# Patent

## Title
Reverse-turn mimetics and method relating thereto

## 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 over a wide range of fields, including use as diagnostic and therapeutic agents. Libraries containing the reverse-turn mimetic structures of this invention are also disclosed as well as methods for screening the same to identify biologically active members. The invention also relates to the use of such compounds 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.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 10/826,972, filed Apr. 16, 2004, which is a continuation-in-part of co-pending U.S. patent application Ser. No. 10/803,179 filed Mar. 17, 2004, 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). This application also 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.


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 (“μ”) 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-3β 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):




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):




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):




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):




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.

In certain embodiments, the compounds of this invention have the following general formula (VI):




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.

The present invention is also related to prodrugs using the libraries containing one or more compounds of formula (I). A prodrug is typically designed to release the active drug in the body during or after absorption by enzymatic and/or chemical hydrolysis. The prodrug approach is an effective means of improving the oral bioavailability or i.v. administration of poorly water-soluble drugs by chemical derivatization to more water-soluble compounds. The most commonly used prodrug approach for increasing aqueous solubility of drugs containing a hydroxyl group is to produce esters containing an ionizable group; e.g., phosphate group, carboxylate group, alkylamino group (Fleisher et al., Advanced Drug Delivery Reviews, 115-130, 1996; Davis et al., Cancer Res., 7247-7253, 2002, Golik et al., Bioorg. Med. Chem. Lett., 1837-1842, 1996).
In certain embodiments, the prodrugs of the present invention have the following general formula (VII):

(VI)—Y—R10 

wherein (VI) is general formula (VI) as described above; Y is oxygen, sulfur, or nitrogen of a group selected from Ra, Rb, Rc, X1, X2 and X3;

R10 is phosphate, hemisuccinate, hemimalate, phosphoryloxymethyloxycarbonyl, dimethylaminoacetate, dimethylaminoalkylcarbamates, hydroxyalkyls, amino acid, glycosyl, substituted or unsubstituted piperidine oxycarbonyl, or a salt thereof; and wherein the prodrugs are capable of serving as a substrate for a phosphatase or a carboxylase and are thereby converted to compounds having general formula (VI).
In some embodiments, R10 of the general formula (VII) is not an amino acid group or a phospho-amino acid group.
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, LXXLI or FXXFF 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 analsis 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 Technolgy). 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.
FIG. 18 shows the changes of concentrations of prodrug A and its parent compound in mouse plasma with the increase of time after i.v. bolus injection of prodrug A. Square: parent compound; Diamond: prodrug A.



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):




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, diC1-4alkylguanidinoC2-5alkyl, 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 C1-4alkl (where the imidazole sustituents 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



—CH2CH(CH3)2
Leucine



—CH(CH3)CH2CH3
Isoleucine



—(CH2)4NH3+
Lysine



—(CH2)3NHC(NH2)NH2+
Arginine



 







Histidine



 



—CH2COO−
Aspartic acid



—CH2CH2COO
Glutamic acid



—CH2CONH2
Asparagine



—CH2CH2CONH2
Glutamine



 







Phenylalanine



 







Tyrosine



 







Tryptophan



 



—CH2SH
Cysteine



—CH2CH2SCH3
Methionine



—CH2OH
Serine



—CH(OH)CH3
Threonine



 







Proline



 







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, —CONR, —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):




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):




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):




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:




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 Figure. 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:




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′:




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′:





wherein R2, R4 and P are as defined above,

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




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 prepartion 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.




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:




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

In another aspect of this invention, prodrugs derived from compounds having general formula (I) are disclosed. The prodrugs generally increase aqueous solubility and thus bioavailability of compounds having general formula (I). In certain embodiments, the prodrugs of the present invention have the following general formula (VII):

(VI)—Y—R10 

wherein (VI) is general formula (VI) as described above; Y is oxygen, sulfur, or nitrogen of a group selected from Ra, Rb, Rc, X1, X2 and X3; R10 is phosphate, hemisuccinate, hemimalate, phosphoryloxymethyloxycarbonyl, dimethylaminoacetate, dimethylaminoalkylcarbamate, hydroxyalkyl, amino acid, glycosyl, substituted or unsubstituted piperidine oxycarbonyl, or a salt thereof; and wherein the prodrugs are capable of serving as a substrate for a phosphatase or a carboxylase and are thereby converted to compounds having general formula (VI). In some embodiments, R10 of the general formula (VII) is not an amino acid group or a phospho-amino acid group.

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:




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 Isocynate)
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 isocynate (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 pro duct 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








 













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-benzy
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-benzy
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-benzy
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-benzy
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


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
86


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
Styrylmethy
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)-α-
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)-α-
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)-α-
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)-α-
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)-α-
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)-α-
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)-α-
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)-60 -
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






Indoleethyl)amino


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)amio
570
571


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






Indoleethyl)amino


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






Indoleethyl)amino


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
(Tetrahydrofuran-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






Indoleethyl)amino


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
(Tetrahydrofuran-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






Indoleethyl)amino


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
(Tetrahydrofuran-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






Indoleethyl)amino


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
(Tetrahydrofuran-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






Indoleethyl)amino


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
(Tetrahydrofuran-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






Indoleethyl)amino


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
(Tetrahydrofuran-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
2-Pipendin-1-yl-
(S)-4-HO-benzyl
Methyl
ethylamino
604
605



3,4-Cl2-benzyl


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-
593
594






Aminophenethylamino


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
Dimethoxybenzylamino
624
625


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-






isoindol-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-1 yl-
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-Pyndylmethyl
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-CO2H-CH2O-
Methyl
Benzylamino
622
623




benzyl


557
Naphthyl-1-ylmethyl
1-Naphthylamino-
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-1-yl-
3,4-(OH)2-benzyl
Methyl
Benzylamino
581
582



methyl


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



aminobenzyl


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
6093,4-F2-2-
4-HO-benzyl
Methyl
Benzylamino
571
572



6-(CH3)-2-



Dimethylaminobenzyl


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



Dimethylaminobenzyl
















TABLE 2B







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








 












Mol.
M + H


No
MOLSTRUCTURE
Weight
(MS)













802




480
481


 


803




430
431


 


804




416
417


 


805




464
465


 


806




430
431


 


807




430
431


 


808




448
448


 


809




416
417


 


810




431
432


 


811




446
447


 


812




450
451


 


813




515
516


 


814




582
583


 


815




532
533


 


816




518
519


 


817




566
567


 


818




532
533


 


819




532
533


 


820




550
551


 


821




518
519


 


822




534
535


 


823




548
549


 


824




552
553


 


825




617
618


 


826




542
543


 


827




492
493


 


828




478
479


 


829




526
527


 


830




492
493


 


831




492
493


 


832




510
511


 


833




478
479


 


834




494
495


 


835




508
509


 


836




512
513


 


837




577
578


 


838




468
469


 


839




516
517


 


840




482
483


 


841




482
483


 


842




468
469


 


843




484
485


 


844




498
499


 


845




502
503


 


846




567
568


 


847




508
509


 


848




458
459


 


849




444
445


 


850




492
493


 


851




458
459


 


852




458
459


 


853




476
477


 


854




444
445


 


855




460
461


 


856




474
475


 


857




478
479


 


858




543
544


 


859




494
495


 


860




444
445


 


861




430
431


 


862




478
479


 


863




444
445


 


864




444
445


 


865




462
463


 


866




430
431


 


867




446
447


 


868




460
461


 


869




464
465


 


870




529
530


 


871




558
559


 


872




508
509


 


873




494
495


 


874




542
543


 


875




508
509


 


876




508
509


 


877




526
527


 


878




494
495


 


879




510
511


 


880




524
525


 


881




528
529


 


882




593
594


 


883




432
433


 


884




480
481


 


885




446
447


 


886




446
447


 


887




464
465


 


888




432
433


 


889




447
448


 


890




462
463


 


891




466
467


 


892




531
532


 


893




558
559


 


894




508
509


 


895




494
495


 


896




542
543


 


897




508
509


 


898




508
509


 


899




526
527


 


900




494
495


 


901




510
511


 


902




524
525


 


903




528
529


 


904




593
594


 


905




544
545


 


906




494
495


 


907




480
481


 


908




528
529


 


909




494
495


 


910




494
495


 


911




512
513


 


912




480
481


 


913




496
497


 


914




510
511


 


915




514
515


 


916




579
580


 


917




464
465


 


918




450
451


 


919




498
499


 


920




464
465


 


921




464
465


 


922




482
483


 


923




450
451


 


924




466
467


 


925




480
481


 


926




484
485


 


927




549
550


 


928




480
481


 


929




430
431


 


930




416
417


 


931




464
465


 


932




430
431


 


933




430
431


 


934




448
449


 


935




416
417


 


936




431
432


 


937




446
447


 


938




450
451


 


939




515
516


 


940




504
505


 


941




454
455


 


942




440
441


 


943




488
489


 


944




454
455


 


945




454
455


 


946




472
473


 


947




440
441


 


948




455
456


 


949




470
471


 


950




474
475


 


951




539
540


 


952




604
605


 


953




554
555


 


954




540
541


 


955




588
589


 


956




554
555


 


957




554
555


 


958




572
573


 


959




540
541


 


960




556
557


 


961




570
571


 


962




574
575


 


963




639
640


 


964




528
529


 


965




478
479


 


966




464
465


 


967




512
513


 


968




478
479


 


969




478
479


 


970




496
497


 


971




464
465


 


972




480
481


 


973




494
495


 


974




498
499


 


975




563
564


 


976




582
583


 


977




532
533


 


978




518
519


 


979




566
567


 


980




532
533


 


981




532
533


 


982




551
552


 


983




518
519


 


984




534
535


 


985




548
549


 


986




552
553


 


987




618
619


 


988




482
483


 


989




432
433


 


990




418
419


 


991




466
467


 


992




432
433


 


993




432
433


 


994




450
451


 


995




418
419


 


996




433
434


 


997




447
448


 


998




452
453


 


999




517
518


 


1000




548
549


 


1001




498
499


 


1002




484
485


 


1003




532
535


 


1004




498
499


 


1005




498
499


 


1006




516
517


 


1007




484
485


 


1008




500
501


 


1009




514
515


 


1010




518
519


 


1011




583
584


 


1012




532
533


 


1013




518
519


 


1014




566
567


 


1015




532
533


 


1016




532
533


 


1017




551
552


 


1018




518
519


 


1019




534
535


 


1020




548
549


 


1021




552
553


 


1022




618
619


 


1023




528
529


 


1024




478
479


 


1025




464
465


 


1026




512
513


 


1027




478
479


 


1028




478
479


 


1029




496
497


 


1030




464
465


 


1031




480
481


 


1032




494
495


 


1033




498
499


 


1034




563
564


 


1035




528
529


 


1036




478
479


 


1037




464
465


 


1038




512
513


 


1039




478
479


 


1040




478
479


 


1041




496
497


 


1042




464
465


 


1043




480
481


 


1044




494
495


 


1045




498
499


 


1046




563
564


 


1047




556
557


 


1048




506
507


 


1049




492
493


 


1050




540
541


 


1051




506
507


 


1052




506
507


 


1053




524
525


 


1054




492
493


 


1055




508
509


 


1056




522
523


 


1057




526
527


 


1058




591
592


 


1059




546
547


 


1060




496
497


 


1061




482
483


 


1062




530
531


 


1063




496
497


 


1064




496
497


 


1065




514
515


 


1066




482
483


 


1067




498
499


 


1068




512
513


 


1069




516
517


 


1070




581
582


 


1071




528
529


 


1072




478
479


 


1073




464
465


 


1074




512
513


 


1075




478
479


 


1076




478
479


 


1077




496
497


 


1078




464
465


 


1079




480
481


 


1080




494
495


 


1081




498
499


 


1082




563
564


 


1083




514
515


 


1084




500
501


 


1085




548
549


 


1086




514
515


 


1087




514
515


 


1088




532
533


 


1089




500
501


 


1090




516
517


 


1091




530
531


 


1092




534
535


 


1093




599
600


 


1094




520
521


 


1095




470
471


 


1096




456
457


 


1097




504
505


 


1098




470
471


 


1099




470
471


 


1100




488
489


 


1101




456
457


 


1102




472
473


 


1103




486
487


 


1104




490
491


 


1105




555
556


 


1106




496
497


 


1107




482
483


 


1108




530
531


 


1109




496
497


 


1110




496
497


 


1111




514
515


 


1112




482
483


 


1113




498
499


 


1114




512
513


 


1115




516
517


 


1116




581
582


 


1117




542
543


 


1118




492
493


 


1119




478
479


 


1120




526
527


 


1121




492
493


 


1122




492
493


 


1123




510
511


 


1124




478
479


 


1125




494
495


 


1126




508
509


 


1127




512
513


 


1128




577
578


 


1129




550
551


 


1130




500
501


 


1131




486
487


 


1132




534
535


 


1133




500
501


 


1134




500
501


 


1135




518
519


 


1136




486
487


 


1137




501
502


 


1138




516
517


 


1139




520
521


 


1140




585
586


 


1141




588
589


 


1142




538
539


 


1143




524
525


 


1144




572
573


 


1145




538
539


 


1146




538
539


 


1147




556
557


 


1148




524
525


 


1149




540
541


 


1150




554
555


 


1151




558
559


 


1152




623
624


 


1153




508
509


 


1154




458
459


 


1155




444
445


 


1156




492
493


 


1157




458
459


 


1158




458
459


 


1159




476
477


 


1160




444
445


 


1161




460
461


 


1162




474
475


 


1163




478
479


 


1164




543
544


 


1165




618
619


 


1166




568
569


 


1167




554
555


 


1168




602
603


 


1169




568
569


 


1170




568
569


 


1171




586
587


 


1172




554
555


 


1173




570
571


 


1174




584
585


 


1175




588
589


 


1176




653
654


 


1177




494
495


 


1178




444
445


 


1179




430
431


 


1180




478
479


 


1181




444
445


 


1182




444
445


 


1183




462
463


 


1184




430
431


 


1185




446
447


 


1186




460
461


 


1187




464
465


 


1188




529
530


 


1189




506
507


 


1190




456
457


 


1191




442
443


 


1192




490
491


 


1193




456
457


 


1194




456
457


 


1195




474
475


 


1196




442
443


 


1197




458
459


 


1198




472
473


 


1199




476
477


 


1200




541
542


 


1201




592
593


 


1202




542
543


 


1203




528
529


 


1204




576
577


 


1205




542
543


 


1206




542
543


 


1207




561
562


 


1208




528
529


 


1209




544
545


 


1210




558
559


 


1211




562
563


 


1212




628
629


 


1213




538
539


 


1214




488
489


 


1215




474
475


 


1216




522
523


 


1217




488
489


 


1218




488
489


 


1219




506
507


 


1220




474
475


 


1221




490
491


 


1222




504
505


 


1223




508
509


 


1224




573
574


 


1225




510
511


 


1226




558
559


 


1227




524
525


 


1228




524
525


 


1229




510
511


 


1230




526
527


 


1231




540
541


 


1232




544
545


 


1233




609
610


 


1234




548
549


 


1235




498
499


 


1236




484
485


 


1237




532
533


 


1238




498
499


 


1239




498
499


 


1240




516
517


 


1241




484
485


 


1242




500
501


 


1243




514
515


 


1244




518
519


 


1245




583
584


 


1246




534
535


 


1247




484
485


 


1248




470
471


 


1249




518
519


 


1250




484
485


 


1251




484
485


 


1252




502
503


 


1253




470
471


 


1254




486
487


 


1255




500
501


 


1256




504
505


 


1257




569
570


 


1258




536
537


 


1259




486
487


 


1260




472
473


 


1261




520
521


 


1262




486
487


 


1263




486
487


 


1264




504
505


 


1265




472
473


 


1266




488
489


 


1267




502
503


 


1268




506
507


 


1269




571
572


 


1270




558
559


 


1271




508
509


 


1272




494
495


 


1273




542
543


 


1274




508
509


 


1275




508
509


 


1276




526
527


 


1277




494
495


 


1278




510
511


 


1279




524
525


 


1280




528
529


 


1281




593
594


 


1282




506
507


 


1283




456
457


 


1284




442
443


 


1285




490
491


 


1286




456
457


 


1287




456
457


 


1288




474
475


 


1289




442
443


 


1290




457
458


 


1291




472
473


 


1292




476
477


 


1293




541
542


 


1294




572
573


 


1295




522
523


 


1296




508
509


 


1297




556
557


 


1298




522
523


 


1299




522
523


 


1300




540
541


 


1301




508
509


 


1302




524
525


 


1303




538
539


 


1304




542
543


 


1305




607
608


 


1306




576
577


 


1307




526
527


 


1308




512
513


 


1309




560
561


 


1310




526
527


 


1311




526
527


 


1312




544
545


 


1313




512
513


 


1314




528
529


 


1315




542
543


 


1316




546
547


 


1317




611
612


 


1318




576
577


 


1319




526
527


 


1320




512
513


 


1321




560
561


 


1322




526
527


 


1323




526
527


 


1324




544
545


 


1325




512
513


 


1326




528
529


 


1327




542
543


 


1328




546
543


 


1329




611
612


 


1330




576
577


 


1331




526
527


 


1332




512
513


 


1333




560
561


 


1334




526
527


 


1335




526
527


 


1336




544
545


 


1337




512
513


 


1338




528
529


 


1339




542
543


 


1340




546
547


 


1341




611
612


 


1342




492
493


 


1343




442
443


 


1344




428
429


 


1345




476
477


 


1346




442
443


 


1347




442
443


 


1348




460
461


 


1349




428
429


 


1350




444
445


 


1351




458
459


 


1352




462
463


 


1353




527
528


 


1354




522
523


 


1355




472
473


 


1356




458
459


 


1357




506
507


 


1358




472
473


 


1359




472
473


 


1360




490
491


 


1361




458
459


 


1362




474
475


 


1363




488
489


 


1364




492
493


 


1365




557
558


 


1366




504
505


 


1367




454
455


 


1368




440
441


 


1369




488
489


 


1370




454
455


 


1371




454
455


 


1372




472
473


 


1373




440
441


 


1374




456
457


 


1375




470
471


 


1376




474
475


 


1377




539
540


 


1378




606
607


 


1379




556
557


 


1380




542
543


 


1381




590
591


 


1382




556
557


 


1383




556
557


 


1384




574
575


 


1385




542
543


 


1386




558
559


 


1387




572
573


 


1388




576
577


 


1389




641
642


 


1390




566
567


 


1391




516
517


 


1392




502
503


 


1393




550
551


 


1394




516
517


 


1395




516
517


 


1396




534
535


 


1397




572
573


 


1398




518
518


 


1399




532
533


 


1400




536
537


 


1401




601
602


 


1402




556
557


 


1403




506
507


 


1404




492
493


 


1405




540
541


 


1406




506
507


 


1407




506
507


 


1408




524
525


 


1409




492
493


 


1410




508
509


 


1411




522
523


 


1412




526
527


 


1413




591
592


 


1414




532
533


 


1415




582
583


 


1416




568
569


 


1417




516
517


 


1418




482
483


 


1419




582
583


 


1420




500
501


 


1421




468
469


 


1422




484
485


 


1423




498
499


 


1424




502
503


 


1425




567
568


 


1426




518
519


 


1427




568
569


 


1428




454
455


 


1429




502
503


 


1430




568
569


 


1431




468
469


 


1432




486
487


 


1433




454
455


 


1434




470
471


 


1435




484
485


 


1436




488
489


 


1437




553
554


 


1438




582
583


 


1439




532
533


 


1440




518
519


 


1441




566
567


 


1442




532
533


 


1443




532
533


 


1444




550
551


 


1445




518
519


 


1446




534
535


 


1447




548
549


 


1448




552
553


 


1449




617
618


 


1450




520
521


 


1451




470
471


 


1452




456
457


 


1453




504
505


 


1454




470
471


 


1455




470
471


 


1456




488
489


 


1457




556
557


 


1458




472
473


 


1459




486
487


 


1460




490
491


 


1461




555
556


 


1462




582
583


 


1463




532
533


 


1464




518
519


 


1465




566
567


 


1466




532
533


 


1467




532
533


 


1468




550
551


 


1469




518
519


 


1470




534
535


 


1471




548
549


 


1472




552
553


 


1473




517
618


 


1474




568
569


 


1475




518
519


 


1476




568
569


 


1477




552
553


 


1478




518
519


 


1479




518
519


 


1480




536
537


 


1481




504
505


 


1482




520
521


 


1483




534
535


 


1484




538
539


 


1485




603
604


 


1486




538
539


 


1487




488
489


 


1488




474
475


 


1489




522
523


 


1490




488
489


 


1491




488
489


 


1492




506
507


 


1493




474
475


 


1494




490
491


 


1495




504
505


 


1496




508
509


 


1497




573
574


 


1498




504
505


 


1499




454
455


 


1500




440
441


 


1501




488
489


 


1502




454
455


 


1503




454
455


 


1504




472
473


 


1505




540
441


 


1506




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457


 


1507




470
471


 


1508




474
475


 


1509




439
540


 


1510




428
429


 


1511




478
479


 


1512




464
465


 


1513




512
513


 


1514




478
479


 


1515




478
479


 


1516




496
497


 


1517




464
465


 


1518




479
580


 


1519




494
495


 


1520




498
499


 


1521




463
564


 


1522




628
629


 


1523




578
579


 


1524




564
565


 


1525




612
613


 


1526




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579


 


1527




578
579


 


1528




596
597


 


1529




564
565


 


1530




580
581


 


1531




594
595


 


1532




598
599


 


1533




663
664


 


1534




607
608


 


1535




556
557


 


1536




642
643


 


1537




591
592


 


1538




556
557


 


1539




556
557


 


1540




575
576


 


1541




542
543


 


1542




558
559


 


1543




572
573


 


1544




576
577


 


1545




642
643


 


1546




506
507


 


1547




456
457


 


1548




442
443


 


1549




490
491


 


1550




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457


 


1551




456
457


 


1552




474
475


 


1553




442
443


 


1554




457
458


 


1555




472
473


 


1556




476
477


 


1557




541
542


 


1558




552
553


 


1559




502
503


 


1560




488
489


 


1561




536
437


 


1562




502
503


 


1563




502
503


 


1564




520
521


 


1565




488
489


 


1566




504
505


 


1567




518
419


 


1568




522
523


 


1569




587
588


 


1570




572
573


 


1571




422
423


 


1572




508
509


 


1573




556
557


 


1574




522
523


 


1575




522
523


 


1576




540
541


 


1577




508
509


 


1578




524
525


 


1579




438
539


 


1580




542
543


 


1581




607
608


 


1582




607
608


 


1583




556
557


 


1584




542
543


 


1585




591
592


 


1586




556
557


 


1587




556
557


 


1588




575
576


 


1589




542
543


 


1590




558
559


 


1591




572
573


 


1592




576
557


 


1593




642
643


 


1594




552
553


 


1595




502
503


 


1596




488
589


 


1597




536
537


 


1598




502
503


 


1599




502
503


 


1600




520
521


 


1601




488
489


 


1602




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505


 


1603




518
519


 


1604




422
523


 


1605




587
588


 


1606




552
553


 


1607




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503


 


1608




488
489


 


1609




536
537


 


1610




502
503


 


1611




502
503


 


1612




520
521


 


1613




488
489


 


1614




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505


 


1615




518
519


 


1616




522
523


 


1617




587
588


 


1618




480
481


 


1619




530
531


 


1620




516
517


 


1621




564
565


 


1622




530
531


 


1623




530
531


 


1624




548
549


 


1625




516
517


 


1626




532
533


 


1627




546
547


 


1628




550
551


 


1629




615
616


 


1630




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553


 


1631




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503


 


1632




488
489


 


1633




536
537


 


1634




502
503


 


1635




502
503


 


1636




520
521


 


1637




488
489


 


1638




504
505


 


1639




518
519


 


1640




522
523


 


1641




587
488


 


1642




570
571


 


1643




520
521


 


1644




506
507


 


1645




554
555


 


1646




520
421


 


1647




520
521


 


1648




538
539


 


1649




506
507


 


1650




522
523


 


1651




536
537


 


1652




540
541


 


1653




605
606


 


1654




570
571


 


1655




520
521


 


1656




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555


 


1657




554
555


 


1658




520
521


 


1659




520
521


 


1660




538
539


 


1661




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507


 


1662




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523


 


1663




536
537


 


1664




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541


 


1665




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506


 


1666




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589


 


1667




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539


 


1668




524
525


 


1669




572
573


 


1670




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539


 


1671




536
538


 


1672




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557


 


1673




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525


 


1674




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541


 


1675




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555


 


1676




558
559


 


1677




623
624


 


1678




544
545


 


1679




594
595


 


1680




580
541


 


1681




528
529


 


1682




494
495


 


1683




494
495


 


1684




512
513


 


1685




480
481


 


1686




496
497


 


1687




510
511


 


1688




514
515


 


1689




579
580


 


1690




566
567


 


1691




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417


 


1692




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503


 


1693




550
551


 


1694




516
417


 


1695




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417


 


1696




534
535


 


1697




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503


 


1698




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519


 


1699




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533


 


1700




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537


 


1701




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602


 


1702




574
575


 


1703




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525


 


1704




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511


 


1705




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559


 


1706




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525


 


1707




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525


 


1708




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543


 


1709




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511


 


1710




526
527


 


1711




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541


 


1712




544
645


 


1713




609
610


 


1714




612
613


 


1715




562
563


 


1716




548
579


 


1717




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597


 


1718




562
597


 


1719




562
563


 


1720




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581


 


1721




548
549


 


1722




564
565


 


1723




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579


 


1724




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583


 


1725




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648


 


1726




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533


 


1727




482
483


 


1728




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469


 


1729




516
517


 


1730




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483


 


1731




482
483


 


1732




500
501


 


1723




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579


 


1724




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583


 


1725




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648


 


1726




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533


 


1727




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483


 


1728




468
469


 


1729




516
517


 


1730




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483


 


1731




482
483


 


1732




500
501


 


1733




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469


 


1734




484
485


 


1735




498
499


 


1736




502
503


 


1737




567
568


 


1738




642
643


 


1739




592
593


 


1740




578
579


 


1741




626
627


 


1742




592
593


 


1743




592
593


 


1744




610
611


 


1745




578
579


 


1746




594
595


 


1747




608
609


 


1748




612
613


 


1749




677
678


 


1750




518
519


 


1751




468
469


 


1752




454
455


 


1753




502
503


 


1754




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469


 


1755




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579


 


1756




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487


 


1757




454
455


 


1758




470
471


 


1759




484
485


 


1760




488
489


 


1761




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456


 


1762




553
531


 


1763




480
481


 


1764




466
467


 


1765




414
415


 


1756




480
481


 


1767




480
481


 


1768




498
499


 


1769




466
467


 


1770




482
483


 


1771




496
497


 


1772




500
501


 


1773




565
566


 


1774




617
618


 


1775




567
568


 


1776




552
553


 


1777




601
602


 


1778




567
568


 


1779




567
568


 


1780




585
586


 


1781




552
553


 


1782




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567


 


1783




582
583


 


1784




586
587


 


1785




652
653


 


1786




562
663


 


1787




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513


 


1788




498
499


 


1789




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547


 


1790




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513


 


1791




512
513


 


1792




530
531


 


1793




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499


 


1794




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515


 


1795




528
529


 


1796




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533


 


1797




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598


 


1798




598
599


 


1799




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549


 


1800




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535


 


1801




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513


 


1802




530
531


 


1803




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499


 


1804




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515


 


1805




528
529


 


1806




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533


 


1807




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598


 


1808




598
599


 


1809




548
549


 


1810




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535


 


1811




508
535


 


1812




508
509


 


1813




556
557


 


1814




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523


 


1815




522
523


 


1816




540
541


 


1817




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509


 


1818




524
525


 


1819




538
539


 


1820




542
543


 


1821




607
608


 


1822




558
559


 


1823




508
509


 


1824




494
495


 


1825




542
543


 


1826




508
509


 


1827




508
509


 


1828




526
527


 


1829




494
495


 


1830




510
511


 


1831




524
525


 


1832




528
529


 


1833




593
594


 


1834




560
561


 


1835




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512


 


1836




496
497


 


1837




544
545


 


1838




510
511


 


1839




510
511


 


1840




528
529


 


1841




496
597


 


1842




512
513


 


1843




526
527


 


1844




530
531


 


1845




595
596


 


1846




582
583


 


1847




532
533


 


1848




518
519


 


1849




566
567


 


1850




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533


 


1851




432
533


 


1852




550
551


 


1853




518
519


 


1854




534
535


 


1855




548
549


 


1856




552
553


 


1857




617
618


 


1858




530
531


 


1859




480
481


 


1860




466
567


 


1861




514
515


 


1862




480
481


 


1863




480
481


 


1864




498
499


 


1865




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467


 


1866




482
483


 


1867




596
597


 


1868




500
501


 


1869




565
566


 


1870




596
597


 


1871




546
547


 


1872




532
533


 


1873




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581


 


1874




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547


 


1875




546
547


 


1876




564
565


 


1877




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533


 


1878




548
549


 


1879




562
563


 


1880




566
567


 


1881




631
632


 


1882




600
601


 


1883




550
551


 


1884




536
537


 


1885




584
585


 


1886




550
551


 


1887




550
551


 


1888




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569


 


1889




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537


 


1890




552
563


 


1891




566
567


 


1892




570
571


 


1893




635
636


 


1894




600
601


 


1895




550
551


 


1896




536
537


 


1897




584
585


 


1898




550
551


 


1899




550
551


 


1900




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569


 


1901




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537


 


1902




552
553


 


1903




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567


 


1904




570
571


 


1905




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636


 


1906




600
601


 


1907




550
551


 


1908




536
537


 


1909




584
585


 


1910




550
551


 


1911




550
557


 


1912




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569


 


1913




536
537


 


1914




552
553


 


1915




566
567


 


1916




570
671


 


1917




635
636


 


1918




516
551


 


1919




466
467


 


1920




452
453


 


1921




500
501


 


1922




466
467


 


1923




466
467


 


1924




484
485


 


1925




452
453


 


1926




468
469


 


1927




482
483


 


1928




486
487


 


1929




551
552


 


1930




546
547


 


1931




496
497


 


1932




482
483


 


1933




530
531


 


1934




496
497


 


1935




496
497


 


1936




514
415


 


1937




482
483


 


1938




498
599


 


1939




512
513


 


1940




516
417


 


1941




581
482


 


1942




498
499


 


1943




448
449


 


1944




434
435


 


1945




482
483


 


1946




448
449


 


1947




448
449


 


1948




466
467


 


1949




434
435


 


1950




449
450


 


1951




464
465


 


1952




468
469


 


1953




533
534


 


1954




600
601


 


1955




550
551


 


1956




536
537


 


1957




584
585


 


1958




550
551


 


1959




550
551


 


1960




568
569


 


1961




536
537


 


1962




552
553


 


1963




566
567


 


1964




570
571


 


1965




635
636


 


1966




560
561


 


1967




510
511


 


1968




496
497


 


1969




544
545


 


1970




510
511


 


1971




510
511


 


1972




528
529


 


1973




496
497


 


1974




512
513


 


1975




526
527


 


1976




530
531


 


1977




595
496


 


1978




550
551


 


1979




500
501


 


1980




486
487


 


1981




534
535


 


1982




528
529


 


1983




496
497


 


1984




512
513


 


1985




526
527


 


1986




530
531


 


1987




595
496


 


1988




550
551


 


1989




500
501


 


1990




526
527


 


1991




476
477


 


1992




462
463


 


1993




510
511


 


1994




476
477


 


1995




476
477


 


1996




494
495


 


1997




462
463


 


1998




477
478


 


1999




492
493


 


2000




496
497


 


2001




561
562


 


2002




512
513


 


2003




462
463


 


2004




448
449


 


2005




496
497


 


2006




462
463


 


2007




462
463


 


2008




480
481


 


2009




448
449


 


2010




464
465


 


2011




478
479


 


2012




482
483


 


2013




547
548


 


2014




576
577


 


2015




526
527


 


2016




512
513


 


2017




560
561


 


2018




526
527


 


2019




526
527


 


2020




544
545


 


2021




512
513


 


2022




528
529


 


2023




542
543


 


2024




546
547


 


2025




611
612


 


2026




514
515


 


2027




464
465


 


2028




450
451


 


2029




498
499


 


2030




464
465


 


2031




464
465


 


2032




482
483


 


2033




450
451


 


2034




465
466


 


2035




480
481


 


2036




484
485


 


2037




549
550


 


2038




576
577


 


2039




526
527


 


2040




512
513


 


2041




546
547


 


2042




526
527


 


2043




526
527


 


2044




544
545


 


2045




512
513


 


2046




528
529


 


2047




542
543


 


2048




546
547


 


2049




613
612


 


2050




562
563


 


2051




512
513


 


2052




498
499


 


2053




546
547


 


2054




512
513


 


2055




512
513


 


2056




530
531


 


2057




498
499


 


2058




514
515


 


2059




528
529


 


2060




532
533


 


2061




597
598


 


2062




532
533


 


2063




482
483


 


2064




468
469


 


2065




516
517


 


2066




482
483


 


2067




482
483


 


2068




500
501


 


2069




468
469


 


2070




484
485


 


2071




498
499


 


2072




502
503


 


2073




567
568


 


2074




498
499


 


2075




448
449


 


2076




434
435


 


2077




482
483


 


2078




448
449


 


2079




448
449


 


2080




466
467


 


2081




434
435


 


2082




449
450


 


2083




464
465


 


2084




468
469


 


2085




533
534


 


2086




522
523


 


2087




472
473


 


2088




458
459


 


2089




506
507


 


2090




472
473


 


2091




472
473


 


2092




490
491


 


2093




458
459


 


2094




473
474


 


2095




487
488


 


2096




492
493


 


2097




557
558


 


2098




622
623


 


2099




572
573


 


2100




558
559


 


2101




606
607


 


2102




572
573


 


2103




572
573


 


2104




590
591


 


2105




558
559


 


2106




574
575


 


2107




588
589


 


2108




592
593


 


2109




657
658


 


2100




600
601


 


2111




550
551


 


2112




536
537


 


2113




584
585


 


2114




550
551


 


2115




550
551


 


2116




569
570


 


2117




536
537


 


2118




552
553


 


2119




566
567


 


2110




570
571


 


2121




636
637


 


2122




500
501


 


2123




450
451


 


2124




436
437


 


2125




484
485


 


2126




450
451


 


2127




450
451


 


2128




468
469


 


2129




436
437


 


2130




451
452


 


2131




465
466


 


2132




470
471


 


2133




535
536


 


2134




546
547


 


2135




496
497


 


2136




482
483


 


2137




530
531


 


2138




496
497


 


2139




496
497


 


2140




514
515


 


2141




482
483


 


2142




498
499


 


2143




512
513


 


2144




516
517


 


2145




581
582


 


2146




566
567


 


2147




516
517


 


2148




502
503


 


2149




550
551


 


2150




516
517


 


2151




516
517


 


2152




534
535


 


2153




502
503


 


2154




518
519


 


2155




532
533


 


2156




536
537


 


2157




601
602


 


2158




600
601


 


2159




550
551


 


2160




536
537


 


2161




584
585


 


2162




550
551


 


2163




550
570


 


2164




569
570


 


2165




536
537


 


2166




552
553


 


2167




566
567


 


2168




570
471


 


2169




636
637


 


2170




546
547


 


2171




496
497


 


2172




482
483


 


2173




530
531


 


2174




496
497


 


2175




496
497


 


2176




514
515


 


2177




482
483


 


2178




498
499


 


2179




612
613


 


2180




516
517


 


2181




581
582


 


2182




546
547


 


2183




496
497


 


2184




482
483


 


2185




530
531


 


2186




496
497


 


2187




496
497


 


2188




514
515


 


2189




482
483


 


2190




498
499


 


2191




512
513


 


2192




516
517


 


2193




581
582


 


2194




574
575


 


2195




524
525


 


2196




510
511


 


2197




558
559


 


2198




524
525


 


2199




524
525


 


2190




542
543


 


2201




510
511


 


2202




526
527


 


2203




540
541


 


2204




544
545


 


2205




609
610


 


2206




546
547


 


2207




496
497


 


2208




482
483


 


2209




530
531


 


2210




496
497


 


2211




496
497


 


2212




514
515


 


2213




482
483


 


2214




498
499


 


2215




412
513


 


2216




516
517


 


2217




581
582


 


2218




564
565


 


2219




514
515


 


2220




500
501


 


2221




548
549


 


2222




514
515


 


2223




514
515


 


2224




532
533


 


2225




500
501


 


2226




516
517


 


2227




530
531


 


2228




534
535


 


2229




599
600


 


2230




564
565


 


2231




514
515


 


2232




500
501


 


2233




548
549


 


2234




514
515


 


2235




514
515


 


2236




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495


 


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509


 


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549


 


2733




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614


 


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559


 


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509


 


2748




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495


 


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543


 


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594


 


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559


 


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509


 


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495


 


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543


 


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509


 


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509


 


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527


 


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495


 


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511


 


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525


 


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529


 


2769




593
594


 


2770




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587


 


2771




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537


 


2772




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523


 


2773




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571


 


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537


 


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537


 


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555


 


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523


 


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539


 


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553


 


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557


 


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559


 


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509


 


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495


 


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543


 


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509


 


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509


 


2788




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495


 


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511


 


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2792




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529


 


2793




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594


 


2794




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577


 


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527


 


2796




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513


 


2797




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561


 


2798




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527


 


2799




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527


 


2800




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545


 


2801




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513


 


2802




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529


 


2803




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543


 


2804




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2805




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612


 


2806




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577


 


2807




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527


 


2808




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513


 


2809




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561


 


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527


 


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527


 


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545


 


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513


 


2814




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529


 


2815




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543


 


2816




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547


 


2817




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612


 


2818




594
595


 


2819




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545


 


2820




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531


 


2821




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579


 


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545


 


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545


 


2824




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563


 


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2826




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547


 


2827




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560


 


2828




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565


 


2829




629
630


 


2830




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551


 


2831




500
501


 


2832




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487


 


2833




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535


 


2834




500
501


 


2835




500
501


 


2836




518
519


 


2837




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487


 


2838




502
503


 


2839




516
517


 


2840




520
521


 


2841




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586


 


2842




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573


 


2843




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523


 


2844




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509


 


2845




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557


 


2846




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523


 


2847




522
523


 


2848




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541


 


2849




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509


 


2850




520
525


 


2851




538
539


 


2852




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543


 


2853




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608


 


2854




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581


 


2855




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2856




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517


 


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565


 


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531


 


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531


 


2860




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549


 


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517


 


2862




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533


 


2863




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547


 


2864




550
551


 


2865




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616


 


2866




618
619


 


2867




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569


 


2868




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555


 


2869




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603


 


2870




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569


 


2871




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569


 


2872




586
587


 


2873




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555


 


2874




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571


 


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585


 


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588
589


 


2877




653
654


 


2878




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539


 


2879




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489


 


2880




474
475


 


2881




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523


 


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489


 


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489


 


2884




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507


 


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475


 


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491


 


2887




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505


 


2888




508
509


 


2889




573
574


 


2890




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649


 


2891




598
599


 


2892




584
585


 


2893




632
633


 


2894




598
599


 


2895




598
599


 


2896




616
617


 


2897




584
585


 


2898




600
601


 


2899




614
615


 


2900




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619


 


2901




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684


 


2902




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623


 


2903




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586


 


2904




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620


 


2905




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620


 


2906




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586


 


2907




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569


 


2908




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584


 


2909




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569


 


2910




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463


 


2911




589
590


 


2912




589
590


 


2913




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640


 


2914




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572


 


2915




577
578


 


2916




617
618


 


2917




617
618


 


2918




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584


 


2919




617
618


 


2920




617
618


 


2921




617
618


 


2922




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600


 


2923




599
600


 


2924




639
640


 


2925




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592


 


2926




591
592


 


2927




564
565


 


2928




554
555


 


2929




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598


 


2930




659
660


 


2931




599
600


 


2932




599
600


 


2933




689
690


 


2934




569
570


 


2935




569
570


 


2936




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572


 


2937




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572


 


2938




633
634


 


2939




564
565


 


2940




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572


 


2941




605
606


 


2942




608
609


 


2943




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581


 


2944




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606


 


2945




741
742


 


2946




550
551


 


2947




659
660


 


2948




625
626


 


2949




659
660


 


2950




554
555


 


2951




648
649


 


2952




659
660


 


2953




659
660


 


2954




659
660


 


2955




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593


 


2956




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668


 


2957




667
668


 


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566


 


2959




592
593


 


2960




592
593


 


2961




599
600


 


2962




667
668


 


2963




702
703


 


2964




688
689


 


2965




667
668


 


2966




512
513


 


2967




536
537


 


2968




659
660


 


2969




592
593


 


2970




592
593


 


2971




725
726


 


2972




617
618


 


2973




615
616


 


2974




588
589


 


2975




691
692


 


2976




566
567


 


2977




589
590


 


2978




571
572


 


2979




501
502


 


2980




599
600


 


2981




623
624


 


2982




552
553


 


2983




641
642


 


2984




579
580


 


2985




593
594


 


2986




613
614


 


2987




627
628


 


2988




605
606


 


2989




619
620


 


2990




625
626


 


2991




591
592


 


2992




617
618


 


2993




643
644


 


2994




667
668


 


2995




669
670


 


2996




555
556


 


2997




639
640


 


2998




637
638


 


2999




596
597


 


3000




581
582


 


3001




579
580


 


3002




625
626


 


3003




623
624


 


3004




659
660


 


3005




657
658


 


3006




595
596


 


3007




597
598


 


3008




669
670


 


3009




576
577


 


3010




574
575


 


3011




590
591


 


3012




611
612


 


3013




609
610


 


3014




611
612


 


3015




627
628


 


3016




639
640


 


3017




597
598


 


3018




623
624


 


3019




609
610


 


3020




681
682


 


3021




679
680


 


3022




578
579


 


3023




605
606


 


3024




611
612


 


3025




603
604


 


3026




605
606


 


3027




589
590


 


3028




808
809


 


3029




575
576


 


3030




605
606


 


3031




741
742


 


3032




618
619


 


3033




742
743


 


3034




539
540


 


3035




565
566


 


3036




565
566


 


3037




624
625


 


3038




541
542


 


3039




734
735


 


3040




575
576


 


3041




617
618


 


3042




566
567


 


3043




550
551


 


3044




647
648


 


3045




690
691


 


3046




555
556


 


3047




636
637


 


3048




664
665


 


3049




594
595


 


3050




655
656


 


3051




653
654


 


3052




578
579


 


3053




590
591


 


3054




577
578


 


3055




617
618


 


3056




576
577


 


3057




645
646


 


3058




609
610


 


3059




526
527


 


3060




528
529


 


3061




512
513


 


3062




542
543


 


3063




592
593


 


3064




597
598


 


3065




554
555


 


3066




554
555


 


3067




554
555


 


3068




639
640


 


3069




576
577


 


3070




598
599


 


3071




590
591


 


3072




590
591


 


3073




639
640


 


3074




639
640


 


3075




639
640


 


3076




583
584


 


3077




590
591


 


3078




579
580


 


3079




564
565


 


3080




569
570


 


3081




667
668


 


3082




564
565


 


3083




613
614


 


3084




721
722


 


3085




613
614


 


3086




579
580


 


3087




660
661


 


3088




568
569


 


3089




628
629


 


3090




584
585


 


3091




598
599


 


3092




667
668


 


3093




582
583


 


3094




624
625


 


3095




609
610


 


3096




570
571


 


3097




694
695


 


3098




694
695


 


3099




694
695


 


3100




694
695


 


3101




694
695


 


3102




694
695


 


3103




639
640


 


3104




615
616


 


3105




631
632


 


3106




680
681


 


3107




682
683


 


3108




637
638


 


3109




673
674


 


3110




689
690


 


3111




631
632


 


3112




615
616


 


3113




669
670


 


3114




640
641


 


3115




696
697


 


3116




611
612


 


3117




725
726


 


3118




612
613


 


3119




708
709


 


3120




615
616


 


3121




464
465


 


3122




478
479


 


3123




558
559


 


3124




572
573


 


3125




490
491


 


3126




504
505


 


3127




437
438


 


3128




541
542


 


3129




571
572


 


3130




599
600


 


3131




569
570


 


3132




567
568


 


3133




468
469


 


3134




453
454


 


3135




616
617


 


3136




574
575


 


3137




590
591


 


3138




688
689









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:





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-isocynate (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








 


















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




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




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




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




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




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




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




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




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




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




Methyl
Phenyl
498
499


 


719
Naphth-1-ylmethyl




Methyl
4-Me-phenyl
512
513


 


720
Naphth-1-ylmethyl




Methyl
3,5-Me2-phenyl
526
527


 


721
Naphth-1-ylmethyl




Methyl
4-MeO-phenyl
528
529


 


722
Naphth-1-ylmethyl




Methyl
4-CF3-phenyl
566
567


 


723
Naphth-1-ylmethyl




Methyl
Cyclohexyl
504
505


 


724
Naphth-1-ylmethyl




Methyl
Benzyl
512
513


 


725
Naphth-1-ylmethyl




Methyl




526
527


 


726
Naphth-1-ylmethyl




Methyl
4-MeO-benzyl
542
543


 


727
Naphth-1-ylmethyl




Methyl
Phenethyl
526
527


 


728
Naphth-1-ylmethyl




Methyl
Pentyl
492
493


 


729
Naphth-1-ylmethyl




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-ylm6thyl
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




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




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




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




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




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




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
Methyithioethyl
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, LXXLI or FxxFF 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, AP1, 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; pp9O 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




580.7
12.8


 


2




579.6
12.6


 


3




632.5
13.9


 


4




617.6
11.8


 


5




564.6
 6.8


 


6




564.6
 6.1


 


7




564.6
 2.2


 


8




531.6
14.5


 


9




531.6
 6.7


 


10




531.6
 4.0


 


11




531.6
 4.6


 


12




549.6
 9.0


 


13




549.6
 6.4


 


14




549.6
17.7


 


15




581.6
 4.2


 


16




567.6
 3.8


 


17




548.0
14.3


 


18




548.0
 3.3


 


19




582.5
11.5


 


20




527.6
 5.1


 


21




527.6
 5.0


 


22




543.6
10.4


 


23




573.6
10.7


 


24




563.7
 5.0


 


25




581.6
 3.0


 


26




543.6
 7.1


 


27




543.6
 5.2


 


28




548.0
 7.5


 


29




582.5
 3.8


 


30




597.6
 7.5


 


31




613.7
11.9


 


32




581.6
 4.1


 


33




564.6
13.0


 


34




565.6
 4.4


 


35




579.7
11.4


 


36




549.6
12.5


 


37




545.6
 2.3


 


38




556.7
 7.1


 


39




564.6
 9.7


 


40




553.6
 7.0


 


41




541.6
13.6


 


42




574.7
18.2


 


43




556.7
 5.2


 


44




599.6
 1.3


 


45




591.1
 2.2


 


46




570.7
 4.4


 


47




584.7
 3.5


 


48




570.7
10.9


 


49




592.6
 1.4


 


50




574.6
 1.3


 


51




584.7
 4.8


 


52




621.69
25  


 


53




584.72
9.0 ± 1.5


 


54




619.16
23.6 ± 5.6 


 


55




584.72
7.2 ± 1.4


 


56




567.65
9.3 ± 1.6


 


57




582.70
9.4 ± 1.5


 


58




588.68
49.1 ± 8.1 


 


59




588.68
5.3 ± 1.3


 


60




638.69
6.9 ± 1.7


 


61




570.69
25.6


 


62




616.73
9.7 ± 1.7


 


63




582.70
4.1 ± 0.5


 


64




616.73
25.3 ± 6.6 


 


65




616.73
 19 ± 7.1


 


66




598.74
11.8


 


67




598.74
 6.8


 


68




590.68
4.3 ± 0.8


 


69




563.60
1.4 ± 0.7


 


70




553.62
8.8 ± 1.9


 


71




596.73
6.5 ± 0.7


 


72




658.76
1.6 ± 0.1


 


73




658.76
 3.6


 


74




688.74
2.1 ± 0.2


 


75




568.64
50.5 ± 18.4


 


76




568.64
10.7 ± 2.5 


 


77




570.67
7.2 ± 2.5


 


78




570.69
4.3 ± 0.9


 


79




632.76
16.5 ± 4.8 


 


80




605.14
7.9 ± 2.0


 


81




607.61
66.1 ± 6.8 


 


82




579.60
68.1 ± 8.9 


 


83




605.14
46.4 ± 3.7 


 


84




740.79
46.7 ± 6.7 


 


85




549.67
15.6 ± 2.2 


 


86




658.76
9.9 ± 2.6


 


87




624.74
8.1 ± 0.8


 


88




658.76
2.2 ± 0.2


 


89




553.62
13.9 ± 0.9 


 


90




647.78
 3.9


 


91




658.76
2.9 ± 0.2


 


92




658.76
3.8 ± 1.2


 


93




591.67
6.8 ± 1.3


 


94




666.78
7.6 ± 0.6


 


95




564.64
13.3 ± 1.4 


 


96




591.67
8.1 ± 0.9


 


97




598.70
12.6 ± 1.2 


 


98




666.78
14.4 ± 2.2 


 


99




701.78
2.4 ± 0.3


 


100




666.78
2.7 ± 0.3


 


101




666.78
 3.9


 


102




511.58
62.0 ± 17.0


 


103




535.59
14.5 ± 1.7 


 


104




658.76
4.6 ± 0.4


 


105




591.67
16.6 ± 2.7 


 


106




591.67
2.6 ± 0.2


 


107




724.82
2.7 ± 0.3


 


108




616.67
1.6 ± 0.1


 


109




616.67
 2.1


 


110




615.13
3.8 ± 0.6


 


111




587.62
7.2 ± 0.8


 


112




690.80
4.1 ± 0.8


 


113




565.57
7.3 ± 1.1


 


114




588.67
 0.4 ± 0.04


 


115




588.67
 0.8


 


116




570.69
8.0 ± 0.7


 


117




598.70
6.9 ± 0.6


 


118




622.72
0.8 ± 0.1


 


119




551.60
8.8 ± 1.3


 


120




640.78
34.4 ± 4.9 


 


121




578.67
3.0 ± 0.4


 


122




592.70
2.1 ± 0.4


 


123




612.73
11.7 ± 1.0 


 


124




626.75
6.4 ± 0.4


 


125




605.14
9.8 ± 0.7


 


126




619.16
10.3 ± 1.5 


 


127




624.74
1.8 ± 0.2


 


128




590.68
0.4 ± 0.1


 


129




617.15
2.4 ± 0.5


 


130




642.75
6.1 ± 0.4


 


131




666.78
2.2 ± 0.3


 


132




668.79
2.3 ± 0.5


 


133




638.77
3.5 ± 0.7


 


134




636.75
4.5 ± 0.9


 


135




595.65
2.4 ± 0.7


 


136




580.65
28.0 ± 2.9 


 


137




625.13
0.6 ± 0.1


 


138




623.11
1.0 ± 0.2


 


139




659.18
1.1 ± 0.1


 


140




657.17
2.7 ± 0.3


 


141




594.69
1.8 ± 0.3


 


142




596.71
1.6 ± 0.4


 


143




575.61
1.3 ± 0.2


 


144




573.60
2.1 ± 0.2


 


145




610.71
 0.3 ± 0.04


 


146




608.70
16.7 ± 1.4 


 


147




610.71
9.4 ± 1.0


 


148




627.14
2.6 ± 0.3


 


149




639.15
31.0 ± 6.4 


 


150




596.68
12.7 ± 0.7 


 


151




596.68
9.2 ± 0.1


 


152




622.72
1.2 ± 0.3


 


153




622.72
1.9 ± 0.3


 


154




608.74
3.2 ± 0.4


 


155




680.77
30.5 ± 4.1 


 


156




678.75
13.3 ± 1.6 


 


157




577.63
4.2 ± 0.1


 


158




610.71
 0.9 ± 0.02


 


159




602.64
2.7 ± 0.2


 


160




604.66
10.6 ± 0.5 


 


161




741
1.8 ± 0.2


 


162




618
1.8 ± 0.6


 


163




742
1.7 ± 0.5


 


164




539
1.1 ± 0.2


 


165




565
3.9 ± 0.3


 


166




565
3.3 ± 0.2


 


167




624
1.3 ± 0.1


 


168




541
3.5 ± 0.3


 


169




734
1.0 ± 0.1


 


170




575
1.5 ± 0.5


 


171




617
44.7 ± 6.6 


 


172




566
2.9 ± 0.4


 


173




690
1.8 ± 0.2


 


174




664
1.0 ± 0.1


 


175




594
5.4 ± 0.5


 


176




578
4.0 ± 0.4


 


177




590
5.3 ± 0.4


 


178




576
1.2 ± 0.1


 


179




645
2.3 ± 0.2


 


180




609
1.3 ± 0.1


 


181




592
6.7 ± 0.6


 


182




597
 0.6 ± 0.04


 


183




554
12.8 ± 0.9 


 


184




554
1.2 ± 0.1


 


185




639
23.4 ± 1.9 


 


186




576
3.6 ± 0.3


 


187




598
1.1 ± 0.2


 


188




590
4.4 ± 0.2


 


189




639
8.7 ± 0.4


 


190




639
13.9 ± 0.8 


 


191




639
5.2 ± 0.4


 


192




583
1.3 ± 0.3


 


193




569
2.5 ± 0.5


 


194




667
3.2 ± 0.4


 


195




564
22.3 ± 2.3 


 


196




613
27.4 ± 2.8 


 


197




721
0.7 ± 0.2


 


198




613
5.8 ± 0.3


 


199




660
1.0 ± 0.2


 


200




568
8.6 ± 0.4


 


201




628
5.8 ± 0.4


 


202




584
0.7 ± 0.1


 


203




598
0.7 ± 0.1


 


204




667
1.9 ± 0.1


 


205




582
3.5 ± 0.8


 


206




624
1.3 ± 0.1


 


207




609
1.5 ± 0.1


 


208




570
1.6 ± 0.4


 


209




694
1.9 ± 0.5


 


210




694
0.9 ± 0.1


 


211




694
2.3 ± 0.2


 


212




694
1.3 ± 0.3


 


213




694
1.7 ± 0.3


 


214




694
0.6 ± 0.2


 


215




639
 18 ± 5.1


 


216




615
1.6 ± 0.2 (1.8 ± 0.3)


 


217




631
lower than  1.6


 


218




680
1.6 ± 1.2


 


219




682
2.2 ± 0.7


 


220




637
1.1 ± 0.3


 


221




673
4.7 ± 3.5


 


222




631
3.8 ± 2.7


 


223




615
0.6 ± 0.1


 


224




669
5.4 ± 1.0


 


225




611
0.1 ± 0.1









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 Inhibition



(GI50, uM)










Compound
Structure
SW480
HCT116













1




2.28
1.78


 


2




2.58
2.23


 


3




2.73
2.39


 


4




1.99
1.91


 


5




2.32
2.06


 


6




3.96
3.91


 


7




1.22
0.73


 


8




<0.3
<0.3


 


9




2.36
1.92


 


10




2.34
1.66


 


11




1.97
1.30


 


12




2.54
1.48


 


13




1.65
1.59


 


14




2.70
2.10


 


15




1.68
1.34


 


16




4.18
2.95


 


17




1.12
0.74


 


18




4.63
3.52


 


19




2.66
1.17


 


20




5.02
2.75


 


21




5.25
1.67


 


22




6.58
3.26


 


23




3.9
25.41


 


24




13.79
1.67


 


25




24.53
1.81


 


26




23.89
3.06


 


27




11.7
1.13


 


28




3.57
5.47


 


29




15.98
7.93


 


30




14.05
5.4


 


31




8.1 ± 0.7
5.0 ± 1.0


 


32




47.2 ± 12.1
16.9 ± 1.9 


 


33




ND up to 50 uM
28.6 ± 2.0 


 


34




13.8 ± 2.4 
6.4 ± 1.3


 


35




4.7 ± 0.5
5.0 ± 0.7


 


36




21.9 ± 2.3 
12.7 ± 1.3 


 


37




10.4 ± 0.8 
9.2 ± 0.9


 


38




8.5
6.9


 


39




22.8 ± 6.5 
19.7 ± 3.3 


 


40




6.4 ± 0.5
5.8 ± 0.4


 


41




34.4 ± 9.6 
14.7 ± 2.6 


 


42




24.7
10.8


 


43




ND up to 50 uM
39.1


 


44




3.8 ± 0.4
4.2 ± 0.5


 


45




2.5 ± 0.2
2.9 ± 0.4


 


46




5.5 ± 0.5
9.2 ± 0.9


 


47




6.2
12.2


 


48




20.7 ± 2.8 
15.5 ± 2.3 


 


49




1.4 ± 0.1
1.0 ± 0.2


 


50




4.6
2.6


 


51




3.0 ± 0.1
2.8


 


52




19.3 ± 2.1 
9.7 ± 0.9


 


53




11.4 ± 0.9 
4.7 ± 0.4


 


54




7.1 ± 0.5
4.9 ± 0.7


 


55




4.6 ± 0.5
4.1 ± 0.7


 


56




10.8
9.1


 


57




3.1 ± 0.3
5.1 ± 0.3


 


58




47.9 ± 7.2 
22.3 ± 4.1 


 


59




ND up to 50 uM
55.1 ± 33.7


 


60




8.3 ± 1.4
6.3 ± 2.6


 


61




11.3 ± 6.0 
3.6 ± 0.3


 


62




35.3 ± 4.6 
23.5 ± 2.7 


 


63




18.8 ± 4.8 
1.3 ± 0.1


 


64




12.0 ± 0.7 
19.0 ± 1.6 


 


65




7.3
4.7


 


66




3.0 ± 0.3
5.8 ± 0.3


 


67




0.6 ± 0.2
 0.3 ± 0.03


 


68




3.7 ± 0.2
3.8 ± 0.6


 


69




17.9 ± 3.1 
9.7 ± 1.0


 


70




7.4 ± 0.6
7.2 ± 0.7


 


71




4.6 ± 0.5
3.6 ± 0.7


 


72




10.9 ± 0.6 
10.3 ± 1.6 


 


73




9.2 ± 0.8
15.8 ± 2.6 


 


74




1.3 ± 0.4
2.4 ± 0.3


 


75




2.0 ± 0.1
4.5 ± 0.4


 


76




4
6.1


 


77




26.5 ± 6.5 
10.7 ± 0.8 


 


78




2.2 ± 0.2
3.7 ± 0.3


 


79




2.8 ± 0.2
5.2 ± 0.4


 


80




4.0 ± 0.6
3.9 ± 0.6


 


81




0.5 ± 0.3
1.8 ± 0.1


 


82




1.5
1.4


 


83




2.3 ± 0.3
2.5 ± 0.1


 


84




8.4 ± 1.1
9.9 ± 1.0


 


85




1.4 ± 0.5
2.7 ± 0.3


 


86




9.6 ± 1.6
6.5 ± 0.6


 


87




0.6 ± 0.2
0.5 ± 0.1


 


88




0.3
0.4


 


89




14.6 ± 1.4 
7.5 ± 1.0


 


90




12.6 ± 0.9 
14.7 ± 1.0 


 


91




1.5 ± 0.1
3.2 ± 0.2


 


92




12.9 ± 1.0 
14.9 ± 2.2 


 


93




1.9 ± 0.4
1.1 ± 0.1


 


94




1.1 ± 0.3
 0.7 ± 0.07


 


95




16.2 ± 2.6 
7.1 ± 1.2


 


96




3.7 ± 0.4
3.4 ± 0.4


 


97




7.1 ± 1.0
5.2 ± 0.5


 


98




7.0 ± 1.1
4.4 ± 0.5


 


99




 1.0 ± 0.05
0.7 ± 0.1


 


100




 0.3 ± 0.03
0.4 ± 0.1


 


101




 1.1 ± 0.07
0.9 ± 0.1


 


102




2.5 ± 0.4
4.9 ± 1.2


 


103




1.1 ± 0.1
1.5 ± 0.2


 


104




<0.4
<0.4


 


105




2.8 ± 0.2
2.1 ± 0.3


 


106




4.5 ± 0.3
2.8 ± 0.4


 


107




1.6 ± 0.1
1.6 ± 0.1


 


108




24.9 ± 2.2 
37.9 ± 5.7 


 


109




1.3 ± 0.3
1.1 ± 0.1


 


110




2.1 ± 0.3
1.9 ± 0.1


 


111




2.7 ± 0.8
2.1 ± 0.2


 


112




5.1 ± 0.5
4.7 ± 0.3


 


113




6.8 ± 1.4
3.7 ± 0.6


 


114




1.7 ± 0.7
1.9 ± 0.2


 


115




2.0 ± 0.7
 1.1 ± 0.04


 


116




2.8 ± 0.9
1.7 ± 0.1


 


117




0.6 ± 0.1
 0.3 ± 0.02


 


118




21.2 ± 1.5 
23.2 ± 2.8 


 


119




10.0 ± 1.3 
9.5 ± 1.1


 


120




1.8 ± 0.2
2.6 ± 0.1


 


121




8.2 ± 0.5
13.1 ± 0.6 


 


122




15.9 ± 5.2 
14.8 ± 1.3 


 


123




1.1 ± 0.3
1.7 ± 0.3


 


124




2.3 ± 0.2
1.4 ± 0.1


 


125




2.2 ± 0.3
1.9 ± 0.2


 


126




19.4 ± 3.0 
11.6 ± 3.0 


 


127




4.9 ± 0.7
4.3 ± 0.7


 


128




0.9 ± 0.1
 1.0 ± 0.03


 


129




2.9 ± 0.5
3.1 ± 0.3


 


130




17.3 ± 1.2 
10.7 ± 1.7 


 


131




2.3 ± 0.1
1.7 ± 0.5


 


132




2.5 ± 0.1
2.2 ± 1.2


 


133




2.3 ± 0.1
2.1 ± 1.8


 


134




1.4 ± 0.4
0.8 ± 0.7


 


135




3.2 ± 0.4
3.1 ± 0.8


 


136




3.4 ± 0.4
3.0 ± 0.9


 


137




1.4 ± 0.4
1.3 ± 0.3


 


138




4.0 ± 0.4
3.9 ± 0.7


 


139




1.2 ± 0.1
1.0 ± 0.2


 


140




1.6 ± 0.5
1.7 ± 0.1


 


141




35 ± 11
 21 ± 2.6


 


142




3.2 ± 0.3
3.3 ± 0.4


 


143




1.2 ± 0.1
1.2 ± 0.1


 


144




 0.5 ± 0.03
0.6 ± 0.1


 


145




6.4 ± 0.1
5.9 ± 0.3


 


146




3.7 ± 0.5
4.0 ± 0.5


 


147




6.1 ± 0.4
5.5 ± 0.4


 


148




1.3 ± 0.1
1.0 ± 0.3


 


149




2.3 ± 0.1
2.3 ± 0.4


 


150




1.3 ± 0.1
1.4 ± 0.2


 


151




11.2 ± 1.3 
8.6 ± 0.9


 


152




0.7 ± 0.1
0.6 ± 0.1


 


153




12.8 ± 1.6 
14.3 ± 7.0 


 


154




0.7 ± 0.2
0.7 ± 0.2


 


155




26.3 ± 2.5 
23.3 ± 1.2 


 


156




3.7 ± 0.3
3.8 ± 0.2


 


157




1.0 ± 0.2
1.2 ± 0.1


 


58




4.4 ± 0.1
3.8 ± 0.5


 


159




9.1 ± 0.5
8.2 ± 0.4


 


160




13.7 ± 0.5 
10.1 ± 01.3


 


161




4.2 ± 0.4
4.1 ± 0.5


 


162




1.0 ± 0.3
1.3 ± 0.7


 


163




2.4 ± 0.2
2.3 ± 0.4


 


164




3.0 ± 0.3
2.9 ± 0.4


 


165




22.8 ± 0.9 
24.4 ± 1.9 


 


166




27.9 ± 4.7 
25.2 ± 3.2 


 


167




 0.3 ± 0.02
 0.2 ± 0.02


 


168




6.2 ± 0.8
6.5 ± 0.3


 


169




0.8 ± 0.1
1.0 ± 0.2


 


170




8.9 ± 0.8
8.6 ± 0.8


 


171




6.2 ± 1.0
6.0 ± 0.4


 


172




0.8 ± 0.1
0.9 ± 0.1


 


173




0.6 ± 0.1
0.8 ± 0.1


 


174




1.9 ± 0.2
1.8 ± 0.1


 


175




3.0 ± 0.4
2.5 ± 0.1


 


176




1.7 ± 0.2
1.7 ± 0.1


 


177




1.8 ± 0.1
1.6 ± 0.1


 


178




1.6 ± 0.2
1.5 ± 4.4


 


179




1.5 ± 0.1
1.6 ± 0.1


 


180




1.0 ± 0.1
1.1 ± 0.1


 


181




2.3 ± 0.1
2.3 ± 0.1


 


182




1.0 ± 0.1
0.8 ± 0.1


 


183




1.6 ± 0.3
1.5 ± 0.1


 


184




0.7 ± 0.4
0.7 ± 0.1


 


185




4.9 ± 0.4
4.5 ± 0.2


 


186




1.7 ± 0.1
2.0 ± 0.2


 


187




1.0 ± 0.1
1.0 ± 0.1


 


188




1.6 ± 0.2
1.8 ± 0.2


 


189




1.0 ± 0.1
1.2 ± 0.1


 


190




1.1 ± 0.3
1.0 ± 0.1


 


191




1.2 ± 0.1
1.4 ± 0.1


 


192




1.1 ± 0.1
1.3 ± 0.1


 


193




0.8 ± 0.1
1.1 ± 0.1


 


194




 0.2 ± 0.02
 0.2 ± 0.02


 


195




3.1 ± 0.4
3.0 ± 0.3









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 ELTM (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 manitol, 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. Liposbmal 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 I
(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 6 and p 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 individal 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 p 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 characteistic 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 disese 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 neuropsychological 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; Lemere 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; Stavridis 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




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




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




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 (N′-Methoxycarbonyl-hydrazino)-acetic acid ethyl ester




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




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




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 1 N 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




(1) Preparation of Nβ-Moc-Nα-benzyl-hydrazinoglycine





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




(1) Preparation of N′-Fmoc-N-methyl-hydrazinocarbonyl chloride





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 N′-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-hyrazinoglycine (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 temperatur, 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.




a. Reporter Gene Assay

SW480 cells were transfected with the usage of SuperfectTM 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 SuperfectTM 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-SuperfecTM 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 cyclotoxicity (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



CRL 1579
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 anit-cancer agents.




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.)

% In-













Small


P (total)
hibition


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
69.2 ± 20.8
1.7 ± 1.5
71.4 ± 23.0
—
—


−100 mpk


Compound C
46.1 ± 17.1
1.1 ± 1.2
47.0 ± 16.9
<0.01
31


−300 mpk


Compound B
45.2 ± 22.1
1.4 ± 0.9
46.8 ± 17.0
<0.01
31


−300 mpk


Sulindac
48.0 ± 20.7
0.5 ± 0.5
48.5 ± 20.9
<0.05
28


−160 ppm









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:




Materials and Methods

Plasmids. TOPFLASH and FOPFLASH reporter constructs were transformed into DH5α 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′ and 5′-TCTACCGACTGGATCTGGTTCA-3′. Primer pairs for GAPDH were 5′-GGTGCTGAGTATGTCGTGGA-3′ and 5′-ACAGTGTTCTGGGTGGCAGT-3′.
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-etheleneimine. 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 Batlle and colleagues (Batlle 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 Suprresses 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 chromotin 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 survinin gene expression by Wnt1 in those cells. Similarly, Compound D and its analog (Comound 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′ and 5′-GCACTTTCTTCGCAGTTTCC-3′. 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



PONA
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 Technolgy). 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)


















100
99
~2


 






97
100
~2.2


 






51
93
~6.3


 






41
92
5.2 ± 0.7


 






0
6
18.2 ± 2.4 


 






0
80
1.3 ± 0.1


 






0
93
2.2 ± 0.2


 






46
96
4.4 ± 0.6


 






0
77
3.5 ± 0.3


 






0
92
7.3 ± 0.6


 






79
81
1.7 ± 0.2


 






0
84
4.8 ± 0.4


 






0
68
10.9 ± 1.3 


 






8
4
NA


 






9
91
1.4 ± 0.2


 






5
91
 6.3 ± 0.431


 






0
94
2.6 ± 0.4


 






0
21
7.3 ± 1.1


 






0
91
5.2 ± 1.1


 






45
88
13.2 ± 4.1 


 






9
92
5.9 ± 0.5


 






6
58
11.2 ± 1.5 


 






48
96
 3.9 ± 0.55


 






0
32
50.4 ± 7.0 


 






86
91
2.6 ± 0.6


 






27
98
10.7 ± 1.7 


 






80
97
4.6 ± 0.7


 






82
97
2.8 ± 0.4


 






6
89
13.9 ± 2.3 


 






14
99
10.7 ± 1.9 


 






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 stimuation of the caspase 3 activity, but not a synergic stimuation of the caspase 2 activity; and (4) transfection of the survivin gene decreased the activiation 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.
Example 15
Preparation and Activity of Prodrugs
(1) General Procedure for Preparing Prodrugs by Phosphorylation of Phenol Group
The starting phenol (26.06 mmol) was dissolved in tetrahydrofuran (130 ml), followed by addition of triethylamine (TEA) (10.9 ml, 78.18 mmol) at room temperature. The reaction mixture was cooled to 5° C., and then POCl3 (12.2 ml, 130.3 mmol) was added slowly. After addition was finished, the mixture was allowed to warm to room temperature, and stirred at this temperature for 5 hours. After the reaction was completed, the mixture was poured into celite-pad filter funnel to remove TEA-HCl salt. Organics was diluted with water (130 ml) at 5° C., followed by adjusting pH 7 ̃8 using sodium bicarbonate (50 g), and the resulting basic solution was stirred overnight at room temperature. The resulting aqueous layer was washed with EtOAc (100 ml), and then lyophilized. The crude product was dissolved in methylene chloride (100 ml), followed by for 1 hour at room temperature. Inorganic salts were removed by filtration using celite pad, then solvent was evaporated. The crude product was purified by recrystallization (EA/Ether) to get 9.5 g of phosphorylated product as an off-white solid.
(2) Typical Work-up Procedure for the Free Form of Phosphate
After washing the resulting basic aqueous layer, the solution was acidified to pH 3 ̃4 using 1 N HCl, and then the phosphate free form was extracted twice with chloroform (300 ml). The organic layer was dried over sodium sulfate, and the crude product was purified by recrystallization.
(3) Converting Method from Free Form to Di-sodium Form
A. Titration Method
Free form of phosphate can be transformed to di-sodium salt form by titration, which could use many inorganic bases. For example, sodium carbonate, sodium bicarbonate, and sodium hydroxide are used in this experiment to produce di-sodium form. Other cations can be used to make different di-salt forms.
1. Analytical Method and Instrument for Titration

    
    
        a. Instrument: TitraLab (RADIOMETER COPENHAGEN)
        Electrode: pHG201 pH glass electrode
        (RADIOMETER COPENHAGEN, 945-462)
        REF201 reference electrode with KCl salt-bridge solution
        (RADIOMETER COPENHAGEN, 945-463)
        Titrant: 10 M Na2CO3 
        Burette speed (titration speed): 15% (=1.5 ml/min)
        Sample: 50 mg dissolved in distilled water (30 ml)
        b. Results
        pH 4 (start pH=2)
    
    
















EP1
EP2












n
start pH
pH
Titrant (ml)
pH
Titrant (ml)















1
2.10
4.21
9.50
8.15
19.03


2
2.08
4.26
10.28
8.02
19.12


Mean
2.09
4.24
9.89
8.09
19.08









B. Using Organic Sodium Donor

The basic drawback of titration using inorganic base is that the water must be used for the solvent. So searching the sodium donor dissolved freely in normal organic solvent is the easiest way to solve the problem. Several reagents such as sodium acetate and sodium ethylhexanoic acid were tested and found to be useful for making a di-sodium salt form.
Table 10 shows compounds for bioactivity test selected from the prodrugs of the present invention and IC50 values thereof, which are measured by the reporter gene assay (RGA) and oncogenic activity by MTS or Sulforhodamine B assay as described in Example 6. The compound numbers on Table 10 are unrelated to those in Table 4 or 5.







TABLE 10







THE REPORTER GENE ASSAY AND ONCOGENIC ACTIVITY BY MTS OR SULFORHODAMINE B


ASSAY FOR SELECTED PRODRUG COMPOUNDS









Assay












RGA,
RGA,





TopF
Survivin
MTS, SW480
MTS, HCT116



IC50,
IC50,
(uM)
(uM)














No
Structure
uM
uM
LD50
GI50
LD50
GI50

















1




4.2
6.4
17.0
2.0
16.1
2.2


 


2




3.5
5.7
 8.2
3.1
23.2
6.6


 


3




11.5

ND up to 50 uM
3.0
41.9
3.1


 


4




7.3
6.5
ND up to 50 uM
6.9
49.3
11.4


 


5




26.0

34.0
5.2
ND up to 50 uM
16.5


 


6




0.8
0.1
 9.2
0.5
 6.4
0.4


 


7




2.3
1.0
12.9
2.2
12.0
1.8


 


8




1.4
0.9
21.6
2.1
23.2
1.9


 


9




9.6
6.0
ND up to 50 uM
7.6
ND up to 50 uM
14.7


 


10




2.8
1.7
 9.4
0.9
 7.9
0.8


 


11




10.3
6.7
ND up to 50 uM
6.5
ND up to 50 uM
6.3


 


12




1.0
0.7
ND up to 50 uM
1.0
19.3
1.2


 


13




1.8
0.9
21.1
2.3
20.0
1.7


 


14




1.7
1.2
21.1
2.3
16.0
2.1









Example 16
Solubility of Selected Prodrugs
General Procedure for Solubility Test of Prodrugs
About 2 mg of each prodrug was dissolved in 1 ml of JP1 or JP2 solution as indicated below. Incubating at a temperature of 37° C., 200 ul of samples were withdrawn at 0 hour, 2 hour and 20 hour. Withdrawn samples were filtered through 0.45 μm syringe filters and analyzed by HPLC system.
Composition of Artificial Gastro-Intestinal Fluids (JP1, JP2)











JP1
JP2


















PH
 1.2
pH
6.8


NaCI
 2.0 g
0.2 M KH2PO4
250 ml


10% HCI
24.0 ml
0.2N NaOH
118 ml


Distilled H2O
Adjusted to 1 L
Distilled H2O
Adjusted to 1 L









Table 11 below shows the results of solubility test of selected prodrugs. The compound numbers on Table 11 are unrelated to those in Table 4, 5 or 10.







TABLE 11







AQUEOUS SOLUBILITY FOR SELECTED PRODRUG COMPOUNDS









Solubility (37° C., ug/mL)



0 hr, 2 hr, 20 hr










No
Structure
JP1 (pH 1.2)
JP2 (pH 6.8)













1




60.1 87.3 92.8
1797 1867 1894


 


2




122 173 160
1950 1939 1940


 


3




1878 1971 2036
1325 1902 2005


 


4




554 646 756
1982 2014 2030


 


5




406 532 684
1761 1778 1758


 


6




1453 1724 1787
1829 1864 1867


 


7




309 446 521
2145 2221 2239


 


8




671 775 921
2295 2317 2272


 


9




2251 2275 2403
2322 2353 2421


 


10




2292 2274 2327
2028 2055 2027


 


11




2006 2000 1998
1636 1654 1651









Example 17
Preparation of Dimethyl-carbamic acid 4-[2-allyl-1benzylcarbamoyl-8-(2,4-difluoro-benzyl)-4,7-dioxo-octahydro-pyrazino[2,1-c][1,2,4]triazin-6-ylmethyl]-phenyl ester




To a stirred solution of starting material (SM) (8.0 g, 13.9 mmol) and potassium carbonate (5.8 g, 41.7 mmol) in dimethylformamide was added dimethylcarbamyl chloride (3.0 g, 27.8 mmol). The reaction mixture was stirred overnight and then dissolved in EtOAc, washed with water five times. The combined organic layer was washed with brine, dried over sodium sulfate, concentrated in vacuo. The residue was chromatographed on silica gel with neat EtOAc to afford product (4.2 g, 32%). The data from analyzing the resulting product by mass spectrometry and NMR are:
MS (ESI): m/e 647 (M+1), 669 (M+Na). 1H-NMR (300 MHz, CDCl3) δ 7.27-7.39 (6H, m), 6.78-7.13 (6H, m), 6.68 (1H, t, J=6.0 Hz), 5.61-5.70 (2H, m), 5.34 (1H, t, J=6.0 Hz), 5.06-5.20 (2H, m), 4.31-4.69 (4H, m), 3.24-3.58 (8H, m), 3.07 (3H, s), and 2.99 (3H, s).
Example 18
Preparation of Carbonic acid 4-[2-Allyl-1-Benzylcarbamoyl-8-(2,4-difluorobenzyl)-4,7-dioxo-octahydro-pyrazino[2,1-c][1,2,4]triazin-6-ylmethyl]-phenyl ester 4-nitro-phenyl ester (2)




To a stirred solution of 2-Allyl-8-(2,4-difluoro-benzyl)-6-(4-hydroxybenzyl)-4,7-dioxohexahydro-pyrazino[2,1-c][1,2,4]triazine-1-carboxylic acid benzylamine (1) (2 g, 3.47 mmol) in THF (40 ml) was added Triethylamine (0.97 ml, 6.95 mmol) and 4-nitrophenylchloroformate (0.7 g, 3.47 mmol). After stirring at room temperature for overnight, the solvent was removed under reduced pressure. The crude compound was purified by chromatography (Hexane, EtOAc 1:1) to give the compound (1.59 g, 62%). The data from analyzing the purified compound by TLC system and NMR are:
TLC System: Rf=0.3 (n-Hexane:EtOAc 1:1) 1H NMR (300 MHz, CDCl3): δ 3.25-3.60 (m, 8H), 4.35 (dd, J=14.9 Hz, 5.7 Hz, 1H), 4.43 (dd, J=14.8 Hz, 6.1 Hz, 1H), 4.52 (d, 14.5 Hz, 1H), 4.68 (d, 14.1H), 5.10 (d, 17.1 Hz, 1H), 5.21 (d, 10.3 Hz, 1H), 5.34 (t, J=6.1 Hz, 1H), 5.58 (dd, J=11.1 Hz, 4.1 Hz, 1H), 5.67 (m, 1H), 6.71 (t, J=6.1 Hz, NH), 6.75-6.98 (m, 2H), 7.13 (d, J=8.4 Hz, 2H), 7.21 (d, J=8.7 Hz, 2H), 7.23-7.39 (m, 6H), 7.46 (d, J=9.5 Hz, 2H), and 8.30 (d, J=9.5 Hz, 2H).
Example 19
Preparation of (2-Dimethylamino-ethyl)-Carbamic acid 4-[2-Allyl-1-benzylcarbamoyl-8-(2,4-difluoro-benzyl)-4,7-dioxo-octahydro-pyrazine[2,1-c][1,2,4]triazine-6-ylmethyl]-phenyl ester hydrochloride salt (3)
To a stirred solution of carbonic acid 4-[2-allyl-1-benzylcarbamoyl-8-(2,4-difluoro-benzyl)-4,7-dioxo-octahydro-pyrazino[2,1-c][1,2,4]triazin-6-ylmethyl]-phenyl ester 4-nitro-phenyl ester (2) (1.2 g, 1.62 mmol) in DMF (25 ml) was added N,N-Dimethylethylenediamine (0.26 ml, 2.43 mmol). After stirring at room temperature for overnight, the solvent was removed under reduced pressure. The residue was diluted with EtOAc and washed with water, brine. The organic layer was dried with Na2SO4 and concentrated in vacuo. The crude compound was purified by chromatography (n-Hexane:EtOAc, 1:1; EtOAc; CH2Cl2:MeOH, 9:1).
The compound was poured with water and was maintained pH 5-6 with 1 N aq. HCl to make HCl salt and then lyophilized to get the compound (0.75 g, 60%). The data from analyzing the resulting compound by TLC system and NMR are:
TLC System: Rf=0.35 (CH2Cl2:MeOH, 9:1) ESI-MS: M+H+ 690.39 1H NMR (300 MHz, CDCl3): δ 2.56 (d, J=4.6 Hz, 6H), 3.18 (bm, 4H), 3.41-3.58 (m, 4H), 3.75 (t, J=10.3 Hz, 1H), 4.21 (bt, 2H), 4.35 (d, J=14.9 Hz 1H), 4.70 (d, J=14.9 Hz 1H), 5.05 (m, 1H), 5.12 (m, 1H), 5.42 (m, 6H), 5.80 (m, 1H), 6.91 (d, J=7.6 Hz, 2H), 7.04 (d, J=7.3, 2H), 7.09 (m, 1H), 7.18-7.26 (m, 6H), 7.31 (d, J=6.9 Hz, 2H), 7.33 (m, 1H), 7.84 (bt, NH), 7.98 (bt, NH), 10.7 (bs, 1H)
Example 20
Mouse In Vivo PK Study of Prodrug a after Single I.V. Bolus Injection
Animal Experiment
Drugs were prepared 10 mg/kg/5 ml in 10% Tween 80. Studies were performed in ICR mice. After i.v. bolus injection through the tail vein, blood samples were acquired from inferior vena cava at several time points and separated to plasma by centrifugation. Plasma samples were preserved at −20° C. until they were analyzed. Bleeding time points were 3, 6, 9, 17, 34, 67, 134, 202, 302 min. N=4.
Sample Preparation
For calibration curve, 98 ul aliquots of control mouse plasma were added 2 ul of drug stock solution and added 2 ul internal standard stock solution, 5 ug/ml of internal standard. Final concentrations of calibration samples were 1, 10, 100, 1000 ng/ml and 10 ug/ml. For plasma sample from animal experiment, 100 ul of plasma were added 2 ul of internal standard. Then, all the samples were added 500 ul of acetonitrile, 500 ul of ethylacetate and 100 ul of DW. Samples were mixed for 10 min and centrifuged. The supernatants were transferred another tubes and evaporated. Adding 200 ul of 40% acetonitrile, they were reconstituted and analyzed by LC-MS system.
Results
The changes of concentrations of prodrug A and its parent compound in mouse plasma with the increase of time after i.v. bolus injection of prodrug A are shown in FIG. 18. Square: parent compound; Diamond: prodrug A.




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 having the following general formula (VII)


—Y—R10  (VI)

wherein (VI) is the formula:




wherein when not linked to R10,
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; and

Wherein one of Ra, Rb, Rc, X1, X2, and X3 is linked to R10 via Y,
Y is an oxygen, sulfur, or nitrogen in Ra, Rb, and Rc, or an oxygen in X1, X2 and X3; and
Y—R10 is hemimalate, dimethylaminoalkylcarbamates, or a salt thereof; or R10 is hydroxyalkyl, glycosyl, substituted or unsubstituted piperidine oxycarbonyl, or a salt thereof.

2. A pharmaceutical composition comprising a compound according to claim 1 and pharmaceutically acceptable carrier.

3. The compound of claim 1, wherein when not linked to R10,
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; a naphthyl group; a quinolinyl group; an indazolyl group; or a benzpyrazolyl group; an isoquinolinyl group; and
Rb is phenyl, pyridyl or piperidyl, all of which may be substituted with one or more substituents selected from a group consisting of halide, hydroxy, cyano, lower alkyl, and lower alkoxy groups.