HK1080868A - Novel compounds, compositions as carriers for steroid/non-steroid anti-inflammatory, antineoplastic and antiviral active molecules - Google Patents

Description

New compounds, compositions as carriers for steroid/non-steroid anti-inflammatory, anti-tumor and anti-viral active molecules

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Priority

This application claims 60/395,190 priority from U.S. provisional application filed on 8/7/2002, the entire contents of which are incorporated herein by reference.

Summary of The Invention

The present invention relates to:

(a) a novel compound represented by structure I:

wherein M represents a macrolide subunit with accumulation properties in inflammatory cells, V represents an anti-inflammatory compound, which may be a steroidal or non-steroidal subunit or an antineoplastic drug subunit or an antiviral compound subunit, and L represents a linker covalently linking M and V;

(b) pharmaceutically acceptable salts, prodrugs and solvates of said compounds;

(c) processes and intermediates for the preparation of such compounds; and

(d) their use in the treatment of inflammatory, neoplastic and viral diseases and conditions in humans and animals.

Such compounds may be used as prodrugs, which transport the compound into target cells and release the compound in such target cells at concentrations higher than would normally be achieved with the compound alone; or such compounds may be active in hybrid form as administered. These compounds and their use are thus a reaction to the technical problem of increasing the effectiveness and/or efficacy and/or reducing the side effects of one or more of the above-mentioned active ingredients by preferentially transporting them in hybridized form to or in the vicinity of the target cell.

Background

Anti-inflammatory drugs can be traditionally classified into steroids and non-steroids. Steroidal anti-inflammatory compounds remain the most effective drugs for the treatment of inflammatory diseases and conditions such as: asthma, chronic obstructive pulmonary disease; inflammatory nasal diseases such as allergic rhinitis, nasal polyps; bowel diseases, such as crohn's disease, colitis, ulcerative colitis; skin inflammation such as eczema, psoriasis, allergic dermatitis, neurodermatitis, pruritus, conjunctivitis; autoimmune diseases such as rheumatoid arthritis; and transplant immunosuppression. In addition, steroids are used as adjuvant chemotherapeutic agents in the treatment of a variety of malignancies, including leukemias, lymphomas, myelomas, and other hematopoietic malignancies. In addition to excellent efficacy and effectiveness, such drugs present a number of adverse side effects such as carbohydrate metabolism, calcium absorption, secretion of endogenous corticosteroids and physiological functions of the pituitary, adrenal cortex and thymus. Recently developed steroids are highly effective in inflammatory diseases and processes because they inhibit many inflammatory mediators with reduced systemic side effects. Patent applications WO 94/13690, WO 94/14834, WO 92/13873 and WO 92/13872 describe so-called "soft" steroids or hydrolysable corticosteroids for topical administration at the site of inflammation, while their systemic side effects are reduced by the instability of the "soft" steroid in serum, where the active steroid is rapidly hydrolysed to an inactive form. However, no ideal steroids have been found which have no adverse effects in long-term and sustained therapy as required for the control of diseases such as asthma or crohn's disease. Thus, there is an urgent need for steroids with improved therapeutic properties and/or fewer or milder side effects.

Nonsteroidal anti-inflammatory drugs with different mechanisms of action act on specific mediators of inflammation, thereby providing a therapeutic effect. Due to differences not only in the mechanism of action, but also in the specific inflammation mediators inhibited, steroidal and non-steroidal drugs have different anti-inflammatory profiles, whereby certain drugs may be more suitable than others for a specific condition. Furthermore, most non-steroidal anti-inflammatory drugs are not absolutely specific and their use is accompanied by adverse side effects when used in larger doses or for long periods of time. Many non-steroidal anti-inflammatory drugs are known to act as inhibitors of the endogenous COX-1 enzyme, which is extremely important in maintaining gastric mucosal integrity. Thus, the use of these drugs often results in damage to the gastric mucosa or even bleeding. (Warner T.D. Proc. Natl.Acad.Sci.U.S.A.)1999, 96, 7563-7568, Proc. Natl.Acad.Sci.U.S.A.). Thus, agents that selectively inhibit COX-2, but not COX-1, are preferred for the treatment of inflammatory diseases. In addition, certain anti-inflammatory compounds (such as theophylline) are known to have a very narrow therapeutic index, which is limited in their use.

Recently, the FDA approved the nonsteroidal anti-inflammatory drug celecoxib (celecoxib) that specifically blocks COX-2 for the treatment of rheumatoid arthritis (Luong et al, Ann. Pharmacother., 2000, 34, 743-. COX-2 is also obtained in many cancers and precancerous lesions, and is expressed and there is established evidence that selective COX-2 inhibitors may be useful in the treatment and prevention of colorectal and other cancers (Taketo, M.M., J.Natl.cancer Inst.)1998, 90, 1609-.

In 1975, TNF- α was defined as a serum factor that is endotoxin induced and causes tumor necrosis in vitro and in vivo (Carswell EA et al, Proc. Natl. Acad. Sci. U.S.A.; 1975, 72: 3666-) -3670). In addition to anti-tumor effects, TNF- α has many other biological effects that are important in homeostasis and pathophysiological conditions. The main sources of TNF-alpha are monocyte-macrophages, T-lymphocytes and mast cells.

The discovery that anti-TNF-. alpha.antibodies (cA2) have therapeutic effects on patients with Rheumatoid Arthritis (RA) (Elliott M et al, Lancet (Lancet), 1994, 344: 1105-1110) has led to an increased interest in finding new TNF-. alpha.inhibitors that can act as effective agents for RA. Rheumatoid arthritis is an autoimmune chronic inflammatory disease characterized by irreversible pathological changes in the joints. In addition to RA therapy, TNF- α antagonists may be used in a number of pathological conditions and diseases such as spondylitis, osteoarthritis, gout and other arthritic diseases, sepsis, septic shock, toxic shock syndrome, atopic dermatitis, contact dermatitis, psoriasis, glomerulonephritis, lupus erythematosus, scleroderma, asthma, cachexia, chronic obstructive non-dysfunction, congestive heart failure, insulin resistance, pulmonary fibrosis, multiple sclerosis, crohn's disease, ulcerative colitis, viral infections and AIDS.

Two different retroviruses, Human Immunodeficiency Virus (HIV) type 1 (HIV-1) or type 2 (HIV-2), are etiologically associated with an immunoreactive disease, acquired immunodeficiency syndrome (AIDS). HIV seropositive individuals begin to be asymptomatic, and typically develop Complex Symptoms (ARCs) associated with AIDS, followed by progression to AIDS. Affected individuals exhibit severe immunosuppression which predisposes them to debilitating and ultimately fatal opportunistic infections.

AIDS is the result of the HIV-1 or HIV-2 virus following its complex viral life cycle. The life cycle of a virion involves the virion attaching itself to a host human T-4 lymphocyte immune cell by binding a glycoprotein on the surface of the virion's protective envelope to the CD4 glycoprotein on the lymphocyte. Once attached, the virion sheds its glycoprotein coat, penetrates the host cell membrane, and does not coat its RNA. The virion enzyme reverse transcriptase directs the process of transcription of RNA into single-stranded DNA. The viral RNA is degraded and a second DNA strand is generated. At this point the double stranded DNA is integrated into human cellular genes and these genes are used for viral replication. The RNA polymerase transcribes the integrated viral DNA into viral mRNA. Viral RNA is translated into the precursor gag-pol fusion polyprotein. The polyprotein is then cleaved by the HIV protease to yield the mature viral proteins. Thus, the HIV protease is involved in regulating a cascade of cleavage processes that result in the maturation of viral particles into a virus that is capable of being completely infectious.

The typical human immune system responsible for killing invading virions is heavily loaded because the virus infects and kills immune system T cells. Furthermore, the enzyme used to generate the new virion particle, viral reverse transcriptase, is not very specific and produces transcription errors that result in continuous changes of glycoproteins on the surface of the viral protective envelope. This lack of specificity reduces the effectiveness of the immune system, as antibodies specifically raised against one glycoprotein may have no effect on another glycoprotein, thereby reducing the amount of antibodies available to combat the virus. The virus continues to replicate and the immune response system continues to decline. In most cases, HIV begins to develop an opportunistic infection by weakening the host immune system without therapeutic intervention. Death can result if no antiviral or immunomodulatory or both are administered.

Hepatitis is an inflammation of the liver that is primarily caused by viruses and is generally less caused by certain drugs or toxins (e.g., alcohols). Viral infections are usually obtained by exposure to contaminated blood. Those most likely to be exposed to viruses are intravenous drug users who share contaminated needles, however, the disease can also be spread by the systemic exposure to humans with some form of hepatitis. In some cases, health care workers who come in contact with contaminated blood and people who need repeated transfusions have become infected with some form of hepatitis.

Three major types of viral hepatitis have been identified, namely hepatitis a, hepatitis b and hepatitis c, although at least four other viruses can cause hepatitis. Hepatitis a is a highly infectious form of hepatitis and is the most common form of the disease. Hepatitis a is usually transmitted through contaminated food or water. Symptoms of hepatitis a are generally similar to those of intestinal influenza and most people with hepatitis a will be well-rehabilitated.

Acute hepatitis b is probably the most severe form of viral liver infection. The symptoms are very similar to hepatitis a, but the symptoms are more severe and longer lasting. Initial symptoms of hepatitis a and hepatitis b include poor appetite, nausea, vomiting, and fever. In the later stages of hepatitis, the urine becomes dark and persistent or recurrent jaundice occurs. About 20% of hepatitis cases eventually progress to cirrhosis (liver scarring). Cirrhosis, a consequence of hepatitis, can be diagnosed by blood tests evaluating liver function. The liver affected by cirrhosis is also eventually weakened. Hepatitis c, the main pathogen being non-a and non-b, is thought to share common symptoms with hepatitis a and b and patients develop chronic infections that can ultimately lead to cirrhosis of the liver.

Neoplastic diseases are a common cause of death due to autonomous uncontrolled cell division. This splitting may be caused by the following factors:

1. mutations in genes caused by carcinogens;

2. a virus;

3. external signals that activate mitosis in certain cell types.

Tumor diseases are treated with various mitotic and cellular metabolic inhibitors. However, specificity has become a major problem with the use of anticancer drugs. In the case of anti-cancer drugs, the drug needs to distinguish between cancerous and non-cancerous host cells. The bulk of anticancer drugs does not differ at this level. In general, anticancer drugs have negative blood effects (e.g., termination of mitosis and breakdown of components formed in bone marrow and lymphoid tissues) and immunosuppressive effects (e.g., suppressed cell counts) as well as severe effects on epithelial tissues (e.g., intestinal mucosa), reproductive tissues (e.g., spermatogenic abnormalities), and nervous system. Calibresi and b.a. chabner: goodman and Gilman "Pharmacological Basis for Therapeutics" (Pergamon Press, 8 th edition) (pp.1209-1216). The successful use of chemotherapeutic agents as anticancer agents is also hampered by the phenomenon of multi-drug resistance, which is the development of resistance to a wide range of structurally unrelated cytotoxic anticancer compounds. Gerlach et al, Cancer research (Cancer Surveys), 5: 25-46(1986). The main reason for progressive drug resistance at the time of diagnosis may be due to a small population of drug resistant cells (e.g., mutant cells) within the tumor. Goldie and Andrew j. coldman, Cancer Research, 44: 3643-3653(1984). Treatment of such tumors with a single drug first results in remission in which the tumor shrinks in size as a result of killing of the primary drug-sensitive cells. As the drug sensitive cells are killed, the remaining drug resistant cells continue to multiply and ultimately control the tumor cell population. Finally, the treatment of cancer is hampered by the presence of significant heterogeneity even in the same type of cancer. For example, certain cancers have the ability to invade tissues and exhibit growth invasive processes characterized by metastasis. These tumors are generally very poor after healing for the patient and there is still no means to identify and distinguish such tumors from non-invasive cancers, and it is difficult for clinicians to alter and/or optimize the therapy. What is needed is a specific anti-cancer means that is reliable for a variety of tumor types and particularly suitable for invasive tumors. It is important that such treatment be effective with minimal host toxicity. Therefore, the ability of cells to reduce the intracellular amount of active drug must be overcome to improve cell targeting and/or improve the pharmacokinetics of anticancer drugs.

Macrolides, such as macrolide antibiotics, preferably accumulate in different cells of the subject to whom such molecules are administered, especially in phagocytic cells, such as mononuclear peripheral blood cells, polymorphonuclear cells, peritoneal and alveolar macrophages; and fluid surrounding the bronchoalveolar epithelium (Glaude R.P. et al antibacterial and chemotherapy (Antimicrob. AgentsChemother.), 1989, 33, 277-; Olsen K.M. et al antibacterial and chemotherapy (Antimicrob. AgentsChemother.) 1996, 40, 2582-. In addition, the relatively weak inflammatory action of certain macrolides is described. For example, the anti-inflammatory effects of erythromycin derivatives (Labro M.T., J.Antimicrob.Chemother., 1998, 41, 37-46; WO 00/42055) and azithromycin derivatives (EP 0283055) have recently been described. The anti-inflammatory effects of certain macrolides are also known from in vitro and in vivo studies in experimental animal models, such as zimosane-induced peritonitis in mice (Mikasa et al J. antibacterial and chemotherapy., (J. Antimicrob. Chemother.). 1992, 30, 339-400348) and endotoxin-induced tracheal neutrophil accumulation in rats (J. Immunol.). 1997, 159, 3395-4005). The modulatory effects of macrolides on cytokines such as interleukin 8(IL-8) (am. J. Respir. Crit. Care Med. 1997, 156, 266-271) or interleukin 5(IL-5) (EP 0775489 and EP 0771564) are also known. In addition, the favourable pharmacokinetic profile of macrolides can increase the tissue concentration of the compound, e.g. in the liver, or increase the leukocyte/plasma ratio (Girard A.E. et al, antibacterial and chemotherapy 3119871948-54; Widlfeuera. et al, antibacterial and chemotherapy 401996, 75-79).

In order to obtain compounds with improved/new activity properties for the diseases for which selective activity is desired, several different active substances are attached to different types of linkers. Hybrids/conjugates/chimeras of erythromycin a derivatives and nucleobases (uracil and thymine) or thymine-derived nucleosides have been reported (Costa a.m. et al tetrahedron letters 41, 2000, 3371-3375). However, such constructs do not exhibit activity/selectivity for the desired target. Furthermore, no macrolide constructs with peptide-type linker have been reported.

The peptide linker introduced into our hybrid molecule as a linker enables the molecule to act as a prodrug, releasing the V moiety by specific lysosomal cleavage within the target cell. Similar linkers have been described for other small molecules (in our case represented by hybrids of anti-inflammatory, anti-tumor and anti-viral compounds) and macromolecules or polymers (Duncan R. et al: Robinson J. R. and Lee V.H. (eds.). basis and use for Controlled Drug Delivery (Controlled Drug Delivery: Fundamental and Applications), 2 nd edition, 1987581-containing 607, Subr V. et al, J. Controlled Release research (J. Controlled Rel.) 181992123-containing 132).

Detailed Description

The compounds represented by formula I differ from the compounds of the prior art in that their structure enables them to accumulate in organs targeted at affecting the inflammatory immune response or the tumour or infection to be treated and in cells in which they occur. The intracellular concentration of the inhibitory compound in tumor cells that are sensitive to radiation can also be increased. The therapeutic effect of the compounds of formula I is produced by macrolide moiety M which has the pharmacokinetic properties of accumulating in cells of the immune system, in particular phagocytes. It enables the compounds of formula I to act primarily by "controlling" the supplementation of the very inflammatory intracellular macrolides at the site of inflammation or infection or malignancy where the active ingredient may exert its activity, rather than only at the site of inflammation or tumor or infection. In this manner, the adverse systemic side effects of steroids or non-steroidal anti-inflammatory substances or anticancer or antiviral compounds are significantly reduced or even eliminated (it is noted that steroids are used as an adjuvant therapy for the treatment of malignancies and the present invention also concerns steroids with antitumor applications). Following local or systemic administration, the hybrid molecules of the invention (and/or their constituents, if releasable) rapidly accumulate in the site of inflammation or tumor or in or near infected cells.

We have recently discovered certain compounds of formula I that exert improved therapeutic effects in such inflammatory diseases, disorders and conditions:

the symbol M in the above structure represents a macrolide subunit having an accumulation property in inflammatory cells, S represents an anti-inflammatory steroid subunit, and L represents a linker covalently linking M and S. Compounds with a steroid subunit S linked via a chain L to the N/9a position of 9-dihydro-9-deoxy-9 a-aza-9 a-homoerythromycin or to the C/3 position of a des-cladinosyl azithromycin derivative or to the C/2' position of a desosaminidyl sugar (desozaminosar) are described in our commonly pending International patent application PCT/HR02/00001, the entire contents of which are incorporated herein by reference. However, hybrid compounds with a steroid subunit linked to a peptide linker at the N/9a position of 9-dihydro-9-deoxo-9 a-aza-9 a-homoerythromycin, which also have the above-mentioned therapeutic effects, have not been described so far. Hybrid compounds with a non-steroidal/anti-tumoral/antiviral subunit linked to a peptide linker in the N/9a position are also not described. Hybrid compounds with a macrolide linked to an antiviral or antitumor subunit are also not described. All such compounds are the subject of the present application.

The present invention relates to:

(a) novel "hybrid" compounds represented by the general formula I:

wherein M represents a macrolide subunit with cumulative properties in inflammatory cells, V represents an anti-inflammatory steroidal or non-steroidal subunit or an anti-tumor or anti-viral subunit as defined below, and L represents a linker linking M and V;

(b) compositions comprising one or more of the foregoing compounds in an amount effective to be anti-inflammatory or anti-malignant or anti-viral, and thereby treat disorders and conditions involving inflammation, malignancy, or viral infection in a mammal, including a human; and

(c) methods of using these compounds to treat such disorders and conditions.

The compounds of the present invention advantageously provide improved therapeutic effects and/or improved side effect profiles.

Suitable macrolide subunits for the hybrid compounds of the present invention may be selected from, but are not limited to, polylactone ring molecules, where "one" refers to a carbon or heteroatom in the ring, and "many" is macrolides with an atomic number greater than about 10, preferably 10 to about 50, more preferably 12-, 14-, 15-, 16-, 17-, and 18-membered lactone rings. Particular preference is given to 14-and 15-membered ring macrolide subunits, most preferred are azithromycin and derivatives thereof and erythromycin and derivatives thereof.

More specific non-limiting examples of molecules from which the macrolide subunits may be selected are as follows:

(i) a macrolide antibiotic comprising: azalides (azalides) such as erythromycin, dirithromycin, azithromycin, 9-dihydro-9-deoxy-9 a-aza-9 a-homoerythromycin, HMR 3004, HMR3647, HMR 3787, josamycin, erythromycylamine (erythromycin), ABT 773, fludromycin, clarithromycin, tylosin, tilmicosin, oleandomycin, desmycosin (desmycosin), CP-163505, roxithromycin, meiocarycin, and lovastatin, and derivatives thereof such as ketolides (e.g., 3-one); lactams (e.g., 8 a-or 9 a-lactams) and derivatives lacking one or more sugar moieties.

(ii) Macrolide immunosuppressants such as FK 506, cyclosporin, amphotericin, and rapamycin;

(iii) macrolide antifungals with host cell inhibitory properties, such as bafilomycin, canavalin (concanamycin), nystatin, natamycin, clindamycin, filipin (filipin), ruscogycin (efruscomycin), trichostatin (trichomycin).

Methods for the synthesis of the above-mentioned not commercially available macrolides and synthetic procedures for macrolides are generally known to those skilled in the art or may be found in the following references: denisa et al, "communications in bioorganic and medicinal chemistry," & Med.chem.Lett)1999, 9, 3075-3080; agourida C. et al J.Chem.Chem. (J.Med.chem.)1998, 41, 4080-; and EP-00680967 (1998); sun Or Y, et al J.Med.chem.)2000, 43, 1045-; US-05747467 (1998); McFarland J.W. et al J.Pharmacochemistry 1997, 40, 1041-1045; denis A. et al, Bioorganic and pharmaceutical chemistry communications (Bioorg. & Med. chem. Lett)1998, 8, 2427-; WO-09951616 (1999); lartey et al journal of medicinal chemistry (J.Med.chem.)1995, 38, 1793-1798; EP 0984019; WO 98/56801, the entire contents of which are incorporated herein by reference.

Other suitable macrolides are known, some of which are disclosed in the following documents: bryskier, A.J., et al, "Macrolides, chemical, pharmacological and Clinical applications" (macromolecules, Chemistry, Pharmacology and Clinical uses); arnette Blackwell: paris, 1993, pp 485-491, 14(R) -hydroxycladamycin, erythromycin-11, 12-carbonate, tri-O-acetyloleandomycin, spiramycin, albomycin, midecamycin, rasaramycin, the entire contents of which are incorporated herein by reference; ma, Z, et al, Current medicinal chemistry-Anti-Infective Agents, 2002, 1, 15-34, the entire contents of which are also incorporated by reference; picromycin, natamycin, HMR-3562, CP-654743, CP-605006, TE-802, TE-935, TE-943, TE-806, 6, 11-bridged ketolides, CP-544372, FMA-199, A-179461; see in particular: structures and derivatives of 14-and 16-membered ring macrolides described by Bryskier et al, at page 487-491; and Ma et al, noting all structural tables and all reaction schemes. All such macrolides conjugated to Ve are within the scope of the invention. The macrolide compounds specifically named or referred to above are commercially available or their synthetic methods are known.

Importantly, the macrolide subunits are derived from macrolides that have the property of accumulating within cells of the immune system, especially phagocytes, that recruit to sites of inflammation. Most of the lactone ring compounds defined above are known to possess this property. For example, 14-membered macrolides such as erythromycin and derivatives thereof, 15-membered macrolides such as azithromycin and derivatives thereof and 8 a-and 9 a-lactams and derivatives thereof, 16-membered macrolides such as tilmicosin and desmycosin and spiramycin.

Other examples of macrolides that accumulate in a particular cell type can be found in the following references: pascal A. et al, "clinical microbial infection" (Clin. Microbiol. infection.) -2001, 7, 65-69. ("Uptake and intracellular activity of ketolide HMR3647 in human phagocytic and non-phagocytic cells"); hand W.L. et al, J.Antimiob.Agents, 2001, 18, 419-425. ("Properties and mechanisms of Azithromycin accumulation and efflux in human polymorphonuclear leukocytes" (Characteristics and mechanisms of Azithromycin accumulation and efflux in human polymorphonuclear leukocytes)); amsden G.W. J.Antimicrobial Agents, 2001, 18, 11-15 ("New Generation antibiotics: tissue-directed antibiotics"); johnson J.D. et al, J.Lab.Clin.Med., 1980, 95, 429-439. ("Antibiotic uptake by alveolar macrophages"); wildfuer A. et al, antibacterial and chemotherapy (antibiotic. Agents Chemother.)1996, 40, 75-79. ("Uptake of azithromycin by various cells under in vivo conditions and its intracellular activity" (Uptake of azithromycin by cells and its intracellular activity)); scorneaux B, et al poultry science (Poult. Sci.)1998, 77, 1510-1521. ("Intracellular accumulation, subcellular distribution and efflux of tilmicosin in chicken phagocytes" (Intracellular accumulation, subcellular preservation, and efflux of in chicken phagemids)); MtairagE.M. et al, J.Antimicrob.Chemother, 1994, 33, 523-536. (Investigation of uptake of human neutrophil erythromycins and erythromycylamine uptake in vitro by human neutrophiles in vitro) (invitations of dirithromycin and erythromycylamine uptake by human neutrophiles in vitro)); anderson R, et al, J.Antibacteri & chemotherapy (J.Antimicrob.Chemother.)1988, 22, 923-933. ("in-vitro evaluation of cellular uptake of the novel macrolide antibacterial clarithromycin (A-56268, TE-031) and in vitro evaluation of the biological activity in phagocytic cells" (A-56268, TE-031), a new macrooligomeric antibacterial agent); tasaka Y et al, J.J.Antibiott 1988, 41, 836-840. ("Lotacmycin uptake by alveolar macrophages" (Rokitamycin uptake); harf R, et al, J.Antibacteri & chemotherapy (J.Antimicrob.Chemother.)1988, 22, 135-140. ("Spiramycin uptake by alveolar macrophages" (Spiramycin uptake by byalvor macrocages)), the entire contents of these documents are incorporated herein by reference. Various macrolide ligation complexes that may also be used in the context of the present invention are described in U.S. provisional applications 60/94,671 and 60/394,670 filed on 8.7.2002, the entire contents of which are incorporated herein by reference.

In addition, cells of the immune system that replenish the site of inflammation,In particular the presence of cumulative activity within phagocytes, is readily determined by one of ordinary skill in the art of the present invention using one of the well-known assays for this purpose. For example, Olsen, K.M. et al, antibacterial and chemotherapy (anitmicrob. Agents) can be used&Chemother.)1996, 40, 2582, 2585. Briefly, test cells, such as polymorphonuclear leukocytes, can be obtained from venous blood of healthy volunteers by Ficoll-Hypaque centrifugation, followed by 2% dextran sedimentation. Erythrocytes were removed by osmotic lysis and PMNs were evaluated by trypan blue exclusion. Alternatively, other cell fractions may be isolated and tested in a similar manner. Mixing tritiated macrolide compound (e.g. 10. mu.M) with 2.5X 10⁶The cells were incubated together for 120 min (37 ℃, 5% CO)₂90% relative humidity) and then by centrifugation, for example by a silicone oil-paraffin layer (86 vol%: 14 vol%) cells were removed from the compound-containing supernatant. For example, the amount of compound is determined by scintillation counting, and a score significantly above background indicates that the macrolide accumulates in the test cells. See Bryskier et al macrolide, chemical, pharmacological and Clinical applications (Mcrrolides, Chemistry, Pharmacology and Clinical Use); arnette Blackwell: paris, 1993pp 375 + 386, page 381, column 2, line 3. On the other hand, the amount of the compound can be measured by HPLC without radiolabeling the compound.

Other assay methods that can be used are disclosed in "macrolide, chemical, pharmacological and Clinical applications" by Bryskier, a.j., et al (Mcrolides, Chemistry, pharmacological and Clinical uses); arnette Blackwell: paris, 1993pp 375-. See in particular the phagocyte uptake assay on page 380-381 and the tables on pages 381, 383 and 385 and pages 382 and 383 for a detailed description of macrolide uptake and localization.

The present invention relates in certain preferred embodiments to compounds represented by formula I, salts and solvates thereof, wherein M particularly represents a macrolide subunit of a 14-or 15-membered lactone ring, most preferably a compound represented by formula II:

wherein:

(i) z and W are independently:

or a bond, wherein:

R_tand R_sIndependently hydrogen or alkyl (preferably methyl or H);

R_Mis OH, OR^PAlkoxy or substituted alkoxy (Syn or Anti configuration or mixtures thereof);

R_Nis H, R^PAlkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl or-C (═ X) -NR_tR_s；

X is O or S;

provided that Z and W cannot be both

Or a bond;

(ii) u and Y are independently H, halogen, alkyl or hydroxyalkyl (preferably H, methyl or hydroxymethyl);

(iii)R¹is hydroxy, OR^P、-O-S²Or ═ O;

(iv)S¹a sugar moiety (e.g., a desosamine (desozamine) group) at the C/5 position of the glycosidic ring of the general formula:

wherein:

R⁸and R⁹Are both hydrogen or together form a bond or R⁹Is hydrogen and R⁸is-N (CH)₃)R^yWherein:

R^yis R^P、R^Zor-C (O) R^ZWherein R is^ZIs hydrogen or cycloalkyl (preferably cyclohexyl) or alkyl (preferably C)₁-C₇Alkyl) or alkenyl (preferably C)₂-C₇Alkenyl) or alkynyl (preferably C)₂-C₇-alkynyl) aryl or heteroaryl or by C₁-C₇-alkyl or C₂-C₇-alkenyl or C₂-C₇Alkyl (R) substituted by alkynyl, aryl or heteroaryl^yPreferably hydrogen, methyl or ethyl);

R¹⁰is hydrogen or R^P；

(v)S²A sugar moiety at the C/3 position of the glycosidic ring of the formula (e.g., cladinosyl):

wherein:

R^3’is hydrogen or methyl; and R is¹¹And R¹²Independently of one another is hydrogen, R¹¹Can be R^POr R¹¹And R¹²Together form a bond;

(vi)R²is H, hydroxy, OR^PAlkoxy (preferably C)₁-C₄Alkoxy, most preferably methoxy), substituted alkoxy;

(vii) a is H or methyl;

(viii) b is methyl or epoxy;

(ix) e is H or halogen (preferably fluorine);

(x)R³is hydroxy, OR^POr alkoxy (preferably C)₁-C₄Alkoxy, most preferably methoxy), substituted alkoxy, or R³Can be reacted with R⁵Combined to form "bridges" (e.g. cyclic carbonates orCyclic carbamate); or if W or Z isThen R³Is a group that can be combined with W or Z to form a "bridge" (e.g., a cyclic carbamate);

(xi)R⁴is C₁-C₄Alkyl (preferably methyl);

(xii)R⁵is hydrogen, hydroxy, OR^P、C₁-C₄Alkoxy, substituted alkoxy or may be substituted with R³Groups that combine to form a bridge (e.g., a cyclic carbonate or a cyclic carbamate);

(xiii)R⁶is H or C₁-C₄Alkyl (preferably methyl or ethyl);

wherein subunit M contains a linking position through which it is linked to subunit D via linker L; the attachment position is located on one or more of the following groups:

a is at S¹、S²Or any reactive hydroxyl, N or epoxy group on the aglycone oxygen, with the proviso that S²(or provided that S is²And S¹) Is cracked;

b Activity on Z or W > N-R_N、-NR_tR_sOr ═ O groups;

c is located at R¹、R²、R³And R⁵A reactive hydroxyl group on any one of them;

d may be first derivatized to hydroxy or-NR_tR_sA group, and then any other group attached to all or part of L (e.g., OH → O → epoxy →

One or more R^PThe groups may be present independently on a macrolide subunit of formula II, wherein R^PRepresents a protecting group which may be selected from alkyl (preferably methyl), alkanoyl (preferably ethyl)Acyl), alkoxycarbonyl (preferably methoxycarbonyl or tert-butoxycarbonyl), arylmethoxycarbonyl (preferably benzyloxycarbonyl), aroyl (preferably benzoyl), aralkyl (preferably benzyl), alkylsilyl (preferably trimethylsilyl) or alkylsilylalkoxyalkyl (preferably trimethylsilylethoxymethyl). The amino protecting group may be removed by conventional techniques. Thus, it is possible to remove, for example, acyl groups, such as alkanoyl, alkoxycarbonyl or aroyl groups, by solvolysis, for example by hydrolysis under acidic or basic conditions. The arylmethoxycarbonyl (benzyloxycarbonyl) group can be cleaved by hydrogenolysis in the presence of a catalyst such as palladium on activated carbon.

L may be selected as a linker represented by formula IV:

X¹-(CH₂)_m-Q-(CH₂)_n-X² IV

wherein:

X¹is selected from-CH₂-, -OC (═ O) -, -C (═ O) -, -NO-, -OC (═ O) NH-, or-C (═ O) NH-;

X²selected from-NH-, -CH₂-, -NHC (═ O) -, -OC (═ O) -, -C (═ O) -, or-O;

q is-NH-or-CH₂-or is absent;

wherein-CH₂-or-NH-groups may each optionally be substituted by C₁-C₇Alkyl radical, C₂-C₇-alkenyl, C₂-C₇Alkynyl, C (O) R^X、C(O)OR^X、C(O)NHR^XIs substituted in which R^XCan be C₁-C₇-alkyl, aryl or heteroaryl;

(symbol)mandnindependently an integer of from 0 to 4,

provided that if Q ═ NH, then n cannot be 0.

L represents a polypeptide linker having from about 2 to about 50 amino acids linked to each other, preferably a tripeptide or tetrapeptide such as:

Gly-Phe-Leu, Gly-Gly-Phe, Gly-Phe-Gly, Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Ala-Val-Ala, Gly-Gly-Phe-Leu, Gly-Phe-Leu-Gly, Gly-Phe-Ala-Leu, Ala-Leu-Ala-Leu, Gly-Phe-Phe-Leu-Leu, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Ala-Gly-Val-Phe, Gly-Phe-Phe-Phe-Gly, and the like, and is not limited thereto.

Preferably L is represented by the formula Gly- (W)_p-Gly, wherein p is an integer from 0 to 3 and W is any amino acid or any combination of amino acids.

In the case where V is a steroidal or non-steroidal anti-inflammatory subunit,

l is only a peptide linker.

V may represent an anti-inflammatory steroidal or non-steroidal compound subunit or an anti-tumour or anti-viral compound subunit.

When V is a steroid subunit, formula X is preferred:

(ii) wherein:

R^aand R^bIndependently of one another, hydrogen or halogen;

R^cis hydroxy, alkoxy (preferably methoxy), alkyl, thiocarbamoyl, carbamoyl or a bond;

R^dand R^eIndependently of one another are hydrogen, OH, CH₃Or C₁-C₄Alkoxy (preferably methoxy or n-propoxy) or each a group or a valence which forms a 1, 3-dioxolane ring with another group (optionally mono-or disubstituted with alkyl or alkenyl), preferably a2, 2-dimethyl or 2-monopropyl or trans-propenyl ring;

R^fis hydrogen, hydroxy, chlorine or carbon attached theretoO where the atoms together form a keto group;

R^jis hydrogen or chlorine

And pharmaceutically acceptable salts and solvates thereof.

In another aspect, the invention is a steroid subunit as disclosed in WO 94/14834, wherein they carry the group > CH-S (O)_n-R^cInstead of the group > CH-C (O) -R^cWherein n is an integer of 0 to 2. See WO 94/14834, the entire content of which is incorporated by reference, in particular pp 2-3.

More generally, steroids used as a subunit source of steroids include, but are not limited to, corticosteroids (such as glucocorticoids and mineralocorticoids) and androgens. Non-limiting examples of corticosteroids include cortisol, cortisone, clobetasol, hydrocortisone, fludrocortisone, fludrocortole, flumethasone, flunisolide, fluocinolone, fluocortole, fluorometholone, prednisone, prednisolone, 6-alpha-methylprednisolone, triamcinolone, alclometasone, beclomethasone, betamethasone, budesonide, dexamethasone, amcinonide, codeazole, desonide, desoximetasone, diflucortolone, flucolone and dichloropine, fleriuplinene, fluticasone, halcinonide, methylprednisolone, paramethasone, pinazoline, prednisone, tixolone, triamcinolone, and their acidic derivatives, such as acetic acid, propionic acid, dipropionic acid, valeric acid, phosphoric acid, isonicotinic acid, mesotrionic acid, tebutatee, and hemisuccinic acid derivatives).

V may be a nonsteroidal anti-inflammatory subunit, i.e. part of a nonsteroidal anti-inflammatory drug (NSAID), including: those that inhibit cyclooxygenase enzymes that lead to the biosynthesis of prostaglandins and certain self-hormone inhibitors, including inhibitors of various isoenzymes of various cyclooxygenase enzymes (including, but not limited to, cyclooxygenase-1 and-2); and as cyclooxygenase and lipoxygenase inhibitors non-steroidal anti-inflammatory drugs (NSAIDs) such as the commercially available NSAIDs aceclofenac, acemetacin, acetaminophen, acexamol, acetylsalicylic acid, acetyl-salicyl-2-amino-4-methylpyridine-acid, 5-aminoacetylsalicylic acid, alclofenac, amfenac, aminoantipyrine, ampiroxicam, animalidine, benzydac, benoxaprofen, bermoprofen, alpha-bisabolol (alpha-bisabolol), bromfenac, 5-bromosalicylic acid acetate, bromsalicin, bucloxic acid, bufen, carprofen, celecoxib, chromoglycate, cinnamyl, clinacalandac, clopidogenic acid, diclofenac sodium, diflunisal, ditalole, droxaxicam, enfenamic acid, doxetanide, etofenamate, felbinac, Fenbufen, fenclorac, fendulcoside, fenoprofen, fentiazac, feuddol, flufenac, flufenamic acid, flunixin, flurbiprofen, glutetacin, glycol salicylate, ibufenac, ibuprofen, ibupren, indomethacin, indoprofen, triazolic acid, isoxacin, isoxicam, ketoprofen, ketorolac, lornoxicam, loxoprofen, methofenamic acid, mefenamic acid, meloxicam, 5-aminosalicylic acid, methyloxazinic acid, moxazolac, montelukast, nabumetone, naproxen, niflumic acid, nimesulide, olsalazine, oxaprozin, oxybutylazone, paracetamol, pamabramide, perisoxazole, phenylacetylsalicylate (phenyl-aceyl-acetyl salicylate), phenylbutazone, piroxicam, flufenamic acid, ketoprofen, flufenamic acid, Pranoprofen, protazonic acid, reservatol, acealicylamine, salicylamide-O-acetic acid (salcylamide-O-acetyl acid), salicylyl sulfate (salcylsulphrenic acid), salicin, salicylamide, salsalate, sulindac, suprofen, suxibutazone, tamoxifen, tenoxicam, tiaprofenic acid, tiaramide, ticlopidine, tenolidine, tolfenamic acid, tolmetin, tropemin, bibutyric acid, simoprofen, zaltoprofen, zomepirac, tomopuro, zafirlukast, and cyclosporine. Other NSAIDs and specific NSAID compounds are disclosed in US patent US 6,297,260, which is incorporated by reference in its entirety (especially in its general formula of claim 1 and the specific list of NSAIDs contained therein); and thiazulidene NSAIDs are disclosed in international patent application WO 01/87890, the entire contents of which are incorporated herein by reference).

Preferred NSAIDs are acetylsalicylic acid, indomethacin, naproxen, ibuprofen, flurbiprofen, ketoprofen, sulindac, etodolac, ketorolac, suprofen, flunixin, diclofenac sodium and tolmetin.

V may represent an antiviral compound including acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine, ritonavir, indinavir, nevirapine, zidovudine, didanosine, stavudine, abacavir, zalcitabine, amprenavir, ribavirin and adamantane, but is not limited thereto. Preferred antiviral compounds are zidovudine, didanosine and stavudine. V anti-inflammatory agents represent antineoplastic agents including but not limited to bicalutamide, camptothecin, estramustine phosphate, flutamide, mechlorethamine, thiotepa, ifosfamide, hydroxyurea, bleomycin, paclitaxel, lomustine, irinotecan, methotrexate, vinorelbine, anastrozole (anastrazole), floxuridine, melphalan, vincristine, vinblastine, mitomycin, nandrolone, goserelin, leuprolide, triptorelin, aminoglutethimide, mitotane, cisplatin, chlorambucil, pentostatin, cladribine, busulfan, etoposide, mitoxantrone, idarubicin, cyclophosphamide, mercaptopurine, thioguanine, cytarabine (cytarabine), cyclophosphamide, doxorubicin, daunorubicin, teniposide, and tamoxifen. Preferred antineoplastic agents are methotrexate, paclitaxel, camptothecin, doxorubicin, taxotere and topotecan.

The bold-faced bonds in the general formulae contained herein are indicated to be on the paper level; dashed bonds (dash-draw bonds) are drawn to indicate bonds that are at the paper level, while dashed lines (dash lines) indicate bonds that may be at or above the paper level. The parallel solid lines and the dotted lines represent single bonds or double bonds. Unless otherwise indicated herein, the following terms have the meanings explained below for them:

"alkyl" refers to a straight or branched chain of 1 to 10 carbon atoms, more preferably 1 to 6 carbon atomsA chain saturated monovalent hydrocarbon group. Preferred straight or branched alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl. Most preferred is methyl. Alkyl groups may be substituted with up to 5 substituents, including halogen (preferably fluoro or chloro), hydroxy, alkoxy (preferably methoxy or ethoxy), acyl, acylamino, cyano, amino, N- (C)₁-C₄) Alkylamino (preferably N-methylamino or N-ethylamino), N-di (C)₁-C₄-alkyl) amino (preferably dimethylamino or diethylamino), aryl (preferably phenyl) or heteroaryl, thiocarbonylamino, acyloxy, amino, amidino, alkylamidino, thioamidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aryl, heteroaryl, aryloxy, aryloxyaryl, nitro, carboxyl, carboxyalkyl, carboxyl substituted alkyl, carboxyl-cycloalkyl, carboxyl substituted cycloalkyl, carboxyaryl, carboxyl substituted aryl, carboxyheteroaryl, carboxyl substituted heteroaryl, carboxyheterocyclyl, carboxyl substituted heterocyclyl, cycloalkyl, cycloalkoxy, heteroaryloxy, heterocyclyloxy and oxycarbonylamino. Such substituted alkyl groups are within the definition of "alkyl" in the present invention. The definition of alkyl groups according to the invention can be extended to other groups bearing alkyl moieties, such as alkoxy groups.

"alkenyl" refers to a straight or branched chain monovalent hydrocarbon radical of 2 to 10, and preferably 2 to 6, carbon atoms containing at least one carbon-carbon double bond. Alkenyl groups may be substituted with the same groups as alkyl groups and such optionally substituted alkenyl groups are included in the term "alkenyl". Preferred are ethenyl, propenyl, butenyl and cyclohexenyl.

"alkynyl" refers to a straight or branched chain monovalent hydrocarbon radical having from 2 to 10, and preferably from 2 to 6, carbon atoms and containing at least one, and preferably no more than three, carbon-carbon triple bonds. Alkynyl groups may be substituted with the same groups as alkyl and these substituted groups are within the definition of alkynyl. Ethynyl, propynyl and butynyl are preferred.

"cycloalkyl" refers to a cyclic group having 3 to 8 carbon atoms containing a single ring optionally fused to an aryl or heteroaryl group. Cycloalkyl groups may be substituted with substituents described below for "aryl" and substituted cycloalkyl groups fall within the definition of "cycloalkyl". Preferred cycloalkyl groups are cyclopentyl and cyclohexyl.

"aryl" refers to an unsaturated aromatic carbocyclic group containing a single ring, such as phenyl or a multiple fused ring, such as naphthyl, having 6 to 14 carbon atoms. The aryl group may optionally be further fused with aliphatic or aryl groups or may be substituted with one or more substituents, such as halogen (fluoro, chloro and/or bromo), hydroxy, C₁-C₇Alkyl radical, C₁-C₇Alkoxy or aryloxy radical, C₁-C₇Alkylthio or arylthio, alkylsulfonyl, cyano or primary or non-primary amino.

"heteroaryl" refers to a monocyclic or bicyclic aromatic ring containing 2 to 10 carbon atoms and 1 to 4 heteroatoms, such as O, S or N. The heteroaryl ring may be optionally fused to another heteroaryl, aryl, or aliphatic cyclic group. Examples of such are furan, thiophene, pyrrole, imidazole, indole, pyridine, oxazole, thiazole, pyrrole, pyrazole, tetrazole, pyrimidine, pyrazine and triazine, preferably furan, pyrrole, pyridine and indole. The term includes groups substituted with the same substituents as described for aryl above.

"Heterocyclyl" refers to a saturated or unsaturated group containing a single or multiple ring system and 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from nitrogen, sulfur or oxygen, wherein in the fused ring system, another or other ring may be aryl or heteroaryl. Heterocyclic groups may be substituted with substituents as described for alkyl groups and heterocyclic groups substituted thereby are within the scope of this definition.

"amino acid" refers to any compound containing an amino group and a carboxylic acid group. The amino group may be present at a position adjacent to the carboxyl function, such as an alpha-amino acid, or at any position within the molecule. Amino acids may also contain other functional groups such as amino, sulfur, carboxyl, formamide, imidazole, and the like. The amino acid may be a synthetic or naturally occurring or modified naturally occurring amino acid, such as norvaline or norleucine.

The symbol K sometimes refers to the moiety of the L group attached to M or V when the context requires.

In preparing compounds having particular pharmacological activity represented by structure I, certain novel compounds are prepared as intermediates for preparing compounds having pharmacological activity. The invention also relates to such intermediates.

The invention also includes pharmaceutically acceptable salts of the compounds of the invention. Pharmaceutically suitable salts of the compounds of the invention include those with inorganic acids (e.g. hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric or sulfuric) or organic acids (For exampleTartaric acid, acetic acid, methanesulfonic acid, trifluoroacetic acid, citric acid, maleic acid, lactic acid, fumaric acid, benzoic acid, succinic acid, methanesulfonic acid, oxalic acid, and p-toluenesulfonic acid).

The invention also includes prodrugs of the compounds of formula I, i.e., compounds that release the active parent drug of formula (I) in vivo upon administration to a mammalian subject. Prodrugs of compounds of formula I are prepared by modifying functional groups present on the compounds of formula I in such a way that the modifications are cleaved in vivo to release the parent compound. Prodrugs include compounds of formula I wherein a hydroxy, amino or carboxyl group of the compound of formula I is bonded to any group which can be cleaved in vivo to regenerate the hydroxy, amino or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, esters of the compounds of formula I (e.g., acetate, formate, and benzoate derivatives) or any other derivative that is converted to the active parent drug upon exposure to physiological pH or by the action of enzymes. In the context of this paragraph, "prodrug" does not refer to a hybrid of the invention that releases the V moiety (as described elsewhere in this specification), but rather refers to a derivative of the V moiety that can release a macrolide or remain linked to a macrolide. The V moiety derivative in the attached or released state may then be converted to the active parent drug in free form or attached to the macrolide.

The present invention also includes solvates (preferably hydrates) of the compounds of formula I or salts thereof.

The compounds of the formula I bear one or more chiralitiesCenter of a shipAnd they may also have geometric isomers depending on the nature of the substituents. Isomers that differ in the arrangement of atoms in space are referred to as "stereoisomers". Stereoisomers that are not mirror images of each other are referred to as "diastereomers" and those that are not mirror images of each other are referred to as "enantiomers". When a compound has a chiral center, it is possible for a pair of enantiomers to occur. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and described by the R-and S-sequence rules of Cahn and Prelog or by the way the molecule rotates about the plane of polarization and are designated dextrorotatory or levorotatory (i.e., referred to as (+) or (-) -isomers, respectively). The chiral compounds may exist as individual enantiomers or as mixtures of enantiomers. Mixtures containing equal proportions of enantiomers are referred to as "racemic mixtures". The present invention includes all individual isomers of the compounds of formula I. The description or naming of a particular compound in the specification and claims is intended to include the individual enantiomers and mixtures thereof, otherwise racemates thereof. Methods for determining the stereochemistry of stereoisomers and resolution thereof are well known in the art.

The invention also includes the syn-anti type stereoisomers encountered and mixtures thereof, when an oxime or similar group is present. The group of the highest Cahn IngoldPrelog precedence order rule attached to the terminal double bond atom of the oxime is compared to the hydroxyl group of the oxime. This stereoisomer is designated Z (zusammen ═ co) or Syn, provided that the oxime hydroxy group is located on the same side of the reference plane through the C ═ N double bond as the most preferential group; the other stereoisomer was designated as E (entgegen ═ opposite) or Anti.

"pharmaceutically acceptable excipient" refers to an excipient used in the preparation of pharmaceutical compositions that are generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use and human pharmaceutical use. As used herein, "pharmaceutically acceptable excipient" includes one and more than one such excipient.

A "Treating" or "treatment" state, disorder or condition includes:

(1) preventing or delaying the manifestation of clinical symptoms of a condition, disorder or condition that occurs in a mammal who may have or be predisposed to said condition, disorder or condition but does not yet develop or develop clinical or subclinical symptoms of said condition, disorder or condition;

(2) inhibiting a condition, disorder or condition, i.e., arresting or slowing the onset of a disease or at least one clinical or subclinical symptom thereof; or

(3) Alleviating the disease, i.e., the regression of the state, disorder or condition, or at least one clinical or subclinical symptom thereof.

The benefit to the treated subject is either statistically significant or at least perceptible to the patient or clinician.

A "therapeutically effective amount" refers to an amount sufficient to effect such treatment (as that phrase is defined above) when a therapeutic state, disorder or condition is administered to a mammal. The "therapeutically effective amount" will vary with the compound, the disease and its severity and the physical condition and responsiveness of the mammal being treated.

Four traditional symptoms of acute inflammation are redness, warming, swelling and pain in the affected area and loss of function of the affected organ.

Inflammatory symptoms and signs associated with a particular condition include:

rheumatoid arthritis-involved joint pain, swelling, warmth and tenderness; systemic stiffness and morning stiffness;

insulin-dependent diabetes mellitus-insulitis; the disease can lead to a variety of complications associated with inflammatory components, including retinopathy, neuropathy, nephropathy; coronary artery disease, peripheral vascular disease, and cerebrovascular disease;

autoimmune thyroiditis-weakness, constipation, shortness of breath, facial, hand and foot edema (puffiness), peripheral edema, bradyarrhythmias;

multiple sclerosis-spasticity, blurred vision, dizziness, weakness of limbs, paresthesia;

uveal retinitis-decreased night vision, decreased peripheral vision;

lupus erythematosus-joint pain, rash, photosensitivity, fever, myalgia, hand and foot edema, urinalysis abnormalities (hematuria, cylindruria (cylindaria), proteinuria), glomerulonephritis, cognitive dysfunction, vascular thrombosis, pericarditis;

scleroderma-raynaud's disease; swelling of hands, arms, legs and face; thickening of the skin; pain in the fingers and knees, swelling stiffness, gastrointestinal dysfunction, restrictive lung disease; pericarditis; renal failure;

other arthritic diseases with an inflammatory component, such as rheumatoid spondylitis, osteoarthritis, septic arthritis and polyarthritis-fever, pain, swelling, tenderness;

other inflammatory brain diseases such as meningitis, alzheimer's disease, AIDS dementia encephalitis-photophobia, cognitive dysfunction, memory loss;

other inflammatory ocular inflammations, such as retinitis-vision loss;

inflammatory skin diseases such as eczema, other dermatitis (e.g. atopic dermatitis, contact dermatitis), psoriasis, UV-radiation induced burns (sun rays and similar UV sources) -erythema, pain, desquamation, swelling, tenderness;

inflammatory bowel diseases, such as crohn's disease, ulcerative colitis-pain, diarrhea, constipation, rectal bleeding, fever, arthritis;

asthma-shortness of breath, wheezing;

other allergic diseases such as allergic rhinitis-sneezing, itching, rhinorrhea;

diseases associated with acute asthma, such as impairment-sensory loss, loss of movement, loss of cognition after stroke;

cardiac tissue damage due to myocardial ischemia-pain, shortness of breath;

lung injury, such as that which occurs in adult respiratory distress syndrome-shortness of breath, hyperventilation, decreased oxygenation, lung infiltration;

inflammation with infection, such as sepsis, septic shock, toxic shock syndrome-fever, respiratory failure, tachycardia, hypotension, leukocytosis;

other inflammatory diseases associated with specific organs or tissues, such as nephritis (e.g. glomerulonephritis) -oliguria, urinalysis abnormalities;

adnexitis-fever, pain, tenderness, leukocytosis;

gout-involved joint pain, tenderness, swelling and elevated uric acid of erythema, serum and/or urine;

cholecystitis-abdominal pain and tenderness, fever, nausea, leukocytosis;

chronic obstructive pulmonary disease-shortness of breath, wheezing;

congestive heart failure-shortness of breath, rale, peripheral edema;

type II diabetes mellitus-advanced organ complications including cardiovascular, ocular, renal, and peripheral vascular disease;

pulmonary fibrosis-hyperventilation, shortness of breath, decreased oxygenation;

vascular diseases such as atherosclerosis and restenosis-pain, loss of sensation, decreased pulse, loss of function;

and alloimmune-pain, tenderness, fever leading to graft rejection.

Subclinical symptoms include, but are not limited to, diagnostic markers for inflammation, whose manifestation may precede clinical manifestation. One type of subclinical condition is an immunological condition, such as the invasion or accumulation of proinflammatory lymphoid cells in an organ or tissue or the presence of activated proinflammatory lymphoid cells local to or surrounding a pathogen or antigen recognized to be specific for an organ or tissue. Activation of lymphoid cells can be determined by techniques well known in the art.

A therapeutically effective amount of an active ingredient "delivered" to a specific site in a host refers to a blood concentration of the active ingredient that results in a therapeutically effective site. This may be achieved, for example, by administering the active ingredient to the host locally or systemically. Specific viral diseases include viral hepatitis (type A, type B, type C, type E), influenza, viral pneumonia, viral bronchitis, herpes infection (herpes simplex virus, Epstein-Barr virus (infectious mononucleosis), herpes zoster), poliomyelitis, AIDS (HIV infection), adult T-cell leukemia (ATL), papilloma, measles, rubella, acute eruption of children, infectious erythema, viral encephalitis, viral myelitis, cytomegalovirus infection, mumps, varicella, rabies, viral enteritis, viral myocarditis, viral pericarditis, etc.

Symptoms and signs of viral infection associated with a particular condition include: viral load, viral replication, viral activity, viremia, viral specific antigens, viral RNA or DNA, reverse transcriptase activity, antiviral CTL activity in the host, T-cell or CD4+ cell count (for HIV). Cancers include, but are not limited to, the following: non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, leukemia, fibroblasts, kidney cancer, melanoma, prostate cancer, CNS cancer, bone/muscle, lymphoma and leukemia.

Neoplasia symptoms and signs associated with a particular condition include: tumor burden, tumor size, organ weight affected, tumor recurrence, subject survival time, time and extent of remission, cancer cell growth, cancer cell survival, apoptosis index, degree and proportion of metastasis, biomarkers associated with a particular type of neoplasia, proliferation markers, activation of associated oncogenes, dysregulation of tumor-associated receptor function, tumor-specific antigens, and tumor-associated angiogenesis.

Identifying subclinical symptoms of viral infection or neoplasia in or around activated pro-inflammatory lymphoid cells having a pathogen or antigen specific for an organ or tissue. Activation of lymphoid cells can be determined by techniques well known in the art. Other subclinical symptoms include the presence and/or amount of various surrogate markers (such as those in the above categories) prior to any clinical sign of disease development. In addition, infections and neoplasias are usually accompanied by an increase in the activity of the immune system at the site of the tumor, so that inflammatory signs are also suitable here.

Among the compounds represented by the general formula II, preferred are:

z and W together are-N (R)_N)C(O)-、-C(O)N(R_N)-、＞C-NR_sR_t、-C(O)-、＞C-N-R_M-、-CH₂NR_N-or-NR_NCH₂-, most preferably-NCH₃CH₂-、-NHCH₂-、-CH₂NH-、-C(O)NH、-NHCO-；

R_s、R_tIs methyl or H;

R_Mis OH or methoxy;

x is O;

R_Nis H, methyl or-C (═ X) -NR_sR_t；

A is H or methyl;

u, Y is H, F, methyl or hydroxymethyl;

R¹is hydroxy, -O-S²Or ═ O;

R²is H, hydroxy or methoxy;

R³is OH, methoxy or a group which forms a cyclic carbamate bridge with W or Z;

R⁴is methyl;

R⁵is H, OH methoxy or with R³A group forming a cyclic carbonate or cyclic carbamate bridge;

the connecting link being either through the nitrogen of Z in the N/9a or N/8a position or through both positions being at S²R at C/4' position of sugar¹²Carbon or R of¹¹Oxygen of (2);

R⁶is H, methyl or ethyl;

R⁸is H, N (CH)₃)₂、NH(CH₃)N(CH₃)CH₂CH₃；

R⁹Is H;

the attachment position is preferably in the C/3 position or via S¹Amino at C/3' or C/11 or W or Z of the sugar or by S²Sugar C/4 "position.

Also preferred are compounds of formula I wherein M has formula II and: (i) z is NCH₃W is CH₂，R²Is a hydroxyl group; or (ii) Z is NH, W is ═ CO and R²Is methoxy. (the compounds described in this paragraph may or may not meet the preferred conditions described above for the remainder of the previous section, but preferably they meet these conditions.)

Another aspect of the invention relates to a process for the preparation of the compounds represented by formula I. In general, the compounds of formula I can be obtained by: first attaching one end of the chain to the macrolide and then attaching the other end of the chain to V; or first linking one end of the chain to V and then linking the other end of the chain to the macrolide; or finally, one part of the chain is linked to the macrolide and the other part of the chain is linked to V, and then the ends of the chain parts are chemically linked to form the chain L.

One skilled in the art will appreciate the need to use protected derivatives of the intermediates used to prepare the compounds of formula I. The functional groups can be protected and deprotected by methods well known in the art. The hydroxy or amino group may be protected by any hydroxy or amino protecting group, for example, as Green t.w.; wuts p.g.m. protective groups in Organic Synthesis (protective groups in Organic Synthesis): john Wiley and Sons, New York, 1999. See also the discussion above regarding protecting groups of formula I. The amino protecting group may be removed by conventional techniques. For example, acyl groups such as alkanoyl, alkoxycarbonyl and aroyl groups may be removed by solvolysis, for example by hydrolysis under acidic or basic conditions. Arylmethoxycarbonyl (e.g. benzyloxycarbonyl) may be cleaved by hydrogenolysis in the presence of a catalyst such as palladium on activated carbon.

More specifically, the compounds of formula I can be prepared by the following method:

a) compounds of formula I may be produced by reacting a compound of formula VI with the free amino group of a macrolide represented by formula VIIa, wherein X is²is-NHC (O) -,

wherein the structural formula of formula VI is as follows:

wherein L is¹Represents a leaving group (such as a hydroxyl group),

and the formula of formula VIIa is as follows:

wherein K is part of a linker molecule L linked to the macrolide subunit.

The reaction is generally carried out with acidic derivatives having the ability to activate carboxylic acid groups, such as halides, mixed anhydrides and especially carbodiimides, such as- (3-dimethylaminopropyl) -3-ethyl-carbocyclic diimine (EDC) and benzotriazoles. The reaction is carried out at room temperature under an inert gas atmosphere, such as nitrogen or argon, in the presence of a base, such as an organic base (e.g., triethylamine). The reaction may take several hours to several days to complete.

For example, when L is-K-NH₂When this is the case, the > N-K-NH group on the macrolide ring may be derivatized to form the > N-K-NH₂And reacting the derivative macrolide with the compound of formula VI as shown below to produce the compound of formula I.

This reaction can be carried out, for example, when the > NH group on the macrolide subunit of formula II is attached at the C/3' or N/9a position.

The compounds represented by formula VI are commercially available or they may be derived from subunit V by methods including one of those well known in the art.

The preparation of the starting macrolide of formula VIIa is described in PCT HR 02/0001, the entire contents of which are incorporated by reference. See also U.S. Pat. No. 4,474,768 and Bright, G.M. et al, J.Antibiot, 1988, 41, 1029-1047, each of which is incorporated herein by reference in its entirety.

b) Compounds of formula I may be produced by reacting a compound of formula VI with the free hydroxyl group of a macrolide of formula VIIb, wherein X²is-OC (O) -:

the reaction is generally carried out with acidic derivatives having the ability to activate carboxylic acid groups, such as halides (such as ethylene dichloride EDC)), mixed anhydrides and in particular carbodiimides. The reaction is generally carried out at room temperature under an inert gas atmosphere such as nitrogen or argon. The reaction may take several hours to several days to complete.

For example, when linking moiety L is-K-O-, compounds of formula I may be formed by (1) derivatizing a > NH group on the macrolide ring to form a > N-K-OH group and (2) reacting the derivatized macrolide with a compound of formula VI as shown below:

the linking group-K-OH may be attached to the secondary nitrogen atom of the macrolide as described below. By reacting macrolides with alkenoyl derivatives, such as CH₂＝CH(CH₂)_m-2C (O) O-alkyl (e.g., methacrylate). Such as then using a metal hydride (e.g., liAlH)₄) Reduction of the ester group (i.e., -C (O) O-alkyl) in an anhydrous organic solvent provides a macrolide (i.e., M-K-OH) containing the linking group-K-OH. The reduction reaction is generally carried out at low temperature and preferably at 0 ℃ or below 0 ℃.

This reaction can also be carried out, for example, when the > NH group is attached at the C/3' or N/9a position of the macrolide subunit represented by formula II.

The starting macrolides of formula VIIb are known compounds or may be obtained following procedures described for analogous compounds, such as those described in Costa, A.M., et al, Tetrahedron Letters 2000, 41, 3371-3375, which is incorporated herein by reference.

c) Compounds of formula I may be prepared by reacting a macrolide of formula VIIc with a compound of formula VIb, wherein X¹is-OC (O) -, Q-NH-and X²is-NHC (O) -,

wherein the formula of formula VIIc is as follows:

wherein the formula of formula VIb is as follows:

thereby obtaining:

this reaction can be carried out, for example, when the OH group is attached at the C/6 or C/4 "position of the macrolide subunit represented by formula II.

The macrolide represented by formula VIIc is formed by reacting the corresponding haloalkanoyl chloride with the free OH on the macrolide.

By reacting a suitable amine (containing a linker-K-NH)₂) With a compound of formula VI to produce a compound of formula VIb.

d) Compounds of formula I may be prepared by reacting a macrolide of formula VIId with a compound of formula VIb, wherein X is¹is-OC (O) NH-and X²is-NHC (O) -.

This reaction can be carried out, for example, when two free OH groups are attached at the C/11 or C/12 position of the macrolide subunit represented by formula II.

The reactant macrolide represented by formula VIId can be produced by reacting an ethyl carbonate on a macrolide subunit containing two ortho hydroxyl substituents;

e) compounds of formula I may be prepared by reacting a macrolide of formula VIIe with a compound of formula VIa, wherein X is¹is-CH₂-, Q is-NH-and X²is-NHC (O) -.

This reaction can be carried out, for example, when the OH group is attached at the C/4 "position of the macrolide subunit represented by formula II.

The reactant macrolide represented by formula VIIe may be produced by the following steps: by oxidation of the corresponding hydroxyl-containing macrolides to give substituents (Structural formula (xxxvi); conversion into epoxy groupsStructural formula (xxxvi); and cleaving the epoxy groups with a suitable reactant (e.g., ethylenediamine).

f) Can be prepared by reacting a compound containing a leaving group L²A macrolide represented by formula VIIf (such as Br) and a subunit V represented by formula VIc are reacted as shown below to prepare a compound represented by formula I.

For example, it is possible to cleave the sugar group attached at the C/3-position of the macrolide subunit represented by formula VIIf and then to react said macrolide with L³-K-L²Preparation of the starting macrolide of formula VIIf by reaction of reagents, wherein L²And L³Is a leaving group.

g) Can be prepared by reacting a compound containing a leaving group L²A macrolide represented by formula VIIg (such as Br) and subunit V of formula VIc are reacted as shown below to prepare a compound represented by formula I.

For example, a macrolide having a free OH group attached to the C/2' position of the macrolide subunit represented by formula II can be reacted with a macrolide of formula L³-C(O)-K-L²Preparation of the starting macrolide of formula VIIg by reaction of reagents, where L²And L³Is a leaving group.

h) It is also possible to use a compound containing a leaving group L²A macrolide of formula VIIh (such as Br) and subunit V of formula VIc are reacted as shown below to prepare the compound of formula I.

i) The compounds of the formula I can also be prepared by reacting a subunit V of the formula VIIIa, prepared from a subunit V containing a free hydroxyl group, with a macrolide of the formula VIIa as shown below (Huang C.M. et al, Chem. Biol.2000, 7, 453-461; hess S, et al, Bioorganic and pharmaceutical chemistry (Bioorg. Med. chem.)2001, 9, 1279-1291).

j) Compounds of the formula I can also be prepared by reacting subunits V of the formula VIIIb (Pandori M.W. et al "chemistry and biology" (Chem. & Biol.)2002, 9, 567-573) or VIIIc (Hess S. et al "Bio-organic and pharmaceutical chemistry" (Bioorg. & Med. Chem.)2001, 9, 1279-1291) prepared from subunits V containing a free amino group with a macrolide of the formula VIIa as shown below.

The following examples illustrate the preparation of the compounds of formula I and do not limit the invention in any way.

Paclitaxel succinate scheme 1 can be prepared according to the procedures described by Huang C.M. et al, Chemistry & Biology, 7, 2000, 453-461.

Gamma-methyl-N' - [4- [ N- [ (2, 4-diamino-6-pteridinyl) methyl ] -N-methylamino ] benzoyl ] -L-glutamate (2) (scheme 2) can be prepared according to the procedures described in Kra lovec J. et al, J.Pharmacol.Chem.32, 1989, 2426-2431.

camptothecin-20-O-hemisuccinate can be prepared according to patent application US4943579 (scheme 3).

5' -O-succinylzidovudine (m ═ 1) can be prepared according to the procedure described by Giammoma G, et al, J.Control. Release 54, 1998, 321-331 (scheme 4).

Scheme 1: synthesis of paclitaxel-macrolide hybrids

Scheme 2: synthesis of methotrexate-macrolide hybrids

Scheme 3: synthesis of camptothecin-macrolide hybrids

Scheme 4: synthesis of zidovudine (AZT) -macrolide hybrids

Traditionally, 16-membered ring macrolides have been divided into subfamilies based on the substitution pattern of aglycones. The main prototypes of this family are represented by leucomycin, spiramycin and tylosin.

Tylosin is a representative 16-membered macrolide containing a highly substituted aglycone with two double bonds (tylonolide) and a third sugar substituent (β -D-mycinose) and a disaccharide linked to the 5-hydroxy group. Hydrolysis of mycarose from the disaccharide gives desmycosyl-tylosin (desmycosin).

Possible modification positions on desmycosin tylosin:

for example, 16-membered ring macrolide hybrids can be prepared by reductive amination of the aldehyde group at the C-20 position.

The reaction can also be used for 17-membered azalides such as 8 a-aza-homodesmycosin and its derivatives (such as di-and tetrahydro derivatives).

On the other hand, derivatization of 16-membered ring macrolides may be carried out by conversion of the double bond (e.g. epoxidation) and cleavage of the epoxy group using suitable reactants, such as diamines, to give the macrolide (M-O-NH-K-NH)₂) To proceed with.

The ketone at the C/9 position can also be modified with hydroxylamine hydrochloride to give an oxime and then reduced to an amine.

When L is a peptide linker, the synthetic route for the compounds of formula I is illustrated in the following examples (V is a steroidal, non-steroidal anti-inflammatory, antiviral and antitumor subunit with a free amino group):

the synthetic route for the compounds of formula I is illustrated in the following examples when L is a peptide linker (V is a steroidal, non-steroidal anti-inflammatory, antiviral and antitumor subunit with a free carboxyl group):

salts of the compounds represented by formula I can be prepared by using generally well-known procedures such as: for example, reacting a compound of structure I with the corresponding base or acid in a suitable solvent or solvent mixture, such as ethers (diethyl ether) or alcohols (ethanol, propanol or isopropanol).

Another aspect of the invention relates to the use of compounds of formula I for the treatment of inflammatory diseases, disorders and conditions characterized by or associated with unwanted inflammatory immune responses, especially all diseases and conditions (anti-cancer, anti-viral) induced by or associated with excessive secretion of TNF-alpha and IL-1.

A therapeutically effective amount of a compound of the invention can be determined by methods well known in the art. Since the compounds of the present invention can be delivered to the desired site more efficiently than the corresponding individual drugs, smaller amounts of the compounds than the molar amount of the anti-inflammatory, anti-tumor and anti-viral drugs can be administered while still achieving the same therapeutic effect. Furthermore, since the active ingredient of the invention is taken up by the target, it is no longer in contact with other tissues, and its administration is expected to produce fewer side effects, which allows an increase in the maximally tolerated anti-inflammatory, antitumor and antiviral doses. Therefore, the following table is used as a guide only. The therapeutically effective dose threshold of the compound, pharmaceutically acceptable salt thereof, solvate thereof, or prodrug thereof is generally equal to or less than the therapeutically effective dose of the anti-inflammatory, anti-neoplastic and anti-viral drugs on a molar basis. The broad and preferred effective amounts of the compounds, pharmaceutically acceptable salts thereof, solvates thereof, or prodrugs thereof are shown in the following table.

	Amount of compound, pharmaceutically acceptable salt thereof, solvate thereof or prodrug thereof
				mg/kg body weight/day drug (single administration)	Mu mol/kg body weight/day hybrid or drug
Width of	About 0.001 to about 1000	About 0.004 to about 4000
			Preference is given to	About 0.01 to about 100	About 0.04 to about 400
More preferably	About 1 to about 100	About 4 to about 400
			Most preferably	About 3 to about 30	About 12 to about 120

Thus, for example, the preferred dose range for indomethacin is 50-200 mg/day, corresponding to the range of 140-560. mu. mol/day. The same molar based range of 140-560mol of the hybrid compound of the invention is the starting point for determining the preferred dosage range. Refinements to this approach are also within the purview of those skilled in the art.

Furthermore, the present invention relates to pharmaceutical compositions comprising an effective amount of a compound of the present invention and a pharmaceutically acceptable excipient, such as a carrier or diluent.

The preparation of the pharmaceutical composition of the present invention includes the steps of mixing, granulating, tableting, and dissolving the components. The chemical carrier may be in solid or liquid form. The solid carrier may be lactose, sucrose, talc, gelatin, agar, pectin, magnesium stearate, fatty acid, but is not limited thereto. The liquid carrier may be: syrup; oils such as olive oil, sunflower seed oil or soybean oil; water; or physiological saline, but not limited thereto. Similarly, the carrier may also contain ingredients which provide sustained release of the active ingredient, such as glyceryl stearate or glyceryl distearate. Pharmaceutical compositions in several dosage forms can be prepared. If a solid carrier is used, the dosage forms may include, but are not limited to, tablets, capsules, hard capsules, powders or granules that can be orally administered. The amount of solid carrier may vary, but is primarily in the range of 25mg-1 g. If a liquid carrier is used, the formulation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid or non-aqueous liquid suspension.

The compounds of the invention may be administered topically or systemically, e.g., orally, parenterally, subcutaneously, mucosally, e.g., orally, intranasally, intrarectally, and intravaginally. "parenteral" refers to the intravenous, intramuscular, or subcutaneous routes. Corresponding formulations of the compounds of the invention may be used for the prevention and treatment (prevention, delay, inhibition or alleviation) of several disorders (diseases and other pathological inflammatory conditions) caused by or associated with abnormal or unwanted (excessive, unregulated or unregulated) inflammatory immune responses, including the production of inflammatory cytokines or other inflammatory mediators, including but not limited to TNF- α and IL-1 β. These diseases include: autoimmune diseases such as rheumatoid arthritis, insulin-dependent diabetes mellitus, autoimmune thyroiditis, multiple sclerosis, uveal retinitis, lupus erythematosus, scleroderma; other arthritic conditions with an inflammatory component such as rheumatoid spondylitis, osteoarthritis, septic arthritis and polyarthritis; other inflammatory brain diseases such as meningitis, alzheimer's disease, AIDS dementia encephalitis; other inflammatory ocular inflammations, such as retinitis; inflammatory skin diseases such as eczema, other dermatitis (e.g. atopic dermatitis, contact dermatitis), psoriasis, UV-radiation induced burns (sun rays and similar UV sources); inflammatory bowel diseases such as crohn's disease, ulcerative colitis; asthma; other allergic diseases such as allergic rhinitis; diseases associated with acute asthma, such as brain injury following stroke; cardiac tissue damage due to myocardial ischemia; lung injury, such as that which occurs in adult respiratory distress syndrome; inflammation with infection, such as sepsis, septic shock, toxic shock syndrome; other inflammatory diseases associated with specific organs or tissues, such as nephritis (e.g., glomerulonephritis); adnexitis; gout; cholecystitis (cholecystitis); chronic obstructive pulmonary disease; congestive heart failure; type II diabetes; pulmonary fibrosis; vascular diseases such as atherosclerosis and restenosis; and alloimmunization resulting in graft rejection.

The biological activity of the compounds of the invention was determined in the following in vitro and in vivo experiments:

2. assays for binding to human glucocorticoid receptor

The gene for the human glucocorticoid receptor alpha isoform (EMBL acc. No. m10901) was cloned by reverse polymerase chain reaction. Total RNA was obtained from human peripheral blood lymphocytes following the manufacturer's (Qiagen) instructions, transcribed into cDNA at 50 ℃ for 45 minutes with AMV reverse transcriptase (Roche), and specific primers were used:

1)5 'ATATGGATCCCTGATGGACTCCAAAGAATCATTAACTCC 3'; and

2)5′ATATCTCGAGGGCAGTCACTTTTGATGAAACAGAAG3′

and pfx polymerase (Invitrogen),

the PCR conditions were: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and annealing at 68 ℃ for 3 minutes for a total of 36 cycles; the final extension step was carried out at 68 ℃ for 7 minutes. The resulting reaction product was cloned into XhoI/BamHI of Bluescript KS plasmid (Stratagene), sequenced by dideoxyfluorescence with M13 and M13rev primers (Microsynth), and then cloned into pcDNA3.1hygro (+) plasmid (Invitrogen Life T)Technologies) XhoI/BamHI sites. Will be 1 × 10⁵COS-1 cells were seeded in 12-well plates (Falcon) in DMEM medium (Invitrogen Life technologies) containing 10% FBS (Biowhitaker) and at 37 ℃ and containing 5% CO₂The culture was carried out in a gaseous atmosphere until the fusion was 70%. The medium was removed and 1. mu.g of DNA, 7. mu.l of PLUS reagent and 2. mu.l of Lipofectamine (Life technologies) in 500. mu.l DMEM were added to each well. At 37 ℃ and with 5% CO₂The cells were incubated and the same volume of 20% FBS/DMEM was added after 5 hours. After 24 hours, the medium was completely changed. At 48 hours after transfection, different concentrations of the test compound and 24nM in DMEM medium were added³H]Dexamethasone (Pharmacia). Cells were incubated at 37 ℃ and 5% CO₂Is incubated for 90 minutes in a gaseous environment, washed 3 times with PBS buffer (Sigma), cooled to 4 ℃ (pH 7.4) and then lysed in Tris buffer (pH 8.0) (Sigma) with 0.2% sds (Sigma). After addition of UltimaGold XR (Packard) scintillation fluid, residual radioactivity was read using a tricarb (Packard) β -scintillation counter.

3. Assay for inhibition of proliferation of mouse T-cell hybridoma 13 as a result of apoptosis induction

Triplicate dilutions of test steroids in RPMI medium (Instituted of Immunology, Zagreb) were prepared in 96-well plates using 10% FBS. 20000 cells/well are added to the compound solution at 37 ℃ and containing 5% CO₂Is maintained in the gas atmosphere of (1), and then 1. mu. Ci [ alpha ], [ alpha ] is added³H]Thymidine (Pharmacia) and the mixture was incubated for an additional 3 hours. Cells were harvested by applying vacuum with GF/C filters (Packard). Add 30. mu.l Microscint O scintillant (Packard) to each well and measure the radioactivity introduced using a beta scintillation counter (Packard). Specificity of glucocorticoid-induced apoptosis was confirmed by antagonism of proliferation inhibition using mifepristone (Sigma).

Mouse model of eosinophilia

Male Balb/C mice weighing 20-25g were randomly grouped and sensitized by intraperitoneal injection of ovalbumin (OVA, Sigma) on days 0 and 14. On day 20, mice were challenged with i.n. (intranasal) administration of OVA (positive control or experimental group) or PBS (negative control). 48 hours after intranasal administration of OVA, the animals were anesthetized and the lungs were rinsed with 1mL of PBS. Cells were separated in a Cytospin 3 cytocentrifuge (Shandon). Cells were stained with Diff-quick (Dade) and the percentage of eosinophils was determined by differential counting of at least 100 cells.

Fluticasone and beclomethasone were used as standard anti-inflammatory substances.

Different doses of the compound were administered daily, intranasally or intraperitoneally, and challenge tests were performed 2 days later and until the test was complete. The compounds are administered as a suspension in carboxymethyl cellulose or in lactose solution.

Model for rat corticosterone inhibition and thymus gland volume reduction

Male Wistar rats weighing 200-250g were randomly grouped. Test compounds and standard glucocorticoids were administered once daily for 3 days by the subcutaneous route. On day 3, rats were stressed with cold stress (4 ℃,1 hour), anesthetized with thiopental (Pliva Inc.) and bled with heparin. The entire thymus was removed from each animal and weighed immediately. The plasma was stored at-70 ℃ until detection. Corticosterone was extracted from 1mL plasma or from a standard dilution of corticosterone in PBS with chloroform (5mL), the interfering compounds were washed with 0.1M NaOH and sulfuric acid was added: h₂O∶C₂H₅OH is 8: 2: 1. After 60 minutes the fluorescence was measured and the excitation/emission wavelength was 470/530.

b) Measurement of TNF-alpha and IL-1 beta secretion in human peripheral blood mononuclear cells in vitro

By using Ficoll-Paque^TMPeripheral Blood Mononuclear Cells (PBMC) were prepared from heparinized whole blood after PBMC separation by Plus (Amersham-Pharmacia). For the determination of TNF-. alpha.levels, a total volume of 200. mu.l of 3.5-5X 10 was added in RPMI 1640 medium in microtiter plates (96-well, Falcon) with a flat bottom⁴Culture of individual cellAfter 18-24 hours of culture, the medium was supplemented with 10% heat-inactivated human AB serum (Croatian Centre For TransfusedMedicine, Zagreb), 100 units/ml penicillin, 100mg/ml streptomycin, and 20mM HEPES (Invitrogen Life Technologies). At 37 ℃ and with 5% CO₂And the cells were incubated at 90% humidity. In the negative control, cells were cultured only in medium (NC), whereas in the positive control TNF- α secretion was stimulated by addition of 1. mu.g/ml lipopolysaccharide (LPS, E.coli serotype 0111: B4, SIGMA) (PC) and the effect of the test substances on TNF- α secretion was determined after their addition to cell cultures stimulated with LPS (TS). By ELISA, according to the manufacturer (R)&D Systems) to determine the level of TNF-alpha in the cell supernatant. The test sensitivity was < 3pg/ml TNF-. alpha.. Determination of IL-1. beta. levels as described for the TNF-. alpha.assay, using only 1X 10⁵Individual cells/well and 0.1ng/ml LPS. By ELISA (R)&D Systems) to determine IL-1 β levels. The percent inhibition of TNF- α or IL-1 β production was calculated by the following equation:

inhibition [% 1- (TS-NC)/(PC-NC) ] × 100.

The IC50 value was defined as the concentration of the substance that inhibited TNF-. alpha.production by 50%. Compounds exhibiting IC-50 at concentrations of 20. mu.M or below 20. mu.M are considered active. IC-50 was calculated using Graph Padprism software.

c) Determination of TNF-alpha secretion in RAW 264.7 cells

Cells were grown at 37 ℃ with 5% CO₂In DMEM medium (Invitrogen Life Technologies) 10% Fetal Bovine Serum (FBS) at 90% humidity. 20000 cells/well plates were fixed in 96 well plates (Falcon). In the negative control, the cells were cultured in medium (NC) only, whereas in the positive control, TNF- α secretion was stimulated by addition of 500pg/ml lipopolysaccharide (LPS, E.coli serotype 0111: B4, SIGMA) (PC) and the effect of the test substances on TNF- α secretion was determined after their addition to the cell culture stimulated with LPS (TS). By ELISA, according to the manufacturer (R)&D Systems) water for the determination of TNF-alpha in cell supernatantsAnd (7) flattening. The percent inhibition of TNF- α production was calculated by the following equation:

inhibition [% 1- (TS-NC)/(PC-NC) ] × 100.

The IC-50 value is defined as the concentration of the substance that inhibits 50% of TNF-. alpha.production. Compounds exhibiting an IC-50 at concentrations of 10. mu.M or below 10. mu.M are considered active.

Human prostaglandin-H synthase-1 (hPGH-1) and human prostacyclin-H synthase-2 (hPGH-2) inhibition assays

The genes encoding hPGH-1 and hPGH-2 were amplified using PCR using Platinumpfx DNA polymerase (Invitrogen Life Technologies) from a human placental cDNA library (Stratagene). The primer sequence for PGH-1 was: 5'

ATATAAGCTTGCGCCATGAGCCGGAGTCTTC3 'and 5'

ATATGGATCCTCAGAGCTCTGTGGATGGTCGC 3'; the primer sequence for hPGH-2 was:

5 ' ATATAAGCTTGCTGCG ATGCTCGCCCGC3 ' and 5 '

ATATGGATCCCTACAGTTCAGTTCAGTCGAACGTTC 3'. Cloning of the PCR product into

The HindIII and BamHI restriction sites of pcDNA3.1Hygro (+) plasmid (Invitrogen Life Technologies) were verified by sequencing.

COS-7 cells (ATCC) were transfected with 5% CO at 37 deg.C₂Cells were grown to medium and fully confluent in 10% Fetal Bovine Serum (FBS) in DMEM medium (Invitrogen Life Technologies) in 24-well plates (Falcon) at 90% humidity. Mu.g of plasmid DNA (pcDNA Hygro 3.1(+) containing PGH-1 or PGH-2 gene or pcDNA Hygro 3.1(+) as a negative control sample) was combined with 1, 5. mu.l of Lipofectamine 2000(Invitrogen Life Technologies) according to the manufacturer's recommendations. 24-48 hours after transfection, test compounds in DMEM were added to the cells, but the medium was not removed and after 40 minutes arachidonic acid (Sigma) was added to a final concentration of 20. mu.M. After 30 minutes, the supernatant was taken outThe solution was then assayed for PGE-2 using the PGE-2 assay kit (Cayman) according to the manufacturer's instructions. No PGE-2 production was detected in the negative control.

The% inhibition was calculated by the following equation:

in vivo model of LPS-induced excessive TNF- α secretion in mice

TNF- α secretion in mice was induced as already described above (Badger AM et al, J.Pharmac.and env.Therap., 1996, 279: 1453-1461). In this experiment, 8-12 week old male Balb/C mice divided into groups of 6-10 animals were used. Animals were treated with solvent only oral administration (negative and positive controls) or the substance solution was given 30 minutes prior to treatment with LPS (E.coli serotype 0111: B4, Sigma) at a dose of 1-25. mu.g/animal. Animals were euthanized after 2 hours by intraperitoneal injection of roumpun (bayer) and ketamine hydrochloride (Parke-Davis). Blood samples from each animal were taken into "vacutainer" tubes (becton dickinson) and plasma was isolated according to the manufacturer's recommendations. TNF-. alpha.levels in plasma were determined by ELISA (Biosource, R & D Systems) according to the method described by the manufacturer. The test sensitivity was < 3pg/ml TNF-. alpha.. The percent inhibition of TNF- α production was calculated by the following equation:

inhibition [% 1- (TS-NC)/(PC-NC) ]. 100.

Compounds that exhibit 30% or more than 30% inhibition of TNF- α production at a dose of 10mg/kg are considered active.

e) Writhing test for analgesic activity

In this experiment, pain was induced by injecting a stimulant, most commonly acetic acid, into the abdominal cavity of mice. Animals have a characteristic writhing response with the name of the test assigned (Collier HOJ et al, drugs and chemotherapy (Pharmac. Chemothers.), 1968, 32: 295-259; Fukawa K et al, J. Pharmac. meth., 1980, 4: 251-259; Schweizer A et al, Effect of active Agents (Agents Actions), 1988, 23: 29-31). This assay is suitable for determining the analgesic activity of a compound. The method comprises the following steps: male Balb/C mice (Charles River, Italy) 8-12 weeks old were used. The control group was orally administered with methylcellulose 30 minutes before intraperitoneal administration of 0.6% strength acetic acid, while the test group was orally administered with standard (acetylsalicylic acid) or test substance in methylcellulose 30 minutes before intraperitoneal administration of 0.6% acetic acid (0.1 ml/10g in volume). The mice were each placed in a glass funnel and the number of writhes was recorded per animal over a 20 minute period. The percentage of writhing inhibition was calculated according to the following equation:

inhibition [% ], [% ] (number of twists in the control group-average value of the number of twists in the test group)/number of twists in the control group X100.

Compounds that exhibit the same activity or better than acetylsalicylic acid are considered active.

In vivo model of LPS-induced mouse shock

Male Balb/C mice (Charles River, Italy) 8-12 weeks old were used. LPS from Serratia autohesia (Sigma, 1-6136) was isolated by dilution with sterile saline. LPS injections were first given intradermally at a dose of 4. mu.g/mouse. After 18-24 hours, LPS was administered intravenously at a dose of 200. mu.g/mouse. Two LPS injections were given to the control group in the manner described above. The substances were administered orally to the test groups half an hour before each LPS administration. Survival was observed after 24 hours.

Compounds that produce a survival rate of 40% or better at a dose of 30mg/kg are considered active.

Compounds are considered to be active if they show statistical significance (p < 0.05 by Student's t-test) in at least two of the above assays. The molar amount of compound used is below the threshold amount (about 30 μm) for macrolides that exert mild anti-inflammatory effects as reported in the literature.

(ii) In vitro assay for screening for inhibition of HIV replication

HIV-1 was transfected into the T4-cell line. MT-4(Koyanagi et al, J.cancer, int. J.cancer, 36, 445-451, 1985) was previously shown to be highly sensitive to HIV for use as a target cell line. Inhibition of HIV-induced cytopathic effects was used as an endpoint. The survival of HIV-and mock-infected cells was estimated by in situ reduction of MTT using spectrophotometry. The 50% cytotoxic concentration was defined as the concentration of compound that reduced the absorbance of the mock-infected control sample by 50%. The percent protection achieved by the compounds in HIV-infected cells was calculated as the optical density determined in HIV-infected cells using the indicated concentrations of the test compounds. The ratio of cytotoxic effect to protective effect was determined.

(iii) In vitro assay for screening for inhibition of HCV replication

The activity of the novel compounds was determined using a modification of the method reported by Lohmann et al [ V.Lohmann et al Science 1999 285 110-.

Cell lines containing HCV replicon were used to demonstrate the ability of the novel compounds to inhibit HCV replicon RNA replication in cells. Inhibition of HCV replicon RNA replication reduces replicon RNA in cells, and this reduction can be measured using methods that specifically quantify such RNA.

This assay is based on the concept of using a reporter gene as a simple readout of intracellular HCV replicon RNA levels. For this purpose, the Renilla luciferase gene was introduced into the first open reading frame of the replicon construct NK5.1 immediately after the Internal Ribosome Entry Site (IRES) sequence (Krieger et al, J.Virol., 75: 4614) and fused with the Neomycin Phosphotransferase (NPTII) gene by means of the self-cleaving peptide 2A from foot-and-mouth disease virus (Ryan & Drew, EMBO Vol 13: 928-channel 933). Following in vitro transcription, RNA was electroporated into human hepatoma Huh7 cells and G418-resistant colonies were isolated and expanded. It was confirmed that the stably selected cell line contained the replicating HCV subgenomic RNA, and that the Renilla luciferase activity expressed by the replicon reflected its RNA level in the cells.

For the experimental procedure, Renilla luciferase HCV replicon cells cultured in Dulbecco's MEM Invitrogen cat No. 31966-021 containing 5% Fetal Calf Serum (FCS) (Invitrogen cat No.10106-169) were fixed onto 96-well plates at 5000 cells/well plate and incubated overnight. After 24 hours, chemical compounds at various dilutions in growth medium were added to the cells, followed by further incubation at 37 ℃ for 3 days. The plate assay was performed in duplicate, one opaque white and one clear, in order to measure the activity and cytotoxicity of the compounds in parallel, thereby ensuring that the observed activity was not due to a reduction in cell proliferation.

At the end of the incubation, cells on white plates were collected and assayed for luciferase activity by using the dual-luciferase reporter assay system (Promega cat No. eb1960). All reagents described in the following paragraphs were included in the manufacturer's kit and were used in the preparation of the reagents as indicated by the manufacturer. Briefly, cells were washed with 20. mu.L of PBS (phosphate buffered saline; pH 7.0) per well and lysed with 25. mu.L of 1 × passive lysis buffer, after which they were incubated at room temperature for 20 minutes. To each well 100 microliters of LAR II reagent was added. The plate was then inserted into a microplate luminometer (Packard) and 100 μ L of Stop & Glo reagent was injected into each well and luminescence was measured. The drug concentration IC50 required to reduce replicon levels by 50% relative to control values for untreated cells can be calculated from a plot of percent reduction in luciferase activity versus drug concentration.

In vitro assay for antitumor Activity

The EL-4 cell line was used for screening anti-tumor activity in vitro. Cells were grown at 37 ℃ with 5% CO₂And DMEM medium (Invitrogen) supplemented with 10% fbs (Invitrogen) at 90% relative humidity. The assay was performed on 96-well plates, with dilutions of compounds from 10-5 to 10-10M and 30000 cells/well used. After 24 hours of treatment, add³H-labeled thymidine (Amersham) for 4 hours. The cells were collected using a cell collector (Packard) using a GF/C filter (Packard). Scintillation fluid (Microscint 20, Packard) was added and the scintillation counted.

Inhibition was calculated as follows: inhibition = (1-CPM sample/CPM positive control) × 100. L1210 intraperitoneal tumor model

DBA2 mice (male 9-12 weeks, 20-30g) were injected intraperitoneally with 106 viable cells on day 0. Animals were then treated with cisplatin or the hybrid compound intraperitoneally on days 1, 2 and 3 with single or multiple doses. The units were weighed daily and observed 2 times daily for signs of tumor growth. Animals were sacrificed if the body weight dropped below 80% of the original body weight or if other severe toxicological problems were observed. Changes in the overall anatomy were noted at the end of the experiment. B16 melanoma intraperitoneally model

Male C57 BL/6J mice were inoculated intraperitoneally (i.p.) with 106 viable B16F10 cells. Cells were injected on day 0 and then intraperitoneally as single or multiple doses of free cisplatin or the hybrid compound on the following 1 day. Animals were monitored as described above.

Subcutaneous model of B16 melanoma

Male C57 BL/6J mice were inoculated subcutaneously (s.c.) with 105 viable B16F10 cells. Establishing the tumor to an area of about 50-70mm²Measured on the basis of the product of biorthogonal diameter. Animals bearing subcutaneous tumors were treated by intraperitoneal or intravenous injection of 2,5, 10, 15mg Pt/kg free cisplatin or hybrid compound 19-21.

Synthetic methods and examples

Preparation of intermediates

Intermediate A

2-Chlorotrityl chloride resin (1 eq. 0,5mmol, 600mg) was placed on a glass cartridge fitted with a coarse frit filter and swollen in DCM for 10 min. The resin was then filtered and washed 3 times with DCM. After washing with DMF, the resin was loaded with N-R-Fmoc-glycine by adding 2.0mL of a 0.6M solution of the amino acid in DMF and 0,630mL of N, N-Diisopropylethylamine (DIPEA) and mixed. After 5 minutes, 0,315ml of DIPEA were added. After 50 minutes of mixing, methanol was added in an amount of 0,5 ml. After 10 min, the resin was filtered and washed 10 times with DCM, DMF and methanol.

Deprotection of the amino acid

Different solutions of piperidine in DMF were prepared and poured onto beads as follows:

5% piperidine/DMF 10 min (about 10ml)

30% piperidine/DMF 15 min (about 10ml)

50% piperidine/DMF 30 min (about 10ml)

After deprotection, the resin was washed with DMF.

The second amino acid Fmoc-leucine (1060mg, 3mmol) and 2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluorophosphate (HBTU) were dissolved in 3mL DMF and 0.216mL (5mmol) DIPEA was added rapidly and simultaneously to the monomer/resin mixture in the reaction tube.

End capping:

a solution of 10eq (2,04ml) acetic anhydride and 10eq (3,48ml) DIPEA in 5ml DMF was prepared. 2,5ml of this solution were added to the reaction mixture for 5 minutes. An additional section was followed to deprotect the second amino acid with piperidine in DMF:

30% piperidine/DMF 2 min (about 10ml)

30% piperidine/DMF 2 min (about 10ml)

30% piperidine/DMF 5 min (about 10ml)

30% piperidine/DMF 5 min (about 10ml)

And washed with large amounts of DMF.

The same coupling deprotection procedure was repeated for the following amino acids:

1162mg, 3mmol of Fmoc-phenylalanine as the third amino acid

HBTU，1081mg/3ml DMF

DIPEA，0,87ml；

And the fourth amino acid Fmoc-glycine, 892mg, 3mmol

HBTU，1081mg/3ml DMF

DI PEA，0,87ml；

Followed by filtration and washing with DMF.

To the tetrapeptide/resin mixture in the reaction tube was added a mixture of dexamethasone acid (567mg, 3eq), HBTU (540mg, 3,8eq) and 0,435ml DIPEA in 3ml DMF, mixed and kept overnight.

End capping:

a solution of 0,5ml acetic anhydride and 0,5ml DIPEA in 3ml DMF was added to the reaction mixture for 5 minutes, followed by washing with DCM, DMF and MeOH and drying under vacuum.

Cleavage from resins

10ml of a 50% trifluoroacetic acid in DCM solution was poured onto the beads and mixed for 15 minutes. Remove the reagents by filtration and wash the beads 2x with DCM. The same procedure was repeated using 10ml of acid. The collected solvent was evaporated to remove excess TFA and a quantity of ether was added. 60,1mg of intermediate A are isolated. MS (m/z): 753.3[ MH]⁺

Example I

Compound 1 (dexamethasone-Gly-Phe-Leu-Gly-azithromycin)

Intermediate A (57 mg; 0.076mmole) was dissolved in dry gas under an inert atmosphereDry CH₂Cl₂(5mL) and cooled at 0 ℃. 0.115mL of N, N-diisopropylethylamine and 20,5mg of 1-hydroxybenzotriazole were added, followed by the addition of the compounds 9-deoxo-9 a-aza-9 a- (γ -aminopropyl) -9 a-homoerythromycin A (60 mg; 0.076mmole) and 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride (57,6mg, 0,30 mmol). The reaction mixture was stirred at room temperature for 24 hours in a stream of argon, then evaporated to a small volume under reduced pressure and purified on a silica gel column (eluent: CHCl)₃∶CH₃OH∶NH₄OH 6: 1: 0.1). 14mg of Compound 1 are obtained;

MS(m/z)：1527，3[MH]⁺.IR(cm^-1)/KBr：3415，2969，2939，2874，1664，1528，1458，1378，1262，1168，1107，1054，1013，959，894，803，702.

intermediate B

2-Chlorotrityl chloride resin (1 eq. 0,5mmol, 600mg) was placed on a glass cartridge fitted with a coarse frit filter and swollen in DCM for 10 min. The resin was then filtered and washed 3 times with DCM. After washing with DMF, the resin was loaded with N-R-Fmoc-glycine by adding 2.0mL of a 0.6M solution of the amino acid in DMF and 0,630mL of N, N-Diisopropylethylamine (DIPEA) and mixed. After 5 minutes, 0,315ml of DIPEA were added. After 50 minutes of mixing, methanol was added in an amount of 0,5 ml. After 10 min, the resin was filtered and washed 10 times with DCM, DMF and methanol.

Deprotection of the amino acid

Different solutions of piperidine in DMF were prepared and poured onto beads as follows:

5% piperidine/DMF 10 min (about 10ml)

30% piperidine/DMF 15 min (about 10ml)

50% piperidine/DMF 30 min (about 10ml)

After deprotection, the resin was washed with DMF.

The second amino acid, Fmoc-leucine (1060mg, 3mmol) and 2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluorophosphate (HBTU) were dissolved in 3mL DMF and 0.216mL (5mmol) of DIPEA was added rapidly to the monomer/resin mixture in the reaction tube.

End capping:

a solution of 10eq (2,04ml) acetic anhydride and 10eq (3,48ml) DIPEA in 5ml DMF was prepared. 2,5ml of this solution were added to the reaction mixture for 5 minutes.

The second amino acid was deprotected with piperidine in DMF as follows:

30% piperidine/DMF 2 min (about 10ml)

30% piperidine/DMF 2 min (about 10ml)

30% piperidine/DMF 5 min (about 10ml)

30% piperidine/DMF 5 min (approx. l0ml)

And washed with large amounts of DMF.

The same coupling deprotection procedure was repeated for the following amino acids:

1162mg, 3mmol of Fmoc-phenylalanine as the third amino acid

HBTU，1081mg/3ml DMF

DIPEA，0,87ml；

And a fourth amino acid

Fmoc-glycine 892mg, 3mmol

HBTU，1081mg/3ml DMF

DIPEA，0,87ml；

The Fmoc protecting group was not removed only after coupling the fourth amino acid.

After filtration and washing with DMF, the product was removed from the resin:

10ml of a solution of 50% trifluoroacetic acid in DCM was poured onto the beads and mixed for 15 minutes. Remove the reagents by filtration and wash the beads 2x with DCM. The same procedure was repeated using 10ml of acid. The collected solvent was evaporated to remove excess TFA and a quantity of ether was added. 109,1mg of intermediate B1 were isolated. MS (m/z): 715, 6(MH)⁺

Intermediate B1

2-Chlorotrityl chloride resin (1eq ═ 0,352mmol, 326mg) was placed on a glass cartridge fitted with a coarse frit filter and swollen in DCM for 10 min. The resin was then filtered and washed 3 times with DCM. After washing with DMF, the resin was loaded with compound B1 by adding 259mg (0,422mmol) and 0,150mL of N, N-Diisopropylethylamine (DIPEA) dissolved in 2mL of DMF and mixed. After 5 minutes, 0,230ml of DIPEA were added. After 50 minutes of mixing, 0,355ml of methanol were added. After 10 min, the resin was filtered and washed 10 times with DCM, DMF and methanol.

Deprotection of the amino acid

Different solutions of piperidine in DMF were prepared and poured onto beads as follows:

5% piperidine/DMF 10 min (about 10ml)

30% piperidine/DMF 15 min (about 10ml)

50% piperidine/DMF 30 min (about 10ml)

After deprotection, the resin was washed with DMF.

377mg (1,055mmol) of indomethacin and 533mg (1,41mmol) of 2- (1-H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluorophosphate (HBTU) were dissolved in 3mL of DMF and 0.602mL (3,52mmol) of DIPEA was rapidly added while adding to the polymer/resin mixture in the reaction tube.

End capping: a solution of 0,5ml acetic anhydride and 0,5ml DIPEA in 3ml DMF was added to the reaction mixture for 5 minutes, filtered and washed with DCM, DMF and MeOH.

Cleavage from resins

10ml of a 50% trifluoroacetic acid in DCM solution was poured onto the beads and mixed for 15 minutes. Remove the reagents by filtration and wash the beads 2x with DCM. The same procedure was repeated using 10ml of acid. The collected solvent was evaporated to remove excess TFA and a quantity of ether was added. 210,8mg of the product, intermediate B, were isolated. MS (m/z): 732, 66[ MH]⁺

Example II

Compound 2 (indometacin-Gly-Phe-Leu-Gly-azithromycin)

Intermediate B (200 mg; 0,27mmol) was dissolved in dry CH under an inert gas atmosphere₂Cl₂(5 mL). 0.416mL (2,14mmol) of N, N-diisopropylethylamine and 74mg (0,55mmol) of 1-hydroxybenzotriazole are added, followed by the addition of the compound 9-deoxo-9 a-aza-9 a- (γ -aminopropyl) -9 a-homoerythromycin A (216,6 mg; 0.27 mmol) and 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride (188mg, 1,09 mmol). The reaction mixture was stirred at room temperature for 24 hours in a stream of argon, then evaporated to a small volume under reduced pressure and purified with a silica gel column (eluent: CHCl)₃∶CH₃OH∶NH₄OH 6: 1: 0.1). To yield 70mg of compound 2; MS (m/z): 1505, 8[ MH]⁺.IR(cm^-1)/KBr：3654，3633，3425，3084，2970，2936，1720，1652，1637，1545，1439，l368，1309，1230，1179，1111，1089，1055，1013，867，803，739，700，643.

Example III

Following the general procedure of example I and substituting the appropriate reactants therein, the following compounds were obtained:

compound 3: r₁＝F；R₂＝F；R₃＝OH

Compound 4: r₁＝F；R₂＝H；R₃＝H

Compound 5: r₁＝F；R₂＝F；R₃＝H

Compound 6: r₁＝H；R₂＝F；R₃＝OH

Example IV

Following the general procedure of example II and substituting the appropriate reactants therein, the following compounds were obtained:

V-Gly-Phe-Leu-Gly-M

example V

Compound 19

To succinic acid paclitaxel (1 eq) in dry CH under argon atmosphere₂Cl₂To the solution (5ml) were added 9 equivalents of triethylamine, 2 equivalents of 1-hydroxybenzotriazole, 1 equivalent of 9-deoxo-9 a-aza-9 a- (γ -aminopropyl) -9 a-homoerythromycin AAnd 4 equivalents of 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride. The reaction mixture was stirred at room temperature for 24 hours in a stream of argon, then evaporated to a small volume under reduced pressure and purified with a silica gel column using chloroform, methanol and ammonia as eluents. The chromatographic product is characterized by the following structural formula:

example VI

Compound 20

To succinic acid camptothecin (1 equivalent) in dry CH under argon atmosphere₂Cl₂To the solution in (5ml) were added 9 equivalents of triethylamine, 2 equivalents of 1-hydroxybenzotriazole, 1 equivalent of 9-deoxo-9 a-aza-9 a- (γ -aminopropyl) -9 a-homoerythromycin A and 4 equivalents of 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride. The reaction mixture was stirred at room temperature for 24 hours in a stream of argon, then evaporated to a small volume under reduced pressure and purified with a silica gel column using chloroform, methanol and ammonia as eluents. The chromatographic product is characterized by the following structural formula:

example VII

Compound 21

To a solution of γ -methyl-N' - [4- [ N- [ (2, 4-diamino-6-pteridinyl) methyl ] -N-methylamino ] benzoyl ] -L-glutamate (1 equivalent) in dry DMF was added 2 equivalents of 1, 1-carbonyldiimidazole (in 5ml DMF) under an argon atmosphere. The reaction mixture was stirred at-5 ℃ for 24 hours, then 1 equivalent of compound 9-deoxy-9 a-aza-9 a- (γ -hydroxypropyl) -9 a-homoerythromycin a in dry DMF was added. The reaction mixture was heated at 100 ℃ for 48 hours, then evaporated and purified with a silica gel column using chloroform, methanol and ammonia as eluents. The chromatographic product is characterized by the following structural formula:

example VIII

Compound 22

To 5' -O-succinylzidovudine (1 equivalent) in dry CH under argon atmosphere₂Cl₂To the solution in (5ml) were added 9 equivalents of triethylamine, 2 equivalents of 1-hydroxybenzotriazole, 1 equivalent of 9-deoxo-9 a-aza-9 a- (γ -aminopropyl) -9 a-homoerythromycin A and 4 equivalents of 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride. The reaction mixture was stirred at room temperature for 24 hours in a stream of argon, then evaporated to a small volume under reduced pressure and purified with a silica gel column using chloroform, methanol and ammonia as eluents. The chromatographic product is characterized by the following structural formula:

abbreviations:

pyr: pyridine compound

NEt₃: triethylamine

4-PP: 4-pyrrolopyridines

DMAP: 2, 6-dimethylaminopyridine

DIPEA: n, N' -diisopropylethylamine

DMF: dimethyl formamide

TFA: trifluoroacetic acid

Claims (52)

1. A compound of the general formula I and pharmaceutically acceptable salts and solvates thereof and the individual diastereomers thereof:

wherein:

m represents a macrolide subunit with the property of accumulation in inflammatory cells;

v is an anti-inflammatory steroidal or non-steroidal anti-inflammatory subunit or an anti-tumor subunit or an anti-viral subunit, and

l is a linker molecule covalently linked to each of M and V.

2. A compound according to claim 1, wherein M represents a group of formula II:

wherein:

(i) z and W are independently: > C ═ O, > CH₂、＞CH-NR_tR_s、＞N-R_NOr > C ═ N-R_MOr a bond, wherein:

R_tand R_sIndependently hydrogen or alkyl;

R_Mis hydroxy, alkoxy, substituted alkoxy OR OR^P；

R_NIs hydrogen, R^PAlkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl or-C (X) -NR_tR_s(ii) a Wherein X is ═ O or ═ S;

provided that Z and W cannot be simultaneously > C ═ O, > CH₂、＞CH-NR_tR_s、＞N-R_NOr > C ═ N-R_MOr a bond;

(ii) u and Y are independently hydrogen, halogen, alkyl or hydroxyalkyl;

(iii)R¹is hydroxy, OR^P、-O-S²A group or ═ O;

(iv)S¹a sugar moiety of the formula:

wherein:

R⁸and R⁹Are both hydrogen or together form a bond, or R⁹Is hydrogen and R⁸is-N (CH)₃)R^yWherein:

R^yis R^P、R^Zor-C (O) R^ZWherein R is^ZIs hydrogen or alkyl or alkenyl or alkynyl or cycloalkyl or aryl or heteroaryl or is substituted by C₂-C₇Alkyl radical, C₂-C₇-alkenyl, C₂-C₇-alkynyl, aryl or heteroaryl substituted alkyl;

R¹⁰is hydrogen or R^P；

(v)S²A sugar moiety of the formula:

wherein:

R^3’is hydrogen or methyl;

R¹¹is hydrogen, R^POr O-R¹¹Is a reaction with R¹²And a group forming > C ═ O or epoxy groups with C/4 "carbon atoms;

R¹²is hydrogen or with O-R¹¹And a group forming > C ═ O or epoxy groups with C/4 "carbon atoms;

(vi)R²is hydrogen, hydroxy, OR^POr an alkoxy group;

(vii) a is hydrogen or methyl;

(viii) b is methyl or epoxy;

(ix) e is hydrogen or halogen;

(x)R³is hydroxy, OR^PAlkoxy or R³Is a reaction with R⁵And a group which forms a cyclic carbonate or cyclic carbamate with the C/11 and C/12 carbon atoms; or if W or Z is > N-R_NThen R³Is a group which forms a cyclic carbamate with W or Z;

(xi)R⁴is C₁-C₄An alkyl group;

(xii)R⁵is hydrogen, hydroxy, OR^P、C₁-C₄-alkoxy or with R³And a group which forms a cyclic carbonate or cyclic carbamate with the C/11 and C/12 carbon atoms;

(xiii)R⁶is hydrogen or C₁-C₄-an alkyl group;

wherein M contains a linking position through which it is linked to V via a linker L; provided that the attachment position is located on one or more of the following groups:

a) at S¹、S²Or any reactive hydroxyl, nitrogen or epoxy group on the aglycone oxygen, with the proviso that S¹Or/and S²Is cracked;

b) activity at Z or W > N-R_Nor-NR_tR_sOr ═ O;

c) at R¹、R²、R³And R⁵A reactive hydroxyl group on any one of them;

d) may be first derivatized to hydroxy or-NR_tR_sAny other group of groups; and is

R^PIs a hydroxyl or amino protecting group.

3. A compound as claimed in claim 1, wherein L represents a member of group IV:

X¹-(CH₂)_m-Q-(CH₂)_n-X² IV

wherein:

X¹is selected from-CH₂-, -C (O) -, OC (O) -, N-O-, -OC (O) NH-or-C (O) NH-;

X²is-NH-or-NHC (O) -, -OC (O) -, -C (O) -, -O or-CH₂-；

Q is-NH-or-CH₂-or is absent;

wherein-CH₂-or-NH-groups may each optionally be substituted by C₁-C₇Alkyl radical, C₂-C₇-alkenyl, C₂-C₇Alkynyl, C (O) R^X、C(O)OR^X、C(O)NHR^XIs substituted in which R^XCan be C₁-C₇-alkyl, aryl or heteroaryl;

(symbol)mandnindependently an integer from 0 to 4, with the proviso that if Q is NH then n cannot be 0;

and V is only an anti-tumor subunit or an anti-viral subunit.

4. The compound of claim 1, wherein L represents a peptide linker comprising a polypeptide of about 2 to about 50 amino acids.

5. A compound as claimed in claim 1, wherein V represents a member of group X of formula:

wherein:

R^aand R^bIndependently represents hydrogen or halogen;

R^cis hydroxy, alkoxy, alkyl, thiocarbamoyl, carbamoyl or a bond;

R^dand R^eIndependently represent hydrogen, hydroxy, methyl or C₁-C₄-alkoxy or each is a group or a bond to another forming 1, 3-dioxolane;

R^fis hydrogen, hydroxy, chlorine or forms a keto group with the carbon atom to which it is attached;

R^jis hydrogen or halogen.

6. The compound of claim 1, wherein V is derived from an NSAID selected from the group consisting of aceclofenac, acemetacin, acetaminophen, acexamol, acetylsalicylic acid, acetyl-salicyl-2-amino-4-methylpyridine-acid, 5-aminoacetylsalicylic acid, alclofenac, amprofen, amfenac, aminoantipyrine, ampiroxicam, animalidine, bendazac, benoxaprofen, bermoprofen, α -bisabolol, bromfenac, 5-bromosalicylic acid acetate, bromosalicin, bucloxic acid, bufen, carprofen, celecoxib, chromoglycate, cinnamyl, clindanac, clopidogrel, diclofenac sodium, diflunisal, ditalozole, droxicam, enfenamic acid, etodolac, etofenamate, felbinac, fenbufen, clofenac, fenflurazoc, fenol, fenoprofen, fenol, fenoprofen, fenclofenamic acid, fenclofenac, fenoprofen, Fentiazac acid, feprenol, flufenac, flufenamic acid, flunixin, flurbiprofen, glutametacin, glycol salicylate, ibufenac, ibuprofen, ibuprophenil, indomethacin, indoprofen, triazolic acid, isoxepac, isoxicam, ketoprofen, ketorolac, lornoxicam, loxoprofen, methofenamic acid, mefenamic acid, meloxicam, 5-aminosalicylic acid, toluic acid, mofetil, lummestranol, nabumetone, naproxen, niflumic acid, nimesulide, oxalazine, oxaprozin, oxybutyzone, acetaminophen, pamabramide, perisoxazole, phenylacetylsalicylate, phenylbutazone, phenylsalicylate, pirazolac, piroprofen, pranoprofen, protazofampriazone, verapamazine, verapamazinic acid, verapametamide, salicylamide-O-acetate-amide, Salicylyl sulfate, salicin, salicylamide, salsalate, sulindac, suprofen, suxibutazone, tamoxifen, tenoxicam, tiaprofenic acid, tiaramide, ticlopridine, tenoritidine, tolfenamic acid, tolmetin, tropesin, biphenyl butyric acid, simofen, zaltoprofen, zomepirac, tomopuro, zafirlukast, and cyclosporine.

7. The compound of claim 1, wherein V is derived from an antineoplastic compound selected from the group consisting of bicalutamide, camptothecin, estramustine phosphate, flutamide, mechlorethamine, thiotepa, ifosfamide, hydroxyurea, bleomycin, paclitaxel, lomustine, irinotecan, methotrexate, vinorelbine, anastrozole, floxuridine, melphalan, vincristine, vinblastine, mitomycin, nandrolone, goserelin, leuprolide, triptorelin, aminoglutethimide, mitotane, cisplatin, chlorambucil, pentostatin, cladribine, busulfan, etoposide, mitoxantrone, idarubicin, cyclophosphamide, mercaptopurine, thioguanine, cytarabine, cyclophosphamide, doxorubicin, daunorubicin, teniposide, tamoxifen, taxotere and topotecan.

8. The compound of claim 1 wherein V is derived from an antiviral compound selected from the group consisting of acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine, ritonavir, indinavir, nevirapine, zidovudine, didanosine, stavudine, abacavir, zalcitabine, amprenavir, ribavirin and adamantane.

9. The compound of claim 2, wherein Z and W together are: -N (CH)₃)-CH₂-、-NH-CH₂-、-CH₂-NH-, -C (O) -NH-or-NH-C (O) -;

a and B are methyl;

e is hydrogen;

R²is hydroxy or methoxy;

S¹represents a deoxyglycosamine sugar wherein R⁸Selected from hydrogen, methyl, amino, C₁-C₆Alkylamino or C₁-C₆A dialkylamino group;

R⁹and R¹⁰Is hydrogen;

R¹is hydroxy or O-S²Group of which S²Represents cladinose, wherein:

R¹¹is hydrogen or O-R¹¹Is a reaction with R¹²And a group forming > C ═ or epoxy groups with C/4 "carbon atoms; r¹²Is hydrogen or with O-R¹¹And a group forming > C ═ O or epoxy groups with C/4 "carbon atoms;

R¹³is methyl;

u is hydrogen;

y is methyl;

R₆is hydroxy, methyl or ethyl;

R⁵is hydrogen, hydroxy, methoxy or with R³And a group forming a cyclic carbonate or cyclic carbamate bridge with the C/11 and C/12 carbon atoms;

R³is hydroxy or a group which forms a cyclic carbamate bridge with W or Z, or R³Is a reaction with R⁵And a group forming a cyclic carbonate or cyclic carbamate bridge with the C/11 and C/12 carbon atoms;

R⁴is methyl;

provided that the bond is either via the nitrogen of Z in the N/9a position or via both S²R at C/4' position of sugar¹²Carbon or R of¹¹Oxygen of (2).

10. The compound of claim 3, wherein:

X¹is-CH₂-or-oc (o) -;

X²is-NHC (O) -;

q is-NH-or absent.

11. The compound of claim 6, wherein:

v is derived from an NSAID selected from the group consisting of S- (+) -ibuprofen, indomethacin, flurbiprofen, naproxen, ketoprofen, acetylsalicylic acid, sulindac, etodolac, ketorolac, suprofen, flunixin, diclofenac sodium, and tolmetin sodium.

12. The compound of claim 7, wherein:

v is derived from an anti-tumor compound selected from the group consisting of methotrexate, paclitaxel, camptothecin, and doxorubicin.

13. The compound of claim 8, wherein:

v is derived from an antiviral compound selected from the group consisting of zidovudine, didanosine and stavudine.

14. A compound of the formula:

15. a compound of the formula:

16. a compound of the formula:

17. a compound of the formula:

18. a compound of the formula:

19. a compound of the formula:

20. a compound of the formula:

21. a compound of the formula:

22. a compound of the formula:

23. a compound of the formula:

24. a compound of the formula:

25. a compound of the formula:

26. a compound of the formula:

27. a compound of the formula:

28. a compound of the formula:

29. a compound of the formula:

30. a compound of the formula:

31. a compound of the formula:

32. a compound of the formula:

33. a compound of the formula:

34. a compound of the formula:

35. a compound of the formula:

36. process for the preparation of compounds of the general formula I

The method comprises the following steps:

a) to obtain wherein X²A compound of formula I which is-NHC (O) -by reacting a compound of formula VI with a free amino group of a macrolide represented by formula VIIa,

wherein formula VI is as follows:

wherein L is¹Represents a leaving group, and represents a leaving group,

and formula VIIa is as follows:

b) is composed ofObtain wherein X²A compound of formula I which is-OC (O) -by reacting a compound of formula VI with the free hydroxyl group of a macrolide represented by formula VIIb,

c) to obtain wherein X¹is-OC (O) -, Q is-NH-and X²A compound of formula I which is-NHC (O) -a macrolide represented by formula VIIc:

reacting with a free amino group of a compound represented by the formula VIb,

d) to obtain wherein X¹is-OC (O) NH-and X²A compound of formula I which is-NHC (O) -a macrolide represented by formula VIId

Reacting with a free amino group of a compound represented by formula VIb;

e) to obtain wherein X¹is-CH₂-, Q is-NH-and X²A compound of formula I which is-NHC (O) -a macrolide represented by formula VIIe:

with a compound of formula VI;

f) to obtain any compound L of the formula I, a leaving group L is added²Is reacted with a free carboxylic acid of a non-steroidal anti-inflammatory subunit of formula VIc, wherein formula VIIf, formula VIIg, formula vih is as follows:

wherein formula VIc is as follows:

37. a pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable salt or solvate of said compound, and a pharmaceutically acceptable diluent or carrier.

38. A method of treating inflammatory diseases, disorders and conditions characterized by or associated with an undesirable inflammatory immune response, particularly diseases and conditions induced by or associated with excessive TNF- α and IL-1 secretion, comprising administering to a subject suffering from one of said disorders or conditions a compound of claim 1.

39. A method of treating an inflammatory disorder or an immune or allergic disorder associated with leukocyte infiltration into inflamed tissue in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof.

40. The method of claim 39, wherein said condition or disorder is selected from the group consisting of asthma, adult respiratory distress syndrome, bronchitis, and cystic fibrosis.

41. The method of claim 39, wherein said inflammatory condition or disorder is selected from the group consisting of inflammatory conditions or immune disorders of the lung, joints, eyes, intestines, skin, and heart.

42. The method of claim 39, wherein said inflammatory condition or disorder is selected from the group consisting of asthma, adult respiratory distress syndrome, bronchitis, cystic fibrosis, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, uveitis, conjunctivitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, distal proctitis, psoriasis, eczema, dermatitis, coronary infarct lesions, chronic inflammation, endotoxic shock and smooth muscle proliferative disorders.

43. A method of reducing inflammation in a diseased organ or tissue, comprising delivering to said organ or tissue a therapeutically effective amount of a compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof.

44. A method of treating viral diseases, disorders, and conditions comprising administering to a subject suffering from one of said diseases or disorders an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof.

45. The method of claim 44, wherein the viral disease is HIV.

46. A method of reducing signs or symptoms or markers of a viral infection comprising administering to a subject in which said signs or symptoms or markers are present a therapeutically effective amount of a compound of claim 1.

47. A method of treating a symptom or sign or marker of a viral infection comprising administering to a subject in which said sign or symptom or marker is present a therapeutically effective amount of a compound of claim 1.

48. The method of claim 47, wherein the symptom or indication is selected from the group consisting of viral load, viral replication, viral activity, viremia, viral-specific antigens, viral RNA, viral DNA, reverse transcriptase activity, antiviral cytotoxic cell activity, and T-cell or CD4+ cell count of the subject.

49. A method of treating a symptom or indication or marker of neoplasia comprising administering to a subject in which the symptom or indication is present a therapeutically effective amount of a compound of claim 1.

50. The method of claim 49, wherein the symptom or indication of neoplasia is selected from the group consisting of tumor burden, tumor size, diseased organ weight, tumor recurrence, survival time, length or extent of subject remission, cancer cell growth, cancer cell survival, an apoptosis index, degree of metastasis or rate of metastasis, biomarkers associated with a particular type of neoplasia, proliferation markers, activation of tumor-associated oncogene regulatory disorders associated with receptor function, tumor-specific antigens, and tumor-associated angiogenesis.

51. A method of treating neoplasia comprising administering to a subject having neoplasia a therapeutically effective amount of a compound of claim 1.

52. The compound of claim 4, wherein the polypeptide is selected from the group consisting of Gly-Phe-Leu, Gly-Gly-Phe, Gly-Phe-Gly, Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Ala-Val-Ala, Gly-Gly-Phe-Leu, Gly-Phe-Leu-Gly, Gly-Phe-Ala-Leu, Ala-Leu-Ala-Leu, Gly-Phe-Phe-Leu, Gly-Leu-Leu-Gly, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, Ala-Gly-Val-Phe, and Gly-Phe-Phe-Gly.