Crystalline forms and salts of muscarinic receptor agonists

Abstract

The present invention relates to compounds and their salts that are muscarinic receptor agonists and are useful for treating muscarinic receptor-mediated diseases.Also provided are crystalline forms of the compounds and their salts; pharmaceutical compositions containing the compounds and their salts or their crystalline forms; therapeutic uses of the compounds and their salts or their crystalline forms; methods for synthesizing them; and intermediates useful in the methods of synthesis.The present invention provides compounds that have selective activity as muscarinic M1 and / or M4 receptor agonists.More specifically, the present invention provides compounds that exhibit selectivity for M4 receptor over M2 and M3 receptor subtypes.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of GB2211399.7, filed August 04, 2022 (04 / 08 / 2022), GB2305444.8, filed April 13, 2023 (13 / 04 / 2023), and GB2309615.9, filed June 26, 2023 (26 / 06 / 2023), the contents of which are incorporated herein by reference in their entireties. Technical Field

[0002] The present invention relates to compounds and salts thereof that are muscarinic receptor agonists and are useful for treating muscarinic receptor-mediated diseases. Also provided are crystalline forms of the compounds and salts thereof; pharmaceutical compositions containing the compounds and salts thereof or their crystalline forms; therapeutic uses of the compounds and salts thereof or their crystalline forms; methods for synthesizing them; and intermediates useful in the methods for said synthesis. [Background technology]

[0003] Background of the Invention Muscarinic acetylcholine receptors (mAChRs) are members of the G protein-coupled receptor superfamily that mediate the actions of the neurotransmitter acetylcholine in both the central and peripheral nervous systems. Five mAChR subtypes, M1–M5, have been cloned. M1 mAChRs are primarily expressed postsynaptically in the cortex, hippocampus, striatum, and thalamus. M2 mAChRs are primarily located in the brainstem and thalamus, but also in the cortex, hippocampus, and striatum, where they reside at cholinergic synaptic terminals (Langmead et al., 2008 Br J Pharmacol). However, M2 mAChRs are also expressed peripherally on the surface of cardiac tissue (where they mediate vagal innervation of the heart), as well as in smooth muscle and exocrine glands. M3 mAChRs are expressed at relatively low levels in the CNS, but are widely expressed in smooth muscle and glandular tissues such as sweat and salivary glands (Langmead et al., 2008 Br J Pharmacol).

[0004] Muscarinic receptors in the central nervous system, particularly M1 mAChRs, play an important role in mediating higher-level cognitive processing. Diseases associated with cognitive impairment, such as Alzheimer's disease, are accompanied by loss of cholinergic neurons in the basal forebrain (Whitehouse et al., 1982 Science). In schizophrenia, which is also characterized by cognitive impairment, mAChR density is reduced in the frontal cortex, hippocampus, and caudate putamen of schizophrenic subjects (Dean et al., 2002 Mol Psychiatry). Furthermore, animal models have shown that blockade or lesion of central cholinergic pathways results in profound agnosia, and nonselective mAChR antagonists induce psychotomimetic effects in patients with neuropathology. Cholinergic replacement therapy has largely been based on the use of acetylcholinesterase inhibitors, which prevent the breakdown of endogenous acetylcholine. In the clinic, these compounds demonstrate efficacy against symptomatic cognitive decline but cause dose-limiting side effects due to stimulation of peripheral M2 and M3 mAChRs, including impaired gastrointestinal motility, bradycardia, nausea, and vomiting (http: / / www.drugs.com / pro / donepezil.html; http: / / www.drugs.com / pro / rivastigmine.html).

[0005] Further discovery efforts have focused on identifying direct M1 mAChR agonists that target cognitive enhancement. These efforts have led to the identification of a range of agonists, exemplified by compounds such as xanomeline, AF267B, sabcomeline, miramelin, and cevimeline. Many of these compounds have been shown to be highly effective in preclinical models of cognition in both rodents and / or nonhuman primates. Miramelin has demonstrated efficacy in rodents against scopolamine-induced deficits in working and spatial memory. Sabcomeline has demonstrated efficacy in marmosets in visual object recognition tasks, and xanomeline reversed mAChR antagonist-induced deficits in cognitive performance in a passive avoidance paradigm.

[0006] Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting older adults (26.6 million cases worldwide in 2006), resulting in severe memory loss and cognitive impairment. While the etiology of the disease is complex, it is characterized by two prominent brain sequelae: the aggregation of amyloid plaques, primarily composed of amyloid-β peptide (Aβ), and neurofibrillary tangles formed by hyperphosphorylated tau protein. Aβ accumulation is considered a central feature in AD progression, and therefore, many putative therapies for AD treatment currently target the inhibition of Aβ production. Aβ results from the proteolytic cleavage of membrane-bound amyloid precursor protein (APP). APP is processed via two pathways: the nonamyloidogenic pathway and the amyloidogenic pathway. While cleavage of APP by γ-secretase is common to both pathways, in the former, APP undergoes α-secretase cleavage to yield soluble APPα. A cleavage site exists within the Aβ sequence, thereby preventing its formation. However, in the amyloidogenic pathway, APP undergoes β-secretase cleavage, generating soluble APPβ and Aβ. In vitro studies have shown that mAChR agonists can promote the processing of APP toward the soluble, nonamyloidogenic pathway. In vivo studies have shown that the mAChR agonist AF267B modified disease-like pathology in 3xTgAD transgenic mice, a model of various components of Alzheimer's disease (Caccamo et al., 2006 Neuron). Finally, the mAChR agonist cevimeline was shown to result in a small but significant reduction in cerebrospinal fluid levels of Aβ in Alzheimer's patients, thus demonstrating its potential disease-modifying efficacy (Nitsch et al., 2000 Neurol).

[0007] Furthermore, preclinical studies suggest that mAChR agonists exhibit an atypical antipsychotic-like profile across a range of preclinical paradigms. The mAChR agonist xanomeline reverses several dopamine-driven behaviors, including amphetamine-induced locomotion in rats, apomorphine-induced climbing in mice, dopamine agonist-induced rotational movement in unilaterally 6-OH-DA-lesioned rats, and amphetamine-induced hyperlocomotion in monkeys (without EPS challenge). Xanomeline also inhibits A10 but not A9 dopamine cell firing and conditioned avoidance in rats, and induces c-fos expression in the prefrontal cortex and nucleus accumbens but not in the striatum. These data all suggest an atypical antipsychotic-like profile (Mirza et al., 1999 CNS Drug Rev). Muscarinic receptors are also involved in the neurobiology of addiction. The reinforcing effects of cocaine and other addictive substances are mediated by the mesolimbic dopamine system, where behavioral and neurochemical studies have shown that cholinergic muscarinic receptor subtypes play a key role in regulating dopaminergic neurotransmission. For example, M(4)(- / -) mice demonstrated significantly enhanced reward-driven behavior following exposure to cocaine (Schmidt et al. Psychopharmacology (2011) Aug; 216(3):367-78). Furthermore, xanomeline has been shown to block the effects of cocaine in these models.

[0008] Muscarinic receptors are also involved in the control of movement and may represent novel treatments for movement disorders such as Parkinson's disease, ADHD, Huntington's disease, Tourette's syndrome, and other syndromes associated with dopaminergic dysfunction as the underlying pathogenic factor-driven disorder.

[0009] Xanomeline, sabcomeline, miramelin, and cevimeline are all in various stages of clinical development for the treatment of Alzheimer's disease and / or schizophrenia. Phase II clinical trials using xanomeline demonstrated efficacy across various cognitive symptom domains, including behavioral disturbances and hallucinations associated with Alzheimer's disease (Bodick et al., 1997 Arch Neurol). This compound has also been evaluated in a small Phase II study for schizophrenia, resulting in significant reductions in positive and negative symptoms compared with placebo controls (Shekhar et al., 2008 Am J Psych). However, in all clinical trials, xanomeline and other related mAChR agonists demonstrated unacceptable safety margins for cholinergic side effects, including nausea, gastrointestinal pain, diarrhea, diaphoresis (excessive sweating), hypersalivation (excessive salivation), syncope, and bradycardia.

[0010] Muscarinic receptors are involved in central and peripheral pain. Pain can be classified into three different types: acute, inflammatory, and neuropathic. Acute pain serves an important defensive function, keeping the organism safe from stimuli that could cause tissue damage, but postoperative pain management is necessary. Inflammatory pain can arise for a variety of reasons, including tissue injury, autoimmune responses, and pathogen invasion, and is triggered by the action of inflammatory mediators such as neuropeptides and prostaglandins, which result in neuronal inflammation and pain. Neuropathic pain is associated with abnormal somatic sensations of pain in response to non-painful stimuli. Neuropathic pain is associated with several different diseases / injuries, such as spinal cord injury, multiple sclerosis, diabetes (diabetic neuropathy), and viral infections (such as HIV or herpes). Neuropathic pain is also common in cancer, both as a result of disease or chemotherapy side effects.

[0011] Activation of muscarinic receptors has been shown to provide analgesia in several pain conditions, due to receptor activation in the spinal cord and higher pain centers in the brain. Increasing endogenous levels of acetylcholine with acetylcholinesterase inhibitors and direct activation of muscarinic receptors with agonists or allosteric modulators have been shown to have analgesic activity. In contrast, blocking muscarinic receptors with antagonists or using knockout mice increases pain sensitivity. Evidence for the role of M1 receptors in pain is reviewed by D.F. Fiorino and M. Garcia-Guzman, 2012.

[0012] More recently, a few compounds have been identified that show improved selectivity for the M1 mAChR subtype over peripherally expressed mAChR subtypes (Bridges et al., 2008 Bioorg Med Chem Lett; Johnson et al., 2010 Bioorg Med Chem Lett; Budzik et al., 2010 ACS Med Chem Lett). Despite the increased level of selectivity for the M3 mAChR subtype, some of these compounds maintain significant agonist activity at both this subtype and the M2 mAChR subtype. Herein, we describe compounds that show a high level of selectivity for the M1 and / or M4 mAChR over the M2 and M3 receptor subtypes.

[0013] WO2015 / 118342 discloses muscarinic agonist compounds. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2015 / 118342 [Non-patent literature]

[0015] [Non-Patent Document 1] Langmead et al., 2008 Br J Pharmacol [Non-Patent Document 2] Langmead et al., 2008 Br J Pharmacol [Non-Patent Document 3] Whitehouse et al., 1982 Science [Non-Patent Document 4] Dean et al., 2002 Mol Psychiatry [Non-Patent Document 5] Caccamo et al., 2006 Neuron [Non-Patent Document 6] Nitsch et al., 2000 Neurol [Non-Patent Document 7] Mirza et al., 1999 CNS Drug Rev [Non-Patent Document 8] Schmidt et al Psychopharmacology (2011) Aug; 216(3):367-78 [Non-Patent Document 9] Bodick et al., 1997 Arch Neurol [Non-Patent Document 10] Shekhar et al., 2008 Am J Psych [Non-Patent Document 11] Bridges et al., 2008 Bioorg Med Chem Lett [Non-Patent Document 12] Johnson et al., 2010 Bioorg Med Chem Lett [Non-Patent Document 13] Budzik et al., 2010 ACS Med Chem Lett [Summary of the Invention] [Problems to be Solved by the Invention]

[0016] The present invention The present invention provides compounds that have selective activity as muscarinic M1 and / or M4 receptor agonists. More particularly, the present invention provides compounds that exhibit selectivity for the M4 receptor over the M2 and M3 receptor subtypes. [Means for solving the problem]

[0017] Thus, a compound of formula (1): [ka] or a salt thereof is provided.

[0018] Compound of formula (2a): [ka] (wherein X represents a salt) is further provided.

[0019] Compound of formula (2b): [ka] (wherein X represents a salt) is further provided.

[0020] Compound of formula (3a): [ka] is further provided.

[0021] Compound of formula (3b): [ka]

[0022] is further provided. Compound of formula (4a): [ka] is further provided.

[0023] Compound of formula (4b): [ka] is further provided.

[0024] Compound of formula (5a): [ka] is further provided.

[0025] Compound of formula (5b): [ka] is further provided.

[0026] Compound of formula (6a): [ka] is further provided.

[0027] Compound of formula (6b): [ka] is further provided.

[0028] Compound of formula (7): [ka] or a salt thereof is further provided.

[0029] Compound of formula (8): [ka] or a salt thereof is further provided.

[0030] Salts of compounds of formula (1) are also provided.

[0031] The compounds of formula (1) can form pharmaceutically acceptable salts.

[0032] The compounds of formula (1) can form acid addition salts.

[0033] The compound of formula (1) can form a citrate salt.

[0034] The compound of formula (1) can form a citrate monohydrate salt.

[0035] The compound of formula (1) can form a fumarate salt.

[0036] The compound of formula (1) can form an HCl salt.

[0037] The compound of formula (1) can form a phosphate salt.

[0038] The compound of formula (1) can form a sulfate salt.

[0039] In the compounds of formula (2a) and (2b), X can be a pharmaceutically acceptable acid.

[0040] In the compounds of formula (2a) and (2b), X can be an acid.

[0041] In the compounds of formula (2a) and (2b), X can be citric acid.

[0042] In the compounds of formula (2a) and (2b), X can be citric acid monohydrate.

[0043] In the compounds of formula (2a) and (2b), X can be fumaric acid.

[0044] In the compounds of formula (2a) and (2b), X can be HCl.

[0045] In the compounds of formula (2a) and (2b), X can be a phosphate.

[0046] In the compounds of formula (2a) and (2b), X can be sulfate.

[0047] In acid addition salts, the acid is 2,5-Dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, butanedioic acid (succinic acid), citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), hydroxyacetic acid (glycolic acid), hydrochloric acid (HCl), maleic acid, malic acid (L), malonic acid, N-acetylglycine (aceturic acid), nicotinic acid, orthophosphoric acid (phosphoric acid), oxoglutaric acid (ketoglutaric acid), p-toluenesulfonic acid (p-TSA), pyroglutamic acid (L), sulfuric acid, and tartaric acid (L). You can choose from:

[0048] The compound can be ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0049] The compound can be a salt of ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0050] The compound can be a pharmaceutically acceptable salt of ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0051] The compound can be an acid addition salt of ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0052] The compound can be ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate.

[0053] The compound can be ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate.

[0054] The compound can be (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate.

[0055] The compound can be ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride.

[0056] The compound can be a salt of ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0057] The compound can be a pharmaceutically acceptable salt of ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0058] The compound can be an acid addition salt of ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0059] The compound can be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate.

[0060] The compound can be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate.

[0061] The compound can be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate.

[0062] The compound can be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride.

[0063] Also provided are synthetic methods, including those that preferentially favor the formation of compounds and intermediates with a desired stereochemistry. For example, the methods can employ stereoselective reduction of a ketone to provide an alcohol or stereoselective reductive amination of the ketone.

[0064] Synthetic methods are provided that follow the general scheme below: [ka] In the formula, Q is [ka] is selected from R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group; X represents a salt.

[0065] Synthetic methods are provided that follow the general scheme below: [ka] In the formula, Z is [ka] is selected from R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group; X represents a salt.

[0066] The compounds of the present invention can be prepared by the following general scheme: [ka] can be prepared in accordance with In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represent any suitable leaving group.

[0067] Similarly, the following general scheme: [ka] A synthetic method according to In the formula, Q is [ka] is selected from R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group; X represents a salt.

[0068] The compounds of the present invention can be prepared by the following general scheme: [ka] can be prepared in accordance with In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group; X represents a salt.

[0069] Similarly, the following general scheme: [ka] A synthetic method according to In the formula, Q is [ka] is selected from R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, the catalyst is a hydrogenation catalyst; X represents a salt.

[0070] The compounds of the present invention can be prepared by the following general scheme: [ka] can be prepared in accordance with In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6is a cycloalkyl group, the catalyst is a hydrogenation catalyst; X represents a salt.

[0071] In the methods described herein, Q is [ka] Q can be expressed as [ka] Q can be expressed as [ka] A particular method is to select a compound in which Q is: [ka] The present invention includes compounds in which:

[0072] In the methods described herein, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 R is a cycloalkyl group. 1 R may be ethyl or t-butyl. 1 R may be t-butyl. 1 may be ethyl.

[0073] In the methods described herein, R 2 R, together with the oxygen atom to which it is attached, represents any suitable leaving group. 2 may be tosyl.

[0074] In the processes described herein, the catalyst refers to a hydrogenation catalyst. The catalyst may be any hydrogenation catalyst capable of catalyzing the transformation shown in the relevant synthesis scheme. The catalyst may be Pd / BaSO4. The catalyst may be Pd / BaSO4 (10 mol%).

[0075] In the methods described herein, X represents a salt. X can be a pharmaceutically acceptable salt. X can be an acid addition salt. X can be a citrate salt. X can be a citrate monohydrate salt.

[0076] The compound can be represented by the following scheme: [ka] It can be prepared in accordance with

[0077] The compound can be represented by the following scheme: [ka] [ka] It can be prepared in accordance with

[0078] The compound can be represented by the following scheme: [ka] It can be prepared in accordance with

[0079] The compound can be represented by the following scheme: [ka] It can be prepared in accordance with

[0080] Similarly, the following scheme: Main chain: [ka] Side chain: [ka] A synthetic method according to the present invention is provided.

[0081] Similarly, the following scheme: [ka] A synthetic method according to the present invention is provided. [Brief explanation of the drawings]

[0082] [Figure 1] For example, the XRPD pattern of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate, as exemplified in Synthesis Example 6.

[0083] [Figure 2] TG / DTA thermogram of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate, as exemplified, for example, in Synthesis Example 6, analyzed from 25° C. to 300° C. at 10° C. per minute.

[0084] [Figure 3] For example, a photomicrograph of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate, as exemplified in Synthesis Example 6.

[0085] [Figure 4] For example, the 1H NMR spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate, as illustrated in Synthesis Example 6.

[0086] [Figure 5] For example, the DVS isotherm of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate, as exemplified in Synthesis Example 6.

[0087] [Figure 6] XRPD traces showing (top) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl salt, (middle) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate, and (bottom) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl salt citrate, as exemplified, for example, in Synthesis Example 6.

[0088] [Figure 7] For example, the XRPD pattern of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride, as exemplified in Synthesis Example 7.

[0089] [Figure 8] TG / DTA thermogram of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride, as exemplified, for example, in Synthesis Example 7, analyzed from 25° C. to 300° C. at 10° C. per minute.

[0090] [Figure 9] DSC thermogram of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride, for example, as exemplified in Synthesis Example 7, analyzed from 25° C. to 300° C. at 10° C. per minute.

[0091] [Figure 10]For example, a photomicrograph of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride, as exemplified in Synthesis Example 7.

[0092] [Figure 11] For example, the 1H NMR spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride, as exemplified in Synthesis Example 7.

[0093] [Figure 12] For example, the DVS isotherm of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride, as exemplified in Synthesis Example 7.

[0094] [Figure 13] XRPD traces showing (top) cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride after DVS, e.g., as exemplified in Synthesis Example 7; (middle) cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride before DVS, e.g., as exemplified in Synthesis Example 7; and (bottom) known alternative HCl salt forms.

[0095] [Figure 14] For example, the XRPD pattern of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate, as exemplified in Synthesis Example 8.

[0096] [Figure 15]TG / DTA thermogram of cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate, as exemplified, for example, in Synthesis Example 8, analyzed from 25°C to 300°C at 10°C per minute.

[0097] [Figure 16] For example, a photomicrograph of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate, as exemplified in Synthesis Example 8.

[0098] [Figure 17] For example, the 1H NMR spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate, as illustrated in Synthesis Example 8.

[0099] [Figure 18] For example, the DVS isotherm of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate, as exemplified in Synthesis Example 8.

[0100] [Figure 19] XRPD traces showing, after DVS, (top) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (free base), (middle) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate, as exemplified, for example, in Synthesis Example 8, and (bottom) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate, as exemplified, for example, in Synthesis Example 8.

[0101] [Figure 20] XRPD traces of (top) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate, e.g., as exemplified in Synthesis Example 8, before humidity stressing, and (bottom) the sample after humidity stressing.

[0102] [Figure 21] XRPD traces of (top) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate, as exemplified, for example, in Synthesis Example 6, before humidity stressing, and (bottom) the sample after humidity stressing.

[0103] [Figure 22] XRPD traces showing (top) cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride before humidity stress, e.g., as exemplified in Synthesis Example 7, (middle) the sample after humidity stress, and (bottom) another known HCl salt form.

[0104] [Figure 23] XRPD traces showing, for example, (top) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate, as exemplified in Synthesis Example 8; (middle) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate (water-soluble sample), as exemplified in Synthesis Example 8; and (bottom) fumaric acid.

[0105] [Figure 24]For example, the H NMR spectrum of ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate, as illustrated in Synthesis Example 5.

[0106] [Figure 25] For example, the XRPD pattern of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate, which is exemplified in Synthesis Examples 1 to 3.

[0107] [Figure 26] TGA / DSC thermogram of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate, as exemplified, for example, in Synthesis Examples 1-3, analyzed from 25°C to 300°C at 10°C per minute.

[0108] [Figure 27] For example, DVS isotherm plot of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate, as exemplified in Synthesis Examples 1-3.

[0109] [Figure 28] For example, a photomicrograph of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate, as exemplified in Synthesis Examples 1-3.

[0110] [Figure 29]SEM micrographs of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate, as exemplified in Synthesis Examples 1 to 3. Top: particle aggregates; bottom: single crystals. Scale bar indicates 50 μm.

[0111] [Figure 30] For example, the 1H NMR spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate, as illustrated in Synthesis Examples 1 to 3.

[0112] [Figure 31] For example, an expanded region of the H NMR spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate, as exemplified in Synthesis Examples 1-3.

[0113] [Figure 32] For example, the XRPD pattern of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate free base, as exemplified in Synthesis Example 4.

[0114] [Figure 33] For example, TGA / DSC thermogram of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate free base, as exemplified in Synthesis Example 4.

[0115] [Figure 34]1H NMR spectrum of tert-butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate.

[0116] [Figure 35] 1H NMR spectrum of tert-butyl cis-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate.

[0117] [Figure 36] 1H-1H NOESY assignment of tert-butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate. Top: NOE cross-peak assignments; Bottom: Expanded region of the 2D-NOESY spectrum.

[0118] [Figure 37] 1H-1H NOESY assignment of tert-butyl cis-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate. Top: NOE cross-peak assignments; Bottom: Expanded region of the 2D-NOESY spectrum. DETAILED DESCRIPTION OF THE INVENTION

[0119] Detailed Description The present invention provides compounds and salts thereof that have selective activity as muscarinic M1 and / or M4 receptor agonists. More particularly, the present invention provides compounds that exhibit selectivity for the M4 receptor over the M2 and M3 receptor subtypes.

[0120] The following compounds are muscarinic M1 and / or M4 receptor agonists: ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (structure shown below); ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (structure shown below); and ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (structure shown below).

[0121] Each of these compounds and their salts is useful for treating muscarinic receptor-mediated diseases and conditions such as, for example, schizophrenia, Alzheimer's disease, Alzheimer's disease psychosis, and bipolar disorder (see, e.g., WO2015 / 118342A1, the disclosure of which is incorporated herein by reference in its entirety). [ka] Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate [ka] Ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate [ka] Ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate Methods for preparing compounds

[0122] The present application provides methods for preparing the compounds and salts thereof described herein.

[0123] Accordingly, there is provided a method for preparing a compound of formula (1), (7) or (8) or a salt thereof, or a crystalline form thereof, comprising reacting a compound of formula (I) below: [ka] with a compound of formula (II) or a salt thereof: [ka] to produce a compound of formula (III) or a salt thereof: [ka]

[0023] A method is provided herein, comprising obtaining (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group; (a)Q 1 is -H, and Q 2 teeth, [ka] Selected from or (b)Q 1 HA-OR 3 and Q 2 HA-OR 3 and R 3 is a linear or branched C 1~6 is an alkyl group, or (c)Q 1 and Q 2 together form the group -O-(CH2) m -O-, and m is an integer selected from 2, 3 and 4.

[0124] In some embodiments, R 1 is a linear or branched C 1~6 In some embodiments, R 1 is ethyl or t-butyl. In some embodiments, R 1 is t-butyl. In some embodiments, R 1 is ethyl.

[0125] In some embodiments, R 2 together with the oxygen atom to which it is attached represents a sulfonate ester leaving group. In some embodiments, R 2 together with the oxygen atom to which it is attached represent an arylsulfonate group. In some embodiments, R 2 together with the oxygen atom to which it is attached represents a tosylate group. Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (compounds of formulas (1), (2b), (3b), (4b), (5b), and (6b))

[0126] Similarly, there is provided a method for preparing a compound of formula (1) or a salt thereof, or a crystalline form thereof, comprising reacting a compound of formula (I) below: [ka] with a compound of the following formula (IIa) or a salt thereof: [ka] to produce a compound of formula (IIIa) or a salt thereof: [ka]

[0023] Provided herein is a method comprising the steps of: (In the formula, R 1 is a linear or branched C 1~6Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group).

[0127] Similarly, there is provided a method for preparing a compound of formula (1) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] under reductive amination conditions to give a compound of formula (IIa) or a salt thereof: [ka] to produce a compound of formula (IIIa) or a salt thereof: [ka]

[0023] Provided herein is a method comprising the steps of: (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group).

[0128] Similarly, there is provided a method for preparing a compound of formula (1) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with a deprotecting acid to give a compound of formula (V) or a salt thereof: [ka]

[0023] A method is provided herein, comprising obtaining (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group).

[0129] Similarly, there is provided a method for preparing a compound of formula (1) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with ethyl chloroformate to give a compound of formula (1) or a salt thereof: [ka] A method is provided herein that includes obtaining:

[0130] In some embodiments, the compound of formula (1) prepared by the methods described hereinabove is a compound of formula (2b) or a crystalline form thereof: [ka] and In the formula, X represents a salt.

[0131] In some embodiments, the compound of formula (1) prepared by the methods described hereinabove is a compound of formula (3b) or a crystalline form thereof: [ka] is.

[0132] In some embodiments, the compound of formula (1) prepared by the methods described hereinabove is a compound of formula (4b) or a crystalline form thereof: [ka] is.

[0133] In some embodiments, the compound of formula (1) prepared by the methods described hereinabove is a compound of formula (5b) or a crystalline form thereof: [ka] is.

[0134] In some embodiments, the compound of formula (1) prepared by the methods described hereinabove is a compound of formula (6b) or a crystalline form thereof: [ka] is.

[0135] Similarly, there is provided a method for preparing a compound of formula (4b) or a crystalline form thereof, comprising reacting a compound of formula (4b): [ka] ethanol, water, citric acid and a compound of formula (1): [ka]

[0013] Provided herein are methods comprising forming a crystalline crystalline product from a crystallization solution comprising:

[0136] Similarly, there is provided a method for preparing a compound of formula (4b) or a crystalline form thereof, comprising the steps of: (ia) a compound of formula (I): [ka] with a compound of the following formula (IIa) or a salt thereof: [ka] to produce a compound of formula (IIIa) or a salt thereof: [ka] Steps to get (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represent any suitable leaving group), or (ib) a compound of formula (IV): [ka] under reductive amination conditions to give a compound of formula (IIa) or a salt thereof: [ka] to produce a compound of formula (IIIa) or a salt thereof: [ka] Steps to get (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group), then (ii) a compound of formula (IIIa) or a salt thereof: [ka] with a deprotecting acid to give a compound of formula (V) or a salt thereof: [ka] Then, (iii) a compound of the following formula (V) or a salt thereof: [ka] with ethyl chloroformate to give a compound of formula (1) or a salt thereof: [ka] Then, (iv) Compound of formula (4b): [ka] ethanol, water, citric acid and a compound of formula (1): [ka] forming from a solution comprising A method is provided herein, comprising:

[0137] In some embodiments, R 1 is a linear or branched C 1~6 In some embodiments, R 1 is ethyl or t-butyl. In some embodiments, R 1 is t-butyl. In some embodiments, R 1 is ethyl.

[0138] In some embodiments, R 2 together with the oxygen atom to which it is attached represents a sulfonate ester leaving group. In some embodiments, R 2 together with the oxygen atom to which it is attached represent an arylsulfonate group. In some embodiments, R 2 together with the oxygen atom to which it is attached represents a tosylate group.

[0139] In some embodiments, the reductive amination conditions include a reducing acid and a reducing agent.

[0140] In some embodiments, the reductive amination conditions include H2 and palladium. In some embodiments, the reductive amination conditions include H2, palladium on barium sulfate, and molecular sieves. In some embodiments, the reductive amination conditions include H2 at about 1000 psi, palladium on barium sulfate (reduced), and molecular sieves.

[0141] In some embodiments, the reductive amination conditions include a reducing acid having a pKa of about 2.0 to about 3.5. In some embodiments, the reductive amination includes a reducing acid selected from citric acid, tartaric acid, pivalic acid, 2,2-dimethylsuccinic acid, acetic acid, di-p-toluoyltartaric acid, difluoroacetic acid, phosphoric acid, and mixtures thereof. In some embodiments, the reductive amination includes a reducing acid that is citric acid. In some embodiments, the reductive amination includes a reducing acid that is tartaric acid. In some embodiments, the reductive amination includes a reducing acid that is a mixture of citric acid and tartaric acid.

[0142] In some embodiments, the reductive amination conditions include reacting at a temperature less than about 40° C. In some embodiments, the reductive amination conditions include reacting at a first temperature for a first period of time, followed by reacting at a second temperature for a second period of time. In some embodiments, the second temperature is higher than the first temperature. In some embodiments, the reductive amination conditions include reacting at room temperature (about 23° C.) for about 1 day, followed by reacting at about 35° C. for about 1 day.

[0143] In some embodiments, the compound of formula (IIa) is a salt. In some embodiments, the compound of formula (IIa) is a tartrate salt.

[0144] In some embodiments, the deprotecting acid is hydrochloric acid. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by the reaction between acetyl chloride and a protic solvent. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by the reaction between acetyl chloride and ethanol.

[0145] In some embodiments, the compound of Formula (V) is a salt. In some embodiments, the compound of Formula (V) is a triacid salt. In some embodiments, the compound of Formula (V) is a hydrochloride salt. In some embodiments, the compound of Formula (V) is a trihydrochloride salt.

[0146] In some embodiments, the crystallization solution comprises ethanol and water in a ratio of about 100:1 to about 100:10 by volume, the crystallization solution comprises ethanol and water in a ratio of about 100:2 to about 100:5 by volume, in some embodiments, the crystallization solution comprises ethanol and water in a ratio of about 100:2.7 by volume, in some embodiments, the crystallization solution comprises ethanol and water in a ratio of about 100:5 by volume.

[0147] In some embodiments, the crystallization solution comprises citric acid and a compound of Formula (1) in a ratio of about 1:1 to about 1.3:1. In some embodiments, the crystallization solution comprises citric acid and a compound of Formula (1) in a ratio of about 1:1 to about 1.2:1. In some embodiments, the crystallization solution comprises citric acid and a compound of Formula (1) in a ratio of about 1:1 to about 1.1:1. In some embodiments, the crystallization solution comprises citric acid and a compound of Formula (1) in a ratio of about 1:1. In some embodiments, the crystallization solution comprises citric acid and a compound of Formula (1) in a ratio of about 1.08:1.

[0148] In some embodiments, seeds of the crystalline form of the compound of formula (4b) are added to the crystallization solution. Ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (compound of formula (7))

[0149] Similarly, there is provided a method for preparing a compound of formula (7) or a salt thereof, or a crystalline form thereof, comprising reacting a compound of formula (I) below: [ka] with a compound of the following formula (IIb) or a salt thereof: [ka] to produce a compound of formula (IIIb) or a salt thereof: [ka]

[0023] A method is provided herein, comprising obtaining (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group).

[0150] Similarly, there is provided a method for preparing a compound of formula (7) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with a deprotecting acid to give a compound of formula (VI) or a salt thereof: [ka]

[0023] A method is provided herein, comprising obtaining (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group).

[0151] In some embodiments, the reaction is carried out at a temperature below 20°C.

[0152] Similarly, there is provided a method for preparing a compound of formula (7) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with ethyl chloroformate to give a compound of formula (VII) or a salt thereof [ka] A method is provided herein that includes obtaining:

[0153] Similarly, there is provided a method for preparing a compound of formula (7) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with a ketal deprotecting acid to give a compound of formula (VIII) [ka] A method is provided herein that includes obtaining:

[0154] Similarly, there is provided a method for preparing a compound of formula (7) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] under reducing conditions to form a compound of formula (IX) or a salt thereof: [ka] to produce a compound of formula (7) or a salt thereof: [ka] A method is provided herein that includes obtaining:

[0155] Similarly, there is provided a method for preparing a compound of formula (7) or a salt thereof, or a crystalline form thereof, comprising the steps of: (i) a compound of formula (I): [ka] with a compound of the following formula (IIb) or a salt thereof: [ka] to produce a compound of formula (IIIa) or a salt thereof: [ka] Steps to get (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group), then (ii) a compound of the following formula (IIIb) or a salt thereof: [ka] with a deprotecting acid to give a compound of formula (VI) or a salt thereof [ka] Then, (iii) a compound of formula (VI) or a salt thereof: [ka] with ethyl chloroformate to give a compound of formula (VII) or a salt thereof [ka] Then, (iv) A compound of the following formula (VII) or a salt thereof: [ka] with a ketal deprotecting acid to give a compound of formula (VIII) [ka] Then, (v) A compound of the following formula (VIII) or a salt thereof: [ka] under reducing conditions to form a compound of formula (IX) or a salt thereof: [ka] to produce a compound of formula (7) or a salt thereof: [ka] A method is provided herein that includes obtaining:

[0156] In some embodiments, R 1 is a linear or branched C 1~6 In some embodiments, R 1 is ethyl or t-butyl. In some embodiments, R 1 is t-butyl. In some embodiments, R 1 is ethyl.

[0157] In some embodiments, R 2 together with the oxygen atom to which it is attached represents a sulfonate ester leaving group. In some embodiments, R 2 together with the oxygen atom to which it is attached represent an arylsulfonate group. In some embodiments, R 2 together with the oxygen atom to which it is attached represents a tosylate group.

[0158] In some embodiments, the reaction between a compound of Formula (I) and a compound of Formula (IIb), or a salt thereof, is heated to between about 80°C and about 110°C.

[0159] In some embodiments, the deprotecting acid is hydrochloric acid. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by the reaction between acetyl chloride and a protic solvent. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by the reaction between acetyl chloride and ethanol.

[0160] In some embodiments, the compound of formula (VI) is a salt. In some embodiments, the compound of formula (VI) is a salt of a diacid. In some embodiments, the compound of formula (VI) is a hydrochloride salt. In some embodiments, the compound of formula (VI) is a dihydrochloride salt.

[0161] In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate further comprises a base. In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate further comprises triethylamine.

[0162] In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate is carried out at a temperature of less than 20°C.

[0163] In some embodiments, the ketal deprotecting acid is hydrochloric acid. In some embodiments, the ketal deprotecting acid is aqueous hydrochloric acid.

[0164] In some embodiments, the reducing conditions comprise a borohydride reagent, hi some embodiments, the reducing conditions comprise sodium triacetoxyborohydride.

[0165] In some embodiments, the reaction between a compound of Formula (VIII) or a salt thereof and a compound of Formula (IX) is carried out at a temperature of less than 20°C. Ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (compound of formula (8))

[0166] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising reacting a compound of formula (I) below: [ka] with a compound of the following formula (IIb) or a salt thereof: [ka] to produce a compound of formula (IIIb) or a salt thereof: [ka]

[0023] A method is provided herein, comprising obtaining (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group).

[0167] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with an acid to form a salt of formula (IIIc) [ka]

[0023] A method is provided herein, comprising obtaining (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, X represents salt).

[0168] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with a deprotecting acid to give a compound of formula (VI) or a salt thereof [ka]

[0023] A method is provided herein, comprising obtaining (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group).

[0169] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with ethyl chloroformate to give a compound of formula (VII) or a salt thereof [ka] A method is provided herein that includes obtaining:

[0170] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] with a ketal deprotecting acid to give a compound of formula (VIII) [ka] A method is provided herein that includes obtaining:

[0171] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising the step of: [ka] under reducing conditions to form a compound of formula (X) or a salt thereof: [ka] to produce a compound of formula (8) or a salt thereof: [ka] Provided herein is a method comprising the steps of: obtaining

[0172] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising: [ka] from a recrystallization solution.

[0173] Similarly, there is provided a method for preparing a compound of formula (8) or a salt thereof, or a crystalline form thereof, comprising the steps of: (i) a compound of formula (I): [ka] with a compound of the following formula (IIb) or a salt thereof: [ka] to produce a compound of formula (IIIa) or a salt thereof: [ka] Steps to get (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group), then (ii) optionally a compound of formula (IIIb): [ka] with a salt-forming acid to form a salt of formula (IIIc) [ka] Steps to get (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, X represents a salt), then (iii) a compound of the following formula (IIIb) or a salt thereof: [ka] with a deprotecting acid to give a compound of formula (VI) or a salt thereof [ka] Then, (iv) A compound of formula (VI) or a salt thereof: [ka] with ethyl chloroformate to give a compound of formula (VII) or a salt thereof [ka] Then, (v) A compound of the following formula (VII) or a salt thereof: [ka] with a ketal deprotecting acid to give a compound of formula (VIII) [ka] Then, (vi) A compound of the following formula (VIII) or a salt thereof: [ka] under reducing conditions to form a compound of formula (X) or a salt thereof: [ka] to produce a compound of formula (8) or a salt thereof: [ka] A method is provided herein that includes obtaining:

[0174] In some embodiments, R 1 is a linear or branched C 1~6 In some embodiments, R 1 is ethyl or t-butyl. In some embodiments, R 1 is t-butyl. In some embodiments, R 1 is ethyl.

[0175] In some embodiments, R 2 together with the oxygen atom to which it is attached represents a sulfonate ester leaving group. In some embodiments, R 2 together with the oxygen atom to which it is attached represent an arylsulfonate group. In some embodiments, R 2 together with the oxygen atom to which it is attached represents a tosylate group.

[0176] In some embodiments, the reaction between a compound of Formula (I) and a compound of Formula (IIb), or a salt thereof, is heated to between about 80°C and about 110°C.

[0177] In some embodiments, the salt-forming acid is hydrochloric acid and X represents the hydrochloride salt.

[0178] In some embodiments, the deprotecting acid is hydrochloric acid. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by the reaction between acetyl chloride and a protic solvent. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by the reaction between acetyl chloride and ethanol.

[0179] In some embodiments, the compound of formula (VI) is a salt. In some embodiments, the compound of formula (VI) is a salt of a diacid. In some embodiments, the compound of formula (VI) is a hydrochloride salt. In some embodiments, the compound of formula (VI) is a dihydrochloride salt.

[0180] In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate further comprises a base. In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate further comprises triethylamine.

[0181] In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate is carried out at a temperature of less than 20°C.

[0182] In some embodiments, the ketal deprotecting acid is hydrochloric acid. In some embodiments, the ketal deprotecting acid is aqueous hydrochloric acid.

[0183] In some embodiments, the reducing conditions comprise a borohydride reagent, hi some embodiments, the reducing conditions comprise sodium triacetoxyborohydride.

[0184] In some embodiments, the reaction between a compound of Formula (VIII) or a salt thereof and a compound of Formula (X) is carried out at a temperature of less than 30°C.

[0185] In some embodiments, the recrystallization solution comprises a mixture of TBME and heptane. In some embodiments, the recrystallization solution comprises a mixture of TBME and heptane in a ratio of about 2:1. In some embodiments, the recrystallization solution comprises a mixture of TBME and heptane in a ratio of about 11:5. Intermediate Compounds Useful in the Process for Preparing the Compounds of the Invention

[0186] The present application also provides intermediate compounds useful in the production of the compounds described herein or salts thereof.

[0187] Thus, the present application relates to a compound of formula (I): [ka] provide more (In the formula, R 1 is a linear or branched C 1~6 Alkyl group, or C 3~6 is a cycloalkyl group, R 2 together with the oxygen atom to which it is attached represents any suitable leaving group).

[0188] In some embodiments, R 1 is a linear or branched C 1~6 In some embodiments, R 1 is ethyl or t-butyl. In some embodiments, R 1 is t-butyl. In some embodiments, R 1 is ethyl.

[0189] In some embodiments, R 2 together with the oxygen atom to which it is attached represents a sulfonate ester leaving group. In some embodiments, R 2 together with the oxygen atom to which it is attached represent an arylsulfonate group. In some embodiments, R 2 together with the oxygen atom to which it is attached represents a tosylate group.

[0190] The present application also relates to a compound of formula (III) or a salt thereof: [ka] provide (In the formula, R 1 is methyl, or linear or branched C 3~6 Alkyl group, or C 3~6 is a cycloalkyl group, (a)Q 1 is -H, and Q 2 teeth, [ka] Selected from or (b)Q 1 HA-OR 3 and Q 2 HA-OR 3 and R 3 is a linear or branched C 1~6 is an alkyl group, or (c)Q 1 and Q 2 together form the group -O-(CH2) m -O-, and m is an integer selected from 2, 3 and 4.

[0191] The present application also relates to a compound of formula (IIIa) or a salt thereof: [ka] provide (R 1 is methyl, or linear or branched C 3~6 Alkyl group, or C 3~6 is a cycloalkyl group).

[0192] In some embodiments, R 1 is methyl, or linear or branched C 3~6 In some embodiments, R 1 is t-butyl.

[0193] In some embodiments, the compound of Formula (IIIa) is a salt. In some embodiments, the compound of Formula (IIIa) is a citrate salt. In some embodiments, the compound of Formula (IIIa) is a hydrochloride salt.

[0194] The present application also relates to a compound of formula (IIIb) or a salt thereof: [ka] provide (In the formula, R 1 is a linear or branched C 1~6Alkyl group, or C 3~6 is a cycloalkyl group).

[0195] In some embodiments, R 1 is a linear or branched C 1~6 In some embodiments, R 1 is ethyl or t-butyl. In some embodiments, R 1 is t-butyl. In some embodiments, R 1 is ethyl.

[0196] The present application also relates to a compound of formula (V) or a salt thereof: [ka] to provide.

[0197] In some embodiments, the compound of Formula (V) is a salt. In some embodiments, the compound of Formula (V) is a triacid salt. In some embodiments, the compound of Formula (V) is a hydrochloride salt. In some embodiments, the compound of Formula (V) is a trihydrochloride salt.

[0198] The present application also relates to a compound of formula (VI) or a salt thereof: [ka] to provide.

[0199] In some embodiments, the compound of formula (VI) is a salt. In some embodiments, the compound of formula (VI) is a salt of a diacid. In some embodiments, the compound of formula (VI) is a hydrochloride salt. In some embodiments, the compound of formula (VI) is a dihydrochloride salt.

[0200] The present application also relates to a compound of formula (VII) or a salt thereof: [ka] to provide.

[0201] The present application also relates to a compound of formula (VIII) or a salt thereof: [ka] to provide. Crystalline morphology

[0202] The present application also provides crystalline forms of the compounds described herein or salts thereof. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl ester

[0203] The present application provides at least one crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate or a salt thereof. In some embodiments, the present application provides at least one crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate. In some embodiments, the present application provides at least one crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, wherein the salt is selected from the group consisting of citrate, hydrochloride, and fumarate.

[0204] In some embodiments, the present application provides at least one crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate. In some embodiments, the present application provides at least one crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate. In some embodiments, the present application provides at least one crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate hydrate. In some embodiments, the present application provides at least one crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate. In some embodiments, the present application provides at least one crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride. In some embodiments, the present application provides at least one crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate

[0205] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DSC thermogram having an endothermic peak with an onset at about 176° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DSC thermogram substantially as shown in FIG.

[0206] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram with a weight loss of about 3.3% from room temperature to about 110° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram with a weight loss of about 27.4% from about 170° C. to 210° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram substantially as shown in FIG.

[0207] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least one XRPD peak selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least two XRPD peaks selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least three XRPD peaks selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least four XRPD peaks selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta.In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least five XRPD peaks selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta. In some embodiments, the crystalline form is characterized by at least six XRPD peaks selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least seven XRPD peaks selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least eight XRPD peaks selected from 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° in terms of two-theta.In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by XRPD peaks at 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, 22.7°±0.2°, and 24.1°±0.2° two-theta.

[0208] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least one XRPD peak selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° in two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least two XRPD peaks selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° in two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least three XRPD peaks selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° two-theta.

[0209] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 11.3°±0.2°, 19.2°±0.2°, and 20.5°±0.2° in two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, and 20.5°±0.2° in two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, and 19.2°±0.2° in two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° in two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by four XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° two-theta.

[0210] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 15.0°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 15.0°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, and 22.3°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 15.0°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, and 22.7°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 15.0°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, and 24.1°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, and 22.3°±0.2° two-theta.In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, and 22.7°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, and 24.1°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, and 22.7°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.3°±0.2°, and 24.1°±0.2° two-theta. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, 20.5°±0.2°, 22.7°±0.2°, and 24.1°±0.2° two-theta.

[0211] In some embodiments, the crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by an XRPD spectrum substantially as shown in FIG. 25.

[0212] In any one of the above embodiments, the crystalline form may be prepared by any one of the methods described herein. In any one of the above embodiments, the crystalline form may be substantially isolated.

[0213] In any one of the above embodiments, the salt is cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate salt. In any one of the above embodiments, the salt is cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate salt hydrate. In any one of the above embodiments, the salt is cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate salt monohydrate. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate

[0214] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DTA thermogram having an endothermic peak with an onset at about 174° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DTA thermogram substantially as shown in Figure 2. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram with a weight loss of about 33% from about 140° C. to about 220° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram substantially as shown in FIG.

[0215] In some embodiments, the crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate has an XRPD spectrum substantially as shown in FIG. 1.

[0216] In any one of the above embodiments, the crystalline form may be prepared by any one of the methods described herein. In any one of the above embodiments, the crystalline form may be substantially isolated.

[0217] In any one of the above embodiments, the salt is cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate. In any one of the above embodiments, the salt is cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate. Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride

[0218] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DSC thermogram having an endothermic peak with an onset at about 234° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DTA thermogram substantially as shown in Figure 8. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram with a weight loss from room temperature to about 200° C. that is a loss of about 3.6%. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram substantially as shown in FIG.

[0219] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by an XRPD spectrum substantially as shown in FIG.

[0220] In any one of the above embodiments, the crystalline form may be prepared by any one of the methods described herein. In any one of the above embodiments, the crystalline form may be substantially isolated. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate

[0221] In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DSC thermogram having an endothermic peak with an onset at about 175° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DTA thermogram substantially as shown in Figure 15. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram with substantially no weight loss from room temperature to about 180° C. In some embodiments, the crystalline form of the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram substantially as shown in FIG. 15.

[0222] In some embodiments, the crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is characterized by an XRPD spectrum substantially as shown in FIG. 14.

[0223] In any one of the above embodiments, the crystalline form may be prepared by any one of the methods described herein. In any one of the above embodiments, the crystalline form may be substantially isolated. Ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate

[0224] The present application also provides at least one crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate or a salt thereof. In some embodiments, the present application provides at least one crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (free base). In some embodiments, the present application provides at least one crystalline form of a salt of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate.

[0225] In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DSC thermogram having an endothermic peak with an onset at about 99° C. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by a DSC thermogram substantially as shown in FIG. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram with a weight loss from room temperature to about 125° C. that is a loss of about 0.34%. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by a TGA thermogram substantially as shown in FIG.

[0226] In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least one XRPD peak selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least two XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least three XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least four XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta.In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least five XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least six XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least seven XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta.

[0227] In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least one XRPD peak selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least two XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by at least three XRPD peaks selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2° two-theta.

[0228] In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 6.1°±0.2°, 17.3°±0.2°, and 17.6°±0.2° in two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, and 17.6°±0.2° in two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, and 17.3°±0.2° in two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by three XRPD peaks at 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2° in two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by four XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2° two-theta.

[0229] In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, and 19.1°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, and 19.7°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 18.4°±0.2°, and 21.3°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 19.1°±0.2°, and 19.7°±0.2° two-theta. In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 19.1°±0.2°, and 21.3°±0.2° two-theta.In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by six XRPD peaks at 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, 17.6°±0.2°, 19.7°±0.2°, and 21.3°±0.2° two-theta.

[0230] In some embodiments, the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is characterized by an XRPD spectrum substantially as shown in FIG.

[0231] In any one of the above embodiments, the crystalline form may be prepared by any one of the methods described herein. In any one of the above embodiments, the crystalline form may be substantially isolated. alternative name

[0232] As used herein, C 19 H 30 A compound of the structural formula shown below, with a molecular formula of N4O2 and a molecular weight of 346.5 g / mol: [ka] The following names and identifiers: It may be referred to as ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate; ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate; and ethyl 2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate (named by ChemDraw Professional v.21.0.0.28, PerkinElmer Informatics, Inc.).

[0233] As used herein, C 19 H 30 A compound of formula (1) shown below, having a molecular formula of N4O2 and a molecular weight of 346.5 g / mol: [ka] The following names and identifiers: Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate;Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate;Ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate; Ethyl (2r,4s)-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate (named by ChemDraw Professional v.21.0.0.28, PerkinElmer Informatics, Inc.) and It is sometimes referred to by its CAS RN: 1803346-98-6.

[0234] As used herein, a compound of formula (3a) shown below: [ka] The molar ratio between ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate and citric acid was 1:1, and C 25 H 38 N4O9(C 19 H 30 N4O2·C6H8O7) and a molecular weight of 538.6 g / mol) is sometimes referred to by the following names and identifiers: 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate; 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate; 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate (1:1); and Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate (1:1).

[0235] As used herein, a compound of formula (3b) shown below: [ka] The molar ratio between ethyl (cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate) and citric acid was 1:1, and C 25 H 38 N4O9(C 19 H 30N4O2·C6H8O7) and a molecular weight of 538.6 g / mol) is sometimes referred to by the following names and identifiers: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate; (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate, citrate (1:1); and (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citrate salt (1:1).

[0236] As used herein, a compound of formula (4a) shown below: [ka] The molar ratio between ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citric acid, and water was 1:1:1, and C 25 H 40 N4O 10 (C 19 H 30 N4O2·C6H8O7·H2O) and a molecular weight of 556.6 g / mol) is sometimes referred to by the following names and identifiers: 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate; Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate; Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate, citrate (1:1) hydrate; and Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citrate salt (1:1) hydrate.

[0237] As used herein, a compound of formula (4b) shown below: [ka] The molar ratio between ethyl (cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citric acid, and water was 1:1:1, and C 25 H 40 N4O 10 (C 19 H 30 N4O2·C6H8O7·H2O) and a molecular weight of 556.6 g / mol) is sometimes referred to by the following names and identifiers: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate; (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate, citrate (1:1) hydrate; (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citrate (1:1) hydrate; and CAS RN:2920742-36-3.

[0238] As used herein, a compound of formula (5a) shown below: [ka] The molar ratio between ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate and hydrochloric acid was 1:1, and C 19 H 31 ClN4O2(C 19 H 30 N4O2·HCl) and a molecular weight of 382.9 g / mol) is sometimes referred to by the following names and identifiers: Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride; Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride; Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate, hydrochloride (1:1); Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, hydrochloride (1:1).

[0239] As used herein, a compound of formula (5b) shown below: [ka] The molar ratio between ethyl (cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate and hydrochloric acid was 1:1, and C 19 H 31 ClN4O2(C 19 H 30 N4O2·HCl) and a molecular weight of 382.9 g / mol) is sometimes referred to by the following names and identifiers: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl ester hydrochloride; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl hydrochloride; (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl salt; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate, hydrochloride (1:1); and (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, hydrochloride (1:1).

[0240] As used herein, a compound of structural formula (6a) shown below: [ka] The molar ratio between ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate and fumaric acid was 1:1, and C 23 H 34 N4O6(C 19 H30 N4O2·C4H4O4) and a molecular weight of 462.6 g / mol) is sometimes referred to by the following names and identifiers: 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate; 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate; Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate, fumarate (1:1); and Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, fumarate (1:1).

[0241] As used herein, a compound of formula (6b) shown below: [ka] The molar ratio between ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate and fumaric acid was 1:1, and C 23 H 34 N4O6(C 19 H 30 N4O2·C4H4O4) and a molecular weight of 462.6 g / mol) is sometimes referred to by the following names and identifiers: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate; (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate; cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate, ethyl fumarate (1:1); and (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate (1:1).

[0242] As used herein, C 20 H 33 The compound of formula (7) shown below, having a molecular formula of F2N3O3 and a molecular weight of 401.5 g / mol: [ka] may be referred to by the following names and identifiers: Ethyl cis-2-[4-[(2R)-4,4-difluoro-2-(hydroxymethyl)-1-pyrrolidinyl]-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate; Ethyl cis-2-[4-[(2R)-4,4-difluoro-2-(hydroxymethyl)-1-pyrrolidinyl]piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate; Ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate; ethyl (2r,4S)-2-(4-((R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate (named by ChemDraw Professional v.21.0.0.28, PerkinElmer Informatics, Inc.); and CAS RN: 1803349-72-5.

[0243] As used herein, C 20 H 34 A compound of formula (8) shown below, having a molecular formula of FN3O3 and a molecular weight of 383.5 g / mol: [ka] may be referred to by the following names and identifiers: Ethyl cis-2-[4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)-1-pyrrolidinyl]-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylate; Ethyl cis-2-[4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)-1-pyrrolidinyl]piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate; Ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate; ethyl (2r,4S)-2-(4-((2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate (named by ChemDraw Professional v.21.0.0.28, PerkinElmer Informatics, Inc.); and CAS RN: 1803346-48-6. definition

[0244] For the purposes of this application, the following definitions apply unless otherwise indicated.

[0245] The term "treatment," with respect to the use of a compound of Formula (1), Formula (2a), Formula (2b), Formula (3a), Formula (3b), Formula (4a), Formula (4b), Formula (5a), Formula (5b), Formula (6a), or Formula (6b), is used to describe any form of intervention in which a compound is administered to a subject suffering from, or at risk of, or potentially at risk for the disease or disorder in question. Thus, the term "treatment" encompasses both prophylactic (preventative) treatment and treatment in which measurable or detectable symptoms of the disease or disorder are present.

[0246] The term "effective therapeutic amount" as used herein (e.g., in relation to the method of treating a disease or condition) refers to the amount of a compound that is effective to produce a desired therapeutic effect.For example, when the condition is pain, an effective therapeutic amount is an amount that is sufficient to achieve a desired level of pain relief.A desired level of pain relief can be, for example, the complete elimination of pain or a reduction in the severity of pain.

[0247] The term "suitable leaving group" describes a group that is suitable to function in this manner in a given reaction. For example, the leaving groups described herein are suitable for bimolecular nucleophilic substitution (S N2) reaction. The term leaving group is defined in the IUPAC Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"), edited by A. D. McNaught and A. Wilkinson, Blackwell Scientific Publications, Oxford (1997), online edition (2019-) written by S. J. Chalk. ISBN 0-9678550-9-8. https: / / doi.org / 10.1351 / goldbook. Suitable leaving groups include sulfonate ester leaving groups (e.g., arylsulfonate esters, alkylsulfonate esters, haloalkylsulfonate esters), halogens (e.g., -I, -Br, -Cl), trialkylamines, dinitrogen, dialkyl ethers, aryliodonium salts, and arylsulfonium salts. In some embodiments, the leaving group is a nucleofuge. In some embodiments, the leaving group is an arylsulfonate ester. In some embodiments, the leaving group is a tosylate group. salt

[0248] The compounds described herein can exist in the form of salts, for example, acid addition salts, or in certain cases, salts of organic and inorganic bases, such as carboxylates, sulfonates, and phosphates.All such salts are within the scope of the present invention, and when referring to a compound, it includes the salt form of the compound defined herein.The salt is usually an acid addition salt.

[0249] The salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with the appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

[0250] Acid addition salts can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts that fall within the scope of the present invention include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane, cyclohexane, cyclohexane-10-sulfonic acid ... Glucosaminoglycans, thiaminic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, iodine acid), Hydrogenated lactic acid, isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobioic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmit ... Included are mono- or di-salts formed with acids selected from the group consisting of moic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic, and valeric acids, as well as acylated amino acids and cation exchange resins.

[0251] In particular, acid addition salts may be formed with acids selected from 2,5-dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, butanedioic acid (succinic acid), citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), hydroxyacetic acid (glycolic acid), hydrochloric acid (HCl), maleic acid, malic acid (L), malonic acid, N-acetylglycine (aceturic acid), nicotinic acid, orthophosphoric acid (phosphoric acid), oxoglutaric acid (ketoglutaric acid), p-toluenesulfonic acid (p-TSA), pyroglutamic acid (L), sulfuric acid, and tartaric acid (L).

[0252] Amine functional groups in the compounds described herein may be reacted with alkylating agents to form quaternary ammonium salts, for example, according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[0253] The compounds of the present invention can exist as mono- or di-salts depending on the pKa of the acid from which the salt is formed.

[0254] The salt forms of the compounds of the present invention are usually pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19. However, non-pharmaceutically acceptable salts may also be prepared as intermediates, which can then be converted to pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salt forms, which may be useful, for example, in the purification or separation of the compounds of the present invention, also form part of the present invention. optical isomer

[0255] Unless the context otherwise requires, when a compound of the invention contains one or more chiral centers and can exist in the form of two or more optical isomers, a reference to the compound includes all optical isomers thereof (e.g., enantiomers, epimers, diastereoisomers, cis-trans positional isomers of cyclic functional groups), either as an individual optical isomer, or as a mixture (e.g., a racemic mixture), or as two or more optical isomers.

[0256] Optical isomers may be characterized and identified by their optical activity (i.e., as + and - isomers, or d and l isomers), or they may be characterized with respect to their absolute stereochemistry using the "R and S" nomenclature developed by Cahn, Ingold, and Prelog, Advanced Organic Chemistry by Jerry March, 4 th Edition, John Wiley & Sons, New York, 1992, pages 109-114, and also Cahn, Ingold & Prelog, Angew. Chem. Int. Ed. Engl., 1966, 5, 385-415. Compounds featuring cyclic moieties may be characterized using the "cis-trans" nomenclature. Optical isomers can be separated by several techniques, including chiral chromatography (chromatography on a chiral support), and such techniques are well known to those skilled in the art. As an alternative to chiral chromatography, optical isomers can be separated by forming diastereomeric salts with chiral acids such as (+)-tartaric acid, (-)-pyroglutamic acid, (-)-di-toluoyl-L-tartaric acid, (+)-mandelic acid, (-)-malic acid, and (-)-camphorsulfonic acid, followed by separation of the diastereoisomers by preferential crystallization, followed by desalting to give the individual enantiomers of the free base.

[0257] When the compounds of the invention exist as two or more optical isomers, one of the enantiomers in a pair may exhibit advantages over the other enantiomer, for example, with respect to biological activity. Thus, in certain circumstances, only one of the pair of enantiomers, or only one of the diastereoisomers, may be desirable for use as a therapeutic agent.

[0258] Accordingly, the present invention provides a composition comprising a compound having one or more chiral centers, wherein at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound exists as a single optical isomer (e.g., enantiomer or diastereomer).

[0259] In one typical embodiment, 99% or more (eg, substantially all) of the total amount of the compound (or compound for use) is present as a single optical isomer.

[0260] For example, in one embodiment, the compound exists as a single enantiomer. In another embodiment, the compound exists as a single diastereoisomer.

[0261] The present invention also provides mixtures of optical isomers, which may be racemic or non-racemic. Thus, the present invention provides: A compound in the form of a racemic mixture of optical isomers. A compound in the form of a non-racemic mixture of optical isomers. cis-trans isomerism

[0262] Further provided herein are compositions comprising compounds having one or more regions of restricted rotation resulting in cis-trans isomerism, wherein at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compounds exist as a single stereoisomer of a cis-trans pair.

[0263] In one typical embodiment, 99% or more (eg, substantially all) of the total amount of the compound (or compound for use) is present as a single cis-trans isomer.

[0264] For example, in one embodiment, the compound exists as a cis isomer. In another embodiment, the compound exists as a trans isomer (e.g., an intermediate compound).

[0265] The present invention also provides mixtures of cis-trans isomers. Isotopes

[0266] The compounds of the present invention may contain one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of that element. For example, a reference to hydrogen includes within its scope: 1 H, 2 H(D) and 3 Similarly, when carbon and oxygen are mentioned, the following are included within their scope: 12 C. 13 C and 14 C, and 16 O and 18 Contains O.

[0267] In a similar manner, when a particular functional group is mentioned, isotopic variations are also included within its scope unless the context indicates otherwise. For example, when an alkyl group such as an ethyl group is mentioned, variations in which one or more hydrogen atoms in the group are in the form of a deuterium or tritium isotope are also encompassed, such as, for example, an ethyl group in which all five hydrogen atoms are deuterium isotopes (perdeuterioethyl group).

[0268] The isotope may be radioactive or non-radioactive. The compound may not contain a radioactive isotope. Such a compound is preferred for therapeutic use. However, the compound may contain one or more radioactive isotopes. Compounds containing such radioactive isotopes may be useful in diagnostic situations. solvate

[0269] The compounds of the present invention may form solvates. Preferred solvates are those formed by incorporating molecules of a non-toxic, pharmaceutically acceptable solvent (hereinafter referred to as a solvating solvent) into the solid-state structure (e.g., crystalline structure) of the compounds of the present invention. Examples of such solvents include water, alcohols (such as ethanol, isopropanol, and butanol), and dimethyl sulfoxide. Solvates can be prepared by recrystallization with a solvent or mixture of solvents containing a solvating solvent for the compounds of the present invention. Whether a solvate is formed in any given example can be determined by analyzing the crystals of the compound using well-known standard techniques, such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray crystallography. The solvate can be a stoichiometric or non-stoichiometric solvate. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates, and dihydrates.

[0270] Thus, the present invention provides: The compound in the form of a solvate. The compound wherein the solvate is a hydrate.

[0271] For a more detailed discussion of solvates and methods used to make and characterize solvates, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, 1999, ISBN 0-967-06710-3, published by SSCI, Inc., West Lafayette, IN, USA.

[0272] Alternatively, the compounds of the invention may be anhydrous rather than existing as a hydrate. Accordingly, the invention provides compounds of the invention in anhydrous form (e.g., anhydrous crystalline form). Crystalline and amorphous forms

[0273] The compound may exist in a crystalline or non-crystalline (e.g., amorphous) state. Whether a compound exists in a crystalline state can be easily determined by standard techniques such as X-ray powder diffraction (XRPD). Crystals and their crystalline structures can be characterized using several techniques, including single-crystal X-ray crystallography, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), and infrared spectroscopy, such as Fourier transform infrared spectroscopy (FTIR). The behavior of crystals under various humidity conditions can be analyzed by gravimetric vapor sorption studies, and also by XRPD. The crystal structure of a compound can be determined by X-ray crystallography, which can be performed in accordance with conventional methods, such as those described herein and in Fundamentals of Crystallography, C. Giacovazzo, H. L. Monaco, D. Viterbo, F. Scordari, G. Gilli, G. Zanotti and M. Catti, (International Union of Crystallography / Oxford University Press, 1992 ISBN 0-19-855578-4 (p / b), 0-19-85579-2 (h / b)). This technique involves the analysis and interpretation of X-ray diffraction of a single crystal. In amorphous solids, the three-dimensional structure that is typically present in crystalline forms does not exist; in amorphous forms, the positions of molecules relative to each other are essentially random; see, for example, Hancock et al. J. Pharm. Sci. (1997), 86, 1).

[0274] Other physical characterization methods such as differential scanning calorimetry (DSC), differential thermal analysis (DTA), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), etc. may help to further characterize the form and determine stability and solvent / moisture content.

[0275] XRPD patterns of reflections (peaks) are generally considered fingerprint regions of specific crystalline forms. It is well known that the relative intensities of XRPD peaks can vary widely depending on, among other things, sample preparation techniques, crystal size distribution, various filters used, sample mounting procedures, and the specific instrument used. In some cases, new peaks may be observed or existing peaks may disappear, depending on the type or settings of the instrument. As used herein, the term "peak" refers to a reflection having a relative height / intensity of at least about 4% of the maximum peak height / intensity. Furthermore, instrument variables and other factors can affect 2-theta values. Thus, peak assignments such as those reported herein can vary by plus or minus about 0.2° (2-theta), and the terms "substantially" and "about" as used in the XRPD context herein are intended to encompass such variations.

[0276] Similarly, temperature readings associated with DSC, TGA, or other thermal experiments can vary by about ±3°C depending on the instrument, specific settings, sample preparation, etc. Thus, for the crystalline forms provided herein, DSC thermograms shown "substantially" in either the figures or the term "about" are understood to accommodate such variations. As used herein, the term "no substantial weight loss" means a weight loss of less than 0.25%.

[0277] Different forms of the same substance may have different bulk properties, such as those related to hygroscopicity, solubility, stability, etc. Forms with high melting points may have good thermodynamic stability, which is advantageous for extending the shelf life of drug formulations containing the solid form. Forms with lower melting points may be less thermodynamically stable, but they may be advantageous in that they have high aqueous solubility, which may lead to improved drug bioavailability. Forms with low hygroscopicity may be desirable for stability against heat and humidity, and may be resistant to degradation during long-term storage.

[0278] Thus, the present invention provides: A compound in crystalline form. The following compounds: (a) 50% to 100% crystalline, more particularly, at least 50% crystalline, or at least 60% crystalline, or at least 70% crystalline, or at least 80% crystalline, or at least 90% crystalline, or at least 95% crystalline, or at least 98% crystalline, or at least 99% crystalline, or at least 99.5% crystalline, or at least 99.9% crystalline, e.g., 100% crystalline. A compound in amorphous form.

[0279] In some embodiments, the crystalline forms provided herein are substantially isolated. "Substantially isolated" means that the compound or crystalline form is at least partially or substantially isolated from the environment in which it was formed or detected. Partial isolation can include, for example, compositions enriched in the salts described herein. Substantial isolation can include compositions containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the crystalline forms described herein or salts thereof.

[0280] In some embodiments, the crystalline form of a compound or salt described herein comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the crystalline form of such compound or salt described herein. Prodrug

[0281] The compounds of the present invention may be supplied in the form of a prodrug. By "prodrug" is meant, for example, any compound that is converted in vivo into a biologically active compound of the present invention.

[0282] For example, some prodrugs can be esters of the active compound (e.g., physiologically acceptable metabolically labile esters). During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters can be formed, for example, by esterification of any hydroxyl groups present in the parent compound, optionally with prior protection of any other reactive groups present in the parent compound, followed by deprotection, if necessary.

[0283] Similarly, some prodrugs are activated by enzymes to yield the active compound or a compound that, upon further chemical reaction, yields the active compound (such as, for example, ADEPT, GDEPT, LIDEPT, etc.) For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

[0284] Thus, the present invention provides prodrugs of the compounds of the present invention, which contain functional groups that can be transformed under physiological conditions to form hydroxyl or amino groups. Complexes and inclusion compounds

[0285] Also included are complexes of compounds of the invention (eg, inclusion complexes or compounds with compounds such as cyclodextrins, or complexes with metals).

[0286] Thus, the present invention provides compounds in the form of complexes or clathrates. Biological Activities and Therapeutic Uses

[0287] The compounds of the present invention have activity as muscarinic M4 receptor agonists (WO2015 / 118342).

[0288] A significant advantage of certain compounds of the invention is that they are highly selective for the M4 receptor over the M1, M2, and M3 receptor subtypes. For example, compounds of the invention typically have a pEC50 activity of at least 6 for the M4 receptor in the functional assay described in Example A of WO2015 / 118342. 50 values, and E higher than 70 max While these compounds have pEC values of less than 5 when tested against M1, M2 and M3 subtypes in the functional assay of Example A of WO2015 / 118342, 50 value and less than 20% E max It can have a value.

[0289] With respect to the compounds of the present invention, the present invention further provides: A compound for use in medicine. A compound for use as a muscarinic M4 receptor agonist. A pEC50 of greater than 6 for the M4 receptor in the assay of Example A herein or an assay substantially similar thereto. 50 and at least 70 E max A compound which is a muscarinic M4 receptor agonist having the formula: pEC greater than 7.0 50 A compound which is a muscarinic M4 receptor agonist having the formula: E of at least 70 for M4 receptors max A compound having the formula:

[0290] A compound selective for the muscarinic M4 receptor compared to the M1, M2 and M3 receptors. pEC<5 for muscarinic M1, M2, and M3 receptor subtypes 50 and E below 30 max A compound having the formula: A compound for use in the treatment of a disease or condition mediated by the muscarinic M4 receptor. pEC greater than 7.0 50 A compound which is a muscarinic M1 receptor agonist having the formula: E of at least 70 for M1 receptors max A compound having the formula: A compound selective for muscarinic M1 receptors compared with M2 and M3 receptors. pEC<5 for muscarinic M2 and M3 receptor subtypes 50 and E below 30 max A compound having the formula: A compound for use in the treatment of a disease or condition mediated by the muscarinic M1 receptor. A compound for use in the treatment of a disease or condition mediated by the muscarinic M1 receptor or the muscarinic M4 receptor.

[0291] Due to their muscarinic M4 receptor agonist activity, the compounds of the present invention can be used to treat Alzheimer's disease, dementia with Lewy bodies, schizophrenia and other psychotic disorders, cognitive disorders, and other diseases mediated by the muscarinic M4 receptor, and can also be used to treat various types of pain.

[0292] Thus, with respect to the compounds of the invention, the invention further provides: a compound for use in the treatment of a cognitive or psychotic disorder.

[0293] Compounds for use in the treatment of cognitive impairment or psychotic disorders (cognitive impairment or psychotic disorders include cognitive impairment, mild cognitive impairment (MCI), (including amnestic MCI and non-amnestic MCI, including mild cognitive impairment due to Alzheimer's disease and / or prodromal Alzheimer's disease), frontotemporal dementia, vascular dementia, dementia with Lewy bodies, presenile dementia, senile dementia, Friederich's ataxia, Down syndrome, Huntington's chorea, hyperkinesia, mania, Tourette's syndrome, and Alzheimer's disease (fda.gov / downloads / Drugs / GuidanceComplianceRegulatoryInformation / Guidances / UCM596728).(including prodromal Alzheimer's disease, and early Alzheimer's disease stages 1, 2, and 3, as defined by the U.S. Food and Drug Administration's "Early Alzheimer's disease: Developing Drugs for Treatment," available at www.fda.gov / health / alzheimer's-diseases / .pdf), progressive supranuclear palsy, impairment of cognitive function (including attention, adaptation, learning disorders, memory (i.e., memory impairment, amnesia, amnesia, transient global amnesia syndrome, and age-associated memory impairment) and language function); cognitive impairment as a result of stroke, other dementia conditions such as Huntington's disease, Pick's disease, AIDS-related dementia, or multi-infarct dementia, alcoholic dementia, hypothyroidism-related dementia, and dementia associated with other degenerative diseases (e.g., cerebellar atrophy and amyotrophic lateral sclerosis); other acute or subacute conditions that may cause cognitive decline, such as delirium or depression (pseudocognitive state), trauma, head injury, age-related cognitive decline, stroke, neurodegeneration, drug-induced conditions, dementia, (including, arising from, or related to conditions selected from transtoxicants, age-related cognitive impairment, autism-related cognitive impairment, Down's syndrome, agnosia associated with psychosis and post-electroconvulsive therapy-related cognitive impairment; cognitive impairment due to drug abuse or drug withdrawal, including nicotine, cannabis, amphetamines, and cocaine; attention deficit hyperactivity disorder (ADHD) and movement disorders such as Parkinson's disease, neuroleptic-induced parkinsonism and tardive dyskinesia; schizophrenia, schizophreniform disorders, psychotic depression, mania, acute mania, delusions, hallucinatory disorders and delusional disorders, personality disorders, obsessive-compulsive disorder, schizotypal disorder, delusional disorder, psychosis due to malignancy, metabolic disorders, endocrine disorders or narcolepsy, psychosis due to drug abuse or drug withdrawal, bipolar disorder, and schizoaffective disorder).

[0294] Compounds for use in the treatment of cognitive impairment or psychotic disorders (cognitive impairment or psychotic disorders include cognitive impairment, mild cognitive impairment (MCI), (including amnestic MCI and non-amnestic MCI, including mild cognitive impairment due to Alzheimer's disease and / or prodromal Alzheimer's disease), frontotemporal dementia, vascular dementia, dementia with Lewy bodies, presenile dementia, senile dementia, Friederich's ataxia, Down syndrome, Huntington's chorea, hyperkinesia, mania, Tourette's syndrome, and Alzheimer's disease (fda.gov / downloads / Drugs / GuidanceComplianceRegulatoryInformation / Guidances / UCM596728).(including prodromal Alzheimer's disease, and early Alzheimer's disease stages 1, 2, and 3, as defined by the U.S. Food and Drug Administration's "Early Alzheimer's disease: Developing Drugs for Treatment," available at www.FDA.org.uk / treatment / treatment), progressive supranuclear palsy, cognitive impairment (including attention, adaptation, learning disorders, memory (i.e., memory impairment, amnesia, amnesia, transient global amnesia syndrome, and age-associated memory impairment) and language functions); cognitive impairment as a result of stroke, Huntington's disease, Pick's disease, AIDS-related dementia, or other dementia conditions such as multi-infarct dementia, alcoholic dementia, hypothyroidism-related dementia, and dementia associated with other degenerative diseases (e.g., cerebellar atrophy and amyotrophic lateral sclerosis); other acute or subacute conditions that may cause cognitive decline, such as delirium or depression (pseudocognitive states), trauma, head injury, age-related cognitive decline, stroke, neurodegeneration, drug-induced states, neurotoxic agents, age-related cognitive impairment due to drug abuse or drug withdrawal, including nicotine, cannabis, amphetamines, and cocaine; attention deficit hyperactivity disorder (ADHD) and movement disorders such as Parkinson's disease, neuroleptic-induced parkinsonism, and tardive dyskinesia; schizophrenia, schizophreniform disorders, psychotic depression, mania, acute mania, delusions, hallucinatory disorders, and delusional disorders; personality disorders, obsessive-compulsive disorder, schizotypal disorder, delusional disorder, mental illness due to malignancy, metabolic disorders, endocrine disorders, or narcolepsy; mental illness due to drug abuse or drug withdrawal; bipolar disorder, schizoaffective disorder, and Alzheimer's disease.

[0295] A compound for use in the treatment of schizophrenia, Alzheimer's disease, Alzheimer's psychosis or bipolar disorder.

[0296] The compound for use in the treatment of schizophrenia, Alzheimer's psychosis or bipolar disorder.

[0297] A compound for use in the treatment of Alzheimer's disease.

[0298] A compound for use in the treatment of dementia with Lewy bodies.

[0299] A compound for use in the treatment of schizophrenia.

[0300] A compound for use in the treatment of bipolar disorder.

[0301] A compound for use in the treatment of Alzheimer's disease psychiatric disorders.

[0302] A method of treating cognitive impairment in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention.

[0303] A method of treating a cognitive disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the cognitive disorder includes, results from, or is associated with a condition as defined above.

[0304] A method for treating cognitive impairment in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), the method comprising administering a therapeutically effective dose of a compound of the invention, wherein the cognitive impairment results from or is associated with Alzheimer's disease.

[0305] A method of treating a cognitive disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the cognitive disorder is dementia with Lewy bodies.

[0306] A method for treating a cognitive disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the cognitive disorder is schizophrenia.

[0307] A method for treating a cognitive disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the cognitive disorder is bipolar disorder.

[0308] A method for treating cognitive impairment in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the present invention, wherein the cognitive impairment is Alzheimer's disease psychiatric disorder.

[0309] Use of a compound of the present invention for the manufacture of a medicament for treating cognitive disorders.

[0310] Use of a compound of the invention for the manufacture of a medicament for the treatment of cognitive disorders, wherein the cognitive disorders include, arise from or are associated with the conditions defined above.

[0311] Use of a compound of the invention for the manufacture of a medicament for treating cognitive impairment, wherein the cognitive impairment includes, results from, or is associated with Alzheimer's disease.

[0312] Use of a compound of the invention for the manufacture of a medicament for treating a cognitive disorder, wherein the cognitive disorder includes, results from, or is associated with dementia with Lewy bodies.

[0313] Use of a compound of the invention for the manufacture of a medicament for treating a cognitive disorder, wherein the cognitive disorder includes, results from or is associated with schizophrenia.

[0314] Use of a compound of the invention for the manufacture of a medicament for treating a cognitive disorder, wherein the cognitive disorder includes, results from or is associated with bipolar disorder.

[0315] Use of a compound of the invention for the manufacture of a medicament for treating cognitive impairment, wherein the cognitive impairment includes, results from or is associated with Alzheimer's disease psychiatric disorder.

[0316] A method of treating a psychotic disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention.

[0317] A method of treating a psychotic disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the psychotic disorder comprises, arises from or is associated with a condition as defined above.

[0318] A method of treating a psychotic disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), the method comprising administering a therapeutically effective dose of a compound of the invention, wherein the psychotic disorder results from or is associated with Alzheimer's disease.

[0319] A method of treating a psychotic disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the psychotic disorder is dementia with Lewy bodies.

[0320] A method of treating a psychotic disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the psychotic disorder is schizophrenia.

[0321] A method of treating a psychotic disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the psychotic disorder is bipolar disorder.

[0322] A method of treating a psychotic disorder in a subject (e.g., a human, e.g., a mammalian patient such as a human in need of such treatment), comprising administering a therapeutically effective dose of a compound of the invention, wherein the psychotic disorder is Alzheimer's disease.

[0323] Use of a compound of the present invention for the manufacture of a medicament for the treatment of a psychotic disorder.

[0324] Use of a compound of the invention for the manufacture of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder comprises, arises from or is associated with a condition as defined above.

[0325] Use of a compound of the invention for the manufacture of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder includes, arises from or is associated with Alzheimer's disease.

[0326] Use of a compound of the invention for the manufacture of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder includes, arises from, or is associated with dementia with Lewy bodies.

[0327] Use of a compound of the invention for the manufacture of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder includes, arises from or is associated with schizophrenia.

[0328] Use of a compound of the invention for the manufacture of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder includes, arises from or is associated with bipolar disorder.

[0329] Use of a compound of the invention for the manufacture of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder includes, arises from or is associated with Alzheimer's psychosis.

[0330] Compounds for treating or reducing the severity of acute, chronic, neuropathic or inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, general neuralgia, visceral pain, osteoarthritis pain, postherpetic neuralgia, diabetic neuropathy, radicular pain, sciatica, lower back pain, head or neck pain, severe or intractable pain, nociceptive pain, breakthrough pain, post-operative pain or cancer pain.

[0331] A method for treating or lessening the severity of acute, chronic, neuropathic or inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, general neuralgia, visceral pain, osteoarthritic pain, postherpetic neuralgia, diabetic neuropathy, radicular pain, sciatica, lower back pain, head or neck pain, severe or intractable pain, nociceptive pain, breakthrough pain, post-operative pain or cancer pain, comprising the administration of a therapeutically effective dose of a compound of the invention.

[0332] Compounds for the treatment of peripheral disorders such as lowering intraocular pressure in glaucoma, and for the treatment of dry eye and dry mouth, including Sjogren's syndrome.

[0333] A method for the treatment of peripheral disorders such as lowering intraocular pressure in glaucoma, and for the treatment of dry eye and dry mouth, including Sjogren's syndrome, comprising the administration of a therapeutically effective dose of a compound of the present invention.

[0334] Use of a compound of the invention for the manufacture of a medicament for treating or reducing the severity of acute, chronic, neurological or inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, general neuralgia, visceral pain, osteoarthritis pain, postherpetic neuralgia, diabetic neuropathy, radicular pain, sciatica, lower back pain, head or neck pain, severe or intractable pain, nociceptive pain, breakthrough pain, post-operative pain or cancer pain, or treating peripheral disorders such as reducing intraocular pressure in glaucoma, and for treating dry eye and dry mouth, including Sjogren's syndrome.

[0335] For example, use of the compounds of the present invention to treat skin lesions due to pemphigus vulgaris, dermatitis herpetiformis, pemphigoid and other blistering skin conditions.

[0336] Use of the compounds of the present invention to treat, prevent, ameliorate or reverse conditions associated with altered gastrointestinal function and motility, such as functional dyspepsia, irritable bowel syndrome, gastroesophageal acid reflux (GER) and esophageal motility disorders, symptoms of gastroparesis and chronic diarrhea.

[0337] Use of the compounds of the present invention to treat olfactory dysfunction such as Bosmer-Henkin-Christiansen syndrome, chemical poisoning (e.g., selenium and silver), hypopituitarism, Kallmann syndrome, skull fractures, tumor treatment, and hypothyroidism.

[0338] Use of the compounds of the present invention to treat addiction.

[0339] Use of the compounds of the present invention to treat movement disorders such as Parkinson's disease, ADHD, Huntington's disease, Tourette's syndrome, and other syndromes associated with dopaminergic dysfunction as the underlying pathogenic factor-driven disease.

[0340] Use of the compounds of the invention to treat behavioral and psychological symptoms of dementia (BPSD; including agitation, verbal aggression, physical aggression, depression, anxiety, abnormal motor behavior, elevated mood, irritability, apathy, disinhibition, impulsivity, delusions, hallucinations, sleep changes and appetite changes). Methods for preparing the compounds of the present invention

[0341] The compounds of the present invention can be prepared in accordance with synthetic methods known to those skilled in the art and methods described herein. The compounds can also be prepared in accordance with the methods described in WO2015 / 118342. Once formed, a compound of the present invention, or a protected derivative thereof, can be converted into another compound of the present invention by methods well known to those skilled in the art. For example, a compound of the present invention can be converted into one salt form or another salt form of a compound of the present invention. Examples of such synthetic procedures are described in standard textbooks such as "Advanced Organic Chemistry and Organic Syntheses" (see references above) or "Fiesers' Reagents for Organic Synthesis," Volumes 1-17, John Wiley, edited by Mary Fieser (ISBN: 0-471-58283-2).

[0342] It may be necessary to protect one or more groups to prevent reactions from occurring at undesirable locations on the molecule. Examples of protecting groups and methods for protecting and deprotecting functional groups can be found in Protective Groups in Organic Synthesis (T. Greene and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).

[0343] The compounds made by the methods described above can be isolated and purified by any of a variety of methods well known to those skilled in the art, examples of which include recrystallization and chromatographic techniques such as column chromatography (e.g., flash chromatography) and HPLC.

[0344] As used herein, the term "reacting" is used as known in the art and generally refers to bringing together chemical reagents in a manner that allows them to interact at a molecular level to achieve a chemical or physical transformation. In some embodiments, the reaction involves two reagents, and one or more equivalents of the second reagent are used relative to the first reagent. The reaction steps of the methods described herein can be carried out for a time and under conditions suitable to prepare the identified product.

[0345] The reactions of the methods described herein can be carried out in a suitable solvent, which can be easily selected by those skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, which can range, for example, from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or in a mixture of more than one solvent. A suitable solvent for a particular reaction step can be selected depending on the particular reaction step.

[0346] Suitable solvents can include halogenated solvents such as carbon tetrachloride, bromodichloromethane, dibromochloromethane, bromoform, chloroform, bromochloromethane, dibromomethane, butyl chloride, dichloromethane, tetrachloroethylene, trichloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethane, 2-chloropropane, 1,2-dichloroethane, 1,2-dibromoethane, hexafluorobenzene, 1,2,4-trichlorobenzene, 1,2-dichlorobenzene, chlorobenzene, fluorobenzene, mixtures thereof, and the like.

[0347] Suitable ether solvents include dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether, anisole, t-butyl methyl ether, mixtures thereof, and the like.

[0348] Suitable protic solvents can include, by way of example and without limitation, water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butyl alcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenol, or glycerol.

[0349] Suitable aprotic solvents can include, by way of example and without limitation, tetrahydrofuran (THF), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP), formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethyl sulfoxide, propionitrile, ethyl formate, methyl acetate, hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate, sulfolane, N,N-dimethylpropionamide, tetramethylurea, nitromethane, nitrobenzene, or hexamethylphosphoramide.

[0350] Suitable hydrocarbon solvents include benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o- or p-xylene, octane, indane, nonane or naphthalene.

[0351] Suitable aqueous buffer solvents include phosphate buffer, tris buffer, barbital buffer, BES (N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer and MOPS (3-(N-morpholino)propanesulfonic acid) buffer.

[0352] The reactions of the methods described herein can be carried out at a suitable temperature, which can be easily determined by those skilled in the art. The reaction temperature depends, for example, on the melting and boiling points of the reagents and solvents, if any; the thermodynamics of the reaction (e.g., a highly exothermic reaction may need to be carried out at a low temperature); the kinetics of the reaction (e.g., a high activation energy barrier may require a high temperature); and the temperature range above which any enzyme components are active / denatured.

[0353] The expressions "ambient temperature" and "room temperature" or "rt", as used herein, are understood in the art and generally refer to a temperature, e.g., a reaction temperature, which is about the temperature of the room in which the reaction is carried out, e.g., a temperature of about 20°C to about 30°C.

[0354] The reactions of the processes described herein can be carried out in air or under an inert atmosphere. Typically, reactions involving reagents or products that are substantially reactive with air can be carried out using air-sensitive synthetic techniques well known to those skilled in the art. Ketoreductase enzymes

[0355] Ketoreductases (KREDs, also called "alcohol dehydrogenases," ADHs, or "carbonyl reductases") catalyze the reduction of aldehydes and ketones to the corresponding primary and secondary alcohols, respectively.

[0356] The reduction catalyzed by KREDs requires a reduced cofactor as the electron donor: some KREDs use reduced nicotinamide adenine dinucleotide (NADH) as a cofactor, others use reduced nicotinamide adenine dinucleotide phosphate (NADPH), and some ketoreductases accept both NADH and NADPH.

[0357] The use of KRED in vitro in the reduction process is + to NADPH, or NAD + A cofactor regeneration system is required to regenerate NADH from ATP. Common cofactor regeneration systems are glucose dehydrogenase (GDH), which takes glucose as a feedstock, or formate dehydrogenase, which takes formate as a feedstock. These cofactor regeneration systems can be used in conjunction with KRED.

[0358] KREDs are ubiquitous enzymes found in all kingdoms of life. Known commercially available KREDs are derived from horse liver (HLADH), baker's yeast (YADH), and from bacteria such as Thermoanaerobium brockii (TBADH) and Lactobacillus kefir (LKADH).

[0359] For industrial applications, it is desirable to use KREDs with high specific activity and stereoselectivity. Another important criterion for the industrial use of KREDs is long-term process stability, which often correlates with high stability at high temperatures and high solvent stability. It may also be desirable to use KREDs with high stereospecificity.

[0360] Enzyme A described herein and used in the methods described herein is SEQ ID NO:1: [ka] It is a ketoreductase enzyme (KRED) having the amino acid sequence:

[0361] Enzyme A can be produced by methods known in the art. For example, methods for expressing enzymes in cellular (e.g., microbial) expression systems are well known and routine to those skilled in the art.

[0362] Enzyme A can be provided by or produced from a host cell (e.g., a microbial cell such as E. coli) that contains a polynucleotide and / or expression vector encoding Enzyme A. The host cell can be TOP10 E. coli. Enzyme A can be produced using the methods disclosed in the Examples.

[0363] The polynucleotide may be DNA or RNA. The polynucleotide may be single-stranded or double-stranded. The polynucleotide may be provided in isolated / purified form or may be provided within a host cell.

[0364] Because the codons corresponding to various amino acids are known, the availability of a polypeptide sequence provides a description of all polynucleotides capable of encoding the polypeptide of interest. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the creation of a large number of nucleic acids, all of which encode the disclosed enzymes. Thus, once a particular amino acid sequence is identified, one skilled in the art can create any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates all possible variations of polynucleotides that can be created by selecting combinations based on possible codon choices.

[0365] Enzyme A is SEQ ID NO:2: [ka] It can be encoded by a polynucleotide and / or expression vector comprising a nucleotide sequence according to

[0366] The polynucleotide can consist of SEQ ID NO:2.

[0367] The polynucleotide can be operably linked to one or more heterologous regulatory or control sequences that control gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered ketoreductase can be introduced into a suitable host cell to express the corresponding ketoreductase. The polynucleotide encoding the ketoreductase enzyme can be codon-optimized for optimal production from the host organism selected for expression. For example, preferred codons used in bacteria are used to express genes in bacteria, preferred codons used in yeast are used to express in yeast, and preferred codons used in mammals are used to express in mammalian cells.

[0368] The polynucleotides encoding the disclosed enzymes can be manipulated in various ways to express the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide before its insertion into the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel, F. ed., Greene Pub. Associates, 1998, updated to 2006.

[0369] The control sequence may be an appropriate promoter sequence, which may be obtained from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (Villa-Kamaroff et al., 1978, Proc. Natl Acad. Sci. USA 75: 3727-3731) and the tac promoter (DeBoer et al., (1983, Proc. Natl. Acad. Sci. USA 80:21-25). Additional promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and Sambrook et al. (supra).

[0370] The control sequence may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide that directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of a nucleic acid sequence may naturally contain a signal peptide coding region linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. A foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.

[0371] Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region to enhance secretion of the polypeptide, although any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used.

[0372] Any suitable vector, promoter, enhancer and stop codon known in the art can be used to express the polypeptide from the vector described herein. The vector can be a plasmid, a phage, a MAC, a virus, etc. The plasmid can be a pET28a plasmid (see Nature 2020; https: / / doi.org / 10.1038 / s42003-020-0939-8).

[0373] Polynucleotides and / or expression vectors can be synthesized by conventional DNA synthesis techniques. If the sequence of the engineered polypeptide is known, polynucleotides encoding the enzyme can be prepared by standard solid-phase methods in accordance with known synthesis methods. Fragments of up to about 100 bases can be synthesized separately and then linked (e.g., by enzymatic or chemical ligation, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the disclosed polynucleotides can be prepared by chemical synthesis using, for example, the classical phosphoramidite method described by Beaucage et al., 1981, Tet Lett 22:1859-69, or the method described by Matthes et al., 1984, EMBO J. 3:801-05, which is typically performed in automated synthesis. Oligonucleotides can be synthesized in accordance with the phosphoramidite method, for example, in an automated DNA synthesizer, purified, annealed, ligated, and cloned into an appropriate vector. Additionally, essentially any nucleic acid can be obtained from any of a variety of commercial suppliers, such as The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc. Chicago, IL, Operon Technologies Inc., Alameda, CA, and many others. Pharmaceutical preparations

[0374] While it is possible for the active compound to be administered alone, it is preferable to supply it as a pharmaceutical composition (eg, formulation).

[0375] Thus, there is provided a pharmaceutical composition comprising at least one compound of the present invention together with at least one pharmaceutically acceptable excipient.

[0376] The composition may be a tablet composition. The composition may be a capsule composition.

[0377] Pharmaceutically acceptable excipients can be selected from, for example, carriers (e.g., solid, liquid, or semi-solid carriers), adjuvants, diluents (e.g., solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and co-solvents), granulating agents, binders, flow aids, coating agents, release-controlling agents (e.g., release-suppressing or retarding polymers, or waxes), binding agents, disintegrants, buffering agents, lubricants, preservatives, antifungal and antibacterial agents, antioxidants, buffering agents, tonicity adjusting agents, thickeners, flavoring agents, sweetening agents, pigments, plasticizers, taste-masking agents, stabilizers, or any other additives conventionally used in pharmaceutical compositions.

[0378] The term "pharmaceutically acceptable," as used herein, means compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, and that are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Each excipient must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0379] Pharmaceutical compositions containing the compounds of the present invention can be formulated according to known techniques, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

[0380] The pharmaceutical composition can be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, sublingual, ocular, otic, rectal, vaginal, or transdermal administration. Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers, or patches such as buccal patches. Tablet compositions can contain a unit dose of the active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, for example, lactose, sucrose, sorbitol, or mannitol; and / or a non-sugar-derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or its derivatives, such as microcrystalline cellulose (MCC), methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and starch, such as corn starch. Tablets may also contain such standard ingredients as binders and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g., swellable cross-linked polymers such as cross-linked carboxymethylcellulose), lubricants (e.g., stearates), preservatives (e.g., parabens), antioxidants (e.g., BHT), buffering agents (e.g., phosphate or citrate buffers), and effervescent agents such as citrate / bicarbonate mixtures. Such additives are well known and need not be discussed in detail here. Tablets may be designed to release drug upon contact with gastric fluids (immediate-release tablets) or to release over an extended period of time or in a controlled manner upon contact with specific regions of the GI gastrointestinal tract (controlled-release tablets).

[0381] The pharmaceutical compositions typically contain approximately 1% (w / w) to approximately 95%, preferably % (w / w), of the active ingredient and 99% (w / w) to 5% (w / w) of a pharmaceutically acceptable additive (e.g., as defined above), or a combination of such additives. Preferably, the compositions contain approximately 20% (w / w) to approximately 90% (w / w) of the active ingredient and 80% (w / w) to 10% of a pharmaceutical additive, or a combination of additives. The pharmaceutical compositions contain approximately 1% to approximately 95%, preferably approximately 20% to approximately 90%, of the active ingredient. Pharmaceutical compositions according to the present invention may be in unit dose form, such as in the form of ampoules, vials, suppositories, pre-filled syringes, dragees, powders, tablets, or capsules.

[0382] Tablets and capsules may contain, for example, 0-20% disintegrant, 0-5% lubricant, 0-5% flow aid, and / or 0-99% (w / w) filler or bulking agent (depending on drug dose). Tablets and capsules may contain 0-10% (w / w) polymer binder, 0-5% (w / w) antioxidant, and 0-5% (w / w) pigment. In addition, slow-release tablets typically contain 0-99% (w / w) release-controlling (e.g., retarding) polymer (depending on dose). Film coatings for tablets or capsules typically contain 0-10% (w / w) polymer, 0-3% (w / w) pigment, and / or 0-2% (w / w) plasticizer.

[0383] Parenteral formulations typically contain 0-20% (w / w) buffer, 0-50% (w / w) cosolvent, and / or 0-99% (w / w) water for injection (WFI) (depending on dose, if lyophilized). Formulations for intramuscular depots may also contain 0-99% (w / w) oil.

[0384] The pharmaceutical formulations may be supplied to the patient in a "patient pack" containing an entire course of treatment in a single package, usually a blister pack.

[0385] The compounds of the present invention are generally supplied in unit dosage form and, thus, typically contain sufficient compound to achieve a desired level of biological activity. For example, a formulation may contain 1 nanogram to 2 grams of active ingredient, e.g., 1 nanogram to 2 milligrams of active ingredient. Within these ranges, particular subranges of compound are 0.1 milligram to 2 grams of active ingredient (more generally, 10 milligrams to 1 gram, e.g., 50 milligrams to 500 milligrams) or 1 microgram to 20 milligrams (e.g., 1 microgram to 10 milligrams, e.g., 0.1 milligram to 2 milligrams of active ingredient).

[0386] For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more typically from 10 milligrams to 1 gram, for example, 50 milligrams to 1 gram, for example, 100 milligrams to 1 gram of active compound.

[0387] The active compound is administered to a patient (e.g., a human or animal patient) in need thereof in an amount sufficient to achieve the desired therapeutic effect (an effective amount). The exact amount of compound to be administered can be determined by a supervising physician according to standard procedures. [Example]

[0388] The present invention will now be exemplified by reference to certain embodiments and methods of synthesis described in the following non-limiting example compounds: The biological activity of one isomer of ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate has been demonstrated in WO2015 / 118342. General Methods and Materials

[0389] The following general methods and materials are illustrative of the methods and materials used during the processes described herein below. NMR

[0390] NMR analyses were performed on a Bruker 400 Avance III NMR spectrometer. X-ray powder diffraction (XRPD):

[0391] XRPD analysis was performed on a Rigaku Miniflex 600 powder X-ray diffractometer, serial number BD70000351-01. For analysis, approximately 0.5–1 mg of sample was added to a PXRD zero-background sample holder. A sheet of weighing paper was used to gently compress the powder, and the sample holder was placed in the sample changer. Run parameters were: Miniflex counter detector, Kb filter (x2), scan axis theta / 2theta, continuous mode, start (degrees) 2.0, end (degrees) 45.0, step (degrees) 0.020, speed (degrees / min) 10.0, spin on, voltage (kV) 40, current (mA) 15.

[0392] It is understood that peak intensities can vary from diffractogram to diffractogram for the same crystalline form based on any number of factors known to those skilled in the art, such as preferred orientation effects, preparation techniques, sample mounting procedures, and the equipment used. In some cases, peak intensities can vary rather dramatically. Therefore, the diffraction peak intensities shown herein are exemplary, and identical diffraction peak intensities are not necessarily required. One skilled in the art can easily compare the diffractograms presented herein with diffractograms generated for unknown crystalline forms to determine whether the diffractograms characterize the same crystalline form as presented herein, or a different form. thermal analysis

[0393] Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) analyses were performed on a TA Instruments Discovery 2500 calorimeter (DSC) with serial number 2500-00547 and a Discovery 5500 (TGA) with serial number 5500-0126.

[0394] For TGA analysis, standard aluminum sample pans were placed into platinum TGA pans and blanks were tared in the instrument. Approximately 1-5 mg of sample was added to the standard aluminum pan and analyzed at 10°C / min up to 400°C.

[0395] Regarding DSC analysis, T ゼロ Bread and T ゼロ The weight of the lid was obtained and recorded. Approximately 1 to 3 mg of sample was ゼロ Weigh into a pan and ゼロ The lid was pressed down. The pan was transferred to the DSC autosampler for analysis. The analysis method was a 10°C / min ramp to 225°C. A reference pan was prepared in the absence of the sample using the same procedure.

[0396] For DSC and TGA, the temperatures observed for thermal events depend on sample purity and may also depend on the rate of temperature change and sample preparation technique and the instrument used. Thus, values reported herein for DSC thermograms may vary by plus or minus 3°C. For example, "about 176°C" means 176°C ± 3°C. Values reported herein for DSC thermograms may also vary by plus or minus 10 joules per gram (i.e., ± 10 joules / gram). Hygroscopic

[0397] Dynamic vapor sorption (DVS) analysis was performed on a Discovery SA-0180 vapor sorption analyzer (DVS) with serial number 958000.901. For DVS analysis, approximately 3-10 mg of sample was placed in a quartz crucible. The crucible was then transferred to the sorption analyzer for drying at 60 °C for approximately 1 hour, and then analyzed at 25 °C from 0% RH to 90% RH with a 0.01% stabilization standard in 10% RH steps within 240 minutes. Microscopy Polarized Light Microscopy (PLM) and Scanning Electron Microscopy (SEM):

[0398] Imaging was performed with a Leica DM LM / P microscope equipped with a DMC2900 camera. For analysis, approximately 1 mg of sample was added to a microscope slide. The slide was then placed in the microscope and polarized light images were captured.

[0399] Scanning electron microscopy (SEM) was used to evaluate the geometry and particle size. Images were taken using a Phenom ProX scanning electron microscope with serial number MVE052018-0480-S. The sample was attached to an area of an SEM mount using carbon conductive double-coated tape. The mount was tapped to remove excess particles and then sputter-coated with gold using a Cressington 108. Imaging was performed at 5-15 kV using an SED detector.

[0400] Chiral RP-HPLC method: System: Shimadzu LC-40 Column: Phenomenex Lux i-Amylose3 250 x 4.6 mm 5 μm Mobile phase: Water and acetonitrile mixture containing 0.1% phosphoric acid (60 / 40 water / acetonitrile). Flow rate: 1mL / min. Column temperature: 40°C. Run time: 15 minutes. Detection: A refractive index detector (RID) was used.

[0401] Retention time: [Table 2]

[0402] Sample preparation: 0.5 mL of reaction sample was added with 0.5 mL of acetonitrile and mixed until homogeneous. The sample was placed in an ultrasonic bath for 5 minutes and then centrifuged at 12,000 x g for 5 minutes. Aliquots of the prepared sample were transferred to glass vials and analyzed using chiral RP-HPLC methods.

[0403] Calculation of diastereomeric excess: Diastereomeric excess (%de) was calculated using the following formula:

number

[0404] A photometric assay was used to screen a range of KRED enzymes for their utility in converting formula (B) to formula (A). 288 diverse KRED enzymes were provided as dried enzyme formulations in 96-well microtiter plates. The enzymes were dissolved in appropriate buffer solutions to produce the clear solutions required for photometric measurements and screening. Photometric measurements recorded a decrease in absorbance at 340 nm, corresponding to the enzyme's consumption of the NADPH cofactor in the reduction reaction.

[0405] The collected data was analyzed to select enzymes that either showed a negative slope over the 10 minute reaction time (the lowest value was selected first) or a rapid decline over 10 minutes with a total absolute absorbance below 0.7. A low initial absorbance is indicative of a very active enzyme, which consumes cofactor faster than the technician can transfer the MTP to the MTP reader.

[0406] Those enzymes that met the selection criteria were subjected to 0.5 mL biocatalysis reactions and validation by HPLC using chiral RP-HPLC methods.

[0407] Biocatalysis conditions: Ketones 20g / L (88mM) NADP 5mM DMSO 5% (v / v) 2mM MgCl2 Glucose 178mM (2 equivalents) GDH (glucose dehydrogenase, CDX-901, Codexis Inc.) 1g / L Potassium phosphate buffer 100mM, pH 7.0 0.5mL, 30℃, 1000rpm shaking, 24h reaction time

[0408] As shown in Table 1 below, Enzyme A significantly outperformed the next best enzymes (Enzymes B-F) in terms of conversion of the starting material, while maintaining the highest observed stereoselectivity (diastereomeric excess) for the trans-alcohol (Eq. (A)). [Table 1-1]

[0409] Generation of synthetic DNA encoding enzyme A A synthetic DNA having SEQ ID NO:2 and encoding Enzyme A was synthesized with codon optimization for E. coli expression.

[0410] SEQ ID NO: 2 [ka] Enzyme A production

[0411] The synthetic DNA (SEQ ID NO:2) and pET28a were digested with NdeI and XhoI restriction endonucleases, purified, and ligated together using T4 DNA ligase. The resulting recombinant molecule was then transformed into E. coli strain TOP10, and the desired expression construct was verified by Sanger DNA sequencing. After transforming the sequence-verified plasmid DNA into the E. coli protein expression host BL21(DE3), a single colony was used to inoculate 10 mL of LBP growth medium (supplemented with 10 g / L plant peptone, 10 g / L NaCl, and 5 g / L yeast extract, 35 μg / mL kanamycin) and grown at 37°C for 16 hours with shaking at 200 rpm. This starter culture was then used to inoculate 1 L of TB growth medium (supplemented with 12 g / L plant peptone, 12 g / L glycerol, 9.96 g / L disodium hydrogen phosphate, 20.4 g / L sodium dihydrogen phosphate, and 35 μg / mL kanamycin) in a 2 L baffled glass Erlenmeyer flask, which was incubated at 28° C. for 16 hours to allow expression of Enzyme A. The culture was then transferred to two 500 mL centrifuge pots (Nalgene®) and centrifuged at 6000 g / 4° C. in an SLA3000 rotor using a Sorvall RC-5C centrifuge. The bacterial pellet was then resuspended in the pot with 5 times the pellet weight of 50 mM sodium phosphate buffer (pH 7.5). After thorough resuspension, the bacterial suspension was transferred to a 100 mL glass beaker for cell disruption, which was performed using an MSE Soniprep 150 sonicator equipped with a 9.5 mm ultrasonic probe for a total of 8 minutes (4 x 2 minute intervals at 16 microns, with a 4 minute incubation time between cycles) at 0°C. The disrupted cell suspension was then transferred to a 50 mL (Nalgene®) centrifuge pot and centrifuged at 13,000 x g for 45 minutes in a Fiberlite™ F13-1 X60cy rotor. After centrifugation, the supernatant (approximately 40 mL) was carefully transferred to a 100 mL plastic container and placed at -20°C for 16 hours.The frozen Enzyme A solution was then freeze-dried by lyophilization (Edwards SuperModulo) for 2 days, maintaining a pressure of less than 1 mbar throughout. The dried enzyme cake was thoroughly comminuted by crushing with a steel spatula into a free-flowing homogenous powder and stored at -20°C between uses. Preparation of tert-butyl trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate [ka]

[0412] 1 mg of enzyme A was placed in a 2 mL reaction tube. The tube was filled with 5 mM nicotinamide adenine dinucleotide phosphate (NADP) in 100 mM potassium phosphate buffer. + A 0.5 mL reaction mix consisting of 100 mM ethanol, 2 mM magnesium chloride hexahydrate, 178 mM glucose anhydrous, 1 g / L glucose dehydrogenase (CDX-901, Codexis Inc.), 5% (v / v) dimethyl sulfoxide (DMSO), and 20 g / L tert-butyl 2-oxo-6-azaspiro[3.4]octane-6-carboxylate was added. The reaction tube was shaken at 1000 rpm at 30 °C for 24 h. The reaction was analyzed using chiral HPLC, which showed a conversion ranging from 98.7% to 100% and a diastereomeric excess of 97% relative to trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate. Synthesis method

[0413] The compounds of Examples 1 to 6 were prepared according to the following methods. [ka]

[0414] Synthesis Example 1: Synthesis of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate

[0415] Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate can be prepared according to the following scheme: Using the above method, approximately 5 kg (free base equivalent) of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate was prepared. [ka] [ka]

[0416] Process description: Step 0 1. Charge EtOH (7 volumes) to the reactor. 2. Charge pyrazolepiperidine dihydrochloride (1 equivalent) to the reactor. 3. Adjust the temperature to 10-15°C. 4. Charge 33% aqueous NaOH (1 volume) to the reactor and maintain the temperature at <30°C. 5. Stir the contents for 2-3 hours at 20-25°C. 6. Filter the contents of the reactor. 7. EtOH (2 volumes) is charged to the reactor and used to rinse the cake. 8. The combined filtrates are recharged to the reactor and concentrated in vacuo at 40-45°C to approximately 5 volumes. 9. Charge toluene (5 volumes) to the reactor and reduce under vacuum to approximately 4-5 volumes at 40-45°C. 10. Charge toluene (5 volumes) to the reactor and reduce under vacuum to approximately 4-5 volumes at 40-45°C. 11. Charge toluene (5 volumes) to the reactor and reduce under vacuum to approximately 4-5 volumes at 40-45°C. 12. Charge toluene (5 volumes) to the reactor. 13. Filter the contents of the reactor to clean the receiving flask. 14. Toluene (2 volumes) is charged to the reactor and used to rinse the cake. 15. Recharge the combined filtrate into the clean reactor and reduce in vacuo to a minimum volume at 40-45°C.

[0417] Step 1 1. Charge DCM (10 volumes) to the reactor. 2. Charge the spiro alcohol (1 equivalent) to the reactor. 3. Charge pyridine (3 equivalents) to the reactor. 4. Adjust the internal temperature to 20-25°C. 5. Charge tosyl anhydride (1.5 eq) to the reactor and maintain a temperature of <35° C. Aim for a temperature of approximately 30° C. during the addition. 6. Stir the contents for at least 2 hours at 20-25°C. 8. Quench the reaction with 10% citric acid solution (5 volumes), ensuring that the temperature does not exceed SOX [mild exotherm]. 9. Stir for 15 minutes and let settle for 15 minutes. 10. Drain the lower organic layer into a holding vessel [containing the product].

[0418] 11. Drain the upper aqueous layer into a holding vessel. 12. Recharge the organic layer from point 10 into the reactor. 13. Charge 10% citric acid solution (5 volumes) to the reactor. 14. Stir for 15 minutes and let settle for 15 minutes. 15. Drain the lower organic layer into a holding vessel [containing the product]. 16. Drain the upper aqueous layer into a holding vessel. 17. Recharge the organic layer from point 15 into the reactor. 18. Add saturated NaHCO3 solution (5 volumes) to the reactor. [Caution: CO2 evolution] 19. Stir for 15 minutes and let settle for 15 minutes. 20. Drain the lower organic layer into a holding vessel [containing the product].

[0419] 21. Drain the upper aqueous layer into a holding vessel. 22. The organic layer from point 20 is recharged into the reactor. 23. Add saturated NaHCO3 solution (5 volumes) to the reactor. [Caution: CO2 generation] 24. Stir for 15 minutes and let settle for 15 minutes. 25. Drain the lower organic layer into a holding vessel [containing the product]. 26. Drain the upper aqueous layer into a holding vessel. 27. The organic portion from point 25 [orange / brown solution] was recharged to the reactor and reduced in vacuo to approximately 5 volumes at 35-40°C. 28. Cyclohexane (5 volumes) was charged to the reactor and reduced in vacuo to approximately 5 volumes at 35-40°C. 29. Cyclohexane (5 volumes) was charged to the reactor and reduced in vacuo to approximately 5 volumes at 35-40°C. 30. Cyclohexane (5 volumes) was charged to the reactor and reduced in vacuo to approximately 5 volumes at 35-40°C.

[0420] 31. Chemist Check 1: DCM content by NMR. Target: <0.2% w / w remaining relative to cyclohexane peak. If fail, repeat cyclohexane strip. 32. Charge cyclohexane (5 volumes) to the reactor and adjust the reactor contents to 10-15°C at 6°C / hr and hold for 1-2 hours. 33. Chemist's Verification 2: Confirmation of Crystallization. Objective: Recrystallize the product. 34. Filter the contents of the reactor. 35. Cyclohexane (2 volumes) is charged to the reactor and used to rinse the cake. 36. Cyclohexane (2 volumes) is charged to the reactor and used to rinse the cake. 37. Transfer the solid to an oven and dry at 35-40°C under vacuum with a nitrogen bleed for a minimum of 12 hours.

[0421] Step 2 1. Chemist's Verification 1: Strip Gravimetric Assay of Piperidine Compounds in Toluene Solution. 2. Charge the reactor with a toluene solution of piperidine compound (4 equivalents based on the result in point 1). 3. Concentrate under vacuum at 40-45°C to approximately 4-5 volumes. 4. Charge NMP (2.5 vol) to the reactor. 5. Concentrate under vacuum at 40-45°C to approximately 5-7 volumes. 6. Charge the spiro alcohol tosylate (1 equivalent) to the reactor. 7. Adjust the contents to 97-102°C and stir under distillation conditions for at least 36 hours under a vacuum of 180-200 mbar [orange solution]. 8. IPC1: Reaction complete by HPLC. Target <3% area of residual spiro alcohol tosylate ester. 9. Adjust the contents to 20-25°C. 10. Charge EtOAc (4 volumes) to the reactor.

[0422] 11. Charge water (4 volumes) to the reactor and maintain a temperature of <25°C [slight exotherm]. 12. Stir for 15 minutes and let settle for 15 minutes. 13. Drain the lower aqueous layer into a holding vessel. 14. Drain the upper organic layer into a holding vessel. [Contains product] 15. Recharge the aqueous layer from point 13 into the reactor. 16. Charge EtOAc (4 volumes) to the reactor. 17. Stir for 15 minutes and let settle for 15 minutes. 18. Drain the lower aqueous layer into a holding vessel. 19. Drain the upper organic layer into a holding vessel. [Contains product] 20. Recharge the aqueous layer from point 18 into the reactor.

[0423] 21. Charge EtOAc (4 volumes) to the reactor. 22. Stir for 15 minutes and let settle for 15 minutes. 23. Drain the lower aqueous layer into a holding vessel. 24. Drain the upper organic layer into a holding vessel. [Contains product] 25. Recharge the aqueous layer from point 23 into the reactor. 26. Charge EtOAc (4 volumes) to the reactor. 27. Stir for 15 minutes and let settle for 15 minutes. 28. Drain the lower aqueous layer into a holding vessel. 29. Recharge the organic layers from points 14, 19 and 24 into the reactor. 30. Charge water (5 volumes) to the reactor.

[0424] 31. Stir for 15 minutes and let settle for 15 minutes. 32. Drain the lower aqueous layer into a holding vessel. 33. Charge water (5 volumes) to the reactor. 34. Stir for 15 minutes and let settle for 15 minutes. 35. Drain the lower aqueous layer into a holding vessel. 36. Charge 2M NaCl (5 volumes) to the reactor. 37. Stir for 15 minutes and let settle for 15 minutes. 38. Drain the lower aqueous layer into a holding vessel. 39. Charge 2M NaCl (5 volumes) to the reactor. 40. Stir for 15 minutes and let settle for 15 minutes.

[0425] 41. Drain the lower aqueous layer into a holding vessel. 42. Charge 5% citric acid solution (5 volumes) into the reactor. 43. Stir for 15 minutes and let settle for 15 minutes. 44. Drain the lower aqueous layer into a holding vessel [containing the product]. 45. Charge 5% citric acid solution (5 volumes) into the reactor. 46. Stir for 15 minutes and let settle for 15 minutes. 47. Drain the lower aqueous layer into a holding vessel [containing the product]. 48. Drain the upper organic layer into a holding vessel. 49. Recharge the aqueous layers from points 44 and 47 into the reactor. 50. Carefully charge sodium carbonate (1 wt) to the reactor [slight exotherm - easy to control with rate of addition] and maintain temperature <30°C until pH >8. [Caution: CO2 gas evolution, foaming]

[0426] 51. Chemist Check 2: Check for basic pH. Goal: pH > 8. If fail, add more sodium carbonate. 52. Charge EtOAc (5 volumes) to the reactor. 53. Stir for 15 minutes and allow to settle for 15 minutes. 54. Drain the lower aqueous layer into a holding vessel. 55. Drain the organic layer into a holding vessel. [Contains product] 56. Recharge the aqueous layer from point 54 into the reactor. 57. Charge EtOAc (5 volumes) to the reactor. 58. Stir for 15 minutes and let settle for 15 minutes. 59. Drain the lower aqueous layer into a holding vessel. 60. Drain the organic layer into a holding vessel. [Contains product]

[0427] 61. Recharge the aqueous layer from point 59 into the reactor. 62. Charge EtOAc (5 volumes) to the reactor. 63. Stir for 15 minutes and allow to settle for 15 minutes. 64. Drain the lower aqueous layer into a holding vessel. 65. Recharge the organic fractions from points 55 and 60 into the reactor. 66. Concentrate under vacuum at 35-40°C to approximately 4-5 volumes. 67. Cyclohexane (5 volumes) is charged to a reactor and reduced under vacuum to approximately 4-5 volumes at 35-40°C. 68. Cyclohexane (5 volumes) is charged to a reactor and reduced under vacuum to approximately 4-5 volumes at 35-40°C. 69. Cyclohexane (5 volumes) is charged to a reactor and reduced under vacuum to approximately 4-5 volumes at 35-40°C. 70. Chemist Check 3: EtOAc content by NMR. Target: 1% w / w. If fail, repeat cyclohexane strip.

[0428] 71. Charge cyclohexane (5 volumes) to the reactor and adjust the reactor contents to 35-40°C, then cool at 6°C / hr to 10-15°C and hold for 1-2 hours. 72. Chemist Identification 4: Identifying Crystallization. Objective: Observe the suspension. 73. Filter the contents of the reactor. 74. Cyclohexane (2 volumes) is charged to the reactor and used to rinse the cake. 75. Cyclohexane (2 volumes) is charged to the reactor and used to rinse the cake. 76. Transfer the solid to an oven and dry at 35-40°C under vacuum with a nitrogen bleed for a minimum of 12 hours.

[0429] (Step 3 was not performed due to process improvement)

[0430] Step 4 1. Charge ethanol (5 volumes) to the reactor. 2. Adjust the reactor contents to -5 to 0°C. 3. Charge acetyl chloride (6 eq.) to the reactor and maintain a temperature of <15°C [colorless solution]. 4. Charge the Boc-product of Step 2 (1 equivalent) to the reactor [eventually forming an orange solution, then a beige suspension]. 5. Adjust the reactor contents to 20-25°C and stir for at least 18 hours. 6. IPC1: Reaction complete by HPLC. Target <0.5% residual SM. 7. Charge TBME (10 vol) to the reactor over at least 15 minutes [agitation may need to be increased briefly during addition]. 8. Agitate the reactor contents for 1 hour ± 15 minutes. 9. Filter the suspension. The solid is hygroscopic - filter under a stream of nitrogen, do not suck dry. 10. Charge TBME (5 volumes) to the reactor and use to wash the filter cake. Filter under a stream of nitrogen and do not suck dry. 11. Charge TBME (5 volumes) to the reactor and use to wash the filter cake. Filter under a stream of nitrogen, do not suck dry. Immediately transfer to the oven. 12. Transfer to an oven and dry at 35-40°C under vacuum with a nitrogen bleed for a minimum of 12 hours.

[0431] Step 5 1. Charge dichloromethane (8 volumes) to the reactor. 2. Charge triethylamine (4.5 equivalents) to the reactor. 3. Adjust the reactor contents to -5 to 0°C. 4. Charge the product of Step 4 (1 equiv.) in small portions to the reactor, maintaining the temperature at <10° C. [beige suspension]. 5. Charge ethyl chloroformate (1.5 equiv.) to the reactor, maintaining the temperature at <10° C. [large exotherm, quickly dissipates, beige suspension]. 6. Adjust the contents to 20-25°C and stir for at least 2 hours. 7. IPC1: Reaction complete by HPLC. Aim for <0.5% residual SM. If not, stir for an additional 2 hours. 8. Slowly charge water (3 volumes) and maintain temperature at <25°C. [A clear biphasic layer will form. Minimal exotherm will occur.] 9. Stir for 15 minutes and let settle for 15 minutes. 10. Drain the lower organic layer into a holding vessel [containing the product].

[0432] 11. Drain the upper aqueous layer into a holding vessel. 12. Recharge the organic layer from point 10 into the reactor. 13. Charge 2M NaCl (4 volumes) to the reactor. 14. Stir for 15 minutes and let settle for 15 minutes. 15. Drain the lower organic layer into a holding vessel [containing the product]. 16. Drain the upper aqueous layer into a holding vessel. 17. Charge the organic layer from point 15 to a clean reactor and concentrate in vacuo at 40-45°C to approximately 4 volumes. 18. Charge EtOH (5 volumes) to the reactor and concentrate in vacuo at 40-45°C to approximately 4 volumes. 19. Charge EtOH (5 volumes) to the reactor and concentrate in vacuo at 40-45°C to approximately 4 volumes. 20. Chemist Check 1: DCM content by NMR. Target is <0.2% w / w DCM with respect to ethanol. If fail, repeat ethanol strip.

[0433] 21. Chemist's Verification 2: Use a strip gravimetric assay of the ethanol solution to determine the amount of product present, and therefore in the next step charge citric acid, ethanol, water and citrate seed crystals. 22. Adjust the reaction to 8 volumes (based on the results of Chemist's Check 2) of 5% water in ethanol. 23. Charge citric acid (1 equivalent based on the results of Chemist's Check 2) to the reactor. 24. Adjust the contents to 20-25°C and stir for 30 minutes to 1 hour [a beige suspension will form]. 25. Adjust the contents to 55-60°C and stir for at least 1 hour [usually dissolves at 50-55°C]. 26. Chemist Check 3: Dissolution. Goal: Formation of solution [orange solution]. If fail, add 5% water in ethanol in 0.25 volume increments and hold for 30 minutes until dissolution is achieved. 27. Using a heated hose, polish filter the solution into a clean reactor [Caution: precipitates at 40°C]. 28. Adjust the contents to 55-60°C and stir for at least 1 hour. 29. Adjust the contents to 40-45°C over 3 hours and seed with 0.5% w / w product citrate. 30. Stir at 40-45°C for 2 hours.

[0434] 31. Adjust contents to 0-5°C over at least 12 hours [beige suspension]. 32. Chemist Identification 4: Precipitation. Objective: Formation of a suspension. 33. IRC2: Form by XRD. Target: Form 2. If fail, seed with a further portion of 0.5% w / w Form 2 of the citrate salt, stir for at least 18 hours and resample for IRC2. 34. Filter the suspension. 35. Charge ethanol (2 volumes) to the reactor. 36. Adjust the temperature to 0-5°C and use it to wash the cake. 37. Charge ethanol (2 volumes) to the reactor. 38. Adjust the temperature to 0-5°C and use it to wash the cake. 39. TBME (5 vol) is charged to the reactor and used to wash the cake. 40. TBME (5 vol) is charged to the reactor and used to wash the cake. 41. IPC3: Wet cake purity by HPLC. Target: >97.0% area. RRT 0.56 < 0.29%, RRT 0.89 < 0.56%, RRT 0.92 < 0.37%, RRT 0.95 < 3.6%, RRT 1.23 < 0.20%. All other individual impurities < 0.15%. 42. Transfer the solid to a vacuum oven and dry at 20-25°C for at least 8 hours and up to 114 hours.

[0435] Synthesis Example 2: Alternative Synthesis of Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate Citrate Monohydrate [ka] Step 1. tert-Butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate [ka] tert-Butyl trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate is dissolved in dichloromethane. Pyridine is added, followed by the addition of tosyl anhydride in small portions. The reaction is stirred until complete, and the organic and aqueous layers are allowed to settle. The organic phase (containing tert-butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate) is washed sequentially with aqueous citric acid and aqueous sodium bicarbonate. The solution containing tert-butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate is solvent exchanged with cyclohexane. The organic layer is then cooled to precipitate the product, and the crystallized material is isolated by filtration. The cake is then washed with cyclohexane and dried.

[0436] Alternative route to tert-butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate [ka] tert-Butyl 2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate was dissolved in DCM (10 V) and pyridine (3 eq.) was added. The solution was stirred at room temperature for 15 min. Tosyl anhydride (1.5 eq.) was added and the reaction mixture was stirred at room temperature for 2 h. An additional portion of tosyl anhydride (0.12 eq.) was added and the reaction mixture was stirred at room temperature for 18 h. The reaction was quenched with 10% aqueous citric acid (5 V) and the organic and aqueous phases were allowed to settle. The organic phase was separated and washed with 10% aqueous citric acid (5 V) and a saturated solution of NaHCO3 (2 x 5 V). The organics were removed by distillation under vacuum to give the crude product.

[0437] The crude product was purified by flash column chromatography (silica gel) using 0-50% EtOAc / hexanes, monitoring the eluate at a wavelength of 254 nm. Once peaks were visible on the chromatogram, the flow rate was reduced by a factor of four, and two fractions corresponding to the cis and trans products were collected (upper and lower, designated by the relative positions of their corresponding spots on the thin-layer chromatography). The 1H NMR spectra of the products are shown in Figure 34 (trans-tosylate, "upper" fraction) and Figure 35 (cis-tosylate, "lower" fraction).

[0438] 1 H- 1 By H nuclear Overhauser effect spectroscopy (NOESY), the upper fraction was assigned to the desired trans isomer and the lower fraction to the undesired cis isomer. The NOESY spectra and assignments are shown in Figure 36 (trans-tosylate, "upper" fraction) and Figure 37 (cis-tosylate, "lower" fraction).

[0439] Step 2. tert-Butyl cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate [ka] 4-(1-methyl-1H-pyrazol-5-yl)piperidine dihydrochloride is suspended in ethanol, followed by the addition of aqueous sodium hydroxide. The suspension is stirred to destroy the HCl salt, and the mixture is then filtered. The cake is washed with ethanol, and the product solution is replaced with toluene. The toluene layer (containing 4-(1-methyl-1H-pyrazol-5-yl)piperidine free base) is then filtered and concentrated. NMP and tert-butyl (2s,4r)-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate are added to the toluene solution of 4-(1-methyl-1H-pyrazol-5-yl)piperidine free base. The solution is heated, and the remaining toluene is distilled under vacuum. The reaction is continued to heat until deemed complete. The product is then diluted with ethyl acetate, and the organics (containing tert-butyl cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate) are washed with water and brine. The product is extracted from the organic layer into a solution of citric acid. The pH of the aqueous layer (containing the product) is adjusted, and the product is extracted into ethyl acetate. The product is displaced into cyclohexane, cooled to induce crystallization, and filtered. The cake is washed with cyclohexane and dried to give tert-butyl cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate.

[0440] Step 3. cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride [ka] Acetyl chloride is stirred in cold ethanol to generate HCl in situ. To this solution is added tert-butyl cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate. The resulting suspension is stirred until deprotection is achieved. MTBE is added to the resulting suspension, and the solid is stirred and then collected by filtration. The cake is washed with MTBE and dried under vacuum to give cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane as the trihydrochloride salt.

[0441] Step 4. Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride [ka] Triethylamine is dissolved in dichloromethane and cooled. To this solution, cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride is added portionwise. The mixture is stirred and ethyl chloroformate is added. The mixture is heated and stirred until the reaction is complete. The organic layer (containing the product) is washed successively with water and a solution of sodium chloride. The product is replaced with ethanol, followed by the addition of anhydrous HCl. The resulting suspension is heated until dissolution occurs, then cooled to initiate crystallization. The solid is stirred, then isolated by filtration, washed with MTBE, and then dried.

[0442] Step 5. Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate [ka] A suspension of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl ester hydrochloride is formed in dichloromethane. The mixture is stirred and aqueous sodium bicarbonate is added. The mixture is stirred until all solids are dissolved. The organic layer (containing the product) is washed successively with water and a solution of sodium chloride. The product is replaced with ethanol, followed by the addition of citric acid and water. The resulting suspension is heated until dissolved and then cooled to initiate crystallization. The solid is stirred, then isolated by filtration, washed with MTBE, and then dried to give cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl ester citrate monohydrate.

[0443] Synthesis Example 3. Alternative Further Synthesis of Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate Citrate Monohydrate [ka] Step 1: tert-butyl cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate [ka] tert-Butyl 2-oxo-6-azaspiro[3.4]octane-6-carboxylate (75.0 g, 328 mmol), 4-(1-methyl-1H-pyrazol-5-yl)piperidine tartrate (132.1 g, 377.0 mmol), molecular sieves (124.0 g, 1041 mmol) (3 Å), citric acid (130.0 g, 676.6 mmol), and methanol (686.5 mL) were sequentially charged to a 1500 mL bomb. Pd (5.0 wt%) on BaSO4 (5:95, palladium:barium sulfate, 69.74 g, 32.77 mmol) (Aldrich, reduced form) was added, and hydrogen was applied to 994 psi. The reaction mixture was stirred at 23° C. for 24 h, then the temperature was raised to 35° C. for an additional 22 h. The hydrogen pressure was then vented and the catalyst was removed by filtration through a 1500 mL "M" frit. The filter cake was washed with MeOH (811.4 g).

[0444] The clear yellow filtrate was concentrated to a solution (538.9 g) by rotary evaporation. While maintaining the temperature below 35° C. with an ice bath, water (251.1 g) was added and the pH was adjusted to 13.64 with potassium hydroxide (256.8 g, 2060 mmol). The resulting milky mixture was concentrated (813 g) on a rotary evaporator, and 2-methoxy-2-methylpropane (600 g) was added. The phases were separated, and the organics were washed with water (250 g). The aqueous phase was subsequently back-extracted with 2-methoxy-2-methylpropane (223.0 g).

[0445] The combined organic phases were concentrated to an oil (188.6 g) on a rotary evaporator. Isopropyl alcohol (473.7 g) was added and concentrated to a yellow oil, from which the free base of the title compound (cis-2-(tert-butyl 4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate) precipitated after approximately 30 minutes at room temperature in a yield of 65.59 g. HPLC showed 16 area % of the undesired trans isomer, resulting in an 81.4% yield of the desired cis isomer. The title compound was used in the next step without further purification.

[0446] During process development, a variety of reduction conditions were found to provide moderate to fair selectivity to the desired cis isomer and moderate to excellent conversion of the starting material; see, for example, Table A below for selected conditions. [Table 3]

[0447] Step 2: cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl citrate [ka] At 22°C, isopropyl alcohol (1300.2 g, 21636 mmol) was added to the product of Step 1 to form a solution with a pH of 9.46. Freshly prepared 33.3% citric acid in ethanol (77.14 g, 133.7 mmol) was added at 24.2°C to give a yellow solution with a pH of 6.68. After 10 minutes, the solution was seeded with cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate tert-butyl citrate crystals (0.517 g). After an additional hour, the pH was 6.79, and 33.3% citric acid in ethanol (51.26 g, 88.84 mmol) was added dropwise. After an additional hour, the pH reached 6.45, and 33.3% citric acid in ethanol (48.0 g, 83.3 mmol) was added dropwise. After an additional 3 hours and 15 minutes, the pH reached 6.1, and crystals were isolated by vacuum filtration through a 3 L "C" frit. The isolated crystals were washed with isopropyl alcohol (229.5 g) and dried overnight under nitrogen pressure to give the title compound (cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl citrate) in 162.55 g yield (162.03 g, corrected for seed). HPLC analysis indicated 7.5 area % trans isomer. NMR indicated 4.11 wt % IPA and 1.00 equivalents of citric acid.

[0448] HPLC of the filtrate showed a trans:cis ratio of 68.2:31.8 (4.0% filtrate loss). LCMS (ES+): Found 375.4 [M+H + ](cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate tert-butyl); (ES-): Found 191.1 [MH - ](citric acid)

[0449] Step 3: cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate tert-butyl ester hydrochloride [ka] A 2000 mL round-bottom flask was charged with cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate tert-butyl citrate (161.34 g, 244.21 mmol; 7.4% trans isomer), 2-methoxy-2-methylpropane (556.8 g, 6317 mmol), and water (399.4 g, 22170 mmol), resulting in a biphasic solution. The pH of the resulting solution was adjusted from 3.44 to 13.49 with potassium hydroxide (106.8 g, 856.6 mmol). The phases were separated, and the organics were washed with water (103.9 g). The aqueous phase was subsequently back-extracted with 2-methoxy-2-methylpropane (196.0 g). During phase separation, a small amount of black interphase solid transferred to the water. The combined organics were concentrated to an oil (146.83 g), and isopropyl alcohol (840.5 g) was added. To this solution was added 22.31 wt% hydrogen chloride (15.20 g, 93.01 mmol; 5.53 M in isopropyl alcohol) to lower the pH from 10.41 to 6.32. The resulting solution was seeded with cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate tert-butyl hydrochloride (0.21 g), and a slurry immediately began to form. After 10 minutes, the pH was 6.45, and 22.31 wt% hydrogen chloride in isopropyl alcohol (14.6 g, 89.3 mmol) was added dropwise over 10 minutes. After an additional 15 minutes, the pH reached 6.2, and 22.31 wt% hydrogen chloride in isopropyl alcohol (15.0 g, 91.8 mmol) was added dropwise over 10 minutes, pausing the addition at a pH range of 1.0 to 4.0. The mixture was placed in an ice bath for 1 hour and 15 minutes to cool to 3.0°C. The slurry was then poured into a 3000 mL "C" frit and allowed to settle, followed by vacuum filtration.The filter cake was washed with isopropyl alcohol (300 mL) and then dried under nitrogen pressure for 2.5 days to give the title compound (cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate tert-butyl hydrochloride) in 95.55 g yield (95.34 g, corrected for seed content). HPLC analysis showed 97.10 wt % as the HCl salt, containing 0.20 area % as the undesired trans isomer. NMR showed 0.35 wt % isopropyl alcohol.

[0450] HPLC of the filtrate showed a cis:trans ratio of 43.4:56.6 (5.52% filtrate loss). LCMS (ES+): Found 375.4 [M+H + ]

[0451] Step 4: cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride [ka] A 2000 mL round-bottom flask was charged with ethanol (340.90 g) and placed in an ice-water bath. Acetyl chloride (92.53 g, 1179 mmol) was added over 15 minutes, maintaining the temperature below 30°C (a vigorous exotherm occurred during the addition of acetyl chloride). The ice-water bath was removed, and after 45 minutes at a reaction temperature of 20°C, tert-butyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride (93.43 g, 220.7 mmol; 0.20 area % trans) was added, rinsing in with ethanol (100.54 g). The mixture was stirred at room temperature (22.9°C) for 22 hours. The resulting slurry of white crystals indicated complete conversion of the starting material (<0.1% starting material by HPLC). 2-Methoxy-2-methylpropane (812 mL) was added in small portions over 35 minutes. The resulting mixture was stirred for 15 minutes. The slurry was poured into a 600 mL "C" frit and filtered to yield large white crystals. The filter cake was washed with 2-methoxy-2-methylpropane (84.46 g) and then dried under nitrogen pressure to afford the title compound (cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride) in 90.17 g yield (determined to be 93.96% pure, assuming 100% yield). HPLC indicated a purity of 99.74 area %. NMR indicated 0.84 wt % ethanol as the only residual solvent. LCMS (ES+): Found 275.4 [M+H + ]

[0452] Step 5. Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate [ka] A 2000 mL round-bottom flask was charged with cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane trihydrochloride (88.3 g, 216 mmol) and methylene chloride (858 g). The resulting slurry was cooled to 11° C. in an ice bath. Triethylamine (169 mL, 1210 mmol) was added over 6 minutes while maintaining the temperature below 21° C. The reaction mixture was brought to 16° C., and ethyl chloroformate (28.80 g, 265.4 mmol) was added over 7 minutes while maintaining the temperature below 28° C. After stirring for 10 minutes, HPLC analysis indicated the reaction was complete. Water (150.0 g) was added, and the mixture was stirred at 17° C. for 15 minutes, and then the phases were separated. Water (150 g) was added to the organic phase, and the pH was adjusted from 8.33 to 12.2 with sodium hydroxide (17.7 g). The phases were separated, and the organics were washed with water (150 g). The three aqueous fractions were sequentially back-extracted with methylene chloride (250 mL). Note that sodium hydroxide (4.50 g, 56.2 mmol) was added to the sodium hydroxide wash fraction to adjust the pH from 9.6 to 12.81. The combined organic fractions were concentrated to an oil (113.3 g), and after adding ethanol (157.5 g), the mixture was again concentrated to an oil (98.3 g). Ethanol (884.3 g) was again added, followed by water (30.67 g, 1702 mmol) and citric acid (44.90 g, 233.7 mmol). The resulting solution was seeded with crystals of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate (0.35 g) and stirred at room temperature overnight, resulting in a slurry or large crystals. The slurry was placed in an ice-water bath, and the mixture was cooled to 1–2°C for 2 hours and 15 minutes. The crystals were isolated by vacuum filtration through a 600 mL "C" frit and then washed with a mixture of ethanol (123.5 g, 2681 mmol) and water (8.2 g, 460 mmol) at 0°C, followed by 2-methoxy-2-methylpropane (167 g, 1890 mmol) at 0°C.The product was dried under nitrogen pressure overnight to give the title compound (cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate) in 110.24 g yield (109.89 g, corrected for seed). HPLC showed 99.7 area % of the target (210 nm). The trans isomer was less than 0.05%.

[0453] HPLC analysis of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate standard shows 101.3 wt%. Karl Fischer titration shows 3.31 wt% water (theory = 3.24 wt% for monohydrate). NMR shows 0.10 wt% ethanol as the only residual solvent.

[0454] HPLC shows 8.5% product in the filtrate and washings.

[0455] The NMR spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate produced by this method is shown in Figures 30 and 31. LCMS (ES+): Found 347.3 [M+H + ] (cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl); (ES-): Found 191.1 [MH - ](citric acid)

[0456] Synthesis Example 4: Synthesis of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate Ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate can be prepared according to the following scheme: Using the process described, 91 g of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate was prepared.

[0457] The bold numbers in the processes described below correspond to the intermediate compounds depicted in the schemes below. Main chain: [ka] Side chain: [ka]

[0458] Step 1 A RB flask was charged with Compound-1 (190 g, 1.0 equiv.) and DCM (10 mL) at 25-30°C under nitrogen, followed by pyridine (3.0 equiv.). The reaction mixture was stirred at room temperature for 10 minutes, and (Ts)O (1.5 equiv.) was added while controlling the temperature below 30°C. The reaction mixture was stirred at room temperature for 1.5 hours. HPLC analysis confirmed the reaction mixture.

[0459] Post-processing: The reaction mixture was quenched with 10% citric acid (5V) and stirred for 15-20 minutes. The aqueous layer was separated and the DCM layer was washed with 10% citric acid (2 x 5V) followed by saturated NaHCO3 solution (2 x 5V). The organic layer was concentrated under vacuum below 45 °C to constant weight to give 305.0 g of crude product.

[0460] purification: The crude material was subjected to silica gel column chromatography (by using 710.0 g of 60-120 mesh silica gel for absorbing the crude material, a column bed packed with 3.0 Kg of 60-120 mesh silica gel with n-hexane, and the column eluted with 12% ethyl acetate in n-hexane) to give 150.0 g of compound-2 in 47% yield. 1 H NMR (400 MHz, DMSO): δ ppm 1.35 (s, 9 H), 1.77 (m, 2 H), 2.00 (m, 2 H), 2.21 (m, 2 H), 2.45 (s, 3 H), 3.13 (m, 4 H), 4.90 (m, 1 H), 7.48 (m, 2 H), 7.80 (m, 2 H).

[0461] Step 2 An RB flask was charged with NMP (2.5 V) and Compound-2 (100.0 g, 1.0 equiv.) under nitrogen. The reaction mixture was then stirred for 30 minutes until a clear solution was formed. Compound-3 (piperidone ethylene ketal) (4.0 equiv.) was added to the RB flask over 10 minutes, and the reaction mixture was then heated to 95-105°C over 1 hour and maintained at the same temperature for 32 hours under nitrogen. The reaction mixture was monitored by HPLC.

[0462] Post-processing: The reaction mixture was cooled to 40-45°C over 1 hour, and ethyl acetate (5V) was added to bring the reaction mixture to room temperature. Water (5V) was added to the reaction mixture by controlling the exotherm below 30-35°C and stirred for 30 minutes. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 x 5V). The combined organic layers were washed with water (2 x 5V) and brine (2 x 5V). The organic layer was extracted with 5% citric acid (3 x 5V). The combined citric acid layers were cooled to 0-5°C and basified with solid Na2CO3 to pH 8-9. The product from the aqueous layer was then extracted with ethyl acetate (3 x 5V). The combined ethyl acetate layers were concentrated under vacuum to 1V at below 45°C and stripped to constant weight with n-heptane (2 x 5V). Crude weight: 84.0g

[0463] purification: The crude material (84.0 g) and n-heptane (5 V) were charged into an RB flask and heated to 80-90 °C for 1 h to obtain a clear solution. The reaction mixture was maintained at 80-90 °C for 30 min, cooled to room temperature, and the solid suspension was filtered, washing the bed with n-heptane (5 V). The solid was dried under vacuum below 45 °C to constant weight, affording 64.0 g of compound-4 in 69% yield. 1 H NMR (400 MHz, DMSO): δ ppm 1.44 (s, 9 H), 1.57 (m, 4 H), 1.79 (m, 4 H), 1.97 (m, 2 H), 2.27 (br, 4 H), 2.68 (m, 1 H), 3.12 (m, 2 H), 3.25 (m, 2 H), 3.82 (s, 4 H).

[0464] Step 3 Ethanol (7V) was charged to an RB flask and cooled to -5 to 0°C over 1 hour. Then, acetyl chloride (6.0 equiv.) was added under nitrogen for 10 minutes by controlling the exotherm below 20°C (a clear solution was observed after the addition was complete). Compound-4 (60.0 g, 1.0 equiv.) was added in small portions (5 lots) to the RB flask within 10 minutes, maintaining the temperature below 20°C, and a solid suspension was observed (a pale yellow to white suspension). The reaction mixture was allowed to reach room temperature and maintained at room temperature for 16 hours. The reaction mixture was monitored using TLC. (TLC system: 10% MeOH in DCM, Compound-4 at 0.5 RF and Compound-5 at 0.2 RF)

[0465] Post-processing: MTBE (10 V) was added to the RB flask and stirred for 1 h. The resulting solid was filtered under N2 atmosphere, the bed was washed with MTBE (5 V), and the solid was then dried under vacuum at 35-40 °C to constant weight to give 52.3 g of compound-5 in 94% yield. 1 H NMR (400 MHz, DMSO): δ ppm 1.81 (m, 4 H), 2.10 (m, 2 H), 2.20 (m, 2 H), 2.50 (m, 2 H), 2.75 (m, 2 H), 3.09 (m, 4 H), 3.30 (m, 2 H), 3.42 (m, 1 H), 3.72 (m, 1 H), 3.93 (s, 4 H), 9.24 (s, 2 H), 11.66 (br, 1H).

[0466] Step 4 A RB flask was charged with DCM (8V) and triethylamine (3.5 equiv.) and cooled to 0-5°C under nitrogen. Compound-5 (50.0 g, 1.0 equiv.) was then added over 5 min, maintaining the temperature at 0-5°C (a slurry was observed). Ethyl chloroformate (1.5 equiv.) was then added dropwise to the reaction mixture within 10 min (a slight exotherm was observed, and the temperature was maintained below 20°C), resulting in a solid suspension. The reaction mixture was stirred for 2-3 h at room temperature. HPLC was used to monitor the reaction mixture.

[0467] Post-processing: Water (3 V) was added and stirred for 30 min, the aqueous layer was separated, and the DCM layer was washed with brine solution (2 × 5 V). The organic layer was concentrated under vacuum below 45 °C and exchanged with n-heptane (2 × 5 V) to constant weight to give 44.4 g of Compound-6 in 89% yield. 1 H NMR (400 MHz, DMSO): δ ppm 1.17 (m, 3 H), 1.59 (m, 4 H), 1.82 (m, 4 H), 2.15 (m, 2 H), 2.30 (br, 4 H), 2.70 (br, 1 H), 3.15 (s, 2 H), 3.33 (m, 2 H), 3.97 (m, 4 H), 4.02 (m, 2 H).

[0468] Step 5 In an RB flask, compound-6 (50.0 g, 1.0 equiv.) and concentrated HCl (20 V) in DCM (10 V) were charged, and the reaction mixture was stirred at room temperature for 4-5 h. The reaction was monitored by HPLC. The DCM layer was separated, and the pH of the aqueous layer was adjusted to 8-9 using solid Na2CO3 at below 15-20 °C, and the reaction mixture was extracted with DCM (2 × 10 V). The combined DCM layers were distilled under vacuum at below 45 °C until constant weight.

[0469] To the crude material was added DCM (10V) followed by concentrated HCl (20V) and the resulting biphasic layer was stirred at room temperature for 4-5 hours. The reaction was monitored by HPLC.

[0470] The DCM layer was separated, the pH of the aqueous layer was adjusted to 8-9 using solid Na2CO3 below 15-20°C, and the reaction mixture was extracted with DCM (2 x 10V). The combined DCM layers were distilled under vacuum below 45°C until constant weight. Keto intermediate weight: 40.0g

[0471] The keto intermediate (40.0 g) was taken in a RBF, and the W211a intermediate (1.3 equiv.) and acetic acid (3.0 equiv.) in DCM (15 V) were added. The reaction mixture was stirred under nitrogen at room temperature for 24 h. The mixture was cooled to 0-5 °C over 1 h, and NaBH(OAc) (2.0 equiv.) was added in small portions (5-6 lots) by controlling the exotherm to less than 10-15 °C. The reaction mixture was allowed to warm to room temperature over 1 h and maintained at room temperature for 4 h. The reaction mixture was monitored by TLC. (TLC system: 50% acetone in hexane: keto intermediate at 0.8 RF and product at 0.2 RF)

[0472] Post-processing: The reaction mixture was quenched with 5% citric acid solution (5V), and the DCM layer was extracted with 5% citric acid solution (3 x 5V). The combined citric acid layers were adjusted to pH 8-9 using solid Na2CO3, charged with DCM (10V), stirred for 10 minutes, and filtered through Celite. The filtrate was transferred to a separatory funnel, and the DCM layer was separated. This aqueous layer was extracted with DCM (4 x 5V). The combined DCM layers were washed with water (4 x 10V), and the solvent was concentrated under vacuum to constant weight below 45 °C to give a gum. Weight of gum: 52.0g

[0473] The crude material (52.0 g) was dissolved in DCM (10 V) and charcoal (0.2×) was added at room temperature. The reaction mixture was stirred for 1 h. The reaction mixture was filtered through Celite and the bed was washed with DCM (5 V). This charcoal treatment was repeated three more times on the filtrate, and the final filtrate was distilled under vacuum and exchanged with n-heptane (2×5 V) at below 45° C. until constant weight was obtained, yielding 48.0 g of crude product.

[0474] purification: The crude material from the two batches, 48.0 g and 75.0 g, was combined in n-heptane (10 V) and heated to 50° C. for 1 hour and maintained at the same temperature for 1 hour. The reaction mixture was cooled to room temperature over 1 hour and stirred at room temperature for 1 hour. The solid was collected by filtration and washed with n-heptane (5 V). The solid was dried under vacuum at less than 45° C. to a constant weight. This n-heptane slurry process was repeated two more times to give 91.0 g of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate ethyl product as an off-white solid (59% overall yield for two steps). 1H NMR(400 MHz, DMSO): δ ppm 1.15 (m, 3 H), 1.30 (m, 2 H), 1.65 (m, 4 H), 1.80 (m, 3 H), 2.00 (m, 2 H), 2.09 (m, 2 H), 2.29 (m, 1 H), 2.35 (m, 1 H), 2.62 (m, 2 H), 2.79 (m, 2 H), 2.97 (m, 1 H), 3.03 (m, 4 H), 3.27 (m, 3 H), 3.445 (m, 1 H), 4.01 (m, 2 H), 4.60 (m, 1 H).

[0475] Side chain Step 6: In an RB flask under nitrogen, compound-7 (190.0 g, 1.0 equiv.) was taken up in DCM (10 V) and cooled to -78 °C. DAST (2.5 equiv.) was added dropwise at -78 °C over 20-25 min (exotherm controlled below -70 °C). After addition, the reaction mixture was allowed to warm to room temperature over 2 h and stirred at room temperature for 16 h. The progress of the reaction was monitored by HPLC.

[0476] Post-processing: After completion of the reaction, the reaction mixture was cooled to 0-5°C over 30 min, diluted with DCM (5 V), and quenched by slowly adding a saturated solution of sodium bicarbonate (35 V) dropwise at 0-5°C until the pH reached 7-8 (by controlling the temperature below 10°C). The reaction mixture was stirred to room temperature over 1 h, stirred at room temperature for 30 min, the two layers were separated, and then the aqueous layer was extracted with DCM (2 x 10 V). The combined organic layers were washed with water (10 V). The solvent was concentrated under vacuum below 45°C to constant weight, affording 201.5 g of compound-8 in 97% yield. 1 HNMR (400 MHz, CDCl3); δ ppm 1.30 (m, 9 H), 2.50 (m, 1 H), 2.76 (m, 1 H), 3.85 (m, 5 H). 4.49 (m, 1 H).

[0477] Step 7 Compound-8 (201.0 g, 1.0 equiv.) and THF (5 V) were added to an RB flask under nitrogen, followed by cooling to 0-5°C over 30 min. LiBH4 (2.0 equiv.) was then added in small portions (3 lots) over 20 min at 0-5°C (the exotherm was controlled below 5°C). The reaction mixture was stirred at 0-5°C for 2.5 h. The reaction was monitored by HPLC.

[0478] Post-processing: After completion of the reaction, the reaction mixture was diluted with DCM (5 V) and quenched with saturated sodium bicarbonate (10 V) solution over 1 h by controlling the exotherm below 15 °C. The two layers were separated, and the aqueous layer was extracted with DCM (2 × 10 V). The combined organic layers were washed with water (10 V) and then brine (10 V). The solvent was concentrated under vacuum below 45 °C to constant weight to give 178.5 g of crude Compound-8 in 99% yield. 1 HNMR (400 MHz, DMSO): δ ppm 1.42 (s, 9 H), 2.33 (m, 1 H), 3.50 (m, 3 H), 3.60 (m, 1 H), 3.84 (m, 1 H), 4.98(s, 1 H).

[0479] Step 8 In an RB flask under nitrogen, Compound-9 (178.0 g, 1.0 equiv.) and 1,4-dioxane (3 V) were added and stirred for 10 minutes. The reaction mixture was cooled to 10-15°C over 1 hour, and 4M HCl in 1,4-dioxane (5 V) was added dropwise over 40 minutes at 10-15°C. The reaction mixture was stirred to room temperature and continued stirring at room temperature for 16 hours. The progress of the reaction was monitored by TLC. (TLC system: 50% EtOAc in hexane: Compound-9 in 0.5 RF, W221a intermediate in 0.1 RF).

[0480] Post-processing: After completion of the reaction mixture, the solvent was concentrated under vacuum under nitrogen at below 45°C and exchanged with acetone (2 x 3V) to give a residue. Acetone (4V) was added to the residue and stirred at room temperature for 30 minutes. The resulting solid was collected by filtration and washed with acetone (2V) under nitrogen. The solid was dried under vacuum at below 45°C to a constant weight to give 110.0 g of the W211a intermediate in 84% yield. 1 HNMR (400 MHz, DMSO): δ ppm 2.33 (m, 1 H), 2.53 (m, 1 H), 3.60 (m, 4 H), 3.90 (m, 1 H), 5.43 (br, 1 H), 9.97 (br, 2 H).

[0481] Synthesis Example 5: Synthesis of ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate Ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate can be prepared according to the following scheme: Using the method described, approximately 1.4 kg of ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate was prepared. [ka] [ka]

[0482] Step 1 1. Charge DCM (10 volumes) to the reactor. 2. Charge spiro alcohol (1 equivalent) to the reactor [light yellow solution]. 3. Charge pyridine (3 equivalents) to the reactor. 4. Adjust the internal temperature to 20-25°C. 5. Charge tosyl anhydride (1.5 eq) to the reactor [moderate exotherm, forming an orange / brown solution], maintaining the temperature at ≦35° C. Aim for a temperature of approximately 30° C. during the addition. 6. Stir the contents for at least 2 hours at 20-25°C. 7. IPC1: Reaction completion by HPLC. Target is ≦0.5% area of residual spiro alcohol. 8. Quench the reaction with 10% citric acid solution (5 volumes), ensuring the temperature does not exceed 30°C [mild exotherm]. 9. Stir for 15 minutes and let settle for 15 minutes. 10. Drain the lower organic layer into a holding vessel [containing the product].

[0483] 11. Drain the upper aqueous layer into a holding vessel. 12. Recharge the organic layer from point 10 into the reactor. 13. Charge 10% citric acid solution (5 volumes) to the reactor. 14. Stir for 15 minutes and let settle for 15 minutes. 15. Drain the lower organic layer into a holding vessel [containing the product]. 16. Drain the upper aqueous layer into a holding vessel. 17. Recharge the organic layer from point 15 into the reactor. 18. Add saturated NaHCO3 solution (5 volumes) to the reactor. [Caution: CO2 evolution] 19. Stir for 15 minutes and let settle for 15 minutes. 20. Drain the lower organic layer into a holding vessel [containing the product].

[0484] 21. Drain the upper aqueous layer into a holding vessel. 22. The organic layer from point 20 is recharged into the reactor. 23. Add saturated NaHCO3 solution (5 volumes) to the reactor. [Caution: CO2 generation] 24. Stir for 15 minutes and let settle for 15 minutes. 25. Drain the lower organic layer into a holding vessel [containing the product]. 26. Drain the upper aqueous layer into a holding vessel. 27. The organic portion from point 25 [orange / brown solution] was concentrated in vacuo on a rotovap at 35-40° C. to remove DCM. 28. The flask was charged with toluene (2 volumes) and concentrated in vacuo on a rotovap at 35-40°C. 29. The flask was charged with toluene (2 volumes) and concentrated in vacuo on a rotovap at 35-40° C. Concentration was continued for at least 1 hour after solvent distillation ceased to ensure optimal drying [beige / yellow solid]. 30. Chemist Check 1: DCM content by NMR. Target: ≦0.1% w / w residual. If fail, repeat toluene strip. 31. Transfer to an oven and dry at 35-40°C under vacuum with a nitrogen bleed for a minimum of 12 hours. Break the material thoroughly using a suitable unused scoop and continue drying until a constant weight is achieved (net weight difference of <1.0% between successive drying runs separated by a minimum of 1 hour). If dryness is not achieved within 48 hours, contact the project chemist for advice.

[0485] Step 2 1. Charge NMP (2.5 vol) to the reactor. 2. Charge the spiro alcohol tosylate (1 equivalent) into the reactor. 3. Charge piperidone ethylene ketal (4 equivalents) to the reactor. 4. Adjust the contents to 97-102°C and stir for at least 36 hours [orange solution]. 5. IPC1: Reaction completion by HPLC. Target is ≤3% area of residual spiro alcohol tosylate. 6. Adjust the contents to 20-25°C. 7. Charge EtOAc (4 volumes) to the reactor. 8. Charge water (4 volumes) to the reactor and maintain a temperature of ≦25° C. [slight exotherm]. 9. Stir for 15 minutes and let settle for 15 minutes. 10. Drain the lower aqueous layer into a holding vessel.

[0486] 11. Drain the upper organic layer into a holding vessel [containing product]. 12. Recharge the aqueous layer from point 10 into the reactor. 13. Charge EtOAc (4 volumes) to the reactor. 14. Stir for 15 minutes and let settle for 15 minutes. 15. Drain the lower aqueous layer into a holding vessel. 16. Drain the upper organic layer into a holding vessel. [Contains product] 17. Recharge the aqueous layer from point 15 into the reactor. 18. Charge EtOAc (4 volumes) to the reactor. 19. Stir for 15 minutes and let settle for 15 minutes. 20. Drain the lower aqueous layer into a holding vessel.

[0487] 21. Drain the upper organic layer into a holding vessel. [Contains product] 22. Recharge the aqueous layer from point 20 into the reactor. 23. Charge EtOAc (4 volumes) to the reactor. 24. Stir for 15 minutes and let settle for 15 minutes. 25. Drain the lower aqueous layer into a holding vessel. 26. Drain the upper organic layer into a holding vessel. [Contains product] 27. Recharge the organic layers from points 11, 16, 21 and 26 into the reactor. 28. Charge water (5 volumes) to the reactor. 29. Stir for 15 minutes and let settle for 15 minutes. 30. Drain the lower aqueous layer into a holding vessel.

[0488] 31. Charge water (5 volumes) to the reactor. 32. Stir for 15 minutes and let settle for 15 minutes. 33. Drain the lower aqueous layer into a holding vessel. 34. Charge 2M NaCl (5 volumes) to the reactor. 35. Stir for 15 minutes and let settle for 15 minutes. 36. Drain the lower aqueous layer into a holding vessel. 37. Charge 2M NaCl (5 volumes) to the reactor. 38. Stir for 15 minutes and let settle for 15 minutes. 39. Drain the lower aqueous layer into a holding vessel. 40. Charge 5% citric acid solution (5 volumes) into the reactor.

[0489] 41. Stir for 15 minutes and let settle for 15 minutes. 42. Drain the lower aqueous layer into a holding vessel [containing the product]. 43. Charge 5% citric acid solution (5 volumes) into the reactor. 44. Stir for 15 minutes and allow to settle for 15 minutes. 45. Drain the lower aqueous layer into a holding vessel. [Contains product] 46. Drain the upper organic layer into a holding vessel. 47. Recharge the aqueous layers from points 42 and 45 into the reactor. 48. Carefully charge sodium carbonate (0.67 wt) to the reactor until pH ≥ 8 and maintain temperature ≤ 30°C [slight exotherm - easily controlled by rate of addition] [Caution: CO2 gas evolution, foaming] 49. Chemist Check 1 - Verify basic pH. Goal: pH ≥ 8. 50. Charge EtOAc (5 volumes) to the reactor.

[0490] 51. Stir for 15 minutes and allow to settle for 15 minutes. 52. Drain the lower aqueous layer into a holding vessel. 53. Drain the organic layer into a holding vessel. [Contains product] 54. Recharge the aqueous layer from point 52 into the reactor. 55. Charge EtOAc (5 volumes) to the reactor. 56. Stir for 15 minutes and let settle for 15 minutes. 57. Drain the lower aqueous layer into a holding vessel. 58. Drain the organic layer into a holding vessel. [Contains product] 59. Recharge the aqueous layer from point 57 into the reactor. 60. Charge EtOAc (5 volumes) to the reactor.

[0491] 61. Stir for 15 minutes and allow to settle for 15 minutes. 62. Drain the lower aqueous layer into a holding vessel. 63. Drain the organic layer into a holding vessel. [Contains product] 64. Charge anhydrous MgSO4 (0.2 wt) to the holding vessel containing the organic layer from points 53, 58 and 63. 65. Stir the holding container until it becomes flocculent. 66. The suspension is filtered through a sinter to remove the MgSO4. 67. Wash the cake with EtOAc (2 volumes). 68. The combined filtrate was concentrated in vacuo on a rotovap at 35-40° C. to remove EtOAc. After solvent stopped distilling from the flask, drying was continued for at least 18 hours [yellow / beige solid]. 69. Continue drying until a constant weight is achieved (≤1.0% net weight difference between successive dryings separated by a minimum of 1 hour). If drying is not achieved within 48 hours, contact the project chemist for advice.

[0492] Step 3 1. Charge methanol (5 volumes) to the reactor. 2. Adjust the reactor contents to 0-5°C. 3. Charge acetyl chloride (1.01 eq) to the reactor and maintain the temperature at ≦20° C. [large exotherm, dissipates quickly]. 4. Stir the contents of the reactor for at least 15 minutes at 20-25°C. 5. Drain the solution from the reactor into a holding vessel [colorless solution]. 6. Charge methanol (5 volumes) to the reactor. 7. Charge the product of Step 2 (1 equivalent) to the reactor [yellow / orange solution]. 8. Adjust the reactor contents to 10-15°C. 9. Charge the preformed methanolic HCl solution from point 5 into the reactor over at least 15 minutes, maintaining the temperature at ≦25° C. [slight exotherm]. 10. Adjust the contents to 20-25°C and stir for at least 2 hours.

[0493] 11. Concentrate the reaction mixture in vacuo at 40-45°C to give a beige solid. 12. Charge IPA (5 vol) to an RBF and concentrate in vacuo at 40-45°C to give a beige solid. 13. Charge IPA (2 volumes) to the reactor. 14. Add the wet solid product from point 12. 15. Add IPA (1 volume) from the previous step to the rotovap flask as a rinse and transfer the suspension to the reactor. 16. Adjust the contents to 60-65°C and stir for at least 1 hour [beige suspension]. 17. Adjust the contents to 0-5°C (target 4°C) over at least 10 hours and stir at this temperature for at least 1 hour [beige suspension]. 18. Filter the suspension. The solid is slightly hygroscopic - suck dry under a stream of nitrogen for no more than 5 minutes. 19. Charge TBME (2 volumes) to the reactor and use to wash the cake. The solid is slightly hygroscopic - suck dry under a stream of nitrogen for no more than 5 minutes. 20. Charge TBME (2 volumes) to the reactor and use to wash the cake. The solid is slightly hygroscopic - suck dry under a stream of nitrogen for no more than 5 minutes. Store under a nitrogen blanket while awaiting the results of IPC1. 21. IPC1: Diastereomeric purity by HPLC. Target: ≥ 98% area 22. Transfer to an oven and dry at 35-40°C under vacuum with a nitrogen bleed for a minimum of 12 hours. Break the material thoroughly using an appropriate unused scoop and continue drying until a constant weight is achieved (≤1.0% net weight difference between successive drying runs separated by a minimum of 1 hour). If dryness is not achieved within 48 hours, contact the project chemist for advice.

[0494] Step 4 1. Charge ethanol (7 volumes) to the reactor. 2. Adjust the reactor contents to -5 to 0°C. 3. Charge acetyl chloride (5 eq) to the reactor and maintain the temperature at ≦20° C. [colorless solution]. 4. Charge the product of Step 3 (1 equivalent) to the reactor [orange solution]. 5. Adjust the reactor contents to 20-25°C and stir for at least 18 hours [orange solution]. 6. IPC1: Reaction completion by HPLC. Target is ≦0.5% residual SM. 7. The reaction mixture was transferred to a rotovap and concentrated in vacuo at 35-40°C. 8. Toluene (5 volumes) was charged to the flask and concentrated in vacuo on a rotovap at 35-40°C. Concentration was continued for at least 1 hour after solvent distillation ceased to ensure optimal drying. [Tan-colored solid] 9. Transfer the solid to an oven and dry at 35-40°C under vacuum with a nitrogen bleed for a minimum of 12 hours. Break the material thoroughly using an appropriate unused scoop and continue drying until a constant weight is achieved (a net weight difference of ≤1.0% between successive drying runs separated by a minimum of 1 hour). If dryness is not achieved within 48 hours, contact the project chemist for advice.

[0495] Step 5 1. Charge dichloromethane (8 volumes) to the reactor. 2. Charge triethylamine (3.5 equivalents) to the reactor. 3. Adjust the reactor contents to -5 to 0°C. 4. Charge the product of Step 4 (1 equiv.) in small portions to the reactor, maintaining the temperature at ≦20° C. [beige suspension]. 5. Charge ethyl chloroformate (1.5 equiv.) to the reactor and maintain the temperature at ≦20° C. [large exotherm, quickly dissipates. Beige suspension]. 6. Adjust the contents to 20-25°C and stir for at least 2 hours. 7. IPC1: Reaction complete by HPLC. Target is ≦0.5% residual SM. If not, stir for an additional 2 hours. 8. The reaction was quenched slowly by adding water (3 volumes) [forming a clear biphasic layer]. 9. Stir for 15 minutes and let settle for 15 minutes. 10. Drain the lower organic layer into a holding vessel [containing the product].

[0496] 11. Drain the upper aqueous layer into a holding vessel. 12. Recharge the organic layer from point 10 into the reactor. 13. Charge 2M NaCl (4 volumes) to the reactor. 14. Stir for 15 minutes and let settle for 15 minutes. 15. Drain the lower organic layer into a holding vessel [containing the product]. 16. Drain the upper aqueous layer into a holding vessel. 17. The organic layer from point 15 was concentrated in vacuo on a rotovap at 40-45° C. to remove DCM. 18. Transfer the solid to a vacuum oven and dry at 35-40°C for at least 8 hours and up to 64 hours. Break the material thoroughly using an appropriate unused scoop and continue drying until a constant weight is achieved (a net weight difference of ≤1.0% between successive drying runs separated by a minimum of 1 hour). If dryness is not achieved within 48 hours, contact the project chemist for advice.

[0497] Step 6 1. Charge 2M HCl (10 volumes) to the reactor. 2. Charge SM (1 equivalent) into the reactor [beige suspension]. 3. Adjust the reactor contents to 60-65°C and stir for 2-3 hours. 4. Adjust the contents to 20-25°C. 5. Chemist's confirmation 1: Reaction completion by HPLC. Objective: FIO. 6. Charge sodium carbonate (1.3 wt) to the reactor and maintain the temperature at ≦30° C. [slight exotherm, CO2 evolution controlled by addition rate]. 7. Charge dichloromethane (5 volumes). 8. Stir for 15 minutes and let settle for 15 minutes. 9. Drain the lower organic layer into a holding vessel [containing the product]. 10. Charge dichloromethane (5 volumes).

[0498] 11. Stir for 15 minutes and let settle for 15 minutes. 12. Drain the lower organic layer into a holding vessel [containing the product]. 13. Drain the upper aqueous layer into a holding vessel. 14. Recharge the organic layers from points 9 and 12 into the reactor. 15. Charge 2M HCl (5 volumes) to the reactor. 16. Stir for 15 minutes and let settle for 15 minutes. 17. Drain the lower organic layer into a holding vessel. 18. Drain the upper aqueous layer into a holding vessel [containing the product]. 19. Recharge the organic layer from point 17 into the reactor. 20. Charge 2M HCl (5 volumes) to the reactor.

[0499] 21. Stir for 15 minutes and let settle for 15 minutes. 22. Drain the lower organic layer into a holding vessel. 23. Drain the upper aqueous layer into a holding vessel [containing the product]. 24. Recharge the aqueous layers from points 18 and 23 into the reactor. 25. Adjust the reactor contents to 60-65°C and stir for 2-3 hours. 26. Adjust the contents to 20-25°C. 27. IPC1: Reaction completion by HPLC. Target: ≤0.5% SM remaining. 28. Charge sodium carbonate (1.3 wt) to the reactor and maintain the temperature at ≦30°C [slight exotherm, CO2 evolution controlled by addition rate]. 29. Charge dichloromethane (5 volumes). 30. Stir for 15 minutes and let settle for 15 minutes.

[0500] 31. Drain the lower organic layer into a holding vessel [containing the product]. 32. Charge dichloromethane (5 volumes). 33. Stir for 15 minutes and let settle for 15 minutes. 34. Drain the lower organic layer into a holding vessel [containing the product]. 35. Drain the upper aqueous layer into a holding vessel. 36. Charge anhydrous MgSO4 (0.2 wt) to the holding vessel containing the organic layer from points 31 and 34. 37. Stir the holding container until it becomes flocculent. 38. The suspension is filtered through a sinter to remove the MgSO4. 39. Wash the cake with dichloromethane (2 volumes). 40. Chemist's Check 2: Strip gravimetric assay of dichloromethane solution; determine amount of piperidone intermediate present and therefore subsequent inputs to this step.

[0501] 41. Charge ((2R,4R)-4-fluoropyrrolidin-2-yl)methanol hydrochloride (1.3 equivalents) to the reactor. 42. Acetic acid (3 equivalents) is charged to the reactor. 43. Adjust the reactor contents to 20-25°C and stir for at least 24 hours. 44. Charge sodium triacetoxyborohydride (2 equivalents) to the reactor and maintain the temperature at ≦30°C. 45. Adjust the reactor contents to 20-25°C and stir for at least 4 hours. 46. IPC2: Reaction complete by HPLC. Target: ≦1.0% piperidone remaining. If fail, loop in more sodium triacetoxyborohydride. 47. Charge 5% citric acid solution (5 volumes) into the reactor. 48. Stir for 15 minutes and let settle for 15 minutes. 49. Drain the lower organic layer into a holding vessel. 50. Drain the upper aqueous layer into a holding vessel [containing the product].

[0502] 51. Recharge the organic layer from point 49 into the reactor. 52. Charge 5% citric acid solution (5 volumes) into the reactor. 53. Stir for 15 minutes and allow to settle for 15 minutes. 54. Drain the lower organic layer into a holding vessel. 55. Drain the upper aqueous layer into a holding vessel [containing the product]. 56. Recharge the aqueous layers from points 50 and 55 into the reactor. 57. Charge sodium carbonate (2 wt) to the reactor and maintain the temperature at ≦30°C [slight exotherm, CO2 evolution controlled by addition rate]. 58. Charge dichloromethane (5 volumes). 59. Stir for 15 minutes and let settle for 15 minutes. 60. Drain the lower organic layer into a holding vessel [containing the product].

[0503] 61. Charge dichloromethane (5 volumes). 62. Stir for 15 minutes and let settle for 15 minutes. 63. Drain the lower organic layer into a holding vessel [containing the product]. 64. Drain the upper aqueous layer into a holding vessel. 65. Recharge the organic layers from points 60 and 63 into the reactor. 66. Charge purified water (5 volumes) to the reactor. 67. Stir for 15 minutes and allow to settle for 15 minutes. 68. Drain the lower organic layer into a holding vessel [containing the product]. 69. Drain the upper aqueous layer into a holding vessel. 70. Recharge the organic layer from point 68 into the reactor.

[0504] 71. Charge purified water (5 volumes) to the reactor. 72. Stir for 15 minutes and allow to settle for 15 minutes. 73. Drain the lower organic layer into a holding vessel [containing the product]. 74. Drain the upper aqueous layer into a holding vessel. 75. Chemist's Check 3: Removal of residual fluoroprolinol from organic layer. If unsuccessful, repeat water wash. 76. Charge anhydrous MgSO4 (0.2 wt) to the holding vessel containing the organic layer from point 73. 77. Stir the holding container until it becomes flocculent. 78. The suspension is filtered through a sinter to remove the MgSO4. 79. Wash the cake with dichloromethane (2 volumes). 80. The filtrate and washings from points 78 and 79 were concentrated in vacuo on a rotovap at 35-40°C to remove DCM. Concentration was continued for at least 1 hour after solvent distillation ceased to ensure optimal drying. The product was an oil that slowly crystallized on standing [beige / yellow solid].

[0505] Step 7 (Recrystallization / Trituration) 1. Charge TBME (11 volumes) to the reactor. 2. Charge heptane (5 volumes) to the reactor. 3. Charge the product of Step 6 (1 equivalent) to the reactor. 4. Adjust the contents to 50-60°C. 5. Chemist Check 1: Check for dissolution. Goal is to achieve dissolution. If fail, add the minimum amount of 11:5 TBME:heptane mixture needed to achieve dissolution. 6. Adjust the contents to 35-40°C and seed with 0.1% w / w Form C. 7. Adjust the contents to 20-25°C and hold for at least 12 hours. 8. Filter the suspension. 9. Charge 2:1 heptane:TBME (2 volumes) to the reactor and use to wash the cake. 10. Transfer the solid to a vacuum oven and dry at 35-40°C for at least 8 hours and up to 64 hours. Break the material thoroughly using an appropriate unused scoop and continue drying until a constant weight is achieved (a net weight difference of ≤1.0% between successive drying runs separated by a minimum of 1 hour). If dryness is not achieved within 48 hours, contact the project chemist for advice.

[0506] Step 0: Recrystallization of Fluoroprolinol HCl Reagent [ka] 1. Charge ethanol (8 volumes) to the reactor. 2. Charge Fluoroprolinol HCl (1 equivalent) to the reactor. 3. Adjust the contents to 75-80°C. 4. Chemist Check 1: Check for dissolution. The goal is to achieve dissolution. If not, add the minimum amount of ethanol necessary to achieve dissolution. 5. Adjust the contents to 20-25°C and hold for at least 12 hours. 6. Filter the suspension. 7. TBME (1 volume) is charged to the reactor and used to wash the cake. 8. Transfer the solid to a vacuum oven and dry at 35-40°C for at least 8 hours and up to 64 hours. Break the material thoroughly using an appropriate unused scoop and continue drying until a constant weight is achieved (a net weight difference of ≤1.0% between successive drying runs separated by a minimum of 1 hour). If dryness is not achieved within 48 hours, contact the project chemist for advice.

[0507] The product (cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate) 1 H-NMR is shown in Figure 24.

[0508] Synthesis Example 6: Synthesis of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate (alternative route to the citrate salt) Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate (citric acid salt) was prepared by precipitation from 2-butanol. Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (free base) (234.7 mg) and citric acid (130.6 mg, approximately 1 mol. equivalent) were placed in a 4 mL vial. 2-BuOH (3 volumes) was added, and the mixture was stirred and heated to 60°C for approximately 2 hours. The temperature was reduced by 5°C increments (approximately every 10 minutes) until ambient temperature was reached. The resulting solid was isolated by vacuum filtration. The solid was washed with MTBE (2 x ca. 5 volumes) and air-dried for ca. 1.5 hours before being transferred to a pre-weighed vial. The solid was dried in a vacuum oven at 40°C until a constant weight was reached. The citrate salt was isolated in ca. 75% yield.

[0509] Synthesis Example 7: Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride (HCl salt) Acetyl chloride (0.44 mL, 5.98 mmol) was added to EtOH (10.25 mL) at 0 °C. After 10 min at this temperature, this solution was added to cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (2.05 g, 5.92 mmol) in EtOH (10.25 mL) at 0 °C. The reaction mixture was stirred overnight at 0 °C and then concentrated to dryness. 2-BuOH (6.15 mL) was added to the residue, and the mixture was heated (110 °C) until complete dissolution was achieved. The reaction mixture was held at this temperature for 10 min and then cooled to 20–25 °C over 3 h. Diethyl ether (8 mL) was added to the thick suspension to aid stirring. After an additional 10 min at 20-25°C, the suspension was filtered and suction dried on a sintered funnel for 2 h. The solid was then transferred to a vacuum oven where it was dried overnight at 40°C to give the HCl salt (1.497 g, 3.91 mmol, 66% yield) as an off-white solid.

[0510] Synthesis Example 8: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate (fumaric acid salt) Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate (fumarate salt) was prepared by precipitation from ethanol (EtOH). Ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (240.0 mg) and fumaric acid (80.1 mg, approximately 1 mol. equivalent) were placed in a 4 mL vial. EtOH (2.5 volumes) was added, and the mixture was stirred and heated to 45°C for approximately 2 hours. After this time, the temperature was reduced by 5°C increments (approximately every 10 minutes) until ambient temperature was reached. The resulting solid was isolated by vacuum filtration. The solid was washed twice with EtOH (approximately 5 volumes) each time. The solid was allowed to air dry on the filter for approximately 1.5 hours before being transferred to a pre-weighed vial. The solid was further dried in a vacuum oven at 40°C until a constant weight was reached. The fumarate salt was produced in approximately 34% yield.

[0511] Example A Salt screening The suitability of the physical properties of several salt forms of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate was evaluated for further development with the goal of finding salts with desirable properties (e.g., reduced hygroscopicity, solubility, crystallinity, physical stability, etc.) that would support later-stage clinical formulation. The following acids were considered: 2,5-dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, butanedioic acid (succinic acid), citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), hydroxyacetic acid (glycolic acid), hydrochloric acid (HCl), maleic acid, malic acid (L), malonic acid, N-acetylglycine (aceturic acid), nicotinic acid, orthophosphoric acid (phosphoric acid), oxoglutaric acid (ketoglutaric acid), p-toluenesulfonic acid (p-TSA), pyroglutamic acid (L), sulfuric acid, and tartaric acid (L).

[0512] Experiments were performed at 20-30 mg scales with 1:1, 1:2, 2:1, and 4:1 stoichiometries (API:acid). To either a solution of the salt former or the solvent-free salt former, a solution of the free base in the selected solvent was added. To a 5 mL (ACN, EtOH, THF, ACN / water, EtOH / water, or THF / water) or 20 mL (MeOH) volumetric flask, cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (free base) was added and the volume was adjusted to form a 1.0 M or 0.1 M solution. This was sonicated in a water bath to ensure complete dissolution.

[0513] The salts were subjected to several testing and analytical techniques, including NMR and XRPD analysis, humidity stress testing, and dynamic vapor sorption (DVS) testing. Crystallization, degree of crystallinity, water solubility, and ease of manufacture were also evaluated. The fumarate, citrate, and HCl salts exhibited the best properties and were therefore selected for further study. Characterization of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate (citrate salt)

[0514] The XRPD pattern obtained for the citrate salt (Synthesis Example 6), Pattern B, is shown in Figure 1. The XRPD pattern indicates a crystalline material.

[0515] Thermogravimetric analysis / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and associated % weight change of the citrate salt (Figure 2). TG / DTA showed a weight loss of approximately 33% between approximately 140°C and approximately 220°C, most likely due to the loss of citric acid. A second weight loss above 220°C corresponds to the onset of decomposition of the material. A broad endotherm was observed with an onset temperature of approximately 174°C. This analysis indicated that the citrate salt was anhydrous.

[0516] Polarized light microscopy (Figure 3) showed that the material consisted of a crystalline solid with irregular geometry.

[0517] The 1H NMR spectrum (Figure 4) was consistent with the molecular structure, and minimal amounts of solvent were detected (<0.1 molar equivalents of 2-BuOH). The API:acid ratio was 1:1.

[0518] The hygroscopic and sorption properties of the citrate salt were determined using dynamic vapor sorption (DVS). The resulting isotherm is shown in Figure 5. Prior to sorption and desorption, the sample was dried at 0% RH. The isotherm showed that the material gradually took up moisture, resulting in a weight gain from 0% to 80% RH, followed by a sharper weight gain between 80% and 90% RH. The isotherm showed that the total weight gain observed between ambient (i.e., 40% RH) and 80% RH was approximately 0.8%, which, based on the European Pharmacopoeia classification, Table 1, indicates that the sample is only slightly hygroscopic. Very little hysteresis was observed between the sorption and desorption curves, and all of the added moisture was lost by 0% RH. XRPD analysis (Figure 6) performed after DVS showed that the citrate salt remained in Pattern B. Characterization of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate

[0519] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate was obtained by the process of Synthesis Examples 1, 2, and 3. A representative powder X-ray powder diffraction (XRPD) pattern is shown in Figure 25, and Table B shows the corresponding peaks and intensities. The XRPD diffractogram in Figure 25 indicated that the sample was crystalline and exhibited a pattern consistent with that of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate monohydrate. [Table 4]

[0520] Thermogravimetric analysis / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and associated % weight change of the citrate monohydrate salt (Figure 26). TG / DTA showed a weight loss step of approximately 0.155% from room temperature to approximately 65°C, followed by a 3.188% weight loss step from approximately 65 to 110°C, which appears to be related to a broad endothermic event with an onset of approximately 88.15°C. This is likely due to the dehydration / loss of bound hydrate (theoretical stoichiometry of monohydrate - 3.14%) with a loss / change in crystal structure. This event was followed by a second weight step of 27.399% from approximately 170 to 210°C, which appears to be related to an overlapping endothermic event with a sharp endotherm with an onset of approximately 175.48°C. This is likely due to the melting of the anhydrous form with loss of acid (theoretical stoichiometry of citrate - 25.7%) followed by rapid decomposition.

[0521] Four samples of similar weight were prepared and analyzed by DSC at various ramp rates (5.0°C / min, 10.0°C / min, 20.0°C / min, and 50.0°C / min) to examine the thermal behavior of these materials. The onset of dehydration / melting appears to be dependent on heating rate, ranging from about 79°C to 103°C. A second, sharper endothermic event appears somewhat dependent on heating rate and is observed to range from about 174.1°C to 178.5°C, with an average onset of melting of 176.4°C ± 2°C.

[0522] The hygroscopic and sorption properties of the citrate monohydrate salt were determined using dynamic vapor sorption (DVS). The resulting isotherm is shown in Figure 27. The above sorption isotherm suggests that the citrate monohydrate salt is slightly hygroscopic, with approximately 0.770% water adsorbed at 25°C / 80% RH, based on the European Pharmacopoeia classification, Table 1. The moisture content remains relatively constant at low humidity (likely recovering losses from the drying step). Diffractograms taken before and after DVS suggest no morphological changes due to the humidity change.

[0523] Primary particles of citrate monohydrate are observed as prismatic / plate-like particles generally less than 50 μm in size, which generally form aggregates larger than 100 μm. Representative images of aggregates by polarized light microscopy (PLM) and scanning electron microscopy (SEM), as well as single particles by SEM, are shown in Figures 28 and 29. Characterization of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride (HCl salt)

[0524] The XRPD pattern obtained for ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride, shown in Figure 7, indicated a crystalline material.

[0525] Thermogravimetric analysis / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and associated percent weight change of cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride (Figure 8). From approximately 25°C to 200°C, a weight loss of 3.6% was observed, corresponding to the loss of approximately 0.75 moles of water, indicating that the HCl salt contains water. A second weight loss above 210°C corresponds to the onset of decomposition of this material. An endotherm was observed with an onset temperature of approximately 234°C.

[0526] The DSC thermogram obtained for ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride is shown in Figure 9. The melting point could not be determined because decomposition began before melting.

[0527] Polarized light microscopy (Figure 10) showed that the material consisted of a crystalline solid with irregular geometry.

[0528] The 1H NMR spectrum (Figure 11) was consistent with the molecular structure, with minimal solvent detected.

[0529] Dynamic vapor sorption (DVS) was used to determine the hygroscopicity and adsorption properties of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl hydrochloride. The resulting isotherms are shown in Figure 12. Prior to sorption and desorption, the sample was dried at 0% RH. The isotherms indicated that the material gradually took up water between 0 and 60% RH, then exhibited a higher rate of weight gain between 60% and 70% RH. The rate of weight gain decreased between 70% and 80% RH and increased again between 80 and 90% RH. The isotherms indicated that the total weight gain observed between ambient (i.e., 40% RH) and 80% RH was approximately 7.5% w / w, indicating that the sample was hygroscopic based on the European Pharmacopoeia classification, Table 1. The desorption rate was comparable to the sorption rate between 90 and 70% RH. However, hysteresis was observed between the sorption and desorption plots between 70 and 0% RH, with approximately 3% water remaining at 0% RH. XRPD analysis (Figure 13) performed after DVS indicated that cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride was converted to the known alternative HCl salt form. These results suggest that conversion to the alternative HCl salt material occurs between 60 and 70% RH. Characterization of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate (fumarate salt)

[0530] The XRPD pattern obtained for cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate, Pattern A, is shown in Figure 14. The XRPD pattern indicates a crystalline material.

[0531] Thermogravimetric analysis / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and associated percent weight change of cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate (Figure 15). No weight loss was observed between approximately 25°C and 180°C, indicating that cis-ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate is an anhydrous product. A second weight loss above 180°C corresponds to the onset of decomposition of this material. An endotherm, most likely due to melting, was observed with an onset temperature of approximately 175°C.

[0532] Polarized light microscopy (Figure 16) showed that the material was crystalline and consisted of fine particles that tended to form aggregates / aggregates.

[0533] The 1H NMR spectrum (Figure 17) was consistent with the molecular structure and no solvent was detected. The API:acid ratio was determined to be 1:1.35.

[0534] Dynamic vapor sorption (DVS) was used to determine the hygroscopicity and adsorption properties of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate. The resulting isotherm is shown in Figure 18. Prior to sorption and desorption, the sample was dried at 0% RH. The isotherm indicated that the material exhibited gradual water uptake between 0% and 60% RH, followed by more pronounced water uptake between 60% and 80% RH, and a sharper uptake between 80 and 90% RH. The isotherm indicated that the total weight increase observed between ambient (i.e., 40% RH) and 80% RH was 0.3%, indicating that the sample was slightly hygroscopic based on the European Pharmacopoeia classification, Table 1. There was little hysteresis between the sorption and desorption curves, and all of the added moisture was lost by 0% RH. XRPD analysis was performed after DVS (Figure 19). The diffractogram showed that cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate remained in pattern A. Characterization of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (free base)

[0535] A representative powder X-ray powder diffraction (XRPD) pattern for ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (free base) is shown in Figure 32, and Table C lists the corresponding peaks and intensities. The XRPD diffractogram in Figure 32 suggests that the sample is crystalline. [Table 5]

[0536] Thermogravimetric / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and associated % weight change of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate (Figure 33). TG / DTA showed a weight loss step of approximately 0.335% from room temperature to approximately 125°C, and a broad endothermic event with an onset of approximately 98.93°C.

[0537] Dynamic vapor sorption (DVS) was used to determine the hygroscopicity and adsorption of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate. Sorption isotherms suggest that the crystalline form of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate is slightly hygroscopic (based on the European Pharmacopoeia classification, Table 1), with approximately 0.669% water adsorbed at 25 °C / 80% RH. Diffractograms obtained before and after DVS suggest no morphological change due to humidity changes.

[0538] Crystalline material of ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate was observed by SEM as irregular / slightly plate-like particles, generally 20-100 μm in size. The fine particles appeared as agglomerates on the surface of larger particles with a druzy geometry. Salt humidity stress load

[0539] Samples of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate (Fumarate Salt), cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate (Citrate Salt), and cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride (HCl Salt) were placed in a relative humidity chamber at 40° C. / 75% RH for 7 days. XRPD analysis of the stressed samples did not detect any change in form for the fumarate and citrate salts (Figures 20 and 21), but the HCl salt showed conversion to another HCl salt form (Figure 22). Salt solubility in water

[0540] The aqueous solubilities of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl fumarate (fumarate salt) and cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate ethyl citrate (citrate salt) were assessed by addition of aliquots. The fumarate salt was almost completely dissolved at approximately 320 mg / mL, and the pH of the solution was approximately 4-5. However, upon addition of additional water, solids precipitated. These solids were analyzed by XRPD and determined to be fumaric acid (Figure 23). This suggests that the fumarate salt was converted to the free base and fumaric acid in water. The citrate salt exhibited an aqueous solubility of approximately 850 to 1700 mg / mL, and the pH of the solution was approximately 3 to 4. The aqueous solubility of the HCl salt was approximately 340 to 390 mg / mL. [Table 1-2]

[0541] Biological data Biological Example 1 Phospho-ERK1 / 2 assay Functional assays were performed using the Alphascreen Surefire phospho-ERK1 / 2 assay (Crouch & Osmond, Comb. Chem. High Throughput Screen, 2008). ERK1 / 2 phosphorylation is a downstream consequence of both Gq / 11 and Gi / o protein-coupled receptor activation, making it suitable for evaluation of M1, M3 (Gq / 11-coupled) and M2, M4 receptors (Gi / o-coupled) rather than using different assay formats for different receptor subtypes.

[0542] CHO cells stably expressing human muscarinic M1, M2, M3, or M4 receptors were plated (25K / well) in 96-well tissue culture plates in MEM-alpha + 10% dialyzed FBS. Once cells adhered, they were serum-starved overnight. Agonist stimulation was performed by adding 5 μL of agonist to the cells for 5 minutes (37°C). The medium was removed, and 50 μL of lysis buffer was added. After 15 minutes, 4 μL of sample was transferred to a 384-well plate, and 7 μL of detection mix was added. The plates were incubated in the dark with gentle agitation for 2 hours, and then read on a PHERAstar plate reader. The resulting data for each receptor subtype determined pEC 50 and E max values were calculated.

[0543] Activity data for ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, ethyl cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate, and ethyl cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate are shown in Table D below. [Table 6]

[0544] equivalent The foregoing examples are presented for the purpose of illustrating the present invention and should not be construed as imposing any limitations on the scope of the invention. It will be readily apparent that numerous modifications and variations can be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and variations are intended to be encompassed by this application.

Claims

1. Compound of formula (1): 【172】 Or its salt.

2. A pharmaceutically acceptable salt of the compound of claim 1.

3. 10. An acid addition salt of the compound of claim 1.

4. The acid is selected from the group consisting of: 2,5-Dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, butanedioic acid (succinic acid), citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), hydroxyacetic acid (glycolic acid), hydrochloric acid (HCl), maleic acid, malic acid (L), malonic acid, N-acetylglycine (aceturic acid), nicotinic acid, orthophosphoric acid (phosphoric acid), oxoglutaric acid (ketoglutaric acid), p-toluenesulfonic acid (p-TSA), pyroglutamic acid (L), and sulfuric and tartaric acids (L).

4. The acid addition salt of claim 3, selected from:

5. The acid is selected from the group consisting of: Hydrochloric acid (HCl), fumaric acid and citric acid 4. The acid addition salt of claim 3, selected from:

6. 10. The citrate salt of the compound of claim 1.

7. 10. The citrate monohydrate salt of the compound of claim 1.

8. Compound of formula (3b): 【Chemistry 173】 2. The compound of claim 1, wherein:

9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. A compound or composition according to any one of claims 1 to 9 for use in medicine.

11. 10. A compound or composition according to any one of claims 1 to 9 for use in the treatment of a cognitive or psychotic disorder, or in the treatment or reduction of the severity of acute, chronic, neuropathic or inflammatory pain.

12. 12. The compound for use according to claim 11, wherein the disorder is Alzheimer's disease, dementia with Lewy bodies or schizophrenia.

13. Steps below 【Chemical 174】 10. A method of synthesizing the compound of claim 1, comprising: In the formula, R １ is a linear or branched C １～６ alkyl group, or C ３～６ is a cycloalkyl group, and R ２ together with the oxygen atom to which it is attached represents any suitable leaving group.

14. Steps below 【Chemistry 175】 10. A method of synthesizing the compound of claim 1, comprising: In the formula, R １ is a linear or branched C １～６ alkyl group, or C ３～６ is a cycloalkyl group, R ２ together with the oxygen atom to which it is attached represents any suitable leaving group.

15. Steps below 【176】 10. A method of synthesizing the compound of claim 1, comprising: In the formula, R １ is a linear or branched C １～６ alkyl group, or C ３～６ is a cycloalkyl group, The method wherein the catalyst is a hydrogenation catalyst.

16. R １ The method of any one of claims 13 to 15, wherein is ethyl or t-butyl.

17. R ２ The method of claim 13 or 14, wherein is tosyl.

18. below: 【Chemical 177】 【Chemical 178】 10. A method for synthesizing the compound of claim 1, comprising:

19. below: 【179】 10. A method for synthesizing the compound of claim 1, comprising:

20. below: 【Chemistry 180】 10. A method for synthesizing the compound of claim 1, comprising:

21. below: 【Chemistry 181】 10. A method for synthesizing the compound of claim 1, comprising:

22. Steps below: 【Chemistry 182】 A synthesis method comprising: In the formula, Z is 【Chemistry 183】 is selected from R １ is a linear or branched C １～６ alkyl group, or C ３～６ is a cycloalkyl group, R ２ together with the oxygen atom to which it is attached represents any suitable leaving group.

23. Z is, 【Chemistry 184】 23. The method of claim 22, wherein:

24. Z is, 【Chemistry 185】 23. The method of claim 22, wherein:

25. A crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate or a salt thereof, prepared by the method of any one of claims 13 to 21.

26. A crystalline form of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate or a salt thereof.

27. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

28. 28. The crystalline form of claim 27, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is the citrate salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

29. 29. The crystalline form of any one of claims 27 or 28, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate hydrate.

30. 30. The crystalline form of any one of claims 27 to 29, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate.

31. 29. The crystalline form of claim 27 or 28, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate.

32. 31. The crystalline form of any one of claims 25 to 30, characterized by a DSC thermogram having an endothermic peak with an onset at about 176°C.

33. 31. The crystalline form of any one of claims 25 to 30, characterized by a DSC thermogram substantially as shown in Figure 26.

34. 34. The crystalline form of any one of claims 25-30, 32 and 33, characterized by a TGA thermogram with a weight loss of about 3.3% from room temperature to about 110°C.

35. 34. The crystalline form of any one of claims 25-30, 32 and 33, characterized by a TGA thermogram substantially as shown in Figure 26.

36. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by at least one XRPD peak selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2° and 20.5°±0.2° two-theta.

37. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by at least two XRPD peaks selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2° and 20.5°±0.2° two-theta.

38. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by at least three XRPD peaks selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2° and 20.5°±0.2° two-theta.

39. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by three XRPD peaks at 11.3°±0.2°, 19.2°±0.2° and 20.5°±0.2° two-theta.

40. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by three XRPD peaks at 11.3°±0.2°, 18.4°±0.2° and 20.5°±0.2° two-theta.

41. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by three XRPD peaks at 11.3°±0.2°, 18.4°±0.2° and 19.2°±0.2° two-theta.

42. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by three XRPD peaks at 18.4°±0.2°, 19.2°±0.2° and 20.5°±0.2° two-theta.

43. 36. The crystalline form of any one of claims 25-30 and 32-35, characterized by four XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2° and 20.5°±0.2° two-theta.

44. 26. The crystalline form of any one of claims 25-30 and 32-35, characterized by an XRPD spectrum substantially as shown in Figure 25.

45. (1) an XRPD spectrum substantially as shown in Figure 25; (2) a DSC thermogram substantially as shown in FIG. 26; and (3) TGA thermogram substantially as shown in FIG. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

46. (1) at least one XRPD peak selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° two-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

47. (1) at least two XRPD peaks selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° in 2-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

48. (1) at least three XRPD peaks selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° in two-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

49. (1) three XRPD peaks at 11.3°±0.2°, 19.2°±0.2°, and 20.5°±0.2° two-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

50. (1) three XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, and 20.5°±0.2° 2-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

51. (1) three XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, and 19.2°±0.2° two-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

52. (1) three XRPD peaks at 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° two-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

53. (1) four XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2° two-theta; (2) a DSC thermogram having an endothermic peak with an onset at about 176°C; and (3) TGA thermogram showing a weight loss of approximately 3.3% from room temperature to approximately 110°C. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

54. 54. The crystalline form of any one of claims 45 to 53, wherein the crystalline form has characteristic (1).

55. The crystalline form of any one of claims 45 to 53, wherein the crystalline form has characteristic (2).

56. The crystalline form of any one of claims 45 to 53, wherein the crystalline form has characteristic (3).

57. 54. The crystalline form of any one of claims 45 to 53, wherein the crystalline form has characteristics (1) and (2).

58. 54. The crystalline form of any one of claims 45 to 53, wherein the crystalline form has characteristics (1) and (3).

59. The crystalline form of any one of claims 45 to 53, wherein the crystalline form has characteristics (2) and (3).

60. 54. The crystalline form of any one of claims 45 to 53, wherein the crystalline form has characteristics (1), (2), and (3).

61. 61. The crystalline form of any one of claims 45 to 60, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is the citrate salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

62. 62. The crystalline form of any one of claims 45 to 61, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate hydrate.

63. 63. The crystalline form of any one of claims 45 to 62, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate.

64. (1) an XRPD spectrum substantially as shown in FIG. 1; (2) a DTA thermogram substantially as shown in FIG. 2; and (3) TGA thermogram substantially as shown in FIG. A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

65. (1) an XRPD spectrum substantially as shown in FIG. 1; (2) a DTA thermogram having an endothermic peak with an onset at about 174°C; and (3) TGA thermogram with weight loss of about 33% from about 140°C to about 220°C A crystalline form of a salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, having at least one characteristic selected from the group consisting of:

66. 66. The crystalline form of claim 64 or 65, wherein the crystalline form has characteristic (1).

67. 66. The crystalline form of claim 64 or 65, wherein the crystalline form has characteristic (2).

68. 66. The crystalline form of claim 64 or 65, wherein the crystalline form has characteristic (3).

69. 66. The crystalline form of claim 64 or 65, wherein the crystalline form has characteristics (1) and (2).

70. 66. The crystalline form of claim 64 or 65, wherein the crystalline form has characteristics (1) and (3).

71. 66. The crystalline form of claim 64 or 65, wherein the crystalline form has characteristics (2) and (3).

72. 66. The crystalline form of claim 64 or 65, wherein the crystalline form has characteristics (1), (2), and (3).

73. 73. The crystalline form of any one of claims 64 to 72, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is the citrate salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

74. 74. The crystalline form of any one of claims 64 to 73, wherein the salt of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate is ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate.

75. 75. A pharmaceutical composition comprising the crystalline form of any one of claims 25 to 74 and a pharmaceutically acceptable excipient.

76. Compound of formula (4b): 【Chemistry 186】 The compound according to any one of claims 1 to 6,

77. Muscarine M ４ for use in treating a disease or condition mediated by a receptor, A compound or salt according to any one of claims 1 to 8 or 76, or A crystalline form according to any one of claims 25 to 74, or 76. The pharmaceutical composition of claim 9 or 75.

78. for use in the treatment of cognitive or psychotic disorders, A compound or salt according to any one of claims 1 to 8 or 76, or A crystalline form according to any one of claims 25 to 74, or 76. The pharmaceutical composition of claim 9 or 75.

79. 10. A compound or salt according to any one of claims 1 to 8 or 76, or a crystalline form according to any one of claims 25 to 74, or a pharmaceutical composition according to claim 9 or 75, for use in the treatment of a cognitive or psychotic disorder, wherein the cognitive or psychotic disorder is Cognitive impairment, mild cognitive impairment (MCI), frontotemporal dementia, vascular dementia, dementia with Lewy bodies, presenile dementia, senile dementia, Friedreich's ataxia, Down's syndrome, Huntington's chorea, hyperkinesia, mania, Tourette's syndrome, Alzheimer's disease, progressive supranuclear palsy, impairment of cognitive function including attention, adaptation, learning disabilities, memory and language functions; cognitive impairment as a result of stroke, Huntington's disease, Pick's disease, AIDS-related dementia, or other dementia conditions such as multi-infarct dementia, alcoholic dementia, hypothyroidism-related dementia, and dementia associated with other degenerative diseases (such as cerebellar atrophy and amyotrophic lateral sclerosis); other acute or subacute conditions that may cause cognitive decline such as delirium or depression, trauma, head injury, age-related cognitive decline, stroke, neurodegenerative disorders cognitive impairment due to gender, drug-induced states, neurotoxic agents, age-related cognitive impairment, autism-related cognitive impairment, Down's syndrome, agnosia associated with psychiatric disorders and post-electroconvulsive therapy related cognitive impairment; cognitive impairment due to drug abuse or drug withdrawal including nicotine, cannabis, amphetamines, cocaine, attention deficit hyperactivity disorder (ADHD) and movement disorders such as Parkinson's disease, neuroleptic-induced parkinsonism and tardive dyskinesia, schizophrenia, schizophreniform disorders, psychotic depression, mania, acute mania, delusions, hallucinatory disorders and delusional disorders, personality disorders, obsessive-compulsive disorder, schizotypal disorder, delusional disorder, psychiatric disorders due to malignancy, metabolic disorders, endocrine disorders or narcolepsy, psychiatric disorders due to drug abuse or drug withdrawal, bipolar disorder, schizoaffective disorder and Alzheimer's disease A compound or salt, or crystalline form, or pharmaceutical composition, which is, comprises, results from, or relates to a state selected from:

80. for use in the treatment of schizophrenia, Alzheimer's disease, or bipolar disorder A compound or salt according to any one of claims 1 to 8 or 76, or A crystalline form according to any one of claims 25 to 74, or 76. The pharmaceutical composition of claim 9 or 75.

81. for use in the treatment of schizophrenia A compound or salt according to any one of claims 1 to 8 or 76, or A crystalline form according to any one of claims 25 to 74, or 76. The pharmaceutical composition of claim 9 or 75.

82. for use in the treatment of bipolar disorder A compound or salt according to any one of claims 1 to 8 or 76, or A crystalline form according to any one of claims 25 to 74, or 76. The pharmaceutical composition of claim 9 or 75.

83. for use in treating Alzheimer's disease psychiatric disorders A compound or salt according to any one of claims 1 to 8 or 76, or A crystalline form according to any one of claims 25 to 74, or 76. The pharmaceutical composition of claim 9 or 75.

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