Synthesis method

Abstract

This disclosure relates to a process for preparing a compound of formula (A) that is useful as an intermediate in the preparation of mAChR agonist compounds; and to a composition comprising the compound of formula (A). One aspect of the present disclosure is a process for preparing a trans-alcohol compound of formula (A), comprising the step of reducing a compound of formula (B) in the presence of a ketoreductase enzyme having SEQ ID NO: 1 to provide the compound of formula (A). In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of NADPH as a cofactor.

Description

[Technical Field]

[0001] (Technical field) This disclosure relates to a method for synthesizing intermediate compounds in the synthesis of muscarinic acetylcholine receptor (mAChR) agonists, and more particularly to an enzymatic process that utilizes ketoreductase to perform stereoselective reduction from ketones to alcohols.

[0002] (Sequence Listing) This application includes a sequence listing electronically submitted as an XML file named "46696-0184WO1 _ST26_SL.xml." This XML file, created on July 31, 2024, is 3,828 bytes in size. The material contained in this XML file is incorporated herein by reference in its entirety. [Background technology]

[0003] (Technical background) Muscarinic acetylcholine receptors (mAChRs) are members of the G protein-coupled receptor superfamily that mediate the action of the neurotransmitter acetylcholine in both the central and peripheral nervous systems. Five mAChR subtypes have been cloned (M1-M5).

[0004] Muscarinic receptors in the central nervous system (particularly M1 mAChRs) play a central role in mediating higher-order cognitive processing. Cognitive impairment-related diseases (e.g., Alzheimer's disease) are associated with loss of cholinergic neurons in the basal forebrain (Whitehouse et al., 1982 Science). In schizophrenia (also characterized by cognitive impairment), mAChR density is reduced in the prefrontal cortex, hippocampus, and caudate nucleus putamen of schizophrenic subjects (Dean et al., 2002 Mol Psychiatry). Furthermore, in animal models, blockade or damage to central cholinergic pathways results in severe cognitive deficits. Non-selective mAChR antagonists have been shown to induce psychopathic effects in psychotic patients. Cholinergic replacement therapy has primarily relied on the use of acetylcholinesterase inhibitors to prevent the breakdown of endogenous acetylcholine. These compounds demonstrated efficacy against symptomatic cognitive decline in clinical settings, but they caused dose-limiting side effects (including gastrointestinal motility disorders, bradycardia, nausea, and vomiting) resulting from stimulation of peripheral M2 mAChR and peripheral M3 mAChR.

[0005] Alzheimer's disease (AD) is the most common neurodegenerative disorder (26.6 million people worldwide in 2006), affecting older adults and resulting in severe memory loss and cognitive impairment. While the etiology of the disease is complex, it is characterized by two distinctive brain sequelae: amyloid plaques (primarily composed of amyloid-beta peptide (Aβ)) and neurofibrillary tangles (formed by hyperphosphorylated tau protein). Aβ accumulation is considered a central feature in the progression of AD, and therefore, many presumptive therapies for treating AD currently target the inhibition of Aβ production. Aβ originates from the cleavage of 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 located within the Aβ sequence, thereby preventing its formation. However, in the amyloid-forming pathway, APP is cleaved by β-secretase to produce soluble APPβ, which in turn produces Aβ. In vitro studies have shown that mAChR agonists can facilitate the processing of soluble APP into the non-amyloid-forming pathway.

[0006] Preclinical studies have suggested that mAChR agonists exhibit an atypical antipsychotic-like profile within a certain preclinical paradigm. Muscarinic receptors are also linked to the neurobiology of addiction. Behavioral and neurochemical studies have shown that the reinforcing effects of cocaine and other addictive substances are mediated by the mesolimbic dopaminergic system, where cholinergic muscarinic receptor subtypes play a crucial role in regulating dopaminergic neurotransmission.

[0007] Muscarinic receptors are also involved in motor control and may offer novel treatments for movement disorders (e.g., Parkinson's disease, ADHD, Huntington's disease, Tourette's syndrome, and other syndromes associated with dopaminergic dysfunction as an underlying pathogenic factor causing the disease).

[0008] The mAChR agonists xanomeline, subcomeline, miramelin, and cevimeline are all progressing through various stages of clinical development for the treatment of Alzheimer's disease and / or schizophrenia. However, in all clinical trials, xanomeline and other related mAChR agonists have shown unacceptable safety margins regarding cholinergic side effects (including nausea, gastrointestinal pain, diarrhea, excessive sweating, excessive salivation, syncope, and bradycardia).

[0009] Muscarinic receptors are involved in central and peripheral pain. Pain can be classified into three distinct types: acute pain, inflammatory pain, and neuropathic pain. Activation of muscarinic receptors has been shown to be analgesic across many pain states through activation of receptors in the spinal cord and higher pain centers in the brain. Increasing endogenous levels of acetylcholine through acetylcholinesterase inhibitors has been shown to impair the analgesic activity of direct activation of muscarinic receptors with agonists or allosteric modulators. In contrast, blockade of muscarinic receptors with antagonists, or blockade using knockout mice, increases pain sensitivity. Evidence for the role of M1 receptors in pain is outlined by D.F. Fiorino and M. Garcia-Guzman, 2012.

[0010] More recently, a small number of compounds showing improved selectivity for the M1 mAChR subtype over peripherally expressed mAChR subtypes have been identified (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 their improved levels of selectivity over the M3 mAChR subtype, some of these compounds retain significant agonist activity at both this subtype and the M2 mAChR subtype.

[0011] WO2015 / 118342 describes certain mAChR agonist compounds, including a compound having the following structure:

Chemical Formula

Chemical Formula

Prior Art Literature

Patent Literature

[0012]

Patent Literature 1

Non-Patent Literature

[0013]

Non-Patent Literature 1

[0014] In this specification, the inventors of the present invention have defined formula (A): [ka] The synthesis of trans-alcohol compounds is described; The trans-alcohol compound of formula (A) is an intermediate for an improved synthetic pathway to mAChR agonist compounds, which exhibits a higher level of selectivity for M1 mAChR and / or M4 mAChR than for M2 and M3 receptor subtypes.

[0015] summary One aspect of this disclosure is formula (A): [ka] A process for preparing a trans-alcohol compound, Formula (B): [ka] The step of reducing the compound in the presence of a ketoreductase enzyme having SEQ ID NO: 1 to provide the compound of formula (A). This is a process that includes [something].

[0016] In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of NADPH as a cofactor.

[0017] In one embodiment, the NADPH cofactor is NADP + It is provided by a cofactor regeneration system containing glucose dehydrogenase and glucose.

[0018] In one embodiment, NADP + It is present at a concentration of approximately 5 mM; Glucose dehydrogenase is present at a concentration of approximately 1 g / L; and Glucose is present at a concentration of approximately 178 mM.

[0019] In one embodiment, NADP + It is present at a concentration of approximately 15 mM; Glucose dehydrogenase is present at a concentration of approximately 0.45 g / L; and Glucose is present at a concentration of approximately 247 mM.

[0020] In one embodiment, the compound of formula (B) is present at a concentration of approximately 88 mM. In another embodiment, the compound of formula (B) is present at a concentration of approximately 198 mM.

[0021] In one embodiment, the ketoreductase enzyme having SEQ ID NO: 1 is present at a concentration of approximately 1 g / L. In another embodiment, the ketoreductase enzyme is present at a concentration of approximately 2 g / L.

[0022] In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of potassium phosphate buffer. In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of approximately 100 mM potassium phosphate buffer. In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of approximately 100 mM potassium phosphate buffer at a pH of approximately 7.0.

[0023] In one embodiment, The step of reducing the compound of formula (B) is (a) NADPH as a cofactor; (b) Approximately 100 mM potassium phosphate buffer with a pH of approximately 7.0 The procedure was performed in the presence of NADP, and the NADPH cofactor was used. +It is provided by a cofactor regeneration system containing glucose dehydrogenase and glucose.

[0024] In one embodiment, NADP + It is present at a concentration of approximately 5 mM; Glucose dehydrogenase is present at a concentration of approximately 1 g / L; Glucose is present at a concentration of approximately 178 mM; The compound of formula (B) is present at a concentration of approximately 88 mM; and The ketoreductase enzyme with sequence number 1 is present at a concentration of approximately 1 g / L.

[0025] In one embodiment, NADP + It is present at a concentration of approximately 15 mM; Glucose dehydrogenase is present at a concentration of approximately 0.45 g / L; Glucose is present at a concentration of approximately 247 mM; The compound of formula (B) is present at a concentration of approximately 198 mM; and The ketoreductase enzyme with sequence number 1 is present at a concentration of approximately 1 g / L.

[0026] In one embodiment, the step of reducing the compound of formula (B) includes a step of heating at approximately 30°C. In another embodiment, the step of reducing the compound of formula (B) includes a step of stirring for approximately 20 to 24 hours.

[0027] Another aspect of this disclosure is formula (A): [ka] A composition comprising the compound, The compound of formula (A) is present in a diastereomer excess of approximately 96% de or approximately 97% de. It is a composition.

[0028] In one embodiment, the compound of formula (A) is present in a diastereomer excess of approximately 96% de.

[0029] In one embodiment, the compound of formula (A) is present in a diastereomeric excess of about 97% de.

[0030] (Detailed Description) (Process for Preparing Compounds) The present application provides a process for preparing intermediate compounds that are useful in the preparation of mAChR agonist compounds (e.g., the mAChR agonist compounds described in WO2015 / 118342) and salts thereof.

[0031] The trans-alcohol intermediate described herein has the following formula (A):

Chemical Formula

[0032] The synthetic route to mAChR agonist compounds utilizing the trans-alcohol intermediate compounds described herein avoids the need to separate stereoisomers by preparative HPLC, and therefore has improved developability for large-scale synthesis.

[0033] For example, an mAChR agonist compound (e.g., the mAChR agonist compound described in WO2015 / 118342) can be produced via a synthetic route comprising the steps outlined in the following scheme:

Chemical Formula

Chemical Formula

Chemical Formula

[0034] Therefore, equation (A): [ka] A process for preparing a trans-alcohol compound, Formula (B): [ka] The step of reducing the compound in the presence of a ketoreductase enzyme having SEQ ID NO: 1 to provide the compound of formula (A). Processes including the above are provided herein.

[0035] In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of NADPH as a cofactor.

[0036] In one embodiment, the NADPH cofactor is NADP + It is provided by a cofactor regeneration system containing glucose dehydrogenase and glucose.

[0037] In one embodiment, NADP + It is present at a concentration of approximately 5 mM to approximately 15 mM. In one embodiment, NADP + It is present at a concentration of approximately 5 mM. In one embodiment, NADP + It is present at a concentration of approximately 15 mM.

[0038] In one embodiment, glucose dehydrogenase is present at a concentration of approximately 1 g / L to approximately 0.45 g / L. In one embodiment, glucose dehydrogenase is present at a concentration of approximately 1 g / L. In one embodiment, glucose dehydrogenase is present at a concentration of approximately 0.45 g / L.

[0039] In one embodiment, glucose is present at a concentration of approximately 178 mM to approximately 247 mM. In one embodiment, glucose is present at a concentration of approximately 178 mM. In one embodiment, glucose is present at a concentration of approximately 247 mM.

[0040] In one embodiment, NADP + It is present at a concentration of approximately 5 mM; Glucose dehydrogenase is present at a concentration of approximately 1 g / L; and Glucose is present at a concentration of approximately 178 mM.

[0041] In one embodiment, NADP + It is present at a concentration of approximately 15 mM; Glucose dehydrogenase is present at a concentration of approximately 0.45 g / L; and Glucose is present at a concentration of approximately 247 mM.

[0042] In one embodiment, the compound of formula (B) is present at a concentration of about 88 mM to about 198 mM. In one embodiment, the compound of formula (B) is present at a concentration of about 88 mM. In one embodiment, the compound of formula (B) is present at a concentration of about 198 mM.

[0043] In one embodiment, the ketoreductase enzyme having SEQ ID NO: 1 is present at a concentration of approximately 1 g / L.

[0044] In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of potassium phosphate buffer. In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of about 100 mM potassium phosphate buffer. In one embodiment, the step of reducing the compound of formula (B) is carried out in the presence of about 100 mM potassium phosphate buffer (pH about 7.0).

[0045] In one embodiment, The step of reducing the compound of formula (B) is (a) NADPH as a cofactor; (b) Approximately 100 mM potassium phosphate buffer with a pH of approximately 7.0 The procedure was performed in the presence of NADP, and the NADPH cofactor was used. + It is provided by a cofactor regeneration system containing glucose dehydrogenase and glucose.

[0046] In one embodiment, NADP + It is present at a concentration of approximately 5 mM; Glucose dehydrogenase is present at a concentration of approximately 1 g / L; Glucose is present at a concentration of approximately 178 mM; The compound of formula (B) is present at a concentration of approximately 88 mM; and The ketoreductase enzyme with sequence number 1 is present at a concentration of approximately 1 g / L.

[0047] In one embodiment, NADP + It is present at a concentration of approximately 15 mM; Glucose dehydrogenase is present at a concentration of approximately 0.45 g / L; Glucose is present at a concentration of approximately 247 mM; The compound of formula (B) is present at a concentration of approximately 198 mM; and The ketoreductase enzyme with sequence number 1 is present at a concentration of approximately 1 g / L.

[0048] In one embodiment, the step of reducing the compound of formula (B) includes a step of heating at approximately 30°C. In another embodiment, the step of reducing the compound of formula (B) includes a step of stirring for approximately 20 to 24 hours.

[0049] The compound of formula (A) is a useful intermediate compound in the synthesis of mAChR agonist compounds described in WO2015 / 118342.

[0050] The process described herein also provides the compound of formula (A) with particularly high levels of diastereomer purity.

[0051] Therefore, also described herein is formula (A): prepared by any one of the processes described herein above: [ka] It is a compound of [the compound].

[0052] In one embodiment, the compound of formula (A) has a diastereomer excess of about 96%de.

[0053] In one embodiment, the compound of formula (A) has a diastereomer excess of about 97%de.

[0054] Formula (A): [ka] A composition comprising the compound, Compositions in which the compound of formula (A) is present in a diastereomer excess of about 96% de or about 97% de are also described herein.

[0055] In one embodiment, the compound of formula (A) is present in a diastereomer excess of approximately 96% de.

[0056] In one embodiment, the compound of formula (A) is present in a diastereomer excess of approximately 97% de.

[0057] One or more embodiments of the present disclosure may be combined with other embodiments of the present disclosure. [Modes for carrying out the invention]

[0058] (definition) Where values ​​described herein are expressed as approximations using the antecedent “approximately,” it is understood that the term “approximately” means ±10% of the stated value. Furthermore, it will be understood that the particular value may form a different embodiment.

[0059] As used herein, the term “react” is used as is known in the art and generally refers to bringing together chemical reagents in such a manner that they enable their interactions at the molecular level and achieve a chemical or physical transformation. Certain types of reactions may be described more specifically; for example, a reduction reaction (e.g., the formal addition of dihydrogen across a double bond) may be described using the term “reduce” (e.g., reducing a ketone (>C=O) to a secondary alcohol (>C(H)-OH)).

[0060] (Cis-trans heterosexual) The compounds described herein (e.g., intermediate compounds) contain one or more bound rotational regions that result in cis-trans isomerism. Cis-trans isomers may also be referred to herein as diastereomers.

[0061] Accordingly, compounds produced by the methods described herein (e.g., intermediate compounds) may be produced such that at least 55% of the compound (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98%) exists as a single cis-trans stereoisomer.

[0062] In a typical embodiment, 99% or more (e.g., substantially all) of the total amount of the compound (e.g., an mAChR agonist compound; an intermediate compound) exists as a single cis-trans isomer.

[0063] For example, in one embodiment, the compound exists as a trans isomer (e.g., an intermediate compound).

[0064] Also described herein are mixtures of cis-trans isomers produced by the processes described herein.

[0065] (Isotopes) The compounds described herein (e.g., intermediate compounds) may include one or more isotopic substitutions, and references to specific elements include all isotopes of that element within that range. For example, references to hydrogen include all isotopes within that range. 1 H, 2 H(D), and 3 It contains H(T). Similarly, references to carbon are within that scope. 12 C, 13 C and 14 C, and the reference to oxygen, is within that scope. 16 O and 18 Each contains O.

[0066] In a similar manner, references to specific functional groups also include isotopic variations within their scope unless the context indicates otherwise. For example, a reference to an alkyl group (e.g., an ethyl group) also covers variations in which one or more of the hydrogen atoms in the group are in the form of a deuterium or tritium isotope (e.g., in an ethyl group where all five hydrogen atoms are in the form of a deuterium isotope (perjuuteroethyl group)).

[0067] The isotope may be radioactive or non-radioactive. The above compound may not contain a radioactive isotope. Such a compound is preferred for therapeutic use. However, the above compound may contain one or more radioactive isotopes. Compounds containing such radioactive isotopes may be useful in diagnostic situations.

[0068] (Solvate) The compounds described herein may form solvates. Preferred solvates are those formed by the incorporation of molecules of a non-toxic, pharmaceutically acceptable solvent (hereinafter referred to as the solvating solvent) into the solid-state structure (e.g., crystalline structure) of the compounds disclosed herein. Examples of such solvents include water, alcohols (e.g., ethanol, isopropanol, and butanol), and dimethyl sulfoxides. Solvates may be prepared by recrystallizing the compounds disclosed herein in a mixture of solvents containing the solvent or its solvating solvent. Whether a solvate has been formed in any given case may be determined by subjecting crystals of the compound to analysis using well-known and standard techniques (e.g., thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray crystallography). The solvates may be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, examples of which include hemihydrates, monohydrates, and dihydrates.

[0069] Therefore, what is also described herein is, It is an intermediate compound in the form of a solvate. An intermediate compound that exists in the form of a solvate, the solvate being a hydrate.

[0070] For a more detailed discussion of solvates and the methods used to produce and characterize them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, SSCI, Inc. (West Lafayette, IN, USA), 1999, ISBN 0-967-06710-3.

[0071] Alternatively, the compounds described herein may exist as anhydrous forms rather than hydrates. Therefore, the intermediate compounds described herein are also described in anhydrous form (e.g., anhydrous crystalline form).

[0072] (Ketoreductase enzyme) Ketereductase (KRED), also known as "alcohol dehydrogenase" (ADH) or "carbonyl reductase," catalyzes the reduction of aldehydes to their corresponding primary alcohols and ketones to their corresponding secondary alcohols.

[0073] KRED-catalyzed reductions require 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.

[0074] In vitro use of KRED in the reduction process converts NADPH to NADP. + To play from, or NADH to NAD + To regenerate from KRED, a cofactor regeneration system is required. Common cofactor regeneration systems are glucose dehydrogenase (GDH), which accepts glucose as a donor, or formate dehydrogenase, which accepts formate as a donor. These cofactor regeneration systems can be used in combination with KRED.

[0075] KRED is a ubiquitous enzyme found in all biological systems. Commonly available commercially produced KRED is derived from horse liver (HLADH), baker's yeast (YADH), and bacteria (e.g., Thermoanaerobium brockii (TBADH) and Lactobacillus kefir (LKADH)).

[0076] For industrial applications, it is preferable to use KRED with high specific activity and stereoselectivity. Another important criterion for the industrial use of KRED is long-term process stability, which often correlates with high stability at high temperatures and high solvent stability. Using KRED with high stereospecificity may also be desirable.

[0077] Enzyme A (described herein and used in the processes described herein) is Sequence ID No. 1: [ka] It is a ketoreductase enzyme (KRED) with the following amino acid sequence.

[0078] Enzyme A can be produced by methods known in the art. For example, methods for expressing enzymes in cellular (e.g., microbial) expression systems are well known and common to those skilled in the art.

[0079] Enzyme A may be provided by or produced from a host cell (e.g., a microbial cell, e.g., E. coli) containing a polynucleotide and / or expression vector encoding enzyme A. The host cell may be TOP10 E. coli. Enzyme A may be produced using the methods disclosed in the examples.

[0080] The polynucleotides described above may be DNA or RNA. The polynucleotides may be single-stranded or double-stranded. The polynucleotides may be supplied in an isolated / purified form or within host cells.

[0081] Due to the knowledge of codons corresponding to various amino acids, the availability of polypeptide sequences provides a description of all polynucleotides capable of encoding the polypeptide of interest. The degeneracy of genetic coding (the same amino acid being encoded by alternative or synonymous codons) allows for the generation of a vast number of nucleic acids, all of which encode the disclosed enzymes. Thus, by identifying a particular amino acid sequence, those skilled in the art can generate any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, the disclosure specifically considers all possible variations of polynucleotides that can be generated by selecting combinations based on possible codon selection.

[0082] Enzyme A is sequence number 2: [ka] This can be encoded by a polynucleotide and / or expression vector containing the nucleotide sequence related to this.

[0083] The above polynucleotide may consist of sequence number 2.

[0084] The polynucleotides described above can be functionally ligated to one or more heterologous regulatory or control sequences that control gene expression in order to produce recombinant polynucleotides capable of expressing their polypeptides. Expression constructs containing heterologous polynucleotides encoding the engineered ketoreductase can be introduced into suitable host cells to express the corresponding ketoreductase. The polynucleotides encoding the ketoreductase enzyme can be codon-optimized for optimal production from a host organism selected for expression. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells.

[0085] The polynucleotides encoding the disclosed enzymes can be manipulated in various ways to provide expression of their polypeptides. Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotides before inserting them into the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidelines are provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press; and in updated versions up to Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, 2006.

[0086] The above regulatory sequence could be a suitable promoter sequence, which may be derived from a gene encoding an extracellular or intracellular polypeptide that is either homogeneous or heterogeneous to the host cell. Suitable promoters for directing the transcription of the nucleic acid constructs of this disclosure for bacterial host cells include the promoter obtained from the lac operon of E. coli, the promoter obtained from the agarase gene (dagA) of Streptomyces coelicolor, the promoter obtained from the levanscuase gene (sacB) of Bacillus subtilis, the promoter obtained from the α-amylase gene (amyL) of Bacillus licheniformis, the promoter obtained from the maltose-producing amylase gene (amyM) of Bacillus stearothermophilus, the promoter obtained from the α-amylase gene (amyQ) of Bacillus amyloliquefaciens, the promoter obtained from the penicillinase gene (penP) of Bacillus licheniformis, the promoters obtained from the xylA and xylB genes of Bacillus subtilis, and the promoter obtained from prokaryotic β-lactamase genes (Villa-Kamaroff et al., 1978, Proc. Natl Acad. Sci. USA). Examples include promoters derived from the 75:3727-3731) and the tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80:21-25). Further promoters are described in "Useful proteins from recombinant bacteria", Scientific American, 1980, 242:74-94; and Sambrook et al. (above).

[0087] The above-mentioned regulatory sequences may also be signal peptide coding regions that encode an amino acid sequence linked to the amino terminus of a polypeptide and direct the encoded polypeptide toward the cell's secretory pathway. The 5' end of the coding sequence of a nucleic acid sequence may intrinsically contain a signal peptide coding region that is naturally linked to a segment of the coding region encoding the secreted polypeptide by a translation read frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region heterogeneous to that coding sequence. This heterogeneous signal peptide coding region may be required if the coding sequence does not naturally contain a signal peptide coding region.

[0088] Alternatively, the heterologous signal peptide coding regions described above may simply replace the native signal peptide coding regions to enhance polypeptide secretion. However, any signal peptide coding region that directs the expressed polypeptide to the secretory pathway of a select host cell may be used.

[0089] Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a polypeptide from the vector relating to this disclosure. The vector may be a plasmid, phage, MAC, virus, etc. The plasmid may be the pET28a plasmid (see Nature 2020 (https: / / doi.org / 10.1038 / s42003-020-0939-8)).

[0090] The polynucleotides and / or expression vectors described above can be synthesized by conventional DNA synthesis techniques. If the sequence of the manipulated polypeptide is known, the polynucleotide encoding the enzyme can be prepared by standard solid-phase methods according to known synthetic methods. Fragments of up to approximately 100 bases can be synthesized individually and then ligated (e.g., by enzymatic or chemical litigation methods, or by polymerase-mediated methods) to form any desired continuous sequence. For example, the disclosed polynucleotides can be prepared by chemical synthesis using, for example, the classical phosphoramidite method described by Beaucage et al., 1981, Tet Lett 22:1859-69, or the method described by Matthes et al., 1984, EMBO J.3:801-05, for example, that these methods are typically carried out by automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, ligated, and cloned into appropriate vectors, for example, in an automated DNA synthesizer. Furthermore, virtually any nucleic acid can be obtained from one of various commercial sources (e.g., The Midland Certified Reagent Company (Midland, TX), The Great American Gene Company (Ramona, CA), ExpressGen Inc. (Chicago, IL), Operon Technologies Inc. (Alameda, CA), and many others).

[0091] (protecting group) In the processes described herein, it may be necessary to protect one or more groups to prevent reactions from occurring at undesirable locations on the molecule. Examples of protecting groups, as well as 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).

[0092] (purification) Compounds prepared by the methods described herein may be isolated and purified by any of the various methods well known to those skilled in the art, examples of such methods include recrystallization techniques and chromatographic techniques (e.g., column chromatography (e.g., flash chromatography) and HPLC).

[0093] (Reaction solvent and reaction conditions) As discussed above in this specification, the term “reacting” is used as is known in the art. In some embodiments, the reacting involves two reagents, where one or more equivalents of the second reagent are used relative to the first reagent. The reacting step of the processes described herein may be carried out for a suitable time under conditions suitable for preparing the specified product.

[0094] The reactions of the processes described herein may be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents may be substantially inactive with both the starting materials (reactants), intermediates, and products at the temperature in which the reaction is carried out (for example, a temperature ranging from the freezing point to the boiling point of the solvent). A given reaction may be carried out in one solvent or in a mixture of more than one solvent. Depending on the particular reaction step, a solvent suitable for a particular step may be selected.

[0095] Suitable solvents 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, and mixtures thereof.

[0096] Suitable ether solvents include dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether, anisole, t-butyl methyl ether, and mixtures thereof.

[0097] 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, i-butyl alcohol, t-butyl alcohol, 2-ethoxyethanol, diethylene glycol, 1-pentanol, 2-pentanol, or 3-pentanol, neopentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenol, or glycerol.

[0098] 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-methylpyrrolidinone (NMP), formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethyl sulfoxide (DMSO), propionitrile, ethyl formate, methyl acetate, hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate, sulfolane, N,N-dimethylpropionamide, tetramethylurea, nitromethane, nitrobenzene, or hexamethylphosphoramide.

[0099] Suitable hydrocarbon solvents include benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-xylene, o-xylene, or p-xylene, octane, indan, nonane, or naphthalene.

[0100] Suitable aqueous buffering 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.

[0101] The reactions of the processes described herein may be carried out at appropriate temperatures that can be readily determined by those skilled in the art. The reaction temperature depends, for example, on the melting and boiling points of the reagents and solvents (if any); the thermodynamics of the reaction (e.g., highly exothermic reactions may need to be carried out at lower temperatures); the kinetics of the reaction (e.g., high activation energy barriers may require higher temperatures); and the temperature range in which any enzyme component is active / at which any enzyme component is denatured.

[0102] As used herein, the terms “ambient temperature” and “room temperature” or “rt” are understood in the art and generally refer to a temperature that is near the temperature of the room in which the reaction is carried out (e.g., reaction temperature) (e.g., a temperature of about 20°C to about 30°C).

[0103] The reactions of the processes described herein may be carried out in air or under an inert atmosphere. Typically, reactions involving reagents or products that are substantially reactive with air may be carried out using air-sensitive synthesis techniques that are well known to those skilled in the art. [Examples]

[0104] (Examples) This disclosure is described herein by reference to, but is not limited to, the specific embodiments described in the following example compounds and methods of synthesis.

[0105] (General methods and materials) The following general methods and materials are illustrative examples of methods and materials used in the processes described below.

[0106] (Chiral RP-HPLC method) System: Shimadzu LC-40 Column: Phenomenex Lux i-Amylose3 250×4.6mm 5μm Mobile phase: A mixture of water and acetonitrile containing 0.1% phosphoric acid (60 / 40 water / acetonitrile). Flow rate: 1mL / min. Column temperature: 40°C. Runtime: 15 minutes. Detection: A differential refractive index detector (RID) was used. Retention time: [Table A]

[0107] (Sample preparation): 0.5 mL of reaction sample was mixed with 0.5 mL of acetonitrile until homogeneous. The sample was placed in an ultrasonic bath for 5 minutes, and then centrifuged at 12.000 × g for 5 minutes. Aliquots of the prepared sample were transferred to glass vials and analyzed using chiral RP-HPLC.

[0108] (Calculation of diastereomer excess rate) The diastereomer excess rate (%de) is given by the following equation:

number

[0109] (Example 1: Screening of KRED enzyme) A series of KRED enzymes were screened for their usefulness in the conversion from formula (B) to formula (A) using a photometric assay. 288 diverse KRED enzymes were provided as dry enzyme preparations in 96 microtiter plates. These enzymes were solubilized in appropriate buffer solutions to produce clear solutions necessary for photometric measurements and screening. Photometric measurements recorded a decrease in absorbance at 340 nm, corresponding to enzymes consuming NADPH cofactors for the reduction reaction.

[0110] The collected data was analyzed, and enzymes were selected. These enzymes exhibited either a negative gradient over a 10-minute reaction time (the smallest number was selected first), or an overall absolute absorbance lower than 0.7 and a rapid decrease over 10 minutes (decreasing abruption). Low initial absorbance is an indicator of a highly active enzyme, which consumes cofactors faster than the technician can transfer its MTP to the MTP leader.

[0111] Enzymes that met the selection criteria were subjected to a 0.5 mL biocatalytic reaction and HPLC confirmation using the chiral RP-HPLC method.

[0112] Biocatalytic conditions: • Ketones 20g / L (88mM) NADP 5mM DMSO 5% (v / v) • MgCl 22mM • Glucose 178 mM (2 equivalents) • GDH (glucose dehydrogenase, CDX-901, Codexis Inc.) 1g / L • Potassium phosphate buffer 100mM pH 7.0 • 0.5 mL, 30°C, shaking at 1000 rpm, reaction time 24 hours.

[0113] As shown in Table 1 below, enzyme A significantly outperformed the second-best enzymes (enzymes B-F) in the conversion of the starting material, while simultaneously maintaining the highest level of stereoselectivity (diastereomer excess) observed for its trans alcohol (formula (A)). [Table 1]

[0114] (Example 2: Production of synthetic DNA encoding enzyme A) Synthetic DNA encoding enzyme A, which has sequence number 2, was synthesized after codon optimization for E. coli expression. [ka]

[0115] (Example 3: Production of Enzyme A) The above synthetic DNA (SEQ ID NO: 2) and pET28a were digested with NdeI restriction endonuclease and XhoI restriction endonuclease, purified, and ligated together using T4 DNA ligase. Subsequently, the resulting recombinant molecules were transformed into E. coli TOP10 strains, and the desired expression construct was confirmed by Sanger DNA sequencing. After transforming the sequenced plasmid DNA into E. coli protein expression host BL21 (DE3), single colonies were inoculated into 10 mL of LBP growth medium (10 g / L vegetable peptone, 10 g / L NaCl, and 5 g / L yeast extract, supplemented with 35 μg / mL kanamycin), and cultured at 37°C for 16 hours with shaking at 200 rpm. Subsequently, this starter culture was inoculated into 1 L of TB growth medium (12 g / L vegetable peptone, 12 g / L glycerol, 9.96 g / L disodium phosphate, 20.4 g / L monosodium phosphate, supplemented with 35 μg / mL kanamycin) in a 2 L baffled glass Erlenmeyer flask, and incubated at 28°C for 16 hours to induce enzyme A expression. The culture was then transferred to two 500 mL centrifuge bottles (Nalgene®) and centrifuged at 6000 g / 4°C in an SLA3000 rotor using a Sorvall RC-5C centrifuge. The bacterial pellet was then resuspended in the bottles with 5 times the weight of the pellet in 50 mM sodium phosphate buffer (pH 7.5). After complete resuspension, the bacterial suspension was transferred to a 100 mL glass beaker for cell disruption. Cell disruption was achieved using an MSE Soniprep 150 sonicator equipped with a 9.5 mm sonication probe at 0°C for a total of 8 minutes (four 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 (Nalgene®) centrifuge bottle and centrifuged at 13000 × g for 45 minutes in a Fiberlite® F13-1 X60cy rotor. After centrifugation, the supernatant (approximately 40 mL) was carefully transferred to a 100 mL plastic container and left at -20°C for 16 hours.Subsequently, the frozen enzyme A solution was subjected to freeze-drying (Edwards SuperModulo) over two days, maintaining a pressure of less than 1 mbar throughout. The dried enzyme cake was then completely pulverized into a uniform, easily flowable powder by crushing it with a steel spatula and stored at -20°C until use.

[0116] (Example 4: Preparation of tert-butyl trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate on an analytical scale) [ka] 1 mg of enzyme A was placed in a 2 mL reaction tube. In the same tube, 5 mM nicotinamide adenine dinucleotide phosphate (NADP) was added in 100 mM potassium phosphate buffer. + A 0.5 mL reaction mixture consisting of trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate tert-butyl was added, comprising 2 mM magnesium chloride hexahydrate, 178 mM glucose anhydride, 1 g / L glucose dehydrogenase (CDX-901, Codexis Inc.), 5% (v / v) dimethyl sulfoxide (DMSO), and 20 g / L 2-oxo-6-azaspiro[3.4]octane-6-carboxylate tert-butyl. The reaction tube was shaken at 1000 rpm and 30°C for 24 hours. The reaction was analyzed using chiral HPLC, which showed a conversion range of 98.7% to 100%, and a diastereomer excess of 97% de for trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate tert-butyl.

[0117] (Example 5: Lab bench-scale preparation of trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate tert-butyl (20 g / L substrate)) [ka] Each 100mL shot bottle contains 50mg of enzyme A (1g / L), 50mg of glucose dehydrogenase (1g / L, CDX-901, Codexis Inc.), and 197mg of nicotinamide adenine dinucleotide phosphate (5mM, NADP). + ), 20 mg magnesium chloride hexahydrate (2 mM) and 1.6 g glucose anhydrous (178 mM) were added. Under magnetic stirring at 300 rpm, the bottle was filled with 47.5 mL of 100 mM potassium phosphate buffer (pH 7.0), and if necessary, the temperature was adjusted to 30°C and the pH to 7. To the stirred solution, 1 g of 2-oxo-6-azaspiro[3.4]octane-6-carboxylate tert-butyl (20 g / L, 88 mM) was added in 2.5 mL of dimethyl sulfoxide (5% v / v, DMSO). After 20 hours, the reaction product was analyzed by chiral RP-HPLC, which showed 100% conversion and a diastereomer excess of 96% de for trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate tert-butyl.

[0118] (Example 6: Lab bench-scale preparation of trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate tert-butyl (45 g / L substrate)) [ka] The following ingredients were added to a 100 mL shot bottle: 50 mg enzyme A (1 g / L), 22.5 mg glucose dehydrogenase (0.45 g / L, CDX-901, Codexis Inc.), and 0.56 g nicotinamide adenine dinucleotide phosphate (11.3 g / L, 15 mM, NADP). +), 20 mg magnesium chloride hexahydrate (2 mM) and 2.2 g glucose anhydrous (247 mM). Under magnetic stirring at 300 rpm, 47.5 mL of 100 mM potassium phosphate buffer (pH 7.0) was packed into the bottle, and if necessary, the temperature was adjusted to 30°C and the pH to 7. To the stirred solution, 2.25 g of 2-oxo-6-azaspiro[3.4]octane-6-carboxylate tert-butyl (45 g / L, 198 mM) was added in 2.5 mL of dimethyl sulfoxide (5% v / v, DMSO). The pH of the reaction was kept constant at 7 by adding sodium hydroxide solution. After 24 hours, the reaction was analyzed using chiral HPLC, which showed a 67% conversion and a 97% de diastereomer excess for trans-2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate tert-butyl.

[0119] (Equal portions) The above-described embodiments are presented for illustrative purposes and should not be construed as imposing any limitation on the scope of the Disclosure. It will be readily apparent that numerous modifications and variations of the specific embodiments of the Disclosure illustrated above and in the embodiments can be made without departing from the underlying principles of the Disclosure. All such modifications and variations are intended to be encompassed by this application.

Claims

1. Formula (A): 【Chemistry 21】 A process for preparing a trans-alcohol compound, Formula (B): 【Chemistry 22】 A step to provide a compound of formula (A) by reducing the compound in the presence of a ketoreductase enzyme having sequence number 1. A process that includes this.

2. The process according to claim 1, wherein the step of reducing the compound of formula (B) is carried out in the presence of NADPH as a cofactor.

3. The aforementioned NADPH cofactor is NADP ＋ The process according to claim 2, provided by a cofactor regeneration system comprising glucose dehydrogenase and glucose.

4. The aforementioned NADP ＋ It is present at a concentration of approximately 5 mM; The glucose dehydrogenase is present at a concentration of approximately 1 g / L; and The glucose is present at a concentration of approximately 178 mM. The process according to claim 3.

5. The process according to any one of claims 1 to 4, wherein the compound of formula (B) is present at a concentration of about 88 mM.

6. The aforementioned NADP ＋ It is present at a concentration of approximately 15 mM; The glucose dehydrogenase is present at a concentration of approximately 0.45 g / L; and The glucose is present at a concentration of approximately 247 mM. The process according to claim 3.

7. The process according to any one of claims 1 to 3 or 6, wherein the compound of formula (B) is present at a concentration of about 198 mM.

8. The process according to any one of claims 1 to 7, wherein in the step of reducing the compound of formula (B), the ketoreductase enzyme having sequence number 1 is present at a concentration of about 1 g / L.

9. The process according to any one of claims 1 to 9, wherein the step of reducing the compound of formula (B) is carried out in the presence of about 100 mM potassium phosphate buffer at pH about 7.

0.

10. The step of reducing the compound of formula (B) is, (a) NADPH as a cofactor; (b) Approximately 100 mM potassium phosphate buffer with a pH of approximately 7.0 It was carried out in the presence of The NADPH cofactor is, (i) NADP ＋ and; (ii) with glucose dehydrogenase; (iii) glucose and The process according to claim 1, provided by a cofactor regeneration system including the following:

11. The aforementioned NADP ＋ It is present at a concentration of approximately 5 mM; The glucose dehydrogenase is present at a concentration of approximately 1 g / L; The glucose is present at a concentration of approximately 178 mM; The compound of formula (B) is present at a concentration of approximately 88 mM; and The ketoreductase enzyme having Sequence ID No. 1 is present at a concentration of approximately 1 g / L. The process according to claim 10.

12. The aforementioned NADP ＋ It is present at a concentration of approximately 15 mM; The glucose dehydrogenase is present at a concentration of approximately 0.45 g / L; The glucose is present at a concentration of approximately 247 mM; The compound of formula (B) is present at a concentration of approximately 198 mM; and The ketoreductase enzyme having Sequence ID No. 1 is present at a concentration of approximately 1 g / L. The process according to claim 10.

13. The process according to any one of claims 1 to 12, wherein the step of reducing the compound of formula (B) includes a step of heating at approximately 30°C.

14. The process according to any one of claims 1 to 13, wherein the step of reducing the compound of formula (B) includes a step of stirring for about 20 hours to about 24 hours.

15. Formula (A): 【Chemistry 23】 A composition comprising the compound, The compound of formula (A) is present in a diastereomer excess of approximately 96% de or approximately 97% de. composition.

16. The composition according to claim 15, wherein the compound of formula (A) is present in a diastereomer excess of about 96% de.

17. The composition according to claim 15, wherein the compound of formula (A) is present in a diastereomer excess of about 97% de.

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