeutic utility in the amelioration of disease and disorders mediated by complement and/or immune activi¬ ty. In specific embodiments, they may be used for the 0 treatment of autoimmune disease or the many diseases associated with the "inappropriate" activation of the complement system. In specific embodiments, a compound of the invention has greater activity than the natural prod¬ uct K76. 5 The invention further provides improved synthetic routes for the preparation of the compounds. In one embodiment of the present invention, compounds are provided which have selectivity in inhibition of C5a release. In particular, such compounds can have utility in limiting the extent of trauma-induced tissue destruc¬ tion, in the prevention and/or treatment of adult respira¬ tory distress syndrome and damage induced by ischemic heart conditions. In other specific embodiments, compounds of the invention which exhibit immunosuppressive activity can be used in the prevention and/or treatment of autoimmune disease or the rejection of transplanted organs and/or tissues. A further embodiment of this invention includes the combined therapy that can be obtained by treating patients with disorders that are routinely treated with thrombolyt- ic agents such as tissue plasminogen activator, streptoki- nase or urokinase (e.σ. myocardial infarction patients) with a combination of the compounds of this invention and the routinely administered thrombolytic compounds. The present invention is also directed to pharmaceu¬ tical compositions comprising such compounds or the salts thereof. 3.1 DEFINITIONS As used herein, the following abbreviations and terms shall have the meanings indicated: n-BuLi = n-butyllithium t-BuLi = tert-butyllithium t-BuSLi = lithium tert-butylthiolate C = complement CHO = Chinese hamster ovary CPM = counts per minute Et 2 0 = diethyl ether hexamethylphosphoric triamide immunoglobulin G isopropanol infrared the monocarboxylie acid derivative of K-76 the sodium salt of the monocarboxylic acid derivative of K-76 0 LAH lithium aluminum hydride MOM methoxymethyl group NK natural killer NMR nuclear magnetic resonance PBL peripheral blood 5 lymphocyte(s) PCC pyridinium chlorochromate PHA phytohemagglutinin PMN polymorphonuclear cells RLi alkyllithium 0 THF tetrahydrofuran TLC thin layer chromatography TMEDA N,N,N• ,N•-tetramethyl- ethylenediamine TMS tetramethylsilane 5 TriMEDA N,N,N• trimethy1ethylenediamine The term "bioisosteric" group, as used in the present invention, describes an alternative chemical group whose ^ electronic configuration is substantially analogous with the group to be replaced such that the polarity and charge of the whole molecule do not change. However, variations in the size, number of atoms or electron structure of the bioisosteric group (or "bioisostere") are permitted which 5 variations may affect its function. Bioisosteres may be acidic (e.g., capable of releasing a proton and, subse¬ quently, bearing a negative charge), basic (e.g., capable of being protonated and, subsequently, bearing a positive charge) or neutral (e.g., not normally capable of func- 5 tioning as an acidic or basic group) . Unless otherwise stated or indicated, the term "al¬ kyl" as used herein refers to methyl, ethyl, and n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl groups. The term "alkanol" denotes a compound derived from coupling an alkyl group and hydroxyl radical. Simi¬ larly, the term "alkoxy" refers to methoxy, ethoxy, n-pro- pyloxy, isopropyloxy, n-, iso-, sec, and tert-butoxy, phenoxy, benzyloxy groups and substituted derivatives thereof. The term "lower" refers to the numerical range of 1 to 4 carbon atoms and includes linear or branched skeletons. Unless otherwise stated or indicated, the term "halo¬ gen" as used herein includes fluorine, chlorine, bromine, and iodine. Unless otherwise stated or indicated, a given struc¬ ture, formula, or nomenclature for the substituted dihy- drobenzofuran analogs of this invention shall subsume all stereoisomers thereof. Unless otherwise stated or indicated, a reference made to a final compound of the invention which is a carboxylic acid, is also meant to include the salt form of such carboxylic acid such as alkali and alkaline-earth metal salts obtained therefrom. 4. BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 illustrates the infrared spectrum of com¬ pound lla. The spectrum is presented for the compound pelletized in potassium bromide. FIGURE 2 demonstrates the inhibition of complement peptide C5a or C3a production by compound lla. The inhi- bition (as described in Section 6.6, infra) of C5a and C3a production is shown as a function of compound lla concen¬ tration. FIGURE 3 demonstrates the inhibition of complement- mediated hemolysis by compound lla. Inhibition of comple¬ ment-mediated hemolysis, assayed as described in Section 6.6.2 infra, is shown as a function of compound lla con¬ centration. FIGURE 4 demonstrates the inhibition of proliferation of peripheral blood lymphocytes (PBL) by compound lla. 3 H-Thymidine incorporation by PHA-stimulated PBL is shown as a function of compound lla concentration. FIGURE 5 demonstrates the inhibition of proliferation of PBL by compound lla. 3 H-Thymidine incorporation by antiCD3 antibody-stimulated PBL is shown as a function of compound lla concentration. FIGURE 6 demonstrates the inhibition of interleukin-2 receptor (IL-2R) release from PBL by compound lla. The level of IL-2R in the supernatant of PBL cultures stimu- lated with anti-CD3 antibody, in the presence of compound lla, was measured by use of an enzyme-linked immunosorbent assay, and is shown as a function of compound lla concen¬ tration. FIGURE 7 shows the inhibition of CD8 protein release from PBL by compound lla. The level of CD8 in the super¬ natant of PBL cultures stimulated with anti-CD8 antibody, in the presence of compound lla, was measured by use of an enzyme-linked immunosorbent assay, and is shown as a function of compound lla concentration. FIGURE 8 demonstrates the inhibition of complement- mediated hemolysis by the disubstituted spirobenzofuran compounds 62, 66, and 68. Inhibition is shown as a func¬ tion of compound concentration . - 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compounds which inhibit complement and/or possess immunosuppressive activ¬ ity. The compounds of the invention contain an aromatic ring and are substituted dihydrobenzofurans, spirobenzonfuran-2(3H)-cycolakanes, and their open chain intermediates. In particular, such compounds can be partial analogs of the fungal metabolite K-76. The complement inhibitors of the invention inhibit C5 activation, that is, the proteolytic generation of bioactive complement fragments C5a and C5b from C5. Such compounds have value in the treatment or prevention of diseases or disorders associated with undesirable or inappropriate activation of the complement system. In specific embodiments, the compounds of the invention can be used in the treatment of inflammatory disorders. They may also be used for the treatment of cardiovascular disease. The present invention also relates to compounds which possess immunosuppressive activity. In particular, such compounds inhibit immune responses. In specific embodi¬ ments, the compounds of the invention can inhibit the killing activity of mononuclear cells, lymphocyte prolif¬ eration and/or activation. The immunosuppressive com- pounds of the invention can be valuable in the treatment of various immune disorders. Furthermore, the compounds of the invention may possess one or more of the K-76-like activities described supra in Section 2.4 and in the references cited therein. The compounds of the present invention, and the intermediates and methods used in their preparation, are described in detail below. 5.1. COMPOUNDS WHICH INHIBIT COMPLEMENT AND/OR SUPPRESS IMMUNE ACTIVITY The present invention relates to organic compounds which inhibit complement and/or suppress immune activity. The organic compounds of the invention comprise substi¬ tuted dihydrobenzofurans of the general formula 3 and substituted spirobenzofuran-2(3H)-cycloalkanes of the general formula 4. The groups represented by R and R^ t include, among others, hydrogen and linear or branched lower alkyl groups having 1 to 4 carbon atoms as defined previously in Section 3.1, supra. In addition, R ! -l_ 4 may each independently represent halogen, amino, amidic, hydroxy1, hydroxyalkyl, alkyloxy, nitro, formyl, acetal, carboxylic acid, trifluoroacetyl, N-substituted lower alkyl carbamoyl, substituted vinyl having up to 10 carbon atoms, an alkylidene group having up to 20 carbon atoms, an aliphatic acyl, a substituted aliphatic acyl, an aromatic acyl, a substituted aromatic acyl, a sulfamoyl, an aminomethyl, N-(lower alkyl) minomethyl, a N, N-di(lower alkyl) aminomethyl, a heterocyclic ring bearing at least one heteroatom selected from nitrogen, oxygen or sulfur (e.g., a tetrazole or oxazoline group), an N- acylcarbamoyl , an a idino or a hydrazide. Moreover, R R 2 and the carbon atoms to which they are attached may to- gether form a five-or six-membered ring (e.g., a cyclic anhydride, such as a phthalic anyhydride derivative; a lactone; or a hydroxy-substituted lactone) . Other groups which may be represented independently by R 3 and ^ include hydrocarbons of 4 to 24 carbon atoms 5which may be of medium-length, long-chain, linear, branched, cyclic, saturated, unsaturated, unsubstituted, or heteroatom substituted. Moreover R 3 , 4 , and the carbon atom to which they are attached may form a cyclic hydro¬ carbon group of 5-24 carbon atoms which may include a five-, six-, or seven-membered saturated or unsaturated ring comprised exclusively of carbon and hydrogen, or in combination with a heteroatom. The ring may be unsub¬ stituted or may contain extra-cyclic heteroatom or hydro¬ carbon substituents. This invention also relates to synthetic open chain intermediate compounds of the general formula 5 wherein R, R lf and R 2 are defined as above for formulae 3 and 4. In addition, R 5 represents hydrogen, lower alkyl groups, or suitable hydroxyl protecting groups such as methoxymethyl, tetrahydropyranyl, 2-methoxypropyl, 2-methoxyethoxymethyl, triarylmethyl, benzyl, methylthiomethyl, or tert-butyl- dimethylsilyl group. The Rg group encompasses chemical groups represented by R^ , as defined above for formulae 3 and 4 as well as substituent cyclohexenylmethyl (5a) , limonenyl (5b) , and carvone-derived diol acetonide (5c) groups. Other compounds may also be derived from intermediates 5 and 5a-c, which are, in turn, converted to products of general formulae 3 or 4 as discussed in the following sections. Table V lists representative com¬ pounds which comprise the general formula 4 of the present invention; this list is not intended to be comprehensive. * Substituents on the spirocyclohexane ring may also be present; e.g., 10-isopropenyl. The substituted dihydrobenzofuran and spiro- benzofuran-2(3H)-cyclohexane compounds of the present invention of the general formulae 3, 4, the synthetic intermediates of the general formula 5, and the salts thereof exhibit complement inhibition, as manifested by inhibition of complement-mediated C5a production and/or inhibition of complement-mediated hemolysis. The comple¬ ment-inhibitory properties of the compounds of the inven¬ tion can be evaluated by modification of known techniques, e.σ.. the assay described in Section 6.6.1, infra. Treatment of the compounds of the present invention with appropriate basic reagents provides pharmaceutically acceptable salts thereof. When a carboxylic acid group is present in the compounds of the invention, a pharmaceuti- scally acceptable ester can also be prepared by treatment with a suitable esterifying group under appropriate condi¬ tions. 5.2. SYNTHETIC PROCESSES Processes are provided which comprise chemical steps for the synthesis of the compounds of the invention. Such processes of the invention are diagrammed in detail in Scheme 1 (See infra Section 5.2.2). The syn¬ thetic scheme depicted in Scheme 1 provides a shorter and more flexible route than one based on the syntheses of K-76 (Corey and Das J. Am. Chem. Soc. 1982, 104, 5551; McMurray et al. J. Am.Chem. Soc. 1985, 107, 2712). Retro- synthetic evaluation of all the final compounds produced according to Scheme 1 ultimately results in two fragments; * an aliphatic and an aromatic portion. These two portions can be joined by using regioselective ortho-lithiation and subsequent alkylation. Two different strategies can then be employed for the production of the final compound of the invention: initial cyclization followed by aromatic functionalization or vice versa. 5.2.1. PREPARATION OF COMPOUNDS OF GENERAL FORMULA 5 The intermediates of the general formula 5 of the invention can be prepared by various methods depending on the types of substituents present therein. The aliphatic substituent, Rή, of compounds of the general formula 5 are derived from suitable alkylating agents. For example, l-bromomethylcyclohexene, 6a, can be used as the starting material for the preparation of compound 5a. Compound 6a can in turn be prepared from cyclohexanone or directly from alkyl cyclohexene- carboxylate, according to a known precess (Wheeler, O.H. and Lerner, I. J. Am. Chem. Soc. 1956, 78, 63; Adams, R. 5 and Thai, A.F. Org. Synth. 1932, 1, 270; Lythgoe, B. et al. J. Chem. Soc. 1956, 406). In a particular example, cyclohexanone is converted to its cyanohydrin, then dehy¬ drated to the cyanoalkene, followed by alcoholysis to an alkyl cycloalkenecarboxylate. Lithium aluminum hydride 0reduction of the ester followed by halogenation with phosphorus trihalide provides compound 6a. Limonenyl chloride, 6b, is obtained readily from limonenyl alcohol by the action of triphenylphosphine in excess halogenated solvent. This conversion provides 5optically active allylic halide from optically active limonenyl alcohol. A procedure analogous to that devel¬ oped by Crawford and co-workers (J. Am. Chem. Soc. 1972, 94, 4298) is used to prepare optically active alcohol by the sequential lithiation of commercially available homo- 0 chiral limonene, oxygenation, and reduction of the resul¬ tant hydroperoxide with aqueous sodium sulfite. A third halointermediate, 6c, is derived from a multistep sequence starting from optically active R-(-)- or S-(+)-carvone. The steps of the synthesis involve 5 reduction of the a,β-unsaturated ketone to the allylic alcohol, epoxidation, reduction to the diol, acetonide formation, and treatment of the unsaturated acetonide with calcium hypochlorite to yield the allylic chloride. The coupling reaction is generally carried out immediately ^ after purification of these unstable allylic halides. 5 •• " " O 6b ^ The aromatic segment of the compounds of the general formula 5 is obtained from substituted alkoxyphenols or resorcinols. For example, 3-methoxymethoxyanisole, 7, is obtained from the reaction of 3-methoxyphenol with chloro¬ methyl methyl ether in a stirred suspension of anhydrous potassium carbonate in acetonitrile (Rail, G.J. H. et al. Tet. Lett. 1976, 1033). Anhydrous conditions and the proper ratio of substrate to solvent are critical to the success of this reaction. For reactions employing quanti¬ ties of reagents above 5 grams, a useful alternative procedure uses the preformed sodium aryloxide, chloromethyl methyl ether, and dimethylformade as solvent (See, Rail, G.J.H. et al. supra) . The group R of formulae 5 and 5a-c should be stable o cleavage under the conditions used to hydrolyze or remove the protecting group R 5 and the subsequent cycliza- tion of the resulting free phenol according to steps 3 or 4 of Scheme 1. In addition, R 5 is preferably a potentially chelating group which can promote the regioselective ortho-metalation required to introduce substituents R 2 and Re of formula 5. Compound 7 embodies the preferred pro¬ tecting groups for resorcinol (R = methyl and R 5 = methoxy- methyl) . The methoxymethyl or MOM ethers can be easily removed in the presence of methyl ethers (Narasimhan, N.S. et al. Synthesis 1979, 906), and the ortho directing power of the MOM group has been shown to be greater than that of a methyl ether (Ronald R.C. Tet. Lett. 1975, 3973). In an alternative embodiment, the commercially avail¬ able compound 3,5-dimethoxybenzyl alcohol, 8, can be used as the aromatic segment for coupling to the allylic halide (See Scheme 2, infra.. The efficacy of the coupling reaction between the metalated aromatic segment and the aliphatic group is greatly influenced by a number of factors i•ncluding the degree of aggregation of the metalating species, typically an alkyllithiu reagent (Gschwend, H.W. and Rodriguez, H.R. Org. React. 1979 , 26, 1) . The choice of solvent and any additives such as N, N, N', N•-tetramethylethylene- diamine (TMEDA) or hexamethylphosphoric triamide (HMPA) , in turn, strongly influences this aggregation state. Solvent(s) , additive(s) , temperature, reaction time, and reagent stoichiometries for coupling the respective substrates should be optimized. Suitable solvents include 5anhydrous aprotic organic solvents such as tetrahydrofuran (THF) , diethyl ether (Et 2 0) , dioxane, and diglyme. Sur¬ prisingly, we have discovered that dry hexane is a pre¬ ferred solvent for selective metalation ortho to a chelat- ing group like OMOM and lithioalkoxymethyl (kinetic condi- IQtions), while THF/TMEDA solvent mixtures, generally, provide for metalation at the thermodynamic site, between the two inductively electron-withdrawing methoxy groups in 8, for example. In one particular embodiment, n-butyllithium (n-BuLi) in THF/TMEDA or hexane/TMEDA 15 allows for the coupling of aromatic segment 7 with allylic bromide 6a; however, only THF/TMEDA allows coupling of 6a with arpmatic segment 8 at the desired 4-position (thermo¬ dynamic) . When the coupling between 6a and 8 is carried out in hexane, lithiation and coupling occurs in the 02-position of 8 (the kinetically favored position) . The aromatic substrate is dissolved in the solvent such as dry THF and the like. The TMEDA is then added and is preferably approximately equimolar to the amount of alkyllithium used. In a preferred embodiment, the amount 5 of alkyllithium (RLi) slowly added at 0°C is approximately 1.1 equivalents per equivalent of a compound of the type such as 7 and is approximately 2.2 equivalents per equiva¬ lent of a substrate such as 8 that has an additional acidic hydrogen. Moreover, the addition of approximately " 1.2 equivalents of a copper salt per equivalent of the lithiated aromatic substrate is preferred to obtain opti¬ mum yields from the coupling reaction. Most sources of copper(I) are suitable including cuprous bromide dimethyl- sulfide, tetrakis (acetonitrile) copper(I) tetrafluoro- borate, and the cuprous halides. The use of cuprous iodide is preferred. Careful control of temperature is important for the success of the aromatic alkylation. While the lithiation is carried out at zero degree to ambient temperature, the subsequent reactions are carried out at low temperature. In a preferred example, the aryllithium reagent is cooled to -78°C followed by the addition of copper(I) salt. The resulting mixture is subsequently stirred at -40°C, a 10temperature at which the corresponding arylcuprate species can form and is relatively stable. The copper reagent is then recooled to -78°C prior to the addition of the elec- trophile. Carrying out the the addition at higher temper¬ atures can diminish the yields of coupled products such as 155. It is important that all manipulations discussed supra be performed in the absence of oxygen and moisture, and are preferably carried out under an inert atmosphere such as dry nitrogen or argon. The product mixture which is obtained from the cou- 0pling reaction can then be worked up by washing several times with a moderately basic aqueous solution, e.g.. saturated aqueous sodium bicarbonate (NaHC0 3 ) . The organic phase can be dried by shaking with a drying agent such as magnesium sulfate, filtered from any solids, and concen- 5 trated under a mild vaccum to provide the crude product. Further purification may then be effected by procedures typically employed in the art (e.g.. fractional distilla¬ tion, fractional crystallization, or chromatographic separation) . 0 5.2.2. PREPARATION OF COMPOUNDS OF THE GENERAL FORMULAE (3 AND 4. Substituted dihydrobenzofuran derivatives of the general formula 3 with substituents OR and 1 - 4 as defined 5 supra in Section 5.1 can be obtained by the metalation and subsequent alkylation of coupled intermediates such as those described in the preceding section, followed by hydrolysis of the protecting groups and cyclizaiton of the ene-phenol. This synthetic pathway is illustrated in Scheme 1, infra. Scheme 1 11 14a (a) .3uli,TH. TWEDA; (b)Cul; (c)R»X: (d) " feuLi, r-«n_/TMEDA; <*)E * (R 2 ); P.H * . iOH; (s)Amt»r*y«rβin; (h) __JSU HM=>A Scheme 1 .cont'd) 20c 23a ( 3. RR,- H.R 2 -COOH.R 3 . Ci-j. R_ ■ .-maihyleyeol-zan-i-v: ) (a) "BuU. THFTMEDA; (b) Cu': 'c) R j X; (d) "BuU UtxanaTMEDA; (β)E* (R2); (!) H * . -τD . (ς) Ambβiyβ r-βin; (h) *___£___. HfcPA; (1) aq 40% OH In a preferred embodiment, the protected resorcinol is lithiated and alkylated in step 1 of Scheme 1 using the allylic bromide 6a as the electrophile. The resulting coupled product 9a can then be dissolved in dry hexane and treated dropwise with alkyllithium in the presence of TMEDA (step 2) at 0°C. Cooling the reaσtion mixture to -78°C and addition of the desired electrophile introduces the R_ substituent at this stage. Generally, the MOM protecting group of intermediates such as 9, with or without the R 2 substituent, may be hydrolyzed by stirring the compound in 5% hydrochloric acid (HCl)/ether mixtures overnight. Deprotection to the free phenol may also be effected by a solvent mixture comprised of 4 N HCl/'PrOH, especially for carboxaldehyde containing derivatives, which need to be stirred for several days. The cyclization step can be achieved most conveniently by stirring the substrate in the presence of Amberlyst resin in a non-polar solvent followed by filtra¬ tion. In this particular process, the resin is washed with fresh solvent, and the filtrates are combined and concentrated to give the desired crude cyclized product in good yield. In the particular case where R 2 = -COOH in intermediate 10a (Scheme 1, step 2, E + = C0 2 ) , the cycliza¬ tion of the phenol (steps 3 and 4) can be achieved by stirring with Amberlyst resin in benzene for 24-72 hours at room temperature. Some difficulty is encountered in the cyclization of intermediates with hydroxymethyl substituents (e.g., R 2 = -CH 2 OH) . A different route is preferred for synthesis of cyclized products with this substituent (see below) . As discussed supra, a variety of R 2 substituents may be introduced before the cyclization step. For example, addition of the following electrophiles to the aryllithium species produces the indicated functional group (these examples serve only to illustrate a possible technique and are not meant to be limiting) : carbon dioxide (carboxyl) ; ethyl chloroformate or diethyl carbonate (carbethoxy) ; dimethylformide (formyl) ; methylisocyanate (N-methyl- carbamoyl) ; tert-butylisocyanate (N-tert-butylcarbamoyl) ; paraformaldehyde (hydroxymethyl) ; trifluoroacetic anhy¬ dride (trifluoroacetyl) ; bromine (bromide) ; chlorine (chloride) ; iodine (iodide) ; substituted vinyl iodides (substituted vinyl group) . Other compounds may, in turn, be obtained by modification of these functional groups by known methods. The cyclized methyl ether derivatives of the type, 11, can be converted to the free phenols, 14 (Scheme 1, step 5) , by known methods. These procedures include but are not limited to treatment of the methyl ether with boron trihalides (McOmie, J.F.W. and West, D.E. Org. Synth. 1973, 5, 412; Grieco, P.A. et al. J. Org. Chem. 1975, 40 1450) , boron trifluoride in the presence of thiols (Fujita, E. et al. Ibid. 1979, 44., 1661; Fujita, E. J. Chem. Soc. Perkin Trans. 1 1976, 44, 4444)-, and lithium tert-butylthiolate salts (t-BuSLi) in hexamethylphosphoric triamide (McMurry, J.E. and Erion, M.D. J. Am. Chem. Soc. 1985, 107, 2712). The use. of t-BuSLi is preferred. Open-chain coupled products such as 9b or 9c (See Scheme 1) give rise to compounds such as 21 or 25, respec¬ tively. The latter intermediates, wherein the olefinic bond involved in the cyclization is exocyclic, or other derivatives containing simple unsaturated aliphatic groups, produce dihydrobenzofuran derivatives disubsti- tuted at the 2-position upon deprotection and treatment with Amberlyst ion-exchange resin (See e.g., 20). t 20c R j - COOEl Slightly more complex analogs containing a diol group, such as 24 or 27, infra. can be obtained in this manner. The use of 3,5-dimethoxybenzyl alcohol, 8, as the starting aromatic component has the advantage of having the R, substituent already in place. Compound 8 is dis¬ solved in THF and is treated at 0°C with 2.0 equivalents of n-butyllithium in the presence of TMEDA (2.0 equiv) . The benzyl alcohol is lithiated predominantly at the 4-position after about 2 hours. Formation of the arylcup¬ rate at -40°C and addition of the alkylating agent com¬ pletes the synthesis of 13 (Scheme 2, step 1) with a small amount of the isomer 13' also being formed. Fractional crystallizaiton of the product mixture provides pure 13. As previously alluded to, cyclization of interme¬ diates with hydroxymethyl substituents can be problematic. In one embodiment, a remedy to this problem involves converting the benzyl alcohol 13c to the formyl compound 13b (See Scheme 2, step 2). This procedure can be accom¬ plished by a Swern oxidation or, more preferrably, by using pyridinium chlorochromate in "buffered" methylene chloride. One of the symmetrical methyl ethers is then selectively cleaved (step 3) and cyclized in the normal fashion to yield the 6-substituted formyl analog 30b. Oxidation of the formyl group to the carboxylic acid (e.g.. 30a or 3la) can be effected by a number of reagents either before or after the cyclization of step 4. Suit- able inorganic oxidizing agents include but are not re¬ stricted to permanganate salts, manganese oxide, chromic acid, chromate salts, silver oxide, silver nitrate, nickel peroxide, and cerium salts such as cerium sulfate, cerium oxide and cerium perchlorate. Silver oxide is preferred. The benzyl alcohol, 30c (4, R = CH 3 , R x = CH 2 0H, and R_ = H) , is obtained by reduction of 30b using, for example, such reducing agents as lithium aluminum hydride, or diborane, preferably in an appropriate solvent such as ether , e.g. , THF, dioxane, or diethyl ether. Sche e 2 13 (major) 13' (minor) 13c 13b 15b 30b 31b 31a (a) "BuU. THFTMEDA; (b)Cul; (c)R»X: (e CC. NaOAe. CH 2 CI 2 : (•) 'BuSU. HMPA; (T) Aπ_____y_l ream; (g) AςjO. aq.5% NaOH Even more complex dihydrobenzofuran-based compounds can be obtained by further modification of the analogs described above. Any of the compounds containing the methyl ether groups at the 4-position of the dihydrobenzo- 5furan skeleton can be cleaved by one of the boron or thiolate reagents mentioned supra. Sometimes, as in the limonene series, it may be desirable to carry out the demethylation of the methyl ether with the carboxylic acid group at the 7-position protected as its alkyl ester (See, e.g., Scheme 1). Open positions in the phenyl ring may be metalated and functionalized as disclosed previously. The double bond of compound 20a can be hydroxylated by transi¬ tion metal oxide reagents such as potassium permanganate or osmium tetroxide. The combination of methylether cleaveage and hydroxylation of 20a, for example, would give compound 24a. Alternatively, compound 24a may be obtained by demethylation of intermediate 27c to the free phenol. In those instances, as in here, where an alkyl ester is employed to protect the carboxylic acid group at the 7-position, an alkaline, hydrolysis step may be neces¬ sary to obtain the free carboxylic acid (Scheme 1, step i) . Other modifications can be envisioned which do not depart significantly from the scope and essence of the products of the present invention. Transformations such as epoxidation of the double bond, hydroxylation at the allylic position of the double bond, oxidate cleavage of the double bond, hydroformylations, and modifications of the products obtained therefrom are but a few non-limiting examples. 5.2.3. PREPARATION OF COMPOUNDS OF GENERAL FORMULA 6-CARB0XYL-4-SUBSTITUTED SPIRO[BENZOFURAN -2.3H.-CYCLOHEXANES1 6-carboxyl spiro[benzofuran-2(3H)-cyclohexane] mole¬ cules that are also substituted at the 4 position to form a series of ether substituted derivatives can be prepared as shown in Schemes 3 and 4 below and as more fully de- scribed in Section 7 infra. Sc eme 3 44a R-CΞ, 44b R-<_H„CE ' _ J Scheme 4 PCS __L g ) SO » ι-_ These compounds and the salts thereof exhibit complement inhibition as manifested by inhibition of complement mediated hemolysis. Other complement-inhibitory proper¬ ties of these compounds can be evaluated by known tech- niques as described in Sections 6.6.1 and 10.2 to 10.4 and by numerous complement assay techniques that are known in the art. Substituents for R in position 4 of compounds of the general formula 4 include a hydrogen atom or a lower alkyl group (e.g. CH 3 -, CH 3 CH 2 -, n-Bu-) , a functionalized lower alkyl group (e.g. HOCH 2 CH 2 -) , a benzyl or substitut¬ ed benzyl group, a phenyl or substituted phenyl group fe.q. C 6 H 5 CH 2 -, Cft" . p-N0 2 C 6 H 4 -, p-CHOC 6 H 4 -, p-N0 2 C 6 H 4 -, and p-NH 2 C 6 H 4 -) . In addition the OR at position 4 of compounds of the general formula 4 can instead be H-. Preferred substitutions at position 4 are exemplified by compounds 44b, 55c and 55e, which are more effective in inhibiting complement mediated hemolysis than is K76COOH. 5.2.4. PREPARATION OF COMPOUNDS OF GENERAL FORMULA - 6, 7 -DISUBSTITUTED -4-METHOXYSPIRO .BENZOFURAN-2f3H.-CYCLOHEXANES " ) 6 ,7-disubstituted spiro[benzofuran-2(3H)-cyclohexane] molecules can be prepared as shown in Schemes 5 and 6 below and as more fully described in Section 8 infra. Scheme 5 56 58 59 65 67 64 63 Scheme 6 W~ (295. eorr.. 63 ~ SMi I9C. f*I - co lib 65 6S The naturally occurring compound K76 has disubstituted formyl groups in positions 6 and 7 of the ring D as shown below. Its oxidized derivative K76COOH has a carboxyl group at position 6 and a formyl group at position 7. R The compounds of this section differ from 76, since they represent disubstituted BCD analogues lacking the A ring structure of K76. In addition the compounds of this section can also be substituted at position 4 of the D ring to form trisubstituted BCD analogues. These di- and tri-substituted compounds and the salts thereof exhibit complement inhibition as manifested by inhibition of complement mediated hemolysis. Other complement-inhibito¬ ry properties of these compounds can be evaluated by known techniques as described in Sections 6.6.1 and 10.2 to 10.4 and by numerous complement assay techniques that are known 5 in the art. The j and R- groups can be any combination of the following: a hydrogen atom, a carboxylic acid group, a formyl group, a hydroxymethyl group, an N-(lower alkyl) carbamoyl group, a trifluoroacetyl group, a carbalkoxy 0 group, a halide group, a vinyl group, a substituted vinyl group having up to 10 carbon atoms, an alkylidene group having up to 20 carbons, an aliphatic acyl group, a sub¬ stituted aliphatic acyl group, an aromatic group, a sub¬ stituted aromatic acyl group, a trifluoroacetyl group, a 5 sulfamoyl group, an N-acylcarbamoyl group, a tetrazole group, a tertiary aliphatic amine group, an oxazoline group, an amidine group, or a hydrazone group. Specific substitutions for Rl and R2 groups include -CHO, -CH 2 OH, -COOH, COCF 3 , S0 2 NH 2 and tetrazole, oxazo- 0 line, imide or CH 2 NMe 2 derivatives. Substitutions at posi¬ tions 6 and 7 can be cyclic compounds as exemplified by compounds 62 and 68. The presence of polar groups in the 6 and 7 positions appears to affect complement inhibition activity, perhaps because such polar groups interact with 5 regions of the complement receptors. Specific compounds include compounds of general formula 4 in which R can vary generally as described above; R j is a carboxyl group or a bioisosteric acid group (such as sulfonamide, imide, or tetrazole) or a bioisos- υ teric basic group (such as a tertiary aliphatic amine, oxazoline, amidine, or hydrazone) , or a bioisosteric neutral group (such as trifluoroacetyl) ; R 2 is a formyl group or a bioisosteric group such as methyl ketone (ace- tyl) , other alkyl ketone, aryl ketone or other similar 5 group. A preferred organic compound of the compositions of this invention is the 4,6,7 trisubstituted spiro[benzofuran-2(3)H cyclohexane] such as compound 68 (6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclo- 5 hexane]) and 6-carboxy-7-formyl-4-phenoxy-spiro[benzo- furan-2(3H)-cyclohexane] . In a specific embodiment, compound 68 exhibits a relatively large amount of com¬ plement inhibition in the hemolysis assay. Even more inhibitory compounds can be produced by combining the optimal subsitutions at the 4 position with the 6,7 disub- stitutions present in 68. In a preferred embodiment, a phenoxy group is substituted at the 4 position. 5.3. DEMONSTRATION OF COMPLEMENT INHIBITION The compounds of the invention can be assayed by any techniques known in the art in order to demonstrate their complement inhibiting activity. Such assays include but are not limited to the following in vitro tests for the ability to inhibit complement system activity or to selec- tively inhibit the generation of complement-derived peptides (See Sections 6.6 and 10.3 for specific exam¬ ples) : (i) measurement of inhibition of complement-me¬ diated lysis of red blood cells (hemolysis) ; (ii) measurement of ability to inhibit formation of C5a and C5a des Arg and/or measurement of ability to inhibit formation of C3a and C3a des Arg; (iii) inhibition of alternative pathway mediated hemolysis. Those compounds which are demonstrated to have sig¬ nificant complement-inhibiting activity can be therapeuti- cally valuable for the treatment or prevention of diseases or such as those described in Section 5.5, infra. 5.4. IMMUNOSUPPRESSIVE ACTIVITY The compounds of the present invention can inhibit immune activity. In particular, the compounds of the invention inhibit cell-mediated immune function. For example, the compounds can suppress natural killer activi¬ ty, inhibit the proliferation of peripheral blood lympho¬ cytes, and/or inhibit the activation of T lymphocytes in PBL culture. Any procedure known in the art may be employed to demonstrate immunosuppressive activity. Such procedures include but are not limited to in vitro assays for inhibi¬ tion of natural killer lysis of target cells, inhibition of proliferation of peripheral blood lymphocytes or inhi¬ bition of cell surface interleukin-2 receptor expression. Specific embodiments of assay procedures which can be used are detailed in the examples sections infra (See Subsec¬ tions 6.7.1 through 6.7.4). 5.5. THERAPEUTIC USES OF THE COMPOUNDS OF THE INVENTION The compounds of the invention which exhibit com¬ plement and/or immune activity inhibition have therapeutic value in the prevention or treatment of various immune or inflammatory diseases or disorders. The compounds of the invention may be administered to a patient for treatment of an immune disorder involving undesirable or inappropri¬ ate complement activity. In particular, an effective dose of an inhibitive compound of the invention may be thera- peutically applied to ameliorate or to prevent a detrimen- tal effect caused by the activity of a component of the complement sys.tem (e.g. , C5a) or an inappropriately reac¬ tive immune system. An effective dose of the compound of the invention for the treatment of a disorder involving undesirable or inappropriate complement activity or an immune disorder can be determined by standard means known in the art taking into account routine safety studies, toxicity studies, dose concentration studies and method of delivery, e.g., bolus, continuous or repeated. In a particular embodiment, a dose of about 0.01 to about 500 mg/kg can be administered. The diseases or disorders which may be treated by the compounds of the invention include but are not limited to those listed in Table III, supra. In particular, those disorders associated with extended zones of tissue de- struction due to burn- or myocardial infarct-induced trauma, and adult respiratory distress syndrome (ARDS) , also known as shock lung, can be treated by administration of an effective amount of the compounds. Detrimental nonspecific activation of the complement system, or unfavorable activation by the alternative pathway, can also be prevented or treated by compounds of the invention. In specific embodiments, such compounds can ameliorate the acute pathological changes induced by specific or non-specific proteolytic processing of C5. The compounds of the invention may also be used to modulate biologic or immune functions directly or indi¬ rectly mediated by the complement system, which can in¬ clude but are not limited to those functions listed in Tables I and II, supra. and the in vivo correlates of the in vitro functions therein. In particular embodiments, the inhibitive compounds can be used to treat inflammation associated with, for example, kidney stones, systemic lupus erythematosis (SLE) , nephrotoxic glomeronephritis, or multiple sclerosis (See, e.g., Experimental Allergic Encephalomyelitis. A Useful Model for Multiple Sclerosis, A Satellite Confer¬ ence of the International Society of Neurochemists, July 16-19, 1983, University of Washington, Seattle, Washing¬ ton; Miyazaki, W. et al. Microbiol. Immunol. 1980, 24, 1091; Konno, S. and Tsurutuji, S. Sr. J. Pharmacol. 1983, 80, 269). In yet another embodiment, the compounds of the invention can be administered for treatment of tissue damage due to myocardial ischemia and reperfusion, result¬ ing from neutrophils attracted by and activated by the complement system. The compounds of the invention may also be adminis¬ tered for the prevention or treatment of diseases or disorders caused or accompanied by increased lymphocyte or natural killer activity, including but not limited to atrophic gastritus, thyroiditis, allergic encephalomyeli- tits, gastric mucosa, thyrotoxicosis, autoimmune hemolytic anemia, pemphigus vulgaris, sympathetic opthalmia, de- layed-type hypersensitivity, rejection of allografts, graft-host reaction, organ transplant rejection, other autoimmune disorders, and drug allergies. They can also be used to alleviate the adverse effects of complement activation caused by therapeutic intervention such as 'tissue plasminogen activator therapy or cardiopulmonary bypass. Pharmaceutical compositions comprising the inhibitive compounds or the salts thereof are provided by the present invention. Such compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. Such a carrier includes but is not limited to saline, buffered saline, dextrose, and water. A pharmaceutical kit comprising one or more containers filled with one or more of the ingredients of the pharma- ceutical composition is also within the scope of the invention. Various delivery systems (e.g.. encapsulation in liposomes, icroparticles, or microcapsules, conjugation to specific molecules) are known and can be used for therapeutic delivery of the compounds. Methods of admin- istration include but are not limited to oral, intrader- al, transdermal, intravenous, subcutaneous, intramuscu¬ lar, intraperitoneal, and intranasal routes. Such adminis¬ tration can be done in either bolus or repeat doses or 5 continuously by infusion for instance. A further embodiment of this invention includes the combined therapy that can be obtained by treating patients with disorders (e.g. myocardial infarction patients) that are routinely treated with thrombolytic agents such as tissue plasminogen activator, streptokinase or urokinase with a combination of the compounds of this invention and the routinely administered thrombolytic compounds or a fibrinolytically active fragment, derivative, or modified version thereof. The usefulness of such a combined thera- PY derives from the observation that the complement system is activated in disorders such as myocardial infarction or bypass surgery. The efficacy of a combined treatment could be substantially better than the thrombolytic treat¬ ment alone due to the ability of the complement inhibitory compounds to modulate the inappropriate and damaging complement activation. The administration of the throm¬ bolytic and complement inhibitory compounds can be simul¬ taneous or sequential or in different dose forms including combinations of oral dose forms with injectables to name just a few. The invention can be better understood by referring to the following examples which are given for illustrative purposes only and are not meant to limit the invention. 6. EXAMPLES 6.1. 2-(1•-CYCLOHEXENYL)METHYL-3- METHOXYMETHOXYANISOLE (9 . The 3-methoxymethoxyanisole, 7, was obtained in 96% yield by the following procedure. A mixture of finely powdered anhdryous potassium carbonate (K 2 C0 3 ; 2.0 equiv) and 3-methoxyphenol (1.0 equiv) in dry acetonitrile was stirred at 0°C for 15 minutes under nitrogen. To ensure that the reaction pH remained above six, 100 ml of aceto¬ nitrile were used per gram of phenol substrate. A - catalytic amount of 18-crown-6 (0.12 equiv) was added, and the mixture was stirred an additional 15 minutes at 0°C. Neat chloromethyl methyl ether (1.5 equiv) was then intro¬ duced slowly. The suspension was allowed to warm up to ambient temperature and stirred for 6 hours. After this 10 time, the mixture was recooled to 0°C, and one-half the original quantities of K 2 C0 3 , 18-crown-6, and CH 3 0CH 2 C1 were added. After another 4 hours of stirring at room tempera¬ ture, the suspension was filtered and the filtrate was concentrated under reduced pressure. The residue was 15 dissolved in ethyl either (Et 2 0) , washed with 5% sodium hydroxide (3 x 50 ml) , concentrated in vacuo, and dis¬ tilled under reduced pressure. Compound 7 was obtained as a clear colorless liquid. Bp 45°C (0.15 mm Hg) . *H NMR (90 MHz, deuteriochloroform) δ 7.16 (IH, m) , 6.61 (3H, m) , 205.16 (2H, S) , 3.79 (3H, s) , and 3.49 (3H, s) in ppm downfield from TMS. ,3 C NMR (CDC1 3 ) δ 160.5,m 158.2, 129.7, 108.2, 107.3, 102.5, 94.3, 55.9, and 55.1 in ppitv downfield from TMS. n-BuLi (1.1 equiv) was added slowly to a THF solution 25 of compound 7 (1.0 equiv) and TMEDA (1.1 equiv), at 0°C under a nitrogen atmosphere. The solution was stirred at room temperature for 2-5 hours and then cooled to -78°C. Cuprous iodide (1.2 equiv.) was added all at once. The light gray suspension was warmed up to -40°C, and after -" stirring for 1.5 hours, turned into a green-gray color. The copper reagent was cooled to -78°C and allowed to react with a THF solution of freshly-prepared allylic bromide 6a (1.3 equiv). The reaction mixture was allowed to warm up to ambient temperature gradually and stirred 35 for up to 72 hours. The mixture was quenched and washed with a saturated aqueous solution of sodium bicarbonate until the aqueous layer became colorless. The organic layer was dried by passage through a plug of potassium carbonate and concentrated in vacuo to give a dark orange c oil. The crude product was distilled under reduced pres¬ sure to provide compound 9a in 69% yield. Bp 105°C (0.15 mm Hg) . Anal. Caqlcd. for C^H^: C, 73.25; H, 8.45. Found: C, 73.13; H, 8.50. l E NMR (90 MHz, CDCL 3 ) δ 7.03 (IH, t, J = 8Hz) , 6.71 (IH, d, J = 8 Hz) , 6.54 (IH, d, J 0 = 8 Hz), 5.23 (IH, broad s) , 5.13 (2H, s) , 3.77 (3H, s) , 3.43 (3H, s) 3,32 (3H, broad s) , 1.95 (4H, m) , and 1.57 (4H, m) in ppm downfield from TMS. 13 C NMR (75 MHz, CDC1 3 ) S 158.5, 155.8, 136.3, 126.7, 120.0, 118.1, 107.0, 104.6, 94.3, 55.8 (2C'S), 30.9, 28.8, 25.3, 23.1, and 22.6 ppm downfield from TMS. IR (neat) 2940, 2840, 1595, 1470, 1440, 1260, 1160, 1105, 1070, and 1025 cm' 1 . 6.2. 3-(1 • -CYCLOHEXENYL)METHYL-2-HYDR0XY-4- METHOXYBEN20IC ACID (12a. • Compound 9a was metalated at the 4-position by the following procedure. A hexane solution of 9a (1.0 equiv) and TMEDA (1.1 equiv) was treated with a hexane solution of n-BuLi (1.1 equiv) added gradually at 0°C under an atmosphere of nitrogen. The solution was stirred at room temperature for 3 hours and then cooled to -78°C. Li-9a The aryllithium reagent Li-9a prepared by the above route was then exposed to a stream of dried carbon dioxide gas bubbled through the -78°C solution for 0.5 hour. Carrying out this reaction at 0°C cuts the resulting yield in half. The mixture was allowed to warm up to room temperature while maintaining a steady stream of gas. The - mixture was poured into water and extracted a few times with 5% aqueous sodium hydroxide, then acidified to pH 1 with concentrated hydrochloric acid, and then extracted into ether. The crude product was back-extracted into Et 2 0 and the combined organic layers dried over magnesium sulfate. The solvent was evaporated and the residue redissolved in a minimum of boiling ether. The warm solution was allowed to cool slightly, and then hexane was added to the point of cloudiness. The mixture was then allowed to stand in the freezer. An off-white solid with a melting point of 161-163°C was harvested (66% yield). Anal. Calcd. for C 15 H 18 0 4 : C, 68.69; H, 6.92. Found: C, 68.75; H, 6.95. >H NMR (90 MHz, acetone-d δ 7.81 (IH, d, J = 9 Hz), 6.62 (IH, d, J = 9 Hz), 5.26 (IH, broad s) , 4.26 (2H, broad s) , 3.90 (3H, s) , 3.29 (2H, broad s) , 2.01 (4H, m) , and 1.57 (4H, m) in ppm downfield from TMS. 13 C NMR (acetone-d δ 173.0, 164.3, 162.1 (2C's), 136.5, 130.6, 121.0, 115.9, 106.5 (2C's), 103.3, 56.3, 30.0, 29.4, 25.8, 23.8, 23.2 (2C's). IR (KBr) 1650, 1610, 1500, 1455, 1265, 1185, and 1090 cm" 1 . 6.3. 3-(1'-CYCLOHEXENYL)METHYL-2-HYDROXY-4- METHOXYBENZALDEHYDE .12b. A solution of aryllithium reagent Li-9a, prepared in situ by the procedure described in Section 6.2, was cooled to -12°C and treated with neat N,N'-dimethylformamide (1.5 equiv) added all at once. The mixture was stirred at room temperature for 3 hours. The mixture was then poured into water, saturated with sodium chloride, and extracted with Et 2 0. The organic layers were combined, dried (MgS0 4 ) , and concentrated in vacuo; the residue was recystallized from ether-hexane (See Section 6.2). The product was purified by column chromatography (silica, ether/hexane eluent) . Mp 48-49°C. Anal.Calcd. for Cι 5 H 18 0 3 : C, 73.15; H, 7.37. Found: C, 73.22; H, 7.40. l H NMR (CDC1 3 ) δ 11.42 (IH, broad s) , 9.64 (IH, s) , 7.33 (IH, d) , 6.51 (IH, d) , 5.23 (IH, broad s) , 3.83 (3H, s) , 3.26 (2H, broad s) , 1.96 (4H, m) , and 1.56 (4H, m) in ppm downfield from TMS. I3 C NMR (CDC1 3 ) 194.7, 164.6, 161.3, 135.5, 133.8, 120.6, 115.9, 115.6, 103.1, 55.9, 29.9, 28.8, 25.2, 23.0, and 22.4 in ppm downfield from TMS. IR (KBr) 2930, 2840, 1625, 1495, 1255, 1100, 800, and 640 cm "1 . 6.4. 7-CARBOXY-4-METHOXYSPIRO[BENZOFURAN- 2f3H.-CYCLOHEXANE! .lla. A benzene solution of compound 12a (Section 6.2) was treated with dry Amberlyst ion-exchange resin (approxi¬ mately 3-4 g per gram of substrate, dried at 110°C under high vacuum) added in one portion. The mixture was stirred for up to 24 hours at room temperature and then filtered. The resin beads were washed thoroughly with fresh benzene and methylene chloride. The filtrates were combined, washed with water, dried over magnesium sulfate, and concentrated in vacuo. The product lla was purified in 85% yield by column chromatography. Mp 192-194°C. Anal. Calcd. for C I5 H 18 0 4 : C, 68.69; H, 6.92. Found: C, 68.52; H, 6.99. l κ NMR (CDC1 3 ) δ broad roll centered at about 12.0 (IH) , 7.85 (IH, d, J = 9 Hz) , 6.50 (IH, d, J = 9 Hz), 3.89 (3H, s) , 2.94 (2H, s) , and 1.69 (10H, broad m) in ppm downfield from TMS. I3 C NMR (CDC1 3 ) 164.5, 160.6, 158.7, 132.7, 113.6, 106.2, 104.5, 94.2, 55.7, 38.0, 37.1 (2C's), 24.9, and 23.2 (2C's) in ppm downfield from TMS. FIGURE 1 shows the infrared (KBr) spectrum. IR (KBr) 1660, 1615, 1445, 1435, 1385, and 1100 cm '1 . Cyclization was also accomplished by heating the phenol precursor in either a 50/50 4N hydrochloric acid/isopropanol solvent mixture or a boron trifluoride etherate solution in THF. This latter cyclization procedure is not recommended for any phenol precursor other than 12a, however. 11 6.5. 7-F0RMYL-4-METH0XYSPIR0[BENZOFURAN- 2 f3H. -CYCLOHEXANE1 (lib) The phenol precursor 12b was treated with Amber¬ lyst resin as described in the previous section. The product lib was isolated and purified by column chromatog¬ raphy (83% yield). Mp 62-63°C. Anal. Calcd. for C 15 H 18 0 3 : C, 73.15; H, 7.37. Found: C, 73.22; H, 7.40. *H NMR (CDCL 3 ) δ 10.16 (IH, s) , 7.66 (IH, d, J = 9 Hz), 6.46 (IH, d, J = 9 HZ), 3.88 (3H, s) , 2.88 (2H, s) , 1.4-2.0 (10H, broad m) . Compound lib is easily transformed to lie by a metal hydride reduction step or other means well-known in the art. 6.6. COMPLEMENT INHIBITION The dihydrobenzofuran and spirobenzo-furancyclohexane compounds of the general formulae 3, 4, the synthetic intermediates of the general formula 5, and the salts thereof of the present invention were assayed for their ability to inhibit complement by the methods described infra. 6.6.1. DEMONSTRATION OF INHIBITION OF C3a AND C5a PRODUCTION The ability to inhibit complement was tested by assaying for specific inhibition of C3a and C5a produc- tion. For all experiments, a single human serum pool, to be used as a source of complement, was aliquoted and stored frozen at -70°C. Human IgG was heat-aggregated, aliquoted, and stored frozen at -70°C. For each experi¬ ment, serum aliquots were equilibrated at 37°C with vary- ing concentrations of the compounds tested. The classical complement pathway was initiated by the addition of aggre¬ gated human IgG. Control samples containing no IgG were always included. After a fixed reaction time of 10 min¬ utes (determined in an earlier time-course study to pro- vide a convenient time interval during which the produc¬ tion of C5a or C3a is nearly complete, i.e., greater than 90%) , the levels of the released complement peptides (C5a or C3a) were determined by radioimmunoassay using commer¬ cially available radioimmunoassay (RIA) kits (C5a RIA, UpJohn Cat. No. 3250-02; C3a RIA, UpJohn Cat. No. 3245-01; C5a RIA, Amersham Cat. No. RPA.520; C3a RIA, Amersham RPA.518) . Since a competitive immunoassay was used, complement peptide (C5a and C3a) concentrations varied inversely with the counts. The Counts Bound (CB) for a sample was de¬ fined as the total counts (in counts per minute, cpm) measured in the pellet minus the counts measured in a non¬ specific binding (NSB) control. The NSB control was a sample containing only tracer peptide ( 125 I-labelled) and second precipitating antiserum; it contained no C5a- or C3a-specific antiserum. The y-axis in FIGURE 2 represents the fraction inhi¬ bition. The fraction inhibition is equal to the Counts Bound (CB) for a "sample," less the CB in the "sample with no added compound," divided by the CB for the "no IgG control" less the CB in the "sample with no added com¬ pound." INHIBITION = ..CB sample. - .CB no compound.1 [ (CB no IgG) - (CB no compound) ] When C5a production is inhibited at concentra- tions of added compound at which C3a production is unaf¬ fected, the data suggest that complement inhibition is directed toward the C5 activation step. FIGURE 2 is representative of the data plots obtained from the assays described above. The results are summarized in Table VI . . . . . . . and demonstrate that the complement inhibition activities exhibited by some of the compounds appear to be directed toward inhibition of C5 activation, and that the inhibito¬ ry activities are comparable to that of K-76 COONa itself. * The concentration of compound required to inhibit C3a or C5a production 50% relative to control samples which contained no test compound. b The inhibition observed at this concentration was less than about 50%. c Only marginal inhibition was observed at a concentration of about 8.5 mM. 6.6.2. DEMONSTRATION OF INHIBITION OF COMPLEMENT-MEDIATED HEMOLYSIS The ability to inhibit complement was also tested by assaying for inhibition of complement-mediated red cell lysis (hemolysis) . The inhibition of hemolysis was deter- mined as a function of compound concentration. The com¬ pounds to be tested were diluted in 0.1 M Hepes buffer (0.15 N NaCl, pH 7.4), and 50 μl were added to each well of a V-bottom microtiter plate. Human serum, used as the complement source, was diluted 1 to 500 in Hepes buffer, and 50 μl were added to each well. Next, commercially available sheep erythrocytes with anti-sheep antibody (Diamedix Cat. No. 789-001) were used as received and added at 100 μl/well to initiate the complement pathway leading to hemolysis. The plate was incubated for 60 minutes at 37°C and then centrifuged at 500 x g for 10 minutes. The supernatants were removed and placed in a flat-bottom microtiter plate. The extent of hemolysis was measured as a function of the sample absorbance at 410 nm. * The maximal absorbance (corresponding to maximal hemoly¬ sis) , ,,^, was obtained from the absorbance value of an erythrocyte sample containing only human serum, A S less the absorbance of a sample containing only the red cells, A Q . Thus, A- ^ = A s - A Q . The difference between the absorbance of an erythrocyte sample containing both human serum and inhibitive compound, and the absorbance of a cell sample containing inhibitive compound only, was defined as A j^pk . The inhibition, IH, was expressed as the fraction (A ^ - ,^)/A ^ , and IH 50 was defined as the concentration of inhibitive compound required to produce a value of IH = 1/2. FIGURE 3 shows the plot of inhibition, IH, versus the concentration of lla added to the cell samples. The concentration of lla corresponding to IH 50 is approxi- mately 0.9 mM. Table VII summarizes the results of sever- al assays using some of the compounds of the present invention and shows their effectiveness in inhibition of hemolysis. TABLE VII INHIBITION OF COMPLEMENT-MEDIATED HEMOLYSIS Compound lla 12a 14a 15a (+)-20a (-)-20a (+)-23a (-)-23a 30a 3la K-76 COONa - *The concentration of compound required to produce a value for hemolysis inhibition (IH, as defined supra) of 1/2. 6.7. IMMUNOSUPPRESSIVE ACTIVITIES The dihydrobenzofuran and spirobenzofuranc- yclohexane compounds of the general formulae 3, 4, inter¬ mediate 5, and the salts thereof of the present invention were tested for their ability to inhibit cell-mediated immune activity. The specific activities demonstrated by the tested compounds included the inhibition of natural killer (NK) activity, the inhibition of peripheral blood lymphocyte (PBL) proliferation, and the inhibition of cell surface interleukin-2 receptor expression as described infra. In addition, the ability of compound lla to inhib¬ it the proliferation of Chinese hamster ovary (CHO) cells was tested; no inhibition of CHO cells was observed, suggesting that the compounds inhibitory activities were specific to the lymphoid cells of the immune system. The experiments were conducted using in vitro assays for determining dose-dependent effects of the compounds of the invention on immune function. 6.7.1. DEMONSTRATION OF INHIBITION OF NATURAL KILLER ACTIVITY Compounds were tested for their effects on the ability of peripheral blood mononuclear cells to lyse NK- sensitive target cells, K562. NK activity was determined using 51 Cr-labeled K562 erythromyeloid leukemia cells as targets, and normal peripheral blood mononuclear cells as effector cells, in a four hour cytotoxicity assay. Effec¬ tor cells were isolated from fresh blood by Ficoll-Hypaque ' gradient centrifugation, and 100 μl of a 5 x 10 5 cell/well suspension were added to each well of a V-bottom micro¬ titer plate. The compound lla was diluted in RPMI 1640 medium containing 10% fetal calf serum and dispensed at 100 μl per well. Target cells (K562) were labeled for 30 minutes with 100 μCi of 51 Cr, washed thoroughly and dis¬ pensed in a 20 μl volume at 10 4 cells/well. These cell concentrations resulted in an effector-to-target cell ratio of 50:1. The microtiter plate was centrifuged at 50 x g for 5 minutes and incubated in a humidified chamber with 5% C0 2 at 37°C. After 4 hours, 100 μl were removed from each well and the radioactivity was measured with a LKB 1275 gamma counter. The percent specific lysis was calculated as follows: % specific lysis = [(EXP - SR)/(TOTAL - B) ] X 100 where EXP (experimental value) was obtained using effector and target cells; SR (the spontaneous release) was ob¬ tained from target cells incubated with media alone; TOTAL release was obtained by hypotonic lysis in water; and B represents instrumental background. Means were calculated from quadruplicate wells and the standard deviations never exceeded 10%. Viability of the effector cells incubated with the test compound was determined by trypan blue exclusion. The results of the inhibition of natural killer activity for 7-carboxy-4-methoxyspiro[benzofuran-2(3H)- cyclohexane] (lla) sodium salt are summarized in Table VIII. TABLE VIII INHIBITION OF NATURAL KILLER ACTIVITY BY 7- CARBOXY-4-METHOXYSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE] (lla) , SODIUM SALT The data listed in Table VIII indicate that compound lla can, at appropriate concentrations, effectively inhibit natural killer activity. 6.7.2. DEMONSTRATION OF INHIBITION OF PROLIFERATION OF PERIPHERAL BLOOD LYMPHOCYTES The proliferation of human peripheral blood lympho¬ cytes (PBL) in response to phytohemagglutinin (PHA, Well¬ come) or anti-CD3 monoclonal antibody (OKT-3, Ortho) was assessed by the incorporation of 3 H-thymidine. Compound 10 . lla was diluted in culture media to the desired concentra¬ tions, human PBL were added to a final concentration of 10 6 cells/ml, and then either PHA (Wellcome) or anti-CD3 (OKT3, Ortho) antibody (final concentration, 1 mg/ml) was l .b.. added to initiate proliferation. The final volume per sample was 100 μl. The cells were incubated for 72 hours after stimulation, pulsed with 1 μCi 3 H-thymidine per sample for 4 hours, harvested, and counted in a scintilla¬ tion counter. Separate experiments, conducted on samples 0 exposed only to varying amounts of lla without the stimu¬ lant, showed that the PBL were viable, as determined visually by trypan blue exclusion, in the concentration, ranges of lla used above. The results (Figs. 4, 5) showed that compound lla inhibited PBL proliferation in a dose- 5 dependent manner. Half-maximal inhibition was observed at about 0.5 mM of lla when cultures were stimulated with either PHA (Fig. 4) or anti-CD3 antibody (Fig. 5) . Be¬ cause cell viability was unaffected by the presence of lla (see supra) , the inhibition apparently was not due to 0 cytotoxic effects. Additional compounds of the invention were tested for their immunosuppressive activities, with the results presented in Table IX. • The concentration of compound required to inhibit PBL proliferation 50% relative to control samples which con¬ tained no test compound. 15. The results (Table IX) revealed that each of the tested compounds inhibited PBL proliferation. Release of cell surface interleukin-2 receptor 0 (IL-2R) or CD8 antigen from lymphocytes is a correlate of T cell activation (Rubin et al. J. Immunol. 1985, 135, 3172- 77; Rubin et al. Fed. proc. 1985 44, 946; Fujimoto, J. et al. J. EXP. Med. 1983, 159. 752-66; Tomkinson, B. et al. 2d Annual Conference on Clinical Immunol. Washington, 5D.C, October 30, 1987). In some experiments, the level of IL-2R or CD8 protein released into the supernatant of the PBL cultures was assessed by removing aliquots there¬ from just prior to pulsing with 3 H-thymidine. Commercially available enzyme immunoassay kits (CELLFREE™ IL-2R, Cat. 0 No. CK1020, T Cell Sciences, Inc., Cambridge, MA, or CELL- FREE™ T8/CD8, Cat. No. CK1040, T Cell Sciences) were used to determine the levels of the two analytes. The results using compound lla showed that lla was an inhibitor of both IL-2R (Fig. 6) and CD8 (Fig. 7) protein release, in 5 stimulated PBL cultures. 6.7.3. DEMONSTRATION OF INHIBITION OF CELL SURFACE INTERLEUKIN-2 RECEPTOR EXPRESSION The interleukin-2 receptor (IL-2R) is not detectable on the surface of resting T cells. Upon activation by 5specific antigens or mitogens, T cell proliferation is mediated by an autocrine mechanism whereby activated cells secrete interleukin-2 and express cell surface IL-2R (Meuer, S.C. et al. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1509; Tsudo, M., et al. J. Exp. Med. 1984, 160, 612- 10617; Waldmann, T.A. , et al. J. Exp. Med. 1984, 160, 1450- 1466) . Compounds were tested for their ability to inhibit T cell activation, as indicated by inhibition of cell-sur¬ face IL-2R expression. PBL cultures (1.5 ml; 24-well 15plates) were stimulated with PHA (1 μg/ml) for 72 hours in the absence or presence of varying amounts of compound lla. Subsequently, the cells were stained using fluores- cein isothiocyanate (FITC)-labeled anti-IL-2R antibody (Act-T-Set IL-2R, Cat. No. AA2009, T Cell Sciences, Inc., 0 Cambridge, MA) and analyzed by flow cytometry (Ortho System 30) (Table X) . 5 TABLE X INHIBITION OF CELL SURFACE IL-2R EXPRESSION BY 7-CARBOXY-4-METHOXYSPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE] .lla.. SODIUM SALT Concentration of lla .mM. 0 0.44 1.76 Based upon a total cell count of 5000. The results listed in Table X indicate a dramatic reduction in the number of PBL expressing the IL-2R in the presence of lla. We also observed that the amount of IL- 2R cell surface expression was reduced in the presence of lla. The data thus demonstrate that compound lla signifi¬ cantly suppresses IL-2R cell-surface expression, indicat¬ ing that this compound can effectively inhibit T cell activation in PBL cultures. 6.7.4. LACK OF INHIBITION OF CHO CELL PROLIFERATION The specificity of compound lla's inhibitory activity upon immune cells was demonstrated by assaying for lla's ability to inhibit the proliferation of Chinese hamster ovary (CHO) cells. Non-confluent CHO cells were allowed to proliferate for 4 hours in a 96-well plate at a volume of 50 μl per well in the absence or presence of compound lla at a concentration of 1.8 mM. The cells were pulsed with 0.5 μCi of 3 H-thymidine per sample for 4 hours., har- vested, and the amount of incorporated 3 H-thymidine was determined by scintillation counting. The CHO cells allowed to proliferate in the absence of added compound yielded a value of 57025 (±7530) CPM. The value for the 5sample grown in the presence of lla was 52549 (±6272) CPM. These values revealed no statistical difference in the proliferation rate of the two samples. Because the CHO cell line is of the non-lymphoid type, this result sug¬ gests that the compounds of the present invention do not 1 0 generally inhibit mammalian cell proliferation, but inhib¬ it activation and/or proliferation of PBL cultures. EXAMPLES OF 6-CARB0XYL-4-SUBSTITUTED- SPIROrBENZOFURAN-2.3H.-CYCLOHEXANES1 15 7.1. 6-CARBOXYL-4-METHOXYSPIRO[BENZOFURAN- 2(3H)-CYCLOHEXANE] SODIUM SALT (44a) AND 6-CARBOXYLATE-4-ETHOXY-SPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] SODIUM SALT f44b. Preparation of 33a-b. Methyl 3,5-dihydroxybenzoate 20 (io g, 59.5 mmol) in dry acetone (200 ml) was added to dry K 2 C0 3 (8.2 g, 59.4 mmol). Dimethysulfate (for 33a; 7.5 g, 59.5 mmol) was added to the mixture, which was refluxed for 24 h. The mixture was filtered. Ether (200 ml) was added to the solution and was extracted with H 2 0 (100 ml x 252) . The organic solution was dried over MgS0 4 ; after remov¬ al of solvent, a crude product (9.0 g) was purified by chromatography (1:19 EtOAc/CH 2 Cl 2 ) to afford 33a as a white solid, yield 3.9 g (36%). mp 95-97°C; TLC R f 0.58 (1:9 EtOAc/CH 2 Cl 2 ) . NMR (CDC1 3 ) δ 7.26-7.13 (m,2H), 6.63 (m,IH) , 0 5.90 (s,lH,OH), 3.90 (s,3H), 3.80 (s,3H). For 33b: The procedure of 33a was modified by using diethyl sulfate as alkylating agent. 33b: mp 98-100°C; yield, 33%; TLC R f 0.51 (1:9 EtOAc/CH 2 Cl 2 ) . NMR (CDC1 3 ) δ 7.16-7.14 (m, 2H) , 5 6. 62 (m, lH) , 5. 70 (s , lH, OH) , 4 . 07-4 . 00 (q, 2H) , 3 . 90 (S , 1H) , 1. 41-1. 38 (t, 3H) . Preparation of 34a-b. NaH (50% in mineral oil; 2.2g, 91.7 mmol) was washed with hexane (40 ml x 2) under N 2 . Dry 5DMF (50 ml) was added and the mixture was cooled to 0°C. A solution of 33a (7.6 g, 41.7 mmol) in dry DMF (40 ml) was added slowly to the mixture, and then stirred for 1 h at 25°C. The mixture was recooled to 0°C, and a solution of MOMC1 (3.7 g, 45.8 mmol) in dry DMF (20 ml) was added 1θdropwise. The mixture was stirred at 25°C for 3 h. Ether (200 ml) was added to the mixture and washed with H 2 0 (100 ml x 4) . The ether portion was dried (MgS0 4 ) and concen¬ trated to give a pale yellow liquid as 34a; yield 8.2 g (87%). TLC R f 0.44 (CH 2 C1 2 ) . IR (neat) 2950, 1740, 1600, 151460, 1430, 1320, 1240, 1150, 1060, 1020, 770 cm" 1 . NMR (CDC1 3 ) Δ7.32-7.31 (m,lH), 7.24-7.23 (m,lH), 6.79 (m,lH), 5.19 (s,2H), 3.90 (s,2H), 3.83 (s,3H), 3.48 (s,3H). For 34b: Procedure for 34a was modified. Yield, 95%; TLC R f 0.42 (CH 2 C1 2 ) . IR (neat) 2980, 1725, 1600, 1450, 1300, 20 1240, 1150, 1060, 1030, 770 CUT 1 . NMR (CDC1 3 ) δ 7.30-7.29 (m,lH), 7.23-7.22 (HI,1H), 6.79-6.78 (m,lH), 5.19 (s,2H), 4.09-4.02 (q,2H), 3.90 (s,3H), 3.48 (s,3H), 1.44-1.39 (t,3H). Preparation of 35a-b. LiAlH 4 (1.7 g, 44.2 mmol) was 25 suspended in dry THF (150 ml) under N 2 and cooled to 0°C. A solution of 34a (10 g, 44.2 mmol) in THF (20 ml) was added dropwise. The mixture was stirred for 3 h at 25°C. Excess LiAlH 4 was carefully decomposed by additional ice. The mixture was filtered through silica gel and rinsed 30 with ether (150 ml) . The solution was washed with H 2 0 (50 ml x 2) , dried and concentrated to give a colorless liquid as 35a, yield 6.7g (76%). TLC R f 0.38 (1:4 EtOAc/CH 2 Cl 2 ) . IR (neat) 3400, 2920, 1600, 1460, 1290, 1150, 1050, 925, 35840cm" 1 . NMR (CDC1 3 ) Δ6.67-6.50 (m,3H), 5.16 (s,2H), 4.65 (s,2H), s,3H), 3.50 (s,3H), 2.56 (s,H,OH) . 35b was pre¬ pared by the same procedure that used for 35a: yield, 90-92%; TLC R f 0.52 (1:4 Et 2 0/CH 2 C1 2 ) . IR (neat) 3400, 2940, 1600, 1460, 1390, 1280, 1210, 1155, 1140, 1030, 925, 850 5cm" 1 . NMR (CDC1 3 ) δ 6.80-6.57 (m,3H), 5.27 (s,2H), 4.70 (s,2H), 4.20-3.97 (q,2H), 3.50 (s,3H), 2.00 (s,H,OH) , 1.50-1.33 (t,3H). Preparation of 36a-b. To a solution of 35a (2.2 g, 11.1 mmol) and imidazole (1.51 g, 22.2 mmol) in CH 2 C1 2 (50 10ml), was added slowly TBDMSCl (2.0 g, 13.3 mmol) dissolved in CH 2 C1 2 (10 ml) . The mixture was stirred at 25°C for 3 h. The mixture was washed with H 2 0 (100 ml x 2) , dried with (MgS0 4 ) and concentrated to give a colorless liquid, 3.3 g (94%) as 36a. TLC R f 0.8 (4:6 hexane/CH 2 Cl 2 ) . IR (neat) 152940, 1600, 1460, 1365, 1300, 1250, 1210, 1190, 1150, 1100, 1055, 1025, 925, 835, 765 cm' 1 . NMR (CDCl 3 ) δ 6.62-6.61 (m,lH), 6.58-6.56 (m,lH), 6.49-6.47 (t,lH), 5.15 (s,2H), 4.69 (s,2H), 3.78 (s,3H), 3.47 (s,3H), 0.95 (s,9H), 0.10 (s,6H). For 36b: It was prepared by the 20 procedure that used for 36a; yield, 90-95%. IR (neat) 2940, 1600, 1460, 1390, 1370, 1290, 1250, 1150, 1100, 1030, 940, 840, 775 cm "1 . NMR (CDCl 3 ) δ 6.61-6.59 (m,lH), 6.56-6.54 (m,lH), 6.48-6.47 (t,IH) , 5.15 (s,2H), 4.67 (S,2H), 4.04-3.97 (q,2H) , 3.47 (S,3H), 1.43-1.37 (t,3H) , 25 0.94 (S,9H), 0.10 (S,6H). Preparation of 38a-b. To a solution of 36a (8.2 g, 26.2 mmol) and TMEDA (4.6 g, 39.4 mmol) in dry THF (200 ml) under N 2 at 0°C, n-BuLi (12 ml, 28.8 mmol) was added slowly. The solution was stirred 30 min at 0°C and then 30 1.5 h at 25°C. The solution was recooled to -78°C, Cul (7.5 g, 39.4 mmol) was added in one portion under positive N 2 stream. The mixture was stirred for 1.5 h at -78°C to -40°C. The mixture was recooled to -78°C, bromide 37 (5.5 _ 35_ g, 31.5 mmol) in THF (20 ml) was added dropwise, and the mixture was stirred 4 h from -78°C to 25°C. Ether (200 ml) was added to the solution and washed with 20% NH 4 0H solu¬ tion until the aqueous layer was no longer blue, dried and concentrated to give a brown liquid (10.4 g) . Purification 5by chromatography (1:19 EtOAc/hexane) afforded a colorless liquid as product 38a (8.7 g, 82%). TLC R f 0.70 (1:9 EtOAc/hexane). IR (neat) 2930, 1610, 1590, 1460, 1430, 1260, 1200, 1100, 1080, 1030, 840, 780 cm 1 . NMR (CDC1 3 ) δ 6.68 (S,1H), 6.61 (S,1H), 5.22-5.18 (m,lH), 5.13 (s,2H), 104.71 (S,2H), 3.79 (s,3H), 3.44 (s,3H), 3.26 (S,2H), 1.97-1.88 (m,4H), 1.62-1.46 (m,4H), 0.94 (s,9H), 0.10 (s,6H). For 38b: Preparation was essentially that used for 38a. Yield, 60-62%. TLC R f 0.66 (1:9 EtOAc/hexane). IR (neat) 2940, 1610, 1590, 1440, 1390, 1370, 1260, 1200, 151150, 1100, 840, 770 cm "1 . NMR (CDC1 3 ) δ 6.67 (s,lH), 6.58 (S,1H), 5.25-5.22 (m,lH), 5.13 (s,2H), 4.69 (s,2H), 4.03- 3.96 (q,2H), 3.44 (s,3H), 3.28 (s,2H), 2.00-1.89 (m,4H), 1.76-1.47 (m,4H), 1.39-1.35 (t,3H), 0.94 (s,9H), 0.10 (s,6H) . 20 Preparation of 39a-b. To the solution of 38a (7.2 g, 14.7 mmol) in THF (100 ml), (Bu) 4 NF (1.0 M in THF, 21 ml, 21 mmol) was added slowly at 25°C. Solution was stirred at 25°C for 2 h. Ether (100 ml) was added to the solution, then washed with 5% HCl (20 ml) and H 2 0 (100 ml x 2) . The 25 organic layer was dried and concentrated to give a color¬ less liquid as 39a, yield 6.0 g (90%). TLC R f 0.53 (2:3 EtOAc/hexane). IR (neat) 3380, 3920, 1610, 1590, 1450, 1430, 1400, 1200, 1160, 1110, 1080, 1030, 960, 925, 830 cm 1 . NMR (CDC1 3 ) δ 6.70 (s,lH), 6.61 (s,lH), 5.21-5.18 30 (m,lH), 5.14 (S,2H), 4.62 (s,2H), 3.79 (s,3H), 3.44 (s,3H), 3.26 (S,2H), 2.00-1.87 (m,4H), 1.62-1.48 (m,4H). For 39b: Preparation was essentially that used for 39a. Yield, 95%. TLC R f 0.58 (2:3 EtOAc/hexane). IR (neat) „ 3400, 2940, 1610, 1590, 1440, 1390, 1200, 1150, 1120, 35 1070, 1030, 925, 820 cm ' 1 . NMR (CDC1 3 ) δ 6.69 (s,lH), 6.58 (S,1H), 5.30-5.25 (m,IH) , 5.14 (s,2H), 4.59 (s,2H), 4.03- 3.96 (q,2H), 3.44 (s,3H), 3.28 (s,2H) , 2.00-1.88 (m,4H), 1.60-1.46 (m,4H), 1.39-1.35 (t,3H). 5 Preparation of 0a-b. To a mixture of PCC (5.5 g, 25.6 mmol) in CH 2 C1 2 (100 ml) at 25°C was added 39a (4.6 g, 15.7 mmol) in CH 2 C1 2 (50 ml) slowly. The mixture was stirred at 25°C for 4 h more. The mixture was filtered through silica gel and rinsed with EtOAc (40 ml) . The 10solution was washed with H 2 0 (50 ml), dried (MgS0 4 ) and concentrated to give a yellow liquid which then was puri¬ fied by chromatography (3:7 EtOAc/hexane) to afford a pale yellow liquid as 40a, yield 4.2 g (92%). TLC R f 0.66 (3:7 EtOAc/hexane). IR (neat) 2920, 1690, 1580, 1450, 1430, 151380, 1300, 1200, 1150, 1110, 1070, 1020, 920, 840, 740, 720 cm' 1 . NMR (CDC1 3 ) δ 9.89 (S,1H), 7.23 (s,lH), 7.10 (S,1H), 5.22 (S,2H), 5.20-5.18 (m,IH) , 3.86 (s,3H), 3.46 (S,3H), 3.33 (S,2H), 1.98-1.88 (m,4H), 1.61-1.48 (m,4H). For 40b: Procedure of preparation was essentially that 20used for 40a. Yield, 75-81%. TLC R f 0.56 (1:4 EtOAC/hexane) . IR (neat) 2920, 1700, 1585, 1440, 1380, 1310, 1200, 1160, 1110, 1070, 1030, 960, 920, 845, 740, 720 cm' 1 . NMR (CDC1 3 ) δ 9.88 (s,lH), 7.22 (s,lH), 7.08 (S,1H), 5.25-5.22 (m,lH), 5.21 (S,2H), 4.12-4.05 (q,2H) , 25 3.47 (S,3H), 3.36 (s,2H), 2.00-1.89 (m,4H), 1.60-1.38 (m,4H), 1.43-1.39 (t,3H). Preparation of 4la-b. To a solution of 40a (4.2 g, 14.5 mmol) in 2-propanol (30 ml) and THF (15 ml) at 0°C was added slowly aq. 4N HCl solution (40 ml, 160 mmol) in 30 2-propanol (10 ml) . The solution was then stirred at 25°C for 16 h or until disappearance of 40a (by TLC) . The solution was extracted with ether (50 ml x 3) . The ether solution was washed with H 2 0 (30 ml) , then dried and con¬ centrated. The crude product was purified by chromatogra- 5 phy (3:7 EtOAc/hexane) to afford 4la as white solid, yield 3 g (84%). mp 153-155°C. TLC R f 0.60 (3:7 EtOAc/hexane) . NMR (CDC1 3 ) δ 9.90 (S,1H), 7.04 (m,2H), 5.78 (s,lH,OH), 5.68-5.65 (m,2H), 3.90 (S,3H), 3.48 (s,2H), 2.09-2.01 5(m,4H), 1.96-1.90 (m,4H). For 41b: Procedure of prepara¬ tion was essentially that used for 41a. Yield, 76%; TLC R f 0.34 (1:4 EtOAc/hexane). NMR (CDC1 3 ) δ 9.86 (s,lH), 7.00 (m,2H), 5.80 (S,1H,0H), 5.70-5.66 (m,2H), 4.13-4.06 (q,2H), 3.47 (s,2H), 2.05-2.02 (m,2H), 1.92-1.88 (m, 2H) , 101.63-1.54 (m,4H), 1.46-1.41 (t,3H). Preparation of 42a-b. Amberlyst 15 (10 g) was added in one portion to the solution of 41a (3.3 g, 13.4 mmol) in CH 2 C1 2 (100 ml) . The mixture was stirred at 25°C for 6 h. The mixture was filtered and the solution was washed 15with H 2 0 (100 ml), dried and concentrated to give a crude product (3 g) which then was purified by chromatography (1:4 EtOAc/hexane) to afford 42a as a white solid (2.5 g, 76%). TLC R f 0.57 (1:4 EtOAc/hexane). IR (KBr) 2930, 1690, 1600, 1430, 1390, 1350, 1325, 1220, 1120, 1030, 920, 830, 0 805, 745 cm" 1 . NMR (CDC1 3 ) δ 9.85 (S,1H), 6.93 (s,lH), 6.89 (S,1H), 3.88 (S,3H), 2.94 (S,2H), 1.86-1.64 (m,6H), 1.55-1.45 (m,4H). 13 C NMR (CDC1 3 ) δ 191.77, 160.53, 156.90, 138.19, 121.25, 105.65, 102.81, 90.36, 55.58, 38.48, 37.23, 25.05, 22.95. For 42b: Procedure of preparation was 25 essentially that used for 42a. mp 105-106°C. Yield, 82%; TLC R f 0.55 (1:4 EtOAc/hexane) . NMR (CDC1 3 ) δ 9.84 (s,lH), 6.91 (S,1H), 6.87 (s,lH), 4.15-4.08 (q,2H), 2.95 (s,2H), 1.86-1.68 (m,6H), 1.53-1.45 (m,4H), 1.45-1.40 (t,3H). Preparation of 43a-b. To a mixture of aq. 2N NaOH 0 . . (30 ml) containing Ag 2 0 (2.4 g, 10.2 mmol) at 50°C, a solu¬ tion of 42a (1 g, 4.1 mmol) in EtOH (1 ml) and THF (5 ml) was added slowly. The mixture was stirred for 6 h at 50°C. The mixture was filtered and the aq. solution was washed _ with ether (50 ml) . The aq. solution then was cooled to 5 0°C, and acidified with cone. HCl solution. The white precipitate that resulted was extracted with ether, dried and concentrated to afford a white solid as 43a, yield 0.75g (71%). TLC R f 0.62 (1:9:10 MeOH/CH 2 Cl 2 /EtOAc) . NMR 5 (CDC1 3 ) δ 7.16 (S,2H), 3.88 (s,3H), 2.94 (s,2H), 1.87-1.65 (m,6H), 1.55-1.47 (m,4H) . For 43b: Procedure for prepara¬ tion was essentially that used for 43a. mp 190-192°C. TLC f 0.62 (1:9:10 MeOH/CH 2 Cl 2 /EtOAC) ; yield,69%. IR (KBr) 3300-2400, 1675, 1595, 1430, 1350, 1325, 1265, 1210, 1120, 101100, 1030, 955, 860, 770, 740 cm- 1 . NMR (CDC1 3 ) «S 7.14 (s,2H), 4.15-4.08 (q,2H), 2.95 (S,2H), 1.86-1.65 (m,6H) , 1.60-1.45 (m,4H), 1.45-1.40 (t,3H). 13 C NMR (CDC1 3 ) δ 172.17, 160.08, 155.61, 130.19, 120.54, 105.39, 104.77, 90.04, 63.80, 38.49, 37.24, 25.09, 22.99, 14.84. Anal. 15 Calcd for C 16 H 20 0 4 : C, 69.54; H, 7.30. Found: C,69_40; H.7.35. Preparation of 44a-b. Sodium hydride (50% in mineral oil, 0.58 g, 24.2 mmol) was washed with hexane (30 ml) under N 2 , then dry ether (50 ml) was added. A solution of 0 43a (3.2 g, 12.2 mmol) in ether (150 ml) was added to above mixture slowly at 25°C. The mixture was stirred at 25°C for 6 h and a white precipitate resulted. The mixture was extracted with H 2 0 (50 ml) . The aqueous solution was collected and freeze dried to give a white solid (3.2 g, 25 92%) . NMR (D 2 0) δ 7.10 (S,1H), 6.92 (S,1H), 3.85 (S,3H) , 2.82 (S,2H), 1.70-1.54 (m,6H), 1.44-1.33 (m,4H). For 44b: Procedure for preparation was essentially that used for 44a. Yield, 75%. NMR (D 2 0) δ 7.09 (s,lH), 6.90 (S,1H), 4.19-4.12 (q,2H) , 2.89 (s,2H), 1.77-1.60 0 (m,6H), 1.50-1.40 (m,4H), 1.40-1.36 (t,3H) . 7.2. .6-FORMYL-4-HYDROXYSPIRO[BENZOFURAN- 2.3H. -CYCLOHEXANE! .52. Preparation of 45. The procedure for the preparation 5 of 45 was essentially that used for 34 except that two molar equivalent of NaH and MOMC1 were used. Yield was 83%; TLC R f 0.65 (1:1 ether/hexane) . Preparation of 46. The procedure for preparation of 46 was essentially that used for 35. Yield was 89%; TLC f 50.27 (1:1 ether/hexane). Preparation of 47. The procedure for preparation was essentially that used for 36. 47: colorless liquid; yield, 83-98%; TLC R f 0.80 (1:1 ether/hexane). IR (neat) 2960, 1600, 1470, 1400, 1370, 1290, 1260, 1140, 1080, 1040, 930, 10840, 780 cm "1 . NMR (CDC1 3 ) δ 6.92-6.72 (m,3H), 5.26 (s,4H), 4.80 (s,2H), 3.57 (s,6H), 1.00 (s,9H), 0.13 (s,6H). Preparation of 48. Procedure for preparation of 48 was essentially that used for 38. Crude product was puri¬ fied by chromatography (1:19 EtOAc/hexane). 48: a clear 15 liquid; yield, 63-79%; TLC R f 0.46 (1:19 EtOAc/hexane). IR (neat) 2940, 1615, 1590, 1455, 1440, 1400, 1370, 1260, 1160, 1100, 1050, 930, 840, 780 cm" 1 . NMR (CDC1 3 ) δ 6.76 (s,2H), 5.25-5.22 (m,lH), 5.15 (s,4H), 4.70 (s,2H), 3.45 (S,6H), 3.30 (S,2H), 2.10-1.96 (m,4H), 1.95-1.88 (m,4H), 200.95 (S,9H), 0.11 (S,6H). Preparation of 49. Procedure for preparation 49 was essentially that used for 39. Yield, 95-100%; TLC R f 0.45 (3:7 EtOAc/CH 2 Cl 2 ) . IR (neat) 3400, 2930, 1620, 1590, 1440, 1290, 1190, 1155, 1110, 1040, 950, 920, 840 cm' 1 . NMR 25 (CDC1 3 ) δ 6.78 (S,2H), 5.24-5.20 (m,lH), 5.17 (s,4H), 4.63 (s,2H), 3.45 (S,6H), 3.30 (s,2H), 2.10-1.88 (m,4H), 1.73 (s,lH,OH), 1.63-1.48 (m,4H). Preparation of 50. Procedure for preparation 50 was essentially that used for 40. Crude product was purified 30 by chromatography (3:7 ether/hexane). Yield, 70-76%; TLC R f 0.43 (3:7 ether/hexane). IR (neat) 2930, 1700, 1585, 1435, 1380, 1290, 1155, 1105, 1050, 950, 925 cm" 1 . NMR (CDC1 3 ) δ 9.89 (S,1H), 7.29 (s,2H), 5.23-5.18 (m,5H), 3.46 „35 (S,6H), 3.37 (S,2H), 2.00-1.95 (m,4H), 1.94-1.87 (m,4H). 13 C NMR (CDCl j ) δ 191. 65 , 156.49 , 135.74 , 135.54, 126.38 , 121.11, 108.79 , 94.39 , 51. 15, 31. 61, 28 . 95 , 25.30 , 23 . 10 , 22.46. Preparation of 51. Procedure for preparation 51 was 5essentially that used for 41. Crude product was purified by chromatography (3:7 EtOAc/hexane). It should be men¬ tioned that a large amount of expected product decomposed or was trapped on silica gel to give a low yield (28%) . For 51: mp 130-132°C. TLC R f 0.41 (3:7 EtOAc/hexane) . IR 10 (KBr) 3460, 3420, 2940, 1690, 1670, 1595, 1450, 1410, 1340, 1200, 1170, 1140, 1080, 1050, 1035, 1010, 840, 790, 740 cm' 1 . NMR (CDC1 3 ) δ 9.83 (S,1H), 6.96 (s,2H), 5.72-5.68 (m,lH), 5.67 (s,2H,OH) , 3.47 (S,2H) , 2.08-2.01 (m,2H), 2.00-1.92 (m,2H), 1.67-1.53 (m,4H) . 15 Preparation of 52. Procedure of preparation 52 was essentially that used for 42. Crude product was purified by chromatography with 3:7 (EtOAc/hexane) as eluent. For 52: TLC R f 0.53(3:7 EtOAc/hexane) . IR (KBr) 3260, 2940, 1680, 1590, 1460, 1400, 1310, 1280, 1250, 1210, 1175, 201110, 1060, 1000, 930, 920, 840, 800, 740 cm" 1 . NMR (CDC1 3 ) δ 9.81 (S, IH), 6.88 (s,lH), 6.86 (S,1H), 2.97 (s,2H), 1.89-1.65 (m,6H), 1.58-1.45 (m,4H). 7.3. 4-BENZYLOXY-6-FORMYL-SPIRO 5 .BENZOFURAN-2.3H.-CYCLOHEXANE. fS3a. Preparation of 53a. Sodium hydride (50% in mineral oil, 62 g, 2.58 mmol) was washed with hexane (20 ml) under N 2 . Dry DMF (5 ml) was added and a solution of 52 (0.30 g, 1.29 mmol) in DMF (10 ml) was added slowly at 25°C. After 0 stirring for 30 min, a solution of benzyl bromide (0.22 g, 1.29 mmol) in DMF (5 ml) was added dropwise. The mixture was warmed to 50°C and stirred for 2 h more. Ether (50 ml) was added to the mixture which was then washed with H 2 0 (50 ml x 3) , dried and concentrated to afford a crude product (0.4 g) . Purification by chromatography (1:4 Et 2 0/hexane) gave a pale yellow liquid as 53a, yield 0.3 g (71%). TLC R f 0.45 (1:4 Et 2 0/hexane) . IR (neat) 2940, 2870, 1695, 1600, 1440, 1390, 1355, 1330, 1220, 1210, 1150, 1100, 1045, 920, 830, 805, 750, 700 cm" 1 . NMR (CDC1 3 ) δ 9.85 (s,lH), 57.46-7.34 (m,5H), 7.01 (S,1H), 6.91 (s,lH), 5.14 (s,2H), 2.98 (s,2H), 1.87-1.64 (m,6H), 1.56-1.42 (m,4H). 7.4. 4-N-BUTYLOXY-6-FORMYL-[NBU-]SPIRO [BENZOFURAN-2.3H.-CYCLOHEXANE! .53b. Preparation of 53b. Sodium hydride (50% in mineral oil, 62 mg, 2.58 mmol) was washed with hexane (30 ml) under N 2 , then dry DMF (5 ml) was added. To the mixture, a solution of 52 (0.3 g, 1.29 mmol) in dry DMF (10 ml) was added dropwise. The mixture was stirred for 30 min. A solution of n-butyl p-toluenesulfonate (0.29 g, 1.29 mmol) in DMF (5 ml) was added dropwise. The mixture was warmed to 50°C and stirred for 2 h. Ether (50 ml) was added to the mixture which was washed with H 2 0 (50 ml x 2) , dried and concentrated to give a crude product. Purification by chromatography (1:4 ether/hexane) afforded a pale yellow liquid as 53b, yield 0.27g (73%). TLC R f 0.58 (ether/hexane). IR (neat) 2940, 2860, 1690, 1600, 1430, 1380, 1320, 1210, 1100, 1030, 920, 825, 800, 745 cm' 1 . NMR (CDC1 3 ) δ 9.84 (S,2H), 6.92 (S,1H), 6.87 (S,1H), 4.07-4.03 (t,2H), 2.94 (s,2H), 1.87-1.64 (m,8H), 1.58-1.43 (m,6H), 1.01-0.96 (t,3H). 7.5. 6-FORMYL-4-PHENOXYSPIRO[BENZOFURAN- 2.3H.-CYCLOHEXANE. (53c. Preparation of 53c. Under N 2 condition, a solution of 52 (0.4 g, 1.72 mmol) in CH 2 C1 2 was added to a mixture of triphenylbismuth diacetate (1.16 g, 2.08 mmol) and copper powder (11 mg, 0.17 mmol) in CH 2 C1 2 (50 ml). The mixture was stirred at 25°C for 24 h, then filtered through silica gel and rinsed with 10% EtOAc in CH 2 C1 2 solution (20 ml) . The solution was washed with H 2 0 (50 ml) , dried and concen¬ trated to give the crude product (0.65 g) , which then was purified by chromatograhy (1:4 ether/hexane) to afford a colorless liquid as 53c; yield 0.4 g (75%). TLC R f 0.59 (1:4 ether/hexane). IR (neat) 3060, 2940, 2860, 1695, 1580, 1485, 1430, 1310, 1215, 1050, 1020, 850, 800, 755, 690 cm "1 . NMR (CDC1 3 ) δ 7.40-6.89 (m,7H) , 2.93 (s,2H), 1.90-1.63 (m,6H), 1.55-1.42 (m,4H). 7.6. 6-FORMYL-4-(2 '-HYDROXYETHYLOXY) SPIR0[BENZ0FURAN-2(3H)- CYCLOHEXANE! (53d) Preparation of 53d. Sodium hydride (50% in mineral oil, 0.10 g, 2.15 mmol) was washed with hexane (50 ml) under N 2 . Dry DMF (15 ml) was added and a solution of 52 (0.50 g, 1.29 mmol) in DMF (10 ml) was added slowly at 25°C. After stirring for 30 min, a solution of 2-[(tertbutyldimethylsilyl)oxy]ethyl bromide in DMF (5 ml) was added dropwise. The mixture was warmed to 50°C and stirred for 3 h more. Ether (50 ml) was added to the mixture, which was then washed with water (50 ml x 3) . The ether layer was collected, dried and concentrated to give a crude product (0.9 g) . Purification by chromatog¬ raphy (1.5:8.5 ether/hexane) gave a pale yellow solid 0.70g (83%). TLC R f 0.50 (1:4 ether/hexane). mp 74-75°C. IR (KBR) 2940, 2860, 1695, 1600, 1440, 1390, 1330, 1220, 1140, 1110, 1095, 960, 830, 780 cm "1 . NMR (CDC1 3 ) δ 9.84 (S,1H), 6.93 (S,1H), 6.89 (s,lH), 4.13 (t,2H), 3.98 (t,2H), 2.95 (S,2H), 1.87-1.45 (m,10H), 0.91 (S,9H), 0.10(s,6H). The above product (0.31 g, 0.79 mmol) was dissolved in THF (5 ml), then tetrabutylammonium fluoride (1 ml, 1.0 mmol, 1.0 M in THF solution) was added, solution was stirred at 25°C for 2 h. Ether (30 ml) was added, and the solution was washed with water (50 ml) . The ether .layer was collected, dried and concentrated to afford a pale yellow liquid. Purification by chromatography (1:4 hexane/ether) gave a pale yellow liquid as 53d. Yield, 0.17g (77%). TLC R f 0.34 (1:4 hexane/ether). IR (neat) 53450(b), 2940, 2870, 1690, 1600, 1440, 1390, 1325, 1220, 1130-1070(b) , 1035, 925, 830, 810, 755cm" 1 . NMR (CDC1 3 ) δ 89.84 (S,1H), 6.93 (S,1H), 6.91 (S,1H), 4.18 (t,2H), 4.00 (t,2H), 2.97 (s,2H), 1.87-1.64 (m,6H), 1.49 (s,b,4H). 10 7.7. 6-FORMYL-4-P-NITROPHENOXYSPIRO ΓBENZOFURAN-2(3H.-CYCLOHEXANE! .53e. Preparation of 53e. Sodium hydride (50% in mineral oil, 0.10 g, 4.58 mmol) was washed with hexane (20 ml) under nitrogen, and dry DMF (50 ml) was added. To the . 15-mixture, a solution of 52 (0.50 g, 2.15 mmol) in DMF (15 ml) was addded slowly at 25°C. The mixture was stirred for 30 min. A solution of l-fluoro-4-nitrobenzene in DMF (10 ml) was added slowly. The mixture was then warmed to 35°C and stirred for 3 h. Ether (100 ml) was added to the 2o mixture after cooling to 25°C which was washed with water (50 ml x 3) . Ether layer was collected, dried and concen¬ trated to give a crude product (0.79 g) . Purification by chromatography (1:9 EtOAc/hexane) afforded a yellow sticky oil as expected product 53e. Yield, 0.41g (54%). TLC R f 250.32 (1:4 EtOAc/hexane). IR (neat) 2940, 2860, 1695, 1570, 1515, 1490, 1430, 1345, 1310, 1240, 1165, 1110, 1065, 1050, 1025, 920, 865, 850, 800, 750, 745 cm" 1 . NMR (CDC1 3 ) δ 9.86(S,1H), 8.26-8.23 (d,lH), 7.06 (s,lH), 7.06-7.03 (m,3H), 2.86 (s,2H), 1.90-1.40 (m,10H). 30 7.8. 6-FORMYL-4-P-FORMYLPHENOXYSPIRO fBENZOFURAN-2.3H.-CYCLOHEXANE! (53f. Preparation of 53f. Sodium hydride (50% in mineral oil, 103 mg, 4.31 mmol) was washed with hexane (20 ml) 5 under N 2 . Dry DMF (30 ml) was added and a solution of 52 (0.40 g, 1.72 mmol) in DMF (5 ml) was added slowly at 25°C, and a solution of p-fluorobenzaldehyde in DMF (5 ml) was added. The mixture was warmed to 50°C and stirred for 24 h. After cooling to 25°C, ether (100 ml) was added, the 5solution was washed with water (30 ml x 3) . The ether layer was colleσted, dried and concentrated to give a crude product. Purification by chromatography (1:9 EtOAc/hexane) gave a pale yellow solid as expected product 53f. Yield, 0.31 g (53%). 53f: mp 127-129°C. TLC f 0.53 10 (1:4 EtOAc/hexane). IR (KBR) 2940, 2840, 1680, 1560, 1500, 1440, 1390, 1320, 1220, 1170, 1050, 840, 800CT-1. NMR (CDC1 3 ) δ 9.95 (S,1H), 9.85 (S,1H), 7.90-7.87 (d,2H), 7.12 (S,1H), 7.10-7.07 (d,2H), 7.04 (S,1H), 2.87 (s,2H) , 1.90-1.40 (m, 10H) . 15 7.9. 6-FORMYLSPIRO[BENZOFURAN- 2(3H.-CYCLOHEXANE] (53q. Preparation of 53g. Pyridine (0.34 g, 4.31 mmol) and 52 (0.50 g, 2.15 mmol) were dissolved in methylene 20 chloride (50 ml) and cooled at 0°C under N 2 . Triflie (trifluoromethanesulfonic) anhydride (0.40 ml, 2.37 mmol) was introduced dropwise by syringe. The solution was stirred for 1 h at 0°C. The solution was then washed with 5% aq. HCl (10 ml) and water (30 ml x 3) , dried and -- concentrated to give a crude product, which was purified by chromatography (1:9 EtOAc/hexane) to afford the expect¬ ed product phenol triflate 52. Yield, 0.70 g (90%). Phenol triflate 52: TLC R f 0.74 (1:4 EtOAc/hexane). IR (neat) 2940, 2860, 1710, 1625, 1590, 1430, 1300, 1250-1200(b) , 30 1140, 1010, 980, 920, 850, 830, 800, 770 cm" 1 . NMR ((CDC1 3 ) δ 9.89 (S,1H), 7.25 (s,2H), 3.12 (S,2H), 1.92-1.44 (m,- 10H) . Phenol triflate 52 (1.00 g, 2.1 mmol), triphenylphosphine (58 mg, 0.22 mmol), palladium acetate 5 (25 mg, 0.11 mmol) and triethylamine (1.66 g, 16.4 mmol) were dissolved in DMF (10 ml) and 96% formic acid (0.50 g, 10.8 mmol) in DMF (2 ml) was added. The mixture was stirred at 60°C for 2 h under N 2 . The mixture was diluted 5with water (50 ml) and extracted with ether (50 ml x 3). The ether layer was collected, dried and concentrated to give a brown residue. The crude product was purified by chromatagraphy (1:9 EtOAc/hexane) to give the expected product 53g as a pale yellow oil. Yield, 0.29 g (49%). 1053g: TLC R f 0.69 (1:9 EtOAc/hexane). IR (neat) 2940, 2860, 1690, 1585, 1490, 1440, 1340, 1310, 1270, 1250, 1150, 1030, 950, 920, 870, 800, 785 cm "1 . NMR (CDC1 3 ) δ 9.90 (S,1H), 7.36-7.33 (d,lH), 7.28-7.26 (d,lH), 7.22 (S,1H), 3.02 (S,1H), 1.89-1.43(m, 10H) . 15 7.10. 4-BENZYLOXY-6-CARBOXYLSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (54a) ; 4-N-BUTYLOXY-6-CARBOXYLSPIRO [BENZOFURAN-2 (3H)-CYCLOHEXANE] (54b) ; 6-CARBOXYL-4-BHENOXYSPIRO [BENZOFURAN-2(3H)-CYCLO HEXANE] (54c); 0 6-CARB0XYL-4-(2 '-HYDROXYETHYLOXYSPIRO [BENZOFURAN-2 (3H)-CYCLOHEXANE] (54d) ; AND 6-CARBOXYL-4-P-NITROPHENOXYSPIRO fBENZOFURAN-2 (3H. -CYCLOHEXANE! (54eV ' Preparation of 54a-e, Procedure for preparation of these compounds was essentially that used for 43. For 54a: 5 mp 173-175°C; yield, 74-83%; TLC R f 0.56 (1:4 hexane/ether). IR (KBr) 3100-2500, 1680, 1600, 1420, 1320, 1215, 1095, 950, 735cm "1 . NMR (CDC1 3 ) δ 7.46-7.34 (m,5H), 7.26 (S,1H), 7.18 (s,lH), 5.13 (s,2H), 2.97 (s,2H), 1.86-1.65 (m,6H), 1.48 (s,br,4H). Anal. Calcd for 0 C 21 H 22 0 4 : C, 74.53; H, 6.55. Found: C,74.44; H, 6.57. For 54b: mp 145-147°C; yield, 81%; TLC R f 0.62 (1:4 hexane/ether). IR (KBr) 3100-2500, 1670, 1600, 1425, 1320, 1275, 1210, 1100, 1030, 940, 865, 765, 745cm "1 . NMR (CDC1 3 ) δ 7.14 (S,2H), 4.06-4.02 (t,2H), 2.94 (s,2H), 5 1.86-1.65 (m,8H), 1.56-1.44 (m, 6H) , 1.01-0.97 (t,3H). Anal. Calcd for C^H^: C, 71.01; H, 7.95. Found: C, 71.01; H, 7.97. For 54c: mp 173-175°C; yield, 71%; TLC R f 0.69 (1:4 5 hexane/ether) . IR (KBr) 3100-2500, 1680, 1580, 1480, 1420, 1310, 1205, 1050, 950, 865, 765, 685cm "1 . NMR (CDC1 3 ) δ 7.37-6.97 (m,7h), 2.90 (S,2H), 1.90-1.60 (m,6H), 1.50-1.40 (m,4H) . 13 C NMR (CDC1 3 ) δ 171.45, 160.88, 156.32, 153.39, 130.62, 129.86, 123.74, 123.56, 118.44, 112.37, 106.59, 1090.34, 38.70, 37.11, 25.02, 22.90. Anal. Calcd for C 2 oH 20 O 4 : C,74.05; H, 6.22. Found: C, 73.97; H, 6.25. For 54d: Reaction was stirred for 8 h at 50°C. 54d: mp 163-164°C; TLC R f 0.72 (1:19 MeOH/ ether ) ; yield, 94%. IR (KBr) 3600-3300(b) , 2940, 2860, 1690, 1600, 1430, 1325, 151320, 1115, 1070, 960, 755, 725 cm "1 . NMR (CDC1 3 ) δ 57.15 (S,1H), 7.14 (S,1H), 4.18 (t,2H), 3.99 (t,2H), 2.96 (s,2H) 1.87-1.65 (m,6H), 1.49 (s,b, 4H) . 13 C NMR (CDC1 3 ) δ 171.24, 160.26, 155.23, 130.28, 120.61, 105.44, 105.36, 90.21, 69.46, 61.43, 38.48, 37.23, 25.05, 22.96. Anal. Calcd for 0 C 16 H 20 O 5 : C, 65.74; H,6.90. Found: C, 65.58; H,6.96. For 54e: Mixture was stirred for 24 h at 50°C. Yield, 56%. 54e: mp 190-19l°C; TLC R f 0.80 (ether). IR (KBr) 3100-2500(b) , 1690, 1585, 1525, 1490, 1425, 1390, 1310, 1230, 1170, 1115, 1070, 1025, 960, 860, 850, 770, 755 cm "1 5 NMR ((CDC1 3 ) δ 8.25-8.22 (d,2H), 7.36 (s,lH), 7.29 (s,lH) , 7.04-7.01 (d,2H), 2.84 (s,2H), 1.90-1.40 (m,10H). 13 C NMR (CDC1 3 ) δ 170.45, 161.92, 161.35, 150.96, 143.03, 131.22, 126.12, 124.98, 116.93, 113.98, 108.45, 90.76, 38.66, 37.05, 24.91, 22.81. Anal. Calcd. for C j oH^NOg: C, 65.03; H, 5.18; N,3.79. Found: C, 64.96; H, 5.23; N, 3.71. 7.11. 6-CARBOXYLSPIRO[BENZOFURAN- 2 (3H)-CYCLOHEXANE] (54g) ; AND 4-P-AMINOPHENOXY-6-CARBOXYLSPIRO [BENZOFURAN-2 (3H)-CYCLOHEXANE] (54h) ; 6-CARBOXYL-4-P-CARBOXYLPHENOXYSPIRO .BENZOFURAN-2.3H. -CYCLOHEXANE! .54i. 5 54g: Mixture was stirred at 60°C for 4 h. Yield, 84%. mp 154-155°C. TLC R f 0.82 (ether). IR (KBR) 3200-2500(b) , 1680(b), 1590, 1440, 1390(b), 1090, 1030, 950, 780, 770cm" 1 . NMR (CDC1 3 ) δ 7.62-7.60 (d,lH), 7.44 (s,lH), 7.21-7.19 (d,lH), 3.01 (S,1H), 1.89-1.43 (m,10H). Anal. 10 Calcd for C M H W 0 3 : C, 72.39; H,6.94. Found: C, 72.31; H, 6.95. 54h: 54e (30 mg, 0.81 mmol) dissolved in MeOH (10 ml) and 10% Pd-C (20 mg) was added. H 2 gas was bubbled through the mixture for 5 h at 25°C. The mixture was filtered and 15 MeOH was removed to give a brown oil. The crude product was purified by preparative TLC plate (1:19 MeOH/CH 2 Cl 2 ) to afford a pale yellow solid as product 54h. Yield, 10 mg (36%). 54h: mp 193-195°C. TLC R f 0.49 (1:9 MeOH/CH 2 Cl 2 ) . IR • (KBr) 3380, 3320, 2940. 2860, 1700, 1600, 1510, 1420, 20 , 1320, 1290, 1270, 1210, 1200, 1050, 950, 920, 770cm" 1 . NMR (CDC1 3 ) δ 7.15 (S,1H), 7.00 (S,1H), 6.87-6.84 (d,2H), 6.87-6.84 (d,2H), 2.94 (S,2H), 1.88-1.43 (m,10H). MS m/z (relative intensity) 339(100), 322(2), 296(5), 282(7), 258(29), 217(61), 196(11), 167(15), 156(7), 130(9), 108(29), 93(95). 54i. Procedure for preparation of these compounds was essentially that used for 43. Mixture was stirred for 24 h at 60°C. The crude product was treated with CH 2 N 2 30 which then was purified by chromatography (1:4 EtOAc/hex¬ ane) to give the dimethyl ester of 54i. Yield, 0.18g (51%). Dimethyl ester of 54i: IR (neat) 2940, 2860, 1750, 1590, 1500, 1430, 1350, 1310, 1280, 1250, 1220, 1160, 1110, 1080, 1070, 1050, 1030, 1000, 920, 880, 850, 770, 35690 cm "1 . NMR (CDC1 3 ) δ 8.03-8.00 (d,2H), 7.26 (s,lH), 7.21 (S, 1H) , 6. 98-6. 95 (d, 2H) , 3 . 91 (s , 3H) , 3 . 86 (s , 3H) , 2. 82 (S , 2H) , 1.88-1. 39 (m, 10H) . The dimethyl ester of 54i (0. 15 g, 0.38 mmol) was dissolved in THF (10 ml) , then a solution of 2N NaOH (5 ml) was added. The solution was warmed to 50°C and stirred for 3 h. After cooling to 25°C, the aq. solution was rinsed with ether (20 ml) and the aq. solution was acidi¬ fied with cone. HCl. A white precipitate resulted, which was extracted with ether (50 ml x 2) . After the precipi- 10 tate was dried and concentrated, a white solid was result¬ ed as product 54i. Yield, 0.13 g (93%). 54i mp 278-280°C. TLC R f 0.29 (1:9 MeOH/CH 2 C1 2 ) . IR (KBr) 3200-2400(b) , 1690, 1605, 1590, 1505, 1425, 1315, 1280, 1220, 1170, 1050, 950, 915, 880, 850, 770, 740, 720cm "1 . NMR (MEOH-d 4 ) δ 8.05-8.02 15 (d,2H), 7.18 (S,1H), 7.15 (s,lH), 7.03-7.00 (d,2H) , 2.84 (s,2H), 1.86-1.43 (m,10H) . 13 C NMR (MeOH-d 4 ) δ 169.21, 168.97, 162.48, 162.13, 153.28, 133.91, 133.13, 126.71, 124.86, 118.09, 114.23, 108.04, 91.47, 39.76, 37.98, 26.09, 23.90. Anal. Calcd for C-_ll_-0-i C, 68.47; H, 5.47. 20 Found: C, 68.22; H, 5.48. 7.12. SODIUM SALTS OF 4-BENZYLOXY-6-CARBOXYLSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55a) ; 4-N-BUTYLOXY-6-CARBOXYLSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55b) ; 25 6-CARBOXYL-4-PHENOXYSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55c) ; 6-CARBOXYL-4-(2 » -HYDROXYETHYLOXYSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55d) ; 6-CARBOXYL-4-P-NITROPHENOXYSPIRO [BENZOFURAN-2(3H)-CYCLOHEXANE] (55e) ; 6-CARBOXYLSPIRO[BENZOFURAN-2(3H)- ^ CYCLOHEXANE] (55g) ; AND U 6-CARBOXYL-4-P-CARBOXYLPHENOXYSPIRO .BENZOFURAN-2.3H.-CYCLOHEXANE! f55i. Preparation of 55a-e,g,i. Procedure for preparation of these compounds was essentially that used for 44. NMR spectra for 55a-g are listed as below. 55a: NMR (D 2 0) δ 35 7.21-6.88 (S,7H), 4.85 (S,2H), 2.56 (s,2H), 1.43-1.06 (m,10H). 55b: NMR (D 2 0) δ 7.10 (S,1H), 6.90 (S,1H), 4.13 (t,2H), 2.93 (S,2H), 1.79-1.67 (m,8H) , 1.50-1.41 (m,6H), 0.93 (t,3H). 55c: NMR (D 2 0) δ 7.20-6.80 (m,7H), 2.61 (s,2H), 1.60-1.12 (m,10H) . 55d: NMR (D 2 0) δ 7.07 (s,lH), 56.92 (S,1H), 4.18 (t,2H), 3.93 (t,2H), 2.96 (s,2H), 1.68 (s, b, 6H) , 1.44(S, b, 4H) . 55e: NMR (D 2 0) δ 8.10-8.07 (d,2H), 7.13 (s,lH), 7.11 (s,lH), 6.98-6.95 (d,2H), 2.68 (S,2H), 1.70-1.23 (m,10H). 55i: NMR (D 2 0) 7.90-7.87 (d,2H), 7.14 (S,1H), 7.12 (S, IH) , 7.00-6.97 (d,2H), 2.76 (s,2H), 101.70-1.27 (m,10H). 55g: NMR (D 2 0) δ 7.45-7.42 (d,lH), 7.23-7.20 (m,2H), 2.93 (s,2H), 1.65 (s, b, 6H) , 1.41 (s,b, 4H) . 13 C NMR (D 2 0) δ 177.63, 160.05, 139.25, 133.68, 127.83, 124.60, 112.09, 93.64, 42.45, 38.96, 27.15, 25.37. 15 8. EXAMPLES OF 6,7-DISUBSTITUTED -4-METHOXYSPIRO[BENZOFURAN- 2.3H.-CYCLOHEXANES1 The melting points were measured with a Thomas Hoover apparatus, and are uncorrected; the infrared spectra were obtained with the aid of a Perkin Elmer 28IB spectropho- 0 . tometer, the \H NMR spectra were recorded in deuterio- chloroform, unless another solvent is specified, either at 90 MHz, in a Varian EM 390 apparatus, or at 300 MHz in a Varian VXR 300 spectrometer. The 13 C NMR were recorded at 75.4 MHz in a Varian VXR 300 spectrometer. All the sig¬ nals are reported in ppm, downfield from tetramethyl- silane, used as internal reference. The multiplicities of the signals are abbreviated as: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, b=broad signal. The 0 low resolution mass spectra were obtained from a Finnigan 3221-F200 spectrograph, at 70 eV. The elemental analyses were done by Atlantic Mirolab, Inc. (Norcross, GA) . The HRMS was obtained at Massachusetts Institute of Technolo¬ gy- 5 All the solvents and chemicals used were of good quality. In several cases, some of them were purified and/or dried following preestablished procedures. All the reactions were carried out under a dry nitrogen 5atmosphere. The column chromatographies were done using MN Silica gel 60, under atmospheric pressure, using dif¬ ferent solvent systems (hexane-ether, hexane-ethyl ace¬ tate) , in which increasing quantities of the second sol¬ vent were periodically added to the first solvent, or on a 10 chromatotron. One percent acetic acid was added during the chromatographies of carboxylic acids. 8.1. SYNTHESIS OF 7-CARBOXY-6-FORMYL-4-METHOXY SPIROrBENXOFURAN-2fDH.-CYCLOHEXANE (62. Preparation of 58: A solution of n-butyllithium in 15 hexane (32.67 ml, 65.34 mmol) was added dropwise to a cold solution (0°C) of 3,5-dimethoxybenzyl alcohol (56, 5 g, 29.7 mmol) and TMEDA (5.67 ml, 35.64 mmol) in THF (300 ml) . After stirring at 0°C for 15 min and at room tempera- 2 0 ture for 90 min, the reaction was cooled to -45°C and copper (I) iodide was added (7054 mg, 37.13 mmol). One hour later, the electrophile 57 [prepared as described in J. Heterocyclic. Chem. 26, 879-891 (1989)] was added via syringe. After stirring overnight at room temperature, 25 concentrated ammonium hydroxide (100 ml) was added, and the reaction products were extracted with ethyl acetate (4 x 150 ml) . The combined organic phases were dried over MgS0 4 , the solvent was evaporated in vacuo and the remain¬ ing oil was chromatographed yielding 58 (6678 mg, 25.49 30 mmol, 86%) as a viscous oil, which crystalized on stand¬ ing. This was recrystallized (hexane ether) giving a white solid mp. 61-63°C; IR (KBr): 3290, 2930, 1590, 1460, 1425, 1210, 1140, y 1120 cm "1 ; l K NMR: 1.56 (m, 4H) , 1.93 (m, 4H) , 2.62 (bs, IH, exc) , 3.26 (bs, 2H) , 3.79 (s, 6H) , 35 4.59 (s,2H), 5.20 (bs, IH) , and 6.54 (s, IH) ; 13 C NMR: 22.5, 23.1, 25.3, 28.8, 30.6, 55.8, 65.7, 102.5, 116.6, 119.9, 136.3, 139.8, and 158.5; Elemental analysis: calcd. C=73.25, H=8.45; obsd. C=73.21, H=8.52. Preparation of 59: A solution of n-butyllithium in hexane (4.2 ml, 8.4 mmol) was added to compound 58 (1000 mg, 3.82 mmol) in hexane (50 ml) and TMEDA (0.69 ml, 4.58 mmol) . After stirring at 0°C for 15 min and at room tem¬ perature for 90 min, the resulting suspension was cooled to -78°C and dry carbon dioxide was bubbled for 1 hr at -78°C and during 1 hr at room temperature. After adding 2N NaOH (25 ml) , the unreacted material was extracted with ether and the extracts were discarded. The aqueous phase was acidified with 6N HCl and the reaction products were extraced with ether (4 x 50 ml) . After drying (MgSO 4 ) and concentration in vacuo of the organic phase, 59 (727 mg, 2.52 mmol, 66%) was recovered as a solid mp. 103-104.5°C (recrystallized from hexane-ether) ; IR (KBr): 3000-2820, 1740, 1600, 1460, 1420, 1340, 1240, 1200, 1090, 1015, and 940 cm" 1 ; *H NMR: 1.48-2.00 (m, 8H) , 3.30 (s, 2H) , 3.88 (s, 3H) , 4.03 (s, 3H), 5.19 (bs, IH) , 5.19 (s, 2H) , and 6.64 (s, IH) ; 13 C NMR: 22.5, 23.1, 25.3, 28.9, 31.0, 56.2, 62.6, 68.8, 98.7,109.8, 121.0, 123.0, 136.2, 148.8, 158.4 and 164.3; Elemental analysis: calcd. C=70.83, H=6.94; C=70.77, H=6.97. Preparation of 60: A solution of 59 (470 mg, 1.63 mmol) was demethylated until no more starting material was left, according to the TLC. The reaction products were extracted with ethyl acetate (4 x 40 ml) , then dried (MgS04) , concentrated in vacuo and purified to yield 60 (332 mg, 1.21 mmol, 74%) as a white solid mp. 150-151.5°C (recrystallized from hexane-ether) ; IR (KBr) : 3420, 3000-2800, 1720, 1600, 1465, 1345, 1280, 1250, 1195 and 1075; Η NMR: 1.45-2.00 (m, 8H) , 3.27 (s, 2H) , 3.88 (s, 3H) , 5.2 (s, 2H) , 6.5 (s, IH) and 7.71 (s, IH) ; 13 C NMR: 22.4 , 23 .0 , 25.2 , 28 .7 , 30.2 , 56.2 , 70. 4 , 96. 1, 104.2 , 115. 5 , 120. 9 , 135. 4 , 146. 2 , 155. 0 , 165. 2 and 172 . 8. Preparation of 61: Amberlyst 15 (290 mg, 2 g/mmol) was added to a solution of 60 (40 mg, 0.146 mmol) in dry methylene chloride (2.9 ml, 20 ml/mmol) after stirring overnight at room temperature, the solvent was filtered through a short column of silica gel, the solids were washed with ethyl acetate and the filtrate was filtered through silica gel. The combined filtrates were concen- trated in vacuo and chromatographed, affording 61 (38 mg, 0.139 mmol, 95%) as a solid mp. 132-134°C (recrystallized from hexane-ether) ; IR (KBr): 2980-2820, 1740, 1610, 1440, 1330, 1280, 1260, 1235, 1205, 1145, 1080, 1010, 930 and 780 cm' 1 , X H NMR: 1.35-2.00 (m, 10H) , 2.89 (s, 2H) , 3.91 (s, 3H) , 5.20 (s, 2H) and 6.46 (s, IH) ; 13 C NMR: 23.1, 25.0, 37.1, 55.9, 69.8, 93.7, 96.0, 102.0, 114.8, 150.2, 157.8, 162.1 and 169.6; Elemental analysis: calcd. C=70.04, H=6.62; Obsd. C=70.11, H=6.62. Preparation of 62: Compound 61 (211 mg, 0.77 mmol) was dissolved in THF (4 ml) and the resulting solution was added to a solution of ION NaOH (6 ml, 60 mmol) and FTBA (1 ml, 1 mmol) . Potassium permanganate was (KMn0 4 ) added to the system until no more starting material was observed by TLC. The excess of KMn0 4 was destroyed with sodium sulfite and the reaction was acidified with 6N H 2 S0 4 . The organic material was extracted with ethyl acetate (4 x 50 ml) , the extracts were dried (MgS0 4 ) , concentrated in vacuo and chromatographed yielding 62 (120 mg, 0.41 mmol, 53%) as a white solid mp. 128-130°C (recrystallized from hexane-ether); IR (KBr): 2950, 2850, 1830, 1770, 1625, 1455, 1340, 1270, 1210, 1145, 990, 985 and 745 cm" 1 ,* ~R NMR: 1.35-1.95 (m, 10H) , 2.86 (s, 2H) , 3.91 (s, 3H) , 5.90 (bs, IH) , 6.53 (S,1H) and 6.61 (s, IH) ; 13 C NMR: 23.1, 24.9, 37.0, 37.1, 56.0, 93.9, 97.3, 98.0, 102.6, 116.4, 150.1, 157.3, and 162.1 and 167.6; Elemental analysis: calcd. C=66.20, H=6.25; obsd. C=66.03, H=6.18. 8.2. SYNTHESIS OF 6-CARBOXY-7-FORMYL-4-METHOXY- SPIRO[BENZOFURAN-2(3H)-CYCLOHEXANE (68) AND 5 6,7-DICARBOXYL-4-METHOXYSPIRO [BENZOFURAN-2(3H. -CYCLOHEXANE! .66. Preparation of 65: A solution of n-butyllithium in hexane (2.20 ml, 4.95 mmol) was added to a solution of N, N, N' trimethylethylenediamine (0.65 ml, 5.09 mmol) in THF 10 (6 ml) at -20°C (CC1 4 /C0 2 ) . After 30 min, lib (1170 mg, 4.76 mmol, prepared as described in section 6.8 supra) in THF (4 ml) was added dropwise, followed 30 min later by n-BuLi (6.35 ml, 14.28 mmol). The resulting system was kept at -20°C for 24 h, and DMF was added (02.20 ml, 28.56 15mmol). After 24 h reaction period, the reaction products were partitioned between ether (4 x 50 ml) and brine (50 ml), chromatography of the extracts gave 65 (1135 mg, 4.14 mmol, 87%) as a solid mp. 129-131°C (recrystallized from hexane-ether); IR (KBr): 3000-2840, 1670, 1600, 1470, 201425, 1390, 1320, 1280, 1260, 1210, 1130, 1030, 890, 850, 770, 700 and 620 cm "1 ; -K NMR: 1.40-1.93 (S, 10H) , 2.93 (s, 2H) , 3.94 (S, 3H) 7.04 (s, IH) , 10.35 (s, IH) and 10.70 (S, IH) ; 13 C NMR: 22.9, 24.9, 37.2, 37.7, 56.0, 93.0, 103.6, 113.7, 120.2, 138.7, 160.3, 164.7, 188.6 and 192.6; 25 Elemental analysis: calcd. C=70.06, H=6.61; obsd. C=69.84, H=6.65. Preparation of 68: A 4 N solution of potassium hydroxide (52.92 mmol) was added dropwise to a stirred solution of compound 65 (2900 mg, 1058 mmol) in THF (10 30 . . . . . ml) and water (7 ml) containing dissolved silver nitrate (3777 mg, 22.22 mmol, 1.05 eq.) at room temperature. The reaction system was protected from direct light. After stirring for 2 h at room temperature, the solids were filtered and extensively washed with distilled water. 2N 35 H 2 S0 4 was added until pH 3, and the reaction products were extracted with ether (3 x 150 ml) , washed with brine (2 x 25 ml) and dried (MgS0 4 ) . Chromatography of the reaction products allowed the recovery of the starting material 5 (1823 mg, 63%), compound 68 (326 mg, 1.12 mmol, 11%, 29% corrected yield) and compound 66 (614 mg, 2.01 mmol, 19%, 51%). Compound 68: mp: 148-150°C (rec. from hexane-ether); IR (KBr): 3440, 3000-2840, 1740, 1630, 1450, 1345, 1290, 1270, 1150, 1105, 1010, 910, 860, 770, 690, cm" 1 ; H NMR 10 (DMSO d 6 ) : 1.30-1.52 (m, 4H) , 1.62-1.81 (m, 6H) , 2.91 (s, 2H) , 3.87 (s, 3H) , 6.55 (bs, IH) , 6.85 (s, IH) , and 7.95 (bs, IH) ; 13 C NMR (DMSO d 6 ) : 22.52, 24.47, 36.57, 37.56, 55.86, 91.90, 96.33, 98.34, 120.60, 121.48, 128.99, 154.0- 3, 158.28, 168.42; MS (e/m, %) : 290 (M+, 89), 289 (72), 15272 (100), 271 (88), 244 (65), 215 (78), 192 (98), 191 (95), 190 (65), 165 (93), 164 (82), 79 (89). Elemental analysis: calcd. C=66.20, H=6.25; obsd. C=66.19, H=6.26. Direct Preparation of 66: To a solution 65 (153 mg, 0.56 mmol) in ethanol (5 ml), was added a solution of 20 silver nitrate (222 mg, 1.30 mmol) in distilled water (1 ml), followed by KOH (3 ml, 2.99 mmol). The system was stirred overnight at room temperature, shielded from the light, then it was filtered and the residue was carefully washed with water. The combined aqueous phases were 25 extracted with ether, the aqueous phase was then acidified and extracted with ether (3 x 25 ml) ; the combined organ¬ ic phases were dried and chromatographed to afford 66 (156 mg, 0.51 mmol, 91%) as a white solid mp. 188-190°C (recrys¬ tallized from acetone); IR (KBr): 3500-2400, 3000-2850, 30 1700, 1610, 1410, 1330, 1290, 1130, 1040, 1000, 930, 855, 750 and 660 cm' 1 : ~K NMR: (acetone d 6 ) : 1.40-1.90 (m, 10H) , 2.93 (s, 2H) , 3.91 (s, 3H) and 6.95 (s, 2H) ; 13 C NMR: (acetone d,;) : 23.5, 25.6, 37.6, 38.7, 56.1, 91.7, 105.1, __ 111.6, 118.8, 133.0, 157.8, 158.8, 167.2 and 186.1; Ele- 5 mental analysis: calcd. C=62.74, H=5.92; obsd. C=62.66, H=6.01. 8.3. SYNTHESIS OF 4-METHOXY-5a,6,8,8a- TETRAHYDROSPIRO[BENZO[2,1-b:3,4-C•] 5 DIFURAN-2(8H)CYCLOHEXAN]-6,8-DIONE; 6,7-DIHYDROXYMETHYL-4-METHOXYSPIRO [BENZOFURAN-2(3H)CYCLOHEXANE] ; AND 4-METHOXYSPIRO[BENZO[2,1-B:3,4-C'] DIFURAN-2.3H.-CYCLOHEXAN!-6 (8H.-ONE Preparation of 63: A cold and stirred solution of 62 10 (150 mg, 0.52 mmol) in acetone (10 ml) was treated with excess of Jones reagent (Bowden et al., 1946, J. Chem. Soc. 39; Bowers et al., 1953, J. Chem. Soc, 2548) until the starting material was completely converted (TLC) . The excess reagent was destroyed with isopropanol, brine was 15 added (30 ml) and the reaction products were extracted with ethyl aσetate (4 x 50 ml) the extracts were dried (MgS0 4 ) , σonσentrated in vaσuo and σhromatograhphed yield¬ ing 63 (134 mg, 0.46 mmol, 89%) as a white solid mp. 197-198.5°C (reσrystallized from aσetone) ; IR (KBr): 3400, 202940, 2860, 1740, 1620, 1450, 1330, 1235, 1140, 1090, 1020, 930 and 760 σm" 1 ; l H NMR (aσetone d 6 ) : 1.45-1.95 (m, 10H) , 3.03 (s, 2H) , 4.08 (S, 3H) , 7.15 (s, IH) ; 13 C NMR (aσetone d 6 ) : 23.5, 25.5, 37.7, 38.3, 57.2, 95.6, 102.1, 123.9, 134.9, 158.4, 161.2, 163.8 and 164.2; MS (m/Z, %) : 25 288 (M+, 40), 287 (32), 231 (40), 208 (49), 207 (52), 85 (27), 81 (100), 77 (36), 71 (28), 69 (42), 57 (67) and 55 (61); HRMS: expected 288.0998 for C 16 H 16 O s , obsd.: 288.0999. Preparation of 64: A solution of 61 (300 mg, 1.09 mmol) in THF (15 ml) was treated for 3 h with LAH (slight 30 excess). After adding Na 2 SO 4 .10H 2 O and ether, the reaction produσts were filtered, affording the diol 64 (260 mg, 0.94 mmol, 86%); Η NMR: 1.40-1.90 (m, 10H) , 2.93 (s, 2H) , 3.83 (S, 3H) , 4.66 (S, 2H) , 4.70 (S, 2H) and 6.47 (s, IH) . 5 Preparation of 67: To a solution of 64 (260 mg, 0.935 mmol) in dry methylene chloride (15 ml) BaMn0 4 (1197 mg, 4.68 mmol) was added and the resulting suspension was stirred for 3 days at room temperature. After recovering the reaction products by partition between brine (30 ml) and ethyl acetate (3 x 50 ml) it was observed that this was an equimolecular mixture of 67 and 65. 9. COMPLEMENT INHIBITION BY DI- AND TRI- SUBSTITUTED SPIRO[BENZOFURAN- 2 (3H.-CYCLOHEXANES! The 4 substituted spirobenzofuran σompounds of exam¬ ple 7 and the disubstituted spirobenzofuran σompounds of example 8 were tested for their capacity to inhibit complement-mediated lysis of sheep red blood cells (SRBC) as described in Section 6.6.2 supra with the modification that the human serum used as the complement source was diluted to 1 to 100 in Hepes buffer, instead of 1 to 125. The results of the hemolysis assays are shown in FIGURE 8 and Tables XI and XII. Table XI Inhibition of Hemolysis by 6,7-disubstituted -4-methoxy spirofbenzofuran-2 (3H)-σyσlohexanes] * The σonσentration of σompound (± standard deviation) required to produσe a value for hemolysis inhibition of 20 0.5 as desσribed in Seσtion 6.36.2 supra. 25 30 5 Table XII Inhibition of Hemolysis by 6-carboxyl-4-substituted s o benzofuran-2 3H -c clohex n * The conσentration of σompound (± standard deviation) required to produσe a value for hemolysis inhibition of 5 0.5 as desσribed in Seσtion 6.36.2 supra. ** The sodium salt of 4-p-aminophenoxy- σarboxyspiro[benzofuran-2(3H)-σyσlohexane] 0 By σomparing the inhibition of hemolysis by σompounds 62, 66 and 68 as shown in figure 8 and Table XI, it σan be seen that the partiσular arrangement of substitutents at positions 6 and 7 of the benzofuran ring is important for anti-hemolytiσ aσtivity. By σomparing IH 50 values for the 5 position 4-substituted series as shown in Table XII" (for example, the values for 55c, 55e, and 55h) , improvements in anti-hemolytic aσtivity σan also be obtained through the optimal σhoiσe of the R substituent at position 4. In addition, several of the σompounds (see for example, 5 σompounds 55c, 55e, 55h and 68 in particular) are more effective in inhibiting complement-mediated hemolysis than is K76COOH. It is anticipated that optimal substituents at position 4, combined with an optimal pair of sub¬ stituents at position 6 and 7, will result in even more 10 potent complement inhibitors. 10. IN VITRO AND IN VIVO INHIBITION OF COMPLEMENT BY DI- AND TRI-SUBSTITUTED SPIROrBENZOFURAN-2f3H.-CYCLOHEXANES! 10.1. INTRODUCTION 15 The role of complement in hyperacute graft rejeσtion has been demonstrated in animal models of both sensitized allograft (Kneσhtle et al., J. Heart Trans¬ plant.. 1983, 4:541; Pruitt and Bollinger, J. Surer. Res.. 20 1991, 50.:350-355; as well as xenograft transplantation (Adaσhi et al., Transplant Proσ.. 1987, .119:1145-1148; Migagawa et al.. Transplantation. 1988, 46.:825; Prewitt et al., 1991, Transplantation 52:868-873). We have σhosen an established disσordant xenograft model, a guinea pig-to- 25 rat σardiaσ transplant, to assess the effeσts of the substituted dihydrobenzofurans on hyperaσute rejection in vivo. 10.2. INHIBITION IN VITRO OF RAT AND HUMAN COMPLEMENT 30 Compounds 68 and 44a were prepared and charac¬ terized as described in seσtions 8.2 and 7.1, respeσtive- ly. These σompounds were demonstrated to inhibit σomple¬ ment-mediated hemolysis using human serum as a σomplement sourσe, as desσribed in seσtion 6.6.2 and Seσtion 9, 5 supra. Compounds 68 and 44a were also demonstrated to inhibit rat σomplement using the methods of seσtion 6.6.2, exσept that rat serum was substituted for human serum as the σomplement sourσe. A soluble human reσombinant σom¬ plement reσeptor type 1 (sCRl) was prepared as desσribed (Weisman et al., Sσience, 1990, 249:146-151; Yeh et al., J. Immunol. 1991; and International Patent Publiσations No. WO 89/09220 and WO 9/05047, both entitled "The human C3b/C4b reσeptor (CR1)" Fearon et al.) and used as a positive σontrol the in vitro assays. The results of 10 these studies are summarized in Table XIII. Table XIII Inhibition of rat or human σomplement-mediated 15 hemolysis by substituted spiro[benzofuran-2(3H) σyσlohexanes] and by sCRl 20 *The σonσentration of σompound required to produσe a value -- for hemolysis inhibition of 0.5 as desσribed in Seσtion 6.6.2, supra. As seen in Table XIII, the human sCRl protein inhib¬ its rat and human σomplement with similar potenσy in this 30 assay. The σompounds, on the other hand, more effeσtively inhibit human than rat σomplement. Twenty-five- to 70- fold higher σonσentrations of σompound 68 were required to inhibit rat σomplement-mediated lysis than were required to inhibit human σomplement-mediated lysis to similar -- levels. 10.3.INHIBITION OF ALTERNATIVE PATHWAY- MEDIATED HEMOLYSIS The inhibition of sheep red blood σell lysis as desσribed in section 6.6.2 predominantly reflects on classiσal σomplement pathway aσtivation sinσe it is initi¬ ated by the antigen-antibody σomplexes on the surfaσe of the sensitized erythroσytes. The assay probably inσludes a minor σontribution from alternative pathway aσtivation as well. The capacity of the substituted dihydrobenzofur¬ ans to inhibit the alternative σomplement pathway was assessed using the modified method of Platts-Mills and Ishizaka, 1974, J. Immunol.. 113:348-358. Essentially, rabbit erythroσytes were lysed using human serum as σom- plement in gelatin veronal buffer (GVB, Sigma, St. Louis, MO) with added EGTA (ethylene glyσol-bis[,3-aminoethyl etherJN f N f N^N^tetraaσetiσ aσid) and Mg 2+ to 8 mM and 2 mM, respeσtively. Rabbit erythroσytes (4.5 x 10 6 σells/ml) , human serum (1 in 24 dilution) , and the σompounds to be tested were inσubated one hour at 37°C in a V-bottom miσrotiter plate, cells pelleted by σentrifugation, the supernatants transferred to a flat-bottom miσrotiter plate, and the absorbanσe at 410 nm determined. Samples were paired with identiσal σontrols laσking human serum (σomplement-independent lysis) . Both samples and σontrols were run in tripliσate. Control values were subtraσted from sample values and the fraσtional inhibition was determined relative to the uninhibited (no added σompound) sample. Results are reported as the σonσentration of σompound yielding fifty per σent (50%) inhibition, AH 50 . The AH 50 values for several of the σompounds are inσluded along with the results of other jLn vitro assays in Table XIV. As seen in Table XIV, σompound 68 inhibited the classical pathway more effectively than K76COOH, but was somewhat less potent than K76COOH in inhibiting alterna- tive σomplement pathway. Compound 44a inhibited the σlassiσal pathways as well as K76COOH, but was σlearly less potent than K76COOH or 68 in inhibiting the alterna¬ tive pathway. 10.4. INHIBITION OF C3b PROTEOLYSIS BY FACTOR I K76COOH has been shown to inhibit the proteoly¬ sis of C3b by faσtor I and the σofaσtor protein H (Hong et al., 1981, J. Immunol., 127:104-108. The σapaσity of the substituted dihydrobenzofurans to inhibit the speσifiσ degradation of the C3b α-σhain by faσtor I was assessed on sodium dodeσylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Wong et al. , 1985, J. Immunol. Methods, 82:303- 313; Weisman et al., 1990, Science, 249:146-151) using either factor H or sCRl as cofactor. Purified C3b at 1.2 μM, factor I (Cytotech, San Diego, CA) at 0.1 μM, sCRl at 8 nM as a cofactor, and test compound at varying con- σentrations, were inσubated for 60 minutes at 37°C. The reaσtions were stopped by the addition of reduσing SDS- PAGE sample buffer (Laemmli, 1970, Nature, 227:680-685) subjeσted to eleσtrophoresis on 4-20% polyaσrylamide gradient gels, visualized with Coomassie Blue stain, and quantitated by densitometry. Controls laσking faσtor I and σontrols with faσtor I but laσking test σompounds were inσluded. Results are reported as the σonσentration of test σompound whiσh inhibited approximately fifty perσent (50%) of the C3b α-σhain degradation, IC 50 . The results for several σompounds are inσluded in Table XIV. TABLE XIV In vitro assays of σomplement inhibition by substi¬ tuted spiro[benzofuran-2(3H)-σyσlohexanes] . Concentration* (μM) yielding 50% inhibition. *Va ues reporte are e mean ± s an ar deviation; sample number, n) Compound 68 and K76COOH were equally effective in inhibit¬ ing proteolysis of C3b by factor I using sCRl as a σofaσ¬ tor. 10.5. GUINEA PIG-TO-RAT CARDIAC TRANSPLANT The effeσts of compounds 68 and 44a on hyper- acute graft rejection were tested in a model of discordant xenografting. The methods were the same as desσribed previously (Prewitt et al., 1991, Transplantation 52:868- 873) exσept that the test σompounds were substituted for the aσtive agent (sCRl) of the earlier study. As indiσat- ed in the earlier study, male Lewis rats, 12-16 weeks of age, and male Hartley guinea pigs, 4-10 weeks of age (Charles River, Wilmington, MA) served as σardiac xeno¬ graft recipients and donors, respeσtively. Under Halo- thane anesthesia, heterotopic σardiaσ xenotransplantation was performed by a modifiσation of the microvascular teσhnique of Ono and Lindsey, 1990, J. Thoraσ. Cardiovas. Surg. , 57:225, with anastomosis of the donor aorta to the recipient infrarenal aorta and the donor pulmonary artery to the recipient inferior vena cava. Cardiaσ xenografts were evaluated visually over the first 30 minutes follow¬ ing reperfusion and then, following abdominal σlosure, every 10 minutes by palpation until rejeσtion. Rejeσtion was defined as total σessation of σardiac xenograft con¬ traction and was confirmed by direct visualization and histological examination. The σompounds 68 and 44a were prepared as 14 mg/ l solutions in phosphate buffered saline (PBS) and were administered as a 2.0 ml intravenous bolus into the reσipient inferior vena σava, superior to the σaval σross σlamp, immediately prior to σardiac xeno- graft reperfusion. Control animals received similar volumes of PBS. Three (n=3) σontrol animals were tested σonσurrent with the animals reσeiving σompound 68; data from historiσ σontrols (n=13) subjeσted to the same proσe¬ dure and previously reported (Pruitt et al., 1991, Trans- * plantation 52:868-873) are also inσluded for σomparison. Table XV inσludes the xenograft survival times (minutes) for individual animals and the means and standard errors of the mean (SEM) for the various groups. As σan be seen in the table, σompound 68 treatment resulted in prolonged graft survival times in 2 of the 3 animals tested relative to σontrols. TABLE XV Guinea pig-to-rat σardiaσ transplant The present invention is not to be limited in sσope by the foregoing examples, whiσh are intended as purely illustrative of speσifiσ aspeσts of the invention and any σompounds that are funσtionally equivalent are within the sσope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing desσription and aσσompanying drawings. Suσh modifiσations are intended to fall within the sσope of the appended σlaims. Various referenσes are σited herein, the disσlosures of whiσh are inσorporated by referenσe herein in their entireties.

Claims: WHAT IS CLAIMED IS: 1. A σompound of the general formula, 4: 4 in whiσh R represents a hydrogen atom, a lower alkyl group, a substituted lower alkyl group, a benzyl group, a substituted benzyl group, a phenyl group or a substituted phenyl group; R, and R 2 represent independently, a σarboxylic acid group, a formyl group, a hydroxymethyl group, a N-(lower alkyl)σarbamoyl group, a trifluoroaσetyl group, a halide group, a vinyl group, a substituted vinyl group having up to 10 carbon atoms, an alkylidene group having up to 20 carbon atoms, an aliphatiσ acyl group, a substituted aliphatic acyl group, an aromatic acyl group, a substituted aromatic acyl group, a sulfamoyl group, an aminomethyl group, a N-(lower alkyl)aminomethyl group, a N,N-di(lower alkyl)aminomethyl group, a heteroσyσliσ ring, an N-(aσyl)carbamoyl group, an amidino group or a hydrazide group; R x and R 2 together with the carbon atoms to which they are attached may also represent a cyσlic anhydride or lactone; or a pharmaceutically acσeptable acid or base addition salt or ester thereof. 2. The compound of claim 1 which is selected from the group σonsisting of 6,7-diσarboxy-4- methoxyspiro[benzofuran-2(3H)-σyσlohexane] , 6,7-diformyl- 4- ethoxyspiro[benzofuran-2(3H)-cyclohexane] , 6,7- di(hydroxymethyl)-4-me hoxyspiro(benzofuran-2(3H)- cylcohexane] , 4-methoxy-3,6-dihydrospiro[benzo[2,1-b:3,4- c']difuran-2(8H)-cyclohexane]-8-one, 7-carboxy-6-formyl-4- methoxyspiro [benzofuran-2(3H)-cyclohexane] , 4- 5 methoxy,5a,6,8,8a-tetrahydrospiro[benzo[2,1-b:3,4- c 1 ]difuran-2(8H)-cyclohexan]-6,8-dione, and 6-carboxy-7- hydroxymethyl-4-methoxyspiro[benzofuran-2(3H)- σyσlohexane]laσtone. O 3. The compound of claim 1 which is selected from the group consisting of 6-carboxy-7-formyl-4- methoxyspiro[brenzofuran-2(3H)-cyclohexane] and 6-carboxy- 7-formyl-4-phenoxyspiro[benzofuran-2(3H)-cyclohexane] . 4. The compound of the general formula, 4: in which R represents a hydrogen atom, a lower alkyl group, a substituted lower alkyl group, a benzyl group, a substituted benzyl group, a phenyl group or a substituted phenyl group; : represents a carboxylic acid group, or a bioisosteric acid or base group; R 2 represents a formyl group or a bioisosteric neutral group; or a pharmaceutically acceptable acid or base addition salt or ester thereof. 5. The σompound of σlaim 4 in whiσh said bioisosteriσ base group is a σarbamoyl group, a sulfamoyl group, an N-aσylσarbamoyl group or a tetrazole ring. 6. The σompound of σlaim 4 in which said bioisosteric base is an aminomethyl group, a N- (lower alkyl)aminomethyl group, a N.N-di(lower alkly)aminomethyl group, an oxazoline ring, an amidino group or a hydrazide group. 7. The compound of claim 4 in which said bioisosteric neutral group is an aliphatic acyl group, a substituted aliphyatic aσyl group, an aromatiσ aσyl group, or a substituted aromatiσ aσyl group. 8. A σompound of the general formula, 4: 4 in whiσh R represents a hydrogen atom, a lower alkyl group, a substituted lower alkyl group, a benzyl group, a substituted benzyl group, a phenyl group or a substituted phenyl group; R 2 represents a hydrogen atom; R ! represents a formyl group, σarboxylic acid group; or a pharmaceuti- σally aσσepta le aσid or base addition salt or ester thereof . 9. The σompound of σlaim 8 whiσh is 6-σarboxy-4- m ' ethoxyspiro [benzofuran-2 ( 3H) -σyclohexane] . 10. The compound of claim 8 which is selected from the group consisting of 6-carboxy-4- ethoxyspiro[benzofuran-3(3H)-σyσlohexane] , 6-formyl-4- hydroxyspiro[benzofuran-2(3H)-σyσlohexane], 4-benzyloxy-6- 5 formylspiro[(benzofuran-2(3H)-cylcohexane] , 4-butyloxy-6- formylspiro[benzofuran-2(3H)-σyσlohexane], 6-formyl-4- phenoxyspiro[benzofuran-2(3H)-σyσlohexane], 6-formyl-4-(2- hydroxyethyloxy)spiro[benzofuran-2(3H)-σyclohexane], 6- formyl-4-(p-nitrophenoxy)sprio[benzofuran-2(3H)- σyσlohexane], 6-formyl-4-(p_-for_nylphenoxy)spiro [benzofuran-2(3H)-σyσlohexane], 6-formylspiro[benzofuran- 2(3H)-σylohexane], 4-benzyloxy-6-σarboxyspiro[benzofuran- 2(3H)-σyclohexane] , 4-butyloxy-6-carboxyspiro[benzofuran- 2(3H)-cyclohexane], 6-carboxy-4-phenoxyspiro[benzofuran- 2(3H)-σyσlohexane], 6-σarboxy-4-(2-hydroxyethyloxy)spiro [benzofuran-2(3H)-cyσlohexane], 6-σarboxy-4-(p_- nitrophenoxy)spiro[benzofuran-2(3H)-σyσlohexane] , 6- carboxyspiro[benzofuran-2(3H)cyclohexine] , 6-carboxy-4- (__- . a inophenoxy)spiro[benzofuran-2(3H)-σyclohexane, and 6- carboxy-4-(p_-σarboxyphenoxy)spiro[benzofuran-2(3H)- σyσlohexane] . 11. A σompound of the general formula, 4: 4 in which R represents a hydrogen atom, a lower alkyl group, a substituted lower alkyl group, a benzyl group, a substituted benzyl group, a phenyl group or a substituted phenyl group; R j and R 2 represent independently a hydrogen atom, a σarboxyliσ aσid group, a formyl group, a hydroxymethyl group, a N-(lower alkyl)σarbamoyl group, a trifluoroaσetyl group, a halide group, a vinyl group, a substituted vinyl group having up to 10 σarbon atoms, an alkylidene group having up to 20 σarbon atoms, an aliphatiσ aσyl group, a substituted aliphatiσ aσyl group, an aromatiσ aσyl group, a substituted aromatiσ aσyl group, a sulfamoyl group, an aminomethyl group, a N-(lower alkyl)aminomethyl group, a N f N-di(lower alkyl)aminomethyl group, a heteroσyσliσ ring, an N-(aσyl)σarbamoyl group, an amidino group or a hydrazide group; Rj and R 2 together with the σarbon atoms to whiσh they are attaσhed may also represent a σyclic anhydride or lactone; or a pharmaceuti- cally aσσeptable aσid or base addition salt or ester thereof, provided that Rj and R 2 do not both represent hydrogen atoms. 12. A pharmaceutical composition for use in a method ' of treating a patient with an immune disorder or a disorder involving undesirable or inappropriate complement aσtivity σomprising an effeσtive amount of the σompound of σlaim 1, 3, 4, 8, 9 or 11. 13. The σomposition of σlaim 12 in whiσh the immune disorder or disorder involving undesirable or inappropriate σomplement aσtivity is seleσted from the group σonsisting of soft tissue destruσtion due to burn, myoσardial infarσt induσed trauma, adult respiratory distress syndrome, and myoσardial isσhmia and reperfusion; speσifiσ and non-speσifiσ proteolytiσ proσessing of C5; inflammation assoσiated with kidney stones, systemiσ lupus erythematosis, nephrotoxiσ glomeronephritis, and multiple sclerosis; atrophic gastritus, thyroiditis, allergiσ enσephalomyelitis, gastriσ muσosa, thyrotoxiσosis, autoimmune hemolytic anemia, pemphigus vulgaris, sympathetic opthalmia, delayed-type hypersensitivity, autoimmune disorders and drug allergies; and tissue plasminogen activator therapy and cardiopulmonary bypass. 5 14. A pharmaσeutiσal σomposition for use in a method of treating a patient with graft rejeσtion, graft-host reaσtion, or organ transplant rejeσtion σomprising an effeσtive amount of the σompound of σlaim 1, 3, 4, 8, 9 or 1011. 15. The σomposition of σlaim 15 in which the graft is a xenograft or an allograft. 15 16. The composition of claim 15 in which the graft is a heart transplant. 0 5 0

Inventors: BRADBURY BARTON J, IP STEPHEN H, KAUFMAN TEODORO S, LEE CHEW, MARSH HENRY C JR, SINDELAR ROBERT D, BRADBURY, BARTON, J., IP, STEPHEN, H., KAUFMAN, TEODORO, S., LEE, CHEW, MARSH, HENRY, C., JR., SINDELAR, ROBERT, D.
Applicants: T CELL SCIENCES INC, UNIV MISSISSIPPI, T CELL SCIENCES, INC., THE UNIVERSITY OF MISSISSIPPI
Application year: 1991
Main classification: A01N43
IPC codes: A61K31, A61P37, C07D307, C07D493