(12) United States Patent (10) Patent No.: US 9,682,928 B2 Partridge Et Al

USOO9682928B2

(12) United States Patent (10) Patent No.: US 9,682,928 B2 Partridge et al. (45) Date of Patent: Jun. 20, 2017

(54) BUMETANIDE ANALOGS, COMPOSITIONS (2013.01); C07D 24I/04 (2013.01); C07D AND METHODS OF USE 261/02 (2013.01); C07D 263/04 (2013.01); C07D 265/30 (2013.01); C07D 275/02 (71) Applicant: NeuroPro Therapeutics, Inc., Bahama, (2013.01); C07D 277/04 (2013.01); C07D NC (US) 279/12 (2013.01); C07D 295/084 (2013.01); (72) Inventors: John J. Partridge, Chapel Hill, NC C07D 307/52 (2013.01) (US); Daryl W. Hochman, Bahama, (58) Field of Classification Search NC (US) CPC C07C 311/39; A61K 31/4355; C07D 211/44; C07D 231/04; C07D 233/02: C07D (73) Assignee: NeuroPro Therapeutics, Inc., Bahama, 237/02: C07D 239/04; C07D 241/04; NC (US) C07D 261/02: C07D 263/04; C07D 265/30; C07D 275/02; C07D 277/04; (*) Notice: Subject to any disclaimer, the term of this C07D 279/12: C07D 295/084; C07D patent is extended or adjusted under 35 307/52 U.S.C. 154(b) by 0 days. See application file for complete search history. (21) Appl. No.: 14/472,181 (56) References Cited (22) Filed: Aug. 28, 2014 U.S. PATENT DOCUMENTS (65) Prior Publication Data 6,495,601 B1 12/2002 Hochman US 2015/OO8O35O A1 Mar. 19, 2015 2002fOO82252 A1 6/2002 Hochman 2005/0267103 A1 12/2005 Hochman Related U.S. Application Data 2006.0035914 A1 2/2006 Hochman 2006.0089350 A1 4/2006 Hochman et al. (63) Continuation of application No. 13/138,282, filed as 2007/O149526 A1 6/2007 Hochman et al. application No. PCT/US2010/000170 on Jan. 22, 2009/0258844 A1 10, 2009 Hochman et al. 2012,0004225 A1 1/2012 Wanaski et al. 2010, now abandoned. 2012fO234721 A1 9/2012 Hochman et al. (60) Provisional application No. 61/257.238, filed on Nov. 2, 2009, provisional application No. 61/146,336, filed Primary Examiner — Rebecca Anderson on Jan. 22, 2009. (74) Attorney, Agent, or Firm — Brinks Gilson & Lione (51) Int. C. CO7D 295/084 (2006.01) (57) ABSTRACT C07C3II/39 (2006.01) A6 IK 3/4355 (2006.01) The present invention provides bumetanide analogs and CO7D 2II/44 (2006.01) compositions comprising Such analogs. The present inven CO7D 23/04 (2006.01) tion also provides pharmaceutical compositions containing CO7D 233/02 (2006.01) these bumetanide analogs and methods for their use. These C07D 237/02 (2006.01) analogs are particularly useful for the treatment and/or CO7D 239/04 (2006.01) prophylaxis of conditions that involve the NaKCl co CO7D 24I/04 (2006.01) transporter or GABA receptor including but not limited to CO7D 26/02 (2006.01) addictive disorders, Alzheimer's Disease, anxiety disorders, CO7D 263/04 (2006.01) ascites, autism, bipolar disorder, cancer, depression, CO7D 265/30 (2006.01) endothelial corneal dystrophy, edema, epilepsy, glaucoma. C07D 275/02 (2006.01) Huntington's Disease, insomnia, ischemia, migraine, C07D 277/04 (2006.01) migraine with aura, neuropathic pain, nociceptive neuralgia, C07D 279/12 (2006.01) nociceptive pain, ocular diseases, pain, Parkinson's disease, C07D 307/52 (2006.01) personality disorders, postherpetic neuralgia, psychosis, (52) U.S. C. Schizophrenia, seizure disorders, tinnitus, and withdrawal CPC ...... C07C3II/39 (2013.01); A61K 31/4355 syndromes. (2013.01); C07D 2II/44 (2013.01); C07D 231/04 (2013.01); C07D 233/02 (2013.01); C07D 237/02 (2013.01); C07D 239/04 1 Claim, 52 Drawing Sheets U.S. Patent Jun. 20, 2017 Sheet 1 of 52 US 9,682,928 B2

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O OH S

PaSo or Lawesson's Reagent -e HNO2S n-rch, HNO2S 1N1ch, "O burnetanide dithiobunetanide CHSH, H' (bumetanide dithioacid)

S SCH

CHSH, H' ^ 1n 1 a H2S, CHOH, H2NOS HN CH 3 ill base “O morpholine

methyl dithiobumetamide (bumetanide dithioester) morpholine r O S OCH ^ S N - HN - morpholine HNO2S 1 CH, H2NO2S 1ch,

O-methylthiobumetanide bumetanide N-morpholinylthioamide (bumetanide O-thioester)

FIGURE 2 U.S. Patent Jun. 20, 2017 Sheet 3 of 52 US 9,682,928 B2

1Y. S SH S N-N/

1) SOCl. 2) tetrahydrooxazole -e H2NO2S 1ch, H2NO2S -N1ch, O “O dithicountietaride buTetanide N-tetrahydrooxazolylthioannide (bumetanide dithioacid) S SH SS -N-No

1) SOC2 2) tetrahydroisoxazole

dithioburmetanide (bumelanide dithioacid) ^ S SH S N -

1) SOC 2) morpholine - N 1N1-ch HNO2S 1N1ch "C (burmetanidedithiounetanide dithioacid) bumetanide N-morpholinylthioamide

S SH S. N

1) SOCl2 2) thiomorpholine -aa-e- HNO2S 1ch, HNO2S 1CH, “O O (b. Riata) burnetanide N-thiomorpholinyithioamide FIGURE 3 U.S. Patent Jun. 20, 2017 Sheet 4 of 52 US 9,682,928 B2

S SH S N - r 1) SOC2 2) 14-piperazines HNOS 1CH, HNOS 1N1ch, O dithioburnetanide bunetanide N-piperazinylthioamide & analogs (burmetamide dithioacid)

1) SOCl2 2) tetrahydropyrimidines HNO2S 1N1ch, HNOS 1N1ch, "O “O (i.i.aifa) burmetanide N-tetrahydropyrimidinylthioamide & analogs S SH S O R 1) SOCI 2) tetrahydropyridazines ------e. H2NO2S 1 CH, H2NOS 1 CH, “O O dithiobumetanide burnetaride N-tetrahydropyridazinylthioamide & analogs (butaetanide dithioacid)

1) SOC 2) 4-piperadone 1N1a HNO2S CH3 H2NOS N1N1 ch

dithioburetaride bumetanide dithioacid) burnetanide N-4-piperadonylthioamide FIGURE 4 U.S. Patent Jun. 20, 2017 Sheet 5 of 52 US 9,682,928 B2

?yr S SH S N-M 1) SOCl2 2) tetrahydroimidazoles (imadazolidines) -a- HNO2S 1CH, HNO2S N 1N1ch, “O O dithioburnetanide bumetanide N-tetrahydroimidazolylthioamide & analogs (burmetanide dithioacid) S. SH 1) S N-NR 1) SOCl2 2) pyrazolidines -ap HNO2S O -N1ch, HNOS 1N1N CH

dithioburmetanide bumetanide N-pyrazolidinylthioamide & analogs (burmetanide dithioacid)

S SH S N-N/ 1) SOC2 2) thiazolidine H-p

HNO2S N11H ch H2NO2S N1N1Nch,H

dithioburetanide bumetanide N-thiazolidinythioamide (burnetanide dithioacid)

S SH S N-s 1) SOC2 2) isothiazolidine -a-robin H2NO2S 1CH, HNO2S 1CH, O. C O (burmetanidedithioburmetanide dithioacid) burnetanide N-isothiazolidinylthioamide- iazolidi FIGURES U.S. Patent Jun. 20, 2017 Sheet 6 of 52 US 9,682,928 B2

S SH S N JNCH, 1) SOCl. 2) cis-2,6-dimethylmorpholine -- HNO2S 3. in-rich, HNO2S 1N1-ch, “O “O dithioburmetanide bumetanide N-cis-2,6-dimethylmorpholinylthioamide (bumetanide dithioacid) HC

S Sh 1) SOC2 2) trans-2,6-dimethylmorpholine H2NO2S1 3. 1CH, HNO2S 1.CH, “O “O dithioburmetamide buretanide N-trans-2,6-dimethylmorpholinylthioanide (burnetanide dithioacid) H3C

1) SOC 2) cis-2,6-dimethylthiomorpholine -He 1Yrch, H2NO2S in-rch, "C “O ditoburetanide bumetanide N-cis-2,6-dimethylthionorpholinylthioamide (bumetanide dithioacid) HC

S SH 1) SOCI H2NO2S1 3. ^-rch, 2) trans-2,6-dimethylthiomorpholine

dithioburmetanide bumetanide N-trans-2,6-dimethylthiomorpholinylthioamide (burmetanide dithioacid) FIGURE 6 U.S. Patent Jun. 20, 2017 Sheet 7 Of 52 US 9,682,928 B2

S Sh S Js R = lower alkyl, cycloaky, aralkyl, 1) SOCl2 aryl, heteroary, heterocycly, acy, 2) 2-methyl-1,4-piperazines thioacyl, Suhony sufonamide, etc -H-e HNO2S 1ch, HNO2S 1ch, “O O dithioburnetaride bumetanide N-3-methylpiperazinylthioamide & analogs (bumetanide dithioacid) (racemate, R- and S-enantiomers) m HC wsCH3

S SH S N 1 ) SOCl2 rs G alkyl, E. aralkyi, 2) 2,2-dimethylmethyl-1,4-piperazines a. o: E. H2NOS 1 CH, HNO2S 1N1ch, O "O dithioburmetamide bumetanide N-3,3-dimethylpiperazinylthioamide & analogs (bumetanide dithioacid) HC -- NR S Sh S N UNch, 1) SOC r = lower alkyl, cycloaky, aralkyl, 2) cis-2,6-dimethylmethyl-1,4-piperazines thioacyl,aryl, heteruary, sulfonyl, heteocyclyl, sulfonamide, acyl, etc N1a1nch, HNO2S 1N1ch,

dithioburmetanide burnetanide N-cis-3,5-dimethylpiperazinylthioamide & analogs (burmetanide dithioacid) HC r S Sh S U-Nc, 2)trans-2,6-dimethylmethyl-1,4-piperazines1) SOCl. E.rs: E. E. alkyi, E. E. aratkyi, 1N1c. -l-al-e- H2NO2S N 3 HNO2S 1ch, O C “O dithioburetanide (burnetanide dithioacid) bunetanide N-trans-3,5-dimethylpiperazinylthioamide & analogs FIGURE 7 U.S. Patent Jun. 20, 2017 Sheet 8 of 52 US 9,682,928 B2

FIGURE 8

Direct Syntheses of a Thioamide Analog PSs benzene, room temp. Hall-Heine Schwarz, G., Org. Syn., Coll. Vol. III, 1955, 332-334. PaSo, NahCO3, CH3CN O Heroes- O - Alper, H., et al., Angew. Chem, - int. Ed., 1978, 17,689. S N (31-56%) PS1, solvent, ultrasound cat. -e- HNO2S N 1N1- C3 66,633555.Raucher, S. and Klein, P., J. HNOS N 1n 1 CH O (80-95%) O

O –HaLawesson's Reagent O Lawesson, S.-O., et al., Org. Syn., Coll. bumetanide N-morpholinyamide Wol. VI, 1990, 372-375: bumetanide N-morpholinylthioanide (NTP-2024) A. Preparation; B. Thiolactam Formation; (thioamide analog of NTP-2024) and references 26-30. Lawesson's Reagent -e- Lawesson, S.-O., et al., Bull. Soc. Chim. Belg., 1978, 87,223, 229, 293,299, and 525. Davy's Reagent me Davy, H., Sulfur Lett., 1985, 3, 39-44 and related references

Thioamide and Thiolactam Review - "Synthesis of Thioamides and Thiolactams", Schaumann, E. in Comprehensive Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, Chapter 24, Pergamon Press, Oxford, 1991, pp.419-434.

Lawesson's Reagent Additional References - Lawesson, S.-O. et al., Org. Syn., Col. Vol.VII, 1990, 372-375 and references therein; Fieser's Reagents for Org. Syn., Vol. 8, 1980, 327; ibid, Vol. 9, 1981, 49-50; ibid, Vol. 10, 1982, 39; ibid, Vol. 11, 1984, 54-55; ibid, Vol. 12, 1986, 59; ibid, Vol. 13, 1988, 38-39; ibid, Vol. 15, 1990, 37,329; and ibid, Vol. 16, 1992, 37-38.

Davy's Reagent References - Davy, H., JCS, Chem. Commun., 1982, 457; Davy, H., Sulfur Lett., 1985, 3, 39-44; Davy, H., Chem. & Ind., 1985, 824; Davy, H., J. Chem. Res. (S), 1985, 272; Davy, H., J. Chem. Res. (M), 1985, 2701 2712. hioester and hiolactone Review - "Synthesis of Thioesters and Thiolactones", Voss, J. in Comprehensive Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, Chapter 2.5, Pergamon Press, Oxford, 1991, pp.435-460.

Selenoesters and Selenoamide Review - "Synthesis of Selenoesters of All Oxidation States", Ogawa, A. and Sonoda, N. in Comprehensive Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, Chapter 2.6, Pergamon Press, Oxford, 1991, pp.461-484. U.S. Patent Jun. 20, 2017 Sheet 9 of 52 US 9,682,928 B2

FIGURE 9

Direct Syntheses of a Selenoamide Analog

o, --O ReleastP.Ses, BacOs, xylene, reflux subr O P.Ses, pyridine, reflux HNOS 1N1ch, vi, Sr.'s Nos s-rch, O O

O -o-Woollins Reagent O Woollins, J. D., et al., Chemistry burnetanide N-morpholinylamide Europe J., 2005, 11, 6221-6227. bunetanide N-Torpholinylselenoanide (NTP-2024) (selenoamide analog of NTP-2024)

Selengesters and Selenoanide Review - "Synthesis of Selenoesters of All Oxidation States". Ogawa, A. and Sonoda, N. in Comprehensive Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6. Chapter 2.6. Pergamon Press, Oxford, 1991, pp.461-484 (especially pp. 478-481).

Woollins Reagent References - (PhPSe2). References - Woollins, J. D., et al., Chem. Eur. J., 2005, 11, 6221-6227 and Woollins references therein.

Thioester and hiolactone Review - "Synthesis of Thioeste S and Thiolactones", Voss, J. in Comprehensive Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, Chapter 2.5, Pergamon Press, Oxford, 1991, pp.435-460.

Lawesson's Reagent Additional References - Lawesson, S.-O., et al., Org. Syn., Coll. Vol.V., 1990.372-375 and references therein; Fieser's Reagents for Org. Syn., Vol. 8, 1980, 327; ibid, Vol. 9, 1981, 49-50; ibid, Vol. 10, 1982, 39; ibid, Wol. 11, 1984, 54-55; ibid, Vol. 12, 1986, 59; ibid, Vol. 13, 1988, 38-39, ibid, Vol. 15, 1990, 37, 329; and ibid, Vol. 16, 1992, 37-38. See selenium analog reagents,

Davy's Reagent References- Davy, H., JCS, Chern. Commun., 1982, 457; Davy, H., Sulfur Lett., 1985, 3, 39-44. Davy, H., Chem. & timd., 1985, 824; Davy, H., J. Chem. Res. (S), 1985, 272; Davy, H., J. Chem. Res. (M), 1985. 2701-2712. See selenium analog reagents. U.S. Patent US 9,682,928 B2

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FIGURE 46C US 9,682,928 B2 1. 2 BUMETANIDE ANALOGS, COMPOSITIONS no change in transmembrane potential. For this reason, these AND METHODS OF USE transporters are known as electroneutral cation-Cl coupled cotransporters. In addition to being heavily implicated in ion CROSS-REFERENCE TO RELATED absorptive and secretory mechanisms, electroneutral cation APPLICATIONS Cl coupled cotransporters play a key role in maintenance and regulation of cell Volume in both epithelial and nonepi This Application is a continuation of U.S. application Ser. thelial cells. Because Na" influx and K" efflux by electroneu No. 13/138,282 filed Jan. 21, 2011, which is a U.S. National tral cotransporters are rapidly corrected by NaK-ATPase, Phase Application of International Patent Application No. the net effect of its activity is Cl movement inside or outside PCT/US2010/000170 filed Jan. 22, 2010, which claims 10 cells. This is known to be accompanied by changes in cell priority to U.S. Provisional Patent Application Nos. 61/146, volume. Finally, a variety of new physiological roles for 336 filed Jan. 22, 2009, and 61/257,238, filed Nov. 2, 2009. electroneutral cotransporters are emerging (e.g., regulation The disclosures of the related and priority patent applica of intraneuronal CI concentration and thus modulation of tions are hereby incorporated by reference in their entireties. neurotransmission.) Gamba (2005) “Molecular Physiology 15 and Pathophysiology of Electroneutral Cation-Chloride FIELD OF THE INVENTION Cotransporters.” Physiol. Rev. 85: 423-493. Four groups of electroneutral cotransporter systems (also The present invention relates to bumetanide, furosemide, known as “symporters') have been functionally identified piretanide, azosemide, and torsemide analogs, including based on cation(s) coupled with chloride, stoichiometry of derivatives, positional isomers, and prodrugs thereof, com transport process, and sensitivity to inhibitors. These sys positions comprising the same and methods of making and tems include: (1) the benzothiadiazine (or thiazide)-sensitive using the same. The present invention also relates to phar NaCl cotransporter, (2) the sulfamoylbenzoic (or maceutical compositions containing derivatives (including bumetanide) sensitive NaK2Cl cotransporters; (3) the prodrugs) of these compounds and methods for using deriva sulfamoylbenzoic (or bumetanide) sensitive NaCl cotrans tives of these compounds. Such derivatives (including pro 25 porters; and (4) the dihydroindenyloxyalkanoic acid drugs) of bumetanide, furosemide, piretanide, azosemide, (DIOA)-sensitive KCl cotransporter. There is some over and torsemide are particularly useful for the treatment and/or lap in sensitivity to inhibitors in the last two groups because prophylaxis of diseases, disorders, and conditions that Na"K2Cl and KCl cotransporters can be inhibited by involve the Na"K"Cl co-transporters (NKCC1 or NKCC2 high concentration of DIOA or loop diuretics, respectively; or combinations thereof) including but not limited to addic 30 however, affinity for inhibitor and the cation coupled with tive disorders, anxiety disorders, ascites, bipolar disorder, chloride clearly differentiate between both groups of trans cancer, depression, edema, endothelial corneal dystrophy, porters. Physiological evidence for these transport mecha epilepsy, glaucoma, ischemia, migraine, neuropathic pain, nisms became available at the beginning of the 1980s, and nociceptive neuralgia, ocular diseases, pain, postherpetic a remarkable amount of information was generated in the neuralgia, and schizophrenia. Compounds described herein 35 following years by characterizing these transport systems in are also particularly useful for the treatment and/or prophy many different cells and experimental conditions. Gamba laxis of diseases, disorders, and conditions that involve the (2005) “Molecular Physiology and Pathophysiology of GABA receptor including but not limited to Alzheimer's Electroneutral Cation-Chloride Cotransporters.” Physiol. Disease, addictive disorders, anxiety disorders, autism, Rev. 85: 423-493. bipolar disorder, depression, epilepsy, Huntington's Disease, 40 One isoform of the Na"K"Cl cotransporter (NKCC) insomnia, migraine, migraine with aura, neuropathic pain, NKCC1 is widely distributed throughout the body. NKCC1 nociceptive pain, pain, Parkinson's disease, personality dis transports Sodium, potassium, and chloride into the cell. orders, psychosis, Schizophrenia, seizure disorders, tinnitus, NKCC1 is also found throughout the nervous system where and withdrawal syndromes. it is expressed on astrocytes, oligodendrocytes, and 45 Schwann cells. Lenart, et al. (2004) The Journal of Neuro BACKGROUND OF THE INVENTION science 24(43): 9585-9597. Another isoform, NKCC2 is found primarily in the kidney, where it serves to extract Na"K"Cl Co-Transporters sodium, potassium, and chloride from the urine. Haas (1994) In absorptive and secretory epithelia, transcellular ion “The Na-K C1 cotransporters.” Am J Physiol Cell transport depends on specific plasma membrane proteins for 50 Physiol 267: C869-C885. Bumetanide, furosemide, pire mediating ion entry into and exit from cells. In basolateral tanide, azosemide, and torsemide are loop diuretics that have membrane of almost all epithelia (with exception of chor a potent diuretic effect. Loop diuretics are antagonists of the oidal plexus), sodium exit and potassium entrance occur Na"K"Cl cotransporter (e.g., NKCC2) in the thick ascend through NaK-ATPase, generating electrochemical gradi ing limb of the loop of Henle and act to inhibit sodium and ents that constitute a driving force for Na' influx and K" 55 chloride reabsorption by competing for the Cl binding site. efflux. Transport of these ions following their gradients can See also Russell (January 2000) *Sodium-Potassium-Chlo be accomplished by specific ion channels, allowing mem ride Cotransport.” Physioglocal Reviews 80C1): 211-275. brane passage of ions alone or by transporters in which Na' The regulation of Cl transport into and out of cells plays or K' transport is accompanied by other ions or solutes by a critical role in the maintenance of intracellular volume and means of several different solute transporters. These mem 60 the excitability of GABA responsive neurons regulated by at brane proteins are known as secondary transporters because least two ion cotransporters: Cl influx is mediated by the ion or molecule translocation is not dependent on ATP NKCC1 which mediates the CIT influx and KCC1 or KCC2 hydrolysis but rather on gradients generated by primary which mediates the Cl efflux. Kahle, et al. (2004) Proc. transporters. A secondary transport mechanism that is very Natl. Acad. Sci. USA 102(46): 16783-16788. The mainte active in transcellular ion transport in epithelial cells is one 65 nance of intra- and extracellular electrolyte homeostasis is in which cations (Na" or K) are coupled with chloride, with required for a wide range of essential physiologic processes, a stoichiometry of 1:1; therefore, ion translocation produces including general functions (e.g., maintenance of proper cell US 9,682,928 B2 3 4 Volume), specialized cell functions (e.g., control of neuronal where approximately 30% of all synapses use GABA as a excitability), and global functions (e.g., regulation of blood transmitter. There are three classes of GABA receptors: pressure.) This homeostasis is achieved via the regulated GABA (ligand-gated ion channel), GABA (G protein movement of Na', K", and Clacross cell membranes by ion coupled receptor), and GABA (ligand-gated ion channel). channels, cotransporters, exchangers, and pumps that Chloride flux into the cell results from the activation of execute transmembrane electrolyte flux. Kahle, et al. (2004) GABA receptors by the binding of GABA molecules, Proc. Natl. Acad. Sci. USA 102(46): 16783-16788. hyperpolarizing the resting membrane potential and decreas The predominant mechanism by which intracellular vol ing the chances of the post-synaptic neuron propagating an ume is maintained in cells in response to changes in extra action potential. GABA receptors are pentameric and cellular tonicity is the raising or lowering of intracellular Cl 10 approximately 19 GABA receptor subunits have been cloned concentration (Cl), thereby minimizing transmembrane from mammals (6C., 3 B, 3y, 1 6, 1e, 10, 17t, and 3p subunits). water flux. ICl is modulated by altering the balance The heterogeneity of GABA subunits is further increased by between C1 entry and exit. The major mediator of C1 entry alternate splicing (e.g., Y2 short and Y2 long are the two is NKCC1 and Cl exit is largely mediated by KCC1. These major splice variants of the Y2). In general, a functional cotransporters are both regulated by extracellular tonicity: 15 GABA receptor requires 2C. Subunits, 2B Subunits and a hypertonicity activates NKCC1 and inhibits KCC1, whereas third “regulatory” subunit (usually Y or ö). WO 2009/ hypotonicity has the opposite effect. Kahle, et al. (2004) 100040. The specific subunit combination determines the Proc. Natl. Acad. Sci. USA 102(46): 16783-16788. pharmacological and ligand binding properties of the An analogous system plays a key role in the control of GABA receptor. The most abundant subunit combination neuronal excitability. In the adult brain, GABA is the major found in the CNS is C. B.Y. This subtype represents inhibitory neurotransmitter. If ICl), is below its equilibrium approximately 40% of GABA receptors in the brain and it potential, C1 enters the cell, resulting in hyperpolarization is expressed throughout the CNS and is located on post and inhibition. If ICl), is above its equilibrium potential, synaptic cells. WO 2007/002359. GABA induces Cl efflux, depolarization, and neuronal The GABA receptors are the targets of a wide range of excitation. The importance of Cl, regulation has been 25 therapeutic and clinically relevant compounds including recognized with the discovery that GABA neurotransmis benzodiazepines, barbiturates, neurosteroids, ethanol, cer sion is not uniformly inhibitory; it is predominantly excit tain intravenous anesthetics, and Subtype specific modula atory in the neonatal period. Similarly, neurons of the tors (e.g., Zolpidem.) These compounds serve as anxiolytics, Suprachiasmatic nucleus show circadian variation in their sedative/hypnotics, anti-epileptic drugs (AED), and memory response to GABA, demonstrating the ability to dynamically 30 enhancers. Many of these therapeutics show efficacy but regulate C1. Finally, GABA neurotransmission in the cause side effects due to unwanted effects at C. and/or C. peripheral nervous system is predominantly excitatory. GABA variants or due to low therapeutic index. For Variation in ICl), in these neurons is determined by mecha example, benzodiazepines such as diazepam (VALIUM) are nisms highly similar to those governing cell Volume. Cl excellent anxiolytics but cause unwanted sedative effects influx largely occurs via NKCC1, whereas Cl efflux is 35 when used clinically. WO 2007/002359. mediated via the neuronal-specific K-Cl cotransporter At a cellular level, GABA receptors are expressed both KCC2. Kahle, et al. (2004) Proc. Natl. Acad. Sci. USA at both post-synaptic and extra-synaptic sites (parasynaptic) 102(46): 16783-16788. where they respond to large changes in GABA concentration The mediators of transcellular CI cotransport (Na—Cl caused by release of the neurotransmitter into the synaptic cotransporter, NKCC1, NKCC2, KCC1, and KCC2) are all 40 space, and extra-synaptically where the receptors respond to related members of the SLC12A family of cation/Cl lower concentrations of GABA that “leak” from synaptic cotransporters; each takes advantage of inward Na" or junctions. The post-synaptic receptors respond to acute outward K gradients to move C1 into or out of cells, changes in neuronal firing whereas the extrasynaptic recep respectively. The importance of this family of transporters is tors are responsible for maintaining overall tone of neuronal underscored by their use as pharmacologic targets (thiazide 45 networks. WO 2009/100040. Tonic inhibition is generated diuretics act at NCC, and loop diuretics act at NKCC2), and by the persistent activation of extrasynapatic (perisynaptic) that their mutation results in diverse diseases. Disruption of GABA receptors and regulates the excitability of individual NKCC1 in mouse leads to hearing loss, altered pain per neurons and neural networks. Jia, et al. et al. (2008) The ception, neuronal excitability, and altered blood pressure. Journal of Pharmacology and Experimental Therapeutics Kahle, et al. (2004) Proc. Natl. Acad. Sci. USA 102(46): 50 326(2): 475482. 16783-16788. Presynaptic GABA receptors situated at extrasynaptic Major advances have been made in the past decade in sites may comprise CfBö and CeB6 isoforms. The extrasyn molecular identification and characterization of Solute car aptic Cafö and CBö GABA receptor isoforms show riers. As of 2005, the Human Genome Organization marked sensitivity to GABA, alcohol, and anesthetics, Sug (HUGO) Nomenclature Committee Database recognizes 43 55 gesting that receptors may present a critical site for regu solute carries (SLC) families, which include a total of 298 lating synaptic function in the developing brain in both transporter genes encoding for uniporters (passive transport physiological and pathological situations. Xiao, et al. et al. ers), cotransporters (coupled transporters), antiporters (ex (2007) “Presynaptic GABA receptors facilitate GABAer changers), vesicular transporters, and mitochondrial trans gic transmission to dopaminergic neurons in the ventral porters. This amount of solute carrier genes represents ~1% 60 tegmental area of young rats.” J. Physiol. 580(Pt.3):731-43. of the total pool of genes that have been calculated to For example, temporal lobe epilepsy (TLE), Parkinson's compose human genome. Gamba (2005) “Molecular Physi disease (PD) and Huntington's disease (HD) are neurode ology and Pathophysiology of Electroneutral Cation-Chlo generative disorders that involve disruptions in GABA sig ride Cotransporters.” Physiol. Rev. 85: 423-493. naling. GABA is the major inhibitory neurotransmitter in the GABA Receptors 65 central nervous system (CNS). TLE seizures reflect excess Gamma-aminobutyric acid (GABA) is the major inhibi excitation, which may result from local inhibitory circuit tory neurotransmitter in the central nervous system (CNS) dysfunction. PD devastates the input to striatal GABAergic US 9,682,928 B2 5 6 neurones and HD destroys striatal GABAergic neurones. The three major hallmarks in the brain that are associated Controlling GABA delivery to specific brain areas should with the disease processes of AD include amyloid plaques benefit each of these diseases. Directing GABA synthesis, and neurofibrillary tangles. Amyloid plaques comprise frag degradation, transport or receptors can control GABA sig ments of B-amyloid peptide mixed with a collection of naling. New drugs targeting GABA synthesis, release, and additional proteins, and remnants of neurons. Neurofibril binding may be used for improved therapeutics treatments lary tangles (NFTS) are found inside neurons and comprise for epilepsy and both Parkinson's and Huntington's disease. tau protein. As neurons die throughout the brain, the affected Kleppner and Tobin (2001) “GABA signaling: therapeutic regions begin to atrophy. By the final stage of AD, damage targets for epilepsy, Parkinson's disease and Huntington's is widespread and brain tissue has shrunk significantly. disease.” Expert Opin Ther Targets. 5(2):219-39. See also 10 NINDS Alzheimer's Disease Information Page (Sep. 23. Shumate, et al. et al. (1998) Epilepsy Research (32): 114 2009). 128; Fritschy (2008) Frontiers in Molecular Neuroscience Currently there are no medicines that can slow the pro 1(5): 1-5; Roberts, et al. et al. (2006) The Journal of gression of AD. However, four FDA-approved medications Biological Chemistry 281 (40): 29431-29435; and Roberts, are used to treat AD symptoms. Unfortunately these medi et al. et al. PNAS 102(33): 11894-11899. 15 cations will not stop or reverse AD, and they appear to help Addictive Disorders individuals for only a few months to a few years. Donepezil Addictive and/or compulsive disorders, such as eating (ARICEPT), rivastigmine (EXELON), and galantamine disorders (including obesity), addiction/physical depen (REMINYL) are prescribed to treat mild to moderate AD dence to stimulants, narcotics (e.g., cocaine, heroin) seda symptoms. Donepezil was recently approved to treat severe tives/hypnotics, and opioids including alcoholism and Smok AD as well. The newest AD medication is memantine ing are major public health problem that impact Society on (NAMENDA), which is prescribed to treat moderate to multiple levels. It has been estimated that substance abuse severe AD symptoms. NINDS Alzheimer's Disease Infor alone costs the United States more than $484 billion per mation Page (Sep. 23, 2009). Treatment of a transgenic year. mouse model of Alzheimer's Disease with picrotoxin, a The alcohol-sensitive CfBö GABA receptor has also 25 GABA antagonist showed improved cognitive functions in been postulated to be involved in alcohol addiction (alco the mice. Yoshiike, et al. et al. (Aug. 21, 2008) "GABA holism). For example, reduced expression of GABA recep receptor-mediated acceleration of aging-associated memory tors comprising an O. Subunit in the nucleus accumbens decline in APP/PSI mice and its pharmacological treatment (NAc) decreased the free consumption and preference for by picrotoxin.” PLoS One. 3(8):e3029. Additionally, the alcohol in rats. Further, the nucleus accumbens contributes 30 expression of NKCC1 has been found to be elevated in to the rewarding and reinforcing effects of drugs including Alzheimer's Disease patients. Johanson, et al. (2004) Cere alcohol suggesting that the GABA receptor, specifically the brospinal Fluid Research 1:3. Therefore novel therapies Ofö isoform, in the NAc is an important mediator of based on the regulation of GABA receptor activity may alcohol self-administration. Rewal, et al. et al. (Jan. 14. relieve the symptoms of AD. 2009) The Journal of Neuroscience 29(2): 543-549. 35 Anxiety Disorders Although most GABA receptor Subunit combinations Anxiety disorders are classified into several subtypes: can be activated by high (anesthetic) alcohol concentrations, anxiety, acute anxiety, panic disorder, Social anxiety disor so far only very specific GABA receptor subunit combi der, obsessive compulsive disorder (OCD), post-traumatic nations (containing the Ö as well as the B. Subunit) exhibit stress disorder (PTSD), generalized anxiety disorder, and dose-dependencies that mirror blood alcohol levels associ 40 specific phobia. American Psychiatric Association. Diagnos ated with mild to moderate intoxication in humans. GABA tic and Statistical Manual of Mental Disorders, 4" edition receptors containing the 8 subunit are located either outside (1994). or in the perimeter of synapses, but not in the Sub-synaptic As a group, the anxiety disorders have the highest preva membrane. WO 2007/002359. lence in the U.S. of all psychiatric disorders. Kessler, et al. Current strategies for the treatment of additive disorders 45 et al. (1994) “Lifetime and 12-month prevalence of DSM include psychological counseling and Support, use of thera III-R psychiatric disorders in the United States: Results from peutic agents, or a combination of both. A variety of agents the National Comorbidity Survey.” Arch Gen Psychiatry known to affect the central nervous system have been used 51:8-19. Anxiety disorders afflict 15.7 million people in the in various contexts to treat a number of indications related United States each year, and 30 million people in the United directly or indirectly to addictive behaviors but a great need 50 States at some point in their lives. Lepine (2002) “The remains for improved addictive disorder therapeutics. Epidemiology of Anxiety Disorders: Prevalence and Soci GABA specific agents that have action at the nucleaus etal Costs. J. Clin. Psychiatry. 63: Suppl 14:4-8. accumbens might be effective therapies for addictive behav Several animal models have been developed which are iors. recognized in the art as being predictive of anxiolytic Alzheimer's Disease 55 activity. These include the fear-potentiated startle model, Alzheimer's disease (AD) is an age-related, non-revers described by Davis in Psychopharmacology 62:1; 1979, ible brain disorder that develops over a period of years. Behav. Neurosci. 100:814; 1986 and TiPS, January 1992 Initially, people experience memory loss and confusion, Vol. 13, 35-41, the elevated plus model described by Lister which may be mistaken for the kinds of memory changes in Psychopharmacol. 92: 180-185; 1987, and the well-known that are sometimes associated with normal aging. However, 60 punished—responding (conflict) model, described, for the symptoms of AD gradually lead to behavior and per example, in “Psychopharmacology of Anxiolytics and Anti Sonality changes, a decline in cognitive abilities such as depressants’, edited by S. E. File, pp. 131-153, Raven Press, decision-making and language skills, and problems recog New York, 1991. nizing family and friends. AD ultimately leads to a severe Anxiety disorders are generally treated with drugs and loss of mental function. AD is the most common cause of 65 psychotherapy. The most commonly prescribed drugs for all dementia among people age 65 and older. NINDS Alzheim anxiety types are the benzodiazepines, other psychoactive er's Disease Information Page (Sep. 23, 2009). drugs such as selective serotonin reuptake inhibitors (SSRI) US 9,682,928 B2 7 8 are also used. However, while these drugs show efficacy, "Decreased GABA receptors and benzodiazepine binding both benzodiazepines and SSRIs are also show adverse sites in the anterior cingulate cortex in autism.” Autism Res. effects during treatment. Denys and de Geus (August 2005) 2009 August; 204): 205-19. Therefore, therapeutics that tar “Predictors of Pharmacotherapy Response in Anxiety Dis get the GABA receptor may be useful in treating autism. orders. Curr Psychiatry Rep. 7(4): 252-7. For example, 5 Bipolar Disorder numerous side effects are associated with long-term use of Bipolar disorder, also known as manic-depressive illness, SSRIs, such as sexual dysfunction and weight gain. is a brain disorder that causes unusual shifts in a person’s Hirschfeld (2003) “Long-term Side Effects of SSRIs: Sexual mood, energy, and ability to function. They can result in Dysfunction and Weight Gain.” J. Clin. Psychiatry. 64: damaged relationships, poor job or school performance, and Suppl 18: 20-4. Further, existing drugs targeting postsynap 10 even suicide. About 5.7 million American adults or about 2.6 tic type GABA receptors produce undesirable results percent of the population age 18 and older have bipolar because they indiscriminately target most of the GABA disorder in any given year. Bipolar disorder typically devel receptors in the brain. WO 2007/136838. In view of nonre ops in late adolescence or early adulthood. However, some sponders and deleterious side effects, a great need exists for people have their first symptoms during childhood, and improved anxiety therapeutics. 15 Some develop them late in life. It is often not recognized as Ascites an illness, and people may suffer for years before it is Ascites is excess fluid in the space between the tissues properly diagnosed and treated. National Institute of Mental lining the abdomen and abdominal organs (the peritoneal Health “Bipolar Disorder” (2008) Complete Publication. cavity) typically caused by liver disease. Disorders that may Bipolar disorder causes dramatic mood Swings—from be associated with ascites include: cirrhosis, hepatitis, portal overly “high” and/or irritable to sad and hopeless, and then vein thrombosis, constrictive pericarditis, congestive heart back again, often with periods of normal mood in between. failure, liver cancer, ovarian cancer, protein-losing enteropa Severe changes in energy and behavior go along with these thy, nephrotic syndrome, and pancreatitis. Some agents are changes in mood. The periods of highs and lows are called available for the treatment of ascites, for example, furo episodes of mania and depression. National Institute of semide but a great need remains for improved ascites 25 Mental Health “Bipolar Disorder” (2008) Complete Publi therapeutics. See Shiozaki, et al. (2006) J. Physiol. Sci. cation. 56(6): 401–406. Signs and symptoms of mania (or a manic episode) Autism include: increased energy, activity, and restlessness; exces Autism spectrum disorder (ASD) is a range of complex sively “high, overly good, euphoric mood; extreme irrita neurodevelopment disorders, characterized by Social impair 30 bility; racing thoughts and talking very fast, jumping from ments, communication difficulties, and restricted, repetitive, one idea to another, distractibility, difficulty concentrating: and stereotyped patterns of behavior. Autistic disorder, little sleep needed; unrealistic beliefs in one's abilities and Sometimes called autism or classical ASD, is the most severe powers; poor judgment; spending sprees; a lasting period of form of ASD, while other conditions along the spectrum behavior that is different from usual; increased sexual drive; include a milder form known as Asperger syndrome, the rare 35 drug abuse, particularly cocaine, alcohol, and sleeping medi condition called Rett syndrome, and childhood disintegra cations; provocative, intrusive, or aggressive behavior, and/ tive disorder and pervasive developmental disorder not or denial that anything is wrong. A manic episode is diag otherwise specified (usually referred to as PDD-NOS). nosed if elevated mood occurs with three or more of the Although ASD varies significantly in character and severity, other symptoms most of the day, nearly every day, for 1 it occurs in all ethnic and Socioeconomic groups and affects 40 week or longer. National Institute of Mental Health “Bipolar every age group. Experts estimate that three to six people out Disorder” (2008) Complete Publication. ofevery 1,000 may develop ASD. Males are four times more Signs and symptoms of depression (or a depressive epi likely to have ASD than females. NINDS Autism Fact Sheet sode) include: lasting sad, anxious, or empty mood; feelings (September 2009). Furthermore, various GABA A subunit of hopelessness or pessimism; feelings of guilt, worthless types, such as C. Variant sequences and C isoforms have 45 ness, or helplessness; loss of interest or pleasure in activities been linked to autism. WO 2009/100040. There is no cure once enjoyed, including sex; decreased energy, a feeling of for ASD, thus there exists a great need for therapeutics to fatigue or of being “slowed down'; difficulty concentrating, treat autism. remembering, making decisions; restlessness or irritability; Multiple lines of evidence, including genetic and imaging sleeping too much, or cannot sleep; change in appetite studies, Suggest that the anterior cingulate cortex and 50 and/or unintended weight loss or gain; chronic pain or other gamma-amino-butyric acid (GABA) system may be affected persistent bodily-symptoms that are not caused by physical in autism. Compared to controls, the autistic patients show illness or injury; and/or thoughts of death or Suicide, or a significant decrease in the mean density of GABA Suicide attempts. A depressive episode is diagnosed if five or receptors in the Supragranular (46.8%) and infragranular more of these symptoms last most of the day, nearly every (20.2%) layers of the anterior cingulate cortex (ACC) and in 55 day, for a period of 2 weeks or longer; National Institute of the density of benzodiazepine binding sites in the Supra Mental Health “Bipolar Disorder” (2008) Complete Publi granular (28.9%) and infragranular (16.4%) lamina. In addi cation. tion, a trend for a decrease in the density of benzodiazepine A mild to moderate level of mania is called hypomania. sites was found in the infragranular layers (17.19%) in the Hypomania may feel good to the person who experiences it autism group. These findings suggest that in the autistic 60 and may even be associated with good functioning and group this downregulation of both benzodiazepine sites and enhanced productivity. Thus even when family and friends GABA receptors in the ACC may be the result of increased learn to recognize the mood Swings as possible bipolar GABA innervation and/or release disturbing the delicate disorder, the person may deny that anything is wrong. excitation/inhibition balance of principal neurons as well as Without proper treatment, however, hypomania can become their output to key limbic cortical targets. These distur 65 severe mania in Some people or can Switch into depression. bances may underlie the core alterations in Socio-emotional In some people, however, symptoms of mania and depres behaviors in autism. Oblak, et al. et al. (August 2009) sion may occur together in what is called a mixed bipolar US 9,682,928 B2 9 10 state. Symptoms of a mixed State often include agitation, rhythm in hippocampus, which is thought to play roles in trouble sleeping, significant change in appetite, psychosis, spatial memory and in the induction of synaptic plasticity and Suicidal thinking. A person may have a very sad, (Heurta and Lisman (1995) Neuron. 15: 1053-63; Heurta hopeless mood while at the same time feeling extremely and Lisman (1996) J. Neurophysiol. 75: 877-84; O'Keefe energized. (1993) Curr. Opin. Neurobiol. 3: 917-24). To date, most The classic form of the illness, which involves recurrent research on the processes underlying the generation and episodes of mania and depression, is called bipolar I disor maintenance of spontaneous synchronized activity has der. Some people, however, never develop severe mania but focused on synaptic mechanisms. However, there is evi instead experience milder episodes of hypomania that alter dence that nonsynaptic mechanisms may also play important nate with depression; this form of the illness is called bipolar 10 roles in the modulation of synchronization in normal and II disorder. When four or more episodes of illness occur pathological activities in the central nervous system. within a 12-month period, a person is said to have rapid Depression cycling bipolar disorder. Some people experience multiple Depression is a common but serious illness, the most episodes within a single week, or even within a single day. common are major depressive disorder and dysthymic dis Rapid cycling tends to develop later in the course of illness 15 order. Major depressive disorder, also called major depres and is more common among women than among men. Sion, is characterized by a combination of symptoms that Medications known as “mood stabilizers' usually are interfere with a person’s ability to work, sleep, study, eat, prescribed to help control bipolar disorder (e.g. lithium or and enjoy once-pleasurable activities. Major depression is valproic acid-DEPAKOTE/VALPROATE). In addition to disabling and prevents a person from functioning normally. medication, psychosocial treatments—including certain An episode of major depression may occur only once in a forms of psychotherapy, are often used to treat bipolar person’s lifetime, but more often, it recurs throughout a disorders. Depending on the medication, side effects include person’s life. National Institute of Mental Health “Depres weight gain, nausea, tremor, reduced sexual drive or perfor sion” (2008) Complete Publication. mance, anxiety, hair loss, movement problems, or dry The forms of depression include: mouth. Lithium treatment can cause low thyroid levels, 25 Dysthymic disorder, also called dysthymia, is character resulting in the need for thyroid Supplementation. Addition ized by long-term (two years or longer) but less severe ally, Valproate R may lead to adverse hormone changes in symptoms that may not disable a person but can prevent one teenage girls and polycystic ovary syndrome in women who from functioning normally or feeling well. People with began taking the medication before age 20. Further, women dysthymia may also experience one or more episodes of suffering bipolar disorder who wish to conceive, or who 30 major depression during their lifetimes. become pregnant, face special challenges due to the possible Psychotic depression, which occurs when a severe depres harmful effects of existing mood stabilizing medications on sive illness is accompanied by some form of psychosis, such the developing fetus and the nursing infant. National Insti as a break with reality, hallucinations, and delusions. Post tute of Mental Health “Bipolar Disorder” (2008) Complete partum-depression, which is diagnosed if a new mother Publication. Improved bipolar disorder therapeutics may be 35 develops a major depressive episode within one month after developed that act to increase GABA activity. delivery. It is estimated that 10 to 15 percent of women Postmortem and genetic studies have linked neuropsychi experience postpartum depression after giving birth. atric disorders including Schizophrenia and bipolar disorder Seasonal affective disorder (SAD), which is characterized with GABAergic neurotransmission and various specific by the onset of a depressive illness during the winter months, GABA receptor subunits. Further, GABA receptor-asso 40 when there is less natural Sunlight. The depression generally ciated proteins involved in GABA receptor trafficking, lifts during spring and summer. SAD may be effectively targeting, clustering, and anchoring that often carry out these treated with light therapy, but nearly half of those with SAD functions in a Subtype-specific manner. Charych, et al. do not respond to light therapy alone. Antidepressant medi (2009) “GABA receptors and their associated proteins: cation and psychotherapy can reduce SAD symptoms, either implications in the etiology and treatment of Schizophrenia 45 alone or in combination with light therapy. National Institute and related disorders. Neuropharmacology. 57(5-6): 481-95. of Mental Health “Depression” (2008) Complete Publica Therefore, GABA receptor specific therapeutics that tion. improve inhibition may be beneficial because bipolar dis Depression can be treated with a number of methods. The ease is a state of alterations of abnormal inhibition/excitation most common treatments are medication and psychotherapy. without adequate inhibition. 50 Antidepressants work to normalize neurotransmitters, nota Cognition, Learning, and Memory bly serotonin, norepinephrine, and dopamine. The newest The cognitive abilities of mammals are thought to be and among the most popular types of antidepressant medi dependent on cortical processing. It has generally been cations are called selective serotonin reuptake inhibitors accepted that the most relevant parameters for describing (SSRIs). SSRIs include fluoxetine (ProzacR), citalopram and understanding cortical function are the spatio-temporal 55 (CelexaR), sertraline (Zoloft(R), and several others. Sero patterns of activity. In particular, long-term potentiation and tonin and norepinephrine reuptake inhibitors (SNRIs) are long-term depression have been implicated in memory and similar to SSRIs and include venlafaxine (Effexor R) and learning and may play a role in cognition. Oscillatory and duloxetine (Cymbalta(R). SSRIs and SNRIs are more popu synchronized activities in the brains of mammals have been lar than the older classes of antidepressants, such as tricy correlated with distinct behavioral states. 60 clics-named for their chemical structure- and monoamine Synchronization of spontaneous neuronal firing activity is oxidase inhibitors (MAOIs) because they tend to have fewer thought to be an important feature of a number of normal side effects. However, medications affect everyone differ and pathophysiological processes in the central nervous ently-no one-size-fits-all approach to medication exists. system. Examples include synchronized oscillations of National Institute of Mental Health “Depression” (2008) population activity Such as gamma rhythms in the neocortex, 65 Complete Publication. which are thought to be involved in cognition (Singer and For all classes of antidepressants, patients can experience Gray (1995) Annu. Rev. Neurosci. 18: 855-86), and theta side effects. Antidepressants may cause mild and often US 9,682,928 B2 11 12 temporary side effects in Some people, but they are usually sis. This procedure was found to induce seizures in epileptic not long-term. The most common side effects associated patients, but rarely in non-epileptic individuals. Garland, et with SSRIs and SNRIs include: headache, nausea, insomnia, al. (1943) Lancet 2: 566. Status epilepticus can be blocked nervousness, agitation, and sexual problems. National Insti in kainic acid-treated rats by intravenous injection of man tute of Mental Health “Depression” (2008) Complete Pub nitol. Baran, et al. (1987) Neuroscience 21: 679. This effect lication. Tricyclic antidepressants also can cause side effects is similar to that achieved by intravenous injection of urea in including: dry mouth, constipation, bladder problems, human patients. Carter (1962) Epilepsia 3: 198. The treat sexual problems, blurred vision, and daytime drowsiness. ment in each of these cases increases the osmolarity of the Additionally, patients taking MAOIs must adhere to signifi blood and extracellular fluid, resulting in water efflux from cant food and medicinal restrictions to avoid potentially 10 the cells and an increase in extracellular space in the brain. serious interactions. They must avoid certain foods that Acetazolamide (ACZ), a diuretic with a different mechanism contain high levels of the chemical tyramine, which is found of action (inhibition of carbonic anhydrase), has been stud in many cheeses, wines and pickles, and some medications ied experimentally as an anticonvulsant (White, et al. (1986) including decongestants. MAOIs interact with tyramine in Advance Neurol. 44: 695; Guillaume, at al. (1991) Epilepsia Such a way that may cause a sharp increase in blood 15 32: 10) and used clinically on a limited basis (Tanimukai, et pressure, which could lead to a stroke. National Institute of al. (1965) Biochem. Pharm. 14:961; Forsythe, et al. (1981) Mental Health “Depression” (2008) Complete Publication. Develop. Med. Child Neurol. 23: 761). Although its mecha GABA is involved in both clinical depression and in nism of anticonvulsant action has not been determined, ACZ animal models of depression. Kram, et al. (October 2000) does have a clear effect on the cerebral extracellular space. “Effects of learned helplessness on brain GABA receptors.” Traditional anti-epileptic drugs exert their principal effect Neuroscience Research 38(2): 193-198. Therefore improved through one of three mechanisms: (a) inhibition of repeti depression therapeutics based on the GABAergic system tive, high-frequency neuronal firing by blocking Voltage may provide better medication. dependent sodium channels; (b) potentiation of Y-aminobu Epilepsy tyric acid (gamma-aminobutyric acid, GABA)-mediated Epilepsy is characterized by abnormal discharges of cere 25 postsynaptic inhibition; and (c) blockade of T-type calcium bral neurons and is typically manifested as various types of channels. seizures. Epileptiform activity is identified with spontane Many current anti-epileptic drug therapies exert their ously occurring synchronized discharges of neuronal popu pharmacological effects on all brain cells, regardless of their lations that can be measured using electrophysiological involvement in seizure activity. Common side effects are techniques. Epilepsy is one of the most common neurologi 30 over-sedation, dizziness, loss of memory and liver damage. cal disorders, affecting about 1% of the population. There Furthermore, 20-30% of epilepsy patients are refractory to are various forms of epilepsy, including idiopathic, symp current therapy. Therefore there is a great need for improved tomatic, and cryptogenic. Genetic predisposition is thought epilepsy therapeutics to reduce both morbidity and mortal to be the predominant etiologic factor in idiopathic epilepsy. ity. Symptomatic epilepsy usually develops as a result of a 35 Glaucoma structural abnormality in the brain. Glaucoma is a group of diseases that can damage the eye's Status epilepticus is a particularly severe form of seizure, optic nerve and result in vision loss and blindness. Glau which is manifested as multiple seizures that persist for a coma occurs when the normal fluid pressure inside the eyes significant length of time, or serial seizures without any slowly rises. Open-angle glaucoma is the most common recovery of consciousness between seizures. The overall 40 form. Other types of glaucoma include: (1) low-tension or mortality rate among adults with status epilepticus is normal-tension glaucoma; (2) angle-closure glaucoma; (3) approximately 20 percent. Patients who have a first episode congenital glaucoma; (4) secondary glaucomas; and (5) are at substantial risk for future episodes and for the devel pigmentary glaucoma including neovascular glaucoma. opment of chronic epilepsy. The frequency of status epilep Everyone is at risk for glaucoma but some populations are ticus in the United States is approximately 150,000 cases per 45 at higher risk than others including African Americans over year, with approximately 55,000 deaths being associated age 40, everyone over age 60, and people with a family with status epilepticus annually. Sirven and Waterhouse history of glaucoma. National Eye Institute Glaucoma Fact (2003) “Management of Status Epilepticus' American Fam Sheet (2008). ily Physician 68: 469-476. Acute processes that are associ Glaucoma is usually detected through a comprehensive ated with status epilepticus include intractable epilepsy, 50 eye exam that includes: (a) visual acuity test; (b) visual field metabolic disturbances (e.g., electrolyte abnormalities, renal test; (c) dilated eye exam; (d) tonometry; and (e) pachym failure, and sepsis), central nervous system infection (men etry. Current glaucoma treatments include medicines, laser ingitis or encephalitis), stroke, degenerative diseases, head trabeculoplasty, conventional Surgery, or a combination of trauma, drug toxicity, and hypoxia. The fundamental patho any of these, however there is a great need for improved physiology of status epilepticus involves a failure of mecha 55 glaucoma therapeutics. National Eye Institute Glaucoma nisms that normally abort an isolated seizure. This failure Fact Sheet (2008). can arise from abnormally persistent, excessive excitation or Huntington's Disease ineffective recruitment of inhibition. Studies have shown Huntington's disease (HD) results from neuronal degen that excessive activation of excitatory amino acid receptors eration leading to uncontrolled movements, loss of intellec can cause prolonged seizures and Suggest that excitatory 60 tual faculties, and emotional disturbance. HD is an auto amino acids may play a causative role. Status epilepticus can Somal dominant disease caused by a CAG expansion in the also be caused by penicillin and related compounds that Htt gene that leads to a poly-glutamine expansion in the antagonize the effects of Y-aminobutyric acid (GABA), the disease protein huntingtin. GABAergic interneurons are primary inhibitory neurotransmitter in the brain. particularly sensitive to the accumulation of mutant hun One early diagnostic procedure for epilepsy involved the 65 tingtin and die early in the development of HD. Some early oral administration of large quantities of water together with symptoms of HD are mood Swings, depression, irritability or injections of vasopressin to prevent the accompanying diure trouble driving, learning new things, remembering a fact, or US 9,682,928 B2 13 14 making a decision. As the disease progresses, concentration relative shortage of the blood Supply to an organ. This can on intellectual tasks becomes increasingly difficult, and the cause necrosis (e.g., cell death). In aerobic tissues such as patient may have difficulty feeding himself or herself and heart and brain, at body temperature necrosis due to isch Swallowing. The rate of disease progression and the age of emia usually takes about 3–4 hours before becoming irre onset vary from person to person. A genetic test, coupled 5 versible. Later, more damage occurs due to the accumulation with a complete medical history and neurological and labo of metabolic wastes due to lack of adequate blood Supply to ratory tests, helps physicians diagnose HD. Presymptomic the tissue. Complete cessation of oxygenation of such organs testing is available for individuals who are at risk for for more than 20 minutes typically results in irreversible carrying the HD gene. In 1 to 3 percent of individuals with damage. HD, no family history of HD can be found. NINDS Publi 10 Inhibition of NKCC1 activity with bumetanide and furo cation “Huntington's Disease: Hope Through Research” semide significantly reduces the infarct Volume and cerebral (2009). edema following cerebral focal ischemia Suggesting that Physicians prescribe a number of medications to help NKCC1 antagonists may be useful in treating ischemia. control emotional and movement problems associated with Chen and Sun (2005) “The role of Na—K C1 co-trans HD. In August 2008 the U.S. Food and Drug Administration 15 porter in cerebral ischemia. Neurol. Res. 27(3): 280-286. approved tetrabenazine to treat Huntington's chorea (the The typical treatment of ischemia involves “clot-buster involuntary writhing movements), making it the first drug drugs (e.g., AlteplaseR) usually given for stroke and heart approved for use in the United States to treat the disease. attack within this time period. However, restoration of blood However, drugs used to treat the symptoms of HD have side flow after a period of ischemia can actually be more dam effects such as fatigue, restlessness, or hyperexcitability. aging than the ischemia because reintroduction of oxygen NINDS Publication “Huntington's Disease: Hope Through causes a greater production of damaging free radicals, Research” (2009). resulting in reperfusion injury. Necrosis is greatly acceler Huntington's disease (HD) is a neurodegenerative disor ated in reperfusion injury and therefore a great need exists der that involves disruptions in GABA signaling. GABA is for improved ischemia therapeutics. the major inhibitory neurotransmitter in the central nervous 25 Migraine system (CNS). HD destroys striatal GABAergic neurons. Migraine headaches afflict 10-20% of the U.S. population, Directing GABA synthesis, degradation, transport, or recep with an estimated loss of 64 million workdays annually. tors can control GABA signaling and so drugs that target Migraine headache is characterized by pulsating head pain these aspects of GABA metabolism may be used for that is episodic, unilateral or bilateral, lasting from 4 to 72 improved therapeutic treatments, for Huntington's disease. 30 hours and often associated with nausea, vomiting, and Kleppner and Tobin (2001) “GABA signaling: therapeutic hypersensitivity to light and/or sound. When accompanied targets for epilepsy, Parkinson's disease and Huntington’s by premonitory symptoms, such as visual, sensory, speech or disease.” Expert Opin Ther Targets. 5(2):219-39. motor symptoms, the headache is referred to as “migraine Insomnia with aura,” formerly known as classic migraine. When not Insomnia is a symptom of any of several sleep disorders, 35 accompanied by Such symptoms, the headache is referred to characterized by persistent difficulty falling asleep or staying as “migraine without aura.” formerly known as common asleep despite the opportunity. NHLBI Diseases and Con migraine. Both types evidence a strong genetic component, ditions Index Insomnia (October 2009). and both are three times more common in women than men. Although there are several different degrees of insomnia, The precise etiology of migraine has yet to be determined. three types of insomnia have been clearly identified: tran 40 It has been theorized that persons prone to migraine have a sient, acute, and chronic. Transient insomnia lasts from days reduced threshold for neuronal excitability, possibly due to to weeks. It can be caused by another disorder, by changes reduced activity of the inhibitory neurotransmitter Y-amin in the sleep environment, by the timing of sleep, severe obutyric acid (GABA). GABA normally inhibits the activity depression, or by stress. Its consequences—sleepiness and of the neurotransmitters serotonin (5-HT) and glutamate, impaired psychomotor performance—are similar to those of 45 both of which appear to be involved in migraine attacks. The sleep deprivation. Acute insomnia is the inability to consis excitatory neurotransmitter glutamate is implicated in an tently sleep well for a period of between three weeks to six electrical phenomenon called cortical spreading depression, months. Chronic insomnia lasts for years at a time. It can be which can initiate a migraine attack, while serotonin is caused by another disorder, or it can be a primary disorder. implicated in vascular changes that occur as the migraine Its effects can vary according to its causes. They might 50 progresses. include sleepiness, muscular fatigue, hallucinations, and/or It has been Suggested that cortical spreading depression mental fatigue; but people with chronic insomnia often show (CSD) underlies migraines including migraines with visual increased alertness. NHLBI Diseases and Conditions Index aura. It is also believed that CSD underlies migraine as part Insomnia (October 2009). of the trigeminal pain circuit. CSD is characterized by a Current insomnia drug therapies that target the GABA 55 short burst of intense depolarization in the occipital cortex, receptor, hypnotics (e.g., benzodiazepines) may have unde followed by a wave of neuronal silence and diminished sirable side effects, therefore a great need exists for evoked potentials that advance anteriorly across the Surface improved insomnia therapeutics with reduced side effects. of the cerebral cortex. Enhanced excitability of the occipital Ischemia cortex neurons has been proposed as the basis for CSD. The Ischemia is a restriction in blood Supply, generally due to 60 visual cortex may have a lower threshold for excitability and factors in the blood vessels, with resultant damage or therefore is most prone to CSD. It has been suggested that dysfunction of tissue due to inadequate oxygenation and mitochondrial disorders, magnesium deficiency, and abnor lack of nutrients of the tissue. Insufficient blood supply mality of presynaptic calcium channels may be responsible causes tissue to become hypoxic, or, if no oxygen is Supplied for neuronal hyperexcitability. Welch (1997) “Pathogenesis at all, anoxic. In contrast with hypoxia, a more general term 65 of Migraine.” Seminars in Neurobiol. 17: 4. During a denoting a shortage of oxygen (usually a result of lack of spreading depression event, profound ionic perturbations oxygen in the air being breathed), ischemia is an absolute or occur, which include interstitial acidification, extracellular US 9,682,928 B2 15 16 potassium accumulation, and redistribution of sodium and due to trauma, diabetes, infection with herpes Zoster chloride ions to intracellular compartments. In addition, (shingles), HIV/AIDS (peripheral neuropathies), late-stage prolonged glial Swelling occurs as a homeostatic response to cancer, amputation (including mastectomy), carpal tunnel altered ionic extracellular fluid composition, and interstitial syndrome, chronic alcohol use, exposure to radiation, and as neurotransmitter and fatty acid accumulation. Studies have an unintended side-effect of neurotoxic treatment agents, shown that furosemide inhibits regenerative cortical spread Such as certain anti-HIV and chemotherapeutic drugs. ing depression in anaesthetized cats. Read, et al. (1997) In contrast to nociceptive pain, neuropathic pain is fre Cephalagia 17: 826. quently described as “burning,” “electric.” “tingling,” or Drug therapy is tailored to the severity and frequency of “shooting in nature. It is often characterized by chronic migraine headaches. For occasional attacks, acute treatment 10 allodynia (pain resulting from a stimulus that does not may be indicated, but for attacks occurring two or more ordinarily elicit a painful response, such as light touch) and times per month, or when attacks greatly impact the patients hyperalgesia (increased sensitivity to a normally painful daily life, prophylactic therapy may be indicated. The side stimulus), and may persist for months or years beyond the effects of acute and prophylactic treatment agents including apparent healing of any damaged tissues. serotonin acting agents, beta-blockers, tricyclic antidepres 15 Taxol-induced peripheral neuropathy model (TIPN) is an sants, anticonvulsants, and botulinum toxin type Ainjections art-accepted animal model of neuropathic pain. Cavaletti, et can limit their use. al. (May 1995) “Experimental peripheral neuropathy GABA modulates nociceptive input to the trigeminocer induced in adult rats by repeated intraperitoneal administra vical complex mainly through GABA receptors. Therefore tion of taxol.” Exp Neurol. 133(1): 64-72. Injection of GABA receptors may provide a target for the development Taxol.R intraperitoneally to female Wistar rats induced a of new therapeutic agents for both acute and prophylactic peripheral neuropathy that resembles neuropathy in humans. treatment of headaches including migraines. Storer, et al. Id. at pages 64; 69. An additional animal model of neuro (2001) “GABA receptors modulate trigeminovascular noci pathic pain comprises the single or five intraperitoneal ceptive neurotransmission in the trigeminocervical com administration of Taxol.R (32 mg/kg) to male Sprague plex.' BrJ Pharmacol. 134(4): 896-904. 25 Dawley rats. Authier, et al. (Dec. 29, 2000) “Description of Nociceptive Pain a short-term Taxol-induced nociceptive neuropathy in rats.” Nociceptive pain occurs in response to the activation of a Brain Res. 887(2): 239-49. specific Subset of peripheral sensory neurons, the nocicep In a spinal cord injury (SCI) model of neuropathic pain, tors. It is generally acute (with the exception of arthritic bumetanide, a NKCC1 antagonist, showed an analgesic pain), self-limiting and serves a protective biological func 30 effect suggesting that normal or elevated NKCC1 activity tion by acting as a warning of on-going tissue damage. It is plays a role in the development and maintenance of SCI typically well localized and often has an aching or throbbing induced neuropathic pain. Cramer, et al. (2008) “The role of quality. Examples of include post-operative pain, sprains, cation-dependent chloride transporters in neuropathic pain bone fractures, burns, bumps, bruises, inflammation (from following spinal cord injury. Molecular Pain 4:36. an infection or arthritic disorder), obstructions, and myofas 35 Neuropathic pain is difficult to treat. Analgesic drugs that cial pain. Nociceptors are the nerves that sense and respond are effective against nociceptive pain (e.g., opioid narcotics to parts of the body that Suffer from damage. They signal and non-steroidal anti-inflammatory drugs) are rarely effec tissue irritation, impending injury, or actual injury. When tive against neuropathic pain. Similarly, drugs that have activated, they transmit pain signals (via the peripheral activity in neuropathic pain are not usually effective against nerves as well as the spinal cord) to the brain. The pain is 40 nociceptive pain. The standard drugs that have been used to typically well localized, constant, and often with an aching treat neuropathic pain appear to often act selectively to or throbbing quality. Visceral pain is the Subtype of nocice relieve certain symptoms but not others in a given patient ptive pain that involves the internal organs. It tends to be (e.g., relief of allodynia, but not hyperalgesia). Bennett episodic and poorly localized. (1998) Hosp. Pract. (Off Ed). 33: 95-98. Treatment agents Nociceptive pain is usually treated with opioids and/or 45 typically employed in the management of neuropathic pain non-steroidal anti-inflammatory drugs (NSAIDS) but due to include tricylic antidepressants (e.g., amitriptyline, imip low efficacy, unacceptable and even severe side effects, and ramine, desimipramine, and clomipramine), Systemic local addiction potential, their use can be limited. GABA recep anesthetics, and anti-epileptic drugs (AED) (e.g., phenytoin, tors are a target for therapeutics to treat nociceptive pain. carbamazepine, Valproic acid, clonazepam, gabapentin, and Hara, et al. (2004) “The Interaction Between Gamma 50 pregabalin (LYRICAR)). See Lowther (September/October Aminobutyric Acid Agonists and Diltiazem in Visceral 2005) “Pharmacotherapy Update from the Department of Antinociception in Rats' Anesth Analg 98: 1380-1384 Pharmacy” Vol. VIII, No. 5. Common side effects include reports that the combination of GABA agonists and L-type over-sedation, dizziness, loss of memory and liver damage. calcium channel blockers may be used to reduce visceral Further, although traditionally not considered useful for the pain. However, GABA agonists are known to have side 55 treatment of neuropathic pain, recent studies from geneti effects, including sedation, dizziness, euphoria, nausea, and cally modified mice indicate that agents targeting only a blurred vision. Therefore a great need exists for nociceptive subset of benzodiazepine (GABA) receptors may provide pain therapeutics. pronounced antihyperalgesic activity against inflammatory Neuropathic Pain and neuropathic pain. Zeilhofer, et al. (2009) “Subtype Neuropathic pain and nociceptive pain differ in their 60 selective GABA receptor mimetics—novel antihyperalge etiology, pathophysiology, diagnosis, and treatment. Neuro sic agents?” J Mol Med 87:465-469. Therefore a great need pathic pain is a common type of chronic, non-malignant exists for improved neuropathic pain therapeutics. pain, which is the result of an injury or malfunction in the Neurotoxicity peripheral or central nervous system and serves no protec A variety of chemical and biological agents, as well as tive biological function. It is estimated to affect more than 65 Some infectious agents, have neurotoxic effects. A common 1.6 million people in the U.S. population. Neuropathic pain example is the pathophysiological effect of acute ethanol has many different etiologies, and may occur, for example, ingestion. Episodic ethanol intoxications and withdrawals, US 9,682,928 B2 17 18 characteristic of binge alcoholism, result in brain damage. However, the somata of putative horizontal cells displayed Animal models designed to mimic the effects of alcohol in only NKCC immunoreactivity and many bipolar cells were the human have demonstrated that a single dose of ethanol only immunopositive for KCC2. In the outer retina, appli given for 5-10 Successive days results in neurodegeneration cation of bumetanide, a specific inhibitor of NKCC activity, in the entorhinal cortex, dentate gyrus and olfactory bulbs, 5 (1) increased the steady-state extracellular concentration of accompanied by cerebrocortical edema and electrolyte (Na K" (K'I()) and enhanced the light-induced decrease in the and K) accumulation. As with other neurodegenerative K" (), (2) increased the spIII photoreceptor-dependent conditions, research has focused primarily on synaptically component of the ERG, and (3) reduced the extracellular based excitotoxic events involving excessive glutamatergic space Volume. In contrast, in the outer retina, application of 10 furosemide, a specific inhibitor of KCC activity, decreased activity, increased intracellular calcium and decreased sPIII and the light-induced reduction in K" (), but had little y-aminobutyric acid. A great need exists for improved neu effect on steady-state K(). In the inner retina, bumetanide rotoxicity therapeutics. increased the Sustained component of the light-induced Postherpetic Neuralgia increase in K(). These findings thus indicate that NKCC Postherpetic neuralgia (e.g., shingles, herpes Zoster) is a 15 and KCC2 control the K" () and extracellular space vol painful condition affecting the nerve fibers and skin. Pos ume in the retina in addition to regulating GABA- and therpetic neuralgia is a complication of shingles, a second glycine-mediated synaptic transmission. In addition, the outbreak of the varicella-Zoster virus, which initially causes anatomical and electrophysiological results together Suggest chickenpox. During an initial infection of chickenpox, some that all of the major neuronal types in the retina are influ of the virus remains in the body, lying dormant inside nerve enced by chloride cotransporter activity. Dmitriev, et al. cells. Years later, the virus may reactivate, causing shingles. (July-August 2007) “Multiple functions of cation-chloride Once reactivated, the virus travels along nerve fibers causing cotransporters in the fish retina. Vis Neurosci 24(4): 635 pain. When the virus reaches the skin, it produces a rash and 45. blisters. A case of shingles (herpes Zoster) usually heals The bumetanide-sensitive NaK2Cl cotransporter within a month. Some patients continue to feel pain long (NKCC) also clearly contributes to the Cl uptake into the after the rash and blisters heal—a type of pain called 25 pigmented epithelium (PE). This work reinforces the general postherpetic neuralgia. A variety of treatments for posther consensus that active secretion of Cl is the major driving petic neuralgia exist, although some do not experience force of aqueous humor formation in mammalian eye and complete relief from pain. further substantiates the existence of species differences in Postherpetic neuralgia results when nerve fibers are dam the mechanism that accomplishes transepithelial Cl trans aged during an outbreak of shingles. These damaged nerves 30 port. Kong, et al. (December 2006) “Chloride secretion by Suffer causing chronic, often excruciating pain that may porcine ciliary epithelium: New insight into species simi persist for months—or even years—in the area where larities and differences in aqueous humor formation.” Invest shingles first occurred. This complication of shingles occurs Ophthalmol Vis Sci. 47(12): 5428-36. much more frequently in older adults. About 50 percent of Additionally, cation-chloride cotransporters are involved adults older than 60 experience postherpetic neuralgia after in retinal function by mediating neural computation in the shingles, whereas only 10 percent of all people with shingles 35 retina. The directional responses of DS ganglion cells are do. The symptoms of postherpetic neuralgia are generally mediated in part by the directional release of gamma limited to the area of the skin where the shingles outbreak aminobutyric acid from starburst dendrites and that the first occurred including sharp and jabbing, burning, or deep asymmetric distribution of two cotransporters (K'Cl and aching pain; extreme sensitivity to touch and tempera cotransporter and NaK"Cl cotransporter) along starburst ture change; itching and numbness; and headaches. In rare 40 cell dendrites mediates direction selectivity. Gavrikov, et al. cases, patients might also experience muscle weakness or (Dec. 23, 2003) “Cation-chloride cotransporters mediate paralysis—if the nerves involved also control muscle move neural computation in the retina.” Proc Natl AcadSci USA ment. A great need exists for improved postherpetic neural 100(26): 16047-52: gia therapeutics. Further, the function of retina depends on cation chloride Ocular Diseases (e.g., Vision Disorders, Ophthalmic Dis 45 transporters regulating GABA. In particular, different cation eases) chloride cotransporters in retinal neurons allow for opposite It is estimated that the lifetime costs for all people with responses to GABA. Thus, in the retina, the opposite effects vision impairment who were born in 2000 will total S2.5 of GABA on different cell types and on different cellular billion (2003 dollars). See generally, Centers for Disease regions are probably primarily determined by the differential Control and Prevention, Economic Costs Associated with 50 targeting of these two chloride transporters. See e.g., Bar Mental Retardation, Cerebral Palsy, Hearing Loss, & Vision bour, et al. (May 1991) “Electrogenic uptake of glutamate Impairment, United States, 2003, MMWR 2004: 53:57-9. and aspartate into glial cells isolated from the salamander These costs include both direct and indirect costs. Direct (Ambystoma) retina.” J. Physiol. 436: 169-193; Keller, et al. medical costs, such as doctor visits, prescription drugs, and (January 1988) “Regulation of intracellular pH in cultured inpatient hospital stays, make up 6% of these costs. Direct bovine retinal pigment epithelial cells. Pflugers Arch. 411 nonmedical expenses. Such as home modifications and spe 55 (1): 47-52; and Vardi, et al. (Oct. 15, 2000) “Evidence that cial education, make up 16% of the costs. Indirect costs, different cation chloride cotransporters in retinal neurons which include the value of lost wages when a person dies allow opposite responses to GABA.” Journal of Neurosci early, cannot work, or is, limited in the amount or type of ence 20(20): 7657-63. See also, Basu, et al. (November work he or she can do, make up 77% of the costs. These 1998) “Proton-driven dipeptide uptake in primary cultured estimates do not include other expenses, such as hospital 60 rabbit conjunctival epithelial cells.” Invest Ophthalmol Vis outpatient visits, emergency department visits, and family Sci. 39(12): 2365-73; Cia, et al. (March 2005) Voltage-gated out-of-pocket expenses. The actual economic costs of vision channels and calcium homeostasis in mammalian rod pho impairment are, therefore, even higher than what is gener toreceptors. J Neurophysiol. 93(3): 1468-75; Do, et al. (June ally reported. U.S. Pat. No. 7.251,528. 2006) “Swelling-activated Cl channels support Cl secre Both NKCC and KCC2 are expressed in the outer and 65 tion by bovine ciliary epithelium.” Invest Ophthalmol Vis inner plexiform layers and colocalized in many-putative Sci. 47(6): 2576-82; Hunt, et al. (November 2005) “Aber amacrine cells and in cells of the ganglion cell layer. rant retinal projections in congenitally deaf mice: how are US 9,682,928 B2 19 20 phenotypic characteristics specified in development and peutic treatments for Parkinson's disease. Kleppner and evolution?” Anat RecA Discov Mol Cell Evol Biol. 287(1): Tobin (2001) “GABA signaling: therapeutic targets for epi 1051-66; MacLeish and Nurse (July 2007) “Ion channel lepsy, Parkinson's disease and Huntington's disease.” compartments in photoreceptors: evidence from salamander Expert Opin. Ther. Targets. 5(2):219-39. rods with intact- and ablated terminals. J Neurophysiol. Schizophrenia 98(1): 86-95; Mito, et al. (March 1993) “Calcium-dependent Schizophrenia is a chronic, severe, and disabling brain regulation of cation transport in cultured human nonpig disorder that affects about 1.1 percent of the U.S. population mented ciliary epithelial cells.” Am J Physiol. 264(3 Pt 1): age 18 and older in a given year. People with Schizophrenia C519-26: Moody (1984) “Effects of intracellular H" on the sometimes hear voices others do not hear, believe that others electrical properties of excitable cells.” Annu Rev Neurosci 10 are broadcasting their thoughts to the world, or become 7: 257-78; Mroz and Lechene (November 1993) “Extracel convinced that others are plotting to harm them. These lular N-methyl-D-glucamine leads to loss of hair-cell experiences can make them fearful and withdrawn and cause sodium, potassium, and chloride.” Hear Res. 7002): 146-50; difficulties when they try to have relationships with others. Schnetkamp (May 8, 1980) “Ion selectivity of the cation National Institute of Mental Health “Schizophrenia' website transport system of isolated intact cattle rod outer segments: 15 (2008). evidence for a direct communication between the rod plasma Symptoms usually develop in men in their late teens or membrane and the rod disk membranes. Biochim Biophys early twenties and women in the twenties and thirties, but in Acta. 598(1): 66-90; and Uhl and Desel (August 1989) rare cases, can appear in childhood. They can include “Optical probes of intradiskal processes in rod photorecep hallucinations, delusions, disordered thinking, movement tors. II: Light-scattering study of ATP-dependent light reac disorders, flat affect, social withdrawal, and cognitive defi tions.” J Photochem Photobiol B. 3(4): 549-64. cits. No cause of Schizophrenia has been determined nor is Accordingly, a number of vision-threatening disorders of there any curative therapy; however, antipsychotics are used the eye presently do not have any effective therapies. One in the treatment of symptoms. National Institute of Mental major problem in treatment of Such diseases is the inability Health “Schizophrenia” website (2008). to deliver therapeutic agents into the eye and maintain them Further, schizophrenia is associated with both decreased there at therapeutically effective concentrations. Therefore a 25 numbers and abnormalities in the distribution of GABAergic great need exists for therapeutics to treat ocular diseases. neurons in the cortex, particularly in the cortical laminae. Parkinson's Disease Kaplan & Sadock's Comprehensive Textbook of Psychiatry Parkinson's disease (PD) belongs to a group of conditions (7' Ed) (2008). In the postmortem studies of schizophrenics, called motor system disorders, which result from the loss of antipsychotic naive Schizophrenics, and non Schizophrenic dopamine-producing brain cells. The four primary symp 30 controls, show a significant decrease in the number of toms of PD are tremor, or trembling in hands, arms, legs, GABA containing inter neurons, and a lessened amount of jaw, and face; rigidity, or stiffness of the limbs and trunk; GABA production within these inter neurons in both of the bradykinesia, or slowness of movement; and postural insta schizophrenic groups. Nestler (1997) “Schizophrenia. An bility, or impaired balance and coordination. As these symp emerging pathophysiology. Nature 385(66.17): 578-9. There toms become more pronounced, patients may have difficulty fore therapeutic agents that target the GABA system may be walking, talking, or completing other simple tasks. PD 35 useful in treating schizophrenia. usually affects people over the age of 50. Early symptoms of Tinnitus PD are subtle and occur gradually. Other symptoms may Tinnitus is the perception of Sound within the human ear include depression and other emotional changes; difficulty in in the absence of corresponding external Sound. Tinnitus is Swallowing, chewing, and speaking, urinary problems or not a disease but a symptom resulting from a range of constipation: skin problems; and sleep disruptions. NINDS 40 underlying causes that can include ear infections, foreign Parkinson's Disease Information Page (Sep. 23, 2009). objects or wax in the ear, nose allergies that prevent (or At present, there is no cure for PD, but a variety of induce) fluid drain and cause wax build-up, and injury from medications provide dramatic relief from the symptoms. loud noises. Tinnitus can also be caused by hearing impair Usually, patients are given levodopa combined with carbi ment and as a side-effect of Some medications. Some cases dopa. Carbidopa delays the conversion of levodopa into 45 of tinnitus are medically unexplained. dopamine until it reaches the brain. Nerve cells can use Tinnitus can be perceived in one or both ears or in the levodopa to make dopamine and replenish the brain's dwin head. It is usually described as a ringing noise, but in some dling Supply. Although levodopa-helps at least three-quar patients it takes the form of a high pitched whining, buZZing, ters of parkinsonian cases, not all symptoms respond equally hissing, Screaming, humming, singing or whistling Sound, or to the drug. Bradykinesia and rigidity respond best, while 50 as ticking, clicking, roaring, “crickets' or “tree frogs' or tremor may be only marginally reduced. Problems with "locusts.' tunes, songs, or beeping. It has also been balance and other symptoms may not be alleviated at all. described as a “whooshing sound, as of wind or waves. Anticholinergics may help control tremorand rigidity. Other Tinnitus can be intermittent or it can be continuous in which drugs, such as bromocriptine, pramipexole, and ropinirole, case it can be the cause of great distress. In some individuals, mimic the role of dopamine in the brain, causing the neurons the intensity of tinnitus can be changed by shoulder, head, to react asthey would to dopamine. An antiviral drug; 55 tongue, jaw, or eye movements. To date, no satisfactory amantadine, also appears to reduce symptoms. In May 2006, therapeutics exists for tinnitus. the FDA approved rasagiline (AZILECTR) to be used along Partial deafferentation produces a loss of tonic inhibition with levodopa for patients with advanced PD or as a in the auditory system that may lead to inappropriate neu single-drug treatment for early PD. NINDS Parkinson's roplastic changes eventually expressed as the pathophysiol Disease Information Page (Sep. 23, 2009). 60 ogy of tinnitus. The pathological down-regulation of GABA Parkinson's disease (PD) is a neurodegenerative disorder provides a potential mechanism for this loss of inhibition. that involves disruptions in GABA signaling. GABA is the For example, in an animal model of tinnitus, vigabatrin, a major inhibitory neurotransmitter in the central nervous GABA agonist, completely and reversibly eliminated the system (CNS). PD destroys the input to striatal GABAergic psychophysical evidence of tinnitus. Brozoski. et al. (2007) neurons. Targeting GABA synthesis, degradation, transport, 65 Vigabatrin, a GABA Transaminase Inhibitor, Reversibly or receptors with new therapeutics may control GABA Eliminates Tinnitus in an Animal Model. J Assoc Res signaling, and therefore may be used for improved thera Otolaryngol. 8(1): 105-118. Further, the disruption of the US 9,682,928 B2 21 NKCC1 gene in mice causes hearing loss. Kahle, et al. (2004) Proc. Natl. Acad. Sci. USA 102(46): 16783-16788. I Therefore, therapeutics targeting GABAergic system and/or O RR NKCC1 may be useful in the treatment of tinnitus. Withdrawal Syndrome Withdrawal syndrome is generally associated with abnor mal physical or psychological features that follow the abrupt discontinuation of a drug (e.g., medications, recreational RsRNOS 1-1ch, drugs, and/or alcohol) that has the capability of producing 10 R3 physical dependence. (e.g., alcohol withdrawal syndrome, II nicotine withdrawal syndrome, opioid withdrawal Syn S RR2 drome, benzodiazepine withdrawal syndrome, methadone withdrawal syndrome, SSRI discontinuation syndrome, hydrocodone withdrawal syndrome). Common withdrawal 15 symptoms include Sweating, tremor, vomiting, anxiety, insomnia, and muscle pain. There are different stages of withdrawal. Generally, a person will start to feel worse and RsRNOS 1-1, worse, hit a plateau, and then the symptoms begin to R3 dissipate. However, withdrawal from certain drugs (e.g., III benzodiazepines, alcohol) can be fatal and therefore the O RR2 abrupt discontinuation of any type of drug is not recom N Y mended. O Further, many additions involve compounds which affect the GABAerigic system including but not limited to alcohol 25 and benzodiazepines. Therefore, when a person ceases use RsRNOS of the compound, the GABAergic system is involved in the R3 symptoms of withdrawal syndrome. Nutt and Lingford IV Hughes (2008) "Addiction: the clinical interface.” British S RR2 Journal of Pharmacology 154(2): 397-405. Therefore agents 30 that act on the GABAergic system may provide therapeutics N Y to treat withdrawal syndromes. O Accordingly, there is a continuing need for compositions and methods for treatment and/or prophylaxis of diseases, 35 RsRNOS disorders, and conditions that involve the Na"K"Cl co transporters (e.g., NKCC1 and NKCC2) including but not R3 limited to addictive disorders, anxiety disorders, ascites, V bipolar disorder, cancer, endothelial corneal dystrophy, O RR edema, depression, epilepsy, glaucoma, ischemia, migraine, 40 neuropathic pain, nociceptive neuralgia, ocular diseases, pain, postherpetic neuralgia, and Schizophrenia. Addition ally; there is a continuing need for compositions and meth ods for treatment and/or prophylaxis of diseases, disorders, RsRNOS N and conditions that involve the GABA receptors including 45 but not limited to Alzheimer's Disease, addictive disorders, R3 anxiety disorders, autism, bipolar disorder, depression, epi VI lepsy, Huntington's Disease, insomnia, migraine, neuro S RR pathic pain, nociceptive pain, pain, Parkinson's disease, personality disorders, psychosis, Schizophrenia, seizure dis 50 orders, tinnitus, and withdrawal syndromes.

SUMMARY OF THE INVENTION RsRNOS N R3 The present invention provides compounds according to 55 Formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, or a pharmaceutically acceptable salt, Solvate, tautomer or XXIV. XXV, and/or XXVI described herein, which are hydrate thereof, wherein analogs of bumetanide, furosemide, piretanide, azosemide, 60 R is not present, H, O or S, and torsemide, including derivatives and prodrugs thereof. R is not present, H or when R is O or S, R, is selected The compounds of the present invention antagonize NKCC1 from the group consisting of hydrogen, alkyl, aralkyl, and/or GABA receptors. The compounds of the present aryl, alkylaminodialky, alkylcarbonylaminodialkyl, invention may also be useful in the treatment of conditions alkyloxycarbonylalkyl, alkylcarbonyloxyalkyl, alkylal that involve NKCC1 and/or GABA receptors. 65 dehyde, alkylketoalkyl, alkylamide, alkarylamide, ary Embodiments of the present invention provide com lamide, an alkylammonium group, alkycarboxylic acid, pounds according to formula I, II, III, IV, V and/or VI: alkylheteroaryl, alkylhydroxy, a biocompatible poly US 9,682,928 B2 23 24 mer Such as alkyloxy(polyalkyloxy)alkyhydroxyl, a polyethylene glycol (PEG), polyethylene glycol ester VIII (PEG ester) and a polyethylene glycol ester (PEG R ester), methyloxyalkyl, methyloxyalkaryl, methylithio- X alkyl and methylthioalkaryl, unsubstituted or substi- 5 n tuted, and when R is not present, R. Selected from the group consisting of hydrogen N,N-dialkylamino, N.N- SO 21 dialkarylamino, N,N-diarylamino, N-alkyl-N-alkary- HN1 lamino, N-alkaryl-N-arylamino, N-alkaryl-N-ary- 1. HN CH lamino, unsubstituted or substituted; 10 HN O R is selected from the group consistion of aryl, halo, hydroxyl, alkoxy, and aryloxy, unsubstituted or Substi- 1. tuted; and H3C CH3 R, and Rs are each independently selected from the group is consisting of hydrogen, alkylaminodialkyl, carbonylal- or a pharmaceutically acceptable salt, Solvate, tautomer or kyl, carbonylalkaryl, carbonylaryl, and salts thereof hydrate thereof, wherein Such as Sodium, potassium, calcium, ammonium, tri- R, is not present or selected from the group consisting of alkylarylammonium and tetraalkylammonium salts, hydrogen, alkyloxycarbonylalkyl, alkylaminocarbo with the following provisos in some embodiments: Rs 20 nylalkyl, alkylaminodialkyl, alkylhydroxy, a biocom of formula I is not phenyloxy when R is O and R. R. patible polymer Such as alkyloxy(polyalkyloxy)alkyl and Rs are H. more specifically, in some embodiments, hydroxyl, a polyethylene glycol (PEG), a polyethylene the compound of formula I is not bumetanide; R of glycol ester (PEG ester) and a polyethylene glycol ether f la III is not Cl, when R, is O and R. R. and R (PEG ether), methyloxyalkyl, methyloxyalkaryl, meth ormula 111 1s not ul. When K 1s U and K. K. and Ks ylthioalkyl and methylthioalkaryl, unsubstituted or are H. more specifically, in Some embodiments, the 25 Substituted; and compound of formula III is not furosemide; R of X is a halide such as bromide, chloride, fluoride, iodide formula III is not methyl when R is O. R. is C1, and Ra or an anionic moiety Such as mesylate or tosylate: and Rs are H. more specifically, in some embodiments, alternatively, X is not present and the compound forms the compound of formula III is not furosemide methyl 30 an “inner or Zwitterionic salt (where R, is H. See esther; R of formula V is not phenyloxy when R is O structure VIIa below), with the proviso that, in some and R. R. and Rs are H. more specifically, in some embodiments, R, is always present and X is not embodiments, the compound of formula V is not pire- present and that an acidic proton has been removed tanide. from the nitrogen flanked by the carbonyl and sulfonyl Embodiments of the present invention provide com- moieties. More specifically, in some embodiments, the pounds according to formula VII: compound of formula VIII is not torsemide.

VII VIIIa NN H \ 40 R1 2 N GNN

S. S ne.3 . .Ns 2 "YÖH, 61 O aO HN CH3 RsRNOS R3

50 or a pharmaceutically acceptable salt, Solvate, tautomer or In one aspect, the invention also relates to a compound of hydrate thereof, wherein the formula IX: R. R. and Rs are defined above; and R is selected from the group consisting of alkyloxycar bonylalkyl, alkylaminocarbonylalkyl, alkylaminodi- 55 IX alkyl, alkylhydroxy, a biocompatible polymer Such as R6 alkyloxy(polyalkyloxy)alkylhydroxyl, a polyethylene Y y glycol (PEG), a polyethylene glycol ester (PEG ester) YR. O N and a polyethylene glycol ether (PEG ether), methyl- H oxyalkyl, methyloxyalkaryl, methylthioalkyl and meth- 60 N S. ylthioalkaryl, unsubstituted or substituted, with the proviso that, in some embodiments, R of formula VII H2NN is not Cl, when R. Rs and R are H. more specifically, S in some embodiments, the compound of formula VII is / \, C not aZosemide. 65 Embodiments of the present invention further provide compounds according to formula VIII: or a pharmaceutically acceptable salt thereof, US 9,682,928 B2 25 26 wherein: 4-pyridylmethyl. 2-(4-(2-aminoethyl)piperazyn-1- Y is oxygen, Sulfur, or selenium; yl)ethyl or a Sultamyl group of the formula Z is oxygen, Sulfur, or nitrogen; R is alkyl, alkylene, alkylcyano, alkylhalo, alkaryl, alky lheterocycloalkyl, or alkylaminodialkyl: R is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky lheterocycloalkyl; or ~x. R and R, together with the atom to which they are attached, form a heterocycloalkyl group; provided that: 10 or if Z is oxygen or Sulfur, then R, is not present; if Y is oxygen and Z is sulfur, then R is not methyl; if Y is sulfur and Z is sulfur, then R is not methyl or if Y is oxygen and Z is nitrogen, then Rs and Ro are not alkylaminodialkyl, both ethyl or benzyl; or if Y is oxygen and Z is oxygen, then R is not methyl, if Y is oxygen and Z is nitrogen, then Rs and Ro, ethyl, butyl, methylcyano, unsubstituted benzyl, 15 together with the nitrogen atom to which they are chloromethyl, 2.2.2-trichloroethyl, ethyl-N-mor attached, do not form a piperazin-1-yl group, a pholinyl, N,N-dimethyl-2-ethylamino, N,N-dim 4-methylpiperazin-1-yl group, or a N-morpholinyl ethyl-3-propylamino or (CH)2C=CHCHCH-(E)- group. (CH)C—CHCH ; In still another aspect, the invention relates to a compound if Y is oxygen, Z is nitrogen, and R, is hydrogen, then of the formula XI-XIII: R is not 2-(4-piperazin-1-yl)ethyl 3-pyridylmethyl, unsubstituted benzyl, or n-butyl: if Y is oxygen and Z is nitrogen, then R and R, are not XI both unsubstituted benzyl or ethyl. 25 In another aspect, the invention relates to a compound of the formula X:

XII

35 S.

XIII

45 or a pharmaceutically acceptable salt thereof, wherein: N Y is oxygen, Sulfur, or selenium; R13 Z is oxygen, Sulfur, or nitrogen; 50 Rs is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl, Ro is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky or a pharmaceutically acceptable salt thereof. lheterocycloalkyl; or wherein: Rs and Ro, together with the atom to which they are 55 Y is O, S, or Se: attached, form a heterocycloalkyl group; R is H, OR, SR, wherein R is hydrogen, alkyl, Ro is hydrogen or alkyl; aralkyl, aryl, alkylaminodialkyl, alkylcarbonylamino provided that: dialkyl, alkyloxycarbonylalkyl, alkylcarbonyloxyalkyl, if Z is oxygen or Sulfur, then Ro is not present; alkylaldehyde, alkylketoalkyl, alkylamide, alkarylam if Z is oxygen and Rs is hydrogen, then Rio is not 60 ide, arylamide, an alkylammonium group, alkylcarbox hydrogen or methyl; ylic acid, alkylheteroaryl, alkylhydroxy, a biocompat if Y is oxygen and Z is oxygen, then Rs is not methyl, ible polymer Such as alkyloxy(polyalkyloxy) unsubstituted benzyl, butyl, or methylcyano; alkylhydroxyl, a polyethylene glycol (PEG), a if Y is oxygen, Z is nitrogen, and Ro is hydrogen, then polyethylene glycol ester (PEG ester) and a polyethyl Ra is not hydrogen, 2-(4-methylpiperazin-1-yl)ethyl, 65 ene glycol ether (PEG ether), methyloxyalkyl, methyl 3-(4-methylpiperazin-1-yl)propyl, 2-(N-morpholi oxyalkaryl, methylthioalkyl or methylthioalkaryl, nyl)ethyl, 2-pyridylmethyl, 3-pyridylmethyl, unsubstituted or substituted, or US 9,682,928 B2 27 28 R is N,N-dialkylamino, N,N-dialkarylamino, N,N-dia R is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky rylamino, N-alkyl-N-alkarylamino, N-alkyl-N-ary lheterocycloalkyl; or lamino, or N-alkaryl-N-arylamino, unsubstituted or Ro and R, together with the atom to which they are Substituted; attached, form a heterocycloalkyl group; and R is aryl, halo, hydroxy, alkoxy, or aryloxy, unsubsti tuted or substituted; and R and R are each independently hydrogen, alkyl, Ra and Rs are each independently hydrogen, alkylam alkenyl, alkoxy, alkaryloxyalkyl, or alkaryl. inodialkyl, carbonylalkyl, carbonylalkaryl, or carbo The present invention also provides compounds that are nylaryl. derivatives, including prodrugs thereof, of bumetanide. In still another aspect, the invention relates to a compound 10 Embodiments of the present invention provide a compound of the formula XIV: according to Formula XVI:

XIV 15

Y Z n R17 R19 ul 1. Ns 1n-1\ 18 MV H N \ O O. d) 25

(3-Aminosulfonyl-5-N,N-dibutylamino-4-phenoxy 30 benzoic acid) or a pharmaceutically acceptable salt thereof, wherein: A compound of the formula XVII: Y is oxygen, Sulfur, or selenium; Z is oxygen, Sulfur, or nitrogen; R is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, 35 aryl, alkaryl, alkylheterocycloalkyl, or alkylaminodi N XVII alkyl: HN1 N R7 is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky N / lheterocycloalkyl; or Yi N Re and R7, together with the atom to which they are 40 attached, form a heterocycloalkyl group; and Rs and Ro are each independently hydrogen or alkyl; O provided that if Z is oxygen or Sulfur, then R, is not R6 \, 1n-1\ present. 45 In another aspect, the invention relates to a compound of S-R62 \, O R60 the formula XV:

R20 50 Y Z n or a pharmaceutically acceptable salt thereof, R21 O N wherein: H N S. 55 Y is —(CH), , wherein X is either 0, 1, or 2: R23 Ro is hydrogen or alkyl:

R.1Ns22 R is hydrogen, lower alkyl, lower alkenyl, alkaryl, MV alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky O O C laminoalkyl, heterocycloalkyl, or heteroaryl; and 60 R is hydrogen, lower alkyl, lower alkenyl, alkaryl, or a pharmaceutically acceptable salt thereof, alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky wherein: laminoalkyl, heterocycloalkyl, or heteroaryl, or R and Y is oxygen, Sulfur, or selenium; Re, taken together with the nitrogen atom to which Z is oxygen, Sulfur, or nitrogen; 65 they are attached form a 4-8 member cycle which can Ro is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, be substituted or unsubstituted and can have one or alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl, more heteroatoms. US 9,682,928 B2 29 30 A compound of the formula XVIII: A compound of the formula XX:

XVIII N a XX 4. O R67

10 R VS N 1N1N "DN 1. \ K O R60 R62 O O R60

15 or a pharmaceutically acceptable salt thereof, wherein: or a pharmaceutically acceptable salt thereof, Y is —(CH), , wherein X is either 0, 1, or 2; wherein: Ro is hydrogen or alkyl; Ro is hydrogen, alkyl, aralkyl, or heteroarylalkyl; R is hydrogen, lower alkyl, lower alkenyl, alkaryl, R is hydrogen, lower alkyl, lower alkenyl, alkaryl, alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky 25 alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky laminoalkyl, heterocycloalkyl, or heteroaryl; and laminoalkyl, heterocycloalkyl, or heteroaryl; R is hydrogen, lower alkyl, lower alkenyl, alkaryl, R is hydrogen, lower alkyl, lower alkenyl, alkaryl, alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky laminoalkyl, heterocycloalkyl, or heteroaryl, or R and laminoalkyl, heterocycloalkyl, or heteroaryl, or R and Re, taken together with the nitrogen atom to which 30 they are attached form a 4-8 member cycle which can R, taken together with the nitrogen atom to which be substituted or unsubstituted and can have one or they are attached form a 4-8 member cycle which can be substituted or unsubstituted and can have one or more heteroatoms. more heteroatoms; and A compound of the formula XIX: 35 R7 is hydrogen, hydroxy, or amino. A compound of the formula XXI: O O XIX SR 40 XXI

O R6 \, 1N-1S in 1 V R1 O d) 45

50 or a pharmaceutically acceptable salt thereof, wherein: R is hydrogen, lower alkyl, lower alkenyl, alkaryl, alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky laminoalkyl, heterocycloalkyl, or heteroaryl; 55 or a pharmaceutically acceptable salt thereof, R is hydrogen, lower alkyl, lower alkenyl, alkaryl, wherein: alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky laminoalkyl, heterocycloalkyl, or heteroaryl, or R and Ro is hydrogen, alkyl, aralkyl, or heteroarylalkyl; Resis —SO-alkyl or —N(R)—Ro: R, taken together with the nitrogen atom to which 60 they are attached form a 4-8 member cycle which can Ro is hydrogen, aryl, heteroaryl, cycloalkenyl, be substituted or unsubstituted and can have one or cycloalkyl, heterocycloalkyl, alkylcycloalkenyl, alkyl more heteroatoms; and cycloalkyl, alkylaryl, alkylheteroaryl or alkylheterocy R is alkyl, alkylcyano, aryl, heteroaryl, -CH2—C cloalkyl; and (O)—N(R)-Rs, or —CH2—O—C(O)—R. 65 Ro is hydrogen or Rao and Rio, together with the nitrogen wherein R. Ras, and Rae are each independently atom to which they are attached, form a heterocy alkyl. cloalkyl. US 9,682,928 B2 31 32 A compound of the formula XXII: A compound of the formula XXIV:

XXII XXIV

O R6 \, N 1N1N 10 R6 \, n N1 V 'NN1 V O R72 R60 R1 O () R62 or a pharmaceutically acceptable salt thereof, 15 wherein: Ro is hydrogen, alkyl, aralkyl, or heteroarylalkyl; or a pharmaceutically acceptable salt thereof, R is hydrogen, lower alkyl, lower alkenyl, alkaryl, wherein: alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky R is hydrogen, lower alkyl, lower alkenyl, alkaryl, laminoalkyl, heterocycloalkyl, or heteroaryl; alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky R is hydrogen, lower alkyl, lower alkenyl, alkaryl, laminoalkyl, heterocycloalkyl, or heteroaryl; alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky R is hydrogen, lower alkyl, lower alkenyl, alkaryl, laminoalkyl, heterocycloalkyl, or heteroaryl, or R and alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky R, taken together with the nitrogen atom to which laminoalkyl, heterocycloalkyl, or heteroaryl, or R and 25 R, taken together with the nitrogen atom to which they are attached form a 4-8 member cycle which can they are attached form a 4-8 member cycle which can be substituted or unsubstituted and can have one or be substituted or unsubstituted and can have one or more heteroatoms; more heteroatoms; R7 is hydroxy or —N(R-7)—R; R7, is hydroxy or —N(R)-Rs: R7 is halo, aryl, arylamino, heteroarylamino, heterocy R7s is —N(Rs)—Rs or heterocycloalkyl, cloalkyl, heteroaryl, heteroaryloxy, heterocycloalkoxy, 30 Ro is alkyl, alkaryl, or alkylheteroaryl; or alkylthio; and Rso and Rs are each independently hydrogen, alkyl, R7 and R-7 are each independently alkyl. alkaryl, or alkylheteroaryl; and A compound of the formula XXIII: Rs is hydrogen, alkyl, aryl or Rs and Rs, together with the nitrogen atom to which they are attached, form a 35 heterocycloalkyl. XXIII A compound of the formula XXV:

XXV 40 N R60 21 H

R61 VS 1)N-1N Srir,O / \, R83 45 R62DN1 \,O () or a pharmaceutically acceptable salt thereof, wherein: 50 Ro is hydrogen, alkyl, aralkyl, or heteroarylalkyl; and or a pharmaceutically acceptable salt thereof, Rs is halo, aryl, heteroaryl, arylamino, heteroarylamino, wherein: aryloxy, heteroaryloxy, arylthio, or heteroarylthio. Ro is hydrogen, alkyl, aralkyl, or heteroarylalkyl; A compound of the formula XXV: R is hydrogen, lower alkyl, lower alkenyl, alkaryl, alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky 55 laminoalkyl, heterocycloalkyl, or heteroaryl; XXVI R is hydrogen, lower alkyl, lower alkenyl, alkaryl, Y Zs alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky na laminoalkyl, heterocycloalkyl, or heteroaryl, or R and R85 Re, taken together with the nitrogen atom to which 60 Rs6 they are attached form a 4-8 member cycle which can be substituted or unsubstituted and can have one or s more heteroatoms; -N R-7s is alkyl, aryl, cycloalkenyl, cycloalkyl, heteroaryl, R61 AVS heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, or 65 O O R72 dialkylaminoalkyl; and R7 is hydrogen, alkyl, aralkyl, or heteroarylalkyl. US 9,682,928 B2 33 34 or a pharmaceutically acceptable salt thereof, wherein: wherein: R is selected from the group consisting of aryl, halo, Y is oxygen, Sulfur, or selenium; hydroxy, alkoxy, and aryloxy, unsubstituted or Substi Z is oxygen, Sulfur, or nitrogen; tuted; R is hydrogen, lower alkyl, lower alkenyl, alkaryl, Ra and Rs are each independently selected from the group alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky consisting of hydrogen, alkylaminodialkyl, carbonylal laminoalkyl, heterocycloalkyl, or heteroaryl; kyl, carbonylalkaryl, carbonylaryl and salts thereof; R is hydrogen, lower alkyl, lower alkenyl, alkaryl, and alkylheteroaryl, aryl, cycloalkenyl, cycloalkyl, dialky R is selected from the group consisting of alkyloxycar laminoalkyl, heterocycloalkyl, or heteroaryl, or R and 10 bonylalkyl, alkylaminocarbonylalkyl, alkylaminodi R, taken together with the nitrogen atom to which alkyl, alkylhydroxy, a biocompatible polymer Such as they are attached form a 4-8 member cycle which can alkyloxy(polyalkyloxy)alkylhydroxyl, a polyethylene be substituted or unsubstituted and can have one or glycol (PEG), a polyethylene glycol ester (PEG ester) more heteroatoms; and a polyethylene glycol ether (PEG ether), methyl R7 is halo, aryl, arylamino, heteroarylamino, heterocy 15 oxyalkyl, methyloxyalkaryl, methylthioalkyl and meth cloalkyl, heteroaryl, heteroaryloxy, heterocycloalkoxy, ylthioalkaryl, unsubstituted or substituted. or alkythio; In yet another aspect, the invention relates to a method for Rs is alkenyl, alkyl, alkylcyano, alkylhalo, alkaryl, alky treating addictive disorders, Alzheimer's Disease, anxiety lheteroaryl, alkylcycloalkenyl, alkylcycloalkyl, alkyl disorders, ascites, autism, bipolar disorder, cancer, depres heterocycloalkyl, or alkylaminodialkyl; Sion, edema, endothelial corneal dystrophy, epilepsy, glau Rss is hydrogen, alkenyl, alkyl, alkylcyano, alkylhalo, coma, Huntington's Disease, insomnia, ischemia, migraine alkaryl, alkylheteroaryl, alkylcycloalkenyl, alkylcy with aura, migraine, neuropathic pain, nociceptive neural cloalkyl, alkylheterocycloalkyl, or alkylaminodialkyl; gia, nociceptive pain, ocular diseases, pain, Parkinson's Rs is —N(Rs.)—Rs, cycloalkenyl, cycloalkyl, or het 25 disease, personality disorders, postherpetic neuralgia, psy erocycloalkyl, and chosis, Schizophrenia, seizure disorders, tinnitus, or with Rs7 and Rss are each independently hydrogen, alkyl, drawal syndromes comprising administering an effective alkaryl, or alkylheteroaryl, or Rs7 and Rss, together with the nitrogen atom to which they are attached, form amount of a compound of the formula VIII: a heterocycloalkyl, provided that if Z is oxygen or 30 Sulfur, Rss is not present. VIII Methods of Use R Embodiments of the present invention provide methods of xe N making the compounds including analogs of bumetanide, GN furosemide, piretanide, aZosemide, and torsemide, including 35 derivatives and prodrugs thereof described herein and fur H H ther provide intermediate compounds formed through the H3C N Ns 2 synthetic methods described herein to provide the compound Srir, of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, ÖH, 6 O O HN CH3 XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, 40 XXIV. XXV, and/or XXVI. In still another aspect, the invention relates to a method for treating addictive disorders, Alzheimer's Disease, anxi ety disorders, ascites, autism, bipolar disorder, cancer, or a pharmaceutically acceptable salt or a Zwitterion depression, edema, endothelial corneal dystrophy, epilepsy, 45 thereof, wherein: glaucoma, Huntington's Disease, insomnia, ischemia, R, is selected from the group consisting of hydrogen, migraine with aura, migraine, neuropathic pain, nociceptive alkyloxycarbonylalkyl, alkylaminocarbonylalkyl, alky neuralgia, nociceptive pain, ocular diseases, pain, Parkin laminodialkyl, alkylhydroxy, a biocompatible polymer son's disease, personality disorders, postherpetic neuralgia, Such as allyloxy(polyalkyloxy)alkylhydroxyl, a poly psychosis, Schizophrenia, seizure disorders, tinnitus, or 50 ethylene glycol (PEG), a polyethylene glycol ester withdrawal syndromes comprising administering an effec (PEG ester) and a polyethylene glycol ether (PEG tive amount of a compound of the formula VII: ether), methyloxyalkyl, methyloxyalkaryl, methylthio alkyl and methylthioalkaryl, unsubstituted or substi tuted; and VII 55 X is a halide or an anionic moiety; or NN X is not present. N In still another aspect, the invention relates to a method R1 2 S N for treating addictive disorders, Alzheimer's Disease, anxi H ety disorders, ascites, autism, bipolar disorder, cancer, N S. 60 depression, edema, endothelial corneal dystrophy, epilepsy, glaucoma, Huntington's Disease, insomnia, ischemia, N migraine with aura, migraine, neuropathic pain, nociceptive R1 Ns MV neuralgia, nociceptive pain, ocular diseases, pain, Parkin O O R3 son's disease, personality disorders, postherpetic neuralgia, 65 psychosis, Schizophrenia, seizure disorders, tinnitus, or withdrawal syndromes comprising administering an effec or a pharmaceutically acceptable salt thereof, tive amount of a compound of the formula IX: US 9,682,928 B2 35 36

IX R6 IX Y Z YR. (1 N Y Z H YR, 91 N N S. H N S.

HN 10 /\,M \, C

or a pharmaceutically acceptable salt thereof, 15 or a pharmaceutically acceptable salt thereof, wherein: wherein: Y is oxygen, Sulfur, or selenium; Y is oxygen, Sulfur, or selenium; Z is oxygen, Sulfur, or nitrogen; Z is oxygen, Sulfur, or nitrogen; R is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, alkaryl, alkylheterocycloalkyl or alkylaminodialkyl; R is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, and alkaryl, alkylheterocycloalkyl or alkylaminodialkyl; and R, is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky lheterocycloalkyl; or R, is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky lheterocycloalkyl; or R and R-7, together with the atom to which they are 25 attached, form a heterocycloalkyl group; provided that R and R-7, together with the atom to which they are if Z is oxygen or Sulfur, then R, is not present. attached, form a heterocycloalkyl group; provided that In a preferred mode, the compound administered for this if Z is oxygen or sulfur, then R, is not present. In still another aspect, the invention relates to a method of method meets the provisos recited above. 30 In another aspect, the invention relates to a method of inhibiting the NKCC2 (CCC2, BSC1) isoform of the Na"K" inhibiting the Na"K"Cl cotransporters comprising admin Cl cotransporters comprising administering an effective istering a therapeutically effective amount of a compound of amount of a compound of the formula IX: the formula IX: 35 IX IX R6 Y Z NR, 91 N 40 H N S.

HN 45 /\, C or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, wherein: 50 wherein: Y is oxygen, Sulfur, or selenium; Y is oxygen, Sulfur, or selenium; Z is oxygen, Sulfur, or nitrogen; Z is oxygen, Sulfur, or nitrogen; R is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, R is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, alkaryl, alkylheterocycloalkyl or alkylaminodialkyl; alkaryl, alkylheterocycloalkyl or alkylaminodialkyl; 55 and and R is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky R, is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky lheterocycloalkyl; or lheterocycloalkyl; or R and R, together with the atom to which they are R and R-7, together with the atom to which they are attached, form a heterocycloalkyl group; provided that 60 attached, form a heterocycloalkyl group; provided that if Z is oxygen or sulfur, then R, is not present. if Z is oxygen or Sulfur, then R, is not present. In yet another aspect, the invention relates to a method of In another aspect, the invention relates to a method of inhibiting the NKCC 1 (CCC1, BSC2) isoform of the inhibiting both the NKCC1 (CCC1, BSC2) isoform and the Na"K"C1 cotransporters comprising administering a thera- 65 NKCC2 (CCC2, BSC1) isoform of the Na"K"Cl cotrans peutically effective amount of a compound of the formula porters comprising administering a therapeutically effective IX: amount of a compound of the formula IX: US 9,682,928 B2 37 38 provided that if Z is oxygen or Sulfur, then Ro is not IX present. R6 In a preferred mode, the compound administered for this Y Z method meets the provisos recited above. YR, O N In still another aspect, the invention relates to a method of H inhibiting the Na"K"Cl cotransporters comprising admin N S. istering a therapeutically effective amount of a compound of the formula X:

10 X Rs or a pharmaceutically acceptable salt thereof, Y Z YR, wherein: 15 Y is oxygen, Sulfur, or selenium; Z is oxygen, Sulfur, or nitrogen; R is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, alkaryl, alkylheterocycloalkyl or alkylaminodialkyl; HN Ns N 1N1N and MV R is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky O O. d) R10 lheterocycloalkyl; or R and R-7, together with the atom to which they are attached, form a heterocycloalkyl group; provided that if Z is oxygen or sulfur, then R, is not present. 25 In yet another aspect, the invention relates to a method for treating addictive disorders, Alzheimer's Disease, anxiety or a pharmaceutically acceptable salt thereof, disorders, ascites, autism, bipolar disorder, cancer, depres wherein: Sion, edema, endothelial corneal dystrophy, epilepsy, glau 30 Y is oxygen, Sulfur, or selenium; coma, Huntington's Disease, insomnia, ischemia, migraine Z is oxygen, Sulfur, or nitrogen; with aura, migraine, neuropathic pain, nociceptive neural Rs is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, gia, nociceptive pain, ocular diseases, pain, Parkinson's alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl; disease, personality disorders, postherpetic neuralgia, psy Ro is hydrogen, alkyl, alkaryl, dialkylaminoalkyl or alky chosis, Schizophrenia, seizure disorders, tinnitus, or with 35 lheterocycloalkyl; or drawal syndromes comprising administering an effective Rs and R, together with the atom to which they are amount of a compound of the formula X: attached, form a heterocycloalkyl group; and Ro is hydrogen or alkyl; provided that if Z is oxygen or sulfur, then R is not 40 present. In yet another aspect, the invention relates to a method of inhibiting the NKCC 1 (CCC1, BSC2) isoform of the Na"K"Cl cotransporters comprising administering a thera 45 peutically effective amount of a compound of the formula X:

X Rs 50 Y Z YR,

55 HN 1\-1N MV or a pharmaceutically acceptable salt thereof, O O O R10 wherein: Y is oxygen, Sulfur, or selenium; Z is oxygen, Sulfur, or nitrogen; Rs is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, 60 alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl, Ro is hydrogen, alkyl, alkaryl, dialkylaminoalkyl or alky lheterocycloalkyl; or or a pharmaceutically acceptable salt thereof, Rs and R, together with the atom to which they are 65 wherein: attached, form a heterocycloalkyl group; and Y is oxygen, Sulfur, or selenium; Ro is hydrogen or alkyl; Z is oxygen, Sulfur, or nitrogen; US 9,682,928 B2 39 40 Rs is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, or a pharmaceutically acceptable salt thereof, alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl, wherein: Ro is hydrogen, alkyl, alkaryl, dialkylaminoalkyl or alky Y is oxygen, Sulfur, or selenium; lheterocycloalkyl; or Z is oxygen, Sulfur, or nitrogen; Rs and Ro, together with the atom to which they are 5 attached, form a heterocycloalkyl group; and Rs is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, Ro is hydrogen or alkyl; alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl; provided that if Z is oxygen or sulfur, then R is not Ro is hydrogen, alkyl, alkaryl, dialkylaminoalkyl or alky present. lheterocycloalkyl; or In another aspect, the invention relates to a method of Rs and Ro, together with the atom to which they are inhibiting the NKCC2 (CCC2, BSC1) isoform of the Na"K" 10 attached, form a heterocycloalkyl group; and Cl cotransporters comprising administering a therapeuti Ro is hydrogen or alkyl: cally effective amount of a compound of the formula X: provided that if Z is oxygen or Sulfur, then Ro is not present. In still another aspect, the invention relates to a method x 15 for treating addictive disorders, Alzheimer's Disease, anxi Rs ety disorders, ascites, autism, bipolar disorder, cancer, Y } n depression, edema, endothelial corneal dystrophy, epilepsy, Ro glaucoma, Huntington's Disease, insomnia, ischemia, migraine with aura, migraine, neuropathic pain, nociceptive 2O neuralgia, nociceptive pain, ocular diseases, pain, Parkin son's disease, personality disorders, postherpetic neuralgia, HN N 1Y-1N psychosis, Schizophrenia, seizure disorders, tinnitus, or MV withdrawal syndromes comprising administering an effec O O. d) R10 25 tive amount of a compound of the formulae XI-XIII:

30 or a pharmaceutically acceptable salt thereof, wherein: Y is oxygen, Sulfur, or selenium; Z is oxygen, Sulfur, or nitrogen; 35 Rs is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl, XII R is hydrogen, alkyl, alkaryl, dialkylaminoalkyl or alky lheterocycloalkyl; or Rs and R, together with the atom to which they are S. attached, form a heterocycloalkyl group; and 40 Ro is hydrogen or alkyl: provided that if Z is oxygen or Sulfur, then Ro is not present. In still another aspect, the invention relates to a method of inhibiting both the NKCC1 (CCC1, BSC2) isoform and the 45 XIII NKCC2 (CCC2, BSC1) isoform of the Na"K"CI cotrans porters comprising administering a therapeutically effective amount of a compound of the formula X:

50 X Rs N Y } R13 n Ro 55 or a pharmaceutically acceptable salt thereof. wherein: Y is O, S, or Se: HN Ns N 1N1N R is H, OR2, SR, wherein R is hydrogen, alkyl, M \ 60 aralkyl, aryl, alkylaminodialkyl, alkylcarbonylamino O O. d) R10 dialkyl, alkyloxycarbonylalkyl, alkylcarbonyloxyalkyl, alkylaldehyde, alkylketoalkyl, alkylamide, alkarylam ide, arylamide, an alkylammonium group, alkylcarbox ylic acid, alkylheteroaryl, alkylhydroxy, a biocompat 65 ible polymer Such as alkyloxy(polyalkyloxy) alkylhydroxyl, a polyethylene glycol (PEG), a polyethylene glycol ester (PEG ester) and a polyethyl US 9,682,928 B2 41 42 ene glycol ether (PEG ether), methyloxyalkyl, methyl oxyalkaryl, methylthioalkyl or methylthioalkaryl, XV unsubstituted or substituted, or R20 R is N,N-dialkylamino, N,N-dialkarylamino, N,N-dia Y. Z rylamino, N-alkyl-N-alkarylamino, N-alkyl-N-ary n H lamino, or N-alkaryl-N-arylamino, unsubstituted or N S. Substituted; R23 R is aryl, halo, hydroxy, alkoxy, or aryloxy, unsubsti tuted or substituted; and 10 Rs6 N. Ra and Rs are each independently hydrogen, alkylam 22 MV inodialkyl, carbonylalkyl, carbonylalkaryl, or carbo O O C nylaryl. In still another aspect, the invention relates to a method or a pharmaceutically acceptable salt thereof, for treating addictive disorders, Alzheimer's Disease, anxi 15 wherein: ety disorders, ascites, autism, bipolar disorder, cancer, depression, edema, endothelial corneal dystrophy, epilepsy, Y is oxygen, Sulfur, or selenium; glaucoma, Huntington's Disease, insomnia, ischemia, Z is oxygen, Sulfur, or nitrogen; migraine with aura, migraine, neuropathic pain, nociceptive Ro is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, neuralgia, nociceptive pain, ocular diseases, pain, Parkin alkaryl, alkylheterocycloalkyl, or alkylaminodialkyl; son's disease, personality disorders, postherpetic neuralgia, R is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky psychosis, Schizophrenia, seizure disorders, tinnitus, or lheterocycloalkyl; or withdrawal syndromes comprising administering an effec Ro and R, together with the atom to which they are tive amount of a compound of the formula XIV: attached, form a heterocycloalkyl group; and R and R2 are each independently hydrogen, alkyl, 25 alkenyl, alkoxy, alkaryloxyalkyl, or alkaryl. XIV The present invention also provides compounds that are R16 derivatives, including prodrugs thereof, of bumetanide. Y Z Embodiments of the present invention provide a compound n R17 30 according to Formula XVI:

R19

1 n 1N1N MV H 35 O O. d)

or a pharmaceutically acceptable salt thereof, (3-Aminosulfonyl-5-N,N-dibutylamino-4- wherein: phenoxybenzoic acid) Y is oxygen, Sulfur, or selenium; 45 Z is oxygen, Sulfur, or nitrogen; R is hydrogen, alkyl, alkylene, alkylcyano, alkylhalo, The present invention provides methods for treating a aryl, alkaryl, alkylheterocycloalkyl, or alkylaminodi condition selected from the group consisting of addictive alkyl: disorders, Alzheimer's Disease, anxiety disorders, ascites, R7 is hydrogen, alkyl, alkaryl, alkylaminodialkyl or alky 50 autism, bipolar disorder, cancer, depression, endothelial lheterocycloalkyl; or corneal dystrophy, edema, epilepsy, glaucoma, Huntington's Re and R7, together with the atom to which they are Disease, insomnia, ischemia, migraine, migraine with aura, attached, form a heterocycloalkyl group; and neuropathic pain, nociceptive neuralgia, nociceptive pain, Rs and R are each independently hydrogen or alkyl: ocular diseases, pain, Parkinson's disease, personality dis provided that if Z is oxygen or Sulfur, then Ro is not 55 orders, postherpetic neuralgia, psychosis, Schizophrenia, Sei present. Zure disorders, tinnitus, and withdrawal syndromes compris In another aspect, the invention relates to a method for ing administering an effective amount of a compound of treating addictive disorders, Alzheimer's Disease, anxiety Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, disorders, ascites, autism, bipolar disorder, cancer, depres XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, Sion, edema, endothelial corneal dystrophy, epilepsy, glau 60 XXIV. XXV, and/or XXVI. In a preferred embodiment, coma, Huntington's Disease, insomnia, ischemia, migraine these compounds are selective antagonists of NKCC1 and/or with aura, migraine, neuropathic pain, nociceptive neural particular GABA receptors. gia, nociceptive pain, ocular diseases, pain, Parkinson's The present invention also provides methods of using the disease, personality disorders, postherpetic neuralgia, psy compounds of formulas I-XXVI for treating disorders chosis, Schizophrenia, seizure disorders, tinnitus, or with 65 involving the Na"K"Cl co-transporters including but not drawal syndromes comprising administering an effective limited to addictive disorders (e.g., compulsive disorders, amount of a compound of the formula XV: eating disorders (e.g., obesity), addiction to narcotics/physi US 9,682,928 B2 43 44 cal dependence, alcohol addiction, narcotic addiction, formation (e.g., glaucoma)), pain (e.g., chronic inflamma cocaine addiction, heroin addiction, opiate addiction, alco tory pain, pain associated with arthritis, fibromyalgia, back holism, and Smoking); anxiety disorders (e.g., anxiety, acute pain, cancer-associated pain, chemotherapy-induced neu anxiety, panic disorder, Social anxiety disorder, obsessive ropathy, chemotherapy-induced peripheral neuropathy, HIV compulsive disorder (OCD), post-traumatic stress disorder 5 treatment induced neuropathy. HIV-treatment induced neu (PTSD), generalized anxiety disorder, and specific phobia); ralgia, pain associated with digestive disease, pain ascites (e.g., peritoneal cavity fluid, peritoneal fluid excess, associated with Crohn's disease, pain associated with auto hydroperitoneum, abdominal dropsy, cancer related to immune disease, pain associated with endocrine disease, ascites, tumors related to ascites); bipolar disorder (e.g., pain associated with diabetic neuropathy, pain associated manic-depressive illness, manic phase, depressive phase, 10 with shingles or herpes Zoster, phantom limb pain, sponta mixed bipolar state, bipolar I disorder, bipolar II disorder, neous pain, chronic post-Surgical pain, chronic temporo rapid-cycling bipolar disorder, bipolar I disorder, bipolar II mandibular pain, causalgia, postherpetic neuralgia, AIDS disorder, rapid-cycling bipolar disorder); cancer (e.g., related pain, complex regional pain syndromes type I and II, tumors, cancer related to ascites, tumors related to ascites); trigeminal neuralgia, chronic back pain, pain associated with depression (e.g., psychotic depression, postpartum depres- 15 spinal cord injury and/or recurrent acute pain); postherpetic Sion, seasonal affective disorder (SAD), cortical spreading neuralgia (e.g., shingles, herpes Zoster); and Schizophrenia. depression, dysthymia (mild depression)): edema (e.g., cen In a preferred embodiment, these compounds are selective tral nervous system edema); endothelial corneal dystrophy antagonists of NKCC1. (e.g., post-chamber ocular diseases); epilepsy (e.g., seizures, The present invention also provides methods of using the epileptic seizures, a seizure cluster, an acute seizure (e.g., 20 compounds of formulas I-XXVI for treating disorders status epilepticus), seizure disorder, and other neurological involving a GABA receptor including but not limited to disorders involving seizures (e.g., cerebral palsy, Ohtahara Alzheimer's Disease (AD), addictive disorders (e.g., com Syndrome)); glaucoma (e.g., increased intraocular pressure, pulsive disorders, eating disorders (e.g., obesity, anorexia angle-closure glaucoma, neovascular glaucoma, open-angle nervosa, bulimia), addiction to narcotics/physical depen glaucoma); ischemia (e.g., cardiac ischemia (myocardial 25 dence, alcohol addiction, narcotic addiction, cocaine addic ischemia), intestinal ischemia, mesenteric artery ischemia tion, heroin addiction, opiate addiction, alcoholism, and (acute mesenteric ischemia), hepatic ischemia, and cerebral Smoking); anxiety disorders (e.g., anxiety, acute anxiety, ischemia (brain ischemia)); migraine (e.g., migraine includ panic disorder, Social anxiety disorder, obsessive compul ing headache, migraine variant, migraine headache, cervical sive disorder (OCD), post-traumatic stress disorder (PTSD), migraine syndrome, acute confusional migraine, migraine 30 generalized anxiety disorder, and specific phobia); autism with aura, migraine without aura); neuropathic pain (e.g., (e.g., Autism spectrum disorder (ASD)); bipolar disorder diabetic neuropathy, diabetic neuropathy, nerve injury, nerve (e.g., manic-depressive illness, manic phase, depressive tract injury, neuropathic pain associated with visceral and/or phase, mixed bipolar state, bipolar I disorder, bipolar II Somatic pain, peripheral neuropathy, chemotherapy-induced disorder, rapid-cycling bipolar disorder, bipolar I disorder, neuropathy, chemotherapy-induced peripheral neuropathy, 35 bipolar II disorder); depression (e.g., psychotic depression, neuralgia, polyneuropathy, mononeuropathy, mononeuritis postpartum depression, seasonal affective disorder (SAD), multiplex, autonomic neuropathy, symmetrical peripheral cortical spreading depression, dysthymia (mild depression)); neuropathy, radiculopathy, large fiber peripheral neuropathy, epilepsy (e.g., seizures, epileptic seizures, a seizure cluster, Small fiber peripheral neuropathy, idiopathic neuropathic an acute seizure (e.g., status epilepticus), seizure disorder, pain); nociceptive neuralgia; ocular diseases (e.g., diseases 40 and other neurological disorders involving seizures (e.g., of retina-retinal detachment and injury response; diseases of cerebral palsy, Ohtahara Syndrome)); Huntington's Disease electrical transmission between various retinal elements (HD) (e.g., Huntington's chorea); insomnia, migraine (e.g., Such as rods, cones, amacrine and horizontal cells, activity migraine including headache, migraine variant, migraine of retinal ganglion cells, dysfunction of Miller (glial) cells, headache, cervical migraine syndrome, acute confusional abnormal function of the retinal pigment epithelium; dys- 45 migraine, migraine with aura, migraine without aura, function of formation of the retina in development and the chronic migraine, transformed migraine); neuropathic pain appropriate maintenance of neural connections following (e.g., diabetic neuropathy, cluster headache, nerve injury, maturation and development; regulation of normal electro nerve tract injury, neuropathic pain associated with visceral lyte homeostasis in various chorioretinal and vitreoretinal and/or somatic pain, peripheral neuropathy, chemotherapy diseases; abnormal function of Miller cells in diabetic 50 induced neuropathy, chemotherapy-induced peripheral neu retinopathy; loss of normal electrical activity in degenerative ropathy, HIV-treatment induced neuropathy, HIV-treatment diseases of retina, inherited and those of unknown etiology; induced neuralgia, neuralgia, polyneuropathy, mononeu inflammatory diseases and conditions of the eye such as ropathy, mononeuritis multiplex, autonomic neuropathy, chorioretinitis, multiple Sclerosis; infectious processes in the symmetrical peripheral neuropathy, radiculopathy, large eye with abnormal inflammatory and injury responses; 55 fiber peripheral neuropathy, small fiber peripheral neuropa uveitis; abnormal function of Miller cells of retina and thy, idiopathic neuropathic pain); nociceptive pain; pain disease thereof; dysfunction of RPE-retinal pigment epithe (e.g., acute pain, chronic inflammatory pain, pain associated lium (e.g., diseases of RPE); endothelial (posterior) corneal with arthritis, fibromyalgia, back pain, cancer-associated dystrophies, which result from primary endothelial dysfunc pain, chemotherapy-induced neuropathy, chemotherapy-in tion, (e.g., Fuchs endothelial corneal dystrophy (FECD), 60 duced peripheral neuropathy, pain associated with digestive posterior polymorphous corneal dystrophy (PPCD) and con disease, pain associated with Crohn's disease, pain associ genital hereditary endothelial dystrophy (CHED)); retinitis ated with autoimmune disease, pain associated with endo pigmentosa; age-related macular degeneration (e.g., dry crine disease, pain associated with diabetic neuropathy, pain age-related macular degeneration, exudative age-related associated with shingles or herpes Zoster, phantom limb macular degeneration, and myopic degeneration); retinopa- 65 pain, spontaneous pain, chronic post-Surgical pain, chronic thy (e.g., diabetic retinopathy, proliferative vitreoretinopa temporomandibular pain, causalgia, postherpetic neuralgia, thy, and toxic retinopathy) and diseases of aqueous humor AIDS-related pain, complex regional pain syndromes type I US 9,682,928 B2 45 46 and II, trigeminal neuralgia, chronic back pain, pain asso angle-closure glaucoma, neovascular glaucoma, open-angle ciated with spinal cord injury, incisional post operative, glaucoma); ischemia (e.g., cardiac ischemia (myocardial trauma associated, burns, recurrent acute pain, head pain, ischemia), intestinal ischemia, mesenteric artery ischemia headache, nonmigrainous, specific non-migraine head pains, (acute mesenteric ischemia), hepatic ischemia, and cerebral tic dolureaux, postherpetic neuralgia, ice pick headache); ischemia (brain ischemia)); migraine (e.g., migraine includ Parkinson's disease, personality disorders, psychosis, Sei ing headache, migraine variant, migraine headache, cervical Zure disorders, personality disorders, Schizophrenia, tinni migraine syndrome, acute confusional migraine, migraine tus, and withdrawal syndromes (e.g., alcohol withdrawal with aura, migraine without aura); neuropathic pain (e.g., syndrome, nicotine withdrawal syndrome, opioid with diabetic neuropathy, diabetic neuropathy, nerve injury, nerve drawal syndrome, benzodiazepine withdrawal syndrome, 10 tract injury, neuropathic pain associated with visceral and/or methadone withdrawal syndrome, SSRI discontinuation Somatic pain, peripheral neuropathy, chemotherapy-induced syndrome, hydrocodone withdrawal syndrome, cocaine neuropathy, chemotherapy-induced peripheral neuropathy, withdrawal syndrome, heroin withdrawal syndrome). In a HIV-treatment induced neuropathy, HIV-treatment induced preferred embodiment, these compounds are selective neuralgia, neuralgia, polyneuropathy, mononeuropathy, antagonists of GABA receptors. In a preferred embodi 15 mononeuritis multiplex, autonomic neuropathy, symmetri ment, these compounds are selective antagonists of GABA cal peripheral neuropathy, radiculopathy, large fiber periph receptors comprising an O. Subunit. In a more preferred eral neuropathy, Small fiber peripheral neuropathy, idio embodiment, these compounds are selective antagonists of pathic neuropathic pain); nociceptive neuralgia: ocular GABA receptors comprising an Os Subunit. In a still more diseases (e.g., diseases of retina-retinal detachment and preferred embodiment, these compounds are selective injury response; diseases of electrical transmission between antagonists of GABA receptors comprising an O. Subunit. various retinal elements such as rods, cones, amacrine and The present invention further provides methods for treat horizontal cells, activity of retinal ganglion cells, dysfunc ing a patient diagnosed with risk factors for a condition tion of Müller (glial) cells, abnormal function of the retinal selected from the group consisting of addictive disorders, pigment epithelium; dysfunction of formation of the retina Alzheimer's Disease, anxiety disorders, ascites, autism, 25 in development and the appropriate maintenance of neural bipolar disorder, cancer, depression, endothelial corneal connections following maturation and development; regu dystrophy, edema, epilepsy, glaucoma, Huntington's Dis lation of normal electrolyte homeostasis in various chori ease, insomnia, ischemia, migraine, migraine with aura, oretinal and vitreoretinal diseases; abnormal function of neuropathic pain, nociceptive neuralgia, nociceptive pain, Müller cells in diabetic retinopathy; loss of normal electrical ocular diseases, pain, Parkinson's disease, personality dis 30 activity in degenerative diseases of retina, inherited and orders, postherpetic neuralgia, psychosis, Schizophrenia, Sei those of unknown etiology; inflammatory diseases and con Zure disorders, tinnitus, and withdrawal syndromes compris ditions of the eye such as chorioretinitis, multiple sclerosis: ing administering an effective amount of a compound of infectious processes in the eye with abnormal inflammatory Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and injury responses: uveitis; abnormal function of Müller XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, 35 cells of retina and disease thereof dysfunction of RPE XXIV. XXV, and/or XXVI. In a preferred embodiment, retinal pigment epithelium (e.g., diseases of RPE); endothe these compounds are selective antagonists of NKCC1 and/or lial (posterior) corneal dystrophies, which result from pri GABA receptors. mary endothelial dysfunction, (e.g., Fuchs The present invention still further provides methods of endothelialcorneal dystrophy (FECD), posterior polymor using the compounds of formulas I-XXVI for treating a 40 phous corneal dystrophy (PPCD) and congenital, hereditary patient diagnosed with risk factors for disorders involving endothelial dystrophy (CHED)); retinitis pigmentosa; age the NaKCl co-transporters including but not limited to related macular degeneration (e.g., dry age-related macular addictive disorders (e.g., compulsive disorders, eating dis degeneration, exudative age-related macular degeneration, orders (e.g., obesity), addiction to narcotics/physical depen and myopic degeneration); retinopathy (e.g., diabetic retin dence, alcohol addiction, narcotic addiction, cocaine addic 45 opathy, proliferative vitreoretinopathy, and toxic retinopa tion, heroin addiction, opiate addiction, alcoholism, and thy) and diseases of aqueous humor formation (e.g., glau Smoking); anxiety disorders (e.g., anxiety, acute anxiety, coma)); pain (e.g., chronic inflammatory pain, pain panic disorder, Social anxiety disorder, obsessive compul associated with arthritis, fibromyalgia, back pain, cancer sive disorder (OCD), post-traumatic stress disorder (PTSD), associated pain, chemotherapy-induced neuropathy, chemo generalized anxiety disorder, and specific phobia); ascites 50 therapy-induced peripheral neuropathy, pain associated with (e.g., peritoneal cavity fluid, peritoneal fluid excess, hydro digestive disease, pain associated with Crohn's disease, pain peritoneum, abdominal dropsy, cancer related to ascites, associated with autoimmune disease, pain associated with tumors related to ascites); bipolar disorder (e.g., manic endocrine disease, pain associated with diabetic neuropathy, depressive illness, manic phase, depressive phase, mixed pain associated with shingles or herpes Zoster, phantom limb bipolar state, bipolar I disorder, bipolar II disorder, rapid 55 pain, spontaneous pain, chronic post-Surgical pain, chronic cycling bipolar disorder, bipolar I disorder, bipolar II disor temporomandibular pain, causalgia, postherpetic neuralgia, der, rapid-cycling bipolar disorder); cancer (e.g., tumors, AIDS-related pain, complex regional pain syndromes type I cancer related to ascites, tumors related to ascites); depres and II, trigeminal neuralgia, chronic back pain, pain asso sion (e.g., psychotic depression, postpartum depression, ciated with spinal cord injury and/or recurrent acute pain); seasonal affective disorder (SAD), cortical spreading 60 postherpetic neuralgia (e.g., shingles, herpes Zoster); and depression, dysthymia (mild depression)): edema (e.g., cen Schizophrenia. In a preferred embodiment, these compounds tral nervous system edema); endothelial corneal dystrophy are selective antagonists of NKCC1. (e.g., post-chamber ocular diseases); epilepsy (e.g., seizures, The present invention still further provides methods of epileptic seizures, a seizure cluster, an acute seizure (e.g., using the compounds of formulas I-XXVI for treating a status epilepticus), seizure disorder, and other neurological 65 patient diagnosed with risk factors for disorders involving a disorders involving seizures (e.g., cerebral palsy, Ohtahara GABA receptor including but not limited to Alzheimer's Syndrome)); glaucoma (e.g., increased intraocular pressure, Disease (AD), addictive disorders (e.g., compulsive disor US 9,682,928 B2 47 48 ders, eating disorders (e.g., obesity, anorexia nervosa, buli Embodiments of the present invention provide a pharma mia), addiction to narcotics/physical dependence, alcohol ceutical composition comprising a compound of Formula I, addiction, narcotic addiction, cocaine addiction, heroin II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, addiction, opiate addiction, alcoholism, and Smoking); anxi XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, ety disorders (e.g., anxiety, acute anxiety, panic disorder, XXV, and/or XXVI, a pharmaceutically acceptable salt, Social anxiety disorder, obsessive compulsive disorder Solvate, tautomer, hydrate, or combination thereof and a (OCD), post-traumatic stress disorder (PTSD), generalized pharmaceutically acceptable carrier, excipient, or diluent. anxiety disorder, and specific phobia); autism (e.g., Autism Embodiments of the present invention provide methods of spectrum disorder (ASD)); bipolar disorder (e.g., manic making the compounds including compounds described 10 herein and further provide intermediate compounds formed depressive illness, manic phase, depressive phase, mixed through the synthetic methods described herein to provide bipolar state, bipolar I disorder, bipolar II disorder, rapid the compounds of Formula I, II, III, IV, V, VI, VII, VIII, IX, cycling bipolar disorder, bipolar I disorder, bipolar II disor X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, der); depression (e.g., psychotic depression, postpartum XXI, XXII, XXIII, XXIV. XXV, and/or XXVI. depression, seasonal affective disorder (SAD), cortical 15 Embodiments of the present invention provide kits spreading depression, dysthymia (mild depression)); epi including the compounds including compounds described lepsy (e.g., seizures, epileptic seizures, a seizure cluster, an herein. These kits may be used in the treatment methods acute seizure (e.g., status epilepticus), seizure disorder, and disclosed herein. In another embodiment, the kits may other neurological disorders involving seizures (e.g., cere include instructions, directions, labels, warnings, or infor bral palsy, Ohtahara Syndrome)); Huntington's Disease mation pamphlets. (HD) (e.g., Huntington's chorea); insomnia, migraine (e.g., Embodiments of the present invention further provide migraine including headache, migraine variant, migraine methods of treating a disease, condition, or disorder, espe headache, cervical migraine syndrome, acute confusional cially the disorders described herein. migraine, migraine with aura, migraine without aura, Embodiments of the present invention provide uses of the chronic migraine, transformed migraine); neuropathic pain 25 compounds including compounds described herein for the (e.g., diabetic neuropathy, cluster headache, nerve injury, preparation of a medicament for carrying out the aforemen nerve tract injury, neuropathic pain associated with visceral tioned utilities. and/or somatic pain, peripheral neuropathy, chemotherapy In addition, compounds of the present invention may be induced neuropathy, chemotherapy-induced peripheral neu used for the regulation of psychiatric disorders and neuro ropathy. HIV-treatment induced neuropathy, HIV-treatment 30 logical disorders and to modulate neuronal synchronization induced neuralgia, neuralgia, polyneuropathy, mononeu as well as improve CNS function. In some embodiments, the ropathy, mononeuritis multiplex, autonomic neuropathy, compound of the prodrug is provided in an amount effective symmetrical peripheral neuropathy, radiculopathy, large for regulating a CNS disorder, and/or a disorder involving fiber peripheral neuropathy, small fiber peripheral neuropa the NaKCl co-transporters and/or a disorder involving thy, idiopathic neuropathic pain); nociceptive pain; pain 35 the GABA receptor. In particular embodiments, the CNS (e.g., acute pain, chronic inflammatory pain, pain associated disorder is Alzheimer's Disease, addictive disorders, anxiety with arthritis, fibromyalgia, back pain, cancer-associated disorders, autism, bipolar disorder, depression, epilepsy, pain, chemotherapy-induced neuropathy, chemotherapy-in Huntington's Disease, insomnia, migraine, migraine with duced peripheral neuropathy, pain associated with digestive aura, neuropathic pain, nociceptive pain, pain, Parkinson's disease, pain associated with Crohn's disease, pain associ 40 disease, personality disorders, psychosis, Schizophrenia, Sei ated with autoimmune disease, pain associated with endo Zure disorders, tinnitus, or withdrawal syndromes. crine disease, pain associated with diabetic neuropathy, pain In a further embodiment, the compounds described herein associated with shingles or herpes Zoster, phantom limb may be used in methods of neuroprotection (e.g., reducing pain, spontaneous pain, chronic post-Surgical pain, chronic damage following stroke, head trauma, reducing damage temporomandibular pain, causalgia, postherpetic neuralgia, 45 from neurodegenerative diseases like Alzheimer's disease AIDS-related pain, complex regional pain syndromes type I Huntington's Disease, and Parkinson's disease) and to and II, trigeminal neuralgia, chronic back pain, pain asso reduce neurotoxicity (e.g., damage from ethanol). ciated with spinal cord injury, incisional post operative, In another embodiment, the compounds described herein trauma associated, burns, recurrent acute pain, head pain, may be used in methods to improve cognition, learning, or headache, nonmigrainous, specific non-migraine head pains, 50 memory. Alternatively, the compounds described herein tic dolureaux, postherpetic neuralgia, ice pick headache); may be used as cognition, learning, or memory aids. Parkinson's disease, personality disorders, psychosis, Sei In one embodiment, the compounds described herein Zure disorders, personality disorders, Schizophrenia, tinni modulate or improve the function of the retina in ocular tus, and withdrawal syndromes (e.g., alcohol withdrawal diseases with inappropriate retinal activity. In another syndrome, nicotine withdrawal syndrome, opioid with 55 embodiment, the compounds described herein regulate the drawal syndrome, benzodiazepine withdrawal syndrome, intracellular pH of retinal pigment epithelial cells. In another methadone withdrawal syndrome, SSRI discontinuation embodiment, the compounds described herein regulate, syndrome, hydrocodone withdrawal syndrome, cocaine change, or modulate the chloride concentration to regulate withdrawal syndrome, heroin withdrawal syndrome). In a glial cell function of retina. preferred embodiment, these compounds are selective 60 In a preferred embodiment, the compounds described antagonists of GABA receptors. In a preferred embodi herein show improved CNS pharmacologic properties and ment, these compounds are selective antagonists of GABA increased transit across the blood-brain barrier (BBB). receptors comprising an C. subunit. In a more preferred In a preferred embodiment, the compounds described embodiment, these compounds are selective antagonists of herein show differential activity with stronger effect on the GABA receptors comprising an Os Subunit. In a still more 65 central nervous system and less diuretic effects. For preferred embodiment, these compounds are selective example, the compounds described herein may be used in antagonists of GABA receptors comprising an O. Subunit. long-term (maintenance) therapy without significant diuretic US 9,682,928 B2 49 50 effect. Also, the analogs of bumetanide, furosemide, pire partition coefficient (e.g., the distribution of a compound tanide, azosemide, and torsemide described herein may be between water and octanol). In further embodiments, the used in combination therapy with diuretics because of their compounds of the present invention may result in fewer lack of diuretic effect. Additionally, the compounds undesirable side effects when employed in the regulatory, described herein do not interfere with diuretics or cause (i.e., preventive, management), and/or treatment, methods severe side effects when administered in conjunction with or described herein. concurrently with a diuretic. In some embodiments, the level of diuresis that occurs In a preferred embodiment, the compounds described following administration of an effective amount of a com herein are targeted to be more specific for the treatment of pound provided below as Formula I-XXVI, is less than one or more of the described conditions and diseases. 10 about 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or Further, these compounds show less instance of effects on 10% of that which occurs following administration of an systems other than the targeted systems. Also the com effective amount of the parent molecule from which the pounds described herein have fewer or no side effects when compound is derived. For example, the compound may be used in combination with other drugs (e.g., fewer undesir less diuretic than the parent molecule when administered at able medication interactions). Additionally, the compounds 15 the same mg/kg dose. Alternatively, the compound may be described herein, especially the analogs and prodrugs may more potent than the parent molecule from which it is be dosed at high levels (>100 mg) with no adverse effects derived, so that a smaller dose of the compound may be (e.g., the compounds are well-tolerated at high dosages). required for effective relief of symptoms, and thus, may In a preferred embodiment, the compounds described elicit less of a diuretic effect. Similarly, the compound may herein act to modulate the anion levels in a central nervous have a longer duration of effect in treating disorders than the system (CNS) cell (e.g., glia and neurons). In a more parent molecule. Accordingly, compounds of the present preferred embodiment, the compounds described herein act invention may be administered less frequently than the to modulate the intracellular chloride levels in a CNS cell. parent molecule, and thus may lead to a lower total diuretic In yet-another embodiment, analogs (including prodrugs) of effect within any given period of time. bumetanide, furosemide, piretanide, azosemide, and 25 In a preferred embodiment, the compounds described torsemide described herein decrease the intracellular chlo herein show selective activity with stronger effect on the ride level in a CNS cell. central nervous system and limited, minimal, or no diuretic In another embodiment, the compounds described herein effects. The preferred analogs of bumetanide, furosemide, decrease the intracellular chloride level (concentration) in a piretanide, aZosemide, or torsemide will produce less than CNS cell wherein said CNS cell is a spinal cord neural cell, 30 one-fifth, more preferably less than one-seventh, of the a glial cell (e.g., an oligodendrocyte, a Schwann cell, an diuretic effect produced by the parent compound. For astrocyte, a microglial cell) or a neuron (e.g., medium spiny example, the compounds described herein (including prod GABA-ergic neuron or a large cholinergic neuron). rugs thereof) may typically be used in long-term therapy In another embodiment, the compounds described herein (e.g., maintenance therapy) without significant diuretic may be administered in combination with a second agent. 35 effect. Also, the analogs of bumetanide, furosemide, pire In a preferred embodiment, the compounds described tanide, azosemide, and torsemide described herein may be herein may be administered prophylactically to prevent, used in combination therapy with diuretics because they lessen the severity of, or delay the onset of symptoms of the produce minimal diuretic effect. Additionally, the com conditions described herein. In another embodiment, the pounds described herein (including prodrugs thereof) do not compounds described herein may be administered prophy 40 interfere with diuretics or cause severe side effects when lactically to prevent, lessen the severity of, or delay the administered in conjunction with or concurrently with a reoccurrence of the conditions described herein. diuretic. In a preferred embodiment, the compounds described The compounds of the present invention of Formulae I, II, herein are targeted to be more specific for the treatment of III, IV, V, VI, VII, VIII, IX, X, XI, XII, XI, XIV, XV, XVI, one or more of the described conditions and diseases. 45 XVII, XV, XIX, XX, XXI, XXII, XXIII, XXIV. XXV, and/or Further, these compounds show less instance of effects on XXVI described herein may be used for the regulation, systems other than the targeted systems. Also the com including prevention, management and treatment, of a range pounds described herein have fewer or no side effects when of conditions including, but not limited to disorders that used in combination with other drugs (e.g., fewer undesir involve at least one of the Na"K"Cl cotransporters. able medication interactions). Additionally, the compounds 50 Embodiments of the present invention encompass com described herein, especially the analogs and prodrugs may pounds (including prodrugs thereof) described herein which be dosed at high levels (e.g., >100 mg/kg) with no or modulate, regulate, inhibit, stimulate, activate, and/or bind minimal adverse effects (e.g., the compounds are well to electroneutral cation-chloride cotransporter co-transport tolerated at high dosages). ers including but not limited to NaCl cotransporters (e.g., Selective Activity 55 thiazide-sensitive Na"Cl cotransporters); apical Embodiments of the present invention provide com bumetanide-sensitive Na"K"Cl cotransporters (e.g., pounds (including analogs and prodrugs) capable of passage NKCC2); basolateral bumetanide-sensitive Na"K"Cl across the blood-brain barrier comprising a compound of cotransporters (e.g., NKCC1); and KCl cotransporters Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XII, (e.g., KCC1, KCC2, KCC3, KCC4). In a preferred embodi XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, 60 ment, the electroneutral cation-chloride cotransporter is a XXIV. XXV, and/or XXVI, or a pharmaceutically acceptable bumetanide-sensitive NaKCl cotransporter (e.g., salt, solvate, tautomer or hydrate thereof. In some embodi NKCC1, NKCC2). ments, compounds of the present invention may have In some embodiments, compounds of the present inven increased lipophilicity and/or reduced diuretic effects com tion may have increased lipophilicity and/or reduced diuretic pared to the diuretic or diuretic-like compounds from which 65 effects compared to the diuretic or diuretic-like compounds they are derived. The lipiphilicity can be measured by from which they are derived. The lipiphilicity can be mea determining the hydrophile-lipophile balance (HLB) or the sured by determining the hydrophile-lipophile balance US 9,682,928 B2 51 52 (HLB) or the partition coefficient (e.g., the distribution of a of a compound of the Formulae I-XXVI or a pharmaceuti compound between water and octanol). In further embodi cally acceptable salt thereof. In another embodiment, the ments, the compounds of the present invention may result in invention comprises a method for antagonizing parasynaptic fewer undesirable side effects when employed in the regu GABA receptors comprising an O. Subunit comprising latory (i.e., preventive, management) and/or treatment meth 5 administering a composition comprising an effective amount ods described herein. of a compound of the Formulae I-XXVI or a pharmaceuti In one embodiment, the invention comprises a method of cally acceptable salt thereof. In yet another embodiment, the inhibiting apical bumetanide-sensitive Na"K"C' cotrans invention comprises a method for antagonizing parasynaptic porters (e.g., NKCC2) comprising administering a compo GABA receptors comprising an as subunit comprising sition comprising an effective amount of a compound of the 10 administering a composition comprising an effective amount Formulae I-XXVI or a pharmaceutically acceptable salt of a compound of the Formulae I-XXVI or a pharmaceuti thereof. In another embodiment, the invention comprises a cally acceptable salt thereof. In a further embodiment, the method of inhibiting basolateral bumetanide-sensitive invention comprises a method for antagonizing parasynaptic Na"K"Cl cotransporters (e.g., NKCC1) comprising admin GABA receptors comprising an as subunit comprising istering a composition comprising an effective amount of a 15 administering a composition comprising an effective amount compound of the Formulae I-XXVI or a pharmaceutically of a compound of the Formulae I-XXVI or a pharmaceuti acceptable salt thereof. In another embodiment, the inven cally acceptable salt thereof. tion comprises a method of inhibiting basolateral In another embodiment, the invention comprises a method bumetanide-sensitive Na"K"Cl cotransporters (e.g., for antagonizing parasynaptic GABA receptors comprising NKCC1) and apical bumetanide-sensitive NaK"Cl an C. Subunit comprising administering a composition com cotransporters (e.g., NKCC2) comprising administering a prising a compound described herein, wherein the antago composition comprising an effective amount of a compound nism of GABA receptors with an C. Subunit is no more than of the Formulae I-XXVI or a pharmaceutically acceptable 50%, more preferably nor more than 25%, or even more salt thereof. preferably no more than 15% of the effect on a GABA In a preferred embodiments, the invention comprises a 25 receptor with an O. receptor. In yet another embodiment, the method of inhibiting basolateral bumetanide-sensitive invention comprises a method for antagonizing parasynaptic Na"K"Cl cotransporters (e.g., NKCC1) comprising admin GABA receptors comprising an Os subunit comprising istering a composition comprising a compound described administering a composition comprising a compound herein, wherein the inhibition of apical bumetanide-sensitive described herein, wherein the antagonism of a GABA Na"K"Cl cotransporters (e.g., NKCC2) is no more than 30 receptor with an O. subunit is no more than 50%, more 50%, more preferably nor more than 25%, or even more preferably nor more than 25%, or even more preferably no preferably no more than 15% of the effect on basolateral more than 15% of the effect on a GABA receptor with an bumetanide-sensitive Na"K"Cl cotransporters (e.g., Cs receptor. In a further embodiment, the invention com NKCC1). In yet another embodiment, the invention com prises a method for antagonizing parasynaptic GABA prises a method of inhibiting apical bumetanide-sensitive 35 receptors comprising an O. Subunit comprising administer Na"K"Cl cotransporters (e.g., NKCC2) comprising admin ing a composition comprising a compound described herein, istering a composition comprising a compound described wherein the antagonism of a GABA receptor with an a, herein, wherein the inhibition of basolateral bumetanide subunit is no more than 50%, more preferably nor more than sensitive NaKCl cotransporters (e.g., NKCC1) is no 25%, or even more preferably no more than 15% of the more than 50%, more preferably nor more than 25%, or even 40 effect on a GABA receptor with an O. receptor. more preferably no more than 15% of the effect on apical The compounds described herein may preferentially bind bumetanide-sensitive Na"K"Cl cotransporters (e.g., GABA receptors. In one embodiment, the compounds NKCC2). Some preferred bumetanide, furosemide, pire described herein may preferentially bind GABA receptors tanide, azosemide, and torsemide analogs and derivatives, comprising an O. Subunit. In another embodiment, the described herein may not act on the GABA receptor or 45 compounds described herein may preferentially bind show only minimal activity toward GABA receptors. GABA receptors comprising an Os Subunit. In a further The compounds of the present invention of Formulae I, II, embodiment, the compounds described herein may prefer III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, entially bind GABA receptors comprising an C. Subunit. In XVII, XVI, XIX, XX, XXI, XXII, XXIII, XXIV, and/or another embodiment, the compounds described herein may XXV described herein may be used for the regulation, 50 preferentially bind GABA receptors comprising an O. including prevention, management and treatment, of a range subunit. In a further embodiment, the compounds described of conditions including, but not limited to disorders that herein may preferentially bind GABA receptors compris involve at least one of the GABA receptor. ing an O2 subunit. In another embodiment, compounds described herein may The compounds described herein may have antagonistic show selective effect on a subset of GABA receptors in the 55 effects on GABA receptors located parasynaptically. In one CNS and less of the side-effects usually associated with embodiment, the compounds described herein may have agents that act on GABA receptors. For example, com antagonistic effects on GABA receptors comprising an O. pounds described herein exhibit less sedation and less Sup Subunit located parasynaptically. In another embodiment, pression of respiration, cognition, or motor function. In the compounds described herein may have an antagonistic another embodiment, compounds described herein may 60 effect on GABA receptors comprising an as Subunit located show a selective effect on GABA receptors comprising an parasynaptically. In a further embodiment, the compounds Cls subunit or an O. Subunit. In another embodiment, com described herein may have an antagonistic effect on GABA pounds described herein show a selective effect on GABA receptors comprising an O. Subunit located parasynaptically. receptors comprising an C. Subunit. Preferential binding of the compounds of this invention In one embodiment, the invention comprises a method for 65 may be reflected in the effective concentration (ECso), i.e., antagonizing parasynaptic GABA receptors comprising the concentration of the compound in vitro at which the administering a composition comprising an effective amount antagonist effect is half the maximal antagonism demon US 9,682,928 B2 53 54 strated by the respective compound on the particular recep and some acyclic thioamides, see U.S. Patent Application tor. In particular, more preferred compounds of this inven Publication No. 2007/014.9526A1. tion will be those whose ECso for GABA receptors with C. FIG. 8 is a scheme showing alternative methods for subunits are no more than 50%, more preferably nor more obtaining bumetanide N-morpholinothioamide. See than 25%, or even more preferably no more than 15% of the Schwarz, G., Org. Syn., Coll. Vol. III, 1955, 332-334; Alper, ECso of the same compound for GABA receptors having C. H., et al., Angew. Chem. Int. Ed., 1978, 17,689. (31-56%); subunits. Even more preferred are compounds whose ECso Raucher, S. and Klein, P. J. Org. Chem., 1981, 46, 3558. for C-containing GABA receptors is no more than 50%, (80-95%); Lawesson, S.-O., et al., Org. Syn., Coll. Vol. VII, more preferably nor more than 25%, or even more prefer 1990, 372-375: A. Preparation; B. Thiolactam Formation; as ably no more than 15% of the ECso for C-containing 10 well as and references 26-30 therein; Davy, H., Sulfur Lett. GABA receptors. Preferential binding for other receptors 1985, 3, 39-44 and related references: “Synthesis of Thio can be characterized similarly. amides and Thiolactams. Schaumann, E. in Comprehensive The compounds described herein are effective in humans Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, and animals to decrease seizures, decrease pain responses, Chapter 2.4, Pergamon Press, Oxford, 1991, pp. 419-434; and decrease migraine in humans and animal models. For 15 Lawesson, S.-O., et al., Org. Syn., Coll. Vol. VII, 1990, example, compounds described herein may preferentially 372-375 and references therein; Fieser’s Reagents for Org. bind to GABA receptor Subtypes and have an antagonistic Syn., Vol. 8, 1980, 327; ibid, Vol. 9, 1981, 49-50; ibid, Vol. effect on GABA receptors that is different from classic 10, 1982, 39; ibid, Vol. 11, 1984, 54-55; ibid, Vol. 12, 1986, benzodiazepine and barbiturate mechanisms. Unlike many 59; ibid, Vol. 13, 1988, 38-39; ibid, Vol. 15, 1990, 37, 329; bumetanide, furosemide, piretanide, azosemide, and and ibid, Vol. 16, 1992, 37-38; Davy, H., JCS, Chem. torsemide analogs and derivatives, compounds described Commun., 1982, 457; Davy, H., Sulfur Lett., 1985, 3,39-44: herein may not act on the NaK2Cl cotransporter (NKCC1 Davy, H., Chem. & Ind., 1985, 824; Davy, H., J. Chem. Res. or NKCC2). Unlike bumetanide, furosemide, piretanide, (S), 1985, 272; Davy, H., J. Chem. Res. (M), 1985, 2701 aZosemide, and torsemide, compounds described herein 2712: “Synthesis of Thioesters and Thiolactones'', Voss, J. in (e.g., NTP-2014) do not elicit diuresis in animal models. For 25 Comprehensive Organic Synthesis, Trost, B. M. Editor-in example, compounds described (e.g., NTP-2014) herein do Chief, Volume 6, Chapter 2.5, Pergamon Press, Oxford, not increase urine output, Sodium excretion, or potassium 1991, pp. 435-460; “Synthesis of Selenoesters of All Oxi excretion. dation States, Ogawa, A. and Sonoda, N. in Comprehensive Embodiments of the present invention further provide Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, methods of treating a disease, condition, or disorder 30 Chapter 2.6, Pergamon Press, Oxford, 1991, pp. 461–484. described herein. In an embodiment, the present invention FIG. 9 is a scheme showing alternative methods for provides methods for the use of the compounds described obtaining bumetanide N-morpholinoselenoamide. See Rae, herein for the manufacture of a composition for the treat I. D. and Wade, M. J., Int. J. Sulfur Chem., 1976, 8, 519; ment of a disease, condition, or disorder described herein. In Voss, J. and Bruhn, F.-R., Liebigs Ann. Chem., 1979, 1931; another embodiment, the present invention provides for 35 Woollins, J. D., et al., Chemistry Europe J., 2005, 11, compositions for use in treating a disease, condition, or 6221-6227: “Synthesis of Selenoesters of All Oxidation disorder described herein comprising a compound described States'. Ogawa, A. and Sonoda, N. in Comprehensive herein. Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, The foregoing and other objects and aspects of the present Chapter 2.6, Pergamon Press, Oxford, 1991, pp. 461-484; invention are explained in greater detail in reference to the 40 Woollins, J. D., et al., Chem. Eur, J., 2005, 11, 6221-6227 drawing and description set forth herein. and Woollins references therein: “Synthesis of Thioamides and Thiolactams'. Schaumann, E. in Comprehensive BRIEF DESCRIPTION OF THE DRAWINGS Organic Synthesis, Trost, B. M. Editor-in-Chief, Volume 6, Chapter 2.4, Pergamon Press, Oxford, 1991, pp. 419-434; FIG. 1 presents a graph depicting the results of 45 Lawesson, S.-O., et al., Org. Syn., Coll. Vol. VII, 1990, bumetanide analogs on the difference in startle amplitude in 372-375 and references therein; Fieser’s Reagents for Org. comparison to control as a measure of the ability of the Syn., Vol. 8, 1980, 327; ibid, Vol. 9, 1981, 49-50; ibid, Vol. bumetanide analogs to alleviate anxiety. See EXAMPLE 10, 1982, 39; ibid, Vol. 11, 1984, 54-55; ibid, Vol. 12, 1986, 137 59; ibid, Vol. 13, 1988, 38-39; ibid, Vol. 15, 1990, 37, 329; FIG. 2 is a scheme showing different pathways for the 50 and ibid, Vol. 16, 1992, 37-38; Davy, H., JCS, Chem. synthesis of the thiobumetanide analog comprising a cyclic Commun., 1982, 457; Davy, H., Sulfur Lett., 1985, 3,39-44: amide, which is bumetanide N-morpholinothioamide NTP Davy, H., Chem. & Ind., 1985, 824; Davy, H., J. Chem. Res. 2024. See Lawesson, S.-O., et al., Org. Syn, Coll. Vol. VII, (S), 1985, 272; Davy, H., J. Chem. Res. (M), 1985, 2701 1990, 372-375 and references cited therein. 2712. FIGS. 3-7 are schemes showing syntheses for thiobu 55 FIG. 10 illustrates an exemplary reaction scheme showing metanide analogs comprising a cyclic amide. Thioamide the butylation reaction of the monobutyl methyl ester start formation can occur in two steps via a) conversion of the ing material using butyraldehyde in the presence of Sodium carboxylic acid into the corresponding acid halide with triacetoxy borohydide. This reaction was carried out in reagents such as thionyl chloride and b) by direct reaction of dichloroethane in the presence of acetic acid. The resulting the acid halide with a primary or secondary amine. The 60 dibutyl methyl ester can be subsequently hydrolyzed under, thioamide formation can also occur in one step via 1-ethyl e.g., acidic conditions to give the dibutylated benzoic acid 3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy product shown after step 3) of the reaction scheme. benzotriazole (EDC-HOBt) peptide-type coupling of the Bumetanide methyl ester can be?is treated with n-butyral carboxylic acid and the amine. For each of the syntheses dehyde in the presence of a catalytic amount of acetic acid shown in FIG. 2 the thioamide bond can be formed in one 65 in dichloromethane at 0°C. to form the Schiff base in situ. step using an EDC-HOBt "peptide-type' coupling. For the To this mixture was then added sodium triacetoxyborohy synthesis of dithiobumetanide (i.e., bumetanide dithioacid) dride to reduce the Schiff base under reductive alkylating US 9,682,928 B2 55 56 conditions to add a second n-butyl group at the 3N-position FIG. 17 A-G show the effect of furosemide on stimulation of the bumetanide ester. This di-n-butylated ester can be?is evoked after discharge activity in rat hippocampal slices. then submitted to ester hydrolysis conditions to produce FIG. 18A-R show furosemide blockade of spontaneous 3N-(n-butyl)bumetanide—a.k.a. 3-(n-butyl) bumetanide epileptiform burst discharges across a spectrum of in vitro and more formally named: 3-(di-n-butylamino)-4-phenoxy models. 5-sulfamoylbenzoic acid. FIG. 19A-H show furosemide blockade of kainic acid FIG. 11 depicts the effects of NTP compounds and evoked electrical “status epilepticus' in urethane-anesthe bumetanide on seizure induction in the rat following kainic tized rats, with EKG recordings shown in the upper traces acid injections. Rats were subjected to kainic acid (KA, 15 and cortical EEG recordings shown in the bottom traces. 10 FIG. 20 shows that significantly less freezing was mg/kg) injections i.p. and treated with DMSO (vehicle), observed in animals treated with either bumetanide or furo NTP compounds (20 umoles/kg), and bumetanide (100 semide than in animals receiving vehicle alone in a test of mg/kg) and examined for seizure induction. Seizure induc contextual fear conditioning in rats. tion was graded as described in EXAMPLE 143. NTP-1 FIG. 21 shows baseline startle amplitudes in a fear (NTP-2011); NTP-2 (NTP-2022); NTP-3 (bumet); NTP-4 15 potentiated startle test in rats, which can be tested with either (NTP-2015); NTP-5 (mannitol); NTP-6 (NTP-2007); NTP-7 bumetanide or furosemide. (NTP-2024); NTP-8 (NTP-2012); NTP-9 (NTP-2008); and FIG.22 shows the amplitude of response in rats on startle Bumet (bumetanide). alone trials determined immediately following administra FIG. 12 depicts the effects of NTP compounds and tion of either DMSO alone, bumetanide, or furosemide. bumetanide on seizure severity (activity) in the rat following FIG. 23 shows the difference score (startle alone-fear kainic acid injections. Rats were Subjected to kainic acid potentiated startle) on the test day in rats treated with either (KA, 15 mg/kg) i.p. injections and treated with DMSO DMSO, bumetanide, or furosemide (vehicle), NTP compounds (20 umoles/kg) and bumetanide FIG. 24 shows the startle alone amplitude in rats one week (100 mg/kg) and examined for seizure severity. Seizure after administration of either DMSO, bumetanide, or furo severity was graded as described in EXAMPLE 143. NTP-1 25 semide. (NTP-2011); NTP-2 (NTP-2022); NTP-3 (bumet); NTP-4 FIG. 25 shows the difference score in rats one week after (NTP-2015); NTP-5 (mannitol); NTP-6 (NTP-2007); NTP-7 administration of either DMSO, bumetanide or furosemide. (NTP-2024); NTP-8 (NTP-2012); NTP-9 (NTP-2008); and FIG. 26 shows the difference score (startle alone-fear Burnet (bumetanide). potentiated startle) on the test day in rats treated with one of FIG. 13 depicts the quantification of epileptiform activity. 30 the following bumetanide analogs: bumetanide N,N-dieth See Haglund and Hochman (Feb. 23, 2005) J. Neurphysiol. ylglycolamide ester (referred to as 2MIK053); bumetanide 94: 907-918. methyl ester (referred to as 3MIK054); bumetanide N,N- FIG. 14A-B shows that furosemide blocks spontaneous dimethylglycolamide ester (referred to as 3MIK069-11): interictal spiking in human Subjects. See Haglund and Hoch bumetanide morpholinodethyl ester (referred to as man (Feb. 23, 2005) J. Neurophysiol. 94: 907-918. 35 3MIK066-4); bumetanide pivaxetil ester (referred to as FIGS. 15A-B is a description of the quantification of 3MIK069-12); bumetanide cyanomethyl ester (referred to as epileptiform activity. FIG. 15A shows the after-discharge 3MIK047); bumetanide dibenzylamide (referred to as activity elicited by 4 seconds of electrical stimulation to the 3MIK065); and bumetanide 3-(dimethylaminopropyl) ester primate sensory cortex. FIG. 15B shows a typical spike (referred to as 3MIK066-5). The vehicle was DMSO. amplitude of all the spikes comprising the burst. The area of 40 FIG. 27 illustrates cumulative urine volume. The animals envelope combines a measurement of duration and spike were fasted overnight prior to dosing and had no access to amplitude. This is of particular relevance because some drinking water prior to any pretreatment. Food and water times the duration can be dramatically reduced by a treat were withheld through the terminal sample collection or for ment, leaving spikes essentially the same size (hence the the first 6 hours of blood sample collection, where appli spike height measurement fails in this case). Occasionally, 45 cable. Prior to test article administration all animals were the spike amplitude is greatly diminished by a treatment, but pretreated with a single IP dose of piperonyl butoxide low-amplitude rumbling lasting many seconds can be elic (“PBX). At approximately 5 to 6 minutes prior to dosing all ited by electrical stimulation (hence the duration measure animals received a single oral (PO) gavage dose of 0.9% ment fails in this case). Thus the area of the envelope Sodium Chloride for Injection, USP, at a dose volume of 15 captures both types of changes simultaneously, and can be 50 mL/kg. The vehicle, DMSO, and the test articles, NTP-2024 thought of as the average spike amplitude multiplied by the and NTP-2006, were administered via a single IP dose at a duration (but over very short duration steps to approximate dose volume of 1 ml/kg. the area-integral). See Haglund and Hochman (Feb. 23. FIG. 28 illustrates cumulative sodium excretion. The 2005) J. Neurophysiol. 94: 907-918. animals were fasted overnight prior to dosing and had no FIG. 16 shows the results of a formalin paw test where 55 access to drinking water prior to any pretreatment. Food and analogs of bumetanide, furosemide, piretanide, azosemide, water were withheld through the terminal sample collection and torsemide NTP-2006, NTP-2024, and NTP-1007 all or for the first 6 hours of blood sample collection, where show anti-inflammatory and analgesic effects in a model of applicable. Prior to test article administration all animals neuropathic pain. VEH=Vehicle: NTP-2006, NTP-2024, and were pretreated with a single IP dose of piperonyl butoxide NTP-1007 were administered in three doses: 50 umole/kg 60 (“PBX). At approximately 5 to 6 minutes prior to dosing all (23 mg/kg for NTP-2006: 21 mg/kg for NTP-2024; and 19 animals received a single oral (PO) gavage dose of 0.9% mg/kg for NTP-1007): 250 mole/kg (119 mg/kg for NTP Sodium Chloride for Injection, USP, at a dose volume of 15 2006: 108 mg/kg for NTP-2024; and 217 mg/kg for NTP mL/kg. The vehicle, DMSO, and the test articles, NTP-2024 1007); and 500 umole/kg (239 mg/kg for NTP-2006: 217 and NTP-2006, were administered via a single IP dose at a mg/kg for NTP-2024; and 193 mg/kg for NTP-1007). 65 dose volume of 1 mL/kg. GBP gabapentin which was administered at 584 umole/kg FIG. 29 illustrates cumulative potassium excretion. The (100 mg/kg). (* p<0.05) (** p-0.01) (*** p-0.001) animals were fasted overnight prior to dosing and had no US 9,682,928 B2 57 58 access to drinking water prior to any pretreatment. Food and (Zolpidem) a sleep aid, and (C) VALIUM (diazepam) an water were withheld through the terminal sample collection anxiolytic drug, all increase both the amplitude and time or for the first 6 hours of blood sample collection, where course of inhibitory currents in C. Subunit containing applicable. Prior to test article administration all animals GABA receptors. were pretreated with a single IP dose of piperonyl butoxide FIG. 37A-C illustrates that select bumetanide derivative, (“PBX). At approximately 5 to 6 minutes prior to dosing all NTP-2014, showed an inhibition of currents in C. GABA animals received a single oral (PO) gavage dose of 0.9% receptor isoform at: (A) 1 M GABA, (B) 10 uM GABA, and (C) 100 uMGABA. Higher GABA concentrations (e.g., Sodium Chloride for Injection, USP, at a dose volume of 15 100 uMGABA) leads to a greater inhibition suggesting that mL/kg. The vehicle, DMSO, and the test articles, NTP-2024 NTP-2014 acts a noncompetitive inhibitor of these GABA and NTP-2006, were administered via a single IP dose at a 10 receptors. dose volume of 1 mL/kg. FIG. 38 is a schematic illustration of a possible mecha FIG. 30 illustrates decreased cumulative duration of hip nism for the action of compounds described herein that pocampal discharges in a model of mesial temporal lobe selectively antagonize parasynaptic C. GABA receptor iso epilepsy following administration of bumetanide, furo forms in GABAergic interneurons. In this suggested mecha semide, piretanide, azosemide, and torsemide esters and 15 nism, (1) GABA is released from the pre-synaptic terminal amide analogs (e.g., NTP-2014, NTP-2026, NTP-2024, by activated inhibitory neurons, (2) GABA binds to post NTP-2006, NTP-1007, and NTP-1003). The results below synaptic GABA receptors that activates them thereby are normalized to the pre-dose condition for each of the increasing inhibition (e.g., hyperpolarization of the post treatment group including DMSO and saline vehicle con synaptic neuron), (3) GABA also binds to parasynaptic (e.g., trols. Doses of test article compounds were 50 mg/kg for presynaptic and extrasynaptic) GABA receptors, (4) in one NTP-2014 and NTP-2026 and 150 mg/kg for NTP-2024, possible mechanism of action, compounds described herein NTP-2006, NTP-1007, and NTP-1003. (e.g., NTP-2014) selectively binds to parasynaptic C. Vari FIG. 31 illustrates decreased cumulative number of hip ant GABA receptors (inhibiting the negative feedback pocampal discharges in a model of mesial temporal lobe loop), thus increasing GABA release. This leads to the epilepsy following administration of bumetanide, furo 25 restoration of the balance of excitation and inhibition by semide, piretanide, azosemide, and torsemide esters and increasing the inhibitory stimulus applied to post-synaptic UOS. amide analogs (e.g., NTP-2014, NTP-2026, NTP-2024, FIG. 39A-C illustrates the effect of NTP-2014 on the NTP-2006, NTP-1007, and NTP-1003). The results below current in the C. BY GABA receptor isoform. The ampli are normalized to the pre-dose condition for each of the tude of the current was not increased as agonist agents like treatment group including DMSO and saline vehicle con 30 benzodiazepines do, in fact it was decreased at concentra trols. Doses of test article compounds were 50 mg/kg for tions of GABA that are found in high firing rates. NTP-2014 and NTP-2026 and 150 mg/kg for NTP-2024, FIG. 40A-B illustrates the effect of NTP-2014 on the NTP-2006, NTP-1007, and NTP-1003. current in C/C/C/Cls GABA receptor isoforms. In FIG. 32 illustrates that a select bumetanide derivative, GABA receptor isoforms containing C. C., C.s subunit NTP-2014, was tested for the ability to inhibit NKCC. In 35 subtypes, 10 uMNTP-2014 inhibited these isoforms at high two different in vitro cell reporter systems tested (A7r5 GABA concentrations. immortalized cells and primary cell cultures), NTP-2014 FIG. 41A-C illustrates the inhibiting effect of NTP-2014 showed no inhibition of NKCC1 chloride transport. Even in on the current in C or C containing GABA receptor the presence of a co-transporter activating mitogen (e.g., isoforms: (A) CBY at 0.1 uMGABA, 1 uM GABA, and FGF), NTP-2014 did not inhibit NKCC1 or NKCC2 (data 40 10 uM GABA and (B) CfBY at 0.1 uM GABA, 1 uM not shown). GABA, and 10 uM GABA. FIG. 41C illustrates that the FIG. 33A-C illustrate that select bumetanide derivative, ECso 20.2 uM for NTP-2014 in C or in C containing NTP-2014, was tested for its effect on urine output (33A), GABA receptor isoforms. sodium excretion (33B), and potassium excretion (33C). FIG. 42A-C illustrates a comparison of bumetanide (A) NTP-2014 does not increase urine output, sodium excretion, 45 and NTP-2014 (B) on the inhibition of O?3y GABA and potassium excretion. receptor isoforms at 100 uM GABA. In contrast to NTP 2014, bumetanide had no-effect on the GABA receptor FIG. 34A-B illustrates that select bumetanide derivative, isoform up to a concentration of 100 uM. This effect is NTP-2014, showed a marked increase in spontaneous IPSCs shown graphically in FIG. 42C, where NTP-2014 shows an in a hippocampal slice model where NTP-2014 showed a ICso 16 uM. significant decrease in the time between inhibitory events 50 FIG. 43 illustrates that in a mouse model of severe (e.g., increased frequency of events). NTP-2014 decreased neuropathic pain (e.g. late phase formalin paw model), the mean interval between inhibitory events from 257 ms to multiple NTP compounds were more effective or compa 205 ms (FIG. 34A) and 535 to 401 ms (FIG. 34B) in two rable to gabapentin at relieving pain. experiments. FIG. 44A-D illustrates that NTP-2014 showed effective FIG. 35A-B illustrates that select bumetanide derivative, 55 analgesic effects in a mouse model of nociceptive pain (e.g., NTP-2014, showed a marked increase in miniature IPSCs in tail flick test). a hippocampal slice model where NTP-2014 showed a FIG. 45 illustrates that NTP-2014 showed effective anal significant decrease in the time between inhibitory events gesic effects in a model of chemotherapy-induced (pacli (e.g., increased frequency of events). NTP-2014 decreased taxol) neuropathic pain. the mean interval between inhibitory events from 315 ms to 60 FIG. 46A-C illustrates that NTP-2014 showed effective 162 ms (FIG. 35A) and 361 to 160 ms (FIG. 35B) in two analgesic effects in a mouse, Chung model of neuropathic experiments. pa1n. FIG. 36A-C illustrates that classic GABAergic drugs (e.g., benzodiazepines) are agonists that increase both the DETAILED DESCRIPTION amplitude and time course of inhibitory currents in al 65 Subunit containing GABA receptors. As shown in (A) The foregoing and other aspects of the present invention FIRISUM (clobazam) an anticonvulsant, (B) AMBIEN will now be described in more detail with respect to embodi US 9,682,928 B2 59 60 ments described herein. It should be appreciated that the “Alkaryl' as used herein refers broadly to a straight or invention can be embodied in different forms and should not branched chain, Saturated hydrocarbon radical bonded to an be construed as limited to the embodiments set forth herein. aryl group. Examples of alkaryl groups include, but are not Rather, these embodiments are provided so that this disclo limited to, benzyl, 4-chlorobenzyl, methylbenzyl, dimethyl sure will be thorough and complete, and will fully convey benzyl, ethylphenyl, propyl-(4-nitrophenyl), and the like. the scope of the invention to those skilled in the art. Such alkaryl groups may be optionally substituted described Definitions herein. The terminology used in the description of the invention “Alkylene' as used herein refers broadly to a straight or herein is for the purpose of describing particular embodi branched chain having two terminal monovalent radical ments only and is not intended to be limiting of the inven 10 centers derived by the removal of one hydrogen atom from tion. As used in the description of the embodiments of the each of the two terminal carbon atoms of straight-chain invention and the appended claims, the singular forms “a.” parent alkane. “an and “the are intended to include the plural forms as "Aryl or “Ar” as used herein refers broadly to an well, unless the context clearly indicates otherwise. Also, as aromatic group or to an optionally Substituted aromatic used herein, “and/or refers to and encompasses any and all 15 group fused to one or more optionally substituted aromatic possible combinations of one or more of the associated listed groups, optionally Substituted with Suitable Substituents items. Furthermore, “about, as used herein when referring including, but not limited to, lower alkyl, lower alkoxy, to a measurable value Such as an amount of a compound, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfo dose, time, temperature is meant to encompass variations of nyl, Oxo, hydroxy, mercapto, amino optionally Substituted 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified by alkyl, carboxy, carbamoyl optionally substituted by alkyl, amount. Unless otherwise defined, all terms, including tech aminosulfonyl optionally Substituted by alkyl, acyl, aroyl, nical and Scientific terms used in the description, have the heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycar same meaning as commonly understood by one of ordinary bonyl, nitro, cyano, halogen, or lower perfluoroalkyl, mul skill in the art to which this invention belongs. tiple degrees of substitution being allowed. Examples of aryl All publications, patent applications, patents, and other 25 include, but are not limited to, phenyl, 2-naphthyl, 1-naph references mentioned herein are incorporated by reference in thyl, and the like. their entirety. "Alkoxy” as used herein alone or as part of another group, “Administration' as used herein, refers broadly to any refers broadly to an alkyl group, as defined herein, appended means by which a composition is given to a patient. A to the parent molecular moiety through an oxy group. In preferred route of administration is oral, and unless other 30 Some embodiments, the alkyl group can be interrupted by wise indicated, any reference herein to “administration one or more heteroatoms (e.g., O, S, or N). Examples of includes "oral administration.” alkoxy include, but are not limited to, methoxy, ethoxy, “Alkenyl as used herein, refers broadly to a straight or propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexy branched chain hydrocarbon radical having one or more loxy, ethyloxyethyl, and the like. double bonds and containing from 2 to 20 carbon atoms. 35 “Alkaryloxy” or "oxyalkaryl as used herein refers Examples of alkenyl groups include propenyl, butenyl, broadly to the group —O-alkyl-aryl wherein Ar is aryl. pentenyl, and the like. “Cycloalkenyl' or “cyclic alkenyl as Examples include, but are not limited to, benzyloxy, oxy used herein refers to carbocycles containing no heteroatoms, benzyl, 2-naphthyloxy, and oxy-2-naphthyl. and includes mono-, bi-, and tricyclic Saturated carbocycles, “Alkaryloxyalkyl or “alkyloxyalkaryl as used herein as well as fused rings systems. Examples of cycloalkenyl 40 refers broadly to the group -alkyl-O-alkyl-aryl wherein Aris groups include cyclopropenyl, cyclopentenyl, cyclohexenyl, aryl. Examples include, but are not limited to benzyloxy cyclopentadienyl, cyclohexadienyl, and the like. Such alk ethyl. enyl and cycloalkenyl groups may be optionally Substituted “Analogs,” as used herein, refer broadly to the modifica as described herein. tion or Substitution of one or more chemical moieties on a “Alkyl as used herein refers broadly to a straight or 45 parent compound and may include derivatives, positional branched chain saturated hydrocarbon radical. “Alkyl also isomers, and prodrugs of the parent compound. refers broadly to cyclic (i.e., cycloalkyl) alkyl groups. "Aryloxy” as used herein refers broadly to the group Examples of alkyl groups include, but are not limited to, —ArO wherein Ar is aryl or heteroaryl. Examples include, straight chained alkyl groups including methyl, ethyl, n-pro but are not limited to, phenoxy, benzyloxy, and 2-maphth pyl. n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and 50 yloxy. branched alkyl groups including isopropyl, tert-butyl, iso “Amino” as used herein refers broadly to NH in which amyl, neopentyl, iso-amyl, and the like. "Cycloalkyl or one or both of the hydrogen atoms may optionally be “cyclic alkyl as used herein refers to carbocycles contain replaced by alkyl or aryl or one of each, optionally Substi ing no heteroatoms, and includes mono-, bi- and tricyclic tuted. saturated carbocycles, as well as fused ring systems. 55 “Alkylthio’ or “thioalkyl, as used herein alone or as part Examples of cycloalkyl include cyclopropyl, cyclobutyl, of another group, refers broadly to an alkyl group, as defined cyclopentyl, cyclohexyl, cycloheptyl, and the like. The herein, appended to the parent molecular moiety through a cycloalkyl can be substituted or unsubstituted, and cyclic sulfur moiety. Representative examples of alkylthio include, alkyl groups including cyclopropyl, cyclopentyl, cyclo but are not limited to, methylthio, thiomethyl, ethylthio. hexyl, cycloheptyl, and the like. Such alkyl groups may be 60 thioethyl, n-propylthio, thio-n-propyl, isopropylthio, thio optionally substituted as described herein. isopropyl. n-butylthio, thio-n-butyl, and the like. “Alkylcyano” refers broadly to a straight or branched "Arylthio’ or “thioaryl, as used herein refers broadly to chain, Saturated or partially unsaturated hydrocarbon radical the group —ArS wherein Ar is aryl. Examples include, but bonded to a cyano (i.e., C=N) group. are not limited to, phenylthio, thiophenyl, 2-naphthylthio. “Alkylhalo refers broadly to a straight or branched chain, 65 and thio-2-naphthyl. saturated or partially unsaturated hydrocarbon radical “Alkarylthio’ or “thioalkaryl” as used herein refers bonded to a halogen (e.g., fluoro, chloro, bromo, and iodo). broadly to the group —S-alkyl-aryl wherein Ar is aryl. US 9,682,928 B2 61 62 Examples include, but are not limited to, benzylthio, thio carbon. The five-membered rings have two double bounds, benzyl, 2-naphthylthio, and thio-2-naphthyl. and the six-membered rings have three double bonds. The “Alkylheterocycloalkyl as used herein refers to as used heteroaryl group can be monocyclic or bicyclic (fused or herein refers broadly to a straight or branched chain, Satu non-fused). Examples of monocyclic heteroaryl groups rated hydrocarbon radical bonded to a heterocycloalkyl include furanyl, thiophene-yl, pyrrolyl, oxazolyl, thiazolyl, group. imidazolyl pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, “Biocompatible polymeras used herein refers broadly to thiazolyl, thiadiazolyl, pyridinyl, pyridaZinyl, pyrimidinyl, a polymer moiety that is Substantially non-toxic and does not pyrazinyl, triazinyl, and the like. Examples of bicyclic tend to produce Substantial immune responses, clotting or heteroaryl groups include indolizinyl, indolyl, isoindolyl, other undesirable effects. Accordingly to some embodiments 10 benzofuranyl, benzothiophene-yl, indazolyl, benzimida of the present invention, polyalkylene glycol is a biocom Zolyl, benzthiazolyl, purinyl, quinolinyl, isoquinolinyl, cin patible polymer where, as used herein, polyalkylene glycol nolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, napthy refers to straight or branched polyalkylene glycol polymers ridinyl, pteridinyl, and the like. The heteroaryl group can be Such as polyethylene glycol, polypropylene glycol, and substituted or unsubstituted. polybutylene glycol, and further includes the monoalkyle 15 “Heterocycloalkyl as used herein refers to a cycloalkyl ther of the polyalkylene glycol. In some embodiments of the group where at least one of the carbon atoms in the ring is present invention, the polyalkylene glycol polymer is a replaced by a heteroatom (e.g., O, S, or N). The heterocy lower alkyl polyalkylene glycol moiety such as a polyeth cloalkyl group can be monocyclic or bicyclic (fused or ylene glycol moiety (PEG), a polypropylene glycol moiety, non-fused). Examples of monocyclic heterocycloalkyl or a polybutylene glycol moiety. PEG has the formula groups include aZetidinyl, pyrrolidinyl, piperidinyl, homopi —HO(CHCHO),H, where n can range from about 1 to peridinyl, piperazinyl, morpholinyl, thiomorpholinyl, about 4000 or more. In some embodiments, n is 1 to 100, and 1-oxothiomorpholinyl, 1,1-dioxothiomorpholinyl, tetrahy in other embodiments, n is 5 to 30. The PEG moiety can be drooxazolyl, tetrahydroisoxazolyl, tetrahydroimidazolyl, linear or branched. In further embodiments, PEG can be tetrahydropyrazolyl, tetrahydrothiazolidinyl, tetrahydroiso attached to groups such as hydroxyl, alkyl, aryl, acyl, or 25 thiazolidinyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, ester. In some embodiments, PEG can be an alkoxy PEG, 4-piperadonyl, and the like. Examples of bicyclic non-fused such as methoxy-PEG (or mPEG), where one terminus is a heterocycloalkyl groups include quinuclidinyl, adamantyl, relatively inert alkoxy group, while the other terminus is a 2-azobicyclo[3.2.1]octyl, and the like. hydroxyl group. Examples of fused heterocycloalkyl groups include any of “Bioavailability’, as used herein, refers broadly to the 30 the aforementioned monocyclic heterocycloalkyl groups availability of a drug to an animal following administration fused with another cycloalkyl or heterocycloalkyl group. and may be used interchangeably with "systemic exposure” The heterocycloalkyl group can be substituted or unsubsti (e.g., the bioavailability of a drug is expressed as the tuted. systemic exposure of a cell to drugs). “Hydrate” as used herein refers broadly to the compound “Carboxy” as used herein refers broadly to the group 35 when the solvent is water. —CO.H. “Increased' or “increase' as used herein, refers broadly to “Cycloalkyl as used herein refers to carbocycles con a quantified change in a measurable quality that is larger taining no heteroatoms, and includes mono-, bi- and tricyclic than the margin of error inherent in the measurement tech saturated carbocycles, as well as fused ring systems. nique, preferably an increase by about 2-fold or greater Examples of cycloalkyl include cyclopropyl, cyclobutyl, 40 relative to a control measurement, more preferably an cyclopentyl, cyclohexyl, and the like. The cycloalkyl can be increase by about 5-fold or greater, and most preferably an substituted or unsubstituted. increase by about 10-fold or greater. In particular, the term “Effective amount’ or “effective,” as used herein, refers “increase,” as used herein, refers broadly to make greater, as broadly to a dose that causes a relief of symptoms of a in number, size, strength, or quality; add to; and/or augment. disease or disorder as noted through clinical testing and 45 “Increase, as used herein, also encompasses expand, evaluation, patient observation, and/or the like. “Effective extend, prolong, augment, enlarge, grow, develop, and/or amount’ or “effective” further can further designate a dose Swell. “Increase,” as used herein, additionally encompasses that causes a detectable change in biological or chemical where a given parameter (e.g., level, amount, size, scope, activity. The detectable changes may be detected and/or duration, weight) is greater, as in number, size, strength, or further quantified by one skilled in the art for the relevant 50 quality, than it once was. Furthermore, the “increase' in any mechanism or process. Moreover, “effective amount’ or number, size, strength, or quality of a given parameter may “effective' can designate an amount that maintains a desired be determined as between to two or more time points, physiological state, i.e., reduces or prevents significant especially if before or after a treatment, event, or adminis decline and/or promotes improvement in the condition of tration of an agent or composition. Further, “increase' refers interest. As is generally understood in the art, the dosage will 55 broadly to significant or detectable, functionally, analyti vary depending on the administration routes, symptoms, and cally, and/or clinically, changes in the number, size, strength, body weight of the patient but also depending upon the or quality of a given parameter in question. compound being administered. “Mammal’ as used herein, refers broadly to any and all "Halo' as used herein refers broadly to bromo, chloro, warm-blooded vertebrate animals of the class Mammalia, fluoro, or iodo. Alternatively, the term “halide” as used 60 including humans, characterized by a covering of hair on the herein refers broadly to bromide, chloride, fluoride, or skin and, in the female, milk-producing mammary glands for iodide. nourishing the young. Examples of mammals include but are “Hydroxy' as used herein refers broadly to the group not limited to alpacas, armadillos, capybaras, cats, chimpan OH. Zees, chinchillas, cattle, dogs, goats, gorillas, hamsters, “Heteroaryl as used herein refers to an aromatic five- or 65 horses, humans, lemurs, llamas, mice, non-human primates, six-membered ring where at least one atom consists of a pigs, rats, sheep, shrews, and tapirs. Mammals include but heteroatom (e.g., O, S, or N), and the remaining atoms are are not limited to bovine, canine, equine, feline, murine, US 9,682,928 B2 63 64 ovine, porcine, primate, and rodent species. Mammal also “Protective,” as used herein, refers broadly to reducing includes any and all those listed on the Mammal Species of the incidence or severity of the disease in a patient. Protec the World maintained by the National Museum of Natural tive, as used herein, refers broadly to inhibiting the disease, History, Smithsonian Institution in Washington D.C., hereby arresting the development of the disease or its clinical incorporated by reference in its entirety. symptoms, and/or causing regression of the disease or its “N-oxide’ or "amine N-oxide’ as used herein refers clinical symptoms. Prevention also preferably includes pre broadly to a chemical structure having an N-O bond where venting or reducing incidence or severity of disease in a the nitrogen is positively charged and the oxygen is nega patient. tively charged. “Protective effect amount,” as used herein, refers broadly 10 to the amount of a compound that, when administered to a “N-substituted sulfonamide' as used herein refers broadly patient reduces the severity of the incidence of signs and/or to a chemical structure having the -SO. NH(R) group. In symptoms, slows the development of the incidence of signs this context, the R-group includes, but is not limited to lower alkyl (e.g., C-C alkyl), lower alkenyl (e.g., C-C alkenyl), and/or symptoms, prevents the development of the incidence alkaryl, aryl, cycloalkenyl, cycloalkyl, dialkylaminoalkyl, of signs and/or symptoms. The “protective effective 15 amount may vary depending on the disease and its severity heterocycloalkyl, and heteroaryl. and the age, weight, medical history, predisposition to “N,N-disubstituted sulfonamide' as used herein refers conditions, preexisting conditions, of the patient to be broadly to a chemical structure having the —SO. NRR' treated. group. In this context, the R and R' are the same or different “Quaternary ammonium' as used herein refers broadly to and are independently lower alkyl, lower alkenyl, alkaryl, a chemical structure having four bonds to the nitrogen with aryl, cycloalkenyl, cycloalkyl, dialkylaminoalkyl, heterocy a positive charge on the nitrogen in the "onium’ state, i.e., cloalkyl, heteroaryl or taken together with the nitrogen atom "RN" or "quaternary nitrogen,” wherein R is an organic to which they are attached form a 4-8 member cycle which Substituent such as alkyl or aryl. The term "quaternary can be substituted or unsubstituted and can have one or more ammonium salt as used herein refers broadly to the asso heteroatoms (e.g., N, O, or S). 25 ciation of the quaternary ammonium cation with an anion. “Parasynaptic” as used herein, refers broadly to receptors “Signs of disease, as used herein, refers broadly to any (e.g., GABA receptors) located outside or in the perimeter abnormality indicative of disease, discoverable on exami of the synapse (e.g., synaptic cleft). Also, “parasynaptic' nation of the patient; an objective indication of disease, in refers broadly to any receptors located perisynaptically, contrast to a symptom, which is a subjective indication of extrasynaptically, and presynaptically. 30 disease. “Patient as used herein, refers broadly to any animal who “Symptoms of disease as used herein, refers broadly to is in need of treatment either to alleviate a disease state or any morbid phenomenon or departure from the normal in to prevent the occurrence or reoccurrence of a disease state. structure, function, or sensation, experienced by the patient Also, “Patient’ as used herein, refers broadly to any animal and indicative of disease. who has risk factors, a history of disease, Susceptibility, 35 “Subjects” as used herein, refers broadly to anyone suit symptoms, signs, was previously diagnosed, is at risk for, or able to be treated according to the present invention include, is a member of a patient population for a disease. The patient but are not limited to, avian and mammalian Subjects, and may be a clinical patient Such as a human or a veterinary are preferably mammalian. Mammals of the present inven patient such as a companion, domesticated, livestock, exotic, tion include, but are not limited to, canines, felines, bovines, or Zoo animal. Animals may be mammals, reptiles, birds, 40 caprines, equines, Ovines, porcines, rodents (e.g., rats and amphibians, or invertebrates. mice), lagomorphs, primates, humans, and the like, and “Pharmaceutically acceptable salt as used herein, refers mammals in utero. Any mammalian Subject in need of being broadly to a salt form of a compound permitting its use or treated according to the present invention is suitable. Human formulation as a pharmaceutical and which retains the Subjects are preferred. Human Subjects of both genders and biological effectiveness of the free acid and base of the 45 at any stage of development (i.e., neonate, infant, juvenile, specified compound and that is not biologically or otherwise adolescent, adult) can be treated according to the present undesirable. invention. “Prophylactically effective amount’ as used herein, refers The present invention can also be carried out on animal broadly to the amount of a compound that, when adminis Subjects, particularly mammalian Subjects such as mice, rats, tered to a patient for prophylaxis of a disease or prevention 50 dogs, cats, cattle, goats, sheep, and horses for veterinary of the reoccurrence of a disease, is sufficient to effect such purposes, and for drug screening and drug development prophylaxis for the disease or reoccurrence. The prophylac purposes. The present invention can also be carried out on tically effective amount may be an amount effective to avians including chickens, ducks, turkeys, geese, quail, prevent the incidence of signs and/or symptoms. The “pro pheasant, ratites (e.g., ostrich) and domesticated birds (e.g., phylactically effective amount may vary depending on the 55 parrots and canaries), and birds in ovo. “Subjects” is used disease and its severity and the age, weight, medical history, interchangeably with “patients.” predisposition to conditions, preexisting conditions, of the “Solvate” as used herein refers broadly is intended to refer patient to be treated. to a pharmaceutically acceptable Solvate form of a specified “Prophylaxis.” as used herein, refers broadly to a course compound that retains the biological effectiveness of Such of therapy where signs and/or symptoms are not present in 60 compound, for example, resulting from a physical associa the patient, are in remission, or were previously present in a tion of the compound with one or more solvent molecules. patient. Prophylaxis includes preventing disease occurring Examples of Solvates, without limitation, include com Subsequent to treatment of a disease in a patient. Further, pounds of the invention in combination with water, 1-pro prevention includes treating patients who may potentially panol. 2-propanol, ethanol, methanol, DMSO, ethyl acetate, develop the disease, especially patients who are susceptible 65 acetic acid, or ethanolamine. to the disease (e.g., members of a patent population, those “Substituted as used herein refers broadly to replacement with risk factors, or at risk for developing the disease). of one or more of the hydrogen atoms of the group replaced US 9,682,928 B2 65 66 by substituents known to those skilled in the art and resulting porters, it is intended that the severity of the disorder or the in a stable compound as described below. Examples of symptoms of the disorder are reduced, or the disorder is Suitable replacement groups include, but are not limited to, partially or entirely eliminated, as compared to that which alkyl, acyl, alkenyl, alkynyl cycloalkyl, aryl, alkaryl, would occur in the absence of treatment. Treatment does not hydroxy, thio, alkoxy, aryloxy, acyl, amino, amido, carboxy, require the achievement of a complete cure of the disorder. carboxyalkyl, thiocarboxyalkyl, carboxyaryl, thiocarbox By the terms “preventing or “prevention' of the disorder yaryl, halo, Oxo, mercapto, Sulfinyl, Sulfonyl, Sulfonamido, involving the NaKCl co-transporters, it is intended that amidino, carbamoyl, cycloalkyl, heterocycloalkyl, dialky the inventive methods eliminate or reduce the incidence or laminoalkyl, carboxylic acid, carboxamido, haloalkyl, diha onset of the disorder, as compared to that which would occur loalkyl, trihaloalkyl, trihaloalkoxy, alkylthio, aralkyl, alkyl 10 in the absence of treatment. Alternatively stated, the present methods slow, delay, control, or decrease the likelihood or Sulfonyl, arylthio, amino, alkylamino, dialkylamino, probability of the disorder in the subject, as compared to that guanidino, ureido, nitro and the like. Substitutions are per which would occur in the absence of treatment. Further, the missible when Such combinations result in compounds terms “treating or “treatment of a disorder involving the stable for the intended purpose. For example, substitutions GABA receptor, are intended that the severity of the are permissible when the resultant compound is sufficiently 15 disorder or the symptoms of the disorder are reduced, or the robust to survive isolation to a useful degree of purity from disorder is partially or entirely eliminated, as compared to a reaction mixture, and formulation into a therapeutic or that which would occur in the absence of treatment. Treat diagnostic agent or reagent. ment does not require the achievement of a complete cure of “Therapy’ or “therapeutic' as used herein refers broadly the disorder. to treating a disease, arresting or reducing the development “Therapeutically effective amount’ as used herein, refers of the disease or its clinical symptoms, and/or relieving the broadly to the amount of a compound that, when adminis disease, causing regression of the disease or its clinical tered to a patient for treating a disease, is sufficient to effect symptoms. Therapy encompasses prophylaxis, prevention, such treatment for the disease. Therapeutically effective treatment, cure, regimen, remedy, minimization, reduction, amount can be an amount effective for prophylaxis, and/or alleviation, and/or providing relief from a disease, signs, 25 an amount effective for prevention. Therapeutically effective and/or symptoms of a disease. Therapy encompasses an amount can be an amount effective to reduce, an amount alleviation of signs and/or symptoms in patients with ongo effective to prevent the incidence of signs/symptoms, to ing disease signs and/or symptoms (e.g., pain, inflamma reduce the severity of the incidence of signs/symptoms, to tion.) Therapy also encompasses “prophylaxis' and “pre eliminate the incidence of signs/symptoms, to slow the vention.” Prophylaxis includes preventing disease occurring 30 development of the incidence of signs/symptoms, to prevent Subsequent to treatment of a disease in a patient or reducing the development of the incidence of signs/symptoms, and/or the incidence or severity of the disease in a patient. The term effect prophylaxis of the incidence of signs/symptoms. The “reduced.” for purpose of therapy, refers broadly to the “therapeutically effective amount' can vary depending on clinical significant reduction in signs and/or symptoms. the disease and its severity and the age, weight, medical Therapy includes treating relapses or recurrent signs and/or 35 history, predisposition to conditions, preexisting conditions, symptoms (e.g., of pain.) Therapy encompasses but is not of the patient to be treated. The term “effective amount” is limited to precluding the appearance of signs and/or symp taken to be synonymous with “therapeutically effective toms anytime as well as reducing existing signs and/or amount for purposes of this invention. symptoms and eliminating existing signs and/or symptoms. 1. Compounds Therapy includes treating chronic disease (“maintenance') 40 According to Some embodiments, the present invention and acute disease. provides novel compounds. Thus, any of the R groups as Therapy can be for patients with risk factors, at risk defined herein can be excluded or modified in order to patients in a Susceptible population, patients with a history exclude a known compound and/or provide a novel com of disease, patients with symptoms, patients with signs, pound. Compounds of the present invention can include patients with signs but no symptoms, and patients with 45 compounds according to formula I, II, III, IV, V, VI, VII, symptoms but no signs. Therapy can also be for patients VIII, IX, X, XI, XII, XII, XIV, XV, and/or XVI: without risk factors, not at risk, patients not in a susceptible population, patients no history of disease, patients with no symptoms, patients without signs. Therapy can alleviate, allay, abate, assuage, curtail, decrease, ease, lessen, lighten, 50 O RR2 make better, make healthy, mitigate, mollify, pacify, relieve, rehabilitate, remedy, repair, and/or soothea disease, disease signs, and/or disease symptoms. “Treating or “treatment,” as used herein, refers broadly to a course of therapy where signs and/or symptoms are 55 present in the patient. The term “reduced.” for purpose of RsRNOSsRNO H therapy, refers broadly to clinically significant reduction in R3 signs and/or symptoms. Treatment includes treating chronic II disease (“maintenance') and acute disease. Treatment can be S RR for patients with risk factors, at risk patients in a susceptible 60 population, patients with a history of disease, and/or patients with symptoms, patients with signs. Treatment can alleviate, allay, abate, assuage, curtail, decrease, ease, lessen, lighten, make better, make healthy, mitigate, mollify, pacify, relieve, rehabilitate, remedy, repair, and/or soothea disease, disease 65 RsRNOS 1n 1ch, signs, and/or disease symptoms. By the terms “treating” or R3 “treatment of a disorder involving the Na"K"Cl co-trans US 9,682,928 B2 67 68 -continued phenyloxy when R is O and R. R. and Rs are H. more III specifically, in Some embodiments, the compound of for O RR mula I is not bumetanide; R of formula III is not Cl, when R is O and R. R. and Rs are H. more specifically, in some embodiments, the compound of formula III is not furo semide; R of formula III is not methyl when R is O, R is Cl, and Ra and Rs are H. more specifically, in some embodi RsRNOS ments, the compound of formula III is not furosemide methyl ester, R of formula V is not phenyloxy when R is R3 s 10 O and R. R. and Rs are H. more specifically, in some IV embodiments, the compound of formula V is not piretanide. S RR In some embodiments of the present invention, the com pound of formula I can be bumetanide, bumetanide alde N O hyde, bumetanide methyl ester, bumetanide cyanomethyl 15 ester, bumetanide ethyl ester, bumetanide isoamyl ester, bumetanide octyl ester, bumetanide benzyl ester, RsRNOS bumetanide dibenzylamide, bumetanide diethylamide, bumetanide morpholinoethyl ester, bumetanide 3-(dimeth R3 s ylaminopropyl) ester, bumetanide N,N-diethylglycolamido V ester, bumetanide N,N-dimethylglycolamido ester, O RR2 bumetanide pivaxetil ester, bumetanide propaxetil ester, bumetanide methoxy(polyethyleneoxy)-ethyl ester, bumetanide benzyltrimethylammonium salt and bumetanide cetyltrimethylammonium salt. In particular embodiments, 25 the compound is not bumetanide. RsRNOS N In other embodiments of the present invention, the com pound of formula I can be bumetanide —(C=O) SH R3 and thioacid, bumetanide S-methylthioester, bumetanide S-cya VI nomethyl thioester, bumetanide S-ethyl thioester, S RR2 30 bumetanide S-isoamyl thioester, bumetanide S-octyl thio ester, bumetanide S-benzyl thioester, bumetanide S-(mor pholinoethyl)thioester, bumetanide S-3-(dimethylamino propyl) thioester, bumetanide S (N.N- diethylglycolamido)thioester, bumetanide S (N.N- RsRNOS N 35 dimethylglycolamido)thioester, bumetanide S-pivaxetil thioester, bumetanide S-propaxetil thioester, bumetanide R3 s S-methoxy(polyethyleneoxy), -ethylthioester, bumetanide —(C=O)—S benzyltrimethylammonium thioacid salt and bumetanide —(C=O)—S cetyltrimethylammo or a pharmaceutically acceptable salt, Solvate, tautomer or 40 nium thioacid salt. hydrate thereof, wherein In some embodiments of the present invention, the com R is not present, H, O or S; pound of formula II can be metastable bumetanide —(C— R is not present, H or when R is O or S, R is selected S)—OH thioacid, bumetanide O-methyl thioester, from the group consisting of hydrogen, alkyl, aralkyl, aryl, bumetanide O-cyanomethyl thioester, bumetanide O-ethyl alkylaminodialkyl, alkylcarbonylaminodialkyl, alkyloxycar 45 thioester, bumetanide O-isoamyl thioester, bumetanide bonylalkyl, alkylcarbonyloxyalkyl, alkylaldehyde, alkylke O-octyl thioester, bumetanide O-benzyl thioester, toalkyl, alkylamide, alkarylamide, arylamide, an alkylam bumetanide O-(morpholinoethyl)thioester, bumetanide monium group, alkylcarboxylic acid, alkylheteroaryl, O-3-(dimethylaminopropyl)thioester, bumetanide O (N, alkylhydroxy, a biocompatible polymer Such as alkyloxy N-diethylglycolamido)thioester, bumetanide, O—(N,N-di (polyalkyloxy)alkylhydroxyl, a polyethylene glycol (PEG), 50 methylglycolamido)thioester, bumetanide O-pivaxetil thio a polyethylene glycol ester (PEG ester) and a polyethylene ester, bumetanide O-propaxetil thioester, bumetanide glycol ether (PEG ether), methyloxyalkyl, methyloxy O-methoxy(polyethyleneoxy)-ethyl thioester, alkaryl, methylthioalkyl and methylthioalkaryl, unsubsti bumetanide —(C=S)—O— benzyltrimethyl ammonium tuted or substituted, and when R is not present, R is thioacid salt and bumetanide (C S)—O— cetyltrimeth selected from the group consisting of hydrogen, N,N-dialky 55 ylammonium thioacid salt. lamino, N,N-dialkarylamino, N,N-diarylamino, N-alkyl-N- In some embodiments of the present invention, the com alkarylamino, N-alkyl-N-arylamino, N-alkaryl-N-ary pound of formula H can be bumetanide thioaldehyde, lamino, unsubstituted or substituted; bumetanide —(C=S)—SH dithioacid, bumetanide methyl R is selected from the group consisting of aryl, halo, dithioester, bumetanide cyanomethyl dithioester, hydroxy, alkoxy, and aryloxy, unsubstituted or Substituted; 60 bumetanide ethyl dithioester, bumetanide isoamyl dithio and ester, bumetanide octyl dithioester, bumetanide benzyl dith R and Rs are each independently selected from the group ioester, bumetanide dibenzylthioamide, bumetanide diethyl consisting of hydrogen, alkylaminodialkyl, carbonylalkyl, thioamide, bumetanide morpholinoethyl dithioester, carbonylalkaryl, carbonylaryl, and salts thereof Such as bumetanide 3-(dimethylaminopropyl)dithioester, Sodium, potassium, calcium, ammonium, trialkylarylammo 65 bumetanide N,N-diethylglycolamido dithioester, nium and tetraalkylammonium salts, with the following bumetanide N,N-dimethylglycolamido dithioester, provisos in Some embodiments: R of formula I is not bumetanide pivaxetil dithioester, bumetanide propaxetil US 9,682,928 B2 69 70 dithioester, bumetanide methoxy (polyethylencoxy)-1-ethyl octyl ester, piretanide benzyl ester, piretanide dibenzylam dithioester, bumetanide benzyltrimethylammonium dithio ide, piretanide diethylamide, piretanide morpholinoethyl acid salt and bumetanide cetyltrimethylammonium dithio ester, piretanide 3-(dimethylaminopropyl) ester, piretanide acid salt. N,N-diethylglycolamide ester, piretanide dimethylglycol In other embodiments of the present invention, the com amide ester, piretanide pivaxetil ester, piretanide propaxetil pound of formula III can be furosemide, furosemide alde ester, piretanide methoxy(polyethyleneoxy)-ethyl ester, hyde, furosemide methyl ester, furosemide cyanomethyl piretanide benzyltrimethylammonium salt and piretanide ester, furosemide ethyl ester, furosemide isoamyl ester, cetyltrimethylammonium salt. In particular embodiments, furosemide octyl ester, furosemide benzyl ester, furosemide the compound is not piretanide. Piretanide monoalkyl (Sub dibenzylamide, furosemide diethylamide, furosemide mor 10 stituted and unsubstituted) amides and thioamides and phar pholinoethyl ester, furosemide 3-(dimethylaminopropyl) maceutically acceptable salts thereof are also contemplated. ester, furosemide N,N-diethylglycolamido ester, furosemide Non-limiting examples of such piretanide monoalkyl amides N,N-dimethylglycolamido ester, furosemide pivaxetil ester, include piretanide monobenzylamide, piretanide monoeth furosemide propaxetil ester, furosemide methoxy (polyeth ylamide, piretanide monobenzylthioamide, piretanide yleneoxy)-ethyl ester, furosemide benzyltrimethylammo 15 monoethylthioamide, and pharmaceutically acceptable salts nium acid salt and furosemide cetyltrimethylammonium thereof. acid salt. In particular embodiments, the compound is not In some embodiments of the present invention, the com furosemide. pound of formula V can be piretanide —(C=O) SH In further embodiments of the present invention, the thioacid, piretanide S-methyl thioester, piretanide S-cya compound of formula III can be furosemide —(C=O)— nomethyl thioester, piretanide S-ethyl thioester, piretanide SHI thioacid, furosemide S-methyl thioester, furosemide S-isoamyl thioester, piretanide S-octyl thioester, piretanide S-cyanomethylthioester, furosemide S-ethylthioester, furo S-benzyl thioester, piretanide S-(morpholinoethyl)thioester, semide S-isoamyl thioester, furosemide S-octyl thioester, piretanide S-3-(dimethylaminopropyl)thioester, piretanide furosemide S-benzyl thioester, furosemide S-(morpholino S—(N,N-diethylglycolamido)thioester, piretanide S (N.N- ethyl)thioester, furosemide S-3-(dimethylaminopropyl) 25 dimethylglycolamido)thioester, piretanide S-pivaxetil thio thioester, furosemide S (N,N-diethylglycolamido)thio ester, piretanide S-propaxetil thioester, piretanide ester, furosemide S (N,N-dimethylglycolamido)thioester, S-methoxy (polyethyleneoxy)--ethylthioester, piretanide furosemide S-pivaxetil thioester, furosemide S-propaxetil —(C=O)—S benzyltrimethylammonium thioacid salt thioester, furosemide S-methoxy(polyethyleneoxy)-- and piretanide —(C=O)—S— cetyltrimethylammonium ethylthioester, furosemide —(C=O)—S" benzyltrimeth 30 thioacid salt. ylammonium thioacid salt and furosemide —(C=O)—S" In further embodiments of the present invention, the cetyltrimethylammonium thioacid salt. compound of formula VI can be metastable piretanide In other embodiments of the present invention, the com —(C=S)—OH thioacid, piretanide O-methyl thioester, pound of formula IV can be metastable furosemide piretanide O-cyanomethylthioester, piretanide O-ethylthio —(C=S)—OH thioacid, furosemide O-methyl thioester, 35 ester, piretanide O-isoamyl thioester, piretanide O-octyl furosemide O-cyanomethyl thioester, furosemide O-ethyl thioester, piretanide O-benzyl thioester, piretanide O-(mor thioester, furosemide O-isoamyl thioester, furosemide O-oc pholinoethyl)thioester, piretanide O-3-(dimethylaminopro tyl thioester, furosemide O-benzyl thioester, furosemide pyl) thioester, piretanide O (N,N-diethylglycolamido) O-(morpholinoethyl)thioester, furosemide O-3-(dimethyl thioester, piretanide, O—(N,N-dimethylglycolamido) aminopropyl)thioester, furosemide O (N,N-diethylglyco 40 thioester, piretanide O-pivaxetil thioester, piretanide lamido)thioester, furosemide O (N,N-dimethylglyco O-propaxetil thioester, piretanide O-methoxy (polyethyl lamido)thioester, furosemide O-pivaxetil thioester, eneoxy)--ethyl thioester, piretanide —(C=S)—O— furosemide O-propaxetil thioester, furosemide O-methoxy benzyltrimethylammonium thioacid salt and piretanide (polyethyleneoxy)-1-ethyl thioester, furosemide —(C=S)—O— cetyltrimethylammonium thioacid salt. —(C=S)—O— benzyltrimethylammonium thioacid salt 45 In some embodiments of the present invention, the com and furosemide —(C=S)—O—cetyltrimethylammonium pound of formula VI can be piretanide thioaldehyde, pire thioacid salt. tanide —(C=S)—SH dithioacid, piretanide methyl dith In further embodiments of the present invention, the ioester, piretanide cyanomethyl dithioester, piretanide ethyl compound of formula IV can be furosemide thioaldehyde, dithioester, piretanide isoamyl dithioester, piretanide octyl furosemide —(C=S)—SH dithioacid, furosemide methyl 50 dithioester, piretanide benzyl dithioester, piretanide diben dithioester, furosemide cyanomethyl dithioester, furosemide Zylthioamide, piretanide diethylthioamide, piretanide mor ethyl dithioester, furosemide isoamyl dithioester, furo pholinoethyl dithioester, piretanide 3-(dimethylaminopro semide octyl dithioester, furosemide benzyl dithioester, pyl)dithioester, piretanide N,N-diethylglycolamido furosemide dibenzylamide, furosemide diethylamide, furo dithioester, piretanide N,N-dimethylglycolamido dithio semide dibenzylthioamide, furosemide diethylthioamide, 55 ester, piretanide pivaxetil dithioester, piretanide propaxetil furosemide morpholinoethyl dithioester, furosemide 3-(di dithioester, piretanide methoxy(polyethyleneoxy)--ethyl methylaminopropyl)dithioester, furosemide N,N-diethylgly dithioester, piretanide benzyltrimethylammonium dithioacid colamido dithioester, furosemide N,N-dimethylglycolamido salt and piretanide cetyltrimethylammonium dithioacid salt. dithioester, furosemide pivaxetil dithioester, furosemide Piretanide monoalkyl (substituted and unsubstituted)thio propaxetil dithioester, furosemide methoxy(polyethylene 60 amides and pharmaceutically acceptable salts thereof are oxy)-ethyl dithioester, furosemide benzyltrimethylammo also contemplated. Non-limiting examples of Such pire nium dithioacid salt and furosemide cetyltrimethylammo tanide monoalkyl thioamides include piretanide monoben nium dithioacid salt. Zylthioamide, piretanide monoethylthioamide, and pharma In still further embodiments of the present invention, the ceutically acceptable salts thereof. compound of formula V can be piretanide, piretanide alde 65 In other embodiments of the present invention, the com hyde, piretanide methyl ester, piretanide cyanomethyl ester, pound of formula VII can be tetrazolyl-substituted azosemi piretanide ethyl ester, piretanide isoamyl ester, piretanide des (e.g. methoxymethyl tetrazolyl-substituted azosemides, US 9,682,928 B2 71 72 methylthiomethyl tetrazolyl-substituted azosemides and -continued N-mPEG350-tetrazolyl-substituted azosemides), azosemide O O benzyltrimethylammonium salt, and/or azosemide cetylt rimethylammonium salt. ty/ OH In some embodiments of the present invention, the com pound of formula VIII can be pyridine-substituted torsemide A quaternary ammonium salts or the corresponding inner salts NH (Zwitterions). Examples include, but are not limited to, N-1N1 methoxymethyl pyridinium torsemide salts, methylthiom 10 ethyl pyridinium torsemide salts, and N-mPEG350-pyri O O dinium torsemide Salts. Embodiments of the present invention further provide "a/ OH intermediate compounds formed through the synthetic meth W ods described herein to provide the compounds of Formula 15 I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XVI, s XVII, XVIII, XVI, XIX, XX, XXI, XXII, XXIII, XXIV, N N-1-N-1 XXV, and/or XXVI. The intermediate compounds may O O possess utility as therapeutic agents for the range of indica tions described herein and/or reagents for further synthesis "A/ OH methods and reactions. As noted previously, any of the R groups as defined herein can be excluded from the compounds of the present inven tion, particularly with reference to denoting novel com 25 C. N-1-N-1 pounds of the present invention. O O In some embodiments, the present invention encompasses the following compounds, including esters, amides, N-Sub ty/ OH stituted sulfonamides and N,N-disubstituted sulfonamides 30 thereof: e CS. O N-1N1 O 35 "./ 4. OH H2NN / / OH F N-1N1 40 r O O su N-11 O "./ 4. OH H2NN / 45 M OH O C N-1-N-1 r O O 50 itsu N-1-N-1 ". 4. HN/ O / OH / OH Br 55

N-1-N-1 N HN H n-1N1 O O O H.N./ 60 / OH H.N./ / OH

65 US 9,682,928 B2 73 74 -continued -continued O O O O HN N4 H.N./ / OH 5 / OH 21 s- N11 N-1-N-1 O O 10 H.N./ / OH jo 15 In other embodiments, the present invention encompasses N HN the following compounds, including esters and amides H n1N1 thereof: O O H2NN 4. / OH 2O o O HNN 4. --O N / OH HN H n-1N1 25 O O O H2NN 4. HN / OH n1N1 O N / - - O 30 HNN 4.O O

O O N-1-N-1O O OH H.N./ 35 O y/ OH N-1-N-1 O N-1N1 40 O O O O HNN M H2NN 4. 7. OH M OH O O 45 O S / N-1-N-1

O O O O H.N./ 50 1N 4.

S O 55 - HN n-1N1 HN n-1N1 O H.N./ O O OM OH 60 1NA OH S ? NN-1-N-1 Y weat YO 65 HN n-1N1 US 9,682,928 B2 75 76 -continued -continued O HN OH an/O O O O OH N-1-N-1 O 10 N-1-N-1 O 15 N1 N 4. OCrsO O OH O N-1-N-1 N-1N1 O 25 \a( O

1N1 N 4. 30 OCrO O OH N-1-1 O 35 N-1-N-1 S \4.C O

40 O O OCrsO HN/ N-1-N-1 Ot OH 45 O

N-11 O O O N/ 50

O OH OCrO O 55 N-1-N-1 N-1-N-1 HN A \ s 29 O \== O 60

OCrsO 65 N-1N1 US 9,682,928 B2 77 78 -continued -continued

O 10 N-11

15 co N-11

HN HN 30 O O

HN 40 HN

O O 45

50 HN HN

O O 55

HN 60

65 US 9,682,928 B2 79 80 -continued -continued O H2N2 NsM W O M M 5 O N HN N^ OH O N

O N-1-N-1 10 N-1-N-1 In yet other embodiments, the present invention encom passes the following compounds, including esters, amides, H N N-substituted sulfonamides and N,N-disubstituted sulfona 15 mides thereof: s \ O HN O O OH 2O H.N./ H.N./ M OH / OH

N-1N1 O O

H 25 v O N GS. O

N-1N1 35 N In still other embodiments, the present invention encom- N passes the following compounds: 40 H2NN / - N. / OH

H2NN / HNN/ \ 45 O 'o' O 1

O N11 50 HN / HN1 \ 2'N,7 N/ H2NN / O O S 55 / OH O HN O n1N1 1. O HN N/ 2 60

O O HN n-1N1 65

US 9,682,928 B2 83 84 -continued -continued O O O H.N./ H.N./

OCCO COOO N =-\

OO S In yet other embodiments, the present invention encom O O passes the following compounds, including N-Substituted H.N./ 15 sulfonamides and NN-disubstituted sulfonamides thereof:

O O O 2O H2NN 4. 1

O O O O 25 N-1S-1 H2NN 4.

30 CCC? O O H2NN / 7. N O 35 O O H2NN / O N-1-1

O C HN/ O S 45 OtO H ^\ O H2NN / O OH 50 Nu-1N1

CSN 55 H.N./O O O O / Y\ H.N./ O / OH 60 Nu-1S-1 O US 9,682,928 B2 85 86 -continued -continued HN/ O H2NN / O (S

COrrO CoreO N-1N1 N-1N1 10

O O 15 O O

H.N./ H.N./ K S

OCOriO 2O OCOrrO N-1N1 N-1N1

O O O 30 H.N./ \ H.N./ M N1\- NH M N O H O H O O 35 N-1-N-1 N-1S-1

O O

H2NN/S O 45 OtS.----.O N1H NH)

OCOriO O N n1N1 r 50 55 H.N./O O x(S s HN/ O

OtO r 60 CCOrrO Nu-1-1 N-1-1

65 US 9,682,928 B2 87 88 -continued -continued

EN/ O H2NN S/ O 1.Oy

OCOrr'sO N n1N1 Nu-1S-1 10

15 O O O O H2NN/ H.N./ O H le O OCOriO N-1N1 N-1S-1

O O O O H.N./ 30 H.N./ le OCOroO O 35 N-1S-1 Nu-1N1

O O H.N./ 2N. O

le 45 HN/ O O Nu1n-1 OCOriO 50 N-1a-1

O O 55 HN/ O

60 OCOrr)O N-1-N-1

65 US 9,682,928 B2 89 90 -continued -continued O O O O H.N./ S /N 5 ? OCOrroO O O Nu-1N1 N-1N1 10

O O H.N./ 15

O N-1N1

HN/ O 25

COrrO 30 N-1-1

35 HN/ O O H 40 CorrsO N-1N1 45

In yet other embodiments, the present invention encom 50 passes the following compounds, N-substituted Sulfona mides and N,N-disubstituted sulfonamides thereof:

O O 55 H.N./ /

OOC?,O N-1N1