CN101351208A

CN101351208A - Reducing cellular cholesterol levels and/or treating or preventing phospholipidosis

Info

Publication number: CN101351208A
Application number: CNA2006800498805A
Authority: CN
Inventors: F·马克斯菲尔德; A·索夫
Original assignee: Cornell Research Foundation Inc
Current assignee: Cornell Research Foundation Inc
Priority date: 2005-11-01
Filing date: 2006-11-01
Publication date: 2009-01-21

Abstract

Compounds disclosed herein may be used in disclosed methods for reducing the amount of cholesterol in a cell, for treating a patient suffering from a disorder characterized by cellular accumulation of cholesterol (such as Niemann-Pick Disease Type C or atherosclerosis), and/or for treating or preventing phospholipidosis. In some embodiments, the compounds may include a pyrrolone or triazine moiety.

Description

Reducing cellular cholesterol levels and/or treating or preventing phosphatidic diseases

Cross reference to related applications

[001] This application claims priority from U.S. provisional application 60/732,342 filed on 1/11/2005, U.S. provisional application 60/807,269 filed on 13/7/2006, and U.S. application 11/555,152 filed on 31/10/2006, each of which is incorporated herein by reference.

Statement regarding federally sponsored research or development

[002] Dk27083, the national institute of health foundation, provides partial support for the research conducted to obtain the subject matter disclosed herein. Accordingly, the U.S. government has certain rights in the subject matter of this application.

Background

[003] Modulation of cellular cholesterol levels is essential for proper cellular function and development. Cholesterol levels within a cell are regulated, in part, by the transport of cholesterol between various compartments and membranes. The proper distribution of cholesterol between various cell membranes is important for many biological functions, such as signal transduction and membrane exchange. Cholesterol levels can also be modulated by trafficking extracellular receptors from the cell to the removal of cholesterol. These cholesterol transport mechanisms have been extensively studied, and defects in the regulation of cellular cholesterol levels have been linked to various diseases.

[004] Niemann-pick disease is a genetic class of lipid storage disorders. There are recognized 4 types of niemann-pick disease: A. b, C and type D. Types a and B are caused by a lack of sphingomyelinase activity that can lead to an increase in sphingomyelin in the cell, often leading to cell death. Patients with niemann-pick disease type a often die at 2 to 4 years of age, while patients with type B may survive to late childhood or adulthood. Type D niemann-pick disease (also known as neoscotia variant) is equivalent to type C and occurs in the descendants of the west of neoscotia province.

[005] Niemann-pick disease type C (NPC) is an autosomal recessive inherited disorder that causes an abnormal accumulation of cholesterol and other lipids in many cell types (1, 2). The most severe symptoms are caused by progressive neuronal degeneration, but the liver and other peripheral organs also exhibit defects. Although the time course can be altered, symptoms often occur in early childhood, and the disease is often fatal in the teens. There have been many attempts to develop treatments for NPC (3-8), but there are currently no effective treatments.

[006] Two genes have been associated with NPC deficiency in humans, although the exact mechanism of activity of these proteins is still under investigation. NCP1 is a multi-spanning membrane protein that is normally associated with late endosomes or lysosomes (9), degrades organelles, and hydrolyzes cholesterol esters carried into cells via lipoproteins (10, 11). NPC1 has a sterol-sensitive transmembrane domain similar to that found in the endoplasmic reticulum of cells which responds to changes in cellular cholesterol (12). The NPC1 protein favors bilayer transport of some hydrophobic molecules, but it does not appear to be directly involved in cholesterol transport (13-16). NPC2 is a soluble, luminal protein that is present in late endosomes and binds cholesterol (17-19). NPC2 can freely shuttle free cholesterol to late endosome and lysosome-restricted membranes, with NPC1 apparently functioning at its export to other cellular sites (20). Loss of functional NPC1 or NPC2 causes accumulation of free cholesterol in endocytic organelles with late endosomal and/or lysosomal characteristics. These abnormal organelles referred to herein are lysosome-Like Storage Organelles (LSOs). NPC-associated LSOs are similar to other genetic glycosphingolipid storage disease (often caused by the inability of a particular lipid to metabolize) associated LSOs in that the storage organelles comprise a multilayered internal assembly of membrane bilayers containing cholesterol, sphingomyelin and a large amount of 2- (monoacylglycerol) -phosphate (BMP), also known as lyso-biphospholipid acid (LBPA) (21, 22). Thus, even though these diseases arise from different genetic defects, certain aspects of the cellular phenotype are quite similar.

[007] Several chains of evidence point to a defect in the transport of cholesterol in NPC, although defects in the transport of other esters can also play an important role (23). NPC cells showed abnormally high levels of non-esterified cholesterol, which accumulated mainly at LSOs. Accumulated cholesterol can be detected using phenanthroline, a fluorescent detergent that can bind free cholesterol on the membrane (24). In wild-type cells, excess cholesterol transported from endosomes to cells is either transported from the cell to the extracellular receptor or esterified by acyl-coa: cholesterol Acyltransferase (ACAT), an enzyme located on the endoplasmic reticulum (25). Despite the high content of free cholesterol in LSOs, the plasma membrane of cultured NPC cells has actually a lower cholesterol content than normal cells (26) and a defect in cholesterol efflux to extracellular receptors (27). Furthermore, the transport of lipoprotein-derived cholesterol by ACAT for esterification has a drawback (28, 29). These characteristics indicate that cholesterol efflux from late endosomes is detrimental to NPC cells.

[008] Several different genetic mutations were found in the NPC1 gene, which corresponds to 95% of NPC diseases in humans (13, 30-33). The correlation between molecular defects and the age of onset of severe symptoms is not well defined. The clinical manifestations of NPC disease range from late onset or mild symptoms in adults to early onset acute symptoms in infants (34, 35). This suggests that other factors in the genetic background may partially ameliorate the disease. Also, studies of cultured cells have shown that overexpression of various proteins affecting membrane trafficking can reduce cholesterol accumulation. In particular, overexpression of small regulated guanosine triphosphatases (GTPases), Rab7 and Rab9 (36-38) reduced sterol accumulation in cultured fibroblasts. Since these proteins regulate many aspects of cell membrane trafficking, they are not necessarily good therapeutic targets. However, both the difference in the age of onset and the influence of overexpression of foreign genes in humans suggest that drug therapy may progress to improve symptoms even if the exact role of NPC protein is not restored.

[009] Phosphatidosis is a condition in which there is an excessive accumulation of phospholipids in body tissues. Excessive accumulation of phospholipids is thought to be associated with altered synthesis and/or metabolism of phospholipids. Phosphatidosis occurs when a patient is given a drug. For example, phosphatidic diseases can be caused when humans are amiodarone, perhexiline, fluoxetine, gentamicin. See m.j.reasor et al.exp.biol.med.2001, 226, 825. Since excessive accumulation of phospholipids is a side effect of certain drugs, compositions and methods for treating drug-induced phosphatidic diseases would be highly desirable.

[010] Thus, there is a need for treatment of niemann-pick disease and other diseases caused by a defect in the regulation of cellular cholesterol levels. There is also a need for methods of treating or preventing drug-induced phosphatidic diseases. The present invention fulfills these needs and provides other related advantages.

Disclosure of Invention

[011] One aspect of the present invention relates to compounds and pharmaceutical compositions useful for reducing the amount of cholesterol in cells. In certain examples, the compounds of the present invention include a pyrrole ring or triazine moiety (moiety). Another aspect of the invention relates to a method of treating a patient characterized by cellular accumulation of cholesterol. In certain embodiments, the invention relates to a method of treating niemann-pick disease type C or atherosclerosis. Another aspect of the invention relates to a method of reducing the amount of cholesterol in a cell by exposing the cell to a compound of the invention. In certain examples, the method comprises exposing the cell to a compound comprising an azole ring or triazine moiety. Another aspect of the invention relates to a method of treating or preventing a drug-induced phosphatidic disease.

Drawings

[012] FIG. 1 depicts the results of a Firexate binding assay in which wild-type CHO cells (TRVb1) and NPC1 mutant CHO cells (CT60) were placed in 384-well plates and grown in conventional medium for 48 hours. Cells were washed with PBS, fixed with 1.5% PFA and stained with filipin. The resulting 360/40nm excitation and 480/40nm emission light filtered with a 365DCLP filter yielded images at 2 positions per well at 10 times magnification. (A) Images of philippine stained TRVb1 cells; (B) image of philippine stained CT60 cells. Scale bar 30 μ M. Image analysis was performed using mean philippine intensity and LSO Compartment Ratio (comparative Ratio). (C) A histogram of mean Fireyi intensity values; (D) histogram of LSO compartment proportion values.

[013] FIG. 2 depicts the results of a phenanthroline binding assay, in which cells were fixed with PFA and labeled with phenanthroline. (A) Images at 10 times magnification were obtained using a Discovery-1 autofluorescence microscope and filtering the resulting 360/40nm excitation and 480/40nm emission using a 365DCLP filter. (B) The shadow and background back images are corrected. (C) The high threshold setting was used to identify the LSO compartment. (D) The high threshold setting is used to encompass the entire cellular region. Scale 20 μ M.

[014] FIG. 3 depicts the results of a phenanthroline binding assay, in which CT60 cells were grown overnight in growth medium and treated with solvent (A) or 10. mu.M of a selective compound (1-a-13) (B) in the screening solution. After 20 hours incubation, cells were washed with PBS, fixed with 1.5% PFA and stained with filipin. Images at 10 x magnification were obtained. Scale 25 μ M.

[015] FIG. 4 depicts images of Phenantheine stained CT60 cells affected by the addition of compounds that induce morphological changes and/or increase the intensity of Phenantheine. (A) Compound 1-c-1 resulted in more dispersed fluorescence with no significant change in mean phenanthroline intensity. (B) Compound 1-c-2 induced more compact LSOs, while the phenanthroline intensity did not change significantly. (C) Compound 1-c-3 caused perinuclear aggregates of LSOs in mutant cells to become more dispersed. (D) Compound 1-b-4 caused a significant increase in the intensity of phenanthroline with filamentous or tubular staining. Scale 15 μ M.

[016] FIG. 5 depicts the chemical structures of 14 compounds derived from primary libraries (1-a-1, 1-a-2, 1-a-3, 1-a-4, 1-a-5, 1-a-6, 1-a-7, 1-a-8, 1-a-9, 1-a-10, 1-a-11, 1-a-12, 1-a-13, and 1-a-14). Compounds 1-c-2 and 1-c-3 caused morphological changes, compound 1-b-2 caused an increase in the strength of felipine, and compound 1-b-4 increased the strength of felipine while inducing morphological changes.

[017] FIG. 6 depicts dose response curves for 14 compounds from the primary pool (1-a-1, 1-a-2, 1-a-3, 1-a-4, 1-a-5, 1-a-6, 1-a-7, 1-a-8, 1-a-9, 1-a-10, 1-a-11, 1-a-12, 1-a-13, and 1-a-14). CT60 and CT43 cells were seeded in 384-well plates with growth media. After 24h, compounds were added to final concentrations of 123nM, 370nM, 1.11. mu.M, 3.33. mu.M and 10. mu.M, 4 wells per concentration, and cells were cultured overnight. Cells were washed with PBS, fixed with PFA and stained with filipin. Determination of the proportion of the LSO compartments: (A) CT60 cells (average of 5 experiments) and (B) CT43 cells (average of 3 experiments). The solid line represents the mean of the solvent control; the dashed line represents-3 SD.

[018] FIG. 7 depicts cytotoxicity assays for 14 compounds from primary pools (1-a-1, 1-a-2, 1-a-3, 1-a-4, 1-a-5, 1-a-6, 1-a-7, 1-a-8, 1-a-9, 1-a-10, 1-a-11, 1-a-12, 1-a-13, and 1-a-14). CT60 and CT43 cells were seeded in 384-well plates with growth media. After 24h, compounds were added to final concentrations of 5, 10 and 20 μ M per 4 wells and cells were cultured overnight. An equal amount of dimethylsulfoxide was added to the control wells. After washing the cells with PBS, they were fixed with PFA and stained with the nuclear stain Hoechst 33258. Images at 4 x magnification were obtained using a Discovery-1 autofluorescence microscope and filtering the resulting 360/40nm excitation and 480/40nm emission using a 365DCLP filter. Results of percentage reduction in cell number compared to control, counted per well: (A) CT60 cells (average of 4 experiments) and (B) CT43 cells (average of 3 experiments).

[019] FIG. 8 depicts the chemical structures of 7 compounds (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a-12, 2-a-13, 2-a-15) obtained from secondary libraries.

[020] FIG. 9 depicts the effect of 7 compounds from the secondary library (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a-12, 2-a-13, 2-a-15). The dose dependence was determined as described in the figure. (A) CT60 cells (average of 5 experiments) and (B) CT43 cells (average of 3 experiments). The solid horizontal line indicates the mean of the solvent control; the dotted line indicates the mean-3 SD.

[021] FIG. 10 depicts the cytotoxicity assays of 7 compounds (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a-12, 2-a-13, 2-a-15). Cytotoxicity of 7 selected compounds from the secondary pool was determined by cell counting and LDH release. For cell counts per well, cell counts were performed as described in fig. 7, results of percent reduction in cell number compared to control: (A) CT60 cells, and (B) CT43 cells. For LDH cytotoxicity assays, measurement of cellular LDH released into the medium in the presence of 7 selected compounds obtained from the secondary: (C) CT60 cells, referenced to low (no compound) and high (lysed cells) controls.

[022] FIG. 11 depicts the effect of 7 compounds from the secondary library at different times (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a-12, 2-a-13, 2-a-15). CT60 cells were seeded in 384-well plates with growth medium. After 24h, compounds were added to final concentrations of 1.11, 3.33 and 10 μ M in 4 different wells/concentration and incubated for (a)4 hours, (B)20 hours, and (C)48 hours. Cells were washed with PBS, fixed with PFA and stained with filipin. The resulting 360/40nm excitation and 480/40nm emission light filtered with a 365DCLP filter yielded images at 2 positions per well at 10 times magnification. The LSO compartment ratio was measured (average of 3 different experiments). The solid horizontal line indicates the mean of the solvent control; the dotted line indicates the mean-3 SD.

[023] FIG. 12 depicts the effect of 7 compounds from the secondary pool on U18666A treatment of normal human fibroblasts (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a-12, 2-a-13, 2-a-15). Normal human fibroblasts were grown in 384-well plates in conventional medium for 24 hours, and the cells were then treated with compound U18666A (500nM or 250nM) in screening medium for 4 hours. The cells were then further incubated overnight with various selection compounds (10 μ M) in the continued presence of U18666A. Finally, cells were washed 3 times with PBS, fixed with 1.5% PFA, washed with PBS and stained with filipin. Images at 10 times magnification were obtained using a Discovery1 microscope and analyzed for LSO scale. The solid horizontal lines refer to the average of the various concentrations of Ul8666A treated cells, and the dashed horizontal lines indicate-3 SD.

[024] FIG. 13 depicts increased cholesterol efflux from 25RA CHO cells incubated in 10 μ M concentration of each compound without NPC mutation for the parental cell lines of CT60 and CT43 cell lines.

Detailed Description

[025] One aspect of the invention provides compositions and methods for modulating the level of cellular cholesterol. The compositions of the present invention may be used to treat niemann-pick disease and other diseases associated with a deficiency in the level of cholesterol in regulatory cells. As described above, proper regulation of cellular cholesterol levels is essential for proper cellular function and development. The effect of compounds on cellular cholesterol levels can be determined using the philippine binding method.

[026] An automated screening assay for identifying compounds that are capable of partially reversing the phenotype of niemann-pick disease C (NPC) mutant cells is described. The method is based on the combination of the fluorescent detergent phenanthroline with free cholesterol. In untreated mutant cells, there was a significant amount of free cholesterol compared to the control cell line (42). The free cholesterol is in high concentration in late endosome-associated LSOs, organelles, but may also contain protein markers, which are not usually abundant in late endosomes (47). The molecular defect in NPC is a mutation or lack of one of the two proteins NPC1 and NPC2 associated with late endosomes. These mutations cause a defect in the efflux of cholesterol from the late endosomes, resulting in the high level accumulation of cholesterol in LSOs.

[027] Two screening assays were developed to evaluate the effect test compounds have on modulating the level of cellular cholesterol. The first detection method employs a threshold of Firexate-fluorescence intensity sufficient to determine the area of each image containing cells. Using this method, total phenanthroline fluorescence can be obtained per cell area in each region. The intensity of plasma membrane staining provided a clear difference in cell area above background levels. However, a second test is required because the threshold used to determine the cell area does not clearly distinguish the LSO compartment from other cellular regions. The detection parameter used is the total fluorescence divided by the number of pixels above the threshold. This method was used to evaluate total cholesterol per cell, based on the approximation that the cell area remains constant under various conditions. While this assay is believed to provide a reliable determination of total cholesterol levels per cell, the results of the assay may be affected if the cells significantly diffuse or aggregate in response to a treatment, or if some cholesterol has a different ability to bind to phenanthroline.

[028] Although this assay does not use subcellular information or single cell analysis, it can use automated microscopy. First, the microscope system is a sensitive detector for relatively weak fluorescence of philippines. Second, the measurement is limited to the area of each region containing cells, which reduces the contribution of the background. Finally, dividing the area covered by the cells by the total fluorescence intensity provides a correction for the difference in cell density at the time of measurement.

[029] We have found that the phenanthroline intensity per pixel, which provides sufficient mutant resolution for wild-type cells, is useful as a screening assay. This parameter is used when we adjust the experimental conditions for detection, such as cell density and labeling conditions. It should be noted, however, that this should be taken into account since in some cases only a few standard deviations isolate CT60 cells from the control cell line.

[030] We achieved better discrimination of mutant cells from wild type using the LSO compartment ratio assay, in which a threshold was used to identify the area in each region containing a re-labeled organelle (i.e., LSOs in mutant cells). Because there are sites for cholesterol accumulation in NPC cells, selective measurement of the cholesterol pool is expected to provide better discrimination of the mutant to wild-type cells. This additional sensitivity is useful in determining part of the active ingredient screening assay. The coefficient Z '(46) is a measure of the discriminatory power of the screening assay, and the LSO compartment ratio test has a Z' of 0.61 versus a mean phenanthroline intensity test of 0.22. It is generally considered that a Z' value of greater than 0.5 is sufficient for screening assays.

[031] In the first round of screening experiments we determined a significant decrease in the 10 μ M phenanthroline marker caused by 14 compounds, including 3 compounds that produced a significant decrease at 123 nM. The primary library is composed of 126 template combinations. Some of the compounds were observed to be effective at 123nM, suggesting that it is likely that some of these compounds have high affinity interactions with their targets.

[032] A compound secondary library was screened with Tanimoto similarity coefficients in the range of 0.3 to 0.96 (high coefficients indicate high similarity). The average Tanimoto similarity coefficient is about 0.75. Screening assays use lower doses of test compounds and focus more on non-toxicity than primary library screening. Even though the dose of test compound was reduced from 10 μ M to 1 μ M in the assay of the secondary library, the secondary library still contained a higher fraction (0.18%) of the selected compound than the primary library (0.1%). Thus, the selection of chemicals in the secondary pool results in significant enrichment among the potential multiple candidates (hits). In addition, many selected compounds have greater efficacy and lower toxicity than the compounds obtained from the primary screening. These 7 compounds identified from the secondary library were based on 4 synthetic templates. The compounds 2-a-1, 2-a-9, 2-a-12 and 2-a-13 are based on triazines, and such compounds have been of significant interest in the field of medicinal chemistry. See (48-52).

[033] The 7 compounds selected from the secondary library for further characterization can be roughly divided into two groups. The compounds 2-a-1, 2-a-9, 2-a-12 and 2-a-13 (group I) are based on a core of 1, 3, 5 triazines, which have been of major interest in the field of medicinal chemistry (52-56). The second group of compounds (group II) has a 5-membered ring heterocyclic core (2-a-3: 2-thioxo-1, 3-thiazolidin-4-one derivatives, 2-a-15 containing a methine linked pyrrole and pyrrol-2-one, 2-a-8 containing a 1, 3-thiazole linked to a dihydropyrazole via N). Both groups of compounds have a number of substitutions in the nucleus, with the triazines of group I bearing predominantly aryl or cyclic amines (or a hydrazine group). Group II are also aryl-substituted, wherein compound 2-a-3 is characterized by an interesting partially saturated diethyl-aminonaphthyl moiety attached by a double bond to 2-thioxo-1, 3-thiazolidin-4-one. Compound 2-a-15 has three aromatic rings in an extended conjugated system, while compound 2-a-8 combines 6 different ring systems, 5 of which are aromatic. Both group I and group II compounds appear as conformationally restricted molecules with high unsaturation. Their periphery tends to be very hydrophobic, while their center is more hydrophilic. We note that this feature means that they are sterically amphiphilic (external hydrophobic-internal hydrophilic). Although in some cases hydrophilic groups (notably the nitro group of 2-a-13) are present at the edges, several hydrogen-bonded moieties are uniformly distributed to the core of these structures suggesting that these cores may assist their specific recognition of targets in vivo.

[034] Although this assay was developed for CHO cell lines, it should also be suitable, with minor modifications, for analysis of cholesterol accumulation in other types of cells. A test for cholesterol accumulation is useful not only for NPC but also for other glycolipid storage diseases. Although the biochemical basis of these diseases is different, many of these diseases result in a similar phenotype, including the formation of internal membrane spirocycles in LSOs, which contain sphingomyelin, lyso-diphosphatic acid and cholesterol (53, 54). The methods described herein are not only useful for chemical screening, but also for molecular genetics screening, such as RNAi inhibition and gene expression. Using traditional methods, it has been determined that a few genes can correct NPC phenotype when overexpressed in cells (36, 38, 55). This screen we describe herein can be used for large scale screening of gene expression.

[035] These screening assays also identified compounds that enhanced phenanthroline staining even beyond levels found in NPC mutant cells. Upon further investigation, some compounds that initially showed increased phenanthroline staining were found to be fluorescent in the band overlapping with the phenanthroline spectrum, and their fluorescence may therefore be the basis for increased fluorescence upon detection. However, several non-fluorescent compounds were also found to increase the phenanthroline staining of NPC cells. We have also found that some compounds are capable of producing significant changes in the morphology of the compartment enriched in free cholesterol. In particular, compound 1-c-3 produced a large network of distinct tubular organelles labeled with phenanthroline.

[036] We also measured the cholesterol content of the treated cells by a direct chemical method. Most of the selected compounds did indeed reduce cellular cholesterol at the time of screening, although the 3 compounds initially selected did not show a reduction in cholesterol in one gas chromatographic assay. Therefore, in some cases, the results of the filipin binding experiments from independent chemical analyses should be carefully verified.

[037] Without being bound by a particular theory, it is believed that cholesterol efflux from late endosomes requires several steps. This extravasation, like many steps of intracellular transport, is apparently predominantly non-vesicular (56). NPC2 is presumed to play a role in transporting cholesterol from the hydrolyzed sterol ester site into the limiting membrane (57). NPC1 and other proteins are presumed to contribute to the transport of cholesterol from the limiting membrane to the cytoplasmic carrier. These, not yet molecularly defined, transport cholesterol to the plasma membrane or other organelles (58, 59). Total free cholesterol in organelles can be reduced by increasing efflux to extracellular receptors in the cytoplasm, and/or by esterification of cholesterol by ACAT in the endoplasmic reticulum of the cell. Reducing cholesterol uptake or reducing synthesis can also result in a reduction in cellular cholesterol upon incubation with the compound.

[038] Using an analytical method similar to the mean intensity and LSO detection, we measured the reduction of lipid BMP accumulation in NPC cells. The selected compounds from the secondary pool did not produce a significant reduction in BMP labeling after 16 hours incubation (data not shown). Several selected compounds from secondary pools did cause a reduction in cholesterol accumulation in normal human fibroblasts treated with U18666A, resulting in accumulation of cholesterol in LSOs. This indicates that these compounds are independent of the SCAP mutation or other specific properties of the CHO cell line used for screening.

[039] Without being bound by a particular theory, the effects of the test compounds may act directly on the LSOs, but may also have indirect effects. For example, Rab4, an overexpression of a small GTPase commonly associated with endosome classification or endocytosis salvage compartments, may partially correct the NPC phenotype (60).

[040] The compounds identified in the screening assays were effective in reducing cholesterol accumulation at concentrations that were not toxic to cultured NPC1 cells. Furthermore, several compounds (fig. 13) should be able to effectively reduce the amount of cholesterol in normal cells, as they show an effect of promoting cholesterol efflux in 25RA CHO cells. The compounds of the invention may also be used to study the mechanism by which cells regulate cholesterol levels. For example, the compounds of the invention may be modified with photosensitive groups for labeling bound ligands or attaching biotin for affinity purification. Furthermore, without being bound by a particular theory, the compounds of the present invention may be effective in reducing cholesterol uptake by cells and/or inhibiting cholesterol biosynthesis.

[041]Another aspect of the invention relates to a method for treating or preventing drug-induced phosphatidic disease. When a pharmaceutical formulation is administered to a patientDrug-induced phosphatidic disease can occur as a side effect. For example, the following drugs can cause phosphatidic diseases: ABT-770, AC-3579, amantadine, ambroxol, amikacin, amiodarone, amitriptyline (amitriptilline), AY-9944, azithromycin, benzamide, bociclidine, bromhexine, clorazine, chloroquine, p-chlorobenzylamine, chlorpromazine, citalopram, clorfrex, clomipramine, clozapine, clotrimazine 200-15, cyclizine, DMP-777, erythromycin, fenfluramine, fluoxetine, fluvoxamine, gentamicin, hydroxyzine, IA-3, mipramine, iprindole, LY281389, maprotiline, chlorphenazine, imipramine, NE-10064, netilmicin, norclocycline (norchllorocycline), oxitiline, perhexiline, phentermine, PNU-177864, promethazine, isoproxazine, rmatrizine, temozine, temozolone, 393, temozolone, chlortrimetrex, 393 Tripelennamine (triprenamine), spectinomycin, zimelidine, 1-chloroantiline (1-chloroamipterine) and 4, 4' -diethylaminoethoxyhexylestrol. See m.j.reasor et al.exp.biol.med.2001, 226, 825; m.j.reasor et al.expert opin.drug saf.2006, 5, 567; Lullmann-Rauch R., Drug-induced lysosol Storage Disorders, in L _{YSOSOMES IN}B_{IOLOGY AND} P_ATHOLOGY，Vol.6.，pp.49-130(Dingle et al.eds.，Amsterdam：North-Holland，1979)；Kodavanti et al.Pharmacol.Rev.1990，42，327；M.J.Reasor.Cationic Amphiphilic Drugs，inC_OMPREHENSIVE T_OXICOLOGY，Vol.8，T_{OXICOLOGY OF THE}R_ESPIRATORY S_YSTEMpp.555-566(Sipes et al. eds., New York: Elsevier Science, 1997); and Sawada et al in Toxicol.Sci., 2005, 83, 282 and Toxicol.Sci.2006, 89, 554. A variety of drugs with cationic lipophilic structures can also lead to drug-induced phosphatidic diseases. Such drugs often have a hydrophilic region consisting of at least one primary nitrogen or substituted nitrogen group that is positively charged at physiological pH, and a hydrophobic region containing aromatic and/or cycloaliphatic groups that may be optionally substituted with halogen. See m.j.reasor et al.Exp.Biol.Med.2001，226，825。

[042] Methods for identifying compounds responsible for phosphatidic disease are well known in the art. See e.g. h.sawada et al.toxicol.sci.2005, 83, 282. Since phospholipid excess accumulation is an undesirable side effect of certain drugs, one aspect of the present invention relates to a method of treating or preventing drug-induced phosphatidic diseases by administering to a patient in need thereof a therapeutically effective amount of a compound of any one of formulas I-IX described herein. In some cases, the drug-induced phosphatidic disease in the patient is not caused by compound U-18666A. In some cases, the drug-induced phosphatidopathy in the patient is caused by ABT-770, AC-3579, amantadine, ambroxol, amikacin, amiodarone, amitriptyline, AY-9944, azithromycin, benzamide, bociclidine, bromhexine, clociclazine, chloroquine, p-chlorobenzylamine, chlorpromazine, citalopram, clofosfomesamide, clomipramine, clozapine, Compound 200-15, cyclizine, DMP-777, erythromycin, fenfluramine, fluoxetine, VOFLUORXAMINE, gentamicin, hydroxyzine, IA-3, imipramine, iprindole, LY281389, maprotiline, chlorphenazine, miiline, NE-10064, netilmicin, norclocridine, oxitriptyline, perhexiline, phentermine, PNU-177864, promazine, promethazine, isopropamide, RMI, 10.393, thiamethoxam, methoxib, tilarone, tobramycin, trimipramine, tripperanol, tripelennamide, spectinomycin, zimelidine, 1-chloroamipiline, and 4, 4' -diethylaminoethoxyhexanediestrol. In certain instances, the drug-induced phosphatidic disease in the patient is caused by the administration of amiodarone, perhexiline, azithromycin, fluoxetine, imipramine, clorazine, tamoxifen, or gentamicin.

[043] Another aspect of the invention relates to a method comprising administering to a patient in need thereof a therapeutically effective amount of a first therapeutic agent and a therapeutically effective amount of a second therapeutic agent; wherein the first therapeutic agent is a compound of any one of formulas I-IX as described herein; the second therapeutic agent is an anorectic agent, antianginal agent, antiarrhythmic agent, antibiotic, anticancer agent, antidepressant, antiestrogen, antihistamine, antilipemic agent, antimalarial agent, antineocardial agent, antihypertensive agent, antithrombotic agent, antiviral agent, cholesterol synthesis inhibitor, diazepine atypical antipsychotic agent, histamine H1-blocker, matrix metalloproteinase inhibitor, neutrophil elastase inhibitor, schistosomiasis-killing agent, secretagogue, selective 5 hydroxytryptamine reuptake inhibitor, or tranquilizer that causes drug-induced phosphatidic disease. In some cases, the second therapeutic agent is an antibiotic, an antiarrhythmic, an antidepressant, a histamine H1-blocker, or an anti-cancer drug that causes drug-induced phosphatidic disease.

[044] Another aspect of the invention relates to a method of treating a mammalian cell afflicted with a drug-induced phosphatidic disease, comprising administering to said cell a therapeutically effective amount of a compound of any one of formulae I-IX as described herein. In some cases, the drug-induced phosphatidic disease in the patient is not caused by compound U-18666A. Another aspect of the invention relates to a method of treating a mammalian cell afflicted with a drug-induced phosphatidopathy comprising administering to the cell a therapeutically effective amount of a compound of any of formulae I-IX described herein, wherein the drug-induced phosphatidopathy is at least in part caused by ABT-770, AC-3579, amantadine, ambroxol, amikacin, amiodarone, amitriptyline, AY-9944, azithromycin, benzamide, becoxetine, bromhexine, clorazine, chloroquine, p-chlorobenzylamine, chlorpromazine, citalopram, clofosfamil, clopramine, clozapine, Compounde 200-15, closazine, DMP-777, erythromycin, fenfluramine, fluoxetine, fluvoxamine, gentamycin, hydroxyzine, IA-3, imipramine, iprindole, Ill 281389, maprotiline, chlorphenbutazine, chlorpheniramine, The administration of mepacrine, NE-10064, netilmicin, norclocycline, oxitiline, perhexiline, phentermine, PNU-177864, promethazine, propranolol, RMI 10.393, sertraline, tamoxifen, thiamethoxam, tirarone, tobramycin, trimipramine, triperanol, tripelennamine, spectinomycin, zimelidine, 1-chloroactaline, and 4, 4' -diethylaminoethoxyhexaestrenol.

[045]Preparation of the Compounds of the invention

[046]The compounds depicted in figures 5 and 8 are commercially available. Compounds represented by general formulas I-XXXIX can be prepared from the compounds shown in figures 5 and 8 or other commercially available compounds using synthetic methods known in the art, see, e.g., j. march,Advanced Organic Chemistry，McGraw Hill Book Company，New York，(1992，4^th edition)；Carey，F.A.and Sundberg，R.J.Advanced Organic Chemistry Part B：Reactions and Synthesis，3^rd Ed.；Plenum Press：New York，1990；and Organic Chemistry 2^nded.ed.bruice, p.y.new Jersey: prentice Hall, 1998. Representative synthetic methods are also described below.

[047]A large number of compounds can be prepared by inserting new functional groups on the aromatic rings or modifying existing functional groups located thereon in the compounds shown in fig. 5 and 8. For example, typical functional group manipulations include those by reaction in FeBr₃In the presence of Br₂Treatment of aromatics to introduce bromine by reaction in FeBr₃Treating the aromatic compound with an acid chloride to introduce an acyl group when present, using SnCl in HCl₂Processing the nitroaromatic compound to obtain the aminoaromatic compound. Other functional group manipulations include use of Na/NH₃The aromatic group is reduced to give the cycloolefin or the cycloalkyl compound depending on the reaction conditions. For example, by the influence of carboxylic acids, with Na/NH₃Reduction of 1-a-13 will selectively yield the cyclohexene derivative. See scheme 1. The cyclohexene intermediate can be further reduced to obtain a cyclohexyl derivative. Alternatively, the cyclohexene intermediate may be treated with an oxidant to form an epoxide. The carboxylic acid group may also be converted to an ester by reaction with an alcohol such as methanol or benzyl alcohol in the presence of DCC.

Scheme 1:

[048] as shown in scheme 2, a large number of compounds can be prepared from lactam derivative intermediates using palladium coupling techniques. Palladium coupling reactions are advantageous because they are often performed in high yields and are suitable for use with a variety of functional groups. In addition, considerable amounts of organoboranes are well known and/or commercially available. In addition, a large number of aromatic halides and alkenyl halides are commercially available which can be readily converted to organoborane starting materials for the coupling reaction.

Scheme 2:

[049] various triazine compounds can be prepared using palladium coupling reactions. As shown in scheme 3, the reaction of commercially available triazines with aryl/heteroaryl bromides or iodides can be used to prepare a number of derivatives, all of which can be converted to other compounds by manipulation of the aromatic functionality described above. Notably, palladium coupling of aryl/heteroaryl bromides or iodides can also be used to prepare the various triazinylhydrazones shown in scheme 4.

Scheme 3:

scheme 4:

[050]the inventionMethod (2)

[051] One aspect of the invention relates to a method of treating a patient suffering from a condition characterized by cholesterol cell accumulation, the method comprising the steps of:

[052] administering to a subject in need thereof an effective amount of a compound of any one of formulas I-IX, wherein formula I is represented by:

Wherein,

x is O or-N (R)⁷)-；

Y is N or-C (R)⁸)-；

R¹And R²Independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R³is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R²And R³Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R⁵is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)⁹＝CR⁹)_n-aryl or- (CR)⁹＝CR⁹)_n-a heteroaryl group;

R⁶is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring,the ring has 1 or 2 heteroatoms selected from O, N and S;

R⁷is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

Each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

formula II is represented as:

wherein,

x is O or-N (R)⁶)-；

R¹And R²Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R⁵is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁶is hydrogen, alkyl, heteroalkyl, haloAlkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹And R⁶Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

formula III is represented as:

wherein,

R¹is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or- (C (R) ⁷)₂)_n-(CR⁷＝C(R⁷)₂)；

R²Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or alkyl;

R³is hydrogen, alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸) (ii) a And is

R⁴And R⁵Independently represents H or alkyl; or R⁴And R⁵Together form a bond;

each R⁶And R⁷Independently represents H or alkyl;

each R⁸Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

A¹And A²Independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl,-(CR⁷＝CR⁷) -aryl or- (CR)⁷＝CR⁷) -a heteroaryl group;

formula IV is represented as:

wherein,

a is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R¹is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³＝CR³) -aryl or- (CR)³＝CR³) -a heteroaryl group;

R²is alkyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each R³Independently represents H or alkyl;

R⁴is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

Formula V is represented as:

wherein,

x is O, -N (R)⁵)-、-N(R⁵)C(O)-、-C(O)N(R⁵)-、-OC(O)-、-CO₂-or-N (R)⁵)CO₂-；

Y is O, S or-N (R)⁵)-；

R¹Is a cycloalkenyl group,Heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

R³and R⁴Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VI is represented as:

wherein,

x is O, S or-N (R)⁴)-；

R¹Is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；

R²Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group;

R³is H, alkyl, alkenyl, aryl or heteroaryl; or R²And R³Together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S;

each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VII is represented by:

wherein,

x is O or S;

R¹is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or-C (O) R ⁵；

R²Is H or alkyl;

R³is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R⁴is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

R⁵Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R⁵Is an alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; and is

Each R⁶Independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

formula VIII is represented as:

wherein,

x is O or S;

R¹、R³and A independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R²and R⁴Independently represents H or alkyl;

R⁵is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S;

formula IX is represented as:

wherein,

X¹is-OR⁵、-SR⁵or-N (R)⁵)₂；

Each X²Independently represent O, S or-N (R)⁵)-；

Each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl, -N (R) ⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R)⁵)₂；

R²And R⁴Independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R³is H, alkyl or halogen;

each R⁵Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each R⁶Independently represent alkyl, cycloalkyl, heterocycleAlkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and is

n is 0, 1, 2, 3 or 4.

[053] In certain embodiments, the present invention relates to the aforementioned method, wherein said disorder is niemann-pick disease type C.

[054] In certain embodiments, the present invention relates to the aforementioned method, wherein said disorder is atherosclerosis.

[055] In certain embodiments, the invention relates to the aforementioned method, wherein said disorder is a lysosomal storage disorder caused by a disturbance in sphingolipid or glycosphingolipid metabolism

[056] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I.

[057]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is N; r¹And R²Independently represents an alkyl group, a haloalkyl group or an aryl group; r ³Is an aryl group; or R²And R³Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

[058]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is N; r¹And R²Is an aryl group; r³Is an aryl group; or R²And R³Together form a can be oneOr a 3-to 8-membered ring optionally substituted with a plurality of alkyl, halogen, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

[059]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is-C (R)⁸)-；R¹And R²Independently represents alkyl, heteroalkyl or haloalkyl; r³Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁴Is hydrogen; r⁵Is a heteroaryl group; r⁶Is H or alkyl; r⁷Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁸Is H or alkyl.

[060] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula II.

[061]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula II, X is-N (R)⁷)-；R¹、R²And R⁵Independently represents aryl or heteroaryl; and R is³、R⁴And R⁶Independently represents hydrogen or alkyl.

[062] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III.

[063]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III; r¹、R²、A¹And A²Independently represents aryl or heteroaryl; r³Is hydrogen or alkyl; r⁶Is H or alkyl; l is a bond.

[064]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III; r ¹Is- (C (R)⁷)₂)_n-(CR⁷＝C(R⁷)₂)；R²Is an alkyl group; r³Is alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；R⁴And R⁵Independently represents H or alkyl; or R⁴And R⁵Together form a bond; each R⁶And R⁷Independently represents H or alkyl; each R⁸Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and A is¹And A²Independently represents an alkyl or heteroalkyl group.

[065]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III, wherein R is¹Contains one carboxylic acid group; r¹Is a carboxylic acid substituted aryl group; r¹Is a carboxylic acid substituted phenyl group; and/or R¹Is a phenyl group para-substituted with a carboxylic acid;

[066] in certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IV.

[067]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IV; A. r¹And R⁴Independently represents aryl or heteroaryl; r¹Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³＝CR³) -aryl or- (CR)³＝CR³) -a heteroaryl group; r²Is alkyl or aryl; each R ³Independently represents H or alkyl.

[068] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula V.

[069]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula V; x is-C (O) N (R)⁵) -or-CO₂-; y is O or S; r¹、R³And R⁴Independently represents aryl or heteroaryl; each R²Independently represent H or alkyl, or 2R²Together form ═ O; r³And R⁴Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[070] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI.

[071]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is aryl, heteroaryl, - (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[072]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r ¹Is aryl, R²Is aryl, R³Is H or alkyl.

[073]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is alkoxy-substituted phenyl, R²Is a dialkylamino-substituted phenyl radical, R³Is H.

[074]In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound is aA compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is- (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; r³Is H or alkyl; r²And R³Together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[075] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII.

[076]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Is alkyl, heteroaryl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r ⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, aralkyl or heterocycloalkyl.

[077]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³RepresentsR⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, aralkyl or heterocycloalkyl; m is 0, 1, 2, 3 or 4; each R⁷Independently represents halogen, hydroxy, amino, carboxy, nitro, cyano, alkyl or alkoxy.

[078] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII.

[079]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r ⁵Is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

[080]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Represents

Wherein n is 0, 1, 2, 3 or 4; each R⁷Independently represents halogen, hydroxy, amino, carboxy, nitro, cyano, alkyl or alkoxy.

[081] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IX;

[082]in certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IX; x¹is-N (R)⁵)₂(ii) a Each X²Independently represents O or S; each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, -N (R)⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R)⁵)₂；R²And R⁴Independently represents aryl, heteroaryl, aralkyl or heteroaralkyl; r³Is H, alkyl or halogen; each R⁵Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and n is 0, 1, 2, 3 or 4.

[083] In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound is

[084] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is any one of formulas X-XXXIX as described below.

[085] Another aspect of the invention relates to a method of reducing the amount of cholesterol in a cell, comprising the steps of:

[086] exposing cells of a mammal to a compound of any one of formulae I-IX, wherein formula I is represented as:

wherein,

x is O or-N (R)⁷)-；

Y is N or-C (R)⁸)-；

R¹And R²Each independently represents alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R⁵is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR) ⁹＝CR⁹)_n-aryl or- (CR)⁹＝CR⁹)_n-a heteroaryl group;

R⁶is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

formula II is represented as:

wherein,

x is O or-N (R)⁶)-；

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

formula III is represented as:

wherein,

R¹is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or- (C (R)⁷)₂)_n-(CR⁷＝C(R⁷)₂)；

R³is hydrogen, alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；

each R⁶And R⁷Independently represents H or alkyl;

l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

A¹And A²Independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)⁷＝CR⁷) -aryl or- (CR)⁷＝CR⁷) -a heteroaryl group;

formula IV is represented as:

wherein,

each R³Independently represents H or alkyl;

formula V is represented as:

wherein,

Y is O, S or-N (R)⁵)-；

R¹Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VI is represented as:

wherein,

x is O, S or-N (R)⁴)-；

R¹Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；

R²Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR) ⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group;

each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VII is represented by:

wherein,

x is O or S;

R¹is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or-C (O) R⁵；

R²Is H or alkyl;

R³is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R⁴is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

R⁵Cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl; or R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; and is

formula VIII is represented as:

wherein,

x is O or S;

R²And R⁴Independently represents H or alkyl;

formula IX is represented as:

wherein,

X¹is-OR⁵、-SR⁵or-N (R)⁵)₂；

Each X²Independently represent O, S or-N (R)⁵)-；

Each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl, -N (R)⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R)⁵)₂；

R²And R⁴Each independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R³is H, alkyl or halogen;

each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and is

n is 0, 1, 2, 3 or 4.

[087] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound reduces the amount of cholesterol in the cell by increasing cholesterol efflux from the cell.

[088] In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound reduces the amount of cholesterol by inhibiting cholesterol uptake by the cell.

[089] In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound reduces the amount of cholesterol in the cell by inhibiting cholesterol synthesis in the cell.

[090] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound reduces the amount of cholesterol in the cell by promoting esterification of cholesterol in the cell.

[091] In certain embodiments, the present invention relates to the aforementioned method, wherein the cell is a human cell.

[092] In certain embodiments, the invention relates to the aforementioned method, wherein the cell has a niemann-pick defect type C.

[093] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I.

[094]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is N; r¹And R²Independently represents an alkyl group, a haloalkyl group or an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r ⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

[095]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is N; r¹And R²Is an aryl group; r³Is aryl radical(ii) a Or R²And R³Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

[096]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is-C (R)⁸)-；R¹And R ²Independently represents alkyl, heteroalkyl or haloalkyl; r³Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁴Is hydrogen; r⁵Is a heteroaryl group; r⁶Is H or alkyl; r⁷Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁸Is H or alkyl.

[097] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula II.

[098]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula II, X is-N (R)⁷)-；R¹、R²And R⁵Independently represents aryl or heteroaryl; and R is³、R⁴And R⁶Independently represents hydrogen or alkyl.

[099] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III.

[0100]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III; r¹、R²、A¹And A²Independently represents aryl or heteroaryl; r³Is hydrogen or alkyl; r⁶Is H or alkyl; l is a bond.

[0101]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III; r¹Is- (C (R)⁷)₂)_n-(CR⁷＝C(R⁷)₂)；R²Is an alkyl group; r³Is alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；R⁴And R ⁵Independently represents H or alkyl; or R⁴And R⁵Together form a bond; each R⁶And R⁷Independently represents H or alkyl; each R⁸Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and A is¹And A²Independently represents an alkyl or heteroalkyl group.

[0102]In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound is a compound having formula III, R¹Comprises one carboxylic acid group; r¹Is a carboxylic acid substituted aryl group; r¹Is a carboxylic acid substituted phenyl group; and/or R¹Is a phenyl group para-substituted with a carboxylic acid.

[0103] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IV.

[0104]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IV; A. r¹And R⁴Independently represents aryl or heteroaryl; r¹Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³＝CR³) -aryl or- (CR)³＝CR³) -a heteroaryl group; r²Is alkyl or aryl; each R³Independently represents H or alkyl.

[0105] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula V.

[0106]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula V; x is-C (O) N (R)⁵) -or-CO₂-; y is O or S; r¹、R³And R⁴Independently represents aryl or heteroaryl; each R²Independently represent H or alkyl, or 2R²Together form ═ O; r³And R⁴Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[0107] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI.

[0108]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is aryl, heteroaryl, - (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[0109]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, R²Is aryl, R³Is H or alkyl.

[0110]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r ¹Is alkoxy-substituted phenyl, R²Is a dialkylamino-substituted phenyl radical, R³Is H.

[0111]In some implementationsIn another embodiment, the invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is- (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; r³Is H or alkyl; r²And R³Together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[0112] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII.

[0113]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Is alkyl, heteroaryl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; each R ⁶Independently represents H, alkyl, aryl, aralkyl or heterocycloalkyl.

[0114]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Represents

R⁴Is H, alkyl,-CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, aralkyl or heterocycloalkyl; m is 0, 1, 2, 3 or 4; each R⁷Independently represents halogen, hydroxy, amino, carboxy, nitro, cyano, alkyl or alkoxy.

[0115] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII.

[0116]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

[0117]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII; x is O or S; r ¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Represents

[0118] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IX.

[0119]In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a peptideA compound of formula IX; x¹is-N (R)⁵)₂(ii) a Each X²Independently represents O or S; each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, -N (R)⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R)⁵)₂；R²And R⁴Each independently represents aryl, heteroaryl, aralkyl or heteroaralkyl; r³Is H, alkyl or halogen; each R⁵Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and n is 0, 1, 2, 3 or 4.

[0120] In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound is

[0121] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of any one of formulas X-XXXIX as described below.

[0122] Another aspect of the present invention relates to a method for treating or preventing a drug-induced phosphatidic disease, comprising the steps of:

[0123] administering to a subject in need thereof an effective amount of a compound of any one of formulas I-IX, wherein formula I is represented by:

wherein,

x is O or-N (R)⁷)-；

Y is N or-C (R)⁸)-；

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R⁷Is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹And R⁷Together form a quiltOne or more alkyl, halogen, hydroxy, alkoxy or amino optionally substituted 3-8 membered rings;

each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

formula II is represented as:

wherein,

x is O or-N (R)⁶)-；

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R⁶is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R ¹And R⁶Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

formula III is represented as:

wherein,

R³is hydrogen, alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸) (ii) a And is

each R⁶And R⁷Independently represents H or alkyl;

l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

formula IV is represented as:

wherein,

each R³Independently represents H or alkyl;

formula V is represented as:

wherein,

Y is O, S or-N (R)⁵)-；

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

R³and R⁴Independently represents cycloalkyl, heterocycloalkyl, or,Cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VI is represented as:

wherein,

x is O, S or-N (R)⁴)-；

Each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VII is represented by:

wherein,

x is O or S;

R²Is H or alkyl;

R⁴is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

R⁵Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; and is

formula VIII is represented as:

wherein,

x is O or S;

R²and R⁴Independently represents H or alkyl;

formula IX is represented as:

Wherein,

X¹is-OR⁵、-SR⁵or-N (R)⁵)₂；

Each X²Independently represent O, S or-N (R)⁵)-；

R³is H, alkyl or halogen;

n is 0, 1, 2, 3 or 4.

[0124] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I.

[0125]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is N; r¹And R²Independently represents an alkyl group, a haloalkyl group or an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r ⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

[0126]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is N; r¹And R²Is an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

[0127]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is-C (R) ⁸)-；R¹And R²Independently represents alkyl, heteroalkyl or haloalkyl; r³Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁴Is hydrogen; r⁵Is a heteroaryl group; r⁶Is H or alkyl; r⁷Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁸Is H or alkyl.

[0128] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula II.

[0129]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula II, X is-N (R)⁷)-；R¹、R²And R⁵Independently represents aryl or heteroaryl; and R is³、R⁴And R⁶Independently represents hydrogen or alkyl.

[0130] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III.

[0131]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III; r¹、R²、A¹And A²Independently represents aryl or heteroaryl; r³Is hydrogen or alkyl; r⁶Is H or alkyl; l is a bond.

[0132]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III; r¹Is- (C (R)⁷)₂)_n-(CR⁷＝C(R⁷)₂)；R²Is an alkyl group; r³Is alkyl, -CO₂R⁸or-C (O) N (R) ⁷)(R⁸)；R⁴And R⁵Independently represents H or alkyl; or R⁴And R⁵Together form a bond; each R⁶And R⁷Independently represents H or alkyl; each R⁸Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and A is¹And A²Independently represents an alkyl or heteroalkyl group.

[0133]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula III; r¹Contains one carboxylic acid group; r¹Is a carboxylic acid substituted aryl group; r¹Is a carboxylic acid substituted phenyl group; and/or R¹Is a phenyl group para-substituted with a carboxylic acid.

[0134] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IV.

[0135]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IV; A. r¹And R⁴Independently represents aryl or heteroaryl; r¹Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³＝CR³) -aryl or- (CR)³＝CR³) -a heteroaryl group; r²Is alkyl or aryl; each R³Independently represents H or alkyl.

[0136] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula V.

[0137]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula V; x is-C (O) N (R)⁵) -or-CO₂-; y is O or S; r¹、R³And R⁴Independently represents aryl or heteroaryl; each R²Independently represent H or alkyl, or 2R²Together form ═ O; r³And R⁴Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2;

[0138] in certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI.

[0139]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is aryl, heteroaryl, - (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; r³Is H or alkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[0140]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, R²Is aryl, R³Is H or alkyl.

[0141]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is alkoxy-substituted phenyl, R²Is a dialkylamino-substituted phenyl radical, R³Is H.

[0142]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is- (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; r³Is H or alkyl; r²And R³Together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

[0143] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII.

[0144]In certain embodiments, the present invention relates to the foregoingThe method, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Is alkyl, heteroaryl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁴Is H, alkyl, -CO ₂R⁶or-C (O) N (R)⁶)₂；R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, aralkyl or heterocycloalkyl.

[0145]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³RepresentsR⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂Aryl groups optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, aralkyl or heterocycloalkyl; m is 0, 1, 2, 3 or 4; each R⁷Independently represents halogen, hydroxy, amino, carboxy, nitro, cyano, alkyl or alkoxy.

[0146] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII.

[0147]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

[0148]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Represents

[0149] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IX.

[0150]In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of formula IX; x¹is-N (R)⁵)₂(ii) a Each X²Independently represents O or S; each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, -N (R)⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R)⁵)₂；R²And R⁴Independently represents aryl, heteroaryl, aralkyl or heteroaralkyl; r³Is H, alkyl or halogen; each R⁵Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and n is 0, 1, 2, 3 or 4.

[0151] In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound is

[0152] In certain embodiments, the present invention relates to the aforementioned method, wherein the compound is a compound of any one of formulas X-XXXIX as described below.

[0153]In certain embodiments, the present invention relates to the aforementioned methods, wherein the compound is

[0154] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the patient is a mammal.

[0155] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the patient is a primate, equine, canine, or feline.

[0156] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the patient is a human.

[0157]Compounds and compositions of the invention

[0158] One aspect of the present invention relates to a compound represented by formula X:

wherein,

x is OH or N (R)⁵)₂；

R²And R⁴Independently represents cycloalkenyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl or heteroaryl, said heteroaryl having 1 heteroatom selected from N, O or S;

R³Is H, alkyl or halogen;

each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

n is 0, 1, 2, 3 or 4; and is

With the proviso that when X is NH₂When R is²And R⁴At least one of which is not an aryl group.

In certain embodiments, the present invention relates to the aforementioned compounds, wherein said X is NH₂And R is²Is an aryl group.

In certain embodiments, the present invention relates to the aforementioned compounds, wherein said X is NH₂And R is⁴Is an aryl group.

[0159] Another aspect of the invention relates to a compound represented by formula XI:

wherein,

R¹and R³Independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R²and R⁴Independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; or R¹And R²Together form a 3-8 membered ring; or R³And R⁴Together form a 3-8 membered ring;

R⁵、R⁶、R⁷And R⁸Independently represents hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; or R⁵、R⁶、R⁷And R⁸Together form a group selected from at least one₂-C₆) Alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl, -N (R)⁹)₂、-OR⁹、-C(O)R⁹、-CO₂R⁹Or C (O) N (R)⁹)₂Aryl or heteroaryl substituted with the functional group of (a); and is

Each R⁹Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

[0160]In certain embodiments, the present invention relates to the aforementioned compounds, wherein R is¹And R²Forming a 6-membered ring.

[0161]In certain embodiments, the present invention relates to the aforementioned compounds, wherein R is³And R⁴Forming a 6-membered ring.

[0162]In certain embodiments, the present invention relates to the aforementioned compounds, wherein R is⁵、R⁶、R⁷And R⁸Together forming an aromatic ring.

[0163] Another aspect of the invention relates to a compound represented by formula XII:

wherein,

x is O, S or-N (R)⁴)-；

R¹Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R²is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)⁵＝CR⁵) -aryl or- (CR) ⁵＝CR⁵) -a heteroaryl group;

each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XIII:

wherein,

x is O, S or-N (R)⁷)-；

R⁴Is- (C (R)⁸)₂)_n-(CR⁸＝C(R⁸)₂)；

R⁵Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or- (CR)⁸＝C(R⁸)₂)；

R⁶Is H, alkyl, alkenyl, aryl or heteroaryl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S;

each R⁷And R⁸Independently represents H, alkyl, cycloalkyl or heterocycloalkyl, aralkyl or heteroaralkyl; and is

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XIV:

wherein,

x is O, S or-N (R)¹²)-；

R⁹Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, -aryl-OR¹⁴Heteroaryl, aralkyl or heteroaralkyl;

R¹⁰is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)¹³＝CR¹³) -aryl or- (CR)¹³＝CR¹³) -a heteroaryl group;

R¹¹is H, alkyl, alkenyl, aryl or heteroaryl;

Each R¹²And R¹³Independently represents H, alkyl, aryl or aralkyl;

R¹⁴is heteroalkylHeterocycloalkyl, heteroaryl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl; and is

n is 1, 2, 3, 4 or 5.

[0164]In certain embodiments, the present invention relates to the aforementioned compounds of formula XIII, wherein X is S and R⁴Is an allyl group.

[0165]In certain embodiments, the invention relates to the aforementioned compounds of formula XIII, wherein X is S, R⁴Is allyl and R⁵Is- (CR)⁸＝C(R⁸)₂)。

[0166]In certain embodiments, the present invention relates to the aforementioned compounds of formula XIV, wherein X is S and R⁹is-allyl-OR¹⁴。

[0167]In certain embodiments, the present invention relates to the aforementioned compounds of formula XIV, wherein X is S and R⁹is-allyl-OR¹⁴，R¹⁰Is aryl, and R¹¹Is H.

[0168] Another aspect of the invention relates to a compound represented by formula XIVa:

wherein,

x is O, S or-N (R)¹²)-；

R¹¹is H, alkyl, alkenyl, aryl or heteroaryl; or R ¹⁰And R¹¹Together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S;

each R¹²And R¹³Independently represents H, alkyl, aryl or aralkyl;

n is 1, 2, 3, 4 or 5.

[0169]In certain embodiments, the invention relates to the aforementioned compound of formula XIVa, wherein X is S and R is⁹Is an aryl group.

[0170] Another aspect of the invention relates to a compound represented by formula XV:

wherein,

R²is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

each R³Independently represents H or alkyl; and is

or a compound represented by formula XVI:

wherein,

R⁵is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, - (CR) ⁷＝CR⁷) -aryl or- (CR)⁷＝CR⁷) -a heteroaryl group;

R⁶is alkyl or aryl;

each R⁷Independently represents H or alkyl; and is

R⁸Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

or a compound represented by formula XVII:

wherein,

R⁹is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)¹¹＝CR¹¹) -aryl or- (CR)¹¹＝CR¹¹) -a heteroaryl group;

R¹⁰is alkyl or aryl;

each R¹¹Independently represents H or alkyl; and is

R¹²Is cycloalkyl, heterocycloalkyl, cycloalkenyl,Heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

[0171]In certain embodiments, the present invention relates to the aforementioned compounds of formula XVI, wherein a is heteroaryl and R is⁶Is an aryl group.

[0172]In certain embodiments, the present invention relates to the aforementioned compounds of formula XVI, wherein A is heteroaryl and R is⁶Is an alkyl group.

[0173]In certain embodiments, the present invention relates to the aforementioned compounds of formula XVII, wherein A is heteroaryl and R is⁹Is aryl, and R¹⁰Is an alkyl group.

[0174]In certain embodiments, the present invention relates to the aforementioned compounds of formula XVII, wherein A is heteroaryl and R is ⁹Is aryl, and R¹⁰Is an aryl group.

[0175] Another aspect of the invention relates to a compound represented by formula XVIII:

wherein,

R¹is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl;

R²is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or alkyl;

R³is hydrogen, alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；

each R⁶And R⁷Independently represents H or alkyl;

each R⁸Independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

or a compound represented by formula XIX:

wherein,

R⁹is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or- (C (R)¹⁵)₂)_n-(CR¹⁵＝C(R¹⁵)₂)；

R¹⁰Is an aryl group;

R¹¹is hydrogen, alkyl, -CO₂R¹⁶or-C (O) N (R)¹⁵)(R¹⁶)；

R¹²And R¹³Independently represents H or alkyl; or R¹²And R¹³Together form a bond;

each R¹⁴And R¹⁵Independently represents H or alkyl;

Each R¹⁶Independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

l is a bond,-C(R¹⁵)₂-or- (CR)¹⁵＝CR¹⁵)-；

A³Represents a divalent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)¹⁵＝CR¹⁵) -aryl-or- (CR)¹⁵＝CR¹⁵) -heteroaryl-; and is

A⁴Represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)¹⁵＝CR¹⁵) -aryl or- (CR)¹⁵＝CR¹⁵) -a heteroaryl group;

or a compound represented by formula XX:

wherein,

R¹⁷is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or- (C (R)²³)₂)_n-(CR²³＝C(R²³)₂)；

R¹⁸Is an aryl group;

R¹⁹is hydrogen, alkyl, -CO₂R²⁴or-C (O) N (R)²³)(R²⁴)；

R²⁰And R²¹Independently represents H or alkyl; or R²⁰And R²¹Together form a bond;

each R²²And R²³Independently represents H or alkyl;

each R²⁴Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

l is a bond, -C (R)²³)₂-or- (CR)²³＝CR²³)-；

A⁵Represents a divalent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)²³＝CR²³) -aryl-or- (CR)²³＝CR²³) -heteroaryl-; and is

A⁶Represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, - (CR) ²³＝CR²³) -aryl or- (CR)²³＝CR²³) -a heteroaryl group;

or a compound represented by formula XXI:

wherein,

R²⁵is- (C (R)³¹)₂)_n-(CR³¹＝C(R³¹)₂)；

R²⁶Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R²⁷is hydrogen, alkyl, -CO₂R³²or-C (O) N (R)³¹)(R³²)；

R²⁸And R²⁹Independently represents H or alkyl; or R²⁸And R²⁹Together form a bond;

each R³⁰And R³¹Independently represents H or alkyl;

each R³²Independently represent alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl;

l is a bond, -C (R)³¹)₂-or- (CR)³¹＝CR³¹) -; and is

A⁷And A⁸Independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³¹＝CR³¹) -aryl or- (CR)³¹＝CR³¹) -a heteroaryl group.

[0176]In certain embodiments, the present invention relates to a compound of formula XVIII as described previously, wherein R is¹Is an aryl group.

[0177]In certain embodiments, the present invention relates to a compound of formula XVIII as described previously, wherein R is¹Is aryl, and R⁴And R⁵Together forming a key.

[0178]In certain embodiments, the present invention relates to a compound of formula XVIII as described previously, wherein R is¹Is aryl, R⁴And R⁵Together form a bond, L is a bond, and A¹Is a heteroaryl group.

[0179]In certain embodiments, the present invention relates to a compound of formula XVIII as described previously, wherein R is ¹Is aryl, R⁴And R⁵Together form a bond, L is a bond, A¹Is heteroaryl, and A²Is an aryl group.

[0180]In certain embodiments, the present invention relates to a compound of formula XVIII as described previously, wherein R is¹Contains one carboxylic acid group; r¹Is a carboxylic acid substituted aryl group; r¹Is a carboxylic acid substituted phenyl group; and/or R¹Is a phenyl group para-substituted with a carboxylic acid.

[0181]In certain embodiments, the present invention relates to a compound of formula XX as described previously, wherein R is¹⁷Contains one carboxylic acid group; r¹⁷Is a carboxylic acid substituted aryl group; r¹⁷Is a carboxylic acid substituted phenyl group; and/or R¹⁷Is a phenyl group para-substituted with a carboxylic acid.

[0182]In certain embodiments, the invention relates to the aforementioned formulaXXI of the formula²⁵Is an aryl group.

[0183]In certain embodiments, the present invention relates to a compound of formula XXI as described above, wherein R is²⁵Is aryl, and R²⁷is-CO₂R³²。

[0184]In certain embodiments, the present invention relates to a compound of formula XXI as described above, wherein R is²⁵Is aryl, R²⁷is-CO₂R³²And A is⁷Is a heteroaryl group.

[0185]In certain embodiments, the present invention relates to a compound of formula XXI as described above, wherein R is²⁵Is aryl, R²⁷is-CO₂R³²，A⁷Is heteroaryl and A⁸Is an aryl group.

[0186] Another aspect of the invention relates to a compound represented by formula XXII:

Wherein,

Y is O, S or-N (R)⁵)-；

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

R³independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R⁴independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XXIII:

wherein,

x is O, -N (R)¹⁰)-、-N(R¹⁰)C(O)-、-C(O)N(R¹⁰)-、-OC(O)-，-CO₂-or-N (R)¹⁰)CO₂-；

Y is O, S or-N (R)¹⁰)-；

R⁶Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each R⁷Independently represent H or alkyl, or 2R⁷Together form ═ O;

R⁸is an aryl group;

R⁹represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

each R¹⁰Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XXIV:

wherein,

x is O, -N (R)¹⁵)-、-N(R¹⁵)C(O)-、-C(O)N(R¹⁵)-、-OC(O)-、-CO₂-or-N (R)¹⁵)CO₂-；

Y is O, S or-N (R) ¹⁵)-；

R¹¹Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

each R¹²Independently represent H or alkyl, or 2R⁷Together form ═ O;

R¹³is an aryl group;

R¹⁴represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

each R¹⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5.

[0187]In certain embodiments, the present invention relates to a compound of formula XXII as described above, wherein R is⁴Is aryl, X is NH, and R¹Is an aryl group.

[0188]In certain embodiments, the present invention relates to the aforementioned compounds of formula XXIII, wherein R is⁴Is aryl, X is NH, and R⁶Is an aryl group.

[0189]In certain embodiments, the present invention relates to a compound of formula XXIV as described above, wherein R is¹³Is aryl, R¹⁴Is aryl and X is NH.

[0190] Another aspect of the invention relates to a compound represented by formula XXV:

wherein,

x is O or S;

R¹is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or-C (O) R⁵；

R²Is H or alkyl;

R⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

or a compound represented by formula XXVI:

wherein,

x is O or S;

R⁷is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or-C (O) R¹¹；

R⁸Is H or alkyl;

R⁹is an aryl group;

R¹⁰is H, alkyl or-C (O) N (R)¹²)₂；

R¹¹Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R¹¹Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR¹²、-N(R¹²)₂、-CO₂R¹²、C(O)N(R¹²)₂Aryl groups optionally substituted with cyano or nitro; and is

Each R¹²Independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

or a compound represented by formula XXVII:

wherein,

x is O or S;

R¹³is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or-C (O) R¹⁷；

R¹⁴Is H or alkyl;

R¹⁵is an aryl group;

R¹⁶is H, alkyl, -CO₂R¹⁸or-C (O) N (R) ¹⁸)₂；

R¹⁷Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; and is

Each R¹⁸Independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

or a compound represented by formula XXVIII:

wherein,

x is O or S;

R¹⁹is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or-C (O) R²³；

R²⁰Is H or alkyl;

R²¹is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R²²is alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

R²³Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R²³Is a halogen atom which may be substituted by one OR more alkyl, halogen, -OR¹⁸、-N(R¹⁸)₂、-CO₂R¹⁸、C(O)N(R¹⁸)₂Aryl groups optionally substituted with cyano or nitro; and is

Each R¹⁸Independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

[0191]In certain embodiments, the invention relates to a compound of the aforementioned formula XXV, wherein X is S, R¹is-C (O) R⁵，R²Is H, and R⁴is-CO₂R⁶。

[0192]In certain embodiments, the invention relates to a compound of the aforementioned formula XXVI, wherein X is S, R ⁷is-C (O) R¹¹And R is⁸Is H.

[0193]In certain embodiments, the invention relates to a compound of the aforementioned formula XXVII, wherein X is S, R¹³is-C (O) R¹⁷，R¹⁴Is H, and R¹⁶is-CO₂R¹⁸。

[0194] Another aspect of the invention relates to a compound represented by formula XXIX:

wherein,

x is O;

y is C (R)⁸)-；

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

Each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

or a compound represented by formula XXX:

wherein

X is-N (R)¹⁶)-；

Y is-C (R)¹⁷)-；

R¹⁰And R¹¹Each independently represents alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹²is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹¹And R¹²Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

R¹³is H, alkyl, cycloalkyl, aryl or aralkyl;

R¹⁴is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)¹⁸＝CR¹⁸)_n-aryl or- (CR)¹⁸＝CR¹⁸)_n-a heteroaryl group;

R¹⁵is H or alkyl; or R¹⁴And R¹⁵Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R¹⁶is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R ¹⁰And R¹⁶Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

each R¹⁷And R¹⁸Independently represents H or alkyl; and is

n is 1 or 2.

[0195]In certain embodiments, the present invention relates to compounds of formula XXX as described previously, wherein R is¹⁶、R¹⁰、R¹¹And R¹²Is an aryl group.

[0196]In certain embodiments, the present invention relates to compounds of formula XXX as described previously, wherein R is¹⁶、R¹⁰、R¹¹And R¹²Is aryl, and R¹³Is H.

[0197]In certain embodiments, the present invention relates to compounds of formula XXX as described previously, wherein R is¹⁶、R¹⁰、R¹¹And R¹²Is aryl, R¹³Is H, and R¹⁴Is an alkyl group.

[0198] Another aspect of the invention relates to a compound represented by formula XXXI:

wherein,

R²and R⁴Independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; or R¹And R²Together form a 3-8 membered ring; or R³And R⁴Together form a 3-8 membered ring; and is

Each R⁵Independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

or a compound represented by formula XXXII:

wherein,

R⁶and R⁸Each independently represents alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁷and R⁹Each independently represents hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; or R⁶And R⁷Together form a 3-8 membered ring; or R⁸And R⁹Together form a 3-8 membered ring; and is

Each R¹⁰Independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl.

[0199]In certain embodiments, the invention relates to compounds of formula XXXI as described previouslyIn which R is¹And R²Together form a 7-membered ring, and R³And R⁴Together forming a 7-membered ring.

[0200]In certain embodiments, the present invention relates to compounds of formula XXXI as described previously, wherein R is¹And R²Together form a 7-membered ring, R³And R⁴Together form a 7-membered ring, and R⁵Is an aryl group.

[0201]In certain embodiments, the present invention relates to compounds of formula XXXII as described previously, wherein R is ¹And R²Together form a 7-membered ring, R³And R⁴Together form a 7-membered ring, and R⁵Is an alkyl group.

[0202]In certain embodiments, the present invention relates to compounds of formula XXXI as described previously, wherein R is¹And R³Is an aryl group.

[0203]In certain embodiments, the present invention relates to compounds of formula XXXI as described previously, wherein R is¹、R³And R⁵Is an aryl group.

[0204] Another aspect of the invention relates to a compound represented by formula XXXIII:

wherein,

x is O;

R²and R⁴Independently represents H or alkyl;

R⁵is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S；

Or a compound represented by formula XXXIV:

wherein,

x is S;

R⁶represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R⁸and A independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁷and R⁹Independently represents H or alkyl;

R¹⁰is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S;

or a compound represented by formula XXXV:

wherein,

x is S;

R¹¹And A independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹³represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl；

R¹²And R¹⁴Independently represents H or alkyl;

R¹⁵is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S;

or a compound represented by formula XXXVI:

wherein,

x is S;

R¹⁶and R¹⁸Independently represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

a represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R¹⁷and R¹⁹Independently represents H or alkyl;

R²⁰is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

[0205]In certain embodiments, the present invention relates to a compound of formula XXXIV as described previously, wherein X is O; a is aryl, R¹⁰Is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S, and R⁸Is an aryl group.

[0206]In certain embodiments, the present invention relates to a compound of formula XXXV as described previously, wherein X is S; a is aryl, R¹⁵Is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S, and R ¹¹Is an aryl group.

[0207]In some implementationsIn another embodiment, the invention relates to a compound of formula XXXVI as described above, wherein X is S; r²⁰Is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S, R¹⁸Is aryl, and R¹⁶Is an aryl group.

[0208] Another aspect of the invention relates to a compound represented by formula XXXVII:

wherein,

R¹、R²and R³Independently represents H, aryl, heteroaryl, aralkyl or heteroaralkyl;

a independently represents a monocyclic or bicyclic aryl or heteroaryl group which may be optionally substituted by one or more halogen, alkyl, nitro, amino, aryl, heteroaryl, aralkyl or heteroaralkyl, cycloalkenyl or heterocycloalkenyl groups.

[0209]In certain embodiments, the present invention relates to the aforementioned compounds, wherein R is¹、R²And R³Is H.

[0210]In certain embodiments, the present invention relates to the aforementioned compounds, wherein R is¹、R²And R³Is H, and A is an aryl group substituted with an amino group.

[0211]In certain embodiments, the present invention relates to the aforementioned compounds, wherein R is¹、R²And R³Is H, and A is an aryl group substituted with a nitro group.

[0212]In certain embodiments, the present invention relates to the aforementioned compounds, wherein R is¹、R²And R³Is H and a is an aryl substituted by halogen.

[0213] Another aspect of the invention relates to a compound represented by formula XXXVIII:

wherein,

R¹independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R²and R³Independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and is

Each R⁴Independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl;

or a compound represented by formula XXXIX:

wherein,

R⁵independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁶and R⁷Independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and is

Each R⁸Independently represents H, alkyl, cycloalkyl, aryl, heterocycloalkyl, aralkyl or heteroAn aralkyl group.

[0214]In certain embodiments, the present invention relates to a compound of formula XXXVIII as described previously, wherein R is ¹Is a haloalkyl group.

[0215]In certain embodiments, the present invention relates to a compound of formula XXXVIII as described previously, wherein R is¹Is haloalkyl, and R²And R³Is an aryl group.

[0216]In certain embodiments, the present invention relates to a compound of formula XXXVIII as described previously, wherein R is¹Is haloalkyl, R²And R³Is aryl, and at least one R⁴Is hydrogen.

[0217]In certain embodiments, the invention relates to a compound of formula XXXIX as described previously, wherein R is⁶And R⁷Is an aryl group.

[0218]In certain embodiments, the invention relates to a compound of formula XXXIX as described previously, wherein R is⁶And R⁷Is aryl, and at least one R⁸Is an aryl group.

[0219]In certain embodiments, the invention relates to a compound of formula XXXIX as described previously, wherein R is⁶And R⁷Is aryl, one R⁸Is aryl, one R⁸Is hydrogen.

[0220] Another aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of the general formulae X-XXXIX, wherein the general formulae X-XXXIX are as described above.

[0221]Definition of

[0222] For convenience, some of the terms used in the specification, examples, and appended claims are collected here.

[0223] The term "ACAT" refers to acyl-coa: cholesterol acyltransferase.

[0224] The term "CHO" refers to chinese hamster ovary.

[0225] The term "DMSO" refers to dimethylsulfoxide;

[0226] the term "FBS" refers to fetal bovine serum.

[0227] The term "GC" refers to gas chromatographic analysis.

[0228] The term "HEPES" refers to 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid.

[0229] The term "LBPA" refers to lyso-diphosphatic acid.

[0230] The term "LDL" refers to low density lipoproteins.

[0231] The term "LSO" refers to a lysosomal storage organelle.

[0232] The term "NPC" refers to niemann-pick disease type C.

[0233] The term "PBS" refers to phosphate buffered saline.

[0234] The term "PFA" refers to paraformaldehyde.

[0235] The term "heteroatom" is well known in the art and refers to an atom of any element other than a carbon or hydrogen atom. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur, and selenium.

[0236]The term "alkyl" is art-recognized and includes saturated aliphatic groups including straight-chain alkyl groups, branched-chain alkyl groups, cyclo-alkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, the straight or branched alkyl backbone has about 30 or fewer carbon atoms (e.g., C) ₁-C₃₀Straight chain of (2), C₃-C₃₀Or about 20 or less branches). Likewise, cycloalkyl groups have about 3 to 10 carbon atoms in their ring structure, or about 5, 6, or 7 carbon atoms in their ring structure.

[0237] Unless otherwise indicated for the number of carbon atoms, "lower alkyl" refers to an alkyl group as defined above, but having from 1 to about 10 carbon atoms, alternatively from 1 to about 6 carbon atoms, in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

[0238] The term "heteroalkyl" is art-recognized and includes saturated aliphatic groups containing at least one heteroatom in the chain, including straight-chain alkyl groups containing at least one heteroatom in the chain, branched-chain alkyl groups containing at least one heteroatom in the chain, cyclo-alkyl (alicyclic) groups containing at least one heteroatom in the ring, alkyl-substituted cycloalkyl groups containing at least one heteroatom in the ring, and cycloalkyl-substituted alkyl groups containing at least one heteroatom in the chain. The term "heterocycloalkyl" refers to a cycloalkane (alicyclic) group containing at least one heteroatom in the ring.

[0239] The terms "alkenyl" and "alkynyl" are well known in the art and refer to unsaturated aliphatic groups of similar length to the alkyl groups described above, but containing at least one double or triple bond, respectively. The terms "alkenyl" and "alkynyl" include unsubstituted, unsaturated aliphatic groups, as well as unsaturated aliphatic groups containing one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, carbonyl, and carboxyl.

[0240] The term "cycloalkenyl" refers to an alicyclic group having at least one double bond. The term "cycloalkenyl" includes unsubstituted unsaturated alicyclic groups as well as unsaturated alicyclic groups containing one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, carbonyl, and carboxyl.

[0241] The term "heterocycloalkenyl" refers to an alicyclic group containing at least one double bond and at least one heteroatom selected from the group consisting of N, O and S. The term "heterocycloalkenyl" includes unsubstituted unsaturated alicyclic groups, as well as unsaturated alicyclic groups containing one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, carbonyl, or carboxyl.

[0242]The term "aryl" is art-recognized and refers to 5-, 6-, and 7-membered monocyclic aryl groups whose ring structure is composed of carbon atoms, such as benzene, naphthalene, anthracene, pyrene, and the like. Aromatic hydrocarbonThe aromatic ring may be substituted with a substituent at one or more ring positions. Representative substituents include halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphinate, carbonyl, carboxy, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaromatic moieties (moieity), -CF ₃CN, -CN, etc. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjoining rings (the rings are "fused rings") in which at least one of the rings is aromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.

[0243]The term "heteroaryl" is art-recognized and refers to 5-, 6-, and 7-membered monocyclic aryl groups having 1 to 4 heteroatoms in the ring, such as pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring may be substituted at one or more ring positions with substituents as described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaromatic moieties, -CF ₃CN, -CN, etc. The term "heteroaryl" also includes polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjoining rings (the rings are "fused rings"), at least one of which is heteroaromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.

[0244] The terms ortho, meta, and para are well known in the art and refer to 1, 2-, 1, 3-and 1, 4-disubstituted benzenes, respectively. For example, 1, 2-xylene and ortho-xylene are synonyms.

[0245] The term "aralkyl" is well known in the art and refers to an aryl substituted alkyl group.

[0246] The term "heteroaralkyl" is art-recognized and refers to a heteroaryl-substituted alkyl group.

[0247]The term "heterocycle" or "heterocyclyl" is art-recognized and refers to a 3 to about 10 membered ring structure, or a 3 to about 7 membered ring, which ring structure contains 1 to 4 heteroatoms. Heterocycles may also be polycyclic. Heterocyclic groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, benzopyran, xanthene, phenothiazine, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, naphthyridine, quinoxaline, quinazoline, pteridine, carbazole, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenothiazine, furazan, phenazine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more ring positions with substituents as described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aryl or heterocyclic aromatic moieties, -CF ₃CN, -CN, etc.

[0248]The term "polycyclic" or "polycyclyl" is art-recognized and refers to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbon atoms are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are connected by non-adjacent atoms are referred to as "bridged" rings. Each ring of the plurality of rings may be substituted withSubstituted with substituents as described above, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aryl or heterocyclic aromatic moieties, -CF₃CN, -CN, etc.

[0249] The term "carbocyclic ring" is well known in the art and refers to an aromatic or non-aromatic ring in which each atom in the ring is carbon.

[0250]The term "nitro" is art-recognized and refers to-NO₂(ii) a The term "halogen" is art-recognized and refers to-F, -Cl, -Br, or-I; the term "sulfhydryl" is well known in the art and refers to-SH; the term "hydroxy" refers to-OH. The term "sulfonyl" is art-recognized and refers to-SO ₂ ^-. "halide" refers to the corresponding anion of a halogen, and "halide-like" has the meaning described in Cotton and Wilkinson "Advanced Inorganic Chemistry"page 560 gives a definition.

[0251] The terms "amine" and "amino" are well known in the art and refer to both unsubstituted and substituted amines, such as moieties that can be represented by the general formula:

wherein R50, R51 and R52 each independently represent hydrogen, alkyl, alkenyl, - (CH)₂)_m-R61, or R50 and R51 together with the N atom to which they are attached form a heterocyclic ring having 4 to 8 atoms in the ring structure; r61 represents aryl, cycloalkyl, cycloalkenyl, heterocycle or polycycle; m is 0 or an integer from 1 to 8. In certain embodiments, only one of R50 or R51 may be a carbonyl group, e.g., R50, R51 and nitrogen together do not constitute an imide. In other embodiments, R50 and R51 (and optionally R52) are each independentlyRepresents hydrogen, alkyl, alkenyl or- (CH)₂)_m-R61. Thus, the term "alkylamine" includes an amino group as defined above with a substituted or unsubstituted alkyl group, i.e., at least one of R50 and R51 is an alkyl group.

[0252] The term "amido" is well known in the art and refers to a moiety that can be represented by the general formula:

Wherein R50 is as defined above, R54 represents hydrogen, alkyl, alkenyl or- (CH)₂)_m-R61, wherein m and R61 are as defined above.

[0253] The term "amido" refers in the art to an amino-substituted carbonyl group, including a moiety that can be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of the amides of the present invention do not include imides that may be unstable.

[0254]The term "alkylthio" refers to an alkyl group as defined above having one thio group. In certain embodiments, an "alkylthio" moiety represents-S-alkyl, -S-alkenyl, -S-alkynyl, and-S- (CH)₂)_m-one of R61, wherein m and R61 are as defined above. Representative alkylthio groups include methylthio, ethylthio, and the like.

[0255] The term "carboxy" is well known in the art and includes, for example, moieties that can be represented by the general formula:

wherein X50 is a bond or represents an oxygen or sulfur, R55 and R56 represent hydrogen, alkyl, alkenyl, - (CH)₂)_m-R61 or a pharmaceutically acceptable salt, R56 represents hydrogen, alkyl, alkenyl or- (CH)₂)_m-R61, wherein m and R61 are as defined above. When X50 is oxygen and R55 or R56 is not hydrogen, the formula represents an "ester". When X50 is oxygen and R55 is as defined above, then the moiety is referred to herein as a carboxyl group, particularly when R55 is hydrogen, the formula represents a "carboxylic acid". When X50 is oxygen and R56 is hydrogen, the formula represents "formic acid". In general, when the oxygen atom of the above formula is replaced with sulfur, the formula represents a "thiocarbonyl". When X50 is sulfur and R55 or R56 is not hydrogen, the formula represents a "thioester", and when X50 is sulfur and R55 is hydrogen, the formula represents a "thiocarboxylic acid". When X50 is sulfur and R56 is hydrogen, the formula represents "thiocarboxylic acid". On the other hand, when X50 is a bond and R55 is other than hydrogen, the above formula represents a "keto group". When X50 is a bond and R55 is hydrogen, the above formula represents an "aldehyde group".

[0256]The term "alkoxy" is well known in the art and refers to an alkyl group as described above having an oxy group attached thereto. Typical alkoxy groups include methoxy, ethoxy, propoxy, t-butoxy, and the like. An "ether" is two hydrocarbons covalently linked by one oxygen. Correspondingly, alkyl substituents which are capable of rendering alkyl as ethers are or are analogous to alkoxy, e.g. as defined by-O-alkyl, -O-alkenyl, -O-alkynyl, -O- (CH)₂)_m-one of R61 wherein m and R61 are as described above.

[0257] The term "sulfonate" is well known in the art and refers to a moiety that can be represented by the general formula:

wherein R57 is an electron pair, hydrogen, alkyl, cycloalkyl or aryl.

[0258] The term "sulfate" is well known in the art and refers to a moiety that can be represented by the general formula:

wherein R57 is as defined above.

[0259] The term "sulfonamide" is art-recognized and refers to a moiety that can be represented by the general formula:

wherein R50 and R56 are as defined above.

[0260] The term "sulfamoyl" is art-recognized and refers to a moiety that can be represented by the general formula:

wherein R50 and R51 are as defined above.

[0261] The term "sulfonyl" is art-recognized and refers to a moiety that can be represented by the general formula:

Wherein R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0262] The term "sulfoxido" is art-recognized and refers to a moiety that can be represented by the general formula:

wherein R58 is as defined above.

[0263] The term "phosphoryl" is well known in the art and may be represented by the general formula:

wherein Q50 represents S or O, and R59 represents hydrogen, lower alkyl or aryl. When used to substitute, for example, an alkyl group, the phosphoryl group in a phosphoryl alkyl group may be represented by the general formula:

wherein Q50 and R59 are each independently as defined above, and Q51 represents O, S or N. When Q50 is S, the phosphoryl moiety is a "phosphorothioate".

[0264] The term "phosphoramidite" is well known in the art and can be represented by the general formula:

wherein Q51, R50, R51 and R59 are as defined above.

[0265] The term "phosphoramidite" is well known in the art and can be represented by the following general formula:

wherein Q51, R50, R51 and R59 are as defined above, and R60 represents a lower alkyl group or an aryl group.

[0266] Similar substitutions may be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyl, aminoalkynyl, amidoalkenyl, amidoalkynyl, iminoalkenyl, iminoalkynyl, thioalkenyl, thioalkynyl, carbonyl-substituted alkenyl or alkynyl groups.

[0267] The definition of each expression, such as alkyl, m, n, etc., when it occurs more than once in any structure, is independent of its definition elsewhere in the same structure.

[0268]The term "selenoalkyl" is well known in the art and refers to a seleno-substituted alkyl group. Analogous "selenoethers" substituted on the alkyl radical are selected from the group consisting of-seleno-alkyl, -seleno-alkenyl, -seleno-alkynyl, selenium- (CH)₂)_mOne of R61, m and R61 are as defined above.

[0269] The terms triflyl, tosyl, mesyl, nonamyl are well known in the art and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, perfluorobutanesulfonyl, respectively. The terms triflate, tosylate, mesylate, nonaflatate are well known in the art and refer to trifluoromethanesulfonyl ester, p-toluenesulfonyl ester, methanesulfonyl ester, perfluorobutylsulfonyl ester functional groups, and molecules containing such groups, respectively.

[0270]The abbreviations Me, Et, Ph, Tf, Nf, Ts and MS independently represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, perfluorobutylsulfonyl, p-toluenesulfonyl and methanesulfonyl. Workers of organic chemistry having ordinary skill in the artA broader list of abbreviations is used Journal of Organic Chemistry (Journal of Organic Chemistry)The first period of each volume of (a); this list usually appears under the heading ofList of abbreviation criteriaIn the table (2).

[0271] Certain compounds contained in the components of the present invention may exist in particular geometric or stereoisomeric forms. In addition, the polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis and trans isomers, R-and S-enantiomers, diastereomers, (D) -isomers, (L) -isomers, racemic mixtures thereof, and combinations thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers and mixtures thereof are included in the present invention.

[0272] For example, if a compound of the invention is desired in the form of a particular enantiomer, it may be prepared by asymmetric synthesis or derivatization with chiral auxiliary reagents, wherein the resulting enantiomeric mixture is separated and the auxiliary groups are cleaved to provide the pure desired enantiomer. Alternatively, where a basic functionality, such as an amino group, or an acidic functionality, such as a carboxyl group, is included in the molecule, a salt of an enantiomer may be formed by an acid or base having the appropriate optical activity, and this enantiomer may then be resolved by fractional crystallization and chromatographic means well known in the art, followed by recovery of the pure enantiomer.

[0273] It should be understood that "replace" or "replacing with" encompasses such implicit conditions: such substitution complies with the permitted valences of the substituted atom and substituent, and the substitution results in a stable compound that, for example, does not spontaneously undergo transformations such as rearrangement, cyclization, elimination or other reactions.

[0274] The term "substituted" also includes all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compound substituents. Exemplary substituents include, for example, those described herein above. The permissible substituents may be one or more and may be the same or different for appropriate organic compounds. For purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and/or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. The present invention is not intended to be limited in any way by the permissible substituents of organic compounds.

[0275]The phrase "protecting group" as used herein means a temporary substituent that protects a potentially reactive functional group from undesirable chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl esters of alcohols, and acetals and ketals of aldehydes and ketones. A review of the field of protecting group chemistry has been made (Greene, T.W.; Wuts, P.G.M.protective Groups in organic Synthesis, 2 ^nded., respectively; wiley: new York, 1991). Protected forms of the compounds of the invention are included within the scope of the invention.

[0276]For the purposes of the present invention, the chemical elements are identified in terms of the periodic table of elements, CAS version,handbook of Physics and Chemistry (Handbook of Chemistry and Physics)67 th edition, 1986-87, inner cover.

[0277]Pharmaceutical composition

[0278] In another aspect, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more compounds as described above, formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As detailed below, the pharmaceutically acceptable compositions of the present invention may be in solid or liquid form specifically formulated for administration, including forms suitable for the following: (1) oral administration, e.g., infusion (aqueous or non-aqueous solution or suspension), tablets, e.g., for buccal, sublingual and systemic absorption, boluses, powders, granules, ointments suitable for the tongue; (2) parenteral administration, e.g. by subcutaneous, intramuscular, intravenous or epidural injection, for example in the form of a sterile solution or suspension or sustained release formulation; (3) administration by topical application, for example as a cream, ointment or controlled release patch or spray applied to the skin; (4) intravaginal or intrarectal administration, for example in the form of suppositories, ointments or foams; (5) sublingual administration; (6) administration in the eye; (7) transdermal administration; or (8) nasal administration.

[0279] As used herein, the phrase "therapeutically effective amount" means an amount of a compound, material, or composition comprising a compound of the present invention that produces the desired therapeutic effect on at least a subpopulation of cells of an animal at a reasonable benefit/risk ratio, suitable for any medical treatment.

[0280] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, novel materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0281] The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, for carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be in a sense such that compatibility with the other ingredients of the composition is "acceptable" and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc powder; (8) adjuvants, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) ethylene glycol, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) physiological saline; (18) ringer's solution; (19) ethanol; (20) a pH buffer; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substrates for pharmaceutical formulations.

[0282] As noted above, certain embodiments of the compounds of the present invention may contain a basic functional group, such as amino or alkylamino, and thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" refers in this respect to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts may be formed in situ during the manufacture of the administration vehicle and dosage form, or by separately reacting a pure compound of the invention in free base form with a suitable organic or inorganic acid, and isolating the salt to form it during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, lauric acid, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthalate, mesylate, glucoheptonate, lactobionate, lauryl sulfonate and the like. (see, e.g., Berge et al (1977) "Pharmaceutical Salts", J.pharm.Sci.66: 1-19).

[0283] Pharmaceutically acceptable salts of the subject compounds include conventional non-toxic salts or the quaternary ammonium salts of such compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include salts from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as salts from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-o-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid, oxalic acid, isethionic acid (isothionic) and the like.

[0284] In other instances, the compounds of the present invention may contain one or more acidic functional groups, thereby enabling the formation of pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" refers in this respect to the relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts may be formed in situ during the manufacture of the administration vehicle and dosage form, or by separately reacting the pure compound of the invention in its free acid form with a suitable base, such as a hydroxide, carbonate or bicarbonate salt of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, aluminum salts, and the like. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (see, e.g., the Berge et al article, supra).

[0285] Wetting agents, emulsifiers and lubricating oils, such as sodium lauryl sulfate and magnesium stearate, as well as coloring, releasing, coating, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions of the invention.

[0286] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, dibutyl hydroxyanisole (BHA), di-t-butyl-p-cresol (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0287] Formulations of the invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will depend upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of compound which produces a therapeutic effect. Generally, this amount will range from about 0.1% to about 99% of the active ingredient in 100%, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

[0288] In certain embodiments, the formulations of the present invention comprise an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, polymeric carriers such as polyesters, and polyanhydrides; and a compound of the invention. In certain embodiments, the formulations described above provide the compounds of the present invention with oral bioavailability.

[0289] Methods of preparing these formulations or compositions comprise combining a compound of the invention with a carrier and optionally one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compounds of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0290] Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or an aqueous or non-aqueous suspension, or as an oil-in-water or water-in-oil liquid emulsion, or as a pellet or syrup, or as a lozenge (using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or as an oral wash, each containing a predetermined amount of a compound of the invention as an active ingredient. The compounds of the invention may also be administered in the form of a bolus, electuary or paste.

[0291] In the solid dosage forms for oral administration of the present invention (capsules, tablets, pills, lozenges, powders, granules, trouches, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or calcium hydrogen phosphate and/or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) diluents, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate; (5) cure retarders, such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds and surfactants such as poloxamers and sodium lauryl sulfate; (7) wetting agents, such as cetyl alcohol, glyceryl monostearate, nonionic surfactants; (8) absorbents such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid and mixtures thereof; (10) a colorant; and (11) a controlled release agent such as cross-linked polyvinylpyrrolidone or ethylcellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also include buffering agents. A similar type of solid composition may also be filled into soft and hard shell capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0292] Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared with binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0293] Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as lozenges, capsules, pills and granules, may be embossed or prepared, optionally with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical art. They may also be formulated to provide slow or controlled release of the active ingredient therein by use of, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They can be made to release rapidly, for example by freeze drying. They may be sterilized, for example, by passing through a filter that filters the bacteria, or by adding a sterilizing agent in the form of a sterile solid composition that is soluble in sterile water or other sterile injectable medium at the point of use. These compositions may also optionally contain a light-avoiding agent and may release the active ingredient in a certain part of the gastrointestinal tract only or preferably in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate with one or more of the abovementioned adjuvants.

[0294] Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, peanut, corn germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0295] In addition to inert diluents, oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, preserving, perfuming and preserving agents.

[0296] Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, sorbitol and polyoxyethylene, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0297] The pharmaceutical composition formulation for rectal or vaginal use according to the invention may be a suppository which may be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, suppository wax or salicylic acid and which is solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity to release the active compound.

[0298] Formulations of the invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be suitable.

[0299] Dosage forms for topical or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and snuffs. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0300] Ointments, pastes, creams and gels may contain, in addition to an active compound of the invention, adjuvants, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polypropylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0301] In addition to the active compounds according to the invention, powders and sprays can contain adjuvants such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0302] Transdermal patches have the additional advantage of providing controlled release of the compounds of the present invention to the human body. Such dosage forms may be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may also be used to increase the flux of the compound across the skin. The flux is controlled either by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0303] Ophthalmic formulations, eye ointments, powders, solutions, and the like, are also contemplated within the scope of the present invention.

[0304] Pharmaceutical compositions of the invention suitable for parenteral administration comprise one or more compounds of the invention and one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions prior to use, which may contain sugars, alcohols, antioxidants, buffers, thiobis-dichlorophenol, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0305] Examples of suitable aqueous and nonaqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0306] These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the action of microorganisms on the subject compounds can be ensured by employing various antibacterial and antifungal agents, for example, benzylcarbamate, chlorobutanol, phenol sorbic acid, and the like. It may be desirable to use isotonic agents in the compositions, such as sugars, sodium chloride and the like. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents which delay absorption, such as lead monostearate and gelatin.

[0307] In some cases, it is desirable to slow the absorption of the drug, either subcutaneously or intramuscularly, in order to prolong the effect of the drug. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug depends on its rate of dissolution and thus may depend on the size and crystalline form of the crystals. Alternatively, delayed absorption of a parenterally administered pharmaceutical form is achieved by dissolving or dispersing the drug in an oily vehicle.

[0308] Injectable depot forms are prepared by forming a microcapsule matrix from the subject compounds in biodegradable polymers such as polylactic-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Other examples of biodegradable polymers include polyorthoesters and polyanhydrides. Injectable depot solutions can be achieved by encapsulating the drug in liposomes or microemulsions, which are compatible with human tissue.

[0309] When the compounds of the present invention are administered as pharmaceutical preparations to humans and animals, they may be administered as such, or in the form of a pharmaceutical composition containing, for example, 0.1% to 99% (more preferably 10% to 30%) of the active ingredient together with a pharmaceutically acceptable carrier.

[0310] The formulations of the present invention may be administered orally, by injection, topically or rectally. They are of course administered in a form suitable for each route of administration. For example, they are injected, inhaled in the form of tablets or capsules, injected, infused or inhaled in the form of eye washes, ointments, suppositories; topical administration in the form of a lotion or ointment; rectally in the form of suppositories. Oral administration is preferred.

[0311] As used herein, "parenteral administration" is intended to distinguish administration from other enteral and topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0312] As used herein, "systemic administration" and "peripheral administration" refer to the administration of a compound, drug or other substance indirectly into the central nervous system, such that it enters the body of the patient and is metabolized and other similar processes occur, e.g., subcutaneously.

[0313] These compounds for use in therapy may be administered to humans and other animals by any suitable route of administration, including orally, nasally, e.g., by spraying, rectally, vaginally, by injection, intracisternally, and topically, e.g., by powder, paste, or drops, including buccally and sublingually.

[0314] Regardless of the route of administration chosen, the compounds of the present invention and/or the pharmaceutical compositions of the present invention may be used in a suitable hydrated form and formulated into pharmaceutically acceptable dosage forms by conventional methods well known to those skilled in the art.

[0315] The actual dosage level of the active ingredient in the pharmaceutical composition of the invention may be varied in order to obtain such an amount of active ingredient: it achieves the desired therapeutic response, composition and mode of administration for a particular patient and is non-toxic to the patient.

[0316] The selected dosage level will depend upon a variety of factors including the activity of the particular compound employed in the present invention or an ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the rate and extent of absorption, the period of treatment, other drugs, compounds and/or materials used in conjunction with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0317] A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can initially prescribe a pharmaceutical composition containing a compound of the invention in a lower dose than is required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0318] In general, a suitable daily dosage of a compound of the invention will be the lowest effective dose of such a compound to achieve a therapeutic effect. Such an effective dose will generally depend on the factors described above. In general, dosages of the compounds of the invention for oral, intravenous, intracerebroventricular, and subcutaneous use in humans will range from about 0.0001 to about 100 milligrams per kilogram of body weight per day for analgesic purposes.

[0319] If desired, an effective daily dose of the active compound may be divided into 2, 3, 4, 5, 6 or more divided doses, optionally in unit dose form, administered separately at appropriate intervals throughout the day, the preferred mode of administration being once daily.

[0320] Although the compound of the present invention may be administered alone, it is preferable to administer the compound in the form of a pharmaceutical preparation (composition).

[0321] Like other drugs, the compounds of the invention may be formulated in any form convenient for human or veterinary administration.

[0322] In another aspect, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the subject compounds as described above, together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As detailed below, the pharmaceutical compositions of the present invention may be prepared for administration in particular in solid or liquid form, including forms suitable for administration as follows: (1) oral administration, such as drinkable (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, plasters suitable for the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural space injection, e.g., in the form of sterile solutions or suspensions; (3) topical application, e.g., creams, ointments or sprays applied to the skin, lungs or mucous membranes; or (4) intravaginal or intrarectal administration, e.g., in the form of suppositories, creams or foams; (5) sublingual or buccal administration; (6) intraocular administration; (7) transdermal administration; or (8) nasal administration.

[0323] The term "treatment" is intended to also encompass prevention, treatment, and cure.

[0324] The patient receiving such treatment is any animal in need thereof, including primates, particularly humans and other mammals, such as horses, cattle, pigs and sheep; and poultry and pets in general.

[0325] The compounds of the invention may be administered as such or in admixture with pharmaceutically acceptable carriers or in combination with antibacterial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Thus, combination therapy encompasses such a continuous, simultaneous and separate administration of the active compound followed by subsequent administration of the active compound when the effect of the first administration has not completely disappeared.

[0326] The addition of the active compounds according to the invention to animal feed is preferably effected by: a suitable feed premix containing an effective amount of the active compound is prepared and this premix is mixed into the complete feed.

[0327] Alternatively, a semi-finished concentrate or feed additive containing the active ingredient may be incorporated into the feed. The preparation and administration of such premixes and complete Feeds is described in the literature of the reference (e.g. "Applied Animal Nutrition", w.h.freedman and co., SanFrancisco, u.s.a., 1969 or "Livestock Feeds and Feeds" O and Bbooks, Corvallis, ore., u.s.a., 1977).

[0328] Micelle

[0329] Recently, the pharmaceutical industry has introduced microemulsion technology to improve the bioavailability of some lipophilic (water insoluble) agents. Examples include Trimetrine (Dordunoo, s.k., et al, Drug Development and Industrial Pharmacy, 17(12), 1685-.

[0330] In one aspect of the invention, a formulation comprises micelles formed from a compound of the invention and at least one amphiphilic carrier, wherein the micelles have an average diameter of less than about 100 nm. More preferably, the micelles are provided with an average diameter of less than about 50nm, and most preferably, the micelles are provided with an average diameter of less than about 30nm, or even less than about 20 nm.

[0331] While all suitable amphiphilic carriers are contemplated, the presently preferred carriers are generally those generally recognized as non-toxic (GRAS) that are capable of solubilizing the compounds of the present invention and emulsifying the compounds at a later time upon contact with a complex aqueous phase, such as that found in the human gastrointestinal tract. Generally, amphiphilic components meeting these requirements have an HLB (balance of hydrophilic versus lipophilic) value of 2-20, the structure of which comprises linear aliphatic radicals in the range of C-6 to C-20. Such as polyethylene-glycolated (glycolized) fatty acid glycerides and polypropylene glycols.

[0332] Particularly preferred amphiphilic carriers are saturated and monounsaturated pegylated (polyethylenglycolyzed) fatty acid glycerides, such as those obtained in fully or partially hydrogenated various vegetable oils. Such oils may advantageously be composed of 3-, 2-and mono-fatty acid glycerides and 2-and mono-polyethylene glycol esters of the corresponding fatty acids, with particularly preferred fatty acid compositions including capric acid 4-10%, capric acid 3-9%, lauric acid 40-50%, myristic acid 14-24%, palmitic acid 4-14% and stearic acid 5-15%. Another useful class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, and saturated or monounsaturated fatty acids (span series) or corresponding ethoxy analogs (tween series).

[0333] Commercially available amphiphilic carriers are specifically contemplated and include the glyceryl monostearate series, oleoyl polyisoeglycol glycerides, caprylic capric acid macrogol glycerides, or lauroglycol (both manufactured and sold by Gattefosse Corporation, Saint Priest, france), polyethylene glycol monooleate, polyethylene glycol-dioleate, polyethylene glycol-monolaurate and dilaurate, lecithin, polysorbate 80, and the like (manufactured and sold by a number of companies in the united states and around the world).

[0334] Polymer and method of making same

[0335] Hydrophilic polymers suitable for use in the present invention are those that are readily soluble in water, can be covalently linked to a vesicle-forming lipid, and are tolerable in vivo, non-toxic effects (i.e., biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also known as polylactide), polyglycolic acid (also known as polyglycolide), polylactic-polyglycolic acid copolymers, polyvinyl alcohol. Preferred polymers are those having a molecular weight of from about 100 or 120 daltons up to about 5000 or 10000 daltons, more preferably from about 300 daltons to about 5000 daltons. In a particularly preferred embodiment, the polymer is a polyethylene glycol having a molecular weight of from about 100 to about 5000 daltons, more preferably a molecular weight of from about 300 to about 5000 daltons. In a particularly preferred embodiment, the polymer is polyethylene glycol of 750 daltons (PEG (750)). The polymer may also be defined by the number of monomers therein; in a preferred embodiment of the invention a polymer of at least about 3 monomers is used, for example a PEG polymer (about 150 daltons) containing three monomers.

[0336] Other hydrophilic polymers suitable for use in the present invention include polyvinylpyrrolidone, polymethyloxazoline (polyoxazoline), polyethyloxazoline (polyoxazoline), polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, cellulose derivatives such as hydroxymethylcellulose or hydroxyethylcellulose.

[0337] In certain embodiments, the formulations of the present invention comprise a biocompatible polymer selected from the group consisting of: polyamides, polycarbonates, polyvinyl polymers, polymers of acrylic and methacrylic acid esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and their copolymers, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly (ortho) esters, poly (butyric acid), poly (valeric acid), poly (lactide-caprolactone), polysaccharides, proteins, hyaluronic acid, polynitrile acrylates, and blends, mixtures or copolymers thereof.

[0338] Cyclodextrin

[0339] Cyclodextrins are cyclic oligosaccharides containing 6, 7 or 8 glucose units, designated by the greek letters α, β or γ, respectively. Cyclodextrins with less than 6 glucose units are not known to be present. The glucose units are linked by alpha-1, 4-glucosidic linkages. As a result of the chair-type conformation of one saccharide unit, all secondary hydroxyl groups (at C-2 and C-3) are located on one side of the ring, while all primary hydroxyl groups at C-6 are located on the other side. Thus, the outer surface is hydrophilic, rendering the cyclodextrin water soluble. In contrast, the internal cavities of cyclodextrins are hydrophobic in that they are lined with C-3 and C-5 hydrogen atoms and ether-like oxygen. These matrices allow it to complex with a variety of relatively hydrophobic compounds including, for example, steroid compounds such as 17- β -estradiol (see, e.g., Van Uden al. plant Cell tissue. org. Cult.38: 1-3-113 (1994)). This complexation occurs by van der waals interactions and the formation of hydrogen bonds. For a general review of cyclodextrin chemistry see Wenz, agnew. 803-822(1994).

[0340] The physicochemical properties of cyclodextrin derivatives strongly depend on the kind and degree of substitution. For example, their solubility in water ranges from insoluble (e.g., triacetyl- β -cyclodextrin) to 147% soluble (w/v) (G-2- β -cyclodextrin). In addition, they are soluble in many organic solvents. The nature of cyclodextrins enables the solubility of various formulation components to be controlled by increasing or decreasing their solubility.

[0341] Many cyclodextrins and methods for their preparation have been described. Such as those described by parmeter (i) et al (U.S. patent No. 3,453,259, incorporated herein by reference) and Gramera et al (U.S. patent No. 3,459,731, incorporated herein by reference). Other derivatives include cyclodextrins with cationic properties [ Parmeter (II), U.S. Pat. No. 3,453,257, incorporated herein by reference ], insoluble, crosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788, incorporated herein by reference), and cyclodextrins with anionic properties [ Parmeter (III), U.S. Pat. No. 3,426,011, incorporated herein by reference ]. Wherein carboxylic acids, hypophosphorous acids, phosphorous acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids (thiosulfinic acids), sulfonic acids are added to the cyclodextrin matrix in cyclodextrins having anionic properties [ see Parmeter (III) above ]. Additionally, cyclodextrin sulfoalkylated derivatives have been described by Stella et al. (U.S. Pat. No. 5,134,127, incorporated herein by reference).

[0342] Liposomes

[0343] Liposomes are composed of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes can be characterized by the type and size of the membrane. Small Unilamellar Vesicles (SUVs) have a single membrane and typically range between 0.02 and 0.05 μm in diameter; the unilamellar vesicles (LUVS) are typically larger than 0.05 μm. Oligo-and multilamellar vesicles have multilayer membranes, usually concentric membranes, and are typically greater than 0.1 μm. Liposomes with non-concentric membranes, i.e. several smaller vesicles enclosed in one larger vesicle, are called multivesicles.

[0344] One aspect of the invention relates to formulations comprising liposomes containing one of the compounds of the invention, wherein the liposomal membrane is prepared to provide the liposomes with improved loading capacity. Alternatively or in the alternative, the compounds of the invention may be contained within, or adsorbed onto, the liposomal bilayer of the liposome. The compounds of the invention can be aggregated with lipid surfactants and carried in the internal space of liposomes; in these cases, the liposome membrane is prepared to resist the destructive effect of the active agent-surfactant aggregates.

[0345] According to a specific embodiment of the invention, a lipid bilayer of a liposome comprises a lipid derivatized with polyethylene glycol (PEG), such as PEG chains, extending from the inner surface of the lipid bilayer into the liposome-encapsulated interior space and from the exterior of the lipid bilayer into the surrounding environment.

[0346] The active agent contained in the liposomes of the invention is in solubilized form. The surfactant and aggregates of the active agent (e.g., emulsions or micelles containing the relevant active agent) can be entrapped in the interior space of the liposomes in accordance with the present invention. The surfactant serves to disperse and solubilize the active agent and may be selected from any suitable aliphatic, cycloaliphatic or aromatic surfactant, including but not limited to different chain lengths, biocompatible Lysophosphatidylcholine (LPCS) (e.g., from about c.sub.14 to c.sub.20). Polymer-derivatized lipids, such as PEG-lipids, can also be used for micelle formation because they act to inhibit micelle/membrane fusion, and the addition of the polymer to the surfactant molecule lowers the CMC of the surfactant and aids in micelle formation. Preferred surfactants have a CMC in the micromolar range; higher CMC surfactants can be used to prepare micelles embedded in liposomes of the invention, however, micellar surfactant monomers can affect the stability of the liposome bilayer and are a factor in designing liposomes with a desired stability.

[0347] Liposomes according to the invention can be prepared by any of a variety of techniques well known in the art. See U.S. Pat. nos. 4,235,871; published PCT application WO 96/14057, both incorporated herein by reference; new RRC, liposomees: a practicallappacach, IRL Press, Oxford (1990), pages 33-104; lasic DD, Liposomes from physics to applications, Elsevier Science publishers BV, Amsterdam, 1993.

[0348] For example, liposomes of the invention can be prepared by diffusing a hydrophilic polymer-derivatized lipid into preformed liposomes, such as by exposing the preformed liposomes to micelles of lipid-grafted polymer, wherein the lipid concentration corresponds to the final mole percent of derivatized lipid desired in the liposome. Liposomes containing hydrophilic polymers can also be formed by homogenization, lipid field hydration, or extrusion techniques well known in the art.

[0349] In another exemplary method of preparation, the active agent is first dispersed by sonication in a hydrophobic molecule of lysophosphatidylcholine or other low CMC surfactant (including polymer grafted lipids) that readily solubilizes. The resulting micellar suspension of active agent is then rehydrated with a dry lipid sample containing a suitable molar percentage of polymer-grafted lipid or cholesterol. The lipid and active agent suspension is then formed into liposomes using extrusion techniques well known in the art, and the resulting liposomes are separated from the unencapsulated solution by standard column separation.

[0350] In one aspect of the invention, liposomes are prepared having a substantially uniform size within a selected size range. One effective granulation method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane will correspond approximately to the largest dimension of the liposomes produced by extrusion. See U.S. patent 4,737,323, which is incorporated herein by reference.

[0351] Release modifiers

[0352] The release profile of the formulations of the present invention is dependent on the encapsulating material, the encapsulated drug concentration, and the presence of the release modifier. For example, release can be manipulated to be pH dependent, e.g., using a pH sensitive coating such that it is released only at low pH, e.g., in the stomach, or at higher pH, e.g., in the intestinal tract. An enteric coating may be used to prevent release from occurring until after passage through the stomach. A mixture of various cyanamide coatings or inclusions in different materials can be used to obtain an initial release in the stomach followed by a subsequent release in the intestine. Release can be manipulated by the introduction of salts or pore formers which can enhance water absorption or drug release by diffusion from the capsule. Excipients that modulate the solubility of the drug may also be used to control the release rate. Agents that increase the degradation or release of the matrix from the matrix may also be added. They can be added to the drug, added as a separate phase (i.e. as particles), or co-dissolved in the polymer phase depending on the compound concerned. In all cases, the amount should be between 0.1 and 30% (w/w polymer). Types of degradation promoters include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, triethanolamine, and surfactants such as tween. The pore-forming agent (i.e., water-soluble compound such as inorganic salt and sugar) capable of increasing the microporous structure of the matrix is added in a particulate manner. The range should be between 1 and 30% (w/w polymer).

[0353] Drug absorption can also be manipulated by varying the residence time of the particles in the intestinal tract. This can be achieved, for example, by: the particles are coated with a mucoadhesive polymer, or are selected as encapsulating material. Examples include most polymers with free carboxyl groups, such as chitosan, cellulose, and in particular polyacrylates (polyacrylate as used herein refers to polymers containing both acrylate groups and modified acrylate groups, such as cyanoacrylate binders and methacrylates).

[0354] Combinatorial libraries

[0355] The subject compounds can be synthesized using the combinatorial synthesis methods described in this section. Combinatorial libraries of compounds can be used to screen for drug, pesticide, or other biologically or medically relevant active substances or material-related properties. A combinatorial library for the purposes of the present invention is a mixture of chemically related compounds which can be co-screened for a desired property; such a chemical combination library may be liquid or covalently attached to a solid support. The preparation of many related compounds in a single reaction can greatly reduce and simplify the number of screening procedures that need to be performed. Screening for suitable biological, pharmaceutical, pesticidal or physical properties can be carried out by conventional methods.

[0356] Diversity in a library of compounds can be created at a variety of different levels. For example, the base aromatic group used in the combinatorial approach may be a multiplicity of aromatic nuclei, such as variations in ring structure, and/or may vary with respect to other substituents.

[0357]A variety of techniques are available in the art for generating libraries of compounds of combinations of small organic molecules. See, for example, Blondelle et al (1995)Trends Anal.Chem.14: 83; U.S. patents 5,359,115 and 5,362,899 to Affymax: U.S. Pat. No. 5,288,514 to Ellman: PCT publication WO 94/08051 to Still et al, which is incorporated herein by reference; chen et al (1994)JACS116: 2661: kerr et al (1993)JACS115: 252; PCT publications WO92/10092, WO93/09668, and WO 91/07087; PCT publication WO93/20242 to Lerner et al, each of which is incorporated herein by reference). Thus, libraries of various compounds on the order of about 16 to 1,000,000 or even more entities can be synthesized and screened for a particular activity or property.

[0358] In an exemplary embodiment, a library of substituted multimers (diversomers) compounds can be synthesized using the techniques described by Still et al in PCT publication WO 94/08051 using the subject reaction, e.g., via a hydrolytic or photolytic group attached to a polymer bead, e.g., localized at a position on a substrate. According to Still et al, libraries of compounds are synthesized on a set of beads, each bead comprising a set of tags that identify a particular multiplex on the bead. In one embodiment particularly suited for the discovery of enzyme inhibitors, the beads may be dispersed on a permeable membrane surface and the plurality released from the beads by cleavage of the linkage to the beads. The released multiplex from each bead will diffuse across the membrane to the assay region where it will interact with an enzyme sample. Detailed descriptions of some of the combinatorial approaches are provided below.

[0359]A. Direct characterization

[0360] One trend in the field of combinatorial chemistry is, for example, the sensitivity of technologies such as Mass Spectrometry (MS), which can be used to characterize sub-femtomolar quantities of compounds and directly determine the chemical structure of a compound selected from a combinatorial library of compounds. For example, if the library of compounds is provided on an insoluble support matrix, the individual compounds may be first released from the support and characterized by MS. In other embodiments, as part of the MS sample preparation technique, an MS technique such as MALDI may be used to release the compound from the support, particularly when initially a labile bond links the compound to the support. For example, a bead selected from a library of compounds may be irradiated in a MALDI step to release a diversity from a support, and the diversity ionized for MS analysis.

[0361]B. Multi-center synthesis method

[0362]The compound libraries of the subject methods can be in a multicenter compound library format. Briefly, Geysen and coworkers (Geysen et al (1984)PNAS81: 3998-. The Geysen technique allows the synthesis and screening of thousands of compounds per week using a multi-center approach, and the linked compounds can be reused in many assays. Some suitable linkers (linker moieties) may also be hung on the pins so that the compound may be cleaved from the support after synthesis for identification of purification and further evaluation (c.f., Bray et al (1990) Tetrahedron Lett31：5811-5814；Valerio et al.(1991)Anal Biochem 197：168-177；Brayet al.(1991)Tetrahedron Lett 32：6163-6166)。

[0363]C. Split-join-recombine

[0364]In yet another embodiment, a diverse library of compounds can be provided on a set of beads using a split-ligation-recombination strategy (see, e.g., Houghten (1985)PNAS82: 5131-; us patent 4,631,211, 5,440,016, 5,480,971, which is incorporated herein by reference). Briefly, as the name implies, in each synthetic step where degeneracy is introduced into the library of compounds, the beads are divided into separate groups, the number of groups equals the number of different substituents to be added at a particular position in the library of compounds, different substituents are paired with each reaction, and the beads are recombined into one library for the next iteration.

[0365]In one embodiment, the separation-ligation-regenerationThe strategy of incorporation can be carried out using a similar process first developed by Houghten, known as "tea bag", in which the synthesis of the compound takes place inside a resin-sealed porous polypropylene bag (Houghten et al (1986)PNAS82: 5131-5135). By placing these bags in a suitable reaction solution, the substituents are attached to the compound-bearing resin, and all common steps, such as washing and deprotection of the resin, take place simultaneously in one reaction vessel. At the end of the synthesis, each bag contains a single compound.

[0366]D. Combinatorial compound libraries by photoalignable, spatially localizable parallel chemical synthesis

[0367]A combinatorial synthetic scheme in which a compound is identified by its localization on a synthetic substrate is referred to as sterically locatable synthesis. In one embodiment, the combinatorial process is controlled by controlling the addition of chemical reagents to specific sites on the solid support (Dower et al (1991)Annu Rep Med Chem 26：271-280；Fodor，S.P.A.(1991)Science251: 767; U.S. Pat. No. 5,143,854 to Pirrung et al (1992), which is incorporated herein by reference; jacobs et al (1994)Trends Biotechnol12: 19-26). The spatial resolution of photolithography provides miniaturization. This technique can be performed by using a protection/deprotection reaction with a photosensitive protection group.

[0368]The main point of this technique is in Gallop et al (1994)J Med Chem37: 1233 and 1251. A synthetic substrate was prepared for attachment by covalent attachment of a photosensitive Nitrodimethoxybenzyloxycarbonyl (NVCO) protected amino linker or other photosensitive linker. Light is used to selectively activate a particular area of the synthetic substrate for attachment. Removal of the photosensitive protecting group by light (deprotection) leads to activation of the selected region. After activation, first a set of amino acid analogs, each bearing a photo-protecting group on the terminal amino group, is exposed to the entire surface. The connection occurs only in the areas located by the light in the previous step. After the reaction had stopped, the plates were washed And the substrate is irradiated by a second set of masks, activating different areas for reaction with a second protective member (building block). The pattern of the mask and the sequence of the reactants determine the products and their location. Since photolithography is used in this process, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be located with the appropriate resolution. The site of each compound is precisely known; thus, its interaction with other molecules can be directly assessed.

[0369] In a photo-localized chemical synthesis, the product depends on the pattern of illumination and the order of addition of reactants. By changing the illumination pattern, a large number of different sets of test compounds can be synthesized simultaneously; this feature leads to the creation of many different masking strategies.

[0370]E. Encoded combinatorial libraries

[0371] In another embodiment, the subject methods employ a library of compounds having an encoded tagging system. Recent advances in the identification of active compounds from combinatorial libraries have employed a variety of chemical indexing systems that utilize tags that uniquely encode the reaction steps (and by inference the structures they carry) that a given bead undergoes. Conceptually, this approach mimics phage display libraries in that the activity is derived from the expressed polypeptide, but the structure of the active polypeptide is deduced from the corresponding genomic DNA sequence. The first code of the synthetic combinatorial library uses DNA as a code. Various other forms of coding have been reported, including coding with sequencable bioligomers (e.g., oligonucleotides and polypeptides), as well as binary coding with an added non-sequenceable label.

[0372]1. Labelling with a sequenceable biomoiigomer

[0373]The principle of using oligonucleotide-coded combinatorial libraries of synthetic compounds was described in 1992 (Brenner et al (1992) PNAS 89: 5381-5383), and an example of such libraries appeared in the second year (Need)les et al.(1993)PNAS90: 10700-10704). A nominal 7⁷The polypeptides of the combinatorial library of (═ 823543) include all combinations of Arg, Gln, Phe, Lys, Val, D-Val and Thr (three letter amino acid code), each of which is encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), the library being prepared by synthesis of a series of alternating rounds of polypeptides and oligonucleotides on a solid support. In this work, amine-attached functional groups on beads are specifically distinguished from the synthesis of polypeptides or oligonucleotides by simultaneously incubating the beads with reagents that generate protected OH groups for oligonucleotide synthesis and protected NH for polypeptide synthesis₂(here, in a ratio of 1: 20). When complete, each tag contains 69-mers with 14 units carrying the code. The bead-linked compound library is incubated with fluorescently labeled antibodies and beads containing linked strongly fluorescent antibodies are sorted by Fluorescence Activated Cytometry (FACS). The DNA tags were amplified by PCR, sequenced, and predicted for the synthesized polypeptide. By these techniques, a library of compounds can be derived for use in the subject methods, wherein the oligonucleotide sequences of the tags identify the sequential combinatorial reactions that a particular bead undergoes, thereby providing identification of the compounds on the bead.

[0374] The use of oligonucleotide tags allows for very sensitive label analysis. Even so, this approach requires careful selection of the orthogonal set of protecting groups required for the alternating synthesis of the label and the library member of compounds. Furthermore, the chemical instability of the tag, particularly the anomeric linkage of the phosphate group and the sugar, may limit the choice of reagents and conditions that can be used for the synthesis of non-oligomeric libraries. In a preferred embodiment, the compound library employs a linker that allows selective stripping of the test compound library members for use in the assay.

[0375]Polypeptides are also used as marker molecules for combinatorial libraries. Two exemplary methods have been described in the art, both of which employ branched linkers attached to a solid phase, wherein the coding and ligand chains are made in turn. In the first method (Kerr JM et al (1993)J Am Chem Soc115: 2529-2531), the synthetic orthogonality is achieved by employing acid-labile protection for the coding strand and base-labile protection for the compound strand.

[0376]In another alternative approach (Nikolaiev et al (1993)Pept Res6: 161-170) using a branched linker such that both the coding unit and the test compound can be attached to the same functional group of the resin. In one embodiment, a cleavable linker may be placed between the branch point and the bead, such that one molecule released by the cleavage contains the code and the compound (Ptek et al (1991) Tetrahedron Lett32: 3891-3894). In another embodiment, a cleavable linker may allow the test compound to be selectively separated from the bead, leaving the code behind. The latter configuration is particularly valuable because it allows screening of test compounds without interference from the coding group. Examples of independent fragmentation and sequence analysis of members of peptide libraries and their corresponding tags in the art have demonstrated that this tag can accurately predict the structure of a polypeptide.

[0377]2. Non-sequenceable markers: binary coding

[0378]An alternative format for encoding test compound libraries employs a set of non-sequenceable electrophoretic marker molecules used as a binary code (Ohlmeyer et al, (1993)PNAS90: 10922-10926). An exemplary label is a halogenated aromatic alkyl ether, which is detectable at less than femtomole levels using Electron Capture Gas Chromatography (ECGC) due to its trimethylsilyl ether. The variation of the length of the alkyl chain and the nature and position of the aromatic halides allows the synthesis of at least 40 such tags, which in principle can encode 2⁴⁰(e.g., up to 10)¹²) Different molecules are used. In the initial report (Ohlmeyer et al, supra), the tag was bound to about 1% of available amine groups in a polypeptide library via a photocleavable o-nitrobenzene linker. This approach is convenient when preparing libraries of compounds that are polypeptide-like or other combinations of amine-containing molecules. However, a more general system has been developed that allows encoding virtually anything Combinatorial libraries of compounds. Here, the compound is attached to a solid support via a photocleavable linker, and the label is inserted onto the bead matrix via a catechol ether linker via a polyacetylene (Nestler et al (1994)J Org Chem59: 4723-4724). This orthogonal binding strategy allows selective detachment of the library members for detection in solution and decoding by ECGC after oxidative detachment of the label set.

[0379]Although there are several amide-linked compound libraries in the art that employ binary coding with electrophoretic labels attached to amine groups, directly attaching these labels to the bead matrix provides greater versatility in the structures that can be prepared in coding combinatorial libraries. In this way the tags and their linkers are as close to inert as the bead matrix. Binary coded combinatorial compound libraries have been reported in which the electrophoretic label is attached directly to a solid phase (Ohlmeyer et al (1995)PNAS92: 6027-6031) and provides guidance for the generation of the library of host compounds. The two libraries are constructed using an orthogonal ligation strategy in which the library members are attached to a solid support via a photoactive linker and the tags are ligated by strong oxidative cleavage of a cross-linking agent. Members of the library of compounds can be used in multiplex assays because they can be repeatedly and locally eluted lustering from the solid support. Continuous light elution also allows for a very high throughput iterative screening strategy: first, multiple beads are placed in a 96-well microplate; second, the compound moieties are separated and transferred to a test plate; third, a metal binding assay determines active pores; fourth, the respective beads are individually rearranged into a new microplate; fifth, individual active compounds are identified; sixth, the structure is decoded.

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Examples

[0442] The present invention now will be generally described and will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

[0443]Example 1: firexate binding assay

[0444] We have developed an assay for correcting the phenotype of NPC in CHO cell lines using filipin, which is capable of binding to non-esterified cholesterol and has been used for visualization of the free cholesterol content in NPC cells. The initial screening was performed in CT60 cells, which expressed the gain of a mutant hamster NPC1 protein and a functional gene mutation in SCAP (42). These cells are unable to transport low density lipoprotein-derived cholesterol out of the late endosome, and a significant amount of cholesterol accumulates in the LSO compartment. FIGS. 1A-B show images of phenanthroline staining in a control CHO cell line TRVb1(45) (FIG. 1A) and in CT60 cells (FIG. 1B). It can be seen that CT60 cells showed more phenanthripine staining than control cells and that the fluorescence in CT60 cells was concentrated in the nuclear-surrounding organelles.

[0445] For screening, images were acquired using a Discovery-1 automated microscope system with a 10-fold objective lens and the background and shading were corrected as described in the methods. Two thresholds were set for philippine staining, a low threshold including all cellular regions, and a high threshold for intense philippine staining in perinuclear LSOs. The set threshold was used for analysis of 64 images of unprocessed CT60 cells in 32 wells of each plate. It was found that the same threshold value can be used for multiple plates in one experiment, but the threshold value will vary from experiment to experiment in different ways. In low-threshold imaging, we compared cell images after threshold based on transmitted light images, and these thresholds provided good agreement with transmitted light cell boundaries.

[0446] As a simple measure of the dye intensity of philippine we measured the intensity of philippine per pixel above a threshold. Conditions for filipin labeling (concentration and time) were adjusted to optimize the differentiation between CT60 cells and control TRVb1 cells. As shown in fig. 1C, this simple method provides a relatively high degree of discrimination between CT60 cells and TRVb1 cells. The method for screening is characterized by a statistical parameter Z' (46):

Z′＝1-(3σ_c++3σ_c-)/|μ_c+-μ_c-|

[0447]wherein sigma_c+And σ_c-Is the Standard Deviation (SD) of the positive and negative control data sets and collects σ_c+And σ_c-Is the average of the positive and negative controls. The Z' value calculated using CT60 for the mean filipin intensity of TRVb1 cells was 0.22, which is generally considered inappropriate for large scale screening because the two distributions are expected to overlap when a large number of wells are screened.

[0448] Although intensity measurements may be useful, it appears that more image information may provide better discrimination of wild type versus NPC cells. To this end, we utilized the spatial distribution of the filipin markers of LSOs, which are concentrated near the nucleus (fig. 1B). We apply a second higher threshold to these bright clusters. An example of applying these thresholds is shown in fig. 2. Then, we measured the total fluorescence intensity of the selected LSO (high threshold, fig. 2C) divided by the number of total pixels in the cell (low threshold, fig. 2D). As shown in figure 1, this LSO compartment assay gave a stronger discrimination of CT60 versus the Z' value of 0.61 for control cells.

[0449] Using the screening technique described above, we screened a compound library of 14956 compounds added to the cells for 16 hours at a final concentration of 10 μ M, using one well for each compound. Prior to the application of these compounds, cells were grown in normal tissue culture medium containing 10% FBS, and therefore, LSOs were saturated with cholesterol (fig. 1B). Cells were then image processed and analyzed using the mean filipin intensity detection and the LSO compartment assay. In general, both assays identify similar compounds that reduce the labeling of phenanthroline.

[0450] Wells with mean phenanthroline intensities above 3SD from the mean of solvent treated cells were further investigated. Images of these selected holes are visually detectable and if we take pictures from the second location, none of the sites showing weak focus (about 0.3% of the total) are re-analyzed. Wells with low cell numbers were considered a toxicity indication, and these cytotoxic compounds were not followed further. We also studied low magnification images of the arrangement in rows and columns of the plate to look for patterns in cell number or brightness that might indicate one of the mechanical errors in the automatic pipetting step. In one example, a band pattern was seen in a set of 8 plates, and these compounds were rescreened after pipette issues were identified.

[0451] From this initial screen, we found that 133 compounds reduced the mean filipin intensity by more than 3SD, and 23 compounds increased the mean filipin intensity by more than 3 SD. Visual inspection of the images also showed that 19 compounds were able to produce morphological changes in the phenanthroline staining pattern, which did not meet our criteria for reducing mean phenanthroline intensity.

[0452] These 175 compounds were then rescreened at 10 μ M under the same conditions as the initial screen, except that each compound was placed in two wells per plate and 2 plates were screened in parallel. Both the mean Firexate strength and the LSO ratio were determined. The selection of 14 compounds from the rescreen resulted in a reproducible decrease in phenanthroline staining at 10 μ M, and 8 compounds were found to reproducibly increase phenanthroline staining. The 9 compounds were found to alter the morphological distribution of felipine. Figure 3 shows images of the screening plates of solvent-treated control wells and compound-treated wells showing reduced philippine staining.

[0453] Figure 4 shows the effect of 4 compounds on the morphological changes observed by philippine staining. These cells showed rearrangement, indicating that cholesterol had been altered to a different interval, or that the morphology of the LSOs itself had changed (compound 1-C-3, fig. 4C). The effect of compound 1-b-4 (FIG. 4D) was very strong as observed by the bright vortex of the phenanthroline staining. The effect of compound 1-b-4 was similar to that of normal human fibroblasts (data not shown), indicating that this response is not associated with the NPC phenotype of the cells. These compounds, which increase the apparent phenanthroline staining and cause morphological changes, were not further investigated in this study.

[0454] The structures of 14 compounds that reduce the phenanthroline labeling and 2 compounds that cause significant morphological changes are shown in fig. 5. Compounds 1-c-2 and 1-c-3 caused morphological changes, and compounds 1-b-2 and 1-b-4 increased the phenanthroline strength.

[0455] Materials and methods for screening

[0456] Materials: cell growth medium Hams F12 and Fetal Bovine Serum (FBS) were purchased from Invitrogen (Carlsbad, CA). All other chemicals, including dimethyl sulfoxide (DMSO), filipin/Paraformaldehyde (PFA) and Hoechst 33258 were purchased from sigma chemicals (st. Libraries of compounds for screening were purchased from chemical university, Inc (San Diego, CA). Metamorph image analysis software was from Molecular Devices Corporation (Downnington, Pa.).

[0457]Cell culture: NPC1 cell lines, CT60 and CT43 were provided by T.Y.Chang ((Dartmouth Medical School, Hanover, NH.) these cell lines were derived from a parental cell line, 25RA, a CHO cell line containing gain function mutations in SREBP lytic activator protein (SCAP) (42). CT60 and CT43 cells were grown in Hams F12 medium supplemented with 1% penicillin/streptomycin (PS), 2g/LGlucose, 10% FBS-containing 1.176 g/l sodium bicarbonate [ Medium A ] ]In a single bed containing 5% CO₂Was maintained at 37 ℃ in a humidified incubator. For screening, CT60 cells (650 cells/well) or CT43 cells (700 cells/well) in 30 μ l growth medium a containing 10% FBS were seeded in Costar 384-well black polystyrene flat, clear-bottomed tissue culture treated plates, respectively (Corning, inc., NY), to obtain-80% fusion when the cells were analyzed.

[0458] Normal human fibroblasts (GM5659E) were grown in MEM with 1% P/S and 10% FBS. For microscopic examination, fibroblasts were plated in normal growth medium in 35mm glass-bottom dishes or in 384-well plates.

[0459]Compound addition: compounds in the compound library were formatted for screening in a high throughput screening machine at the university of rockhner. Cells were treated with compounds from the chemical library for 1 day after plating. Using a Packard MiniTrak^TMRobotic liquid handling System, we added 0.1. mu.l (5mM stored in DMSO) to 25. mu.l of each compound consisting of Medium A and 1% FBS and 20mM2- [4- (hydroxyethyl) -1-piperazinyl]Ethanesulfonic acid (HEPES) in screening medium S in Falcon 384-well V-bottomed polypropylene plates. To obtain a final concentration of-10. mu.M, 23. mu.l of the premix compound was dispensed into plates containing cells and 30. mu.l of medium A. For primary screening, 352 test compounds were added to each plate, and the remaining 32 wells were supplemented with DMSO alone as a control. All plates with compound added were incubated at 37 ℃ for 16 h. The plate was then washed 3 times with Phosphate Buffered Saline (PBS) pH 7.4 using a Bio-Tek Elx405 plate washer (Bio-Tek Instruments Inc., Winooski, VT). For each wash cycle, 70 μ l of PBS was dispensed and aspirated to a residual volume of 16 μ l per well. Finally, cells were fixed with 1.5% PFA in PBS for 20 min at room temperature and then washed 3 more times with PBS.

[0460] And (4) carrying out fluorescent labeling. For fixed cells, phenanthroline was added to PBS at room temperature over 45 minutes to a final concentration of 50 μ g/ml for labeling free cholesterol. Cells were finally washed 3 times with PBS and images were obtained immediately after labeling.

[0461] Fluorescence microscopy: model Discovery-1 automated fluorescence microscope from Molecular Devices Corporation, equipped with xenon arc lamp (Perkinelmer, CA), Nikon 10X plane Fluor 0.3NA objective and a photometrics CoolSnaPQ camera (1392X 1040 pixels; Roper Scientific, Tucson, AZ) was used to acquire images. The phenanthroline images were obtained using an 60/40nm excitation and 480/40nm emission filter with a 365dclp (dichoric Long pass) filter. The image files are stored on the local host computer before being transferred to a server.

[0462] The plates were transported from the plate storage using a CRS CatALyst Express robot (Thermo Electron Corp). Images were acquired at 2 locations per well, each approximately 50 μm from the center of the well, and the exposure time for each image was 75ms using a 2 x 2 frame union method. Different methods are used for automatic focusing for primary and secondary screening. In the primary screening, using the image-based focusing and MetaMorph auto-focusing algorithms, each well was focused over a range of + -150 μm and each site per well was focused over a range of + -20 μm. The image used for focusing was passed through a 15ms exposure time using an 8 x 8 frame union method to reduce photobleaching. For secondary screening, laser-based autofocus (LAF v.2 from Molecular Devices) was used to find the bottom of the plate. Image-based focusing is used to determine the offset between the bottom of the plate and the cells, and then each position is refocused beyond a 20 μm range. Regardless of the focusing method, the acquisition time per plate is 60-75 minutes. 696 x 520 pixel image is obtained with 12 intensity bits per pixel. Each pixel is 1.25 × 1.2 μm in the object.

[0463] Image analysis: images of phenanthroline stained cells were analyzed using Metamorph Discovery-1 image analysis software. Two different image analysis methods were developed: (1) mean filipin intensity detection and (2) LSO compartment ratio detection. First, to correct for shading, an image is created by averaging all the images obtained from one panel and smoothing the averaged image with a low pass filter. Each pixel of the image is then multiplied by the average intensity of the shadow image and the resulting pixel intensity is again divided by the shadow image pixel by pixel. The background was subtracted from the shadow-corrected image by determining the 5 th percentile intensity value and subtracting this value from every pixel in the image. At the plating density used, at least 5% of the image area in all regions is free of cells. Next, two different thresholds are applied to these philippine images. First, a low threshold is set for including all the areas occupied by cells. This screening value was used to conform the outline of the cell to the outline of the transmitted light image of the cell. Second, by selecting the bright areas of the philippine staining, a higher threshold was set for the bright stained areas in CT60 and/or CT43 cells, with the objective of mainly identifying the LSOs of the perinuclear regions of the cells. For mean-philippine intensity determination, using the low threshold alone, we determined the total philippine intensity above the low threshold divided by the number of pixels above the low threshold per region. This gives a mean phenanthroline intensity for each cellular region. For the LSO compartment ratio, we selectively measured the total Fireyi intensity above the high threshold of the region divided by the number of pixels at the low threshold. This gives a measure of the total intensity of LSO phenanthroline per cell area.

[0464] Normalized values were obtained by dividing the value obtained in the presence of the solvent control in each plate by the value obtained in the presence of the compound. Similar methods were used to analyze the effect of compounds on the retention of cholesterol in LSOs caused by treatment of human fibroblasts with U18666A.

[0465] Immunofluorescence: CT60 cells grew up to 70% confluency in the dish at the bottom of the coverslip. After 24h, compound (10 μ M) was added to the cells in screening medium containing 20mM HEPES. After 16-22h treatment with compound, cells were washed 3 times with PBS and fixed with 3.3% PFA for 20 min at room temperature. The cells were then treated with 50mM ammonium chloride for 10 minutes and washed 3 times with PBS. After blocking with 0.1% bovine serum albumin for 20 min, cells were washed with PBS, permeabilized with 0.05% saponin and incubated with anti-LBPA (1: 100) for 30 min at room temperature (J.Gruenberg, Univ.of Geneva contribution). The cells were then washed 3 times with PBS and incubated with goat anti-mouse IgG-Alexa546 (1: 200) and 100. mu.g/ml phenanthroline for 30 min. Finally, cells were washed 3 times with PBS and images were obtained under a Leica DMIRB microscope (Leica mikrosculated System GmbH, Germany) equipped with a Princeton instruments (Princeton, NJ) cooled CCD camera and metamorph imaging System software driven Leica DMIRB microscope. All image acquisitions used an oil immersion objective (25 x, 1.4 NA). Alexa 546-Tf was imaged with a standard rhodamine filter cube, and philippine was imaged with a Leica a4 cube [360nm (40nm passband) excitation filter and 470nm (40nm bandpass) emission filter ]. Image analysis was performed using Metamorph Discovery-1 image analysis software using an image analysis algorithm for screening to assess the concentration of cholesterol and LBPA in the presence and absence of compound.

[0466]Example 2: dose-dependent assay

[0467] Dose-dependence: using the same method as used for the screening, compounds were tested at 10. mu.M, 3.33. mu.M, 1.11. mu.M, 370nM and 123nM, respectively, in 4 wells. The amount required for one run of the selected compounds was purchased from Chemical university and 10mM stock solutions were prepared in DMSO. Secondary storage plates (20. mu.M, 6.66. mu.M, 2.22. mu.M, 740nM and 246nM) were prepared at 2-fold concentrations of compound in the screening medium. To obtain the final concentration, 30. mu.l of this secondary stock was added to each well of cells containing 30. mu.l of growth medium A containing 10% FBS. The final concentration of dimethyl sulfoxide (0.2% v/v) was the same in all wells. Cells were plated at 650 cells/well in costar 384-well plates containing 30 μ l of growth medium a containing 10% FBS per well. After incubation for 20 hours in the presence of compound, cells were washed with PBS, fixed with PFA and stained with filipin as described in screening assays. Dose-dependence of 14 selected compounds from the primary compound library was determined at least 5 times in separate experiments on CT60 cells and at least 3 times on CT43 cells. Dose-dependence of 7 selected compounds from the secondary compound library was determined at least 3 times in independent experiments at CT60 cells and at least 2 times at CT43 cells.

[0468] The dose response curves for 14 selected compounds identical to those in the screening assay in one assay are shown in FIGS. 6A-B. Three of the compounds reduced the phenanthroline staining to 3SD below the mean of solvent treatment in 1.1 μm CT60 cells, and the same compounds treated cells were less than 123nM below the mean of solvent treatment by 2SD above (fig. 6A). Compound 1-a-14 has an unusual dose response curve, which may be associated with its cytotoxic effect at high concentrations. To examine whether this effect was specific for the CT60 cell line, we also tested the dose response of the 14 selected compounds to CT43 cells, another NPC1 mutant cell line from blastocyte strain 25 RA. Most compounds were effective below the untreated mean of 10 μ M by 3SD or more, but at lower concentrations were ineffective on CT43 cells (FIG. 6B). It is interesting to note that the general trends for the effect of these 14 compounds in these two cell lines are similar, although the general compounds are more effective on CT60 cells at a given dose.

[0469]Example 3: time course detection

[0470] The effect of these compounds at 1.11, 3.33 and 10 μ M concentrations for 4, 20, 48h was determined using a method similar to dose-dependent. On the first day, CT60 cells were seeded at 600 cells/well in growth medium in 3 384-well plates. To ensure that the same cell density is maintained at the final time point, the compounds are added in a time sequence. After overnight incubation, compounds diluted in medium S in the first plate (48h time point) were added to the wells to reach final concentrations of 1.11, 3.33 and 10 μ M. After 52h of cell seeding, compound was added to the second plate in a similar manner and incubated for 20 h. Finally, after 68h of cell seeding, compound was added to the third plate in a similar manner and incubated for 4 h. All three plates were washed 3 times with PBS, fixed with 1.5% PFA and stained with 50. mu.g/ml Phellin. 4 wells were measured per condition in each experiment, repeated 3 times per experiment for CT60 and CT43 (data for CT43 not shown).

[0471]Example 4: toxicity detection

[0472] To measure toxicity, we treated CT60 and CY43 cells with compound at 5, 10 and 20 μ M for 24 hours. The number of cells per well was compared to control cells treated with DMSO (fig. 7). After 24h incubation, most compounds did not cause a significant reduction in cell number at 10 μ M. Compound 1-a-14 caused a 50% reduction in the number of CT60 cells and an 80% reduction in CT43 cells at 10. mu.M. Compounds 1-a-4, 5, 6, 8 and 13 were partially toxic, as indicated by a 20-30% reduction in cell number at 10. mu.M after 24 hours.

[0473]Methods for cell counting: compound was added in parallel four replicates at concentrations of 0 (dimethylsulfoxide solvent control), 5, 10 and 20 μm to CT60 and CT43 cells seeded in 384-well plates using a similar method of dose-dependent detection except that the cells were nuclear stained with Hoechst 33258. The final concentration of dimethyl sulfoxide in the wells was 0.2%. For control cells, the same amount of dimethyl sulfoxide was added to the cells. Cells were cultured for 24, 48 and 72 hours. After each time point, cells were washed and then fixed with 1.5% PFA. After washing the cells 3 times with PBS, the cells were stained for nuclei in PBS with 5. mu.g/ml Hoechst 33258(25mg/ml DMSO stock) for 45 minutes at room temperature. Finally, cells were washed 3 times with PBS and images were obtained using a Nikon 4X Plan Apo 0.2NA eyepiece. For Hoechst imaging, we used the same filter settings as for felipine. At 12 intensity bits per pixel, we have collected one image of 520 × 696 pixels per well. Each pixel is 3.125 × 3.125 μm in 13 subjects. Using the Integrated MorphometryAnalysis function of MetaMorph, cells were identified by each plate in an interactive manner to standard single cells Nuclear region (-200 μm)²) To count. The amount of standard regions per object that exceed a threshold is determined and the total amount of standard regions per image is used as the cell count. The percentage reduction in cell number compared to DMSO control was calculated at each concentration and time point.

[0474]Example 5: determination of cholesterol by gas chromatography

[0475] The cholesterol-lowering effect of the 14 selected compounds, as determined by the phenanthroline markers, was measured by an alternative chemical method, which included GC separation of solvent-extracted cellular lipids (43). Under the same conditions as for the screening assay, CT60 cells were treated with compound at 10 μ M. The FC concentration per cell was determined by gas chromatography, and all the values obtained were compared with the determination of the mean filipin intensity (table 1). As shown in table 1, most of the 14 selected compounds caused a relatively mild decrease in the total FC concentration of the cells, as measured by the mean filipin method. Selecting from the first screening a selection of subjects having variable effects on cellular cholesterol; including some compounds that significantly increase free cholesterol in the cell.

[0476] Table 1 free cholesterol content of C T60 cells tested by mean filipin intensity and gas chromatography after treatment with 14 selected compounds from a pool of primary compounds.

Compound number	Score of mean Firepin intensity control	μ g determined by G C¹Fraction of FC/. mu.g protein control
Compound number	Score of mean Firepin intensity control	μ g determined by G C¹Fraction of FC/. mu.g protein control	1-a-1	0.87±0.04	1.02±0.03
1-a-2	0.88±0.04	1.15±0.05	1-a-1	0.87±0.04	1.02±0.03
1-a-2	0.88±0.04	1.15±0.05	1-a-3	0.95±0.04	1.27±0.07
1-a-4	1.01±0.04	0.87±0.07	1-a-3	0.95±0.04	1.27±0.07
1-a-4	1.01±0.04	0.87±0.07	1-a-5	0.89±0.04	1.18±0.06
1-a-6	0.92±0.04	1.37±0.01	1-a-5	0.89±0.04	1.18±0.06
1-a-6	0.92±0.04	1.37±0.01	1-a-7	0.89±0.04	1.04±0.13
1-a-8	0.90±0.04	1.31±0.10	1-a-7	0.89±0.04	1.04±0.13
1-a-8	0.90±0.04	1.31±0.10	1-a-9	0.99±0.04	0.86±0.07
1-a-10	0.96±0.04	1.23±0.08	1-a-9	0.99±0.04	0.86±0.07
1-a-10	0.96±0.04	1.23±0.08	1-a-11	0.90±0.04	0.82±0.07
1-a-12	0.91±0.04	0.73±0.03	1-a-11	0.90±0.04	0.82±0.07
1-a-12	0.91±0.04	0.73±0.03	1-a-13	0.97±0.04	1.54±0.11
1-a-14	1.24±0.04	1.44±0.16	1-a-13	0.97±0.04	1.54±0.11

¹FC-free cholesterol

[0477] CT60 or CT43 cells were seeded in 6-well plates on the first day. The selection compound was added to the cells at a concentration of 10 μm. The cells were cultured for 24 hours. Extracting cell lipid with n-hexane and isopropanol (3: 2V/V). The lipid extract was dried and resuspended in n-hexane and subsequently separated on a gas chromatograph using beta-sitosterol as internal standard.

[0478] The method comprises the following steps: CT60 cells were plated in 6-well plates in Ham's F-12/1.176 g/L sodium bicarbonate/2 g/L glucose/10% FBS containing medium. After 24 hours of incubation, the cells were treated with 10 μ M of each compound and the medium was changed to contain Ham's F-12/1.176 g/L sodium bicarbonate/2 g/L glucose/5.5% FBS/20mM HEPES. The media in each well was changed so that the compounds after addition to the cells had similar experimental conditions as used for the screening. After 18h of compound treatment, cells were washed 2 times with Hank's Balanced Salt Solution (HBSS). The cellular lipids were extracted with n-hexane/isopropanol (3: 2v/v) (43), dried and resuspended in n-hexane, and then separated on a Gas Chromatograph (GC) using the following conditions. A Hewlett packard gas chromatography model HP 5890 series II (Palo Alto, CA) was used to separate free cholesterol equipped with a flame ionization detector, a separation-non-separation syringe, and a 15 m x 0.53 mm HP-5 capillary column coated with a 5% phenylmethylsiloxane film having a thickness of 1.5 μm. The injection temperature was maintained at 255 ℃ and the furnace temperature isothermally controlled at 260 ℃ using helium as the mobile phase at a flow rate of 30 mL/min. Free Cholesterol (FC) was quantified using β -sitosterol as an internal standard to correct for lipid loss during extraction.

[0479]Example 6: screening of Secondary Compound libraries

[0480] A secondary library containing 3962 compounds was evaluated. These compounds were selected on the basis of chemical similarity with respect to Tanimoto coefficient (47).

[0481] We screened these 3962 compounds using a method similar to the primary library screening except that the compounds were initially screened at both 10 μ M and 1 μ M concentrations. Each compound was added to a single well and images were taken at 2 positions per well and averaged to one value. 2 complete screens of the secondary pool were performed at two concentrations. Images were analyzed using two methods, mean phenanthroline intensity and LSO compartment ratio. At 10 μ M, we found 574 compounds to be lower than control 3SD when measured using one assay and 34 compounds to be lower than control 3SD when measured using both assays. At 1 μ M, we found 202 compounds to be lower than control 3SD using at least one assay and 6 compounds to be lower than control 3SD using 2 assays.

[0482] We chose 202 compounds at will and rescreen 2 times at 1. mu.M, 300nM and 100 nM. We added the compound to 4 different wells at each concentration and averaged the measurements of the images at 2 positions per well. We obtained 8 values for each compound from each assay at 1 μ M, 300nM and 100nM by 2 screens. For each assay (mean phenanthroline intensity and LSO compartment ratio) we have a total of 10 values of 1 μ M (2 from the first screen and 8 randomly selected) and 8 values of 300nM and 100 nM. Based on these data, we selected 7 compounds for further analysis, whose chemical structures are shown in FIG. 8.

[0483] The dose response curves for the 7 selected compounds are shown in figure 9. The data show that 4 compounds (2-a-8, 2-a-9, 2-a-12 and 2-a-13) showed a reduction of more than 3SD in the LSO compartment assay of 370nM, and 3 compounds (2-a-8, 2-a-9 and 2-a-12) also showed an effect on C T60 cells at 123nM, lower than the solvent control 2 SD. Most of these compounds were also effective on CT43 cells as candidates from the first round (fig. 9B).

[0484]Example 7: absorption of low density lipoproteins

[0485] CT60 cells were grown to 70% confluence in 96-well specialized optical culture plates (Corning, inc., Corning, NY). After 24 hours, the cells were incubated with DiI-LDL (6mg/ml) and a selection compound (10. mu.M) in screening medium supplemented with 20mM HEPES. Each compound was added to 8 wells and equal amounts of dimethyl sulfoxide and DiI-LDL were added to control wells. After 20h, cells were washed 3 times with PBS, fixed with 1.5% PFA for 20 min, and stained with 50. mu.g/ml Phellin for 45 min. Images were obtained at 20-fold magnification using a Discovery-1 model autofluorescence microscope. Images of DiI-LDL were obtained using a 535nm/40nm excitation filter and a 610nm/60nm bandpass with a Chroma 51001bs DiChronic filter. The filipin image was obtained as described above. Images were acquired at 4 sites per well, yielding 32 images per compound. The background and shading correction for this DiI-LDL image was as described above. A low threshold was set to determine cell area from the philippine images. Finally, the mean DiI-LDL intensity was measured for each cellular region.

[0486] As shown in Table 2, all of the selected compounds from the secondary pool except 2-a-15 caused a decrease in the uptake of low density lipoprotein over a 20 hour incubation. This would require further work to determine if this is a primary effect of certain compounds, or if the reduction in low density lipoprotein uptake is secondary to the release of cholesterol from LSOs. Although, as a consequence of a point mutation in one of the SCAP alleles, CT60 cells were partially defective in SCAP function, it is expected that the cells will respond to increased cholesterol by decreasing the expression of LDL receptors.

[0487] Table 2 absorption of DiI-LDL by CT60 cells after treatment with 7 selected compounds from the secondary pool.

Compound numbering	Cell area fraction of DiI intensity/control
Compound numbering	Cell area fraction of DiI intensity/control	2-a-1	0.43±0.01
2-a-3	0.62±0.01	2-a-1	0.43±0.01
2-a-3	0.62±0.01	2-a-8	0.77±0.01
2-a-9	0.85±0.01	2-a-8	0.77±0.01
2-a-9	0.85±0.01	2-a-12	0.72±0.01
2-a-13	0.72±0.01	2-a-12	0.72±0.01
2-a-13	0.72±0.01	2-a-15	1.00±0.01

[0488] Cells were incubated with DiI-LDL (6. mu.g/ml) in the presence of compound (10. mu.M) for 20 hours. The intensity per unit cell area is measured as described in the methods. Values were normalized to the intensity per cell area of control (solvent treated) cells. Values are based on 32 images from 8 wells per condition. + -SEM.

[0489]Example 8: toxicity assay of compounds from the secondary library.

[0490] FIG. 10 shows the toxicity assay results for 7 selected compounds from the secondary library. After 24h, Compound 2-a-12 caused a 75% reduction in cell number at 20 μm for both CT60 and CT43 cells. Other compounds did not or caused only a slight reduction in cells under these conditions, while they reduced phenanthroline staining. Cytotoxicity of these compounds was assessed by measuring LDH released into the medium. As shown in fig. 10C, the cytotoxic effect measured by this method was less than the reduction obtained from cell counts, indicating that these compounds may slow down cell growth without causing cell death after 24 hours.

[0491] The time course of the decrease in the proportion of LSO compartments at concentrations of 1.1, 3.3 and 10. mu.M is shown in FIG. 11. After 4 hours of treatment, compound 2-a-3 showed a decrease in the proportion of LSO compartments of more than 3 SD. Over 20 hours, all compounds showed a decrease in the proportion of the LSO compartment at 10. mu.M, and several compounds were effective at 1.11 and 3.33. mu.M. The effect of reducing the proportion of LSO compartments is generally retained after 48 hours of treatment.

[0492] Six of the 7 compounds selected from the secondary pool had low toxicity and significantly reduced FC not only in LSOs but also in whole cells. Effective concentrations of several of these compounds were below 0.5. mu.M. Table 3 summarizes the effect of these 6 selected compounds from the secondary pool in terms of cholesterol reduction as well as the toxic effect.

[0493] Table 3 summary of the effect of 7 compounds from the secondary pool on CT60 cells

Compound number.	Minimum effective dose (LSO measurement) (-3SD @20h)	Mean Firexate measurement @10 μ M fraction of dose control	g FC¹Fraction of/. mu.g protein (GC) @ 10. mu.M dose control	Fraction of protein content (μ g/well) control	Cell count @10 μ M fraction of dose control after 24h
Compound number.	Minimum effective dose (LSO measurement) (-3SD @20h)	Mean Firexate measurement @10 μ M fraction of dose control		Fraction of protein content (μ g/well) control	Cell count @10 μ M fraction of dose control after 24h	2-a-1	3.33μM	0.74±0.02	0.65±0.02	0.94±0.03	1.00
2-a-3	3.33μM	0.75±0.01	0.81±0.03	0.92±0.04	1.00	2-a-1	3.33μM	0.74±0.02	0.65±0.02	0.94±0.03	1.00
2-a-3	3.33μM	0.75±0.01	0.81±0.03	0.92±0.04	1.00	2-a-8	123nM	0.74±0.01	0.76±0.03	0.94±0.04	0.96
2-a-9	370nM	0.74±0.01	0.75±0.04	0.94±0.05	0.90	2-a-8	123nM	0.74±0.01	0.76±0.03	0.94±0.04	0.96
2-a-9	370nM	0.74±0.01	0.75±0.04	0.94±0.05	0.90	2-a-12	370nM	0.87±0.01	---	---	0.67
2-a-13	370nM	0.75±0.02	0.79±0.04	0.97±0.03	1.00	2-a-12	370nM	0.87±0.01	---	---	0.67
2-a-13	370nM	0.75±0.02	0.79±0.04	0.97±0.03	1.00	2-a-15	3.33μM	0.79±0.01	0.89±0.03	0.84±0.02	0.94

¹FC-free cholesterol

[0494]Example 9: determination of the cytotoxicity of LDH.

[0495] The cytotoxic effect of the selected compounds was determined using the LDH release assay kit (Roche Diagnostic GmbH, Penzberg, Germany) according to the manufacturer's instructions. CT60 cells were plated at 3500 cells/well in 96-well plates (Costar, Corning inc., Corning, NY) and incubated for 24 hours. Using a similar method to the dose-dependent assay, compounds were added in parallel to 3 at concentrations of 0 (dimethylsulfoxide solvent control), 5, 10 and 20 μ M, respectively, to CY60 cells. After 24h of treatment, 100 μ l of tissue culture supernatant was removed and LDH activity was determined by measuring absorbance at 492nm using a SpectraMax M2 fluorescence plate reader (Molecular Devices inc., Sunnyvale, CA). The experiment was repeated 3 times, thus obtaining an average of 9 data points.

[0496] As shown in fig. 10C, the cytotoxicity measured by this method was less than the decrease measured by cell count, indicating that these compounds may slow down cell growth without causing cell death after 24 hours.

[0497]Example 10: measurement of fibroblast cells in normal human body.

[0498] All the above results were from CHO cells with a mutation in one of the SCAPs in addition to the NPC1 gene mutation. To determine if the compound might have an effect on other cell types, we treated normal human fibroblasts with compound U18666A, which caused cholesterol accumulation similar to that shown by NPC cell lines (NL Jacobs et al, J Lipid Res.1997 Oct; 38 (10): 1973-87). We found that treatment of cells with 250nM or 500nM U18666A caused a significant increase in cholesterol accumulation as measured by the philippine staining (data not shown) and LSO compartment ratio method (fig. 12). When cells were treated with selected compounds from the secondary pool, several of these compounds caused a significant decrease in phenanthroline staining (fig. 12). In particular, compound 2-a-1 caused a sharp decrease in phenanthroline staining.

[0499]Example 11: assay for 25RACHO cells

[0500]25RACHO cells were incubated with compounds 2-a-1, 2-a-3, 2-a-8, 2-a-9 and 2-a-13 at a concentration of 10. mu.M. Cell load³H cholesterol, then followed (chase) overnight in the presence or absence of various compounds. The extracellular medium is removed and the radioactivity released from the cells is measured. The cells are then lysed and the radioactivity still remaining in the cells is measured. The data (FIG. 13) show that these compounds promote cholesterol efflux from 25-RA cells, a CHO cell line without Niemann-pick's disease, which was used to select blast lines for CT43 and CT60 cells that we screened above. 25-RA cells have a defect in SCAP that alters cholesterol homeostasis, including increasing cholesterol synthesis.

[0501]Example 12: lysosomal inhibition of acid lipase

[0502] Certain compounds disclosed herein may have therapeutic effects by inhibiting Lysosomal Acid Lipase (LAL). In particular compounds 1-a-4, 1-a-11, 1-a-14, 2-a-3, 2-a-8, 2-a-9, 2-a-13, 2-a-15, and other compounds within the general structural range defining the named compounds can inhibit the hydrolysis of cholesterol esters by LAL.

Claims

1. A method for treating a patient suffering from a condition characterized by cholesterol cell accumulation comprising the steps of:

administering to a patient in need thereof a therapeutically effective amount of a compound of any one of formulas I-IX, wherein formula I is represented by:

wherein,

x is O or-N (R)⁷)-；

Y is N or-C (R)⁸)-；

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

formula II is represented as:

wherein,

x is O or-N (R)⁶)-；

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

formula III is represented as:

wherein,

R³is hydrogen, alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；

each R⁶And R⁷Independently represents H or alkyl;

each R⁸Independently represents alkyl, cycloalkyl, aryl, heteroalkyl, aralkyl or heteroaralkyl;

l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

formula IV is represented as:

wherein,

Each R³Independently represents H or alkyl; and is

formula V is represented as:

wherein,

Y is O, S or-N (R)⁵)-；

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VI is represented as:

wherein,

x is O, S or-N (R)⁴)-；

each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VII is represented by:

wherein,

x is O or S;

R²Is H or alkyl;

R³is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or an optionally substituted bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R⁴is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

R⁵Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R⁵Is substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; and is

formula VIII is represented as:

wherein,

x is O or S;

R²and R⁴Independently represents H or alkyl;

R⁵is an optionally substituted monocyclic or bicyclic ring having a structure selected from O,1, 2 or 3 heteroatoms of N and S;

formula IX is represented as:

wherein,

X¹is-OR⁵、-SR⁵or-N (R)⁵)₂；

Each X²Independently represent O, S or-N (R)⁵)-；

R³is H, alkyl or halogen;

n is 0, 1, 2, 3 or 4.

2. The method of claim 1, wherein the disorder is niemann-pick disease type C.

3. The method of claim 1, wherein the disorder is atherosclerosis.

4. The method of claim 1, wherein said disorder is a lysosomal storage disorder caused by a disturbance in sphingolipid or glycosphingolipid metabolism.

5. The method of claim 1, wherein the compound is a compound of formula I.

6. The method of claim 1, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is N; r¹And R²Independently represents an alkyl group, a haloalkyl group or an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r ⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

7. The method of claim 1, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is N; r¹And R²Is an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; and R is⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

8. The method of claim 1, wherein the compound is of formula IThe compound, X is-N (R)⁷) -; y is-C (R)⁸)-；R¹And R²Independently represents alkyl, heteroalkyl or halogenated alkyl; r ³Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁴Is hydrogen; r⁵Is a heteroaryl group; r⁶Is H or alkyl; r⁷Is hydrogen, alkyl, heteroalkyl, or haloalkyl; and R is⁸Is H or alkyl.

9. The method of claim 1, wherein the compound is a compound of formula II.

10. The method of claim 1, wherein the compound is a compound of formula II, X is-N (R)⁶)-；R¹、R²And R⁵Independently represents aryl or heteroaryl; and R is³、R⁴And R⁶Independently represents hydrogen or alkyl.

11. The method of claim 1, wherein the compound is a compound of formula III.

12. The method of claim 1, wherein the compound is a compound of formula III; r¹、R²、A¹And A²Independently represents aryl or heteroaryl; r³Is hydrogen or alkyl; r⁶Is H or alkyl; and L is a bond.

13. The method of claim 1, wherein the compound is a compound of formula III; r¹Is- (C (R)⁷)₂)_n-(CR⁷＝C(R⁷)₂)；R²Is an alkyl group; r³Is alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；R⁴And R⁵Independently represents H or alkyl; or R⁴And R⁵Together form a bond; each R⁶And R⁷Independently represents H or alkyl; each R⁸Independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; l is a bond, -C (R) ⁷)₂-or- (CR)⁷＝CR⁷) -; and A is¹And A²Independently represents an aryl or heteroaryl group.

14. The method of claim 1, wherein the compound is a compound of formula IV.

15. The method of claim 1, wherein the compound is a compound of formula IV; A. r¹And R⁴Independently represents aryl or heteroaryl; r¹Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³＝CR³) -aryl or- (CR)³＝CR³) -a heteroaryl group; r²Is alkyl or aryl; and each R³Independently represents H or alkyl.

16. The method of claim 1, wherein the compound is a compound of formula V.

17. The method of claim 1, wherein the compound is a compound of formula V; x is-C (O) N (R)⁵) -or-CO₂-; y is O or S; r¹、R³And R⁴Independently represents aryl or heteroaryl; each R²Independently represent H or alkyl, or 2R²Together form ═ O; r³And R⁴Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

18. The method of claim 1, wherein the compound is a compound of formula VI.

19. The method of claim 1, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is aryl, heteroaryl, - (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

20. The method of claim 1, wherein the compound is a compound of formula VII.

21. The method of claim 1, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Is aryl, heteroaryl or an optionally substituted bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

22. The method of claim 1, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Represents

R⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is a substituted OR unsubstituted alkyl, halogen, -OR ⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; m is 0, 1, 2, 3 or 4; each R⁷Independently represents halogen, hydroxy, amino, carboxy, nitro, cyano, alkyl or alkoxy.

23. The method of claim 1, wherein the compound is a compound of formula VIII.

24. The method of claim 1, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Is an optionally substituted bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

25. The method of claim 1, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Represents

26. The method of claim 1, wherein the compound is a compound of formula IX.

27. The method of claim 1, wherein the compound is a compound of formula IX; x¹is-N (R)⁵)₂(ii) a Each X²Independently represents O or S; each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, -N (R)⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R)⁵)₂；R²And R⁴Independently represents aryl, heteroaryl, aralkyl or heteroaralkyl; r³Is H, alkyl or halogen; each R⁵Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and n is 0, 1, 2, 3 or 4.

28. The method of claim 1, wherein the compound is:

29. a method of reducing the amount of cholesterol in a cell comprising the steps of:

exposing mammalian cells to a compound of any one of formulas I-IX, wherein formula I is represented as:

wherein,

x is O or-N (R)⁷)-；

Y is N or-C (R)⁸)-；

R³is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R ²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

formula II is represented as:

wherein,

x is O or-N (R)⁶)-；

R¹And R²Independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl,Heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R³is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R ²And R³Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

formula III is represented as:

wherein,

R³is hydrogen, alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；

each R⁶And R⁷Independently represents H or alkyl;

L is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

formula IV is represented as:

wherein,

each R³Independently represents H or alkyl; and is

formula V is represented as:

wherein,

Y is O, S or-N (R)⁵)-；

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VI is represented as:

wherein,

x is O, S or-N (R)⁴)-；

each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VII is represented by:

wherein,

x is O or S;

R²Is H or alkyl;

R³is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or an optionally substituted bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R⁴is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

R⁵Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R⁵Is optionally substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; and is

formula VIII is represented as:

wherein,

x is O or S;

R²and R⁴Independently represents H or alkyl;

formula IX is represented as:

wherein,

X¹is-OR⁵、-SR⁵or-N (R)⁵)₂；

Each X²Independently represent O, S or-N (R)⁵)-；

R³is H, alkyl or halogen;

n is 0, 1, 2, 3 or 4.

30. The method of claim 29, wherein the compound reduces the amount of cholesterol in the cell by increasing cholesterol efflux from the cell.

31. The method of claim 29, wherein the compound reduces the amount of cholesterol in the cell by inhibiting cholesterol uptake by the cell.

32. The method of claim 29, wherein the compound reduces the amount of cholesterol by inhibiting cholesterol synthesis by the cell.

33. The method of claim 29, wherein the compound reduces the amount of cholesterol in the cell by promoting esterification of cholesterol in the cell.

34. The method of claim 29, wherein the cell is a human cell.

35. The method of claim 29, wherein the cell has a niemann-pick type C defect.

36. The method of claim 29, wherein the compound is a compound of formula I.

37. The method of claim 29, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is N; r¹And R²Independently represents an alkyl group, a haloalkyl group or an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r ⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; and R is⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

38. The method of claim 29, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is N; r¹And R²Is an aryl group; r³Is an aryl group; or R²And R³Together forming a group which may be substituted by one or more alkyl, halogen, hydroxy, alkoxy or amino groupsAn optionally substituted 3-8 membered ring; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; and R is⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

39. The method of claim 29, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is-C (R)⁸)-；R¹And R²Independently represents alkyl, heteroalkyl or haloalkyl; r³Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r ⁴Is hydrogen; r⁵Is a heteroaryl group; r⁶Is H or alkyl; r⁷Is hydrogen, alkyl, heteroalkyl, or haloalkyl; and R is⁸Is H or alkyl.

40. The method of claim 29, wherein the compound is a compound of formula II.

41. The method of claim 29, wherein said compound is a compound of formula II, X is-N (R)⁶)-；R¹、R²And R⁵Independently represents aryl or heteroaryl; and R is³、R⁴And R⁶Independently represents hydrogen or alkyl.

42. The method of claim 29, wherein the compound is a compound of formula III.

43. The method of claim 29, wherein the compound is a compound of formula III; r¹、R²、A¹And A²Independently represents aryl or heteroaryl; r³Is hydrogen or alkyl; r⁶Is H or alkyl; and L is a bond.

44. The method of claim 29, wherein the compound is a compound of formula III; r¹Is- (C (R)⁷)₂)_n-(CR⁷＝C(R⁷)₂)；R²Is an alkyl group; r³Is alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；R⁴And R⁵Independently represents H or alkyl; or R⁴And R⁵Together form a bond; each R⁶And R⁷Independently represents H or alkyl; each R⁸Independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; l is a bond, -C (R) ⁷)₂-or- (CR)⁷＝CR⁷) -; and A is¹And A²Independently represents an aryl or heteroaryl group.

45. The method of claim 29, wherein the compound is a compound of formula IV.

46. The method of claim 29, wherein the compound is a compound of formula IV; A. r¹And R⁴Independently represents aryl or heteroaryl; r¹Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³＝CR³) -aryl or- (CR)³＝CR³) -a heteroaryl group; r²Is alkyl or aryl; each R³Independently represents H or alkyl.

47. The method of claim 29, wherein the compound is a compound of formula V.

48. The method of claim 29, wherein the compound is a compound of formula V; x is-C (O) N (R)⁵) -or-CO₂-; y is O or S; r¹、R³And R⁴Independently represents aryl or heteroaryl; each R²Independently represent H or alkyl, or 2R²Together form ═ O; r³And R⁴Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

49. The method of claim 29, wherein the compound is a compound of formula VI.

50. The method of claim 29, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is aryl, heteroaryl, - (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; r³Is H or alkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

51. The method of claim 29, wherein said compound is a compound of formula VII.

52. The method of claim 29, wherein said compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Is aryl, heteroaryl or an optionally substituted bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is optionally substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; and each R⁶Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

53. The method of claim 29, wherein said compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Represents

R⁴Is H, alkyl, -CO ₂R⁶or-C (O) N (R)⁶)₂；R⁵Is optionally substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; m is 0, 1, 2, 3 or 4; and each R⁷Independently represents halogen, hydroxy, amino, carboxy, nitro, cyano, alkyl or alkoxy.

54. The method of claim 29, wherein the compound is a compound of formula VIII.

55. The method of claim 29, wherein said compound is a compound of formula VIII; x is O or S; (ii) a R¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; and R is⁵Is an optionally substituted bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

56. The method of claim 29, wherein said compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Represents

Wherein n is 0, 1, 2, 3 or 4; and each R⁷Independently represents halogen, hydroxy, amino, carboxy, nitro, cyano, alkyl or alkoxy.

57. The method of claim 29, wherein the compound is a compound of formula IX.

58. The method of claim 29, wherein the compound is a compound of formula IX; x¹is-N (R)⁵)₂(ii) a Each X²Independently represents O or S; each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, -N (R)⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R)⁵)₂；R²And R⁴Independently represents aryl, heteroaryl, aralkyl or heteroaralkyl; r³Is H, alkyl or halogen; each R⁵Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and n is 0, 1, 2, 3 or 4.

59. The method of claim 29, wherein the compound is:

60. a compound represented by formula X:

wherein,

x is OH or N (R)⁵)₂；

R²And R⁴Independently represents cycloalkenyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl having 1 heteroatom selected from N, O or S;

R³Is H, alkyl or halogen;

n is 0, 1, 2, 3 or 4; and is

In which X is NH₂When R is²And R⁴At least one of which is not an aryl group.

61. The compound of claim 60, wherein X is NH₂And R is²Is an aryl group.

62. The compound of claim 60, wherein X is NH₂And R is⁴Is an aryl group.

63. A compound represented by formula XI:

wherein,

R⁵、R⁶、R⁷and R⁸Independently represents hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; or R⁵、R⁶、R⁷And R⁸Together form an aryl or heteroaryl group substituted with at least one functional group selected from (C) ₂-C₆) Alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl, -N (R)⁹)₂、-OR⁹、-C(O)R⁹、-CO₂R⁹Or C (O) N (R)⁹)₂(ii) a And is

64. The compound of claim 63, wherein R¹And R²Forming a 6-membered ring.

65. The compound of claim 63, wherein R³And R⁴Forming a 6-membered ring.

66. The compound of claim 63, wherein R⁵、R⁶、R⁷And R⁸Together forming an aromatic ring.

67. A compound represented by formula XII:

wherein,

x is O, S or-N (R)⁴)-；

each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XIII:

Wherein,

x is O, S or-N (R)⁷)-；

R⁴Is- (C (R)⁸)₂)_n-(CR⁸＝C(R⁸)₂)；

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XIV:

wherein,

x is O, S or-N (R)¹²)-；

R¹¹is H, alkyl, alkenyl, aryl or heteroaryl;

each R¹²And R¹³Independently represents H, alkyl, aryl or aralkyl;

R¹⁴is heteroalkyl, heterocycloalkyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; and is

n is 1, 2, 3, 4 or 5.

68. The compound of formula XIII according to claim 67, wherein X is S and R⁴Is an allyl group.

69. The compound of formula XIII according to claim 67, in which X is S, R ⁴Is allyl and R⁵Is- (CR)⁸＝C(R⁸)₂)。

70. A compound of formula XIV as claimed in claim 67 wherein X is S and R⁹is-aryl-OR¹⁴。

71. A compound represented by formula XV:

wherein,

each R³Independently represents H, alkyl; and is

or a compound represented by formula XVI:

wherein,

R⁵is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, - (CR)⁷＝CR⁷) -aryl or- (CR)⁷＝CR⁷) -a heteroaryl group;

R⁶is alkyl or aryl;

each R⁷Independently represents H or alkyl; and is

or a compound represented by formula XVII:

Wherein,

R¹⁰is alkyl or aryl;

each R¹¹Independently represents H or alkyl; and is

R¹²Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl.

72. A compound of formula XVI as claimed in claim 71, wherein A is heteroaryl and R⁶Is an aryl group.

73. A compound of formula XVI as claimed in claim 71, wherein A is heteroaryl and R⁶Is an alkyl group.

74. A compound of formula XVII as claimed in claim 71, wherein A is heteroaryl and R⁹Is aryl, and R¹⁰Is an alkyl group.

75. A compound of formula XVII as claimed in claim 71, wherein A is heteroaryl and R⁹Is aryl, and R¹⁰Is an aryl group.

76. A compound represented by formula XVIII:

wherein,

R³is hydrogen, alkyl, -CO ₂R⁸or-C (O) N (R)⁷)(R⁸)；

each R⁶And R⁷Independently represents H or alkyl;

l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

or a compound represented by formula XIX:

wherein,

R¹⁰Is an aryl group;

R¹¹is hydrogen, alkyl, -CO₂R¹⁶or-C (O) N (R)¹⁵)(R¹⁶)；

each R¹⁴And R¹⁵Independently represents H or alkyl;

l is a bond, -C (R)¹⁵)₂-or- (CR)¹⁵＝CR¹⁵)-；

A⁴Represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR) ¹⁵＝CR¹⁵) -aryl or- (CR)¹⁵＝CR¹⁵) -a heteroaryl group;

or a compound represented by formula XX:

wherein,

R¹⁸Is an aryl group;

R¹⁹is hydrogen, alkyl, -CO₂R²⁴or-C (O) N (R)²³)(R²⁴)；

R²⁰And R²¹Independently represents H or alkyl;or R²⁰And R²¹Together form a bond;

each R²²And R²³Independently represents H or alkyl;

l is a bond, -C (R)²³)₂-or- (CR)²³＝CR²³)-；

A⁶Represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, - (CR)²³＝CR²³) -aryl or- (CR)²³＝CR²³) -a heteroaryl group;

or a compound represented by formula XXI:

wherein,

R²⁵is- (C (R)³¹)₂)_n-(CR³¹＝C(R³¹)₂)；

R²⁷is hydrogen, alkyl, -CO₂R³²or-C (O) N (R)³¹)(R³²)；

each R³⁰And R³¹Independently represents H or alkyl;

Each R³²Independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

l is oneBond, -C (R)³¹)₂-or- (CR)³¹＝CR³¹) -; and is

77. A compound of formula XVIII as claimed in claim 76, wherein R¹Is an aryl group.

78. A compound of formula XVIII as claimed in claim 76, wherein R¹Is aryl, and R⁴And R⁵Together forming a key.

79. A compound of formula XVIII as claimed in claim 76, wherein R¹Is aryl, R⁴And R⁵Together form a bond, L is a bond, and A¹Is a heteroaryl group.

80. A compound of formula XVIII as claimed in claim 76, wherein R¹Is aryl, R⁴And R⁵Together form a bond, L is a bond, A¹Is heteroaryl, and A²Is an aryl group.

81. A compound of formula XXI as claimed in claim 76, wherein R is²⁵Is an allyl group.

82. A compound of formula XXI as claimed in claim 76, wherein R is²⁵Is allyl, and R²⁷is-CO₂R³²。

83. A compound of formula XXI as claimed in claim 76, wherein R is²⁵Is allyl, R²⁷is-CO₂R³²And A is⁷Is a heteroaryl group.

84. A compound of formula XXI as claimed in claim 76, wherein R is ²⁵Is allyl, R²⁷is-CO₂R³²，A⁷Is heteroaryl and A⁸Is an aryl group.

85. A compound represented by formula XXII:

wherein,

Y is O, S or-N (R)⁵)-；

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

R³represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R⁴represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XXIII:

wherein,

Y is O, S or-N (R)¹⁰)-；

each R⁷Independently represent H or alkyl, or 2R⁷Together form ═ O;

R⁸is an aryl group;

each R¹⁰Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

or a compound represented by formula XXIV:

Wherein,

Y is O, S or-N (R)¹⁵)-；

R¹¹Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

each R¹²Independently represent H or alkyl, or 2R¹²Together form ═ O;

R¹³represents an aryl group;

R¹⁴represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aryl, aralkyl or heteroaralkyl;

each R¹⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

86. the process of claim 85A compound of the formula XXII, wherein R⁴Is aryl, X is NH, and R¹Is an aryl group.

87. A compound of formula XXIII as claimed in claim 85, wherein R is⁹Is aryl, X is NH, and R⁶Is an aryl group.

88. A compound of formula XXIV as claimed in claim 85, wherein R is¹³Is aryl, R¹⁴Is aryl and X is NH.

89. A compound represented by formula XXV:

wherein,

x is O or S;

R²Is H or alkyl;

R³is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or an optionally substituted bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R⁴is H, alkyl, -CO ₂R⁶or-C (O) N (R)⁶)₂；

or a compound represented by formula XXVI:

wherein,

x is O or S;

R⁸Is H or alkyl;

R⁹is an aryl group;

R¹⁰is H, alkyl or-C (O) N (R)¹²)₂；

R¹¹Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R¹¹Is substituted by one OR more alkyl, halogen, -OR¹²、-N(R¹²)₂、-CO₂R¹²、C(O)N(R¹²)₂An aryl group optionally substituted with cyano or nitro; and is

or a compound represented by formula XXVII:

wherein,

x is O or S;

R¹⁴Is H or alkyl;

R¹⁵is an aryl group;

R¹⁶is H, alkyl, -CO₂R¹⁸or-C (O) N (R)¹⁸)₂；

or a compound represented by formula XXVIII:

XXVIII

wherein,

x is O or S;

R²⁰Is H or alkyl;

R²¹is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or an optionally substituted bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R²²is alkyl, -CO₂R¹⁸or-C (O) N (R)¹⁸)₂；

R²³Is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R²³Is optionally substituted by one OR more alkyl, halogen, -OR¹⁸，-N(R¹⁸)₂、-CO₂R¹⁸、C(O)N(R¹⁸)₂An aryl group optionally substituted with cyano or nitro; and is

90. A compound of formula XXV as claimed in claim 89, wherein X is S, R¹is-C (O) R⁵，R²Is H, and R⁴is-CO₂R⁶。

91. A compound of formula XXVI as claimed in claim 89, wherein X is S, R⁷is-C (O) R¹¹And R is⁸Is H.

92. A compound of formula XXVII as claimed in claim 89 wherein X is S, R¹³is-C (O) R ¹⁷，R¹⁴Is H, and R¹⁶is-CO₂R¹⁸。

93. A compound represented by formula XXIX:

wherein,

x is O;

y is-C (R)⁸)-；

R³is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

or a compound represented by formula XXX:

wherein,

x is-N (R)¹⁶)-；

Y is-C (R)¹⁷)-；

R¹⁰And R¹¹Independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R¹²Is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹¹And R¹²Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

R¹³is H, alkyl, cycloalkyl, aryl or aralkyl;

R¹⁴is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, - (CR)¹⁸＝CR¹⁸)_n-aryl or- (CR)¹⁸＝CR¹⁸)_n-a heteroaryl group;

R¹⁵is H or alkyl; or R¹⁴And R¹⁵Together form a monocyclic or bicyclic ring which may be optionally substituted, said ring having 1 or 2 heteroatoms selected from O, N and S;

R¹⁶is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹⁰And R¹⁶Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

each R¹⁷And R¹⁸Independently represents H or alkyl;and is

n is 1 or 2.

94. The compound of formula XXX as claimed in claim 93, wherein R¹⁶、R¹⁰、R¹¹And R¹²Is an alkyl group.

95. The compound of formula XXX as claimed in claim 93, wherein R ¹⁶、R¹⁰、R¹¹And R¹²Is an alkyl group, and R¹³Is H.

96. The compound of formula XXX as claimed in claim 93, wherein R¹⁶、R¹⁰、R¹¹And R¹²Is alkyl, R¹³Is H, and R¹⁴Is an aryl group.

97. A compound represented by formula XXXI:

wherein,

R⁵Independently represents cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

or a compound represented by formula XXXII:

wherein,

R⁶and R⁸Independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁷and R⁹Independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; or R ⁶And R⁷Together form a 3-8 membered ring; or R⁸And R⁹Together form a 3-8 membered ring; and is

R¹⁰Independently represents H, alkyl, cycloalkyl, heteroaryl, aralkyl or heteroaralkyl.

98. The compound of formula XXXI as claimed in claim 97, wherein R¹And R²Together form a 7-membered ring, and R³And R⁴Together forming a 7-membered ring.

99. The compound of formula XXXI as claimed in claim 97, wherein R¹And R²Together form a 7-membered ring, R³And R⁴Together form a 7-membered ring, and R⁵Is an aryl group.

100. The compound of formula XXXII as claimed in claim 97, wherein R¹And R²Together form a 7-membered ring, R³And R⁴Together form a 7-membered ring, and R⁵Is an alkyl group.

101. The compound of formula XXXI as claimed in claim 97, wherein R¹And R³Is an aryl group.

102. The compound of formula XXXI as claimed in claim 97, wherein R¹、R³And R⁵Is an aryl group.

103. A compound represented by formula XXXIII:

wherein,

x is O;

R²and R⁴Independently represents H or alkyl;

Or a compound represented by formula XXXIV:

wherein,

x is S;

R⁷and R⁹Independently represents H or alkyl;

or a compound represented by formula XXXV:

wherein,

x is S;

R¹³represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R¹²and R¹⁴Independently represents H or alkyl;

or a compound represented by formula XXXVI:

wherein,

x is S;

R¹⁷and R¹⁹Independently represents H or alkyl;

104. A compound of formula XXXIV as described in claim 103 wherein X is S; a is aryl, R¹⁰Is a bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S, and R⁸Is an aryl group.

105. A compound of formula XXXV as claimed in claim 103 wherein X is S; a is aryl; r¹⁵Is a bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and SAnd R is¹¹Is an aryl group.

106. The compound of formula XXXVI recited in claim 103 wherein X is S; r²⁰Is a bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S; r¹⁸Is aryl, and R¹⁶Is an aryl group.

107. A compound represented by formula XXXVII:

wherein,

R¹、R²and R³Independently represents H, aryl, heteroaryl, aralkyl or heteroaralkyl; and is

A is independently a monocyclic or bicyclic aryl or heteroaryl group substituted with halo, alkyl, nitro, amino, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocycloalkyl.

108. The compound of claim 107, wherein R¹、R²And R³Is H.

109. The compound of claim 107, wherein R¹、R²And R³Is H and a is a monocyclic aryl substituted by one amino group.

110. The compound of claim 107, wherein R¹、R²And R³Is H and a is a monocyclic aryl substituted by one nitro group.

111. The compound of claim 107, wherein R¹、R²And R³Is H, and A is substituted by a halogenA monocyclic aryl group substituted with an aryl group.

112. A compound represented by formula XXXVIII:

XXXVIII

wherein,

R¹represents alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁴Independently represents H, alkyl, cycloalkyl, heteroaryl, aralkyl or heteroaralkyl;

or a compound represented by formula XXXIX:

wherein,

R⁵represents alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R⁶and R⁷Each independently represents hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and is

R⁸Independently represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

113. The compound of formula XXXVIII as claimed in claim 112, whereinR¹Is a haloalkyl group.

114. The compound of formula XXXVIII as claimed in claim 112, wherein R¹Is haloalkyl, and R²And R³Is an aryl group.

115. The compound of formula XXXVIII as claimed in claim 112, wherein R¹Is haloalkyl, R²And R³Is aryl, and at least one R⁴Is hydrogen.

116. The compound of formula XXXIX as claimed in claim 112, wherein R is⁶And R⁷Is an aryl group.

117. The compound of formula XXXIX as claimed in claim 112, wherein R is⁶And R⁷Is an aryl group; and at least one R⁸Is an aryl group.

118. The compound of formula XXXIX as claimed in claim 112, wherein R is⁶And R⁷Is aryl, one R⁸Is aryl, one R⁸Is hydrogen.

119. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of claims 60 to 118.

120. A method of treating or preventing drug-induced phosphatidic disease, comprising the steps of:

administering to a patient in need thereof a therapeutically effective amount of a compound of any one of formulas I through IX, wherein formula I is represented by:

Wherein,

x is O or-N (R)⁷)-；

Y is N or-C (R)⁸)-；

R³is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy, or amino groups;

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R⁷is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; or R¹And R⁷Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups;

Each R⁸And R⁹Independently represents H or alkyl; and is

n is 1 or 2;

formula II is represented as:

wherein,

x is O or-N (R)⁶)-；

R⁴is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

formula III is represented as:

wherein,

R³is hydrogen, alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸) (ii) a And is

each R⁶And R⁷Independently represents H or alkyl;

l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and is

formula IV is represented as:

wherein,

each R³Independently represents H or alkyl; and is

Formula V is represented as:

wherein,

x is O, -N (R)⁵)-、-N(R⁵)C(O)-、-C(O)N(R⁵)-、-OC(O)-、-CO₂-or-N (R)⁵)CO₂-

Y is O, S or-N (R)⁵)-；

each R²Independently represent H or alkyl, or 2R²Together form ═ O;

each R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VI is represented as:

VI

wherein,

x is O, S or-N (R)⁴)-；

each R⁴And R⁵Independently represents H, alkyl, aryl or aralkyl; and is

n is 1, 2, 3, 4 or 5;

formula VII is represented by:

wherein,

x is O or S;

R²Is H or alkyl;

R⁴is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；

formula VIII is represented as:

wherein,

x is O or S;

R²and R⁴Independently represents H or alkyl;

R⁵is an can be controlledAn optionally substituted monocyclic or bicyclic ring, said ring having 1, 2 or 3 heteroatoms selected from O, N and S; and is

Formula IX is represented as:

wherein,

X¹is-OR⁵、-SR⁵or-N (R)⁵)₂；

Each X²Independently represent O, S or-N (R)⁵)-；

R³is H, alkyl or halogen;

n is 0, 1, 2, 3 or 4.

121. The method of claim 120, wherein the compound is a compound of formula I.

122. The method of claim 120, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is N; r¹And R²Independently represents an alkyl group, a haloalkyl group or an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; and R is⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

123. The method of claim 120, wherein the compound is a compound of formula I, X is-N (R)⁷) -; y is N; r¹And R²Is an aryl group; r³Is an aryl group; or R²And R³Together form a 3-8 membered ring optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups; r⁴Is hydrogen; r⁵Is heterocycloalkyl or aryl; r⁶Is H or alkyl; or R⁵And R⁶Together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; and R is⁷Is hydrogen; or R¹And R⁷Together form a 3-8 membered ring which may be optionally substituted with one or more alkyl, halo, hydroxy, alkoxy or amino groups.

124. The method of claim 120, wherein the compound is a compound of formula I, X is O or-N (R)⁷) -; y is-C (R)⁸)-；R¹And R²Independently represents alkyl, heteroalkyl or haloalkyl; r³Is hydrogen, alkyl, heteroalkyl, or haloalkyl; r⁴Is hydrogen; r⁵Is a heteroaryl group; r⁶Is H or alkyl; r⁷Is hydrogen, alkyl, heteroalkyl, or haloalkyl; and R is⁸Is H or alkyl.

125. The method of claim 120, wherein the compound is a compound of formula II.

126. The method of claim 120, wherein the compound is a compound of formula II, X is-N (R) ⁶)-；R¹、R²And R⁵Independently represents aryl or heteroaryl; and R is³、R⁴And R⁶Independently represents hydrogen or alkyl.

127. The method of claim 120, wherein the compound is a compound of formula III.

128. The method of claim 120, wherein the compound is a compound of formula III; r¹、R²、A¹And A²Independently represents aryl or heteroaryl; r³Is hydrogen or alkyl; r⁶Is H or alkyl; and L is a bond.

129. The method of claim 120, wherein the compound is a compound of formula III; r¹Is- (C (R)⁷)₂)_n-(CR⁷＝C(R⁷)₂)；R²Is an alkyl group; r³Is alkyl, -CO₂R⁸or-C (O) N (R)⁷)(R⁸)；R⁴And R⁵Independently represents H or alkyl; or R⁴And R⁵Together form a bond; each R⁶And R⁷Independently represents H or alkyl; each R⁸Independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; l is a bond, -C (R)⁷)₂-or- (CR)⁷＝CR⁷) -; and A is¹And A²Independently represents an alkyl or heteroalkyl group.

130. The method of claim 120, wherein the compound is a compound of formula IV.

131. The method of claim 120, wherein the compound is a compound of formula IV; A. r¹And R⁴Independently represents aryl or heteroaryl; r ¹Is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, - (CR)³＝CR³) -aryl or- (CR)³＝CR³) -a heteroaryl group; r²Is alkyl or aryl; and each R³Independently represents H or alkyl.

132. The method of claim 120, wherein the compound is a compound of formula V.

133. The method of claim 120, wherein the compound is a compound of formula V; x is-C (O) N (R)⁵) -or-CO₂-; y is O or S; r¹、R³And R⁴Independently represents aryl or heteroaryl; each R²Independently represent H or alkyl, or 2R²Together form ═ O; r³And R⁴Independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁵Independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

134. The method of claim 120, wherein the compound is a compound of formula VI.

135. The method of claim 120, wherein the compound is a compound of formula VI; x is S; r¹Is aryl, heteroaryl or- (C (R)⁵)₂)_n-(CR⁵＝C(R⁵)₂)；R²Is aryl, heteroaryl, - (CR)⁵＝CR⁵) -aryl or- (CR)⁵＝CR⁵) -a heteroaryl group; each R⁵Independently represent H, alkylAryl or aralkyl; and n is 1 or 2.

136. The method of claim 120, wherein the compound is a compound of formula VII.

137. The method of claim 120, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Is aryl, heteroaryl or an optionally substituted bicyclic ring having 1 or 2 heteroatoms selected from O, N and S; r⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; and each R⁶Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

138. The method of claim 120, wherein the compound is a compound of formula VII; x is O or S; r¹Is aryl, heteroaryl or-C (O) R⁵；R²Is H or alkyl; r³Represents

R⁴Is H, alkyl, -CO₂R⁶or-C (O) N (R)⁶)₂；R⁵Is substituted by one OR more alkyl, halogen, -OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂An aryl group optionally substituted with cyano or nitro; each R⁶Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; m is 0, 1, 2, 3 or 4; and each R⁷Independently represent halogen, hydroxy, amino, carboxy, nitro, cyano Alkyl or alkoxy.

139. The method of claim 120, wherein the compound is a compound of formula VIII.

140. The method of claim 120, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; and R is⁵Is an optionally substituted bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

141. The method of claim 120, wherein the compound is a compound of formula VIII; x is O or S; r¹、R³And A independently represents aryl or heteroaryl; r²And R⁴Independently represents H or alkyl; r⁵Represents

142. The method of claim 120, wherein the compound is a compound of formula IX.

143. The method of claim 120, wherein the compound is a compound of formula IX; x¹is-N (R)⁵)₂(ii) a Each X²Independently represents O or S; each R¹Independently represents alkyl, halogen, nitro, cyano, alkoxy, -N (R)⁵)₂、-OH、-C(O)R⁶、-CO₂R⁵Or C (O) N (R) ⁵)₂；R²And R⁴Independently represent aryl, heteroaryl, aralkylA heteroaryl or a heteroarylalkyl group; r³Is H, alkyl or halogen; each R⁵Independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each R⁶Independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and n is 0, 1, 2, 3 or 4.

144. The method of claim 1, wherein the compound is:

145. the method of claim 120, wherein the patient is a mammal.

146. The method of claim 120, wherein the patient is a human.

147. The method of claim 1, 29, or 120, wherein said compound is a compound of formula III, R¹Contains one carboxylic acid group; r¹Is a carboxylic acid substituted aryl group; r¹Is a carboxylic acid substituted phenyl group; and/or R¹Is a phenyl group para-substituted by a carboxylic acid.

148. A compound having the formula XVIII as claimed in claim 76, wherein R¹Contains one carboxylic acid group; r¹Is a carboxylic acid substituted aryl group; r¹Is a carboxylic acid substituted phenyl group; and/or R¹Is a phenyl group para-substituted by a carboxylic acid.

149. A compound of formula XX according to claim 76, wherein R ¹⁷Contains one carboxylic acid group; r¹⁷Is a carboxylic acid substituted aryl group; r¹⁷Is a carboxylic acid substituted phenyl group; and/or R¹⁷Is a phenyl group para-substituted by a carboxylic acid.