US 20090005386A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0005386 A1 Abbott et al. (43) Pub. Date: Jan. 1, 2009

(54) METHODS FOR MODULATING ION Related U.S. Application Data CHANNELS (60) Provisional application No. 60/654,531, filed on Feb. (76) Inventors: Geoffrey W. Abbott, Crompond, 18, 2005. NY (US); Robert Engel, Carle O O Place, NY (US); JaimeLee Iolani Publication Classification Rizzo, Glen Cove, NY (US) (51) Int. Cl. A 6LX 3L/21995 (2006.01) Correspondence Address: A6IP 2L/00 (2006.01) Irving N. Feit (52) U.S. Cl...... S14/249 Hoffmann & Baron 6900 Jericho Turnpike (57) ABSTRACT Syosset, NY 11791 (US) In one embodiment, the invention provides an ion having the (21) Appl. No.: 11/884,628 formula: (I) In another embodiment, the invention provides a method for modulating potassium, Sodium, and cyclic nucle (22) PCT Filed: Feb. 17, 2006 otide-modulated ion channels in a mammal in need thereof. In a further embodiment, the invention provides a method for (86). PCT No.: PCT/US2006/005750 modulating ligand-gated ion channels or transient receptor potential channels in a mammal in need thereof. The methods S371 (c)(1), comprise administering an ion having the formula described (2), (4) Date: Jun. 3, 2008 above. GE) BrO N NM (C12) JE188 Patent Application Publication Jan. 1, 2009 Sheet 1 of 5 US 2009/000538.6 A1

FIG. 1A

FIG. 1C 1.O

0.8

0.4

O.2

O.O 10 1OO JE188, uM Patent Application Publication Jan. 1, 2009 Sheet 2 of 5 US 2009/000538.6 A1 FIG. 2A

FIG. 2C 1.O

O.8

0.6 I/Imax 0.4

0.2

O.O 0.01 0.1 1 10 TA279, M Patent Application Publication Jan. 1, 2009 Sheet 3 of 5 US 2009/000538.6 A1 FIG. 3A 4. Ny-i.e. a 6 ei.e. a effice (vica) efécieu) 12 (i.e. e.

2 Kv2.1 Kv3.4 (A1-28) KCNG4

'drug?control 2

2 Patent Application Publication Jan. 1, 2009 Sheet 4 of 5 US 2009/000538.6 A1 FIG. 4A

GE) ()G) 2C O -ICl. G) 2C O ( G) G) ) (SGED GE)3)

ortho (JSG17) meta (TA37) para (JC,638.2a)

FIG. 4B

2 Kv2.1 "Kv3.4 (A1-28) KCNO)4

'drug control 1

Ortho meta para relative positions of didabco groups on aromatic ring Patent Application Publication Jan. 1, 2009 Sheet 5 of 5 US 2009/000538.6 A1 FIG. 5

current, LA

E 250 LM TG28 time, minutes O Washout

FIG. 6

-20 voltage, mV Control D. JC279.3a (opener) o JC638.2a (blocker) US 2009/000538.6 A1 Jan. 1, 2009

METHODS FOR MODULATING ON or branched, and wherein one or more carbon atoms of the CHANNELS acyclic hydrocarbon group is optionally Substituted by one or more heteroatoms, wherein no heteroatom is bound to: another heteroatom, 0001. This application asserts priority to U.S. Provisional V when V is N or N, Application Ser. No. 60/654,531 filed on Feb. 18, 2005, the specification of which is hereby incorporated by reference in V? when V is N', its entirety. V when V is N', or V when V* is N or N'; and BACKGROUND OF THE INVENTION provided that at least one of V, V, V, and V represents N'. 0002 Ion channels are transmembrane proteins which 0006. In another embodiment, the invention provides a generally catalyze the transport of ions across cell mem method for modulating potassium, Sodium, and cyclic nucle branes. The ion channels participate in processes as diverseas otide-modulated ion channels in a mammal in need thereof. the generation and timing of action potentials, synaptic trans The method comprises administering to the mammal an missions, secretion of hormones, contraction of muscles, etc. effective amount of an ion as described above. 0003. The family of cation channels typically mediates 0007. In yet another embodiment, the invention provides a electrical activity. This family includes potassium, Sodium method for modulating ligand-gated ion channels or transient and cyclic nucleotide-modulated ion channels. It is reported receptor potential channels in a mammal in need thereof. The that these ion channels play an important role in many physi method comprises administering to the mammal an effective ological processes, including the regulation of heart rate, amount of an ion as described above. muscle contraction, neurotransmitter release, neuronal excit ability, cell proliferation, etc. Thus, aberrations in the activity BRIEF DESCRIPTION OF THE FIGURES of these channels are responsible for many diseases and con ditions. 0008 FIG. 1. MiRP2 alters Kv2.1 block by JE 188. (A) 0004. Accordingly, it would be beneficial to modulate ion The structure of compound JE 188. (B) Representative traces channels in diseases and conditions characterized by aberrant of current recorded in Xenopus oocytes using TEVC, and activity in potassium, Sodium and cyclic nucleotide-modu voltage protocol in upper left. Oocytes were injected with lated ion channels. Thus, there is a need for a method for cRNA encoding Kv2.1+/-cRNA encoding MiRP2, as indi modulating potassium, Sodium and cyclic nucleotide-modu cated. Scale as indicated in lower left. Control trace (solid lated ion channels in mammals suffering from Such diseases line) was recorded after 5 pulses in 4 mM KCl bath medium and conditions. (pH 7.4) in the absence of drug. Dashed line indicates a trace during pulsing in the presence of 250 uMJE188. (C) Mean SUMMARY OF THE INVENTION current inhibition assessed by comparison of peak current at various drug concentrations with peak current before drug 0005. The above objectives have been met by the present application (n=5-6 oocytes per point; error bars indicate invention which provides, in one embodiment, an ion com SEM). Drug block was reversible upon washout, and traces prising the following formula: that did not recover to control peak current (+/-10%) upon washout were discarded. 0009 FIG.2. TA279 is a potent blocker of Kv2.1 channels. (1) (A) The structure of compound TA 279. (B) Representative traces of currents recorded in Xenopus oocytes using two electrode Voltage clamp, and Voltage protocol in upper left. Oocytes were injected with cRNA encoding Kv2.1+/-cRNA encoding MiRP2, as indicated. Scale as indicated in lower left. Control trace (solid line) was recorded after 5 pulses in N 3 / | N/6 normal 4 mM KC1 bath medium (pH 7.4). Dashed line indi R R m3 cates equilibrium trace during pulsing in the presence of 5uM TA279. (C) Mean current inhibition assessed by comparison wherein: of peak current at various drug concentrations with peak each of R', R. R. R. R. R. independently of each other, current before drug application (n=4-6 oocytes per point; represents an acyclic hydrocarbon group having one to four error bars indicate SEM). Drug block was reversible upon carbon atoms; washout and traces that did not recover to control peak current each of X and Y independently represents a carbocyclic or (+/-10%) upon washout were discarded. heterocyclic, saturated or unsaturated, Substituted or unsub (0010 FIG. 3. Didabco strings differentiate between dif stituted ring or fused ring system; an acyclic hydrocarbon ferent channels, and can block or open. (A) Structures of six group having one to twenty six carbons atoms; or a combi didabco Strings, differing only in the number of CH2 groups in nation thereof; the string (4-12). (B) Effects of each of the six didabco strings m1, m2, and m3 independently represent 0 or 1; (at 1 mM, externally applied via the bath) on peak K" currents V and V* independently represent C, N, or N: recorded at 0 mV during 0.1 Hz, 2s pulses from -80 mVusing V and V independently represent C or N“: TEVC in whole oocytes injected with cRNA for Kv2.1, Z represents the number of positive charges in the ion and has Kv3.4 or KCNO4. A Kv3.4 variant lacking the inactivation a value of at least 1; ball domain (deletion 1-28) was used in this study. Values wherein any of said acyclic hydrocarbon groups is saturated shown are compared to pre-drug level (-1) and are the mean or unsaturated, Substituted or unsubstituted, straight-chained of the peak current recorded at equilibrium from 2-6 experi US 2009/000538.6 A1 Jan. 1, 2009

ments. Error bars indicate SEM. Thus values <1 indicate 0017. The symbol Z represents the total number of positive channel “block.” values > 1 indicate channel opening by the charges in the ion. Positive charge Z results from positively compound. charged nitrogen atoms (N) in the ion. The positively 0011 FIG. 4. Aromatic ring-separated didabcostings dif charged nitrogenatoms in the ion include those as provided in ferentiate between different channels. (A) Structures of three V, V, V, and V. didabco strings, differing only in the positioning of the dabco 0018. At least one of V, V, V, and V4 in the ion repre groups around an aromatic ring. (B) Effects of each of the sent N. Accordingly, Z is required to represent at least 1. For three didabco strings (at 1 mM, externally applied) on peak example, when m3 is 1, then at least one of V, V, V, and V4 K" currents recorded at 0 mV using TEVC in Xenopus represent N. Similarly, when m3 is 0, then at least one of V' oocytes expressing Kv2.1, Kv3.4 lacking 1-28, or KCNO4. and V represent N". Values shown are compared to pre-drug level (=1) and are the (0019 R', R. R. R. R. R. X and Y may independently mean value from 2-8 experiments. Error bars indicate SEM. represent an acyclic hydrocarbon group. 0012 FIG. 5. TG28 (the didabco string with 12 CH 0020. The acyclic hydrocarbon group contains a minimum groups shown in FIG. 3) blocks and partially recovers Kv2.1 of one carbon atom. Preferably, the acyclic hydrocarbon wash-in, opens Kv2.1 during wash-out. Peak currents (filled group independently in each of R', R. R. R. R. R. X and squares) recorded at 0 mV during Successive 0.1 Hz, 2 S Y contains a minimum of two carbon atoms. Preferably, the pulses from -80 to 0 mV recorded in Xenopus oocytes using acyclic hydrocarbon group in X contains a minimum of eight two-electrode Voltage clamp, and Voltage protocol in upper or nine carbon atoms, for example, —(CH2) - or —(CH2) left, during three cycles of wash-in (upper Solid line) and o groupS. washout (upper open line) of 250 uMTG28 in 4 mMKCl bath 0021. The acyclic hydrocarbon group contains a maxi medium (pH 7.4). Oocytes were injected with cRNA encod mum of twenty six carbon atoms. Preferably, the acyclic ing Kv2.1. Four phases are highlighted with letters a-d on the hydrocarbon group independently in each of R', R. R. R. final cycle of the three: a rapid block during wash-in, b. slow R contains a maximum of four carbonatoms. Preferably, the partial recovery of current during wash-in, c. rapid recovery acyclic hydrocarbon group in X contains a maximum often or and overshoot during wash-out, and d. Slow relaxation to twelve carbon atoms, for example, —(CH2) - or —(CH2) original (pre-drug) current during wash-out. 12 - groupS. 0013 FIG. 6. Opening and block of Kv3.4 by two didabco 0022. The acyclic hydrocarbon group may be saturated compounds show voltage-dependency. JC279.3a is the did and straight-chained, i.e., a straight-chained alkyl group. abco string with 10 CH groups shown in FIG. 3. JC638.2a is Some examples of straight-chained alkyl groups include the para didabco string shown in FIG. 4. Normalized I/V methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl. relationship for A1-28 Kv3.4 currents in oocytes in the dodecyl, hexadecyl, eicosyl, docosyl, and hexacosyl. absence (control) or presence as indicated of 1 mMJC279.3a 0023. Alternatively, the acyclic hydrocarbon group may or JC638.2a. Peak currents during 2 s pulses to voltages be saturated and branched, i.e., a branched alkyl group. Some between -80 and +60 mV (protocol upper left insert) were examples of branched alkyl groups include iso-propyl, iso recorded, before and after drug was washed in during Succes butyl, sec-butyl, t-butyl, di-(t-butyl)methyl, 3-ethyl-2,3-dim sive 0.1 Hz, 2 s pulses from -80 to 0 mV until equilibrium ethylhexyl, and 4-(1,1-dimethylethyl)heptyl. block or opening was observed. Points are means from n=3- 0024. The acyclic hydrocarbon group may alternatively be 16 oocytes; error bars indicate SEM. unsaturated. Unsaturated hydrocarbons include, for example, alkenyl, alkynyl, and combinations thereof, i.e., enynes. DETAILED DESCRIPTION OF THE INVENTION 0025. The unsaturated acyclic hydrocarbon group may be straight-chained. Some examples of straight-chained alkenyl 0014. The invention is based on the surprising discovery groups include vinyl, allyl, 2-butenyl, 3-butenyl, 1-hexenyl, by the inventors that certain positively charged ions modulate 1.3-hexadienyl, 9-decenyl, 1.3.5-hexatrienyl, oleyl, linole potassium, sodium, and cyclic nucleotide-modulated ion nyl, palmitoleyl, and arachidonyl. Some examples of straight channels. chained alkynyl groups include acetylenyl, propargyl, and butynyl. Some examples of straight-chained enynes include Ions hex-1-en-3-ynyl and hexa-1,5-dien-3-ynyl. 0026. Alternatively, the unsaturated acyclic hydrocarbon 0015. In one aspect, the present invention relates to a posi group may be branched. Some examples of branched alkenyl tively charged ion comprising the following formula: groups include 2-methylene-3-butenyl and 2,3-dimethyl-but 2-enyl. An example of a branched alkynyl group includes 2,5-dimethyl-hex-3-ynyl. (1) 0027 X and Y may also independently represent a ring. The ring may be, for example, a four, five, six, seven, or eight member ring. 0028. The ring may be saturated or unsaturated. An unsat urated ring contains at least one double bond. For example, a five member ring can have one or two double bonds, and a N / | N/ seven member ring can have one to three double bonds. m3 0029. In one embodiment, the ring is a carbocyclic ring. The carbocyclic ring may be saturated. Some examples of I0016. In formula (1), V' and V* independently represent Suitable Saturated carbocyclic rings include cyclobutane, C, N, or N“; and V and V independently represent C or N“. cyclopentane, cyclohexane, cycloheptane, and cyclooctane At least one of V, V, V, and V represents N'. r1ngS. US 2009/000538.6 A1 Jan. 1, 2009

0030 Alternatively, the carbocyclic ring may be unsatur 0040 Alternatively, the heterocyclic ring may be unsatur ated. The unsaturated carbocyclic rings may be either aro ated. The unsaturated heterocyclic rings may be either aro matic, i.e., “aryl' or 'arenyl or non-aromatic. matic, i.e., "heteroaryl' or "heteroarenyl, or non-aromatic. 0031 Examples of unsaturated carbocyclic rings include 0041. Examples of unsaturated heterocyclic rings contain cyclopentene, cyclohexene, cycloheptene, cyclopentadiene, ing one or more nitrogen heteroatoms include pyrrole, pyri 1.3-cyclohexadiene, 1,4-cyclohexadiene, 1.3-cycloheptadi dine, pyrazole, pyrazine, pyrimidine, imidazole, and triazine ene, cycloheptatriene, and cyclooctadiene rings. Preferably, rings. Examples of unsaturated heterocyclic rings containing the unsaturated carbocyclic ring is a benzene ring, i.e., phenyl one or more oxygen heteroatoms include furan, pyran, and or phenylene. 1,3-dioxolerings. Examples of unsaturated heterocyclic rings 0032. Any of the acyclic and carbocyclic hydrocarbon containing one or more Sulfur heteroatoms include thiophene, groups described above may have one or more carbon atoms thiopyran, 1.3-dithiole, and 1,3-dithiine rings. Examples of substituted by one or more heteroatoms. Some preferred het unsaturated heterocyclic rings containing a combination of eroatoms include, for example, nitrogen (N), oxygen (O), heteroatoms include oxazole, thiazole, and oxathiole rings. sulfur (S), and combinations thereof. 0042 Any of the carbocyclic and heterocyclic rings for X 0033. The nitrogen and sulfur heteroatoms may also con and Y described above may also be polycyclic. Some tain additional Substituents. For example, a nitrogen heteroa examples of polycyclic carbocyclic ring systems include tom may be an amine oxide or oxime. A Sulfur heteroatom bicyclo[2.2.2]octane and bicyclo[3.3.3 undecane. Some may be a Sulfoxide or Sulfone. In addition, where the nitrogen examples of polycyclic heterocyclic ring systems include is not double bonded within a ring or an acyclic chain, the 1,4-diazabicyclo[2.2.2]octane, 1-aza-4-azonia-bicyclo2.2. nitrogen may be substituted by any suitable group. Such as, 2octane, 1,4-diazoniabicyclo2.2.2]octane, 1,5-diaza-bicy for example, H and lower alkyl. clo3.3.3 undecane, 1-aza-5-azonia-bicyclo[3.3.3 undecane, 0034 Since any of the nitrogen heteroatoms discussed and 1,5-diazaoniabicyclo[3.3.3 undecane. above may be positively charged, any such positively charged 0043. In addition, any of the carbocyclic and heterocyclic nitrogen heteroatoms may contribute to the positive charge rings described above may be fused to one or more, typically (Z). Accordingly, a positive charge Z of 4, or higher than 4. one or two, other rings to make a fused ring system. 0044. In one embodiment, the fused rings are all carbocy may be due in part to positively charged nitrogen heteroat clic and saturated. Some examples of such fused ring systems OS. include decahydronaphthalene, tetradecahydroanthracene, 0035. The acyclic and carbocyclic hydrocarbon groups tetradecahydrophenanthrene and hexadecahydropyrene may be substituted with more than one heteroatom as long as fused rings. In another embodiment, the carbocyclic fused the resulting heteroatom-substituted hydrocarbon is stable. ring system is composed of a combination of Saturated and The stable heteroatom-substituted acyclic or carbocyclic unsaturated rings. Some examples of such fused ring systems hydrocarbons do not have one heteroatom bound to another include bicyclo4.3.0non-3-ene, bicyclo[4.4.0dec-8-ene, heteroatom. Moreover, no heteroatom is bound to: V' when and bicyclo4.4.0dec-7,9-diene fused rings. V' is NorN"; V when V is N.V. when VisN"; or V when 0045. In another embodiment, all of the fused carbocyclic V is N or N. Some examples of unstable heteroatom-sub rings are unsaturated. The unsaturated carbocyclic rings may stituted hydrocarbons not considered herein include hydra also be aromatic. Some examples of unsaturated carbocyclic Zines, peroxides, and disulfides. fused ring systems which are also aromatic include naphtha 0036. The term “lower alkyl refers to an acyclic hydro lene, phenanthrene, anthracene, triphenylene, aZulene, chry carbon group having one to six carbon atoms. Lower alkyl sene, pyrene, and biphenylene fused rings. groups may be branched or unbranched, and saturated or 0046. In yet another embodiment, the fused ring system is unsaturated. Some examples of Saturated lower alkyl groups composed of a mixture of carbocyclic and heterocyclic rings. include methyl, ethyl, n-propyl, isopropyl. n-butyl, sec-butyl, Some examples of Such fused ring systems include indoline, i-butyl, t-butyl, n-pentyl, n-hexyl, 4-methyl-2-pentyl, and so quinoline, isoquinoline, phthalazine, benzimidazole, ben on. Some examples of unsaturated lower alkyl groups include Zothiazole, benzisoxazole, benzodioxole, quinoxaline, vinyl, propenyl, isopropenyl, propargyl, and so on. quinazoline, benzoxazine, cinnoline, acridine, and phenazine 0037 Accordingly, in one embodiment, the acyclic hydro fused rings. carbon group is heteroatom-Substituted to form a polyalkyle 0047. In yet another embodiment, the fused ring system is neoxide. Some examples of polyalkyleneoxides include composed of only heterocyclic rings. Examples of such fused polymethyleneoxide, polyethyleneoxide, polypropylenenox ring systems include pteridine, purine, 1.8-naphthyridine, ide, and combinations thereof. 1,8.9-triazaanthracene, 1,5-diazabicyclo4.3.0non-5-ene 0038. When one or more carbon atoms of the carbocyclic and thieno3.2-bfuran fused rings. rings described above are substituted by one or more heteroa 0048. In formula (1), X and Y may also independently toms described above, a heterocyclic ring is formed. Accord represent a combination of any of the ring or fused ring ingly, X and Y may also be heterocyclic rings. systems described above and any of the acyclic hydrocarbon 0039. The heterocyclic ring may be saturated. Examples groups described above. For example, the foregoing groups of saturated heterocyclic rings containing one or more nitro gen heteroatoms include pyrrolidine, piperidine, imidazoli may representa combination of an alkyl or alkenyl group with dine, N,N-dimethylimidazolidine, pyrazolidine, piperazine, an aryl or heteroaryl ring. homopiperazine, and hexahydro-1,3,5-triazine rings. 0049. Some examples of such combination groups for X Examples of Saturated heterocyclic rings containing one or include benzyl ( CH-CH ), O-tolyl ( CHs-o- more oxygen heteroatoms include tetrahydrofliran, tetrahy CH ), o-xylyl (o-CH-CH CH ), m-xylyl dropyran, and 1,4-dioxane rings. Examples of Saturated het (m-CH CH-CH2—), p-xylyl (p-CH CH erocyclic rings containing one or more Sulfur heteroatoms CH ), dimethylpyridinyl, e.g., lutidinyl (—CH2— include tetrahydrothiophene and 1,4-dithiane rings. (NCH)—CH2—), and dimethylpyrazinyl (e.g., —CH2— Examples of Saturated heterocyclic rings containing a com (NCHN)—CH2—). Some examples of such combination bination of heteroatoms include 13-oxazolidine, 1,3-thiazo groups for Y include benzyl ( CH-CHs), o-tolyl lidine, 1.3-oxathiolane, and morpholine rings. (—CHs-o-CH), o-xylyl (o-CH CH-CH-), m-xylyl US 2009/000538.6 A1 Jan. 1, 2009

(m-CH2—CH CH), p-xylyl (p-CH CH, CH), erably, in represents an integer from 1 to 10. More preferably, methylpyridinyl (—CH2—(NCH)), and N-methylpyrazi in represents an integer from 1 to 3. Even more preferably, n is nyl ( NCHN CH). 2 in the formula C.H., for all three of R', R, and R, as 0050 Additional examples of combination groups suit shown in formula (1 m) below: able for X or Y include 1,2-dimethylphenyl, 1,2-dimeth ylphenylene, 1,4-diisopropylphenyl, 1,4-dimethylphenylene, (1m) 1.2-dimethylbenzyl, cumenyl, 2-methylpyridinyl, 2-eth +1 ylpyrazinyl, 2-methylimidazolyl, and 2-propylbenzimida N Nt-X Zole. 0051. Any of the groups thus far described may be either unsubstituted, or substituted with one or more substituents. 0.058 Informula (1 m), X represents preferably an acyclic Some examples of substituents include —OR, NOR7), hydrocarbon group having one to twenty six carbons atoms. —C(O)N(R) - SR", halo, and COOR7. Halo includes, More preferably, X represents an acyclic hydrocarbon group for example, F, Cl, and Br. R7 independently represents H. having ten to eighteen carbonatoms. Even more preferably, X phenyl, or a lower alkyl group as previously defined. represents an acyclic hydrocarbon group having twelve to 0052. In formula (1), V and V* independently represent sixteen carbon atoms. Some particularly preferred ions are C, N, or N, and V and V independently represent C or N“. given below in formulas (1n) and (1p). The subscript m1 represents the presence or absence of the Y group bound to V. The subscript m2 represents the presence (1n) or absence of the Y group bound to V. The subscript m3 +1 represents the presence or absence of the polycyclic ring system containing V and V*. The subscripts m1, m2, and m3 N N'-(CH2)CH independently represent 1 when any of the mentioned groups are present, or represent 0 when any of the mentioned groups 1-aza-4-azonia-4-dodecylbicyclo2.2.2]octane (positively are absent. charged ion component of JE188) 0053. When V' represents C or N", any one of R', R, or R may be bound to the C or N' with a double bond. In such (1p) a case, V may be C or N' without requiring V' to be bound to +1 Y, i.e., m1 in (Y), is 0. Similarly, when V represents C or N", V does not require a bond to Yifany one of R. R. or R is bound to V4 with a double bond, i.e., m2 in (Y), would be N 7'-chlich O. 1-aza-4-azonia-4-hexadecylbicyclo2.2.2]octane (positively 0054 However, preferably, when Vl represents C or N. charged ion component of TA279) then none of R', R, or R is bound to V' with a double bond, and thus, m1 in (Y), is 1 when V' represents C or N'. 0059. In another embodiment of formula (1), m3 is 1. Similarly, it is preferable that when V represents C or N', When m3 is 1, then X is a linking group. Accordingly, in one then none of R. R. or R is bound to V4 with a double bond, embodiment, when m3 is 1, the ion may be represented by the and thus, m2 in (Y), is 1 when V represents C or N". generic formula (1b): 0055. In one embodiment of formula (1), m3 is 0. When m3 is 0, then X is a non-linking group. Accordingly, when m3 (1b) is 0, the ion may be represented by formula (1a) below. In formula (1a), R. R. R. X, Y, Z, and ml are as previously defined. (1a) RI N / N 2-ye

0060 Informula (1b), R', R,R,R,R,R, X,Y, V, V, N / V, V, m1, m2, and Z are as previously defined. I0056. In a further embodiment to formula (1a), one of V' 0061. In formula (1b), V and V may both represent N“, and V representsN", Zrepresents 1, andm' is 0. For example, and VandV may both represent C. A representative formula V may represent N and V may represent N as shown in in accordance with the foregoing embodiment is given as formula (1k) below: formula (1 c): (1k) R1 (1c) R R4 +z N / Y- r 2- R5 Y 0057. Inafurther embodiment to formula (1k), R', R, and Rare independently represented by the formula C.H. Pref US 2009/000538.6 A1 Jan. 1, 2009

0062 Alternatively, V and V* may both represent N', 0067. Alternatively, V° and V may both represent N', while V represents C and V represents N. A representative while V' represents C and V represents N. A representative formula in accordance with the foregoing embodiment is formula in accordance with the foregoing embodiment is given as formula (1 d): given as formula (li): (1d) (1i) R1 4 +z v-KC--/ N4N / 0063. In formula (1b), V and V may both represent N', R3 R6 and V and V* may both represent C. A representative formula in accordance with the foregoing embodiment is given as 0068 Informula (1b), V, V, and V may all represent N', formula (1e): while V represents C. A representative formula in accor dance with the foregoing embodiment is given as formula (1e)

(1) R4 +z

0064. Alternatively, V and V may both represent N“, while V represents C and V represents N. A representative -(C-C) formula in accordance with the foregoing embodiment is N. N. given as formula (1f): 0069. Alternatively, V and V may both represent N“, while V and V* independently represent N or N. A repre (1f) sentative formula in accordance with the foregoing embodi ment is given as formula (2):

(2)

0065. In formula (1b), V and V* may both represent N', and V and V may both represent C. A representative formula N / N / in accordance with the foregoing embodiment is given as formula (1g): 0070. In formula (2) above, R', R. R. R. R. R. X, Y, m1, and m2 are as previously defined. The Superscripts a and (1g) d independently represent 0 or 1. 4 (0071. In a preferred embodiment, each of R', R. R. R. R, and R' in formula (2) above are independently repre sented by the formula CH, wherein in represents an integer from 1 to 26. More preferably, n is an integer from 1 to 10. Even more preferably, n is an integer from 1 to 4. Even more preferably, n is an integer from 1 to 3. Even more preferably, 0066. In formula (1b), V and V may both represent N“, n is 2 in the formula C.H., for all six of R',R,R,R,Rand and V and V* may both represent C. A representative formula R. A representative formula in accordance with the forego in accordance with the foregoing embodiment is given as ing embodiment is given as formula (3): formula (1h): (3) (1h) N-1 -N d-(Y) N / NN 1/

0072. In a further preferred embodiment of formula (3), a and d are both 0 and m1 and m2 are both 0. In the foregoing embodiment, each bicyclic ring system in formula (3) repre US 2009/000538.6 A1 Jan. 1, 2009 sents a 4-aza-1-azoniabicyclo2.2.2]octane ring system. A containing groups for X include the Xylyl groups. A repre representative formula in accordance with the foregoing sentative formula in accordance with the foregoing embodi embodiment is given as formula (3a): ment is given as formula (4):

(3a) (4) +2 +2

N N -X- N N N-HC/FVCH-N N N 7 S 7 SCV 731 0073. In a further preferred embodiment of formula (3a), X represents an acyclic hydrocarbon group as previously 0079. In a particularly preferred ion of formula (4), X defined. In a preferred embodiment, the acyclic hydrocarbon represents p-xylyl. The ion, C.C.'-bis(4-aza-1-azoniabicyclo group for X has four to twelve carbon atoms. For example, 2.2.2]octanyl)p-Xylene, corresponds to the following for Some particularly preferred hydrocarbon groups for X mula (5): include (CH) , (CH) , (CH) , and —(CH2)2 -. Accordingly, some particularly preferred ions are represented by the following formulas: (5)

N N"-HC CH2 -N N (3.b) N 7 0080. In another embodiment of formula (3), V, V and Vall represent N“, while V represents N. A representative formula in accordance with the foregoing embodiment is given as formula (6): 1.8-bis(4-aza-1-azoniabicyclo2.2.2]octanyl)octane 0074 (6) R1 R4 / / (3.c) y-néN-x- N

I0081. In another embodiment of formula (3), all of V, V, V, and V represent N". A representative formula in accor 0075 1.9-bis(4-aza-1-azoniabicyclo[2.2.2]octanyl) dance with the foregoing embodiment is given as formula (7): Ola (7) (3d) +2

N R3/ N R6 /

0076 1,10-bis(4-aza-1-azoniabicyclo2.2.2]octanyl) de I0082 For the purpose of clarity, the ion shown in any of the formulas thus far shown may be ascribed the symbol (I). The CaC ion (I) according to any of these formulas requires, by neces sity, to be associated with a negatively charged counteranion or counteranions (W) to make a neutral compound. I0083. Any counteranion may be useful for the purposes of the present invention. A particular counteranion may be cho Sen for any number of reasons including its optimizing effect on the function of the ion, and/or its effect on solubility, density, ease of manufacture, cost, and so on, of the com pound that includes the ion. 0077. 1,12-bis(4-aza-1-azoniabicyclo[2.2.2]octanyl) 0084. The counteranion may be singly negatively charged, dodecane i.e., a simple counteranion. Some examples of suitable simple 0078. In another preferred embodiment of formula (3a). X counteranions include chloride, perchlorate, Sulfate, nitrate, contains a phenylene ring. Particularly preferred phenylene and tetrafluoroborate. US 2009/000538.6 A1 Jan. 1, 2009

0085 Alternatively, the counteranion may be, for sodium channels (ENaC). Any epithelia sodium channel example, doubly negatively or triply negatively charged, i.e., known to those in the art can be modulated in accordance with a dianion or trianion. Some examples of Suitable counter the method of the invention. For example, the epithelial dianions include carbonate, oxide, Sulfide, oxalate, fumarate, Sodium channel can comprise an alpha, beta, gamma and/or terephthalate, malonate, maleate. Succinate, catecholate, delta subunit. ascorbate, and a doubly deprotonated porphyrin. Some 0095. In yet another embodiment, the ion channel is a examples of Suitable counter trianions include phosphate, cyclic nucleotide-modulated ion channel. Any type of cyclic citrate, ascorbate, 1,3,5-benzenetricarboxylate, 1,3,5-ben nucleotide-modulated ion channel may be modulated in Zenetriolate, 2-carboxymalonate, and erythritolate. accordance with the method of the present invention. Examples of cyclic nucleotide-modulated ion channels 0.086 From the above, it is evident that numerous ion include the cyclic nucleotide gated (CNG) channels and the counteranion combinations are possible. Accordingly, the hyperpolarization-activated cyclic nucleotide gated channels ion-counteranion combinations are appropriately described (HCN). by the following formula: (0096. Examples of CNG channels include CNGA1, I'W', (8) CNGA2, CNGA3, CNGA4, CNGB1 and CNGB3. Examples 0087 Informula (8), the symbol I represents an ion of the of HCN channels include HCN1, HCN2, HCN3 and HCN4. present invention in accordance with any of the formulas thus TABLE 1 far shown. W represents any suitable counteranion, such as those described above. Zis the charge of the ion as previously Pore-forming K channel C-Subunits described. r is the charge of the counteranion. By the rules of chemistry, counter charges are balanced in formula (8) Subclass Channel Gene according to the formula uZrt. Six transmembrane domains, one pore Voltage-gated (shaker) Kv1.1 KCNA1 Method for Modulating Kv1.2 KCNA2 Kv1.3 KCNA3 0088. In another aspect, the present invention provides a Kv1.4 KCNA4 method for modulating potassium, sodium and cyclic nucle Kv1.5 KCNAS otide-modulated ion channels in a mammal in need thereof. Kv1.6 KCNA6 The method comprises administering to the mammal an Kv1.7 KCNA7 effective amount of an ion as described above. Voltage-gated (shab) Kv2.1 KCNB1 Kv2.2 KCNB2 0089. In one embodiment, the ion channel is a potassium Voltage-gated (shaw) Kv3.1 KCNC1 ion channel. Any type of potassium ion channel may be Kv3.2 KCNC2 modulated in accordance with the method of the present Kv3.3 KCNC3 Kv3.4 KCNC4 invention. Examples of potassium ion channels include pore Voltage-gated (shal) Kv4.1 KCND1 forming C-Subunit channels with or without modulatory Kv4.2 KCND2 B-subunits. Kv4.3 KCND3 0090 The pore-forming C-subunit group of potassium Voltage-gated (silent) Kws.1 KCNE1 Kw8.1 KCNG1 channels typically contains either 1) six transmembrane seg Kw8.2 KCNG2 ments and one pore-forming region, 2) two transmembrane Kw8.3 KCNG3 segments and one pore-forming region, or 3) four transmem Kw8.1 Kw).2 KCNS1 brane segments and two pore-forming regions. The C-Subunit Kw).2 KCNS2 group can be further sub-divided based on the channels defin Kw).3 KCNS3 ing property (e.g., Voltage-gated, calcium-activated, inward, Voltage-gated (EAG) EAG KCNH1 rectifier, ATP-sensitive, etc.) and sequence similarity (e.g., EAG2 ERG1 KCNH2 Kv1.1, Kv1.2, etc.). Examples of pore-forming C-Subunit ERG2 potassium channels are shown below in Table 1. ERG3 0091. The non pore-forming (modulatory) B-subunit ELK1 KCNH4 group of potassium channels includes single transmembrane ELK2 KCNH3 ELK3 spanning proteins, such as Mink and Mink-related proteins Voltage-gated (KvLQT related) KCNQ1 KCNQ1 (MiRPs), and large ATP-binding cassette (ABC) transport KCNQ2 KCNQ2 related proteins, such as the Sulfonylurea receptors. Examples KCNQ3 KCZQ3 of modulatory B-subunits are shown below in Table 2. KCNQ4 KCNQ4 KCNQ5 KCNQ5 0092 A potassium ion channel comprising any of the sub Ca”-activated (large conductance) Slo-1 KCNMA1 units shown in Table 1 and Table 2 can be modulated in Slo-2 KCNMA2 accordance with the method of the present invention. Slo-3 KCNMA3 Ca”-activated (intermediate conductance) KCA KCNN4 0093. In another embodiment, the ion channel is a sodium Ca"activated (small conductance) SK1 KCNN1 ion channel. Any type of Sodium ion channel can be modu SK2 KCNN2 lated in accordance with the method of the present invention. SK3 KCNN3 An example of a class of sodium channel useful in the method Two transmembrane domains, one pore of the present invention is the Voltage-gated sodium channel. Inward rectifier (ROMK1) Kir1.1 KCN1 Examples of Voltage-gated Sodium channels include Na1.1, Inward rectifier Kir2.1 KCNV2 Na1.2, Na, 1.3, Na1.4, Na1.5, Na1.6, Na1.7, Na, 1.8 and Kir2.2 KCNJ12 Na1.9. Kir2.3 KCN4 0094. Another example of a class of sodium channel use Kir2.4 KCN14 ful in the method of the present invention is the epithelia US 2009/000538.6 A1 Jan. 1, 2009

cys-loop ligand gated ion channels include nicotinic recep TABLE 1-continued tors, 5-hydroxytryptamine receptor type 3 receptors, and the Zinc activated ion channel. Examples of anion specific cys Pore-forming K channel Ci-subunits loop ligand gated ion channels include Y-aminobutyric acid Subclass Channel Gene type A (GABA, which includes p subunits sometimes referred to as GABA), and glycine receptors. Inward rectifier (G-protein regulated) Kir3.1 KCNJ3 Kir3.2 KCNJ6 0100 Any ATP gated ion channel can be modulated in Kir3.3 KCNJ9 accordance with the method of the present invention. Kir3.4 KCNJS Examples of ATPgated ion channel include ATP2X receptors Inward rectifier Kir4.1 KCNJ10 and CFTR. Kir4.2 KCNJ15 KirS.1 KCN16 0101 Any glutamate activated cationic channel can be Kirf.1 KCNJ13 modulated in accordance with the method of the present Inward rectifier (ATP sensitive) Kiré.1 KCN8 invention. Examples of glutamate activated cationic cationic Kiró.2 KCNJ11 channels include NMDA receptors, AMPA receptors, and Four transmembrane domains, two pores Kainate receptors. Two Pore TWIK-1 KCNK1 0102. In another embodiment, the ion channel is a tran TWIK-2 KCNK6 sient receptor potential (Trp) ion channel. Any type of Trpion KCNK7 KCNK7 channel may be modulated in accordance with the method of TASK KCNK3 the present invention. Examples of Trpion channels include TASK-2 KCNKS TASK-3 KCNK9 Trpc (e.g., Trpc1, Trpc2, Trpc3, Trpc4, Trpc5 Trpcó, Trpc7. TASK-5 KCNK15 etc.), TrpV (e.g., TrpV1, TrpV2, TrpV3, TrpV4, TrpV5, Trpv6. TRAAK KCNK4 etc), Trpm (e.g., Trpm 1, Trpm2, Trpm3, Trpm4, Trpm5. TREK-1 KCNK2 Trpmé, Trpm7, Trpm8, etc), ANKTM1, Trpp (e.g., Trpp2. TREK-2 KCNK10 THIK-1 KCNK13 Trpp3, Trpp5, etc), and Trpml (e.g., Troml1, Trpm 12, Trpiml3, THIK-2 KCNK12 etc). TALK-1 KCNK16 TALK-2 KCNK17 Administration 0103) In one aspect of the invention, the effective amount of an ion administered in accordance with the method of the TABLE 2 invention is any amount effective for modulating a potassium, Sodium, or cyclic nucleotide-modulated ion channel. In Modulatory K. channel B-subunits another aspect of the invention, the effective amount of anion Name Known partners Gene administered in accordance with the method of the invention is any amount effective for modulating a ligand-gated ion Mink KCNQ1, ERG1 KCNE1 Mirp1 ERG1, KCNQ2, KCNQ3, Kv4.2 KCNE2 channel (e.g., cys-loop ligand gated, ATP gated, glutamate Mirp2 KCNQ1, KCNQ4, Kv3.4 KCNE3 activated cationic gated) or Trp ion channel. An ion is con Mirp3 KCNE4 sidered to modulate the ion channel if the ion opens, or BkB1 Slo-1 KCNMB1 BKB2 Slo-1 KCNMB2 conversely blocks, the ion channel. BKB3 Slo-1 KCNMB3 0104. In one embodiment, the administered ion opens an BKB4 Slo-1 KCNMB4 ion channel. The term “open’ as used herein refers to an Kv31 Kv1.x increase of current through an ion channel. The increase in Kv|B2 Kv1.x KCNAB1 Kv|B3 Kv1.x KCNAB2 current may be any statistically significant increase occurring KCHIP1 Kv4.x KCNIP1 upon ion administration. For example, the increase in current KCHIP2 Kv4.x KCNIP2 may be at least about 10%, preferably at least about 25%, KCHIP3 Kv4.x more preferably at least about 50%, even more preferably at KCHAP Kv2.1, Kv4.3 SUR1 Kir6.1, Kir6.2 ABCC8 least about 75%, and even more preferably at least about 90%. SUR2 Kir6.1, Kir6.2 ABCC9 In a preferred embodiment, the administered ion opensanion channel in a mammal Suffering from a disease or condition which would benefit from opening of the ion channel, i.e., 0097. In a further aspect, the present invention provides a from increased current through the channel. method for modulating a ligand-gated ion channel and tran 0105. In another embodiment, the administered ion blocks sient receptor potential (Trp) ion channel in a mammal in the ion channel. The term “block' as used herein refers to the need thereof. The method comprises administering to the physical occlusion of the ion channel pore by the ion of the mammal an effective amount of an ion as described above. invention, thereby inhibiting current flow. The inhibition of 0098. In one embodiment, the ion channel is a ligand current may be any statistically significant decrease of current gated ion channel. Any type of ligand-gated ion channel may occurring upon ion administration. For example, the current be modulated in accordance with the method of the present flow may be inhibited by at least about 10%, preferably at invention. Examples of ligand-gated channels include cys least about 25%, more preferably at least about 50%, even loop ligand gated ion channels, ATP gated ion channels, and more preferably at least about 75%, and even more preferably glutamate activated cationic channels. at least about 90%. Optimally, the ion blocks an ion channel 0099. Any Cys-loop ligand gated ion channel can be in a mammal Suffering from a disease or condition which modulated in accordance with the method of the present would benefit from a blocked ion channel. invention. The cys-loop ligand gated ion channels can be 0106. In one aspect of the invention, mammals in need of cation specific or anion specific. Examples of cation specific modulating ion channels are those mammals Suffering from a US 2009/000538.6 A1 Jan. 1, 2009

disease or condition characterized by aberrant potassium, subject to be treated, the disease or condition to be treated, etc. Sodium, or cyclic nucleotide-modulated ion channel activity. The appropriate amount of the ion can readily be determined In another aspect of the invention, mammals in need of modu by those skilled in the art during preclinical and clinical trials. lating ion channels are those mammals suffering from a dis 0119 The minimum amount of an ion administered to a ease or condition characterized by aberrant ligand-gated ion mammal is the lowest amount capable of modulating a potas channels or Trpion channels. Such diseases and conditions sium, Sodium, or cyclic nucleotide-modulated channel. The are well known to those skilled in the art. maximum amount administered to a mammal is the highest 0107 Examples of such diseases or conditions include, effective amount that does not cause undesirable side effects. but are not limited to, cardiovascular diseases and conditions, I0120 Any mammal can be treated in accordance with the immune system diseases and conditions, Smooth muscle dis method of the present invention. Mammals include, for orders, central and peripheral nervous system diseases and example, humans, baboons, and other primates, as well as pet disorders, skeletal muscle diseases and disorders, endocrine animals such as dogs and cats, laboratory animals such as rats disorders (e.g., disorders associated with hormone secretion), and mice, and farm animals such as horses, sheep and cows. and cell proliferative disorders, and degenerative disorders. I0121. An ion useful in the method of the present invention 0108. Other diseases and conditions include hyper-re may be administered by any method known in the art. Some flexic bladder conditions, baldness, deafness, headaches, examples of Suitable modes of administration include oral anxiety, and aberrant excitability in afferents sensitized by and systemic administration. Systemic administration can be chronic inflammation. enteral or parenteral, e.g., administered intravenously; intra 0109 Examples of cardiovascular diseases and conditions muscularly; Subcutaneously, as injectable Solutions or Sus include long QT syndrome, Brugada syndrome, arrhythmia, pensions; intraperitoneally; or rectally. Liquid or Solid (e.g., atrial fibrillation, atrial flutter, Torsades de Pointes, heart fail tablets, gelatin capsules) formulations can be employed. ure, congestive heart failure, ischemia, Ventricular fibrilla I0122) An ion may be administered to a mammal by con tion, Ventricular tachycardia and coronary artery disease. trolled release, as is known in the art. Controlled release 0110. Examples of immune system diseases and condi administration is a method of drug delivery to achieve a tions include delayed type-hypersensitivity, autoimmune dis certain level of the drug over a particular period of time. The orders, autoimmune encephalomyelitis, immunosuppres level of ion over a particular period of time is typically mea Sion, diseases and conditions characterized by immune cell Sured by plasma or serum concentrations. Suitable controlled activation and/or proliferation. Immunosuppression includes release formulations include delayed, Sustained, and imme the Suppression of the immune system via, for instance, diate (i.e., instantaneous) release. Methods for controlled administration of therapeutic agents, such as is generally used release of compounds are well known in the art, and are in organ transplantation. described in, for example, international patent application 0111 Examples of nervous system diseases and condi PCT/US02/10748, and U.S. Pat. Nos. 5,567,439; 6,838,094; tions include stroke, cerebral ischemia, dementia, cognition disorders, sleep disorders, depression, musculoskeletal con 6,863,902; and 6,905,708. ditions, epilepsy, Alzheimer's disease, Parkinson's disease, I0123. Other routes of administration include oral, topical, and abnormal regulation of neuronal excitability. intrabronchial, or intranasal administration. For oral admin 0112 Examples of smooth muscle disorders include istration, liquid or Solid formulations may be used. Some hypertension, angina, asthma, urinary incontinence, prema examples of formulations suitable for oral administration ture labor, and hypoxia. The hypertension can be any type of include tablets, gelatin capsules, pills, troches, elixirs, Sus hypertension. For example, the hypertension can be pulmo pensions, syrups, and wafers. Intrabronchial administration nary hypertension, cardiovascular hypertension, and hyper can include an inhaler spray. For intranasal administration, tension of the kidneys. administration of an ion can be accomplished by a nebulizer 0113. Examples of hormones which can contribute to an or liquid mist. endocrine disorder include insulin, prolactin, growth hor 0.124. The ion can be formulated in a suitable pharmaceu mone, and cholecystokinin. For example, a disorder involv tical carrier. In this specification, a pharmaceutical carrier is ing the hormone insulin is diabetes. considered to be synonymous with a vehicle oran excipient as 0114 Examples of cell proliferative disorders include is understood by practitioners in the art. Examples of carriers cancer, fibrosis, atherosclerosis and restenosis. include starch, milk, Sugar, certain types of clay, gelatin, 0115. Another disease includes cystic fibrosis. For Stearic acid or salts thereof, magnesium or calcium Stearate, example the ATP gated ion channel, CFTR is reported to be talc, vegetable fats or oils, gums and glycols. involved in cystic fibrosis. 0.125. The ion can be formulated into a composition con 0116 Trp channels are involved in sensory transduction taining one or more of the following: a stabilizer, a Surfactant, (e.g., temperature, touch, pain, taste, hearing, etc.) and a preferably a nonionic Surfactant, and optionally a salt and/or variety of other physiological functions. Examples of condi a buffering agent. tions associated with aberrant Trp channel function include 0.126 The stabilizer may, for example, be an amino acid, glomerulosclerosis and polycystic kidney disease. Such as for instance, glycine; or an oligosaccharide, such as 0117 Effective amounts of an ion to be administered are for example, Sucrose, tetralose, lactose or a dextran. Alterna those amounts which are capable of modulating potassium, tively, the stabilizer may be a Sugar alcohol. Such as for Sodium, or cyclic nucleotide-modulated ion channels. Opti instance, mannitol; or a combination thereof. Preferably the mally, Such amounts impart a beneficial effect on the mam stabilizer or combination of stabilizers constitutes from about mal. 0.1% to about 10% weight for weight of the ion. 0118. The administered amount of anion will vary accord I0127. The surfactant is preferably a nonionic surfactant, ing to numerous factors that are well known in the art, Such as Such as a polysorbate. Some examples of suitable Surfactants the particular ion utilized, the mode of application, particular include Tween 20, Tween 80: a polyethylene glycol or a US 2009/000538.6 A1 Jan. 1, 2009 polyoxyethylene polyoxypropylene glycol, Such as Pluronic F-68 at from about 0.001% (w/v) to about 10% (w/v). -continued 0128. The salt or buffering agent may be any salt or buff 2X ering agent. Suitable salts include, for example, sodium or A w A w potassium chloride. Suitable buffers include, for example, sodium or potassium bicarbonate or biphosphate. Preferably, the buffering agent maintains the pH of the ion formulation in the range of about 5.5 to about 7.5. The salt and/or buffering agent is also useful to maintain the osmolality at a level (Class B salts) suitable for administration to a mammal. Preferably the salt or buffering agent is present at a roughly isotonic concentration of about 150 mM to about 300 mM. In Schematic 2, a didabco species was first generated by 0129. The ion can beformulated into a composition which reaction of an excess of dabco in ethyl acetate with the appro may additionally contain one or more conventional additives. priate C,c)-dihaloalkane. The resultant material was then Some examples of such additives include a solubilizer such taken sequentially in limited reaction with a single equivalent as, for example, glycerol; an antioxidant such as for example, of an appropriately Substituted 1-haloalkane in ethyl acetate benzalkonium chloride (a mixture of quaternary ammonium followed by capping with the desired simple terminal haloal compounds, known as "quart'), benzyl alcohol, chloretone or kane. chlorobutanol; anaesthetic agent such as for example a mor 0.134 Members of Structural Class C contain two dabco phine derivative; or an isotonic agent etc., Such as described units separated by carbon chains and an aromatic ring, with above. As a further precaution against oxidation or other the Substituents of the aromatic ring being in ortho-, meta spoilage, the composition may be stored under nitrogen gas in and para-relationships (see schematic 3). vials sealed with impermeable stoppers. 0130. Examples have been set forth below for the purpose of illustration and to describe the best mode of the invention Schematic 3 at the present time. However, the scope of this invention is not to be in any way limited by the examples set forth herein. CHBr BrCH1 V EXAMPLES bromomethyl groups o-, m-, p ethyl acetate Example 1 2 Br Representative Syntheses of Didabco Compounds 0131 Dabco was purchased from Sigma-Aldrich Co. (St. Louis, Mo.). Generally, dabco is taken in reaction with a Suitably functionalized haloalkane in an inert solvent. Such as C.C.'-(1-azonia-4-aza-bicyclo[2.2.2]octyl)xylene ethanol, unless limited reaction on the dabco is desired in (Ortho-, meta-, or para which instances ethyl acetate is used. (Class C salts) 0132) Three structural classes of dabco derivatives were synthesized. Members of structural class A bear a lipophilic In Schematic 3, the procedure is similar to that of schematic 2. chain of variable length attached through one of the nitrogens In Schematic 3, the reaction starts with the ortho, meta, and of dabco (see Schematic 1). para-bis(halomethyl)benzene reagents. Schematic 1. 0.135 Synthesized compounds were determined to be pure due to the lack of detectable contaminants by NMR analysis. 0.136 For example, a didabco compound containing the -ethyl He- acetate Nw N-CH(CH2)CHX- 2(CH2)CH3 ion corresponding to formula (3b), i.e., 1.8-bis(4-aza-1-aZo niabicyclo2.2.2]octanyl)octane, was prepared as follows: 1-alkyl-1-azonia-4- Two equivalents of DABCO (1,4-diazabicyclo[2.2.2]octane) azabicyclo2.2.2]octane halide was dissolved in 100 mL of acetonitrile with stirring at room temperature. To the DABCO solution was added one equiva (Class A salts) lent of 1,8-dichlorooctane while stirring. The resulting reac In Schematic 1, limited reaction of dabco with a single equiva tion mixture was stirred at room temperature for one day, lent of an appropriately substituted 1-haloalkane in ethyl forming a white precipitate. The white precipitate, 1.8-bis(4- acetate was performed followed by capping with the desired aza-1-azoniabicyclo2.2.2]octanyl)octane, was then col simple terminal haloalkane. lected by suction filtration and washed with acetonitrile 0133 Members of Structural Class B contain two dabco (3x50 mL) and dried under high vacuum. units with a hydrocarbon spacer (see Schematic 2). Example 2 Schematic 2 Synthesis of C.C.'-bis(4-aza-1-azoniabicyclo2.2.2 octanyl)p-Xylen w ethyl acetate 0.137. A didabco compound containing the ion corre sponding to formula (5), i.e., C.C.'-bis(4-aza-1-azoniabicyclo US 2009/000538.6 A1 Jan. 1, 2009

2.2.2]octanyl)-xylene, was prepared as follows: Two Drug inhibition constants are calculated after generating a equivalents of DABCO (1,4-diazabicyclo[2.2.2]octane) was dose response curve by fitting with a logistic dose-response dissolved in 100 mL of acetonitrile with stirring at room function (see equation (i) below). Typically, 5-20 cells per temperature. To the DABCO solution was added one equiva group are recorded from for heterologous expression experi lent of C.C.'-dibromo-p-xylene while stirring. The resulting ments. Values are to be stated as meantSEM with statistical reaction mixture was stirred at room temperature for one day, significance set at p-0.05 (unpaired students t-test, ANOVA forming a white precipitate. The white precipitate, C.C.'-bis or the Mann-Whitney Rank Sum Test as appropriate). (4-aza-1-azoniabicyclo2.2.2]octanyl)p-Xylene, was then 0142. Injected oocytes, bathed in bath solution containing collected by suction filtration and washed with acetonitrile 4 mM KCl unless otherwise stated, are impaled with current (3x50 mL) and dried under high vacuum and Voltage electrodes and clamped at-80 mV holding poten tial using a Warner OC-725C amplifier. For single-voltage Example 3 dose-response assessment, currents are recorded for several pulses (each of 1-5s at a frequency of 0.1 Hz, depending upon General Materials and Methods channel type) in the absence of drug, until equilibrium current 0.138. The ion channel subunit genes are in expression level is reached, and then during further pulses with bath vectors that facilitate in vitro transcription of channel cRNA application of various concentrations of drug, each until equi using the Ambion mMessage mMachine in vitro transcription librium current inhibition is observed, with a washout phase kit. cDNA constructs are column-purified, linearized with the in between to eliminate the possibility of channel run-down appropriate restriction enzyme that cuts a single site follow and other non-drug effects. Ooctyes in which currents do not ing the polyA tail, then purified by precipitation and tran return to within 10% of pre-drug level after washout are scribed in vitro using a promoter, such as T7, T3 and SP6. discarded. Drug concentrations are chosen to ensure inhibi Transcribed cRNA is purified by precipitation and resus tion values between 0 and 90-100% with at least 3 values pended in RNAse-free water ready for injection. Each tran within the linear portion of the curve so that the inhibition scription can provide enough cFNA for several months of constant (Ki) can be calculated accurately by fitting with a experiments. logistic dose-response function (formula (i)) using the Origin 0139 Stage V-VI Xenopus oocytes are separated into 6.1 graph package. Small groups using forceps, defolliculated by agitation in 0.143 Kv2.1 mutants were constructed using the Quick Ca"-free Ringer solution containing collagenase for one Change Multi Site-Directed Mutagenesis Kit (Stratagene, La hour, washed, then stored at 15° C. in full Ringers solution Jolla, Calif.). Mutant Kv2.1 genes (in the pGA1 oocyte before injection.cRNA sufficient to give 1-10 LA of current at expression vector for oocyte expression) were sequenced in physiological Voltages for each channel type is injected into their entirety to confirm appropriate mutations and check for Xenopus oocytes (selected by appearance for overall health inadvertent mutations, and then Subcloned into a wild-type and uniformity of color) using a Sutter manual injector (typi pGA1 backbone. For oocyte expression, Kv3.4 was in pRF1, cally 0.1-5 ngcRNA in a volume of 25-50nl). The oocytes are Kv4.2 was in pRAT. cRNA transcripts were produced from then stored for 1-3 days at 15° C. before TEVC recording. SacII-linearized (Kv2.1), Mlullinearized (Kv3.4) and Not1 Beta Subunit cRNA is co-injected as appropriate, typically 2 linearized (Kv4.2) DNA templates using the T3 (Kv2.1), SP6 ng per oocyte. (Kv3.4) and T7 (Kv4.2) mMessage mMachine kits (Ambion, 0140 Whole cell oocyte experiments are performed using Austin, Tex.). cRNA was quantified by spectrophotometry a Warner OC-725C amplifier and pClamp9 software with and its size integrity verified by gel electophoresis. Defoli oocytes bathed in a small-volume Warner oocyte bath and culated stage V and VI Xenopus laevis oocytes (purchased viewed with a Fisher dissection microscope. Bath solution for from Nasco, Grand Island, N.Y.) were injected with 5 ng of whole cell oocyte recordings, e.g., for potassium channel cRNA encoding Kv2.1, Kv3.4 or Kv4.2. Chinese Hamster analysis, is generally (in mM): 96 NaCl. 4 KC1, 1 MgCl, 0.3 Ovary (CHO) cells were transfected with 0.25 ug Kv2.1 CaCl, 10 HEPES (Ph74); whole-cell oocyteTEVC pipettes cDNA, 2 ugblank plasmid and 2 ug Green fluorescent Protein are filled with 3 M KC1. Oocyte patches are studies using an in pBOB using Supetfect transfection reagent (Qiagen) 24 Axon Instruments small-volume bath, viewed with an Olym hours before whole-cell voltage clamp studies. pus IX50 inverted microscope, and recordings made using an 0144) Whole-cell two-electrode voltage-clamp (TEVC) Axon Multiclamp 700 A Amplifier and pCLAMP9 software recordings were performed on X laevis oocytes expressing (AXon Instruments). Extracellular Solution, e.g., for potas cloned Kv2.1, Kv3.4 or Kv4.2 using a Warner OC-725C sium channel analysis, is generally (in mM): 135 NaCl, 5 amplifier and pClamp9 (Axon Instruments, Foster City, KC1, 1.2MgCl, 5 HEPES, 2.5 CaCl, 10 D-glucose (Ph 7.4). Calif.) software. Oocytes were bathed in a small-volume Intracellular Solution, e.g., for potassium channel analysis, is Warner oocyte bath and viewed with a Fisher dissection generally (in mM): 10 NaCl, 117 KC1, 2 MgCl, 11 HEPES, microscope. Bath solution for whole-cell oocyte recordings 11 EGTA, ICaCl (Ph 7.2). Patch pipettes are approximately was (in mM):96 NaCl, 4KC1,0.7 MgCl, 1 CaCl, 10 HEPES 3-5 MS2 resistance when filled with recording solution. (pH 7.4); whole-cell oocyte TEVC pipettes were of 0.2-2 M 0141 Quantitative aspects of ion action on ion channels resistance when filled with 3 MKC1. Experiments were per are compared using standard pulse protocols. Pulse times and formed 24–48 hours after cRNA injection. For assessment of Voltages will vary according to which currents are being current-Voltage relationships in the absence or presence of studied. I/V and G/V curves are typically fit to a standard dabco derivatives, oocytes were held at -80 mV, stepped for 2 Boltzmann function: 1/(1+exp(V-V)/V}, where V is seconds at voltages between -80 mV and +60 mV in 20 mV the half-maximal Voltage of activation and V the slopefactor, steps (Kv2.1) or between -80 and +50 mV in 10 mV steps for quantification and comparison. Kinetics of drug effects (Kv3.4), then held for 500 ms at -40 mV before returning to upon wash-in are generally fit with single or multiple expo the holding potential. For assessment of block during wash-in nential functions and values for t and amplitude reported. of dabco derivatives at a single Voltage, oocytes were held at US 2009/000538.6 A1 Jan. 1, 2009

-80 mV, and repetitively stepped to 0 mV for 2 s with an with MiRP2 in rat brain. The sensitivities of two diammo interpulse interval of 10 s or 60s as indicated. A range of nium compounds (JE188 and TA279) were analyzed by func concentrations of dabco or dabco derivatives were washed in tional expression in Xenopus oocytes using two-electrode via the bath solution during repetitive single-Voltage pulses voltage clamp (TEVC), and bath application of the com (after an initial period of 5 pulses with control solution to pounds at various concentrations. Dose-responsecurves were ensure current stabilization) until equilibrium block was fitter with a logistic dose-response function: achieved, to establish dose-responses. Inhibited currents are compared to the current at the end of 5-pulse control period, y=A=(A/1=(Xixo') Equation (i) thus some bar graphs show control current values slightly less than 1, and with an error bar, to show how much currents deviate in the absence of drug. Washout was performed after GE) BrO the highest concentration of blocker was washed in. In cases where washout was achieved, only those recordings in which current returned to within 10% of original current level were used for construction of dose-response curves. For cysteine protection experiments using the K356C variant of Kv2.1, a JE188 dabco derivative (JC638.2a) was washed in to equilibrium block, followed by wash-in of a solution containing both JC638.2a and MTSET until no further changes in current were seen; following this the solution was washed out with standard bath Solution. As controls for this protection analy sis, JC638.2a or MTSET were washed in alone until equilib rium block, then washed out. 0145 For whole-cell voltage clamp studies of CHO cells, 0148 JE 188 compound, when applied externally to whole bath solution was (in mM): 135 NaCl, 5 KC1, 1.2 MgCl, 5 oocytes via the bath, inhibited Kv2.1 currents with or without HEPES, 2.5 CaCl, and 10 D-glucose, pH 7.4. Pipettes were MiRP2 (see FIG. 1). MiRP2 caused a two-fold decrease in 3-5 MQ resistance when filled with intracellular solution sensitivity (Kv2.1: x=23.3+2.1 uM, p=1.4+0.1; MiRP2 containing (in mM): 10 NaCl, 117 KC1, 2MgCl, 11 HEPES, Kv2.1: x=49.2+8.2 uM, p=1...6+0.2). This shift is in contrast 11 EGTA, and 1 CaCl, pH 7.2. For experiments with extra to the two-fold increase in tetraethylammonium affinity cellular drug application, cells were held at -80 mV and observed for MiRP2-KV2.1 channels compared to Kv2.1 subjected to 2 sec test pulses from to 0 mV every 30 s. alone. Importantly, Kv2.1 currents are 500-fold more sensi Whole-cell patch clamp recordings were performed at 22-25° tive to block by JE188 than to block by tetraethylammonium. C. using an IX50 inverted microscope equipped with epifluo 0149 TA279 was also applied externally to whole oocytes rescence optics for GFP detection (Olympus), a Multiclamp expressing Kv2.1 with or without MiRPs, via the bath 700 A Amplifier, a Digidata 1300 Analogue/Digital converter medium (FIG. 2). TA279 has a longer carbon string (C16 and pClamp9 Software (AXon Instruments). Leak and liquid versus C12). Application of various doses of TA279 and junction potentials (<4 mV.) were not compensated for when fitting of the dose response curve for current inhibition generating current-Voltage relationships. For intracellular showed that MiRP2 did not significantly alter inhibition of drug application, the tip of the recording electrode was first Kv2.1 channels by TA279 (Kv2.1: X=1.0+0.8 uM, p=0. filled with drug-free intracellular solution, the electrode was 8+0.1 MiRP2-Kv2.1: x=1.3+0.4 uM, p=0.8+0.2). Signifi then back-filled with intracellular solution containing TEA cantly, Kv2.1 currents are 10,000-fold more sensitive to (20 mM), TA279 (5 mM) or JC638.2C (0.5 mM). Cells were TA279 than to tetraethylammonium, and 20-fold more sen held at -80 mV and subjected to 2 sec test pulses from -60 sitive to TA279 than to JE188. mV to +60 mV in 10 mV increments, followed by a 1 sec tail pulse to -30 mV. After an initial recording which we denoted Example 5 time 0, a second recording was performed after 5 minutes. 0146 Data analysis: Current-voltage relationships were Didabco Strings are a Class of KV Channel-Reactive obtained by measuring peak current during depolarizing Agents pulses. Inhibition constants for DABCO blockers were cal culated after generating a dose response curve by fitting with 0150. We performed a screen to examine the effects of a logistic dose-response function y=A+A-A/1+(X/X). didabco strings (e.g., compounds with a dabco group at either where A is the maximum response plateau, A is the mini end, differing from one another in the number of CH groups mum response plateau, X is drug concentrator. X is the ICs. separating the two dabco groups) (see FIG. 3). Structure I and p the Hill slope. Data analysis was performed using below represents a dabco group. CLAMPFIT 9 (Axon Instruments, Foster City, Calif.) and tabulated in Excel 5.0. Graphs were generated using Origin 6.1. Data are expressed as mean-SEM, with n specifying the Structure I number of independent experiments. Statistical significance was assessed by one-way ANOVA with p-0.05 being indica tive of significance. Example 4 0151. Three representative KV channels were tested: 1) Kv2.1 Channels are Inhibited by Two Monocationic Kv2.1 (a low tetraethylammonium-affinity delayed rectifier Compounds highly expressed in mammalian brain and heart), 2) Kv3.4 (a 0147 We explored blocking effects on Kv2.1 with and high-tetraethylammonium-affinity A-type channel for con without the MiRP2 ancillary subunit. Kv2.1 can co-assemble trolling skeletal muscle excitability and neuronal refractori US 2009/000538.6 A1 Jan. 1, 2009

ness) and 3) KCNO4 (a mid-range tetraethylammonium-af in rodent heart, and generates an O2-sensitive current in pull finity channel associated with inherited deafness and monary arteries. Kv3.4 is a fast-inactivating KV channel involved in diseases involving neuronal hyperexcitability). expressed primarily in the brain and skeletal muscle. Co 0152 The data demonstrate that various didabco strings assembly of Kv3.4 with delayed rectifier Kv3 subfamily a possess several notable properties. These properties include: Subunits in the brain is thought to generate heteromeric chan (i) differentiation between the three channel types; (ii) varied nels with differing inactivation rates, perhaps enhancing fir effects depending on chain length; and (iii) a capacity to ing rate of fast-spilding neurons. In skeletal muscle, Kv3.4 open, i.e. increase KV current in Some cases, while blocking forms complexes with the MiRP2 ancillary subunit to control in others. myocyte excitability and repolarization. 0153. In a further experiment, we tested the same three Kv 0159 Bath-applied DABCO, at 1 mM had no effects on channels for sensitivity to members of another class of did the magnitude or kinetics of either Kv2.1 or Kv3.4 currents abco compounds. The didabco groups in these compounds heterologously expressed in X. laevis oocytes, analyzed by are separated by an aromatic ring, and they differ only in the two-electrode voltage clamp (TEVC). Next, the sensitivities relative positioning of the dabco groups on that ring (ortho, of Kv2.1 and Kv3.4 to two DABCO-derived monostrings, meta, para) (FIG. 4A). The compounds each behave distinctly JE188 and TA279, were analyzed by functional expression in from one another and show channel-type dependence of X. laevis oocytes using TEVC and bath application of the block. One compound (ortho JSG 17) is a KCNQ4-specific compounds at various concentrations. opener (1 mM, within the group of three channels tested, FIG. (0160. The JE18812-carbon monostring DABCO com 4B). pound significantly inhibited both Kv2.1 and Kv3.4 channels at 50 uM, with 250 uM producing almost complete inhibition Example 6 in both cases. Fitting of dose response curves with a logistic The TG28 Didabco String and Kv2.1 dose response function yielded JE188 an inhibition constants ICs of 23.3+2.1 uM, slope=1.4+0.1 for Kv2.1; and an ICs of 0154 TG28 is a didabco string with 12 CH groups sepa 34.2+10 uM, slope=0.7+0.1 for Kv3.4. rating the two dabco groups, which was shown in FIG. 3 to 0.161 Recording of current-voltage families for both block Kv2.1. Further review of this block revealed a striking channel types in the presence and absence of JE 188 showed effect: rapid reduction of current (block) followed by slow, voltage dependence of block in both cases such that inhibition partial restoration of current (partial opening). Subsequent was much less pronounced at more positive Voltages. For washout of TG28 resulted in a rapid increase of Kv2.1 Kv2.1 channels, mean inhibition was 3-fold more at 0 mV. current to a level, on average, two-fold larger than the pre than at +60 mV; for Kv3.4 channels mean inhibition was drug current, then a slow decrease to pre-drug current levels 2-fold more at 0 mV than at +50 mV. For Kv3.4 channels, (FIG. 5). crossover of the N-type inactivation occurred with applica O155 Based on the data, TG28 is a Kv2.1 blocker from the tion of 50 uMJE188, suggesting either slowing of inactiva outside, but a Kv2.1 opener from the inside. Alternatively, tion or partial drug unbinding at depolarized Voltages. there are two different external TG28 binding sites on Kv2.1 (0162. The TA279 16-carbon monostring DABCO com with different binding affinities and functional consequences. pound is a potent compound, inhibiting both Kv2.1 and Kv3.4 channels at 1 LM, with 5 mM producing almost complete Example 7 inhibition in both cases. Fitting of dose response curves with Didabco Compounds Act Within the KV Channel a logistic dose response function yielded an ICso of 1.9-3.6 Permeation Pathway uM, slope=0.6+0.4 for Kv2.1; and an ICs of 0.6+0.1 uM, slope=2.8+1.1 for Kv3.4. These values indicate that Kv2.1 0156 To investigate the mechanism of KV channel block channels are 3,000-fold more sensitive to block by externally or opening by didabco compounds, the current-Voltage rela applied TA279 than to block by externally-applied TEA, and tionship (I/V curve) of A1-28 Kv3.4 channels expressed in Kv3.4 channels are 500-fold more sensitive. Block of Kv2.1 Xenopus oocytes was assessed using TEVC recording in the and Kv3.4 was again Voltage-dependent; mean inhibition of absence and presence of a didabco opener (JC279.3a, the 10 Kv2.1 channels was 3-fold more at 0 mV than at +60 mV; for CH2-group didabco from FIG. 3) or a didabco blocker Kv3.4 channels mean inhibition was 1.5-fold more at 0 mV. (JC638.2a, the para didabco from FIG. 4). than at +50 mV. For Kv3.4 channels, N-type inactivation was (O157 FIG. 6 shows that block or opening of Kv3.4 by relatively less complete (as a proportion ofpeak current) in the JC279.3a or JC638.2a is voltage-dependent. Specifically, presence of TA279. block or opening of Kv3.4 channels by these two didabco 0163. It was considered possible that unlike TEA, exter compounds was attenuated at more depolarized (positive) nally-applied TA279 might cross the plasma membrane to Voltages. access a higher-affinity internal site. TEA has a higher-affin ity site in the innerpore of KV channels, but can only access it Example 8 when applied internally. Indeed, TA279 washout was much slower than typically observed for TEA, taking several min Kv2.1 and Kv3.4 Channels are Differentially Inhib utes, suggesting the possibility of it crossing the plasma mem ited by DABCO Monostrings brane to exert its action. 0158 Blocking effects of non-substituted DABCO on (0164. To assess at which side this DABCO derivative acts, Kv2.1 and Kv3.4 potassium channels were examined. Kv2.1 TA279 was applied extracellularly (via the bath) and intrac is a delayed rectifier KV channel expressed in mammalian ellularly (via the recording electrode) to Kv2.1 channels heart, brain and other excitable tissues. In particular, Kv2.1 expressed in CHO cells. Extracellular application of 500 nM facilitates dynamic control of neuronal excitability, forms a TA279 produced a mean current inhibition of 59+4% at 0 mV component of the IK cardiac myocyte repolarization current (n=3), indicating ~4-fold higher sensitivity than Kv2.1 chan US 2009/000538.6 A1 Jan. 1, 2009

nels expressed in oocytes, a common phenomenon because Kv2.1 and Kv3.4, was also screened for sensitivity to repre oocytes are thought to sequester some drugs, decreasing sentative DABCO derivatives. Kv4.2 generates A-type cur apparent sensitivity. Intracellular application of a ten-fold rents in mammalian brain and generates the cardiac I current higher concentration of TA279 (5 uM) produced no signifi in some species. As with previous reports for TEA, Kv4.2 was cant block at 0 mV, either at 0 or 5 minutes after attaining the relatively insensitive to DABCO derivatives, showing 3% whole-cell configuration, compared to control currents in block with 100 uMJE188, 3% block with 1 mM TG27, and which no drug was added to the electrode, as assessed by 38% block with 0.5 mM JC638.2C. Thus, DABCO com one-way ANOVA (n=4). As a positive control. 20 mMtetra pounds, in particular simple monostrings and di)ABCO ethylammonium (TEA) was shown to produce ~85% block at strings, can be used to differentiate between transient outward 0 mV at time 0 and ~88% block after 5 minutes. These data currents generated by Kv3.4 versus Kv4.2, and at much lower demonstrates that the high affinity site of TA279 is accessed concentrations than, for example, TEA. This property may from outside the cell and does not require TA279 to cross the prove useful in distinguishing between different A-type cur plasma membrane. rents in the brain in regions where both Kv3.4 and Kv4.2 are (0165 Simple diDABCO strings, with a DABCO group at expressed. Such as the hippocampus. each end separated by a variable-length hydrocarbon chain, also inhibited both Kv2.1 and Kv3.4 channels but with lower Example 9 affinity to the two monostrings tested. TG26 (4-carbon string) was less effective than TG27 (8-carbon string) at inhibiting JC638.2a Binds at a Similar External Site to TEA in either channel at a concentration of 1 mM and in the case of Kv2.1 both compounds Kv2.1 was more sensitive than Kv3.4. 0169 Generation of V curves from current-voltage fami lies recorded in the presence or absence of DABCO derived compounds Such as JC638.2a indicated Voltage dependence of block. This may suggest that DABCO derivatives block within the ion conduction pathway, as observed for TEA. To explore this hypothesis further block of wild-type and mutant Kv2.1 channels by JC638.2C. was analyzed. First, JC638.2C. was applied intracellularly to Kv2.1 channels expressed in CHO cells, via the recording electrode. As with TA279, intra cellularly applied JC638.2C. (0.5 mM) produced no signifi cant block at 0 mV when compared to control recordings with no drug in the recording electrode, as assessed by one-way ANOVA (n=4-5) whereas inparallel positive control experi ments intracellularly applied TEA (20 mM) produced 70% and 80% block at 0 mV at 0 and 5 minutes respectively (n=3-5). This suggested that JC638.2C-blocked via an exter nal, not internal, site. 0166 Construction of dose response curves yielded an (0170 Effects of the Y380T mutation in Kv2.1 on block by ICs for TG27 of 270.5+66.4 uM, slope=2.1+0.8 for Kv2.1; JC.638.2C. was compared. It was found that the mutation did Kv3.4 appeared somewhat less sensitive but an accurate fit of not alter block by JC638.2C, giving an ICs of 185+41 uM. the dose response curve could not be achieved because appli slope=1.3, not significantly different from wild-type. cation of TG27 at concentrations above 1 mM began to intro (0171 Methanethiosulfonate (MTS) forms the basis for a duce nonspecific leak. Similarly, construction of dose group of compounds that are able to bind irreversibly to the responses for TG26 was not possible because doses required free sulfhydryl group of cysteine residues that line the pore of to produce >50% block were toxic to the oocytes. channels. Introduction of a cysteine at position 356 in Kv2.1 0167 Even greater differentiation between the potency of made this position modifiable with MTSET resulting in per similar compounds for a given channel was observed with manent fractional block. However, when TEA was adminis diDABCO compounds separated by an aromatic group. tered before MFSET this ability to permanently modify the Three such compounds of similar structure except for the introduced cysteine was lost. Thus, TEA protects Kv2.1 chan relative positioning of the DABCO groups on the aromatic nels from MTSET modification, suggesting an external TEA were assessed: JSG 17, TA37 and JC638.2O, which contain binding site further from the pore than Kv2.1 residue K56, DABCO groups in ortho, meta and para positions, respec more distal from the pore thanY380. The ability of JC638.2o. tively, around the central aromatic ring (FIG. 4A). For both to prevent modification by MTSET at Kv2.1-K356C, because Kv2.1 and Kv3.4, only the para compound, JC638.2C. of similarities between the effects of some DABCO com showed significant inhibition at 1 mM, indicating the critical pounds and TEA, was investigated. When JC638.2C. was nature of the shape of the compound in determining block. administered alone, the K356C mutant was inhibited by JC638.2C. did not, however, significantly differentiate 62-3%; this block was reversed with washout. When MTSET between Kv2.1 and Kv3.4 in terms of sensitivity. Dose was used to modify the introduced cysteine, currents were response curves yielded an ICs for JC638.2C. of 185.7+17 inhibited 56.3%; washout here was incomplete, leaving uM, slope=1.4+0.1 for Kv2.1; and an ICs of 129+20.7 LM, 244% current inhibition remaining. Pre-application of JC638. slope=1.3+0.2 for Kv3.4. As with the DABCO monostrings, 2C. prevented permanent modification of Kv2.1-K356C: co JC638.2O. showed voltage dependent block, with inhibition application of JC638.2 (and MTSET inhibited K356C-Kv2.1 being attenuated at more positive Voltages. current by 84+1%, completely reversible with washout. 0168 The sensitivity of Kv4.2, a fast-inactivating KV These effects are similar to those previously observed for channel with relatively low sensitivity to TEA compared to protection from MTSET modification of Kv2.1-K356C by US 2009/000538.6 A1 Jan. 1, 2009

TEA, and together with the lack of effect of intracellularly 7. A method according to claim 6, wherein X represents an applied JC638.2O, suggest that JC638.2C. binds to a similar acyclic hydrocarbon group having ten to eighteen carbon external site in Kv2.1 to TEA. atOmS. What is claimed is: 8. A method according to claim 7, wherein the ion is rep 1. A method for modulating potassium, Sodium, and cyclic resented by: nucleotide-modulated ion channels in a mammal in need (1n) thereof, the method comprising administering to the mammal -- an effective amount of an ion comprising the following for mula: N N'-(CH2)CH.

-z (1) 9. A method according to claim 7, wherein the ion is rep R1 R4 resented by: (1p) N / N / -- m3 N N'- (CH2)15CH3. wherein: 10. A method according to claim 1, wherein Z represents 2, each of R', R. R. R. R. R. independently of each other, 3, or 4. represents an acyclic hydrocarbon group having one to 11. A method according to claim 10, wherein the ion is four carbon atoms; represented by: each of X and Y independently represents a carbocyclic or heterocyclic, Saturated or unsaturated, Substituted or unsubstituted ring or fused ring system; an acyclic (2) hydrocarbon group having one to twenty six carbons atoms; or a combination thereof; m1, m2, and m3 independently represent 0 or 1; V and V* independently represent C, N, or N'; V and V independently represent C or N“: Z represents the number of positive charges in the ion and has a value of at least 1: wherein a and d independently represent 0 or 1; wherein any of said acyclic hydrocarbon groups is satu R", R. R. R. R. R. X, Y, m1, and m2 are as defined in rated or unsaturated, Substituted or unsubstituted, claim 1; and straight-chained or branched, and wherein one or more carbon atoms of the acyclic hydrocarbon group is Z represents 2, 3, or 4. optionally substituted by one or more heteroatoms, 12. A method according to claim 11, wherein R', R. R. wherein no heteroatom is bound to: R. R. and R. are independently represented by the formula another heteroatom, CH, wherein in represents an integer from 1 to 3. V when V is N or N, 13. A method according to claim 12, wherein the ion is V when V is N, represented by: V when V is N', or V when V is N or N; and provided that at least one of V, V, V, and V represents N'. (3) 2. A method according to claim 1, wherein one of V, V, V3, and V4 represents N' and Z represents 1. (Y)-N N-X-N 3. A method according to claim 2, wherein m3 is 0. N 7 4. A method according to claim 3, wherein m' is 0, V1 represents N, and V represents N'. 5. A method according to claim 4, wherein R', R, and R' wherein a and d independently represent 0 or 1; are independently represented by the formula CH, wherein X, Y, m1, and m2 are as defined in claim 1; and in represents an integer from 1 to 3. Z represents 2, 3, or 4. 6. A method according to claim 5, wherein the ion is rep 14. A method according to claim 13, wherein the ion is resented by: represented by:

(1m) (3a)

wherein X represents an acyclic hydrocarbon group having one to twenty six carbons atoms. wherein X is as defined in claim 1. US 2009/000538.6 A1 Jan. 1, 2009

15. A method according to claim 14, wherein X represents 31. A method for modulating ligand-gated ion channels or an acyclic, Saturated or unsaturated. Substituted or unsubsti transient receptor potential channels in a mammal in need tuted, straight-chained or branched hydrocarbon group hav thereof, the method comprising administering to the mammal ing four to twelve carbon atoms. an effective amount of an ion comprising the following for 16. A method according to claim 15, wherein X represents mula: —(CH) . 17. A method according to claim 15, wherein X represents —(CH2)9—. -z (1) 18. A method according to claim 15, wherein X represents R1 R4 —(CH2)o . 19. A method according to claim 15, wherein X represents —(CH2)2 -. N / N / 20. A method according to claim 14, wherein X comprises m3 a phenylene ring. 21. A method according to claim 20, wherein the ion is wherein: represented by each of R',R,R,R,R,R, independently of each other, represents an acyclic hydrocarbon group having one to four carbon atoms; (4) each of X and Y independently represents a carbocyclic or heterocyclic, Saturated or unsaturated, Substituted or unsubstituted ring or fused ring system; an acyclic N N-HCG y-CH-N N. hydrocarbon group having one to twenty six carbons atoms; or a combination thereof, m1, m2, and m3 independently represent 0 or 1; V and V* independently represent C, N, or N': 22. A method according to claim 21, wherein the ion is V and Vindependently represent C or N“: represented by Z represents the number of positive charges in the ion and has a value of at least 1: wherein any of said acyclic hydrocarbon groups is satu -2 (5) rated or unsaturated, Substituted or unsubstituted, straight-chained or branched, and wherein one or more carbon atoms of the acyclic hydrocarbon group is N N-1 N "-HC CH2 -NN 7N. optionally substituted by one or more heteroatoms, N / wherein no heteroatom is bound to: another heteroatom, 23. A method according to claim 1, wherein the ion channel V when V is N' or is a potassium ion channel. V when V is N, 24. A method according to claim 23, wherein the potassium V when V is N', or ion channel is Voltage gated. V when V is N or N; and 25. A method according to claim 1, wherein the potassium provided that at least one of V, V, V, and V represents N'. ion channel is ATP gated. 32. A method according to claim 31, wherein the channel is 26. A method according to claim 23, wherein the potassium a ligand-gated ion channel. ion channel is calcium activated. 33. A method according to claim 32, wherein the ligand 27. A method according to claim 23, wherein the potassium gated ion channel is a cys-loop ligand gated channel. ion channel is an inward rectifier. 34. A method according to claim 32, wherein the ligand 28. A method according to claim 23, wherein the potassium gated ion channel is an ATP gated channel. ion channel is any one of the potassium ion channels shown in 35. A method according to claim 32, wherein the ligand Table 1 and Table 2. gated ion channel is a glutamate activated cationic channel. 29. A method according to claim 1, wherein the ion channel 36. A method according to claim 31, wherein the channel is is a sodium ion channel. a transient receptor potential channel. 30. A method according to claim 1, wherein the ion channel is a cyclic nucleotide-modulated ion channel. c c c c c