HK1115296A - Prophylactic and therapeutic treatment of infectious and other diseases with immunoeffector compounds - Google Patents

Description

Prophylactic and therapeutic treatment of infectious and other diseases with immune effector compounds

The present application is a divisional application of the invention patent application having application number 02828435.6 and application date of 2002, 2.4.

Background

The innate immune system coordinates the inflammatory response to pathogens through a system that the subject distinguishes between self and non-self pathways, the receptors recognizing molecular species that are uniquely synthesized by microorganisms. These species are sometimes referred to as pathogen-associated molecular patterns (PAMPs) and include, for example, Lipopolysaccharide (LPS), peptidoglycan, lipotecoic acids, and Bacterial Lipoproteins (BLPs).

LPS, recognized by the innate immune system, is a massive outer cell wall component from gram-negative bacteria. Although the chemical structure of LPS has been known for some time, the molecular basis for the recognition of LPS by serum proteins and/or cells has only begun to be elucidated. In a recent series of reports, a family of receptors known as Toll-like receptors (TLRs) has been implicated in a strong innate immune response to LPS and other microbial components. TLRs are membrane proteins with a single transmembrane domain. The cytoplasmic domain is about 200 amino acids and is similar to the cytoplasmic domain of the IL-1 receptor. The extracellular domain is relatively large (about 550-980 amino acids) and may contain multiple ligand-binding sites.

The importance of TLRs in LPS immune responses has been specifically shown in at least two Toll-like receptors, Tlr2 and Tlr 4. For example, transfection studies with embryonic kidney cells have shown that human Tlr2 is sufficient to provide a response to LPS (Yang et al, Nature 395: 284-288 (1998); Kirschning et al J.exp Med.11: 2091-97 (1998)). Strong LPS reactions appear to require both LPS-binding protein (LBP) and CD14 linked to LPS with high affinity. Direct binding of LPS to Tlr2 was observed at relatively low affinity, suggesting that accessory proteins may contribute to the in vivo binding and/or activation of Tlr2 by LPS.

The importance of Tlr4 in LPS immune responses was demonstrated together with the positional cloning in LPS mutant murine strains. Two mutant alleles of the murine lps gene have been identified, a semi-dominant allele present in the C3H/HeJ strain and a second recessive allele present in the C57BL/10ScN and C57BL/10ScCr strains. Mice homozygous for the LPS mutant allele are susceptible to infection by gram-negative bacteria and are resistant to LPS-induced septic shock. The lps locus from these species was cloned and shown that this mutation altered the murine Tlr4 gene in both cases (Portorak et al, science 282: 2085-. From these reports, it can be concluded that Tlr4 is required for LPS reactions.

The biologically active endotoxin substructure of LPS is lipid-a, a phosphorylated, multiply fatty acid-acylated glucosamine disaccharide, used to immobilize the entire structure in the outer membrane of gram-negative bacteria. We have previously reported that the toxic effects of lipid A can be ameliorated by selective chemical modification of lipid A to yield monophosphoryl lipid A compounds (MPL)^®An immunostimulant; corixa; seattle, WA). The preparation and use of MPL for vaccine adjuvants and other applications has been described^®Immunostimulants, and structurally similar compounds (see, e.g., U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094; 4,987,237; Johnson et al, J Med Chem 42: 4640-,editing; plenum: new York, 495-. In particular, these and other references indicate that MPL^®Immunostimulants and related compounds have significant adjuvant activity for increasing humoral and/or cell-mediated immunity to antigens if used in vaccine formulations with proteinaceous and carbohydrate antigens.

Has been described as structurally similar to MPL^®Synthetic mono-and disaccharide molecules called Aminoalkyl Glucosaminide Phosphates (AGPs) of immunostimulants, see, e.g., u.s. patent No.6,113,918, u.s. patent No.6,303,347, and WO98/50399 (published 12/10 1998). These compounds retain significant adjuvant properties when formulated with antigens in vaccine compositions and have similar or improved toxicity profiles compared to monophosphoryl lipid a. These compounds have been described as being incorporated into vaccine formulations with antigen (U.S. patent No.6,113,918) and as single therapeutic agents in the absence of antigen, WO 01/90129 (published 11/29/2001).

Cyclic aminoalkyl glucosaminide phosphates or "cyclic AGPs" have been described in PCT patent application No. PCT/US 01/24284. These cyclic AGPs are potent immune effector molecules that increase both humoral and cell-mediated immune responses to vaccine antigens. The term "cyclic AGP" as used herein refers to an azacycloalkyl or (azacycloalkyl) alkyl glucosaminide phosphate in which 2-deoxy-2-amino-b-D-glucopyranose (glucosamine) is glycosidically linked to an azacycloalkyl or (azacycloalkyl) alkyl (aglycon) group.

The present invention provides a single therapeutic agent (monetherapies) formulated and administered in the absence of foreign antigens for the prophylactic and/or therapeutic treatment of plant and animal diseases and conditions, such as infectious diseases, autoimmunity and allergic reactions.

The monotherapeutic agents of the invention comprise one or more cyclic AGPs. These and other aspects of the invention will become apparent from the following detailed description and the accompanying drawings.

Summary of the invention

In one aspect, the present invention provides methods for treating, ameliorating, or substantially preventing a disease or condition in an animal by administering an effective amount of a compound having formula (I):

wherein X is-O-or-NH-and Y is-O-or-S-; r¹，R²And R³Are each independently (C)₂-C₂₀) Acyl groups, including saturated, unsaturated and branched acyl groups; r⁴is-H or-PO₃R⁷R⁸Wherein R is⁷And R⁸Are each independently H or (C)₁-C₄) An aliphatic group; r⁵is-H, -CH₃or-PO₃R⁹R¹⁰Wherein R is⁹And R¹⁰Are each independently selected from-H and (C)₁-C₄) An aliphatic group; r⁶Independently selected from H, OH, (C)₁-C₄) Oxyaliphatic radical, -PO₃R¹¹R¹²，-OPO₃R¹¹R¹²，-SO₃R¹¹，-OSO₃R¹¹，-NR¹¹R¹²，-SR¹¹，-CN，-NO₂，-CHO，-CO₂R¹¹and-CONR¹¹R¹²Wherein R is¹¹And R¹²Are each independently selected from H and (C)₁-C₄) An aliphatic group; provided that R is⁴And R⁵One is a phosphorus-containing group and if R is⁴is-PO₃R⁷R⁸，R⁵Is not-PO₃R⁹R¹⁰Wherein^*1-3"and^**"denotes a chiral center; wherein the subscripts n, m, p and q are each independently an integer of from 0 to 6, provided that the sum of p and m is from 0 to 6.

In some embodiments, the compounds of the present invention comprise-O-, R-on X and Y⁴Is PO₃R⁷R⁸，R⁵And R⁶Is H, and the subscripts n, m, p, and q are integers from 0 to 3. In a more preferred embodiment, R⁷And R⁸is-H. In one embodiment, subscript n is 1, subscript m is 2, and subscripts p and q are 0. In other embodiments, R¹，R²And R³Is (C)₆-C₁₄) Acyl radical, (C)₆-C₁₂) Or (C)₆-C₈) An acyl group, in a particular embodiment, provides (C)₆-C₁₂) An acyl group. Another embodiment provides that the method comprises the step of,^*1-3in the R configuration, Y is in the equivalant position, and^**is in the S configuration.

Illustrative embodiments include (N- [ (R) -3-tetradecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-tetradecanoyloxytetradecanoyl ] -beta-D-glucopyranoside and pharmaceutically acceptable salts thereof, formula (II),

n- [ (R) -3-dodecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-dodecanoyloxytetradecanoyl ] - β -D-glucopyranoside and pharmaceutically acceptable salts thereof; a compound of the formula (III),

and N- [ (R) -3-decanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino ] -3-O- [ (R) -3-decanoyloxytetradecanoyl ] - β -D-glucopyranoside and pharmaceutically acceptable salts thereof; formula (IV).

In certain illustrative aspects of the invention, the above methods are used to treat, ameliorate or substantially prevent infectious diseases, autoimmune diseases and allergic reactions.

In other aspects, the invention provides pharmaceutical compositions comprising one or more of the above compounds in a suitable excipient, formulated and/or administered in the absence of a foreign antigen.

Description of exemplary embodiments of the invention

Illustrative prophylactic and therapeutic uses

The present invention broadly relates to prophylactic and therapeutic methods for treating certain diseases and other medical conditions by administering an effective amount of one or more of the compounds described herein or a pharmaceutical composition comprising one or more of these compounds. Although certain cyclic AGP compounds have been described as useful as adjuvants in combination with foreign administered antigens in vaccine formulations, and for certain other applications, the present invention provides novel therapeutic approaches that employ these compounds preferably in monotherapy applications, i.e., in the absence of a foreign administered antigen.

In one aspect, the present invention provides a method of treating, ameliorating and/or substantially preventing an infectious disease in a eukaryotic subject, particularly an animal, preferably a human. Given the importance of TLR-mediated signaling in the innate immune response to microbial attack, the ability to selectively and with minimal toxicity stimulate these pathways provides an effective approach to a wide variety of infectious agents for prophylactic and/or therapeutic treatment modalities.

The methods described herein are applicable to virtually any kind of infectious agent, including bacteria, viruses, parasites, and fungi. Illustratively, the invention is useful for the prophylactic and/or therapeutic treatment of bacterial infections from species of the genera Pseudomonas, Escherichia, Klebsiella, Enterobacter, Proteus, Serratia, Candida, Staphylococcus, Streptococcus, Chlamydia, Mycoplasma, Bacillus, and various other genera. Illustrative viral conditions that can be treated in accordance with the present invention include, for example, those caused by influenza virus, adenovirus, parainfluenza virus, rhinovirus, Respiratory Syncytial Virus (RSVs), herpes virus, cytomegalovirus, hepatitis viruses, such as, hepatitis B virus and hepatitis C virus, and others. Illustrative fungi include, for example, Aspergillus, Candida albicans, Cryptococcus neoformans, Coccidioides immitus, and others.

In one illustrative embodiment, the invention provides methods for treating a subject, particularly an immunodeficient subject, who has suffered from or is at risk of suffering from an infection, such as a nosocomial bacterial and viral infection. About 2 million out of 4 million hospitalized individuals per year develop nosocomial infections during their hospitalization, and about 1% of them, or about 400,000 patients develop nosocomial pneumonia with over 7000 deaths. This makes nosocomial pneumonia a leading cause of death for hospital-acquired infections. Thus, this embodiment satisfies the clear need for an effective prophylactic approach to the management of nosocomial infections.

In a related embodiment, the invention provides prophylactic treatment of immunocompromised patients, such as HIV-positive patients, who have developed or may develop pneumonia from an opportunistic infection or from reactivation from a suppressed or occult infection. In 1992, an infection with Pneumocystis carinii (Pneumocystis carinii) was reported in approximately 20,000 cases of AIDS patients in the United states alone. In addition, 60-70% of all AIDS patients sometimes develop Pneumocystis carinii during their disease. Thus, the present invention in this embodiment provides an effective prophylactic method for this at risk population.

In another related embodiment, the methods of the invention are used to treat populations of patients with other immune defects and/or those that may be afflicted with an infectious disease, including, for example, patients with cystic fibrosis, chronic obstructive pulmonary disease, and other immune defects and/or with long term hospitalization.

In another embodiment, the infectious disease of the invention is a chronic infection. The chronic infection comprises chronic hepatitis, human papilloma virus, oral or vaginal candidiasis, periodontal disease or chronic sinusitis due to fungal colonization.

In another aspect of the invention, the compounds described herein are used in a method of treating, ameliorating or substantially preventing allergic conditions and symptoms, such as sinusitis, chronic rhinosinusitis, asthma, atopic dermatitis, and psoriasis. This protocol is based, at least in part, on the ability of these compounds to activate cytokine production by target cells, and can compete with a stereotyped allergic cytokine response characterized by IL-4 production or hyper-responsiveness to IL-4 activity. Administration of certain compounds disclosed herein results in IFN- γ and IL-12 expression from antigen processing and presenting cells, as well as other cells, resulting in down-regulation of cytokines associated with allergic reactions, such as IL-4, 5, 6, 10 and 13.

In another aspect of the invention, these compounds are used in methods of treating autoimmune diseases and conditions. The compounds for use in this embodiment are typically selected from those capable of antagonizing, inhibiting or otherwise negatively modulating one or more Toll-like receptors, particularly Tlr2 and/or Tlr4, such that the autoimmune response associated with a given condition is ameliorated or substantially prevented. Illustratively, the methods provided by this embodiment can be used to treat conditions such as inflammatory bowel disease, rheumatoid arthritis, chronic arthritis, multiple sclerosis, and psoriasis.

While not wishing to be bound by theory, it is believed that the efficacy of the prophylactic and therapeutic uses described above is based, at least in part, on the involvement of these compounds in the modulation of Toll-like receptor activity. In particular, Toll-like receptors Tlr2, Tlr4, and the like are believed to be specifically activated, competitively inhibited, or otherwise affected by non-toxic LPS derivatives and mimetics disclosed herein. Thus, the methods of the present invention provide an effective and selective pathway for modulating the innate immune response pathway in animals without the toxicity often associated with natural bacterial components that normally stimulate such pathways.

Exemplary cyclic AGP compounds

Illustrative compounds for the above prophylactic and therapeutic uses include compounds having the formula I:

and pharmaceutically acceptable salts thereof, wherein X is-O-or-NH-and Y is-O-or-S-; r¹，R²And R³Are each independently (C)₂-C₂₀) Acyl groups, including saturated, unsaturated and branched acyl groups; r⁴is-H or-PO₃R⁷R⁸Wherein R is⁷And R⁸Are each independently H or (C)₁-C₄) An aliphatic group; r⁵is-H, -CH₃or-PO₃R⁹R¹⁰Wherein R is⁹And R¹⁰Are each independently selected from-H and (C)₁-C₄) An aliphatic group; r⁶Independently selected from H, OH, (C)₁-C₄) Oxyaliphatic radical, -PO₃R¹¹R¹²、-OPO₃R¹¹R¹²、-SO₃R¹¹、-OSO₃R¹¹、-NR¹¹R¹²、-SR¹¹、-CN、-NO₂、-CHO、-CO₂R¹¹and-CONR¹¹R¹²Wherein R is¹²And R¹²Are each independently selected from H and (C)₁-C₄) An aliphatic group; provided that R is⁴And R⁵One is a phosphorus-containing group and when R is⁴is-PO₃R⁷R⁸When R is⁵Is not-PO₃R⁹R¹⁰Wherein^*1-3"and^**"denotes a chiral center; wherein each of the subscripts n, m, p and q is independently an integer of from 0 to 6, provided that the sum of p and m is from 0 to 6.

Although the hexopyranoside in formula I is given in the glucose configuration, other glycosides are also within the scope of the invention. For example, glucopyranosides, including other hexopyranosides (e.g., allose, altrose, mannoside, gulose, idoside, galactosides, talose) are within the scope of the invention.

In the above general formula, the symbol^*1″，″^*2"and^*3"the configuration of the 3' -stereocenters (stereogenic centers) to which normal fatty acyl residues are attached is R or S, but R is preferred. To which R is directly or indirectly attached⁶And the carbon atom of the cyclic aglycone unit of the glucosamine unit (marked ″)^**") may be R or S. In the above formula, Y may be in a horizontal or axial position, but is preferably horizontal. All stereoisomers, enantiomers, diastereomers and mixtures thereof are contemplated as being within the scope of the present invention.

In an illustrative embodiment of the invention, X and Y are-O-, R⁴Is phosphono, R⁵And R⁶Is H, and subscripts n, m, p, and q are integers from 0 to 3, and more preferably from 0 to 2. In an exemplary embodiment, the integer n is 1, the integer m is 2, and the integers p and q are 0. In this embodiment, the compounds of the invention are 2-pyrrolidinylmethyl β -D-glucosaminide 4-phosphate having the general formula (V):

in another illustrative embodiment of the invention, R of formula (III)¹，R²And R³Is a tetradecanoyl residue and the 3' -stereo to which they are attachedConfiguration of the center () "^*1-3") is R, Y is in the horizontal position, and the pyrrolidine stereocenter (")^**") is S.

Other exemplary embodiments include N- [ (R) -3-tetradecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-tetradecanoyloxytetradecanoyl ] -beta-D-glucopyranoside, and pharmaceutically acceptable salts thereof, formula (II):

n- [ (R) -3-dodecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-dodecanoyloxytetradecanoyl ] - β -D-glucopyranoside and pharmaceutically acceptable salts thereof; in the formula (III), the compound is shown in the formula,

and N- [ (R) -3-decanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino ] -3-O- [ (R) -3-decanoyloxytetradecanoyl ] -beta-D-glucopyranoside and pharmaceutically acceptable salts thereof; formula IV.

The compounds of the invention can be used as described in Johnson et al, bioorg.med.chem.lett.9: 2273(1999) and PCT/WO98/50399 and the references therein. In general, the synthetic methods described in the above references can be widely used to prepare compounds having different acyl groups and subclasses. For example, certain compounds useful in the present invention are described in U.S. provisional application No.60/223,056 and international application PCT/US 01/24284. In general, the synthetic methods described in the above-referenced documents herein, and other synthetic methods otherwise well known in the art, are widely applicable for the preparation of these compounds. For example, in the manufacture of compounds having different acyl groups and substituents, those skilled in the art will appreciate that the various methods described therein can be altered to use alternative acylating agents, or can be initiated with commercially available materials having suitable acyl groups attached.

The term "acyl" refers to those groups derived from aliphatic organic acids by removal of the hydroxyl portion of the acid. Thus, acyl is meant to include, for example, acetyl, propionyl, butyryl, decanoyl, and pivaloyl.

″(C₂-C₂₀) Acyl "is an acyl group having 2 to 20 carbons. Similarly, (C)₆-C₁₄)、(C₆-C₁₂)、(C₉-C₁₂) And (C)₆-C₈) Acyl is an acyl group having 6 to 14 carbons, 6 to 12 carbons, 9 to 12 carbons, and 6 to 8 carbons, respectively. The term "acyl" also includes such groups having typical substituents such as hydroxyl, ketone, and the like.

Unless otherwise indicated, the term "aliphatic" by itself or as part of another substituent means a straight or branched chain or cyclic hydrocarbon moiety, including moieties containing both cyclic and chain members, which may be fully saturated or mono-or polyunsaturated, having the specified number of carbon atoms (i.e., C)₁-C₄Meaning 1-4 carbons). Examples of the saturated hydrocarbon group include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclopropyl, cyclopropylmethyl, methylene, ethylene, and n-butylene. Unsaturated alkyl groups are groups having one or more double and/or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 1-propynyl and 2- (butadienyl).

The term "oxyaliphatic" refers to those groups having an aliphatic group attached to the remainder of the molecule through an oxygen atom.

The above terms (e.g., "aliphatic", "acyl") are meant to include both substituted and unsubstituted forms of the indicated moiety, respectively. Preferred substituents for each group are given below.

Substituents for aliphatic groups may be various groups selected from: -OR ', - (O), (S), (NR ', - (N-OR ', - (NR ' R ', - (SR ',), - (S), -NR ' R ', - (NR ' R *, -oc (O) R ', - (c) (O) R ', - (CO) R ', - (O) R ', (S '), - (S) (-R ', (S) (-R ') R ', (S) (-c) R ', (S), (c) R ', (S), (₂R′、-CONR′R″、-OC(O)NR′R″、-NR″C(O)R′、-NR′-C(O)NR″R*、-NR″C(O)₂R′、-NH-C(NH₂)＝NH、-NR′C(NH₂)＝NH、-NH-C(NH₂)＝NR′、-S(O)R′、-S(O)₂R′、-S(O)₂NR' R ", -CN and-NO₂The number is zero to (2m '+ 1), where m' is the total number of carbon atoms in these groups. R ', R' and R * each independently mean hydrogen and unsubstituted (C)₁-C₈) An aliphatic group. If R 'and R' are attached to the same nitrogen atom, they may be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR' R "is meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will appreciate that the term "aliphatic" is meant to include, for example, haloalkyl (e.g., -CF)₃and-CH₂CF₃) Such groups and the like.

Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. In the compounds having halogen substituents, the halogens may be the same or different.

The term "pharmaceutically acceptable salt" is meant to include salts of the active compounds prepared using relatively non-toxic acids or bases, depending on the particular substituents on the compounds described herein. If the compounds of the invention contain relatively acidic functionalities, base addition salts can be obtained by adding the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. If the compounds of the invention contain relatively basic functionalities, acid addition salts may be obtained by adding the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydriodic acid, or phosphorous acid and the like, as well as salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts and the like, and salts and the like of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge, s.m. et al, "drug salts", Journal of Pharmaceutical Science (Journal of Pharmaceutical Science), 1977, 66, 1-19). Certain specific compounds of the present invention comprise both basic and acidic functionalities, enabling the compounds to be converted into base or acid addition salts.

The neutral form of these compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent forms of the compounds differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are identical to the parent forms of the compounds for purposes of the present invention.

In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Alternatively, prodrugs can be converted to the compounds of the present invention by chemical or biochemical means in an ex vivo environment. For example, a prodrug can be slowly converted to a compound of the invention when placed in a transdermal patch (transdermal patchreservoir) by a suitable enzyme or chemical reagent.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms (including hydrated forms). In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses to which the invention pertains and are intended to be within the scope of the invention.

Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers are included within the scope of the present invention.

The compounds of the present invention may also contain an abnormal proportion of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be substituted with radioactive isotopes, such as tritium(s) ((iii))³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) And performing radioactive labeling. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

Illustrative pharmaceutical compositions and delivery thereof

In another embodiment, the invention relates to pharmaceutical compositions comprising one or more compounds of the invention formulated and administered in the absence of foreign antigens, i.e., in combination with pharmaceutically-acceptable carriers or excipients, for monotherapy applications. These pharmaceutical compositions can be used alone, or in combination with one or more other treatment regimens for administration to cells, tissues, animals or plants. In many of these embodiments, the pharmaceutical compositions of the present invention comprise one or more compounds described herein.

The phrase "pharmaceutically acceptable" refers to molecular bodies and compositions that do not produce allergic or similar untoward reactions when administered to a human. As used herein, "carrier" or "excipient" includes any and all solvents, dispersion media, carriers, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

Illustrative carriers for formulating pharmaceutical compositions include, for example, water-in-oil or oil-in-water emulsions, aqueous compositions with or without organic co-solvents suitable for Intravenous (IV) use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microparticles, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.

In certain embodiments, the pharmaceutical composition comprises one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents and/or preservatives.

For some applications, aqueous formulations are preferred, especially those comprising an effective amount of one or more surfactants. For example, the composition may be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacylphosphatidylglycerols, such as Dimyristoylphosphatidylglycerol (DPMG), Dipalmitoylphosphatidylglycerol (DPPG), and Distearoylphosphatidylglycerol (DSPG), diacylphosphatidylcholines, such as Dimyristoylphosphatidylcholine (DPMC), Dipalmitoylphosphatidylcholine (DPPC), and Distearoylphosphatidylcholine (DSPC); diacylphosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacylphosphatidylethanolamines such as Dimyristoylphosphatidylethanolamine (DPME), Dipalmitoylphosphatidylethanolamine (DPPE), and Distearoylphosphatidylethanolamine (DSPE). Typically, the surfactant in the aqueous formulation: the mono-/disaccharide molar ratio is from about 10: 1 to about 1: 10, more typically from about 5: 1 to about 1: 5, but any effective amount of surfactant can be used in the aqueous formulation to best suit the particular target of interest.

As used herein, an "effective amount" is an amount that exhibits a response that is greater than that of the vehicle or negative control. As discussed above, the precise dose of a compound of the present invention administered to a patient will depend on the route of administration, the pharmaceutical composition, and the patient.

The compounds and pharmaceutical compositions of the present invention can be formulated for essentially any route of administration, e.g., injection, inhalation by the oral or intranasal route, rectal, vaginal or intratracheal instillation, ingestion, or transdermal or transmucosal route, and the like. Thus, the therapeutic effects obtainable by the methods and compositions of the present invention may be, for example, systemic, local, tissue-specific, etc., depending on the particular requirements of a given context of the invention.

Illustrative formulations may be formulated and administered parenterally, i.e., intraperitoneally, subcutaneously, intramuscularly, or intravenously. An illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP water for injection (WFI). Other illustrative carriers include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion. Pharmaceutically acceptable parenteral solvents are generally selected such that they provide a solution or dispersion that can be filtered through a 0.22 micron filter without removal of the active ingredient.

Illustrative examples of carriers for subcutaneous or intramuscular use include 1-2 or 1-4 mixtures of Phosphate Buffered Saline (PBS) solution, 5% glucose (in WFI) and 0.01-0.1% triethanolamine (in 5% glucose) or 0.9% sodium chloride (in USP WFI), or 10% USP ethanol, 40% propylene glycol and the balance acceptable isotonic solution such as 5% glucose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine (in USP WFI) and 1-10% squalene or a parenteral vegetable oil-in-water emulsion.

Examples of carriers for administration via mucosal surfaces depend on the particular route, e.g., oral, sublingual, intranasal, etc. If administered orally, illustrative examples include pharmaceutical grades of mannitol, starch, lactose, magnesium stearate, Sodium saccharin (Sodium saccharate), cellulose, magnesium carbonate, and analogs thereof, with mannitol being preferred. If administered intranasally, illustrative examples include powder suspensions of polyethylene glycols, phospholipids, glycols and glycolipids, sucrose, and/or methylcellulose, with or without bulking agents such as lactose and preservatives such as benzalkonium chloride, EDTA. In a particularly illustrative embodiment, the phospholipid 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is used as an about 0.01-0.2% isotonic aqueous carrier for intranasal administration of a compound of the invention at a concentration of about 0.1-3.0 mg/ml.

If administered by inhalation, illustrative carriers include polyethylene glycol or glycols, DPPC, methylcellulose, powdered dispersions, and preservatives, with polyethylene glycol and DPPC being preferred. In many cases, the compound is preferably in aerosolized form when administered by inhalation. Illustratively, delivery may be by use of a disposable delivery device, a nebulizer, a breath activated powder inhaler, an aerosol Metered Dose Inhaler (MDI), or any other of the many nebulizer delivery devices available in the art. Alternatively, a stopcock (mist tent) may be used or administered directly through an endotracheal tube. Delivery via intratracheal or nasopharyngeal modes is effective for some conditions.

Those skilled in the art will recognize that the foregoing description is illustrative and not exhaustive. Indeed, many other formulation techniques and pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as are studies of appropriate dosing and treatment regimens for use of the particular compositions described herein in various treatment regimens.

The compounds may be evaluated in a variety of assay formats, including those described herein, to identify and select those having the properties most suitable for a given application of the invention. For example, animal models can be used to confirm and evaluate the cytokine release profile in the systemic circulation following administration of a cyclic AGP compound. In addition, there are a variety of in vitro and in vivo models for examining the immune response to different antigenic components for changes in one or more aspects, thus identifying the compounds most useful for eliciting a particular immune response of interest. For example, the compounds can be contacted with target cells, such as macrophages, dendritic cells or Langerhans cells, in vitro, and the cytokines produced can be measured. In addition, gene expression arrays can be used to identify specific pathways that are activated or inhibited by a particular cyclic AGP of interest.

It is understood that, if desired, the compounds disclosed herein can be administered in conjunction with other therapeutic regimens, such as antimicrobial, antiviral, and antifungal compounds or agents, various DNA-based therapeutic agents, RNA-based therapeutic agents, polypeptide-based therapeutic agents, and/or with other immune effectors. In fact, essentially any other component can be included as long as the other component does not cause significant side effects when in contact with the target cell or host tissue. The composition may thus be delivered with various other agents as needed or desired for the particular embodiment of the invention being practiced.

Illustratively, the pharmaceutical compositions of the invention may include or be used in conjunction with DNA encoding one or more therapeutic proteins, antisense RNAs, ribozymes or the like. The DNA may be present in any of a variety of delivery systems known to those of ordinary skill in the art including nucleic acid expression systems, bacterial and viral expression systems. Many gene delivery technologies are well known in the art, such as, for example, Rolland, crit. Rev. therapy. drug Carriers Systems 15: 143, 198, 1998, and those described in the references cited therein. Suitable nucleic acid expression systems include DNA sequences (e.g., suitable promoters and termination signals) required for expression in a patient. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), typically involving the use of a nonpathogenic (defective) replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al, proc.natl.acad.sci.usa86: 317 and 321, 1989; flexner et al, ann.n.y.acad.sci.569: 86-103, 1989; flexner et al, vaccine 8: 17-21, 1990; U.S. patent nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. patent nos. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; berkner, biotechnology 6: 616-627, 1988; rosenfeld et al, science 252: 431-434, 1991; kolls et al, proc.natl.acad.sci.usa 91: 215-; Kass-Eisler et al, proc.natl.acad.sci.usa90: 11498. 11502, 1993; guzman et al, cycle 88: 2838 2848, 1993; and Guzman et al, cir. Res.73: 1202-1207, 1993 techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art.

DNA may also be "naked," for example as described by Ulmer et al, science 259: 1745. cndot. 1749, 1993 and published by Cohen, science 259: 1691 1692, 1993. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently delivered into the cells. It is clear that the pharmaceutical composition of the invention may comprise both a polynucleotide and a protein component.

Any of a variety of other immunostimulants may be included in the compositions of the present invention. For example, cytokines such as GM-CSF, interferons or interleukins further modulate the immune response of interest. For example, in certain embodiments, additional components may be included in the composition to further increase the induction of high levels of Th 1-type cytokines (e.g., IFN-. gamma., TNF. alpha., IL-2, and IL-12). In addition, or in addition thereto, high levels of Th 2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) may be desirable in certain therapeutic settings. The levels of these cytokines can be readily assessed using standard assays. For a review of the cytokine family, see Mosmann and Coffman, ann.rev.immunol.7: 145-173, 1989.

Illustrative compositions for inducing Th 1-type cytokines include, for example, combinations of CpG-containing oligonucleotides (wherein the CpG dinucleotide is unmethylated) as described in WO96/02555, WO 99/33488 and U.S. patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, in Sato et al, science 273: 352, 1996. Other suitable immunostimulants include saponins such as QS21(Aquila biopharmaceuticals Inc., Framingham, MA), GPI-100(Marciani et al, vaccine 18: 3141, 2000, U.S. Pat. No.6,080,725) and related saponin derivatives and mimetics thereof.

Other illustrative immunostimulants that may be used in conjunction with the present invention include Montanide ISA720(Seppic, France), SAF (Chiron, California, USA), ISCOMS (CSL), MF-59(Chiron), SBAS series adjuvants (e.g., SBAS-2 or SBAS-4, available from Smith KlineBeecham, Rixendart, Belgium), and Enhanzyn^TMImmunostimulants (Corixa, Hamilton, MT). Polyoxyethylene ether immunostimulants are described in WO99/52549A 1.

The invention is further illustrated by the following non-limiting examples.

Examples

Example 1

Preparation of N- [ (R) -3-tetradecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-tetradecanoyloxytetradecanoyl ] -beta-D-glucopyranoside triethylammonium salt: triethylammonium salt of a compound having formula (II)

(1a) To 2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl group]A solution of-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) - β -D-glucopyranosyl bromide (1.05g, 0.81mmol) in dry 1, 2-dichloroethane (10mL) was added 4 Å molecular sieves (0.5g), dry CaSO₄(2.2g, 16mmol), and N- [ (R) -3-tetradecanoyloxytetradecanoyl group]- (S) -2-pyrrolidinemethanol (0.40g, 0.75 mmol). The resulting mixture was stirred at room temperature 1h, using Hg (CN)₂(1.02g, 4.05mmol) and heated to reflux in the dark for 16 h. The cooled reaction mixture is treated with CH₂Cl₂Diluted and filtered. The filtrate was washed with 1N aq KI and dried (Na)₂SO₄) And concentrating. Flash chromatography on silica gel (gradient elution, 15 → 20% EtOAc/hexanes) afforded 0.605g (43%) of N- [ (R) -3-tetradecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl]-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) - β -D-glucopyranoside, an amorphous solid.

(1b) A solution of the compound prepared in (1a) above (0.50g, 0.29mmol) in AGOH (10mL) was treated with zinc powder (0.98g, 15mmol) in three equal portions over 1-h at 60 ℃. The cooled reaction mixture was sonicated, filtered through a pad of Celite, and concentrated. The residue obtained is in CH₂Cl₂And saturated aq NaHCO₃Divided among them and layered. The organic layer was dried (Na)₂SO₄) And concentrating. The resulting crude amino alcohol and (R) -3-tetradecanoyloxytetradecanoic acid (0.155g, 0.34mmol) in CH₂Cl₂(3.5mL) was stirred with powdered 4 Å molecular sieve (0.25g) for 0.5h and then treated with 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (0.11g, 0.44 mmol). The resulting mixture was stirred at room temperature for 8h, filtered through Celite, and concentrated. Flash chromatography on silica gel with 50% EtOAc/hexanes provided 0.355g (68%) of N- [ (R) -3-tetradecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-2- [ (R) -3-tetradecanoyloxytetradecanoylamino]-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl group]- β -D-glucopyranoside, a colorless syrup.

(1c) A solution of the compound prepared in (1b) above (0.300g, 0.166mmol) in a mixture of AcOH (1mL) and tetrahydrofuran (9mL) in PtO₂(0.15g) was hydrogenated in the presence of hydrogen at room temperature and 70psig for 18 h. The reaction mixture was diluted with 2: 1CHCl₃MeOH (50mL) dilution and simple sonication. CollectingCatalyst and 2: 1CHCl₃MeOH wash, then concentrate the combined filtrate and wash. Use of CHCl on silica gel₃-MeOH-H₂O-Et₃Flash chromatography on N (90: 10: 0.5) gave the partially purified product, which was then dissolved in ice-cold 2: 1CHCl₃MeOH (30mL) and ice-cold 0.1N aq HCl (12 mL). The organic phase was filtered and extracted from 2% aq Et₃Freeze-drying in N (5mL, pyrogen-free) gave 0.228g (79%) of N- [ (R) -3-tetradecanoyloxytetradecanoyl group]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino]-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl group]- β -D-glucopyranoside triethylammonium salt, a colorless powder:

mp 67-70 ℃; IR 3306, 2955, 2923, 2853, 1736, 1732, 1644, 1548, 1466, 1378, 1245, 1177, 1110, 1053, 844cm^-1；¹H NMR(CDCl₃-CD₃OD)δ0.88(m，18H)，1.0-1.2.05(mH)，2.20-2.70(m，12H)，3.06(q，6H，J＝7.2Hz)，3.3-325(mH)，4.52(d，1H，J＝8Hz)，5.05-5.28(m，4H)，7.44(d，1H，J＝9Hz)；¹³C NMR(CDCl₃)δ173.3，173.0，170.3，169.6，168.6，101.8，100.4，75.8，72.5，72.4，70.9，70.8，70.3，70.2，69.9，69.3，67.9，66.6，56.5，56.3，54.5，47.4，45.8，44.6，41.4，41.0，39.7，39.2，39.0，34.5，34.3，34.1，32.0，29.7，29.4，28.1，27.3，25.7，25.3，25.2，25.1，24.0，22.7，21.6，14.1，8.6.

Analytical calculation C₁₀₁H₁₉₄N₃O₁₇P·H₂O: c, 68.47; h, 11.15; n, 2.37; p, 1.75. found: c, 68.79; h, 11.00; n, 2.24; p, 1.97.

Example 2:

preparation of N- [ (R) -3-dodecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-dodecanoyloxytetradecanoyl ] - β -D-glucopyranoside triethylammonium salt); triethylammonium salts of formula (III).

(2a) To 2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]A solution of-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) -alpha-D-glucopyranosyl bromide (1.60g, 1.27mmol) in dry 1, 2-dichloroethane (3.2mL) was added 4 Å molecular sieves (0.6g), dry CaSO₄(1.0g, 7.3mmol), and N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinemethanol (0.58g, 1.14 mmol). The resulting mixture was stirred at room temperature for 1h with Hg (CN)₂(0.58g, 2.3mmol) and heated to reflux in the dark for 6 h. The cooled reaction mixture is treated with CH₂Cl₂Diluted and filtered through celite bed. The filtrate was washed with 1N aq KI and dried (Na)₂SO₄) And concentrating. Flash chromatography on silica gel (gradient elution, 25 → 35% EtOAc/hexane) afforded 1.72g (82%) of N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) - β -D-glucopyranoside, a colorless oil.

(2b) A solution of the compound prepared in (2a) above (1.58g, 0.806mmol) in AcOH (40mL) was treated with zinc powder (2.6g, 40mmol) in three equal portions over 1 hour at 60 ℃. The cooled reaction mixture was sonicated, filtered through a pad of Celite, and concentrated. The resulting residue was purified in EtOAc and saturated aq NaHCO₃Divided and layered in between. The organic layer was washed with brine and dried (Na)₂SO₄) And concentrated to give 1.3g of a white solid. The resulting crude amino alcohol and (R) -3-dodecanoyloxytetradecanoic acid (0.45g, 1.05mmol) in CH₂Cl₂(20mL) was treated with 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (0.30g, 1.21 mmol). The resulting mixture was stirred at room temperature for 18h and concentrated. On silica gel with 40 → 50% EtOAcHexane flash chromatography gave 0.89g (56%) of N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-2- [ (R) -3-dodecanoyloxytetradecanoylamino]-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]-beta-D-glucopyranoside, a white foam material.

(2c) A solution of the compound prepared in (2b) above (0.75g, 0.44mmol) in a mixture of AcOH (4.5mL) and tetrahydrofuran (45mL) in PtO₂(0.45g) was hydrogenated in the presence of hydrogen at room temperature and 70psig for 18 h. The reaction mixture was diluted with 2: 1CHCl₃MeOH (35mL) dilution and simple sonication. The catalyst was collected and used in 2: 1CHCl₃MeOH wash, then concentrate the combined filtrate and wash. On silica gel with CHCl₃-MeOH-H₂O-Et₃N (gradient elution; 96: 4: 0.3 → 90: 10: 0.5) was subjected to flash chromatography to give a partially purified product (0.51g), which was dissolved in ice-cold 2: 1CHCl₃MeOH (50mL) and washed with ice cold 0.1Naq HCl (20 mL). The organic phase was filtered and concentrated. The resulting white wax was washed from 2% aqEt₃Freeze-drying in N (70mL, pyrogen-free) gave 0.54g (78%) of N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinomethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxy tetradecanoylamino]-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]- β -D-glucopyranoside triethylammonium salt, a white powder:

mp 146-; IR (film) 3292, 3100, 2958, 2922, 2852, 1739, 1731, 1659, 1651, 1644, 1562, 1555, 1468, 1455, 1433, 1377, 1339, 1310, 1253, 1238, 1183, 1160, 1107, 1080, 1047, 960, 856, 722cm^-1；¹H NMR(CDCl₃-CD₃OD)δ0.88(m，18H)，1.0-2.10(mH)，2.20-2.75(m，12H)，3.04(q，6H，J＝7.2Hz)，3.3-4.3(mH)，4.45(d，1H，J＝8.5Hz)，5.0-5.28(m，4H)；¹³C NMR(CDCl₃)δ173.9，173.4，173.2，170.6，170.1，169.2，101.4，75.5，74.0，70.8，70.7，70.2，68.5，60.5，56.6，53.6，47.4，45.6，40.9，39.6，38.8，34.5，34.3，34.2，34.1，31.9，29.7，29.6，29.5，29.4，29.4，29.3，29.2，27.3，25.2，25.0，23.6，22.7，21.6，14.0，8.3.

MALDI-MS calculated value [ M + Na ]]⁺1590.1900, found 1590.1866; analytical calculation C₉₅H₁₈₂N₃O₁₇P·3H₂O: c, 66.20; h, 10.99; n, 2.44. found: c, 66.36; h, 10.69; and N, 2.15.

Example 3:

preparing N- [ (R) -3-decanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino ] -3-O- [ (R) -3-decanoyloxytetradecanoyl ] -beta-D-glucopyranoside triethylammonium salt; triethylammonium salts of formula (IV).

(3a) To 2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-decanoyloxytetradecanoyl group]A solution of-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) -alpha-D-glucopyranosyl bromide (1.70g, 1.38mmol) in dry 1, 2-dichloroethane (3.5mL) was added 4 Å molecular sieves (0.6g), dry CaSO₄(1.2g, 8.8mmol), and N- [ (R) -3-decanoyloxytetradecanoyl group]- (S) -2-pyrrolidinemethanol (0.60g, 1.24 mmol). The resulting mixture was stirred at room temperature for 1h with Hg (CN)₂(0.63g, 2.5mmol) and heated to reflux in the dark for 6 h. The cooled reaction mixture is treated with CH₂Cl₂Diluted and filtered through celite bed. The filtrate was washed with 1N aq KI and dried (Na)₂SO₄) And concentrating. Flash chromatography on silica gel (gradient elution, 25 → 40% EtOAc/hexanes) afforded 1.82g (80%) of N- [ (R) -3-decanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-decanoyloxytetradecanoyl]-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) - β -D-glucopyranoside, a colorless oil.

(3b) A solution of the compound prepared in (3a) above (1.67g, 1.02mmol) in AcOH (50mL) was treated with zinc powder (3.33g, 51mmol) in three equal portions at 60 ℃ for 1 h. The cooled reaction mixture was sonicated, filtered through a pad of Celite, and concentrated. The resulting residue was purified in EtOAc and saturated aq NaHCO₃Divided and layered in between. The organic layer was washed with brine and dried (Na)₂SO₄) And concentrated to give 1.25g of a white solid. The resulting crude amino alcohol and (R) -3-decanoyloxytetradecanoic acid (0.53g, 1.33mmol) were added to CH₂Cl₂(20mL) was treated with 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (0.38g, 1.53 mmol). The resulting mixture was stirred at room temperature for 18h and concentrated. Flash chromatography on silica gel with 40 → 50% EtOAc/hexanes afforded 1.23g (74%) of N- [ (R) -3-decanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-2- [ (R) -3-decanoyloxytetradecanoylamino]-3-O- [ (R) -3-decanoyloxytetradecanoyl group]-beta-D-glucopyranoside, a white foam material.

(3c) A solution of the compound prepared in (3b) above (1.07g, 0.654mmol) in a mixture of AcOH (6.5mL) and tetrahydrofuran (65mL) in PtO₂(0.66g) was hydrogenated in the presence of hydrogen at room temperature and 70psig for 18 h. The reaction mixture was diluted with 2: 1CHCl₃MeOH (50mL) dilution and simple sonication. The catalyst was collected and used in 2: 1CHCl₃MeOH wash, then concentrate the combined filtrate and wash. The resulting waxy solid was lyophilized from 2% aq triethylamine to give about 1g of crude triethylammonium salt as a white powder. On silica gel with CHCl₃-MeOH-H₂O-Et₃Flash chromatography of N (gradient elution; 96: 4: 0.3 → 88: 12: 1: 0.6) afforded the partially purified product (0.84g), which was dissolved in ice-cold 2: 1CHCl₃MeOH (168mL) and ice-cold 0.1N aq HCl (67 mL). The organic phase was filtered and concentrated. The resulting white wax (about 0.7g) was removed from 2% aq Et₃Freeze-drying in N (70mL, pyrogen-free) gave 0.79g (79%) of N- [ (R) -3-decanoyloxytetradecanoyl group]- (S) -2-pyrrolidinomethyl-2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino]-3-O- [ (R) -3-decanoyloxytetradecanoyl group]- β -D-glucopyranoside triethylammonium salt, a white powder:

mp 121-122 ℃; IR (film) 3287, 3093, 2961, 2913, 2850, 1745, 1738, 1732, 1716, 1666, 1660, 1651, 1644, 1635, 1565, 1556, 1538, 1470, 1455, 1434, 1416, 1378, 1337, 1311, 1248, 1184, 1104, 1081, 1021, 964, 721cm^-1；¹H NMR(CDCl₃-CD₃OD)δ0.88(m，18H)，1.0-2.05(mH)，2.20-2.75(m，12H)，3.04(q，6H，J＝7.2Hz)，3.3-4.3(mH)，4.45(d，1H，J＝8.5Hz)，5.0-5.28(m，4H)；¹³C NMR(CDCl₃)δ173.7，173.4，173.2，170.5，170.1，169.1，101.4，75.6，74.0，70.8，70.2，68.7，60.4，56.6，53.8，47.4，45.6，41.0，39.6，38.9，34.5，34.3，34.2，34.1，31.9，29.7，29.6，29.5，29.4，29.4，29.3，29.2，27.3，25.3，25.0，23.7，22.7，21.6，14.1，8.4.

MALDI-MS calculated value [ M + Na ]]⁺1506.0961, found 1506.1008; analytical calculation C₈₉H₁₇₀N₃O₁₇P: c, 67.43; h, 10.81; n, 2.65. found: c, 67.26; h, 10.85; and N, 2.47.

Example 4

Monocyte hyperplasia listeria attack mouse model

This example provides an experiment to evaluate the induction of non-specific resistance when a mouse model is challenged with Listeria monocytogenes using the compounds prepared in examples 1, 2 and 3. Mice (5/group) were treated intravenously with 1 μ g of cyclic AGP or MPL dissolved in 0.2% Triethanolamine (TEOA). Two days later, the mice used about 10⁵Listeria monocytogenes serotype 10403 (stock culture provided by JoryBaldridge, university of washington, Pullman, WA.) was challenged intravenously. Two days after challenge, mice were sacrificed and each assayed by applying 10-fold serial dilutions of spleen homogenate onto tryptic soy agar platesNumber of Colony Forming Units (CFUs) in spleen of individual mice. The degree of protection afforded by a given AGP or MPL was calculated by subtracting the mean of bacteria/spleen (log10 values) in the group of mice treated with a given compound from the mean of bacteria/spleen (log10 values) in the control group "sham" treated with vehicle (0.2% TEOA) prior to challenge with listeria monocytogenes.

Of the compounds tested, the compound of example 3 was most active, inducing a level of protection equivalent to MPL (approximately 0.9 log10 units). The compound of example 2 induced a slightly lower level of protection, and the compound of example 1 was the least protective (0.7 and 0.2 log units, respectively).

Example 5

Prevention of lethal influenza challenge by prophylactic administration of cyclic AGPs

This example provides an experiment to evaluate the protective effect of cyclic AGP-treated mice against lethal influenza challenge. BALB/c mice (10 mice/group) were treated intranasally with 20 μ g of the compound of examples 1, 2 and 3, or with MPL 48 hours prior to a lethal intranasal challenge with influenza A/HK/68(5 LD 50). Protection was assessed by survival 21 days after challenge, observation of clinical symptoms (ruffles, arched posture and dyspnea), and prevention of weight loss.

As seen from the listeria model, the compounds of examples 2 and 3 provided enhanced protection compared to the vehicle control. Mice treated with the compound of example 3 had 60% survival; the survival rate was 40% for the treatment with the compound of example 2 and 30% for the treatment with MPL. Mice treated with the compound of example 1 did not survive. These data indicate that the compound of example 3 provides excellent protection, followed by the compounds of examples 2 and 1.

All publications and patent applications cited in this specification are herein incorporated by reference in their entirety. Although the foregoing invention has been described in some detail for purposes of illustration and for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (43)

1. Use of one or more compounds having the formula:

wherein X is selected from-O-and-NH-;

y is selected from-O-and-S-;

R¹，R²and R³Are each independently (C)₂-C₂₀) An acyl group;

R⁴selected from-H and-PO₃R⁷R⁸Wherein R is⁷And R⁸Are each independently selected from-H and (C)₁-C₄) An aliphatic group;

R⁵is selected from-H, -CH₃and-PO₃R⁹R¹⁰Wherein R is⁹And R¹⁰Are each independently selected from-H and (C)₁-C₄) An aliphatic group;

R⁶is selected from H, OH, (C)₁-C₄) Oxyaliphatic radical, -PO₃R¹¹R¹²，-OPO₃R¹¹R¹²，-SO₃R¹¹，-OSO₃R¹¹，-NR¹¹R¹²，-SR¹¹，-CN，-NO₂，-CHO，-CO₂R¹¹and-CONR¹¹R¹²Wherein R is¹¹And R¹²Are each independently selected from H and (C)₁-C₄) An aliphatic radical, provided that R⁴And R⁵One is a phosphorus-containing group and when R is⁴is-PO₃R⁷R⁸When R is⁵Is not-PO₃R⁹R¹⁰；

Wherein^*1″，″^*2″，″^*3"and^**"denotes a chiral center;

wherein n is 1, m is 2, and p and q are 0.

2. The use of claim 1, wherein X and Y are-O-, R⁴Is PO₃R⁷R⁸，R⁵And R⁶Is H.

3. The use of claim 2, wherein R⁷And R⁸is-H.

4. The use of claim 1, wherein R¹，R²And R³Are respectively C₆-C₁₄An acyl group.

5. The use of claim 1, wherein R¹，R²And R³Are respectively C₆-C₁₂An acyl group.

6. The use of claim 1, wherein R¹，R²And R³Are decanoyl residues, respectively.

7. The use of claim 1, wherein R¹，R²And R³Are each a dodecanoyl residue.

8. The use of claim 1, wherein R¹，R²And R³Each is a tetradecanoyl residue.

9. The use of claim 1, wherein^*1、^*2And^*3is in the R configuration.

10. Use according to claim 1, wherein Y is in the equatorial position.

11. The use of claim 1, wherein^**Is in the S configuration.

12. The use of claim 1, wherein^*1、^*2And^*3is of the R configuration, wherein Y is in the equatorial position, and wherein^**Is in the S configuration.

13. The use of claim 1, wherein the infectious disease is caused by a bacterium, virus, parasite or fungus.

14. The use of claim 13, wherein the bacterium is a gram-negative bacterium or a gram-positive bacterium.

15. The use of claim 13, wherein the infectious disease is caused by a bacterium selected from the group consisting of pseudomonas, escherichia, klebsiella, enterobacter, proteus, serratia, candida, bacillus, and staphylococcus.

16. The use of claim 15, wherein the infectious disease is pneumonia.

17. The use of claim 16, wherein the pneumonia is nosocomial pneumonia.

18. The use of claim 17, wherein the pneumonia is in an HIV positive patient.

19. The use of claim 1, wherein the infectious disease is a chronic infection.

20. The use of claim 19, wherein the chronic infection comprises chronic hepatitis, human papilloma virus, oral or vaginal candidiasis, periodontal disease or chronic sinusitis due to fungal colonization.

21. The use of claim 1, wherein the allergic condition is selected from the group consisting of asthma, atopic dermatitis, seasonal allergy and chronic rhinosinusitis.

22. The use of claim 1, wherein the autoimmune disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, chronic arthritis, multiple sclerosis and psoriasis.

23. The use of claim 1, wherein said compound is administered to said animal by a route selected from the group consisting of parenteral, oral, intravenous, infusion, intranasal, inhalation, transdermal and transmucosal.

24. Use of one or more compounds of the formula:

wherein X is selected from-O-and-NH-;

y is selected from-O-and-S-;

R¹，R²and R³Are each independently selected from (C)₂-C₂₀) An acyl group;

R⁴selected from-H and-PO₃R⁷R⁸Wherein R is⁷And R⁸Are each independently selected from-H and (C)₁-C₄) An aliphatic group;

R⁵is selected from-H, -CH₃and-PO₃R⁹R¹⁰Wherein R is⁹And R¹⁰Are each independently selected from-H and (C)₁-C₄) An aliphatic group;

R⁶is selected from H, OH, (C)₁-C₄) Oxyaliphatic radical, -PO₃R¹¹R¹²，-OPO₃R¹¹R¹²，-SO₃R¹¹，-OSO₃R¹¹，-NR¹¹R¹²，-SR¹¹，-CN，-NO₂，-CHO，-CO₂R¹¹and-CONR¹¹R¹²Wherein R is¹¹And R¹²Are each independently selected from H and (C)₁-C₄) An aliphatic radical, provided that R⁴And R⁵One is a phosphorus-containing group and when R is⁴is-PO₃R⁷R⁸When R is⁵Is not-PO₃R⁹R¹⁰；

Wherein^*1″，″^*2″，″^*3", and^**"denotes a chiral center;

wherein n is 1, m is 2, and p and q are 0.

25. The use of claim 24, wherein X and Y are-O-, R⁴Is PO₃R⁷R⁸，R⁵And R⁶Is H.

26. The use of claim 25, wherein R⁷And R⁸is-H.

27. The use of claim 24, wherein R¹，R²And R³Are respectively C₆-C₁₄An acyl group.

28. The use of claim 24, wherein R¹，R²(ii) a And R³Are respectively C₆-C₁₂An acyl group.

29. The use of claim 24, wherein R¹，R²And R³Are decanoyl residues, respectively.

30. The use of claim 24, wherein R¹，R²And R³Are each a dodecanoyl residue.

31. The use of claim 24, wherein R¹，R²And R³Each is a tetradecanoyl residue.

32. The use of claim 24, wherein^*1、^*2And^*3is in the R configuration.

33. The use of claim 24, wherein Y is in the equatorial position.

34. The use of claim 24, wherein^**Is in the S configuration.

35. The use of claim 24, wherein^*1，^*2And are and^*3is of the R configuration, wherein Y is in the equatorial position, and wherein^**Is in the S configuration.

36. The use of claim 24, wherein the infectious disease is caused by bacteria.

37. The use of claim 36, wherein the bacterium is a gram-negative bacterium, or a gram-positive bacterium.

38. The use of claim 36, wherein the infectious disease is caused by a bacterium selected from the group consisting of pseudomonas, escherichia, klebsiella, enterobacter, proteus, serratia, candida, bacillus, and staphylococcus.

39. The use of claim 38, wherein the infectious disease is pneumonia.

40. The use of claim 39, wherein the pneumonia is nosocomial pneumonia.

41. Use of a cyclic AGP in the manufacture of a medicament formulated and administered in the absence of a foreign antigen for ameliorating or substantially preventing an infectious disease, autoimmune disease, or allergic condition in a subject.

42. The use of claim 41 wherein the cyclic AGP is N- [ (R) -3-dodecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxy-tetradecanoylamino ] -3-O- [ (R) -3-dodecanoyloxytetradecanoyl ] - β -D-glucopyranoside or a pharmaceutically acceptable salt thereof.

43. The use of claim 41 wherein the cyclic AGP is N- [ (R) -3-decanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino ] -3-O- [ (R) -3-decanoyloxytetradecanoyl ] - β -D-glucopyranoside or a pharmaceutically acceptable salt thereof.