WO2003026657A1 - Composes utiles pour le traitement de la douleur - Google Patents

Composes utiles pour le traitement de la douleur Download PDF

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Publication number
WO2003026657A1
WO2003026657A1 PCT/US2002/030215 US0230215W WO03026657A1 WO 2003026657 A1 WO2003026657 A1 WO 2003026657A1 US 0230215 W US0230215 W US 0230215W WO 03026657 A1 WO03026657 A1 WO 03026657A1
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WIPO (PCT)
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receptor
human
compound
binding affinity
guanidine
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PCT/US2002/030215
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English (en)
Inventor
Carlos C. Forray
Douglas Craig
Joel Kawakami
Michael J. Konkel
Lakmal W. Boteju
John M. Wetzel
Stewart A. Nobel
Honghe Wan
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Synaptic Pharmaceutical Corporation
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Publication of WO2003026657A1 publication Critical patent/WO2003026657A1/fr

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    • 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
    • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
    • 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
    • A61K31/47—Quinolines; Isoquinolines

Definitions

  • Neuroregulators comprise a diverse group of natural products that subserve or modulate communication in the nervous system. They include, but are not limited to, neuropeptides, amino acids, biogenic amines, lipids and lipid metabolites, and other metabolic byproducts. These neuroregulators interact with one or more specific types of cell surface receptors to activate one or more biological responses from within the cell by transducing signals from the receptor to the inside of the cell.
  • G- protein coupled receptors GPCRs
  • GPCRs represent a major class of cell surface receptors with which many neurotransmitters interact to mediate their effects. GPCRs are predicted to have seven membrane-spanning domains and are coupled to their effectors via G-proteins linking receptor activation with intracellular biochemical sequel such as stimulation of adenylyl cyclase.
  • Neuropeptide FF is an octapeptide isolated from bovine brain in 1985 by Yang et al . using antibodies to the molluscan neuropeptide FMRFamide (FMRFa) . F RFamide- like immmunoreactivity was observed in rat brain, spinal cord, and pituitary, suggesting the existence of mammalian ho ologs of the FMRFa family of invertebrate peptides.
  • FMRFa molluscan neuropeptide FMRFamide
  • NPFF is also called F8Fand.de and morphine modulating peptide, whereas NPAF is also called A18Famide in the literature.
  • NPFF and NPAF are encoded from the same gene, and cleaved from a common precursor protein (Vilim and Ziff 1995) .
  • NPFF neuropeptide FF
  • NPFF-1 and NPFF-2 Two known receptor subtypes for NPFF receptor subtypes (NPFF-1 and NPFF-2) were discovered and cloned from rat and human tissues (PCT International Publication No. WO 00/18438).
  • NPFF-1 and NPFF-2 Two NPFF receptor subtypes (NPFF-1 and NPFF-2) were discovered and cloned from rat and human tissues.
  • PCT International Publication No. WO 00/18438 The localization of protein and mRNA for these two receptors indicates that they may have utility as targets for drugs to treat a variety of disorders including, but not limited to, disorders of electrolyte balance, diabetes, respiratory disorders, gastrointestinal disorders, depression, phobias, anxiety, mood disorders, cognition/memory disorders, obesity, pain, alertness/sedation, lower urinary tract disorders and cardiovascular indications .
  • NPFF neuropeptide FF
  • endogenous NPFF is an endogenous modulator of opioid systems with effects on morphine analgesia, tolerance, and withdrawal (Panula et al . 1996 Rou y and Zajac, 1998) .
  • NPFF appears to represent an endogenous "anti-opioid" system in the CNS, acting at specific high-affinity receptors that are distinct from opioid receptors (Payza et al . 1993, Raffa et al. 1994).
  • Endogenous NPFF has been suggested to play a role in morphine tolerance: agonists of NPFF precipitate "morphine abstinence syndrome" (symptoms of morphine withdrawal) in morphine-dependent animals (Malin et al .
  • NPFF neuropeptide FF
  • NPFF peptides to modulate the opioid system raised the possibility that NPFF interacts directly with opiate receptors.
  • radioligand binding assays using a tyrosine-substituted NPFF analog [ 125 I]Y8Fa demonstrate that NPFF acts through specific high affinity binding sites distinct from opiate receptors (Allard et al. 1989, 1992, Gouarderes et al . 1998, Panula at al . 1987) that are sensitive to inhibition by guanine nucleotides (Payza et al . 1993).
  • NPFF and related peptidic agonists exhibit direct analgesic activity in some animal models.
  • NPFF has been shown to produce analgesia in the rat tail-flick and paw pressure models, upon intrathecal administration
  • SLAAPQRF-amide isolated from rat brain and spinal cord
  • NPFF neuropeptide FF
  • NPFF neuropeptide FF
  • PFRF-amide PFRF-amide
  • NPFF and related peptides have a number of other biological activities that may be therapeutically relevant.
  • NPFF and FMRFamide have been shown to reduce deprivation- and morphine-induced feeding in rats
  • NPFF receptors may be important targets in the treatment of eating disorders. Effects on feeding behavior are further supported by findings that demonstrate NPFF-like immunoreactive neurons, as well as NPFF1 receptor mRNA, localize to the hypothalamus (Panula, et al. 1996, Bonini at al, 2000).
  • the NPFF 1-selective ligand, BIBP 3226 which is also a neuropeptide Y Yl antagonist, blocks feeding through a nonspecific mechanism, not secondary to inhibition of Yl (Morgan et al. 1998). These data suggest that feeding behavior may be regulated through a NPFFl receptor mechanism. FMRFamide has also been shown to produce antipsychotic (Muthal et al .
  • NPFF receptors may be valuable targets for the treatment of psychosis and anxiety.
  • Kavaliers and Colwell (1993) have shown that i.c.v. administered NPFF has a biphasic effect of spatial learning in mice: low doses improve and high doses impair learning. This suggests the possibility that different NPFF receptor subtypes may have opposite roles in some types of learning behavior.
  • NPFF is known to have indirect effects on water and electrolyte balance. Arima et al. (1996) have shown that NPFF will reduce the increase in vasopressin release produced by salt loading or hypovolemia.
  • NPFF neuropeptide FF
  • A-18-Famide have been shown to produce significant inhibition of glucose- and arginine-induced insulin release in rats (Fehmann et al. 1990).
  • NPFF and analogs have been reported on intestinal motility in mice (Gicquel et al. 1993) and guinea pigs (Demichel et al .
  • NPFF neuropeptide FF
  • NPFF receptors are potential targets for drugs to treat Gl motility disorders, including irritable bowel syndrome.
  • NPFF has been shown to precipitate nicotine abstinence syndrome in a rodent model, raising the possibility that nicotine dependence may be attenuated by measures which inactivate NPFF (Malin et al . 1996) .
  • NPFF receptor antagonists may be of use for this purpose.
  • NPFF is known to elicit two acute cardiovascular responses when administered peripherally: elevation of blood pressure and heart rate (Allard et al . 1995, Laguzzi et al . 1996) . These actions may be mediated peripherally, centrally, or both.
  • agents acting at NPFF receptors may be of value in the treatment of hypertension or hypotension.
  • Described herein are unique sulfonamido-peptidomimetic ligands which are either agonists and/or antagonists at one or more NPFF receptor subtypes . Also described herein are quinazolino- and quinoiino-guanidine containing compounds that are the first known small molecule (non- peptide/non-peptoid) ligands (either agonists and/or antagonists) at the neuropeptide NPFF1 and NPFF2 receptors .
  • NPFF agonists and/or antagonists have great potential as being therapeutically useful agents for the treatment of a diverse array of clinically relevant human disorders.
  • NPFF agonists may have therapeutic potential, among others, for the treatment of pain, memory loss, circadian rhythm disorders, and micturition disorders.
  • Cloned receptor subtypes of NPFF and the development of high-efficiency in vitro assays, both for binding and receptor activation, has aided the discovery and development of novel NPFF ligands in our hands.
  • it is practically possible to design a molecule that is an agonist at one NPFF subtype, and an antagonist at the other (s) . This concept of a dual-acting molecule provides an attractive means of designing drugs that can treat multiple disorders.
  • These molecules may be used by themselves as drugs or as valuable tools for the design of drugs for the treatment of various clinical abnormalities in a subject wherein the abnormality is alleviated by increasing or decreasing the activity of a mammalian NPFF receptor which comprises administering to the subject an amount of a compound which is an antagonist or agonist of mammalian NPFF receptors to effect a treatment of the abnormality.
  • the abnormality can be a lower urinary tract disorder, such as interstitial cystitis or urinary incontinence, such as urge incontinence or stress incontinence particularly urge incontinence, a regulation of a steroid hormone disorder, an epinephrine release disorder, a gastrointestinal disorder, irritable bowel syndrome, a cardiovascular disorder, an electrolyte balance disorder, diuresis, hypertension, hypotension, diabetes, hypoglycemia, a respiratory disorder, asthma, a reproductive function disorder, an immune disorder, an endocrine disorder, a musculoskeletal disorder, a neuroendocrine disorder, a cognitive disorder, a memory disorder, a sensory modulation and transmission disorder, a motor coordination disorder, a sensory integration disorder, a motor integration disorder, a dopaminergic function disorder, an appetite disorder, an eating disorder, obesity, a serotonergic function disorder, an olfaction disorder, nasal congestion, a sympathetic innervation disorder, an affective disorder, pain, psychotic behavior, morph
  • the present invention provides a method of treating pain in a subject which comprises administering to the subject an amount of a compound effective to treat pain in the subject, wherein the compound binds to a NPFF1 receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor.
  • the invention also provides a method of treating a urinary disorder in a subject which comprises administering to the subject an amount of a compound effective to treat the urinary disorder in the subject, wherein the compound binds to a NPFF1 receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor.
  • the present invention further provides a method of treating pain in a subject which comprises administering to the subject an amount of a compound effective to treat pain in the subject, wherein the compound binds to a NPFF2 receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF1 receptor.
  • the invention also provides a method of treating a urinary disorder in a subject which comprises administering to the subject an amount of a compound effective to treat the urinary disorder in the subject, wherein the compound binds to a NPFF2 receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF1 receptor.
  • Figure 1A-1B Correlation between binding affinities at human and rat recombinant Neuropeptide ⁇ FF (NPFFl and NPFF2) receptors.
  • the binding affinities (pKi values) for 18 compounds were tested at rat NPFF (rNPFF) receptors and plotted against the pKi values for the same 18 compounds tested at human NPFF (hNPFF) receptors.
  • Figure 2 Effect of compound 4006A on bladder activity in the anesthetized rat. Rhythmic elevations in bladder pressure, resulting from distension induced contractions, were unaffected by i.v. administration of physiological saline. In contrast, the NPFF receptor ligand compound 4006A produced immediate inhibition of bladder activity, which persisted for 12 min.
  • Figure 3 Effect of compound 4005A on bladder activity in the anesthetized rat. Rhythmic elevations in bladder pressure, resulting from distension induced contractions, were unaffected by i.v. administration of physiological saline. In contrast, the NPFF receptor ligand compound 4005A produced immediate inhibition of bladder activity, which persisted for 35 min. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a method of treating pain in a subject which comprises administering to the subject an amount of a compound effective to treat pain in the subject, wherein the compound binds to a NPFFl receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor.
  • the compound binds to the NPFFl receptor with a binding affinity greater than 25-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor. In a further embodiment, the compound binds to the NPFFl receptor with a binding affinity greater than 50-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor.
  • the invention also provides a method of treating a urinary disorder in a subject which comprises administering to the subject an amount of a compound effective to treat the urinary disorder in the subject, wherein the compound binds to a NPFFl receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor.
  • the urinary disorder is urinary incontinence.
  • the urinary incontinence is urge incontinence or stress incontinence.
  • the urinary disorder is urinary retention.
  • the compound binds to the NPFFl receptor with a binding affinity greater than 25-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor. In a further embodiment, the compound binds to the NPFFl receptor with a binding affinity greater than 50-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor .
  • the invention further provides a method of treating an abnormality mediated by a NPFFl receptor in a subject which comprises administering to the subject an amount of a compound effective to treat the abnormality in the subject, wherein the compound binds to the NPFFl receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor.
  • the abnormality is an eating disorder, obesity, a psychotic disorder, anxiety, a learning disorder, a memory disorder, an electrolyte balance disorder, diuresis, diabetes, an intestinal motility disorder, irritable bowel syndrome, nicotine addiction, or a cardiovascular disorder.
  • the abnormality is a lower urinary tract disorder, interstitial cystitis, a steroid hormone disorder, an epinephrine release disorder, a gastrointestinal disorder, hypoglycemia, a respiratory disorder, asthma, a reproductive function disorder, an immune disorder, an endocrine disorder, a musculoskeletal disorder, a neuroendocrine disorder, a cognitive disorder, a sensory modulation and transmission disorder, a motor coordination disorder, a sensory integration disorder, a motor integration disorder, a dopaminergic function disorder, an appetite disorder, a serotonergic function disorder, an olfaction disorder, nasal congestion, a sympathetic innervation disorder, an affective disorder, morphine tolerance, opiate addiction, or migraine.
  • the compound binds to the NPFFl receptor with a binding affinity greater than 25-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor. In a further embodiment, the compound binds to the NPFFl receptor with a binding affinity greater than 50-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor .
  • the subject is a human being and the NPFFl receptor is the human NPFFl receptor and the NPFF2 receptor is the human NPFF2 receptor.
  • the compound is an agonist at the NPFFl receptor and an agonist at the NPFF2 receptor. In one embodiment of any of the methods described herein, the compound is an antagonist at the NPFFl receptor and an antagonist at the NPFF2 receptor. In one embodiment of any of the methods described herein, the compound is an agonist at the NPFFl receptor and an antagonist at the NPFF2 receptor. In one embodiment of any of the methods described herein, the compound is an antagonist at the NPFFl receptor and an agonist at the NPFF2 receptor.
  • the compound binds to the human NPFFl receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to each of a human ⁇ 1A adrenoceptor, a human ⁇ 1B adrenoceptor, and a human 1D adrenoceptor.
  • the compound binds to the human NPFFl receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to each of a human -,- adrenoceptor, a human ⁇ 2B adrenoceptor and a human ⁇ , c adrenoceptor.
  • the compound binds to the human NPFFl receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human dopamine D-, receptor.
  • the compound binds to the human NPFFl receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human histamine H 2 receptor.
  • the compound binds to the human NPFFl receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human NMDA receptor.
  • the compound binds to the human NPFFl receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human norepinephrine transporter or to a human serotonin transporter .
  • the compound binds to the human NPFFl receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to each of a human neuropeptide Yl receptor, a human neuropeptide Y2 receptor, a human neuropeptide Y4 receptor, and a human neuropeptide Y5 receptor.
  • the invention also provides a method of treating pain in a subject which comprises administering to the subject an amount of a compound effective to treat pain in the subject, wherein the compound binds to a NPFF2 receptor with a binding- affinity greater than • ten-fold higher than the binding affinity with which the compound binds to a NPFFl receptor.
  • the ' compound binds to the NPFF2 receptor with a binding affinity greater than 25-fold higher than the binding affinity with which the compound binds to a NPFFl receptor. In a further embodiment, the compound binds to the NPFF2 receptor with a' binding affinity greater than 50-fold higher than the binding affinity with which the compound binds to a NPFFl receptor.
  • the invention also provides a method of treating a urinary disorder in a subject which comprises administering to the subject an amount of a compound effective to treat the urinary disorder in the subject, wherein the compound binds to a NPFF2 receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFFl receptor.
  • the urinary disorder is urinary incontinence.
  • the urinary incontinence is urge incontinence or, stress incontinence.
  • the urinary disorder is urinary retention.
  • the compound binds to the NPFF2 receptor with a binding affinity greater than 25-fold higher than the binding affinity with which the compound binds to a NPFFl receptor. In a further embodiment, the compound binds to the NPFF2 receptor with a binding affinity greater than 50-fold higher than the binding affinity with which the compound binds to a NPFFl receptor .
  • the invention further provides a method of treating an abnormality mediated by a NPFF2 receptor in a subject which comprises administering to the subject an amount of a compound effective to treat the abnormality in the subject, wherein the compound binds to the NPFF2 receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a NPFF2 receptor.
  • the abnormality is an eating disorder, obesity, a psychotic disorder, anxiety, a learning disorder, a memory disorder, an electrolyte balance disorder, diuresis, diabetes, an intestinal motility disorder, irritable bowel syndrome, nicotine addiction, or a cardiovascular disorder.
  • the abnormality is a lower urinary tract disorder, interstitial cystitis, a steroid hormone disorder, an epinephrine release disorder, a gastrointestinal disorder, hypoglycemia, a respiratory disorder, asthma, a reproductive function disorder, an immune disorder, an endocrine disorder, a musculoskeletal disorder, a neuroendocrine disorder, a cognitive disorder, a sensory modulation and transmission disorder, a motor coordination disorder, a sensory integration disorder, a motor integration disorder, a dopaminergic function disorder, an appetite disorder, a serotonergic function disorder, an olfaction disorder, nasal congestion, a sympathetic innervation disorder, an affective disorder, morphine tolerance, opiate addiction, or migraine.
  • the compound binds to the NPFF2 receptor with a binding affinity greater than 25-fold higher than the binding affinity with which the compound binds to a NPFFl receptor. In a further embodiment, the compound binds to the NPFF2 receptor with a binding affinity greater than 50-fold higher than the binding affinity with which the compound binds to a NPFFl receptor.
  • the subject is a human being and the NPFFl receptor is the human NPFFl receptor and the NPFF2 receptor is the human NPFF2 receptor.
  • the compound is an agonist at the NPFFl receptor and an agonist at the NPFF2 receptor. In one embodiment, the compound is an antagonist at the NPFFl receptor and an antagonist at the NPFF2 receptor. In one embodiment, the compound is an agonist at the NPFFl receptor and an antagonist at the NPFF2 receptor. In one embodiment, the compound is an antagonist at the NPFFl receptor and an agonist at the NPFF2 receptor.
  • the compound binds to the human NPFF2 receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to each of a human a 1? adrenoceptor, a human ⁇ 1Er adrenoceptor, and a human ⁇ 1D adrenoceptor.
  • the compound binds to the human NPFF2 receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to each of a human _- adrenoceptor, a human P adrenoceptor and a human ⁇ : adrenoceptor .
  • the compound binds to the human NPFF2 receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human dopamine D receptor.
  • the compound binds to the human NPFF2 receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human histamine Hj receptor.
  • the compound binds to the human NPFF2 receptor with a
  • binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human NMDA receptor.
  • the compound binds to the human NPFF2 receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to a human norepinephrine transporter or to a human serotonin transporter .
  • the compound binds to the human NPFF2 receptor with a binding affinity at least 10-fold higher than the binding affinity with which the compound binds to .each of a human neuropeptide Yl receptor, a human neuropeptide Y2 receptor, a human neuropeptide Y4 receptor, and a human neuropeptide Y5 receptor.
  • the compound binds to a NPFF receptor with a binding affinity greater than 10-fold higher than the binding affinity with which it binds to any of the non- NPFF receptors described herein. In further embodiments of any of the methods described herein, the compound binds to a NPFF receptor with a binding affinity greater than 10-fold higher than the binding affinity with which it binds to a human norepinephrine transporter or to a human serotonin transporter. Examples of the binding characteristics of such compounds are shown in Table 8.
  • enantiomers, diastereomers and double bond regioisomers and stereoisomers exist. This invention contemplates racemic mixtures of compounds as well as isolated enantiomers. This invention also contemplates mixtures of diastereomers, double bond regioisomers or stereoisomers as well as isolated diastereomers or double bond regioisomers or stereoisomers.
  • the small molecule compounds disclosed herein are the first known (non-peptide/non-peptoid) ligands (either antagonists or agonists) at the neuropeptide FF(NPFF) receptor (s) .
  • agonist is used throughout this application to indicate a compound which increases the activity of any of the receptors of the subject invention.
  • antagonist is used throughout this application to indicate a compound which binds to, but does not increase the activity of, any of the receptors of the subject invention.
  • the activity of a G-protein coupled receptor such as the polypeptides disclosed herein may be measured using any of a variety of functional assays in which activation of the receptor in question results in an observable change in the level of some second messenger system, including, but not limited to, adenylate cyclase, calcium mobilization, arachidonic acid release, ion channel activity, inositol phospholipid hydrolysis or guanylyl cyclase.
  • Heterologous expression systems utilizing appropriate host cells to express the nucleic acid of the subject invention are used to obtain the desired second messenger coupling. Receptor activity may also be assayed in an oocyte expression system.
  • pharmaceutically acceptable carrier means any of the standard pharmaceutically acceptable carriers. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions.
  • the formulations of the present invention can be solutions, suspensions, emulsions, syrups, elixirs, capsules, tablets, and the like.
  • the compositions may contain a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, or the like.
  • the formulations can also be lyophilized, and/or may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "Remington's Pharmaceutical Science", 17th Ed., 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
  • the formulations can include powdered carriers, such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Further, tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. The formulations can also contain coloring and flavoring to enhance patient acceptance. The formulations can also include any of disintegrants, lubricants, plasticizers, colorants, and dosing vehicles.
  • water a suitable oil, saline, aqueous dextrose (glucose) , and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration contain preferably a water soluble salt of the active ingredient, suitable stabilizing agents, and, if necessary, buffer substances.
  • Antioxidants such as, for example, sodium bisulfate, sodium sulfite, citric acid and its salts, sodium EDTA, ascorbic acid, and the like can be used either alone or in combination with other suitable antioxidants or stabilizing agents typically employed in the pharmaceutical compositions.
  • parenteral solutions can contain preservatives, such as, for example, benzalkonium chloride, methyl- or propyl-paraben, chlorobutanol and the like.
  • terapéuticaally effective amount means that amount of a compound that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease, disorder, or abnormality being treated.
  • subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
  • compositions In order for a composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine the toxicity in a suitable animal model; the dosage of the composition (s) , and the concentration of components in the composition; and the timing of administration in order to maximize the response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, the present disclosure and the documents cited herein.
  • the present invention includes within its scope prodrugs of the compounds of this inventions.
  • prodrugs will be functional derivatives of the compounds of the invention which are readily convertible in vivo into the required compound.
  • a prodrug of the quinazolino- and quinolino-guanidines may have an acyl group attached to any of the three nitrogens of the guanidine, forming an N-acyl guanidine.
  • administering shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient.
  • Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985. Included in this invention are pharmaceutically acceptable salts and complexes of all of the compounds described herein.
  • the salts include, but are not limited to, the following acids and bases: Inorganic acids which include hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and boric acid; organic acids which include acetic acid, trifluoroacetic acid, formic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzoic acid, glycolic acid, lactic acid, and mandelic acid; inorganic bases include ammonia and hydrazine; and organic bases which include methylamine, ethylamine, hydroxyethylamine, propylamine, dimethylamine, diethylamine , t rime t hylamine , triethylamine, ethylenediamine, hydroethylamine, morpholine, piperazine, and guanidine.
  • This invention further provides for the hydrates and polymorphs of all of the compounds described herein.
  • the present invention further includes metabolites of the compounds of the present invention.
  • Metabolites include active species produced upon introduction of compounds of this invention into the biological milieu.
  • MOPAC Mated Oligonucleotide Primed Amplification of cDNA lOOng of rat genomic DNA (Clonetech, Palo Alto, CA) was used for degenerate MOPAC PCR using Taq DNA polymerase (Boehringer-Mannheim, Indianapolis, IN) and the following degenerate oligonucleotides: JAB126, designed based on an alignment of the sixth transmembrane domain of more than 180 members of the rhodopsin superfamily of G protein-coupled receptors; and JAB108, designed based on an alignment of the seventh transmembrane domain of the same rhodopsin superfamily.
  • the conditions for the MOPAC PCR reaction were as follows : 3 minute hold at 94°C; 10 cycles of 1 minute at 94°C, 1 minute 45 seconds at 44°C, 2 minutes at 72°C; 30 cycles of 94°C for 1 minute, 49°C for 1 minute 45 seconds, 2 minutes at 72 °C; 4 minute hold at 72 °C; 4°C until ready for agarose gel electrophoresis.
  • the products were run on a 1% agarose TAE gel and bands of the expected size ( ⁇ 150bp) were cut from the gel, purified using the QIAQUICK gel extraction kit (QIAGEN, Chatsworth, CA) , and subcloned into the TA cloning vector (Invitrogen, San Diego, CA) .
  • White (insert-containing) colonies were picked and subjected to PCR using pCR2.1 vector primers JABl and JAB2 using the Expand Long Template PCR System and the following protocol: 94 °C hold for 3 minutes; 35 cycles of 94°C for 1 minute, 68 °C for 1 minute 15 seconds ; 2 minute hold at 68 °C, 4°C hold until products were ready for purification.
  • PCR products were purified by isopropanol precipitation (10 ⁇ l PCR product, 18 ⁇ l low TE, 10.5 ⁇ l 2M NaClO, and 21.5 ⁇ l isopropanol) and sequenced using the ABI Big Dye cycle sequencing protocol and ABI 377 sequencers (ABI, Foster City, CA) . Nucleotide and amino acid sequence analyses were performed using the Wisconsin Package (GCG, Genetics Computer Group, Madison, WI) .
  • GCG Genetics Computer Group, Madison, WI
  • MPR3-RGEN-31 and MPR3-RGEN-45 Two PCR products produced from rat genomic cDNA (MPR3-RGEN-31 and MPR3-RGEN-45) were determined to be identical clones of a novel G protein-coupled receptor-like sequence based on database searches and its homology to other known G protein-coupled receptors ( ⁇ 30-40% amino acid identity to dopamine D2 , orexin, galanin, angiotensin 1 and 5-HT 2b receptors) .
  • This novel sequence was designated SN0RF2.
  • This insert is about 2.8 kb in length with an approximately 200 bp 5' untranslated region, a 1296 bp coding region, and a 1.3 kb 3 ' untranslated region.
  • the clone is also in the correct orientation for expression in the mammalian expression vector pEXJ.T7.
  • This construct of SNORF2 in pEXJ.T7 was designated BN-6.
  • the full length SNORF2 was determined to be most like the orexin 1 receptor (45% DNA identity, 35% amino acid identity) , orexin 2 receptor (40% DNA identity, 32% amino acid identity), and NPY2 receptor (47% DNA identity, 29% amino acid identity) , although several other G protein-coupled receptors also displayed significant homology.
  • NPFFl receptor gene The full-length, intronless version of the human NPFFl receptor gene may be isolated using standard molecular biology techniques and approaches such as those briefly described below:
  • the full-length sequence may be obtained by sequencing this cosmid clone with additional sequencing -primers. Since at least two introns are present in this gene, one in the amino terminus and one just after the third transmembrane domain, the full-length intronless gene may be obtained from cDNA using standard molecular biology techniques. For example, a forward PCR primer designed in the 5'UT and a reverse PCR primer designed in the 3'UT may be used to amplify a full-length, intronless gene from cDNA. RT-PCR localization has identified several human tissues which could be used for this purpose, including cerebellum, spinal cord, hippocampus, lung and kidney. Standard molecular biology techniques could be used to subclone this gene into a mammalian expression vector.
  • Approach #2 Standard molecular biology techniques could be used to screen commercial human cDNA phage libraries by hybridization under high stringency with a J2 P-labeled oligonucleotide probe, BB609, corresponding to the 2/3 loop of the PLC29b clone.
  • BB609 J2 P-labeled oligonucleotide probe
  • RNAFFl sequence BB629, forward primer in TMI , and A71, reverse primer in TMIV.
  • a full-length clone could be isolated by Southern hybridization of colony lifts of positive pools with a 32 P-labeled oligonucleotide probe, BB609, corresponding to the 2/3 loop of the PLC29b clone.
  • Approach #3 As yet another alternative method, one could utilize 3' and 5' RACE to generate PCR products from human cDNA expressing human NPFFl (for example, cerebellum, spinal cord, hippocampus, lung and kidney), which contain the additional sequences of human NPFFl.
  • human cDNA expressing human NPFFl for example, cerebellum, spinal cord, hippocampus, lung and kidney
  • a reverse primer derived from PLC29b between the amino terminus and TM IV could be used to amplify the additional amino terminus sequence for hNPFFl .
  • a forward primer derived from PLC29b between the amino terminus and TM IV could be used to amplify the additional 3' sequence for hNPFFl, including TMs 5-7 and the COOH terminus.
  • RACE PCR product could then be sequenced to determine the missing sequence.
  • This new sequence could then be used to design a forward PCR primer in the 5 ' UT and a reverse primer in the 3 ' UT .
  • These primers could then be used to amplify a full-length hNPFFl clone from human cDNA sources known to express NPFFl (for example, cerebellum, spinal cord, hippocampus, lung and kidney) . Additional details can be found in PCT International Publication No. WO 00/18438, the disclosure of which is hereby incorporated by reference in its entirety into this application. Clonin ⁇ of human NPFFl receptor
  • hNPFFl human NPFFl receptor from the initiating methionine to TMIV was determined to be present in .
  • plc29b a partial clone, found in a Synaptic Pharmaceutical Corporation in-house database.
  • a human cosmid library (Stratagene) was screened with a 32 P-labeled probe (BB609) corresponding to the II/III loop of plc29b. Partial DNA sequencing of one positive clone from this library, COS28a revealed similar sequence as had been previously shown for plc29b, with an intron downstream of TMIII.
  • COS28a was amplified with a vector primer and BB702, BB703 or BB704, forward primers ' in ' TMIV. DNA sequencing of these PCR products resulted in the identification of TMIV through the stop codon.
  • an in-house human spinal cord library was screened by PCR using a forward primer in the region of the initiating methionine (BB729) and a reverse primer corresponding to TMIV (BB728) .
  • BB729 initiating methionine
  • BB728 reverse primer corresponding to TMIV
  • W4 One positive pool, W4 , was subdivided and a positive sub-pool was screened by colony hybridization with a 32 P- labeled probe from TMII, BB676.
  • Plasmid DNA was isolated for clone W4-18-4, renamed B098, and DNA sequencing revealed that it was full-length but in the wrong orientation for expression in the expression vector pEXJ.
  • B098 was amplified with BB757, a forward primer at the initiating methionine which contained an upstream BamHI site, and BB758, a reverse primer at the stop codon which contained a EcoRI site.
  • the products from 3 independent PCR reactions were ligated into pcDNA3.1+ and transformed into DH5 ⁇ cells.
  • the sequence of one of these transformants, 3.3 was identical to the hNPFFl sequence previously determined from the consensus of B098, C0S28a and plc29b. Clone 3.3 was renamed BO102.
  • the hNPFFl clone contains an open reading frame with 1293 nucleotides and predicts a protein of 430 amino acids. Hydrophobicity analysis reveals seven hydrophobic domains which are presumed to be transmembrane domains. The sequence of hNPFFl was determined to be most similar to the rat NPFFl (86% nucleotide identity, 87% amino acid identity) and human NPFF2 (56% nucleotide identity, 49% amino acid identity. The human NPFFl receptor also shares
  • rNPFFl rat NPFFl
  • EST accesion number AA449919
  • AA449919 is a 532 bp sequence annotated in Genbank as "Soares total fetus Nb2HF8 9w Homo sapiens cDNA clone 788698 5' similar to SW:NYR DROME P25931 NEUROPEPTIDE Y RECEPTOR," which when translated corresponds to the region between the first extracellular loop and the beginning of the sixth transmembrane domain of rNPFFl.
  • AA449919 GAP analysis of AA449919 with rNPFFl indicated that there is 57% DNA identity and a 50% amino acid identity between the two receptor sequences over this region.
  • AA449919 displays 60% DNA identity and 59% amino acid identity over the region that overlaps with the known sequence for hNPFFl (first extracellular loop to TM4) , while over the same range rNPFFl is 62% and 61% identical to AA449919 at the DNA and amino acid levels, respectively.
  • hNPFFl and rNPFFl share 86% DNA identity and 92 % amino acid identity over this region.
  • NPFF-like NPFF2
  • RACE Rapid Amplification of cDNA ends
  • 5 ⁇ l template human spleen Marathon-Ready cDNA was amplified with oligonucleotide primers JAB256 and API, the Expand Long DNA Template PCR System (Boehringer-Mannheim, Indianapolis, IN) and the following PCR protocol were used: 94°C hold for 3 mintites; 5 cycles of 94°C for 30 seconds, 72°C for 4 minutes; 5 cycles of 94°C for 30 seconds, 70°C for 4 minutes; 30 cycles of 94°C for 30 seconds, 68°C for 4 minutes; 68°C hold for 4 minutes; 4°C hold until products were ready to be loaded on a gel.
  • hNPFF2 human spinal cord cDNA was amplified in eight independent PCR reactions using the Expand Long Template PCR System with buffer I (four of the eight reactions) or buffer 3 (4 reactions) and two oligonucleotide primers with restriction sites incorporated into their 5' ends: BB675 is a forward primer upstream of the initiating methionine and contains a BamHI site, and BB663.
  • the PCR conditions for this reaction were as follows: 9 °C hold for 5 minutes; 37 cycles of 9 °C for 30 seconds, 64°C for 30 seconds, 68 °C for 2 minutes; a 7 minute hold at 68°C, and a 4°C hold until products were ready to be loaded on a gel .
  • the products were electrophoresed on a 1% agarose TAE gel, and a band of approximately 1.35 kb was cut and purified using the QIAQUICK gel extraction kit.
  • the purified bands of seven of the eight reactions were cut with BamHI and EcoRI, gel purified again using the same method, and ligated into pcDNA3.1(+) (Invitrogen, Carlsbad, CA) . Eighteen colonies from the subsequent transformations were picked and determined to be positive for NPFF-like by PCR. Eight of these 18 clones were fully sequenced, and one of these,
  • NPFF-like in oocytes For expression of NPFF-like in oocytes, one ul of each of these eight ligations of the BB675-BB663 PCR product into pcDNA3.1(+) was subjected to PCR with AN35, a pcDNA3.1 primer at the CMV promoter site, and the 3' NPFF-like primer BB663 using the Expand Long Template PCR System and the following PCR protocol: 94 °C hold for 3 minutes; 37 cycles of 94°C for 30 seconds, 65°C for 30 seconds, 68°C for 2 minutes; a 7 minute hold at 68 °C, and a 4°C hold until products were ready for in vitro transcription. Of the seven PCR reactions, six yielded products of the expected size.
  • mRNA transcripts were generated as described for NPFFl, using PCR products from ligation reactions or linearized DNA from B089 as DNA templates. Oocytes were injected with 5-50 ng NPFF2 mRNA and incubated as previously described.
  • rat genomic DNA (Clontech, Palo Alto, CA)
  • rat hypothalamic cDNA or rat spinal cord cDNA was amplified with a forward PCR primer corresponding to TMIV of human NPFF2 (JAB307) and a reverse primer corresponding to TMVI of human NPFF2 (JAB 306) .
  • PCR was performed with the Expand Long Template PCR System (Roche Molecular Biochemicals, Indianapolis, IN) under the following conditions: 1 minute at 94°C, 2 minutes at 50°C, 2 minutes at 68°C for 40 cycles, with a pre- and post-incubation of 3 minutes at 94°C and 4 minutes at 68°C respectively.
  • RNA sequence for rat NPFF2 was obtained using primers designed against the human NPFF2 NH 2 and COOH termini along with PCR primers designed against the rat NPFF2 fragment.
  • PCR was performed on rat spinal cord cDNA with BB665, a sense primer just upstream of TMI in human NPFF2, and BB795, an antisense primer in the second extracellular loop of the rat NPFF2.
  • PCR was performed on rat spinal cord cDNA with BB793, a sense primer from the third intracellular loop in rat NPFF2 , and BB668, an antisense primer just downstream from TMVII in human NPFF2.
  • PCR was performed using the Expand Long Template PCR System (Roche Biochemicals, Indianapolis, IN) with buffer 2 (NH 2 terminal) or buffer 1 (COOH terminal) and the following conditions: 30 seconds at 94°C, 30 seconds at 42°C (NH 2 terminal) or 50°C (COOH terminal), 1.5 minutes at 68°C for 40 cycles, with a pre- and post-incubation of 3 minutes at 94''C and 4 minutes at 68°C respectively.
  • a 500 bp band from the NH 2 terminal PCR and a 300 bp band from the COOH terminal PCR were isolated from a TAE gel, purified using the QIAQUICK gel extraction kit (QIAGEN, Chatsworth, CA) , and sequenced on both strands as described above.
  • a rat • liver genomic phage library (2.75 million recombinants, Stratagene, LaJolla, CA) was screened using a ";: P-labeled oligonucleotide probe, BB712 , corresponding to the second extracellular loop and TMV of the rat NPFF2 fragment above.
  • Hybridization of nitrocellulose filter overlays of the plates was performed at high stringency: 42"C in a solution containing 50% formamide, 5x SSC (IX SSC is 0.15M sodium chloride, 0.015M sodium citrate), lx Denhardt ' s solution (0.02% polyvinylpyrrolindone, 0.02% Ficoll, 0.02% bovine serum albumin), 7 mM Tris and 25 ⁇ g/ml sonicated salmon sperm DNA.
  • the filters were washed at 55°C in 0. lx SSC containing 0.1% sodium dodecyl sulfate and exposed at -70°C to Kodak BioMax MS film in the presence of an intensifying screen.
  • rNPFF2-l, rNPFF2-4 and rNPFF2-6 were isolated on a tertiary plating.
  • Sequencing with HK137, a sense primer from TMV of the rat NPFF2 fragment revealed the sequence for TMVII, the COOH terminus and some 3 ' UT .
  • Sequencing with HK139, an antisense primer from TMII of rNPFF2 revealed the presence an intron upstream of TMII.
  • NPFF2 The full-length NPFF2 was amplified from rat spinal cord cDNA using a sense primer in the 5 ' UT (HK146, also incorporating a BamHI restriction site) and an antisense primer from the 3 ' UT (HK147, also incorporating a Bs tXI restriction site) and the Expand Long Template PCR System (Roche Molecular Biochemicals, Indianapolis, IN) using buffer 2 and the following PCR conditions: 30 seconds at 94"C, 2.5 minutes at 68°C for 32 cycles, with a pre- and post-incubation of 5 minutes at 94°C and 7 minutes at 68"C, respectively. Products from 5 independent PCR reactions were gel-purified. 1 ⁇ l of each reaction was used as a template to re-amplify the product using the same PCR conditions. The products were digested with BamHI and BstXI and ligated into a modified pcDNA3.1 vector
  • COS-7 cells are grown on 150 mm plates in DMEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% C0 2 .
  • Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4 days .
  • Human embryonic kidney 293 cells (HEK-293 cells) are grown on 150 mm plates in DMEM with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% C0 2 . Stock plates of 293 cells are trypsinized and split 1:6 every 3-4 days.
  • Mouse fibroblast LM(tk-) cells are grown on 150 mm plates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% C0 .
  • Stock plates of LM(tk-) cells are trypsinized and split 1:10 every 3-4 days.
  • CHO cells Chinese hamster ovary (CHO) cells were grown on 150 mm plates in HAM's F-12 medium with supplements (10% bovine calf serum, 4 mM L-glutamine and 100 units/ml penicillin/ 100 ug/ml streptomycin) at 37°C, 5% C0 2 . Stock plates of CHO cells are trypsinized and split 1:8 every 3-4 days.
  • Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm plates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% C02. Stock plates of NIH-3T3 cells are trypsinized and split 1:15 every 3-4 days.
  • DMEM Dulbecco's Modified Eagle Medium
  • Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue culture dishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C, no C0 2 .
  • High Five insect cells are grown on 150 mm tissue culture dishes in Ex-Cell 400TM medium supplemented with L-Glutamine, also at 27°C, no CO, .
  • Transient transfection Receptors studied may be transiently transfected into COS-7 cells by the DEAE-dextran method using 1 ⁇ g of DNA /10 ,J cells (Cullen, 1987) .
  • Schneider 2 Drosophila cells may be cotransfected with vectors containing the receptor gene under control of a promoter which is active in insect cells, and a selectable resistance gene, eg., the G418 resistant neomycin gene, for expression of the polypeptides disclosed herein.
  • Stable transfection DNA encoding the human receptors disclosed herein may be co-transfected with a G-418 resistant gene into the human embryonic kidney 293 cell line by a calcium phosphate transfection method (Cullen, 1987) . Stably transfected cells are selected with G-418.
  • LM(tk-) cells stably transfected with the DNA encoding the human receptor disclosed herein may be routinely converted from an adherent monolayer to a viable suspension.
  • Adherent cells are harvested with trypsin at the point of confluence, resuspended in a minimal volume of complete DMEM for a cell count, and further diluted to a concentration of 10 6 cells/ml in suspension media (10% bovine calf serum, 10% 10X Medium 199 (Gibco) , 9 mM NaHC0 3 , 25 mM glucose, 2 mM L-glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin, and 0.05% methyl cellulose) .
  • Cell suspensions are maintained in a shaking incubator at 37°C, 5% • C0 2 for 24 hours.
  • Membranes harvested from cells grown in this manner may be stored as large, uniform batches in liquid nitrogen.
  • cells may be returned to adherent cell culture in complete DMEM by distribution into 96-well microtiter plates coated with poly-D-lysine (0.01 mg/ml) followed by incubation at 37°C, 5% C0 2 for 24 hours.
  • the coding region of DNA encoding the human receptors disclosed herein may be subcloned into pBlueBacIII into existing restriction sites or sites engineered into sequences 5 ' and 3 ' to the coding region of the polypeptides.
  • 0.5 ⁇ g of viral DNA (BaculoGold) and 3 ⁇ g of DNA construct encoding a polypeptide may be co-transfected into 2 x 10 6 Spodoptera frugiperda insect Sf9 cells by the calcium phosphate co-precipitation method, as outlined by Pharmingen (in "Baculovirus Expression Vector System: Procedures and Methods Manual") . The cells then are incubated for 5 days at 27°C.
  • the supernatant of the co-transfection plate may be collected by centrifugation and the recombinant virus plaque purified.
  • the procedure to infect cells with virus, to prepare stocks of virus and to titer the virus stocks are as described in Pharmingen' s manual.
  • Cells may be screened for the presence of endogenous human receptor using radioligand binding or functional assays . Cells with either no or a low level of the endogenous human receptors disclosed herein present may be transfected with the human receptors.
  • Transfected cells from culture flasks are scraped into 5 ml of 20 mM Tris-HCl, 5mM EDTA, pH 7.5, and lysed by sonication.
  • the cell lysates are centrifuged at 1000 rpm for 5 min. at 4°C, and the supernatant is centrifuged at 30,000 x g for 20 min. at 4°C.
  • the pellet is suspended in binding buffer (50 mM Tris-HCl, 60 mM NaCl, 1 mM MgCl , 33 ⁇ M EDTA, 33 ⁇ M EGTA at pH 7.4 supplemented with 0.2% BSA, 2 ⁇ g/ml aprotinin, and 20 ⁇ M bestatin) .
  • Optimal membrane suspension dilutions defined as the protein concentration required to bind less than 10% of the added radioligand, are added to 96-well polpropylene microtiter plates containing J H-labeled compound, unlabeled compounds, and binding buffer to a final volume of 250 ⁇ l .
  • membrane preparations are incubated in the presence of increasing concentrations of [ 3 H] -labeled compound.
  • binding affinities of the different compounds are determined in equilibrium competition binding assays, using [ 125 I] -labeled compound in the presence of ten to twelve different concentrations of the displacing ligands.
  • Competition assay 50pM radioligand, 10 - 12 points. Binding reaction mixtures are incubated for 2 hr at 25 °C, and the reaction stopped by filtration through a double layer of GF filters treated with 0.1% polyethyleneimine, using a cell harvester. Wash buffer: 50mM Tris-HCl, 0.1% BSA. Radioactivity may be measured by scintillation counting and data are analyzed by a computerized non- linear regression program.
  • Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of l ⁇ M final concentration unlabeled. Protein concentration may be measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard.
  • Plasmids encoding the NPFF receptors have been deposited with the American Type Culture Collection (ATCC) , 10801 University Boulevard., Manassas, Virginia 20110-2209, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These plasmids comprise regulatory elements necessary for expression of DNA in a cell operatively linked to DNA encoding the NPFF receptor so as to permit expression thereof.
  • ATCC American Type Culture Collection
  • Plasmids pEXJ-rNPFFl and pWE15-hNPFFl were deposited on September 9, 1998, with the American Type Culture Collection (ATCC) , 10801 University Boulevard., Manassas, Virginia 20110-2209, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and were accorded ATCC Accession Nos. 203184 and 203183, respectively. Plasmid pCDNA3. l-hNPFF2b was deposited on September 22, 1998, with the American Type Culture Collection (ATCC) , 10801 University Boulevard., Manassas, Virginia 20110-2209, U.S.A.
  • Plasmid pcDNA3.1-hNPFFl was deposited on January 21, 1999, with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, Virginia 20110-2209, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Accession No. 203605. Plasmid pcDNA3.
  • NPFP receptors may be used for -the treatment of pain, lower urinary tract disorders, obesity, as well as other indications.
  • the design of such compounds can be optimized by determining their binding interactions at the native serotonin (5HT) and norepinephrine (NE) transporters.
  • the NPFF compound (s) would optimally not bind at the following receptors due to possible side effects: human ⁇ 1A adrenergic, human ⁇ 1B adrenergic, human ⁇ 1D adrenergic, human ⁇ 2A adrenergic, human 2B adrenergic, and human a 2c adrenergic receptors; human neuropeptide Y (NPY) Yl, Y2, Y4, and Y5 receptors; and the N-methyl-D-aspartate (NMDA) receptor channel complex.
  • NPY neuropeptide Y
  • NMDA N-methyl-D-aspartate
  • the binding properties of compounds at different receptors were determined using cultured cell lines that selectively express the receptor of interest.
  • Cell lines were prepared by transfecting the cloned cDNA or cloned genomic DNA or constructs containing both genomic DNA and cDNA encoding the receptors.
  • the methods to obtain the cDNA of the receptors, express said receptors in heterologous systems, and carry out assays to determine binding affinity are described herein below.
  • the binding interactions of compounds at different transporters were determined using tissue preparations and specific assays as described herein below.
  • LM(tk-) cell lines stably transfected with the genes encoding the la , lb , and ⁇ ld receptors were used.
  • the nomenclature describing the x receptors was changed recently, such that the receptor formerly designated ⁇ la is now designated ld , and the receptor formerly designated ⁇ lc is now designated ⁇ la .
  • the cell lines expressing these receptors were deposited with the ATCC before the nomenclature change and reflect the subtype designations formerly assigned to these receptors.
  • the cell line expressing the receptor described herein as the ⁇ la receptor was deposited with the ATCC on September 25, 1992, under ATCC Accession No. CRL 11140 with the designation L- ⁇ lc .
  • the cell line expressing receptor described herein as the ⁇ ld receptor was deposited with the ATCC on September 25, 1992, under ATCC Accession No. CRL 11138 with the designation L- ⁇ 1A .
  • the cell line expressing the ⁇ lb receptor is designated L- ⁇ 1B , and was deposited on September 25, 1992, under ATCC Accession No. CRL 11139.
  • Binding assays using the ⁇ 1A and ⁇ 1B adrenergic receptors may be carried out according to the procedures described in U.S. Patent No. 5,780,485, the disclosure of which is hereby incorporated by reference in its entirety into this application.
  • Binding assays for the human ⁇ 1D adrenergic receptor may be carried out according to- the procedures described in U.S. Patent No. 6,156,518, the disclosure of which is hereby incorporated by reference in its entirety into this application.
  • LM(tk-) cell lines stably transfected with the genes encoding the 2Ar ot 2B r and 2c receptors were used.
  • the cell line expressing the 2A receptor is designated L- ⁇ 2A , and was deposited on November 6, 1992, under ATCC Accession No. CRL ' 11180.
  • the cell line expressing the ⁇ 2B receptor is designated L-NGC- ⁇ 2B , and was deposited on October 25, 1989, under ATCC Accession No. CRL 10275.
  • the cell line expressing the ⁇ 2C receptor is designated L- ⁇ 2C , and was deposited on November 6, 1992, under ATCC Accession No.
  • CRL-11181 Cell lysates were prepared as described herin, and suspended in 25mM glycylglycine buffer (pH 7.6 at room temperature) . Equilibrium competition binding assay were performed using [ : 'H] rauwolscine (0.5nM), and nonspecific binding was determined by incubation with lO ⁇ M phentolamine . The bound radioligand was separated by filtration through GF/B filters using a cell harvester.
  • Binding assays using the ⁇ 2 adrenergic receptors may be carried out according to the procedures described in U.S. Patent No. 5,780,485, the disclosure of which is hereby incorporated by reference in its entirety into this application.
  • Human Histamine H ⁇ Receptor The coding sequence of the human histamine H x receptor, homologous to the bovine H ⁇ receptor, is obtained from a human hippocampal cDNA library, and is cloned into the eukaryotic expression vector pCEXV-3.
  • the plasmid DNA for the H x receptor is designated pcEXV-Hl, and was deposited on November 6, 1992 under ATCC Accession No. 75346. This construct is transfected into COS-7 cells by the DEAE-dextran method. Cells are harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM EDTA, pH 7.5.
  • the cell lysates are centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant is centrifuged at 30,000 x g for 20 min. at 4°C.
  • the pellet is suspended in 37.8 mM NaHP0 4 , 12.2 mM KH 2 P0 4 , pH 7.5.
  • the binding of the histamine H x antagonist [ ; ⁇ ]mepyramine (InM, specific activity: 24.8 Ci/mM) is done in a final volume of 0.25 ml and incubated at room temperature for 60 min. Nonspecific binding is determined in the presence of 10 ⁇ M mepyramine .
  • the bound radioligand is separated by filtration through GF/B filters using a cell harvester.
  • Human Dopamine D 2 Receptors The potency of compounds at the D2 receptor is determined using membrane preparations from COS-7 cells transfected with the gene encoding the human D 2 receptor.
  • the coding region for the human D2 receptor is obtained from a human striatum cDNA library, and cloned into the cloning site of PCDNA 1 eukariotic expression vector.
  • the plasmid DNA for the D 2 receptor is designated pcEXV-D2, and was deposited on November 6, 1992 under ATCC Accession No. ATC 75344. This construct is transfected into COS-7 cells by the DEAE-dextran method.
  • Cells are harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM EDTA, pH 7.5.
  • the cell lysates are centrifuged at 1000 rpm for 5 minutes at 4°C, and the supernatant is centrifuged at 30,000 x g for 20 minutes at 4°C.
  • the pellet is suspended in 50 mM Tris-HCl (pH 7.4) containing ImM EDTA, 5mM KCl, 1.5mM CaCl 2 , 4mM MgCl 2 , and 0.1% ascorbic acid.
  • the cell lysates are incubated with [3H] spiperone (2nM) , using lO ⁇ M (+) Butaclamol to determine nonspecific binding.
  • Neuropeptide receptors Stably transfected cell lines which may be used for binding experiments include, for the Yl receptor, 293-hYl-5 (deposited June 4, 1996, under ATCC
  • Binding assays using the NPY receptors may be carried out according to the procedures described in U.S. Patent No. 5,602,024, the disclosure of which is hereby incorporated by reference in its entirety into this application.
  • NMDA Receptor Channels The methods to determine binding affinity at native N-methyl-D-aspartate (NMDA) receptor channels are described in Wong E.H. et al . (1988), the disclosure of which is hereby incorporated by reference in its entirety into this application.
  • Transporters The binding properties of compounds were evaluated at native, tissue-derived transporters, namely serotonin (5HT) transporter and norepinephrine (NE) transporter, according to protocols described ' in Owens (1997), the disclosure of which is hereby incorporated by reference in its entirety into this application.
  • tissue-derived transporters namely serotonin (5HT) transporter and norepinephrine (NE) transporter
  • TLC Thin-layer chromatography
  • Method D In a flask equipped with a magnetic stirrer, a solution of 6-bromo-2-fluorobenzoic acid (l.OOg, 4.57 mmol) dissolved in anhydrous ethyl ether (7 mL) was cooled to -78°C using a dry ice-acetone bath. Methyl lithium was then added dropwise (6.8 mL of a 1.4 M solution in ethyl ether, 9.59 mmol) . The reaction was further stirred at -78°C for 5 min followed by warming to -r.t. by removing the dry ice- acetone bath.
  • Compound 4002A (class: Quinolino-guanidine; synthesized using Method J) . Name: N- ( 6-ethyl-4-methyl-2-quinolinyl) guanidine .
  • Compound 1007A (class: Quinazolino-guanidine; Purchased from Sigma) .
  • N- (4-methyl-2-quinolinyl) guanidine was made in the same manner as N- (6-ethyl-4-methyl-2-quinolinyl) guanidine (see Example 3) except that 2-chloro-4-methylquinoline was used in place of 2-chloro-6-ethyl-4-methylquinoline .
  • Compound 6001A (class: Quinolino-guanidine; synthesized using Method J (67% yield))
  • N- ( , 7-dimethyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2-quinolinyl) guanidine (see Example 3) except that 3-methylaniline was used in place of 4-ethylaniline .
  • Compound 4006A (Class: Quinolino-guanidine; synthesized using Method J (17% yield) )
  • N- (4-ethyl-7-methyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2-quinolinyl) guanidine
  • N- ( 4 , 8-dimethyl-2-quinolinyl) guanidine was made in the same manner as N- (6-ethyl-4-methyl-2-quinolinyl) guanidine (see Example 3) except that 2-chloro-4 , 8-dimethylquinoline was used in place of 2-chloro-6-ethyl-4-methylquinoline .
  • Compound 6002A class: Quinolino-guanidine; synthesized using Method J.(20% yield)
  • N- ( 6-chloro-4-methyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2- quinolinyl) guanidine (see Example 3) except that 2,6- dichloro-4-methylquinoline was used in place of 2-chloro- 6-ethyl-4-methylquinoline .
  • Compound 4005A (class: Quinolino-guanidine; synthesized using Method J (42-71% yield)).
  • N- (1-methylbenzo [f] quinolin-3-yl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2-quinolinyl) guanidine (see Example 3) except that 3-chloro-l- methylbenzo[f ] quinoline was used in place of 2-chloro-6- ethyl-4-methylquinoline .
  • N- ( 6-methoxy-4-methyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2- quinolinyl) guanidine (see Example 3) except that 2-chloro- 6-methoxy-4-methylquinoline was used in place of 2-chloro- 6-ethyl-4-methylquinoline .
  • Compound 4004A (class: Quinolino-guanidine; synthesized using Method J (13% yield) ) .
  • N- (4, 5, 7-trimethyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2-quinolinyl) guanidine (see Example 3) except that 3, 5-dimethylaniline was used in place of 4-ethylaniline.
  • Compound 4008A (class: Quinolino-guanidine; synthesized using Method J (7% yield) ) .
  • N- ( 4 , 5, 7-trimethyl-2-quinolinyl) guanidine N- ( 4 , 5, 7-trimethyl-2-quinolinyl) guanidine .
  • N- (4, 6-dimethyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2-quinolinyl) guanidine (see Example 3) except that 4-methylaniline was used in place of 4-ethylaniline.
  • Compound 4001A (class: Quinolino-guanidine; synthesized using Method J (5% yield) ) . Name: N- ( 4 , 6-dimethyl-2-quinolinyl) guanidine .
  • N- ( 4 -methyl- 6-phenyl-2-quinolinyl ) guanidine was made in t h e s ame manner a s N- ( 6-ethyl-4 -methyl-2 - quinolinyl) guanidine (see Example 3) except that 2-chloro- 4-methyl-6-phenylquinoline was used in place of 2-chloro- 6-ethyl-4-methylquinoline .
  • Compound 4003A (class: Quinolino-guanidine; synthesized using Method J (28% yield) ) .
  • N- (7-ethyl-4-methyl-2-quinazolinyl ) guanidine was made in the same manner as N- ( 6-ethyl- -methyl-2- quinolinyl) guanidine (see Example 3) except that 3- ethylaniline was used in place of 4-ethylaniline.
  • Compound 1020A (class: Quinazolino-guanidine; synthesized using Method C (52% yield) ) .
  • N- (7-fluoro-4-methyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2- quinolinyl) guanidine (see Example 3) except that 3- f luoroaniline was used in place of 4-ethylaniline.
  • Compound 4007A (class: Quinolino-guanidine; synthesized using Method J (36% yield) ) .
  • Compound 1002A (class: Quinazolino-guanidine).
  • N- ( 4 , 6-dimethyl-2-quinazolinyl) guanidine N- ( 4 , 6-dimethyl-2-quinazolinyl) guanidine .
  • Tripos A compound purchased from Tripos was found to have the wrong structure assignment and to contain an impurity. Tripos' incorrect structure assignment was 2- [(4,7- dimethyl-2-quinazolinyl) amino] -4-quinazolinol .
  • the sample was determined to be a mixture of ⁇ - (4 , 6-dimethyl-2-quinazolinyl) guanidine and methyl 2- aminobenzoate, which was separated by preparative TLC to afford pure ⁇ - ( 4, 6-dimethyl-2-quinazolinyl) guanidine .
  • N- ( 6, 7-difluoro-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1, steps B and C) except that 3, 4-difluoroaniline . was used in place of 3,4- dibutoxyaniline .
  • Compound 1019A (class: Quinolino-guanidine; synthesized using Method J (42% yield)).
  • N- (7-bromo-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 3- bromoaniline was used in place of 3, 4-dibutoxyaniline .
  • N- ( 6-bromo-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- bromoaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1026A (class: Quinazolino-guanidine; synthesized using Methods C (4% yield) ) . Name: N- ( 6-bromo-4-methyl-2-quinazolinyl) guanidine .
  • N- [4 -methyl-7- (trifluoromethoxy) -2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl ) guanidine (see Example 1) except that 3- trifluoromethoxyaniline was used in place of 3,4- dibutoxyaniline .
  • N- ( 6-chloro-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- chloroaniline- was used in place of 3, 4-dibutoxyaniline .
  • Compound 1013A ame N- ( 6-chloro-4-methyl-2-quinazolinyl ) guanidine
  • N- (6-methoxy-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- methoxyaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1011A (class: Quinazolino-guanidine; synthesized using Method C (13% yield)).
  • N- (7-isopropyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 3- isopropylaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1021A (class: Quinazolino-guanidine; synthesized using Method C (85%), except that reverse phase (C18) column chromatography eluting with acetonitrile was used in place of normal phase) .
  • N- [ -methyl-6- (trifluoromethoxy) -2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- trifluoromethoxyaniline was used in place of 3,4- dibutoxyaniline .
  • N- (4-methyl-6-pentyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- pentylaniline was used in place of 3, 4-dibutoxyaniline .
  • N- ( 4 , 6, 7-trimethyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2-
  • Compound 1015A (class: Quinazolino-guanidine; synthesized 15 using Method C (12% yield)).
  • N- ( 4 , 6, 7-trimethyl-2-quinazolinyl ) guanidine N- ( 4 , 6, 7-trimethyl-2-quinazolinyl ) guanidine .
  • N- [ 6- (benzyloxy) -4-methyl-2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- 5 benzyloxyaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1028A (class: Quinazolino-guanidine; synthesized using Method C (6% yield) ) .
  • N- [7- (1-hydroxyethyl) -4-methyl-2-quinazolinyl] guanidine was made in the same manner as N- (6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 3-(l- hydroxyethyl) aniline was used in place of 3,4- dibutoxyaniline .
  • N- ( 6-ethyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy ⁇ 4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- ethylaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1003A (class: Quinazolino-guanidine; synthesized using Method C (7% yield) ) . Name: N- (6-ethyl-4-methyl-2-quinazolinyl) guanidine .
  • N- (6-sec-butyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4-sec- butylaniline was used in place of 3 , 4-dibutoxyaniline .
  • Compound 2002A (class: Quinazolino-guanidine; synthesized using Method C (36% yield) ) .
  • N- (4-methylfuro [2 , 3-g] quinazolin-2-yl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 5- nitro- [2, 3] -benzofuran was used in place of 1, 2-dibutoxy- 4 -nitrobenzene .
  • N- ( 4-methylfuro [2, 3-g] quinazolin-2-yl) guanidine (class: Quinazolino-guanidine; synthesized using Method C (85% yield) ) .
  • N- ( 6-butoxy-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- butoxyaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1012A (class: Quinazolino-guanidine; synthesized using Method C (12% yield) ) . Name : N- ( 6-butoxy-4-methyl-2-quinazolinyl) guanidine .
  • Compound 1032A (class: Quinazolino-guanidine; synthesized using Method C (11% yield)).
  • N- (6-cyclohexyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- cyclohexylaniline was used in place of 3,4- dibutoxyaniline .
  • Compound 1029A (class: Quinazolino-guanidine; synthesized using Method C (14% yield) ) .
  • N- [ 4-methyl-6- (pentyloxy) -2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- pentyloxyaniline was used in place of 3, 4-dibutoxyaniline .
  • Pentyl 2, 2, 4-trimethyl-l, 2-dihydro-6-quinolinyl ether
  • N- [4-methyl-6- (4-methylphenoxy) -2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- (4- methylphenoxy) aniline was used in place of 3,4- dibutoxyaniline .
  • N- ( 6- tert-butyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 6-tert- butylaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1004A (class: Quinazolino-guanidine; synthesized using Method C (45% yield) .
  • N- (7-ethoxy-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- 5 quinazolinyl) guanidine (see Example 1) except that 3- ethoxyaniline was used in place of 3, 4-dibutoxyaniline .
  • N- [7- ( ert-butyl) -4-methyl-2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 3-tert- butylaniline was used in place of 3, 4-dibutoxyaniline .
  • N- ( 6-hydroxy-4 , 7-dimethyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 6- nitro-3, 4-dihydro-l (2H) -naphthalenone was used in place of 1, 2-dibutoxy-4-nitrobenzene .
  • Compound 1017A (class: Quinazolino-guanidine; synthesized using methods B & C (28% yield over 2 steps) ) .
  • N- ( 6-methoxy-4 , 7-dimethyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- methoxyaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1016A (class: Quinazolino-guanidine; synthesized using Method C (41% yield)).
  • N- ( 4-methyl-8, 9-dihydrobenzo [g] quinazolin-2-yl) guanidine was made in the same manner as N- (6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 7- nitro-1-tetralone was used in place of 1, 2-dibutoxy-4- nitrobenzene .
  • Compound 1037A (class: Quinazolino-guanidine; synthesized using Method C (11% yield)).
  • N- ( 4 -methyl -7 , 8-dihydro- 6 H- cyclopenta [g] quinazolin-2- yl) guanidine was made in the same manner as N-(6,7- dibutoxy-4-methyl-2-quinazolinyl) guanidine (see Example 1) except that 5-aminoindane was used in place of 3,4- dibutoxyaniline .
  • Compound 1038A (class: Quinazolino-guanidine; synthesized using Method C (18% yield)).
  • Example 1 except that 4- [ (5-phenoxypentyl) oxy] aniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1005A (class: Quinazolino-guanidine; synthesized using Method C (12% yield) ) .
  • N- ( 6-butyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- butylaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 2004A (class: Quinazolino-guanidine; synthesized using Method C (44% yield)).
  • N- ( 6-benzyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- benzylaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 2003A (class: Quinazolino-guanidine; synthesizec using Method C (19% yield)).
  • N- ( 6-hexyl-4-methyl-2-quinazolinyl) guanidine was made ii the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- hexylaniline was used in place of 3, 4-dibutoxyaniline .
  • N- [7- (benzyloxy) -4-methyl-2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 3-
  • N- ( 6-heptyl-4-methyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- heptylaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 2006A (class: Quinazolino-guanidine; synthesized using Method C (18% yield)).
  • N- ( 4-methyl-6-pentyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2- quinolinyl) guanidine (see Example 3) except that 4- pentylaniline was used in place of 4-ethylaniline.
  • Compound 5002A (class: Quinolino-guanidine; synthesized using Method J (2% yield) ) .
  • N- (4-methyl-6-propyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- propylaniline was used in place of 3, 4-dibutoxyaniline .
  • N- (4-methyl-6-phenyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- phenylaniline was used in place of 3, 4-dibutoxyaniline .
  • Compound 1010A (class: Quinazolino-guanidine; synthesized using Method C (3% yield) ) .
  • N- ( 4-methyl-6-octyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 4- octylaniline was used in place of 3, -dibutoxyaniline .
  • Compound 1009A (class: Quinazolino-guanidine; synthesized using Method C (12% yield)) .
  • N- ( 6-hexyl-4 -methyl-2-quinolinyl ) guanidine was made in the same manner as N- ( 6-ethyl-4 -methyl-2-quinolinyl ) guanidine ( see Example 3 ) except that 4 -hexylaniline was used in place of 4-ethylaniline .
  • Name N- ( 4-hexylphenyl) -3-oxobutanamide .
  • Compound 5003A (class: Quinolino-guanidine; synthesized using Method J (10% yield) ) .
  • N - ( 6- [1- (4-hydroxyl-pentyl) ] -4-methyl-2- quinazolino) guanidine was made in the same manner as N- ( 6- ethyl-4-methyl-2-quinazolino) guanidine (see Example 1) except that 5- (4-aminophenyl) -2-pentanol was used in place of 4-ethylaniline.
  • N- ( 6-butyl-4-methyl-2-quinolinyl) guanidine was made in the same manner as N- ( 6-ethyl-4-methyl-2-quinolinyl) guanidine (see Example 3) except that 4-butylaniline was used in place of 4-ethylaniline.
  • AJ- (4-methyl-7-phenyl-2-quinazolinyl) guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 3- phenylaniline was used in place of 3, 4-dibutoxyaniline .
  • N- [ -methy1-7- (isopropoxy) -2-quinazolinyl] guanidine was made in the same manner as N- ( 6, 7-dibutoxy-4-methyl-2- quinazolinyl) guanidine (see Example 1) except that 3- isopropoxyaniline was used in place of 3,4- dibutoxyaniline .
  • the compounds of the present invention may be synthesized by the routes shown in Schemes 4 and 5 , or with appropriate modifications as described herein.
  • Method 1, and Method 2 the product is isolated at the end of the synthesis, and purified by a suitable procedure such as high performance liquid chromatography (HPLC) , crystallization, column chromatography, thin layer chromatography, etc. While preferred reactants have been identified herein, it is further contemplated that the present invention would include chemical equivalents to each reactant(s) specifically enumerated in this disclosure .
  • Rink amide MBHA resin (1.85g, lmmol , 0.54mmol/g, Novabiochem, San Diego, CA, #01-64-0013) was swelled in a mixture of N, N-dimethylformamide (DMF), and N- methylpyrrolidone (NMP) (1:1, 25mL) in a glass column with a sintered glass frit, on a platform shaker, for lOmin. The solvents were drained and the resin was treated with 30% piperidine in DMF (25mL) for 5 min. and the liquid was drained. The piperidine treatment was repeated for 25 min. The resin was then washed, for 5min.
  • DMF N, N-dimethylformamide
  • NMP N- methylpyrrolidone
  • H-Arginine (diZ) -phenylalaninamide .HCl (N ⁇ -Boc) arginine (diZ) -phenylalaninamide (3.3g), was dissolved in THF (20mL) , and treated with 4M HCl in dioxane (20mL) for 20 min. The solvent was evaporated to dryness . The residue was treated with anhydrous ether and triturated. The precipitated product was filtered and washed with ether, and vacuum dried: 2.15g (72%) .
  • 1-naphthalenesulfonyl chloride (2eq. ) was coupled with H-Arginine (diZ) -phenylalaninamide .HCl, with 4 eq. of triethylamine in THF for 4-6 h.
  • the reaction ixtur was evaporated to dryness, and partitioned between ethyl acetate and sat. aq. NaHC0 3 .
  • the ethyl acetate layer was washed with water, sat. aq. NaCl and dried (Na 2 S0 4 ) . Filtration and evaporation of the ethyl acetate led to the protected compound.
  • the Z groups were removed by hydrogenation with Pd /C (5%) as the catalyst, in ethanol, with 0.5% V/V cone HCl.
  • the product was purified by using reverse phase preparative HPLC (250 x 22.5mm, Primesphere C18-HC) with a gradient of 10% - 70% acetonitrile (0.1% TFA) in water (0.1% TFA) over 30 min
  • N-amido-substituted products (where R3 and R4 in the generic structure is a substituent other than H)
  • R3 and R4 in the generic structure is a substituent other than H
  • modifying procedure 1 to accommodate the incorporation of R3 or R4 via alkylation or reductive coupling.
  • the resin is treated with the appropriate alkyl halide (0.9eq.), in DMF or dichloromethane, with 2 - 3eq. of triethylamine for 3- 4h.
  • the synthesis can be achieved by starting with the protected phenylalanine attached to Wang resin or 2-chlorotrityl chloride resin. Cleavage with the TFA cocktail after the synthesis is complete gives the product with the C-terminal acid.
  • the cleavage solution is neutralized with pyridine in methanol, and evaporated.
  • the crude compound containing a C-terminal acid is then coupled to an appropriate amine ((R8) 2 NH) by using a coupling procedure similar to that described in Method 2, to give the substituted amide.
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 18.9min;
  • This compound was synthesized as described in Method 1, except that 3-nitrophenylsulfonyl chloride (442 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that 4-nitrophenylsulfonyl chloride (442 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that 4-chlorophenylsulf onyl chloride (422.14 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that 2-bromophenylsulfonyl chloride (511.04 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that 4-methylphenylsulfonyl chloride (381.3 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that phenylsulfonyl chloride (353.24 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that 2, 4-dichlorophenylsulfonyl chloride (491.02 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride .
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 19.9 min;
  • This compound was synthesized as described in Method 1, except that ⁇ -toluenesulfonyl chloride (379.3 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 21.7 min;
  • This compound was synthesized as described in Method 1, except that 4-iodophenylsulfonyl chloride (605.04 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 19.7 min;
  • This compound was synthesized as described in Method 1, except that 2-naphthalenesulfonyl chloride (453.36 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 19.0 min;
  • This compound was synthesized as described in Method 1, except that 3 , 4-dimethoxyphenylsulfonyl chloride (473.36 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride .
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 14.9 min;
  • This compound was synthesized as described in Method 1, except that 4-chloro-3-nitrophenylsulfonyl chloride (512.14 mg, 2 mmol) was used in place of 1- naphthalenesulfonyl chloride.
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 19.9 min;
  • This compound was synthesized as described in Method 1, except that 2 , 4-dinitrophenylsulfonyl chloride (533.24 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • HPLC Primesphere C-18 reverse phase column 4.6 x 250mm, 10 - 56% acetonitrile (0.1% TFA) in water (0.1% TFA) over 24 min, flow rate 1 mL / min, detection at 220nm, retention time 19.9 min;
  • This compound was synthesized as described in Method 1, except that 3 -chloro-4 -fluorophenylsulfonyl chloride (458.12 mg, 2 mmol) was used in place of l- naphthalenesulf onyl chloride .
  • This compound was synthesized as described in Method 1, except that 2-Nitro-4-trif luoromethylphenylsulf onyl chloride (579.24 mg, 2 mmol) was used in place of 1- naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that 2 , 6-dichlorophenylsulfonyl chloride (491.02 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride .
  • This compound was synthesized as described in Method 1, except that 3- (2 , 5-dichlorothiophene) sulfonyl chloride (503.08 mg, 2 mmol) was used in place of 1- naphthalenesulfonyl chloride .
  • This compound was synthesized as described in Method 1, except that 3-methyl-6-methoxyphenylsulfonyl chloride (441.36 mg, 2 mmol) was used in place of 1- naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that 2 , 5-dichlorophenylsulfonyl chloride (491.02 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride .
  • This compound was synthesized as described in Method 1, except that 3 , 4-dichlorophenylsulf onyl chloride (491.02 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride .
  • This compound was synthesized as described in Method 1, except that pentafluorophenylsulfonyl chloride (533.14 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride .
  • This compound was synthesized as described in Method 1, except that 2-nitrophenylsulfonyl chloride (443.24 mg, 2 mmol) was used in place of 1-naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that (D) Phenylalanine was used in place of (L) Phenylalanine, and 2-naphthalenesulfonyl chloride (453.36 mg, 2 mmol) was used in place of 1- naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that (D) Arginine (Pbf) was used in place of (L) Arginine (Pbf) .
  • This compound was synthesized as described in Method 1, except that (D) Phenylalanine was used to substitute (L) Phenylalanine, and 2 , 6-difluorophenylsulfonyl chloride (425.20 mg, 2 mmol) was used in place of 1- naphthalenesulfonyl chloride.
  • This compound was synthesized as described in Method 1, except that (D) Phenylalanine was used to substitute (L) Phenylalanine, and 4-fluorophenylsulfonyl chloride (389.22 mg, 2 mmol) was used in place of 1- naphthalenesulfonyl chloride.
  • the binding data reflect competitive displacement of ( [ ⁇ :5 I]lDMeNPFF) .
  • Agonist potency is the concentration of a compound required to elicit 50% of maximum response. Intrinsic activity of a compound is measured as the percent of maximum response elicited by the ligand, neuropeptide FF.
  • Arginine or Phenylalanine residues were changed to their corresponding D-isomer. This modification is expected to further improve the stability of these compounds against enzymatic degradation. Binding and functional activities of these compounds at rat NPFFl and NPFF2 receptors are shown in Table 5.
  • Table 8 shows the cross-reactivity of NPFP compounds.
  • the binding affinity (Ki) of these compounds were tested according to the protocols described herein at the following receptors; human ⁇ 1A , ⁇ 1B , ⁇ 1D , ⁇ 2ft , 2B , and ⁇ 2c adrenergic receptors; human Yl, Y2, Y , and Y5 receptors; and N-Methyl-D-aspartic acid (NMDA) receptor channels.
  • the binding interactions of these compounds were additionally tested at the norepinephrine (NE) transporter (NE uptake) and serotonin (5-hydroxytryptamine (5HT) ) transporter (5HT uptake) according to protocols described herein
  • DIRC extension-induced rhythmic contraction'
  • This model is widely considered to be predictive for the actions of drugs to treat human urge incontinence (also refered to as detrusor instability or unstable bladder) .
  • drugs that are active in this model which also are used therapeutically in humans include oxybutynin and baclofen (Morikawa et al, 1992); imipramine and nortriptyline (Pietra et al, 1990) ; and nifedipine and terodiline (Guarneri et al, 1993) .
  • results presented herein represent the first demonstration that synthetic ligands which are active as agonists at the NPFF2 receptor inhibit the micturition reflex. In this regard their actions mimic the action of the endogenous peptide ligand NPFF.
  • the ability of these compounds to inhibit the micturition reflex in this model can be taken as an indication that they will be effective in the treatment of urge incontinence in humans.
  • an agonist as a ligand has an intrinsic activity (IA) >15%, while an antagonist as a ligand has a Ki ⁇ 1.2 mM and an intrinsic activity (IA) ⁇ 15% at the rat cloned neuropeptide FF (NPFF) receptors.
  • IA intrinsic activity
  • NPFF rat cloned neuropeptide FF
  • Compounds 2001A to 2006A, and 5001A to 5003A are quinolino-guanidines that are concurrently agonists at both the NPFFl and NPFF2 receptors; compounds
  • 1001B to 1008B, 1010B to 1017B, 1019B, 1021B to 1033B, and 2003B are sulfonylamides that are concurrently agonists at both the NPFFl and NPFF2 receptors;
  • Compounds 1001A to 1039A, and 4001A to 4009A are quinazolino-guanidines that are antagonists at the NPFFl receptor and agonists at the NPFF2 receptor;
  • compound 3001B is a sulfonylamide that is an antagonist at the NPFFl receptor, and an agonist at the NPFF2 receptor;
  • Compounds 3001 A, and 6001A to 6003A are quinolino- guanidines that are concurrently antagonists at both the NPFFl and NPFF2 receptors.
  • Compounds that are agonists at the NPFF2 receptor are suitable for treating incontinence, and also pain.
  • Compounds that are concurrently agonists at both the NPFFl and NPFF2 receptors are suitable for treating incontinence, and also pain.
  • Neuropeptide FF reduces food intake in rats. Peptides 17 (2) :353-354, 1996.
  • FMRFamide-related peptides including the mammalian-derived FaRPs F-8- Famide (NPFF) and A-18-Famide, for opioid mu, delta, kappa 1, kappa 2a, or kappa 2b receptors.
  • NPFF mammalian-derived FaRPs F-8- Famide
  • A-18-Famide for opioid mu, delta, kappa 1, kappa 2a, or kappa 2b receptors.
  • NPFF Mammalian Neuropeptide FF

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Abstract

La présente invention concerne des méthodes de traitement de la douleur, de l'incontinence urinaire et d'autres anomalies qui sont induites par le récepteur NPFF, ces méthodes consistant à administrer à un individu, une quantité thérapeutiquement efficace d'un composé chimique agissant au niveau du récepteur NPFF1, du récepteur NPFF2 ou bien au niveau des deux récepteurs NPFF1 et NPFF2.
PCT/US2002/030215 2001-09-24 2002-09-24 Composes utiles pour le traitement de la douleur WO2003026657A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007504176A (ja) * 2003-09-05 2007-03-01 アクテリオン　ファマシューティカルズ　リミテッド グアニジン誘導体
JP2007507472A (ja) * 2003-10-06 2007-03-29 ブルスター，ジークフリート ソマトスタチン受容体サブタイプ４（ｓｓｔｒ４）及び１（ｓｓｔｒ１）に作用するスルホニルアミノ−ペプチド模倣薬
US7381818B2 (en) 2003-10-28 2008-06-03 Epoch Biosciences, Inc. Fluorescent probes containing 5′-minor groove binder, fluorophore and quenching moieties and methods of use thereof
WO2024003535A2 (fr) 2022-06-27 2024-01-04 University Of Dundee Composés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3149113A (en) * 1959-04-22 1964-09-15 Monsanto Chemicals Preparation of 2-guanidinoquinazoline compounds

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3149113A (en) * 1959-04-22 1964-09-15 Monsanto Chemicals Preparation of 2-guanidinoquinazoline compounds

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007504176A (ja) * 2003-09-05 2007-03-01 アクテリオン　ファマシューティカルズ　リミテッド グアニジン誘導体
JP2007507472A (ja) * 2003-10-06 2007-03-29 ブルスター，ジークフリート ソマトスタチン受容体サブタイプ４（ｓｓｔｒ４）及び１（ｓｓｔｒ１）に作用するスルホニルアミノ−ペプチド模倣薬
US7381818B2 (en) 2003-10-28 2008-06-03 Epoch Biosciences, Inc. Fluorescent probes containing 5′-minor groove binder, fluorophore and quenching moieties and methods of use thereof
US7759126B2 (en) 2003-10-28 2010-07-20 Elitech Holding B.V. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for amplification (or PCR) analysis
WO2024003535A2 (fr) 2022-06-27 2024-01-04 University Of Dundee Composés

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