Nitrogen-containing heterocyclic compounds and their uses

Abstract

The present invention relates to a nitrogen-containing heterocyclic compound derivative and its use. The compound according to the present invention can effectively improve synaptic plasticity without side effects, thereby exerting therapeutic effects on various neurological disorders such as neurodegenerative diseases, neurodegenerative pathologies, neuropsychiatric disorders, memory loss, cognitive dysfunction / injury, and extinction learning disorders.

Description

[Technical Field]

[0001] The present invention relates to nitrogen-containing heterocyclic compounds and their uses. More specifically, the present invention relates to compounds, compositions or methods for the prevention or treatment of neurological or neurodegenerative disorders. [Background technology]

[0002] Synaptic plasticity and critical periods

[0003] Neural networks are networks of neurons. Synapses are nodes within neural networks where neurons exchange information with each other. Within a network, synapses are "plastic" because their number and strength change continuously throughout life as synaptic inputs are added, removed, or refined in response to ongoing neural activity. Synaptic plasticity refers to this activity-dependent transformation of synapses, manifested by changes in the strength and / or efficiency of synaptic transmission.

[0004] Higher-order functions such as learning, cognition, synthesis, judgment, art, and creativity depend on the ability to form neural networks (called cortical neural networks) in the cerebral cortex. Synaptic plasticity of cortical neural networks reaches its peak during the early postnatal period, known as the critical period. After the critical period, cortical synaptic plasticity gradually declines with age, eventually reaching a state where experience no longer actively modulates the cerebral cortex. Interestingly, however, recent studies have shown that under certain conditions, synaptic plasticity can be restored to a high level even after the critical period has ended: in animal models, peripheral nerve injury reactivated synaptic plasticity in the somatosensory thalamocortical circuit that transmits peripheral sensory signals to the cerebral cortex (Yu et al., 2012, Neuron. 74:731-42; Petrus et al., 2014, Neuron. 81:664-73; Gao et al., 2014, J Neurosci. 34:10770-9).

[0005] Decreased synaptic plasticity is also associated with the degeneration of adult brain function. In particular, synaptic plasticity in thalamocortical circuits is severely restricted with age. As mentioned above, if it is possible to resume critical periods, reactivating or increasing cortical synaptic plasticity through therapeutic means may be useful for treating diseases caused by reduced synaptic plasticity.

[0006] Histone deacetylase inhibitors

[0007] Most DNA in cells exists packaged in a denser, more compacted form known as chromatin. Compaction occurs by wrapping DNA around a group of proteins called histones. The degree of chromatin compaction depends on the level of histone acetylation. Generally, histone acetyltransferases (HATs) acetylate DNA, allowing access, while histone deacetylases (HDACs) deacetylate DNA, blocking access. Because transcription can occur only when access is permitted, chromatin compaction acts as a regulatory mechanism for gene expression (Verdin and Ott, 2015, Nat Rev Mol Cell Biol 16:258-264).

[0008] To date, 11 types of mammalian HDACs have been identified. These types are broadly divided into four classes based on the degree of homology with yeast HDACs. Class I includes HDACs 1, 2, 3, and 8. Class II HDACs are divided into class IIa (HDACs 4, 5, 7, and 9) and class IIb (HDACs 6 and 10) based on structural similarity. Class III HDACs include SIRT1-7, and class IV includes HDAC11.

[0009] Class I HDACs are primarily restricted to the nucleus, whereas class II HDACs shuttle between the nucleus and cytoplasm through phosphorylation of specific serine residues. The N-terminal domain of HDACs interacts with multiple tissue-specific transcription factors and acts as a transcription inhibitor. The C-terminal domain of HDACs possesses deacetylase enzyme activity. However, class IIa HDACs lack deacetylase activity due to loss-of-function mutations in the C-terminal catalytic domain in which a tyrosine (Tyr) residue is replaced by a histidine (His).

[0010] HDAC4

[0011] HDAC4 is expressed throughout the body. HDAC4 is particularly highly expressed in the brain, heart, and skeletal muscle (Grozinger et al., 1999, Proc. Natl. Acad. Sci. USA 96, 4868-4873; Darcy et al., 2010, J. Comp. Neurol. 518, 722-740). In the brain, HDAC4 levels peak immediately after birth (Sando et al., 2012, Cell 151, 821-834). HDAC4 expression was detected in corticotropin-releasing hormone (CRH) neurons (100±0%), oxytocin neurons (98±2%), vasopressin neurons (100±0%), orexin neurons (100±0%), AgRP neurons (100±0%), POMC neurons (92±4%), histamine neurons (85±7%), dopamine neurons (100±0%), serotonin neurons (93±2%), and noradrenergic neurons (100±0%) (Takase et al., 2013, PloS ONE 8:e58473).

[0012] Cytoplasmic HDAC4 rapidly translocates to the nucleus in response to apoptosis-inducing conditions such as low potassium or excitotoxic glutamate. Ectopic nuclear overexpression of HDAC4 promotes neuronal death and inhibits the transcriptional activity of survival factors such as MEF2 and CREB (Bolger and Yao, 2005, J Neurosci. 2005; 25:9544-9553). HDAC4-containing neurons are uniform in size and widely distributed throughout the neuropil, including the paraventricular nucleus (PVN), lateral hypothalamus (LHA), hypothalamic arcuate nucleus (ARC), tuberomammillary nucleus (TMN), dorsal raphe (DR), and locus coeruleus (LC). Notably, HDAC4 colocalizes with PSD95 (Mielcarek et al., 2013, PLoS Biol. 11:e1001717; Takase et al., 2013, PLoS ONE 8:e58473), suggesting a role for HDAC4 in synaptic plasticity (Sando et al., 2012, Cell 151, 821-834; Mielcarek et al., 2013, PLoS Biol. 11:e1001717).

[0013] In an animal model, tissue-specific deletion of HDAC4 in the rat hippocampus eliminated long-lasting stress-induced behavioral changes (Sailaja et al., 2012, Proc. Natl. Acad. Sci. USA 109, E3687-E3695). Overexpression of HDAC4 accelerated the death of cerebellar granule neurons by increasing their susceptibility to H2O2 injury through inhibition of peroxisome proliferator-activated receptor α (PPARα) activity (Bolger and Yao, 2005, J. Neurosci. 25, 9544-9553; Li et al., 2012, Nat. Med. 18, 783-790; Sando et al., 2012; Cell 151, 821-834; Yang et al., 2011, FEBS Lett. 585, 761-766). Overexpression of HDAC4 in the rat hippocampus induced depression-like behaviors (Sarkar et al., 2014, Neuropsychopharmacology 39, 2221-2232).

[0014] In a Drosophila model, overexpression of HDAC4 in adult mushroom body neurons, a structure important for memory formation, caused impairment of long-term courtship memory but not short-term memory (Fitzsimons et al., 2013, PloS ONE 8: e83903). Subsequent studies investigating the relationship between HDAC4 subcellular distribution showed that deficits in mushroom body morphogenesis, eye development, and long-term memory were associated with nuclear HDAC4 abundance (Main et al., 2021, Front. Mol. Neurosci. 14:616642).

[0015] In humans, increased levels of HDAC4 have been identified in postmortem brains of patients with frontotemporal lobar degeneration (FTLD) (Yeh et al., 2013, Brain Res. 1504, 16-24; Whitehouse et al., 2014, Neuropathol. Appl. Neurobiol. 41, 245-257). HDAC4 has been identified as a component of Lewy bodies in brains with Parkinson's disease and as a component of intranuclear inclusions in neuronal intranuclear inclusion disease (Takahashi-Fujigasaki and Fujigasaki, 2006, Neuropathol. Appl. Neurobiol. 32, 562-566). HDAC4 has been observed to be significantly overexpressed in specific cortical regions of patients with autism (Nardone et al., 2014, Transl. Psychiatry 4:e433).

[0016] HDAC inhibitors and synaptic plasticity

[0017] Histone modifications have been proposed as a potential method for restoring cortical plasticity in adulthood. Valproic acid is a known histone deacetylase (HDAC) inhibitor. In a clinical study in which participants took valproic acid capsules, the critical period was reopened, enabling absolute pitch learning, previously only possible during the critical period, in adults (Gervain et al., 2013, Front Syst Neurosci. 7:102). When adult rats were exposed to an enriched environment compared with normal cage conditions, total H3 acetylation in the visual cortex increased compared to controls, and ocular dominance plasticity, which is typically only observed during the critical period, was also observed after the critical period (Baroncelli L et al., 2016, J Neurosci. 36:3430-40).

[0018] Treatment with the HDAC inhibitor SAHA (suberoylanilide hydroxamic acid) increased H3 acetylation in the visual cortex to levels typically observed in young rats, particularly at the P3 promoter of brain-derived neurotrophic factor (BNDF), which is known to be important for developmental synaptic plasticity and long-term memory in the visual cortex (Baroncelli L et al., 2016, J Neurosci. 36:3430-40). Increased BDNF expression was also observed in neurospheres (Bagheri et al., 2019, Int. J. Mol. Sci. 20: 1109).

[0019] SAHA and other HDAC inhibitors have been shown to be effective in ameliorating symptoms in mouse models of Huntington's disease (HD) (Ferrante et al., 2003; Hockly et al., 2003, J. Biol. Chem. 278, 16059-16072; Gardian et al., 2005, J. Biol. Chem. 280, 556-563). HDAC4 expression correlates with the length of polyglutamine residues expressed in HD. This is colocalized with cytoplasmic inclusions and a wide range of peripheral organ pathologies, such as skeletal muscle atrophy and heart failure, which are manifested in Huntington's disease (Zielonka et al., 2014, Front. Physiol. 5:380; Mielcarek et al., 2014, PLoS Genet. 10:e1004550; Mielcarek et al., 2014, PLoS ONE 9:e108961; Zielonka et al., 2014, Exp. Clin. Cardiol. 20, 2547-2554). Reducing HDAC4 levels in the R6 / 2 mouse model of Huntington's disease delayed the formation of cytoplasmic aggregates in various brain regions, improved motor coordination and neurological phenotypes, and extended lifespan (Mielcarek et al., 2013, PLoS Biol. 11:e1001717).

[0020] Interestingly, SAHA treatment in R6 / 2 mice restored cortical BDNF transcript levels (Mielcarek et al., 2011, PloS ONE 6:e27746). Because HDAC4 and HDAC5 can inhibit specific BDNF transcripts, and this effect has been shown to be reversed by SAHA treatment, the memory-enhancing and neuroprotective effects of HDAC inhibitors may be associated with increased BDNF expression (Koppel and Timmusk, 2013, Neuropharmacology 75, 106-11).

[0021] HDAC4 plays a central role in brain physiology and may be a useful target for treating various neurodegenerative disorders. However, currently available HDAC inhibitors inhibit a broad range of HDACs, making them unsuitable for inhibiting specific HDACs. SAHA increases synaptic plasticity, but causes serious side effects, including fatigue, nausea, diarrhea, and thrombocytopenia, making SAHA unsuitable for human use (Meghan E. et al., 2011, Bone. 48: 1117-1126).

[0022] Therefore, it would be useful to develop selective HDAC inhibitors for targeted therapy. Summary of the Invention [Problem to be solved by the invention]

[0023] An object of the present invention is to solve all of the above problems.

[0024] An object of the present invention is to provide a nitrogen-containing heterocyclic compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for improving synaptic plasticity.

[0025] Another object of the present invention is to provide a pharmaceutical composition for treating or preventing a neurological disorder or a neurodegenerative disorder, which comprises, as an active ingredient, a nitrogen-containing heterocyclic compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for improving synaptic plasticity.

[0026] Another object of the present invention is to provide a method for treating or preventing a neurological or neurodegenerative disorder, which comprises administering to a subject a nitrogen-containing heterocyclic compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for improving synaptic plasticity.

[0027] Another object of the present invention is to provide a pharmaceutical composition for combined administration containing a nitrogen-containing heterocyclic compound for improving synaptic plasticity, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof and a second therapeutic agent, and a combined administration method using the same.

[0028] The objects of the present invention are not limited to the above objects, but will become clearer in the following description and be realized by the means and combinations thereof set forth in the claims. [Means for solving the problem]

[0029] A typical configuration of the present invention for achieving the above object is as follows.

[0030] According to one aspect of the present invention, there is provided a compound represented by the following chemical formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0031] JPEG2025511114000002.jpg43115

[0032] R 1 is C 1-6 Alkyl, C 3-12 cycloalkyl, aryl, or heteroaryl, and one or more hydrogen atoms in the group are unsubstituted or each independently substituted with a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, nitro, one or more C 1-6 alkyl-substituted or unsubstituted amino, or hydroxy-substituted;

[0033] A is C=O or a bond;

[0034] Q is -(CR 4 R 5 ) m - or a bond, where R 4 and R 5 are independently hydrogen or C 1-6 alkyl, and m is an integer from 1 to 6;

[0035] R 2 is hydrogen, C 1-6 alkyl or aryl,

[0036] R 3 is hydrogen, carboxyl, N-hydroxycarboxamido, or carboxamido, and one or more hydrogens in said group are unsubstituted or C 1-6 is substituted with alkyl,

[0037] n is 0 or 1.

[0038] In one embodiment, R 1 is C 1-6 Alkyl, C 3-12 cycloalkyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl, and one or more hydrogen atoms in the group are unsubstituted or each independently substituted with a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, nitro, one or more C 1-6 It may be substituted with alkyl or unsubstituted amino, or hydroxy.

[0039] In one embodiment, R 1 may be methyl, cyclohexyl, phenyl, 4-methoxyphenyl, 4-(tert-butyl)phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 3,4-dichlorophenyl, 2-chloro-4-fluorophenyl, p-tolyl, m-tolyl, pyridin-4-yl, thiophen-2-yl, furan-2-yl, pyridin-3-yl, pyridin-2-yl, pyrimidin-5-yl, pyrimidin-2-yl, 4-nitrophenyl, 4-aminophenyl, 3-nitrophenyl, 3-aminophenyl, 4-methoxy-pyridin-3-yl, 6-methoxy-pyridin-3-yl, 4-(dimethylamino)phenyl, or 4-hydroxy-phenyl.

[0040] In one embodiment, R 2 is H or C 1-6 It may also be alkyl.

[0041] In one embodiment, R 3 may be H, carboxyl, methylcarboxyl, ethylcarboxyl, N-hydroxycarboxamide, carboxamide, or sodium carboxylate.

[0042] According to another aspect of the present invention, there is provided a compound represented by the following chemical formula (2-A) or (2-B), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0043] JPEG2025511114000003.jpg32146

[0044] In the above formula,

[0045] R 4 is C 1-6 Alkyl, C 3-12 cycloalkyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl, and one or more hydrogen atoms in the group are unsubstituted or each independently substituted with a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, nitro, one or more C 1-6 substituted with alkyl-substituted or unsubstituted amino or hydroxy;

[0046] R 5 and R 6 are each independently hydrogen or C 1-6 It is alkyl.

[0047] According to another aspect of the present invention, there is provided a compound represented by the following chemical formula (4), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0048] JPEG2025511114000004.jpg43115

[0049] In the above formula,

[0050] R 2 is hydrogen, C 1-6 alkyl or aryl,

[0051] R 3 is hydrogen, carboxyl, N-hydroxycarboxamido, or carboxamido, and one or more hydrogens in said group are unsubstituted or C 1-6 is substituted with alkyl,

[0052] n is 0 or 1.

[0053] According to another aspect of the present invention, there is provided a compound represented by the following chemical formula (5), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0054] JPEG2025511114000005.jpg46126

[0055] In the above formula,

[0056] R 2 is hydrogen, C 1-6 alkyl or aryl,

[0057] R 4 is phenyl or phenyl substituted with one or more halogens,

[0058] n is 0 or 1.

[0059] In one embodiment, the compound may be a compound selected from the compounds presented in Tables 2 to 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0060] In one embodiment, the compound may be BnH12001 or an enantiomer thereof.

[0061] In one embodiment, the stereoisomers may be optical isomers.

[0062] In one embodiment, the compound may be an inhibitor of HDAC, hi another embodiment, the HDAC may be HDAC4.

[0063] In one embodiment, the compound may increase synaptic plasticity.

[0064] In accordance with yet another aspect of the present invention, there is provided a pharmaceutical composition comprising the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0065] In one embodiment, the composition may be for the prevention or treatment of a neurological or neurodegenerative disorder.

[0066] In other embodiments, the compound, its stereoisomer, or its pharmaceutically acceptable salt can be delivered to the brain at a concentration of about 0.1 to about 3 μM.

[0067] In another embodiment, the disorder may be Alzheimer's disease.

[0068] In another embodiment, the disorder may be PTSD.

[0069] In one embodiment, the composition can further comprise a second therapeutic agent.

[0070] In other embodiments, the second therapeutic agent may be aducanumab.

[0071] According to yet another aspect of the present invention, there is provided a method for preventing or treating a neurological or neurodegenerative disorder, comprising administering the compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0072] According to yet another aspect of the present invention, there is provided a use of the compound, its stereoisomer, or its pharmaceutically acceptable salt as a medicine or for the manufacture of a medicine.

[0073] According to one embodiment, the use is for reactivating or increasing brain synaptic plasticity.

[0074] According to another embodiment, the use is for the prevention or treatment of a neurological or neurodegenerative disorder. [Effects of the Invention]

[0075] The compounds according to the present invention can effectively improve or reactivate synaptic plasticity without side effects, thereby exerting therapeutic effects on various neurological or neurodegenerative disorders. [Brief explanation of the drawings]

[0076] [Figure 1a] 1 shows the results of measuring cell viability in response to administration of BnH12001 (0, 0.1, 1, 10, 100 μM) in the human cell line SH-SY5Y.

[0077] [Figure 1b] 1 shows the results of measuring cell viability in response to administration of BnH12001 (0, 0.1, 1, 10, 100 μM) in the human cell line 293T.

[0078] [Figure 2] 2 shows the results of measuring the expression of BDNF and GluN2B after treating 293T cells with the compound BnH12001.

[0079] [Figure 3a] 1 shows the results of measuring the expression of HDAC4, BDNF, and GluN2B after treating SH-SY5Y cells with the compound BnH12001. [Figure 3b] 1 shows the results of measuring the expression of HDAC4, BDNF, and GluN2B after treating SH-SY5Y cells with the compound BnH12001. [Figure 3c] 1 shows the results of measuring the expression of HDAC4, BDNF, and GluN2B after treating SH-SY5Y cells with the compound BnH12001.

[0080] [Figure 4a] The figure shows the site of osmotic pump (Os pump) injection in mouse experiments.

[0081] [Figure 4b] The patch recording sites in the mouse experiments are shown.

[0082] [Figure 4c] The results of measuring the excitation / inhibition balance in the mouse barrel cortex (layer 4) in response to BnH12001 administration (Os pump, ventricular injection, 3 μM) are shown.

[0083] [Figure 4d] This shows changes in thalamocortical (TC) excitatory postsynaptic current (EPSC) potency in the mouse barrel cortex (layer 4) in response to BnH12001 administration (Os pump, intravenous injection, 3 μM).

[0084] [Figure 5a] The results of measuring the excitation / inhibition balance in the mouse barrel cortex (layer 4) in response to oral administration of BnH12001 (1 mg / kg) are shown.

[0085] [Figure 5b] 1 shows changes in TC EPSC potency in mouse barrel cortex (layer 4) in response to oral administration of BnH12001 (1 mg / kg).

[0086] [Figure 5c] Changes in TC EPSC potency in mouse barrel cortex (layer 4) in response to administration of BnH12001 at various doses (0.03, 0.1, 0.3, 1, 3, 10 mg / kg) are shown.

[0087] [Figure 6a] 1 shows the dosing schedule for analyzing the efficacy of BnH12001 enantiomer (3S,6R, 98% or greater) administered alone or in combination with aducanumab to improve in vitro synaptic plasticity in an animal model of Alzheimer's disease.

[0088] [Figure 6b] 1 shows LTP measurements of field excitatory postsynaptic potentials in the hippocampus of a mouse model of Alzheimer's disease in response to administration of the BnH12001 enantiomer (3S,6R, 98% or greater) alone or in combination with aducanumab.

[0089] [Figure 6c] Figure 1 shows the excitation / inhibition balance (right) and changes in TC EPSC potency (left) in the sensory cortex of a mouse model of Alzheimer's disease in response to administration of the BnH12001 enantiomer (3S,6R, 98% or greater) alone or in combination with aducanumab.

[0090] [Figure 7] 1 shows the binding characteristics of BnH12001 to HDAC4.

[0091] [Figure 8a] 1 shows the knockdown effect of HDAC4 shRNA on the expression of HDAC4, BDNF, and GluN2B in the human cell line SH-SY5Y. [Figure 8b] 1 shows the knockdown effect of HDAC4 shRNA on the expression of HDAC4, BDNF, and GluN2B in the human cell line SH-SY5Y.

[0092] [Figure 9a] The effect of BnH12001 on the expression of BDNF and GluN2B is shown. [Figure 9b] The effect of BnH12001 on the expression of BDNF and GluN2B is shown. [Figure 9c]The effect of BnH12001 on the expression of BDNF and GluN2B is shown.

[0093] [Figure 10a] The time course effect of BnH12001 on HDAC4 expression is shown. [Figure 10b] The time course effect of BnH12001 on HDAC4 expression is shown.

[0094] [Figure 11a] 1 shows the effect of BnH12001 on thalamocortical EPSC potency. [Figure 11b] 1 shows the effect of BnH12001 on thalamocortical EPSC potency.

[0095] [Figure 12a] 1 shows changes in TC EPSC potency in mouse barrel cortex (layer 4) in response to oral administration (1 mg / kg) of BnH12001 enantiomer (3R,6S, 98%<) and BnH12001 enantiomer (3S,6R, 98%<).

[0096] [Figure 12b] The results of measuring the excitation / inhibition balance in the mouse barrel cortex (layer 4) following oral administration (1 mg / kg) of BnH12001 enantiomer (3R,6S, 98%<) and BnH12001 enantiomer (3S,6R, 98%<) are shown.

[0097] [Figure 13a] 1 shows the effect of BnH12001 enantiomers (3S,6R, 98% or more) on the expression of BDNF and GluN2B in primary cultured cortical neurons.

[0098] [Figure 13b] 1 shows the effect of BnH12001 enantiomers (3S, 6R, 98% or more) on the expression of HDAC4, BDNF, and GluN2B in 8-week-old C57BL / 6J mice.

[0099] [Figure 13c] The effect of BnH12001 enantiomers (3S,6R, 98% or more) on the expression of GluN2B in 13-month-old C57BL / 6J mice is shown. DETAILED DESCRIPTION OF THE INVENTION

[0100] The detailed description of the present invention that follows will be described with reference to particular embodiments in which the invention may be practiced and with reference to certain drawings, but the invention is not limited thereto, but rather only by the appended claims, along with the full scope of equivalents to which such claims are entitled when properly written. Terms presented in describing the present invention should generally be understood in their ordinary sense, unless otherwise indicated, and apply to all such terms as used herein, not just to the aspect or embodiment of the invention for which the term is defined.

[0101] Throughout the detailed description of the present invention, when compositions and kits are described as having or comprising particular components, it is contemplated that there are additional compositions and kits of the present invention that consist essentially of or consist of the recited components.

[0102] In applications where an element or component is referred to as being included / included in or selected from a list of mentioned elements or components, it is to be understood that the element or component can be any one of the mentioned elements or components, or that the element or component can be selected from the group consisting of the mentioned elements or components.

[0103] It should be understood that the order of steps or sequence for performing certain operations is immaterial so long as the invention remains operable. Moreover, two or more steps or operations may be conducted simultaneously.

[0104] At various places in the present specification, variables or parameters are disclosed as groups or ranges, and the detailed description is specifically intended to include all individual members of such groups and ranges.

[0105] In this specification, examples or exemplary language, such as "such as" or "including," is merely intended to better describe the invention and does not limit the scope of the invention unless claimed. Nothing in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0106] I. Definition

[0107] The singular terms "a," "an," and "the" are used herein to refer to the plural (ie, to at least one) as well, unless the context requires otherwise.

[0108] "And / or" is used herein to mean "and" or "or" unless otherwise indicated.

[0109] "At least one" includes each and every recited object after the expression individually, and various combinations of two or more of the recited objects, unless otherwise understood from context and usage.

[0110] "Comprise," "including," "have," "having," "containing," "including" (including grammatical equivalents thereof) should generally be understood to be open-ended and non-limiting, e.g., not excluding additional, unmentioned elements or steps unless otherwise specifically stated or understood from the context.

[0111] "About" means approximately, in the vicinity, roughly, or to the extent of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries around the stated numerical values. In general, the term "about" is used herein to modify a numerical value around the stated value by a variance of 10%.

[0112] The terms "individual," "patient," and "subject" are used interchangeably and include any animal, including mammals (e.g., mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, including humans).

[0113] "Cure," "treat," or "treatment" includes any effect that results in the improvement of a disease, illness, disorder, etc., such as alleviating, reducing, modulating, ameliorating, mitigating, or eliminating, or ameliorating the symptoms thereof. Treatment may be to cure, improve, or at least partially reverse the disorder. In certain embodiments, treatment is to cure the disease.

[0114] "Active agent" is used to refer to a compound having biological activity or a pharmaceutically acceptable salt thereof. In some embodiments, an "active agent" is a compound having pharmaceutical utility or a pharmaceutically acceptable salt thereof. For example, the active agent may be an anti-neurodegenerative therapeutic agent.

[0115] "Compound" means a chemical compound having a structure depicted by a chemical formula set forth herein, a stereoisomer thereof (including a mixture of stereoisomers), or a pharmaceutically acceptable salt thereof.

[0116] "Pharmaceutically acceptable" includes molecular entities and dosage forms that do not produce adverse, allergic or other abnormal reactions when administered appropriately to animals or humans.

[0117] "Pharmaceutically acceptable salts" refer to salts that retain the biological effectiveness and properties of the free base or free acid. For example, pharmaceutically acceptable salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts, etc. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethylamine, lysine, arginine, N-ethylpiperidine, piperidine, piperazine, etc.

[0118] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that aid in the administration and absorption of an active agent by a subject and can be included in the compositions of the present invention without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, coloring agents, and the like. Such preparations can be sterilized and, if desired, can be mixed with auxiliary substances that do not adversely react with the compounds of the present invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring agents, and / or aromatic substances. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0119] Exemplary pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; synthetic oils; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; phosphate buffers; emulsifiers such as TWEENs®; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffers.

[0120] A "pharmaceutical composition" or "pharmaceutical dosage form" refers to a combination of an active agent with an inert or active carrier.

[0121] An "effective amount" refers to the amount of a compound or composition (e.g., a compound or composition of the present invention) sufficient to achieve a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular dosage form or route of administration.

[0122] "Administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intraspinal, intracranial, intraventricular, intranasal, or subcutaneous administration to a subject, or implantation of a sustained-release device, e.g., a mini-Os pump. Administration can be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. "Coadministration" refers to administration of a composition described herein simultaneously with, immediately before, or immediately after the administration of one or more additional therapies (e.g., anti-cancer agents, chemotherapeutic agents, or agents for treating neurodegenerative diseases). The compounds of the present invention can be administered alone or co-administered to a patient. Co-administration is intended to include simultaneous or sequential administration of compounds individually or in combination (one or more compounds or formulations). Thus, formulations can also be combined with other active agents if desired (e.g., to reduce metabolic degradation).

[0123] The pharmaceutical dosage forms of the present invention can be administered to mammals, e.g., humans, but also to other mammals, e.g., animals in need of veterinary treatment, such as livestock (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0124] The "cognitive function" of a subject can be defined as intellectual activity or function. Examples of intellectual activity or function include, but are not limited to, attention, processing speed, learning and memory, executive function, verbal fluency, and working memory. For example, the composition of the present invention can improve cognitive function if it improves one or more intellectual activities or functions in a subject with impaired cognitive function.

[0125] "Histone deacetylase" or "HDAC" is intended to refer to any member of a family of enzymes that remove Nε-acetyl groups from the ε-amino groups of lysine residues of proteins (e.g., histones or tubulin). Unless the context indicates otherwise, the term "histone" refers to any histone protein, including H1, H2A, H2B, H3, H4, and H5 from any species.

[0126] "HDAC-mediated disease or disorder" or "histone deacetylase-mediated disease or disorder" refers to a disease or disorder in which HDAC and / or HDAC action is important or necessary, for example, for the onset, progression, or development.

[0127] "HDAC enzymatic activity inhibition" or "HDAC inhibition" means reducing the catalytic activity of HDAC or reducing the activity of HDAC unrelated to catalytic activity.

[0128] "Neurological disorders" include neurodegenerative diseases, neuropsychiatric disorders, memory loss, cognitive impairment / damage, and extinction learning disorders.

[0129] "Neurodegenerative disease" means any disorder that can be reversed, prevented, managed, treated, ameliorated, or eliminated by agents that stimulate the generation of new neurons.

[0130] "Memory" refers to the ability to retrieve information about past events and knowledge.

[0131] "Age-related memory loss," as further defined herein, refers to any continuum of conditions characterized by a deterioration in neurological function that does not rise to the level of dementia, as defined in the Diagnostic and Statistical Manual of Mental Disorders: 4th Edition of the American Psychiatric Association (DSM-IV, 1994).

[0132] "Injury-related memory loss" refers to memory loss where there is brain damage and possibly neurological damage as well.

[0133] "Impaired cognitive function" refers to cognitive function that is not as robust as that observed in age-matched normal subjects, and includes states of cognitive decline.

[0134] As used herein, unless otherwise specified, "alkyl" generally refers to C1 to C6 10 and specifically includes methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. This term includes all substituted and unsubstituted alkyl groups, particularly halogenated alkyl groups, and more particularly fluorinated alkyl groups. Non-limiting examples of moieties with which the alkyl group may be substituted are selected from the group consisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, thiol, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, optionally unprotected or protected, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, incorporated herein by reference.

[0135] As used herein, unless otherwise specified, "aryl" refers to an aromatic monocyclic or polycyclic ring system containing 6 to 14 carbon atoms, specifically including phenyl, biphenyl, or naphthyl, preferably phenyl. The term includes all substituted and unsubstituted moieties. Aryl groups may be substituted with any of the described moieties, including, but not limited to, one or more moieties selected from the group consisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, thiol, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, optionally unprotected or protected, as known to those skilled in the art, e.g., as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, incorporated herein by reference.

[0136] As used herein, unless otherwise specified, "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system having 5 to 14 ring atoms, wherein 1 to 4 of the ring atoms are independently O, N, or S, and the remaining ring atoms are carbon atoms, and preferably an aromatic monocyclic ring system having 5 or 6 ring atoms, wherein 1 to 4 of the ring atoms are independently O, N, or S, and the remaining ring atoms are carbon atoms, and specifically includes pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, thiophenyl, imidazolyl, or oxazolyl. Heteroaryl groups may be substituted with any of the described moieties, including, but not limited to, one or more moieties selected from the group consisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, thiol, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, optionally unprotected or protected, as known to those of skill in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, incorporated herein by reference.

[0137] II. Active Compounds

[0138] In one aspect of the present invention, there is provided a compound of the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0139] JPEG2025511114000006.jpg40109

[0140] In the above formula,

[0141] R 1 is C 1-6 Alkyl, C 3-12cycloalkyl, aryl, or heteroaryl, and one or more hydrogen atoms in the group are unsubstituted or each independently substituted with a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, nitro, one or more C 1-6 may be substituted with alkyl-substituted or unsubstituted amino or hydroxy;

[0142] A is C=O or a bond;

[0143] Q is -(CR 4 R 5 ) m - or a bond, where R 4 and R 5 are independently hydrogen or C 1-6 alkyl, and m is an integer from 1 to 6;

[0144] R 2 is hydrogen, C 1-6 alkyl or aryl,

[0145] R 3 is hydrogen, carboxyl, N-hydroxycarboxamido, or carboxamido, and one or more hydrogens in said group are unsubstituted or C 1-6 may be substituted with alkyl,

[0146] n is 0 or 1.

[0147] In one embodiment, the stereoisomer may be an optical isomer. Preferably, the compound may be in the form of the (3R,6S)-enantiomer or the (3S,6R)-enantiomer.

[0148] In one embodiment, R 1 is C 1-6 Alkyl, C 3-12 It may be cycloalkyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl. 1One or more hydrogen atoms in the group may each independently be a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, nitro, one or more C 1-6 It may be substituted with alkyl-substituted or unsubstituted amino or hydroxy. Preferably, R 1 One or more hydrogen atoms in the group may each independently be a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, or one or more C 1-6 It may be substituted with alkyl or unsubstituted amino.

[0149] In one embodiment, R 1 is C 1-6 Alkyl, C 3-12 Cycloalkyl, phenyl, phenyl substituted with one or more halogens, one or more C 1-6 Phenyl substituted with alkyl, one or more halogens and one or more C 1-6 Phenyl substituted with alkyl, one or more C 1-6 Phenyl substituted with alkoxy, unsubstituted or one or more C 1-6 Phenyl substituted with one or more amino groups substituted with alkyl, phenyl substituted with one or more nitro groups, pyridinyl, one or more C 1-6 It may be pyridinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl substituted with alkoxy.

[0150] In other embodiments, R 1may be methyl, cyclohexyl, phenyl, 4-methoxyphenyl, 4-(tert-butyl)phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 3,4-dichlorophenyl, 2-chloro-4-fluorophenyl, p-tolyl, m-tolyl, pyridin-4-yl, thiophen-2-yl, furan-2-yl, pyridin-3-yl, pyridin-2-yl, pyrimidin-5-yl, pyrimidin-2-yl, 4-nitrophenyl, 4-aminophenyl, 3-nitrophenyl, 3-aminophenyl, 4-methoxy-pyridin-3-yl, 6-methoxy-pyridin-3-yl, 4-(dimethylamino)phenyl, or 4-hydroxy-phenyl.

[0151] In one embodiment, R 2 is H or C 1-6 It may also be alkyl.

[0152] In one embodiment, R 3 may be H, carboxyl, methylcarboxyl, ethylcarboxyl, N-hydroxycarboxamide, carboxamide, or sodium carboxylate.

[0153] In one embodiment, A and Q may not simultaneously be bonds.

[0154] In one embodiment, m may be an integer from 1 to 6, an integer from 1 to 3, or an integer from 1 to 2.

[0155] In one embodiment, a compound of formula (2-A) or (2-B) can be provided:

[0156] JPEG2025511114000007.jpg28144

[0157] In the above formula,

[0158] R 4 is C 1-6 Alkyl, C 3-12cycloalkyl, aryl, or heteroaryl, and one or more hydrogen atoms in the group are unsubstituted or each independently substituted with a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, nitro, one or more C 1-6 may be substituted with alkyl-substituted or unsubstituted amino or hydroxy, R 5 and R 6 are each independently hydrogen or C 1-6 It is alkyl.

[0159] In one embodiment, R 4 is C 1-6 Alkyl, C 3-12 It may be cycloalkyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl. 4 One or more hydrogen atoms in the group may each independently be a halogen atom, C 1-6 Alkyl, C 1-6 Alkoxy, nitro, one or more C 1-6 It may be substituted with alkyl-substituted or unsubstituted amino or hydroxy.

[0160] In one embodiment, R 4 is C 1-6 Alkyl, C 3-12 Cycloalkyl, phenyl, phenyl substituted with one or more halogens, one or more C 1-6 Phenyl substituted with alkyl, one or more halogens and one or more C 1-6 Phenyl substituted with alkyl, one or more C 1-6 Phenyl substituted with alkoxy, unsubstituted or one or more C 1-6 Phenyl substituted with one or more amino groups substituted with alkyl, phenyl substituted with one or more nitro groups, pyridinyl, one or more C 1-6 It may be pyridinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl substituted with alkoxy.

[0161] In other embodiments, R4 may be methyl, cyclohexyl, phenyl, 4-methoxyphenyl, 4-(tert-butyl)phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 3,4-dichlorophenyl, 2-chloro-4-fluorophenyl, p-tolyl, m-tolyl, pyridin-4-yl, thiophen-2-yl, furan-2-yl, pyridin-3-yl, pyridin-2-yl, pyrimidin-5-yl, pyrimidin-2-yl, 4-nitrophenyl, 4-aminophenyl, 3-nitrophenyl, 3-aminophenyl, 4-methoxy-pyridin-3-yl, 6-methoxy-pyridin-3-yl, 4-(dimethylamino)phenyl, or 4-hydroxy-phenyl.

[0162] In one embodiment, a compound of formula (4) can be provided:

[0163] JPEG2025511114000008.jpg4165

[0164] In the above formula,

[0165] R 2 is hydrogen, C 1-6 alkyl or aryl,

[0166] R 3 is hydrogen, carboxyl, N-hydroxycarboxamido, or carboxamido, and one or more hydrogens in said group are unsubstituted or C 1-6 may be substituted with alkyl,

[0167] n is 0 or 1.

[0168] Preferably, R 2 is hydrogen or C 1-6 It may also be alkyl.

[0169] In one embodiment, a compound of formula (5) can be provided:

[0170] JPEG2025511114000009.jpg44120

[0171] In the above formula,

[0172] R 2 is hydrogen, C 1-6 alkyl or aryl,

[0173] R 4 is phenyl or phenyl substituted with one or more halogens, preferably 2-chloro-4-fluorophenyl;

[0174] n is 0 or 1.

[0175] Preferably, R 2 is hydrogen or C 1-6 It may also be alkyl.

[0176] In one embodiment, the compound of formula (1) may be provided in the form of a pharmaceutically acceptable salt thereof. Preferably, the salt may be a sodium salt.

[0177] In one embodiment, compounds of Formula (1) include, but are not limited to, the compounds set forth in Table 1 below. BnH12001 set forth in Table 1 below will be used interchangeably hereinafter as "BnH12001 (racemic)" or "12-004."

[0178] [Table 1]

[0179] JPEG2025511114000011.jpg215145

[0180] JPEG2025511114000012.jpg215145

[0181]

[0182] In one aspect, the compounds and compositions described herein may be inhibitors of HDAC. Preferably, the HDAC is HDAC4.

[0183] In other embodiments, the compounds and compositions described herein are agents for modulating synaptic plasticity. Preferably, the synaptic plasticity is synaptic plasticity in the barrel cortex (e.g., layer 4) of the brain.

[0184] III. Pharmaceutical Compositions

[0185] In general, the compounds described herein can be formulated into pharmaceutical compositions using techniques known to those skilled in the art. The concentration of the active compound in the pharmaceutical composition will vary depending on the absorption, inactivation, and excretion rates of the drug, as well as other factors known to those skilled in the art. It should be noted that dosage values ​​will also vary depending on the severity of the condition to be alleviated. For any particular subject, the specific dosage regimen will be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and it should be further understood that the concentration ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions. The active ingredient can be administered at once or divided into multiple smaller doses to be administered at varying time intervals.

[0186] The concentration of inhibitor that reduces HDAC activity to 50% of the activity of uninhibited HDAC is called IC 50 In some embodiments, the reduction in HDAC activity is determined as an IC value of less than 100 nanomolar. In some embodiments, the reduction in HDAC activity is at least 50%, e.g., at least about 75%, e.g., at least about 90%. In some embodiments, the reduction in HDAC activity is at least 95%, e.g., at least 99%. In some embodiments, the compounds described herein have an IC value of less than 100 nanomolar. 50 In some embodiments, the compounds described herein have an IC value of 100 nanomolar to 1 micromolar. 50In some embodiments, the compounds described herein have an IC value of 1 to 25 micromolar. 50 It has a value.

[0187] In some embodiments, such inhibition is specific, i.e., the histone deacetylase inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a protein at a concentration lower than the concentration of the inhibitor required to produce another unrelated biological effect. In some embodiments, the concentration of the inhibitor required for histone deacetylase inhibitory activity is at least 2-fold lower, e.g., at least 5-fold lower, e.g., at least 10-fold lower, e.g., at least 20-fold lower, than the concentration required to produce the unrelated biological effect.

[0188] In some embodiments, the pharmaceutical composition comprises about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg, about 2000 mg, about 2250 mg, about 2500 mg, about 2750 mg, or about 3000 mg of the compound.

[0189] In some embodiments, the pharmaceutical composition contains from about 0.01 mg to about 125 mg, preferably from about 1 mg to about 50 mg, per kg of subject body weight per day, one or more times per day to achieve the desired therapeutic effect.

[0190] In certain embodiments, the composition contains, for example, 0.0001 to 99.9 wt %, preferably 0.001 to 50 wt %, of the compound based on the weight of the total composition. The content ratio is a value based on the dry weight from which the solvent has been removed.

[0191] In certain embodiments, the composition comprises a 5% (w / v), 10% (w / v), 15% (w / v), 20% (w / v), 25% (w / v), 30% (w / v), 35% (w / v), or 40% (w / v) aqueous solution of one or more compounds. In some embodiments, an effective amount of the composition comprises a 25% (w / v) solution of one or more compounds.

[0192] In certain embodiments, the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable excipients, hi some embodiments, the one or more pharmaceutically acceptable excipients are selected from the group comprising a diluent, a buffer, a preservative, a stabilizer, a solubilizer, or any combination thereof.

[0193] In certain embodiments, the pharmaceutical composition can be formulated for administration as a liquid dosage form suitable for oral, intracavity, intradermal, intramuscular, intraspinal, intravenous, subcutaneous, or intracerebroventricular administration.

[0194] In certain embodiments, the liquid dosage form of the pharmaceutical composition as described herein further comprises a diluent. In some embodiments, the inert diluent is saline.

[0195] In certain embodiments, the liquid dosage form of the pharmaceutical composition as described herein further comprises a buffer. For example, buffers suitable for use in the present invention include all organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-fumarate mixture), and the like. Examples of buffers include, but are not limited to, dibasic fumarate and disodium fumarate mixtures, monobasic fumarate and disodium fumarate mixtures, gluconate buffers (e.g., gluconic acid and sodium gluconate mixtures, gluconic acid and sodium hydroxide mixtures, gluconic acid and potassium gluconate mixtures), oxalate buffers (e.g., oxalic acid and sodium oxalate mixtures, oxalic acid and sodium hydroxide mixtures, oxalic acid and potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid and sodium lactate mixtures, lactic acid and sodium hydroxide mixtures, lactic acid and potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid and sodium acetate mixtures, acetic acid and sodium hydroxide mixtures, etc.). Phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can also be used.

[0196] In certain embodiments, the liquid dosage form of the pharmaceutical composition as described herein further comprises a preservative.For example, preservatives suitable for use in the present invention include, but are not limited to, phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens (e.g., methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol).

[0197] In certain embodiments, the liquid dosage form of the pharmaceutical composition as described herein further comprises a stabilizer.For example, suitable stabilizers include, but are not limited to, polyhydric sugar alcohols, trihydric or higher sugar alcohols, amino acids, organic sugars or sugar alcohols, polyvinylpyrrolidone monosaccharides, trisaccharides, polysaccharides, proteins, sulfur-containing reducing agents, amino acid polymers, and polyethylene glycol.

[0198] In certain embodiments, the liquid dosage form of the pharmaceutical composition described herein further comprises a solubilizing agent. In some embodiments, the solubilizing agent is an ionic surfactant. Examples of non-ionic surfactants include, but are not limited to, polysorbates, poloxamers, pluronic polyols, and polyoxyethylene sorbitan monoethers.

[0199] In certain embodiments, the pharmaceutical compositions disclosed herein can be prepared for storage as lyophilized dosage forms or aqueous solutions by mixing the compositions with any pharmaceutically acceptable carrier, excipient, or stabilizer typically used in the art (e.g., buffering agents, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives).

[0200] Where the compound is administered orally, it can be formulated with a pharmaceutically acceptable excipient in the form of a pill, capsule, cachet, tablet, etc.

[0201] Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersing agent. Molded tablets can be prepared by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets can optionally be coated or scored, and are optionally formulated to provide slow or controlled release of the active ingredient. Tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, e.g., gelatin capsules, syrups, or elixirs can be prepared for oral use. Dosage forms of the compounds of the present invention intended for oral use can be prepared by any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more ingredients, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable formulation. Tablets containing the active ingredient are suitably mixed with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture. Such excipients may include, for example, inert diluents such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be coated by known techniques, including uncoated or microencapsulated, to delay disintegration and absorption in the gastrointestinal tract and provide a sustained action over a longer period of time. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate may be used alone or with a wax.

[0202] In certain embodiments, the pharmaceutical compositions herein are administered to a subject by, for example, oral, intracavity, intradermal, intramuscular, intraspinal, intravenous, subcutaneous, or intracerebroventricular administration.

[0203] IV. Biological Assay

[0204] Methods for selecting compounds useful in the present invention are described herein.

[0205] In one embodiment, the compound of the present invention is selected for its ability to interact with HDAC.In one embodiment, HDAC is HDAC4.In another embodiment, the binding of the compound to HDAC is measured by surface plasmon resonance assay.In one embodiment, the compound is BnH12001 or its enantiomer.

[0206] In one embodiment, the compound is selected for its ability to increase the expression of BDNF and / or GluN2B in targeted neurons. In one embodiment, the compound is selected for its ability to increase the expression of BDNF in targeted neurons. In yet another embodiment, the compound is selected for its ability to increase the expression of GluN2B in targeted neurons. In one embodiment, the expression level is measured using RT-PCR. In one embodiment, the compound is selected from the group consisting of BnH12001 or an enantiomer thereof, BnH12005, BnH12006, BnH12007, BnH12009, BnH12010, BnH12016, BnH12018, BnH12025, BnH12038, BnH12039, BnH12060, BnH12061, BnH12063, BnH12065, BnH12068, BnH12069, BnH12070, BnH12073, BnH12074, BnH12079, and BnH12096. Preferably, the compound is BnH12001 or an enantiomer thereof.

[0207] In one embodiment, the compounds of the present invention are selected for their non-toxicity to human cells. In one embodiment, the compounds of the present invention are selected for their lack of significant effect on cell viability of the human cell line SH-SY5Y. In yet another embodiment, the compounds of the present invention are selected for their lack of significant effect on cell viability of the human cell line 293T. In one embodiment, the compounds do not reduce cell viability of SH-SY5Y or 293T at concentrations of about 0.1, 1, 10, or 100 μM. In one embodiment, the compound may be one selected from the compounds listed in Table 5. Preferably, the compound is BnH12001 or an enantiomer thereof.

[0208] In one embodiment, the compounds of the present invention are selected for their ability to increase potency, as measured by Tc EPSC potency in rodent barrel cortex (layer 4). In one embodiment, the compounds increase potency, as measured by Os pump delivery via ventricular infusion, at concentrations of about 0.1 to about 5 μM. In yet another embodiment, the concentration may be about 0.1 to about 3 μM. In yet another embodiment, the concentration may be about 3 μM. In yet another embodiment, the concentration is about 0.03, 0.1, 0.3, 1, 3, or 10 mg / kg. In one embodiment, the compound is BnH12001 or an enantiomer thereof.

[0209] Detailed examples of methods useful for selecting compounds of the present invention are described herein.

[0210] V. Therapeutic Uses of Compounds

[0211] The compound of the present invention can be useful for inhibiting at least one HDAC type.In one aspect, the compound described herein can be therapeutically useful, because certain HDACs are known to be involved in the biological pathway that governs higher brain function.Therefore, the compound described herein can be useful when brain pathology is related to or mediated by HDAC.

[0212] Recent reports have detailed the importance of histone acetylation in central nervous system (CNS) functions such as neuronal differentiation, memory formation, drug addiction, and depression (Citrome, Psychopharmacol. Bull. 2003, 37, Suppl. 2, 74-88; Johannessen, CNS Drug Rev. 2003, 9, 199-216; Tsankova et al., 2006, Nat. Neurosci. 9, 519-525).

[0213] Neurological disorders are diseases that have a component of dysfunction of the central or peripheral nervous system. Neurological disorders can result from developmental abnormalities, disease, genetic defects, injury, or toxins that cause disruption of the structure or function of the nervous system. Such disorders can affect the central nervous system (e.g., brain, brainstem, and cerebellum), the peripheral nervous system (e.g., cranial nerves, spinal nerves, and sympathetic and parasympathetic nervous systems), and / or the autonomic nervous system (e.g., the part of the nervous system that regulates involuntary actions and is divided into the sympathetic and parasympathetic nervous systems).

[0214] Therefore, provided herein is a method for treating or preventing the neurological or neurodegenerative disorders mediated by HDAC in a subject in need of treatment or prevention, comprising administering to the subject a therapeutically effective amount of at least one compound as described herein.In some embodiments, the compound described herein can be useful for regulating the activity of class IIa HDAC.In some embodiments, the compound is an inhibitor of HDAC4.

[0215] Most neurodegenerative disorders are caused by the degeneration of neurons in the brain. Neurons containing HDAC4 are widely distributed in the brain (Mielcarek et al., 2013, PLoS Biol. 11:e1001717; Takase et al., 2013, PLoS ONE 8:e58473). Therefore, a treatment such as the present invention that modulates the activity of HDAC4 in the brain can be useful for any neurodegenerative disease of the brain, because the intervention mechanism contemplated by the present invention can target HDAC4 in neurons regardless of the observed brain pathology. There is no neurodegenerative disease for which the compounds of the present invention are not useful. The compounds of the present invention are not bound by current understanding of the molecular mechanisms involving HDAC4 in brain neurons. Thus, the compounds of the present invention can be useful for any chronic or acute neurodegenerative disorder, as long as the neurons affected by the pathological symptoms of the disease express HDAC4.

[0216] Examples of chronic neurodegenerative disorders include: chronic neurodegenerative diseases, such as familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively), familial and sporadic Parkinson's disease, Huntington's disease, familial and sporadic Alzheimer's disease, multiple sclerosis, muscular dystrophy, olivopontocerebellar atrophy, multiple system atrophy, Wilson's disease, progressive supranuclear palsy, diffuse Lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, striatonigral degeneration, torsion dystonia, familial tremor, Down syndrome, Tourette's syndrome, Hallervorden-Spatz disease, diabetic peripheral neuropathy, dementia pugilistica, AIDS dementia, age-related dementia, age-related memory impairment, and amyloidosis-related neurodegenerative diseases, such as diseases induced by prion protein (PrP) associated with transmissible spongiform encephalopathies (Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, scrapie, and kuru), and diseases caused by excess accumulation of cystatin C (hereditary cystatin C angiopathy). Examples of acute neurodegenerative disorders include traumatic brain injury (e.g., surgery-related brain injury), cerebral edema, peripheral nerve injury, spinal cord injury, Leigh's disease, Guillain-Barré syndrome, lysosomal storage disorders such as lipofuscinosis, Alper's disease, restless legs syndrome, dizziness due to CNS degeneration, etc.; pathologies occurring with chronic alcohol or drug abuse, including, for example, degeneration of neurons in the locus coeruleus and cerebellum, and drug-induced movement disorders; pathologies occurring with aging, including degeneration of cerebellar and cortical neurons leading to cognitive and motor impairments; pathologies occurring with chronic amphetamine abuse, including degeneration of basal ganglia neurons leading to movement disorders; pathological changes due to local trauma, such as stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic encephalopathy, hyperglycemia, hypoglycemia, or direct trauma; pathologies occurring with negative side effects of therapeutic drugs and therapies (e.g., NMD of glutamate receptors). Degeneration of cingulate and entorhinal cortex neurons in response to doses of A-class antagonist anticonvulsants) and Bernice-Korsakoff-related dementia.

[0217] In addition to acute or chronic degenerative disorders, the compounds of the present invention can be useful for treating other types of brain diseases, as long as the affected neurons are partially regulated by HDAC4.Examples of such diseases include: neuropsychiatric disorders related to cognition, dementia, social behavior, anxiety or other personality disorders, such as schizophrenia, delirium, attention deficit disorder (ADD), bipolar disorder, obsessive-compulsive disorder or eating disorder; fear and anxiety disorders, such as panic disorder, various phobias, post-traumatic stress disorder (PTSD), psychogenic erectile dysfunction or insomnia; various forms of addiction; and mood disorders, such as depression.

[0218] The types of diseases that can benefit from the use of the compounds described herein are not limited to brain diseases. Any neurological disorder in which affected neurons are regulated by HDAC4 can potentially benefit from the compounds of the present invention. As described herein, Alzheimer's disease and PTSD, although their pathology and known causes of the disease, are significantly different, have benefited from the treatment of the compounds described herein. In the case of Alzheimer's disease, BnH12 series compounds have reduced disease symptoms and improved memory. Examples of neurological disorders that can benefit from the compounds of the present invention include: peripheral nervous system diseases caused by degeneration or injury, such as ataxia or trauma; and acute or chronic degenerative diseases of the eye, such as glaucoma.

[0219] To the extent that the plasticity of affected neurons is partially regulated by HDAC4, memory loss or cognitive decline can also benefit from treatment with the compounds of the present invention. Examples of such disorders include: memory loss / impairment, disorders associated with cognitive functions such as creativity, problem-solving, and intuition.

[0220] Memory includes short-term memory (also called working or recent memory) and long-term memory. Short-term memory involves recent events, while long-term memory is associated with recalling events from the more distant past. Methods for assessing memory recall ability are known to those skilled in the art and can include routine cognitive tests.

[0221] Age-related memory loss is characterized by objective memory loss at an older age compared to when the subject was younger, but cognitive test performance is within the normal range for the subject's age. Subjects with age-related memory loss score within the normal range on standardized diagnostic tests for dementia, as described in DSM-IV. Furthermore, DSM-IV provides separate diagnostic criteria for a disorder called age-related cognitive decline. Age-related memory loss can include brain mass loss, gyral atrophy, ventricular enlargement, and selective loss of neurons in various brain regions. For some embodiments of the present invention, more progressive forms of memory loss are also included in the definition of age-related memory disorder. Thus, individuals with above-normal memory loss and cognitive impairment but scoring below the diagnostic threshold for Frank dementia may be considered to have mild neurocognitive impairment, mild cognitive impairment, senile amnesia, benign senile amnesia, early dementia, provisional dementia, etc. Such subjects may be somewhat more likely to develop Frank dementia in later life (see also US Patent Application No. 2006 / 008517).

[0222] Symptoms associated with age-related memory loss include, but are not limited to, alterations in biochemical markers associated with the aging brain, such as IL-1β, IFN-γ, p-JNK, and p-ERK, evidenced by a decline in long-term potentiation, memory, and learning, and a decline in synaptic activity or function, such as synaptic plasticity. In some embodiments, the compounds of the present invention modulate the activity of IL-1β, IFN-γ, p-JNK, or p-ERK.

[0223] Methods for improving memory can include memory access resetting and memory retrieval. As used herein, the term access resetting refers to increasing memory retrieval. Without being bound by the mechanism of action, it is believed that the compounds of the present invention are effective in increasing memory retrieval by resetting synaptic networks. The process of resetting synaptic networks can include increasing the number of active brain synapses and / or reversing neuronal loss.

[0224] In some cases, learning is referred to as memory.Learning is different from memory improvement and refers to the ability to generate new memories that did not exist before.In some embodiments, to test the ability of a compound to affect learning ability rather than the ability of a subject to recall old memories, at least one compound of the present invention is administered before or at the same time as memory generation.In some embodiments, to test the ability of a compound to affect the recall of previously generated memories, at least one compound described herein is administered after memory generation, preferably after memory loss.

[0225] Also provided is a method for improving learning and memory formation in a subject suffering from memory loss, comprising administering to the subject an effective amount of at least one compound described herein, which method may be equally useful for improving learning and memory formation in normal subjects.

[0226] Neurogenesis, or the birth of new nerve cells, was previously thought to occur only in developing organisms. However, recent studies have demonstrated that neurogenesis continues into and throughout adulthood. Ongoing neurogenesis is thought to be an important mechanism underlying neuroplasticity, which allows organisms to adapt to environmental changes and influences learning and memory throughout life. In one aspect, the present invention includes a method for increasing synaptic density in a subject, comprising administering to a subject in need thereof a compound of the present invention, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof. In one aspect, the present invention includes a method for increasing synaptic plasticity in a subject, comprising administering to a subject in need thereof a compound of the present invention, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof. In one aspect, the present invention includes a method for increasing neuronal dendritic density in a subject, comprising administering to a subject in need thereof a compound of the present invention, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0227] The present invention provides a method for improving memory in a subject with memory impairment. Examples of types of memory impairment include Alzheimer's disease, absent-minded professor, amnesia, memory loss, anticipatory amnesia, blackout (alcohol-related amnesia), Brom's syndrome, childhood amnesia, false memory syndrome, dissociative fugue state, excess memory, Korsakoff's syndrome, partial amnesia, memory disbelief syndrome, post-traumatic amnesia, prosopamnesia, psychogenic amnesia, suppressed memory, retrograde amnesia, Ribot's Law, selective amnesia, source amnesia, source monitoring error, seven sins of memory, tip of the tongue, transient epileptic amnesia, transient global amnesia, and twilight sleep.

[0228] In one embodiment, the memory disorder is Alzheimer's disease. Such a method comprises selectively administering an inhibitor and monitoring the subject to confirm the recovery of previously lost memory. The subject can be monitored by routine tests known in the art.

[0229] In another embodiment, the subject with Alzheimer's disease is a subject suffering from end-stage Alzheimer's disease. Many compounds proposed for the treatment of Alzheimer's disease are designed to prevent plaque buildup and treat the early stages of the disease. The compounds of the present invention are useful for treating both early and late-stage dementia because they improve memory and cognition rather than simply preventing plaque buildup.

[0230] In one embodiment, the disease or disorder mediated by HDAC is cognitive dysfunction / injury.Therefore, also provided is the method for promoting cognitive function in the subject suffering from cognitive dysfunction / injury, comprising administering to subject an effective amount of at least one compound as described herein.The present invention also provides the method for promoting cognitive function in normal subject.

[0231] In some cases, cognitive function is reduced by about 5%, about 10%, about 30%, or more compared to cognitive function measured in age-matched normal subjects. Cognitive function can be enhanced to any detectable degree, but in humans, preferably is enhanced sufficiently to enable the injured subject to carry out the daily activities of normal life.

[0232] Cognitive function can be assessed and optionally defined by one or more tests or assays of cognitive function. Non-limiting examples of cognitive tests or assays include the CANTAB (see, e.g., Fray et al. "CANTAB battery: proposed utility in neurotoxicology." Neurotoxicol Teratol 1996; 18(4):499-504), the Stroop Test, Trail Making, Wechsler Digit Span, or the CogState computerized cognitive test (see, e.g., Dehaene et al. "Reward-dependent learning in neural networks for planning and decision making." Brain Res. Brain Res. 2000; 126:21729; Iverson et al. "Interpreting change on the WAIS-III / WMS-I11 in clinical samples." Arch Clin Neuropsychol. 2001; 16(2):183-91; and Weaver et al. "Mild memory impairment in healthy older adults is distinct from normal aging." Cogn. 2006; 60(2):146-55). The methods of the present invention can be used to promote cognitive function in a normal subject, treat / cure, alleviate / ameliorate a subject with cognitive impairment, or prevent cognitive impairment in a subject. As used herein, a normal subject is a subject who has not been diagnosed with a disorder associated with cognitive impairment.

[0233] In some embodiments, the cognitive dysfunction or damage is associated with, but is not limited to, Alzheimer's disease, Huntington's disease, seizure-induced memory loss, schizophrenia, Rubinstein-Taybi syndrome, Rett syndrome, Fragile X syndrome, dementia with Lewy bodies, vascular dementia, bipolar disorder and social, cognitive and learning disorders associated with autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), dyslexia, learning disabilities, traumatic head injury, stroke-induced cognitive and motor disorders, traumatic brain injury, neurodegeneration and neuronal loss-mediated cognitive disorders, and attention deficit disorder.

[0234] In some embodiments, the cognitive dysfunction or damage is associated with, but is not limited to, anxiety disorders, conditioned fear responses, panic disorders, obsessive-compulsive disorders, post-traumatic stress disorders, phobias, social anxiety disorders, substance dependence recovery, or age-associated memory impairment (AAMI), and age-related cognitive decline (ARCD).

[0235] It has been demonstrated that administration of HDAC inhibitors sodium butyrate or trichostatin A promotes fear extinction in mice, and this promotion reflects that induced by commonly used behavioral manipulations, and is consistent with other studies showing the role of the hippocampus in the extinction of contextual fear (reference [Lattal, et al., 2007, Behav. Neurosci. 121, 5, 1125-1131]). In one embodiment, the disease or disorder mediated by HDAC is extinction learning disorder, for example, fear extinction deficiency.Therefore, also provided is a method for treating or preventing extinction learning disorder in a subject, comprising administering an effective amount of at least one compound described herein to a subject suffering from extinction learning disorder.

[0236] The compounds of the present invention can be used to facilitate the psychological process of extinction learning, thereby being useful in treating, alleviating, and / or preventing neuropsychiatric and other related disorders. Unlike traditional anxiolytics, which are administered chronically and treat the physiological symptoms of anxiety, the compounds of the present invention can be used long-term or short-term in conjunction with a second therapy, e.g., psychotherapy.

[0237] Although not thoroughly investigated herein, HDAC4 mediates gene expression in other cell types, including immune cells.The compounds of the present invention are not limited to the current understanding of the molecular mechanisms involved in HDAC4 in immune cells or other types of cells found in cancer.Thus, the compounds of the present invention can be useful for any disease, including hyperproliferative diseases, such as malignant tumors of various origins; immune system diseases, such as autoimmune diseases, inflammation or allergies; infectious diseases; transplant rejection; and fibrotic diseases.

[0238] In some embodiments, the disease or disorder mediated by HDAC is an immune disorder that can be co-treated with TNFα or other immunomodulatory agents when a therapeutically effective amount of at least one compound as described herein is administered to the subject.Therefore, also provided is a method for treating an immune disorder mediated by HDAC in a subject, comprising administering a therapeutically effective amount of at least one compound as described herein to a subject in need of treatment for an immune disorder before, during, or after treatment with TNFα or other immunomodulatory agents.

[0239] In some embodiments, HDAC-mediated diseases or disorders include disorders that can also be treated by progenitor / stem cell-based therapy, such as: diabetes-related disorders (organ failure, cirrhosis and hepatitis); central nervous system (CNS) disorders associated with dysregulation of progenitor cells in the brain (e.g., PTSD); tumors (e.g., retinoblastoma); disorders that affect oligodendrocyte progenitor cells (e.g., astrocytoma and ventricular cell tumor); multiple sclerosis; demyelinating disorders such as leukodystrophy; neurological disorders associated with white matter loss; cerebellar disorders such as ataxia; and olfactory progenitor cell disorders (e.g., anosmia).Therefore, also provided is a method for treating HDAC-mediated disorders in a subject, comprising administering a therapeutically effective amount of at least one compound described herein to a subject in need of HDAC-mediated disorder treatment before, during, or after treatment with progenitor / stem cell-based therapy.

[0240] In some embodiments, the disease or disorder mediated by HDAC comprises blood pressure disorders associated with nitric oxide (NO) regulation (e.g., hypertension, erectile dysfunction, asthma; and ocular disorders such as glaucoma).Therefore, also provided is a method for treating blood pressure disorders associated with HDAC-mediated nitric oxide (NO) regulation in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound described herein.

[0241] In some embodiments, the disease or disorder is a cardiac hypertrophy disorder.Therefore, also provided is a method for treating a cardiac hypertrophy disorder mediated by HDAC in a subject, comprising administering a therapeutically effective amount of at least one compound described herein to the subject in need of treatment of a cardiac hypertrophy disorder mediated by HDAC.

[0242] In some embodiments, the HDAC-mediated disease or disorder includes hematological disorders, such as thalassemia, anemia, and sickle cell anemia. Accordingly, there is also provided a method for treating an HDAC-mediated blood disorder in a subject, comprising administering a therapeutically effective amount of at least one compound described herein to a subject in need of such treatment.

[0243] In some embodiments, the disease or disorder mediated by HDAC comprises a metabolic disease, such as prediabetes or diabetes (type 1 or type 2). Accordingly, methods of treating a metabolic disease in a subject, including administering a therapeutically effective amount of at least one compound described herein to a subject in need of treatment for a metabolic disease, such as prediabetes or diabetes (type 1 or type 2) mediated by HDAC, are also provided.

[0244] In one aspect, the present invention provides a pharmaceutical composition further comprising a second therapeutic agent for treating a disease or disorder mediated by HDAC in a subject suffering from the disease or disorder mediated by HDAC.In some embodiments, the second therapeutic agent is formulated to treat a disease or disorder mediated by HDAC, preferably HDAC4.

[0245] Also provided are methods for treating an HDAC-mediated disease or disorder in a subject suffering from an HDAC-mediated disease or disorder, wherein at least one compound described herein, or a pharmaceutically acceptable salt thereof, is administered to the subject along with one or more additional active agents.

[0246] In some embodiments, the second therapeutic agent is selected from the group consisting of donepezil, rivastigmine, galantamine, memantine, verubecestat, solanezumab, bapineuzumab, aducanumab, lecanemab, tideglusib, epothilone D, and ABBV-8E12.

[0247] In some embodiments, the second therapeutic agent is selected from the group consisting of a cholinesterase inhibitor, an NMDA receptor antagonist, a humanized antibody targeting tau protein, a humanized antibody targeting amyloid beta protein, and a BACE inhibitor.

[0248] Exemplary treatments encompassed by the present invention include combination therapy. For example, the combination therapy may be a drug therapy (i.e., a compound of the present invention) and behavioral therapy. Behavioral therapy includes, but is not limited to, electroconvulsive seizure therapy, exercise, group therapy, conversation therapy, or conditioning. In yet another embodiment, the behavioral therapy is cognitive behavioral therapy. Examples of behavioral therapies that can be used in the present method are described, for example, in Cognitive-Behavioral Therapies by K. Dobson, ed., Guilford Publications, Inc., 2002 and The New Handbook of Cognitive Therapy: Basics and Beyond by Judith S.S. Beck, Guilford Publications, Inc., 1995, both of which are incorporated herein by reference in their entireties. Any pharmaceutically active ingredient recognized by those skilled in the art as a pharmacological agent that enhances learning or conditioning can be used in the method of the present invention. For example, one class of pharmaceutically active ingredients contemplated herein includes compounds that increase norepinephrine levels in the brain. Such compounds include compounds that act as norepinephrine reuptake inhibitors, such as tomoxetine, reboxetine, duloxetine, venlafaxine, and milnacipran, and compounds that induce the release of norepinephrine, such as amphetamine, dextroamphetamine, pemoline, and methylphenidate.Another class of such pharmacoactive ingredients is compounds that increase the level of acetylcholine in the brain, including, for example, compounds that block the breakdown of acetylcholine.Examples of such compounds include donepezil HCl or Aricept, which inhibit cholinesterase activity. TM and tacrine.

[0249] A method for combination therapy can include administering to a subject one or more sessions of a combination therapy protocol, wherein the combination therapy protocol includes acute administration of a therapeutically effective amount of a compound of the present invention that improves learning or conditioning, along with a psychotherapy session. In one embodiment, exposure to the compound occurs within about 24 hours, preferably within about 12 hours, more preferably within about 6 hours, before starting the psychotherapy session. A full course of treatment for a neuropsychiatric disorder involves at least one session of such a combination therapy protocol.

[0250] The present invention will now be described in more detail by the following examples, which are presented to aid in the understanding of the present invention and are not intended to, and should not be construed as, limiting its scope in any way.

[0251] Methods for obtaining the compounds described herein, or pharmaceutically acceptable salts thereof, will be apparent to those skilled in the art, and suitable procedures are described, for example, in the preparations below and in the references cited therein.

[0252] VI. Methods for producing the active compounds

[0253] Methods for synthesizing the compounds of the present invention are provided herein. The names and structures of specific exemplified compounds are provided in Tables 2-4.

[0254] [Table 2]

[0255] JPEG2025511114000014.jpg211146

[0256] JPEG2025511114000015.jpg211146

[0257] JPEG2025511114000016.jpg54146

[0258]

[0259] [Table 3]

[0260] JPEG2025511114000018.jpg211146

[0261] JPEG2025511114000019.jpg158146

[0262]

[0263] [Table 4]

[0264] JPEG2025511114000021.jpg212146

[0265] JPEG2025511114000022.jpg126146

[0266]

[0267] Methods for obtaining the compounds described herein, their stereoisomers, or pharmaceutically acceptable salts thereof will be apparent to those of ordinary skill in the art, and suitable procedures are described, for example, in the preparations and examples below and in the references cited therein.

[0268] Manufacturing example

[0269] Preparation Example 1. Preparation of active compound according to representative reaction scheme 1

[0270] [Reaction Scheme 1]

[0271] JPEG2025511114000023.jpg33143

[0272] JPEG2025511114000024.jpg177146

[0273] JPEG2025511114000025.jpg176146

[0274] (1) Synthesis of compound BnH12001 enantiomer (3R,6S, 98% or more)

[0275] (3R,6S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0276] Step 1: Synthesis of methyl (3R,6S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (compound BnH12020)

[0277] To a solution of 2-chloro-4-fluorophenylacetic acid (2.22 g, 11.8 mmol) in DMF (40.0 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (1.94 g, 12.4 mmol), DIPEA (3.08 mL, 17.7 mmol), HOBt (1.91 g, 14.1 mmol), and EDC·HCl (2.19 g, 14.1 mmol). The mixture was stirred at room temperature for 3 hours and 30 minutes. The mixture was diluted with EtOAc (40.0 mL) and washed with saturated aqueous NH₄Cl (40.0 mL) and brine (40.0 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane: EtOAc = 3:1 to 2:1) to give the desired compound (3.28 g, 85%) as a colorless oil.

[0278] 1H NMR (400 MHz, Chloroform-d) δ 7.14 (dd, J = 8.5, 2.6 Hz, 1H), 6.96 (td, J = 8.3, 2.6 Hz, 1H), 4.97 (s, 0.5H), 4.80 (d, J = 13.8 Hz, 0.5H), 4.16 (s, 0.5H), 3.90 (d, J = 13.9 Hz, 0.5H), 3.82 - 3.71 (m, 2H), 3.24 (t, J = 13.1 Hz, 0.5H), 2.81 (t, J = 12.9 Hz, 0.5H), 2.38 (d, J = 35.9 Hz, 1H), 1.98 (d, J = 13.2 Hz, 1H), 1.90 - 1.75 (m, 1H), 1.64 (s, 2H), 1.28 - 1.16 (m, 3H).

[0279] Step 2: Synthesis of compound BnH12001 enantiomer (3S,6R)

[0280] To a solution of methyl (3R,6S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (3.20 g, 9.76 mmol) in THF (33.7 mL) was added 1N NaOH (14.6 mL, 14.6 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (15.0 mL) and washed twice with DCM (30.0 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (30.0 mL). The organic layer was washed with brine (30.0 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. EtO (30.0 mL) was added to the residue and stirred at room temperature for 1 hour. The precipitate was filtered and washed with Et2O (10.0 mL) to give compound BnH12001 enantiomer (3S,6R) (2.75 g, 89%) as a white solid.

[0281] 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 7.40 (dd, J = 8.9, 2.6 Hz, 1H), 7.34 (dt, J = 15.2, 7.6 Hz, 1H), 7.16 (td, J = 8.5, 2.7 Hz, 1H), 4.70 (s, 0.5H), 4.49 (d, J = 13.5 Hz, 0.5H), 4.27 (s, 0.5H), 3.97 - 3.81 (m, 1H), 3.81 - 3.68 (m, 1.5H), 3.14 (t, J = 12.8 Hz, 0.5H), 2.64 (t, J = 12.6 Hz, 0.5H), 2.40 - 2.31 (m, 0.5H), 2.21 (s, 0.5H), 1.84 (d, J = 12.2 Hz, 1H), 1.76 - 1.60 (m, 2H), 1.55 (s, 1H), 1.14 (dd, J = 56.8, 6.9Hz, 3H).

[0282] (2) Synthesis of compound BnH12006

[0283] (3R,6S)-1-(2-cyclohexylacetyl)-6-methylpiperidine-3-carboxylic acid

[0284] Step 1: Synthesis of methyl (3R,6S)-1-(2-cyclohexylacetyl)-6-methylpiperidine-3-carboxylate

[0285] To a solution of cyclohexylacetic acid (100 mg, 0.703 mmol) in DMF (2.34 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (116 mg, 0.738 mmol), DIPEA (0.184 mL, 1.06 mmol), HOBt (114 mg, 0.844 mmol), and EDC·HCl (131 mg, 0.844 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 3:1 to 2:1) to give the desired compound (147 mg, 74%) as a colorless oil.

[0286] Step 2: Synthesis of compound BnH12006

[0287] To a solution of methyl (3R,6S)-1-(2-cyclohexylacetyl)-6-methylpiperidine-3-carboxylate (147 mg, 0.522 mmol) in THF (1.80 mL) was added 1N NaOH (0.784 mL, 0.784 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1–2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO (DCM:MeOH = 95:5 to 90:10) to afford compound BnH12006 (102 mg, 73%) as a white solid.

[0288] 1H NMR (400 MHz, Chloroform-d) δ 4.99 (s, 0.5H), 4.81 (s, 0.5H), 4.19 (s, 0.5H), 3.91 (s, 0.5H), 3.22 (s, 0.5H), 2.75 (s, 0.5H), 2.48 - 2.36 (m, 1H), 2.22 (s, 2H), 1.98 (s, 1H), 1.90 - 1.58 (m, 11H), 1.34 - 1.05 (m, 8H), 0.96 (d, J = 12.0 Hz, 2H).

[0289] (3) Synthesis of compound BnH12007

[0290] (3R,6S)-6-Methyl-1-(2-phenylacetyl)piperidine-3-carboxylic acid

[0291] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-phenylacetyl)piperidine-3-carboxylate

[0292] To a solution of phenylacetic acid (100 mg, 0.734 mmol) in DMF (2.45 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (121 mg, 0.771 mmol), DIPEA (0.192 mL, 1.10 mmol), HOBt (119 mg, 0.881 mmol), and EDC·HCl (137 mg, 0.881 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 3:1 to 2:1) to give the desired compound (135 mg, 66%) as a colorless oil.

[0293] Step 2: Synthesis of compound BnH12007

[0294] To a solution of methyl (3R,6S)-6-methyl-1-(2-phenylacetyl)piperidine-3-carboxylate (135 mg, 0.490 mmol) in THF (1.69 mL) was added 1N NaOH (0.735 mL, 0.735 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO (DCM:MeOH = 95:5 to 90:10) to afford compound BnH12007 (105 mg, 82%) as a white solid.

[0295] 1H NMR (400 MHz, Chloroform-d) δ 7.32 (t, J = 7.5 Hz, 2H), 7.28 - 7.20 (m, 3H), 4.97 (s, 0.5H), 4.82 (d, J = 13.7 Hz, 0.5H), 4.17 (s, 0.5H), 3.91 (d, J = 13.7 Hz, 0.5H), 3.76 (s, 2H), 3.12 (t, J = 12.9 Hz, 0.5H), 2.77 (t, J = 12.7 Hz, 0.5H), 2.39 (s, 0.5H), 2.05 (s, 0.5H), 1.90 (d, J = 13.2 Hz, 1H), 1.77 (d, J = 14.3 Hz, 1H), 1.66 - 1.36 (m, 2H), 1.17 - 1.06 (m, 3H).

[0296] (4) Synthesis of compound BnH12008

[0297] (3R,6S)-6-Methyl-1-propionylpiperidine-3-carboxylic acid

[0298] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-propionylpiperidine-3-carboxylate

[0299] To a solution of propionic acid (0.051 mL, 0.675 mmol) in DMF (2.25 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (111 mg, 0.709 mmol), DIPEA (0.178 mL, 1.01 mmol), HOBt (109 mg, 0.810 mmol), and EDC·HCl (126 mg, 0.810 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 3:1 to 2:1) to give the desired compound (83 mg, 57%) as a colorless oil.

[0300] Step 2: Synthesis of compound BnH12008

[0301] To a solution of methyl (3R,6S)-6-methyl-1-propionylpiperidine-3-carboxylate (80.0 mg, 0.375 mmol) in THF (1.29 mL) was added 1N NaOH (0.563 mL, 0.563 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO (DCM:MeOH = 95:5 to 90:10) to afford compound BnH12008 (43.0 mg, 57%) as a white solid.

[0302] 1H NMR (400 MHz, Chloroform-d) δ 4.96 (s, 0.5H), 4.81 (s, 0.5H), 4.17 (s, 0.5H), 3.89 (s, 0.5H), 3.22 (s, 0.5H), 2.77 (s, 0.5H), 2.50 - 2.28 (m, 4H), 2.00 (d, J = 13.6 Hz, 1H), 1.84 (qd, J = 12.1, 5.5 Hz, 2H), 1.67 (s, 3H), 1.30 - 1.06 (m, 9H).

[0303] (5) Synthesis of compound BnH12011

[0304] (3R,6S)-1-(2-chloro-4-fluorobenzoyl)-6-methylpiperidine-3-carboxylic acid

[0305] Step 1: Synthesis of methyl (3R,6S)-1-(2-chloro-4-fluorobenzoyl)-6-methylpiperidine-3-carboxylate

[0306] To a solution of 2-chloro-4-fluorobenzoic acid (100 mg, 0.573 mmol) in DMF (1.91 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (94.6 mg, 0.602 mmol), DIPEA (0.150 mL, 0.859 mmol), HOBt (92.9 mg, 0.687 mmol), and EDC·HCl (107 mg, 0.687 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with EtOAc (3.00 mL) and washed with saturated aqueous NH₄Cl (3.00 mL) and brine (3.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (123 mg, 71%) as a colorless oil.

[0307] Step 2: Synthesis of compound BnH12011

[0308] To a solution of methyl (3R,6S)-1-(2-chloro-4-fluorobenzoyl)-6-methylpiperidine-3-carboxylate (123.0 mg, 0.392 mmol) in THF (1.35 mL) was added 1N NaOH (0.588 mL, 0.588 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to provide compound BnH12011 (87.0 mg, 74%) as a white solid.

[0309] 1H NMR (400 MHz, Chloroform-d) δ 7.22 (s, 1H), 7.20 - 7.11 (m, 1H), 7.04 (td, J = 8.3, 2.5 Hz, 1H), 5.10 (s, 0.5H), 4.92 (d, J = 12.9 Hz, 0.5H), 3.69 (s, 0.5H), 3.44 - 3.28 (m, 1H), 3.17 (s, 0.5H), 2.99 (dt, J = 26.2, 12.9 Hz, 0.5H), 2.71 - 2.52 (m, 1H), 2.35 (d, J = 6.7 Hz, 0.5H), 2.04 (d, J = 11.9 Hz, 1H), 1.93 - 1.69 (m, 2H), 1.60 (d, J = 11.8 Hz, 1H), 1.32 - 1.14 (m, 3H).

[0310] (6) Synthesis of compound BnH12023

[0311] (3R,6S)-1-(2-(4-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0312] Step 1: Synthesis of methyl (3R,6S)-1-(2-(4-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0313] To a solution of 2-(4-chlorophenyl)acetic acid (100 mg, 0.586 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (96.8 mg, 0.616 mmol), DIPEA (0.153 mL, 0.879 mmol), HOBt (95.1 mg, 0.703 mmol), and EDC·HCl (109 mg, 0.703 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH4Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane:EtOAc = 2:1) to give the desired compound (81.5 mg, 44%) as a pale yellow oil.

[0314] Step 2: Synthesis of compound BnH12023

[0315] To a solution of methyl (3R,6S)-1-(2-(4-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (70.0 mg, 0.226 mmol) in THF (1.00 mL) was added 1N NaOH (0.339 mL, 0.339 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to provide compound BnH12023 (57.0 mg, 85%) as a white solid.

[0316] 1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.23 (dd, J = 18.1, 8.1 Hz, 2H), 4.72 (s, 0.5H), 4.51 (dd, J = 13.3, 4.2 Hz, 0.5H), 4.23 (s, 0.5H), 3.87 (d, J = 13.6 Hz, 0.5H), 3.74 (q, J = 12.6, 9.6 Hz, 2H), 3.02 (t, J = 12.9 Hz, 0.5H), 2.60 (t, J = 12.8 Hz, 0.5H), 2.17 (tt, J = 11.9, 4.3 Hz, 1H), 1.81 - 1.77 (m, 1H), 1.70 - 1.60 (m, 3H), 1.51 - 1.48 (m, 2H), 1.05 (dd, J = 12.5, 6.8 Hz, 3H).

[0317] (7) Synthesis of compound BnH12024

[0318] (3R,6S)-6-Methyl-1-(2-(p-tolyl)acetyl)piperidine-3-carboxylic acid

[0319] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(p-tolyl)acetyl)piperidine-3-carboxylate

[0320] To a solution of p-tolylacetic acid (100 mg, 0.666 mmol) in DMF (2.22 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (110 mg, 0.699 mmol), DIPEA (0.174 mL, 0.999 mmol), HOBt (108 mg, 0.799 mmol), and EDC·HCl (124 mg, 0.799 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 2:1 to 1:1) to give the desired compound (156 mg, 85%) as a colorless oil.

[0321] Step 2: Synthesis of compound BnH12024

[0322] To a solution of methyl (3R,6S)-6-methyl-1-(2-(p-tolyl)acetyl)piperidine-3-carboxylate (156 mg, 0.539 mmol) in THF (1.86 mL) was added 1N NaOH (0.809 mL, 0.809 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (1.50 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12024 (135 mg, 90%) as a white solid.

[0323] 1H NMR (400 MHz, Chloroform-d) δ 7.26 (s, 1H), 7.12 (s, 2H), 4.96 (s, 0.5H), 4.81 (d, J = 13.7 Hz, 0.5H), 4.17 (s, 0.5H), 3.91 (d, J = 14.0 Hz, 0.5H), 3.71 (s, 2H), 3.11 (t, J = 12.9 Hz, 0.5H), 2.76 (t, J = 12.8 Hz, 0.5H), 2.38 (s, 0.5H), 2.32 (s, 3H), 2.09 (d, J = 9.0 Hz, 0.5H), 1.90 (dd, J = 13.2, 4.0 Hz, 1H), 1.77 (d, J = 13.5 Hz, 1H), 1.67 - 1.34 (m, 2H), 1.14 - 1.03 (m, 3H).

[0324] (8) Synthesis of compound BnH12025

[0325] (3R,6S)-1-(2-(4-methoxyphenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0326] Step 1: Synthesis of methyl (3R,6S)-1-(2-(4-methoxyphenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0327] To a solution of 4-methoxyphenylacetic acid (100 mg, 0.602 mmol) in DMF (2.01 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (99.3 mg, 0.632 mmol), DIPEA (0.157 mL, 0.903 mmol), HOBt (97.6 mg, 0.722 mmol), and EDC·HCl (112 mg, 0.722 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 2:1 to 1:1) to give the desired compound (147 mg, 80%) as a colorless oil.

[0328] Step 2: Synthesis of compound BnH12025

[0329] To a solution of methyl (3R,6S)-1-(2-(4-methoxyphenyl)acetyl)-6-methylpiperidine-3-carboxylate (145 mg, 0.475 mmol) in THF (1.64 mL) was added 1N NaOH (0.712 mL, 0.712 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (1.50 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to provide compound BnH12025 (114 mg, 82%) as a white solid.

[0330] 1H NMR (400 MHz, Chloroform-d) δ 7.19 - 7.12 (m, 2H), 6.85 (d, J = 8.3 Hz, 2H), 4.96 (s, 0.5H), 4.80 (d, J = 13.7 Hz, 0.5H), 4.17 (s, 0.5H), 3.92 (d, J = 14.0 Hz, 0.5H), 3.79 (s, 3H), 3.69 (s, 2H), 3.12 (t, J = 12.9 Hz, 0.5H), 2.77 (t, J = 12.8 Hz, 0.5H), 2.36 (d, J = 13.1 Hz, 0.5H), 2.09 (d, J = 7.1 Hz, 0.5H), 1.90 (dd, J = 13.5, 3.9 Hz, 1H), 1.77 (q, J = 12.5 Hz, 1H), 1.64 - 1.38 (m, 2H), 1.12 (t, J = 8.2 Hz, 3H).

[0331] (9) Synthesis of compound BnH12026

[0332] (3R,6S)-1-(2-(3-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0333] Step 1: Synthesis of methyl (3R,6S)-1-(2-(3-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0334] To a solution of 2-(3-chlorophenyl)acetic acid (100 mg, 0.586 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (96.8 mg, 0.616 mmol), DIPEA (0.153 mL, 0.879 mmol), HOBt (95.1 mg, 0.703 mmol), and EDC·HCl (109 mg, 0.703 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH4Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane:EtOAc = 2:1) to give the desired compound (123 mg, 67%) as a pale yellow oil.

[0335] Step 2: Synthesis of compound BnH12026

[0336] To a solution of methyl (3R,6S)-1-(2-(3-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (100 mg, 0.323 mmol) in THF (1.50 mL) was added 1N NaOH (0.484 mL, 0.484 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to provide compound BnH12026 (73.8 mg, 77%) as a white solid.

[0337] 1H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 1H), 7.32 (dt, J = 17.1, 8.8 Hz, 3H), 7.18 (dd, J = 17.6, 7.3 Hz, 1H), 4.73 (s, 0.5H), 4.52 (d, J = 13.4 Hz, 0.5H), 4.25 (s, 0.5H), 3.90 (d, J = 14.0 Hz, 0.5H), 3.77 (q, J = 13.0, 10.3 Hz, 2H), 3.04 (t, J = 13.1 Hz, 0.5H), 2.62 (t, J = 12.8Hz, 0.5H), 2.18 (t, J = 13.1 Hz, 1H), 1.81 (d, J = 12.7 Hz, 1H), 1.67 (d, J = 12.8 Hz, 1H), 1.53 (s, 2H), 1.07 (dd, J = 15.6, 7.1 Hz, 3H).

[0338] (10) Synthesis of compound BnH12027

[0339] (3R,6S)-1-(2-(2-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0340] Step 1: Synthesis of methyl (3R,6S)-1-(2-(2-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0341] To a solution of 2-chlorophenylacetic acid (100 mg, 0.586 mmol) in DMF (1.95 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (96.8 mg, 0.616 mmol), DIPEA (0.153 mL, 0.879 mmol), HOBt (95.1 mg, 0.703 mmol), and EDC·HCl (109 mg, 0.703 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with EtOAc (3.00 mL) and washed with saturated aqueous NH4Cl (3.00 mL) and brine (3.00 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane:EtOAc = 2:1 to 1:1) to give the desired compound (148 mg, 85%) as a colorless oil.

[0342] Step 2: Synthesis of compound BnH12027

[0343] To a solution of methyl (3R,6S)-1-(2-(2-chlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (145 mg, 0.468 mmol) in THF (1.61 mL) was added 1N NaOH (0.702 mL, 0.702 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (1.50 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12027 (122 mg, 88%) as a white solid.

[0344] 1H NMR (400 MHz, Chloroform-d) δ 7.38 (dd, J = 7.0, 2.3 Hz, 1H), 7.29 (s, 1H), 7.21 (ddd, J = 9.7, 6.7, 2.4 Hz, 2H), 4.98 (s, 0.5H), 4.82 (d, J = 13.8 Hz, 0.5H), 4.13 (s, 0.5H), 3.86 (dd, J = 20.1, 13.7 Hz, 2.5H), 3.21 (t, J = 12.9 Hz, 0.5H), 2.81 (t, J = 12.8 Hz, 0.5H), 2.42 (s, 0.5H), 2.28 (s, 0.5H), 1.99 - 1.91 (m, 1H), 1.82 (dd, J = 13.1, 6.6 Hz, 1H), 1.72 - 1.51 (m, 2H), 1.18 (t, J = 6.7 Hz, 3H).

[0345] (11) Synthesis of compound BnH12028

[0346] (3R,6S)-1-(2-(3,4-dichlorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0347] Step 1: Synthesis of methyl (3R,6S)-1-(2-(3,4-dichlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0348] To a solution of 2-(3,4-dichlorophenyl)acetic acid (100 mg, 0.488 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (80.5 mg, 0.512 mmol), DIPEA (0.127 mL, 0.732 mmol), HOBt (79.1 mg, 0.585 mmol), and EDC·HCl (90.9 mg, 0.585 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (120 mg, 71%) as a gray oil.

[0349] Step 2: Synthesis of compound BnH12028

[0350] To a solution of methyl (3R,6S)-1-(2-(3,4-dichlorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (110 mg, 0.320 mmol) in THF (2.00 mL) was added 1N NaOH (0.479 mL, 0.479 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12028 (103 mg, 97%) as a white solid.

[0351] 1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 7.56 - 7.46 (m, 2H), 7.23 - 7.19 (m, 1H), 4.71 (s, 0.5H), 4.50 (dd, J = 13.5, 4.3 Hz, 0.5H), 4.25 (s, 0.5H), 3.89 (dd, J = 14.4, 4.1 Hz, 0.5H), 3.84 - 3.69 (m, 2H), 3.06 (t, J = 12.9 Hz, 0.5H), 2.62 (t, J = 12.8 Hz, 0.5H), 2.28 - 2.16 (m, 1H), 1.83 - 1.78 (m, 1H), 1.72 - 1.62 (m, 1H) 1.57 - 1.50 (m, 2H), 1.09 (dd, J = 28.9, 6.9 Hz, 3H).

[0352] (12) Synthesis of compound BnH12029

[0353] (3R,6S)-1-(2-(4-(tert-butyl)phenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0354] Step 1: Synthesis of methyl (3R,6S)-1-(2-(4-(tert-butyl)phenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0355] To a solution of 2-(4-(tert-butyl)phenyl)acetic acid (100 mg, 0.520 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (85.9 mg, 0.546 mmol), DIPEA (0.136 mL, 0.780 mmol), HOBt (84.3 mg, 0.624 mmol), and EDC·HCl (96.9 mg, 0.624 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane: EtOAc = 2:1) to give the desired compound (114 mg, 66%) as a yellow oil.

[0356] Step 2: Synthesis of compound BnH12029

[0357] To a solution of methyl (3R,6S)-1-(2-(4-(tert-butyl)phenyl)acetyl)-6-methylpiperidine-3-carboxylate (95.0 mg, 0.287 mmol) in THF (2.00 mL) was added 1N NaOH (0.430 mL, 0.430 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12029 (78.2 mg, 86%) as a white solid.

[0358] 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 7.32 (d, J = 7.8 Hz, 2H), 7.12 (dd, J = 17.8, 7.9 Hz, 2H), 4.73 (s, 0.5H), 4.55 - 4.50 (m, 0.5H), 4.24 (s, 1H), 3.89 - 3.85 (m, 0.5H), 3.67 (q, J = 15.4 Hz, 2H), 3.00 (t, J = 12.8 Hz, 0.5H), 2.59 (t, J = 12.7 Hz, 0.5H), 2.18 - 2.13 (m, 1H), 1.80 - 1.76 (m, 1H), 1.69 - 1.60 (m, 1H), 1.51 - 1.49 (m, 2H), 1.26 (s, 9H), 1.04 (d, J = 6.9 Hz, 3H).

[0359] (13) Synthesis of compound BnH12030

[0360] (3R,6S)-6-Methyl-1-(2-(m-tolyl)acetyl)piperidine-3-carboxylic acid

[0361] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(m-tolyl)acetyl)piperidine-3-carboxylate

[0362] To a solution of 2-(m-tolyl)acetic acid (100 mg, 0.666 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (110 mg, 0.699 mmol), DIPEA (0.174 mL, 0.999 mmol), HOBt (108 mg, 0.799 mmol), and EDC·HCl (124 mg, 0.799 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH4Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane:EtOAc = 2:1) to give the desired compound (88.0 mg, 45%) as a colorless oil.

[0363] Step 2: Synthesis of compound BnH12030

[0364] To a solution of methyl (3R,6S)-6-methyl-1-(2-(m-tolyl)acetyl)piperidine-3-carboxylate (70.0 mg, 0.242 mmol) in THF (1.00 mL) was added 1N NaOH (0.363 mL, 0.363 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12030 (26.7 mg, 40%) as a white solid.

[0365] 1H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.04 - 6.96 (m, 3H), 4.73 (s, 0.5H), 4.53 (dd, J = 13.1, 4.1 Hz, 0.5H), 4.22 (s, 0.5H), 3.88 (dd, J = 9.0, 3.9 Hz, 0.5H), 3.73 - 3.62 (m, 2H), 2.98 (t, J = 12.8 Hz, 0.5H), 2.59 (t, J = 12.7 Hz, 0.5H), 2.27 (s, 3H), 2.19 - 2.13 (m, 0.5H), 2.09 - 1.99 (m, 0.5H), 1.77 (d, J = 12.6 Hz, 1H), 1.69 - 1.58 (m, 1H), 1.49 - 1.44 (m, 2H), 1.04 (d, J = 6.9 Hz, 3H).

[0366] (14) Synthesis of compound BnH12031

[0367] (3R,6S)-1-(2-(4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0368] Step 1: Synthesis of methyl (3R,6S)-1-(2-(4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0369] To a solution of 2-(4-fluorophenyl)acetic acid (100 mg, 0.649 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (107 mg, 0.681 mmol), DIPEA (0.170 mL, 0.973 mmol), HOBt (105 mg, 0.779 mmol), and EDC·HCl (121 mg, 0.779 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH4Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane:EtOAc = 2:1) to give the desired compound (112 mg, 59%) as a yellow oil.

[0370] Step 2: Synthesis of compound BnH12031

[0371] To a solution of methyl (3R,6S)-1-(2-(4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (100 mg, 0.341 mmol) in THF (2.00 mL) was added 1N NaOH (0.511 mL, 0.511 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to provide compound BnH12031 (33.1 mg, 34%) as a white solid.

[0372] 1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 7.24 (dt, J = 18.2, 6.8 Hz, 2H), 7.12 (t, J = 8.7 Hz, 2H), 4.72 (s, 0.5H), 4.52 (dd, J = 15.1, 3.7 Hz, 0.5H), 4.24 (s, 0.5H), 3.88 (dd, J = 14.8, 3.9 Hz, 0.5H), 3.77 - 3.66 (m, 2H), 3.01 (t, J = 12.8 Hz, 0.5H), 2.60 (t, J = 12.7 Hz, 0.5H), 2.16 (tt, J = 12.8, 5.0 Hz, 1H), 1.81 - 1.76 (m, 1H), 1.70 - 1.60 (m, 1H), 1.51 - 1.47 (s, 2H), 1.05 (t, J = 7.6 Hz, 3H).

[0373] (15) Synthesis of compound BnH12032

[0374] (3R,6S)-1-(2-(4-bromophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0375] Step 1: Synthesis of methyl (3R,6S)-1-(2-(4-bromophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0376] To a solution of 2-(4-bromophenyl)acetic acid (100 mg, 0.465 mmol) in DMF (2.50 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (76.8 mg, 0.488 mmol), DIPEA (0.122 mL, 0.698 mmol), HOBt (75.4 mg, 0.558 mmol), and EDC·HCl (86.6 mg, 0.558 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (110 mg, 66%) as a gray oil.

[0377] Step 2: Synthesis of compound BnH12032

[0378] To a solution of methyl (3R,6S)-1-(2-(4-bromophenyl)acetyl)-6-methylpiperidine-3-carboxylate (105 mg, 0.296 mmol) in THF (2.00 mL) was added 1N NaOH (0.445 mL, 0.445 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to provide compound BnH12032 (36.0 mg, 35%) as a white solid.

[0379] 1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.17 (dd, J = 17.8, 8.0 Hz, 2H), 4.72 (s, 0.5H), 4.51 (dd, J = 13.3, 4.3 Hz, 0.5H), 4.23 (s, 0.5H), 3.87 (dd, J = 14.5, 4.2 Hz, 0.5H), 3.89 - 3.64 (m, 2H), 3.02 (t, J = 12.8 Hz, 0.5H), 2.60 (t, J = 12.7 Hz, 0.5H), 2.21 - 2.14 (m, 1H), 1.82 - 1.76 (m, 1H), 1.66 (qd, J = 11.9, 4.5 Hz, 1H), 1.52 - 1.48 (m, 2H), 1.06 (dd, J = 13.8, 6.9 Hz, 3H).

[0380] (16) Synthesis of compound BnH12033

[0381] Sodium (3R,6S)-6-methyl-1-(2-(pyridin-4-yl)acetyl)piperidine-3-carboxylate

[0382] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(pyridin-4-yl)acetyl)piperidine-3-carboxylate

[0383] To a solution of 2-(pyridin-4-yl)acetic acid hydrochloride (100 mg, 0.576 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (95.1 mg, 0.605 mmol), DIPEA (0.301 mL, 1.73 mmol), HOBt (93.4 mg, 0.691 mmol), and EDC·HCl (107 mg, 0.691 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 1:1) to give the desired compound (145 mg, 84%) as a yellow oil.

[0384] Step 2: Synthesis of compound BnH12033

[0385] To a solution of methyl (3R,6S)-1-(2-(furan-2-yl)acetyl)-6-methylpiperidine-3-carboxylate (140 mg, 0.528 mmol) in THF (2.50 mL) was added 1N NaOH (0.792 mL, 0.792 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was concentrated under reduced pressure, and the residue was purified by column chromatography on C18 silica (HO:MeOH = 0-50%) to give compound BnH12033 (16.0 mg, 28%) as a white solid.

[0386] 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 5.7 Hz, 2H), 7.23 (dd, J = 18.2, 5.0 Hz, 2H), 4.69 (s, 0.5H), 4.43 (d, J = 13.6 Hz, 0.5H), 4.14 (s, 0.5H), 3.82 - 3.66 (m, 3H), 2.95 (t, J = 13.0 Hz, 0.5H), 1.83 - 1.72 (m, 2H), 1.56 - 1.47 (m, 3H), 1.06 (dd, J = 19.3, 6.8 Hz, 3H).

[0387] (17) Synthesis of compound BnH12034

[0388] (3R,6S)-6-Methyl-1-(2-(thiophen-2-yl)acetyl)piperidine-3-carboxylic acid

[0389] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(thiophen-2-yl)acetyl)piperidine-3-carboxylate

[0390] To a solution of 2-(thiophen-2-yl)acetic acid (100 mg, 0.703 mmol) in DMF (3.50 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (116 mg, 0.739 mmol), DIPEA (0.184 mL, 1.06 mmol), HOBt (114 mg, 0.844 mmol), and EDC·HCl (131 mg, 0.84 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (152 mg, 76%) as a yellow oil.

[0391] Step 2: Synthesis of compound BnH12034

[0392] To a solution of methyl (3R,6S)-6-methyl-1-(2-(thiophen-2-yl)acetyl)piperidine-3-carboxylate (148 mg, 0.526 mmol) in THF (3.00 mL) was added 1N NaOH (0.789 mL, 0.789 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12034 (90.3 mg, 64%) as a white solid.

[0393] 1H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 7.37 (dd, J = 5.0, 1.4 Hz, 1H), 6.96 - 6.88 (m, 2H), 4.72 (s, 0.5H), 4.50 (dd, J = 14.3, 4.1 Hz, 0.5H), 4.28 (s, 0.5H), 4.01 - 3.89 (m, 2.5H), 3.05 (t, J = 12.9 Hz, 0.5H), 2.62 (t, J = 12.7 Hz, 0.5H), 2.25 - 2.13 (m, 1H), 1.82 - 1.77 (m, 1H), 1.71 - 1.61 (m, 1H), 1.55 - 1.50 (m, 2H), 1.07 (dd, J = 20.3, 6.9 Hz, 3H).

[0394] (18) Synthesis of compound BnH12035

[0395] (3R,6S)-1-(2-(furan-2-yl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0396] Step 1: Synthesis of methyl (3R,6S)-1-(2-(furan-2-yl)acetyl)-6-methylpiperidine-3-carboxylate

[0397] To a solution of 2-(furan-2-yl)acetic acid (100 mg, 0.793 mmol) in DMF (4.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (131 mg, 0.833 mmol), DIPEA (0.207 mL, 1.19 mmol), HOBt (129 mg, 0.952 mmol), and EDC·HCl (148 mg, 0.95 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (153 mg, 72%) as a yellow oil.

[0398] Step 2: Synthesis of compound BnH12035

[0399] To a solution of methyl (3R,6S)-1-(2-(furan-2-yl)acetyl)-6-methylpiperidine-3-carboxylate (140 mg, 0.528 mmol) in THF (2.50 mL) was added 1N NaOH (0.792 mL, 0.792 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to provide compound BnH12035 (100 mg, 75%) as a white solid.

[0400] 1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 7.54 (d, J = 1.8 Hz, 1H), 6.38 (t, J = 2.5 Hz, 1H), 6.18 (dd, J = 24.1, 3.2 Hz, 1H), 4.70 (t, J = 5.6 Hz, 0.5H), 4.49 (dd, J = 13.4, 4.3 Hz, 0.5H), 4.25 (dd, J = 8.4, 4.5 Hz, 0.5H), 3.89 (dd, J = 13.9, 4.2 Hz, 0.5H), 3.83 - 3.73 (m, 2H), 3.10 - 3.04 (m, 0.5H), 2.61 (t, J = 12.7 Hz, 0.5H), 2.28 - 2.14 (m, 1H), 1.84 - 1.78 (m, 1H), 1.72 - 1.49 (m, 2H), 1.07 (dd, J = 19.2, 6.9 Hz, 3H).

[0401] (19) Synthesis of compound BnH12038

[0402] Sodium (3R,6S)-6-methyl-1-(2-(pyridin-3-yl)acetyl)piperidine-3-carboxylate

[0403] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(pyridin-3-yl)acetyl)piperidine-3-carboxylate

[0404] To a solution of 2-(pyridin-3-yl)acetic acid hydrochloride (100 mg, 0.576 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (95.1 mg, 0.605 mmol), DIPEA (0.301 mL, 1.73 mmol), HOBt (93.4 mg, 0.691 mmol), and EDC·HCl (107 mg, 0.691 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (DCM:MeOH = 0-5%) to give the desired compound (76.2 mg, 47%) as a yellow oil.

[0405] Step 2: Synthesis of compound BnH12038

[0406] To a solution of methyl (3R,6S)-6-methyl-1-(2-(pyridin-3-yl)acetyl)piperidine-3-carboxylate (75.0 mg, 0.271 mmol) in THF (2.00 mL) was added 1N NaOH (0.407 mL, 0.407 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was concentrated under reduced pressure, and the residue was purified by column chromatography on C18 silica (HO:MeOH = 0-50%) to give compound BnH12038 (26.7 mg, 34%) as a pale yellow solid.

[0407] 1H NMR (400 MHz, DMSO-d6) δ 8.43 - 8.38 (m, 2H), 7.60 (ddt, J = 14.5, 7.8, 2.0 Hz, 1H), 7.31 (dt, J = 8.1, 4.4 Hz, 1H), 4.68 (t, J = 5.8 Hz, 0.5H), 4.42 (dd, J = 14.2, 3.9 Hz, 0.5H), 4.21 (t, J = 6.3 Hz, 0.5H), 3.85 - 3.64 (m, 2.5H), 2.97 (dd, J = 13.9, 11.9 Hz, 0.5H), 2.54 - 2.48 (m, 0.5H), 1.86 - 1.69 (m, 2H), 1.61 - 1.42 (m, 3H), 1.06 (dd, J = 30.0, 6.9 Hz, 3H).

[0408] (20) Synthesis of compound BnH12039

[0409] Sodium (3R,6S)-6-methyl-1-(2-(pyridin-2-yl)acetyl)piperidine-3-carboxylate

[0410] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(pyridin-2-yl)acetyl)piperidine-3-carboxylate

[0411] To a solution of 2-(pyridin-2-yl)acetic acid hydrochloride (100 mg, 0.576 mmol) in DMF (3.00 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (95.1 mg, 0.605 mmol), DIPEA (0.301 mL, 1.73 mmol), HOBt (93.4 mg, 0.691 mmol), and EDC·HCl (107 mg, 0.691 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (DCM:MeOH = 0-5%) to give the desired compound (71.2 mg, 44%) as a yellow oil.

[0412] Step 2: Synthesis of compound BnH12039

[0413] To a solution of methyl (3R,6S)-6-methyl-1-(2-(pyridin-3-yl)acetyl)piperidine-3-carboxylate (75.0 mg, 0.271 mmol) in THF (2.00 mL) was added 1N NaOH (0.407 mL, 0.407 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was concentrated under reduced pressure, and the residue was purified by column chromatography on C18 silica (HO:MeOH = 0-50%) to give compound BnH12039 (66.1 mg, 98%) as a white solid.

[0414] 1H NMR (400 MHz, DMSO-d6) δ 8.43 - 8.38 (m, 2H), 7.60 (ddt, J = 14.5, 7.8, 2.0 Hz, 1H), 7.31 (dt, J = 8.1, 4.4 Hz, 1H), 4.68 (t, J = 5.8 Hz, 0.5H), 4.42 (dd, J = 14.2, 3.9 Hz, 0.5H), 4.21 (t, J = 6.3 Hz, 0.5H), 3.85 - 3.64 (m, 2.5H), 2.97 (dd, J = 13.9, 12.0 Hz, 0.5H), 2.54 - 2.48 (m, 0.5H), 1.86 - 1.69 (m, 2H), 1.61 - 1.42 (m, 3H), 1.06 (dd, J = 30.0, 6.9 Hz, 3H).

[0415] (21) Synthesis of compound BnH12040

[0416] (3R,6S)-6-Methyl-1-(2-(pyrimidin-5-yl)acetyl)piperidine-3-carboxylic acid

[0417] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(pyrimidin-5-yl)acetyl)piperidine-3-carboxylate

[0418] To a solution of 2-(pyrimidin-5-yl)acetic acid (100 mg, 0.724 mmol) in DMF (3.50 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (120 mg, 0.760 mL), DIPEA (0.378 mL, 2.17 mmol), HOBt (117 mg, 0.869 mmol), and EDC·HCl (135 mg, 0.869 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (DCM:MeOH = 0-5%) to give the desired compound (72.8 mg, 36%) as a yellow oil.

[0419] Step 2: Synthesis of compound BnH12040

[0420] To a solution of methyl (3R,6S)-6-methyl-1-(2-(pyrimidin-5-yl)acetyl)piperidine-3-carboxylate (65.0 mg, 0.234 mmol) in THF (1.00 mL) was added 1N NaOH (0.352 mL, 0.352 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl to pH 4 at 0 °C and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12040 (35.1 mg, 56%) as a white solid.

[0421] 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 9.04 (s, 1H), 8.62 (d, J = 12.7 Hz, 2H), 4.70 (s, 0.5H), 4.52 - 4.47 (m, 0.5H), 4.32 (s, 0.5H), 3.99 - 3.69 (m, 2.5H), 3.15 (t, J = 12.9 Hz, 0.5H), 2.65 (t, J = 12.8 Hz, 0.5H), 2.45 - 2.38 (m, 0.5H), 2.26 - 2.18 (m, 0.5H), 1.86 - 1.83 (m, 1H), 1.73 - 1.54 (m, 3H), 1.14 (dd, J = 58.2, 6.9 Hz, 3H).

[0422] (22) Synthesis of compound BnH12041

[0423] (3R,6S)-6-Methyl-1-(2-(pyrimidin-2-yl)acetyl)piperidine-3-carboxylic acid

[0424] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(pyrimidin-2-yl)acetyl)piperidine-3-carboxylate

[0425] To a solution of 2-(pyrimidin-2-yl)acetic acid (100 mg, 0.724 mmol) in DMF (3.50 mL) was added methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (120 mg, 0.760 mL), DIPEA (0.378 mL, 2.17 mmol), HOBt (117 mg, 0.869 mmol), and EDC·HCl (135 mg, 0.869 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (DCM:MeOH = 0-5%) to give the desired compound (50.0 mg, 24%) as a pale yellow oil.

[0426] Step 2: Synthesis of compound BnH12041

[0427] To a solution of methyl (3R,6S)-6-methyl-1-(2-(pyrimidin-2-yl)acetyl)piperidine-3-carboxylate (45.0 mg, 0.162 mmol) in THF (1.00 mL) was added 1N NaOH (0.243 mL, 0.243 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl to pH 4 at 0 °C and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12041 (22.1 mg, 51%) as a yellow sticky solid.

[0428] 1 H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 8.75 (t, J = 4.7 Hz, 2H), 7.39 (q, J = 4.6 Hz, 1H), 4.71 (s, 0.5H), 4.52 - 4.48 (m, 0.5H), 4.26 (s, 0.5H), 4.10 - 3.86 (m, 2H), 3.88 (d, J = 14.8 Hz, 0.5H), 3.07 (t, J = 12.8 Hz, 0.5H), 2.68 - 2.60 (m, 0.5H), 1.84 - 1.80 (m, 1H), 1.73 - 1.61 (m, 4H), 1.09 (dd, J = 11.9, 6.9 Hz, 3H).

[0429] (23) Synthesis of compound BnH12065

[0430] (3R,6S)-1-Benzoyl-6-methylpiperidine-3-carboxylic acid

[0431] Step 1: Synthesis of methyl (3R,6S)-1-benzoyl-6-methylpiperidine-3-carboxylate

[0432] To a solution of benzoic acid (100 mg, 0.819 mmol) in DMF (2.73 mL) was added methyl (3R,6S)-6-methylpiperidine-3-carboxylate (135 mg, 0.860 mmol), DIPEA (0.215 mL, 1.23 mmol), HOBt (133 mg, 0.983 mmol), and EDC·HCl (153 mg, 0.983 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 3:1 to 2:1) to give the desired compound (150 mg, 70.1%) as a colorless oil.

[0433] Step 2: Synthesis of compound BnH12065

[0434] To a solution of methyl (3R,6S)-1-benzoyl-6-methylpiperidine-3-carboxylate (150 mg, 0.574 mmol) in THF (1.98 mL) was added 1N NaOH (0.861 mL, 0.861 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12065 (121 mg, 80.7%) as a white solid.

[0435] 1 H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 7.45-7.43 (q, J = 3.2 Hz, 3H), 7.37-7.34 (q, J = 3.2 Hz, 2H), 2.95 (s, 1H), 2.44-2.36 (q, J = 11.2 Hz, 2H), 1.89-1.54 (m, 5H), 1.17-1.11 (m, 3H).

[0436] (24) Synthesis of compound BnH12069

[0437] (3R,6S)-6-Methyl-1-(2-(4-nitrophenyl)acetyl)piperidine-3-carboxylic acid

[0438] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(4-nitrophenyl)acetyl)piperidine-3-carboxylate

[0439] To a solution of 2-(4-nitrophenyl)acetic acid (200 mg, 1.10 mmol) in DMF (3.68 mL) was added methyl (3R,6S)-6-methylpiperidine-3-carboxylate (182 mg, 1.16 mmol), DIPEA (0.289 mL, 1.66 mmol), HOBt (179 mg, 1.33 mmol), and EDC·HCl (206 mg, 1.33 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (8.00 mL) and washed with saturated aqueous NH₄Cl (8.00 mL) and brine (8.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 2:1 to 1:1) to give the desired compound (180 mg, 50.9%) as a pale yellow oil.

[0440] Step 2: Synthesis of compound BnH12069

[0441] To a solution of methyl (3R,6S)-6-methyl-1-(2-(4-nitrophenyl)acetyl)piperidine-3-carboxylate (180 mg, 0.562 mmol) in THF (1.94 mL) was added 1N NaOH (0.843 mL, 0.843 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (6.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12069 (166 mg, 96.4%) as a white solid.

[0442] 1 H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 8.19-8.17 (d, J = 8.5 Hz, 2H), 7.53-7.47 (dd, J = 17.2, 8.3 Hz, 2H), 4.72 (s, 0.5H), 4.54 - 4.50 (d, 0.5H), 4.26 (s, 0.5H), 4.00-3.84 (q, J = 15.9, 15.4 Hz, 2.5H), 3.10-3.04 (t, J = 12.9 Hz, 0.5H), 2.67-2.60 (t, J = 12.8 Hz, 0.5H), 2.30-2.19 (m, 1H), 1.84-1.78 (td, J = 13.9, 3.6 Hz, 1H), 1.71-1.62 (m, 1H), 1.55 (s, 2H), 1.14-1.05 (dd, J = 26.7, 6.9 Hz, 3H).

[0443] (25) Synthesis of compound BnH12070

[0444] (3R,6S)-1-(2-(4-aminophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0445] Step 1: Synthesis of compound BnH12070

[0446] To a solution of BnH12068 (92.0 mg, 0.300 mmol) in MeOH (2.00 mL) was added 10 wt% palladium on carbon (9.20 mg) under N2 gas in an ice-water bath at 0 °C. The mixture was degassed and purged with H2 gas. After removing the cooling bath, the mixture was vigorously stirred at room temperature under H2 gas for 1 h 30 min. The reaction mixture was filtered through a Celite pad and washed with MeOH (5.00 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (DCM:MeOH = 0-10%) to give compound BnH12070 (46.0 mg, 55.4%) as a pale yellow solid.

[0447] 1 H NMR (400 MHz, DMSO-d6) δ 12.26 (s, 1H), 6.88-6.81 (dd, J = 17.1, 7.9 Hz, 2H), 6.50-6.47 (m, 2H), 4.91 (s, 2H), 4.71 (s, 0.5H), 4.52-4.48 (m, 0.5H), 4.19 (s, 0.5H), 3.86-3.83 (d, J = 13.9 Hz, 0.5H), 3.54-3.46 (m, J = 5.4 Hz, 2H), 2.97-2.90 (t, J = 12.9 Hz, 0.5H), 2.58-2.55 (d, J = 12.7 Hz, 0.5H), 2.14-2.00 (m, 1H), 1.76-1.72 (m, 1H), 1.63-1.60 (d, J = 13.2 Hz, 1H), 1.48-1.41 (d, J = 16.0 Hz, 2H), 1.02-0.99 (t, J = 6.1Hz, 3H).

[0448] (26) Synthesis of compound BnH12073

[0449] (3R,6S)-6-Methyl-1-(2-(3-nitrophenyl)acetyl)piperidine-3-carboxylic acid

[0450] Step 1: Synthesis of methyl (3R,6S)-6-methyl-1-(2-(3-nitrophenyl)acetyl)piperidine-3-carboxylate

[0451] To a solution of 2-(3-nitrophenyl)acetic acid (200 mg, 1.10 mmol) in DMF (3.68 mL) was added methyl (3R,6S)-6-methylpiperidine-3-carboxylate (182 mg, 1.16 mmol), DIPEA (0.289 mL, 1.66 mmol), HOBt (179 mg, 1.33 mmol), and EDC·HCl (206 mg, 1.33 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (8.00 mL) and washed with saturated aqueous NH₄Cl (8.00 mL) and brine (8.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 2:1 to 1:1) to give the desired compound (210 mg, 59.4%) as a pale yellow oil.

[0452] Step 2: Synthesis of compound BnH12073

[0453] To a solution of methyl (3R,6S)-6-methyl-1-(2-(3-nitrophenyl)acetyl)piperidine-3-carboxylate (210 mg, 0.656 mmol) in THF (2.26 mL) was added 1N NaOH (0.983 mL, 0.983 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (6.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12073 (173 mg, 86.2%) as a white solid.

[0454] 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.13-8.09 (m, 2H), 7.70-7.58 (m, 2H), 4.72 (s, 0.5H), 4.53-4.49 (m, 0.5H), 4.30 (s, 0.5H), 4.01-3.84 (m, 2.5H), 3.12-3.05 (t, J = 12.8 Hz, 0.5H), 2.67-2.60 (m, 0.5H), 2.33-2.20 (m, 1H), 1.83-1.80 (d, J = 11.9 Hz, 1H), 1.69-1.63 (d, J = 11.2 Hz, 1H), 1.57-1.53 ​​(d, J = 16.0 Hz, 2H), 1.16-1.05 (dd, J = 34.1, 6.9 Hz, 3H).

[0455] (27) Synthesis of compound BnH12074

[0456] (3R,6S)-1-(2-(4-aminophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0457] Step 1: Synthesis of compound BnH12074

[0458] To a solution of BnH12073 (98.0 mg, 0.320 mmol) in MeOH:THF (1:1, 2.14 mL) was added 10 wt% palladium on carbon (9.80 mg) under N2 gas in an ice-water bath at 0 °C. The mixture was degassed and purged with H2 gas. After removing the cooling bath, the mixture was vigorously stirred at room temperature under H2 gas for 2 h. The reaction mixture was filtered through a Celite pad and washed with MeOH (5.00 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (DCM:MeOH = 0-10%) to give compound BnH12074 (52.0 mg, 58.8%) as a pale yellow solid.

[0459] 1H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H), 6.95-6.90 (td, J = 7.8, 4.1 Hz, 1H), 6.43-6.35 (m, 3H), 5.02 (s, 2H), 4.74-4.72 (s, 0.5H), 4.55-4.51 (d, 0.5H), 4.15 (s, 0.5H), 3.83-3.79 (d, J = 14.4 Hz, 0.5H), 3.58-3.48 (m, 2H), 2.98-2.92 (t, J = 12.9 Hz, 0.5H), 2.60-2.54 (t, J = 12.7 Hz, 0.5H), 2.18-2.09 (m, 1H), 1.78-1.74 (d, J = 13.0 Hz, 1H), 1.67-1.62 (m, J = 13.6 Hz, 1H), 1.50-1.44 (d, J = 27.9 Hz, 2H), 1.04-1.00 (dd, J = 10.0, 6.8 Hz, 3H).

[0460] (28) Synthesis of compound BnH12075

[0461] (3R,6S)-1-(2-(6-methoxypyridin-3-yl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0462] Step 1: Synthesis of methyl (3R,6S)-1-(2-(6-methoxypyridin-3-yl)acetyl)-6-methylpiperidine-3-carboxylate

[0463] To a solution of 2-(6-methoxypyridin-3-yl)acetic acid (100 mg, 0.598 mmol) in DMF (1.99 mL) was added methyl (3R,6S)-6-methylpiperidine-3-carboxylate (98.8 mg, 0.628 mmol), DIPEA (0.157 mL, 0.897 mmol), HOBt (97.0 mg, 0.718 mmol), and EDC·HCl (111 mg, 0.718 mmol). The mixture was stirred at room temperature for 3 h. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane: EtOAc = 1:1) to give the desired compound (87.0 mg, 47.5%) as a colorless oil.

[0464] Step 2: Synthesis of compound BnH12075

[0465] To a solution of methyl (3R,6S)-1-(2-(6-methoxypyridin-3-yl)acetyl)-6-methylpiperidine-3-carboxylate (87.0 mg, 0.284 mmol) in THF (0.979 mL) was added 1N NaOH (0.426 mL, 0.426 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 3-4 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12075 (51.4 mg, 61.9%) as a white solid.

[0466] 1H NMR (400 MHz, DMSO-d6) δ 12.43 (s, 1H), 7.99-7.95 (d, J = 16.8 Hz, 1H), 7.55-7.49 (dt, J = 9.9, 5.5 Hz, 1H), 6.76-6.74 (d, J = 8.5 Hz, 1H), 4.71 (s, 0.5H), 4.52-4.49 (d, J = 13.6 Hz, 0.5H), 4.28 (s, 0.5H), 3.94-3.91 (d, J = 13.9 Hz, 0.5H), 3.74-3.59 (m, 2H), 3.09-3.02 (t, J = 12.9 Hz, 0.5H), 2.64-2.57 (t, J = 12.8 Hz, 0.5H), 2.26-2.17 (d, J = 33.1 Hz, 1H), 1.82-1.79 (d, J = 13.2 Hz, 1H), 1.67-1.61 (d, J = 12.6 Hz, 1H), 1.56-1.53 ​​(m, 2H), 1.13-1.03 (dd, J = 32.5, 6.9 Hz, 3H).

[0467] (29) Synthesis of compound BnH12076

[0468] (3R,6S)-1-(2-(4-(dimethylamino)phenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0469] Step 1: Synthesis of methyl (3R,6S)-1-(2-(4-(dimethylamino)phenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0470] To a solution of 2-(4-(dimethylamino)phenyl)acetic acid (100 mg, 0.558 mmol) in DMF (1.86 mL) was added methyl (3R,6S)-6-methylpiperidine-3-carboxylate (92.1 mg, 0.586 mmol), DIPEA (0.146 mL, 0.837 mmol), HOBt (90.5 mg, 0.670 mmol), and EDC·HCl (104 mg, 0.670 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane: EtOAc = 2:1 to 1:1) to give the desired compound (142 mg, 79.9%) as a pale yellow oil.

[0471] Step 2: Synthesis of compound BnH12076

[0472] To a solution of methyl (3R,6S)-1-(2-(4-(dimethylamino)phenyl)acetyl)-6-methylpiperidine-3-carboxylate (142 mg, 0.446 mmol) in THF (1.54 mL) was added 1N NaOH (0.669 mL, 0.669 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 3-4 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12076 (69.0 mg, 50.8%) as a white solid.

[0473] 1H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 1H), 7.05-6.99 (dd, J = 18.3, 8.1 Hz, 2H), 6.68-6.66 (d, J = 8.2 Hz, 2H), 4.72 (s, 0.5H), 4.54-4.50 (m, 0.5H), 4.23-4.20 (d, J = 9.0 Hz, 0.5H), 3.88-3.85 (d, J = 13.9 Hz, 0.5H), 3.62-3.52 (m, 2H), 2.98-2.91 (t, J = 12.7 Hz, 0.5H), 2.85 (s, 6H), 2.60-2.53 (t, J = 12.6 Hz, 0.5H), 2.13-2.09 (m, 1H), 1.78-1.74 (d, J = 12.7 Hz, 1H), 1.68-1.58 (q, J = 13.0 Hz, 1H), 1.50-1.40 (d, J = 16.3 Hz, 2H), 1.03-1.00 (dd, J = 7.2, 2.9 Hz, 3H).

[0474] (30) Synthesis of compound BnH12079

[0475] (3R,6S)-1-(2-(4-hydroxyphenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0476] Step 1: Synthesis of 2-(4-hydroxyphenyl)acetic acid

[0477] To a suspension of 2-(4-hydroxyphenyl)acetic acid (500 mg, 3.01 mmol) in DCM (5.02 mL) was added 1 M BBr solution (6.02 mL, 6.02 mmol) under N gas at −78 °C and stirred for 5 h while warming from −78 °C to −15 °C. The reaction mixture was quenched with HO (7.50 mL), and the resulting solid was filtered and washed with DCM (5.00 mL) to give the desired compound (128 mg, 28.0%) as a pale pink solid.

[0478] Step 2: Synthesis of methyl (3R,6S)-1-(2-(4-hydroxyphenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0479] To a solution of 2-(4-hydroxyphenyl)acetic acid (72.0 mg, 0.473 mmol) in DMF (1.58 mL) was added methyl (3R,6S)-6-methylpiperidine-3-carboxylate (78.1 mg, 0.497 mmol), DIPEA (0.124 mL, 0.710 mmol), HOBt (76.7 mg, 0.568 mmol), and EDC·HCl (88.2 mg, 0.568 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (DCM:MeOH = 0-2%) to give the desired compound (78.0 mg, 56.6%) as a pale yellow sticky solid.

[0480] Step 3: Synthesis of compound BnH12079

[0481] To a solution of methyl (3R,6S)-1-(2-(4-hydroxyphenyl)acetyl)-6-methylpiperidine-3-carboxylate (78.0 mg, 0.268 mmol) in THF (0.923 mL) was added 1N NaOH (0.402 mL, 0.402 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (4.00 mL) and washed three times with DCM (4.00 mL × 3). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (4.00 mL). The organic layer was washed with brine (4.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12079 (69.0 mg, 50.8%) as a white solid.

[0482] 1H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H), 9.25 (s, 1H), 7.05-6.97 (dd, J = 17.6, 7.9 Hz, 2H), 6.70-6.68 (d, J = 7.9 Hz, 2H), 4.73 (s, 0.5H), 4.55-4.50 (m, 0.5H), 4.21 (s, 0.5H), 3.88-3.84 (d, J = 13.8 Hz, 0.5H), 3.64-3.54 (d, J = 6.2 Hz, 2H), 3.00-2.94 (t, J = 12.9Hz, 0.5H), 2.61-2.55 (d, J = 12.5 Hz, 0.5H), 2.16-2.07 (m, 1H), 1.79-1.75 (d, J = 12.9 Hz, 1H), 1.65-1.62 (d, J = 13.2 Hz, 1H), 1.50-1.47 (d, J = 15.1 Hz, 2H), 1.04-1.01 (t, J = 5.5 Hz, 3H).

[0483] Preparation Example 2. Preparation of active compound according to representative reaction scheme 2

[0484] [Reaction Scheme 2]

[0485] JPEG2025511114000026.jpg27144

[0486] JPEG2025511114000027.jpg181120

[0487] (1) Synthesis of compound BnH12001 (12-004)

[0488] 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0489] JPEG2025511114000028.jpg27146

[0490] Step 1: Synthesis of 6-methylpiperidine-3-carboxylic acid (c)

[0491] To a solution of 6-methylnicotinic acid (500 mg, 3.65 mmol) in EtOH (6.63 mL) were added acetic acid (0.663 mL, 3.65 mmol) and Pd / C (272 mg, 2.55 mmol). The reaction mixture was degassed and purged with H gas three times. The mixture was then stirred at room temperature for 3 days and at 50 °C for 16 h. After cooling to room temperature, the mixture was filtered through Celite and washed with MeOH (10.0 mL). The filtrate was concentrated under reduced pressure and used without further purification.

[0492] Step 2: Methyl 6-methylpiperidine-3-carboxylate (2-1-12-004)

[0493] To a solution of 6-methylpiperidine-3-carboxylic acid (crude) in MeOH (8.10 mL) was added SOCl (0.798 mL, 10.9 mmol) dropwise at 0 °C and refluxed at 70 °C for 2 h. The mixture was concentrated, and the residue was dissolved in EtOAc (10.0 mL) and washed with saturated NaHCO (10.0 mL). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure to give methyl 6-methylpiperidine-3-carboxylate (180 mg, 31%, 2 steps) as a brown oil. The desired compound was used without further purification.

[0494] Step 3: Synthesis of methyl 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0495] To a solution of 2-chloro-4-fluorophenylacetic acid (200 mg, 1.06 mmol) in DMF (3.53 mL) was added methyl 6-methylpiperidine-3-carboxylate (175 mg, 1.11 mmol), DIPEA (0.277 mL, 1.59 mmol), HOBt (172 mg, 1.27 mmol), and EDC·HCl (198 mg, 1.27 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 3:1 to 2:1) to give the desired compound (227 mg, 65%) as a colorless oil.

[0496] Step 4: Synthesis of Compound 12-004

[0497] To a solution of methyl 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate (220 mg, 0.702 mmol) in THF (2.42 mL) was added 1N NaOH (1.05 mL, 1.05 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with HO (3.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was washed with brine (5.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure. EtO (5.00 mL) was added to the residue and stirred at room temperature for 1 h. The precipitate was filtered and washed with EtO (2.00 mL) to give compound 12-004 (150 mg, 71%) as a white solid.

[0498] 1H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H) 7.43 - 7.22 (m, 2H), 7.21 - 7.10 (m, 1H), 4.66 (d, J = 29.7 Hz, 0.7H), 4.49 (d, J = 12.5 Hz, 0.3H), 4.27 (s, 0.3H), 4.03 (d, J = 7.2 Hz, 0.5H), 3.97 - 3.68 (m, 2.5H), 3.14 (t, J = 12.9 Hz, 0.5H), 2.71 - 2.57 (m, 1H), 2.34 (d, J = 8.9 Hz, 0.25H), 2.21 (s, 0.25H), 1.87 (t, J = 16.7 Hz, 2H), 1.77 - 1.50 (m, 2H), 1.41 - 1.34 (m, 0.5H), 1.27 - 1.00 (m, 3.5H).

[0499] (2) Synthesis of compound BnH12004

[0500] (R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)piperidine-3-carboxylic acid

[0501] Step 1: Synthesis of methyl (R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)piperidine-3-carboxylate

[0502] To a solution of 2-chloro-4-fluorophenylacetic acid (100 mg, 0.530 mmol) in DMF (1.77 mL) was added methyl (R)-piperidine-3-carboxylate (79.7 mg, 0.557 mmol), DIPEA (0.139 mL, 0.795 mmol), HOBt (86.0 mg, 0.636 mmol), and EDC·HCl (98.8 mg, 0.636 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (3.00 mL) and washed with saturated aqueous NH₄Cl (3.00 mL) and brine (3.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 3:1 to 2:1) to give the desired compound (128 mg, 76%) as a colorless oil.

[0503] Step 2: Synthesis of compound BnH12004

[0504] To a solution of methyl (R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)piperidine-3-carboxylate (128 mg, 0.408 mmol) in THF (1.41 mL) was added 1N NaOH (0.612 mL, 0.612 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (1.50 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO (DCM:MeOH = 95:5 to 90:10) to afford compound BnH12004 (83.0 mg, 67%) as a white solid.

[0505] 1H NMR (400 MHz, DMSO-d6) δ 7.39 (dt, J = 9.0, 3.1 Hz, 1H), 7.32 (ddd, J = 11.3, 8.6, 6.3 Hz, 1H), 7.16 (td, J = 8.5, 2.7 Hz, 1H), 4.42 - 4.31 (m, 0.5H), 3.87 - 3.76 (m, 4H), 3.53 - 3.41 (m, 2H), 3.12 - 3.02 (m, 1.5H), 2.78 (dd, J = 12.9, 10.6 Hz, 0.5H), 2.31 - 2.21 (m, 0.5H), 2.02 - 1.87 (m, 1H), 1.70 (d, J = 11.9 Hz, 1H), 1.57 (dt, J = 11.6, 6.0 Hz, 1H), 1.49 - 1.30 (m, 1H).

[0506] (3) Synthesis of compound BnH12005

[0507] (S)-2-(2-chloro-4-fluorophenyl)-1-(2-methylpiperidin-1-yl)ethan-1-one

[0508] Step 1: Synthesis of compound BnH12005

[0509] To a solution of 2-chloro-4-fluorophenylacetic acid (100 mg, 0.530 mmol) in DMF (1.77 mL) was added (S)-2-methylpiperidine (55.2 mg, 0.557 mmol), DIPEA (0.139 mL, 0.795 mmol), HOBt (86.0 mg, 0.636 mmol), and EDC·HCl (98.8 mg, 0.636 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (3.00 mL) and washed with saturated aqueous NH₄Cl (3.00 mL) and brine (3.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 3:1 to 2:1) to afford compound BnH12005 (112 mg, 78%) as a colorless oil.

[0510] 1H NMR (400 MHz, DMSO-d6) δ 7.39 (dd, J = 8.9, 2.7 Hz, 1H), 7.33 (s, 1H), 7.16 (td, J = 8.5, 2.7 Hz, 1H), 4.70 (s, 0.5H), 4.25 (s, 0.5H), 3.77 (d, J = 39.4 Hz, 2.5H), 3.14 (d, J = 14.1 Hz, 0.5H), 2.71 - 2.60 (m, 0.5H), 1.68 - 1.45 (m, 5H), 1.38 - 1.04 (m, 4H).

[0511] (4) Synthesis of compound BnH12012

[0512] (2-(2-chloro-4-fluorophenyl)acetyl)-D-proline

[0513] Step 1: Synthesis of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-D-prolinate

[0514] To a solution of 2-chloro-4-fluorophenylacetic acid (100 mg, 0.530 mmol) in DMF (1.77 mL) was added D-prolinate (82.2 mg, 0.636 mmol), DIPEA (0.140 mL, 0.795 mmol), HOBt (86.0 mg, 0.636 mmol), and EDC·HCl (99.0 mg, 0.636 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (3.00 mL) and washed with saturated aqueous NH₄Cl (3.00 mL) and brine (3.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (112 mg, 70%) as a colorless oil.

[0515] Step 2: Synthesis of compound BnH12012

[0516] To a solution of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-D-prolinate (112 mg, 0.357 mmol) in THF (1.23 mL) was added 1N NaOH (0.535 mL, 0.535 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12012 (24.0 mg, 22%) as a white solid.

[0517] 1H NMR (400 MHz, Chloroform-d) δ 7.31 (dd, J = 8.6, 5.9 Hz, 1H), 7.16 (dd, J = 8.4, 2.6 Hz, 1H), 6.99 (td, J = 8.3, 2.7 Hz, 1H), 4.68 - 4.62 (m, 1H), 3.80 (d, J = 2.8 Hz, 2H), 3.68 (ddd, J = 10.7, 7.4, 3.6 Hz, 1H), 3.63 - 3.55 (m, 1H), 2.55 - 2.47 (m, 1H), 2.20 - 1.96 (m, 3H).

[0518] (5) Synthesis of compound BnH12013

[0519] (2-(2-chloro-4-fluorophenyl)acetyl)-L-proline

[0520] Step 1: Synthesis of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-L-prolinate

[0521] To a solution of 2-chloro-4-fluorophenylacetic acid (100 mg, 0.530 mmol) in DMF (1.77 mL) was added methyl L-prolinate (71.9 mg, 0.557 mmol), DIPEA (0.140 mL, 0.795 mmol), HOBt (86.0 mg, 0.636 mmol), and EDC·HCl (99.0 mg, 0.636 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (3.00 mL) and washed with saturated aqueous NH₄Cl (3.00 mL) and brine (3.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (126 mg, 79%) as a colorless oil.

[0522] Step 2: Synthesis of compound BnH12013

[0523] To a solution of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-L-prolinate (126 mg, 0.420 mmol) in THF (1.45 mL) was added 1N NaOH (0.631 mL, 0.631 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12013 (26.0 mg, 21%) as a white solid.

[0524] 1H NMR (400 MHz, Chloroform-d) δ 7.30 (dd, J = 8.6, 5.9 Hz, 1H), 7.16 (dd, J = 8.4, 2.6 Hz, 1H), 7.00 (td, J = 8.3, 2.7 Hz, 1H), 4.68 - 4.62 (m, 1H), 3.80 (d, J = 2.7 Hz, 2H), 3.68 (td, J = 8.9, 7.6, 3.5 Hz, 1H), 3.63 - 3.55 (m, 1H), 2.57 - 2.46 (m, 1H), 2.20 - 1.90 (m, 3H).

[0525] Preparation Example 3. Preparation of active compound according to representative reaction scheme 4

[0526] [Reaction Scheme 4]

[0527] JPEG2025511114000029.jpg28146

[0528] JPEG2025511114000030.jpg152146

[0529] (1) Synthesis of compound BnH12014

[0530] (R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-N-hydroxypyrrolidine-2-carboxamide

[0531] Step 1: Synthesis of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-D-prolinate

[0532] To a solution of 2-chloro-4-fluoroacetic acid (100 mg, 0.530 mmol) in DMF (1.77 mL) was added methyl D-prolinate (105 mg, 0.636 mmol), DIPEA (0.139 mL, 0.795 mmol), HOBt (86.0 mg, 0.636 mmol), and EDC·HCl (98.8 mg, 0.636 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (113 mg, 71%) as a colorless oil.

[0533] Step 2: Synthesis of compound BnH12014

[0534] To a solution of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-D-prolinate (100 mg, 0.334 mmol) in MeOH (1.50 mL) and THF (1.50 mL) was added hydroxylamine hydrochloride (0.197 mL, 3.34 mmol) and KOH (37.4 mg, 0.667 mmol). The mixture was stirred at room temperature for 2 hours. 1N HCl was added to the mixture to adjust the pH to 1-2, and the mixture was extracted with DCM (4.00 mL × 5). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on NH-SiO2 (DCM:MeOH = 9:1) to give compound BnH12014 (25.0 mg, 24%) as a white solid.

[0535] 1H NMR (400 MHz, Chloroform-d) δ 9.92 (s, 1H), 7.34 (dd, J = 8.5, 6.0 Hz, 1H), 7.14 (dd, J = 8.5, 2.6 Hz, 1H), 6.99 (td, J = 8.3, 2.7 Hz, 1H), 4.52 (d, J = 7.8 Hz, 1H), 3.72 (s, 2H), 3.56 (dd, J = 17.2, 8.7 Hz, 2H), 2.42 (s, 1H), 2.22 - 2.09 (m, 1H), 2.08 - 1.90 (m, 2H).

[0536] (2) Synthesis of compound BnH12016

[0537] (S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-N-hydroxypyrrolidine-2-carboxamide

[0538] Step 1: Synthesis of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-L-prolinate

[0539] To a solution of 2-chloro-4-fluoroacetic acid (100 mg, 0.530 mmol) in DMF (1.77 mL) was added methyl L-prolinate (105 mg, 0.636 mmol), DIPEA (0.139 mL, 0.795 mmol), HOBt (86.0 mg, 0.636 mmol), and EDC·HCl (98.8 mg, 0.636 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (128 mg, 80%) as a colorless oil.

[0540] Step 2: Synthesis of compound BnH12016

[0541] To a solution of methyl (2-(2-chloro-4-fluorophenyl)acetyl)-L-prolinate (128 mg, 0.427 mmol) in MeOH (1.50 mL) and THF (1.50 mL) was added hydroxylamine hydrochloride (59.4 mg, 0.854 mmol) and KOH (95.85 mg, 1.71 mmol). The mixture was stirred at room temperature for 17 hours. 1N HCl was added to the mixture to adjust the pH to 1-2, and the mixture was extracted with DCM (4.00 mL × 5). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on NH-SiO2 (DCM:MeOH = 9:1) to give compound BnH12016 (19.0 mg, 14%) as a white solid.

[0542] 1H NMR (400 MHz, Chloroform-d) δ 9.87 (s, 1H), 7.35 - 7.27 (m, 1H), 7.15 (dd, J = 8.4, 2.6 Hz, 1H), 6.99 (td, J = 8.3, 2.7 Hz, 1H), 4.54 (d, J = 8.1 Hz, 1H), 3.73 (d, J = 2.6 Hz, 2H), 3.66 - 3.49 (m, 2H), 2.46 (s, 1H), 2.17 (dd, J = 17.6, 8.5 Hz, 1H), 2.09 - 1.89 (m, 2H).

[0543] (3) Synthesis of compound BnH12009

[0544] (3R,6S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-N-hydroxy-6-methylpiperidine-3-carboxamide

[0545] Step 1: Synthesis of compound BnH12009

[0546] To a solution of the compound BnH12001 enantiomer (3S,6R) (60.0 mg, 0.191 mmol) in THF (0.956 mL) was added CDI (46.5 mg, 0.287 mmol) and stirred at room temperature for 1 hour. Hydroxylamine hydrochloride (39.9 mg, 0.574 mol) was added to the mixture, and the mixture was stirred at room temperature for 17 hours. The mixture was diluted with DCM (2.00 mL) and washed with 5% aqueous KHSO (2.00 mL). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on NH-SiO (DCM:MeOH = 10:1) to give compound BnH12009 (32.0 mg, 50%) as a white solid.

[0547] 1H NMR (400 MHz, Chloroform-d) δ 9.58 (s, 1H), 7.24 (dd, J = 8.7, 6.1 Hz, 2H), 7.13 (dd, J = 8.4, 2.6 Hz, 1H), 6.96 (td, J = 8.2, 2.6 Hz, 1H), 4.54 (d, J = 13.4 Hz, 0.5H), 4.18 (s, 0.5H), 3.89 - 3.61 (m, 2H), 3.49 (s, 2H), 2.87 (t, J = 12.4 Hz, 0.5H), 2.07 (dd, J = 29.7, 12.3 Hz, 2H), 1.68 (d, J = 23.3 Hz, 3H), 1.36 - 1.18 (m, 4H).

[0548] (4) Synthesis of compound BnH12018

[0549] (R)-1-Benzoyl-N-hydroxypyrrolidine-2-carboxamide

[0550] Step 1: Synthesis of methyl benzoyl D-prolinate

[0551] To a solution of benzoic acid (100 mg, 0.819 mmol) in DMF (2.73 mL) was added methyl D-prolinate (163 mg, 0.983 mmol), DIPEA (0.215 mL, 1.23 mmol), HOBt (133 mg, 0.983 mmol), and EDC·HCl (153 mg, 0.983 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (126 mg, 66%) as a colorless oil.

[0552] Step 2: Synthesis of compound BnH12018

[0553] To a solution of methyl benzoyl-D-prolinate (124 mg, 0.414 mmol) in MeOH (3.00 mL) and THF (3.00 mL) was added hydroxylamine hydrochloride (0.244 mL, 4.14 mmol) and KOH (46.4 mg, 0.827 mmol). The mixture was stirred at room temperature for 2 hours. 1N HCl was added to the mixture to adjust the pH to 1-2, and the mixture was extracted with DCM (4.00 mL × 5). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on NH-SiO2 (DCM:MeOH = 9:1) to give compound BnH12018 (28.0 mg, 28%) as a white solid.

[0554] 1H NMR (400 MHz, Chloroform-d) δ 10.04 (s, 1H), 7.52 (d, J = 7.3 Hz, 2H), 7.44 (dq, J = 14.3, 7.3 Hz, 3H), 4.71 (s, 1H), 3.60 - 3.48 (m, 2H), 2.54 (d, J = 6.4 Hz, 1H), 2.12 (d, J = 13.3 Hz, 2H), 1.93 - 1.81 (m, 1H).

[0555] (5) Synthesis of compound BnH12019

[0556] (S)-1-Benzoyl-N-hydroxypyrrolidine-2-carboxamide

[0557] Step 1: Synthesis of methylbenzoyl-L-prolinate

[0558] To a solution of benzoic acid (100 mg, 0.819 mmol) in DMF (2.73 mL) was added methyl L-prolinate (163 mg, 0.983 mmol), DIPEA (0.215 mL, 1.23 mmol), HOBt (133 mg, 0.983 mmol), and EDC·HCl (153 mg, 0.983 mmol). The mixture was stirred at room temperature for 3 h 30 min. The mixture was diluted with EtOAc (4.00 mL) and washed with saturated aqueous NH₄Cl (4.00 mL) and brine (4.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (145 mg, 75%) as a colorless oil.

[0559] Step 2: Synthesis of compound BnH12019

[0560] To a solution of methyl benzoyl-L-prolinate (140 mg, 0.600 mmol) in MeOH (3.00 mL) and THF (3.00 mL) was added hydroxylamine hydrochloride (0.354 mL, 6.00 mmol) and KOH (67.4 mg, 1.20 mmol). The mixture was stirred at room temperature for 2 hours. 1N HCl was added to the mixture to adjust the pH to 1-2, and the mixture was extracted with DCM (4.00 mL × 5). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on NH-SiO2 (DCM:MeOH = 9:1) to give compound BnH12019 (38.0 mg, 27%) as a white solid.

[0561] 1H NMR (400 MHz, Chloroform-d) δ 10.01 (s, 1H), 7.52 (d, J = 7.2 Hz, 2H), 7.44 (dq, J = 14.5, 7.2 Hz, 3H), 4.71 (t, J = 6.4 Hz, 1H), 3.55 (dt, J = 11.5, 6.2 Hz, 2H), 2.54 (d, J = 6.4 Hz, 1H), 2.10 (dp, J = 13.9, 7.1 Hz, 2H), 1.93 - 1.82 (m, 1H).

[0562] Preparation Example 4. Preparation of active compound according to representative reaction scheme 5

[0563] [Reaction Scheme 5]

[0564] JPEG2025511114000031.jpg34147

[0565] JPEG2025511114000032.jpg69129

[0566] (1) Synthesis of compound BnH12022

[0567] (3R,6S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxamide

[0568] Step 1: Synthesis of compound BnH12022

[0569] To a suspension of the compound BnH12001 enantiomer (3S,6R) (100 mg, 0.319 mmol) in MeCN (3.43 mL) was added CDI (77.5 mg, 0.478 mmol) and stirred at room temperature for 20 minutes. NHOH (0.186 mL, 4.78 mmol) was added to the mixture, and the mixture was stirred at room temperature for 17 hours. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated NHCl (4.00 mL), 10% citric acid (4.00 mL), and brine (4.00 mL). The organic layer was dried over MgSO, filtered, and concentrated. The residue was purified by column chromatography (5% MeOH in DCM) to give compound BnH12022 (84 mg, 84%) as a white solid.

[0570] 1H NMR (400 MHz, DMSO-d6) δ 7.43 - 7.26 (m, 3H), 7.16 (tt, J = 8.6, 2.3 Hz, 1H), 6.89 (d, J = 25.2 Hz, 1H), 4.69 (s, 0.5H), 4.32 (dd, J = 20.4, 8.7 Hz, 1H), 3.80 (ddd, J = 36.3, 31.6, 16.2 Hz, 2.5H), 3.19 - 3.08 (m, 0.5H), 2.65 (t, J = 12.6 Hz, 0.5H), 2.33 - 2.21 (m, 0.5H), 2.19 - 2.03 (m, 0.5H), 1.76 - 1.67 (m, 2H), 1.63 - 1.58 (m, 1H), 1.54 (s, 1H), 1.14 (dd, J = 61.2, 6.9 Hz, 3H).

[0571] Preparation Example 5. Preparation of active compound according to representative reaction scheme 6

[0572] [Reaction Scheme 6]

[0573] JPEG2025511114000033.jpg60142

[0574] JPEG2025511114000034.jpg115130

[0575] (1) Synthesis of compound BnH12060

[0576] (S)-1-(2-methylpiperidin-1-yl)-2-(pyridin-3-yl)ethan-1-one

[0577] Step 1: Synthesis of compound BnH12060

[0578] To a solution of 3-pyridylacetic acid (100 mg, 0.729 mmol) in DMF (2.43 mL) was added (S)-2-methylpiperidine (75.9 mg, 0.766 mmol), DIPEA (0.191 mL, 1.09 mmol), HOBt (118 mg, 0.875 mmol), and EDC·HCl (136 mg, 0.875 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (0–5% MeOH in DCM) to afford compound BnH12060 (54.2 mg, 34%) as an orange oil.

[0579] 1H NMR (400 MHz, Chloroform-d) δ 8.54-8.51 (dt, J = 6.3, 3.1 Hz, 2H), 7.69 (s, 1H), 7.33-7.30 (dd, J = 7.9, 4.8 Hz, 1H), 4.96 (s, 0.5H), 4.56-4.52 (d, J = 13.6 Hz, 0.5H), 4.19 (s, 0.5H), 3.80-3.73 (m, 2H), 3.67-3.63 (d, J = 14.1 Hz, 0.5H), 3.17-3.10 (t, J = 13.5 Hz, 0.5H), 2.75-2.68 (t, J = 13.4 Hz, 0.5H), 1.63-1.56 (d, J = 29.3 Hz, 5H), 1.36-1.28 (d, J = 34.4 Hz, 1H), 1.21-1.16 (d, J = 8.2 Hz, 3H).

[0580] (2) Synthesis of compound BnH12061

[0581] (S)-1-(2-methylpiperidin-1-yl)-2-phenylethan-1-one

[0582] Step 1: Synthesis of compound BnH12061

[0583] To a solution of 2-phenylacetic acid (100 mg, 0.734 mmol) in DMF (2.45 mL) was added (S)-2-methylpiperidine (76.5 mg, 0.771 mmol), DIPEA (0.192 mL, 1.10 mmol), HOBt (119 mg, 0.881 mmol), and EDC·HCl (137 mg, 0.881 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to afford compound BnH12061 (58.7 mg, 37%) as a colorless oil.

[0584] 1H NMR (400 MHz, Chloroform-d) δ 7.35 - 7.23 (m, 5H), 5.01-4.97 (d, J = 8.0 Hz, 0.5H), 4.58-4.55 (d, J = 13.6 Hz, 0.5H), 4.17-4.13 (d, J = 9.5 Hz, 0.5H), 3.74 (s, 2H), 3.65-3.61 (d, J = 13.7 Hz, 0.5H), 3.06-2.99 (t, J = 13.5 Hz, 0.5H), 2.71-2.65 (t, J = 13.5 Hz, 0.5H), 1.61-1.44 (m, 6H), 1.16-1.09 (m, 3H).

[0585] Preparation Example 6. Preparation of active compound according to representative reaction scheme 7 (anhydrous form)

[0586] [Reaction Scheme 7]

[0587] JPEG2025511114000035.jpg38146

[0588] JPEG2025511114000036.jpg162131

[0589] (1) Synthesis of compound BnH12062

[0590] (3S,6R)-6-Methyl-1-(2-phenylacetyl)piperidine-3-carboxylic acid

[0591] Step 1: Synthesis of methyl (3S,6R)-6-methyl-1-(2-phenylacetyl)piperidine-3-carboxylate

[0592] To a solution of 2-phenylacetic acid (100 mg, 0.734 mmol) in DMF (2.45 mL) was added methyl (3S,6R)-methyl-6-methylpiperidine-3-carboxylate (anhydrous form) (121 mg, 0.771 mmol), DIPEA (0.192 mL, 1.10 mmol), HOBt (119 mg, 0.881 mmol), and EDC·HCl (137 mg, 0.881 mmol). The mixture was stirred at room temperature for 3 h. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 3:1 to 2:1) to give the desired compound (110 mg, 54%) as a colorless oil.

[0593] Step 2: Synthesis of compound BnH12062

[0594] To a solution of methyl (3S,6R)-6-methyl-1-(2-phenylacetyl)piperidine-3-carboxylate (105 mg, 0.381 mmol) in THF (1.32 mL) was added 1N NaOH (0.572 mL, 0.572 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12062 (50.5 mg, 51%) as a white solid.

[0595] 1H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 1H), 7.33-7.30 (t, J = 7.4 Hz, 2H), 7.25-7.19 (dd, J = 16.7, 7.8 Hz, 3H), 4.74 (s, 0.5H), 4.55-4.52 (d, J = 13.4 Hz, 0.5H), 4.24 (s, 0.5H), 3.90-3.86 (d, J = 13.8 Hz, 0.5H), 3.78-3.68 (t, J = 5.8 Hz, 2H), 2.99 (t, J = 12.8 Hz, 0.5H), 2.63-2.57 (d, J = 12.5 Hz, 0.5H), 2.17-2.08 (m, 1H), 1.79-1.76 (d, J = 12.9 Hz, 1H), 1.69-1.59 (m, 1H), 1.52-1.48 (d, J = 13.9 Hz, 2H), 1.05-1.03 (d, J = 6.8 Hz, 3H).

[0596] (2) Synthesis of compound BnH12063

[0597] (3S,6R)-1-(2-(4-methoxyphenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0598] Step 1: Synthesis of methyl (3S,6R)-1-(2-(4-methoxyphenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0599] To a solution of 2-(4-methoxyphenyl)acetic acid (100 mg, 0.602 mmol) in DMF (2.01 mL) was added methyl (3S,6R)-6-methylpiperidine-3-carboxylate (anhydrous form) (99.3 mg, 0.632 mmol), DIPEA (0.158 mL, 0.903 mmol), HOBt (97.6 mg, 0.722 mmol), and EDC·HCl (112 mg, 0.722 mmol). The mixture was stirred at room temperature for 1 h 30 min. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane: EtOAc = 2:1) to give the desired compound (85.2 mg, 46%) as a colorless oil.

[0600] Step 2: Synthesis of compound BnH12063

[0601] To a solution of methyl (3S,6R)-1-(2-(4-methoxyphenyl)acetyl)-6-methylpiperidine-3-carboxylate (85.2 mg, 0.279 mmol) in THF (1.44 mL) was added 1N NaOH (0.419 mL, 0.419 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (2.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was washed with brine (3.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure to provide compound BnH12063 (65.1 mg, 80%) as a white solid.

[0602] 1H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 1H), 7.16-7.10 (dd, J = 18.6, 8.1 Hz, 2H), 6.88-6.86 (d, J = 8.5 Hz, 2H), 4.73 (s, 0.5H), 4.54 - 4.50 (m, 0.5H), 4.23 (s, 0.5H), 3.89-3.86 (d, J = 14.4 Hz, 0.5H), 3.73 (s, 3H), 3.66-3.60 (m, 2H), 3.01-2.95 (t, J = 12.8 Hz, 0.5H), 2.62-2.59 (d, J = 12.4 Hz, 0.5H), 2.15 - 2.09 (m, 1H), 1.80-1.76 (d, J = 11.8 Hz, 1H), 1.69-1.63 (m, J = 13.4 Hz, 1H), 1.51-1.43 (m, J = 13.5 Hz, 2H), 1.03 (d, J = 6.9 Hz, 3H).

[0603] (3) Synthesis of compound BnH12068

[0604] Sodium (3S,6R)-6-methyl-1-(2-(pyridin-3-yl)acetyl)piperidine-3-carboxylate

[0605] Step 1: Synthesis of methyl (3S,6R)-6-methyl-1-(2-(pyridin-3-yl)acetyl)piperidine-3-carboxylate

[0606] To a solution of 2-(pyridin-3-yl)acetic acid (100 mg, 0.729 mmol) in DMF (2.43 mL) was added methyl (3S,6R)-6-methylpiperidine-3-carboxylate (anhydrous form) (120 mg, 0.766 mmol), DIPEA (0.191 mL, 1.09 mmol), HOBt (118 mg, 0.875 mmol), and EDC·HCl (136 mg, 0.875 mmol). The mixture was stirred at room temperature for 2 h. The mixture was diluted with EtOAc (5.00 mL) and washed with saturated aqueous NH₄Cl (5.00 mL) and brine (5.00 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (DCM:MeOH = 0-5%) to give the desired compound (112 mg, 56%) as a pale yellow oil.

[0607] Step 2: Synthesis of compound BnH12068

[0608] To a solution of methyl (3S,6R)-6-methyl-1-(2-(pyridin-3-yl)acetyl)piperidine-3-carboxylate (112 mg, 0.405 mmol) in THF (2.10 mL) was added 1N NaOH (0.608 mL, 0.608 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was concentrated under reduced pressure. The residue was purified by column chromatography on C18-SiO2 (HO:MeOH = 1:0 to 4:1) to give compound BnH12068 (91.0 mg, 79%) as a pale blue solid.

[0609] 1H NMR (400 MHz, DMSO-d6) δ 8.43-8.39 (m, 2H), 7.63-7.57 (dd, J = 15.5, 7.8 Hz, 1H), 7.34-7.30 (dt, J = 8.2, 4.4 Hz, 1H), 4.68 (s, 0.5H), 4.42-4.38 (d, J = 14.4 Hz, 0.5H), 4.20 (s, 0.5H), 3.81-3.68 (m, 2.5H), 2.99-2.92 (t, J = 12.8 Hz, 0.5H), 2.68 (s, 0.5H), 1.78-1.66 (dd, J = 18.2, 8.2 Hz, 2H), 1.56-1.43 (m, 3H), 1.11-1.02 (dd, J = 30.2, 6.9 Hz, 3H).

[0610] Preparation Example 7. Preparation of active compounds by methods other than the representative reaction scheme

[0611] (1) Synthesis of BnH12001

[0612] 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0613] JPEG2025511114000037.jpg73143

[0614] Step 1: Compound 1 (200 mg, 1.06 mmol) was dissolved in 5 mL of dichloromethane (DCM), and oxalyl chloride (136 μL, 1.59 mmol) was added dropwise at 0° C. for 5 min, followed by the addition of dimethylformamide (DMF) (1 drop) along with a catalyst. After stirring at room temperature for 6 h, the mixture was concentrated under reduced pressure to give 219 mg of compound 2. The reaction was carried on to the next step without further purification.

[0615] Step 2: Compound 3 (166 mg, 1.06 mmol) was dissolved in 10 mL of DCM, and triethylamine (295 μL, 2.12 mmol) was added at 0 °C. Compound 2 (219 mg, 1.06 mmol) was dissolved in 5 mL of DCM and added dropwise at 0 °C, followed by stirring at room temperature for 2 h. The reaction mixture was worked up with DCM and water, and then purified using a column filled with silica gel 60 (0.040-0.063 mm) (1.09385.9025, Merck) to give 131 mg of compound 4 (38% yield).

[0616] Step 3: Compound 4 (131 mg, 400 μmol) was dissolved in 5 mL of tetrahydrofuran (THF) / methanol (1:1). 1N NaOH (800 μL, 800 μmol) was added and stirred at room temperature for 12 hours. After the reaction was completed, the pH was adjusted to 3 with 1N HCl, extracted with ethyl acetate, and concentrated under reduced pressure. 34 mg of the target compound 5 was obtained from the resulting reaction mixture using prep TLC (27% yield). The identity was confirmed by NMR spectroscopy: 1H NMR (CDCl3, 400 MHz): δ 7.22 (1H, m), 7.08 (1H, m), 6.91 (1H, bs), 4.75 (1H, m), 3.86 (1H, m), 3.71 (2H, m), 3.01 (1H, m), 2.28 (1H, m), 1.91 (1H, bs), 1.74 (1H, m), 1.58 (1H, bs), 1.13 (3H, m).

[0617] (2) Synthesis of BnH12001 enantiomer (3S,6R, 98% or more)

[0618] JPEG2025511114000038.jpg44145

[0619] Step 1: Preparation of methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0620] 2-(2-Chloro-4-fluorophenyl)acetic acid (1.7 kg), (3S,6R)-methyl 6-methylpiperidine-3-carboxylate (1.05 eq.), DIPEA (1.5 eq.), HOBt (0.3 eq.), and EDC·HCl (1.2 eq.) were added to a solution of 2-MeTHF (10 V). The mixture was heated at 25°C to 35°C for 1.3 h. The mixture was washed twice with HO (5 V) to give methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate in 99.6% purity.

[0621] Step 2: Preparation of BnH12001 enantiomer (3S,6R, 98% or more)

[0622] Methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate was scaled up to 2.953 kg. Methyl (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate was added to 2-MeTHF (10 V) along with 1.3 eq. of 1 N NaOH. The mixture was heated at 25° C. for 3 h. Crystallization from 2-MeTHF / n-heptane yielded 2.754 kg of (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid with less than 0.1% impurities.

[0623] Step 3: Purification of BnH12001 enantiomers (3S,6R, 98% or more)

[0624] (3S,6R)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid (2.57 kg) was added to EA (5 V) and n-heptane (15 V). The mixture was heated from 0° C. to 45° C. to produce 2.404 kg of a dry cake as a white solid.

[0625] (3) Synthesis of compound BnH12010

[0626] (3R,6S)-1-(2-chloro-4-fluorophenethyl)-6-methylpiperidine-3-carboxylic acid

[0627] JPEG2025511114000039.jpg60143

[0628] Step 1: 2-chloro-4-fluorophenethyl 4-methylbenzenesulfonate (e)

[0629] To a solution of p-toluenesulfonyl chloride (240 mg, 2.21 mmol), DMAP (24.5 mg, 0.200 mmol), and TEA (0.503 mL, 3.61 mmol) in DCM (3.00 mL) was added 2-(2-chloro-4-fluorophenyl)ethanol (350 mg, 2.001 mmol) at 0 °C and stirred at room temperature for 17 h. The mixture was diluted with EtOAc (5.00 mL) and washed with HO (5.00 mL × 3). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane: EtOAc = 10:1) to give the desired compound (523 mg, 79%) as a colorless oil.

[0630] Step 2: Methyl (3R,6S)-1-(2-chloro-4-fluorophenethyl)-6-methylpiperidine-3-carboxylate (f)

[0631] To a solution of 2-chloro-4-fluorophenethyl 4-methylbenzenesulfonate (200 mg, 0.608 mmol) in DMF (3.00 mL) was added K2CO3 (252 mg, 1.83 mmol) and methyl (3R,6S)-rel-6-methyl-3-piperidinecarboxylate (76.5 mg, 0.487 mmol). The mixture was stirred at 110 °C for 17 h. The mixture was diluted with EtOAc (4.00 mL) and washed with H2O (4.00 mL × 3). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane: EtOAc = 10:1) to give the desired compound (63.0 mg, 33%) as a colorless oil.

[0632] Step 3: Synthesis of compound BnH12010

[0633] To a solution of methyl (3R,6S)-1-(2-chloro-4-fluorophenethyl)-6-methylpiperidine-3-carboxylate (60.0 mg, 0.191 mmol) in THF (0.659 mL) was added 1N NaOH (0.287 mL, 0.287 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with HO (1.50 mL) and washed twice with DCM (2.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (2.00 mL). The organic layer was washed with brine (2.00 mL), dried over MgSO, filtered, and concentrated under reduced pressure. EtO (1.00 mL) was added to the residue and stirred at room temperature for 30 minutes. The precipitate was filtered and washed with Et2O (2.00 mL) to give compound BnH12010 (42.0 mg, 73%) as a white solid.

[0634] 1H NMR (400 MHz, Chloroform-d) δ 7.21 (dd, J = 8.5, 6.1 Hz, 1H), 7.12 (dd, J = 8.5, 2.7 Hz, 1H), 6.92 (td, J = 8.3, 2.7 Hz, 1H), 4.41 (s, 1H), 4.30 (t, J = 6.7 Hz, 2H), 4.21 (s, 1H), 3.06 (t, J = 6.7 Hz, 2H), 2.94 (dd, J = 13.7, 12.0 Hz, 1H), 2.39 (d, J = 13.1 Hz, 1H), 1.95 (t, J = 7.9 Hz, 1H), 1.84 - 1.52 (m, 3H), 1.12 (d, J = 6.9 Hz, 3H).

[0635] (4) Synthesis of compound BnH12021

[0636] Ethyl (3R,6S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0637] JPEG2025511114000040.jpg26144

[0638] Step 1: Synthesis of ethyl (3R,6S)-1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylate

[0639] To a solution of the compound BnH12001 enantiomer (3S,6R) (70.0 mg, 0.223 mmol) in DCM (1.01 mL) was added a catalytic amount of DMF and oxalyl chloride (0.057 mL, 0.669 mmol). The mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. The residue was dissolved in DCM (1.01 mL) and EtOH (0.065 mL, 1.12 mmol) was added. The mixture was stirred at room temperature for 1 hour and washed with saturated aqueous NaHCO3 (2.00 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane: EtOAc = 3:1) to give compound BnH12021 (37.0 mg, 48%) as a colorless oil.

[0640] 1H NMR (400 MHz, Chloroform-d) δ 7.14 (dd, J = 8.9, 2.6 Hz, 1H), 6.96 (td, J = 8.4, 2.7 Hz, 1H), 4.96 (s, 0.5H), 4.78 (d, J = 13.4 Hz, 0.5H), 4.15 (q, J = 7.2 Hz, 2.5H), 3.88 - 3.69 (m, 2.5H), 3.21 (t, J = 13.0 Hz, 0.5H), 2.79 (t, J = 12.9 Hz, 0.5H), 2.36 (d, J = 12.6 Hz, 0.5H), 2.23 (d, J = 11.2 Hz, 0.5H), 1.93 (d, J = 13.0 Hz, 1H), 1.78 (d, J = 14.8 Hz, 1H), 1.71 - 1.61 (m, 2H), 1.34 - 1.09 (m, 6H).

[0641] (5) Synthesis of compound BnH12094

[0642] 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-methylpiperidine-3-carboxylic acid

[0643] JPEG2025511114000041.jpg56144

[0644] Step 1: Synthesis of ethyl 6-ethylnicotinate (o)

[0645] To a solution of ethyl 6-chloronicotinate (2.20 g, 11.9 mmol) in anhydrous THF (60.0 mL) were added Fe(acac) (460 mg, 1.30 mmol) and NMP (1.60 mL, 16.6 mmol). The mixture was cooled to -78 °C, and ethylmagnesium bromide was added dropwise. After stirring at -78 °C for 1 h, the reaction mixture was quenched with HO (50.0 mL) at 0 °C and extracted with EtOAc (50.0 mL). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO (n-hexane: EtOAc = 1:0 to 9:1) to give the desired compound (2.04 g, 96%) as a pale yellow oil.

[0646] Step 2: Ethyl 6-ethylpiperidine-3-carboxylate (p)

[0647] To a solution of ethyl 6-ethylnicotinate (2.04 g, 11.4 mmol) in EtOH (20.0 mL) were added acetic acid (2.07 mL, 11.4 mol) and Pd / C (848 mg, 7.97 mmol). The mixture was purged with H gas three times and stirred under H gas at 65 °C for 24 h. After cooling to room temperature, the reaction mixture was filtered through Celite and washed with MeOH (30.0 mL). The filtrate was concentrated under reduced pressure and used without further purification.

[0648] Step 3: Ethyl 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-ethylpiperidine-3-carboxylate (BnH12094-1)

[0649] To a solution of 2-(2-chloro-4-fluorophenyl)acetic acid (450 mg, 2.39 mmol) in DMF (8.00 mL) was added ethyl 6-ethylpiperidine-3-carboxylate (464 mg, 2.51 mmol), DIPEA (0.623 mL, 3.58 mmol), HOBt (387 mg, 2.86 mmol), and EDC·HCl (549 mg, 2.86 mmol). The mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc (20.0 mL) and washed with saturated aqueous NH4Cl (20.0 mL) and brine (20.0 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (n-hexane:EtOAc = 1:0 to 2:1 and 0 to 3% MeOH in DCM) to give the desired compound (121 mg, 24%, 2 steps) as a colorless oil.

[0650] Step 4: Synthesis of compound BnH12094

[0651] To a solution of ethyl 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-ethylpiperidine-3-carboxylate (115 mg, 0.323 mmol) in THF (3.00 mL) was added 1N NaOH (0.970 mL, 0.970 mmol). The mixture was stirred at 50 °C for 17 h. The reaction mixture was diluted with HO (5.00 mL) and washed twice with DCM (5.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (5.00 mL). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was dissolved in EtOAc (2.00 mL), and n-hexane (10.0 mL) was added. The resulting solid was filtered and washed with n-hexane (5.00 mL) to give compound BnH12094 (82.0 mg, 77%) as a white solid.

[0652] 1H NMR (400 MHz, DMSO-d6) δ 12.43 (s, 1H), 7.42 - 7.23 (m, 2H), 7.17 (td, J = 8.5, 2.8 Hz, 1H), 4.54 - 4.47 (m, 1H), 3.96 - 3.70 (m, 2.5H), 3.25 - 3.21 (m, 0.5H), 3.07 (t, J = 12.9 Hz, 0.5H), 2.66 - 2.53 (m, 1H), 2.39 - 2.33 (m, 0.5H), 2.24 - 2.18 (m, 0.5H), 1.84 - 1.40 (m, 5.5H), 0.82 (dt, J = 42.2, 7.3 Hz, 3H).

[0653] (6) Synthesis of compound BnH12096

[0654] 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-isopropylpiperidine-3-carboxylic acid

[0655] JPEG2025511114000042.jpg54144

[0656] Step 1: Ethyl 6-isopropylnicotinate (r)

[0657] To a solution of ethyl 6-chloronicotinate (300 mg, 1.62 mmol) in anhydrous THF (8.00 mL) was added Fe(acac) (62.8 mg, 0.178 mmol) and NMP (0.218 mL, 2.26 mmol). The mixture was cooled to −78 °C, and isopropylmagnesium bromide was added dropwise. After stirring at −78 °C for 1 h, the reaction mixture was quenched with HO (10.0 mL) at 0 °C and extracted with EtOAc (10.0 mL). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO (DCM:n-hexane = 1:1 and n-hexane:EtOAc = 9:1) to give the desired compound (170 mg, 54%) as a pale yellow oil.

[0658] Step 2: Ethyl 6-isopropylpiperidine-3-carboxylate(s)

[0659] To a solution of ethyl 6-isopropylnicotinate (165 mg, 0.854 mmol) in EtOH (2.00 mL) were added acetic acid (0.155 mL, 0.854 mol) and Pd / C (63.6 mg, 0.598 mmol). The mixture was purged with H gas three times and stirred under H gas at 65 °C for 17 h. After cooling to room temperature, the reaction mixture was filtered through Celite and washed with MeOH (5.00 mL). The filtrate was concentrated under reduced pressure and used without further purification.

[0660] Step 3: Ethyl 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-isopropylpiperidine-3-carboxylate (BnH12096-1)

[0661] To a solution of 2-(2-chloro-4-fluorophenyl)acetic acid (270 mg, 1.43 mmol) in DMF (5.00 mL) was added ethyl 6-isopropylpiperidine-3-carboxylate (300 mg, 1.51 mmol), DIPEA (0.375 mL, 2.15 mmol), HOBt (233 mg, 1.72 mmol), and EDC·HCl (330 mg, 1.72 mmol). The mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (15.0 mL) and washed with saturated aqueous NH₄Cl (15.0 mL) and brine (15.0 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on SiO₂ (n-hexane:EtOAc = 4:1 to 2:1) to give the desired compound (176 mg, 54%, 2 steps) as a pale yellow oil.

[0662] Step 4: Synthesis of compound BnH12096

[0663] To a solution of ethyl 1-(2-(2-chloro-4-fluorophenyl)acetyl)-6-isopropylpiperidine-3-carboxylate (176 mg, 0.476 mmol) in THF (1.64 mL) was added 1N NaOH (0.714 mL, 0.714 mmol). The mixture was stirred at 50 °C for 4 h. The reaction mixture was diluted with HO (3.00 mL) and washed twice with DCM (3.00 mL × 2). The aqueous layer was acidified with 1N HCl at 0 °C to pH 1-2 and extracted with EtOAc (3.00 mL). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. EtO (4.00 mL) was added to the residue and stirred at room temperature for 30 min. The precipitate was filtered and washed with EtO (3.0 mL) to give compound BnH12096 (102 mg, 63%) as a white solid.

[0664] 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 7.42 - 7.23 (m, 2H), 7.17 (td, J = 8.5, 2.8 Hz, 1H), 4.54 (dd, J = 13.2, 4.3 Hz, 0.5H), 4.23 - 4.13 (m, 0.5H), 4.01 - 3.67 (m, 2.5H), 3.57 (dd, J = 10.7, 4.5 Hz, 0.5H), 3.05 (dd, J = 14.1, 11.9 Hz, 0.5H), 2.45 - 2.31 (m, 0.5H), 2.30 - 1.96 (m, 1.5H), 1.87 (dd, J = 28.7, 12.7 Hz, 2H), 1.74 - 1.58 (m, 1H), 1.55 - 1.32 (m, 1H), 1.06 - 0.66 (m, 6H).

[0665] Example

[0666] The compounds obtained in the above Preparation Examples were subjected to biological assays as follows.

[0667] Example 1. Cell culture and maintenance

[0668] Human undifferentiated neuroblastoma cell line SH-SY5Y and human embryonic kidney cell line (HEK)-293T were purchased from the Korean Cell Line Bank (Korea) and Takara (Japan), respectively. Cells were grown in HyClone Minimal Essential Medium (MEM) and Dulbecco's Modified Eagle Medium (DMEM), respectively, supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 mg / mL). Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were passaged up to 10 times. Cells were detached using 0.05% trypsin-EDTA solution and seeded onto new culture dishes for maintenance.

[0669] Primary cortical neuron cells were isolated from cerebral cortices of 18-day-pregnant mouse fetuses and incubated for 30 minutes in prep media containing 0.25% trypsin and 1dL DNase (mM: NaCl 107.5, NaH2PO4 1, NaHCO3 3.5, glucose 11, HEPS 1%, penicillin-streptomycin 1%). Single cells were then dissociated and seeded. Primary cortical neuron cells were grown in Neurobasal medium supplemented with 1% (v / v) B27, 1% penicillin (100 U / mL), streptomycin (100 mg / mL), and 1% Glutamax. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Drug efficacy was evaluated after 14 days of culture.

[0670] Example 2. Cytotoxicity test

[0671] To investigate the cytotoxicity of the compounds described herein in human cell lines, SH-SY5Y and 293T cells were used. Approximately 8,000 cells (for 293T) and 15,000 cells (for SH-SY5Y) were plated per well of a 96-well plate (SPL Life Sciences Co., Ltd., Pocheon, Korea). After 24 hours of incubation at 37°C in a humidified atmosphere containing 5% CO2, the culture medium was replaced with a series of concentrations of the test compound diluted in the culture medium. The incubation time was 24 hours, and six replicates were performed for each measurement. Finally, 10 μL of CCK-8 reagent (Dojindo Laboratories, Rockville, MD, USA) was added to each well. After 3 minutes of incubation at 37°C, the OD at 450 nm was measured using a multifunction microplate reader (Tecan Infinite M200 Pro). These measurements were compared with those of the control group to calculate cell viability, which was expressed as a percentage of the control group. IC was calculated using SPSS software. 50 To do this, the absorbance of blank wells containing growth medium alone was subtracted from the values ​​for wells containing cells. The background (vehicle) reading was set to 100% viability, and the viability of each test solution was then calculated.

[0672] Cell viability (%)=[(As-Ab) / (Ac-Ab)]×100

[0673] Data are presented as means and standard errors of the means (SEM). Statistical analysis was performed by one-way ANOVA with Tukey's post-hoc test (GraphPad Prism 5 software package, version 5.02, GraphPad Software Inc., USA). Significance was accepted at p < 0.05.

[0674] Example 3. Measurement of expression levels of BDNF and / or GluN2B

[0675] Example 3.1. RT-PCR

[0676] RT-PCR was used to measure the levels of BDNF and GluN2B in response to the compounds of the present invention. 293T cells were detached using 0.05% trypsin-EDTA solution and seeded into 24-well or 6-well plates for RNA analysis. After 24 hours of incubation at 37°C in a humidified atmosphere containing 5% CO2, the culture medium was replaced with medium containing a range of concentrations of the test compound diluted in DMEM without FBS. A time-course experiment was performed. The same amount of DMSO was used as a negative control.

[0677] After compound treatment, RNA was isolated from the cells. After removing the growth medium, 1 × 10 5 ~10 7 0.3-0.4 mL of TRIzol per cell TM The cells were lysed by adding TRIzol directly to the culture dish. After incubation at 4°C for 10 minutes, the sample was centrifuged at 12,000 × g for 10 minutes at 4°C. The total RNA precipitate formed a white gel-like pellet at the bottom of the tube. After discarding the supernatant, the pellet was lysed in 1 mL of TRIzol, which was used for lysis. TM The samples were resuspended in 1 mL of 75% ethanol per 1000 μL of RNA. After brief vortexing, the samples were centrifuged at 7500 × g for 5 minutes at 4°C. The supernatant was discarded with a micropipette and allowed to dry under vacuum or in air for 5–10 minutes. The pellet was resuspended in 20–50 μL of RNase-free water. The RNA concentration was determined using Thermo Scientific TM μDrop TM The A230 / 260 and A260 / 280 ratios were measured to determine RNA quality.

[0678] On ice, experimental RNA (up to 1 μg in 4 μl) and 1 μl (100 pmol) of oligo dT primer were combined in nuclease-free water to a final volume of 5 μl per RT reaction. After tightly sealing each RNA tube, the tubes were placed in a PCR machine at 70°C for 5 minutes. After removing the tubes, they were immediately cooled in ice water for at least 5 minutes. Each tube was centrifuged in a microcentrifuge for 10 seconds to collect any condensate and maintain the original volume. The tubes were kept closed on ice until the reverse transcription reaction mixture was added. The reverse transcription reaction mixture was prepared as follows:

[0679] JPEG2025511114000043.jpg100145

[0680] Reverse transcription was performed using Improm-II Reverse Transcriptase (A3803) (Promega) and Recombinant RNasin Ribonuclease Inhibitor (N2515) (Promega) according to the manufacturer's instructions. A 15 μl aliquot of the reverse transcription reaction mixture was added to each reaction tube on ice, and the tubes were placed back into the PCR machine. PCR conditions were as follows:

[0681] 25°C, 5 min (annealing) → 42°C, 60 min (extension) → 70°C, 15 min (RT inactivation) → 4°C hold

[0682] PCR products were analyzed by agarose gel electrophoresis. A housekeeping gene (ACTIN) was used as a PCR control. Replicate samples confirmed the validity of the changes observed at the RNA level. The primer sequences used were as follows:

[0683] JPEG2025511114000044.jpg79146

[0684] Example 3.2. Western blotting

[0685] Western blot analysis was used to measure the amount of GluN2B reacting with the compounds of the present invention. Fetal cerebral cortices were excised from 17-day-pregnant mice, dissociated into single cells, cultured for 13 days, and treated with BnH drugs for 24 hours. Protein lysates were prepared from the drug-treated cells using RIPA protein lysis buffer containing protease and phosphate inhibitors. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology) with bovine serum albumin as the standard. Equal amounts of protein (20 μg per lane) were separated by electrophoresis on an SDS-polyacrylamide gel at 90 V for 2 hours, then transferred to a PVDF membrane at 100 V for 1 hour. The primary reaction was performed with GluN2B antibody (ab65783, Abcam) at 4°C for 1 day, followed by three 10-minute washes with 1X PBT. The secondary reaction was performed with anti-mouse antibody (sc-2357, Santa Cruz) for 1 hour, followed by three 10-minute washes with 1X PBT. GluN2B protein expression was confirmed by Western blotting as described above. Each blot was then reprobed with anti-β-actin antibody (sc-47778, Santa Cruz). The housekeeping protein β-actin was used as a normalization control.

[0686] As a result, as shown in Table 6, changes in GluN2B protein were confirmed with various BnH12 series compounds.

[0687] Data analysis

[0688] The vehicle was set to 1, and the increase or decrease in the BnH12 series was confirmed (N per group was 3 or more).

[0689] Example 4. Synaptic function test

[0690] Example 4.1. Os pump implantation

[0691] Eight-week-old male C57BL / 6J mice were anesthetized with isoflurane during surgery. Subsequently, the mice were placed in a stereotaxic frame (RWD Life Science, China). The parietal and frontal bones were exposed through a thalamotomy incision in the scalp from the midline from eye level to the occipital prominence. After the skull was exposed, a small burr hole was drilled. An infusion cannula was placed at the following coordinates: (mouse: vertex -3.30, lateral -1.50, and ventral 0.7 mm). Sterile saline solution (+ / - the compound of the present invention) was perfused through the infusion cannula at a constant flow rate of 0.5 μL / h using a mini-Os pump (ALZET Osmotic Pumps, DURECT Corporation, CA, USA). Implantation was performed in layer 4 of the eastern barrel cortex. To estimate the extent of diffusion of the infused compound in layer 4 of the barrel cortex during pump infusion, methylene blue was first injected to stain the diffusion area. On average, methylene blue diffused up to 2000.0 μm from the injection site (total area of ​​barrel cortex is approximately 1500 μm). Based on this analysis, barrel cortex area was recorded.

[0692] Example 4.2. Oral Administration of Compounds

[0693] A stock solution of 2 mg of BnH12001 was prepared by dissolving it in 800 μl of DMSO. For oral administration, 200 μl of BnH12001 DMSO stock, PEG400, and saline were mixed at a ratio of 5:55:40, and administered per animal. For the control group, 200 μl of vehicle, a 5:55:40 mixture of DMSO, PEG400, and saline, was administered per animal. In single-drug administration studies, the compound was administered at a dose of 1 mg / kg once daily for approximately 11–12 days. A dose-response study was performed by administering the compound at doses of 0.03, 0.1, 0.3, 1, 3, and 10 mg / kg once daily for approximately 11–12 days. Five mice were used for each compound-treated and control group.

[0694] Example 4.3. Intraperitoneal injection of compounds

[0695] The compound was diluted to 25 mg / kg using DMSO:PEG400:saline (5:55:40). The compound was administered intraperitoneally (IP) at 10:00 AM daily for approximately 11-12 days (intraperitoneal volume: 200 μl). The control group was administered intraperitoneally in the same manner using vehicle (DMSO:PEG400:saline (5:55:40)). Five mice were used for each compound-administered and control group.

[0696] Example 4.4. Intravenous Infusion of Compounds

[0697] Aducanumab or control antibody was used as a stock solution at 1 mg / ml. For intravenous injection, the tail vein injection method was used. Aducanumab was administered at a dose of 5 mg / kg. The control antibody was administered using an antibody inactivated with aducanumab at a dose of 5 mg / kg. In the drug injection study, the compound was administered once daily for approximately 11 to 13 days. Five mice were used for each compound-treated and control group.

[0698] Example 4.5. Thalamocortical (TC) or Hippocampal Slice Preparation

[0699] For preparation of TC or hippocampal slices, 9- to 10-week-old male C57BL / 6J mice were anesthetized with isoflurane. Subsequently, the brains were rapidly cooled by gentle cardiac perfusion with ice-cold sucrose artificial cerebrospinal fluid (aCSF). The brains were isolated and placed in ice-cold sucrose aCSF. Paracoronal slices were prepared at a 50° angle relative to the midline with a 10° ramp. Slices were then incubated in aCSF at 35°C for 30 minutes to recover. Slices were then incubated in aCSF for 1-4 hours at room temperature (23-25°C) before being placed in the experimental recording chamber. Standard aCSF was saturated with 95% O2 / 5% CO2. Mouse brains were isolated and sectioned at 400 µm at a 50° angle (55° for mice) and attached to a vibratome disc with the sectioned section at the bottom. Brain slices were incubated in aCSF solution for 30 min at 35°C or incubated in aCSF solution for 1 h at room temperature.

[0700] Composition of sucrose-based aCSF

[0701] JPEG2025511114000045.jpg171145

[0702] Recording aCSF composition (room temperature: RT)

[0703] JPEG2025511114000046.jpg171145

[0704] Example 4.6. Electrophysiological experiments on barrel cortex

[0705] All electrophysiological experiments were performed at 27–29°C. For electrophysiological experiments, electrodes with resistances of 3.5–4.2 mΩ were used, and stimulation was delivered to the ventral posteromedial nucleus (VPM) using concentric bipolar electrodes (FHC, Bowdoin, ME, USA). Under 4× magnification and differential interference contrast (DIC) optics, the somatosensory cortex was identified by the presence of barrel cortex and the ability to evoke brief, constant field excitatory postsynaptic potentials (fEPSPs) upon VPM stimulation. Whole-cell voltage-clamp recordings were performed on stellate cells (SC) in layer IV of the somatosensory barrel cortex using infrared illumination and differential interference contrast (DIC) optics. The composition of the whole-cell recording solution is presented below:

[0706] Whole Cell Recording Internal Solution

[0707] JPEG2025511114000047.jpg150145

[0708] Whole-cell recordings were made using a Multiclamp 700A (Molecular Devices, Sunnyvale, CA, USA) digitized at 10 kHz and filtered at 2 kHz. During recording, input and series resistances were monitored as previously described (Isaac, Crair, Nicoll, & Malenka, 1997, Silent synapses during development of thalamocortical inputs, DOI: 10.1016 / s0896-6273(00)80267-6). TC EPSCs were elicited by VB stimulation at 0.1 Hz and were recognized as monosynaptic if they exhibited short, constant latencies that did not change with increasing stimulus intensity, as previously described (Feldman, Nicoll, Malenka, & Isaac, 1998, Synaptic plasticity at thalamocortical synapses in developing rat somatosensory cortex: LTP, LTD, and silent synapses, https: / / doi.org / 10.1002 / (SICI)1097-4695(199910)41:1<92::AID-NEU12>3.0.CO;2-U; Isaac et al., 1997, Silent Synapses during Development of Thalamocortical Inputs, https: / / doi.org / 10.1016 / S0896-6273(00)80267-6).

[0709] For the minimum stimulation protocol (potency), the VB thalamus stimulation intensity was adjusted to determine the lowest intensity that elicited a mixture of synaptic responses and failures. The failure rate was calculated as the number of failures divided by the total number of attempts. Potency was calculated as the mean EPSC peak amplitude, excluding failures (see Chittajallu & Isaac, 2010, Emergence of cortical inhibition by coordinated sensory-driven plasticity at distinct synaptic loci, DOI: 10.1038 / nn.2639; Isaac et al., 1997, Silent Synapses during Development of Thalamocortical Inputs, https: / / doi.org / 10.1016 / S0896-6273(00)80267-6; Stevens & Wang, 1995, Facilitation and depression at single central synapses, DOI: 10.1016 / 0896-6273(95)90223-6).

[0710] The criteria for single-axon stimulation were the presence or absence of synaptic events and the absence of changes in the mean amplitude of EPSCs evoked by small increases in stimulation intensity, as previously reported (Chittajallu and Isaac, 2010, Emergence of cortical inhibition by coordinated sensory-driven plasticity at distinct synaptic loci, DOI: 10.1038 / nn.2639; Gil et al., 1999, Efficacy of Thalamocortical and Intracortical Synaptic Connections: Quanta, Innervation, and Reliability, https: / / doi.org / 10.1016 / S0896-6273(00)80788-6). Measurement parameters were as follows:

[0711] Measurement parameters

[0712] JPEG2025511114000048.jpg44146

[0713] Excitatory postsynaptic potentials (EPSCs) were recorded using the whole-cell patch clamp technique with a patch clamp amplifier (Molecular Devices, USA). The measurement electrode was a borosilicate glass capillary with a resistance of 3.5–4.2 MΩ when filled with the electrode's internal solution. The brain slice in the chamber was placed on a vertical microscope, fixed with an anchor, and gravity-driven aCSF solution was allowed to flow at a rate of 5–10 mL / min. The stimulator electrode was placed at the junction between thalamic fibers and the S1 barrel cortex (layer 4), and cells within that region were located and recorded. After whole-cell patching, the membrane voltage was clamped at -70 mV (to measure AMPA currents). EPSCs were confirmed by applying a stimulus, and the stimulator was manipulated to determine the minimum stimulus intensity at which the current was switched on and off at 50%. The recording time was 3 minutes 20 seconds or 8 minutes 20 seconds, and the amplitude was measured for each trace (20 or 50 traces in total). For potency, EPSC size was set to 50–100 pA at -70 mV, and five traces were measured at 0 mV.

[0714] Example 4.7. Electrophysiological experiments on the hippocampus

[0715] Field recordings were performed using a concentric bipolar electrode positioned in the stratum radiatum of the CA1 region using an extracellular glass pipette (3.5 MΩ) filled with aCSF. Specifically, fEPSPs were elicited from Schaffer collateral (SC) / commissural fibers using a concentric bipolar electrode positioned 200–300 μm from the recording pipette. Stimulation was delivered via a bipolar electrode (FHC, Bowdoin, Maine, USA) placed in the Schaffer collateral-CA1 (SC). The SC circuit was visualized using differential interference contrast (DIC) microscopy at 4x magnification and confirmed by the ability to evoke brief, constant field excitatory postsynaptic potentials (fEPSPs) at CA1 synapses by SC input stimulation. For all fEPSP experiments, a test stimulus (30 μA) was measured before the start of every experiment, and the test pulse stimulation intensity that evoked 50% of the maximum fEPSP was used. Baseline synaptic responses were recorded for 30 min, followed by induction of long-term potentiation (LTP) with high-frequency stimulation (HFS, 100 Hz, 4 trains, 1 s duration, 20 s inter-train interval). Recordings were made every 10 s for 1 h using a multiclamp 700A amplifier (Molecular Devices, San Jose, CA, USA) digitized at 10 kHz on a Digidata 1440 and filtered at 2 kHz, and pClamp 10.0 software (Molecular Devices, San Jose, CA, USA). Measurement parameters were as follows:

[0716] Measurement parameters

[0717] JPEG2025511114000049.jpg66145

[0718] Excitatory postsynaptic potentials (EPSPs) were recorded using the whole-cell patch clamp technique with a patch clamp amplifier (Molecular Devices, USA). The measurement electrodes were borosilicate glass capillaries with a resistance of 3.5–4.2 MΩ when filled with the electrode's internal solution. The brain slices in the chamber were placed on a vertical microscope, fixed with anchors, and gravity-driven aCSF solution was allowed to flow through them at a rate of 5–10 mL / min. The stimulator electrode was placed in the SC region, and connections within the CA1 region were located and recorded. As the stimulation intensity increased, recording points were recorded until either no fEPSP increase or a pop spike occurred (I / O curve). Baseline synaptic responses were recorded for 20 min, followed by induction of long-term potentiation (LTP) by high-frequency stimulation (HFS, 100 Hz, 4 trains, 1 s duration, 20 s inter-train interval / TBS; 100 Hz, 10 trains, 50 ms duration, 200 ms inter-train interval) followed by recording for 60 min (LTP).

[0719] Example 4.8. Data Analysis

[0720] Potency analysis was performed using the Clampfit program (Axon Instruments). A baseline was established by specifying an interval between 0 and 100 ms. Traces were averaged by selecting "On" currents appearing between 110 and 200 ms. The negative current amplitude (pA) of the averaged trace was confirmed.

[0721] The GABA / AMPA ratio was analyzed using Clampfit. For AMPA currents, a baseline was set by specifying an interval between 0 and 100 ms. Traces from 110 to 200 ms were averaged. The negative current amplitude (pA) of the averaged trace was confirmed. For GABA currents, a baseline was set by specifying an interval between 0 and 110 ms. Traces from 110 to 300 ms were averaged. The positive current amplitude (pA) of the averaged trace was confirmed.

[0722] I / O curves were calculated using Clampfit. Baselines were set at intervals between 0 and 6 ms. Five traces recorded from the same stimulus were averaged. The amplitude of the fiber volley and the 20-80% rising slope of the fEPSP were analyzed. For LTP, baselines were set at intervals between 0 and 6 ms. The 20-80% rising slope of the fEPSP was analyzed before and after LTP induction using HFS (or TBS).

[0723] Example 4.9. Statistical Analysis

[0724] All data are presented as mean ± SEM, and n indicates the number of cells used in each experiment. Generally, one slice was used from each animal; one recording was made from each slice. To compare mean values ​​from two groups, an unpaired Student's t-test was used; for three or more groups, a one-way analysis of variance (ANOVA) was used. A p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using Prism 9.0 (GraphPad Prism 9.1.2, GraphPad Software Inc., USA).

[0725] Dose-response curves were constructed using Graphpad Prism 9 according to the manufacturer's guidelines.

[0726] Example 4.10. Western Blotting

[0727] For thalamocortical (TC) slice preparation, 9- to 10-week-old male C57BL / 6J rodents (mice) were anesthetized with isoflurane. Subsequently, the brains were rapidly cooled by transcardial perfusion with ice-cold sucrose artificial cerebrospinal fluid (aCSF). The brains were isolated and placed in ice-cold sucrose aCSF. Paracoronal slices were prepared at a 50° angle relative to the midline on a 10° ramp. Slices were then incubated in aCSF at 35°C for 30 min to recover.

[0728] Slices were subsequently incubated in aCSF for 1–4 h at room temperature (23–25°C) before being placed in the experimental recording chamber. Standard aCSF was saturated with 95% O2 / 5% CO2.

[0729] Composition of sucrose-based aCSF

[0730] JPEG2025511114000050.jpg161145

[0731] Composition of recording aCSF (room temperature: RT)

[0732] JPEG2025511114000051.jpg162146

[0733] All electrophysiological experiments were performed at 27–29°C. Rodents (mice) were perfused with blood from a sucrose-based aCSF solution (4°C) using gravity. Mouse brains were isolated and sectioned at 400 μm at a 50° angle (55° for mice) and attached to a vibratome disc with the sectioned section facing downward. Brain slices were incubated in a 35°C aCSF solution for 30 min or at RT for 1 h. Mouse cortical tissue was prepared according to the previously described protocol and subsequently homogenized and lysed for protein extraction. Protein lysates were prepared from the tissue using NP40 (FNN0021, Invitrogen) protein lysis buffer containing protease and phosphate inhibitors according to the manufacturer's instructions. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology) with bovine serum albumin as the standard. Equal amounts of protein (20 μg per lane) were separated by electrophoresis on 8% or 12% SDS-polyacrylamide gels and then blotted onto PVDF membranes (GE Healthcare). To block nonspecific binding, the membranes were incubated with 5% nonfat dry milk in PBST (150 mM NaCl, 50 mM Tris, pH 7.4, 0.05% Tween 20) for 1 hour at room temperature. Anti-HDAC4 (SAB4300413, Sigma-Aldrich), anti-BDNF (ANT-010, Alomone Las), and anti-NMDAR2B (ab65783, Abcam plc) antibodies were incubated overnight at 4°C in 2% blocking solution. After antibody incubation and several rinses with PBST, the membranes were incubated with a mouse anti-rabbit IgG-HRP conjugated secondary antibody (sc-2357, Santa Cruz) for 1 hour. Each blot was then re-probed using an anti-β-actin antibody (sc-47778, Santa Cruz). The housekeeping protein β-actin was detected as a normalization control. The antibodies used in the methods described herein are as follows:

[0734] JPEG2025511114000052.jpg90146

[0735] Example 5. Therapeutic efficacy in PTSD

[0736] Example 5.1. Animal acquisition and care

[0737] Animals were obtained from DBL. Animals were obtained 7 days before the start of the study. Twenty male SD rats, approximately 7 weeks old, were used. The animals were maintained under normal environmental conditions with a 12 / 12-hour light / dark cycle in a colony room and had free access to food and water. The rats were allowed to adapt to their new housing cages for 7 days. All studies were performed using these animals. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize animal suffering. Body weights were measured every 3 days from the 7th day onwards.

[0738] Example 5.2. Rat modeling of PTSD

[0739] The protocol involves two exposures to foot shock without an opportunity for escape.

[0740] Context A (Day -1)

[0741] Body weight was measured 3 hours before the experiment. The temperature of the experimental room was maintained at 22°C. The animals were placed in a dark experimental room for 10 minutes to allow adaptation. Each animal was placed in the first room. After 2 minutes, the door separating the two compartments was opened, allowing the animals to move freely between the two compartments. After 5 minutes, 15 foot shocks of 1 mA / 2 seconds were administered at random intervals (90 to 270 seconds). After the test, the animals were left in the box for 1 minute and then moved to the colony room.

[0742] Context B (Day 0)

[0743] Body weight was measured 3 hours before the experiment. The temperature of the experimental room was maintained at 22°C. The animals were placed in a dark experimental room for 10 minutes to allow adaptation. The animals were placed in the first compartment, which had sandpaper attached. At this time, the door was kept closed so that the animals could not cross into the second compartment. After 10 minutes, the animals were given three shocks of 2 mA / sec. After the test, the animals were left in the box for 1 minute and then moved to the colony room.

[0744] compound injection

[0745] The compound was diluted to 25 mg / kg using DMSO:PEG400:saline (5:55:40). From the 7th day to the 24th day, the compound was intraperitoneally administered every day at 10 am (the intraperitoneal administration volume was 200 μl). The control group was also intraperitoneally injected in the same manner using vehicle (DMSO:PEG400:saline (5:55:40)).

[0746] Checking the freezing

[0747] To confirm freezing, on the 7th day after the first footshock protocol, rats were placed in the first compartment of the apparatus with sandpaper for 5 min (without footshock). Freezing behavior was recorded using the shutavoid_v1803 program (Panlab, Spain) according to the manufacturer's guidelines. The door remained closed as in Context B. Freezing was defined as the absence of skeletal muscle movement in the rat. Animals with a freezing time of 240 s or less were excluded. Excluded animals were sacrificed using CO2.

[0748] Data analysis

[0749] A camera was placed in front of the transparent acrylic panel of the electronic box to monitor the movements of all animals. Freezing was monitored for all groups every three days starting on the seventh day. Each animal was filmed for five minutes. The freezing time for each animal was determined based on the footage. The freezing time on the seventh day was set to 100%, and the freezing time for each day of the week was then converted into a percentage.

[0750] Example 6. Therapeutic efficacy in AD

[0751] Example 6.1. Animal Acquisition and Care

[0752] APP / PS1 mice, an animal model of Alzheimer's disease, were obtained from Taconic Bioscience. Animals were obtained 7 days before the start of the study. Twenty male mice, approximately 15 months old, were used. The animals were maintained in a colony room under normal environmental conditions with a 12 / 12-hour light / dark cycle and had free access to food and water. The mice were allowed to adapt to their new housing cages for 7 days. All studies were performed using these animals. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize animal suffering. Body weights were measured every 3 days from the 7th day onwards.

[0753] Example 7. Selection of BnH12001

[0754] Example 7.1. In vitro cytotoxicity

[0755] The cytotoxicity of the compounds was evaluated by treating cells with BnH12001 (racemate) and measuring cell viability.

[0756] Cell viability was measured in response to BnH12001 (0, 0.1, 1, 10, and 100 μM) treatment in the human cell lines 293T and SH-SY5Y, and the results showed that there was no significant change in cell viability across the entire concentration range, confirming the cytotoxic safety of BnH12001 (Figure 1a and Figure 1b).

[0757] Example 7.2. Analysis of the effects of HDAC4 depletion on gene expression in vitro

[0758] The BnH12 series drugs of the present invention were synthesized as compounds targeting HDAC4, which inhibits BDNF expression. Therefore, we investigated whether BDNF expression increases due to HDAC4 degradation by BnH12001 treatment. Furthermore, since increased BDNF expression is known to increase GluN2B expression, we also measured GluN2B mRNA levels in addition to BDNF to assess the HDAC4 degradation efficacy of BnH12001.

[0759] BDNF and GluN2B mRNA levels were measured 6 hours after treatment of 293T cells with BnH12001 (racemate). Results showed that the expression of BDNF and GluN2B was significantly increased compared to the control group, indicating that BnH12001 effectively degrades HDAC4, thereby inducing increased expression of BDNF and GluN2B, demonstrating that BnH12001 exerts its predicted efficacy in vitro (Figure 2).

[0760] When BnH12001 (racemate) was applied to primary cultured cortical neurons using the same experimental procedure as described above, HDAC4 protein expression was significantly inhibited and BDNF and GluN2B expression was significantly increased (Figure 3).

[0761] Example 7.3. Analysis of the efficacy of enhancing synaptic plasticity in vitro

[0762] After administration of BnH12001 (racemate), the potency and GABA / AMPA ratio were measured in the barrel cortex to confirm the efficacy of the compound, and the effective concentration was determined through a dose-response curve of BnH12001.

[0763] To this end, 8-week-old C57BL / 6J mice were divided into a control group (vehicle-treated group) and an experimental group (BnH12001-treated group), each consisting of five mice. After Os pump injection surgery, BnH12001 was directly infused into layer 4 of the barrel cortex using an Os pump at a total dose of 3 μM for two consecutive weeks (Figures 4a and 4b).

[0764] Administration of BnH12001 compound significantly increased excitatory TC synaptic potency (TC potency) in the mouse barrel cortex (layer 4), as confirmed by measuring the GABA / AMPA ratio (Figure 4c), while maintaining the excitation / inhibition balance in the barrel cortex (Figure 4d). Since an increase in TC potency indicates increased synaptic plasticity, these results demonstrate that BnH12001 administration increases TC synaptic potency in the sensory cortex while maintaining the excitation / inhibition balance, indicating that BnH12001 is effective in improving cognitive performance.

[0765] Eight-week-old C57BL / 6J mice were orally administered a dose of 1 mg / kg. Five mice were administered in each control and compound-treated group. The results confirmed that BnH12001 administration increased TC synaptic potency in the sensory cortex while maintaining the excitatory / inhibitory balance (Figures 5a and 5b). We also observed changes in TC EPSC potency in the mouse barrel cortex (layer) in response to BnH12001 administration at various doses (0.03, 0.1, 0.3, 1, 3, and 10 mg / kg). Five mice were administered at each concentration in the control and compound-treated groups. The EC50 for BnH12001 was confirmed to be 0.3023 (Figure 5c).

[0766] Example 7.4. Analysis of the efficacy of enhancing synaptic plasticity in vitro in animal models of Alzheimer's disease

[0767] We investigated the efficacy of BnH12001 enantiomer (3S,6R, 98% or more) administered alone or in combination with aducanumab (Creative Biolabs) in APP / PS1 mice (Taconic Bioscience), an animal model of Alzheimer's disease (15-month-old). An isotype antibody was used as a control antibody. The administration schedule was as shown in Figure 6a (for intravenous administration, see Example 4.4). Five mice were used for each administration group.

[0768] Changes in the slope of field excitatory postsynaptic potential (fEPSP) in the CA1 region of the Schaffer collateral circuit in the hippocampus confirmed that BnH12001 enantiomer (3S,6R, 98% or more) alone, aducanumab alone, and the combination of BnH12001 enantiomer (3S,6R, 98% or more) and aducanumab all increased synaptic efficacy (Fig. 6b). Furthermore, measurements of fEPSP LTP in the CA1 region of the Schaffer collateral circuit in the hippocampus confirmed a significant increase in synaptic long-term potentiation (LTP) in the excitatory SC pathway (Fig. 6b). Because increases in fEPSP slope and LTP indicate increased synaptic plasticity, these results demonstrate that BnH12001 enantiomer (3S,6R, 98% or more) alone and in combination with aducanumab increases SC pathway synaptic potency in the hippocampus, indicating that BnH12001 enantiomer (3S,6R, 98% or more) alone and in combination with aducanumab are effective in treating Alzheimer's disease.

[0769] On the other hand, we attempted to confirm the efficacy of the compound against Alzheimer's disease by administering the BnH12001 enantiomer (3S,6R, 98%<) alone or in combination with aducanumab to APP / PS1 mice, and then measuring the potency and GABA / AMPA ratio in the sensory cortex. In particular, we attempted to compare the effects of the BnH12001 enantiomer (3S,6R, 98%<) and aducanumab administered alone and in combination.

[0770] When BnH12001 enantiomer (3S,6R, 98% or more) was administered alone or in combination with aducanumab to APP / PS1 mice, it increased excitatory TC synaptic potency (TC potency) (Figure 6c, left panel) while maintaining excitation / inhibition balance, as confirmed by measuring the GABA / AMPA ratio (Figure 6c, right panel). Because increased TC potency indicates increased synaptic plasticity, these results demonstrate that BnH12001 enantiomer (3S,6R, 98% or more) alone or in combination with aducanumab increases TC synaptic potency in the sensory cortex while maintaining excitation / inhibition balance, indicating that BnH12001 enantiomer (3S,6R, 98% or more) alone or in combination with aducanumab is effective in treating Alzheimer's disease. In this case, the combination of BnH12001 enantiomer (3S,6R, 98%<) and aducanumab showed significantly superior efficacy compared to the administration of either BnH12001 enantiomer (3S,6R, 98%<) or aducanumab alone.

[0771] Example 8. Surface plasmon resonance assay

[0772] The compounds of the present invention were selected for their ability to interact with HDAC4. To carry out the test, a surface plasmon resonance assay was used. Samples were prepared and loaded onto a Biacore T200 as follows:

[0773] JPEG2025511114000053.jpg82146

[0774] Samples were immobilized under the following conditions: Protein stock was diluted with 10 mM sodium acetate. 1 M ethanolamine (pH 8.5) was used as a blocking agent.

[0775] JPEG2025511114000054.jpg32146

[0776] FIG. 7 shows the binding kinetics of BnH12001 to HDAC4.

[0777] Example 9. Effect of compounds on HDAC catalytic activity

[0778] Compounds (racemate) were resuspended in 100 mM DMSO and tested at 10-volume IC concentrations against HDACs 1-9 and 11 in 3-fold serial dilutions starting at 100 μM. 50 As reference compounds, Trichostatin A (TSA) and TMP269 were tested at 10-volume IC50 in 3-fold serial dilutions starting from 10 μM or 1 μM. 50 Tested with.

[0779] A 10 μM fluorescent peptide of p53 residues 379-382 (RHK(Ac)AMC) was used as a substrate for HDACs 1, 2, 3, and 6. A 50 μM fluorescent HDAC class 2a substrate (trifluoroacetyl lysine) was used as a substrate for HDACs 4, 5, 7, 9, and 11. A 100 μM fluorescent peptide of p53 residues 379-382 (RHK(Ac)K(Ac)AMC) was used as a substrate for HDAC 8.

[0780] IC using GraphPad Prism 4 program based on sigmoidal dose-response equation 50 Values ​​were calculated and the concentration of the blank (DMSO) value was entered as 1.00E-12 M for curve fitting. Curve fitting was performed for compound datasets that showed less than 65% enzyme activity at the highest concentration of compound tested.

[0781] Experiments were performed in duplicate. BnH12001 did not show any inhibitory effect on the catalytic activity of HDACs 1-9 and 11.

[0782] Example 10. Expression of BDNF and GluN2B and effects on thalamocortical EPSC potency

[0783] Inhibition of HDAC4 protein expression by transfection with lentiviral HDAC4 shRNA significantly increased BDNF and GluN2B expression in SH-SY5Y cells (Figures 8a and 8b). Similarly, BnH12001 (racemate) (1 μM) increased BDNF and GluN2B expression in SH-SY5Y cells after 6 and 24 hours of administration (Figures 9a and 9b). BnH12001 significantly inhibited HDAC4 protein expression in the mouse barrel cortex after oral and intraperitoneal administration, respectively, and this inhibitory effect was maintained for at least 9 days (Figures 10a and 10b).

[0784] We investigated the effect of BnH12001 on thalamocortical EPSC potency and the involvement of BDNF-TrkB signaling in this regard. As shown in Figure 11a, A1-B, application of BDNF to the mouse barrel cortex using an Alzet osmotic pump significantly increased TC EPSC potency without altering the IPSC / EPSC ratio. This BDNF-induced increase in TC EPSC potency was almost completely prevented by application of cyclotraxin B (1 μM) (Figure 11a, A1-B). Similarly, orally applied BnH12001 (3 μM) significantly increased TC EPSC potency in layer 4 barrel cortex (Figure 11b, A1-B), and this increase was almost completely inhibited by application of cyclotraxin B (1 μM) (Figure 11b, C1-D).

[0785] Example 11. Confirmation of efficacy of BnH12001 enantiomers

[0786] Example 11.1. Analysis of the efficacy of enhancing synaptic plasticity in vitro

[0787] After oral administration of BnH12001 enantiomer (3R,6S, 98%<) and BnH12001 enantiomer (3S,6R, 98%<), potency and GABA / AMPA ratio in the barrel cortex were measured to confirm the efficacy of these compounds.

[0788] Eight-week-old C57BL / 6J mice were orally administered 1 mg / kg of BnH12001 (racemate), BnH12001 enantiomer (3R,6S, 98% or more), and BnH12001 enantiomer (3S,6R, 98% or more). Five mice were administered each in the control (vehicle) and compound-treated groups. Results confirmed that both the racemate and the enantiomers increased TC synaptic potency in the sensory cortex while maintaining the excitatory / inhibitory balance. Both enantiomers exhibited superior efficacy compared to the racemate, with the BnH12001 enantiomer (3S,6R, 98% or more) exhibiting the greatest efficacy (Figures 12a and 12b).

[0789] Example 11.2. BDNF and GluN2B Expression and Effects on Thalamocortical EPSC Potency

[0790] BnH12001 enantiomer (3S,6R, 98%<) (1 μM) increased BDNF and GluN2B expression in primary cultured cortical neurons (DIV 14) after 24 h treatment (FIG. 13a).

[0791] In addition, 8-week-old C57BL / 6J mice were orally administered the BnH12001 enantiomer (3S,6R, 98% or more) at a dose of 1 mg / kg and sacrificed 2 days later to observe changes in HDAC4, BDNF, and GluN2B expression. The results showed that BnH12001 enantiomer (3S,6R, 98% or more) effectively degraded HDAC4 and significantly increased BDNF and GluN2B expression compared with the untreated group (vehicle). Therefore, the BnH12001 enantiomer (3S,6R, 98% or more) demonstrated excellent in vivo efficacy (Figure 13b).

[0792] On the other hand, 13-month-old C57BL / 6J mice were orally administered the BnH12001 enantiomer (3S,6R, 98% or more) at a dose of 1 mg / kg and sacrificed 2 days later to observe changes in GluN2B expression. The results confirmed that administration of the BnH12001 enantiomer (3S,6R, 98% or more) significantly increased GluN2B expression compared to the untreated group (vehicle) (Figure 13c).

[0793] Example 12. In vitro cytotoxicity of various BnH12 series compounds

[0794] The cytotoxicity of the compounds was evaluated by treating cells with various BnH12 series compounds and measuring cell viability using the experimental procedure described in Example 2. As a result, for almost all compounds, cell viability of 85% or more was observed at a concentration of 10 μM, and even when the concentration was increased to 100 μM, cell viability of 80% or more was observed.

[0795] [Table 5]

[0796] JPEG2025511114000056.jpg214140

[0797] JPEG2025511114000057.jpg135144

[0798]

[0799] The cell viability was measured using 293T cell line at various compound concentrations (10 or 100 μM). The results showed that most compounds did not significantly affect cell viability within the two concentration ranges.

[0800] Example 13. Confirmation of the efficacy of various BnH12 series compounds

[0801] Using the experimental procedures described in Example 3, we investigated whether various BnH12 series compounds increase GluN2B expression, and the results are shown in Table 6. These in vitro experimental results are shown relative to the effect of the group without compound treatment (vehicle), which was set at 1.

[0802] [Table 6]

[0803] JPEG2025511114000059.jpg69146

[0804]

[0805] As can be seen from Table 6, the representative compounds of the present invention showed superior efficacy compared to the vehicle, especially BnH12001 enantiomer (3S,6R, 98% or more), BnH12018, BnH12025, BnH12038, BnH12060, BnH12061, BnH12063, BnH12069, BnH12073, BnH12074, and BnH12096.

Claims

1. A compound represented by the following chemical formula (1), a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: R １ is C １－６ Alkyl, C ３－１２ cycloalkyl, aryl, or heteroaryl, and one or more hydrogen atoms in the group are unsubstituted or each independently substituted with a halogen atom, C １－６ Alkyl, C １－６ Alkoxy, nitro, one or more C １－６ an alkyl-substituted or unsubstituted amino or a hydroxy-substituted A is C=O or a bond; Q is -(CR ４ R ５ ) ｍ - or a bond, where R ４ and R ５ are each independently hydrogen or C １－６ alkyl, m is an integer from 1 to 6, R ２ is hydrogen, C １－６ alkyl or aryl; R ３ is hydrogen, carboxyl, N-hydroxycarboxamide, or carboxamide, and one or more hydrogens in the group are unsubstituted or C １－６ is substituted with alkyl, n is 0 or 1.

2. R １ is C １－６ Alkyl, C ３－１２ cycloalkyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl, and one or more hydrogen atoms of the groups are unsubstituted or each independently a halogen atom, C １－６ Alkyl, C １－６ Alkoxy, nitro, one or more C １－６ alkyl-substituted or unsubstituted amino, or hydroxy-substituted; 2. The compound of claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

3. R １ is methyl, cyclohexyl, phenyl, 4-methoxyphenyl, 4-(tert-butyl)phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 3,4-dichlorophenyl, 2-chloro-4-fluorophenyl, p-tolyl, m-tolyl, pyridin-4-yl, thiophen-2-yl, furan-2-yl, pyridin-3-yl, pyridin-2-yl, pyrimidin-5-yl, pyrimidin-2-yl, 4-nitrophenyl, 4-aminophenyl, 3-nitrophenyl, 3-aminophenyl, 4-methoxy-pyridin-3-yl, 6-methoxy-pyridin-3-yl, 4-(dimethylamino)phenyl, or 4-hydroxy-phenyl; 2. The compound of claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

4. R ２ is H or C １－６ is alkyl, 2. The compound of claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

5. R ３ is H, carboxyl, methylcarboxyl, ethylcarboxyl, N-hydroxycarboxamide, carboxamide, or sodium carboxylate; 2. The compound of claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

6. A compound represented by the following chemical formula (2-A) or (2-B), a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: In the above formula, R ４ is C １－６ Alkyl, C ３－１２ cycloalkyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrole, furanyl, or thiophenyl, and one or more hydrogen atoms of the groups are unsubstituted or each independently a halogen atom, C １－６ Alkyl, C １－６ Alkoxy, nitro, one or more C １－６ substituted with alkyl or unsubstituted amino or hydroxy; R ５ and R ６ are each independently hydrogen or C １－６ It is an alkyl.

7. A compound represented by the following chemical formula (4), a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: In the above formula, R ２ is hydrogen, C １－６ alkyl or aryl; R ３ is hydrogen, carboxyl, N-hydroxycarboxamide, or carboxamide, and one or more hydrogens in the group are unsubstituted or C １－６ is substituted with alkyl, n is 0 or 1.

8. A compound represented by the following chemical formula (5), a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: In the above formula, R ２ is hydrogen, C １－６ alkyl or aryl; R ４ is phenyl or phenyl substituted with one or more halogens; n is 0 or 1.

9. The compound according to claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of: 。

10. The compound is BnH12001 or an enantiomer thereof.

10. The compound according to claim 9.

11. The stereoisomer is an optical isomer.

2. The compound of claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

12. The compound is an inhibitor of HDAC.

2. The compound of claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

13. The HDAC is HDAC4.

13. The compound of claim 12, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

14. The compound increases synaptic plasticity.

2. The compound of claim 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof as an active ingredient. Pharmaceutical compositions.

16. The composition is for the prevention or treatment of a neurological or neurodegenerative disorder. The pharmaceutical composition of claim 15.

17. The compound, its stereoisomer, or its pharma- ceutically acceptable salt is delivered to the brain at a concentration of about 0.1 to about 3 μM.

17. The pharmaceutical composition of claim 16.

18. The disorder is Alzheimer's disease.

17. The pharmaceutical composition of claim 16.

19. The disorder is PTSD.

17. The pharmaceutical composition of claim 16.

20. further comprising a second therapeutic agent, The pharmaceutical composition of claim 15.

21. The second therapeutic agent is aducanumab.

21. The pharmaceutical composition of claim 20.

22. A method for the prevention or treatment of a neurological or neurodegenerative disorder, comprising administering to an individual in need thereof a compound according to any one of claims 1 to 14, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

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