MX2008004951A - Methods and compositions for the treatment of neuropsychiatric and addictive disorders - Google Patents

Abstract

The present invention relates to methods and compositions for treating neuropathic pain and neuropsychiatric disorders by administering agents that are effective in reducing the effective amount, inactivating, and/or inhibiting the activity of a Na+-K+-2Cl-(NKCC) cotransporter. In certain embodiments, the Na+-K+-2Cl-co-transporter is NKCC1.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF NEUROPSYCHIATRIC AND ADDICTIVE ALTERATIONS TECHNICAL FIELD OF THE INVENTION The present invention is concerned with methods and compositions for the treatment of selected conditions of the central and peripheral nervous systems using non-synaptic mechanisms. More specifically, the present invention is concerned with methods and compositions for the treatment of neuropsychiatric and addictive disorders by administering agents that modulate the expression and / or activity of co-transporters of sodium-potassium fluoride.

BACKGROUND OF THE INVENTION Neuropathic pain and nociceptive pain differ in their etiology, pathophysiology, diagnosis and treatment. Nociceptive pain occurs in response to the activation of a specific subset of peripheral sensory neurons, the nociceptors. It is generally acute (with the exception of arthritic pain), self-limiting, and serves as a biological receptor function by acting as a warning of tissue damage. It is commonly well localized and often has a bereavement or pulse quality. Examples of nociceptive pain include postoperative pain, dislocations, bone fractures, burns, bumps, contusions, inflammation (of an infection or arthritic alteration), obstructions and myofacial pain. Nociceptive pain can usually be treated with opioids and non-spheroidal anti-inflammatory drugs (NSAIDs). Neuropathic pain is a common type of chronic non-malignant pain that is the result of an injury or malfunction in the central or peripheral nervous system and does not serve any biological protective function. It is estimated that it affects more than 1.6 million people in the population of the United States of America. Neuropathic pain has many different etiologies and can occur, for example, due to trauma, diabetes, infection with herpes zoster (shingles), peripheral neuropathies of HIV / AIDS, late stage cancer, amputation (including mastectomy), carpal tunnel, chronic alcohol use, exposure to radiation and as an unwanted side effect of neurotoxic treatment agents, such as certain anti-HIV and chemotherapeutic drugs. In contrast to nociceptive pain, neuropathic pain is often described as "burning," "electric," "tingling," or "shooting" in nature. It is frequently characterized by chronic allodynia (defined as pain resulting from a stimulus that does not ordinarily produce a painful response, such as a light touch) and hyperalgesia (defined as an increased sensitivity to a normally painful stimulus) and may persist for months or years longer. beyond the obvious healing of any damaged tissues. Neuropathic pain is difficult to treat. Analgesic drugs that are effective against normal pain (eg, narcotic opioids and non-spheroidal anti-inflammatory drugs) are rarely effective against neuropathic pain. Similarly, drugs that are active in neuropathic pain are not usually effective against nociceptive pain. Standard drugs that have been used to treat neuropathic pain often seem to act selectively to relieve certain symptoms but not others in a given patient (eg, relieving allodynia but not hyperalgesia). For this reason, it has been suggested that successful therapy may require the use of multiple different combinations of drugs and individualized therapy (see, for example, Bennett, Hosp. Pract. (Off Ed.) 33: 95-98, 1998). Treatment agents commonly employed in the management of neuropathic pain include tricyclic antidepressants (eg, amitriptyline, imipramine, desipramine, and clomipramine), systemic local anesthetics, and anticonvulsants (such as phenytoin, carbamazepine, valproic acid, clonazepam, and gabapentin). Many anticonvulsants originally developed for the treatment of epilepsy and other alterations and attacks have found application in the treatment of non-epileptic conditions, which include neuropathic pain, mood disturbances (such as bipolar affective disorder) and schizophrenia (for a review of the use of antiepileptic drugs in the treatment of non-epileptic conditions, see Rogawski and Loscher, Nat. Medicine, 10 : 685-692, 2004). Thus, it has been suggested that epilepsy, neuropathic pain and affective alterations have a common pathophysiological mechanism (Rogawski & amp; amp; amp; amp;; Loscher, ibid; Ruscheweyh & Sandkuhler, Pain 105: 327-338, 2003), that is, a pathological increase in neuronal excitability, with an inappropriately corresponding high frequency of spontaneous firing of neurons. However, only some and not all antiepileptic drugs are effective in the treatment of neuropathic pain and in addition such antiepileptic drugs are effective only in certain subsets of patients with neuropathic pain (McCleane, Expert, Opin, Pharmacother, 5: 1299-1312, 2004). Epilepsy is characterized by abnormal discharges of brain neurons and is commonly manifested as several types of attacks. The epileptiform activity is identified with synchronized discharges that occur spontaneously with neuronal populations that can be measured using electrophysiological techniques. This synchronized activity, which distinguishes epileptiform activity from non-epileptiform activity, is termed "hypersynchronization" because it describes the state in which individual neurons become increasingly likely to discharge in a blocked manner over time. Hyperinchromised activity is commonly induced in experimental models of epilepsy either by increased or decreasing excitatory synaptic currents and therefore it was assumed that hyperexcitability per se was the defining element involved in the generation and maintenance of epileptiform activity. Similarly, it was believed that neuropathic pain involved the conversion of neurons involved in the transmission of pain from a state of normal sensitivity to one of hypersensitivity (Costigan &Woolf, Jnl.Pain 1: 35-44, 2000). The focus on the development of treatments for both epilepsy and neuropathic pain has thus been in the suppression of neuronal excitability either by: (a) suppressing the generation of action potential; (b) increase the inhibitory synaptic transmission or (c) decrease the excitatory synaptic transmission. However, it has been shown that hypersynchronous epileptiform activity can be dissociated from hyperexcitability and that in furosemide chloride cation co-transport inhibitor reversibly blocks increased discharges without reducing hyperexcited synaptic responses (Hochman et al., Science 270: 99-102). , nineteen ninety five) . Both the abnormal expression of sodium channel genes (Waxman, Pain 6: S133-140, 1999; Waxman et al., Proc. Nati. Acad.

Sci USA 96: 7635-7639, 1999) and pacemaker channels (Chaplan et al., J. Neurosci, 23: 1169-1178, 2003) are believed to play a role in the molecular basis of neuropathic pain. Neuropsychiatric disorders, in which alterations of anxiety are included, are treated in general by consulting room and / or with drugs. Many of the drugs currently used in the treatment of such alterations have significant negative side effects, such as tendencies to induce dependence. The chloride cation co-transporters (CCC) are important regulatory regulators of neuronal chloride concentration that are thought to influence cell-to-cell communication as well as various aspects of neuronal development, plasticity and trauma. The CCC gene family consists of three broad groups: Na + -Cl co-transporters (NCC), K + -C1 co-transporters (KCC) and Na + -K + -2C1 co-transporters (NKCC). NKCC isoforms have been identified NKCC1 is found in a wide variety of secretory epithelium and non-epithelial cells, whereas NKCC2 is mainly expressed in the kidney.For a review of the structure, function and regulation of NKCC1, see Haas and Forbush, Annu., Rev. Physiol. 62: 515-534, 2000. Randall et al., Have identified two linked variants of the Slcl2a2 gene encoding NKCC1, referred to as NKCCla and NKCClb { Am. J. Physiol. 273 (Cell Physiol. 42): C1267-1277, 1997.) NKCC1 is a gene that has 27 exons, whereas the NKCClb variant misses exon 21. The joint NKCClb variable is expressed mainly in the brain.It is thought that NKCClb may be more of 10% active compared to NKVVla, either in its proportional present in a much less in the brain than its NKCCla. It has been suggested that differential binding of the NKCC1 transcript may play a regulatory role in human tissues (Vibat et al., Biochem 298: 218-230, 2001). The co-transport of Na-K-Cl in all cells and tissues is inhibited by diuretic loops, including furosemide, bumetanide and benzmetanide. Mice expelled by Na-K-2C1 co-transporter have been shown to have impaired nociception phenotypes as well as abnormal walking and locomotion (Sung et al., Jnl, Neurosci, 20: 7531-7538, 2000). Delpire and Mount have suggested that NKCC1 may be involved in the perception of pain. { Ann. Rev. Physiol. 64: 803-843, 2002). Laird et al. recently described studies demonstrating reduced pulsation hyperalgesia in mice expelled with NKCC1 compared to wild type mice and heterozygous mice. { Neurosci. Letts. 361: 200-203, 2004). However, in this acute pain model no difference in punctuated hyperalgesia was observed between the three groups of mice. Morales-Aza et al. Have suggested that, in arthritis, the altered expression of NKCC1 and the K-Cl co-transporter KCC2 may contribute to the control of spinal cord excitability and may thus represent therapeutic targets for the treatment of inflammatory pain. (Neurobiol, Dis 17: 62-69, 2004). Granados-Soto et al. Have described studies in rats in which formalin-induced nociception was reduced by administration of the NKCC inhibitors bumetanide, furosemide or piretanide (Pain 114: 231-238, 2005). While the model of acute pain induced by formalin is used extensively, it is believed to have little relevance to chronic pain conditions (Alker et al., Mol.Med. Today 5: 319-321, 1999). The co-treatment of brain damage induced by exposure to episodic alcohol with an NMDA receptor antagonist, NMDA-free receptor and Ca2 + channel antagonists together with furosemide has been shown to produce alcohol-dependent cerebrocortical damage by 75-85%, while which prevents brain hydration and electrolyte elevations (Collins et al, FASEB J., 12: 221-230, 1998). The authors stated that the results suggest that furosemide and related agents may be useful as neuroprotective agents in alcohol abuse. illis et al., have published studies indicating that nedocromil sodium, furosemide and bumetanide inhibit sensory nerve activation by reducing sting and histamine-induced information responses in the human skin in vivo. Espinosa et al. and Ahmad et al., have previously suggested that furosemide could be useful in the treatment of certain types of epilepsy. { Spanish Medicine 61: 280-281, 1969; and Brit. J. Clin. Pharmacol. 3: 621-625, 1976). As with epilepsy, the pharmacological intervention approach in neuropathic pain has been to reduce neuronal hyperexcitability. Most of the agents currently used to treat neuropathic pain point to synaptic activity in excitatory pathways, for example, modulating the release or activity of excitatory neurotransmitters, potentiating inhibitory pathways, blocking the ion channels illustrated in the generation of impulses and / or act as membrane stabilizers. Conventional agents and therapeutic methods for the treatment of neuropathic pain and neuropsychiatric disorders thus reduce neuronal excitability and inhibit synaptic firing. A serious deficiency of these therapies is that they are non-selective and exert their actions in both normal and abnormal neuronal populations. This leads to negative and unwanted side effects, which can affect normal CNS functions, such as knowledge, learning and memory and produce adverse psychological and psychological effects in the treated patient. Common side effects include over-sedation, dizziness, memory loss and liver damage. Accordingly, there is a continuing need for methods and compositions for the treatment of neuronal disorders that disrupt the hyper-synchronized neuronal activity without diminishing the neuronal excitability and spontaneous synchronization required for the normal functioning of the central and peripheral nervous systems. Addictive alterations, such as eating disorders (in which obesity is included), addiction to narcotics, sexual addiction, alcoholism and smoking, are a major public health problem that impacts society at multiple levels. It has been estimated that substance abuse costs the United States of America more than 484 billion per year. Current strategies for the treatment of addictive disorders include psychological counseling and support, use of therapeutic agents or a combination of both. It is known that a variety of agents affecting the central nervous system have been used in various contexts to treat a number of directly or indirectly stored indications with addictive behaviors. For example, the combination of phentermine and fenflurmin) was used for many years to exert an anorectic effect to treat obesity. Topiramate is an anticonvulsant that was originally developed as an anti-diabetic agent and is approved for use in the treatment of epileptic seizures in adults and children. It is a GABA receptor agonist and has sodium channel blockage activity. Studies on the specificity of topiramate in the treatment of alcohol dependence showed that oral administration of topiramate led to a decrease in strong drinking and alcohol cravings, with a concurrent increase in days of abstinence improved liver infusions (Johnson et al. Lancet, 361: 1677-85, 2003). It has also been shown that topiramate is effective in the treatment and alteration of eating by gluttony associated with obesity (McElroy et al., Am. J. Psychiatry 160: 255-261, 2003; McElroy et al., J. Clin. : 1463-9, 2004), and bipolar alteration (Suppes, J. Clin. Psychopharmacol., 22: 599-609, 2002). More recently, it has been suggested that topiramate may be an effective treatment for obesity.

Brief description of the invention The treatment compositions and methods of treatment of the present invention are useful for the treatment of conditions, in which addictive alterations and neuropsychiatric alterations are included, such as bipolar alterations, anxiety alterations, (in which panic, social anxiety disorder, obsessive-compulsive disorder, post-traumatic stress disorder, generalized anxiety disorder and specific phobia are induced (American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4th edition - Text Revision, 2000)), depression and schizophrenia, which are characterized by neuronal hypersynchrony. The compositions and methods of the invention can be used to reduce the neuronal hypersynchrony associated with addictive and / or neuropsychiatric disorders without suppressing their neuronal excitability, thereby avoiding the undesirable side effects frequently associated with agents currently used for the treatment of addictive alterations and alterations. neuropsychiatric Addictive alterations include eating disorders (which include obesity and feeding by buddha), alcoholism, sexual addiction, addiction to narcotics and smoking, addiction to exercise and games. As used herein, the term "effective alteration" is defined as an alteration characterized by an uncontrollable compulsion to repeat a treatment regardless of its consequences. The compositions and methods of the invention can be employed to reduce the neuronal hypersynchrony associated with such connections without suppressing neuronal excitability, thereby avoiding undesirable side effects. The methods and compositions disclosed herein in general involve non-synaptic mechanisms and modulate, in general, reduce the synchronization of the activity of the neuronal population. The synchronization of the activity of the neuronal population is by manipulating the concentrations and anion gradients in the central and / or peripheral nervous systems. More specifically, the compositions of the invention are capable of reducing an effective amount, inactivating and / or inhibiting the activity of a Na-K ^ -2C1 co-transporter (NKCC). Especially preferred treatment agents of the present invention exhibit a high degree of NKCC co-transporter antagonistic activity in cells of the central and / or peripheral nervous system, for example, glial cells, Schwann cells and / or neuronal cell populations exhibit a lower degree of activity of renal cell populations. In one embodiment, the compositions of the invention are capable of reducing the effective amount, inactivating and / or inhibiting the activity of the NKCC1 co-transporter. NKCC1 antagonists are especially preferred treatment agents for use in the methods of the invention. NKCC co-transporter antagonists that can be usefully employed in the treatment compositions of the invention include, but are not limited to, SNK-targeted NKCC co-transporter antagonists such as furosemide, bunetamide, ethacrynic acid, torsemide, azosemide. muzolimine, piretanide, tripamide and the like, as well as thiazide and thiazide-like diuretics, such as bendroflumethiazide, benzthiazide, chlorothiazide, chlorhydrothiazide, hydroflumethiazide, methyl clotiazide, polythiazide, trichloromethazine, chlorthalidone, indapamide, metolazone and kinetazone, together with analogues and functional derivatives of such components. Analogs of NKCC SNC-targeted co-transporter antagonists such as furosemide, bumetanide, piretanide, azosemide and torsemide can be usefully employed in the compositions and methods of the invention and include those provided below as Formulas I-VII. It is believed that such analogs have increased lipophilicity and reduced diuretic effects compared to NKCC SNC co-transporter antagonists from which they are derived and thus result in fewer undesirable side effects when employed in the methods of treatment of the invention. In certain embodiments, compounds are provided according to formulas I, II, III, IV, V and / or VI: III or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein: Ri is not present, is H, 0 or S; R 2 is not present, is H or when Ri is O or S, R 2 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, alkylaminodialkyl, alkylcarbonylamino-dialkyl, alkyloxycarbonylalkyl, alkylcarbonyloxyalkyl, alkylaldehyde, alkylketoalkyl, alkylamide, alkarylamide , arylamide, an alkylammonium group, alkylcarboxylic acid, alkylheteroaryl, alkylhydroxy, a biocompatible polymer such as alkyloxy (polyalkyloxy) -alkylhydroxyl, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) and a polyethylene glycol ether (ether). PEG), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl, unsubstituted or substituted and when Ri is not present, R 2 is selected from the group consisting of hydrogen, N, N-dialkylamino, N, N-dialcarylamino, N, -diarylamino, N -alkyl-N-alkarylamino, N-alkyl-N-arylamino, N-alkaryl-N-arylamino unsubstituted or substituted; R3 is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy, unsubstituted or substituted; and R4 and R5 are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, carbonylalkyl, carbonylalkyl, carbonylaryl and salts thereof such as sodium, potassium, calcium, ammonium, tialkarylammonium and tetraalkylammonium salts, with the following conditions in some embodiments: R3 of formula I is not phenyloxy when Ri is O and R2, R4 and 5 are H, more specifically, in some embodiments, the compound of formula I is not bumetanide; R3 of formula III is not Cl, when Ri is O and R2, R4 and R5 are H, more specifically, in some embodiments, the compound of formula III is not furosemide; R2 of formula III is not methyl when Ri is O, R3 is Cl and R (and R5 are H, more specifically, in some embodiments, the compound of formula III is not furosemide methyl ester; R3 of formula V is not phenyloxy when Ri is 0 and R2, 4 and R5 are H, more specifically, in some embodiments, the compound of formula V is not pyretanide Additional moieties of the present invention provide compounds according to formula VII: or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein: R2, R4 and R5 are defined above and R6 is selected from the group consisting of alkyloxycarbonylalkyl, alkylaminocarbonylalkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible polymer such as alkyloxy (polyalkyloxy) ) -alkylhydroxyl, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) and a polyethylene glycol (PEG ether), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and rathylthioalkyl ether, unsubstituted or substituted, with the proviso that, in some embodiments, R3 is not Cl, when R4, R5 and Rg are H, more specifically, in some embodiments, the compound of formula VII is not azosemide. In still further embodiments of the present invention there are provided compounds according to formula VIII: or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof; wherein: R7 is not present or is selected from the group consisting of hydrogen, alkyloxycarbonylalkyl, alkylaminocarbonylalkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible polymer such as alkyloxy- (polyalkyloxy) alkylhydroxy, a polyethylene glycol (PEG), a polyethylene glycol ester ( PEG ester) and an ether of polyethylene glycol (PEG ether), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl, unsubstituted or substituted and X is a halide such as a portion of bromide, chloride, fluoride, iodide or an anionic portion such as mesylate or tosylate; alternatively, X is not present and the compound forms an "internal" salt or suteryonic salt (where R7 is H), with the proviso that, in some embodiments, R7 is always present and X is not present. More specifically, in some embodiments, the compound of formula VIII is not torsemide. Modalities of the present invention provide drugs capable of passing through the blood-brain barrier comprising a compound of formula I, II, III, IV. V, VI, VII and / or VIII, or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof. In some embodiments, the prodrug compound is provided in an amount effective to regulate a CNS alteration. In particular modalities, the CNS alteration is epilepsy, anxiety, neuropathic pain, neural function, drug addiction / physical dependence and / or migraines. Modalities of the present invention provide a pharmaceutical composition comprising a compound of formulas I, II, III, IV. V, VI, VII and / or VIII, or a salt, solvate, tautomer, pharmaceutically acceptable hydrate or combination thereof and a pharmaceutically acceptable carrier, excipient or diluent. In certain embodiments, the compound of the pharmaceutical composition is present in an amount effective to regulate a CNS alteration. In particular modalities, the CNS alteration is an addictive alteration, a neuropsychiatric alteration or neuropathic pain. The embodiments of the present invention provide for the use of compounds described above for the preparation of a medicament for treatment and / or prevention of a CNS disorder selected from the group consisting of: addictive alterations, neuropsychiatric disorders and neuropathic pain. In one embodiment, the level of diuresis that occurs following the administration of an effective amount of an analogue provided below as Formulas I-VIII is less than 99%, 90%, 80%, 70%, 60%, 50% , 40%, 30%, 20% or 10% of that which occurs immediately after the administration of an effective amount of the original molecule from which the analogue is derived. For example, the analog may be less diuretic than the original molecule when administered at the same mg / kg dose. Alternatively, the analog may be more potent than the original molecule from which it is derived, such that a smaller dose of the analog is required for effective relief of symptoms, thereby producing less of a diuretic effect. Similarly, the analog may have a longer effect relationship in the treatment of alterations than the original molecule, such that the analog may be administered less frequently than the original molecule, thus leading to a lower total diuretic effect within a given period of time. Other treatment agents that can be usefully employed in the compositions and methods of the invention include but are not limited to: antibodies or antigen binding fragments thereof, which specifically bind to NKCCl; solvable NKCC1 ligands, small molecule inhibitors of NKCCl; antisense oligonucleotides to NKCCl; small interfering RNA molecules NKCCl-specific (siRNA or iRNA) and soluble NKCCl molecules designed. Preferably, such antibodies or antigen binding elements thereof and small molecule inhibitors of NKCCl specifically bind to the NKCCl domains involved in the bimetanide linkage as described for example in Haas and Forbush II, Annu. Rev. Physiol. 62: 515-534, 2000. The sequence of polypeptides for human NKCCl is provided in SEQ ID NO: 1 with the corresponding cDNA sequence being provided in SEQ ID NO: 2. Since the methods and treatment agents of the present invention employ "non-synaptic" mechanisms, little or no suppression of neuronal excitability is presented. More specifically, the treatment agents of the invention cause little (less than a 1% change in comparison to pre-administration levels) or no suppression of action potential generation or excitatory synaptic transmission. Indeed, a slight increase in neuronal stability may occur after the administration of certain of the inventive treatment agents. This is in stark contrast to conventional anti-epileptic drugs currently used in the treatment of neuropathic pain, which can suppress neuronal excitability. The methods and agents of treatment of the present invention affect the synchronization or relative synchrony of the activity of the neuronal population. Preferred methods and treatment agents modulate the extracellular concentration of anionic chloride and / or gradients in the central and peripheral nervous systems to reduce neuronal synchronization or relative synchrony without substantially affecting neuronal excitability. In another aspect, the present invention relates to methods and agents for alleviating neuropathic pain or abnormal pain perception, by applying or modulating spontaneous hyper-synchronized bursts or bursts of neuronal activity and the propagation of action potential or impulse conduction in certain nerve cells and fibers of the peripheral nervous system, for example, primary sensory afferent fibers, pain fibers, dorsal horn neurons, and supraesinal sensory and pain pathways. In another aspect, the present invention is concerned with methods and agents for the treatment, improvement and / or prevention of neuropsychiatric disorders and addictive alterations. In one aspect, the present invention is concerned with methods and agents for the treatment or prevention of neuropsychiatric disorders and addictive and / or compulsive disorders by affecting or modulating the spontaneous hyper-synchronized bursts of neuronal activity and the propagation of action potentials or impulse conduction. in certain cells and nerve fibers of the peripheral nervous system, for example, primary sensory afferent fibers, pain fibers, dorsal horn neurons, and supraspinal sensory and pain pathways. The treatment agents of the invention can be used in combination with other known treatment agents, such as those currently used in the treatment of neuropsychiatric disorders. Those of skill in the art will appreciate that the combination of a treatment agent of the present invention with another known treatment agent may involve both synaptic and non-synaptic mechanisms. The compositions and methods of treatment of the present invention may be used therapeutically and episodically following the onset of symptoms or prophylactically, prior to the onset of specific symptoms. In certain embodiments, the methods of the invention for the treatment of addictive disorders involve the administration of a treatment agent comprising a diuretic (eg, a loop diuretic such as furosemide)., toracemide or bumetanide or a thiazide or thiazide-like diuretic), in combination with one or more antidiuretic components, in order to counteract undesirable diuretic effects of the primary treatment agent. Negative side effects that can be avoided by such methods include loss of body water and depletion of electrolytes (such as potassium, magnesium, calcium and thiamine) and vitamin B. Antidiuretic components that can be usefully employed in such methods include, for example, antidiuretic hormones, such as vasopressin, which increases the reabsorption of water by the kidneys and salts and electrolytes, which act to replenish lost ions due to diuresis. Preferred mode, the diuretic treatment agent and the anti-diuretic component are combined together in a composition formulated as a liquid beverage, food or food supplement. In certain embodiments, the treatment agents employed in the methods of the invention are capable of crossing the brain-blood barrier and / or are administered using administration systems that facilitate the administration of the agents to the central nervous system. For example, various blood-brain barrier (BBB) permeability enhancers can be used, if desired, to transiently and reversibly increase the permeability of the blood-brain barrier to a treatment agent. Such BBB permeability-enhancing agents may include leukotrienes, bradykinin agonists, histamine, strong-bond disruptors (eg, zonulin), hyperosmotic solutions (eg, mannitol), cytoskeletal contraction agents, short-chain alkyl glycerols (eg, 1-0-pent ilglycerol) and others that are currently known in the art. The above-mentioned aspects and additional aspects of the present invention, together with the way to obtain them, will be better understood with reference to the following more detailed description. All references disclosed herein are incorporated herein by reference in their entirety as if each were incorporated individually.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A, 1A1, IB, 1B1, 1C, 1C1 and ID show the effect of furosemide on the stimulation evoked after the discharge activity in rat hippocampal joints. Figures 2A-2R show the blockade of furosemide in spontaneous epileptiform burst discharges through a spectrum of in vitro models.

The . Figures 3A-3H show the furosemide blockade of the electrical "epilepticus" status evoked by clinical acid in urethane-anesthetized rats, with upper trace EKG records and cortical EEG records shown in bottom tracings. Figures 4A and 4B show a schematic diagram of ion co-transport under reduced chloride concentration conditions. Figure 5 shows that significantly less paralysis was observed in animals treated with either bumetanide or furosemide than in animals receiving the vehicle alone in a conditioning test to contextual fear in rats. Figure 6 shows reference surprise amplitudes in a fear-enhanced surprise test in rats. Figure 7 shows the response amplitude in rats in surprise tests alone determined immediately after administration of either DMSO or alone, bumetanide or furosemide. Figure 8 shows the difference score (surprise-only surprise-enhanced fear) on the test day in rats treated with either DMSO, bumetanide or furosemide. Figure 9 shows the extent of surprise alone in rats one week after administration of either D SO, bumetanide or furosemide. Figure 10 shows the reference score in rats after administration of either DSMO, bumetanide or furosemide. Figure 11 shows the difference score percent (surprise alone - fear-enhanced surprise) on the test day in rats treated with either one of the following bumetanide analogs: bumetanide 3- (dimethylaminopropyl) ester; Benzyltrimethylammonium salt of bumetanide; bumetanide dibenzylamide; bumetanide cyanomethyl ester; , N-methylglycolamide ester of bumetanide; N, N-dimethylglycolamide bumetanide ester; morpholinomethyl ester of bumetanide, pivaxethyl ester of bumetanide; bumetanide methyl ester; bimetanide diethylamide and bumetanide benzyl ester. The vehicle was DMSO.

DETAILED DESCRIPTION OF THE INVENTION As discussed above, preferred agents and methods of treatment of the present invention, for use in the treatment of neuropathic pain, addictive alterations and / or neuropsychiatric disorders, modulate or disrupt the synchrony of neuronal population activity in Enhanced synchronization areas by reducing the activity of NKCC co-transporters. As described in detail hereinafter and illustrated in the examples, the movement of ions and modulation of ionic gradients by means of ion-dependent co-transporters, preferably co-transporters dependent on cation-chloride, is critical for the regulation of neuronal synchronization. It has been thought that for a long time the chloride co-transport function is mainly directed to the movement of chloride out of the cells. The sodium-independent transporter, which has been shown to be neuronally located, moves the chloride ions out of the neurons. Blocking this carrier, such as by administration of the furosemide bottle diuretic, leads to hyperexcitability, which is the short-term response of chloride cation co-transporters such as furosemide. However, the long-term response to furosemide shows that the sodium-dependent movement of chloride ions, moderated by the co-transporter of Na + -K + -2C1"glial associated NKCC1, plays an active role in blocking the neuronal synchronization, without affecting the excitability and cellular activity evoked by stimulation Haglund and Hochman have shown that furosemide is able to block epileptic activity in humans while not affecting normal brain activity (J. Neurophysiol. (Feb. 23)., 2005) doi: 10.1152 / j n .009 4.200). These results provide support for the belief that the methods and compositions of the invention can be effectively employed in the treatment of neuropathic pain, neuropsychiatric disorders and addictive and / or compulsive disorders without giving treatment to undesirable side effects frequently observed with conventional treatments. . As discussed above, the NKCC1 splice variant referred to as NKCClb is more active than the NKCCla variant. A central or peripheral nervous system that expresses a little more percentage of NKCClb and may thus be more prone to alterations such as neuropathic pain and epilepsy. Similarly, a treatment agent that is more specific for NKCClb compared to NKCCla may be more effective in the treatment of such alterations. The methods of the invention can be used for the treatment and / or prophylaxis of neuropathic pain which have, for example, the following etiologies: alcohol abuse; diabetes; eosinophilia-myalgia syndrome; Guillain-Barre syndrome, exposure to heavy metals such as arsenic, lead, mercury and thallium; HIV / AIDS; exposure to anti-HIV / AIDS drugs; malignant tumors; medications that include amiodarone, aurothioglucose, cisplatin, dapsone, stavudine, zalcitabine, didanosine, sidulfiram, FK506, hydralazine, isoniazid, metronidazole, nitrofurantoin, paclitaxel, phenytoin, and vincristine; monoclonal gammopathies; multiple sclerosis; central post-stroke pain, post-herpetic neulangia; trauma in which include carpal tunnel syndrome, cervical or lumbar radiculopathy, complex regional pain syndrome, spinal cord injury and stump pain; trigeminal neuralgia; vasculitis; vitamin B6 magnesium deficiency and certain vitamin deficiencies (B12, Bl, B6, E). Neuropsychiatric disorders that can be effectively treated using the methods of the invention include but are not limited to, bipolar disorders, anxiety alterations, panic disorders, depression, schizophrenia, obsessive-compulsive disorders and post-traumatic stress syndrome. Anxiety disorders are classified into several subtypes: panic disorder, social anxiety disorder, obsessive-compulsive disorder, post-traumatic stress disorder, generalized anxiety disorder and specific phobia (American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4a Edition, 2000). The methods of the invention can be used for the treatment and / or prophylaxis of addictive and / or compulsive alterations such as: eating disorders, in which obesity and feeding by gluttony are included; alcoholism, addiction to narcotics and smoking. Compositions that can be effectively employed in the methods of the invention are capable of reducing the effective amount, deactivating and / or inhibiting the activity of a co-transporter of Na + -K + -2C1 (NKCC). Preferably, such compositions are capable of reducing the effective amount, of inactivating and / or inhibiting the activity of the NKCC1 co-transporter. In certain embodiments, the compositions of the invention comprise at least one treatment agent selected from the group consisting of: NKCC1 antagonists (which include but are not limited to, small molecule inhibitors of NKCC1, antibodies or fragments of antigen link thereof, which bind specifically to NKCC1 and soluble NKCC1 ligands); antisense oligonucleotides NKCC1; small NKCCl-specific interfering RNA molecules (siRNA or RNAi) and soluble NKCC1 molecules designed. In preferred embodiments, the treatment agent is selected from the group consisting of: SNC-targeted co-transporter NKCC antagonists, such as furosernide, bumetanide, ethacrynic acid, torsemide, azosemide, muzolimine, piretanide, tripamide and the like; thiazide and thiazide-like diuretics, such as bendroglimetiazide, benzthiazide, chlorotiazide, hydrochlorothiazide, hydro-flumetiazide, methylclothiazide, polythiazide, trichlormethiazide, chlorthalidone, indapamide, metolazone and kinetazone and analogs and functional derivatives of such components. According to some embodiments, the methods of the present invention employ new compounds. Thus, any of the R groups as defined herein may be excluded or modified in order to exclude a known compound and / or provide a new compound. In certain embodiments, the methods disclosed herein employ compounds that have the following structures: I, or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein: Ri is not present, is H, O or S; R2 is not present, is H or when Ri is O or S, R2 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, alkylaminodialkyl, alkylcarbonyl-aminodialkyl, alkyloxycarbonylalkyl, alkylcarbonyloxylalkyl, alkylaldehyde, alkylketoalkyl, alkylamide, alkarylamide , arylamide, an alkylammonium group, alkylcarboxylic acid, alkylheteroaryl, alkylhydroxy, a biocompatible polymer such as alkyloxy (poly-alkyloxy) alkylhydroxy, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) and a polyethylene glycol ether (ether) of PEG), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl, unsubstituted or substituted and when Ri is not present, R 2 is selected from the group consisting of hydrogen, N, dialkylamino, N, N-dialcarylamino, N, N-diarylamino, N-alkyl-N-alkarylamino, N-alkyl-N-arylamino, N-alkaryl-N-arylamino unsubstituted or substituted; R3 is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy, unsubstituted or substituted; and R4 and R5 are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, carbonylalkyl, carbonylalcaryl, carbonylaryl and salts thereof such as sodium, potassium, calcium, ammonium, trialkylarylammonium and tetraalkylammonium salts, with the following conditions in some embodiments: R3 of formula I is not phenyloxy when Rx is 0 and R2, R4 and R5 are H, more specifically, in some embodiments, the compound of formula I is not bumetanide; R3 of formula III is not Cl, when Ri is O and R2, R4 and R5 are H, more specifically, in some embodiments, the compound of formula III is not furosemide; R2 of formula III is not methyl when Ri is O, R3 is Cl and R4 and R5 are H, more specifically, in some embodiments, the compound of formula III is not furosemide methyl ester; R3 of formula V is not phenyloxy when Ri is 0 and R2, R4 and R5 are H, more specifically, in some embodiments, the compound of formula V is not pyretanide. In some embodiments of the present invention, the compound of formula I may be bumetanide, bumetanide aldehyde, bumetanide methyl ester, bimetanide cyanomethyl ester, bumetanide ethylester, bumetanide isoamyl ester, bumetanide ocylester, bumetanide benzyl ester, bumetanide dibenzylamide, diethylamide bumetanide, morpholinosteril ester of bumetanide, 3- (dimethylaminopropyl) ester of bumetanide, N, N-diethylglycolamidoester of bumetanide, N, N-dimethyl-glycolamidoester of bumetanide, pivaxet ester of bumetanide, propaxet ester of bumetanide, methoxy (polyethyleneoxy) ni- bumetanide ethyl ester, benzyltrimethalmmonium salt of bumetanide, and cet iltrimet iammonium salt of bumetanide. In particular embodiments, the compound is not bumetanide. In other embodiments of the present invention, the compound of formula I can be thioacid of bumetanide [- (C = 0) -SH], S-methyl thioester of bumetanide, S-cyanomethylthioester of bumetanide, S-methylthioether of bumetanide, S-isoamyl amioester of bumetanide, S-octyl ioester of bumetanide, S-benzylthioester of bumetanide, S- (morpholinoethyl) thioester of bumetanide, S- [3- (dimethyl-aminopropyl)] thioester of bumetanide, S- (N, N-diethylglycol- amido) thioester of bumetamide, S- (N, N-dimethylglycol-amido) thioester of bumetamide, S-pivaxetyl thioester of bumetamide, S-propaxetyl thioester of bumetamide, S- [methoxy (polyethyleneoxy) n -! - ethyl] thioester of bumetamide, [- (C = 0) -S ~] thiocacid benzyltrimethylammonium salt of bumetamine and thioacid salt of [- (C = 0) -S] cetyltrimethylammonium salt of bumetamine. In some embodiments of the present invention, the compound of formula II can be bumetanide thioate [- (C = S) -OH] metastable, O-met ilt ioester of bumetanide, 0-cyanomethylthioester of bumetanide, 0-et ilthioester of bumetanide, O-isoamyl thioester of bumetanide, 0-octyl thioether of bumetanide, Bumetanide O-benzylthioether, 0- (morpholinoethyl) thioether of bumetanide, 0- [3- (dimethylaminopropyl)] thioester of bumetanide, 0- (N, N-diethylglycolamido) thioester of bumetanide, bumetanide, 0- (, N -dimet ilglicolamiodo) thioester of bumetanide, 0-pivaxet iltioester of bumetanide, 0-propaxetiltioester of bumetanide, 0- [methoxy (polyethyleneoxy) ni-ethyl] thioester of bumetanide, thioacid salt of [- (C = S) -0] benzyltrimethylammonium bimetanide and thioacid salt of [- (C = S) -0] cetyltrimethylammonium bumetanide. In some embodiments of the present invention, the compound of formula II can be thioaldehyde of bumetanide, dithioacid [~ (C = S) -SH] of bumetanide, methyldithioester of bumetanide, cyanomethyldithioester of bumetanide, ethyldithioester of bumetanide, isoamyldithioester of bumetanide, butetanide octyldithioester, bumetanide benzyldithioester, bumetanide dibenzylthioamide, bumetanide diethylstiamide, bumetanide morpholinoethyldithioester, bumetanide 3- (dimethylaminopropyl) dithioester, bumetanide N, N-diethylglycolamidodithioester, bumetanide N, N-dimethylglycolamidodithioester, pivaxethyl-dithioester of bumetanide, bumetanide propaxethylthioester, 0- [methoxy (polyethyleneoxy) ni-ethyl] dithioester of bumetanide, benzyltrimethylammonium dithioacid salt of bumetanide and butetanide cetyltrimethylammonium dithioacid salt. In other embodiments of the present invention, the compound of formula III can be furosemide, furosemide aldehyde, furosemide methyl ester, furosemide cyanomethyl ester, furosemide ethyl ester, furosemide ioamyl ester, ochilyester <; of furosemide, furosemide benzyl ester, furosemide morpholinester, furosemide 3- (dimethylaminopropyl) ether, furosemide N-diethylglycolamidoester, furosemide N, N-dimethylglycolaminoester, furosemide pivaxetlester, furosemide propaxetilyester, metoci (polyethylenenoxy) ni furosemide ethyl ether, furosemide benzyltrimethylammonium salt and furosemide cetyltrimethylammonium salt. In particular embodiments, the compound is not furosemide.

In further embodiments of the present invention, the compound of formula II can be [- (C = 0) -SH] thioacid of furosemide, S-methylthioester of furosemide, S-cyanomethylthioester of furosemide, S-ethylthioester of furosemide, S-isoamymiyoster of firosemide, S-octyl thioester of furosemide, S-benzyl thioester of furosemide, S- (morpholinoethyl) thioester of furosemide, S- [3- (dimethylaminopropyl)] thioester of furosemide, S- (N, N-diethylglycolamido) thioester of furosemide, S- (N, -dimethylglycolamido) thioester of furosemide, S-pivaxetyl thioester of furosemide, S-propaxethylthioester. of furosemide, S- [methoxy (polyethyleneoxy) n -! - ethyl] thioester of furosemide, thioacid salt of [- (C = 0) -S] benzyltrimethylammonium furosemide and thioacid salt of [- (C = 0) -S] cetyltrimethylammonium furosemide. In other embodiments of the present invention, the compound of formula IV may be the [- (C = 0) -OH ~] thioacid of metastable furosemide, furosemide O-methylthioester, furosemide 0-cyanomethylthioester, furosemide 0-ethyl thioester, O-isoamyl thioester of furosemide, O-octyl thioester of furosemide, O-benzyl thioester of furosemide, 0- (morpholinoethyl) thioester of furosemide, 0- [3- (dimethylaminopropyl)] thioester of furosemide, 0- (N, -diethylglycolamido) thioester furosemide, furosemide 0- (N, N-dimethylglycolamido) thioester, furosemide O-pivaxetyl thioester, furosemide O-propaxetyl thioester, furosemide 0- [methoxy (polyethyleneoxy) ni-ethyl] thioester, thioacid salt of [- (C = S) -0 ~] benzyltrimethylammonium furosemide and thioacid salt of furosemide [- (C = S) -0 ~] cetyltrimethylammonium. In further embodiments of the present invention, the compound of formula IV can be thioaldehyde furosemide, [- (C = S) -SH] dithioacid furosemide, methyldithioester furosemide, cyanomethyldithioester furosemide. Furosemide ethyldithioester, furosemide isoamyldithioester, furosemide octyldithioester, furosemide benzyldithioester, furosemide dibenzyl thioamide, furosemide diethyl amide, furosemide morpholinoethyl iodithioester, furosemide (3-dimethylaminopropyl) dithioester, furosemide N, N-diethylglycolamidodithioester, N, N-dimethylglycolamidodithioester from furosemide, pivaxet iodithioester from furosemide, propaxethyldithioester from furosemide, methoxy (polyethyleneoxy) n-α-ethyldithioester from furosemide, dithioacid salt from benzyltrimethylammonium from furosemide and dithioacid salt from cetyltrimethylammonium from furosemide. In still further embodiments of the present invention, the compound of formula C can be piretanide, pyretanide methyl ester aldehyde, piretanide cyanomethyl ester, piretanide ethyl ester, piretanide isoamyl ester, piretanide octyl ester, piretanide benzyl ester, piretanide dibenzylamide, diethylamide of pyretanide, morpholinoethexester of piretanide, (3-dimethylaminopropyl) ester of piretanide,?,? - diethylglycolamidoester of piretanide, N, N-dimethylglycolamidoester of piretanide, pivaxetilester of piretanide, propaxetilester of piretanide, methoxy (polyethyleneoxy)? -? - ethyl ester of piretanide, benzyltrimethylammonium salt of piretanide and cetyltrimethylammonium salt of piretanide. In particular embodiments, the compound is not pyretinide. In some embodiments of the present invention, the compound of formula V may be [- (C = 0) -SH] thioacid of piretanide, S-methylthioester of piretanide, S-cyanomethylthioester of piretanide, S-etiol thioester of piretanide, S- pyretamide isoamythioester, pyretanide S-octylthioester, pyretanide S-benzyl thioester, pyretanide S- (morpholinoethyl) thioester, pyretanide S- [3- (dimethylaminopropyl)] thioester, pyretanide S- (N, N-diethylglycolamido) thioester) , S- (N, N-dimethylglycolamido) thioester of piretanide, S-pivaxetilothioester of piretanide, S-propaxetilothioster of piretanide, S- [methoxy (polyethyleneoxy) ni-ethyl] thioester of piretanide, thioacid salt of [- (C = 0) -S-] of benzyltrimethylammonium of piretanide and thioacid salt of [- (C = 0) -S ~] of cetyltrimethylammonium of piretanide. In embodiments of the present invention, the compound of formula VI can be thioacid [- (C = 0) -OH "] of metastable piretanide, pyretanide O-methylthioester, pyritlanide O-cyanomethylthioester, pyretanide O-ethylthioester, pyretanide O-isoamyl thioester, O- pyrithanide octylthioester, pyretanide O-benzyl thioester, pyretanide 0- (morpholinoethyl) thioester, pyretanide 0- [3- (dimethylaminopropyl)] thioester, pyretanide 0- (N, N-diethylglycolamido) thioester, p-0- (N, N-dimethylglycolamido) thioester of piretanide, O-pivaxetyl thioester of piretanide, O-propaxethyl thioester of piretanide, 0- [methoxy (polyethyleneoxy) n-x-ethyl] thioester of piretanide, thioacid salt of [- (C = S) -0] of piretanide benzyltrimethylammonium and thioacid salt of [- (C = S) -0"] cetyltrimethylammonium piretanide. In some embodiments, the compound of formula VI can be piretanide thioaldehyde, dithioacid of [(C = S) -0H "] of piretanide, pycytane-methyldithioester, pyranthanide cyanomethyldithioester, pyretanide isoamyldithioester, pyrytanide octyldithioester, pyrimetidine benzyldithioester, dibenzyldithioamide of piretanide, piretanide diethylthioamide, piretanide morpholinoethyldithioester, pyretanide 3- (dimethylaminopropyl) dithioester, pyrimetidine N, N-diethylglycolamidodithioester, pyretanide N, N-dimethylglycolamidodithioester, piretanide pivaxethyldithioester, pyrethanide propaxethyldithioester, methoxy (polyethyleneoxy) ni- piretanide ethyl phthioether, pyrithanolamide benzyltrimethylammonium dithioacid salt and pyrithanolamide cetyltrimethylammonium dithioacid salt In certain embodiments, the methods disclosed herein employ compounds having the following structure: VII or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein: R3, R4 and R5 are defined above and R¾ is selected from the group consisting of alkyloxycarbonylalkyl, alkylaminocarbonylalkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible polymer such as alkyloxy (polyalkyloxy) -alkylhydroxyl, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) or a polyethylene glycol ether (PEG ether), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl, unsubstituted or substituted, with the proviso that , in some embodiments, R3 of formula VII is not Cl, when R4, R5 and R6 are H, more specifically, in some embodiments, the compound of formula VII is not azosemide.

In certain embodiments, the compounds of formula VII can be tetrazolyl-substituted azosemides (such as methoxymethyltetrazolyl-substituted azosemides, methylthiomethyltetrazolyl-substituted azosemides and N-mPEG350-tetrazolyl-substituted azosemides), azenemide benzyltrimethylammonium salt and / or cetyltrimethylammonium salt of azosemide In still further embodiments, the methods disclosed herein employ compounds according to formula VIII VIII or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein: R7 is not present or is selected from the group consisting of hydrogen, alkyloxycarbonylalkyl, alkylaminocarbonylalkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible polymer such as alkyloxy (polyalkyloxy) alkylhydroxyl, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) and an ether of polyethylene glycol (PEG ether), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl, unsubstituted or substituted and X "is a halide such as bromide, chloride, fluoride, iodide or an anionic portion such as a mesylate or tosylate, alternatively, X "is not present and the compound forms an" inner "salt or suteryonic salt (wherein R7 is H), with the proviso that, in some embodiments, R7 is always present and X "is not present.More specifically, in some embodiments, the compound of formula VIII is not torsem In some embodiments, the compounds of formula VIII may be quaternary ammonium salts of pyridin-substituted torsemide or the corresponding internal salts (sutures). Examples include, but are not limited to, salts of methoxymethylpyridinium torsemide, salts of methylthiomethylpyridinium torsemide and salts of N-mPEG350-pyridinium torsemide. Modalities of the present invention further provide intermediate compounds formed by means of the synthetic methods described herein to provide compounds of formulas I, II, III, IV, V, VI, VII and / or VIII. Intermediate compounds may possess utility as therapeutic agents for the range of indications described herein and / or reagents for additional synthesis methods and reactions. As previously indicated, any of the R groups as defined herein may be excluded from the compounds of the present invention, particularly with reference to denoting new compounds of the present invention. The term "aryl" or Ar as used herein refers to an aromatic group, a heteroaryl group or an optionally substituted aromatic group or heteroaryl group fused to one or more optionally substituted aromatic groups or heteroaryl groups, optionally substituted with appropriate substituents , wherein, but not limited to, lower alkyl, lower alkoxy, lower alkylsulfamyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, tetrazolyl, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, acyl, aroyl, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, or perfluoroalkyl, multiple degrees of substitution are allowed. Examples of aryl include but are not limited to phenyl, 2-naphthyl, 1-nagthyl, 4-pyridyl and the like. The term "halo" as used herein refers to bromine, chlorine, fluoro or iodine. Alternatively, the term "halide" as used herein refers to bromide, chloride, fluoride or iodide. The term "hydroxy" refers to the -OH group.

The term "alkoxy" as used herein, alone or as part of another group, refers to an alkyl group, as defined herein, appended to the original molecular portion by means of an oxy group. Representative examples of alkoxy include but are not limited to methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like. The terms "alkaryloxy" or "oleylalcaryl" as used herein refer to the -O-alkyl-aryl group wherein Ar is aryl. Examples include but are not limited to benzyloxy, oxylbenzyl, 2-naphthyloxy and oxy-2-naphthyl. The term "aryloxy" as used herein is assigned to the group -ArO wherein Ar is aryl or heteroaryl. Examples include but are not limited to phenoxy, benzyloxy and 2-naphthyloxy. The term "amino" as used herein refers to -NH2 in which one or both hydrogen atoms may optionally be replaced by alkyl or aryl and one of each, optionally substituted. The terms "alkylthio" or thioalkyl "as used herein, alone or as part of another group, refers to an alkyl group, as defined herein, appended to the original molecular portion by means of a sulfur moiety. Representative examples of alkylthio include but are not limited to methylthio, thiomethyl, ethylthio, thioethyl, n-propylthio, thio-n-propyl, isopropylthio, thio-isopropyl, n-butylthio, thio-n-butyl and the like. "arylthio" and "thioaryl" as used herein, refer to the group ArS, where Ar is aryl, Examples include, but are not limited to, phenylthio, thiophenyl, 2-naphthylthio and thio-2-naphthyl. "Alkylthio" or "thioalkaryl" as used herein refers to the S-alkyl-aryl group in which Ar is aryl, Examples include, but are not limited to, benzylthio, thiobenzyl, 2-naphthylthio and thio-2- Naphthyl The term "carboxy" as used herein refers to the group -CO2H.The term "ammonium c "uaternario" as used herein refers to a chemical structure having four bonds to nitrogen with a positive charge on nitrogen in the "onium" state, ie, "R4N +" or "nitrogen ^ quaternary", where R is an organic substituent, such as alkyl or aryl. The term "quaternary ammonium salt" as used herein, refers to the association of the quaternary ammonium cation with an anion. The term "substituted" as used herein, refers to the replacement of one or more of the hydrogen atoms of the group replaced by substituents known to those experienced in the art and which result in a stable compound as described below in the present.

Examples of suitable replacement groups include but are not limited to alkyl, acyl, alkenyl, alkynyl, cycloalkium, aryl, alkaryl, hydroxy, thio, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl, thiocarboxyalkyl, carboxyryl, thiocarboxiaryl, halo, oxo, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl, cycloalkyl, heterocycloalkyl, dialkylaminoalkyl, carboxylic acid, carboxamido, haloalkyl, dihaloalkyl, trihaloalkyl, trihaloalkoxy, alkylthio, aralkyl,. alkylsulfonyl, arylthio, amino, alkylamino, dialkylamino, guanidino, ureido, nitro and the like. Substitutions are permissible when such combinations result in stable compounds for the intended purpose. For example, substitutions are permissible when the resulting compound is sufficiently robust to survive isolation to a useful degree of purity of a reaction mixture and formulation to a therapeutic or diagnostic agent or reagent. The term "effective amount" or "effective" is intended to designate a dose that causes relief of symptoms of a disease and disorder as denoted by clinical testing and evaluation or observation of the patient and / or the like. "Effective" or "effective" amount may also designate a dose that causes a detectable change in biological or chemical activity, detectable changes may be detected and / or additionally quantified by that experienced in the art by the relevant mechanism or process., "effective amount" or "effective" can designate an amount that maintains a desired physiological state, that is, reduces or prevents a significant decrease and / or promotes improvement in the condition of interest. As is generally understood in the art, the dosage will vary depending on routes of administration, symptoms and body weight of the patient, but also depending on the compound being administered. The term "solvate" as used herein, is intended to refer to a pharmaceutically acceptable solvate form or a specified compound that retains the biological effectiveness of such a compound, e.g., resulting from a physical association of the compound with one or more molecules of solvents. Solvate examples include, without limitation, compounds of the invention in combination with water, 1-propanol, 2-propanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid or ethanolamine. The term "hydrate" as used herein refers to the compound when the solvent is water. The term "biocompatible polymer" as used herein refers to a polymer portion that is substantially non-toxic and does not tend to produce substantial immune responses, coagulation or other undesirable effects. According to some embodiments of the present invention, the polyalkylene glycol is a biocompatible polymer wherein, as used herein, the polyalkylene glycol refers to straight or branched chain polyalkylene glycol polymers such as polyethylene glycol, polypropylene glycol or polybutylene glycol and further includes the monoalkylether of the polyalkylene glycol. In some embodiments of the present invention, the polyalkylene glycol polymer is a polyalkylene glycol lower alkyl portion such as a polyethylene glycol (PEG) portion, a polypropylene glycol portion or a polybutylene glycol portion. PEG has the formula -HO (CH2CH2O) nH, wherein n can range from about 1 to about 4000 or more. In some embodiments, n is from 1 to 100 and in other embodiments, n is from 5 to 30. The PEG portion may be linear or branched. In further embodiments, PEG can be attached to groups such as hydroxyl, alkyl, aryl, acyl or ester. In some embodiments, PEG may be a PEG alkoxy, such as methoxy-PEG (or mPEG), wherein one term is a relatively inert alkoxy group, while the other term is a hydroxyl group.

Synthesis Methods The compounds of the formulas I-VIII can be synthesized using traditional synthesis techniques well known to those skilled in the art. More specific synthesis routes are described later in the present. The bumetanide analogs are simplified by reacting the carboxylic acid portion of bumetanide with various reagents. For example, bumetanide can undergo esterification via reaction with alcohols, in which linear, branched, substituted or unsubstituted alcohols are included. Bumetanide may also be alkylated via reaction with unsubstituted and substituted unsubstituted alkyl halides and aryl halides, which include chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, N- dietary ilacetamide and the like. PEG-type esters can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000-OT and the like. "Axetil" type esters can also be formed by alkylation using alkyl halides such as chloromethyl piovalate or chloromethyl propylate. Bumetanide can also undergo amidation by reaction with appropriate substituted or unsubstituted alkylamines or arylamines, either after conversion to the acid chloride or by using an acxtivator such as l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). . The bumetanide can also be reacted with a quaternary ammonium hydroxide, such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide to form quaternary ammonium salts of bumetanide. As used herein, a co-transporter is electroneutral, which moves equal amounts of ionic species charged oppositely from one side of one membrane to another. As used herein, a co. Cation-chloride transporter refers to a co-transporter that makes move one or several cations with an equal number of chloride ions. Exemplary chloride cation co-transporters include, but are not limited to, the co-transporter Na + K + 2C1"diuretic-sensitive loop in the brain (NKCC1) and the co-transporter Na + Cl" thiazide-sensitive (NCC). Discussions regarding the molecular classification of cation-chloride co-transporters, their physiology and pharmacology can be found in Mount et al. J Exp Biol 201: 2091-2102, 1998 and Russell JM. Physiol Rev. January 2000; 80 (1): 211-76. The cerebrospinal-specific co-transporter NKCC1 is an isoform of its kidney analog NKCC2. Furosemide and bumetanide are classic examples of NKCC antagonists. The thiazide-sensitive co-transporter is antagonized by thiazide diuretics. Exemplary thiazide diuretics include but are not limited to chlorothiazide, hydroxychlorothiazide and benzthiazide. The modification of the diuretic compound or diuretic-like compound may include reacting the diuretic or diuretic-like compound with and / or functional compound selected from the group consisting of an aluminum hydride, alkyl halide, alcohol, aldehyde, alkaryl halide, mono- and dialkylamino, mono- and dialcarylamine and mono- and diarylamine and quaternary ammonium salt, unsubstituted or substituted or combinations thereof. Non-limiting examples of compounds that can be used as starting material are exemplified below. bumetanide Furosemide Indi ce Merck 13a Merck Index 13th edition. 2001 1471 edition. 2001 4330 Merck Index 13a Indi ce Merck 13th edition. 2001 7575. edition, 2001, 924. loresamide Indi ce Merck 13th edition, 2001, 9629.

The compounds of formulas I, II, III, IV, V, VI, VII and / or VIII can be synthesized using traditional synthesis techniques well known to those skilled in the art. More specific synthesis routes are described later in the present.

A. Bumetanide analogues, thiobumetanide analogues and dithiobumetanide analogues 1. Thiobumetanide and dithiobumetanide thiobumetanide [- (C = 0VSH1 thiobumetanide C = S> O H) bumetanide thioacid dithioacid of [- (C = O SH] bumetanide thioacid of [- (C = 3> 0H] bumetanide The thiobumetanide analogs are synthesized by reacting the carboxylic acid portion of bumetanide from various reagents. For example, bumetanide can undergo conversion to the corresponding thioacid by treatment with thionyl chloride to form the corresponding bumetanide acid chloride followed by reaction with sodium hydrogen sulfide to give thiobumetanide [~ (C = 0) -SH], also known as thioacid of [- (C = 0) -SH] of bumetanide by the methodology of Noble, P. and Tarbell, DS, Qrg. Synth., Coll. Vol. IV, John iley & Sons, Inc., New York, 1963, 924-927. See Reaction Scheme 1. Thiobumetanide can undergo conversion to the corresponding bumetanide thioacid chloride with thionyl chloride, followed by the treatment of thioacid chloride with sodium hydrogen sulfide to give dithiobumetanide [- (C = S) -SH], also known as dithioacid of [~ (C = S) -SH] of bumetanide by similar methodology. The reaction of the thioacid chloride of bumetanide with secondary amines will give the corresponding bumetanide thioamides. Bumetanide can also react with phosphorus pentasulfide to produce bumetanide phyto-acid. For reviews of this body of chemistry see "Thioacyl Halides", "Thiocarboxylic 0-Acid Esters" and "Dithiocarboxylic Acid Esters", all by Glass, RS in: Science of Synthesis, (Charette, AB, Ed.), Volume 22, Thieme Chemistry, 2005, Chapters 22.1.2, 22.1.3 and 22.1.4 and references thereof. See also "Synthesis of Thioamides and Thiolactams", Schaumann, E., in: Comprehensive Organic Synthesis, (Trost, B. M. and Fleming, I., Eds.), Permagon Press, 1991, Volume 6, Chapter 2.4, pp. 450-460 and reference thereof.

Reaction Scheme 1: Synthesis of thiobumetanide. { thioacid of [- (C = 0) -SH] of bumetanide alkaline metal salt of thiobumetanide thiobumetanide. { thioacid of [- (C = 0) -SH] of bumetanide) 2. Bumetanide and S-Thiobumetanide Analogs The bumetanide analogs are synthesized by reacting the carboxylic acid portion of bumetanide with various reagents. For example, bumetanide can undergo esterification via reaction with alcohols, in which linear, branched, substituted or unsubstituted alcohols are included. Bumetanide or thiobumetanide can also be alkylated via reaction with unsubstituted and substituted alkyl halides and alkyl halides, which include chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, -diet ilacetamide and the like. PEG-type esters can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000-OT and the like. "Axetyl" esters can also be formed by alkylation by using alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Bumetanide can also undergo amidation by reaction with appropriate substituted or unsubstituted alkylamines or arylamines, either after conversion to the acid chloride or by using an activator such as l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Bumetanide or thiobumetanide can also be reacted with a quaternary amino hydroxide, such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form quaternary amino salts of bumetanide or thiobumetanide. Reaction Schemes 2, 3 and 4 present synthesis schemes of some exemplary compounds according to formula I.

Reaction Scheme 2: Synthesis of exemplary compounds according to Formula I Reaction Scheme 3. Synthesis of compounds exemplifying agreement with Formula I to R = benzyl, cetyl, methyl, ethyl .... R '= methyl, ethyl, propyl, ... 6 thioesters thioo "asethyl" of bumetanide R "= methyl, ethyl, propyl ... R = H, methyl; R "= methyl, ethyl, propyl, ... R '= H, methyl, ethyl, t-butyl Bumetanide salts, thiobumetanide and thiobumetanide esters and should easily undergo acid catalyzed hydrolysis and base catalyzed hydrolysis to produce bumetanide molecule containing carboxylic acid by methods well known in the art. { See Yang, W. and Drueckhammer, D.G., J. Amer. Chem. Soc, 2001, 123 (44), 11004-11009 and references herein). . { See Reaction Scheme).

Reaction Scheme 4. Hydrolysis of bumetanide, thiobumetanide and S-thiobumetanide esters salts of bumetanide esters of S-thioburnetanide (thioesters of S-burnetanide) bumetanide 3. Thiobumetanide Analogs O-Substituents and Dithiobumetanide Analogs Bumetanide can undergo conversion to the corresponding thioacid by treatment with thionyl chloride to form the corresponding acid chloride followed by reaction with sodium hydroxide or sodium hydrogen sulfide to give 0-thiobumetanide and meta-stable dithiobumetanide using the methodology of Noble, P. and Tarbell, DS, Org. Synth , Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927. . { See Reaction Schemes 5 and 6).

Reaction Scheme 5. Synthesis of metastable thiobumetanide. { thioacid of [- (C = S) -OH] of bumetanide} alkaline metal salt of thiobumetanide isomeric thiobumetanide isomer. { dithioacid of ((C = S) -OH) of bumetanide. "metastable" Reaction Scheme 6. Synthesis of dithiobumetanide, thioacid of [- (C = S) -SH] of bumetanide. alkali metal salt of dithiobumetanide dithiobumetanide. { dithioacid of [- (OS) -SH] of bumetanide} The dithiobumetanide analogs are in turn synthesized by reacting the thiocarboxylic acid portion of S-thiobumetanide with various reagents. For example, S-thiobumetanide can undergo esterification via reaction with alcohols and thiols, in which linear, branched, substituted or unsubstituted alcohols and thiols are included. S-thiobumetanide can also be activated via reaction with unsubstituted and substituted unsubstituted alkyl halides and alkaryl halides, which include chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-, N -diethylacetamide and the like. PEG-type esters can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl analogs or tosylates such as eO-PEG1000-OT and the like. "Axetil" type esters can also be formed by alkylation by using alkyl halides such as chloromethyl pivalate or chloromethyl propionate. The S-thiobumetanide may also be reacted with a quaternary ammonium hydroxide, such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form quaternary ammonium salts of thiobumetanide. See Reaction Schemes 7, 8 and 9, which show some exemplary compounds according to formula II.

Reaction Scheme 7. Synthesis of exemplary compounds according to Formula II alkyl esters of thiobumetanide 1a drtio nest R = benzyl. cetyl, methyl, ethyl R '= methyl, ethyl, propyl R "= methyl, ethyl, propyl R" = methyl, ethyl, propyl Reaction Scheme 8. Synthesis of exemplary compounds according to Formula II th quaternary ammonium salts of thiobumetanide R = benzyl, cetyl. methyl, ethyl thioesters "axetil" type of bumetanide R '= methyl, ethyl, propyl R = H, methyl; R "= methyl, ethyl, propyl R' = H, methyl, ethyl, t-butyl R" = methyl, ethyl , propyl Thiobumetanide, thiobumetanide amides, O-bumetanide esters and dithiobumetanide esters should readily undergo acid catalyzed hydrolysis and base catalyzed hydrolysis to produce the bumetanide molecule containing carboxylic acid by methods well known in the art. { See Yang, W. and Drueckhammer, D. G., J. Amer. Chem. Soc, 2001, 123 (44), 11004-11009 and references therein). For additional reviews of this body of chemistry, see "Thioacyl Halides", "Thiocarboxylic O-Acid Esters" and "Dithiocarboxylic Acid Esters", all by Glass, RS in: Science of Synthesis, (Charette, AB, Ed.), Volume 22, Thieme Chemistry, 2005, Chapters 22.1.2, 22.1.3 and 22.1.4 and references therein. See also "Synthesis of Thioamides and Thiolactams", Schaumann, E., in: Comprehensive Organic Synthesis, (Trost, B. M. and Fleming, I., Eds.), Permagon Press, 1991, Volume 6, Chapter 2.4, pp. 450-460 and references therein. . { See Reaction Scheme 9).

Reaction scheme 9. Hydrolysis of thiobumetanide, amines of thiobume añida, esters of O-thiobumetamide and esters of diiobumetanide thiobumetanide bumetanide amides (bumetanide thioamides) B. Furosemide analogs, thiofurosemide analogs and dithio-sulpho-amide analogues 1. Tiofurosemide and dithio-furosemide lyofurosemida [- (C = 0 SH) thiofurosemide [C = S 0H1] furosemide dioxyacid of [- (C = 0) -SH] furosemide thioacid of [- (C = S) -OH] furosemide The thiofurosemide analogues are synthesized by reacting the carboxylic acid portion of furosemide with various reagents. For example, furosemide may undergo conversion to the corresponding thioacid by treatment with thionyl chloride to form the corresponding furosemide acid chloride followed by reaction with sodium hydrogen sulfide to give thiofurosemide [- (C = 0-SH], also known as [ - (C = 0-SH] thioacid of furosemide by the methodology of Noble, P. and Tarbell, DS, Qrg. Synth., -Coll. Vol. IV, John Wiley &Sons, Inc., New York, 1963, 924-927. {See Reaction Scheme 10.) Thiofurosemide may undergo conversion to the corresponding furosemide thioacid chloride with thionyl chloride, followed by treatment of thioacid chloride with sodium hydrogen sulphide to give dithio-furosemide [- (C = S-SH], also known as furosemide dithioacid [- (C = S-SH], by similar methodology. {See Reaction Scheme 10).

The reaction of thioacid chloride of furosemide with secondary amines will give the corresponding furosemide thioamides. Furosemide may also undergo reaction with phosphorus pentasulfide to produce furosemide dithioacid. For reviews of this body of chemistry, see "Thioacyl Halides", "Thiocarboxylic O-Acid Esters" and "Dithiocarboxylic Acid Esters", all by Glass, RS in Science of Synthesis, (Charette, AB, Ed.), Volume 22, Thieme Chemistry, 2005, Chapters 22.1.2, 22.1.3 and 22.1.4 and references therein. See also "Synthesis of Thioamides and Thiolactams", Schaumann, E., in: Comprehensive Organic Synthesis, (Trost, B. M. and Fleming, I., Eds.), Permagon Press, 1991, Volume 6, Chapter 2.4, pp. 450-460 and references therein.

Diagram of Reaction 10. Synthesis of thiofurosemide. { thioacid of [- (C = 0) -SH] furosemide} furosemide furosemide acid chloride alkaline metal of thiofurosemide xioTurosemiaa. { thioacid of [(C = 0) SH] furosemide) 2. Furosemide and S-furosemide analogues Furosemide analogs are synthesized by methods analogous to those used in the synthesis of the burnetanide analogues. Furosemide can undergo esterification via reaction with alcohols, in which linear, branched, substituted or unsubstituted alcohol are included. Furosemide or thiofurosemide may also be alkylated via reaction with unsubstituted and substituted unsubstituted alkyl halides and alkaryl halides including, for example, chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro -N, -diethylacetamide and the like. PEG-type esters can be bonded by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-C and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000-OT and the like. "Axetyl" esters can also be formed by alkylation by using alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Furosemide can also undergo amidation by reaction with appropriate substituted or unsubstituted alkylamines or arylamines, either after conversion to the acid chloride or by using an activator such as l-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC). Furosemide or thiofurosemide can also be reacted with a quaternary ammonium hydroxide, such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form quaternary ammonium salts of furosemide or thiofurosemide. Reaction Schemes 11, 12 and 13 present some exemplary compounds according to Formula III.

Reaction Scheme 11, Synthesis of exemplary compounds according to Formula III a Reaction Scheme 12. Synthesis of exemplary compounds according to Formula III 2 thiobumetanide alkyl esters etanid 6 th axetil thioesters of bumetanide R = H. methyl, 5a quaternary ammonium salts of bumetanide R '= H. methyl, ethyl, t-butyl R = benzyl, cetyl, methyl, ethyl R' = methyl, ethyl, propyl R "= methyl, ethyl, propyl R" = methyl, ethyl, propyl Salts of thiofurosemide and S-thiofurosemide esters should readily undergo acid catalyzed hydrolysis and base catalyzed hydrolysis to produce the furosemide molecule containing carboxylic acid by methods well known in the art (see, Yang, W. any Drueckhammer, DG, J. Amer. Chem. Soc, 2001, 123 (44), 11004-11009 and references therein). (See Reaction Scheme 13).

Reaction Scheme 13. hydrolysis of thiofurosemide salts and thiofurosemide esters furosemide 3. O-substituted thiofurosemide and dithio-furosemide analogues Furosemide may undergo conversion to the corresponding thioacid by treatment with thionyl chloride to form the corresponding acid chloride followed by reaction with sodium hydroxide or sodium hydrogen sulfide to give O-thiofurosemide and dithiofurosemide by the methodology of Noble, P. and Tarbell, DS, Org. Synth , Coll. Vol. IV, John iley & Sons, Inc., New York, 1963, 924-927. . { See Schemes of. Reaction 14 and 15).

Diagram of Reaction 14. Synthesis of metastable thiofurosemide. { thioacid of [- (C = S) -OH]} of furosemide metallic salt of the isomeric isoférico isoférico thiodurosemide isodium 1 dithioacid of [- (C = S) -OH] furosemide} "metastable" Diagram of Reaction 15. Synthesis of diisofurosemide. { dithioacid of [- (C = S) SH] dithioacid} ti ofu rose measure acid chloride of thiofurosemide dithiofurosemide metallic salt of thiofurosemide alkali. { dithioacid of [(C = S) -SH] furosemide} The thiofurosemide analogues are in turn synthesized by reacting the thiocarboxylic acid portion of thiofurosemide with various reagents. For example, thiofurosemide can undergo esterification via reaction with alcohols or thiols, which include linear, branched, substituted or unsubstituted alcohols and thiols. S-thiofurosemide may also be alkylated via reaction with unsubstituted and substituted unsubstituted alkyl halides and alkaryl halides, which include chloracetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, - diethylacetamide and the like. PEG-type esters can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-C1 and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000-OT and the like. "Axetyl" esters can also be formed by alkylation by using alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Thiofurosemide can also be reacted with a quaternary ammonium hydroxide, such as a benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form quaternary ammonium salts of thiofurosemide. Reaction Schemes 14, 15, 16, 17 and 18 present synthetic schemes of some exemplary compounds according to Formula IV.

Reaction Scheme 16. Synthesis of exemplary compounds according to Formula IV R = methyl: = methyl, ethyl, propyl R '= methyl, ethyl, t-butyl Reaction Scheme 17. Synthesis of exemplary compounds according to Formula IV 6 thioesters '3xethyl' of furosemide R "= methyl, ethyl, propyl, R = H, methyl; R '= H. methyl, ethyl, t-butyl, ...

Thiofurosemioda, thiofurosemide amides and S-thiofurosemide esters should readily undergo acid catalyzed hydrolysis or base catalyzed hydrolysis to produce the furosemide molecule containing the carboxylic acid by methods well known in the art (see Yang, W. and Drueckhammer , DG, J. Amer. Chem. Soc., 2001, 123 (44), 11004-11009 and references therein). For additional reviews of this body of chemistry, see "Thioacyl Halides", "Thiocarboxylic O-Acid Esters" and "Dithiocarboxylic Acid Esters", all by Glass, R. S. in: Science of Synthesis, (Charette, A. B., Ed.), Volume 22, Thieme Chemistry, 2005, Chapters 22.1.2, 22.1.3 and 22.1.4 and references therein. See also "Synthesis of Thioamides and Thiolactams", Schaumann, E., in :, Comprehensive Organic Synthesis, (Trost, B. M. and Fleming, I., Eds.), Permagon Press, 1991, Volume 6, Chapter 2.4, pp. 450-460 and references therein. (See Reaction Scheme 18).

Diagram of Reaction 18. Hydrolysis of thiofurosemide, amides of thiofurosemide and esters of S-thiofurosemide esters of thiofurosemide thiofurosemide (thioesters of furosemide) Amides of triofurosemide furosemide (thioamides of furosemide) C. Piretanide Analogs and Tiopyrethanide Analogs 1. Tiopyrethanide and Ditopyrethanide thiopiretanide [- (C = 0) -SH] thiopytantanide [- (C = S SH) pyrithanide thioacid dioxide of [- (C = 0) -SH] pyrithanide thioacid of [- (C = S) -SH] of py etanide The piretanide analogs are synthesized by reacting the carboxylic acid portion of piretanide with various reagents. For example, the piretanide can undergo conversion to the corresponding thioacid by treatment with thionyl chloride to form the corresponding piretanide acid chloride followed by reaction with sodium hydrogen sulfide to give thiopiretanide [- (C = 0) -SH], also known as thioacid of [- (C = 0) -SH] of piretanide by the methodology of Noble, P. and Tarbell, DSr > Org. Synth , Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927. See Reaction Scheme 19. Thiopyrethanide may undergo conversion to the corresponding thio-acid chloride of thio-acid with thionyl chloride, followed by treatment of thioacid chloride with sodium hydrogen sulfide to give dithiopyrethanide [- (C = S) -SH], also known as [~ (C = S) -SH] dithioacid of piretanide by similar methodology. The reaction of thio-acid chloride of piretanide with secondary amines will give the corresponding piretanide thioarnides. The piretanide can also undergo reaction with phosphorus pentasulfide to produce pyretanide dithioacid. For reviews of this body of chemicals see "Thioacyl Halides", "Thiocarboxylic O-Acid Esters" and "Dithiocarboxylic Acid Esters", all by Glass, RS In: Science of Synthesis, (Charette, AB, Ed.), Volume 22, Thieme Chemistry, 2005, Chapters 22.1.2, 22.1.3 and 22.1.4 and references therein. See also "Synthesis of Thioamides and Thiolactams", Schaumann, E., in: Comprehensive Organic Synthesis, (Trost, B. M. and Fleming, I., Eds.), Permagon Press, 1991, Volume 6, Chapter 2.4, pp. 450-460 and references therein.

Diagram of Reaction 19. Synthesis of tiopiretañida. { thioacid of [- (C = 0) -SH] of piretanide} piretanide piretanide acid chloride tiopiretanide alkali metal salt thioiretanide. { thioacid of | - (C = 0) -SH] of piretanide! 2. Piretanide and analogs of S-thioiretanide The piretanide can undergo esterification via reaction with alcohols, in which linear, branched, substituted or unsubstituted alcohols are included. The piretanide or thioiretanide can also be alkylated via reaction with unsubstituted and unsubstituted alkyl halides and substituted alkaryl halides, which include benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, N- diethylacetamide and the like. PEG-type esters can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl or alkyloxy (polyalkyloxy) alkyl tosylates such as MeO-PEG1000-OT and the like. "Axetyl" esters can also be formed by alkylation by using alkyl halides such as chloromethyl pivalate or chloromethyl propionate. The piretanide can also undergo amidation by reaction with appropriate substituted or unsubstituted alkylamines or arylamines, either after conversion to the acid chloride by using an activator such as l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). The piretanide or thioiretanide can also be reacted with a quaternary aluminum hydroxide, such as benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form quaternary ammonium salts of piretanide or thioiretanide. Reaction Schemes 19, 20, 21 and 22 present synthesis schemes of some exemplary compounds according to formula V.

Scheme of reaction 20. Synthesis exemplary compounds according to Formula V 1 ? alkyl esters of piretanide iretanide R = methyl, ethyl, propyl, piretanide 6 esters "axetil" of pyretanide 5 salts of quaternary ammonium of pyretanide R = H, methyl; R = benzyl. cetyl. methyl, ethyl. ... 4 simple pyretanide amides R '= H, methyl, ethyl, t-butyl R' = methyl, ethyl, propyl R = H. methyl, ethyl, benzyl R "= methyl, ethyl, propyl R '= H. methyl, ethyl, benzyl R "= methyl, ethyl, propyl Reaction Scheme 21. Synthesis of exemplary compounds according to Formula V nest The salts of tiopiretanide and S-thiopiretanide esters should readily undergo hydrolysis catalyzed by acid and hydrolysis catalyzed by base to produce the bumetanide molecule containing the carboxylic acid by methods well known in the art. { See Yang, W., Drueckhammer D.G., J. Amer. Chem. Soc, 2001, 123 (44), 11004-11009 and references therein). (See Reaction Scheme 22).

Reaction Scheme 22. Hydrolysis of tiopyrethanide salts and S-thiopytantanide esters piretanide 3. O-substituted analogs of thioiretanide and di-pyrethanedide analogs The pyretanide can undergo conversion to the corresponding thioacid by treatment with thionyl chloride to form the corresponding acid chloride followed by reaction with sodium hydroxide or sodium hydrogen sulfide to give 0-t metastable iopiretanida and dith iopiretanida using the methodology of Noble, P. and Tarbell, DS, Org. Synth , Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927. See Reaction Schemes 23 and 24.

Diagram of Reaction 23. Synthesis of metastable tiopiretanide. { [- (C = S) -OH] thioacid of piretanide} tiopiretanida? =? or chlorophyll of tiopyrethanide acid dithiopyrethanide Y = S alkaline metal salt of isoprotic isopropanol isopropanol. { [-C = S) -0H] dithioacid of pyretanide) "metastable" Reaction scheme 24. Synthesis of dithiopyrethanide. { piretanide dithioacid [- (C = S) -SH]} alkali metal salt of dithiopyrethanide dithiopyrethanide. { [- (C = S) -SH] dithioacid of piretanide} The tiopiretanide analogs are synthesized by methods analogous to those used in the synthesis of the piretanide analogues. Specifically, the thioiretanide may undergo esterification via reaction with thiols, in which linear, branched, substituted or unsubstituted thiols are included. The tioioethanide can also be alkylated via reaction with unsubstituted and substituted unsubstituted alkyl halides and substituted alkaryl halides, including chloroacetonitrile, benzyl chloride, 1- (dimethylamino) propyl chloride, 2-chloro-N, - diethylacetamide and the like. PEG-type esters can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000OT and the like. "Axetyl" thioesters can also be formed by alkylation using alkyl halides such as chloromethyl pivalate or chloromethyl propionate. The tiopiretanide can also be reacted with a quaternary ammonium hydroxide, such as trimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form quaternary ammonium salts of thioiretanide. Reaction Schemes 12, 24, 25, 26 and 27 present some exemplary compounds according to formula VI.

Reaction Scheme 25. Synthesis of similar compounds according to Formula VI.

R = benzyl, cetyl, methyl, ethyl, ... 6th thioesters "axetil" type of R '= methyl, ethyl, propyl, ... piretanide R "= methyl, ethyl, propyl, ... R = H, methyl R '= H, methyl, ethyl, t-butyl, R' "= methyl, ethyl, propyl, ...

Reaction scheme 26. Synthesis of exemplary compounds according to Formula VI 2a alkyl thioesters of piretanide thiopyrethanide R = methyl, ethyl, propyl, i-propyl, butyl, i-butyl -CH2CH2 (OCH2CH2) n. ,-Y aryl and heteroaryl thioesleres of piretanide R = phenyl. benzyl, phenethyl, 2-pyridyl-3-pyridyl, ... (m = 0, 1, 2) ethanide R = H, methyl, ethyl, benzyl, R '= H, methyl, ethyl, benzyl, p-quaternary ammonium salts of piretanide thioesters type "axetil" R = benzyl, cetyl, methyl, ethyl, ... R = H, methyl; R '= methyl, ethyl, propyl, ... R "= methyl, ethyl, propyl, ... R' = H, methyl, ethyl, t-butyl, R" '= methyl, ethyl, propyl, ...

The thioiretanide, tiopyrethanide amides and thiopiretanide esters should readily undergo acid catalyzed hydrolysis and base catalyzed hydrolysis to produce the carboxylic acid containing the pyretanide molecule by methods well known in the art. { See Yang, W. and Drueckhammer, D.G., J. Amer. Chem. Soc, 2001, 123 (44), 11004-11009 and references thereof). For additional reviews of this body of chemistry, see "Thioacyl Halides", "Thiocarboxylic O-Acid Esters" and "Dithiocarboxylic Acid Esters", all by Glass, RS in Science of Synthesis, (Charette, AB, Ed.), Volume 22 , Thieme Chemistry, 2005, Chapters 22.1.2, 22.1.3 and 22.1.4 and references therein. See also "Synthesis of Thioamides and Thiolactams", Schaumann, E., in Comprehensive Organic Synthesis, (Trost, B. M. and Eleming, I., Eds.), Permagon Press, 1991, Volume 6, Chapter 2.4, pp. 450-460 and references therein. (See Reaction Scheme 27).

Scheme of Reaction 27. Hydrolysis of tiopiretanide, tiopiretanide amides and tiopiretanide esters pyrimethamide thioiretanide amides (piretanide thioamides) D. Azosemide analogs Azosemide analogs are synthesized by reacting several reagents with the tetrazolyl portion of azosemide. Azosemide can undergo hudroxyalkylation with the addition of an aldehyde, by which a hydroxyalkyl functionality is formed. An alcohol can optionally be reacted together with the aldehyde to obtain an ether. An alkylthiol can optionally be added with the aldehyde to form a thioether. The azosemide can also be alkylated by the addition of appropriate alkyl halides or alkaryl halides, which include alkyl or alkaryl halides comprising an ether or thioether bond, such as methylchloromethyl ether and benzylchloromethylthioether. Peak ethers which can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000OT and the like. "Axetyl" type analogues can also be formed by addition of alkyl or alkaryl halides, such as alkyl pivalate or chloromethyl propionate. The azosemide can also be reacted with a quaternary ammonium salt, such as benzylammonium bromide and base such as sodium hydroxide or cetyltrimethylammonium bromide and base such as sodium hydroxide, in order to form a quaternary ammonium salt of azosemide . Reaction Scheme 28 below presents a synthesis scheme of some exemplary compounds according to formula VII.

Reaction Scheme 28. Synthesis of exemplary compounds according to Formula VII 2 N-substituted alkaneside azosemide 1 azosemide alkylator route) to ethers R = H, methyl, ethyl, ... N-substituted X (CH2) mOR, Et3N base N = N alkylation route to N-substituted thioethers X (CH2) mSR, Et3N base route of N-alkylation H, NO, S XCH (R) OCOR ', formation Cl Et3N salt base. quaternary azosemide ethers R N * Br 'N-substituted NaOH R = methyl, ethyl, benzyl,. -CH2CH2 (OCH2CH2) n.1-Y gone R = benzyl. cetyl, methyl, ethyl. ... 6 azosemite type "axetil" N-substituted R = H. methyl R '= H. methyl, ethyl, t-butyl E. Torsemide analogues Torsemide analogues (also known as torasemide) are synthesized by reacting several reagents with the pyridine portion of torsemide. the torsemide can undergo alkylation by the addition of appropriate alkyl or alkaryl halides, in which benzyl chloride is included to form N-substituted quaternary ammonium salts. Alkyl halides and alkaryl halides comprising an ether linkage, in which metylchloromethyl ether and benzylchloromethyl ether are included, can be used to form N-substituted ether quaternary ammonium salts. Alkyl halides and alkaryl halides comprising a thioester linkage, in which are included metylchloromethanethioether and benzylchloromethylthioether, can be used to form N-substituted thioether quaternary ammonium salts. Quaternary ammonium salts containing PEG-type ether can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl semantics or tosylates such as MeO-PEG1000-OT and the like . "Axetyl" quaternary ammonium salts can also be formed via the addition of alkyl halides such as chloromethyl pivalate or chloromethyl propionate. Reaction Scheme 29 below presents a synthesis scheme of some exemplary compounds according to formula VIII.

Reaction Scheme 29. Synthesis of Exemplary Compounds According to Formula VIII Route F. Benzaldehyde, Bumetanide, Poretanide and Furosemide Analogs The bumetanide, piretanide and furosemide substituted benzoic acids can be selectively reduced to the corresponding bumetanide aldehyde, piretanide aldehyde and aldehyde of furosemide using ammonium-substituted ammonium hydrides such as bis (4-methylpiperazinyl) aluminum hydride by literature methods. See Muraki, M. and Mukiayama, T., Chem. Letters, 1974, 1447; Muraki, M. and Mukiayama, T., Chem. Letters, 1975, 215; and Hubert, T., D., Eyman, D. P. and iemer, D. F., J. Org. Chem., 1984, 2279. (See Reaction Scheme 30). It is well known that the more lipophilic the benzaldehydes are easily oxidized in the air, the more hydrophilic the benzoic acids and the benzaldehydes are also metabolized into the corresponding benzoic acids in vivo via the use of NADPH co-factor and with a number of enzymes P450 oxidants.

Reaction Scheme 30. If thesis of benzaldehyde analogues exemplary bumetanide, pi ethanide and furosemide. "pyretanide aldehyde" piretanide 1 -, R2 = HF R3 = 0-aryl, R "= R5 = H For reduction methods used to convert benzoic acids to the corresponding benzaldehydes, see Muraki, M. and Miukiyama, T. , Chem. Letters, 1974, 1447, ibid, 1975, 215; Hubert, T. D., Eyman, D.P. and Wierner, D. F. , J. Org. Chem., 1984, 2279. Lipophilic thiobenzaldehydes can also be prepared from the corresponding benzaldehydes by agents in the treatment including hydrogen sulphide and diphosphorus pentasulfide (See Smith, MB and March, J., March 's Advanced Organic Chemistry, 5th edition, 2001, John Wiley &Sons, Inc., New York, Part 2, Chapter 16, pp. 1185-1186 C. Sulfur Nucleophiles, Section 16-10, The Addition of H2S and Thiols to Carbonyl Compounds .). { See Reaction Scheme 31). In turn, these thiobenzaldehydes are easily converted back to the corresponding benzaldehydes under hydrolytic conditions. It is well known that more lipophilic benzaldehydes are easily oxidized in the air to the more hydrophilic benzoic acids and that the benzaldehydes are also metabolized to the corresponding benzoic acids in vivo, via the use of the NADPH co-factor with a number of enzymes of 450 oxidants. A similar mechanism can be applied for the conversions of thiobenzaldehyde to thiobenzoic acids and then benzoic acids.

Reaction scheme 31. Synthesis of exemplary thiobenzaldehyde analogs of bumetanide, piretanide and furosemide "furosemide thioaldehyde" G. PEG analogs of bumetanide, piretanide and furosemide and their counterparts of thioacid thiobumetanide, thioiretanide and thiofurosemide, dithiobumetanide, dithiopyrethanide and dithiofurosemide PEG esters of bumetanide, furosemide and piretanide can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl such as MeO-PEG350-C1 and alkyloxy (polyalkyloxy) alkyl esters or tosylates such as MeO-PEG1000-OT and the like. . { See Reaction Scheme 32).

Reaction Scheme 32. Synthesis of exemplary polyethylene glycol esters of bumetanide, furosemide and piretanide bumetanide "PEG esters of bumetanide" R2 = OCH ^ H ^ OCH ^ H ^ n.i-Y R3 = 0-aryl. RA = R5 = H m = 1 - 5, n = 1 - 100 "PEG furosemide esters" R2 = OCH2CH2 (OCH2CH2) n., - And furosemide R3 = chloride, R < j = R5 = H m = 1 - 5. n = 1 - 100"PEG esters of piretanide" R2 = OCH2CH2 (OCH2CH2) n.1-Y R3 = O-aryl, ¾ = R5 = H m = 1 - 5, n = 1 - 100 PEG-X is X- (CH2) m (OCH2CH2) nYY, wherein X is halo or another leaving group (mesylate "OMs", tosylate "OTs") and Y is OH or an alcohol protecting group such as an alkyl group , an aryl group, an acyl group or an ester group and wherein m = 1-5 and n = 1-100. PEG esters of thiobumetanide, thiofurosemide and thioiretanide can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000-OT and the similar. . { See Reaction Scheme 33).

Diagram of Reaction 33. Synthesis of exemplary polyethylene glycol thioesters of thiobumetanide, thiofurosemide and thiopiretamide thiobumetanide "PEG thioesters of bumetanide" R2 = OCH2CH2 (OCH2CH2) n., - and R3 = 0-aryl. R = R5 = H m = 1 - 5, n = 1 - 100 'PEG thioesters of furosemide "R2 = OCH2CH2 (OCH2CH2) n., - Y thiofurosemide R3 = chloride R" = R5 = H m = 1-5, n = 1 - 100 "PEG thioesters of piretanide" thiopiretanide R2 = OCH2CH2 (OCH2CH2) n., - Y R3 = 0-aryl, R "= R5 = H m = 1-5, n = 1 - 100 PEG-X is X- (CH2) m (OCH2CH2) nYY, wherein X is halo or another leaving group (mesylate "OMs", tosylate "OTs") and Y is OH or an alcohol protecting group such as an alkyl group, an aryl group, an acyl group or an ester group and where m = 1-5 and n = 1-100. The PEG esters of dithiobumetanide, dithio-sulosemide and dithiopyrethanide can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and the alkyloxy (polyalkyloxy) alkyl analogues or tosylates such as MeO-PEG1000-OT and the similar. . { See Reaction Scheme 3).

Reaction Scheme 34. Synthesis of exemplary polyethylene glycol dithioesters of dithiobumetanide, dithiopurosemide and dithiopyrethanide "PEG thioesters of piretanide" dithiopyrethanide R3 = OCH2CH2 (OCH2CH2) n., - Y R3 = 0-aryl, R "= R5 = H m = 1 - 5, n = 1 - 100 PEG-X is X- (CH2) m (OCH2CH2) nYY, wherein X is halo or another leaving group (mesylate "OMs", tosylate "OTs") and Y is OH or an alcohol protecting group such as an alkyl group , an aryl group, an acyl group or an ester group and wherein m = 1-5 and n = 1-100.

H. Analogs PEG types of azosemide and torsemide PEG-type azosemide and torsemide esters can be formed by alkylation using alkyloxy (polyalkyloxy) alkyl halides such as MeO-PEG350-Cl and alkyloxy (polyalkyloxy) alkyl analogs or tosylates such as MeO -PEG1000-OT and the like. . { See Reaction Scheme 35).

Reaction Scheme 35. Synthesis of exemplary alkyl polyethylene glycol ethers of azosemide and torsemide "methyl PEG azosemide azosemide ethers" Re = (CH2) mOCH3CH2 (OCH2CH2), rY R3 = chloride, R "= R5 = H m = 1 - 5. n = 1 - 100 "quaternary ammonium torsemide salts of methyl PEG ether of torsemide" m = 1 - 5, n = 1 - 100 PEG-X is X- (CH2) m (OCH2CH2) niY, wherein X is halo or another leaving group (mesylate "OMs", tosylate "OTs") and Y is OH or an alcohol protecting group such as an alkyl group , an aryl group, an acyl group or an ester group and wherein m = 1-5 and n = 1-100. Starting materials for synthesizing compounds of the present invention may further include compounds described in U.S. Patent No. 3,634,583 issued to Feit; U.S. Patent No. 3,806,534 issued to Fiet; U.S. Patent No. 3,058,882 issued to Struem et al .; U.S. Patent No. 4,010,273 issued to Bormann; U.S. Patent No. 3,665,002 issued to Popelak and U.S. Patent No. 3,665,002 issued to Delarge. The compounds of the present invention can include isomers, tauromers, suteions, enantiomers, diastereomers, racemates or stereochemistry mixtures thereof. the term "isomers" as used herein is reeffered to compounds having the same number and class of atoms and here the same molecular weight, but differ with respect to the arrangement or configuration of the atoms in space. Additionally, the term "isomers" includes stereoisomers and geometric isomers. The terms "stereoisomers" or "optical isomers" as used herein refer to a stable isomer having at least one chiral atom or restricted rotation giving rise to perpendicular symmetric planes (e.g., certain biphenyls, alenes and spiro compounds). ) that can rotate the polarized light in the plane. Due to asymmetric centers and other chemical structure may exist in any of the compounds of the present invention that can give rise to stereoisomerism, the invention contemplates stereoisomers and mixtures thereof. The compounds of the present invention and their salts may include asymmetric carbon atoms and may therefore exist as individual stereoisomers, racemates and as mixtures of enantiomers and diastereoisomers. Commonly, such compounds will be prepared as a racemic mixture. If desired, however, such compounds can be prepared or isolated as esters of pure isomers, that is, as individual enantiomers or diastereomers or as stereoisomer-enriched mixtures. Tautomers are easily interconvertible constitutional isomers and there is a change in connectivity of a ligand as in the keto and enol forms of ethyl acetoacetate (the present invention includes tautomers of any such compound). The suteions are internal salts of bipolar compounds that have acidic and basic groups in the same molecule. At neutral pH, the cation and anion of most of the sutures are equally ionized. The present invention further provides prodrugs which comprise the compounds described herein. The term "prodrug" is intended to refer to a compound that is converted under physiological conditions, by solvolysis or metabolically, to a specified compound that is pharmaceutically / pharmacologically active. The prodrug may be a compound of the present invention that has been chemically derived in such a way that. (i) it retains some, all or none of the biactivity of its original drug compound and (ii) is metabolized in a subject to produce the original drug compound. The prodrug of the present invention can also be a "partial prodrug" in which the compound has been chemically derivatized such that: (i) it retains some, all or none of the bioactivity of its original drug compound and (ii) is metabolized in a subject to produce a biologically active derivative of the compound. The prodrugs can be formed using a hydrolysable coupling to the compounds described herein. An additional discussion of prodrugs can be found in Ettmayer et al. J. Med. Chem. 47 (10): 2394-2404 (2004). The prodrugs of the present invention are able to pass through the blood-brain barrier and can undergo hydrolysis by SNC esterases to provide the active compound. In addition, the prodrugs provided herein may also exhibit improved bioavailability, improved aqueous stability, improved passive intestinal absorption, moderate intestinal absorption by improved transporter, protection against accelerated metabolism, selective tissue feeding and / or passive enrichment in target tissue. . The prodrugs of the present invention may include compounds according to formula I, II, III, IV and / or V described herein. The prodrugs of the present invention may further include bumetanide, bumetanide dibenzyllanide, bumetanide diethylamide, bumetanide morpholinoethyl esters, bumetanide 3- (dimethylaminopropyl) ester, bumetanide N, N-diethylglycolamide ester, bumetanide dimethylglycolamide ester, bumetanide pivaxethyl esters, furosemide, furosemide ethylester, furosemide cyanomethyl ester, furosemide benzyl ester, furosemide morpholinoethyl esters, furosemide 3- (dimethylaminopropyl) ester, furosemide N, N-diethylglycolamide ester, furosemide dibenzynalenide, furosemide benzyltrimethylammonium salt, furosemide cetyltrimethylammonium salt, N , Furosemide N-dimethylglycolamide ester, furosemide pivaxetylester, furosemide propaxetylester, piretanide, piretanide ethylester, piretanide cyanomethylester, piretanide benzyl ester, piretanide morpholinoethyl esters, pyretanide 3- (dimethylaminopropyl) ester, N, -diethylglycolamide ester of piretanide, die piretanide thylamide, piretanide dibenzillanide, piretanide benzyltrimethylammonium salt, piretanide cetyltrimethylammonium salt, piretanide -dimethylglycolamide ester, piretanide pivaxetilyester, piretanide propaxetylester, tetrazolyl-substituted azosemide, pyridinium-substituted torsemide salts (also called quaternary pyridine-substituted-quaternary ammonium salts), also as similar derivatives of indacrinone and ozolinone. See Reaction Schemes previously presented.

In addition, as shown in the previously presented Reaction Schemes, the prodrugs can be formed by attaching compatible polymers, such as those previously described in which polyethylene glycol (PEG) is included to compounds of the present invention using degradable linkages under physiological conditions . See also Schacht, E.H. et al. Poly (ethylene glycol) Chemistry and Biological Applications, American Chemical Society, San Francisco, CA 297-315 (1997). The annexation of PEG to proteins can be used to reduce the immunogenicity and / or prolong the half-life of the compounds provided herein. Any conventional PEG coating method can be employed, provided that the PEG coated agent retains the pharmaceutical activity. The compositions of the present invention are suitable for human and veterinary applications and are preferably administered as pharmaceutical compositions. The pharmaceutical compositions comprise one or more treatment agents or a pharmaceutically acceptable salt thereof and a physiologically acceptable carrier. A pharmaceutically acceptable salt, as used herein, refers to a salt form of a compound that allows its use or combination as a pharmaceutical and that retains the biological activity of the free acid and base of the specified compound and is not biologically or another undesirable way. Examples of such salts are described in Handbook of Pharmaceutical Salts: Properties, Selection and Use, Wermuth, C.G. and Stahl, P.H. (eds.), Wiley-Verlag Helvetica Acta, Zürich, 2002 [ISBN 3-906390-26-8]. Examples of such salts include alkali metal salts and addition salts of free acids and bases. Examples of pharmaceutically acceptable salts include, but are not limited to, sulfates, pyrosulfates, bisulfates, sulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, , isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butin-4-fioates, hexin-1, 6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates , xulenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, α-hydroxybutyrates, glycolates, tartrates, methanesulfonates, ethanesulfonates, propansulfonates, toluenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates and mandelates. The pharmaceutical compositions of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more treatment agents of the present invention can be combined with another agent, in a treatment combination and administered according to a treatment regimen of the present invention. Such combinations can be administered as separate compositions, combined for administration to a complementary delivery system or formulated in a combined composition, such as a mixture or a fusion compound. Additionally, the combination of the aforementioned treatment may include an agent that improves the permeability of BBB and / or a hyperosmotic agent. The carriers and additives used for such pharmaceutical compositions can take a variety of forms depending on the mode of anticipated administration. Thus, compositions for oral administration may for example be solid preparations such as tablets, sugar-coated tablets, hard capsules, soft capsules, granules, powders and the like, with appropriate carriers and additives which are starches, sugars, binders, diluent, agents of granulation, lubricants, disintegrating agents and the like. Due to its ease of use and compliance with the highest patient, tablets and capsules represent advantageous oral dosage forms for many medical conditions. Similarly, compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, elixirs and the like with suitable carriers and additives which are water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents and the like. . Typical preparations for parenteral administration comprise the active ingredient with a carrier such as sterile water or parenterally acceptable oil which include polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil with other additives to aid in solubility or preservation They can also be included. In case of a solution, it can be lyophilized to a powder and then reconstituted immediately before use. For dispersions and suspensions, suitable carriers and additives include aqueous gums, cellulose, silicates or oils. The pharmaceutical compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, nasal, inhalation (for example, via an aerosol), buccal (for example, sub-lingual), vaginal, topical (this is, both on the surface of the skin and mucosal, in which airway surfaces are included), transfomeric and parenteral administration (eg, subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intrathecal, intracerebral, intracranial, int raarterial or intravenous), although the most appropriate route in any given case will depend on the nature and severity of the condition being treated and the nature of the particular active agent that is used. The pharmaceutical compositions of the present invention are particularly suitable for oral, sublingual, parenteral, implantation, nasal and inhalation administration. The compositions for injection will include the active ingredient together with appropriate carriers which include propylene glycol-alcohol-water, isotonic water, sterile water for injection (USP), emulPhor ™ -alcohol-water, cremophor-EL ™ or other suitable suitable carriers for those experienced in art. These carriers can be used alone or in combination with other conventional silubilization agents such as ethanol, a glycol or other agents known to those skilled in the art. When the compounds of the present invention are to be applied in the form of solutions or injections, the compounds can be used in dissolving or suspending in any conventional diluent wherein the compounds of the present invention are to be applied in the form of solutions or injections, the compounds can be used by dissolution or solution in any conventional diluent. The diluents may include, for example, physiological saline, Ringer's solution, an aqueous glucose solution, an aqueous dextrose solution, an alcohol, a fatty acid ester, glycerol, a glycol, an oil derived from plant or animal sources. , a paraffin and the like. These preparations can be prepared according to any conventional method known to those skilled in the art. Compositions for nasal administration can be formulated as aerosols, drops, powders and gels. Aerosol formulations commonly comprise a solution or fine suspension of the active ingredient in a physiologically acceptable aqueous or non-aqueous solvent. Such formulations are commonly presented in amounts of a single dose or multiple doses in sterile form in a sealed container. The sealed container can be a cartridge or filled for use with a spray device. Alternatively, the sealed container may be a unit assortment device such as a single-use nasal inhaler.pump atomizer or an aerosol dispenser equipped with a metering valve adjusted to provide a therapeutically effective amount, which is designed for recording once the content has been used completely. When the dosage form comprises an aerosol dispenser, it will contain a propellant such as a compressed gas, air as an example or an organic propellant in which a fluorochlorohydrocarbon or fluorohydrocarbon is included. Suitable compositions for buccal or sublingual administration include tablets, troches and lozenges, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth or gelatin and glycerin. Compositions for rectal administration include suppositories that contain a conventional suppository base such as cocoa butter. Suitable compositions for transdermal administration include ointments, gels and patches. Other compositions known to those skilled in the art can also be applied for percutaneous or subcutaneous administration such as casts. In addition, in preparations such as pharmaceutical compositions comprising the active ingredient or ingredients or mixtures with components necessary for the formation of the composition, other pharmacologically acceptable conventional additives may be incorporated, for example, excipients, stabilizers, antiseptics, wetting agents, agents emulsifiers, lubricants, sweetening agents, coloring agents, flavoring agents, isotonicity agents, pH regulating agents, antioxidants and the like. As the additives, there may be mentioned, for example, starch, sucrose, fructose, dextrose, lactose, glucose, mannitol, sorbitol, precipitated decalcium carbonate, crystalline cellulose, carbopymethylcellulose, dextrin, gelatin, acacia, EDTA, magnesium stearate, talc. , hydroxypropylmethylcellulose, sodium metabisulfite and the like.

In additional embodiments, the present invention provides kits that include one or more containers that comprise pharmaceutical dosage units that comprise an effective amount of one or more compounds of the present invention. When aqueous suspensions or elixirs are desired for oral administration, the essential active ingredient therein can be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water. , ethanol, propylene glycol, glycerin and combinations thereof. The compositions described herein can be administered as part of a sustained release formulation. Such formulations can in general be prepared using well known technology and administered for example, by oral, rectal or transdermal delivery systems or by implants of a therapeutic formulation or reagent in one or more desired target site (s). . Sustained release formulations may contain a treatment composition comprising a treatment agent of the invention alone or in combination with a second treatment agent, dispersed in a carrier matrix and / or contained within a reservoir surrounded by a membrane that controls the speed. Carriers for use in such formulations are biocompatible and can also be biodegradable. According to one embodiment, the sustained release formulation provides a relatively constant level of release of active composition. According to another embodiment, the sustained release formulation is contained in a device that may be actuated by the subject or medical personnel, at the onset of certain symptoms, for example, to deliver predetermined dosages of the treatment composition. The amount of the treatment composition contained within a sustained release formulation depends on the site of implantation, the expected rate and duration of release and the nature of the condition to be treated or prevented. In certain embodiments, the compositions of the present invention for the treatment of neuropathic pain and neuropsychiatric disorders are administered using a formulation and administration regimen that facilitates the release of the treatment composition (s) to the central nervous system. Treatment compositions, such as NKCC1 antagonists can be formulated to facilitate crossing the blood-brain barrier as described above or can be co-administered with an agent that crosses the blood-brain barrier. The treatment compositions can be administered in liposome formulations, for example, which cross the blood-brain barrier or can be co-administered with other compounds, such as bradykinins, bradykinin analogs or derivatives or other compounds such as SERAPORT ™, that cross the blood-brain barrier. Alternatively, the treatment compositions of the present invention can be administered using a spinal shunt that places the treatment composition directly into the circulating cerebrospinal fluid. For some treatment conditions, there may be transient or permanent interruptions of the blood-brain barrier and the specialized formulation of the treatment composition to cross the blood-brain barrier may not be necessary. It has been determined, for example, that an i.v. of 20 mg of furosemide reduces or ablates both spontaneous interictal activity and epileptiform activity evoked by electrical stimulation in human patients who are refractory to antiepileptic drugs (AED) or. (Haglund &Hochman J. Neurophysiol. (Feb. 23, 2005) doi: 10.1152 / j n .0094 .2004). The routes and frequency of administration of the therapeutic compositions disclosed herein, also as dosages, vary according to the indication and from one individual to another and can be easily determined by the physician from information that is generally available and by monitoring patients and adjusting dosages and compliance treatment regimens using the standard techniques. In general, appropriate dosages and appropriate treatment regimen provide the active composition (s) in an amount sufficient to provide therapeutic and / or prophylactic benefit. Dosages and treatment regimens can be established by monitoring improved clinical outcomes in treated patients compared to untreated patients. The term "effective amount" or "effective" is intended to designate a dose that causes a relief of symptoms of a disease or disorder, as noted in clinical trials and evaluation, observation of the patient and / or the like. "Effective amount" or "effective" may also determine a dose that causes a detectable change in biological or chemical activity. The detectable changes can be detected and / or quantified additionally by that experienced in the art for the relevant mechanism or process. In addition, "effective amount" or "effective" can designate an amount that maintains a desired physiological state, that is, reduces or prevents significant reduction and / or promotes improvement in the condition of interest. Dosages and therapeutically effective treatment regimens will depend on the condition, the severity of the condition and the general condition of the patient being treated. Since the pharmacokinetics and pharmacodynamics of the treatment compositions of the present invention vary in different patients, a preferred method for determining a therapeutically effective dosage in a patient is to gradually scale up the dosage and monitor the clinical and laboratory indications. For combination therapy, the two or more agents are coadministered, such that each of the agents is present in a therapeutically effective amount for a sufficient time to produce a therapeutic or prophylactic effect. The term "co-administration" is intended to encompass the simultaneous or sequential administration of two or more agents in the same formulation, or unit dosage form or in separate formulations. Dosages and appropriate treatment regimens for treatment of acute episodic conditions, chronic conditions or prophylaxis will necessarily vary to accommodate the patient's condition. By way of example, for the treatment of neuropathic pain, furosemide can be administered to a patient in amounts of 10-40 mg at a frequency of 1-3 times per day, preferably in an amount of 40 mg three times a day. In an alternative setting, bumetanide can be administered orally for the treatment of neuropathic pain in amounts of 1-10 mg at a frequency of 1-3 times a day. Those skilled in the art will appreciate that smaller doses may be employed, for example, in pediatric applications. In further embodiments, bumetanide analogs according to the present invention can be administered in amounts of 1.5 to 6 mg daily, for example a tablet or capsule three times a day. In some embodiments, furosemide analogs according to the present invention can be administered in amounts of 60 to 240 mg / day, eg, a tablet or capsule three times a day. In other embodiments, piretanide analogs according to the present invention can be administered in amounts of 10 to 20 mg daily, for example, a tablet or capsule once a day. In some embodiments, azosemide analogs according to the present invention can be administered in an amount of 60 mg per day. In other embodiments, torsemide analogs according to the present invention can be administered in amounts of 10 to 20 mg daily, for example, a tablet or capsule once a day. It should be noted that lower doses can be administered, particularly for i.v. administration. The methods and systems of the present invention can also be used to evaluate candidate compounds and candidate treatment regimens for the treatment and / or prophylaxis of neuropathic pain and neuropsychiatric disorders. Various techniques can be employed to generate candidate compounds that potentially have the desired NKCC1 co-transporter antagonist activity. Candidate compounds can be generated using procedures well known to those skilled in the art of synthetic organic chemistry. Expression-activity relationships and molecular modeling techniques are useful for the purpose of modifying known N-CC1 antagonists such as furosemide, bumetanide, ethanidic acid and related compounds, to confer the desired activities and specificities. Methods for selecting candidate compounds for desired activities are described in U.S. Patent Nos. 5,902, 732; 5,976,825; 6,096,510 and 6,319,682, which are incorporated herein by reference in their entirety. Candidate compounds can be selected for NKCC1 antagonist activity using the screening methods of the present invention with various cell types in culture such as glial cells, neuronal cells, kidney cells and the like or in animal models in situ. Selection techniques for identifying chloride co-transporter antagonist activity, for example, may involve altering the ion balance of the extracellular space in the tissue culture sample or in situ in an animal model, by producing a chloride concentration anionic higher than "normal". The geometrical and / or optical properties of the cell or tissue sample subject to this altered ionic balance were determined and the candidate agents are administered. Following administration of the candidate agents, the corresponding geometrical and / or optical properties of the cell or tissue sample are monitored to determine if the ionic imbalance persists or if the cells responded to the alteration of the ionic imbalances in the extracellular space and intracellular. If the ionic imbalance persists, the candidate agent is probably a chloride co-transporter agonist. By selecting using various types of cells or tissues, candidate compounds that have a high level of co-antagonist activity. -Chial chloride transporter and glial cells that have a reduced level of neuronal cell chloride and renal cell co-transporter antagonist activity can be identified. Similarly, effects on different types of cell and tissue systems can be determined. Additionally, the efficacy of the candidate compounds can be determined by simulating or inducing a condition, such as neuropathic pain, in situ in an animal model, monitoring the geometrical and / or optical properties of the cell or tissue sample during stimulation of the condition, administering the candidate compound, then monitoring the geometrical and / or optical properties of the cell or tissue sample following administration of the candidate compound and comparing the geometrical and / or optical properties of the cell or tissue sample to determine the effect of the candidate compound. Tests of the efficacy of treatment compositions for neuropathic pain relief can be carried out using well-known methods and animal models, such as that described in Bennett, Hosp. Pract (Off Ed). 33: 95-98, 1998. As discussed above, compositions for use in the methods of the invention may comprise a treatment agent selected from the group consisting of: antibodies or antigen binding fragments thereof, which are linked specifically to NKCCl; soluble ligands that bind to NKCCl; antisense oligonucleotides to NKCCl and small interferential RNA molecules (siRNA or RNAi) that are specific for NKCCl. Antibodies that specifically bind to NKCCl are known in the art and include those available from Alpha Diagnostic International, Inc. (San Antonio, TX 78238). An "antigen binding site" or "antigen binding fragment" of an antibody refers to that part of the antibody that participates in the antigen binding. The antigen binding site is formed by amino acid residues of N-terminal ("V") variable regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as "hypervariable regions" that are interposed between more conservative bleaching stretches known as "structure regions" or "FR". Thus, the term "FR" refers to amino acid sequences that are naturally found between and adjacent to hypervariable regions in immunoglobulin. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are arranged in three-dimensional space to form an antigen binding surface. The antigen binding surface is complementary to the three-dimensional surface of a linked antigen and the three hypervariable regions of each of the heavy and light chains are referred to as "regions determining complementarity" or "CDR". A number of molecules are known in the art to comprise antigen binding sites capable of exhibiting the binding properties of an antibody molecule. For example, the proteolytic enzyme papain preferably cleaves IgG molecules to produce several fragments, two of which (the "F (ab)" fragments) each comprise a covalent heterodimer and includes an intact antigen binding site. The pepsin enzyme is able to cleave IgG molecules to provide several fragments, in which the "F (ab ') 2" fragment comprising both antigen binding sites is included. An "Fv" fragment can be produced by preferential proteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, but are more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH:: VL heterodimer that includes an antigen binding site that defines much of the antigen recognition and binding capabilities of the natural antibody molecule (Inbar et al., Proc. Nati. Acad. Sci. USA 69: 2659-2662, 1972; Hochman et al. Biochem 15: 2706-2710, 1976; and Ehrlich et al. Biochem 19: 4091-4096, 1980). Humanized antibodies that bind specifically to NKCC1 can also be employed in the methods of the invention. A number of humanized antibody molecules comprising an antigen binding site derived from a non-human immunoglobulin have been described, in the. which include chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al., Nature 349: 293-299, 1991; Lobuglio et al., Proc. Nati. Acad. Sci. USA 86: 220 -4224, 1989; Shaw et al., J Immunol., 138: 4534-4538, 1987; and Brown et al., Cancer Res. 47: 3577-3583, 1987); Rodent CDRs inserted into a human support FR before fusion with a constant domain of the appropriate human antibody (Riechmann et al., Nature 332: 323-327, 1988; Verhoeyen et al., Science 239: 1534-1536, 1988; Jones et al., Nature 321: 522-525, 1986); and rodent CDRs supported by recombinantly designed FRs (European Patent Publication No. 519,596, published December 23, 1992). These "humanized" molecules are designed to minimize undesirable immune responses to rodent antihuman antibody molecules that limit the duration and effectiveness of therapeutic applications of those portions in human receptors. Modulation of the affinity of NKCC1 can be effected alternatively by reducing or inhibiting the expression of the polypeptide, which can be obtained by interfering with the transcription and / or translation of corresponding polynucleotide. Expression of the polypeptide can be inhibited for example by introducing antisense expression vectors, antisense oligodeoxyribonucleotides, antisense phosphorothioate oligodeoxyribonucleotides, antisense oligonucleotides or antisense phosphorothioate oligonucleotides or by other means well known in the art. All such antisense polynucleotides are collectively referred to herein as "antisense oligonucleotides". The antisense oligonucleotides for use in the methods of the invention are sufficiently complementary to the NKCC1 polynucleotide to bind specifically to the polynucleotide. The sequence of an antisense oligonucleotide need not be 100% complementary to that of the polynucleotide in order that the antisense oligonucleotide be effective in the methods of the invention. Rather, an antisense oligonucleotide is sufficiently complementary when the binding of the antisense oligonucleotide to the polynucleotide interferes with the normal function of the polynucleotide to cause a loss of utility and when non-specific binding of the oligonucleotide to other non-target sequences is avoided. The design of appropriate antisense oligonucleotides is well known in the art. Oligonucleotides that are complementary to the 5 'end of the message, for example, the 5' untranslated high sequence and including the AUG start codon, must work more efficiently to inhibit translation. however, complementary oligonucleotides to either the untranslated 5 'or 3' non-coding regions of the target polynucleotide may also be employed. Cell permeation and antisense oligonucleotide activity can be improved by appropriate chemical modifications, such as the use of C-5-phenoxazine-substituted phenoxazine oligonucleotides (Flanagan et al., Wat. Biotechnol., 17: 48-52, 1999) or 2'-0- (2-methoxy) -ethyl (2'-MOE) -oligoucleotides (Zhang et al., Nat. Biotechnol.18: 862-867, 2000). The use of techniques involving antisense oligonucleotides is well known in the art and is described, for example, in Robinson-Benion et al. (Methods in Enzymol, 254: 363-375, 1995) and Kawasaki et al. [Artific. Organs 20: 836-848, 1996). Expression of the NKCC1 polypeptide can also be specifically suppressed by methods such as RNA interference (RNAi). A review of this technique is found in Science, 288: 1370-1372, 2000. Briefly, additional genetic suppression methods, employing RNA or antisense DNA, operate by binding to the reverse sequence of a gene of interest in such a way that the link interferes with subsequent similar processes and therefore it blocks the synthesis of the corresponding protein. RNAi operates at the post-translational level and is sequence specific, but suppresses gene expression much more efficiently. Exemplary methods for controlling or modifying gene expression are provided in WO 99/49029, O 99/53050 and WO 01/75164, the disclosures of which are incorporated by reference. In these methods, the silencing of the post-translation gene is effected by a process of sequence-specific RNA degradation that results in rapid degradation of transcript-related genes. Studies have shown that double-stranded RNA can act as a mediator of sequence-specific gene silencing. { see for example, Montgomery and Fire, Trends in Genetics, 14: 255-258, 1998). Genetic constructor that produce transcripts with self-complementary regions are particularly efficient in gene silencing. It has been shown that one or more ribonucleases specifically bind to and cleave double-stranded RNAs into short fragments. The ribonuclease (s) remains (are) associated with these fragments, which in turn specifically bind to complementary mRNA, that is, they bind specifically to the strand of mRNA transcribed for the gene of interest. The mRNA for the gene is also degraded by the ribonuclease (s) in short fragments, thereby eliminating the translation and expression of the gene. Additionally, an RNA polymerase can act to facilitate the synthesis of numerous copies of the short fragments, which exponentially increases the efficiency of the system. A unique aspect of RNAi is that silencing is not limited to the cells where it is initiated. The effects of genetic silencing can be disseminated to other parts of an organism. The NKCCl polynucleotide can thus be employed to generate gene silencing constructs and / or gene-specific self-complementary double-stranded RNA sequences that can be employed in the methods of the invention using methods of administration known in the art. A genetic costructo can be used to express the self-complementary RNA sequences. Alternatively, the cells can be contacted with gene-specific double-stranded RNA molecules, such that the RNA molecules are internalized to the cell cytoplasm to exert a gene silencing effect. The double-stranded RNA must have sufficient homology to the NKCCl gene to modulate RNAi if it affects the expression of non-target genes. The double-stranded DNA is at least 20 nucleotides in length and is preferably 21-23 nucleotides in length.

Preferably, the double-stranded RNA corresponds specifically to a polynucleotide of the present invention. The use of small interfering RNA molecules (siRNA) of 21-23 nucleotides in length to suppress gene expression in martyr cells is described in O 01/75164. Tools for designing optimal inhibitory siRNAs include those available from DNAengine Inc. (Seattle, WA). An RNAi technique employs genetic constructors within which sense and antisense sequences are placed in regions that blanch an intron sequence in proper splice orientation with donor and acceptor splice sites. Alternatively, spacer sequences of various lengths can be employed to separate self-complementary regions of sequence in the construct. During the processing of the genetic construct transcript, the intron sequences are spliced, allowing sense and antisense sequences, as well as splice and junction sequences, to bind to form double-stranded RNA. Then the selected ribonucleases are ligated to and cleaved in double-stranded RNA, initiating by this the cascade of events that lead to the degradation of specific mRNA gene sequences and silencing of specific genes. For in vivo uses a genetic construct, antisense oligonucleotide or RNA molecule can be administered by various methods recognized in the art (see for example, Rolland, Crit., Rev. Therap, Drug Carrier Systems 15: 143-198, 1998 and references cited). Both viral and non-viral administration methods have been used for gene therapy. Useful viral vectors include, for example, adenoviruses, adeno-associated viruses (AAV), retroviruses, vaccinia viruses and avian poxviruses. Improvements have been made in the efficiency and targeting of genes to tumor cells with adenoviral vectors, for example, by coupling adenoviruses to N-polylysine complexes and strategies that exploit receptor-modulated endocytosis for selective approach (see, for example, Curiel et al., Hum. Gene Ther., 3: 147-154, 1992; and Cristiano &Curiel, Cancer Gene Ther. 3: 49-57, 1996). Non-viral methods for administering polynucleotides are reviewed in Chang and Seymour, (Eds) Curr. Opin. Mol. Ther., Vol. 2, 2000. These methods include contacting cells with naked DNA, cationic liposomes, or polynucleotide polyplexes with cationic polymers and dendrimers for systemic administration (Chang and Seymour, Ibid.). Liposomes can be modified by the incorporation of ligands that recognize cell surface receptors and allow the targeting of specific receptors for receptor-mediated endocytosis (see, for example, Xu et al., Mol. Genet. Metab., 64: 193-197; 1998; and Xu et al., Hum. Gene Ther., 10: 2941-2952, 1999). Tumor targeting bacteria, such as Salmonella, are potentially useful for administering genes to tumors followed by systemic administration (Low et al., Nat. Biotechnol., 27: 37-41, 1999). Bacteria can be designed ex vivo to penetrate and deliver DNA with high efficiency to, for example, mammalian epithelial cells in vivo (see, eg, Grillot-Courvalin et al., Wat Biotechnol 16: 862-866, 1998) . Oligonucleotides stabilized by degradation can be encapsulated in liposomes and released to patients by injection either intravenously or directly to a target site (e.g., the origin of neuropathic pain). Alternatively, retroviral vectors or adenoviral vectors or antisense RNA that express naked DNA for the polypeptides of the invention can be administered to patients. Appropriate techniques for use in such methods are well known in the art. The compositions and methods of treatment of the present invention have been described above with respect to certain preferred embodiments. The Examples described below describe the results of specific experiments and are not intended to limit the invention in any way.

EXAMPLE 1 Methyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide methyl ester) To a suspension of bumetanide (1.2 g, 3.29 mmol) in methanol (12 ml) under nitrogen atmosphere was added a mixture of thionyl chloride ( 70 μ?) In methanol (6 ml) for 5 minutes. After stirring for 5 minutes, the reaction mixture became soluble. The reaction is stirred for an additional 30 minutes, at which time the reaction was complete by thin layer chromatography (TLC). The methanol was removed under reduced pressure and the residue was brought into ethyl acetate and washed with saturated sodium bicarbonate, water and brine. The ethyl acetate was dried under anhydrous magnesium sulfate and concentrated in a yield of 1.1 g (89%) of methyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate as a white solid. Using similar methodology, bimetanide ethyl ester, bumetanide isoamyl ester, bumetanide octyl ester and bumetanide benzyl ester can be prepared.

EXAMPLE 2 3-Aminosulfonyl-5-buylamino-4-phenoxy-iobenzoic acid (- (C = Q) -SH thiobumetanide thioacid, bumetanide) Bumetanide can be reacted with thionyl chloride to make the corresponding acid chloride which is then it can react with sodium hydrogen sulfide to give aminosulfonyl-5-butylamino-4-phenoxythiobenzoic acid (thiobumetanide thioacid, S-bumetanide). using the methodology of Noble, P. and Tarbell, D. S., Org. Synth., Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927.

EXAMPLE 3 3-Aminosulfonyl-5-butylamino-4-phenoxythiobenzoic acid (- (C = Q) -SH thiobumetanide thioacid, bumetanide) Bumetanide methyl ester can be reacted with hydrogen sulphide or sodium hydrogen sulfide to give, followed by acidification , 3-aminosulfonyl-5-butylamino-4-phenoxythiobenzoic acid (thiobumetanide, bumetanide acid).

EXAMPLE 4 Thiomethyl 3-aminosulfonyl-5-butylamino-4-phenoxythiobenzoate (S-methyl bumetanide thioester) Similar to Example 1, bumetanide can be reacted with a catalytic amount of thionyl chloride in methyntiol (methyl mercaptan) to give thiomethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate. Using the similar methodology with bumetanide and the corresponding thiols, S-bumetanide ethyl thioester, S-bumetanide isoamyl ester, S-bumetanide octyl thioester and S-bumetanide benzyl thioester can be prepared. Using the similar methodology with dithiobumetanide and the corresponding alcohols, O-ethyl thioester of bumetanide, O-isoamyl thioester of bumetanide, O-octyl thioester of bumetanide and O-benzyl thioester of bumetanide can be prepared.

EXAMPLE 5 3-Aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoic acid (- (C = S) -SH dithiobutane acid, bumetanide) Thiobumetanide can be reacted with thionyl chloride to make the corresponding thioacid chloride which is then it can react with sodium hydrogen sulphide to give 3-aminosulgonyl-5-butylamino-4-phenoxydithiobenzoic acid (dithiobumetanide dithioacid, bumetanide), by the methodology of Noble, P. and Tarbell, DS, Org. Synth., Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927.

EXAMPLE 6 Methyl 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate (bumetanide methyldithioester) Similarly to Example 1, dithiobumetanide can be reacted with a catalytic amount of thionyl chloride in methyntiol (methyl mercaptan) to give methyl 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate. Using similar methodology of bumetanide ethyldithioether, bumetanide isoamyldithioether, bumetanide octyldithioether and bumetanide methyldithioether can be prepared.

EXAMPLE 7 Cyanomethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide cyanomethyl ester) Bumetanide (1.0 g, 2.7 mmol) was dissolved in dimethylformamide (DMF) and chloroacetonitrile (195 ul), 2.7 mmol) was added followed by triethylamine (465 ul). The reaction was heated at 100 ° C for 12 hours, TLC and liquid chromatography-coupled mass spectrometry (LC / MS) indicated that the reaction was complete. The reaction was cooled to room temperature brought into dichloromethane and washed with water, saturated ammonium chloride and reduced to a watery paste. Water (25 ml) was added to the watery paste and the crude product was purified as a decolorized white solid. The 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate of cyanomethyl (850 mg) was obtained via recrystallization from acetonitrile. Using similar methodology, the butetanide ethyl ether, bumetanide isoamyl ester, bumetanide octyl ester and bumetanide methyl ester can be prepared.

EXAMPLE 8 Benzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate (bumetanide benzyl ester) Bumetanide (1.15 g, 3.15 mmol) was dissolved in dimethylformamide (DMF, 10 mL) and benzyl chloride (400 ul, 2.8 mmol) followed by triethylamine (480 ul). The reaction was heated at 80 ° C for 12 hours. TLC and LV / MS indicated that the reaction was complete. The reaction was cooled to room temperature brought into dichloromethane and washed with water, saturated with saturated ammonium chloride and concentrated to a slurry. To the suspension was added water (25 ml), the resulting solids were filtered and dried in a vacuum oven at 50 ° C for 12 hours to yield 1.0 g (80%) of 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate of benzyl.

EXAMPLE 9 2- (4- morpholino) ethyl 3-aminosulfonyl-5-butylamino-4-phenyloxybenzoate (bumetanide morpholinoethyl ester) Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (DMF, 12 ml) and hydrochloride was added of 4- (2-chloroethyl) morpholine (675 mg, 6.62 mmol) followed by triethylamine (1 mL) and sodium iodide (500 mg, 3.33 mmol). The reaction was heated at 95 ° C for 8 hours, TLC and LC / MS indicated that the reaction was complete. The reaction was cooled to room temperature brought into dichloromethane and washed with water, saturated ammonium chloride and concentrated to dryness. After purification via biotage column chromatography, the puirified eluate, on evaporation under vacuum, gave 2- (4-morpholino) ethyl 3- aminosulfonyl-5-butylamino-4-phenoxybenzoate as a white solid (600 mg, 62%). %).

EXAMPLE 10 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate of 3- (N, N-dimethylaminopropyl) [3- (dimethylminopropyl) ester of bumetanide] Similar to Example 54, bumetanide can be reacted with chloride hydrochloride of 3- (dimethylamino) propyl, triethylamino and sodium iodide in dimethylformamide (DMF) to produce 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate of 3- (N, N-dimethylaminopropyl).

EXAMPLE 11 3-Aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate of 3- (N, N-dimethylaminopropyl) [3- (dimethylaminopropyl) dithioester of bumetanide] In a similar manner to Example 10, dithiobumetanide can be reacted with 3- (dimethylamino) propyl chloride hydrochloride, triethylamine and sodium iodide in dimethylformamide (DMF) to give 3- (N, -dimethylaminopropyl) 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate.

EXAMPLE 12 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate of N, N-dimethylaminocarbonylmethyl (?,? -diethylglycolamidoester of bumetanide) Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (12 ml) and 2-chloro- N, N-diethylacetamide (500 mg, 3.35 mmol) was added followed by triethylamine (0.68 ml) and sodium iodide (500 mg, 3.33 mmol). The reaction was heated at 95 ° C for 8 hours, TLC and LC / MS indicated that the reaction was complete. The reaction was cooled to room temperature brought into dichloromethane and washed with water, saturated ammonium chloride and reduced to a thick slurry. To the suspension was added water (25 ml), the resulting solids precipitated from the solution. The product was filtered and dried in a vacuum oven at 50 ° C for 12 hours to yield 1.0 g of N, N-diethylaminocarbonylmethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

EXAMPLE 13 N, N-Diethyl 3-aminosulonyl-5-butylamino-4-phenoxybenzamide (bumetanide diethylamide) Bumetanide (1.16 g, 3.2 mmol) was dissolved in dichloromethane (10 ml) and l-ethyl-3- (3 -dimethylaminopropyl) carbodiimide (EDC, 690 mg, 3.6 mmol) was added and after 5 minutes N-hydroxybenzotriaxol (HOBt, 498 mg, 3.6 mmol) was added and the solution allowed to stir for an additional 5 minutes. Diethylamine (332 ul, 3.2 mmol) was added and the reaction was stirred for 2 hours. The reaction was washed with saturated sodium bicarbonate, water, brine and dried with magnesium sulfate. The dichloromethane was removed under reduced pressure to yield 860 mg (65%) of pure N, N-diethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzamide.

EXAMPLE 14, N-Diethyl 3-aminosulfonyl-5-butylamino-4-phenoxythiobenzamide (bumetanide diethylthioamide) Similar to Example 5, dithiobumetanide can be reacted with thionyl chloride to give thioacid chloride, which can be made reacting with diethylamine to produce?,? - diethyl 3-aminosulfonyl-5-butylamino-4-phenoxythiobenzamide.

EXAMPLE 15?,? - dibenzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzamide (bumetanide dibenzylamide) Bumetanide (960 mg, 2.6 mmol) was dissolved in dimethylformamide (DMF, 10 ml) and l-ethyl-3- ( 3-dimethylaminopropyl) carbodiimide (EDC, 560 mg, 3.6 mmol) was added and after 10 minutes 1-hydroxybenzotriazole (HOBt, 392 mg, 2.9 mmol) was added and the solution allowed to stir for an additional 10 minutes. Dibenzylamine (1 ml, 5.2 mmol) was added and the reaction was stirred for 2 hours, in which the reaction was complete by LC / MS. The reaction was poured into saturated ammonium chloride (20 mL) and extracted with methyl acetate (2 x 100 mL). The ethyl acetate was washed with saturated sodium bicarbonate, water, brine and dried over anhydrous sodium sulfate. The ethyl acetate was removed under reduced pressure to yield 1.0 g (75%) of β, β-dibenzyl 3-aminosulgonyl-5-butylamino-4-phenoxybenzamide as a white solid.

EXAMPLE 16 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate benzyltrimethylammonium (Benzyltrimethylammonium salt of bumetanide) To a solution of benzyltrimethylammonium hydroxide (451 mg, 2.7 mmol) in water (10 ml) was added bumetanide (1 g, 2.7 g. mmol) in a period of 5 minutes. The reaction mixture became clear after 10 minutes of stirring. The water was removed under reduced pressure to produce a crude colorless oil. The crude product was obtained from recrystallization and the oil with water and heptane to yield 690 mg of 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate benzyltrimethylammonium as light pink crystals.

EXAMPLE 17 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate of Cetyltrimethylammonium (Cetyltrimethylammonium salt of bumetanide) Similarly to Example 16, bumetanide can be reacted with cetyltrimethylammonium hydroxide in water to produce 3-aminosulfonyl-5-but Cetyltrimethylammonium-4-phenoxybenzoate.

EXAMPLE 18 N, N-dimethylaminocarbonylmethyl (?,? -dimethylglycolamidoester of bumetanide) 3-aminosulfonyl-5-butylamino-5-butylamino-4-phenoxybenzoate Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (DMF, 10 ml) and 2 g. -chloro-N, N-dimethylacetamide (410 ul, 3.9 mmol) followed by triethylamine (0.70 ml) and sodium iodide (545 mg, 3.6 mmol) The reaction was heated at 50 ° C for 10 hours, the TLC and LC / MS indicated that the reaction was complete The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate, water and brine and dried over anhydrous magnesium sulfate. under reduced pressure and the product was purified via flash chromatography to yield 685 mg (60%) of N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

EXAMPLE 19 3-aminosulfonyl-5-b-tilaniino-4-phenoxybenzoate of t-butylcarbonyloxymethyl (bumetanide pivaxethyl esters) Bumetanide (1.2 g, 3.29 mmol) was dissolved in dimethylformamide (DMF, 10 ml) and chloromethyl pivalate was added (575 ul, 3.9 mmol) followed by triethylamine (0.70 ml) and sodium iodide (545 mg, 3.6 mmol). The reaction was heated at 50 ° C for 10 hours, TLC and LC / MS indicated that the reaction was complete. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate, water and brine and dried over anhydrous magnesium sulfate. The ethyl acetate was removed under reduced pressure and the product was purified via flash chromatography to yield 653 mg (60%) of pure t-butylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

EXAMPLE 20 T-Butylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate (Bumetanide Pivaxethyldithioester) In a manner similar to Example 19, dithiobumetanide is reacted with chloromethyl pivalate, triethylamine and sodium iodide in dimethylformamide ( DMF) to produce t-butylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate.

EXAMPLE 21 Ethylcarbonyloxymethyl 3-aminosulphonyl-5-butylamino-4-phenoxybenzoate (Bumetanide Propaxyethyl ester) In a manner similar to Example 19, bumetanide can be reacted with chloromethyl propionate, triethylamine and sodium iodide in dimethylformamide (DMF) to produce Ethylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.

EXAMPLE 22 Ethylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate (Bumetanide Propaxethyldithioester) Similarly to Example 21, dithiobumetanide can be reacted with chloromethyl propionate, triethylamine and sodium iodide in dimethylformamide (DMF) to produce ethylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate.

EXAMPLE 23 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate methyl (pyretanide methyl ester) In a similar manner to Example 1, the piretanide can be reacted with thionyl chloride and methanol to produce 3-aminosulfonyl- methyl-phenoxy-5- (1-pyrrolidinyl) benzoate. Using similar methodology, the piretanide ethyl ester, piretanide isoamyl ester, piretanide octyl ester and piretanide benzyl ester can be prepared.

+++ EXAMPLE 24 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -thiobenzoic acid (thioiretanide- (C = 0) -SH pyoethanide thioacid) The piretanide can be reacted with thionyl chloride to manufacture the corresponding acid chloride and then it can be reacted with sodium hydrogen sulphide to give 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -thiobenzoic acid (thioiretanide, thio-acid of S-piretanide) by the Noble methodology, P. and Tarbell, DS, Org. Synth , Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927.

EXAMPLE 25 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -thiobenzoic acid (thio-pyrethanedide- (C = 0) -SH piretanide thioacid) Pyretanide methyl ester can be reacted with hydrogen sulphide or sodium hydrogen sulfide to give 3-aminosul-fonyl-4-phenoxy-5- (1-pyrrolidinyl) -thiobenzoic acid (thioiretanide, thio-acid of S-piretanide).

EXAMPLE 26 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -thiomethylbenzoate (S-methylthioester of piretanide) Similarly to Example 1, the piretanide can be reacted with a catalytic amount of thionyl chloride in metantiol (methyl mercaptan) to give thiomethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate. Using similar methodology with piretanide and the corresponding thiols, S-ethyl thioester of piretanide, S-isoamyl thioester of piretanide, S-octyl thioester of piretanide and S-benzyl thioester of piretanide can be prepared. Using similar methodology with dithiopyrethanide and the corresponding alcohols, pyretanide O-ethyl thioester, pyretanide 0-isoamyl thioester, pyretanide O-octyl thioester and pyretanide O-benzyl thioester can be prepared.

EXAMPLE 27 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -dithiobenzoic acid (pyththine-dithioxyethanide- (C = S) -SH dithioacid) Thiopyrethanide can be reacted with thionyl chloride to make the corresponding chloro-thioacid which can then be reacted with sodium hydrogen sulfide to give 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -dithiobenzoic acid (dithiopyrethanide, pyretanide dithioacid) by the methodology of Noble, P. and Tarbell, D. S., Org. Synth., Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927.

EXAMPLE 2 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -d-thiobenzoate methyl (pyretanide methyldithioester) Similarly to Example 1, the dithiopyrethanide can be reacted with a catalytic amount of thionyl chloriro in metantiol ( methyl mercaptan) to give methyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -dithiobenzoate. Using similar methodology, the pyrithanide ethyldithioester, pyretanide isoamidithiolyester, pyretanide octyl idioester and pyretanide benzyldithioester can be prepared.

EXAMPLE 29 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoic acid cyanomethyl ester (cyanomethylester of piretanide) In a similar manner to Example 7, the piretanide can be reacted with chloroacetonitrile and triethylamine in DMF to produce 3-aminosulfonyl Cyanomethyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate.

EXAMPLE 30 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzyl benzoate (piretanide benzyl ester) In a similar manner to Example 8, the piretanide can be reacted with benzyl chloride and triethylamine in DMF to produce -aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzyl benzoate.

EXAMPLE 31 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoic acid 2- (4-morpholino) ethyl ester (pyretanide morpholinoethyl esters) In a similar manner to Example 9, the pyretanide can be reacted with hydrochloride 2 - (4-chloroethyl) morpholino, triethylamine and sodium iodide in DMF to produce 2- (4-morpholine) ethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate.

EXAMPLE 32 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoic acid 3- (N, N-dimethylaminopropyl) [3- (dimethylaminopropyl) pyretanide ester] In a similar manner to Example 54, the pyretanide can be reacting with 3- (dimethylamino) propyl chloride hydrochloride, triethylamine and sodium iodide in dimethylformamide (DMF) to produce 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate of 3- (N, N-dimethylaminopropyl).

EXAMPLE 33 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -dithiobenzoate of 3- (N, -dimethylaminopropyl) [3- (dimethylaminopropyl) dithioester of pyretanide] Similar to Example 32, dithiopyrethanide can be made react with chloride hydrochloride 3- (dimethylamino) propyl, triethylamine and sodium iodide in dimethylformamide (DMF) to produce 3-aminosulfonyl-4 -phenoxy-5- (1-pyrrolidinyl) -ditiobenzoato 3- (N, N- dimethylaminopropyl).

EXAMPLE 34 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoic acid ester of pyridine, [beta] -diethylaminocarbonylmethyl [, -diethylglycolamidoester] In a similar manner to Example 12, the piretanide can be reacted with hydrochloride 2- chloro N, N-diethylacetamide, triethylamine and sodium iodide in dimethylformamide (DMF) to produce the 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate of N, N-diethylaminocarbonylmethyl.

EXAMPLE 35 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoic acid N, N-diethyl ester (pyretanide diethylamide) Similar to Example 13, the piretanide can be reacted with EDC, HOBt and diethylamine in DMF to produce _N, N-diethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzamide.

EXAMPLE 36 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoic acid N, N-diethyl ester (pyrethanide diethylthioamide) In a similar manner to Example 35, the dithiopyrethanide can be reacted with EDC, HOBt and diethylamine in DMF to produce N, N-diethyl 3-aminosulfonyl-phenoxy-5- (1-pyrrolidinyl) -thiobenzamide.

EXAMPLE 37 3-Aminosulfonyl-4-phenoxy-5- (l-pyrrolidinyl) -benzoate of N, N-dibenzyl (piretanide dibenzylamide) In a similar manner to Example 15, the piretanide can be reacted with EDC, HOBt and dibenzylamine in DMF to produce, N-dibenzyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzamide.

EXAMPLE 38 3-aminosulfonyl-4-phenoxy-5- (l-pyrrolidinyl) benzoate rimetilamonio benzyl (benzyltrimethylammonium salt of piretanide) similar to Example 16 way, piretanide can be reacted with benzyltrimethylammonium hydroxide to produce 3- aminosulfonyl-4-phenoxy-5- (l-pyrrolidinyl) -benzyltrimethylammonium benzoate.

EXAMPLE 39 3-Aminosulfonyl-4-phenoxy-5- (l-pyrrolidinyl) -benzoate of cetyltrimethylammonium (cetyltrimethylammonium salt of piretanide) In a similar manner to Example 17, the piretanide can be reacted with cetyltrimethylammonium hydroxide in water to produce the Cetyltrimethylammonium 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate.

EXAMPLE 0 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate of N, N-dimethylaminocarbonylmethyl (β, β-dimethylglycolamide of piretanide) In a similar manner to Example 18, the piretanide can be reacted with 2- chloro-N, N-dimethylacetamide, triethylamine and sodium iodide in DMF to produce the N, N-dimethylaminocarbonylmethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate.

EXAMPLE 41 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -b-butylaminocarbonyloxymethylbenzoate (piretanide pivaxetilyester) In a similar manner to Example 19, the piretanide can be reacted with chloromethyl pivalate, triethylamine and sodium iodide. sodium in DMF to produce the 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate of t-butylcarbonyloxymethyl.

EXAMPLE 42 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -dithiobenzoate of t-butylaminocarbonyloxymethyl (Pivaxethyldithioise of pyretanide) Similar to Example 41, dithiopyrethanide can be reacted with chloromethyl pivalate, triethylamine and iodide of sodium in DMF to produce 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -dithiobenzoate of t-butylcarbonyloxymethyl.

EXAMPLE 43 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -ethylcarbonyloxymethylbenzoate (Piretanide Propaxetilyester) In a similar manner to Example 21, the piretanide can be reacted with chloromethyl propionate, triethylamine and sodium iodide in DMF to produce ethylcarbonyloxymethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) -benzoate.

EXAMPLE 44 Ethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzoate (furosemide ethyl ester) The method of Bundgaard, H., Norgaard, T. and Nielsen, N. M can be used. . , Int. J. Pha rmaceutics, 1988, 42, 217-224, to prepare ethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzoate, mp 163-165 ° C . Using similar methodology furosemide methyl ester, furosemide isoamyl ester, furosemide octyl ester and furosemide benzyl ester can be prepared.

EXAMPLE 45 Methyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzoate (furosemide methyl ester) The method of Bundgaard, H., Norgaard, T. and Nielsen, N. M may be employed. . , Int. J. Pharmaceutics, 1988, 42, 217-224, to prepare methyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzoate.

EXAMPLE 46 5-aminosul onyl-4-chloro-2- [(2-furanylmethyl) amino] thiobenzoic acid (thiofurosemide, - (C = Q) -SH furosemide thioacid) Furosemide can be reacted with thionyl chloride to manufacture the corresponding acid chloride which can then be reacted with sodium hydrogen sulphide to give 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] thiobenzoic acid (thiofurosemide, thioacid of S-furosemide) by methodology of Noble, P. and Tarbell, DS, Org. Synth., Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927.

EXAMPLE 47 5-Aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -iobenzoic acid (thiofurosemide, - (C = Q) -SH furosemide thioacid) The furosemide methyl ester can be reacted with hydrogen sulphide or Sodium hydrogen sulfide to give, followed by acidification, 3-aminosulfonyl acid .5. butylamino-4-phenoxythiobenzoic acid (thiofurosemide, thioacid of S-furosemide).

EXAMPLE 48 Thiomethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate acid (furosemide S-methyl thioester) Similar to Example 1, bumetanide can be reacted with a catalytic amount of chloride of thionyl in methyntiol (methyl mercaptan) to give thiomethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate. Using similar methodology with furosemide and the corresponding thiols, the furosemide S-ethylthioester, furosemide S-isoamyl ester, furosemide S-octyl thioester and furosemide S-benzyl thioester can be prepared. Using similar methodology with dit iofurosemide and the corresponding alcohols, furosemide O-et ilthioester, furosemide 0-isoamylester, furosemide O-octyl thioester and furosemide 0-benzyl thioester can be prepared.

EXAMPLE 49 5-Aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -di-benzoic acid (dithio-furosemide, - (C = S) -SH-dithioacid furosemide) Thiofurosemide can be reacted with thionyl chloride to manufacture the corresponding thioacid chloride which can then be reacted with sodium hydrogen sulphide to give 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -dithiobenzoic acid (dithio-furosemide, furosemide dithioacid) by methodology de Noble, P. and Tarbell, DS, Org. Synth., Coll. Vol. IV, John Wiley & Sons, Inc., New York, 1963, 924-927.

EXAMPLE 50 Methyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -diiobenzoate (furosemide methyldithioester) Similarly to Example 1, the dithio-furosemide can be reacted with a catalytic amount of sodium chloride. thionyl in methyntiol (methyl mercaptan) to give methyl 5-aminosulfonyl-4-chloro-2 - [(2-furanylmethyl) amino] dithiobenzoate. Using similar methodology the diethyldithioether of furosemide, S-isoamyl ester of furosemide, octyl ioester of furosemide and benzyl thioester of furosemide can be prepared.

EXAMPLE 51 5-Aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzoic acid cyanomethyl ester (furosemide cyanomethyl ester) In a similar manner to Example 7, furosemide can. reacting chloroacetonitrile triethylamine and DMF to produce the cyanomethyl 5-aminosulfonyl-4-chloro-2 [(2-furanylmethyl) amino] benzoate.

EXAMPLE 52 5-Aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzyl benzoate (furosemide benzyl ester) Similarly to Example 8, furosemide can be reacted with benzyl chloride and triethylamine in DMF to produce benzyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate.

EXAMPLE 53 2- (4-morpholino) ethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzoate (furosemide morpholinoethyl ester) The method of Mork, N., Bundgaard, H., Shalmi, M. and Christensen, S., Int. J. Pharmaceutics, 1990, 60, 163-169, to prepare 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] -benzoate 2- (4-morpholino) ethyl, melting point 134-135 ° C.

EXAMPLE 54 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of N, N-dimethylaminopropyl) [3- (dimethylaminopropyl) ester of furosemide] The method of Mork, N., Bundgaard can be employed , H., Shalmi, M. and Christensen, S., Int. J. Pharmaceutics, 1990, 60, 163-169, to prepare 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] enzoate of N, N-dimethylaminopropyl), melting point 212-213 ° C.

EXAMPLE 55 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] dithiobenzoate of 3- (N, N-dimethylaminopropyl) [3- (dimethylaminopropyl) dithioester of furosemide] Similarly to Example 54, dithio-furosemide can be reacted with 3- (dimethylamino) propyl chloride hydrochloride, triethylamine and sodium iodide in dimethylformamide (DMF) to produce 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] dithiobenzoate 3- (N, N-dimethylaminopropyl).

EXAMPLE 56 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of furosemide, N-diethylaminocarbonylmethyl [?,? -diethylglycolamidoester] The method of Mork, N. , Bundgaard, H., Shalmi, M. and Christensen, S., Int. J. Pharmaceutics, 1990, 60, 163-169, to prepare 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] ] N, N-diethylaminocarbonylmethyl benzoate, melting point 135-136 ° C.

EXAMPLE 57?,? - Diethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzamide (furosemide diethylamide) Similar to Example 13, furosemide can be reacted with EDC, HOBt and diethylamine in DMF to produce?,? - diethyl 5-aminosulfonyl-chloro-2- [(2-furanylmethyl) amino] benzamide.

EXAMPLE 58?,? - Diethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzamide (furosemide diethylthioamide) Similar to Example 57, the dithio-furosemide can be reacted with EDC, HOBt and diethylamine in DMF to produce?,? - diethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] thiobenzamide.

EXAMPLE 59.?,? - dibenzyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzamide (furosemide dibenzylamide) In a similar manner to Example 15, furosemide can be reacted with EDC, HOBt and dibenzylamine in DMF to produce N, N-diethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzamide.

EXAMPLE 60 5-Aminosulonyl-4-chloro-2- [(2-furanylmethyl) amino] benzyltrimethylammonium benzoate (furosemide benzyltrimethylammonium salt) In a similar manner to Example 16, furosemide can be reacted with benzyltrimethylammonium hydroxide to produce 5-Aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate benzyltrimethylammonium.

EXAMPLE 61 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of cemtrimethylammonium (cetyltrimethylammonium salt of furosemide) Similar to Example 17, furosemide can be reacted with cetyltrimethylammonium hydroxide to produce 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate. of cetyltrimethylammonium.

EXAMPLE 62 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of, N-dimethylaminocarbonylmethyl (?,? -dimethylglycolamidoester of furosemide) The method of Bundgaard, H., Norgaard, T. and Nielsen, NM, Int. J. Pharmaceutics, 1988, 42, 217-224, can be used to prepare the 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of N, N-dimethylaminocarbonylmethyl, melting point 193-194 ° C.

EXAMPLE 63 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] t-butylcarbonyloxymethyl benzoate (furosemide pivaxetilyester) The method of Mork, N., Bundgaard, H., Shalmi, M. and Christensen, S., Int. J. Pharmaceutics, 1990, 60, 163-169, can be used to prepare t-butylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate.

EXAMPLE 64 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] dithiobenzoate of t-butylcarbonyloxymethyl (furosemide pivaxetidithioester) Similarly to Example 63, dithio-furosemide can be reacted with chloromethyl pivalate, triethylamine and sodium iodide in dimethylformamide (DMF) to produce t-butylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] dithiobenzoate.

EXAMPLE 65 Ethylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] dithiobenzoate (Furosemide propamethyl ester) The method of Mork, N. , Bundgaard, H., Shalmi, M. and Christensen, S., Int. J. Pharmaceutics, 1990, 60, 163-169, can be used to prepare ethylcarbonyloxymethyl 5-aminosulfonyl-4-chloro-2- [(2-furanyl-ethyl) amino] dithiobenzoate, melting point 141-142 ° C.

EXAMPLE 66 5- [1- (t-Butylcarbonyloxymethyl) -lH-tetrazol-5-yl] -2-chloro-4- [(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide) In a similar manner to Example 19 , azosemide is reacted with chloromethyl piyalate,. triethylamine and sodium iodide in DMF to produce 5- [1- (t-butylcarbonyloxymethyl) -lH-tetrazol-5-yl] -2-chloro-4- [(2-thienylmethyl) amino] benzenesulfonamide.

EXAMPLE 67 2-Chloro-5- [1- (ethylcarbonyloxymethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide) Similar to Example 19, azosemide can reacting with chloromethyl propionate, triethylamine and sodium iodide in DMF to produce 2-chloro-5- [1- (ethylcarbonylocimethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide.

EXAMPLE 68 2-Chloro-5- [1- (hydroxymethyl) -1H-tetrazol-5-yl] -4- [(2-thienylmethyl) mino] benzenesulfonamide (azoside tetrazolyl-uiside) Azosemide can be reacted with formaldehyde in methylene chloride, mixtures of methylene chloride-DMF or DMF to produce 2-chloro-5- [1- (hydroxymethyl) -1H-tetrazol-5-yl] -4- [. (2-thienylmethyl) amino] benzenesulfonamide.

EXAMPLE 69 2-Chloro-5- [1- (methoxymethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide (tetrazolyl-substituted azosemide) Azosemide can be reacted with formaldehyde, methanol and a strong acid in methylene chloride, mixtures of methylene chloride-DMF or DMF to produce 2-chloro-5- [1- (methoxymethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino) ] benzenesulfonamide.

EXAMPLE 70 2-Chloro-5- [1- (methylthiomethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide (tetrazoyl-substituted azosemide) Azosemide can be reacted with formaldehyde, methanediol and a strong acid in rnetylene chloride, mixtures of methylene chloride-D F or DMF to produce 2-chloro-5- [1- (methylthiomethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide.

EXAMPLE 71 2-Chloro-5- [1- (benzyloxymethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide (tetrazoyl-substituted azosemide) Azosemide is reacted with benzyl chloromethyl ether, triethylamine and sodium iodide in DMF to produce 2-chloro-5- [1- (benzyloxymethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide.

EXAMPLE 72 2-Chloro-5- [1- (benzyloxymethyl) -1H-tetrazol-5-yl] -4- [(2-thienylmethyl) mino] benzenesulfonamide salt (benzyltrimethylammonium salt of azosemide) Similar to Example 16, azosemide is reacted with benzyltrimethylammonium hydroxide in water to yield the benzyltrimethylammonium salt of 2-chloro-5- [1- (benzyloxymethyl) lH-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] ] benzenesulfonamide.

EXAMPLE 73 Cetyltrimethylammonium salt of 2-chloro-5- [1- (benzyloxymethyl) -1H-tetrazol-5-yl] -4- [(2-thienylmethyl) amino] benzenesulfonamide (cetyltrimethylammonium salt of azosemide) Similar to Example 16 , azosemide is reacted with cetyltrimethylammonium hydroxide in water to produce the cetyltrimethylammonium salt of 2-chloro-5- [1- (benzyloxymethyl) lH-tetrazol-5-yl] -4 - [(2-thienylmethyl) amino] benzenesulfonamide.

EXAMPLE 74 3- Isobutylcarbonyloxymethochloride of 3-isopropylcarbamyl-sulfonamido-4- (3'-methylphenyl) aminopyridinium (Pyridinium-substituted-torsemide salt) In a manner similar to Example 19, the torsemide is reacted with chloromethyl pivalate, triethylamine and sodium iodide in DMF to produce 3-isopropylcarbamylsulfonamido-4- (3'-methylphenyl) aminopyridinium t-butylcarbonyloxymethochloride and a certain amount of 3-isopropylcarbamyl sulfonamido-4- (3'-methylgenyl) aminopyridinium t-burylcarbonyloxymethoiodide.

EXAMPLE 75 3-isopropylcarbamyl sulfonamido-4- (3'-methylphenyl) aminopyridinium ethylcarbonyloxymethochloride (Pyridinium-substituted torsemide salt) In a similar manner to Example 19, the torsemide is reacted with chloromethyl propionate, triethylamine and sodium iodide in DMF for producing 3-isopropylcarbamyl sulfonamido-4- (3'-methylphenyl) aminopyridinium ethylcarbonyloxymethochloride and a certain amount of 3-isopropylcarbamyl sulfonamido-4- (3'-methylphenyl) aminopyridinium ethylcarbonylmethoiodide.

EXAMPLE 76 3-Isopropylcarbamyl sulfonamido-4- (3'-methylphenyl) aminopyridinium benzoloxymethoxide (Pyridinium-substituted torsemide salt) Similar to Example 8, the torsemide can be reacted with benzylchloromethyl ether and triethylamine in DMF to produce 3-isopropylcarbamyl sulfonamido benzyloxymethochloride -4- (3 '-methylphenyl) aminopyridinium.

EXAMPLE 77 3-Isopropylcarbamylsulfonamido-4- (3'-methylphenyl) aminopyridinium methoxymethoxide (Pyridinium-substituted torsemide salt) In a similar manner to Example 8, the torsemide is reacted with methylchloromethyl ether and triethylamine and in DMF to produce methoxymethochloride 3- isopropylcarbamysulfonamido-4- (3'-methylphenyl) aminopyridinium.

EXAMPLE 78 3-Isopropylcarbamyl sulfonamido-4- (3'-methylphenyl) aminopyridinium phenylidene (pyridinium-substituted torsemide salt) In a similar manner to Example 8, the torsemide can be reacted with benzyl chloride and triethylamine in DMF to produce phenylmethochloride of 3-isopropylcarbamyl-sulfonamido-4- (3'-methylphenyl) aminopyridinium.

EXAMPLE 79 3-Isopropylcarbamyl sulfonamido-4- (3'-methylphenyl) aminopyridinium benzylthiomethochloride (pyridinium-substituted torsemide salt) In a similar manner to Example 8, the torsemide can be reacted with benzyl thioether chloride and triethylamine in DMF to produce benzylthiamethochloride of 3 -isopropylcarbamyl-sulfonamido-4- (3'-methylphenyl) aminopyridinium.

EXAMPLE 80 3-Isopropylcarbamyl sulfonamido-4- (3'-methylphenyl) aminopyridinium methylthiomethochloride (pyridinium-substituted torsemide salt) In a similar manner to Example 8, the torsemide can be reacted with methylchloromethylthioether chloride and triethylamine in DMF to produce methylthiamethochloride 3 - isopropylcarbamylsulfonamido-4 - (3'-methyl phenyl) aminopyridinium.

EXAMPLE 81 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate methoxy (polyethyleneoxy) ni-ethyl (bumetanide esters mPEG350) Similar to Example 8, bumetanide is reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF to produce methoxy (polyethyleneoxy) ni-ethyl 3-aminosulfonyl-5-but i lamino-4-phenoxybenzoate wherein n is in the range of 7- 8 EXAMPLE 82 Methoxy (polyethyleneoxy) ni-ethyl thiobutene (thioesters of S-bumetanide mPEG350) Similarly to Example 8, thiobumetanide is reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF to produce methoxy (polyethyleneoxy) ni-ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate wherein n is in the range of 7- 8 EXAMPLE 83 Methoxy (polyethyleneoxy) n-ethyl (bumetanide esters mPEGlOOO) 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate Similar to Example 8, thiobumetanide can be reacted with MeO-PEG1000-OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce methoxy (polyethyleneoxy)? -? - ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate wherein n is in the range of 19-24.

EXAMPLE 84 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate methoxy (polyethyleneoxy) ni-ethyl (dithioesters of bumetanide mPEGlOOO) Similar to Example 8, dithiobumetanide can be reacted with MeO-PEG1000-OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce methoxy (polyethyleneoxy) ni-ethyl 3-aminosulfonyl-5-butylamino-4-phenoxy-dithiobenzoate wherein n is in the range from. 19-24.

EXAMPLE 85 3-aminosulfonyl-4-phenoxy-5- (l-pyrrolidinyl) benzoate of methoxy (polyethyleneoxy) ni-ethyl (mPEG350 pyretanide esters) In a similar manner to Example 8, the piretanide can be reacted with MeO-PEG350- Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triet ilamine in DMF to produce methoxy (polyethyleneoxy) n-i-ethyl 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate where n is in the range of 7-8. Similarly, mPEG350 dithioesters of bumetanide can be formed with dithiobumetanide, MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF.

EXAMPLE 86 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate of methoxy (polyethyleneoxy) ni-ethyl (mPEG350 thioesters of S-piretanide) Similar to Example 8, the thiopytanide can be reacted with MeO- PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF to produce methoxy (polyethyleneoxy) n-3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) thiobenzoate) ethyl wherein n is in the range of 7-8.

EXAMPLE 87 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate of methoxy (polyethyleneoxy) ni-ethyl (mPEGlOOO pyretanide esters) In a similar manner to Example 8, the piretanide can be reacted with MeO-PEG1000- OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce methoxy (polyethyleneoxy) ni-ethyl 3-aminosulfonyl-phenoxy-5- (1-pyrrolidinyl) benzoate wherein n is in the range of 19-24. Similarly, mPEGlOOO thioesters of S-piretanide can be formed with S-thiopiretanide, MeO-PEG1000-OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF.

EXAMPLE 88 3-Aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzoate of methoxy (polyethyleneoxy) n-i-ethyl (mPEGlOOO di-esters of pyretanide) Similar to Example 8, dithiopyrethanide can be reacted with MeO-PEG1000-OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce 3-aminosulfonyl-4-phenoxy-5- (1- pyrrolidinyl) dithiobenzoate of methoxy (polyethyleneoxy) ni-ethyl, wherein n is in the range of 19-24. Similarly, mPEGlOOO dithioesters of piretanide can be formed with dithiopyrethanide, MeO-PEG1000-OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF.

EXAMPLE 89 5-aminosulfonyl-4-chloro-2 - [(2-furanylmethyl) amino] benzoate of methoxy (polyethyleneoxy) ni-ethyl (mPEG350 esters of furosemide) Similar to Example 8, furosemide can be reacted with eO -PEG1000-OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce methoxy 5-aminosulfonyl-4-chloro-2- [(2-furanimethyl) amino] benzoate ( polyethyleneoxy) n-i_ethyl wherein n is in the range of 7-8.

EXAMPLE 90 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of methoxy (polyethyleneoxy) ni-ethyl (mPEG350 thioesters of S-furosemide) Similar to Example 8, thiofurosemide can be reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF to produce 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] thiobenzoate methoxy (polyethyleneoxy) n_i-ethyl, where n is in the range of 7-8.

EXAMPLE 91 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of methoxy (polyethyleneoxy) ni-ethyl (mPEGlOOO furosemide esters) Similar to Example 8, furosemide can be reacted with MeO -PEG100-OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce methoxy 5-aminosulfonyl-chloro-2- [(2-furanylmethyl) amino] benzoate (polyethyleneoxy) ) n_i-ethyl where n is in the range of 19-24.

EXAMPLE 92 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzoate of methoxy (polyethyleneoxy) ni-ethyl (mPEGlOOO dithioesters of furosemide) In a similar manner to Example 8, dithiofurosemide can be reacted with MeO-PEG100-OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce 5-aminosulfonyl-chloro-2- [(2-furanylmethyl) amino] dithiobenzoate methoxy ( polyethyleneoxy)? _? - ethyl, wherein n is in the range of 19-24. Similarly, mPEG350 dithioesters of furosemide can be formed with dithiofurosemide, MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF.

EXAMPLE 93 5- [1- [Methoxy (polyethyleneoxy) n-i-ethyl] -lH-tetrazol-5-yl] -2-chloro-4- [(2-thienylmethyl) amino] benzenesulfonamides (N-mPEG350-tetrazolyl-substituted azosemides) ) Similarly to Example 8, azosemide can be reacted with MeO-PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DF to produce 5- [1- [ methoxy (polyethyleneoxy) ni-ethyl] -lH-tetrazol-5-yl] -2-chloro-4 - [(2-thienylmethyl) amino] benzenesulfonamides, wherein n is in the range of 7-8.

EXAMPLE 94 5- [1- [Methoxy (polyethyleneoxy) ni-ethyl] -lH-tetrazol-5-yl] -2-chloro-4- [(2-thienylmethyl) amino] benzenesulfonamides (N-mPEG1000-tetrazolyl-substituted azosemides ) Similarly to Example 8, azosemide can be reacted with MeO-PEG1000-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce 5- [1- [ methoxy (polyethyleneoxy) ni-ethyl] -lH-tetrazol-5-yl] -2-chloro-4- [(2-thienylmethyl) amino] benzenesulfonamides, wherein n is in the range of 19-24.

EXAMPLE 95 Methoxy (polyethyleneoxy) n-1-ethochlorides of 3-isopropylcarbamyl-sulfonamido-4- (3'-methylphenyl) aminopyridinium (Torsemide salts Nm-PEG350-pyridinium) In a similar manner to Example 8, the torsemide can be reacted with MeO -PEG350-Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF to produce methoxy (polyethyleneoxy) ni-ethochlorides of 3-isopropylcarbamyl-sulfonamido-4- (3'-methylphenyl) aminopyridinium, where n is in the range of 7-8.

EXAMPLE 96 3-Isopropylcarbamylsulfonamido-4- (3'-methylphenyl) aminopyridinium methoxy (polyethyleneoxy) ni-ethochlorides (Nm-PEG1000-pyridinium torsemide salts) Similarly to Example 8, the torsemide can be reacted with MeO- PEG1000-OT (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in DMF to produce 3-isopropylcarbamyl-sulfonamido-4- (3'-methylphenyl) aminopyridinium methoxy (polyethyleneoxy) n-1-etochlorides, where n is in the range of 19-24.

EXAMPLE 97 3-aminosulfonyl-5-butylamino-4-phenoxybenzaldehyde (bumetanide aldehyde) By the method of Muraki and Mukiayama (Chem. Letters, 1974, 1447 and Chem. Letters, 1975, 215), bumetanide can be reacted with bis (4-methylpiperazinyl) aluminum hydride to produce 3-aminosulfonyl-5-butylamino-4-phenoxybenzaldehyde.

EXAMPLE 98 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzaldehyde (piretanide aldehyde) By the method of Muraki and Mukiayama. { Chem. Letters, 1974, 1447 and Chem. Letters, 1975, 215), the piretanide can be reacted with bis (4-methylpiperazinyl) aluminum hydride to produce 3-aminosulfonyl-4-phenoxy-5- (1-pyrrolidinyl) benzaldehyde EXAMPLE 99 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) amino] benzaldehyde (furosemide aldehyde) By the method of Muraki and Mukiayama. { Chem. Letters, 1974, 1447 and Chem. Letters, 1975, 215), furosemide can be reacted with bis (4-methylpiperazinyl) aluminum hydride to produce 5-aminosulfonyl-4-chloro-2- [(2-furanylmethyl) ) amino] benzaldehyde.

EXAMPLE 100 Effects of furosemide on epileptiform discharges in hippocampal cuts During these studies, spontaneous epileptiform activity was produced by a variety of treatments. Sprague-Dawley rats (males and females, 25-35 days old) were decapitated, the upper part of the skull was quickly collected and the brain cooled with icy oxygenated cutting medium. The cutting medium was an artificial cerebrospinal fluid based on sucrose (sACSF) consisting of 220 mM sucrose, 3 mM KCI, 1.25 mM NaH2P04, 2 mM gSO4, 26 mM NaHCO3, 2 mM CaCl2 and 10 mM dextrose (295-305). mOsm). A hemisphere of brain containing hippocampus was blocked and glued (cyanoacrylate adhesive) to the stage of a vibrocorter (Frederick Haer, Brunsick, ME). Horizontal or transverse slices of 400 μp ?, thickness were cut in oxygenated cutting medium at 4 ° C (95% 02; 5% C02). The sections were immediately transferred to a retention chamber where they were immersed in an oxygen bath medium (ACSF) consisting of 124 mM NaCl, 3 mM KCI, 125 mM NaH2P04, 2 mM MgSO4, 26 mM NaHCO3, 2 mM CaCl2 and 10 mM dextrose (295-305 mOsm). The cuts or slices were kept at room temperature for at least 45 minutes before being transferred to a dip-style recording chamber (all other experiments). In the recording chamber, the cuts were fused with oxygenated record cuts at 34-35 ° C. All animal procedures were carried out in accordance with the animal care guidelines of NIH and the University of Washington. In most of the cutting experiments, simultaneous extracellular field electrode recordings of CAI and CA3 areas were obtained. A bipolar tungsten stimulator electrode was placed in the collaterals of Schaffer to evoke field responses synoptically driven in CAI. The stimuli consisted of pulses of 100-300 ^ iseg duration at an intensity of four times the population peak threshold. After discharges were provoked by a train of two seconds of such stimuli provided at 60 Hz. Bursts similar to spontaneous interictals were observed in the slices treated by the following modifications or additions to the bath medium: 10 mM potassium (6 slices or cuts; animals; average - 81 bursts / minute); 4-aminopyridine 200-300μ? (4 slices, two animals, average -33 bursts / minute); bicuculina 50-100 μ? (4 slices, three animals, average - 14 bursts / minute); M Mg + (one hour of infusion - 3 slices, two animals, average - 20 bursts / minute or three hours of infusion - two slices, two animals); zero calcium / 6 mM KCl and 2 mM EGTA (4 slices, three animals). In all treatments, furosemide was added to the recording medium once a consistent level of bursts was established. In the first of these procedures, episodes of two discharges were provoked by electrical stimulation of Schaffer collaterals (Stasheff et al., Brain Res. 344: 296, 1985) and the extracellular field response was monitored in the pyramidal cell region of CAI (13 slices, 8 animals) . The concentration of Mg ++ in the bath medium was reduced to 0.9 mM and then the discharges were evoked by stimulation at 60 Hz for 2 seconds at an intensity of 4 times the stimulation peak threshold (the peak intensity of the population peak varied). between 20-150 μ? a pulse duration of 100-300 ^ iseg). The tissue is allowed to recover for 10 minutes between stimulation tests. In each experiment, the initial response in CAI to the synaptic input was first tested by recording the field potential evoked by a single pulse. In the control condition, the collateral stimulation of Schaffer evoked a single population peak (Figure 1A, insert). Tetanic stimulation evoked approximately 30 seconds after discharge (Figure 1A, left) associated with a large change in intrinsic signal (Figure 1A, right). For the imaging of intrinsic optical signals, the tissue was placed in a perfusion chamber located at the stage of a vertical microscope and illuminated with a white light beam (tungsten filament light and lens system; Finger Inc.) directed through the microscope condenser. The light was controlled and regulated (power supply - Lamda Inc.) to minimize fluctuations and was filtered (695 nm long passage) in such a way that the slice or cut was transilluminated with long wavelengths (red). The field of vision and amplification were determined by the choice of microscope objectives (4X to monitor all slices). Image-frames were acquired with a camera of charge-coupled devices (CCD) (Dage MTI Inc.) at 30 Hz and were digitized at 8 bits with a spatial resolution of 512 x 480 pixels using an Imaging Technology imaging system Inc. Series 151; The gains and displacements of the control box-camera and the A / D board were adjusted to optimize the sensitivity of the system. The physical elements of image formation were controlled by a compatible 486-PC computer. To increase the signal / noise, an averaged image was composed from 16 individual frame images, integrated in 0.5 seconds and averaged together. An experimental series commonly involved the continuous acquisition of a series of images averaged over a period of several minutes, at least 10 of these combined images were acquired as control images before stimulation. Pseudo-colored images were calculated by subtracting the first control image from subsequently acquired images and assigning a color look-up table to the pixel values. For these images, usually a linear low pass filter was used to remove the high frequency noise and a linear histogram sketch was used to map the pixel values in the dynamic range of the system. All the operations in these images were linear, in such a way that quantitative information was preserved. The noise was defined as the standard deviation of AR / R fluctuations of the sequence of control images within a given acquisition series, where AR / R represents the magnitude of the change in light transmission through the tissue. Delta R / R was calculated by forming all the difference images and dividing the first control image: (subsequent image - first control image) / first control image. The noise was always < 0.01 for each of the sequences of the chosen sequence images. The absolute change in light transmission through the tissue was filmed during some experiments by acquiring images after placing neutral density filters between the camera and the light source. On average, the electronic components of the camera and the electronic components of the imaging system amplified the signal 10 times before digitization, such that the peak absolute changes in light transmission through the tissues were usually between 1 and 2%. The grayscale photo shown in Figure ID is a video image of a typical hippocampal slice in the recording chamber. The fine gold wire mesh that was used to hold the fabric in place can be seen as dark lines running diagonally through the slice. A stimulating electrode can be seen in the upper right part of the stratum radiatum of CAI. The registration electrode (too thin to be observed in the photo) was inserted at the point indicated by the white arrow. Figure 1A illustrates that two seconds of stimulation at 60 Hz produced after discharge activity and shows an episode after typical redlipping recorded by the extracellular electrode. The internal phase of Figure 1A shows the CAI field response to a single 200-second test pulse (artifact on the arrow) administered to the Schaffer collaterals. Figure 1A1 shows a map of the peak change in optical transmission through tissue evoked through stimulation collateral of Schaffer. The region of maximum optical change corresponds to the apical and basal dendritic regions of CAI either on one side or the other of the stimulating electrode. Figure IB illustrates traces of samples that show responses to stimulation after 20 minutes of perfusion with medium containing 2.5 mM furosemide. Both the electrical activity after discharge (shown in Figure IB) and the optical changes evoked by stimulation (shown in Figure 1B1) were blocked. Nevertheless, there was a hyper-excitable field response (multiple population peaks) to the test pulse (internal). Figures 1C and 1C1 illustrate that restoration of initial response patterns was seen after 45 minutes of perfusion with normal bath medium. The opposing effects of furosemide blockade of post-discharges evoked by stimulation and a concomitant increase in synaptic response to a prieba-ompulse illustrate the two key outcomes: (1) epileptiform activity blocked by furosemide and (2) synchronization (as reflected) by spontaneous epileptiform activity) and excitability (as reflected by the response to a single synaptic input) were dissociated. The experiments in which the dose dependence of furosemide was examined determined that a minimum concentration of 1.25 mM was required to block the two discharges and optical changes.

EXAMPLE 101 Effects of furosemide on epileptiform discharges in hippocampal sections subjected to perfusion with bath medium with high content of -K + (10 mM). Rat hippocampal slices, prepared as described above, were perfused with a solution of high K + content until extensive periods of spontaneous interictal-like bursts were recorded simultaneously in the CA3 pyramidal cell regions (traces above9 and CAI (traces lower) (Figures 2a and 2B) After 15 minutes of perfusion with medium containing furosemide (furosemide 2.5 mM), the bursts were increased in magnitude (Figures 2A and 2B), however, after 45 minutes of perfusion of furosemide, the bursts increased in magnitude (Figures 2C and 2D), however, after 45 minutes of perfusion of furosemide, the bursts were reversibly blocked (Figure 2E, 2F, 2G and 2H). of perfusion of furosemide, the synaptic response to a single test impulse administered to the Schaffer collaterals was either unchanged or improved (d atos not shown). It is possible that the initial increase in discharge amplitude reflected a decrease induced by furosemide in inhibition (Misgeld et al., Science 232: 1413, 1986, Thompson et al., J. Neurophysiol., 60: 105, 1988, Thompson and Gáhwiler, J. Neuropysiol 61: 512, 1989, and Pearce, Neuron 10: 189, 1993). These were previously reported (Pearce, Neuron 10: 189, 1993). It has been previously reported (Pearce, Neuron 10: 189, 1993) that furosemide blocks a component of inhibitory currents in hippocampal slices in a latency (<15 minutes) similar to the time to initiate the increased excitability seen herein. The longer latency required for the furosemide blocking of the spontaneous burst could correspond to the additional time required for a sufficient blockade of the furosemide sensitive cell regulation mechanisms under conditions of high -K + content. After testing the effects of furosemide on slices or sections subjected to perfusion with high content of -K +, similar studies were performed with a variety of other in vitro models commonly studied for epileptiform discharges (Galvan et al., Brain Res. 241: 75 , 1982, Schwart zkroin and Prince, Brain Res.183: 61, 1980, Anderson et al., Brain Res. 398: 215, 1986, and Zhang et al., Epilepsy Res. 20: 105, 1995). After prolonged exposure (2-3 hours) to a magnesium-free medium (0-Mg ++), slices or slices were shown to develop epileptiform discharges that are resistant to common clinically used anticonvulsant drugs (Zhang et al., Epilepsy Res. 20: 105, 1995). Records of the entorinal cortex (Figure 21) and subiculum (not shown) showed that after three hours of perfusion with 0-Mg ++ medium, slices or slices developed burst patterns that appeared similar to bursts "resistant" to resistant anticonvulsant "previously described, one hour after the addition of furosemide to the bath medium, these bursts were blocked (Figure 2J), furosemide also blocked spontaneous burst discharges observed with the following additions / modifications to the bath medium: (1) addition α -aminopyridine 200-300 μ? (4-AP, a potassium channel blocker) (Figures 2K and 2L); (2) addition of the GABA antagonist bicuculline a, 50-100 μ? (Figures 2M and 2N); (3) removal of magnesium (0-Mg ++) - one hour of perfusion (Figures 20 and 2P) and (4) removal of extracellular chelation plus calcium (0-Ca ++) (Figures 2Q and 2R). manipulations, patterns similar to spontaneous interictal fu were registered simultaneously from CAI and CA3 (Figures 2K, 2L, 2M and 2N show only the trace of CA3 and Figures 20, 2P, 2Q and 2R show only the trace of CAI). In the 0-Ca ++ experiments, furosemide 5 mM blocked the bursts with a latency of 15-20 minutes. For all other protocols, bursts were blocked with 2.5 mM furosemide with a latency of 20-60 minutes. Furosemide reversibly blocked the spontaneous burst activity of both CAI and CA3 in all experiments (Figures 2L, 2N, 2P and 2R).

EXAMPLE 102 Effects of furosemide on epileptiform activity induced by i.v. of Cainic Acid in Anaesthetized Rats This example illustrates an in vivo model in which the epileptiform activity was induced by i.v. from cainic acid (KA) to anesthetized rats (Lothman et al., Neurology 31: 806, 1981). The results are illustrated in Figures 3A-3H. Sprague-Dawley rats (4 animals, weights 250-270 g) were anesthetized by urethane (1.25 g / kg i.p.) and anesthesia was maintained by additional urethane injections (0.25 g / kg i.p.) as necessary. The body temperature was monitored using a rectal temperature probe and maintained at 35-37 ° C with a heating pad, the heart rate (EKG) was continuously monitored. The jugular vein was cannulated aside for intravenous drug administration. The rats were placed in a stereotaxic opf device (with the upper part of the skull level) and a bipolar stainless steel electrode insulated 0.5 mm from the tip was inserted to a depth of 0.5-1.2 mm from the cortical surface to record the electroencephalographic agility (EEG) to the fronto-parietal cortex. In some experiments, a pipette containing 2M NaCl was lowered to a depth of 2.5-3.0 mm to record hippocampal EEG. The data recorded on VHS videotape and analyzed offline. Following the surgical preparation and electrode placement, the animals are allowed to recover for 30 minutes before the experiments were initiated with an injection of cainic acid (10-12 mg / kg, i.v.). The intense attack activity, an increased heart rate and rapid movements of the vibrissae were induced with a latency of approximately 30 minutes. Once the stable electrical attack was evident, furosemide was administered in boluses of 20 mg / kg every 30 minutes to a total of three injections. The experiments were terminated with the intravenous administration of urethane. The care of the animals was in accordance with NIH guidelines and approved by the Animal Care Committee of the University of Washington. Figures 3A-3H show the furosemide blocking of electric "status epilepticus" evoked by cainic acid in urethane-anesthetized rats. The EKG records are shown as the upper traces and the EEG records are shown as the lower traces. In this model, an intense electrical discharge ("status epilecticus" electrical) was recorded from the cortex (or deep hippocampal electrodes) 30-60 minutes after the injection of KA (10-12 mg / kg) (Figures 3C and 3D). Control experiments (and previous reports, Lothman et al., Neurology, 31: 806, 1981) showed that this status-like activity was maintained for more than three hours. Subsequent intravenous furosemide injections (cumulative dose: 40-60 mg / kg) blocked the attack activity with a latency of 30-45 minutes, often producing a relatively flat EEG (Figures 3E, 3F, 3G and 3H). Even 90 minutes after the injection of furosemide, the cortical activity remained close to normal reference levels (ie, those observed before the KA injections and furosemide injections). Studies in the pharmacokinetics of furosemide in rat indicate that the dosages used in this example were very well below the toxic levels (Hammarlund and Paalzow, Biopharmaceutics Drug Disposition, 3: 345, 1982).

Experimental methods for Examples 103-106 Hippocampal slices or slices were prepared from adult Sprague-Dawley rats as previously described. Transverse hippocampal slices of 100 μp? of thickness were cut with a vibrating cutter. The slices commonly contained the entire hippocampus and subiculum. After cutting, the slices were stored in an oxygenated retention chamber at room temperature for at least one hour before registration. All records were acquired in an inferia chamber with oxygenated artificial cerebral spinal fluid (ACSF) (95% 02, <5% C02) at 34 ° -35 ° C. Normal ACSF (in mmol / L) contained: NaCl 124, KC1 3, NaH2P04 1.25, MgS04 1.2, NaHC03 26, CaCl2 2 and dextrose 10. Sharp electrodes for intracellular recordings of pyramidal cells of CAI and CA3 were filled with potassium acetate 4M. The field records of the body layers of CAI and CA3 cells were acquired with low resistance glass electrodes filled with 2M NaCl. For stimulation of the Schaffer collateral path or spin, a small monopolar tungsten electrode was placed on the surface of the slice. The activities evoked by stimulation and spontaneous field and intracellular recordings were digitized (eurocorder, Neurodata Instruments, New York, NY) and stored on videotape. AxoScope programming elements (Axon Instruments) on a personal computer were used for offline analysis of the data. In some experiments, normal or medium medium with low chloride content was used that contains vicuculin (20 μ?), 4-aminopyridine (4-AP) (100 μ?) Or high content of -K + (7.5 or 12 mM). In all experiments, solutions with low chloride content (7 and 21 mM [C1 ~] 0) were prepared by equimolar replacement of NaCl with Na + -gluconate (Sigma). All solutions were prepared in such a way that they had a pH of about 7.4 and an osmolarity of 590-300 mOsm at 35 ° C and at the equilibrium of carbooxygenation with 95% 02/5% C02. After placement in the interface chamber, the slices were perfused at approximately 1 ml / min. At this flow rate, it took 8-10 minutes for the changes in the perfusion media to be consummated. All the times reported here have taken this delay into account and have an error of approximately + 2 minutes.

EXAMPLE 103 Synchronization or timing of cessation of spontaneous epileptiform bursts in areas in CAI and CA3 The relative contributions of the factors that modulate synchronized activity vary between the CAI and CA3 areas. these factors include differences in local circuits and region-specific differences in cell packaging and volume fraction in cell spaces. If the anti-epileptic effects of anion or chloride cotransport antagonism are due to a disharmonization in the neuronal discharge timing, it could be expected that the chloride co-transport blockade differentially adapts the CAI and CA3 areas. To test this, a series of experiments were carried out to characterize differences in the timing or timing of blocking spontaneous epileptiform activity in the CAI and CA3 areas. Field activity was recorded simultaneously in areas CAI and CA3 (approximately halfway between the nearest and most distant extension of the CA3 region) and spontaneous bursts were induced by treatment with - [K +] 0 (12 μ ?; n = 12 ), bicuculline (20 mM, n = 12) and 4-AP (100 μ ?; n = 5). A single electrical stimulus was fed to the Schaffer collaterals, halfway between the CAI and CA3 areas, every 30 seconds, such that the field response in the CAI and CA3 areas could be monitored throughout the duration of each experiment. . In all experiments, at least 20 minutes of continuous spontaneous epileptiform bursts were observed before commutation to low [C1 ~] 0 (21 mM) or medium containing furosemide (2.5 mM). In all cases, after exposure of 30-40 minutes to furosemide or medium with low chlorine content, spontaneous gusts ceased in the area of CAI before the bursts ceased in the CA3 area. The commonly observed time sequence of events included an initial increase in the frequency and amplitude of bursts of the spontaneous field events, then a reduction in the amplitude of the burst discharges that was faster in CAI than in CA3. After CAI became silent, CA3 continued to discharge for 5-10 minutes, until it no longer exhibited spontaneous epileptiform events. This temporal pattern of cessation of bursts was observed with all treatments that induce epileptiforms tested, regardless of whether the agent used to block the spontaneous bursts was furosemide or medium with low content of [C1 ~] 0. In all the stages of these experiments, the stimulation of Schaffer collaterals evoked hyperexcitated responses in both of the body layers of CAI and CA3 cells. Immediately after the spontaneous gusts were blocked in both CAI and CA3 areas, hyperexcited population peaks could still be evoked.

The possibility was considered that the observed cessation of bursts in CAI before CA3 was an artifact of the organization of synaptic contacts between these areas in relation to the choice of recording sites. It is known that the projections of the various sub-regions of CA3 end in an organized manner in CAI; CA3 cells closest to the dentate virus (proximal CA3) tend to project more strongly to the distant portions of CAI (near the subicular border), whereas CA3 projections that arise from cells located more distantly in CA3 end up more strongly in portions of CAI located closest to the CA2 border. If the cessation of bursts occurs in the different subregions of CA3 at different times, the results of the previous set of experiments could arise not as a difference between CAI and CA3, but rather as a function of the variability in activity of bursts through of the CA3 subregions. This possibility was tested in three experiments. Immediately after the spontaneous bursts ceased in CAI, the CA3 field was studied with a recording electrode. Records from different CA3 sites (from the portions closest to the most distant portions of CA3), showed that all subregions of the CA3 area spontaneously burst during the time that CAI was silent. The observation that CA3 continued to spontaneously discharge after CAI became silent was unexpected since population discharges in CA3 are generally thought to evoke CAI discharges by means of excitatory synaptic transmission. As previously described, single-impulse stimuli fed into Schaffer collaterals still evoked multiple population peaks in CAI even after spontaneous bursts were blocked; thus, the excitatory synaptic transmissions hyperexcited at the synapse of CA3 to CAI was intact. Given this sustained efficiency of synaptic transmission and continuous spontaneous field discharges in CA3, it was postulated that the spontaneous burst cells in CAI was due to a decrease in the timing of the incoming excitatory pulse. Furthermore, since the spontaneous epileptiform discharge in CA3 also eventually ceased, perhaps this process of desynchronization occurred at different times in the two hippocampal subfields.

EXAMPLE 104 Effect of antagonism of chloride co-transport in synchronization of field population discharges of CAI and CA3 The observation of Example 103 suggested a temporal relationship between the exposure time to medium containing low content of [C1"] 0 or furosemide and the characteristics of the activity of spontaneous bursts.In addition, this relationship was different between the CAI and CA3 areas In order to better characterize the temporal relationships, the occurrences of CAI action potentials and the events of CAI were compared. population peak in the field response of the CAI and CA3 subfields during the discharge of spontaneous bursts and evoked by stimulation.Intracellular records were obtained from CAI pyramidal cells, with intracellular electrode placed near (<100 μ ??) to the electrode of CAI field The slice or cut was stimulated every 20 seconds with a single stimulus fed to the collaterals of Schaffer. Since continuous spontaneous bursts were established for at least 20 minutes, the bath medium was connected to a low-content medium of [C1"] 0 containing bicuculline. After approximately 20 minutes, the frequency and amplitude of bursts was at its maximum. Simultaneous and intracellular field records during this time showed that the CAI and intracell field records were closely synchronized with the CA3 field downloads. during each spontaneous discharge, the CA3 field response preceded the CAI download for several milliseconds. During events evoked by stimulation, the action potential discharges of the CAI pyramidal cell were closely synchronized to both of the field discharges of CA3 and CAI. With continuous exposure to a medium with low content of [C1"] 0, the latency between the spontaneous discharges of the CAI and CA3 areas increased, with a maximum latency of 30-40 milliseconds that occurs after 30-40 minutes of exposure to the medium with low content of chloride containing bicuculline During this time, the amplitude of both of the spontaneous field discharges of CAI and CA3 decreased.The discharges evoked by stimulation during this time started the discharges that occur spontaneously in morphology and relative latency However, the depolarization evoked by the initial stimulus of the neuron (supposedly the monosynaptic EPSP) began without any significant increase in latency The time interval during which these data were acquired corresponds to the time immediately before of cessation of spontaneous bursts in CAI After 40-50 minutes of perfusion with low-content medium gone from [C1 ~] 0, spontaneous gusts were almost abolished in CAI but were unaffected in CA3. Schaffer's collateral stimulation during this time showed that the monosynaptically triggered responses of the CAI pyramidal cells occurred without any significant increase in latency, but that the evoked field responses of stimulation were almost abolished. The time interval during which these data were acquired correspond to the moments immediately before the cessation of spontaneous bursts in CA3. After prolonged expulsion to a medium containing low [C1 ~] 0 content, large increments (> 30 milliseconds) developed in the latency between the Schaffer collateral stimulation and the consequent CA3 field discharge. Inevitably, no field response could be evoked by the collateral stimulation of Schaffer in either the CAI or CA3 areas. However, the release of action potential of the CAI pyramidal cells in response to the collateral stimulation of Schaffer could be evoked with little change in response latency. Of course, for the entire duration of the experiments (greater than two hours), potential discharges of action of the CAI pyramidal cells could be evoked to a short latency by means of Schaffer collateral stimulation. In addition, although the hyperexcited discharges evoked by CA3 stimulation were inevitably blocked after prolonged exposure to medium of low [C1 ~] 0 content, the antidromic response in CA3 appeared to be conserved.

EXAMPLE 105 Effects of antagonism of co-transport of chloride on the synchronization of burst discharges in pyramidal CAI cells The above data suggest that the disappearance of field responses may be due to a desynchronization of the presence of action potentials between neurons . That is, although the synoptically driven excitation of the CAI pyramidal cells was not conserved, the synchrony of action potential among the neural population of CAI was not sufficient to add to a measurable CD field response. In order to test this, paired intracellular registrations of CAI pyramidal cells were acquired simultaneously with the CAI field responses. In these experiments, both the intracellular electrodes and the field registration electrodes were placed within 200 μp \ each other. During the period of maximum spontaneous activity induced by the low-content medium of [C1"] 0 containing bicuculline, the records showed that the action potentials between pairs of CAI neurons and the CAI field discharges were strongly synchronized during the Spontaneous discharges and shocks evoked by stimulation After prolonged exposure to medium with low content of [C1 ~] 0, when the amplitude of the field discharge of CAI began to expand and decrease, both of the spontaneous discharges and discharges evoked by stimulation showed a synchronous desynchronization in the presence of action potentials between pairs of CAI neurons and between the action potentials and the field responses.This desynchronization was coincident with the suppression of field amplitude of CAI. that the spontaneous gusts in CAI ceased, a significant increase in latency had developed re the collateral stimulation of Schaffer and the field discharge of CAI. At this time, the paired intracellular recordings showed a spectacular desynchronization in the synchronization of action potential discharges between pairs of neurons and between the presence of action potentials and the field discharges evoked by Schaffer collateral stimulation]. It is possible that the observed desynchronization of the CAI action potential discharge is due to the randomization of mechanisms necessary for the generation of synoptically driven action potential, such as a disruption in synaptic release synchronization or random conduction failures in processes neuronal If this were the case, one would expect that the presence of action potentials between a given pair of neurons would vary randomly from one stimulus to another. This was tested by comparing the patterns of action potential discharges of neuronal pairs between multiple consecutive stimuli of the Schaffer collaterals. During each stimulation event, the action potentials occurred at almost identical times to each other and showed an almost identical burst morphology from stimulation to stimulation. It was also modified to see if the presence of action potentials between a given pair of neurons during simultaneous field discharges was fixed over time. The patterns of action potential discharges in a given pair of CAI neurons were compared between consecutive spontaneous field bursts during the time when the presence of action potentials was clearly disengaged. Just as in the case of action potential discharges evoked by discharge stimulation described above, the action potentials generated during a spontaneous population discharge occurred at almost identical times to each other and showed an almost identical burst morphology from a spontaneous discharge to The next.

EXAMPLE 106 Effects of treatment with low chloride content with spontaneous synaptic activity It is possible that the anti-epileptic effects associated with the antagonism of co-transport of chlorine are moderated by some action on the release of the transmitter. The blocking of chloride co-transport could alter the amount or synchronization of the transmitter released from the terminals, thus affecting neuronal synchronization. To test whether exposure to low content of [C1"] 0 affected the mechanisms associated with the release of the transmitter, intracellular CAI responses were simultaneously recorded with CAI and CA3 field responses during a treatment that dramatically increased the spontaneous synaptic release of the transmitter of the presynaptic terminals The increased spontaneous release of the transmitter was induced by treatment with 4-AP (100 μ?) After 40 minutes of exposure to a medium containing 4-AP, spontaneous synchronized bursts were recorded in CAI and CA3 areas Switching to medium with low content of [C1"] 0 containing 4-AP initially led, as previously demonstrated, to improved spontaneous bursts. Intercellular records with high grain content showed that spontaneous high-amplitude synaptic activity was produced by 4-AP treatment. Additional exposure to the medium with low chloride content blocked the discharge of spontaneous bursts in CAI, although CA3 continued to discharge spontaneously. At this time, the CAI intercellular records showed that the spontaneous synaptic noise was further increased and remained so for a prolonged exposure time to medium with low chloride content containing 4-AP. These data suggest that the mechanisms responsible for the terminal synaptic release are not adversely affected by exposure to low chloride content in a manner that could explain the blocking of spontaneous bursts induced by 4-AP in CAI. These results also eliminate the possibility that the effects of exposure to low content of [C1"] 0 are due to alterations in dendritic properties in CAI that as they had their effectiveness in producing PSP to the soma.

EXPERIMENTAL METHODS FOR EXAMPLES 107 TO 111 In all of the following experiments, [C1"] 0 was reduced by equimolar replacement of NaCl with Na + -gluconate.Calconate was used rather than other starch replacements for several reasons. Patch-clamp tests have shown that gluconate appears to be virtually imperme- ent to chloride channels, while other anions (including sulfate, isethionate and acetate) are permeable to varying degrees.Secondly, the transport of extracellular potassium through the co- NKCC1 glial transport is blocked when the extracellular chloride is replaced by gluconate but is not completely blocked when it is replaced with isethionate, since this furosemide-sensitive co-transporter plays a significant role in cell swelling and volume changes of the extracellular space (ECS), it is desired to use the appropriate anion replacement, in such a way that the treatment effects would be comparable to previous furosemide experiments (Hochman et al. Science, 270: 99-102, 1995; U.S. Patent No. 5,902,732). Thirdly, formate, acetate and propionate generate weak acids when used as Cl substitutes and lead to an early drop in intracellular pH, gluconate remains extracellular and has not been reported to induce intracellular pH shifts. Fourth, for comparison purposes, we want to use the same anion replacement that had been used in previous studies that examines the effects of low - [C1 ~] 0 content on the changes evoked by ECS activity. Certain replacements of anions may chelate calcium, although subsequent work has failed to demonstrate any significant anionic ability to chelate calcium, there is still some concern in the literature regarding this issue. following experiments, since resting membrane potentials remained normal and synaptic responses (for their post, hyperexcitable synaptic responses) could be produced even after several hours of exposure to the medium in which - [C1 ~] 0 had been reduced by gluconate substitution. In addition, it was confirmed that the concentration of calcium in the medium of low content of - [C1 ~] 0 was identical to that in the control medium by measurements made with selective Ca2 + microelectrodes. Adult Sprague-Dawley rats were prepared as previously described. Briefly, transverse hippocampal slices, 400 μ? T? of thickness, were placed using a vibrating cutter. Slices or cuts commonly contained the entire hippocampus and subiculum. After cutting, the slices were stored in an oxygenated retention chamber for at least one hour before registration. All records were acquired in an inferred type chamber with artificial cerebral spinal fluid (95% 02/5% C02) (ACSF) at 34-35 ° F. The normal ACSF contained (in mmol / L): NaCl 124, KC1 3, NaH2P04 1.25, NaHC03 26, CaCl2 2 and dextrose 10. In some experiments, normal or medium medium with low chloride content was used containing bicuculline (20 μ ?), 4-AP (100 μ?), Or high K + content (12 mM). Solutions of low chloride content (7, 16 and 21 mM [C1"] 0) were prepared by equimolar replacement of NaCl with Na + -gluconate (Sigma Chemical Co., San Luis, MO) All solutions were prepared in such a manner which had a pH of about 7.4 and an osmolarity of 290-300 mOsm at 35 ° C and at the equilibrium of carboxynation with 95% 02/5% C02.Acute electrodes filled with 4M potassium acetate were used for intracellular cell registration pyramidal CAI Field records of cell body layers CAI or CA3 were acquired with low resistance glass electrodes filled with NaCl (2M) For stimulation of the Schaffer collateral path, a small monopolar electrode was placed on the surface of the slices midway between the CAI and CA3 areas. Spontaneous activities and activities evoked by stimulation of intracellular field records were digitized (Neurocorder, Neurodata Instruments, New York, NY) and stored on video tape. Programming elements AxoScope (Axon Instruments Inc.) on a PC computer was used for offline analysis of the data. Ion-selective microelectrodes were manufactured according to standard methods well known in the art. Double barrel pipettes were pulled and broken to a tip diameter of approximately 3.0 μ? T ?. The reference barrel was filled with ACSF and the other barrel was silanized and the tip refilled with a selective K + resin (Corning 477317). The rest of the silanized barrel was filled with KC1 (40 mM). Each barrel was conductive, via Ag / AgCl wires to a high impedance dual differential amplifier (PI FD223). Each ion-selective microelectrode was calibrated by using solutions of known ionic composition and was considered appropriate if it was characterized by an almost Nernstian dependent response and if it remained stable throughout the duration of the experiment. After placement in the interphase chamber, the slices were super-fused at approximately 1 ml / minute. At this flow rate, it took approximately 8-10 minutes for changes in perfusion media to be completed. All reported times have taken this time delay into account and have an error of approximately + 2 minutes.

EXAMPLE 107 Effects of low content of - [C1"] 0 in CAI field records Other studies have shown that prolonged exposure of cortical and hippocampal slices at low content of - [C1 ~] 0 does not affect the intrinsic and synaptic properties such as input resistance, membrane potential and rest, generation of action potential induced by depolarization or excitatory synaptic action.A previous study has also partially characterized the epileptogenic properties of exposure to low content of - [C1"] 0 to CAI area of the hippocampus. The following studies carried out to observe the times of onset and possible cessation of hyperexcitability and hypersynchrony induced by low content of - [C1 ~] 0. the slices (n = 6) were initially perfused with normal medium until stable intracellular and field recordings were established in a CAI pyramidal cell and the CAI cell body layer, respectively. In two experiments, the same cell was retained for the entire duration of the experiment (greater than 2 hours). In the remaining experiments, (n = 4) the initial intracellular record was lost during the sequence of medium changes and additional records acquired from different cells. The patterns of neuronal activity in these experiments were identical to those observed when a single cell was observed. The field and intracellular electrodes were always placed in close proximity to each other (< 200 μ? P?). In each case, after approximately 15-20 minutes of exposure to the medium of low [C1 ~] 0 (7 mM) content, spontaneous bursts developed, first at the cellular level and then in the field. This, spontaneous field activity, represents the discharge of synchronized bursts in a large population of neurons, around 5-10 minutes, time after which the field registration became evident. When the field became silent for the first time, the cell continued to discharge spontaneously. This result suggests that the population activity had "desynchronized" while the ability of the individual cells to discharge had not deteriorated. After approximately 30 minutes of exposure to the medium of low [C1-] 0 content, the intracellular record showed that the cells continued to discharge spontaneously although the field remained silent. The response of the cell to intracellular current injection at two points in time showed that the cells' ability to generate action potentials had not been impaired by exposure to low content of [C1"] 0.

In addition, the electrical stimulation in the stratum radiatum of CAI produced burst shocks, indicating that a hyperexcitable state was maintained in the tissue.

EXAMPLE 108 Effects of low content of [C1 ~] 0 on epileptiform activity induced by high content of [K +] Q in CAI The previous set of experiments demonstrated that the exposure of tissues to medium with low content of [C1 ~] 0 induced a brief period of bursts of spontaneous field potential when they ceased within 10 minutes. If it is considered that a reduction of [C1 ~] 0 is unavoidably capable of blocking spontaneous epileptiform bursts (ie, synchronized), then these results suggest anti-epileptic effects would probably be observable only after this initial period of bursts had ceased. The temporal effects of treatment with low content of [C1"] 0 in the activity of bursts with high content of [K +] 0 were examined The slices (n = 12) were exposed to the medium in which [+] 0 had been increased to 12 mM and the field potentials were recorded with a field electrode in the CAI cell body layers.Fire bursts of spontaneous field potentials were observed for at least 20 minutes and then the slices were exposed to the medium in which [K + ] 0 was maintained at 12 mM, but [C1 ~] 0 was reduced to 21 mM. In the course of 15-20 minutes after the tissue was exposed to medium with high content of [K +] O / low content of [C1 ~] 0. In the course of 15-20 minutes after the tissue was exposed to the medium of low content of - [Cl] O / high content of - [K +] 0-, the amplitude of bursts was increased in each event and each: field event it had a longer duration. After a brief period after this facilitated field activity (lasting 5-10 minutes), the bursts stopped. To test if this blockage was reversible, after at least 10 minutes of silence of field potential, it was switched back to high content of - [K +] 0- with [Cl] 0 normal. The bursts returned in the course of 20-40 minutes. In each experiment, the CAI field response to the collateral stimulation of Schaffer was monitored. The largest field responses were recorded just before the cessation of spontaneous gusts, during the period when spontaneous gusts had the greatest amplitude. Even after blockage of spontaneous bursts, however, multiple population peaks were produced by collateral Schaffer stimulation, indicating that the synaptic transmission was intact and that the tissue remained hyperexcitable. In four slices, intracellular records of CAI pyramidal cells were acquired together with the CAI field record. During the bursts period spontaneous induced by high content of - [K +] 0-, hyperpolarization current was injected into the cell in such a way that post-synaptic potentials (PSP) could be observed best. After blocking with low content of - [Cl] 0- spontaneous bursts, action potentials that spontaneously and PSP were still observed. These observations also support the view that synaptic activity, per se, was not blocked by treatment with low content of - [Cl] 0.

EXAMPLE 109 Blocking with low content of - [Cl] Q of activity epileptiform induced by 4-AP, high content of - [K +] Q and bicuculline in CAI and CA3 Then it was tested whether the treatment with low Content of - [Cl] 0 could block epileptiform activity in the CAI and CA3 areas, which was produced by different pharmacological treatments, as has been demonstrated for treatment with furosemide. For this set of experiments, it is chosen to test the effects of treatment with low content of - [Cl] 0 in the spontaneous bursts that had been induced by high content of ~ [K +] 0 (12 mM) (n = 5), 4-AP (100 μ?) (n = 4) and bicuculline (20 and 100 μ?) (n = 5). In each set of experiments, field responses were recorded simultaneously from the CAI and CA3 areas and in each case, the spontaneous epileptiform activity in both CAI and CA3 areas, was reversibly blocked in 30 minutes after [Cl] 0 in the perfusion medium had been reduced to 21 mM. These data suggest that, like furosemide, the low content of - [Cl] 0 reversibly blocks spontaneous bursts in several of the most commonly studied in vitro models of epileptiform activity.

EXAMPLE 110 Comparison between low content of - [Cl] Q and furosemide in the blockade of epileptiform activity induced by high content de - [K +] 0- The data from previous sets of experiments are consistent with the hypothesis that anti¬ subjects Epileptic patients with both low content of - [Cl] 0 and furosemide are moderated by their actions in the same physiological mechanisms. To further test this hypothesis, the temporal sequence of effects of low content of - [Cl] 0 (n = 12) and furosemide (2.5 and 5 mM) (n = 4) in bursts induced by high content of - [K +] 0- as recorded with a field electrode in CAI. It was found that both the low content of - [Cl] 0 as the furosemide treatment induced a similar temporal sequence of effects: a short initial period of increased amplitude of field activity and then blocking (reversible) spontaneous field activity. In both cases, the electrical stimulation of Schaffer collaterals produced hyperexcited responses even after the spontaneous bursts had been blocked.

EXAMPLE 111 Consequences of prolonged exposure to medium with low content of - [C1-] Q with [K +] 0 varied In the preceding experiments, field activity was monitored in some slices for > 1 hour after the spontaneous gusts had been blocked by the exposure to low content of - [Cl] 0. After such exposure to low content of - [Cl] 0 prolonged, spontaneous long-term depolarizing shifts developed. The morphology and frequency of these late field events appeared to be related to extracellular potassium and chloride concentrations. Motivated by these observations, a set of experiments were carried out in which they were systematically varied [Cl] 0 and [K +] 0 and the effects of these ion changes were observed in spontaneous field events that occur late. In the first set of experiments, the slices were exposed to medium having low content of - [C1] 0 (7 mM) and - [K +] 0 normal (3 mM) (n = 6). After exposure of 50-70 minutes to this medium, spontaneous events were recorded in the CAI area; these events appeared as negative displacements of 5-10 mV in the CD field, with the first episode that lasts 30-60 seconds. Each subsequent episode was longer than the previous one. This observation suggested that ion-homeostatic mechanisms were diminished over time as a result of ion concentrations in the bath medium. In some experiments (n = 2) in which these negative CD field shifts had been induced, intracellular recordings of CAI pyramidal cells were acquired simultaneously with CAI field records. For these experiments, intracell and field records were acquired close to each other (<; 200 μt). Before each negative field shift (10-20 seconds), the neuron begins to depolarize. Cellular depolarization was indicated by a decrease in resting membrane potential, an increase in the frequency of spontaneous firing and a reduction in amplitude of action power. Coinciding with the onset of the negative field shift, the cells became sufficiently depolarized, so that they were unsuitable for spontaneous firing action potentials or action potentials produced by current (not shown). Since neuronal depolarization began 10-20 seconds before the field shift field, it may be that a gradual increase in extracellular potassium results in the depolarization of a neuronal population, thus initiating these field events.

Such an increase in [K +] 0 could be due to alterations in the chloride-dependent glial co-transport mechanisms that normally move potassium from the extracellular spaces to the intracellular spaces. To test if the increments in [K +] 0 preceded these negative field shifts (and chloride depolarization in parallel), were made experiments (n = 2) in which a microelectrode K + -selective was used to record changes in [K +] 0.

In each experiment, the K + -selective microelectrode and a field electrode were placed in a pyramidal layer of CAI close to each other (<200 μp?) And a stimulation pulse was administered to the Schaffer collaterals every 20 seconds, such so that the magnitude of the population peak could be monitored. Multiple negative field shifts that occur spontaneously were evoked by perfusion with medium of low content of - [Cl O] (7 mM). Each event was associated with a significant increase in [K +] 0, with the increase of [K +] 0 that starts several seconds before the start of a negative field shift. A increment of 1.5-2.0 mM slow in [K +] 0 occurred in a time interval of approximately 1-2 minutes before the start of each event. The field responses evoked by stimulation increased slowly in amplitude over time, along with the [K +] 0 increased, until just before the negative field shift. + In a second set of experiments (n = 4), [K] 0 was increased to 12 mM and [Cl-] 0 was increased to 16 mM. After exposure of 50-90 minutes to this medium, slow oscillations were recorded in the CAI area. These oscillations were characterized by negative CD displacements of 5-10 mV and the field potential and had a periodicity of approximately 1 cycle / 40 seconds. Initially, these oscillations occurred intermittently and had an irregular morphology. Over time, these oscillations became continuous and developed a 'regular waveform. On exposure to furosemide (2.5 mM), the amplitude of the oscillations was gradually decreased and the frequency increased until the oscillations were completely blocked. Such oscillations induced by low content of - [C1] 0 - in tissue slices had not been previously reported. However, the temporal characteristics of the oscillatory events bear a striking similarity to the oscillations of - [C1] 0 - induced by low content of [K +] 0 that were previously described in a purely axonal preparation.

In a third set of experiments (n = 5) [Cl] 0 it was further increased to 21 mM and [K +] 0 was reduced back to 3 mM. In these experiments, negative displacements that occur infrequently, individually, in the field potential developed over the course of 40-70 minutes (data not shown). These events (5-10 mV) that last 40-60 seconds, occurred at random intervals and maintained a relatively constant duration throughout the experiment. These events could sometimes be produced by a single electrical stimulus administered to the Schaffer collaterals. Finally, a final set of experiments (n = ), [C1 ~] 0 was maintained at 21 mM and [K +] 0 was raised to 12 mM. In these experiments, spontaneous field events that occur late were not observed during the course of the experiments (2-3 hours).

EXAMPLE 112 Changes in [K +] p during exposure to low chloride content Adult 'Sprague-Dawley rats were prepared as previously described. Transverse hippocampal cuts, 400 μ ?? of thickness, were cut with a vibratory cutter and stored in an oxygenated retention chamber for 1 hour before registration. A dip-type camera was used to the microelectrode K + -selective logs. Slices were perfused with oxygenated artificial cerebrospinal fluid (ACSF) (95% 02/5% CO2) at 34-35 ° C. Normal ACSF contained 10 mM dextrose, 124 mM NaCl, 3 mM KC1, 1.25 mM aH2P0, MgSC >; 4 1.2 mM, NaHCC > 3 26 mM and CaCl2 2 m. In some experiments, normal or medium medium with low chloride content was used. contains -aminopyridine (4-AP) at 100 μ ?. Solutions with low chloride content (21 mM [Cl] n) were prepared by equimolar replacement of NaCl with Na + -gluconate (Sigma Chemical Co.). Field records of the cell body layers of CAI or CA3 were acquired with low resistance glass electrodes filled with NaCl (2M.) For stimulation of the Schaffer collateral path, a monopolar stainless steel electrode was placed on the surface of slice in half between the areas of CAI and CA3. All records were digitized (Neurorocorder, Neurodata Instruments, New York, NY) and stored on videotape.

K + -selective microelectrodes were manufactured according to standard methods. Briefly, the reference barrel of a double-barrel pipette was filled with ACSF and the other barrel was silanized and the tip refilled with KC1 with K + -selective resin (Corning 477317.) - The ion-selective microelectrodes were calibrated and considered appropriate if they had a Nernstiana slope response and remained stable throughout the duration of the experiment. It has been shown that exposure of hippocampal slices to medium with low content of - [Cl-] n includes a sequence of temporally-dependent changes in the activity of pyramidal CAI cells, with three characteristic phases, as described above. In brief, exposure to the medium with low content of - [Cl-] n results in a brief period of increased hyperexcitability and spontaneous epileptiform discharge. With additional exposure to the medium with low content of - [Cl] Q, the spontaneous epileptiform activity is blocked, but the cellular hyperexcitability remains and the trigger times of action potential become less synchronized with each other. Finally, with prolonged exposure, the action potential trigger times become sufficiently desynchronized, in such a way that the field response evoked by stimulation completely disappears, yet the individual cells continue to show evoked responses monosynaptically to the collateral stimulation of Schaffer . The following results demonstrate that the anti-epileptic effects of furosemide on chloride co-transport antagonism are independent of direct actions in excitatory synaptic transmission and are a consequence of a desynchronization of population activity with any associated decreases in excitability.

In six hippocampal slices, K + -selective and field microelectrodes were placed in the cell body layer of CAI and a stimulating electrode was placed in the Schaffer collateral path and single impulse stimuli (300 μe) were administered every 20 seconds. After a stable reference [K +] n was observed for at least 20 minutes, perfusion was switched to low-content medium de - [Cl]? · In the course of 1-2 minutes of exposure to medium with low content of - [Cl] Q, the field responses are they became hyperexcitable as the [K +] n began to rise. After approximately 4-5 minutes of exposure to medium with low content of - [Cl] Q, the magnitude of the field response decreased until until it was abolished completely. The corresponding record of [K +] n showed that potassium began to rise immediately after the exposure to medium with low content of - [Cl] o, and that the peak of this elevation of [K +] n corresponded in time to the maximally hyperexcitable CAI field response. Coincident with the reduction of the magnitude of the response of field, the [K +] n began to decrease until after exposure 8-10 minutes in the medium with low content of - [Cl] o, became constant for the rest of the experiment at 1.8-2.5 mM above the control levels. Four slices were switched back to the control medium and allowed to fully recover. Then the experiment was repeated with the K + microelectrode -selective placed in the stratum Radiatum A similar sequence of changes in [K +] n was observed in the dendritic layer, with the values of [K +] n being 0.2-0.3 mM lower than those observed in the cell body layers. In four hippocampal slices, the responses of changes evoked by stimulation in [K +] or between control conditions and after the CAI field response was abolished completely by exposure to low content of - [Cl ] 0 were compared. In each slice, the [K +] n-selective measurements were first acquired in the cell body layer and then after allowing full recovery in the control medium, the experiment was repeated with the K + electrode -selective moved to stratum radiatum. Each stimulation test consisted of a 10 Hz salvo fed to the Schaffer collateral for 5 seconds. The peak elevations in [K +] or were similar between control conditions and after prolonged exposure to low medium content of - [Cl] Q and between the cell body and the dendritic layers. However, the recovery times observed after prolonged exposure to low content of - [Cl] o were significantly longer than those observed during control conditions. These results show that the administration of furosemide results in [K +] or increased extracellular spaces. Exposure of brain tissue to the medium with low content of - [Cl] n immediately induces a elevation in [K +] or by 1-2 mM, which remained throughout the duration of exposure and coincided with the initial increase in excitability the inevitable abolition of the CAI field response. This loss of field response of CAI during exposure to low content of - [Cl it is most likely due to the desynchronization of neuronal firing times. Significantly, the increases evoked by stimulation in [K + lo, both in the cell body and dendritic layers were almost identical before and after the blockage with low content of - [Cl] n of the CAI field response. These data suggest that the synaptic impulse evoked by comparable stimulation and generation of action potential occurred under control conditions and after blocking with low [Cl] n content of the field.

Together these data demonstrate that the antiepileptic and desynchronizing effects of the co-transporter chloride antagonist, furosemide, are independent of direct actions on excitatory synaptic transmission and are a consequence of a desynchronization of population activity without decrease in excitability.

EXAMPLE 113 Changes in extracellular pH during exposure to low chloride content Antagonists of the anion / chloride dependent co-transporter, such as furosemide and low content of - [Cl-] n, may affect extracellular pH transients that could contribute to maintenance of synchronized population activity. Rabbit brain and hippocampal slices were prepared as described in Example 80, except that NaHCO 3 was replaced by equimolar amounts of HEPES (26 nM) and an interphase type chamber was used. In four hippocampal brain slices continuous spontaneous bursts were produced by exposure to medium containing 4-AP 100 μ, as described in Example 13. Field records were simultaneously acquired from the cell body layers in the CAI areas and CA3. A stimulus administered every 30 seconds to the collaterals of Schaffer throughout the duration of the experiments. After at least 20 minutes a continuous burst was observed, the slices were exposed to HEPES medium containing 4-AP nominally free of bicarbonate. There were no significant changes observed in spontaneous field responses or field responses evoked by stimulus resulting from prolonged exposure (0.2 hours) to the HEPES medium. After the slices had been exposed for at least 2 hours to the medium of HEPES, the perfusion was switched to HEPES medium containing 4-AP- in which the [Cl] n had been reduced to 21 mM. Exposure to HEPES medium of low content of - [Cl] Q induced identical sequences of events and in the same time course as previously observed with the medium containing - [Cl] o of low content of NaHC03 ~. After complete blockage of spontaneous bursts, the perfusion medium was switched back to the middle of HEPES with [Cl] or normal- In the course of 20-40 minutes, spontaneous gusts resumed. At the time when the spontaneous gusts had resumed, the slices had been perfused with HEPES medium nominally free of carbonate for more than 3 hours. These data suggest that the actions of chloride co-transport antagonism in synchronization and excitability are independent of the effects on extracellular pH dynamics. Figure 4 illustrates a schematic model of ion co-transport under reduced [Cl] conditions. Figure A, left panel, shows that the chloride gradient necessary for the generation of IPSP in neurons is maintained by ion efflux through a K + co-transporter, Cl furosemide-sensitive. Under normal conditions, a high concentration of intracellular potassium (maintained by the pump of 3Na +, 2K + -ATPase) serves as the driving force for the extrusion of Cl against its concentration gradient. In glial cells, as shown in the right panel of Figure 4A, the movement of ions through the furosemide-sensitive NKCC co-transporter is from extracellular spaces to intracellular spaces. The ion gradients required for this co-transport are maintained, in part, by the "transmembrane solid cycle": sodium ions taken to glial cells by means of the NKCC co-transporter are continuously extruded by the 3Na pump, 2K, -ATPase, in such a way that a low concentration of intracellular sodium is maintained.

The velocity and direction of the ion flow through the furosemide-dependent co-transporters are functionally proportional to their ion-product differences written as + - + - + [K] ix [Cl] i - [K] ox [Cl] o) for neuronal co-transport of K, + + - 2 + Cl and as [Na] ix [K] ix [Cl] i - [Na] ox + - 2 [K] or x [Cl] o) for co-transport of glial NKCC. The sign of these ion-product differences shows the direction of ion transport, the positive is from intracellular to extracellular spaces. Figure 4B shows a schematic phenomenological model that explains the emergence of late spontaneous field events that arise as a result of the prolonged exposure to low content of - [Cl] o. The ion-product differences for neurons and glia are denoted as QN and QG, respectively. Under control conditions (1), the ion-product differences for neurons are such so that K + and Cl are co-transported from intracellular to extracellular spaces (QN > 0); the differences in ion-products for glial cells are such that Na +, K + and Cl are co-transported from ECS to compartments intracellular (QG < 0.) When [Cl] o is reduced (2), the ion-product differences are altered in such a way that the neuronal efflux of KCl is increased; however, the glial ion co-transport is inverted (QG> 0), such that there is a net efflux of KCl and NaCl from intracellular to extracellular spaces. These changes result in the accumulation of extracellular potassium over time. inevitably, [K +] or reaches a level that induces the depolarization of neuronal populations, resulting in an even larger accumulation of [K +] or. This large accumulation of extracellular ions serves to reverse the ion-product differences such that KCl is moved from extracellular to intracellular spaces (QN <0, QG <0) (3.) Additional clearance of extracellular potassium inevitably restores Transmembrane ion gradients at initial conditions. Through cycles in this process, repetitive negative field events are generated.

EXAMPLE 114 Therapeutic Effectiveness of Furosemide in the Relief of Pain Symptoms in an Animal Model of Neuropathic Pain The ability of furosemide to relieve pain will be examined in rodents using the Chung model of neuropathic pain (see, for example, Walker et al. Mol. Med. Today 5: 319-321, 1999). Sixteen adult male Long-Evans rats will be used in this study. All rats will receive a spinal ligation of nerve L5 as detailed below. Eight of the sixteen rats will receive an injection (intravenous) of furosemide and the remaining eight will receive intravenous injection of vehicle only. The pain threshold will be determined immediately using the mechanical room abandonment test. Differences in pain thresholds between the two groups will be compared. If furosemide alleviates pain, the furosemide treatment group will exhibit a higher pain threshold than the group that received the vehicle.

Model of Chung Neuropathy Spinal nerve ligation was performed under isoflouran anesthesia with animals placed in the prone position to access the left spinal nerves L4-L6. Under amplification, approximately one third of the transverse process is removed. The spinal nerve L5 is carefully identified and dissected free of the adjacent spinal nerve L4 and then tightly ligated using a 6-0 suture. The wound is treated with an antiseptic solution, the muscular layer is sutured and the incision is closed with wound fasteners. Behavioral testing of the mechanical leg abandonment threshold takes place within 3-7 days of the incision. Briefly, the animals are placed inside the • Plexiglas camera (20 x 10.5 x 40.5 era) and they are allowed to become accustomed for 15 minutes. The camera is placed on top of a mesh screen, in such a way that mechanical stimuli can be administered to the plantar surface of both hind legs. The mechanical threshold measurements for each hind paw are obtained using an up / down method with eight von Frey monofilaments (5, 7, 13, 26, 43, 64, 106 and 202 mN). Each test begins with a von Frey force of 13 mN fed to the right hind leg for approximately 1 second and then to the left hind leg. If there is no retreat response, the next highest force is administered. If there is an answer, the next lower force is administered. This procedure is carried out until no response is made to the highest force (202 mN) or until four stimuli are administered immediately after the initial response. The leg withdrawal threshold of 50% for each leg is calculated using the following formula: [Xth] log = [vFrjlog + ky where [vFr] is the strength of the last von Frey monofilament used, k = 0.2268 which is the average interval (in logarithmic units) between the von Frey monofilaments and y is a value that depends on the withdrawal response pattern. If an animal does not respond to the highest von Frey hair (202 mN), then y = 1.00 and the mechanical leg withdrawal response of 50% for that leg is calculated to be 340.5 mN. The mechanical leg withdrawal threshold tests are performed three times and the 50% withdrawal values are averaged in all three tests to determine the average mechanical leg withdrawal threshold for the right and left leg for each animal.

EXAMPLE 115 Therapeutic Efficacy of Furosemide and Bumetanide for Alleviating Intense Anxiety Symptoms or Post-Traumatic Stress Alteration The therapeutic utility of furosemide and bumetanide in the treatment of post-traumatic stress disorder is examined by determining the ability of these compounds to relieve intense anxiety in conditioning contextual fear in rats. Contextual fear conditioning involves mating of an aversive event, in this case moderate foot shock, with a distinctive environment. The intensity of fear memory is determined using paralysis, a defensive reaction typical of species in rats, marked by complete immobility, except by breathing. If the rats are placed in a distinctive environment and are subjected to shock immediately they do not learn to be afraid of the context. However, if you allow them to explore the distinctive environment some time before the immediate shock, they show intense anxiety and fear when they are placed back into the same environment. You can take advantage of this fact and by dividing by contextual contextual fear conditioning into two phases, you can separately study treatment effects on memory for the context (specifically a hippocampus-based process) of learning the association between the context and shock or experience the aversiveness of the shock (which depends on the emotional response circuits in which amygdala are included). The post-traumatic stress syndrome (PTSD) in humans has been shown to be related to the emotional response circuits in the tonsils and for this reason contextual memory conditioning is a widely accepted model for PTSD. The experiment employed 24 rats. Each rat received a single 5 minute exploration episode of a new small environment. 72 hours later they were placed in the same environment and immediately received two moderate leg crashes (1 milliamper) separated by 53 seconds. 24 hours later, 8 of the rats received an injection (I.V.) of furosemide (100 mg / kg) in vehicle (DIVISO) and 8 of the rats were injected I.V. with bumetanide (50 mg / kg) in vehicle (DMSO.) The remaining 8 rats received an injection of DMSO alone. Each rat was placed again in the same environment for 8 minutes during which the paralysis was measured, as a Pavlov conditioned fear index. Four identical cameras (20 X 20 X 15 cm) were used. All aspects of synchronization and event control were under microcomputer control (MedPC, MedAssociates Inc., Vermont, USA.). Paralysis measurement was carried out by means of an elevated video camera connected to the microcomputer and was automatically screened using a piece of TM programming specialty elements, FreezeFrame (OER Inc., Reston, VA). The total paralysis time was analyzed in a one-way analysis of variance (ANOVA) test, with drug dose as the factor within groups. As shown in Figure 5, significantly less paralysis was observed in animals treated with either bumetanide or furosemide than in animals receiving vehicle alone, indicating that bumetanide and furosemide can be effectively used in post-stress alteration treatment. -traumatic EXAMPLE 116 Therapeutic Efficacy of Furosemide and Bumetanide for Alleviating Anxiety The therapeutic efficacy of furosemide and bumetanide for relieving anxiety was examined by evaluating the effects of these compounds on the fear-enhanced surprise test (SPF) in rats. This test is commonly used to distinguish effects of anxiolytic drugs from non-specific effects, such as sedation / muscle relaxation. Twenty-four rats received a 30-minute habituation period to the FPS apparatus. Twenty-four hours later, reference surprise amplitudes were collected. Then the rats were divided into three paired groups based on reference surprise amplitudes. One of the rats exhibited a reference surprise significantly higher than the others and was excluded from the experiments. Groups 1 and 2 therefore consisted of 8 rats each, Group 3 consisted of 7 rats. Following the reference surprise amplitude collection, 20 light / shock matings were administered in two sessions on two consecutive days (that is, 10 light / shock pairings per day). On the final day (day 5), groups 2 and 3 received one injection (i.v.) either furosemide (100 mg / kg) or bumetanide (70 mg / kg) in vehicle (DMSO) and group 1 received vehicle only. Immediately following the injections, the amplitudes of surprise were determined during surprise tests alone and tests of surprise more fear (light followed by surprise). The surprise potentiated by fear (amplitudes of light + surprise minus amplitudes of surprise alone) was compared between the treatment groups. The animals were trained and tested on four identical stabilizer devices (Med-Associates.) Briefly, each rat was placed in a small Plexiglas cylinder. The floor of each stabilometer consisted of four 18 mm diameter stainless steel bars separated by 18 mm through which the shock could be administered. The movements of the cylinder result in the displacement of an accelerometer, where the resulting voltage is proportional to the speed of the displacement of the cage. The surprise amplitude is defined as the maximum accelerometer voltage that occurs during the first 0.25 seconds after the surprise stimulus is administered. The analog output of the accelerometer was amplified, digitized ++++ in one. scale of 0-4096 units and stored in a microcomputer. Each stabilizer was enclosed in an attenuating box of ventilated light sound. All sound level measurements were made with a Sound Level Meter Precision. The noise of a fan attached to the side wall of each wooden box produced an overall background noise level of 64 dB. The surprise stimulus was a burst of 50 ms of white noise (rise-decay time of 5 ms) generated by a white noise generator. The visual conditioned stimulus employed was illumination of a light bulb adjacent to the source of white noise. The unconditioned stimulus was a 0.6 mA foot shock with a duration of 0.5 seconds, generated by four constant current shock devices located outside the chamber. The presentation and sequencing of all the stimuli were under the control of the microcomputer. The FPS procedures consisted of 5 days of testing; during days 1 and 2 reference surprise responses were collected, day 3 and 4 light pairing / shock were administered, day 5 tests for fear-boosted surprise were carried out. Mating: In the first two days all rats were placed in the Plexiglas cylinders and 3 minutes later presented with 30 stimuli - from surprise to an inter-stimulus interval of 30 seconds. An intensity of 105 dB was used. The average surprise amplitude through the 30 surprise stimuli on the second day was used to assign rats to. treatment groups with similar means. Training: In the next 2 days, the rats were placed in the Plexiglas cylinders. Each day after 3 minutes after entry, 10 CS-shock matings were administered. The shock was administered during the last 0.5 seconds of the CS of 3.7 seconds at an average inter-test interval of 4 minutes (interval, 3-5 min.) Tests: The rats were placed in the same surprise boxes where they were trained and after 3 minutes were presented with 18 stimuli that produce surprise (all at 105 dB). These initial surprise stimuli were used to again accustom the rats to acoustic surprise stimuli. Thirty seconds after the last of these stimuli, each animal received 60 surprise stimuli with half of the stimuli presented alone (surprise tests alone) and the other half presented 3.2 seconds from the beginning of the CS of 3.7 seconds (CS- tests). surprise) All surprise stimuli are presented at an inter-stimulus interval mean of 30 seconds that vary randomly between 20 and 40 seconds. Measurements: The treatment groups were compared in the difference of surprise amplitude between the CS-surprise and surprise alone (fear-enhancing) tests. Figure 6 shows the reference surprise amplitudes for each group of rats determined before the test day. Figure 7 shows the amplitude of response in surprise tests alone, determined on the test day immediately following the injection of either DMSO alone, bumetanide or furosemide. Figure 8 shows the difference score (surprise alone - surprise boosted by fear) on the test day. As shown in the figures, a statistically lower difference score was significantly observed in rats treated with either furosemide or bumetanide than in rats treated with vehicle alone, indicating that both furosemide and bumetanide are effective in reducing anxiety. Figures 9 and 10 show the surprise amplitude alone and the difference score, respectively, one week after treatment with either furosemide or bumetanide. It was found that animals treated with either furosemide or bumetanide have a higher difference score than animals treated with vehicle alone. However, since the error bars are so large for the animals treated per vehicle, the data do not imply any statistically significant difference between vehicle and bumetanide, with possibly a small difference between vehicle and furosemide.

EXAMPLE 117 Therapeutic Efficacy of Bumetanide Analytes to Relieve Anxiety The therapeutic efficacy of various bumetanide analogs for relieving anxiety was examined using the fear-enhanced surprise test (SPF) in rats as described above. Figure 11 shows the percent difference score (surprise alone - surprise boosted by fear) on the test day in rats treated with one of the following bumetanide analogs: bumetanide 3- (dimethylaminopropyl) ester; benzyltrimethylammonium salt of bumetanide; bumetanide dibenzyl amide; bumetanide cyanomethyl ester; N, N-diethylglyconamide ester of bumetanide; ?,? - Bumetanide dimethylglycolamide ester; morpholinodiet bumetanide ester; pivaxetil ester of bumetanide; methyl ester of bumetanide; bumetanide diethylamide; and bumetanide benzyl ester. The vehicle was DMSO. The number of animals tested for each bumetanide analog is shown in Table 1.

Table 1 As can be seen from Figure 11, the percentage difference score obtained after the administration of most of the bumetanide analogs was significantly lower than that observed immediately after the administration of either vehicle alone or bumetanide, demonstrating that these Analogs can be effectively used to reduce anxiety. In addition, several of the bumetanide analogs were observed to have significantly diuretic effects significantly lower than those generated associated with either furosemide or bumetanide. While the present invention has been described with reference to the specific embodiments thereof, it will be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to suit a particular situation, material, composition of matter, method, step or method steps, for use in carrying out the present invention. It is intended that such modifications be within the scope of the claims appended hereto. All patents and publications cited herein and PCT Application WO 00/37616, published June 29, 2000, are specifically incorporated by reference in their entirety. SEQ ID NO: 1-2 are summarized in the attached Sequence Listing. The codes for the polynucleotide and polypeptide sequences used in the attached Sequence Listing conform to the standard of WIPO ST.25 (1988), Appendix 2.

Claims (29)

1. CLAIMS 1. A method for the treatment of an alteration in a mammalian subject, characterized in that it comprises administering an effective amount of a composition comprising a + + Co-transporter antagonist of Na K 2 Cl to the subject, wherein the alteration is selected from the group consisting of: addictive alterations and neuropsychiatric disorders.
2. 2. The method according to claim 1, characterized in that the co-transporter antagonist of Na + K + 2C1 reduces or blocks discharges of neuronal population hypersynchronized by non-synaptic effects.
3. 3. The method according to claim 1, characterized in that the co-transporter antagonist of Na + K + 2C1 is a co-transporter antagonist of NKCCl.
4. 4. The method of compliance with the claim 1, characterized in that the co-transporter antagonist of Na + K + 2C1 is a SNK-targeted NKCC co-transporter antagonist.
5. 5. The method according to claim 4, characterized in that the co-transporter antagonist of Na + K + 2C1 is selected from the group consisting of: furosemide; bumetanide; ethacrynic acid; torsemide; azosemide; muzolimine; pyretanide; tripamide; and analogs and functional derivatives thereof.
6. 6. The method according to claim 1, characterized in that the co-transporter antagonist of Na + K + 2C1 is selected from the group consisting of: thiazide; and thiazide-like diuretics.
7. 7. The method of compliance with the claim 1, characterized in that the co-transporter antagonist of Na + K + 2C1 is a compound selected from the group consisting of the following: or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein: Ri is not present, is H, O or S; R 2 is not present, is H or when Ri is O or
8. S, R 2 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, alkylaminodialkyl, alkylcarbonylamino-dialkyl, alkyloxycarbonylalkyl, alkylcarbonyloxyl-alkyl, alkylaldehyde, alkylketoalkyl, alkylamide, alkarylamide , arylamide, an alkylammonium group, alkylcarboxylic acid, alkylheteroaryl, alkylhydroxy, a biocompatible polymer such as alkyloxy (polyalkyloxy) -alkylhydroxyl, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) and a polyethylene glycol ether (ether) of PEG), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl, unsubstituted or substituted and when Ri is not present, R 2 is selected from the group consisting of hydrogen, N, N-dialkylamino, N, N-dialcarylamino, N, N-diarylamino , N-alkyl-N-alkarylamino, N-alkyl-N-arylamino, N-alkaryl-N-arylamino unsubstituted or substituted; R3 is selected from the group consisting of aryl, halo, hydroxy, alkoxy and aryloxy, unsubstituted or substituted; Y R4 and R5 are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, carbonylalkyl, carbonylalcaryl, carbonylaryl and salts thereof such as sodium, potassium, calcium, ammonium, trialkylarylammonium and tetraalkylammonium salts, with the following conditions: R3 Formula I is an unsubstituted aryloxy when Ri is O and R2, R4 and R5 are H; R3 of formula III is not Cl, when Ri is O and R2, R and Rs are H; R2 of formula III is not methyl when Ri is O, R3 is Cl and R4 and R5 are H and R3 of formula V is unsubstituted aryloxy when Ri is O and R2, R < And Rs are H. The method according to claim 7, characterized in that the compound is selected from the group consisting of bumetanide aldehyde, bumetanide methyl ester, bumetanide cyanomethyl ester, bumetanide ethyl ester, isoamyl ester of bumetanide, bumetanide, butetanide octyl ester, bumetanide benzyl ester, bumetanide dibenzylamide, bumetanide diethylamide, bumetanide morfolemethyl ester, bumetanide-3- (dimethylaminopropyl) ester, bumetanide-N, N-N-diethylglycolamido ester, bumetanide dimethyl-il-glycolamido ester, pivaxetil ester of bumetanide, propamethyl ester of bumetanide, methoxy (polyethyleneoxy) nl-ethyl ester of bumetanide, benzyltrimethylammonium salt of bumetanide and salt of cet ilt rimet ilammonium of bumetanide.
9. 9. The method of "compliance with the claim 7, characterized in that the compound is selected from the group consisting of methyl thioester of bumetanide, cyanomethyl thioester of bumetanide, ethyl thioester of bumetanide, isoamyl thioester of bumetanide, octyl thioester of bumetanide, benzyl thioester of bumetanide, mofolinoethyl thioester of bumetanide, 3- (dimethylaminopropyl) thioester of bumetanide, N, N-diethylglycolamide thioester of bumetanide, dimethylglycolamide thioester of bumetanide, pivaxetil thioester of bumetanide, propaxethyl thioester of bumetanide, methoxy (polyethyleneoxy) nl-ethyl thioester of bumetanide, thioacid of bumetanide (thiobumetanide), salt of benzyltrimethylammonium thioacid of bumetanide and thioacid salt of cetyltrimeti lammonium of bumetanide.
10. The method according to claim 7, characterized in that the compound is selected from the group consisting of metastable bumetanide thioacid, bumetanide O-methyl thioester, bumetanide O-cyanomethyl thioester, bumetanide 0-ethyl thioester, 0- isoamyl thioester of bumetanide, 0-octyl thioester of bumetanide, 0-benzyl thioester of bumetanide, 0- (morpholinoethyl) thioester of bumetanide, 0- [3- (dimethylaminopropyl)] thioester of bumetanide, 0- (N, N-diethylglycolamido) thioester of bumetanide, 0- (dimethylglycolamido) thioester of bumetanide, 0-pivaxetil thioester of bumetanide, 0-propaxetil thioester of bumetanide, 0- [methoxy (polyethyleneoxy) nl-ethyl] thioester of bumetanide, thioacid salt of benzyltrimethylammonium of bumetanide and thioacid salt of bumetanide cetyltrimethylammonium.
11. The method according to claim 7, characterized in that the compound is selected from the group consisting of bumetanide thioaldehyde, bumetanide dithioacid, bumetanide methyl dithioester, bumetanide cyanomethyl dithioester, bumetanide ethyl dithioester, bumetanide isoamyldithioester, butetanide octyl dithioester, bumetanide benzyl dithioester, bumetanide dibenzylthioamide, bumetanide diethyl thioamide, bumetanide morpholinoethyl dithioester, bumetanide 3- (dimethylaminopropyl) dithioester, bumetanide diethylglycollarate butetanide dithioester, bumetanide dimethylglycolamido dithioester, bumetanide pivaxetil dithioester, propaxethyl bumetanide dithioester, methoxy (polyethyleneoxy) nl-ethyl dithioester of bumetanide, dithioacid salt of benzyltrimethylammonium of bumetanide and dithioacid salt of cetyltrimethylammonium of bumetanide.
12. The method according to claim 7, characterized in that the compound is selected from the group consisting of furosemide methyl ester, furosemide cyanoraethyl ester, furosemide ethyl ester, furosemide isoamyl ester, furosemide octyl ester, benzyl ester furosemide, furosemide morpholinoethyl ester, furosemide 3- (dimethylaminopropyl) ester, furosemide N, N-diethylglycolamido ester, furosemide dimethylglycolamido ester, furosemide pivaxetil ester, furosemide propaxethyl ester, furosemide methoxy (polyethyleneoxy) nyl ethyl ester , furosemide benzyltrimetyl ammonium acid salt and furosemide cetyltrimethylammonium acid salt.
13. 13. The method according to the claim 7, characterized in that the compound is selected from the group consisting of furosemide thioacid, furosemide S-methyl thioester, furosemide S-cyanomethyl thioester, furosemide S-ethyl thioester, furosemide S-isoamyl thioester, furosemide S-octyl thioester furosemide, S-benzyl thioester of furosemide, S- (raorfolinoethyl) thioester of furosemide, S- [3- (dimethylaminopropyl)] thioester of furosemide, S- (N, N-diethylglycolamido) thioester of furosemide, S- (dimethylglycole) thioester of furosemide, furosemide S-pivaxetil thioester, furosemide S-propaxethyl thioester, furosemide S- [methoxy (polyethyleneoxy) nl-ethyl] thioester, furosemide benzyltrimethylammonium thioacid salt and furosemide cet iltrimethylammonium thioacid salt.
14. The method according to claim 7, characterized in that the compound is selected from the group consisting of metastable furosemide thioacidO-methyl thioester of furosemide, O-cyanomethyl thioester of furosemide, 0-ethyl thioester of furosemide, 0-isoamyl thioester of furosemide, 0-octyl thioester of furosemide, 0-benzyl thioester of furosemide, 0- (morpholinoethyl) thioester of furosemide, 0- [3- (dimethylaminopropyl)] thioester of furosemide, 0- (N, N-diethylglycolamido) thioester of furosemide, 0- (dimethylglycolamido) thioester of furosemide, 0-pivaxetil thioester of furosemide, 0-propaxethyl thioester of furosemide , Furosemide 0- [methoxy (polyethyleneoxy) ni-ethyl] thioester, furosemide benzyltrimethylammonium thioacid salt and furosemide cetyltrimethylammonium thioacid salt.
15. The method according to claim 7, characterized in that the compound is selected from the group consisting of furosemide thioaldehyde, furosemide dithioacid, furosemide methyl dithioester, furosemide cyanomethyl dithioester, furosemide ethyl dithioester, furosemide isoamyl dithioester, octyl furosemide dithioester, furosemide benzyl dithioester, furosemide dibenzyl thioamide, furosemide d.iet iltioamide, furosemide morpholinoethyl dithioester, furosemide 3- (dimethylaminopropyl) dithioester, furosemide N-diethylglycolamido dithioester, furosemide dimethylglycolamido dithioester, pivaxetil furosemide dithioester, furosemide propaxethyl dithioester, furosemide methyloxy (polyethyleneoxy) nl-ethyl dithioester, furosemide benzyltrimethylammonium dithioacid salt and furosemide cetyltrimethylammonium dithioacid salt.
16. 16. The method according to claim 7, characterized in that the compound is selected from the group consisting of, piretanide aldehyde, piretanide methyl ester, piretanide cyanomethyl ester, piretanide ethyl ester, piretanide isoamyl ester, piretanide, phenytanide benzyl ester, piretanide dibenzyl amide, piretanide diethylamide, piretanide morpholinoethyl ester, piretanide 3- (dimethylaminopropyl) ester, N, N-diethyl ilganolamide piretanide ester, piretanide dimethylglycolamide ester, piretanethyl ester piretanide, propaxethyl Pyretanide ester, pyroxymethoxy (polyethyleneoxy) nl-ethyl ester, pyretanide benzyltrimethylammonium salt and piretanide cetyltrimethylammonium salt.
17. 17. The method according to claim 7, characterized in that the compound is selected from the group consisting of piretanide thioacid, S-methyl thioester of piretanide, S-cyanomethyl thioester of piretanide, S-ethyl thioester of piretanide, S-isoamyl thioester of piretanide, S-octyl thioester of piretanide, S-benzyl thioester of piretanide, S- (morpholinoethyl) thioester of piretanide, S- [3- (dimethylaminopropyl)] thioester of piretanide, S- (N, N-diethylglycolamido) thioester of piretanide, S- (dimethylglycolamido) thioester of piretanide, S-pivaxetil thioester of piretanide, S-propaxetil thioester of piretanide, S- [methoxy (polyethyleneoxy) ni-ethyl] thioester of piretanide, thioacid salt of benzyltrimethylammonium of piretanide and salt of thioacid of cetyltrimethylammonium of piretanide.
18. 18. The method of compliance with the claim 7, characterized in that the compound is selected from the group consisting of metastable piretanide thioacid, pyretanide O-methyl thioester, pyretanide O-cyanomethyl thioester, piretanide 0-ethyl thioester, pyretanide O-isoamyl thioester, O-octyl thioester of piretanide, O-benzyl thioester of piretanide, 0- (morpholinoethyl) thioester of piretanide, 0- [3- (dimethylaminopropyl)] thioester of piretanide, 0- (N, N-diethylglycolamido) thioester of piretanide, 0- (dimethylglycolamid) thioester of piretanide, O-pivaxetil thioester of piretanide, 0-propaxetil thioester of pyretanide, 0- [methoxy (polyethyleneoxy) n-l-ethyl] thioester of piretanide, thioacid salt of benzyltrimethylammonium of piretanide and thioacid salt of cetyltrimethylammonium of piretanide.
19. 19. The method according to claim 7, characterized in that the compound is selected from the group consisting of piretanide thioaldehyde, piretanide dithioacid, piretanide methyl dithioester, piretanide cyanomethyl dithioester, piretanide ethyl dithioester, piretanide isoamyldithioester, Piretanide octyl dithioester, Piretanide benzyl dithioester, Piretanide dibenzylthioamide, Piretanide diethylthioamide, Piretanide morpholinoethyl dithioester, Piretanide 3- (dimethylaminopropyl) dithioester, Piretanide N, N-diethylglycolamido dithioester, Piretanide dimethylglycolamido dithioester, Pivaxetil dithioester piretanide, propanethyl dithioester of piretanide, methoxy (polyethyleneoxy) nl-ethyl dithioester of piretanide, thioacid salt of benzyltrimethylammonium of piretanide and dithioacid salt of cetyltrimethylammonium of piretanide.
20. 20. The method according to claim 1, characterized in that the co-transporter antagonist of Na + K + 2C1 is a compound of formula VII: N = N or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein R3 is selected from the group consisting of unsubstituted or substituted aryl, halo, hydroxy, alkoxy and aryloxy; R4 and R5 are each independently selected from the group consisting of hydrogen, alkylaminodialkyl, carbonylalkyl, carbonylalkaline, carbonylaryl and salts thereof; R¾ is selected from the group consisting of alkyloxycarbonylalkyl, alkylaminocarbonylalkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible polymer such as alkyloxy (polyalkyloxy) alkylhydroxyl, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) and a polyethylene glycol ether (ether) of PEG), methyloxyalkyl, methyloxyalkaryl, methythoxyalkyl and methylthioalkaryl, unsubstituted or substituted, with the proviso that R3 is not Cl, when R4, R5 and Rg are H.
21. The method according to claim 20, characterized because the compound is selected from the group consisting of tetrazolyl-substituted azozemide, benzoyl-rimethylammonium salt of azozemide and cet-iltrimet-ammonium salt of azozemide.
22. 22. The method of compliance with the claim 1, characterized in that the antagonist of co-t carrier of Na + K + 2C1 is a compound of formula VIII: VIII or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof, wherein: R7 is selected from the group consisting of alkyloxycarbonylalkyl, alkylaminocarbonylalkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible polymer such as alkyloxy (polyalkyloxy) alkylhydroxy, a polyethylene glycol (PEG) ), an ester of polyethylene glycol (PEG ester) and an ether of polyethylene glycol (PEG ether), methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl, unsubstituted or substituted; and X is a halide portion or an anionic portion or alternatively, X is not present.
23. 23. The method according to claim 22, characterized in that the compound is a quaternary ammonium salt of pyridine-substituted torsemide.
24. 24. The method according to any of claims 1-23, characterized in that the antagonist of Na + K + 2C1 co-transporter modulates the composition of the extracellular ion and chloride gradients in the tissue of the nervous system.
25. 25. The method according to any of claims 1-23, characterized in that the composition is administered orally, sublingually, nasally, transdermally, intravenously, intrathecally or by inhalation.
26. 26. The method according to any of claims 1-25, characterized in that the alteration is a neuropsychiatric alteration selected from the group consisting of: bipolar alterations; anxiety disorders; depression and schizophrenia.
27. 27. The method according to any of claims 1-26, characterized in that the alteration is an anxiety disorder selected from the group consisting of: panic disorder; alteration of social anxiety; obsessive-compulsive disorder; post-traumatic stress disorder; alteration of generalized anxiety and specific phobia.
28. 28. The method according to any of claims 1-25, characterized in that the alteration is an addictive alteration selected from the group consisting of: feeding alterations; alcoholism; addiction to narcotics and smoking.
29. 29. The method according to any of claims 1-25 and 28, characterized in that the alteration is a feeding alteration selected from the group consisting of: obesity and feeding by gluttony.