Patent Application
20150246140
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 24, 2015, is named CTH-01501_SL.txt and is 40,857 bytes in size.
More than 30 million Americans suffer from chronic sleep disorders such as narcolepsy and insomnia. Safe, effective, non-addictive, and long-lasting drugs, however, are substantially unavailable. For example, a recent, large longitudinal study found that the use of sleep aids significantly increases patient mortality (Kripke, D. F. et al., British Medical Journal 2:e000850 (2012)). Barbiturates, for example, have addictive tendencies and can be lethal at high dosages. Benzodiazepines, such as temazepam, are less effective and tend to lose their sedative effect with continued use. Nonbenzodiazepines, such as the GABAA receptor agonist zolpidem, are similarly less effective and also lose their sedative effect with continued use. In comparison, gammahydroxybutyrate (GHB) is a powerful muscle relaxant that is effective for treating narcolepsy, but GHB has a short duration of action of about two to three hours and a propensity for abuse. Despite the pressing need for new agents to treat sleep disorders, relatively little is known of the genetic circuits regulating sleep and wakefulness.
In some aspects, the invention relates to methods of treating conditions associated with sleep disorders, comprising administering an agent that modulates the activity of a neuropeptide receptor or a neuropeptide receptor ligand. In some aspects, the invention relates to methods of screening for agents that modulate the activity of a neuropeptide receptor in an organism, optionally using cells or organisms comprising a genetic modification that affects a neuropeptide signaling pathway. In some aspects, the invention relates to methods of characterizing a condition associated with a sleep disorder comprising sequencing a nucleic acid encoding either a neuropeptide receptor or a neuropeptide receptor ligand and comparing the sequence with a reference sequence.
Neuropeptides play a crucial role in modulating the nervous system through combinatorial inputs with multiple receptors. This modularity of these ligand/receptor systems provides a flexible information-processing and transmission network to translate internal and external signals into behavioral outputs such as feeding, locomotion, pain perception, and mechano-, chemo-, and thermo-sensation.
In vertebrates, numerous neuropeptide/receptor systems have been reported to modulate sleep (see generally Raffa, Peptides 9(4), 915 (1988); Prober et al., J. Neurosci. 26(51), 13400 (2006)), although a universal signaling pathway has not been previously reported. For example, the role of the neuropeptide Y (NPY) system in sleep is elusive and may be state-dependent and species-dependent. The NPY system possesses dual functions in promoting sleep and wakefulness in mammals, depending on the site and vehicle of NPY introduction. In C. elegans, the neuropeptide-like-protein NLP-22 regulates a sleep-like state and the RFamide peptides flp-18 and flp-21 in combination with GPCRs NPR-4/5 and NPR-1 modulate foraging and lethargus, respectively. In comparison, the vertebrate RFamide peptides modulate feeding and reproduction. The instant invention relates to the finding that the ALA-synthesized RFamide peptides can induce sleep via GPCRs, and that homologous vertebrate peptides function similarly. Specifically, agonists of the G-protein coupled receptors (GPCRs) neuropeptide FF receptor 1 (NPFFR1), neuropeptide FF receptor 2 (NPFFR2), neuropeptide Y receptor type 1 (NPY1R), neuropeptide Y receptor type 2 (NPY2R), and tachykinin receptor (TACR) can induce sleep in a subject and antagonists can induce wakefulness.
Previously, it was found that epidermal growth factor (EGF) signaling regulates sleep in a wide range of organisms (see, e.g., Kramer A., et al. Science 294:2511-15 (2001)). In some aspects, the instant invention relates to the finding that the downstream mediators of the EGF sleep pathway are neuromodulators comprising versatile neuropeptide ligand/receptor systems that can promote and maintain sleep by dampening sensory and motor abilities, and yet permit fast reversibility when perturbed by sensory stimulation. Additionally, some aspects of the invention relate to the finding that the functions of these ligand/receptor systems are conserved through evolution from nematodes through vertebrates.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “about” refers to a range of values of plus or minus 10% of a specified value. For example, the phrase “about 100” includes plus or minus 10% of 100, i.e., from 90 to 110, unless clearly contradicted by context.
Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.
As used herein, the terms “effective amount” and “therapeutically effective amount” mean a dosage sufficient to produce a desired result, e.g., to induce sleep in a subject, to increase wakefulness in a subject, or to sedate a subject.
The term “prevent” is art-recognized, and when used in relation to a condition, such as sleep, is well understood in the art, and includes administration of a composition which reduces the likelihood of, or delays the onset of, the condition in a subject relative to a subject which does not receive the composition. Thus, prevention of sleep includes, for example, reducing the likelihood that a subject receiving the composition will sleep relative to a subject that does not receive the composition, and/or delaying the onset of sleep, on average, in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount. Similarly, prevention of insomnia includes, for example, increasing the likelihood that a subject receiving the composition will sleep relative to a subject that does not receive the composition, and/or inducing sleep in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
The terms “neuropeptide FF receptor 1” and “NPFFR1” refer to vertebrate NPFFR1. In some embodiments, NPFFR1 refers to homologs of human NPFFR1. In some embodiments, NPFFR1 refers to orthologs of human NPFFR1. For example, NPFFR1 may refer to Danio rerio NPFFR-112.
The terms “neuropeptide FF receptor 2” and “NPFFR2” refer to vertebrate NPFFR2. In some embodiments, NPFFR2 refers to homologs of human NPFFR2. In some embodiments, NPFFR2 refers to orthologs of human NPFFR2. For example, NPFFR2 may refer to Danio rerio NPFFR-2.1 or NPFFR-2.2.
The terms “neuropeptide Y receptor 1” and “NPY1R” (“NPYR1”) refer to vertebrate NPY1R. In some embodiments, NPY1R refers to homologs of human NPY1R. In some embodiments, NPY1R refers to orthologs of human NPY1R. For example, NPY1R may refer to Danio rerio NPY1R.
The terms “neuropeptide Y receptor 2” and “NPY2R” (“NPYR2”) refer to vertebrate NPY2R. In some embodiments, NPY2R refers to homologs of human NPY2R. In some embodiments, NPY2R refers to orthologs of human NPY2R. For example, NPY2R may refer to Danio rerio NPY2R.
The terms “tachykinin receptor” and “TACR” refer to vertebrate TACR. In some embodiments, TACR refers to homologs of human TACR. In some embodiments, TACR refers to orthologs of human TACR. For example, TACR may refer to Danio rerio TACR.
As used herein, the terms “treat”, “treating”, and “treatment” include inhibiting the condition, e.g., reducing the onset or symptoms of a condition, disorder, or disease, such as insomnia, fatigue, or narcolepsy. These terms also encompass therapy. Treatment means any manner in which the symptoms of a condition, disorder, or disease are ameliorated or otherwise beneficially altered. Preferably, the subject in need of such treatment is a mammal, more preferably a human.
In some aspects, the invention relates to a method for regulating sleep in a subject, comprising administering to the subject a composition comprising an agent that modulates the activity of a G-protein coupled receptor (GPCR) selected from neuropeptide FF receptor 1 (NPFFR1), neuropeptide FF receptor 2 (NPFFR2), neuropeptide Y receptor type 1 (NPY1R), neuropeptide Y receptor type 2 (NPY2R), and tachykinin receptor (TACR).
In some embodiments, the subject is human and the GPCR is a human GPCR.
In some embodiments, regulating sleep in the subject comprises inducing sleep in the subject. The invention may relate to a method of sedating the subject. In other embodiments, regulating sleep in the subject comprises increasing wakefulness in a subject. For example, either sleep or wakefulness may be induced in the subject by administering an agonist or antagonist of the GPCR, respectively.
In some embodiments, the invention relates to a method for inducing sleep in a subject, comprising administering to the subject a composition comprising a neuropeptide FF receptor 1 (NPFFR1) agonist. In some embodiments, the invention relates to a method for inducing wakefulness in a subject, comprising administering to the subject a composition comprising a neuropeptide FF receptor 1 (NPFFR1) antagonist.
An agent that modulates the activity of a GPCR may be selected from agonists or antagonists of the GPCR. The agent may be a ligand of the GPCR, e.g., the agent may specifically bind to the GPCR. For example, the agent may bind to an extracellular domain of the GPCR. Similarly, the agent may bind to the transmembrane or cytosolic domain of the GPCR, or the agent may bind to residues in or near the extracellular/transmembrane domains of the GPCR.
An agonist of a GPCR induces a signal transduction pathway mediated by the GPCR upon binding to the GPCR. Accordingly, an agonist modulates the activity of a GPCR by inducing at least one signal transduction pathway mediated by the GPCR, e.g., relative to the level of signaling mediated by the GPCR in the absence of the agonist. For example, an agonist might be a naturally-occurring GPCR ligand or a derivative of a naturally-occurring GPCR ligand, or an agonist may mimic the function of a naturally-occurring GPCR ligand. Similarly, an agonist may induce a conformation of the GPCR that increases the signaling of a GPCR-ligand complex upon binding to the GPCR, e.g., by binding to an allosteric site.
An antagonist of a GPCR inhibits a signal transduction pathway mediated by the GPCR upon binding to the GPCR. Accordingly, an antagonist modulates the activity of a GPCR by inhibiting at least one signal transduction pathway mediated by the GPCR, e.g., relative to the level of signaling mediated by the GPCR in the absence of the antagonist. For example, an antagonist may block a naturally-occurring GPCR ligand from binding to the GPCR, or an antagonist may induce a conformation of the GPCR that reduces the signaling of the GPCR upon binding to the GPCR, e.g., by binding to an allosteric site.
In some embodiments, the GPCR is NPFFR1 or NPFFR2, preferably human NPFFR1 or human NPFFR2. The agent may modulate the activity of more than one GPCR. For example, the agent may modulate the activity of NPFFR2 and either NPY1R and/or NPY2R. The GPCR may be NPFFR1.
In some embodiments, the agent is selected from neuropeptide FF (NPFF), RFamide-related peptide 1 (RFRP1), RFamide-related peptide 2 (RFRP2), or RFamide-related peptide (RFRP3). In some embodiments, the agent is not NPFF, RFRP1, RFRP2, or RFRP3. In some embodiments, the agent is a peptide or a derivative of a peptide. The agent may be a small molecule, e.g., having a molecular weight less than 2000 amu, such as less than 1000 amu, less than 500 amu, less than 400 amu, or less than 300 amu.
In some embodiments, the subject has been diagnosed with a sleep disorder. The subject may have been diagnosed with a psychological condition. The subject may have fatigue, attention deficit hyperactivity disorder, depression, bipolar disorder, schizophrenia, autism, and/or an eating disorder. In some embodiments, the subject has not been diagnosed with a sleep disorder. The subject may or may not have been diagnosed with a psychological condition.
The neuropeptide signaling pathways are conserved throughout vertebrates (
Administration may comprise, for example, oral administration. Accordingly, the composition may be formulated for oral administration.
In certain aspects, the invention relates to a method of characterizing a sleep disorder in a subject, the method comprising: obtaining a sample of a nucleic acid from the subject, wherein the nucleic acid encodes a NPFFR1, NPFFR2, NPY1R, NPY2R, TACR, NPFF, RFRP1, RFRP2, and/or RFRP3 gene; sequencing the nucleic acid; comparing the nucleotide sequence encoded by the nucleic acid with a reference nucleotide sequence from a subject that does not have a sleep disorder; and diagnosing the subject with a sleep disorder if the nucleotide sequence is different from the reference nucleotide sequence. In certain aspects, the invention relates to a method of characterizing a sleep disorder in a subject, the method comprising: obtaining a sample of a nucleic acid from the subject, wherein the nucleic acid encodes a NPFFR1, NPFFR2, NPY1R, NPY2R, TACR, NPFF, RFRP1, RFRP2, and/or RFRP3 gene; sequencing the nucleic acid; comparing the amino acid sequence encoded by the nucleic acid with a reference amino acid sequence from a subject that does not have a sleep disorder; and diagnosing the subject with a sleep disorder (or even a particular sleep disorder) if the amino acid sequence is different from the reference amino acid sequence. Preferably, the subject is human and the nucleic acid encodes a human gene.
The sample may be obtained directly from the subject or indirectly, e.g., from a physician or an archive. The subject may be diagnosed with a sleep disorder, for example, if the nucleic acid encodes a nucleotide or amino acid sequence for NPFFR1, NPFFR2, NPY1R, NPY2R, and/or TACR that prevents the expression of a full-length protein (e.g., the sequence comprises a truncation mutation), prevents the proper folding of the protein, prevents the protein from localizing to the extracellular membrane, prevents binding to a ligand, prevents the binding of a ligand from initiating a signaling pathway, prevents an interaction with one or more signaling molecules, decreases the stability of the protein, and/or targets the protein for degradation. The subject may be diagnosed with a sleep disorder if the nucleotide sequence comprises a mutation in a promoter or enhancer region that affects the transcription of the protein, e.g., by increasing or decreasing the rate at which a gene encoding the protein is transcribed. The subject may be diagnosed with a sleep disorder, for example, if the nucleic acid encodes an amino acid sequence for NPFF, RFRP1, RFRP2, and/or RFRP3 that prevents the expression of a full-length peptide (e.g., the sequence comprises a truncation mutation), prevents the post-translational modification of the peptide (e.g., prevents the cleavage of a precursor protein or peptide into RFRP1, RFRP2, and/or RFRP3), prevents cells from secreting the peptide, decreases the half-life of the peptide, and/or alters the binding affinity of the peptide for one or more GPCRs.
Methods for Identifying Agonists and Antagonists of NPFFR1, NPFFR2, NPY1R, NPY2R, and/or TACR
In some aspects, the invention relates to a method of identifying whether a compound affects a condition in a subject, comprising: administering the compound to a test subject and monitoring the activity of the test subject, wherein the test subject comprises a genetic modification that reduces the expression of NPFFR1, NPFFR2, NPY1R, NPY2R, and/or TACR, and/or a homolog or ortholog of any one of NPFFR1, NPFFR2, NPY1R, NPY2R and/or TACR; administering the compound to a control subject and monitoring the activity of the control subject, wherein the control subject does not comprise the genetic modification; comparing the activity of the control subject and the test subject; and identifying whether the compound affects the condition, wherein the condition is selected from fatigue, sleep, wakefulness, attention deficit hyperactivity disorder, chronic fatigue syndrome, depression, bipolar disorder, schizophrenia, autism, and an eating disorder. Identifying whether the compound affects the condition may comprise determining whether the activity of the test subject is different from the activity of the control subject, e.g., whether the test subject sleeps more or less than the control subject or whether the test subject is more or less active than the control subject. For example, the activity may be sleep and monitoring the activity may comprise measuring duration of sleep. The condition may be sleep or wakefulness, e.g., the method may comprise identifying whether a compound affects sleep or wakefulness in the subject.
The subject may be a vertebrate such as a zebrafish or mouse. The genetic modification may be a knockout or knockdown mutation of a gene encoding NPFFR1, NPFFR2, NPY1R, NPY2R, and/or TACR, and/or a homolog or ortholog of any one of NPFFR1, NPFFR2, NPY1R, NPY2R and/or TACR.
In certain aspects, the invention relates to a method of identifying whether a compound affects a condition in a subject, comprising: administering the compound to a test subject and monitoring the activity of the test subject, wherein the test subject is Caenorhabditis elegans comprising a genetic modification that reduces the expression of neuropeptide receptor 7 (NPR-7) or neuropeptide receptor 22 (NPR-22); administering the compound to a control subject and monitoring the activity of the control subject, wherein the control subject is Caenorhabditis elegans and the control subject does not comprise the genetic modification; comparing the activity of the control subject and the test subject; and identifying whether the compound affects the condition. Identifying whether the compound affects the condition may comprise determining whether the activity of the test subject is different from the activity of the control subject. The activity may be, for example, feeding quiescence, locomotion quiescence, pumping, or response latency. The condition may be sleep or wakefulness.
Neuropeptide receptor ligands are well known in the art (see generally Mankus, J. V. & C. F. McCurdy, Future Med Chem 4(9):1085-92 (2012)). For example, the agent may be RF9 (Formula V; 2-Adamantanecarbonyl-Arg-Phe-NH2) or GR231118 (CAS 158859-98-4). RF9 is an antagonist of NPFFRs, e.g., RF9 is a NPFFR1 antagonist and a NPFFR2 antagonist, and RF9 also specifically binds to other GPCRs, e.g., NPY1R. GR231118 is a NPY1R antagonist, and GR231118 also specifically binds to other GPCRs, e.g., NPY4R. The agent may be BIBP-3226 (Formula VI; N-[(1R)]-4-[(Aminoiminomethyl)amino-1-[[[(4-hydroxyphenyl)methyl]amino]carbonyl]butyl-α-phenylbenzeneacetamide trifluoroacetate), which is an antagonist of NPY1R and NPFFRs. The agent may be dNPA (d-Asn-Pro-(NHMe)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2), which is a NPFFR2 agonist. Other neuropeptide receptor ligands are well known in the art (see, e.g., U.S. Pat. No. 7,727,979; U.S. Pat. No. 7,544,691; U.S. Pat. No. 6,960,574; U.S. Patent Application Publication No. 2007/0123510; U.S. Patent Application Publication No. 2005/0136444; U.S. Patent Application Publication No. 2003/0176314; U.S. Patent Application Publication No. 2003/0139431; PCT Patent Application Publication No. WO 2009/038012; PCT Patent Application Publication No. WO 2004/080965; PCT Patent Application Publication No. WO 2003/026657, hereby incorporated by reference, especially for the compounds disclosed therein).
In some embodiments, the agent comprises a guanidino group. For example, the agent may comprise an arginine moiety. In some embodiments, the agent does not comprise a guanidino group. The agent may comprise a phenylalanine moiety and/or an adamantane moiety.
The agent may be selected from N-(4,6-dimethyl-2-quinolinyl)guanidine and N-(4,7-dimethyl-2-quinolinyl)guanidine, which are antagonists of NPFFR1 and agonists of NPFFR2 (U.S. Patent Application Publication No. 2003/0139431, hereby incorporated by reference, especially for the compounds disclosed therein). The agent may be selected from N-(6-benzyl-4-methyl-2-quinazolinyl)guanidine, N-(6-butyl-4-methyl-2-quinazolinyl)guanidine, N-(6-hexyl-4-methyl-2-quinazolinyl)guanidine, N-(4-methyl-6-pentyl-2-quinolinyl)guanidine, and N-(6-butyl-4-methyl-2-quinolinyl)guanidine, which are agonists of both NPFFR1 and NPFFR2 (U.S. Patent Application Publication No. 2003/0139431, hereby incorporated by reference, especially for the compounds disclosed therein). The compound may be selected from N-(4-methyl-2-quinolinyl)guanidine, N-(4-ethyl-7-methyl-2-quinolinyl)guanidine, and N-(4,8-dimethyl-2-quinolinyl)guanidine, which are antagonists of both NPFFR1 and NPFFR2 (U.S. Patent Application Publication No. 2003/0139431, hereby incorporated by reference, especially for the compounds disclosed therein).
The agent may be selected from N-(5-ethyl-5-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-tert-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-isopropyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5-butyl-5,6,7,8-tetrahydro-4H-cycloheptathiazol-2-yl)-guanidine, N-(4-ethyl-4-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(5-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-propyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-cyclohex-1-enyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-sec-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-isobutyl-4-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-tert-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(1,1-dimethyl-propyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-[6-(3-methoxy-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(6-thiophene-2-yl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(4-fluorophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(4-allyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(3-fluorophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(6-cyano-6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6,6-diphenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(4-methoxy-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(5-phenyl-5,6,7,8-tetrahydro-4H-cycloheptathiazol-2-yl)-guanidine, N-(6-benzo[1,3]dioxol-5-yl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(4-cyanophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(4-benzyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5-methyl-5-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(3,5-bis-trifluoromethylphenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(6-o-tolyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-m-tolyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(2-ethyl-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-[6-(4-chlorophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-[6-(4-benzyloxy-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl-4-spiro-cyclohexane)-guanidine, N-(6-p-tolyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5,5-dimethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6,6-dimethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-methyl-4-propyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(7-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4,4-dimethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, and N-(6-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine (U.S. Pat. No. 7,727,979, hereby incorporated by reference, especially for the compounds disclosed therein).
The agent may be selected from 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxylic acid tert-butyl ester, N-(5-hexyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-[5-(2-cyclohexyl-ethyl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, N-(5-ethyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxylic acid butyl ester, N-[5-(propane-2-sulphonyl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, N-(5-phenylacetyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxylic acid benzyl ester, N-(5-pentanoyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-thiocarboxylic acid propyl amide, N-[5-(2-propyl-pentanoyl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, N-(5-benzyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-(5-prop-2-ynyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-(5-cyclopropanecarbonyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-[5-(butane-1-sulphonyl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, N-(5-isobutyryl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-[5-(2,2-dimethyl-propionyl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-thiocarboxylic acid benzyl amide, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxylic acid tert-butyl amide, N-(5-but-3-enoyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-(5-benzyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-c]azepine-2-yl)-guanidine, 3-(2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-yl)-propionic acid ethyl ester, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxylic acid pentyl amide, N-[5-(2-methoxy-acetyl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, N-(5-cyclopropylmethyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-(5-methanesulphonyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, N-[5-(3-methyl-butyryl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-thiocarboxylic acid-(2-methoxy-1-methyl-ethyl)-amide, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxylic acid phenyl amide, [3-(2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-yl)-3-oxo-propyl]-carbamic acid tert-butyl ester, N-[5-(4-dimethylamino-butyryl)-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl]-guanidine, N-(5-propyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]pyridine-2-yl)-guanidine, 2-guanidino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-thiocarboxylic acid isopropyl amide, N-(4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, (2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-yl)-ethyl acetate ethyl ester, N-(4-hydroxymethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-tosyloxymethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-azidomethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-aminomethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-acetylaminomethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5-ethyl-5-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5,5-dimethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5,5-dimethyl-6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-tert-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-isopropyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5,5,7-trimethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6,6-dimethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5-butyl-5,6,7,8-tetrahydro-4H-cycloheptathiazol-2-yl)-guanidine, N-(4-ethyl-4-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(5-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-methyl-4-propyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-propyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-cyclohex-1-enyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-sec-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-isobutyl-4-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine N-(6-tert-butyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, 2-guanidino-6-phenyl-4,5,6,7-tetrahydro-benzothiazole-6-carboxylic acid ethyl ester, N-[6-(1,1-dimethyl-propyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(7-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(3-methoxy-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(6-thiophene-2-yl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5,5,7,7-tetramethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(4-fluorophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-6-carboxylic acid ethyl ester N-(4,4-dimethyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(4,5,6,7-tetrahydro-benzothiazole-2-yl-4-spiro-cyclohexane)-guanidine, N-(5,6,7,8-tetrahydro-4H-cycloheptathiazol-2-yl)-guanidine, N-(4-allyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-methyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(3-fluorophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(6-cyano-6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine N-(4-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine N-(6,6-diphenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine N-[6-(4-methoxy-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(5-phenyl-5,6,7,8-tetrahydro-4H-cycloheptathiazol-2-yl)-guanidine N-(6,7-dihydro-4H-pyrano[4,3-d]thiazol-2-yl)-guanidine, N-(6-benzo[1,3]dioxol-5-yl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-6-carboxylic acid propyl amide, N-[6-(4-cyanophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(4-benzyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(5-methyl-5-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(3,5-to-trifluoromethylphenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-(6-o-tolyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-(6-m-tolyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, N-[6-(2-ethyl-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, N-[6-(4-chlorophenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid benzyl amide, N-(5,6-dihydro-4H-cyclopentathiazol-2-yl)-guanidine, N-[6-(4-benzyloxy-phenyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid methyl phenethyl amide, N-(6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl-4-spiro-cyclohexane)-guanidine, N-(6-p-tolyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid-(3-methyl-butyl)-amide, N-(4-tert-butyl-6-phenyl-4,5,6,7-tetrahydro-benzothiazole-2-yl)-guanidine 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-6-carboxylic acid phenyl amide, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid butyl ethyl amide, N-[4-(2-cyano-ethyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid ethyl ester, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid dipropyl amide, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid phenyl amide, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-6-carboxylic acid allyl amide, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid propyl amide, N-[4-(piperidine-1-carbonyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid allyl amide, 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-6-carboxylic acid-(3-methyl-butyl)-amide, N-[4-(morpholine-4-carbonyl)-4,5,6,7-tetrahydro-benzothiazole-2-yl]-guanidine, and 2-guanidino-4,5,6,7-tetrahydro-benzothiazole-4-carboxylic acid diisopropyl amide (U.S. Pat. No. 7,727,979, hereby incorporated by reference, especially for the compounds disclosed therein).
The agent may be selected from 1-(4-Fluorobenzylideneamino)guanidine, 1-[3-(Trifluoromethyl)benzylideneamino]guanidine, 1-[1-(3-Bromophenyl)ethylideneamino]guanidine, 1-(5-Fluoro-2-nitrobenzylideneamino)guanidine, 1-[(Benzo[1,3]dioxol-5-yl methylideneamino]guanidine, 1-[(Anthracen-9-yl)methylideneamino]guanidine, 1-(3,5-Dimethoxybenzylideneamino)guanidine, 1-(2,4-Dichlorobenzylideneamino)guanidine, 1-(3-Fluoro-4-methoxybenzylideneamino)guanidine, 1-(3-Bromo-4-fluorobenzylideneamino)guanidine, 1-(3,4,5-Trimethoxybenzylideneamino)guanidine, 1-(4-Fluoro-3-methylbenzylideneamino)guanidine, 1-(3-Chloro-4-fluorobenzylideneamino)guanidine, 1-(3-Bromo-4-methoxybenzylideneamino)guanidine, 1-(2,5-Difluorobenzylideneamino)guanidine, 1-(2,4-Difluorobenzylideneamino)guanidine, 1-(2,3-Dichlorobenzylideneamino)guanidine, 1-(4-Bromo-2-fluorobenzylideneamino)guanidine, 1-(4-Phenylbenzylideneamino)guanidine, 1-(4-Phenoxybenzylideneamino)guanidine, 1-(3-Phenoxybenzylideneamino)guanidine, 1-(3,5-Di-tert-butyl-2-hydroxybenzylideneamino)guanidine, 1-(2,3,5-Trichlorobenzylideneamino)guanidine, 1-(3,5-Dibromo-4-hydroxybenzylideneamino)guanidine, 1-(4-Isopropoxybenzylideneamino)guanidine, 1-(3,4-Diethoxybenzylideneamino)guanidine, 1-(3,5-Difluorobenzylideneamino)guanidine, 1-(3,4-Dibromobenzylideneamino)guanidine, 1-(3,4-Dibromobenzylideneamino)guanidine, 1-(4-Chloro-3-fluorobenzylideneamino)guanidine, 1-(3-Chloro-4-hydroxybenzylideneamino)guanidine, 1-(4-Fluoro-3-nitrobenzylideneamino)guanidine, 1-(3,5-Dimethyl-4-hydroxybenzylideneamino)guanidine, 1-(4-Methoxy-2,3-dimethylbenzylideneamino)guanidine, 1-[4-Chloro-3-(trifluoromethyl)benzylideneamino]guanidine, 1-(3-Bromo-4,5-dimethoxybenzylideneamino)guanidine, 1-[3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylideneamino]guanidine, [(Cyclohexylphenylmethylideneamino]guanidine, 1-[1-(2,3-Dihydro-benzo[1,4]dioxin-6-yl)ethylideneamino]guanidine, 1-(4-Benzyloxy-3-chlorobenzylideneamino)guanidine, 1-(4-Allyloxy-3-chlorobenzylideneamino)guanidine, 1-(3-Chloro-4-methoxybenzylideneamino)guanidine, 1-[3-Chloro-4-(4-cyanobutoxyl)benzylideneamino]guanidine, 1-[3-Chloro-4-(3-phenoxypropoxyl)benzylideneamino]guanidine, 1-[3-Chloro-4-(2-phenylethoxyl)benzylideneamino]guanidine, 1-(3-Chloro-4-hexyloxybenzylideneamino)guanidine, 1-(3-Chloro-4-propoxyobenzylideneamino)guanidine, 1-[3-Chloro-4-(2-methylpropoxyl)benzylideneamino]guanidine, 1-[3-Chloro-4-(4-methylpentoxyl)benzylideneamino]guanidine, 1-[3-Chloro-4-(4-cyclohexylmethoxyl)benzylideneamino])guanidine, 1-[3-Chloro-4-(2-ethylbutoxyl)benzylideneamino]guanidine, 1-(3-Chloro-4-octyloxybenzylideneamino)guanidine, 1-[3-Chloro-4-(2-ethoxy-ethoxy)benzylideneamino])guanidine, 1-(2-Phenylbenzylideneamino)guanidine, 1-(3,4-Dichlorophenyl)-1-(propylideneaminoguanidine), 1-[4-(2-Fluorophenyl)benzylideneamino]guanidine, 1-[3-(2-Trifluoromethylphenyl)benzylideneamino]guanidine, 1-(5-Chloro-2,3-dimethoxybenzylideneamino)guanidine, 1-[2-Fluoro-4-(trifluoromethyl)benzylideneamino]guanidine, 1-[2,4-Bis(trifluoromethyl)benzylideneamino]guanidine, 1-[2,3-Difluoro-4-(trifluoromethyl)benzylideneamino]guanidine, 1-[3-Fluoro-4-(trifluoromethyl)benzylideneamino]guanidine, 1-[3-Nitro-4-(trifluoromethyl)benzylideneamino]guanidine, 1-[2-Fluoro-3-(trifluoromethyl)benzylideneamino]guanidine, 1-[2-Fluoro-5-(trifluoromethyl)benzylideneamino]guanidine, 1-[3-Fluoro-5-(trifluoromethyl)benzylideneamino]guanidine, 1-[4-Fluoro-3-(trifluoromethyl)benzylideneamino]guanidine, 1-[2-Chloro-5-(trifluoromethyl)benzylideneamino]guanidine, 1-[2-Chloro-3-(trifluoromethyl)benzylideneamino]guanidine, 1-[3-Chloro-2-fluoro-5-(trifluoromethyl)benzylideneamino]guanidine, 1-[(4-Fluoro-1-naphthalen-1-yl)methylideneamino]guanidine, 1-[4-Methoxy-3-(trifluoromethyl)benzylideneamino]guanidine, 1-[2-Methoxy-5-(trifluoromethyl)benzylideneamino]guanidine, 1-[Naphthalen-2-yl-methylideneamino]guanidine, 1-[5-Bromo-2-ethoxybenzylideneamino]guanidine, 1-[2,4-Dimethylbenzylideneamino]guanidine, 1-[4-Chloro-3-nitrobenzylideneamino]guanidine, 1-(4-Benzyloxy-2-hydroxybenzylideneamino)guanidine, 1-[(1H-Indol-5-yl)methylideneamino]guanidine, 1-(4-Butoxybenzylideneamino)guanidine, 1-(4-Cyanobenzylideneamino)guanidine, 1-(2,5-Dimethoxybenzylideneamino)guanidine, 1-(2-Benzyloxy-3-methoxybenzylideneamino)guanidine, 1-[1-(2-Methoxy-naphthalen-1-yl)methylideneamino]guanidine, 1-(4-Hydroxy-3-methoxy-5-nitrobenzylideneamino)guanidine, 1-(3,4-Dihydroxybenzylideneamino)guanidine, 1-(3-Bromobenzylideneamino)guanidine, 1-(3,5-Dibromobenzylideneamino)guanidine, 1-[1-(3,4-Dichlorophenyl)ethylideneamino]guanidine, 1-(4-n-Hexyloxybenzylideneamino)guanidine, 1-(3,4-Dibenzyloxybenzylideneamino)guanidine, 1-[(6-Bromobenzo[1,3]dioxol-5-yl)methylideneamino]guanidine, 1-[1-(4-Bromophenyl)ethylideneamino]guanidine, 1-[1-(3-Methylphenyl)ethylideneamino]guanidine, 1-(3-Methylbenzylideneamino)guanidine, 1-(3,4-Dimethylbenzylideneamino)guanidine, 1-[1-(4-Ethylphenyl)ethylideneamino]guanidine, 1-[1-(3,4-Dimethylphenyl)ethylideneamino]guanidine, 1-(4-n-pentylbenzylideneamino)guanidine, 1-[1-(4-n-Heptylphenyl)ethylideneamino]guanidine, 1-[1-(5,6,7,8-Tetrahydronaphthalen-2-yl)ethylideneamino]guanidine, 1-(4-Ethylbenzylideneamino)guanidine, 1-[1-(2-Bromophenyl)ethylideneamino]guanidine, 1-{1-[3-(Trifluoromethyl)phenyl]ethylideneamino}guanidine, 1-{1-[3,5-Bis-(trifluoromethyl)phenyl]ethylideneamino}guanidine, 1-[1-(2,5-Dimethoxyphenyl)ethylideneamino]guanidine, 1-[1-(2-Hydroxy-4-methoxyphenyl)ethylideneamino]guanidine, 1-[1-(4-Benzyloxy-2-hydroxy-3-methylphenyl)ethylideneamino]guanidine, 1-[1-(Benzo[1,3]dioxol-5-yl)ethylideneamino]guanidine, 1-(3,4-Dichlorobenzylideneamino)guanidine, 1-[1-(4-Dimethylaminophenyl)pentylideneamino]guanidine, 1-{4-[Ethyl-(2-hydroxyethyl)amino]-2-methylbenzylideneamino}guanidine, 1-(4-Diethylamino-2-hydroxybenzylideneamino)guanidine, 1-(4-Diethylaminobenzylideneamino)guanidine, 1-[1-(4-Piperidin-1-yl-phenyl)ethylideneamino]guanidine, 1-{4-[Methyl-(2-cyanoethyl)amino]benzylideneamino}guanidine, 1-{4-[Methyl-(2-hydroxyethyl)amino]benzylideneamino}guanidine, 1-(4-Di-n-butylaminobenzylideneamino)guanidine, 1-(2-Methoxy-4-N,N-diethylaminobenzylideneamino)guanidine, 1-(3-Cyanobenzylideneamino)guanidine, 1-[(4-Trifluoromethyl)benzylideneamino]guanidine, 1-(2,4-Dimethoxybenzylideneamino)guanidine, 1-(2,3-Dimethoxybenzylideneamino)guanidine, 1-(4-Ethoxybenzylideneamino)guanidine, 1-(4-n-Propoxybenzylideneamino)guanidine, 1-(2,3,6-Trichlorobenzylideneamino)guanidine, 1-(4-Chlorobenzylideneamino)guanidine, 1-(5-Bromo-2-fluorobenzylideneamino)guanidine, 1-(2-Bromo-5-fluorobenzylideneamino)guanidine, 1-(3-Chlorobenzylideneamino)guanidine, 1-(3-Fluorobenzylideneamino)guanidine, 1-(2,3,4-Trimethoxybenzalideneamino)guanidine, 1-(3,5-Bistrifluoromethylbenzylideneamino)guanidine, 1-(5-Bromo-2,4-dimethoxybenzylideneamino)guanidine, and 1-[(5-(2-(Trifluoromethyl)phenyl)-furan-2-yl)-methyleneamino]guanidine (U.S. Patent Application No. 2005/0136444, hereby incorporated by reference, especially for the compounds disclosed therein). The compound may be selected from 1-(3,4-Dichlorobenzylideneamino)guanidine, 1-(3,4-Dibromobenzylideneamino)guanidine, and 1-(3-Bromo-4,5-dimethoxybenzylideneamino)guanidine (U.S. Patent Application No. 2005/0136444, hereby incorporated by reference, especially for the compounds disclosed therein).
The agent may be selected from N-(4-methyl-6-propyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-isopropyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4,5-dimethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-tert-butyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-8-phenyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-6-phenyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-[6-(1,1-dimethyl-propyl)-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl]-guanidine, N-(8-tert-butyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4,6-dimethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-yl)-guanidine, N-(4-methyl-5,6,7,8,9,10-hexahydro-cyclooctapyrimidin-2-yl)-guanidine, N-(8-sec-butyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4,8-dimethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(8-allyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-6,7-dihydro-5H-cyclopentapyrimidin-2-yl)-guanidine, N-(8-cyclohex-1-enyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-isopropyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-tert-butyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-propyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-phenyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, or N-(6-tert-butyl-4-trifluoromethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-[8-(2-cyano-ethyl)-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl]-guanidine, 2-guanidino-4-methyl-7,8-dihydro-5H-pyrido[4,3-d]pyrimidine-6-carboxylic acid tert-butyl ester, N-(6-phenyl-4-trifluoromethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-isopropyl-4-trifluoromethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(5,6,7,8-tetrahydro-quinolin-2-yl)-guanidine, N-(6-phenyl-5,6,7,8-tetrahydro-quinoline-2-yl)-guanidine, N-(5,6,7,8-tetrahydro-quinoxalin-2-yl)-guanidine, N-(6-phenyl-5,6,7,8-tetrahydro-quinoxalin-2-yl)-guanidine, N-(7-phenyl-5,6,7,8-tetrahydro-quinoxalin-2-yl)-guanidine, 6,7,8-tetrahydro-benzo[1,2,4]triazin-3-yl)-guanidine, N-(7-phenyl-5,6,7,8-tetrahydro-benzo[1,2,4]triazin-3-yl)-guanidine, and N-(6-phenyl-5,6,7,8-tetrahydro-benzo[1,2,4]triazin-3-yl)-guanidine (U.S. Pat. No. 7,544,691, hereby incorporated by reference, especially for the compounds disclosed therein). For example, the agent may be N-(6-isopropyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-6-propyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, or N-(6-tert-butyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine (U.S. Pat. No. 7,544,691, hereby incorporated by reference, especially for the compounds disclosed therein).
The compound may be selected from N-(4-methyl-6-propyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(6-isopropyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4,5-dimethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine and N-(6-tert-butyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-8-phenyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(4-methyl-6-phenyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-[6-(1,1-dimethyl-propyl)-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl]guanidine, N-(8-tert-butyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(4,6-dimethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-yl) guanidine, N-(4-methyl-5,6,7,8,9,10-hexahydro-cyclooctapyrimidin-2-yl)-guanidine, N-(8-sec-Butyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(4,8-dimethyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(8-Allyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(6-Isopropyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-tert-butyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(4-methyl-6,7-dihydro-5H-cyclopentapyrimidin-2-yl)-guanidine, N-(6-propyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(8-Cyclohex-1-enyl-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine, N-(6-tert-Butyl-4-trifluoromethyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-(6-phenyl-4-trifluoromethyl-5,6,7,8-tetrahydro-quinazolin-2-yl) guanidine, N-[8-(2-cyano-ethyl)-4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl]-guanidine, N-(6-Isopropyl-4-trifluoromethyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-guanidine; 2-guanidino-4-methyl-7,8-dihydro-5H-pyrido[4,3-d]pyrimidine-6-carboxylic acid tert-butyl ester, N-(5,6,7,8-tetrahydro-quinolin-2-yl) guanidine, N-(6-phenyl-5,6,7,8-tetrahydro-quinolin-2-yl) guanidine, N-(5,6,7,8-tetrahydro-quinoxalin-2-yl) guanidine, N-(6-phenyl-5,6,7,8-tetrahydro-quinoxalin-2-yl)-guanidine, N-(7-phenyl-5,6,7,8-tetrahydro-quinoxalin-2-yl)-guanidine-5,6,7,8-tetrahydro-benzo[1,2,4]triazin-3-yl)-guanidine; N-(7-phenyl-5,6,7,8-tetrahydro-benzo[1,2,4]triazin-3-yl)-guanidine, and N-(6-phenyl-5,6,7,8-tetrahydro-benzo[1,2,4]triazin-3-yl)-guanidine (U.S. Pat. No. 7,544,691, hereby incorporated by reference, especially for the compounds disclosed therein).
The agent may be selected from any one of Formulas I-X, or a derivative, ester, or salt thereof:
For example, the agent may be a molecule disclosed in PCT Patent Application Publication No. WO 2009/038012 (hereby incorporated by reference, especially for the compounds disclosed therein), including molecules similar to the molecule depicted in Formula VII. Similarly, the agent may be a molecule disclosed in U.S. Patent Application Publication No. U.S. 2003/0139431 (hereby incorporated by reference, especially for the compounds disclosed therein), including molecules similar to the molecules depicted in Formulas VIII-X.
Certain embodiments of the present invention are well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention.
The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, and published patent applications as cited throughout this application) are hereby expressly incorporated by reference. When definitions of terms in documents that are incorporated by reference herein conflict with those used herein, the definitions used herein govern.
Strains were grown, unless indicated otherwise, on nematode growth medium (NGM) 2% agar plate seeded with E. coli strain OP50 and maintained at 20° C. under standard animal husbandry conditions (Brenner, Genetics 77, 71 (1974)).
The wild-type worm strain was the Bristol variety, N2. Mutation strains were obtained from the Caenorhabditis Genetics Center (CGC) including RB761 npr-7(ok527) X and RB1405 npr-22(ok1598) IV, which were provided by the C. elegans Gene Knockout Project at OMRF (http://www.mutantfactory.ouhsc.edu/), and VC1971 flp-14(gk3109) III, which was provided by the C. elegans Reverse Genetics Core Facility at the University of British Columbia (http://www.celeganskoconsortium.omrf.org). Mutation strain FX02427flp-13(tm2427) IV was obtained from the National Bioresource Project.
Mutant Strains:
flp-13(tm2427) is a 382 by deletion (89:291) starting in the first intron to part of the second intron that removes the second exon and causes a frame shift at amino acid residue 31. This mutation deletes the C-terminus of the propeptide and completely abolishes all peptide cleavage sites and synthesis of all FLP-13 peptides.
flp-24(gk3109) is a 1180 by deletion (92:210) starting in the first intron to the 3′ intergenic sequence that removes the second exon and 3′UTR of flp-24. This deletion truncates the protein at amino acid residue 29, removing the majority of the propeptide, all post-translational processing sites, and the FLP-24 peptide.
npr-22(ok1598) is a 2528 by deletion that erases exons 2 to 5 of isoform a and exons 2 to 6 of isoform b, causing protein truncation before the first transmembrane domain and resulting in a null mutation of both isoforms.
npr-7(ok527) Xis a 1211 by deletion that erases half of exon 3 and the entire exon 4.
Conditional expression of cDNAs was achieved by generating a fusion of the coding sequence of a gene under study to the hsp-16.41 promoter (Lesa and Sternberg, Mol. Biol. Cell 8, 779-793 (1997)). A synthetic DNA fragment consisting of the hsp-16.41 promoter, DNA coding sequence and its endogenous 3′UTR was generated using fusion PCR techniques (Hobert, BioTechniques 32, 728 (2002)). N2 animals of mixed stages were harvested for RNA extraction and subsequently reverse-transcribed into cDNA for amplification of flp-13 cDNA and flp-24 cDNA. The 3′UTR regions were amplified from N2 wild-type mixed stage animal genomic lysates. The hsp-16.41 promoter region was amplified from plasmid ppD49.83 and the PCR product was verified by DNA sequencing. The cDNAs and 3′UTR genomic DNAs amplified were sequenced and blasted against the predictions from www.wormbase.org. The final fusion PCR product was sequenced and blasted against the predicted DNA sequence for the conditional gene overexpression assay.
hs:FLP-24 overexpression DNA was used as a backbone to introduce exogenous zebrafish RF-amides in C. elegans. The FLP-24 peptide was replaced by either zebrafish NPFF (DrNPFF) or zebrafish RFRP-1 (DrRFRP-1), keeping the endogenous FLP-24 signal peptide, propeptide, cleavage sites, and 3′UTR intact.
Primer sequences used in the generation of the above fusions were as follows:
To synthesize the fusion PCR fragments for heat shock overexpression, the following primer pairs were used:
To identify neuropeptides that mediate ALA-dependent sleep, micro-dissected GFP-labeled ALA neurons during late L4-stage were profiled (
All conserved sequences were amplified from N2 wild-type mixed stage worm genomic lysates. The pes-10, green fluorescent protein (GFP), and unc-54 3′UTR sequence was amplified from pD97.78. The mCherry sequence was amplified from pAM-31.1.
Primer sequences used in the generation of the above fusions are listed as follows:
Transgenic strains were generated by injecting the fusion DNA fragments into the gonads of young adult hermaphrodites along with a fluorescent co-injection marker and a carrier (Mello and Fire, Methods Cell Biol. 48, 451 (1995)). In order to control for variation between transgenes, at least two independent lines from each injection were used for behavioral assays and expression patterns.
TB513: dpy-20(e2017); chIs513[ceh-14::GFP, dpy-20(+)]
PS5009: pha-1(e2123ts); him-5(e1490); syEx723[hsp16-41: lin-3C cDNA(10 ng/μl)+myo2:GFP(10 ng/μl)+pha-1(+)(pbx-1)(90 ng/μl)+bluescript(90 ng/μl)]
PS6562 syEX1285[hsp16-41:flp-13(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6563 syEX1286[hsp16-41:flp-24(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6564 syEX1287[hsp16-41:flp-13; flp-24(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6565 RB1405 npr-22(ok1598)+syEX1288[hsp16-41:flp-13; flp-24(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6566, PS5009 syIs197, him-5(e1490); FX02427 flp-13(tm2427)
PS6567, PS5009 syIs197, him-5(e1490); VC9171 flp-24(gk3109)
PS6568, PS5009 syIs197, him-5(e1490); RB1405 npr-22(ok1598)
PS6569 syEx1285, syIs197, him-5(1490); ceh-14(ch3) X
PS6570 syEx1286, syIs197, him-5(1490); ceh-14(ch3) X
PS6571 syEX1294[hsp16-41:flp-7(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6572 syEX1295[Pflp-13::GFP(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6573 syEX1296[Pflp-24::GFP(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6574 syEX1297[Pnpr-22::GFP(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6575 syEX1298[Prab-3:npr-22::GFP(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6576 syEX1299[Pmyo-3:npr-22::GFP(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6577 syEX1300[hsp16-41:DrRFRP-1(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
PS6578 syEX1301[hsp16-41:DrNPFF-1(10 ng/μl), PmyosdsRed(10 ng/μl), KS+(90 ng/μl)]
Individual ALA neurons from wild-type larvae of strain TB513 at the mid-L4 larval stage were hand-picked and glued on an agar pad for micro-dissection (Schwarz et. al., Proc. Natl. Acad. Sci. U.S.A. 109(40), 16246 (2012)). Isolated individual ALA neurons tagged by GFP were collected with an unpolished patch-clamp tube that served as a pipette, transferred to a prelubricated eppendorf tube, and snap-frozen with liquid nitrogen. Frozen tubes with individual ALA neurons were kept at −70° C. until amplification.
RT-PCR of individual neurons was performed essentially as in Schwarz et al. (Proc. Natl. Acad. Sci. U.S.A. 109(40), 16246 (2012)). To obtain RNA-seq data by Illumina sequencing, aliquots of individual RT-PCR reactions were pooled into two pools (4 cells and 5 cells). Existing whole wild-type larval RNAseq data (Schwarz et al., Proc. Natl. Acad. Sci. U.S.A. 109(40), 16246 (2012)) was used as a control for housekeeping versus ALA-enriched genes. Expression values for genes were defined by pooling reads from both wild-type mid-L4 ALA neuron sets into a single set of expression values, doing likewise for both wild-type larval RNA-seq sets, and computing ALA/larval ratios of gene activity. After quality filtering and truncation but before mapping, RNA-seq data from the two pools of wild-type ALA comprised 1,164,892,280 nt in 30,655,060 reads and 1,520,526,262 nt in 40,013,849 reads (all reads were single-end and 38 nt). Of these, 25.2% could be mapped to WS190 protein-coding gene models (i.e., 17,798,207 out of 70,668,909 reads). The expression of 7,698 and 4,068 genes were detected in the two pools separately, and 8,133 genes collectively were detected.
Deep sequencing analysis allowed the detection of 8,113 protein-coding genes, with eight genes encoding neuropeptides that had greater than 17-fold enrichment in ALA relative to whole larval transcriptome (
To identify if these ALA-enriched neuropeptides mediate sleep, the neuropeptides were examined to determine whether they could confer resistance to EGF/stress-induced sleep. Ten young adult hermaphrodites were hand selected and transferred by a platinum pick to a NGM plate coated with a thin lawn of OP50 bacteria. The plates were sealed with parafilm and placed in a 35° C. water bath for 30 min, recovered at 20° C. for 10 min before examination for feeding for 60 min, as indicated by pharyngeal pumping activity. Each animal was examined under high magnification on a stereomicroscope for 5 sec for feeding, indicated by the movement of the grinder in the posterior pharyngeal bulb. Typically, animals with no observed movement were scored as quiescence for feeding behavior.
Mutants of all four highest scoring ALA-synthesized neuropeptides were resistant to EGF/stress-induced sleep to varying degrees: nlp-8 exhibited the highest resistance, followed by flp-13, flp-24 and flp-7 mutants. By contrast, resistance in other mutants of ALA-synthesized neuropeptides were not observed, namely, flp-10, flp-19 and ins-27 (
To characterize the role of ALA-synthesized neuropeptides in inducing C. elegans sleep, three physiological behavior measurements were assessed: locomotor cessation, feeding cessation, and decreased sensory arousal to stimulation (
Neuropeptide Heat-Activated Gain-of-Function Quiescence Assays.
Young adult hermaphrodites were hand selected and transferred by a platinum pick to NGM plates coated with a thin lawn of OP50 bacteria. Each plate hosted approximately 20 animals. Heat shock assays were performed as described by Van Buskirk & Sternberg (Development 137(12), 2065 (2010)). Briefly, the plates were sealed with parafilm and placed in a 33° C. water bath for 30 min, returned to 20° C. for 2 h, and scored for suppression of locomotion and feeding, and for response latency to sensory and mechanical stimulations.
Feeding Behavior.
Each animal was examined for feeding, indicated by the movement of the grinder in the posterior pharyngeal bulb under high magnification on a stereomicroscope for 5 sec. Typically, animals with no movement observed were scored as quiescent for feeding behavior.
Locomotion.
For locomotion analysis, animals recovered from heat-activation were placed on a Leica stereomicroscope base with a Unibrain camera, illuminated with continuous white light, and imaged for 4 minutes to track forward and backward movements. The centroid velocity plot was calculated over a 2-s interval (Van Burskirk & Sternberg, Development 137(12), 2065 (2010)).
Of the four neuropeptides conditionally expressed under a heat-shock promoter, three were sufficient to induce sleep in typically active young adults (
In agreement with the mild resistance to stress-induced sleep in flp-7 mutants, mild induction of sleep by overexpressing FLP-7 peptides was also observed (
(
Chemical Response Assay.
C. elegans move immediately backward when presented with 30% 1-octanol; however, such withdrawal behavior may be delayed due to decreased sensory arousal during sleep. For the chemical response assay, animals recovered from heat-activation were presented with 30% 1-octanol (Raizen et al., Nature 451(7178), 569 (2008)). 1-octanol was diluted to a final concentration of 30% with Ethanol (volume:volume). Briefly, an eyebrow hair dipped in 30% 1-octanol was presented to the nose of each worm within the length of a pharynx. The time required for the worm to move backward for the length of a pharynx was recorded and documented as response latency. All comparison between treatments and genotypes were made on the same day. Similar to sleeping animals, hs:FLP-13 and hs:FLP-24 animals exhibited delayed withdrawal behavior when presented with 1-octanol (
Lethargus Analysis.
For lethargus analysis, mid-L4 animals were handpicked and tracked for movement over the next 7 h. The animals were inspected for vulval eversion at the end of recording to ensure that they had reached the young adult stage. Movement was quantified by calculating the centroid velocity (Van Burskirk & Sternberg, Development 137(12), 2065 (2010)).
Mechanical Response Assay.
For the mechanical response assay, animals were perturbed by harsh touch with the tip of a platinum worm pick on the nose or tail of the worm. Animals were recorded for forward and backward movements under a Leica stereomicroscope with a Unibrain camera.
Neuropeptides exert their effects predominantly through G-protein-coupled receptors (GPCR). Four ALA-expressed FLP genes, flp-7, flp-9, flp-13, flp-22, can activate the GPCR NPR-22 in vitro (Table 3), suggesting that NPR-22 might act as a receptor for ALA neuropeptides. Thus, the responses of npr-22 and mutants of paralogous receptors to EGF/stress-induced sleep were investigated. Heat-stressed mutants defective in npr-3, npr-7, or npr-22 displayed reduced sleep relative to wild type animals (
C.elegans RFamide peptides that can activate
Next, the ambulatory effects were assayed and robust locomotor rescue was observed in npr-7 mutants with hs:FLP-13 peptides, and partial rescue was observed for hs:FLP-24, suggesting that NPR-7 is likely a receptor of FLP-13 peptides. Additionally, npr-22 was found to be partially required to mediate locomotion suppression by all peptides tested (
NPR-7 shares high amino acid sequence similarity with human FF1 receptor (NPFFR1) and NPY1R, while the two isoforms of NPR-22 are highly similar in sequence with human FF1 receptor NPFFR1, and NPY1R (Table 4). Reciprocal BLAST against vertebrate receptors revealed that NPR-7 and NPR-22 are likely orthologs of human FF1 and zebrafish npffr1. The bioactive peptide sequence of FF1 receptor ligands generated by the prepropeptide genes NPVF and NPFF, with FLP-13 and FLP-24 peptides were aligned, and a conserved carboxy terminus and similar hydrophobic backbone between FLP peptides and RFRP-1 was observed (
To determine whether RFRP neuropeptides regulate sleep/wake states in diurnal vertebrates, transgenic zebrafish were generated under the control of a heat-shock promoter that enabled the conditional overexpression of the RFRP precursor polypeptide (hs:RFRP), which becomes proteolytically processed into mature RFRP-1, RFRP-2, and RFRP-3 peptides (
These findings depict a previously unknown signaling pathway in vertebrate sleep regulation mediated by the FF1 receptor (e.g., NPFFR1) and its RFRP ligands. Further, zebrafish FF1 is likely the functional equivalent of nematode NPR-7, as ligands of both receptors induce sleep, and thus nematodes can be used for modeling NPFFR1 and similar GPCR-mediated sleep pathways.
Two-tail p-values were calculated using InStat software (GraphPad). Means were compared using an unpaired t-test with Welch's correction in the case of unequal variances.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims priority to U.S. Provisional Patent Application No. 61/938,046, filed on Feb. 10, 2014, hereby incorporated by reference in its entirety.
This invention was made with government support under NS070911, GM084389, and DA031367, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61938046 | Feb 2014 | US |
