CN101351208A - Reduce cellular cholesterol levels and/or treat or prevent phospholipid disorders - Google Patents

Abstract

The compounds disclosed in this specification can be used in the methods disclosed in this specification for reducing the amount of cholesterol in cells, for treating patients with disorders characterized by cellular accumulation of cholesterol (such as Niemann-Pick type C disease or atherosclerosis) and/or treat or prevent phospholipid disease. In some embodiments, these compounds may include pyrrolidone or triazine moieties.

Description

Reduce cellular cholesterol levels and/or treat or prevent phospholipid disorders

Cross-references to related applications

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

Statement on Federally Funded Research and Development

[002] National Institutes of Health Grant No. DK27083 provided partial support for research leading to the subject matter disclosed in this application. Accordingly, the United States Government has certain rights in the subject matter of this application.

Background technique

[003] Regulation 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. 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 regulated by transport from cells to extracellular receptors that remove cholesterol. These cholesterol transport mechanisms have been extensively studied, and defects in regulating cellular cholesterol levels have been linked to various diseases.

[004] Niemann-Pick disease is a class of inherited lipid storage disorders. Four types of Niemann-Pick disease are recognized: types A, B, C, and D. Types A and B are caused by a lack of sphingomyelinase activity that leads to increased sphingomyelin in cells, usually leading to cell death. People with type A Niemann-Pick disease tend to die between the ages of 2 and 4, whereas those with type B may survive into late childhood or into adulthood. Type D Niemann-Pick disease (also known as the Nova Scotia variant) is the equivalent of type C and occurs in descendants of western Nova Scotia.

[005] Niemann-Pick disease type C (NPC) is an autosomal recessive disorder that causes an abnormal accumulation of cholesterol and other lipids in many types of cells (1,2 ). The most severe symptoms are caused by progressive neuronal degeneration, but the liver and other peripheral organs also show defects. Although the time course can be altered, symptoms tend to occur in early childhood and the disease is usually fatal in the teens. There have been many attempts to develop treatments for NPC (3-8), but currently there are 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 being investigated. NCP1 is a multispan membrane protein that normally associates with late endosomes or lysosomes (9), degrades organelles, and hydrolyzes cholesteryl esters brought into cells by lipoproteins (10, 11). NPC1 has a sterol-sensitive transmembrane domain similar to proteins found in the endoplasmic reticulum that respond to changes in cellular cholesterol (12). The NPC1 protein facilitates bilayer transport of some hydrophobic molecules, but it does not appear to be directly involved in cholesterol transport (13-16). NPC2 is a soluble lumen protein found in late endosomes and binds cholesterol (17-19). NPC2 can freely shuttle free cholesterol to late endosomes and lysosome-restricted membranes, where NPC1 apparently plays a role in its export to other cellular sites (20). Loss of functional NPC1 or NPC2 causes accumulation of free cholesterol in endocytic organelles characteristic of late endosomes and/or lysosomes. These abnormal organelles referred to here are lysosome-like storage organelles (LSOs). The LSOs associated with NPC are similar to those associated with other inherited glycosphingolipid storage disorders (often caused by the inability to metabolize a specific lipid) in that the storage organelles contain a multilayered interior of a membrane bilayer In whorls, the bilayer contains cholesterol, sphingomyelin, and large amounts of 2-(monoacylglycerol)-phosphate (BMP), also known as lyso-bisphosphatidic acid (LBPA) (21, 22). Thus, even though these diseases arise from different genetic defects, some aspects of the cellular phenotype are quite similar.

[007] Several lines of evidence point to a defect in the transport of cholesterol in NPC, although defects in the transport of other esters may also play an important role (23). NPC cells display abnormally high levels of non-esterified cholesterol, which accumulates mainly in LSOs. Accumulated cholesterol can be detected using filipin, a fluorescent detergent capable of binding free cholesterol on membranes (24). In wild-type cells, excess cholesterol transported from endosomes is either transported from the cell to extracellular receptors or esterified by acyl-CoA:cholesterol acyltransferase (ACAT), an endoplasmic Enzymes online (25). Despite the high content of free cholesterol in LSOs, the plasma membrane of cultured NPC cells actually has lower cholesterol content than normal cells (26) and defects in cholesterol efflux to extracellular receptors (27). Furthermore, there is a drawback to the delivery of lipoprotein-derived cholesterol for esterification by ACAT (28, 29). These features suggest that cholesterol efflux from late endosomes is detrimental to NPC cells.

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

[009] A phospholipidosis is a condition in which phospholipids accumulate excessively in body tissues. Excessive accumulation of phospholipids is thought to be related to changes in phospholipid synthesis and/or metabolism. Phospholipid disorders can occur when a patient is given certain drugs. For example, amiodarone, perhexiline, fluoxetine, and gentamicin can cause phospholipidosis when administered to humans. 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 phospholipidopathy would be highly desirable.

[010] Accordingly, there is a need for treatments for Niemann-Pick disease and other diseases caused by defects in the regulation of cellular cholesterol levels. There is also a need for methods of treating or preventing drug-induced phospholipidosis. The present invention fulfills these needs and has other related advantages.

Contents of the invention

[011] One aspect of the present invention pertains to compounds and pharmaceutical compositions useful for lowering the amount of cholesterol in a cell. In certain instances, compounds of the invention include a pyrrole ring or triazine 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 containing an pyrrole ring or triazine moiety. Another aspect of the invention relates to a method of treating or preventing drug-induced phospholipidosis.

Description of drawings

What Fig. 1 described is the result of filipin binding assay, wherein the CHO cell (CT60) of wild-type CHO cell (TRVb1) and NPC1 mutant type is put into 384 well plates and grows in conventional culture medium 48 Hour. Cells were washed with PBS, fixed with 1.5% PFA, and stained with filipin. The 360/40nm excitation and 480/40nm emission filtered by 365DCLP filter were imaged at 10X magnification at 2 positions per well. (A) Image of TRVb1 cells stained with filipin; (B) Image of CT60 cells stained with filipin. Bar = 30 μΜ. Image analysis was performed using mean filipin intensity and LSO compartment ratio (Compartment Ratio). (C) Histogram of mean filipin intensity values; (D) Histogram of LSO compartment proportion values.

[013] Figure 2 depicts the results of a filipin binding assay in which cells were fixed with PFA and labeled with filipin. (A) Images obtained at 10x magnification using a Discovery-1 automated fluorescence microscope and filtering the 360/40nm excitation and 480/40nm emission light using a 365DCLP filter. (B) Image after correcting shadows and background. (C) High threshold settings were used to identify LSO compartments. (D) A high threshold setting was used to include the entire cell area. Bar = 20 μΜ.

What Fig. 3 depicts is the result of filipin binding assay, wherein CT60 cells are grown overnight in growth medium and are treated in screening solution with solvent (A) or 10 μ M selected compound (1-a-13) (B) . After 20 hours of incubation, the cells were washed with PBS, fixed with 1.5% PFA, and stained with filipin. Acquire images at 10X magnification. Bar = 25 μΜ.

[015] Figure 4 depicts images of filipin-stained CT60 cells affected by the addition of compounds that induce morphological changes and/or increase the intensity of filipin. (A) Compound 1-c-1 resulted in more diffuse fluorescence with no significant change in mean filipin intensity. (B) Compound 1-c-2 induces more compact LSOs without significant changes in filipin intensity. (C) Compound 1-c-3 caused the perinuclear accumulation of LSOs to become more dispersed in mutant cells. (D) Compound 1-b-4 caused a significant increase in filipin intensity with filamentous or tubular staining. Bar = 15 μM.

Fig. 5 has described the chemical structure (1-a-1, 1-a-2, 1-a-3, 1-a-4, 1-a-5 of 14 kinds of compounds obtained from primary library , 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 filipin potency, and compound 1-b-4 increased filipin potency while inducing morphological changes.

Fig. 6 has described 14 kinds of dose-response curves (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 medium. After 24 hours, the compounds were added to the final concentrations of 123nM, 370nM, 1.11μM, 3.33μM and 10μM, 4 wells for each concentration, and the cells were cultured overnight. Cells were washed with PBS, fixed with PFA, and stained with filipin. Determination of the ratio 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.

Fig. 7 has described 14 kinds of cytotoxicity analysis (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 medium. After 24 hours, the compound was added to the final concentration of 5, 10 and 20 μM, 4 wells for each concentration, and the cells were cultured overnight. The same amount of DMSO was added to the control wells. Cells were washed with PBS, fixed with PFA, and stained with nuclear stain Hoechst33258. Images were acquired at 4X magnification using a Discovery-1 automated fluorescence microscope and filtering the resulting 360/40 nm excitation and 480/40 nm emission light using a 365 DCLP filter. Cells per well were counted and the results of percentage reduction in cell number compared to control: (A) CT60 cells (average of 4 experiments) and (B) CT43 cells (average of 3 experiments).

What Fig. 8 described is the chemical structure (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a- 12, 2-a-13, 2-a-15).

What Fig. 9 described is the influence (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a-12 of 7 kinds of compounds obtained from secondary library , 2-a-13, 2-a-15). 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 dashed line indicates the mean-3 SD.

What Fig. 10 described is the cytotoxicity analysis (2-a-1, 2-a-3, 2-a-8, 2-a-9, 2-a-12, 2-a- 13, 2-a-15). The cytotoxicity of seven selected compounds obtained from the secondary library was determined by cell count and LDH release. For cell counts per well, cell counts were performed as described in Figure 7, results of percent reduction in cell number compared to control: (A) CT60 cells, and (B) CT43 cells. For the LDH cytotoxicity assay, measurement of cellular LDH released into culture medium in the presence of seven selected compounds from secondary: (C) CT60 cells, reference low (no compound) and high (dissolved cells) control.

What Fig. 11 described is the influence (2-a-1, 2-a-3, 2-a-8, 2-a-9, 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 24 h, compounds were added to final concentrations of 1.11, 3.33 and 10 μΜ in 4 different wells/concentrations 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 360/40nm excitation and 480/40nm emission filtered by 365DCLP filter were imaged at 10X magnification at 2 positions per well. The ratio of LSO compartments was measured (average of 3 different experiments). The solid horizontal line indicates the mean of the solvent control; the dashed line indicates the mean-3 SD.

What Fig. 12 described is the influence (2-a-1, 2-a-3, 2-a-8, 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 a 384-well plate with conventional medium for 24 hours, and then the cells were treated with compound U18666A (500 nM or 250 nM) in selection medium for 4 hours. Cells were then further incubated overnight with various selected compounds (10 [mu]M) in the continued presence of U18666A. Finally, the cells were washed 3 times with PBS, fixed with 1.5% PFA, washed with PBS and stained with filipin. Images were acquired at 10X magnification and analyzed for LSO ratio using a Discovery1 microscope. Solid horizontal lines refer to the mean of cells treated with each concentration of U18666A, dashed horizontal lines indicate -3 SD.

[024] FIG. 13 depicts increased cholesterol efflux from 25RA CHO cells, parental cell lines without NPC mutation, for CT60 and CT43 cell lines incubated with various compounds at 10 μM concentration.

Detailed ways

[025] One aspect of the invention provides compositions and methods for modulating levels of cellular cholesterol. The compositions of the present invention can be used to treat Niemann-Pick disease and other diseases associated with defects in the regulation of cellular cholesterol levels. As noted 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 filipin binding assay.

[026] Described herein is an automated screening assay to identify compounds capable of partially reversing the phenotype of Niemann-Pick disease type C (NPC) mutant cells. The method is based on the binding of filipin, a fluorescent detergent, to free cholesterol. In untreated mutant cells, there was a large amount of free cholesterol compared to control cell lines (42). This free cholesterol is highly concentrated in LSOs, organelles associated with late endosomes, but can also contain protein markers, which are generally not abundant in late endosomes (47). The molecular defect in NPC is a mutation or absence of one of two proteins, NPC1 and NPC2, associated with late endosomes. These mutations cause defective cholesterol efflux from late endosomes, leading to high levels of cholesterol accumulation in LSOs.

[027] Two screening assays were developed to evaluate the effect that test compounds have on modulating cellular cholesterol levels. The first detection method employs a filipin-fluorescence intensity threshold sufficient to determine the area of each image containing cells. Using this method, total filipin fluorescence can be obtained per cell area in each region. The intensity of plasma membrane staining provides a clear difference in the area of cells above background levels. But a second assay is needed because the threshold used to determine cell area does not clearly distinguish the LSO compartment from other cellular areas. The detection parameter used was the total fluorescence divided by the number of pixels above the threshold. This method is used to estimate total cholesterol per cell, based on the approximation that cell area remains constant under various conditions. Although this assay is considered to provide a reliable measure of total cholesterol content per cell, it may be useful if cells spread or aggregate significantly in response to a treatment, or if some cholesterol has a differential ability to bind filipin , the test results may be affected,.

[028] Although this assay does not use subcellular information or single cell analysis, it can be analyzed using automated microscopy. First, the microscope system is a sensitive detector for the relatively weak filipin fluorescence. Second, measurements are limited to the area of each region containing cells, which reduces background contributions. Finally, dividing the area covered by cells by the total fluorescence intensity provides a correction for differences in cell density when measured.

[029] We have found that filipin intensities per pixel that provide sufficient resolution of mutant versus wild-type cells are useful as a screening assay. This parameter was adopted when we adjusted the experimental conditions for the assay, such as cell density and labeling conditions. It should be noted, however, that caution should be exercised as in some cases CT60 cells were isolated from control cell lines by only a small standard deviation.

[030] We achieved good discrimination of wild-type versus mutant cells using the LSO compartment ratio assay, in which a threshold was used to identify the area in each region containing heavily labeled organelles (i.e., the LSOs). Because there are sites of cholesterol accumulation in NPC cells, selective measurement of cholesterol pools is expected to provide better discrimination of mutant versus wild-type cells. This additional sensitivity is useful in identifying partial active ingredient screening assays. The coefficient Z' (46) is a measure of the discriminative power of the screening assay, and the LSO compartment ratio assay had a Z' of 0.61, compared to 0.22 for the mean filipin intensity assay. Z' values greater than 0.5 are generally considered adequate for screening assays.

[031] In the first round of screening experiments, we identified 14 compounds that caused significant reductions in 10 [mu]M filipin labeling, including 3 compounds that produced significant reductions at 123 nM. The primary library was synthesized by combining 126 templates. Some compounds were observed to be effective at 123 nM, suggesting that it is possible that some of these compounds have high affinity interactions with their targets.

[032] A secondary library of compounds was screened with a Tanimoto similarity coefficient ranging from 0.3 to 0.96 (a high coefficient indicates high similarity). The average Tanimoto similarity coefficient is about 0.75. Screening assays use lower doses of test compounds and place greater emphasis on nontoxicity than primary library screening. Even though the dose of test compounds was reduced from 10 μM to 1 μM in the assay of the secondary library, the secondary library still contained a higher fraction (0.18%) of selected compounds compared to the primary library (0.1%). Thus, selection of chemicals in secondary libraries results in significant enrichment among potentially multiple hits. In addition, many of the selected compounds had greater efficacy and lower toxicity than those obtained from the primary screen. The 7 compounds identified from the secondary library were based on 4 synthetic templates. Compounds 2-a-1, 2-a-9, 2-a-12 and 2-a-13 are based on triazines and this class of compounds has been of great interest in the field of medicinal chemistry. See (48-52).

[033] The seven compounds selected from the secondary library for further characterization can be roughly divided into two groups. Compounds 2-a-1, 2-a-9, 2-a-12 and 2-a-13 (Group I) are based on a 1,3,5 triazine core, and this class of compounds has always been an important topic in the field of medicinal chemistry. of important interest (52-56). The second group of compounds (group II) has a 5-membered heterocyclic core (2-a-3: 2-thio-1,3-thiazolidin-4-one derivative, 2-a-15 contains a methine 2-a-8 contains 1,3-thiazole N-linked to a dihydropyrazole). These two groups of compounds are heavily substituted on the core, and the triazines in group I mainly have aryl groups or cyclic amines (or have a hydrazine group). Group II is also aryl substitution, where compound 2-a-3 features an interesting partially saturated diethyl-amino group attached via a double bond to 2-thioxo-1,3-thiazolidin-4-one naphthyl moiety. Compound 2-a-15 has three aromatic rings in an expanded 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 to be conformationally restricted molecules with a high degree of unsaturation. Their periphery tends to be very hydrophobic, while their center is more hydrophilic. We note that this feature implies that they are a kind of steric amphipathic (hydrophobic on the outside - hydrophilic on the inside). Although in some cases there are hydrophilic groups at the edges (notably the nitro group of 2-a-13), the consistent distribution of several hydrogen-bonded moieties towards the core of these structures suggests that these The core assists their specific recognition of targets in vivo.

[034] Although this assay was developed for use in CHO cell lines, with minor modifications it should also be applicable to the analysis of cholesterol accumulation in other cell types. A test for cholesterol accumulation would be 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 inner membrane helices in LSOs that contain sphingomyelin, lyso-diphosphatidic acid, and cholesterol (53, 54). The method described here can be used not only for chemical screens, but also for molecular genetic screens, such as RNAi inhibition and gene expression. Using traditional methods, a small number of genes have been identified that can correct the NPC phenotype when overexpressed in cells (36, 38, 55). The screens we describe here can be used for large-scale gene expression screens.

[035] These screening assays also identified compounds that enhanced filipin staining even beyond the levels seen in NPC mutant cells. Upon further investigation, some compounds initially shown to increase filipin staining were found to fluoresce in bands overlapping with the filipin spectrum, and thus their fluorescence may be the basis for increased fluorescence when detected. However, several non-fluorescent compounds were also found to increase filipin staining in NPC cells. We also found that some compounds produced significant changes in the morphology of free cholesterol-enriched compartments. In particular, compound 1-c-3 produced a large network of distinct tubular organelles marked by filipin.

[036] We also measured the cholesterol content of treated cells by a direct chemical method. Most of the selected compounds did reduce cellular cholesterol at the time of the screen, although the first 3 compounds selected did not show cholesterol reduction in a gas chromatographic analysis. Therefore, in some cases, results of filipin binding assays obtained from independent chemical assays should be carefully verified.

[037] Without being bound by a particular theory, it is believed that the extravasation of cholesterol from late endosomes requires several steps. This spillover, like many steps in intracellular transport, is apparently predominantly non-vesicular (56). NPC2 is speculated to play a role in the transport of cholesterol from the site of hydrolysis of sterol esters to the limiting membrane (57). It is speculated that NPC1 and other proteins facilitate the transport of cholesterol from the boundary membrane to the cytoplasmic carrier. These as yet molecularly identified carriers transport cholesterol to the plasma membrane or other organelles (58, 59). Total free cholesterol in organelles can be reduced by increasing spillover to extracellular receptors in the cytoplasm, and/or by esterification of cholesterol by ACAT in the endoplasmic reticulum. Decreased cholesterol uptake or decreased synthesis can also result in a decrease in cellular cholesterol upon incubation with the compound.

[038] Using assays similar to mean intensity and LSO assays, we determined the reduction of lipid BMPs aggregated in NPC cells. Selected compounds from the secondary library did not produce a significant reduction in BMP markers after 16 hours of incubation (data not shown). Several selected compounds from the secondary library did cause a decrease in cholesterol accumulation in U18666A-treated normal human fibroblasts, resulting in cholesterol accumulation in LSOs. This suggests that these compounds are not dependent on the SCAP mutation or other special properties of the CHO cell lines used for the screen.

[039] Without being bound by a particular theory, the effects of the test compounds may be directly on the LSOs, but there may also be indirect effects. For example, overexpression of Rab4, a small GTPase normally associated with endosomal sorting or endocytic recycling compartments, can partially correct the NPC phenotype (60).

[040] Compounds identified in the screening assay were effective in reducing cholesterol accumulation at concentrations that were not toxic to cultured NPC1 cells. In addition, several compounds (Fig. 13) should be able to effectively reduce the amount of cholesterol in normal cells, since they were shown to promote cholesterol extravasation in 25RA CHO cells. The compounds of the invention can also be used to study the mechanisms by which cells regulate cholesterol levels. For example, compounds of the present invention can be modified with photosensitive groups for labeling bound ligands or linked with biotin for affinity purification. Furthermore, without being bound by a particular theory, the compounds of the present invention are effective in reducing cholesterol uptake by cells and/or inhibiting cholesterol biosynthesis.

[041] Another aspect of the invention pertains to methods for treating or preventing drug-induced phospholipidopathy. Drug-induced phospholipidopathy can occur as a side effect when a pharmaceutical preparation is administered to a patient. For example, the following drugs can cause phospholipidosis: ABT-770, AC-3579, amantadine, ambroxol, amikacin, amiodarone, amitryptilline, AY-9944, azithromycin, benzo Formamide, Poxidine, Bromhexine, Chlorcyclazine, Chloroquine, Chlorphentermine, Chlorpromazine, Citalopram, Cloforex, Clomipramine, Clozapine, Compound 200-15 , cyclizine, DMP-777, erythromycin, fenfluramine, fluoxetine, fluvoxamine, gentamicin, hydroxyzine, IA-3, imipramine, iprindole, LY281389 , maprotiline, meclizine, mepalene, NE-10064, netilmicin, norchlorcyclizine (norchlorcyclizine), oxitriptyline, perhexiline, phentermine, PNU-177864, Promethazine, promethazine, propranolol, RM110.393, sertraline, tamoxifen, thioridazine, tilarone, tobramycin, trimipramine, tripalamol, tripyramine (triperennamine), spectinomycin, zimelidine, 1-chloroamitriptyline (1-chloroamitryptiline) and 4,4'-diethylaminoethoxyhexestrol. See MJ Reasor et al _. 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; MJReasor.Cationic Amphiphilic Drugs, in C _OMPREHENSIVE T _OXICOLOGY , Vol.8, T _{OXICOLOGY OF THE} R _ESPIRATORY S _YSTEM pp.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 cause drug-induced phospholipidopathy. Such drugs often have a hydrophilic region consisting of at least one primary or substituted nitrogen group positively charged at physiological pH, and an aromatic and/or cycloaliphatic group optionally substituted by halogen. the hydrophobic region. See MJReasor et al. Exp. Biol. Med. 2001, 226, 825.

[042] Methods for identifying compounds that cause phospholipidopathy are well known in the art. See eg H. Sawada et al. Toxicol. Sci. 2005, 83, 282. Since excessive accumulation of phospholipids is an undesirable side effect of certain drugs, one aspect of the present invention relates to a method for treating phospholipids by administering to a patient in need thereof a therapeutically effective amount of any one of the compounds of formulas I-IX described herein. Or a method of preventing drug-induced phospholipidosis. In certain instances, the patient's drug-induced phospholipidopathy was not caused by compound U-18666A. In certain instances, the patient's drug-induced phospholipidopathy was caused by ABT-770, AC-3579, amantadine, ambroxol, amikacin, amiodarone, amitriptyline, AY-9944, Azithromycin, benzamide, poxidine, bromhexine, chlorcyclizine, chloroquine, chlorphentermine, chlorpromazine, citalopram, cloforex, clomipramine, clozapine, compound 200-15, cyclizine, DMP-777, erythromycin, fenfluramine, fluoxetine, fluvoxamine, gentamicin, hydroxyzine, IA-3, imipramine, ipridin Indole, LY281389, maprotiline, meclizine, mepalene, NE-10064, netilmicin, norclocyclizine, oxitriptyline, perhexiline, phentermine, PNU-177864, Promethazine, promethazine, propranolol, RMI 10.393, sertraline, tamoxifen, thioridazine, tilarone, tobramycin, trimipramine, tripalarol, tripyramine, promethazine caused by administration of spectinomycin, zimelidine, 1-chloroamitriptyline and 4,4'-diethylaminoethoxyhexestrol. In certain instances, the patient's drug-induced phospholipidopathy was caused by administration of amiodarone, perhexiline, azithromycin, fluoxetine, imipramine, chlorcyclazine, tamoxifen, or gentamicin. caused by the drug.

[043] Another aspect of the present invention relates to a method comprising administering to a patient in need 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 the Any compound of the general formula I-IX; the second therapeutic agent is an anorectic agent, antianginal drug, antiarrhythmic drug, antibiotic, anticancer agent, antidepressant, antiestrogens , antihistamines, antilipids, antimalarials, antinausea, antitension, antithrombotics, antivirals, cholesterol synthesis inhibitors, diazepines, atypical antipsychotics, histamine H1-blockers stagnant agents, matrix metalloproteinase inhibitors, neutrophil elastase inhibitors, schistosomimetics, secretagogues, selective serotonin reuptake inhibitors, or tranquilizers that cause drug-induced phospholipidosis. In certain instances, the second therapeutic agent is an antibiotic, antiarrhythmic, antidepressant, histamine H1-blocker, or anticancer drug that causes drug-induced phospholipidosis.

[044] Another aspect of the present invention relates to a method for treating a mammalian cell of drug-induced phospholipidosis, comprising administering any one of the general formulas I-IX described in this specification to a therapeutically effective amount of the cell compound. In certain instances, the patient's drug-induced phospholipidopathy was not caused by compound U-18666A. Another aspect of the present invention relates to a method for treating mammalian cells with drug-induced phospholipidosis, comprising administering to the cells a therapeutically effective amount of any compound of the general formula I-IX described in this specification, Among them, drug-induced phospholipidosis is caused at least in part by ABT-770, AC-3579, amantadine, ambroxol, amikacin, amiodarone, amitriptyline, AY-9944, azithromycin, benzamide , poxidine, bromhexine, chlorocyclazine, chloroquine, p-chlorphentermine, chlorpromazine, citalopram, cloforex, clomipramine, clozapine, compound 200-15, race Crezine, DMP-777, erythromycin, fenfluramine, fluoxetine, fluvoxamine, gentamicin, hydroxyzine, IA-3, imipramine, iprindole, LY281389, horse Protriptyline, Meclizine, Mepalene, NE-10064, Netilmicin, Norclocyclizine, Oxitriptyline, Perhexiline, Phentermine, PNU-177864, Promethazine, Promethazine Zine, propranolol, RMI 10.393, sertraline, tamoxifen, thioridazine, tilarone, tobramycin, trimipramine, tripalarol, tripyramine, spectinomycin, azithromycin caused by the administration of meridine, 1-chloroamitriptyline and 4,4'-diethylaminoethoxyhexestrol.

[045] Preparation of Compounds of the Invention

[046] The compounds described in Figures 5 and 8 are commercially available. Using synthetic methods known in the art, compounds represented by general formula I-XXXIX can be prepared from compounds shown in Figures 5 and 8 or other commercially available compounds, see e.g. J. March, Advanced Organic Chemistry , McGraw Hill Book Company , New York, (1992, ^{4 th} edition); Carey, FA and Sundberg, RJ Advanced Organic Chemistry Part B: Reactions and Synthesis, 3 ^rd Ed.; Plenum Press: New York, 1990; and Organic Chemistry 2 ^nd Ed. Ed. Bruice , PY 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 in the compounds shown in Figures 5 and 8 or by modifying existing functional groups located thereon. For example, typical functional group manipulations include introducing bromine by treating the aromatic compound with _Br2 in the presence of _FeBr3 , introducing an acyl group by treating the aromatic compound with an acid chloride in the presence of _FeBr3 , introducing an acyl group by treating the aromatic compound with an acid chloride in the presence of FeBr3, using _SnCl2 in HCl Treatment of nitroaromatics gives aminoaromatics. Other functional group manipulations include reduction of aromatic groups with Na/ _NH3 to give cycloalkenes or cycloalkyl compounds depending on the reaction conditions. For example, reduction of 1-a-13 with Na/ _NH3 will selectively give cyclohexene derivatives due to the effect of carboxylic acid. See option 1. The cyclohexene intermediate can be further reduced to give a cyclohexyl derivative. Alternatively, the cyclohexene intermediate can be treated with an oxidizing agent to form an epoxide. The carboxylic acid group can also be converted to an ester by reaction with an alcohol such as methanol or benzyl alcohol in the presence of DCC.

plan 1:

[048] As shown in Scheme 2, a large number of compounds can be prepared from lactam derivative intermediates using the palladium coupling technique. Palladium coupling reactions are advantageous because they often proceed in high yields and are applicable to a wide variety of functional groups. Furthermore, considerable quantities of organoboranes are known and/or commercially available. In addition, a large number of aromatic and alkenyl halides are commercially available, which can be easily converted into organoborane starting materials for coupling reactions.

Scenario 2:

[049] A variety of 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 transformed into other compounds through the above-mentioned aromatic functional groups manipulation. Notably, palladium coupling of aryl/heteroaryl bromides or iodides can also be used to prepare various triazinylhydrazones as shown in Scheme 4.

Option 3:

Option 4:

[050] The method of the present invention

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

Give any one compound in the formula I-IX of patient's effective dose needed, and wherein formula I is expressed as:

in,

X is O or -N(R ⁷ )-;

Y is N or -C(R ⁸ )-;

R ^{and R} independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or Heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ² and R ³ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

^R is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R ⁵ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁹ =CR ⁹ ) _n -aryl, or -(CR ⁹ =CR ⁹ ) _n -heteroaryl;

R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring, the ring has 1 or 2 heteroatoms selected from O, N and S;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

each R ⁸ and R ⁹ independently represent H or alkyl; and

n is 1 or 2;

Formula II is expressed as:

in,

X is O or -N(R ⁶ )-;

R ^{and R} independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ² and R ³ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

^R is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

Formula III is expressed as:

in,

R ¹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ⁷ ) ₂ ) _n -(CR ⁷ = C(R ⁷ ) ₂ );

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or alkyl;

R ³ is hydrogen, alkyl, -CO ₂ R ⁸ or -C(O)N(R ⁷ )(R ⁸ ); and

R ⁴ and R ⁵ independently represent H or an alkyl group; or R ⁴ and R ⁵ together form a bond;

Each R ⁶ and R ⁷ independently represent H or an alkyl group;

each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

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

A ¹ and A ² independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁷ =CR ⁷ )-aryl or -(CR ⁷ = CR ⁷ )-heteroaryl;

Formula IV is expressed as:

in,

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

R ¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³ =CR ³ )-aryl or -(CR ³ =CR ³ )-hetero Aryl;

^R is alkyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each ^R independently represents H or an alkyl group;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula V is expressed as:

in,

X is O, -N(R ⁵ )-, -N(R ⁵ )C(O)-, -C(O)N(R ⁵ )-, -OC(O)-, -CO ₂ -, or -N (R ⁵ )CO ₂ -;

Y is O, S or -N(R ⁵ )-;

^R is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Each R ² independently represents H or an alkyl group, or 2 R ² together form =O;

R ^{and R} independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each ^R independently represents H, alkyl, aryl or aralkyl; and

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

Formula VI is expressed as:

in,

X is O, S or -N(R ⁴ )-;

R ¹ is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ =C(R ⁵ ) ₂ ) ;

R ² is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-hetero Aryl;

R ³ is H, alkyl, alkenyl, aryl or heteroaryl; or R ² and R ³ together form an optionally substituted monocyclic or bicyclic ring having O selected from O, N and S , 1 or 2 heteroatoms;

each R ^{and R} independently represent H, alkyl, aryl or aralkyl; and

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

Formula VII is expressed as:

in,

X is O or S;

R ¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -C(O)R ⁵ ;

R ² is H or alkyl;

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

R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ;

R ⁵ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R ⁵ is a group that can be replaced by any one or more of alkyl, halogen, -OR ⁶ , -N(R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl groups; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula VIII is represented as:

in,

X is O or S;

R ¹ , R ³ and A independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R ² and R ⁴ independently represent H or an alkyl group;

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

Formula IX is expressed as:

in,

X ¹ is -OR ⁵ , -SR ⁵ or -N(R ⁵ ) ₂ ;

Each X ² independently represents O, S or -N(R ⁵ )-;

Each ^R independently represents alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl Alkyl, -N(R ⁵ ) ₂ , -OH, -C(O)R ⁶ , -CO ₂ R ⁵ or C(O)N(R ⁵ ) ₂ ;

R ^and R ^{independently} represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R ³ is H, alkyl or halogen;

each ^R independently represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

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

[053] In certain embodiments, the present invention relates to the aforementioned method, wherein said condition is Niemann-Picker disease type C.

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

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

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

[057] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is O or -N(R ⁷ )-; Y is N; R ¹ and R ² independently represent alkyl, haloalkyl or aryl; ^R3 is aryl; or ^R2 and ^R3 together form a 3 which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino -8-membered ring; R ⁴ is hydrogen; R ⁵ is heterocycloalkyl or aryl; R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring, said ^The ring has 1 or 2 heteroatoms selected from O, N and ^S ; ^R is hydrogen; Choose a substituted 3-8 membered ring.

[058] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is -N(R ⁷ )-; Y is N; R ¹ and R ² are aryl R is an aryl group; or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one ^{or more} alkyl, halogen, hydroxyl, alkoxy or amino; R is hydrogen; R ⁵ is heterocycloalkyl or aryl; R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring having a ring selected from O, N and S 1 or 2 heteroatoms; ^R7 is hydrogen; or ^R1 and ^R7 together form a 3-8 membered ring optionally substituted with one or more alkyl, halogen, hydroxy, alkoxy or amino groups.

[059] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is -N(R ⁷ )-; Y is -C(R ⁸ )-; R ¹ and ^R2 independently represent alkyl, heteroalkyl or haloalkyl; ^R3 is hydrogen, alkyl, heteroalkyl or haloalkyl; ^R4 is hydrogen; ^R5 is heteroaryl; ^R6 is H or alkane R ⁷ is hydrogen, alkyl, heteroalkyl or haloalkyl; R ⁸ is H or alkyl.

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

[061] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula II, X is -N(R ⁷ )-; R ¹ , R ² and R ⁵ independently represent aryl or heteroaryl; and R ³ , R ⁴ and R ⁶ independently represent hydrogen or alkyl.

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

[063] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III; R ¹ , R ² , A ¹ and A ² independently represent aryl or heteroaryl; ^R3 is hydrogen or alkyl; ^R6 is H or alkyl; L is a bond.

[064] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III; R ¹ is -(C(R ⁷ ) ₂ ) _n -(CR ⁷ =C(R ⁷ ) ₂ ); R ² is an alkyl group; R ³ is an alkyl group, -CO ₂ R ⁸ or -C(O)N(R ⁷ )(R ⁸ ); R ⁴ and R ⁵ independently represent H or an alkyl group or R ⁴ and R ⁵ together form a bond; each R ⁶ and R ⁷ independently represent H or an alkyl group; each R ⁸ independently represent an alkyl, cycloalkyl, heterocycloalkyl, aryl, hetero Aryl, aralkyl or heteroaralkyl; L is a bond, -C(R ⁷ ) ₂ - or -(CR ⁷ =CR ⁷ )-; and A ¹ and A ² independently represent alkyl or heteroalkane base.

[065] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III, wherein ^R comprises a carboxylic acid group; ^R is a carboxylic acid substituted aryl; R ¹ is a carboxylic acid substituted phenyl group; and/or R ¹ is a carboxylic acid para-substituted phenyl group;

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

[067] In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula IV; A, R ¹ and R ⁴ independently represent aryl or heteroaryl; R ¹ is ring Alkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³ =CR ³ )-aryl or -(CR ³ =CR ³ )-heteroaryl; R ² is alkyl or aryl; each R ³ independently represents H or alkyl.

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

[069] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula V; X is -C(O)N(R ⁵ )- or -CO ₂ -; Y is O or S; R ¹ , R ³ and R ⁴ independently represent aryl or heteroaryl; each R ² independently represents H or alkyl, or 2 R ² together form =O; R ³ and R ⁴ independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each ^R independently 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 said compound is a compound of Formula VI.

[071] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, heteroaryl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ =C(R ⁵ ) ₂ ); R ² is aryl, heteroaryl, -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-heteroaryl each R ^{independently} 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 said compound is a compound of formula VI; X is S; R ¹ is aryl, R ² is aryl, R ³ is H or alkyl.

[073] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is alkoxy substituted phenyl, R ² is dialkyl Amino substituted phenyl, R ³ is H.

[074] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, heteroaryl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ =C(R ⁵ ) ₂ ); R ² is -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-heteroaryl; R ³ is H or alkyl R ^{and R} together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S; each ^R independently 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 said compound is a compound of formula VII.

[076] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VII; X is O or S; ^R is aryl, heteroaryl or -C(O ) R ⁵ ; R ² is H or alkyl; R ³ is alkyl, heteroaryl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heterocyclic rings selected from O, N and S atom; R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ; R ⁵ is a group that can be replaced by one or more alkyl, halogen, -OR ⁶ , -N( R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl group; each R ⁶ independently represents H, alkyl, aryl , aralkyl or heterocycloalkyl.

[077] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VII; X is O or S; ^R is aryl, heteroaryl or -C(O )R ⁵ ; R ² is H or alkyl; R ³ represents R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ; R ⁵ is a group that can be replaced by one or more alkyl, halogen, -OR ⁶ , -N(R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl group; each R ⁶ independently represents H, alkyl, aryl, aryl Alkyl or heterocycloalkyl; m is 0, 1, 2, 3 or 4; each R ^{independently} represents halogen, hydroxyl, amino, carboxyl, nitro, cyano, alkyl or alkoxy.

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

[079] In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VIII; X is O or S; R ¹ , R ³ and A independently represent aryl or heteroaryl R ² and R ⁴ independently represent H or alkyl; R ⁵ is an optionally substituted monocyclic or bicyclic ring with 1, 2 or 3 heteroatoms selected from O, N and S.

其中n是0、1、2、3或4；每个R⁷独立地代表卤素、羟基、氨基、羧基、硝基、氰基、烷基或烷氧基。[080] In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VIII; X is O or S; R ¹ , R ³ and A independently represent aryl or heteroaryl group; R ² and R ⁴ independently represent H or alkyl; R ⁵ represents

wherein n is 0, 1, 2, 3 or 4; each R ^{independently} represents halogen, hydroxy, amino, carboxyl, nitro, cyano, alkyl or alkoxy.

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

[082] In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula IX; X ¹ is -N(R ⁵ ) ₂ ; Each X ² independently represents O or S ; each R ¹ independently represents alkyl, halogen, nitro, cyano, alkoxy, -N(R ⁵ ) ₂ , -OH, -C(O)R ⁶ , -CO ₂ R ⁵ or C( O) N(R ⁵ ) ₂ ; R ² and R ⁴ independently represent aryl, heteroaryl, aralkyl or heteroaralkyl; R ³ is H, alkyl or halogen; each R ⁵ independently represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; each ^R independently 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 method, wherein said compound is

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

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

[086] Mammalian cells are exposed to a compound of any one of formulas I-IX, wherein formula I is represented as:

in,

X is O or -N(R ⁷ )-;

Y is N or -C(R ⁸ )-;

R ^{and R} independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ² and R ³ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

^R is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R ⁵ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁹ =CR ⁹ ) _n -aryl, or -(CR ⁹ =CR ⁹ ) _n -heteroaryl;

R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring, the ring has 1 or 2 heteroatoms selected from O, N and S;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

each R ⁸ and R ⁹ independently represent H or alkyl; and

n is 1 or 2;

Formula II is expressed as:

in,

X is O or -N(R ⁶ )-;

R ^{and R} independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ² and R ³ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

^R is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

Formula III is expressed as:

in,

R ¹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ⁷ ) ₂ ) _n -(CR ⁷ = C(R ⁷ ) ₂ );

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or alkyl;

R ³ is hydrogen, alkyl, -CO ₂ R ⁸ or -C(O)N(R ⁷ )(R ⁸ );

R ⁴ and R ⁵ independently represent H or an alkyl group; or R ⁴ and R ⁵ together form a bond;

Each R ⁶ and R ⁷ independently represent H or an alkyl group;

each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

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

A ¹ and A ² independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁷ =CR ⁷ )-aryl or -(CR ⁷ = CR ⁷ )-heteroaryl;

Formula IV is expressed as:

in,

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

R ¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³ =CR ³ )-aryl or -(CR ³ =CR ³ )-hetero Aryl;

^R is alkyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each R ^{independently} represents H or an alkyl group;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula V is expressed as:

in,

X is O, -N(R ⁵ )-, -N(R ⁵ )C(O)-, -C(O)N(R ⁵ )-, -OC(O)-, -CO ₂ -, or -N (R ⁵ )CO ₂ -;

Y is O, S or -N(R ⁵ )-;

^R is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Each R ² independently represents H or an alkyl group, or 2 R ² together form =O;

R ^{and R} independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each ^R independently represents H, alkyl, aryl or aralkyl; and

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

Formula VI is expressed as:

in,

X is O, S or -N(R ⁴ )-;

R ¹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ = C(R ⁵ ) ₂ );

R ² is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-hetero Aryl;

R ³ is H, alkyl, alkenyl, aryl or heteroaryl; or R ² and R ³ together form an optionally substituted monocyclic or bicyclic ring having O selected from O, N and S , 1 or 2 heteroatoms;

each R ^{and R} independently represent H, alkyl, aryl or aralkyl; and

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

Formula VII is expressed as:

in,

X is O or S;

R ¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -C(O)R ⁵ ;

R ² is H or alkyl;

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

R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ;

R ⁵ cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R ⁵ is a group that can be replaced by one or more alkyl, halogen, -OR ⁶ , -N(R ⁶ ) _2. -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl groups; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula VIII is represented as:

in,

X is O or S;

R ¹ , R ³ and A independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R ² and R ⁴ independently represent H or an alkyl group;

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

Formula IX is expressed as:

in,

X ¹ is -OR ⁵ , -SR ⁵ or -N(R ⁵ ) ₂ ;

Each X ² independently represents O, S or -N(R ⁵ )-;

Each ^R independently represents alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl Alkyl, -N(R ⁵ ) ₂ , -OH, -C(O)R ⁶ , -CO ₂ R ⁵ or C(O)N(R ⁵ ) ₂ ;

R ² and R ⁴ independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R ³ is H, alkyl or halogen;

each ^R independently represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

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

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

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

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

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

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

[092] In certain embodiments, the present invention relates to the aforementioned method, wherein said cell has a type C Niemann-Picker deficiency.

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

[094] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is O or -N(R ⁷ )-; Y is N; R ¹ and R ² independently represent alkyl, haloalkyl or aryl; ^R3 is aryl; or ^R2 and ^R3 together form a 3 which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino -8-membered ring; R ⁴ is hydrogen; R ⁵ is heterocycloalkyl or aryl; R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring, said ^The ring ^has 1 or 2 heteroatoms selected from O, N and S; ^R is hydrogen; Choose a substituted 3-8 membered ring.

[095] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is -N(R ⁷ )-; Y is N; R ¹ and R ² are aromatic R is an aryl group; or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one ^{or more} alkyl, halogen, hydroxyl, alkoxy or amino; R is hydrogen; R ⁵ is heterocycloalkyl or aryl; R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring having a ring selected from O, N and S 1 or 2 heteroatoms; ^R7 is hydrogen; or ^R1 and ^R7 together form a 3-8 membered ring optionally substituted by one or more alkyl, halogen, hydroxy, alkoxy or amino groups.

[096] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is -N(R ⁷ )-; Y is -C(R ⁸ )-; R ¹ and ^R2 independently represent alkyl, heteroalkyl or haloalkyl; ^R3 is hydrogen, alkyl, heteroalkyl or haloalkyl; ^R4 is hydrogen; ^R5 is heteroaryl; ^R6 is H or alkane R ⁷ is hydrogen, alkyl, heteroalkyl or haloalkyl; R ⁸ is H or alkyl.

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

[098] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula II, X is -N(R ⁷ )-; R ¹ , R ² and R ⁵ independently represent aryl or heteroaryl; and R ³ , R ⁴ and R ⁶ independently represent hydrogen or alkyl.

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

[0100] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III; R ¹ , R ² , A ¹ and A ² independently represent aryl or heteroaryl; ^R3 is hydrogen or alkyl; ^R6 is H or alkyl; L is a bond.

[0101] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III; R ¹ is -(C(R ⁷ ) ₂ ) _n -(CR ⁷ =C(R ⁷ ) ₂ ); R ² is an alkyl group; R ³ is an alkyl group, -CO ₂ R ⁸ or -C(O)N(R ⁷ )(R ⁸ ); R ⁴ and R ⁵ independently represent H or an alkyl group or R ⁴ and R ⁵ together form a bond; each R ⁶ and R ⁷ independently represent H or an alkyl group; each R ⁸ independently represent an alkyl, cycloalkyl, heterocycloalkyl, aryl, hetero Aryl, aralkyl or heteroaralkyl; L is a bond, -C(R ⁷ ) ₂ - or -(CR ⁷ =CR ⁷ )-; and A ¹ and A ² independently represent alkyl or heteroalkane base.

[0102] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III, ^R comprising a carboxylic acid group; ^R being a carboxylic acid substituted aryl; ^R being a carboxylic acid substituted phenyl group; and/or R ¹ is a carboxylic acid para-substituted phenyl group.

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

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula IV; A, R ¹ and R ⁴ independently represent aryl or heteroaryl; R ¹ is ring Alkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³ =CR ³ )-aryl or -(CR ³ =CR ³ )-heteroaryl; R ² is alkyl or aryl; each R ³ independently represents H or alkyl.

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

[0106] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula V; X is -C(O)N(R ⁵ )- or -CO ₂ -; Y is O or S; R ¹ , R ³ and R ⁴ independently represent aryl or heteroaryl; each R ² independently represents H or alkyl, or 2 R ² together form =O; R ³ and R ⁴ independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each ^R independently 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 said compound is a compound of Formula VI.

[0108] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, heteroaryl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ =C(R ⁵ ) ₂ ); R ² is aryl, heteroaryl, -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-heteroaryl each R ^{independently} represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, R ² is aryl, R ³ is H or alkyl.

[0110] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; ^R is phenyl substituted with alkoxy, ^R is dialkyl Amino substituted phenyl, R ³ is H.

[0111] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, heteroaryl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ =C(R ⁵ ) ₂ ); R ² is -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-heteroaryl; R ³ is H or alkyl R ^{and R} together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S; each ^R independently 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 said compound is a compound of formula VII.

[0113] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VII; X is O or S; ^R is aryl, heteroaryl or -C(O ) R ⁵ ; R ² is H or alkyl; R ³ is alkyl, heteroaryl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heterocyclic rings selected from O, N and S atom; R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ; R ⁵ is a group that can be replaced by one or more alkyl, halogen, -OR ⁶ , -N( R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl group; each R ⁶ independently represents H, alkyl, aryl , aralkyl or heterocycloalkyl.

R⁴是H、烷基、-CO₂R⁶或-C(O)N(R⁶)₂；R⁵是一个可以被一个或多个烷基、卤素、-OR⁶、-N(R⁶)₂、-CO₂R⁶、C(O)N(R⁶)₂、氰基或硝基任选取代的芳基基团；每个R⁶独立地代表H、烷基、芳基、芳烷基或杂环烷基；m是0、1、2、3或4；每个R⁷独立地代表卤素、羟基、氨基、羧基、硝基、氰基、烷基或烷氧基。In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a kind of compound of formula VII; X is O or S; ^R is aryl, heteroaryl or-C(O )R ⁵ ; R ² is H or alkyl; R ³ represents

R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ; R ⁵ is a group that can be replaced by one or more alkyl, halogen, -OR ⁶ , -N(R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl group; each R ⁶ independently represents H, alkyl, aryl, aryl Alkyl or heterocycloalkyl; m is 0, 1, 2, 3 or 4; each R ^{independently} represents halogen, hydroxyl, amino, carboxyl, nitro, cyano, alkyl or alkoxy.

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

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VIII; X is O or S; R ¹ , R ³ and A independently represent aryl or heteroaryl R ² and R ⁴ independently represent H or alkyl; R ⁵ is an optionally substituted monocyclic or bicyclic ring with 1, 2 or 3 heteroatoms selected from O, N and S.

其中n是0、1、2、3或4；每个R⁷独立地代表卤素、羟基、氨基、羧基、硝基、氰基、烷基或烷氧基。In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VIII; X is O or S; R ¹ , R ³ and A independently represent aryl or heteroaryl group; R ² and R ⁴ independently represent H or alkyl; R ⁵ represents

wherein n is 0, 1, 2, 3 or 4; each R ^{independently} represents halogen, hydroxy, amino, carboxyl, nitro, cyano, alkyl or alkoxy.

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

[0119] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula IX; X ¹ is -N(R ⁵ ) ₂ ; Each X ² independently represents O or S ; each R ¹ independently represents alkyl, halogen, nitro, cyano, alkoxy, -N(R ⁵ ) ₂ , -OH, -C(O)R ⁶ , -CO ₂ R ⁵ or C( O) N(R ⁵ ) ₂ ; R ² and R ⁴ independently represent aryl, heteroaryl, aralkyl or heteroaralkyl; R ³ is H, alkyl or halogen; each R ⁵ independently represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl and n is 0, 1, 2, 3 or 4.

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

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

Another aspect of the present invention relates to a method for treating or preventing drug-induced phospholipidosis, comprising the steps of:

Give a compound of any one of the formula I-IX of effective dose to the patient in need, wherein formula I is represented as:

in,

X is O or -N(R ⁷ )-;

Y is N or -C(R ⁸ )-;

R ^{and R} independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or Heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ² and R ³ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

^R is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R ⁵ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁹ =CR ⁹ ) _n -aryl, or -(CR ⁹ =CR ⁹ ) _n -heteroaryl;

R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring, the ring has 1 or 2 heteroatoms selected from O, N and S;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

each R ⁸ and R ⁹ independently represent H or alkyl; and

n is 1 or 2;

Formula II is expressed as:

in,

X is O or -N(R ⁶ )-;

R ^{and R} independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ² and R ³ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

^R is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

Formula III is expressed as:

in,

R ¹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ⁷ ) ₂ ) _n -(CR ⁷ = C(R ⁷ ) ₂ );

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or alkyl;

R ³ is hydrogen, alkyl, -CO ₂ R ⁸ or -C(O)N(R ⁷ )(R ⁸ ); and

R ⁴ and R ⁵ independently represent H or an alkyl group; or R ⁴ and R ⁵ together form a bond;

Each R ⁶ and R ⁷ independently represent H or an alkyl group;

each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

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

A ¹ and A ² independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁷ =CR ⁷ )-aryl or -(CR ⁷ = CR ⁷ )-heteroaryl;

Formula IV is expressed as:

in,

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

R ¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³ =CR ³ )-aryl or -(CR ³ =CR ³ )-hetero Aryl;

^R is alkyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each R ^{independently} represents H or an alkyl group;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula V is expressed as:

in,

X is O, -N(R ⁵ )-, -N(R ⁵ )C(O)-, -C(O)N(R ⁵ )-, -OC(O)-, -CO ₂ -, or -N (R ⁵ )CO ₂ -;

Y is O, S or -N(R ⁵ )-;

^R is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Each R ² independently represents H or an alkyl group, or 2 R ² together form =O;

R ^{and R} independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each ^R independently represents H, alkyl, aryl or aralkyl; and

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

Formula VI is expressed as:

in,

X is O, S or -N(R ⁴ )-;

R ¹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ = C(R ⁵ ) ₂ );

R ² is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-hetero Aryl;

R ³ is H, alkyl, alkenyl, aryl or heteroaryl; or R ² and R ³ together form an optionally substituted monocyclic or bicyclic ring having O selected from O, N and S , 1 or 2 heteroatoms;

each R ^{and R} independently represent H, alkyl, aryl or aralkyl; and

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

Formula VII is expressed as:

in,

X is O or S;

R ¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -C(O)R ⁵ ;

R ² is H or alkyl;

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

R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ;

R ⁵ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl; or R ⁵ is a group that may be replaced by one or more alkyl, halogen, -OR ⁶ , -N(R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl groups; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Formula VIII is represented as:

in,

X is O or S;

R ¹ , R ³ and A independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R ² and R ⁴ independently represent H or an alkyl group;

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

Formula IX is expressed as:

in,

X ¹ is -OR ⁵ , -SR ⁵ or -N(R ⁵ ) ₂ ;

Each X ² independently represents O, S or -N(R ⁵ )-;

Each ^R independently represents alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl Alkyl, -N(R ⁵ ) ₂ , -OH, -C(O)R ⁶ , -CO ₂ R ⁵ or C(O)N(R ⁵ ) ₂ ;

R ^and R ^{independently} represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R ³ is H, alkyl or halogen;

each ^R independently represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

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

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

[0125] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is O or -N(R ⁷ )-; Y is N; R ¹ and R ² independently represent alkyl, haloalkyl or aryl; ^R3 is aryl; or ^R2 and ^R3 together form a 3 which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino -8-membered ring; R ⁴ is hydrogen; R ⁵ is heterocycloalkyl or aryl; R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring, said ^The ring has 1 or 2 heteroatoms selected from O, N and ^S ; ^R is hydrogen; Choose a substituted 3-8 membered ring.

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula I, X is -N(R ⁷ )-; Y is N; R ¹ and R ² are aryl R is an aryl group; or ^R and ^R together form a 3-8 membered ring which may be optionally substituted by one ^{or more} alkyl, halogen, hydroxyl, alkoxy or amino; R is hydrogen; R ⁵ is heterocycloalkyl or aryl; R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring having a ring selected from O, N and S 1 or 2 heteroatoms; ^R7 is hydrogen; or ^R1 and ^R7 together form a 3-8 membered ring optionally substituted with one or more alkyl, halogen, hydroxy, alkoxy or amino groups.

[0127] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula I, X is -N(R ⁷ )-; Y is -C(R ⁸ )-; R ¹ and ^R2 independently represent alkyl, heteroalkyl or haloalkyl; ^R3 is hydrogen, alkyl, heteroalkyl or haloalkyl; ^R4 is hydrogen; ^{R5 is heteroaryl; R6 is} H or alkane R ⁷ is hydrogen, alkyl, heteroalkyl or haloalkyl; R ⁸ is H or alkyl.

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

[0129] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula II, X is -N(R ⁷ )-; R ¹ , R ² and R ⁵ independently represent aryl or heteroaryl; and R ³ , R ⁴ and R ⁶ independently represent hydrogen or alkyl.

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

[0131] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III; R ¹ , R ² , A ¹ and A ² independently represent aryl or heteroaryl; ^R3 is hydrogen or alkyl; ^R6 is H or alkyl; L is a bond.

[0132] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III; R ¹ is -(C(R ⁷ ) ₂ ) _n -(CR ⁷ =C(R ⁷ ) ₂ ); R ² is an alkyl group; R ³ is an alkyl group, -CO ₂ R ⁸ or -C(O)N(R ⁷ )(R ⁸ ); R ⁴ and R ⁵ independently represent H or an alkyl group or R ⁴ and R ⁵ together form a bond; each R ⁶ and R ⁷ independently represent H or an alkyl group; each R ⁸ independently represent an alkyl, cycloalkyl, heterocycloalkyl, aryl, hetero Aryl, aralkyl or heteroaralkyl; L is a bond, -C(R ⁷ ) ₂ - or -(CR ⁷ =CR ⁷ )-; and A ¹ and A ² independently represent alkyl or heteroalkane base.

[0133] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula III; ^R contains a carboxylic acid group; ^R is a carboxylic acid substituted aryl group; R ¹ is a carboxylic acid substituted phenyl; and/or R ¹ is a carboxylic acid para-substituted phenyl.

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

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula IV; A, R ¹ and R ⁴ independently represent aryl or heteroaryl; R ¹ is ring Alkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³ =CR ³ )-aryl or -(CR ³ =CR ³ )-heteroaryl; R ² is alkyl or aryl; each R ³ independently represents H or alkyl.

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

[0137] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula V; X is -C(O)N(R ⁵ )- or -CO ₂ -; Y is O or S; R ¹ , R ³ and R ⁴ independently represent aryl or heteroaryl; each R ² independently represents H or alkyl, or 2 R ² together form =O; R ³ and R ⁴ independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl; each ^R independently 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 said compound is a compound of Formula VI.

[0139] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, heteroaryl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ =C(R ⁵ ) ₂ ); R ² is aryl, heteroaryl, -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-heteroaryl R ³ is H or alkyl; each R ⁵ independently represents H, alkyl, aryl or aralkyl; and n is 1 or 2.

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, R ² is aryl, R ³ is H or alkyl.

[0141] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; ^R is phenyl substituted with alkoxy, ^R is dialkyl Amino substituted phenyl, R ³ is H.

[0142] In certain embodiments, the present invention relates to the aforementioned method, wherein said compound is a compound of formula VI; X is S; R ¹ is aryl, heteroaryl or -(C(R ⁵ ) ₂ ) _n -(CR ⁵ =C(R ⁵ ) ₂ ); R ² is -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-heteroaryl; R ³ is H or alkyl R ^{and R} together form an optionally substituted monocyclic or bicyclic ring having 0, 1 or 2 heteroatoms selected from O, N and S; each ^R independently 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 said compound is a compound of formula VII.

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a kind of compound of formula VII; X is O or S; ^R is aryl, heteroaryl or-C(O ) R ⁵ ; R ² is H or alkyl; R ³ is alkyl, heteroaryl or an optionally substituted monocyclic or bicyclic ring having 1 or 2 heterocyclic rings selected from O, N and S atom; R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ; R ⁵ is a group that can be replaced by one or more alkyl, halogen, -OR ⁶ , -N( R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl group; each R ⁶ independently represents H, alkyl, aryl , aralkyl or heterocycloalkyl.

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a kind of compound of formula VII; X is O or S; ^R is aryl, heteroaryl or-C(O )R ⁵ ; R ² is H or alkyl; R ³ represents R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ; R ⁵ is a group that can be replaced by one or more alkyl, halogen, -OR ⁶ , -N(R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl groups; each R ⁶ independently represents H, alkyl, aryl, aryl Alkyl or heterocycloalkyl; m is 0, 1, 2, 3 or 4; each R ^{independently} represents halogen, hydroxyl, amino, carboxyl, nitro, cyano, alkyl or alkoxy.

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

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VIII; X is O or S; R ¹ , R ³ and A independently represent aryl or heteroaryl R ² and R ⁴ independently represent H or alkyl; R ⁵ is an optionally substituted monocyclic or bicyclic ring with 1, 2 or 3 heteroatoms selected from O, N and S.

其中n是0、1、2、3或4；每个R⁷独立地代表卤素、羟基、氨基、羧基、硝基、氰基、烷基或烷氧基。In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula VIII; X is O or S; R ¹ , R ³ and A independently represent aryl or heteroaryl group; R ² and R ⁴ independently represent H or alkyl; R ⁵ represents

wherein n is 0, 1, 2, 3 or 4; each R ^{independently} represents halogen, hydroxy, amino, carboxyl, nitro, cyano, alkyl or alkoxy.

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

In certain embodiments, the present invention relates to aforementioned method, wherein said compound is a compound of formula IX; X ¹ is -N(R ⁵ ) ₂ ; Each X ² independently represents O or S ; each R ¹ independently represents alkyl, halogen, nitro, cyano, alkoxy, -N(R ⁵ ) ₂ , -OH, -C(O)R ⁶ , -CO ₂ R ⁵ or C( O) N(R ⁵ ) ₂ ; R ² and R ⁴ independently represent aryl, heteroaryl, aralkyl or heteroaralkyl; R ³ is H, alkyl or halogen; each R ⁵ independently represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; each ^R independently 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 method, wherein said compound is

[0152] In certain embodiments, the present invention relates to the aforementioned method, wherein said 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 method, wherein said compound is

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

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

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

Compounds and compositions of the present invention

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

in,

X is OH or N(R ⁵ ) ₂ ;

Each ^R independently represents alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl Alkyl, -N(R ⁵ ) ₂ , -OH, -C(O)R ⁶ , -CO ₂ R ⁵ or C(O)N(R ⁵ ) ₂ ;

R ² and R ⁴ independently represent cycloalkenyl, heterocycloalkenyl, aryl, aralkyl, heteroarylalkyl or heteroaryl, the heteroaryl has 1 heteroaryl selected from N, O or S atom;

R ³ is H, alkyl or halogen;

each ^R independently represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

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

with the proviso that when X is _NH2 , at least one of ^R2 and ^R4 is not aryl.

In certain embodiments, the present invention relates to the aforementioned compound, wherein said X is NH ₂ and R ² is aryl.

In certain embodiments, the present invention relates to the aforementioned compound, wherein said X is NH ₂ and R ⁴ is aryl.

Another aspect of the present invention relates to a compound represented by formula XI:

in,

R ^{and R} independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or Heteroaralkyl;

R ^and R ^{independently} represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroarylalkyl; or R ¹ and R ² form a 3-8 membered ring together; or R ³ and R ⁴ form a 3-8 membered ring together;

R ⁵ , R ⁶ , R ⁷ and R ⁸ independently represent hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; or R ⁵ , R ⁶ , R ⁷ and R ⁸ together form a group consisting of at least one member selected from (C ₂ -C ₆ ) alkyl, halogen, nitro, cyano, alkoxy, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, aryl, hetero Functional groups of aryl, aralkyl or heteroaralkyl, -N(R ⁹ ) ₂ , -OR ⁹ , -C(O)R ⁹ , -CO ₂ R ⁹ or C(O)N(R ⁹ ) ₂ substituted aryl or heteroaryl; and

Each ^R independently represents H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

[0160] In certain embodiments, the present invention relates to the aforementioned compounds, wherein R ^{and R} form a 6-membered ring.

[0161] In certain embodiments, the present invention relates to aforementioned compounds, wherein R ³ and R ⁴ form a 6-membered ring.

[0162] In certain embodiments, the present invention relates to the aforementioned compound, wherein R ⁵ , R ⁶ , R ⁷ and R ⁸ together form an aromatic ring.

Another aspect of the present invention relates to a compound represented by formula XII:

in,

X is O, S or -N(R ⁴ )-;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl;

R ² is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁵ =CR ⁵ )-aryl or -(CR ⁵ =CR ⁵ )-hetero Aryl;

R ³ is H, alkyl, alkenyl, aryl or heteroaryl; or R ² and R ³ together form an optionally substituted monocyclic or bicyclic ring having O selected from O, N and S , 1 or 2 heteroatoms;

each R ^{and R} independently represent H, alkyl, aryl or aralkyl; and

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

Or a compound represented by formula XIII:

in,

X is O, S or -N(R ⁷ )-;

R ⁴ is -(C(R ⁸ ) ₂ ) _n -(CR ⁸ =C(R ⁸ ) ₂ );

R ⁵ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(CR ⁸ =C(R ⁸ ) ₂ );

^R is H, alkyl, alkenyl, aryl or heteroaryl; or R and ^R together form an optionally substituted monocyclic ^or bicyclic ring having O selected from O, N and S , 1 or 2 heteroatoms;

Each ^R and ^R independently represents H, alkyl, cycloalkyl or heterocycloalkyl, aralkyl or heteroaralkyl; and

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

Or a compound represented by formula XIV:

in,

X is O, S or -N(R ¹² )-;

R ⁹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, -aryl-OR ¹⁴ , heteroaryl, aralkyl, or heteroaralkyl;

R ¹⁰ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ¹³ =CR ¹³ )-aryl or -(CR ¹³ =CR ¹³ )-hetero Aryl;

R ¹¹ is H, alkyl, alkenyl, aryl or heteroaryl;

Each R ¹² and R ¹³ independently represent H, alkyl, aryl or aralkyl;

R is ^heteroalkyl , heterocycloalkyl, heteroaryl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl; and

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

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

[0165] In certain embodiments, the present invention relates to compounds of the aforementioned formula XIII, wherein X is S, R ⁴ is allyl and R ⁵ is -(CR ⁸ =C(R ⁸ ) ₂ ).

[0166] In certain embodiments, the present invention relates to compounds of the aforementioned formula XIV, wherein X is S and ^R9 is -allyl- ^OR14 .

[0167] In certain embodiments, the present invention relates to compounds of the aforementioned formula XIV, wherein X is S and R ⁹ is -allyl-OR ¹⁴ , R ¹⁰ is aryl, and R ¹¹ is H.

Another aspect of the present invention relates to a compound represented by formula XIVa:

in,

X is O, S or -N(R ¹² )-;

R ⁹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, -aryl-OR ¹⁴ , heteroaryl, aralkyl, or heteroaralkyl;

R ¹⁰ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ¹³ =CR ¹³ )-aryl or -(CR ¹³ =CR ¹³ )-hetero Aryl;

R ¹¹ is H, alkyl, alkenyl, aryl or heteroaryl; or R ¹⁰ and R ¹¹ together form an optionally substituted monocyclic or bicyclic ring having O selected from O, N and S , 1 or 2 heteroatoms;

Each R ¹² and R ¹³ independently represent H, alkyl, aryl or aralkyl;

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

[0169] In certain embodiments, the present invention relates to compounds of the aforementioned formula XIVa, wherein X is S ^and R is aryl.

Another aspect of the present invention relates to a compound represented by formula XV:

in,

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

R ¹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³ =CR ³ )-aryl or -(CR ³ =CR ³ )-hetero Aryl;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl;

each ^R independently represents H or alkyl; and

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Or a compound represented by formula XVI:

in,

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

R ⁵ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁷ =CR ⁷ )-aryl or -(CR ⁷ =CR ⁷ )-heteroaryl;

^R is alkyl or aryl;

each ^R independently represents H or alkyl; and

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Or a compound represented by formula XVII:

in,

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

R ⁹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ¹¹ =CR ¹¹ )-aryl or -(CR ¹¹ =CR ¹¹ )-hetero Aryl;

R ¹⁰ is alkyl or aryl;

each ^R independently represents H or alkyl; and

R ¹² is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

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

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

[0173] In certain embodiments, the present invention relates to compounds of the aforementioned formula XVII, wherein A is heteroaryl, R ⁹ is aryl, and R ¹⁰ is alkyl.

[0174] In certain embodiments, the present invention relates to compounds of the aforementioned formula XVII, wherein A is heteroaryl, R ⁹ is aryl, and R ¹⁰ is aryl.

Another aspect of the present invention relates to a compound represented by formula XVIII:

in,

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl;

^R is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, or alkyl;

R ³ is hydrogen, alkyl, -CO ₂ R ⁸ or -C(O)N(R ⁷ )(R ⁸ );

R ⁴ and R ⁵ independently represent H or an alkyl group; or R ⁴ and R ⁵ together form a bond;

Each R ⁶ and R ⁷ independently represent H or an alkyl group;

Each ^R independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

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

A ¹ and A ² independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁷ =CR ⁷ )-aryl or -(CR ⁷ = CR ⁷ )-heteroaryl;

Or a compound represented by formula XIX:

in,

R ⁹ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ¹⁵ ) ₂ ) _n -(CR ¹⁵ ═C (R ¹⁵ ) ₂ );

R ¹⁰ is aryl;

R ¹¹ is hydrogen, alkyl, -CO ₂ R ¹⁶ or -C(O)N(R ¹⁵ )(R ¹⁶ );

R ¹² and R ¹³ independently represent H or an alkyl group; or R ¹² and R ¹³ together form a bond;

Each R ¹⁴ and R ¹⁵ independently represents H or an alkyl group;

each ^R independently represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

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

A ³ represents a divalent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ¹⁵ =CR ¹⁵ )-aryl- or -(CR ¹⁵ = CR ¹⁵ )-heteroaryl-; and

A ⁴ represents cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ¹⁵ =CR ¹⁵ )-aryl or -(CR ¹⁵ =CR ¹⁵ )-hetero Aryl;

Or a compound represented by formula XX:

in,

R ¹⁷ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl or -(C(R ²³ ) ₂ ) _n -(CR ²³ = C(R ²³ ) ₂ );

R ¹⁸ is aryl;

R ¹⁹ is hydrogen, alkyl, -CO ₂ R ²⁴ or -C(O)N(R ²³ )(R ²⁴ );

R ²⁰ and R ²¹ independently represent H or an alkyl group; or R ²⁰ and R ²¹ together form a bond;

each R ²² and R ²³ independently represent H or an alkyl group;

each ^R independently represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

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

A ⁵ represents a divalent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ²³ =CR ²³ )-aryl- or -(CR ²³ = CR ²³ )-heteroaryl-; and

A ⁶ represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, -(CR ²³ =CR ²³ )-aryl or -(CR ²³ =CR ²³ )-heteroaryl;

Or a compound represented by formula XXI:

in,

R ²⁵ is -(C(R ³¹ ) ₂ ) _n -(CR ³¹ =C(R ³¹ ) ₂ );

R is ^cycloalkyl , heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl;

R ²⁷ is hydrogen, alkyl, -CO ₂ R ³² or -C(O)N(R ³¹ )(R ³² );

R ²⁸ and R ²⁹ independently represent H or an alkyl group; or R ²⁸ and R ²⁹ together form a bond;

Each R ³⁰ and R ³¹ independently represents H or an alkyl group;

Each R ^{independently} represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl;

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

A ⁷ and A ⁸ independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ³¹ =CR ³¹ )-aryl or -(CR ³¹ = CR ³¹ )-heteroaryl.

[0176] In certain embodiments, the present invention relates to compounds of the aforementioned formula XVIII, wherein R ¹ is aryl.

[0177] In certain embodiments, the present invention relates to compounds of the aforementioned formula XVIII, wherein R ¹ is aryl, and R ⁴ and R ⁵ together form a bond.

[0178] In certain embodiments, the present invention relates to compounds of the aforementioned formula XVIII, wherein ^R is aryl, ^R and R together form a bond, L is a ^bond , and ^A is heteroaryl.

[0179] In certain embodiments, the present invention relates to a compound of the aforementioned formula XVIII, wherein ^R is aryl, ^R and ^R form a bond together, L is a bond, ^A is heteroaryl, and A ² is aryl.

In certain embodiments, the present invention relates to the compound of aforementioned formula XVIII, wherein R ¹ comprises a carboxylic acid group; R ¹ is an aryl group substituted by a carboxylic acid; R ¹ is a benzene substituted by a carboxylic acid and/or R ¹ is a carboxylic acid para-substituted phenyl group.

In certain embodiments, the present invention relates to the compound of aforementioned formula XX, wherein R ¹⁷ comprises a carboxylic acid group; R ¹⁷ is a carboxylic acid substituted aryl group; R ¹⁷ is a carboxylic acid substituted benzene and/or R ¹⁷ is a carboxylic acid para-substituted phenyl group.

[0182] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXI, wherein R ²⁵ is aryl.

[0183] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXI, wherein R ²⁵ is aryl, and R ²⁷ is -CO ₂ R ³² .

[0184] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXI, wherein R ²⁵ is aryl, R ²⁷ is -CO ₂ R ³² , and A ⁷ is heteroaryl.

[0185] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXI, wherein R ²⁵ is aryl, R ²⁷ is -CO ₂ R ³² , A ⁷ is heteroaryl and A ⁸ is aryl.

Another aspect of the present invention relates to a compound represented by formula XXII:

in,

X is O, -N(R ⁵ )-, -N(R ⁵ )C(O)-, -C(O)N(R ⁵ )-, -OC(O)-, -CO ₂ -, or -N (R ⁵ )CO ₂ -;

Y is O, S or -N(R ⁵ )-;

^R is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Each R ² independently represents H or an alkyl group, or 2 R ² together form =O;

^R independently represent cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

^R independently represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

each ^R independently represents H, alkyl, aryl or aralkyl; and

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

Or a compound represented by formula XXIII:

in,

X is O, -N(R ¹⁰ )-, -N(R ¹⁰ )C(O)-, -C(O)N(R ¹⁰ )-, -OC(O)-, -CO ₂ - or -N (R ¹⁰ )CO ₂ -;

Y is O, S or -N(R ¹⁰ )-;

^R is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Each R ⁷ independently represents H or an alkyl group, or 2 R ⁷ together form =O;

R ⁸ is aryl;

^R represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

each ^R independently represents H, alkyl, aryl, or aralkyl; and

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

Or a compound represented by formula XXIV:

in,

X is O, -N(R ¹⁵ )-, -N(R ¹⁵ )C(O)-, -C(O)N(R ¹⁵ )-, -OC(O)-, -CO ₂ -, or -N (R ¹⁵ )CO ₂ -;

Y is O, S or -N(R ¹⁵ )-;

R is ^cycloalkenyl , heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl;

Each R ¹² independently represents H or an alkyl group, or 2 R ⁷ together form =O;

R ¹³ is aryl;

^R represents cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

each ^R independently represents H, alkyl, aryl, or aralkyl; and

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

[0187] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXII, wherein ^R is aryl, X is NH, and ^R is aryl.

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

[0189] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXIV, wherein R ¹³ is aryl, R ¹⁴ is aryl, and X is NH.

Another aspect of the present invention relates to a compound represented by formula XXV:

in,

X is O or S;

R ¹ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, heteroaralkyl, or -C(O)R ⁵ ;

R ² is H or alkyl;

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

R ⁴ is H, alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ;

R ⁵ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl; or R ⁵ is a group that may be replaced by one or more alkyl, halogen, -OR ⁶ , -N(R ⁶ ) ₂ , -CO ₂ R ⁶ , C(O)N(R ⁶ ) ₂ , cyano or nitro optionally substituted aryl groups; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Or a compound represented by formula XXVI:

in,

X is O or S;

R ⁷ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -C(O)R ¹¹ ;

R ⁸ is H or alkyl;

^R9 is aryl;

R ¹⁰ is H, alkyl or -C(O)N(R ¹² ) ₂ ;

R ¹¹ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R ¹¹ is a group that may be replaced by one or more alkyl, halogen, -OR ¹² , -N(R ¹² ) ₂ , -CO ₂ R ¹² , C(O)N(R ¹² ) ₂ , cyano or nitro optionally substituted aryl groups; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Or a compound represented by formula XXVII:

in,

X is O or S;

R ¹³ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -C(O)R ¹⁷ ;

R ¹⁴ is H or alkyl;

R ¹⁵ is aryl;

R ¹⁶ is H, alkyl, -CO ₂ R ¹⁸ or -C(O)N(R ¹⁸ ) ₂ ;

R is ^cycloalkenyl , heterocycloalkenyl, heteroaryl, aralkyl, or heteroaralkyl; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Or a compound represented by formula XXVIII:

in,

X is O or S;

R ¹⁹ is cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -C(O)R ²³ ;

R ²⁰ is H or alkyl;

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

R ²² is alkyl, -CO ₂ R ⁶ or -C(O)N(R ⁶ ) ₂ ;

R ²³ is cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl; or R ²³ is a group that may be replaced by one or more alkyl, halogen, -OR ¹⁸ , -N(R ¹⁸ ) ₂ , -CO ₂ R ¹⁸ , C(O)N(R ¹⁸ ) ₂ , cyano or nitro optionally substituted aryl groups; and

Each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

[0191] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXV, wherein X is S, R ¹ is -C(O)R ⁵ , R ² is H, and R ⁴ is -CO ₂ R ⁶ .

[0192] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXVI, wherein X is S, R ⁷ is -C(O)R ¹¹ , and R ⁸ is H.

In certain embodiments, the present invention relates to the aforementioned compound of formula XXVII, wherein X is S, R ¹³ is -C(O)R ¹⁷ , R ¹⁴ is H, and R ¹⁶ is -CO ₂ R ¹⁸ .

Another aspect of the present invention relates to a compound represented by formula XXIX:

in,

X is O;

Y is C(R ⁸ )-;

R ^{and R} independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or Heteroaralkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ² and R ³ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

^R is hydrogen, alkyl, cycloalkyl, aryl or aralkyl;

R ⁵ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ⁹ =CR ⁹ ) _n -aryl, or -(CR ⁹ =CR ⁹ ) _n -heteroaryl;

R ⁶ is H or alkyl; or R ⁵ and R ⁶ together form an optionally substituted monocyclic or bicyclic ring, the ring has 1 or 2 heteroatoms selected from O, N and S;

each R ⁸ and R ⁹ independently represent H or alkyl; and

n is 1 or 2;

Or a compound represented by formula XXX:

in

X is -N(R ¹⁶ )-;

Y is -C(R ¹⁷ )-;

R ¹⁰ and R ¹¹ independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroarylalkyl;

^R is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ¹¹ and R ¹² together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

R ¹³ is H, alkyl, cycloalkyl, aryl or aralkyl;

R ¹⁴ is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -(CR ¹⁸ ═CR ¹⁸ ) _n -aryl, or -(CR ¹⁸ =CR ¹⁸ ) _n -heteroaryl;

R ¹⁵ is H or alkyl; or R ¹⁴ and R ¹⁵ together form an optionally substituted monocyclic or bicyclic ring having 1 or 2 heteroatoms selected from O, N and S;

R ¹⁶ is hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl or R ¹⁰ and R ¹⁶ together form a 3-8 membered ring which may be optionally substituted by one or more alkyl, halogen, hydroxyl, alkoxy or amino groups;

each R ¹⁷ and R ¹⁸ independently represent H or alkyl; and

n is 1 or 2.

[0195] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXX, wherein R ¹⁶ , R ¹⁰ , R ¹¹ and R ¹² are aryl.

[0196] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXX, wherein R ¹⁶ , R ¹⁰ , R ¹¹ and R ¹² are aryl, and R ¹³ is H.

[0197] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXX, wherein R ¹⁶ , R ¹⁰ , R ¹¹ and R ¹² are aryl, R ¹³ is H, and R ¹⁴ is alkyl.

Another aspect of the present invention relates to a compound represented by formula XXXI:

in,

R ^{and R} independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or Heteroaralkyl;

R ^and R ^{independently} represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroarylalkyl; or R ¹ and R ² together form a 3-8 membered ring; or R ³ and R ⁴ together form a 3-8 membered ring; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

Or a compound represented by formula XXXII:

in,

R and ^R independently represent alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl ^, heteroaryl, aralkyl or heteroarylalkyl;

R ⁷ and R ⁹ independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aryl Alkyl or heteroaralkyl; or R ⁶ and R ⁷ together form a 3-8 membered ring; or R ⁸ and R ⁹ together form a 3-8 membered ring; and

Each R ¹⁰ independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aralkyl, or heteroaralkyl.

[0199] In certain embodiments, ^the present invention relates to compounds of the aforementioned formula XXXI, wherein R and ^R form a 7-membered ring together, and R ^{and R} form a 7-membered ring together.

In certain embodiments, the present invention relates to the compound of aforementioned formula XXXI, wherein R ¹ and R ² form a 7-membered ring together, R ³ and R ⁴ form a 7-membered ring together, and R ⁵ is aromatic base.

In certain embodiments, the present invention relates to the compound of aforementioned formula XXXII, wherein R ¹ and R ² form a 7-membered ring together, R ³ and R ⁴ form a 7-membered ring together, and R ⁵ is alkane base.

[0202] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXI, wherein ^R and ^R are aryl.

[0203] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXI, wherein R ¹ , R ³ and R ⁵ are aryl.

Another aspect of the present invention relates to a compound represented by formula XXXIII:

in,

X is O;

R ¹ , R ³ and A independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R ² and R ⁴ independently represent H or an alkyl group;

^R is 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:

in,

X is S;

^R represents cycloalkenyl, heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R and A independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, ^aralkyl or heteroaralkyl;

R ⁷ and R ⁹ independently represent H or an alkyl group;

R ¹⁰ is 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:

in,

X is S;

R and A independently represent cycloalkenyl, heterocycloalkenyl ^, aryl, heteroaryl, aralkyl or heteroaralkyl;

R represents ^cycloalkenyl , heterocycloalkenyl, heteroaryl, aralkyl or heteroaralkyl;

R ¹² and R ¹⁴ independently represent H or an alkyl group;

^R is 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:

in,

X is S;

R ¹⁶ and R ¹⁸ independently represent cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

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

R ¹⁷ and R ¹⁹ independently represent H or an alkyl group;

R ²⁰ is an optionally substituted monocyclic or bicyclic ring having 1, 2 or 3 heteroatoms selected from O, N and S.

In certain embodiments, the present invention relates to the compound of aforementioned formula XXXIV, wherein X is O; A is aryl, ^R is an optionally substituted monocyclic or bicyclic, and the ring has the group selected from 1, 2 or 3 heteroatoms of O, N and S, and R ⁸ is aryl.

In certain embodiments, the present invention relates to the compound of aforementioned formula XXXV, wherein X is S; A is aryl, R is an optionally substituted ^monocyclic or bicyclic, and said ring has 1, 2, or 3 heteroatoms of O, N, and S, and R ¹¹ is aryl.

In certain embodiments, the present invention relates to the compound of aforementioned formula XXXVI, wherein X is S; R ²⁰ is an optionally substituted monocyclic or bicyclic ring, said ring has a compound selected from O, N and S 1, 2 or 3 heteroatoms, R ¹⁸ is aryl, and R ¹⁶ is aryl.

Another aspect of the present invention relates to a compound represented by formula XXXVII:

in,

R ¹ , R ² and R ³ independently represent H, aryl, heteroaryl, aralkyl or heteroaralkyl;

A independently represents a single group optionally substituted by one or more halogen, alkyl, nitro, amino, aryl, heteroaryl, aralkyl or heteroaralkyl, cycloalkenyl or heterocycloalkenyl Cyclic or bicyclic aryl or heteroaryl.

[0209] In certain embodiments, the present invention relates to the aforementioned compound, wherein R ¹ , R ² and R ³ are H.

[0210] In certain embodiments, the present invention relates to the aforementioned compounds, wherein R ¹ , R ² and R ³ are H, and A is an aryl group substituted with amino.

[0211] In certain embodiments, the present invention relates to the aforementioned compounds, wherein R ¹ , R ² and R ³ are H, and A is an aryl group substituted with nitro.

[0212] In certain embodiments, the present invention relates to the aforementioned compounds, wherein R ¹ , R ² and R ³ are H, and A is an aryl group substituted with halogen.

Another aspect of the present invention relates to a compound represented by formula XXXVIII:

in,

^R independently represents alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaryl base;

R ^{and R} independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl radical or heteroarylalkyl; and

each ^R independently represents H, alkyl, cycloalkyl, heterocycloalkyl, aralkyl, or heteroaralkyl;

Or a compound represented by formula XXXIX:

in,

^R independently represents alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaryl base;

R and ^R independently represent hydrogen, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl ^, aralkyl radical or heteroarylalkyl; and

Each ^R independently represents H, alkyl, cycloalkyl, aryl, heterocycloalkyl, aralkyl, or heteroaralkyl.

[0214] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXVIII, wherein ^R is haloalkyl.

[0215] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXVIII, wherein ^R is haloalkyl, and R ^{and R} are aryl.

[0216] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXVIII, wherein ^R is haloalkyl, R ^{and R} are aryl, and at least one ^R is hydrogen.

[0217] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXIX, wherein R ^{and R} are aryl.

[0218] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXIX, wherein R ^{and R} are aryl, and at least one ^R is aryl.

[ ⁰²¹⁹ ] In certain embodiments, the present invention relates to compounds of the aforementioned formula XXXIX, wherein R and ^R are aryl, one ^R is aryl, and one ^R is hydrogen.

Another aspect of the present invention relates to a pharmaceutical composition containing a pharmaceutically acceptable excipient and a compound of any one of the general formula X-XXXIX, wherein the general formula X -XXXIX as above.

[0221] Definition

[0222] For convenience, certain 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.

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-bis phosphatidic acid.

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

[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 art recognized 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.

The term "alkyl" is art-recognized and includes saturated aliphatic groups, including straight chain alkyl, branched chain alkyl, cycloalkane (alicyclic) group, alkyl substituted cycloalkyl and ring Alkyl substituted alkyl. In certain embodiments, the linear or branched alkyl backbone has about 30 or fewer carbon atoms (e.g., C ₁ -C ₃₀ for straight chains, C ₃ -C ₃₀ for branched chains), or About 20 or less. Likewise, cycloalkyl groups have about 3-10 carbon atoms in their ring structure, or about 5, 6 or 7 carbon atoms in the ring structure.

Unless otherwise stated to the number of carbon atoms, "lower alkyl" refers to an alkyl group as defined above, but having 1 to about 10 carbon atoms in its main chain structure, or 1 to about 6 carbon atoms . Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

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, containing at least one heteroatom in the chain branched chain alkyl groups of atoms, cycloalkane (alicyclic) groups containing at least one heteroatom in the ring, alkyl-substituted cycloalkyl groups containing at least one heteroatom in the ring, and cycloalkanes containing at least one heteroatom in the chain substituted alkyl groups. The term "heterocycloalkyl" refers to a cycloalkane (alicyclic) group containing at least one heteroatom in the ring.

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

[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 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 N, O and S. The term "heterocycloalkenyl" includes unsubstituted unsaturated alicyclic groups, and unsaturated alicyclic groups containing one or more substituents selected from halogen, alkyl, alkoxy, carbonyl, or carboxyl.

[0242] The term "aryl" is art-recognized and refers to 5-, 6-, and 7-membered monocyclic aromatic groups whose ring structure is composed of carbon atoms, such as benzene, naphthalene, anthracene, pyrene, and the like. Aromatic rings may be substituted by substituents at one or more ring sites. Representative substituents include halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, phosphonate , phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonylamino, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaromatic moiety, _-CF3 , -CN, etc. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbon atoms are shared by two adjacent rings (these rings are "fused rings"), wherein at least One ring is aromatic, for example, the other ring can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and/or heterocyclic.

The term "heteroaryl" is art-recognized and refers to 5, 6 and 7 membered monocyclic aromatic groups with 1 to 4 heteroatoms in the ring, such as pyrrole, furan, thiophene, imidazole, oxazole, thiazoles , Triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and so on. Those aryl groups having heteroatoms in the ring structure may also be referred to as "arylheterocycles" or "heteroaromatics". Aromatic rings may be substituted at one or more ring positions by substituents as described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy , amino, nitro, mercapto, imino, amido, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonylamino, ketone, aldehyde , ester, heterocyclyl, aryl or heteroaryl ring moiety, _-CF3 , -CN, and the like. The term "heteroaryl" also includes polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjacent rings (these rings are "fused rings"), Where at least one ring is a heteroaryl ring, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and/or heterocyclic.

[0244] The terms ortho, meta and para are art-recognized 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 art recognized 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 terms "heterocycle" or "heterocyclyl" are art-recognized and refer to 3 to about 10 membered ring structures, or 3 to about 7 membered rings, the ring structures of which contain 1 to 4 heteroatoms. Heterocycles can also be polycyclic. Heterocyclic groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, benzopyran, xanthene, phenothioxine, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, Pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, naphthyridine, naphthyridine, quinoxaline, quinazoline , quinazoline, pteridine, carbazole, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenpyrazine, phenothiazine, furazan, phenpyrazine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, Oxazoles, piperidines, piperazines, morpholines, lactones, lactams such as azetidinone and pyrrolidone, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more ring sites by substituents as described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, Alkoxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonylamino, Ketones, aldehydes, esters, heterocyclyls, aryl or heterocyclic aromatic moieties, _-CF3 , -CN, and the like.

The term "polycyclic" or "polycyclyl" is art-recognized and refers to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and/or heterocyclic Cyclic group) in which two or more carbon atoms are common to two adjacent rings, for example, these rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each ring of the polycyclic ring may be substituted by a substituent as described above, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, mercapto, Imino, amido, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaromatic part, _-CF3 , -CN, etc.

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

The term "nitro" is known in the art and refers to -NO ₂ ; the term "halogen" is known in the art and refers to -F, -Cl, -Br or -I; the term "mercapto" is known in the art refers to -SH; the term "hydroxyl" refers to -OH. The term "sulfonyl" is art recognized and refers to _-SO2- ^. "Halide" refers to the corresponding anion of a halogen, and "halide-like" has the definition given at page 560 of " Advanced Inorganic Chemistry ", edited by Cotton and Wilkinson.

The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines, such as moieties which may 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 form a heterocycle together with the N atom to which they are connected, and the ring structure of the heterocycle It has 4 to 8 atoms; R61 represents aryl, cycloalkyl, cycloalkenyl, heterocyclic or polycyclic; m is 0 or an integer from 1 to 8. In certain embodiments, only one of R50 or R51 may be a carbonyl group, eg, R50, R51 and nitrogen together do not form an imide. In other embodiments, R50 and R51 (optionally, and R52) each independently represent hydrogen, alkyl, alkenyl or -(CH ₂ ) _m -R61. Thus, the term "alkylamine" includes an amino group as defined above bearing a substituted or unsubstituted alkyl group, ie at least one of R50 and R51 is an alkyl group.

The term "amido" is art-recognized and refers to a moiety which can be represented by the following 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 amino-substituted carbonyl groups, including a moiety which may be represented by the general formula:

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

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

The term "carboxy" is art-recognized and includes, for example, moieties that can be represented by the general formula:

Among them, 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, the moiety herein refers to carboxyl, especially when R55 is hydrogen, the general formula represents "carboxylic acid". When X50 is oxygen and R56 is hydrogen, the general formula represents "formic acid". In general, when the oxygen atom of the above formula is replaced by sulfur, the formula represents "thiocarbonyl". When X50 is sulfur and R55 or R56 is not hydrogen, the general formula represents "thioesters" and when X50 is sulfur and R55 is hydrogen, the general formula represents "thiocarboxylic acids". When X50 is sulfur and R56 is hydrogen, the general formula represents "thioformic acid". On the other hand, when X50 is a bond and R55 is other than hydrogen, the above general formula represents "keto". When X50 is a bond and R55 is hydrogen, the above general formula represents an "aldehyde group".

[0256] The term "alkoxy" is art-recognized and refers to an alkyl group as described above to which is appended an oxy group. Typical alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Correspondingly, the alkyl substituent capable of making the alkyl an ether is or is similar to an alkoxy group, for example, -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH ₂ ) _m - represented by one of R61, wherein m and R61 are as described above.

The term "sulfonate" is art recognized and refers to a moiety that can be represented by the general formula:

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

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

wherein R57 is as defined above.

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.

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.

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.

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.

The term "phosphoryl" is well known in the art and can be represented by the following general formula:

wherein Q50 represents S or O, and R59 represents hydrogen, lower alkyl or aryl. When used to replace, for example, an alkyl group, the phosphoryl group in a phosphorylalkyl group can 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 "phosphorothioate".

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.

The term "phosphonamidite" is 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 lower alkyl or aryl.

Similar substitutions can be used for alkenyl and alkynyl to generate, for example, aminoalkenyl, aminoalkynyl, amidoalkenyl, amidoalkynyl, iminoalkenyl, iminoalkynyl, thioalkenyl , thioalkynyl, carbonyl-substituted alkenyl or alkynyl.

[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 "selenyl" is art-recognized and refers to a selenyl-substituted alkyl group. Similar "selenoethers" substituted on the alkyl group are selected from -selenium-alkyl, -selenium-alkenyl, -selenium-alkynyl, selenium-(CH ₂ ) _m -R61, m and R61 are as above defined.

[0269] The terms triflyl, tosyl, mesyl, nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, perfluorobutylsulfonyl, respectively. The terms triflate, tosylate, mesylate, nonaflate are art-recognized and refer to trifluoromethanesulfonyl ester, p-toluenesulfonyl ester, methanesulfonyl ester, perfluorobutylsulfonyl ester functional groups and molecules containing said groups, respectively .

The abbreviations Me, Et, Ph, Tf, Nf, Ts and MS independently represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, perfluorobutylsulfonyl, p-toluenesulfonyl and methanesulfonyl . A more extensive list of abbreviations used by organic chemists of ordinary skill in the field appears in the first issue of each volume of the Journal of Organic Chemistry ; this list usually appears in a document titled Abbreviations Standard list table.

[0271] Certain compounds contained in the compositions 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 , their racemic mixtures, and combinations thereof, all belong to the scope of the invention. Additional asymmetric carbon atoms may be present in substituents, such as in alkyl groups. All these isomers and mixtures thereof are included in the present invention.

[0272] For example, if a specific enantiomeric form of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting enantiomeric mixture is separated and the auxiliary group is Fraction to provide the pure desired enantiomer. Alternatively, where the molecule contains a basic functional group such as an amino group, or an acidic functional group such as a carboxyl group, the salts of the enantiomers can be formed with acids or bases of appropriate optical activity, followed by fractional crystallization and Chromatographic means are used to resolve this enantiomer and the pure enantiomer is subsequently recovered.

[0273] It should be understood that "substituted" or "substituted with" includes the implied condition that the substitution complies with the permitted valences of the substituted atom and substituent, and that the substitution result is a stable A compound, eg, which does not spontaneously undergo transformations, such as rearrangements, cyclizations, eliminations, or other reactions.

[0274] The term "substituted" also includes all permissible substituents of organic compounds. In a broad aspect, the 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 the same or different for appropriate organic compounds. For the purposes of this invention, a heteroatom such as nitrogen may have hydrogen substituents and/or any permissible organic compound substituents described herein to satisfy the valence of the heteroatom. This 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 which protects a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl esters of alcohols, acetals and ketals of aldehydes and ketones. The field of protecting group chemistry has been reviewed (Greene, TW; Wuts, PGM Protective Groups in Organic Synthesis, ^2nd ed.; Wiley: New York, 1991). Protected forms of the compounds of the invention are included within the scope of the invention.

[0276] For purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS Edition, Handbook of Chemistry and Physics , 67th Edition, 1986-87, inside cover.

[0277] Pharmaceutical composition

[0278] In another aspect, the present invention provides a pharmaceutically acceptable composition comprising one or more compounds as described above in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable Carriers (additives) and/or diluents for formulation. As detailed below, the pharmaceutically acceptable compositions of the present invention may be in solid or liquid form specially formulated for administration, including forms suitable for: (1) oral administration, for example, infusion (aqueous or Aqueous solution or suspension), tablets, e.g. for buccal, sublingual and systemic absorption, boluses, powders, granules, ointment for tongue; (2) parenteral administration, e.g. subcutaneous, intramuscular , intravenous or epidural injection, for example in the form of a sterile solution or suspension or sustained release formulation; (3) topical administration, for example as a cream, ointment or controlled release patch or spray applied to the skin (4) intravaginally or rectally, for example, in the form of a suppository, ointment, or foam; (5) sublingually; (6) intraocularly; (7) transdermally; or (8) ) nasal administration.

[0279] The phrase "therapeutically effective amount" as used herein means the amount of a compound, material or composition containing a compound of the present invention which is at a reasonable benefit/risk ratio for an animal of at least A subpopulation of cells can produce the desired therapeutic effect, suitable for any medical treatment.

The phrase "pharmaceutically acceptable" used herein refers to compounds, new materials, ingredients and/or dosage forms within the scope of reasonable medical judgment, suitable for contact with human and animal tissues, without excessive toxicity, irritation sex, anaphylaxis, or other problems or complications, there is 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 (such as lubricants, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent-encapsulating materials for carrying or transporting a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the composition and not injurious to the patient. Some examples of materials that can be used 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) Tragacanth powder; (5) Malt; (6) Gelatin; (7) Talc; (8) Excipients 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) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol , mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) physiological saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer; (21) polyester, polycarbonate and/or polyanhydride and (22) other non-toxic compatible bases 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, therefore, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts", in this context, refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds of the present invention. These salts can 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 so that it can be formed in a subsequent purification process. . 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, Naphthalene Dicarboxylate, Methanesulfonate, Glucoheptonate, Lacturonate, Lauryl Sulfonate and more. (See, eg, 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 quaternary ammonium salts of these compounds, eg, derived from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, and those derived from organic acids such as acetic, propionic, succinic, glycolic , 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 - salts of o-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid and the like.

[0284] In other instances, the compounds of the present invention may contain one or more acidic functional groups capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in this context refers to the relatively non-toxic, inorganic and organic base addition salts of the compounds of the present invention. These salts can be formed in situ during the manufacture of the administration vehicle and dosage form, or from the separate reaction of a pure compound of the invention in its free acid form and a suitable base, such as a hydroxide of a pharmaceutically acceptable metal cation, Carbonates or bicarbonates, ammonia, or pharmaceutically acceptable organic primary, secondary, or tertiary amines. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, aluminum salts, and the like. Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. (see, eg, the aforementioned article by Berge et al.).

Wetting agents, emulsifiers and lubricating oils, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring and fragrance agents, preservatives and Antioxidants may also be present in the compositions of the present invention.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants , such as ascorbyl palmitate, butylated hydroxyanisole (BHA), di-tert-butyl-p-cresol (BHT), lecithin, propyl gallate, α-tocopherol, etc.; (3) metal chelating agents, such as Citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0287] Formulations of the present 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 which can be combined with a carrier material to produce a single dosage form will depend upon the host being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.1% to about 99% active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

[0288] In certain embodiments, the formulations of the invention contain 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 agent; and a compound of the invention. In certain embodiments, the formulations described above render the compounds of the invention orally bioavailable.

[0289] Methods of preparing these formulations or compositions comprise bringing into association a compound of the invention and the carrier and, optionally, one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing the compound of the present invention into association 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 elixir or syrup, or as a lozenge (using an inert base materials such as gelatin and glycerin, or sucrose and acacia) and/or as a mouthwash, each of which contains a predetermined amount of the compound of the present invention as an active ingredient. The compounds of this invention may also be administered as a bolus, electuary or poultice.

In solid dosage forms (capsules, tablets, pills, lozenges, powders, granules, trouches, etc.) for oral administration of the present invention, the active ingredient and one or more pharmaceutically acceptable carriers Mixtures such as sodium citrate or dibasic calcium phosphate and/or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, dextrose, mannitol, and/or silicic acid; (2) binders , such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and/or gum arabic; (3) diluents, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca Starch, alginic acid, certain silicates, sodium carbonate; (5) curing retardants, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamer and ten Sodium dialkyl sulfate; (7) wetting agents such as cetyl alcohol, glyceryl monostearate, nonionic surfactants; (8) absorbents such as kaolin and bentonite; (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) coloring agents and (11) a controlled release agent, such as cross-linked polyvinylpyrrolidone or ethyl cellulose. As with capsules, tablets and pills, the pharmaceutical composition may also include buffering agents. Solid compositions of a similar type can also be filled in soft and hard shell capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0292] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Tablets may be compressed with binders (such as gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (such as sodium starch glycolate or croscarmellose sodium), Surfactant or dispersant preparation. Molded tablets may be made 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 this invention, such as lozenges, capsules, pills, and granules, can be compressed or prepared, optionally with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical art. They can also be formulated to provide slow or controlled release of the active ingredient therein, by using, for example, hydroxypropylmethylcellulose in varying ratios to provide the desired release profile, using other polymer matrices, liposomes and/or microparticles. ball. They can be formulated for immediate release, eg freeze-dried. They can be sterilized, for example, by passing through a bacteria-retaining filter, or by adding sterilizing agents in the form of sterile solid compositions dissolved in sterile water or other sterile injectable medium just before use. These compositions may also optionally contain opacifying agents and may release the active ingredient(s) only, or preferably, in a certain part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0294] Liquid dosage forms for oral administration of the compounds of this invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , propylene glycol, 1,3-butene glycol, oils (especially cottonseed, peanut, corn germ, olive, castor, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acids of sorbitan Esters, and mixtures thereof.

[0295] Besides inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, colors, preservatives, fragrances and preservatives.

Suspensions may contain, in addition to the active ingredient, suspending agents, such as ethoxylated isostearyl alcohol, sorbitol and polyoxyethylene, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide , bentonite, agar and tragacanth, and mixtures thereof.

[0297] The pharmaceutical composition formulation for rectal or vaginal use of the present invention may be a suppository prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating adjuvants or carriers. For preparation, such excipients or carriers are, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylic acid, and are solid at room temperature but liquid at body temperature and thus can melt in the rectum or vaginal cavity to release the active compounds.

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

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

Ointments, creams, creams and gels may contain, in addition to the active compounds of the invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polypropylene glycol, organic Silicon, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0301] Powders and sprays can contain, in addition to the active compounds of this invention, excipients 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 added advantage of providing controlled release of the compounds of the invention to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in the proper medium. Absorption enhancers can 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, ophthalmic ointments, powders, solutions, and the like are also contemplated by the present invention.

The pharmaceutical composition of the present invention suitable for parenteral administration includes one or more compounds of the present invention and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions Or emulsion, or sterile powder that can be reconstituted into a sterile injectable solution or dispersion before use, it may contain sugars, alcohols, antioxidants, buffers, thiobisdichlorophene, Isotonic solutes for the blood of the body, or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) 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 maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0306] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms on 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, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the 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, slowing the absorption of subcutaneous or intramuscular drugs may be desirable in order to prolong the effect of the drug. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The rate of absorption of a drug depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or dispersing the drug in an oily vehicle.

[0308] Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include polyorthoesters and polyanhydrides. Injectable depot forms are achieved by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.

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

[0310] The formulations of the 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 eyewashes, ointments, suppositories; topically in the form of lotions or ointments; rectally in the form of suppositories. Oral administration is preferred.

[0311] "parenteral administration" as used herein refers to a mode of administration different from other enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal , Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injections and infusions.

[0312] As used herein, "systemic administration" and "peripheral administration" refer to administration of a compound, drug or other substance that does not directly enter the central nervous system so that it enters the patient's body and is metabolized and otherwise A similar procedure, such as subcutaneous administration.

The compounds used in therapy may be administered to humans and other animals by any suitable route of administration, including orally, nasally, e.g. by spray, rectally, vaginally, by injection, intracranally and topically, e.g. by powder , ointment or drops, including buccal and sublingual.

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

[0315] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied in order to obtain an amount of the active ingredient that achieves the desired therapeutic response, composition, and mode of administration for a particular patient, and is not harmful to the patient. toxicity.

[0316] The selected dosage level will depend on a variety of factors including the activity of the particular compound employed in the invention, or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the absorption The rate and extent of treatment, the duration of treatment, other drugs, compounds and/or materials used in combination with the specific compound employed, the age, sex, weight, condition, general health and previous medication history of the patient being treated, and the field of medicine known similar factors.

[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 may initially prescribe pharmaceutical compositions containing a compound of the invention at lower dosages than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

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

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

[0320] Although the compounds of the present invention may be administered alone, it is preferred that such compounds be administered in the form of pharmaceutical formulations (compositions).

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

[0322] In another aspect, the present invention provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more of the subject compounds as described above, formulated together with one or more of the pharmaceutically Acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical composition of the present invention can be specially prepared for administration in solid or liquid form, including the following administration forms: (1) oral administration, such as drink (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, ointment for tongue application; (2) parenteral administration, e.g., subcutaneous, intramuscular, intravenous or epidural injection, e.g. as a sterile solution or (3) topical administration, such as a cream, ointment or spray for application to the skin, lungs or mucous membranes; or (4) vaginal or rectal administration, such as in suppository, ointment or foam form; ( 5) Sublingual or buccal administration; (6) Intraocular administration; (7) Transdermal administration; or (8) Nasal administration.

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

[0324] Patients receiving this treatment are any animal in need, including primates, especially humans and other mammals such as horses, cows, pigs and sheep; and poultry and pets in general.

[0325] The compound of the present invention can be administered by itself or mixed with a pharmaceutically acceptable carrier, and can also be administered in combination with antibacterial drugs, such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Accordingly, combination therapy includes such sequential, simultaneous and separate administrations of subsequent administrations of the active compounds that have not completely worn off the effects of the first administration.

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

[0327] Alternatively, a semi-finished concentrate or feed additive containing the active ingredient may be mixed into the feed. The preparation and administration of such premixes and complete feeds are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" O and Bbooks, Corvallis, Ore., U.S.A., 1977).

[0328] Micelles

[0329] Recently, the pharmaceutical industry has introduced microemulsion technology to increase the bioavailability of some lipophilic (water insoluble) agents. Examples include Trimetrine (Dordunoo, S.K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991 and REV 5901 (Sheen, P.C., et al., J Pharm Sci 80(7), 712-714 , 1991). Among other things, microemulsions provide enhanced bioavailability through preferential direct absorption into the lymphatic system rather than the circulatory system, thereby bypassing the liver and preventing the compound from being destroyed in the hepatobiliary circulation.

[0330] In one aspect of the invention, formulations comprise micelles of 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 provided micelles have an average diameter of less than about 50 nm, most preferably provided micelles have an average diameter of less than about 30 nm, or even less than about 20 nm.

[0331] While all suitable amphiphilic carriers are considered, presently preferred carriers are generally those generally recognized as non-toxic (GRAS), which are capable of solubilizing the compounds of the invention and which are later combined with a complexed aqueous phase (if present) emulsifies the compound on contact with the human gastrointestinal tract). Typically, amphiphilic components meeting these requirements have an HLB (hydrophilic to lipophilic balance) value of 2-20 and a structure comprising linear aliphatic radicals ranging from C-6 to C-20. Examples are polyethylene-glycolized fatty acid glycerides and polypropylene glycol.

[0332] Particularly preferred amphiphilic carriers are saturated and monounsaturated polyethyleneglycolyzed (polyethyleneglycolyzed) fatty acid glycerides, such as those obtained from fully or partially hydrogenated various vegetable oils. This oil may advantageously consist of 3-, 2- and mono-glycerides of fatty acids and 2- and monopolyethylene glycol esters of the corresponding fatty acids, a particularly preferred fatty acid composition comprising 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 class of useful amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, and saturated or monounsaturated fatty acids (Span series) or the corresponding ethoxylated analogs (Tween series).

Commercially available amphiphilic carriers are particularly contemplated and include the glyceryl monostearate series, oleoylpolyisoglycolglycerides, caprylic caprate macrogolglycerides, or lauroglycol (both manufactured by Gattefosse Corporation of France). , manufactured and sold by Saint Priest), polyethylene glycol monooleic acid, polyethylene glycol-dioleic acid, polyethylene glycol-monolauric acid and dilauric acid, lecithin, polysorbate 80, etc. (made of polysorbate manufactured and sold by companies in the United States and around the world).

Polymer

[0335] Hydrophilic polymers suitable for use in the present invention are those that are readily soluble in water, can be covalently attached to a vesicle-forming lipid, and are tolerated in vivo without toxic effects (i.e., biophase Compatible) polymer. Suitable polymers include polyethylene glycol (PEG), polylactic acid (also known as polylactide), polyglycolic acid (also known as polyglycolide), poly(lactic-co-polyglycolic acid), polyvinyl alcohol. Preferred polymers have 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 750 Dalton polyethylene glycol (PEG(750)). Polymers can also be defined by the number of monomers therein; a preferred embodiment of the invention uses polymers of at least about 3 monomers, such as PEG polymers (about 150 Daltons) containing three monomers.

Other suitable hydrophilic polymers for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide , polydimethylacrylamide, cellulose derivatives such as hydroxymethylcellulose or hydroxyethylcellulose.

[0337] In certain embodiments, the formulations of the present invention include a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyolefins, acrylic and methacrylate polymers , polyethylene polymers, polyglycolide, polysiloxane, polyurethane and their copolymers, cellulose, polypropylene, polyethylene, polystyrene, lactic acid and glycolic acid polymers, polyanhydrides, poly(o- ) esters, poly(butyric acid), poly(valeric acid), poly(lactide-caprolactone), polysaccharides, proteins, hyaluronic acid, polycyanoacrylates, and blends, mixtures or copolymers thereof things.

[0338] Cyclodextrin

[0339] Cyclodextrins are cyclic oligosaccharides containing 6, 7 or 8 glucose units, named by the Greek letters alpha, beta or gamma, respectively. Cyclodextrins with less than 6 glucose units are not known to exist. Glucose units are linked by alpha-1,4-glucosidic linkages. As a result of the chair conformation of a sugar unit, all secondary hydroxyl groups (at C-2 and C-3) are on one side of the ring, while all primary hydroxyl groups at C-6 are on the other. Therefore, the external surface is hydrophilic, making cyclodextrins water-soluble. In contrast, the inner cavities of cyclodextrins are hydrophobic because they are lined with C-3 and C-5 hydrogen atoms and ether-like oxygens. These matrices allow it to complex with a variety of relatively hydrophobic compounds, including, for example, steroid compounds such as 17-β-estradiol (see, for example, Van Uden et al. Plant Cell Tiss. 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 the chemical properties of cyclodextrins see Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822 (1994).

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

[0341] A number of cyclodextrins and methods for their preparation have been described. For example, the electrically neutral cyclodextrins described by Parmeter (I) et al. (US Patent No. 3,453,259, incorporated herein by reference) and Gramera et al. (US Patent No. 3,459,731, incorporated herein by reference). Other derivatives include cyclodextrins with cationic properties [Parmeter (II), U.S. Patent No. 3,453,257, incorporated herein by reference], insoluble cross-linked cyclodextrins (Solms, U.S. Patent No. 3,420,788, incorporated herein by reference), and Cyclodextrins with anionic properties [Parmeter (III), US Patent No. 3,426,011, incorporated herein by reference]. Among the cyclodextrins with anionic properties, carboxylic acid, hypophosphorous acid, phosphorous acid, phosphonic acid, phosphoric acid, thiophosphonic acid, thiosulphinic acid, and sulfonic acid are attached to the cyclodextrin parent [ See Parmeter (III) above]. In addition, cyclodextrin sulfoalkylated derivatives have been described by Stella et al. (US Patent No. 5,134,127, incorporated herein by reference).

[0342] Liposomes

[0343] Liposomes consist of at least one lipid bilayer membrane that encloses an aqueous interior compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically range in diameter between 0.02 and 0.05 μm; large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large oligolamellar and multilamellar vesicles have multiple, usually concentric, membranes and are usually larger than 0.1 μm. Liposomes with a non-concentric membrane, that is, several smaller vesicles encapsulated within one larger vesicle, are called multivesicular.

[0344] One aspect of the present invention pertains to formulations comprising liposomes containing a compound of the present invention, wherein the liposome membrane is prepared to provide liposomes with increased loading capacity. Alternatively or alternatively, the compounds of the invention may be contained within the liposome bilayer of liposomes, or adsorbed on the liposome bilayer of liposomes. Compounds of the invention can be aggregated with lipid surfactants and carried within the liposome interior space; in these cases, the liposome membrane is prepared to resist the disruptive effects of the active agent-surfactant aggregates.

According to a particular embodiment of the invention, the lipid bilayer of a liposome comprises 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 extend from the exterior of the lipid bilayer into the surrounding environment.

[0346] The active agent contained in the liposomes of the invention is in dissolved form. Aggregates of surfactants and active agents (such as emulsions or micelles containing related active agents) can be embedded in the inner space of liposomes according to the invention. The surfactant acts to disperse and solubilize the active agent and may be selected from any suitable aliphatic, cycloaliphatic or aromatic surfactants including, but not limited to, biocompatible lysophospholipids of varying chain lengths Choline (LPCS) (eg from about C.sub.14 to C.sub.20). Polymer-derived lipids such as PEG-lipids can also be used for micelle formation as they act to inhibit micelle/membrane fusion and adding polymers to surfactant molecules reduces the CMC of the surfactant and helps in the formation of micelles. 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 may affect lipid The stability of the body bilayer and is a factor in designing liposomes for an ideal stability.

[0347] Liposomes according to the invention can be prepared by any of a variety of techniques known in the art. See U.S. Patent No. 4,235,871; Published PCT Application WO 96/14057, both incorporated herein by reference; New RRC, Liposomes: A practical approach, 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-derived lipid into preformed liposomes, such as by exposing the preformed liposomes to lipid grafting In micelles composed of polymers, the lipid concentration corresponds to the final molar percentage of derivatized lipids required in the liposomes. 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 in a lysophosphatidylcholine or other low CMC surfactant (including polymer-grafted lipids) that readily solubilizes hydrophobic molecules by ultrasound. middle. The resulting micellar suspension of active agent is then used to rehydrate a dry lipid sample containing an appropriate 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 to have an approximately uniform size within a selected size range. An efficient granulation method involves extruding an aqueous suspension of liposomes through a series of polycarbonate membranes of selected uniform pore size; the pore size of the membrane will roughly correspond to that of the liposomes produced by extrusion. biggest size. See US Patent 4,737,323, which is incorporated herein by reference.

[0351] Release modulator

[0352] The release profile of the formulations of the invention is dependent on the encapsulating material, the concentration of the encapsulated drug, and the presence of a release modifier. For example, release can be manipulated to be pH dependent, eg, using a pH sensitive coating such that release is only at low pH values such as in the stomach, or at higher pH values such as in the intestinal tract. An enteric coating can be used to prevent release until after passage through the stomach. Various coatings or mixtures of cyanamides encapsulated in different materials can be used to obtain initial release in the stomach followed by subsequent release in the intestinal tract. Release can be manipulated by introducing salts or pore formers that enhance water uptake or drug release by diffusion from the capsule. Excipients capable of modulating drug solubility can also be used to control the rate of release. Agents that increase matrix degradation or release from the matrix may also be added. They can be added to the drug, added as a separate phase (ie as particles), or co-dissolved in the polymer phase depending on the compound involved. In all cases the amount should be between 0.1 and 30% (w/w polymer). Types of degradation accelerators 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 sulfuric acid Protamine, spermine, choline, ethanolamine, diethanolamine, triethanolamine and surfactants such as Tween.RTM and Pluronic.RTM. Pore formers (ie, water-soluble compounds such as inorganic salts and sugars) capable of increasing the microporous structure of the matrix are added in particulate form. The range should be between 1 and 30% (w/w polymer).

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

Combinatorial Libraries

[0355] The subject compounds can be synthesized using the combinatorial syntheses described in this section. Combinatorial libraries of compounds can be used to screen drugs, pesticides, 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 that can be screened together for a desired property; such a compound library can be liquid or covalently attached to a solid support. Preparation of many related compounds in a single reaction can greatly reduce and simplify the number of screening processes that need to be performed. Screening for suitable biological, pharmaceutical, pesticide or physical properties can be performed by conventional methods.

[0356] Diversity in compound libraries can be created at various levels. For example, the base aromatic group used in the combinatorial approach may have a diversity of aromatic nuclei, such as variations in ring structure, and/or may vary with respect to other substituents.

[0357] Various techniques are available in the art for generating combinatorial compound libraries of small organic molecules. See, eg, Trends Anal. Chem. 14:83 by Blondelle et al. (1995); U.S. Patents 5,359,115 and 5,362,899 by Affymax; U.S. Patent 5,288,514 by Ellman; PCT Publication WO 94/08051 by Still et al., all of which are incorporated by reference This specification; Chen et al. (1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT Publications WO92/10092, WO93/09668 and WO91/07087; PCT Publication WO93/20242 by Lerner et al, where are incorporated into this specification by reference). Thus, libraries of various compounds on the order of about 16 to 1,000,000 or more diverse compounds can be synthesized and screened for specific activities or properties.

[0358] In an exemplary embodiment, a library of substituted diversomers compounds can be synthesized using the techniques described in PCT Publication WO 94/08051 by Still et al., for example by a hydrolysis or photo The cleavage group is attached to the polymer bead, for example positioned at one position on the substrate. According to the technique of Still et al., compound libraries are synthesized on a set of beads, each bead including a set of tags that identify a particular diversity on the bead. In an embodiment particularly suitable for the discovery of enzyme inhibitors, beads can be dispersed on the surface of a permeable membrane, and the multimers released from the beads by cleavage of the attachment to the beads. Diversity released from each bead will diffuse across the membrane to the assay zone where it will interact with an enzyme sample. A detailed description of some combination methods is provided below.

A. Direct Characterization

A developing trend in the field of combinatorial chemistry is the sensitivity of, for example, techniques such as mass spectrometry (MS), which can be used to characterize compounds in sub-femtomolar The chemical structure of the compound. For example, if a compound library is provided on an insoluble support matrix, individual compounds can first be released from the support and characterized by MS. In other embodiments, as part of the MS sample preparation technique, MS techniques such as MALDI can be used to release the compound from the support, especially when a labile bond was originally used to link the compound to the support. For example, a bead selected from a compound library can be irradiated in a MALDI step to release diversity from the support and ionize the diversity for MS analysis.

B. Multicenter Synthesis

[0362] The compound libraries of the subject methods may be in a multi-center compound library format. Briefly, Geysen and coworkers (Geysen et al. (1984) PNAS 81:3998-4002) introduced a method for generating compound libraries by means of a polyacrylic Parallel synthesis on polyethylene pins of sticks. The Geysen technology uses a multi-center approach to synthesize and screen thousands of compounds per week, and linked compounds can be reused in many assays. Some suitable linker moieties can also be suspended to the pins, so that the compound can be cleaved from the carrier after synthesis for purification identification and further evaluation (cf, Bray et al. (1990) Tetrahedron Lett 31 : 5811-5814; Valerio et al. (1991) Anal Biochem 197: 168-177; Bray et al. (1991) Tetrahedron Lett 32: 6163-6166).

C. Separation-connection-recombination

In yet another embodiment, a split-join-recombine strategy can be used to provide a diverse library of compounds on a set of beads (see, for example, Houghten (1985) PNAS 82:5131-5135; U.S. Patent 4,631,211, 5,440,016, 5,480,971, which are incorporated herein by reference). Briefly, as the name implies, at each synthetic step where degeneracy is introduced into the compound library, the beads are divided into independent groups equal to the number of different substituents to be added at a particular position in the compound library The number of different substituents was paired with each reaction, and the beads recombined into one library for the next replicate.

[0365] In one embodiment, the split-attach-recombination strategy can be performed using a similar method first developed by Houghten called the "tea bag", wherein the synthesis of the compound occurs inside a resin-sealed porous polypropylene bag. Internal (Houghten et al. (1986) PNAS 82:5131-5135). By placing these bags in a suitable reaction solution, the substituents are attached to the compound-loaded resin, while all common steps, such as washing and deprotection of the resin, occur simultaneously in one reactor. At the end of the synthesis, each bag contains a single compound.

D. Combinatorial compound libraries by photodirected, spatially localizable parallel chemical synthesis

[0367] A scheme of combinatorial synthesis in which a compound is identified by its location on a synthetic substrate is called spatially localizable synthesis. In one embodiment, the combinatorial process is performed 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, SPA (1991) Science 251:767; Pirrung et al. (1992) US Patent No. 5,143,854, incorporated herein by reference; Jacobs et al. (1994) Trends Biotechnol 12:19-26). The spatial resolution of photolithography offers miniaturization. This technique can be performed by using protection/deprotection reactions with photosensitive protection groups.

[0368] The gist of this technique is described in Gallop et al. (1994) J Med Chem 37: 1233-1251. A synthetic substrate is prepared for attachment by covalent attachment of a photosensitive nitrobenzyloxycarbonyl (NVCO) protected amino linker or other photosensitive linker. Light is used to selectively activate a specific area of the synthetic substrate for attachment. Removal of photosensitive protecting groups by light (deprotection) results in activation of selected regions. After activation, first a set of amino acid analogs is exposed across the surface, each bearing a photosensitive protecting group on the terminal amino group. Connections occur only in areas located by light in previous steps. After the reaction has ceased, the plate is washed and the substrate is irradiated through a second set of masks, activating different regions for reaction with a second protected building block. The pattern of masks and the sequence of reactants define the products and their locations. Since light lithography is utilized in this process, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be mapped with suitable resolution. The site of each compound is known precisely; therefore, its interactions with other molecules can be directly evaluated.

[0369] In a photodirected chemical synthesis, the product depends on the pattern of illumination and the order of addition of the reactants. By varying the illumination pattern, many different sets of test compounds can be synthesized simultaneously; this feature leads to the generation of many different masking strategies.

E. Encoded Combinatorial Libraries

[0371] In another embodiment, the subject methods employ a library of compounds possessing an encoded marker system. Recent advances in the identification of active compounds from combinatorial libraries employ a variety of chemical indexing systems that utilize tags that uniquely encode the reaction steps a given bead underwent (and, inferred, the structure it carries) . Conceptually, this approach mimics phage display libraries, where activity is derived from expressed polypeptides, but the structure of the active polypeptide is deduced from the corresponding genomic DNA sequence. The first codes for synthetic combinatorial libraries used DNA as the code. Various other forms of encoding have been reported, including encoding with sequenceable biooligomeric species (eg, oligonucleotides and polypeptides), and binary encoding with additional non-sequenable tags.

1. Labeling with sequenceable biooligomeric polymers

[0373] The principle of a synthetic compound library utilizing oligonucleotide coding combinations was described in 1992 (Brenner et al. (1992) PNAS 89:5381-5383), and an example of such a compound library appeared the following year (Needles et al. (1993) PNAS 90: 10700-10704). A nominally ⁷⁷ (=823543) combinatorial library of polypeptides includes all combinations of Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acid codes), each of which is represented by a specific Dinucleotide codes (TA, TC, CT, AT, TT, CA, and AC, respectively), the library is 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 polypeptide or oligonucleotide synthesis by simultaneously incubating the beads with reagents that generate protection for oligonucleotide synthesis. OH group and protected _NH2 (here, in a ratio of 1:20) for polypeptide synthesis. When completed, each label contained 69-mers, of which 14 units were coded. The bead-linked compound library was incubated with fluorescently labeled antibodies, and the beads containing linked strongly fluorescent antibodies were sorted by fluorescence-activated cytometry (FACS). DNA tags are amplified by PCR, sequenced, and synthesized peptides are predicted. Through these techniques, compound libraries for use in the subject methods can be derived, wherein the oligonucleotide sequence of the tag identifies the sequential combinatorial reactions that a particular bead undergoes, thereby providing identification of the compound on the bead.

[0374] The use of oligonucleotide tags allows very sensitive marker analysis. Even so, the approach requires careful selection of an orthogonal set of protecting groups required for the alternate synthesis of tags and compound library members. In addition, the chemical instability of tags, especially anomeric attachment of phosphate groups and sugars, may limit the choice of reagents and conditions that can be used for the synthesis of non-oligomeric libraries. In preferred embodiments, the compound library employs linkers that allow selective stripping of test compound library members for use in the assay.

[0375] Polypeptides have also been used as marker molecules for combinatorial libraries. Two exemplary methods have been described in the art, both employing branched linkers attached to a solid phase, where the coding and ligand strands are fabricated alternately. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115:2529-2531), synthetic orthogonality is achieved by using acid-labile protection for the coding strand and Alkaline instability protection.

[0376] In another alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170), a branched linker is used so that both the coding unit and the test compound are attached to the same functional group of the resin superior. In one embodiment, a cleavable linker can be placed between the branch point and the bead so that the cleavage releases a molecule containing the code and compound (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894) . In another embodiment, a cleavable linker allows the test compound to be selectively separated from the bead, leaving behind a code. The latter configuration is particularly valuable because it allows screening of test compounds without interference from coding groups. Sequence analyzes of independent fragments and peptide library members and their corresponding tags have been demonstrated in the art to accurately predict the structure of polypeptides.

2. Non-sequenceable markers: binary encoding

[0378] An alternative format for encoding a library of test compounds employs a set of non-sequenceable electrophoretic marker molecules used as a binary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926) . An exemplary label is a haloarylalkyl ether, which is detectable at sub-femtomolar levels by electron capture gas chromatography (ECGC) due to its trimethylsilyl ether. Variations in the length of the alkyl chain and the nature and position of the aromatic halide allow the synthesis of at least 40 such tags, which could in principle encode 2 ⁴⁰ (eg, up to 10 ¹² ) different molecules. In the original report (Ohlmeyer et al., supra) tags were attached to about 1% of the available amine groups in a polypeptide repertoire via a photocleavable o-nitrophenyl linker. This approach is convenient when preparing compound libraries of peptide-like or other combinatorial amine-containing molecules. However, a more general system has been developed that allows encoding of virtually any combinatorial library of compounds. Here, the compound will be attached to the solid support via a photocleavable linker, and the label will be inserted into the bead matrix via a catechol ether linker through the polyyne (Nestler et al. (1994) J Org Chem 59:4723-4724). This orthogonal binding strategy allows selective detachment of compound library members for detection in solution and decoding by ECGC following oxidative detachment of the marker set.

[0379] While there are several amide-linked compound libraries in the art using binary codes with electrophoretic labels attached to amine groups, direct attachment of these labels to bead matrices provides a more robust structure that can be prepared in coded combinatorial libraries. Great versatility. Attached in this manner, the tags and their linkers are as nearly inert as the bead matrix. Binary-encoded combinatorial compound libraries have been reported in which electrophoretic labels are directly attached to a solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031 ) and provide guidance for generating subject compound libraries. The two compound libraries were constructed using an orthogonal ligation strategy, in which the compound library members were connected to the solid-phase support through a photosensitive linker, and the tags were connected by strong oxidative cleavage of the cross-linker. Because the members of the compound library can be reproducibly and locally photoeluted from the solid support, the members of the compound library can be used for multiplex detection. Continuous light elution also allows for a very high-throughput iterative screening strategy: first, multiplex beads are placed in a 96-well microplate; second, compound fractions are isolated and transferred to assay plates; third, a metal Binding assays identify active wells; fourth, corresponding beads are individually rearranged into new microwell plates; fifth, individual active compounds are identified; and sixth, structures are decoded.

[0380] References

1.Pentchev, P., Vanier, MT., Suzuki, K., and Patterson, MC.1995.Niemann-Pick Disease type C: Cellular cholesterol lipidosis.New York: McGraw-Hill.2625-2639 pp.

2.Li, H., Repa, J.J., Valasek, M.A., Beltroy, E.P., Turley, S.D., German, D.C., and Dietschy, J.M.2005.Molecular, anatomical, and biochemical events associated with neurodegeneration in mice with Niemann-Pick type C disease. J Neuropathol Exp Neurol 64: 323-333.

3.Patterson, M.C., and Platt, F.2004.Therapy ofNiemann-Pick disease, type C.Biochim Biophys Acta 1685:77-82.

4.Hsich, G., Sena-Esteves, M., and Breakefield, X.O.2002.Critical issues in gene therapy for neurologic disease. Hum Gene Ther13:579-604.

5.Liscum, L., Arnio, E., Anthony, M., Howley, A., Sturley, S.L., and Agler, M.2002.Identification of a pharmaceutical compound that partially corrects the Niemann-Pick C phenotype incultured cells. J Lipid Res 43: 1708-1717.

6.Bascunan-Castillo, E.C., Erickson, R.P., Howison, C.M., Hunter, R.J., Heidenreich, R.H., Hicks, C., Trouard, T.P., and Gillies, R.J.2004.Tamoxifen and vitamin E treatments delay symptoms in the mouse model of Niemann-Pick C.J Appl Genet 45:461-467.

7.Zervas, M., Somers, K.L., Thrall, M.A., and Walkley, S.U.2001.Critical role for glycosphingolipids in Niemann-Pick diseasetype C.Curr Biol 11:1283-1287.

8.Griffin, L.D., Gong, W., Verot, L., and Mellon, S.H.2004.Niemann-Pick type C disease involves disrupted neurosteroidogenesis and responds to allopregnanol ketone.Nat Med 10:704-711.

9.Vanier, M.T., and Suzuki, K.1998.Recent advances inelucidating Niemann-Pick C disease.Brain Pathol 8:163-174.

10.Goldstein, J.L., and Brown, M.S.1992.Lipoprotein receptors and the control of plasma LDL cholesterol levels.Eur HeartJ 13 Suppl B: 34-36.

11.Garver, W.S., and Heidenreich, R.A.2002.TheNiemann-Pick C proteins and trafficking of cholesterol through thelate endosomal/lysosomal system.Curr Mol Med 2:485-505.

12.Carstea, E.D., Morris, J.A., Coleman, K.G., Loftus, S.K., Zhang, D., Cummings, C., Gu, J., Rosenfeld, M.A., Pavan, W.J., Krizman, D.B., et al .1997.Niemann-Pick C1 disease gene:homology to mediators of cholesterol homeostasis.Science 277:228-231.

13.Liscum, L.2000.Niemann-Pick type C mutations causelipid traffic jam.Traffic 1:218-225.

14.Puri, V., Watanabe, R., Dominguez, M., Sun, X., Wheatley, C.L., Marks, D.L., and Pagano, R.E.1999.Cholesterol modulates membrane traffic along the endocytic pathway insphingolipid-storage diseases . Nat Cell Biol 1:386-388.

15.Ioannou, Y.A.2000.The structure and function of theNiemann-Pick C1 protein.Mol Genet Metab 71:175-181.

16.Scott, C., and Ioannou, Y.A.2004.The NPC1 protein:structure implies function.Biochim Biophys Acta 1685:8-13.

17.Vanier, M.T., and Millat, G.2004.Structure and function ofthe NPC2 protein.Biochim Biophys Acta 1685:14-21.

18.Friedland, N., Liou, H.L., Lobel, P., and Stock, A.M.2003.Structure of a cholesterol-binding protein deficiency in Niemann-Picktype C2 disease.Proc Natl Acad Sci U S A 100:2512 -2517.

19.Okamura, N., Kiuchi, S., Tamba, M., Kashima, T., Hiramoto, S., Baba, T., Dacheux, F., Dacheux, J.L., Sugita, Y., and Jin , Y.Z.1999. A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol. Biochim Biophys Acta 1438: 377-387.

20.Zhang, M., Sun, M., Dwyer, N.K., Comly, M.E., Patel, S.C., Sundaram, R., Hanover, J.A., and Blanchette-Mackie, E.J.2003.Differential trafficking of the Niemann- Pick C1 and 2 protein highlights distinct roles in late endocytic lipid trafficking. Acta Paediatr Suppl 92:63-73; discussion 45.

21.Sun, X., Marks, D.L., Park, W.D., Wheatley, C.L., Puri, V., O'Brien, J.F., Kraft, D.L., Lundquist, P.A., Patterson, M.C., Pagano, R.E., et al.2001.Niemann-Pick C variant detection by alteredsphingolipid trafficing and correlation with mutations within a specific domain of NPC1.Am J Hum Genet 68:1361-1372.

22.Kobayashi, T., Beuchat, M.H., Lindsay, M., Frias, S., Palmiter, R.D., Sakufaba, H., Parton, R.G., and Gruenberg, J.1999.Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat Cell Biol 1:113-118.

23.Mukherjee, S., and Maxfield, F.R.2004.Lipid and cholesterol trafficking in NPC.Biochim Biophys Acta 1685:28-37.

24.Bornig, H., and Geyer, G.1974.Staining of cholesterol with the fluorescent antibiotic " filipin ". Acta Histochem 50: 110-115.

25.Maxfield, F.R., and Wustner, D.2002.Intracellularcholesterol transport.J Clin Invest 110:891-898.

26.Lange, Y., Ye, J., and Steck, T.L.1998.Circulation ofcholesterol between lysosomes and the plasma membrane.J BiolChem 273:18915-18922.

27.Chen, W., Sun, Y., Welch, C., Gorelik, A., Leventhal, A.R., Tabas, I., and Tall, A.R.2001.Preferential ATP-binding cassettetransporter A1-mediated cholesterol efflux from lateendosomes/lysosomes. J Biol Chem 276: 43564-43569.

28.Pentchev, P.G., Comly, M.E., Kruth, H.S., Vanier, M.T., Wenger, D.A., Patel, S., and Brady, R.O.1985.A defect in cholesterol olesterification in Niemann-Pick disease (type C)patients .Proc NatlAcad Sci U S A 82:8247-8251.

29.Lin, S., Lu, X., Chang, C.C., and Chang, T.Y.2003.Humanacyl-coenzyme A: cholesterol acyltransferase expressed in chinesehamster ovary cells: membrane topology and active site location. MolBiol Cell 14: 2447 -2460.

30.Park, W.D., O'Brien, J.F., Lundquist, P.A., Kraft, D.L., Vockley, C.W., Karnes, P.S., Patterson, M.C., and Snow, K.2003.Identification of 58 novel mutations in Niemann- Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1. Hum Mutat 22: 313-325.

31.Yang, C.C., Su, Y.N., Chiou, P.C., Fietz, M.J., Yu, C.L., Hwu, W.L., and Lee, M.J.2005.Six novel NPC1 mutations in Chinese patients with Niemann-Pick disease type C.J Neurol yurosurgPs 76: 592-595.

32.Vanier, M.T., and Millat, G.2003.Niemann-Pick diseasetype C.Clin Genet 64:269-281.

33.Di Leo, E., Panico, F., Tarugi, P., Battisti, C., Federico, A., and Calandra, S.2004.A point mutation in the lariat branch pointof intron 6 of NPC1 as the cause of abnormal pre-mRNA splicing in Niemann-Pick type C disease. Hum Mutat 24:440.

34.Patterson, M.C., Vanier, M.T., Suzuki, K, Morris, J.A., Cartsea, E., Neufeld, E.B., Blanehette-Mackie, E.J., Pentchev, P.G.2001.Niemann-Pick type C: a lipid trafficking disorder. In Themetabolic and molecular bases of inherited disease. B.A. Scriver SR, Valle D, Sly WS, Childs B, Kinzler KW, Vogelstein B, editor. NewYork: McGraw Hill. 3611-3633.

35.Meiner, V., Shpitzen, S., Mandel, H., Klar, A., Ben-Neriah, Z., Zlotogora, J., Sagi, M., Lossos, A., Bargal, R ., Sury, V., et al. 2001. Clinical-biochemical correlation in molecularly characterized patients with Niemann-Pick type C. Genet Med 3: 343-348.

36.Choudhury, A., Dominguez, M., Puri, V., Sharma, D.K., Narita, K., Wheatley, C.L., Marks, D.L., and Pagano, R.E.2002.Rabproteins mediate Golgi transport of caveola- internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick Ccells. J Clin Invest 109: 1541-1550.

37.Blom, T.S., Linder, M.D., Snow, K., Pihko, H., Hess, M.W., Jokitalo, E., Veckman, V., Syvanen, A.C., and Ik ketone n, E.2003. Defective endocytic trafficing of NPC1 and NPC2 underlying infantile Niemann-Pick type C disease. Hum Mol Genet 12:257-272.

38.Walter, M., Davies, J.P., and Ioannou, Y.A.2003.Telomerase immortalization upregulates Rab9 expression and restores LDL cholesterol egress from Niemann-Pick C1 lateendosomes.J Lipid Res 44:243-253.

39.Maxfield, F.R., and McGraw, T.E.2004.Endocyticrecycling.Nat Rev Mol Cell Biol 5:121-132.

40.Mukherjee, S., and Maxfield, F.R.2004.Membranedomains.Annu Rev Cell Dev Biol 20:839-866.

41.Prinz, W.2002.Cholesterol trafficking in the secretory and endocytic systems.Semin Cell Dev Biol 13:197-203.

42.Cruz, J.C., Sugii, S., Yu, C., and Chang, T.Y.2000.Role ofNiemann-Pick type C1 protein in intracellular trafficking of lowdensity lipoprotein-derived cholesterol.J Biol Chem 275:4013-4021 .

43.Shiratori, Y., Okwu, A.K., and Tabas, I.1994.Freecholesterol loading of macrophages stimulates phosphatidylcholinebiosynthesis and up-regulation of CTP: phosphocholinecytidylyltransferase.J Biol Chem 269:113387-113

44.Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J.1951.Protein measurement with the Folin phenol reagent.J BiolChem 193:265-275.

45.McGraw, T.E., Greenfield, L., and Maxfield, F.R.1987.Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.J Cell Biol 105:207-214.

46.Zhang, J.H., Chung, T.D., and Oldenburg, K.R.1999.ASimple Statistical Parameter for Use in Evaluation and Validation ofHigh Throughput Screening Assays.J Biomol Screen 4:67-73.

47.Willet P., B., JM., Down, GM.1998.Chemical SimilaritySearching.J.Chem.Inf.Comput.Sci 38:983-996.

48. Zhang, M., Dwyer, N.K., Neufeld, E.B., Love, D.C., Coke y, A., Comly, M., Patel, S., Watari, H., Strauss, J.F., 3rd, Pentchev, P.G., et al. 2001. Sterol-modulated glycolipid sorting occurs inniemann-pick C1 late endosomes. J Biol Chem 276: 3417-3425.

49.Boven, L.A., van Meurs, M., Boot, R.G., Mehta, A., Boon, L., Aerts, J.M., and Laman, J.D.2004.Gaucher cells demonstrate adistinct macrophage phenotype and resemble alternatively activatedmacrophages. Am J Clin Pathol 122:359-369.

50.Platt, F.M., Neises, G.R., Reinkensmeier, G., Townsend, M.J., Perry, V.H., Proia, R.L., Winchester, B., Dwe k, R.A., and Butters, T.D.1997.Prevention of lysosomal storage in Tay-Sachsmice treated with N-butyldeoxynojirimycin. Science 276: 428-431.

51.Neufeld, E.B., Stonik, J.A., Demosky, S.J., Jr., Knapper, C.L., Combs, C.A., Coke, A., Comly, M., Dwyer, N., Blanchette-Mackie, J ., Remaley, A.T., et al.2004.The ABCA1transporter modulates late endocytic trafficing: insights from the correction of the genetic defect in Tangier disease.J Biol Chem279:15571-15578.

52.Brzozowski, Z.1998.2-Mercapto-N-(azolyl)benzenesulfonamides.V.Syntheses, anti-HIVand anticancer activity of some4-chloro-2-mercapto-5-methyl-N-(1,2,4 -triazolo[4,3-a]pyrid-3-yl)benzenesulfonamides.Acta Pol Pharm55:375-379.

53.Brzozowski, Z., Saczewski, F., and Gdaniec, M.2000.Synthesis, structural characterization and antitumor activity of novel2, 4-diamino-1, 3, 5-triazine derivatives.Eur J Med Chem35: 1053-1064.

54.Schatzberg, A.F.2004.Employing pharmacological treatment of bipolar disorder to greatest effect.J Clin Psychiatry 65 Suppl15: 15-20.

55.Mohr, R., Busehauer, A., and Schunack, W.1986.[H2-antihistaminics.31.1,2,5-Triazine-2,4-diamines and-2,4,6-triamines with H2-antagonistic activity]. Arch Pharm (Weinheim) 319: 878-885.

56.Chambers, M.S., Atack, J.R., Carling, R.W., Collinson, N., Cook, S.M., Dawson, G.R., Ferris, P., Hobbs, S.C., O'Connor, D., Marshall, G. , et al.2004.An orally bioavailable,functionally selective inverse agonist at the benzodiazepine site of GABAA alpha5 receptors with cognition enhancing properties.J Med Chem 47:5829-5832.

57.Hao, M., Lin, S.X., Karylowski, O.J., Wustner, D., McGraw, T.E., and Maxfield, F.R.2002.Vesicular and non-vesicular steroltransport in living cells.The endocytic recycling compartment is a large sterol storage organelle. J Biol Chem 277: 609-617.

58.Sleat, D.E., Wiseman, J.A., El-Banna, M., Price, S.M., Verot, L., Shen, M.M., Tint, G.S., Vanier, M.T., Walkley, S.U., and Lobel, P. 2004. Genetic evidence for nonredundant functional cooperation between NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci U S A101: 5886-5891.

59.Strauss, J.F., 3rd, Kishida, T., Christenson, L.K., Fujimoto, T., and Hiroi, H.2003.START domain proteins and the intracellulartrafficking of cholesterol in steroidogenic cells.Mol Cell Endocrinol202:59- 65.

60.Tall, A.R.2003.Role of ABCA1 in cellular cholesterol effluxand reverse cholesterol transport.Arterioscler Thromb Vasc Biol23:710-711.

61.Choudhury, A., Sharma, D.K., Marks, D.L., and Pagano, R.E.2004.Elevated endosomal cholesterol levels in Niemann-Pickcells inhibit rab4and perturb membrane recycling.Mol Biol Cell15:4500-4511.

Example

[0442] Now that the present invention is generally described, it will be more readily understood by reference to the following examples, which are intended to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

Example 1: Filipin Binding Detection

[0444] We developed an assay for correcting the phenotype of NPC in CHO cell lines using filipin, which binds to non-esterified cholesterol, and has been used to visualize free cholesterol content in NPC cells. The initial screen was performed in CT60 cells expressing a mutated hamster NPC1 protein and a gain-of-function gene mutation in SCAP (42). These cells are unable to transport LDL-derived cholesterol out of late endosomes, and large amounts of cholesterol accumulate in the LSO compartment. Figures 1A-B show images of filipin staining in a control CHO cell line TRVbl(45) (Figure 1A) and in CT60 cells (Figure 1B). It can be seen that CT60 cells show more filipin staining than control cells and the fluorescence in CT60 cells is localized in the organelles around the nucleus.

[0445] For screening, images were acquired using a Discovery-1 automated microscope system with a 10X objective and corrected for background and shading as described in Methods. Two thresholds were set for filipin staining, a low threshold encompassing all cellular regions and a high threshold for intense filipin staining in perinuclear LSOs. The set threshold was used for the analysis of 64 images of untreated CT60 cells in 32 wells per plate. It was found that the same threshold could be used for multiple plates in an experiment, but the threshold would vary with experiments performed in different ways. In low-threshold development, we compared thresholded cell images based on transmitted-light images, and these thresholds provided good agreement with transmitted-light cell boundaries.

[0446] As a simple measure of filipin staining intensity, we measured filipin intensity per pixel above a threshold. Filipin labeling (concentration and time) conditions were adjusted to optimize differentiation between CT60 cells and control TRVb1 cells. As shown in Figure 1C, this simple approach provided a fairly high degree of discrimination between CT60 cells and TRVb1 cells. This method for screening is characterized by the statistical parameter Z' (46):

Z'＝1-(3σ _c+ +3σ _c- )/|μ _c+ -μ _c- |

[0447] where σc ₊ and _σc- are the standard deviations (SD) of the positive and negative control data sets and _σc+ and _σc- are the mean values of the positive and negative controls. The Z' value calculated using the mean filipin intensity of CT60 versus 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] While intensity detection may be useful, it appears that more image information may provide better discrimination of wild type versus NPC cells. To this end, we exploited the spatial distribution of filipin labeling 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 Figure 2. We then measured the total fluorescence intensity of selected LSOs (high threshold, Figure 2C) divided by the number of total pixels in the cell (low threshold, Figure 2D). As shown in Figure 1, this LSO compartmental assay gave stronger discrimination of CT60 versus control cells with a Z' value of 0.61.

[0449] Using the screening techniques described above, we screened a compound library of 14,956 compounds added to cells for 16 hours at a final concentration of 10 μΜ using one well for each compound. Prior to application of these compounds, cells were grown in normal tissue culture medium containing 10% FBS, thus, LSOs were saturated with cholesterol (Fig. 1B). Cells were then imaged and analyzed using mean filipin intensity detection and LSO compartmental assays. In general, both assays identified similar compounds that reduced filipin labeling.

[0450] Wells with mean filipin intensity exceeding 3SD from the mean of solvent-treated cells were further investigated. The images of these selected wells were visually detectable, and none of the sites showing weak focus (approximately 0.3% of the total) were reanalyzed if we were to acquire images from a second location. Wells with low cell numbers were considered an indication of toxicity, and these cytotoxic compounds were not followed up further. We also studied low-magnification images of the arrangement in the 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 automated pipetting step. In one instance, a band pattern was seen in a set of 8 plates, and the compounds were rescreened after a pipette issue was identified.

[0451] From this initial screen, we found 133 compounds that decreased mean filipin intensity by more than 3 SD, and 23 compounds that increased mean filipin intensity by more than 3 SD. Visual inspection of the images also revealed morphological changes that 19 compounds could produce in filipin staining patterns that did not meet our criteria for reduced mean filipin intensity.

[0452] These 175 compounds were then re-screened at 10 μΜ 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 mean filipin intensity and LSO ratio values were determined. Fourteen compounds were selected from the rescreen to reproducibly reduce filipin staining at 10 μM, and 8 compounds were found to reproducibly increase filipin staining. Nine compounds were found to alter the morphological distribution of filipin. Figure 3 shows images of screening plates of solvent-treated control wells and compound-treated wells showing reduced filipin staining.

[0453] Figure 4 shows the effect of 4 compounds in causing the morphological changes observed by filipin staining. These cells showed rearrangements, suggesting that cholesterol had been altered to different compartments, or that the morphology of the LSOs themselves had been altered (compound 1-c-3, Figure 4C). The effect of compound 1-b-4 (Fig. 4D) was very strong, as observed by the bright swirls of filipin staining. The effect of compound 1-b-4 was similar to that of normal human fibroblasts (data not shown), suggesting that this response is independent of the NPC phenotype of the cells. These compounds, which increased apparent filipin staining and caused morphological changes, were not further investigated in this study.

[0454] The structures of the 14 compounds that reduced filipin labeling and the 2 compounds that caused significant morphological changes are shown in Figure 5. Compounds 1-c-2 and 1-c-3 caused morphological changes, and compounds 1-b-2 and 1-b-4 increased filipin potency.

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 chemical reagents, including dimethyl sulfoxide (DMSO), filipin/paraformaldehyde (PFA) and Hoechst 33258 were purchased from Sigma Chemicals (St. Louis, MO). Compound libraries used for screening were purchased from Chemical Diversity, Inc. (San Diego, CA). Metamorph image analysis software was from Molecular Devices Corporation (Downington, PA).

Cell culture: NPC1 cell lines, CT60 and CT43 are provided by TYChang ((Dartmouth Medical School, Hanover, NH). These cell lines are from the parental cell line 25RA, which is a protein containing in SREBP cleavage activator (SCAP). Gain-of-function mutant CHO cell lines (42). CT60 and CT43 cells were grown in Hams F12 medium supplemented with 1% penicillin/streptomycin (PS), 2 g/L glucose, 1.176 g of 10% FBS /L sodium bicarbonate [medium A], maintained at 37°C in a humidified incubator containing 5% CO ₂ . For screening, CT60 cells (650 cells) in 30 μl growth medium A containing 10% FBS /well) or CT43 cells (700 cells/well) were seeded in Costar 384-well black polystyrene flat, transparent-bottomed tissue culture-treated plates (Corning, Inc., NY) to be analyzed when the cells were ~80% fusion was obtained when .

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

[0459] Compound Addition: Compounds in the compound library were formatted for screening in the Rockefeller University High Throughput Screener. Cells were treated with compounds from the chemical library for 1 day after plating. Using the Packard MiniTrak ^™ robotic liquid handling system, we added 0.1 μl (5 mM in DMSO) to 25 μl of each compound consisting of medium A with 1% FBS and 20 mM 2-[4-(hydroxyethyl)-1-piperone In selection medium S composed of azinyl]ethanesulfonic acid (HEPES) in Falcon 384-well V-bottom polypropylene plates. To obtain ~10 μΜ final concentration, 23 μl of premixed compounds were dispensed into plates containing cells and 30 μl medium A. For the initial screening, 352 test compounds were added to each plate, and the remaining 32 wells were added with DMSO only as a control. All compound added plates were incubated at 37°C for 16h. Then, they were washed 3 times with pH 7.4 phosphate buffered saline (PBS) using a Bio-Tek Elx405 plate washer (Bio-Tek Instruments Inc., Winooski, VT). For each wash cycle, 70 μl of PBS was dispensed and then aspirated to a residual volume of 16 μl per well. Finally, cells were fixed with 1.5% PFA in PBS for 20 minutes at room temperature, and then washed 3 more times with PBS.

[0460] Fluorescent labeling. For fixed cells, filipin was added to a final concentration of 50 μg/ml in PBS over 45 min at room temperature for labeling of free cholesterol. Cells were finally washed 3 times with PBS and images were acquired immediately after labeling.

Fluorescence Microscope: An automated fluorescence microscope Discovery-1 from Molecular Devices Corporation was used to acquire images, equipped with a xenon arc lamp (PerkinElmer, CA), a Nikon 10X Plan Fluor 0.3NA objective, and a PhotometricsCoolSnapHQ camera (1392 x 1040 pixels ; Roper Scientific, Tucson, AZ). Filipin images were acquired using a 60/40nm excitation and 480/40nm emission filter with a 365DCLP (DiChroic Long Pass) filter. Image files are stored on the local host computer before being transferred to a server.

[0462] The CRS CataLyst Express robot (Thermo Electron Corp) was used to transport the plates from the plate depository. Images were acquired at 2 positions per well, each position was approximately 50 μm away from the center of the well, using a 2×2 box-merge method, and the exposure time for each image was 75 ms. Different methods are used for autofocusing for primary and secondary screening. In the initial screening, each well was focused over a range of ±150 μm and each site of each well was focused over a range of ±20 μm using image-based focusing and MetaMorph autofocus algorithms. Images for focus were binned using 8 x 8 with a 15 ms exposure time 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 each position is then refocused over a 20 μm range. Regardless of the focusing method, the acquisition time per plate was 60-75 minutes. 696 × 520 pixel images were acquired with 12 intensity bits per pixel. Each pixel is 1.25 x 1.2 μm in the object.

[0463] Image Analysis: Images of Filipin-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 was created by averaging all the images obtained from a plate 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 then divided pixel by pixel by the shadow image. Background was subtracted from the shading corrected image by determining the 5th percentile intensity value and subtracting this value from each pixel in the image. All regions had at least 5% of the image area free of cells at the plating density used. Next, two different thresholds were applied to these filipin images. First, set a low threshold for including all regions occupied by cells. Use this filter value to align the outline of the cell with the profile of the transmitted light image of the cell. Second, a higher threshold was set for brightly stained areas in CT60 and/or Ct43 cells by selecting filipin-stained bright areas, with the aim of primarily identifying LSOs in the perinuclear regions of cells. For mean filipin intensity determination, using the low threshold alone, we determined the total filipin intensity above the low threshold divided by the number of pixels above the low threshold per region. This gives an average filipin intensity per cell area. For LSO compartment proportions, we selectively measured the total filipin intensity above the high threshold divided by the number of pixels at the low threshold in that region. This gives a measure of the total intensity of LSO filipin per cell area.

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

[0465] Immunofluorescence: CT60 cells grew to 70% confluency in culture dishes on the bottom of coverslips. After 24 h, compounds (10 [mu]M) were added to the cells in selection medium containing 20 mM HEPES. After 16-22 h of compound treatment, the cells were washed 3 times with PBS and fixed with 3.3% PFA for 20 min at room temperature. Cells were then treated with 50 mM ammonium chloride for 10 minutes and washed 3 times with PBS. After blocking with 0.1% bovine serum albumin for 20 minutes, the cells were washed with PBS, permeabilized with 0.05% saponin and incubated with anti-LBPA (1:100) for 30 minutes at room temperature (J.Gruenberg, Univ.of Geneva contribute). Then the cells were washed 3 times with PBS, and incubated with goat anti-mouse IgG-Alexa546 (1:200) and 100 μg/ml filipin for 30 minutes. Finally, cells were washed 3 times with PBS and images were acquired under a Leica DMIRB microscope (Leica Mikroscope und Systeme GmbH, Germany) equipped with a Princeton Instruments (Princeton, NJ) cooled CCD camera and MetaMorph Imaging System software driven. All images were acquired using an oil immersion objective (25×, 1.4NA). The Alexa 546-Tf was imaged with a standard rhodamine filter cube and the Filippin was imaged with a Leica A4 cube [360nm (40nm passband) excitation filter and 470nm (40nm bandpass) emission filter]. Image analysis was performed using the Metamorph Discovery-1 image analysis software using image analysis algorithms for screening to evaluate the concentrations of cholesterol and LBPA in the presence and absence of compounds.

Example 2: Dose-dependent detection

[0467] Dose dependence: Compounds were tested in 4 wells at 10 μM, 3.33 μM, 1.11 μM, 370 nM and 123 nM using the same method as used for the screening. Amounts of selected compounds required for one run were purchased from Chemical Diversity and 10 mM stock solutions were prepared in DMSO. Secondary stock plates of 2X concentrations of compounds (20 μM, 6.66 μM, 2.22 μM, 740 nM and 246 nM) were prepared in screening media. To obtain the final concentration, 30 μl of this secondary stock was added to cells containing 30 μl per well of growth medium A containing 10% FBS. The final concentration of dimethylsulfoxide (0.2% v/v) was the same in all wells. Cells were plated at 650 cells/well into a costar 384-well plate containing 30 μl per well of growth medium A containing 10% FBS. After incubation for 20 hr in the presence of compounds, cells were washed with PBS, fixed with PFA and stained with filipin as described for the screening assay. The dose dependence of 14 selected compounds from the primary compound library was determined in independent experiments at least 5 times in CT60 cells and at least 3 times in CT43 cells. The dose dependence of 7 selected compounds from the secondary compound library was determined in independent experiments at least 3 times in CT60 cells and at least 2 times in CT43 cells.

[0468] In one assay, the dose-response curves for the same 14 selected compounds as in the screening assay are shown in Figures 6A-B. Three of these compounds reduced filipin staining in CT60 cells at 1.1 μm to 3 SD below the mean value of solvent treatment, and the same compound-treated cells were more than 2 SD below the mean value of solvent treatment at 123 nM ( FIG. 6A ). Compound 1-a-14 has an unusual dose-response curve, which may be related to its cytotoxicity at high concentrations. To test whether this effect was specific to the CT60 cell line, we also examined the dose response of these 14 selected compounds in CT43 cells, another NPC1-mutated cell line derived from the parental cell line 25RA. Most compounds were efficacious more than 3 SD below the untreated mean at 10 [mu]M, but lost their effect on CT43 cells at lower concentrations (Fig. 6B). It is interesting to note that the general trend of the effects of these 14 compounds was similar in the two cell lines, although in general the compounds were more effective at a given dose on CT60 cells.

Example 3: Time course detection

[0470] Using a similar dose-dependent approach, the effects of these compounds at 1.11, 3.33 and 10 μΜ concentrations of treatment for 4, 20, 48 h were determined. On the first day, CT60 cells were seeded in growth medium in three 384-well plates at 600 cells/well. To ensure that the same cell density was maintained at the final time point, compounds were added in chronological order. After overnight incubation, compounds diluted in medium S from the first plate (48h time point) were added to the wells to achieve final concentrations of 1.11, 3.33 and 10 μΜ. After 52h of cell seeding, compounds were added to the second plate in a similar manner and incubated for 20h. Finally, 68 hours after the cells were seeded, the compounds were added to the third plate in a similar manner and incubated for 4 hours. All three plates were washed 3 times with PBS, fixed with 1.5% PFA and stained with 50 μg/ml filipin. Four wells per condition were measured in each experiment and each experiment was replicated 3 times for CT60 and CT43 (data for CT43 not shown).

Example 4: Toxicity Detection

[0472] To measure toxicity, we treated CT60 and CY43 cells with compounds at 5, 10 and 20 μΜ for 24 hours. The number of cells per well was compared to control cells treated with DMSO (Figure 7). After 24h of incubation, most compounds did not cause a significant decrease in the number of cells at 10 μM. Compound 1-a-14 at 10 μM caused a 50% reduction in the number of CT60 cells and an 80% reduction in the number of CT43 cells. 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.

The method that is used for cell counting: use the similar method of dose-dependent detection method, the compound is added in 384 holes with the concentration of 0 (dimethyl sulfoxide solvent control group), 5, 10 and 20 μ m in parallel four times Plates of CT60 and CT43 cells differ in that the cells are nuclear stained with Hoechst 33258. The final concentration of dimethylsulfoxide was 0.2% in the wells. For control cells, an equal amount of DMSO 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 cells were washed 3 times with PBS, nuclei were stained with 5 μg/ml Hoechst 33258 (25 mg/ml DMSO stock) in PBS for 45 minutes at room temperature. Finally, cells were washed 3 times with PBS, and images were acquired with a Nikon 4X Plan Apo 0.2NA eyepiece. For imaging of Hoechst, we used the same filter settings as Filipin. At 12 intensity bits per pixel, we acquired a 520 × 696 pixel image per well. Each pixel is 3.125 x 3.125 μm in 13 objects. Using the Integrated Morphometry Analysis function of MetaMorph, cells were counted by interactively defining a standard mononuclear area (~200 μm ² ) per plate. The amount of standard area per target above a threshold was determined, and the total amount of standard area per image was used as a cell count. The percent reduction in cell number compared to DMSO control was calculated at each concentration and time point.

Example 5: Determination of Cholesterol by Gas Chromatography

[0475] The cholesterol-lowering effect of 14 selected compounds identified by filipin labeling was measured by an alternative chemical method involving GC separation of solvent-extracted cellular lipids (43). CT60 cells were treated with compounds at 10 [mu]M under the same conditions as the screening assay. The concentration of FC per cell was determined by gas chromatography, and all 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 cells, as measured by the mean Filippin method. The hits from the first screen had variable effects on cellular cholesterol; including some compounds that significantly increased free cholesterol in cells.

Table 1 The CT 60 cells detected by average filipin intensity and gas chromatography are treated with free cholesterol after being selected from 14 kinds of selected compounds in the primary compound library.

Compound No. Score of mean filipin intensity controls Fraction of μg ¹ FC/μg protein control determined by GC 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-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-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-13 0.97±0.04 1.54±0.11 1-a-14 1.24±0.04 1.44±0.16

¹ FC-free cholesterol

[0477] CT60 or CT43 cells were seeded in 6-well plates on the first day. Selected compounds were added to cells at a concentration of 10 μM. Cells were cultured for 24 hours. Cellular lipids were extracted with n-hexane:isopropanol (3:2 V/V). Lipid extracts were dried and resuspended in n-hexane, followed by separation on a gas chromatograph using β-sitosterol as an internal standard.

[0478] Methods: CT60 cells were seeded in 6-well plates containing Ham's F-12/1.176 g/L sodium bicarbonate/2 g/L glucose/10% FBS medium. After 24 hours of incubation, cells were treated with 10 μM of each compound, and the medium was replaced with Ham’s F-12/1.176 g/L NaHCO3/2 g/L Glucose/5.5% FBS/20 mM HEPES. The change of medium in each well is to have similar experimental conditions to those used for screening after the compound is added to the cells. After compound treatment for 18 h, the cells were washed twice with Hank's balanced salt solution (HBSS). Cellular lipids were extracted with n-hexane/isopropanol (3:2 v/v) (43), dried and resuspended in n-hexane, and then separated on a gas chromatograph (GC) using the following conditions. A Hewlett Packard gas chromatograph model HP 5890 Series II (Palo Alto, CA) was used to separate free cholesterol, equipped with a flame ionization detector, split-non-separated injector, and coated with 5% phenyl A 15 m x 0.53 mm HP-5 capillary column of methylsiloxane film. The injection temperature was maintained at 255°C, the furnace temperature was isothermally controlled at 260°C, and helium was used 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.

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 in terms of 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 two concentrations of 10 μM and 1 μM. Each compound was added to a single well and images were acquired at 2 positions per well and averaged into one value. Two complete screens of the secondary library were performed at two concentrations. Images were analyzed using both mean filipin intensity and LSO compartment ratio. At 10 [mu]M, we found 574 compounds 3SD below the control by one assay and 34 compounds below 3SD of the control by both assays. At 1 μM, we found 202 compounds 3SD below control with at least one assay and 6 compounds 3SD below control with 2 methods.

[0482] We randomly selected these 202 compounds and rescreened twice at 1 μM, 300 nM and 100 nM. We added compounds at each concentration in 4 different wells and averaged the measurements from the images at 2 positions in each well. Through 2 screens we obtained a total of 8 values per compound from each assay at 1 μM, 300 nM and 100 nM. For each analytical method (average filipin intensity and LSO compartment ratio) we had a total of 10 values at 1 μM (2 from the first screen and 8 randomly selected) and 8 values at 300 nM and 100 nM. Based on these data, we selected 7 compounds for further analysis, and their chemical structures are shown in Figure 8.

[0483] Dose-response curves for seven selected compounds are shown in Figure 9. The data showed that 4 compounds (2-a-8, 2-a-9, 2-a-12 and 2-a-13) exhibited more than 3 SD reduction in the LSO compartment assay at 370 nM, and 3 Compounds (2-a-8, 2-a-9 and 2-a-12) also showed an effect on CT 60 cells lower than solvent control 2SD at 123nM. Most of these compounds were also active on CT43 cells as hits from the first round (Fig. 9B).

Example 7: Absorption of LDL

[0485] CT60 cells were grown to 70% confluency in 96-well specialized optical culture plates (Corning, Inc., Corning, NY). After 24 hours, the cells were incubated with DiI-LDL (6 mg/ml) and the selected compound (10 μM) in selection medium supplemented with 20 mM HEPES. Each compound was added to 8 wells, and equal amounts of DMSO and DiI-LDL were added to control wells. After 20 h, the cells were washed 3 times with PBS, fixed with 1.5% PFA for 20 minutes, and stained with 50 μg/ml filipin for 45 minutes. Images were acquired at 20X magnification using a Discovery-1 automated fluorescence microscope. Images of DiI-LDL were acquired using a 535nm/40nm excitation filter and a 610nm/60nm bandpass with a Chroma 51001bs DiChroic filter. Filipin images were obtained as described above. Images were acquired at 4 sites per well, yielding 32 images per compound. The DiI-LDL images were background and shading corrected as described above. Set a low threshold to determine the cell area from the filipin image. Finally, the mean DiI-LDL intensity per cell area was measured.

[0486] As shown in Table 2, all of the selected compounds from the secondary library except 2-a-15 caused a reduction in LDL uptake after 20 hours of incubation. This will require further work to determine whether this is a primary effect of certain compounds, or whether the reduction in LDL uptake is secondary to the release of cholesterol from LSOs. Although CT60 cells are partially defective in SCAP function as a consequence of a point mutation in one SCAP allele, it was expected that the cells would respond to increased cholesterol by reducing LDL receptor expression.

The uptake of DiI-LDL after table 2 CT60 cells are treated with 7 kinds of selected compounds from secondary library.

Compound No. Cell area fraction of DiI intensity/control 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-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 μg/ml) in the presence of compound (10 μM) for 20 hours. Intensity per unit cell area was measured as described in Methods. Values were normalized to intensity per cell area versus control (solvent treated) cells. Values are based on 32 images from 8 wells per condition. ±SEM.

Example 8: Toxicity determination of compounds from secondary libraries.

[0490] Figure 10 shows the results of toxicity assays for seven selected compounds from the secondary library. After 24h, compound 2-a-12 caused a 75% reduction in cell number in both CT60 and CT43 cells at 20 μm. The other compounds had no or only slight reduction in cells under these conditions, whereas they reduced filipin staining. Cytotoxicity of these compounds was assessed by measuring LDH released into the medium. As shown in Figure 10C, the cytotoxicity measured by this method was less than the reduction obtained by cell counts, suggesting that these compounds may slow cell growth after 24 hours without causing cell death.

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

[0492] Six of the seven compounds selected from the secondary library had low toxicity and significantly reduced FC not only in LSOs but also in whole cells. Several of these compounds were effective at concentrations below 0.5 [mu]M. Table 3 summarizes the cholesterol-lowering and toxic effects of these six selected compounds from the secondary library.

Table 3 is from the generalization of 7 kinds of compounds of secondary storehouse to CT60 cell influence

Compound number. Minimum effective dose (LSO measurement) (-3SD@20h) Fraction of mean filipin measurements @ 10 μM dose control Fraction of g FC ¹ /μg protein (GC)@10 μM dose control Fraction of protein content (μg/well) control Fraction of cell counts @ 10 μM dose control after 24 h 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-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

¹ FC-free cholesterol

Example 9: Cytotoxicity assay of LDH.

[0495] Cytotoxicity of selected compounds was determined using the LDH Release Assay Kit (Roche Diagnostic GmbH, Penzberg, Germany) according to the manufacturer's instructions. CT60 cells were seeded in 96-well plates (Costar, Corning Inc., Corning, NY) at 3500 cells/well and incubated for 24 hours. Using a method similar to the dose-dependent assay, the compound was added to CY60 cells in three parallels at concentrations of 0 (DMSO solvent control), 5, 10 and 20 μM, respectively. After 24 h of treatment, 100 μl of tissue culture supernatant was removed, and the LDH activity was determined by measuring the absorbance at 492 nm 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 Figure 10C, the cytotoxicity measured by this method was less than the reduction measured by cell counting, suggesting that these compounds may slow cell growth after 24 hours without causing cell death.

Example 10: Assays on normal human fibroblasts.

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

Example 11: Determination of 25RACHO cells

[0500] 25 RACHO 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 μM. Cells were loaded ^with3H cholesterol and then chased overnight in the presence or absence of various compounds. The medium outside the cells is removed and the radioactivity released from the cells is measured. The cells are then lysed, and the radioactivity that remains in the cells is measured. The data (FIG. 13) indicate that these compounds promote cholesterol efflux from 25-RA cells, a Niemann-Pick disease-free CHO cell line that was used to select CT43 and Mother cell line of CT60 cells. 25-RA cells have a defect in SCAP that alters cholesterol homeostasis, including increased cholesterol synthesis.

Example 12: Inhibition of Lysosome Acid Lipase

[0502] Certain compounds disclosed herein may have therapeutic effects through inhibition of 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 structure defining the named compound can inhibit the hydrolysis of cholesteryl esters by LAL.

Claims (149)

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)-；

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 having 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;

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 or heteroaralkyl;

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,

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)；

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, heteroalkyl, 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, - (CR)⁷＝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; and is

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 cycloalkenyl, 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, 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;

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) R⁵；

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

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 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)-；

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

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

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

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)-；

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 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;

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;

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 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;

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)；

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 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,

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; and is

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 cycloalkenyl, 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, 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;

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) R⁵；

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

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

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；

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, aralkyl, heteroaralkyl, or heteroaryl having 1 heteroatom selected from N, O or S;

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;

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,

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 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

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

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)-；

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

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;

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 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,

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

each R³Independently represents H, alkyl; and is

R⁴Is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

or a compound represented by formula XVI:

wherein,

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

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,

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

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, 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,

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

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

R³is hydrogen, alkyl, -CO ₂R⁸or-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 is

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

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 (R)¹⁵)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 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

l is oneBond, -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.

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,

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

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,

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 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;

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

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；

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

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

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 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

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:

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 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

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

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)-；

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 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 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;

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,

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; and is

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;

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;

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.

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;

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

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)-；

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 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 having 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 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;

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 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, 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, - (CR)⁷＝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; and is

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 cycloalkenyl, 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:

VI

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;

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, 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

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 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)-；

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

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

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

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.

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