Agent 2 for use in treating tissue damage

Abstract

An agent for use in medicine, comprising a compound of formula (I): (wherein Ar is an aryl linker group, such as, for example, a 1,4-phenyl group), or a respective pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof. The compound of formula (I) is an inhibitor of human C-reactive protein (CRP) and can be used to treat medical conditions mediated by CRP. Also provided are methods for making the compound of formula (I) and chemical intermediates thereof. [Formula 1] JPEG2023534884000044.jpg77170

Description

[Technical Field]

[0001] The present invention relates to agents that specifically bind to C-reactive protein (CRP) in vivo, thereby inhibiting the binding of CRP to autologous cellular and tissue ligands, and compositions containing such agents for use in the treatment or prevention of tissue injury, particularly ischemic, traumatic, infectious, inflammatory, and neoplastic conditions. [Background technology]

[0002] C-reactive protein (CRP) is a normal plasma protein of the pentraxin protein family, the other member of which is serum amyloid P component (SAP) (1). CRP is a classic acute-phase protein, and its circulating concentrations increase dramatically in response to tissue injury, infection, inflammation, and most forms of cancer. In most conditions, the CRP levels achieved closely correlate with the extent and activity of the disease. CRP is a calcium-dependent ligand-binding protein that binds with highest affinity to phosphocholine residues but also binds a variety of other ligands, both autologous and exogenous in origin. Autologous ligands include native and modified plasma lipoproteins, damaged cell membranes, several different phospholipids and related compounds, and small nuclear ribonucleoprotein particles. Exogenous ligands include some glycans, phospholipids, and other components of microorganisms, such as capsular and somatic components of bacteria, fungi, and parasites, as well as plant products. CRP bound to macromolecular ligands activates the classical complement pathway via Clq, leading to the activation and fixation of C3, the major adhesion molecule of the complement system, the production of the major chemotactic factors C3a and C5a, and the initiation of the terminal lytic phase, C5–C9.

[0003] In addition to closely reflecting the extent and activity of any disease process that triggers increased CRP production, higher circulating CRP concentrations also significantly predict disease progression, the development of complications, and clinical outcomes. The widespread clinical observation of this association across a wide range of diseases is consistent with CRP playing a pathogenic role in exacerbating tissue damage and, therefore, disease severity. CRP does not bind to normal, healthy cells, but strongly binds to ligands exposed on dead and damaged cells, subsequently activating complement. While CRP-mediated complement activation can contribute to the clearance of cellular debris from tissues and host defense against some microorganisms, it is clear that complement activation can also cause severe tissue damage, similar to many antibody-mediated hypersensitivity reactions.

[0004] The complement-dependent pathogenicity of human CRP was first confirmed experimentally by demonstrating that administration of human CRP to rats undergoing coronary artery ligation increased the size of the resulting acute myocardial infarction (2). Human CRP and activated rat complement were deposited within and around the infarct, and the exacerbation of tissue damage was undoubtedly complement-dependent. Similar observations were made in the rat middle cerebral artery occlusion model of stroke (3). Subsequently, several independent groups have made comparable observations in a range of different animal models.

[0005] The design of the first small molecule CRP binding inhibitor, bis(phosphocholine)hexane (BPC6), for in vivo use allowed definitive confirmation of the pathogenic role of human CRP in exacerbating tissue damage after ischemic infarction (2). Administration of this compound to rats that underwent coronary artery ligation and received human CRP completely abolished the increased damage that occurred in animals treated with human CRP, which received no treatment. Subsequently, bis(phosphocholine)octane (BPC8) was found to be a more potent inhibitor of CRP binding in vitro and had the same protective effect against human CRP pathogenesis in a rat acute myocardial infarction model involving ischemia-reperfusion, as well as after terminal coronary artery ligation (Pepys, unpublished observations). Thus, human CRP was validated as a therapeutic target, demonstrating the efficacy of intervention with small molecule CRP binding inhibitors.

[0006] These observations pave the way for novel approaches to reducing disease severity in a wide variety of tissue-damaging conditions in which circulating concentrations of CRP are elevated. It seems clear that inhibiting CRP binding in vivo will neither prevent nor cure a wide variety of diseases with vastly different etiologies. However, reducing the extent, severity, and duration of tissue damage and extending the survival of patients with heart attack, stroke, rheumatoid arthritis, and other chronic inflammatory diseases of unknown etiology, burns, bacterial and viral infections, or cancer cachexia, as well as numerous other conditions, remains an urgent and significant unmet medical need.

[0007] WO03 / 097104A1 describes an agent that binds to CRP and inhibits CRP binding or other ligands. The agent comprises multiple ligands covalently linked to form a complex with multiple C-reactive protein (CRP) molecules, where (i) at least two of the ligands are the same or different and are capable of binding to a ligand-binding site present on the CRP molecule; or (ii) at least one of the ligands is capable of binding to a ligand-binding site present on the CRP molecule, and at least one other of the ligands is capable of binding to a ligand-binding site present on a serum amyloid P component (SAP) molecule. A preferred ligand for CRP is a bis(phosphocholine) ligand, and an exemplary compound designated BPC8 has the following formula (BPC8):

[0008] [ka]

[0009] The number 8 in BPC8 refers to the n-octyl linker group in the formula above. Corresponding compounds BPC6, BPC7, etc., with n-hexyl, n-heptyl, etc. linker groups are also disclosed.

[0010] BPC6 and BPC8 strongly bind to CRP and crosslink pairs of natural pentameric protein molecules. BPC6 and BPC8 completely eliminated the harmful effects of human CRP in a rat model of acute myocardial infarction (4, and Pepys et al., unpublished observations). However, large-scale synthesis and purification of bis(phosphocholine)alkane compounds are difficult.

[0011] Thus, there remains a need for agents or compounds for use in treating medical conditions exacerbated by CRP that are more easily prepared and offer improved properties over compounds described in the prior art. Summary of the Invention

[0012] In a first aspect, the present invention provides an agent for use in medicine, the agent having formula (I): [ka] wherein Ar is an aryl linker group such as 1,4-phenyl. and respective pharmaceutically acceptable salts, solvates, prodrugs, or derivatives thereof.

[0013] Suitably, the compounds of formula (I) are inhibitors of human C-reactive protein (CRP).

[0014] In a second aspect, the present invention provides an agent according to the first aspect of the invention for use in the treatment or prevention of tissue damage in a subject having an inflammatory and / or tissue damaging condition. In a further aspect, the present invention provides a pharmaceutical composition comprising an agent according to the first aspect of the invention in admixture with one or more pharmaceutically acceptable excipients, diluents or carriers.

[0015] In a further aspect, the present invention provides a method for preparing a compound of formula (I), comprising reacting a compound of formula (III): [ka] (In the formula, R 1 is a carboxyl protecting group. with a compound of formula (IV-A) or formula (IV-B): [ka] to form a compound of formula (V), [ka] Next, R 1 cleaving the protecting group to form a compound of formula (I).

[0016] In a further aspect, the present invention provides a compound of formula (III) or a salt thereof with an optically active organic acid compound such as (1S)-(+)-10-camphorsulfonic acid. DETAILED DESCRIPTION OF THE INVENTION

[0017] In a first aspect, the present invention provides an agent for use in medicine, the agent having formula (I): [ka] wherein Ar is an aryl linker group. and its respective pharmaceutically acceptable salts, solvates, prodrugs, or derivatives thereof.

[0018] The Ar linker group is preferably a monocyclic, bicyclic, or fused bicyclic aryl group optionally containing one, two, or three heteroatoms in the aromatic ring, preferably selected from N or S. The Ar linker group preferably contains 4 to 12 carbon atoms in the aromatic ring (i.e., excluding carbon atoms of optional substituents). The aromatic ring of the Ar group is connected to the palindromic terminal group of the compound of Formula (I) via an amide bond, as shown in Formula (I). Preferably, the bond angle between the two Ar-CO bonds is approximately 180 degrees. Thus, for example, when Ar is a single six-membered aromatic ring, such as a phenyl group, the bond is preferably located at the para position (1,4) of the ring. The resulting steric relationship is believed to position the quinuclidinyl terminal group appropriately for binding to the corresponding acceptor group of CRP.

[0019] In one embodiment, the Ar group is selected from 1,4-phenyl, 2,6-naphthyl, or 4,4'-biphenyl, or a group of the same ring system containing 1, 2, or 3 heteroatoms in the ring (e.g., 2,6-pyridyl instead of 1,4-phenyl), in each case the aromatic ring may be substituted with one or more substituents R, as defined below.

[0020] In these embodiments, the linker group Ar has the formula Ar-I to Ar-VI: [ka] wherein R represents one or more optional substituents on the aryl ring. Suitably, R may be selected from halogen, hydroxy, cyano, -CONH2, or C1-C5 (cyclo)alkyl or C1-C5 (cyclo)alkoxy, where the alkyl group is optionally substituted with a phenyl group (e.g., R is -O-benzyl) or one or more halogen atoms, such as trifluoromethyl. More suitably, R may be C1-C4 alkyl or C1-C4 alkoxy, such as methyl. Suitably, there are zero, one, or two R substituents on the aryl linker, more suitably there are zero or one R substituents, and in some cases there are no R substituents. In certain embodiments, the Ar linker group is a 1,4-phenyl linker group having zero, one, or two R substituents.

[0021] In one embodiment, the aryl linker group Ar is represented by formula Ar-VII to Ar-XVI: [ka] The group consisting of:

[0022] In one embodiment of particular interest, the compound of formula (I) has the following formula (II): [ka] It has.

[0023] The compound of formula (II) is also referred to herein synonymously as P2B-B, or APL-2191, or the compound of Example 1.

[0024] The compounds of formula (I) and formula (II) are R,R,R,R stereoisomers. Other stereoisomers of this structure have been found to be less active. The S,S,S,S isomer is believed to be the most active alternative stereoisomer.

[0025] Preferably, the diastereomeric purity of the (R,R,R,R) stereoisomer in the agent of the present invention is at least about 50% by weight, preferably at least about 60%, more preferably at least about 75%, even more preferably at least about 90%, and most preferably at least about 98%. That is, the amount of the (R,R,R,R) stereoisomer preferably exceeds the amount of all other stereoisomers of the compound present in the agent. Most preferably, at least about 98% by weight of all stereoisomers of the compound present in the agent is the R,R,R,R stereoisomer.

[0026] The compounds of formula (I) and formula (II), in crystalline or dissolved form, may exist in zwitterionic form (COO-QNH+), and such zwitterionic forms are hereby encompassed within the definitions of formula (I) and formula (II) above. Similarly, the definitions of formula (I) and formula (II) encompass all crystalline forms and polymorphs of said compounds.

[0027] The bivalent ligand compound of formula (I) was found to bind strongly to human CRP in vitro and in vivo, forming a stable complex of pairs of native pentameric CRP molecules crosslinked by up to five ligand molecules. Because the ligand-binding pocket of each CRP protomer is shielded and the entire binding (B) face of each CRP pentamer is completely occluded in this complex, CRP cannot mediate tissue-damaging effects in vivo. Furthermore, dissociation of individual noncovalently associated protomers of native CRP from within the CRP-ligand complex is completely inhibited under physiological conditions.

[0028] Preferably, the compounds of formula (I) have an IC of about 20 μM or less, preferably about 10 μM or less, more preferably about 5 μM or less, or most preferably about 1 μM or less. 50 It is an inhibitor of human C-reactive protein (CRP) with IC 50 to measure the IC of a particular compound within a specific range. 50 Suitable methods for determining whether a patient has a genomic DNA fragment are described herein.

[0029] In a second aspect, the present invention provides an agent according to the invention for use in the treatment or prevention of a medical condition mediated by CRP. In another aspect, the present invention provides the use of an agent according to the first aspect of the invention for the manufacture of a medicament for the treatment or prevention of a medical condition mediated by CRP.

[0030] The agents of the present invention, including compounds of formula (I), can be administered simultaneously, simultaneously, separately, or sequentially with one or more other pharmaceutically active agents, such as anti-inflammatory agents such as corticosteroids; antiviral, antibacterial, antifungal, or antiparasitic agents; inhibitors / antagonists of pro-inflammatory cytokines such as IL-1, IL-6, and TNF; anticoagulants; and inhibitors of complement activation or biologically active fragments thereof.

[0031] The present invention further provides a method for treating a medical condition mediated by CRP in a patient in need thereof, comprising administering to the patient a therapeutic amount of an agent according to the present invention or a pharmaceutical composition according to the present invention.

[0032] In one embodiment, the inflammatory and / or tissue damage condition comprises one or more of acute coronary syndrome, unstable angina, plaque rupture, and / or early atherothrombosis.

[0033] In one embodiment, the inflammatory and / or tissue damage condition is selected from an infectious disease, an allergic complication of an infectious disease, an inflammatory disease, ischemic or other necrosis, traumatic tissue injury, and malignant neoplasia. For example, the condition may be an infectious disease selected from a bacterial infection, including sepsis, a viral infection, a fungal infection, and a parasitic infection.

[0034] In one embodiment, the condition is an inflammatory disease selected from rheumatoid arthritis, juvenile chronic (rheumatoid) arthritis, ankylosing spondylitis, psoriatic arthritis, systemic vasculitis, polymyalgia rheumatica, Reiter's disease, Crohn's disease, and familial Mediterranean fever, and other autoinflammatory conditions.

[0035] In one embodiment, the condition is tissue necrosis selected from myocardial infarction, ischemic stroke, tumor embolism, and acute pancreatitis.

[0036] In one embodiment, the condition is a trauma selected from elective surgery, burns, chemical injuries, fractures, and compression injuries.

[0037] In one embodiment, the condition is a malignant neoplasia selected from lymphoma, Hodgkin's disease, carcinoma, and sarcoma.

[0038] In one embodiment, the condition is an allergic complication of an infection selected from rheumatic fever, glomerulonephritis, and erythema nodosum leprosum.

[0039] In one embodiment, the condition is infection or a complication of infection with a severe acute respiratory syndrome (SARS) coronavirus, particularly SAR-SCoV2.

[0040] Preferably, the method comprises administering to the patient an agent of the present invention in an amount sufficient to bind all soluble CRP in the circulation and extracellular tissue fluids, for example, the amount may be sufficient to bind at least about 70% of the available CRP, preferably at least about 90% of the available CRP, and optimally 95%, 99%, or 100% of the available CRP.

[0041] In a further aspect, the present invention provides a pharmaceutical composition comprising an agent according to the first aspect of the invention in admixture with one or more pharmaceutically acceptable excipients, diluents or carriers.

[0042] Pharmaceutical compositions can be formulated containing an agent of the present invention or a pharmaceutically acceptable salt, ester, or prodrug thereof, optionally incorporating a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). As used herein, "pharmaceutically acceptable salt" refers to a salt of a compound of Formula (I) with an anion or cation known and accepted in the art for the formation of salts for pharmaceutical use. Acid addition salts can be formed, for example, by mixing a solution of the agent with a solution of a pharmaceutically acceptable non-toxic acid, including, but not limited to, hydrochloric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. When the agent has a carboxylic acid group, the present invention also contemplates salts thereof, preferably non-toxic, pharmaceutically acceptable salts thereof, including, but not limited to, sodium, potassium, calcium, and quaternary ammonium salts thereof. In a particularly preferred embodiment, the salt is a salt with HCl, particularly a 0.2HCl salt.

[0043] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro, ed., 1985). The choice of pharmaceutical carrier, excipient, or diluent can be selected according to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may optionally contain suitable binders, lubricants, suspending agents, coating agents, solubilizing agents as, or in addition to, the carrier, excipient, or diluent.

[0044] Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition. Antioxidants and suspending agents may also be used.

[0045] Pharmaceutical compositions may also be in the form of prodrugs, containing an agent or derivative thereof that becomes active only upon metabolism by the recipient. The exact nature and amounts of the components of such pharmaceutical compositions can be determined empirically and will depend, in part, on the route of administration of the composition. Where appropriate, pharmaceutical compositions of the present invention can be administered by inhalation, in the form of a suppository or pessary, topically (including ophthalmically) in the form of a lotion, solution, cream, ointment, or powder, by use of a skin patch, orally in the form of a tablet containing excipients such as starch or lactose, or in capsules or ovules, either alone or mixed with excipients, or in the form of an elixir, solution, or suspension containing flavoring or coloring agents, or pharmaceutical compositions of the present invention can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intraarterially.

[0046] Liquid forms into which the compositions of the present invention can be incorporated for administration by injection include edible oils such as cottonseed oil, sesame oil, coconut oil, and peanut oil, as well as aqueous emulsions with elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone, and gelatin.

[0047] For parenteral administration, the compositions are best used in the form of a sterile aqueous solution which may contain other substances, for example, buffers to adjust the pH, or enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, the compositions can be administered in the form of tablets or lozenges which can be formulated in conventional manner.

[0048] The use of the compounds of the present invention aims to saturate all circulating and other soluble CRP molecules in the body with the ligand drug. The required daily dose of drug is preferably one that provides at least about 1 mole of drug, more preferably at least about 5 moles of drug, per mole of native pentameric CRP with which it is complexed.

[0049] The exact form of the pharmaceutical composition and its dosage may also depend on the subject to be treated, including body weight, route of administration, and disease state, which may be determined as a matter of routine concern to one skilled in the art.

[0050] In a further aspect, the present invention provides a process for preparing a compound of formula (I) as defined in any one of claims 1 to 6, comprising reacting a compound of formula (III): [ka] (In the formula, R 1 is a carboxyl protecting group. with a compound of formula (IV-A) or formula (IV-B): [ka] to form a compound of formula (V): [ka] followed by the formation of a compound of R 1 cleaving the protecting group to form a compound of formula (I).

[0051] Protecting group R 1 may be any of the protecting groups conventionally used to protect carboxyl groups during peptide synthesis from amino acids. For example, the protecting group R 1 can be selected from C1-C5 alkyl, trityl, 2,4-dimethoxybenzyl (DMB), benzyl, or 9-fluorenylmethyl. 1 is a C1-C5 alkyl, particularly a methyl group.

[0052] The step of reacting a compound of formula (III) with a compound of formula (IV-A) to form a compound of formula (V) can be carried out by any of the methods conventionally used to form amide bonds in peptide synthesis. For example, the -COOH groups in a compound of formula (IV) can be activated by converting them to esters of strong acids or groups of formula -COX, where X is a leaving group readily displaced by nucleophilic substitution, such as chloro, alkylsulfonate, or toluenesulfonate, followed by nucleophilic reaction with a primary amine group in a compound of formula (III). In other embodiments, activation of the carboxylic acid can be carried out using either a phosphate-containing reagent, a triazine-based reagent, a carbodiimide-based reagent, or a hydroxybenzotriazole-based reagent in the presence of an organic solvent and a base. Preferred conditions include TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) with diisopropylethylamine in MeCN at room temperature.

[0053] Alternatively, compounds of formula (I) can be prepared using the bis-acid chloride of compound (IV-B). Typical reaction conditions include warming to 30° C. in chloroform for 16 hours.

[0054] Compounds of formula (III) and formula (IV-A / B) are either commercially available, prepared according to the methods described herein, or prepared according to the literature.

[0055] Finally, the carboxylate group of the compound of formula (V) is deprotected by any of the methods known in the art, for example, to remove the group -COOR 1 When is an alkyl ester such as a methyl ester, the ester can be hydrolyzed under mildly basic conditions such as 10% KOH (aq) for 1 hour at 50° C., followed by neutralization with formic acid at pH 4-5.

[0056] In one embodiment, the method further comprises preparing a compound of formula (III) by a process comprising reacting a compound of formula (VII) with a compound of formula (VIII). [ka] (wherein L represents a leaving group, i.e., a weakly basic group that is easily displaced by nucleophilic substitution.) Suitable leaving groups L include bromo, iodo, alkylsulfonate, and phenylsulfonate groups, such as p-bromophenylsulfonate. R 1 is a carboxyl protecting group as defined above. The reaction is preferably carried out in an aprotic solvent in the presence of a strong non-nucleophilic base. For example, the strong base may be potassium bis(trimethylsilyl)amide, KHMDS, and the solvent may be toluene / THF. The reaction proceeds by nucleophilic substitution to give compounds of formula (IX-A) and formula (IX-B): [ka] This forms a mixture of stereoisomers.

[0057] The synthesis of the compound of formula (III) then involves hydrolyzing and separating the mixture of stereoisomers to isolate the compound of formula (III) or its salt with an optically active organic acid compound. Hydrolysis can be carried out under mild acidic conditions, for example, with HO in the presence of (1S)-10-camphorsulfonic acid. The salt of formula (III).2CSA preferentially precipitates from the mixture. Other chiral organic acids commonly used to separate enantiomers may be suitable, such as (2S,3S)-tartaric acid, (R)-malic acid, or (-)-(R)-mandelic acid.

[0058] In a further aspect, the present invention provides a compound of formula (III): [ka] (In the formula, R 1 is a carboxyl protecting group as defined above. or a salt of a compound of formula (III) with an optically active organic acid compound as defined above.

[0059] In one embodiment, the compound according to this aspect is a salt of the compound of formula (III) with (1S)-(+)-10-camphorsulfonic acid (CSA), particularly the 0.2CSA salt. [Example]

[0060] The invention will now be described by reference to specific embodiments described in the following examples. Compounds are named using conventional IUPAC nomenclature or by chemical supplier names, but the invention is not limited thereto.

[0061] The following synthetic procedures are provided to illustrate the methods used. The precursors used in a given preparation or step may not necessarily originate from the individual batch synthesized according to the steps in the description given.

[0062] [Analysis method] Where analytical data are cited in the examples and preparations, one of the following analytical methods was used unless otherwise specified. (NMR): 400MHz Bruker Avance III and Bruker Avance Neo.

[0063] (LC-MS or HPLC method): Method 1: MS instrument type: SHIMADZU LC-MS-2020; column: Kinetex EVO C18 30 × 2.1 mm, 5 μm; mobile phase A: 0.0375% TFA (vol / vol) in water; B: 0.01875% TFA (vol / vol) in acetonitrile; gradient: 0.0 min 0% B → 0.8 min 60% B → 1.20 min 60% B → 1.21 min 0% B → 1.55 min 0% B; flow rate: 1.5 mL / min; oven temperature: 50 °C; PDA detection: 220 nm and 254 nm.

[0064] Method 2: MS instrument type: Agilent 1200 LC / G1956A MSD; Column: Kinetex EVO C18 2.1 × 30 mm, 5 μm; Mobile phase A: 0.0375% TFA (vol / vol) in water; B: 0.01875% TFA (vol / vol) in acetonitrile; Gradient: 0.0 min 90% B → 0.35 min 90% B; Flow rate: 1.5 mL / min; Oven temperature: 50 °C; DAD: 100–1000.

[0065] Method 3: HPLC instrument type: SHIMADZU LC-AB; column: Kinetex C18 LC column 4.6 × 50 mm, 5 μm; mobile phase A: 0.0375% TFA (vol / vol) in water; B: 0.01875% TFA (vol / vol) in acetonitrile; gradient: 0.0 min 0% B → 4.20 min 60% B → 5.30 min 60% B → 5.31 min 0% B → 6.00 min 0% B; flow rate: 1.5 mL / min; oven temperature: 50 °C; PDA detection: PDA (220 nm, 215 nm, and 254 nm).

[0066] Method 4: MS instrument type: SHIMADZU LC-MS-2020; column: Kinetex EVO C18 30 × 2.1 mm, 5 μm; mobile phase A: 0.0375% TFA (vol / vol) in water; B: 0.01875% TFA (vol / vol) in acetonitrile; gradient: 0.0 min 0% B → 3.0 min 60% B → 3.50 min 60% B → 3.51 min 0% B → 4.00 min 0% B; flow rate: 0.8 mL / min; oven temperature: 50 °C; PDA detection: 220 nm and 254 nm.

[0067] Method 5: MS instrument type: SHIMADZU LC-MS-2020; column: Kinetex EVO C18 2.1 × 30 mm, 5 μm; mobile phase A: 0.025% NH₃·H₂O in water (vol / vol); B: acetonitrile; gradient: 0.0 min 0% B → 0.8 min 60% B → 1.20 min 60% B → 1.21 min 0% B → 1.55 min 0% B; flow rate: 1.5 mL / min; oven temperature: 40 °C; PDA detection: 220 nm and 254 nm.

[0068] Method 6: HPLC instrument type: SHIMADZU LC-20AB; column: Kinetex C18 LC column 4.6 × 50 mm, 5 μm; mobile phase A: 0.0375% TFA (vol / vol) in water; B: 0.01875% TFA (vol / vol) in acetonitrile; gradient: 0.0 min 0% B → 4.20 min 30% B → 5.30 min 30% B → 5.31 min 0% B → 6.00 min 0% B; flow rate: 1.5 mL / min; oven temperature: 50 °C; PDA detection: PDA (220 nm, 215 nm, and 254 nm).

[0069] Method 7: MS instrument type: SHIMADZU LC-20AB; column: Kinetex C18 LC column 4.6 × 50 mm, 5 μm; mobile phase A: 0.0375% TFA (vol / vol) in water; B: 0.01875% TFA (vol / vol) in acetonitrile; gradient: 0.0 min 0% B → 2.40 min 30% B → 3.70 min 30% B → 3.71 min 0% B → 4.00 min 0% B; flow rate: 1 mL / min; oven temperature: 50 °C; PDA detection: 220 nm and 254 nm.

[0070] Method 8: MS instrument type: Agilent 1100 LC & Agilent G1956A; Column: Waters XSelect HSS T3 3.5 μm 4.6 × 50 mm; Mobile phase A: 0.0375% TFA (v / v) in water; B: 0.01875% TFA (v / v) in acetonitrile; Gradient: 0.0 min 0% B → 5.00 min 30% B → 6.00 min 100% B → 6.50 min 100% B → 6.51 min 0% B → 7.00 min 0% B; Flow rate: 1 mL / min; Oven temperature: 40 °C; PDA detection: 220 nm and 254 nm.

[0071] Method 9: MS instrument type: SHIMADZU LCMS-2020; column: Kinetex EVO C18 2.1 × 30 mm, 5 μm; mobile phase A: 0.025% NH₃·H₂O in water (vol / vol); B: acetonitrile; gradient: 0.0 min 5% B → 0.8 min 95% B → 1.2 min 95% B → 1.21 min 5% B → 1.55 min 5% B; flow rate: 1.5 mL / min; oven temperature: 40 °C; UV detection: 220 nm and 254 nm.

[0072] Method 10: MS instrument type: Agilent 1100 LC & Agilent G1956A, column: K Waters XSelect HSS T3 3.5 μm 4.6 × 50 mm, mobile phase A: 0.0375% TFA (v / v) in water, B: 0.01875% TFA (v / v) in acetonitrile, gradient: 0.0 min 0% B → 5 min 30% B → 6 min 100% B → 6.5 min 100% B → 6.51 min 0% B, flow rate: 0.6 mL / min, oven temperature: 40 °C; UV detection: 220 nm and 254 nm.

[0073] HPLC method 1: MS instrument type: SHIMADZU LC-20AB, column: XBridge® C18 3.5 μm 4.6 × 150 mm, mobile phase A: 0.0375% TFA (vol / vol) in water, B: 0.01875% TFA (vol / vol) in acetonitrile, gradient: 0.0 min 0% B → 10.0 min 60% B → 15.0 min 60% B → 15.01 min 0% B → 15.02 min 0% B → 20.0 min 0% B, flow rate: 1.0 mL / min, oven temperature: 40 °C; UV detection: 220 nm, 215 nm, and 254 nm.

[0074] [Abbreviation] Where the following abbreviations are used, the following meanings apply: ACN or MeCN is acetonitrile. CDCl3 is deuterated chloroform. CSA is camphor-10-sulfonic acid. D2O is heavy water. DCM is dichloromethane. DIPEA or DIEA is N,N-diisopropylethylamine. DMAP is 4-(dimethylamino)pyridine. DMSO is dimethyl sulfoxide. EA is ethyl acetate. EtOH is ethanol. FA is formic acid. H2O is water. HCl is hydrochloric acid. HPLC is high performance liquid chromatography. IPA is isopropyl alcohol. KHMDS is potassium bis(trimethylsilyl)amide. KOH is potassium hydroxide. LCMS is liquid chromatography mass spectrometry. MeOH is methanol. MTBE is methyl tertbutyl ether. N2 is nitrogen. Na2SO4 is sodium sulfate. NH3 is ammonia. NH4HCO3 is ammonium bicarbonate. NMR is nuclear magnetic resonance. PDA is a photodiode array detector. SFC is supercritical fluid chromatography. TBTU is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate. TEA is triethylamine. TFA is trifluoroacetic acid THF is tetrahydrofuran. TLC is thin layer chromatography.

[0075] [Preparation] (Preparation of Compound 2: [(3S)-quinuclidin-3-yl]4-bromobenzenesulfonate)

[0076] [ka] To a solution of (3S)-quinuclidin-3-ol (2.00 g, 15.73 mmol, 1.00 equiv), DMAP (19.21 mg, 157.30 μmol, 0.01 equiv), and TEA (4.78 g, 47.19 mmol, 6.54 mL, 3.00 equiv) in DCM (40.00 mL) was added 4-bromobenzenesulfonyl chloride (6.03 g, 23.60 mmol, 1.50 equiv) at 0 °C. The mixture was stirred at 20 °C for 16 h. The mixture was washed with saturated NaHCO (100 mL). The saturated NaHCO layer was extracted with EA (100 mL × 2). The combined organic phase was dried over NaSO and concentrated under reduced pressure to give the crude product as a yellow oil. The yellow oil was purified by chromatography on silica gel eluting with DCM:MeOH=30:1 to give [(3S)-quinuclidin-3-yl]4-bromobenzenesulfonate (3.20 g, 8.32 mmol, 52.88% yield, 90% purity) as a yellow solid. 1 HNMR analysis was performed. 1 H NMR: (400MHz, CDCl3) δ = 7.81 - 7.57 (m, 4H), 4.65 - 4.49 (m, 1H), 3.04 (dd, J=8.4, 15.2 Hz, 1H), 2.89 -2.48 (m, 5H), 1.93 ( d, J=2.8 Hz, 1H), 1.80 - 1.71 (m, 1H), 1.61 (tdd, J=4.6, 9.5, 13.9 Hz, 1H), 1.47 - 1.22 (m, 2H).

[0077] Preparation of diastereomers 4A and 4B <Isomer 4A: (R)-methyl 2-((diphenylmethylene)amino)-2-((3R)-quinuclidin-3-yl)acetate> <Isomer 4B: (S)-methyl 2-((diphenylmethylene)amino)-2-((3R)-quinuclidin-3-yl)acetate>

[0078] [ka] To a solution of (3S)-quinuclidin-3-yl 4-bromobenzenesulfonate (63 g, 182 mmol) and methyl 2-[(diphenylmethylidene)amino]acetate (92.2 g, 364 mmol) in toluene (578 mL) and THF (186 mL) was added KHMDS (0.70 M in toluene, 520 mL) under N2, and the reaction was stirred at 65 °C for 12 h. The reaction mixture was cooled, poured into water (1.00 L), and ethyl acetate (1500 mL) was added. The phases were separated, and the aqueous phase was extracted with ethyl acetate (3 × 1.00 L). The organic phase was washed with saturated brine (2 × 500 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude mixture (113 g) was obtained as a dark brown oil and used directly in the next step. Selected NMR data for the crude material indicated dr(R,R)(R,S)=2.3:1. 1 H NMR: 400 MHz, DMSO-d6: crude selected δ ppm 4.08 (d, J = 8.8 Hz, 2H), 3.95 (d, J = 10.2 Hz, 1H).

[0079] 5.2 Preparation of (+)-CSA salt (R,R) (R)-Methyl 2-amino-2-((3R)-quinuclidin-3-yl)acetate bis(((1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methanesulfonate)

[0080] [ka] To a solution of the crude reaction mixture of 4A and 4B (105 g, 177 mmol) in IPA (700 mL) was added HO (3.21 g, 178 mmol) and the reaction was warmed to 45 °C. A solution of (+)CSA (103 g, 442 mmol) in IPA (300 mL) was added and the reaction was continued to stir at 45 °C for 12 h. The reaction mixture was cooled to 25 °C and filtered to give a white solid. The solid was washed with IPA (100 mL) and MTBE (100 mL) and dried under vacuum to give the title compound as a white solid (62.0 g, 93.5 mmol, 52.9% yield). 1 H NMR: 400 MHz, DMSO-d6: δ ppm 9.62 - 9.58 (br, s, 1H), 8.51 (br, s, 3H), 4.25 - 4.22 (d, J = 10.4 Hz, 1H), 3.78 (s, 3H), 3.25 - 3.23 (m, 5H), 2.90 - 2.86 (d, J = 14.8 Hz, 2H), 2.66 (m, 2H), 2.41 - 2.37 (d, J = 14.8 Hz, 3H), 1.95 - 1.93 (m, 2H), 1.85 - 1.78 (m, 11H), 1.30 - 1.27 (m, 4H), 1.04 (s, 6H), 0.74 (s, 6H).

[0081] (Confirmation of the stereochemistry of compound 5.2(+)-CSA salt (R,R)) 20 mg of compound 4A was dissolved in 1.3 mL of dichloromethane / cyclohexane / methanol (5:5:3). The solution was kept in a semi-sealed 4 mL vial and allowed to slowly evaporate at room temperature. Crystals were observed on day 2 and selected for X-ray crystallography.

[0082] The crystals were colorless needles with dimensions of 0.10 × 0.02 × 0.02 mm. The symmetry of the crystal structure was determined using a Rigaku Oxford Diffraction XtaLAB Synergy 4 circle diffractometer equipped with a HyPix-6000HE area detector: a = 7.0236(2) Å, b = 26.8204(6) Å, c = 18.0068(5) Å, α = 90°, β = 99.114(3)°, γ = 90°, V = 3349.22(16) Å. 3 , Z=4, Dc=1.315g / cm 3 , F(000)=1424.0, μ(Cu Kα)=1.918mm -1 , and T = 293(2) K, the crystal was assigned to the monoclinic space group P21. Cryogenic system: Oxford Cryostream 800Cu: λ = 1.54184 Å, 50 W, microfocus source with multilayer mirror (μ-CMF). Distance from crystal to CCD detector: d = 35 mm. Tube voltage: 50 kV. Tube current: 1 mA. 5. The absolute configuration of the 2(+)-CSA salt was assigned to (R,R).

[0083] Alternatively, compound 4A(R,R) could be isolated according to the following method.

[0084] [ka] The crude reaction mixture of 4A and 4B was purified by silica gel column chromatography eluting with EA:MeOH (40:1 to 10:1) to give a mixture of diastereomers. The diastereomers were separated by chiral preparative SFC (column: DAICEL CHIRALPAK IG (250 mm × 50 mm, 10 μm); mobile phase: [0.1% NH₃·H₂O in EtOH]; B%: 45%; 320 min) to give compound 4A (6.00 g, 16.5 mmol) as a brown oil and compound 4B (RS) (9.00 g, 24.8 mmol) as two diastereomers. <Diastereomer 1 (RR): (R)-methyl 2-((diphenylmethylene)amino)-2-((3R)-quinuclidin-3-yl)acetate> 1 H NMR: 400 MHz (DMSO-d): δ ppm: 7.64-7.34 (m, 8H), 7.25-7.09 (m, 2H), 4.09 (d, J = 8.8 Hz, 1H), 3.60 (s, 3H), 2.92-2.77 (m, 1H), 2.73-2.60 (m, 2H), 2.55 (br d, J = 6.4 Hz, 1H), 2.43-2.21 (m, 3H), 1.66-1.33 (m, 3H), 1.19 (br d, J = 5.2 Hz, 2H). SFC: Rt = 1.633 min, 100% <Diastereomer 2 (RS): (S)-methyl 2-((diphenylmethylene)amino)-2-((3R)-quinuclidin-3-yl)acetate> 1 H NMR 400 MHz (DMSO-d6): δ ppm: 7.62-7.35 (m, 8H), 7.18 (dd, J = 1.6, 7.4 Hz, 2H), 3.96 (d, J = 10.0 Hz, 1H), 3.65 (s, 3H), 2.95-2.80 (m, 1H), 2.65 (br t, J = 7.6 Hz, 2H), 2.48 (br s, 1H), 2.4 -2.23 (m, 2H), 2.17 (br dd, J = 7.2, 13.6 Hz, 1H), 1.65-1.40 (m, 3H), 1.13-1.05 (m, 1H), 0.96-0.78 (m, 1H). SFC: Rt = 1.854 min, 100%

[0085] Alternatively, compounds 4A and 4B could be separated using preparative TLC as follows. The mixture of 4A and 4B was purified by preparative TLC (EtOAc:MeOH (NH3, 7M) = 10:1) to give 202.25 mg of 4A (purity 91.7%, 98.6% ee.) as a yellow solid and 114.50 mg of 4B (purity 97.7%, 94.3% ee.) as a yellow oil. LCMS MS m / z 363.2 [M+H] + 4A: 1 H NMR: (400MHz, CDCl3): δ ppm 7.66 - 7.58 (m, 2H), 7.52 - 7.45 (m, 3H), 7.43 - 7.31 (m, 3H), 7.19 (dd, J=1.6, 7.4Hz, 2H), 4.22 (d, J=8.3 Hz, 1H), 3.73 - 3.68 (m, 3H), 3.17 - 3.03 (m, 1H), 2.92 - 2.82 (m, 2H), 2.64 - 2.51 (m, 3H), 1.77 - 1.55(m, 3H), 1.52 - 1.40 (m, 1H), 1.37 - 1.25 (m, 1H). 4B: 1 H NMR: (400MHz, CDCl3): δ ppm 7.65 - 7.56 (m, 2H), 7.54 - 7.44 (m, 3H), 7.43 - 7.29 (m, 3H), 7.20 (dd, J=2.9, 6.4Hz, 2H), 4.08 (d, J=10.0 Hz, 1H), 3.76 - 3.72 (m, 3H), 3.24 - 3.09 (m, 1H), 3.00 - 2.80 (m, 2H), 2.61 - 2.38 (m, 3H), 1.81 - 1.58(m, 3H), 1.28 - 1.15 (m, 1H), 1.12 - 1.00 (m, 1H)

[0086] Alternatively, the HCl salt of compound 5 was obtained according to the following procedure. To a solution of the above prepared 4A stereoisomer (390.00 mg, 1.08 mmol) in THF (6 mL) was added HCl (12 M (aqueous), 780.09 μL, purity 37%) at 0° C. The mixture was stirred at 0° C. for 1 h. The mixture was concentrated to remove THF. To the residue were added methyl tert-butyl ether (20 mL) and water (20 mL). The aqueous layer was concentrated under reduced pressure to give (R)-methyl 2-amino-2-((3R)-quinuclidin-3-yl)acetate (250.00 mg, crude, diHCl salt) as a yellow solid.

[0087] Preparation of Compound 5 Free Parent from Compound 5 CSA Salt <(R)-methyl 2-amino-2-((3R)-quinuclidin-3-yl)acetate> [ka] To a suspension of Ambersep 900 (470 g) in MeOH (900 mL) was added methyl (2R)-2-amino-2-[(3R)-1-azabicyclo[2.2.2]octan-3-yl]acetate bis(+)camphorsulfonate (Preparation 1, 47.0 g, 70.9 mmol) and the mixture was stirred under N for 1 h at 20° C. The reaction mixture was filtered and concentrated in vacuo to give the title compound (11.0 g, 55.5 mmol, 78.3% yield) as a yellow oil. 1 H NMR: 400 MHz (DMSO-d): δ ppm: 7.64-7.34 (m, 8H), 7.25-7.09 (m, 2H), 4.09 (d, J = 8.8 Hz, 1H), 3.60 (s, 3H), 2.92-2.77 (m, 1H), 2.73-2.60 (m, 2H), 2.55 (br d, J = 6.4 Hz, 1H), 2.43-2.21 (m, 3H), 1.66-1.33 (m, 3H), 1.19 (br d, J = 5.2 Hz, 2H).

[0088] The hydrochloride salt of compound 5 could also be converted to the free parent using the Ambersep 900 method described above.

[0089] [Synthesis of Examples] [Example 1] (APL-2191 P2B_B) <(R,2R,2'R)-2,2'-(terephthaloylbis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0090] [ka] Step 1: To methyl (2R)-2-amino-2-[(3R)-1-azabicyclo[2.2.2]octan-3-yl]acetate (Preparation 2, 330 g, 333 mmol, 20% solution in MeCN) and benzene-1,4-dicarboxylic acid (20.50 g, 123 mmol) in MeCN (1.30 L) under N was added TBTU (88.2 g, 275 mmol) followed by DIEA (65.3 g, 505 mmol, 88.0 mL). The reaction was stirred at 25 °C for 12 h. The reaction mixture was concentrated in vacuo to give a crude yellow oil, which was used directly in the next step.

[0091] Step 2: To a solution of the crude reaction mixture from Step 1 (64.9 g, 123 mmol) in IPA (1.07 L) was added KOH (69.2 g, 123 mmol, 1.07 L, 10% aqueous solution), and the reaction was stirred at 50 °C for 1 h under N2. The reaction mixture was filtered, and the mother liquor was extracted with ethyl acetate (2 × 300 mL). The aqueous layer was adjusted to pH = 4-5 with formic acid and stirred for 12 h. The resulting white solid was filtered and stirred in water (740 mL) for 2 h at 90 °C before being cooled to 25 °C. The solid was filtered, washed with water (2 × 300 mL), and dried under vacuum to give the title compound as a white solid (31.4 g, 48.6 mmol, 39.5% yield). 1 H NMR 400 MHz (DO): δ ppm: 7.84 (s, 4H), 4.53 (br d, J = 10.8 Hz, 2H), 3.54 - 3.36 (m, 2H), 3.35 - 3.26 (m, 8H), 3.07 (br dd, J = 7.6, 12.2 Hz, 2H), 2.53 - 2.51 (m, 2H), 2.19 - 1.98 (m, 4H), 1.94 - 1.91 (m, 6H). LCMS (Method 1): Rt = 2.275 min, MS m / z [M+H] + 499.4, theoretical mass: 498.6 HPLC (Method 1): Rt = 3.908 min, 99.7%

[0092] Elemental analysis: C 45.89%; H 7.96%; N 8.18%, theoretical + 10H2O: C 46.01%; H 8.02%; N 8.25%. 15 mg of compound 4A was dissolved in 1.2 ml of ethanol / H2O (1:1) at 60°C. The solution was filtered through a 0.45 μm microporous filter and kept in a sealed 4 ml vial at room temperature. Needle-shaped crystals were observed in the solution and selected for X-ray crystallography.

[0093] The crystals were colorless needles with dimensions of 0.30 × 0.04 × 0.04 mm. The symmetry of the crystal structure was determined using a Rigaku Oxford Diffraction XtaLAB Synergy 4 circle diffractometer equipped with a HyPix-6000HE area detector: a = 15.9948(2) Å, b = 22.5673(3) Å, c = 9.5013(2) Å, α = 90°, β = 90°, γ = 90°, V = 3429.58(10) Å, Z = 4, Dc = 1.280 g / cm, F(000) = 1424.0, μ(CuKα) = 0.889 mm. -1 Using the parameters of , and T = 110(14) K, the crystal was assigned to the orthorhombic space group C2221. Cryogenic system: Oxford Cryostream 800Cu: λ = 1.54184 Å, 50 W, microfocus light source with multilayer mirror (μ-CMF). Distance from crystal to CCD detector: d = 35 mm. Tube voltage: 50 kV. Tube current: 1 mA. The absolute configuration of Example 1 was assigned to be (R,R,R,R).

[0094] Example 1 Preparation of HCl Salt To a suspension of APL-2191 (30.9 g, 45.5 mmol, 1 equiv., 10H2O) in HO (760 mL) and EtOH (760 mL), HCl (12 M, 7.61 mL, 2.01 equiv.) was added at 25 °C and stirred for 12 h. The reaction mixture was concentrated in vacuo to afford APL-2191·2HCl (28.2 g, 39.0 mmol, 85.7% yield, 10H2O) as a crystalline off-white solid. LCMS (Method 1): Rt = 2.300 min, MS m / z 250.1 [M+H / 2] + HPLC (method 2): Rt = 3.889 min, 99.3% 1 H NMR 400 MHz (DO): δ ppm: 7.85 (s, 4H), 4.67 (d, J = 11.2 Hz, 2H), 3.63 - 3.50 (m, 2H), 3.41 - 3.22 (m, 8H), 3.10 (ddd, J = 1.8, 6.8, 13.2 Hz, 2H), 2.73 - 2.58 (m, 2H), 2.30 - 2.16 (m, 4H), 2.10 - 1.88 (m, 6H).

[0095] Additionally, Example 1 could be prepared according to the following procedure. Step 1: To a solution of (R)-methyl 2-amino-2-((3R)-quinuclidin-3-yl)acetate (100.00 mg, 504.39 μmol) in CHCl (8.00 mL) was added benzene-1,4-dicarbonyl chloride (51.20 mg, 252.19 μmol, 0.50 equiv) at 30° C. The mixture was stirred at 30° C. for 16 h. The mixture was concentrated to give the crude product as a white solid (180.00 mg, crude, HCl salt). LCMS: MS m / z 527.5 [M+N] +

[0096] Step 2: To a solution of the bismethyl ester (180.00 mg, 341.80 μmol) in THF (2.00 mL) was added LiOH (48.00 mg, 2.00 mmol) in water (2 mL) at 25° C. The mixture was stirred at 25° C. for 1 h. The mixture was concentrated under reduced pressure to remove THF. 1 M HCl (aq) was added to the mixture to pH=3. The mixture was concentrated under reduced pressure. The residue was dissolved in MeOH (5 mL) and purified by preparative HPLC (TFA) to give Example 1 (24.20 mg, 48.05 μmol, 14.06% yield, 99% purity) as a white solid. 1 H NMR 400 MHz (DO): δ ppm 7.77 (s, 4H), 4.62 (d, J=11.2 Hz, 2H), 3.50 (br t, J=10.9 Hz, 2H), 3.41 - 3.14(m, 8H), 3.11 - 2.96 (m, 2H), 2.65 - 2.57 (m, 2H), 2.32 - 2.07 (m, 4H), 2.05 - 1.79 (m, 6H). LCMS: Rt = 5.91, MS m / z 501.1 [M+H] + , theoretical mass: 500.2

[0097] The following examples were prepared using the same procedure as described in Example 1 (see General Methods below), using the appropriate dicarboxylic acid and compound 5(R,R) as described in each example. The examples were purified as described individually in Step 1 and Step 2.

[0098] [Basic Methods of Examples 2 to 11]:

[0099] [ka] Step 1: To a solution of compound 5 (2.70 equiv.) in ACN (10 V) was added TBTU (2.23 equiv.) and the appropriate carboxylic acid (1 equiv.) under nitrogen at 20° C. DIEA (4.11 equiv.) was added to the mixture, and the mixture was stirred under N for 6 h at 20° C. The reaction mixture was concentrated in vacuo and purified as described for each example.

[0100] Step 2: To a solution of the bis-methyl ester (1.00 equiv.) in IPA (20.0 V) was added aqueous KOH (10.0%, 10.0 equiv.) at 20° C. The mixture was stirred at 50° C. for 1 h, cooled to room temperature, and purified as described for each example.

[0101] [Example 2] (APL-6968) <(R,2R,2'R)-2,2'-((pyridine-2,5-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0102] [ka] Example 2 was prepared according to the general method using pyridine-2,5-dicarboxylic acid. Step 1: The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 × 30 mm, 3 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 1%–20%, 7 min) to give the bismethyl ester (330 mg, 524 μmol, 43.8% yield, 91.2% purity, FA) as a white solid. 1 H NMR 400 MHz (CDCl3): δ ppm: 8.93 (br s, 1H), 8.82 - 8.60 (m, 2H), 8.50 - 8.40 (m, 2H), 8.26 - 8.24 (m, 1H), 8.02 - 8.01 (m, 1H), 4.92 - 4.84 (m, 1H), 4.82 - 4.73 (m, 1H), 3.80 (d, J = 14.0 Hz, 6H), 3.28 - 3.12 (m, 7H), 2.31 - 2.12 (m, 12H), 1.99 - 1.77 (m, 10H). LCMS (Method 1) Rt = 0.685 min, MS m / z [M+H] + 528.2

[0103] ステップ2: The residue was fractionated and HPLC was performed: Waters Atlantis T3 150 × 30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 1% ~ 20%, 10 minutes) was purified, and Example 2 (74.0 mg, 132 μmol, purity 97.4%, FA) was obtained as a white solid. MS (Method 8): MS m / z 499.9 [M+H] + Theoretical mass: 499.2 HPLC (Method 1): Rt = 2.31 min 1 H NMR 400 MHz (D2O): δ ppm: 8.97 (d, J = 1.6 Hz, 1H), 8.43 (s, 1H), 8.33 - 8.31 (m, 1H), 8.13 (d, J = 8.0 Hz, 1H), 4.56 (dd, J = 8.4, 10.4 Hz, 2H), 3.60 - 3.52 (m, 2H), 3.39 - 3.24 (m, 8H), 3.13 - 3.04 (m, 2H), 2.62 - 2.51 (m, 2H), 2.28 - 2.19 (m, 4H), 2.05 - 1.89 (m, 6H).

[0104] [Example 3] (APL-6969) <(R,2R,2'R)-2,2'-((pyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0105] [ka] Example 3 was prepared according to the general method using pyrazine-2,5-dicarboxylic acid. Step 1: The residue was purified by preparative HPLC (column: Waters Xbridge 150 × 25 mm, 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 14%–44%, 9 min) to give the bismethyl ester (90.0 mg, 124 μmol, 10.4% yield, 72.7% purity) as a white solid. LCMS (Method 1): Rt = 0.704 min, MS m / z 529.3 [M+H] +

[0106] Step 2: The mixture was filtered, FA (aqueous solution, 20% in water) was added to adjust the mixture to pH = 7-8, and the mixture was purified by preparative HPLC (column: Waters Xbridge 150 × 25 mm, 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 1%-10%, 9 min) to give Example 3 (51.0 mg, 98.0 μmol, yield 57.5%, purity 96.0%) as a white solid. MS (Method 2): MS [M+H] + 501.1, theoretical mass: 500.2 HPLC (Method 3): Rt = 0.824 min 1 H NMR 400 MHz (DO): δ ppm: 9.21 (s, 2H), 8.38 (br s, 4H), 4.55 (d, J = 3.60 Hz, 2H), 3.53 - 3.48 (m, 2H), 3.40 - 3.20 (m, 8H), 3.10 - 3.01 (m, 2H), 2.63 - 2.52 (m, 2H), 2.21 (br s, 4H), 2.07 - 1.85 (m, 6H).

[0107] [Example 4] (APL-6970) <(R,2R,2'R)-2,2'-((pyridazine-3,6-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0108] [ka] Example 4 was prepared according to the general method using pyridazine-3,6-dicarboxylic acid. Step 1: The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 × 30 mm, 3 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 5%–35%, 8 min) to give the bismethyl ester (110 mg, 144 μmol, yield 22.0%, purity 81.1%) as a white solid. 1 H NMR 400 MHz (CDCl3): δ ppm: 7.86 (d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.65 (dd, J = 1.6, 8.0 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 6.55 - 6.50 (m, 2H), 4.96 - 4.91 (m, 2H), 3.79 (s, 6H), 3.16 - 3.05 (m, 3H), 3.02 - 2.69 (m, 11H), 2.31 (s, 3H), 2.09 - 1.87 (m, 9H), 1.79 - 1.63 (m, 5H). LCMS (Method 1): Rt = 0.807 min, MS m / z 617.3

[0109] Step 2: The residue was purified by preparative HPLC (column: Waters Atlantis T3 150 × 30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 1% to 20%, 10 min) to give Example 4 (87.0 mg, 154 μmol, yield 32.7%, purity 97.3%, FA) as a white solid. LCMS (Method 8): Rt = 2.296 min, MS m / z 501.4 [M+H] + , theoretical mass: 500.3 1 H NMR 400 MHz (DO): δ ppm: 8.41 (s, 2H), 8.35 (s, 0.25H), 4.62 (d, J = 10.8 Hz, 2H), 3.62 - 3.52 (m, 2H), 3.42 - 3.22 (m, 8H), 3.19 - 3.09 (m, 2H), 2.68 - 2.55 (m, 2H), 2.30 - 2.18 (m, 4H), 2.09 - 1.86 (m, 6H).

[0110] [Example 5] (APL-6971) <(R)-2-(4'-(((R)-carboxy((R)-quinuclidin-3-yl)methyl)carbamoyl)-[1,1'-biphenyl]-4-ylcarboxamido)-2-((R)-quinuclidin-3-yl)acetic acid>

[0111] [ka] Example 5 was prepared according to the general method using [1,1'-biphenyl]-4,4'-dicarboxylic acid. Step 1: The crude material was obtained as a colorless liquid and used directly in the next step. LCMS (Method 1) Rt = 0.789 min, MS m / z 603.4 [M+H] +

[0112] Step 2: The mixture was filtered, and FA (aqueous solution, 20% in water) was added to adjust the mixture to pH = 7 - 8. Purification by preparative HPLC (column: Waters Xbridge 15 x 25 mm, 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 1% - 10%, 9 min) gave Example 5 (37.0 mg, 63.7 μmol, 15.5% yield, 99.0% purity) as a white solid. LCMS (Method 4): Rt = 1.43 min, MS m / z 575.3 [M+H] + , theoretical mass: 574.2 1 H NMR 400 MHz (DO): δ ppm: 7.89 - 7.77 (m, 8H), 4.55 (d, J = 10.8 Hz, 2H), 3.59 - 3.50 (m, 2H), 3.42 - 3.18 (m, 8H), 3.13 - 3.02 (m, 2H), 2.58 - 2.48 (m, 2H), 2.30 - 2.15 (m, 4H), 2.09 - 1.84 (m, 6H).

[0113] [Example 6] (APL-6972) <(R)-2-(4'-(((R)-carboxy((R)-quinuclidin-3-yl)methyl)carbamoyl)-2'-methyl-[1,1'-biphenyl]-4-ylcarboxamido)-2-((R)-quinuclidin-3-yl)acetic acid>

[0114] [ka] Example 6 was prepared according to the general method using 2-methyl-[1,1'-biphenyl]-4,4'-dicarboxylic acid. Step 1: The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 × 30 mm, 3 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 5%–35%, 8 min) to give the bisester (110 mg, 144 μmol, yield 22.0%, purity 81.1%) as a white solid. LC-MS (Method 1): Rt = 0.807 min, MS m / z 617.3 [M+H] + 1 H NMR 400 MHz (CDCl3): δ ppm: 7.86 (d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.65 (dd, J = 1.6, 8.0 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 6.55 - 6.50 (m, 2H), 4.96 - 4.91 (m, 2H), 3.79 (s, 6H), 3.16 - 3.05 (m, 3H), 3.02 - 2.69 (m, 11H), 2.31 (s, 3H), 2.09 - 1.87 (m, 9H), 1.79 - 1.63 (m, 5H).

[0115] Step 2: The residue was purified by preparative HPLC (column: Waters Atlantis T3 150 × 30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 1% to 20%, 10 min) to give Example 6 (FA salt, 16 mg, 5.44 μmol, yield 4.13%, purity 95.0%) as a white solid. LCMS (Method 4): Rt = 1.597 min, MS m / z 589.3 [M+H] + , theoretical mass: 588.3 1 H NMR 400 MHz (DO+DMSO): δ ppm: 8.24 (s, 1H), 7.90 - 7.80 (m, 2H), 7.71 (s, 1H), 7.68 - 7.62 (m, 1H), 7.43 (br d, J = 8.4 Hz, 2H), 7.34 - 7.27 (m, 1H), 3.35 - 3.32 (m, 2H), 3.26 - 3.05 (m, 9H), 2.91 - 2.83 (m, 2H), 2.22 (s, 3H), 2.15 - 2.07 (m, 4H), 1.92 - 1.68 (m, 7H).

[0116] [Example 7] (APL-6973) <(R,2R,2'R)-2,2'-((naphthalene-2,6-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0117] [ka] Example 7 was prepared according to general method 1 using naphthalene-2,6-dicarboxylic acid. Step 1: The residue was purified by preparative HPLC (column: 3_Phenomenex Luna C18 75 × 30 mm, 3 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%–35%, 7 min), and the mixture was lyophilized to give the bisester (160 mg, 277 μmol, 60.0% yield) as a white solid. LC-MS (Method 1): Rt = 0.770 min, MS m / z [M+H] + 577.4

[0118] Step 2: The mixture was filtered, FA (aqueous solution, 20% in water) was added, and the mixture was adjusted to pH = 7-8. The residue was purified by preparative HPLC (column: Waters Xbridge 150 * 25mm *5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 1% to 10%, 9 min) to give Example 7 (35.0 mg, 62.0 μmol, yield 22.0%, purity 96.0%) as a white solid. LCMS (Method 5): Rt = 0.282 min, MS m / z 549.1 [M+H] + , theoretical mass: 548.2 HPLC (Method 6): Rt = 1.624 min 1 H NMR 400 MHz (DO): δ ppm: 8.23 ​​(s, 2H), 7.95 (d, J = 10.0 Hz, 2H), 7.77 (d, J = 10.0 Hz, 2H), 4.59 (d, J = 11.2 Hz, 2H), 3.64 - 3.52 (m, 2H), 3.44 - 3.23 (m, 10H), 3.15 - 3.04 (m, 2H), 2.63 - 2.52 (m, 2H), 2.31 - 2.18 (m, 5H), 2.09 - 1.86 (m, 7H).

[0119] [Example 8] (APL-6974) <(R,2R,2'R)-2,2'-((2,5-dimethylterephthaloyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0120] [ka] Example 8 was prepared according to the general method using 2,5-dimethylbenzene-1,4-dicarboxylic acid. Step 1: The crude product was triturated with ACN (5 mL) and MeOH (3 mL) for 10 min at 20° C. and filtered to give the bis ester (114 mg, 185 μmol, 17.9% yield, 90.1% purity) as a white solid. LCMS (Method 1): Rt = 0.771 min, MS m / z 555.3 [M+H]+ , 1 H NMR 400 MHz (DMSO): δ ppm: 8.75 (d, J = 6.8 Hz, 1H), 7.18 (br s, 1H), 4.50 - 4.46 (m, 1H), 3.68 (s, 3H), 3.17 - 2.91 (s, 12H), 2.78-2.68 (m, 1H), 2.30 (s, 3H), 2.20 - 2.19 (m, 1H), 1.96 - 1.90 (m, 1H), 1.83 - 1.68 (m, 2H), 1.74 - 1.53 (m, 2H), 1.16 (d, J = 6.0 Hz, 1H).

[0121] ステップ2: The residue was fractionated and HPLC was performed: Waters Atlantis T3 150×30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 1%~20%, 10 minutes) was refined and obtained in Example 8 (10.0 mg, 17.1 μmol, yield 9.50%, purity 98.1%, FA) and a white solid was obtained. LCMS (Method 8): Rt = 2.773 min, MS m / z 527.3 [M+H] + Theoretical mass: 526.3 1 H NMR 400 MHz (D2O+DMSO): δ ppm: 7.16 (s, 2H), 4.34 (br d, J = 10.4 Hz, 2H), 3.41 - 3.33 (m, 2H), 3.22 - 3.10 (m, 7H), 2.94 - 2.88 (m, 2H), 2.36 - 2.27 (m, 3H), 2.21 (s, 6H), 2.16 - 2.01 (m, 4H), 1.90 - 1.70 (m, 6H). LCMS m / z 527.3 [M+H] + , theoretical mass: 526.3, Rt = 2.77 minutes, 100%

[0122] [Example 9] (APL-6975) <(R,2R,2'R)-2,2'-((2-methylterephthaloyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0123] [ka] Example 9 was prepared according to the general method using 2-methylbenzene-1,4-dicarboxylic acid. Step 1: The residue was purified by preparative HPLC (column: Phenomenex luna C18 150 × 25 mm, 10 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 0% to 20%, 10 min) to give the bisester (500 mg, 647 μmol, yield 23.3%, purity 70.0%) as a white solid. LCMS (Method 1): Rt = 0.707 min, MS m / z 541.2 [M+H] +

[0124] Step 2: The mixture was filtered, and FA (aqueous solution, 20% in water) was added to adjust the mixture to pH = 7 - 8. The mixture was purified by preparative HPLC (column: Waters Xbridge 150 × 25 mm, 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 1% - 10%, 9 min) to give Example 9 (116 mg, 202.26 μmol, 54.67% yield, 97.4% purity, FA) as a white solid. LCMS (Method 8): Rt = 2.353 min, MS m / z 513.0 [M+H] + , theoretical mass: 512.3 1 H NMR 400 MHz (DO): δ ppm: 8.38 (m, 1H), 7.64 - 7.59 (m, 2H), 7.42 (d, J = 8.0 Hz, 1H), 4.53 - 4.48 (m, 2H), 3.60 - 3.49 (m, 2H), 3.39 - 3.21 (m, 8H), 3.12 - 2.99 (m, 2H), 2.54 - 2.40 (m, 2H), 2.35 (s, 3H), 2.30 - 2.14 (m, 4H), 2.04 - 1.86 (m, 6H).

[0125] [Example 10] (APL-6976) <(R,2R,2'R)-2,2'-((2,5-bis(benzyloxy)terephthaloyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)>

[0126] [ka] Example 10 was prepared following the general procedure using 2,5-bis(benzyloxy)benzene-1,4-dicarboxylic acid. Step 1: The residue was purified by preparative HPLC (column: Phenomenex luna C18 150 × 25 mm, 10 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 11%–41%, 10 min) to give the bisester (180 mg, 211 μmol, 39.9% yield, 92.1% purity, FA) as a white solid. 1 H NMR 400 MHz (CDCl3): δ ppm: 8.52 (d, J = 8.4 Hz, 2H), 8.37 (s, 1H), 7.93 (s, 2H), 7.58 - 7.40 (m, 10H), 5.27 - 5.17 (m, 4H), 4.79 - 4.75 (m, 2H), 3.69 (s, 6H), 3.34 - 3.14 (m, 6H), 3.07 - 2.95 (m, 2H), 2.90 - 2.75 (m, 4H), 2.11 - 2.01 (m, 2H), 1.99 - 1.88 (m, 6H), 1.85 - 1.70 (m, 4H).

[0127] ステップ2: The residue was fractionated and HPLC was performed: Phenomenex luna C18 150×25mm, 10μm; mobile phase: [water (0.225% FA)-ACN]; B%: 1%~30%, 10 minutes) was refined and obtained in Example 10 (67.0 mg, 87.6 μmol, yield 40.4%, purity 99.0%, FA) and a white solid was obtained. LCMS (Method 1): Rt = 0.799 min, MS m / z 711.3 [M+H] + Theoretical mass: 710.33 HPLC (Method 7): Rt = 2.519 min. 1 H NMR 400 MHz (D2O): δ ppm: 7.64 (s, 2H), 7.59 - 7.50 (m, 10H), 5.25 - 5.17 (m, 4H), 4.57 (d, J = 10.4 Hz, 2H), 3.28 - 3.10 (m, 6H), 2.98 - 2.88 (m, 2H), 2.75 - 2.67 (m, 2H), 2.47 - 2.35 (m, 2H), 2.13 - 2.02 (m, 6H), 2.08 - 1.87 (m, 2H), 1.86 - 1.73 (m, 4H).

[0128] [Example 11] (P2B-E(APL numberなし)) 2-[[3-[[Carboxy-[(3R)-quinuclidin-3-yl]methyl]carbamoyl]benzoyl]amino]-2-[(3R)-quinuclidin-3-yl]acetic acid

[0129] [ka] Step 1: To a solution of methyl 2-amino-2-[(3R)-quinuclidin-3-yl]acetate (100.00 mg, 504.39 μmol) in CHCl (4 mL) was added benzene-1,3-dicarbonyl chloride (51.20 mg, 252.20 μmol) at 30° C. The mixture was stirred at 30° C. for 16 hours. The mixture was concentrated under reduced pressure to give the crude product as a yellow solid. LCMS: Rt = 0.881 min, MS m / z 527.3 [M+H] +

[0130] Step 2: To a solution of the bismethyl ester (165.00 mg, 313.31 μmol) in THF (4 mL) was added LiOH (96.00 mg, 4.01 mmol, 12.79) in HO (4 mL) at 30° C. The mixture was stirred at 30° C. for 2 h. The mixture was concentrated under reduced pressure to remove THF. Water (10 mL) and 1 M HCl (aq) were added to the residue to pH=2. The mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by preparative HPLC to give Example 11 (37.20 mg, 63.79 μmol, 40.72% yield, 98% purity, 2HCl) as a white solid. 1 H NMR 400 MHz (DO): δ ppm: 8.10 - 8.01 (m, 1H), 7.88 (dd, J=1.7, 7.8 Hz, 2H), 7.54 (t, J=7.8 Hz, 1H), 4.62(d, J=11.0 Hz, 2H), 3.59 - 3.50 (m, 2H), 3.38 - 3.15 (m, 8H), 3.11 - 2.99 (m, 2H), 2.71 - 2.54 (m, 2H), 2.26 - 2.07 (m, 4H), 2.06- 1.78 (m, 6H). LCMS Rt = 5.9 min, MS m / z = 499.3 [M+H] + , theoretical mass: 498

[0131] [Biological Assay] (MIRA Immunoturbidimetric Assay) The CRP immunoturbidimetric assay on the Roche COBAS MIRA Plus automated analyzer utilizes two differently sized latex particles covalently coupled to two different monoclonal antibodies with specificity for different CRP epitopes (5). This assay has been validated by Roche for the measurement of native pentameric CRP, exhibiting high sensitivity and specificity, a high upper limit of detection, and was calibrated against a standard generated in our laboratory. Unexpectedly, one of the antibodies in the assay binds to an epitope present on the ligand-binding B-face of CRP. Therefore, if the binding pocket is occupied by a ligand or blocked, for example, by complex formation between the B-faces of the pentamer, this assay cannot detect CRP. However, CRP detection can be demonstrated in other types of assays in which antibodies binding to different epitopes are used. Bivalent compounds, such as BPC8 and APL-2191, have been designed to crosslink pairs of CRP pentamers. Therefore, inhibition of CRP recognition in the MIRA assay is a useful tool for monitoring the effectiveness and potency of complex formation between such ligands and CRP (6).

[0132] CRP concentrations were measured in the presence and absence of ligand using a COBAS MIRA automated analyzer. Concentrated Tris-calcium buffer (x10 TC) was prepared in MilliQ water from trishydroxymethylamine (100 mM), calcium chloride (20 mM), and sodium chloride (1.4 M). The pH was adjusted to 8.0 using HCl, and sodium azide was added (0.1% w / v). The buffer was stored at 4°C. A 10x diluted working buffer (TC) was prepared by diluting 100 ml of x10 concentrated buffer with 900 ml of MilliQ water. Human CRP was isolated, purified, and characterized as previously reported (6-9) and stored frozen at -80°C. Stock CRP was thawed at 37°C as needed, and the prepared working dilutions were maintained at 4°C throughout the experiment. CRP concentrations were calculated as A after correcting for absorbance at 320 nm (light scattering). 280 The concentration of human CRP was determined spectrophotometrically (Beckman Coulter DU650) in a quartz cuvette with a 1 cm path length by measuring the absorbance coefficient of human CRP, A (1%, 1 cm) = 17.5 (10). Human CRP at approximately 90 μg / ml (0.78 μM pentamer) in TC buffer was prepared from a stock solution, and 75 μl aliquots were used in the assay. Compounds were supplied as solids by Wuxi AppTec (Wuhan, China). Compounds were dissolved in TC buffer at appropriate concentrations up to 10 mM depending on solubility (labeled S1). They were then serially diluted 1:2 with TC buffer (100 μl ligand + 200 μl TC) to prepare up to nine dilutions, S2–S10. A TC buffer control (S0) was included in each assay. A 15 μl volume of each ligand solution was incubated with 75 μl of CRP at room temperature for 1 h. The final concentrations were 0.73 μM native pentameric CRP and 0.03 μM ligands S1 to S10. r This corresponded to a ratio of 0.04. If a compound had low solubility in TC buffer, a lower stock concentration (from 0.6 mM) was used, corresponding to a final top assay concentration of 100 µM.

[0133] Data are expressed as CRP measurements (mg / L) against final total ligand concentration (μM) and plotted using Sigmaplot (V14) to fit a four-parameter logistic curve y = min + (max - min) / (1 + (x / EC 50 ) - Hill slope) to calculate EC 50 was calculated. Where appropriate, samples were also measured in whole normal human serum after spiking with known amounts of human CRP. All compounds were assayed against a highly purified preparation of bis(phosphocholine)octane (BPC8) prepared by Carbogen AMCIS AG and diluted in sterile water to a concentration of 10 mM, which was stored at -80°C. Solutions were diluted with TC buffer where appropriate.

[0134] [Table 1]

[0135] An example of formula (I) is the RR,RR stereoisomer. Other stereoisomers of this structure are less active or inactive. The SS,SS isomer is the most active alternative isomer (designated QA,QA quinuclidine; amino acids: SS,SS IC50 34.4 μM, RS,RS IC50>1000 μM, SR,SR IC50>1000 μM, RS,RR>1000 μM). Those skilled in the art can prepare other isomers using the desired stereoisomer and following the methods described above, using appropriate protecting group strategies.

[0136] Each document cited herein (the "Application Citations") and each document cited or referenced in the Application Citations, and any manufacturer's specifications or instructions for any product mentioned in this specification and any documents incorporated herein, are hereby incorporated by reference in their entirety, and the technology in each document incorporated by reference in its entirety may be used in the practice of this invention.

[0137] This invention claims priority to UK patent application GB2002299.2 filed February 19, 2020, the entire contents of which are incorporated herein by reference.

[0138] [Table 2]

Claims

1. Formula (I): 【Chemistry 1】 wherein Ar is an aryl linker group. and the respective pharmaceutically acceptable salts or solvates thereof.

2. The linker group Ar is represented by the following formulae Ar-I to Ar-VI: 【Chemistry 2】 is selected from the group consisting of In the formula, R is halogen, hydroxy, cyano, or —CONH ２ or C1-C5 (cyclo) alkyl or C1-C5 (cyclo) alkoxy, wherein the alkyl group is optionally substituted with a phenyl group or with one or more halogen atoms.

3. The aryl linker group Ar is represented by the following formulae Ar-VII to Ar-XVI: 【Transformation 3】 3. The compound of claim 2 selected from the group consisting of:

4. 4. The compound of any one of claims 1 to 3, having a diastereomeric purity for the (R,R,R,R) stereoisomer of at least about 50% by weight, preferably at least about 60% by weight, more preferably at least about 75% by weight, even more preferably at least about 90% by weight, and most preferably at least about 98% by weight.

5. The compound of formula (I) is represented by the following formula (II): 【Chemistry 4】 The compound according to any one of claims 1 to 4, having the formula:

6. 6. The compound of any one of claims 1 to 5, wherein the compound of formula (I) is a hydrochloride salt, in particular a 2HCl salt.

7. The compounds of formula (I) have an IC of about 20 μM or less, even more preferably about 10 μM or less, or about 5 μM or less, or about 1 μM or less. ５０ 7. The compound according to any one of claims 1 to 6, which is an inhibitor of human C-reactive protein (CRP), having the formula:

8. A compound according to any one of claims 1 to 7 for use in medicine.

9. A compound according to any one of claims 1 to 7 for use in the treatment or prevention of tissue damage in a subject having an inflammatory and / or tissue damaging condition.

10. 10. The compound of claim 9, wherein the inflammatory and / or tissue damage condition comprises one or more of acute coronary syndrome, unstable angina, plaque rupture, and / or early atherothrombosis.

11. The compound of claim 10, wherein the inflammatory and / or tissue damaging condition is selected from infectious diseases, allergic complications of infectious diseases, inflammatory diseases, ischemic or other necrosis, traumatic tissue injury, and malignant neoplasia.

12. 12. The compound of claim 11, wherein the condition is an infection selected from a bacterial infection, including sepsis, a viral infection, e.g., a Severe Acute Respiratory Syndrome (SARS) virus infection, such as SARS-Cov-2 infection, a fungal infection, and a parasitic infection.

13. 10. The compound of claim 9, wherein the condition is an inflammatory disease selected from rheumatoid arthritis, juvenile chronic (rheumatoid) arthritis, ankylosing spondylitis, psoriatic arthritis, systemic vasculitis, polymyalgia rheumatica, Reiter's disease, Crohn's disease, and familial Mediterranean fever, and other autoinflammatory conditions.

14. 10. The compound of claim 9, wherein the condition is tissue necrosis selected from myocardial infarction, ischemic stroke, tumor embolism, and acute pancreatitis.

15. 10. The compound of claim 9, wherein the condition is a trauma selected from elective surgery, burns, chemical injuries, fractures, and compression injuries.

16. 10. The compound of claim 9, wherein the condition is a malignant neoplasia selected from lymphoma, Hodgkin's disease, carcinoma, and sarcoma.

17. 10. The compound of claim 9, wherein the condition is an allergic complication of an infection selected from rheumatic fever, glomerulonephritis, and erythema nodosum leprosum.

18. A composition comprising a compound according to any one of claims 1 to 7 in admixture with one or more pharmaceutically acceptable excipients, diluents or carriers.

19. A process for preparing a compound of formula (I) as defined in any one of claims 1 to 7, comprising the step of reacting a compound of formula (III): 【Transformation 5】 (In the formula, R １ is a carboxyl protecting group. with a compound of formula (IV-A) or formula (IV-B): 【Transformation 6】 to form a compound of formula (V): 【Transformation 7】 forming a compound of Next, the R １ cleaving the protecting group to form a compound of formula (I).

20. Formula (III): 【Transformation 8】 (In the formula, R １ is a carboxyl protecting group, and R １ is selected from C1-C5 alkyl, trityl, 2,4-dimethoxybenzyl (DMB), benzyl, or 9-fluorenylmethyl. or a salt thereof with an optically active organic acid compound.

21. 21. The compound according to claim 20, which is a salt of the compound of formula (III) and (1S)-(+)-10-camphorsulfonic acid.

22. The protecting group R １ is methyl.

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