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Definitions

• the invention relates to products that inhibit calpain-2 function, and methods for specifically inhibiting calpain-2 activation or activity, and to methods of treating and preventing neurodegenerative diseases that are susceptible to treatment with molecules that interfere with calpain-2 function.
• TBI Traumatic Brain Injury
• calpain- 1 and calpain-2 play opposite functions in both synaptic plasticity and neurodegeneration. While calpain-1 is required for the induction of synaptic plasticity, calpain-2 limits the extent of synaptic plasticity during the minutes following the induction event (Wang et al., 2014); likewise, calpain-1 is neuroprotective and calpain-2 is neurodegenerative (Wang et al., 2013).
• Calpain-1 activation is linked to synaptic NMDA receptor stimulation, which accounts for its necessary role in long term potentiation (LTP) induction. It is also involved in neuroprotection elicited by synaptic NMDA receptor stimulation.
• LTP long term potentiation
• calpain-2 is linked to extrasynaptic NMDA receptor stimulation and is involved in neurodegeneration. Calpain-2 is also activated by BDNF->ERK -mediated phosphorylation and limits the extent of LTP following theta-burst stimulation (TBS).
• TBS ta-burst stimulation
• Selective calpain-2 inhibitors could be used not only for TBI, but also for a number of acute indications associated with neuronal death, including stroke, concussion, intracerebral hemorrhage, acute glaucoma, and spinal cord injury. They could also be used to prevent neurodegeneration elicited by seizure activity and could therefore be useful to prevent epileptogenesis.
• compounds which are selective inhibitors of calpain-2 are provided.
• Preferred compounds can be useful to treat acute neurodegeneration.
• R 1 is a non-hydrogen substituent such as C1-C6 alkyl, halogen, cyano, nitro, Ci-C 6 alkoxy, -CO(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), -CONH(CH 2 ) m N(R a )(R b ), -CONH-CH(R c )(R d ) , - (CH 2 ) m N(R a )(R b ), or -0(CH 2 ) m 0H;
• R a , R b , R c , and R d are independently hydrogen or unsubstituted C1-C6 alkyl, which may be linear or branched alkyl; n is independently an integer from 0 (where the ring A is unsubstituted) to the value permitted by the valence of the ring such as 5 where A is phenyl; m is independently an integer from 0 to 6; L 1 and L 2 are each the same or different optionally substituted alkylene having one to 6 carbons (e.g. -(03 ⁇ 4)r where p is 1 to 6 and each carbon may have zero, one or two non-hydrogen substituents),
• R 2 is non-hydrogen substituent such as optionally substituted C 1 -C 6 alkyl
• R 4 and R 5 are independently hydrogen, or unsubstituted C 1 -C 6 alkyl such as methyl; and pharmaceutically acceptable salts thereof.
• R a and R b are independently hydrogen or unsubstituted C 1 -C 6 alkyl.
• R a and R b are independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl).
• R a and R b are independently hydrogen or methyl.
• R a and R b are hydrogen.
• R a and R b are methyl.
• R a is hydrogen and R b is methyl.
• R a and R b are independently hydrogen or isopropyl.
• R a is hydrogen and R b is isopropyl.
• R c and R d are independently hydrogen or unsubstituted C 1 -C 6 alkyl.
• R c and R d are independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl).
• R c and R d are independently hydrogen or methyl.
• R c and R d are hydrogen.
• R c and R d are methyl.
• R c is hydrogen and R d is methyl.
• R 4 and R 5 are independently hydrogen, methyl or ethyl. In a particular aspect, R 4 and R 5 are methyl.
• L 1 and L 2 are unsubstituted alkylene such as methylene (-CH2-) and ethylene (-CH2-CH2-).
• the group A is carbocyclic aryl such as phenyl or a heteroaryl with one of more nitrogen ring members such as optionally substituted pyridinyl or optionally substituted pyrazinyl.
• n may be 0, 1, 2, or 3, such as 0 or 1, or 1.
• the compound may be a racemate including:
• the compound has preferably the formula (IIA):
• R 1 is -CO(CH 2 ) m N(R a )(R b ) wherein m is 0 or 1, preferably m is 0, and R a and R b are hydrogen or methyl.
• R 1 is -CONH 2 , or -CONHCH 3 .
• R 1 is -0(CH 2 ) m N(R a )(R b ), wherein m is 1, 2, or 3, preferably m is 2, and R a and R b are hydrogen or methyl.
• R 1 is -OCFhCFhNXCFb ⁇ or - OCH 2 CH 2 NHCH 3 .
• R 1 is -CONH(CH2) m N(R a )(R b ), wherein m is 1, 2, or 3, preferably m is 2, and R a and R b are hydrogen, methyl or isopropyl.
• R 1 is - CONHCH 2 CH 2 NHCH(CH3) 2 , -CONHCH 2 CH 2 N(CH3)2, or -CONHCH 2 CH 2 NH(CH3).
• R 1 is -CONH-CH(R c )(R d ) wherein R c and R d are independently hydrogen or methyl.
• R 1 is -CONHCH(CH 3 ) 2 , or -CONHCH 2 CH 3.
• R 1 is -(CH2) m N(R a )(R b ), wherein m is 1, 2, or 3, preferably m is 1, and R a and R b are hydrogen or methyl.
• R 1 is -CH2NH2 , -CFbNF ⁇ CFb), or . - CH 2 N(CH 3 )2.
• R 1 is -0(CH 2 ) m 0H, wherein m is 1, 2, or 3, preferably m is 2.
• R 1 is -OCH2CH2OH.
• the compound may be a racemate including:
• the compound has preferably the formula (IIIA):
• R 1A is cyano (-CN) or unsubstituted alkyl such as methyl and R 1B is C1-C 6 alkoxy, preferably -OCH3.
• the compound may be a racemate including:
• compounds of the following Formula (X) are provided: wherein:
• A is C 1 -C 6 alkyl, carboxyl (-C(O)O-), aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;
• B is carbocyclic aryl, heteroaryl, cycloalkyl, or heterocycloalkyl
• L 1 is a bond, or substituted or unsubstituted C 1 -C 6 alkylene
• L 2 is a bond, substituted or unsubstituted C 1 -C 6 alkylene, or -S(0) 2 -,
• Each R 1 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 ) mR
• R 2 is unsubstituted C 1 -C 6 alkyl
• Each R 6 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 ) mR
• R a , R b , R c , and R d are independently hydrogen, C1-C6 alkyl that may be optionally substituted with halogen, -OH, amine, or unsubstituted C3-6 cycloalkyl;
• X is halogen; n is independently an integer from 0 to 12; m is independently an integer from 0 to 6; k is independently an integer from 0 to 12; and pharmaceutically acceptable salts thereof.
• A is phenyl and L 1 is -(CTEjp- wherein p is 1 to 4.
• the compound has the following Formula (XI).
• L 2 , R 6 and k are as defined above n is an integer of 0 to 5.
• n is an integer of 0 to 5.
• -L 2 -B- is
• the compound has the following Formula (Xl-a),
• R 2 , L 2 , and R 6 are as defined above k is an integer of 0 to 5.
• the compound has the following Formula (Xl-b),
• R 2 , L 2 , and R 6 are as defined above k is an integer of 0 to 4.
• the compound has the following Formula (XI-c), and R 6 are as defined above k is an integer of 0 to 3.
• the compound has the following Formula (Xl-d), and R 6 are as defined above k is an integer of 0 to 6.
• L 1 is a bond, methylene, or ethylene and A is Ci-4 alkyl, cycloalkyl, or heterocycloalkyl.
• -L'-A-R 1 is
• the compound has the following Formula (XII),
• R 6 are as defined above k is an integer of 0 to 5 and n is an integer of 0 to 5.
• Each R 1 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) -C(0)0CH(R c )(R d ) -(CH 2 ) m N (R a )(R b ), - (CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 )mR c ,
• R 2 is unsubstituted C 1 -C 6 alkyl
• Each R 6 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 ) m N(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 ) m
• R a , R b , R c , and R d are independently hydrogen, C 1 -C 6 alkyl that may be optionally substituted with halogen, -OH, amine, or unsubstituted C 3-6 cycloalkyl;
• X is halogen; n is independently an integer from 0 to 5; m is independently an integer from 0 to 6; k is independently an integer from 0 to 5; p is independently an integer from 0 to 6; and pharmaceutically acceptable salts thereof.
• Each R 1 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 ) mR
• R 2 is unsubstituted C1-C6 alkyl
• Each R 6 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 ) mR
• R a , R b , R c , and R d are independently hydrogen, C1-C6 alkyl that may be optionally substituted with halogen, -OH, amine, or unsubstituted C3-6 cycloalkyl;
• X is halogen; n is independently an integer from 0 to 5; m is independently an integer from 0 to 6; k is independently an integer from 0 to 5; p is independently an integer from 0 to 6; and pharmaceutically acceptable salts thereof.
• each R a , R b , R c , and R d is independently hydrogen, methyl, ethyl, propyl or isopropyl.
• the compound may be a racemate including:
• the compound has preferably the structure of,
• compositions comprising said compounds, and methods of treating neurodegenerative disorders with said compounds are also provided.
• methods are provided for treating traumatic brain injury (TBI) in a patient and the methods include administering to a patient in need thereof an effective amount of a compound or composition described herein.
• methods are provided for treating a subject suffering from a disorder or symptom associated with neuronal cell death by administering to the subject an effective amount of a compound or composition as described herein.
• the subject may be identified as suffering from a particular disease or disorder such as stroke, concussion, intracerebral hemorrhage, epilepsy, acute glaucoma, and spinal cord injury.
• the compound or composition is administered via a method selected from the group consisting of oral administration, intravenous injection, subcutaneous injection, intranasal delivery, or intraci sternal injection.
• Methods of treatment comprises administering to a subject such as a mammal, particularly a primate including a human, an effective amount of one or more compounds as disclosed herein.
• a subject such as a mammal, particularly a primate including a human
• an effective amount of one or more compounds as disclosed herein A suitable subject may be identified and selected for treatment.
• the one or more compounds disclosed herein then may be administered to the identified subject.
• FIG. 1 shows assay results showing selectivity in vitro of NA112 for calpain-2 vs calpain-1.
• FIG. 2 shows assay results showing selectivity in vivo of NA112 for calpain-2 vs calpain-1.
• FIG. 3 shows in vivo efficacy of NA112 in DMSO solution administered 24 h after TBI plus i.p. injection ofNA112 at 0.1 or 1.0 mg/kg
• FIG. 4 shows the number of TUNEL-labelled degenerating cells in the ipsilateral side of the brain where indicated doses of NA101 (C2I) were injected intraperitoneally to WT mice at 1 h after TBI and the cells were analyzed 24 h after TBI.
• FIG. 5 shows the number of TUNEL-labelled degenerating cells in the ipsilateral side of the brain where indicated doses of NA112 (C12) were injected intraperitoneally to WT mice at 1 h after TBI and the cells were analyzed 24 h after TBI.
• FIG. 6 shows stability of NA112 in mouse plasma.
• FIG. 7 shows stability of NA112 in mouse liver homogenate with an estimated half- life.
• FIG. 8 shows activity ofNA112A (S-S isomer) against calpain-2 and calpain-1.
• FIG. 9 shows that NA112A (S-S isomer) became inactive in mouse plasma.
• FIG. 10A shows assay results showing selectivity in vitro of NA112 in liposomal formation for calpain-2 vs calpain-1.
• FIG. 10B shows values obtained with NA112 dissolved in DMSO are shown for comparison to FIG. 10 A.
• FIG. 11 shows in vivo efficacy of NA112 in liposomes vs DMSO.
• FIG. 12 shows in vivo efficacy of NA112 in liposomes and images from two different animals are shown to illustrate the decrease in TUNEL staining in NA112-treated mice.
• FIG. 13 shows a graph with the numbers of TUNEL-positive cells in images similar to those shown in FIG.12.
• FIG. 14 shows changes in NA112 plasma concentration at various times after intravenous injection.
• FIG. 15 shows changes in NA112 brain concentration at various times after intravenous injection.
• FIG. 16 shows changes in NA112 plasma concentrations fitted with the 2-compartment model.
• FIG. 17 shows the assay results of selectivity of NA184 for calpain-2 vs calpain-1.
• FIG. 18 shows rapid epimerization of NA184A in PBS.
• FIG. 19 shows results of NA184 IC50 for calpain-1 activity and calpain-2 activity in WT and calpain-1 KO mice.
• FIG. 20 shows in vivo efficacy with quantification of TUNEL staining 24 h after TBI plus i.p. injection of WT mice with NA184 at indicated doses 1 h after TBI.
• FIGS. 21 A and 21B show that NA84 significantly inhibited calpain-2 under these conditions equally well in male and female mice and rats.
• FIGS. 22A, 22B, and 22C show thatNA184 significantly prevented cell death in cortex and to the same extent in male and female rats when injected twice at 1 and 8 h after TBI.
• FIG. 23 shows correlation between calpain-2 activity and brain cell death in rats.
• R 1 , n, L 1 , R 2 , L 2 , R 4 and R 5 are as defined above.
• R 1 is absent (n is 0 and the A ring does not contain any non-hydrogen substituents), alkyl, alkoxy or halogen
• A is carbocyclic aryl such as phenyl or heteroaryl
• L 1 and L 2 are each unsubstituted alkylene, particularly methylene (-CH2-)
• R 4 and R 5 are independently hydrogen, or unsubstituted C1-C6 alkyl such as methyl.
• R 4 and R 5 are independently hydrogen, methyl or ethyl. In a particular aspect, R 4 and R 5 are methyl.
• Exemplary preferred A-L 1 - groups include the following:
• the chiral carbon most adjacent L 1 has an (S) configuration.
• the chiral carbon most adjacent to L 1 has an (R) configuration.
• the chiral carbon most adjacent to L 2 has an (S) configuration.
• the chiral carbon most adjacent to L 2 has an (R) configuration.
• Compounds of the invention may be utilized as racemic or optically enriched mixtures.
• Particularly preferred compounds of the invention are compounds analog of NA112, which may have the following formula (II) or (III).
• R 1A is cyano, or unsubstituted C 1 -C 6 alkyl
• R 1B is C 1 -C 6 alkoxy.
• R 1A is cyano (-CN) or unsubstituted alkyl such as methyl and R 1B is C 1 -C 6 alkoxy, preferably -OCH 3.
• Particularly preferred compound, NA112 has the following structure.
• A is C 1 -C 6 alkyl, carboxyl (-C(O)O-), aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;
• B is carbocyclic aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;
• L 1 is a bond, or substituted or unsubstituted C 1 -C 6 alkylene
• L 2 is a bond, substituted or unsubstituted C1-C 6 alkylene, or -S(0)2-,
• Each R 1 is a non-hydrogen substituent such as C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 ) m N(R a )C(0)R b , -(CH
• R 2 is non-hydrogen substituent such as optionally substituted C1-C6 alkyl
• Each R 6 is independently a non-hydrogen substituent such as C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl -C(0)(CH 2 ) m N(R a )(R b ), - 0(CH 2 ) m N(R a )(R b ), -CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) -C(0)0CH(R c )(R d ) - (CH 2 )mN(R a )(R b ), -(CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R
• R a , R b , R c , and R d are independently hydrogen, C 1 -C 6 alkyl that may be optionally substituted with halogen, -OH, amine, or unsubstituted C 3-6 cycloalkyl;
• the compound of Formula (X) may be a racemate including
• R 2 , L 1 , L 2 , R 6 , n and k are as defined above.
• L 1 is optionally substituted C1-C6 alkylene having (e.g. -(03 ⁇ 4)r where p is 1 to 6 and each carbon may have zero, one or two non-hydrogen substituents),
• L 2 is optionally substituted alkylene having one to 6 carbons (e.g. -(CH 2 )p where p is 1 to 6 and each carbon may have zero, one or two non-hydrogen substituents), or -S(0)2-.
• R 2 is unsubstituted C 1 -C 6 alkyl.
• R 2 is a linear unsubstituted C 1 -C 6 alkyl, or branched C 3 -C 6 alkyl e.g., isopropyl, isobutyl or t-butyl.
• R a and R b are independently hydrogen, methyl, ethyl, propyl or isopropyl.
• R c and R d are independently hydrogen or methyl.
• R a and R b are independently hydrogen or unsubstituted C 1 -C 6 alkyl.
• R a and R b are independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl).
• R a and R b are independently hydrogen or methyl.
• R a and R b are hydrogen.
• R a and R b are methyl.
• R a is hydrogen and R b is methyl.
• R a and R b are independently hydrogen or isopropyl.
• R a is hydrogen and R b is isopropyl.
• R c and R d are independently hydrogen or unsubstituted C 1 -C 6 alkyl.
• R c and R d are independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl).
• R c and R d are independently hydrogen or methyl.
• R c and R d are hydrogen.
• R c and R d are methyl.
• R c is hydrogen and R d is methyl.
• A is phenyl and L 1 is -(03 ⁇ 4) r - and p is 0 to 6 (when p is 0, L 1 is a bond).
• p is 1 to 6.
• the compound may have a Formula (XI). wherein B, R 1 , p, R 2 , L 2 , R 6 and k are as defined above n is an integer of 0 to 5.
• the compound of Formula (XI) may be a racemate including
• R 6 , n, p, and k are as defined above.
• the compound may have a Formula (Xl-a).
• R 2 , and R 6 are as defined above k is an integer of 0 to 5.
• the compound of Formula (CI-a) may be a racemate including and R 6 are as defined above.
• n is 0.
• the compound is
• n 1 to 2.
• the compound is
• the compound may have a Formula (Xl-b). and R 6 are as defined above n is an integer of 0 to 5 and k is an integer of 0 to 4.
• the compound of Formula (CI-b) may be a racemate including
• the compound is
• the compound may have a Formula (XI-c). and R 6 are as defined above k is an integer of 0 to 3.
• the compound of Formula (XI-c) may be a racemate including
• the compound may have a Formula (Xl-d),
• R 6 are as defined above k is an integer of 0 to 6.
• the compound of Formula (XI-d) may be a racemate including are as defined above.
• L 1 is a bond, methylene, or ethylene and A is C1-4 alkyl, cycloalkyl (e.g., adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or heterocycloalkyl (e.g., 5 to 12 membered heterocycloalkylene).
• cycloalkyl e.g., adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl
• heterocycloalkyl e.g., 5 to 12 membered heterocycloalkylene
• the -L'-A-R 1 is
• the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• -L 2 -B is .
• the compound has a Formula (XII).
• R 1 , p, R 2 , and R 6 are as defined above n is an integer of 0 to 5 and k is an integer of 0 to 5.
• the compound of Formula (XII) may be a racemate including are as defined above.
• two R 6 together with atoms attached thereto are joined to form a cycloalkyl, or heterocycloalkyl.
• the compound of Formula (XII) may have the structure of
• the compound has a Formula (XIII), wherein:
• Each R 1 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 ) mR
• R 2 is unsubstituted C1-C6 alkyl
• Each R 6 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) -C(0)0CH(R c )(R d ) -(CH 2 ) m N (R a )(R b ), - (CH 2 )mN(R a )C(0)R b , -(CH 2 )mN(R a )C(0)0R b ,-0(CH 2 )mR c , -0(CH 2 )mR c , -
• R a , R b , R c , and R d are independently hydrogen, C1-C6 alkyl that may be optionally substituted with halogen, -OH, amine, or unsubstituted C3-6 cycloalkyl;
• X is halogen; n is independently an integer from 0 to 5; m is independently an integer from 0 to 6; k is independently an integer from 0 to 5; p is independently an integer from 0 to 6; and pharmaceutically acceptable salts thereof.
• the compound of Formula (XIII) may be a racemate including
• the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• the compound may have a Formula (XIV), wherein:
• Each R 1 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N (R a )(R b ), - (CH 2 ) m N(R a )C(0)R b , -(CH 2 ) m N(R a )C(0)0R b ,-0(CH 2 ) m R c , -0(CH 2 )
• R 2 is unsubstituted C1-C6 alkyl
• Each R 6 is independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl -C(0)(CH 2 ) m N(R a )(R b ), -0(CH 2 ) m N(R a )(R b ), - CONH(CH 2 ) m N(R a )(R b ), -C(0)NH-CH(R c )(R d ) , -C(0)0CH(R c )(R d ) , -(CH 2 ) m N(R a )(R b ), - (CH 2 ) m N(R a )C(0)R b , -(CH 2 ) m N(R a )C(0)0R b ,-0(CH 2 ) m R c , -0(CH 2 )
• R a , R b , R c , and R d are independently hydrogen, C1-C6 alkyl that may be optionally substituted with halogen, -OH, amine, or unsubstituted C3-6 cycloalkyl;
• X is halogen; n is independently an integer from 0 to 5; m is independently an integer from 0 to 6; k is independently an integer from 0 to 5; p is independently an integer from 0 to 6; and pharmaceutically acceptable salts thereof.
• the compound of Formula (XIV) may be a racemate including
• the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• Preferred compound, NA184 has the following structure.
• the compound may be a racemate including: Particularly preferred compound, NA184, is S-S isomer having the following structure
• a “calpain-2 selective inhibitor” or a “selective calpain-2 inhibitor” as referred to herein is a compound with a calpain-2 inhibition constant (Ki) lower than its Ki for calpain-1.
• a calpain-2 selective inhibitor is a compound with a Ki for calpain-2 that is 2-fold to 10-fold lower than its Ki for calpain-1.
• a calpain-2 selective inhibitor is a compound with an IC 50 value for calpain-2 that is 10-50-fold lower than its IC 50 for calpain-1 in an in situ assay.
• IC 50 values for NA112 on the activity of in situ calpain-1 and calpain-2 activities were measured (Wang et al., 2014).
• the selectivity of NA112 for calpain-2, measured as a ratio of IC 50 calpain- 1/ IC 50 calpain-2 was about 13.
• structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.
• Alkyl refers to a saturated, straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, having from one to twelve carbon atoms (C1-C12 alkyl), from one to eight carbon atoms (Ci-Cs alkyl) or from one to six carbon atoms (C1-C6 alkyl), and which is attached to the rest of the molecule by a single bond.
• alkyl groups include methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.
• Alkylene or “alkylene chain” refers to a straight or branched divalent hydrocarbon (alkyl) chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, respectively. Alkylenes can have from one to twelve carbon atoms, e.g., methylene, ethylene, propylene, n-butylene, and the like.
• alkylene chain is attached to the rest of the molecule through a single or double bond.
• the points of attachment of the alkylene chain to the rest of the molecule can be through one carbon or any two carbons within the chain.
• Optionally substituted alkylene refers to alkylene or substituted alkylene.
• Alkoxy refers to a radical of the formula -OR a where R a is an alkyl having the indicated number of carbon atoms as defined above.
• alkoxy groups include without limitation -O-methyl (methoxy), -O-ethyl (ethoxy), -O-propyl (propoxy), -O-isopropyl (iso propoxy) and the like.
• Cycloalkyl and heterocycloalkyl by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
• heterocycloalkyl examples include, but are not limited to, l-(l,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2- piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran- 3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.
• a “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.
• Carbocyclic aryl or “cycloalkyl” refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring, but without any hetero (N, O or S) ring members in the aromatic ring.
• Exemplary carbocyclic aryls are hydrocarbon ring system radical comprising hydrogen and 6 to 9 carbon atoms and at least one aromatic ring; hydrocarbon ring system radical comprising hydrogen and 9 to 12 carbon atoms and at least one aromatic ring; hydrocarbon ring system radical comprising hydrogen and 12 to 15 carbon atoms and at least one aromatic ring; or hydrocarbon ring system radical comprising hydrogen and 15 to 18 carbon atoms and at least one aromatic ring.
• the carbocyclic aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
• Carbocyclic aryl radicals include, but are not limited to, carbocyclic aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.
• “Optionally substituted carbocyclic aryl” refers to an unsubstituted carbocyclic aryl group or a substituted carbocylic aryl group.
• a cycloalkyl is a cycloalkenyl.
• the term “cycloalkenyl” is used in accordance with its plain ordinary meaning.
• a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system.
• monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl.
• bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings.
• bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (Ctfrj w , where w is 1, 2, or 3).
• Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbomenyl and bicyclo[2.2.2]oct 2 enyl.
• fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
• the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring.
• cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia.
• multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
• multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
• multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
• a heterocycloalkyl is a heterocyclyl.
• heterocyclyl as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle.
• the heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
• the 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S.
• the 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
• the 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S.
• the heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle.
• heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3- dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl,
• the heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl.
• the heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.
• bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3- dihydrobenzofuran-3-yl, indolin-l-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-lH-indolyl, and octahydrobenzofuranyl.
• heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
• the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.
• Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
• multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring.
• multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
• multicyclic heterocyclyl groups include, but are not limited to lOH-phenothiazin-10-yl, 9,10-dihydroacridin-9-yl, 9,10- dihydroacridin-10-yl, lOH-phenoxazin-10-yl, 10,1 l-dihydro-5H-dibenzo[b,f]azepin-5-yl, l,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro-lH-carbazol-9-yl.
• halo or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl.
• halo(Ci-C4)alkyl includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-triiluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
• aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
• a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring.
• heteroaryl refers to a 5- to 14-membered ring system radical comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring.
• the heteroaryl radical may be a stable 5-12 membered ring, a stable 5-10 membered ring, a stable 5-9 membered ring, a stable 5-8 membered ring, a stable 5-7 membered ring, or a stable 6 membered ring that comprises at least 1 heteroatom, at least 2 heteroatoms, at least 3 heteroatoms, at least 4 heteroatoms, at least 5 heteroatoms or at least 6 heteroatoms.
• Heteroaryls may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized.
• the heteroatom may be a member of an aromatic or non-aromatic ring, provided at least one ring in the heteroaryl is aromatic.
• Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl,
• Various compounds and substituents that are "optionally substituted” or “substituted” may be suitably substituted at one or more available positions by, but not limited to, halogen (F, Cl, Br, I); nitro; hydroxy; amino; alkyl such as C1-C4 alkyl; alkenyl such as C2-C8 alkenyl; alkoxy e.g. C1-C6 alkxoy, alkylamino such as Ci-Cs alkylamino; carbocyclic aryl such as phenyl, naphthyl, anthracenyl, etc; heteroaryl; and the like.
• halogen F, Cl, Br, I
• nitro hydroxy
• amino alkyl
• alkenyl such as C2-C8 alkenyl
• alkoxy e.g. C1-C6 alkxoy
• alkylamino such as Ci-Cs alkylamino
• carbocyclic aryl such as
• compositions of the invention comprise NA112 or NA184, and a pharmaceutically acceptable excipient.
• Excipients used in pharmaceutical composition of the invention are safe and provide the appropriate delivery for the desired route of administration, of an effective amount ofNA112, orNA184.
• a compound of the invention can be formulated as a pharmaceutical dosage form and administered to a subject in need of treatment, for example, a mammal, such as a human patient, in a variety of forms adapted to the chosen route of administration.
• the compositions of the present invention may be administered in a variety of different ways, including oral administration, intravenous injection, intramuscular injection, subcutaneous injection or by intranasal delivery.
• the compounds may be included in solutions, suspensions and other dosage forms adapted for intravenous or subcutaneous injection.
• Solutions of the compounds of the invention can be prepared in water or a physiologically acceptable buffer, optionally mixed with a nontoxic surfactant, including cyclodextrins. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, liposomes, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the compounds of the invention which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions.
• the liquid carrier can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the compounds of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
• Useful dosages of compounds of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
• the amount of the compounds of the invention required for use in treatment will vary depending on the particular therapeutic agent, the composition, if there is one, that comprises the therapeutic agent, the route of administration, the nature of the condition being treated and the age and condition of the patient, and will be ultimately at the discretion of the attendant physician or clinician.
• a therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See, e.g ., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, an effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutic dose can also be selected by analogy to dosages for comparable therapeutic agents.
• Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
• salts are meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
• base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
• pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
• acid addition salts can be obtained by contacting the neutral 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, lactic, mandelic, phthalic, benzenesulfonic, p-tolyl sulfonic, citric, tartaric, oxalic, methanesulfonic, and the like.
• inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic
• 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 el al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
• Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
• the compounds of the present disclosure may exist as salts, such as with pharmaceutically acceptable acids.
• the present disclosure includes such salts.
• Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art.
• the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
• the parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.
• Methods provided herein are methods for treating traumatic brain injury (TBI) in a patient.
• Preferred method includes administering to a patient in need thereof an effective amount of a compound or composition as described herein.
• a method of treating a subject suffering from a disorder or symptom associated with neuronal cell death includes administering to the subject an effective amount of a compound or composition as described herein.
• the subject is suffering from stroke, concussion, intracerebral hemorrhage, acute glaucoma, seizure activity and/or spinal cord injury.
• the patient has been identified as suffering or susceptible to a disorder or symptom associated with neuronal cell death and the compound is administered to the identified subject.
• the compound or composition is administered via a method selected from the group consisting of oral administration, intravitreal injection, intraocular injection, intraocular perfusion, periocular injection and sub-Tenon injection.
• the subject is a human.
• the compound NA110 can be synthesized according to Scheme 1.
• Step 1 Preparation of tert-butyl (l-hydroxybutan-2-yl)carbamate
• Step 3 Preparation of tert-butyl (l-cyano-l-hydroxybutan-2-yl)carbamate
• Step 5 Preparation of methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4- methylpentanamido)-2-hydroxypentanoate hoc O 300
• Step 6 Preparation of 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2- hydroxypentanoic acid (Intermediate A) intermediate A Methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2- hydroxypentanoate (8.6 g) was dissolved in a mixture of 1M NaOH (30 ml) and THF (30 ml) and stirred overnight (LCMS control). Then the solution was diluted with ethyl acetate (200 ml) and 0.5M aqueous HCL (200 ml).
• Step 1 Preparation of tert-butyl ((2S)-l-((l-(3,5-dimethoxybenzylamino)-2-hydroxy-l- oxopentan-3-yl)amino)-4-methyl-l-oxopentan-2-yl)carbamate
• Step 2 Preparation of (2S)-2-amino-N-(l-(3,5-dimethoxybenzylamino)-2-hydroxy-l- oxopentan-3-yl)-4-methylpentanamide (Intermediate B)
• Step 2 Oxidation to (2S)-2-(3-benzylureido)-N-(l-((3,5-dimethoxybenzyl)amino)-l,2- dioxopentan-3-yl)-4-methylpentanamide (Compound NA112)
• the hydroxy compound (4.2 g) was dissolved/suspended in dichloromethane (150 mL) and treated with Dess-Martin periodinane (6.7g) stirring at room temperature for 2 h (absence of starting material on LC). Then the reaction mixture was partitioned between saturated bicarbonate solution (300 ml) and ethyl acetate (300 ml). The aqueous layer was extracted twice more with ethyl acetate (2* 100ml) and the combined organic layers are washed with water (100 ml), dried, filtered, and concentrated to dryness. The residue was then purified by column chromatography to afford 1.8 g ( ⁇ 70% LCMS purity) of target compound.
• Calpain activity in cerebellar P2 fractions was measured 24 h after traumatic brain injury (TBI) in adult wild-type mice and the assay results for calpain-1 and calpain -2 are shown in FIG. 2.
• TBI traumatic brain injury
• 0.1 or 1 mg/kg of NA112 was injected intraperitoneally 1 h after TBI.
• Each measurement of in vivo selectivity was obtained as follows:
• Calpain-1 activity Calpain activity with 20 pM Ca 2+ . Normalized to Vehicle.
• Calpain-2 activity Calpain activity with 5 mM Ca 2+ minus Calpain activity with 20 mM Ca 2+ . Results are means ⁇ S.E.M. of 3 experiments.
• Indicated doses of NA101 (C2I, FIG 4) or NA112 (C12; FIG. 5) were injected intraperitoneally to WT mice at 1 h after TBI.
• the number of TUNEL-labelled degenerating cells in the ipsilateral side of the brain was analyzed 24 h after TBI.
• NA1 12 in b-cyclodextrin formulation demonstrated good plasma stability with an estimated half-life of 17 hours (FIG. 6).
• NA112 C12, 0.2 mM
• NA1 12 in B-cyclodextrin formulation demonstrated good stability in mouse liver homogenate with an estimated half-life of NA112 was 15 hours (FIG. 7). Aliquots were taken and the degree of inhibition of purified human calpain-2 was measured. Results were normalized to the maximum degree of inhibition (100%) measured at tO.
• Example 8 Separation of Isomers and activities thereof
• NA112A Like in NA101, there are 2 chiral centers for NA112. NA112A, where chiral center 1 is the S- form and chiral center 2 is the S- form was separated from the S-R- form (NA112B) using methods that are well-known methods for separating diastereoisomers.
• NA112A S-S isomer
• NA112B S-R stereoisomer
• the inhibitory activity of the compounds NA112A (S-S isomer) and NA112B (S-R stereoisomer) against calpain-1 and calpain-2 were determined.
• the NA112B compound (S- R stereoisomer) had no inhibitory activity at the highest concentration tested, 3 mM.
• NA112A S-S isomer
• FIG. 8 NA112A (S-S isomer) showed the expected inhibitory activity against calpain-2 and calpain-1.
• NA1 12A S-S stereoisomer was incubated in mouse plasma at 37 °C to determine whether it undergoes epimerization and therefore becomes inactive. As shown in FIG. 9,
• NA1 12A rapidly inactivated in mouse plasma.
• NA112A in B-cyclodextrin formulation (10 mM) was incubated in mouse plasma at 37 °C for the indicated periods of time. Aliquots were taken and the degree of inhibition of purified human calpain-2 was measured. Results were normalized to the maximum degree of inhibition (50%) measured at tO.
• Liposomes were prepared as follows:
• composition (Each for 1 ml of liposomal formulation)
• DMPC Dimyristoyl phosphatidylcholine
• DMPG Dimyristoyl phosphatidylcholine
• DMPG- Na Dimyristoyl phosphatidylcholine
• Aqueous medium Phosphate Buffered Saline pH 6.8 - 1ml
• Various concentrations of NA112 in the liposomal formulation were incubated with human calpain-1 (purified from erythrocytes, ecalpain-1) or a recombinant human calpain-2 (hcalpain-2) and calpain activity assayed as in FIG. 1.
• IC 50 were determined and transformed into Ki values (FIG. 10A, 10B). Values previously obtained with NA112 dissolved in DMSO are shown for comparison (Fig. 10B).
• Example 11 NA112 In vivo efficacy in liposome formulation
• Calpain activity in cerebellar P2 fractions was measured 24 h after TBI in adult WT mice, and vehicle or 0.1 or 1 mg/kg of NA112 (12) dissolved in DMSO or liposomal formulation (lipo) was injected intraperitoneally 1 h after TBI.
• vehicle or 0.1 or 1 mg/kg of NA112 (12) dissolved in DMSO or liposomal formulation (lipo) was injected intraperitoneally 1 h after TBI.
• lipo liposomal formulation
• Calpain-1 activity Calpain activity with 20 mM Ca 2+ , normalized to Vehicle.
• Calpain-2 activity Calpain activity with 5 mM Ca 2+ minus Calpain activity with 20 pM Ca 2+ , then normalized to vehicle.
• DMSO or liposomes Vehicle (DMSO or liposomes) or 0.1 or 1 mg/kg of NA112 (12) dissolved in DMSO or liposomal formulation (lipo) was injected intraperitoneally 1 h after TBI in WT mice. Calpain activity was measured 24 h later in cerebellar P2 fractions. Calpain-1 activity was measured in the presence of 20 pM calcium and calpain-2 activity was measured as the difference between calpain activity measured in the presence of 5 mM calcium and that measured in the presence of 20 pM calcium (FIG. 11). In both cases, calpain activity was normalized to the values measured in vehicle-treated mice. Results are means ⁇ S.E.M. of 3 animals. Note that NA112 produced the same degree of calpain-2 inhibition whether it is dissolved in DMSO or liposomes.
• NA1 12 was prepared in liposomes at a concentration of 1.5 mg/ml. Mice were injected in the tail vein with 200 pi of NA112 in liposomes (corresponding to a dose of 10 mg/kg) and were sacrificed at the following time-points: 1, 5, 15, 30 min, 1, 2, 4, 8, 16 and 24 h. Blood was collected and plasma rapidly prepared by centrifugation. Brains were also collected. NA112 in plasma and in brain homogenates was assayed with LC/Ms and the sensitivity of the assay was 1 ng/ml. Experiments were performed in duplicate (A1 and A2 in Table 3). Results were averaged for preparing the figure and calculating the half-life of NA112 in plasma and in brain.
• Table 3 Plasma concentration of NA112 at various times after iv injection.
• FIG. 14 shows changes in NA112 plasma concentration at various times after iv injection and the data in table 3 are plotted as a function of time.
• FIG. 15 shows changes in NA112 brain concentration at various times after intravenous injection. Data from table 4 are plotted as a function of time. While the curve is not easily fitted with a 2-compartment model, the slower component appears to have a half-life of about 3 h.
• FIG. 16 A two-compartment model was used to analyze the data from FIG. 14. This assumes that there is a rapid distribution of the drug from the plasma to various organs, followed by a slower elimination of the drug form the plasma. In this case, the curve is well-fitted with the 2- compartment model (FIG. 16), indicating a half-life in the plasma of 7.8 h. In FIG. 16, changes in NA112 plasma concentrations fitted with the 2-compartment model.
• R 1 is -OR 4 or -OR 5 in Formula (I)
• R 2 is same as R 2 in Formula (I)
• n is an inteter of 0 to 5
• k is an integer of 1 to 5
• Methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-hydroxypentanoate (previously described in W02020/037012) is Boc-deprotected to afford methyl 3-((S)-2- (amino)-4-methylpentanamido)-2-hydroxypentanoate hydrochloride salt.
• the free base of the amino group is then reacted with an ⁇ -substituted) benzylisocyanate or equivalent reagent to construct the desired methyl 3-((S)-2-(3- (R 2 -substituted)benzylureido)-4-methylpentanamido)- 2-hydroxypentanoate.
• the methyl ester of this intermediate is hydrolyzed to the corresponding acid, 3-((S)-2-(3-(R 2 -substituted)benzylureido)-4-methylpentanamido)-2-hydroxypentanoic acid.
• the acid is then functionalized with a series of (Ri-substituted)benzylamines to afford the desired N-(Ri-substituted)benzyl-3-((S)-2-(3-(R 2 -substituted)benzylureido)-4-methylpentan- amido)-2-hydroxypentanamide which is oxidized at the secondary alcohol position to afford the final products, the N-(Ri-substituted)benzyl-3-((S)-2-(3-(R 2 -substituted)benzylureido)-4- methylpentanamido)-2-oxopentanamides.
• Step 1 Preparation of methyl 3- -2-amino-4-methylpentanamido)-2-hvdroxy- pentanoate
• Step 2 Preparation of methyl 3- -2-(3-benzylureido)-4-methylpentanamido)-2- hydroxypentanoate
• Methyl 3-((S)-2-(amino)-4-methylpentanamido)-2-hydroxypentanoate hydrochloride salt (3.2 g) with some dioxane from previous step was dissolved in CH3CN / THF (1/1) mixture (80 ml) and Et3N (2 ml) was added in one portion.
• Benzylisocyanate (1.33 g, 1.2 equiv) was added in one portion, and the reaction mixture was stirred for 4 h at room temperature (LCMS control).
• Step 3 Preparation of 3- -2-(3-benzylureido)-4-methylpentanamido)-2-hvdroxy- pentanoic acid
• Step 5 Preparation of (2S)-2-(3-benzylureido)-N-(T-(Y3-fluoro-2-methoxybenzv0aminoV L2-dioxopentan-3-vD-4-methylpentanamide (N
• Step 1 Preparation of (2S)-2-(3-benzylureidoVN-(2-hvdroxy-l-(Y2-methoxy-3-methyl- benzvDamino)-l-oxopentan-3-vD-4-methylpentanamide
• Step 2 Preparation of (2S)-2-(3-benzylureidoVN-(T-(Y2-methoxy-3-methylbenzvD- amino)-L2-dioxopentan-3-vD-4-methylpentanamide (NSN23482)
• Step 1 Preparation of (2S)-2-(3-benzylureido)-N-(T-(Y3-chloro-2-methoxy- benzv0amino)-2-hvdroxy-l-oxopentan-3-v0-4-methylpentanamide
• Step 2 Preparation of (2S)-2-(3-benzylureido)-N-(T-(Y3-chloro-2-methoxy- benzvDamino)-L2-dioxopentan-3-vD-4-methylpentanamide (NA184)
• Step 4 Preparation of (2S)-2-(3-benzylureido)-N-(T-(Y2-cvano-3-methylbenzv0amino)-2- hydroxy- 1 -oxopentan-3 -vD-4-methylpentanamide
• Step 5 Preparation of (2S)-2-(3-benzylureido)-N-(T-(Y2-cvano-3-methylbenzv0aminoV E2-dioxopentan-3-vD-4-methylpentanamide (NSN23500)
• R 1 is -OR 4 or -OR 5 in Formula (I)
• R 2 is same as R 2 in Formula (I)
• n is an inteter of 0 to 5
• k is an integer of 1 to 5
• commercially available 2- aminobutan-l-ol is transformed into the key Intermediate A, 3-((S)-2-((tert- butoxycarbonyl)amino)-4-methylpentanamido)-2-hydroxypentanoic acid.
• Intermediate A is functionalized on the acid terminus with the desired (Ri-substituted)benzyl amine and then after deprotection of the Boc-protected amine, affords Intermediate B, (2S)-2-amino-N-(l-((Ri- substituted)benzylamino)-2-hydroxy-l-oxopentan-3-yl)-4-methylpentanamide.
• Intermediate B is then transformed into a series of ureas by reacting the desired (R2-substituted) benzylamine- derived isocyanate or equivalent reagent.
• Step 1 Preparation of 3-((SV2-((tert-butoxycarbonv0amino)-4-methylpentanamido)-2- hydroxypentanoic acid
• Step 2 Preparation of tert-butyl (Y2S)-1 -i( ⁇ -((3.5-dimethoxybenzyl )amino)-2-hvdroxy- l - oxopentan-3-vDamino)-4-methyl-l-oxopentan-2-vDcarbamate
• Step 3 Preparation of (2S)-2-amino-N- 3.5-dimethoxybenzyl )amino)-2-hvdroxy- l - oxopentan-3-vD-4-methylpentanamide
• Step 4 Preparation of methyl 4-(T5S)- l 2-(3.5-dimethoxyphenyl )-8-ethyl-9-hvdroxy-5- isobutyl-3.6. 10-trioxo-2.4.7.1 1 -tetraazadodecyl ibenzoate (2S)-2-amino-N-(l-((3,5-dimethoxybenzyl)amino)-2-hydroxy-l-oxopentan-3-yl)-4-methyl- pentanamide, (0.42 g, HC1 salt) was dissolved in dioxane (10 mL) and Et3N (0.3 mL) and methyl 4-(isocyanatomethyl)benzoate (0.23 g) were added.
• Step 5 Preparation of 4-(T5S)- l 2-(3.5-di ethoxyphenyl)-8-ethyl-9-hvdroxy-5-isobutyl-
• Step 6 Preparation of 4-(T5S)- l 2-(3.5-di ethoxyphenyl)-8-ethyl-9-hvdroxy-5-isobutyl-
• Step 7 Preparation of 4-(Y5S)-12-(3.5-dimethoxyphenvO-8-ethyl-5-isobutyl-3.6.9.10- tetraoxo-2.4.7. 1 1 -tetraazadodecyl )-N-(2-(isopropylamino) ethvnbenzamide
• Example 14-2 Alternate Order of Steps Produces Anomalous Cyclized Products If the oxidation of the secondary alcohol is carried out earlier in the sequence, unexpected anomalous cyclized products are obtained.
• Step 3 Preparation of tert-butyl (T2S)- 1 -(Tl -((3.5-dimethoxybenzyl )amino)- l .2- dioxopentan-3-vDamino)-4-methyl-l-oxopentan-2-vDcarbamate
• Step 4 Preparation of (2S)-2-amino-N-(l -((3.5-dimethoxybenzyl )amino)-l 2- dioxopentan-3-vD-4-methylpentanamide and (6R)-N-(3.5-dimethoxybenzyl )-3-ethyl-6- isobutyl-5-oxo-3A5.6-tetrahvdropyrazine-2-carboxamide
• Step 5 Preparation of N-(3.5-dimethoxybenzvO-3-(YS)-4-isobutyl-2.5-dioxo- imidazolidin-l-vD-2-oxopentanamide (NSN23499) and N-(3.5-dimethoxybenzvD-3- ethyl-5-hvdroxy-6-isobutylpyrazine-2-carboxamide (NSN23490)
• N-(3,5- dimethoxybenzyl)-3-((S)-4-isobutyl-2,5-dioxoimidazolidin-l-yl)-2-oxopentanamide (NSN23499) and N-(3,5-dimethoxybenzyl)-3-ethyl-5-hydroxy-6-isobutylpyrazine-2-carbox- amide (NSN23491).
• Step 1 Preparation of i2S)-2-(YN-benzylsulfamoyl )amino)-N-( l -((3.5-dimethoxybenzyl )- amino)-2-hvdroxy-l-oxopentan-3-vD-4-methylpentanamide
• Step 2 Preparation of (2S)-2-((N-benzylsulfamoyl )amino)-N-( l -((3.5-dimethoxy- benzvDamino)- 1 2-dioxopentan-3 -vD-4-methylpentanamide(N SN23501 )
• (2S)-2-((N-benzylsulfamoyl)amino)-N-(l-((3,5-dimethoxybenzyl) _, amino)-2-hydroxy-l- oxopentan-3-yl)-4-methylpentanamide 150 mg was dissolved in CTLCN (10 mL) and 1 drop of water, pyridine (150 pL), and DMSO (100 pL) were added. Then Dess-Martin Periodinane (150 mg) was added and the reaction stirred at room temperature for 2 h. The mixture was quenched by the addition of saturated aqueous NaHCCb (20 ml) and extracted with ethyl acetate (3 x 10 ml).
• Example 14-5-1 Preparation ofN-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)- ethoxy)benzyl)ureido)-4-methylpentanamido)-2-hydroxypentanamide
• Step 1 Preparation of tert-butyl (4-(2-(dimethylamino)ethoxy)benzvDcarbamate
• Step 2 Preparation of N-(3.5-dimethoxybenzvD-3-(YS)-2-(3-(4-(2-(dimethylaminoV ethoxy)benzvDureido)-4-methylpentanamido)-2-hvdroxypentanamide
• Step 3 Preparation of N-(3.5-dimethoxybenzvO-3-(YS)-2-(3-(4-(2-(dimethylaminoV ethoxy)benzvDureido)-4-methylpentanamido)-2-oxopentanamide(NSN23483)
• Step 1 Preparation of tert-butyl (4-((dimethylamino)methv0benzv0carbamate l-(4-(aminomethyl)phenyl)-N,N-dimethylmethanamine (0.33 g) was dissolved in THF (5 ml) and Boc-anhydride (0.45g) was added. The mixture was stirred at room temperature for 2 h and evaporated to dryness. The residue was purified by column silica gel chromatography to give pure tert-butyl (4-((dimethylamino)methyl)benzyl)carbamate (0.45 g, 85%) which was used directly in the next step.
• Step 2 Preparation of N-(3.5-dimethoxybenzvD-3-(YS)-2-(3-(4-(Ydimethylamino) rnethvDbenzvDureido)-4-methylpentanamido)-2-hvdroxypentanamide
• Boc-protected amine, tert-butyl (4-((dimethylamino)methyl)benzyl)carbamate (1.0 mmol) and 2- chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml).
• Triflic anhydride (1.5 mmol) was added dropwise over 5 min.
• (2S)-2- amino-N-(l-((3,5-dimethoxybenzyl)amino)-2-hydroxy-l-oxopentan-3-yl)-4-methylpentanamide hydrochloride salt (0.3 mmol) and triethylamine (3.0 mmol) were added to the resulting mixture.
• Step 3 Preparation of N-(3.5-dimethoxybenzvO-3-(YS)-2-(3-(4-(Ydimethylamino)methvO- benzvDureido)-4-methylpentanamido)-2-oxopentanamide
• Example 14-6 Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10- tetraoxo-2,4,7,ll-tetraazadodecyl)-N-isopropylbenzamide (NSN23488)
• Step 2 Preparation of 4-(T5S)- l 2-(3.5-di ethoxyphenyl)-8-ethyl-9-hvdroxy-5-isobutyl- 3.6. 10-trioxo-2.4.7. 1 1 -tetraazadodecyl )-N-isopropylbenzamide
• Boc-protected amine tert-butyl (4-(isopropylcarbamoyl)benzyl)carbamate (1.0 mmol) and 2- chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml). Triflic anhydride (1.5 mmol) was added dropwise over 5 min. After stirring for 1 h at room temperature (2S)-2-amino- N-(l-((3,5-dimethoxybenzyl)amino)-2-hydroxy-l-oxopentan-3-yl)-4-methylpentanamide hydrochloride salt (0.3 mmol) and triethylamine (3.0 mmol) were added to the reaction mixture.
• Step 3 Preparation of 4-((5S)-12-(3.5-dimethoxyphenvD-8-ethyl-5-isobutyl-3.6.9.10- tetraoxo-2.4.7.11-tetraazadodecvD-N-isopropylbenzamide (NSN23488)
• Step 1 Preparation of tert-butyl (4-cvano-2-methoxybenzvDcarbamate
• Step 2 Preparation of (2S)-2-(3-(4-cvano-2-methoxybenzyl )ureido)-N-( l -((3.5-di- methoxybenzvDamino)-2-hvdroxy-l-oxopentan-3-vD-4-methylpentanamide
• Boc-protected amine, tert-butyl (4-cyano-2-methoxybenzyl)carbamate (1.0 mmol) and 2- chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml).
• Triflic anhydride (1.5 mmol) was added dropwise over 5 min.
• (2S)-2- amino-N-(l-((3,5-dimethoxybenzyl)amino)-2-hydroxy-l-oxopentan-3-yl)-4-methylpentanamide hydrochloride salt (0.3 mmol) and triethylamine (3.0 mmol) were added to the reaction mixture.
• Step 3 Preparation of (2S)-2-(3-(4-cvano-2-methoxybenzyl )ureido)-N-( l -((3.5-di- methoxybenzv0amino)-L2-di oxopentan-3 -nP-4-methylpentanamide (NSN23489)
• Example 14-8 preparation of n-(3,5-dimethoxybenzyl)-3-((s)-2-(3-(2-methoxy-4- methylbenzyl)ureido)-4-methylpentanamido)-2-oxopentanamide(NSN23490)
• Step 1 Preparation of tert-butyl (2-methoxy-4-methylbenzvDcarbamate
• Step 2 Preparation of N-(3.5-dimethoxybenzvD-2-hvdroxy-3- -2-(3-(2-methoxy-4- methylbenzv0ureido)-4-methylpentanamido)pentanamide
• Boc-protected amine, tert-butyl (2-methoxy-4-methylbenzyl)carbamate (1.0 mmol) and 2- chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml).
• Triflic anhydride(1.5 mmol) was added dropwise over 5 min.
• (2S)-2- amino-N-(l-((3,5-dimethoxybenzyl)amino)-2-hydroxy-l-oxopentan-3-yl)-4-methylpentanamide hydrochloride salt (0.3 mmol) and triethylamine (3.0 mmol) were added to the reaction mixture.
• Step 3 Preparation of N-(3.5-dimethoxybenzvO-3-(YSV2-(3-(2-methoxy-4-methyl- benzvDureido)-4-methylpentanamido)-2-oxopentanamide(NSN23490)
• Step 1 Preparation of tert-butyl (4-carbamoylbenzvDcarbamate
• Step 2 Preparation of 4-(T5S)- l 2-(3.5-di ethoxyphenyl)-8-ethyl-9-hvdroxy-5-isobutyl- 3.6.10-trioxo-2A7.11-tetraazadodecvDbenzamide and (2S)-2-(3-(4- cvanobenzyl )ureido)-N-( l -((3.5-dimethoxybenzyl ) amino)-2-hvdroxy-l-oxopentan-3-vD- 4-methylpentanamide triphosgene (300 mg) and triethylamine (0.8 mL). The mixture was stirred for 30 min at room temperature.
• Step 3 Preparation of (2S)-2-(3-(4-cvanobenzyl )ureido)-N-(l -((3.5-dimethoxybenzyl )- amino)- 1.2-di oxopentan-3 -nP-4-methylpentanamide (NSN23492)
• Example 14-10 Preparation of Preparation ofN-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2- hydroxyethoxy) benzyl) ureido)-4-methylpentanamido)-2-oxopentanamide
• Step 1 Preparation of 4-(2-(Ytert-butyldimethylsilyl)oxy) ethoxy) benzonitrile
• Step 4 Preparation of (2S)-2-(3-(4-(2-((tert-butyldimethylsilvDoxy)ethoxy)benzvD- ureidol-N-fl -((3.5-dimethoxybenzyl )amino)-2-hvdroxy-l -oxopentan-3-yl )-4- methylpentanamide
• Boc-protected amine, tert-butyl (4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzyl)carbamate (1.0 mmol) and 2-chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml). Triflic anhydride (1.5 mmol) was added dropwise over 5 min.
• Step 5 Preparation of N-(3.5-dimethoxybenzvO-3-(YS)-2-(3-(4-(2-hvdroxyethoxy)- benzyl) ureido)-4-methylpentanamido)-2-oxopentanamide
• the crude ketone was dissolved in THF (5 ml). To this solution was added TBAF (0.5 ml, 1M in THF) and the mixture was stirred overnight at room temperature (LC control of TBS deprotection). After completion of the deprotection, the mixture was diluted with water (20 ml) and extracted with ethyl acetate (3 x 20 ml). The organics were evaporated to dryness.
• Example 14-11 Additional Analogs Prepared by Example 14
• NA184A S-S isomer
• NA184B S-R stereoisomer
• the NA184B compound had no inhibitory activity at the highest concentration tested, 3 mM.
• NA184A S-S isomer
• NA184A S-S stereoisomer
• WT or calpain-1 KO mouse cerebellar P2 homogenate were used for measuring inhibitory activity of compounds against the mouse endogenous calpain-l/-2.
• Each reaction contains 100 ul of WT or calpain-1 KO mouse cerebellar P2 homogenate + 0, 20 or 2000 uM Ca 2+ + 0 - 10,000 nM NA184.
• Calpain-1 activity calpain activity under 20 uM Ca 2+ in WT mice
• Calpain-2 activity calpain activity under 2000 uM Ca 2+ in calpain-1 KO mice Results are shown in FIG. 19, and the data were used to calculate the IC50 shown in Table 7.
• Example 18 In vivo Efficacy in a rat model of TBI in males and females.

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