WO2012142459A1 - Isoindoline compounds for the treatment of spinal muscular atrophy and other uses - Google Patents

Abstract

Disclosed are compounds of Formula I Formula (I) in which n and R1?-R4? are defined herein. Also disclosed is a method of treating spinal muscular atrophy, as well as methods of using such compounds to increase SMN expression, increase EAAT2 expression, or increase the expression of a nucleic acid that encodes a translational stop codon introduced directly or indirectly by mutation or frameshift.

Description

ISOINDOLINE COMPOUNDS FOR THE TREATMENT OF SPINAL MUSCULAR ATROPHY AND OTHER USES

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 1/475,541, filed April 14, 201 1, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Spinal Muscular Atrophy (SMA) is a paralyzing and often fatal disease of infants and children. To date there is no effective treatment for SMA. This disease is caused by mutations that reduce the level of survival motor neuron protein (SMN), resulting in the loss of motor neurons in the central nervous system. Drugs that increase SMN expression are expected to be useful in the prevention and treatment of SMA. Prior studies in cultured cells have shown that indoprofen, a previously marketed non-steroidal anti-inflammatory drug (NSAID), increases the level of expression of SMN protein via an unknown mechanism (Lunn et al., Chem. & Biol. , 1 1 : 1489- 1493(2004)). Indoprofen also was shown to increase survival of SMA model mouse fetuses when administered in utero. The mechanism of regulation of SMN expression is not thought to be related to indoprofen' s NSAID activity since not all of the NSAIDs tested increase SMN expression. One possible mechanism of action is increased protein translation, as it has been shown that the level of the SMN protein can be increased by drugs that cause translational read-through of nonsense stop codons (Wolstencroft et al., Hum. Mol. Genet., 14:1199-210 (2005)). However, indoprofen is not a useful drug for SMA because it is only weakly active in increasing SMN expression, does not enter the brain at sufficient levels, and has lethal side effects due to its cyclooxygenase (Cox) inhibitory activity.

[0003] Accordingly, there is a need for new compounds, compositions, and methods that can address these problems.

BRIEF SUMMARY OF THE INVENTION

[0004] The invention provides a compound or pharmaceutically acceptable salt of the Formula I:

(I) wherein n and R^!-R⁴ are as described herein, as well as a method for preparing a compound of Formula I and intermediate compounds useful in such a method.

[0005] The invention also provides a method of increasing SMN expression in a cell comprising administering a compound of the invention to a cell comprising a nucleic acid encoding SMN, whereby expression of SMN is increased. The invention further provides a method of treating a disease or condition associated with underexpression of SMN in a mammal, particularly spinal muscular atrophy (SMA), comprising administering a compound of the invention to the mammal, whereupon SMA is treated.

[0006] In another aspect, the invention provides a method of increasing the expression of excitatory amino acid transporter (EAAT2) in a cell comprising administering a compound of the invention to a cell comprising a nucleic acid that encodes EAAT2, whereby expression of EAAT2 is increased. The invention further provides a method of treating a disease or condition associated with underexpression of EAAT2 in a mammal, particularly a

neurological disease, comprising administering a compound of the invention to the mammal, whereupon the neurological disorder is treated or prevented,

[0007] In still another aspect, the invention provides a method of altering the expression in a cell of a nucleic acid that encodes a translational stop codon, the method comprising administering a compound of the invention to a cell comprising a nucleic acid that encodes a translational stop codon, whereby expression of the nucleic acid is altered, e.g., increased. The invention further provides a method of treating a disease or condition associated with the occurrence of a translational stop codon in a mammal, the method comprising administering a compound of the invention to the mammal, whereupon the disease or condition is treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a bar graph representation of the results from a Western blot analysis showing SMN protein levels in patient fibroblasts cultured in vitro in the presence and absence of Compound 2, as described herein.

[0009] Figure 2 is a line graph showing PK data in rat and dog for Compound 2, as described herein.

[0010] Figure 3 is a bar graph representation of the results of an ELISA analysis showing SMN protein levels in C/C SMA mouse liver after in vivo BID or TID administration of vehicle or vehicle plus Compound 2, as described herein.

DETAILED DESCRIPTION OF THE INVENTION

[0011] In one as ect, the present invention provides a compound of Formula I:

(I) or salt thereof, wherein:

n is 0, 1, 2, or 3;

R¹ is a Q-Cg alkyl, C₂-Cg alkenyl, C₂-C₈ alkynyl, Q-Cg alkoxy, amino, Cs-Cg cycloalkyl, C₃-C₈ heterocycloaikyl, aryl, or heteroaryl, any of which are optionally substituted with one or more of CpC₈ alkyl, C₃-C₆ cycloalkyl, Ci-C_¾ alkoxy, Ci-C₈ haloalkoxy, C C_s haloalkyl, halogen, -CN, -C(0)OR^s, -C(0)NR⁵(R⁶), -N0₂, -S0₂NR⁵(R⁶), - NR⁷(S0₂)R⁸, -NR⁵C(0)NR⁶R⁷, amino, Q-C₄ alkylamino, Ci-C₄ dialkylamino, C₃-Q;

cycloalkylamino, aryl, heteroaryl, or heterocycloaikyl (e.g., substituted with 1-5 such groups, 1-3 such groups, or even a single such group);

R² is H or Ci-Cg alkyl;

or R^l and R² can be taken together with the atoms to which they are attached to form a 5- or 6-membered lactam ring, optionally comprising a second heteroatom in the lactam ring selected from the group consisting of N, O, and S; R³ is H, hydroxyl, Ci-C₈ alkyl, C₂-C₃ alkenyl, C₃-C₈ cycloaikyl, Ci-C₈ alkoxy, C₃-C₈ cycloalkyloxy, Ci-C₈ haloalkoxy, Q-Cg haloalkyl, halogen, alkylsulfonyl, -CN, -N0₂, - S0₂NR^s(R⁶), -NR⁷(S0₂)R⁸, -NR⁵C(0)R⁶, -NR⁵C(0)NR R⁷, amino, C_rC₄ alkylamino, C₃-C₆ cycloalkylamino, aryl, heteroaryl, or heterocycloalkyl;

R⁴ is H, C_rCg alkyl, C₃-C₆ cycloaikyl, C C_s alkoxy, C_rC₈ haloalkoxy, Ct-C₈ haloalkyl, halogen, -CN, -C(0)OR⁵, -C(0)NR⁵(R⁶), -N0₂, -S0₂NR^s(R⁶), -NR⁷(S0₂)R⁸, - NR⁵C(0)NR⁶R⁷, amino, C1 -C4 alkylamino, C3-C6 cycloalkylamino, aryl, heteroaryl, or heterocycloalkyl;

R⁵, R⁶, and R⁷ are independently H, Ci-C₈ alkyl, C₃-C₆ cycloaikyl, aryl, or heteroaryl; and

R⁸ is Ci-Cs alkyl, C₃-C₆ cycloaikyl, aryl, or heteroaryl.

[0012] The invention encompasses all compounds described by Formula I, and salts thereof, without limitation. However, for the purposes of further illustration, additional aspects and embodiments of the invention are discussed herein.

[0013] In one embodiment, R^! is Cj-Cg alkyl, haloalkyl, C₃-C₆ cycloaikyl, alkoxy, aminoalkyl, alkylamino, dialkylamino, halo alkylamino, and C₃-C₆ heterocycloalkyl, wherein the cycloaikyl and heterocycloalkyl are optionally substituted with C C₈ alkyl or -C(0)OR⁵. Non-limiting examples of suitable R¹ groups include methyl, methoxyl, and any of the following:

[0014] R² can be H or any branched or straight chain -Q alkyl, without limitation, including a C_rC₆ alkyl, C₁ -C4 alkyl, or Ci-C₂ alkyl.

[0015] Alternatively, R¹ and R² can be taken together with the atoms to which they are attached to form a 5- or 6-niembered lactam ring, optionally comprising a second heteroatom in the lactam ring selected from the group consisting of N, O, and S. Thus, for instance, the compound can be of Formula II:

or a salt thereof, wherein m is 1 or 2, and X is CH_2! O, N, or S.

[0016] R³ and R⁴ can be any group as previously defined, without limitation. However, according to certain aspects of the invention, R³ is H or halogen and/or R⁴ is H. Thus, for instance, the compound can be of Formula III:

or a salt thereof, wherein R , R , and R are as previously defined. According to yet another aspect of the invention, R is hydrogen or halogen.

[0017] Specific examples of compounds of the invention include, without limitation, those provided in Table 1.

Table 1

Table 1

Table 1

[0018] Preferred compounds of the invention are able to penetrate the blood-brain barrier so as to accumulate in therapeutically effective amounts (e.g., result in a brain to plasma ratio of about 20% or greater), and/or do not have significant cyclooxygenase (Cox) inhibitory activity (e.g., have less than toxic levels of Cox inhibitory activity at concentrations predicted to be therapeutic).

[0019] As used herein, unless otherwise specified, the term "alkyl" means a saturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C1-C20, Cj-Cio, Cj-C4, etc.). Representative saturated straight chain alkyls include methyl, ethyl, ^-propyl, -butyl, fl-pentyl, rc-hexyl, ίί-heptyl, R-octyl, /j-nonyl and n- decyl; while representative saturated branched alkyls include isopropyl, sec-butyl, isobutyl, (erf-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3- dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4- dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3- dimethylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2- ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2- methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. An alkyl group can be unsubstituted or substituted.

[0020] The term "cycloalkyl," as used herein, means a cyclic alkyl moiety containing from, for example, 3 to 6 carbon atoms, preferably from 5 to 6 carbon atoms. Examples of such moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0021] The term "heterocycloalkyl" means a cycloalkyl moiety having one or more heteroatoms selected from nitrogen, sulfur, and/or oxygen. Preferably, a heterocycloalkyl is a 5 or 6-membered monocyclic ring and contains one, two, or three heteroatoms selected from nitrogen, oxygen, and/or sulfur. The heterocycloalkyl can be attached to the parent structure through a carbon atom or through any heteroatom of the heterocycloalkyl that results in a stable structure. Examples of such heterocyclic rings are pyrrolinyl, pyranyl, piperidyl, tetrahydrofuranyl, tetrahydrothiopheneyl, morpholinyl, dioxanyl, or dioxoianyl.

[0022] As used herein, unless otherwise specified, the term "alkoxy" means -O-(alkyl), wherein alkyl is defined above. The term "cycloalkyloxy" means -O-(cycloalkyl), wherein cycloalkyl is defined above. Furthermore, as used herein, the term "haloalkoxy" means an alkoxy substituted with one or more halogens, wherein alkoxy and halogen are defined as above.

[0023] As used herein, unless otherwise specified, the term "alkylamino" means

-NH(alkyl) or -N(alkyl)(alkyl), wherein alkyl is defined above. As used herein, unless otherwise specified, the term "cycloaikylamino" means -NH(cycio alkyl) or

-N(cycloalkyl)(cycloalkyl), wherein cycloalkyl is defined above.

[0024] As used herein, unless otherwise specified, the term "alkenyl group" means a straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C2-C₂o, C₂-Cio, C₂-C₄, etc.) and including at least one carbon-carbon double bond. Representative straight chain and branched alkenyls include vinyl, allyl, 1-butenyl, 2- butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-methyl-2-butenyl, 2,3- dimethyl-2-butenyl, 1-hexenyl, 2-hexenyL 3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1- octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl, 3- decenyl, and the like. Any unsaturated group (double bond) of an alkenyl can be

unconjugated or conjugated to another unsaturated group. An alkenyl group can be unsubstituted or substituted.

[0025] As used herein, unless otherwise specified the term "aikynyl group" means a straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C2-C20, C₂-Cio, C₂-C6, etc.), and including at least one carbon-carbon triple bond. Representative straight chain and branched alkynyls include -acetylenyl, -propynyl, -1- butynyl, -2-butynyl, - 1-pentynyl, -2-pentynyl, -3-methyl-l -butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl, -2-octynyl, -7- octynyl, -1-nonynyl, -2-nonynyl, -8-nonynyl, -1-decynyl, -2-decynyl, -9-decynyl, and the like. Any unsaturated group (triple bond) of an aikynyl can be unconjugated or conjugated to another unsaturated group. An aikynyl group can be unsubstituted or substituted. [0026] As used herein, unless otherwise specified, the term "halogen" or "halo" means fluoro, chloro, bromo, or iodo. Furthermore, unless otherwise specified, the term "haloalkyl" means an alkyl substituted with one or more halogens, wherein alkyl and halogen are defined as above.

[0027] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic moiety, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, naphthyl, anthracenyl, pyrenyl, and the like. An aryl moiety generally contains from, for example, 6 to 30 carbon atoms, preferably from 6 to 18 carbon atoms, more preferably from 6 to 14 carbon atoms and most preferably from 6 to 10 carbon atoms. It is understood that the term aryl includes carbocyclic moieties that are planar and comprise 4n+2 π electrons, according to Hiickel's Rule, wherein n = 1, 2, or 3.

[0028] The terms "cycloalkylaryl" refer to an aryl, as defined herein, that is substituted with a cycloalkyl group, as defined herein, or as a fused ring, e.g., benzo. Examples of cycloalkylaryl groups include 5-, 6-, 7-, or 8-1,2,3,4-tetrahydronaphthalenyl and 4-, 5-, 6-, or 7-2,3 -dihydro- 1 H-indeny 1.

[0029] The term "heteroaryl" refers to aromatic 5 or 6 membered monocyclic groups, 9 or 10 membered bicyclic groups, and 1 1 to 14 membered tricyclic aryl groups having one or more heteroatoms (O, S, or N). Each ring of the heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be quaternized. Illustrative examples of heteroaryl groups are pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1 ,2,3,)- and (l,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thiopheneyl, isothiazolyl, thiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrro!o[2,3-c]pyridinyl, pyrrolo[3,2- c pyridinyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, pyrrolo[3,2-d]pyrimidinyl, and py rro lo [2 , 3 -d]pyrimidiny 1.

[0030] As used herein, unless otherwise specified, the term "substituted" means a group substituted by one or more substituents, such as, alkyl, alkenyl, alkynyl, cycloalkyl, aroyl, halo, haloalkyl (e.g., trifiuoromethyl), haloalkoxy (e.g., trifluoromethoxy), hydroxy, alkoxy, alkylthioether, cycloalkyloxy, heterocylooxy, oxo, alkanoyl, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, heterocyclo, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, aryl alkylamino, cycloalkylamino, heterocycloamino, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocyclothio, alkylthiono, arylthiono, aryiaikylthiono, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido (e.g., -S0₂N¾), substituted sulfonamido, nitro, cyano, carboxy, carbamyl (e.g., -CONH2), substituted carbamyl (e.g., -CONH-alkyl, -CONH-aryl, -CONH-arylalkyl, or instances where there are two substituents on the nitrogen selected from alkyl or arylalkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino, substituted or unsubstituted

heterocycloalkyl, and substituted or unsubstituted heteroaryl.

[0031] Whenever a range of the number of atoms in a structure is indicated (e.g., a Cj-Cg, Ci-C₆, C1-C4, or C[-C3 alkyl, haloalkyl, alkylamino, alkenyl, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1-8 carbon atoms (e.g., Q-Cg), 1-6 carbon atoms (e.g., C[-C₆)₅ 1-4 carbon atoms (e.g., Q-C₄), 1-3 carbon atoms (e.g., C1-C3), or 2-8 carbon atoms (e.g., C₂-Ca) as used with respect to any chemical group (e.g., alkyl, haloalkyl, alkylamino, alkenyl, etc.) referenced herein

encompasses and specifically describes 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, as appropriate, as well as any sub-range thereof (e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms,

1- 5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms, 2-3 carbon atoms,

2- 4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms,

3- 4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms,

4- 5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 5-6 carbon atoms,

5- 7 carbon atoms, 5-8 carbon atoms, 6-7 carbon atoms, or 6-8 carbon atoms, as appropriate).

General Synthesis

[0032J The compounds of the invention described herein can be prepared by any suitable technique. According to one aspect of the invention, a compound of Formula I or salt thereof, or intermediate compound associated therewith, is prepared by a method comprising one or more of the following steps: (1) nitration of isobenzofuran-l(3H)-one to 6- nitroisobenzofuran-l(3H)-one; (2) converting 6-nitroisobenzofuran-l(3H)~one to methyl-2- (chloromethyl)-5-nitrobenzoate by opening the lactone ring; (3) addition of quinolin-6-amme to methyl-2-(chloromethyl)-5-nitrobenzoate with formation of the salt of the lactam 6-nitro-2- (quinolin-6-yl)isoindolin-l-one; (4) reduction to form intermediate I-li; (5) acylation to form a compound of Formula I; and (6) formation of pharmaceutically acceptable salts of Formula 1. This method is illustrated in Scheme 1, below. The intermediate compounds of Scheme 1, as well as each individual process step for preparing the intermediate compounds, is considered to be an additional aspect of the invention. Thus, provided herein is a compound of Scheme 1, particularly a compound of Formula I-li, including salts thereof. Also provided herein is a method of preparing a compound of Formula I or salt thereof, or an intermediate compound of Scheme 1, including salts thereof, comprising one or more of steps (1) through (6) of the method described above and illustrated in Scheme 1. Scheme 1 can be modified to introduce a substituent identified as R⁴ above by having a suitable R⁴ group present on the quinoline-6 -amine reagent.

SCHEME 1

[0033] According to another aspect of the invention, a compound of Formula I or salt thereof, or intermediate compound associated therewith, is prepared by a method comprising one or more of the following steps: (1) ethylation of the acid group of 2-methyl-5- nitrobenzoic acid (e.g., with SOCl₂ in EtOH); (2) reduction of the nitrate group (e.g., with ¾, 10% Pd/C, in EtOH); (3) replacement of the amine to halogen, e.g., bromide (e.g., with NaN0₂ and HBr in ¾0 then CuBr and HBr in H₂0); (4) halogenation, e.g., bromination, of the 2-methyl (e.g., with NBS, benzoyl peroxide, and CC1₄); (5) addition of quinolin-6-amine (e.g., with DIPEA and EtOH) to form 6-halogen-2-(qumolin-6-yl)isoindolin-l-one; (6) addition of R¹(CH₂)_nC(0)NHR² to 6-halogen-2-(quinolin-6-yl)isoindolin-l-one (e.g., with (lR,2R)-Nl,N2-dimethylcycIohexane-l,2-diamine, Cul, and ¾C0₃ in toluene) to form a compound of Formula I; and (7) formation of pharmaceutically acceptable salts of Formula I (e.g., with 2 N HCI in ether, MeOH, and dioxane to form the HC1 salt). This method is illustrated in Scheme 2, below. The intermediate compounds of Scheme 2, as well as each individual process step for preparing the intermediate compounds, is considered to be an additional aspect of the invention. Thus, provided herein is a compound of Scheme 2, including salts thereof. Also provided herein is a method of preparing a compound of Formula I or salt thereof, or an intermediate compound of Scheme 2, including salts thereof, comprising one or more of steps (1) through (7) of the method described above and illustrated in Scheme 2. Scheme 2 can be modified to introduce a substituent identified as R⁴ above by having a suitable R⁴ group present on the quinoline-6-amine reagent. Also, R' and R² may be taken together to form the lactam for the addition in step (6).

Γ f 0034] According to another aspect of the invention, a compound of Formula I or salt thereof, or intermediate compound associated therewith, is prepared by a method comprising one or more of the following steps: (1 ) acylation of the amine of intermediate I- 1 i of Scheme 1 with 3-halogen-propanoyl halide to form intermediate I-3i; (2) replacement of the halogen with an amine to form a compound of Formula I; and (3) formation of pharmaceutically acceptable salts of Formula I (e.g., using 2 N HC1 to form the HC1 salt). This method is illustrated in Scheme 3, below. The intermediate compounds of Scheme 3, as well as each individual process step for preparing the intermediate compounds, is considered to be an additional aspect of the invention. Thus, provided herein is a compound of Scheme 3, particularly a compound of Formula I-3i, including salts thereof. Also provided herein is a method of preparing a compound of Formula I or salt thereof, or an intermediate compound of Scheme 3, including salts thereof, comprising one or more of steps (1) through (3) of the method described above and illustrated in Scheme 3. Scheme 3 can be modified to introduce a substituent identified as R⁴ above by having a suitable R⁴ group present on the quinoline of intermediate I-li. SCHEME 3

I

[0035] According to another aspect of the invention, a compound of Formula I or salt thereof, or intermediate compound associated therewith, is prepared by a method comprising one or more of the following steps: (1) conversion of the amine of intermediate I-li to isocyanate (e.g., with diphosgene) to form intermediate I-4i; (2) addition of an amine to form a compound of Formula I; and (3) formation of pharmaceutically acceptable salts of Formula I (e.g., using 2 N HC1 to form the HC1 salt). This method is illustrated in Scheme 4, below. The intermediate compounds of Scheme 4, as well as each individual process step for preparing the intermediate compounds, is considered to be an additional aspect of the invention. Thus, provided herein is a compound of Scheme 4, particularly a compound of Formula I-4i, including salts thereof. Also provided herein is a method of preparing a compound of Formula I or salt thereof, or an intermediate compound of Scheme 4, including salts thereof, comprising one or more of steps (1) through (3) of the method described above and illustrated in Scheme 4. Scheme 4 can be modified to introduce a substituent identified as R⁴ above by having a suitable R⁴ group present on the quinoline of intermediate I-li.

SCHEME 4

I [0036] According to another aspect of the invention, a compound of Formula I or salt thereof, or intermediate compound associated therewith, is prepared by a method comprising one or more of the following steps: (1) bromination of 4-fluoro-3-methoxybenzoic acid (e.g., with Br₂); (2) ethylation of the acid group (e.g., with H₂S0₄ and EtOH); (3) conversion of the bromide to CN (e.g., with CuCN); (4) formation of the lactam 5-fluoro-6-methoxyisoindolin- 1-one (e.g., using 10% Pd/C); (5) addition of 6-bromoquinoline (e.g., using Pd₂(dba)3, Xantphos, Cs₂C0₃, and dioxane) to form intermediate I-5i; (6) conversion of the methoxyl to hydroxyl (e.g., using 48% HBr and HO Ac) to form intermediate I-5ii (which can be the salt, e.g., HBr salt); (7) addition of N-phenyl-bis(trifluoromethansulfonimide) to form

intermediate I-Siii; (8) addition of R¹(CH₂)₁₁C(0)NHR² (e.g., using Pd₂(dba)₃, 2-di-tert- butylphosphino-3,4,5,6-tetramethyl-2',4',6'-triisopropyl-l, -biphenyl_? K₃P0_4j tBuOH, and molecular sieves) to form a compound of Formula I; (9) formation of pharmaceutically acceptable salts of Formula I (e.g., with 2 N HC1 to form the HC1 salt). This method is illustrated in Scheme 5, below. The intermediate compounds of Scheme 5, as well as each individual process step for preparing the intermediate compounds, is considered to be an additional aspect of the invention. Thus, provided herein is a compound of Scheme 5, particularly compounds of Formula I-5i, I-5ii, and I-5iii, including salts thereof. Also provided herein is a method of preparing a compound of Formula I or salt thereof, or an intermediate compound of Scheme 5, including salts thereof, comprising one or more of steps (1) through (9) of the method described above and illustrated in Scheme 5. Scheme 5 can be modified to introduce a substituent identified as R⁴ above by having a suitable R⁴ group present on the 6-bromoquinoline reagent.

SCHEME 5

[0037] According to another aspect of the invention, a compound of Formula I or salt thereof, or intermediate compound associated therewith, is prepared by a method comprising one or more of the following steps: (1) amidation of the amine of intermediate I-li with RCH(NHFmoc)C(0)Cl (e.g., in DIPEA and THF) to form intermediate I-6i; (2) replacement of the Fmoc protecting group with hydrogen (e.g., with piperidine) to form a compound of Formula I; and (3) formation of pharmaceutically acceptable salts of Formula I (e.g., using 2 N HC1). This method is illustrated in Scheme 6, below. The intermediate compounds of Scheme 6, as well as each individual process step for preparing the intermediate compounds, is considered to be an additional aspect of the invention. Thus, provided herein is a compound of Scheme 6, particularly a compound of Formula I-6i, including salts thereof. Also provided herein is a method of preparing a compound of Formula I or salt thereof, or an intermediate compound of Scheme 6, including salts thereof, comprising one or more of steps (1) through (3) of the method described above and illustrated in Scheme 6. Scheme 6 can be modified to introduce a substituent identified as R⁴ above by having a suitable R⁴ group present on the quinoline of intermediate I-li.

SCHEME 6

NHFmoc

I

[0038] The pharmaceutically acceptable salts of the compounds of Formula I can be any pharmaceutically acceptable salt, and can be prepared from a non-salt compound of Formula I in any suitable manner. Typically, such salts can be prepared from a compound using relatively nontoxic acids or bases, depending on the particular starting "parent" compound. Base addition salts of parent compounds with relatively acidic functionalities can be prepared by contacting the free acid form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium salts, and the like. Acid addition salts of parent compounds having relatively basic functionalities can be obtained by contacting the free base form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic. or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al, Journal of Pharmaceutical Science, 66: 1-19 (1977)). Compounds of the invention may contain both basic and acidic functionalities, which allow the compounds to be converted into either base or acid addition salts. The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.

Pharmaceutical Composition

[0039] Any one or more of the compounds of the invention described herein can be formulated as a pharmaceutical composition comprising at least one compound of the invention and a pharmaceutically acceptable carrier. The carrier can be any of those conventionally used and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of

administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical composition, the compounds of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0040] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[0041] The choice of carrier will be determined in part by the particular compound of the invention and other active agents or drugs used, as well as by the particular method used to administer the compound. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present inventive methods. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the compound of the invention are known, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective response than another route.

[0042] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0043] Injectable formulations are among those formulations that are suitable in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (See, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compounds of the invention can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol,

dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l ,3-dioxolane-4-methanol, ethers, such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0044] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, com, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0045] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts. Suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyi ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkano lam ides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-p-aminopropionates, and 2-alkyl -imidazoline quaternary ammonium salts, and (e) mixtures thereof

[0046] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi- dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0047] Topical formulations are well-known to those of skill in the art. Such

formulations are particularly suitable in the context of the present invention for application to the skin. [0048] The compounds of the invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa.

[0049] Additionally, the compounds of the invention, or compositions comprising such compounds, can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0050] One of ordinary skill in the art will readily appreciate that the compounds of the invention described herein can be modified in any number of ways to increase the therapeutic efficacy of the compound. For instance, the compound could be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds to targeting moieties is known in the art. The term "targeting moiety" as used herein, refers to any molecule or agent that specifically recognizes and binds to a cell-surface receptor, such that the targeting moiety directs the delivery of the compound to a population of cells on which surface the receptor is expressed. Targeting moieties include, but are not limited to, antibodies, or fragments thereof, peptides, hormones, growth factors, cytokines, and any other naturally- or non-naturally-existing ligands, which bind to cell surface receptors. The term "linker" as used herein, refers to any agent or molecule that bridges the compound to the targeting moiety. One of ordinary skill in the art recognizes that sites on the compounds, which are not necessary for the function of the compound, are ideal sites for attaching a linker and/or a targeting moiety, provided that the linker and/or targeting moiety, once attached to the compound, do(es) not interfere with the function of the compound.

[0051] Alternatively, the compounds of the invention described herein can be formulated as a depot drug form, such that the manner in which the compound of the invention is released into the body to which it is administered is controlled with respect to time and location within the body (see, e.g., U.S. Patent 4,450,150). Depot forms of compounds of the invention can be, for example, an implantable composition comprising the compound and a porous material, such as a polymer, wherein the compound is encapsulated by or diffused throughout the porous material. The depot is then implanted into the desired location within the body and the compound is released from the implant at a predetermined rate by diffusing through the porous material.

[0052] In some contexts, the compounds of the invention can be advantageously administered via an implanted pump that allows intrathecal delivery. Such a delivery method is especially useful for delivery of drugs to the CNS when the drugs administered do not otherwise sufficiently penetrate the blood-brain barrier.

[0053] The pharmaceutical composition can additionally comprise other

pharmaceutically active agents or drugs. Examples of such other pharmaceutically active agents or drugs that can be suitable for use in combination with one or more compounds of the invention include drugs that enhance translational read-through (e.g., WO 2004/009558, gentamycin, or other aminoglycoside antibiotics), drugs that inhibit one or more histone deacetylase enzymes (e.g., valproate, phenylbutyrate, or hydroxyurea), or drugs that increase SM expression via other mechanisms. The compounds of the invention also can be administered or formulated in combination with anticancer agents or other antibiotics.

Suitable anticancer agents include, without limitation, alkylating agents; nitrogen mustards; folate antagonists; purine antagonists; pyrimidine antagonists; spindle poisons;

topoisomerase inhibitors; apoptosis inducing agents; angiogenesis inhibitors;

podophyllotoxins; nitrosoureas; cisplatin; carboplatin; interferon; asparginase; tamoxifen; leuprolide; flutamide; megestrol; mitomycin; bleomycin; doxorubicin; irinotecan; and taxol. Antibiotics include macrolides (e.g., tobramycin), cephalosporins (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime, or cefadroxil), clarithromycin, erythromycin, penicillins (e.g., penicillin V), and quinolones (e. g., ofloxacin, ciprofloxacin, or norfloxacin).

[0054] The pharmaceutical composition can comprise an amount or dose of a compound of the invention sufficient to affect a therapeutic or prophylactic response in the host over a reasonable time frame. The appropriate dose will depend upon the nature and severity of the disease or affliction to be treated or prevented, as well as by other factors. For instance, the dose also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular compound. Ultimately, the attending physician will decide the dosage of the compound of the present invention with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated. Typical doses might be, for example, 0.1 mg to 1 g daily, such as 5 mg to 500 mg daily.

Methods of Use

[0055] The compounds can be used for any purpose including, without limitation, the treatment, prevention, or diagnosis of a disease or condition, the screening of compounds that can be used to treat, prevent, or diagnose a disease or condition, or the research of the underlying mechanisms or causes of a disease or condition, which research can be used, for example, in the development of methods to treat, prevent, or diagnose the disease or condition.

[0056] Use of the compounds of the invention may comprise administration of a compound to a cell. The compounds of the invention described herein can be administered to a cell in vitro. As used herein, the term "m vitro" means that the cell is not in a living organism. The compounds of the invention also can be administered to a cell in vivo. In vivo administration to a cell of an organism can be facilitated by administration of the compound to the host organism itself. Hosts include, for example, bacteria, yeast, fungi, plants, and mammals. Preferably, the host is a mammal. For purposes of the present invention, mammals include, but are not limited to, the order Rodentia, such as mice, and the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order

Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simioids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human. Furthermore, the host can be the unborn offspring of any of the forgoing hosts, especially mammals (e.g., humans), in which case any screening of the host or cells of the host, or administration of compounds to the host or cells of the host, can be performed in utero.

[0057] Without wishing to be bound by any particular theory, it is believed that the compounds of the invention are particularly useful with respect to (a) diseases and conditions involving the under-expression of survival motor neuron protein (SMN), (b) diseases and conditions that can be ameliorated by modulating protein translation, e.g. read through of stop codons UAA, UAG, UGA) or shifting the translational reading frame, and (c) diseases and conditions associated with elevated glutamate levels in the central nervous system (CNS) and/or that could be ameliorated by increases in glutamate transporter (e.g., EAAT2) expression.

[0058] The terms "treat" and "prevent," as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the diseases described herein being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

A. Diseases and Conditions Involving Under-Expression of Survival Motor

Neuron Protein

[0059] Preferred compounds of the invention can be used to increase SMN expression in a cell that comprises an SMN2 gene, particularly a cell that does not comprise an SM 1 gene, or that comprises a defective or deleted SMN1 gene. Thus, one aspect of the invention provides a method of increasing SMN expression in a cell comprising administering a compound of the invention to a cell comprising a nucleic acid that encodes SMN2, desirably a cell that comprises a defective or deleted SMN1 gene, whereby SMN expression is increased.

[0060] SMN1 and SMN2 refer to two different genes that each encode SMN protein, but that differ by a single base pair. The base pair change in SMN2 results in decreased expression of SMN due to an alternative splicing event that deletes exon 7, the exon containing the normal signals for termination of protein translation. Without these signals, protein translation terminates at stop codons encountered a few amino acids downstream, creating a protein with an abnormal C terminus. The resulting SMN protein, termed SMN Δ7, is unstable, thus expression of SMN2 produces low levels of SMN protein. Because SMN2 is typically not properly expressed, SMN protein is produced primarily by SMN1. Most patients that exhibit under-expression of SMN have a defective or mutant SMN1 gene, but an intact SMN2 gene. Without wishing to be bound by any particular theory, it is believed that the compounds of the invention increase the expression of SMN2 via a post-transcriptional mechanism, possibly by suppressing the effects of the abnormal translational termination of SMN Δ7. For example, it is believed that compounds of the invention may stimulate translational read through or frameshift at the abnormal SMN Δ7 termination stop codon, resulting in an extended C terminus and stabilization of the SMN Δ7 protein.

[0061] The increase in expression of SMN after administration of a compound of the invention can be any increase as compared to the expression level of SMN in the cell in the absence of the compound of the invention. Typically, the cell will have a defective or mutant SM l protein and/or reduced levels of SMN protein expression in the absence of a compound of the invention. Methods for detecting and measuring increased SMN

expression, particularly through expression of SMN2, are known in the art and described herein.

[0062] The ceil to which the compound of the invention is administered preferably is in a host. Suitable hosts are as previously described herein. The host is desirably a mammal, especially a human. The method of this aspect of the invention is most suitable for use in conjunction with a host that is afflicted with a disease or at risk for developing a disease, such as a disease associated with the under-expression of SMN. Such diseases include, for example, spinal muscular atrophy (SMA). Preferably, one or more symptoms of the disease are prevented, reduced, or eliminated subsequent to administration of at least one compound of the invention, thereby effectively treating or preventing the disease to at least some degree. Accordingly, the invention provides a method of treating spinal muscular atrophy (SMA) in a mammal comprising administering a therapeutically effective amount of at least one compound of the invention to the mammal, whereupon SMA is treated.

B. Other Diseases and Conditions Ameliorated by Modulating Protein Translation

[0063] In a related aspect, the invention provides a method of altering in a cell the expression of a nucleic acid that encodes a pathological translational modulating signal. The pathological translational modulating signal can be a normally occurring stop codon or a stop codon introduced directly or indirectly by mutation, particularly a nonsense stop codon. The modulating signal can also be a normally occurring signal for programmed translational frame shift or a frameshift signal introduced directly or indirectly by mutation. The method comprises administering a compound or pharmaceutically acceptable salt of the invention to a cell comprising a nucleic acid that encodes a pathological translational modulating signal, particularly a stop codon or frame shift signal, whereby expression of the nucleic acid is altered. Without wishing to be bound by any particular theory, it is believed that the compounds of the invention permit translational (ribosomal) read through of stop codons, especially those introduced directly or indirectly by mutation. According to a preferred aspect of the invention, the stop codon is one that is introduced directly or indirectly by a mutation, and the compound of the invention permits translational read -through of the stop codon, preferably without interfering with the effect of normal stop codons (e.g., stop codons not introduced directly or indirectly by mutation, which do not interfere with the production of a full-length protein). The compounds, thus, increase expression of the protein products of such nucleic acids. Compounds of the invention may also modulate gene expression through effects on naturally occurring stop codons,

[0064] As used herein, the term "translational stop codon introduced directly or indirectly by mutation" means any stop codon that results in the premature or otherwise unusual termination of translation and consequential production of a truncated gene product or protein. Translational stop codons introduced directly or indirectly by mutation include, for example, those that result in a gene product that has reduced stability or reduced activity (or no activity) as compared to the normal, full-length protein product, or results in the reduction or complete absence of a protein product. Translational stop codons introduced directly or indirectly by mutation also include, for example, those that result in a mRNA that is a target of nonsense-mediated RNA decay. The translational stop codon can be present in the DNA or RNA of any type of cell, and can arise through any type of mutagenesis event. For example, the nucleic acid that encodes the translational stop codon can be, without limitation, a defective SMN1 gene or a defective or normal SMN2 gene, or any transcript thereof. In particular, the alternative splicing event that occurs during SMN2 expression, which results in the deletion of S N exon 7, also creates a nonsense stop codon in the protein reading frame. The nucleic acid comprising the stop codon can be endogenous to the cell, or a nucleic acid introduced into the cell, for example, by a virus. Without wishing to be bound by any particular theory, it is believed that a compound of the invention can increase expression of DNA or RNA by suppressing the effects of the nonsense stop codons, possibly by allowing translational read-through of the stop codon or by causing a shift in the translational reading frame or by suppressing nonsense-mediated mRNA decay.

[0065] An appropriate screening assay can be employed to determine whether a cell or host comprises a nucleic acid that encodes a translational stop codon introduced directly or indirectly by mutation. For instance, the DNA or RNA of a cell (e.g., a cell of a host or an organism that is pathogenic to the host) can be sequenced or subjected to Southern Blot, polymerase chain reaction (PCR), use of the Short Tandem Repeat (STR), or polymorphic length restriction fragments (RFLP) analysis to determine if a nonsense mutation is present. Alternatively, it can be determined if a cell (e.g., a cell of a host) expresses altered levels of a protein encoded by the nucleic acid using western blot or other immunoassays. Other methods of determining whether a nucleic acid that encodes a premature translational stop codon are available.

[0066] Ribosomal frameshifting is used by many RNA viruses to regulate the expression of viral genes. Frameshifting may involve the use of pseudoknot structures to induce -1 frameshifts or +1 frameshifts during translation (e.g.,flaviviridae, retroviridae,

coronaviridae, astroviridae, alphaviridae, arteriviridae, and orthomyxoviridae), the use of internal ribosomal entry sites (IRES) that facilitate the translation of multiple proteins from polycistronic RNAs (Q.g.,flavivindae,picornaviridae, coronaviridae, retroviridae, togaviridae, and the rhabdoviridae), or other regulatory mechanisms to express their genes that might be expected to be affected by modulation the fidelity of ribosomes including translational termination-re- initiation. Additional mechanisms utilized by vimses suppress the utilization of stop codons by interfering with cellular factors required for their recognition. Without wishing to be bound by any particular theory, the compounds of the present invention may act through any of these mechanisms.

[0067] Without wishing to be bound by any particular theory, it is believed that the compounds of the invention also modulate protein translation by permitting translational (ribosomal) frame shift in response to frame shift signals, whether naturally occurring or introduced directly or indirectly by mutation. According to a preferred aspect of the invention, the frame shift signal is one that occurs naturally as a programmed frame shift signal to regulate production of alternative proteins from a single mRNA, and the compound of the invention alters the frequency with which the ribosome shifts the reading frame in response to the signal. The compounds, thus, alter the relative expression of the alternative protein products of such nucleic acids. Compounds of the invention may also modulate gene expression through effects on frame shift signals introduced directly or indirectly by mutation. The frame shift signal can be present in the DNA or RNA of any type of cell or a nucleic acid introduced into the cell, for example, by a virus. Without wishing to be bound by any particular theory, it is believed that a compound of the invention can inhibit viral replication by altering the translation of viral mRNA in response to programmed frame shift signals, resulting in an improper balance of viral protein production.

[0068] The cell to which the compound is administered can be in a host, desirably a mammal or human. The method of this aspect of the invention is most suitable for use in conjunction with a host that is afflicted with, or at risk for developing, a disease, associated with a pathological translational modulating signal, such as a stop codon or frame shift signal.

[0069] The types of diseases associated with pathological translational modulating signals include, but are not limited to, genetic diseases, autoimmune diseases, blood diseases, collagen diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, pulmonary diseases, inflammatory diseases, central nervous system diseases, infectious diseases (e.g., bacterial, viral, and fungal), and cancers (including tumors and other cancers), especially cancers associated with p53 mutations. Specific examples of such diseases are set forth below, without limitation. Furthermore, the invention is in no way limited to the above discussed mechanisms of action, and the compounds of the inventions can be used to treat any such diseases, regardless of the mechanism(s) by which the compounds act.

[0070] Specific examples of genetic diseases include, without limitation, SMA, amyloidosis, hemophilia, Alzheimer's disease, Tay Sachs disease, Niemann Pick disease (e.g., Type C), atherosclerosis, giantism, dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, Parkinson's disease, Huntington's disease, cystic fibrosis, muscular dystrophy (e.g., Duchenne muscular dystrophy), heart disease, kidney stones, Rett syndrome, ataxia- telangiecstasia, familial hypercholesterolemia, retinitis pigmentosa, and Marfan syndrome.

[0071] Specific examples of inflammatory and autoimmune diseases include, without limitation, arthritis, rheumatoid arthritis, osteoarthritis, and graft versus host disease.

[0072] Specific examples of blood diseases include, without limitation, hemophilia, Von Willebrand disease, thalassemia (e.g., /-thalassemia), and kidney stones.

[0073] Specific examples of collagen diseases include, without limitation, osteogenesis imperfect, and cirrhosis.

[0074] Specific examples of central nervous system diseases include, without limitation, multiple sclerosis, muscular dystrophy (e.g., Duchenne muscular dystrophy), Niemann Pick disease (e.g., Type C), Alzheimer's disease, Huntington's disease, Tay Sachs disease, late infantile neuronal ceroidlipofuscinosis (LINCL), Leber's hereditary optic neuropathy, and Parkinson's disease. [0075] Specific examples of infectious diseases include, without limitation, infection by a virus of ^' flaviviridae, retroviridae, coronaviridae, astroviridae, alphaviridae, arteriviridae, orthomyxoviridae, picornaviridae, togaviridae, and r abdoviridae. Specific examples also include Human Immunodeficiency Virus infection (HIV/AIDS), viral hepatitis (e.g., hepatitis A, B, C, B with D, E, F, and/or G), Human Herpes virus (HHV) infection (including herpes zoster), Human Papilloma Virus (HPV) infection, Varicella zoster virus (VZV) infection, severe acute respiratory syndrome (SARS), herpes simplex (e.g., HSV-1, HSV-2) infection, and Pseudomon s aeruginosa infection. Any type of HIV can be treated, but preferably an HIV-1 and/or HIV-2 infection is treated. The method also encompasses infection by an HIV group (e.g., groups M, N, and/or O), and subtype (e.g., clades A, B, C, D, E, EA, F, and/or G), human T cell leukemia virus type I infection, human T cell leukemia virus type II infection, human coronavirus infection, transmissible gastroenteritis virus infection, berne virus infection, human astro virus serotype- 1 infection, rous sarcoma virus infection, human coronavirus infection, transmissible gastroenteritis virus infection, infectious bronchitis virus infection, and simian retrovirus type I infection. The method also encompasses infection by feline immunodeficiency virus, mouse mammary tumor virus, Giardia lamblia virus, Saccharomyces cerevisiae dsR A virus L-A, S. cerevisiae ds RNA virus LI , bacteriophage T7, bacteriophage lambda, red clover necrotic mosaic virus, barley yellow dwarf virus, beet western yellow virus, potato leaf roll virus, and equine artertitis virus,

[0076] A compound of the invention can be combined with other well-known HIV therapies and prophylactic vaccines already in use. The combination of the compound of the invention can generate an additive or a synergistic effect with current treatments. The compound of the invention can be combined with other HIV and AIDS therapies and vaccines, such as highly active antiretroviral therapy (HAART), which comprises a combination of protease inhibitors and reverse transcriptase inhibitors, azidothymidine (AZT), structured treatment interruptions of HAART, cytokine immune enhancement therapy (e.g., interleukin (IL)-2, IL-12, CD40L + IL-12, IL-7, HIV protease inhibitors (e.g., ritonavir, indinavir, and nelfinavir, etc.), and interferons (IFNs), cell replacement therapy, recombinant viral vector vaccines, DNA vaccines, inactivated virus preparations, immunosuppressive agents, such as Cyclosporin A, and cyanovirin therapy (see, e.g., U.S. Patent No. 6,015,876 and International Patent Application Publication No. WO 03/072594). Such therapies can be administered in the manner already in use for the known treatment providing a therapeutic or prophylactic effect (see, e.g., Silvestri et al. Immune Intervention in AIDS. In: Immunology of Infectious Disease, H.E. Kauffman, A. Sher, and R. Ahmed eds., ASM Press, Washington DC (2002)).

[0077] A compound of the invention can be used in combination with one or more other antiviral agents, such as VX-497 (merimepodib, Vertex Pharmaceuticals), VX-498 (Vertex Pharmaceuticals), Levovirin, Viramidine, Ceplene (maxamine), XTL-001 and XTL-002 (XTL Biopharmaceuticals), abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol atazanavir, atripla, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine. The combination of the compound of the invention and one or more antiviral agents can generate an additive or a synergistic effect with current treatments.

[0078] Specific examples of cancers include cancer of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart or adrenals. More particularly, cancers include solid tumor, sarcoma, carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, a blood-born tumor, acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblasts leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, or multiple myeloma. See, e.g., Harrison's Principles of Internal Medicine, Eugene Braumvald et al., eds., pp. 491-762 (15th ed. 2001).

[0079] Diseases, such as cancers, associated with p53 mutations that result in a translational stop codon include, but are not limited to, the diseases and mutations described in Masuda et al, Tokai JExp Clin Med., 25 (2): 69-77 (2000); Oh et al., Mol Cells, 10 (3): 275-80 (2000); Li et al., Lab Invest, 80 (4): 493-9 (2000); Yang et al., Zhonghua Zhong Liu Za Zhi, 21 (2): 114-8 (1999); Finkeistein et ζΙ., ΜοΙ Diagn., 3 (1): 37-41 (1998); Kajiyama et al., Dis Esophagus., 11 (4): 279-83 (1998); Kawamura et al., Leuk Res., 23 (2): 115-26 (1999); Radig et al., Hum Pathol, 29 (1 1): 1310-6 (1998); Schuyer et al., Int J Cancer, 76 (3): 299-303 (1998); Wang-Gohrke et al., Oncol Rep., 5 (1): 65-8 (1998); Fulop et al., J ReprodMed., 43 (2): 1 19-27 (1998); Ninomiya et al., J Dermatol Set., 14 (3): 173-8 (1997); Hsieh et al., Cancer Lett, 100 (1- 2): 107-13 (1996); Rail et al., Pancreas., 12 (1): 0-7 (1996); Fukutomi et al., NipponRinsho, 53 (1 1): 2764-8 (1995); Frebourg et al., Am J Hum Genet, 56 (3): 608-15 (1995); Dove et al., Cancer Surv., 25: 335-55 (1995); Adamson et al., Br J Haematol., 89 (1): 61-6 (1995); Grayson et al., Am J Pediatr Hematol Oncol, 16 (4): 341 -7 (1994); Lepelley etal., Leukemia, 8 (8): 1342-9 (1994); Mclntyre et al., J Clin Oncol, 12 (5): 925-30 (1994); Horio et al, Oncogene, 9 (4): 1231-5 (1994); Nakainura et al, Jpn J Cancer Res., 83 (12): 1293-8 (1992); Davidoff et al, Oncogene, 7 (1): 127-33 (1992); and ishioka etal., Biochem Biophys Res Comm n., 177 (3): 901-6 (1991). Each of these references is incorporated herein by reference.

[0080] Preferably, one or more symptoms of the disease are prevented, reduced, or eliminated subsequent to administration of the compound of the invention, thereby effectively treating or preventing the disease to at least some degree. Accordingly, the method of the invention can be used to treat or prevent such a disease.

C. Diseases and Conditions Ameliorated by Increasing EAAT2 Expression

[0081] In yet another related aspect, the invention provides a method of increasing the expression of excitatory amino acid transporter (EAAT2) in a cell comprising administering a compound of the invention to a cell comprising a nucleic acid that encodes EAAT2, whereby expression of EAAT2 is increased. Certain diseases (e.g., multiple sclerosis, muscular dystrophy, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), and epilepsy) and other conditions, such as stroke or other trauma to the CNS, are associated with elevated glutamate levels in the CNS that can be toxic, leading to brain damage or even death. Cellular uptake of glutamate via the EAAT2 transporter is responsible, at least in part, for maintaining appropriately low glutamate levels in the CNS. Without wishing to be bound by any particular theory, it is believed that the compounds of the invention can activate or enhance EAAT2 expression, thereby

advantageously lowering glutamate levels in the CNS. Methods of detecting and measuring increased EAAT2 expression are known in the art and described, for instance, in Rothstein et al., Nature, 43 (3): 73-7 (2005).

[0082] The cell to which the compound of the invention is administered preferably is in a host. Suitable hosts are as previously described herein. The host is desirably a mammal, especially a human. The method of this aspect of the invention is most suitable for use in conjunction with a host that is afflicted with a disease or condition, or at risk for developing a disease or condition, associated with decreased expression of EAAT2 or elevated glutamate levels in the CNS. Such diseases and conditions that have been associated with decreased expression of EAAT2 or elevated glutamate levels include, for example, SMA, stroke, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and epilepsy. Furthermore, the invention is in no way limited to the foregoing mechanism and the compounds described herein can be used to treat any of the disclosed diseases, regardless of the mechanism(s) by which the compounds act. Preferably, one or more symptoms of the disease or condition are prevented, reduced, or eliminated subsequent to administration of the compound of the invention, thereby effectively treating or preventing the disease or condition to at least some degree. Accordingly, the invention provides a method of treating or preventing a neurological disorder selected from the group consisting of SMA, stroke, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and epilepsy in a mammal comprising administering a therapeutically effective amount of at least one compound of the invention to the mammal, whereupon the neurological disorder is treated or prevented. The treatment of the neurological disorder can be effected, for example, by increasing the expression of EAAT2. EXAMPLES

[0083] The following examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way.

EXAMPLE 1

[0084] This examples illustrates general preparation methods of N-(3-oxo-2-(quinolin-6- yl)isoindolin-5-yl)amides.

[0085] Step A: Synthesis of 6-nitrobenzofuran-l(3H)-one

[0086] To an ice-cold stirred solution of phthalide (40.0 g, 298 mmol) in sulfuric acid (50.0 mL) was added potassium nitrate (30.4 g, 300 mmol) dissolved in sulfuric acid (80 mL) via dropwise addition. The reaction was stirred and allowed to warm to room temperature over 5 h. The resulting mixture was poured into ice-water, and the precipitate collected was recrystallized from ethanol (3 L) to afford 6-nirobenzofuran-l(3H)-one (50.4 g, 94%) as an off-white solid (98% of the desired regioisomer by Ή NMR): 1H NMR (500 MHz, CDC1₃) δ 8.77 (d, J= 1.5 Hz, 1H), 8.58 (dd, J= 8.5, 2.0 Hz, 1H), 7.72 (d, J- 8.5 Hz, 1H), 5.45 (s, 2H).

[0087] Step B: Synthesis of methyl 2-(chloromethyi)-5~nitrobenzoate

[0088] To a suspension of 6-nitroisobenzofuran-l(3H)-one (9,0 g, 50.0 mmol) in thionyl chloride (7.3 mL, 100.0 mmol) was added boron trifluoride diethyl etherate (0.37 mL, 3.0 mmol) followed by benzyltriethylammonium chloride (0.687 g, 3.0 mmol). The reaction was heated at 80 °C for 2.5 d. After this time, the reaction was then cooled to room temperature and concentrated in vacuo to remove most of the thionyl chloride. The remaining reaction mixture was diluted with dichloromethane (50 mL) and cooled to 0 °C. To this solution was added methanol (5.0 mL, 123.0 mmol) followed by the slow dropwise addition of NjV- diisoproylethylamine (9.0 mL, 51.6 mmol) not allowing the reaction to go above 10 °C. After complete addition, the reaction was allowed to warm to room temperature and stirred for 2 h. At this time, the reaction was further diluted with dichloromethane and washed with saturated sodium bicarbonate and brine. The organic layer was dried (sodium sulfate), filtered and concentrated in vacuo to give a dark red oil. Purification by flash

chromatography (silica, 0% to 20% ethyl acetate/hexanes) afforded methyl 2-(chloromethyl)-

5- nitrobenzoate (8.2 g, 71%) as a white solid: Ή NMR (300 MHz, CDC1₃) δ 8.83 (d, J= 2.4 Hz, 1H), 8.38 (dd, J= 8.5, 2.5 Hz, 1H), 7.82 (d, J- 8.6 Hz, 1H), 5.12 (s, 2H), 4.00 (s, 3H).

[0089] Step C: Synthesis of 6-nitro-2-(quinolin-6-yl)isoindolin-l-one

[0090] A mixture of methyl 2-(chloromethyl)-5-nitrobenzoate (35.0 g, 152,4 mmol) and

6- aminoquinoline (22.2 g, 152.4 mmol) with a catalytic amount of sodium iodide (1.14 g, 7.62 mmol) and dimethylaminopyridine (0.931 g, 7.62 mmol) in acetonitrile (600 mL) was stirred with mechanical stirrer and heated at 75-80 °C for 65 h. The mixture was then treated with trifluoroacetic acid (35.1 g, 305 mmol) and the mixture was heated at 80 °C for 3 h. After this time, the reaction was diluted with ethanol and cooled with an ice-bath. The resulting precipitate was collected by filtration, washed with ethanol, and dried to afford 6- nitro-2-(quinolin-6-yl)isoindolin-l-one hydrochloric acid salt (37.9 g, 73%) as a light brown solid: ^!H NMR (500 MHz, DMSO-c¾) δ 9.09 (d, J= 3.8 Hz, 1H), 8.85 (d, J- 7.9 Hz, 1H), 8.69 (dd, J= 9.3, 2.2 Hz, 1H), 8.62 (d, J= 1.9 Hz, 1H), 8.57 (dd, J= 8.3, 2.1 Hz, 1 H), 8.50 (d, J= 1.9 Hz, 1H), 8.32 (d, J= 9.3 Hz, 1H), 8.03 (d, J= 8.3 Hz, 1H), 7.87 (dd, J= 8.2, 4.8 Hz, 1H), 5.37 (s, 2H); ESI MS m/z 306 [M + H]⁺.

[0091] Step D: Synthesis of 6-amino-2-(quinolin-6-yi)isoindolin-l-one

[0092] A suspension of 6-nitro-2-(quinolin-6-yl)isoindolin-l-one hydrochloric acid salt

(20.5 g, 60.0 mmol) and acetic acid (36.0 g, 600 mmol) in ethanol (600 mL) and water (120 mL) was stirred with mechanical stirrer and heated to reflux. To this refluxing mixture was added iron powder (8.46 g, 150 mmol) and the mixture was heated at reflux for 1.5 h. The mixture was then cooled to room temperature. To this mixture was added 480 mL of 6 N hydrochloric acid and the mixture was stirred at room temperature overnight. The resulting precipitate was collected by filtration, washed with water and ethanol. The filter cake was stirred with sodium bicarbonate solution overnight. The resulting precipitate was collected by filtration, washed with water and tetrahydrofuran, and dried under vacuum at 70 °C overnight to afford 6-amino-2-(quinolin-6-yl)isoindolin-l-one (16.7 g, >100%). This material was further triturated with water and ethanol. The resulting precipitate was collected by filtration, washed with water and ethanol, and dried under vacuum at 70 °C to afford 6- amino-2-(quinolin-6-yl)isoindolin-l-one (14.4 g, 87%) as an off-white solid: 1H NMR (500 MHz, DMSO-i¾) 5 8.83 (dd, J= 4.1, 1.3 Hz, 1H), 8.48 (dd, J- 9.2, 2.5 Hz, 1H), 8.35-8.33 (m, 2H), 8.06 (d, J= 9.2 Hz, 1H), 7.53 (dd, J= 8.3, 4.2 Hz, 1H), 7.32 (d, J= 8.1 Hz, 1H), 6.97 (d, J= 2.0 Hz, 1H), 6.91 (dd, J- 8.1, 2.1 Hz, 1H), 5.43 (s, 2H), 4.97 (s, 2H); ESI MS m/z 276 [M + H]⁺.

[0093] Step E: General Synthesis of Amides

[0094] To an ice-cooled suspension of 6-amino-2-(quinolin-6-yl)isoindolin-l-one and N,iV-diisopropylethyl amine (1.5 eq) in tetrahydrofuran was added an acid chloride (1.2 eq). The mixture was stirred at room temperature for 2 h. To this mixture was then added a solution of sodium bicarbonate in water. The resulting precipitate was collected by filtration, washed with water and tetrahydrofuran, and dried under vacuum at 70 °C to afford the crude product. This material was further triturated with water. The resulting precipitate was collected by filtration, washed with water and ethanol, and dried under vacuum at 70 °C to afford the desired amide as an off-white solid, which was carried onto the next step without further purification.

[0095 Ste F: General S nthesis of H drochloric Acid Salts

[0096] To a suspension of the amide from the previous step in methanol was added the 2 N hydrochloric acid in diethyl ether (10 eq). The mixture was stined at room temperature for 2 h. To this mixture was then added additional diethyl ether to facilitate precipitation. The resulting precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum at 70 °C to afford the desired hydrochloric acid salt. [0097] Representative examples prepared using these procedures are shown in Table 2 below along with mass spectral data and melting points.

Tabid

EXAMPLE 2

[0098] This example illustrates preparation of N-(3-oxo-2-(quinolin-6-yl)isoindolin-5- yl)isobutyramide.

Compound 2

[0099] Steps A through D of Example 1 were followed.

[00100] Step E: Synthesis of N-[3-oxo-2-(quinoline-6-yl)isoindolin-5-yl]isobutyramide

[00101] To an ice-cooled suspension of 6-amino-2-(quinoiin-6-yl)isoindolin-l-one (14.3 g, 51.9 mmol) and N,N-diisopropylethylamine (10.1 g, 77.9 mmol) in tetrahydrofuran (500 mL) was added isobutyryl chloride (6.77 g, 63.3 mmol). The mixture was stirred at room temperature for 2 h. To this mixture was then added a solution of sodium bicarbonate (8.74 g, 104 mmol) in water (200 mL). The resulting precipitate was collected by filtration, washed with water and tetrahydrofuran, and dried under vacuum at 70 °C to afford iV-[3-oxo- 2-(quinoline-6-yl)isoindolin-5-yl]isobutyramide (18.5 g, >100%). This material was further triturated with water. The resulting precipitate was collected by filtration, washed with water and ethanol, and dried under vacuum at 70 °C to afford V-[3-oxo-2-(quinoline-6- yl)isoindolin-5-yl]isobutyramide (15.5 g, 87%) as an off-white solid: ^lH NMR (500 MHz, DMSO-i/e) δ 10.12 (s, 1H), 8.85 (dd, J = 4.1, 1.5 Hz, 1H), 8.49 (dd, J= 9.2, 2.4 Hz, 1H), 8.39-8.31 (m, 2H), 8.21 (d, J= 1.6 Hz, 1H), 8.09 (d, J = 9.2 Hz, 1H), 7.83 (dd, J= 8.2, 1.9 Hz, 1H), 7.63 (d, J= 8.2 Hz, 1H), 7.54 (dd, J= 8.3, 4.1 Hz, lH), 5.12 (s, 2H), 2.64 (h, J- 6.8 Hz, 1H), 1.14 (d, J = 6.8 Hz, 6H); ESI MS m/z 346 [M + H]⁺.

EXAMPLE 3

[00102] This example demonstrates the ability of compounds of the invention to increase SMN-containing gem particles in fibroblasts from SMA patients.

[0100] SMN protein can be found in punctate nuclear particles called "gems" (Liu and Dreyfuss, EMBO J . 15 (14): 3555-3565 (1996)). In fibroblast cells derived from SMA patients, the number of gems corresponds to disease severity as follows: severe type 1 patients have approximately 5 gems per 100 nuclei; mild type 3 patients have 20-50 gems per 100 nuclei; normal individuals have approximately 100-150 gems per 100 nuclei (Coovert et ai., Hum Mol Genet, 6 (8): 1205-1214 (1997), Young et al., Exp Cell Res, 265 (2): 252-261 (2001)).

[0101] Gems are detectable by immunohistochemistry and gem counts in patient fibroblasts have been used to test the ability of compounds to increase SMN protein levels (Mattis et al., Hum Genet, 120: 589-601 (2006)). Following these methods, fibroblast cells from a type 1 SMA patient (3813 cells, Coriell Cell Repositories) were treated for 48 hours with a compound of the invention. The cells were then fixed and incubated with a monoclonal antibody against SMN (4B7, Wolstencroft et al., Hum Mol Genet, 14: 1199-1210 (2005)). A FITC-conjugated anti-mouse secondary antibody was used to visualize the labeled gems. Positive controls in these experiments were provided by treating fibroblasts from the same patient with 5000 μΜ valproic acid and 60 μΜ vanadate. As shown in Table 3, treatment of the cells with compounds of the invention increased the number of SMN- containing gems in patient fibroblasts. Table 3

EXAMPLE 4

[0102] This example demonstrates the ability of a compound of the invention to increase total cellular SMN protein in fibroblasts from SMA patients.

[0103] Primary fibroblasts derived from a Type I SMA patient (3813; Coriell Cell Repositories) were cultured alone or with Compound 2 at a concentration of 30 nM for 48 hours. Western blots were performed using standard methods. Monoclonal SMN anti-SMN antibody 4B7 (Wolstencroft et al., Hum Mil Genet, 14:1199-1210 (2005)) was used to identify SMN protein. Tubulin was used as an internal normalization standard, and DMSO treatment served as the negative control. Extracts of fibroblasts from a normal carrier individual (3814) were run for comparison. The results are presented in Figure 1.

[0104] Treatment with the drug increased SMN expression by 32% as compared to treatment with DMSO alone, which was a statistically significant increase. Increasing expression to a level that is intermediate between that seen in severe type I patients and normal carrier individuals is expected to have clinical benefit in SMA patients.

EXAMPLE 5

[0105] This example demonstrates the ability of compounds of the invention to increase SMN reporter gene expression in cervical carcinoma cell lines.

[0106] Cervical carcinoma cell lines were transformed with a SMN2-linked luciferase- reporter gene construct. The reporter is designed to detect shifts in the alternative splicing of SMN2 sequences. In particular, the reporter is constructed as a fusion of the alternatively spliced sequences of the SMN2 gene and the luciferase gene. Luciferase sequences are in the correct transiational reading frame only when the SMN2 sequences are spliced according to the normal SMN1 mechanism (i.e., splicing to include exon 7). The SMN-luciferase fusion transcript produced in this assay is predominantly exon 7-deleted, therefore little Iuciferase is typically produced. If splicing excludes exon 7 in transcripts from this construct, productive expression of Iuciferase protein requires a shift in the ribosomal reading frame between the S N-encoding RNA sequence and the iuciferase- encoding RNA sequence. Treatment with compounds of the invention does not result in increased inclusion of exon 7 in transcripts in the assay, thus increases in Iuciferase expression in response to compound treatment suggests increased ribosomal frame shifting upstream of Iuciferase. Compounds were tested in the assay by administering the compound to the cell line and detecting an increase in the

Iuciferase activity. The assay is described in greater detail in Zhang et al., Gene Ther,, 8: 1532-8 (2001). Compounds were administered as described in Lunn et al, Chem. & Biol, 1 1 : 1489-1493 (2004).

[0107] The results are reported in Table 4 as a fold-increase over a baseline reading of transformed cells without any test compound. The μΜ EC₅o value also is reported in Table 4 for some exemplary compounds of the invention.

Table 4

[0108] As the results show, many of the tested compounds increased luciferase activity in the assay. However, these compounds did not increase luciferase by changing the splicing pattern of the SMN-luciferase fusion m NA. As a result, the luciferase coding sequences are preceded by one or more stop codons and are out of frame with the SMN sequences in this message. Therefore, increases in luciferase production in this assay suggest that the compounds of the invention can alter translation, e.g., by stimulating ribosomal stop codon read through or frameshifting. In addition, the effects of indoprofen and its analogs active in this assay have been shown to extend to an increase in production of SMN from the endogenous SMN2 gene in SMA patient fibroblasts. Thus, the increased luciferase activity in the assay indicates that the compounds of the invention can be used to increase expression of SMN from the endogenous SMN2 gene. EXAMPLE 6

[0109] This example demonstrates microsomal stability of several of the compounds of the invention.

[0110] Compounds were incubated in in vitro preparations of liver microsomes derived from human, cynomolgous monkey, Sprague Dawley rat, and beagle dog sources. Testing was conducted according to standard commercial procedures (Cerep Inc. catalog assay numbers 0416, 0527, 0528, and 0526 for the four respective species). The percentage of compound remaining was measured at 60 minutes. The results are presented in Table 5. 'ND' indicates a test that was not done. The results demonstrate the ability of compounds to withstand microsomal metabolism in vitro under stringent conditions, which is a predictor of favorable in vivo PK properties.

Table 5

Compound % Remaining % Remaining % Remaining % Remaining No. Human Cynomolgous Rat Beagle

1 95 60 76 ND

2 92 58 88 102

3 102 89 94 ND

5 86 ND ND ND

6 46 ND ND ND

7 104 72 81 ND

8 106 69 63 ND

9 84 56 69 ND

12 86 ND 84 ND

14 93 81 95 ND

16 39 30 88 73

17 52 27 46 62

18 81 7 49 72

19 99 65 83 102

20 8 44 7 ND

21 5 2 11 23

22 88 ND ND 83

23 87 ND 85 78

24 42 ND 21 2

25 100 52 66 93

26 30 9 39 87 EXAMPLE 7

[0111] This example illustrates the pharmacokinetic properties of a compound of the invention.

[0112] Compound 2 was administered as a single oral dose, by gavage, to Sprague- Dawley rats and beagle dogs. Adult male rats (n=3), with indwelling jugular cannulas, received an oral dose of 10 mg/kg as a 1 mg/mL suspension in 0.5% methylcellulose 400. Adult male beagle dogs (n=3) received a single 10 mg/kg dose of 5 mg/mL solution in propylene glycol and PEG-400 (50:50, by volume) following pretreatment with pentagatrin (6 micorgram/kg) 30 minutes prior to drug dosing. Plasma samples were analyzed for drug by LC-MS.

[0113] The results are presented in Figure 2. Drug exposure is shown following single oral dose administration at 10 mg/kg in rat and dog. Drug was measured in plasma samples taken at successive time points following administration. The black line indicates the estimated EC₅₀ for this compound as determined by Western blot analysis of extracts from patient fibroblasts following in vitro drug treatment. The estimate of the in vitro EC50 of 10 nM was corrected for the anticipated fraction of drug bound to protein, based on independent measurements of plasma protein binding for this compound. The

pharmacokinetic profiles demonstrate that, at an oral dose of 10 mg/kg, drug plasma levels are maintained at or above the estimated EC50 for this compound for approximately 6 hours in rat and 16 hours in dog.

[0114] In a separate study, the brain-to-plasma ratio of drug following oral delivery in mice was 0.38. In this study, drug was administered as a single oral dose to adult male FVB mice at lOOmg/kg in a suspension of 0.5% HPMC. The brain-to-plasma ratio was calculated from AUC values.

EXAMPLE 8

[0115] This example illustrates use of a compound of the invention to increase SMN protein levels in vivo.

[0116] Non-congenic C/C SMA mice were treated by oral gavage with Compound 2 at 45 mg/kg solubilized in vehicle (propylene glycol (PG)-polyethylene glycol 400 (50:50)) or vehicle alone. Mice were provided from a colony supported by the SMA Foundation at Psychogenics (Tarrytown, NY, USA). Treatment was BID for 13.5 days, starting in 14- week-old mice, or TID for 5.3 days, starting in 18 week-old mice. Mice were sacrificed at 2 hours following the final dose, and the livers were removed. Tissue homogenization and

SMN protein determinations were performed using an SMN ELISA kit (ADI-900-209 from

Enzo Life Sciences, Farmingdale, NY). Tissues were tested in duplicate.

[0117] The results are shown in Figure 3, Results are reported as picogram of SMN per milligram of total protein. Asterisks designate treatment groups for which the SMN protein component was significantly different from that for the vehicle control (P>0.05, one-sided t- test).

[0118] As the results show, SMN protein levels in the livers of the treated mice increased with drug treatment as compared to control mice. SMN increases in the liver were statistically significant in both sets of drug-treated mice. With BID treatment, a statistically significant increase of 16% was seen. With TID treatment, drug-treated mice showed a 75% increase in SMN protein in the liver.

[0119] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0120] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0121] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A com ound, of Formula I :

CO

or salt thereof, wherein:

n is 0, 1 , 2, or 3;

R¹ is a Ci-Cg alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, Ci-Cg alkoxy, amino, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, aryl, or heteroaryl, any of which are optionally substituted with one or more of -Cg alkyl, C3-C6 cycloalkyl, Ci-Cg alkoxy, Cj-C₈ haloalkoxy, C_rC₈ haloalkyl, halogen, -CN, -C(O)0R^s, -C(0)NR⁵(R⁶), -N0₂, -S0₂NR⁵(R⁶), - NR⁷(S0₂)R⁸, -NR⁵C(0)NR⁶R⁷, amino, C1-C4 alkylamino, C1-C4 dialkylamino, C₃-C₆ cycloalkylamino, aryl, heteroaryl, or heterocycloalkyl;

R² is H or d-C₈ alkyl;

or R and R can be taken together with the atoms to which they are attached to form a 5- or 6-membered lactam ring, optionally comprising a second heteroatom in the lactam ring selected from the group consisting of N, O, and S;

R³ is H, hydroxyl, C C₈ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, Ci-C_e alkoxy, C₃-C₈ cycloalkyloxy, C^Cg haloalkoxy, Ci-C₈ haloalkyl, halogen, alkylsulfonyl, -CN, -NO2, - S0₂NR⁵(R⁶), -NR⁷(S0₂)R⁸, -NR⁵C(0)R⁶, -NR⁵C(0)NR⁶R⁷, amino, C1-C4 alkylamino, C₃-C₆ cycloalkylamino, aryl, heteroaryl, or heterocycloalkyl;

R⁴ is H, Ci-Cs alkyl, C₃-C₆ cycloalkyl, C,-C₃ alkoxy, C_rC₈ haloalkoxy, Q-Q haloalkyl, halogen, -CN, -C(0)OR⁵, -C(0)NR⁵(R⁶), -N0₂, -S0₂NR⁵(R⁶), -NR⁷(S0₂)R⁸, - NR C(0)NR R , amino, C1-C4 alkylamino, C3-C6 cycloalkylamino, aryl, heteroaryl, or heterocycloalkyl;

R⁵, R⁶, and R⁷ are independently H, CpCg alkyl, C₃-C₆ cycloalkyl, aryl, or heteroaryl; and

R⁸ is Ci-Cg alkyl, C3-C6 cycloalkyl, aryl, or heteroaryl.

2. The compound of claim 1, wherein R¹ is Ci-Cg alkyl, haloalkyl, C₃-C₆ cycioalkyL alkoxy, aminoalkyl, alkylamino, dialkylamino, haloalkyl amino, and C₃-C₆ heterocycloalkyl, wherein the cycloalkyi and heterocycloalkyl are optionally substituted with Ci-Ce alkyl or -C(0)OR⁵.

3. The compound or pharmaceutically acceptable salt of any of claim 1 or 2, wherein R^s is methyl, methoxy,

4. The compound of any preceding claim, wherein R is H.

5. The compound of claim 1, wherein R and R are taken together with the atoms to which they are attached to form a 5- or 6-membered lactam ring, optionally comprising a second heteroatom in the lactam ring selected from the group consisting of N, O, and S.

6. The compound of any preceding claim, wherein R⁴ is H.

7. The compound of any preceding claim, wherein R³ is H or halogen.

8. A compound of Table 1 or a salt thereof.

9. A composition comprising at least one compound of any preceding claim and a pharmaceutically acceptable carrier.

10. A method of increasing survival motor neuron protein (SMN) expression in a cell comprising administering a compound of any of claims 1-8 to a cell comprising a nucleic acid encoding SMN, whereby SMN expression is increased.

1 1. The method of claim 10, wherein the cell is in a host, and the host is a mammal.

12. The method of claim 1 1 , wherein the mammal is a human afflicted with a disease associated with under-expression of SMN.

13. The method of claim 12, wherein the disease is spinal muscular atrophy

(SMA).

14. A method of treating spinal muscular atrophy (SMA) in a mammal comprising administering a therapeutically effective amount of at least one compound or

pharmaceutically acceptable salt of any of claims 1-8 to the mammal, whereupon SMA is treated.

15. A method of increasing in a cell the expression of a nucleic acid that encodes a translational stop codon, the method comprising administering a compound or

pharmaceutically acceptable salt of any of claims 1-8 to a cell comprising a nucleic acid that encodes a translational stop codon, whereby expression of the nucleic acid is increased.

16. The method of claim 15, wherein the stop codon has been introduced directly or indirectly by mutation.

17. The method of claim 16, wherein the host is a mammal.

18. The method of claim 17, wherein the mammal is a human afflicted with a disease associated with the translational stop codon introduced directly or indirectly by mutation.

19. The method of claim 18, wherein the disease is cancer, diabetes, cystic fibrosis, Rett syndrome, ataxia-telangiecstasia, or muscular dystrophy.

20. A method of altering in a cell the expression of a nucleic acid that encodes a translational frameshift signal, the method comprising administering a compound or pharmaceutically acceptable salt of any of claims 1-8 to a cell comprising a nucleic acid that encodes a translational frameshift signal, whereby expression of the nucleic acid is altered.

21. The method of claim 20, wherein the translational frameshift signal is within a nucleic acid introduced in the cell by a virus.

22. The method of claim 21, wherein the virus is human immunodeficiency virus type I, human immunodeficiency virus type II, feline immunodeficiency virus, rous sarcoma virus, mouse mammary tumor virus, simian retrovirus type I, human T cell leukemia virus type I. human T cell leukemia virus type II, infectious bronchitis virus, human coronavirus, transmissible gastroenteritis virus, berne virus, equine artertitis virus, human astrovirus serotype- 1, Giardia lamblia virus, Saccharomyces cerevisiae dsRNA virus L-A, S. cerevisiae ds RNA virus LI, bacteriophage T7, bacteriophage lambda, barley yellow dwarf virus, beet western yellow virus, potato leaf roll virus, severe acute respiratory syndrome caronavirus, herpes simplex virus, or red clover necrotic mosaic virus, hepatitis virus, Varicella zoster virus, or human papilloma virus.

23. A method of increasing the expression of excitatory amino acid transporter (EAAT2) in a cell comprising administering to a cell comprising a nucleic acid that encodes EAAT2 a compound of any of claims 1-8, whereby the expression of EAAT2 is increased.

24. The method of claim 23, wherein the cell is in a host, and the host is a mammal.

25. The method of claim 24, wherein the mammal is a human that has an elevated level of glutamate in the central nervous system.

26. The method of claim 25, wherein the mammal is a human that has suffered stroke or trauma to an area of the central nervous system.

27. The method of claim 26, wherein the mammal is a human that is afflicted with a disease, optionally, a neurodegenerative disease.

28. The method of claim 27, wherein the disease is SMA, stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington's disease, or epilepsy.

29. A method of treating or preventing a neurological disorder selected from the group consisting of SMA, stroke, Parkinson's disease, Alzheimer's disease, ALS, MS, Huntington's disease, and epilepsy in a mammal comprising administering a therapeutically effective amount of at least one compound of any of claims 1-8 to the mammal, whereupon the neurological disorder is treated or prevented.

30. A method of treating or preventing an infectious disease in a mammal comprising administering a therapeutically effective amount of at least one compound or pharmaceutically acceptable salt of any of claims 1-8 to the mammal, whereupon the infectious disease is treated or prevented. 1. The method of claim 30, wherein the infectious disease is viral.

32. The method of claim 31 , wherein the infectious disease is Human

Immunodeficiency Virus infection (HIV/ AIDS), viral hepatitis, Human Herpes virus (HHV) infection, Human Papilloma Virus (HPV) infection, Herpes Simplex Virus infection, human T cell leukemia virus type I infection, human T cell leukemia virus type II infection, human coronavirus infection, transmissible gastroenteritis virus infection, berne virus infection, human astrovirus serotype- 1 infection, severe acute respiratory syndrome coronavirus infection, or Varicella zoster virus.

33. The method of claim 30, wherein the infectious disease is severe acute respiratory syndrome (SARS), herpes zoster, or Pseudomonas aeruginosa infection.

34. A compound or pharmaceutically acceptable salt of any of claims 1-8 for use in increasing survival motor neuron protein (SMN) expression in a cell comprising a nucleic acid encoding SMN.

35. A compound or pharmaceutically acceptable salt of any of claims 1-8 for use in treating spinal muscular atrophy (SMA) in a mammal.

36. A compound or pharmaceutically acceptable salt of any of claims 1-8 for use in increasing the expression of a nucleic acid that encodes a translational stop codon in a cell comprising a nucleic acid that encodes a translational stop codon.

37. A compound or pharmaceutically acceptable salt of any of claims 1-8 for use in altering the expression of a nucleic acid that encodes a translational frameshift signal in a cell comprising a nucleic acid that encodes a translational frameshift signal.

38. A compound of any of claims 1-8 for use in increasing the expression of excitatory amino acid transporter (EAAT2) in a cell comprising a nucleic acid that encodes EAAT2.

39. A compound of any of claims 1-8 for use in treating or preventing in a mammal a neurological disorder selected from the group consisting of SMA, stroke, Parkinson's disease, Alzheimer's disease, ALS, MS, Huntington's disease, and epilepsy.