Peptides for activating cell signaling in osteoprogenitor cells

Abstract

The present invention provides a compound and a pharmaceutical composition of a peptidomimetic ligand. The peptidomimetic ligand can be conjugated with a phosphonate drug. Since the compound and the pharmaceutical composition of the present invention are specific to α4β1 integrin and bone surface on mesenchymal stem cells, they can be used to treat osteoporosis and promote bone growth.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 700,743, filed on July 19, 2018, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] STATEMENT AS TO RIGHTS TO INVENTS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under Grant No. AR061366 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Osteoporosis is a disease that causes increased bone fragility due to estrogen deficiency and aging. It is a major public health problem, with nearly 50% of white women and 25% of white men at risk for an osteoporotic fracture in their lifetime (National Osteoporosis Foundation publication). In addition, more than 2 million osteoporotic fractures occur in the United States each year, with 27% of vertebral fractures and 14% of hip fractures. Therefore, especially with the rapid global growth of our aging population, osteoporosis represents a significant health concern.

[0006] Aging is related to the reduction of bone marrow skeletal osteoprogenitor cells and the appropriate microenvironment supporting these osteoprogenitor cells to differentiate into osteoblasts to form bone. Along with aging, the decline of the number of mesenchymal stem cells (MSC) in the bone marrow leads to a reduction in osteogenesis, and may be the most important factor leading to a reduction in bone formation and an increase in bone fragility (Heersche, JN, CG Bellows and Y.Ishida, J Prosthet Dent, 1998, 79 (1): pp. 14-6; Ettinger, MP, Arch Intern Med, 2003. 163 (18): pp. 2237-46). Currently, almost all treatments for osteoporosis are to reduce bone loss by reducing osteoclast bone resorption, thereby preventing further destruction of bone. Importantly, this type of anti-resorptive drug cannot restore the lost bone structure. Current drugs that enhance bone formation include hPTH(1-34) (teriparatide), PTHrp (abaloparatide), and Evenity (romosozumab) and have been approved by the FDA for the treatment of osteoporosis. Abaloparatide may be more effective than teriparatide and has a more rapid onset of fracture reduction compared with teriparatide in the setting of uncoupling of bone remodeling. In preclinical studies, treatment with teriparatide and abaloparatide was limited to 2 years, and a black box warning was noted regarding the risk of osteosarcoma. Teriparatide and abaloparatide are not recommended for use in patients at high risk of osteosarcoma, with a history of radiation, primary hyperparathyroidism, and any form of secondary hyperparathyroidism. In addition, all current drug agents can reduce the risk of vertebral fractures by approximately 50% or more, but their efforts in reducing hip fractures remain low.

[0007] The treatment mode of targeting bone formation by increasing the number and / or activity of osteoblasts may be a more attractive method for enhancing bone formation and promoting bone regeneration. Although bone regeneration by inducing osteogenesis from MSC is a reasonable strategy for treating osteoporosis, because MSC cannot migrate to the bone surface, systemic infusion of MSC in vivo fails to promote the osteogenic response of bone, which is the main clinical problem for MSC transplantation (Gao, J., et al., Cells Tissues Organs, 2001.169 (1): 12-20 pages; Meyerrose, TE, et al., Stem Cells, 2007, 25 (1): 220-7 pages). In addition, the transplantation of MSC requires the use of chemotherapy and / or radiation donor ablation, which may cause concomitant damage to endogenous mesenchymal cells (Bacigalupo, A., Best Pract Res Clin Haematol, 2004, 17 (3): 387-99 pages).

[0008] Cell adhesion is a process by which cells contact each other, migrate toward a specific target or are positioned in the extracellular matrix. Cell adhesion constitutes one of the basic mechanisms behind many biological phenomena. Studies on the molecular basis of cell adhesion have shown that various cell surface macromolecules (collectively referred to as cell adhesion molecules or receptors) mediate cell-cell and cell-matrix interactions. For example, members of the cell surface receptor integrin family mediate cell-cell and cell-matrix interactions and regulate cell motility, migration, survival and proliferation (Hynes, Cell, 69: 11-25 (1992); Hynes, Cell, 1110: 673-687 (2002)). Integrin is a non-covalent heterodimer complex consisting of two subunits α and β. There are at least 18 different α subunits and at least 8 different β subunits.

[0009] Mesenchymal stem cells within the bone marrow have multi-lineage potential and represent a mixture of precursors of mesenchymal-derived cell types including osteoblasts, chondrocytes, and adipocytes (Owen, M. et al., Ciba Found Symp, 1988, 136: 42-60; Bruder, SP, et al., J Cell Biochem, 1994, 56(3): 283-94; Prockop, DJ, Science, 1997, 276(5309): 71-4). Osteocytes at all stages of maturation are critically dependent on cell-matrix and cell-cell interactions (Mukherjee, S., et al., J Clin Invest, 2008, 118(2): 491-504; Grzesik, WJ and P.G. Robey, J Bone Miner Res, 1994, 9(4): 487-96; Vukicevic, S., et al., Cell, 1990, 63(2): 437-45; Mbalaviele, G., et al., J Bone Miner Res, 2006, 21(12): 1821-7). Bone marrow is the location of committed osteoblast progenitor cells, and osteogenic differentiation is the default pathway for MSC lineage commitment (Halleux, C., et al., J Musculoskelet Neuronal Interact, 2001, 2(1): p. 71-6; Muraglia, A., et al., J Cell Sci, 2000, 113(Pt 7): p. 1161-6). Mobilizing osteoblast progenitor cells to the bone surface is a key step in osteoblast maturation and formation of mineralized tissue (Adams, GB, et al., Nature, 2006, 439(7076): p. 599-603; Chen, XD, et al., J Bone Miner Res, 2007, 22(12): p. 1943-56). Once osteoblast progenitor cells are "committed" to the bone surface, they synthesize a series of proteins including osteocalcin, osteopontin, bone sialoprotein, osteonectin, collagen-I and fibronectin, which will further enhance the adhesion and maturation of osteoblasts (Gronthos, S., et al., Periodontol 2000, 2006, 41: pp. 188-95; Gronthos, S., et al., Bone, 2001, 28(2): pp. 174-81; Gronthos, S., et al., J Bone Miner Res, 1997, 12(8): pp. 1189-97). These interactions are mainly mediated by transmembrane integrin receptors, which mainly use arginine-glycine-aspartic acid (RGD) sequences to recognize and bind specific ligands.MSCs express integrins α1, α2, α3, α4, α6, α11, CD51 (integrin αV) and CD29 (integrin β1) (Brooke, G., et al., Stem Cells Dev, 2008). It is reported that integrins α1β1, α2β1, α. v β1, α v β5, α5β1 and α4β1 are expressed in osteoblasts (Grzesik, WJ and Robey, PG, J Bone Miner Res, 1994, 9(4): 487-96; Gronthos, S., et al., Bone, 2001, 28(2): 174-81; Gronthos, S., et al., J Bone Miner Res, 1997. 12(8): 1189-97; Cowles, EA, LL Brailey, and GA Gronowicz, J Biomed Mater Res, 2000, 52(4): 725-37). Overexpression of α4 integrin on MSCs has been reported to increase MSC homing to bone (Mukherjee, S., et al., J Clin Invest, 2008, 118(2): 491-504).

[0010] Bisphosphonates are widely used to treat osteoporosis. Such drugs are also used as "vehicles" for delivering bone-targeted drugs to bone tissue as prodrugs based on their bisphosphonate moiety. Bisphosphonates have been used to deliver sustained-release diclofenac (nonsteroidal anti-inflammatory drug) to the bones of rats (Hirabayashi, H., et al., J Control Release, 2001, 70 (1-2): pp. 183-91). The bisphosphonate dose required for this drug delivery is typically 10 to 100 times lower than the dose required for treating osteoporosis, hypocalcemia, Paget's disease or metastatic bone cancer.

[0011] It is well known that bone formation is beneficial in the treatment of a variety of different diseases in mammals, including simple aging, bone degeneration and osteoporosis, fracture healing, fusion or joint fixation, osteogenesis imperfecta, etc., as well as for the successful installation of various medical orthopedic and periodontal implants, such as screws, rods, titanium cages for spinal fusion, hip joints, knee joints, ankle joints, shoulder joints, dental plates and rods, etc.

[0012] The use of cathepsin K inhibitors and TGF-beta binding proteins, etc., to increase bone mineralization for the treatment of diseases characterized at least in part by increased bone resorption (e.g., osteopenia, bone fractures, osteoporosis, arthritis, tumor metastasis, Paget's disease and other metabolic bone disorders) is well known, as shown by U.S. Publication No. 2004 / 0235728, published on November 25, 2004, to Selwyn Aubrey Stoch, and U.S. Patent No. 6,489,445, published on January 15, 2004, to Mary E. Brunkow et al., and U.S. Publication No. 2004 / 0009535 published on January 15, 2004. In Brunkow's '445 patent and '535 publication, TGF-beta binding proteins include Sost polypeptides (full length and short peptides) antibodies that interfere with the interaction between the TGF-beta binding protein sclerostin and members of the TGF-beta superfamily (particularly bone morphogenetic protein). A new family of human TGF-beta binding proteins and nucleic acids encoding them are described in Brunkow'445 patent. The protein binds to at least human bone morphogenetic protein-5 and human bone morphogenetic protein-6. The aforementioned diseases are due to systemic loss of bone mineral, so the administration of antibody therapeutics is for systemic (whole body) increase in bone mineral density.

[0013] U.S. Publication No. 2006 / 0165799, published on July 27, 2006, teaches a bone filling composition for stimulating bone formation and bone consolidation comprising biocompatible calcium sulfate and an adhesive biopolymer. The composition is intended to be applied to the missing portion of an injured bone without spreading to surrounding organs.

[0014] U.S. Publication No. 2005 / 025604, published November 17, 2005, shows induction of bone formation by mechanically inducing an increase in osteoblast activity and increasing systemic blood concentrations of a bone anabolic agent, including optionally increasing systemic blood concentrations of an anti-bone resorptive agent.

[0015] U.S. Patent No. 7,576,175, issued on August 18, 2009, shows α4β1 integrin ligands that exhibit high binding affinity, specificity, and stability. The ligand comprises a peptide having "n" independently selected amino acids, wherein at least one amino acid is an unnatural amino acid or a D-amino acid, and wherein n is an integer from 3 to 20.

[0016] U.S. Publication No. 2010 / 0021379, published on January 28, 2010, shows antibody conjugates comprising a targeting agent covalently linked to an antibody or fragment thereof. The targeting agent comprises a ligand comprising a peptide or peptidomimetic specific for an integrin receptor, such as α4β1 integrin.

[0017] What is needed in the art are new compositions and methods for treating osteoporosis and promoting bone growth. Summary of the invention

[0018] In one embodiment, the invention provides a compound according to formula I:

[0019]

[0020] or a pharmaceutically acceptable salt thereof,

[0021] in:

[0022] R 1 Selected from –OH, –NH2 and -LD;

[0023] R 2 Selected from H, C 1-6 Alkyl and C 3-8 Cycloalkyl;

[0024] Each R 3 and R 4 are independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Haloalkyl;

[0025] X 1 , X 2 and X 3 are independently selected from amino acid residues;

[0026] X 4 Selected from (N 6 -modified) lysine residues, citrulline residues, homocitrulline residues, leucine residues, (N-methyl)leucine residues, isoleucine residues, (N-methyl)isoleucine residues and homophenylalanine residues;

[0027] L is the connector;

[0028] D is a phosphonate drug; and

[0029] The subscripts m, n, p and q are each independently an integer from 0 to 4.

[0030] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound as described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0031] In another embodiment, the present invention provides a method for treating osteoporosis. The method comprises administering to an individual in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof.

[0032] In another embodiment, the present invention provides a method for promoting bone growth. The method comprises administering to an individual in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof.

[0033] In another embodiment, the present invention provides a method for treating low bone mass. The method comprises administering to an individual in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof.

[0034] In another embodiment, the invention provides a method for treating a disease or condition characterized by secondary low bone mass. The method comprises administering to an individual in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A flow chart for screening cell signaling activators with affinity for osteoblast-specific transcription factor (osterix)+ osteoprogenitor cells is shown.

[0036] Figure 2 Lymphocyte binding is shown. Peripheral mononuclear cells were extracted from blood and incubated for 1 hour with beads displaying scrambled, YLL3 or YLL8 peptides.

[0037] Figure 3 Comparison of the binding affinity of YLL3, YLL8 and LLP2A peptides to osteoprogenitor cells after 10 days of culture.

[0038] Figure 4 is a comparison of the activation of Akt signaling in osteoprogenitor cells by YLL3, YLL8 and LLP2A peptides after 10 days of culture.

[0039] Figure 5 Comparison of several YLL peptides on osteogenesis in osteoprogenitor cells. Alkaline phosphatase levels were measured after 14 days. After 21 days, osteoblast maturation and mineralization were measured using Alizarin red staining.

[0040] FIG. 6A to FIG. 6D It was shown that YLL3 and YLL8 stimulated osteogenesis in vitro. Bone marrow stromal cells were incubated with beads displaying autofluorescence quenching of YLL3 or YLL8 peptides in osteogenic medium. Fig. 6AQuantitative measurement of alkaline phosphatase (ALP) levels at day 10 or Alizarin Red (AR) levels at day 21 for YLL3 and YLL8 beads are shown. Figure 6B Representative images of ALP staining in BMSCs cultured with beads displaying YLL3 or YLL8 at day 10 are shown. Figure 6C Representative images of AR staining in BMSCs cultured with beads displaying YLL3 or YLL8 at day 21 are shown. Fig.6D The results show that in osteogenic medium with 6×10 -8 Results of BMSCs cultured with YLL3 and YLL8 peptides for 10 or 21 days to measure ALP levels on day 10 and AR levels on day 21. *, p<0.05 vs. control.

[0041] 7A to 7C Activation of Akt signaling using YLL8 is shown. Fig. 7A is the layout of the Akt signaling array. Figure 7B The results show that the osteogenic medium with 6x10 -8 BMSCs were cultured with either M peptide or hPTH(1-34) for three days, and then the cell lysates were used to perform Akt arrays. Figure 7C Shown from Figure 7B Quantitative pixel density of selected wells in .

[0042] Figure 8 Comparison of the effects of YLL3, YLL8 and LLP2A peptides on bone mass in young mice.

[0043] 9A to 9E The in vivo anabolic effects of YLL3 and YLL8 on bone mass in young mice are shown. Two-month-old female and male mice were treated with PBS control, 50 μg / kg of YLL3 or YLL8, or 25 μg / kg of PTH, subcutaneously, 5 times a week for 21 days (n=4-6 / group). All mice received calcein at -7 days and -2 days before euthanasia. Trabecular bone volume and surface-based bone formation were measured at the distal femur, and cortical bone volume and bone formation were measured at the mid-femur ( Fig.9A , Fig. 9B and Fig. 9C ). Fig.9D Representative images of trabecular bone samples taken from male mice are shown. Fig.9E Representative images of cortical bone samples taken from male mice are shown. *, p<0.05 vs. PBS.

[0044] FIG. 10A to FIG. 1 OD shows the effect of YLL8 on bone formation in vivo. Four-month-old female and male mice were treated with PBS control, 5 μg / kg (~6×10-9 M) YLL8 or 25 μg / kg PTH, subcutaneously injected, 5 times a week, for 21 days (n=4-6 / group). All mice received alizarin red and calcein on days -7 and -2 before euthanasia. Fig. 10A Shown are trabecular bone volume and surface-based bone formation measured in the distal femur metaphysis (DFM). Fig. 10B Representative images of DFM trabecular areas in female mice are shown. Fig. 10C Representative images of DFM trabecular areas in male mice are shown. Figure 10D shows cortical bone mass measured by micro-CT in the mid-femur; bone strength was obtained by three-point bending of the femur. *, p<0.05 vs. PBS.

[0045] Fig.11 The anabolic effects of YLL on trabecular bone volume, osteocalcin and CTX-1 in young mice are shown. Four-month-old female and male mice were treated with PBS control, 5 μg / kg of YLL3, 5 μg / kg of YLL8 or 25 μg / kg of PTH, subcutaneous injection, 5 times a week, for 28 days (n=4-6 / group). Trabecular bone volume was obtained by micro-CT scanning of the distal femur. Osteocalcin and CTX-1 were measured in serum.

[0046] Fig.12 Expression of Prx1 and osteoblast-specific transcription factor in bone is shown. Representative distal femurs are shown for Prx1-GFP (green, left) and osteoblast-specific transcription factor-mCherry (red, right) mice.

[0047] FIG. 13A to FIG. 13D Callus mineralization using YLL3 in fracture healing is shown. Female Prx1-GFP / ERT mice were fractured at two months of age and received tamoxifen at a dose of 10 mg / kg for three days. YLL3 or YLL8 was given at 10 μg / kg and hPTH (1-34) was given at 50 μg / kg, subcutaneously, 5 times a week for 10 or 21 days. Alizarin red was given -1 day before euthanasia. Fig.13A Representative femoral fracture images from Prx1-GFP / ERT mice at day 10 are shown. Fig. 13B Shown are representative callus images from the outer edge of the callus in the indicated treatment groups at day 10 after fracture, as well as representative micro-CT thickness maps of callus, callus bone volume, and bone mineral content from the indicated treatment groups at day 10 after fracture. Fig. 13C Representative fractured femur images from Prx1-GFP / ERT mice at day 21 are shown. Fig.13DRepresentative callus images of the outer edge of the callus from the indicated treatment groups at day 21 after fracture, and representative micro-CT thickness maps of the callus, callus bone volume, and bone mineral content from the indicated treatment groups at day 21 after fracture are shown.

[0048] Fig.14 Synthetic schemes of YLL3-Ale and YLL8-Ale conjugates are shown.

[0049] FIG. 15A to FIG. 15C Two-month-old female mice treated with: PBS control; 100 μg / kg or 300 μg / kg of YLL3-Aln or YLL8-Aln, subcutaneous injection, once every two weeks, 2 doses; or 500 μg / kg of YLL3-Aln or YLL8-Aln, one intravenous dose. Mice were euthanized on day 28 (n=4-5 / group). Fig.15A Quantified trabecular bone volume and trabecular bone thickness are shown. Fig. 15B Representative 3D images of trabecular regions taken from the distal femur in the indicated treatment groups are shown. Fig. 15C Shown are midshaft femoral cortical bone volume and cortical bone thickness measured in femurs by micro-CT representative images of the midshaft femoral cortex in the indicated treatment groups. *, p<0.05 vs. PBS.

[0050] Fig.16 Two-month-old female mice (n=4-6 / group) treated with PBS control, 100 μg / kg or 300 μg / kg of YLL3-Aln or YLL8-Aln, respectively, subcutaneously, once every two weeks, for 28 days are shown. Trabecular bone formation was measured in the distal femoral metaphysis. *, p<0.05, relative to PBS. Arrows show double-labeled bone surfaces. DETAILED DESCRIPTION

[0051] The present invention provides peptidomimetic ligand compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds. The peptidomimetic ligands can also be conjugated with phosphonate drugs such as alendronate. The compounds and pharmaceutical compositions of the present invention can be used to promote bone growth, treat osteoporosis, treat low bone mass, and treat diseases or conditions characterized by secondary low bone mass due to their ability to activate Akt signaling.

[0052] I. Definitions

[0053] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.

[0054] As used herein, the term "Ale," "Aln," or "Alen" refers to alendronate.

[0055] When substituent groups are designated by their conventional chemical formula written from left to right, they also encompass chemically identical substituents that would result from writing the structure from right to left, for example, -CH2O- is equivalent to -OCH2-.

[0056] As used herein, the terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0057] As used herein, the term "alkyl" refers to a straight or branched saturated aliphatic group having a specified number of carbon atoms. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, etc.

[0058] As used herein, the term "haloalkyl" refers to an alkyl group as defined above in which some or all of the hydrogen atoms are replaced by halogen atoms. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term "perfluoro" defines a compound or group having at least two available hydrogens replaced by fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0059] As used herein, the term "heteroalkyl" refers to an alkyl group having 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms may also be useful, including but not limited to B, Al, Si and P. The heteroatoms may also be oxidized, such as but not limited to -S(O)- and -S(O)2-. For example, heteroalkyl groups may include ethers, thioethers, alkyl-amines and alkyl-thiols.

[0060] As used herein, the term "alkoxy" refers to an alkyl group containing an oxygen atom, such as methoxy, ethoxy and the like.

[0061] As used herein, the term "aryl" refers to an aromatic ring assembly of a monocyclic or fused bicyclic, tricyclic or more rings containing 6 to 16 ring carbon atoms. For example, aryl can be phenyl, benzyl or naphthyl, preferably phenyl. "Arylene" means a divalent group derived from an aryl. Aryl can be selected from one, two or three groups of alkyl, alkoxy, aryl, hydroxyl, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene, monosubstituted, disubstituted or trisubstituted; all of which are optionally further substituted, for example as defined above; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl. Alkynedioxy is a divalent substituent connected to two adjacent carbon atoms of phenyl, such as methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent connected to two adjacent carbon atoms of phenyl, such as oxyethylene or oxypropylene. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0062] As used herein, the term "heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly comprising 5 to 16 ring atoms, wherein 1 to 4 ring atoms are heteroatoms. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolyl, isoquinolyl, benzothienyl, benzofuranyl, furyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl or any other (especially monosubstituted or disubstituted) group substituted by, for example, alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolyl preferably represents 2-, 3- or 4-quinolyl. Isoquinolyl preferably represents 1-, 3- or 4-isoquinolyl. Benzopyranyl and benzothiopyranyl preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, and most preferably represents 4-thiazolyl. Triazolyl is preferably 1-(1,2,4-triazolyl), 2-(1,2,4-triazolyl) or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

[0063] Substituents for the aryl and heteroaryl groups are varied and are selected from: -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO2, -C02R', -CONR'R", -C(O)R', -OC(O)NR'R", -NR"C(O)R', -NR"C(O)2R', -NR'-C(O)NR"R"', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH 2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and wherein R', R" and R'" are independently selected from hydrogen, C1-C8 alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0064] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic, fused bicyclic or bridged polycyclic ring assembly containing 3 to 12 ring atoms or the specified number of atoms. 3-8 Cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and up to cyclooctyl.

[0065] As used herein, each of the above terms (eg, "alkyl," "aryl," and "heteroaryl") are meant to include both substituted and unsubstituted forms of the designated radical.

[0066] Substituents for the hydrocarbyl and heterohydrocarbyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC( R', -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR(SO2)R', -CN and -NO2, and their number is zero to (2m'+1), where m' is the total number of carbon atoms in such a group. R', R", R"' and R"" are each independently selected from hydrogen, C1-C8 alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstituted aryl)oxy-(C1-C4)alkyl. When a compound of the invention includes more than one R group, for example, each R group is independently selected as each R', R", R"' and R"" group (when more than one of these groups is present). When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0067] As used herein, the term "peptide" refers to a compound consisting of a single chain of D- or L-amino acids or a mixture of D- or L-amino acids connected by peptide bonds. Typically, the length of a peptide is about 2 to about 50 amino acids. Preferably, the length of the peptide of the present invention is about 2 to about 25 amino acids, more preferably 3 to 20 amino acids in length, and most preferably 3 to 10 amino acids in length.

[0068] As used herein, the term "amino acid" refers to naturally occurring, unnatural, and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0069] As used herein, the term "naturally occurring amino acids" refers to those amino acids encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0070] As used herein, the term "unnatural amino acid" includes, but is not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N- or D-configurations that function in a manner similar to the naturally occurring amino acids. 6 - Modified lysine and N-methyl amino acids. Unnatural amino acids are not encoded by the genetic code and may, but need not, have the same basic structure as a naturally occurring amino acid.

[0071] As used herein, the term "amino acid analogue" refers to a compound having the same basic chemical structure (i.e., an alpha carbon linked to a hydrogen, a carboxyl group, an amino group, and an R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

[0072] As used herein, the term "amino acid mimetic" refers to a compound that has a structure that is different from the general chemical structure of an amino acid but functions in a manner similar to naturally occurring amino acids. Suitable amino acid mimetics include, but are not limited to, β-amino acids and γ-amino acids. In β-amino acids, the amino group is bonded to the β-carbon atom of the carboxyl group so that there are two carbon atoms between the amino group and the carboxyl group. In γ-amino acids, the amino group is bonded to the γ-carbon atom of the carboxyl group so that there are three carbon atoms between the amino group and the carboxyl group. Suitable R groups for β-amino acids or γ-amino acids include, but are not limited to, side chains present in naturally occurring amino acids and non-natural amino acids.

[0073] As used herein, the term “ 6 "-modified lysine" refers to a non-natural amino acid based on lysine, wherein the nitrogen atom H2N-CH2-CH2-CH2-CH2- of the R-group side chain of lysine is modified by a suitable substituent as described above, such as a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. For example, the nitrogen atom of the R-group side chain of lysine can be substituted by a 3-(pyridin-3-yl)propionyl or a 3-(pyridin-3-yl)prop-2-enoyl group.

[0074] Amino acids can be characterized by at least one of several properties. For example, amino acids can be positively charged, negatively charged, hydrophilic, or hydrophobic.

[0075] As used herein, the term "positively charged amino acid" refers to those amino acids with basic or positively charged side chains at pH values ​​below the pKa, including but not limited to Lys, Arg, His, HoArg, Agp, Agb, Dab, Dap and Orn and stereoisomers thereof. Basic amino acids are generally represented by the symbol "X + "express.

[0076] As used herein, the term "negatively charged amino acid" refers to an amino acid with an acidic or negatively charged side chain at a pH above the pKa, and includes, but is not limited to, Asp, Glu, Aad, Bec, and stereoisomers thereof. Acidic amino acids are generally denoted by the symbol "X - " indicates. Those skilled in the art will appreciate that other basic amino acids and acidic amino acids are known in the art.

[0077] As used herein, the term "neutral charged amino acid" refers to those amino acids having side chains with a neutral charge at a pH value equal to the pKa.

[0078] As used herein, the term "hydrophilic amino acid" refers to those amino acids having polar and uncharged side chains, including but not limited to Asn, Ser, Thr, and GIn.

[0079] As used herein, the term "hydrophobic amino acid" refers to those amino acids with hydrophobic side chains, including but not limited to Val, Leu, Ile, Met and Phe. In some embodiments, the hydrophobic amino acid is selected from proline, proline analogs and stereoisomers thereof. In some embodiments, the proline analog is hydroxyproline.

[0080] As used herein, the term "D-amino acid" refers to the D stereoisomer of an amino acid. The letters D and L are commonly used in the art to refer to stereoisomers of amino acids. D-amino acids are those amino acids that can be synthesized from the dextrorotatory isomer of glyceraldehyde, i.e., D-glyceraldehyde. Similarly, L-amino acids are those amino acids that can be synthesized from the levorotatory isomer of glyceraldehyde, i.e., L-glyceraldehyde.

[0081] Amino acids may be referred to herein by either the well-known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0082] With respect to amino acid sequences, one skilled in the art will recognize that a single substitution, deletion or addition to a nucleic acid, peptide, polypeptide or protein sequence (which alters, adds or deletes a single amino acid or a small number of amino acids in the encoded sequence) is a "conservatively modified variant" where the alteration results in the replacement of an amino acid with a chemically similar amino acid (i.e., hydrophobic, hydrophilic, positively charged, neutral, negatively charged). The chemically similar amino acids include, but are not limited to, naturally occurring amino acids such as L-amino acids, stereoisomers of naturally occurring amino acids such as D-amino acids, and non-natural amino acids such as amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycine and N-methyl amino acids. Exemplary hydrophobic amino acids include valine, leucine, isoleucine, methionine, phenylalanine and tryptophan. Exemplary aromatic amino acids include phenylalanine, tyrosine and tryptophan. Exemplary aliphatic amino acids include serine and threonine. Exemplary basic amino acids include lysine, arginine and histidine. Exemplary amino acids with carboxylate side chains include aspartate and glutamate. Exemplary amino acids with carboxamide side chains include asparagine and glutamine. Providing functionally similar amino acid conservative substitution tables is well known in the art. Such conservatively modified variants are supplements to polymorphic variants of the present invention, interspecific homologues and allelotrope, and do not exclude polymorphic variants of the present invention, interspecific homologues and allelotrope.

[0083] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, such substitutions can be made: wherein an aliphatic amino acid (e.g., G, A, I, L, or V) is replaced by another member of the group. Similarly, aliphatic polar uncharged groups, such as C, S, T, M, N, or Q, can be replaced by another member of the group; basic residues such as K, R, or H can be replaced with each other. In some embodiments, amino acids with acidic side chains, such as E or D, can be replaced by their uncharged counterparts, such as Q or N, respectively; or vice versa. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions of each other:

[0084] 1) Alanine (A), glycine (G);

[0085] 2) Aspartic acid (D), glutamic acid (E);

[0086] 3) Asparagine (N), glutamine (Q);

[0087] 4) Arginine (R), Lysine (K);

[0088] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0089] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0090] 7) serine (S), threonine (T); and

[0091] 8) Cysteine ​​(C), Methionine (M)

[0092] (See, e.g., Creighton, Proteins (1984)).

[0093] As used herein, the term "joint" refers to a portion having one or more different reactive functional groups that allows a portion such as a peptide to be covalently linked to a chelating agent. In some embodiments, the linking portion has two different reactive functional groups, i.e., a heterobifunctional joint. Suitable joints include, but are not limited to, those available from Pierce Biotechnology, Inc. (Rockford, IL). In some embodiments, the joint provides a carboxyl group for the connection of the chelating agent and an amino group for the connection of the peptide. However, it will be appreciated by those skilled in the art that any reactive functional group may be present on the joint as long as it is compatible with the functional group on the portion to be covalently linked.

[0094] The linkers that can be used in the present invention include those with one or more different reactive functional groups, which allow a part such as a peptide to be covalently linked to a chelator. The linking part has two or more different reactive functional groups. In some cases, a multivalent linker can be used. Suitable linkers include, but are not limited to, those available from Pierce Biotechnology, Inc. (Rockford, IL). In some embodiments, the linker provides a carboxyl group for the connection of the chelator and an amino group for the connection of the peptide. However, it is understood by those skilled in the art that any reactive functional group may be present on the linker as long as it is compatible with the functional group on the part to be covalently linked. As used herein, the term "chelator-linker conjugate" refers to a chelator covalently linked to a linker. Such a chelator-linker conjugate can be connected to a peptide through a functional group present on the linker. Some suitable linkers include, but are not limited to, β-alanine, 2,2'-ethylenedioxybis(ethylamine)monosuccinamide (Ebes) and bis(Ebes)-Lys. Other suitable linkers include those with biotin. Additional linkers can be found in Bioconjugate Techniques, Greg T. Hermanson, Academic Press, 2nd edition, 2008 (incorporated herein by reference in its entirety).

[0095] As used herein, the term "salt" refers to an acid salt or a basic salt of a compound used in the methods of the present invention. Illustrative examples of pharmaceutically acceptable salts are inorganic acid (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.) salts, organic carboxylic acid (acetic acid, propionic acid, glutamic acid, citric acid, etc.), organic sulfonic acid (methanesulfonic acid) salts, quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. It is understood that pharmaceutically acceptable salts are non-toxic. Additional information about suitable pharmaceutically acceptable salts can be found, for example, in R EMINGTON :T HE S CIENCE AND P RACTICE OF P HARMARCY , 21st Edition, Copyright 2006, Lippincott Williams & Wilkins, Philadelphia PA ("Remington"), which is incorporated herein by reference.

[0096] Pharmaceutically acceptable salts of the acidic compounds of the present invention include salts formed with bases, i.e., cationic salts, such as alkali metal salts and alkaline earth metal salts, such as sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, and ammonium salts, such as ammonium salts, trimethylammonium salts, diethylammonium salts and tris-(hydroxymethyl)-methyl-ammonium salts.

[0097] As used herein, the term "hydrate" refers to a compound complexed with at least one water molecule. The compounds of the present invention may be complexed with 1 to 10 water molecules.

[0098] As used herein, the terms "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that facilitate administration of an active agent to an individual and are absorbed by it. "Pharmaceutically acceptable excipient" refers to an excipient that can be included in the composition of the present invention and does not produce significant adverse toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline solution, lactated Ringer's solution, regular sucrose, regular glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, and pigments, etc. Those skilled in the art will recognize that other pharmaceutical excipients can be used in the present invention.

[0099] As used herein, the term "osteoporosis" refers to a disease that results in increased bone fragility and / or decreased or reduced bone density due to any determined or undetermined cause or condition. "Low bone mass" or "osteopenia" is a condition, not a disease, that may develop into osteoporosis once bone density continues to decrease over time. Low bone mass is characterized by a T score of -1 to -2.15. Osteoporosis is characterized by a T score of less than -2.15. Among the specific causes or conditions targeted by the methods of the present invention are primary osteoporosis associated with menopause (natural, premature or surgical), aging, or both, as well as secondary osteoporosis or secondary low bone mass associated with medical conditions (e.g., Paget's disease, chronic kidney disease, amenorrhea caused by eating disorders, transplants, hyperthyroidism, hyperparathyroidism) or the use of certain medications (e.g., various cancer chemotherapies, gonadotropin-releasing hormone agonists, medroxyprogesterone acetate for birth control, corticosteroids, anticonvulsants, etc.).

[0100] As used herein, the phrase "Akt signaling" refers to any of a number of biochemical pathways involving the enzymatic activity of the serine / threonine-specific protein kinase Akt (also known as protein kinase B or PKB). Akt itself is activated after phosphorylation by phosphatidylinositol 3-kinase (PI3K) and other kinases. Activated Akt phosphorylates many cellular targets, thereby regulating processes including, but not limited to, cell viability, angiogenesis, osteogenesis, and cell metabolism associated with cell viability.

[0101] As used herein, the term "isomers" refers to compounds that have the same chemical formula but are distinguishable in structure.

[0102] As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and which are readily converted from one isomeric form to another.

[0103] As used herein, the term "patient" or "subject" refers to a living organism suffering from or prone to a condition that can be treated by administering a pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, and other non-mammals.

[0104] As used herein, the term "therapeutically effective amount" refers to the amount of a conjugated functional agent or pharmaceutical composition that can be used to treat or improve an identified disease or condition or that exhibits a detectable therapeutic effect or inhibitory effect. The effect can be detected by any assay method known in the art.

[0105] As used herein, the term "treat" refers to any indication of successful treatment or improvement of an injury, pathology, or condition, including any objective or subjective parameter, such as ablation; remission; lessening of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the end point of degeneration less debilitating; improving the patient's physical or mental health. Treatment or improvement of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and / or psychiatric assessment. For example, the methods of the present invention successfully treat delirium in a patient by reducing the incidence of impaired consciousness or cognition.

[0106] As used herein, the term "disorder" or "condition" refers to a state or health condition in a patient or individual that can be treated with the peptidomimetic ligand compounds of the invention.

[0107] As used herein, the term "one", "an" or "a kind" when used for a group of substituents or "substituent groups" herein means at least one / one. For example, when a compound is substituted with an alkyl or aryl group, the compound is optionally substituted with at least one alkyl and / or at least one aryl group, wherein each alkyl and / or aryl group is optionally different. In another example, when a compound is substituted with a substituent group, the compound is substituted with at least one substituent group, wherein each substituent group is optionally different. When a group can be substituted with one or more of a plurality of substituent groups, such substituent groups are selected to conform to the principles of chemical bonding and to produce such compounds: it is not inherently unstable and / or is not known by those of ordinary skill in the art to be unstable under environmental conditions such as aqueous, neutral or physiological conditions.

[0108] II. Peptidomimetic Ligand and Phosphonate Drug Conjugates

[0109] In some embodiments, the present invention provides a peptidomimetic ligand compound of Formula I:

[0110]

[0111] or a pharmaceutically acceptable salt thereof, wherein R 1 Selected from –OH, –NH2 and –LD. 2Selected from H, C 1-6 Alkyl and C 3-8 Cyclic hydrocarbon group. Each R 3 and R 4 are independently selected from H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Haloalkyl. 1 , X 2 and X 3 are independently selected from amino acid residues. 4 Selected from (N 6 -modified) lysine residues, citrulline residues, homocitrulline residues, leucine residues, (N-methyl)leucine residues, isoleucine residues, (N-methyl)isoleucine residues and homophenylalanine residues. L is a linker. D is a phosphonate drug. Subscripts m, n, p and q are each independently an integer from 0 to 4.

[0112] In some embodiments, R 2 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, branched hexyl, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane. In some embodiments, R 2 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched-chain pentyl, n-hexyl, and branched-chain hexyl. 2 is selected from H, methyl, ethyl, n-propyl, and isopropyl. In some embodiments, subscript n is 0, 1, or 2. In some embodiments, subscript n is 1 or 2. In some embodiments, subscript n is 1. In some embodiments, subscript n is 1 or 2 and R 2 is selected from the group consisting of H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, subscript n is 1 or 2 and R 2 is selected from H and methyl. In some embodiments, subscript n is 1 and R 2 For H.

[0113] In some embodiments, each R 3 and R 4 R is independently selected from H, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl and branched hexyl. 3 and R 4 R is independently selected from H, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl and isopropyl. 3 and R 4 is independently selected from H, chlorine, bromine, iodine and methyl.3 H and R 4 is selected from H, chlorine, bromine and methyl. 3 H and R 4 In some embodiments, subscript p is 0 and subscript q is 0, 1, 2, or 3. In some embodiments, subscript p is 0 and subscript q is 1 or 2. In some embodiments, subscript p is 0 and subscript q is 0. In some embodiments, subscript p is 0, subscript q is 1, and R 4 It is methyl.

[0114] In some embodiments, X 1 , X 2 and X 3 is an independently selected amino acid residue, including but not limited to naturally occurring amino acids; D-amino acids; non-natural amino acids, including but not limited to amino acid analogs in L-configuration or D-configuration, amino acid mimetics, N-substituted glycine, N-methyl amino acids, phenylalanine analogs, and derivatives of lysine (Lys), ornithine (Orn) and α,γ-diaminobutyric acid (Dbu); hydrophilic amino acids; hydrophobic amino acids; positively charged amino acids; and negatively charged amino acids. In some embodiments, the subscript m is 0, 1, 2, or 3. In some embodiments, the subscript m is 0, 1, or 2. When the subscript m is 2, the moiety -(X 1 ) m -For –X 1a -X 1b -, where X 1a and X 1b Also as described above and below for X 1 , X 2 and X 3 Independently selected amino acid residues are defined.

[0115] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.

[0116] Stereoisomers of naturally occurring α-amino acids include, but are not limited to, D-amino acids and L-amino acids. D-amino acids suitable for use in the present invention include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof. In some embodiments, the D-amino acid is selected from D-α-amino acids, D-β-amino acids, D-γ-amino acids, and combinations thereof. In some embodiments, the D-α-amino acids are selected from stereoisomers of naturally occurring α-amino acids, non-natural D-α-amino acids, and combinations thereof.

[0117] Unnatural amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, N-substituted glycine, N-methyl amino acids, phenylalanine analogs, and derivatives of lysine (Lys), ornithine (Orn) and α,γ-diaminobutyric acid (Dbu) that function in a manner similar to naturally occurring amino acids. Unnatural amino acids are not encoded by the genetic code and may, but do not necessarily, have the same basic structure or function as naturally occurring amino acids.

[0118] Unnatural amino acids for use in the present invention include, but are not limited to, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, ornithine, pentylglycine, pipecolic acid, thioproline, aminophenylalanine, hydroxytyrosine, and aminotyrosine.

[0119] In some other embodiments, the unnatural amino acid is selected from 1-aminocyclopentane-1-carboxylic acid (Acp), 1-aminocyclobutane-1-carboxylic acid (Acb), 1-aminocyclopropane-1-carboxylic acid (Acpc), citrulline (Cit), homocitrulline (HoCit), α-aminoadipic acid (Aad), 3-(4-pyridyl)alanine (4-Pal), 3-(3-pyridyl)alanine (3-Pal), propargylglycine (Pra), α-aminoisobutyric acid (Aib), α-aminobutyric acid (Abu), , norvaline (Nva), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dbu), α-tert-butylglycine (Bug), 3,5-dinitrotyrosine Tyr (3,5-diNO2), norleucine (Nle), 3-(2-naphthyl)alanine (Nal-2), 3-(1-naphthyl)alanine (Nal-1), cyclohexylalanine (Cha), di-n-propylglycine (Dpg), cyclopropylalanine (Cpa), homoleucine (Hle), homoserine (HoSe r), homoarginine (Har), homocysteine ​​(Hcy), methionine sulfoxide (Met(O)), methionine methylsulfonium (Met(S-Me)), α-cyclohexylglycine (Chg), 3-benzo-thienylalanine (Bta), taurine (Tau), hydroxyproline (Hyp), O-benzyl-hydroxyproline (Hyp(Bzl)), homoproline (HoPro), β-homoproline (βHoPro), thiazolidine-4-carboxylic acid (Thz), pipecolic acid (Nip), isonepiperidinic acid (IsoNip), 3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one (Cptd), tetrahydro-isoquinoline-3-carboxylic acid (3-Tic), 5H-thiazolo[3,2-a]pyridine-3-carboxylic acid (Btd), 3-aminobenzoic acid (3-Abz), 3-(2-thienyl)alanine (2-Thi), 3-(3-thienyl)alanine (3-Thi), α-aminosuberic acid (Asu), diethylglycine (Deg), 4-amino-4-carboxy-1,1-Dioxo-tetrahydrothiopyran (Acdt), 1-amino-1-(4-hydroxycyclohexyl)carboxylic acid (Ahch), 1-amino-1-(4-ketocyclohexyl)carboxylic acid (Akch), 4-amino-4-carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr(3-NO2)), 1-amino-1-cyclohexanecarboxylic acid (Ach), 1-amino-1-(3-piperidinyl)carboxylic acid (3-Apc), 1-amino-1-(4-piperidinyl)carboxylic acid (4-Apc), 2-amino-3-(4-piperidinyl)propionic acid (4-App), 2-aminoindan-2-carboxylic acid (Aic), 2-amino-2-naphthylacetic acid (Ana), (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid (Ppca), 4-thiazolylalanine (Tha), 2-aminooctanoic acid (A oa), 2-aminoheptanoic acid (Aha), ornithine (Orn), azetidine-2-carboxylic acid (Aca), α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (Acdi), thiazolidine-2-carboxylic acid (Thz(2-COOH)), allylglycine (Agl), 4-cyano-2-aminobutyric acid (Cab), 2-pyridylalanine (2-Pal), 2-quinolylalanine (2-quinoylalanine) (2-Qal), cyclobutylalanine (Cba), phenylalanine analogs, lysine derivatives, ornithine (Orn) derivatives, α, γ-diaminobutyric acid Dbu derivatives, stereoisomers thereof, and combinations thereof (see, Liu and Lam, Anal. Biochem., 295: 9-16 (2001)). Therefore, the non-natural α-amino acid exists in the form of a non-natural L-α-amino acid, a non-natural D-α-amino acid, or a combination thereof. ,

[0120] In some embodiments, the non-natural amino acid residue is selected from the following compounds of Table 1:

[0121] Table 1. Unnatural amino acids useful in the present invention.

[0122]

[0123] Suitable amino acid analogs include, but are not limited to, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Suitable amino acid mimetics include, but are not limited to, β-amino acids and γ-amino acids. Suitable R groups for β-amino acids or γ-amino acids include, but are not limited to, side chains present in naturally occurring amino acids and non-natural amino acids.

[0124] N-substituted glycines suitable for use in the present invention include, but are not limited to, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-(2-methoxyethyl)glycine, N-benzylglycine, (S)-N-(1-phenylethyl)glycine, N-cyclohexylmethylglycine, N-(2-phenylethyl)glycine, N-(3-phenylpropyl)glycine, N-(6-aminogalactosyl)glycine, N-(2-(3'-indolylethyl)glycine, N-(2-(p-methoxyphenylethyl))glycine, N-(2-(p-chlorophenylethyl))glycine, and N-[2-(p-hydroxyphenylethyl)]glycine. N-substituted glycine oligomers, referred to herein as "peptoids," have been shown to be protease resistant (Miller et al., Drug Dev. Res., 35:20-32 (1995). Thus, peptoids comprising at least one unnatural α-amino acid, D-amino acid, or a combination thereof are within the scope of the present invention.

[0125] Suitable N-methyl amino acids include N-methyl-Ala, N-methyl-Cys, N-methyl-Asp, N-methyl-Glu, N-methyl-Phe, N-methyl-Gly, N-methyl-His, N-methyl-Ile, N-methyl-Arg, N-methyl-Lys, N-methyl-Leu, N-methyl-Met, N-methyl-Asn, N-methyl-Gln, N-methyl-Ser, N-methyl-Thr, N-methyl-Val, N-methyl-Trp, N-methyl-Tyr, N-methyl-Acp, N-methyl-Acb, N-methyl-Acpc, N-methyl-Cit, N-methyl-HoCit, N-methyl-Aad, N-methyl-4-Pa l, N-methyl-3-Pal, N-methyl-Pra, N-methyl-Aib, N-methyl-Abu, N-methyl-Nva, N-methyl-Dpr, N-methyl-Dbu, N-methyl-Nle, N-methyl-Nal-2, N-methyl-Nal-1, N-methyl-Cha, N-methyl-Cpa, N-methyl-Hle, N-methyl-HoSer, N-methyl-Har, N-methyl-Hcy, N-methyl-Chg, N-methyl-Bta, N-methyl-2-Thi, N-methyl-3-Thi, N-methyl-Asu, N-methyl-Acdt, N-methyl-Ahch, N-methyl-Akch, N-methyl-Actp, N-methyl-Ty r(3-NO2), N-methyl-Ach, N-methyl-3-Apc, N-methyl-4-Apc, N-methyl-4-App, N-methyl-Tha, N-methyl-Aoa, N-methyl-Aha, N-methyl-Orn, N-methyl-Aca, N-methyl-Agl, N-methyl-Cab, N-methyl-2-Pal, N-methyl-Cba, N-methyl-HoPhe, N-methyl-Phg, N-methyl-Phe(4-NH2), N-methyl-4-Phe(4-Me), N-methyl-Phe(4-F), N-methyl-Phe(4-Cl), N-methyl-Phe(2-Br), N-methyl-Phe(3-Br), N-methyl N-methyl-Phe(4-Br), N-methyl-Phe(3-CF3), N-methyl-Phe(4-CF3), N-methyl-Phe(4-NO2), N-methyl-Phe(4-CN), N-methyl-Bpa, N-methyl-Phg(4-Cl), N-methyl-Phg(4-Br), N-methyl-Tyr(Me), N-methyl-Lys38, N-methyl-Lys27, N-methyl-Lys73, N-methyl-Lys55, N-methyl-Lys28, N-methyl-Lys72, N-methyl-Lys12, N-methyl-Lys123, N-methyl-Lys63, N-methyl-Lys124, N-methyl-Lys82,N-methyl-Lys31, N-methyl-Lys15, N-methyl-Lys125, N-methyl-Lys43, N-methyl-Lys24, N-methyl-Lys5, N-methyl-Lys4, N-methyl-Lys50, N-methyl-Lys81, N-methyl-Orn38, N-methyl-Orn27, N-methyl-Orn73, N-methyl-Orn55, N-methyl-Orn28, N-methyl-Orn72, N-methyl-Orn12, N-methyl-Orn123, N-methyl-Orn63, N-methyl-Orn124, N-methyl-Orn82, N-methyl-Orn31, N-methyl-Orn15, N-methyl-Orn125, N-methyl-Orn43, N-methyl-Orn24 、N-methyl-Orn5、N-methyl-Orn4、N-methyl-Orn50、N-methyl-Orn81、N-methyl-Dbu38、N-methyl-Dbu27、N-methyl-Dbu73、N-methyl-Dbu55、N-methyl-Dbu28、N-methyl-Dbu72、N-methyl-Dbu12、N-methyl-Dbu123、N-methyl-Dbu63、N-methyl-Dbu124、N-methyl-Dbu82、N-methyl-Dbu31、N-methyl-Dbu15、N-methyl-Dbu125、N-methyl-Dbu43、N-methyl-Dbu24、N-methyl-Dbu5、N-methyl-Dbu4、N-methyl-Dbu50、N-methyl-Dbu81、stereoisomers thereof, and combinations thereof. 、

[0126] Suitable phenylalanine analogs that can be used in the present invention include, but are not limited to, homophenylalanine (HoPhe), phenylglycine (Phg), 3,3-diphenylalanine (Dpa), 4-aminophenylalanine (Phe(4-NH2)), 2-methylphenylalanine (Phe(2-Me)), 3-methylphenylalanine (Phe(3-Me)), 4-methylphenylalanine (Phe(4-Me)), 4-azidophenylalanine (Phe(4-N3)), 2-fluorophenylalanine (Phe(2-F)), 3-fluorophenylalanine (Phe(3-F)), 4-fluorophenylalanine (Phe(4-N3)). Phe(4-F)), 2-chlorophenylalanine (Phe(2-Cl)), 3-chlorophenylalanine (Phe(3-Cl)), 4-chlorophenylalanine (Phe(4-Cl)), 2-bromophenylalanine (Phe(2-Br)), 3-bromophenylalanine (Phe(3-Br)), 4-bromophenylalanine (Phe(4-Br)), 2-iodophenylalanine (Phe(2-I)), 3-iodophenylalanine (Phe(3-I)), 4-iodophenylalanine (Phe(4-I)), 2-trifluoromethylphenylalanine (Phe(2-CF3)), 3-trifluoromethylphenylalanine (P ... Alanine (Phe (3-CF3)), 4-trifluoromethylphenylalanine (Phe (4-CF3)), 2-methoxyphenylalanine (Phe (2-OMe)), 3-methoxyphenylalanine (Phe (3-OMe)), 2-nitrophenylalanine (Phe (2-NO2)), 3-nitrophenylalanine (Phe (3-NO2)), 4-nitrophenylalanine (Phe (4-NO2)), 2-cyanophenylalanine (Phe (2-CN)), 3-cyanophenylalanine (Phe (3-CN)), 4-cyanophenylalanine (Phe (4-CN)), 3, 4-dimethoxyphenylalanine (Phe(3,4-diOMe)), 3,4-difluorophenylalanine (Phe(3,4-diF)), 3,5-difluorophenylalanine (Phe(3,5-diF)), 2,4-dichlorophenylalanine (Phe(2,4-diCl)), 3,4-dichlorophenylalanine (Phe(3,4-diCl)), 4-benzoylphenylalanine (Bpa), 4-carboxyphenylalanine (Phe(4-COOH)), 4,4'-diphenylalanine (Bip), 2,3,4,5,6-pentafluorophenylalanine (Phe(F5)), 3,4,5-trifluorophenylalanine (Phe(F3)), 4-chlorophenylglycine (Phg(4-Cl)), 2-chlorophenylglycine (Phg(2-Cl)), 3-chlorophenylglycine (Phg(3-Cl)), 4-bromophenylglycine (Phg(4-Br)), 2-bromophenylglycine (Phg(2-Br)), 3-bromophenylglycine (Phg(3-Br)), 4-ethylphenylalanine (Phe(4-Et)), 4- Ethoxyphenylalanine (Phe(4-OEt)), 4-butoxyphenylalanine (Phe(4-OBu)), O-methyltyrosine (Tyr(Me)), O-benzyltyrosine (Tyr(Bzl)), 3,5-dibromotyrosine (Tyr(diBr)), 3,5-diiodotyrosine (Tyr(diI)), homotyrosine (HoTyr), 3-chlorotyrosine (Tyr(3-Cl)), stereoisomers thereof, and combinations thereof.

[0127] Suitable derivatives of lysine (Lys), ornithine (Orn) and α,γ-diaminobutyric acid (Dbu) that can be used in the present invention include, but are not limited to, Lys38, Lys27, Lys73, Lys55, Lys28, Lys72, Lys12, Lys123, Lys63, Lys124, Lys82, Lys31, Lys15, Lys125, Lys43, Lys24, Lys5, Lys4, Lys50, Lys81, Orn38, Orn27, Orn73, Orn55, Orn28, Orn72, Orn1 2, Orn123, Orn63, Orn124, Orn82, Orn31, Orn15, Orn125, Orn43, Orn24, Orn5, Orn4, Orn50, Orn81, Dbu38, Dbu27, Dbu73, Dbu55, Dbu28, Dbu72, Dbu12, Dbu123, Dbu63, Dbu124, Dbu82, Dbu31, Dbu15, Dbu125, Dbu43, Dbu24, Dbu5, Dbu4, Dbu50, Dbu81, stereoisomers thereof, and combinations thereof. The derivatives of Orn and Dbu are similar to lysine derivatives having corresponding carboxylic acids connected to the side chains of Orn and Dbu, respectively. The structures of Lys, Orn and Dbu derivatives are disclosed in U.S. Patent No. 7,576,175, which is incorporated herein by reference.

[0128] Suitable hydrophobic amino acids for use in the present invention include, but are not limited to, leucine (Leu), leucine analogs, phenylalanine (Phe), phenylalanine analogs, proline (Pro), proline analogs, valine (Val), isoleucine (Ile), glycine (Gly), alanine (Ala), Met, norvaline (Nva), norleucine (Nle), 1-aminocyclopropane-1-carboxylic acid (Acpc), 1-aminocyclobutane-1-carboxylic acid (Acb), α-cyclohexylglycine (Chg), cyclohexylalanine (Cha), propargylglycine (Pra), cyclopropylalanine (Cpa), homoleucine (Hle), α-aminoisobutyric acid (Aib), α-aminobutyric acid (Abu), 3-(2-thienyl)alanine (2-Thi), 3-(3-thienyl)alanine (3-Thi), 3-(3-pyridyl)alanine (3-Pa l), 3-(2-naphthyl)alanine (Nal-2), 2-amino-2-naphthylacetic acid (Ana), tyrosine (Tyr), 3,5-dinitrotyrosine (Tyr(3,5-diNO2)), diethylglycine (Deg), 4-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran (Acdt), 1-amino-1-(4-hydroxycyclohexyl)carboxylic acid (Ahch), 1-amino-1-(4-ketocyclohexyl)carboxylic acid (Ahch), Acid (Akch), 4-amino-4-carboxytetrahydropyran (Actp), 3-nitrotyrosine (Tyr(3-NO2)), 1-amino-1-cyclohexanecarboxylic acid (Ach), 2-aminoindan-2-carboxylic acid (Aic), (2S, 5R)-5-phenylpyrrolidine-2-carboxylic acid (Ppca), 4-thiazolylalanine (Tha), 2-aminooctanoic acid (Aoa), 2-aminoheptanoic acid (Aha), and stereoisomers thereof. In some embodiments, the proline analog is hydroxyproline.

[0129] In some embodiments, the hydrophobic amino acid is selected from the compounds of Table 2:

[0130] Table 1. Hydrophobic amino acids useful in the present invention.

[0131]

[0132] In some embodiments, the hydrophobic amino acid is selected from the compounds of Table 3:

[0133] Table 3. Hydrophobic amino acids useful in the present invention.

[0134]

[0135] Suitable hydrophilic amino acids that can be used in the present invention include, but are not limited to, Asn, Ser, Thr, Gln, and stereoisomers thereof. Suitable positively charged amino acids that can be used in the present invention include, but are not limited to, Lys, Arg, His, HoArg, Agp, Agb, Dab, Dap, Orn, and stereoisomers thereof. Suitable negatively charged amino acids that can be used in the present invention include, but are not limited to, aspartic acid, glutamic acid, α-aminoadipic acid, α-aminosuberic acid, homoaspartic acid, γ-carboxy-glutamic acid, 4-carboxyphenylalanine, and stereoisomers thereof. In other embodiments, negatively charged amino acids are selected from Aad, Bec, and Bmc.

[0136] In some embodiments, X 1 Can be an unnatural amino acid, a hydrophobic amino acid, a positively charged amino acid or a negatively charged amino acid. In some embodiments, X 2 X can be a hydrophilic amino acid, a hydrophobic amino acid, or a negatively charged amino acid. 2 X may be a negatively charged amino acid residue, a hydrophilic amino acid residue, a hydroxyproline (Hyp) residue, or a 1-amino-1-cyclohexanecarboxylic acid (Ach) residue. 3 It can be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid. In some embodiments, when the subscript m is 2, and the part -(X 1 ) m -For –X 1a -X 1b -When, X 1a can be an unnatural amino acid, a hydrophobic amino acid or a negatively charged amino acid; and X 1b It can be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid.

[0137] In some embodiments, X 4 Selected from N 6 -(3-(pyridin-3-yl)propionyl)-lysine (Lys12), (E)-N 6 -(3-(pyridin-3-yl)acryloyl)-lysine (Lys38), citrulline (Cit), homocitrulline (HoCit), leucine (Leu), N-methyl-leucine (N-MeLeu), isoleucine (Ile), N-methyl-isoleucine (N-MeIle), and homophenylalanine (HoPhe). In some embodiments, X 4 Is citrulline (Cit) or N 6 -(3-(Pyridin-3-yl)propionyl)-lysine (Lys12).

[0138] In some embodiments, the compound has a structure according to Formula Ia:

[0139]

[0140] or a pharmaceutically acceptable salt thereof.

[0141] In some embodiments, the compound has a structure according to Formula Ib:

[0142]

[0143] In some embodiments, the compound has a structure according to Formula Ic:

[0144]

[0145] In some embodiments, the compound has a structure according to Formula II:

[0146]

[0147] or a pharmaceutically acceptable salt thereof.

[0148] In some embodiments, the present invention provides compounds of Formula I, Formula Ia, Formula Ib, Formula Ic, or Formula II, wherein X 1 It can be an unnatural amino acid, a hydrophobic amino acid, a positively charged amino acid or a negatively charged amino acid; X 2 Can be a hydrophilic amino acid, a hydrophobic amino acid, or an amino acid with a negative charge; X 3 can be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid; and X 4 Can be N 6 -(3-(pyridin-3-yl)propionyl)-lysine (Lys12), (E)-N 6 -(3-(pyridin-3-yl)acryloyl)-lysine (Lys38), citrulline (Cit), homocitrulline (HoCit), leucine (Leu), N-methyl-leucine (N-MeLeu), isoleucine (Ile), N-methyl-isoleucine (N-MeIle) and homophenylalanine (HoPhe). In some embodiments, when the subscript m is 2, and the moiety -(X 1 ) m -For –X 1a -X 1b -When, X 1a can be an unnatural amino acid, a hydrophobic amino acid or a negatively charged amino acid; and X 1b It can be a hydrophilic amino acid, a hydrophobic amino acid or a negatively charged amino acid.

[0149] In some embodiments, the present invention provides compounds of Formula I, Formula Ia, Formula Ib, Formula Ic, or Formula II, wherein X 1 X may be proline (Pro), α-aminoadipic acid (Aad), valine (Val), arginine (Arg), 1-amino-1-(4-piperidinyl)carboxylic acid (4-Apc), D-histidine (D-His), D-O-methyltyrosine (D-Tyr(Me)), ornithine (Orn), α-aminoisobutyric acid (Aib), tyrosine (Tyr), homocitrulline (HoCit), norleucine (Nle), aspartic acid (Asp), isoleucine (Ile) or D-leucine (D-Leu); 2 X may be aspartic acid (Asp), isoleucine (Ile), serine (Ser), glutamic acid (Glu), D-glutamic acid (D-Glu), D-propargylglycine (D-Pra), 1-amino-1-cyclohexanecarboxylic acid (Ach), 1-aminocyclopropane-1-carboxylic acid (Acpc), 4-methylphenylalanine (Phe(4-Me)), cyclohexylalanine (Cha), α-aminoadipic acid (Aad), hydroxyproline (Hyp), 2-aminoindan-2-carboxylic acid (Aic), glutamine (Gln), or D-α-cyclohexylglycine (D-Chg); 3 can be glutamic acid (Glu), aspartic acid (Asp), α-aminoadipic acid (Aad), isoleucine (Ile), serine (Ser) or norleucine (Nle); and X 4 Can be N 6 -(3-(pyridin-3-yl)propionyl)-lysine (Lys12), (E)-N 6 -(3-(pyridin-3-yl)acryloyl)-lysine (Lys38), citrulline (Cit), homocitrulline (HoCit), leucine (Leu), N-methyl-leucine (N-MeLeu), isoleucine (Ile), N-methyl-isoleucine (N-MeIle) and homophenylalanine (HoPhe). In some embodiments, when the subscript m is 2, and the moiety -(X 1 ) m -For –X 1a -X 1b -When, X 1a can be homocitrulline (HoCit), isoleucine (Ile), D-leucine (D-Leu) or aspartic acid (Asp); and X 1b It may be aspartic acid (Asp), threonine (Thr) or D-methyltyrosine (D-Tyr(Me)).

[0150] In some embodiments, the present invention provides compounds of Formula I, Formula Ia, Formula Ib, Formula Ic, or Formula II, wherein the subscript m is 0, and the moiety -X 4 -X 3 -X 2 – is –Lys12-Aad-Ach–, –Ile-Glu-Acpc– or –HoPhe-Nle-Glu–. In some embodiments, the present invention provides a compound of Formula I, Formula Ia, Formula Ib or Formula II, wherein the subscript m is 1, and the moiety –X 4 -X 3 -X 2 -X 1 – for –N-MeIle-Glu-Asp-Pro–, –Lys12-Glu-Ile-Aad–, –Cit-Glu-Ser-Val–, –Lys12-Glu-Glu-Arg –, –N-MeLeu-Asp-D-Pra-4-Apc–, –Lys38-Glu-Ile-Aad–, –Lys38-Glu-Glu-Arg–, –HoCit-Ile-P he(4-Me)-D-His–, –Leu-Ser-Cha-D-Tyr(Me)–, –HoCit-Glu-Glu-Orn–, –Ile-Glu-Aad-Aib–, –Lys38-Aad-Phe(4-Me)-Tyr–, –Lys38-Aad-Aic-Nle–, –Ile-Aad-Ser-Asp–, or –Ile-Aad-Ser-Tyr–. In some embodiments, the present invention provides a compound of Formula I, Formula Ia, Formula Ib, Formula Ic, or Formula II, wherein the subscript m is 2, and the moiety –X 4 -X 3 -X 2 -X 1a -X 1b – is –Lys12-Aad-Hyp-HoCit-Asp–, –HoCit-Aad-Gln-Ile-Asp–, –HoCit-Aad-D-Glu-D-Leu-Thr–, or –HoCit-Aad-D-Chg-Asp-D-Tyr(Me)–. In some embodiments, the present invention provides a compound of Formula I, Formula Ia, or Formula II, or a pharmaceutically acceptable salt thereof, wherein the subscript m is 0, and the moiety –X 4 -X 3 -X 2 - is -Lys12-Aad-Ach-. In some embodiments, the present invention provides a compound of Formula I, Formula Ia or Formula II or a pharmaceutically acceptable salt thereof, wherein the subscript m is 1, and the moiety -X 4 -X 3-X 2 -X 1 – is –Cit-Glu-Ser-Val–.

[0151] In some embodiments, the compound of Formula Ib is selected from:

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] In some embodiments, the compound of Formula Ic is selected from:

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] The L part of formula I, formula Ia and formula II can be any suitable joint, including but not limited to joints with one or more different reactive functional groups, which allow parts such as peptides, monomers and polymers to be covalently connected. The connecting part may have two or more different reactive functional groups. In some cases, a multivalent joint can be used, and a variety of peptides and / or a variety of active agents of the present invention can be connected via a joint. Suitable joints include but are not limited to those available from Pierce Biotechnology, Inc. (Rockford, IL). It is understood by those skilled in the art that any reactive functional group may be present on the joint, as long as it is compatible with the functional group on the part to be covalently connected. Some suitable joints include but are not limited to β-alanine, 2,2'-ethylenedioxybis(ethylamine)monosuccinamide (Ebes), bis(Ebes)-Lys and polyethylene glycol. Other suitable joints include affinity-based joints (e.g., joints comprising biotin) that bind to the part via non-covalent interactions. Other suitable linkers include cleavable linkers, such as disulfide linkers, such as 4-((2-((2-aminoethyl)disulfanyl)ethyl)amino)-4-oxobutanoic acid, which can be cleaved under reducing conditions, or peptide linkers that can be cleaved by the action of proteases.

[0165] Those of ordinary skill in the art will recognize that other joints are also feasible for the compounds of the present invention. Many such joints can be found in "Bioconjugate Techniques", Academic Press, San Diego, 1996 by Greg T Hermanson, or prepared by the techniques described therein, which are incorporated herein by reference. In addition, those of ordinary skill in the art will recognize that other joints can be prepared based on the Click chemical synthesis technology described in Kolb, HC, Finn, MG, Sharpless, KB, Angew. Chem. Int'l. Ed. 40 (11): 2004–2021 (2001), which are incorporated herein by reference. Joints that can be used in the present invention include those based on Ebes and PEG moieties. The joint may include 0 to 6 Ebes or PEG groups. Ebes and PEG groups can be conjugated by the techniques cited above. In some embodiments, the joint L of Formula I, Formula Ia, and Formula II includes at least one of N-(8-amino-3,6-dioxa-octyl) succinamic acid (Ebes) and polyethylene glycol (PEG).

[0166] In some other embodiments, the linker L of Formula I, Formula Ia, and Formula II is selected from:

[0167]

[0168]

[0169]

[0170]

[0171] Where k is 0 to 6.

[0172] The D part of formula I, formula Ia and formula II can be any suitable phosphonate drug, including but not limited to monophosphonate, bisphosphonate and triphosphonate. In some embodiments, the D part of formula I, formula Ia and formula II is a phosphonate or bisphosphonate compound. In some embodiments, the D part of formula I, formula Ia and formula II is a bisphosphonate compound. In some embodiments, the D part is a bisphosphonate compound, such as but not limited to etidronate (Didronel), clodronate (Bonefos, Loron), tiludronate (Skelid), pamidronate (APD, Aredia), neridronate, olpadronate, alendronate (Fosamax), ibandronate (Boniva), risedronate (Actonel) and zoledronate (Zometa). Other bisphosphonates are described in more detail below. Those skilled in the art will appreciate that other bisphosphonates can also be used in the present invention.

[0173] In some embodiments, the D moiety of Formula I, Formula Ia, and Formula II has the following formula:

[0174]

[0175] Where R 5 is selected from H, OH and halogen; R 6 Selected from H and C 1-6 alkyl; and the subscript t is 1 to 6.

[0176] In some other embodiments, the D moiety of Formula I, Formula Ia, and Formula II has a formula selected from:

[0177]

[0178] In some embodiments, the compound of Formula I, Formula Ia, and Formula II is selected from:

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] In some embodiments, the compound of Formula I, Formula Ia, and Formula II is selected from:

[0185]

[0186] In some embodiments, salts, hydrates, solvates, prodrug forms, isomers, and metabolites of compounds of Formula I, Formula Ia, and Formula II are provided.

[0187] In some embodiments, the present invention provides isomers or pharmaceutically acceptable salts of compounds of Formula I, Formula Ia and Formula II. Salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, phosphonic acids, isonicotinates, lactates, salicylates, citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Other salts include, but are not limited to, salts with inorganic bases, including alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; aluminum salts; and ammonium salts such as ammonium, trimethyl-ammonium, diethylammonium and tris-(hydroxymethyl)-methyl-ammonium salts. Other salts with organic bases include salts with diethylamine, diethanolamine, meglumine and N,N'-dibenzylethylenediamine. Acid addition salts, such as inorganic acids, organic carboxylic acids and organic sulfonic acids, such as hydrochloric acid, methanesulfonic acid, maleic acid are also possible, provided that a basic group such as pyridyl forms part of the structure.

[0188] Neutral forms of compounds of Formula I, Formula Ia and Formula II can be regenerated by contacting a pharmaceutically acceptable salt of Formula I, Formula Ia or Formula II with a base or acid and isolating the parent compound in a conventional manner. The parent forms of compounds of Formula I, Formula Ia and Formula II differ from the various salt forms in certain physical properties (e.g., solubility in polar solvents), but in other respects, these salts are equivalent to the parent forms of the compounds for the purposes of the present invention.

[0189] Some compounds of the present invention may exist in non-solvated form and solvated form (including hydrated form). Typically, the solvated form is equivalent to the non-solvated form and is encompassed within the scope of the present invention. Some compounds of the present invention may exist in multiple crystalline forms or amorphous forms. Typically, all physical forms are equivalent for the intended use of the present invention and are intended to fall within the scope of the present invention.

[0190] Certain compounds of the invention possess asymmetric carbon atoms (optical centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms, which can be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids and, individual isomers are encompassed within the scope of the invention. The compounds of the invention do not include those known in the art to be too unstable to be synthesized and / or isolated. The invention is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0191] The present invention also provides compounds in the form of prodrugs. Prodrugs of the compounds described herein are those compounds that are susceptible to chemical changes under physiological conditions to provide compounds of the present invention. In addition, prodrugs can be converted into compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, when the prodrug is placed in a transdermal patch reservoir together with a suitable enzyme or chemical reagent, the prodrug can be slowly converted into a compound of the present invention.

[0192] Compound of the present invention can be synthesized by multiple methods known to those skilled in the art (see Comprehensive Organic Transformations of Richard C.Larock, 1989) or by appropriate combinations of commonly known synthetic methods. The technology that can be used to synthesize compound of the present invention is obvious to those skilled in the art, and is easily available. The following discussion is provided to illustrate some methods that can be used to make multiple methods of the compounds of the present invention. However, this discussion is not intended to limit the scope of the reaction or reaction sequence that can be used to prepare the compounds of the present invention. It will be appreciated by those skilled in the art that other methods of preparing compounds can be used for the present invention.

[0193] The "one-bead one-compound" (OBOC) combinatorial library approach was first reported in 1991 (Lam et al., Nature, 1991, 354: pp. 82-4). Essentially, when the "split-mix" synthesis method (Lam et al., id; Houghten et al., Nature, 1991, 354: pp. 84-6; Furka et al., Int. J. Peptide Protein Res., 1991, 37: pp. 487-93) is used to generate combinatorial libraries, each bead expresses only one chemical entity (Lam et al., id; Lam et al., Chem. Rev., 1997, 97: pp. 411-48). Random libraries of millions of beads can then be screened in parallel for specific receptor molecules (e.g., receptors, antibodies, enzymes, viruses, whole cells, etc.). Positive beads were physically separated for structure determination by microsequencing using automated Edman degradation (Lam et al., Nature, 1991, 354: p. 82-4).

[0194] The one-bead-one-compound (OBOC) combinatorial library approach synthesizes millions of compounds so that each bead displays only one compound. An example of a compound identified by the OBOC combinatorial library approach is LLP2A, which specifically binds to integrin α4β1 (IC 50 =2pM). LLP2A, when conjugated with a near-infrared fluorescent dye, can be used to image cells expressing α4β1 with high sensitivity and specificity to direct therapeutic compounds to lymphomas expressing α4β1 (Peng, L., et al., Nat Chem Biol, 2006, 2(7): pp. 381-9). This ligand is known to direct compounds to lymphomas expressing α4β1 (Peng, L., et al., Nat Chem Biol, 2006, 2(7): pp. 381-9; Peng, L., et al., Mol Cancer Ther, 2008, 7(2): pp. 432-7; Aina, OH, et al., Mol Pharm, 2007. 4(5): pp. 631-51; Aina, OH, et al., Mol Cancer Ther, 2005. 4(5): pp. 806-13).

[0195] The compounds described herein can be used in combination with other active agents known to be useful for treating osteoporosis or promoting bone growth, or can be used in combination with adjuvants that may be ineffective alone but can contribute to the efficacy of the active agent. In some embodiments, the co-administration of the compound with other agents herein includes administering an active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20 or 24 hours of the second active agent. Co-administration includes administering two active agents simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20 or 30 minutes between each other) or in any order. In some embodiments, co-administration can be accomplished by co-preparation, that is, preparing a single pharmaceutical composition including two active agents. In other embodiments, the active agent can be prepared separately. In another embodiment, the active agent and / or the adjuvant can be connected or conjugated to each other.

[0196] III. Pharmaceutical Compositions and Methods of Administration

[0197] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient.

[0198] The compounds of the present invention can be prepared and administered in a variety of oral, parenteral and topical dosage forms. Oral preparations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc. suitable for ingestion by patients. The compounds of the present invention can also be administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intraduodenal or intraperitoneal. Similarly, the compounds described herein can be administered by inhalation, such as intranasal administration. In addition, the compounds of the present invention can be administered transdermally. The compounds of the present invention can also be administered by the following: intraocular, intravaginal and intrarectal routes (including suppositories, insufflations, powders and aerosols) (e.g., steroid inhalers, see Rohatagi, J. Clin. Pharmacol. 35: 1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75: 107-111, 1995).

[0199] The composition typically comprises a conventional pharmaceutical carrier or excipient, and may additionally comprise other drugs, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, etc. In some embodiments, the composition will comprise from about 0.01% by weight to about 90% by weight (e.g., from about 0.1% by weight to about 75% by weight, or from about 0.1% by weight to about 50% by weight, or from about 0.1% by weight to about 10% by weight) of the peptidomimetic ligand compound, with the remainder being made up of a suitable pharmaceutical carrier and / or excipient. Suitable excipients may be customized for specific compositions and for example for routes of administration of methods well known in the art described by the above-mentioned Remington.

[0200] For oral administration, the composition can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders and sustained-release formulations. Suitable excipients for oral administration include pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0201] In some embodiments, the pharmaceutical composition takes the form of a pill, tablet or capsule, and thus, the composition may include any of the following together with the ligand or a combination of ligands: a diluent, such as lactose, sucrose, dicalcium phosphate, etc.; a disintegrant, such as starch or its derivatives; a lubricant, such as magnesium stearate, etc.; and a binder, such as starch, gum arabic, polyvinyl pyrrolidone, gelatin, cellulose and its derivatives. The ligand may also be formulated as a suppository placed in a carrier such as polyethylene glycol (PEG).

[0202] Liquid compositions can be prepared by dissolving or dispersing the ligand or combination of ligands and optionally one or more pharmaceutically acceptable excipients in a carrier such as saline (e.g., 0.9% w / v sodium chloride), aqueous dextrose, glycerol, ethanol, etc. to form a solution or suspension, e.g., for oral, topical or intravenous administration. The ligands of the invention can also be formulated as retention enemas.

[0203] For topical application, the composition of the present invention can be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments and transdermal patches. In order to deliver by inhalation, the composition can be delivered in dry powder or liquid form by a sprayer. For parenteral administration, the composition can be in the form of sterile injectable solutions and sterile packaged powders. In some embodiments, injectable solutions can be prepared at a pH of about 4.5 to about 7.5.

[0204] The compositions of the present invention may also be provided in lyophilized form. Such compositions may include a buffer, such as bicarbonate, for reconstitution prior to administration, or the buffer may be included in a lyophilized composition reconstituted, for example, with water. The lyophilized composition may also include a suitable vasoconstrictor, such as epinephrine. The lyophilized composition may be provided in a syringe, optionally packaged with a buffer for reconstitution, so that the reconstituted composition may be administered immediately to the patient.

[0205] In powders, the carrier is a finely divided solid with which the finely divided active ingredient is mixed. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted into the desired shape and size. Suitable solid excipients include, but are not limited to, magnesium carbonate; magnesium stearate; calcium phosphate; calcium silicate; talc; pectin; dextran, dextrin and cyclodextrin inclusion complexes; low melting waxes; cocoa butter; carbohydrates; sugars, including but not limited to lactose, dextrose, sucrose, mannitol or sorbitol; starches, including but not limited to those from corn, wheat, rice, potato or other plants; celluloses, such as methylcellulose, hydroxypropylmethyl-cellulose or sodium carboxymethylcellulose; and gums, including gum arabic, tragacanth and acacia; and proteins, including but not limited to gelatin, collagen; microcrystalline cellulose, water, saline, syrup, ethylcellulose and polyacrylic acids, such as Carbopol, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.; lubricants; mineral oils; wetting agents; emulsifiers; suspending agents; preservatives, such as methyl-hydroxy-benzoate, ethyl-hydroxy-benzoate and propyl-hydroxy-benzoate (i.e., p-hydroxybenzoate); pH adjusters, such as inorganic and organic acids and bases; sweeteners; and flavoring agents; biodegradable polymer beads. If necessary, disintegrators or solubilizers, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, alginate or its salt, such as sodium alginate, can be added.

[0206] The pharmaceutically acceptable carrier may include a physiologically acceptable compound that acts, for example, to stabilize the compound of the present invention or to regulate its absorption, or other excipients as needed. Physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. Those skilled in the art will appreciate that the selection of pharmaceutically acceptable carriers including physiologically acceptable compounds depends, for example, on the route of administration of the compound of the present invention and the specific physiological and chemical characteristics of the compound of the present invention. Typically, such carriers are nontoxic to the recipient at the dose and concentration employed. Typically, the preparation of such compositions requires the therapeutic agent to be combined with a buffer, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates (including glucose, maltose, sucrose or dextrin), chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin is an exemplary appropriate diluent.

[0207] The dragee cores are provided with a suitable coating, for example a concentrated sugar solution, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or dragee coating for product identification or to characterize the amount (i.e., dosage) of the active compound. The pharmaceutical preparations of the invention may also be used orally using, for example, push-fit capsules made of gelatin and soft sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules may contain the compounds of the invention mixed with fillers or binders (e.g., lactose or starch), lubricants (e.g., talc or magnesium stearate) and optional stabilizers. In soft capsules, the compounds of the invention may be dissolved or suspended in a suitable liquid (e.g., fatty oils, liquid paraffin or liquid polyethylene glycol with or without stabilizers).

[0208] Some sustained release embodiments include biodegradable and / or slowly dissolving polymeric substances. Such polymeric substances include polyvinyl pyrrolidone, low molecular weight and medium molecular weight hydroxypropyl cellulose and hydroxypropyl methylcellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl starch, potassium methacrylate divinylbenzene copolymer, polyvinyl alcohol, starch, starch derivatives, microcrystalline cellulose, ethyl cellulose, methyl cellulose and cellulose derivatives, β-cyclodextrin, poly (methyl vinyl ether / maleic anhydride), dextran, scierozlucans, mannan, xanthan gum, alginic acid and its derivatives, dextrin derivatives, glyceryl monostearate, semi-synthetic glycerides, glyceryl palmitostearate, glyceryl behenate, polyvinyl pyrrolidone, gelatin, magnesium stearate, stearic acid, sodium stearate, talc, sodium benzoate, boric acid and colloidal silicon dioxide.

[0209] The sustained-release agent of the present invention can also include auxiliary materials, such as starch, pregelatinized starch, calcium phosphate mannitol, lactose, sucrose, glucose, sorbitol, microcrystalline cellulose, gelatin, polyvinyl pyrrolidone, methylcellulose, starch solution, ethylcellulose, gum arabic, tragacanth, magnesium stearate, stearic acid, colloidal silicon dioxide, glyceryl monostearate, hydrogenated castor oil, wax and mono-, di- and tri-substituted glycerides. The sustained-release agent can also be prepared according to the general description in WO94 / 06416.

[0210] To prepare suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active ingredient is dispersed uniformly therein by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and then to solidify.

[0211] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0212] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavoring agents, stabilizers and thickeners as needed. Aqueous suspensions suitable for oral use can be prepared by dispersing the fine active ingredient in water with the following materials: viscous substances such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum arabic, and dispersants or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of oxyalkylene with fatty acids (e.g., polyoxyethylene stearate), condensation products of oxyethylene with long-chain aliphatic alcohols (e.g., heptadecanethenoxycetanol), condensation products of oxyethylene with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate) or condensation products of oxyethylene with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose, aspartame or saccharin. The osmolarity of the formulation may be adjusted.

[0213] Also included are solid form preparations that are intended to be converted into liquid form preparations for oral administration before use. Such liquid forms include solutions, suspensions and emulsions. These preparations can also include, for example, coloring agents, correctives, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc., except that the active ingredient.

[0214] Oily suspensions can be prepared by suspending the compound of the present invention in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or in a mineral oil (e.g., liquid paraffin) or a mixture of these. Oily suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners (e.g., glycerol, sorbitol, or sucrose) may be added to provide a palatable oral preparation. These preparations may be preserved by adding antioxidants such as ascorbic acid. As an example of an injectable oil carrier, see Minto, J.Pharmacol.Exp.Ther.281:93-102,1997. The pharmaceutical preparations of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil or a mineral oil as described above, or a mixture of these. Suitable emulsifiers include naturally occurring gums such as gum arabic and gum tragacanth; naturally occurring phospholipids such as soy lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweeteners and flavoring agents, such as syrups and elixir formulations. Such formulations may also contain a demulcent, a preservative, or a coloring agent.

[0215] In some embodiments, the pharmaceutical preparation is a unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human individuals and other mammals (e.g., dogs), each unit containing a predetermined amount of active substance (e.g., ampoule) calculated to produce the desired onset, tolerance, and / or therapeutic effect together with a suitable pharmaceutical excipient. In addition, a composition of higher concentration can be prepared, from which a more diluted unit dose composition can then be prepared. Therefore, a composition of higher concentration will contain substantially more than, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times the amount of ligand or ligand combination. In such a form, the preparation is subdivided into a unit dose containing an appropriate amount of active ingredient. The unit dosage form can be a packaged preparation, and the packaging contains a discrete amount of preparations, such as packaged tablets, capsules, and powders in vials or ampoules. Similarly, the unit dosage form itself can be a capsule, tablet, cachet, or lozenge, or can be an appropriate number of any of these in packaged form. If desired, the composition can also contain other compatible therapeutic agents. Certain pharmaceutical formulations may deliver the compounds of the invention as sustained release formulations.

[0216] IV. Administration and Methods of Treatment

[0217] Can be used as required with suitable pharmaceutical excipient by part of the present invention by any acceptable mode of administration.Therefore, use can be for example intravenous, local, subcutaneous, percutaneous, transdermal, intramuscular, oral, intraarticular, parenteral, intraarteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectum, vaginal or by suction.Also can be directly applied to bone surface and / or applied to the tissue around bone.Preparation can take above-mentioned any solid form, semisolid form or liquid dosage form, such as, for example tablet, pill, capsule, powder, solution, suspension, emulsion, suppository, retention enema, cream, ointment, emulsion, gel, aerosol etc.

[0218] Typically, the pharmaceutical preparation is delivered to mammals, including humans and non-human mammals. Non-human mammals treated using the methods of the present invention include domestic animals (i.e., dogs, cats, mice, rodents, and lagomorphs) and agricultural animals (cattle, horses, sheep, pigs). Local increase in bone can be used for fracture healing, fusion (arthrodesis), orthopedic reconstruction, and periodontal repair. Systemic increase in bone can be used to treat low bone mass, i.e., osteopenia.

[0219] Typically, the dosage will be effectively delivered to one or more suitable sites by picomolar to micromolar concentrations of the ligand. However, it will be appreciated by those of ordinary skill in the art that the dosage administered will vary depending on many factors, including but not limited to the specific ligand or ligand group to be administered, mode of administration, type of administration (e.g., imaging, treatment), age of the patient, and the patient's physical condition. Preferably, the minimum dose and concentration required to produce the desired result should be used. For children, the elderly, weak patients, and patients suffering from heart and / or liver disease, the dosage should be appropriately adjusted. Further guidance can be obtained from studies using experimental animal models to assess dosage as known in the art. However, the cell binding affinity and specificity of the increase associated with the ligand of the present invention allow for a larger safety range of dosage concentration and repeated administration.

[0220] In some embodiments, a therapeutically effective amount is an amount that activates Akt signaling in an individual. Akt signaling and activation thereof can be used, for example, 7A to 7C The Akt signaling array shown is evaluated in bone marrow stromal cells. Typically, the peptidomimetic ligand compound will be administered in a dosage range of about 0.01 mg to about 1000 mg per kg of individual body weight (i.e., about 0.01 mg / kg to 1000 mg / kg). The dosage of the compound can be, for example, about 0.01 mg / kg to 1000 mg / kg, or about 0.1 mg / kg to 250 mg / kg, or about 0.2 mg / kg to 100 mg / kg. The dosage of compound can be about 0.1mg / kg, 0.2mg / kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 1mg / kg, 5mg / kg, 10mg / kg, 25mg / kg, 50mg / kg, 75mg / kg, 100mg / kg, 200mg / kg, 300mg / kg, 400mg / kg, 500mg / kg, 600mg / kg, 700mg / kg, 800mg / kg, 900mg / kg or 1000mg / kg. Dosage can be according to the needs of the patient, the severity of the disease treated and the specific preparation used and change. The size of dosage will also be determined by the presence, nature and degree of any adverse side effects accompanied by the administration of drugs in a specific patient. The total dose can be divided and applied in batches over a period of time, to be suitable for treating the patient's condition or disease.

[0221] Compound can be administered for a period of time, and this time will change according to the overall condition of the individual of the character of specific disease, its severity and being administered compound.Compositions comprising the combination of ligand of the present invention or ligand can be repeatedly administered, for example at least 2,3,4,5,6,7,8 or more times, or compositions can be administered by continuous infusion.Administration can be carried out for example every hour, every 2 hours, 3 hours, 4 hours, 6 hours, 8 hours or twice a day (including every 12 hours), or any interval thereof.Administration can be carried out once a day or once every 36 hours or 48 hours or once a month or several months.After treatment, the change of individual condition and the alleviation of symptoms of disease can be monitored.In the case that individual does not have obvious reaction to specific dosage level, the dosage of compound can be increased, or if the alleviation of symptoms of disease is observed, or if the disease has been treated, or unacceptable side effects occur under specific dosage, then dosage can be reduced.

[0222] In implementing the method of the present invention, the pharmaceutical composition can be used alone or in combination with other therapeutic agents or diagnostic agents. The other drugs used in the combination scheme of the present invention can be used separately, or one or more drugs used in the combination scheme can be used together, for example, in the form of a mixture. If one or more drugs are used alone, the timing and schedule of administration of each drug may be different. Other therapeutic agents or diagnostic agents can be used simultaneously, separately or at different times with the compound of the present invention.

[0223] In some embodiments, the present invention provides a method for promoting systemic bone growth. Systemic bone growth refers to the growth of bones throughout an individual, and may affect all bones in an individual's body. An individual who needs systemic bone growth may suffer from a variety of diseases and disease states. In some embodiments, an individual suffers from a low bone mass phenotypic disease. Low bone mass can be determined by a variety of methods known to those skilled in the art. For example, low bone mass is characterized by a T value less than about -1. Low bone mass phenotypic diseases may include osteoporosis, osteopenia, and osteoporosis-pseudoglioma syndrome (OPPG). In some other embodiments, low bone mass phenotypic diseases may be osteopenia or osteoporosis-pseudoglioma syndrome (OPPG). In some other embodiments, the present invention provides a method for treating low bone mass by administering a therapeutically effective amount of the compounds of the present invention to an individual in need thereof.

[0224] After administration of the compounds of the present invention, systemic bone growth can be determined by a variety of methods, such as improvements in bone density. Bone density can be measured by a variety of different methods, including T scores and Z scores. The T score is the number of standard deviations above or below the mean value of a healthy 30-year-old adult of the same gender as the patient. Low bone mass is characterized by a T score of -1 to -2.15. Osteoporosis is characterized by a T score of less than -2.15. The Z score is the number of standard deviations above or below the mean value for the patient's age and gender. An improvement in the T score or Z score indicates bone growth. Bone density can be measured at various locations of the skeleton, such as the spine or hip. Those skilled in the art will appreciate that other methods for determining bone density can be used in the present invention.

[0225] In some embodiments, the present invention provides a method of treating osteoporosis, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof.

[0226] The present invention also provides methods of treating diseases characterized by secondary induced osteoporosis ("secondary low bone mass"), but not limited to, osteomalacia, polyostotic fibrous dysplasia, Paget's disease, rheumatoid arthritis, zero gravity, osteoarthritis, prolonged inactivity or immobility, osteomyelitis, celiac disease, Crohn's disease, ulcerative colitis, inflammatory bowel disease, gastrectomy, secondary induced osteoporosis, amenorrhea, Cushing's disease, Cushing's syndrome, diabetes mellitus, diabetes, eating disorders, hyperparathyroidism, hyperthyroidism, hyperprolactinemia, Kleinefelter's syndrome, thyroid disease, Turner's syndrome, steroid-induced osteoporosis, epilepsy or depression-induced osteoporosis, immobility, arthritis, cancer-induced secondary osteoporosis low bone mass, gonadotropin-releasing hormone agonist-induced low bone mass, thyroid drug-induced low bone mass, dilantin (phenytoin), sodium valproate-induced low bone mass, chemotherapy-induced low bone mass, immunosuppressant-induced low bone mass, blood thinner-induced low bone mass, Graves' disease, juvenile rheumatoid arthritis, malabsorption syndrome, anorexia nervosa, kidney disease, anticonvulsant therapy (e.g., for epilepsy), corticosteroid therapy (e.g., for rheumatoid arthritis, asthma), immunosuppressive therapy (e.g., for cancer), inadequate nutrition (especially calcium, vitamin D), amenorrhea (absence of menstruation) due to excessive exercise, smoking and alcohol abuse, pregnancy-related osteoporosis, copper deficiency, diaminoaciduria type 2, Werner syndrome, Hajdu-Cheney syndrome, juvenile hyperostosis corticalis deformans juvenilis, methylmalonic aciduria type 2, cystathionine beta-synthase deficiency, exemestane, hyperimmunoglobulin E (IgE) syndrome, hemochromatosis, Singleton Merten syndrome, beta-thalassemia (homozygous), reflex sympathetic osteodystrophy, sarcoidosis, Winchester syndrome, Hallerman-Streiff syndrome (HSS), cyproterone acetate, glycerol kinase deficiency, Bonnet-Dechaume-Blanc syndrome, prednisolone, heparin, osteodysplastic senile cutis, Torg osteolysis syndrome, orchiectomy, Fabry disease, pseudoprogeria syndrome, Wolcott-Rallison syndrome, ankylosing spondylitis, myeloma, systemic infantile hyalinization infantilehyalinosis), Albright hereditary osteodystrophy, autoimmune lymphoproliferative syndrome, Brown-Sequard syndrome, Diamond-Blackfan anemia, galactorrhea-hyperprolactinemia,Gonadal dysgenesis, kidney disease, Menkes disease, menopause, neuritis, ovarian insufficiency due to FSH resistance, familial ovarian insufficiency, premature aging, primary biliary cirrhosis, prolactinoma, familial prolactinoma, renal osteodystrophy, ulcerative colitis, underweight, Werner syndrome, bone tumors, bone cancer, brittle bone disease, osteogenesis imperfecta, and osteogenesis imperfecta tarda. Other conditions include bone damage, such as fractured or weakened bones, or bones damaged by radiation therapy. Those skilled in the art will appreciate that other types of conditions, diseases, and treatments can also lead to osteoporosis. ,

[0227] Glucocorticoids are a class of corticosteroids, which are steroid hormones that are commonly used to treat diseases or conditions associated with an overactive immune system, such as asthma, allergies, asthma, autoimmune diseases (e.g., Graves' disease, rheumatoid arthritis, lupus, inflammatory bowel disease, etc.) and sepsis. However, the use of steroids or glucocorticoids may lead to rapid bone loss and a high risk of prone fractures. In some cases, steroids or glucocorticoids (i.e., glucocorticoids) can cause osteonecrosis, the details of which are described below.

[0228] In some embodiments, the present invention provides a method for treating steroid-induced bone loss by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. In some embodiments, the present invention provides a method for treating glucocorticoid-induced bone loss by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. In some embodiments, the present invention provides a method for treating steroid-induced osteoporosis by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. In some embodiments, the present invention provides a method for treating glucocorticoid-induced osteoporosis by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need.

[0229] Osteonecrosis, also known as avascular necrosis or aseptic necrosis, is the death of bone cells in bones due to reduced blood flow. If left untreated, the death of bone cells in bones may lead to collapse of the bone area, which may then lead to degenerative arthritis of the joints near the bones. Osteonecrosis most often affects the hip and knee, but may also affect the shoulder, wrist, hand, ankle, foot and jaw. Osteonecrosis may have a variety of causes, including traumatic and non-traumatic causes. Typically in traumatic osteonecrosis, severe trauma to the bone interrupts the blood supply to the bone. Non-traumatic osteonecrosis may be caused by: certain drugs, such as corticosteroids (e.g., prednisone, cortisone, dexamethasone or methylprednisolone), especially when high doses of drugs are administered for a long time; due to excessive drinking; from radiotherapy; or because of disease or condition. See, for example, Xie et al., 2015, Journal of Orthopaedic Translation, 3: 58-70, which is incorporated herein by reference.

[0230] In some embodiments, the present invention provides a method for treating osteonecrosis by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. In some embodiments, the present invention provides a method for treating post-traumatic osteonecrosis (e.g., osteonecrosis occurring after a fracture or dislocation of a bone) by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. In some embodiments, the present invention provides a method for treating non-traumatic osteonecrosis by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. In some embodiments, the present invention provides a method for treating steroid-induced osteonecrosis (e.g., high-dose steroid-induced osteonecrosis or glucocorticoid-induced osteonecrosis), alcohol-induced osteonecrosis, or smoking-induced osteonecrosis by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. In some embodiments, the present invention provides a method of increasing vascular density in osteonecrotic tissue by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need thereof. In some embodiments, the present invention provides a method of preventing or reducing cell death in osteonecrotic tissue by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0231] In some other embodiments, the present invention provides a method for treating secondary induced osteonecrosis by administering a therapeutically effective amount of a compound of Formula I, Formula Ia, Formula Ib, Formula II, or a pharmaceutically acceptable salt thereof to an individual in need. Examples of diseases or conditions that can induce osteonecrosis (i.e., secondary osteonecrosis) include, but are not limited to: Legg-Calvé-Perthes disease, Caisson disease, sickle cell disease, post-radiation, chemotherapy, arterial disease, Gaucher disease, lipid disorders, connective tissue disease, pancreatitis, kidney disease, liver disease, or lupus. In some embodiments, osteonecrosis is idiopathic osteonecrosis.

[0232] The invention also provides methods of treating patient populations characterized by damaged bones, such as fractured bones or bones damaged by radiation, as well as children for whom osteoporosis medications are contraindicated.

[0233] In some embodiments, the present invention provides a method for promoting bone growth by administering a therapeutically effective amount of a compound of the present invention to an individual in need thereof. Bone growth can be measured in a variety of ways known to those skilled in the art. The method for measuring bone growth includes but is not limited to Uct (micro-CT), dual X-ray absorption (bone density), ultrasound, QCT, SPA, DPA, DXR, SEXA, QUS, X-rays, using human eyes during surgical procedures, Alizarin Red S, serum osteocalcin, serum alkaline phosphatase, serum bone Gla protein (BGP), bone mineral content, serum calcium, serum phosphorus, tantalum markers and serum IGF-1.

[0234] Many indicators of bone growth can be used to measure bone growth, including bone density. In some embodiments, bone growth can be demonstrated by a 0.1% increase in bone density. In other embodiments, bone growth can be demonstrated by a 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more increase in bone density. Those skilled in the art will appreciate that bone growth is local, systemic, or both.

[0235] In some other embodiments, the methods of the present invention promote bone growth by administering a compound of the present invention, such as a compound of Formula I. The administration of the compound of the present invention can promote local bone growth and / or systemic bone growth. In some embodiments, the administration of the compound of the present invention promotes systemic bone growth. Bone growth can be achieved by increasing bone mineral content, increasing bone density and / or the growth of new bone. In other embodiments, local administration of the compounds of the present invention and drugs achieves systemic bone growth.

[0236] V. Examples

[0237] Example 1: Screening for cell signaling with affinity for osteoblast-specific transcription factors + osteoprogenitor cells Activator

[0238] The method for discovering osteogenic-specific peptides targeting osteocytes with cell-binding affinity and intracellular signaling involves bead screening of a focused-bead-one-compound (OBOC) peptide library designed based on the integrin α4β1 motif. The synthetic method used to develop the focused OBOC combinatorial library is shown below in Scheme 1.

[0239] Solution 1

[0240]

[0241] In brief, TentaGel beads (1.0 g, loading 0.26 mmol / g) were swollen in DMF (20 mL) for 3 hours. The resin was divided into 43 equal parts and placed in 43 disposable polypropylene columns with polyethylene glass frits. 43 different Fmoc-amino acids (Table 4) (4 equivalents) were dissolved in DMF solutions of 6-Cl HOBt (4 equivalents) and DIC (4 equivalents) respectively, and added to 43 columns (each column only receives one amino acid). The coupling was carried out for 2 hours at room temperature. After filtering, the beads were merged, mixed and washed 3 times with DMF, MeOH and DMF respectively. Fmoc-deprotection (5 minutes, 15 minutes) was carried out on the beads with 20% 4-methylpiperidine. After washing with DMF, MeOH and DMF, the beads were mixed with 1g blank TentaGel beads. Using the same split synthesis method as described above, 2 g of the combined beads were sequentially coupled with 30 Fmoc-amino acids at the X2 position (Table 5), 8 Fmoc-amino acids at the X3 position (Table 6) and 18 Fmoc-amino acids at the X4 position (Table 7), respectively. After Fmoc-deprotection, the beads were washed with DMF, MeOH, DCM and dried thoroughly in vacuo.

[0242] Table 4. Amino acids for position X1.

[0243]

[0244]

[0245] Table 5. Amino acids for position X2.

[0246]

[0247]

[0248] Table 6. Amino acids for position X3.

[0249]

[0250] Table 7. Amino acids for position X4.

[0251]

[0252]

[0253] Then, as described in the reference (Liu R, Marik J, Lam KS. J Am Chem Soc. 2002 Jul 3; 124 (26): 7678-80.), two-layer beads were prepared using a biphasic solvent method. Briefly, the dried beads were swollen in water for 1 day. The water was removed by filtration, and a solution of Fmoc-OSu (17.5 mg, 0.052 mmol, 0.1 equivalent relative to the beads) in DCM / diethyl ether (150 mL, 55 / 45) was added to the wet beads, followed by DIEA (36 μL, 0.208 mmol). The mixture was vigorously shaken at room temperature for 45 minutes. The liquid was removed by filtration. The beads were washed 3 times each with DMF, MeOH and DCM. A DCM solution of Boc anhydride (567 mg, 2.6 mmol) and DIEA (906 μL, 5.2 mmol) was added to the beads and rotated for 1 hour. The Kaiser test was negative, indicating that the coupling was completed. Fmoc on the surface of the beads was removed with 20% 4-methylpiperidine (5 minutes, 15 minutes). The beads were washed and coupled with 4-[(N'-2-methylphenyl)ureido]-phenylacetic acid (UPA) (4 equivalents) in DMF in the presence of 6-Cl HOBt (4 equivalents) and DIC (4 equivalents). The coupling was confirmed to be completed within 4 hours by a negative Kaiser test. After washing with DMF, methanol and DCM, the beads were then dried under vacuum for 1 hour. Side chain deprotection was achieved using a mixture of 82.5% TFA: 5% phenol: 5% thioanisole: 5% water: 2.5% TIS. After neutralization twice with 2% DIEA / DMF, the resin was washed with DMF, MeOH, DCM, DMF, DMF / water, water and PBS in sequence. The bead library was stored in 70% ethanol.

[0254] like Figure 1 As shown, the OBOC combinatorial library was screened and peptides that showed affinity for target cells and were used as activators or inhibitors of target cell signaling pathways (one bead / one compound / two functions) were identified (see Liu, R. et al. Curr Opin Chem Biol., 2017, 38: 117-26). Bone marrow stromal cells (BMSCs) were obtained from mice and maintained in mesenchymal maintenance medium for 7 days to 2 weeks (about 3 generations), changed to osteogenic medium for 3 days, and then incubated with the focused integrin library for 1 hour. The cells and beads were then fixed and stained for phosphorylated Atk, which is a signal transduction pathway that can promote cell survival and growth in response to extracellular signals (see Manning BD et al., Cell. 2007, 129: 1261-74). Positive beads were identified as having cell binding and stained with green fluorescence (p-Akt+) ( Figure 1 , step A). ​​Positive beads were manually picked out and microsequencing was performed using Edman degradation (see Liu and Lam, Anal. Biochem. 2001, 295: 9-16). Each peptide on the bead was resynthesized with a fluorescent quencher (nitrotyrosine) residue inside the bead to quench the autofluorescence of the bead. These beads were incubated with osteoprogenitor cells (OPCs) obtained from osteoblast-specific transcription factor-mCherry reporter mice. Osteoblast-specific transcription factors are expressed by osteochondroprogenitor cells and are generally considered to be early markers of osteoblast maturation, as described in Mizoguchi T et al. Developmental cell. 2014, 29, 340-9 and Liu Y et al., PLoS One. 2013, 8: e71318. Positive beads that passed the second round of screening were identified by their binding affinity to osteoblast-specific transcription factor + cells ( Figure 1 Step B). Confirm activation of Akt binding again ( Figure 1 , step C). Using this method, 22 peptides (Table 8) were identified as Akt signaling activators with high affinity for osteoblast-specific transcription factor + cells ( Figure 1 , step D). It was confirmed that the lead peptides YLL3 and YLL8 have high binding affinity for osteoblast-specific transcription factor cells and have an in vitro osteogenic effect. The beads showing YLL3 and YLL8 also showed low affinity for lymphocytes ( Figure 2 ).

[0255] Example 2: Synthesis of YLL ligand

[0256] YLL3, YLL8 and other ligands listed in Table 8 were prepared using commercially available starting materials and methods known in the art. Typically, YLL ligands were prepared via Fmoc solid phase peptide synthesis using 4-[(N'-2-methylphenyl)ureido]phenylacetic acid (UPA), Rink Amide MBHA resin and related protected amino acid components as described in International Publication No. WO 2012 / 031228A2.

[0257] Solid phase synthesis of YLL3 and YLL8

[0258] YLL8 was synthesized using standard solid phase peptide synthesis methods on Rink amide MBHA resin (GL Biochem, Shanghai, China). Briefly, Rink amide MBHA resin (0.5 g, 0.325 mmol, loading 0.65 mmol / g) was swollen in DMF for 2 hours, and then Fmoc-deprotected twice (5 minutes, 15 minutes) with 20% 4-methylpiperidine in DMF. The beads were washed with DMF (3×10 mL), MeOH (3×10 mL) and DMF (3×10 mL). Fmoc-Ach-OH (0.365 g, 0.975 mmol) was dissolved in a DMF solution of 6-Cl HOBt (0.165 g, 0.975 mmol) and DIC (152 μL, 0.975 mmol) and then added to the beads. The coupling was performed at room temperature for 2 hours. After filtration, the beads were washed 3 times with DMF (3×10 mL), MeOH (3×10 mL) and DMF (3×10 mL). The Fmoc deprotection group was removed twice with 20% 4-methylpiperidine (5 minutes, 15 minutes). After washing with DMF, MeOH and DMF, the beads were then subjected to additional coupling and deprotection cycles stepwise with Fmoc-Aad (tBu) and Fmoc-Lys (A12) in the same manner as described above. After removing Fmoc, a DMF solution of 4-[(N'-2-methylphenyl)ureido]phenylacetic acid (UPA, 0.923 g, 3.25 mmol), HOBt (0.498 g, 3.25 mmol) and DIC (509 μL, 3.25 mmol) was added to the beads. The reaction was carried out at room temperature overnight. The beads were washed with DMF (5×5 mL), MeOH (3×5 mL) and DCM (3×5 mL). The beads were then dried in vacuo for 1 hour before a cleavage mixture of 95% TFA: 2.5% water: 2.5% TIS was added. The cleavage reaction was allowed to proceed for 2 hours at room temperature. The liquid was collected and concentrated. The crude product was precipitated with diethyl ether and purified using preparative RP-HPLC. The fractions were collected and lyophilized to give the designed product YLL8, MALDI-TOF MS: 813.30 Daltons.

[0259] YLL3 was synthesized using a similar method to YLL8, but the following different building blocks Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cit-OH and UPA were coupled to the beads in sequence. Crude YLL3 was cleaved from the beads and purified by RP-HPLC. MALDI-TOF MS: 756.25 Daltons.

[0260] Table 8. YLL ligands.

[0261]

[0262]

[0263]

[0264] Example 3: In vitro osteogenic effect of YLL peptide

[0265] The lead peptides YLL3 and YLL8 have high binding affinity for osteoblast-specific transcription factor cells and exhibit in vitro osteogenic effects, such as Figure 3 and Figure 4 Mouse bone marrow stromal cells (BMSCs) were obtained from osteoblast-specific transcription factor-mCherry transgenic mice and cultured with focused library beads encoding YLL3, YLL8, and LLP2A peptides in osteogenic medium for 10 days and fixed. Osteoblast-specific transcription factor (OSX+) marks osteochondrogenic progenitor cells, and positive cells appear red ( Figure 3 ). Mouse BMSCs obtained from normal mice were incubated with focused library beads displaying LLP2A, YLL3, or YLL8 peptides and cultured in osteogenic medium for 10 days. Cells with beads were stained for anti-Akt-Alexa488. Positive cells showed activation of Atk and were stained green ( Figure 4 The focused library beads showing several peptides of Table 4 were also incubated with mouse BMSCs obtained from conventional mice and cultured in osteogenic medium for 10 days. Alkaline phosphatase levels corresponding to osteogenic differentiation and osteoblast maturation were measured on day 14. Alizarin red, a measure of osteoblast maturation and mineralization, was measured on day 21 ( Figure 5 ).

[0266] In additional in vitro osteogenesis studies, osteogenic differentiation (alkaline phosphatase, ALP activity) of YLL3 and YLL8 was measured at day 10, and osteoblast maturation (Alizarin Red staining) was measured at day 21. Beads displaying peptides YLL3 or YLL8 increased ALP levels ( Fig. 6A and Figure 6B ) and induced higher mineralized nodule formation ( Fig. 6A and Figure 6C ). Note that cells are localized to or around the beads and deposited minerals ( Figure 6C These results were confirmed by adding the peptides directly to osteoblast differentiation cultures ( Fig.6D ).

[0267] The specificity of YLL in activating Akt signaling was then investigated by Western blot analysis using an Akt signaling array. -8 M) were incubated with BMSCs in osteogenic medium for three days to confirm the activation of Akt pathway ( Fig. 7A ). Human PTH (1-34) (6x10 -8 M) was used as a positive control. Figure 7B As shown, compared with the control and YLL3, YLL8 has very similar characteristics to PTH in activating members of the Akt signaling pathway, including phosphorylation of Akt, phosphoinositide-dependent kinase 1 (ERK1 / 2), p53, PDK1), p53, 4EBP1 and BCL2, associated agonist of cell death (BAD), and RIK1 ( Figure 7B , Figure 7C ))YLL3 activated p53 and Akt ( Figure 7B , 7C). These results suggest that osteoblast-targeted peptides (e.g., YLL8) increase osteoblast differentiation and maturation through signaling pathways that promote cell growth or pro-surviving mechanisms.

[0268] Example 4: Anabolic Effects of YLL Peptide in Vivo

[0269] In a study to determine whether YLL peptides affect bone metabolism in vivo, female and male mice aged two months were treated with PBS control, YLL3, YLL8, or LLP2A at 50 μg / kg intravenously on day 1. hPTH (1-34) was administered subcutaneously at 25 μg / kg, 5 times per week for 21 days (n=4-6 / group). Trabecular and cortical bone volumes of the distal femur and cortical bone volumes of the distal and mid-femur were measured by microCT ( Figure 8 ).

[0270] In additional studies to determine whether YLL affects bone metabolism in vivo, YLL3 and YLL8 were injected into 2-month-old mice at 25 μg / kg or 50 μg / kg, subcutaneously (SC), 5 times a week for 21 days. hPTH (1-34) was injected subcutaneously at 25 μg / kg, 5 times a week as a positive control. The results from the 50 μg / kg group are shown in Figure 9. Daily injections of both YLL peptides for 21 days did not change body weight or cause any visible side effects. In females, YLL3 and YLL8 increased trabecular bone volume by approximately 13% (p<0.05, relative to PBS) and 8% ( Fig.9A ), which was associated with a 60% (p < 0.05, relative to PBS) and 86% ( Fig. 9B and Fig.9D In males, YLL3 and YLL8 increased trabecular bone volume by approximately 15% (p < 0.05, relative to PBS) and 6% ( Fig.9A ), which was associated with a 53% (p<0.05, relative to PBS) and 50% (p<0.05, relative to PBS) increase in trabecular bone formation rate ( Fig.9A and Fig.9D In males, both YLL3 and YLL8 increased cortical bone volume by approximately 15%, and higher bone formation was observed on the periosteal surface ( Fig. 9C and Fig.9E ). Note that YLL3 increases intracortical bone remodeling ( Fig.9E ). These results suggest that both YLL3 and YLL8 stimulate bone formation at both trabecular and cortical surfaces and are comparable to the anabolic effects of daily PTH treatment.

[0271] In another similar study to determine whether YLL affects bone metabolism in vivo, YLL3 and YLL8 were injected subcutaneously (sc) at 5 μg / kg, 5 times a week for 21 days (n=5-7 / group) into 4-month-old female and male mice. hPTH (1-34) was injected subcutaneously at 25 μg / kg, 5 times a week as a positive control. Daily injections of YLL8 for 21 days did not change body weight or cause any obvious side effects. Both YLL8 and hPTH (1-34) increased femoral trabecular bone volume in female mice by approximately 130% compared to PBS-treated mice. In male mice, YLL8 increased femoral trabecular bone volume by approximately 70%, while hPTH (1-34) increased femoral trabecular bone volume by approximately 30% ( Fig. 10A The mineral apposition rate, a parameter corresponding to osteoblast activity, increased by more than 70% in females and by approximately 30% in males, resulting in an overall increase in the rate of surface-based bone formation ( Fig. 10A , Fig. 10BThe anabolic effect of YLL was confirmed by an increase in trabecular bone mass measured by micro-CT and serum levels of osteocalcin in the distal femoral metaphysis ( Fig.11 Bone resorption measured by serum CTX1 showed that, especially in female mice, PTH increased bone resorption by 200% from the PBS-treated group, whereas YLLL did not significantly alter CTX1 ( Fig.11 ). Daily YLL3 and YLL8 treatment for 21 days increased cortical bone mass in males and increased bone strength in females and males, which was comparable to daily hPTH (1-34) injections (Figure 10D). These results indicate that YLL3 and YLL8 induce uncoupling of bone formation and resorption and increase bone mass.

[0272] Example 5: Mineralization Potential During Fracture Repair Using YLL Peptide

[0273] The following experiments were performed to determine if the YLL peptide accelerates fracture repair. The recruitment and activation of endogenous osteoblast lineage cells during fracture healing is a critical step in repairing fractures. Since inducible osteoblast-specific transcription factor-reporter mice are not directly commercially available, inducible Prx1-CreERT-GFP mice were used to track the recruitment and osteogenic differentiation of osteoprogenitor cells after fracture and YLL treatment. In uninjured mice, Prx1 marks osteoprogenitor cells at the growth plate and alongside the trabecular and intracortical bone surfaces, similar to where osteoblast-specific transcription factors are expressed in bone ( Fig.12 A closed, stable mid-femoral fracture model was used to follow the movement of osteoblast lineage cells contributing to intramembranous or endochondral bone formation in this model. Human PTH (1-34) was used as a positive control (50 μg / kg, 5 times per week). On day 10 after fracture, the presence of Prx1 / GFP expressing cells was observed in the callus, some co-localizing with Alizarin Red, indicating active bone apposition from those neighboring cells ( Fig.13A In YLL8- and PTH-treated mice, the number of Prx1+ cells was greatly increased, and some colocalized with mineral deposits ( Fig.13A ). Almost all Prx1+ green cells overlapped with Alizarin Red, indicating high osteoblast differentiation and mineral absorption in these cells after YLL3 treatment ( Fig.13A ), resulting in 100% higher callus bone volume compared with PBS-treated mice on day 10 ( Fig. 13B Further dynamic histomorphometric studies using fluorescent dyes to label mineralization at day 21 after fracture showed that nearly all Prx1+ and its progeny cells overlapped with AR mineral deposition in all groups. In YLL3-treated mice, Prx1+ and its progeny cells were activated and promoted callus formation, co-localizing within the regenerating bone callus region ( Fig. 13CQuantitative measurements by micro-CT scanning confirmed higher callus mineral content in YLL3-treated mice at day 21 after fracture ( Fig.13D These data suggest that a similar anabolic mechanism activates osteoblast lineage cells via YLL8 and PTH during fracture healing. YLL3 injection greatly activated osteogenic differentiation of osteoprogenitor cells and induced significantly higher callus mineralization, which accelerated fracture repair.

[0274] Example 6: Synthesis of YLL3-alendronate (YLL3-Ale) and YLL8-alendronate (YLL8-Ale)

[0275] To ensure that the anabolic effect was bone specific, the YLL3 and YLL8 peptides were conjugated to alendronate.

[0276]

[0277]

[0278] YLL3-alendronate (YLL3-Ale) and YLL8-alendronate (YLL8-Ale) were prepared by linking alendronate-maleimide (Ale-Mal) (generated by conjugation of alendronate and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC)) to D-cysteine, lysine, two N-(8-amino-3,6-dioxa-octyl) succinamide linkers and YLL3 or YLL8, as described above. Fig.14 As shown in and further described in International Publication No. WO 2012 / 031228 A2, or as described below.

[0279] Generally, the synthesis of YLL8-Aln involves three steps.

[0280] Step 1. Solid phase synthesis of YLL8-Lys(D-Cys)

[0281] The synthetic method for synthesizing YLL8-Lys(D-Cys) is shown in Scheme 2 below. Rink amide MBHA resin (0.5 g, 0.325 mmol, loading 0.65 mmol / g) was swollen in DMF for 3 hours. Fmoc was deprotected twice with 20% 4-methylpiperidine in DMF (5 minutes and 15 minutes, respectively). After filtration, the beads were then washed with DMF (3×10 mL), methanol (MeOH) (3×10 mL) and DMF (3×10 mL), respectively. Fmoc-Lys(Dde)-OH (0.519 g, 0.975 mmol) was dissolved in a DMF (8 mL) solution of 6-Cl HOBt (0.165 g, 0.975 mmol) and DIC (152 μL, 0.975 mmol) and then added to the suspension of beads. The coupling was performed at room temperature overnight. After filtration, the beads were washed with DMF (3×10 mL), MeOH (3×10 mL) and DMF (3×10 mL), respectively. Fmoc was deprotected twice (5 minutes and 15 minutes, respectively) with 20% 4-methylpiperidine in DMF (8 mL). Fmoc-linker (0.612 g, 1.3 mmol) was dissolved in a DMF (8 mL) solution of HOBt (0.22 g, 1.3 mmol) and DIC (201 μL, 1.3 mmol) and then added to the suspension of beads. Coupling was performed at room temperature until the Kaiser test was negative. After filtration, the beads were washed with DMF (3×10 mL), MeOH (3×10 mL) and DMF (3×10 mL), respectively. Fmoc was deprotected twice (5 minutes and 15 minutes, respectively) with 20% 4-methylpiperidine in DMF (8 mL). The Fmoc-linker (0.612 g, 1.3 mmol) was dissolved in a solution of HOBt (0.22 g, 1.3 mmol) and DIC (201 μL, 1.3 mmol) in DMF (8 mL) and then added to the suspension of beads. The coupling was carried out at room temperature until the Kaiser test was negative. After filtration, the beads were washed with DMF (3×10 mL), MeOH (3×10 mL) and DMF (3×10 mL), respectively. Fmoc was deprotected twice (5 minutes and 15 minutes, respectively) with 20% 4-methylpiperidine in DMF (8 mL). After filtration, the beads were washed with DMF (3×10 mL), MeOH (3×10 mL) and DMF (3×10 mL), respectively. Fmoc-Ach-OH (0.365 g, 0.975 mmol) was dissolved in a DMF solution of 6-Cl HOBt (0.165 g, 0.975 mmol) and DIC (152 μL, 0.975 mmol) and then added to the beads. The coupling was performed at room temperature for 2 hours.

[0282] Solution 2

[0283]

[0284] After filtration, the beads were washed with DMF (3×10 mL), MeOH (3×10 mL), and DMF (3×10 mL), respectively. The Fmoc deprotection group was removed twice with 20% 4-methylpiperidine (5 minutes and 15 minutes, respectively). After washing with DMF, MeOH, and DMF, the beads were then subjected to additional coupling and deprotection cycles stepwise with Fmoc-Aad(OtBu)-OH, Fmoc-Lys(A12), and UPA in the same manner as described above.

[0285] The Dde protecting group was removed twice with 2% hydrazine monohydrate in DMF (5 minutes and 10 minutes respectively), and the beads were washed with DMF, MeOH and DMF, and then Boc-D-Cys(Trt)-OH (4 equivalents for the resin, 220 mg, 1.3 mmol), HOBt (0.176 g, 1.3 mmol) and DIC (201 μL, 1.3 mmol) were coupled in DMF (8 mL). The coupling reaction was carried out at room temperature until the Kaiser test was negative (4 hours to overnight). The beads were thoroughly washed with DMF, MeOH and DCM, respectively, and then dried under vacuum for 1 hour, and then a cleavage mixture (8 mL) of 82.5% trifluoroacetic acid (TFA): 5% thioanisole: 5% phenol: 5% water: 2.5% triisopropylsilane (TIS) (v / v) was added. The cleavage reaction was carried out at room temperature for 2-3 hours. The off-white crude product was precipitated and washed with cold diethyl ether. The purity was determined by analytical HPLC and the crude product was used in the next step without further purification.

[0286] Step 2. Synthesis of Aln-Mal

[0287] The synthetic method for synthesizing Aln-Mal is shown in Scheme 3 below. Alendronate disodium salt (35.4 mg, 0.1208 mmol) (freeze-dried powder from aqueous alendronic acid solution and 2 equivalents of NaOH) was dissolved in 0.1M PBS (15 mL) (containing 10 mM EDTA), pH 7.5. The aqueous solution was then cooled in an ice-water bath, and a solution of sulfo-SMCC (58 mg, 0.133 mmol) in water (14 mL) was added dropwise. After the addition was complete, the resulting solution was allowed to warm to room temperature while stirring for 2 hours to produce an Aln-Mal solution, which was used for conjugation in the next step without purification.

[0288] Solution 3

[0289]

[0290] Step 3. Conjugation of YLL8-Aln

[0291] The conjugation method used to synthesize YLL8-Aln is shown in Scheme 4 below. The Aln-Mal solution prepared in step 2 was cooled with an ice-water bath, then acetonitrile (8 mL) was added and a solution of LLP2A-Lys(D-Cys) (200 mg, 0.133 mmol) in a small amount (~4 mL) of 50% acetonitrile / water was added dropwise. The pH was adjusted to 6 to 7 with aqueous NaHCO3. The resulting mixture was stirred for 1 hour and then warmed to room temperature. After a negative Ellman test, the solution was lyophilized. The resulting powder was redissolved in a small amount of 50% acetonitrile / water and purified by reverse phase high performance liquid chromatography (RP-HPLC); Vydac C18 column (10 μm, 22×250 mm), buffer A: 0.1% TFA / H2O, buffer B: 0.1% TFA / acetonitrile. Gradient: 10% ACN, 1 min; 40% ACN, 50 min; 100% ACN, 3 min. The collected eluate was lyophilized to obtain an off-white powder YLL8-Aln, MALDI-TOF MS: 1972.75 Daltons. YLL3-Aln was synthesized by conjugation of YLL3-Lys (D-Cys) and Aln-Mal using a method similar to YLL8-Aln. MALDI-TOF MS: 1915.76 Daltons.

[0292] Solution 4

[0293]

[0294] In some cases, the YLL-Ale compound is a single organic molecule consisting of a highly derivatized synthetic peptidomimetic portion (UPA-YLL portion) with high affinity and specificity for OPCs linked to the bone-targeting bisphosphonate alendronate via a hydrophilic linker, wherein the bisphosphonate portion is linked to the linker portion via a copper-free Click reaction of DBCO with N3-modified alendronate (Aln-N3), which is prepared from alendronate and 4-azidobutyric acid NHS ester (N3-NHS).

[0295] Example 7: Alendronate-conjugated YLL8 increases bone formation and bone mass

[0296] Next, the effect of alendronate conjugated peptide on bone metabolism was assessed. Two-month-old female mice were treated with PBS controls, YLL3-Ale or YLL8-Ale, with 100 μg / kg (25nmol) or 300 μg / kg (75nmol), subcutaneously, once every other week, or a 500 μg / kg IV dose. Each mouse was injected twice. The total cumulative dose was 200 μg / kg / month to 600 μg / kg / month (25nmol to 150nmol), respectively. Alendronate concentration is about 1 / 10 to 1 / 5 of the compound. YLL-Ale injection does not change body weight or cause any visible side effects. Although the peptide YLL3 alone shows anabolic effects, compared with the PBS control, the YLL3-Ale at the two doses used fails to achieve statistically significant differences in bone formation or bone mass (Figure 15). In contrast, YLL8-Ale increased trabecular bone volume by more than 20% (p < 0.05, vs. PBS) at 300 μg / kg × 2 SC or 500 μg / kg × 1 IV, which was consistent with increased trabecular thickness (30%, p < 0.05, vs. PBS) ( Fig.15A and Fig. 15B ) and amount (28%, p < 0.05, relative to PBS). Both 100 μg / kg and 300 μg / kg doses of YLL8-Ale increased cortical bone volume by approximately 16% and cortical thickness by approximately 15% ( Fig. 15C YLL8-Aln increased osteoblast number and activity (higher double-labeled surface and mineral deposition rate on both trabecular and cortical bone surfaces, such as Fig.16 The results showed that bisphosphonate conjugation of YLL8 peptide reduced the frequency of administration and maintained its anabolic effect. More specifically, these results showed that YLL8-Aln increased osteoblast activity, stimulated bone formation, and increased trabecular and cortical bone mass with a total of 600 μg / kg / month for 2 doses or 500 μg / kg per month for one dose.

[0297] As described herein, dual affinity and functional screening methods were used to discover "osteoblast-specific" peptides. The osteogenic peptide YLL8 has a high affinity for osteoblast-specific transcription factor + cells and activates phosphorylation of Akt (a pro-survival signal for osteoblastic osteoprogenitor cells). Both YLL3 and YLL8 can increase the differentiation and maturation of osteoblasts in vitro. Short-term (3 weeks) daily low-dose injections of YLL3 and YLL8 induce bone anabolic effects comparable to PTH (1-34), increasing the rate of mineral deposition (which corresponds to osteoblast activity) and the rate of bone formation. Compared with PTH (1-34), daily injections of YLL do not significantly affect bone resorption. This uncoupling of bone remodeling leads to rapid model-dependent bone increase and rapid increase in bone strength in trabecular and cortical bones, which is higher than that of mice treated with hPTH (1-34). Daily injections of YLL, especially YLL3, for 21 days greatly increased callus formation and mineralization during fracture repair. The conjugation of YLL8 to alendronate reduces the injection frequency, but shows a similar degree of bone increase in trabecular bone and cortical bone. Compared with other bone anabolic drugs (e.g., large molecular weight proteins), the size of the YLL peptide is relatively small (e.g., three to four synthetic amino acids), which makes the manufacture and large-scale production of drugs and / or pharmaceutical preparations based on YLL peptides easier, as well as its easier metabolic decomposition. YLL can be used as a "therapeutic peptide" or used together with a bone binder (affiliated agent) to enhance its bone specificity.

[0298] Current drug treatment options for osteoporosis generally include antiresorptive and anabolic therapies. For example, recombinant human PTH (1-34) (teriparatide) is known for its bone anabolic effects and consists of 34 amino acids, which are the biologically active portion of the hormone. PTHrP (abalotriptide), which was recently approved by the FDA for the treatment of osteoporosis, is a 139-173-amino acid protein homologous to the N-terminus of PTH. Both hPTH (1-34) and PTHrp have significant anabolic effects on bone, but also have other broad physiological effects expressed in various tissues. In addition to their effects on bone and joint development, PTH and PTHrp also act in an autocrine / paracrine manner to regulate calcein metabolism and organogenesis, such as mammary gland development. Sclerostin antibodies are another bone anabolic agent that may be more specific to bone cells. Sclerostin is primarily expressed by osteocytes in bone. However, sclerostin can also be expressed in cartilage and lymphocytes, which may lead to extra-skeletal side effects. Therefore, the research described in this article has focused on the development of osteoanabolic drugs that can increase both trabecular and cortical bone mass.

[0299] The canonical Wnt / β-catenin signaling pathway plays a key role in bone formation and is an active drug target for the study / discovery of bone anabolic therapeutics. The studies described herein focused on Akt activation because it is one of the lead kinases activated by α4β1 integrin upon binding to osteoprogenitor cells. Likewise, the focus was on activating osteoprogenitor cells and their osteogenic potential, rather than inducing mitogens or inducing new bone formation, which is typically observed when using Wnt-targeted or growth factors. To verify the effects of osteogenic YLL peptides on osteoblast differentiation, ALP and AR staining were performed, reflecting the initial and final stages of osteoblast differentiation, respectively. ALP and AR staining studies showed that YLL3 and YLL8 enhanced osteogenic differentiation of osteoprogenitor cells in vitro. Activation of the Akt signaling pathway was also confirmed. Similar to PTH, YLL8 exhibited a broader effect on Akt pathway activation, while YLL3 was more specific for Atk activation. The studies described herein also showed that injection of YLL3 and YLL8 resulted in an increased rate of mineral deposition, which indicates activated osteoblast function. YLL3 and YLL8 injections also resulted in increased bone formation, bone mass and bone strength in young mice. Short-term treatment studies (i.e., 21 days) showed that the anabolic effects of YLL on male mice were generally higher than those on female mice, which may be due to the fact that young males have higher osteogenesis than females, and high estrogen levels during growth may have a negative impact on bone formation in females.

[0300] The YLL peptide was conjugated to the bone-targeted drug alendronate. In this case, alendronate was used as a "carrier" for bone-specific delivery of the peptide. To further determine the enhanced bone-targeting effect of YLL, in vivo experiments were conducted and showed that two subcutaneous doses or one intravenous dose of YLL8-Aln increased trabecular bone mass and cortical bone mass. Overall, these studies show that YLL8 can stimulate osteoblast differentiation in vitro and in vivo, demonstrating the efficacy of YLL8-Aln on bone formation. Dynamic bone morphometry and histological analysis confirmed that YLL8 significantly promoted bone formation by enhancing osteoblast activity. Surprisingly, when YLL3 was conjugated to alendronate, YLL3-Aln lost its anabolic effect on bone. This may be due to conformational changes in the structural configuration of YLL3 after Aln conjugation. Increasing the dose and / or dosing frequency of YLL3-Aln may produce the anabolic effect observed for the unconjugated YLL3 peptide. Alternatively, the conjugation method can be modified so that YLL3 is cleaved from the alendronate conjugate and released upon reaching the bone.

[0301] In addition to aging and osteoporosis, nearly 8 million adults experience fractures each year in the United States alone, of which approximately 5% to 13% result in nonunion or delayed union. Current therapies for the treatment of nonunion of fractures mainly include bone transplantation and the use of recombinant bone morphogenetic protein (BMP). The significant complication rate of bone transplantation ranges from 10% to 25% and is associated with inconsistent results. BMP treatment requires orthopedic surgery before BMP delivery. In addition, BMP may cause inflammation, poor mineralization and is expensive. The development of osteogenic anabolic agents that can increase bone mineralization and accelerate fracture repair is an ongoing work. To this end, daily injections of YLL3 led to the development of mature callus in the early stage (10 days after fracture) and produced greater callus mineral density in the later stage (21 days after fracture). Since the initial stage of fracture repair is the recruitment and activation of osteoprogenitor cells, the results showed that mice treated with YLL3 had a large amount of minerals absorbed by osteoprogenitor cells in the early stage, indicating that the activation of these endogenous osteoblasts is being recruited to the fracture site. Compared to the more pronounced formation of woven bone in PBS-treated controls, YLL3-treated mice developed well-mineralized lamellar tissue by day 21. Fracture healing was observed by day 21 after fracture, which was not observed until 35 to 45 days in mice and was superior to daily injections of PTH used at 50 μg / kg. These fracture studies did not include measurements of overall bone strength, but increases in bone mineral density measured by microCT are a major determinant of bone strength, suggesting that the higher bone strength may result from higher bone mineral density following YLL3 treatment.

[0302] In summary, the screening method was used to identify osteoprogenitor cell-specific targeting osteogenic peptides through a pro-cell surviving mechanism. The same "one-entity tri-action" screening principle can be used to screen antagonists or agonist drugs targeting other specific signals. Two peptides targeting osteoprogenitor cells, YLL3 and YLL8, increased the differentiation and maturation of osteoblasts in vitro and increased the activity of osteoblasts in vivo. Improved bone specificity was also observed using peptide-bisphosphonate conjugates, which maintained anabolic effects at lower drug administration frequencies. Osteogenic peptides show bone growth properties and can be used as therapeutic agents for the treatment of osteoporosis and / or accelerated fracture repair.

[0303] Although the foregoing has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be appreciated by those skilled in the art that certain changes and modifications may be made within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was incorporated by reference individually.

Claims

1. A compound having a formula selected from the following:

2. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

3. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating osteoporosis.

4. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for promoting bone growth.

5. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating low bone mass.

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