Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/64—Cyclic peptides containing only normal peptide links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the field of organic chemistry and medicine.
• the invention provides novel macrocyclic compounds based on the core structure of Sansalvamide A (San A), and methods for their preparation and use.
• compounds of the present invention comprise cyclic pentapeptides, their pharmaceutically acceptable salts and hydrate forms, and derivatives thereof, and pharmaceutical formulations comprising these compositions.
• Such compounds possess anticancer activity and activity comprising anti-cell-proliferative, anti- cell migration and/or apoptotic (promoting) activity, and are therefore useful in methods of treatment of a human or an animal body.
• the invention also relates to processes for the manufacture of said cyclic pentapeptides, to pharmaceutical compositions comprising them, and to their use in the manufacture of medicaments for use in the production of an anticancer effect in a warm-blooded animal such as man.
• the invention also provides methods of using said cyclic pentapeptides or pharmaceutical compositions to treat, prevent (prophylaxis of) or ameliorate cancers, including, but not limited to, colon cancer such as MSS and MSI colon cancers, pancreatic cancer, rectal cancer, breast cancer, prostate cancer, and melanoma.
• Sansalvamide A is a lipophilic depsipeptide marine natural product isolated from a marine fungus (Fusarium ssp.), which has been shown to exhibit cytotoxic activity in several cancer cell lines. (Fenical et al., Tetrahedron Lett. 1999, 40, 2913-16). In a mechanism of action study in the poxvirus molluscum contagiosum virus (MCV), San A was shown to be an inhibitor of a virus-encoded topoisomerase I. (Hwang et al., Molecular Pharmacology 1999, 55, 1049-1053). Unlike most Topo I inhibitors, San A does not stabilize Topo I- DNA covalent complexes.
• the macrocyclic peptides exhibit favorable biological, chemical, and physical properties. Cyclic peptides are more hydrophobic and are able to penetrate cell membranes faster than linear peptides, thus providing improved oral availability. Further, the cyclic nature of these compounds restricts bond rotation and provides a more rigid three-dimensional structure than linear peptides or other small molecule drugs. Finally, cyclic peptides are resistant to degradation by proteases, leading to longer half-lives in vivo. Such compounds are useful for developing therapeutic agents because of their diverse functionalization, defined three- dimensional conformations, and extended half-lives compared to linear peptides. San A is composed of four hydrophobic amino acids and one hydrophobic hydroxy- acid.
• Compounds of the present invention are cyclic pentapeptide analogs of San A, where the hydroxy acid in position 4 is exchanged for an amino acid.
• Colon carcinoma is one of the most common human cancers; pancreatic cancer is somewhat less common but more lethal than colon cancer. Both diseases have been considered for years as among the most drug resistant types of cancers. Pancreatic cancer is the fifth most deadly cancer in the U.S. Only 10% of patients are eligible for surgery, fewer than 20% of pancreatic cancers respond to the drug of choice (2,2-difluorodeoxycytidine; Gemzar), and the mortality rate is 95% in 5 years. Recently several new drugs, specifically oxaliplatin, bevucizumab, cetuximab and the tyrosine kinases inhibitors, have improved survival; however, current therapy is far from acceptable. In spite of significant research efforts, few truly novel classes of compounds have been identified that have activity against these 2 types of tumors. Thus, there is an urgent medical need to develop more effective drugs for the treatment of colon and pancreatic cancer.
• Carcinogenesis in the colon/rectum is thought to occur through two different pathways.
• the existing model suggests that 80-85% of colon cancers involve chromosomal instability, where point mutations are found in loci within RAS, p53, and other checkpoint proteins. (Boland et al., Gastroenterology 2000, 118, S115-S128; Carethers et al., Gastroenterology 1999, 117, 123-131).
• the remaining 15-20% of colon cancers involve a loss in the DNA mismatch repair system, which leads to point mutations in repetitive sequences. These repetitive sequences are known as microsatellites, and occur in several important growth regulators.
• MSI colon cancers are resistant to current chemotherapeutic drugs and MSS colon cancers are treated with a relatively toxic drug.
• MSS colon cancers are known to respond to chemotherapeutic drugs.
• MSI colon cancers do not respond to 5-FU, or to current chemotherapeutic drugs, finding new structures that target both cancer pathways would be very valuable.
• San A is known to inhibit Topoisomerase I activity, which is important for DNA replication, repair, and transcription. (Hwang et al., Molecular Pharmacology
• Hsp90 Heat shock protein, Hsp90, functions as a molecular chaperone for intracellular signaling molecules.
• Hsp90 There are two isoforms of Hsp90, alpha and beta. Because it folds, assembles, and stabilizes proteins that regulate the growth of cancer cells, both Hsp90 isoforms are up-regulated in most cancers.
• the N-terminal domain contains the ATP binding site, which is the binding site for compounds targeting Hsp90 that are currently in clinical trials. Inhibitors of Hsp90 successfully stop cancer cell growth; thus they have outstanding potential as anticancer therapeutics.
• N-methyl moieties were responsible for activity in cancer cell lines. See Liu et al., /. Med. Chem. 2005; 48:3630-38.
• the invention provides novel cyclic pentapeptides, and methods for their preparation and use as anti-cell proliferative and/or anticancer agents; thus, the invention also provides pharmaceutical preparations and formulations comprising compounds of this invention.
• compounds of the present invention comprise cyclic peptides related to San A, which have a cyclic peptide backbone comprising five amino acid residues.
• compounds of the invention represent a novel structural class that targets cancers, including pancreatic cancers, colon cancers such as MSS and MSI forms of colon cancer, and other cancers and cell proliferative conditions.
• compositions and formulations comprising one or more compositions of this invention, e.g., the cyclic pentapeptides of this invention, and a pharmaceutically acceptable excipient.
• Such compositions are useful for the treatment of cell proliferative diseases and conditions, such as cancers, for example, pancreatic cancer and colon cancer, e.g., MSS colon cancer or MSI colon cancer, rectal cancer, breast cancer, prostate cancer, and/or melanoma.
• the invention provides a cyclic pentapeptide of formula (I):
• each of R 1 -, R 2 -, R 3 -, R 4 -, and Rs- independently represents H, or C1-C4 alkyl; and wherein R 1 - may cyclize with R 1 to form a 5-10 membered azacyclic ring;
• R 1 represents a C1-C4 alkyl, C5-C12 arylalkyl, C5-C12 heteroarylalkyl, or C1-C6 aminoalkyl group, each of which may be optionally substituted; or R 1 may cyclize with R 1 - to form a 5-10 membered azacyclic ring; and each of R 2 , R 3 , R 4 , and R 5 independently represents H, or C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 cycloalkylalkyl, C1-C8 heterocyclylalkyl, C1-C6 aminoalkyl, C5-C12 arylalkyl, or a heteroform of one of these, each of which may be optionally substituted; with the proviso that the compound of formula (I) is not cyclo[-Phe-Leu-Val-Leu-Leu-] or cyclo[-pBrP
• the invention provides a cyclic peptoid of formula (II):
• each of R 11 , R 12 , R 13 , R 14 and R 15 is independently selected from H, or C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C6 aminoalkyl, C5-C12 arylalkyl, or a heteroform of one of these, each of which may be optionally substituted.
• the invention provides pharmaceutical compositions comprising one of more cyclic pentapeptides of formula (I) and (II), or a pharmaceutically acceptable salt or hydrate form thereof, and at least one pharmaceutically acceptable excipient.
• the invention provides processes for the manufacture of cyclic pentapeptides of formula (I) and to pharmaceutical compositions comprising them, and to their use in the manufacture of medicaments for use in the production of an anticancer effect in a warm-blooded animal such as man.
• the invention provides methods of treating, ameliorating or preventing (prophylaxis of) (including preventing a recurrence of) a cancer, such as colon cancers, e.g.,
• MSS or MSI colon cancers pancreatic cancer, rectal cancer, breast cancer, prostate cancer, and melanoma, by administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition of formula (I) or formula (II).
• the invention provides modified forms of compounds of formula (I) and (II), including peptides labeled with biotin or a fluorophoric groups, such as rhodamine, as well as peptides coupled to stabilizing or targeting agents, and to methods of using these compounds and formulations.
• biotinylated peptides of the present invention are useful for example in affinity assays. Fluorescently labeled peptides are useful, for example, to study the mechanism of action of compounds of the invention.
• biotin or rhodamine are linked to a lysine residue in the cyclic pentapeptides backbone via an alkylene or heteroalkylene linkage.
• the invention provides methods to synthesize compounds of formula (I) and formula (II), and/or their pharmaceutically acceptable salt or hydrate forms.
• kits comprising compositions of the invention (e.g., the pharmaceutical compositions, formulations), including instruction means for practicing the methods of the invention.
• the present invention provides novel cyclic pentapeptides comprising both D- and L- amino acids in their cyclic backbone. These cyclic pentapeptides generally have good aqueous solubility and enhanced stability over their linear counterparts. Additionally, the incorporation of unnatural amino acid residues into the cyclic peptide backbones, particularly the inclusion of D- amino acids, further enhances their stability against proteases.
• the compounds of the invention possess a unique chemical structure and represent a novel class of anticancer and anti-cell growth/ cell proliferative therapeutic agents. In one aspect, these compounds demonstrate cytotoxicity against a variety of cancer cell lines. In one aspect, compounds of the invention are cytotoxic against cancer cells, including colon cancer such as colon cancer MSS cells and cell lines, colon cancer MSI cells and cell lines
• chemotherapeutically resistant strains pancreatic cancer cells and cell lines, rectal cancer cells and cell lines and breast cancer cells and cell lines.
• compounds of the invention demonstrate cytotoxicity against chemotherapeutically resistant MSI colon cancer cells and pancreatic cancer cells, difficult-to-cure cancers for which no effective treatments are currently available.
• These cyclic pentapeptides demonstrate cytotoxicity comparable to 5-FU against MSS colon cancer. Further, the cyclic pentapeptides are also potent against MSI colon cancer.
• affinity chromatography experiments biotin-labeled cyclic pentapeptides of the present invention were surprisingly found to bind to Hsp90.
• compounds of the invention appear to bind to a unique region on the C-terminus of Hsp90.
• compounds of the present invention may demonstrate their anticancer effects by interaction with Hsp90, a well-established oncogenic, representing an innovative approach towards treatment of these cancers.
• the compounds of the present invention target drug-resistant cancers, e.g., colon and pancreatic cancer cells.
• incorporation of a single N-methyl and/or a single D-amino acid leads to significantly improved potency against a cancer, e.g., a colon and pancreatic cancer cell in vivo or a cell lines.
• compounds wherein a single L- amino acid is exchanged with a D-amino acid at amino acid residue 2 and/or 3 and/or 5 exhibited significantly enhanced potency against colon cancer cell lines relative to the corresponding peptides comprising all-L amino acids (including those that comprise N-methyl moieties) or all-D amino acids.
• San A derivatives comprising a single D-amino acid exhibit excellent "drug-like" potency as antitumor agents, and this structure- activity relationship (SAR) is general for the two types of colon cancers (MSS and MSI).
• the invention provides a use of at least one compound of the invention, or a compound made by a method of the invention, for the preparation of a pharmaceutical or a veterinary composition.
• the invention provides a use of at least one compound of the invention, or a compound made by a method of the invention, for the preparation of a pharmaceutical or a veterinary composition to treat, ameliorate or prevent a skin condition, psoriasis, a hormone-dependent tumor or a hormone-influenced non-malignant disorder, benign prostate hyperplasia (BPH), endometriosis; a disease or condition having an inflammatory component, an autoimmune disease, rheumatoid arthritis, an infectious disease, diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, psoriasis, a cancer, a lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix
• Figure 1 shows representative examples of cyclic pentapeptides which comprise only L- amino acid residues in their cyclic peptide backbones.
• Figure 2 shows representative examples of cyclic pentapeptides which comprise only D- amino acid residues in their cyclic peptide backbones.
• Figure 3 shows representative examples of cyclic pentapeptides which comprise one D- amino acid residue and four other amino acid residues in their cyclic peptide backbones.
• Figure 4 shows representative examples of cyclic pentapeptides which comprise more than one D-amino acid residues and one or more other amino acid residues in their cyclic peptide backbones.
• Figure 5 shows representative examples of additional compounds of the invention, including cyclic pentapeptides which comprise all-L amino acids, as well as examples having one or more D-amino acid residues.
• Figure 6 shows the inhibitory activities of various cyclic pentapeptides against HT-29 (MSS colon), SW-480 (MSS colon), HCT-116 (MSI colon), and PL-45 (pancreatic) cancer cell lines.
• Figure 7 shows compounds with changes at position 1 run in three cell lines: HCT-116 and HCT- 15 colon cancer and PL-45 pancreatic cancer cell lines. Data represents results from at least 3 separate experiments and each performed in quadruplicate. Margin of error is + 5%.
• Figure 8 shows compounds with changes at position 2 run in three cell lines: HCT-116 and HCT- 15 colon cancer and PL-45 pancreatic cancer cell lines. Data represents results from at least 3 separate experiments and each performed in quadruplicate. Margin of error is + 5%.
• Figure 9 shows compounds with changes at position 3 run in three cell lines: HCT-116 and HCT- 15 colon cancer and PL-45 pancreatic cancer cell lines. Data represents results from at least 3 separate experiments and each performed in quadruplicate. Margin of error is + 5%.
• Figure 10 shows compounds with changes at position 4 run in three cell lines: HCT-116 and HCT-15 colon cancer and PL-45 pancreatic cancer cell lines. Data represents results from at least 3 separate experiments and each performed in quadruplicate. Margin of error is + 5%.
• Figure 11 shows compounds with changes at position 5 run in three cell lines: HCT-116 and HCT-15 colon cancer and PL-45 pancreatic cancer cell lines. Data represents results from at least 3 separate experiments and each performed in quadruplicate. Margin of error is + 5%.
• Figure 12 shows compounds comprising all L- or all D-amino acids combined with N-methylated amino acids run in three cell lines: HCT-116 and HCT-15 colon cancer and PL-45 pancreatic cancer cell lines. Data represents results from at least 3 separate experiments and each performed in quadruplicate. Margin of error is + 5%.
• Figure 13 shows IC 50 S of compounds run in three cell lines: HCT-116 and HCT-15 (colon) and PL-45 (pancreatic). Data represents results from at least 3 separate experiments and each performed in quadruplicate. Margin of error is + 5%. 200 ⁇ M is the outside limit of detection.
• Figure 15 shows results from the Annexin V assay.
• Panel 15(a) shows cells + 1% DMSO only at 90 minutes.
• Panel 15(b) show cells + 1% DMSO + 50 ⁇ M compound (55) at 90 minutes.
• Figure 16 shows fluorophore-labeled compound (24) incubated with PL-45 cancer cells.
• Dapi was used to stain the nucleus and the picture using the Dapi wavelength filter was overlaid on top of the picture using the Rhodamine wavelength filter (16b).
• Panel 16(a) shows 24- fluorophore is visible in cells.
• Panel 16(b) shows a Dapi filtered view of cells overlaid on top of the Rhodamine filtered view.
• compositions of the invention have anti-cancer activity, anti-cell-proliferative, anti-cell migration and/or apoptotic activity.
• compounds of the invention are useful in methods of treatment for a subject afflicted with any disease or condition comprising cell proliferation, e.g., a cancer or an infection that results in unwanted cell growth.
• the invention also provides methods for the preparation of novel cyclic pentapeptides, and pharmaceutically acceptable formulations thereof, and to their use as pharmaceuticals, e.g., as anti-cell growth agents, as anticancer agents and the like.
• Use of the pharmaceuticals of the invention can be for ameliorating (treating) any disease or condition comprising cell proliferation, e.g., a cancer or an infection, or for ameliorating or preventing (prophylaxis of) (including preventing a recurrence of) their onset or recurrence, or for ameliorating or preventing side effects such as unwanted cell proliferation or hyperplasia.
• the invention also provides processes for the manufacture of cyclic pentapeptides of this invention, to pharmaceutical compositions (e.g., formulations) comprising them and to their use in the manufacture of medicaments for use in the production of an anticancer, anti-cell- proliferation/migration and/or apoptotic effects in any individual, e.g., any warm-blooded animal such as man or animal, including veterinary uses.
• pharmaceutical compositions e.g., formulations
• cyclic pentapeptides and pharmaceutical compositions of this invention can be useful to treat, prevent or ameliorate cancers, including, but not limited to, MSS and MSI colon cancers, pancreatic cancer, rectal cancer, breast cancer, prostate cancer, brain cancer, liver cancer, and/or melanoma or any other skin cancer, leukemias, and the like.
• cancers including, but not limited to, MSS and MSI colon cancers, pancreatic cancer, rectal cancer, breast cancer, prostate cancer, brain cancer, liver cancer, and/or melanoma or any other skin cancer, leukemias, and the like.
• the cyclic pentapeptides and pharmaceutical compositions of this invention can be useful to treat, prevent or ameliorate any cell proliferative condition, e.g., a skin condition such as psoriasis, or a hormone-dependent tumor or a hormone-influenced non-malignant disorder such as benign prostate hyperplasia (BPH) and endometriosis; or any disease or condition having an inflammatory component, e.g., an autoimmune disease such as rheumatoid arthritis, or an infectious disease; including treating, preventing or ameliorating any disease states associated with unwanted angiogenesis and/or cellular proliferation, such as diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, and psoriasis.
• a cell proliferative condition e.g., a skin condition such as psoriasis, or a hormone-dependent tumor or a hormone-influenced non-malignant disorder such as benign prostate hyperplasia (BPH)
• Cancers that can be treated, prevented or ameliorated by using compositions of this invention include lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (
• the term “residue” refers to a particular amino acid that is incorporated into the pentapeptide backbone of the present invention.
• the amino acid residue bearing the substituent R 1 in formula (I) is sometimes referred to herein as “residue 1" or alternatively as “position 1".
• the amino acid residue bearing the substituent R 2 is sometimes referred to herein as “residue 2" or “position 2”, and so on around the pentapeptide ring, up to the amino acid bearing substituent R 5 , which is sometimes referred to herein as “residue 5" or "position 5".
• alkyl includes straight-chain, branched-chain and cyclic monovalent hydrocarbyl radicals, and combinations of these, which contain only C and H when they are unsubstituted.
• Alkyl groups may be optionally unsaturated, such as in alkenyl or alkynyl groups. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like.
• the total number of carbon atoms in each such group is sometimes described herein, e.g., when the group can contain up to ten carbon atoms it may be described as 1-lOC or as Cl-ClO or as Cl-10 or as C 1-1 O.
• alkenyl and alkynyl groups are defined similarly to alkyl groups, and include straight-chain, branched-chain and cyclic monovalent hydrocarbyl radicals, and combinations of these, which contain only C and H when they are unsubstituted.
• alkenyl groups contain one or more carbon-carbon double bonds
• alkynyl groups contain one or more carbon-carbon triple bonds.
• the alkyl, alkenyl and alkynyl substituents of the invention contain 1-8C (alkyl) or 2-8C (alkenyl or alkynyl). Preferably they contain 1-4C (alkyl) or 2-4C (alkenyl or alkynyl).
• Alkyl, alkenyl and alkynyl groups are often substituted to the extent that such substitution makes sense chemically.
• Heteroalkyl “heteroalkenyl”, and “hetero alkynyl” and the like are defined similarly to the corresponding hydrocarbyl (alkyl, alkenyl and alkynyl) groups, but the 'hetero' terms refer to groups that contain one or more heteroatoms selected from O, S and N and combinations thereof, within the backbone residue.
• heteroatoms typically N, O and S
• the numbers describing the group though still written as e.g.
• C1-C6 represent the sum of the number of carbon atoms in the group plus the number of such heteroatoms that are included as replacements for carbon atoms in the ring or chain being described.
• Such heteroalkyl groups may be optionally substituted with the same substituents as alkyl groups.
• N the nitrogen atom may be present as NH or it may be substituted if the heteroalkyl or similar group is described as optionally substituted.
• S the sulfur atom may optionally be oxidized to SO or SO 2 unless otherwise indicated.
• such groups do not include more than two contiguous heteroatoms as part of the heteroalkyl chain, although an oxo group may be present on N or S as in a nitro or sulfonyl group.
• alkyl in one aspect includes cycloalkyl and cycloalkylalkyl groups, the term
• cycloalkyl may be used herein to specifically describe a carbocyclic non-aromatic group that is connected via a ring carbon atom, and "cycloalkylalkyl” may be used to describe a carbocyclic non-aromatic group that is connected to the base molecule through an alkyl linker.
• cyclohexylalanine (Cha) comprises a cycloalkylalkyl substituent.
• heterocyclyl may be used to describe a non-aromatic cyclic group that contains at least one heteroatom as a ring member and that is connected to the molecule via a ring atom of the cyclic group, which may be C or N; and “heterocyclylalkyl” may be used to describe such a group that is connected to another molecule through an alkyl linker.
• the sizes and substituents that are suitable for the cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the same as those described above for alkyl groups. Where an alkyl group is substituted with an aryl or heteroaryl group, it is referred to as an arylalkyl or heteroarylalkyl substituent.
• an "aromatic” moiety or “aryl” moiety refers to a monocyclic or fused bicyclic moiety having the well-known characteristics of aromaticity; examples include phenyl and naphthyl.
• “heteroaromatic” and “heteroaryl” refer to such monocyclic or fused bicyclic ring systems which contain as ring members one or more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits aromaticity in 5-membered rings as well as 6-membered rings.
• Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, imidazolyl, triazolyl, thiadiazolyl, oxadiazolyl, and tetrazolyl rings, and the fused bicyclic moieties formed by fusing one of these monocyclic groups with a phenyl ring or with any of the heteroaromatic monocyclic groups to form a C8- ClO bicyclic group such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolinyl, quinolinyl, benzothiazolyl, benzofuranyl,
• any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition. It also includes bicyclic groups where at least one ring has the characteristics of aromaticity, even though it may be fused to a nonaromatic ring.
• the ring systems contain 5-12 ring member atoms.
• the monocyclic heteroaryl groups contain 5-6 ring members, and the bicyclic heteroaryls contain 8-10 ring members.
• Aryl and heteroaryl moieties may be substituted with a variety of substituents which are known in the art.
• Preferred substituents include, but are not limited to, halo, C1-C8 alkyl, -NO 2 , -CN, -OR', -SR', -COOR', -C(O)NR' 2 , and -NR' 2 , where each R' independently represents H, C1-C4 alkyl or C5-C12 arylalkyl, or a heteroform of one of these.
• arylalkyl and “heteroarylalkyl” refer to aromatic and heteroaromatic ring systems which are bonded to their attachment point through a linking group such as an alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers.
• a linking group such as an alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers.
• the linker is C1-C8 alkyl or a hetero form thereof.
• These linkers may also include a carbonyl group, thus making them able to provide substituents as an acyl or heteroacyl moieties.
• Heteroarylalkyl refers to a moiety comprising an aryl group that is attached through a linking group, and differs from “arylalkyl” in that at least one ring atom of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O and S.
• an aryl or heteroaryl ring in an arylalkyl or heteroarylalkyl group may be optionally substituted on the aromatic portion with the same substituents described above for aryl groups.
• an arylalkyl group includes a phenyl ring and a heteroarylalkyl group includes a C5-C6 monocyclic or C8-C10 fused bicyclic heteroaromatic ring, each of which may be optionally substituted with the groups defined above for aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups, where the alkyl groups can optionally cyclize to form a ring, and wherein the alkyl or heteroalkyl groups may be optionally fluorinated.
• the arylalkyl or heteroarylalkyl ring comprises a phenol or an indole ring.
• Preferred substituents on phenyl include
• Arylalkyl and “heteroarylalkyl” groups are described by the total number of carbon atoms in the ring and alkylene or similar linker. Thus a benzyl group is a C7-arylalkyl group, and phenethyl is a C8-arylalkyl group.
• Alkylene in one aspect refers to a divalent hydrocarbyl group; because it is divalent, it can link two other groups together.
• n 1-8 and preferably n is 1-4
• an alkylene can also be substituted by other groups, and can be of other lengths, and the open valences need not be at opposite ends of a chain.
• -CH(Me)- and -C(Me) 2 - may also be referred to as alkylenes, as can a cyclic group such as cyclopropan- 1,1-diyl.
• a three-atom linker that is an alkylene group refers to a divalent group in which the available valences for attachment to other groups are separated by three atoms such as -(CH 2 ) 3 -, i.e., the specified length represents the number of atoms linking the attachment points rather than the total number of atoms in the hydrocarbyl group: -C(Me) 2 - would thus be a one-atom linker, since the available valences are separated by only one atom.
• the alkylene may contain one or more double or triple bonds.
• "Heteroalkylene” in one aspect is defined similarly to the corresponding alkylene groups, but the 'hetero' terms refer to groups that contain one or more heteroatoms selected from O, S and N and combinations thereof, within the backbone residue; thus at least one carbon atom of a corresponding alkylene group is replaced by one of the specified heteroatoms to form a heteroalkylene group.
• an "aminoalkyl” group refers to a C1-C6 alkyl group that is substituted with at least one amine group having the formula -NR2, where each R is independently H, Cl- C8 alkyl, C5-C12 aryl and C5-C12 arylalkyl, or a heteroform of one of these.
• Such aminoalkyl groups may be optionally substituted on the alkyl portion with one or more other groups suitable as substituents for an alkyl group.
• the aminoalkyl substituent is a 1- aminoalkyl group such as a 1-aminomethyl, 1-aminoethyl, 1-aminopropyl or 1-aminobutyl group.
• the aminoalkyl group may comprise a protected amine.
• suitable amine protecting groups may vary depending on the functionality present in the particular monomer.
• protected amines may include, for example, carbamates (e.g. tert-butoxycarbonyl, benzyloxycarbonyl, fluorenylmethyloxy- carbonyl, allyloxycarbonyl or (trialkylsilyl)ethoxycarbonyl), carboxamides (e.g. formyl, acyl or trifluoroacetyl), sulfonamides, phthalimides, Schiff base derivatives, and the like.
• carbamates e.g. tert-butoxycarbonyl, benzyloxycarbonyl, fluorenylmethyloxy- carbonyl, allyloxycarbonyl or (trialkylsilyl)ethoxycarbonyl
• carboxamides e.g. formyl, acyl or trifluoroacetyl
• an aminoalkyl group may be coupled through an alkylene or heteroalkylene linker to a group such as biotin, or a fluorophore-containing group, such as rhodamine, and such compounds may be useful for screening or mechanistic studies.
• Heteroform in one aspect refers to a derivative of a group such as an alkyl, aryl, or acyl, wherein at least one carbon atom of the designated carbocyclic group has been replaced by a heteroatom selected from N, O and S.
• the heteroforms of alkyl, alkenyl, alkynyl, acyl, aryl, and arylalkyl are heteroalkyl, heteroalkenyl, heteroalkynyl, heteroacyl, heteroaryl, and heteroarylalkyl, respectively. It is understood that no more than two N, O or S atoms are ordinarily connected sequentially, except where an oxo group is attached to N or S to form a nitro or sulfonyl group.
• Halo in one aspect includes fluoro, chloro, bromo and iodo. Fluoro and chloro are often preferred.
• Amino in one aspect refers to NR' 2 wherein each R' is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl group or a heteroform of one of these groups, as defined above, each of which may be optionally substituted with the substituents described herein as suitable for the corresponding type of group.
• the two R' groups on one nitrogen atom may be linked together to form an azacyclic ring.
• an 'azacyclic' group refers to a heterocyclic group containing at least one nitrogen atom as a ring atom, wherein the group is attached to the base molecule through a nitrogen atom of the azacyclic group.
• azacyclic groups are 3-8 membered monocyclic rings or 8-12 membered bicyclic fused ring systems, and may be saturated, unsaturated or aromatic and may contain a total of 1-3 heteroatoms independently selected from N, O and S as ring members.
• an azacyclic ring may comprise a nitrogen-containing ring fused to a phenyl ring.
• the unnatural amino acid "Tic" comprises a tetrahydroisoquinoline ring, which represents a 10-membered fused bicyclic azacyclic group.
• a “therapeutically effective amount” means that amount of a drug or pharmaceutical agent that will elicit desired therapeutic effect, biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician.
• therapeutically effective amount means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
• the term also includes within its scope amounts effective to enhance normal physiological function.
• subject refers to a human or other warm-blooded animal subject.
• peptide and polypeptide are used interchangeably and refer to a compound made up of a chain of amino acid residues linked by peptide bonds. Unless otherwise indicated, the sequence for peptides is given in the order from the amino terminus to the carboxyl terminus. In certain embodiments, one or more amino acids in the peptide are D- amino acids.
• the cyclic pentapeptides of the invention have the structures and/or sequences described herein with at least one conservative amino acid substitution, where such compounds retain their activity, e.g., retain their anticancer activity and activity comprising anti- cell-proliferative, anti-cell migration and/or apoptotic (promoting) activity.
• the "conservative amino acid substitutions" are substitutions which do not result in a significant change in the activity or tertiary structure of a selected polypeptide or protein.
• the substitutions typically involve replacing a selected amino acid residue with a different residue having similar physico-chemical properties. Groupings of amino acids by physico-chemical properties are known to those of skill in the art.
• the "conservative amino acid substitutions” comprises exchange of residues between families of amino acid residues having similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), neutral polar side chains (e.g., asparagine, cysteine, glutamine, serine, threonine, tyrosine), neutral nonpolar side chains (e.g., alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine), beta-branched side chains (e.g., isoleucine, threonine, valine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
• basic side chains e.g., lysine, arginine, histidine
• references herein to cyclic pentapeptides are also meant to include their stereoisomers, pharmaceutically acceptable salts and hydrate or solvate forms, as well as pharmaceutical compositions and formulations thereof.
• therapeutically active metabolites where the metabolites themselves fall within the scope of the claimed invention, are also compounds of the current invention.
• Prodrugs which are compounds that are converted to the claimed compounds as they are being administered to a patient or after they have been administered to a patient, are also compounds of this invention.
• the cyclic pentapeptides of the present invention have a cyclic peptide backbone which comprises five amino acid residues.
• the compounds of the invention can comprise natural and/or unnatural amino acids. Suitable unnatural amino acids can include, but are not limited to, D-amino acids and N-alkylated amino acids, especially N-methylated amino acids.
• D-amino acids may be denoted herein as, for example, DXaa, D-Xaa or (D)-Xaa
• N-alkylated amino acids may be referred to herein as NR"Xaa, where R" corresponds to the N- alkyl substituent and Xaa corresponds to the particular amino acid residue.
• N- methyl valine may sometimes be referred to herein as NMeVaI
• (D)-valine may sometimes be referred to herein as DVaI.
• amino acid is used in the conventional sense to refer to an organic chemical compound comprising at least one amino group (i.e., — NH2 or — NRNH) and at least one carboxylic acid group (i.e., — COOH).
• an amino group may be a substituted amino group (i.e., -NRNH, where RN IS a nitrogen substituent), for example, as in the case of proline.
• amino acids are often denoted herein as AA, or as H — AA — OH, where the initial — H is part of an amino group, and the final — OH is part of a carboxylic acid group.
• Amino acids may often be conveniently further classified according to their structure, for example, as alpha-amino acids, beta-amino acids, and the like.
• alpha amino acid is used in the conventional sense to refer to amino acids in which at least one carboxylic acid group (i.e., — COOH) and at least one amino group (i.e., — NH2or — NRNH) are directly attached to a single carbon atom (designated the alpha carbon) and may be conveniently denoted HNRN — CRARB — COOH, wherein RN, RA and RB are substituents.
• Two or more of the substituents RN, RA and RB may together form a single multivalent substituent, thus a cyclic alpha amino acid.
• RN and RA together form the single divalent substituent — CH2CH2CH2 —
• RB is — H.
• the alpha carbon will be chiral (i.e., R or S), and the alpha-amino acid will be optically active.
• R or S the alpha-amino acid
• glycine for which RA and RB are both — H
• alanine for which RA is — CH3 and RB is — H
• D-alanine or L-alanine is optically active and may be in D- or L-forms, denoted D-alanine or L-alanine, respectively.
• the alpha carbon of D-alanine is in the R configuration whereas the alpha carbon of L-alanine is in the S configuration.
• alpha-amino acids Of the wide variety of alpha-amino acids known, only about twenty are naturally occurring. Naturally occurring alpha-amino acids are often denoted HNRN — CHR — COOH (since RB is — H) where RN denotes a nitrogen substituent and R denotes an amino acid substituent (often referred to as an amino acid sidechain).
• amino acid substituents include those substituents found in the twenty naturally occurring alpha amino acids, such as, for example, — H (glycine, G, GIy), — CH3 (alanine, A, Ala), — CH2OH (serine, S, Ser), -CH(CH 3 )OH (threonine, T, Thr), CH2SH (cysteine, C, Cys), and — CH2C6 Hs (phenylalanine, F, Phe).
• Other examples of amino acid substituents include those which are structurally similar to those substituents found in the naturally occurring amino acids, such as, for example, — CH2CH2OH (homoserine) and — CH2CH2SH (homocysteine).
• the naturally occurring amino acids are often represented by a three letter code or a one-letter code.
• the three-letter and one-letter codes for the twenty naturally occurring acids are well established in the art, and the standard conventions are used herein.
• amino acids In addition to an alpha carboxylic acid group (i.e., — COOH) and an alpha amino group (i.e., — NH2or — NRNH), many amino acids have additional functional groups. Lysine, for which the amino acid substituent, R, is — (CH2)4NH2, has an additional amino group (i.e., — NH2). Aspartic acid and glutamic acid, for which the amino acid substituents, R, are —
• amino acids have other additional functional groups, including, for example, thioether groups (e.g., in methionine), phenol groups (e.g., in tyrosine), amide groups (e.g., in glutamine), and heterocyclic groups (e.g., in histidine).
• thioether groups e.g., in methionine
• phenol groups e.g., in tyrosine
• amide groups e.g., in glutamine
• heterocyclic groups e.g., in histidine
• alpha amino acids In addition to the twenty naturally occurring amino acids, several other classes of alpha amino acids are also known. Examples of these other classes include D-amino acids, N ⁇ alkyl amino acids, alpha-alkyl amino acids, cyclic amino acids, chimeric amino acids, and miscellaneous amino acids. These non-natural amino acids have been widely used to modify bioactive polypeptides to enhance resistance to proteolytic degradation and/or to impart conformational constraints to improve biological activity (Hruby et al., Biochem. J. (1990) 268:249-262; Hruby and Bonner, Methods in Molecular Biology (1994) 35:201-240).
• N ⁇ -alkyl amino acids are the N ⁇ -methyl amino acids, such as, N ⁇ -methyl glycine (i.e., NMeGIy, sarcosine, Sar), N ⁇ -methyl alanine (i.e., NMeAIa), and N ⁇ -methyl lysine (i.e., NMeLys).
• N ⁇ -methyl glycine i.e., NMeGIy, sarcosine, Sar
• N ⁇ -methyl alanine i.e., NMeAIa
• N ⁇ -methyl lysine i.e., NMeLys
• other N ⁇ -methyl amino acids including N ⁇ -methyl valine (i.e., NMeVaI), N ⁇ -methyl leucine (i.e., NMeLeu), and N ⁇ -methyl phenylalanine (i.e., NMePhe).
• alpha- alkyl amino acids examples include alpha- aminoisobutyric acid (i.e., Aib), diethylglycine (i.e., Deg), diphenylglycine (i.e., Dpg), alpha-methyl proline (i.e., (ocMe)Pro), and alpha-methyl valine (i.e., ( ⁇ Me)Val) (Balaram, Pure & Appl. Chem. (1992) 64:1061-1066; Toniolo et al., Biopolymers (1993) 33:1061-1072; Hinds et al., J. Med. Chem. (1991) 34:1777- 1789).
• alpha- aminoisobutyric acid i.e., Aib
• Deg diethylglycine
• Dpg diphenylglycine
• alpha-methyl proline i.e., (ocMe)Pro
• alpha-methyl valine i.e
• cyclic amino acids include 1-amino-l -cyclopropane carboxylic acid, 1- amino-1-cyclopentane carboxylic acid (i.e., cyclic leucine), aminoindane carboxylic acid (i.e., Ind), tetrahydroisoquinolinecarboxylic acid (i.e., Tic) and tetrahydrocarbolinecarboxylic acid (i.e., Tea) (Toniolo, C, Int. J. Peptide Protein Res. (1990) 35:287-300; Burgess, K., Ho, K. K., and Pal, B. J. Am. Chem. Soc. (1995) 117:3808-3819).
• alkenyl and alkynyl containing amino acids such as propargylglycine, dehydroalanine, and the like.
• chimeric amino acids include penicillamine (i.e., Pen), combination of cysteine with valine, and 4-mercaptoproline (i.e., Mpt), combination of proline and homocysteine.
• miscellaneous alpha-amino acids include ornithine (i.e., Orn), 2-naphthylalanine (i.e., 2-Nal), phenylglycine (i.e., Phg), t-butylglycine (i.e., tBug), alpha-ethylglycine (i.e., (ocEt)Gly), alpha-n- propylglycine (i.e., ( ⁇ Pr)Gly), alpha-n-butylglycine (i.e., nBug), O-benzylserine (i.e., (OBzl)Ser), p-bromophenylalanine (i.e., pBrPhe), cyclohexylalanine (i.e., Cha), and alpha- amino-2-thiophenepropionic acid (i.e., Thi).
• ornithine i.e., Or
• alpha-amino acids other such as beta amino acids
• beta amino acids can also be used in the present invention.
• these other amino acids include 2-aminobenzoic acid (i.e., Abz), .beta-aminopropanoic acid (i.e., .beta-Apr), .gamma-aminobutyric acid (i.e., gamma-Abu), and 6-aminohexanoic acid (i.e., epsilon-Ahx).
• 2-aminobenzoic acid i.e., Abz
• .beta-aminopropanoic acid i.e., .beta-Apr
• gamma-aminobutyric acid i.e., gamma-Abu
• 6-aminohexanoic acid i.e., epsilon-Ahx
• the cyclic pentapeptide of the present invention may be synthesized by synthesizing a linear peptide with the same peptide sequence and then cyclizing the linear peptide.
• amino acid-containing species e.g., polypeptides
• a wide variety of protecting groups and strategies are known in the art.
• an alpha-amino group i.e., — NH2
• a 9 fluorenylmethyloxycarbonyl group i.e., Fmoc; as — NHFmoc
• Pmc 2,2,5,7, 8-pentamethylchroman-6-sulfonyl group
• Mtr 4-methoxy-2,3,6-trimethylbenzenesulfonyl group
• Mts mesitylene-2-sulfonyl group
• the carboxamide groups of asparagine and glutamine may be protected with a trityl group (i.e., — C(C6H5)3, Tr; as -CONHTr).
• the side chain carboxylic acid groups of aspartic and glutamic acid may be protected with a t-butyl group (i.e., — C(CH3)3, tBu; as -COOtBu) or a cyclohexyl group (i.e., — C6H11, cHx; as —
• carboxylic acid groups such as terminal carboxylic acid groups, may be protected with a methyl group (i.e., — CH3, as — C00CH3), an ethyl group (i.e., — CH2CH3, as COOCH2CH3), or a benzyl group (i.e., — CH2C6H5, as — COOCH2C6H5).
• a methyl group i.e., — CH3, as — C00CH3
• an ethyl group i.e., — CH2CH3, as COOCH2CH3
• a benzyl group i.e., — CH2C6H5, as — COOCH2C6H5
• the thiol group of cysteine i.e., — SH
• a t-butylthio group i.e., — SC(CH3)3, tBuS; as — SStBu
• a trityl group i.e., — C(C6H5)3, Tr; as — STr
• the imidazole group of histidine may be protected with a trityl group (i.e., — C(C6H5)3, Tr; as — STr).
• the hydroxyl groups of homoserine, serine and threonine may be protected with a t-butyl group (i.e., — C(CH3)3, tBu; as — OtBu), a trityl group (i.e., — C(C6H5)3, Tr; as — OTr), or a t-butyldimethylsilyl group (i.e., — Si(CH3)2(C(CH3)3), TBDMS; as — OTBDMS).
• the indole nitrogen of tryptophan may be protected with a trityl group (i.e., — C(C6H5)3, Tr).
• the hydroxyl group of tyrosine i.e., — H
• a trityl group i.e., — C(C6H5)3, Tr; as — OTr.
• a carboxylic acid group i.e., — COOH
• an amino group i.e., — NRRH
• a polypeptide e.g., a "2-mer" of the two amino acids serine and cysteine (wherein the carboxylic acid group of serine and the amino group of cysteine have formed a peptide linkage) may conveniently be represented as H-Ser — Cys — OH or H — S — C — OH, or, more simply, as Ser — Cys, S — C, or SC.
• the amino acid moieties of a polypeptide are often referred to as amino acid residues.
• the invention provides a compound of formula (I):
• each of R 1 -, R 2 -, R 3 -, R 4 -, and Rs- independently represents H, or C1-C4 alkyl or C1-C4 heteroalkyl.
• each of R 1 -, R 2 -, R 3 -, R 4 -, and R 5 - independently represents H or methyl.
• any one of R 1 -, R 2 -, R 3 -, R 4 -, and R 5 is methyl, and the other four of R 1 -, R 2 -, R 3 -, R 4 -, and R 5 are H.
• each of R 1 -, R 2 -, R 3 -, R 4 -, and R 5 is H.
• R 1 - may cyclize with R 1 to form a 5-10 membered azacyclic ring.
• R 1 - may cyclize with R 1 to form a tetrahydroisoquinoline ring.
• R 1 represents a C5-C12 arylalkyl, C5-C12 heteroarylalkyl, or C1-C6 aminoalkyl group, each of which may be optionally substituted. In certain embodiments, R 1 represents an optionally substituted C5-C12 arylalkyl group. In preferred embodiments, R 1 represents CH 2 ArX, where Ar represents a phenyl ring and X is selected from H, halo, OH and C1-C4 alkoxy. In some embodiments, R 1 may cyclize with R 1 - to form a 5-10 membered azacylic ring.
• each of R 2 , R 3 , R 4 , and R 5 independently represents H, or C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 cycloalkylalkyl, C1-C6 aminoalkyl, C5- C12 arylalkyl, or a heteroform of one of these, each of which may be optionally substituted.
• each of R 2 , R 3 , R 4 , and R 5 independently represents H, methyl, ethyl, n- propyl, isopropyl, n-butyl, sec -butyl, isobutyl, tert-butyl, CH 2 -cyclohexyl, CH 2 OH, CH 2 OBzI, or CH 2 ArX, where Ar represents a phenyl ring and X is selected from H, halo, OH, and C1-C4 alkoxy.
• the carbon atom bearing R 1 has the (R)-configuration and the carbon atoms bearing R 2 , R 3 , R 4 , and R 5 have the (S)-configuration.
• R 1 is CH 2 ArX, where Ar represents a phenyl ring and X is selected from H, OH, OMe, Br, or Cl.
• R 2 is sometimes a C1-C4 alkyl or C5-C12 arylalkyl group.
• the carbon atom bearing R 2 has the (R)-configuration and the carbon atoms bearing R 1 , R 3 , R 4 , and R 5 have the (S)-configuration.
• R 2 is benzyl or isobutyl, and R 2 - is H or Me.
• the carbon atom bearing R 2 has the (R)-configuration and comprises a phenyl ring, optionally substituted with OH, Br or Cl.
• R 3 is a C1-C4 alkyl group. In certain embodiments, R 3 is isopropyl. In preferred embodiments, the carbon atom bearing R 3 has the (R)-configuration. In further embodiments, the carbon atom bearing R 3 has the (R)-configuration and the carbon atoms bearing R 1 , R 2 , R 4 , and R 5 have the (S)-configuration. In some such embodiments, R 3 is isopropyl or isobutyl, and R 3 - is H or Me.
• R 4 is a C1-C4 alkyl group. In preferred embodiments, R 4 comprises a hydrophobic group.
• R 5 is a C1-C4 alkyl group. In preferred embodiments, R 5 is an isobutyl group. In certain embodiments, the carbon atom bearing R 5 has the (R) -configuration. In further embodiments, the carbon atom bearing R 5 has the (R)-configuration and the carbon atoms bearing R 1 , R 2 , R 3 , and R 4 have the (S)-configuration. In some such embodiments, R 5 is isopropyl or isobutyl, and R 5 is H or Me.
• two or more of the carbon atoms bearing R 1 , R 2 , R 3 , R 4 and R 5 have the (R)-configuration. In some embodiments, each of the carbon atoms bearing R 1 , R 2 , R 3 , R 4 and R 5 has the (R)-configuration.
• the cyclic pentapeptide of formula (I) comprises only L-amino acid residues in its cyclic peptide backbone.
• Nonlimiting examples of cyclic L-pentapeptides are shown in Figure 1.
• the cyclic pentapeptide of formula (I) comprises only D-amino acid residues in its cyclic peptide backbone.
• Nonlimiting examples of cyclic D-pentapeptides are shown in Figure 2.
• the cyclic pentapeptide of formula (I) comprises both D- and L-amino acid residues in the cyclic peptide backbone.
• the cyclic pentapeptide may comprise one, two, three, or four D-amino acid residues in the cyclic peptide backbone.
• Nonlimiting examples of cyclic pentapeptides of formula (I) having a single D-amino acid residue are shown in Figure 3.
• Non-limiting examples having two or more D-amino acids are shown in Figure 4.
• Additional non-limiting examples of cyclic pentapeptides comprising all L-amino acids, or comprising one or more D-amino acid residues in the cyclic backbone are shown in Figure 5.
• the compounds of formula (I) comprise a single D- amino acid and four L-amino acids in the cyclic peptide backbone.
• the (D)-amino acid comprises residue 1, residue 2 or residue 3.
• R 1 comprises an optionally substituted C5-C12 arylalkyl group; in specific embodiments, residue 1 is (D)-tyrosine or (D)-phenylalanine.
• R 2 comprises an optionally substituted C5-C12 arylalkyl group; in specific embodiments residue 2 is (D)-N-methyl-phenylanine.
• R 3 comprises an optionally substituted C1-C4 alkyl group; in specific embodiments residue 3 is (D)-valine or (D)-N-methyl-valine.
• R 5 comprises an optionally substituted C1-C4 alkyl group; in specific embodiments, residue 5 is N-methyl-leucine or (D)-N-methyl-leucine.
• the compound of formula (I) comprises two or more D-amino acids and the remainder L-amino acids.
• residues 1 and 5 comprise D- amino acids.
• residues 4 and 5 comprise D-amino acids.
• the invention provides a cyclic pentapeptoid of formula (II):
• each of R 11 , R 12 , R 13 , R 14 and R 15 is independently selected from H, or C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 cycloalkylalkyl, C1-C6 aminoalkyl, C5-C12 arylalkyl, or a heteroform of one of these, each of which may be optionally substituted.
• each of R 11 and R 15 comprises a C5-C12 arylalkyl or C1-C8 cycloalkylalkyl group; each of R 12 and R 13 comprises a C1-C8 alkyl or C5-C12 arylalkyl group; and R 14 comprises a C1-C8 alkyl, C1-C8 heterocyclylalkyl, or C5-C12 arylalkyl group.
• the invention provides pharmaceutical compositions comprising one of more cyclic pentapeptides of formula (I) or cyclic pentapeptoids of formula (II), or their pharmaceutically acceptable salt or hydrate forms, and at least one pharmaceutically acceptable excipient.
• the invention provides methods of treating, preventing or ameliorating a cell proliferative disease or condition, e.g., a cancer, comprising providing a pharmaceutical composition of the invention; and administering a therapeutically effective amount of a pharmaceutical composition of formula (I) or a pharmaceutical composition of formula (II) to a patient in need thereof, thereby treating the desired condition, as described herein.
• this invention encompasses methods of treating, preventing, or ameliorating cancer using compounds or compositions of the invention, or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, or prodrugs thereof.
• the invention provides pharmaceutical compositions and methods of using such compositions to treat cancers, including but not limited to, colon cancers such as MSS colon cancer or MSI colon cancer, pancreatic cancer, rectal cancer, breast cancer, prostate cancer, and melanoma; or any cell proliferative condition, such as benign prostate hyperplasia (BPH) and endometriosis; or any disease or condition having an inflammatory component, e.g., an autoimmune disease such as rheumatoid arthritis, or an infectious disease; including treating, preventing or ameliorating any disease states associated with unwanted angiogenesis and/or cellular proliferation, such as diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, and psoriasis.
• colon cancers such as MSS colon cancer or MSI colon cancer
• pancreatic cancer rectal cancer
• breast cancer prostate cancer
• prostate cancer and melanoma
• any cell proliferative condition such as benign prostate hyperplasia (BPH) and
• the invention also provides methods to use the compounds and pharmaceutical compositions of the invention in the manufacture of medicaments for use in the production of an anti-cell proliferative effect, e.g., an anti-anticancer effect, in a warm-blooded animal such as man or animal (a veterinary indication).
• an anti-cell proliferative effect e.g., an anti-anticancer effect
• Uses can be to treat or prevent, or prevent recurrence, or ameliorate symptoms, of any cell proliferative condition, such as a cancer, a benign prostate hyperplasia (BPH), endometriosis; an inflammatory or autoimmune disease such as rheumatoid arthritis, an infectious disease, unwanted angiogenesis and/or cellular proliferation, diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis and/or psoriasis.
• a cancer a benign prostate hyperplasia (BPH)
• BPH benign prostate hyperplasia
• endometriosis an inflammatory or autoimmune disease such as rheumatoid arthritis, an infectious disease, unwanted angiogenesis and/or cellular proliferation, diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis and/or psoriasis.
• the invention provides modified forms of compounds of formula (I) and (II), including peptides and peptoids labeled with biotin or a fluorophoric groups, such as rhodamine, as well as peptides coupled to stabilizing or targeting agents, and to methods of making and using these compounds and formulations.
• biotinylated peptides of the present invention are useful for affinity assays. Fluorescently labeled peptides are useful for studying the intracellular localization of compounds of the invention.
• the invention provides methods to synthesize compounds of formula (I) and formula (II), and/or their pharmaceutically acceptable salt or hydrate forms by macrocyclization of a linear pentapeptide or linear pentapeptoid precursor.
• kits comprising at least one composition of formula (I) or formula (II) (e.g., the pharmaceutical compositions or dietary supplements of the invention), including instruction means for practicing the methods of the invention (e.g., directions as to indications, dosages, routes and methods of administration).
• the cyclic pentapeptides described herein may have asymmetric centers. It is understood, that whether a chiral center in an isomer is "R” or “S” depends on the chemical nature of the substituents of the chiral center. All configurations of compounds of the invention are considered part of the invention. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms.
• optically active forms such as by resolution of racemic forms or by synthesis from optically active starting materials.
• Mixtures of isomers of the compounds of the examples or chiral precursors thereof can be separated into individual isomers according to methods which are known per se, e.g. fractional crystallization, adsorption chromatography or other suitable separation processes.
• Resulting racemates can be separated into antipodes in the usual manner after introduction of suitable salt-forming groupings, e.g. by forming a mixture of diastereoisomeric salts with optically active salt-forming agents, separating the mixture into diastereomeric salts and converting the separated salts into the free compounds.
• the enantiomeric forms may also be separated by fractionation through chiral high pressure liquid chromatography columns.
• Many geometric isomers of olefins and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention.
• Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
• the cyclic pentapeptide is provided as a pharmaceutically acceptable salt for enhancing pharmacological properties.
• pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• pharmaceutically acceptable salts in one aspect, refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids.
• Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Salts in the solid form may exist in more than one crystal structure, and may also be in the form of hydrates.
• Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
• basic ion exchange resins such as
• salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
• acids include acetic, 2-acetoxybenzoic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethane disulfonic, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfamic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.
• the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418.
• the invention provides parenteral formulations comprising a pharmaceutical composition of the invention.
• the invention provides enteral formulations comprising a pharmaceutical composition of the invention.
• the invention provides methods for treating cancer comprising providing a pharmaceutical composition of the invention; and administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof, thereby treating the desired condition, as described herein.
• a therapeutically effective amount of a compound of the present invention will depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian.
• an effective amount of a compound of Formula (I) or (II) for the treatment of, for example colon or pancreatic cancer will generally be in the range of 0.1 to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 1 to 10 mg/kg body weight per day.
• the actual amount per day would usually be from 70 to 700 mg and this amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same.
• An effective amount of a salt or solvate, or physiologically functional derivative thereof may be determined as a proportion of the effective amount of the compound of Formula (I) or (II) per se. It is envisaged that similar dosages would be appropriate for treatment of the other conditions referred to herein.
• this invention encompasses methods of treating, preventing, and ameliorating, cancer using compounds of the invention, or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, or prodrugs thereof.
• Cancers may be solid or blood-borne. Examples of cancer include, but are not limited to, cancers of the skin, such as melanoma; lymph node; breast; cervix; uterus; gastrointestinal tract; lung; ovary; prostate; colon; rectum; mouth; brain; head and neck; throat; testes; kidney; pancreas; bone; spleen; liver; bladder; larynx; nasal passages; AIDS-related cancers; endometrial tumors; sarcomas, e.g.
• compounds of the invention can be used for treating, preventing or ameliorating either primary or metastatic tumors.
• compounds of the invention are used to treat MSS and MSI colon cancers, pancreatic cancer, rectal cancer, breast cancer, prostate cancer, and melanoma.
• compounds of the invention are also useful in the treatment of other cell proliferative disorders such as psoriasis, vascular smooth cell proliferation associated with atherosclerosis, post-surgical stenosis and restenosis, and in the treatment of Alzheimer's disease.
• the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
• a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
• the pharmaceutical compositions used in the methods of the invention may be formulated in any way, and administered by any means known in the art.
• compositions of the present invention can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like.
• Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
• a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
• Such an effective dose will generally depend upon the factors described above.
• the compounds may be used in an amount of from about 0.01 mg to about 2000 mg per day, and can be adjusted in a conventional fashion (e.g., the same amount administered each day of the treatment, prevention or management period), in cycles (e.g., one week on, one week off), or in an amount that increases or decreases over the course of treatment, prevention, or management.
• the dose can be from about 0.1 mg to about 1000 mg, from about 0.1 mg to about 500 mg, from about 0.1 mg to about 100 mg, from about 0.1 mg to about 50 mg, from about 0.1 mg to 10 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 500 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, from about 1 mg to about 10 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 500 mg, from about 10 mg to 100 mg, from about 10 mg to 50 mg, from about 50 mg to about 500 mg, from about 50 mg to 200 mg, or from about 100 mg to 300 mg per day.
• the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
• the compound is administered as one dose per day.
• the compound is administered continuously, as through intravenous or other routes.
• the compound is administered less frequently than daily, such as weekly or less.
• composition While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
• the compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable excipients, including for example diluents and/or carriers.
• the subject receiving this treatment is any animal in need, including primates, in particular humans, and other mammals.
• compositions or dietary supplements may be prepared according to any method known to the art for the manufacture of pharmaceuticals.
• Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
• a formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture, as further described herein.
• invention provides a pharmaceutical composition or dietary supplements comprising compositions of the invention formulated as a tablet, gel, geltab, pill, implant, liquid, spray, powder, food, feed pellet, as an injectable formulation or as an encapsulated formulation, lotion, patch or inhalant.
• compositions of the invention can be chemically modified to produce a protected form that possesses better specific activity, prolonged half-life, and/or reduced immunogenicity in vivo, e.g., the composition can be chemically modified formulated or modified by glycosylation, pegylation (modified with polyethylene glycol (PEG), activated PEG, or equivalent), encapsulation with liposomes or equivalent, encapsulated in nanostructures (e.g., nanotubules, nano- or microcapsules), or combinations thereof, or equivalents thereof, e.g., as described by Wang (2005) MoI Genet Metab. 86(l-2):134-140. Epub 2005 JuI 11.
• the polypeptide is chemically conjugated with activated PEG, or, 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, e.g., as described by Ikeda (2005) Amino Acids 29(3):283-287. Epub 2005 Jun 28.
• tablets may be coated by standard aqueous or nonaqueous techniques.
• Such compositions and preparations should contain at least 0.1 percent of active compound.
• the percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit.
• the amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained.
• the active compounds can also be administered intranasally as, for example, liquid drops or spray.
• the tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch or gelatin; dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin.
• a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil.
• tablets may be coated with shellac, sugar or both.
• a syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
• the invention also provides biocompatible matrices such as sol-gels encapsulating a composition of the invention for use as pharmaceutical composition, e.g., including silica-based (e.g., oxysilane) sol-gel matrices.
• biocompatible matrices such as sol-gels encapsulating a composition of the invention for use as pharmaceutical composition, e.g., including silica-based (e.g., oxysilane) sol-gel matrices.
• the invention also provides nano- or microcapsules comprising a composition of the invention for use as pharmaceutical composition or dietary supplements.
• compositions and dietary supplements used in the methods of the invention can be administered by any means known in the art, e.g., parenterally, topically, orally, or by local administration, such as by aerosol or transdermally.
• compositions and dietary supplements can be prepared according to any method known to the art for the manufacture of pharmaceuticals and dietary supplements.
• Such drugs and dietary supplements can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
• a formulation (which includes "dietary supplements") can be admixtured with nontoxic pharmaceutically or orally acceptable excipients which are suitable for manufacture.
• Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
• compositions and dietary supplements for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals and dietary supplements to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
• Pharmaceutical preparations and dietary supplements for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
• Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen.
• Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets.
• Aqueous suspensions of the invention can an active agent comprising a composition of the invention in admixture with excipients suitable for the manufacture of aqueous suspensions.
• Such excipients include a suspending agent, such as sodium carboxymethyl-cellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbit
• the aqueous suspension can 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.
• preservatives such as ethyl or n-propyl p-hydroxybenzoate
• coloring agents such as a coloring agent
• flavoring agents such as sucrose, aspartame or saccharin
• sweetening agents such as sucrose, aspartame or saccharin.
• Formulations can be adjusted for osmolality.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• Oil-based pharmaceuticals are particularly useful for administration of hydrophobic formulations or active agents of the invention (a composition of the invention).
• Oil-based suspensions can be formulated by suspending an active agent (e.g., a composition of the invention) in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these.
• an active agent e.g., a composition of the invention
• a vegetable oil such as arachis oil, olive oil, sesame oil or coconut oil
• a mineral oil such as liquid paraffin
• the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
• Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose.
• a palatable oral preparation such as glycerol, sorbitol or sucrose.
• an antioxidant such as ascorbic acid.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
• intravenous chemotherapy may be administered intravenously.
• intravenous chemotherapy may be given on either an inpatient or outpatient basis.
• various systems may be surgically inserted into the vasculature to maintain access. Commonly used systems include the Hickman line, the Port-a-Cath or the PICC line. These systems result in a lower infection risk, reduce the incidence of phlebitis or extravasation, and abolish the need for repeated insertion of peripheral cannulae.
• the pharmaceutical compounds and dietary supplements can also be delivered as microspheres for slow release in the body.
• microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
• the pharmaceutical compounds, formulations and dietary supplements of the invention can be lyophilized.
• the invention provides a stable lyophilized formulation comprising a composition of the invention, which can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof.
• a process for preparing a stable lyophilized formulation can include the equivalent of lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. patent app. no. 20040028670.
• compositions e.g., formulations, including dietary supplements
• liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587.
• the compounds of the present invention may administered either as single agents or, alternatively, in combination with known anticancer treatments such as radiation therapy or a chemotherapy regimen in combination with cytostatic or cytotoxic agents.
• the above compounds can be administered in combination with one or more chemotherapeutic agents, including but not limited to, taxanes (e.g., paclitaxel, Taxol ® , docataxel), topoisomerase I inhibitors (e.g., camptothecin, topotecan, irinotecan), CPT-Il, anthracycline glycosides (e.g., daunorubicin, doxorubicin or epirubicin), topoisomerase II inhibitors (e.g., etoposide, teniposide), vinca alkaloids (e.g., navelbine, vinblastine, vinblastine, vindesine, vinorelbine), nucleoside agents (e.g., gemcitabine, flurorura), chemo
• the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). In one aspect additional therapeutic agents which are normally administered to treat or prevent a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated".
• a combination treatment of the present invention as defined herein may be achieved by way of the simultaneous, sequential or separate administration of the individual components of said treatment.
• Such other drugs may be administered by a route and in an amount commonly used therefor.
• a pharmaceutical composition in unit dosage form containing such other drugs and the cyclic pentapeptide is preferred.
• the combination therapy also includes therapies in which the cyclic pentapeptide and one or more other drugs are administered on different schedules.
• the drugs may be combined into a single combination tablet or other oral dosage form, or the drugs may be packaged together as separate tablets or other oral dosage forms.
• the pharmaceutical compositions of the present invention include those that contain one or more other active ingredients, in addition to a cyclic pentapeptide.
• the compound of the present invention and the other active ingredients may be used in lower doses than when each is used singly.
• the use of such combination therapies could provide additive or synergistic anticancer effects.
• Combination of two or more drugs in therapy may result in one of three outcomes: (1) additive, i.e., the effect of the combination is be equal to the sum of the effects of each drug when administered alone; (2) synergistic, i.e., the effect of the combination is greater than the sum of the effects of each drug when administered alone; or (3) antagonistic, i.e., the effect of the combination is less than the sum of the effects of each drug when administered alone.
• This approach provides a reliable, high-yielding route for preparation of the cyclic pentapeptides of the present invention.
• the approach involves two fragments, a tripeptide fragment comprising amino acid residues 1, 2 and 3, and a dipeptide fragment comprising amino acid residues 4 and 5.
• the route is amenable to inserting desired amino acids systematically within the cyclic pentapeptide backbone.
• the route was also designed to facilitate large-scale synthesis for extensive biological studies. Cyclization to form large macrocycles is usually very challenging, and typically the yields are low.
• high-yielding cyclization conditions were developed which provided the final macrocycles in good yields.
• Scheme 1 The approach described in Scheme 1 is suitable for the preparation of cyclic pentapeptides containing diverse functionality, including for example aliphatic, aromatic and polar sidechains, as well as the incorporation of both natural and unnatural amino acids, including for example, D-amino acids and N-alkylated amino acids, at various positions around the pentapeptide backbone.
• Scheme 1 Synthesis of Macrocyclic Pentapeptides
• R 1 - R 5 are alpha substituents on amino acids residues 1 -5 respectively
• Peptide derivatives of formula (I) may also be prepared using approaches familiar to those skilled in the art of peptide chemistry or simple modifications of those approaches.
• Scheme 1 exemplifies the use of Boc-protecting group, one of skill in the art would readily appreciate that other protecting group strategies could be employed to provide fully deprotected linear pentapeptides which represent the penultimate cyclization precursors.
• the first subset comprises only L-amino acids; non-limiting examples are shown in Figure 1.
• the second subset comprises only D-amino acids; non- limiting examples are shown in Figure 2.
• the third subset comprises both L- and D-amino acids.
• Non-limiting examples comprising a single D-amino acid are shown in Figure 3.
• Non- limiting examples comprising two or more D-amino acids are shown in Figure 4. Additional compounds of the invention are shown in Figure 5.
• Linear peptides often have problems with solubility, and degradation within cells. Macrocyclic peptides show improved cellular stability over linear peptides, but often have difficulty being soluble at an appropriate level needed for a commercial drug.
• a recent solution to these two problems is the use of peptidomimetics, known as "peptoids", are peptide-like compounds where functionality is located on the amide nitrogen rather than on the alpha carbon of the amino acid. Such compounds, which formally comprise N-alkylated glycine residues, have improved solubility and stability within cells.
• Peptoids of the invention may be prepared as shown in Scheme 2.
• Peptoids are synthesized using the Zuckermann and Moos method where the synthesis of each N-substituted glycine is built in two steps. (Zuckermann, et al., /. Am. Chem. Soc. 1992, 114, 10646-10647). The first involves an acylation step and the second step involves a nucleophilic displacement. Starting with a Wang resin and a halo acid attached to the resin, coupling of residues 11 in parallel provides the first N-substituted glycine.
• cyclic pentapeptoids are designed to "match" the active structures identified in the cyclic pentapeptide series.
• Peptoid derivatives of formula (II) may be prepared as shown in Scheme 2. These cyclic pentapeptoid compounds allow examination of the impact of chirality, the positioning of the sidechain relative to the amide carbonyl, the necessity for aromatic versus alkyl, and/or polar versus non-polar side chains, and the relevance of size or "fit”.
• CoMFA models were generated using the NMR data and HCTl 16, HCTl 5 and PL45 screening data sets. The CoMFA models were trained using the natural logarithmic quantity log [inh. %]. CoMFA modeling provides a projection of a pharmacophore map that is generated by fitting the experimental inhibition data using a partial least squares fit. The pharmacophore maps for each cell line were almost identical, indicating the compounds were most likely presenting the amino acid side chains in the same conformation in each cell line.
• Solid-state structures for compounds of the invention are determined using small molecule X-ray crystallization.
• the coupling agents 2( 1 -H-benzotriazole- 1 -yl)- 1 , 1 ,3-tetramethyl-uronium tetrafluoroborate (TBTU) and PyBROP were purchased from NovaBiochem.
• DEPBT 3(diethoxyphosphoryloxy)-l,2,3-benzotriazone-4(3H)] was purchased from Aldrich.
• Acids were deprotected using 4 equivalents of lithium hydroxide (or until pH ⁇ 11) in methanol (0.1M).
• the peptide was placed in a flask, along with lithium hydroxide and methanol and stirred overnight. Within 21 hours the acid was usually deprotected.
• the aqueous solution was extracted three times with methylene chloride, and the combined organic layer was dried, filtered and concentrated in vacuo.
• the tripeptide fragment (Scheme 1), comprising residues 1-3, was synthesized as follows. Residue 1 was coupled to the N-Boc protected residue 2 to give the Boc-protected 1-2 dipeptide in 80-94% yield. Deprotection of the amine on residue 2 using TFA gave the free dipeptide amines in quantitative yields. Coupling of the dipeptide to residue 3 gave the desired tripeptide in good yields (80%-95%). The synthesis of dipeptide fragment comprising residues 4-5, was synthesized as follows. Residue 4 was coupled to Boc- protected residue 5 to give the Boc-protected 4-5 dipeptide in 90-95% yield.
• the amine was deprotected on tripeptide Fragment 1 using TFA and the acid was deprotected in dipeptide Fragment 2 using lithium hydroxide. Fragment 1 and Fragment 2 were coupled using multiple coupling agents yielding linear pentapeptides in 66-90% yields.
• compound 3 has the sequence cyclo[-Phe-Xaa-Val-Leu-Leu-], where Phe corresponds to residue 1, and the residue in position 2 (i.e., Xaa-2) is NMeLeu.
• Macrocyclic peptoids are synthesized using the Zuckermann and Moos method where the synthesis of each N-substituted glycine is built in two steps.
• a Wang resin is acylated with an alpha-halo acid, and the halo substituent is displaced by coupling of the amines corresponding to residue 11, in parallel, to provide the first N-substituted glycine.
• Coupling of bromoacetic acid using HATU in DMF, and subsequent coupling of the second amine monomer (residue 12) provides the second N-substituted glycine. Repeating the process using amines 13, 14, and 15 (corresponding to residues 13, 14, and 15) gives the linear pentapeptoid.
• 3 H-thymidine incorporation assays involved culturing cells in 96 well plates at a concentration of 3000 cells/well. After incubation for approximately 16 hours, the media was replaced with fresh media both with and without the addition of compounds, to give 1% DMSO in wells. Cells were then incubated for 56 hours, whereupon 3 H-thymidine was added for 16 hours (thus cells were incubated with compound for a total of 72 hours). Cells were then washed, fixed, solubilized and the DNA was isolated and counted in a scintillation counter using standard approaches.
• pancreatic cancer cells were seeded in tissue culture plates in media. The plates were incubated in CO 2 for 6 hours at 37 0 C to allow cells to attach to the plate. The serum containing media was removed, and fresh serum and media were added. The cells were then incubated for 24 hours. Next, 50 ⁇ M compound (55) was added to the cells, which were incubated for two time points: 1.5 hours and 3 hours (additional time points and concentrations are planned, but for purposes of gathering preliminary data these were chosen). Two control experiments were run at each time point (cells with no DMSO), and cells with 1% DMSO but no compound. The cells were then rinsed with binding buffer, the buffer was removed, and the cells were resuspended.
• Annexin V and propidium iodide (PI) were added to the cells.
• Three controls were run: annexin V only, PI only, and both annexin V and PI to mixtures of the cells from all six experiments.
• the six experiments involved three experiments at each time point: a) cells only, b) cells + 1% DMSO, c) cells + 1% DMSO + compound and 3 hour experiments included the same three experiments. All nine experiments were then analyzed by flow cytometry and the results are shown in Figures 15(a) and 15(b). It is important to note that both a) cells and b) cells + 1% DMSO gave the same results, indicating DMSO had no impact on apoptosis.
• Biotinylated compounds 24-Biotin and (27-Biotin) were used in affinity purification assays to isolate proteins from HCT-116 and PL-45 cells that bind to these compounds. After incubation of the cell lysates with the biotinylated compounds, streptavidin resin was added and the unbound proteins were removed by washing 5 times with buffer. Re- suspension of the resin in buffer followed by incubation of the resin-bound (27-Biotin)-protein complex with non- biotinylated (27) allowed for competitive elution of protein targets from the resin (27-Biotin).

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