Calpain-2 inhibitor compounds and therapeutic methods

Abstract

The present invention provides a compound that is a selective inhibitor of calpain-2, and a method for treating neurodegenerative diseases by administering the compound. [Solution] A compound of formula (I) or (X) is provided, which includes the treatment of disorders such as disorders or symptoms related to neuronal cell death. Preferred compounds may be useful for treating acute neurodegeneration. JPEG2026053525000291.jpg35125 (I) JPEG2026053525000292.jpg30146

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 975,644, filed Feb. 12, 2020, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Field of the Invention The present invention relates to products that inhibit the function of calpain - 2, as well as methods for specifically inhibiting the activation or activity of calpain - 2, and methods for treating and preventing neurodegenerative diseases that are susceptible to the therapeutic effects of molecules that interfere with the function of calpain - 2.

Background Art

[0003] Many studies have shown that calpain, a calcium - dependent protease, is involved in neurodegeneration in both acute and chronic animal models of neurodegeneration. In particular, calpain has been shown to play an important role in neuronal degeneration and axonal injury following traumatic brain injury (TBI). Although much has been learned over the past few decades about the mechanisms underlying the neuropathology caused by TBI, most treatments for TBI target the symptoms following TBI, particularly neurobehavioral disorders, and there are few attempts to provide neuroprotection. <000092​Calpain-1 and calpain-2, the two major calpain isoforms in the brain, play opposing roles in both synaptic plasticity and neurodegeneration. Calpain-1 is required for the induction of synaptic plasticity, while calpain-2 limits the degree of synaptic plasticity for several minutes following the induction event (Wang et al., 2014). Similarly, calpain-1 is neuroprotective, while calpain-2 is neurodegenerative (Wang et al., 2013). These dual and opposing functions of calpain-1 / 2, as well as the lack of selective inhibitors for these two calpain isoforms, have contributed to previous difficulties in developing calpain inhibitors for translational applications, particularly for the prevention of neurodegeneration. Calpain-1 activation is associated with synaptic NMDA receptor stimulation, which explains its necessary role in long-term potentiation (LTP) induction. It is also involved in neuroprotection induced by synaptic NMDA receptor stimulation. On the other hand, calpain-2 is associated with extrasynaptic NMDA receptor stimulation and is involved in neurodegeneration. Calpain-2 is also activated by BDNF->ERK-mediated phosphorylation and limits the degree of LTP after Θ burst stimulation (TBS). Therefore, selective calpain-2 inhibitors can be both neuroprotective and cognitive enhancers. Selective calpain-2 inhibitors can be used not only for TBI but also for several acute indications associated with neuronal death, including stroke, concussion, intracerebral hemorrhage, acute glaucoma, and spinal cord injury. They can also be used to prevent neurodegeneration induced by seizure activity and therefore may be useful in preventing epileptic seizures. [Overview of the project] [Means for solving the problem]

[0005] In one embodiment, a compound that is a selective inhibitor of calpain-2 is provided.

[0006] Preferred compounds may be useful for treating acute neurodegeneration.

[0007] In certain embodiments, compounds of the following formula (I): [Chemical formula] [In the formula, A is a carbocyclic aryl or heteroaryl, R 2 is a C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, -CO(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -CONH-CH(R​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​and pharmaceutically acceptable salts thereof are provided.

[0008] In certain preferred embodiments, R a and R b R is independently hydrogen or an unsubstituted C1-C6 alkyl group. In certain embodiments, R a and R b R is independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl). In certain embodiments, R a and R b R is independently hydrogen or methyl. In certain embodiments, R a and R b is hydrogen. In certain embodiments, R a and R b is methyl. In certain embodiments, R a is hydrogen, R b is methyl. In certain embodiments, R a and R b R is independently hydrogen or isopropyl. In certain embodiments, R a is hydrogen, R b It is isopropyl.

[0009] In certain preferred embodiments, R c and R d R is independently hydrogen or an unsubstituted C1-C6 alkyl group. In certain embodiments, R c and R d R is independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl). In certain embodiments, R c and R d R is independently hydrogen or methyl. In certain embodiments, R c and R d is hydrogen. In certain embodiments, R c and R d is methyl. In certain embodiments, R c is hydrogen, R d It is methyl.

[0010] In certain preferred embodiments, R 4 and R 5is independently hydrogen, methyl or ethyl. In certain embodiments, R 4 and R 5 are methyl.

[0011] In a preferred embodiment, one or both of L 1 and L 2 is an unsubstituted alkylene such as methylene (-CH2-) and ethylene (-CH2-CH2-).

[0012] In a further preferred embodiment, group A is a carbocyclic aryl such as phenyl, or a heteroaryl having one or more nitrogen ring members such as optionally substituted pyridinyl or optionally substituted pyrazinyl.

[0013] In certain embodiments, n can be 0, 1, 2, or 3, such as 0 or 1, or 1.

[0014] In certain preferred embodiments, compounds having the structure of formula (II) are provided.

Chemical formula

[0015] The compound can be a racemate comprising the following.

Chemical formula

[0016] The compound preferably has formula (IIA).

Chemical formula

[0017] In certain embodiments, R 1 is -CO(CH2) m N(R a )(R b ), wherein m is 0 or 1, preferably m is 0, and R a and R bis hydrogen or methyl. For example, R 1 is -CONH2 or -CONHCH3.

[0018] In certain embodiments, R 1 is -O(CH2) m N(R a )(R b ), where m is 1, 2, or 3, preferably m is 2, and R a and R b are hydrogen or methyl. For example, R 1 is -OCH2CH2N(CH3)2 or -OCH2CH2NHCH3.

[0019] In certain embodiments, R 1 is -CONH(CH2) m N(R a )(R b ), where m is 1, 2, or 3, preferably m is 2, and R a and R <000008​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ For example, R 1 is -CH2NH 2、 -CH2NH(CH3) or -CH2N(CH3)2.

[0022] In certain embodiments, R 1 is -O(CH2) m OH, m is 1, 2, or 3, and preferably m is 2. For example, R 1 is -OCH2CH2OH.

[0023] In certain preferred embodiments, compounds having the structure of formula (III) are provided,

Chemical Structure

[0024] The compound can be a racemate comprising the following.

Chemical Structure

[0025] The compound preferably has formula (IIIA).

Chemical Structure

[0026] In certain embodiments, R 1A is cyano (-CN) or unsubstituted alkyl such as methyl, and R 1B is C1-C6 alkoxy, preferably -OCH3.

[0027] In certain preferred embodiments, compounds having the following structure are provided.

Chemical Structure

[0028] The compound may be a racemic mixture containing the following: [ka]

[0029] The compound is preferably [ka] It has the structure of [the object].

[0030] In certain embodiments, compounds of the following formula (X): [ka] [In the formula, A is a C1-C6 alkyl, carboxyl(-C(O)O-), aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group. B is a carbocyclic aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, L 1 These are bonded, substituted, or unsubstituted C1-C6 alkylenes. L 2 These are bonded, substituted, or unsubstituted C1-C6 alkylenes or -S(O)2-, Each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 It is an unsubstituted C1-C6 alkyl, Each R 6 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, and heterocycloalkyl-C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m It is OH, or -O(Ph)X, 2 R 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a , R b , R c , and R dThese are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 12. m is an independent integer between 0 and 6. k is an integer between 0 and 12, independently. and pharmaceutically acceptable salts thereof are provided.

[0031] In certain preferred embodiments, A is phenyl, and L 1 ha-(CH2) p - and p is 1 to 4.

[0032] The compound has the following formula (XI).

[0033] [ka] B, R 1 ,NPR 2 , L 2 , R 6 And k are as defined above. n is an integer from 0 to 5.

[0034] In certain preferred embodiments, -L 2 -B- is [ka] That is the case.

[0035] The compound has the following formula (XI-a). [ka] R 1 ,NPR 2 , L 2 , R 6 It is defined as above. k is an integer from 0 to 5.

[0036] The compound has the following formula (XI-b). [ka] R 1 ,NPR 2 , L 2 , R 6 It is defined as above. k is an integer from 0 to 4.

[0037] The compound has the following formula (XI-c). [ka] R 1 ,NPR 2 , L 2 , R 6 It is defined as above. k is an integer between 0 and 3.

[0038] The compound has the following formula (XI-d). [ka] R 1 ,NPR 2 , L 2 , R 6 It is defined as above. k is an integer from 0 to 6.

[0039] In certain preferred embodiments, L 1 is a bond, methylene, or ethylene, and A is C 1-4 It is alkyl, cycloalkyl, or heterocycloalkyl.

[0040] In particular, in certain preferred embodiments, -L 1 -AR 1 teeth [ka] That is the case.

[0041] In certain preferred embodiments, the compound has the following formula (XII). [ka] R 1 ,NPR 2 , L 2 , R 6 As defined above, k is an integer between 0 and 5, and n is an integer between 0 and 5.

[0042] In certain embodiments, the compound of the following formula (XIII): [ka] [In the formula, Each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 It is an unsubstituted C1-C6 alkyl, Each R 6These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, and heterocycloalkyl-C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m It is OH, or -O(Ph)X, Two R's 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a ,R b ,R c , and R d These are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 5. m is an independent integer between 0 and 6. k is an independent integer between 0 and 5. p is an independent integer between 0 and 6. and pharmaceutically acceptable salts thereof are provided.

[0043] In certain embodiments, compounds of the following formula (XIV): [ka] [In the formula, each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 It is an unsubstituted C1-C6 alkyl, Each R 6 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, and heterocycloalkyl-C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(Rb ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m It is OH, or -O(Ph)X, Two R's 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a ,R b ,R c , and R d These are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 5. m is an independent integer between 0 and 6. k is an independent integer between 0 and 5. p is an independent integer between 0 and 6. and pharmaceutically acceptable salts thereof are provided.

[0044] In a preferred embodiment, each R a , R b , R c , and R d These are independently hydrogen, methyl, ethyl, propyl, or isopropyl.

[0045] In certain preferred embodiments, compounds having the following structure are provided. [ka]

[0046] The compound may be a racemic mixture containing the following: [ka]

[0047] The compound is preferably [ka] It has the structure of [the object].

[0048] Pharmaceutical compositions comprising the compound and methods for treating neurodegenerative disorders with the compound are also provided. In certain embodiments, a method for treating traumatic brain injury (TBI) in a patient is provided, the method comprising administering an effective amount of the compound or composition described herein to a patient in need thereof. In certain embodiments, a method is provided for treating a subject suffering from a disorder or condition associated with neuronal cell death by administering an effective amount of the compound or composition described herein to the subject. For example, the subject may be identified as suffering from certain diseases or disorders such as stroke, concussion, intracerebral hemorrhage, epilepsy, acute glaucoma, and spinal cord injury. The compound or composition is administered by a method selected from the group consisting of oral administration, intravenous injection, subcutaneous injection, intranasal delivery, or intracisional injection.

[0049] The treatment method generally involves administering an effective amount of one or more compounds disclosed herein to a target, such as a mammal, particularly a primate including humans. A suitable target may be identified and selected for treatment. Then, one or more compounds disclosed herein may be administered to the identified target.

[0050] Other aspects of the present invention are disclosed below. [Brief explanation of the drawing]

[0051] [Figure 1]The assay results demonstrating the in vitro selectivity of NA112 of calpain-2 over calpain-1 are shown. [Figure 2] The assay results demonstrating the in vivo selectivity of NA112 of calpain-2 over calpain-1 are shown. [Figure 3] This study demonstrates the in vivo efficacy of NA112 in DMSO solution administered 24 hours after intraperitoneal injection of NA112 at a dose of TBI + 0.1 mg / kg or 1.0 mg / kg. [Figure 4] The number of TUNEL-labeled degenerated cells in the ipsilateral brain was shown, obtained by intraperitoneal injection of the indicated dose of NA101(C2I) into WT mice 1 hour after TBI, and cell analysis 24 hours after TBI. [Figure 5] The number of TUNEL-labeled degenerated cells in the ipsilateral brain was shown, obtained by intraperitoneal injection of the indicated dose of NA112(C12) into WT mice 1 hour after TBI, and cell analysis 24 hours after TBI. [Figure 6] This demonstrates the stability of NA112 in mouse plasma. [Figure 7] This demonstrates the stability of NA112 in mouse liver homogenate with an estimated half-life. [Figure 8] This shows the activity of NA112A (SS isomer) against calpain-2 and calpain-1. [Figure 9] This indicates that NA112A (SS isomer) was inactivated in mouse plasma. [Figure 10A] The assay results show the in vitro selectivity of NA112 in ribosome formation of calpain-2 relative to calpain-1. [Figure 10B] For comparison with Figure 10A, the values ​​obtained using NA112 dissolved in DMSO are shown. [Figure 11] This demonstrates the in vivo efficacy of liposomal NA112 against DMSO. [Figure 12] The in vivo efficacy of NA112 in liposomes is demonstrated, and images from two different animals illustrate the reduction in TUNEL staining in NA112-treated mice. [Figure 13] A graph showing the number of TUNEL-positive cells in images similar to those shown in Figure 12 is presented. [Figure 14] This shows the changes in NA112 plasma concentration at various time points after intravenous injection. [Figure 15] This shows the changes in NA112 brain concentration at various time points after intravenous injection. [Figure 16] This shows the changes in NA112 plasma concentration using a two-compartment model. [Figure 17] The assay results for the selectivity of NA184 of calpain-2 over calpain-1 are shown. [Figure 18] This demonstrates the rapid epimerization of NA184A in PBS. [Figure 19] The results of NA184 IC50 for calpain-1 and calpain-2 activity in WT and calpain-1 KO mice are shown. [Figure 20] This figure shows the in vivo efficacy of intraperitoneal injection of NA184 at the indicated dose 1 hour after TBI+TBI in WT mice, as measured by TUNEL staining at 24 hours. [Figure 21] Figures 21A and 21B show that NA84 significantly inhibited calpain-2 equally well in male and female mice and rats under these conditions. [Figure 22] Figures 22A, 22B, and 22C show that NA184 significantly prevented cortical cell death when injected twice, 1 hour and 8 hours after TBI, and prevented cell death to a similar degree in male and female rats. [Figure 23] This study demonstrates a correlation between calpain-2 activity and brain cell death in rats. [Modes for carrying out the invention]

[0052] compound As will be discussed, in one embodiment, a compound of the following formula (I) is provided, [ka] In the formula, A, R 1 ,n,L 1 , R 2 , L 2 , R 4 and R 5 It is defined above. In certain embodiments, preferably R 1 Either does not exist (n is 0 and the A ring does not contain a non-hydrogen substituent), is alkyl, alkoxy or halogen, and A is a carbocyclic aryl such as phenyl or heteroaryl, L 1 and L 2 Each of these is an unsubstituted alkylene, particularly methylene (-CH2-), and R 4 and R 5 These are independently hydrogen or an unsubstituted C1-C6 alkyl such as methyl.

[0053] In certain preferred embodiments, R 4 and R 5 R is independently hydrogen, methyl, or ethyl. In certain embodiments, R 4 and R 5 It is methyl.

[0054] Exemplary Preferred AL 1 -The following are some of the basics: [ka]

[0055] The above also applies to other L 1 A preferred A group has a linker.

[0056] In a particular preferred embodiment, L 1 The chiral carbon closest to it has the (S) configuration. In certain embodiments, L 1 The chiral carbon closest to it has the (R) configuration.

[0057] In a particular preferred embodiment, L 2 The chiral carbon closest to it has the (S) configuration. In certain embodiments, L2 The chiral carbon closest to it has the (R) configuration.

[0058] The compounds of the present invention can be used as racemic mixtures or optically enriched mixtures.

[0059] Particularly preferred compounds of the present invention are compound analogs of NA112 that may have the following formula (II) or (III). [ka]

[0060] In certain aspects, R 1A R is a cyano or unsubstituted C1-C6 alkyl, 1B is a C1-C6 alkoxy. In certain embodiments, R 1A R is an unsubstituted alkyl such as cyano(-CN) or methyl, 1B The C1-C6 alkoxy is preferably -OCH3.

[0061] A particularly preferred compound, NA112, has the following structure. [ka]

[0062] In another embodiment, the compound of formula (X) [ka] [In the formula, A is a C1-C6 alkyl, carboxyl(-C(O)O-), aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group. B is a carbocyclic aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, L 1 These are bonded, substituted, or unsubstituted C1-C6 alkylenes. L 2These are bonded, substituted, or unsubstituted C1-C6 alkylenes or -S(O)2-, Each R 1 These are non-hydrogen substituents, such as C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, and heterocycloalkyl-C(O)(CH2). m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 This is a non-hydrogen substituent such as a C1-C6 alkyl group, which may be substituted depending on the circumstances. Each R 6 These are independently non-hydrogen substituents, such as C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, and heterocycloalkyl-C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(Rc )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m It is OH, or -O(Ph)X, Two R's 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a , R b , R c , and R d These are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is independently a value ranging from 0 (ring A is unsubstituted) to a value allowed by the valence of the ring, for example, 5 when A is phenyl. m is an independent integer between 0 and 6. k is an integer, independently ranging from 0 (ring B is unsubstituted) to a value allowed by the valence of the ring, e.g., 5, where B is phenyl. and pharmaceutically acceptable salts thereof are provided.

[0063] In certain embodiments, the compound of formula (X) may be a racemic mixture comprising the following: [ka] A, B, R 1 , R 2 , L 1 , L 2, R 6 n and k are as defined above.

[0064] In a particular embodiment, L 1 (For example, -(CH2) p The molecule is a substituted C1-C6 alkylene having p = 1 to 6, and each carbon atom may have 0, 1, or 2 nonhydrogen substituents. In a particular embodiment, L 2 This is an optionally substituted alkylene having 1 to 6 carbon atoms (e.g., -(CH2) p p is 1 to 6, and each carbon may have 0, 1 or 2 nonhydrogen substituents), or -S(O)2-.

[0065] In a particular embodiment, R 2 R is an unsubstituted C1-C6 alkyl group. For example, R 2 These are linear unsubstituted C1-C6 alkyl or branched C3-C6 alkyl, such as isopropyl, isobutyl, or t-butyl.

[0066] In certain aspects, R a and R b These are independently hydrogen, methyl, ethyl, propyl, or isopropyl.

[0067] In a particular embodiment, R c and R d These are independently hydrogen or methyl.

[0068] In certain preferred embodiments, R a and R b R is independently hydrogen or an unsubstituted C1-C6 alkyl group. In certain embodiments, R a and R b R is independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl). In certain embodiments, R a and R b R is independently hydrogen or methyl. In certain embodiments, R a and R bis hydrogen. In certain embodiments, R a and R b is methyl. In certain embodiments, R a is hydrogen, R b is methyl. In certain embodiments, R a and R b R is independently hydrogen or isopropyl. In certain embodiments, R a is hydrogen, R b It is isopropyl.

[0069] In certain preferred embodiments, R c and R d R is independently hydrogen or an unsubstituted C1-C6 alkyl group. In certain embodiments, R c and R d R is independently hydrogen, methyl, ethyl, or propyl (e.g., isopropyl). In certain embodiments, R c and R d R is independently hydrogen or methyl. In certain embodiments, R c and R d is hydrogen. In certain embodiments, R c and R d is methyl. In certain embodiments, R c is hydrogen, R d It is methyl.

[0070] In certain embodiments, A is phenyl, and L 1 ha-(CH2) p - and p is 0 to 6 (if p is 0, L 1 (This is a bond). Preferably, p is 1 to 6.

[0071] The compound may have formula (XI).

[0072] [ka] B, R 1 , p, R 2 , L 2 , R 6 And k are as defined above. n is an integer from 0 to 5.

[0073] In certain embodiments, the compound of formula (XI) may be a racemic mixture comprising the following: [ka] B, R 1 , R 2 , L 1 , L 2 , R 6 n, p, and k are as defined above.

[0074] In certain embodiments, -L 2 -B- is as follows: [ka]

[0075] The compound may have formula (XI-a). [ka] R 1 ,NPR 2 , and R 6 It is defined as above. k is an integer from 0 to 5.

[0076] In certain embodiments, the compound of formula (XI-a) may be a racemic mixture comprising the following: [ka] R 1 ,NPR 2 , k and R 6 It is defined as above.

[0077] In certain embodiments, n is 0 in formula (XI-a). For example, the compound is as follows: [ka] [ka] [ka] [ka] [ka]

[0078] In certain embodiments, n in formula (XI-a) is between 1 and 2. For example, the compounds are as follows: [ka] [ka] [ka] [ka]

[0079] The compound may have formula (XI-b). [ka] R 1 , p, R 2 and R 6 The definition is as shown above. n is an integer from 0 to 5, and k is an integer from 0 to 4.

[0080] In certain embodiments, the compound of formula (XI-b) may be a racemic mixture comprising the following: [ka] R 1 n, k, p, R 2 and R 6 It is defined as above.

[0081] For example, the compounds are as follows: [ka]

[0082] The compound may have formula (XI-c). [ka] R 1 ,NPR 2 , and R 6 It is defined as above. k is an integer between 0 and 3.

[0083] In certain embodiments, the compound of formula (XI-c) may be a racemic mixture comprising the following: [ka] R 1 n, k, p, R 2 and R 6 It is defined as above.

[0084] For example, compounds [ka] That is the case.

[0085] The compound may have formula (XI-d). [ka] R 1 ,NPR 2 , and R 6 It is defined as above. k is an integer from 0 to 6.

[0086] In certain embodiments, the compound of formula (XI-d) may be a racemic mixture comprising the following: [ka] R 1 n, k, p, R 2 and R 6It is defined as above.

[0087] For example, compounds [ka] That is the case.

[0088] In a particular embodiment, L 1 A is a bond, methylene or ethylene, and A is C 1-4 These are alkyl, cycloalkyl (e.g., adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) or heterocycloalkyl (e.g., 5-12 membered heterocycloalkylene).

[0089] In certain embodiments, -L 1 -AR 1 The following applies: [ka]

[0090] For example, the compounds are as follows: [ka] [ka] [ka]

[0091] In certain embodiments, -L 2 -B is [ka] That is the case.

[0092] In certain embodiments, the compound has formula (XII). [ka] R1 , p, R 2 and R 6 As defined above, n is an integer between 0 and 5, and k is an integer between 0 and 5.

[0093] In certain embodiments, the compound of formula (XII) may be a racemic mixture comprising the following: [ka] R 1 , n, p, k, R 2 and R 6 It is defined as above.

[0094] In a particular embodiment, two R 6 It combines with the atom it is bonded to to form a cycloalkyl or heterocycloalkyl group.

[0095] The compound of formula (XII) may have the following structure. [ka]

[0096] In one embodiment, the compound is given by formula (XIII), [ka] [In the formula, Each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c)(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 It is an unsubstituted C1-C6 alkyl, Each R 6 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, and heterocycloalkyl-C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m It is OH, or -O(Ph)X, Two R's 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a ,R b ,R c , and R d These are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 5. m is an independent integer between 0 and 6. k is an independent integer between 0 and 5. p is an independent integer between 0 and 6. and pharmaceutically acceptable salts thereof are provided.

[0097] In certain embodiments, the compound of formula (XIII) may be a racemic mixture comprising the following: [ka] R 1 n, k, p, R 2 and R 6 It is defined as above.

[0098] For example, the compounds are as follows: [ka]

[0099] In one embodiment, the compound is given by formula (XIV) [ka] [In the formula, Each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2)m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 It is an unsubstituted C1-C6 alkyl, Each R 6 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, and heterocycloalkyl-C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) mIt is OH, or -O(Ph)X, Two R's 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a , R b , R c , and R d These are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 5. m is an independent integer between 0 and 6. k is an independent integer between 0 and 5. p is an independent integer between 0 and 6. and pharmaceutically acceptable salts thereof are provided.

[0100] In certain embodiments, the compound of formula (XIV) may be a racemic mixture comprising the following: [ka]

[0101] For example, the compounds are as follows: [ka]

[0102] The preferred compound NA184 has the following structure. [ka]

[0103] The compound may be a racemic mixture containing the following: [ka]

[0104] A particularly preferred compound, NA184, is an SS isomer having the following structure. [ka]

[0105] These compounds may be calpain-2 selective inhibitors. As used herein, “calpain-2 selective inhibitor” or “selective calpain-2 inhibitor” refers to a compound having a calpain-2 inhibitory constant (Ki) lower than its Ki for calpain-1. For example, a calpain-2 selective inhibitor is a compound having a Ki for calpain-2 that is 2 to 10 times lower than its Ki for calpain-1. Preferably, the calpain-2 selective inhibitor is the IC of calpain-1 in an in situ assay. 50 ICs with a calpain-2 content 10 to 50 times lower than 50 It is a compound that has a value. For example, the IC of NA112 on the activity of in situ calpain-1 and calpain-2 activity. 50 The value was measured (Wang et al., 2014). IC 50 Calpain-1 / IC 50 The selectivity of NA112 for calpain-2, measured as a ratio of calpain-2, was approximately 13.

[0106] The compounds of the present invention include enantiomers, racemates, stereoisomers, and individual isomers having an asymmetric carbon atom (optical center or chiral center) and which can be defined as (R)- or (S)-isomers with respect to absolute stereochemistry, and these are encompassed within the scope of the present invention. The present invention means that the above-mentioned racemates and optically pure forms of the compounds are included. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents, or they can be decomposed using conventional techniques.

[0107] Unless otherwise specified, the structures shown herein also include all stereochemical forms of the structure, i.e., the R and S configurations of each chiral center. Therefore, single stereoisomers of the compound, as well as enantiomers and diastereomer mixtures, are within the scope of the present invention.

[0108] "Alkyl" is composed only of carbon and hydrogen atoms, and has 1 to 12 carbon atoms (C1-C 12 Alkyl groups refer to saturated linear or branched hydrocarbon chain radicals having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl) that are bonded to the rest of the molecule by single bonds. Examples of alkyl groups include methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, and 2-methylhexyl.

[0109] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon (alkyl) chain composed solely of carbon and hydrogen, linking the rest of the molecule to a radical group. Alkylenes can have 1 to 12 carbon atoms, such as methylene, ethylene, propylene, and n-butylene. Alkylene chains are bonded to the rest of the molecule via single or double bonds. The bonding points of the alkylene chain to the rest of the molecule can be via one carbon or any two carbons within the chain. "Optionally substituted alkylene" refers to alkylene or substituted alkylene.

[0110] "alkoxy" is the formula -OR a It refers to the base of R a This is an alkyl group having the number of carbon atoms shown above. Examples of alkoxy groups, but are not limited to, include -O-methyl (methoxy), -O-ethyl (ethoxy), -O-propyl (propoxy), and -O-isopropyl (isopropoxy).

[0111] "Cycloalkyl" and "heterocycloalkyl," either by themselves or in combination with other terms, mean the cyclic versions of "alkyl" and "heteroalkyl," respectively, unless otherwise specified. Cycloalkyl and heterocycloalkyl are not aromatic. Furthermore, in the case of heterocycloalkyl, the heteroatom can occupy a position where the heterocycle is bonded to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-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, and 2-piperazinyl. "Cycloalkylene" and "heterocycloalkylene" refer to divalent groups derived from cycloalkyl and heterocycloalkyl, respectively, either alone or as part of another substituent.

[0112] A "carbocyclic aryl" or "cycloalkyl" refers to a hydrocarbon ring radical that contains hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring, but the aromatic ring does not contain a hetero(N, O, or S) ring member. Exemplary carbocyclic aryls are hydrocarbon ring radicals containing hydrogen, 6 to 9 carbon atoms, and at least one aromatic ring; hydrocarbon ring radicals containing hydrogen, 9 to 12 carbon atoms, and at least one aromatic ring; hydrocarbon ring radicals containing hydrogen, 12 to 15 carbon atoms, and at least one aromatic ring; or hydrocarbon ring radicals containing hydrogen, 15 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, carbocyclic aryl radicals can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which may include condensed or bridging ring systems. Carbocyclic aryl radicals include, but are not limited to, carbocyclic aryl radicals derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, 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 carbocyclic aryl group.

[0113] Cycloalkyl is cycloalkenyl. The term "cycloalkenyl" is used according to its simple, ordinary meaning. In embodiments, a cycloalkenyl is a monocyclic, bicyclic, or polycyclic cycloalkenyl ring system. In embodiments, a monocyclic cycloalkenyl ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, such a group being unsaturated (i.e., containing at least one cyclic carbon-carbon double bond) but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, a bicyclic cycloalkenyl ring is a bridged monocyclic ring or a fused bicyclic ring. In embodiments, a bridged monocyclic ring contains a monocyclic cycloalkenyl ring, where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of 1 to 3 additional carbon atoms (i.e., form (CH2) w(The bridging group is w, where w is 1, 2, or 3). Typical examples of bicyclic cycloalkenyls include, but are not limited to, norborneyl and bicyclo[2.2.2]oct2 enyl. In embodiments, the condensed bicyclic cycloalkenyl ring system includes a monocyclic cycloalkenyl ring condensed to any of phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocyclyl, or monocyclic heteroaryl. In embodiments, the bridging or condensed bicyclic cycloalkenyl is bonded to the parent molecule via any carbon atoms contained within the monocyclic cycloalkenyl ring. In embodiments, the cycloalkenyl group is optionally substituted with one or two groups that are independently oxo or thia. In the embodiment, the polycyclic cycloalkenyl ring includes a monocyclic cycloalkenyl ring (base ring) condensed to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclil, or (ii) two ring systems independently selected from the group consisting of phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclil. In the embodiment, the polycyclic cycloalkenyl is bonded to the parent molecule via any carbon atom contained within the base ring. In the embodiment, the polycyclic cycloalkenyl ring includes a monocyclic cycloalkenyl ring (basic ring) condensed to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclil, or (ii) two ring systems independently selected from the group consisting of phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclil.

[0114] Heterocycloalkyls are heterocyclyls. As used herein, the term “heterocyclyl” means monocyclic, bicyclic, or polycyclic heterocycles. A 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, and the ring is saturated or unsaturated but not aromatic. A 3 or 4-membered ring contains one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring may contain 0 or 1 double bond and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. A 6 or 7-membered ring contains 0, 1, or 2 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. A heterocyclyl monocyclic heterocycle is bonded to its parent molecule via any carbon or nitrogen atom contained within the heterocyclyl monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include azetidinyl, azepanyl, azilidinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, This includes, but is not limited to, oxazolidinyl, piperadinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianil. A heterocyclyl bicyclic heterocycle is a monocyclic heterocycle condensed with any of phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocycle, or monocyclic heteroaryl. A heterocyclyl bicyclic heterocycle is bonded to the parent molecule via any carbon or nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.Representative examples of bicyclic heterocyclils include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl. In embodiments, the heterocyclil group is optionally substituted with one or two groups that are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or 6-membered monocyclic heterocyclyl ring condensed 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 with one or two groups that are independently oxo or thia. A polycyclic heterocyclyl ring system is a monocyclic heterocyclyl ring (base ring) condensed with either (i) one ring system selected from the group consisting of bicyclic aryls, bicyclic heteroaryls, bicyclic cycloalkyls, bicyclic cycloalkenyls, and bicyclic heterocyclyls, or (ii) two other ring systems independently selected from the group consisting of phenyls, bicyclic aryls, monocyclic or bicyclic heteroaryls, monocyclic or bicyclic cycloalkyls, monocyclic or bicyclic cycloalkenyls, and monocyclic or bicyclic heterocyclyls. The polycyclic heterocyclyl is bonded to the parent molecule via any carbon or nitrogen atom contained within the base ring. In the embodiment, the polycyclic heterocyclyl ring system is a monocyclic heterocyclyl ring (basic ring) condensed with either (i) one ring system selected from the group consisting of bicyclic aryls, bicyclic heteroaryls, bicyclic cycloalkyls, bicyclic cycloalkenyls, and bicyclic heterocyclyls, or (ii) two other ring systems independently selected from the group consisting of phenyls, monocyclic heteroaryls, monocyclic cycloalkyls, monocyclic cycloalkenyls, and monocyclic heterocyclyls.Examples of polycyclic heterocyclyl groups include, but are not limited to, 10H-phenothiazine-10-yl, 9,10-dihydroacridine-9-yl, 9,10-dihydroacridine-10-yl, 10H-phenoxazine-10-yl, 10,11-dihydro-5H-dibenzo[b,f]azepine-5-yl, 1,2,3,4-tetrahydropyrido[4,3-g]isoquinoline-2-yl, 12H-benzo[b]phenoxazine-12-yl, and dodecahydro-1H-carbazole-9-yl.

[0115] The terms "halo" or "halogen" mean a fluorine, chlorine, bromine, or iodine atom, either by itself or as part of another substituent, unless otherwise specified. Furthermore, terms such as "haloalkyl" include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, and 3-bromopropyl.

[0116] The term "aryl" means, unless otherwise specified, a polyunsaturated aromatic hydrocarbon substituent, which may be monocyclic, fused together (i.e., fused ring aryl), or covalently polycyclic (preferably 1 to 3 rings). A fused ring aryl refers to multiple fused rings, where at least one of the fused rings is an aryl ring.

[0117] The term "heteroaryl" refers to a 5-14 membered ring radical comprising a hydrogen atom, 1-13 carbon atoms, 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For the purposes of the present invention, a 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, and may contain 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. A heteroaryl may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, and may include condensed or bridging ring systems. The nitrogen, carbon, or sulfur atom in the heteroaryl radical may optionally be oxidized, and the nitrogen atom may optionally be quaternized. The heteroatoms may be members of an aromatic or non-aromatic ring, insofar as at least one ring in the heteroaryl is aromatic.Examples, though not limited to them, include: azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxolyl, benzofuranil, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanil, benzonaphthofuranil, benzoxazolyl, benzodioxolyl, benzodioxynil, benzopyranil, benzopyranonil, benzofuranil, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, sinnolinil, dibenzofuranil, dibenzothiophenyl, furanil, furanonil, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl This includes isoindolyl, indolinyl, isoindolyl, isoquinolyl, indolidinyl, isoxazolyl, naphthilidinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxyranil, 1-oxidepyridinyl, 1-oxidepyrimidinyl, 1-oxidepyradinyl, 1-oxidepyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxadinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinyl, pyrimidinyl, pyridadinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl).

[0118] symbol" [ka] The symbol '' indicates the bonding point of the chemical part to the rest of the molecule or chemical formula.

[0119] Various compounds and substituents that are "optionally substituted" or "substituted" may be appropriately substituted at one or more available positions by, but are not limited to, halogens (F, Cl, Br, I); nitro; hydroxy; amino; alkyls such as C1-C4 alkyls; alkenyls such as C2-C8 alkenyls; alkoxys, e.g., C1-C6 alkoxy; alkylaminos, e.g., C1-C8 alkylaminos; carbocyclic aryls such as phenyl, naphthyl, anthracenyl; heteroaryls, etc.

[0120] Compositions, pharmaceutical compositions and formulations The pharmaceutical composition of the present invention comprises NA112 or NA184 and a pharmaceutically acceptable excipient. The excipient used in the pharmaceutical composition of the present invention is safe and provides appropriate delivery of an effective amount of NA112 or NA184 for a desired route of administration.

[0121] The compounds of the present invention described above can be formulated as pharmaceutical dosage forms and administered to subjects in need of treatment, such as mammals including human patients, in various forms suitable for a selected route of administration. The compositions of the present invention can be administered by a variety of different methods, including oral administration, intravenous injection, intramuscular injection, subcutaneous injection, or intranasal delivery. For example, the compounds may be contained in solutions, suspensions, and other dosage forms suitable for intravenous or subcutaneous injection.

[0122] Solutions of the compounds of the present invention can be prepared in water or a physiologically acceptable buffer and may be mixed with a non-toxic surfactant, possibly containing cyclodextrin. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, liposomes, mixtures thereof, and oils. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth.

[0123] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the compounds of the present invention that are suitable for the immediate preparation of sterile injectable solutions or injectable solutions or dispersions. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers may be solvents or liquid dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Prevention of microbial action may be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Often, it is preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. Sustained absorption of the injectable composition may be achieved by the use of absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0124] The sterile injection solution is prepared by incorporating the required amount of the compound of the present invention, along with various other components listed above as needed, into a suitable solvent, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient and any additional desired components present in the previously sterile filtered solution.

[0125] Useful dosages of the compounds of the present invention can be determined by comparing their in vitro and in vivo activities in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art. See, for example, U.S. Patent No. 4,938,949. The amount of the compounds of the present invention required for therapeutic use will vary depending on the specific therapeutic agent, the composition if one contains the therapeutic agent, the route of administration, the nature of the condition being treated, and the patient's age and condition, and is ultimately at the discretion of the attending physician or clinician.

[0126] The therapeutically effective dose can be determined empirically by conventional procedures known to those skilled in the art. For example, see The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, the effective dose can be initially estimated using either a cell culture assay or a suitable animal model. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans. Therapeutic doses can also be selected in the same way as doses of equivalent therapeutic agents.

[0127] The specific mode of administration and medication regimen will be selected by the attending physician, taking into account the details of the case (e.g., the patient, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-day doses of the compound over a period of several days, several months, or even several years.

[0128] The term "pharmaceutically acceptable salt" means that, depending on the specific substituents found in the compounds described herein, salts of the active compound prepared with a relatively non-toxic acid or base may be obtained. If the compounds of this disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compound 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 or magnesium salts, or similar salts. If the compounds of this disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compound 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 such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monocarbonate, phosphoric acid, monohydrogen-phosphoric acid, dihydrogen-phosphoric acid, sulfuric acid, monohydrogen-sulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, and methanesulfonic acid. Salts of amino acids such as alginates, and salts of organic acids such as glucuronic acid or galacturonic acid are also included (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). The specific compounds of this disclosure contain both basic and acidic functional groups that enable the conversion of the compound into either a base addition salt or an acid addition salt.

[0129] Accordingly, the compounds of this disclosure may exist as salts with pharmaceutically acceptable acids, etc. This disclosure includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobroms, phosphates, sulfates, methanesulfons, nitrates, maleates, acetates, citrates, fumarates, propions, tartrates (e.g., (+)-tartrate, (-)-tartrate, 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, etc.). These salts may be prepared by methods known to those skilled in the art.

[0130] The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound by conventional methods. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0131] method The methods provided herein are methods for treating traumatic brain injury (TBI) in patients. A preferred method involves administering an effective amount of the compound or composition described herein to a patient who requires it.

[0132] In one embodiment, a method is provided for treating a subject suffering from a disorder or condition related to neuronal cell death, the method comprising administering an effective amount of the compound or composition described herein to the subject.

[0133] In certain embodiments, the subject is suffering from a stroke, concussion, intracerebral hemorrhage, acute glaucoma, seizure activity, and / or spinal cord injury.

[0134] In certain embodiments, a patient is identified as having or being susceptible to a disorder or condition related to neuronal cell death, and the compound is administered to the identified subject.

[0135] In certain embodiments, the compound or composition is administered by a method selected from the group consisting of oral administration, intravitreal injection, intraocular injection, intraocular irrigation, periorbital injection, and sub-Tenon's capsule injection.

[0136] In certain embodiments, the subject is a human being. [Examples]

[0137] Example 1: Synthesis of compound NA112 [ka] Compound NA110, (S)-2-(3-benzylureido)-N-((S)-1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide, can be synthesized according to Scheme 1. [ka]

[0138] Preparation of intermediate A [ka]

[0139] Preparation of 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid (Intermediate A) [ka]

[0140] Step 1: Preparation of tert-butyl(1-hydroxybutan-2-yl)carbamate [ka] 10 g of 2-aminobutan-1-ol was dissolved in 300 mL of chloroform and treated with 25 g of di-tert-butyl dicarbonate and 200 mL of sodium hydroxide solution (2 M). After stirring overnight at room temperature, the solvent was removed, and the residue was purified by flash chromatography (hexane / ethyl acetate 0-50%) to obtain tert-butyl(1-hydroxybutan-2-yl)carbamate (18 g, yield 86%).

[0141] Step 2: Preparation of tert-butyl(1-oxobutan-2-yl)carbamate [ka] DMSO (25 ml) was added at -78°C to a stirred solution of oxalyl chloride (13 ml) in CH2Cl2 (200 mL). After stirring for 10 minutes, tert-butyl(1-hydroxybutan-2-yl)carbamate (18 g) in CH2Cl2 (100 mL) was added dropwise, and the resulting mixture was stirred for 30 minutes. Next, Et3N (50 ml) was added, the reaction mixture was warmed to room temperature, and stirred for another 30 minutes. Then, water (200 mL) was added, the reaction mixture was extracted with CH2Cl2 (3 × 100 mL), the combined organic extract was dried, concentrated in vacuum to obtain a residue, which was purified by column silica gel chromatography (hexane / ethyl acetate 0-20%) to obtain tert-butyl(1-oxobutan-2-yl)carbamate (10.6 g, 58%).

[0142] Step 3: Preparation of tert-butyl(1-cyano-1-hydroxybutan-2-yl)carbamate [ka] 10.6 g of tert-butyl(1-oxobutan-2-yl)carbamate was dissolved in 200 mL of dioxane and cooled to 0°C for 10 minutes. At this point, 24 g of NaHSO3 in 100 mL of water was added. The reaction mixture was stirred at 0°C for 10 minutes, 13 g of KCN in 100 mL of water was added, and the solution was stirred overnight. The reaction mixture was diluted with 1 L of ethyl acetate, and the organic layer was work-treated by washing it three times with saturated sodium bicarbonate (3 × 100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to dryness to obtain 9.7 g of tert-butyl(1-cyano-1-hydroxybutan-2-yl)carbamate (80%).

[0143] Step 4: Preparation of methyl 3-amino-2-hydroxypentanoate [ka] 9.7 g of tert-butyl(1-cyano-1-hydroxybutan-2-yl)carbamate was dissolved in 200 ml of dry MeOH, and HCl gas (prepared by replacing the reaction H2SO4 with NaCl) was bubbling into the solution until the gas was absorbed / dissolved (4-5 hours, LC-MS control). The solution was then refluxed for approximately 16 hours, evaporated to dryness, and dried overnight under high vacuum. The crude product (8.5 g) was used in the next step without further purification.

[0144] Step 5: Preparation of methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoate [ka] Crude methyl 3-amino-2-hydroxypentanoate HCl salt (8.5 g) was suspended in acetonitrile (300 mL), treated with triethylamine (20 mL), HATU (19 g), and subsequently BOC-leucine hydrate (11.5 g), and the mixture was stirred overnight at room temperature (LC-MS control). Next, the mixture was evaporated, diluted with ethyl acetate (300 mL), and washed with 0.1 M aqueous HCl (2 × 100 mL). The organic matter was evaporated, and the crude product was purified by column silica gel chromatography (hexane / RINKAN, 0 to 30%) to obtain a mixture of four diastereomers. Yield 8.6 g (52%).

[0145] Step 6: Preparation of 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid (intermediate A) [ka] Methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoate (8.6 g) was dissolved in a mixture of 1 M NaOH (30 ml) and THF (30 ml) and stirred overnight (LC-MS control). The solution was then diluted with ethyl acetate (200 ml) and 0.5 M aqueous HCl (200 ml). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 × 50 ml). The combined extracts were dried (MgSO4), filtered, and evaporated to dryness to obtain 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid (6.62 g, yield 80%).

[0146] Preparation of (2S)-2-amino-N-(1-((2,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka]

[0147] Step 1: Preparation of tert-butyl((2S)-1-((1-(3,5-dimethoxybenzylamino)-2-hydroxy-1-oxopentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate [ka] 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid (6.62 g) was dissolved in acetonitrile (100 mL), treated with (3,5-dimethoxyphenyl)methaneamine (3.5 g), HATU (8.7 g), and DIPEA (4.6 ml), and stirred overnight at room temperature. The solution was evaporated to dryness, and the crude product was purified by column silica gel chromatography (hexane-ethyl acetate, 0-100%) to obtain tert-butyl((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (5.4 g, 57%).

[0148] Step 2: Preparation of (2S)-2-amino-N-(1-(3,5-dimethoxybenzylamino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (intermediate B) [ka] 5.4 g of tert-butyl((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate was dissolved in dioxane / HCl (50 mL, 4 M) and stirred at room temperature for 30 minutes. After removing the solvent and subsequently drying under vacuum, pure (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (5.2 g, 100%) was obtained.

[0149] Preparation of (2S)-2-(3-benzylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (compound NA112) [ka]

[0150] Step 1: Acylation of (2S)-2-amino-N-(1-(3,5-dimethoxybenzylamino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide: [ka] (2S)2-amino-N-(1-(3,5-dimethoxybenzylbenzylamino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (5.2 g) was suspended in acetonitrile (50 mL) and treated with benzyl isocyanate (2.5 g, 1.75 equivalents) and triethylamine (6 mL, 4 equivalents). The mixture was stirred at room temperature until LC-MS analysis indicated completion of the reaction. The solvent was evaporated to obtain a residue, which was purified by column silica gel chromatography to obtain the corresponding urea (4.2 g, 65% yield).

[0151] Step 2: Oxidation to (2S)-2-(3-benzylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (compound NA112) [ka] A hydroxy compound (4.2 g) was dissolved / suspended in dichloromethane (150 mL) and treated with Dess-Martin periodinane (6.7 g) with stirring at room temperature for 2 hours (no starting material was detected on LC). Next, the reaction mixture was partitioned into saturated bicarbonate solution (300 ml) and ethyl acetate (300 ml). The aqueous layer was extracted twice more with ethyl acetate (2 × 100 ml), and the combined organic layers were washed with water (100 ml), dried, filtered, concentrated, and dried. The residue was then purified by column chromatography to obtain 1.8 g of the target compound (approximately 70% LC-MS purity). This material was recrystallized from DCM (approximately 20 ml) to obtain 0.835 g (20% yield by LCMS, approximately 97.4% purity) of the target (2S)-2-(3-benzylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (compound NA112).

[0152] Example 2: Calpain assay selectivity - In vitro selectivity Cerebellar homogenates from wild-type (WT) mice or calpain-1 knockout (KO) mice were incubated with 20 μM calcium to activate calpain-1, or with 2 mM calcium to activate calpain-2, increasing the concentration of NA112 (12). Calpain activity was measured by cleavage of succinyl-Leu-Tyr-7-amino-4-methylcoumarin (Suc-Leu-Tyr-AMC) to obtain an increase in fluorescence. Activity was normalized to values ​​measured in the presence of a vehicle (DMSO). The graph in Figure 1 shows the assay results for the selectivity of NA112 of calpain-2 to calpain-1, and the IC of NA112 for calpain-1 and calpain-2 was obtained using the data from Figure 1. 50 I calculated it. [Table 1]

[0153] Example 3: Calpain assay selectivity - in vivo selectivity Calpain activity in the cerebellar P2 fraction was measured 24 hours after traumatic brain injury (TBI) in adult wild-type mice, and the assay results for calpain-1 and calpain-2 are shown in Figure 2. One hour after TBI, 0.1 mg / kg or 1 mg / kg of NA112 was administered intraperitoneally. In vivo selectivity for each measurement was obtained as follows: • Calpain-1 activity: 20 μM Ca 2+ Calpain activity in this case. Normalized for the vehicle. • Calpain-2 activity: 5 mM Ca 2+ Calpain activity minus 20 μM Ca 2+ Calpain activity. The results are the average of three experiments ± SEM.

[0154] Example 4: In vivo efficacy (DMSO solution) Quantification of TUNEL staining 24 hours after intraperitoneal injection of NA112 at TBI + 0.1 mg / kg or 1.0 mg / kg. The total number of TUNEL-positive cells in three coronal sections (bregma 0.50, -0.58, -1.58 mm) from each brain was counted and averaged. Results are mean ± SEM for three animals. *p<0.05 relative to vehicle. One-way ANOVA followed by Bonferroni test.

[0155] Example 5: Comparison of NA101 and NA112 WT mice were intraperitoneally injected with the prescribed dose of NA101 (C2I, Figure 4) or NA112 (C12; Figure 5) one hour after TBI. The number of TUNEL-labeled degenerated cells on the ipsilateral side of the brain was analyzed 24 hours after TBI.

[0156] The following structure shows compound NA101. [ka]

[0157] Example 6: Stability of NA112 in mouse plasma NA112 in β-cyclodextrin formulations showed good plasma stability with an estimated half-life of 17 hours (Figure 6). NA112 (C12, 0.2 mM) was incubated with mouse plasma at 37°C for the indicated period. Aliquots were taken, and the inhibition of purified human calpain-2 was measured. The results were normalized to the maximum inhibition (100%) measured at t0.

[0158] Example 7: Stability of NA112 in mouse liver homogenate NA112 in the β-cyclodextrin preparation showed good stability in mouse liver homogenate, and the estimated half-life of NA112 was 15 hours (Figure 7). Aliquots were taken and the inhibition of purified human calpain-2 was measured. The results were normalized to the maximum inhibition (100%) measured at t0.

[0159] Example 8: Separation of isomers and their activity Similar to NA101, NA112 contains two chiral centers. NA112A, in which chiral center 1 is the S-isomer and chiral center 2 is the S-isomer, was separated from the SR-isomer (NA112B) using a well-known method for separating diastereoisomers. [ka]

[0160] The inhibitory activity of compounds NA112A (SS isomer) and NA112B (SR stereoisomer) against calpain-1 and calpain-2 was determined. Compound NA112B (SR stereoisomer) did not exhibit inhibitory activity at the highest concentration tested, 3 μM. As shown in Figure 8, NA112A (SS isomer) showed the expected inhibitory activity against calpain-2 and calpain-1.

[0161] NA112A (SS stereoisomer) was incubated in mouse plasma at 37°C to determine whether it underwent epimerization and thus became inactive. As shown in Figure 9, NA112A was rapidly inactivated in mouse plasma. NA112A in a β-cyclodextrin preparation (10 μM) was incubated in mouse plasma at 37°C for the instructed period. Aliquots were taken and the inhibition of purified human calpain-2 was measured. The results were normalized to the maximum inhibition (50%) measured at t0.

[0162] Example 9: NA112-liposome formulation Liposomes were prepared as follows: Composition (for 1 ml liposome formulations) NA112-1mg Dimyristoylphosphatidylcholine (DMPC)-15mg 1,2-Dimiristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG-Na) - 5 mg Aqueous medium: Phosphate-buffered saline pH 6.8 - 1 ml

[0163] procedure 1) 2 mg of NA112, 30 mg of DMPC, and 10 mg of DMPG-Na were dissolved in chloroform (700 μL) and methanol (200 μL), the organic solvent was evaporated (this took 1.5 hours), and the mixture was left under vacuum overnight (16 hours). 2) 2 ml of PBS (pH 6.8) was added to the lipid film and vortexed to hydrate the phospholipids. 3) Using a probe sonicator (30% amplitude, 40s pulse), the sample was sonicated for 7 cycles with a cooling pad. (1 cycle: 30% amplitude, 40-second pulse). 4) Particle size, PDI, and zeta potential were measured. 5) The control was prepared as described without using NA112.

[0164] result The characteristics of liposome particles are shown below (Table 2). [Table 2] We determined various parameters that reflect the size of liposome particles.

[0165] Example 10: In vitro selectivity of NA112 in liposome formulations Various concentrations of NA112 in liposomal formulations were incubated with human calpain-1 (e-calpain-1 purified from red blood cells) or recombinant human calpain-2 (h-calpain-2), and calpain activity was assayed as shown in Figure 1. 50 The values ​​were determined and converted to Ki values ​​(Figures 10A and 10B). For comparison, previously obtained values ​​using NA112 dissolved in DMSO are shown (Figure 10B).

[0166] Example 11: In vivo efficacy of NA112 in liposome formulations Calpain activity in the cerebellar P2 fraction was measured 24 hours after TBI in adult WT mice. A vehicle dissolved in DMSO or liposomal formulation (Lipo) or 0.1 mg / kg or 1 mg / kg of NA112(12) was administered intraperitoneally 1 hour after TBI. In vivo selective measurements were obtained as follows: • Calpain-1 activity: 20 μM Ca normalized relative to the vehicle. 2+ Calpain activity. • Calpain-2 activity: 5 mM Ca 2+ Calpain activity minus 20 μM Ca 2+ Calpain activity was then normalized relative to the vehicle.

[0167] One hour after TBI in WT mice, 0.1 mg / kg or 1 mg / kg of NA112 (12) dissolved in a vehicle (DMSO or liposomes) or in DMSO or liposomal formulations (Lipo) was intraperitoneally injected. Calpain activity was measured in the cerebellar P2 fraction 24 hours later. Calpain-1 activity was measured in the presence of 20 μM calcium, and calpain-2 activity was measured as the difference between calpain activity measured in the presence of 5 mM calcium and calpain activity measured in the presence of 20 μM calcium (Figure 11). In both cases, calpain activity was normalized to the value measured in vehicle-treated mice. Results are mean ± SEM of 3 animals. Note that NA112 produced a similar degree of calpain-2 inhibition regardless of whether it was dissolved in DMSO or liposomes.

[0168] One hour after TBI in WT mice, 0.1 mg / kg or 1 mg / kg of NA112 dissolved in either a vehicle (control liposome) or a liposomal formulation was injected. The animals were sacrificed after 24 hours, and the brains were stained with TUNEL to analyze the degree of cell death. To illustrate the decrease in TUNEL staining in NA112-treated mice, images from two different animals are shown in Figure 12.

[0169] The number of TUNEL-positive cells in images similar to those in Figure 12 was quantified (Figure 13). The results in Figure 13 are the mean ± SEM values ​​from three different animals.

[0170] Example 12: PK test in liposome formulations NA112 was prepared in liposomes at a concentration of 1.5 mg / ml. Mice were injected into the tail vein with 200 μl of NA112 in liposomes (corresponding to a dose of 10 mg / kg), and sacrificed at the following time points: 1 min, 5 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, and 24 hours. Blood was collected, and plasma was rapidly prepared by centrifugation. Brain tissue was also collected. NA112 in plasma and brain homogenates was assayed by LC / Ms, with a sensitivity of 1 ng / ml. The experiment was performed in two series (Table 3, A1 and A2). Figures were created, and the results were averaged to calculate the half-lives of NA112 in plasma and brain.

[0171] [Table 3]

[0172] Figure 14 shows the changes in NA112 plasma concentration at various time points after intravenous injection, plotting the data from Table 3 as a function of time.

[0173] [Table 4]

[0174] Figure 15 shows the changes in NA112 brain concentration at various time points after intravenous injection. The data from Table 4 are plotted as a function of time. The curve does not readily fit a two-compartment model, but the slower component appears to have a half-life of approximately 3 hours.

[0175] [Table 5]

[0176] A two-compartment model was used to analyze the data from Figure 14. This assumes rapid distribution of the drug from plasma to various organs, followed by slower elimination of the drug from plasma. In this case, the curve fits well to the two-compartment model (Figure 16), which shows a plasma half-life of 7.8 hours. In Figure 16, the change in NA112 plasma concentration fit well to the two-compartment model.

[0177] The data in Figure 14 fits the two-compartment model equation and shows a very good fit between the predicted and observed values.

[0178] Example 13: Preparation of benzylamide analogs with diversity (right side) [ka] R 1 is the -OR of equation (I) 4 OR 5 That is R 2 R is in equation (I) 2 It is the same as n is an integer between 0 and 5. k is an integer between 1 and 5. Methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoate (previously described in International Publication No. 2020 / 037012) is deprotected by Boc to obtain methyl 3-((S)-2-(amino)-4-methylpentanamide)-2-hydroxypentanoate hydrochloride. The free base of the amino group is then reacted with (R2-substituted) benzyl isocyanate or an equivalent reagent to construct the desired methyl 3-((S)-2-(3-(R2-substituted) benzylureido)-4-methylpentanamide)-2-hydroxypentanoate. The methyl ester of this intermediate is hydrolyzed to the corresponding acid, 3-((S)-2-(3-(R2-substituted) benzylureido)-4-methylpentanamide)-2-hydroxypentanoic acid. Next, the acid is functionalized with a series of (R1-substituted)benzylamines to obtain the desired N-(R1-substituted)benzyl-3-((S)-2-(3-(R2-substituted)benzylureido)-4-methylpentanamide)-2-hydroxypentanamide, which is then oxidized at the secondary alcohol position to obtain the final product, N-(R1-substituted)-benzyl-3-((S)-2-(3-(R2-substituted)benzylureido)-4-methylpentanamide)-2-oxopentanamide.

[0179] Example 13-1 Step 1: Preparation of methyl 3-((S)-2-amino-4-methylpentanamide)-2-hydroxypentanoate [ka] 3.6 g of methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoate was dissolved in dioxane / HCl (50 mL, 4 M) and stirred at room temperature for 1 hour. After removing the solvent and subsequently drying under vacuum, pure methyl 3-((S)-2-amino-4-methylpentanamide)-2-hydroxypentanoate hydrochloride (3.2 g) was obtained. This material was used in the next step without further purification. LCMS[M+H]+=261.3

[0180] Step 2: Preparation of methyl 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoate [ka] 3.2 g of methyl 3-((S)-2-(amino)-4-methylpentanamide)-2-hydroxypentanoate hydrochloride (containing some dioxane from the previous step) was dissolved in 80 ml of CH3CN / THF (1 / 1) mixture, and 2 ml of Et3N was added all at once. 1.33 g (1.2 equivalents) of benzyl isocyanate was added all at once, and the reaction mixture was stirred at room temperature for 4 hours (LC-MS control). The mixture was evaporated to dryness, and the residue was dissolved in ethyl acetate (100 ml). The solution was washed with 0.5 M HCl (20 ml) and saturated NaHCO3 solution (20 ml). The organic matter was evaporated, and the crude material was purified by silica gel flash chromatography to obtain pure methyl 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoate (0.61 g, yield 39%) after evaporation. The material was used in the next step without further purification. LCMS[M+H] + =394.6

[0181] Step 3: Preparation of 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoic acid [ka] Methyl 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoate from the previous step was dissolved in THF / H2O / MeOH (1 / 1 / 0.5, 20 mL), and lithium hydroxide monohydrate (130 mg) was added. The mixture was stirred at room temperature for 6 hours, quenched to approximately pH 3 with 1 M HCl, and then extracted with ELISA (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated to dryness to obtain 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoic acid (0.51 g, approximately 90%) as colorless glass. LCMS[M+H] + =380.4. The material was used in the next process without further refinement.

[0182] Step 4: Preparation of (2S)-2-(3-benzylureido)-N-(1-((3-fluoro-2-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoic acid (113 mg, 1 equivalent) was dissolved in acetonitrile (5 mL), then (3-fluoro-2-methoxyphenyl)methaneamine (1.2 equivalents) was added, followed by HATU (170 mg, 1.5 equivalents) and DIPEA (100 μL). The reaction was completed after 24 hours (LC-MS), and the mixture was then quenched by adding saturated NaHCO3 solution (20 ml). The solution was extracted with ethyl acetate (3 × 10 mL). The combined organic layers were washed with 0.5 M aqueous HCl solution (2 × 5 ml), dried over Na2SO4, and concentrated to dryness. The crude residue (150 mg), (2S)-2-(3-benzylureido)-N-(1-((3-fluoro-2-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide, was used in the next step without further purification. LC-MS[M+H] + = 517.4

[0183] Step 5: Preparation of (2S)-2-(3-benzylureido)-N-(1-((3-fluoro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23482) [ka] Crude (2S)-2-(3-benzylureido)-N-(1-((3-fluoro-2-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (150 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Then, Dess Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 mL) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated to dryness. When the crude residue was subjected to reverse-phase HPLC purification, pure (2S)-2-(3-benzylureido)-N-(1-((3-fluoro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23482) was obtained. Yield after HPLC purification: 11.2 mg (14%) of NSN23482. LCMS[M+H]+=515.3

[0184] Example 13-2 Step 1: Preparation of (2S)-2-(3-benzylureido)-N-(2-hydroxy-1-((2-methoxy-3-methylbenzyl)-amino)-1-oxopentan-3-yl)-4-methylpentanamide [ka] 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoic acid (113 mg, 1 equivalent) was dissolved in acetonitrile (5 mL), then (2-methoxy-3-methylphenyl)-methanamine (1.2 equivalents) was added, followed by HATU (170 mg, 1.5 equivalents) and DIPEA (100 μL). After 24 hours, the reaction was complete (LC-MS). The mixture was quenched by adding saturated NaHCO3 solution (20 ml) and extracted with ethyl acetate (3 × 10 mL). The combined organic layer was washed with 0.5 M aqueous HCl solution (2 × 5 mL), dried over Na₂SO₄, and concentrated to dryness to obtain (2S)-2-(3-benzylureido)-N-(2-hydroxy-1-((2-methoxy-3-methylbenzyl)amino)-1-oxopentan-3-yl)-4-methylpentanamide. The crude product (150 mg) was used in the next step without further purification. LC-MS[M+H] + =513.1

[0185] Step 2: Preparation of (2S)-2-(3-benzylureido)-N-(1-((2-methoxy-3-methylbenzyl)-amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23482) [ka] Crude (2S)-2-(3-benzylureido)-N-(2-hydroxy-1-((2-methoxy-3-methylbenzyl)amino)-1-oxopentan-3-yl)-4-methylpentanamide (150 mg) from the previous step was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Then Des Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 mL). The organic layer was dried over Na2SO4 and concentrated to dryness. When the crude residue was subjected to reverse-phase HPLC purification, pure (2S)-2-(3-benzylureido)-N-(1-((2-methoxy-3-methylbenzyl)-amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23482) was obtained. Yield after HPLC purification: 31 mg (40%) of NSN23482. LCMS[M+H]+=511.2

[0186] Example 13-3 Step 1: Preparation of (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoic acid (113 mg, 1 equivalent) was dissolved in acetonitrile (5 mL), then (3-chloro-2-methoxyphenyl)methaneamine (1.2 equivalents) was added, followed by HATU (170 mg, 1.5 equivalents) and DIPEA (100 μg). After 24 hours, the reaction was complete (LC-MS). The mixture was quenched with saturated NaHCO3 solution (20 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic layers were washed with 0.5 M aqueous HCl solution (2 × 5 ml), dried over Na₂SO₄, and concentrated to dryness to obtain crude (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (150 mg), which was used in the next step without further purification. LCMS[M+H]+=533.5

[0187] Step 2: Preparation of (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NA184) [ka] Crude alcohol (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (150 mg) from the previous step was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Then Des Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 mL). The organic layer was dried over Na2SO4 and concentrated to dryness. Reverse-phase HPLC purification of the crude residue yielded pure (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NA184). Yield after HPLC purification: 51 mg (64%) of NA184. LCMS[M+H] + =531.1

[0188] Example 13-4 Step 1: Preparation of 2-(bromomethyl)-6-methylbenzonitrile [ka] 2,6-dimethylbenzonitrile (1.3 g) was dissolved in CCl4 (50 mL), and NBS (1.8 g) was added to the solution. The mixture was stirred overnight at room temperature (LC control). The mixture was diluted with dichloromethane (100 mL) and washed with saturated NaHCO3 solution (2 × 50 mL). The organic solution was evaporated, and the crude product was purified by flash (silica column) to obtain pure 2-(bromomethyl)-6-methylbenzonitrile (1.26 g, 60%).

[0189] Step 2: Preparation of 2-(azidomethyl)-6-methylbenzonitrile [ka] 1.26 g of 2-(bromomethyl)-6-methylbenzonitrile was dissolved in 30 mL of dry acetonitrile, and 1.2 g of sodium azide was added. The reaction mixture was heated under reflux for 4 hours (TLC control), diluted with 200 mL of cold water, and extracted with ethyl acetate (2 × 30 mL). The combined extract was evaporated to dryness to obtain 1.05 g of 2-(azidomethyl)-6-methylbenzonitrile (100%).

[0190] Step 3: Preparation of 2-(aminomethyl)-6-methylbenzonitrile [ka] 1.05 g of 2-(azidomethyl)-6-methylbenzonitrile was dissolved in 50 mL of methanol, and 1 g of 10% Pd / C (wet) was added. The suspension was stirred at room temperature for 1.5 hours under a hydrogen atmosphere (1.3 atm) (LC and TLC control). After all azides were consumed, the mixture was filtered through Celite and evaporated to dryness. The crude material was purified by flash silica column to obtain pure 2-(aminomethyl)-6-methylbenzonitrile (0.26 g, 30%).

[0191] Step 4: Preparation of (2S)-2-(3-benzylureido)-N-(1-((2-cyano-3-methylbenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] 3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-hydroxypentanoic acid (113 mg, 1 equivalent) was dissolved in acetonitrile (5 mL), then 2-(aminomethyl)-6-methylbenzonitrile (1.2 equivalents) was added, followed by HATU (170 mg, 1.5 equivalents) and DIPEA (100 μL). After 24 hours, the reaction was complete (LC-MS). The mixture was quenched with saturated NaHCO3 solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The combined organic layers were washed with 0.5 M aqueous HCl solution (2 x 5 ml), dried over Na2SO4, and concentrated to dryness to obtain crude (2S)-2-(3-benzylureido)-N-(1-((2-cyano-3-methylbenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (150 mg), which was used in the next step without further purification. LCMS[M+H]+=508.3

[0192] Step 5: Preparation of (2S)-2-(3-benzylureido)-N-(1-((2-cyano-3-methylbenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23500) [ka] Crude alcohol (2S)-2-(3-benzylureido)-N-(1-((2-cyano-3-methylbenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (150 mg) from the previous step was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Then Des Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated aqueous NaHCO3 (20 ml) and extracted with ethyl acetate (3 × 10 mL). The organic layer was dried over Na2SO4 and concentrated to dryness. When the crude residue was subjected to reverse-phase HPLC purification, pure (2S)-2-(3-benzylureido)-N-(1-((2-cyano-3-methylbenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23500) was obtained. Yield after HPLC purification: 28 mg (37%) NSN23500, LCMS[M+H] + = 506.5.

[0193] Example 13-5 The following analogues were prepared using general method #1: [ka] (2S)-2-(3-benzylureido)-N-(1-((2-methoxy-3-methylbenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-fluoro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((2-cyano-3-methylbenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1,2-dioxo-1-((4-(2-oxopyrrolidine-1-yl)benzyl)amino)pentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((4-(isopentyloxy)benzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(4-(1,2,3-thiadiazole-4-yl)benzyl)-3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-benzylureido)-4-methyl-N-(1-(((1-methyl-1H-indazole-6-yl)methyl)amino)-1,2-dioxopentan-3-yl)pentanamide [ka] (2S)-2-(3-benzylureido)-N-(1,2-dioxo-1-((2,4,6-trimethoxybenzyl)amino)pentan-3-yl)-4-methylpentanamide [ka] N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-(((2'-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((4-(tert-butyl)benzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-([1,1'-biphenyl]-4-ylmethyl)-3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(4-(1H-pyrazole-1-yl)benzyl)-3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-benzylureido)-4-methyl-N-(1-(((1-methyl-1H-benzo[d]imidazole-5-yl)methyl)amino)-1,2-dioxopentan-3-yl)pentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-fluorobenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((2-(difluoromethoxy)benzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-methylbenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-4-methyl-N-(1-((2-nitrobenzyl)amino)-1,2-dioxopentan-3-yl)pentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-(((2,3-dihydrobenzo[b][1,4]dioxin-5-yl)methyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((2,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((2,3-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3,4-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((2,4-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((4-fluoro-2-(trifluoromethyl)benzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((4-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-bromobenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((5-bromo-2-fluorobenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-bromo-4-fluorobenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-bromo-4-methylbenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((2-bromobenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((4-bromobenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-bromo-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3,4-diethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2R)-2-(3-benzylureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-(dimethylamino)benzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3-acetamidobenzyl)-3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((2-(dimethylamino)benzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-4-methyl-N-(1-((2-morpholinobenzyl)amino)-1,2-dioxopentan-3-yl)pentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((3-chloro-2-(cyclopropylmethoxy)benzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1,2-dioxo-1-((pyridine-3-ylmethyl)amino)pentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-(((6-methoxypyridine-3-yl)methyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1-((furan-2-ylmethyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1,2-dioxo-1-((((S)-tetrahydrofuran-2-yl)methyl)amino)pentan-3-yl)-4-methylpentanamide Benzylurea-arylsulfonamide analog [ka] (2S)-2-(3-benzylureido)-N-(1-((3-methoxyphenyl)sulfonamide)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-benzylureido)-N-(1,2-dioxo-1-(phenylsulfonamide)pentan-3-yl)-4-methylpentanamide [ka] N-(benzo[d][1,3]dioxol-5-ylsulfonyl)-3-((S)-2-(3-benzylureido)-4-methylpentanamide)-2-oxopentanamide

[0194] Example 14: Preparation of urea analogs with diversity (left side) [ka] R 1 is the -OR of equation (I) 4 OR 5 That is R 2 R is in equation (I) 2It is the same as n is an integer between 0 and 5. k is an integer between 1 and 5. Using a previously reported methodology (International Publication No. 2020 / 037012 pamphlet), commercially available 2-aminobutan-1-ol is converted to a key intermediate A, 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid. Intermediate A is functionalized at the acid terminus with a desired (R1-substituted) benzylamine, and then, after deprotection of the Boc-protecting amine, intermediate B, (2S)-2-amino-N-(1-((R1-substituted)benzylamino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide. Intermediate B is then converted to a series of ureas by reacting with a desired (R2-substituted) benzylamine-derived isocyanate or equivalent reagent. Each member of the series of N-(R1-substituted)-benzyl-3-((S)-2-(3-(R2-substituted)-benzylureido)-4-methylpentanamide)-2-hydroxypentanamide (intermediate C) formed in this manner is oxidized at the secondary hydroxyl group to obtain each final product, N-(R1-substituted)benzyl-3-((S)-2-(3-(R2-substituted)benzylureido)-4-methylpentanamide)-2-oxopentanamide, and product D.

[0195] Example 14-1 Step 1: Preparation of 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid [ka] Methyl 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoate (7.2 g) was dissolved in a mixture of 1 M NaOH (30 ml) and THF (60 ml) and stirred overnight (LC-MS control). The solution was diluted with ethyl acetate (300 ml) and 0.5 M HCl (300 ml). The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (3 × 50 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to dryness to obtain 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid (intermediate A) (6.5 g, yield 94%). LC-MS[M+H] + =347.3

[0196] Step 2: Preparation of tert-butyl((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate [ka] 3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamide)-2-hydroxypentanoic acid (6.5 g) was dissolved in acetonitrile (150 mL), treated with (3,5-dimethoxyphenyl)methaneamine (3.5 g), HATU (8.9 g), and DIPEA (11 mL), and stirred at room temperature for 1 hour (LC-MS control). The mixture was evaporated, extracted with ethyl acetate (200 mL), and washed with 0.5 M HCl (2 × 50 mL). The crude product was purified by flash chromatography (hexane-ethyl acetate, 0-100%) to obtain tert-butyl((2S)-1-((1-((3,5-di-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate as a colorless oil (4.7 g, 50%). LCMS[M+H] + =496.4

[0197] Step 3: Preparation of (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] 3.5 g of tert-butyl((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate was dissolved in dioxane / HCl (30 mL, 4 M) and stirred at room temperature for 1 hour. After removing the solvent and subsequently drying under vacuum, pure (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (2.9 g, 100%) was obtained. LCMS[M+H] + =396.2

[0198] Step 4: Preparation of methyl 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzoate [ka] (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (0.42 g, HCl salt) was dissolved in dioxane (10 mL), and Et3N (0.3 mL) and methyl 4-(isocyanatomethyl)benzoate (0.23 g) were added. The reaction mixture was stirred at room temperature for 4 hours, diluted with saturated NaHCO3 solution (20 mL), and extracted with ethyl acetate (2 × 10 mL). The organic matter was evaporated, and the crude product was purified by flash column chromatography to obtain methyl 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzoate (0.42 g, 72%).

[0199] Step 5: Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzoic acid [ka] Methyl 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-benzoate (0.42 g) was dissolved in a mixture of 1 M NaOH (5 mL) and THF (5 mL) and stirred overnight (LC-MS control). The solution was diluted with ethyl acetate (30 mL) and 0.5 M HCl (30 mL). The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to dryness to obtain 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-benzoic acid (0.3 g, yield 75%).

[0200] Step 6: Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-N-(2-(isopropylamino)ethyl)benzamide [ka] Dissolve 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzoic acid (100 mg, 1 equivalent) in acetonitrile (5 mL), N 1-Isopropylethane-1,2-diamine (1.2 equivalents) was added, followed by HATU (150 mg, 1.5 equivalents) and DIPEA (100 μL). After 24 hours, the reaction was complete (LC-MS). The reaction product was quenched by adding saturated NaHCO3 solution (20 ml), extracted with ethyl acetate (3 × 10 ml), dried over Na2SO4, concentrated, and dried. Crude 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-N-(2-(isopropylamino)ethyl)benzamide (100 mg) was used in the next step without further purification. LC-MS[M+H] + =657.7

[0201] Step 7: Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)-N-(2-(isopropylamino)ethyl)benzamide [ka] Crude 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-N-(2-(isopropyl-amino)ethyl)benzamide (100 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Dess Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by the addition of saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated. LCMS analysis showed a very troublesome over-oxidation reaction mixture. The desired product could not be recovered from the mixture.

[0202] Example 14-2: Alternative sequence of steps for generating abnormal cyclization products If the oxidation of a secondary alcohol occurs early in the process, unexpected and unusual cyclization products can be obtained.

[0203] Alternative step 3: Preparation of tert-butyl((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-1,2-dioxo-pentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate [ka] 1.3 g of tert-butyl((2S)-1-((1-((3,5-di-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-amino)-4-methyl-1-oxopentan-2-yl)carbamate was dissolved in 40 mL of CH3CN. Eight drops of water, 5 equivalents of pyridine, and 4 equivalents of DMSO were added, followed by the addition of 0.95 g (1 equivalent) of Des Martin periodinane in three portions over 1 hour. The reaction mixture was stirred at room temperature for 2 hours, quenched by the addition of 100 mL of saturated aqueous NaHCO3 solution, and extracted with ethyl acetate (3 × 50 mL). The organic layer was dried over Na2SO4 and concentrated to dryness. The crude residue was purified by silica column chromatography to obtain 0.96 g (78%) of pure tert-butyl((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-1,2-dioxo-pentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate. LCMC[M+H] + =494.5

[0204] Alternative step 4: Preparation of (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide and (6R)-N-(3,5-dimethoxybenzyl)-3-ethyl-6-isobutyl-5-oxo-3,4,5,6-tetrahydropyrazine-2-carboxamide [ka] 0.96 g of tert-butyl((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-1,2-dioxo-pentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate was dissolved in dioxane / HCl (15 mL, 4 M) and stirred at room temperature for 1 hour. After removing the solvent and subsequently drying under vacuum, a mixture of the two compounds (0.85 g) was obtained, as determined by LC-MS. The mixture was used in the next step. LC-MS[M+H] + =376.2 and 394.5

[0205] Alternative step 5: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-4-isobutyl-2,5-dioxoimidazolidin-1-yl)-2-oxopentanamide (NSN23499) and N-(3,5-dimethoxybenzyl)-3-ethyl-5-hydroxy-6-isobutylpyrazine-2-carboxamide (NSN23490) [ka] A mixture (0.85 g) of the two compounds ((2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)-amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide and (6R)-N-(3,5-dimethoxybenzyl)-3-ethyl-6-isobutyl-5-oxo-3,4,5,6-tetrahydropyrazine-2-carboxamide) from step 4 above was dissolved in CH3CN / dioxane 1 / 1, 50 mL and treated with Et3N (1.5 mL) and triphosgene (0.4 g). After stirring at room temperature for 0.5 hours, LC analysis showed the presence of two major products (A and B). The mixture was evaporated to dryness, dissolved in ethyl acetate (100 mL), and washed with saturated NaHCO3 (20 mL) and 0.1 M HCl (20 mL). The organic solution was evaporated and subjected to HPLC purification. Two products were isolated and characterized as N-(3,5-dimethoxybenzyl)-3-((S)-4-isobutyl-2,5-dioxoimidazolidine-1-yl)-2-oxopentanamide (NSN23499) and N-(3,5-dimethoxybenzyl)-3-ethyl-5-hydroxy-6-isobutylpyrazine-2-carboxamide (NSN23491). A 47mg, NSN23499;LCMS[M+H]+=420.5 B about 90mg, NSN23491;LCMS[M+H]+=374.4

[0206] Example 14-3 Preparation of N-(2-(diethylamino)ethyl)-4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzamide [ka] Dissolve 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzoic acid (100 mg, 1 equivalent) in acetonitrile (5 mL), N 1 ,N 1-Diethylethane-1,2-diamine (1.2 equivalents) was added, followed by HATU (150 mg, 1.5 equivalents) and DIPEA (100 μL). After 24 hours, the reaction was complete (LC-MS). The reaction product was quenched by adding saturated NaHCO3 solution (20 ml), extracted with ethyl acetate (3 × 10 ml), dried over Na2SO4, concentrated, and dried. Crude N-(2-(diethylamino)ethyl)-4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-benzamide (100 mg) was used in the next step without further purification. LC-MS[M+H] + =671.6

[0207] Preparation of N-(2-(diethylamino)ethyl)-4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)benzamide [ka] Crude N-(2-(diethylamino)ethyl)-4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzamide (120 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Dess Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated. The crude residue was subjected to reverse-phase HPLC purification to obtain pure N-(2-(diethylamino)-ethyl)-4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)benzamide. (2.3 mg, 3%); LCMS[M+H] + =669.6

[0208] Example 14-4: Preparation of sulfamoylamino analogs Step 1: Preparation of (2S)-2-((N-benzylsulfamoyl)amino)-N-(1-((3,5-dimethoxybenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (0.09 g, HCl salt) was dissolved in acetonitrile (10 ml), and Et3N (0.2 ml) and benzyl sulfamoyl chloride (60 mg) were added. The reaction mixture was stirred at room temperature for 4 hours, diluted with saturated NaHCO3 solution (20 ml), and extracted with ethyl acetate (2 × 10 ml). The organic matter was evaporated, and the crude (2S)-2-((N-benzyl sulfamoyl)-amino)-N-(1-((3,5-dimethoxybenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide was used in the next step without further purification. LCMS-565.5

[0209] Step 2: Preparation of (2S)-2-((N-benzylsulfamoyl)amino)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23501) [ka] (2S)-2-((N-benzylsulfamoyl)amino)-N-(1-((3,5-dimethoxybenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (150 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Des Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated to dryness. When the crude residue was subjected to reverse-phase HPLC purification, pure (2S)-2-((N-benzylsulfamoyl)amino)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide was obtained. (15 mg, 13%) NSN23501, LCMS[M+H]+=563.5

[0210] Example 14-5: Preparation of N-(3,5-dimethoxybenzyl)amide analogs Example 14-5-1: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)benzyl)ureido)-4-methylpentanamide)-2-hydroxypentanamide Step 1: Preparation of tert-butyl(4-(2-(dimethylamino)ethoxy)benzyl)carbamate [ka] 2-(4-(aminomethyl)phenoxy)-N,N-dimethylethane-1-amine (0.39 g) was dissolved in THF (5 ml), and anhydrous Boc (0.45 g) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness. The residue was purified by column silica gel chromatography to obtain tert-butyl (4-(2-(dimethylamino)ethoxy)benzyl)carbamate (0.49 g, 83%). The material was used without further purification.

[0211] Step 2: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)-ethoxy)benzyl)ureido)-4-methylpentanamide)-2-hydroxypentanamide [ka] Using the procedure for Boc-protected amines described by Kim and Lee (Tetrahedron Letters 57(2016)4890-4892; Spyropoulos and Kokotos; see also J.Org.Chem.2014,79,4477-4483), tert-butyl(4-(2-(dimethylamino)ethoxy)benzyl)carbamate (1.0 mmol) and 2-chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml). Anhydrous triflulinic acid (1.5 mmol) was added dropwise over 5 minutes. After stirring at room temperature for 1 hour, (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (0.3 mmol) and triethylamine (3.0 mmol) were added to the resulting mixture. After stirring for another hour (LC control), the mixture was diluted with water (20 ml), the layers were separated, and the aqueous layer was extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Crude N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)-benzyl)ureido)-4-methylpentanamide)-2-hydroxypentanamide (approximately 150 mg) was used in the next step without further purification. LCMS[M+H] + =616.6

[0212] Step 3: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)-benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide (NSN23483) [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)benzyl)ureido)-4-methylpentanamide)-2-hydroxypentanamide (150 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Dess Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated aqueous NaHCO3 (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated to dryness. When the crude residue was subjected to reverse-phase HPLC purification, pure N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)-benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide (15 mg, 12%) (NSN23483) was obtained. LCMS[M+H]+=614.2

[0213] Example 14-5-2: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-((dimethylamino)-methyl)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide Step 1: Preparation of tert-butyl(4-((dimethylamino)methyl)benzyl)carbamate [ka] 1-(4-(aminomethyl)phenyl)-N,N-dimethylmethaneamine (0.33 g) was dissolved in THF (5 ml), and anhydrous Boc (0.45 g) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness. The residue was purified by column silica gel chromatography to obtain pure tert-butyl (4-((dimethylamino)methyl)benzyl)carbamate (0.45 g, 85%), which was used directly in the next step.

[0214] Step 2: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-((dimethylamino)methyl)benzyl)ureido)-4-methylpentanamide)-2-hydroxypentanamide [ka] Boc-protecting amine, tert-butyl (4-((dimethylamino)methyl)benzyl)carbamate (1.0 mmol), and 2-chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml). Anhydrous triflulinic acid (1.5 mmol) was added dropwise over 5 minutes. After stirring at room temperature for 1 hour, (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (0.3 mmol) and triethylamine (3.0 mmol) were added to the resulting mixture. After stirring for a further 1 hour (LC control), the mixture was diluted with water (20 ml), the layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Crude N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-((dimethylamino)methyl)benzyl)ureido)-4-methylpentanamide)-2-hydroxypentanamide (approximately 150 mg) was used in the next step without further purification.

[0215] Step 3: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-((dimethylamino)methyl)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-((dimethylamino)methyl)benzyl)ureido)-4-methylpentanamide)-2-hydroxypentanamide (150 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Dess Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated aqueous NaHCO3 (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated. When the crude residue was subjected to reverse-phase HPLC purification, pure N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-((dimethylamino)methyl)-benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide (6 mg, 5%) was obtained. LCMS[M+H]+=584.4

[0216] Example 14-6: Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)-N-isopropylbenzamide (NSN23488) Step 1: Preparation of tert-butyl(4-(isopropylcarbamoyl)benzyl)carbamate [ka] 4-(aminomethyl)-N-isopropylbenzamide (0.38 g) was dissolved in THF (5 ml), and Boc anhydrous (0.45 g) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness. The residue was purified by column silica gel chromatography to obtain tert-butyl(4-(isopropylcarbamoyl)benzyl)carbamate (0.43 g, 74%), which was used directly in the next step.

[0217] Step 2: Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-N-isopropylbenzamide [ka] 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). Anhydrous triflric acid (1.5 mmol) was added dropwise over 5 minutes. After stirring at room temperature for 1 hour, (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (0.3 mmol) and triethylamine (3.0 mmol) were added to the reaction mixture. After stirring for a further 1 hour (LC control), the mixture was diluted with water (20 ml), the layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Crude 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-N-isopropylbenzamide (approximately 150 mg) was used in the next step without further purification.

[0218] Step 3: Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)-N-isopropylbenzamide (NSN23488) [ka] 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)-N-isopropylbenzamide (150 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Dess Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated to dryness. The crude residue was subjected to reverse-phase HPLC purification to obtain pure 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)-N-isopropylbenzamide (18 mg, 15%). NSN23488

[0219] Example 14-7: Preparation of (2S)-2-(3-(4-cyano-2-methoxybenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23489) Step 1: Preparation of tert-butyl(4-cyano-2-methoxybenzyl)carbamate [ka] 4-(aminomethyl)-3-methoxybenzonitrile (0.32 g) was dissolved in THF (5 ml), and anhydrous Boc (0.45 g) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness. The residue was purified by column silica gel chromatography to obtain tert-butyl(4-cyano-2-methoxybenzyl)carbamate (0.41 g, 78%), which was used directly in the next step.

[0220] Step 2: Preparation of (2S)-2-(3-(4-cyano-2-methoxybenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] Boc-protecting amine, tert-butyl(4-cyano-2-methoxybenzyl)carbamate (1.0 mmol), and 2-chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml). Anhydrous triflulinic acid (1.5 mmol) was added dropwise over 5 minutes. After stirring at room temperature for 1 hour, (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (0.3 mmol) and triethylamine (3.0 mmol) were added to the reaction mixture. After stirring for a further 1 hour (LC control), the mixture was diluted with water (20 ml), the layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to dryness under reduced pressure. Crude (2S)-2-(3-(4-cyano-2-methoxybenzyl)ureido)-N-(1-((3,5-di-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (approximately 150 mg) was used in the next step without further purification.

[0221] Step 3: Preparation of (2S)-2-(3-(4-cyano-2-methoxybenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23489) [ka] (2S)-2-(3-(4-cyano-2-methoxybenzyl)ureido)-N-(1-((3,5-di-methoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (150 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Des Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated. Reverse-phase HPLC purification of the crude residue yielded pure (2S)-2-(3-(4-cyano-2-methoxybenzyl)ureido)-N-(1-((3,5-di-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (15 mg, 13%). NSN23489. LCMS[M+H] + = 582.5

[0222] Example 14-8: Preparation of n-(3,5-dimethoxybenzyl)-3-((s)-2-(3-(2-methoxy-4-methylbenzyl)-ureido)-4-methylpentanamide)-2-oxopentanamide (NSN23490) Step 1: Preparation of tert-butyl(2-methoxy-4-methylbenzyl)carbamate [ka] (2-methoxy-4-methylphenyl)methaneamine (0.3g) was dissolved in THF (5ml), and Boc anhydrous (0.45g) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness. The residue was purified by column silica gel chromatography to obtain tert-butyl (2-methoxy-4-methylbenzyl)carbamate (0.39g, 78%), which was used directly in the next step.

[0223] Step 2: Preparation of N-(3,5-dimethoxybenzyl)-2-hydroxy-3-((S)-2-(3-(2-methoxy-4-methylbenzyl)ureido)-4-methylpentanamide)pentanamide [ka] Boc-protecting amine, tert-butyl(2-methoxy-4-methylbenzyl)carbamate (1.0 mmol), and 2-chloropyridine (3.0 mmol) were dissolved in dry dichloromethane (20 ml). Anhydrous triflulinic acid (1.5 mmol) was added dropwise over 5 minutes. After stirring at room temperature for 1 hour, (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (0.3 mmol) and triethylamine (3.0 mmol) were added to the reaction mixture. After stirring for a further 1 hour (LC control), the mixture was diluted with water (20 ml), the layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Crude N-(3,5-dimethoxybenzyl)-2-hydroxy-3-((S)-2-(3-(2-methoxy-4-methylbenzyl)ureido)-4-methylpentanamide)pentanamide (approximately 120 mg) was used in the next step without further purification.

[0224] Step 3: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(2-methoxy-4-methylbenzyl)-ureido)-4-methylpentanamide)-2-oxopentanamide (NSN23490) [ka] N-(3,5-dimethoxybenzyl)-2-hydroxy-3-((S)-2-(3-(2-methoxy-4-methylbenzyl)ureido)-4-methylpentanamide)pentanamide (120 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Dess Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated. The crude residue was subjected to reverse-phase HPLC purification to obtain pure N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(2-methoxy-4-methylbenzyl)-ureido)-4-methylpentanamide)-2-oxopentanamide (3 mg, 3%). NSN23490, LCMS[M+H] + = 571.3

[0225] Example 14-9: Preparation of (2S)-2-(3-(4-cyanobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23492) Step 1: Preparation of tert-butyl(4-carbamoylbenzyl)carbamate [ka] 4-(aminomethyl)benzamide (0.3 g) was dissolved in THF (5 ml), and anhydrous Boc (0.45 g) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness. The residue was purified by column silica gel chromatography to obtain tert-butyl(4-carbamoyl-benzyl)carbamate (0.43 g, 86%), which was used directly in the next step.

[0226] Step 2: Preparation of 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzamide and (2S)-2-(3-(4-cyanobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] To a solution of DCM (5 mL) and THF (5 mL) containing 4-carbamidobenzylamine (300 mg), triphosgene (300 mg) and triethylamine (0.8 mL) were added. The mixture was stirred at room temperature for 30 minutes. Next, (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (200 mg) was added, and the reaction mixture was stirred at room temperature for 45 minutes. LC analysis of the reaction mixture showed the presence of a portion (approximately 5%) of the desired carboxamide product [4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-9-hydroxy-5-isobutyl-3,6,10-trioxo-2,4,7,11-tetraazadodecyl)benzamide], but the main compound was the corresponding cyano derivative (approximately 50%), which is the dehydrated product of carboxamide [(2S)-2-(3-(4-cyanobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide]. The mixture was diluted with water (20 ml), the layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude material (approximately 120 mg) was used in the next step without further purification.

[0227] Step 3: Preparation of (2S)-2-(3-(4-cyanobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)-amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (NSN23492) [ka] A mixture (120 mg) from the previous step, mainly containing (2S)-2-(3-(4-cyanobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide, was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Des Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic layer was dried over Na2SO4 and concentrated to dryness. When the crude residue was subjected to reverse-phase HPLC purification, pure (2S)-2-(3-(4-cyanobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)-amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide (12 mg, 11%) was obtained. NSN23492, LCMS[M+H] + = 552.4

[0228] Example 14-10: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-hydroxyethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide Step 1: Preparation of 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzonitrile [ka] 4-hydroxybenzonitrile (1.2 g) was dissolved in DMF (15 mL), and K2CO3 (2.5 g) and (2-bromoethoxy)-(tert-butyl)dimethylsilane (2.4 g) were added. The reaction mixture was stirred at 50°C for 2 hours, at which point an additional 2.4 g of (2-bromoethoxy)(tert-butyl)dimethylsilane was added. Stirring was continued at 50°C for another 1 hour (LC control), the reaction mixture was cooled to room temperature and diluted with water. The mixture was extracted three times with RINKAN, the organic layer was washed five times with saturated aqueous NaCl, then dried over Na2SO4, concentrated and dried. The crude residue was loaded dry onto silica gel and purified by flash column chromatography to obtain 4-(2-((tert-butyldimethylsilyl)oxy)-ethoxy)-benzonitrile (2.3 g, yield 83%) as colorless crystals.

[0229] Step 2: Preparation of (4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)phenyl)methylamine [ka] 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzonitrile (2.3 g) was dissolved in THF and cooled to 0°C. 15 mL of LiAlH4 (1 M in Et2O) was added dropwise. The reaction mixture was held at 0°C for 45 minutes and quenched by slowly adding saturated sodium sulfate aqueous solution, followed by Et2O. The solid was filtered off, the filtrate was concentrated and purified by flash chromatography to obtain (4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)phenyl)methanamine (1.5 g, yield 64%).

[0230] Step 3: Preparation of tert-butyl(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzyl)carbamate [ka] (4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)phenyl)methaneamine (0.56 g) was dissolved in THF (5 ml), and Boc anhydrous (0.45 g) was added. The mixture was stirred at room temperature for 2 hours and evaporated to dryness. The residue was purified by column silica gel chromatography to obtain tert-butyl(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzyl)carbamate (0.71,93%), which was used directly in the next step.

[0231] Step 4: Preparation of (2S)-2-(3-(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide [ka] Boc-protecting 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). Anhydrous triflulinic acid (1.5 mmol) was added dropwise over 5 minutes. After stirring at room temperature for 1 hour, (2S)-2-amino-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide hydrochloride (0.3 mmol) and triethylamine (3.0 mmol) were added to the reaction mixture. After stirring for a further 1 hour (LC control), the mixture was diluted with water (20 ml), the layers were separated, and the aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Crude (2S)-2-(3-(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (approximately 200 mg) was used in the next step without further purification.

[0232] Step 5: Preparation of N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-hydroxyethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)-amino)-2-hydroxy-1-oxopentan-3-yl)-4-methylpentanamide (200 mg) was dissolved in CH3CN (10 mL), and one drop of water, pyridine (150 μL), and DMSO (100 μL) were added. Next, Des Martin periodinane (150 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was quenched by adding saturated NaHCO3 aqueous solution (20 ml) and extracted with ethyl acetate (3 × 10 ml). The organic matter was evaporated to dryness to obtain the ketone. LCMS[M+H] + =701.7. Crude ketone was dissolved in THF (5 ml). TBAF (0.5 ml, 1 M in THF) was added to this solution, and the mixture was stirred overnight at room temperature (LC control for TBS deprotection). After deprotection was complete, the mixture was diluted with water (20 ml) and extracted with ethyl acetate (3 × 20 ml). The organic matter was evaporated to dryness. When the crude residue was subjected to reverse-phase HPLC purification, pure N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-hydroxyethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide (3 mg, 3%) was obtained. NSN23498, LCMS[M+H]+=587.6

[0233] Examples 14-11: Additional analogues prepared by Example 14 Using the strategies illustrated in General Method #2 and Examples, the following further analogues were prepared: [ka] (2S)-2-(3-benzylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)-N-(2-(isopropylamino)ethyl)benzamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] 4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)-N-isopropylbenzamide [ka] (2S)-2-(3-(4-cyano-2-methoxybenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(2-methoxy-4-methylbenzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-hydroxyethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(4-cyanobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-((dimethylamino)methyl)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(2-(diethylamino)ethyl)-4-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)benzamide [ka] (2S)-2-(3-(2,4-dichlorobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-(3-chlorobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-4-methyl-2-(3(4-methylbenzyl)ureido)pentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(3,4-dichlorobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(2-fluorobenzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-isopropylureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-((3R,5R,7R)-adamantan-1-yl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-(tert-butyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-(4-(4-chlorophenoxy)benzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] tert-butyl(3-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)phenyl)carbamate [ka] tert-butyl(3-((5S)-12-(3,5-dimethoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)benzyl)carbamate [ka] (2S)-2-(3-(2-(difluoromethoxybenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-(2-chloro-6-fluoro-3-methylbenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(2-fluoro-5-(trifluoromethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(2-methoxy-5-(trifluoromethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(6-chloro-2-fluoro-3-methylbenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-(2-bromo-5-fluorobenzyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-methoxycarbonylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-cyclohexylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] Ethyl (((2S)-1-((1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)glycinate [ka] Ethyl(8S)-1-(3,5-dimethoxyphenyl)-5-ethyl-8-isobutyl-3,4,7,10-tetraoxo-2,6,9,11-tetraazatetradecane-14-oate [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-ethylureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-4-methyl-2-(3-((tetrahydro-2H-pyran-4-yl)methyl)ureido)pentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-4-methyl-2-(3-neopentylureido)pentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(cyclopentylmethyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-4-methyl-2-(3-(1-(methylsulfonyl)piperidine-4-yl)ureido)pentanamide)-2-oxopentanamide [ka] (2S)-2-(3-cyclopropylureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((2S)-4-methyl-2-(3-(tetrahydrofuran-3-yl)ureido)pentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3(3-methoxypropyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(cyclohexylmethyl)ureido)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-4-methyl-2-(3-(pyridine-3-yl)ureido)pentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-4-methyl-2-(3-(2-morpholinoethyl)ureido)pentanamide)-2-oxopentanamide [ka] N-(3-chloro-2-methoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] 3-(4-((5S)-12-(3-chloro-2-methoxyphenyl)-8-ethyl-5-isobutyl-3,6,9,10-tetraoxo-2,4,7,11-tetraazadodecyl)phenyl)propanoic acid [ka] N-(3-chloro-2-methoxybenzyl)-3-((S)-2-(3-(cyclohexylmethyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3-chloro-2-methoxybenzyl)-3-((S)-4-methyl-2-(3-(2-morpholinoethyl)ureido)pentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(2-(azepan-1-yl)ethyl)ureido)-N-(1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] N-(3-chloro-2-methoxybenzyl)-3-((S)-4-methyl-2-(3-phenethylureido)pentanamide)-2-oxopentanamide [ka] N-(3-chloro-2-methoxybenzyl)-3-((S)-4-methyl-2-(3-(2-(tetrahydro-2H-pyran-4-yl)ethyl)ureido)pentanamide)-2-oxopentanamide [ka] N-(3-chloro-2-methoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)phenyl)propanamide)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3-chloro-2-methoxybenzyl)-3-((S)-4-methyl-2-(3-phenylpropanamide)pentanamide)-2-oxopentanamide [ka] N-(3,4-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)benzyl)ureido)-4-methylpentanamide)-2-oxopentanamide [ka] (2S)-2-(3-(cyclohexylmethyl)ureido)-N-(1-((3-fluoro-5-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-(3-(cyclohexylmethyl)ureido)-N-(1-((3,4-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide

[0234] Non-ureid analogues [ka] N-(3,4-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)phenyl)propanamide)-4-methylpentanamide)-2-oxopentanamide [ka] N-(3,5-dimethoxybenzyl)-3-((S)-2-(3-(4-(2-(dimethylamino)ethoxy)phenyl)propanamide)-4-methylpentanamide)-2-oxopentanamide

[0235] N-(benzylsulfamoyl)amino analog [ka] (2S)-2-((N-benzylsulfamoyl)amino)-N-(1-((3,5-dimethoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide [ka] (2S)-2-((N-benzylsulfamoyl)amino)-N-(1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide

[0236] Example 15: Calpain assay selectivity - In vitro selectivity Cerebellar homogenates derived from wild-type (WT) mice or calpain-1 knockout (KO) mice were incubated with 20 μM calcium to activate calpain-1, or with 2 mM calcium to activate calpain-2, increasing the concentration of NA184. The graph in Figure 17 shows the assay results for the selectivity of calpain-2 to calpain-1, and the IC50 of NA184 to calpain-1 and calpain-2 was determined using the data from Figure 17. 50 The following was calculated (Table 6). [Table 6]

[0237] Similar to NA112, NA184 contains two chiral centers. The inhibitory activity of compounds NA184A (SS isomer) and NA184B (SR stereoisomer) against erythrocyte human calpain-1 (e-calpain-1) and recombinant human calpain-2 (h-calpain-2) was determined. Compound NA184B (SR stereoisomer) showed no inhibitory activity at the highest concentration tested, 3 μM. NA184A (SS isomer) showed the expected inhibitory activity against calpain-2 and calpain-1. NA184A (SS stereoisomer) was incubated in PBS at 37°C to determine if it underwent epimerization and thus became inactive. As shown in Figure 18, NA184A was rapidly inactivated in PBS.

[0238] Example 16: Calpain assay selectivity - in vivo selectivity WT or calpain-1 KO mouse cerebellar P2 homogenate (crude synaptosome fraction) was used to measure the inhibitory activity of compounds against endogenous mouse calpain-1 / -2. Each reaction involved 100 μm of WT or calpain-1 KO mouse cerebellar P2 homogenate + 0, 20, or 2000 μM of Ca 2+ It contains NA184 at a concentration of +0 to 10,000 nM. Calpain-1 activity = 20 μM Ca in WT mice 2+ Calpain activity under the following conditions Calpain-2 activity = 2000 μM Ca in calpain-1 KO mice 2+ Calpain activity under the following conditions

[0239] The results are shown in Figure 19, and the IC50 shown in Table 7 was calculated using the data. [Table 7]

[0240] Example 17: In vivo efficacy (DMSO solution) Quantification of TUNEL staining in WT mice after intraperitoneal injection of NA184 at the indicated dose 1 hour after TBI and 24 hours after TUNEL staining. The total number of TUNEL-positive cells in three coronal sections (bregma 0.50, -0.58, -1.58 mm) from each brain was counted and averaged. Results are mean ± SEM from 3–6 animals. *p<0.05, **p<0.01 relative to vehicle. One-way ANOVA followed by Bonferroni test (Figure 20). From this curve, the ED50 was estimated to be approximately 0.13 mg / kg.

[0241] Example 18: In vivo efficacy in male and female TBI rat models The inventors also performed the same TBI model in male and female mice and male and female rats. In all cases, NA184 was administered intraperitoneally at a dose of 1 mg / kg. For the rat experiment, NA184 was injected twice, 1 hour and 8 hours after TBI. Animals were sacrificed 24 hours after TBI, and calpain activity was assayed in the brain and cell death was analyzed in the cortex. The results showed that NA184 significantly inhibited calpain-2, rather than calpain-1, equally well and effectively in male and female mice and rats under these conditions (Figures 21A and 21B). Similarly, NA184 significantly prevented cell death in the cortex, to a similar degree in male and female rats (Figures 22A, 22B, and 22C). Furthermore, there was a very significant correlation between calpain-2 activity and cell death in rats (Figure 23). R² was 0.61, and p<0.005. The present invention includes the following embodiments. [1] The compound of the following formula (I), [ka] [In the formula, A is a carbocyclic aryl or heteroaryl, R 1 It is a non-hydrogen substituent, n is an integer ranging from 0 (ring A is unsubstituted) to a value allowed by the valence of A. L 1 and L 2 Each of these is an identical or different possibly substituted alkylene having 1 to 6 carbon atoms. R 2 It is a non-hydrogen substituent, R 4 and R 5 [These are independently hydrogen, or an unsubstituted C1-C6 alkyl such as methyl], and The pharmaceutically acceptable salt. [2] The compound described in [1], wherein A is phenyl. [3] L 1 and L 2The compounds described in [1] or [2], wherein each of the groups is -CH2-. [4] R 4 and R 5 A compound according to any one of the items [1] to [3], wherein the compound is methyl. [5] R 2 A compound according to any one of [1] to [4], wherein is an unsubstituted C1-C6 alkyl group. [6] The following structure: [ka] [In the formula, R 1 C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, -CO(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -CONH-CH(R c )(R d ), -(CH2) m N(R a )(R b ), or -O(CH2) m It is OH, m is an independent integer between 0 and 6. R a , R b , R c , and R d The compound according to any one of [1] to [5], wherein is independently hydrogen or an unsubstituted C1-C6 alkyl, which may be a linear or branched alkyl. [7] R a and R b A compound according to any one of [1] to [6], wherein the compound is independently hydrogen, methyl, ethyl, propyl, or isopropyl. [8] R c and R dA compound according to any one of [1] to [7], wherein the compound is independently hydrogen or methyl. [9] R 1 A compound according to any one of the items [1] to [8], wherein is -CONH2, -OCH2CH2N(CH3)2, -CONHCH2CH2NHCH(CH3)2, -CONHCH(CH3)2, -CH2N(CH3)2, or -CH2N(CH3)2.

[10] The following structure: [ka] [In the formula, R 1A R is a cyano or unsubstituted C1-C6 alkyl, 1B A compound according to any one of [1] to [4], wherein [ is a C1-C6 alkoxy].

[11] R 1B The compound described in

[10] , wherein is -OCH3.

[12] The aforementioned compound, [ka] The compound described in [1].

[13] The compound according to any one of [1] to

[12] , wherein the aforementioned compound is a racemic mixture.

[14] The compound according to any one of [1] to

[13] , wherein the compound exists as an optically enriched mixture.

[15] The following structure: [ka] A compound according to any one of [1] to

[14] , having the following characteristics.

[16] The compound of the following formula (X), [ka] [In the formula, A is a C1-C6 alkyl, carboxyl(-C(O)O-), aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group. B is a carbocyclic aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, L 1 These are bonded, substituted, or unsubstituted C1-C6 alkylenes. L 2 These are bonded, substituted, or unsubstituted C1-C6 alkylenes or -S(O)2-, Each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 It is an unsubstituted C1-C6 alkyl, Each R 6These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m It is OH, or -O(Ph)X, 2 R 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a , R b , R c and R d These are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 12. m is an independent integer between 0 and 6. k is an integer between 0 and 12, independently. and its pharmaceutically acceptable salts.

[17] A is phenyl, L 1ga-(CH2) p - and the compound described in

[16] , wherein p is 1 to 4.

[18] The aforementioned compound is given by the following formula (XI): [ka] [wherein the formula n is an integer between 0 and 5] The compound described in either

[16] or

[17] .

[19] L 2 -B- is, [ka] The compound described in any one of the items

[16] to

[18] .

[20] The aforementioned compound is given by the following formula (XI-a): [ka] A compound according to any one of

[16] to

[18] , having the formula [wherein k is an integer from 0 to 5 and n is an integer from 0 to 5]. [twenty one] The aforementioned compound is given by the following formula (XI-b), [ka] A compound according to any one of

[16] to

[18] , having the formula [wherein k is an integer from 0 to 4 and n is an integer from 0 to 5]. [twenty two] The aforementioned compound is given by the following formula (XI-c): [ka] A compound according to any one of

[16] to

[18] , having the formula [wherein k is an integer from 0 to 3 and n is an integer from 0 to 5]. [twenty three] The aforementioned compound is given by the following formula (XI-d): [ka] A compound according to any one of

[16] to

[18] , having the formula [wherein k is an integer from 0 to 6 and n is an integer from 0 to 5]. [twenty four] L 1 However, the bond is methylene or ethylene, and A is C 1-4 The compound described in

[16] is alkyl, cycloalkyl, or heterocycloalkyl. [twenty five] -L 1 -AR 1 but [ka] The compound described in

[24] .

[26] The aforementioned compound is given by the following formula (XII): [ka] The compound according to

[16] , having [wherein k is an integer from 0 to 5 and n is an integer from 0 to 5].

[27] A compound having the following formula (XIII), [ka] [In the formula, Each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(Rb ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, R 2 It is an unsubstituted C1-C6 alkyl, Each R 6 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m It is OH, or -O(Ph)X, 2 R 6 It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R a , R b , R c and Rd These are independently hydrogen, optionally substituted C1-C6 alkyl, -OH, amine, or unsubstituted C, which may be substituted with halogens. 3-6 It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 5. m is an independent integer between 0 and 6. k is an independent integer between 0 and 5. p is an independent integer between 0 and 6. and its pharmaceutically acceptable salts.

[28] Compounds having the following formula (XIV), [ka] [In the formula, Each R 1 These are independently C1-C6 alkyl, halogen, cyano, nitro, C1-C6 alkoxy, aryl, heterocycloaryl, heterocycloalkyl, and -C(O)(CH2) m N(R a )(R b ), -O(CH2) m N(R a )(R b ), -CONH(CH2) m N(R a )(R b ), -C(O)NH-CH(R c )(R d ), -C(O)OCH(R c )(R d ), -(CH2) m N(R a )(R b ), -(CH2) m N(R a )C(O)R b ,-(CH2) m N(R a )C(O)OR b -O(CH2) m R c -O(CH2) m OH, -S(O)2R b , or -O(Ph)X, 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(O)(CH2) m N(R a )(R b )、-O(CH2) m N(R a )(R b )、-CONH(CH2) m N(R a )(R b )、-C(O)NH-CH(R c )(R d )、-C(O)OCH(R c )(R d )、-(CH2) m N(R a )(R b )、-(CH2) m N(R a )C(O)R b 、-(CH2) m N(R a )C(O)OR b 、-O(CH2) m R c 、-O(CH2) m OH, or -O(Ph)X, two Rs 6 together with the atoms to which they are attached optionally combine to form cycloalkyl or heterocycloalkyl, R a 、R b 、R c and R d are independently hydrogen, optionally substituted C1-C6 alkyl, -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 its pharmaceutically acceptable salts.

[29] Each R a , R b , R c and R d A compound according to any one of [1] to

[28] , wherein the compound is independently hydrogen, methyl, ethyl, propyl, or isopropyl.

[30] The aforementioned compound, [ka] The compound described in

[16] .

[31] A pharmaceutical composition comprising a compound described in any one of items [1] to

[30] and a pharmaceutically acceptable excipient.

[32] A method for treating traumatic brain injury (TBI) in a patient, comprising the step of administering an effective amount of any one of the compounds or compositions described in [1] to

[31] to the patient who requires treatment for TBI.

[33] A method for treating a subject suffering from a disorder or condition related to neuronal cell death, comprising the step of administering an effective amount of any one of the compounds or compositions described in [1] to

[31] to the subject suffering from a disorder or condition related to neuronal cell death.

[34] The method according to

[33] , wherein the subject is suffering from stroke, concussion, intracerebral hemorrhage, acute glaucoma, seizure activity and / or spinal cord injury.

[35] The method according to any one of

[33] to

[34] , wherein the patient is identified as having or being susceptible to a disorder or condition related to neuronal cell death, and the compound is administered to the identified subject.

[36] The method according to any one of

[32] to

[35] , wherein the compound or composition is administered by a method selected from the group consisting of oral administration, intravitreous injection, intraocular injection, intraocular irrigation, periorbital injection and sub-Tenon's capsule injection.

[37] The method according to any one of

[32] to

[36] , wherein the subject is a human.

Claims

1. The compound of the following formula (I), 【Chemistry 1】 [In the formula, A is a carbocyclic aryl or heteroaryl, R １ It is a non-hydrogen substituent, n is an integer ranging from 0 (ring A is unsubstituted) to a value allowed by the valence of A, L １ and L ２ Each of these is an identical or different substituted alkylene having 1 to 6 carbon atoms. R ２ It is a non-hydrogen substituent, R ４ and R ５ These are independently hydrogen, or unsubstituted C such as methyl. １ -C ６ It is alkyl, and The pharmaceutically acceptable salt.

2. The compound according to claim 1, wherein A is phenyl.

3. L １ and L ２ is each -CH ２ -, the compound according to claim 1 or 2.

4. R ４ and R ５ The compound according to any one of claims 1 to 3, wherein is methyl.

5. R ２ is non-substituted C １ -C ６ A compound according to any one of claims 1 to 4, wherein it is alkyl.

6. The following structure: 【Chemistry 2】 [In the formula, R １ is C １ -C ６ Alkyl, halogen, cyano, nitro, C １ -C ６ Alkoxy, -CO(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -O(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -CONH(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -CONH-CH(R ｃ ) (Caution ｄ ), - (CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), or -O(CH ２ ) ｍ OH, m is an independent integer between 0 and 6. R ａ , R ｂ , R ｃ , and R ｄ These are, independently, hydrogen or unsubstituted C １ -C ６ The compound according to any one of claims 1 to 5, having [being alkyl, which may be linear or branched alkyl].

7. R ａ and R ｂ The compound according to any one of claims 1 to 6, wherein is independently hydrogen, methyl, ethyl, propyl, or isopropyl.

8. R ｃ and R ｄ The compound according to any one of claims 1 to 7, wherein is independently hydrogen or methyl.

9. R １ ga-CONH ２ , -OCH ２ CH ２ N(CH ３ ) ２ , -CONHCH ２ CH ２ NHCH(CH ３ ) ２ , -CONHCH(CH ３ ) ２ ien-CH ２ N(CH ３ ) ２ , or -CH ２ N(CH ３ ) ２ The compound according to any one of claims 1 to 8.

10. The following structure: 【Transformation 3】 [In the formula, R １Ａ is cyano or unsubstituted C １ -C ６ It is alkyl, R １Ｂ is C １ -C ６ The compound according to any one of claims 1 to 4, having [being an alkoxy].

11. R １Ｂ ga-OCH ３ The compound according to claim 10.

12. The aforementioned compound, 【Chemistry 4】 The compound according to claim 1.

13. The compound according to any one of claims 1 to 12, wherein the compound is a racemic mixture.

14. The compound according to any one of claims 1 to 13, wherein the compound exists as an optically enriched mixture.

15. The following structure: 【Transformation 5】 A compound according to any one of claims 1 to 14, having the following characteristics.

16. The compound of the following formula (X), 【Transformation 6】 [In the formula, A is C １ -C ６ They are alkyl, carboxyl (-C(O)O-), aryl, heteroaryl, cycloalkyl, or heterocycloalkyl. B is a carbocyclic aryl, heteroaryl, cycloalkyl, or heterocycloalkyl compound. L １ C is a combination, or substitution or non-substitution C. １ -C ６ It is alkylene, L ２ C is bonded, substituted, or unsubstituted. １ -C ６ Alkylene or -S(O) ２ - and Each R １ is independently C １ -C ６ alkyl, halogen, cyano, nitro, C １ -C ６ alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(O)(CH ２ ) ｍ N(R ａ )(R ｂ ), -O(CH ２ ) ｍ N(R ａ )(R ｂ ), -CONH(CH ２ ) ｍ N(R ａ )(R ｂ ), -C(O)NH-CH(R ｃ )(R ｄ ), -C(O)OCH(R ｃ )(R ｄ ), -(CH ２ ) ｍ N(R ａ )(R ｂ ), -(CH ２ ) ｍ N(R ａ )C(O)R ｂ , -(CH ２ ) ｍ N(R ａ )C(O)OR ｂ , -O(CH ２ ) ｍ R ｃ , -O(CH ２ ) ｍ OH, -S(O) ２ R ｂ , or -O(Ph)X, and R ２ is non-substituted C １ -C ６ It is alkyl, Each R ６ Independently, C １ -C ６ Alkyl, halogen, cyano, nitro, C １ -C ６ Alkoxy, aryl, heterocycloaryl, heterocycloalkyl-C(O)(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -O(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -CONH(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -C(O)NH-CH(R ｃ ) (Caution ｄ ), -C(O)OCH(R ｃ ) (Caution ｄ ), - (CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), - (CH ２ ) ｍ N(R) ａ ) C(O)R ｂ ,-(CH ２ ) ｍ N(R) ａ ) C(O)OR ｂ , -O(CH ２ ) ｍ R ｃ , -O(CH ２ ) ｍ It is OH, or -O(Ph)X, Two R ６ It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R ａ , R ｂ , R ｃ and R ｄ C may be independently substituted with hydrogen or halogen. １ -C ６ Alkyl, -OH, amine, or unsubstituted C ３－６ It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 12. m is an independent integer between 0 and 6. k is an independent integer between 0 and 12. and its pharmaceutically acceptable salts.

17. A is phenyl, L １ ga- (CH ２ ) ｐ The compound according to claim 16, wherein p is 1 to 4.

18. The aforementioned compound is given by the following formula (XI): 【Transformation 7】 [wherein the formula n is an integer from 0 to 5] The compound according to any one of claims 16 and 17.

19. L ２ -B- is, 【Transformation 8】 The compound according to any one of claims 16 to 18.

20. The aforementioned compound is given by the following formula (XI-a): 【Chemistry 9】 The compound according to any one of claims 16 to 18, having the formula [wherein k is an integer from 0 to 5 and n is an integer from 0 to 5].

21. The aforementioned compound is given by the following formula (XI-b), 【Chemistry 10】 The compound according to any one of claims 16 to 18, having the formula [wherein k is an integer from 0 to 4 and n is an integer from 0 to 5].

22. The aforementioned compound is given by the following formula (XI-c), 【Chemistry 11】 The compound according to any one of claims 16 to 18, having the formula [wherein k is an integer from 0 to 3 and n is an integer from 0 to 5].

23. The aforementioned compound is given by the following formula (XI-d): 【Chemistry 12】 The compound according to any one of claims 16 to 18, having the formula [wherein k is an integer from 0 to 6 and n is an integer from 0 to 5].

24. L １ However, it is a bond, methylene, or ethylene, and A is C １－４ The compound according to claim 16, wherein it is alkyl, cycloalkyl, or heterocycloalkyl.

25. -L １ -A-R １ but 【Chemistry 13】 The compound according to claim 24.

26. The aforementioned compound is given by the following formula (XII): 【Chemistry 14】 The compound according to claim 16, having [wherein k is an integer from 0 to 5 and n is an integer from 0 to 5].

27. Compounds having the following formula (XIII), 【Chemistry 15】 [In the formula, Each R １ C is independent １ -C ６ Alkyl, halogen, cyano, nitro, C １ -C ６ Alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(O)(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -O(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -CONH(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -C(O)NH-CH(R ｃ ) (Caution ｄ ), -C(O)OCH(R ｃ ) (Caution ｄ ), - (CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), - (CH ２ ) ｍ N(R) ａ ) C(O)R ｂ ,-(CH ２ ) ｍ N(R) ａ ) C(O)OR ｂ , -O(CH ２ ) ｍ R ｃ , -O(CH ２ ) ｍ OH, -S(O) ２ R ｂ , or -O(Ph)X, R ２ is non-substituted C １ -C ６ It is alkyl, Each R ６ Independently, C １ -C ６ Alkyl, halogen, cyano, nitro, C １ -C ６ Alkoxy, aryl, heterocycloaryl, heterocycloalkyl-C(O)(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -O(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -CONH(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -C(O)NH-CH(R ｃ ) (Caution ｄ ), -C(O)OCH(R ｃ ) (Caution ｄ ), - (CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), - (CH ２ ) ｍ N(R) ａ ) C(O)R ｂ ,-(CH ２ ) ｍ N(R) ａ ) C(O)OR ｂ , -O(CH ２ ) ｍ R ｃ , -O(CH ２ ) ｍ It is OH, or -O(Ph)X, Two R ６ It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R ａ , R ｂ , R ｃ and R ｄ C may be independently substituted with hydrogen or halogen. １ -C ６ Alkyl, -OH, amine, or unsubstituted C ３－６ It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 5. m is an independent integer between 0 and 6. k is an independent integer between 0 and 5. p is an independent integer between 0 and 6. and its pharmaceutically acceptable salts.

28. Compounds having the following formula (XIV), 【Chemistry 16】 [In the formula, Each R １ C is independent １ -C ６ Alkyl, halogen, cyano, nitro, C １ -C ６ Alkoxy, aryl, heterocycloaryl, heterocycloalkyl, -C(O)(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -O(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -CONH(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -C(O)NH-CH(R ｃ ) (Caution ｄ ), -C(O)OCH(R ｃ ) (Caution ｄ ), - (CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), - (CH ２ ) ｍ N(R) ａ ) C(O)R ｂ ,-(CH ２ ) ｍ N(R) ａ ) C(O)OR ｂ , -O(CH ２ ) ｍ R ｃ , -O(CH ２ ) ｍ OH, -S(O) ２ R ｂ , or -O(Ph)X, R ２ is non-substituted C １ -C ６ It is alkyl, Each R ６ Independently, C １ -C ６ Alkyl, halogen, cyano, nitro, C １ -C ６ Alkoxy, aryl, heterocycloaryl, heterocycloalkyl-C(O)(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -O(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -CONH(CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), -C(O)NH-CH(R ｃ ) (Caution ｄ ), -C(O)OCH(R ｃ ) (Caution ｄ ), - (CH ２ ) ｍ N(R) ａ ) (Caution ｂ ), - (CH ２ ) ｍ N(R) ａ ) C(O)R ｂ ,-(CH ２ ) ｍ N(R) ａ ) C(O)OR ｂ , -O(CH ２ ) ｍ R ｃ , -O(CH ２ ) ｍ It is OH, or -O(Ph)X, Two R ６ It, together with the atom it is bonded to, and possibly by bonding, to form a cycloalkyl or heterocycloalkyl group. R ａ , R ｂ , R ｃ and R ｄ C may be independently substituted with hydrogen or halogen. １ -C ６ Alkyl, -OH, amine, or unsubstituted C ３－６ It is a cycloalkyl, X is a halogen, n is an independent integer between 0 and 5. m is an independent integer between 0 and 6. k is an independent integer between 0 and 5. p is an independent integer between 0 and 6. and its pharmaceutically acceptable salts.

29. Each R ａ , R ｂ , R ｃ and R ｄ The compound according to any one of claims 1 to 28, wherein is independently hydrogen, methyl, ethyl, propyl, or isopropyl.

30. The aforementioned compound, 【Chemistry 17】 The compound according to claim 16.

31. A pharmaceutical composition comprising a compound according to any one of claims 1 to 30 and a pharmaceutically acceptable excipient.

32. A method for treating traumatic brain injury (TBI) in a patient, comprising the step of administering an effective amount of a compound or composition according to any one of claims 1 to 31 to the patient who requires treatment for traumatic brain injury (TBI).

33. A method for treating a subject suffering from a disorder or condition related to neuronal cell death, comprising the step of administering an effective amount of a compound or composition according to any one of claims 1 to 31 to the subject suffering from a disorder or condition related to neuronal cell death.

34. The method according to claim 33, wherein the subject is suffering from stroke, concussion, intracerebral hemorrhage, acute glaucoma, seizure activity and / or spinal cord injury.

35. The method according to any one of claims 33 to 34, wherein the patient is identified as having or being susceptible to a disorder or condition related to neuronal cell death, and the compound is administered to the identified subject.

36. The method according to any one of claims 32 to 35, wherein the compound or composition is administered by a method selected from the group consisting of oral administration, intravitreal injection, intraocular injection, intraocular irrigation, periorbital injection, and sub-Tenon's capsule injection.

37. The method according to any one of claims 32 to 36, wherein the subject is a human.

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