The invention relates to novel 2-heteroarylcarboxamides, processes for their preparation, and their use for producing medicaments for the treatment and/or prophylaxis of diseases and for improving perception, concentration, learning and/or memory.

The invention relates to novel 2-heteroarylcarboxamides, processes for their preparation, and their use for producing medicaments for the treatment and/or prophylaxis of diseases and for improving perception, concentration, learning and/or memory.
Nicotinic acetylcholine receptors (nAChR) form a large family of ion channels which are activated by the messenger acetylcholine which is produced in the body (Galzi and Changeux, Neuropharmacol. 1995, 34, 563-582). A functional nAChR consists of five subunits which may be different (certain combinations of α1-9 and β1-4,γ,δ,ε subunits) or identical (α7-9). This leads to the formation of a diversity of subtypes which differ in the distribution in the muscles, the nervous system and other organs (McGehee and Role, Annu. Rev. Physiol. 1995, 57, 521-546). Activation of nAChR leads to influx of cations into the cell and to stimulation of nerve cells or muscle cells. Selective activation of individual nAChR subtypes restricts this stimulation to the cell types which have a corresponding subtype and is thus able to avoid unwanted side effects such as, for example, stimulation of nAChR in the muscles. Clinical experiments with nicotine and experiments in various animal models indicate that central nicotinic acetylcholine receptors are involved in learning and memory processes (e.g. Rezvani and Levin, Biol. Psychiatry 2001, 49, 258-267). Nicotinic acetylcholine receptors of the alpha7 subtype (α7 nAChR) have a particularly high concentration in regions of the brain which are important for learning and memory, such as the hippocampus and the cerebral cortex (Séguéla et al., J. Neurosci. 1993, 13, 596-604). The α7 nAChR has a particularly high permeability for calcium ions, increases glutamatergic neurotransmission, influences the growth of axons and, in this way, modulates neuronal plasticity (Broide and Leslie, Mol. NeurobioL 1999, 20, 1-16).
Certain N-(1-azabicyclo[2.2.2]oct-3-yl)heteroaryl carboxamides for the treatment of, inter alia, psychoses are described in DE-A 37 24 059.
N-(Azabicycloalkyl)heteroaryl carboxamides, in particular N-(1-azabicyclo-[2.2.2]oct-4-yl)benzothiophene-3-carboxamides, are disclosed in WO 93/15073 and in EP-A 0 485 962 as intermediates for the synthesis of pharmaceutically active compounds.
1-Azabicycloalkanes and their action on the nicotinic α7-receptor are known from JP 14030084A.
U.S. Pat. No. 4,605,652 and EP-A 0 372 335 disclose, for example, N-(1-azabicyclo[2.2.2]oct-3-yl)thiophene-2-carboxamide and its memory-improving effect.
The present invention relates to compounds of the formula (I)
in which    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen or C1-C6-alkyl,    R3 represents hydrogen, halogen or C1-C6-alkyl,    A represents oxygen or sulfur,and    the ring B represents benzo, pyrido, pyrimido, pyridazo or pyridazino which are optionally substituted by radicals selected from the group consisting of hydrogen, halogen, C1-C6-alkanoyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, C1-C6-acylamino, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-alkylthio, C1-C6-alkylamino, heteroarylcarbonylamino, arylcarbonylamino, C1-C4-alkylsulfonylamino, di-(C1-C4-alkylsulfonyl)amino, arylsulfonylamino, di(arylsulfonyl)amino, C3-C6-cycloalkylcarbonylmethyl, 1,3-dioxapropane-1,3-diyl, amino(hydroxyimino)methyl and benzo,and their salts, solvates and solvates of the salts.
The compounds according to the invention can exist in stereoisomeric forms which are either like image and mirror image (enantiomers) or which are not like image and mirror image (diastereomers). The invention relates both to the enantiomers and diastereomers and to their respective mixtures. These mixtures of the enantiomers and diastereomers can be separated in a known manner into the stereoisomeric uniform components.
Compounds according to the invention can also be present in the form of their salts, solvates or solvates of the salts.
Salts which are preferred for the purposes of the invention are physiologically acceptable salts of the compounds of the invention.
Physiologically acceptable salts of the compounds according to the invention may be acid addition salts of the compounds with mineral acids, carboxylic acids or sulfonic acids. Particularly preferred examples are salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
However, salts which may be mentioned are also salts with conventional bases, such as, for example, alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts) or ammonium salts derived from ammonia or organic amines such as, for example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabiethylamine, 1-ephenamine or N-methylpiperidine.
Solvates is the term used for the purposes of the invention for those forms of the compounds which form a complex with solvent molecules by coordination in the solid or liquid state. Hydrates are a special form of solvates in which the coordination takes place with water.
For the purposes of the present invention, the substituents generally have the following meaning:
C1-C6- and C1-C4-Alkoxy are a straight-chain or branched alkoxy radical having 1 to 6 and 1 to 4 carbon atoms, respectively. Preference is given to a straight-chain or branched alkoxy radical having 1 to 4, particularly preferably 1 to 3, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.C1-C6- and C1-C4-Alkyl are a straight-chain or branched alkyl radical having 1 to 6 and 1 to 4 carbon atoms, respectively. Preference is given to a straight-chain or branched alkyl radical having 1 to 4, particularly preferably 1 to 3, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-pentyl and n-hexyl.(C1-C6)-Alkanoyl is a straight-chain or branched alkyl radical having 1 to 6 carbon atoms which carries a doubly attached oxygen atom in the 1-position and is attached via the 1-position. Preference is given to a straight-chain or branched alkanoyl radical having 1 to 4, particularly preferably 1 to 2, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: formyl, acetyl, propionyl, n-butyryl, isobutyryl, pivaloyl and n-hexanoyl.C1-C6-Alkylamino is a straight-chain or branched alkylamino radical having 1 to 6 carbon atoms, preferably 1 to 4, particularly preferably 1 to 3, carbon atoms. Nonlimiting examples include methylamino, ethylamino, n-propylamino, isopropylamino and tert-butylamino.(C1-C6)-Acylamino is an amino group having a straight-chain or branched alkanoyl substituent which has 1 to 6 carbon atoms and is attached via the carbonyl group. Preference is given to an acylamino radical having 1 to 4, particularly preferably 1 to 2, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: formamido, acetamido, propionamido, n-butyramido and pivaloylamido.C1-C4-Alkylsulfonylamino is a straight-chain or branched alkylsulfonylamino radical having 1 to 4, preferably 1 to 3, carbon atoms. Nonlimiting examples include methylsulfonylamino, ethylsulfonylamino, n-propylsulfonylamino, isopropyl-sulfonylamino, tert-butylsulfonylamino.Arylsulfonylamino is a naphthyl- or phenylsulfonylamino radical and preferably a phenylsulfonylamino radical.C1-C6-Alkylthio is a straight-chain or branched alkylthio radical having 1 to 6 carbon atoms. Preference is given to a straight-chain or branched alkylthio radical having 1 to 4, particularly preferably 1 to 3, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: methylthio, ethylthio, n-propylthio, isopropylthio, tert-butylthio, n-pentylthio and n-hexylthio.Arylcarbonyl is a naphthyl- or phenylcarbonyl radical and preferably a phenylcarbonyl radical (Benzoyl radical).Heteroarylcarbonyl is a heteroarylcarbonyl radical having a 5- to 6-membered, preferably a 5-membered, heteroaryl ring having up to 2 heteroatoms selected from the group consisting of O, S and N. Nonlimiting examples include thienylcarbonyl, furylcarbonyl, pyrrolylcarbonyl, thiazolylcarbonyl, oxazolylcarbonyl, imidazolylcarbonyl, pyridylcarbonyl, pyrimidylcarbonyl.C3-C6-Cycloalkylcarbonylmethyl is a monocyclic cycloalkyl group having 3 to 6 carbon atoms which is attached via a carbonylmethyl group [—C(═O)—CH2—]. Nonlimiting examples include cyclopropylcarbonylmethyl, cyclopentylcarbonylmethyl and cyclohexylcarbonylmethyl.Halogen is fluorine, chlorine, bromine and iodine. Preference is given to fluorine, chlorine and bromine. Particular preference is given to fluorine and chlorine.
When radicals in the compounds according to the invention are optionally substituted, unless indicated otherwise the radicals may have one or more identical or different substituents. Preference is given to radicals substituted by up to three identical or different substituents.
Preference is given to compounds of the formula (I),
in which
    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen or (C1-C6)-alkyl,    R3 represents hydrogen, halogen or (C1-C6)-alkyl,    A represents oxygen or sulfur,and    the ring B represents benzo, pyrido, pyrimido, pyridazo or pyridazino which are optionally substituted by radicals selected from the group consisting of hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1-C6)-alkylthio and benzo.
Particular preference is given to compounds of the formula (I),
in which
    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen,    R3 represents hydrogen, chlorine, fluorine or methyl,    A represents oxygen or sulfur,and    the ring B represents benzo or pyrido, where benzo or pyrido is optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-alkylthio, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl, 1,3-dioxapropane-1,3-diyl, amino(hydroxyimino)methyl and benzo.
Especially preferred are compounds of the formula (Ia)
in which    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen or C1-C6-alkyl,    R3 represents hydrogen, halogen or C1-C6-alkyl,    A represents oxygen or sulfur,and    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, C1-C6-alkoxy, C1-C6-alkylthio, C1-C6-alkylamino, heteroarylcarbonylamino, arylcarbonylamino, C1-C4-alkylsulfonylamino, di(arylsulfonyl)amino, C3-C6-cycloalkylcarbonylmethyl or amino(hydroxyimino)methyl.
Very particular preference is given to compounds of the formula (Ia),
in which
    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen,    R3 represents hydrogen, chlorine, fluorine or methyl,    A represents oxygen or sulfur,and    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, methyl, ethyl, methoxy, ethoxy, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl or amino(hydroxyimino)methyl.
Particularly preferred are compounds of the formula (Ia),
in which
    R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen,    R3 represents hydrogen, chlorine, fluorine or methyl,    A represents oxygen or sulfur,and    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, methyl, ethyl, methoxy, ethoxy, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl or amino(hydroxyimino)methyl.
Also preferred are compounds of the formula (I),
in which
R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,
and R2, R3, A and the ring B are as defined above.
Particularly preferred are compounds of the formula (Ia),
in which
R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,
and R2, R3, A and Z are as defined above.
Also preferred are compounds of the formula (I),
in which
R2 represents hydrogen or methyl,
and R1, R3, A and the ring B are as defined above.
Particularly preferred are compounds of the formula (I),
in which
R2 represents hydrogen,
and R1, R3, A and the ring B are as defined above.
Also preferred are compounds of the formula (I),
in which
R3 represents hydrogen, fluorine, chlorine or methyl,
and R1, R2, A and the ring B are as defined above.
Particularly preferred are compounds of the formula (I),
in which
R3 represents hydrogen or methyl,
and R1, R2, A and the ring B are as defined above.
Very particularly preferred are compounds of the formula (I),
in which
R3 represents hydrogen,
and R1, R2, A and the ring B are as defined above.
Also preferred are compounds of the formula (I),
in which
A represents sulfur,
and R1, R2, R3 and the ring B are as defined above.
Also preferred are compounds of the formula (I),
in which
A represents oxygen,
and R1, R2, R3 and the ring B are as defined above.
Particularly preferred are compounds of the formula (Ia),
in which
A represents sulfur,
and R1, R2, R3 and Z are as defined above.
Also particularly preferred are compounds of the formula (Ia),
in which
A represents oxygen,
and R1, R2, R3 and Z are as defined above.
Also preferred are compounds of the formula (I),
in which
    the ring B represents benzo or pyrido, where benzo and pyrido are optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-alkylthio, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl and amino(hydroxyimino)methyl,and R1, R2, R3 and A are as defined above.
Particularly preferred are compounds of the formula (I),
in which
the ring B represents benzo or pyrido, where benzo and pyrido are optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido and (C1-C4)-alkyl,and R1, R2, R3 and A are as defined above.
Very particular preference is given to compounds of formula (I),
in which
    the ring B represents benzo, where benzo is optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido and (C1-C4)-alkyl,and R1, R2, R3 and A are as defined above.
Also particularly preferred are compounds of the formula (Ia),
in which
    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, methyl, ethyl, methoxy, ethoxy, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl or amino(hydroxyimino)methyl,and R1, R2, R3 and A are as defined above.
Very particular preference is given to combinations of two or more of the preferred ranges mentioned above.
Especially preferred are compounds of the formula (I),
in which
    R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,    R2 and R3 represent hydrogen,    A represents sulfur,and    the ring B represents benzo or pyrido, where benzo and pyrido are optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido and (C1-C4)-alkyl.
The invention relates furthermore to a process for preparing the compounds of the formula (I), characterized in that compounds of the formula (II)R1R2NH  (II),in which R1 and R2 are as defined aboveare reacted with a compound of the formula (III)
in whichR3, A and the ring B are as defined above, andX represents hydroxy or a suitable leaving group,in an inert solvent, if appropriate in the presence of a condensing agent and if appropriate in the presence of a base.
If X represents a leaving group, preference is given to chlorine, mesyloxy and isobutyloxycarbonyloxy, in particular to chlorine.
Inert solvents are, for example, halogenated hydrocarbons, such as methylene chloride, trichloromethane, carbon tetrachloride, trichloroethane, tetrachloroethane, 1,2-dichloroethane or trichloroethylene, ethers, such as diethyl ether, methyl tert-butyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether or diethylene glycol dimethyl ether, hydrocarbons, such as benzene, xylene, toluene, hexane, cyclohexane or mineral oil fractions, or other solvents, such as nitromethane, ethyl acetate, acetone, dimethylformamide, dimethylacetamide, 1,2-dimethoxyethane, dimethyl sulfoxide, acetonitrile or pyridine; preference is given to tetrahydrofuran, dimethylformamide or chloroform.
Condensing agents are, for example, carbodiimides, such as, for example, N,N′-diethyl-, N,N,′-dipropyl-, N,N′-diisopropyl-, N,N′-dicyclohexylcarbodiimide, N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N′-propyloxymethyl-polystyrene (PS-carbodiimide), or carbonyl compounds, such as carbonyldiimidazole, or 1,2-oxazolium compounds, such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulfate or 2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino compounds, such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutyl chloroformate, or bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride or benzotriazolyloxytri(dimethylamino)-phosphonium hexafluorophosphate, or O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), 2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU) or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) or benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), or mixtures of these.
If appropriate, it may be advantageous to use these condensing agents in the presence of an auxiliary nucleophile, such as, for example, 1-hydroxybenzotriazole (HOBt).
Bases are, for example, alkali metal carbonates, such as, for example, sodium carbonate or potassium carbonate or sodium bicarbonate or potassium bicarbonate, or organic bases, such as trialkylamines, for example triethylamine, N-methylmorpholine, N-methylpiperidine, 4-N,N-dimethylaminopyridine or N,N-diisopropylethylamine.
Preference is given to O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) in the presence of N,N-diisopropylethylamine and to the combination of N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt), in each case in dimethylformamide.
The process according to the invention is preferably carried out in a temperature range of from room temperature to 50° C., at atmospheric pressure.
The compounds of the formulae (II) and (III) are known or can be synthesized by known processes from the appropriate starting materials (cf., for example, "Comprehensive Heterocyclic Chemistry", Katritzki et al., Ed.; Elsevier, 1996).
Thus, for example, substituted benzothiophene-2-carboxylic acids can be obtained from appropriately substituted 2-halobenzaldehydes by reaction with methyl mercaptoacetate (see, for example, A. J. Bridges, A. Lee, E. C. Maduakor, Schwartz, Tetrahedron Lett. 1992, 33, 7499) and subsequent hydrolysis of the ester (synthesis scheme 1):

To synthesize the corresponding pyrido derivatives, it is possible to react 2-halo-benzonitrile starting materials with methyl mercaptoacetate to give the 3-aminobenzothiophene-2-carboxylic esters (synthesis scheme 2). The amino function can be removed by diazotization. Finally, the ester is hydrolyzed to give the target compound:

Substituted benzofuran-2-carboxylic acids can be obtained, for example, in accordance with D. Bogdal, M. Warzala, Tetrahedron 2000, 56, 8769 (synthesis scheme 3):

The compounds according to the invention of the formula (I) are suitable for use as medicaments for the treatment and/or prophylaxis of diseases in humans and/or animals.
The compounds according to the invention have an unforeseeable, useful pharmacological activity spectrum.
They act as ligands, in particular as α7-nAChR agonists.
The compounds of the invention can, because of their pharmacological properties, be employed alone or in combination with other active ingredients for the treatment and/or prevention of cognitive impairments, especially of Alzheimer's disease. Because of their selective effect as α7-nAChR agonists, the compounds of the invention are particularly suitable for improving perception, concentration, learning or memory, especially after cognitive impairments like those occurring for example in situations/diseases/syndromes such as mild cognitive impairment, age-associated learning and memory impairments, age-associated memory loss, vascular dementia, craniocerebral trauma, stroke, dementia occurring after strokes (post-stroke dementia), post-traumatic brain syndrome, general concentration impairments, concentration impairments in children with learning and memory problems, attention deficit hyperactivity disorder, Alzheimer's disease, Lewy body dementia, dementia with degeneration of the frontal lobes, including Pick's syndrome, Parkinson's disease, progressive nuclear palsy, dementia with corticobasal degeneration, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia, schizophrenia with dementia or Korsakoffs psychosis.
The compounds of the invention can be employed alone or in combination with other active ingredients for the prophylaxis and treatment of acute and/or chronic pain (for a classification, see "Classification of Chronic Pain, Descriptions of Chronic Pain Syndromes and Definitions of Pain Terms", 2nd edition, Meskey and Begduk, editors; IASP Press, Seattle, 1994), especially for the treatment of cancer-induced pain and chronic neuropathic pain like, for example, that associated with diabetic neuropathy, postherpetic neuralgia, peripheral nerve damage, central pain (for example as a consequence of cerebral ischaemia) and trigeminal neuralgia, and other chronic pain such as, for example, lumbago, backache (low back pain) or rheumatic pain. In addition, these active ingredients are also suitable for the therapy of primary acute pain of any origin and of secondary states of pain resulting therefrom, and for the therapy of states of pain which were formerly acute and have become chronic.
The in vitro effect of the compounds of the invention can be shown in the following assays:
1. Determination of the Affinity of Test Substances for α7-nAChR by inhibition of [3H]-methyllycaconitine Binding to Rat Brain Membranes
The [3H]-methyllycaconitine binding assay is a modification of the method described by Davies et al. in Neuropharmacol. 1999, 38, 679-690.
Rat brain tissue (hippocampus or whole brain) is homogenized in homogenization buffer (10% w/v, 0.32 M sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.01% (w/v) NaN3, pH 7.4, 4° C.) at 600 rpm in a glass homogenizer. The homogenate is centrifuged (1000×g, 4° C., 10 min) and the supernatant is removed. The pellet is resuspended (20% w/v) and the suspension is centrifuged (1000×g, 4° C., 10 min). The two supernatants are combined and centrifuged (15 000×g, 4° C., 30 min). The pellet obtained in this way is referred to as the P2 fraction.
The P2 pellet is washed with binding buffer (50 mM Tris-HCl, 1 mM MgCl2, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, pH 7.4), and centrifuged (15 000×g, 4° C., 30 min), twice.
The P2 membranes are resuspended in binding buffer and incubated in a volume of 250 μl (amount of membrane protein 0.1-0.5 mg) in the presence of 1-5 nM [3H]-methyllycaconitine, 0.1% (w/v) BSA (bovine serum albumin) and various concentrations of the test substance at 21° C. for 2.5 h. Incubation is then carried out in the presence of 1 μM α-bungarotoxin or 100 μM nicotine or 10 μM MLA (methyllycaconitine) to determine the non-specific binding.
The incubation is stopped by adding 4 ml PBS (20 mM Na2HPO4, 5 mM KH2PO4, 150 mM NaCl, pH 7.4, 4° C.) and filtering through type A/E glass fibre filters (Gelman Sciences) which have previously been placed in 0.3% (v/v) polyethyleneimine (PEI) for 3 h. The filters are washed twice with 4 ml of PBS (4° C.), and the bound radioactivity is determined by scintillation measurement. All the assays are carried out in triplicate. The dissociation constant of the test substance Ki was determined from the IC50 of the compounds (concentration of the test substance at which 50% of the ligand bound to the receptor is displaced), the dissociation constant KD and the concentration L of [3H]-methyllycaconitine using the equation Ki=IC50/(1+L/KD).
In place of [3H]-methyllycaconitine it is also possible to employ other α7-nAChR-selective radioligands such as, for example, [125I]-α-bungarotoxin or nonselective nAChR radioligands together with inhibitors of other nAChRs.
Representative in vitro data for the effects of the compounds of the invention are shown in Table A:
TABLE AExampleKi (nM)1942.0353.73663.03780.04237.04658.04875.0513.9583.15920.06250.06420.06555.0682.0732.87422.0766.77720.07880.08031.08828.0
The suitability of the compounds of the invention for the treatment of cognitive impairments can be shown in the following animal models:
2. Object Recognition Test
The object recognition test is a memory test. It measures the ability of rats (and mice) to distinguish between familiar and unfamiliar objects.
The test is carried out as described by Blokland et al., NeuroReport 1998, 9, 4205-4208; A. Ennaceur J. Delacour, Behav. Brain Res. 1988, 31, 47-59; A. Ennaceur K. Meliani, Psychopharmacology 1992, 109, 321-330; and Prickaerts et al., Eur. J. Pharmacol. 1997, 337, 125-136.
In a first run, a rat is confronted in an otherwise empty observation arena of relatively large size by two identical objects. The rat will investigate, i.e. sniff round and touch, both objects extensively. In a second run, after an interval of 24 hours, the rat is put in the observation arena again. One of the familiar objects has now been replaced by a new, unfamiliar object. If a rat recognizes the familiar object, it will concentrate on investigating the unfamiliar object. However, after 24 hours, a rat has normally forgotten which object it investigated in the first run, and it will therefore inspect both objects to the same extent. Administration of a substance with a learning- and memory-improving effect may lead to a rat recognizing the object seen in the first run 24 hours previously as familiar. It will investigate the new, unfamiliar object in more detail than the familiar one. This memory ability is expressed in a discrimination index. A discrimination index of zero means that the rat investigates both objects, the old and the new, for equal times; that is to say it has not recognized the old object and reacts to both objects as if they were new and unfamiliar. A discrimination index greater than zero means that the rat inspects the new object for longer than the old one; that is to say the rat has recognized the old object.
3. Social Recognition Test:
The social recognition test is a test to examine the learning- or memory-improving effect of test substances.
Adult rats housed in groups are placed singly in test cages 30 minutes before the start of the test. Four minutes before the start of the test, the test animal is put in an observation box. After this adaptation time, a juvenile animal is put in with the test animal and the time for which the adult animal investigates the juvenile animal is measured for 2 minutes (trial 1). All behaviours clearly directed at the young animal are measured, i.e. anogenital inspection, pursuit and fur care, during which the old animal is no further than 1 cm from the young animal. The juvenile animal is then taken out, and the adult is left in its test cage (for 24-hour retention, the animal is returned to its home cage). The adult test animal is treated with test substance before or after the first test. Depending on the timing of the treatment, the learning or the storage of the information about the young animal can be influenced by the substance. After a fixed period (retention), the test is repeated (trial 2). A larger difference between the investigation times measured in trials 1 and 2 means that the adult animal has remembered the young animal better.
The compounds of the formula (I) according to the invention are suitable for use as medicaments for humans and animals.
The present invention also includes pharmaceutical preparations which, besides inert, nontoxic, pharmaceutically suitable excipients and carriers, contain one or more compounds of the formula (I), or which consist of one or more compounds of the formula (I), and processes for producing these preparations.
The compounds of the formula (I) are to be present in these preparations in a concentration of from 0.1 to 99.5% by weight, preferably from 0.5 to 95% by weight, of the complete mixture.
Besides the compounds of the formula (I), the pharmaceutical preparations may also contain other active pharmaceutical ingredients.
The abovementioned pharmaceutical preparations can be produced by known methods in a conventional way using, for example, the auxiliary or auxiliaries or excipient(s).
The novel active ingredients can be converted in a known manner into conventional formulations such as tablets, coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, nontoxic, pharmaceutically suitable excipients or solvents. In these cases, the therapeutically active compound should in each case be present in a concentration of about 0.5 to 90% by weight of the entire mixture, i.e. in amounts which are sufficient to reach the stated dose range.
The formulations are produced for example by extending the active ingredients with solvents and/or excipients, where appropriate with use of emulsifiers and/or dispersants, it being possible for example when water is used as diluent where appropriate to use organic solvents as auxiliary solvents.
Administration takes place in a conventional way, preferably orally, transdermally or parenterally, especially perlingually or intravenously. However, it can also take place by inhalation through the mouth or nose, for example with the aid of a spray, or topically via the skin.
It has generally proved advantageous to administer amounts of about 0.001 to 10 mg/kg, on oral administration preferably about 0.005 to 3 mg/kg, of body weight to achieve effective results.
It may, nevertheless, be necessary where appropriate to deviate from the stated amounts, in particular as a function of the body weight or of the mode of administration, of the individual behaviour toward the medicament, the nature of its formulation and the time or interval over which administration takes place. Thus, it may be sufficient in some cases to make do with less than the aforementioned minimum amount, whereas in other cases the stated upper limit must be exceeded. Where larger amounts are administered, it may be advisable to divide these into a plurality of single doses over the day.
Abbreviations:BINAP2,2′-Bis(diphenylphosphino)-1,1′-binaphthylDCIdirect chemical ionization (in MS)DMFN,N-DimethylformamideDMSODimethyl sulfoxideEDCN′-(3-Dimethylaminopropyl)-N-ethylcarbodiimide × HClEDTAEthylenediaminetetraacetic acideq.Equivalent(s)ESIElectrospray ionization (in MS)HATUO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphateHOBt1-Hydroxy-1H-benzotriazole × H2OHPLCHigh pressure/high performance liquid chromatographyLC-MSLiquid chromatography with coupled mass spectroscopyMSMass spectroscopyNMRNuclear magnetic resonance spectroscopyPd2(dba)3Tris(dibenzylideneacetone)dipalladium(0)RTRoom temperature, 20° C.RtRetention time (in HPLC)TBTUO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborateTFATrifluoroacetic acidTHFTetrahydrofuranTrisTris(hydroxymethyl)aminomethaneLC-MS Method A:
MS instrument:Micromass Quattro LCZIonization: ESI positiveHPLC instrument:HP 1100UV detector DAD: 208-400 nmOven temperature: 40° C.Column:Symmetry C 1850 mm × 2.1 mm; 3.5 μmGradient:TimeA:B:Flow rate(min)%%(ml/min)0.0010.090.00.504.0090.010.00.506.0090.010.00.506.1010.090.01.007.5010.090.00.50A: Acetonitrile + 0.1% formic acidB: Water + 0.1% formic acidLC-MS Method B:
MS instrument:Finnigan MAT 900SIonization: ESI positiveHPLC instrument:TSP: P4000, AS3000, UV3000HRUV detector 3000HR: 210 nmOven temperature: 70° C.Column:Symmetry C 18 150 mm × 2.1 mm; 5 μmSupplier:WatersTimeFlow rate(min)A: %B: %C: %(ml/min)Gradient:0 249490.92.595 2.5 2.51.2595 2.5 220.127.116.11 249491.26.5 249491.27 249490.9A: AcetonitrileB: Water + 0.6 g/l 35% strength hydrochloric acidC: WaterLC-MS Method C:
MS instrument:Micromass Platform LCZIonization: ESI positiveHPLC instrument:HP 1100UV detector DAD: 208-400 nmOven temperature: 40° C.Column:Symmetry C 1850 mm × 2.1 mm; 3.5 μmGradientTimeA:B:Flow rate(min)%%(ml/min)0.0010.090.00.504.0090.010.00.506.0090.010.00.506.1010.090.01.007.5010.090.00.50A: Acetonitrile + 0.1% formic acidB: Water + 0.1% formic acidLC-MS Method D:
Instrument: Micromass Platform LCZ, HP1100; Column: Symmetry C18, 50 mm×2.1 mm, 3.5 μm; Mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile+0.05% formic acid; Gradient: 0.0 min 90% A→4.0 min 10% A→6.0 min 10% A; Oven: 40° C.; Flow rate: 0.5 ml/min; UV detection: 208-400 nm.
LC-MS Method E:
Instrument: Micromass Quattro LCZ, HP1100; Column: Uptisphere HDO, 50 mm×2.0 mm, 3 μm; Mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile+0.05% formic acid; Gradient: 0.0 min 100% A→0.2 min 100% A→2.9 min 30% A→3.1 min 10% A→4.5 min 10% A; Oven: 55° C.; Flow rate: 0.8 ml/min; UV detection: 208-400 nm.
LC-MS Method F:
Instrument: Micromass Quattro LCZ, HP1100; Column: Symmetry C18, 50 mm×2.1 mm, 3.5 μm; Mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile+0.05% formic acid; Gradient: 0.0 min 90% A→4.0 min 10% A→6.0 min 10% A; Oven: 40° C.; Flow rate: 0.5 ml/min; UV detection: 208-400 nm.
LC-MS Method G:
MS instrument: Micromass ZQ; HPLC instrument: Waters Alliance 2790; Column: Uptisphere C18, 50 mm×2.0 mm, 3.0 μm; Mobile phase A: acetonitrile+0.05% formic acid, mobile phase B: water+0.05% formic acid; Gradient: 0.0 min 5% A→2.0 min 40% A→4.5 min 90% A→5.5 min 90% A; Oven: 45° C.; Flow rate: 0.0 min 0.75 ml/min→4.5 min 0.75 ml/min→5.5 min 1.25 ml/min; UV detection: 210 nm.
HPLC Method H:
Instrument: HP 1100 with DAD detection; column: Kromasil RP-18, 60 mm×2 mm, 3.5 μm; mobile phase A: 5 ml HClO4/l H2O, mobile phase B: acetonitrile; Gradient: 0 min 2% B, 0.5 min 2% B, 4.5 min 90% B, 6.5 min 90% B; Flow rate: 0.75 ml/min; Temperature: 30° C.; UV detection: 210 nm.
Starting Materials:
General reaction scheme for the synthesis of methyl 1-benzothiophene-2-carboxylates:
General Procedure for the Synthesis of methyl 1-benzothiophene-2-carboxylates:
Under an argon atmosphere, 1.5 equivalents of sodium hydride (60% pure) are initially charged in absolute DMSO (0.60-1.26 M suspension). At room temperature, 1.1 equivalents of methyl mercaptoacetate are slowly added dropwise to the reaction mixture, and it is left to stir at room temperature until evolution of hydrogen ceases (about 15 min). 1.0 equivalents of the appropriate benzaldehyde are dissolved in absolute DMSO (1.60-3.36 M solution) and added at room temperature to the reaction mixture. The reaction mixture is stirred until the reaction is complete (about 5-10 min) and then poured into ice-water. The resulting precipitate is filtered off with suction, dried at 40° C. under reduced pressure overnight and reacted further as crude product.
General Reaction Scheme for the Synthesis of 1-benzothiophene-2-carboxylic acids:
General Procedure for the Synthesis of 1-benzothiophene-2-carboxylic acids:
A mixture of equal parts of THF and 2 N potassium hydroxide solution (0.28-0.47 M solution) is added to the appropriate methyl 1-benzothiophene-2-carboxylate. The reaction mixture is left to stir at room temperature overnight. The THF is removed under reduced pressure and the aqueous reaction mixture is acidified with concentrated hydrochloric acid. The resulting precipitate is filtered off with suction and dried under reduced pressure at 40° C.
General Procedure for Amide Linkage Between 3-quinuclidinamine and 2-benzothiophene- or 2-benzofurancarboxylic acids (Variant A):
1.5 eq. of the appropriate enantiomeric 3-quinuclidinamine hydrochloride are, together with 1 eq. of the carboxylic acid and 1.5 eq. of HATU, initially charged in DMF at 0° C. After addition of 1.5 eq. of N,N-diisopropylethylamine, the mixture is stirred for 30 min. A further 4 eq. of N,N-diisopropylethylamine are added, and the mixture is stirred at RT overnight. Purification is carried out chromatographically.
General Procedure for Amide Linkage Between 3-quinuclidinamine and 2-benzothiophene- or 2-benzofurancarboxylic acids (Variant B):
1.0 eq. of the appropriate enantiomeric 3-quinuclidinamine dihydrochloride are, together with 1 eq. of the carboxylic acid and 1.2 eq. of HATU, initially charged in DMF at 0° C. After addition of 1.2 eq. of N,N-diisopropylethylamine, the mixture is stirred for 30 min. A further 2.4 eq. of N,N-diisopropylethylamine are added, and the mixture is stirred at RT overnight. Purification is carried out chromatographically.
General Procedure for the Synthesis of methyl 3-aminothienopyridine-2-carboxylates:
1.0 equivalents of the appropriate pyridine derivative are dissolved in absolute DMSO (0.93-0.96 M solution), and 2 equivalents of triethylamine are added. After addition of 1 equivalent of methyl mercaptoacetate, the reaction mixture is stirred at 60° C. overnight. The reaction mixture is poured into ice-water and stirred therein. The precipitated solid is filtered off with suction and, if required, purified by column chromatography (silica gel 60, mobile phase toluene/ethyl acetate 20:1 to 5:1).
General Procedure for the Synthesis of methyl thienopyridine-2-carboxylates:
With cooling at −5° C., 1.0 equivalents of the appropriate methyl 3-aminothienopyridine-2-carboxylate is initially charged in 75% strength sulfuric acid (0.33-0.36 M solution). A solution of 3.2 equivalents of sodium nitrite in water (0.92-1.00 M solution) is slowly added dropwise to the reaction mixture such that the temperature of the reaction mixture does not exceed 0° C. The mixture is stirred at 0° C. for 45 min. 60-65 equivalents of ice-cold 50% strength hypophosphoric acid are then added dropwise to the reaction mixture, again in such a way that the temperature does not exceed 0° C. The reaction mixture is stirred at −5° C. for one hour and then kept in the fridge overnight. The reaction mixture is diluted with sodium bicarbonate solution and ethyl acetate, and sodium bicarbonate is added a little at a time until the mixture gives a basic reaction. Work-up is described in the examples below.
General Procedure for the Synthesis of thienopyridine-2-carboxylic acids:
A mixture of equal parts of THF and 2 N potassium hydroxide solution (0.22 M solution) is added to the appropriate methyl thienopyridine-2-carboxylate. The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with water, washed twice with ethyl acetate and acidified with concentrated hydrochloric acid. Work-up is described in the examples below.

Bayer Schering Pharma Aktiengesellschaft


The present invention is based on the finding that nucleic acids containing at least one unmethylated cytosine-guanine (CpG) dinucleotide affect immune responses in a subject. These nucleic acids containing at least one unmethylated cytosine-guanine (CpG) dinucleotide can be used to treat pulmonary disorders having an immunologic component, such as a response to inhaled lipopolysaccharide. The invention provides methods of treating subjects who have or are at risk of having these pulmonary disorders, and methods of altering the immunological components of the pulmonary disorders. The invention also provides pharmaceutical compositions for treating pulmonary disorders that have an immunologic component.

Endotoxin is one of the primary mediators of inflammation released by Gram negative organisms and is an important cause of environmentally induced airway disease, such as ARDS. Inhaled endotoxin can cause airflow obstruction in previously unexposed subjects. Inhaled endotoxin is strongly associated with the development of acute decrements in airflow among cotton workers, wine confinement workers, and poultry workers. The concentration of endotoxin in the bioaerosol appears to be the most important occupational exposure associated with the development and progression of airway disease in agricultural workers (Schwartz, D. A., et al., Am. J. Respir. Crit. Care Med. 152:603-8, 1995).
In addition to being related to several occupational pulmonary diseases, exposure to endotoxin and to its purified derivative lipopolysaccharide (LPS) is also associated with severe asthma. The concentration of endotoxin in the domestic environment adversely affects asthmatics, with higher concentrations of ambient endotoxin associated with greater degrees of airflow obstruction. In addition, asthmnatic individuals develop airflow obstruction at lower concentrations of inhaled endotoxin than normal controls. Exposure-response studies have shown that inhaled lipopolysaccharide (LPS) produces recruitment of neutrophils, activation of macrophages with production and release of pro-inflammatory cytokines, and damage to airway epithelia in a dose-dependent manner. These studies indicate that endotoxin is an important cause of airway disease among exposed individuals.
The acute respiratory distress syndrome (ARDS) is a condition characterized by acute hypoxemia respiratory failure due to pulmonary edema (reviewed in Honing, E. G., and Ingram, R. H., Jr., in: Harrison's Principles of Internal Medicine, 14th Edition, A. S. Fauci, et al. (eds.), McGraw-Hill, N.Y., pp. 1483-1486, 1998; and Goodman, R. B., et al., Am J. Respir. Crit. Care Med. 154:602-11, 1996). ARDS represents a spectrum of responses to acute lung injury (ALI); these response occur as complications of a more widespread systemic response to acute inflammation or injury. ALI develops rapidly after a predisposing condition triggers a systemic inflammatory response and is most strongly associated with conditions that produced direct alveolar injury or direct injury via the pulmonary capillary bed, such as aspiration, diffuse infection, toxic inhalation, direct injury to the alveolar epithelium, or sepsis syndrome. ALI is the consequence of unregulated over-expression of usual systemic inflammatory responses to infection and/or injury. Injury involves the alveolar epithelium and the pulmonary capillary endothelium, and results in a complex cascade of events. Injury is produced by cellular events associated with neutrophils, macrophages, monocytes, and lymphocytes producing various cytokines, in turn producing cellular activation, chemotaxis, and adhesion.
Gram-negative infections are a major cause of morbidity and mortality, especially in hospitalized and immunocompromised patients. (Duma, Am. J. of Med., 78 (Suppl. 6A): 154-164, 1985; and Kreger et al., Am. J. Med., 68:344-355, 1980). Although available antibiotics are generally effective in inhibiting growth of Gram-negative bacteria, they do not neutralize the pathophysiological effects associated with endotoxins. Endotoxin is a heat stable bacterial toxin composed of lipopolysaccharides (LPS) released from the outer membrane of Gram-negative bacteria upon lysis (Shenep et al., J. Infect. Dis., 150(3):380-388, 1984), and is a potent stimulator of the inflammatory response. Endotoxemia occurs when endotoxin enters the bloodstream resulting in a dramatic systemic inflammatory response.
The uptake of oligonucleotides by B lymphocytes has been shown to be regulated by LPS-induced cell activation (Krieg, A. M., et al., Antisense Res. Devel. 1:161, 1991). Many detrimental in vivo effects of LPS have been shown to result from soluble mediators released by inflammatory cells. (Morrison et al., Am. J. Pathol., 93(2):527-617, 1978). Monocytes and neutrophils, which ingest and kill microorganisms, play a key role in this process. Monocytes and neutrophils respond to endotoxin in vivo by releasing soluble proteins with microbicidal, proteolytic, opsonic, pyrogenic, complement-activating and tissue-damaging effects. These factors mediate many of the pathophysiological effects of endotoxin. For example, tumor necrosis factor (TNF), a cytokine released by endotoxin-stimulated monocytes, causes fever, shock, and alterations in glucose metabolism and is a potent stimulator of neutrophils. Other cytokines such as IL-1, IL-6, and IL-8 also mediate many of the pathophysiologic effects of LPS, as well as other pathways involving endothelial cell activation by tissue factor, kininogen, nitric oxide and complement.
Endotoxin-associated disorders result from extra-gastrointestinal exposure to LPS, e.g., administration of LPS-contaminated fluids, inhalation of LPS, or Gram-negative infections. Endotoxin-associated disorders can also result when the natural cellular barrier is injured and the normal Gram-negative flora breach this barrier. For example, endotoxin-associated disorders can occur (a) when there is ischemia of the gastrointestinal tract (e.g, following hemorrhagic shock or during certain surgical procedures), or (b) when systemic or local inflammation causes increased permeability of the gut or lung to endotoxin or Gram-negative organisms. The presence of endotoxin and the resulting inflammatory response may result, for example, in adult respiratory distress syndrome (ARDS), dust-induced airway disease, and exacerbation of asthma, in addition to endotoxemia, systemic inflammatory response syndrome (SIRS), sepsis syndrome, septic shock, disseminated intravascular coagulation (DIC), cardiac dysfunction, organ failure, liver failure (hepatobiliary dysfunction), brain failure (CNS dysfunction), renal failure, multi-organ failure and shock.
Several therapeutic compounds have been developed to inhibit the toxic effects of endotoxin, including antibacterial LPS-binding agents and anti-LPS antibodies, although each has met with limitations. For example, Polymyxin B (PMB) is a basic polypeptide antibiotic which binds to Lipid A, the most toxic and biologically active component of endotoxin. PMB inhibits endotoxin-mediated activation of neutrophil granule release in vitro and is a potential therapeutic agent for Gram-negative infections. However, because of its systemic toxicity, this antibiotic has limited therapeutic use, and is generally used topically. Combination therapy using antibiotics and high doses of methylprednisolone sodium succinate (MPSS) showed more promise as this regimen prevented death in an experimental animal model of Gram-negative sepsis. However, a clinical study using MPSS with antibiotics in treatment of patients having clinical signs of systemic sepsis showed that mortality rates were not significantly different between the treatment and placebo groups (Bone et al., N. Engl. J. Med. 317:653, 1987).

Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders


A cup holder includes one or two supporting members that support a container inserted into a receiving portion of a holder main body, and is arranged in a circumferential direction of the holder main body. A container having the protrusion and recess pattern on a side surface can smoothly be taken out from the receiving portion with a low risk of the supporting members interfering with other members around the holder main body.

A cup holder for retaining a beverage container and the like in a stable state is equipped in a passenger compartment of an automobile. As such a cup holder, ones having a supporting member capable of supporting a side surface of the container inserted into a receiving portion of a holder main body are known. Typically, in the cup holder having the supporting member, the container such as a PET bottle having various dimensions and shapes that is inserted into the receiving portion can be retained in the stable state by the supporting member advancing into the receiving portion. Further, the container such as the PET bottle can be taken out from the receiving portion as a result of the supporting member retracting from the receiving portion.
As shown in FIG. 6, a cup holder 50 disclosed in Japanese patent No. 3725505 includes a holder main body 51 having a receiving portion 51a into which a beverage container B1 is to be inserted, and supporting members 52 capable of advancing into and retracting from the retaining portion 51a of the holder main body 51. Each of the supporting members 52 is provided capable of rotating with an axial line O along a horizontal direction as a center, and is biased such that its portion lower than the axial line O advances into the receiving portion 51a. When the beverage container B1 having a protrusion and recess pattern on a side surface is to be taken out from the receiving portion 51a, a protrusion B1a of the beverage container B1 makes contact with guide portions 52a of the supporting members 52. Each of the guide portions 52a has a shape with which a force F from the protrusion B1a acting on the guide portion 52a is oriented toward a lower side than the axial line O. With the force F being oriented toward the lower side than the axial line O, a moment is exerted on the supporting members 52 in a direction that causes the guide portions 52a to retract from the receiving portion 51a (a counter-clockwise moment). Due to this, the beverage container B1 having the protrusion and recess pattern on the side surface can smoothly be taken out from the receiving portion 51a of the holder main body 51.
As shown in FIG. 7A and FIG. 7B, a cup holder 60 disclosed in Japanese utility model No. 3137651 includes a holder main body 61 having a receiving portion 61a into which a beverage container is to be inserted, and a supporting member 62 capable of advancing into and retracting from the receiving portion 61a of the holder main body 61. The supporting member 52 is configured of an upper piece 621 having its upper end supported rotatably by a side portion of the holder main body 61, a lower piece 622 having its upper end linked rotatably to the upper piece 621 and its lower end sliding on the side portion of the holder main body 61 in an up and down direction, and a spring 63 that is attached to a linking portion of the upper piece 621 and the lower piece 622 and that biases the linking portion to advance in a direction approaching a center axis of the holder main body 61. A stopper surface 621a that restricts a rotation of the lower piece 622 relative to the upper piece 621 to be within a predetermined range is formed at a lower end of the upper piece 621. When the beverage container is inserted into the receiving portion 61a, the linking portion of the upper piece 621 and the lower piece 622 is pressed by a side surface of the beverage container, and the supporting member 62 is retracted to a position shown in FIG. 7B from its most advanced position shown in FIG. 7A.


An unloading device for equipment such as ships, bunkers, storage yards or the like comprises an elevator which has a substantially vertical supporting structure with a lower pickup end and an upper delivery end and a conveyor mounted on the structure with an elevator section for conveying material substantially vertical along the support structure. The lower end of the support structure engages into a rotatable bucket wheel which is mounted on a supporting structure. The bucket wheel is mounted in an inclined position relative to the supporting structure preferably at an angle of 45.degree. to the vertical axis of the supporting structure.

This invention relates in general to unloading devices and in particular to a new and useful loading device for ships, bunkers and storage yards and the like which comprise an elevator having a substantially vertical supporting structure with a lower pickup end on which a bucket wheel is mounted in an inclined position relative to the elevator.

Unloading device for ships, bunkers, storage yards or the like

O & K Orenstein & Koppel Aktiengesellschaft Werk Lubeck


A fuse holder tester (50) is arranged with a plurality of conductive, resiliently mounted contact fingers (51, 52), for making electrical contact with leads (11, 12) embedded in the walls of the fuse holder (10). The tester also includes a spring-loaded probe (58) arranged to make contact with a third lead (13) located at the base of the fuse holder, upon insertion of the tester into the holder. In one example, a testing assembly (70) is provided, having six of the testers (50) capable of being simultaneously inserted into a typical fuse block (30) having six of the fuse holders (10).

Many of the circuits used in telephone central offices have fuse devices located in the circuits to protect the equipment from current surges. Typical of the fuses used in these arrangements is the 70 type, or tubular shaped, fuse (manufactured by Western Electric Company, Incorporated of New York, N.Y.), which is inserted into a fuse holder for connection to the electrical circuit which it protects. The fuse holder has a tubular shaped opening designed to accept the fuse and has two leads embedded in the walls of the opening and one lead coiled at the bottom of the opening.
Frequently, circuits using tubular type fuses must be tested to assure proper electrical continuity. Since the fuse holder for tubular type fuses has two leads embedded in the wall of the tubular opening, making electrical contact with these leads has proved to be physically difficult. In one previous arrangement, a dummy tubular fuse is inserted into each fuse holder opening and, because of the location of the embedded leads, is turned by the test operator until electrical contact is made with each embedded lead for test purposes. However, this arrangement is time consuming and, because of the numerous inserting, turning and pulling out operations of the dummy fuse by the test set operator during the work day, has resulted in operator fatigue and in poor test results.

Apparatus for testing leads of fuse holders


According to one configuration, an example communication system includes a primary wireless station, multiple candidate wireless stations, and a remote network. The primary wireless station is in communication with one or more of the multiple candidate wireless stations. During operation, when the primary wireless station needs to provide one or more communication devices access to a remote network, the primary wireless station monitors presence of the multiple candidate wireless stations. Each of the multiple candidate wireless stations is available to support a wireless backhaul link from the primary wireless station to a remote (such as a core) network. The primary wireless station is operable to select an appropriate candidate wireless station amongst the multiple candidate wireless stations. Via the selected candidate wireless station, the primary wireless station establishes a backhaul link to the remote network.

Conventional wireless networks typically include one or more wireless base stations to provide mobile communication devices access to a remote network such as the Internet.
One type of wireless base station is a so-called CBSD (Citizen Broadband Radio Service Device). Such a system uses one or more wireless channels allocated from a CBRS (Citizens Band Radio Service) to support communications with one or more mobile communication devices. Typically, one or more wireless channels is allocated to a base station by a SAS (Spectrum Access Service) to support communications with one or more mobile communication devices. For example, a base station can be configured to communicate with the SAS to receive notification of the wireless channel allocated for use. SAS communications as well as the data used by Mobile devices transmitted by the base station are transported over a respective Backhaul. User plane data, control plane data, Management traffic and communication with SAS carried over the Backhaul connection with the Radio base station.
Via a communication link such as a backhaul link between the base station and a remote network, the base station provides the one or more mobile communication devices access to a remote network. In other words, in an uplink direction, the base station forwards communications over a backhaul link to target recipients in the remote network. In a reverse direction, such as a downlink direction, the base station receives communications from the remote network over a backhaul link and wirelessly conveys received communications to respective target recipients.

Backhaul selection in a wireless network


The complete cDNA sequence encoding the amino acid sequence corresponding to mammalian Na.sup.+ /nucleoside cotransporter protein (SNST) is disclosed. Methods for obtaining the gene encoding SNST and for obtaining recombinantly produced SNST are described. Antibodies, an inhibitor of nucleoside transport by SNST, and methods for detecting other inhibitors are also described. Methods for inhibiting uptake of nucleosides by SNST using the compositions of the invention are also included.

Cotransporter proteins are membrane-bound proteins that actively transport substances into cells. For example, organic substrates such as sugars, amino acids, carboxylic acids and neurotransmitters, are transported into eucaryotic cells by sodium (Na.sup.+) cotransporter proteins. Some transport proteins have been identified, for example, Na.sup.+ /glucose and Na.sup.+ /proline transporters (Peerce and Wright, Proc. Natl. Acad. Sci. USA 8:2223-2226 (1984); Wright and Peerce, J. Biol. Chem. 259:14993-14996 (1984)), and the brain Na.sup.+ Cl/GABA transporter (Radian et al., J. Biol. Chem. 261:15437-15441 (1986)), and progress has been made in locating their active sites and probing their conformational states (Peerce and Wright, supra; Wright and Peerce, supra; Peerce and Wright, J. Biol. Chem. 259:14105-14112 (1984); J. Biol. Chem. 260:6026-6031 (1985); Proc. Natl. Acad. Sci. USA 83:8092-8096 (1986); Biochem. 26:4272-4276 (1987)).
There appears to be a gene family of sodium (Na+) dependent transporters related to the mammalian intestinal Na+/glucose cotransporter, SGLT1. Na+/glucose cotransporters have been cloned and sequenced from rabbit intestine (Hediger, et al, Nature 330:379-381 (1987)) and kidney (Coady, et al, Am. J. Physiol. 259:C605-C610 (1990)), human intestine (Hediger, et al, Proc. Natl. Acad. Sci. USA 86:5748-5752, and LLC-PK.sup.1 cells (Ohta, et al, Mol. Cell. Biol. 10:6491-6499 (1990)). All of these transporters share greater than 80% identity of amino acid sequence. The only other proteins with significant homology to the mammalian SGLT1 are bacterial Na+-dependent transporters for proline (Nakao, et al, Mol. Gen. Genet. 208:70-75 (1987)) and pantothenate (Jackowski & Alix, Bacteriol. 172:3842-3848 (1990)). This suggests that the relationship between members of this multigene family is at the level of the transport mechanism, the Na+ coupling, rather than the substrate being transported.
Defects in Na.sup.+ -driven transporters may be associated with diseases. For example, a defect in the intestinal brush border Na.sup.+ /glucose cotransporter (SGLT1) is the origin of the congenital glucose-galactose malabsorption syndrome (Turk et al. Nature 350:354-356 (1991)).
It has been difficult to clone mammalian cotransport proteins in part because of the difficulties in purification of low abundance, hydrophobic membrane proteins that constitute less than 0.2% of the membrane protein. Hediger et al., Nature 330:379-381 (1987) described a new strategy for cloning rabbit intestinal Na.sup.+ /glucose cotransporter for expression without the use of antibodies or synthetic oligonucleotide probes.
Nucleosides direct a number of important physiological activities, particularly in the mammalian cardiovascular and central nervous systems. For example, adenosine is a potent vasoactive molecule in the coronary and cerebral vessels and a modulator of potassium and calcium channels in neurons and cardiac muscle (Belardinelli et al., Prog. Cardiovasc. Diseases 32:73-97 (1989)). Furthermore, nucleosides and their analogs are known to be potent cytotoxic and anti-retroviral agents. Nucleosides and their analogs, and nucleoside transport inhibitors are being used or proposed for use as broad spectrum anti-retroviral drugs, anti-cancer drugs, for treating ischemia and reperfusion-induced cell injury in the heart, and anti-arrhythmic drugs. For example, nucleoside transporter proteins may be useful to transport drugs to treat diseases such as AIDS (see, Yarchoan and Broder, New Engl. J. Med. 316:557-564 (1987)). In addition, membrane transport proteins are involved in the regulation of the uptake of nucleosides into cells. Nucleosides are required for normal growth, metabolism and function of cells. Some cells, such as bone marrow, leukocytes and brain cells, are deficient in purine biosynthesis and dependent on uptake of preformed purines.
Phloridzin, (1-[2-.beta.-D-Glucopyranosyloxy)-4,6-dihydroxyphenyl]-3]-4-hydroxyphenyl) -1-propanone, No. 7300, p. 1163, Merck Index, 11th Edition, (1989)) a phloretin-2'-.beta.-glucoside, is a natural substance occurring in all parts of the apple tree, and is known to be a very specific inhibitor of sugar transport in the mammalian intestine and kidney (see Newey et al., J. Physiol. 169:229-236 (1963) and Diedrich, Methods in Enzymology 191:755-780 (1990)). These effects are known to be due to competitive inhibition of active, i.e. sodium dependent, sugar transport. Fifty percent inhibition is achieved by phloridzin concentrations as low as 5.times.10.sup.-6 molar. Phloridzin is very specific for the Na.sup.+ /glucose cotransporter protein because the glucose moiety is recognized by the active site on the membrane transport protein. There are no other known effects of phloridzin on biological. systems.
No other specific inhibitors of sodium dependent cotransporter proteins are known.
Nucleoside cotransporter proteins have been identified (Plageman et al., Biochim. Biophys. Acta. 947:405-554 (1988)), but have not been isolated, cloned and expressed. Therefore, these proteins are available in small quantities only from mammalian cell membranes. Thus, it would be desirable to have available a method for producing practical quantities of nucleoside cotransporter protein for use alone or with drugs that can be delivered into cells by the protein and to identify inhibitors that will specifically block the activity of the cotransporter protein.

The Regents of the University of California Office of Technology Transfer


To enable running in a multiple document load mode, an interface procedure is used to specify more than one bin in a "bins for each set" selection and the desired bin capacity is set in the Bin Capacity selection. From 1 to 6 bins per set may be selected. The operator selects the copy quantity, places the first set of originals in the document handler and presses start print. During this job segment, if the number of copies delivered is equal to the bin capacity, then the sorters will automatically increment to the next bin. Incrementing to the next bin is only dependent on reaching bin capacity and is not related to the loads in the document handler. When copying of the first set of originals is complete, the screen reads: "LOAD MORE ORIGINALS AND PRESS START PRINT OR PRESS END JOB IF JOB DONE". The operator then loads the next set of originals and presses start print. This sequence continues until the last sets of originals is completed, the operator then presses "END JOB". The job is now complete with each output set occupying a group of consecutive bins. The number of bins per group is determined by the value set in "bins for each set".

The invention relates to an unlimited document feeder, and more particularly, to automatically coordinating the use of a plurality of sorter towers with multiple bins to accommodate successive loads of the document feeder to complete a single job requirement.
High volume duplication jobs often have more originals than the document handler can process in one load. However, in general, all copier/duplicator products conclude the job when the last document in the document handler has been copied. These jobs must therefore be broken into a number of "sub" jobs, each having no more originals than the document handler can hold. The result is a manual collation task to build up the final output sets from separate parts. This is very time consuming and offers too much potential for human error.
It would be desirable, therefore, to provide a system where the completion of the last original in the ADH does not terminate the job, but instead the operator is prompted to replace the completed stack of originals with another stack and continue the job.
It is an objective, therefore, of the present invention to provide a new and improved document feeder. It is another object of the present invention to provide a document feeder and control that prompts the operator to complete discrete portions of a total job by separate loadings of the automatic document feeder. It is a further object of the present invention to use from 1 to 6 bins to store each output set to provide a delivery point for multiple original load sets.
There is prior art on the basic concepts of a copier having both an ADH and a sorter, in combination, and with so called "limitless sorting" Limitless sorting is a known copying mode alternately using two sets of sorter bins, one of the sets of bins can be filled while the other set of bins is being unloaded.
Also of particular interest as relating to the subject of "limitless sorting" is U.S. Pat. No. 4,361,320 issued Nov. 30, 1982 to H. Kikuchi, et al. It discloses a single vertical array of bins divided (functionally) into two groups when the number of copies to be collated exceeds the number of bins, thus allowing copying to operate continuously and allowing an operator to remove the collated copies from one group while copies are being collated in the other group. When the number of pages of a document exceeds a predetermined number, the first group is defined to contain more bins than the second group, thereby reducing the number of times each document page must be fed to the copier.


A hybrid system working machine, during a normal working time, operated by a normal operation mode that the output of a power generator and the accumulated power of a capacitor are used while the engine is operated over a high-efficiency range at a low output. Switching of a selector switch to a high-speed running mode position and running operation enable long-distance and high-speed running of an increase in engine output by a high-output operation mode.

A hybrid system working machine is constituted such that an averaged part of the required electric power is supplied from a generator, and when the generator is deficient in the electric power to be supplied, the shortage is supplemented by the electric power stored from a power storage means, whereas when the electric power is supplied in a relatively greater amount from the generator, the surplus electric power is stored (charged) in the power storage means.
Accordingly, the power storage means repeats the charging/discharging, changing the input/output in accordance with a variation in a load.
FIG. 6 shows a general trend of the power variation in a conventional non-hybrid system hydraulic excavator (relationship between the time and a change in the power which is determined from data for an actual machine). Regarding the property of a hydraulic excavator receiving a strong variation in the load, it is shown that either the total power from actuators or the power from a hydraulic pump as a power source for the actuators varies significantly in a short time.
On the other hand, FIG. 7 shows a trend (simulation) of a variation in the power for various leveling works, when a hybrid system excavator is employed.
In the case of such a hybrid system excavator, it is constituted such that the averaged power in the required power supplied from a power source, as shown by the solid line in FIG. 7, is supplied from a generator (one dot line), and that when the power is supplied in a relatively smaller amount from the generator, the shortage from the averaged power is supplemented by the power supplied from a power storage means, whereas when the power is supplied in a relatively larger amount from the generator, the surplus power is stored in the power storage means.
As a result, the output from the power storage means frequently and significantly varies, as indicated by the broken line in FIG. 7, thereby repeating inputting (charging) above the level of 0 kW and outputting (discharging) below the level thereof in a short time.
On the other hand, the excavator is constituted such that an upper rotating body is mounted onto a crawler type lower traveling body, and a excavating apparatus as a main working apparatus is mounted onto the upper rotating body, thereby making it possible either to carry out normal works (excavating, leveling and others) with the aid of the excavating apparatus, or to move in a site in the self-traveling manner with the aid of a crawler type traveling apparatus mounted onto the lower traveling body.
The movement is normally carried out in a small distance, so that it can be achieved by only the power from an engine. However, in a possible case when the excavator has to self-travel at a high speed in a relatively long distance, an assistance of the power storage means is further required.
In such a case, the power storage means has to have a greater capacity, which ensures an output for a long period, and therefore a battery (secondary battery, such as lead-acid battery, nickel hydrogen battery or the like) can be preferably employed for the power storage means.
On contrary, such a battery is operated with the aid of the chemical reaction between an electrolyte and electrodes, and therefore this causes the lifetime to become extremely short, when it is used in a working machine, such as an excavator, wherein the charging/discharging is frequently repeated due to a significant variation in the load. This eventually necessitates the exchange of the battery with high frequency, and leads to an increased cost in operation as well as to a disadvantage resulting from the environment problem due to the abandonment of batteries.
On the other hand, the employment of a capacitor (electrical double layer condenser) for such a power storage means provides a longer lifetime, even if the charging/discharging is frequently repeated. This is due to the fact that the electric charges are physically stored in the capacitor. Nevertheless, a reduced storage capacity provides a rapid consumption of charges, thereby making it difficult to travel at high speed in a long distance as described above. In the case of an empty storage, the movement or traveling has to be carried out, using only the output from the engine, so that the traveling speed is reduced and a long time is required for the movement.
As described in Japanese Unexamined Patent Application Publication No. 2000-295717, when an excavator is constituted such that it includes two type condensers, a battery and a capacitor, and that the condensers may be selectively used in accordance with the working conditions, the above-mentioned problems are solved. In this case, however, the cost of manufacturing the condensers extremely increases, along with increased complexity in the control, so that such a system is not available in the practical usage.

Kobelco Construction Machinery Co., Ltd.


Novel orthoesters are provided which can be used as a 2xe2x80x2-hydroxyl protecting groups or 2xe2x80x2-modification in the synthesis of polymers containing ribonucleic acid (RNA) nucleotides. The RNA comprising the orthoester can be handled and analyzed while 2xe2x80x2-modified, thereby minimizing potential degradation. The orthoester is stable during oligonucleotide synthesis. The orthoester is subsequently modified and can then be removed under mild acidic conditions. The ease and dependability of this process and the quality of the RNA product synthesized with this invention are comparable to that previously associated only with DNA synthesis.

The present invention relates to the field of protecting groups in organic synthesis and, more particularly, to the use of these compounds as ribonucleoside protecting groups and as 2xe2x80x2-modifications. Still more specifically, the protecting groups are used in the synthesis of oligonucleotides containing ribonucleotide subunits.
The ability to routinely synthesize ribonucleic acid (RNA) has become increasingly important as research reveals the multitude of RNA""s biological functions. There are many types of RNA including ribosomal RNA, transfer RNA and messenger RNA. RNA also is important in various structures and functions as well as being a catalyst in enzymatic reactions, as in the case of ribozymes. Because of the important biological roles RNA plays, both known and unknown, it is of considerable utility to be able to synthesize short (2-300 nucleotides) defined sequences of RNA, commonly referred to as RNA oligonucleotides or oligoribonucleotides. Over the past 25 years, many chemical approaches have been explored for synthesizing RNA oligonucleotides. Because deoxyribonucleic acid (DNA) methodologies have progressed more rapidly, the usual strategy for the synthesis of RNA has been to adapt DNA chemistries to RNA synthesis. Consequently, most approaches have focused on retaining the 5xe2x80x2-dimethoxytrityl (DMT) ether and adding a compatible 2xe2x80x2-hydroxyl protecting group such as fluoride-labile silyl ethers, photo-labile moieties, and acid-labile acetals. A delicate balance has been required to successfully utilize the acid-labile 2xe2x80x2-acetals in conjunction with the acid-labile 5xe2x80x2-DMT ether. Therefore, other approaches have involved retaining the 2xe2x80x2-acetal while replacing the 5xe2x80x2-DMT.
The acid-labile acetals have many attractive features. For example, it has been reported that it is possible to chromatograph some 2xe2x80x2-acetal-protected RNA. However, the subsequent removal of these acetals, which are used with the DMT group, requires acidic conditions which subsequently cause degradation of the RNA, or require extremely long periods of time to remove ( greater than 24 hours). Therefore, although it may be possible to safely handle and purify some 2xe2x80x2-acetal-protected RNAs, the harsh conditions required for rapid deprotection may require further purification of the RNA, thereby negating this advantage. Milder conditions can be used but these are inconvenient, requiring more than 24 hours. More-labile acetals can not be used as they would not be sufficiently stable to the DMT acid-deprotection conditions during RNA synthesis.
Of all of the RNA synthesis methods reported to date, only the 5xe2x80x2-DMT-2xe2x80x2-t-butyldimethylsilyl (TBDMS) and the 5xe2x80x2-DMT-2xe2x80x2-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP) chemistries are readily available commercially. Unfortunately, neither of these methods allows RNA synthesis to be as routine and dependable as DNA synthesis. The impediments facing 5xe2x80x2-DMT-2xe2x80x2-FPMP chemistry are related to the problem of balancing two acid-labile protecting groups. One of the major difficulties with the 2xe2x80x2-TBDMS approach is that stepwise coupling yields are only 96-98% under routine conditions compared to  greater than 99% for DNA. These methods enable the synthesis of RNA in acceptable yields and quality, but a high level of skill and significant investments in training and experience are required to deliver adequate results.
One of the most desirable conditions for the final 2xe2x80x2-deprotection of synthesized RNA is an extremely-mildly-acidic aqueous solution. In the optimal scheme, the 2xe2x80x2-protecting groups only have to be stable to withstand oligonucleotide synthesis conditions. Scaringe and Caruthers (U.S. patent application Ser. No. 08/488,878, filed Jun. 9, 1995, now U.S. Pat. No. 5,889,136 and incorporated herein by reference) recently reported a novel RNA synthesis strategy similar to such a scheme. Their investigations led to the development of silyl ethers for protection of the 5xe2x80x2-hydroxyl. However, this 5xe2x80x2-silyl ether oligonucleotide synthesis chemistry was not compatible with mildly-acid-labile 2xe2x80x2-acetals. Acid-labile orthoester protecting groups were investigated and discovered to have potential for use at the 2xe2x80x2-hydroxyl. The 2xe2x80x2-orthoesters that were developed in conjunction with 5xe2x80x2-silyl ethers enabled the synthesis of RNA oligonucleotides. Scaringe and Caruthers disclose specific orthoester protecting groups at the 2xe2x80x2-position of ribonucleotides. However, they do not disclose the orthoesters of the present invention.
The present invention provides an orthoester moiety that serves as a protecting group, particularly for RNA synthesis. Also provided in this invention is RNA comprising the protecting group which possesses novel advantages and useful features, e.g., a modified RNA oligonucleotide that is easily handled and analyzed with minimal concern about degradation. The protecting groups can be readily cleaved ( less than 10 minutes), if so desired, under extremely mild conditions that cause no detectable degradation of the RNA. No prior art anticipates that the following 2xe2x80x2-modification
would be advantageous to RNA synthesis. This 2xe2x80x2-modification is the result of using an orthoester of the following general structure:
where R represents protecting groups which can be removed prior to removing the orthoester.
The prior art has provided several means to synthesize RNA oligonucleotides. However, none have enabled the synthesis, handling, analysis and use of RNA oligonucleotides to be as robust and dependable as DNA synthesis, nor produced RNA comparable to the high quality in which DNA can be produced. No prior art has disclosed a modification of RNA that enables the RNA to be easily handled and then where the modification, e.g., a protecting group, can be removed under mild conditions to yield fully deprotected RNA. The present invention, therefore, provides more robust RNA synthesis methods which consistently produce higher quality RNA on a routine basis.
Accordingly, it is an object of this invention to provide useful protecting groups for the improved synthesis, analysis, handling and use of RNA oligonucleotides or other polymers containing ribonucleotides. It is a further objective of this invention to provide RNA, comprising such protecting groups, that can be easily analyzed, handled and used without requiring extensive safeguards against degradation. The protecting groups can be subsequently removed if desired under extremely mild conditions to yield high-quality fully deprotected RNA. Those skilled in the art can use these protecting groups in other organic synthesis methodologies as well. Therefore, this invention is not limited to the field of nucleic acid and oligonucleotide chemistry.
The present invention achieves these and other objectives by the provision of novel orthoester protecting groups with innovative and useful features. We have developed, for example, the O-bis(2-acetyl-ethoxy)methyl (ACE) orthoester:
that is stable to nucleoside and oligonucleotide synthesis conditions but is modified via ester hydrolysis during base deprotection of the oligonucleotide or polymer. The resulting 2xe2x80x2-bis(2-hydroxyethyl)methyl orthoester protecting group (2xe2x80x2-EG):
is 10 times more labile to acid than it""s precursor, the ACE orthoester. Complete cleavage of the 2xe2x80x2-EG orthoester may be effected using extremely mild conditions (pH 3,  less than 10 min., 55xc2x0 C.). The innovative features of this chemistry have enabled the synthesis of RNA oligonucleotides surpassing results in the prior art in terms of quality.
The orthoesters of the present invention are illustrated as follows:
where R represents protecting groups which can be removed prior to removing the orthoester. An example of an orthoester with appropriate R groups is as follows:
where X1, X2, and X3 are appropriate atoms or ligands. Several suitable orthoesters are illustrated as follows:
The general structure of a ribonucleoside of this invention is illustrated as follows:
where exocyclic amines, 5xe2x80x2-hydroxyl and the 3xe2x80x2-hydroxyl groups are appropriately protected and/or functionalized for use in oligonucleotide synthesis and R represents protecting groups which can be removed while leaving the 2xe2x80x2-orthoester moiety intact. More specifically, the general structure of a ribonucleoside of this invention has the following structure where the R groups from above are acyl protecting groups and X1, X2, and X3 are appropriate atoms or ligands:
The ACE orthoester comprises protecting groups. No prior art describes the use of protected orthoesters in oligonucleotide synthesis. It was not known that an orthoester utilizing, for example, protected ethylene glycol ligands would be stable to oligonucleotide synthesis conditions. It also was not known that orthoesters would be significantly more labile once protecting groups were removed from the ethylene glycol ligands. The novel orthoesters have been utilized for the synthesis of RNA of higher quality than that disclosed in prior art. Furthermore, this invention has made it possible to synthesize, handle, analyze and use RNA with ease comparable to DNA. Some useful aspects of this invention are as follows:
(1) It is possible to analyze and handle RNA while it is still 2xe2x80x2-protected. The structure of a typical ribonucleotide in a polymer of the present invention has the 2xe2x80x2-EG modified structure:
The ability to analyze and handle polymers containing the 2xe2x80x2-EG-modified ribonucleotide subunit is important for two major reasons: (a) The 2xe2x80x2-modified RNA is relatively stable to degradation. Therefore, it is not necessary to observe stringent sterile conditions while handling, analyzing and purifying 2xe2x80x2-modified RNA synthesized using this invention. (b) While the RNA is still 2xe2x80x2-modified, it is possible to analyze and purify the oligonucleotides. Over 200 sequences have been synthesized according to this invention and it has been possible to resolve every oligonucleotide into a major product during analysis. (To those skilled in the art, it is known that a percentage of RNA oligonucleotides, approximately 5-10%, can not be resolved under routine analysis conditions if the RNA is fully deprotected because of strong secondary structures and folding common to RNA.) Thus, it is possible to synthesize RNA sequences without concern over whether it will be possible to analyze the final product.
(2) The RNA quality is consistent with any sequence For comparison, it has been reported that a 27-mer was synthesized with standard 5xe2x80x2-silyl-2xe2x80x2-orthoester chemistry in 35% overall yield. The same 27-mer was synthesized with 5xe2x80x2-DMT-2xe2x80x2-TBDMS chemistry in 45% overall yield. Using a novel orthoester of the invention, a comparable 27-mer was synthesized in  greater than 70% overall yield. A 36-mer described in a following example was routinely synthesized in 65-70% overall yield. Coupling yields of  greater than 99% were possible in  less than 90 seconds. For many applications using RNA synthesized with this invention, it is no longer necessary to purify the RNA after synthesis as the quality of the crude RNA synthesized is sufficient for subsequent use. The high quality and high yields of RNA observed with such short coupling times are comparable with those routinely experienced in DNA synthesis.
(3) The final 2xe2x80x2-deprotection conditions of the RNA oligonucleotide are the mildest ever reported for this application by an order of magnitude. For example, 2xe2x80x2-ACE-uridine has a half life of xcx9c7.5 minutes at pH 2, 25xc2x0 C., which is comparable to previously reported orthoesters and acetals that have been used in oligonucleotide synthesis. However, when the ACE orthoester is modified by ester hydrolysis, the 2xe2x80x2-modified uridine is 10 times more labile with a half life of  less than 45 seconds at pH 2, 25xc2x0 C. Several assays demonstrated that there was no detectable degradation or isomerization under the extremely mild conditions used to 2xe2x80x2-deprotect RNA oligonucleotides synthesized according to this invention.
This invention includes several other protecting groups. For example, the following 2xe2x80x2-acetal protecting group is included within this invention and can be synthesized by those skilled in the art utilizing the present specification and well-known synthetic techniques.
This protecting group, or variations thereof, would exhibit similar properties to the orthoester protecting groups of this invention. Therefore, this invention includes other classes of protecting groups.
The present invention also provides polymers and oligonucleotides that comprise an acid-labile protecting group wherein the half life of the protecting group is as follows: the half life of that protecting group, when on the 2xe2x80x2-hydroxyl of a uridine nucleoside, is  less than 3 minutes at pH 2, 25xc2x0 C.
The invention also provides oligonucleotide comprising an acid-labile 2xe2x80x2-hydroxyl protecting group wherein the half life of the protecting group is as follows: the half life of that protecting group, when on the 2xe2x80x2-hydroxyl of a uridine nucleoside, is  less than 3 minutes at pH 2, 25xc2x0 C.
Further, the invention provides an oligonucleotide comprising an acid-labile 2xe2x80x2-hydroxyl protecting group which is itself protected by a second protecting group, and, wherein upon removal of the second protecting group(s) on the first protecting group, yield an acid-labile first protecting group wherein the half life of the first protecting group is now as follows: the half life of the deprotected first protecting group, when on the 2xe2x80x2-hydroxyl of a uridine nucleoside, is  less than 3 minutes at pH 2, 25xc2x0 C.
The features of the novel orthoesters of the present invention have made it possible to routinely synthesize RNA in high quality. Following synthesis, it is now possible to analyze almost any RNA oligonucleotide. Prior to this invention, this was not always possible. The RNA synthesized with this invention can be handled without the need for sterile conditions. When ready for use, the RNA is easily deprotected under extremely mild conditions that do not degrade the RNA nor contribute any detectable impurities. Oligonucleotides and polymers synthesized with this invention may be used for a wide array of purposes in various applications, including as antisense molecules, enzymatic molecules, diagnostic molecules, therapeutic molecules and research molecules. All of these uses are well known to those skilled in the art. The 2xe2x80x2-modification can be left on for subsequent applications, for example, analysis and purification, or it may be removed, if desired, to yield RNA with 2xe2x80x2-hydroxyl groups.


This invention reveals improvements in a flexible robotic limb which can function as a robotic snake. These improvements are particularly pertinent to miniaturization applications such as catheters or positioners for microsurgery, micro-assembly, micro-manipulation, or micro-exploration. This invention developes detailed new component designs, and it combines the basic structure with systems for sensing, control, and signal transmission. These systems include pressure sensors, length sensors, protective skins, ultrasonic imagers, cutting tools, and multiplexing schemes.

This invention reveals improvements in a flexible robotic limb which can function as a robotic snake, here called a robo-snake. These improvements are particularly pertinent to miniaturization applications such as catheters or positioners for microsurgery, micro-assembly, micro-manipulation, or micro-exploration. This invention features improvements in a prior-art flexible robotic arm of Rennex, U.S. Pat. No. 5,129,279, which issued in July, 1992. This prior-art invention incorporated the following basic structural features. The flexible arm comprised a series of expansible base units which were interconnected by six independently controlled length actuators. This interconnection was accomplished with universal joints. This structure was very versatile is terms of its motion. Each stage could extend, tilt, twist, and expand or contract radially. The combination of stages could position its working end along a tortuous path, and it could self-propel itself along a grid, a tunnel, or a blood vessel. It could also grip objects or position tools or imaging devices. Other features included optimal simplicity of control, ease of construction, lightness, and stiffness.
The most important element of the current invention in its two forms, a robo-snake or a flexible robotic arm, is the length actuators, especially for miniaturization. Inventor Rennex has submitted three patents for length actuators with the same application date as for the current invention. (These serial numbers will be provided.) All three of these attempt to minimize the actuator size. The first is titled "Inchworm Actuator"; it provides for improvements over the prior art of inchworm linear motors to achieve miniature actuators. The second is titled "Micro-Actuator"; it provides for a two-way, micro-machined actuator which can be internally locked at a particular length. The third is titled "Free-Standing Traveling-Wave Actuator"; it provides for a traveling-wave ultrasonic motor of minimal size which can be free-standing as required in the robo-snake. All of these actuators can be configured in such a way that they are "free-wheeling" when the power is cut off, which may be an advantage in terms of safety.


A rake apparatus includes an elongated handle with an upper wall, a peripheral wall, and an open bottom end. A head is attached to the bottom end and has a bottom surface having an opening therein. Each of a plurality of first teeth is attached to the head. An axle is mounted within the opening. A pawl is rotatably mounted on the axle. The pawl includes a first arm and a second arm orientated generally perpendicular to each other. The second arm extends downwardly through the opening. Each of a plurality of second teeth is attached to the second arm. An actuator is coupled to the first arm and is adapted for selectively urging the first arm upwardly. The actuator may be selectively actuated so that the second teeth are moved toward the first teeth.

1. Field of the Invention
The present invention relates to rake devices and more particularly pertains to a new rake device for raking leaves into a pile that may then be picked up using the device.
2. Description of the Prior Art
The use of rake devices is known in the prior art. U.S. Pat. No. 3,601,966 describes a device that includes a pair of sets of teeth that may be moved toward each other by moving an actuator up and down a handle of the device. Another type of rake device is U.S. Pat. No. 6,336,314 again includes an actuator that is slid upwardly or downwardly a handle to move sets of teeth away for toward each other.
While these devices fulfill their respective, particular objectives and requirements, the need remains for a device that includes an actuator that does not require the movement of an actuator upwardly and downwardly along a handle so that the device is simpler to use and requires less effort. This also allows a person to retain their lower hand at fixed position on the handle so that the use of the device for both raking leaves and for lifting leaves is more comfortable.


In a turret type yarn winder, a contact pressure acting on a roller bail by a yarn package is detected and used for controlling the rotation of a turret disc in accordance with the development of yarn layers on a bobbin so that the contact pressure is maintained at a predetermined value. A roller bail (5) detects a pressure acting thereon from a yarn package by a contact pressure sensor (7). If the detected contact pressure is higher than a target value, the turret disc (1) is driven in the direction to reduce the contact pressure. A driving torque of a motor (11) for driving the turret disc (1) is controlled in the normal/reverse direction by a signal corresponding to a deviation between the detected contact pressure and the target value so that the detected contact pressure coincides with the target value.

1. Field of the Invention
The present invention relates to an apparatus for controlling a rotation of a turret disc in a turret type yarn winder based on the detected contact pressure between a yarn package and a roller bail.
2. Description of the Related Arts
A yarn winder has been already disclosed in Japanese Unexamined Patent Publication (Kokai) No. 2-255468, in which a displacement of a roller bail supported by an arm to be in contact with a yarn package is detected by a proximity switch and a turret disc is positively made to rotate in a normal direction based on the detected signal so that an increase of a package diameter is absorbed thereby, while adjusting a contact pressure onto the yarn package, i.e., a pressing force of the roller bail against the yarn package by the adjustment of a spring stiffness.
According to the above turret type yarn winder of the prior art, since the positional control of the roller bail based on the rotation of the turret disc and the adjustment of a contact pressure are carried out independently from each other, it is necessary to vary a spring stiffness and determine conditions by detecting loads (contact pressures) applied thereby on the roller bail at the respective positions at which the control is carried out, whereby a prompt and accurate adjustment is difficult. Particularly, when a plurality of turret type winders are densely arranged in multiple stairs and rows, the contact pressure adjustment is very complicated because it is necessary to determine the contact pressure of the respective winder by the individual mechanical adjustment.


The invention is an atomizing pump for dispensing a quantity of liquid in a fine spray. The pump mechanism is constructed entirely of plastic, including the inlet and outlet valves, the piston, pump housing, and spring. This construction allows the pump to be easily recycled. The pump can also be constructed using two biasing springs. The first spring is used only to return the pump piston; the second spring is used only to bias the outlet valve. In this manner, the person operating the pump only presses downwardly against the force of the first spring, and not the second. As a result of this biasing arrangement, less actuation force is required.

Atomizing pumps which dispense a quantity of liquid in a fine spray are generally constructed with a metallic coil spring which is used to bias the pump piston axially outwardly. More often than not, the spring is located within the pump chamber, or below the pump chamber in a location through which liquid flows on its way to the pump chamber. Examples of pumps which include a metallic coil spring in the pump chamber include U.S. Pat. Nos. 3,211,346; 3,746,260 and 4,113,145. Examples of pumps which include a metallic coil spring in a location through which liquid flows on its way to the pump chamber include U.S. Pat. Nos. 4,025,046; 4,122,982 and 4,389,003. In all of these pumps, the liquid to be dispensed contacts the metallic spring as it passes from the container to the atmosphere via the pump.
Although many atomizing pumps are still manufactured with metallic valve mechanisms, other pumps have been created which eliminate the need for metallic valve mechanisms. An example of the latter is shown in U.S. Pat. Nos. 4,113,145; 4,144,987 and 4,389,003. However, these pumps still include a metallic coil spring which is in contact with the liquid to be dispensed. The contact of liquids with metallic elements, either valves or springs, can result in unwanted chemical reactions which can degrade either the liquid or the metallic elements. Furthermore, the existence of metallic parts within a plastic pump makes the pump difficult to recycle, as the metallic parts must be removed from the plastic before it is remelted.
Another form of atomizing pump which has come into use in recent years is the so-called "precompression" pump. This type of pump has an outlet valve which opens in response to a predetermined minimum pressure in the chamber. This is generally accomplished by including a valve member or stem within the pump chamber which has a net upwardly-facing surface upon which pressure acts. When the pressure acting on this valve member is sufficiently high, it overcomes a biasing spring, pushing the valve member downwardly and opening the outlet valve. Examples of these types of pumps are found in U.S. Pat. Nos. 3,399,836; 4,025,046; 4,144,987 and 4,389,003. These pumps use a single metallic coil spring, which urges the valve member as well as the piston member upwardly. Because this one spring is used to resist pressure action on the valve stem and to return the pump piston, the spring must be sufficiently stiff to provide both functions. However, this high stiffness requires an operator to press down with a fairly high actuation force on the actuator to ensure spraying. High actuation force is sometimes difficult to achieve for the elderly, children and some women; the result is that these precompression pumps are sometimes perceived as difficult to operate by certain people.