Crystalline forms and salts of muscarinic receptor agonists

Abstract

The present invention relates to compounds and salts thereof as muscarinic receptor agonists and for the treatment of muscarinic receptor mediated diseases. The invention also provides crystalline forms of the compounds and salts thereof; pharmaceutical compositions containing said compounds and salts thereof, or their crystalline forms; therapeutic uses of the compounds and salts thereof, or crystalline forms thereof; synthesis methods thereof; and intermediates suitable for use in the synthesis process.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of GB 2211399.7 filed on August 4, 2022 (04 / 08 / 2022), GB 2305444.8 filed on April 13, 2023 (13 / 04 / 2023), and GB 2309615.9 filed on June 26, 2023 (26 / 06 / 2023), the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to compounds and salts thereof that are muscarinic receptor agonists and are suitable for treating diseases mediated by muscarinic receptors. 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 the compounds; and intermediates suitable for the synthetic methods. Background of the Invention

[0005] Muscarinic acetylcholine receptor (mAChR) is a member of the G protein-coupled receptor superfamily, which mediates the effect of neurotransmitter acetylcholine in both the central and peripheral nervous systems. 5 mAChR subtypes (M1 to M5) have been cloned. M1mAChR is mainly expressed postsynaptically in the cortex, hippocampus, striatum and thalamus; M2 mAChR is mainly located in the brainstem and thalamus, but is also expressed on the cholinergic synaptic terminals in the cortex, hippocampus and striatum (Langmead et al., 2008 Br J Pharmacol). However, M2 mAChR is also expressed peripherally in cardiac tissue (wherein M2mAChR mediates vagal innervation of the heart) and smooth muscle and exocrine glands. M3 mAChR is expressed at relatively low levels in the CNS, but is widely expressed in smooth muscle and glandular tissues (such as sweat glands and salivary glands) (Langmead et al., 2008 Br J Pharmacol).

[0006] Muscarinic receptors (especially M1 mAChR) in the central nervous system play a key role in mediating high-level cognitive processing. Diseases associated with cognitive impairment, such as Alzheimer's disease, are accompanied by the 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 prefrontal cortex, hippocampus and caudate nucleus of schizophrenia individuals (Dean et al., 2002 Mol Psychiatry). In addition, in animal models, blocking or damage of central cholinergic pathways leads to serious cognitive deficits, and non-selective mAChR antagonists have been shown to induce psychotomimetic effects in psychotic patients. Cholinergic replacement therapy is largely based on the use of acetylcholinesterase inhibitors to prevent the decomposition of endogenous acetylcholine. These compounds have been clinically shown to be effective for symptomatic cognitive impairment, but they cause dose-limiting side effects due to stimulation of peripheral M2 and M3 mAChRs, including gastrointestinal disturbances, bradycardia, nausea and vomiting (http: / / www.drugs.com / pro / donepezil.html; http: / / www.drugs.com / pro / rivastigmine.html).

[0007] Further discovery work aims to identify direct M1 mAChR agonists whose goal is to improve cognitive function. The result of these efforts is the identification of a series of agonists, exemplary compounds such as xanomeline, AF267B, sabcomeline, milameline and cevimeline. Many of these compounds have been shown to be highly effective in preclinical cognitive models of rodents and / or non-human primates. Milameline has been shown to be effective against scopolamine-induced working memory and spatial memory deficits in rodents; sabcomeline has been shown to be effective in visual object discrimination tasks in marmosets, and xanomeline reverses the deficits in cognitive performance induced by mAChR antagonists in a passive avoidance paradigm.

[0008] Alzheimer's disease (AD) is the most common neurodegenerative disorder (26.6 million people worldwide in 2006) that affects the elderly, leading to severe memory loss and cognitive impairment. The etiology of the disease is complex, but is characterized by two hallmark brain sequelae: aggregates of amyloid plaques composed primarily of the amyloid-β peptide (Aβ), and neurofibrillary tangles formed by hyperphosphorylated tau protein. Accumulation of Aβ is considered a core feature in the progression of AD, and therefore, many putative therapies currently used to treat AD are aimed at inhibiting Aβ production. Aβ is derived from the proteolytic cleavage of the membrane-bound amyloid precursor protein (APP). APP is processed through two pathways, the non-amyloidogenic pathway and the amyloidogenic pathway. Cleavage of APP by γ-secretase is common to both pathways, but in the former APP is cleaved by α-secretase to produce soluble APPα. The cleavage site is within the Aβ sequence, thereby preventing the formation of Aβ. However, in the amyloidogenic pathway, APP is cleaved by β-secretase to produce soluble APPβ and Aβ. In vitro studies have shown that mAChR agonists can promote the processing of APP to the soluble non-amyloidogenic pathway. In vivo studies have shown that the mAChR agonist AF267B changes the disease-like pathology in 3×TgAD transgenic mice (a model of different components of Alzheimer's disease) (Caccamo et al., 2006 Neuron). Finally, the mAChR agonist Cevimeline has been shown to slightly but significantly reduce the level of Aβ in the cerebrospinal fluid of Alzheimer's patients, thereby demonstrating potential disease-modulating efficacy (Nitsch et al., 2000 Neurol).

[0009] In addition, preclinical studies have shown that mAChR agonists demonstrate atypical antipsychotic drug-like features in a series of preclinical paradigms. mAChR agonist Xanomeline reverses the behavior driven by multiple dopamine, including the rat movement induced by amphetamine, the mouse climbing induced by apomorphine, the rat turning over of the unilateral 6-OH-DA damage driven by dopamine agonists and the monkey movement loss control (without EPS tendency) induced by amphetamine. It also shows inhibition of A10 (rather than A9), dopamine cell discharge and conditioned avoidance, and induces c-fos expression in rat prefrontal cortex and nucleus accumbens, rather than c-fos expression in striatum. These data all show that it has atypical antipsychotic drug-like features (Mirza et al., 1999 CNSDrug Rev). Muscarinic receptors also participate 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 an important role in regulating dopaminergic neurotransmission. For example, M(4)(- / -) mice exhibit significantly enhanced reward-driven behavior upon exposure to cocaine (Schmidt et al., Psychopharmacology (2011) Aug; 216(3): 367-78). In addition, xanomeline has been shown to block the effects of cocaine in these models.

[0010] Muscarinic receptors are also involved in the control of movement and potentially represent new therapeutic approaches for the treatment of movement disorders such as Parkinson's disease, ADHD, Huntington's disease, Tourette's syndrome and other syndromes associated with dopaminergic dysfunction as a potential pathogenic factor driving the disease.

[0011] Xanomeline, sabcomeline, miramemline and cevimeline have all been developed into different clinical development stages for the treatment of Alzheimer's disease and / or schizophrenia. Phase II clinical studies of Xanomeline have confirmed its efficacy in various cognitive symptom areas, including behavioral disorders 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 of schizophrenia patients, and when compared with a placebo control group, it significantly alleviates positive and negative symptoms (Shekhar et al., 2008 Am J Psych). However, in all clinical studies, Xanomeline and other related mAChR agonists have shown unacceptable safety limits in terms of cholinergic side effects, including nausea, gastrointestinal pain, diarrhea, sweating (excessive sweating), excessive salivation (excessive salivation), syncope and bradycardia.

[0012] Muscarinic receptors are involved in central and peripheral pain. Pain can be divided into three different types: acute pain, inflammatory pain and neuropathic pain. Acute pain has an important protective function in protecting the organism from stimulation that may cause tissue damage, but postoperative pain needs to be managed. Inflammatory pain may occur due to many reasons (including tissue damage, autoimmune response and pathogen invasion), and is triggered by the action of inflammatory mediators (such as neuropeptides and prostaglandins), resulting in neuronal inflammation and pain. Neuropathic pain is related to abnormal pain sensations for non-painful stimuli. Neuropathic pain is related to a variety of different diseases / traumas, such as spinal cord injury, multiple sclerosis, diabetes (diabetic neuropathy), viral infections (such as HIV or herpes). This is also common in cancer, both as a result of the disease and as a side effect of chemotherapy. Activating muscarinic receptors has been shown to be analgesic in many pain states by activating receptors in higher pain centers in the spinal cord and brain. Increasing endogenous levels of acetylcholine by acetylcholinesterase inhibitors and directly activating muscarinic receptors with agonists or allosteric modulators have been shown to have analgesic activity. Conversely, blocking muscarinic receptors with antagonists or using knockout mice increases pain sensitivity. The evidence for the role of M1 receptors in pain was reviewed in 2012 by DF Fiorino and M. Garcia-Guzman.

[0013] Recently, a small number of compounds have been identified that show 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 relative to the M3 mAChR subtype, some of these compounds retain significant agonist activity at both this subtype and the M2 mAChR subtype. Herein, we describe compounds that show a higher level of selectivity for the M1 and / or M4 mAChRs than for the M2 and M3 receptor subtypes.

[0014] WO2015 / 118342 discloses muscarinic agonist compounds.

[0015] invention

[0016] The present invention provides compounds having selective activity as muscarinic M1 and / or M4 receptor agonists. More specifically, the present invention provides compounds that exhibit selectivity for the M4 receptor relative to the M2 and M3 receptor subtypes.

[0017] Therefore, there is provided a compound of formula (1):

[0018]

[0019] or a salt thereof.

[0020] Additionally provided are compounds of formula (2a):

[0021]

[0022] Wherein X represents salt.

[0023] Additionally provided are compounds of formula (2b):

[0024]

[0025] Wherein X represents salt.

[0026] Additionally provided are compounds of formula (3a):

[0027]

[0028] Additionally provided are compounds of formula (3b):

[0029]

[0030] Additionally provided are compounds of formula (4a):

[0031]

[0032] Additionally provided are compounds of formula (4b):

[0033]

[0034] Additionally provided are compounds of formula (5a):

[0035]

[0036] Additionally provided are compounds of formula (5b):

[0037]

[0038] Additionally provided are compounds of formula (6a):

[0039]

[0040] Additionally provided are compounds of formula (6b):

[0041]

[0042] Additionally provided are compounds of formula (7):

[0043]

[0044] or a salt thereof.

[0045] Additionally provided are compounds of formula (8):

[0046]

[0047] or a salt thereof.

[0048] Also provided are salts of compounds of formula (1).

[0049] The compound of formula (1) can form a pharmaceutically acceptable salt. The compound of formula (1) can form an acid addition salt.

[0050] The compound of formula (1) can form a citrate salt.

[0051] The compound of formula (1) can form a citrate monohydrate.

[0052] The compound of formula (1) can form a fumarate salt.

[0053] The compound of formula (1) can form an HCl salt.

[0054] The compound of formula (1) can form a phosphate.

[0055] The compound of formula (1) can form a sulfate salt.

[0056] In the compounds of formula (2a) and (2b), X may be a pharmaceutically acceptable acid.

[0057] In the compounds of formula (2a) and (2b), X may be an acid.

[0058] In the compounds of formula (2a) and (2b), X may be citric acid.

[0059] In the compounds of formula (2a) and (2b), X may be citric acid monohydrate.

[0060] In the compounds of formula (2a) and (2b), X may be fumaric acid.

[0061] In the compounds of formula (2a) and (2b), X may be HCl.

[0062] In the compounds of formula (2a) and (2b), X may be phosphoric acid.

[0063] In the compounds of formula (2a) and (2b), X may be sulfuric acid.

[0064] In acid addition salts, the acid may be selected from:

[0065] 2,5-dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, succinic acid, citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), glycolic 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).

[0066] The compound may be ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0067] The compound may 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.

[0068] The compound may 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.

[0069] The compound may 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.

[0070] The compound may be (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate.

[0071] The compound may be (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate.

[0072] The compound may be (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate.

[0073] The compound may be (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride.

[0074] The compound may be a salt of ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate.

[0075] The compound may 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.

[0076] The compound may 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.

[0077] The compound may be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate.

[0078] The compound may be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate.

[0079] The compound may be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate.

[0080] The compound may be ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride.

[0081] Also provided are synthetic methods, including those that preferentially favor the formation of compounds and intermediates having desired stereochemistry. For example, the methods may employ stereoselective reduction of ketones to give alcohols, or stereoselective reductive amination of ketones.

[0082] A synthetic method is provided according to the following general scheme:

[0083]

[0084] Where Q is selected from:

[0085]

[0086] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0087] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group; and X represents a salt.

[0088] A synthetic method is provided according to the following general scheme:

[0089]

[0090] Where Z is selected from:

[0091]

[0092] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0093] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group; and X represents a salt.

[0094] The compounds of the present invention can be prepared according to the following general schemes:

[0095]

[0096] Where R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl, and R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group. Also provided is a synthetic method according to the following general scheme:

[0097]

[0098] Where Q is selected from:

[0099]

[0100] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0101] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group; and X represents a salt.

[0102] The compounds of the present invention can be prepared according to the following general schemes:

[0103]

[0104] Where R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0105] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group; and X represents a salt.

[0106] Also provided are synthetic methods according to the following general scheme:

[0107]

[0108] Where Q is selected from:

[0109]

[0110] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0111] Cat. is a hydrogenation catalyst;

[0112] And X represents salt.

[0113] The compounds of the present invention can be prepared according to the following general schemes:

[0114]

[0115] Where R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0116] Cat. is a hydrogenation catalyst;

[0117] And X represents salt.

[0118] In the methods described herein, Q can be Q can be Q can be A specific method includes where Q is of compounds.

[0119] In the methods described herein, R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl. 1 It can be ethyl or tert-butyl. 1 It may be tert-butyl. 1 It may be ethyl.

[0120] In the methods described herein, R 2 Together with the oxygen atom to which it is attached, R represents any suitable leaving group. 2 It may be tosyl.

[0121] In the processes described herein, Cat. represents a hydrogenation catalyst. Cat. can be any hydrogenation catalyst capable of catalyzing the transformations shown in the relevant synthesis schemes. Cat. can be Pd / BaSO4. Cat. can be Pd / BaSO4 (10 mol%).

[0122] 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.

[0123] The compounds can be prepared according to the following scheme:

[0124]

[0125] The compounds can be prepared according to the following scheme:

[0126]

[0127] The compounds can be prepared according to the following scheme:

[0128]

[0129] The compounds can be prepared according to the following scheme:

[0130]

[0131] Also provided is a synthetic method according to the following scheme:

[0132] Main Chain

[0133]

[0134] Sidechain:

[0135]

[0136] Also provided is a synthetic method according to the following scheme:

[0137] BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 : XRPD spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate as shown in Synthesis Example 6.

[0139] 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 analyzed at 10°C / min from 25°C to 300°C as exemplified in Synthesis Example 6.

[0140] Figure 3: A micrograph 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.

[0141] Figure 4 : Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate as exemplified in Synthesis Example 6 1 H NMR spectrum.

[0142] Figure 5 : 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.

[0143] Figure 6 : XRPD traces showing (top) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, (middle) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate as exemplified in, for example, Synthesis Example 6, and (bottom) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate after DVS as exemplified in, for example, Synthesis Example 6.

[0144] Figure 7 : XRPD spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride shown in, for example, Synthesis Example 7.

[0145] 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 analyzed at 10°C / min from 25°C to 300°C as exemplified in Synthesis Example 7.

[0146] Fig. 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 analyzed at 10°C / min from 25°C to 300°C as exemplified in Synthesis Example 7.

[0147] Fig.10: A micrograph 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.

[0148] Fig.11 : Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride as exemplified in Synthesis Example 7 1 H NMR spectrum.

[0149] Fig.12 : 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 shown in, for example, Synthesis Example 7.

[0150] Fig.13 : XRPD traces showing (top) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride after DVS as exemplified in, for example, Synthesis Example 7, (middle) cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride before DVS as exemplified in, for example, Synthesis Example 7, (bottom) known alternative HCl salt forms.

[0151] Fig.14 : The XRPD spectrum of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate shown in, for example, Synthesis Example 8.

[0152] Fig.15 : TG / DTA thermogram of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate analyzed at 10°C / min from 25°C to 300°C as exemplified in Synthesis Example 8.

[0153] Fig.16 : A micrograph 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.

[0154] Fig.17 : Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate as exemplified in Synthesis Example 8 1H NMR spectrum.

[0155] Fig.18 : 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 shown in, for example, Synthesis Example 8.

[0156] Fig.19 : XRPD traces showing (top) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate (free base), (middle) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate as exemplified in, for example, Synthesis Example 8, and (bottom) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate after DVS as exemplified in, for example, Synthesis Example 8.

[0157] Fig. 20 : XRPD traces showing (top) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate before humidity stress as exemplified in, for example, Synthesis Example 8, and (bottom) the sample after humidity stress.

[0158] Fig.21 : XRPD traces showing (top) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate before humidity stress as exemplified in, for example, Synthesis Example 6, and (bottom) the sample after humidity stress.

[0159] Fig. 22 : XRPD traces showing (top) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride before humidity stress as exemplified in, for example, Synthesis Example 7, (middle) the sample after humidity stress and (bottom) a known alternative HCl salt form.

[0160] Fig.23: XRPD trace showing (top) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate trans-fumarate, as exemplified in, for example, Synthesis Example 8, (middle) ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate, as exemplified in, for example, Synthesis Example 8, and (bottom) fumaric acid.

[0161] Fig.24 : For example, the cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester shown in Synthesis Example 5 1 H NMR spectrum.

[0162] Fig.25 : 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 illustrated in, for example, Synthesis Examples 1 to 3.

[0163] Fig.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 analyzed at 10°C / min from 25°C to 300°C as exemplified in, for example, Synthesis Examples 1 to 3.

[0164] Fig. 27 : DVS isotherm of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate illustrated in, for example, Synthesis Examples 1 to 3.

[0165] Fig.28 : A micrograph of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate monohydrate illustrated in, for example, Synthesis Examples 1 to 3.

[0166] Fig.29 : SEM micrograph of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate as exemplified in, for example, Synthesis Examples 1 to 3. Top: Aggregates of particles; Bottom: Single crystals. Scale bar indicates 50 μm.

[0167] Fig.30: 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 illustrated in, for example, Synthesis Examples 1 to 3.

[0168] Fig.31 : Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate as exemplified in Synthesis Examples 1 to 3 1 Zoomed portion of the H-NMR spectrum.

[0169] Fig.32 : XRPD spectrum 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.

[0170] Fig.33 : 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.

[0171] Fig.34 : trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester 1 H NMR spectrum.

[0172] Fig.35 : cis-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester 1 H NMR spectrum.

[0173] Fig.36 :pass 1 H- 1 H NOESY assignments of tert-butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate. Top: assignments of NOE cross peaks; bottom: zoomed portion of the 2D-NOESY spectrum.

[0174] Fig.37 :pass 1 H- 1 H NOESY assignments of tert-butyl cis-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate. Top: assignments of NOE cross peaks; bottom: zoomed portion of the 2D-NOESY spectrum.

[0175] Detailed description

[0176] The present invention provides compounds and salts thereof having selective activity as muscarinic M1 and / or M4 receptor agonists. More specifically, the present invention provides compounds that exhibit selectivity for the M4 receptor relative to the M2 and M3 receptor subtypes.

[0177] The following compounds are muscarinic M1 and / or M4 receptor agonists: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (structure shown below); cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (structure shown below); and cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (structure shown below).

[0178] Each of these compounds and salts thereof are useful, for example, in the treatment of muscarinic receptor-mediated diseases and conditions, such as schizophrenia, Alzheimer's disease, Alzheimer's psychosis, and bipolar disorder (see, e.g., WO 2015 / 118342 A1, the disclosure of which is incorporated herein by reference in its entirety).

[0179]

[0180] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester

[0181]

[0182] Cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester

[0183]

[0184] cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester

[0185] Methods for preparing compounds

[0186] The present application provides methods for preparing the compounds described herein and their salts.

[0187] Thus, provided herein is a method for preparing a compound of formula (1), formula (7) or formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (I):

[0188]

[0189] Reaction with the compound of formula (II) or its salt:

[0190]

[0191] To provide the following steps of providing a compound of formula (III) or a salt thereof:

[0192]

[0193] in:

[0194] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0195] R 2 together with the oxygen atom to which it is attached represents any suitable leaving group; and wherein:

[0197] (a)Q 1 Yes - H and Q 2 Selected from:

[0198] or

[0199] (b)Q 1 For-OR 3 And Q 2 For-OR 3 , where R 3 It is C 1-6 Straight or branched chain alkyl; or

[0200] (c)Q 1 and Q 2 Together they form the group -O-(CH2) m -O-, and m is an integer selected from 2, 3 and 4.

[0201] In some embodiments, R 1 It is C 1-6 In some embodiments, R 1 is ethyl or tert-butyl. 1 In some embodiments, R 1 It is ethyl.

[0202] In some embodiments, R2 Together with the oxygen atom to which it is attached, represents a sulfonate leaving group. 2 Together with the oxygen atom to which it is attached, represents an aryl sulfonate group. 2 Together with the oxygen atom to which it is attached, it represents a tosylate group.

[0203] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (compounds of formula (1), formula (2b), formula (3b), formula (4b), formula (5b), formula (6b))

[0204] Also provided herein is a process for preparing a compound of formula (I) or a salt thereof or a crystalline form thereof, wherein the process comprises reacting a compound of formula (I):

[0205]

[0206] Reaction with the compound of formula (IIa) or its salt:

[0207]

[0208] To provide the following steps of providing a compound of formula (IIIa) or a salt thereof:

[0209]

[0210] in:

[0211] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0212] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group.

[0213] Also provided herein is a method for preparing a compound of formula (I) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (IV):

[0214]

[0215] With the following compound (IIa) or its salt:

[0216]

[0217] The step of reacting under reductive amination conditions to provide a compound of formula (IIIa) or a salt thereof:

[0218]

[0219] Among them, R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl.

[0220] Also provided herein is a method for preparing a compound of formula (1) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (IIIa) or a salt thereof:

[0221]

[0222] Reaction with a deprotecting acid to provide a compound of formula (V) or a salt thereof:

[0223]

[0224] Among them, R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl.

[0225] Also provided herein is a method for preparing a compound of formula (I) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (V) or a salt thereof:

[0226]

[0227] The step of reacting with ethyl chloroformate to provide a compound of formula (1) or a salt thereof:

[0228]

[0229] In some embodiments, the compound of formula (1) prepared by the method described above is a compound of formula (2b) or a crystalline form thereof:

[0230]

[0231] Wherein X represents salt.

[0232] In some embodiments, the compound of formula (1) prepared by the method described above is a compound of formula (3b) or a crystalline form thereof:

[0233]

[0234] In some embodiments, the compound of formula (1) prepared by the method described above is a compound of formula (4b) or a crystalline form thereof:

[0235]

[0236] In some embodiments, the compound of formula (1) prepared by the method described above is a compound of formula (5b) or a crystalline form thereof:

[0237]

[0238] In some embodiments, the compound of formula (1) prepared by the method described above is a compound of formula (6b) or a crystalline form thereof:

[0239]

[0240] Also provided herein is a method for preparing a compound of formula (4b) or a crystalline form thereof, wherein the method comprises the following steps:

[0241]

[0242] The compound of formula (4b) is formed from a crystallization solution comprising ethanol, water, citric acid and the compound of formula (1):

[0243]

[0244] Also provided herein is a method for preparing a compound of formula (4b) or a crystalline form thereof, wherein the method comprises the following steps:

[0245] (ia) making the compound of formula (I):

[0246]

[0247] Reaction with the compound of formula (IIa) or its salt:

[0248]

[0249] To provide the following compound (IIIa) or a salt thereof:

[0250]

[0251] in:

[0252] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0253] R 2 and the oxygen atom to which it is attached represent any suitable leaving group; or (ib) making a compound of formula (IV):

[0254]

[0255] With the following compound (IIa) or its salt:

[0256]

[0257] Reaction under reductive amination conditions provides the following compound of formula (IIIa) or a salt thereof:

[0258]

[0259] Among them, R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl; then (ii) making the following compound (IIIa) or a salt thereof:

[0260]

[0261] Reaction with a deprotecting acid to provide a compound of formula (V) or a salt thereof:

[0262] Then (iii) the compound of the following formula (V) or a salt thereof:

[0263]

[0264] Reaction with ethyl chloroformate to provide a compound of formula (1) or a salt thereof:

[0265] Then

[0266] (iv) a solution comprising ethanol, water, citric acid and a compound of formula (1)

[0267]

[0268] To form a compound of formula (4b):

[0269]

[0270] In some embodiments, R 1 It is C 1-6 In some embodiments, R 1 is ethyl or tert-butyl. 1 In some embodiments, R 1 It is ethyl.

[0271] In some embodiments, R 2 Together with the oxygen atom to which it is attached, represents a sulfonate leaving group. 2 Together with the oxygen atom to which it is attached, represents an aryl sulfonate group. 2 Together with the oxygen atom to which it is attached, it represents a tosylate group.

[0272] In some embodiments, the reductive amination conditions include a reducing acid and a reducing agent.

[0273] In some embodiments, the reductive amination conditions include H2 and palladium. In some embodiments, the reductive amination conditions include H2, palladium / barium sulfate, and molecular sieves. In some embodiments, the reductive amination conditions include H2, palladium / barium sulfate (reduced), and molecular sieves at about 1000 psi.

[0274] 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 the group consisting of citric acid, tartaric acid, trimethylacetic acid, 2,2-dimethylsuccinic acid, acetic acid, di-p-toluoyltartaric acid, difluoroacetic acid, phosphoric acid, 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.

[0275] In some embodiments, the reductive amination conditions include reacting at a temperature below 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.

[0276] In some embodiments, the compound of formula (IIa) is a salt. In some embodiments, the compound of formula (IIa) is a tartrate salt.

[0277] In some embodiments, the deprotecting acid is hydrochloric acid. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by reaction between acetyl chloride and a protic solvent. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by reaction between acetyl chloride and ethanol.

[0278] 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.

[0279] In some embodiments, the crystallization solution comprises ethanol and water in a ratio of about 100:1 to about 100:10 by volume, and 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.

[0280] In some embodiments, the crystallization solution comprises citric acid and the 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 the 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 the 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 the compound of formula (1) in a ratio of about 1:1. In some embodiments, the crystallization solution comprises citric acid and the compound of formula (1) in a ratio of about 1.08:1.

[0281] In some embodiments, seed crystals of the compound of formula (4b) in crystalline form are added to the crystallization solution.

[0282] cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro [3.4] Ethyl octane-6-carboxylate (Compound of formula (7))

[0283] Also provided herein is a method for preparing a compound of formula (7) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (I):

[0284]

[0285] Reaction with the compound of formula (IIb) or its salt:

[0286]

[0287] To provide the following steps of providing a compound of formula (IIIb) or a salt thereof:

[0288]

[0289] in:

[0290] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0291] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group.

[0292] Also provided herein is a method for preparing a compound of formula (7) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (IIIb) or a salt thereof:

[0293]

[0294] The step of reacting with a deprotecting acid to provide a compound of formula (VI) or a salt thereof:

[0295]

[0296] Among them, R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl.

[0297] In some embodiments, the reaction is carried out at a temperature below 20°C.

[0298] Also provided herein is a method for preparing a compound of formula (7) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (VI) or a salt thereof:

[0299]

[0300] The step of reacting with ethyl chloroformate to provide a compound of formula (VII) or a salt thereof:

[0301]

[0302] Also provided herein is a method for preparing a compound of formula (7) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (VII) or a salt thereof:

[0303]

[0304] The step of reacting with a ketal deprotecting acid to provide a compound of formula (VIII):

[0305]

[0306] Also provided herein is a method for preparing a compound of formula (7) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (VIII) or a salt thereof:

[0307]

[0308] Reaction with the compound of the following formula (IX) or its salt under reducing conditions:

[0309]

[0310] The step of providing a compound of formula (7) or a salt thereof:

[0311]

[0312] Also provided herein is a method for preparing a compound of formula (7) or a salt thereof or a crystalline form thereof, wherein the method comprises the following steps:

[0313] (i) making the compound of the following formula (I):

[0314]

[0315] Reaction with the compound of formula (IIb) or its salt:

[0316]

[0317] To provide the following compound (IIIa) or a salt thereof:

[0318]

[0319] in:

[0320] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0321] R 2 together with the oxygen atom to which it is attached, represents any suitable leaving group; then

[0322] (ii) making the following compound (IIIb) or a salt thereof:

[0323]

[0324] Reaction with a deprotecting acid to provide a compound of formula (VI) or a salt thereof:

[0325] Then

[0326] (iii) making the compound of the following formula (VI) or a salt thereof:

[0327]

[0328] Reaction with ethyl chloroformate to provide a compound of formula (VII) or a salt thereof:

[0329] Then

[0330] (iv) making the compound of the following formula (VII) or a salt thereof:

[0331]

[0332] Reaction with the ketal deprotecting acid to provide a compound of formula (VIII):

[0333] Then

[0334] (v) making the compound of the following formula (VIII) or a salt thereof:

[0335]

[0336] Reaction with the compound of the following formula (IX) or its salt under reducing conditions:

[0337]

[0338] To provide a compound of formula (7) or a salt thereof:

[0339]

[0340] In some embodiments, R 1 It is C 1-6 In some embodiments, R 1 is ethyl or tert-butyl. 1 In some embodiments, R 1 It is ethyl.

[0341] In some embodiments, R 2 Together with the oxygen atom to which it is attached, represents a sulfonate leaving group. 2 Together with the oxygen atom to which it is attached, represents an aryl sulfonate group. 2 Together with the oxygen atom to which it is attached, it represents a tosylate group.

[0342] In some embodiments, the reaction between the compound of formula (I) and the compound of formula (lib) or a salt thereof is heated to about 80°C to about 110°C.

[0343] In some embodiments, the deprotecting acid is hydrochloric acid. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by reaction between acetyl chloride and a protic solvent. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by reaction between acetyl chloride and ethanol.

[0344] In some embodiments, the compound of formula (VI) is a salt. In some embodiments, the compound of formula (VI) is a diacid salt. In some embodiments, the compound of formula (VI) is a hydrochloride salt. In some embodiments, the compound of formula (VI) is a dihydrochloride salt.

[0345] In some embodiments, the reaction between the compound of formula (VI) or its salt and ethyl chloroformate further comprises a base. In some embodiments, the reaction between the compound of formula (VI) or its salt and ethyl chloroformate further comprises triethylamine.

[0346] In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate is carried out at a temperature below 20 °C.

[0347] In some embodiments, the ketal deprotecting acid is hydrochloric acid. In some embodiments, the ketal deprotecting acid is aqueous hydrochloric acid.

[0348] In some embodiments, the reducing conditions comprise a borohydride reagent. In some embodiments, the reducing conditions comprise sodium triacetoxyborohydride.

[0349] In some embodiments, the reaction between the compound of formula (VIII) or a salt thereof and the compound of formula (IX) is carried out at a temperature below 20 °C.

[0350] cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro [3.4] Ethyl octane-6-carboxylate (Compound of formula (8))

[0351] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (I):

[0352]

[0353] Reaction with the compound of formula (IIb) or its salt:

[0354]

[0355] To provide the following steps of providing a compound of formula (IIIb) or a salt thereof:

[0356]

[0357] in:

[0358] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0359] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group.

[0360] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (IIIb):

[0361]

[0362] The step of reacting with an acid to provide a salt of formula (IIIc):

[0363]

[0364] in:

[0365] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl; and

[0366] X represents salt.

[0367] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (IIIb) or a salt thereof:

[0368]

[0369] The step of reacting with a deprotecting acid to provide a compound of formula (VI) or a salt thereof:

[0370]

[0371] Among them, R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl.

[0372] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (VI) or a salt thereof:

[0373]

[0374] The step of reacting with ethyl chloroformate to provide a compound of formula (VII) or a salt thereof:

[0375]

[0376] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (VII) or a salt thereof:

[0377]

[0378] The step of reacting with a ketal deprotecting acid to provide a compound of formula (VIII):

[0379]

[0380] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises reacting a compound of formula (VIII) or a salt thereof:

[0381]

[0382] and reacting with a compound of the following formula (X) or a salt thereof under reducing conditions,

[0383]

[0384] The step of providing a compound of formula (8) or a salt thereof:

[0385]

[0386] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises making a compound of formula (8) or a salt thereof:

[0387]

[0388] The step of recrystallization from the recrystallization solution.

[0389] Also provided herein is a method for preparing a compound of formula (8) or a salt thereof or a crystalline form thereof, wherein the method comprises the following steps:

[0390] (i) making the compound of the following formula (I):

[0391]

[0392] Reaction with the compound of formula (IIb) or its salt:

[0393]

[0394] To provide the following compound (IIIa) or a salt thereof:

[0395]

[0396] in:

[0397] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0398] R 2 together with the oxygen atom to which it is attached, represents any suitable leaving group; then

[0399] (ii) optionally, making the following compound of formula (IIIb):

[0400]

[0401] Reaction with a salt-forming acid to provide a salt of formula (IIIc):

[0402]

[0403] in:

[0404] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl; and

[0405] X represents salt; then

[0406] (iii) making the compound of the following formula (IIIb) or a salt thereof:

[0407]

[0408] Reaction with a deprotecting acid to provide a compound of formula (VI) or a salt thereof:

[0409] Then

[0410] (iv) making the compound of the following formula (VI) or a salt thereof:

[0411]

[0412] Reaction with ethyl chloroformate to provide a compound of formula (VII) or a salt thereof:

[0413] Then

[0414] (v) making the compound of the following formula (VII) or a salt thereof:

[0415]

[0416] Reaction with the ketal deprotecting acid to provide a compound of formula (VIII):

[0417] Then

[0418] (vi) making the compound of the following formula (VIII) or a salt thereof:

[0419]

[0420] With the compound of the following formula (X) or a salt thereof:

[0421]

[0422] Reaction under reducing conditions to provide a compound of formula (8) or a salt thereof:

[0423]

[0424] In some embodiments, R 1 It is C 1-6 In some embodiments, R 1 is ethyl or tert-butyl. 1 In some embodiments, R 1 It is ethyl.

[0425] In some embodiments, R 2 Together with the oxygen atom to which it is attached, represents a sulfonate leaving group. 2Together with the oxygen atom to which it is attached, represents an aryl sulfonate group. 2 Together with the oxygen atom to which it is attached, it represents a tosylate group.

[0426] In some embodiments, the reaction between the compound of formula (I) and the compound of formula (lib) or a salt thereof is heated to about 80°C to about 110°C.

[0427] In some embodiments, the salt-forming acid is hydrochloric acid and X represents hydrochloride.

[0428] In some embodiments, the deprotecting acid is hydrochloric acid. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by reaction between acetyl chloride and a protic solvent. In some embodiments, the deprotecting acid is hydrochloric acid generated in situ by reaction between acetyl chloride and ethanol.

[0429] In some embodiments, the compound of formula (VI) is a salt. In some embodiments, the compound of formula (VI) is a diacid salt. In some embodiments, the compound of formula (VI) is a hydrochloride salt. In some embodiments, the compound of formula (VI) is a dihydrochloride salt.

[0430] In some embodiments, the reaction between the compound of formula (VI) or its salt and ethyl chloroformate further comprises a base. In some embodiments, the reaction between the compound of formula (VI) or its salt and ethyl chloroformate further comprises triethylamine.

[0431] In some embodiments, the reaction between the compound of formula (VI) or a salt thereof and ethyl chloroformate is carried out at a temperature below 20 °C.

[0432] In some embodiments, the ketal deprotecting acid is hydrochloric acid. In some embodiments, the ketal deprotecting acid is aqueous hydrochloric acid.

[0433] In some embodiments, the reducing conditions comprise a borohydride reagent. In some embodiments, the reducing conditions comprise sodium triacetoxyborohydride.

[0434] In some embodiments, the reaction between the compound of formula (VIII) or a salt thereof and the compound of formula (X) is carried out at a temperature below 30 °C.

[0435] 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.

[0436] Intermediate compounds suitable for use in the process for preparing the compounds of the present invention

[0437] The present application also provides intermediate compounds useful for preparing the compounds described herein or their salts.

[0438] Therefore, the present application also provides a compound of formula (I):

[0439]

[0440] in:

[0441] R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl;

[0442] R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group.

[0443] In some embodiments, R 1 It is C 1-6 In some embodiments, R 1 is ethyl or tert-butyl. 1 In some embodiments, R 1 It is ethyl.

[0444] In some embodiments, R 2 Together with the oxygen atom to which it is attached, represents a sulfonate leaving group. 2 Together with the oxygen atom to which it is attached, represents an aryl sulfonate group. 2 Together with the oxygen atom to which it is attached, it represents a tosylate group.

[0445] The present application also provides a compound of formula (III) or a salt thereof:

[0446]

[0447] in:

[0448] R 1 Is methyl or C 3-6 Straight or branched chain alkyl, or C 3-6 Cycloalkyl;

[0449] (a)Q 1 Yes - H and Q 2 Selected from:

[0450] or

[0451] (b)Q 1 Yes-OR 3 And Q 2 Yes-OR 3 , and R3 It is C 1-6 Straight or branched chain alkyl; or

[0452] (c)Q 1 and Q 2 Together they form the group -O-(CH2) m -O-, and m is an integer selected from 2, 3 and 4.

[0453] The present application also provides a compound of formula (IIIa) or a salt thereof:

[0454]

[0455] Where R 1 is methyl, or C 3-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl.

[0456] In some embodiments, R 1 is methyl, or C 3-6 In some embodiments, R 1 It is tert-butyl.

[0457] 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.

[0458] The present application also provides a compound of formula (IIIb) or a salt thereof:

[0459]

[0460] Among them, R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 In some embodiments, R 1 It is C 1-6 In some embodiments, R 1 is ethyl or tert-butyl. 1 In some embodiments, R 1 It is ethyl.

[0461] The present application also provides a compound of formula (V) or a salt thereof:

[0462]

[0463] 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.

[0464] The present application also provides a compound of formula (VI) or a salt thereof:

[0465]

[0466] In some embodiments, the compound of formula (VI) is a salt. In some embodiments, the compound of formula (VI) is a diacid salt. In some embodiments, the compound of formula (VI) is a hydrochloride salt. In some embodiments, the compound of formula (VI) is a dihydrochloride salt.

[0467] The present application also provides a compound of formula (VII) or a salt thereof:

[0468]

[0469] The present application also provides a compound of formula (VIII) or a salt thereof:

[0470]

[0471] Crystalline form

[0472] The present application also provides a crystalline form of a compound described herein or a salt thereof.

[0473] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester

[0474] The present application provides at least one crystalline form of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester or its salt. In some embodiments, the present application provides at least one crystalline form of a salt of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester. In some embodiments, the present application provides at least one crystalline form of a salt of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, wherein the salt is selected from citrate, hydrochloride and fumarate.

[0475] In some embodiments, the present application provides at least one crystalline form of the citrate salt of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester. In some embodiments, the present application provides at least one crystalline form of the citrate salt of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester. In some embodiments, the present application provides at least one crystalline form of the citrate salt of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester. In some embodiments, the present application provides at least one crystalline form of the citrate salt of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester. In some embodiments, the present application provides at least one crystalline form of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate. In some embodiments, the present application provides at least one crystalline form of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride. In some embodiments, the present application provides at least one crystalline form of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate.

[0476] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate

[0477] 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 being substantially as Fig.26 DSC thermogram shown in .

[0478] 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 having 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 having 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 having a TGA thermogram with a weight loss of about 27.4% from about 170°C to 210°C. Fig.26 TGA thermogram shown in .

[0479] 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, expressed in 2θ, 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 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, expressed in 2θ, 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 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, expressed in 2θ, 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 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, expressed in 2θ, 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 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, expressed in 2θ, 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 some embodiments, the crystalline form is characterized by at least 6 XRPD peaks, expressed in 2θ, 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 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, expressed in 2θ, 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 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, expressed in 2θ, 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 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°, expressed in 2θ.

[0480] 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, expressed in 2θ, selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°. 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 at least two XRPD peaks, expressed in 2θ, selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°. 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 at least three XRPD peaks, expressed in 2θ, selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°.

[0481] 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, expressed in 2θ, at 11.3°±0.2°, 19.2°±0.2°, and 20.5°±0.2°. 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 three XRPD peaks, expressed in 2θ, at 11.3°±0.2°, 18.4°±0.2°, and 20.5°±0.2°. 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 three XRPD peaks, expressed in 2θ, at 11.3°±0.2°, 18.4°±0.2°, and 19.2°±0.2°. 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 three XRPD peaks, expressed in 2θ, at 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°. In some embodiments, the form of the salt of crystalline cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester is characterized by four XRPD peaks at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°, expressed in 2θ.

[0482] 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°, expressed in 2θ. 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°, expressed in 2θ. 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°, expressed in 2θ. 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°, expressed in 2θ. 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°, expressed in 2θ. 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°, expressed in 2θ.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°, expressed in 2θ. 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°, expressed in 2θ. 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°, expressed in 2θ. 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°, expressed in 2θ.

[0483] 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 being substantially as follows Fig.25 XRPD spectrum shown in .

[0484] In any of the above embodiments, the crystalline form may be prepared by any of the methods described herein. In any of the above embodiments, the crystalline form may be substantially isolated.

[0485] In any 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-carboxylic acid ethyl ester citrate salt. In any 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-carboxylic acid ethyl ester citrate salt hydrate. In any 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-carboxylic acid ethyl ester citrate salt monohydrate.

[0486] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate

[0487] 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 being substantially as 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 having 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 having a weight loss of about 33% from about 140° C. to about 220° C. Figure 2 TGA thermogram shown.

[0488] 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 has substantially Figure 1 XRPD spectrum shown in .

[0489] In any of the above embodiments, the crystalline form may be prepared by any of the methods described herein. In any of the above embodiments, the crystalline form may be substantially isolated.

[0490] In any 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-carboxylic acid ethyl ester citrate. In any 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-carboxylic acid ethyl ester citrate.

[0491] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride

[0492] 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 being substantially as 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 having a TGA thermogram with a weight loss of about 3.6% from room temperature to about 200°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 having a weight loss of about 3.6% from room temperature to about 200°C. Figure 8 TGA thermogram shown in .

[0493] 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 being substantially as follows Figure 7 XRPD spectrum shown in .

[0494] In any of the above embodiments, the crystalline form may be prepared by any of the methods described herein. In any of the above embodiments, the crystalline form may be substantially isolated.

[0495] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate

[0496] 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 being substantially as Fig.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 no significant 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 with no significant weight loss from room temperature to about 180°C. Fig.15 TGA thermogram shown in .

[0497] 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 being substantially as follows Fig.14 XRPD spectrum shown in .

[0498] In any of the above embodiments, the crystalline form may be prepared by any of the methods described herein. In any of the above embodiments, the crystalline form may be substantially isolated.

[0499] Cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester

[0500] The present application also provides at least one crystalline form of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester or its salt. In some embodiments, the present application provides at least one crystalline form of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (free base). In some embodiments, the present application provides at least one crystalline form of a salt of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester.

[0501] 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 being substantially as Fig.33 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 of about 0.34% from room temperature to about 125°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 TGA thermogram with a weight loss of about 0.34% from room temperature to about 125°C. Fig.33 TGA thermogram shown in .

[0502] 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, expressed in 2θ, 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°. 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, expressed in 2θ, 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°. 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, expressed in 2θ, 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°. 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, expressed in 2θ, 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°. 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, expressed in 2θ, 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°.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, expressed in 2θ, 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°. 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, expressed in 2θ, 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°. 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°, expressed in 2θ.

[0503] 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, expressed in 2θ, selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2°. 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, expressed in 2θ, selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2°. 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, expressed in 2θ, selected from 6.1°±0.2°, 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2°.

[0504] 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, expressed in 2θ, at 6.1°±0.2°, 17.3°±0.2°, and 17.6°±0.2°. 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, expressed in 2θ, at 6.1°±0.2°, 14.8°±0.2°, and 17.6°±0.2°. 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, expressed in 2θ, at 6.1°±0.2°, 14.8°±0.2°, and 17.3°±0.2°. 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, expressed in 2θ, at 14.8°±0.2°, 17.3°±0.2°, and 17.6°±0.2°. 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°, expressed in 2θ.

[0505] 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°, expressed in 2θ. 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°, expressed in 2θ. 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°, expressed in 2θ. 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, expressed in 2θ, 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°. 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°, expressed in 2θ. 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°, expressed in 2θ.

[0506] 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 being substantially as Fig.32 XRPD spectrum shown.

[0507] In any of the above embodiments, the crystalline form may be prepared by any of the methods described herein. In any of the above embodiments, the crystalline form may be substantially isolated.

[0508] Alternative Names

[0509] As used herein, a compound having the structural formula shown below:

[0510]

[0511] With C 19 H 30 The molecular formula of N4O2 and its molecular weight of 346.5 g / mol can be referred to by the following names and identifiers:

[0512] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0513] 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester; and

[0514] Ethyl 2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate (as named by ChemDraw Professional v. 21.0.0.28, PerkinElmer Informatics, Inc.).

[0515] As used herein, the compound of formula (1) is as follows:

[0516]

[0517] With C 19 H 30 The molecular formula of N4O2 and its molecular weight of 346.5 g / mol can be referred to by the following names and identifiers:

[0518] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0519] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0520] (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0521] (2r,4s)-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (as named by ChemDraw Professional v.21.0.0.28, PerkinElmer Informatics, Inc.); and

[0522] CAS RN:1803346-98-6.

[0523] As used herein, the compound of formula (3a) is shown below:

[0524]

[0525] 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 is 1:1; and C 25 H 38 N4O9(C 19 H 30 N4O2·C6H8O7) and a molecular weight of 538.6 g / mol; it may be referred to by the following names and identifiers:

[0526] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate;

[0527] 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate;

[0528] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, citrate (1:1); and

[0529] Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citrate (1:1).

[0530] As used herein, the compound of formula (3b) is shown below:

[0531]

[0532] wherein the molar ratio between cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester and citric acid is 1:1; and C 25 H 38 N4O9(C 19 H 30 N4O2·C6H8O7) and a molecular weight of 538.6 g / mol; it may be referred to by the following names and identifiers:

[0533] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate;

[0534] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate;

[0535] (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate;

[0536] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, citrate (1:1); and

[0537] Ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citrate (1:1).

[0538] As used herein, the compound of formula (4a) is shown below:

[0539]

[0540] wherein the molar ratio of ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citric acid and water is 1:1:1; and C 25 H 40 N4O 10 (C 19 H 30N4O2·C6H8O7·H2O) and a molecular weight of 556.6 g / mol; it may be referred to by the following names and identifiers:

[0541] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate;

[0542] 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate;

[0543] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, citrate (1:1), hydrate; and

[0544] Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, citrate (1:1), hydrate.

[0545] As used herein, the compound of formula (4b) is shown below:

[0546]

[0547] wherein the molar ratio of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, citric acid and water is 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; it may be referred to by the following names and identifiers:

[0548] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate;

[0549] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate;

[0550] (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate;

[0551] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, citrate (1:1), hydrate;

[0552] (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, citrate (1:1), hydrate; and

[0553] CAS RN:2920742-36-3.

[0554] As used herein, the compound of formula (5a) is shown below:

[0555]

[0556] wherein 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 is 1:1; and having C 19 H 31 ClN4O2(C 19 H 30 N4O2·HCl) and a molecular weight of 382.9 g / mol; may be 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;

[0557] 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride;

[0558] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, hydrochloride (1:1);

[0559] Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, hydrochloride (1:1).

[0560] As used herein, the compound of formula (5b) is shown below:

[0561]

[0562] wherein the molar ratio between cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester and hydrochloric acid is 1:1; and having C 19 H 31ClN4O2(C 19 H 30 N4O2·HCl) and a molecular weight of 382.9 g / mol; it may be referred to by the following names and identifiers:

[0563] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride;

[0564] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride;

[0565] (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride;

[0566] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, hydrochloride (1:1); and

[0567] Ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, hydrochloride (1:1).

[0568] As used herein, the compound of structural formula (6a) is shown below:

[0569]

[0570] wherein 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 is 1:1; and having C 23 H 34 N4O6(C 19 H 30 N4O2·C4H4O4) and a molecular weight of 462.6 g / mol; it may be referred to by the following names and identifiers:

[0571] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate;

[0572] 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate;

[0573] 2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, fumarate (1:1); and

[0574] Ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, fumarate (1:1).

[0575] As used herein, the compound of formula (6b) is shown below:

[0576]

[0577] wherein the molar ratio of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester to fumaric acid is 1:1; and C 23 H 34 N4O6(C 19 H 30 N4O2·C4H4O4) and a molecular weight of 462.6 g / mol; it may be referred to by the following names and identifiers:

[0578] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate;

[0579] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate;

[0580] (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate;

[0581] cis-2-[4-(1-methyl-1H-pyrazol-5-yl)-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester, fumarate (1:1); and

[0582] Ethyl (2r,4s)-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, fumarate (1:1).

[0583] As used herein, the compound of formula (7) is shown below:

[0584]

[0585] With C 20 H33 The molecular formula of F2N3O3 and its molecular weight of 401.5 g / mol can be referred to by the following names and identifiers:

[0586] cis-2-[4-[(2R)-4,4-difluoro-2-(hydroxymethyl)-1-pyrrolidinyl]-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0587] cis-2-[4-[(2R)-4,4-difluoro-2-(hydroxymethyl)-1-pyrrolidinyl]piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0588] cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0589] (2r,4S)-2-(4-((R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (as named by ChemDraw Professional v.21.0.0.28, PerkinElmer Informatics, Inc.); and

[0590] CAS RN:1803349-72-5.

[0591] As used herein, the compound of formula (8) is shown below:

[0592]

[0593] With C 20 H 34 The molecular formula of FN3O3 and its molecular weight of 383.5 g / mol can be referred to by the following names and identifiers:

[0594] cis-2-[4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)-1-pyrrolidinyl]-1-piperidinyl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0595] Cis-2-[4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)-1-pyrrolidinyl]piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester

[0596] cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester;

[0597] (2r,4S)-2-(4-((2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (as named by ChemDraw Professional v.21.0.0.28, PerkinElmer Informatics, Inc.); and

[0598] CAS RN:1803346-48-6.

[0599] definition

[0600] In this application, unless stated otherwise, the following definitions apply.

[0601] The term "treatment" in connection with the use of a compound of Formula (1), (2a), (2b), (3a), (3b), (4a), (4b), (5a), (5b), (6a), or (6b) is used to describe any form of intervention in which a compound is administered to an individual suffering from, or at risk of, or potentially at risk of suffering from, a disease or condition of interest. Thus, the term "treatment" encompasses both preventative / prophylactic treatment and treatment of measurable or detectable symptoms of a disease or condition.

[0602] As used herein, the term "therapeutically effective amount" (e.g., in connection with a method of treatment of a disease or condition) refers to an amount of a compound effective to produce a desired therapeutic effect. For example, if the condition is pain, a therapeutically effective amount is an amount sufficient to provide a desired level of pain relief. The desired level of pain relief can be, for example, complete elimination of pain or a reduction in the severity of pain.

[0603] The term "suitable leaving group" describes a group that is suitable for acting as a leaving group 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: IUPAC. Compendium of Chemical Terminology, Second Edition (the "Gold Book"). Compiled by ADMcNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). Online version (2019-) is made by SJ Chalk. ISBN 0-9678550-9-8. https: / / doi.org / 10.1351 / goldbook. Suitable leaving groups may include: sulfonate leaving groups (e.g., aryl sulfonate, alkyl sulfonate, haloalkyl sulfonate), halogen (particularly -I, -Br, -Cl), trialkylamine, dinitrogen, dialkyl ether, aryl iodonium salt and aryl sulfonium salt. In some embodiments, the leaving group is a leaving nucleus. In some embodiments, the leaving group is an aryl sulfonate. In some embodiments, the leaving group is a tosylate group.

[0604] Salt

[0605] The compounds described herein may exist in the form of salts, such as acid addition salts, or in some 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 reference to a compound also includes the salt form of the compound as defined herein.

[0606] The salts are typically acid addition salts.

[0607] 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, page 388, August 2002. Generally, such salts can be prepared by reacting the free acid or free base forms of these compounds with an appropriate base or acid in water or an organic solvent or a mixture of the two; generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is used.

[0608] Acid addition salts can be formed with a wide variety of acids (both inorganic and organic). Examples of acid addition salts falling within the scope of the present invention include mono- or di-salts formed with an acid selected from 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, butyric acid, (+) camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, 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, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic 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, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

[0609] In particular, acid addition salts can be formed from an acid selected from the group consisting of 2,5-dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, succinic acid (succinic acid), citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), glycolic 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).

[0610] The amine functional groups in the compounds described herein can form quaternary ammonium salts according to methods well known to those skilled in the art, such as by reaction with an alkylating agent. Such quaternary ammonium compounds are within the scope of the present invention.

[0611] The compounds of the present invention may exist as mono- or di-salts, depending on the pKa of the acid forming the salt.

[0612] The salt forms of the compounds of the invention are generally 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 in the form of intermediates, which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salt forms, which may be used, for example, in the purification or isolation of the compounds of the invention, also form part of the present invention.

[0613] Optical isomers

[0614] Unless the context requires otherwise, when a compound of the invention contains one or more chiral centers and can exist in two or more optical isomeric forms, reference to the compound includes all optical isomeric forms thereof (e.g., enantiomers, epimers, diastereomers, cis-trans geometric isomers of cyclic functional groups), either as an individual optical isomer or as a mixture of two or more optical isomers (e.g., a racemic mixture).

[0615] Optical isomers can be characterized and identified by their optical activity (i.e., as + and - isomers, or d and l isomers), or they can be characterized by their absolute stereochemistry using the "R and S" nomenclature developed by Cahn, Ingold and Prelog, see Advanced Organic Chemistry by Jerry March, 4 th Edition, John Wiley & Sons, New York, 1992, pp. 109-114, see also Cahn, Ingold & Prelog, Angew. Chem. Int. Ed. Engl., 1966, 5, 385-415. Compounds characterized by a cyclic moiety can be characterized using the "cis-trans" nomenclature. In the case of optical isomers, separation can be performed by a variety of 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, separating the diastereomers by preferential crystallization, and then dissociating the salts to give the individual enantiomers of the free base.

[0616] When the compounds of the present invention exist in two or more optical isomeric forms, one enantiomer of a pair of enantiomers may, for example, exhibit advantages over the other enantiomer in terms of biological activity. Therefore, in some cases, it may be desirable to use only one of a pair of enantiomers or to use only one of a plurality of diastereomers as a therapeutic agent.

[0617] Thus, the invention provides compositions containing compounds 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 is present as a single optical isomer (e.g., enantiomer or diastereomer).

[0618] In one general embodiment, 99% or more (eg, substantially all) of the total amount of the compound (or applied compound) is present as a single optical isomer.

[0619] For example, in one embodiment, the compound exists as a single enantiomer. In another embodiment, the compound exists as a single diastereomer.

[0620] The present invention also provides a mixture of optical isomers, which may be racemic or non-racemic. Therefore, the present invention provides:

[0621] A compound in the form of a racemic mixture of optical isomers.

[0622] A compound in the form of a non-racemic mixture of optical isomers.

[0623] Cis-trans isomerism

[0624] Additionally, provided herein are compositions containing compounds having one or more regions of restricted rotation that produce cis-trans isomerism, wherein at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the compound exists as a single cis-trans stereoisomer.

[0625] In one general embodiment, 99% or more (eg, substantially all) of the total amount of the compound (or applied compound) is present as a single cis-trans isomer.

[0626] For example, in one embodiment, the compound exists as a cis isomer. In another embodiment, the compound exists as a trans isomer (eg, an intermediate compound).

[0627] The present invention also provides a mixture of cis-trans isomers.

[0628] isotope

[0629] The compounds of the present invention may contain one or more isotopic substitutions, and references to a particular element include within its scope all isotopes of that element. For example, references to hydrogen include within its scope 1 H. 2 H(D) and 3 H(T). Similarly, references to carbon and oxygen include within their scope 12 C. 13 C and 14 C and 16 O and 18 O.

[0630] In a similar manner, references to specific functional groups also include isotopic variations within their scope unless the context indicates otherwise. For example, references to alkyl groups such as ethyl groups also encompass variations in which one or more hydrogen atoms in the group are in deuterium or tritium isotopic form, such as ethyl groups in which all five hydrogen atoms are in deuterium isotopic form (perdeuterioethyl groups).

[0631] Isotopes may be radioactive or non-radioactive. Compounds may not contain radioactive isotopes. Such compounds are preferably used for therapeutic purposes. However, compounds may contain one or more radioactive isotopes. Compounds containing such radioactive isotopes may be used in diagnostic situations.

[0632] Solvate

[0633] The compounds of the present invention can form solvates. Preferred solvates are solvates formed by incorporating molecules of a nontoxic pharmaceutically acceptable solvent (hereinafter referred to as a solvating solvent) into the solid state structure (e.g., crystal 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 recrystallizing the compounds of the present invention with a solvent or solvent mixture containing a solvating solvent. Whether a solvate has been formed in any given case can be determined by analyzing the crystals of the compound using well-known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray crystallography. Solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates, and dihydrates.

[0634] Therefore, the present invention provides:

[0635] Compounds in the form of solvates.

[0636] The compound wherein the solvate is a hydrate.

[0637] For a more detailed discussion of solvates and methods for preparing and characterizing them, see Bryn et al., Solid-State Chemistry of Drugs, 2nd edition, published by SSCI, Inc., West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0638] Alternatively, the compounds of the invention may be anhydrous rather than existing as hydrates. Accordingly, the invention provides compounds of the invention in an anhydrous form (eg, an anhydrous crystalline form).

[0639] Crystalline and amorphous forms

[0640] Compounds can 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 crystal structures can be characterized using a variety of 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 different humidity conditions can be studied by weight vapor sorption and also analyzed by XRPD. The determination of the crystal structure of a compound can be carried out by X-ray crystallography, which can be carried out according to conventional methods, such as those described herein and those described in Fundamentals of Crystallography, C. Giacovazzo, HL 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 single crystals. In an amorphous solid, there is no three-dimensional structure that is usually present in a crystalline form, and the positions of the molecules in an amorphous form relative to each other are essentially random, see, for example, Hancock et al., J. Pharm. Sci. (1997), 86, 1).

[0641] Other characterization methods, such as differential scanning calorimetry (DSC), differential thermal analysis (DTA), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), etc., can help further identify the form and help determine stability and solvent / water content.

[0642] The XRPD pattern of reflection (peak) is generally considered to be the fingerprint of a specific crystalline form. As is well known, the relative intensity of XRPD peaks can vary widely, especially depending on sample preparation techniques, crystal size distribution, various filters used, sample installation procedures and the specific instrument used. In some cases, depending on the type or setting of the instrument, new peaks or existing peaks can be observed to disappear. As used herein, the term "peak" refers to a reflection with a relative height / intensity of at least about 4% of the maximum peak height / intensity. In addition, instrument changes and other factors can affect 2θ values. Therefore, peak attribution, such as the peak attribution reported herein, can vary plus or minus about 0.2 ° (2θ), and the terms "substantially" and "about" as used herein in the context of XRPD are intended to cover the above-mentioned changes.

[0643] In the same manner, temperature readings associated with DSC, TGA or other thermal experiments may vary by about ±3°C depending on the instrument, specific settings, sample preparation, etc. Therefore, the crystalline forms provided herein or the term "about" "substantially" as shown in any of the accompanying drawings of the DSC thermogram should be understood to accommodate such variations. As used herein, the term "no significant weight loss" means a weight loss of less than 0.25%.

[0644] Different forms of the same substance may have different overall properties related to, for example, hygroscopicity, solubility, stability, etc. Forms with high melting points may have good thermodynamic stability, which is beneficial for extending the shelf life of drug formulations containing the solid form. Forms with lower melting points may have lower thermodynamic stability, but may be advantageous because they have increased water solubility, which may translate into increased drug bioavailability. Weakly hygroscopic forms may be desirable for their stability to heat and humidity, and may be resistant to degradation during long-term storage.

[0645] Therefore, the present invention provides:

[0646] A compound in crystalline form.

[0647] A compound which is:

[0648] (a) 50% to 100% crystalline, and 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, for example 100% crystalline.

[0649] The compound is in amorphous form.

[0650] In some embodiments, the crystalline form provided herein is substantially isolated. "Substantially isolated" means that the compound or crystalline form is at least partially or substantially separated from the environment in which it is formed or detected. Partial separation can include, for example, a composition rich in salts described herein. Substantially separating can include a composition 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 a crystalline form described herein or a salt thereof.

[0651] 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 a compound or salt described herein.

[0652] Prodrug

[0653] The compounds of the present invention may exist in the form of prodrugs."Prodrug"means any compound that is converted in vivo into a biologically active compound of the present invention, for example.

[0654] 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 produce the active drug. Such esters can be formed, for example, by esterification of any hydroxyl groups present in the parent compound, wherein any other reactive groups present in the parent compound are protected in advance as appropriate, and then deprotected if necessary.

[0655] In addition, some prodrugs are activated enzymatically to produce the active compound, or to produce a compound that, after further chemical reaction, produces the active compound (e.g., as in 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.

[0656] Thus, the present invention provides prodrugs of compounds of the present invention, wherein the compounds contain a functional group which can be converted under physiological conditions to form a hydroxyl or amino group.

[0657] Complexes and Clathrates

[0658] Complexes of the compounds of the invention (eg, inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals) are also contemplated.

[0659] Thus, the present invention provides compounds in the form of complexes or clathrates.

[0660] Biological activity and therapeutic uses

[0661] The compounds of the present invention have activity as muscarinic M4 receptor agonists (WO2015 / 118342).

[0662] A significant advantage of certain compounds of the present invention is that they are highly selective for the M4 receptor relative to the M1, M2 and M3 receptor subtypes. For example, although the compounds of the present invention generally have a pEC of at least 6 for the M4 receptor in the functional assay described in Example A of WO2015 / 118342, 50 Value and E greater than 70 max However, in the functional assay of Example A of WO2015 / 118342, when tested against M1, M2 and M3 subtypes, they can have a pEC value of less than 5. 50 value and less than 20% of E max value.

[0663] Regarding the compounds of the present invention, the present invention also provides:

[0664] Compounds used in medicine.

[0665] Compounds that act as muscarinic M4 receptor agonists.

[0666] A compound which is a muscarinic M4 receptor agonist having a pEC of greater than 6 for the M4 receptor in an assay of Example A herein or an assay substantially similar thereto 50 and an E of at least 70 max .

[0667] Compounds having a pEC greater than 7.0 50 A muscarinic M4 receptor agonist.

[0668] Compounds having an E of at least 70 for the M4 receptor max .

[0669] Compounds that are selective for the M4 receptor compared to the muscarinic M1, M2 and M3 receptors.

[0670] Compounds having a pEC of less than 5 for muscarinic M1, M2 and M3 receptor subtypes 50 and E less than 30 max .

[0671] Compounds for use in treating a disease or condition mediated by the muscarinic M4 receptor.

[0672] Compounds having a pEC greater than 7.0 50 A muscarinic M1 receptor agonist.

[0673] Compounds having an E of at least 70 for the M1 receptor max .

[0674] Compounds that are selective for the M1 receptor compared to the muscarinic M2 and M3 receptors.

[0675] Compounds having a pEC of less than 5 for muscarinic M2 and M3 receptor subtypes 50 and E less than 30 max .

[0676] Compounds for use in treating diseases or conditions mediated by muscarinic M1 receptors.

[0677] Compounds for use in treating a disease or condition mediated by a muscarinic M1 receptor or a muscarinic M4 receptor.

[0678] By virtue of their muscarinic M4 receptor agonist activity, the compounds of the present invention can be used to treat Alzheimer's disease, Lewy body dementia, schizophrenia and other psychotic disorders, cognitive disorders and other diseases mediated by muscarinic M4 receptors, and can also be used to treat various types of pain.

[0679] Therefore, with respect to the compounds of the present invention, the present invention also provides:

[0680] Compounds for use in the treatment of cognitive or psychotic disorders.

[0681] Compounds for use in the treatment of cognitive disorders or psychotic disorders, wherein the cognitive disorders or psychotic disorders include, are caused by, or are associated with a condition selected from the group consisting of cognitive impairment, mild cognitive impairment (MCI) (including amnestic MCI and non-amnestic MCI, and including mild cognitive impairment caused by Alzheimer's disease and / or prodromal Alzheimer's disease), 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 (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 "Early Alzheimer's disease: Developing Drugs forTreatment” is available at fda.gov / download / Drugs / GuidanceComplianceRegulatorylnformation / Guidances / UCM596728.pdf), progressive supranuclear palsy, cognitive dysfunction including attention, orientation, learning impairment, memory (i.e., memory impairment, amnesia, amnestic disorder, transient global amnesia syndrome, and age-related memory impairment), and language function; cognitive impairment caused by stroke, Huntington's disease, Pick's disease, AIDS-related dementia or other dementing conditions (such as multi-infarct dementia, alcoholic dementia, hypothyroid dementia, and dementia associated with other degenerative conditions such as cerebellar atrophy and amyotrophic lateral sclerosis); other acute or subacute conditions that can cause cognitive decline, such as delirium or depression (pseudo-dementia conditions); trauma, head trauma, age-related cognitive decline, stroke, nervous system degeneration, drug-induced conditions, neurotoxic agents, age cognitive impairment associated with schizophrenia, cognitive impairment associated with autism, Down syndrome, cognitive deficits associated with psychosis, and cognitive impairment associated with electroconvulsive therapy; cognitive impairment caused by drug abuse or withdrawal including nicotine, marijuana, 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, paranoia, hallucinations and delusional disorders, personality disorders, obsessive-compulsive disorder, schizotypal disorder, delusional disorder, psychosis due to malignant disease, metabolic disorders, endocrine diseases or narcolepsy, psychosis caused by drug abuse or withdrawal, bipolar disorder, and schizoaffective psychotic disorder.

[0682] Compounds for use in the treatment of cognitive disorders or psychotic disorders, wherein the cognitive disorders or psychotic disorders are selected from, including conditions selected from, caused by, or associated with conditions selected from: cognitive impairment, mild cognitive impairment (MCI), (including amnestic MCI and non-amnestic MCI, and 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, Friedreich's ataxia, Down syndrome, Huntington's disease, hyperkinesia, mania, Tourette's syndrome, Alzheimer's disease (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 "Early Alzheimer's disease: Developing Drugs for Treatment" available at fda.gov / download / Drugs / GuidanceComplianceRegulatorylnformation ... dances / UCM596728.pdf), progressive supranuclear palsy, cognitive dysfunction including attention, orientation, learning impairment, memory (i.e., memory impairment, amnesia, amnestic disorder, transient global amnesia syndrome, and age-related memory impairment), and language function; other acute or subacute conditions that may cause cognitive decline, such as delirium or depression (pseudo-dementia conditions); trauma, head trauma, age-related cognitive decline, stroke, nervous system degeneration, drug-induced conditions, neurotoxic agents, age-related cognitive impairment, autism-related cognitive impairment, Down syndrome, cognitive deficits associated with psychosis, and post-electroconvulsive therapy cognitive disorders caused by drug abuse or withdrawal including nicotine, marijuana, 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, paranoia, hallucinations and delusional disorders, personality disorders, obsessive-compulsive disorder, schizophrenia, delusional disorders, psychosis due to malignant disease, metabolic disorders, endocrine diseases or narcolepsy, psychosis caused by drug abuse or withdrawal, bipolar disorder, schizoaffective disorder and Alzheimer's psychosis.

[0683] Compounds for use in the treatment of schizophrenia, Alzheimer's disease, Alzheimer's psychosis, or bipolar disorder.

[0684] Compounds for use in the treatment of schizophrenia, Alzheimer's psychosis or bipolar disorder.

[0685] Compounds for the treatment of Alzheimer's disease.

[0686] Compounds for the treatment of Lewy body dementia.

[0687] Compounds for use in the treatment of schizophrenia.

[0688] Compounds for use in the treatment of bipolar disorder.

[0689] Compounds for the treatment of Alzheimer's psychosis.

[0690] A method of treating cognitive disorders in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), the method comprising administering a therapeutically effective amount of a compound of the invention.

[0691] A method of treating a cognitive disorder in a subject (e.g., a mammalian patient, such as a human, e.g., a human in need of such treatment), the method comprising administering a therapeutically effective dose of a compound of the invention, wherein the cognitive disorder comprises, is caused by, or is associated with a condition as defined above.

[0692] A method of treating a cognitive disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), the method comprising administering a therapeutically effective amount of a compound of the invention, wherein the cognitive disorder is caused by or associated with Alzheimer's disease.

[0693] A method of treating a cognitive disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), the method comprising administering a therapeutically effective amount of a compound of the invention, wherein the cognitive disorder is dementia with Lewy bodies.

[0694] A method of treating a cognitive disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), the method comprising administering a therapeutically effective amount of a compound of the invention, wherein the cognitive disorder is schizophrenia.

[0695] A method of treating a cognitive disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), the method comprising administering a therapeutically effective amount of a compound of the invention, wherein the cognitive disorder is bipolar disorder.

[0696] A method of treating a cognitive disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), the method comprising administering a therapeutically effective amount of a compound of the invention, wherein the cognitive disorder is Alzheimer's psychosis.

[0697] The invention relates to use of the compound of the present invention for preparing a drug for treating cognitive impairment.

[0698] Use of a compound of the invention for the preparation of a medicament for the treatment of cognitive disorders, wherein the cognitive disorders comprise, are caused by or are associated with a condition as defined above.

[0699] The invention relates to a method for preparing a drug for treating cognitive disorders, wherein the cognitive disorders include Alzheimer's disease, are caused by Alzheimer's disease or are related to Alzheimer's disease.

[0700] The invention relates to a method for preparing a medicament for treating cognitive disorders, wherein the cognitive disorders include, are caused by or are associated with Lewy body dementia.

[0701] The invention relates to the use of the compound of the present invention for preparing a medicament for treating cognitive disorders, wherein the cognitive disorders include schizophrenia, are caused by schizophrenia or are related to schizophrenia.

[0702] The invention relates to a method for preparing a drug for treating cognitive disorders, wherein the cognitive disorders include bipolar disorders, are caused by bipolar disorders or are related to bipolar disorders.

[0703] The invention relates to a method for preparing a medicament for treating cognitive disorders, wherein the cognitive disorders include, are caused by or are associated with Alzheimer's psychosis.

[0704] A method of treating a psychotic disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), the method comprising administering a therapeutically effective amount of a compound of the invention.

[0705] A method of treating a psychotic disorder in a subject (e.g., a mammalian patient, such as a human, e.g., 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 comprises, is caused by, or is associated with a condition as defined above.

[0706] A method of treating a psychotic disorder in a subject (e.g., a mammalian patient, such as a human, e.g., a human in need of such treatment), comprising administering a therapeutically effective amount of a compound of the invention, wherein the psychotic disorder is caused by or associated with Alzheimer's disease.

[0707] A method of treating a psychotic disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), comprising administering a therapeutically effective amount of a compound of the invention, wherein the psychotic disorder is dementia with Lewy bodies.

[0708] A method of treating a psychotic disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), comprising administering a therapeutically effective amount of a compound of the invention, wherein the psychotic disorder is schizophrenia.

[0709] A method of treating a psychotic disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), comprising administering a therapeutically effective amount of a compound of the invention, wherein the psychotic disorder is bipolar disorder.

[0710] A method of treating a psychotic disorder in a subject (eg, a mammalian patient, such as a human, eg, a human in need of such treatment), comprising administering a therapeutically effective amount of a compound of the invention, wherein the psychotic disorder is Alzheimer's psychosis.

[0711] The invention relates to the use of the compounds of the invention for the preparation of medicaments for the treatment of psychotic disorders.

[0712] Use of a compound of the invention for the preparation of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder comprises, is caused by or is associated with a condition as defined above.

[0713] The use of the compounds of the present invention for the preparation of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder comprises, is caused by or is associated with Alzheimer's disease.

[0714] Use of the compounds of the present invention for the preparation of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder comprises, is caused by or is associated with Lewy body dementia.

[0715] The use of the compounds of the present invention for the preparation of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder comprises, is caused by or is associated with schizophrenia.

[0716] The use of the compounds of the present invention for the preparation of a medicament for treating a psychotic disorder, wherein the psychotic disorder comprises, is caused by or is associated with a bipolar disorder.

[0717] Use of the compounds of the present invention for the preparation of a medicament for the treatment of a psychotic disorder, wherein the psychotic disorder comprises, is caused by or is associated with Alzheimer's psychosis.

[0718] Compounds for treating or lessening the severity of acute pain, chronic pain, neuralgia or inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, general neuralgia, visceral pain, osteoarthritis pain, postherpetic neuralgia, diabetic neuropathy, radicular pain, sciatica, back pain, head or neck pain, severe or intractable pain, nociceptive pain, breakthrough pain, postoperative pain or cancer pain.

[0719] A method for treating or lessening the severity of acute pain, chronic pain, neuralgia or inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, general neuralgia, visceral pain, osteoarthritis pain, postherpetic neuralgia, diabetic neuropathy, radicular pain, sciatica, back pain, head or neck pain, severe or intractable pain, nociceptive pain, breakthrough pain, postoperative pain or cancer pain, comprising administering a therapeutically effective amount of a compound of the present invention.

[0720] Compounds for the treatment of peripheral disorders, such as reducing intraocular pressure in glaucoma and treating dry eyes and dry mouth including Sjögren's syndrome.

[0721] A method of treating peripheral disorders, such as reducing intraocular pressure in glaucoma and treating dry eyes and dry mouth including Sjögren's syndrome, comprising administering a therapeutically effective amount of a compound of the invention.

[0722] Use of the compounds of the present invention in the preparation of a medicament for treating or alleviating the severity of acute pain, chronic pain, neuralgia or inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, general neuralgia, visceral pain, osteoarthritis pain, postherpetic neuralgia, diabetic neuropathy, radicular pain, sciatica, back pain, head or neck pain, severe or intractable pain, nociceptive pain, breakthrough pain, postoperative pain or cancer pain, or for treating peripheral conditions, such as reducing intraocular pressure in glaucoma and treating dry eyes and dry mouth including Sjögren's syndrome.

[0723] The compounds of the invention are useful for treating skin lesions caused by, for example, pemphigus vulgaris, dermatitis herpetiformis, pemphigoid and other blistering skin conditions.

[0724] The compounds of the invention are useful for treating, preventing, ameliorating or reversing conditions associated with altered gastrointestinal function and motility, such as functional dyspepsia, irritable bowel syndrome, gastroesophageal acid reflux (GER) and esophageal dysmotility, symptoms of gastroparesis and chronic diarrhea.

[0725] The compounds of the invention are useful in the treatment of olfactory dysfunctions such as Bosma-Henkin-Christiansen syndrome, chemical (eg, selenium and silver) poisoning, hypopituitarism, Kallmann Syndrome, skull fractures, tumor treatment, and underactive thyroid gland.

[0726] Use of the compounds of the present invention for treating addiction.

[0727] The compounds of the present invention are useful for the treatment of 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 driving the disease.

[0728] Use of compounds of the invention for treating behavioral and psychiatric symptoms of dementia (BPSD; including agitation, verbal aggression, physical aggression, depression, anxiety, abnormal motor behavior, hyperactivity, irritability, apathy, disinhibition, impulsivity, delusions, hallucinations, sleep changes and appetite changes).

[0729] Process for preparing the compounds of the present invention

[0730] The compounds of the present invention can be prepared according to synthetic methods known to those skilled in the art and the methods described herein. The compound can also be prepared according to the method described in WO2015 / 118342. Once a compound of the present invention or its protected derivative is formed, it can be converted into another compound of the present invention by methods well known to those skilled in the art. For example, the compounds of the present invention can be converted into a salt form or an alternative salt form of the compounds of the present invention. Examples of such synthetic procedures are described in standard texts, such as Advanced Organic Chemistry and Organic Syntheses (see above references) or Fiesers'Reagents for Organic Synthesis, Volumes 1-17, John Wiley, edited by Mary Fieser (ISBN: 0-471-58283-2).

[0731] It may be necessary to protect one or more groups to prevent reactions at undesired 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).

[0732] The compounds prepared by the foregoing methods can be isolated and purified by any of a variety of methods well known to those skilled in the art, and examples of such methods include recrystallization and chromatographic techniques, such as column chromatography (eg flash chromatography) and HPLC.

[0733] As used herein, the term "react" is used as known in the art, and generally refers to bringing together chemical reagents in a manner that allows them to interact at the molecular level to achieve a chemical or physical transformation. In some embodiments, the reaction involves two reagents, wherein 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 period of time and under conditions suitable for preparing the identified product.

[0734] The reaction of the methods described herein can be carried out in a suitable solvent that can be easily selected by a technician in the field of organic synthesis. Suitable solvents can be substantially unreactive with starting materials (reactants), intermediates or products at a temperature for carrying out the reaction (e.g., a temperature in the range of the freezing temperature of the solvent to the boiling temperature of the solvent). A given reaction can be carried out in a solvent or a mixture of more than one solvent. According to the specific reaction steps, a suitable solvent for a specific reaction step can be selected.

[0735] 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.

[0736] 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, tert-butyl methyl ether, mixtures thereof, and the like.

[0737] By way of example, suitable protic solvents may include, but are not limited to, water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 2-ethoxyethanol, diethylene glycol, 1-pentanol, 2-pentanol or 3-pentanol, neopentyl alcohol, tert-amyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenol or glycerol.

[0738] By way of example, suitable aprotic solvents may include, but are not limited to, 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-methylpyrrolidone (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.

[0739] Suitable hydrocarbon solvents include benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o- or p-xylene, octane, indane, nonane or naphthalene.

[0740] 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.

[0741] The reaction of the method described herein can be carried out at an appropriate temperature, which can be easily determined by those skilled in the art. The reaction temperature will depend on, for example, the melting and boiling points of the reagents and solvents (if any); the thermodynamics of the reaction (e.g., a violent exothermic reaction may need to be carried out at a reduced temperature); the kinetics of the reaction (e.g., a high activation barrier may require an elevated temperature); and the temperature range (any enzyme component is active within this temperature range) / the temperature at which any enzyme component denatures.

[0742] The expressions "ambient temperature" and "room temperature" or "rt" used herein are understood in the art and generally refer to a temperature, such as reaction temperature, about the temperature in the room where the reaction is carried out, such as a temperature of about 20°C to about 30°C.

[0743] The reactions of the methods described herein can be carried out in air or under an inert atmosphere. Generally, reactions involving reagents or products that are substantially reactive with air can be carried out using air-sensitive synthesis techniques well known to those skilled in the art.

[0744] Ketoreductase

[0745] Ketoreductase (KRED, also known as 'alcohol dehydrogenase' ADH, or 'carbonyl reductase') catalyzes the reduction of aldehydes and ketones to the corresponding primary and secondary alcohols, respectively.

[0746] The reduction catalyzed by KRED requires a reduced cofactor as an 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.

[0747] In vitro use of KRED in the reduction process requires a cofactor regeneration system to regenerate from NADP + Regeneration of NADPH or from NAD + Regenerate NADH. Common cofactor regeneration systems are glucose dehydrogenase (GDH), which uses glucose as a feedstock, or formate dehydrogenase, which uses formate as a feedstock. These cofactor regeneration systems can be used in conjunction with KRED.

[0748] KRED is a ubiquitous enzyme found in all kingdoms of life. As is well known, commercially available KRED is derived from horse liver (HLADH), baker's yeast (YADH), and bacteria such as Thermoanaerobium brockii (TBADH) and Lactobacillus kefir (LKADH).

[0749] For industrial applications, it is desirable to use KREDs with high specific activity and stereoselectivity. Another important criterion in the industrial use of KREDs is long process stability, which is usually associated with high stability at elevated temperatures and high solvent stability. It may also be desirable to use KREDs with high stereospecificity.

[0750] The enzyme A described herein and used in the methods described herein is a ketoreductase (KRED) having the amino acid sequence SEQ ID NO: 1:

[0751] MNSVQSQGTALITGASSGIGAIYAERLAARGFDLLLVARDKARLDSAASQLRDAHGVQVEVWKADLTQKDDVIKLEQRLRSDSSISSLLINNAGVAADGPLANADMDQLERLIQLNITAVTRLASAAAASFAKAGRGTIINIASVVALFPERFNATYTASKAYVLSLTQSLNAELEGSGVQIQAVLPG

[0752] VTRTEIWERSGIDASGIPAEMVMDAGEMVDAALAGLDQGELVTIPSLPDAGEWQSFVAARHVMAPNLSRSAAAQRYKSGH.

[0753] Enzyme A can be produced by methods known in the art. For example, methods for expressing enzymes in cell (eg, microbial) expression systems are well known and conventional to those skilled in the art.

[0754] Enzyme A can be provided or produced by a host cell (e.g., a microbial cell, such as E. coli) comprising a polynucleotide and / or an 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.

[0755] The polynucleotide can be DNA or RNA. The polynucleotide can be single-stranded or double-stranded. The polynucleotide can be provided in an isolated / purified form, or provided in a host cell.

[0756] Owing to the codons corresponding to various amino acids, the availability of polypeptide sequences provides a description of all polynucleotides capable of encoding polypeptides of the present invention. The degeneracy of the genetic code (wherein the same amino acid is encoded by alternative codons or synonymous codons) allows the generation of extremely large amounts of nucleic acids, all of which encode disclosed enzymes. Therefore, in the case of having identified a specific amino acid sequence, those skilled in the art can prepare 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 each possible variant of the polynucleotides that can be generated by selecting combinations based on possible codon options.

[0757] Enzyme A may be encoded by a polynucleotide and / or an expression vector comprising a nucleotide sequence according to SEQ ID NO: 2:

[0758] ATGAATTCTGTGCAGTCTCAAGGTACGGCTCTGATCACTGGCGCCTCGTCCGGTATCGGTGCGATTTACGCCGAGCGTCTGGCGGCGCGTGGTTTTGATCTGTTGCTGGTGGCCCGTGACAAGGCCCGTCTGGACAGCGCCGCCAGCCAATTGCGCGACGCTCACGGTGTGCAGGTCGAGGTGTGGAAAGCGGATCTGACCCAAAAGGACGACGTGATCAAACTCGAACAGCGCTTGCGCAGCGATTCGAGCATCAGCCTGCTGATTAACAATGCCGGCGTGGCCGCCGACGGCCCGCTGGCCAATGCCGACATGGATCAACTGGAACGCCTGATCCAGTTGAACATCACCGCCGTCACGCGTCTGGCGTCGGCCGCCGCTGCCAGTTTCGCCAAGGCAGGTCGCGGCACGATCATCAACATCGCCTCGGTCGTGGCGCTGTTCCCCGAGCGTTTCAATGCGACCTACACCGCCAGCAAGGCCTATGTGTTGAGTCTGACCCAATCGTTGAACGCGGAGCTCGAAGGCTCCGGCGTGCAGATCCAGGCCGTGCTGCCGGGCGTGACCCGCACTGAAATCTGGGAGCGTTCGGGGATCGACGCCAGTGGCATTCCGGCGGAAATGGTCATGGACGCCGGGGAGATGGTGGATGCCGCCCTGGCCGGTCTGGATCAGGGCGAACTGGTCACCATTCCATCGCTGCCCGATGCCGGCGAATGGCAGTCGTTTGTGGCGGCGCGCCATGTCATGGCGCCGAACCTTTCGCGCAGCGCTGCAGCCCAGCGCTACAAATCAGGTCATTGA。

[0759] The polynucleotide may consist of SEQ ID NO:2.

[0760] The polynucleotide can be operably linked to one or more heterologous regulatory sequences 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 can be codon optimized for optimal production by a host organism selected for expression. For example, preferred codons for use in bacteria are for expressing genes in bacteria; preferred codons for use in yeast are for expression in yeast; and preferred codons for use in mammals are for expression in mammalian cells.

[0761] The polynucleotide encoding the disclosed enzyme can be manipulated in a variety of ways to provide expression of the polypeptide. Depending on the expression vector, the isolated polynucleotide may need or must be manipulated prior to its insertion into the expression vector. The 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.

[0762] 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 Escherichia coli lactose operon, the Streptomyces coelicolor agarohydrolase gene (dagA), the Bacillus subtilis fructosucrase gene (sacB), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (Villa-Kamaroff et al., 1978, Proc. Natl Acad. Sci. USA 75:3727-3731) and tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80:21-25). Other promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al., supra.

[0763] The control sequence may also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region, which is naturally linked to a fragment of the coding region encoding the secreted polypeptide in the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is unrelated to the coding sequence. When the coding sequence does not naturally contain a signal peptide coding region, an exogenous signal peptide coding region may be required.

[0764] Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell may be used.

[0765] Any suitable vector, promoter, enhancer and stop codon known in the art can be used to express polypeptides from the vectors 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).

[0766] Polynucleotide and / or expression vector can be synthesized by conventional DNA synthesis technology.In the case of the sequence of known engineered polypeptide, polynucleotide of encoding enzyme can be prepared by standard solid phase method according to known synthesis method.Fragments up to about 100 bases can be synthesized separately, and then connected (for example, by enzymatic or chemical connection method, or polymerase-mediated method) to form any desired continuous sequence.For example, disclosed polynucleotide can be prepared by chemical synthesis, for example, using Beaucage et al., 1981, the classical phosphoramidite method described by Tet Lett 22:1859-69, or Matthes et al., 1984, the method described by EMBO J.3:801-05, for example, as generally implemented in automated synthesis method.According to the phosphoramidite method, oligonucleotide is synthesized, for example, synthesized, purified, annealed, connected and cloned into suitable vector in automatic DNA synthesizer. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources, 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 the like.

[0767] Pharmaceutical preparations

[0768] While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (eg, formulation).

[0769] Accordingly, there is provided a pharmaceutical composition comprising at least one compound of the invention and at least one pharmaceutically acceptable excipient.

[0770] The composition may be a tablet composition. The composition may be a capsule composition.

[0771] Pharmaceutically acceptable excipients can be selected from, for example, carriers (e.g., solid, liquid or semisolid carriers), adjuvants, diluents (e.g., solid diluents such as fillers or extenders; and liquid diluents such as solvents and co-solvents), granulating agents, binders, glidants, coating agents, controlled release agents (e.g., polymers or waxes that retard or delay release), binders, disintegrants, buffers, lubricants, preservatives, antifungal and antibacterial agents, antioxidants, buffers, tonicity regulators, thickeners, flavoring agents, sweeteners, pigments, plasticizers, taste-masking agents, stabilizers or any other excipients conventionally used in pharmaceutical compositions.

[0772] The term "pharmaceutically acceptable" as used herein means compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of individuals (e.g., human individuals) without excessive toxicity, irritation, allergic response, or other problems or complications, 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.

[0773] Pharmaceutical compositions containing compounds of the invention can be formulated according to known techniques, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

[0774] The pharmaceutical composition may be in any form suitable for oral, parenteral, topical, intranasal, intratracheal, 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 may contain a unit dose of the active compound and 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 cellulose or a derivative thereof, such as microcrystalline cellulose (MCC), methylcellulose, ethylcellulose, hydroxypropylmethylcellulose and starch, such as corn starch. Tablets may also contain standard ingredients such 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), buffers (e.g., phosphate or citrate buffers), and effervescent agents (such as citrate / bicarbonate mixtures). Such excipients are well known and need not be discussed in detail here.

[0775] Tablets may be designed to release the drug upon contact with gastric fluid (immediate release tablets) or to release the drug in a controlled manner over an extended period of time or in a specific area of ​​the gastrointestinal tract (controlled release tablets).

[0776] The pharmaceutical composition generally comprises about 1% (w / w) to about 95% (w / w), preferably % (w / w) of active ingredient and 99% (w / w) to 5% (w / w) of pharmaceutically acceptable excipients (e.g., as defined above) or a combination of such excipients. Preferably, the composition comprises about 20% (w / w) to about 90% (w / w) of active ingredient and 80% (w / w) to 10% of pharmaceutical excipients or a combination of excipients. The pharmaceutical composition comprises about 1% to about 95%, preferably about 20% to about 90% of active ingredient. The pharmaceutical composition according to the present invention can be, for example, in the form of a unit dosage form, such as an ampoule, a vial, a suppository, a prefilled syringe, a dragee, a powder, a tablet or a capsule.

[0777] Tablets and capsules may contain, for example, 0-20% (w / w) of disintegrants, 0-5% (w / w) of lubricants, 0-5% (w / w) of glidants and / or 0-99% (w / w) of fillers and / or bulking agents (depending on the drug dose). They may also contain 0-10% (w / w) of polymer binders, 0-5% (w / w) of antioxidants, 0-5% (w / w) of pigments. In addition, slow-release tablets typically additionally contain 0-99% (w / w) of polymers that control release (e.g., delay) (depending on the dose). The film coating of a tablet or capsule typically contains 0-10% (w / w) of a polymer, 0-3% (w / w) of a pigment and / or 0-2% (w / w) of a plasticizer.

[0778] Parenteral formulations typically contain 0-20% (w / w) buffer, 0-50% (w / w) cosolvents and / or 0-99% (w / w) water for injection (WFI) (depending on dose and whether lyophilized). Formulations for intramuscular depots may also contain 0-99% (w / w) oil.

[0779] Pharmaceutical formulations may be provided to the patient in a "patient pack" containing an entire course of treatment in a single package, usually a blister pack.

[0780] The compounds of the present invention are generally presented in unit dosage form and therefore generally contain enough compound to provide the desired level of biological activity. For example, the formulation may contain 1 nanogram to 2 grams of active ingredient, such as 1 nanogram to 2 milligrams of active ingredient. Within these ranges, specific subranges of the compound are 0.1 milligram to 2 grams of active ingredient (more typically 10 milligrams to 1 gram, such as 50 milligrams to 500 milligrams) or 1 microgram to 20 milligrams (e.g. 1 microgram to 10 milligrams, such as 0.1 milligram to 2 milligrams of active ingredient).

[0781] For oral compositions, unit dosage forms may contain 1 mg to 2 g, more usually 10 mg to 1 g, for example 50 mg to 1 g, for example 100 mg to 1 g, of active compound.

[0782] The active compound is administered to a patient in need thereof (eg, a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect (effective amount). The precise amount of compound administered can be determined by the supervising physician according to standard procedures. Example

[0783] The present invention will now be illustrated by reference to the specific embodiments described in the following example compounds and synthetic methods, but the present invention is not limited thereto. The biological activity of an isomer of ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate was demonstrated in WO2015 / 118342.

[0784] General methods and materials

[0785] The following general methods and materials are illustrative of methods and materials used during the methods described below.

[0786] NMR

[0787] NMR analyses were performed on a Bruker 400 Avance III NMR spectrometer.

[0788] X-Ray Powder Diffraction (XRPD):

[0789] XRPD analysis was performed on a Rigaku powder X-ray diffractometer Miniflex 600 serial number BD70000351-01. For analysis, approximately 0.5 to 1 mg of sample was added to the PXRD zero background sample holder. The powder was gently pressed down with a piece of weighing paper, and the sample holder was placed in the sample changer. Operating parameters: Miniflex counting detector, Kb filter (x2), scanning axis θ / 2θ, continuous mode, start (deg) 2.0, stop (deg) 45.0, step size (deg) 0.020, speed (deg / min) 10.0, rotation-yes, voltage (kV) 40, current (mA) 15.

[0790] It should be understood that based on many factors known to those skilled in the art, such as preferred directional effects, preparation techniques, sample installation procedures, instruments used, etc., for the same crystalline form, the peak intensity can vary from one diffraction pattern to another. In some cases, the peak intensity can vary quite significantly. Therefore, the diffraction peak intensities shown herein are exemplary and do not necessarily require the same diffraction peak intensities. Those skilled in the art will be able to easily compare the diffraction patterns provided herein with the diffraction patterns produced by the unknown crystal form, and confirm whether the diffraction patterns characterize the same crystalline form or different crystalline forms provided herein.

[0791] Thermal Analysis

[0792] Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) analyses were performed on a TA Instruments Discovery 2500 calorimeter with serial number 2500-00547 (DSC) and Discovery 5500 with serial number 5500-0126 (TGA).

[0793] For TGA analysis, a standard aluminum sample pan was placed in a platinum TGA pan and the instrument was tared blank. Approximately 1 to 5 mg of sample was added to the standard aluminum pan and analyzed at 10°C / min up to 400°C.

[0794] For DSC analysis, obtain and record T zero Plate and T zero Weigh about 1 to 3 mg of sample to T zero Plate and press T zero The pan was transferred to the DSC autosampler for analysis. The method used for analysis was a ramp to 225°C at 10°C / min. A reference pan was prepared using the same procedure but without the sample.

[0795] For DSC and TGA, it is known that the temperature observed for a thermal event will depend on sample purity, and may also depend on the rate of temperature change, as well as sample preparation techniques and the instrument used. Therefore, the values ​​reported herein for a DSC thermogram may vary by plus or minus 3°C. For example, "about 176°C" means 176°C ± 3°C. The values ​​reported herein for a DSC thermogram may also vary by plus or minus 10 joules / gram (i.e., ± 10 joules / gram).

[0796] Hygroscopicity

[0797] 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 to 10 mg of sample was placed in a quartz crucible. The crucible was then transferred to the sorption analyzer, dried at 60°C for approximately 1 hour, and subsequently analyzed at 25°C in 10% RH steps for no more than 240 minutes, from 0% RH up to 90% RH, with a stability standard of 0.01%.

[0798] Microscopy

[0799] Polarized Light Microscopy (PLM) and Scanning Electron Microscopy (SEM):

[0800] Imaging was performed on 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 positioned on a microscope where polarization images were captured.

[0801] Scanning electron microscopy (SEM) was used to evaluate morphology and particle size. Images were taken using a Phenom ProX scanning electron microscope with serial number MVE052018-0480-S. The sample was added to a portion of the SEM mounting table using carbon conductive double-coated tape. The mounting table was tapped to remove excess particles and then gold sputter coated with a Cressington 108. Imaging was performed with a SED detector at 5 to 15 kV.

[0802] Chiral RP-HPLC Method :

[0803] System: Shimadzu LC-40

[0804] Column: Phenomenex Lux i-Amylose3 250x 4.6mm 5μm

[0805] Mobile phase: A mixture of water and acetonitrile (60 / 40 water / acetonitrile) with 0.1% phosphoric acid.

[0806] Flow rate: 1mL / min.

[0807] Column temperature: 40°C

[0808] Running time: 15 minutes.

[0809] Detection: A refractive index detector (RID) was used.

[0810] Retention time:

[0811]

[0812] Sample preparation: 0.5 mL of acetonitrile was added to 0.5 mL of the reaction sample 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. An aliquot of the prepared sample was transferred to a glass vial and analyzed using a chiral RP-HPLC method.

[0813] Calculation of diastereomeric excess:

[0814] Diastereomeric excess (%de) was calculated using the following equation:

[0815]

[0816] Among them C 反式异构体 corresponds to the area % of the peak in the HPLC chromatogram generated using the chiral RP-HPLC method and has a retention time of 5.6 min; and C 顺式异构体 Corresponds to the area % of the peak in the HPLC chromatogram generated using the chiral RP-HPLC method and has a retention time of 7.1 min.

[0817] Screening of KRED enzymes

[0818] A series of KRED enzymes were screened for their effectiveness in converting Formula (B) to Formula (A) using photometry. 288 different KRED enzymes were provided as dried enzyme preparations in 96-well microtiter plates. The enzymes were dissolved in appropriate buffer solutions to produce clear solutions necessary for photometric measurements and screening. Photometric measurements recorded a decrease in absorbance at 340 nm, which corresponds to the enzyme consuming the NADPH cofactor for the reduction reaction.

[0819] Analyze the collected data and select the enzyme that either shows a negative slope within 10 minutes of reaction time (select the lowest number first) or shows a total absolute absorbance below 0.7 and aborts the decrease within 10 minutes. A low initial absorbance indicates high enzyme activity that consumes the cofactor faster than the technician can transfer the MTP to the MTP reader.

[0820] Those enzymes that met the selection criteria were provided to 0.5 mL biocatalytic reactions and subjected to HPLC validation using a chiral RP-HPLC method.

[0821] Biocatalytic conditions:

[0822] Ketone 20g / L (88mM)

[0823] NADP 5mM

[0824] DMSO 5%(v / v)

[0825] MgCl2 2mM

[0826] Glucose 178mM (2 equivalents)

[0827] GDH (glucose dehydrogenase, CDX-901, Codexis Inc.) 1 g / L

[0828] Potassium phosphate buffer 100 mM pH 7.0

[0829] 0.5 mL, 30°C, 1000 rpm shaking, 24 h reaction time

[0830] As shown in Table 1 below, Enzyme A significantly outperformed the next best enzyme (Enzyme BF) in converting the starting material while maintaining the highest level of stereoselectivity (diastereomeric excess) observed for the trans alcohol (Formula (A)).

[0831] Table 1

[0832]

[0833] Production of synthetic DNA encoding enzyme A

[0834] A synthetic DNA having SEQ ID NO: 2 and encoding enzyme A was synthesized with codon optimization for E. coli expression.

[0835] SEQ ID NO:2

[0836] ATGAATTCTGTGCAGTCTCAAGGTACGGCTCTGATCACTGGCGCCTCGTCCGGTATCGGTGCGATTTACGCCGAGCGTCTGGCGGCGCGTGGTTTTGATCTGTTGCTGGTGGCCCGTGACAAGGCCCGTCTGGACAGCGCCGCCAGCCAATTGCGCGACGCTCACGGTGTGCAGGTCGAGGTGTGGAAAGCGGATCTGACCCAAAAGGACGACGTGATCAAACTCGAACAGCGCTTGCGCAGCGATTCGAGCATCAGCCTGCTGATTAACAATGCCGGCGTGGCCGCCGACGGCCCGCTGGCCAATGCCGACATGGATCAACTGGAACGCCTGATCCAGTTGAACATCACCGCCGTCACGCGTCTGGCGTCGGCCGCCGCTGCCAGTTTCGCCAAGGCAGGTCGCGGCACGATCATCAACATCGCCTCGGTCGTGGCGCTGTTCCCCGAGCGTTTCAATGCGACCTACACCGCCAGCAAGGCCTATGTGTTGAGTCTGACCCAATCGTTGAACGCGGAGCTCGAAGGCTCCGGCGTGCAGATCCAGGCCGTGCTGCCGGGCGTGACCCGCACTGA AATCTGGGAGCGTTCGGGGATCGACGCCAGTGGCATTCCGGCGGAAATGGTCATGGACGCCGGGGAGATGGTGGATGCCGCCCTGGCCGGTCTGGATCAGGGCGAACTGGTCACCATTCCATCGCTGCCCGATGCCGGCGAATGGCAGTCGTTTGTGGCGGCGCGCCATGTCATGGCGCCGAACCTTTCGCGCAGCGCTGCAGCCCAGCGCTACAAATCAGGTCATTGA

[0837] Production of enzyme A

[0838] 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 molecules were subsequently transformed into E. coli strain TQP10 and the desired expression constructs were verified by Sanger DNA sequencing. After the sequence-verified plasmid DNA was transformed into the E. coli protein expression host BL21 (DE3), a single colony was inoculated with 10 mL of LBP growth medium (10 g / L phytone, 10 g / L NaCl and 5 g / L yeast extract, supplemented with 35 μg / mL kanamycin), and cultured for 16 hours at 37°C with shaking at 200 rpm. The starter culture was then inoculated into 1 L of TB growth medium (12 g / L phytone, 12 g / L glycerol, 9.96 g / L sodium hydrogen phosphate, 20.4 g / L sodium dihydrogen phosphate, supplemented with 35 μg / mL kanamycin) in a 2 L baffled glass conical flask and incubated at 28°C for 16 h to achieve expression of enzyme A. The culture was then transferred to 2 × 500 mL centrifuge tanks. The cells were centrifuged at 6000 g / 4 °C in a SLA3000 rotor using a Sorvall RC-5C centrifuge. The bacterial pellets were then resuspended in their jars with 5x 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 achieved at 0 °C using an MSE Soniprep 150 ultrasonic generator equipped with a 9.5 mm ultrasonic probe for a total of 8 minutes (4 × 2 minute intervals at 16 microns with a 4 minute incubation time between cycles). The disrupted cell suspension was then transferred to a 50 mL Centrifuge tank and in Fiberlite TM The enzyme A solution was then freeze-dried by lyophilization (Edwards SuperModulo) for 2 days, keeping the pressure below 1 mbar. The dried enzyme cake was thoroughly crushed into a free-flowing uniform powder and stored at -20 °C between uses.

[0839] Preparation of trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester

[0840]

[0841] 1 mg of enzyme A was placed in a 2 mL reaction tube. 0.5 mL of a reaction mixture consisting of 5 mM nicotinamide adenine dinucleotide phosphate (NADP) in 100 mM potassium phosphate buffer was added to the tube. + ), 2mM magnesium chloride hexahydrate, 178mM anhydrous glucose, 1g / L glucose dehydrogenase (CDX-901, Codexis Inc.), 5% (v / v) dimethyl sulfoxide (DMSO) and 20g / L 2-oxo-6-azaspiro [3.4] octane-6-carboxylic acid tert-butyl ester. The reaction tube was shaken at 1000rpm and 30°C for 24 hours. The reaction was analyzed using a chiral HPLC method, showing that the conversion rate of trans-2-hydroxy-6-azaspiro [3.4] octane-6-carboxylic acid tert-butyl ester was 98.7%-100%, and the diastereomeric excess was 97% de.

[0842] Synthesis method

[0843] The compounds of Examples 1-6 were prepared according to the following methods.

[0844]

[0845]

[0846] Synthesis Example 1: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6- Synthesis of Ethyl Formate Citrate Monohydrate

[0847] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate can be prepared according to the following scheme. About 5 kg (free base equivalent) of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate was prepared using the described method.

[0848]

[0849]

[0850] Method Description:

[0851] Step 0

[0852] 1. Charge EtOH (7 vol) into the reactor.

[0853] 2. Charge pyrazolidine dihydrochloride (1 equivalent) into the reactor.

[0854] 3. Adjust the temperature to 10-15℃

[0855] 4. Charge 33% NaOH aqueous solution (1 vol) into the reactor, keeping the temperature <30°C.

[0856] 5. Stir the contents at 20 to 25°C for 2 to 3 hours.

[0857] 6. Filter the contents of the reactor.

[0858] 7. EtOH (2 vol) was charged to the reactor and used to rinse the filter cake.

[0859] 8. The combined filtrates were recharged to the reactor and concentrated under vacuum at 40 to 45°C to approximately 5 volumes.

[0860] 9. Toluene (5 vol) was charged to the reactor and reduced to about 4 to 5 volumes under vacuum at 40 to 45 °C.

[0861] 10. Toluene (5 vol) was charged to the reactor and reduced to about 4 to 5 volumes under vacuum at 40 to 45 °C.

[0862] 11. Toluene (5 vol) was charged to the reactor and reduced to about 4 to 5 volumes under vacuum at 40 to 45 °C.

[0863] 12. Toluene (5 vol) was charged into the reactor.

[0864] 13. Filter the contents of the reactor to clean the receiver flask.

[0865] 14. Toluene (2 vol) was charged to the reactor and used to rinse the filter cake.

[0866] 15. The combined filtrates were recharged to the clean reactor and reduced to minimum volume under vacuum at 40 to 45°C.

[0867] Step 1

[0868] 1. Charge DCM (10 vol) into the reactor.

[0869] 2. Charge the spiro alcohol (1 equivalent) into the reactor.

[0870] 3. Pyridine (3 eq.) was charged into the reactor.

[0871] 4. Adjust the internal temperature to 20 to 25°C

[0872] 5. Charge toluenesulfonic anhydride (1.5 eq) into the reactor, maintaining the temperature < 35° C. The target temperature during the addition was approximately 30° C.

[0873] 6. Stir the contents at 20 to 25°C for at least 2 hours

[0874] 8. Quench the reaction with 10% citric acid solution (5 vol), ensuring that the temperature does not exceed SOX [moderate exotherm],

[0875] 9. Stir for 15 minutes and allow to settle for 15 minutes.

[0876] 10. Drain lower organic layer into holding vessel [contains product].

[0877] 11. Drain the upper aqueous layer into a holding container.

[0878] 12. The organic layer from point 10 was recharged into the reactor.

[0879] 13. Charge 10% citric acid solution (5 vol) into the reactor.

[0880] 14. Stir for 15 minutes and allow to settle for 15 minutes.

[0881] 15. Drain the lower organic layer into a holding vessel [containing product].

[0882] 16. Drain the upper aqueous layer into a holding container.

[0883] 17. The organic layer from point 15 is recharged into the reactor.

[0884] 18. Charge saturated NaHCO3 solution (5 vol) into the reactor. [Caution: CO2 release].

[0885] 19. Stir for 15 minutes and allow to settle for 15 minutes.

[0886] 20. Drain the lower organic layer into a holding vessel [containing product].

[0887] 21. Drain the upper aqueous layer into a holding container.

[0888] 22. The organic layer from point 20 is recharged into the reactor.

[0889] 23. Charge saturated NaHCO3 solution (5 vol) into the reactor. [Caution: CO2 release].

[0890] 24. Stir for 15 minutes and allow to settle for 15 minutes.

[0891] 25. Drain the lower organic layer into a holding vessel [containing product].

[0892] 26. Drain the upper aqueous layer into a holding container.

[0893] 27. The organic portion of point 25 [orange / brown solution] was recharged into the reactor and reduced to about 5 vol in vacuum at 35-40°C.

[0894] 28. Cyclohexane (5 vol) was charged to the reactor and reduced to about 5 vol under vacuum at 35-40 °C.

[0895] 29. Cyclohexane (5 vol) was charged to the reactor and reduced to about 5 vol under vacuum at 35-40 °C.

[0896] 30. Cyclohexane (5 vol) was charged to the reactor and reduced to about 5 vol under vacuum at 35-40 °C.

[0897] 31. Chemist Check 1: Determine DCM content by NMR. Target: < 0.2% w / w remaining wrt cyclohexane peak. If failed, repeat cyclohexane stripping.

[0898] 32. Charge cyclohexane (5 vol) into the reactor and adjust the contents of the reactor to 10-15°C at 6°C / hr and hold for 1-2 hours.

[0899] 33. Chemist's Check 2: Crystallization Check. Objective: Recrystallization of the product.

[0900] 34. Filter the contents of the reactor.

[0901] 35. Cyclohexane (2 vol) was charged to the reactor and used to rinse the filter cake.

[0902] 36. Cyclohexane (2 vol) was charged to the reactor and used to rinse the filter cake.

[0903] 37. Transfer the solid to an oven and dry under vacuum at 35-40°C with nitrogen evolution for a minimum of 12 hours.

[0904] Step 2

[0905] 1. Chemist's test 1: Strip weight assay of a toluene solution of a piperidine compound.

[0906] 2. Charge the reactor with a toluene solution of the piperidine compound (4 equivalents, based on the results in point 1).

[0907] 3. Concentrate under vacuum at 40 to 45°C to about 4 to 5 volumes.

[0908] 4. Charge NMP (2.5 vol) into the reactor.

[0909] 5. Concentrate under vacuum at 40 to 45 °C to about 5 to 7 volumes

[0910] 6. Charge spiro alcohol tosylate (1 eq) into the reactor.

[0911] 7. Adjust the contents to 97°C to 102°C and stir under distillation conditions at a vacuum of 180 to 200 mbar for at least 36 hours, [orange solution]

[0912] 8. IPC 1: Determine reaction completion by HPLC. Target <3 area % spiro alcohol tosylate remaining.

[0913] 9. Adjust the contents to 20 to 25°C.

[0914] 10. Charge EtOAc (4 vol) into the reactor

[0915] 11. Charge water (4 vol) into the reactor, keeping the temperature <25°C [small exotherm]

[0916] 12. Stir for 15 minutes and allow to settle for 15 minutes.

[0917] 13. Drain the lower aqueous layer into a holding container.

[0918] 14. Drain the upper organic layer into a holding container. [Contains product]

[0919] 15. The aqueous layer from point 13 was recharged into the reactor.

[0920] 16. Charge EtOAc (4 vol) into the reactor

[0921] 17. Stir for 15 minutes and allow to settle for 15 minutes.

[0922] 18. Drain the lower aqueous layer into a holding container.

[0923] 19. Drain the upper organic layer into a holding container. [Contains product]

[0924] 20. The aqueous layer from point 18 is recharged into the reactor.

[0925] 21. Charge EtOAc (4 vol) into the reactor

[0926] 22. Stir for 15 minutes and allow to settle for 15 minutes.

[0927] 23. Drain the lower aqueous layer into a holding container.

[0928] 24. Drain the upper organic layer into a holding container. [Contains product]

[0929] 25. The aqueous layer from point 23 is recharged into the reactor.

[0930] 26. Charge EtOAc (4 vol) into the reactor

[0931] 27. Stir for 15 minutes and allow to settle for 15 minutes.

[0932] 28. Drain the lower aqueous layer into a holding container.

[0933] 29. The organic layers from points 14, 19 and 24 were recharged into the reactor.

[0934] 30. Charge water (5 vol) into the reactor.

[0935] 31. Stir for 15 minutes and allow to settle for 15 minutes.

[0936] 32. Drain the lower aqueous layer into a holding container.

[0937] 33. Charge water (5 vol) into the reactor.

[0938] 34. Stir for 15 minutes and allow to settle for 15 minutes.

[0939] 35. Drain the lower aqueous layer into a holding container.

[0940] 36. Charge 2M NaCl (5 vol) into the reactor.

[0941] 37. Stir for 15 minutes and allow to settle for 15 minutes.

[0942] 38. Drain the lower aqueous layer into a holding container.

[0943] 39. Charge 2M NaCl (5 vol) into the reactor.

[0944] 40. Stir for 15 minutes and allow to settle for 15 minutes.

[0945] 41. Drain the lower aqueous layer into a holding container.

[0946] 42. Charge 5% citric acid solution (5 vol) into the reactor.

[0947] 43. Stir for 15 minutes and allow to settle for 15 minutes.

[0948] 44. Drain the lower aqueous layer into a holding container. [Contains product]

[0949] 45. Charge 5% citric acid solution (5 vol) into the reactor.

[0950] 46. ​​Stir for 15 minutes and allow to settle for 15 minutes.

[0951] 47. Drain the lower aqueous layer into a holding container. [Contains product]

[0952] 48. Drain the upper organic layer into a holding vessel.

[0953] 49. The aqueous layers from points 44 and 47 are recharged into the reactor.

[0954] 50. Carefully charge the reactor with sodium carbonate (1 wt) [slightly exothermic - easily controlled by addition rate], keeping the temperature < 30°C until pH > 8. [Caution: CO2 gas evolution, bubbling]

[0955] 51. Chemist Check 2: Confirm alkaline pH. Target: pH > 8. If failed add more sodium carbonate

[0956] 52. Charge EtOAc (5 vol) into the reactor

[0957] 53. Stir for 15 minutes and allow to settle for 15 minutes.

[0958] 54. Drain the lower aqueous layer into a holding container.

[0959] 55. Discharge the organic layer to a holding container. [Contains product]

[0960] 56. The aqueous layer from point 54 is recharged into the reactor.

[0961] 57. Charge EtOAc (5 vol) into the reactor.

[0962] 58. Stir for 15 minutes and allow to settle for 15 minutes.

[0963] 59. Drain the lower aqueous layer into a holding container.

[0964] 60. Drain the organic layer into a holding container. [Contains product]

[0965] 61. The aqueous layer from point 59 is recharged into the reactor.

[0966] 62. Charge EtOAc (5 vol) into the reactor.

[0967] 63. Stir for 15 minutes and allow to settle for 15 minutes.

[0968] 64. Drain the lower aqueous layer into a holding vessel.

[0969] 65. The organic fraction from points 55 and 60 is recharged to the reactor.

[0970] 66. Concentrate under vacuum at 35 to 40°C to about 4 to 5 volumes.

[0971] 67. Cyclohexane (5 vol) was charged to the reactor and reduced to about 4 to 5 volumes under vacuum at 35 to 40°C.

[0972] 68. Cyclohexane (5 vol) was charged to the reactor and reduced to about 4 to 5 volumes under vacuum at 35 to 40°C.

[0973] 69. Cyclohexane (5 vol) was charged to the reactor and reduced to about 4 to 5 volumes under vacuum at 35 to 40°C.

[0974] 70. Chemist Check 3: Determine EtOAc content by NMR. Target: ≥ 1% w / w. If failed, repeat cyclohexane stripping.

[0975] 71. Cyclohexane (5 vol) was charged to the reactor and the contents of the reactor were adjusted to 35 to 40°C, then cooled to 10 to 15°C at 6°C / hr and held for 1 to 2 hours.

[0976] 72. Chemist's Check 4: Crystallization Check. Objective: Suspension is observed.

[0977] 73. Filter the contents of the reactor.

[0978] 74. Cyclohexane (2 vol) was charged to the reactor and used to rinse the filter cake.

[0979] 75. Cyclohexane (2 vol) was charged to the reactor and used to rinse the filter cake.

[0980] 76. The solid was transferred to an oven and dried under vacuum at 35 to 40°C with nitrogen evolution for a minimum of 12 hours.

[0981] (Step 3 is not implemented due to method improvement)

[0982] Step 4

[0983] 1. Charge ethanol (5 vol) into the reactor.

[0984] 2. Adjust the contents of the reactor to -5 to 0°C.

[0985] 3. Charge acetyl chloride (6 equivalents) into the reactor and keep the temperature <15°C [colorless solution]

[0986] 4. The Boc-product from step 2 (1 eq.) was charged to the reactor [finally forming an orange solution, then a beige suspension].

[0987] 5. Adjust the contents of the reactor to 20 to 25°C and stir for at least 18 hours.

[0988] 6. IPC 1: Determine reaction completion by HPLC. Target < 0.5% SM remaining.

[0989] 7. Charge TBME (10 vol) into the reactor over at least 15 minutes [temporary increase in stirring may be required during the addition].

[0990] 8. Stir the contents of the reactor for 1 hour ± 15 minutes.

[0991] 9. Filter the suspension. The solid is hygroscopic - filter under a stream of nitrogen and do not drain.

[0992] 10. TBME (5 vol) was charged into the reactor and used to wash the filter cake. Filtered under nitrogen flow and did not drain.

[0993] 11. TBME (5 vol) was charged into the reactor and used to wash the filter cake. Filtered under nitrogen flow without draining. Transferred to oven immediately.

[0994] 12. Transfer to oven and dry under vacuum at 35 to 40°C with nitrogen evolution for a minimum of 12 hours.

[0995] Step 5

[0996] 1. Charge dichloromethane (8 vol) into the reactor.

[0997] 2. Triethylamine (4.5 eq.) was charged into the reactor.

[0998] 3. Adjust the contents of the reactor to -5 to 0°C.

[0999] 4. Charge the product of step 4 (1 equivalent) into the reactor in batches, keeping the temperature <10°C. [Beige suspension]

[1000] 5. Ethyl chloroformate (1.5 equiv.) was charged to the reactor, maintaining the temperature < 10 °C [strong exotherm, rapidly dissipating, beige suspension].

[1001] 6. Adjust the contents to 20 to 25°C and stir for at least 2 hours.

[1002] 7. IPC 1: Determine reaction completion by HPLC. Target < 0.5% SM remaining. If this fails, stir for an additional 2 hours.

[1003] 8. Slowly charge water (3 vol), keeping the temperature <25°C. [Transparent two-phase layer formed, minimal exotherm]

[1004] 9. Stir for 15 minutes and allow to settle for 15 minutes.

[1005] 10. Discharge the lower organic layer into a holding container [containing the product]

[1006] 11. Drain the upper aqueous layer into a holding container.

[1007] 12. The organic layer from point 10 was recharged to the reactor.

[1008] 13. Charge 2M NaCl (4 vol) into the reactor

[1009] 14. Stir for 15 minutes and allow to settle for 15 minutes.

[1010] 15. Discharge the lower organic layer into a holding container [containing product]

[1011] 16. Drain the upper aqueous layer into a holding container.

[1012] 17. The organic layer from point 15 was charged to a clean reactor and concentrated in vacuo at 40 to 45°C to about 4 volumes.

[1013] 18. Charge EtOH (5 vol) into the reactor and concentrate in vacuo at 40 to 45 °C to approximately 4 volumes.

[1014] 19. Charge EtOH (5 vol) into the reactor and concentrate in vacuo at 40 to 45 °C to approximately 4 volumes.

[1015] 20. Chemist Check 1: Determine DCM content by NMR. Target < 0.2% w / w DCM / ethanol. If failed, repeat ethanol stripping.

[1016] 21. Chemist's Check 2: Stripping gravimetric determination of the ethanol solution is used to determine the amount of product present and therefore determine the charge amounts of citric acid, ethanol, water and citrate seeds in the next step.

[1017] 22. Adjust the reaction to 8 volumes (based on results of Chemist Check 2) of 5% water / ethanol.

[1018] 23. Charge citric acid (1 equivalent based on Chemist Check 2) into the reactor.

[1019] 24. Adjust the contents to 20 to 25°C and stir for 30 minutes to 1 hour [a beige suspension will form]

[1020] 25. Adjust the contents to 55 to 60°C and stir for at least 1 hour [usually dissolved at 50 to 55°C]

[1021] 26. Chemist Check 3: Dissolution. Target: Formation of solution [orange solution]. If failed, add incremental 0.25 volume parts of 5% water / ethanol and hold for 30 minutes until dissolution is achieved.

[1022] 27. Fine filter the solution into a clean reactor using a heated hose [Warning: Precipitation at 40°C]

[1023] 28. Adjust the contents to 55 to 60°C and stir for at least 1 hour.

[1024] 29. Adjust contents to 40 to 45°C over 3 hours and seed with 0.5% w / w product citrate.

[1025] 30. Stir at 40 to 45°C for 2 hours.

[1026] 31. Adjust the contents to 0 to 5°C over at least 12 hours [beige suspension].

[1027] 32. Chemist Check 4: Precipitation. Goal: To form a suspension.

[1028] 33. IRC 2: Form determination by XRD. Target: Form 2. If failed, inoculate with another portion of 0.5% w / w citrate Form 2, stir for at least 18 h and resample for IPC 2.

[1029] 34. Filter the suspension.

[1030] 35. Charge ethanol (2 vol) into the reactor.

[1031] 36. Adjust the temperature to 0 to 5°C and use for washing the filter cake.

[1032] 37. Charge ethanol (2 vol) into the reactor.

[1033] 38. Adjust the temperature to 0 to 5°C and use for washing the filter cake.

[1034] 39. TBME (5 vol) was charged into the reactor and used to wash the filter cake.

[1035] 40. TBME (5 vol) was charged to the reactor and used to wash the filter cake.

[1036] 41.IPC 3: 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%

[1037] 42. Transfer the solid to a vacuum oven and dry at 20 to 25°C for a minimum of 8 hours up to a maximum of 114 hours.

[1038] Synthesis Example 2: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6- Alternative Synthesis of Ethyl Formate Citrate Monohydrate

[1039]

[1040] Step 1. tert-Butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate

[1041]

[1042] Trans-2-hydroxy-6-azaspiro [3.4] octane-6-tert-butyl formate is dissolved in dichloromethane. Pyridine is added and toluenesulfonic anhydride is added in batches. The stirring reaction is until completion, and the organic layer and water layer are allowed to stand. The organic phase (containing trans-2-(tosyloxy)-6-azaspiro [3.4] octane-6-tert-butyl formate) is washed with citric acid aqueous solution and sodium bicarbonate aqueous solution successively. The solution solvent containing trans-2-(tosyloxy)-6-azaspiro [3.4] octane-6-tert-butyl formate is replaced with cyclohexane. The organic layer is then cooled to precipitate the product, and the material of crystallization is separated on the filter. The filter cake is then washed with cyclohexane and dried.

[1043] Alternative route to tert-butyl trans-2-(tosyloxy)-6-azaspiro[3.4]octane-6-carboxylate

[1044]

[1045] 2-hydroxy-6-azaspiro [3.4] octane-6-carboxylic acid tert-butyl ester is dissolved in DCM (10V), and pyridine (3 equivalents) is added. The solution is stirred at room temperature for 15 minutes. Toluenesulfonic anhydride (1.5 equivalents) is added, and the reaction mixture is stirred for 2 hours at room temperature. Another portion of toluenesulfonic anhydride (0.12 equivalents) is added, and the reaction mixture is stirred for 18 hours at room temperature. The reaction is quenched with 10% citric acid aqueous solution (5V), and the organic phase and aqueous phase are precipitated. The organic phase is separated and washed with 10% citric acid aqueous solution (5V) and saturated NaHCO solution (2×5V). Organic matter is removed by distillation under vacuum to obtain a crude product.

[1046] The crude product was purified by flash column chromatography (silica gel) 0-50% EtOAc / hexanes, monitoring elution at a wavelength of 254 nm. Once visible peaks appeared on the chromatogram, the flow rate was reduced 4-fold and two fractions (top and bottom, named by the relative positions of their corresponding spots on the thin layer chromatography) corresponding to the cis- and trans-products were collected. The 1H NMR spectrum of the product is shown in Fig.34 (trans-toluenesulfonate, the "top" fraction) and Fig.35 (cis-tosylate, "bottom" fraction).

[1047] The top fraction was designated as the desired trans isomer and 1 H- 1H nuclear Overhauser effect spectroscopy (NOESY) assigned the bottom fraction to the undesired cis isomer. The NOESY spectrum and assignment are shown in Fig.36 (trans-toluenesulfonate, the "top" fraction) and Fig.37 (cis-tosylate, the "bottom" fraction).

[1048] Step 2. cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate Tert-butyl ester

[1049]

[1050] 4-(1-methyl isophthalic acid H-pyrazole-5-yl) piperidine dihydrochloride is suspended in ethanol, then sodium hydroxide aqueous solution is added.The stirred suspension is to crush the HCl salt, and then the mixture is filtered.The filter cake is washed with ethanol, and the product solution is transferred to toluene.Then the toluene layer (containing 4-(1-methyl isophthalic acid H-pyrazole-5-yl) piperidine free alkali) is filtered and concentrated.In the toluene solution of 4-(1-methyl isophthalic acid H-pyrazole-5-yl) piperidine free alkali, NMP and (2s, 4r) -2- (tosyloxy) -6- azaspiro [3.4] octane -6- tert-butyl formates are added.The solution is heated, and the residual toluene is vacuum distilled.The heated reaction is continued until it is considered that the reaction is complete. The product is then diluted with ethyl acetate, and the organic matter (containing cis-2-(4-(1-methyl isophthalic acid H-pyrazole-5-yl) piperidine-1-yl)-6-azaspiro [3.4] octane-6-carboxylic acid tert-butyl ester) is washed with water and brine. The product is extracted into a citric acid solution from the organic layer. The pH of the water layer (containing the product) is adjusted, and the product is extracted into ethyl acetate. The product is transferred to cyclohexane, cooled to induce crystallization, and filtered. The filter cake is washed with cyclohexane and dried to obtain cis-2-(4-(1-methyl isophthalic acid H-pyrazole-5-yl) piperidine-1-yl)-6-azaspiro [3.4] octane-6-carboxylic acid tert-butyl ester.

[1051] Step 3. cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride Salt

[1052] Step 3

[1053]

[1054] Acetyl chloride is added to cold ethanol and stirred to produce HCl in situ. In this solution, cis-2-(4-(1-methyl isophthalic acid H-pyrazole-5-yl) piperidine-1-yl)-6-azaspiro [3.4] octane-6-tert-butyl formate is added. 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 filter cake is washed with MTBE, and vacuum dried to obtain cis-2-(4-(1-methyl isophthalic acid H-pyrazole-5-yl) piperidine-1-yl)-6-azaspiro [3.4] octane, which is trihydrochloride.

[1055] Step 4. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate Ethyl acetate hydrochloride

[1056]

[1057] Triethylamine is dissolved in dichloromethane and cooled. To this solution, cis-2-(4-(1-methyl-1H-pyrazole-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride is added in batches. 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 with water and sodium chloride solution in sequence. The product is transferred to ethanol, and anhydrous HCl is then added. The resulting suspension is heated until dissolved, and then cooled to induce crystallization. The solid is stirred, and then separated by filtration, washed with MTBE, and then dried.

[1058] Step 5. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate Ethyl citrate monohydrate

[1059]

[1060] A suspension of cis-2-[4-(1-methyl-1H-pyrazole-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride is formed in dichloromethane. The mixture is stirred, and an aqueous sodium bicarbonate solution is added. The mixture is stirred until all solids are dissolved. The organic layer (containing the product) is washed with water and sodium chloride solution in sequence. The product is transferred to ethanol, and citric acid and water are then added. The resulting suspension is heated until dissolved, and then cooled to induce crystallization. The solid is stirred, then separated by filtration, washed with MTBE, and then dried to obtain cis-2-[4-(1-methyl-1H-pyrazole-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate.

[1061] Synthesis Example 3. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-yl Alternative Synthesis of Ethyl Formate Citrate Monohydrate

[1062]

[1063] Step 1: cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate Tert-butyl ester

[1064]

[1065] 2-Oxo-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester (75.0 g, 328 mmol), 4-(1-methyl-1H-pyrazol-5-yl)piperidinium tartrate (132.1 g, 377.0 mmol), molecular sieves (124.0 g, 1041 mmol) (3 angstroms), citric acid (130.0 g, 676.6 mmol) and methanol (686.5 mL) were sequentially charged into a 1500 mL high pressure vessel. 5.0 wt.% Pd / BaSO4 (5:95, palladium:barium sulfate, 69.74 g, 32.77 mmol) (Aldrich, reduction) was added, and hydrogen was applied to 994 psi. The reaction mixture was stirred at 23° C. for 24 hours, and then the temperature was raised to 35° C. for another 22 hours. The hydrogen pressure was then vented and the catalyst was removed by filtration over a 1500 mL "M" frit. The filter cake was washed with MeOH (811.4 g).

[1066] The clear yellow filtrate was concentrated to a solution (538.9 g) by rotary evaporation. Water (251.1 g) was added and the pH was adjusted to 13.64 with potassium hydroxide (256.8 g, 2060 mmol) while keeping the temperature below 35 ° C with an ice bath. The resulting milky mixture (813 g) was concentrated on a rotary evaporator and 2-methoxy-2-methylpropane (600 g) was added. The phases were separated and the organic matter was washed with water (250 g). The aqueous phase was stripped continuously with 2-methoxy-2-methylpropane (223.0 g).

[1067] The organic phases were combined and concentrated on a rotary evaporator to an oil (188.6 g). Isopropanol (473.7 g) was added and concentrated to a yellow oil, and after about 30 minutes at room temperature, the title compound (cis-tert-butyl 2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate) free base precipitated in a yield of 65.59 g. HPLC showed 16 area % of the undesired trans isomer, giving the desired cis isomer in 81.4% yield. The title compound was used in the next step without further purification.

[1068] During process development, various reduction conditions were found to provide moderate to reasonable selectivity to the desired cis isomer and moderate to excellent conversion of the starting material, see, for example, selected conditions in Table A below.

[1069] Table A

[1070]

[1071] Step 2: cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate Tert-butyl citrate

[1072]

[1073] Isopropanol (1300.2 g, 21636 mmol) was added to the product of step 1 at 22°C to form a solution with a pH of 9.46. A freshly prepared 33.3% citric acid solution (77.14 g, 133.7 mmol) in ethanol 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 crystals of cis-tert-butyl 2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate citrate (0.517 g). After another hour, the pH was 6.79 and a 33.3% citric acid solution (51.26 g, 88.84 mmol) in ethanol was added dropwise. After an additional hour, the pH was 6.45, and a solution of 33.3% citric acid (48.0 g, 83.3 mmol) in ethanol was added dropwise. After an additional 3 hours and 15 minutes, the pH was 6.1, and the crystals were isolated by vacuum filtration on a 3L "C" frit. The isolated crystals were washed with isopropanol (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 ester citrate) in a yield of 162.55 g (162.03 g corrected for seed crystals). HPLC analysis showed 7.5 area % of the trans isomer; NMR showed 4.11 wt % IPA and 1.00 equivalents of citric acid.

[1074] HPLC of the filtrate showed a trans:cis ratio of 68.2:31.8 (4.0% filtrate loss).

[1075] 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-carboxylic acid tert-butyl ester); (ES-): found 191.1 [MH - ](Citric acid)

[1076] Step 3: cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate Tert-Butyl Acid Hydrochloride

[1077]

[1078] 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-carboxylic acid tert-butyl ester 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) to give 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 organic phase was washed with water (103.9 g). The aqueous phase was stripped continuously with 2-methoxy-2-methylpropane (196.0 g). During the separation of the phases, a small amount of black interphase solid was transferred to the aqueous phase. The combined organics were concentrated to an oil (146.83 g) and isopropanol (840.5 g) was added. 22.31 wt% hydrogen chloride (15.20 g, 93.01 mmol; 5.53 M in isopropanol) was added to the solution and the pH was dropped from 10.41 to 6.32. The resulting solution was seeded with cis-tert-butyl 2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate hydrochloride (0.21 g) and a slurry began to form immediately. After 10 minutes, the pH was 6.45 and a solution of 22.31 wt% hydrogen chloride (14.6 g, 89.3 mmol) in isopropanol was added dropwise over 10 minutes. After another 15 minutes, the pH was 6.2, and a 22.31 wt% solution of hydrogen chloride (15.0 g, 91.8 mmol) in isopropanol was added dropwise over 10 minutes, stopping the addition in the pH range of 1.0-4.0. 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 onto a 3000 mL "C" glass frit and allowed to settle, and then vacuum filtered. The filter cake was washed with isopropanol (300 mL) and then dried under nitrogen pressure for 2.5 days to obtain 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 ester hydrochloride) in an amount of 95.55 g (95.34 g based on seed correction). HPLC analysis showed 97.10 wt% HCl salt, 0.20 area% undesired trans isomer; NMR showed 0.35 wt% isopropanol.

[1079] HPLC of the filtrate showed 43.4:56.6 cis:trans (5.52% filtrate loss).

[1080] LCMS (ES+): found 375.4 [M+H + ]

[1081] Step 4: cis-2-(4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride Salt

[1082]

[1083] Ethanol (340.90g) was charged into a 2000mL round-bottom flask and placed in an ice-water bath. Acetyl chloride (92.53g, 1179mmol) was added within 15 minutes, keeping the temperature below 30°C (violent exotherm during the addition of acetyl chloride). The ice-water bath was removed, and after 45 minutes, cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid tert-butyl ester hydrochloride (93.43g, 220.7mmol; 0.20 area % trans) was added at a reaction temperature of 20°C, and rinsed with ethanol (100.54g). The mixture was stirred for 22 hours at room temperature (22.9°C). The resulting white crystal slurry showed complete conversion of the starting material (<0.1% starting material was shown by HPLC). 2-methoxy-2-methylpropane (812mL) was added in batches within 35 minutes. The obtained mixture was stirred for 15 minutes. The slurry was poured onto 600mL "C" glass frit and filtered to provide large white crystals. The filter cake was washed with 2-methoxy-2-methylpropane (84.46g), then dried under nitrogen pressure to obtain the title compound (cis-2-(4-(1-methyl-1H-pyrazole-5-yl)piperidin-1-yl)-6-azaspiro[3.4]octane trihydrochloride), with an output of 90.17g (specified purity 93.96%, assuming a yield of 100%). HPLC shows 99.74 area % purity; NMR shows 0.84wt% ethanol as only residual solvent).

[1084] LCMS (ES+): found 275.4 [M+H + ]

[1085] Step 5. cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate Ethyl citrate monohydrate

[1086]

[1087] 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 dichloromethane (858 g). The resulting slurry was cooled to 11 ° C with an ice bath. Triethylamine (169 mL, 1210 mmol) was added over 6 minutes while keeping the temperature below 21 ° C. In the reaction mixture at 16 ° C, ethyl chloroformate (28.80 g, 265.4 mmol) was added over 7 minutes while keeping the temperature below 28 ° C. After stirring for 10 minutes, HPLC analysis showed that 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 organic phase was washed with water (150 g). The three aqueous fractions were stripped continuously with dichloromethane (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 ethanol (157.5 g) was added, then concentrated again to an oil (98.3 g). Ethanol (884.3 g) was added again, 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-carboxylic acid ethyl ester citrate monohydrate (0.35 g) and stirred overnight at room temperature to form a slurry or large crystals. The slurry was placed in an ice-water bath to cool the mixture to 1 to 2° C. for 2 hours and 15 minutes. The crystals were isolated by vacuum filtration on a 600 mL "C" frit, then washed with a mixture of ethanol (123.5 g, 2681 mmol) and water (8.2 g, 460 mmol) at 0° C., and then washed with 2-methoxy-2-methylpropane (167 g, 1890 mmol) at 0° C. The product was dried under nitrogen pressure overnight to provide the title compound (cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate) in a yield of 110.24 g (109.89 g corrected based on crystals). HPLC showed 99.7 area % of target (210 nm); less than 0.05% of the trans isomer.

[1088] HPLC vs cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate standard showed 101.3 wt%. Karl Fischer titration showed 3.31 wt% water (theoretical = 3.24 wt% for monohydrate). NMR showed 0.10 wt% ethanol as the only residual solvent.

[1089] HPLC showed 8.5% product in the filtrate and washes.

[1090] The NMR spectrum of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate prepared by this method is shown in Fig.30 and Fig.31 middle.

[1091] 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-carboxylic acid ethyl ester); (ES-): found 191.1 [MH-] (citric acid)

[1092] Synthesis Example 4: cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6- Synthesis of Ethyl Azaspiro[3.4]octane-6-carboxylate

[1093] Cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester can be prepared according to the following scheme. 91 g of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester was prepared using the described method.

[1094] The numbers in bold in the methods described below correspond to the intermediate compounds described in the schemes below.

[1095] Main Chain

[1096]

[1097] Sidechain:

[1098]

[1099] Step 1

[1100] Compound-1 (190 g, 1.0 eq.) and DCM (10 V) were charged into a RB flask at 25-30°C under nitrogen, followed by the addition of pyridine (3.0 eq.). The reaction mixture was stirred at room temperature for 10 min and (Ts)2O (1.5 eq.) was added while the temperature was controlled below 30°C. The reaction mixture was stirred at room temperature for 1.5 h. HPLC check was performed.

[1101] Post-processing:

[1102] The reaction mixture was quenched with 10% citric acid (5V) and stirred for 15 to 20 minutes. The aqueous layer was separated, the DCM layer was washed with 10% citric acid (2×5V), and then the DCM layer was washed with saturated NaHCO3 solution (2×5V). The organic layer was concentrated in vacuo to constant weight at less than 45°C to obtain 305.0 g of crude product.

[1103] purification:

[1104] The crude product was subjected to silica gel column chromatography (by using 710.0 g of 60 to 120 mesh silica gel for adsorption of the crude material, and filling the column bed with 3.0 Kg of 60-120 mesh silica gel / n-hexane, and eluting the column using 12% ethyl acetate / n-hexane) to obtain 150.0 g of compound-2 with a yield of 47%.

[1105] 1 H NMR (400MHz, DMSO): δppm 1.35(s,9H),1.77(m,2H),2.00(m,2H),2.21(m,2H),2.45(s,3H),3.13(m,4H),4.90(m,1H),7.48(m,2H),7.80(m,2H).

[1106] Step 2

[1107] Under nitrogen, NMP (2.5V) and compound-2 (100.0g, 1.0 equivalent) are loaded into a RB flask. The reaction mixture is then stirred for 30 minutes until a clear solution is formed. Compound-3 (piperidone ethylene ketal) (4.0 equivalent) is added to the RB flask over a 10min period, and then the reaction mixture is heated to 95 to 105°C over a 1h period, and kept at the same temperature for 32h under a nitrogen atmosphere. The reaction mixture is monitored by HPLC.

[1108] Post-processing:

[1109] The reaction mixture was cooled to 40 to 45°C over a period of 1 hour, and ethyl acetate (5V) was added, and the reaction mixture was allowed to reach room temperature. By controlling the exotherm to be less than 30 to 35°C, water (5V) was added to the reaction mixture and stirred for 30 minutes. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×5V). The combined organic layer was washed with water (2×5V) and brine (2×5V). The organic layer was extracted with 5% citric acid (3×5V). The combined citric acid layer was cooled to 0 to 5°C and alkalized to pH 8 to 9 with solid Na2CO3. The product was then extracted from the aqueous layer with ethyl acetate (3×5V). The combined ethyl acetate layer was concentrated to 1V under vacuum below 45°C and stripped to constant weight with n-heptane (2×5V).

[1110] Rough weight: 84.0g

[1111] purification:

[1112] The crude material (84.0 g) and n-heptane (5 V) were charged into a RB flask and heated to 80 to 90 ° C in 1 h to obtain a clear solution. The reaction mixture was kept at 80 to 90 ° C for 30 minutes and cooled to room temperature, the solid suspension was filtered and the filter bed was washed with n-heptane (5 V). The solid was dried under vacuum to constant weight at less than 45 ° C to obtain 64.0 g of compound-4 with a yield of 69%.

[1113] 1 H NMR (400MHz, DMSO): δppm 1.44(s,9H),1.57(m,4H),1.79(m,4H),1.97(m,2H),2.27(br,4H),2.68(m,1H),3.12(m,2H),3.25(m,2H),3.82(s,4H).

[1114] Step 3

[1115] Ethanol (7V) was charged into a RB flask and cooled to -5 to 0°C within 1h, then acetyl chloride (6.0 eq) was added to the flask under nitrogen by controlling the exotherm below 20°C over a period of 10 minutes (clear solution was observed after the addition was complete). Compound-4 (60.0 g, 1.0 eq) was added to the RB flask in batches (5 batches) within 10 minutes, keeping the temperature below 20°C, and a solid suspension was observed. (Pale yellow to white suspension). The reaction mixture was allowed to reach room temperature and kept at room temperature for a period of 16 hours. The reaction mixture was monitored using TLC.

[1116] (TLC system: 10% MeOH / DCM, Compound-4@0.5RF and Compound-5@0.2RF)

[1117] Post-processing:

[1118] MTBE (10V) was added to the RB flask and stirred for 1 hour. The obtained solid was filtered under N2 atmosphere, the filter bed was washed with MTBE (5V), and then the solid was dried under vacuum at 35 to 40°C to constant weight to obtain 52.3g of compound-5 with a yield of 94%.

[1119] 1 H NMR (400MHz, DMSO): δppm 1.81(m,4H),2.10(m,2H),2.20(m,2H),2.50(m,2H),2.75(m,2H),3.09(m,4H),3 .30(m,2H),3.42(m,1H),3.72(m,1H),3.93(s,4H),9.24(s,2H),11.66(br,1H).

[1120] Step 4

[1121] DCM (8V) and triethylamine (3.5 equivalents) were loaded into the RB flask and cooled to 0 to 5°C under nitrogen, then compound-5 (50.0 g, 1.0 equivalents) was added to the flask over a period of 5 minutes, keeping the temperature at 0 to 5°C (slurry was observed). Ethyl chloroformate (1.5 equivalents) was then added dropwise to the reaction mixture over 10 minutes (slight exotherm was observed, keeping the temperature below 20°C), and a solid suspension was observed. The reaction mixture was stirred at room temperature for 2 to 3 hours. HPLC was used to monitor the reaction mixture.

[1122] Post-processing:

[1123] Add water (3V) and stir for 30 minutes, separate the aqueous layer and wash the DCM layer with brine solution (2×5V). Concentrate the organic layer under vacuum below 45°C and exchange with n-heptane (2×5V) to constant weight to obtain 44.4g of compound-6 with a yield of 89%.

[1124] 1 H NMR (400MHz, DMSO): δppm 1.17(m,3H),1.59(m,4H),1.82(m,4H),2.15(m,2H),2.30(br,4H),2.70(br,1H),3.15(s,2H),3.33(m,2H),3.97(m,4H),4.02(m,2H).

[1125] Step 5

[1126] In a RB flask were added compound-6 (50.0 g, 1.0 eq.) and concentrated HCl (20 V) in DCM (10 V) and the reaction mixture was stirred at RT for 4 to 5 h. The reaction was monitored by HPLC.

[1127] The DCM layer was separated and 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 (2x10V). The combined DCM layers were vacuum distilled below 45°C to constant weight.

[1128] To the crude was added DCM (10 V) followed by concentrated HCl (20 V) and the resulting two-phase layer was stirred at room temperature for 4 to 5 h. The reaction was monitored by HPLC.

[1129] The DCM layer was separated and 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 (2×10 V). The combined DCM layers were vacuum distilled below 45°C to constant weight.

[1130] Ketone-Int Weight: 40.0g

[1131] Ketone-Int (40.0 g) is placed in RBF and DCM (15 V) containing W211a Int (1.3 equivalents), acetic acid (3.0 equivalents) is added, and the reaction mixture is stirred for 24 hours at room temperature under nitrogen. Cool to 0 to 5 ° C in the time period of 1 h, and by controlling the exothermicity below 10 to 15 ° C, add NaBH (OAc) 3 (2.0 equivalents) in batches (5 to 6 batches). Make the reaction mixture reach room temperature in the time period of 1 h, and keep 4 h at room temperature. Check by TLC.

[1132] (TLC system: 50% acetone / hexane: ketone-Int@0.8RF and product@0.2RF)

[1133] Post-processing:

[1134] The reaction mixture was quenched with 5% citric acid solution (5V), and the DCM layer was extracted with 5% citric acid solution (3×5V). The combined citric acid layer was adjusted to pH 8 to 9 using solid Na2CO3 and loaded with DCM (10V) and stirred for 10 minutes, filtered through diatomaceous earth. The filtrate was placed in a separatory funnel and the DCM layer was separated. The aqueous layer was extracted with DCM (4×5V). The combined DCM layer was washed with water (4×10V) and the solvent was concentrated to constant weight under vacuum below 45°C to obtain a gelatinous material.

[1135] Gel material weight: 52.0g

[1136] The crude material (52.0 g) was dissolved in DCM (10 V) and charcoal (0.2 times) was added thereto at room temperature, and the reaction mixture was stirred for 1 h. The reaction mixture was filtered through celite and the filter bed was washed with DCM (5 V). The charcoal treatment was repeated 3 more times for the filtrate; the final filtrate was distilled and exchanged with n-heptane (2×5 V) to constant weight under vacuum at below 45° C. to obtain 48.0 g of crude product.

[1137] purification:

[1138] The crude material from two batches (i.e. 48.0g and 75.0g) was combined in n-heptane (10V), and heated to 50°C in 1h and kept at the same temperature for 1h. The reaction mixture was cooled to room temperature within 1 hour and stirred at room temperature for 1 hour. The solid was collected by filtration and washed with n-heptane (5V). The solid was dried to constant weight under vacuum below 45°C. The n-heptane slurry treatment was repeated 2 more times to obtain 91.0g of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-ethyl formate product in off-white solid form, and the total yield of the two steps was 59%.

[1139] 1 H NMR (400MHz, DMSO): δppm 1.15(m,3H),1.30(m,2H),1.65(m,4H),1.80(m,3H),2.00(m,2H),2.09(m,2H),2.29(m,1H),2.35(m,1H), 2.62(m,2H),2.79(m,2H),2.97(m,1H),3.03(m,4H),3.27(m,3H),3.445(m,1H),4.01(m,2H),4.60(m,1H).

[1140] Sidechain

[1141] Step 6:

[1142] In a RB flask, compound-7 (190.0 g, 1.0 eq.) was placed in DCM (10 V) under nitrogen and cooled to -78°C, at -78°C DAST (2.5 eq.) was added dropwise over 20 to 25 minutes (exotherm controlled below -70°C). After addition, the reaction mixture was allowed to reach room temperature over a period of 2 hours and stirred at room temperature for 16 hours. The progress of the reaction was monitored by HPLC.

[1143] Post-processing:

[1144] After the reaction is completed, the reaction mixture is cooled to 0 to 5 ° C over a period of 30 min, diluted with DCM (5 V), and slowly dripped with a saturated solution of sodium bicarbonate (35 V) at 0 to 5 ° C (by controlling the temperature to be less than 10 ° C), the reaction mixture is quenched with the saturated solution of sodium bicarbonate until pH reaches 7 to 8. The reaction mixture is stirred to room temperature over a period of 1 h, and stirred at room temperature for 30 min, two layers are separated, and the aqueous layer is then extracted with DCM (2x10 V). The combined organic layer is washed with water (10 V). The solvent is concentrated to constant weight under vacuum below 45 ° C to obtain 201.5 g of compound-8, with a yield of 97%.

[1145] 1 HNMR (400MHz, CDCl3); δppm 1.30(m,9H),2.50(m,1H),2.76(m,1H),3.85(m,5H).4.49(m,1H).

[1146] Step 7

[1147] Compound-8 (201.0 g, 1.0 eq.) and THF (5 V) were added to a RB flask under nitrogen and then cooled to 0 to 5° C. over a period of 30 minutes. LiBH4 (2.0 eq.) was then added in batches (3 batches) at 0 to 5° C. over 20 minutes (exotherm controlled below 5° C.). The reaction mixture was stirred at 0 to 5° C. for 2.5 h. The reaction was monitored by HPLC.

[1148] Post-processing:

[1149] After the reaction is completed, the reaction mixture is diluted with DCM (5V) and quenched with saturated sodium bicarbonate (10V) solution below 15 ° C by controlling heat release in the time period of 1h. Two layers are separated and the aqueous layer is extracted with DCM (2x10V). The combined organic layer is washed with water (10V) and then washed with brine (10V). Below 45 ° C, the solvent is concentrated to constant weight under vacuum to obtain 178.5g of crude compound-8, and the yield is 99%.

[1150] 1 HNMR (400MHz, DMSO): δppm 1.42 (s, 9H), 2.33 (m, 1H), 3.50 (m, 3H), 3.60 (m, 1H), 3.84 (m, 1H), 4.98 (s, 1H).

[1151] Step 8

[1152] Compound-9 (178.0 g, 1.0 eq.) and 1,4-dioxane (3V) were added to the RB flask under nitrogen and stirred for 10 minutes. The reaction mixture was cooled to 10 to 15°C within 1 hour, and a 4M HCl solution in 1,4-dioxane (5V) was added dropwise at 10 to 15°C over a period of 40 minutes. The reaction mixture was stirred to room temperature and continued to stir at room temperature for 16 hours. The reaction progress was monitored by TLC. (TLC system: 50% EtOAc / hexane: Compound-9@0.5RF, W221aint@0.1RF)

[1153] Post-processing:

[1154] After the reaction mixture was completed, the solvent was concentrated under vacuum at below 45°C under nitrogen and exchanged with acetone (2×3V) to obtain 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 constant weight to obtain 110.0g of W211a Int with a yield of 84%.

[1155] 1 HNMR (400MHz, DMSO): δppm 2.33(m,1H), 2.53(m,1H), 3.60(m,4H), 3.90(m,1H), 5.43(br,1H), 9.97(br,2H).

[1156] Synthesis Example 5: cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-nitrogen Synthesis of Heterospiro[3.4]octane-6-carboxylic Acid Ethyl Ester

[1157] Cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester can be prepared according to the following scheme. About 1.4 kg of cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester was prepared using the described method.

[1158]

[1159]

[1160] Step 1 1. Charge DCM (10 vol) into the reactor.

[1161] 2. Charge the spiro alcohol (1 equivalent) into the reactor. [Light yellow solution]

[1162] 3. Pyridine (3 eq.) was charged into the reactor.

[1163] 4. Adjust the internal temperature to 20 to 25°C

[1164] 5. Charge toluenesulfonic anhydride (1.5 equiv.) to reactor [moderate exotherm, orange / brown solution formed], maintaining temperature ≤ 35° C. Target temperature during addition was approximately 30° C.

[1165] 6. Stir the contents at 20 to 25°C for at least 2 hours.

[1166] 7. IPC 1: Determine reaction completion by HPLC. Target ≤ 0.5 area % spiro alcohol remaining.

[1167] 8. Quench the reaction with 10% citric acid solution (5 vol), ensuring the temperature does not exceed 30°C [moderate exotherm].

[1168] 9. Stir for 15 minutes and allow to settle for 15 minutes.

[1169] 10. Discharge the lower organic layer into a holding container [containing the product]

[1170] 11. Drain the upper aqueous layer into a holding container.

[1171] 12. The organic layer from point 10 was recharged to the reactor.

[1172] 13. Charge 10% citric acid solution (5 vol) into the reactor.

[1173] 14. Stir for 15 minutes and allow to settle for 15 minutes.

[1174] 15. Discharge the lower organic layer into a holding container [containing product]

[1175] 16. Drain the upper aqueous layer into a holding container.

[1176] 17. The organic layer from point 15 was recharged to the reactor.

[1177] 18. Charge saturated NaHCO3 solution (5 vol) into the reactor. [Note: CO2 release]

[1178] 19. Stir for 15 minutes and allow to settle for 15 minutes.

[1179] 20. Discharge the lower organic layer into a holding vessel [containing product]

[1180] 21. Drain the upper aqueous layer into a holding container.

[1181] 22. The organic layer from point 20 is recharged to the reactor.

[1182] 23. Charge saturated NaHCO3 solution (5 vol) into the reactor. [Note: CO2 release]

[1183] 24. Stir for 15 minutes and allow to settle for 15 minutes.

[1184] 25. Discharge the lower organic layer into a holding vessel [containing product]

[1185] 26. Drain the upper aqueous layer into a holding container.

[1186] 27. The organic portion from point 25 [orange / brown solution] was concentrated in vacuo on a rotary evaporator at 35 to 40 °C to remove DCM.

[1187] 28. Toluene (2 vol) was charged to the flask and concentrated in vacuo on a rotary evaporator at 35 to 40 °C.

[1188] 29. Charge toluene (2 vol) into a flask and concentrate under vacuum on a rotary evaporator at 35 to 40°C. Continue concentrating for at least 1 hour after the solvent stops distilling to ensure optimal drying. [Beige / yellow solid]

[1189] 30. Chemist Check 1: Determine DCM content by NMR. Target: ≤ 0.1% w / w remaining. If failed, repeat toluene stripping.

[1190] 31. Transfer to an oven and dry under vacuum at 35 to 40°C with nitrogen evolution for a minimum of 12 hours. Crush the material thoroughly using an appropriate unused spoon and continue drying until constant weight is achieved (net weight difference between successive dryings with a minimum interval of 1 hour < 1.0%). If dryness is not achieved within 48 hours, contact the project chemist for advice.

[1191] Step 2

[1192] 1. Charge NMP (2.5 vol) into the reactor.

[1193] 2. Charge spiro alcohol tosylate (1 eq.) into the reactor.

[1194] 3. Charge piperidone ethylene ketal (4 equiv.) into the reactor.

[1195] 4. Adjust the contents to 97 to 102°C and stir for at least 36 hours. [Orange solution]

[1196] 5. IPC 1: Determine reaction completion by HPLC. Target ≤ 3 area % spiro alcohol tosylate remaining.

[1197] 6. Adjust the contents to 20 to 25°C

[1198] 7. Charge EtOAc (4 vol) into the reactor

[1199] 8. Charge water (4 vol) into the reactor, maintaining a temperature of ≤25°C [small exotherm]

[1200] 9. Stir for 15 minutes and allow to settle for 15 minutes.

[1201] 10. Drain the lower aqueous layer into a holding container.

[1202] 11. Drain the upper organic layer into a holding container. [Contains product]

[1203] 12. The aqueous layer from point 10 was recharged to the reactor.

[1204] 13. Charge EtOAc (4 vol) into the reactor

[1205] 14. Stir for 15 minutes and allow to settle for 15 minutes.

[1206] 15. Drain the lower aqueous layer into a holding container.

[1207] 16. Drain the upper organic layer into a holding container. [Contains product]

[1208] 17. The aqueous layer from point 15 was recharged to the reactor.

[1209] 18. Charge EtOAc (4 vol) into the reactor

[1210] 19. Stir for 15 minutes and allow to settle for 15 minutes.

[1211] 20. Drain the lower aqueous layer into a holding container.

[1212] 21. Drain the upper organic layer into a holding container. [Contains product]

[1213] 22. The aqueous layer from point 20 was recharged to the reactor.

[1214] 23. Charge EtOAc (4 vol) into the reactor

[1215] 24. Stir for 15 minutes and allow to settle for 15 minutes.

[1216] 25. Drain the lower aqueous layer into a holding container.

[1217] 26. Drain the upper organic layer into a holding container. [Contains product]

[1218] 27. The organic layers from points 11, 16, 21 and 26 were recharged into the reactor.

[1219] 28. Charge water (5 vol) into the reactor.

[1220] 29. Stir for 15 minutes and allow to settle for 15 minutes.

[1221] 30. Drain the lower aqueous layer into a holding container.

[1222] 31. Charge water (5 vol) into the reactor.

[1223] 32. Stir for 15 minutes and allow to settle for 15 minutes.

[1224] 33. Drain the lower aqueous layer into a holding container.

[1225] 34. Charge 2M NaCl (5 vol) into the reactor.

[1226] 35. Stir for 15 minutes and allow to settle for 15 minutes.

[1227] 36. Drain the lower aqueous layer into a holding container.

[1228] 37. Add 2M NaCl (5 vol) into the reactor.

[1229] 38. Stir for 15 minutes and allow to settle for 15 minutes.

[1230] 39. Drain the lower aqueous layer into a holding container.

[1231] 40. Charge 5% citric acid solution (5 vol) into the reactor.

[1232] 41. Stir for 15 minutes and allow to settle for 15 minutes.

[1233] 42. Drain the lower aqueous layer into a holding container. [Contains product]

[1234] 43. Charge 5% citric acid solution (5 vol) into the reactor.

[1235] 44. Stir for 15 minutes and allow to settle for 15 minutes.

[1236] 45. Drain the lower aqueous layer into a holding container. [Contains product]

[1237] 46. ​​Drain the upper organic layer into a holding vessel.

[1238] 47. The aqueous layers from points 42 and 45 are recharged into the reactor.

[1239] 48. Carefully charge the reactor with sodium carbonate (0.67 wt) [slightly exothermic - easily controlled by rate of addition], maintaining temperature ≤ 30°C until pH ≥ 8. [Caution: CO2 gas evolution, bubbling]

[1240] 49. Chemist Check 1-Confirm alkaline pH. Target: pH ≥ 8.

[1241] 50. Charge EtOAc (5 vol) into the reactor

[1242] 51. Stir for 15 minutes and allow to settle for 15 minutes.

[1243] 52. Drain the lower aqueous layer into a holding container.

[1244] 53. Drain the organic layer into a holding container. [Contains product]

[1245] 54. The aqueous layer from point 52 is recharged to the reactor.

[1246] 55. Charge EtOAc (5 vol) into the reactor

[1247] 56. Stir for 15 minutes and allow to settle for 15 minutes.

[1248] 57. Drain the lower aqueous layer into a holding container.

[1249] 58. Drain the organic layer into a holding container. [Contains product]

[1250] 59. The aqueous layer from point 57 is recharged to the reactor.

[1251] 60. Charge EtOAc (5 vol) into the reactor

[1252] 61. Stir for 15 minutes and allow to settle for 15 minutes.

[1253] 62. Drain the lower aqueous layer into a holding container.

[1254] 63. Drain the organic layer into a holding container. [Contains product]

[1255] 64. Anhydrous MgSO4 (0.2 wt) was charged to the holding vessel containing the organic layers from points 53, 58 and 63.

[1256] 65. Stir the holding container until flocs are formed.

[1257] 66. Filter the suspension through a sinter filter to remove MgSO4.

[1258] 67. Wash the filter cake with EtOAc (2 vol).

[1259] 68. Concentrate the combined filtrates in vacuo on a rotary evaporator at 35 to 40 °C to remove EtOAc. Continue drying for at least 18 hours after the solvent stops distilling from the flask. [Yellow / beige solid]

[1260] 69. Continue drying until constant weight is reached (the net weight difference between successive dryings should be at least 1 hour apart). < 1.0%). If dryness is not achieved within 48 hours, contact project chemist for advice.

[1261] Step 3 1. Charge methanol (5 vol) into the reactor.

[1262] 2. Adjust the contents of the reactor to 0 to 5°C

[1263] 3. Charge acetyl chloride (1.01 equiv) into the reactor, maintaining the temperature ≤ 20°C [strong exotherm, dissipates quickly].

[1264] 4. Stir the contents of the reactor at 20 to 25°C for at least 15 minutes.

[1265] 5. Drain the solution from the reactor into a holding container [colorless solution]

[1266] 6. Charge methanol (5 vol) into the reactor.

[1267] 7. Charge the product of step 2 (1 equivalent) into the reactor [yellow / orange solution]

[1268] 8. Adjust the contents of the reactor to 10 to 15°C.

[1269] 9. Charge the preformed methanolic HCl solution from point 5 to the reactor over at least 15 minutes, maintaining the temperature ≤ 25°C [slight exotherm].

[1270] 10. Adjust the contents to 20 to 25°C and stir for at least 2 hours.

[1271] 11. The reaction mixture was concentrated in vacuo at 40 to 45°C to give a beige solid.

[1272] 12. IPA (5 vol) was charged to RBF and concentrated in vacuo at 40 to 45 °C to give a beige solid.

[1273] 13. Charge IPA (2 vol) into the reactor.

[1274] 14. Add wet product solids from point 12.

[1275] 15. Charge IPA (1 vol) from the previous step into the rotary evaporation flask as a rinse and transfer the suspension to the reactor.

[1276] 16. Adjust the contents to 60 to 65°C and stir for at least 1 hour [beige suspension]

[1277] 17. Adjust the contents to 0 to 5°C (target 4°C) over at least 10 hours and stir at this temperature for at least 1 hour [beige suspension]

[1278] 18. Filter the suspension. Solid is slightly hygroscopic - pump dry under a stream of nitrogen for no longer than 5 minutes.

[1279] 19. TBME (2 vol) was charged to the reactor and used to wash the filter cake. Solids were slightly hygroscopic - pumped dry under nitrogen flow for no longer than 5 minutes.

[1280] 20. Charge TBME (2 vol) to reactor and use to wash filter cake. Solid slightly hygroscopic - pump dry under nitrogen stream for no more than 5 minutes. Store under nitrogen blanket while awaiting IPC 1 results.

[1281] 21.IPC 1: Diastereomeric purity by HPLC. Target: ≥98 area% de.

[1282] 22. Transfer to an oven and dry under vacuum at 35 to 40°C with nitrogen evolution for a minimum of 12 hours. Crush the material thoroughly using an appropriate unused spoon and continue drying until a constant weight is reached (net weight difference between successive dryings with a minimum interval of 1 hour). < 1.0%). If dryness is not achieved within 48 hours, contact project chemist for advice.

[1283] Step 4 1. Charge ethanol (7 vol) into the reactor.

[1284] 2. Adjust the contents of the reactor to -5 to 0°C

[1285] 3. Charge acetyl chloride (5 equivalents) into the reactor and keep the temperature ≤ 20°C [colorless solution]

[1286] 4. Load the product of step 3 (1 equivalent) into the reactor [orange solution]

[1287] 5. Adjust the contents of the reactor to 20 to 25 °C and stir for at least 18 hours [orange solution].

[1288] 6. IPC 1: Determine reaction completion by HPLC. Target ≤ 0.5% SM remaining.

[1289] 7. The reaction mixture was transferred to a rotary evaporator and concentrated under vacuum at 35 to 40 °C.

[1290] 8. Charge toluene (5 vol) into the flask and concentrate under vacuum on a rotary evaporator at 35 to 40°C. Continue concentrating for at least 1 hour after the solvent stops distilling to ensure optimal drying. [Tan solid]

[1291] 9. Transfer the solid to an oven and dry under vacuum at 35 to 40°C with nitrogen evolution for a minimum of 12 hours. Crush the material thoroughly using an appropriate unused spoon and continue drying until a constant weight is reached (net weight difference between successive dryings with a minimum interval of 1 hour). < 1.0%). If dryness is not achieved within 48 hours, contact project chemist for advice.

[1292] Step 5

[1293] 1. Charge dichloromethane (8 vol) into the reactor.

[1294] 2. Charge triethylamine (3.5 eq.) into the reactor.

[1295] 3. Adjust the contents of the reactor to -5 to 0°C

[1296] 4. The product of step 4 (1 equivalent) was added to the reactor in batches, keeping the temperature ≤ 20°C.

[1297] [Beige suspension]

[1298] 5. Ethyl chloroformate (1.5 equiv.) was charged to the reactor, maintaining the temperature ≤ 20°C [strong exotherm, rapidly dissipating, beige suspension].

[1299] 6. Adjust the contents to 20 to 25°C and stir for at least 2 hours.

[1300] 7. IPC 1: Determine reaction completion by HPLC. Target ≤ 0.5% SM remaining. If failed, stir for an additional 2 hours.

[1301] 8. The reaction was quenched slowly by adding water (3 vol). [A clear two-phase layer was formed]

[1302] 9. Stir for 15 minutes and allow to settle for 15 minutes.

[1303] 10. Discharge the lower organic layer into a holding container [containing the product]

[1304] 11. Drain the upper aqueous layer into a holding container.

[1305] 12. The organic layer from point 10 was recharged to the reactor.

[1306] 13. Charge 2M NaCl (4 vol) into the reactor

[1307] 14. Stir for 15 minutes and allow to settle for 15 minutes.

[1308] 15. Discharge the lower organic layer into a holding container [containing product]

[1309] 16. Drain the upper aqueous layer into a holding container.

[1310] 17. The organic layer from point 15 was concentrated in vacuo on a rotary evaporator at 40 to 45°C to remove DCM.

[1311] 18. Transfer the solid to a vacuum oven and dry at 35 to 40°C for a minimum of 8 hours and a maximum of 64 hours. Crush the material thoroughly using a suitable unused spoon and continue drying until a constant weight is reached (the net weight difference between successive dryings should be at least 1 hour apart). < 1.0%). If dryness is not achieved within 48 hours, contact project chemist for advice.

[1312] Step 6 1. Charge 2M HCl (10 vol) into the reactor.

[1313] 2. Charge SM (1 equivalent) into the reactor. [Beige suspension]

[1314] 3. Adjust the contents of the reactor to 60 to 65°C and stir for 2 to 3 hours.

[1315] 4. Adjust the contents to 20 to 25°C

[1316] 5. Chemist Check 1: Confirm reaction completion by HPLC. Target: FIO.

[1317] 6. Charge sodium carbonate (1.3 wt) into the reactor, keeping the temperature ≤ 30°C. [Small exotherm, controlled by addition rate, CO2 release]

[1318] 7. Charge dichloromethane (5 vol).

[1319] 8. Stir for 15 minutes and allow to settle for 15 minutes.

[1320] 9. Discharge the lower organic layer into a holding container [containing the product]

[1321] 10. Charge dichloromethane (5 vol).

[1322] 11. Stir for 15 minutes and allow to settle for 15 minutes.

[1323] 12. Discharge the lower organic layer into a holding container [containing the product]

[1324] 13. Drain the upper aqueous layer into a holding container.

[1325] 14. The organic layers from points 9 and 12 were recharged into the reactor.

[1326] 15. Charge 2M HCl (5 vol) into the reactor

[1327] 16. Stir for 15 minutes and allow to settle for 15 minutes.

[1328] 17. Discharge the lower organic layer into the holding container

[1329] 18. Drain the upper aqueous layer into a holding vessel [containing product]

[1330] 19. The organic layer from point 17 was recharged to the reactor.

[1331] 20. Charge 2M HCl (5 vol) into the reactor

[1332] 21. Stir for 15 minutes and allow to settle for 15 minutes.

[1333] 22. Discharge the lower organic layer into the holding container

[1334] 23. Drain the upper aqueous layer into a holding container [containing product]

[1335] 24. The aqueous layers from points 18 and 23 were recharged into the reactor.

[1336] 25. Adjust the contents of the reactor to 60 to 65°C and stir for 2 to 3 hours.

[1337] 26. Adjust the contents to 20 to 25°C

[1338] 27.IPC 1: Determine reaction completion by HPLC. Target: ≤ 0.5% SM remaining.

[1339] 28. Charge sodium carbonate (1.3 wt) into the reactor, keeping the temperature ≤ 30°C. [Small exotherm, controlled by addition rate, CO2 release]

[1340] 29. Charge dichloromethane (5 vol).

[1341] 30. Stir for 15 minutes and allow to settle for 15 minutes.

[1342] 31. Discharge the lower organic layer into a holding vessel [containing product]

[1343] 32. Charge dichloromethane (5 vol).

[1344] 33. Stir for 15 minutes and allow to settle for 15 minutes.

[1345] 34. Discharge the lower organic layer into a holding vessel [containing product]

[1346] 35. Drain the upper aqueous layer into a holding container.

[1347] 36. Anhydrous MgSO4 (0.2 wt) was charged to the holding vessel containing the organic layers from points 31 and 34.

[1348] 37. Keep stirring the container until flocs are formed.

[1349] 38. Filter the suspension through a sintered filter to remove MgSO4.

[1350] 39. Wash the filter cake with dichloromethane (2 vol).

[1351] 40. Chemist's Check 2: Stripping gravimetric determination of dichloromethane solution; used to determine the amount of piperidinone intermediate present, and therefore determine the subsequent charge for this step.

[1352] 41. ((2R,4R)-4-Fluoropyrrolidin-2-yl)methanol hydrochloride (1.3 equiv.) was charged into the reactor.

[1353] 42. Charge acetic acid (3 eq.) into the reactor.

[1354] 43. Adjust the contents of the reactor to 20 to 25°C and stir for at least 24 hours.

[1355] 44. Sodium triacetoxyborohydride (2 equivalents) was charged into a reactor, maintaining the temperature ≤ 30°C.

[1356] 45. Adjust the contents of the reactor to 20 to 25°C and stir for at least 4 hours.

[1357] 46. ​​IPC 2: Reaction completed by HPLC. Target: ≤ 1.0% piperidinone remaining. Failed Cycle - Charge more sodium triacetoxyborohydride.

[1358] 47. Charge 5% citric acid solution (5 vol) into the reactor

[1359] 48. Stir for 15 minutes and allow to settle for 15 minutes.

[1360] 49. Discharge the lower organic layer into the holding container

[1361] 50. Drain the upper aqueous layer into a holding vessel [containing product]

[1362] 51. The organic layer from point 49 is recharged to the reactor.

[1363] 52. Charge 5% citric acid solution (5 vol) into the reactor

[1364] 53. Stir for 15 minutes and allow to settle for 15 minutes.

[1365] 54. Discharge the lower organic layer into the holding container

[1366] 55. Drain the upper aqueous layer into a holding vessel [containing product]

[1367] 56. The aqueous layers from points 50 and 55 are recharged into the reactor.

[1368] 57. Sodium carbonate (2 wt) was charged into the reactor, and the temperature was maintained at ≤ 30°C [small exotherm, CO2 release was controlled by addition rate]

[1369] 58. Charge dichloromethane (5 vol).

[1370] 59. Stir for 15 minutes and allow to settle for 15 minutes.

[1371] 60. Discharge the lower organic layer into a holding vessel [containing product]

[1372] 61. Charge dichloromethane (5 vol).

[1373] 62. Stir for 15 minutes and allow to settle for 15 minutes.

[1374] 63. Discharge the lower organic layer into a holding vessel [containing product]

[1375] 64. Drain the upper aqueous layer into a holding container.

[1376] 65. The organic layers from points 60 and 63 are recharged into the reactor.

[1377] 66. Add purified water (5 vol) into the reactor

[1378] 67. Stir for 15 minutes and allow to settle for 15 minutes.

[1379] 68. Discharge the lower organic layer into a holding vessel [containing product]

[1380] 69. Drain the upper water layer into a holding container

[1381] 70. The organic layer from point 68 is recharged to the reactor.

[1382] 71. Add purified water (5 vol) into the reactor

[1383] 72. Stir for 15 minutes and allow to settle for 15 minutes.

[1384] 73. Discharge the lower organic layer into a holding vessel [containing product]

[1385] 74. Drain the upper water layer into a holding container

[1386] 75. Chemist Check 3: Remove residual fluoroprolinol from the organic layer. If failed, repeat the water wash.

[1387] 76. Anhydrous MgSO4 (0.2 wt) was charged to the holding vessel containing the organic layer from point 73.

[1388] 77. Keep stirring the container until flocs are formed.

[1389] 78. The suspension was filtered through a sintered filter to remove MgSO4.

[1390] 79. The filter cake was washed with dichloromethane (2 vol).

[1391] 80. The filtrate and washings from points 78 and 79 were concentrated in vacuo on a rotary evaporator at 35 to 40°C to remove the DCM. Concentration was continued for at least 1 hour after the solvent ceased distilling to ensure optimal drying. The product was an oil which slowly crystallized on standing. [Beige / yellow solid]

[1392] Step 7 (recrystallization / grinding)

[1393] 1. TBME (11 vol) was charged into the reactor.

[1394] 2. Charge heptane (5 vol) into the reactor.

[1395] 3. Load the product of step 6 (1 equivalent) into the reactor

[1396] 4. Adjust the contents to 50 to 60°C

[1397] 5. Chemist Check 1: Solubility Check. Achieve target solubility. If failed, add the minimum amount of 11:5 TBME:heptane mixture required to achieve solubility.

[1398] 6. Adjust contents to 35 to 40°C and inoculate with Form C at 0.1% w / w.

[1399] 7. Adjust the contents to 20 to 25°C and maintain for at least 12 hours.

[1400] 8. Filter the suspension.

[1401] 9. 2:1 heptane:TBME (2 vol) was charged to the reactor and used to wash the filter cake.

[1402] 10. Transfer the solid to a vacuum oven and dry at 35 to 40°C for a minimum of 8 hours and a maximum of 64 hours. Use an appropriate unused spoon to crush the material thoroughly and continue drying until a constant weight is reached (the net weight difference between successive dryings should be at least 1 hour apart). < 1.0%). If dryness is not achieved within 48 hours, contact project chemist for advice.

[1403] Step 0: Recrystallization of Fluprolinol HCl Reagent

[1404]

[1405] 1. Charge ethanol (8 vol) into the reactor.

[1406] 2. Charge Fluprolinol HCl (1 equivalent) into the reactor

[1407] 3. Adjust the contents to 75 to 80°C

[1408] 4. Chemist Check 1: Solubility Check. Achieve target solubility. If failed, add the minimum amount of ethanol required to achieve solubility.

[1409] 5. Adjust the contents to 20 to 25°C and maintain for at least 12 hours.

[1410] 6. Filter the suspension.

[1411] 7. TBME (1 vol) was charged into the reactor and used to wash the filter cake.

[1412] 8. Transfer the solid to a vacuum oven and dry at 35 to 40°C for a minimum of 8 hours and a maximum of 64 hours. Use an appropriate unused spoon to crush the material thoroughly and continue drying until a constant weight is reached (the net weight difference between successive dryings should be at least 1 hour apart). < 1.0%). If dryness is not achieved within 48 hours, contact project chemist for advice.

[1413] The product (cis-2-{4-[(2R,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester) 1 H-NMR shows Fig.24 middle.

[1414] Synthesis Example 6: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6- Synthesis of Ethyl Formate Citrate (Citrate Alternative Route)

[1415] Cis-2-[4-(1-methyl-1H-pyrazole-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate (citrate) is prepared by precipitation from 2-butanol. Cis-2-[4-(1-methyl-1H-pyrazole-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (free base) (234.7mg) and citric acid (130.6mg, about 1mol equivalent) are loaded into 4mL vials. 2-BuOH (3 volumes) is added, and the mixture is stirred and heated to 60°C for about 2 hours. The temperature is reduced by 5°C (about 10 minutes each time) until ambient temperature is reached. The obtained solid is separated by vacuum filtration. The solid is washed with MTBE (2x about 5 volumes) and air-dried for about 1.5 hours, and then transferred to a pre-weighed vial. The solid was dried in a vacuum oven at 40°C until constant weight. The citrate salt was isolated in approximately 75% yield.

[1416] Synthesis Example 7: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6- Ethyl formate hydrochloride (HCl salt)

[1417] Acetyl chloride (0.44 mL, 5.98 mmol) was added to EtOH (10.25 mL) at 0°C and after 10 minutes at this temperature, this solution was added to EtOH (10.25 mL) containing cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (2.05 g, 5.92 mmol) 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 until complete dissolution was achieved (110°C). The reaction mixture was maintained at this temperature for 10 min and then allowed to cool to 20 to 25°C over 3h. Diethyl ether (8 mL) was added to the viscous suspension to aid stirring. After another 10 minutes at 20 to 25°C, the suspension was filtered and drained on a sinter funnel for 2 hours. The solid was then transferred to a vacuum oven where it was dried at 40 °C overnight to afford the HCl salt as an off-white solid (1.497 g, 3.91 mmol, 66% yield).

[1418] Synthesis Example 8: cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-yl Ethyl formate fumarate (fumarate)

[1419] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate (fumarate) was prepared by precipitation from ethanol (EtOH). Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (240.0 mg) and fumaric acid (80.1 mg, about 1 mol equivalent) were loaded into a 4 mL vial. EtOH (2.5 volumes) was added, and the mixture was stirred and heated to 45 ° C for about 2 hours. Thereafter, the temperature was reduced by 5 ° C (about 10 minutes each time) until ambient temperature was reached. The obtained solid was separated by vacuum filtration. The solid was washed twice with EtOH (about 5 volumes) each time. The solid was air dried on the filter for about 1.5 hours and then transferred to a pre-weighed vial. The solid was further dried in a vacuum oven at 40°C until constant weight. The fumarate salt was produced in about 34% yield.

[1420] Example A

[1421] Salt screening

[1422] The physical properties of various salt forms of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate were evaluated for further development. The goal was to find salts with the desired properties (e.g., reduced hygroscopicity, solubility, crystallinity, physical stability, etc.) to support subsequent phase clinical formulations. The following acids were studied: 2,5-dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, succinic acid (succinic acid), citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), glycolic 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).

[1423] The experiment was carried out using 1:1, 1:2, 2:1 and 4:1 stoichiometric (API: acid) on a scale of 20 to 30 mg. A solution of the free base in the selected solvent was added to a solution of the salt former or a pure salt former. Cis-2-[4-(1-methyl-1H-pyrazole-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-ethyl carboxylate (free base) was added to a 5 mL (ACN, EtOH, THF, ACN / water EtOH / water or THF / water) or 20 mL (MeOH) volumetric flask and made constant to volume to form a 1.0 M or 0.1 M solution. It was sonicated in a water bath to ensure complete dissolution.

[1424] The salts were subjected to a variety of tests and analytical techniques including: NMR and XRPD analysis, humidity stress testing, dynamic vapor sorption (DVS) testing. Crystallization, crystallinity, water solubility, and ease of manufacturing were also evaluated. The fumarate, citrate, and HCl salts showed the best properties and were therefore selected for further study.

[1425] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester Characterization of Citrate (Citrate Salt)

[1426] The XRPD pattern (pattern B) obtained for citrate (Synthesis Example 6) is shown in Figure 1 Middle. XRPD pattern indicated crystalline material.

[1427] Thermogravimetric / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and the associated weight % change of citrate ( Figure 2 ). TG / DTA showed a weight loss of about 33% between about 140°C and about 220°C, most likely due to loss of citric acid. The second weight loss at temperatures above 220°C corresponds to the onset of decomposition of the material. A broad endothermic peak was observed at an onset temperature of about 174°C. Analysis indicated that the citrate salt was anhydrous.

[1428] Polarized light microscopy ( Figure 3 ) shows that the material consists of a crystalline solid with an irregular morphology.

[1429] 1H NMR spectrum ( Figure 4 ) was consistent with the molecular structure, and very little solvent was detected (<0.1 molar equivalent of 2-BuOH). The API:acid ratio was 1:1.

[1430] Dynamic vapor sorption (DVS) was used to determine the hygroscopic and adsorption properties of citrate. The isotherms obtained are shown in Figure 5 The samples were dried at 0% RH and then subjected to adsorption and desorption. The isotherm plots show that the material exhibits a gradual uptake of moisture, with a weight gain from 0% RH to 80% RH followed by a more dramatic weight gain between 80% and 90% RH. The isotherm plots show that the total weight gain observed between ambient (i.e., 40% RH) and 80% RH is -0.8%, indicating that the sample is slightly hygroscopic (based on Ph. Eur. classification, Table 1). Very little hysteresis is observed between the adsorption and desorption curves, and at 0% RH, all acquired moisture is lost. XRPD analysis after DVS ( Figure 6 ) shows that citrate is still retained as pattern B.

[1431] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester Characterization of Citrate Monohydrate

[1432] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate was obtained by the methods of Synthesis Examples 1, 2 and 3. A representative powder X-ray powder diffraction (XRPD) pattern is shown in Fig.25 , and Table B shows the corresponding peaks and intensities. Fig.25 The XRPD diffraction pattern in indicated that the sample was crystalline and exhibited a pattern consistent with cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate monohydrate.

[1433] Table B

[1434]

[1435] Thermogravimetric / differential thermal analysis (TG / DTA) was performed to determine the thermal profile of citrate monohydrate and the associated weight % change ( Fig.26 ). TG / DTA showed a weight loss phase of about 0.155% from RT to about 65°C, followed by a weight loss phase of 3.188% from about 65 to 110°C, which appeared to be associated with a broad endothermic event with an onset temperature of about 88.15°C. This may be due to dehydration / loss of bound hydrate (theoretical stoichiometric monohydrate - 3.14%) and loss / change of crystal structure. This event was followed by a second weight phase of 27.399% from about 170 to 210°C, which appeared to be associated with an overlapping endothermic event, which included a sharp endotherm with an onset temperature of about 175.48°C. This may be due to melting of the dehydrated form and loss of acid (theoretical stoichiometric citrate - 25.7%), followed by immediate degradation.

[1436] Four samples of similar weight were prepared and analyzed by DSC at different heating rates (5.0°C / min, 10.0°C / min, 20.0°C / min, and 50.0°C / min) to study the thermal behavior of the material. The onset of dehydration / melting appeared to be dependent on the heating rate (in the range from about 79°C to 103°C). The second more intense endothermic event appeared to be somewhat dependent on the heating rate and was observed to range from about 174.1°C to 178.5°C with an average onset melting temperature of 176.4°C ± 2°C.

[1437] Dynamic vapor sorption (DVS) was used to determine the hygroscopic and adsorption properties of citrate monohydrate. The isotherms obtained are shown in Fig. 27The above adsorption isotherms indicate that citrate monohydrate is slightly hygroscopic (based on Ph. Eur. classification, Table 1), adsorbing about 0.770% water at 25°C / 80% RH. However, the moisture content is gained at low humidity (probably recovering losses from the drying step) and remains relatively constant. The diffractograms taken before and after DVS indicate that there is no form change due to humidity changes.

[1438] Primary particles of citrate monohydrate were observed as prismatic / blade-shaped particles with a size typically less than 50 μm, which formed aggregates that were typically larger than 100 μm. Representative images of aggregates measured by polarized light microscopy (PLM) and scanning electron microscopy (SEM) and individual particles measured by SEM are shown in Fig.28 and Fig.29 middle.

[1439] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester Characterization of Hydrochloride (HCl Salt)

[1440] Figure 7 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 indicated crystalline material.

[1441] Thermogravimetric / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and associated weight % change ( ) of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride. Figure 8 ). A 3.6% weight loss was noted from about 25°C to 200°C, which corresponds to a loss of about 0.75 moles of water, indicating that the HCl salt contained moisture. A second weight loss at a temperature above 210°C corresponds to the onset of decomposition of the material. An endotherm was observed at an onset temperature of about 234°C.

[1442] The DSC thermogram of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride is shown in Fig. 9 The melting point cannot be determined because decomposition begins before melting.

[1443] Polarized light microscopy ( Fig.10 ) showed that the material consisted of a crystalline solid with an irregular morphology.

[1444] 1H NMR spectrum ( Fig.11 ) is consistent with the molecular structure, and minimal solvent is detected.

[1445] Dynamic vapor sorption (DVS) was used to determine the hygroscopic and adsorption properties of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate hydrochloride. The isotherms obtained are shown in Fig.12 The samples were dried at 0% RH and then subjected to adsorption and desorption. The isotherms showed that the material showed a gradual uptake of moisture from 0 to 60% RH, followed by a higher rate of weight gain between 60% RH and 70% RH. The rate of weight gain decreased between 70% RH and 80% RH, and increased again between 80% RH and 90% RH. The isotherms showed 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 European Pharmacopoeia classification, Table 1). The desorption rate was comparable to the adsorption rate between 90% RH and 70% RH. However, a hysteresis was observed between the adsorption and desorption curves between 70% RH and 0% RH, and approximately 3% of water was retained at 0% RH. XRPD analysis performed after DVS ( Fig.13 ) show that cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester hydrochloride has been converted to the known alternative HCl salt form. These results indicate that conversion to the alternative HCl salt material occurs between 60% RH and 70% RH.

[1446] Cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester Characterization of Fumarate (Fumarate Salt)

[1447] The obtained XRPD pattern (pattern A) of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate is shown in Fig.14 Medium. XRPD pattern indicated crystalline material.

[1448] Thermogravimetric / differential thermal analysis (TG / DTA) was performed to determine the thermal profile and associated weight % change ( cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate salt Fig.15 ). No weight loss was observed between about 25°C and 180°C, indicating that cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate is anhydrous. The second weight loss at a temperature above 180°C corresponds to the onset of material decomposition. An endotherm most likely due to melting was observed at an onset temperature of about 175°C.

[1449] Polarized light microscopy ( Fig.16 ) show that the material is crystalline and consists of fine particles that tend to form aggregates / agglomerates.

[1450] 1H NMR spectrum ( Fig.17 ) was consistent with the molecular structure, and no solvent was detected. The API:acid ratio was determined to be 1:1.35.

[1451] Dynamic vapor sorption (DVS) was used to determine the hygroscopic and adsorption properties of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate. The isotherms obtained are shown in Fig.18 The samples were dried at 0% RH and then subjected to adsorption and desorption. The isotherms showed that the material exhibited a gradual water uptake between 0% RH and 60% RH, followed by a more significant water uptake between 60% RH and 80% RH, and a more intense uptake between 80% RH and 90% RH. The isotherms showed a total weight gain of 0.3% observed between ambient (i.e., 40% RH) and 80% RH, indicating that the sample was slightly hygroscopic (based on the European Pharmacopoeia classification, Table 1). There was little hysteresis between the adsorption and desorption curves, and all the water gained at 0% RH was lost. XRPD analysis was performed after DVS ( Fig.19 ). The diffraction pattern shows that cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate is retained as pattern A.

[1452] cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro [3.4] Characterization of ethyl octane-6-carboxylate (free base)

[1453] A representative powder X-ray powder diffraction (XRPD) pattern of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester (free base) is shown in Fig.32 , and Table C shows the corresponding peaks and intensities. Fig.32 The XRPD diffraction pattern in indicated that the sample was crystalline.

[1454] Table C

[1455]

[1456] 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. Fig.33TG / DTA showed a weight loss phase of about 0.335% from RT to about 125°C, and a broad endothermic event with an onset of about 98.93°C.

[1457] The hygroscopic and adsorption properties of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester were determined using dynamic vapor sorption (DVS). The adsorption isotherms indicated that the crystalline form of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester was slightly hygroscopic (based on the Ph. Eur. classification, Table 1), adsorbing approximately 0.669% of water at 25°C / 80% RH. The diffraction patterns obtained before and after DVS indicated that there was no form change due to humidity changes.

[1458] The crystalline material of cis-2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester was observed by SEM as irregular / slightly flaky particles with sizes typically ranging from 20 to 100 μm. The fines appeared to be aggregated on larger particles in a cluster-like morphology.

[1459] Humidity stress of salt

[1460] Samples of ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate fumarate (fumarate), ethyl cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate citrate (citrate), and ethyl 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 samples after stressing did not detect a change in form of the fumarate and citrate salts ( Fig. 20 , Fig.21 ), however the HCl salt showed transformation to the alternative HCl salt form ( Fig. 22 ).

[1461] Water solubility of salt

[1462] The water solubility of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester fumarate (fumarate) and cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester citrate (citrate) was evaluated by adding aliquots. The fumarate was almost completely dissolved at about 320 mg / mL, and the solution pH was about 4-5. However, when more water was added, solids precipitated. These solids were analyzed by XRPD and determined to be fumaric acid ( Fig.23 ). This indicates that the fumarate salt is converted to free base and fumaric acid in water. The citrate salt exhibits a water solubility of about 850 to 1700 mg / mL and a solution pH of about 3 to 4. The water solubility of the HCl salt is about 340 to 390 mg / mL.

[1463] Table 1: Hygroscopicity classification (adopted from European Pharmacopoeia and Sihorkar et al. (Pharmaceutical Dev. & Technol.(2013),18(2),348-358)

[1464]

[1465] Biological data

[1466] Biological Example 1

[1467] Phospho-ERK1 / 2 assay

[1468] 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 Gq / 11 and Gi / o protein-coupled receptor activation, making it well suited for the evaluation of M1, M3 (Gq / 11 coupled) and M2, M4 receptors (Gi / o coupled), rather than using different assay formats for different receptor subtypes. CHO cells stably expressing human muscarinic M1, M2, M3 or M4 receptors were seeded (25K / well) on 96-well tissue culture plates containing MEM-α + 10% dialyzed FBS. Once adhered, cells were serum starved overnight. Agonist stimulation was performed by adding 5 μL of agonist to the cells for 5 minutes (37°C). The culture 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 mixture was added. The plates were incubated for 2 hours in the dark with gentle agitation and then read on a PHERAstar plate reader. The pEC was calculated from the resulting data for each receptor subtype. 50 and E max value.

[1469] The activity data for ethyl 2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate, ethyl 2-{4-[(2R)-4,4-difluoro-2-(hydroxymethyl)pyrrolidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate and ethyl 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.

[1470] Table D

[1471]

[1472] Equivalent

[1473] The above-described embodiments are presented for the purpose of illustrating the present invention, and should not be construed as imposing any limitation on the scope of the present invention. It is apparent that, without departing from the principles of the present invention, various modifications and changes may be made to the specific embodiments of the present invention described above and illustrated in the embodiments. All such modifications and changes are intended to be encompassed by the application.

Claims

1. Compound of formula (1): or a salt thereof.

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

3. An acid addition salt of the compound according to claim 1.

4. The acid addition salt according to claim 3, wherein the acid is selected from: 2,5-dihydroxybenzoic acid (gentisic acid), 2-furoic acid, acetic acid, succinic acid (succinic acid), citric acid, ethanesulfonic acid (ESA), fumaric acid, gluconic acid (D), glucuronic acid (D), glycolic 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 acid and tartaric acid (L).

5. The acid addition salt according to claim 3, wherein the acid is selected from: Hydrochloric acid (HCl), fumaric acid, and citric acid.

6. A citrate salt of the compound according to claim 1.

7. Citrate monohydrate of the compound according to claim 1.

8. The compound according to claim 1, which is a compound of formula (3b):

9. A pharmaceutical composition comprising a compound as defined in 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. A compound or composition according to any one of claims 1 to 9 for use in treating cognitive disorders or psychiatric disorders or treating or reducing the severity of acute pain, chronic pain, neuropathic pain or inflammatory pain.

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

13. A method for synthesizing the compound according to claim 1, comprising the following steps: Where R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl, and R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group.

14. A method for synthesizing the compound according to claim 1, comprising the following steps: Where R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl, and R 2 N together with the oxygen atom to which it is attached represents any suitable leaving group.

15. A method for synthesizing the compound according to claim 1, comprising the following steps: Where R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl, and Cat. is a hydrogenation catalyst.

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

17. The method according to claim 13 or 14, wherein R 2 It is tosyl.

18. A method for synthesizing the compound according to claim 1, comprising:

19. A method for synthesizing the compound according to claim 1, comprising:

20. A method for synthesizing the compound according to claim 1, comprising:

21. A method for synthesizing the compound according to claim 1, comprising:

22. A synthesis method comprising the following steps: Where Z is selected from: R 1 It is C 1-6 Straight or branched chain alkyl or C 3-6 Cycloalkyl, and R 2 Together with the oxygen atom to which it is attached, represents any suitable leaving group.

23. The method of claim 22, wherein Z is:

24. The method of claim 22, wherein Z is:

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 process of any one of claims 13 to 21.

26. A crystalline form of cis-2-[4-(1-methyl-1H-pyrazol-5-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylic acid ethyl ester 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. 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. The crystalline form according to 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. The crystalline form according to 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. 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. The crystalline form according to any one of claims 25 to 30, characterized in that The DSC thermogram has an endothermic peak with an onset at about 176°C.

33. The crystalline form according to any one of claims 25 to 30, characterized in that The DSC thermogram is essentially as shown in Figure 26.

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

35. The crystalline form according to any one of claims 25 to 30, 32 and 33, characterized in that The TGA thermogram is essentially as shown in Figure 26.

36. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, at least one XRPD peak is selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°.

37. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, at least two XRPD peaks are selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°.

38. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, at least three XRPD peaks are selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°.

39. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, the three XRPD peaks are at 11.3°±0.2°, 19.2°±0.2°, and 20.5°±0.2°.

40. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, the three XRPD peaks are at 11.3°±0.2°, 18.4°±0.2°, and 20.5°±0.2°.

41. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, the three XRPD peaks are at 11.3°±0.2°, 18.4°±0.2°, and 19.2°±0.2°.

42. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, the three XRPD peaks are at 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°.

43. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that Expressed in 2θ, the four XRPD peaks are at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°.

44. The crystalline form according to any one of claims 25 to 30 and 32 to 35, characterized in that The XRPD spectrum is essentially as shown in Figure 25.

45. 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: (1) an XRPD substantially as shown in Figure 25; (2) a DSC thermogram substantially as shown in FIG. 26 ; and (3) A TGA thermogram substantially as shown in FIG. 26 .

46. ​​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: (1) at least one XRPD peak, expressed in 2θ, selected from the group consisting of 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

47. 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: (1) at least two XRPD peaks, expressed in 2θ, selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

48. 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: (1) at least three XRPD peaks, expressed in 2θ, selected from 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

49. 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: (1) Expressed in 2θ, three XRPD peaks are at 11.3°±0.2°, 19.2°±0.2°, and 20.5°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

50. 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: (1) Expressed in 2θ, three XRPD peaks are at 11.3°±0.2°, 18.4°±0.2°, and 20.5°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

51. 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: (1) Expressed in 2θ, three XRPD peaks are at 11.3°±0.2°, 18.4°±0.2°, and 19.2°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

52. 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: (1) Expressed in 2θ, three XRPD peaks are at 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

53. 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: (1) Expressed in 2θ, the four XRPD peaks are at 11.3°±0.2°, 18.4°±0.2°, 19.2°±0.2°, and 20.5°±0.2°; (2) a DSC thermogram having an endothermic peak with an onset at about 176° C.; and (3) TGA thermogram having a weight loss of about 3.3% from room temperature to about 110°C.

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

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

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

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

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

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

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

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

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

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

64. 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: (1) an XRPD substantially as shown in Figure 1; (2) a DSC thermogram substantially as shown in FIG2 ; and (3) A TGA thermogram substantially as shown in FIG2 .

65. 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: (1) an XRPD substantially as shown in Figure 1; (2) a DSC thermogram having an endothermic peak onset at about 174° C.; and (3) A TGA thermogram having a weight loss of about 33% loss from about 140°C to about 220°C.

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

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

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

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

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

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

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

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

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

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

76. A compound according to any one of claims 1 to 6, which is a compound of formula (4b):

77. A compound or salt according to any one of claims 1 to 8 or 76, or The crystalline form according to any one of claims 25 to 74, or The pharmaceutical composition according to claim 9 or 75, It is useful for treating diseases or conditions mediated by the muscarinic M4 receptor.

78. A compound or salt according to any one of claims 1 to 8 or 76, or The crystalline form according to any one of claims 25 to 74, or The pharmaceutical composition according to claim 9 or 75, It is used to treat cognitive impairment or psychotic disorders.

79. A compound or salt according to any one of claims 1 to 8 or 76, or The crystalline form according to any one of claims 25 to 74, or The pharmaceutical composition according to claim 9 or 75, It is used for treating cognitive disorders or psychotic disorders, wherein the cognitive disorders or psychotic disorders are selected from the following conditions, comprise the following conditions, are caused by the following conditions, or are associated with the following conditions: Cognitive impairment, mild cognitive impairment (MCI), frontotemporal dementia, vascular dementia, dementia with Lewy bodies, presenile dementia, senile dementia, Friedreich's ataxia, Down syndrome, Huntington's disease, hyperkinesia, mania, Tourette's syndrome, Alzheimer's disease, progressive supranuclear palsy, impairment of cognitive function including attention, orientation, learning disabilities, memory and language function; cognitive impairment caused by stroke, Huntington's disease, Pick's disease, AIDS-related dementia or other dementia conditions, such as multi-infarct dementia, alcoholic dementia, hypothyroid dementia and dementia associated with other degenerative disorders such as cerebellar atrophy and amyotrophic lateral sclerosis; other acute or subacute conditions that can cause cognitive impairment, such as delirium or depression, trauma, head trauma, age-related cognitive impairment, stroke , neurological degeneration, drug-induced conditions, neurotoxic agents, age-related cognitive impairment, autism-related cognitive impairment, Down syndrome, cognitive deficits associated with psychosis, and cognitive impairment associated with electroconvulsive therapy; cognitive impairment caused by drug abuse or drug withdrawal including nicotine, marijuana, 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, paranoia, hallucinations and delusional disorders, personality disorders, obsessive-compulsive disorder, schizotypal disorder, delusional disorder, psychosis caused by malignant disease, metabolic disorders, endocrine diseases or narcolepsy, psychosis caused by drug abuse or drug withdrawal, bipolar disorder, schizoaffective disorder and Alzheimer's psychosis.

80. A compound or salt according to any one of claims 1 to 8 or 76, or The crystalline form according to any one of claims 25 to 74, or The pharmaceutical composition according to claim 9 or 75, It is used to treat schizophrenia, Alzheimer's disease, or bipolar disorder.

81. A compound or salt according to any one of claims 1 to 8 or 76, or The crystalline form according to any one of claims 25 to 74, or The pharmaceutical composition according to claim 9 or 75, It is used to treat schizophrenia.

82. A compound or salt according to any one of claims 1 to 8 or 76, or The crystalline form according to any one of claims 25 to 74, or The pharmaceutical composition according to claim 9 or 75, It is used to treat bipolar disorder.

83. A compound or salt according to any one of claims 1 to 8 or 76, or The crystalline form according to any one of claims 25 to 74, or The pharmaceutical composition according to claim 9 or 75, It is used to treat Alzheimer's disease.

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