WO2002039997A2 - Composes modulant ace-2 et procedes d'utilisation associes - Google Patents

Composes modulant ace-2 et procedes d'utilisation associes Download PDF

Info

Publication number
WO2002039997A2
WO2002039997A2 PCT/US2001/045703 US0145703W WO0239997A2 WO 2002039997 A2 WO2002039997 A2 WO 2002039997A2 US 0145703 W US0145703 W US 0145703W WO 0239997 A2 WO0239997 A2 WO 0239997A2
Authority
WO
WIPO (PCT)
Prior art keywords
methyl
carboxy
ethylamino
pentanoic acid
phenyl
Prior art date
Application number
PCT/US2001/045703
Other languages
English (en)
Other versions
WO2002039997A3 (fr
Inventor
Susan L. Acton
Timothy D. Ocain
Alexandra E. Gould
Natalie A. Dales
Bing Guan
James A. Brown
Michael Patane
Vivek J. Kadambi
Michael Solomon
Alain Stricker-Krongrad
Original Assignee
Millennium Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Millennium Pharmaceuticals, Inc. filed Critical Millennium Pharmaceuticals, Inc.
Priority to AU2002239454A priority Critical patent/AU2002239454A1/en
Publication of WO2002039997A2 publication Critical patent/WO2002039997A2/fr
Publication of WO2002039997A3 publication Critical patent/WO2002039997A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/422—Oxazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • Obesity is generally defined as the excessive accumulation of body fat. A body weight 20% over that in standard height-weight tables is widely accepted as an obese condition (except for certain heavily muscled persons). Since 1985 obesity has been recognized as a chronic disease, and is the second leading cause of preventable death in the United States. The increasing prevalence of obesity and preobesity, or overweight, is a major public health issue in the United States. Approximately one tit ⁇ rd of adults are estimated to be obese. According to the U.S. Bureau of the Census, approximately 58 million American adults (26 million men and 32 million women) are obese. Socioepidemiologic studies suggest that age, socioeconomic status, and genetics may be important risk factors.
  • renin- angiotensin-aldosterone system a proteolytic enzyme formed in the granules of the juxtaglomerular apparatus cells of the kidney, catalyzes the conversion of angiotensinogen (a plasma protein) into angiotensin I, a decapeptide.
  • This inactive product is then hydrolyzed by angiotensin converting enzyme (ACE) to an octapeptide, angiotensin II, which is a potent vasoconstrictor and also stimulates the release of aldosterone.
  • ACE angiotensin converting enzyme
  • Aldosterone is an adrenal cortex hormone that promotes the retention of salt and water by the kidneys, and thus increases plasma volume, resulting in an increase in blood pressure.
  • ACE is produced in the endothelium of somatic tissues and in the testis. It is expressed ubiquitously in the vasculature, including highly vascularized organs such as the lung, heart, pancreas, and kidney.
  • ACE also referred to as peptidyl dipeptidase A (EC 3.4.15.1) and kininase II, is a metallopeptidase, more particularly, a zinc dipeptidase which, in addition to angiotensin I, can also hydrolyze other biologically active polypeptides, such as kinins, e.g. , bradykinin.
  • Bradykinin is a vasodilator, which acts, at least in part, by inducing the release of vasodilator prostaglandins, and which is inactivated upon hydrolysis by ACE.
  • ACE activity regulates blood pressure, at least in part by producing angiotensin II, a vasoconstrictor, and by inactivating bradykinin, a vasodilator.
  • ACE plays a pivotal role in the RAS regulation of blood pressure.
  • ACE-2 an ACE-like peptidase
  • ACE-2 differs from ACE in being a carboxypeptidase and the angiotensin I cleavage product is a nonapeptide (Angl-9). Also unlike ACE, ACE-2 expression is highly restricted to the heart, kidney, large intestine, small intestine, adipose tissue and testis. ACE-2, however, is also produced by the endothelium like ACE.
  • the invention pertains, at least in part, to the treatment of ACE-2 associated states, such as body weight disorders, e.g., diabetes, such as obesity, anorexia, and cachexia, by targeting the expression or activity of an ACE homologue, referred to herein as ACE-2.
  • ACE-2 an ACE homologue
  • the invention also relates to compounds that modulate ACE-2 associated states, (e.g., body weight disorders, etc.) via ACE-2 activity.
  • Another aspect of the invention comprises administering, e.g., systemically (.eg., orally) or locally to a subject, an effective amount of an ACE-2 modulating compound.
  • the ACE-2 modulating compound can be an ACE-2 agonist, an ACE-2 inverse agonist, or an ACE-2 antagonist.
  • an ACE-2 inhibitor e.g., antagonist
  • a body weight disorder such as obesity
  • an ACE-2 activator e.g., agonist
  • a body weight disorder such as anorexia or cachexia
  • the invention also encompasses methods of treatment using ACE-2 modulating compounds such as activators (e.g., agonists), inverse agonists, and inhibitors (e.g., antagonists) of ACE-2.
  • ACE-2 modulating compounds include small molecules, and organic and inorganic compounds.
  • the invention pertains, at least in part, to a method for the treatment of a body weight disorder in a subject.
  • the method includes administering to a subject an effective amount of an ACE-2 modulating compound, such that the body weight disorder in the subject is treated.
  • the invention pertains, at least in part, to a method for decreasing the appetite of a subject.
  • the method includes administering to the subject an effective amount of an ACE-2 modulating compound, such that the appetite of the subject is decreased.
  • the invention includes a method for increasing muscle mass in a subject.
  • the method includes administering to a subject an effective amount of an ACE-2 modulating compound, such that the muscle mass of the subject is increased.
  • the invention pertains to a method for decreasing the body fat content of a subject. The method includes administering to the subject an effective amount of an ACE-2 modulating compound, such that the amount of fatty tissue of the subject is decreased.
  • the invention pertains, at least in part, to a method of promoting weight gain in a subject, by administering to the subject an effective amount of an ACE-2 activator (e.g., agonist).
  • an ACE-2 activator e.g., agonist
  • the subject may be suffering from anorexia or cachexia.
  • the invention also pertains at least in part, to a method of reducing body fat in a subject, by administering to the subject an effective amount of an ACE-2 inhibitor (e.g., antagonist).
  • an ACE-2 inhibitor e.g., antagonist
  • the invention pertains to a method for treating diabetes in a subject. The method includes administering to the subject an effective amount of an ACE-2 modulating compound.
  • the invention also includes a method for treating a state associated with lipid metabolism in a subject.
  • the method includes administering to the subject an effective amount of an ACE-2 modulating compound, such that the state is treated.
  • the invention also pertains to a method for treating atherosclerosis in a subject.
  • the method includes administering to the subject an effective amount of an ACE-2 modulating compound.
  • transgenic non-human animals which include (and preferably express) a heterologous form of an ACE-2 gene described herein, or which misexpress an endogenous ACE-2 gene (e.g., an animal in which expression of one or more of the subject ACE-2 genes is disrupted, e.g., a "knock-out" mouse).
  • Such transgenic animals can serve as in vivo models for studying cellular and/or tissue disorders comprising mutated or mis-expressed ACE-2 alleles or for use in drug screening, e.g., to identify modulators of the invention.
  • transgenic animals can be useful for expressing recombinant ACE-2 polypeptides.
  • mice completely lacking functional ACE-2 protein exhibit a significantly lower percentage of body fat and a lower body weight compared to similarly aged wild-type males of the same strain.
  • knock-out mice in which the gene encoding ACE-2 is not biologically active exhibit a significantly lower percentage of body fat and a lower body weight compared to similarly aged wild-type males of the same strain.
  • the invention also relates to assays designed to screen for compounds or compositions that modulate ACE-2 activity, i.e., compounds or compositions that act as activators (e.g., agonists), inhibitors (e.g., antagonists), or inverse agonists of ACE-2, and thereby identify modulators of the invention, e.g., useful for treating body weight disorders.
  • cell-based assays or non-cell-based assays can be used to detect compounds that interact with, e.g., act as a ligand or substrate, of ACE-2.
  • the cell-based assays are advantageous in that they are useful in the identification of compounds that affect ACE-2 biological activity, but do not necessarily interact directly with ACE-2.
  • the invention also relates to assays designed to screen for compounds or compositions that modulate ACE-2 gene expression and thereby identify modulators of the invention.
  • cell-based assays or cell lysate assays e.g., in vitro transcription or translation assays
  • compounds or compositions that modulate ACE-2 transcription e.g., compounds that modulate the expression, production, or activity of transcription factors involved in ACE-2 gene expression; or polynucleotides that form triple helical structures with an ACE-2 regulatory region and inhibit transcription of the ACE-2 gene.
  • cell-based assays or cell-lysate assays can be used to screen for compounds or compositions that modulate translation of ACE-2 transcripts (e.g., antisense and ribozyme molecules).
  • the cell-based assays or cell-lysate assays can be used to test polynucleotide constructs designed to modify the expression of the ACE-2 gene in vivo.
  • constructs include polynucleotide constructs designed for gene therapy useful for the methods described, e.g., expression constructs or gene replacement constructs that place the ACE-2 gene under the control of an inducible promoter system or a constitutive promoter system.
  • ACE-2 therapeutics and/or ACE-2 modulating compounds for treating ACE-2 associated states, such as body weight disorders, which are described in more detail below.
  • ACE-2 therapeutics or ACE-2 modulating compounds include activators (e.g., agonists), inverse agonists, and inhibitors (e.g., antagonists) of ACE-2.
  • ACE-2 modulating compounds include small molecules, organic and inorganic compounds, and peptides and antibodies, as well as nucleotide sequences that can be used to inhibit ACE-2 gene expression (e.g., antisense, triplex DNA, and ribozyme molecules), and gene or regulatory sequence replacement constructs designed to enhance ACE-2 gene expression (e.g., expression constructs that place the ACE-2 gene under the control of a strong promoter system).
  • the invention also pertains to a method for modulating ACE-2, by contacting ACE-2 with an ACE-2 modulating compound, such that ACE-2 is modulated.
  • the ACE-2 modulating compound may be any compound described herein.
  • the invention also encompasses the use of such compounds and compositions, including gene therapy approaches, that modulate ACE-2 activity or ACE-2 gene expression to treat body weight disorders,and other ACE-2 associated states.
  • FIG. 1 is a schematic diagram of the construction of the ACE-2 targeting vector.
  • the exon encoding the active site of ACE-2 was replaced with a pGKNEO/URA3 cassette by homologous recombination in yeast.
  • the 5 'flanking probe identified the presence of wild type and targeted alleles using Southern analysis.
  • ACE-2 is a target for the development of methods of treating body weight disorders, and other ACE-2 associated states.
  • the invention pertains, at least in part, to a method for the treatment of an ACE-
  • the method includes administering to a subject an effective amount of an ACE-2 modulating compound, such that the ACE-2 associated state in the subject is treated.
  • the invention also pertains to compounds and methods which inhibit the activity of ACE-2. The invention is based at least in part on the discovery of a specific role for
  • ACE-2 in body weight regulation and other processes.
  • the ACE-2 gene (GenBank Accession No. 291820) encodes a protein having regions which are significantly homologous to regions of known angiotensin converting enzymes (ACEs).
  • ACEs angiotensin converting enzymes
  • the genes and proteins used in the methods disclosed herein are referred to as Angiotensin Converting Enzyme 2 (ACE-2) genes and proteins, which are described in U.S. Patent No. 6,194,556, and Donoghue, et al, supra, each of which is incorporated herein by reference in its entirety.
  • ACE-2 is a protein having regions which are significantly homologous to regions of known angiotensin converting enzymes (ACEs).
  • the sequence of the full length cDNA encoding ACE-2 was determined from a clone obtained from a cDNA library prepared from mRNA of a human heart of a subject who had congestive heart failure.
  • the cDNA encoding the full length human ACE-2 protein and comprising 5' and 3' untranslated regions is 3396 nucleotides long (SEQ ID NO: 1).
  • the mature ACE-2 protein is 787 amino acids in length (amino acid residues 19 to 805 of the full length peptide shown in SEQ ID NO: 2).
  • the ACE-2 polypeptide is an angiotensin converting enzyme homologue and shares sequence identity with endothelial ACE and testicular ACE.
  • the ACE-2 protein further comprises functional domains shared by ACEs.
  • ACE-2 contains a single catalytic domain from about amino acids 147 to 555 of SEQ ID NO:2, which is encoded by the nucleotide sequence from residues 520 to 1746 of SEQ ID NO: 1 , and referred to herein as the ACE-2 catalytic domain. This domain is approximately 42% identical to each of the two catalytic domains in endothelial ACE (Donoghue, et al. supra).
  • ACE-2 also comprises a zinc binding domain (ZBD) within the catalytic domain from about amino acid 374 to amino acid 378 of SEQ ID NO:2, which is encoded by the nucleotide sequence from nucleotide 1201 to 1215 of SEQ ID NO:l, and referred to herein as a minimum zinc binding domain. It is likely that at least some of the adjacent amino acids participate in binding zinc.
  • the sequence of the minimum zinc binding domain is identical to the zinc binding domain that is present in all ACE proteins which have been identified as being located in the catalytic site of the enzyme (Lattion et al. (1989) FEBS Letters 252:99).
  • ACE-2 As the sequence encompassing amino acids 372 to 381 of SEQ ID NO:2 are conserved in all ACE proteins, it is likely that amino acids 372, 373, 379, 380, and 381 of SEQ ID NO:2 are involved in binding zinc. In addition, all the amino acids which have been reported as interacting with the zinc atom or involved in catalysis in ACE proteins are present in ACE-2. Thus, by analogy, His 374, 378 and Glu 402 are probably the amino acids coordinating the zinc atom, and Glu 375, His 417, and Glu 406 are involved in the catalytic activity of ACE-2. ACE-2 has a hydrophobic region in its C-terminal domain from about amino acid 741 to about amino acid 765.
  • This hydrophobic region is thought to be a transmembrane domain, similar to that present in ACE proteins.
  • a BLAST search (Altschul et al. J. Mol. Biol. (1990) 215:403) of the nucleic acid and the amino acid sequences of ACE-2 revealed that certain portions of the ACE-2 protein and cDNA have a significant homology to certain regions of previously identified angiotensin converting enzymes.
  • Two forms of ACE proteins have been described previously: a larger form, referred to as endothelial or somatic ACE, since it is present in numerous somatic tissues, including vascular endothelium, renal tubular epithelium, ciliated gut epithelium, stimulated macrophages, areas of the brain and testis.
  • the smaller form of ACE is referred to as the testicular form, since it is found essentially only in developing sperm cells in the testis.
  • ACE-2 protein The specific role of the ACE-2 protein in vivo was investigated by engineering ACE-2 "knock out" mice in which most of the endogenous ACE-2 gene coding sequence was deleted, thereby creating mice which are unable to produce biologically active ACE-2 protein.
  • ACE-2 knock out mice human ACE-2 gene sequences were utilized to isolate and clone the murine ACE-2 gene.
  • a murine ACE-2 targeting construct was then generated which was designed to delete the majority of the murine ACE-2 coding sequence upon homologous recombination with the endogenous murine ACE-2 gene.
  • Embryonic stem (ES) cells containing the disrupted ACE-2 gene were produced, isolated and microinjected into murine blastocysts to yield mice chimeric for cells containing a disrupted ACE-2 gene. Offspring of the chimeric mice resulting from germline transmission of the ES genome were obtained and animals heterozygous for the disrupted ACE-2 were identified.
  • mice heterozygous for the ACE-2 disrupted gene were bred together to produce mice homozygous for the ACE-2 mutation.
  • Inactivation of the ACE-2 by gene targeting resulted in, at least in part, male mice that have a " higher ratio of lean to fat tissue, a lower percentage of overall body fat tissue, and a lower overall body weight than wild type counterparts.
  • ACE-2 is capable of hydrolyzing angiotensin I (1-10) by cleaving the C- terminal amino acid (i.e., leucine) -from angiotensin I.
  • the resulting 9-amino acid peptide (“Ang.(l-9)") can be further hydrolyzed by ACE into a 5 -amino acid peptide containing the first five amino acids from angiotensin I.
  • ACE-2 is also capable of cleaving angiotensin II (1-8) to angiotensin 1-7 with a much higher catalytic activity than with angiotensin I as a substrate.
  • ACE-2 also catalyzes the hydrolysis of other peptides, such as (des-Arg9)-bradykinin and Lys-(des-Arg9)-bradykinin, and thus may be involved in regulating blood pressure in a similar manner as endothelial ACE protein.
  • Other biologically active peptides which can be hydrolyzed by ACE-2 include apelin-13 and apelin-36, ⁇ -casomorphin, dynorphin A 1-13, ghrelin, and neurotensin. All of the ACE-2 hydrolytic activity has been found to be carboxypeptidase activity only. Kinetic characterization of the hydrolytic activity of ACE-2 is described in Example 38.
  • Apelin is a peptide that appears to be an endogenous ligand of the G-protein coupled receptor APJ, also known as the angiotensin receptor AT(1), and may play a role in body fluid homeostasis, blood pressure, circadian rhythm, and food and water intake, ⁇ -casomorphin, a hepta-peptide produced during the hydrolysis of casein, has been shown to stimulate food intake in experimental animals.
  • Dynorphin A is an opioid peptide that may have both physiological and pathological roles in acute and chronic pain states.
  • Ghrelin is a recently discovered hormone which appears to be an important regulator of growth hormone secretion and energy homeostasis, and thus may play an important role in obesity and cachexia as well as in the regulation of growth processes.
  • Neurotensin is a tridecapeptide that exhibits selective anatomic and neurochemical interactions with dopaminergic systems and appears to be involved in some of the behavioral properties of psychostimulants.
  • ACE-2 is characterized by the presence of a transmembrane domain in the carboxy terminal portion of the protein. Thus, ACE-2 can be in a membrane bound form. ACE proteins have also been found in a soluble form, which may result either from leakage of the protein from the surface or, from specific hydrolysis by a protease, or the soluble form may be encoded by a differentially spliced mRNA. Accordingly, ACE-2 is believed also to exist in a soluble form.
  • ACE-2 associated state and "ACE-2 associated disorder” include those states which are associated with ACE-2, ACE-2 substrates, or the products of an ACE-2 metabolic pathway.
  • ACE-2 associated states and disorders also include states and disorders which are characterized by aberrant levels of ACE-2 activity, and/or levels of ACE-2 substrate and/or ACE-2 metabolic products.
  • ACE-2 associated states and disorders may include, for example, high blood pressure, high blood pressure related diseases and disorders, and, in particular, arterial hypertension.
  • Other ACE-2 associated states include congestive heart failure (CHF), body weight disorders, neurodegenerative disorders, and diseases associated with peptide hormones or cytokine processing.
  • CHF congestive heart failure
  • Blood pressure refers to the pressure exerted by the blood upon the walls of the blood vessels, e.g., arteries, and is usually measured on the radial artery by means of a sphygmomanometer, and expressed in millimeters of mercury.
  • a normal blood pressure corresponds to a diastolic blood pressure of less than 85 mm Hg
  • a high normal blood pressure corresponds to a diastolic blood pressure between 85 and 89 mm Hg
  • a mild hypertension corresponds to a diastolic blood pressure between 90-104 mm Hg
  • a moderate hypertension corresponds to a diastolic blood pressure between 105 and 114 mm Hg
  • severe hypertension corresponds to a diastolic blood pressure higher than 115 mm Hg.
  • Abnormal blood pressure can also be dete ⁇ nined based on the systolic blood pressure (when the diastolic pressure is less than 90 mm Hg).
  • a normal blood pressure corresponds to a systolic blood pressure of less than 140 mm Hg
  • a borderline systolic hypertension corresponds to a systolic blood pressure between 140 and 159 mm Hg
  • isolated systolic hypertension corresponds to a systolic blood pressure higher than 160 mm Hg.
  • This classification is borrowed from Cecil: Essentials of Medicine, Third Edition by Andreoli et al. W.B. Saunders Company (1993).
  • a diagnosis of hypertension is usually made in an adult over 18 years of age if the average of two or more blood pressure measurements on at least two subsequent visits is 90 mm Hg or higher diastolic or 140 mm Hg systolic. Since children and pregnant women have a lower blood pressure, a blood pressure over 120/80 (i.e., 120 mm Hg systolic blood pressure/80 mm Hg diastolic blood pressure), is considered abnormal.
  • Isolated systolic hypertension refers to a condition in which the systolic blood pressure is greater than 160 mm Hg and the diastolic blood pressure is less than 85 mm Hg. ISH is associated with enhanced morbidity.
  • ACE-2 associated states also include other blood pressure related diseases or conditions, e.g., CHF (congestive heart failure), chronic heart failure, left ventricular hypertrophy, acute heart failure, myocardial infarction, and cardiomyopathy.
  • CHF congestive heart failure
  • chronic heart failure chronic heart failure
  • left ventricular hypertrophy left ventricular hypertrophy
  • acute heart failure myocardial infarction
  • cardiomyopathy cardiomyopathy
  • CHF is characterized by the inability of the left ventricle to maintain a normal blood pressure. This results in a baroflex-mediated reflex increase in sympathetic discharge, which stimulates the myocardium to beat faster and stronger, yet increases peripheral vasoconstriction so that the afterload rises and the load on the failing myocardium augments (Lionel H. Opie, Drugs for the Heart, Third Edition, W.B. Saunders Co., 1991). Excess adrenergic activity also results in enhanced activity of the renin-angiotensin system, further increasing peripheral vascular resistance and contributing to fluid retention (edema) by stimulation of the secretion of aldosterone.
  • angiotensin promotes the release of vasopressin to contribute to abnormal volume regulation and hyponatremia in severe CHF.
  • Overloading of the left ventricle also results in hypertrophy of the ventricular muscle, resulting in a decrease in its contractility, further contributing to the condition.
  • vasodilators such as ACE-inhibitors are efficient in treating CHF and reducing mortality.
  • the present invention contemplates therapeutic methods and compositions in which ACE-inhibiting compounds are administered to a subject concurrently or separately with ACE-2 modulating compounds.
  • ACE inhibiting compounds are particularly preferred therapeutics for treating CHF since they are able to inhibit the deleterious neurohumoral viscious circle involving angiotensin-renin-aldosterone.
  • ACE-2 modulating, e.g., inhibiting, compounds, which also modulate angiotensin hydrolysis will also be useful for treating and preventing CHF.
  • neurodegenerative disorders includes neuropathies, Alzheimer disease, Parkinson's disease, Huntington's disease, amyotropic lateral sclerosis, motor neuron disease, traumatic nerve injury, multiple sclerosis, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis, dysmyelination disease, mitochondrial disease, migrainous disorder, stroke, aging, dementia, peripheral nervous system diseases and mental disorders such as depression and schizophrenia.
  • the term includes disorders which can be treated by administering an effective amount of a compound of the invention.
  • ACE-2 associated states also include states which are associated with regulating cell proliferation, such as smooth cell proliferation. Smooth muscle cell proliferation in the intima of muscular arteries is a primary cause of vascular stenosis in atherosclerosis, after vascular surgery, and after coronary angioplasty. Several animal studies have indicated that the renin-angiotensin system plays an important role in this vascular response to injury.
  • the stimulatory effect of angiotensin II on cell growth and replication in the cardiovascular system which may result in myocardial hypertrophy and hypertrophy or hyperplasia of conduit and resistance vessels in certain subjects is mediated through angiotensin II receptors (subtype ATI) (Rosendorff C. J. Am. Coll. Cardiol. (1996)28: 803).
  • ACE transforming growth factor beta
  • the invention pertains to methods for reducing or inhibiting smooth muscle cell proliferation, comprising administering to a subject an efficient amount of a composition by administering an ACE-2 modulating, e.g., inhibiting, compound.
  • ACE-2 modulating, e.g., inhibiting, compounds may be administered systemically or locally, e.g., at a site of vascular injury.
  • ACE-2 associated states include kidney diseases or disorders, e.g., renal failure.
  • Angiotensin and ACEs are important in the development and for the maintenance of the functional and structural integrity of the adult kidney (see, e.g., Hilgers et al. Semin. Nephrol. (1997) 17:492).
  • Chronic renal disease evolves to end- stage renal failure through events, including enhanced intraglomerular pressure and plasma protein ultrafiltration, mediated at least in part by angiotensin II.
  • ACE inhibitors reduce intracapillary pressure and ameliorate glomerular size-selective function (see, e.g., Ruggenenti and Remuzzi Curr. Opin. Nephrol. Hypertens. (1997) 6:489).
  • ACE-2 modulating compounds may be used for treating and preventing renal diseases or disorders, either alone or in combination with known ACE inhibitors.
  • ACE-2 associated states also include various other hyperadrenergic states, such as acute myocardial infarction (AMI) and some ventricular arrthythmias.
  • the invention further provides methods for treating kinetensin associated conditions.
  • ACE-2 cleaves the C-terminal amino acid (leucine) from kinetensin.
  • Kinetensin is a nine amino acid peptide having the sequence lARRHPYFL (SEQ ID NO: 3), which has been reported to induce a dose-dependent release of histamine from mast cells, as well as induce a dose-dependent increase in vascular permeability when injected intradermally (Sydbom et al.
  • modulating the plasma and/or tissue level of kinetensin such as by modulating the hydrolysis of the C-terminal amino acid from kinetensin, should be useful for treating conditions that are caused by, or contributed to by, an abnormal kinetensin level.
  • Such conditions include those caused by, or contributed to by, an abnormal histamine release from mast cells and/or by an abnormal vascular permeability.
  • ACE-2 associated states include, for example, SIRS (Systemic Inflammatory Response Syndromes), sepsis, polytrauma, inflammatory bowel disease, acute and chronic pain, bone destruction in rheumatoid and osteo arthritis and periodontal disease, dysmenorrhea, premature labor, brain edema following focal injury, diffuse axonal injury, stroke, reperfusion injury and cerebral vasospasm after subarachnoid hemorrhage, allergic disorders including asthma, adult respiratory distress syndrome, wound healing and scar formation.
  • SIRS Systemic Inflammatory Response Syndromes
  • body weight disorder includes disorders or states associated with growth or metabolism of fat tissue including, but not limited to, rapid weight loss or weight gain, obesity, anorexia, cachexia, bulimia, diabetes, generalized or familial partial lipodystrophy (peripheral fat wasting), hypercholesterolemia, hyperlipidemia, and other diseases of aberrant metabolic rate.
  • a symptom of a body weight disorder is an abnormal body weight which can be determined according to the body mass index (BMI), which is the ratio of [body weight in kg] divided by [height in m] 2 .
  • BMI body mass index
  • the percent body fat of said subject is 5% or less, 8% or less, 10% or less, 15% or less, 5% or greater, 10%) or greater, 12.5% or greater, 15% or greater, 17.5% or greater, 20% or greater, 25% or greater, 30% or greater, 35% or greater, 40% or greater, etc.
  • ACE-2 associated states may also include infertility or other disorders relating to gamete maturation.
  • ACE-2 associated states may also include cognitive disorders, and disorders associated with bradykinin and des-Arg bradykinin.
  • the invention also pertains to a method for treating a blood pressure related disease or disorder in a subject.
  • the invention involves administering a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, to the subject, such that the blood pressure related disease or disorder is treated.
  • the invention also pertains to a method of treating chronic heart failure in a subject.
  • the invention includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the chronic heart failure in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the chronic heart failure in the subject is treated.
  • the invention also pertains to a method of treating left ventricular hypertrophy in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the left ventricular hypertrophy in said subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the left ventricular hypertrophy in said subject is treated.
  • the invention also pertains to a method of treating acute heart failure in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that said acute heart failure in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound
  • the invention also pertains to a method of treating cardiomyopathy in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the cardiomyopathy in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the cardiomyopathy in the subject is treated.
  • the invention also pertains to a method of treating congestive heart failure in a subject.
  • the method involves administering to the subject a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the congestive heart failure in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound
  • the invention also pertains to a method of treating arterial hypertension in a subject.
  • the method includes administering to the subject, an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the arterial hypertension in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound
  • the invention also pertains to a method of treating myocardial infarction in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such thatmyocardial infarction in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such thatmyocardial infarction in the subject is treated.
  • the invention also pertains to a method for treating a cell proliferation disorder in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the cell prolideration disorder in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the cell prolideration disorder in the subject is treated.
  • cell proliferation disorders include, for example, cancer.
  • the cell proliferation disorder is a smooth cell proliferation disorder.
  • the invention also pertains to a method for treating vascular stenosis in a subject.
  • the method includes administering to a subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the vascular stenosis in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the vascular stenosis in the subject is treated.
  • the invention also pertains to a method of treating a kidney disease or disorder in a subject.
  • the method includes administering to a subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the kidney disease or disorder in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the kidney disease or disorder in the subject is treated.
  • the invention also pertains to a method of treating a kinetensin associated disorder in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the kinentensin associated disorder in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the kinentensin associated disorder in the subject is treated.
  • kinetensin associated disorders include those caused by, for example, abnormal vascular permeability, local and systemic allergic reactions, exzema, asthma, and anaphylactic shock.
  • the invention also pertains to a method of treating a state associated with inflammation.
  • the method includes administering to a subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the state associated with inflammation in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the state associated with inflammation in the subject is treated.
  • states associated with inflammation include SIRS, polytrauma, inflammatory bowel disease, acute and chronic pain, bone destruction in rheumatoid and osteo arthritis, periodontal disease, dysmeorrhea, premature labor, brain edema following focal injury, diffuse axonal injury, allergic disorders, wound healing, and scar formation.
  • the invention also pertains to a method for treating a neurodegenerative disorder in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the neurodegenerative disease in the subject is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the neurodegenerative disease in the subject is treated.
  • neurodegenerative disorder examples include neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotropic lateral sclerosis, motor neuron disease, traumatic nerve injury, multiple sclerosis, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis, dysmyelination disease, mitochondrial disease, migrainous disorder, stroke, aging, dementia, peripheral nervous system diseases and mental disorders.
  • the neurodegenerative disorder is Alzheimer's disease.
  • the invention also pertains to a method for treating a subject for a stroke.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the subject is treated for the stroke.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound
  • the invention also pertains to a method for treating heart disease in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the subject is treated for heart disease.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the subject is treated for heart disease.
  • the invention pertains to a method for treating diabetes in a subject.
  • the method includes administering to the subject an effective amount of an ACE-2 modulating compound.
  • the diabetes may be or may not be related to a body weight disorder in the subject.
  • the invention also includes a method for treating a state associated with lipid metabolism in a subject.
  • the method includes administering to the subject an effective amount of an ACE-2 modulating compound, such that the state is treated.
  • state associated with lipid metabolism includes disorders and states which are caused or modulated (e.g., increased) by abberant, normal, or undesirable (elevated or depressed) levels of lipid metabolism.
  • states associated lipid metabolism include, for example, obesity, lipidosis, a lipodystrophy, e.g., hyperlipenia, hyperlipidemia, hyperproteinemia, hyperliposis, lipoidosis, and lipolipoidosis.
  • the invention also pertains to a method for treating atherosclerosis in a subject.
  • the method includes administering to the subject an effective amount of an ACE-2 modulating compound.
  • the invention also pertains to a method for treating a disease or state associated with a peptide hormone in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the disease associated with a peptide hormone is treated.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the disease associated with a peptide hormone is treated.
  • diseases or states associated with peptide hormones include inflammation, blood pressure effects, kidney disease, and preterm labor.
  • the invention also pertains to a method for treating a disease associated with cytokine processing in a subject.
  • the method involves administering to a subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that said disease associated with cytokine processing is treated in said subject.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that said disease associated with cytokine processing is treated in said subject.
  • the invention also pertains to the methods of treating any of the above mentioned diseases and disorders by further administering in combination with the compounds of the invention, an ACE inhibitor.
  • ACE inhibitors examples include captopril, enalapril, enalaprilat, zofenopril, ceroanapril, alacepril, benazepril, delapril, quinapril, quinaprilat, moexipril, rentiapril, spirapril, cilazapril, perindopril, fosinopril, linsinopril, ramipril, and trandolapril.
  • the compounds of the invention are also useful for treating ACE associated disorders and diseases.
  • the compounds of the invention which are dual inhibitors of ACE and ACE-2 can be administered to a subject suffering from an ACE associated disorder, such that said ACE associated disorder is treated.
  • treatment includes the application or administration of a therapeutic agent (e.g., ACE-2 modulating compounds) to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease or disorder (e.g., an ACE-2 associated state) or a symptom of a disease or disorder, such that the disease or disorder (or at least one symptom of the disease or disorder) is cured, healed, prevented, alleviated, relieved, altered, remedied, ameliorated, improved or otherwise affected, preferably in an advantageous manner.
  • a therapeutic agent e.g., ACE-2 modulating compounds
  • Therapeutic agents include, but are not limited to ACE-2 modulating compounds such as small molecules, organic compounds, inorganic compounds,, peptides, antibodies, ribozymes and antisense oligonucleotides.
  • ACE-2 modulating compounds such as small molecules, organic compounds, inorganic compounds,, peptides, antibodies, ribozymes and antisense oligonucleotides.
  • administering includes routes of administration which allow the
  • ACE-2 modulating compound to perform its intended function, e.g. modulating, e.g., inhibiting, the function of ACE-2 and/or treating an ACE-2 associated state, e.g., a body weight disorder, etc.
  • routes of administration include parental injection (e.g., subcutaneous, intravenous, and intramuscular), intraperitoneal injection, rectal, occullar, oral, inhalation, and transdermal.
  • the injection can administered by bolus injection or by continuous infusion.
  • the ACE-2 modulating, e.g., inhibiting, compound can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function.
  • the ACE-2 modulating, e.g., inhibiting, compound can be administered alone or with a pharmaceutically acceptable carrier. Further, the ACE-2 modulating, e.g., inhibiting, compound can be administered as a mixture of ACE-2 modulating, e.g., inhibiting, compounds, which also can be coadministered with a pharmaceutically acceptable carrier. The ACE-2 modulating, e.g., inhibiting, compound can be administered prior to the onset of an ACE-2 mediated state, or after the onset of a ACE-2 mediated state. The ACE-2 modulating, e.g., inhibiting, compound also can be administered as a prodrug which is converted to another form in vivo. In certain embodiments, the ACE-2 modulating compound may be administered such that the compound is exposed to the lower gastrointestinal tract (e.g., small and large intestines, bowel, etc.) and has limited systemic absorbtion.
  • the lower gastrointestinal tract e.g., small and large intestines
  • the invention includes methods and compositions for modifying body weight and/or the percentage of body fat and treating body weight disorders, including but not limited to, obesity, cachexia, diabetes, and anorexia, by administering to the subject an effective amount of an ACE-2 modulating compound, such that the body weight disorder is treated or prevented in the subject.
  • An approach which may be used to ameliorate body weight disorders is the administration of ACE-2 modulating compounds, such as ACE-2 inhibitors (e.g., antagonists), activators (e.g., agonists) or inverse agonists, such as, but not limited to compounds of any one of Formulae I-NIII.
  • underweight individuals e.g., an anorexic or cachexic phenotype
  • symptoms of certain body weight disorders such as, for example, cachexia or anorexia, which involve an underweight (e.g., a BMI ⁇ 18.5 kg/m 2 ) phenotype
  • an underweight e.g., a BMI ⁇ 18.5 kg/m 2
  • activators e.g., agonists
  • ACE-2 protein activity can be used therapeutically to promote weight gain and/or increase the percentage of body fat in subjects with an underweight phenotype, e.g., anorexia or cachexia.
  • a BMI 25.0 - 29.9 kg/m 2
  • inhibitors e.g., antagonists
  • ACE-2 protein activity can be used therapeutically to reduce weight gain, enhance weight loss and/or decrease the percentage of body fat in subjects with an overweight or obese phenotype.
  • activators e.g., agonists
  • ACE-2 activity can be used therapeutically to promote weight gain and/or increase the percentage of body fat in subjects with an underweight phenotype, e.g., anorexia or cachexia.
  • Inhibitors e.g., antagonists
  • ACE-2 activity can be used to reduce weight gain, enhance weight loss, and/or reduce the percentage of body fat in subjects with an obese phenotype.
  • the invention also pertains to methods for increasing muscle mass of a subject by administering to the subject an effective amount of an ACE-2 modulating compound, such that the muscle mass of the subject is increased.
  • the language "effective amount" of the compound is that amount necessary or sufficient to treat or prevent an ACE-2 associated state, e.g. prevent or treat the various morphological and somatic symptoms of an ACE-2 associated state, e.g., a body weight disorder.
  • the effective amount can vary, depending on such factors as the size and weight of the subject, the type of illness, or the particular ACE-2 modulating, e.g., inhibiting, compound.
  • the choice of the ACE-2 modulating, e.g., inhibiting, compound can affect what constitutes an "effective amount”.
  • the effective amount of the ACE-2 modulating can be determined through consideration of the toxicity and therapeutic efficacy of the ACE-2 modulating, e.g., inhibiting, compounds by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 5 o (dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 (/ ED 5 o- Compounds which exhibit large therapeutic induces are preferred.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 5 o with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 5 o ( . e.
  • the term "subject" or "patient” includes animals (e.g., mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans) which are capable of suffering from an ACE-2 associated disorder, e.g., a body weight disorder, etc.
  • animals e.g., mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans) which are capable of suffering from an ACE-2 associated disorder, e.g., a body weight disorder, etc.
  • the subject is normal weight, under weight, or over weight subjects as well as transgenic subjects.
  • the subject has a BMI of 18 or less, 18 or greater, 19 or greater, 20 or greater, 21 or greater, 22 or greater, 23 or greater, 24 or greater, 25 or greater, 26 or greater, 27 or greater, 28 or greater, 29 or greater, 30 or greater, 31 or greater, 32 or greater, 33 or greater, 34 or greater, 35 or greater, 36 or greater, 37 or greater, 38 or greater, 39 or greater, 40 or greater, 41 or greater, 42 or greater, 43 or greater, 44 or greater, or 45 or greater.
  • the invention described in the subsections below encompasses screening methods (e.g., assays) for the identification of ACE-2 therapeutics and/or ACE-2 modulating compounds that can be used to treat body weight disorders.
  • ACE-2 therapeutic and ACE-2 modulating compound are used interchangeably herein.
  • the invention also encompasses activators (e.g., agonists), inverse agonists, and inhibitors (e.g., antagonists) of ACE-2, including small molecules, organic and inorganic compounds, peptides and antibodies, as well as nucleotide sequences that can be used to inhibit ACE-2 gene expression (e.g., antisense and ribozyme molecules), and gene or regulatory sequence replacement constructs designed to enhance ACE-2 gene expression (e.g. , expression constructs that place the ACE-2 gene under the control of a strong promoter system).
  • activators e.g., agonists
  • inverse agonists e.g., and inhibitors
  • ACE-2 include small molecules, organic and
  • the invention also pertains to a method of treating an ACE-2 associated state in a subject, by administering to the subject a therapeutically effective amount of an ACE- 2 modulating, e.g., inhibiting, compound, such that the ACE-2 associated state is treated.
  • the invention also pertains, at least in part, to each of the ACE-2 modulating compounds disclosed herein, as well as each of the intermediates and other compounds described in the synthesis of the ACE-2 modulating compounds.
  • the invention also pertains, per se, to pharmaceutical compositions comprising ACE-2 modulating compounds of the invention in combination with a pharmaceutically acceptable carrier.
  • ACE-2 modulating compounds include peptides, antibodies, ribozymes, antisense oligonucleotides, and small molecules.
  • small molecule ACE-2 modulating compounds include compounds of the formula (I):
  • Z is a zinc coordinating moiety and A is an amino-acid mimicking moiety, and pharmaceutically acceptable salts thereof.
  • ACE-2 modulating compound refers to organic compounds, inorganic compounds, peptides, antibodies, ribozymes, antisense oligonucleotides, and small molecules, which modulate, e.g., inhibit, activate, promote, or otherwise alter the activity of ACE-2.
  • ACE-2 modulating compounds include both ACE-2 activators (e.g. , agonists), inverse agonists and inhibitors (e.g., antagonists).
  • the term includes, but is not limited to, compounds of formulae I, II, III, IN, N, VI, Nil and NIII.
  • ACE-2 inhibiting compound includes compounds which reduce the activity of ACE-2, e.g., the ability of ACE-2 to hydrolyze substrate, in vivo or in vitro.
  • the ACE-2 inhibiting compounds are ACE-2 antagonists or inverse agonists.
  • zinc coordinating moiety includes moieties which interact with metals, e.g., zinc, associated with ACE-2. Although not wishing to be bound by theory, it is thought that the zinc coordinating moiety interacts with at least one zinc atom which is associated with the zinc binding domain of ACE-2, as discussed above.
  • Examples of zinc coordinating moieties include, for example, groups which are either capable of coordinating to zinc (e.g., electron donating groups, e.g., an ester, a guanidine, a carboxylic acid, hydroxyalkyl, an alkyl group, an amide, an amine, a hydroxyl, a thiol, a ketone, an aldehyde, carboxylate, sulfonate, sulfide, imidazolyl, or other heterocyclic moieties) or are capable of being converted into groups capable to coordinating to zinc, e.g., cleavable prodrug moieties, cleavable carboxylic acid prodrug moieties, protecting prodrug moieties or ester prodrug capable of releasing the free acid upon administration.
  • groups which are either capable of coordinating to zinc e.g., electron donating groups, e.g., an ester, a guanidine, a carboxylic acid
  • the zinc-coordinating moiety may be a hydrogen atom.
  • the language "zinc coordinating moiety” includes all moieties which coordinate to zinc or other metal atoms associated with ACE-2 and allow the compounds of the invention to perform their intended function, e.g., modulating ACE-2 activity.
  • the term “interact” includes any interactions which allow the compound to perform its intended function. Examples of interactions include ionic interactions, hydrophobic interactions, covalent interactions, hydrogen bond interactions, and combinations thereof.
  • Prodrugs are compounds which are converted in vivo to active forms (see, e.g.,
  • Prodrugs can be used to alter the biodistribution (e.g., to allow compounds which would not typically enter the reactive site of the protease) or the pharmacokinetics for a particular compound.
  • a carboxylic acid group can be esterified, e.g. , with a methyl group or an ethyl group to yield an ester.
  • the ester is administered to a subject, the ester is cleaved, enzymatically or non- enzymatically, reductively, oxidatively, or hydrolytically, to reveal the anionic group.
  • An anionic group can be esterified with moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate compound which subsequently decomposes to yield the active compound.
  • prodrug moieties includes moieties which can be metabolized in vivo to a group capable of coordinating to zinc or another enzyme binding site.
  • the prodrug moieties may be metabolized in vivo by esterases or by other mechanisms.
  • Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1-19).
  • the prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable derivatizing agent.
  • carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst.
  • cleavable prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties, (e.g., ethyl esters, propyl esters, butyl esters, pentyl esters, cyclopentyl esters, hexyl esters, cyclohexyl esters), lower alkenyl esters, dilower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters, acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl
  • the prodrug moiety itself may coordinate to the zinc without being converted prior to coordination.
  • cleavable prodrug moieties may comprise protecting prodrug moieties.
  • substituted includes substituents which can be placed on the moiety and which allow the molecule to perform its intended function. Examples of substituents include alkyl, alkenyl, alkynyl, aryl, NR'R", CN, NO 2 , F, Cl, Br, I, CF 3 , CC1 3 , CHF 2 , CHC1 2 , CONR'R", S(O) ⁇ .
  • R and R" are each independently hydrogen, C1 -C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or optionally substituted aryl.
  • substitutions enhance the ability of the ACE-2 modulating compound to perform its intended function, e.g., modulate ACE-2 activity.
  • protecting prodrug moiety includes moieties attached by a linkage, e.g., a cleavable linkage, to the ACE-2 modulating compound and which can be metabolized in vivo to yield an active drug.
  • protecting prodrug moieties include amino acids (e.g., glycine, alanine), branched or unbranched, substituted or unsubstituted lower alkyls (e.g., methyl, ethyl, propyl groups), lower alkenyl groups, dilower amino-lower alkyl groups, acylamino lower alkyl groups, acyloxy lower alkyl groups, aryl groups, aryl lower alkyl groups, and substituted aryl and aryl lower alkyl groups.
  • amino acids e.g., glycine, alanine
  • substituted or unsubstituted lower alkyls e.g., methyl, ethyl, propyl groups
  • a zinc coordinating moiety of the invention is a moiety of the formula (X):
  • R is hydrogen, carboxylic acid, unsubstituted or substituted lower alkyl esters, lower alkenyl esters, dilower alkyl amino esters, arylaminocarbonyl, aroyl, aryl, alkylaminocarbonyl, aminocarbonyl, lower alkyl amides, dilower alkyl amides, alkenylaminocarboxy, hydroxy, ether, thio, amino, (CH ) ⁇ - 4 SR 7 , a heterocycle, or a cleavable prodrug moiety;
  • R 7 is alkyl, alkenyl, alkynyl, aryl or hydrogen; P 2a and P 2b are each independently selected for each occurrence each independently hydrogen, substituted or unsubstituted, branched, straight chain or cyclic C ⁇ -C 5 alkyl; and q is 0, 1, 2, or 3.
  • R 7 includes moieties of the formulae (XI and XII):
  • P 5 , P 6 , and P 7 are each independently selected from the group consisting of substituted and unsubstituted alkyl, benzyl, phenyl, cycloalkyl, alkenyl, and alkynyl.
  • P 5 include ethyl, cyclopentyl and butyl.
  • P 7 include t-butyl, and examples of P include hydrogen or methyl.
  • P , P , and P can be substituted with any substituent which allows the compound of the invention to perform its intended function, e.g., bind or interact with ACE-2.
  • P 5 , P , and P are selected such that the compound is capable of performing its intended function after being administered to a subject.
  • P , P , and P may be selected such that the compound of the invention is capable of being absorbed by the digestive system after being administered orally.
  • P 5 , P 6 , and P 7 include moieties which allow the compound to perform its intended function in vivo.
  • the R 7 group may be cleaved in vivo to yield a carboxylic acid or carboxylate. Therefore, in one embodiment, P 5 , P , and P 7 may be selected such that they are cleaved in vivo to yield a compound which is capable of performing its intended function, e.g., interact with ACE-2.
  • amino acid mimicking moiety includes moieties which are the same or are structurally similar to an N-linked terminal natural or unnatural amino acid (e.g., leucine, histidine) and which interact with ACE-2 resulting in inhibition or modulation of ACE-2 activity. Although not wishing to be bound by theory, it is thought that the amino acid mimicking moiety may interact with a binding pocket region of ACE-2.
  • the amino acid mimicking moiety is comprised of at least one natural or unnatural amino acid, or a derivative thereof, containing, for example, a charged, an uncharged, a polar or non-polar side chain (e.g., the side chains of alanine, valine, arginine, leucine, isoleucine, phenylalanine, tryptophan, methionine, glycine, serine, proline, threonine, cysteine, tyrosine, histidine, asparagine, glutamine, lysine, glutamic acid, and aspartic acid).
  • a polar or non-polar side chain e.g., the side chains of alanine, valine, arginine, leucine, isoleucine, phenylalanine, tryptophan, methionine, glycine, serine, proline, threonine, cysteine, tyrosine, histidine, asparagine
  • the amino acid mimicking moiety may be comprised of two or more amino acids which interact with ACE-2. These amino acid mimicking moieties may be linked by a functional group, for example, an amine group.
  • the amino acid mimicking moiety may comprise a side chain pocket binding moiety (J-D) and an auxiliary pocket binding moiety (G-M) linked by a group (Q).
  • the group Q may be, for example, a bond, O, S, (CR 3 R 3a ) ⁇ - 3 , CR 3 OR 3a (e.g., CHOH), CR 3 SH (e.g., CHSH), CR 3 NR 3a R 3b (e.g., CR 3 NHR 3a , CHNH 2 , CHNHR 3 , CHNR 3 R 3a ) NR 3 (e.g, NH), O(CR 3 R 3a ) n , (CR 3 R 3a ) n O(CR 3b R 3c ) n , (e.g., (CH 2 )nO(CH 2 ) n ) wherein n is either 0, 1, 2, or 3, and R 3 , R 3a , R 3b , and R 3c are each independently hydrogen, substituted or unsubstituted C ⁇ -C 5 branched or straight chain alkyl, C 2 -C 5 branched or straight chain alkenyl, aryloxycarbony
  • side chain pocket includes a region of ACE-2 which interacts with side chain pocket binding moieties.
  • side chain pocket binding moiety includes moieties which interact with the side chain pocket.
  • side chain pocket binding moieties include hydrogen, branched or straight chain, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, aryl, or heteroaryl moieties or amino acid side chains of a natural or unnatural amino acids.
  • Examples of preferred side chain pocket binding moieties include substituted or unsubstituted, branched or straight chain alkyl groups, and uncharged or charged amino acid side chains such as those of alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, proline, arginine, glutamic acid, aspartic acid, , lysine, histidine and glutamine. Also included are non-natural amino acid side chains, e.g., derived from substituted natural amino acids, analogs of natural amino acids, derivatives of natural amino acids, and other non-naturally occurring amino acids.
  • the side chain pocket binding moieties may also include substituted or unsubstituted heterocyclic moieties which may include imidazoles, thiazoles, pyrazoles, and benzimidiazoles.
  • side chain pocket binding moiety may be of the formula (XIII):
  • R 8 is hydrogen, or alkyl, and optionally may be linked to form a cyclic structure with the D group;
  • J is selected from the group consisting of a bond, O, S, CR 4 SH (e.g., CHSH), CR 4 OH (e.g., CHOH), CR 4b NR 4 R 4a (e.g, CHNH 2 ), NR 4 (e.g., NH), (CR 4 R 4a ) n , (e.g., (CH 2 ) n ), O(CR 4 R 4a ) n (e.g., O(CH 2 ) n ), (CR 4 R 4a ) p O(CR 4a R 4b ) n , (e.g., (CH 2 )pO(CH 2 ) lake), a chain of 1 to 5 atoms (e.g., carbon) optionally substituted by C ⁇ -C 6 alkyl, halogens, wherein n is either 0, 1, 2, or 3, and p is 0, 1, 2, or 3; and R 4 , R 4a , R 4b , and R 4c are each independently
  • D is hydrogen, alkoxy, alkyl, alkenyl, amine, hydroxy, alkynyl, aryl, or heteroaryl, any of which optionally may be branched or substituted.
  • substituted D groups include alkyl amino, alkylhydroxy, alkylthio, alkylphenyl, alkylcycloalkyl, and alkylacetylene.
  • D may be linked to form a ring with G, M, or Q.
  • J is a bond and D is alkyl (e.g., methyl, ethyl, isopropyl, n- propyl, isobutyl, n-butyl, t-butyl, pentyl, etc.) or alkynyl.
  • D may be phenyl, or heteroaryl, e.g., pyridinyl or imidazolyl.
  • J and D are unsubstituted alkyl, alkynyl, aryl, or alkenyl.
  • auxiliary pocket includes a region of ACE-2 which interacts with auxiliary pocket binding moieties.
  • auxiliary pocket binding moiety includes moieties which interact with the auxiliary pocket of ACE-2.
  • auxiliary pocket binding moieties include hydrogen, branched or straight chain, substituted or unsubstituted alkyl, aryl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, or heteroaryl moieties, or amino acid side chains of a natural or unnatural amino acid.
  • auxiliary pocket binding moieties may interact with metals associated with ACE-2, e.g., zinc.
  • auxiliary pocket binding moieties include moieties of the formula
  • linking moiety or “G” includes moieties which link the anchor moiety with the ACE-2 modulating compound.
  • linking moiety includes covalent bonds, C ⁇ -C 6 alkyl, alkenyl, alkynyl, ether, ester, thioether, amine, and carbonyl moieties.
  • the linking moiety is a covalent bond, amine C ⁇ -C 6 , C ⁇ -C 5 , C]-C 4 , C1-C 3 , or C ⁇ -C 2 alkyl.
  • the linking moiety may be substituted with up to three, four, five, or six heteroatoms.
  • anchor moiety or “M” includes moieties which interact with the auxiliary binding pocket of ACE-2. In certain embodiments, these moieties may also interact with metals, e.g., zinc, associated with ACE-2.
  • anchor moiety includes moieties which allow the ACE-2 interacting compound to perform its intended function.
  • anchor moieties include, for example, hydrogen atoms, unsubstituted or substituted alkyl, alkenyl, alkynyl, carbocyclic groups, e.g., substituted or unsubstituted phenyl, naphthyl or heterocyclic (including heteroaryl), e.g., substituted or unsubstituted furanyl, imidazolyl, benzothiophenyl, benzofuranyl, quinolinyl, isoquinolinyl, benzodioxazolyl, benzoxazolyl, benzothiazolyl, benzoimidazolyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, methylenedioxyphenyl, indolyl, thienyl, pyrimidyl, pyrazinyl, pyrazolyl, purinyl, deazapurinyl,, napthridinyl,
  • substituents include alkyl, alkenyl, alkynyl, aryl, NR'R", CN, NO , F, Cl, Br, I, CF 3 , CCI3, CHF 2 , CHC1 2 , CONR'R", S(O) 2 NRR", CHO, S(O) 0 - 2 R ⁇ O(CR'R") 0 . 2 CF 3 , OCCI 3 , SCF 3 , SCCI3, COR', CO 2 R, and OR and wherein R and R" are each independently hydrogen, C ⁇ -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or optionally substituted aryl.
  • the anchor moiety is substituted with at least one subanchor moiety and/or auxiliary subanchor moiety.
  • Each subanchor moiety (“L”) or auxiliary subanchor moiety (“V”) may optionally be linked to the anchor moiety through a sublinking moiety ("K”) or auxiliary sublinking moiety (“W”).
  • Auxiliary subanchor and sublinking moieties include all moieties described below for linking and sublinking moieties.
  • Auxiliary sublinking and auxiliary subanchor moieties generally, are connected to M through at least one heteroatom of M.
  • sublinking and subanchor moieties generally, but not necessarily, are connected to M through a carbon atom of M.
  • anchor moiety includes hydrogen, alkyl, alkenyl, alkynyl, carbocyclic groups, aryl groups, e.g., substituted or unsubstituted phenyl, biphenyl, or heterocyclic, heteroaryl groups e.g., substituted or unsubstituted furanyl, imidazolyl, benzothiophenyl, benzofuranyl, quinolinyl, isoquinolinyl, benzodioxazolyl, benzoxazolyl, benzothiazolyl, benzoimidazolyl, thiazolyl, isothiaozolyl, oxazolyl, isooxazolyl, methylenedioxyphenyl, indolyl, thienyl, pyrimidyl, pyrazinyl, purinyl, or deazapurinyl.
  • the subanchor moiety is aliphatic or aromatic, e.g., unsubstituted or substituted carbocyclic, heterocyclic, or phenyl.
  • the subanchor moiety allows the ACE-2 modulating, e.g. , inhibiting, compound to perform its intended function, e.g., inhibit ACE-2 function.
  • a molecule of the invention may comprise one or more subanchor moieties.
  • sublinking-subanchor moieties include moieties of the formulae: wherein said sublinking and subanchor moieties can be substituted at any position capable of substitution with alkyl, alkenyl, alkynyl, aryl, NR'R", CN, NO 2 , F, Cl, Br, I, CF 3 , CC1 3 , CHF 2 , CHC1 2 , CONR'R", S(O) 2 NRR", CHO, S(O) 0 - 2 R', O(CR'R")o- 2 CF 3 , OCCI3, SCF 3 , SCCI3, COR', CO 2 R', and OR * and wherein R' and R" are each independently hydrogen, C ⁇ -C 6 alkyl, C -C 6 alkenyl, C 2 -C 6 alkynyl or optionally substituted aryl.
  • subanchor moieties include, for example, 4-chlorophenyl, biphenyl, 3-chlorophenyl, 3,5-dichlorophenyl, 3-methylphenyl, 3,5-dimethylphenyl, 3-CF 3 - phenyl, 2,5-dichlorophenyl, m-fluorophenyl, m-iodophenyl, methylenedioxyphenyl, m- trifluoromethylphenyl, 2,3-dichlorophenyl, 2,3-difluorophenyl, 2,3-dimethoxyphenyl, p- fluorophenyl, p-nitrophenyl, p-t-butylphenyl, p-isopropyl phenyl, p- ' trifluoromethoxyphenyl, 3,4-dimethylphenyl, 4-cyanophenyl, 3, 4-chlorophenyl, 4- methylphenyl, and 4-trifluoromethylpheny
  • sublinking moiety or "K” includes moieties which link the subanchor moiety with the anchor moiety.
  • the term “sublinking moiety” includes covalent bonds, C ⁇ -C 6 alkyl, alkenyl, alkynyl, ether, ester, thioether, amine, amide and carbonyl moieties.
  • the linking moiety is a covalent bond, amine or C ⁇ -C 6 alkyl.
  • a subanchor moiety may be connected to the anchor moiety through one or more sublinking moieties. Examples of sublinking moieties also include O, S, CR K OH (e.g. , CHOH), (CR K R K1 ) n -SO 2 , CR K NR K1 R K2 (e.g. , CHNHR K1 , CHNR K1 R , K K2
  • NR e.g., (CH 2 ) n ), O(CR KTRJKK 1 ) n (e.g., O(CH 2 ) n ) (CR K R K1 ) obligeO(CR K2 R K3 ) n , (e.g., (CH 2 ) n O(CH 2 )n), a chain of 1 to 5 atoms optionally substituted by Cj-Cg alkyl, halogens, wherein n is either 0, 1, 2, or 3, and R ⁇ , R K1 , R 2 , and are hydrogen, each independently substituted or unsubstituted Cj-Cg branched or straight chain alkyl, aryl, arylalkyl, substituted or unsubstituted acyl, C3-Cg ring, optionally substituted with up to four heteroatoms.
  • sublinking moiety is a covalent bond, an ether, an amine, or -C 3 alkyl (e.g., -CH 2 -, - CH 2 CH 2 -, etc.).
  • sublinking moieties include CONR ⁇ CRV 1 , CR K R K1 -S(O 2 ), (CR K R K1 )o-3, and (CR K R K1 ) 0 - 2 O(CR K2 R K3 )o- 2 , wherein R ⁇ , R K1 , R M , and R are selected from the group consisting of halogens, hydrogen, and alkyl.
  • K is (CH 2 ), CONHCH 2 , CH 2 -S(O 2 ), (CH 2 ) 2 , or (CH 2 )O(CH 2 ).
  • the anchor moiety includes cyclic moieties of the formula (XV):
  • ⁇ is 0, 1, 2, 3, 4, or 5;
  • Ti, T 2 , T 3 , T 4 , and T 5 are each independently carbon, nitrogen, sulfur, or oxygen, optionally bound to hydrogen or oxygen (e.g., to form a carbonyl or sulfonyl group);
  • T 6 is carbon, nitrogen, sulfur, oxygen or a covalent bond (such that a five membered ring is formed), optionally bound to hydrogen or oxygen (e.g., forming a carbonyl or sulfonyl group) each K is an independently selected sublinking moiety; and each L is an independently selected subanchor moiety.
  • the sublinking and subanchor moieties may be attached to any available atom of the aromatic or heteroaromatic ring. Furthermore, two or more sublinking moieties may be attached to the same subanchor moiety, forming a bicyclic or tricyclic ring system.
  • anchor moieties include moieties wherein T 2 and T 4 are nitrogen, T l5 T 3 and T 5 are carbon and T 6 is a covalent bond.
  • Another example of an anchor moiety includes the moiety wherein T 2 and T 3 are nitrogen, Ti, T 4 , T 5 are each carbon, and T 6 is a covalent bond.
  • Another example of an anchor moiety includes the moiety wherein Ti, T 3 , and T 5 are each carbon, T is nitrogen, and T 4 is sulfur.
  • anchor moieties (“M-K-L”) also include: wherein each K and L are independently selected sublinking and subanchor moieties for each position capable of substitution. In one embodiment, K is a covalent bond and L is a hydrogen atom.
  • N and W are each independently selected auxiliary sublinking and auxiliary subanchor moieties, respectively.
  • auxiliary sublinking moiety or “N” includes sublinking moieties as described previously that when bonded to a heteroatom (e.g., nitrogen, oxygen, sulfur, phosphorous, etc.) result in a compound of the invention which is capable of performing its intended function (e.g., modulate ACE-2).
  • auxiliary subanchor moieties include, but are not limited to sublinking moieties as described above. Examples of auxiliary sublinking moieties (or “N”) include covalent bonds,
  • V is (CH 2 ), CONHCH 2 , CH 2 -S(O 2 ), (CH 2 ) 2 , or (CH 2 )O(CH 2 ).
  • V is a covalent bond.
  • auxiliary subanchor moiety includes, but is not limited to, subanchor moieties as described previously. It also includes moieties that when bonded to auxiliary subanchor moieties result in a compound of the invention which is capable of performing its intended function (e.g., modulate ACE-2).
  • the anchor moiety is substituted or unsubstituted pyrazolyl, thioazolyl, or oxazolyl or imidazolyl.
  • the imidazolyl anchor moiety is substituted with one or more subanchor moieties ("L"), linked to the imidazolyl anchor moiety through sublinking moieties ("K").
  • the imidazolyl anchor moiety is represented by the formula (XVI):
  • each K is an independently selected sublinking moiety
  • each L is an independently selected subanchor moiety.
  • the sublinking and subanchor moieties may be attached to any available atom of the imidazole ring. Furthermore, two or more sublinking moieties may be attached to the same subanchor moiety, forming a bicyclic or tricyclic ring system.
  • K-L when K-L is bonded directly to a heteroatom (e.g., the nitrogen), the K-L may be replaced by V-W, which represents auxiliary sublinking and auxiliary anchor moieties, respectfully.
  • the imidazolyl anchor moiety is of the formula (XVII): (XVII) wherein V and W are, respectively, auxiliary sublinking and auxiliary subanchor moieties as described above.
  • V is alkyl
  • W is cycloalkyl.
  • K is a covalent bond, aminocarbonyl, (CH 2 ) n or (CH 2 )pO(CH 2 ) n , wherein n is 0, 1, 2, 3, 4, or 5 and p is 0, 1, 2, 3, 4, or 5.
  • L is a subanchor moiety, such as, but not limited to, unsubstituted or substituted phenyl, alkyl or cyclic alkyl.
  • L e.g., phenyl or another subanchor moiety
  • substituents include alkyl, alkenyl, alkynyl, aryl, NR'R", CN, NO 2 , F, Cl, Br, I, CF 3 , CC1 3 , CHF 2 , CHC1 2 , CONR'R", S(O)!- 2 NR'R", CHO, O(CR'R")o-3CF 3 , S(O)o- 2 R ⁇ O(CR'R")o-3CCl 3 , SCF 3 , SCC1 3 , COR', CO 2 R', and O wherein R and R" are each independently hydrogen, C1 - C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or optionally substituted aryl.
  • L is phenyl and is substituted with a nitro group.
  • L is 4-chlorophenyl, biphenyl, 3-chlorophenyl, 3,5-dichlorophenyl, 3-methylphenyl, 3,5- dimethylphenyl, 3-CF 3 -phenyl, 2,5-dichlorophenyl, m-fluorophenyl, m-iodophenyl, methylenedioxyphenyl, m-trifluoromethylphenyl, 2,3-dichlorophenyl, 2,3- difluorophenyl, 2,3-dimethoxyphenyl, p-fluorophenyl, p-nitrophenyl, p-t-butylphenyl, p- isopropyl phenyl, p-trifluoromethoxyphenyl, 3,4-dimethylphenyl, 4-cyanophenyl, 3,4- chlorophenyl, 4-methylphenyl
  • anchor moiety is of the formula (XVIII):
  • K is a covalent bond, aminocarbonyl, (CR K R ⁇ ) n (e.g., CH 2 ) n ) or (e.g., (CH 2 ) p O(CH 2 ) n ) wherein n is 0, 1, 2, 3, 4, or 5 and p is 0, 1, 2, 3, 4, or 5.
  • L is a subanchor moiety, such as, but not limited to, unsubstituted or substituted phenyl, alkyl or cyclic alkyl.
  • L e.g., phenyl or another subanchor moiety, can be substituted with one or more substituents that allow the compound to perform its intended function, e.g., modulate ACE-2 activity.
  • substituents include alkyl, alkenyl, alkynyl, aryl, NR'R", CN, NO 2 , F, Cl, Br, I, CF3, CCI3, CHF 2 , CHC1 2 , CONR'R", S(O) ⁇ - 2 NR'R", CHO, O(CR'R")o- 3 CF 3 , S(O) 0 - 2 R', O(CR'R")o- 3 CCl 3 , SCF 3 , SCCI3, COR, CO 2 R', and O wherein R and R" are each independently hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or optionally substituted aryl.
  • phenyl anchor moiety is of the formula (XIX):
  • K is a covalent bond, alkoxy, or oxy and L is substituted or unsubstituted aryl, e.g., phenyl, benzothiophenyl, benzofuranyl, pyridiyl, quinoline, isoquinoline, , etc.
  • L is substituted phenyl, e.g., halogen substituted or alkyl (e.g., methyl).
  • anchor moiety is of the formula (XX):
  • anchor moiety is of the formula (XXI):
  • V and W are, respectively, auxiliary sublinking and auxiliary subanchor moieties as described above.
  • the anchor moiety and/or other portions of the ACE- 2 modulating, e.g., inhibiting, compound may be selected such that the compound is less toxic, e.g., less toxic as measured in the Toxicity Assay.
  • the anchor moiety and/or other portions of the ACE-2 modulating, e.g., inhibiting, compound is selected such that the compound is not substantially metabolized before performing its intended function.
  • the ACE-2 compound may comprise a carbon-carbon bond linking the anchor moiety to the subanchor or sublinking moiety.
  • substantially metabolized includes metabolism of the ACE-2 modulating, e.g., inhibiting, compound which prevents the modulating compound from performing its intended function.
  • the term does not include cleavage or other alteration of prodrug moieties which do not prevent the ACE-2 modulating, e.g., inhibiting, compound from performing its intended function.
  • the ACE-2 modulating compound of the invention may be identified through assays which determine the compound's K[.
  • An example of such an assay is the ACE-2 Competitive Substrate Assay (ACS Assay).
  • the ACS assay determines the ability of a compound to inhibit the activity of ACE-2 by having a test compound compete with a known ACE-2 substrate and determining the amount of known substrate converted by ACE-2 to its cleaved products.
  • the ACE-2 assay is described in greater detail in Example 11.
  • the invention includes ACE-2 modulating, e.g., inhibiting, compounds which interact with ACE-2 with a K, of 50 ⁇ M or less, 40 ⁇ M or less, 20 ⁇ M or less, 10 ⁇ M or less, 1 ⁇ M or less, 0.75 ⁇ M or less, 0.1 ⁇ M or less, 0.50 ⁇ M or less, 0.25 ⁇ M or less.
  • the invention also pertains to an ACE-2 modulating, e.g., inhibiting, compound of the formula (II):
  • the compound is an ACE-2 inhibiting compound.
  • enzyme coordinating moiety includes, preferably, carboxylic acids, hydrogen, and cleavable prodrug moieties. It includes electron rich moieties which are able to coordinate to the enzyme to facilitate inhibition of ACE-2.
  • the enzyme coordinating moiety also includes prodrugs which can be hydrolyzed in vivo to an active form.
  • the language "enzyme coordinating moiety” includes any moiety, which, in vivo and/or in vitro, allows for interactions to occur between the ACE- 2 modulating compound and ACE-2.
  • E is a carboxylic acid or a carboxylic acid equivalent.
  • carboxylic acid equivalent includes moieties which allow the ACE-2 modulating compound to perform its intended function, e.g., modulate ACE-2 function.
  • carboxylic acid equivalents include, for example, SO 3 H, PO 3 H, SO 2 NH 2 , SO 2 NHCH 3 , CONHSO 2 R 5 , SO 2 NHCF 3 , NHSO 2 CH 3 , NHSO 2 CF 3 , CONHOH, CONHCN, and other carboxylic acid isoteres.
  • E is CO 2 R 5 (e.g., CO 2 H), CONR 5 R 5' (e.g., CONH 2 , CONHR 5 ) or hydrogen, wherein R 5 and R 5' are each hydrogen, independently substituted or unsubstituted C ⁇ -C 5 branched or straight chain alkyl, C 2 -C branched or straight chain alkenyl, aryl, C 3 -C 8 ring, optionally substituted with up to four heteroatoms.
  • the term "carboxylic acid equivalent” also includes certain heterocycles which allow the ACE-2 modulating compound to perform its intended function. Examples of heterocyclic carboxylic acid equivalents include tetrazole.
  • E comprises P , linked to Z through a linker comprising
  • P groups include substituted lower alkyl esters, unsubstituted lower alkyl esters, branched lower alkyl esters, lower unsubstituted alkenyl esters, lower substituted alkenyl esters, dilower alkyl amino esters, acyl amino esters, acyloxy lower alkyl esters, unsubstituted aryl esters, substituted aryl esters, aryl lower alkyl esters, substituted aryl lower alkyl esters, amides, lower alkyl amides, dilower alkyl amides, lower alkenyl amides, dilower alkenyl amides, and aryl amides.
  • P 4 include carboxylic acids and (CH ⁇ MSP .
  • P is alkyl, alkenyl, alkynyl, or hydrogen.
  • P 4 is a hydrogen, carboxylic acid, (CH 2 ) ⁇ 4 SP 4 , a cleavable prodrug moiety, carboxylic acid, COOP 4 ', or CONP 4 'P 4 ", wherein P 4' and P 4 are each independently hydrogen, Cj-Cg alkyl, C2-C alkenyl, C2-Cg alkynyl, or optionally substituted aryl.
  • P 3a and P 3 include hydrogen, substituted or unsubstituted, branched, straight chain and cyclic C ⁇ -C 5 alkyl, each independently selected for each occurrence.
  • values of q include 0, 1, 2, and 3.
  • P 3a and P 3 include hydrogen, substituted or unsubstituted, branched, straight chain and cyclic C ⁇ -C 5 alkyl, each independently selected for each occurrence.
  • values of q include 0, 1, 2, and 3.
  • P 3a and P 3 include hydrogen, substituted or unsubstituted, branched, straight chain and cyclic C ⁇ -C 5 alkyl, each independently selected for each occurrence.
  • values of q include 0, 1, 2, and 3.
  • P is not a carboxylic acid.
  • P , and P are each independently selected from the group consisting of substituted and unsubstituted alkyl, benzyl, phenyl, cycloalkyl, alkenyl, and alkynyl.
  • P 5 , P 6 , and P 7 are any substituent which allows the compound of the invention to perform its intended function, e.g., bind or interact with ACE-2.
  • P 5 , P 6 , and P 7 are selected such that the compound is capable of performing its intended function after being administered to a subject.
  • P , P , and P may be selected such that the compound of the invention is capable of being absorbed by the digestive system after being administered orally.
  • P , P , and P include moieties which allow the compound to perform its intended function in vivo.
  • P 4 groups may be cleaved in vivo to yield a carboxylic acid or carboxylate. Therefore, in one embodiment, P 5 , P 6 , and P 7 may be selected such that P 4 is cleaved in vivo to yield a compound which is capable of performing its intended function.
  • the invention pertains to compounds of the formula (III):
  • P 4 is selected from the group consisting of a carboxylic acid, cleavable prodrug moieties, COOP 4' , (CH 2 ) ⁇ - 4 SP 4' , or C(O)NP 4 P 4 ";
  • R 7 is hydrogen, carboxylic acid, unsubstituted or substituted lower alkyl esters, lower alkenyl esters, dilower alkyl amino esters, arylaminocarbonyl, aroyl, aryl,
  • P 4 ', P 4 ", R 7 * and R 7 ' are each independently hydrogen, Ci -Cg alkyl, C 2 -C6 alkenyl, C2-C6 alkynyl or optionally substituted aryl;
  • R is selected from the group consisting of hydrogen, alkyl and a covalent bond to D;
  • R 9 is lower alkyl or hydrogen;
  • Q is a bond, O, S, CR 3 OH (e.g., CHOH), (CR 3 R 3a ) n (e.g., (CH 2 ) n ), CR 3 SH (e.g., CHSH), CR 3 NR 3a R 3b (e.g., CR 3 NH 2 , CHNHR 3 , CHNH 2 , CR 3 NHR 3a ), NR 3 , O(CR 3 R 3a ) n (e.g., O(CH 2 ) n ), CR 3 R 3a ) n O(CR 3b R 3c ) n (e.g., (CH 2 ) consumerO(CH 2 ) fond) 5 wherein n is either 0, 1, 2, or 3, and R 3 , R 3a , R 3b , and R 3c are each independently hydrogen, substituted or unsubstituted C ⁇ -C 6 branched or straight chain alkyl, C 2 -C 6 branched or straight chain alken
  • G is a linking moiety
  • M is an anchor moiety, heterocyclic, carbocyclic, or CONR'R", wherein R and R" are each independently hydrogen, Cj-Cg alkyl, C2-C6 alkenyl, C2-Cg alkynyl or optionally substituted aryl;
  • J is selected from the group consisting of a bond, substituted or unsubstituted alkyl, alkenyl, and alkynyl;
  • D is hydrogen, alkoxy, alkyl, alkenyl, alkynyl, aryl, or optionally linked to G, M, or Q to form a ring; t is 0 or 1 ; p is O, 1, 2, 3, 4, or 5; q is 1, 2, or 3; and enantiomers, diastereomers, mixtures of enantiomers, mixtures of diastereomers, and pharmaceutically acceptable salts thereof.
  • the * carbons are in the S,S configuration.
  • P is a carbonyl moiety bound to R .
  • R 6 include hydroxyl and protecting prodrug moieties.
  • Each or P 2 , P 2b , P 3a , and P 3b are hydrogen.
  • R 7 is hydrogen or a carboxylic acid
  • R 8 is hydrogen
  • p is 0, t is 1
  • q is 0, and Q is NH.
  • the invention pertains to a compound of the formula
  • R .6 is hydroxyl or a protecting prodrug moiety
  • R 7 is an hydrogen atom, carboxylic acid, unsubstituted or substituted, arylaminocarboxy, aroyl, alkylaminocarboxy, aminocarboxy, alkenylaminocarboxy, a protecting prodrug moiety, hydroxyl, heterocycle, alkoxy, ether, thiol, an amine lower alkyl esters, lower alkenyl esters, dilower alkyl amino, aryl, (CH 2 ) ⁇ .
  • SR 7 a heterocycle, or a cleavable prodrug moiety,COOR 7' , CO ⁇ R 7 R 7 " , (CH 2 ) ⁇ - SR r , a heterocycle, or a cleavable prodrug moiety;
  • R 7 ' and R 7 " are each independently hydrogen, Ci-C ⁇ alkyl, C2-C6 alkenyl, C2- Cg alkynyl or optionally substituted aryl;
  • R is hydrogen, or alkyl, and optionally linked to D to form a ring;
  • R 9 is lower alkyl or hydrogen
  • Q is a bond, O, S, CR 3 OH, CR 3 SH, CR 3 NH 2 , CR 3 NR 3a R 3b , NR 3 , (CR 3 R 3a ) n , O(CR 3 R 3a ) n , CR 3 R 3a ) procurO(CR 3b R 3c ) n , wherein n is either 0, 1, 2, or 3, and R 3 , R 3a , R 3b , and R 3c are each independently hydrogen, substituted or unsubstituted -Cs branched or straight chain alkyl, C -C branched or straight chain alkenyl, aryloxycarbonyl, arylaminocarbonyl, arylalkylsulfonyl, arylalkyl, substituted or unsubstituted acyl, aryl, C 3 -C 8 ring, optionally substituted with up to four heteroatoms.
  • G is a linking moiety
  • M is an anchor moiety
  • J is a bond, a substituted or unsubstituted alkyl, alkenyl, or alkynyl moiety
  • D is hydrogen, alkoxy, alkyl, alkenyl, alkynyl, aryl, or heteroaryl, optionally linked to G, M or Q to form a ring; t is 0 or 1 ; p is 0, 1, 2, 3, 4, or 5; and q is 0, 1, 2, or 3.
  • R 6 is hydroxyl, an amino acid (e.g., glycine, alanine), branched or unbranched, substituted or unsubstituted lower alkyls (e.g., methyl, ethyl, propyl groups), lower alkenyl groups (e.g., ethenenyl, propenyl, butenyl, pentenyl), di- lower amino-lower alkyl groups (dimethylamino, diethylamino, diisopropylamino, di-n- propylamino, methylethylamino, methylpropylamino, etc.) acylamino lower alkyl groups, acyloxy lower alkyl groups, aryl groups (e.g., phenyl, furanyl, biphenyl, napthridinyl, pyrazolyl, etc.), aryl lower alkyl groups, and substituted aryl or aryl lower alkyl groups,
  • R 9 is lower alkyl (e.g., methyl, ethyl, propyl, or butyl) or hydrogen.
  • Q is CH 2 , O, or NR 3 .
  • R 3 is, for example, hydrogen, substituted or unsubstituted cyclic, branched or straight chain C ⁇ -C alkyl, C 1 -C 5 alkenyl, acyl, aryloxycarbonyl, arylaminocarbonyl, arylalkylsulfonyl, or aryl.
  • G is a bond, Ci, C 2 , C 3 , C 4 , C 5 or C 6 alkyl, alkenyl, alkynyl, ether, ester, thioether, amine, or carbonyl, optionally substituted with up to three, four, five, or six heteroatoms.
  • M is a hydrogen atom, alkyl (straight, branched or cycloalkyl), alkenyl, alkynyl, heterocyclic, carbocyclic, aryl, e.g., phenyl, biphenyl, heteroaryl, e.g., furanyl, imidazolyl, benzothiophenyl, benzofuranyl, quinolinyl, isoquinolinyl, benzodioxazolyl, benzoxazolyl, benzothiazolyl, benzoimidazolyl, thiazolyl, isothiazolyl, oxazolyl, benzothiazolyl, isooxazolyl, methylenedioxyphenyl, indolyl, thienyl, pyrimidyl, pyrazinyl, pyrazolyl, purinyl, deazapurinyl, naphthyl, napthridinyl, or in
  • R 8 and R 9 are hydrogen or alkyl.
  • R 8 is alkyl and is linked to D to form a ring.
  • R 9 is alkyl and is linked with M to form a ring.
  • D and Q are linked to form a ring.
  • the compounds of formula (IN) are ACE-2 modulating compounds, e.g. , ACE-2 inhibiting compounds.
  • the invention also pertains to compound wherein R is hydrogen or a carboxylic acid; R 8 is hydrogen and R 9 is hydrogen, p is 0, t is 1, q is 0.
  • compounds of the invention include compounds of the formula (VII):
  • Q is CH 2 , O, or ⁇ R 3 ;
  • R 3 is hydrogen, substituted or unsubstituted, branched, cyclic, or straight chain C 1 -C 5 alkyl, -C 5 alkenyl, acyl, arylalkyl, aryloxycarbonyl, arylaminocarbonyl, arylalkylsulfonyl, or aryl;
  • P 4 is a carboxylic acid, substituted or unsubstituted, branched, cyclic, or straight chain, lower alkyl esters, alkenyl esters, dilower alkyl amino esters, (CH 2 ) ⁇ - 4 SP 4 , COOP 4 ', or CONP 4 'P 4 ";
  • P 4 ' and P 4 " are each independently hydrogen, Cj-C6 alkyl, C2-C ⁇ alkenyl, C2- Cg alkynyl or optionally substituted aryl;
  • R 7 is hydrogen, carboxylic acid, unsubstituted or substituted lower alkyl esters, lower alkenyl esters, dilower alkyl amino esters, arylaminocarbonyl, aroyl, aryl, alkylaminocarbonyl, aminocarbonyl, lower alkyl amides, dilower alkyl amides, alkenylaminocarboxy, hydroxy, ether, thio, amino, (CH 2 ) ⁇ - 4 SR 7 , a heterocycle, cleavable prodrug moiety, COOR , CONR R , a heterocycle, or a cleavable prodrug moiety;
  • R 7 ' and R 7 " are each independently hydrogen, Ci-C ⁇ alkyl, C2-C6 alkenyl, C2- C5 alkynyl or optionally substituted aryl; R 7 is alkyl, alkenyl, alkynyl, aryl, or hydrogen;
  • G is a linking moiety, such as, for example, a covalent bond, substituted or unsubstituted, Ci-C ⁇ (e.g., Ci, C 2 , C 3 , C , C 5 or C 6 ) alkyl, alkenyl, alkynyl, heterocyclic, ether, thioether, amine or carbonyl moiety, optionally substituted with up to three, four, five or six heteroatoms; M is an anchor moiety, such as for example, alkyl, hydrogen, heterocyclic, or carbocyclic;
  • J is a bond, substituted or unsubstituted alkyl, alkenyl, or alkynyl;
  • D is alkyl, alkenyl, alkynyl, aryl, alkoxy, heteroaryl, or optionally linked to Q, G or M to form a ring, and enantiomers, diastereomers, mixtures of enantiomers, mixtures of diastereomers, and pharmaceutically acceptable salts thereof.
  • the invention pertains to compounds wherein P 4 comprises a carbonyl group bonded to R .
  • Certain compounds of this embodiment are represented by the formula (VIII):
  • R 6 is -OH or a protecting prodrug moiety
  • R 7 is an hydrogen atom, carboxylic acid, an amide, a protecting prodrug moiety, hydroxyl, thiol, heterocycle (e.g., imidazole, thiazole, oxazole), ether, alkoxy, or an amine
  • Q is CH 2 , O, NH, or NR 3
  • G is a linking moiety as described above, a covalent bond, C ⁇ -C 5 alkyl, alkenyl, alkynyl, ether, thioether, amine, or carbonyl
  • M is an anchor moiety as described above, alkyl, hydrogen, aryl, heteroaryl, heterocyclic, or carbocyclic
  • J is a bond, substituted or substituted alkyl, alkenyl, or alkynyl moiety
  • D is alkyl, alkoxy, alkenyl, alkynyl, aryl, or heteroaryl
  • R 7 is a carboxylic acid group
  • G is a covalent bond, C 1 -C3 alkyl, or aminoalkyl
  • M is phenyl, or heteroaryl (e.g., thienyl, triazolyl, thiazolyl, or imidazolyl).
  • J is a covalent bond or alkynyl.
  • D may be alkyl (e.g., n-propyl, methyl, isopropyl, ethyl, cycloalkyl, or butyl), a side chain of a natural or unnatural amino acid, or heteroaryl (e.g., pyridinyl or imidozlyl).
  • Examples of compounds of the invention which may be prodrugs include compounds of the formula (N):
  • M is carbocyclic, heterocyclic, CO ⁇ R'R", wherein R' and R" are each independently hydrogen, C ⁇ -Cg alkyl, C2-Cg alkenyl, C2-Cg alkynyl or optionally substituted aryl;
  • Q is a bond, O, S, CR 3 OH, CR 3 SH, CR 3 ⁇ R 3a R 3b , NR 3 , (CR 3 R 3a ) n , O(CR 3 R 3b ) n , and (CR 3 R 3a ) procurO(CR 3b R 3c ) n , wherein n is 0, 1, 2, or 3 and R 3 , R 3a , R 3b , and R 3c are each independently hydrogen, substituted or unsubstituted, branched, cyclic, or straight chain C ⁇ -C 6 alkyl, C 2 -C 6 alkenyl, acyl, arylalkyl, aryloxycarbonyl, arylaminocarbonyl, arylalkylsulfonyl, or aryl;
  • K is an independently selected sublinking moiety for each position capable of substitution of M
  • L is an independently selected subanchor moiety selected for each position capable of substitution of M
  • P 4 is a hydrogen, carboxylic acid, (CH 2 ) ⁇ - 4 SP 4 , a cleavable prodrug moiety, carboxylic acid, COOP 4 ', or CONP 'P 4 ";
  • R 7 is hydrogen, carboxylic acid, aroyl, aryl, COOR 7' , C(O)NR 7 'R 7 ", hydroxy, ether, thio, (CH ) ! - 4 SR 7 , a heterocycle, or a cleavable prodrug moiety;
  • P 4 , P 4 , R 7' and R 7 independently hydrogen, Cj-Cg alkyl, C2-Cg alkenyl, C2- C ⁇ alkynyl or optionally substituted aryl; alkyl, alkenyl, alkynyl, or hydrogen; n is O, 1, 2, 3, or 4;
  • J is a bond, substituted or unsubstituted alkyl, alkenyl, or alkynyl
  • D is hydrogen, alkyl, alkenyl, amine, alkoxy, hydroxy, alkynyl, aryl, or heteroaryl, any of which optionally may be branched or substituted
  • t is 0 or 1
  • Q is O, CR 3 J ⁇ R3a , NR', or S M includes anchor moieties, as described previously, such as, for example, moieties of the formulae:
  • K is selected independently for each position capable of substitution from the subanchor moieties described previously;
  • L is selected independently for each position capable of substitution from the subanchor moieties described previously;
  • N is an auxiliary sublinking moiety
  • W is an auxiliary subanchor moiety.
  • carrier includes both cycloalkyl, cycloalkenyl, aryl, biaryl and any ring system which includes one ring composed of carbon atoms bonded to each other.
  • the carbon atoms may be bonded to atoms which are not carbon, as long as at least one ring is formed with the carbon atoms.
  • heterocyclic includes heteroaryls as well as any ring formed which incorporate a heteroatom or an atom which is not carbon.
  • the ring may be saturated or unsaturated and may contain one or more double bonds.
  • auxiliary sublinking moiety or “N” includes sublinking moieties as described previously that when bonded to a heteroatom (e.g., nitrogen, oxygen, sulfur, phosphorous, etc.) result in a compound of the invention which is capable of performing its intended function (e.g., modulate ACE-2).
  • auxiliary subanchor moieties include, but are not limited to sublinking moieties as described above. Examples of auxiliary sublinking moieties (or “N”) include covalent bonds,
  • V is (CH 2 ), CONHCH 2 , CH 2 -S(O 2 ), (CH 2 ) 2 , or (CH 2 )O(CH 2 ).
  • V is a covalent bond.
  • auxiliary subanchor moiety includes, but is not limited to, subanchor moieties as described previously. It also includes moieties that when bonded to auxiliary subanchor moieties result in a compound of the invention which is capable of performing its intended function (e.g., modulate ACE-2).
  • auxiliary subanchor moieties include, for example, alkyl, alkenyl, alkynyl,
  • the auxiliary subanchor moiety can be substituted at any position that allows the auxiliary subanchor moiety to perform its intended function, e.g., allows the compound of the invention to interact with ACE-2.
  • substituents of N and W include hydrogen, chlorine, bromine, fluorine, iodine, nitro, carboxy, substituted or unsubstituted alkoxy, alkyl, and aryl.
  • N-W is
  • M-K-L is
  • M-K-L is N-(2-aminoethyl)-2-aminoethyl
  • sublinking moieties include covalent bonds, and (CR ⁇ K ⁇ R ⁇ 1 )o-2O(CR ,K2 ⁇ R>K3 J )o-2 5 wherein R ⁇ , R K1 , R 1 ⁇ 2 , and R K3 are selected from the group consisting of halogens, hydrogen, and alkyl.
  • K is (CH 2 ), CO ⁇ HCH 2 , CH 2 -S(O 2 ), (CH 2 ) 2 , or (CH 2 )O(CH 2 ).
  • Other sublinking moieties include NR K , O, and S.
  • the subanchor moiety is selected from the group consisting of hydrogen, NH , and substituted and unsubstituted alkyl, aryl, arylalkyl, and arylalkylamino.
  • L can be substituted at any position which allows it to perform its intended function.
  • L can be substituted at any position capable of being substituted. Examples of possible substituents include, but are not limited to, hydrogen, chlorine, bromine, fluorine, iodine, nitro, carboxy, substituted or unsubstituted alkoxy, alkyl, and aryl.
  • M is:
  • K and L are chosen independently for each position capable of substitution.
  • K include covalent bonds, alkyl, alkenyl, alkynyl, or aryl.
  • L include hydrogen atoms, substituted and unsubstituted alkyl, aryl, arylalkylamino, arylalkyl,
  • Each of the subanchor and sublinking moieties shown above can be substituted at each position capable of substitution.
  • substituents of the subanchor and sublinking moieties include chlorine, bromine, fluorine, iodine, nitro, alkoxy (substituted or unsubstituted), cyano, fluoroalkoxy (OCF 3 ), alkyl (substituted or unsubstituted), carboxy, fluoroalkyl (CF 3 ), and substituted or unsubstituted aryl, e.g., benzyl.
  • M is linked to K and/or L through a carbon-carbon bond.
  • K is a covalent bond and L is a hydrogen atom.
  • K is a covalent bond and L is alkyl.
  • n is 1.
  • Q is NH.
  • J is a bond.
  • D is alkyl (e.g., substituted alkyl, e.g., alkyl amino, alkylhydroxy, alkylthio, alkylphenyl, alkylcycloalkyl, or alkylacetylene) or alkoxy.
  • D is alkyl, e.g., -Cs, e.g., isobutyl.
  • P and R are each carboxylic acids
  • both P and R 7 are not both carboxylic acids.
  • P 5 , P 6 , and P 7 are each independently selected from the group consisting of substituted and unsubstituted alkyl, benzyl, phenyl, cycloalkyl, alkenyl, and alkynyl.
  • P 5 , P 6 , and P 7 are any substituent which allows the compound of the invention to perform its intended function, e.g., bind or interact with
  • P , P , and P are selected such that the compound is capable of performing its intended function after being administered to a subject.
  • P 5 , P 6 , and P 7 may be selected such that the compound of the invention is capable of being absorbed by the digestive system after being administered orally.
  • P 5 , P , and P 7 include moieties which allow the compound to perform its intended function in vivo.
  • P and R 7 groups may be cleaved in vivo to yield a carboxylic acid or carboxy late. Therefore, in one embodiment, P 5 , P 6 , and P 7 may be selected such that in vivo P 4 and/or R 7 are selected such that they are cleaved in vivo to yield a compound which is capable of performing its intended function.
  • the invention also pertains to compounds wherein, the * carbons are of the S configuration.
  • the following diagram shows the stereochemistry of four possible stereoisomers:
  • Examples of compounds of the invention of formula (N) include:
  • Compounds of the invention of formula V include, but are not limited to, 2- ⁇ 2-[3-(3,5-Dichloro-benzyl)-3H-imidazol-4-yl]-l-ethoxycarbonyl-ethylamino ⁇ -4- methyl-pentanoic acid; 2- ⁇ 2-[3-(3,5-Dichloro-benzyl)-3H-imidazol-4-yl]-l - ethoxycarbonyl-ethylamino ⁇ -4-methyl-pentanoic acid ethyl ester; 2- ⁇ l-Carboxy-2-[3- (3 ,5 -dichloro-benzy l)-3 H-imidazol-4-yl] -ethylamino ⁇ -4-methyl-pentanoic acid ethyl ester; 2-[l-Ethoxycarbonyl-2-(2-phenyl-thiazol-4-yl)-ethylamino]-4-methyl-p
  • the compounds of the invention also include prodrugs.
  • Prodrugs of the invention may or may not be able to interact with ACE-2 prior to being metabolized in vivo. However, once the compounds of the invention which are prodrugs are metabolized in vivo or in vitro, they are capable of performing their intended function, e.g. , bind or interact with ACE-2.
  • the compounds of the invention are capable of performing their intended function after being orally administered. In order to perform their intended function after oral administration, it is believed that the compounds must be absorbed by a portion of the digestive tract. In one embodiment of the invention, the compounds of the invention are capable of being absorbed by the digestive tract.
  • the prodrug compound is metabolized in vivo or in vitro to yield a compound which is capable of performing its intended function, e.g., bind or interact with ACE-2.
  • the ability of a compound to be metabolized in vivo or in vitro can be determined using methods known in the art. Examples of these methods include exposing the compound of the invention to reactive components present in a subject and analyzing the results of the interaction of the compound of the invention with the reactive components. Examples of reactive components include, for example, enzymes present in vivo, and other species present in vivo which are capable of interacting with and altering the compound of the invention.
  • the compounds of formula NI below may be converted to compounds which are included in formula I-N, Nil, and NIII, above, after being metabolized in vivo, in vitro, or ex vivo.
  • the invention pertains to a method for treating a subject by administering to the subject an effective amount of a compound which is metabolized in vivo to a compound capable of interacting with ACE-2.
  • the compound capable of interacting with ACE-2 is of any one of formulas I-NIII.
  • the invention also pertains to compounds of the formula (NI):
  • M is heterocyclic, carbocyclic, or CONR'R", wherein R and R" are each independently hydrogen, Cj-C ⁇ alkyl, C2-Cg alkenyl, C2-C ⁇ alkynyl or optionally substituted aryl;
  • K is an independently selected subanchor moiety for each position of M which is capable of substitution
  • L is an independently selected subanchor moiety for each position of M which is capable of substitution
  • P 8 is hydrogen or alkyl
  • P 9 is carboxylic acid, unsubstituted or substituted lower alkyl esters, (CH 2 ) ⁇ .
  • SP 9 lower alkenyl esters, dilower alkyl amino esters, amides (lower alkylaminocarbonyl, lower dialkylaminocarbonyl, aminocarbonyl, alkenylaminocarbonyl, dialkenylaminocarbonyl, arylaminocarbonyl, arylalkyl aminocarbonyl, etc.) lower alkyl amides, dilower alkyl amides, or lower alkyl amides;
  • P » ⁇ o is carboxylic acid, unsubstituted or substituted lower alkyl esters, (CH 2 ) ! -
  • lower alkenyl esters dilower alkyl amino esters, amides (lower alkylaminocarbonyl, lower dialkylaminocarbonyl, aminocarbonyl, alkenylaminocarbonyl, dialkenylaminocarbonyl, arylaminocarbonyl, arylalkylaminocarbonyl, etc.), lower alkyl amides, dilower alkyl amides, or lower alkyl amides;
  • P 9 and P 10 are each independently alkyl, alkenyl, alkynyl, aryl, or hydrogen; a is 1, 2, or 3; b is 0 or 1 ; and x is 0, 1, 2, 3, or 4, and enantiomers, diastereomers, mixtures of enantiomers, mixtures of diastereomers, and pharmaceutically acceptable salts thereof.
  • M examples include anchor moieties include those described above, such as, for example, substituted or unsubstituted aryl, heteroaryl, carbocyclic, heterocyclic or amidyl moieties.
  • anchor moieties include those described above, such as, for example, substituted or unsubstituted aryl, heteroaryl, carbocyclic, heterocyclic or amidyl moieties.
  • M includes moieties of the formula:
  • K and L are each independently selected subanchor and sublinking moieties as described above.
  • N and W are each independently selected auxiliary subanchor and sublinking moieties as described above.
  • auxiliary sublinking moieties include covalent bonds, CO ⁇ R v2 CR v R vl , CR v R vl -S(O 2 ), (CR v R vl ) 0 - 3 , and (CR v R vl )o- 2 O(CR V2 R V3 ) 0 . 2 , wherein R ⁇ , R V1 , R V2 , and R V3 are selected from the group consisting of halogens, hydrogen, and alkyl.
  • V is (CH 2 ), CONHCH 2 , CH 2 -S(O 2 ), (CH 2 ) 2 , or (CH )O(CH 2 ).
  • V is a covalent bond.
  • auxiliary subanchor moieties include, for example, alkyl, alkenyl, alkynyl,
  • the auxiliary subanchor moiety can be substituted at any position that allows the auxiliary subanchor moiety to perform its intended function, e.g., allows the compound of the invention to interact with ACE-2.
  • substituents of V and W include hydrogen, chlorine, bromine, fluorine, iodine, nitro, carboxy, substituted or unsubstituted alkoxy, alkyl, and aryl.
  • V-W is
  • M-K-L is
  • M-K-L is N-(2-aminoethyl)-2-aminoethyl
  • sublinking moieties include covalent bonds, CONR K2 CR K R K1 , CR K R K1 -S(O 2 ), (CR K R K1 ) 0 - 3 , and (CR K R K1 ) 0 . 2 O(CR K2 R K3 ) 0 - 2 , wherein
  • R >K , ⁇ RKl , R ,K2 , and R »K3 are selected from the group consisting of halogens, hydrogen, and alkyl.
  • K is (CH 2 ), CONHCH 2 , CH 2 -S(O 2 ), (CH 2 ) 2 , or (CH )O(CH 2 ).
  • Other sublinking moieties include NR K , O, and S.
  • the subanchor moiety is selected from the group consisting of hydrogen, NH 2 , and substituted and unsubstituted alkyl, aryl, arylalkyl, and arylalkylamino.
  • L can be substituted at any position which allows it to perform its intended function.
  • L can be substituted at any position capable of being substituted. Examples of possible substituents include, but are not limited to, hydrogen, chlorine, bromine, fluorine, iodine, nitro, carboxy, substituted or unsubstituted alkoxy, alkyl, and aryl.
  • M is:
  • K and L are chosen independently for each position capable of substitution.
  • K include covalent bonds, alkyl, alkenyl, alkynyl, or aryl.
  • L include hydrogen atoms, substituted and unsubstituted alkyl, aryl, arylalkylamino,
  • M is linked to K and/or L through a carbon-carbon bond.
  • K is a covalent bond and L is a hydrogen atom.
  • K is a covalent bond and L is alkyl.
  • Each of the subanchor, sublinking, auxiliary subanchor, and auxiliary sublinking moieties shown above can be substituted at each position capable of substitution.
  • substituents of the subanchor and sublinking moieties include chlorine, bromine, fluorine, iodine, nitro, alkoxy (e.g., OR, unsubstituted or substituted, e.g., halogenated, e.g., fluoroalkoxy, O(CR'R")o- 3 CF 3 , O(CR'R")o- 3 CCl 3 ), cyano, alkyl (unsubstituted or substituted, e.g., halogenated, e.g., fluoroalkyl, e.g.
  • thioethers e.g., alkylthiols, e.g., substituted or unsubstituted, e.g., SCF 3 , SCCI 3
  • substituted or unsubstituted aryl alkenyl, alkynyl, e.g., benzyl.
  • substituents for the subanchor and the sublinking moieties include other substituents described supra wherein R and R" are each independently hydrogen, Cj-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or optionally substituted aryl.
  • M is bound to the subanchor or sublinking moiety through a carbon atom, e.g., through a carbon-carbon bond, or through another bond which is substantially metabolically stable.
  • x is 1.
  • a is 1.
  • P 9 is substituted or unsubstituted alkyl (e.g., CrC 8 alkyl, e.g., methyl, ethyl, propyl, t-butyl, isobutyl, pentyl, hexyl, etc.), alkylamino, alkylhydroxy, alkyl thio, alkenylphenyl, alkylcycloalkyl, and alkylacetylene.
  • ⁇ is and the * carbons are of the S configuration (as shown)
  • ⁇ is , and the * carbons are of the S configuration (as shown).
  • Examples of compounds of the invention of formula (NI) include:
  • Examples of compounds of formula VI of the invention include, but are not limited to, 3-[3-(3,5-Dichloro-benzyl)-3H-imidazol-4-yl]-2-(4-isobutyl-2-methyl-5-oxo- oxazolidin-3-yl)-propionic acid; 2- ⁇ 4-[3-(3,5-Dichloro-benzyl)-3H-imidazol-4- ylmethyl]-2-methyl-5-oxo-oxazolidin-3-yl ⁇ -4-methyl-pentanoic acid; 3-[3-(3,5- Dichloro-benzyl)-3H-imidazol-4-yl]-2-(4-isobutyl-2-methyl-5-oxo-oxazolidin-3-yl)- propionic acid ethyl ester; 2- ⁇ 4-[3-(3,5-Dichloro-benzyl)-3H-imidazol-4-ylmethyl]-2- metl ⁇
  • the ACE-2 compounds of the invention do not include compounds A, G, H, I, J, or L. In another embodiment, the ACE-2 compounds of the invention do not include BD, 2-(l-carboxy-ethyIamino)-3-(lH-imidazol-4-yl)-propionic acid.
  • Examples of compounds of the invention include: 2- ⁇ l-Carboxy-2-[3-(3-nitro-benzyl)-3H-imidazol-4-yl]-ethylamino ⁇ -4-methyl-pentanoic acid;
  • Examples compounds of the invention include those having the structures shown below:
  • small molecules includes molecules which are capable of being used as therapeutic agents e.g., peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic (including, e.g., heteroorganic and organometallic compounds) and inorganic compounds.
  • the term includes compounds which have a molecular weight of about, for example, 10,000 grams per mole or less, 5,000 grams per mole or less, 2,000 grams per mole or less, or 1 ,000 g/mol grams per mole or less.
  • the small molecule is an organic compound. Examples of small molecules include those described in Formulae 1 -VIII and in Table 2.
  • Organic compounds comprise one or more carbon atoms.
  • the compound is an inorganic compound.
  • Inorganic compounds include compounds which do not comprise a carbon atom.
  • alkyl includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • straight-chain alkyl groups e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octy
  • alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
  • a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C ⁇ -C 6 for straight chain, C 3 -C 6 for branched chain), and more preferably 4 or fewer.
  • preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • C ⁇ -C 6 includes alkyl groups containing 1 to 6 carbon atoms.
  • alkyl includes both "unsubstituted alkyls" and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sul
  • Cycloalkyls can be further substituted, e.g., with the substituents described above.
  • An "alkylaryl” or an “arylalkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • the term “alkyl” also includes the side chains of natural and unnatural amino acids.
  • aryl includes groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • aryl includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g.
  • naphthalene benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinofine, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine.
  • aryl heterocycles may also be referred to as "aryl heterocycles", “heterocycles,” “heteroaryls” or “heteroaromatics”.
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
  • alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
  • alkenyl includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups.
  • alkenyl includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonen
  • alkenyl further includes alkenyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
  • a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-Cg for straight chain, C3-C6 for branched chain).
  • cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • C -C includes alkenyl groups containing 2 to 6 carbon atoms.
  • alkenyl includes both "unsubstituted alkenyls" and “substituted alkenyls”, the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
  • alkynyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.
  • alkynyl includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups.
  • alkynyl further includes alkynyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
  • a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-Cg for straight chain, C3-C6 for branched chain).
  • C 2 -C 6 includes alkynyl groups containing 2 to 6 carbon atoms.
  • alkynyl includes both "unsubstituted alkynyls" and
  • substituted alkynyls refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including, e.g., alkylcarbonylamino, ary
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure.
  • Lower alkenyl and “lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.
  • acyl includes compounds and moieties which contain the acyl radical (CH 3 CO-) or a carbonyl group.
  • substituted acyl includes acyl groups where one or more of the hydrogen atoms are replaced by for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, ary
  • acylamino includes moieties wherein an acyl moiety is bonded to an amino group.
  • the term includes alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
  • aroyl includes compounds and moieties with an aryl or heteroaromatic moiety bound to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
  • alkoxy alkyl includes alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g. , oxygen, nitrogen or sulfur atoms.
  • alkoxy includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom.
  • alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups.
  • substituted alkoxy groups include halogenated alkoxy groups.
  • the alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate
  • amine or "amino” includes compounds where a nitrogen atom is covalently bonded to at least one carbon or heteroatom.
  • alkyl amino includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
  • dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
  • arylamino and “diarylamino” include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively.
  • alkylarylamino alkylaminoaryl or “arylaminoalkyl” refers to an amino group which is bound to at least one alkyl group and at least one aryl group.
  • alkaminoalkyl refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.
  • amide or "aminocarboxy” includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group.
  • alkaminocarboxy groups which include alkyl, alkenyl, or alkynyl groups bound to an amino group bound to a carboxy group. It includes arylaminocarboxy groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group.
  • alkylaminocarboxy include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively, are bound to a nitrogen atom which is in turn bound to the carbon of a carbonyl group.
  • carbonyl or “carboxy” includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom.
  • moieties which contain a carbonyl include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.
  • thiocarbonyl or “thiocarboxy” includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.
  • ether includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms.
  • alkoxyalkyl which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
  • esters includes compounds and moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group.
  • ester includes alkoxycarboxy groups such as mefhoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
  • alkyl, alkenyl, or alkynyl groups are as defined above.
  • thioether includes compounds and moieties which contain a sulfur atom bonded to two different carbon or hetero atoms.
  • examples of thioethers include, but are not limited to alkthioalkyls, alkthioalkenyls, and alkthioalkynyls.
  • alkthioalkyls include compounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bonded to an alkyl group.
  • alkthioalkenyls and alkthioalkynyls refer to compounds or moieties wherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkynyl group.
  • hydroxy or "hydroxyl” includes groups with an -OH or -O " X + , where X* is a counterion.
  • halogen includes fluorine, bromine, chlorine, iodine, etc.
  • perhalogenated generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.
  • polycyclyl or “polycyclic radical” refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the poly cycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl, arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
  • heteroatom includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus. It will be noted that the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof.
  • Scheme 1 depicts a general method of synthesizing compounds of the invention, wherein Q is NH.
  • Resin bound FMOC protected amino acids are commercially available from Novabiochem.
  • the amino terminus is deprotected by treatment with 20% piperidine in DMF for 30 minutes (Scheme 1).
  • the amine (1-1) is then treated with acetic acid in DMF and various commercially available ⁇ -ketoesters.
  • the resulting Schiff base is then treated with a hydride agent such as NaBH CN to be converted into the corresponding secondary amine.
  • the acids are removed from the resin through treatment with a strong acid, e.g., trifluoroacetic acid.
  • the resulting acid (1-2) is esterified through treatment with the corresponding alcohol (1-3).
  • the diastereomers prepared here are separated by conventional means.
  • the thiol compounds of the invention, wherein Q is CH 2 SH, are synthesized by methods well known in the art. A sample synthesis is outlined below in Scheme 2.
  • the thiol is synthesized, for example, by heating thiolacetic acid with an appropriately substituted acrylic acid (2-1) to form the thioester (2-2).
  • the thioester is hydrolyzed to the thiol by treatment with base to give the thiol (2-3).
  • the ester is formed by treating the acid compound with the appropriate alcohol to form the desired ester (2-4).
  • One of skill in the art can separate resulting enantiomers through conventional means such as, for example, chiral HPLC chromatography.
  • the imidazole compounds of the invention are synthesized using the procedure outlined in Scheme 3.
  • the amine (3-1) reacts with the carbonyl of the ⁇ -ketoacid (3-2) to form the Schiff base, which is readily converted into the secondary amine (3-3) by a reducing agent.
  • Substituted imidazole compounds of the invention are synthesized, for example, by treating ⁇ -protected histidine with a suitable halogenenated compound, such as benzyl bromide. The substituted histidine derivatives are then used in place of histidine in the synthesis shown in Scheme 3.
  • a phenyl ACE-2 modulating compound is synthesized by treating an ethyl ester (4-1) with triflic anhydride and 2,6-lutidine in methylene chloride at -78°C. To this mixture, leucine methyl ester can be added forming the secondary amine (4-2). The ester is then treated with aqueous base to form the resulting diacid (4- 3). Further synthetic examples are given in the Example section.
  • Scheme 4 Benzylpyrazole compounds of the invention are synthesized using the methods outlined in Scheme 5.
  • commercially available 3-methylpyrazole (5-1) was oxidized to pyrazole-3 -carboxylic acid and then treated with 2 equivalents of benzyl bromide (Jones, R. G. J. Am. Chem. Soc. 1949, 71, 3994).
  • the resulting 1- benzylpyrazole-3 -carboxylic acid benzyl ester was then reduced with LiAlH and converted to bromide 5-2.
  • Alkylation of Scholkopf s dihydropyrazine with this bromide provided 5-3 as a mixture of diastereomers (3-4 to 1 trans: cis) (Scholkopf, U.
  • Phenylthiazole compounds of the invention are synthesized by preparing amino esters 6-1 and subsequently alkylating the esters with the trifluoromethane sulfonate of leucic acid methyl ester (6-3) (Svete, J.; et al. J. Heterocyclic Chem. 1994, 31, 1259.) Hydrolysis of the esters provides the diacids 6-2 (Scheme 6).
  • K-L alkyl, aryl, heteroaryl, arylalkyl, etc.
  • Thiazoles and oxazoles of the invention are prepared by the method shown in Scheme 7.
  • Amino ester 7-2 is alkylated with the triflate of an appropriate alcohol ester in the presence of 2,6-lutidine, and treated with NaOH in MeOH to provide the diacid 7-3.
  • K-L alkyl, aryl, heteroaryl, arylalkyl, etc.
  • Isoxazolyl compounds of the invention are synthesized, for example, by using the method shown in Scheme 8.
  • Scheme 8 the methyl ester of 2-tert- butoxycarbonylamino-4-oxobutyric acid (8-1) is reacted with hydroxylamine hydrochloride, then with NCS, and finally with an appropriate alkyne in the presence of triethylamine to provide the amino ester 8-3 (Gosselin, F. et al. J. Org. Chem. 1998, 63, 7463; Aicher, T. D. et al. J. Med. Chem. 1998, 41, 4556).
  • This amino ester is then reacted as described previously with an appropriate trifluoromethane sulfonate (8-5). Ester hydrolysis then provides the diacid 8-4.
  • Triazolyl compounds of the invention are synthesized, for example, by the method shown in Scheme 9.
  • Triazole 9-1 is reduced to the alcohol and then converted to the bromide 9-2 (WO 99/43677).
  • Alkylation of Scholkopf s dihydropyrazine with this bromide, hydrolysis of the dihydropyrazine, and reaction with the appropriate triflate as previously described provides diester 9-6. Removal of the SEM protecting group, alkylation with an appropriate alkyl bromide, and ester hydrolysis gives the diacid 9-7.
  • L-K alkyl, aryl, heteroaryl, arylalkyl, etc.
  • Boc group is removed with HCl in dioxane to provide amine 10-2, which is then treated with the appropriate ketoester in the presence of NaB(OAc) H to provide the secondary amine 10-3. Further treatment of this amine with a variety of electrophiles (see conditions i-vii Scheme 10, and Sellier, C. et al. Liebigs Ann.Chem.
  • Scheme 18 depicts another method for synthesizing the thiol alkanoic acids compounds of the invention.
  • Compound 18-1 JOC, 1995, 60, 5157
  • triflate (11-2 as described in Scheme 11 above
  • compound 18-2 upon hydrolysis with sodium hydroxide produces the thiol alkanoic acid compound of the invention, 18-3.
  • Sulfonamide compounds of the invention can be synthesized using the method depicted in Scheme 20.
  • Leucine methyl ester, 19-1 is reacted with a sulfonyl chloride in the presence of triethyl amine (TEA) in dichloromethane (DCM) to give sulfonamide (20-1).
  • TEA triethyl amine
  • DCM dichloromethane
  • 20-1 is then reacted with ethyl iodoacetate and silver oxide in DMF to yield sulfonamide, 20-2.
  • the sulfonamide, 20-2 is then hydrolysed with aqueous sodium hydroxide to give the di-acid, 20-3.
  • N-3 alkylated imidazolyl compoxmds of the invention are synthesized by the route shown in Scheme 23.
  • Boc 2 O (2 eq) is added to a solution of histidine methyl ester 23-1 (1 eq,) in MeOH (0.4 M) and triethylamine (2 eq).
  • the reaction mixture is concentrated and the residue is dissolved in dichloromethane (DCM) and H 2 O.
  • the layers are separated and the organic layer are washed with brine, dried over Na 2 SO 4 , filtered and concentrated to provide a yellow oil. This oil is triturated from hexane to provide the di-Boc protected imidazole 23-2 as white solid.
  • reaction mixture is then concentrated again and diluted with DCM or EtOAc and washed with NaHCO 3 (3x) and brine (lx) then dried over Na 2 SO 4 , filtered and concentrated.
  • the resulting oil is purified by column chromatography (MeOH/DCM) to give the N-3 alkylated imidazole derivative 23-4.
  • the Boc protection is removed from the imidazole derivative 23-4 using HCl in dioxane. Trituration of the resulting di-HCl salt from ethyl acetate (EtOAc) provides a white solid which is suspended in dichloroethane (0.1 M). The keto-ester 23-5 (2 eq) is added to this suspension and stirred for 1 hour. After 1 hour, NaB(OAc) 3 H (3 eq) is added slowly. After 24 hoxxrs, sodium carbonate (NaHCO ) is added to quench the reaction (the pH of the reaction mixture is raised to 8 with saturated NaHCO 3 and stirred for 1 hour). The layers are separated and the aqueous layer is extracted with EtOAc twice.
  • EtOAc ethyl acetate
  • volatile co-solvents such as MeOH, EtOH, MeCN or dioxane may be used to speed the reaction progress. Once the reaction is complete, any volatile co- solvent is evaporated and the mixture is acidified to pH 3-4 using 6N HCl. In most cases, upon acidification the desired mono-ester 24-2 forms a precipitate or a gum. This material is separated, dried and purified either on a flash column or by HPLC.
  • Scheme 25 depicts the synthesis of mono-esters such as 25-4 from intermediate 25-1. Standard hydrolysis of the ester 25-1 followed by Boc removal provides the amino acid 25-2. Reductive amination, at neutral pH, between amine 25-2 and the keto- ester 25-3 provides the mono-ester 25-4.
  • the keto-ester 25-3 is prepared from the keto- acid via a similar procedure as shown in Scheme 24 (Cs 2 CO 3 and the appropriate alkyl or aryl halide in dimethylformamide (DMF)).
  • Di-ester prodrugs such as 26-1 are prepared from the diacid 23-5 via esterification as in Scheme 26 (base such as Cs 2 CO 3 and an alkyl or aryl halide in DMF).
  • base such as Cs 2 CO 3 and an alkyl or aryl halide in DMF.
  • differentially substituted di-esters are prepared via reductive amination of the appropriately substituted coupling partners (Scheme 27).
  • Scheme 27 for example, the amino ethyl ester 25-1 is reacted with the benzyl ester of the keto acid 24-2 to give the di-ester 27-1.
  • Lactone prodrugs such as 28-1 and 28-2 are synthesized from the diacid 23-5 (Scheme 28). Reaction of 23-5 with an aldehyde (such as formaldehyde) provides a mixture of prodrug lactones 28-1 and 28-2. Alternatively, a mono-ester such as 24-2 or 25-4 is treated with an aldehyde such as formaldehyde to give a fully protected prodrugs 29-1 and 29-2 (Scheme 29). (Ref: King, G. A. et al. Org. Prep. Proced. Int. (1997) 29:177-184; Paleo, M. et al. J. Org. Chem. (1997) 62:6862-6869).
  • aldehyde such as formaldehyde
  • Scheme 30 depicts methods of synthesizing aromatic and phenyl derivatives of the compounds of the invention.
  • Alkylated tyrosine derivatives (30-1 (p), 30-2 (m), 30- 3 (o)) are obtained from the tyrosine-leucine derivatives such as 30-6 (Scheme 30).
  • tyrosine methyl ester 30-4 is reacted with the triflate of the leucic ester alcohol 30-5 in DCM and lutidine at -78 C to provide the amino diester 30-6.
  • the substituted tyrosine derivative is assembled prior to triflate displacement (Scheme 31).
  • alkylation of the protected tyrosine derivative 31-1 is performed using similar conditions as above, followed by amine deprotection, thus providing the amino ester 31-2.
  • Triflate displacement, as above, provides the diester 30- 7. Hydrolysis and purification provides diacid 30-8.
  • Diphenyl ether derivatives are synthesized according to Scheme 32.
  • Boc-Tyr-OMe (32-1) is reacted with a boronic acid derivative (32-2) in the presence of Cu(OAc) 2 , pyridine and 4 A sieves to give the diphenyl either which, after Boc deprotection provides the amino ester 32-3.
  • Reaction of the amine 32-3 with the triflate of the leucic ester alcohol (31-5) gives the amino diester 32-4.
  • Hydrolysis of the esters affords the diacid 32-5 which is purified by HPLC. Meta and ortho substituted tyrosine derivatives are prepared similarly via the appropriate starting materials.
  • Substituted phenylalanine derivatives (33-1) are synthesized from the appropriate aryl halide starting material.
  • Esterification of Boc-Phe(4-Br)-OH (33-4) (or Boc-Phe(4-I)-OH) using TMSCHN 2 followed by palladium catalyzed cross coupling provides the 4-substituted amino acid derivative 33-5 (Negishi, E.-I. and King, A. O. J. Org. Chem. (1977) 42:1821; Klement, I. et al Tetrahedron Lett. (1994) 35:1177). Boc deprotection followed by reaction with the triflate of the leucic ester alcohol, as before, provides the diester 33-6.
  • 1-Napthylalanine (34-1) and 2-napthylalanine (34-2) derivatives are available via the routes shown in Schemes 30-33.
  • substituted naphthalene derivatives (34- 3, 34-4) are synthesized from the halogenated (or hydroxyl) naphthalene amino acid derivatives (34-5, 34-6).
  • Amino acid synthesis is achieved via a number of methods (Ref: Hagiwara, D. et al. J. Med. Chem. (1994) 37:2090-2099; Myers, A. G. and Gleason, J. L. Org. Syn (1999) 76:57-76; Myers, A. G., and Yoon, T. Tetrahedron Lett.
  • the invention also pertains to a method of treating an ACE-2 associated state, for example, which is a method for treating a body weight disorder or a state associated with body mass.
  • the method involves administering to a subject a therapeutically effective amount of an ACE-2 modulating, e.g., inhibiting, compound, such that the ACE-2 associated state is treated (e.g., at least one symptom is alleviated).
  • states associated with body mass include lipodystrophy, cachexia
  • the ACE-2 inhibitors can be used to increase muscle mass, and/or decrease body fat.
  • the ACE-2 inhibitors can be administered to healthy individuals or to individuals who wish to alter their body composition, e.g., subjects who wish to increase their body mass.
  • ACE-2 inhibitors can also be used to treat disorders such as obesity and diabetes.
  • the language "in combination with" another therapeutic agent includes co- administration of the ACE-2 modulating compound, (e.g., inhibitor) and another therapeutic agent, administration of the ACE-2 modulating compound first, followed by the other therapeutic agent and administration of the other therapeutic agent first, followed by the ACE-2 modulating, e.g., inhibiting, compound.
  • the other therapeutic agent may be any agent which is known in the art to treat, prevent, or reduce the symptoms of a ACE-2 associated state, e.g., a body weight disorder.
  • the other therapeutic agent may be any agent of benefit to the subject when administered in combination with the administration of an ACE-2 modulating, e.g., inhibiting, compound.
  • the other therapeutic agent may also be an ACE-2 modulating compound.
  • ACE-2 compounds can also be administered in combination with other known therapies for ACE-2 associated states. Other methods of treating ACE-2 associated states, e.g., body weight disorders, are known to those skilled in the art.
  • the other therapeutic agent may be an ACE inhibitor.
  • the ACE-2 inhibitor may be a dual inhibitor of ACE and ACE-2.
  • Dual inhibitors include compounds and combinations of compounds which inhibit both ACE and ACE-2 gene expression or protein activity.
  • Dual inhibitors can be compounds which bind to both ACE and ACE-2 equally well, compounds which bind to ACE preferentially over ACE-2, and compounds which bind to ACE-2 preferentially over ACE.
  • a dual inhibitor has a Kj for ACE of 5 ⁇ M or less (e.g. 4 ⁇ M or less, 3 ⁇ M or less, 2 ⁇ M or less, or, preferably, 1 ⁇ M or less), and a K; of 5 ⁇ M or less for ACE-2.
  • the dual inhibitor compounds of the invention are compounds in Table 2 which are identified by *** in the Human ACE-2 Activity column and * in the ACE Activity column.
  • a dual inhibitor may be subject specific, based on the needs of a particular subject. For example, for a particular condition in a particular subject, it may be advantageous to treat the subject's condition by administering to the subject a dual inhibitor which inhibits ACE and ACE-2 equally. For another subject, it may be advantageous to administer to the subject a dual inhibitor which inhibits ACE at a higher rate than ACE-2. Alternatively, for yet another subject it may be advantageous to administer to the subject a dual inhibitor which inhibits ACE-2 at a higher rate than ACE.
  • the selection of a particular dual inhibitor for a particular subject is based on the response of the subject to an ACE-2 inhibitor, ACE inhibitor, or dual inhibitor. For example, based on the subject's response to an ACE-2 inhibitor, a person skilled in the art may determine that a dual inhibitor and/or ACE inhibitor may be effective to treat a subject's condition. Similarly, based on a subject's response to an ACE inhibitor, a person skilled in the art may determine that a dual inhibitor or a ACE-2 inhibitor may be more effective to treat a subject's condition. Furthermore, based on a subject's response to a dual inhibitor, a person of skill in the art may determine that a particular subject may benefit from increased ACE or ACE-2 inhibition to treat the subj ect ' s condition.
  • the response of a subject to a particular dual inhibitor, ACE inhibitor, or ACE-2 inhibitor can be predicted, e.g., by pharmacogenomics.
  • Pharmacogenomics predicts a subject's response to a particular drug based on the subject's genotype. For example, based on a subject's genotype, an effective dual inhibitor to treat the subject's condition will be administered.
  • the ratio of inhibition of ACE and ACE-2 can be tailored to each subject to increase the efficacy of each subject's treatment.
  • the invention also pertains to a method for increasing body muscle, e.g., increasing muscle mass, in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, such that the body muscle of a subject is increased.
  • the subject is suffering from cachexia, e.g., cachexia resulting from a disease, e.g., cancer or an autoimmune disease, e.g., AIDS.
  • the subject is not suffering from cachexia and desires greater muscle mass, e.g., a person who desires greater muscle mass, e.g., an athlete or body builder.
  • the subject is suffering from decreased muscle mass.
  • the decreased body muscle mass may be due to old age, inactivity, space travel, obesity, etc.
  • the muscle mass of said subject is increased about 1% or greater, about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 35%) or greater, about 40% or greater, about 45% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, about
  • the invention also pertains to a method for decreasing body fat in a subject.
  • the method includes administering to the subject an effective amount of a compound of the invention, e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound, so that the body fat in the subject is decreased.
  • a compound of the invention e.g., a compound of any one of formulae I-NIII, e.g., an ACE-2 modulating, e.g., ACE-2 inhibiting, compound
  • the subject is of normal or above average weight and desires decreased body fat.
  • the subject is obese.
  • the subject is suffering from a disorder of lipid metabolism, e.g., a lipodystrophy or a lipidosis.
  • the percent body fat of the subject is decreased about 1% or greater, about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 35% or greater, about 40% or greater, about 45% or greater, about 50% or greater, about 55% or greater, about 60% or greater, about 65% or greater, about 70% or greater, about 85% or greater, about 90% or greater, about 95% or greater, about 100% or greater, about 125% or greater, about 150% or greater, about 175% or greater, about 200% or greater, about 250% or greater, about 300%) or greater, etc. as compared to the body fat percentage of the subject prior to administration of the ACE-2 modulating compound of the invention.
  • the invention pertains, at least in part, to a method for decreasing the appetite of a subject.
  • the method includes administering to the subject an effective amount of an ACE-2 modulating compound of the invention such that the appetite of the subject is decreased, e.g., suppressed.
  • the invention also relates to a pharmaceutical composition containing a pharmaceutically acceptable carrier and an effective amoxmt of an ACE-2 modulating, e.g., inhibiting, compound to treat an ACE-2 associated state.
  • the invention pertains to pharmaceutical compositions comprising a compound of any one of formulae I-NIII, as described above.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the invention within or to the subject such that it can performs its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • the pharmaceutically acceptable carrier is selected such that the ACE-2 modulating compound of the invention has low systemic absorbtion and relatively high exposure to the gastrointestinal tract (e.g., lower gastrointestinal tract, e.g., bowel, large and small and intestines).
  • the gastrointestinal tract e.g., lower gastrointestinal tract, e.g., bowel, large and small and intestines.
  • certain embodiments of the present compounds can contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids.
  • pharmaceutically acceptable salts in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the invention.
  • salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).
  • the compounds of the invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin
  • Formulations of the invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the invention as an active ingredient.
  • a compound of the invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostea
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert dilutents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert dilutents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the invention to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the d g from subcutaneous or intramuscular injection.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlor
  • a liquid suspension of crystalline or amorphous material having poor water solubility This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
  • the rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered dmg form is accomplished by dissolving or suspending the drag in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • the preparations of the invention may be given orally, parenterally, topically, or rectally. They are of coxrse given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, dmg or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds of the invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this 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 subject, composition, and mode of administration, without being toxic to the subject.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, 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 subject 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.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a compound of the invention While it is possible for a compound of the invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.
  • the regimen of administration can affect what constitutes an effective amount.
  • the ACE-2 modulating e.g., inhibiting
  • several divided dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection.
  • the dosages of the ACE-2 modulating, e.g., inhibiting, compound(s) can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • cell based and non-cell based assays are described that can be used to identify compounds that interact with the ACE-2 nucleic acid or protein, e.g., modulate the expression of the ACE-2 gene and/or activity of the ACE-2 polypeptide.
  • the cell based assays can be used to identify compounds or compositions that affect the biological activity of the ACE-2 polypeptide, whether they bind to ACE-2, or act on ACE-2 target peptides, e.g., ligands or substrates, such as angiotensin I, bradykinin, neurotensin, and kinetensin.
  • Such cell based assays of the invention utilize cells, cell lines, or engineered cells or cell lines that express the ACE-2 polypeptide.
  • the cells can be further engineered to incorporate a reporter molecule linked to an ACE-2 ligand or substrate to aid in the identification of compounds that modulate ACE-2 enzymatic activity.
  • the invention also encompasses the use of cell based assays and cell lysate assays (e.g. , in vitro transcription or translation assays) to screen for compounds or compositions that modulate ACE-2 gene expression and therefor can be used to identify ACE-2 modulating compounds useful for the methods described herein.
  • constructs containing a reporter sequence linked to a regulatory element of the ACE-2 gene can be used in engineered cells, or in cell lysate extracts, to screen for compounds that modulate the expression of the reporter gene product at the level of transcription.
  • assays can be used to identify compounds that modulate the expression or activity of transcription factors involved in ACE-2 gene expression, or to test the activity of triple helix polynucleotides.
  • engineered cells or translation extracts can be used to screen for compoxmds (including antisense and ribozyme constructs) that modulate the translation of ACE-2 mRNA transcripts, and therefore, affect expression of the ACE-2 gene.
  • compoxmds including antisense and ribozyme constructs
  • the invention also encompasses ACE-2 proteins, polypeptides (including soluble ACE-2 polypeptides or peptides), and biologically active fragments thereof, and ACE-2 fusion proteins for use in non-cellular screening assays, for use in generating antibodies, and for the diagnosis and/or treatment of body weight disorders.
  • ACE-2 protein products can also be used to treat body weight disorders, including, for example, obesity, anorexia or cachexia.
  • Such ACE-2 protein products include, but are not limited to, soluble derivatives such as peptides or polypeptides corresponding to truncated ACE-2 polypeptides and ACE-2 fusion protein products (especially ACE-2 -Ig fusion proteins, e.g., fusions of the ACE-2 polypeptide or a domain of the ACE-2 polypeptide, to an IgFc domain).
  • antibodies to ACE-2 or anti-idiotypic antibodies that mimic or interfere with ACE-2 activity can be used to treat body weight disorders such as obesity, anorexia or cachexia.
  • an effective amount of soluble ACE-2 polypeptide, or an ACE-2 fusion protein (e.g., ACE-2 -IgFc) or an anti-idiotypic antibody (or an antigen-binding fragment thereof, e.g., an Fab, F(ab)' 2 , or a single chain antibody, e.g., scFv) that mimics the ACE-2 activity would interact with and thereby "mop up" or "neutralize” endogenous ACE-2 ligand, and prevent or reduce ACE-2 activity, thereby leading to a weight loss or a reduction of a high BMI.
  • nucleotide constmcts encoding such ACE-2 products can be used to genetically engineer host cells to express such ACE-2 products in vivo; these genetically engineered cells can function as "bioreactors" in the body delivering a continuous supply of an ACE-2 polypeptide, an ACE-2 polypeptide fragment, or an ACE-2 fusion protein that will "mop up” or neutralize an ACE-2 ligand.
  • Gene therapy approaches for the modulation of ACE-2 expression and/or activity in the treatment of body weight disorders are within the scope of the invention.
  • nucleotide constmcts encoding functional ACE-2 polypeptides or polypeptide fragments, mutant ACE-2 polypeptides or polypeptide fragments, as well as antisense and ribozyme molecules can be used to modulate ACE-2 gene expression.
  • the invention also encompasses pharmaceutical formulations, methods for the prophylactic or therapeutic treatment of body weight disorders, and kits for the diagnosis or prognosis of body weight disorders.
  • ACE-2 protein The specific role of the ACE-2 protein in vivo was investigated by engineering ACE-2 "knock out" mice in which most of the endogenous ACE-2 gene coding sequence was deleted, thereby creating mice which are unable to produce biologically active ACE-2 protein.
  • ACE-2 knock out mice human ACE-2 gene sequences were utilized to isolate and clone the murine ACE-2 gene.
  • a murine ACE-2 targeting construct was then generated which was designed to delete the majority of the murine ACE-2 coding sequence upon homologous recombination with the endogenous murine ACE-2 gene.
  • Embryonic stem (ES) cells containing the dismpted ACE-2 gene were produced, isolated and microinjected into murine blastocysts to yield mice chimeric for cells containing a disrupted ACE-2 gene. Offspring of the chimeric mice resulting from germline transmission of the ES genome were obtained and animals heterozygous for the dismpted ACE-2 were identified.
  • mice heterozygous for the ACE-2 disrupted gene were bred together to produce mice homozygous for the ACE-2 mutation.
  • Inactivation of the ACE-2 by gene targeting resulted in male mice that have a higher ratio of lean to fat tissue, a lower percentage of overall body fat tissue, and a lower overall body weight than wild type counterparts.
  • Screening Assays for Dmgs Useful in Regulation of Body Weight At least three different assay systems, described in the subsections below, can be designed and used to identify compounds or compositions that modulate ACE-2 activity or ACE-2 gene expression, and therefore, modulate body weight and/or the percentage of body fat.
  • ACE-2 or cells expressing ACE-2 can be packaged in a variety of containers, e.g., vials, tubes, microtitre well plates, bottles, and the like.
  • Other reagents can be included in separate containers and provided with the kit; e.g. , positive controls samples, negative control samples, ACE-2 target peptides (including but not limited to), buffers, cell culture media, etc., and instmctions for use.
  • a cell-based assay system can be used to screen for compounds that modulate the activity of an ACE-2 protein or expression of an ACE- 2 gene and thereby, modulate body weight and/or the percentage of body fat.
  • cells that endogenously express ACE-2 can be used to screen for compounds.
  • cell lines such as 293 cells, COS cells, CHO cells, fibroblasts, and the like, genetically engineered to express ACE-2 can be used for screening purposes.
  • host cells genetically engineered to express a functional enzyme that catalyzes the cleavage of ACE-2 target peptides, e.g., ligands or substrates (e.g., angiotensin I, des-Arg bradykinin, neurotensin, and kinetensin), can be used in the assay; e.g., as measured by production of the cleavage product.
  • a functional enzyme that catalyzes the cleavage of ACE-2 target peptides e.g., ligands or substrates (e.g., angiotensin I, des-Arg bradykinin, neurotensin, and kinetensin)
  • ligands or substrates e.g., angiotensin I, des-Arg bradykinin, neurotensin, and kinetensin
  • the cleavage product of angiotensin I is angiotensin 1-9.
  • the host cells expressing ACE-2, or a biologically active fragment thereof should show significant catalysis of an ACE-2 substrate (e.g., angiotensin I, des-Arg bradykinin, neurotensin, or kinetensin).
  • ACE-2 substrate e.g., angiotensin I, des-Arg bradykinin, neurotensin, or kinetensin.
  • Host cells should preferably possess a number of characteristics, depending on the readout, to maximize the catalytic activity of ACE-2, for example, for detecting the generation of angiotensin 1-9 from the cleavage of angiotensin I.
  • the cells expressing ACE-2 are exposed to a test compound or to vehicle controls (e.g., placebos).
  • ACE-2 substrates e.g., angiotensin I, des-Arg bradykinin, neurotensin, or kinetensin
  • the cells can be assayed for the production of ACE-2 cleavage products and/or the reduction in the amount of ACE-2 substrate, by, for example, mass spectrometry.
  • a construct encoding at least the catalytic domain of ACE-2 is linked to any of a variety of different reporter genes and introduced into an appropriate host cell.
  • reporter genes include, but are not limited to, chloramphenicol acetyltransferase (CAT), luciferase, GUS, growth hormone, and placental alkaline phosphatase (SEAP).
  • CAT chloramphenicol acetyltransferase
  • SEAP placental alkaline phosphatase
  • Alkaline phosphatase assays are particularly useful in the practice of the invention as the enzyme is secreted from the cell.
  • tissue culture supernatant can be assayed for secreted alkaline phosphatase.
  • alkaline phosphatase activity is measurable by calorimetric, bioluminescent or chemilumenscent assays such as those described in Bronstein, I. et al. (1994, Biotechniques 17: 172-177).
  • Such assays provide a simple, sensitive, and easily automatable detection system for pharmaceutical screening.
  • ACE e.g., human testicular ACE (GenBank Accession No. P22966) or human endothelial ACE (GenBank Accession No. P12822)
  • ACE-2 e.g., human testicular ACE (GenBank Accession No. P22966) or human endothelial ACE (GenBank Accession No. P12822)
  • host cells can be genetically engineered to express any of the amino acid sequences for known angiotensin converting enzymes.
  • testicular and endothelial ACE have been described in Ehlers, et al.
  • each of the foregoing sequences can be utilized to engineer a cell or cell line that expresses one of the ACEs for use in screening assays described herein.
  • the activation or inhibition of ACE-2 catalytic activity is compared to the effect of the test compound on the other ACEs.
  • non-cell based assay systems are useful in the identification of compounds that interact with, e.g., bind to, ACE-2.
  • Such compounds may act as inhibitors (e.g., antagonists), inverse agonists, or activators (e.g., agonists) of ACE-2 acti vity and may be used in the treatment of body weight disorders.
  • Isolated membranes can be used to identify compounds that interact with ACE-2.
  • CHO cells are genetically engineered to express ACE-2.
  • Membranes are harvested by standard techniques and used in an in vitro enzymatic assay.
  • An ACE-2 substrate e.g., angiotensin I, a decapeptide; des-Arg bradykinin, an octapeptide; neurotensin, a 13- amino acid peptide; or kinetensin, a nonapeptide
  • cleavage products e.g., angiotensin 1-9, des-Arg bradykinin septapeptide, neurotensin 12-amino acid peptide, or kinetensin octapeptide
  • the reduction of substrate is measured, e.g., by mass spectrometry.
  • membranes can be incubated with a known substrate in the presence or absence of candidate substrate, and the reaction products analyzed by mass spectrometry. In samples containing compounds that compete with the known substrate the amount of the reaction product of the known substrate will be lower compared to the samples containing only the known substrate.
  • recombinantly expressed ACE-2 can be obtained as a secreted product (e.g., from the culture media of transfected cells expressing ACE-2) and utilized in non-cell based assays to identify candidate substrates of ACE-2.
  • the recombinantly expressed ACE-2 polypeptides or fusion proteins containing at least the catalytic domain of ACE-2 can be used in the methods of the non-cell based screening assays described above.
  • the screens may be designed to identify compounds that antagonize the ACE-2 hydrolysis of known ACE-2 substrates, such as angiotensin I, des-Arg bradykinin, neurotensin, and kinetensin.
  • Assays for Compounds or Compositions That Modulate Expression of ACE-2 In vitro cell based assays can be designed to screen for compounds that regulate ACE-2 expression at either the transcriptional or translational level.
  • DNA encoding a reporter molecule can be linked to a regulatory element of the ACE-2 gene and used in appropriate intact cells, cell extracts, or lysates to identify compounds that modulate ACE-2 gene expression.
  • Appropriate cells or cell extracts are prepared from any cell type that normally expresses the ACE-2 gene, thereby ensuring that the cell extracts contain the transcription factors required for in vitro or in vivo transcription.
  • the screen can be used to identify compounds that modulate the expression of the reporter construct. In such screens, the level of reporter gene expression is determined in the presence of the test compound and compared to the level of expression in the absence of the test compound.
  • cells or cell lysates containing ACE-2 transcripts may be tested for modulation of ACE-2 mRNA translation.
  • test compounds are assayed for their ability to modulate the translation of ACE-2 mRNA in in vitro translation assays.
  • Compounds that reduce the level of ACE-2 expression are useful in the treatment of body weight disorders associated with a BMI above normal, such as obesity.
  • those compounds that increase the expression of ACE-2 may be useful for treatment of disorders with a BMI below normal, such as anorexia or cachexia.
  • compounds that affect ACE-2 activity include, but are not limited to, compounds that (i) bind to ACE-2; (ii) inhibit or decrease binding of an ACE-2 substrate to ACE-2; (iii) stimulate, activate or enhance ACE-2 activity (agonists) or decrease or inhibit ACE-2 activity (inverse agonists and antagonists); and (iv) neutralize ACE-2 activity by binding to an ACE-2 substrate.
  • compounds that affect ACE-2 gene activity e.g.
  • ACE-2 gene expression by affecting ACE-2 gene expression, including molecules (e.g., proteins, peptides, and small molecules) that affect transcription or interfere with splicing events so that expression of the full length or a truncated form of ACE-2 is modulated) can also be identified in the screens of the invention.
  • molecules e.g., proteins, peptides, and small molecules
  • assays described herein can also identify compounds that are indirectly modulated by ACE-2 activity. The identification and use of such compounds which affect other events that result from ACE-2 catalytic activity to thereby modulate effects of ACE-2 on the development of body weight disorders are within the scope of the invention. Such compounds can be used as part of a therapeutic method for the treatment of body weight disorders.
  • the compounds which can be screened in accordance with the invention include, but are not limited to peptides and antibodies, and fragments thereof, and other compounds or agents (e.g., peptidomimetics) that bind to the catalytic domain of ACE-2 and either mimic the activity triggered by an ACE-2 substrate (e.g., agonists, activators) or inhibit the activity triggered by the substrate (e.g., antagonists, inhibitors or inverse agonists); as well as peptides and antibodies, and fragments thereof, and other organic compounds that bind to and "neutralize" the ACE-2 substrate; i.e., prevents the substrate from being cleaved by ACE-2.
  • compounds or agents e.g., peptidomimetics
  • Compounds include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries; (see, e.g., Lam, K.S. et al. (1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature
  • combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z. et al. (1993) Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab') 2 , scFv, and Fab expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.
  • phosphopeptides including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z. et al. (1993) Cell 72:767-778
  • antibodies including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotyp
  • small molecules includes molecules which are capable of being used as therapeutic agents e.g., peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic (including, e.g., heteroorganic and organometallic compounds) and inorganic compounds.
  • therapeutic agents e.g., peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic (including, e.g., heteroorganic and organometallic compounds) and inorganic compounds.
  • the term includes compounds which have a molecular weight of about, for example, 10,000 grams per mole or less, 5,000 grams per mole or less, 2,000 grams per mole or less, or 1,000 g/mol grams per mole or less, less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • Small molecules include those as described above.
  • the small molecule is an organic compound. Examples of small molecules include those described in Formulae 1 -VIII and in Table 2.
  • Organic compounds comprise one or more carbon atoms.
  • the compound is an inorganic compound. Inorganic compounds include compounds which do not comprise a carbon atom.
  • test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R.N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • Computer modeling and searching technologies permit identification of compounds, as well as improvement of already identified compounds, that can modulate ACE-2 expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites can be substrate binding sites.
  • the active site can be identified using computer modeling methods and searching techniques known in the art including, for example, methods and techniques based on the amino acid sequences of peptides, the nucleotide sequences of nucleic acids, and structures obtained from studies of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods are useful in the identification of the active site by determining the position of the complexed ligand on the factor.
  • the three dimensional geometric stmcture of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular stmcture. Alternatively, solid or liquid phase NMR can be used to determine certain intra-molecular distances. Any other experimental method of stmcture determination can be used to obtain partial or complete geometric structures. The geometric structures can be measured with a complexed ligand, natural or artificial to increase the accuracy of the active site stmcture determined. If an incomplete or insufficiently accurate stmcture has been obtained, the methods of computer based numerical modeling can be used to complete or improve the accuracy of the stmcture.
  • Any recognized modeling method can be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular stmcture. Such a search seeks compounds having stmctures that match the determined active site stmcture and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. Compounds found from this type of search are potential ACE-2 modulating compounds.
  • these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the structural consequences of the modification can be determined using the experimental and computer modeling methods described above.
  • the altered stmcture can then be compared to the active site structure of the compound to determine if an improved fit or interaction results.
  • systematic variations in composition such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands with a more desirable specificity or activity.
  • Further experimental and computer modeling methods useful in the identification of modulating compounds based upon identification of the active sites of ACE-2, and related transduction and transcription factors will be apparent to those of skill in the art.
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular stmcture. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • Compounds identified using assays such as those described herein are useful, for example, in elaborating the biological function of ACE-2, in particular, in regulating body weight and/or modulating the percentage of body fat, and in methods of treatment for body weight disorders in human subjects.
  • the in vivo efficacy of compounds identified in a screen described herein can be tested in animal model systems for body weight disorders to identify drags, pharmaceuticals, therapeutic methods and compositions, and interventions that can be therapeutically effective.
  • a compound identified by the methods described herein can be tested in an animal model of body weight disorder to determine an effective concentration and treatment regimen to ameliorate the body weight disorder symptoms in the test animals.
  • the response of the animals to the test compound can be monitored by assessing the reversal of symptoms or physiological conditions associated with the body weight disorder being tested.
  • transgenic animals that express the human ACE-2 polypeptides, or fragments thereof, are useful.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees are useful in the generation of transgenic animals.
  • Any technique known in the art can be used to introduce the human ACE-2 transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Hoppe, P.C. and Wagner, 1989, U.S. Pat. No.
  • the invention provides for transgenic animals that carry the ACE-2 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals.
  • the transgene can be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene can be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. ((1992) Proc. Natl. Acad. Sci. USA 89:6232-6236).
  • ACE-2 transgene When the ACE-2 transgene is to be integrated into the chromosomal site of the endogenous ACE-2 gene, the use of gene targeting techniques is preferred. Briefly, when such a technique is to be utilized, vectors containing nucleotide sequences homologous to the endogenous ACE-2 gene and/or sequences flanking the gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the endogenous ACE-2 gene.
  • the transgene can be selectively expressed in a particular cell type with concomitant inactivation of the endogenous ACE-2 gene in only that cell type, by following, for example, the teaching of Gu et al. ((1994) Science 265:103-106).
  • the regulatory sequences required for such a cell-type specific recombination will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • founder animals have been generated, standard techniques such as Southern blot analysis or PCR techniques are used to analyze animal tissues to determine whether integration of the transgene has taken place.
  • level of mRNA expression of the transgene in the tissues of the founder animals can be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of ACE-2 gene-expressing tissue, can also be evaluated immunocytochemically using antibodies specific for the ACE-2 transgene product.
  • ACE-2 protein, polypeptides and peptide fragments, mutated, truncated, or deleted forms of the ACE-2 and/or ACE-2 fusion proteins can be prepared for a variety of uses, including, but not limited to, the generation of antibodies, as reagents in diagnostic assays, the identification of other cellular gene products involved in the regulation of body weight, as reagents in assays for screening for compounds that can be used in the treatment of body weight disorders, and as pharmaceutical reagents useful in the treatment of body weight disorders related to aberrant ACE-2 gene expression or protein activity.
  • ACE-2 The amino acid sequence of ACE-2 is shown in SEQ ID NO: 2.
  • Peptides corresponding to one or more domains of the ACE-2 e.g., the catalytic domain, or the zinc binding domain
  • truncated ACE-2 e.g. , an ACE-2 polypeptide in which a fragment is deleted
  • fusion proteins in which the full length ACE-2, an ACE- 2 peptide, or truncated ACE-2 polypeptide is fused to an unrelated protein are also within the scope of the invention.
  • Such soluble peptides, proteins, fusion proteins, and antibodies (including anti-idiotypic antibodies) that bind to and prevent or inhibit the proteolysis of an ACE-2 ligand by ACE-2 can be used as described herein to effectuate weight loss.
  • peptides corresponding to biologically active fragments of ACE-2 e.g., fragments containing the catalytic domain, or a fragment thereof
  • the entire ACE-2 polypeptide can be fused to another polypeptide (e.g., an IgFc polypeptide).
  • Fusion of an ACE-2 polypeptide, or a biologically active fragment thereof, to an IgFc polypeptide can increase the stability of the preparation, increase the half-life and activity of the ACE-2-Ig fusion protein in vivo, and can confer upon the fusion protein immunoglobulin effector functions (e.g. , binding to an Ig receptor).
  • the Fc region of the Ig portion of the fusion protein can be modified to reduce immunoglobulin effector function.
  • a variety of host expression vector systems are useful for the expression of nucleotide sequences encoding the appropriate regions of the ACE-2 to produce such polypeptides. Where the resulting peptide or polypeptide is a soluble derivative, the peptide or polypeptide can be recovered from the culture media. Where the polypeptide or protein is not secreted, the ACE-2 product can be recovered from the host cell itself. With regard to recombinant ACE-2 expressed in CHO cells, ACE-2 is synthesized as a transmembrane protein, some of which is cleaved posttranslationally to generate a secreted form in vivo and in cell culture (Donoghue, et al. , supra).
  • the host-expression vector systems also encompass engineered host cells that express ACE-2, or biologically active equivalents, in situ, i. e. , anchored in the cell membrane. Purification or enrichment of ACE-2 from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves are also useful in situations where it is important not only to retain the structural and functional characteristics of membrane-bound ACE-2, but to assess biological activity, e.g., in drug screening assays.
  • the host expression vector systems that are useful for purposes of the invention include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing ACE-2 nucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the ACE-2 nucleotide sequences; insect cell systems infected with recombinant vims expression vectors (e.g., baculovims) containing the ACE-2 sequences; plant cell systems infected with recombinant vims expression vectors (e.g., cauliflower mosaic vims, CaMN; tobacco mosaic virus, TMN) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing ACE-2 nucleotide sequences; and mammalian
  • a number of expression vectors can be advantageously selected depending upon the use intended for the ACE-2 gene product being expressed. For example, when a large quantity of such a protein is to be produced for the generation of pharmaceutical compositions of ACE-2 protein or for raising antibodies to the ACE-2 protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther, et al. (1983) ⁇ MBO J.
  • pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed.
  • a system described by Janknecht, et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht, et al. (1991) Proc. ⁇ atl. Acad. Sci. USA 88:8972-8976).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene becomes translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto
  • ⁇ i -nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
  • Autographa calif ornica nuclear polyhedrosis vims (Ac ⁇ PN) is used as a vector to express foreign genes.
  • the virus grows in Spodopterafrugiperda cells.
  • the ACE-2 coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the vims and placed under control of an Ac ⁇ PN promoter (e.g., the polyhedrin promoter).
  • Successful insertion of the ACE-2 gene coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i.e., vims lacking the proteinaceous coat coded for by the polyhedrin gene).
  • the recombinant vimses are then used to infect cells in which the inserted gene is expressed. (See, e.g., Smith, et al. (1983) J. Virol. 46:584; Smith, U.S. Patent No. 4,215,051.)
  • adenovims In mammalian host cells, there are a number of useful viral-based expression systems.
  • the ACE-2 polynucleotide sequence of interest can be ligated to an adenovims transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene is then be inserted in the adenovims genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant vims that is viable and capable of expressing the ACE-2 gene product in infected hosts.
  • Specific initiation signals may also be required for efficient translation of inserted ACE-2 nucleotide sequences, including, e.g., the ATG initiation codon and adjacent sequences.
  • specific initiation signals may also be required for efficient translation of inserted ACE-2 nucleotide sequences, including, e.g., the ATG initiation codon and adjacent sequences.
  • no additional translational control signals may be needed.
  • exogenous translational control signals including, e.g., the ATG initiation codon, must be provided.
  • initiation codon must be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner, et al. (1987) Methods in Enzymol. 153:516-544).
  • a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • Such posttranslational modifications e.g., glycosylation
  • processing e.g. , cleavage
  • Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of proteins and gene products.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Accordingly, it can be advantageous to use eukaiyotic host cells which possess the cellular machinery to enable proper processing of the primary transcript, glycosylation, phosphorylation, or other posttranslational modifications of the gene product.
  • Mammalian host cells which possess such capabilities include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38 cell lines. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the ACE-2 sequences described above may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method is useful in engineering cell lines to express the ACE-2 gene product.
  • Such engineered cell lines are particularly useful in screening and the evaluation of compounds that affect the endogenous activity of the ACE-2 gene product.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al. (1980) Natl. Acad. Sci. USA 77:3567; O'Hare, et al. (1981) Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al. (1981) J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al. (1984) Gene 30:147).
  • Antibodies that specifically recognize one or more epitopes of ACE-2 or conserved variants of ACE-2, or peptide fragments of ACE-2 are also encompassed by the invention.
  • Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the aforementioned.
  • the antibodies of the invention are useful, for example, in the detection of the ACE-2 in a biological sample and therefore, can be utilized as part of a diagnostic or prognostic technique whereby subjects can be tested for abnormal amounts of ACE-2.
  • Such antibodies are also useful in conjunction with, for example, compound screening schemes, e.g., as described herein, for the evaluation of the effect of test compounds on ACE-2 gene expression and/or activity of ACE-2 polypeptides.
  • Such antibodies can be used in conjunction with the gene therapy techniques described, below, e.g. , to evaluate the normal endogenous and/or engineered ACE-2-expressing cells prior to their introduction into the subject.
  • Such antibodies are additionally useful as a reagent for the inhibition of abnormal ACE-2 activity.
  • Such antibodies are therefore useful as part of weight disorder treatment methods.
  • antibody refers to an immunoglobulin molecule and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (/. e. , immunoreacts with) an antigen, such as a ACE-2 molecule.
  • immunologically active portions of immunoglobulin molecules include scFV and dcFV fragments, Fab and F(ab') fragments.
  • the antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric, humanized, fully human, non-human (e.g., murine, rat, rabbit, or goat), or single chain antibody. In a preferred embodiment it has effector function and can fix complement.
  • the antibody can be coupled to a toxin or imaging agent.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of ACE-2 .
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular ACE-2 protein with which it immunoreacts.
  • Polyclonal anti- ACE-2 antibodies can be prepared as described above by immunizing a suitable subject with a ACE-2 immunogen.
  • the anti- ACE-2 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized ACE-2 .
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against ACE-2 can be isolated from the subject (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497 (see also Brown et al. (1981) J. Immunol. 127:539- 46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31 ; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma cell line
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds ACE-2 .
  • Any of the many well known protocols for fusing lymphocytes and immortalized cell lines can be used to generate an anti- ACE-2 monoclonal antibody (see, e.g., Galfre et al.
  • the immortal cell line e.g. , a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (Manassas VA). Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells. Unfused splenocytes die after several days because they are not transformed.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind ACE-2 , e.g., using a standard ELISA assay.
  • a monoclonal anti- ACE-2 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with an ACE-2 polypeptide to thereby identify immunoglobulin library members that bind ACE-2 .
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • Chimeric, humanized, and completely human antibodies are also within the scope of the invention. Chimeric, humanized, but most preferably, completely human antibodies are desirable for applications which include repeated administration, e.g., therapeutic treatment of human subjects, and some diagnostic applications. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; EPO Publication No. 184,187; EPO Publication No. 171,496; EPO Publication No. 173,494; PCT International Publication No. WO
  • Completely human antibodies are particularly desirable for therapeutic treatment of human subjects.
  • Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93); and U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806.
  • companies such as Abgenix, Inc. (Fremont, CA) and Medarex, Inc. (Princeton, NJ), are available to provide human antibodies directed against a selected antigen using technology similar to that described above.
  • Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non- human monoclonal antibody e.g., a murine antibody
  • This technology is described by Jespers et al. (1994) Bio/Technology 72:899-903.
  • a full-length ACE-2 protein, or an antigenic fragment thereof, can be used as an immunogen or can be used to identify anti- ACE-2 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like.
  • the antigenic fragments of ACE-2 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompass an epitope of ACE-2, respectively.
  • the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Fragments of ACE-2 which include, e.g., amino acid residues 147 to 555 of SEQ ID NO:2, can be used as immunogens to make an antibody against the ACE-2 catalytic domain.
  • Antibodies reactive with, or specific for, any of this region, or other regions or domains described herein are provided.
  • the antibody fails to bind to an Fc receptor, e.g., it is of an immunoglobulin class which does not support Fc receptor binding or has been modified, e.g., by deletion or other mutation, such that, is does not have a functional Fc receptor binding region.
  • Preferred epitopes encompassed by the antigenic peptide are regions of ACE-2 which are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • regions of ACE-2 which are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • an Emini surface probability analysis of the human ACE-2 protein sequence can be used to identify the regions that have a particularly high probability of being localized to the surface of the ACE-2 protein, and are thus likely to constitute surface residues useful for targeting antibody production.
  • the antibody binds an epitope on any domain or region on ACE-2 proteins described herein.
  • the anti- ACE-2 antibody can be a single chain antibody.
  • a single-chain antibody (scF V) may be engineered as described, for example, in Colcher, D. et al. , (1999 ) Ann. NY Acad. Sci. 880: 263-80; and Reiter, Y. (1996) Clin. Cancer Res. 2:245- 52.
  • the single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target ACE-2 protein.
  • An anti-ACE-2 antibody can be used to detect ACE-2 protein (e.g. , in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein.
  • Anti- ACE-2 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i. e. , physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoeiythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1, 131 1, 35 S or 3 H.
  • Antibodies to ACE-2 can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" ACE-2, using techniques well known to those skilled in the art. (See, e.g., Greenspan and Bona (1993) FASEB J 7:437-444; and Nissinoff (1991) J. Immunol. 147:2429-2438.)
  • antibodies which bind to the ACE-2 catalytic site and competitively inhibit the binding of ACE-2 substrates can be used to generate anti- idiotypes that "mimic" the ACE-2 catalytic site and, therefore, bind and neutralize ACE- 2 substrates.
  • Such neutralizing anti-idiotypes, or fragments thereof can be used in therapeutic regimens to reduce or inhibit ACE-2 activity and promote a reduction in body weight and/or the percentage of body fat.
  • antibodies to ACE-2 that can act as (e.g., agonists activators) of ACE-2 activity can be generated. Such antibodies that enhance ACE-2 catalytic activity are particularly useful for treating weight disorders such as anorexia and cachexia. Alternatively, antibodies that act as inhibitors (e.g., antagonists) or inverse agonists of ACE-2 activity and inhibit the catalytic activity of ACE-2 are useful in the treatment of weight disorders such as obesity.
  • ACE-2 Gene Therapy Approaches to Controlling ACE-2 Activity and Regulating Body Weight
  • the expression of ACE-2 can be controlled in vivo (e.g., at the transcriptional or translational level) using gene therapy approaches to regulate ACE-2 activity and treat body weight disorders. Several approaches are described below.
  • ACE-2 nucleic acid sequences can be utilized, e.g., in the treatment of body weight disorders, including anorexia and cachexia. Where the cause is a defective ACE-2 gene, treatment can be administered, for example, in the form of gene replacement therapy.
  • one or more copies of a normal ACE-2 gene or a portion of the ACE-2 gene that directs the production of an ACE-2 gene product exhibiting normal function can be inserted into the appropriate cells of a subject, using vectors which include, but are not limited to, adenovims, adeno- associated vims, retroviras, and herpes virus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
  • vectors include, but are not limited to, adenovims, adeno- associated vims, retroviras, and herpes virus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
  • ACE-2 gene expression is limited to the heart (in particular, the endothelium of most intramyocardial vessels, including capillaries, venules, and medium-sized coronary arteries and arterioles), testis, small intestine, large intestine, adipose tissue, and kidney, (in particular, the endothelium and focally in rare smooth muscle cells of medium sized vessels, and proximal tubule epithelial cells), gene replacement therapy techniques can be used to deliver ACE-2 gene sequences to these cell types in subjects by direct administration of ACE-2 gene sequences to the site within these tissues where the ACE-2 gene sequences are expressed.
  • targeted homologous recombination can be utilized to correct the defective endogenous ACE-2 gene in the appropriate tissue.
  • targeted homologous recombination can be used to correct the defect in ES cells in order to generate offspring with a corrected trait.
  • Additional methods which are useful in increasing the overall level of ACE-2 gene expression and/or ACE-2 activity include the introduction of appropriate ACE-2- expressing cells, preferably autologous cells, into a subject at positions and in numbers which are sufficient to ameliorate the symptoms of body weight disorders, such as anorexia and cachexia. Such cells can be either recombinant or non-recombinant.
  • the cells which can be administered to increase the overall level of ACE-2 gene expression in a subject are normal cells, or cells which express the ACE-2 gene, such as the endothelium of most intramyocardial vessels, testis, small intestine, large intestine, adipose tissue, and the endothelium and focally in rare smooth muscle cells of medium sized vessels, and proximal tubule epithelial cells of the kidney.
  • the cells can be delivered directly to the anatomical site, or as part of a tissue graft located at a different site in the body.
  • Such cell-based gene therapy techniques are well known to those skilled in the art. (See, e.g., U.S. Patent No. 5,399,349 and U.S.
  • Patent No. 5,460,959 compounds, identified in the assays described above, that stimulate or enhance the proteolytic activity of ACE-2, can be used to achieve weight gain.
  • the formulation and mode of administration will depend upon the physico-chemical properties of the compoxmd.
  • therapeutic methods to reduce body weight and/or the percentage of body fat can be designed to reduce the level of endogenous ACE-2 gene expression, e.g., using antisense or ribozyme approaches to inhibit or prevent translation of ACE-2 mRNA transcripts; triple helix approaches to inhibit transcription of the ACE-2 gene; or targeted homologous recombination to inactivate or "knock out" the ACE-2 gene or its endogenous promoter.
  • Such gene therapy is useful in the treatment of body weight disorders such as obesity, where the inhibition of ACE-2 expression is designed to reduce body weight and/or the percentage of body fat.
  • ACE-2 gene expression is limited to the heart, kidney, adipose tissue, small intestine, large intestine and testis, delivery techniques can be utilized which enable administration of the antisense, ribozyme or DNA constmcts described herein directly to the tissue containing the target cells.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to mRNA.
  • the antisense oligonucleotides will bind to the complementary mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarily and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • antisense nucleotides complementary to the coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred. Oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs were recently shown to be effective at inhibiting translation of mRNAs as well. (See generally, Wagner, R.
  • oligonucleotides complementary to either the 5'- or 3'- non- translated, non-coding regions of ACE-2 can be used in an antisense approach to inhibit translation of endogenous ACE-2 mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, the results obtained using the antisense oligonucleotide can be compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA. 86:6553-6556; Lemaitre et al. (1987) Proc.
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide can comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguan
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands mn parallel to each other (Gautier et al. (1987) Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al, (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • Oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. ((1988) Nucl. Acids Res. 16:3209)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85:7448-7451), etc.
  • the antisense molecules should be delivered to the cells which express the ACE- 2 in vivo, e.g., heart (in particular, the endothelium of most intramyocardial vessels, including capillaries, venules, and medium-sized coronary arteries and arterioles), testis, small intestine, large intestine, adipose tissue, and kidney, (in particular, the endothelium and focally in rare smooth muscle cells of medium sized vessels, and proximal tubule epithelial cells).
  • ACE- 2 in vivo
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g.
  • antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense molecules linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • modified antisense molecules designed to target the desired cells (e.g., antisense molecules linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • a vector can be constructed to remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bemoist and Chambon (1981) Nature 290:304- 310), the promoter contained in the 3' long terminal repeat of Rous sarcoma vims (Yamamoto, et al. (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et al. (1981) Proc. Natl. Acad. Sci.
  • SV40 early promoter region Bemoist and Chambon (1981) Nature 290:304- 310
  • the promoter contained in the 3' long terminal repeat of Rous sarcoma vims Yamamoto, et al. (1980) Cell 22:787-797
  • any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA constmct which can be introduced directly into the tissue site (e.g., heart (in particular, the endothelium of most intramyocardial vessels, including capillaries, venules, and medium-sized coronary arteries and arterioles), small intestine, large intestine, adipose tissue, testis, and kidney, (in particular, the endothelium and focally in rare smooth muscle cells of medium sized vessels, and proximal tubule epithelial cells)).
  • the tissue site e.g., heart (in particular, the endothelium of most intramyocardial vessels, including capillaries, venules, and medium-sized coronary arteries and arterioles), small intestine, large intestine, adipose tissue, testis, and kidney, (in particular, the endothelium and focally in rare smooth muscle cells
  • Ribozyme molecules designed to catalytically cleave ACE-2 mRNA transcripts can also be used to prevent translation of ACE-2 mRNA and expression of ACE-2 polypeptide.
  • ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy ACE-2 mRNAs
  • the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
  • the target mRNA have the following sequence of two bases: 5'-UG- 3'.
  • the constmction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach (1988) Nature, 334:585-591.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the ACE-2 mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al. (1984) Science, 224:574-578; Zaug and Cech (1986) Science, 231:470-475; Zaug, et al. (1986) Nature, 324:429-433; PCT publication No. WO 88/04300; and Been and Cech (1986) Cell, 47:207-216).
  • Cech-type ribozymes such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al. (1984) Science, 224:574-578; Za
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence where after cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in ACE-2.
  • the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the ACE-2 in vivo, e.g., heart, testis, small intestine, large intestine, adipose tissue, and kidney.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous ACE-2 messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency. Endogenous ACE-2 gene expression can also be reduced by inactivating or
  • ACE-2 "knocking out” the ACE-2 gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al, (1985) Nature 317:230-234; Thomas and Capecchi (1987) Cell 51 :503-512; and Thompson, et al. (1989) Cell 5:313-321; each of which is incorporated herein by reference in its entirety).
  • a mutant, nonfunctional ACE-2 (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous ACE-2 gene can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express ACE-2 in vivo.
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive ACE-2 polypeptide (e.g., see Thomas and Capecchi (1987) and Thompson (1989), supra).
  • ES embryonic stem
  • this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
  • endogenous ACE-2 gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the ACE-2 gene (i. e. , the ACE-2 promoter and/or enhancers) to form triple helical stmctures that prevent transcription of the ACE-2 gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the ACE-2 gene i. e. , the ACE-2 promoter and/or enhancers
  • triple stmctures that prevent transcription of the ACE-2 gene in target cells in the body.
  • soluble ACE-2 polypeptides or fragments thereof, or fusion proteins e.g. fusion Ig molecules can be administered in vivo where they can function as "bioreactors" that deliver a supply of the soluble molecules.
  • soluble ACE-2 polypeptides and fusion proteins when expressed at appropriate concentrations, should neutralize or "mop up" the native ligand for ACE-2. and thus act as inhibitors of ACE-2 activity and promote weight loss and/or reduce the percentage of body fat in a subject.
  • Example 1 Production of ACE and ACE-2 polypeptides ACE and ACE-2 polypeptides were produced in CHO cells as follows.
  • Full length ACE-2 expression vector (pFLA-2) was generated by ligating an EcroRI/Afl III fragment of a 5' RACE clone (cchr002) and an Afl Ill/Not I fragment of the original library clone into the EcoRI/Not I sites of pcDNA3.1 (Invitrogen).
  • An expression vector for secreted ACE-2 (pMD37) was generated by subcloning into pACE-2 a small PCR fragment which has a stop codon inserted after the serine immediately preceding the transmembrane domain.
  • Testicular ACE cDNA was obtained from James Riordan and was subcloned into pcDNA3.1 for expression.
  • CHO Kl cells which are maintained in serum-free Ultra CHO medium (Biowhittaker) at 37 °C in a humidified 5% CO 2 incubator, were seeded at 1 x 10 cells/ 10 cm dish on day 0 and transfected with lipofectamine on day 1 as per manufacturers' instmctions. Briefly, 10 ⁇ g of DNA were combined with 40 ⁇ l of lipofectamine in Opti-MEM medium and the mixture was incubated on the cells for 5-6 hours. Ultra CHO medium was then added to the cells and they were incubated overnight at 37 °C. On day 2, the medium was changed and on day 4 the media were collected. For the examples below, the conditioned media containing the secreted ACE-2 protein was concentrated in Centriplus 30 concentrators (Amicon).
  • the following protocol is an example of a protocol that was used for the purification of ACE-2 for use in the subsequent examples.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Child & Adolescent Psychology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des composés modulant ACE-2, destinés au traitement de problèmes de poids. L'invention concerne également des procédés d'utilisation de ces composés et des compositions pharmaceutiques contenant lesdits composés.
PCT/US2001/045703 2000-11-01 2001-10-31 Composes modulant ace-2 et procedes d'utilisation associes WO2002039997A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002239454A AU2002239454A1 (en) 2000-11-01 2001-10-31 Ace-2 modulating compounds and use thereof

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US70421600A 2000-11-01 2000-11-01
US09/704,216 2000-11-01
US87038201A 2001-05-29 2001-05-29
US09/870,382 2001-05-29
US37174101P 2001-10-19 2001-10-19
US60/371,741 2001-10-19

Publications (2)

Publication Number Publication Date
WO2002039997A2 true WO2002039997A2 (fr) 2002-05-23
WO2002039997A3 WO2002039997A3 (fr) 2002-11-28

Family

ID=27409032

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/045703 WO2002039997A2 (fr) 2000-11-01 2001-10-31 Composes modulant ace-2 et procedes d'utilisation associes

Country Status (2)

Country Link
AU (1) AU2002239454A1 (fr)
WO (1) WO2002039997A2 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6592865B2 (en) 2001-06-04 2003-07-15 Human Genome Sciences, Inc. Methods and compositions for modulating ACE-2 activity
WO2005020977A1 (fr) * 2003-08-21 2005-03-10 Wisconsin Alumni Research Foundation Agents potentialisant la secretion d'insuline a base d'alpha-cetoglutarate
US6900033B2 (en) 2001-06-04 2005-05-31 Human Genome Sciences, Inc. Methods and compositions for modulating ACE-2 activity
US7163952B2 (en) 2001-12-03 2007-01-16 Japan Tobacco Inc. Azole compound and medicinal use thereof
US7271280B2 (en) 2002-03-05 2007-09-18 Sumitomo Chemical Company, Limited Process for preparing a biaryl compound
JP2008542196A (ja) * 2005-05-05 2008-11-27 クロマ セラピューティクス リミテッド カルボキシルエステラーゼにより加水分解可能なアルファアミノ酸エステル−薬剤複合体
WO2009076694A1 (fr) * 2007-12-18 2009-06-25 Apeiron Biologics Forschungs- Und Entwicklungsgesellschaft M.B.H. Traitement de maladies inflammatoires par ace2
WO2009106640A2 (fr) * 2008-02-28 2009-09-03 Maastricht University Dérivés de n-benzyl-imidazole
US8178681B2 (en) 2004-10-28 2012-05-15 Shionogi & Co., Ltd. 3-carbamoyl-2-pyridone derivatives
WO2013086162A1 (fr) * 2011-12-06 2013-06-13 Nova Southeastern University Composés radioiodables de modulation de l'enzyme 2 de conversion de l'angiotensine (ace-2), leur préparation, et procédés pour leur utilisation
WO2015172196A1 (fr) * 2014-05-13 2015-11-19 Monash University Composés hétérocycliques et leur utilisation
CN106478518A (zh) * 2016-09-27 2017-03-08 南通常佑药业科技有限公司 一种庚烯酸环戊酯衍生物的制备方法
WO2019106085A1 (fr) * 2017-11-29 2019-06-06 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Méthode visant à moduler la pigmentation par modulation de l'enzyme de conversion de l'angiotensine 2
CN111087445A (zh) * 2019-10-14 2020-05-01 浙江大学 一种用于ace2活性检测的质谱探针及其制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000724A1 (fr) * 1989-07-06 1991-01-24 Abbott Laboratories Alkynyle amines de regulation de la neurotransmission cholinergique
WO2000018899A2 (fr) * 1998-09-30 2000-04-06 Millennium Pharmaceuticals, Inc. Homologue d'enzyme de conversion d'angiotensine et son utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000724A1 (fr) * 1989-07-06 1991-01-24 Abbott Laboratories Alkynyle amines de regulation de la neurotransmission cholinergique
WO2000018899A2 (fr) * 1998-09-30 2000-04-06 Millennium Pharmaceuticals, Inc. Homologue d'enzyme de conversion d'angiotensine et son utilisation

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6900033B2 (en) 2001-06-04 2005-05-31 Human Genome Sciences, Inc. Methods and compositions for modulating ACE-2 activity
US6592865B2 (en) 2001-06-04 2003-07-15 Human Genome Sciences, Inc. Methods and compositions for modulating ACE-2 activity
US7163952B2 (en) 2001-12-03 2007-01-16 Japan Tobacco Inc. Azole compound and medicinal use thereof
US7271280B2 (en) 2002-03-05 2007-09-18 Sumitomo Chemical Company, Limited Process for preparing a biaryl compound
US7714157B2 (en) 2002-03-05 2010-05-11 Sumitomo Chemical Company, Limited Process for preparing a biaryl compound
US7863301B2 (en) 2003-08-21 2011-01-04 Wisconsin Alumni Research Foundation Potentiators of insulin secretion
WO2005020977A1 (fr) * 2003-08-21 2005-03-10 Wisconsin Alumni Research Foundation Agents potentialisant la secretion d'insuline a base d'alpha-cetoglutarate
US8507557B2 (en) 2003-08-21 2013-08-13 Wisconsin Alumni Research Foundation Potentiators of insulin secretion
US8367666B2 (en) 2004-10-28 2013-02-05 Shionogi & Co., Ltd. 3-carbamoyl-2-pyridone derivatives
US8178681B2 (en) 2004-10-28 2012-05-15 Shionogi & Co., Ltd. 3-carbamoyl-2-pyridone derivatives
JP2008542196A (ja) * 2005-05-05 2008-11-27 クロマ セラピューティクス リミテッド カルボキシルエステラーゼにより加水分解可能なアルファアミノ酸エステル−薬剤複合体
WO2009076694A1 (fr) * 2007-12-18 2009-06-25 Apeiron Biologics Forschungs- Und Entwicklungsgesellschaft M.B.H. Traitement de maladies inflammatoires par ace2
EP3272357A1 (fr) * 2007-12-18 2018-01-24 Apeiron Biologics AG Traitement des maladies inflammatoires
WO2009106640A3 (fr) * 2008-02-28 2009-10-29 Maastricht University Dérivés de n-benzyl-imidazole
WO2009106640A2 (fr) * 2008-02-28 2009-09-03 Maastricht University Dérivés de n-benzyl-imidazole
WO2013086162A1 (fr) * 2011-12-06 2013-06-13 Nova Southeastern University Composés radioiodables de modulation de l'enzyme 2 de conversion de l'angiotensine (ace-2), leur préparation, et procédés pour leur utilisation
WO2015172196A1 (fr) * 2014-05-13 2015-11-19 Monash University Composés hétérocycliques et leur utilisation
CN106478518A (zh) * 2016-09-27 2017-03-08 南通常佑药业科技有限公司 一种庚烯酸环戊酯衍生物的制备方法
WO2019106085A1 (fr) * 2017-11-29 2019-06-06 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Méthode visant à moduler la pigmentation par modulation de l'enzyme de conversion de l'angiotensine 2
US11337909B2 (en) 2017-11-29 2022-05-24 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Methods for modulating pigmentation by angiotensin-converting enzyme 2 modulation
CN111087445A (zh) * 2019-10-14 2020-05-01 浙江大学 一种用于ace2活性检测的质谱探针及其制备方法和应用

Also Published As

Publication number Publication date
WO2002039997A3 (fr) 2002-11-28
AU2002239454A1 (en) 2002-05-27

Similar Documents

Publication Publication Date Title
US7045532B2 (en) ACE-2 modulating compounds and methods of use thereof
US6632830B1 (en) ACE-2 inhibiting compounds and methods of use thereof
WO2002039997A2 (fr) Composes modulant ace-2 et procedes d'utilisation associes
JP5921679B2 (ja) 血漿カリクレインの阻害薬としてのベンジルアミン誘導体
CN101959404B (zh) 单磷酸腺苷活化蛋白激酶调节剂
Iimuro et al. Matrix metalloproteinase gene delivery for liver fibrosis
CN1918131B (zh) 谷氨酰胺酰基环化酶抑制剂
AU2006212772B2 (en) Combination of organic compounds
AU737117B2 (en) Use of matrix metalloproteinase inhibitors for treating neurological disorders and promoting wound healing
RU2132327C1 (ru) Замещенные карбоксамиды и фармацевтическая композиция на их основе
SI9600163A (en) Substituted n-(indole-2-carbonyl)-beta-alanimamides and derivatives as antidiabetic agents
KR100252605B1 (ko) 세포 유착저해제인 3-알킬옥시-, 아릴옥시-, 또는 아릴알킬옥시벤조(b)티오펜-2-카르복스아미드류
KR20010050798A (ko) 말초 또는 중추 신경계 장애의 치료를 위한 매트릭스금속프로테이나제 억제제인 α-술포닐아미노 히드록삼산
MXPA99001974A (es) Metodo para tratar y prevenir fallas cardiacas y dilatacion ventricular.
AU2013291865B2 (en) Thiophene derivatives used in the treatment of diabetes
CA2158760A1 (fr) Inhibiteurs synthetiques de metalloproteases matricielles et utilisations correspondantes
EA009291B1 (ru) Применение эффекторов глутаминил- и глутаматциклаз
KR20050059294A (ko) 11-베타-하이드록시 스테로이드 데하이드로게나제 형태 1및 형태 2의 억제제
CN104755464B (zh) 聚蛋白多糖酶抑制剂
AU2006311723A1 (en) Combination of an angiotensin II receptor blocker, a calcium channel blocker and another active agent
JPH08503475A (ja) 抗変性活性剤としてのカルボキシ−ペプチジル誘導体
US20160068489A1 (en) Amidopyridinol derivative or pharmaceutically acceptable salt thereof and pharmaceutical composition comprising same as active component
HRP970474A2 (en) Compounds for and a method of inhibiting matrix metalloproteinases
EP1666469A1 (fr) Inhibiteur d'inhibiteur-1 activateur de plasmogene
JP2006523698A (ja) Ｐｐａｒ変調薬としての（３−｛３−（２，４−ビス−トリフルオロメチル−ベンジル）−（５−エチル−ピリミジン−２−イル）−アミノ−プロポキシ｝−フェニル）−酢酸および関連化合物、ならびに代謝性障害の処置方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP