Reverse-turn mimetics and method relating thereto

Information

  • Patent Grant
  • 8106049
  • Patent Number
    8,106,049
  • Date Filed
    Thursday, September 3, 2009
    15 years ago
  • Date Issued
    Tuesday, January 31, 2012
    12 years ago
Abstract
Conformationally constrained compounds that mimic the secondary structure of reverse-turn regions of biologically active peptides and proteins are disclosed. Such reverse-turn mimetic structures have utility as diagnostic and therapeutic agents for inhibiting or treating disorders modulated by Wnt-signaling pathway, such as cancer, especially colorectal cancer, restenosis associated with angioplasty, polycystic kidney disease, aberrant angiogenesis disease, rheumatoid arthritis disease, tuberous sclerosis complex, Alzheimer's disease, excess hair growth or loss, or ulcerative colitis. In one embodiment, these reverse-term mimetics have a general structure of formula (VI):
Description
STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200146402C9_SEQUENCE_LISTING.txt. The text file is 3 KB, was created on Jan. 19, 2010, and is being submitted electronically via EFS-Web.


BACKGROUND OF THE INVENTION

1. Field of the Invention


The present invention relates generally to reverse-turn mimetic structures and to a chemical library relating thereto. The invention also relates to applications in the treatment of medical conditions, e.g., cancer diseases, and pharmaceutical compositions comprising the mimetics.


2. Description of the Related Art


Random screening of molecules for possible activity as therapeutic agents has occurred for many years and resulted in a number of important drug discoveries. While advances in molecular biology and computational chemistry have led to increased interest in what has been termed “rational drug design”, such techniques have not proven as fast or reliable as initially predicted. Thus, in recent years there has been a renewed interest and return to random drug screening. To this end, particular strides having been made in new technologies based on the development of combinatorial chemistry libraries, and the screening of such libraries in search for biologically active members.


In general, combinatorial chemistry libraries are simply a collection of molecules. Such libraries vary by the chemical species within the library, as well as the methods employed to both generate the library members and identify which members interact with biological targets of interest. While this field is still young, methods for generating and screening libraries have already become quite diverse and sophisticated. For example, a recent review of various combinatorial chemical libraries has identified a number of such techniques (Dolle, J. Com. Chem., 2(3): 383-433, 2000), including the use of both tagged and untagged library members (Janda, Proc. Natl. Acad. Sci. USA 91:10779-10785, 1994).


Initially, combinatorial chemistry libraries were generally limited to members of peptide or nucleotide origin. To this end, the techniques of Houghten et al. illustrate an example of what is termed a “dual-defined iterative” method to assemble soluble combinatorial peptide libraries via split synthesis techniques (Nature (London) 354:84-86, 1991; Biotechniques 13:412-421, 1992; Bioorg. Med. Chem. Lett. 3:405-412, 1993). By this technique, soluble peptide libraries containing tens of millions of members have been obtained. Such libraries have been shown to be effective in the identification of opioid peptides, such as methionine- and leucine-enkephalin (Dooley and Houghten, Life Sci. 52, 1509-1517, 1993), and a N-acylated peptide library has been used to identify acetalins, which are potent opioid antagonists (Dooley et al., Proc. Natl. Acad. Sci. USA 90:10811-10815, 1993. More recently, an all D-amino acid opioid peptide library has been constructed and screened for analgesic activity against the mu (“p”) opioid receptor (Dooley et al, Science 266:2019-2022, 1994).


While combinatorial libraries containing members of peptide and nucleotide origin are of significant value, there is still a need in the art for libraries containing members of different origin. For example, traditional peptide libraries to a large extent merely vary the amino acid sequence to generate library members. While it is well recognized that the secondary structures of peptides are important to biological activity, such peptide libraries do not impart a constrained secondary structure to its library members.


To this end, some researchers have cyclized peptides with disulfide bridges in an attempt to provide a more constrained secondary structure (Tumelty et al., J. Chem. Soc. 1067-68, 1994; Eichler et al., Peptide Res. 7:300-306, 1994). However, such cyclized peptides are generally still quite flexible and are poorly bioavailable, and thus have met with only limited success.


More recently, non-peptide compounds have been developed which more closely mimic the secondary structure of reverse-turns found in biologically active proteins or peptides. For example, U.S. Pat. No. 5,440,013 to Kahn and published PCT applications nos. WO94/03494, WO01/00210A1, and WO01/16135A2 to Kahn each disclose conformationally constrained, non-peptidic compounds, which mimic the three-dimensional structure of reverse-turns. In addition, U.S. Pat. No. 5,929,237 and its continuation-in-part U.S. Pat. No. 6,013,458, both to Kahn, disclose conformationally constrained compounds which mimic the secondary structure of reverse-turn regions of biologically active peptides and proteins. The synthesis and identification of conformationally constrained, reverse-turn mimetics and their application to diseases were well reviewed by Obrecht (Advances in Med. Chem., 4, 1-68, 1999).


While significant advances have been made in the synthesis and identification of conformationally constrained, reverse-turn mimetics, there remains a need in the art for small molecules which mimic the secondary structure of peptides. There is also a need in the art for libraries containing such members, as well as techniques for synthesizing and screening the library members against targets of interest, particularly biological targets, to identify bioactive library members.


The present invention also fulfills these needs, and provides further related advantages by providing confomationally constrained compounds which mimic the secondary structure of reverse-turn regions of biologically active peptides and proteins.


Wnt signaling pathway regulates a variety of processes including cell growth, oncogenesis, and development (Moon et al., 1997, Trends Genet. 13, 157-162; Miller et al., 1999, Oncogene 18, 7860-7872; Nusse and Varmus, 1992, Cell 69, 1073-1087; Cadigan and Nusse, 1997, Genes Dev. 11, 3286-3305; Peifer and Polakis, 2000 Science 287, 1606-1609; Polakis 2000, Genes Dev. 14, 1837-1851). Wnt signaling pathway has been intensely studied in a variety of organisms. The activation of TCF4/β-catenin mediated transcription by Wnt signal transduction has been found to play a key role in its biological functions (Molenaar et al., 1996, Cell 86:391-399; Gat et al., 1998 Cell 95:605-614; Orford et al., 1999 J. Cell Biol. 146:855-868; Bienz and Clevers, 2000, Cell 103:311-20).


In the absence of Wnt signals, tumor suppressor gene adenomatous polyposis coli (APC) simultaneously interacts with the serine kinase glycogen synthase kinase (GSK)-3β and β-catenin (Su et al., 1993, Science 262, 1734-1737: Yost et al., 1996 Genes Dev. 10, 1443-1454: Hayashi et al., 1997, Proc. Natl. Acad. Sci. USA, 94, 242-247: Sakanaka et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 3020-3023: Sakanaka and William, 1999, J. Biol. Chem 274, 14090-14093). Phosphorylation of APC by GSK-33 regulates the interaction of APC with β-catenin, which in turn may regulate the signaling function of β-catenin (B. Rubinfeld et al., Science 272, 1023, 1996). Wnt signaling stabilizes β-catenin allowing its translocation to the nucleus where it interacts with members of the lymphoid enhancer factor (LEF1)/T-cell factor (TCF4) family of transcription factors (Behrens et al., 1996 Nature 382, 638-642: Hsu et al., 1998, Mol. Cell. Biol. 18, 4807-4818: Roose et all., 1999 Science 285, 1923-1926).


Recently c-myc, a known oncogene, was shown to be a target gene for β-catenin/TCF4-mediated transcription (He et al., 1998 Science 281 1509-1512: Kolligs et al., 1999 Mol. Cell. Biol. 19, 5696-5706). Many other important genes, including cyclin D1, and metalloproteinase, which are also involved in oncogenesis, have been identified to be regulated by TCF4/bata-catenin transcriptional pathway (Crawford et al., 1999, Oncogene 18, 2883-2891: Shtutman et al., 1999, Proc. Natl. Acad. Sci. USA., 11, 5522-5527: Tetsu and McCormick, 1999 Nature, 398, 422-426).


Moreover, overexpression of several downstream mediators of Wnt signaling has been found to regulate apoptosis (Moris et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 7950-7954: He et al., 1999, Cell 99, 335-345: Orford et al, 1999 J. Cell. Biol., 146, 855-868: Strovel and Sussman, 1999, Exp. Cell. Res., 253, 637-648). Overexpression of APC in human colorectal cancer cells induced apoptosis (Moris et al., 1996, Proc. Natl. Acad. Sci. USA., 93, 7950-7954), ectopic expression of β-catenin inhibited apoptosis associated with loss of attachment to extracellular matrix (Orford et al, 1999, J. Cell Biol. 146, 855-868). Inhibition of TCF4/β-catenin transcription by expression of dominant-negative mutant of TCF4 blocked Wnt-1-mediated cell survival and rendered cells sensitive to apoptotic stimuli such as anti-cancer agent (Shaoqiong Chen et al., 2001, J. Cell. Biol., 152, 1, 87-96) and APC mutation inhibits apoptosis by allowing constitutive survivin expression, a well-known anti-apoptotic protein (Tao Zhang et al., 2001, Cancer Research, 62, 8664-8667).


Although mutations in the Wnt gene have not been found in human cancer, a mutation in APC or β-catenin, as is the case in the majority of colorectal tumors, results in inappropriate activation of TCF4, overexpression of c-myc and production of neoplastic growth (Bubinfeld et al, 1997, Science, 275, 1790-1792: Morin et al, 1997, Science, 275, 1787-1790: Casa et al, 1999, Cell. Growth. Differ. 10, 369-376). The tumor suppressor gene (APC) is lost or inactivated in 85% of colorectal cancers and in a variety of other cancers as well (Kinzler and Vogelstein, 1996, Cell 87, 159-170). APC's principal role is that of a negative regulator of the Wnt signal transduction cascade. A center feature of this pathway involves the modulation of the stability and localization of a cytosolic pool of β-catenin by interaction with a large Axin-based complex that includes APC. This interaction results in phosphorylation of β-catenin thereby targeting it for degradation.


CREB binding proteins (CBP)/p300 were identified initially in protein interaction assays, first through its association with the transcription factor CREB (Chrivia et al, 1993, Nature, 365, 855-859) and later through its interaction with the adenoviral-transforming protein E1A (Stein et al., 1990, J. Viol., 64, 4421-4427: Eckner et al., 1994, Genes. Dev., 8, 869-884). CBP had a potential to participate in variety of cellular functions including transcriptional coactivator function (Shikama et al., 1997, Trends. Cell. Biol., 7, 230-236: Janknecht and Hunter, 1996, Nature, 383, 22-23). CBP/p300 potentiates β-catenin-mediated activation of the siamois promoter, a known Wnt target (Hecht et al, 2000, EMBO J. 19, 8, 1839-1850). β-catenin interacts directly with the CREB-binding domain of CBP and β-catenin synergizes with CBP to stimulate the transcriptional activation of TCF4/β-catenin (Ken-Ichi Takemaru and Randall T. Moon, 2000 J. Cell. Biol., 149, 2, 249-254).


BRIEF SUMMARY OF THE INVENTION

From this background, it is seen that TCF4/β-catenin and CBP complex of Wnt pathway can be taken as target molecules for the regulation of cell growth, oncogenesis and apoptosis of cells, etc. Accordingly, the present invention addresses a need for compounds that block TCF4/β-catenin transcriptional pathway by inhibiting CBP, and therefore can be used for treatment of cancer, especially colorectal cancer.


In brief, the present invention is directed to a new type of conformationally constrained compounds, which mimic the secondary structure of reverse-turn regions of biologically active peptides and proteins. This invention also discloses libraries containing such compounds, as well as the synthesis and screening thereof.


The compounds of the present invention have the following general formula (I):




embedded image



wherein A is —(CHR3)— or —(C═O)—, B is —(CHR4)— or —(C═O)—, D is —(CHR5)— or —(C═O)—, E is —(ZR6)— or —(C═O)—, G is —(XR7)n—, —(CHR7)—(NR8)—, —(C═O)—(XR9)—, or —(C═O)—, W is —Y(C═O)—, —(C═O)NH—, —(SO2)— or is absent, Y is oxygen, sulfur, or —NH—, X and Z is independently nitrogen or CH, n=0 or 1; and R1, R2, R3, R4, R5, R6, R7, R8 and R9 are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers thereof.


In an embodiment wherein A is —(CHR3)—, B is —(C═O)—, D is —(CHR5)—, E is —(C═O)—, and G is —(XR7)n—, the compounds of this invention have the following formula (II):




embedded image



wherein W, X, Y and n are as defined above, and R1, R2, R3, R5 and R7 are as defined in the following detailed description.


In an embodiment wherein A is —(C═O)—, B is —(CHR4)—, D is —(C═O)—, E is —(ZR6)—, and G is —(C═O)—(XR9)—, the compounds of this invention have the following formula (III):




embedded image



wherein W, X and Y are as defined above, Z is nitrogen or CH (with the proviso that when Z is CH, then X is nitrogen), and R1, R2, R4, R6 and R9 are as defined in the following detailed description.


In an embodiment wherein A is —(C═O)—, B is —(CHR4)—, D is —(C═O)—, E is —(ZR6)—, and G is —(XR7)n—, the compounds of this invention have the following general formula (IV):




embedded image



wherein W, Y and n are as defined above, Z is nitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen and n is not zero), and R1, R2, R4, R6 and R7, are as defined in the following detailed description.


The present invention is also directed to libraries containing one or more compounds of formula (I) above, as well as methods for synthesizing such libraries and methods for screening the same to identify biologically active compounds. Compositions containing a compound of this invention in combination with a pharmaceutically acceptable carrier or diluent are also disclosed.


The present invention is also related to methods for identifying a biologically active compound using the libraries containing one or more compound of formula (I). In a related aspect, the present invention provides a method for performing a binding assay, comprising (a) providing a composition comprising a first co-activator and an interacting protein, said first co-activator comprising a binding motif of LXXLL (SEQ ID NO: 1), LXXLI (SEQ ID NO:2) or FXXFF (SEQ ID NO:3) wherein X is any amino acid; (b) combining the first co-activator and the interacting protein with a test compound; and (c) detecting alteration in binding between the first co-activator and the interacting protein in the presence of the compound having general formula (I).


The present invention also provides methods for preventing or treating disorders associated with Wnt signaling pathway. Disorders that may be treated or prevented using a compound or composition of the present invention include tumor or cancer (e.g., KSHV-associated tumor), restenosis associated with angioplasty, polycystic kidney disease, aberrant angiogenesis disease, rheumatoid arthritis disease, ulcerative colitis, tuberous sclerosis complex, hair loss, and Alzheimer's disease. Such methods comprise administering to a subject in need thereof a compound or composition of the present invention in an amount effective to achieve the desired outcome.


In a related aspect, the present invention further provides methods for promoting neurite outgrowth, differentiation of a neural stem cell, and apoptosis in cancer cells. Such methods comprise administering to appropriate cells a compound or composition of the present invention in an amount effective to achieve the desired outcome.


These and other aspects of this invention will be apparent upon reference to the attached figure and following detailed description. To this end, various references are set forth herein, which describe in more detail certain procedures, compounds and/or compositions, and are incorporated by reference in their entirety.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 provides a general synthetic scheme for preparing reverse-turn mimetics of the present invention.



FIG. 2 provides a general synthetic scheme for preparing reverse-turn mimetics of the present invention.



FIG. 3 shows a graph based on the measurement of IC50 for Compound A of the present invention using SW480 cells, wherein cell growth inhibition on SW480 cells was measured at various concentrations of Compound A prepared in Example 4 to obtain the IC50 value. Specifically, the degree of inhibition in firefly and renilla luciferase activities by Compound A was determined. As a result, the IC50 of Compound A against SW480 cell growth was found as disclosed in Table 4. Detailed procedures are the same as disclosed in Example 6.



FIG. 4. PC-12 cells were cultured on coated dishes, and differentiated for 10 days in 50 ng/ml nerve growth factor (NGF) (as described in Example 7). (A, B) Vector-transfected PC-12 cells (A) and PC-12 cells overexpressing wt PS-1 (B) exhibit extensive neurite outgrowth after 10 days in NGF. (C)PC-12 cells expressing mutant PS-1/L286V do not display significant neurites under the same culture conditions. (D,E) Immunofluorescence analysis of GAP-43 (as described in Example 7), a molecular marker of neurite outgrowth, demonstrates intense staining for GAP-43 in the neurites (D) of vector-transfected and overexpressing PS-1/WT in PC-12 cells (E). (F) Lack of neurite outgrowth corresponds to weak GAP-43 immunostaining in the mutant cells. Data represent at least two independent experiments. (G) Differentiated cells were transfected with, Topflash, a TCF/β-catenin reporter construct. Cells were lysed, and luciferase activity measured 6 hours post-transfection (as described in Example 7). Data represent the mean of three independent experiments (±SD). Asterisk indicate P<0.05.



FIG. 5. Compound D phenotypically corrects deficient neuronal differentiation in PC-12 overexpressing mutant PS-1/L286V cells. Mutant cells were exposed to 10 μM Compound D, in addition to NGF, during the differentiation period (Misner et al., Proc. Natl. Acad. Sci. USA 98, 11714 (2001)). (A) Neurite elongation and extension are observed in PC-12 cells overexpressing PS-1/L286V upon treatment with Compound D. (B) GAP-43 (green) is significantly elevated in the mutant cells, and is seen in the neurites. (C) Quantitation of neurite outgrowth in PC-12 cells. Number of mutant cells with neurite lengths greater than two cell diameters was less than 10% that of the vector-transfected and overexpressing PS-1/WT in PC-12 cells. Number of mutant PS-1/L286V cells that had the defined neurite lengths was significantly increased, after treatment with 10 μM Compound D. The results are the average (±SD) of three independent determinations. Asterisk indicate P<0.05.



FIG. 6. Ephrin B2 (EphB2) receptor expression. Immunofluorescence analysis and RT-PCR were performed to detect EphB2 receptor expression (as described in Example 7). (A, B) EphB2 receptors are clearly demonstrated in neurites of vector-transfected and overexpressing PS-1/WT cells. The intensity of staining correlates with the high expression level. (C) In contrast, PS-1/L286V PC-12 cells have markedly reduced EphB2 receptor expression. (D) Treatment of mutant cells with Compound D leads to increased EphB2 receptor expression, which is focused at points of neurite outgrowth. (E) Expression of EphB2 receptor has previously been shown to be transcriptionally regulated (Guo et al., J. Neurosci. 17, 4212 (1997)). Lane 1, vector-transfected PC-12 cells, lane 2, overexpressing PS-1/WT cells, lane 3, overexpressing mutant PS-1/L286V cells, lane 4, mutant cells treated with Compound D. RT-PCR analysis indicates message for EphB2 receptor in cells overexpressing mutant PS-1/L286V is decreased compared to those in both the vector-transfected and overexpressing wt PS-1 PC-12 cells. Treatment with 10 μM Compound D upregulates EphB2 message. GAPDH is used an internal control.



FIG. 7. A. Compound D arrests cells in G1. FACS analysis was performed on SW480 (lower panel) and HCT116 (upper panel) cells treated for 24 hours with either Compound D (25 μM) (right) or control (0.5% DMSO (left). 5.5×106 cells were fixed and stained with propidium iodide (PI). B. Compound D selectively activates caspases in colon carcinoma cell lines. SW480 and HCT116 (left graph) cells (105) along with the normal colonocytes CCD18Co (right graph) were treated with either control (0.5% DMSO) or Compound D (25 μM). 24 hours post treatment, cells were lysed and the caspase-3/7 enzymatic activities were measured. Relative fluorescence units (RFU) were calculated by subtracting the unit values of the blank (control, without cells) from the treated samples (Compound D or control) and plotted.



FIG. 8. Compound D reduces colony growth in soft agar in a dose dependent manner. Increasing concentrations of 5-fluorouracil (5-FU) (0.5-32 μM) and Compound D (0.25-5 μM) were added to SW480 (5000 cells/well) of triplicate wells. Cells were washed and suspended in soft agar growth medium. The number of colonies after 8 days (colonies over 60 μM diameter) were counted and plotted against the compound concentration. Mean±SE of three determinations is indicated. The colony number of control in the absence of the compound was 1,637±71.



FIG. 9. A. Compound C reduces tumor growth in nude mouse model. B. Compound C slightly reduces body weight in nude mouse model.



FIG. 10. The survivin transcriptional activity is upregulated by Wnt1, but knout-down by Compound D. Percent luciferase activities were measured in wildtype, CBP+/−, and p300+/−3T3 cells in the absence of Wnt1 and Compound D, or in the presence of Wnt1, Compound D or both.



FIG. 11. Compound A (right graph) and Compound D (left graph) inhibit the activity of a survivin luciferase reporter in SW480 cells. The luciferase activities under the control of the survivin promoter were measured in SW480 cells treated with compound A or Compound D at various concentrations.



FIG. 12. RT-PCR analysis indicates that Compound D treatment decreases the expression level of the survivin gene.



FIG. 13. Compound D decreases the association of various proteins with the survivin promoter. ChIP assays on SW480 cells treated with either Compound D (25 μM) or control (0.5% DMSO) for 18 hours were performed.



FIG. 14. Compound D decreases survivin expression at the translational level. A. Western blot analysis of extracts of cells treated with vehicle (0.5% DMSO) alone, 10 μM or 25 μM Compound D, or 5 μM 5-FU was performed using survivin 6E4 monoclonal antibody (Cell Signaling Technology). B. Survivin immunofluorescence microscopy. Cultured cancer cells were fixed and stained with anti-survivin green. C. Survivin immunofluorescence microscopy. SW480 cells treated with Compound D were fixed and stained with anti-survivin green.



FIG. 15. Compound D activates the caspase 3 activity (but not the caspase 2 activity) via suppression of the survivin expression. Cultured cells with or without transfection of a construct containing the survivin gene were treated with stausporine (0.5 μM), Compound D (2.5 μM or 5.0 μM), or both. The caspase 2 and caspase 3 activities in these cells were measured.



FIG. 16. Compound D promotes cell death via suppression of the survivin expression. Cultured cancer cells with or without transfection of a construct containing the survivin gene were treated with stausporine (0.5 μM), Compound D (5.0 μM), or both. The cell death of these cells was measured.



FIG. 17. Compound D increases the number of cells in G0. Cultured cancer cells with or without transfection of a construct containing the survivin gene were treated with stausporine (0.5 μM), Compound D (5 μM), or both. FACS analysis was performed on these cells and the percentages of cells in G0 are indicated.





DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to conformationally constrained compounds that mimic the secondary structure of reverse-turn regions of biological peptide and proteins (also referred to herein as “reverse-turn mimetics”, and is also directed to chemical libraries relating thereto.


The reverse-turn mimetic structures of the present invention are useful as bioactive agents, including (but not limited to) use as diagnostic, prophylactic and/or therapeutic agents. The reverse-turn mimetic structure libraries of this invention are useful in the identification of bioactive agents having such uses. In the practice of the present invention, the libraries may contain from tens to hundreds to thousands (or greater) of individual reverse-turn structures (also referred to herein as “members”).


In one aspect of the present invention, a reverse-turn mimetic structure is disclosed having the following formula (I):




embedded image



wherein A is —(CHR3)— or —(C═O)—, B is —(CHR4)— or —(C═O)—, D is —(CHR5)— or —(C═O)—, E is —(ZR6)— or —(C═O)—, G is —(XR7)n—, —(CHR7)—(NR8)—, —(C═O)—(XR9)—, or —(C═O)—, W is Y(C═O)—, —(C═O)NH—, —(SO2)— or nothing, Y is oxygen, sulfur, or —NH—, X and Z is independently nitrogen or CH, n=0 or 1; and R1, R2, R3, R4, R5, R6, R7, R8 and R9 are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers thereof.


In one embodiment, R1, R2, R3, R4, R5, R6, R7, R8 and R9 are independently selected from the group consisting of aminoC2-5alkyl, guanidineC2-5alkyl, C1-4alkylguanidinoC2-5alkyl, diC1-4alkylguanidino-C2-5alkyl, amidinoC2-5alkyl, C1-4alkylamidinoC2-5alkyl, diC1-4alkylamidinoC2-5alkyl, C1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl, substituted bis-phenyl methyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, substituted pyridyl, (where the substituents are independently selected from one or more of amino amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC1-4alkyl, substituted pyridylC1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC1-4alkyl, substituted pyrimidylC1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C1-4alkyl, substituted triazin-2-yl-C1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC1-4alkyl, substituted imidazol C1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazolinylC1-4alkyl, N-amidinopiperazinyl-N—C0-4alkyl, hydroxyC2-5alkyl, C1-5alkylaminoC2-5alkyl, hydroxyC2-5alkyl, C1-5alkylaminoC2-5alkyl, C1-5dialkylaminoC2-5alkyl, N-amidinopiperidinylC1-4alkyl and 4-aminocyclohexylC0-2alkyl.


In one embodiment, R1, R2, R6 of E, and R7, R8 and R9 of G are the same or different and represent the remainder of the compound, and R3 of A, R4 of B or R5 of D is selected from an amino acid side chain moiety or derivative thereof. As used herein, the term “remainder of the compound” means any moiety, agent, compound, support, molecule, linker, amino acid, peptide or protein covalently attached to the reverse-turn mimetic structure at R1, R2, R5, R6, R7, R8 and/or R9 positions. This term also includes amino acid side chain moieties and derivatives thereof.


In another embodiment R3 of A, R5 of D, R6 of E, and R7, R8, and R9 of G are the same or different and represent the remainder of the compound, while one or more of, and in one aspect all of, R1, R2 and R4 of B represent an amino acid sidechain. In this case, the term “remainder of the compound” means any moiety, agent, compound, support, molecule, linker, amino acid, peptide or protein covalently attached to the reverse-turn mimetic structure at R3, R5, R6, R7, R8 and/or R9 positions. This term also includes amino acid side chain moieties and derivatives thereof.


As used herein, the term “remainder of the compound” means any moiety, agent, compound, support, molecule, atom, linker, amino acid, peptide or protein covalently attached to the reverse-turn mimetic structure. This term also includes amino acid side chain moieties and derivatives thereof. In one aspect of the invention, any one or more of the R1, R2, R3, R4, R5, R6, R7, R8 and/or R9 positions may represent the remainder of the compound. In one aspect of the invention, one or more of R1, R2 and R4 represents an amino acid side chain moiety or a derivative thereof.


As used herein, the term “amino acid side chain moiety” represents any amino acid side chain moiety present in naturally occurring proteins including (but not limited to) the naturally occurring amino acid side chain moieties identified in Table 1. Other naturally occurring amino acid side chain moieties of this invention include (but are not limited to) the side chain moieties of 3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylysine, γ-carboxyglutamate, phosphotyrosine and phosphoserine. In addition, glycosylated amino acid side chains may also be used in the practice of this invention, including (but not limited to) glycosylated threonine, serine and asparagine.












TABLE 1







Amino Acid Side Chain Moiety
Amino Acid









—H
Glycine



—CH3
Alanine



—CH(CH3)2
Valine



—CH2 CH(CH3)2
Leucine



—CH(CH3)CH2 CH3
Isoleucine



—(CH2)4NH3+
Lysine



—(CH2)3NHC(NH2)NH2+
Arginine









embedded image


Histidine







—CH2COO-
Aspartic acid



—CH2CH2COO-
Glutamic acid



—CH2CONH2
Asparagine



—CH2CH2CONH2
Glutamine









embedded image


Phenylalanine









embedded image


Tyrosine









embedded image


Tryptophan







—CH2SH
Cysteine



—CH2CH2SCH3
Methionine



—CH2OH
Serine



—CH(OH)CH3
Threonine









embedded image


Proline









embedded image


Hydroxyproline










In addition to naturally occurring amino acid side chain moieties, the amino acid side chain moieties of the present invention also include various derivatives thereof. As used herein, a “derivative” of an amino acid side chain moiety includes modifications and/or variations to naturally occurring amino acid side chain moieties. For example, the amino acid side chain moieties of alanine, valine, leucine, isoleucine and phenylalanine may generally be classified as lower chain alkyl, aryl, or arylalkyl moieties. Derivatives of amino acid side chain moieties include other straight chain or branched, cyclic or noncyclic, substituted or unsubstituted, saturated or unsaturated lower chain alkyl, aryl or arylalkyl moieties.


As used herein, “lower chain alkyl moieties” contain from 1-12 carbon atoms, “lower chain aryl moieties” contain from 6-12 carbon atoms and “lower chain aralkyl moieties” contain from 7-12 carbon atoms. Thus, in one embodiment, the amino acid side chain derivative is selected from a C1-12 alkyl, a C6-12 aryl and a C7-12 arylalkyl, and in a more preferred embodiment, from a C1-7 alkyl, a C6-10 aryl and a C7-11 arylalkyl.


Amino side chain derivatives of this invention further include substituted derivatives of lower chain alkyl, aryl, and arylalkyl moieties, wherein the substituent is selected from (but is not limited to) one or more of the following chemical moieties: —OH, —OR, —COOH, —COOR, —CONH2, —NH2, —NHR, —NRR, —SH, —SR, —SO2R, —SO2H, —SOR and halogen (including F, Cl, Br and I), wherein each occurrence of R is independently selected from straight chain or branched, cyclic or noncyclic, substituted or unsubstituted, saturated or unsaturated lower chain alkyl, aryl and aralkyl moieties. Moreover, cyclic lower chain alkyl, aryl and arylalkyl moieties of this invention include naphthalene, as well as heterocyclic compounds such as thiophene, pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine, quinoline, isoquinoline and carbazole. Amino acid side chain derivatives further include heteroalkyl derivatives of the alkyl portion of the lower chain alkyl and aralkyl moieties, including (but not limited to) alkyl and aralkyl phosphonates and silanes.


Representative R1, R2, R3, R4, R5, R6, R7, R8 and R9 moieties specifically include (but are not limited to)-OH, —OR, —COR, —COOR, —CONH2, —CONK, —CONRR, —NH2, —NHR, —NRR, —SO2R and —COSR, wherein each occurrence of R is as defined above.


In a further embodiment, and in addition to being an amino acid side chain moiety or derivative thereof (or the remainder of the compound in the case of R1, R2, R3, R5, R6, R7, R8 and R9), R1, R2, R3, R4, R5, R6, R7, R8 or R9 may be a linker facilitating the linkage of the compound to another moiety or compound. For example, the compounds of this invention may be linked to one or more known compounds, such as biotin, for use in diagnostic or screening assay. Furthermore, R1, R2, R3, R4, R5, R6, R7, R8 or R9 may be a linker joining the compound to a solid support (such as a support used in solid phase peptide synthesis) or alternatively, may be the support itself. In this embodiment, linkage to another moiety or compound, or to a solid support, is preferable at the R1, R2, R7 or R8, or R9 position, and more preferably at the R1 or R2 position.


In the embodiment wherein A is —(CHR3)—, B is —(C═O)—, D is —(CHR5)—, E is —(C═O)—, and G is —(XR7)n—, the reverse turn mimetic compound of this invention has the following formula (II):




embedded image



wherein R1, R2, R3, R5, R7, W, X and n are as defined above. In a preferred embodiment, R1, R2 and R7 represent the remainder of the compound, and R3 or R5 is selected from an amino acid side chain moiety.


In the embodiment wherein A is —(C═O)—, B is —(CHR4)—, D is —(C═O)—, E is —(ZR6)—, G is —(C═O)—(XR9)—, the reverse turn mimetic compound of this invention has the following general formula (III):




embedded image



wherein R1, R2, R4, R6, R9, W and X are as defined above, Z is nitrogen or CH (when Z is CH, then X is nitrogen). In a preferred embodiment, R1, R2, R6 and R9 represent the remainder of the compound, and R4 is selected from an amino acid side chain moiety.


In a more specific embodiment wherein A is —(C═O)—, B is —(CHR4)—, D is —(C═O)—, E is —(ZR6)—, and G is —(XR7)n—, the reverse turn mimetic compound of this invention has the following formula (IV):




embedded image



wherein R1, R2, R4, R6, R7, W, X and n are as defined above, and Z is nitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen and n is not zero). In a preferred embodiment, R1, R2, R6 and R7 represent the remainder of the compound, and R4 is selected from an amino acid side chain moiety. In one aspect, R6 or R7 is selected from an amino acid side chain moiety when Z and X are both CH.


These compounds may be prepared by utilizing appropriate starting component molecules (hereinafter referred to as “component pieces”). Briefly, in the synthesis of reverse-turn mimetic structures having formula (I), first and second component pieces are coupled to form a combined first-second intermediate, if necessary, third and/or fourth component pieces are coupled to form a combined third-fourth intermediate (or, if commercially available, a single third intermediate may be used), the combined first-second intermediate and third-fourth intermediate (or third intermediate) are then coupled to provide a first-second-third-fourth intermediate (or first-second-third intermediate) which is cyclized to yield the reverse-turn mimetic structures of this invention. Alternatively, the reverse-turn mimetic structures of formula (I) may be prepared by sequential coupling of the individual component pieces either stepwise in solution or by solid phase synthesis as commonly practiced in solid phase peptide synthesis.


Specific component pieces and the assembly thereof to prepare compounds of the present invention are illustrated in FIG. 1. For example, a “first component piece” may have the following formula S1:




embedded image



wherein R2 is as defined above, and R is a protective group suitable for use in peptide synthesis, where this protection group may be joined to a polymeric support to enable solid-phase synthesis. Suitable R groups include alkyl groups and, in a preferred embodiment, R is a methyl group. In FIG. 1, one of the R groups is a polymeric (solid) support, indicated by “Pol” in the FIG. Such first component pieces may be readily synthesized by reductive amination of H2N—R2 with CH(OR)2—CHO, or by a displacement reaction between H2N—R2 and CH(OR)2—CH2-LG (wherein LG refers to a leaving group, e.g., a halogen (Hal) group).


A “second component piece” may have the following formula S2:




embedded image



where P is an amino protection group suitable for use in peptide synthesis, L1 is hydroxyl or a carboxyl-activation group, and R4 is as defined above. Preferred protection groups include t-butyl dimethylsilyl (TBDMS), t-butyloxycarbonyl (BOC), methyloxycarbonyl (MOC), 9H-fluorenylmethyloxycarbonyl (FMOC), and allyloxycarbonyl (Alloc). N-Protected amino acids are commercially available; for example, FMOC amino acids are available from a variety of sources. In order for the second component piece to be reactive with the first component piece, L1 is a carboxyl-activation group, and the conversion of carboxyl groups to activated carboxyl groups may be readily achieved by methods known in the art for the activation of carboxyl groups. Suitable activated carboxylic acid groups include acid halides where L1 is a halide such as chloride or bromide, acid anhydrides where L1 is an acyl group such as acetyl, reactive esters such as an N-hydroxysuccinimide esters and pentafluorophenyl esters, and other activated intermediates such as the active intermediate formed in a coupling reaction using a carbodiimide such as dicyclohexylcarbodiimide (DCC). Accordingly, commercially available N-protected amino acids may be converted to carboxylic activated forms by means known to one of skill in the art.


In the case of the azido derivative of an amino acid serving as the second component piece, such compounds may be prepared from the corresponding amino acid by the reaction disclosed by Zaloom et al. (J. Org. Chem. 46:5173-76, 1981).


Alternatively, the first component piece of the invention may have the following formula S1′:




embedded image



wherein R is as defined above and L2 is a leaving group such as halogen atom or tosyl group, and the second component piece of the invention may have the following formula S2′:




embedded image



wherein R2, R4 and P are as defined above,


A “third component piece” of this invention may have the following formula S3:




embedded image



where G, E, L1 and L2 are as defined above. Suitable third component pieces are commercially available from a variety of sources or can be prepared by methods well known in organic chemistry.


In FIG. 1, the compound of formula (1) has —(C═O)— for A, —(CHR4)— for B, —(C═O)— for D, and —(CR6)— for E. Compounds of formula (1) wherein a carbonyl group is at position B and an R group is at position B, i.e., compounds wherein A is —(CHR3)— and B is —(C═O)—, may be prepared in a manner analogous to that shown in FIG. 1, as illustrated in FIG. 2. FIG. 2 also illustrates adding a fourth component piece to the first-second-third component intermediate, rather than attaching the fourth component piece to the third component piece prior to reaction with the first-second intermediate piece. In addition, FIG. 2 illustrates the preparation of compounds of the present invention wherein D is —(CHR5)— (rather than —(C═O)— as in FIG. 1), and E is —(C═O)— (rather than —(CHR6)— as in FIG. 1). Finally, FIG. 2 illustrates the preparation of compounds wherein G is NR7.


Thus, as illustrated above, the reverse-turn mimetic compounds of formula (I) may be synthesized by reacting a first component piece with a second component piece to yield a combined first-second intermediate, followed by reacting the combined first-second intermediate with third component pieces sequentially to provide a combined first-second-third-fourth intermediate, and then cyclizing this intermediate to yield the reverse-turn mimetic structure.


The syntheses of representative component pieces of this invention are described in Preparation Examples and working Examples.


The reverse-turn mimetic structures of formula (III) and (IV) may be made by techniques analogous to the modular component synthesis disclosed above, but with appropriate modifications to the component pieces.


The reverse-turn mimetic structures of the present invention are useful as bioactive agents, such as diagnostic, prophylactic, and therapeutic agents. For example, the reverse-turn mimetic structures of the present invention may be used for modulating a cell signaling transcription factor related peptides in a warm-blooded animal, by a method comprising administering to the animal an effective amount of the compound of formula (I).


Further, the reverse-turn mimetic structures of the present invention may also be effective for inhibiting peptide binding to PTB domains in a warm-blooded animal; for modulating G protein coupled receptor (GPCR) and ion channel in a warm-blooded animal; for modulating cytokines in a warm-blooded animal.


Meanwhile, it has been found that the compounds of the formula (I), especially compounds of formula (VI) are effective for inhibiting or treating disorders modulated by Wnt-signaling pathway, such as cancer, especially colorectal cancer.




embedded image



wherein Ra is a phenyl group; a substituted phenyl group having one or more substituents wherein the one or more substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl, and hydroxyl groups; a benzyl group; a substituted benzyl group with one or more substituents where the one or more substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidazonyl, C1-4alkylamino, C1-4dialkylamino, halogen, perfluoro C1-4alkyl, C1-3alkoxy, nitro, carboxy, cyano, sulfuryl, and hydroxyl group; or a bicyclic aryl group having 8 to 11 ring members, which may have 1 to 3 heteroatoms selected from nitrogen, oxygen or sulfur; Rb is a monocyclic aryl group having 5 to 7 ring members, which may have 1 to 2 heteroatoms selected from nitrogen, oxygen or sulfur, and aryl ring in the compound may have one or more substituents selected from a group consisting of halide, hydroxy, cyano, lower alkyl, and lower alkoxy groups; Rc is a saturated or unsaturated C1-6alkyl, C1-6alkoxy, perfluoro C1-6alkyl group; and X1, X2, and X3 may be the same or different and independently selected from hydrogen, hydroxyl, and halide.


In another aspect, it is an object of the present invention to provide a pharmaceutical composition comprising a safe and effective amount of the compound having general formula (VI) and pharmaceutically acceptable carrier, which can be used for treatment of disorders modulated by Wnt signaling pathway, especially by TCF4-β-catenin-CBP complex.


Further, the present invention is to provide a method for inhibiting the growth of tumor cells by using the above-described composition of the present invention; a method for inducing apoptosis of tumor cells by using the above-described composition of the present invention; a method for treating a disorder modulated by TCF4-β-catenin-CBP complex by using the above-described composition of the present invention; and a method of treating cancer such as colorectal cancer by administering the composition of the present invention together with other anti-cancer agent such as 5-fluorouracil (5-FU), taxol, cisplatin, mitomycin C, tegafur, raltitrexed, capecitabine, and irinotecan, etc.


In a preferred embodiment of the present invention, the compound of the present invention has a (6S,10R)-configuration as follows:




embedded image



wherein Ra and Rb have the same meanings as defined above.


In another aspect of this invention, libraries containing reverse-turn mimetic structures of the present invention are disclosed. Once assembled, the libraries of the present invention may be screened to identify individual members having bioactivity. Such screening of the libraries for bioactive members may involve; for example, evaluating the binding activity of the members of the library or evaluating the effect the library members have on a functional assay. Screening is normally accomplished by contacting the library members (or a subset of library members) with a target of interest, such as, for example, an antibody, enzyme, receptor or cell line. Library members which are capable of interacting with the target of interest, are referred to herein as “bioactive library members” or “bioactive mimetics”. For example, a bioactive mimetic may be a library member which is capable of binding to an antibody or receptor, or which is capable of inhibiting an enzyme, or which is capable of eliciting or antagonizing a functional response associated, for example, with a cell line. In other words, the screening of the libraries of the present invention determines which library members are capable of interacting with one or more biological targets of interest. Furthermore, when interaction does occur, the bioactive mimetic (or mimetics) may then be identified from the library members. The identification of a single (or limited number) of bioactive mimetic(s) from the library yields reverse-turn mimetic structures which are themselves biologically active, and thus are useful as diagnostic, prophylactic or therapeutic agents, and may further be used to significantly advance identification of lead compounds in these fields.


Synthesis of the peptide mimetics of the library of the present invention may be accomplished using known peptide synthesis techniques, in combination with the first, second and third component pieces of this invention. More specifically, any amino acid sequence may be added to the N-terminal and/or C-terminal of the conformationally constrained reverse-turn mimetic. To this end, the mimetics may be synthesized on a solid support (such as PAM resin) by known techniques (see, e.g., John M. Stewart and Janis D. Young, Solid Phase Peptide Synthesis, 1984, Pierce Chemical Comp., Rockford, Ill.) or on a silyl-linked resin by alcohol attachment (see Randolph et al., J. Am Chem. Soc. 117:5712-14, 1995).


In addition, a combination of both solution and solid phase synthesis techniques may be utilized to synthesize the peptide mimetics of this invention. For example, a solid support may be utilized to synthesize the linear peptide sequence up to the point that the conformationally constrained reverse-turn is added to the sequence. A suitable conformationally constrained reverse-turn mimetic structure which has been previously synthesized by solution synthesis techniques may then be added as the next “amino acid” to the solid phase synthesis (i.e., the conformationally constrained reverse-turn mimetic, which has both an N-terminus and a C-terminus, may be utilized as the next amino acid to be added to the linear peptide). Upon incorporation of the conformationally constrained reverse-turn mimetic structures into the sequence, additional amino acids may then be added to complete the peptide bound to the solid support. Alternatively, the linear N-terminus and C-terminus protected peptide sequences may be synthesized on a solid support, removed from the support, and then coupled to the conformationally constrained reverse-turn mimetic structures in solution using known solution coupling techniques.


In another aspect of this invention, methods for constructing the libraries are disclosed. Traditional combinatorial chemistry techniques (see, e.g., Gallop et al., J. Med. Chem. 37:1233-1251, 1994) permit a vast number of compounds to be rapidly prepared by the sequential combination of reagents to a basic molecular scaffold. Combinatorial techniques have been used to construct peptide libraries derived from the naturally occurring amino acids. For example, by taking 20 mixtures of 20 suitably protected and different amino acids and coupling each with one of the 20 amino acids, a library of 400 (i.e., 202) dipeptides is created. Repeating the procedure seven times results in the preparation of a peptide library comprised of about 26 billion (i.e., 208) octapeptides.


Specifically, synthesis of the peptide mimetics of the library of the present invention may be accomplished using known peptide synthesis techniques, for example, the General Scheme of [4,4,0] Reverse-Turn Mimetic Library as follows:




embedded image


Synthesis of the peptide mimetics of the libraries of the present invention was accomplished using a FlexChem Reactor Block which has 96 well plates by known techniques. In the above scheme ‘Pol’ represents a bromoacetal resin (Advanced ChemTech) and detailed procedure is illustrated below.


Step 1


A bromoacetal resin (37 mg, 0.98 mmol/g) and a solution of R2-amine in DMSO (1.4 mL) were placed in a Robbins block (FlexChem) having 96 well plates. The reaction mixture was shaken at 60° C. using a rotating oven [Robbins Scientific] for 12 hours. The resin was washed with DMF, MeOH, and then DCM


Step 2


A solution of commercial available FmocAmino Acids (4 equiv.), PyBob (4 equiv.), HOAt (4 equiv.), and DIEA (12 equiv.) in DMF was added to the resin. After the reaction mixture was shaken for 12 hours at room temperature, the resin was washed with DMF, MeOH, and then DCM.


Step 3


To the resin swollen by DMF before reaction was added 25% piperidine in DMF and the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and the resin was washed with DMF, Methanol, and then DCM. A solution of hydrazine acid (4 equiv.), HOBt (4 equiv.), and DIC (4 equiv.) in DMF was added to the resin and the reaction mixture was shaken for 12 hours at room temperature. The resin was washed with DMF, MeOH, and then DCM.


Step 4a (Where Hydrazine Acid is MOC Carbamate)


The resin obtained in Step 3 was treated with formic acid (1.2 mL each well) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under a reduced pressure using SpeedVac [SAVANT] to give the product as oil. The product was diluted with 50% water/acetonitrile and then lyophilized after freezing.


Step 4b (Where Fmoc Hydrazine Acid is Used to Make Urea Through Isocyanate)


To the resin swollen by DMF before reaction was added 25% piperidine in DMF and the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and the resin was washed with DMF, Methanol, then DCM. To the resin swollen by DCM before reaction was added isocyanate (5 equiv.) in DCM. After the reaction mixture was shaken for 12 hours at room temperature the resin was washed with DMF, MeOH, then DCM. The resin was treated with formic acid (1.2 mL each well) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under a reduced pressure using SpeedVac [SAVANT] to give the product as oil. The product was diluted with 50% water/acetonitrile and then lyophilized after freezing.


Step 4c (Where Fmoc-Hydrazine Acid is Used to Make Urea Through Active Carbamate)


To the resin swollen by DMF before reaction was added 25% piperidine in DMF and the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and the resin was washed with DMF, MeOH, and then DCM. To the resin swollen by DCM before reaction was added p-nitrophenyl chloroformate (5 equiv.) and diisopropyl ethylamine (5 equiv.) in DCM. After the reaction mixture was shaken for 12 hours at room temperature, the resin was washed with DMF, MeOH, and then DCM. To the resin was added primary amines in DCM for 12 hours at room temperature and the resin was washed with DMF, MeOH, and then DCM. After reaction the resin was treated with formic acid (1.2 mL each well) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under a reduced pressure using SpeedVac [SAVANT] to give the product as oil. The product was diluted with 50% water/acetonitrile and then lyophilized after freezing.


To generate these block libraries the key intermediate hydrazine acids were synthesized according to the procedure illustrated in Preparation Examples.


Tables 2A and 2B show a [4,4,0] Reverse turn mimetics library which can be prepared according to the present invention, of which representative preparation is given in Example 4.









TABLE 2A







THE [4,4,0] REVERSE TURN MIMETICS LIBRARY




embedded image


















No
R2
R4
R7
R1—Y′
Mol. Weight
M + H
















1
2,4-Cl2-benzyl
4-HO-benzyl
Allyl
OCH3
533
534


2
2,4-Cl2-benzyl
4-NO2-benzyl
Allyl
OCH3
562
563


3
2,4-Cl2-benzyl
2,4-F2-benzyl
Allyl
OCH3
553
554


4
2,4-Cl2-benzyl
4-Cl-benzyl
Allyl
OCH3
552
553


5
2,4-Cl2-benzyl
2,2-bisphenylethyl
Allyl
OCH3
594
595


6
2,4-Cl2-benzyl
3-t-Bu-4-HO-benzyl
Allyl
OCH3
590
591


7
2,4-Cl2-benzyl
4-Me-benzyl
Allyl
OCH3
531
532


8
2,4-Cl2-benzyl
Cyclohexylmethyl
Allyl
OCH3
523
524


9
2,4-Cl2-benzyl
4-F-benzyl
Allyl
OCH3
535
536


10
2,4-Cl2-benzyl
2-Cl-benzyl
Allyl
OCH3
552
553


11
2,4-Cl2-benzyl
2,4-Cl2-benzyl
Allyl
OCH3
586
587


12
2,4-Cl2-benzyl
Naphth-2-ylmethyl
Allyl
OCH3
567
568


13
2,4-Cl2-benzyl
4-HO-benzyl
Benzyl
OCH3
583
584


14
2,4-Cl2-benzyl
4-NO2-benzyl
Benzyl
OCH3
612
613


15
2,4-Cl2-benzyl
2,4-F2-benzyl
Benzyl
OCH3
603
604


16
2,4-Cl2-benzyl
4-Cl-benzyl
Benzyl
OCH3
602
603


17
2,4-Cl2-benzyl
2,2-bisphenylethyl
Benzyl
OCH3
644
645


18
2,4-Cl2-benzyl
3-t-Bu-4-HO-benzyl
Benzyl
OCH3
640
641


19
2,4-Cl2-benzyl
4-Me-benzyl
Benzyl
OCH3
582
583


20
2,4-Cl2-benzyl
Cyclohexylmethyl
Benzyl
OCH3
574
575


21
2,4-Cl2-benzyl
4-F-benzyl
Benzyl
OCH3
585
586


22
2,4-Cl2-benzyl
2-Cl-benzyl
Benzyl
OCH3
602
603


23
2,4-Cl2-benzyl
2,4-Cl2-benzyl
Benzyl
OCH3
636
637


24
2,4-Cl2-benzyl
Naphth-2-ylmethyl
Benzyl
OCH3
618
619


25
2,4-Cl2-benzyl
4-HO-benzyl
Allyl
OCH3
479
480


26
2,4-Cl2-benzyl
4-NO2-benzyl
Allyl
OCH3
508
509


27
2,4-Cl2-benzyl
2,4-F2-benzyl
Allyl
OCH3
499
500


28
2,4-Cl2-benzyl
4-Cl-benzyl
Allyl
OCH3
497
498


29
Phenethyl
2,2-bisphenylethyl
Allyl
OCH3
539
540


30
Phenethyl
3-t-Bu-4-HO-benzyl
Allyl
OCH3
535
536


31
Phenethyl
4-Me-benzyl
Allyl
OCH3
477
478


32
Phenethyl
Cyclohexylmethyl
Allyl
OCH3
469
470


33
Phenethyl
4-F-benzyl
Allyl
OCH3
481
482


34
Phenethyl
2-Cl-benzyl
Allyl
OCH3
497
498


35
Phenethyl
2,4-Cl2-benzyl
Allyl
OCH3
531
532


36
Phenethyl
Naphth-2-ylmethyl
Allyl
OCH3
513
514


37
Phenethyl
4-HO-benzyl
Benzyl
OCH3
529
530


38
Phenethyl
4-NO2-benzyl
Benzyl
OCH3
558
559


39
Phenethyl
2,4-F2-benzyl
Benzyl
OCH3
549
550


40
Phenethyl
4-Cl-benzyl
Benzyl
OCH3
547
548


41
Phenethyl
2,2-bisphenylethyl
Benzyl
OCH3
589
590


42
Phenethyl
3-t-Bu-4-HO-benzyl
Benzyl
OCH3
585
586


43
Phenethyl
4-Me-benzyl
Benzyl
OCH3
527
528


44
Phenethyl
Cyclohexyl-methyl
Benzyl
OCH3
519
520


45
Phenethyl
4-F-benzyl
Benzyl
OCH3
531
532


46
Phenethyl
2-Cl-benzyl
Benzyl
OCH3
547
548


47
Phenethyl
2,4-Cl2-benzyl
Benzyl
OCH3
582
583


48
Phenethyl
Naphth-2-ylmethyl
Benzyl
OCH3
563
564


49
Phenethyl
4-HO-benzyl
Allyl
OCH3
497
498


50
Phenethyl
4-NO2-benzyl
Allyl
OCH3
526
527


51
Phenethyl
2,4-F2-benzyl
Allyl
OCH3
517
518


52
Phenethyl
4-Cl-benzyl
Allyl
OCH3
515
516


53
4-F-phenylethyl
2,2-bisphenylethyl
Allyl
OCH3
557
558


54
4-F-phenylethyl
3-t-Bu-4-HO-benzyl
Allyl
OCH3
553
554


55
4-F-phenylethyl
4-Me-benzyl
Allyl
OCH3
495
496


56
4-F-phenylethyl
Cyclohexyl-methyl
Allyl
OCH3
487
488


57
4-F-phenylethyl
4-F-benzyl
Allyl
OCH3
499
500


58
4-F-phenylethyl
2-Cl-benzyl
Allyl
OCH3
515
516


59
4-F-phenylethyl
2,4-Cl2-benzyl
Allyl
OCH3
549
550


60
4-F-phenylethyl
Naphth-2-ylmethyl
Allyl
OCH3
531
532


61
4-F-phenylethyl
4-HO-benzyl
Benzyl
OCH3
547
548


62
4-F-phenylethyl
4-NO2-benzyl
Benzyl
OCH3
576
577


63
4-F-phenylethyl
2,4-F2-benzyl
Benzyl
OCH3
567
568


64
4-F-phenylethyl
4-Cl-benzyl
Benzyl
OCH3
565
566


65
4-F-phenylethyl
2,2-bisphenylethyl
Benzyl
OCH3
607
608


66
4-F-phenylethyl
3-t-Bu-4-HO-benzyl
Benzyl
OCH3
603
604


67
4-F-phenylethyl
4-Me-benzyl
Benzyl
OCH3
545
546


68
4-F-phenylethyl
Cyclohexyl-methyl
Benzyl
OCH3
537
538


69
4-F-phenylethyl
4-F-benzyl
Benzyl
OCH3
549
550


70
4-F-phenylethyl
2-Cl-benzyl
Benzyl
OCH3
565
566


71
4-F-phenylethyl
2,4-Cl2-benzyl
Benzyl
OCH3
599
600


72
4-F-phenylethyl
Naphth-2-ylmethyl
Benzyl
OCH3
581
582


73
4-F-phenylethyl
4-HO-benzyl
Allyl
OCH3
509
510


74
4-F-phenylethyl
4-NO2-benzyl
Allyl
OCH3
538
539


75
4-F-phenylethyl
2,4-F2-benzyl
Allyl
OCH3
529
530


76
4-F-phenylethyl
4-Cl-benzyl
Allyl
OCH3
527
528


77
4-MeO-phenylethyl
2,2-bisphenylethyl
Allyl
OCH3
569
570


78
4-MeO-phenylethyl
3-t-Bu-4-HO-benzyl
Allyl
OCH3
565
566


79
4-MeO-phenylethyl
4-Me-benzyl
Allyl
OCH3
507
508


80
4-MeO-phenylethyl
Cyclohexyl-methyl
Allyl
OCH3
499
500


81
4-MeO-phenylethyl
4-F-benzyl
Allyl
OCH3
511
512


82
4-MeO-phenylethyl
2-Cl-benzyl
Allyl
OCH3
527
528


83
4-MeO-phenylethyl
2,4-Cl2-benzyl
Allyl
OCH3
561
562


84
4-MeO-phenylethyl
Naphth-2-ylmethyl
Allyl
OCH3
543
544


85
4-MeO-phenylethyl
4-HO-benzyl
Benzyl
OCH3
559
560


86
4-MeO-phenylethyl
4-NO2-benzyl
Benzyl
OCH3
588
589


87
4-MeO-phenylethyl
2,4-F2-benzyl
Benzyl
OCH3
579
580


88
4-MeO-phenylethyl
4-Cl-benzyl
Benzyl
OCH3
577
578


89
4-MeO-phenylethyl
2,2-bisphenylethyl
Benzyl
OCH3
619
620


90
4-MeO-phenylethyl
3-t-Bu-4-HO-benzyl
Benzyl
OCH3
615
616


91
4-MeO-phenylethyl
4-Me-benzyl
Benzyl
OCH3
557
558


92
4-MeO-phenylethyl
Cyclohexylmethyl
Benzyl
OCH3
549
550


93
4-MeO-phenylethyl
4-F-benzyl
Benzyl
OCH3
561
562


94
4-MeO-phenylethyl
2-Cl-benzyl
Benzyl
OCH3
577
578


95
4-MeO-phenylethyl
2,4-Cl2-benzyl
Benzyl
OCH3
612
613


96
4-MeO-phenylethyl
Naphth-2-ylmethyl
Benzyl
OCH3
593
594


97
Isoamyl
4-HO-benzyl
Styrylmethyl
OCH3
521
522


98
Isoamyl
4-NO2-benzyl
Styrylmethyl
OCH3
550
551


99
Isoamyl
2,4-F2-benzyl
Styrylmethyl
OCH3
541
542


100
Isoamyl
4-Cl-benzyl
Styrylmethyl
OCH3
539
540


101
Isoamyl
2,2-bisphenylethyl
Styrylmethyl
OCH3
581
582


102
Isoamyl
3-t-Bu-4-HO-benzyl
Styrylmethyl
OCH3
497
498


103
Isoamyl
4-Me-benzyl
Styrylmethyl
OCH3
519
520


104
Isoamyl
Cyclohexylmethyl
Styrylmethyl
OCH3
511
512


105
Isoamyl
4-F-benzyl
Styrylmethyl
OCH3
523
524


106
Isoamyl
2-Cl-benzyl
Styrylmethyl
OCH3
539
540


107
Isoamyl
2,4-Cl2-benzyl
Styrylmethyl
OCH3
574
575


108
Isoamyl
Naphth-2-ylmethyl
Styrylmethyl
OCH3
555
556


109
Isoamyl
4-HO-benzyl
2,6-Cl2-benzyl
OCH3
563
564


110
Isoamyl
4-NO2-benzyl
2,6-Cl2-benzyl
OCH3
592
593


111
Isoamyl
2,4-F2-benzyl
2,6-Cl2-benzyl
OCH3
583
584


112
Isoamyl
4-Cl-benzyl
2,6-Cl2-benzyl
OCH3
582
583


113
Isoamyl
2,2-bisphenylethyl
2,6-Cl2-benzyl
OCH3
624
625


114
Isoamyl
3-t-Bu-4-HO-benzyl
2,6-Cl2-benzyl
OCH3
540
541


115
Isoamyl
4-Me-benzyl
2,6-Cl2-benzyl
OCH3
562
563


116
Isoamyl
Cyclohexylmethyl
2,6-Cl2-benzyl
OCH3
554
555


117
Isoamyl
4-F-benzyl
2,6-Cl2-benzyl
OCH3
565
566


118
Isoamyl
2-Cl-benzyl
2,6-Cl2-benzyl
OCH3
582
583


119
Isoamyl
2,4-Cl2-benzyl
2,6-Cl2-benzyl
OCH3
616
617


120
Isoamyl
Naphth-2-ylmethyl
2,6-Cl2-benzyl
OCH3
598
599


121
3-MeO-propyl
4-HO-benzyl
Styrylmethyl
OCH3
523
524


122
3-MeO-propyl
4-NO2-benzyl
Styrylmethyl
OCH3
552
553


123
3-MeO-propyl
2,4-F2-benzyl
Styrylmethyl
OCH3
543
544


124
3-MeO-propyl
4-Cl-benzyl
Styrylmethyl
OCH3
541
542


125
3-MeO-propyl
2,2-bisphenylethyl
Styrylmethyl
OCH3
583
584


126
3-MeO-propyl
3-t-Bu-4-HO-benzyl
Styrylmethyl
OCH3
499
500


127
3-MeO-propyl
4-Me-benzyl
Styrylmethyl
OCH3
521
522


128
3-MeO-propyl
Cyclohexyl-methyl
Styrylmethyl
OCH3
513
514


129
3-MeO-propyl
4-F-benzyl
Styrylmethyl
OCH3
525
526


130
3-MeO-propyl
2-Cl-benzyl
Styrylmethyl
OCH3
541
542


131
3-MeO-propyl
2,4-Cl2-benzyl
Styrylmethyl
OCH3
575
576


132
3-MeO-propyl
Naphth-2-ylmethyl
Styrylmethyl
OCH3
557
558


133
3-MeO-propyl
4-HO-benzyl
2,6-Cl2-benzyl
OCH3
565
566


134
3-MeO-propyl
4-NO2-benzyl
2,6-Cl2-benzyl
OCH3
594
595


135
3-MeO-propyl
2,4-F2-benzyl
2,6-Cl2-benzyl
OCH3
585
586


136
3-MeO-propyl
4-Cl-benzyl
2,6-Cl2-benzyl
OCH3
584
585


137
3-MeO-propyl
2,2-bisphenylethyl
2,6-Cl2-benzyl
OCH3
626
627


138
3-MeO-propyl
3-t-Bu-4-HO-benzyl
2,6-Cl2-benzyl
OCH3
541
542


139
3-MeO-propyl
4-Me-benzyl
2,6-Cl2-benzyl
OCH3
563
564


140
3-MeO-propyl
Cyclohexyl-methyl
2,6-Cl2-benzyl
OCH3
556
557


141
3-MeO-propyl
4-F-benzyl
2,6-Cl2-benzyl
OCH3
567
568


142
3-MeO-propyl
2-Cl-benzyl
2,6-Cl2-benzyl
OCH3
584
585


143
3-MeO-propyl
2,4-Cl2-benzyl
2,6-Cl2-benzyl
OCH3
618
619


144
3-MeO-propyl
Naphth-2-ylmethyl
2,6-Cl2-benzyl
OCH3
600
601


145
4-MeO-phenylethyl
4-HO-benzyl
Styrylmethyl
OCH3
585
586


146
4-MeO-phenylethyl
4-NO2-benzyl
Styrylmethyl
OCH3
614
615


147
4-MeO-phenylethyl
2,4-F2-benzyl
Styrylmethyl
OCH3
605
606


148
4-MeO-phenylethyl
4-Cl-benzyl
Styrylmethyl
OCH3
603
604


149
4-MeO-phenylethyl
2,2-bisphenylethyl
Styrylmethyl
OCH3
645
646


150
4-MeO-phenylethyl
3-t-Bu-4-HO-benzyl
Styrylmethyl
OCH3
561
562


151
4-MeO-phenylethyl
4-Me-benzyl
Styrylmethyl
OCH3
583
584


152
4-MeO-phenylethyl
Cyclohexyl-methyl
Styrylmethyl
OCH3
575
576


153
4-MeO-phenylethyl
4-F-benzyl
Styrylmethyl
OCH3
587
588


154
4-MeO-phenylethyl
2-Cl-benzyl
Styrylmethyl
OCH3
603
604


155
4-MeO-phenylethyl
2,4-Cl2-benzyl
Styrylmethyl
OCH3
638
639


156
4-MeO-phenylethyl
Naphth-2-ylmethyl
Styrylmethyl
OCH3
619
620


157
4-MeO-phenylethyl
4-HO-benzyl
2,6-Cl2-benzyl
OCH3
628
629


158
4-MeO-phenylethyl
4-NO2-benzyl
2,6-Cl2-benzyl
OCH3
657
658


159
4-MeO-phenylethyl
2,4-F2-benzyl
2,6-Cl2-benzyl
OCH3
648
649


160
4-MeO-phenylethyl
4-Cl-benzyl
2,6-Cl2-benzyl
OCH3
646
647


161
4-MeO-phenylethyl
2,2-bisphenylethyl
2,6-Cl2-benzyl
OCH3
688
689


162
4-MeO-phenylethyl
3-t-Bu-4-HO-benzyl
2,6-Cl2-benzyl
OCH3
604
605


163
4-MeO-phenylethyl
4-Me-benzyl
2,6-Cl2-benzyl
OCH3
626
627


164
4-MeO-phenylethyl
Cyclohexylmethyl
2,6-Cl2-benzyl
OCH3
618
619


165
4-MeO-phenylethyl
4-F-benzyl
2,6-Cl2-benzyl
OCH3
630
631


166
4-MeO-phenylethyl
2-Cl-benzyl
2,6-Cl2-benzyl
OCH3
646
647


167
4-MeO-phenylethyl
2,4-Cl2-benzyl
2,6-Cl2-benzyl
OCH3
680
681


168
4-MeO-phenylethyl
Naphth-2-ylmethyl
2,6-Cl2-benzyl
OCH3
662
663


169
Tetrahydrofuran-2-
4-HO-benzyl
Styrylmethyl
OCH3
535
536



ylmethyl







170
Tetrahydrofuran-2-
4-NO2-benzyl
Styrylmethyl
OCH3
564
565



ylmethyl







171
Tetrahydrofuran-2-
2,4-F2-benzyl
Styrylmethyl
OCH3
555
556



ylmethyl







172
Tetrahydrofuran-2-
4-Cl-benzyl
Styrylmethyl
OCH3
553
554



ylmethyl







173
Tetrahydrofuran-2-
2,2-bisphenylethyl
Styrylmethyl
OCH3
595
596



ylmethyl







174
Tetrahydrofuran-2-
3-t-Bu-4-HO-benzyl
Styrylmethyl
OCH3
511
512



ylmethyl







175
Tetrahydrofuran-2-
4-Me-benzyl
Styrylmethyl
OCH3
533
534



ylmethyl







176
Tetrahydrofuran-2-
Cyclohexyl-methyl
Styrylmethyl
OCH3
525
526



ylmethyl







177
Tetrahydrofuran-2-
4-F-benzyl
Styrylmethyl
OCH3
537
538



ylmethyl







178
Tetrahydrofuran-2-
2-Cl-benzyl
Styrylmethyl
OCH3
553
554



ylmethyl







179
Tetrahydrofuran-2-
2,4-Cl2-benzyl
Styrylmethyl
OCH3
588
589



ylmethyl







180
Tetrahydrofuran-2-
Naphth-2-ylmethyl
Styrylmethyl
OCH3
569
570



ylmethyl







181
Tetrahydrofuran-2-
4-HO-benzyl
2,6-Cl2-benzyl
OCH3
577
578



ylmethyl







182
Tetrahydrofuran-2-
4-NO2-benzyl
2,6-Cl2-benzyl
OCH3
606
607



ylmethyl







183
Tetrahydrofuran-2-
2,4-F2-benzyl
2,6-Cl2-benzyl
OCH3
597
598



ylmethyl







184
Tetrahydrofuran-2-
4-Cl-benzyl
2,6-Cl2-benzyl
OCH3
596
597



ylmethyl







185
Tetrahydrofuran-2-
2,2-bisphenylethyl
2,6-Cl2-benzyl
OCH3
638
639



ylmethyl







186
Tetrahydrofuran-2-
3-t-Bu-4-HO-benzyl
2,6-Cl2-benzyl
OCH3
553
554



ylmethyl







187
Tetrahydrofuran-2-
4-Me-benzyl
2,6-Cl2-benzyl
OCH3
575
576



ylmethyl







188
Tetrahydrofuran-2-
Cyclohexyl-methyl
2,6-Cl2-benzyl
OCH3
568
569



ylmethyl







189
Tetrahydrofuran-2-
4-F-benzyl
2,6-Cl2-benzyl
OCH3
579
580



ylmethyl







190
Tetrahydrofuran-2-
2-Cl-benzyl
2,6-Cl2-benzyl
OCH3
596
597



ylmethyl







191
Tetrahydrofuran-2-
2,4-Cl2-benzyl
2,6-Cl2-benzyl
OCH3
630
631



ylmethyl







192
Tetrahydrofuran-2-
Naphth-2-ylmethyl
2,6-Cl2-benzyl
OCH3
612
613



ylmethyl







193
Phenethyl
4-HO-benzyl
Methyl
(4-Me-phenyl)amino
528
529


194
Phenethyl
4-HO-benzyl
Methyl
(4-Cl-phenyl)amino
548
549


195
Phenethyl
4-HO-benzyl
Methyl
Phenylamino
514
515


196
Phenethyl
4-HO-benzyl
Methyl
((R)-a-
542
543






methylbenzyl)amino




197
Phenethyl
4-HO-benzyl
Methyl
Benzylamino
528
529


198
Phenethyl
4-HO-benzyl
Methyl
(4-MeO-phenyl)amino
544
545


199
Phenethyl
4-HO-benzyl
Methyl
(4-Br-phenyl)amino
592
593


200
Phenethyl
4-HO-benzyl
Methyl
(4-CF3-phenyl)amino
582
583


201
Phenethyl
4-HO-benzyl
Methyl
Pentylamino
508
509


202
Phenethyl
4-HO-benzyl
Methyl
(2-Phenylethyl) amino
542
543


203
Phenethyl
4-HO-benzyl
Methyl
(4-MeO-benzyl)amino
558
559


204
Phenethyl
4-HO-benzyl
Methyl
Cyclohexylamino
520
521


205
2,2-bisphenylethyl
4-HO-benzyl
Methyl
(4-Me-phenyl)amino
604
605


206
2,2-bisphenylethyl
4-HO-benzyl
Methyl
(4-Cl-phenyl)amino
624
625


207
2,2-bisphenylethyl
4-HO-benzyl
Methyl
Phenylamino
590
591


208
2,2-bisphenylethyl
4-HO-benzyl
Methyl
((R)-a-
618
619






methylbenzyl)amino




209
2,2-bisphenylethyl
4-HO-benzyl
Methyl
Benzylamino
604
605


210
2,2-bisphenylethyl
4-HO-benzyl
Methyl
(4-MeO-phenyl)amino
620
621


211
2,2-bisphenylethyl
4-HO-benzyl
Methyl
(4-Br-phenyl)amino
669
670


212
2,2-bisphenylethyl
4-HO-benzyl
Methyl
(4-CF3-phenyl)amino
658
659


213
2,2-bisphenylethyl
4-HO-benzyl
Methyl
Pentylamino
584
585


214
2,2-bisphenylethyl
4-HO-benzyl
Methyl
(2-Phenylethyl) amino
618
619


215
2,2-bisphenylethyl
4-HO-benzyl
Methyl
(4-MeO-benzyl)amino
634
635


216
2,2-bisphenylethyl
4-HO-benzyl
Methyl
Cyclohexylamino
596
597


217
Phenethyl
3,4-Cl2-benzyl
Methyl
(4-Me-phenyl)amino
581
582


218
Phenethyl
3,4-Cl2-benzyl
Methyl
(4-Cl-phenyl)amino
601
602


219
Phenethyl
3,4-Cl2-benzyl
Methyl
Phenylamino
566
567


220
Phenethyl
3,4-Cl2-benzyl
Methyl
((R)-a-
595
596






methylbenzyl)amino




221
Phenethyl
3,4-Cl2-benzyl
Methyl
Benzylamino
581
582


222
Phenethyl
3,4-Cl2-benzyl
Methyl
(4-MeO-phenyl)amino
597
598


223
Phenethyl
3,4-Cl2-benzyl
Methyl
(4-Br-phenyl)amino
645
646


224
Phenethyl
3,4-Cl2-benzyl
Methyl
(4-CF3-phenyl)amino
634
635


225
Phenethyl
3,4-Cl2-benzyl
Methyl
Pentylamino
561
562


226
Phenethyl
3,4-Cl2-benzyl
Methyl
(2-Phenylethyl) amino
595
596


227
Phenethyl
3,4-Cl2-benzyl
Methyl
(4-MeO-benzyl)amino
611
612


228
Phenethyl
3,4-Cl2-benzyl
Methyl
Cyclohexylamino
573
574


229
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
(4-Me-phenyl)amino
657
658


230
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
(4-Cl-phenyl)amino
677
678


231
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
Phenylamino
643
644


232
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
((R)-a-
671
672






methylbenzyl)amino




233
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
Benzylamino
657
658


234
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
(4-MeO-phenyl)amino
673
674


235
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
(4-Br-phenyl)amino
721
722


236
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
(4-CF3-phenyl)amino
711
712


237
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
Pentylamino
637
638


238
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
(2-Phenylethyl) amino
671
672


239
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
(4-MeO-benzyl)amino
687
688


240
2,2-bisphenylethyl
3,4-Cl2-benzyl
Methyl
Cyclohexylamino
649
650


241
Isoamyl
4-HO-benzyl
Methyl
(4-Me-phenyl)amino
478
479


242
Isoamyl
4-HO-benzyl
Methyl
(4-Cl-phenyl)amino
498
499


243
Isoamyl
4-HO-benzyl
Methyl
Phenylamino
464
465


244
Isoamyl
4-HO-benzyl
Methyl
((R)-a-
492
493






methylbenzyl)amino




245
Isoamyl
4-HO-benzyl
Methyl
Benzylamino
478
479


246
Isoamyl
4-HO-benzyl
Methyl
(4-MeO-phenyl)amino
494
495


247
Isoamyl
4-HO-benzyl
Methyl
(4-Br-phenyl)amino
542
543


248
Isoamyl
4-HO-benzyl
Methyl
(4-CF3-phenyl)amino
532
533


249
Isoamyl
4-HO-benzyl
Methyl
Pentylamino
458
459


250
Isoamyl
4-HO-benzyl
Methyl
(2-Phenylethyl) amino
492
493


251
Isoamyl
4-HO-benzyl
Methyl
(4-MeO-benzyl)amino
508
509


252
Isoamyl
4-HO-benzyl
Methyl
Cyclohexylamino
470
471


253
Isoamyl
4-HO-benzyl
Methyl
(4-Me-phenyl)amino
554
555


254
Isoamyl
4-HO-benzyl
Methyl
(4-Cl-phenyl)amino
574
575


255
Isoamyl
4-HO-benzyl
Methyl
Phenylamino
540
541


256
Isoamyl
4-HO-benzyl
Methyl
((R)-a-
568
569






methylbenzyl)amino




257
Isoamyl
4-HO-benzyl
Methyl
Benzylamino
554
555


258
Isoamyl
4-HO-benzyl
Methyl
(4-MeO-phenyl)amino
570
571


259
Isoamyl
4-HO-benzyl
Methyl
(4-Br-phenyl)amino
619
620


260
Isoamyl
4-HO-benzyl
Methyl
(4-CF3-phenyl)amino
608
609


261
Isoamyl
4-HO-benzyl
Methyl
Pentylamino
534
535


262
Isoamyl
4-HO-benzyl
Methyl
(2-Phenylethyl) amino
568
569


263
Isoamyl
4-HO-benzyl
Methyl
(4-MeO-benzyl)amino
584
585


264
Isoamyl
4-HO-benzyl
Methyl
Cyclohexylamino
546
547


265
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-Me-phenyl)amino
526
527


266
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-Cl-phenyl)amino
546
547


267
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Phenylamino
512
513


268
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
((R)-a-
540
541






methylbenzyl)amino




269
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Benzylamino
526
527


270
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-MeO-phenyl)amino
542
543


271
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-Br-phenyl)amino
591
592


272
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-CF3-phenyl)amino
580
581


273
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Pentylamino
506
507


274
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(2-Phenylethyl) amino
540
541


275
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-MeO-benzyl)amino
556
557


276
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Cyclohexylamino
518
519


277
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-Me-phenyl)amino
602
603


278
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-Cl-phenyl)amino
622
623


279
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Phenylamino
588
589


280
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
((R)-a-
616
617






methylbenzyl)amino




281
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Benzylamino
602
603


282
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-MeO-phenyl)amino
618
619


283
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-Br-phenyl)amino
667
668


284
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-CF3-phenyl)amino
656
657


285
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Pentylamino
582
583


286
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(2-Phenylethyl)amino
616
617


287
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
(4-MeO-benzyl)amino
632
633


288
4-methylbenzyl
3,4-Cl2-benzyl
Methyl
Cyclohexylamino
594
595


289
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(N-Cbz-3-
751
752






Indoleethypamino




290
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(Naphth-2-
614
615






ylmethyl)amino




291
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(2-Phenylethyl)amino
578
579


292
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
[2-(4-MeO-
608
609






phenyl)ethyl]amino




293
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(3-CF3-benzyl)amino
632
633


294
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(4-MeO-benzyl)amino
594
595


295
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(4-F-phenylethyl)amino
596
597


296
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(3,4-Cl2-benzyl)amino
633
634


297
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(2-HO-ethyl)amino
518
519


298
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(3-MeO-propyl)amino
546
547


299
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(Tetrahydrofuran-2-
558
559






ylmethyl)amino




300
Naphth-1-ylmethyl
4-HO-benzyl
Methyl
(cyclohexylmethyl)amino
570
571


301
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(N-Cbz-3-
779
780






Indoleethypamino




302
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(Naphth-2-
642
643






ylmethyl)amino




303
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(2-Phenylethyl)amino
606
607


304
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
[2-(4-MeO-
636
637






phenyl)ethyl]amino




305
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(3-CF3-benzyl)amino
660
661


306
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(4-MeO-benzyl)amino
622
623


307
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(4-F-phenylethyl)amino
624
625


308
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(3,4-Cl2-benzyl)amino
661
662


309
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(2-HO-ethyl)amino
546
547


310
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(3-MeO-propyl)amino
574
575


311
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(Tetrahydrofuran-2-
586
587






ylmethyl)amino




312
Naphth-1-ylmethyl
4-HO-benzyl
Propyl
(cyclohexylmethyl)amino
598
599


313
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(N-Cbz-3-
771
772






Indoleethypamino




314
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(Naphth-2-
634
635






ylmethyl)amino




315
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(2-Phenylethyl)amino
598
599


316
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
[2-(4-MeO-
628
629






phenyl)ethyl]amino




317
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(3-CF3-benzyl)amino
652
653


318
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(4-MeO-benzyl)amino
614
615


319
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(4-F-phenylethyl)amino
616
617


320
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(3,4-Cl2-benzyl)amino
653
654


321
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(2-HO-ethyl)amino
538
539


322
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(3-MeO-propyl)amino
566
567


323
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(Tetra hyd rofuran-2-
578
579






ylmethyl)amino




324
Naphth-1-ylmethyl
3,4-F2-benzyl
Methyl
(cyclohexylmethyl)amino
590
591


325
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(N-Cbz-3-
799
800






Indoleethypamino




326
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(Naphth-2-
662
663






ylmethyl)amino




327
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(2-Phenylethyl)amino
626
627


328
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
[2-(4-MeO-
656
657






phenyl)ethyl]amino




329
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(3-CF3-benzyl)amino
680
681


330
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(4-MeO-benzyl)amino
642
643


331
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(4-F-phenylethyl)amino
644
645


332
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(3,4-Cl2-benzyl)amino
681
682


333
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(2-HO-ethyl)amino
566
567


334
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(3-MeO-propyl)amino
594
595


335
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(Tetra hyd rofuran-2-
606
607






ylmethyl)amino




336
Naphth-1-ylmethyl
3,4-F2-benzyl
Propyl
(cyclohexylmethyl)amino
618
619


337
Naphth-1-ylmethyl
4-biphenylyl-methyl
Methyl
(N-Cbz-3-
811
812






Indoleethypamino




338
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(Naphth-2-
674
675






ylmethyl)amino




339
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(2-Phenylethyl)amino
638
639


340
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
[2-(4-MeO-
668
669






phenyl)ethyl]amino




341
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(3-CF3-benzyl)amino
692
693


342
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(4-MeO-benzyl)amino
654
655


343
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(4-F-phenylethyl)amino
656
657


344
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(3,4-Cl2-benzyl)amino
693
694


345
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(2-HO-ethyl)amino
578
579


346
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(3-MeO-propyl)amino
606
607


347
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(Tetrahyd rofuran-2-
618
619






ylmethyl)amino




348
Naphth-1-ylmethyl
4-biphenylylmethyl
Methyl
(cyclohexylmethyl)amino
630
631


349
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(N-Cbz-3-
839
840






Indoleethypamino




350
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(Naphth-2-
702
703






ylmethyl)amino




351
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(2-Phenylethyl)amino
666
667


352
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
[2-(4-MeO-
696
697






phenyl)ethyl]amino




353
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(3-CF3-benzyl)amino
720
721


354
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(4-MeO-benzyl)amino
682
683


355
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(4-F-phenylethyl)amino
684
685


356
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(3,4-Cl2-benzyl)amino
721
722


357
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(2-HO-ethyl)amino
606
607


358
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(3-MeO-propyl)amino
634
635


359
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(Tetrahyd rofuran-2-
646
647






ylmethyl)amino




360
Naphth-1-ylmethyl
4-biphenylylmethyl
Propyl
(cyclohexylmethyl)amino
658
659


361
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(N-Cbz-3-
807
808






Indoleethypamino




362
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(Naphth-2-
670
671






ylmethyl)amino




363
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(2-Phenylethyl)amino
634
635


364
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
[2-(4-MeO-
664
665






phenyl)ethyl]amino




365
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(3-CF3-benzyl)amino
688
689


366
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(4-MeO-benzyl)amino
650
651


367
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(4-F-phenylethyl)amino
652
653


368
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(3,4-Cl2-benzyl)amino
689
690


369
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(2-HO-ethyl)amino
574
575


370
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(3-MeO-propyl)amino
602
603


371
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(Tetra hyd rofuran-2-
614
615






ylmethyl)amino




372
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
(cyclohexylmethyl)amino
626
627


373
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(N-Cbz-3-
835
836






Indoleethypamino




374
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(Naphth-2-
698
699






ylmethyl)amino




375
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(2-Phenylethyl)amino
662
663


376
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
[2-(4-MeO-
692
693






phenyl)ethyl]amino




377
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(3-CF3-benzyl)amino
716
717


378
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(4-MeO-benzyl)amino
678
679


379
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(4-F-phenylethyl)amino
680
681


380
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(3,4-Cl2-benzyl)amino
717
718


381
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(2-HO-ethyl)amino
602
603


382
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(3-MeO-propyl)amino
630
631


383
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(Tetrahydrofura n-2-
642
643






ylmethyl)amino




384
Naphth-1-ylmethyl
3-t-Bu-4-HO-benzyl
Propyl
(cyclohexylmethyl)amino
654
655


385
4-Methoxybenzyl
OCH3
5-F-benzyl
OCH3
470
471


386
Naphthyl-1-ylmethyl
4-HO-benzyl
Styrylmethyl
OCH3
591
592


387
Naphthyl-1-ylmethyl
4-NO2-benzyl
Styrylmethyl
OCH3
620
621


388
Naphthyl-1-ylmethyl
3,4-F2-benzyl
Styrylmethyl
OCH3
611
612


389
Naphthyl-1-ylmethyl
4-Cl-benzyl
Styrylmethyl
OCH3
609
610


390
Naphthyl-1-ylmethyl
4-Phenyl-benzyl
Styrylmethyl
OCH3
651
652


391
Naphthyl-1-ylmethyl
3-t-Bu-4-HO-benzyl
Styrylmethyl
OCH3
647
648


392
Naphthyl-1-ylmethyl
4-Methyl-benzyl
Styrylmethyl
OCH3
589
590


393
Naphthyl-1-ylmethyl
Cyclohexylmethyl
Styrylmethyl
OCH3
581
582


394
Naphthyl-1-ylmethyl
4-F-benzyl
Styrylmethyl
OCH3
593
594


395
Naphthyl-1-ylmethyl
2-Cl-benzyl
Styrylmethyl
OCH3
609
610


396
Naphthyl-1-ylmethyl
3,4-Cl2-benzyl
Styrylmethyl
OCH3
644
645


397
Naphthyl-1-ylmethyl
Naphthyl-1-ylmethyl
Styrylmethyl
OCH3
625
626


398
3,4-Cl2-benzyl
4-HO-benzyl
Styrylmethyl
OCH3
610
611


399
3,4-Cl2-benzyl
4-NO2-benzyl
Styrylmethyl
OCH3
639
640


400
3,4-Cl2-benzyl
3,4-F2-benzyl
Styrylmethyl
OCH3
629
630


401
3,4-Cl2-benzyl
4-Cl-benzyl
Styrylmethyl
OCH3
628
629


402
3,4-Cl2-benzyl
4-Phenyl-benzyl
Styrylmethyl
OCH3
670
671


403
3,4-Cl2-benzyl
3-t-Bu-4-HO-benzyl
Styrylmethyl
OCH3
666
667


404
3,4-Cl2-benzyl
4-Methyl-benzyl
Styrylmethyl
OCH3
608
609


405
3,4-Cl2-benzyl
Cyclohexylmethyl
Styrylmethyl
OCH3
600
601


406
3,4-Cl2-benzyl
4-F-benzyl
Styrylmethyl
OCH3
611
612


407
3,4-Cl2-benzyl
2-Cl-benzyl
Styrylmethyl
OCH3
628
629


408
3,4-Cl2-benzyl
3,4-Cl2-benzyl
Styrylmethyl
OCH3
662
663


409
3,4-Cl2-benzyl
Naphthyl-1-ylmethyl
Styrylmethyl
OCH3
644
645


410
Naphthyl-1-ylmethyl
4-HO-benzyl
2,6-Cl2-benzyl
OCH3
634
635


411
Naphthyl-1-ylmethyl
4-NO2-benzyl
2,6-Cl2-benzyl
OCH3
663
664


412
Naphthyl-1-ylmethyl
3,4-F2-benzyl
2,6-Cl2-benzyl
OCH3
654
655


413
Naphthyl-1-ylmethyl
4-Cl-benzyl
2,6-Cl2-benzyl
OCH3
652
653


414
Naphthyl-1-ylmethyl
4-Phenyl-benzyl
2,6-Cl2-benzyl
OCH3
694
695


415
Naphthyl-1-ylmethyl
3-t-Bu-4-HO-benzyl
2,6-Cl2-benzyl
OCH3
690
691


416
Naphthyl-1-ylmethyl
4-Methyl-benzyl
2,6-Cl2-benzyl
OCH3
632
633


417
Naphthyl-1-ylmethyl
Cyclohexylmethyl
2,6-Cl2-benzyl
OCH3
624
625


418
Naphthyl-1-ylmethyl
4-F-benzyl
2,6-Cl2-benzyl
OCH3
636
637


419
Naphthyl-1-ylmethyl
2-Cl-benzyl
2,6-Cl2-benzyl
OCH3
652
653


420
Naphthyl-1-ylmethyl
3,4-Cl2-benzyl
2,6-Cl2-benzyl
OCH3
686
687


421
Naphthyl-1-ylmethyl
Naphthyl-1-ylmethyl
2,6-Cl2-benzyl
OCH3
668
669


422
3,4-Cl2-benzyl
4-HO-benzyl
2,6-Cl2-benzyl
OCH3
652
653


423
3,4-Cl2-benzyl
4-NO2-benzyl
2,6-Cl2-benzyl
OCH3
681
682


424
3,4-Cl2-benzyl
3,4-F2-benzyl
2,6-Cl2-benzyl
OCH3
672
673


425
3,4-Cl2-benzyl
4-Cl-benzyl
2,6-Cl2-benzyl
OCH3
671
672


426
3,4-Cl2-benzyl
4-Phenyl-benzyl
2,6-Cl2-benzyl
OCH3
712
713


427
3,4-Cl2-benzyl
3-t-Bu-4-HO-benzyl
2,6-Cl2-benzyl
OCH3
708
709


428
3,4-Cl2-benzyl
4-Methyl-benzyl
2,6-Cl2-benzyl
OCH3
650
651


429
3,4-Cl2-benzyl
Cyclohexylmethyl
2,6-Cl2-benzyl
OCH3
642
643


430
3,4-Cl2-benzyl
4-F-benzyl
2,6-Cl2-benzyl
OCH3
654
655


431
3,4-Cl2-benzyl
2-Cl-benzyl
2,6-Cl2-benzyl
OCH3
671
672


432
3,4-Cl2-benzyl
3,4-Cl2-benzyl
2,6-Cl2-benzyl
OCH3
705
706


433
3,4-Cl2-benzyl
Naphthyl-1-ylmethyl
2,6-Cl2-benzyl
OCH3
686
687


434
2-Piperidin-1-yl-ethyl
(S)-4-HO-benzyl
Methyl
Benzylamino
535
536


435
3,4-Cl2-benzyl
(S)-4-HO-benzyl
Methyl
2-Piperidin-1-yl-
604
605






ethylamino




436
3,4-Cl2-benzyl
(S)-4-HO-benzyl
Methyl
2-(1-Methyl-pyrrolidin-
604
605






2-yl)-ethylamino




437
3-Pyridylmethyl
(S)-4-HO-benzyl
Methyl
3,4-Cl2-benzylamino
583
584


438
2-Morpholin-4-yl-ethyl
(S)-4-HO-benzyl
Methyl
3,4-Cl2-benzylamino
606
607


439
3,4-Cl2-benzyl
(S)-4-HO-benzyl
Methyl
3-Pyridylmethylamino
583
584


440
3,4-Cl2-benzyl
(S)-4-HO-benzyl
Methyl
2-Morpholin-4-yl-
606
607






ethylamino




441
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
3-Imidazol-1-yl-
582
583






propylamino




442
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-Aminophenethylamino
593
594


443
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
3-Pyridylmethylamino
565
566


444
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
2-(3-Pyridylethyl)amino
579
580


445
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-Pyridylmethylamino
565
566


446
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Benzyloxycarbonylamino
622
623


447
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-F-benzylamino
582
583


448
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-CO2H-benzylamino
608
609


449
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-CF3-benzylamino
632
633


450
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
(S)-alpha-
578
579






methylbenzylamino




451
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
(R)-alpha-
578
579






methylbenzylamino




452
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
2-F-benzylamino
582
583


453
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
2,3-
624
625






Dimethoxybenzylamino




454
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Cyanomethylamino
513
514


455
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Phenylhydrazino
565
566


456
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-Aminobenzylamino
579
580


457
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
(S,S){2-[(2-hydroxy-1-
693
694






methyl-2-phenyl-ethyl)-








methyl-carbamoyl]-








ethyl}-amino




458
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
[4-(1,3-dioxo-1,3-
715
716






dihydro-








isoindo1-2-ylmethyl)-








cyclohexyl]-








methylamino




459
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Indan-1-ylamino
590
591


460
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
PhenylGlycine
622
623


461
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
2,6-F2-benzylamino
600
601


462
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
3-F-benzylamino
582
583


463
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Benzimidazol-2-yl-
604
605






amino




464
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Diphenylmethylamino
640
641


465
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Furan-2-yl-methylamino
554
555


466
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-Dimethylamino-
607
608






benzylamino




467
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Thiofuran-2-yl-
584
585






methylamino




468
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-NO2-benzylamino
609
610


469
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
BnO
565
566


470
4-Methoxy-naphthyl-
4-HO-benzyl
Methyl
Benzylamino
594
595



1-ylmethyl







471
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Phenethyl
563
564


472
Naphthyl-1-ylmethyl
4-Methoxy-benzyl
Methyl
Benzylamino
578
579


473
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
4-CF3-phenylamino
618
619


474
Naphthyl-1-ylmethyl
4-NO2-benzyl
Methyl
4-CF3-phenylamino
647
648


475
Naphthyl-1-ylmethyl
4-NO2-benzyl
Methyl
Benzylamino
593
594


476
Benzyl
Naphthyl-1-ylmethyl
4-CN-benzyl
OCH3
574
575


477
Thiofuran-2-yl-methyl
Naphthyl-1-ylmethyl
4-CN-benzyl
OCH3
594
595


478
4-Dimethylamino-
Naphthyl-1-ylmethyl
4-CN-benzyl
OCH3
617
618



benzyl







479
Phenethyl
Naphthyl-1-ylmethyl
4-CN-benzyl
OCH3
588
589


480
8-Quinoline-1yl-
4-HO-benzyl
Methyl
Benzylamino
565
566



methyl







481
4-Pyridylmethyl
Naphthyl-1-ylmethyl
Benzyl
OCH3
550
551


482
3,4-Dimethoxybenzyl
Naphthyl-1-ylmethyl
Benzyl
OCH3
609
610


483
3,4-Dimethoxy-
Naphthyl-1-ylmethyl
Benzyl
OCH3
623
624



phenethyl







484
Thiofuran-2-yl-methyl
Naphthyl-1-ylmethyl
Benzyl
OCH3
569
570


485
Naphthyl-1-ylmethyl
3-Pyridylmethyl
Methyl
Benzylamino
549
550


486
Naphthyl-1-ylmethyl
Pentafluorobenzyl
Methyl
Benzylamino
638
639


487
Naphthyl-1-ylmethyl
3-F-4-HO-benzyl
Methyl
Benzylamino
582
583


488
4-F-phenethyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
598
599


489
4-Methoxyphenethyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
610
611


490
3,4-Dimethoxy-
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
640
641



phenethyl







491
Naphthyl-1-ylmethyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
616
617


492
3,4-Dimethoxybenzyl
Naphthyl-1-ylmethyl
4-CN-benzyl
OCH3
634
635


493
3,4-Dimethoxy-
Naphthyl-1-ylmethyl
4-CN-benzyl
OCH3
648
649



phenethyl







494
4-Quinoline-1yl-
4-HO-benzyl
Methyl
Benzylamino
565
566



methyl







495
2-Pyridylmethyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
567
568


496
3-Pyridylmethyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
567
568


497
3,4-Dimethoxybenzyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
626
627


498
4-Methyl-benzyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
580
581


499
Thiofuran-2-yl-methyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
572
573


500
4-CF3-benzyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
634
635


501
2,6-F2-benzyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
602
603


502
4-F-benzyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
584
585


503
Thiofuran-2-yl-ethyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
586
587


504
3,4-Cl2-benzyl
4-Methyl-benzyl
Methyl
4-CF3-phenylamino
634
635


505
4-CO2H-Benzyl
4-HO-benzyl
Methyl
Benzylamino
558
559


506
Naphthyl-1-ylmethyl
3-t-Bu-4-HO-benzyl
Methyl
Benzylamino
620
621


507
Naphthyl-1-ylmethyl
3,4-(OH)2-benzyl
Methyl
Benzylamino
580
581


508
2-F-benzyl
4-HO-benzyl
Methyl
Benzylamino
532
533


509
3-F-benzyl
4-HO-benzyl
Methyl
Benzylamino
532
533


510
4-F-benzyl
4-HO-benzyl
Methyl
Benzylamino
532
533


511
2,4-F2-benzyl
4-HO-benzyl
Methyl
Benzylamino
550
551


512
2,6-F2-benzyl
4-HO-benzyl
Methyl
Benzylamino
550
551


513
2,5-F2-benzyl
4-HO-benzyl
Methyl
Benzylamino
550
551


514
3-CF3-benyl
4-HO-benzyl
Methyl
Benzylamino
582
583


515
4-CF3-benyl
4-HO-benzyl
Methyl
Benzylamino
582
583


516
3,4,5-F3-benyl
4-HO-benzyl
Methyl
Benzylamino
568
569


517
2-Cl-benzyl
4-HO-benzyl
Methyl
Benzylamino
548
549


518
3-Cl-benzyl
4-HO-benzyl
Methyl
Benzylamino
548
549


519
2,4-Cl2-benzyl
4-HO-benzyl
Methyl
Benzylamino
582
583


520
(S)-Methylphenyl
4-HO-benzyl
Methyl
Benzylamino
528
529


521
(R)-Methylphenyl
4-HO-benzyl
Methyl
Benzylamino
528
529


522
4-Methyl-benzyl
4-HO-benzyl
Methyl
Benzylamino
528
529


523
4-Methoxybenzyl
4-HO-benzyl
Methyl
Benzylamino
544
545


524
3,4-Dimethoxybenzyl
4-HO-benzyl
Methyl
Benzylamino
574
575


525
Furan-2-yl-
4-HO-benzyl
Methyl
Benzylamino
504
505



methylamino







526
(R)-Methylnaphthyl-1-
4-HO-benzyl
Methyl
Benzylamino
578
579



ylmethyl







527
(S)-Methylnaphthyl-1-
4-HO-benzyl
Methyl
Benzylamino
578
579



ylmethyl







528
Naphthyl-1-ylmethyl
3-Oxy-pyridin-1-
Methyl
Benzylamino
565
566




ylmethyl






529
(R)-alpha-
4-HO-benzyl
Methyl
Benzylamino
578
579



methylbenzyl







530
Naphthyl-2-ylmethyl
4-HO-benzyl
Methyl
Benzylamino
564
565


531
4-F-naphthyl-1-
4-HO-benzyl
Methyl
Benzylamino
582
583



ylmethyl







532
2-Methoxybenzyl
4-HO-benzyl
Methyl
Benzylamino
544
545


533
4-Cl-benzyl
4-HO-benzyl
Methyl
Benzylamino
548
549


534
3,4-Cl2-benzyl
4-HO-benzyl
Methyl
Benzylamino
582
583


535
2-CF3Obenzyl
4-HO-benzyl
Methyl
Benzylamino
598
599


536
2-CF3Sbenzyl
4-HO-benzyl
Methyl
Benzylamino
614
615


537
2-CF3benzyl
4-HO-benzyl
Methyl
Benzylamino
582
583


538
5-Quinoline-1yl-
4-HO-benzyl
Methyl
Benzylamino
565
566



methyl







539
8-Quinoline-1yl-
3-t-Bu-4-HO-benzyl
Methyl
Benzylamino
621
622



methyl







540
8-Quinoline-1yl-
4-NO2-benzyl
Methyl
Benzylamino
594
595



methyl







541
8-Quinoline-1yl-
(1H-Pyrrol-2-yl)-
Methyl
Benzylamino
538
539



methyl
methyl






542
Naphthyl-1-ylmethyl
4-Benzyloxy-
Methyl
Benzylamino
697
698




carbonylaminobenzyl






543
2,3-Cl2-benzyl
4-HO-benzyl
Methyl
Benzylamino
582
583


544
Pentafluorobenzyl
4-HO-benzyl
Methyl
Benzylamino
604
605


545
Benzyl
4-HO-benzyl
Methyl
Benzylamino
514
515


546
Quinoxaline-5yl-
4-HO-benzyl
Methyl
Benzylamino
566
567



methyl







547
8-Quinoline-1yl-
3-Pyridylmethyl
Methyl
Benzylamino
550
551



methyl







548
8-Quinoline-1yl-
Pentafluorobenzyl
Methyl
Benzylamino
639
640



methyl







549
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
Benzylamino(thiourea)
580
581


550
Naphthyl-1-ylmethyl
4-Amino-benzyl
Methyl
Benzylamino
563
564


551
3,4,5-tri-
4-Amino-benzyl
Methyl
Benzylamino
603
604



Methoxybenzyl







552
Naphthyl-1-ylmethyl
4-Pyridylmethyl
Methyl
Benzylamino
549
550


553
Naphthyl-1-ylmethyl
(R) 4-HO-phenyl
Methyl
Benzylamino
550
551


554
2-HO-3-Methoxy-
4-HO-benzyl
Methyl
Benzylamino
560
561



benzyl







555
Naphthyl-1-ylmethyl
3-Nitro-4-HO-
Methyl
Benzylamino
609
610




benzyl






556
Naphthyl-1-ylmethyl
4-CO2—CH2O-
Methyl
Benzylamino
622
623




benzyl






557
Naphthyl-1-ylmethyl
1-Naphtoylamino-
Methyl
Benzylamino
641
642




methyl






558
Naphthyl-1-ylmethyl
4-Oxy-pyridylmethyl
Methyl
Benzylamino
565
566


559
4-F-alpha-
4-HO-benzyl
Methyl
Benzylamino
546
547



methylbenzyl







560
Naphthyl-1-ylmethyl
Benzoylaminoethyl
Methyl
Benzylamino
605
606


561
8-Quinoline-1yl-
3,4-(OH)2-benzyl
Methyl
Benzylamino
581
582



methyl







562
4-N,N-Dimethylamino-
4-HO-benzyl
Methyl
Benzylamino
557
558



benzyl







563
Naphthyl-1-ylmethyl
(R) 4-F-benzyl
Methyl
Benzylamino
609
610


564
Naphthyl-1-ylmethyl
4-HO-benzyl
Methyl
2-Chloroethylamino
536
537


565
Naphthyl-1-ylmethyl
4-HO-phenethyl
Methyl
Benzylamino
578
579


566
4-F-benzyl
3-F,4-HO-benzyl
Methyl
Benzylamino
550
551


567
2,4-F2-benzyl
3-F,4-HO-benzyl
Methyl
Benzylamino
568
569


568
3-CF3benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
568
569


569
(S)-Methylnaphthyl-1-
(R) 4-HO-phenyl
Methyl
Benzylamino
514
515



ylmethyl







570
(R)-Methylnaphthyl-1-
(R) 4-HO-phenyl
Methyl
Benzylamino
514
515



ylmethyl







571
2,3,6-F3-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
554
555


572
3-F-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
518
519


573
4-Cl-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
534
535


574
3-Cl-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
534
535


575
2-Cl-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
534
535


576
3,4-Cl2-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
568
569


577
3-CF3O-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
584
585


578
4-F-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
518
519


579
2,4-F2-benzyl
(R) 4-HO-phenyl
Methyl
Benzylamino
536
537


580
3-(2-Chloro-ethyl)-
4-HO-benzyl
Methyl
Benzylamino
634
635



ureido]-benzyl







581
3-Aminobenzyl
4-HO-benzyl
Methyl
Benzylamino
529
530


582
3-N-
4-HO-benzyl
Methyl
Benzylamino
543
544



Methylaminobenzyl







583
3-N,N-
4-HO-benzyl
Methyl
Benzylamino
557
558



Dimethylaminobenzyl







584
1H-Benzoimidazol-4-
4-HO-benzyl
Methyl
Benzylamino
554
555



ylmethyl







585
2-HO-benzyl
4-HO-benzyl
Methyl
Benzylamino
530
531


586
2-Pyridylmethyl
4-HO-benzyl
Methyl
Benzylamino
515
516


587
4-Pyridylmethyl
4-HO-benzyl
Methyl
Benzylamino
515
516


588
8-quinolin-2-ylmethyl
4-HO-benzyl
Methyl
Benzylamino
565
566


589
8-Benzofuran-4-
4-HO-benzyl
Methyl
Benzylamino
554
555



ylmethyl







590
Naphthyl-1-ylmethyl
4-HO-phenyl
Methyl
Benzylamino
550
551


591
4-F-benzyl
4-HO-phenyl
Methyl
Benzylamino
518
519


592
2,4-F2-benzyl
4-HO-phenyl
Methyl
Benzylamino
536
537


593
(R)-Toluylmethyl
4-HO-benzyl
Methyl
Benzylamino
542
543


594
(S)-Toluylmethyl
4-HO-benzyl
Methyl
Benzylamino
542
543


595
1,2,3,4-tetrahydro-
4-HO-benzyl
Methyl
Benzylamino
554
555



naphthalen-2-yl







596
Naphthyl-1-ylmethyl
3,4-
Methyl
Benzylamino
608
609




Dimethoxybenzyl






597
2-Dimethylamino-6-F-
4-HO-benzyl
Methyl
Benzylamino
575
576



benzyl







598
2-
4-HO-benzyl
Methyl
Benzylamino
557
558



Dimethylaminobenzyl







599
Naphthyl-1-ylmethyl
4-CN-benzyl
Methyl
Benzylamino
573
574


600
4-F-2-CF3-benzyl
4-HO-benzyl
Methyl
Benzylamino
599
600


601
4-Cl-2-
4-HO-benzyl
Methyl
Benzylamino
591
592



Dimethylaminobenzyl







602
3-N,N-
4-HO-benzyl
Methyl
Benzylamino
571
572



Ethylmethyllamino-








benzyl







603
3-Diethylaminobenzyl
4-HO-benzyl
Methyl
Benzylamino
585
586


604
4-Cl-3-
4-HO-benzyl
Methyl
Benzylamino
591
592



Dimethylaminobenzyl







605
4-F-2-
4-HO-benzyl
Methyl
Benzylamino
575
576



Dimethylaminobenzyl







606
3,5-(CH3)2-2-
4-HO-benzyl
Methyl
Benzylamino
585
586



Dimethylamino-benzyl







607
3-(CH3)-2-
4-HO-benzyl
Methyl
Benzylamino
571
572



Dimethylaminobenzyl







608
6-(CH3)-2-
4-HO-benzyl
Methyl
Benzylamino
571
572



Dimethylaminobenzyl







609
3,4-F2-2-
4-HO-benzyl
Methyl
Benzylamino
593
594



Dimethylaminobenzyl
















TABLE 2B







THE [4,4,0]REVERSE TURN MIMETICS LIBRARY




embedded image

















Mol.
M + H


No
MOLSTRUCTURE
Weight
(MS)





802


embedded image


480
481





803


embedded image


430
431





804


embedded image


416
417





805


embedded image


464
465





806


embedded image


430
431





807


embedded image


430
431





808


embedded image


448
449





809


embedded image


416
417





810


embedded image


431
432





811


embedded image


446
447





812


embedded image


450
451





813


embedded image


515
516





814


embedded image


582
583





815


embedded image


532
533





816


embedded image


518
519





817


embedded image


566
567





818


embedded image


532
533





819


embedded image


532
533





820


embedded image


550
551





821


embedded image


518
519





822


embedded image


534
535





823


embedded image


548
549





824


embedded image


552
553





825


embedded image


617
618





826


embedded image


542
543





827


embedded image


492
493





828


embedded image


478
479





829


embedded image


526
527





830


embedded image


492
493





831


embedded image


492
493





832


embedded image


510
511





833


embedded image


478
479





834


embedded image


494
495





835


embedded image


508
509





836


embedded image


512
513





837


embedded image


577
578





838


embedded image


468
469





839


embedded image


516
517





840


embedded image


482
483





841


embedded image


482
483





842


embedded image


468
469





843


embedded image


484
485





844


embedded image


498
499





845


embedded image


502
503





846


embedded image


567
568





847


embedded image


508
509





848


embedded image


458
459





849


embedded image


444
445





850


embedded image


492
493





851


embedded image


458
459





852


embedded image


458
459





853


embedded image


476
477





854


embedded image


444
445





855


embedded image


460
461





856


embedded image


474
475





857


embedded image


478
479





858


embedded image


543
544





859


embedded image


494
495





860


embedded image


444
445





861


embedded image


430
431





862


embedded image


478
479





863


embedded image


444
445





864


embedded image


444
445





865


embedded image


462
463





866


embedded image


430
431





867


embedded image


446
447





868


embedded image


460
461





869


embedded image


464
465





870


embedded image


529
530





871


embedded image


558
559





872


embedded image


508
509





873


embedded image


494
495





874


embedded image


542
543





875


embedded image


508
509





876


embedded image


508
509





877


embedded image


526
527





878


embedded image


494
495





879


embedded image


510
511





880


embedded image


524
525





881


embedded image


528
529





882


embedded image


593
594





883


embedded image


432
433





884


embedded image


480
481





885


embedded image


446
447





886


embedded image


446
447





887


embedded image


464
465





888


embedded image


432
433





889


embedded image


447
448





890


embedded image


462
463





891


embedded image


466
467





892


embedded image


531
532





893


embedded image


558
559





894


embedded image


508
509





895


embedded image


494
495





896


embedded image


542
543





897


embedded image


508
509





898


embedded image


508
509





899


embedded image


526
527





900


embedded image


494
495





901


embedded image


510
511





902


embedded image


524
525





903


embedded image


528
529





904


embedded image


593
594





905


embedded image


544
545





906


embedded image


494
495





907


embedded image


480
481





908


embedded image


528
529





909


embedded image


494
495





910


embedded image


494
495





911


embedded image


512
513





912


embedded image


480
481





913


embedded image


496
497





914


embedded image


510
511





915


embedded image


514
515





916


embedded image


579
580





917


embedded image


464
465





918


embedded image


450
451





919


embedded image


498
499





920


embedded image


464
465





921


embedded image


464
465





922


embedded image


482
483





923


embedded image


450
451





924


embedded image


466
467





925


embedded image


480
481





926


embedded image


484
485





927


embedded image


549
550





928


embedded image


480
481





929


embedded image


430
431





930


embedded image


416
417





931


embedded image


464
465





932


embedded image


430
431





933


embedded image


430
431





934


embedded image


448
449





935


embedded image


416
417





936


embedded image


431
432





937


embedded image


446
447





938


embedded image


450
451





939


embedded image


515
516





940


embedded image


504
505





941


embedded image


454
455





942


embedded image


440
441





943


embedded image


488
489





944


embedded image


454
455





945


embedded image


454
455





946


embedded image


472
473





947


embedded image


440
441





948


embedded image


455
456





949


embedded image


470
471





950


embedded image


474
475





951


embedded image


539
540





952


embedded image


604
605





953


embedded image


554
555





954


embedded image


540
541





955


embedded image


588
589





956


embedded image


554
555





957


embedded image


554
555





958


embedded image


572
573





959


embedded image


540
541





960


embedded image


556
557





961


embedded image


570
571





962


embedded image


545
575





963


embedded image


639
640





964


embedded image


528
529





965


embedded image


478
479





966


embedded image


464
465





967


embedded image


512
513





968


embedded image


478
479





969


embedded image


478
479





970


embedded image


496
497





971


embedded image


464
465





972


embedded image


480
481





973


embedded image


494
495





974


embedded image


498
499





975


embedded image


563
564





976


embedded image


582
583





977


embedded image


532
533





978


embedded image


518
519





979


embedded image


566
567





980


embedded image


532
533





981


embedded image


532
533





982


embedded image


551
552





983


embedded image


518
519





984


embedded image


534
535





985


embedded image


548
549





986


embedded image


552
553





987


embedded image


618
619





988


embedded image


482
483





989


embedded image


432
433





990


embedded image


418
419





991


embedded image


466
467





992


embedded image


432
433





993


embedded image


432
433





994


embedded image


450
451





995


embedded image


418
419





996


embedded image


433
434





997


embedded image


447
448





998


embedded image


452
453





999


embedded image


517
518





1000


embedded image


548
549





1001


embedded image


498
499





1002


embedded image


484
485





1003


embedded image


532
533





1004


embedded image


498
499





1005


embedded image


498
499





1006


embedded image


516
517





1007


embedded image


484
485





1008


embedded image


500
501





1009


embedded image


514
515





1010


embedded image


518
519





1011


embedded image


583
584





1012


embedded image


532
533





1013


embedded image


518
519





1014


embedded image


566
567





1015


embedded image


532
533





1016


embedded image


532
533





1017


embedded image


551
552





1018


embedded image


518
519





1019


embedded image


534
535





1020


embedded image


548
549





1021


embedded image


552
553





1022


embedded image


618
619





1023


embedded image


528
529





1024


embedded image


478
479





1025


embedded image


464
465





1026


embedded image


512
513





1027


embedded image


478
479





1028


embedded image


478
479





1029


embedded image


496
497





1030


embedded image


464
465





1031


embedded image


480
481





1032


embedded image


494
495





1033


embedded image


498
499





1034


embedded image


563
564





1035


embedded image


528
529





1036


embedded image


478
479





1037


embedded image


464
465





1038


embedded image


512
513





1039


embedded image


478
479





1040


embedded image


478
479





1041


embedded image


496
497





1042


embedded image


464
465





1043


embedded image


480
481





1044


embedded image


494
495





1045


embedded image


498
499





1046


embedded image


563
564





1047


embedded image


556
557





1048


embedded image


506
507





1049


embedded image


492
493





1050


embedded image


540
541





1051


embedded image


506
507





1052


embedded image


506
507





1053


embedded image


524
525





1054


embedded image


492
493





1055


embedded image


508
509





1056


embedded image


522
523





1057


embedded image


526
527





1058


embedded image


591
592





1059


embedded image


546
547





1060


embedded image


496
497





1061


embedded image


482
483





1062


embedded image


530
531





1063


embedded image


496
497





1064


embedded image


496
497





1065


embedded image


514
515





1066


embedded image


482
483





1067


embedded image


498
499





1068


embedded image


512
513





1069


embedded image


516
517





1070


embedded image


581
582





1071


embedded image


528
529





1072


embedded image


478
479





1073


embedded image


464
465





1074


embedded image


512
513





1075


embedded image


478
479





1076


embedded image


478
479





1077


embedded image


496
497





1078


embedded image


464
465





1079


embedded image


480
481





1080


embedded image


494
495





1081


embedded image


498
499





1082


embedded image


563
564





1083


embedded image


514
515





1084


embedded image


500
501





1085


embedded image


548
549





1086


embedded image


514
515





1087


embedded image


514
515





1088


embedded image


532
533





1089


embedded image


500
501





1090


embedded image


516
517





1091


embedded image


530
531





1092


embedded image


534
535





1093


embedded image


599
600





1094


embedded image


520
521





1095


embedded image


470
471





1096


embedded image


456
457





1097


embedded image


504
505





1098


embedded image


470
471





1099


embedded image


470
471





1100


embedded image


488
489





1101


embedded image


456
457





1102


embedded image


472
473





1103


embedded image


486
487





1104


embedded image


490
491





1105


embedded image


555
556





1106


embedded image


496
497





1107


embedded image


482
483





1108


embedded image


530
531





1109


embedded image


496
497





1110


embedded image


496
497





1111


embedded image


514
515





1112


embedded image


482
483





1113


embedded image


498
499





1114


embedded image


512
513





1115


embedded image


516
517





1116


embedded image


581
582





1117


embedded image


542
543





1118


embedded image


492
493





1119


embedded image


478
479





1120


embedded image


526
527





1121


embedded image


492
493





1122


embedded image


492
493





1123


embedded image


510
511





1124


embedded image


478
479





1125


embedded image


494
495





1126


embedded image


508
509





1127


embedded image


512
513





1128


embedded image


577
578





1129


embedded image


550
551





1130


embedded image


500
501





1131


embedded image


486
487





1132


embedded image


534
535





1133


embedded image


500
501





1134


embedded image


500
501





1135


embedded image


518
519





1136


embedded image


486
487





1137


embedded image


501
502





1138


embedded image


516
517





1139


embedded image


520
521





1140


embedded image


585
586





1141


embedded image


588
589





1142


embedded image


538
539





1143


embedded image


524
525





1144


embedded image


572
573





1145


embedded image


538
539





1146


embedded image


538
539





1147


embedded image


556
557





1148


embedded image


524
525





1149


embedded image


540
541





1150


embedded image


554
555





1151


embedded image


558
559





1152


embedded image


623
624





1153


embedded image


508
509





1154


embedded image


458
459





1155


embedded image


444
445





1156


embedded image


492
493





1157


embedded image


458
459





1158


embedded image


458
459





1159


embedded image


476
477





1160


embedded image


444
445





1161


embedded image


460
461





1162


embedded image


474
475





1163


embedded image


478
479





1164


embedded image


543
544





1165


embedded image


618
619





1166


embedded image


568
569





1167


embedded image


554
555





1168


embedded image


602
603





1169


embedded image


568
569





1170


embedded image


568
569





1171


embedded image


586
587





1172


embedded image


554
555





1173


embedded image


570
571





1174


embedded image


584
585





1175


embedded image


588
589





1176


embedded image


653
654





1177


embedded image


494
495





1178


embedded image


444
445





1179


embedded image


430
431





1180


embedded image


478
479





1181


embedded image


444
445





1182


embedded image


444
445





1183


embedded image


462
463





1184


embedded image


430
431





1185


embedded image


446
447





1186


embedded image


460
461





1187


embedded image


464
465





1188


embedded image


529
530





1189


embedded image


506
507





1190


embedded image


456
457





1191


embedded image


442
443





1192


embedded image


490
491





1193


embedded image


456
457





1194


embedded image


456
457





1195


embedded image


474
475





1196


embedded image


442
443





1197


embedded image


458
459





1198


embedded image


472
473





1199


embedded image


476
477





1200


embedded image


541
542





1201


embedded image


592
593





1202


embedded image


542
543





1203


embedded image


528
529





1204


embedded image


576
577





1205


embedded image


542
543





1206


embedded image


542
543





1207


embedded image


561
562





1208


embedded image


528
529





1209


embedded image


544
545





1210


embedded image


558
559





1211


embedded image


562
563





1212


embedded image


628
629





1213


embedded image


538
539





1214


embedded image


488
489





1215


embedded image


474
475





1216


embedded image


522
523





1217


embedded image


488
489





1218


embedded image


488
489





1219


embedded image


506
507





1220


embedded image


474
475





1221


embedded image


490
491





1222


embedded image


504
505





1223


embedded image


508
509





1224


embedded image


573
574





1225


embedded image


510
511





1226


embedded image


558
559





1227


embedded image


524
525





1228


embedded image


524
525





1229


embedded image


510
511





1230


embedded image


526
527





1231


embedded image


540
541





1232


embedded image


544
545





1233


embedded image


609
610





1234


embedded image


548
549





1235


embedded image


498
499





1236


embedded image


484
485





1237


embedded image


532
533





1238


embedded image


498
499





1239


embedded image


498
499





1240


embedded image


516
517





1241


embedded image


484
485





1242


embedded image


500
501





1243


embedded image


514
515





1244


embedded image


518
529





1245


embedded image


583
584





1246


embedded image


534
535





1247


embedded image


484
485





1248


embedded image


470
471





1249


embedded image


518
519





1250


embedded image


484
485





1251


embedded image


484
485





1252


embedded image


502
503





1253


embedded image


470
471





1254


embedded image


486
487





1255


embedded image


500
501





1256


embedded image


504
505





1257


embedded image


569
570





1258


embedded image


536
537





1259


embedded image


486
487





1260


embedded image


472
473





1261


embedded image


520
521





1262


embedded image


486
487





1263


embedded image


486
587





1264


embedded image


504
505





1265


embedded image


472
473





1266


embedded image


488
489





1267


embedded image


502
503





1268


embedded image


506
507





1269


embedded image


571
572





1270


embedded image


558
559





1271


embedded image


508
509





1272


embedded image


494
495





1273


embedded image


542
543





1274


embedded image


508
509





1275


embedded image


508
509





1276


embedded image


526
527





1277


embedded image


494
495





1278


embedded image


510
511





1279


embedded image


524
525





1280


embedded image


528
529





1281


embedded image


593
594





1282


embedded image


506
507





1283


embedded image


456
457





1284


embedded image


442
443





1285


embedded image


490
491





1286


embedded image


456
457





1287


embedded image


456
457





1288


embedded image


474
475





1289


embedded image


442
443





1290


embedded image


457
458





1291


embedded image


472
473





1292


embedded image


476
477





1293


embedded image


541
542





1294


embedded image


572
573





1295


embedded image


522
523





1296


embedded image


508
509





1297


embedded image


556
557





1298


embedded image


522
523





1299


embedded image


522
523





1300


embedded image


540
541





1301


embedded image


508
509





1302


embedded image


524
525





1303


embedded image


538
539





1304


embedded image


542
543





1305


embedded image


607
608





1306


embedded image


576
577





1307


embedded image


526
527





1308


embedded image


512
513





1309


embedded image


560
561





1310


embedded image


526
527





1311


embedded image


526
527





1312


embedded image


544
545





1313


embedded image


512
513





1314


embedded image


528
529





1315


embedded image


542
543





1316


embedded image


546
547





1317


embedded image


611
612





1318


embedded image


576
577





1319


embedded image


526
527





1320


embedded image


512
513





1321


embedded image


560
561





1322


embedded image


526
527





1323


embedded image


526
527





1324


embedded image


544
545





1325


embedded image


512
513





1326


embedded image


528
529





1327


embedded image


542
543





1328


embedded image


546
547





1329


embedded image


611
612





1330


embedded image


576
577





1331


embedded image


526
527





1332


embedded image


512
513





1333


embedded image


560
561





1334


embedded image


526
527





1335


embedded image


526
527





1336


embedded image


544
545





1337


embedded image


512
513





1338


embedded image


528
529





1339


embedded image


542
543





1340


embedded image


546
547





1341


embedded image


611
612





1342


embedded image


492
493





1343


embedded image


442
443





1344


embedded image


428
429





1345


embedded image


476
477





1346


embedded image


442
443





1347


embedded image


442
443





1348


embedded image


460
461





1349


embedded image


428
429





1350


embedded image


444
445





1351


embedded image


458
459





1352


embedded image


462
463





1353


embedded image


527
528





1354


embedded image


522
523





1355


embedded image


472
473





1356


embedded image


458
459





1357


embedded image


506
507





1358


embedded image


472
473





1359


embedded image


472
473





1360


embedded image


490
491





1361


embedded image


458
459





1362


embedded image


474
475





1363


embedded image


488
489





1364


embedded image


492
493





1365


embedded image


557
558





1366


embedded image


504
505





1367


embedded image


454
455





1368


embedded image


440
441





1369


embedded image


488
489





1370


embedded image


454
455





1371


embedded image


454
455





1372


embedded image


472
473





1373


embedded image


440
441





1374


embedded image


456
457





1375


embedded image


470
471





1376


embedded image


474
475





1377


embedded image


539
540





1378


embedded image


606
607





1379


embedded image


556
557





1380


embedded image


542
543





1381


embedded image


590
591





1382


embedded image


556
557





1383


embedded image


556
557





1384


embedded image


574
575





1385


embedded image


542
543





1386


embedded image


558
559





1387


embedded image


572
573





1388


embedded image


576
577





1389


embedded image


641
642





1390


embedded image


566
567





1391


embedded image


516
517





1392


embedded image


502
503





1393


embedded image


550
551





1394


embedded image


516
517





1395


embedded image


516
517





1396


embedded image


534
535





1397


embedded image


502
503





1398


embedded image


518
519





1399


embedded image


532
533





1400


embedded image


536
537





1401


embedded image


601
602





1402


embedded image


556
557





1403


embedded image


506
507





1404


embedded image


492
493





1405


embedded image


540
541





1406


embedded image


506
507





1407


embedded image


506
507





1408


embedded image


524
525





1409


embedded image


492
493





1410


embedded image


508
509





1411


embedded image


522
523





1412


embedded image


526
527





1413


embedded image


591
592





1414


embedded image


532
533





1415


embedded image


482
483





1416


embedded image


468
469





1417


embedded image


516
517





1418


embedded image


482
483





1419


embedded image


482
483





1420


embedded image


500
501





1421


embedded image


468
469





1422


embedded image


484
485





1423


embedded image


498
499





1424


embedded image


502
503





1425


embedded image


567
568





1426


embedded image


518
519





1427


embedded image


468
469





1428


embedded image


454
455





1429


embedded image


502
503





1430


embedded image


468
469





1431


embedded image


468
469





1432


embedded image


486
487





1433


embedded image


454
455





1434


embedded image


470
471





1435


embedded image


484
485





1436


embedded image


488
489





1437


embedded image


553
554





1438


embedded image


582
583





1439


embedded image


532
533





1440


embedded image


518
519





1441


embedded image


566
567





1442


embedded image


532
533





1443


embedded image


532
533





1444


embedded image


550
551





1445


embedded image


518
519





1446


embedded image


534
535





1447


embedded image


548
549





1448


embedded image


552
553





1449


embedded image


617
618





1450


embedded image


520
521





1451


embedded image


470
471





1452


embedded image


456
457





1453


embedded image


504
505





1454


embedded image


470
471





1455


embedded image


470
471





1456


embedded image


488
489





1457


embedded image


456
457





1458


embedded image


472
473





1459


embedded image


486
487





1460


embedded image


490
491





1461


embedded image


555
556





1462


embedded image


582
583





1463


embedded image


532
533





1464


embedded image


518
519





1465


embedded image


566
567





1466


embedded image


532
533





1467


embedded image


532
533





1468


embedded image


550
551





1469


embedded image


518
519





1470


embedded image


534
535





1471


embedded image


548
549





1472


embedded image


552
553





1473


embedded image


617
618





1474


embedded image


568
569





1475


embedded image


518
519





1476


embedded image


504
505





1477


embedded image


552
553





1478


embedded image


518
519





1479


embedded image


518
519





1480


embedded image


536
537





1481


embedded image


504
505





1482


embedded image


520
521





1483


embedded image


534
535





1484


embedded image


538
539





1485


embedded image


603
604





1486


embedded image


538
539





1487


embedded image


488
489





1488


embedded image


474
475





1489


embedded image


522
523





1490


embedded image


488
489





1491


embedded image


488
489





1492


embedded image


506
507





1493


embedded image


474
475





1494


embedded image


490
491





1495


embedded image


504
505





1496


embedded image


508
509





1497


embedded image


573
574





1498


embedded image


504
505





1499


embedded image


454
455





1500


embedded image


440
441





1501


embedded image


488
489





1502


embedded image


454
455





1503


embedded image


454
455





1504


embedded image


472
473





1505


embedded image


440
441





1506


embedded image


456
457





1507


embedded image


470
471





1508


embedded image


474
475





1509


embedded image


539
540





1510


embedded image


528
529





1511


embedded image


478
479





1512


embedded image


464
465





1513


embedded image


512
513





1514


embedded image


478
479





1515


embedded image


478
479





1516


embedded image


496
497





1517


embedded image


464
465





1518


embedded image


479
480





1519


embedded image


494
495





1520


embedded image


498
499





1521


embedded image


563
564





1522


embedded image


628
629





1523


embedded image


578
579





1524


embedded image


564
565





1525


embedded image


612
613





1526


embedded image


578
579





1527


embedded image


578
579





1528


embedded image


596
597





1529


embedded image


564
565





1530


embedded image


580
581





1531


embedded image


594
595





1532


embedded image


598
599





1533


embedded image


663
664





1534


embedded image


607
608





1535


embedded image


556
557





1536


embedded image


542
543





1537


embedded image


591
592





1538


embedded image


556
557





1539


embedded image


556
557





1540


embedded image


575
576





1541


embedded image


542
543





1542


embedded image


558
559





1543


embedded image


572
573





1544


embedded image


576
577





1545


embedded image


642
643





1546


embedded image


506
507





1547


embedded image


456
457





1548


embedded image


442
443





1549


embedded image


490
491





1550


embedded image


456
457





1551


embedded image


456
457





1552


embedded image


474
475





1553


embedded image


442
443





1554


embedded image


457
458





1555


embedded image


472
473





1556


embedded image


476
477





1557


embedded image


541
542





1558


embedded image


552
553





1559


embedded image


502
503





1560


embedded image


488
489





1561


embedded image


536
537





1562


embedded image


502
503





1563


embedded image


502
503





1564


embedded image


520
521





1565


embedded image


488
489





1566


embedded image


504
505





1567


embedded image


518
519





1568


embedded image


522
523





1569


embedded image


587
588





1570


embedded image


572
573





1571


embedded image


522
523





1572


embedded image


508
509





1573


embedded image


556
557





1574


embedded image


522
523





1575


embedded image


522
523





1576


embedded image


540
541





1577


embedded image


508
509





1578


embedded image


524
525





1579


embedded image


538
539





1580


embedded image


542
543





1581


embedded image


607
608





1582


embedded image


607
608





1583


embedded image


556
557





1584


embedded image


542
543





1585


embedded image


591
592





1586


embedded image


556
557





1587


embedded image


556
557





1588


embedded image


575
576





1589


embedded image


542
543





1590


embedded image


558
559





1591


embedded image


572
573





1592


embedded image


576
577





1593


embedded image


642
643





1594


embedded image


552
553





1595


embedded image


502
503





1596


embedded image


488
489





1597


embedded image


536
537





1598


embedded image


502
503





1599


embedded image


502
503





1600


embedded image


520
521





1601


embedded image


488
489





1602


embedded image


504
505





1603


embedded image


518
519





1604


embedded image


522
523





1605


embedded image


587
588





1606


embedded image


552
553





1607


embedded image


502
503





1608


embedded image


488
489





1609


embedded image


536
537





1610


embedded image


502
503





1611


embedded image


502
503





1612


embedded image


520
521





1613


embedded image


488
489





1614


embedded image


504
505





1615


embedded image


518
519





1616


embedded image


522
523





1617


embedded image


587
588





1618


embedded image


580
581





1619


embedded image


530
531





1620


embedded image


516
517





1621


embedded image


564
565





1622


embedded image


530
531





1623


embedded image


530
531





1624


embedded image


548
549





1625


embedded image


516
517





1626


embedded image


532
533





1627


embedded image


546
547





1628


embedded image


550
551





1629


embedded image


615
616





1630


embedded image


552
553





1631


embedded image


502
503





1632


embedded image


488
489





1633


embedded image


536
537





1634


embedded image


502
503





1635


embedded image


502
503





1636


embedded image


520
521





1637


embedded image


488
489





1638


embedded image


504
505





1639


embedded image


518
519





1640


embedded image


522
523





1641


embedded image


587
588





1642


embedded image


570
571





1643


embedded image


520
521





1644


embedded image


506
507





1645


embedded image


554
555





1646


embedded image


520
521





1647


embedded image


520
521





1648


embedded image


538
539





1649


embedded image


506
507





1650


embedded image


522
523





1651


embedded image


536
537





1652


embedded image


540
541





1653


embedded image


605
606





1654


embedded image


570
571





1655


embedded image


520
521





1656


embedded image


506
507





1657


embedded image


554
555





1658


embedded image


520
521





1659


embedded image


520
521





1660


embedded image


538
539





1661


embedded image


506
507





1662


embedded image


522
523





1663


embedded image


536
537





1664


embedded image


540
541





1665


embedded image


605
606





1666


embedded image


588
589





1667


embedded image


538
539





1668


embedded image


524
525





1669


embedded image


572
573





1670


embedded image


538
539





1671


embedded image


538
539





1672


embedded image


556
557





1673


embedded image


524
525





1674


embedded image


540
541





1675


embedded image


554
555





1676


embedded image


558
559





1677


embedded image


623
624





1678


embedded image


544
545





1679


embedded image


494
495





1680


embedded image


480
481





1681


embedded image


528
529





1682


embedded image


494
495





1683


embedded image


494
495





1684


embedded image


512
513





1685


embedded image


480
481





1686


embedded image


496
497





1687


embedded image


510
511





1688


embedded image


514
515





1689


embedded image


579
580





1690


embedded image


566
567





1691


embedded image


516
517





1692


embedded image


502
503





1693


embedded image


550
551





1694


embedded image


516
517





1695


embedded image


516
517





1696


embedded image


534
535





1697


embedded image


502
503





1698


embedded image


518
519





1699


embedded image


532
533





1700


embedded image


536
537





1701


embedded image


601
602





1702


embedded image


574
575





1703


embedded image


524
525





1704


embedded image


510
511





1705


embedded image


558
559





1706


embedded image


524
525





1707


embedded image


524
525





1708


embedded image


542
543





1709


embedded image


510
511





1710


embedded image


526
527





1711


embedded image


540
541





1712


embedded image


544
545





1713


embedded image


609
610





1714


embedded image


612
613





1715


embedded image


562
563





1716


embedded image


548
549





1717


embedded image


596
597





1718


embedded image


562
563





1719


embedded image


562
563





1720


embedded image


580
581





1721


embedded image


548
549





1722


embedded image


564
565





1723


embedded image


578
579





1724


embedded image


582
583





1725


embedded image


647
648





1726


embedded image


532
533





1727


embedded image


482
483





1728


embedded image


468
469





1729


embedded image


516
517





1730


embedded image


482
483





1731


embedded image


482
483





1732


embedded image


500
501





1733


embedded image


468
469





1734


embedded image


484
485





1735


embedded image


498
499





1736


embedded image


502
503





1737


embedded image


567
568





1738


embedded image


642
643





1739


embedded image


592
593





1740


embedded image


578
579





1741


embedded image


626
627





1742


embedded image


592
593





1743


embedded image


592
593





1744


embedded image


610
611





1745


embedded image


578
579





1746


embedded image


594
595





1747


embedded image


608
609





1748


embedded image


612
613





1749


embedded image


677
678





1750


embedded image


518
519





1751


embedded image


468
469





1752


embedded image


454
455





1753


embedded image


502
503





1754


embedded image


468
469





1755


embedded image


468
469





1756


embedded image


486
487





1757


embedded image


454
455





1758


embedded image


470
471





1759


embedded image


484
485





1760


embedded image


488
489





1761


embedded image


553
554





1762


embedded image


530
531





1763


embedded image


480
481





1764


embedded image


466
467





1765


embedded image


514
515





1766


embedded image


480
481





1767


embedded image


480
481





1768


embedded image


498
499





1769


embedded image


466
467





1770


embedded image


482
483





1771


embedded image


496
497





1772


embedded image


500
501





1773


embedded image


565
566





1774


embedded image


617
618





1775


embedded image


567
568





1776


embedded image


552
553





1777


embedded image


601
602





1778


embedded image


567
568





1779


embedded image


567
568





1780


embedded image


585
586





1781


embedded image


552
553





1782


embedded image


568
569





1783


embedded image


582
583





1784


embedded image


586
587





1785


embedded image


652
653





1786


embedded image


562
563





1787


embedded image


512
513





1788


embedded image


498
499





1789


embedded image


546
547





1790


embedded image


512
513





1791


embedded image


512
513





1792


embedded image


530
531





1793


embedded image


498
499





1794


embedded image


514
515





1795


embedded image


528
529





1796


embedded image


532
533





1797


embedded image


597
598





1798


embedded image


598
599





1799


embedded image


548
549





1800


embedded image


534
535





1801


embedded image


582
583





1802


embedded image


548
549





1803


embedded image


548
549





1804


embedded image


566
567





1805


embedded image


534
535





1806


embedded image


550
551





1807


embedded image


564
565





1808


embedded image


568
569





1809


embedded image


633
634





1810


embedded image


572
573





1811


embedded image


522
523





1812


embedded image


508
509





1813


embedded image


556
557





1814


embedded image


522
523





1815


embedded image


522
523





1816


embedded image


540
541





1817


embedded image


508
509





1818


embedded image


524
525





1819


embedded image


538
539





1820


embedded image


542
543





1821


embedded image


607
608





1822


embedded image


558
559





1823


embedded image


508
509





1824


embedded image


494
495





1825


embedded image


542
543





1826


embedded image


508
509





1827


embedded image


508
509





1828


embedded image


526
527





1829


embedded image


494
495





1830


embedded image


510
511





1831


embedded image


524
525





1832


embedded image


528
529





1833


embedded image


593
594





1834


embedded image


560
561





1835


embedded image


510
511





1836


embedded image


496
497





1837


embedded image


544
545





1838


embedded image


510
511





1839


embedded image


510
511





1840


embedded image


528
529





1841


embedded image


496
497





1842


embedded image


512
513





1843


embedded image


526
527





1844


embedded image


530
531





1845


embedded image


595
596





1846


embedded image


582
583





1847


embedded image


532
533





1848


embedded image


518
519





1849


embedded image


566
567





1850


embedded image


532
533





1851


embedded image


532
533





1852


embedded image


550
551





1853


embedded image


518
519





1854


embedded image


534
535





1855


embedded image


548
549





1856


embedded image


552
553





1857


embedded image


617
618





1858


embedded image


530
531





1859


embedded image


480
481





1860


embedded image


466
467





1861


embedded image


514
515





1862


embedded image


480
481





1863


embedded image


480
481





1864


embedded image


498
499





1865


embedded image


466
467





1866


embedded image


482
483





1867


embedded image


496
497





1868


embedded image


500
501





1869


embedded image


565
566





1870


embedded image


596
597





1871


embedded image


546
547





1872


embedded image


532
533





1873


embedded image


580
581





1874


embedded image


546
547





1875


embedded image


546
547





1876


embedded image


564
565





1877


embedded image


532
533





1878


embedded image


548
549





1879


embedded image


562
563





1880


embedded image


566
567





1881


embedded image


631
632





1882


embedded image


600
601





1883


embedded image


550
551





1884


embedded image


536
537





1885


embedded image


584
585





1886


embedded image


550
551





1887


embedded image


550
551





1888


embedded image


568
569





1889


embedded image


536
537





1890


embedded image


552
553





1891


embedded image


566
567





1892


embedded image


570
571





1893


embedded image


635
636





1894


embedded image


600
601





1895


embedded image


550
551





1896


embedded image


536
537





1897


embedded image


584
585





1898


embedded image


550
551





1899


embedded image


550
551





1900


embedded image


568
569





1901


embedded image


536
537





1902


embedded image


552
553





1903


embedded image


566
567





1904


embedded image


570
571





1905


embedded image


635
636





1906


embedded image


600
601





1907


embedded image


550
551





1908


embedded image


536
537





1909


embedded image


584
585





1910


embedded image


550
551





1911


embedded image


550
551





1912


embedded image


568
569





1913


embedded image


536
537





1914


embedded image


552
553





1915


embedded image


566
567





1916


embedded image


570
571





1917


embedded image


635
636





1918


embedded image


516
517





1919


embedded image


466
467





1920


embedded image


452
453





1921


embedded image


500
501





1922


embedded image


466
467





1923


embedded image


466
467





1924


embedded image


484
485





1925


embedded image


452
453





1926


embedded image


468
469





1927


embedded image


482
483





1928


embedded image


486
487





1929


embedded image


551
552





1930


embedded image


546
547





1931


embedded image


496
497





1932


embedded image


482
483





1933


embedded image


530
531





1934


embedded image


496
497





1935


embedded image


496
497





1936


embedded image


514
515





1937


embedded image


482
483





1938


embedded image


498
499





1939


embedded image


512
513





1940


embedded image


516
517





1941


embedded image


581
582





1942


embedded image


498
499





1943


embedded image


448
449





1944


embedded image


434
435





1945


embedded image


482
483





1946


embedded image


448
449





1947


embedded image


448
449





1948


embedded image


466
467





1949


embedded image


434
435





1950


embedded image


449
450





1951


embedded image


464
465





1952


embedded image


468
469





1953


embedded image


533
534





1954


embedded image


600
601





1955


embedded image


550
551





1956


embedded image


536
537





1957


embedded image


584
585





1958


embedded image


550
551





1959


embedded image


550
551





1960


embedded image


568
569





1961


embedded image


536
537





1962


embedded image


552
553





1963


embedded image


566
567





1964


embedded image


570
571





1965


embedded image


635
636





1966


embedded image


560
561





1967


embedded image


510
511





1968


embedded image


496
497





1969


embedded image


544
545





1970


embedded image


510
511





1971


embedded image


510
511





1972


embedded image


528
529





1973


embedded image


496
497





1974


embedded image


512
513





1975


embedded image


526
527





1976


embedded image


530
531





1977


embedded image


595
596





1978


embedded image


550
551





1979


embedded image


500
501





1980


embedded image


486
487





1981


embedded image


534
535





1982


embedded image


500
501





1983


embedded image


500
501





1984


embedded image


518
519





1985


embedded image


486
487





1986


embedded image


501
502





1987


embedded image


516
517





1988


embedded image


520
521





1989


embedded image


585
586





1990


embedded image


526
527





1991


embedded image


476
477





1992


embedded image


462
463





1993


embedded image


510
511





1994


embedded image


476
477





1995


embedded image


476
477





1996


embedded image


494
495





1997


embedded image


462
463





1998


embedded image


477
478





1999


embedded image


492
493





2000


embedded image


496
497





2001


embedded image


561
562





2002


embedded image


512
513





2003


embedded image


462
463





2004


embedded image


448
449





2005


embedded image


496
497





2006


embedded image


462
463





2007


embedded image


462
463





2008


embedded image


480
481





2009


embedded image


448
449





2010


embedded image


464
465





2011


embedded image


478
479





2012


embedded image


482
483





2013


embedded image


547
548





2014


embedded image


576
577





2015


embedded image


526
527





2016


embedded image


512
513





2017


embedded image


560
561





2018


embedded image


526
527





2019


embedded image


526
527





2020


embedded image


544
545





2021


embedded image


512
513





2022


embedded image


528
529





2023


embedded image


542
543





2024


embedded image


546
547





2025


embedded image


611
612





2026


embedded image


514
515





2027


embedded image


464
465





2028


embedded image


450
451





2029


embedded image


498
499





2030


embedded image


464
465





2031


embedded image


464
465





2032


embedded image


482
483





2033


embedded image


450
451





2034


embedded image


465
466





2035


embedded image


480
481





2036


embedded image


484
485





2037


embedded image


549
550





2038


embedded image


576
577





2039


embedded image


526
527





2040


embedded image


512
513





2041


embedded image


560
561





2042


embedded image


526
527





2043


embedded image


526
527





2044


embedded image


544
545





2045


embedded image


512
513





2046


embedded image


528
529





2047


embedded image


542
543





2048


embedded image


546
547





2049


embedded image


611
612





2050


embedded image


562
563





2051


embedded image


512
513





2052


embedded image


498
499





2053


embedded image


546
547





2054


embedded image


512
513





2055


embedded image


512
513





2056


embedded image


530
531





2057


embedded image


498
499





2058


embedded image


514
515





2059


embedded image


528
529





2060


embedded image


532
533





2061


embedded image


597
598





2062


embedded image


532
533





2063


embedded image


482
483





2064


embedded image


468
469





2065


embedded image


516
517





2066


embedded image


482
483





2067


embedded image


482
483





2068


embedded image


500
501





2069


embedded image


468
469





2070


embedded image


484
485





2071


embedded image


498
499





2072


embedded image


502
503





2073


embedded image


567
568





2074


embedded image


498
499





2075


embedded image


448
449





2076


embedded image


434
435





2077


embedded image


482
483





2078


embedded image


448
449





2079


embedded image


448
449





2080


embedded image


466
467





2081


embedded image


434
435





2082


embedded image


449
450





2083


embedded image


464
465





2084


embedded image


468
469





2085


embedded image


533
534





2086


embedded image


522
523





2087


embedded image


472
473





2088


embedded image


458
459





2089


embedded image


506
507





2090


embedded image


472
473





2091


embedded image


472
473





2092


embedded image


490
491





2093


embedded image


458
459





2094


embedded image


473
474





2095


embedded image


487
488





2096


embedded image


492
493





2097


embedded image


557
558





2098


embedded image


622
623





2099


embedded image


572
573





2100


embedded image


558
559





2101


embedded image


606
607





2102


embedded image


572
573





2103


embedded image


572
573





2104


embedded image


590
591





2105


embedded image


558
559





2106


embedded image


574
575





2107


embedded image


588
589





2108


embedded image


592
593





2109


embedded image


657
658





2110


embedded image


600
601





2111


embedded image


550
551





2112


embedded image


536
537





2113


embedded image


584
585





2114


embedded image


550
551





2115


embedded image


550
551





2116


embedded image


569
570





2117


embedded image


536
537





2118


embedded image


552
553





2119


embedded image


566
567





2121


embedded image


570
571





2121


embedded image


636
637





2122


embedded image


500
501





2123


embedded image


450
451





2124


embedded image


436
437





2125


embedded image


484
485





2126


embedded image


450
451





2127


embedded image


450
451





2128


embedded image


468
469





2129


embedded image


436
437





2130


embedded image


451
452





2131


embedded image


465
466





2132


embedded image


470
471





2133


embedded image


535
536





2134


embedded image


546
547





2135


embedded image


496
497





2136


embedded image


482
483





2137


embedded image


530
531





2138


embedded image


496
497





2139


embedded image


496
497





2140


embedded image


514
515





2141


embedded image


482
483





2142


embedded image


498
499





2143


embedded image


512
513





2144


embedded image


516
517





2145


embedded image


581
582





2146


embedded image


566
567





2147


embedded image


516
517





2148


embedded image


502
503





2149


embedded image


550
551





2150


embedded image


516
517





2151


embedded image


516
517





2152


embedded image


534
535





2153


embedded image


502
503





2154


embedded image


518
519





2155


embedded image


532
533





2156


embedded image


536
537





2157


embedded image


601
602





2158


embedded image


600
601





2159


embedded image


550
551





2160


embedded image


536
537





2161


embedded image


584
585





2162


embedded image


550
551





2163


embedded image


550
551





2164


embedded image


569
570





2165


embedded image


536
537





2166


embedded image


552
553





2167


embedded image


566
567





2168


embedded image


570
571





2169


embedded image


636
637





2170


embedded image


546
547





2171


embedded image


496
497





2172


embedded image


482
483





2173


embedded image


530
531





2174


embedded image


496
497





2175


embedded image


496
497





2176


embedded image


514
515





2177


embedded image


482
483





2178


embedded image


498
499





2179


embedded image


512
513





2180


embedded image


516
517





2181


embedded image


581
582





2182


embedded image


546
547





2183


embedded image


496
497





2184


embedded image


482
483





2185


embedded image


530
531





2186


embedded image


496
497





2187


embedded image


496
497





2188


embedded image


514
515





2189


embedded image


482
483





2190


embedded image


498
499





2191


embedded image


512
513





2192


embedded image


516
517





2193


embedded image


581
582





2194


embedded image


574
475





2195


embedded image


524
525





2196


embedded image


510
511





2197


embedded image


558
559





2198


embedded image


524
525





2199


embedded image


524
525





2200


embedded image


542
543





2201


embedded image


510
511





2202


embedded image


526
527





2203


embedded image


540
541





2204


embedded image


544
545





2205


embedded image


609
610





2206


embedded image


546
547





2207


embedded image


496
497





2208


embedded image


482
483





2209


embedded image


530
531





2210


embedded image


496
497





2211


embedded image


496
497





2212


embedded image


514
515





2213


embedded image


482
483





2214


embedded image


498
499





2215


embedded image


512
513





2216


embedded image


516
517





2217


embedded image


581
582





2218


embedded image


564
565





2219


embedded image


514
515





2220


embedded image


500
501





2221


embedded image


548
549





2222


embedded image


514
515





2223


embedded image


514
515





2224


embedded image


532
533





2225


embedded image


500
501





2226


embedded image


516
517





2227


embedded image


530
531





2228


embedded image


534
535





2229


embedded image


599
600





2230


embedded image


564
565





2231


embedded image


514
515





2232


embedded image


500
501





2233


embedded image


548
549





2234


embedded image


514
515





2235


embedded image


514
515





2236


embedded image


532
533





2237


embedded image


500
501





2238


embedded image


516
517





2239


embedded image


530
531





2240


embedded image


534
535





2241


embedded image


599
600





2242


embedded image


582
583





2243


embedded image


532
533





2244


embedded image


518
519





2245


embedded image


566
567





2246


embedded image


532
533





2247


embedded image


532
533





2248


embedded image


550
551





2249


embedded image


518
519





2250


embedded image


534
535





2251


embedded image


548
549





2252


embedded image


552
553





2253


embedded image


617
618





2254


embedded image


538
539





2255


embedded image


488
489





2256


embedded image


474
475





2257


embedded image


522
523





2258


embedded image


488
489





2259


embedded image


488
489





2260


embedded image


506
507





2261


embedded image


474
475





2262


embedded image


490
491





2263


embedded image


504
505





2264


embedded image


508
509





2265


embedded image


573
574





2266


embedded image


560
561





2267


embedded image


510
511





2268


embedded image


496
497





2269


embedded image


544
545





2270


embedded image


510
511





2271


embedded image


510
511





2272


embedded image


528
529





2273


embedded image


496
497





2274


embedded image


512
513





2275


embedded image


526
527





2276


embedded image


530
531





2277


embedded image


595
596





2278


embedded image


568
569





2279


embedded image


518
519





2280


embedded image


504
505





2281


embedded image


552
553





2282


embedded image


518
519





2283


embedded image


518
519





2284


embedded image


536
537





2285


embedded image


504
505





2286


embedded image


519
520





2287


embedded image


534
535





2288


embedded image


538
539





2289


embedded image


603
604





2290


embedded image


606
607





2291


embedded image


556
557





2292


embedded image


542
543





2293


embedded image


590
591





2294


embedded image


556
557





2295


embedded image


556
557





2296


embedded image


574
575





2297


embedded image


542
543





2298


embedded image


558
559





2299


embedded image


572
573





2300


embedded image


576
577





2301


embedded image


641
642





2302


embedded image


526
527





2303


embedded image


476
477





2304


embedded image


462
463





2305


embedded image


510
511





2306


embedded image


476
477





2307


embedded image


476
477





2308


embedded image


494
495





2309


embedded image


462
463





2310


embedded image


478
479





2311


embedded image


492
493





2312


embedded image


496
497





2313


embedded image


561
562





2314


embedded image


636
637





2315


embedded image


586
587





2316


embedded image


572
573





2317


embedded image


620
621





2318


embedded image


586
587





2319


embedded image


586
587





2320


embedded image


604
605





2321


embedded image


572
573





2322


embedded image


588
589





2323


embedded image


602
603





2324


embedded image


606
607





2325


embedded image


671
672





2326


embedded image


512
513





2327


embedded image


462
463





2328


embedded image


448
449





2329


embedded image


496
497





2330


embedded image


462
563





2331


embedded image


462
463





2332


embedded image


480
481





2333


embedded image


448
449





2334


embedded image


464
465





2335


embedded image


478
479





2336


embedded image


482
483





2337


embedded image


547
548





2338


embedded image


524
525





2339


embedded image


474
475





2340


embedded image


460
461





2341


embedded image


508
509





2342


embedded image


474
475





2343


embedded image


474
475





2344


embedded image


492
493





2345


embedded image


460
461





2346


embedded image


476
477





2347


embedded image


490
491





2348


embedded image


494
495





2349


embedded image


559
560





2350


embedded image


610
611





2351


embedded image


560
561





2352


embedded image


546
547





2353


embedded image


594
595





2354


embedded image


560
561





2355


embedded image


560
561





2356


embedded image


579
580





2357


embedded image


546
547





2358


embedded image


562
563





2359


embedded image


576
577





2360


embedded image


580
581





2361


embedded image


646
647





2362


embedded image


556
557





2363


embedded image


506
507





2364


embedded image


492
493





2365


embedded image


540
541





2366


embedded image


506
507





2367


embedded image


506
507





2368


embedded image


524
525





2369


embedded image


492
493





2370


embedded image


508
509





2371


embedded image


522
523





2372


embedded image


526
527





2373


embedded image


591
592





2374


embedded image


592
593





2375


embedded image


542
543





2376


embedded image


528
529





2377


embedded image


576
577





2378


embedded image


542
543





2379


embedded image


542
543





2380


embedded image


560
561





2381


embedded image


528
529





2382


embedded image


544
545





2383


embedded image


558
559





2384


embedded image


562
563





2385


embedded image


627
628





2386


embedded image


566
567





2387


embedded image


516
517





2388


embedded image


502
503





2389


embedded image


550
551





2390


embedded image


516
517





2391


embedded image


516
517





2392


embedded image


534
535





2393


embedded image


502
503





2394


embedded image


518
519





2395


embedded image


532
533





2396


embedded image


536
537





2397


embedded image


601
602





2398


embedded image


552
553





2399


embedded image


502
503





2400


embedded image


488
489





2401


embedded image


536
537





2402


embedded image


502
503





2403


embedded image


502
503





2404


embedded image


520
521





2405


embedded image


488
489





2406


embedded image


504
505





2407


embedded image


518
519





2408


embedded image


522
523





2409


embedded image


587
588





2410


embedded image


554
555





2411


embedded image


504
505





2412


embedded image


490
491





2413


embedded image


538
539





2414


embedded image


504
505





2415


embedded image


504
505





2416


embedded image


522
523





2417


embedded image


490
491





2418


embedded image


506
507





2419


embedded image


520
521





2420


embedded image


524
525





2421


embedded image


589
590





2422


embedded image


550
551





2424


embedded image


500
501





2424


embedded image


486
487





2425


embedded image


534
535





2426


embedded image


500
501





2427


embedded image


500
501





2428


embedded image


518
519





2429


embedded image


486
487





2430


embedded image


502
503





2431


embedded image


516
517





2432


embedded image


520
521





2433


embedded image


585
586





2434


embedded image


524
525





2435


embedded image


474
475





2436


embedded image


460
461





2437


embedded image


508
509





2438


embedded image


474
475





2439


embedded image


474
475





2440


embedded image


492
493





2441


embedded image


460
461





2442


embedded image


475
476





2443


embedded image


490
491





2444


embedded image


494
495





2445


embedded image


559
560





2446


embedded image


590
591





2447


embedded image


540
541





2448


embedded image


526
527





2449


embedded image


574
575





2450


embedded image


540
541





2451


embedded image


540
541





2452


embedded image


558
559





2453


embedded image


526
527





2454


embedded image


542
543





2455


embedded image


556
557





2456


embedded image


560
561





2457


embedded image


625
626





2458


embedded image


594
495





2459


embedded image


544
545





2460


embedded image


530/531






2461


embedded image


578
579





2462


embedded image


544
545





2463


embedded image


544
545





2464


embedded image


562
563





2465


embedded image


530
531





2466


embedded image


546
547





2467


embedded image


560
561





2468


embedded image


564
565





2469


embedded image


629
630





2470


embedded image


594
595





2471


embedded image


544
545





2472


embedded image


530
531





2473


embedded image


578
579





2474


embedded image


544
545





2475


embedded image


544
545





2476


embedded image


562
563





2477


embedded image


530
531





2478


embedded image


546
547





2479


embedded image


560
561





2480


embedded image


564
565





2481


embedded image


629
630





2482


embedded image


594
595





2483


embedded image


544
545





2484


embedded image


530
531





2485


embedded image


578
579





2486


embedded image


544
545





2487


embedded image


544
545





2488


embedded image


562
563





2489


embedded image


530
531





2490


embedded image


546
547





2491


embedded image


560
561





2492


embedded image


564/565






2493


embedded image


629
630





2494


embedded image


510
511





2495


embedded image


460
461





2496


embedded image


446
447





2497


embedded image


494
495





2498


embedded image


460
461





2499


embedded image


460
461





2500


embedded image


478
479





2501


embedded image


446
447





2502


embedded image


461
462





2503


embedded image


476
477





2504


embedded image


480
481





2505


embedded image


545
546





2506


embedded image


540
541





2507


embedded image


490
491





2508


embedded image


476
477





2509


embedded image


524
525





2510


embedded image


490
491





2511


embedded image


490
491





2512


embedded image


508
509





2513


embedded image


476
477





2514


embedded image


492
493





2515


embedded image


506
507





2516


embedded image


510
511





2517


embedded image


575
576





2518


embedded image


510
511





2519


embedded image


460
461





2520


embedded image


446
447





2521


embedded image


494
495





2522


embedded image


460
461





2523


embedded image


460
461





2524


embedded image


478
479





2525


embedded image


446
447





2526


embedded image


462
463





2527


embedded image


476
477





2528


embedded image


480
481





2529


embedded image


545
546





2530


embedded image


612
613





2531


embedded image


562
563





2532


embedded image


548
549





2533


embedded image


596
597





2534


embedded image


562
563





2535


embedded image


562
563





2536


embedded image


580
581





2537


embedded image


548
549





2538


embedded image


564
565





2539


embedded image


578
579





2540


embedded image


582
583





2541


embedded image


647
648





2542


embedded image


572
573





2543


embedded image


522
523





2544


embedded image


508
509





2545


embedded image


556
557





2546


embedded image


522
523





2547


embedded image


522
523





2548


embedded image


540
541





2549


embedded image


508
509





2550


embedded image


524
525





2551


embedded image


538
539





2552


embedded image


542
543





2553


embedded image


607
608





2554


embedded image


562
563





2555


embedded image


512
513





2556


embedded image


498
499





2557


embedded image


546
547





2558


embedded image


512
513





2559


embedded image


512
513





2560


embedded image


530
531





2561


embedded image


498
499





2562


embedded image


514
515





2563


embedded image


528
529





2564


embedded image


532
533





2565


embedded image


597
598





2566


embedded image


538
539





2567


embedded image


488
489





2568


embedded image


474
475





2569


embedded image


522/523






2570


embedded image


488
489





2571


embedded image


488
489





2572


embedded image


506
507





2573


embedded image


474
475





2574


embedded image


490
491





2575


embedded image


504
505





2576


embedded image


508
509





2577


embedded image


573
574





2578


embedded image


524
525





2579


embedded image


474
475





2580


embedded image


460
461





2581


embedded image


508
509





2582


embedded image


474
475





2583


embedded image


474
475





2584


embedded image


492
493





2585


embedded image


460
461





2586


embedded image


476
477





2587


embedded image


490
491





2588


embedded image


494
495





2589


embedded image


559
560





2590


embedded image


588
589





2591


embedded image


538
539





2592


embedded image


524
525





2593


embedded image


572
573





2594


embedded image


538
539





2595


embedded image


538
539





2596


embedded image


556
557





2597


embedded image


524
525





2598


embedded image


540
541





2599


embedded image


554
555





2600


embedded image


558
559





2601


embedded image


623
624





2602


embedded image


526
527





2603


embedded image


476
477





2604


embedded image


462
463





2605


embedded image


510
511





2606


embedded image


476
477





2607


embedded image


476
477





2608


embedded image


494
495





2609


embedded image


462
463





2610


embedded image


478
479





2611


embedded image


492
493





2612


embedded image


496
497





2613


embedded image


561
562





2614


embedded image


588
589





2615


embedded image


538
539





2616


embedded image


524
525





2617


embedded image


572
573





2618


embedded image


538
539





2619


embedded image


538
539





2620


embedded image


556
557





2621


embedded image


524
525





2622


embedded image


540
541





2623


embedded image


554
555





2624


embedded image


558
559





2625


embedded image


623
624





2626


embedded image


574
575





2627


embedded image


524
525





2628


embedded image


510
511





2629


embedded image


558
559





2630


embedded image


524
525





2631


embedded image


524
5252





2632


embedded image


542
543





2633


embedded image


510
511





2634


embedded image


526
527





2635


embedded image


540
541





2636


embedded image


544
545





2637


embedded image


609
610





2638


embedded image


544
545





2639


embedded image


494
495





2640


embedded image


480
481





2641


embedded image


528
529





2642


embedded image


494
495





2643


embedded image


494
495





2644


embedded image


512
513





2645


embedded image


480
481





2646


embedded image


496
497





2647


embedded image


510
511





2648


embedded image


514
515





2649


embedded image


579
580





2650


embedded image


510
511





2651


embedded image


460
461





2652


embedded image


446
447





2653


embedded image


494
495





2654


embedded image


460
461





2655


embedded image


460
461





2656


embedded image


478
479





2657


embedded image


446
447





2658


embedded image


462
463





2659


embedded image


476
477





2660


embedded image


480
481





2661


embedded image


545
546





2662


embedded image


534
535





2663


embedded image


484
485





2664


embedded image


470
471





2665


embedded image


518
519





2666


embedded image


484
485





2667


embedded image


484
485





2668


embedded image


502
503





2669


embedded image


470
471





2670


embedded image


486
487





2671


embedded image


500
501





2672


embedded image


504
505





2673


embedded image


569
570





2674


embedded image


634
635





2675


embedded image


584
585





2676


embedded image


570
571





2677


embedded image


618
619





2678


embedded image


584
585





2679


embedded image


584
585





2680


embedded image


602
603





2681


embedded image


570
571





2682


embedded image


586
587





2683


embedded image


600
601





2684


embedded image


604
605





2685


embedded image


669
670





2686


embedded image


613
614





2687


embedded image


563
564





2688


embedded image


548
549





2689


embedded image


597
598





2690


embedded image


563
564





2691


embedded image


563
564





2692


embedded image


581
582





2693


embedded image


548
549





2694


embedded image


564
565





2695


embedded image


578
579





2696


embedded image


582
583





2697


embedded image


648
649





2698


embedded image


512
513





2699


embedded image


462
463





2700


embedded image


448
449





2701


embedded image


496
497





2702


embedded image


462
463





2703


embedded image


462
463





2704


embedded image


480
481





2705


embedded image


448
449





2706


embedded image


463
464





2707


embedded image


478
479





2708


embedded image


482
483





2709


embedded image


547
548





2710


embedded image


558
559





2711


embedded image


508
509





2712


embedded image


494
495





2713


embedded image


542
543





2714


embedded image


508
509





2715


embedded image


508
509





2716


embedded image


526
527





2717


embedded image


494
495





2718


embedded image


510
511





2719


embedded image


524
525





2720


embedded image


528
529





2721


embedded image


593
594





2722


embedded image


578
579





2723


embedded image


528
529





2724


embedded image


514
515





2725


embedded image


562
563





2726


embedded image


528
529





2727


embedded image


528
529





2728


embedded image


546
547





2729


embedded image


514
515





2730


embedded image


530
531





2731


embedded image


544
545





2732


embedded image


548
549





2733


embedded image


613
614





2734


embedded image


613
614





2735


embedded image


563
564





2736


embedded image


548
549





2737


embedded image


597
598





2738


embedded image


563
564





2739


embedded image


563
564





2740


embedded image


581
582





2741


embedded image


548
549





2742


embedded image


564
565





2743


embedded image


578
579





2744


embedded image


582
583





2745


embedded image


648
649





2746


embedded image


558
559





2747


embedded image


508
509





2748


embedded image


494
495





2749


embedded image


542
543





2750


embedded image


508
509





2751


embedded image


508
509





2752


embedded image


526
527





2753


embedded image


494
495





2754


embedded image


510
511





2755


embedded image


524
525





2756


embedded image


528
529





2757


embedded image


593
594





2758


embedded image


558
559





2759


embedded image


508
509





2760


embedded image


494
495





2761


embedded image


542
543





2762


embedded image


508
509





2763


embedded image


508
509





2764


embedded image


526
527





2765


embedded image


494
495





2766


embedded image


510
511





2767


embedded image


524
525





2768


embedded image


528
529





2769


embedded image


593
594





2770


embedded image


586
587





2771


embedded image


536
537





2772


embedded image


522
523





2773


embedded image


570
571





2774


embedded image


536
537





2775


embedded image


536
537





2776


embedded image


554
555





2777


embedded image


522
523





2778


embedded image


538
539





2779


embedded image


552
553





2780


embedded image


556
557





2781


embedded image


621
622





2782


embedded image


558
559





2783


embedded image


508
509





2784


embedded image


494
495





2785


embedded image


542
543





2786


embedded image


508
509





2787


embedded image


508
509





2788


embedded image


526
527





2789


embedded image


494
495





2790


embedded image


510
511





2791


embedded image


524
525





2792


embedded image


528
529





2793


embedded image


593
594





2794


embedded image


576
577





2795


embedded image


526
527





2796


embedded image


512
513





2797


embedded image


560
561





2798


embedded image


526
527





2799


embedded image


526
527





2800


embedded image


544
545





2801


embedded image


512
513





2802


embedded image


528
529





2803


embedded image


542
543





2804


embedded image


546
547





2805


embedded image


611/612






2806


embedded image


576
577





2807


embedded image


526
527





2808


embedded image


512
513





2809


embedded image


560
561





2810


embedded image


526
527





2811


embedded image


526
527





2812


embedded image


544
545





2813


embedded image


512
513





2814


embedded image


528
529





2815


embedded image


542
543





2816


embedded image


546
547





2817


embedded image


611
612





2818


embedded image


594
595





2819


embedded image


544
545





2820


embedded image


530
531





2821


embedded image


578
579





2822


embedded image


544
545





2823


embedded image


544
545





2824


embedded image


562
563





2825


embedded image


530
531





2826


embedded image


546
547





2827


embedded image


560
561





2828


embedded image


564
565





2829


embedded image


629
630





2830


embedded image


550
551





2831


embedded image


500
501





2832


embedded image


486
487





2833


embedded image


534
535





2834


embedded image


500
501





2835


embedded image


500
501





2836


embedded image


518
519





2837


embedded image


486
487





2838


embedded image


502
503





2839


embedded image


516
517





2840


embedded image


520
521





2841


embedded image


585
586





2842


embedded image


572
573





2843


embedded image


522
523





2844


embedded image


508
509





2845


embedded image


556
557





2846


embedded image


522
523





2847


embedded image


522
523





2848


embedded image


540
541





2849


embedded image


508
509





2850


embedded image


524
525





2851


embedded image


538
539





2852


embedded image


542
543





2853


embedded image


607
608





2854


embedded image


580
581





2855


embedded image


530
531





2856


embedded image


516
517





2857


embedded image


564
565





2858


embedded image


530
531





2859


embedded image


530
531





2860


embedded image


548
549





2861


embedded image


516
517





2862


embedded image


532
533





2863


embedded image


546
547





2864


embedded image


550
551





2865


embedded image


615
616





2866


embedded image


618
619





2867


embedded image


568
569





2868


embedded image


554
555





2869


embedded image


602
603





2870


embedded image


568
569





2871


embedded image


568
569





2872


embedded image


586
587





2873


embedded image


554
555





2874


embedded image


570
571





2875


embedded image


584
585





2876


embedded image


588
589





2877


embedded image


653
654





2878


embedded image


538
539





2879


embedded image


488
489





2880


embedded image


474
475





2881


embedded image


522
523





2882


embedded image


488
489





2883


embedded image


488
489





2884


embedded image


506
507





2885


embedded image


474
475





2886


embedded image


490
491





2887


embedded image


504
505





2888


embedded image


508
509





2889


embedded image


573
574





2890


embedded image


648
649





2891


embedded image


598
599





2892


embedded image


584
585





2893


embedded image


632
633





2894


embedded image


598
599





2895


embedded image


598
599





2896


embedded image


616
617





2897


embedded image


584
585





2898


embedded image


600
601





2899


embedded image


614
615





2900


embedded image


618
619





2901


embedded image


683
684





2902


embedded image


622
623





2903


embedded image


585
586





2904


embedded image


619
620





2905


embedded image


619
620





2906


embedded image


585
586





2907


embedded image


568
569





2908


embedded image


583
584





2909


embedded image


568
569





2910


embedded image


462
463





2911


embedded image


589
590





2912


embedded image


589
590





2913


embedded image


639
640





2914


embedded image


571
572





2915


embedded image


577
578





2916


embedded image


617
618





2917


embedded image


617
618





2918


embedded image


583
584





2919


embedded image


617
618





2920


embedded image


617
618





2921


embedded image


617
618





2922


embedded image


599
600





2923


embedded image


599
600





2924


embedded image


639
640





2925


embedded image


591
592





2926


embedded image


591
592





2927


embedded image


564
565





2928


embedded image


554
555





2929


embedded image


597
598





2930


embedded image


659
660





2931


embedded image


599
600





2932


embedded image


599
600





2933


embedded image


689
690





2934


embedded image


569
570





2935


embedded image


569
570





2936


embedded image


571
572





2937


embedded image


571
572





2938


embedded image


633
634





2939


embedded image


564
565





2940


embedded image


571
572





2941


embedded image


605
606





2942


embedded image


608
609





2943


embedded image


580
581





2944


embedded image


605
606





2945


embedded image


741
742





2946


embedded image


550
551





2947


embedded image


659
660





2948


embedded image


625
626





2949


embedded image


659
660





2950


embedded image


554
555





2951


embedded image


648
649





2952


embedded image


659
660





2953


embedded image


659
660





2954


embedded image


659
660





2955


embedded image


592
593





2956


embedded image


667
668





2957


embedded image


667
668





2958


embedded image


565
566





2959


embedded image


592
593





2960


embedded image


592
593





2961


embedded image


599
600





2962


embedded image


667
668





2963


embedded image


702
703





2964


embedded image


688
689





2965


embedded image


667
668





2966


embedded image


512
513





2967


embedded image


536
537





2968


embedded image


659
660





2969


embedded image


592
593





2970


embedded image


592
593





2971


embedded image


725
726





2972


embedded image


617
618





2973


embedded image


615
616





2974


embedded image


588
589





2975


embedded image


691
692





2976


embedded image


566
567





2977


embedded image


589
590





2978


embedded image


571
572





2979


embedded image


501
502





2980


embedded image


599
600





2981


embedded image


623
624





2982


embedded image


552
553





2983


embedded image


641
642





2984


embedded image


579
580





2985


embedded image


593
594





2986


embedded image


613
614





2987


embedded image


627
628





2988


embedded image


605
606





2989


embedded image


619
620





2990


embedded image


625
626





2991


embedded image


591
592





2992


embedded image


617
618





2993


embedded image


643
644





2994


embedded image


667
668





2995


embedded image


669
670





2996


embedded image


555
556





2997


embedded image


639
640





2998


embedded image


637
638





2999


embedded image


596
597





3000


embedded image


581
582





3001


embedded image


579
580





3002


embedded image


625
626





3003


embedded image


623
624





3004


embedded image


659
660





3005


embedded image


657
658





3006


embedded image


595
596





3007


embedded image


597
598





3008


embedded image


669
670





3009


embedded image


576
577





3010


embedded image


574
575





3011


embedded image


590
591





3012


embedded image


611
612





3013


embedded image


609
610





3014


embedded image


611
612





3015


embedded image


627
628





3016


embedded image


639
640





3017


embedded image


597
598





3018


embedded image


623
624





3019


embedded image


609
610





3020


embedded image


681
682





3021


embedded image


679
680





3022


embedded image


578
579





3023


embedded image


605
606





3024


embedded image


611
612





3025


embedded image


603
604





3026


embedded image


605
606










In addition, synthesis of the peptide mimetics of the library of the present invention may be accomplished using the General Scheme of [4,3,0] Reverse-Turn Mimetic Library as follows:




embedded image


Synthesis of the peptide mimetics of the bicyclic template libraries of the present invention was accomplished using FlexChem Reactor Block which has 96 well plate by known techniques. In the above scheme ‘Pol’ represents Bromoacetal resin (Advanced ChemTech) and detailed procedure is illustrated below.


Step 1


The bromoacetal resin (1.6 mmol/g) and a solution of R1 amine in DMSO (2M solution) were placed in 96 well Robbins block (FlexChem). The reaction mixture was shaken at 60° C. using rotating oven [Robbins Scientific] for 12 hours. The resin was washed with DMF, MeOH, and then DCM


Step 2


A solution of commercial available Fmoc-Amino Acids (4 equiv.), PyBob (4 equiv.), HOAt (4 equiv.), and DIEA (12 equiv.) in DMF was added to the resin. After the reaction mixture was shaken for 12 hours at room temperature, the resin was washed with DMF, MeOH, and then DCM.


Step 3


To the resin swollen by DMF before reaction was added 25% piperidine in DMF. After the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and then washed with DMF, Methanol, then DCM. A solution of hydrazine carbamoyl chloride (4 equiv.), HOBt (4 equiv.), and DIC (4 equiv.) in DMF was added to the resin. After the reaction mixture was shaken for 12 hours at room temperature, the resin was washed with DMF, MeOH, and then DCM.


Step 4


To the resin swollen by DMF before reaction was added 25% piperidine in DMF. After the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and then washed with DMF, Methanol, then DCM. To the resin swollen by DCM before reaction was added R1-isocyanate (5 equiv.) in DCM. After the reaction mixture was shaken for 12 hours at room temperature the resin was washed with DMF, MeOH, then DCM.


Step 5


The resin was treated with formic acid (1.2 mL each well) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under reduced pressure using SpeedVac [SAVANT] to give the product as oil. These products were diluted with 50% water/acetonitrile and then lyophilized after freezing.


Table 3 shows a [4,3,0] reverse turn mimetics library which can be prepared according to the present invention, of which representative preparation is given in Example 5.









TABLE 3







THE [4,3,0] REVERSE TURN MIMETICS LIBRARY




embedded image























Mol.



No
R2
R4
R6
R1
Weight
M + H





610
Isoamyl
4-HO-phenyl
Methyl
Phenyl
466
467


611
Isoamyl
4-HO-phenyl
Methyl
4-Me-phenyl
480
481


612
Isoamyl
4-HO-phenyl
Methyl
3,5-Me2-phenyl
494
495


613
Isoamyl
4-HO-phenyl
Methyl
4-MeO-phenyl
496
497


614
Isoamyl
4-HO-phenyl
Methyl
4-CF3-phenyl
534
535


615
Isoamyl
4-HO-phenyl
Methyl
Cyclohexyl
472
473


616
Isoamyl
4-HO-phenyl
Methyl
Benzyl
480
481





617
Isoamyl
4-HO-phenyl
Methyl


embedded image


494
495





618
Isoamyl
4-HO-phenyl
Methyl
4-MeO-benzyl
510
511


619
Isoamyl
4-HO-phenyl
Methyl
Phenethyl
494
495


620
Isoamyl
4-HO-phenyl
Methyl
Pentyl
460
461


621
Isoamyl
4-HO-phenyl
Methyl
Hexyl
474
475


622
Benzyl
4-HO-phenyl
Methyl
Phenyl
486
487


623
Benzyl
4-HO-phenyl
Methyl
4-Me-phenyl
500
501


624
Benzyl
4-HO-phenyl
Methyl
3,5-Me2-phenyl
514
515


625
Benzyl
4-HO-phenyl
Methyl
4-MeO-phenyl
516
517


626
Benzyl
4-HO-phenyl
Methyl
4-CF3-phenyl
554
555


627
Benzyl
4-HO-phenyl
Methyl
Cyclohexyl
492
493


628
Benzyl
4-HO-phenyl
Methyl
Benzyl
500
501





629
Benzyl
4-HO-phenyl
Methyl


embedded image


514
515





630
Benzyl
4-HO-phenyl
Methyl
4-MeO-benzyl
530
531


631
Benzyl
4-HO-phenyl
Methyl
Phenethyl
514
515


632
Benzyl
4-HO-phenyl
Methyl
Pentyl
480
481


633
Benzyl
4-HO-phenyl
Methyl
Hexyl
494
495


634
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
Phenyl
536
537


635
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
4-Me-phenyl
550
551


636
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
3,5-Me2-phenyl
564
565


637
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
4-MeO-phenyl
566
567


638
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
4-CF3-phenyl
604
605


639
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
Cyclohexyl
542
543


640
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
Benzyl
550
551





641
Naphth-1-ylmethyl
4-HO-phenyl
Methyl


embedded image


564
565





642
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
4-MeO-benzyl
580
581


643
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
Phenethyl
564
565


644
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
Pentyl
530
531


645
Naphth-1-ylmethyl
4-HO-phenyl
Methyl
Hexyl
544
545


646
Cyclohexylmethyl
4-HO-phenyl
Methyl
Phenyl
492
493


647
Cyclohexylmethyl
4-HO-phenyl
Methyl
4-Me-phenyl
506
507


648
Cyclohexylmethyl
4-HO-phenyl
MethyL
3,5-Me2-phenyl
520
521


649
Cyclohexylmethyl
4-HO-phenyl
Methyl
4-MeO-phenyl
522
523


650
Cyclohexylmethyl
4-HO-phenyl
Methyl
4-CF3-phenyl
560
561


651
Cyclohexylmethyl
4-HO-phenyl
Methyl
Cyclohexyl
468
469


652
Cyclohexylmethyl
4-HO-phenyl
Methyl
Benzyl
506
507





653
Cyclohexylmethyl
4-HO-phenyl
Methyl


embedded image


520
521





654
Cyclohexylmethyl
4-HO-phenyl
Methyl
4-MeO-benzyl
536
537


655
Cyclohexylmethyl
4-HO-phenyl
Methyl
Phenethyl
520
521


656
Cyclohexylmethyl
4-HO-phenyl
Methyl
Pentyl
486
487


657
Cyclohexylmethyl
4-HO-phenyl
Methyl
Hexyl
500
501


658
4-methylbenzyl
4-HO-phenyl
Methyl
Phenyl
500
501


659
4-methylbenzyl
4-HO-phenyl
Methyl
4-Me-phenyl
514
515


660
4-methylbenzyl
4-HO-phenyl
Methyl
3,5-Me2-phenyl
528
529


661
4-methylbenzyl
4-HO-phenyl
Methyl
4-MeO-phenyl
530
531


662
4-methylbenzyl
4-HO-phenyl
Methyl
4-CF3-phenyl
568
569


663
4-methylbenzyl
4-HO-phenyl
Methyl
Cyclohexyl
506
507


664
4-methylbenzyl
4-HO-phenyl
Methyl
Benzyl
514
515





665
4-methylbenzyl
4-HO-phenyl
Methyl


embedded image


528
529





666
4-methylbenzyl
4-HO-phenyl
Methyl
4-MeO-benzyl
544
545


667
4-methylbenzyl
4-HO-phenyl
Methyl
Phenethyl
528
529


668
4-methylbenzyl
4-HO-phenyl
Methyl
Pentyl
494
495


669
4-methylbenzyl
4-HO-phenyl
Methyl
Hexyl
508
509


670
Methoxypropyl
4-HO-phenyl
Methyl
Phenyl
468
469


671
Methoxypropyl
4-HO-phenyl
Methyl
4-Me-phenyl
482
483


672
Methoxypropyl
4-HO-phenyl
Methyl
3,5-Me2-phenyl
496
497


673
Methoxypropyl
4-HO-phenyl
Methyl
4-MeO-phenyl
498
499


674
Methoxypropyl
4-HO-phenyl
Methyl
4-CF3-phenyl
536
537


675
Methoxypropyl
4-HO-phenyl
Methyl
Cyclohexyl
474
475


676
Methoxypropyl
4-HO-phenyl
Methyl
Benzyl
482
483





677
Methoxypropyl
4-HO-phenyl
Methyl


embedded image


496
497





678
Methoxypropyl
4-HO-phenyl
Methyl
4-MeO-benzyl
512
513


679
Methoxypropyl
4-HO-phenyl
Methyl
Phenethyl
496
497


680
Methoxypropyl
4-HO-phenyl
Methyl
Pentyl
462
463


681
Methoxypropyl
4-HO-phenyl
Methyl
Hexyl
476
477


682
Phenethyl
4-HO-phenyl
Methyl
Phenyl
500
501


683
Phenethyl
4-HO-phenyl
Methyl
4-Me-phenyl
514
515


684
Phenethyl
4-HO-phenyl
Methyl
3,5-Me2-phenyl
528
529


685
Phenethyl
4-HO-phenyl
Methyl
4-MeO-phenyl
530
531


686
Phenethyl
4-HO-phenyl
Methyl
4-CF3-phenyl
568
569


687
Phenethyl
4-HO-phenyl
Methyl
Cyclohexyl
506
507


688
Phenethyl
4-HO-phenyl
Methyl
Benzyl
514
515





689
Phenethyl
4-HO-phenyl
Methyl


embedded image


528
529





690
Phenethyl
4-HO-phenyl
Methyl
4-MeO-benzyl
544
545


691
Phenethyl
4-HO-phenyl
Methyl
Phenethyl
528
529


692
Phenethyl
4-HO-phenyl
Methyl
Pentyl
494
495


693
Phenethyl
4-HO-phenyl
Methyl
Hexyl
508
509


694
2,2-bisphenylethyl
4-HO-phenyl
Methyl
Phenyl
576
577


695
2,2-bisphenylethyl
4-HO-phenyl
Methyl
4-Me-phenyl
590
591


696
2,2-bisphenylethyl
4-HO-phenyl
Methyl
3,5-Me2-phenyl
604
605


697
2,2-bisphenylethyl
4-HO-phenyl
Methyl
4-MeO-phenyl
606
607


698
2,2-bisphenylethyl
4-HO-phenyl
Methyl
4-CF3-phenyl
644
645


699
2,2-bisphenylethyl
4-HO-phenyl
Methyl
Cyclohexyl
582
583


700
2,2-bisphenylethyl
4-HO-phenyl
Methyl
Benzyl
586
587





701
2,2-bisphenylethyl
4-HO-phenyl
Methyl


embedded image


604
605





702
2,2-bisphenylethyl
4-HO-phenyl
Methyl
4-MeO-benzyl
620
621


703
2,2-bisphenylethyl
4-HO-phenyl
Methyl
Phenethyl
604
605


704
2,2-bisphenylethyl
4-HO-phenyl
Methyl
Pentyl
570
571


705
2,2-bisphenylethyl
4-HO-phenyl
Methyl
Hexyl
584
585


706
Naphth-1-ylmethyl
Benzyl
Methyl
Phenyl
520
521


707
Naphth-1-ylmethyl
Benzyl
Methyl
4-Me-phenyl
534
535


708
Naphth-1-ylmethyl
Benzyl
Methyl
3,5-Me2-phenyl
548
549


709
Naphth-1-ylmethyl
Benzyl
Methyl
4-MeO-phenyl
550
551


710
Naphth-1-ylmethyl
Benzyl
Methyl
4-CF3-phenyl
588
589


711
Naphth-1-ylmethyl
Benzyl
Methyl
Cyclohexyl
526
527


712
Naphth-1-ylmethyl
Benzyl
Methyl
Benzyl
534
535





713
Naphth-1-ylmethyl
Benzyl
Methyl


embedded image


548
549





714
Naphth-1-ylmethyl
Benzyl
Methyl
4-MeO-benzyl
564
565


715
Naphth-1-ylmethyl
Benzyl
Methyl
Phenethyl
548
549


716
Naphth-1-ylmethyl
Benzyl
Methyl
Pentyl
514
515


717
Naphth-1-ylmethyl
Benzyl
Methyl
Hexyl
528
529





718
Naphth-1-ylmethyl


embedded image


Methyl
Phenyl
498
499





719
Naphth-1-ylmethyl


embedded image


Methyl
4-Me-phenyl
512
513





720
Naphth-1-ylmethyl


embedded image


Methyl
3,5-Me2-phenyl
526
527





721
Naphth-1-ylmethyl


embedded image


Methyl
4-MeO-phenyl
528
529





722
Naphth-1-ylmethyl


embedded image


Methyl
4-CF3-phenyl
566
567





723
Naphth-1-ylmethyl


embedded image


Methyl
Cyclohexyl
504
505





724
Naphth-1-ymethyl


embedded image


Methyl
Benzyl
512
513





725
Naphth-1-ylmethyl


embedded image


Methyl


embedded image


526
527





726
Naphth-1-ylmethyl


embedded image


Methyl
4-MeO-benzyl
542
543





727
Naphth-1-ylmethyl


embedded image


Methyl
Phenethyl
526
527





728
Naphth-1-ylmethyl


embedded image


Methyl
Pentyl
492
493





729
Naphth-1-ylmethyl


embedded image


Methyl
Hexyl
506
507





730
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
Phenyl
570
571


731
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
4-Me-phenyl
584
585


732
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
3,5-Me2-phenyl
598
599


733
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
4-MeO-phenyl
600
601


734
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
4-CF3-phenyl
638
639


735
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
Cyclohexyl
576
577


736
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
Benzyl
584
585





737
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl


embedded image


598
599





738
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
4-MeO-benzyl
614
615


739
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
Phenethyl
598
599


740
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
Pentyl
564
565


741
Naphth-1-ylmethyl
Naphth-1-ylmethyl
Methyl
Hexyl
578
579


742
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
Phenyl
526
527


743
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
4-Me-phenyl
540
541


744
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
3,5-Me2-phenyl
554
555


745
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
4-MeO-phenyl
556
557


746
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
4-CF3-phenyl
594
595


747
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
Cyclohexyl
532
533


748
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
Benzyl
540
541





749
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl


embedded image


554
555





750
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
4-MeO-benzyl
570
571


751
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
Phenethyl
554
555


752
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
Pentyl
520
521


753
Naphth-1-ylmethyl
Cyclohexylmethyl
Methyl
Hexyl
534
535


754
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
Phenyl
554
555


755
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
4-Me-phenyl
568
569


756
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
3,5-Me2-phenyl
582
583


757
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
4-MeO-phenyl
584
585


758
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
4-CF3-phenyl
622
623


759
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
Cyclohexyl
560
561


760
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
Benzyl
568
569





761
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl


embedded image


582
583





762
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
4-MeO-benzyl
598
599


763
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
Phenethyl
582
583


764
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
Pentyl
548
549


765
Naphth-1-ylmethyl
4-chlorobenzyl
Methyl
Hexyl
562
563


766
Naphth-1-ylmethyl
Methyl
Methyl
Phenyl
444
445


767
Naphth-1-ylmethyl
Methyl
Methyl
4-Me-phenyl
458
459


768
Naphth-1-ylmethyl
Methyl
Methyl
3,5-Me2-phenyl
472
473


769
Naphth-1-ylmethyl
Methyl
Methyl
4-MeO-phenyl
474
475


770
Naphth-1-ylmethyl
Methyl
Methyl
4-CF3-phenyl
512
513


771
Naphth-1-ylmethyl
Methyl
Methyl
Cyclohexyl
450
451


772
Naphth-1-ylmethyl
Methyl
Methyl
Benzyl
458
459





773
Naphth-1-ylmethyl
Methyl
Methyl


embedded image


472
473





774
Naphth-1-ylmethyl
Methyl
Methyl
4-MeO-benzyl
488
489


775
Naphth-1-ylmethyl
Methyl
Methyl
Phenethyl
472
473


776
Naphth-1-ylmethyl
Methyl
Methyl
Pentyl
438
439


777
Naphth-1-ylmethyl
Methyl
Methyl
Hexyl
452
453


778
Naphth-1-ylmethyl
Isobutyl
Methyl
Phenyl
486
487


779
Naphth-1-ylmethyl
Isobutyl
Methyl
4-Me-phenyl
500
501


780
Naphth-1-ylmethyl
Isobutyl
Methyl
3,5-Me2-phenyl
514
515


781
Naphth-1-ylmethyl
Isobutyl
Methyl
4-MeO-phenyl
516
517


782
Naphth-1-ylmethyl
Isobutyl
Methyl
4-CF3-phenyl
554
555


783
Naphth-1-ylmethyl
Isobutyl
Methyl
Cyclohexyl
492
493


784
Naphth-1-ylmethyl
Isobutyl
Methyl
Benzyl
500
501





785
Naphth-1-ylmethyl
Isobutyl
Methyl


embedded image


514
515





786
Naphth-1-ylmethyl
Isobutyl
Methyl
4-MeO-benzyl
530
531


787
Naphth-1-ylmethyl
Isobutyl
Methyl
Phenethyl
514
515


788
Naphth-1-ylmethyl
Isobutyl
Methyl
Pentyl
480
481


789
Naphth-1-ylmethyl
Isobutyl
Methyl
Hexyl
494
495


790
Naphth-1-ylmethyl
Methylthioethyl
Methyl
Phenyl
504
505


791
Naphth-1-ylmethyl
Methylthioethyl
Methyl
4-Me-phenyl
518
519


792
Naphth-1-ylmethyl
Methylthioethyl
Methyl
3,5-Me2-phenyl
532
533


793
Naphth-1-ylmethyl
Methylthioethyl
Methyl
4-MeO-phenyl
534
535


794
Naphth-1-ylmethyl
Methylthioethyl
Methyl
4-CF3-phenyl
572
573


795
Naphth-1-ylmethyl
Methylthioethyl
Methyl
Cyclohexyl
510
511


796
Naphth-1-ylmethyl
Methylthioethyl
Methyl
Benzyl
518
519





797
Naphth-1-ylmethyl
Methylthioethyl
Methyl


embedded image


532
533





798
Naphth-1-ylmethyl
Methylthioethyl
Methyl
4-MeO-benzyl
548
549


799
Naphth-1-ylmethyl
Methylthioethyl
Methyl
Phenethyl
532
533


800
Naphth-1-ylmethyl
Methylthioethyl
Methyl
Pentyl
498
499


801
Naphth-1-ylmethyl
Methylthioethyl
Methyl
Hexyl
512
513









In a further aspect of this invention, the present invention provides methods for screening the libraries for bioactivity and isolating bioactive library members.


In yet another aspect, the present invention provides a method for carrying out a binding assay. The method includes providing a composition that includes a first co-activator, an interacting protein, and a test compound. The amino acid structure of the first co-activator includes a binding motif of LXXLL (SEQ ID NO:1), LXXLI (SEQ ID NO:2) or FXXFF (SEQ ID NO:3) wherein X is any amino acid. The method further includes detecting an alteration in binding between the first co-activator and the interacting protein due to the presence of the compound, and then characterizing the test compound in terms of its effect on the binding.


The assay may be carried out by any means that can measure the effect of a test compound on the binding between two proteins. Many such assays are known in the art and can be utilized in the method of the present invention, including the so-called Two-Hybrid and Split-Hybrid systems.


The Two-Hybrid system, and various means to carry out an assay using this system, are described in, e.g., U.S. Pat. No. 6,410,245. The Split-Hybrid system has been described by, e.g., Hsiu-Ming Shiu et al. Proc. Natl. Acad. Sci. USA, 93:13896-13901, November 1996; and John D. Crispino, et al. Molecular Cell, 3:1-20, February 1999. In the Split-Hybrid system, a fusion protein is utilized where protein X is fused to the lexA DNA binding domains (pLexA) and protein Y is fused to the transcription activator VP16 (pSHM.1-LacZ). Interaction between lexA-X and VP16-Y leads to the expression of the Tetracycline repressor protein (TetR). TetR prevents transcription of the HIS3 reporter gene, making the cells unable to grow on media lacking histidine. Disruption of protein-protein interaction will restore the ability of the cells to grow on such media by shutting down expression of the tetracycline repressor. Accordingly, compounds of the present invention may be added to the growing cells, and if the addition of the compound restores the ability of the cells to grow on the media, the compound may be seen as an effective disruptor of the protein-protein interaction.


The yeast strains required to make the Split-Hybrid system work can be employed with two hybrid LexA/VP16 constructs such as those described by Stanley M. Hollenberg, et al. Molecular and Cellular Biology 15(7):3813-3822, July 1995. A useful modification of the Split-Hybrid system was utilized by Takemaru, K. I. and Moon, R. T. J. of Cell Biol. 149:249-254, 2000.


Other assay formats are also suitable. For example, reporter gene assays for AP-1, ELISA, for example, blocking the production of IL-2 by a T-cell line after stimulation with CD3 and CD28 to look for inhibitors of IL-2 transcription. Direct binding assays (between coactivators and their partners) can be performed by surface plasmon resonance spectroscopy (Biacore, Sweden, manufactures suitable instruments) or ELISA.


Exemplary transcriptional regulators include, without limitation, VP16, VP64, p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol. 23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504. Other exemplary transcription factors include, without limitation, OsGAI, HALF-1, C1, API, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP, and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev. 5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.


In a preferred embodiment, the transcriptional coactivator is a human transcriptional coactivator. In another preferred embodiment, the transcriptional coactivator is a member of the p300/CBP family of co-activators which have histone acetyltransferase activity. p300 is described for example by Eckner et al, 1994 and CBP by Bannister and Kouzarides, 1996. For the purposes of the present invention, reference to p300/CBP refers to human allelic and synthetic variants of p300, and to other mammalian variants and allelic and synthetic variants thereof, as well as fragments of said human and mammalian forms of p300. In one aspect of the assay, the interacting protein is a transcription factor or a second co-activator.


In one aspect of the assay, the interacting protein is any one of RIP140; SRC-1 (NCoA-1); TIF2 (GRIP-1; SRC-2); p (CIP; RAC3; ACTR; AIB-1; TRAM-1; SRC-3); CBP (p300); TRAPs (DRIPs); PGC-1; CARM-1; PRIP (ASC-2; AIB3; RAP250; NRC); GT-198; and SHARP (CoAA; p68; p72). In another aspect of the assay, the interacting protein is any one of TAL 1; p73; MDm2; TBP; HIF-1; Ets-1; RXR; p65; AP-1; Pit-1; HNF-4; Stat2; HPV E2; BRCA1; p45 (NF-E2); c-Jun; c-myb; Tax; Sap 1; YY1; SREBP; ATF-1; ATF-4; Cubitus; Interruptus; Gli3; MRF; AFT-2; JMY; dMad; PyLT: HPV E6; CITTA; Tat; SF-1; E2F; junB; RNA helicase A; C/EBP β; GATA-1; Neuro D; Microphthalimia; E1A; TFIIB; p53; P/CAF; Twist; Myo D; pp 9O RSK; c-Fos; and SV40 Large T. In another aspect of the assay, the interacting protein is any one of ERAP140; RIP140; RIP160; Trip1; SWI1 (SNF); ARA70; RAP46; TIF1; TIF2; GRIP1; and TRAP. In another aspect of the invention, the interacting protein is any one of VP16; VP64; p300; CBP; PCAF; SRC1 PvALF; AtHD2A; ERF-2; OsGAI; HALF-1; C1; AP-1; ARF-5; ARF-6; ARF-7; ARF-8; CPRF1; CPRF4; MYC-RP/GP; and TRAB1. In another aspect of the invention, the first co-activator is CBP or p300.


The test compound is selected from compounds as described herein. For example, compounds having the formula (I), (II), (III), (IV), (VI) and (VIa). Typically, a test compound will be evaluated at several different concentrations, where these concentrations will be selected, in part, based on the conditions of the assay, e.g., the concentrations of the first co-activator and the interacting protein. Concentrations in the range of about 0.1 to 10 μM are typical. In one aspect, the assay evaluates the relative efficacy of two compounds to affect the binding interaction between two proteins, where at least one of those two compounds is a compound of the present invention. The more effective compound can than serve as a reference compound in a study of the relationship between compound structure and compound activity.


The libraries of the present invention were screened for bioactivity by various techniques and methods. In general, the screening assay may be performed by (1) contacting the mimetics of a library with a biological target of interest, such as a receptor, to allow binding between the mimetics of the library and the target to occur, and (2) detecting the binding event by an appropriate assay, such as the calorimetric assay disclosed by Lam et al. (Nature 354:82-84, 1991) or Griminski et al. (Biotechnology 12:1008-1011, 1994) (both of which are incorporated herein by reference). In a preferred embodiment, the library members are in solution and the target is immobilized on a solid phase. Alternatively, the library may be immobilized on a solid phase and may be probed by contacting it with the target in solution.


Table 4 below shows compounds for bioactivity test selected from the library of the present invention and IC50 values thereof, which are measured by the Reporter gene assay as described in Example 6.









TABLE 4







IC50(μM) OF SELECTED LIBRARY COMPOUNDS










No
STRUCTURE
M.W.
IC50(μM)













1


embedded image


580.7
12.8





2


embedded image


579.6
12.6





3


embedded image


632.5
13.9





4


embedded image


617.6
11.8





5


embedded image


564.6
6.8





6


embedded image


564.6
6.1





7


embedded image


564.6
2.2





8


embedded image


531.6
14.5





9


embedded image


531.6
6.7





10


embedded image


531.6
4.0





11


embedded image


531.6
4.6





12


embedded image


549.6
9.0





13


embedded image


549.6
6.4





14


embedded image


549.6
17.7





15


embedded image


581.6
4.2





16


embedded image


567.6
3.8





17


embedded image


548.0
14.3





18


embedded image


548.0
3.3





19


embedded image


582.5
11.5





20


embedded image


527.6
5.1





21


embedded image


527.6
5.0





22


embedded image


543.6
10.4





23


embedded image


573.6
10.7





24


embedded image


563.7
5.0





25


embedded image


581.6
3.0





26


embedded image


543.6
7.1





27


embedded image


543.6
5.2





28


embedded image


548.0
7.5





29


embedded image


582.5
3.8





30


embedded image


597.6
7.5





31


embedded image


613.7
11.9





32


embedded image


581.6
4.1





33


embedded image


564.6
13.0





34


embedded image


565.6
4.4





35


embedded image


579.7
11.4





36


embedded image


549.6
12.5





37


embedded image


545.6
2.3





38


embedded image


556.7
7.1





39


embedded image


564.6
9.7





40


embedded image


553.6
7.0





41


embedded image


541.6
13.6





42


embedded image


574.7
18.2





43


embedded image


556.7
5.2





44


embedded image


599.6
1.3





45


embedded image


591.1
2.2





46


embedded image


570.7
4.4





47


embedded image


584.7
3.5





48


embedded image


570.7
10.9





49


embedded image


592.6
1.4





50


embedded image


574.6
1.3





51


embedded image


584.7
4.8





52


embedded image


621.69
25





53


embedded image


584.72
9.0 ± 1.5





54


embedded image


619.16
23.6 ± 5.6 





55


embedded image


584.72
7.2 ± 1.4





56


embedded image


567.65
9.3 ± 1.6





57


embedded image


582.70
9.4 ± 1.5





58


embedded image


588.68
49.1 ± 8.1 





59


embedded image


588.68
5.3 ± 1.3





60


embedded image


638.69
6.9 ± 1.7





61


embedded image


570.69
25.8





62


embedded image


616.73
9.7 ± 1.7





63


embedded image


582.70
4.1 ± 0.5





64


embedded image


616.73
25.3 ± 6.6 





65


embedded image


616.73
 19 ± 7.1





66


embedded image


598.74
11.8





67


embedded image


598.74
6.8





68


embedded image


590.68
4.3 ± 0.8





69


embedded image


563.60
1.4 ± 0.7





70


embedded image


553.62
8.8 ± 1.9





71


embedded image


596.73
6.5 ± 0.7





72


embedded image


658.76
1.6 ± 0.1





73


embedded image


658.76
3.6





74


embedded image


688.74
2.1 ± 0.2





75


embedded image


568.64
50.5 ± 18.4





76


embedded image


568.64
10.7 ± 2.5 





77


embedded image


570.67
7.2 ± 2.5





78


embedded image


570.69
4.3 ± 0.9





79


embedded image


632.76
16.5 ± 4.8 





80


embedded image


605.14
7.9 ± 2.0





81


embedded image


607.61
66.1 ± 6.8 





82


embedded image


579.60
68.1 ± 8.9 





83


embedded image


605.14
46.4 ± 3.7 





84


embedded image


740.79
46.7 ± 6.7 





85


embedded image


549.67
15.6 ± 2.2 





86


embedded image


658.76
9.9 ± 2.6





87


embedded image


624.74
8.1 ± 0.8





88


embedded image


658.76
2.2 ± 0.2





89


embedded image


553.62
13.9 ± 0.9 





90


embedded image


647.78
3.9





91


embedded image


658.76
2.9 ± 0.2





92


embedded image


658.76
3.8 ± 1.2





93


embedded image


591.67
6.8 ± 1.3





94


embedded image


666.78
7.6 ± 0.6





95


embedded image


564.64
13.3 ± 1.4 





96


embedded image


591.67
8.1 ± 0.9





97


embedded image


598.70
12.6 ± 1.2 





98


embedded image


666.78
14.4 ± 2.2 





99


embedded image


701.78
2.4 ± 0.3





100


embedded image


666.78
2.7 ± 0.3





101


embedded image


666.78
3.9





102


embedded image


511.58
62.0 ± 17.0





103


embedded image


535.59
14.5 ± 1.7 





104


embedded image


658.76
4.6 ± 0.4





105


embedded image


591.67
16.6 ± 2.7 





106


embedded image


591.67
2.6 ± 0.2





107


embedded image


724.82
2.7 ± 0.3





108


embedded image


616.67
1.6 ± 0.1





109


embedded image


616.67
2.1





110


embedded image


615.13
3.8 ± 0.6





111


embedded image


587.62
7.2 ± 0.8





112


embedded image


690.80
4.1 ± 0.8





113


embedded image


565.57
7.3 ± 1.1





114


embedded image


588.67
 0.4 ± 0.04





115


embedded image


588.67
0.8





116


embedded image


570.69
8.0 ± 0.7





117


embedded image


598.70
6.9 ± 0.6





118


embedded image


622.72
0.8 ± 0.1





119


embedded image


551.60
8.8 ± 1.3





120


embedded image


640.78
34.4 ± 4.9 





121


embedded image


578.67
3.0 ± 0.4





122


embedded image


592.70
2.1 ± 0.4





123


embedded image


612.73
11.7 ± 1.0 





124


embedded image


626.75
6.4 ± 0.4





125


embedded image


605.14
9.8 ± 0.7





126


embedded image


619.16
10.3 ± 1.5 





127


embedded image


624.74
1.8 ± 0.2





128


embedded image


590.68
0.4 ± 0.1





129


embedded image


617.15
2.4 ± 0.5





130


embedded image


642.75
6.1 ± 0.4





131


embedded image


666.78
2.2 ± 0.3





132


embedded image


668.79
2.3 ± 0.5





133


embedded image


638.77
3.5 ± 0.7





134


embedded image


636.75
4.5 ± 0.9





135


embedded image


595.65
2.4 ± 0.7





136


embedded image


580.65
28.0 ± 2.9 





137


embedded image


625.13
0.6 ± 0.1





138


embedded image


623.11
1.0 ± 0.2





139


embedded image


659.18
1.1 ± 0.1





140


embedded image


657.17
2.7 ± 0.3





141


embedded image


594.69
1.8 ± 0.3





142


embedded image


596.71
1.6 ± 0.4





143


embedded image


575.61
1.3 ± 0.2





144


embedded image


573.60
2.1 ± 0.2





145


embedded image


610.71
 0.3 ± 0.04





146


embedded image


608.70
16.7 ± 1.4 





147


embedded image


610.71
9.4 ± 1.0





148


embedded image


627.14
2.6 ± 0.3





149


embedded image


639.15
31.0 ± 6.4 





150


embedded image


596.68
12.7 ± 0.7 





151


embedded image


596.68
9.2 ± 0.1





152


embedded image


622.72
1.2 ± 0.3





153


embedded image


622.72
1.9 ± 0.3





154


embedded image


608.74
3.2 ± 0.4





155


embedded image


680.77
30.5 ± 4.1 





156


embedded image


678.75
13.3 ± 1.6 





157


embedded image


577.63
4.2 ± 0.1





158


embedded image


610.71
 0.9 ± 0.02





159


embedded image


602.64
2.7 ± 0.2





160


embedded image


604.66
10.6 ± 0.5 









It has been found according to the present invention that compounds of general formula (I), and especially the compounds of general formula (VI), can inhibit CBP-mediated transcriptional activation in cancer cells due to their specific binding to CBP. This conclusion is supported by immunoprecipitation of CBP of SW480 cells with compounds of the present invention.


The compounds of the present invention can also inhibit the survivin expression in SW480 cells, and therefore, inhibit the oncogenic activity in cancer cells. The compounds of the present invention can be used for inhibiting cancer cells, and thus, would be useful for the regulation of cell growth. Supporting such results, the compounds of the present invention further shows that it can induce the caspase-3 activation in SW480 cells, and therefore, induce the apoptotic activity in cells. The compounds of the present invention can be also advantageously used for inducing apoptosis in cells.


To confirm the oncogenic activity in cancer cell in in vitro MTS cytotoxicity assay was tested by following method.


(1) Cytotoxicity Test


SW480 or HCT116 cells were placed into 96 well microplate (104 cells/well) and incubated for 24 hours at 37° C. The cells were treated with TCF4 compound at various concentrations for 24 hours. 20 μl of MTS solution (Promega) was added into each well and incubated for 2 hours at 37° C. Cell viability was measured by reading the absorbance at 490 nm using microplate reader (Molecular Device) and cytotoxicity of a compound at each concentration was calculated.


(2) Growth Inhibition Assay


SW480 or HCT116 cells were placed into 96 well microplate (104 cells/well) and incubated for 24 hours at 37° C. 20 μl of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) solution (Promega) was added into each well and the absorbance after 2 hour incubation at 37° C. (negative control) was read. And then, the cells were treated with TCF4 compound at various concentrations for 48 hours. 20 μl of MTS solution (Promega) was added into each well and incubated for 2 hour at 37° C. Cell viability was measured by reading the absorbance at 490 nm using a microplate reader (Molecular device) and cytotoxicity of a compound at each concentration was calculated.


The results of oncogenic activity for selected library compounds were shown in the Table 5. The compound numbers is Table 5 are unrelated to the compound numbers in Table 4.









TABLE 5







ONCOGENIC ACTIVITY BY MTS OR SULFORHODAMINE B ASSAY


FOR SELECTED LIBRARY COMPOUNDS











Growth Inhibiton




(GI50, uM)










Compound
Structure
SW480
HCT116













1


embedded image


2.28
1.78





2


embedded image


2.58
2.23





3


embedded image


2.73
2.39





4


embedded image


1.99
1.91





5


embedded image


2.32
2.06





6


embedded image


3.96
3.91





7


embedded image


1.22
0.73





8


embedded image


<0.3
<0.3





9


embedded image


2.36
1.92





10


embedded image


2.34
1.66





11


embedded image


1.97
1.30





12


embedded image


2.54
1.48





13


embedded image


1.65
1.59





14


embedded image


2.70
2.10





15


embedded image


1.68
1.34





16


embedded image


4.18
2.95





17


embedded image


1.12
0.74





18


embedded image


4.63
3.52





19


embedded image


2.66
1.17





20


embedded image


5.02
2.75





21


embedded image


5.25
1.67





22


embedded image


6.58
3.26





23


embedded image


3.9
25.41





24


embedded image


13.79
1.67





25


embedded image


24.53
1.81





26


embedded image


23.89
3.06





27


embedded image


11.7
1.13





28


embedded image


3.57
5.47





29


embedded image


15.98
7.93





30


embedded image


14.05
5.4





31


embedded image


8.1 ± 0.7
5.0 ± 1.0





32


embedded image


47.2 ± 12.1
16.9 ± 1.9 





33


embedded image


ND up to 50 uM
28.6 ± 2.0 





34


embedded image


13.8 ± 2.4 
6.4 ± 1.3





35


embedded image


4.7 ± 0.5
5.0 ± 0.7





36


embedded image


21.9 ± 2.3 
12.7 ± 1.3 





37


embedded image


10.4 ± 0.8 
9.2 ± 0.9





38


embedded image


8.5
6.9





39


embedded image


22.8 ± 6.5 
19.7 ± 3.3 





40


embedded image


6.4 ± 0.5
5.8 ± 0.4





41


embedded image


34.4 ± 9.6 
14.7 ± 2.6 





42


embedded image


24.7
10.8





43


embedded image


ND up to 50 uM
39.1





44


embedded image


3.8 ± 0.4
4.2 ± 0.5





45


embedded image


2.5 ± 0.2
2.9 ± 0.4





46


embedded image


5.5 ± 0.5
9.2 ± 0.9





47


embedded image


6.2
12.2





48


embedded image


20.7 ± 2.8 
15.5 ± 2.3 





49


embedded image


1.4 ± 0.1
1.0 ± 0.2





50


embedded image


4.6
2.6





51


embedded image


3.0 ± 0.1
2.8





52


embedded image


19.3 ± 2.1 
9.7 ± 0.9





53


embedded image


11.4 ± 0.9 
4.7 ± 0.4





54


embedded image


7.1 ± 0.5
4.9 ± 0.7





55


embedded image


4.6 ± 0.5
4.1 ± 0.7





56


embedded image


10.8
9.1





57


embedded image


3.1 ± 0.3
5.1 ± 0.3





58


embedded image


47.9 ± 7.2 
22.3 ± 4.1 





59


embedded image


ND up to 50 uM
55.1 ± 33.7





60


embedded image


8.3 ± 1.4
6.3 ± 2.6





61


embedded image


11.3 ± 6.0 
3.6 ± 0.3





62


embedded image


35.3 ± 4.6 
23.5 ± 2.7 





63


embedded image


18.8 ± 4.8 
1.3 ± 0.1





64


embedded image


12.0 ± 0.7 
19.0 ± 1.6 





65


embedded image


7.3
4.7





66


embedded image


3.0 ± 0.3
5.8 ± 0.3





67


embedded image


0.6 ± 0.2
 0.3 ± 0.03





68


embedded image


3.7 ± 0.2
3.8 ± 0.6





69


embedded image


17.9 ± 3.1 
9.7 ± 1.0





70


embedded image


7.4 ± 0.6
7.2 ± 0.7





71


embedded image


4.6 ± 0.5
3.6 ± 0.7





72


embedded image


10.9 ± 0.6 
10.3 ± 1.6 





73


embedded image


9.2 ± 0.8
15.8 ± 2.6 





74


embedded image


1.3 ± 0.4
2.4 ± 0.3





75


embedded image


2.0 ± 0.1
4.5 ± 0.4





76


embedded image


4
6.1





77


embedded image


26.5 ± 6.5 
10.7 ± 0.8 





78


embedded image


2.2 ± 0.2
3.7 ± 0.3





79


embedded image


2.8 ± 0.2
5.2 ± 0.4





80


embedded image


4.0 ± 0.6
3.9 ± 0.6





81


embedded image


0.5 ± 0.3
1.8 ± 0.1





82


embedded image


1.5
1.4





83


embedded image


2.3 ± 0.3
2.5 ± 0.1





84


embedded image


8.4 ± 1.1
9.9 ± 1.0





85


embedded image


1.4 ± 0.5
2.7 ± 0.3





86


embedded image


9.6 ± 1.6
6.5 ± 0.6





87


embedded image


0.6 ± 0.2
0.5 ± 0.1





88


embedded image


0.3
0.4





89


embedded image


14.6 ± 1.4 
7.5 ± 1.0





90


embedded image


12.6 ± 0.9 
14.7 ± 1.0 





91


embedded image


1.5 ± 0.1
3.2 ± 0.2





92


embedded image


12.9 ± 1.0 
14.9 ± 2.2 





93


embedded image


1.9 ± 0.4
1.1 ± 0.1





94


embedded image


1.1 ± 0.3
 0.7 ± 0.07





95


embedded image


16.2 ± 2.6 
7.1 ± 1.2





96


embedded image


3.7 ± 0.4
3.4 ± 0.4





97


embedded image


7.1 ± 1.0
5.2 ± 0.5





98


embedded image


7.0 ± 1.1
4.4 ± 0.5





99


embedded image


1.0 ± 0.05
0.7 ± 0.1





100


embedded image


 0.3 ± 0.03
0.4 ± 0.1





101


embedded image


 1.1 ± 0.07
 0.9 ± 0.1





102


embedded image


2.5 ± 0.4
4.9 ± 1.2





103


embedded image


1.1 ± 0.1
1.5 ± 0.2





104


embedded image


<0.4
<0.4





105


embedded image


2.8 ± 0.2
2.1 ± 0.3





106


embedded image


4.5 ± 0.3
2.8 ± 0.4





107


embedded image


1.6 ± 0.1
1.6 ± 0.1





108


embedded image


24.9 ± 2.2 
37.9 ± 5.7 





109


embedded image


1.3 ± 0.3
1.1 ± 0.1





110


embedded image


2.1 ± 0.3
1.9 ± 0.1





111


embedded image


2.7 ± 0.8
2.1 ± 0.2





112


embedded image


5.1 ± 0.5
4.7 ± 0.3





113


embedded image


6.8 ± 1.4
3.7 ± 0.6





114


embedded image


1.7 ± 0.7
1.9 ± 0.2





115


embedded image


2.0 ± 0.7
 1.1 ± 0.04





116


embedded image


2.8 ± 0.9
1.7 ± 0.1





117


embedded image


0.6 ± 0.1
 0.3 ± 0.02





118


embedded image


21.2 ± 1.5 
23.2 ± 2.8 





119


embedded image


10.0 ± 1.3 
9.5 ± 1.1





120


embedded image


1.8 ± 0.2
2.6 ± 0.1





121


embedded image


8.2 ± 0.5
13.1 ± 0.6 





122


embedded image


15.9 ± 5.2 
14.8 ± 1.3 





123


embedded image


1.1 ± 0.3
1.7 ± 0.3





124


embedded image


2.3 ± 0.2
1.4 ± 0.1





125


embedded image


2.2 ± 0.3
1.9 ± 0.2





126


embedded image


19.4 ± 3.0 
11.6 ± 3.0 





127


embedded image


4.9 ± 0.7
4.3 ± 0.7





128


embedded image


0.9 ± 0.1
 1.0 ± 0.03





129


embedded image


2.9 ± 0.5
3.1 ± 0.3





130


embedded image


17.3 ± 1.2 
10.7 ± 1.7 









In other aspects the present invention provides pharmaceutical compositions containing a compound having the general formula (I), or the general formula (II), or the general formula (III), or the general formula (IV), or the general formula (VI). These compositions may be used in various methods (e.g., treating cancer or Alzheimer's disease) of the present invention as described in detail below.


The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. In addition, pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.


Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.


Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a compound having general formula (I), (II), (III), (IV), or (VI) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.


Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.


Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.


For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.


Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.


The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.


In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.


It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.


Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.


The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.


For instance, in certain embodiments, a pharmaceutical composition of the present invention is one suitable for oral administration in unit dosage form such as a tablet or capsule that contains from about 1 mg to about 1 g of the compound of this invention. In some other embodiments, a pharmaceutical composition of the present invention is one suitable for intravenous, subcutaneous or intramuscular injection. A patient may receive, for example, an intravenous, subcutaneous or intramuscular dose of about 1 μg/kg to about 1 g/kg of the compound of the present invention. The intravenous, subcutaneous and intramuscular dose may be given by means of a bolus injection or by continuous infusion over a period of time. Alternatively a patient will receive a daily oral dose approximately equivalent to the daily parenteral dose, the composition being administered 1 to 4 times per day.


The following table illustrates representative pharmaceutical dosage forms containing the compound or pharmaceutically-acceptable salt thereof for therapeutics or prophylactic use in humans:


















Tablet 1
mg/tablet







Compound
100



Lactose Ph. Eur.
179



Croscarmellose sodium
12.0



Polyvinylpyrrolidone
6



Magnesium stearate
3.0







Tablet 2
mg/tablet







Compound
50



Lactose Ph. Eur.
229



Croscarmellose sodium
12.0



Polyvinylpyrrolidone
6



Magnesium stearate
3.0







Tablet 3
mg/tablet







Compound
1.0



Lactose Ph. Eur.
92



Croscarmellose sodium
4.0



Polyvinylpyrrolidone
2.0



Magnesium stearate
1.0







Capsule
mg/capsule







Compound
10



Lactose Ph. Eur.
389



Croscarmellose sodium
100



Magnesium stearate
1.0







Injection 1
(50 mg/ml)







Compound
0.5% w/v



Isotonic aqueous solution
to 100%










The pharmaceutical composition containing the compound of general formulae (I) or (II) or (III) or (IV) or (VI) can be used for treatment of disorders modulated by Wnt signaling pathway, especially cancer, more especially colorectal cancer.


In one aspect, the present invention provides compounds that inhibit the binding of a radiolabeled enkephalin derivative to the δ and μ opiate receptors. Accordingly, the reverse-turn mimetics of the present invention may be used as receptor agonists and as potential analgesic agents.


In another aspect, the present invention provides methods for inhibiting tumor growth. Such methods comprise the step of administering to a subject (e.g., a mammalian subject) having a tumor a compound with general formula (I), especially general formula (VI) in an amount effective to inhibit tumor growth. A compound or composition inhibits tumor growth if the tumor sizes are statistically significantly smaller in subjects with the treatment of the compound or composition than those without the treatment.


The inhibitory effect of a particular compound or composition of the present invention on tumor growth may be characterized by any appropriate methods known in the art. For instance, the effect of the compound or composition on survivin expression may be measured. Compounds or compositions down-regulate survivin expression are likely to have inhibitory effects on tumor growth. In addition, assays using tumor cell lines (e.g., soft agar assays using SW480 cells) and animal models for tumor growth (e.g., nude mice grafted with tumor cells and Min mouse model) may also be used to evaluate the inhibitory effect on tumor growth of a given compound or composition as described in detail in the examples. Other exemplary animal models or xenografts for tumor growth include those for breast cancer (Guo et al., Cancer Res. 62: 4678-84, 2002; Lu et al., Breast Cancer Res. Treat. 57: 183-92, 1999), pancreatic cancer (Bouvet et al., Cancer Res. 62: 1534-40, 2002), ovarian tumor (Nilsson et al., Cancer Chemother. Pharmacol. 49: 93-100, 2002; Bao et al., Gynecol. Oncol. 78: 373-9, 2000), melanoma (Demidem et al., Cancer Res. 61: 2294-300, 2001), colorectal cancer (Brown et al., Dig. Dis. Sci. 45: 1578-84, 2000; Tsunoda et al., Anticancer Res. 19: 1149-52, 1999; Cao et al., Clin. Cancer Res. 5: 267-74, 1999; Shawler et al., J. Immunother. Emphasis Tumor Immunol. 17: 201-8, 1995; McGregor et al., Dis. Colon. Rectum. 36: 834-9, 1993; Verstijnen et al., Anticancer Res. 8: 1193-200, 1988), hepatocellular cancer (Labonte et al., Hepatol. Res. 18: 72-85, 2000), and gastric cancer (Takahashi et al., Int. J. Cancer 85: 243-7, 2000).


The compound or composition that inhibits tumor growth may be administrated into a subject with a tumor via an appropriate route depending on, for example, the tissue in which the tumor resides. The appropriate dosage may be determined using knowledge and techniques known in the art as described above. The effect of the treatment of the compound or composition on tumor growth may also be monitored using methods known in the art. For instance, various methods may be used for monitoring the progression and/or growth of colorectal cancer, including colonoscopy, sigmoidoscopy, biopsy, computed tomograph, ultrasound, magnetic resonance imaging, and positron emission tomography. Methods for monitoring the progression and/or growth of ovarian cancer include, for example, ultrasound, computed tomography, magnetic resonance imaging, chest X-ray, laparoscopy, and tissue sampling.


In a related aspect, the present invention provides a method for treating or preventing cancer. Such methods comprise the step of administering to a subject in need thereof a compound or composition having general formula (I), especially the compound of general formular (VI), in an amount effective to treat or prevent cancer in the subject. Treating cancer is understood to encompass reducing or eliminating cancer progression (e.g., cancer growth and metastasis). Preventing cancer is understood to encompass preventing or delaying the onset of cancer. Various types of cancer may be treated or prevented by the present invention. They include, but are not limited to, lung cancer, breast cancer, colorectal cancer, stomach cancer, pancreatic cancer, liver cancer, uterus cancer, ovarian cancer, gliomas, melanoma, lymphoma, and leukemia.


A subject in need of treatment may be a human or non-human primate or other animal with various types of cancer. A subject in need of prevention may be a human or non-human primate or other animal that is at risk for developing cancer. Methods for diagnosing cancer and screening for individuals with high risk of cancer are known in the art and may be used in the present invention. For instance, colorectal cancer may be diagnosized by fecal occult blood test, sigmoidoscopy, colonoscopy, barium enema with air contrast, and virtual colonoscopy. An individual with high risk of colorectal cancer may have one or more colorectal cancer risk factors such as a strong family history of colorectal cancer or polyps, a known family history of hereditary colorectal cancer syndromes, a personal history of adenomatous polyps, and a personal history of chronic inflammatory bowel disease.


A compound with general formula (I) useful in cancer treatment or prevention may be identified by appropriate methods known in the art. Methods that may be used to select compounds for inhibitory effect on tumor growth as described above may also be used. The route of administration, the dosage of a given compound, the effectiveness of the treatment may be determined using knowledge and techniques known in the art. Factors that may be considered in making such a determination include, for example, type and stage of the cancer to be treated.


The compound with general formula (I) useful in cancer treatment and prevention may be administered in combination with an anti-neoplastic agent. An anti-neoplastic agent refers to a compound that inhibits tumor growth. Exemplary anti-neoplastic agents include Fluorouracil; 5-fluoro-2,4(1H,3H)-pyrimidinedione (5-FU), taxol, cisplatin, mitomycin C, tegafur, raltitrexed, capecitabine, and irinotecan (Arango et al., Cancer Research 61, 2001 4910-4915). A compound with general formula (I) administered in combination with an anti-neoplastic agent does not necessarily require that the compound and the anti-neoplastic agent be administered concurrently. The compound and the agent may be administered separately as long as at a time point, they both have effects on same cancer cells.


In a further related aspect, the present invention provides methods for promoting apoptosis in cancer cells. Such methods comprise the step of contacting cancer cells with a compound having general formula (I), especially a compound having general formula (VI), in an amount effective to promote apoptosis in these cells. A compound promotes apoptosis if the number of cancer cells undergoing apoptosis is statistically significantly larger in the presence of the compound than that in the absence of the compound. Such compounds may be identified by methods known in the art (e.g., measuring caspase activities and/or cell death) using cultured cancer cell lines, xenografts, or animal cancer models. Preferably, the compound is more active in promoting apoptosis in cancer cells than in normal cells. Cancer cells treatable by the present method may be from various tissue origins.


In another aspect of the present invention, a method for treating a disorder modulated by Wnt signaling pathway in which the method comprises administering to a patient a safe and effective amount of the compounds having general formula (I), especially the compound of general formula (VI) is disclosed. Pharmaceutical composition containing the compound of the present invention can be also used for this purpose. In this connection, it is found in the present invention that the compounds having general formula (I), especially the compound of general formula (VI) or the pharmaceutical composition containing thereof can be useful for the treatment of disorder modulated by TCF4-β catenin—CBP complex, which is believed to be responsible for initiating the overexpression of cancer cells related to Wnt signaling pathway. Thus, it is another aspect of the present invention to provide a method for the treatment of disorder modulated by TCF4-β catenin—CBP complex, using the compounds having the general formula (I), especially the compound of general formula (VI).


The present invention also provides compounds and methods for inhibiting survivin expression. Survivin is a target gene of the TCF/beta-catenin pathway, and more specifically is a target gene of the TCF/beta-catenin/CBP pathway. It is a member of the IAP (Inhibitor of Apoptosis Protein) family of proteins. Biological activity associated with survivin includes: highly expressed at G2/M, regulating cell cycle entry and exit; associated with microtubule, centrosomes, centromeres and midbody depending upon the phases of the cell cycle; and anti-apoptosis via interacting directly or indirectly with caspases (e.g., caspase 3, 7 and 9). In connection with cancer, survivin is widely and highly expressed in tumor cells, but expressed to little or no extent in normal tissue cells. Also, it has been observed that cancer patients whose tumors expressed survivin had a decreased overall survival. Furthermore, the degree of surviving expression has been correlated with other cancer markers, e.g., Ki67, PNCA, p53, APC, etc.


The effect of a particular compound of the present invention on survivin expression may be characterized by methods known in the art. Such methods include methods for characterizing survivin expression at the transcriptional or translational level. Exemplary methods for characterizing survivin expression at the transcriptional level are: cDNA microarry, reverse transcription-polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), and assays for reporter activities driven by survivin promoter. Exemplary methods for characterizing survivin expression at the translational level are: Western blot analysis, immunochemistry and caspase activities. Detailed descriptions of the above exemplary methods may be found in the examples below.


As described above, the present invention provides methods for inhibiting survivin expression. Such methods comprise the step of contacting a survivin-expressing cell with a compound of the present invention in an amount effective to inhibit survivin expression. A compound inhibits survivin expression if survivin expression in a cell is decreased in the presence of the compound compared to survivin expression in the absence of the compound. Survivin-expressing cells include tumor cells that express, such as cells in or from lung cancer, breast cancer, stomach cancer, pancreatic cancer, liver cancer, uterus cancer, ovarian cancer, gliomas, melanoma, colorectal cancer, lymphoma and leukemia. The step of contacting the survivin-expressing cells with the compound may be performed in vitro, ex vivo, or in vivo. A compound useful in inhibiting survivin expression may be identified, and the effects of a particular compound of the present invention may be characterized, by appropriate methods known in the art, as described in detail above.


Compounds of the present invention have been shown to inhibit the expression of survivin. Blanc-Brude et al., Nat. Medicine 8:987 (2002), have shown that survivin is a critical regulator of smooth muscle cell apoptosis which is important in pathological vessel-wall remodeling. Accordingly, another aspect of the present invention provides a method of treating or preventing restenosis associated with angioplasty comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic of the present invention. In one embodiment the invention treats the restenosis, i.e., administration of a reverse-turn mimetic of the present invention to a subject having restenosis achieves a reduction in the severity, extent, or degree, etc. of the restenosis. In another embodiment the invention prevents the restenosis, i.e., administration of a reverse-turn mimetic of the present invention to a subject that is anticipated to develop new or additional restenosis achieves a reduction in the anticipated severity, extent, or degree, etc. of the restenosis. Optionally, the subject is a mammalian subject.


Compounds of the present invention have been shown to inhibit TCF/B-catenin transcription. Rodova et al., J. Biol. Chem. 277:29577 (2002), have shown that PKD-1 promoter is a target of the B-catenin/TCF pathway. Accordingly, another aspect of the present invention provides a method of treating or preventing polycystic kidney disease comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic of the present invention. In one embodiment the invention treats the polycystic kidney disease, i.e., administration of a reverse-turn mimetic of the present invention to a subject having polycystic kidney disease achieves a reduction in the severity, extent, or degree, etc. of the polycystic kidney disease. In another embodiment the invention prevents polycystic kidney disease, i.e., administration of a reverse-turn mimetic of the present invention to a subject that is anticipated to develop new or additional polycystic kidney disease achieves a reduction in the anticipated severity, extent, or degree, etc. of the polycystic kidney disease. Optionally, the subject is a mammalian subject.


Compounds of the present invention have been shown to inhibit the expression of Wnt signaling. Hanai et al., J. Cell Bio. 158:529 (2002), have shown that endostatin, a known anti-angiogenic factor, inhibits Wnt signaling. Accordingly, another aspect of the present invention provides a method of treating or preventing aberrant angiogenesis disease comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic of the present invention. In one embodiment the invention treats the aberrant angiogenesis disease, i.e., administration of a reverse-turn mimetic of the present invention to a subject having aberrant angiogenesis disease achieves a reduction in the severity, extent, or degree, etc. of the aberrant angiogenesis disease. In another embodiment the invention prevents aberrant angiogenesis disease, i.e., administration of a reverse-turn mimetic of the present invention to a subject that is anticipated to develop new or additional aberrant angiogenesis disease achieves a reduction in the anticipated severity, extent, or degree, etc. of the aberrant angiogenesis disease. Optionally, the subject is a mammalian subject.


Compounds of the present invention have been shown to inhibit the expression of Wnt signalling. Sen et al., P.N.A.S. (USA) 97:2791 (2000), have shown that mammals with rheumatoid arthritis demonstrate increased expression of Wnt and Fz in RA synovial tissue. Accordingly, another aspect of the present invention provides a method of treating or preventing rheumatoid arthritis disease comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic of the present invention. In one embodiment the invention treats the rheumatoid arthritis disease, i.e., administration of a reverse-turn mimetic of the present invention to a subject having rheumatoid arthritis disease achieves a reduction in the severity, extent, or degree, etc. of the rheumatoid arthritis disease. In another embodiment the invention prevents rheumatoid arthritis disease, i.e., administration of a reverse-turn mimetic of the present invention to a subject that is anticipated to develop new or additional rheumatoid arthritis disease achieves a reduction in the anticipated severity, extent, or degree, etc. of the rheumatoid arthritis disease. Optionally, the subject is a mammalian subject.


Compounds of the present invention have been shown to inhibit the expression of Wnt signalling. Uthoff et al., Int. J. Oncol. 19:803 (2001), have shown that differential upregulation of disheveled and fz (Wnt pathway molecules) occurs in ulcerative colitis (compared to Chron's disease patients). Accordingly, another aspect of the present invention provides a method of treating or preventing ulcerative colitis comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic the present invention. In one embodiment the invention treats the ulcerative colitis, i.e., administration of a reverse-turn mimetic of the present invention to a subject having ulcerative colitis achieves a reduction in the severity, extent, or degree, etc. of the ulcerative colitis. In another embodiment the invention prevents ulcerative colitis, i.e., administration of a reverse-turn mimetic of the present invention to a subject that is anticipated to develop new or additional ulcerative colitis achieves a reduction in the anticipated severity, extent, or degree, etc. of the ulcerative colitis. Optionally, the subject is a mammalian subject.


Compounds of the present invention have been shown to inhibit Wnt TCF/catenin signalling. Accordingly, another aspect of the invention provides a method of treating or preventing tuberious sclerosis complex (TSC) comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic the present invention. Subjects having TSC typically develop multiple focal lesions in the brain, heart, kidney and other tissues (see, e.g., Gomez, M. R. Brain Dev. 17(suppl): 55-57 (1995)). Studies in mammalian cells have shown that overexpression of TSC1 (which expresses hamartin) and TSC2 (which expresses tuberin) negatively regulates cell proliferation and induces G1/S arrest (see, e.g., Miloloza, A. et al., Hum. Mol. Genet. 9: 1721-1727 (2000)). Other studies have shown that hamartin and tuberin function at the level of the 3-catenin degradation complex, and more specifically that these proteins negatively regulate beta-catenin stability and activity by participating in the beta-catenin degradation complex (see, e.g., Mak, B. C., et al. J. Biol. Chem. 278(8): 5947-5951, (2003)). Beta-catenin is a 95-kDa protein that participates in cell adhesion through its association with members of the membrane-bound cadherin family, and in cell proliferation and differentiation as a key component of the Wnt/Wingless pathway (see, e.g., Daniels, D. L., et al., Trends Biochem. Sci. 26: 672-678 (2001)). Misregulation of this pathway has been shown to be oncogenic in humans and rodents. The present invention provides compounds that modulate β-catenin activity, and particularly its interactions with other proteins, and accordingly may be used in the treatment of TSC. Thus, in one embodiment the invention treats TSC, i.e., administration of a reverse-turn mimetic of the present invention to a subject having TSC achieves a reduction in the severity, extent, or degree, etc. of the TSC. In another embodiment the invention prevents TSC, i.e., administration of a reverse-turn mimetic of the present invention to a subject that is anticipated to develop new or additional TSC achieves a reduction in the anticipated severity, extent, or degree, etc. of the TSC. Optionally, the subject is a mammalian subject.


Compounds of the present invention have been shown to inhibit the expression of Wnt signalling. The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS) and β-cell malignancies such as primary effusion lymphoma (PEL) and multicentric Castleman's disease. Fujimuro, M. et al., Nature Medicine 9(3):300-306 (2003), have shown that LANA acts to stabilize β-catenin, apparently by redistribtution of the negative regular GSK-3β. The present invention provides compounds and methods for inhibiting β-catenin protein interactions, e.g., β-catenin/TCF complex formation. Thus, the compounds of the present invention thwart the LANA-induced accumulation of β-catenin/TCF complex and, at least in part, the consequences of KSHV infection. Accordingly, another aspect of the present invention provides a method of treating or preventing conditions due to infection by Karposi's sarcoma-associated herpesvirus (KSHV). Such conditions include KSHV-associated tumors, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The method comprises administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic the present invention. In one embodiment the invention treats the KSHV-associated tumor, i.e., administration of a reverse-turn mimetic of the present invention to a subject having a KSHV-associated tumor achieves a reduction in the severity, extent, or degree, etc. of the tumor. In another embodiment the invention prevents a KSHV-associated tumor, i.e., administration of a reverse-turn mimetic of the present invention to a subject that is anticipated to develop new or additional KSHV-associated tumors achieves a reduction in the anticipated severity, extent, or degree, etc. of the tumor. Optionally, the subject is a mammalian subject.


LEF/TCF DNA-binding proteins act in concert with activated β-catenin (the product of Wnt signaling) to transactivate downstream target genes. DasGupta, R. and Fuchs, E. Development 126(20):4557-68 (1999) demonstrated the importance of activated LEF/TCF complexes at distinct times in hair development and cycling when changes in cell fate and differentiation commitments take place. Furthermore, in skin morphogenesis, β-catenin has been shown to be essential for hair follicle formation, its overexpression causing the “furry” phenotype in mice (Gat, U., et al. Cell 95:605-614 (1998) and Fuchs, E. Harvey Lect. 94:47-48 (1999). See also Xia, X. et al. Proc. Natl. Aad. Sci. USA 98:10863-10868 (2001). Compounds of the present invention have been shown to inhibit the expression of Wnt signaling, and interfere with formation of β-catenin complexes. Accordingly, the present invention provides a method for modulating hair growth comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic the present invention, where the amount is effective to modulate hair growth in the subject. Optionally, the subject is a mammalian subject.


The present invention provides compounds useful in treating or preventing Alzheimer's disease. Alzheimer's disease (AD) is a neurodegenerative disease with progressive dementia. This disease is accompanied by three main structural changes in the brain, namely, i) intracellular protein deposits (also known as neurofibrillary tangles, or NFT), ii) extracellular protein deposits termed amyloid plaques that are surrounded by dystrophic neuritis, and iii) diffuse loss of neurons.


The compounds or compositions of the present invention rescue defectes in neuronal differentiation caused by a presenilin-1 mutation and may decrease the number, or rate at which neuronal precursor populations differentiate to neurons in Alzheimer's brains. Presenilins are transmembrane proteins whose functions are related to trafficking, turnover and cleavage of Notch and Amyloid Precursor Protein. Missense mutations in presenilin 1 (PS-1) are associated with early-onset familial Alzheimer's disease (Fraser et al., Biochem. Soc. Symp. 67, 89 (2001)). The compounds of the present invention may be applicable not only to individuals with PS-1 familial Alzheimer's mutations, but also to general Alzheimer's patients.


In addition, the present invention provides a method for treating or preventing Alzheimer's disease comprising administering to a subject in need thereof a safe and effective amount of a reverse-turn mimetic of the present invention, where the amount is effective to treat or prevent Alzheimer's disease in the subject. Treating Alzheimer's disease is understood to encompass reducing or eliminating the manifestation of symptoms characteristic of Alzheimer's disease, or delaying the progression of this disease. Preventing Alzheimer's disease is understood to encompass preventing or delaying the onset of this disease.


A subject in need of treatment may be a human or non-human primate or other animal that is at various stages of Alzheimer's disease. Methods for diagnosing Alzheimer's disease are known in the art (see, e.g., Dinsmore, J. Am. Osteopath. Assoc. 99(9 Suppl.):S1-6, 1999; Kurz et al., J. Neural Transm. Suppl. 62: 127-33, 2002; Storey et al., Front Viosci. 7: e155-84, 2002; Marin et al., Geriatrics 57: 36-40, 2002; Kril and Halliday, Int. Rev. Neurobiol. 48: 167-217, 2001; Gurwitz, Trends Neurosci. 23: 386, 2000; Muller-Spahn and Hock, Eur. Arch. Psychiatry Clin. Neurosci. 249 Suppl. 3: 37-42; Fox and Rossor, Rev. Neuro. (Paris) 155 Suppl. 4: S33-7, 1999), including the use of neuropyschological measures, functional imaging measures, biological markers, and autopsy of brain tissue. A subject in need of prevention may be a human or non-human primate or other animal that is at risk for developing Alzheimer's disease, such as an individual having a mutation of certain genes responsible for this disease (e.g., genes encoding amyloid precursor protein, presenilin 1, and presenilin 2), and/or a gene involved in the pathogenesis of this disease (e.g., apolipoprotein E gene) (Rocchi et al., Brain Res. Bull. 61: 1-24, 2003).


Compounds with structures as set forth in formula (I) may be screened for their activities in treating or preventing Alzheimer's disease by any appropriate methods known in the art. Such screening may be initially performed using in vitro cultured cells (e.g, PC-12 cells as described in Example 8). Compounds capable of rescuing defects in neuronal differentiation caused by a presenilin 1 mutation may be further screened using various animal models for Alzheimer's disease. Alternatively, compounds with structures as set forth in formula (I) may be directedly tested in animal models for Alzheimer's disease. Many model systems are known in the art and may be used in the present invention (see, e.g., Rowan et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358: 821-8, 2003; Lernere et al., Neurochem. Res. 28: 1017-27, 2003; Sant'Angelo et al., Neurochem. Res. 28: 1009-15, 2003; Weiner Harv. Rev. Psychiatry 4: 306-16, 1997). The effects of the selected compounds on treating or preventing Alzheimer's disease may be characterized or monitored by methods known in the art for evaluating the progress of Alzheimer's disease, including those described above for diagnosing this disease.


The present invention also provides methods for promoting neurite outgrowth. Such methods comprise the step of contacting a neuron with a compound according to formula (I) in an amount effective to promote neurite outgrowth. These methods are useful in treating neurodegenerative diseases (e.g., glaucoma, macular degeneration, Parkinson's Disease, and Alzheimer's disease) and injuries to nervous system. A compound promotes neurite outgrowth if the neurite lengths of neurons are statistically significantly longer in the presence of the compound than those in the absence of the compound. Such a compound may be identified using in vitro cultured cells (e.g, PC-12 cells, neuroblastoma B104 cell) (Bitar et al., Cell Tissue Res. 298: 233-42, 1999; Pellitteri et al., Eur. J. Histochem. 45: 367-76, 2001; Satoh et al., Biochem. Biophys. Res. Commun. 258: 50-3, 1999; Hirata and Fujisawa, J. Neurobiol. 32:415-25, 1997; Chauvet et al., Glia 18: 211-23, 1996; Vetter and Bishop, Curr. Biol. 5: 168-78, 1994; Koo et al., Proc. Natl. Acad. Sci. USA 90: 4748-52, 1993; Skubitz et al., J. Cell Biol. 115: 1137-48, 1991; O'Shea et al., Neuron 7: 231-7, 1991; Rydel and Greene, Proc. Natl. Acad. Sci. USA 85: 1257-61, 1988) or using explants (Kato et al., Brain Res. 31: 143-7, 1983; Vanhems et al., Eur. J. Neurosci. 2: 776-82, 1990; Carri et al., Int. J. Dev. Neurosci. 12: 567-78, 1994). Contacting a neuron with a compound according to the present invention may be carried out in vitro or in vivo. The resulting treated neuron, if generated in vitro, may be transplanted into a tissue in need thereof (Lacza et al., Brain Res. Brain Res. Protoc. 11: 145-54, 2003; Chu et al., Neurosci. Lett 343: 129-33, 2003; Fukunaga et al., Cell Transplant 8: 435-41, 1999).


The present invention also provides methods for promoting differentiation of a neural stem cell comprising contacting a neural stem cell with a compound according to formula (I) in an amount effective to promote differentiation of a neural stem cell. Such methods are also useful in treating neurodegenerative diseases (e.g., glaucoma, macular degeneration, Parkinson's Disease, and Alzheimer's disease) and injuries to nervous system. “Neural stem cell” refers to a clonogenic, undifferentiated, multipotent cell capable of differentiating into a neuron, an astrocyte or an oligodendrocyte under appropriate conditions. A compound promotes differentiation of neural stem cells if neural stem cells exhibit a statistically significantly higher degree of differentiation in the presence of the compound than in the absence of the compound. Such a compound may be identified using assays involving in vitro cultured stem cells or animal models (Albranches et al., Biotechnol. Lett. 25: 725-30, 2003; Deng et al., Exp. Neurol. 182: 373-82, 2003; Munoz-Elias et al., Stem Cells 21: 437-48, 2003; Kudo et al., Biochem. Pharmacol. 66: 289-95, 2003; Wan et al., Chin. Med. J. 116: 428-31, 2003; Kawamorita et al., Hum. Cell 15: 178-82, 2002; Stpyridis and Smith, Biochem. Soc. Trans. 31:45-9, 2003; Pachernik et al., Reprod. Nutr. Dev. 42: 317-26, 2002; Fukunaga et al., supra). The neural stem cell may be a cultured stem cell, a stem cell freshly isolated from its source tissue, or a stem cell within its source organism. Thus, contacting the neural stem cell with a compound according to the present invention may be carried out either in vitro (for a cultured or freshly isolated stem cell) or in vivo (for a stem cell within its source organism). The resulting differentiated neural cell, if generated in vitro, may be transplanted into a tissue in need thereof (Lacza et al., supra; Chu et al., supra; Fukunaga et al., supra). Such a tissue includes a brain tissue or other nervous tissue that suffers from a trauma or a neurodegenerative disease.


The following non-limiting examples illustrate the compounds, compositions, and methods of use of this invention.


EXAMPLES
Preparation Example 1
Preparation of (N-Fmoc-N′—R3-hydrazino)-acetic acid
(1) Preparation of N-Fmoc-N′-Methyl Hydrazine



embedded image


2 L, two-neck, round-bottomed-flask was fitted with a glass stopper and a calcium tube. A solution of methylhydrazine sulfate (20 g, 139 mmol, where R3 is methyl) in THF (300 mL) was added and a solution of DiBoc (33 g, 153 mmol) in THF was added. Saturated sodium bicarbonate aqueous solution (500 mL) was added dropwise via addition funnel over 2 hours with vigorous stirring. After 6 hours, a solution of Fmoc-Cl (39 g, 153 mmol) in THF was added slowly. The resulting suspension was stirred for 6 hours at 0° C. The mixture was extracted with ethyl acetate (EA, 500 mL) and the organic layer was retained. The solution was dried with sodium sulfate and evaporated in vacuo. The next step proceeded without purification.


A 1 L, two-necked, round-bottom-flask was fitted with a glass stopper and a calcium tube. A solution of the product from the previous step in MeOH (300 mL) was added and conc. HCl (30 mL, 12 N) was added slowly via addition funnel with magnetic stirring in ice water bath and stirred overnight. The mixture was extracted with EA (1000 mL) and the organic layer was retained. The solution was dried with sodium sulfate and evaporated in vacuo. The residue was purified by recrystallization with n-hexane and EA to give N-Fmoc-N′-methyl hydrazine (32.2 g, 83%). 1HNMR (DMSO-D6) δ 7.90˜7.88 (d, J=6 Hz, 2H), δ 7.73˜7.70 (d, J=9 Hz, 2H), 7.44˜7.31 (m, 4H), 4.52˜4.50 (d, J=6 Hz, 2H), 4.31˜4.26 (t, J=6 Hz, 1H), 2.69 (s, 1H).


(2) Preparation of (N-Fmoc-N′-methyl-hydrazino)-acetic acid t-butyl ester



embedded image


1 L, two-necked, round-bottom-flask was fitted with a glass stopper and reflux condenser connected to a calcium tube. A solution of N-Fmoc-N′-methyl hydrazine (20 g, 75 mmol) in toluene (300 mL) was added. A solution of t-butylbromo acetate (22 g, 111 mmol) in toluene (50 mL) was added slowly. Cs2CO3 (49 g, 149 mmol) was added slowly. NaI (11 g, 74 mmol) was added slowly with vigorous stirring. The reaction mixture was stirred at reflux temperature over 1 day. The product mixture was filtered and extracted with EA (500 mL). The solution was dried over sodium sulfate and evaporated in vacuo. The product was purified by chromatography with hexane:EA=2:1 solution to give (N-Fmoc-N′-methyl-hydrazino)-acetic acid t-butyl ester (19.8 g, 70%).



1H-NMR (CDCl3-d) δ 7.78˜7.75 (d, J=9 Hz, 2H), δ 7.61˜7.59 (d, J=6 Hz, 2H), 7.43˜7.26 (m, 4H), 4.42˜4.40 (d, J=6 Hz, 2H), 4.23 (b, 1H), 3.57 (s, 2H), 2.78 (s, 3H), 1.50 (s, 9H).


(3) Preparation of (N-Fmoc-N′-methyl-hydrazino)-acetic acid



embedded image


1 L, two-neck, round-bottomed-flask was fitted with a glass stopper and reflux condenser connected to a calcium tube. (N-Fmoc-N′-methyl-hydrazino)-acetic acid t-butyl ester (20 g, 52 mmol) was added. A solution of HCl (150 mL, 4 M solution in dioxane) was added slowly with vigorous stirring in an ice water bath. The reaction mixture was stirred at RT over 1 day. The solution was concentrated completely under reduced pressure at 40° C. A saturated aq. NaHCO3 solution (100 mL) was added and the aqueous layer was washed with diethyl ether (100 mL). Conc. HCl was added dropwise slowly at 0° C. (pH 2-3).


The mixture was extracted and the organic layer was retained (500 mL, MC). The solution was dried with sodium sulfate and evaporated in vacuo. The residue was purified by recrystallization with n-hexane and ethyl acetate to give (N-Fmoc-N′-methyl-hydrazino)-acetic acid (12 g, 72%). 1H-NMR (DMSO-d6) δ 12.38 (s, 1H), 8.56 (b, 1H), 7.89˜7.86 (d, J=9 Hz, 2H), 7.70˜7.67 (d, J=9 Hz, 2H), 7.43˜7.29 (m, 4H), 4.29˜4.27 (d, J=6 Hz, 2H), 4.25˜4.20 (t, J=6 Hz, 1H), 3.47 (s, 2H), 2.56 (s, 3H).


Preparation Example 2
Preparation of (N-Moc-N′—R7-hydrazino)-acetic acid
(1) Preparation of (M-Methoxycarbonyl-hydrazino)-acetic acid ethyl ester



embedded image


MOC-NH—NH2 (50 g, 0.55 mol) was dissolved in DMF (300 ml), and then ethyl bromoacetate (68 ml, 0.555 mol) and potassium carbonate (77 g, 0.555 mol) were added to the reaction vessel. The mixture was warmed to 50° C. for 5 hours. After the reaction was completed, the mixture was filtered, and diluted with EtOAc, and washed with brine (3 times). The crude product was purified by column (eluent:Hex/EtOAc=4/1) to provide 72 of colorless oil.


(2) [N—R7—N′-methoxycarbonyl-hydrazino]-acetic acid ethyl ester



embedded image


The ethyl ester (10 g, 0.05 mol), potassium carbonate (6.9 g, 0.05 mol), and R7-bromide (14.1 g, 0.06 mol) were dissolved in DMF (200 ml), and The mixture was warmed to 50° C. for 5 hours. After the reaction was completed, the mixture was filtered, and diluted with EA, and washed with brine (3 times). The crude product was purified by Chromatography (eluent:Hex/EtOAc=4/1).


(3) [N—R7—N′-methoxycarbonyl-hydrazino]-acetic acid



embedded image


The alkylated ethyl ester (9.5 g, 0.03 mol) was dissolved in THF/water (1/1, ml), and added 2N NaOH (28.3 ml) solution at 0° C. The mixture was stirred at RT for 2 hours. After the starting ester was not detected on UV, the solution was diluted with EA, then separated. The aqueous layer was acidified to pH 3-4 by 1N HCl, and the compound was extracted by DCM (3 times). The combined organic layer was dried over MgSO4, and evaporated to give a yellow solid.


Example 1



embedded image


(1) Preparation of Nβ-Moc-Nα-benzyl-hydrazinoglycine



embedded image


This compound was prepared according to literature procedure. (Cheguillaume et. al., Synlett 2000, 3, 331)


(2) Preparation of 1-Methoxycarbonyl-2,8-dibenzyl-6-methyl-4,7-dioxo-hexahydro-pyrazino[2,1-c][1,2,4]triazine

Bromoacetal resin (60 mg, 0.98 mmol/g) and a solution of benzyl amine in DMSO (2.5 ml, 2 M) were placed in vial with screw cap. The reaction mixture was shaken at 60° C. using rotating oven [Robbins Scientific] for 12 hours. The resin was collected by filtration, and washed with DMF, then DCM, to provide a first component piece.


A solution of Fmoc-alanine (4 equiv., commercially available, the second component piece), HATU (PerSeptive Biosystems, 4 equiv.), and DIEA (4 equiv.) in NMP (Advanced ChemTech) was added to the resin. After the reaction mixture was shaken for 4 hours at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


To the resin was added 20% piperidine in DMF. After the reaction mixture was shaken for 8 min at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


A solution of Nβ-Moc-Nα-benzyl-hydrazinoglycine (4 equiv., compound (3) in preparative example 2, where R7 is benzyl, 3rd component piece), HOBT [Advanced ChemTech] (4 equiv.), and DIC (4 equiv.) in DMF was added to the resin prepared above. After the reaction mixture was shaken for 3 hours at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then MeOH. The resin was dried in vacuo at room temperature.


The resin was treated with formic acid (2.5 ml) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under reduced pressure to give the product as an oil. 1H-NMR (400 MHz, CDCl3) δ ppm; 1.51 (d, 3H), 2.99 (m, 1H), 3.39 (d, 1H), 3.69 (m, 1H), 3.75 (m, 1H), 3.82 (s, 3H), 4.02 (d, 1H), 4.24 (d, 1H), 4.39 (d, 1H), 4.75 (d, 1H), 5.14 (q, 1H), 5.58 (dd, 1H), 7.10-7.38 (m, 10H).


Example 2



embedded image


(1) Preparation of N′-Fmoc-N-methyl-hydrazinocarbonyl chloride



embedded image


An ice-cooled biphasic mixture of N-methyl hydrazine carboxylic acid


9H-fluoren-9-ylmethyl ester (107 mg, 0.4 mmol) in 15 ml of CH2Cl2 and 15 ml of saturated aq. NaHCO3 was rapidly stirred while 1.93 M phosgene in toluene (1.03 ml, 2 mmol) was added as a single portion. The reaction mixture was stirred for 30 min, the organic phase was collected, and the aqueous phase was extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to afford 128 mg (97%) of carbamoyl chloride as a foamy solid. [Caution: Phosgene vapor is highly toxic. Use it in a hood]. This product was used for the following solid phase synthesis without further purification.


(2) Preparation of 2,5-Dimethyl-7-benzyl-3,6-dioxo-hexahydro-[1,2,4]triazolo[4,5-a]pyrazine-1-carboxylic acid benzylamide

Bromoacetal resin (30 mg, 0.98 mmol/g) and a solution of benzyl amine in DMSO (1.5 ml, 2 M) were placed in vial with screw cap. The reaction mixture was shaken at 60° C. using rotating oven [Robbins Scientific] for 12 hours. The resin was collected by filtration, and washed with DMF, then DCM, to provide the first component piece.


A solution of Fmoc-alanine (3 equiv., second component piece, commercially available), HATU (PerSeptive Biosystems, 3 equiv.), and DIEA (3 equiv.) in NMP (Advanced ChemTech) was added to the resin. After the reaction mixture was shaken for 4 hours at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF, to thereby add the second component piece to the first component piece.


To the resin was added 20% piperidine in DMF. After the reaction mixture was shaken for 8 min at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


A solution of N′-Fmoc-N-methyl-hydrazinocarbonyl chloride (combined third and fourth component pieces, 5 equiv.) obtained in the above step (1), DIEA (5 equiv.) in DCM was added to the resin prepared above. After the reaction mixture was shaken for 4 hours at room temperature, the resin was collected by filtration and washed with DMF, DCM, and DMF.


To the resin was added 20% piperidine in DMF (10 ml for 1 g of the resin). After the reaction mixture was shaken for 8 min at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


The resin was treated with a mixture of benzyl isocyanate (4 equiv.) and DIEA (4 equiv.) in DCM for 4 hours at room temperature. Then, the resin was collected by filteration and washed with DMF, DCM, and then MeOH. The resin was dried in vacuo at room temperature.


The resin was treated with formic acid for 14 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under reduced pressure to give the product as an oil.



1H-NMR (400 MHz, CDCl3) δ ppm; 1.48 (d, 3H), 2.98 (s, 3H), 3.18 (m, 1H), 3.46 (m, 1H), 4.37-4.74 (m, 5H), 5.66 (dd, 1H), 6.18 (m, 1H), 7.10-7.40 (m, 10H).


Example 3
Preparation of 2,5,7-trimethyl-3,6-dioxo-hexahydro-[1,2,4]triazolo[4,5-a]pyrazine-1-carboxylic acid benzylamide

The title compound is prepared according to the same procedure as described in Example 2, but reacting bromoacetal resin with a solution of methyl amine instead of benzyl amine. 1H-NMR (400 MHz, CDCl3) δ ppm; 1.48 (d, 3H), 2.99 (s, 3H), 3.03 (s, 3H), 3.38 (m, 1H), 3.53 (dd, 1H), 4.36 (dd, 1H), 4.52 (q, 1H), 4.59 (dd, 1H), 5.72 (dd, 1H), 6.19 (br.t, 1H), 7.10-7.38 (m, 5H).


Example 4
Preparation of 2-methyl-5-(p-hydroxyphenylmethyl)-7-naphthylmethyl-3,6-dioxo-hexahydro-[1,2,4]triazolo[4,5-a]pyrazine-1-carboxylic acid benzylamide

Bromoacetal resin (30 mg, 0.98 mmol/g) and a solution of naphthylmethyl amine in DMSO (1.5 ml, 2 M) were placed in vial with screw cap. The reaction mixture was shaken at 60° C. using rotating oven [Robbins Scientific] for 12 hours. The resin was collected by filtration, and washed with DMF, then DCM to provide the first component piece.


A solution of Fmoc-Tyr(OBut)-OH (3 equiv.), HATU (PerSeptive Biosystems, 3 equiv.), and DIEA (3 equiv.) in NMP (Advanced ChemTech) was added to the resin. After the reaction mixture was shaken for 4 hours at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF, to thereby add the second component piece to the first component piece.


To the resin was added 20% piperidine in DMF. After the reaction mixture was shaken for 8 min at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


A solution of W-Fmoc-N-methyl-hydrazinocarbonyl chloride (5 equiv.), DIEA (5 equiv.) in DCM was added to the resin prepared above. After the reaction mixture was shaken for 4 hours at room temperature, the resin was collected by filtration and washed with DMF, DCM, and DMF.


To the resin was added 20% piperidine in DMF (10 ml for 1 g of the resin). After the reaction mixture was shaken for 8 min at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


The resin was treated with a mixture of benzyl isocyanate (4 equiv.) and DIEA (4 equiv.) in DCM for 4 hours at room temperature. Then, the resin was collected by filteration and washed with DMF, DCM, and then MeOH. The resin was dried in vacuo at room temperature.


The resin was treated with formic acid for 14 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under reduced pressure to give the product as an oil.



1H-NMR (400 MHz, CDCl3) δ ppm; 2.80-2.98 (m, 5H), 3.21-3.37 (m, 2H), 4.22-4.52 (m, 2H), 4.59 (t, 1H), 4.71 (d, 1H), 5.02 (dd, 1H), 5.35 (d, 1H), 5.51 (d, 1H), 6.66 (t, 2H), 6.94 (dd, 2H), 7.21-8.21 (m, 12H).


Example 5
Preparation of 2-methyl-6-(p-hydroxyphenylmethyl)-8-naphthyl-4,7-dioxo-hexahydro-pyrazino[2,1-c][1,2,4]triazine-1-carboxylic acid benzylamide

Bromoacetal resin (60 mg, 0.98 mmol/g) and a solution of naphthyl amine in DMSO (2.5 ml, 2 M) were placed in vial with screw cap. The reaction mixture was shaken at 60° C. using rotating oven [Robbins Scientific] for 12 hours. The resin was collected by filtration, and washed with DMF, then DCM.


A solution of Fmoc-Tyr(OBut)-OH (4 equiv.), HATU [PerSeptive Biosystems] (4 equiv.), and DIEA (4 equiv.) in NMP (Advanced ChemTech) was added to the resin. After the reaction mixture was shaken for 4 hours at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


To the resin was added 20% piperidine in DMF. After the reaction mixture was shaken for 8 min at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


A solution of Nβ-Fmoc-Nα-benzyl-hydrazinoglycine (4 equiv.), HOBT [Advanced ChemTech] (4 equiv.), and DIC (4 equiv.) in DMF was added to the resin prepared above. After the reaction mixture was shaken for 3 hours at room temperature, the resin was collected by filtration and washed with DMF, and then DCM. To the resin was added 20% piperidine in DMF (10 ml for 1 g of the resin). After the reaction mixture was shaken for 8 min at room temperature, the resin was collected by filtration and washed with DMF, DCM, and then DMF.


The resin was treated with a mixture of benzyl isocyanate (4 equiv.) and DIEA (4 equiv.) in DCM for 4 hours at room temperature. Then, the resin was collected by filteration and washed with DMF, DCM, and then MeOH. After the resin was dried in vacuo at room temperature, the resin was treated with formic acid (2.5 ml) for 18 hours at room temperature. The resin was removed by filtration, and the filtrate was condensed under reduced pressure to give the product as an oil.



1H-NMR (400 MHz, CDCl3) δ ppm; 2.73 (s, 3H), 3.13 (d, 1H), 3.21-3.38 (m, 3H), 3.55 (d, 1H), 3.75 (t, 1H), 4.22 (dd, 1H), 4.36 (dd, 1H), 4.79 (d, 1H), 5.22 (t, 1H), 5.47 (m, 2H), 6.68 (d, 2H), 6.99 (d, 2H), 7.21-8.21 (m, 12H);


MS (m/z, ESI) 564.1 (MH+) 586.3 (MNa+).


Example 6
Bioassay for the Measurement of IC50 Against SW480 Cells and Cytotoxicity Test on the Cell Lines

The test compound (Compound A) used in this example was prepared in Example 4.




embedded image



a. Reporter Gene Assay


SW480 cells were transfected with the usage of Superfect™ transfect reagent (Qiagen, 301307). Cells were trypsinized briefly 1 day before transfection and plated on 6 well plate (5×105 cells/well) so that they were 50-80% confluent on the day of transfection.


Four microgram (TOPFlash) and one microgram (pRL-null) of DNAs were diluted in 150 μl of serum-free medium, and 30 μl of Superfect™ transfect reagent was added. The DNA-Superfect mixture was incubated at room temperature for 15 min, and then, 1 ml of 10% FBS DMEM was added to this complex for an additional 3 hours of incubation. While complexes were forming, cells were washed with PBS twice without antibiotics.


The DNA-Superfect™ transfect reagent complexes were applied to the cells before incubating at 37° C. at 5% CO2 for 3 hours. After incubation, recovery medium with 10% FBS was added to bring the final volume to 1.18 ml. After 3 hours incubation, the cells were harvested and reseeded to 96 well plate (3×104 cells/well). After overnight incubation at 37° C. at 5% CO2, the cells were treated with Compound A for 24 hours. Finally, the activity was checked by means of luciferase assay (Promega, E1960).



FIG. 3 illustrates the results of the measurement of IC50 of Compound A for SW480 cells.


b. Sulforhodamine B (SRB) Assay


Growth inhibitory effect of Compound A on the cells listed below was measured by the sulforhodamine B assay. SW480 cells in 100 μl media were plated in each well of 96-well plate and allowed to attach for 24 hours. Compound A was added to the wells to produce the desired final concentrations, and the plates were incubated at 37° C. for 48 hours. The cells were then fixed by gentle addition of 100 μl of cold (4° C.) 10% trichloroacetic acid to each well, followed by incubation at 4° C. for 1 hour. Plates were washed with deionized water five times and allowed to air dry. The cells were then stained by addition of 100 μl SRB solution (0.4% SRB(w/v) in 1% acetic acid (v/v)) to wells for 15 min. After staining, the plates were quickly washed five times with 1% acetic acid to remove any unbound dye, and allowed to air dry. Bound dye was solubilized with 10 mmol/L Tris base (pH 10.5) prior to reading the plates. The optical density (OD) was read on a plate reader at a wavelength of 515 nm with Molecular Device. Inhibition of growth was expressed as relative viability (% of control) and GI50 was calculated from concentration-response curves after log/probit transformation.


Table 6 shows in vitro cytotoxicity (SRB) assay data for Compound A obtained in Example 4. The values in Table 6 are in μg/ml.













TABLE 6







Example




Origin
Cell
4
Cisplatin
5-FU



















Colon
T84
1.134
>10
1.816



LOVO
0.532
>10
1.029



HT29
1.694
>10
5.334



DLD-1
1.775
>10
>10



COLO205
1.136
>10
1.130



CACO-2
1.201
>10
0.451



SW480-Kribb
1.137
>10
>10



SW480-CWP
0.980
4.502
>10



SW620
1.426
>10
5.570



KM12
1.451
>10
2.729



HCT15
2.042
>10
1.179



HCT116
0.96
>10
1.039



HCC2998
1.047
>10
5.486



786-0
1.417
3.347
0.584


Leukemia
HL60
1.243
>10
7.010



RPMI8226
1.1.177
>10
>10



K562/VIN
1.640
>10
7.071



K562/ADR
7.682
>10
>10



K562
1.247
>10
6.133


Prostate
PC3
1.207
>10
>10



HT1080
1.469
>10
0.798


Lung
A549
1.386
>10
1.007



NCI H460
1.498
>10
1.397



NCI H23
1.296
5.176
2.254


Renal
293
0.731
6.641
2.015



CAKI-1
0.467
>10
0.925



ACHN
1.263
5.019
5.062


Melanoma
RPMI7951
0.936
5.010
0.920



M14
2.289
3.447
1.225



HMV-II
4.834
3.190
0.695



HMV-I
1.153
5.478
2.110



G361
0.584
4.827
1.539



CRL1579
1.830
0.699
>10



A431
1.083
3.722
0.404



A253
1.398
2.084
2.926



UACC62
0.563
>10
1.093



SK-MEL-28
1.291
>10
>10



SK-MEL-5
0.888
>10
2.434



LOX-IMVI
1.526
>10
>10



A375
1.391
>10
1.464


Breast
MCF7/ADR
9.487
9.907
>10



MCF7
7.355
>10
1.751









Example 7
Min Mouse Model

Selected compounds of the present invention (Compound B and Compound C) were evaluated in the min mouse model to evaluate their efficacy as anti-cancer agents.




embedded image


The min mouse model is a widely used model to test for this type of efficacy. The numbers of polyp formed in small intestine and colon of these mice after various treatments were measured (Table 7). The data shown that both compounds, when administered at about 300 mpk, reduce the number of polyp in min mice compared to those in the control mice treated with vehicle only.









TABLE 7







MIN MOUSE MODEL DATA










Polyp Number (Mean ± S.D.)














Small


P (total)
% Inhibition


Group
Intestine
Colon
Total
Vs. VH
vs. VH





Wild Type
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0




Vehicle
65.8 ± 15.9
1.8 ± 1.5
67.7 ± 15.3




Compound C -100 mpk
69.2 ± 20.8
1.7 ± 1.5
71.4 ± 23.0




Compound C -300 mpk
46.1 ± 17.1
1.1 ± 1.2
47.0 ± 16.9
<0.01
31


Compound B -300 mpk
45.2 ± 22.1
1.4 ± 0.9
46.8 ± 17.0
<0.01
31


Sulindac -160 ppm
48.0 ± 20.7
0.5 ± 0.5
48.5 ± 20.9
<0.05
28









Example 8
Chemogenomic Inhibition of CBP/β-Catenin Interaction Rescues Defects in Neuronal Differentiation Caused by a Presenilin-1 Mutation

The following compound (Compound D) was used in this example:




embedded image



Materials and Methods


Plasmids. TOPFLASH and FOPFLASH reporter constructs were transformed into DH5a competent cells by standard protocol. Plasmids used for transfection assays were isolated and purified using EndoFree Maxi Kit (Qiagen, Valencia, Calif.).


PC-12 Cell Culture. PC-12 cells were maintained in RPMI 1640 supplemented with 10% horse serum, 5% fetal bovine serum, 4.5 g/L glucose, 2 mM L-glutamine, 1.0 mM sodium pyruvate and 10 μg/ml penicillin-streptomycin.


Cell Differentiation. Cell culture dishes were pre-coated overnight with 0.25 mg/ml collagen (Cohesion, Calif.), 10 μg/ml Poly-L-Lysine (Sigma-Aldrich, St. Louis, Mo.) and 12 μg/ml Polyethyleneimine (ICN, La Mesa, Calif.). Cells were cultured on coated dishes at 15,000 cells/cm2, and differentiated into a neuron-like phenotype by incubation in medium with reduced serum (1% fetal bovine serum), containing 50 ng/ml nerve growth factor (NGF) (Sigma-Aldrich) for 10 days. NGF-containing medium was changed every 2-3 days.


Treatment with Compound D. Compound D, a small molecule inhibitor of β-catenin/CBP interaction, was dissolved in DMSO at a stock concentration of 100 mM. Differentiated PC-12/L286V cells were treated with increasing concentrations of this compound for 4 hours. Transfection was then initiated after this treatment period. For cell differentiation experiments, Compound D was added at a concentration of 10 μM, together with NGF, for the entire differentiation period.


Transfection. PC-12 cells were cultured and differentiated on 60-mm dishes. At the end of the 10-day differentiation period, cells were transfected with 2 μg reporter constructs, TOPFLASH and FOPFLASH, per 60-mm dish. Transfections were performed using Superfect (Qiagen) according to manufacturer's instructions.


Luciferase Assays. Cells were lysed, 6 hours after transfections, in 100 μl of Cell Culture Lysis Reagent (Promega, Madison, Wis.), and scraped into microcentrifuge tubes. Tubes were then centrifuged briefly (about 10 seconds) at 12000 rpm to pellet cell debris. Luciferase activity was measured on 20 μl of cell lysate and 100 μl substrate from the Luciferase Assay System (Promega). Luciferase activity was measure using Packard LumiCount. (Hewlett Packard). Quantitation of luciferase was performed in triplicates, and repeated in at least three independent experiments.


Immunofluorescence. Cells were plated at a density of 10,000 cells/cm2 on sterile coated 22×22 mm coverslips in a 6-well culture plate.


Differentiation was initiated, as previously described, for 10 days. The differentiated cells were then fixed in methanol for 15 minutes at −20° C. This is followed by a 15 minutes incubation with PBS+0.1% Triton X-100. The coverslips were incubated with antibodies raised against Ephrin B2 Receptor (Santa Cruz Biotechnology) and Gap-43 (Novus Biologicals) for 40 minutes at 37° C. After a series of washes with PBS-Triton X-100, secondary antibody conjugated to FITC (Jackson ImmunoResearch, Westgrove, Pa.) was applied. All slides images were acquired using a Nikon PCM2000 Laser Scanning Confocal Microscope mounted on a Nikon Eclipse E600 upright microscope (Nikon, Melville, N.Y.). Quantitation of Neurite Outgrowth. Cell counts were taken from six randomly chosen microscopic fields (10×). In each field, total number of cells, as well as cells that displayed neurites greater than twice the length of the cell body was determined. The number of cells with such outgrowths was then expressed as a percentage of the total number of cells. Values obtained were from duplicates of three independent experiments.


RT-PCR. To analyze the mRNA levels for Ephrin B2 (EphB2) receptor, total RNA was isolated using Trizol (Invitrogen-GIBCO-BRL, Baltimore, Md.) from differentiated cells. 2 μg RNA was reverse transcribed in a total volume of 20 μl with random hexamer (50 ng), and using the Superscript II reverse transcription system (Invitrogen-GIBCO-BRL), according to manufacturer's guidelines. PCR was carried out in a 50 μl volume containing 5 μl cDNA, 100 pmol primers, 100 μM dNTPs, 1× Taq buffer and 1.5 mM MgCl2. Reaction mixtures were heated to 80° C. for 10 min, after which Taq was added. cDNAs were amplified for 25 (EphB2 receptor) or 15 (GAPDH) cycles. One round of amplification consisted of 1 min at 94° C., 2 min at 60° C., and 2 min at 72° C., with a final extension time of 10 min at 72° C. The PCR products were resolved and visualized by electrophoresis in a 2% gel, stained with ethidium bromide. EphB2 receptor PCR primers used were, 5′-CACTACTGGACCGCACGATAC-3′ (SEQ ID NO:4) and 5′-TCTACCGACTGGATCTGGTTCA-3′(SEQ ID NO:5). Primer pairs for GAPDH were 5′-GGTGCTGAGTATGTCGTGGA-3′ (SEQ ID NO:6) and 5′-ACAGTGTTCTGGGTGGCAGT-3′(SEQ ID NO:7).


Results


Rat PC-12 cells are derived from the neural crest lineage and upon nerve growth factor (NGF) treatment, undergo differentiation to a neurite-bearing sympathetic-like neuron (Greene and Tischler, Proc Natl Acad Sci USA 73, 2424 (1976)). Utilizing a PC-12 cell based model, the effects of an early-onset FAD associated PS-1 mutation, PS-1/L286V, on TCF/β-catenin mediated transcription and neuronal differentiation were characterized. It has been demonstrated that specifically blocking transcription mediated by TCF/β-catenin/CBP alleviates PS-1 induced defects in neuronal differentiation.


PC-12 cells stably overexpressing either wild type PS-1 (PS-1/WT) or mutant PS-1 (PS-1/L286V) and a vector-transfected control cell line (Guo et al., Neuroreport, 8, 379 (1996)) were plated on dishes coated with collagen, poly-L-lysine and poly-ethyleneimine. Differentiation was induced by treatment with 50 ng/ml of NGF for 10 days. Overexpressing PS-1/WT cells or the vector-transfected cells had extensive neurite formation (similar to PC-12 cell clones from ATCC), whereas the PS-1/L286V mutant cells had only stubby neurite formation (FIG. 4 A-C). Additionally, vector-transfected PC-12 control and PS-1/WT cells displayed extensive expression of the neuronal differentiation marker GAP-43 (Gorgels et al., Neurosci Lett. 83, 59 (1987)) (FIG. 4 D,E), whereas the PS-1/L286V cells were essentially devoid of this marker (FIG. 4 F).


To assess the effects of the PS-1/L286V mutation on canonical Wnt/β-catenin signaling, we transiently transfected NGF treated PC-12 cells with Topflash, a Wnt/β-catenin signaling reporter construct (Morin et al., Science 275, 1787 (1997)). As seen in FIG. 4F, the overexpressing PS-1/WT cells had similar levels of TCF/β-catenin signaling compared to the vector control cells. However, the PS-1/L286V mutant cells displayed significantly (10-fold) increased Topflash expression. In contrast, the negative control reporter construct Fopflash did not show any significant differences.


It was hypothesized that dysregulated TCF/β-catenin signaling in the PS-1/L286V mutant cells was responsible for the defective differentiation and neurite outgrowth. To test this hypothesis, a specific small molecule inhibitor of TCF/β-catenin signaling, Compound D (Emami et al., Cancer Cell, in press), was used. This small molecule selectively blocks the β-catenin/CBP interaction, but not the β-catenin/p300 interaction, thereby interrupting a subset of TCF/β-catenin transcription. Treatment of the PS-1/L286V mutant cells with 10 μM Compound D plus NGF decreased TCF/β-catenin reporter gene transcription, and led to essentially normal neurite outgrowth and differentiation (FIG. 5 A), similar to that seen in the overexpressing PS-1/WT cells (FIGS. 5 A, B), as compared to the untreated cells (FIG. 4 C). Furthermore, PS-1/L286V mutants treated with Compound D showed similar intense GAP-43 staining to the PS-1/WT and vector-transfected cells (FIG. 4 B). To demonstrate that Compound D treated mutant cells develop neurites similar to that of the vector control or PS-1/WT cells, cells that had neurites greater than twice the length of the cell body were counted. Treatment with Compound D substantially increased the percentage of cells bearing neurites to levels similar to that of the vector-transfected and overexpressing PS-1/WT cells (FIG. 5 C). It is concluded that blocking transcription mediated by TCF/β-catenin/CBP corrects many of the phenotypic defects in neurite outgrowth and neuronal differentiation due to the PS-1/L286V mutation.


Ephrin B2 receptors (EphB2) have been implicated in synapse formation (Wilkinson, Nat. Rev. Neurosci. 2, 155 (2001)) and the Ephrin A family has recently been shown to play a role in hippocampal dendritic spine morphology (Murai et al., Nat. Neurosci. 6, 153 (2003)). Focused EphB2 expression was observed, which localized with neuronal processes in the vector and PS-1/WT-transfected cells (FIG. 6 A, B), whereas the PS-1/L286V mutant cells demonstrated very weak and diffuse EphB2 signal (FIG. 6 C). Increased TCF/β-catenin signaling in PS-1/L286V mutant cells manifested itself in decreased EphB2 expression as judged by RT-PCR (FIG. 6 E, lane 3). Furthermore, addition of 10 μM Compound D led to increased EphB2 message (FIG. 6 E, lane 4) as well as EphB2 expression in these cells (FIG. 6 D). These results are consistent with the data of Bathe and colleagues (Battle et al., Cell 111, 251 (2002)) who recently showed that expression of EphB2/EphB3 receptors and their ligand ephrin-B1 is inversely controlled in colonic crypts via TCF/β-catenin transcription, and that proper regulation is important for appropriate cell proliferation, differentiation and sorting. We present evidence that the PS-1/L286V mutation via increased TCF/β-catenin signaling, decreased the expression of EphB2 receptors and this is corrected by Compound D mediated inhibition of the β-catenin/CBP interaction.


Example 9
Compound D Causes a G1/S-Phase Arrest and Activates Caspase Activity

Flow Cytometric Analysis (FACS)


For FACS analysis, approx. 5×106 cells from Compound D-treated or vehicle-treated were fixed with 70% chilled ethanol and stored at −20° C. for at least 30 minutes. The cells were washed once with 1×PBS and incubated with propidium iodine (PI) solution (85 μg/ml propidium iodine, 0.1% Nonidet P-40, 10 mg/ml RNAse) for 30 minutes at room temperature. 10,000 stained cells for each sample were acquired using Beckman Coulter EPICS XL-MCL Flow Cytometry and the percentage of cells in different phase of the cell cycle was determined by Expo32 ADC software (Coulter Corporation, Miami, Fla., 33196).


Caspase-3 Activity Assay


SW480, HCT116, and CCD18Co cells were plated at 105 cells per well (96-well plates) for 24 hours prior to treatment. 25 μM of Compound D or control (0.5% DMSO) was added to each well. 24 hours post treatment, cells were lysed and caspase activity was measured using a caspase-3/7 activity kit (Apo-One Homogeneous caspase-3/7 assay, #G77905, Promega). Relative fluorescence units (RFU) were obtained by subtracting the unit values of the blank (control, without cells) from the experimental measured values.


Compound D Causes a G1/S-Phase Arrest and Activates Caspase Activity


It has been shown that inhibition of the expression of the cyclin D1 gene causes arrest at the G1/S-phase of the cell cycle (Shintani et al., “Infrequent alternations of RB pathway (Rb-p16INK4A-cyclin D1) in adenoid cystic carcinoma of salivary glands,” Anticancer Res. 20:2169-75 (2000)). HCT116 (FIG. 7A, upper panel) and SW480 (FIG. 7A, lower panel) cells were treated with Compound D (25 μM) (FIG. 7A, right) or control (0.5% DMSO) (FIG. 7A, left) for 24 hours. The cells were subsequently stained with propidium iodide (PI) and analyzed for DNA content by FACS cytofluorometry. As expected, the control cells, (FIG. 7A, left), were cycling normally whereas the Compound D treated cells (FIG. 7A, right) showed increased accumulation at G1/S-phase of the cell cycle. Thus, it can be seen that Compound D causes arrest of cells at the G1 phase.


Caspases are cysteine proteases that are generally activated in a given population of cells triggered by apoptotic stimuli. To assess apoptotic induction in SW480, HCT116, and wild-type colonocytes (CCD18Co cells), the cells were treated with either Compound D (25 μM) or control (0.5% DMSO) for 24 hours, followed by an assay for caspase-3/7 activity. As shown in FIG. 7B, Compound D specifically and significantly activated the caspase-3/7 pathway in SW480 and HCT116 cells compared to CCD18Co cells.


Example 10
Compound D Reduces Proliferation of Transformed Colorectal Cells

Soft Agar Assays


The soft agar colony formation assay was conducted with SW480 cells by some modification of the procedure previously described (Moody et al., “A vasoactive intestinal peptide antagonist inhibits non-small cell lung cancer growth,” Proc. Natl. Acad. Sci. USA. 90:4345-49 (1993)).


Each well (35 mm) of a 6-well plate (Nalge Nunc International, Roskide, Denmark) was coated with 1 ml of 0.8% bottom agar in DMEM medium containing 10% fetal bovine serum. After it was solidified, 1 ml of DMEM medium containing 0.4% top agar, 10% fetal bovine serum, compound doubly concentrated, and 5,000 single viable cells was added to each well. The cultures were incubated at 37° C. in humidified 5% CO2 incubator. Colonies in soft agar were monitored daily and photographed after incubation for 8 days. Colonies>60 μm in diameter were counted.


Compound D Reduces Proliferation of Transformed Colorectal Cells


Soft agar colony forming assays were performed using SW480 cells treated with Compound D (0.25-5 μM) and 5-fluorouracil (5-FU) (0.5-32 μM). As shown in FIG. 8A, Compound D shows a dose dependent decrease in the number of colonies formed. IC50 value of Compound D and 5-FU was 0.87±0.11 μM and 1.98±0.17 μM, respectively. Thus, Compound D increased caspase activity and reduced growth in vitro of colorectal cells that are transformed by mutations that activate β-catenin signaling.


Example 11
Compound C Reduces Tumor Growth in Nude Mouse Model

SW620 cells (9×106 cells/mouse) were grafted into nude mice subcutaneously on Day 0. Mice received 200 mg/kg of Compound C intraperitoneally every other day until Day 21 after 4 times of 300 mg/kg every other day starting Day 1. Compound C reduces the tumor growth in the treated mice compared to the vehicle control mice (FIG. 9A), and slightly reduces body weights of the treated mice compared to those of the vehicle control mice (FIG. 9B).


Example 12
Compound D Suppresses Survivin Expression

The effect of Compound D on survivin expression was studied at both transcriptional and translational levels. The methods used at the transcriptional level include cDNA microarray analysis, RT-PCR, survivin reporter assays and chromatin immunoprecipitation (ChIP). The methods used at translational levels include Western blot analysis and immunochemistry.


A plasmid containing luciferase under the control of survivin promoter was constructed and transfected into wild type, CBP+/−, or p300+/−3T3 cells. The results (FIG. 10) show that Wnt 1 stimulates expression of the survivin gene in all three types of cells, whereas Compound D reduces expression of the survivin gene and decreases the stimulation of the survivin gene expression by Wnt1 in those cells. Similarly, Compound D and its analog (Compound A) were shown to inhibit expression of survivin in SW480 cells (FIG. 11).


Real time reverse transcription-PCR analysis was performed according to the protocol provided with the SYBR Green PCR Master Mix Kit (Perkin Elmer Biosystems, Shelton, ST). Total RNA templates for the RT-PCR reactions were extracted with the Rneasy Midi Kit (Qiagen) from cells treated with Compound D (25 μM) or control (0.5% DMSO) 24 hours after treatment. The primers used for the RT-PCR reactions were 5′-AGCCCTTTCTCAAGGACCAC-3′ (SEQ ID NO:8) and 5′-GCACTTTCTTCGCAGTTTCC-3′ (SEQ ID NO:9). Table 8 shows the results of the analysis. A ratio less than 0.5 indicates a significant decrease of gene expression due to the treatment of Compound D, whereas a ratio greater than 1.5 indicates a significant increase of gene expression. A ratio about 1 indicates no change. As indicated in Table 8 and FIG. 12, the expression of the survivin gene is significantly reduced in the presence of Compound D compared to the control.









TABLE 8







Gene Expression with and without Compound D











Ratio




(Treated/DMSO



Gene
Control)














Ubiquitin
0.98



GADPH
0.98



HLAC
1.01



Survivin
0.30



PCNA
0.33



Antigen KI-67
0.45



MIC-1
7.0



GADD-153
7.00










ChIP assays on SW 480 cells treated with either Compound D (25 μM) or control (0.5% DMSO) were performed. As shown in FIG. 13, the survivin promoter is occuried by CBP, β-catenin, Tcf4 and acetylated histone in control treated cells. Treatment with Compound D decreases the association of all these proteins with the survivin promoter.


To characterize the effect of Compound D on the survivin expression at the translational level, Western blot analysis of extracts of cells treated with vehicle (0.5% DMSO) alone, 10 μM or 25 μM Compound D, or 5 μM 5-FU was performed using survivin 6E4 monoclonal antibody (Cell Signaling Technology). The results (FIG. 14A) show that the treatments with Compound D at both concentrations and the treatment with 5-FU reduced the amount of the survivin protein. The treatments with Compound D at both concentrations were more effective in reducing the survivin expression than the treatment with 5-FU, and the treatment with Compound D at the higher concentration (i.e., 25 μM) was most effective.


The effect of Compound D on the survivin expression at the translational level was further characterized using immunofluorescence microscopy. In the absence of Compound D, survivin localizes to the mitotic spindle apparatus, consistent with the notion that survivin is involved in chromosomal separation (FIG. 14B). This expression pattern was not observed in SW480 cells after the treatment of Compound D as little or no survivin protein was detected (FIG. 14C).


Example 13
Effects of Various Compounds on Survivin and TCF4 Expression

The effects of various compounds having general formula (I) on survivin and TCF4 expression were characterized. The results are shown in Table 9.









TABLE 9







Effects of compounds on survivin and TCF4 expression










Survivin % inhibition
TCF4 IC50











5 uM
25 uM
(uM)
















embedded image


100
99
~2  







embedded image


97
100
~2.2







embedded image


51
93
~6.3







embedded image


41
92
5.2 ± 0.7







embedded image


0
6
18.2 ± 2.4 







embedded image


0
80
1.3 ± 0.1







embedded image


0
93
2.2 ± 0.2







embedded image


46
96
4.4 ± 0.6







embedded image


0
77
3.5 ± 0.3







embedded image


0
92
7.3 ± 0.6







embedded image


79
81
1.7 ± 0.2







embedded image


0
84
4.8 ± 0.4







embedded image


0
68
10.9 ± 1.3 







embedded image


8
4
NA







embedded image


9
91
1.4 ± 0.2







embedded image


5
91
 6.3 ± 0.431







embedded image


0
94
2.6 ± 0.4







embedded image


0
21
7.3 ± 1.1







embedded image


0
91
5.2 ± 1.1







embedded image


45
88
13.2 ± 4.1 







embedded image


9
92
5.9 ± 0.5







embedded image


6
58
11.2 ± 1.5 







embedded image


48
96
 3.9 ± 0.55







embedded image


0
32
50.4 ± 7.0 







embedded image


86
91
2.6 ± 0.6







embedded image


27
98
10.7 ± 1.7 







embedded image


80
97
4.6 ± 0.7







embedded image


82
97
2.8 ± 0.4







embedded image


6
89
13.9 ± 2.3 







embedded image


14
99
10.7 ± 1.9 







embedded image


25
44
27.1 ± 4.6 









Example 14
Compound D Promotes Apoptosis Via Suppression of Survivin Expression

To determine the effect of Compound D on apoptosis and the role of survivin in such an effect, the activities of caspases 2 and 3 in cultured tumor cells treated with either Compound D or control were measured. The results (FIG. 15) show that (1) Compound D (at 2.5 μM or 5.0 μM) activated the caspase 3 activity, but not the caspase 2 activity; (2) stausporine (0.5 μM) increased both the caspase 2 and caspase 3 activities; (3) the co-treatment of stausporine and Compound D produced a synergic stimulation of the caspase 3 activity, but not a synergic stimulation of the caspase 2 activity; and (4) transfection of the survivin gene decreased the activation of the caspase 3 activity induced by the treatment of stausporine or Compound D, and the synergic stimulation of the caspase 3 activity induced by the co-treatment of stausporine and Compound D. The above results suggest that Compound D stimulate the caspase 3 activity via suppression of the expression of the survivin gene.


The effect of compound D on apoptosis and the role of survivin in such an effect were further characterized by measuring cell death of cultured tumor cells treated with staurosporine (0.5 μM), Compound D (5.0 μM) or both. The results (FIG. 16) showed that both Compound D and stausporine promote cell death, and that transfection of the survivin gene decreased the increase in cell death induced by the treatment of stausporine, Compound D, or both. The above results suggest that Compound D promote apoptosis via suppression of the expression of the survivin gene.


To determine the effect of Compound D on cell cycle and the role of survivin in such an effect, FACS analysis was performed on cultured tumor cells with or without transfection of a construct containing the survivin gene and further treated with stausporine (0.5 μM), Compound D (5 μM), or both. The results (FIG. 17) show that both stausporine and Compound D increase the number of cells in G0, and that overexpression of survivin in the cells decreases the effect of the treatment of stausporine, Compound D, or both. These results suggest that the effect of Compound D on cell cycle may be at least partially via suppression of the expression of the survivin gene.


It will be appreciated that, although specific embodiments of the invention have been described herein for the purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.


All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. patent application Ser. No. 10/087,443 filed on Mar. 1, 2002, and U.S. patent application Ser. No. 09/976,470 filed on Oct. 12, 2001, are incorporated herein by reference.


From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims
  • 1. A compound of formula (VI):
  • 2. The compound of claim 1, wherein the bicyclic aryl group is not substituted.
  • 3. The compound of claim 1, wherein the bicyclic aryl group is substituted.
  • 4. The compound of claim 3, wherein the bicyclic aryl group is substituted indazolyl.
  • 5. The compound of claim 4 selected from the group consisting of:
  • 6. The compound of claim 3, wherein the bicyclic aryl group is substituted quinolinyl.
  • 7. The compound of claim 6 selected from the group consisting of:
  • 8. The compound of claim 3, wherein the bicyclic aryl group is substituted 3,4-dihydrobenzo[1,4]oxazinyl.
  • 9. The compound of claim 8 selected from the group consisting of:
  • 10. The compound of claim 3, wherein the bicyclic aryl group is substituted naphthyl.
  • 11. The compound of claim 10 being
  • 12. The compound of claim 3, wherein the bicyclic aryl group is substituted benzotriazolyl.
  • 13. The compound of claim 12 selected from the group consisting of:
  • 14. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.
  • 15. The pharmaceutical composition of claim 14, wherein the bicyclic aryl group is not substituted.
  • 16. The pharmaceutical composition of claim 14, wherein the bicyclic aryl group is substituted.
Priority Claims (1)
Number Date Country Kind
PCT/KR02/01901 Oct 2002 WO international
CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 11/242,653, filed Oct. 4, 2005, which is a divisional of U.S. patent application Ser. No. 10/803,179, filed Mar. 17, 2004 (issued U.S. Pat. No. 7,232,822); which is a continuation-in-part of U.S. patent application Ser. No. 10/411,877, filed Apr. 9, 2003 (now abandoned); which is a continuation-in-part of U.S. patent application Ser. No. 10/087,443, filed Mar. 1, 2002 (now abandoned); which is a continuation-in-part of U.S. patent application Ser. No. 09/976,470, filed Oct. 12, 2001 (now abandoned). U.S. patent application Ser. No. 10/411,877, filed Apr. 9, 2003 (now abandoned) claims priority to PCT application serial No. PCT/KR02/01901, filed Oct. 11, 2002. The disclosures of these applications are incorporated herein by reference in their entireties.

US Referenced Citations (39)
Number Name Date Kind
5440013 Kahn Aug 1995 A
5475085 Kahn Dec 1995 A
5618914 Kahn Apr 1997 A
5670155 Kahn Sep 1997 A
5672681 Kahn Sep 1997 A
5693325 Kahn Dec 1997 A
5710245 Kahn Jan 1998 A
5840833 Kahn Nov 1998 A
5859184 Kahn et al. Jan 1999 A
5929237 Kahn Jul 1999 A
6013458 Kahn et al. Jan 2000 A
6020331 Kahn Feb 2000 A
6117896 Qabar et al. Sep 2000 A
6184223 Kahn et al. Feb 2001 B1
6245764 Kahn et al. Jun 2001 B1
6294525 Stasiak et al. Sep 2001 B1
6372744 Qabar et al. Apr 2002 B1
6413963 Kahn et al. Jul 2002 B2
6440955 Stasiak et al. Aug 2002 B1
6548500 Kahn et al. Apr 2003 B2
6762185 Kahn et al. Jul 2004 B1
7232822 Moon et al. Jun 2007 B2
7531320 Kahn et al. May 2009 B2
7563825 Kahn Jul 2009 B1
7566711 Moon et al. Jul 2009 B2
7576084 Moon et al. Aug 2009 B2
7671054 Moon et al. Mar 2010 B1
20010039274 Kahn et al. Nov 2001 A1
20020022620 Kahn et al. Feb 2002 A1
20020065416 Stasiak et al. May 2002 A1
20020068695 Scolastico et al. Jun 2002 A1
20030021773 Moroder et al. Jan 2003 A1
20030027819 Qabar et al. Feb 2003 A1
20030105103 Golebiowski et al. Jun 2003 A1
20040053331 Kahn et al. Mar 2004 A1
20040072831 Moon et al. Apr 2004 A1
20050049234 Deslongchamps et al. Mar 2005 A1
20070128669 Kahn Jun 2007 A1
20070129353 Kahn Jun 2007 A1
Foreign Referenced Citations (15)
Number Date Country
2384126 Mar 2001 CA
0065724 Dec 1982 EP
WO 9403494 Feb 1994 WO
WO 9715577 May 1997 WO
WO 9805333 Feb 1998 WO
WO 9849168 Nov 1998 WO
WO 0100210 Jan 2001 WO
WO 0116135 Mar 2001 WO
WO 03006447 Jan 2003 WO
WO 03031448 Apr 2003 WO
WO 2004072076 Aug 2004 WO
WO 2004072077 Aug 2004 WO
WO 2004093828 Nov 2004 WO
WO 2005116032 Dec 2005 WO
WO2009148192 Dec 2009 WO
Related Publications (1)
Number Date Country
20100120758 A1 May 2010 US
Divisions (1)
Number Date Country
Parent 10803179 Mar 2004 US
Child 11242653 US
Continuations (1)
Number Date Country
Parent 11242653 Oct 2005 US
Child 12553858 US
Continuation in Parts (3)
Number Date Country
Parent 10411877 Apr 2003 US
Child 10803179 US
Parent 10087443 Mar 2002 US
Child 10411877 US
Parent 09976470 Oct 2001 US
Child 10087443 US