AGENTS FOR USE IN THE TREATMENT OF TISSUE DAMAGE 2

Abstract

An agent for use in medicine, wherein the agent comprises a compound of Formula (I): wherein Ar is an aryl linker group, for example a 1,4-phenyl group, including individual pharmaceutically acceptable salts, solvates, prodrugs or derivatives thereof. The compounds of Formula (I) are inhibitors of human C-reactive protein (CRP) and may be used for the treatment of medical conditions mediated by CRP. Also provided are methods of making the compounds of Formula (I) and chemical intermediates thereof.

##STR00001##

Claims

1. An agent for use in medicine, wherein the agent comprises a compound of Formula ##STR00034## wherein Ar is an aryl linker group, including individual pharmaceutically acceptable salts, solvates, prodrugs or derivatives thereof.

2. An agent for use in medicine according to any preceding claim, wherein the linker group Ar is selected from the group consisting of the following general Formulae Ar-I to Ar-VI: ##STR00035## wherein R represents one or more optional substituents on the aryl ring(s) selected from halogen, hydroxy, cyano, —CONH.sub.2, or C1-C5 (cyclo)alkyl or C1-C5 (cyclo)alkoxy wherein the alkyl groups are optionally substituted with a phenyl group or with one or more halogen atoms.

3. An agent for use in medicine according to claim 2, wherein the aryl linker group Ar is selected from the group consisting of groups having formulae Ar-VII to Ar-XVI: ##STR00036##

4. An agent for use in medicine according to any preceding claim, wherein the diastereomeric purity of the (R,R,R,R) isomer is at least about 50% by weight, suitably at least about 60%, more suitably at least about 75%, still more suitably at least about 90%, and most suitably at least about 98%.

5. An agent for use in medicine according to any preceding claim, wherein the compound of Formula (I) has the following Formula (II): ##STR00037##

6. An agent for use in medicine according to any preceding claim, wherein the compound of Formula (I) is a hydrochloride salt, in particular a 0.2HCl salt.

7. An agent for use in medicine according to any preceding claim, wherein the compound of Formula (I) is an inhibitor of human C-reactive protein (CRP) having an IC.sub.50 of about 20 μM or less, still more preferably about 10 μM or less, or about 5 μM or less, or about 1 μM or less.

8. An agent according to any preceding claim, for use in the treatment or prevention of tissue damage in a subject having an inflammatory and/or tissue damaging condition.

9. An agent according to claim 8, wherein the inflammatory and/or tissue damaging condition comprises one or more of acute coronary syndrome, unstable angina, plaque rupture, and/or incipient atherothrombosis.

10. An agent according to claim 9, wherein the inflammatory and/or tissue damaging condition is selected from an infection, an allergic complication of infection, an inflammatory disease, ischemic or other necrosis, traumatic tissue damage and malignant neoplasia.

11. An agent according to claim 10, wherein the condition is an infection selected from a bacterial infection including sepsis, a viral infection for example a severe acute respiratory syndrome (SARS) viral infection such as a SARS-Cov-2 infection, a fungal infection and a parasitic infection.

12. An agent according to claim 8, 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.

13. An agent according to claim 8, wherein the condition is tissue necrosis selected from myocardial infarction, ischaemic stroke, tumour embolization and acute pancreatitis.

14. An agent according to claim 8, wherein the condition is trauma selected from elective surgery, burns, chemical injury, fractures and compression injury.

15. Use according to claim 8, wherein the condition is malignant neoplasia selected from lymphoma, Hodgkin's disease, carcinoma and sarcoma.

16. An agent according to claim 8, wherein the condition is an allergic complication of infection selected from rheumatic fever, glomerulonephritis, and erythema nodosum leprosum.

17. A pharmaceutical composition comprising an agent according to any of claims 1 to 7 in admixture with one or more pharmaceutically acceptable excipients, diluents or carriers.

18. A method of making a compound of Formula (I) as defined in any of claims 1 to 7, comprising the steps of reacting a compound of Formula (III): ##STR00038## wherein R.sup.1 is a carboxyl protecting group, with a compound of Formula (IV-A) or (IV-B): ##STR00039## to form a compound of Formula (V): ##STR00040## followed by cleavage of the R.sup.1 protecting groups to form the compound of Formula (I).

19. A chemical compound of Formula (III): ##STR00041## wherein R.sup.1 is a carboxyl protecting group, or a salt thereof with an optically active organic acid compound.

20. A chemical compound according to claim 19, wherein the compound is a salt of the compound of Formula (III) with (1S)-(+)-10-camphorsulfonic acid.

21. A chemical compound according to claim 19 or 20, wherein the protecting group R.sup.1 is methyl.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0020] In a first aspect, the present invention provides an agent for use in medicine, wherein the agent comprises a compound of Formula (I):

##STR00007##

[0021] wherein Ar is an aryl linker group, including individual pharmaceutically acceptable salts, solvates, prodrugs or derivatives thereof.

[0022] The Ar linker group is suitably a monocyclic, bicyclic, or fused bicyclic aryl group optionally containing 1, 2 or 3 hetero atoms in the aromatic ring(s), the hetero atoms suitably being selected from N or S. The Ar linker group suitably contains from 4 to 12 carbon atoms in the aromatic rings (i.e. excluding carbon atoms in optional substituent groups). The aromatic ring(s) of the Ar group are linked to the palindromic end groups of the compounds of Formula (I) through amide bonds as shown in Formula (I). Suitably, the bond angle between the two Ar-CO bonds is about 180 degrees. Thus, for example, where Ar is a single six-membered aromatic ring such as a phenyl group, the bonds are suitably located para (1,4) on the ring. It appears that the resulting conformational relationship positions the quinuclidinyl end groups appropriately for binding to respective receptors in the CRP.

[0023] In embodiments, the Ar group is selected from 1,4-phenyl, 2,6-naphthyl or 4,4′-biphenyl, or groups of the same ring system containing 1, 2 or 3 heteroatoms in the ring(s), (e.g. 2,6-pyridyl instead of 1,4-phenyl). In each case, the aromatic rings may be substituted with one or more substituent groups R as defined below.

[0024] In these embodiments, the linker group Ar may be selected from the group consisting of the following general Formulae Ar-I to Ar-VI:

##STR00008##

[0025] wherein R represents one or more optional substituents on the aryl ring(s). Suitably, R may be selected from halogen, hydroxy, cyano, —CONH.sub.2, or C1-C5 (cyclo)alkyl or C1-C5 (cyclo)alkoxy wherein the alkyl groups are optionally substituted with a phenyl group (e.g. wherein R is —O-benzyl) or with one or more halogen atoms, for example trifluoromethyl. More suitably, R may be C1-C4 alkyl or C1-C4 alkoxy, for example methyl. Suitably, there are 0, 1 or 2 R substituents on the aryl linker, more suitably 0 or 1 R substituents, and in some cases no R substituents. In specific embodiments, the Ar linker group is a 1,4-phenyl linker group having 0, 1 or 2 R substituents.

[0026] In embodiments, the aryl linker group Ar is selected from the group consisting of groups having formulae Ar-VII to Ar-XVI:

##STR00009##

[0027] In embodiments of particular interest, the compound of Formula (I) has the following Formula

##STR00010##

[0028] This compound of Formula (II) is also referred to herein interchangeably as P2B-B, or as APL-2191, or as the Compound of Example 1.

[0029] The compounds of Formula (I) and (II) are R,R,R,R stereoisomers. The other stereoisomers of this structure have been found to have lesser activity. The S,S,S,S isomer is thought to be the most active alternative stereoisomer.

[0030] Suitably, the diastereomeric purity of the (R,R,R,R) stereoisomer in the agents of the invention is at least about 50% by weight, suitably at least about 60%, more suitably at least about 75%, still more suitably at least about 90%, and most suitably at least about 98%. That is to say, the amount of the (R,R,R,R) stereoisomer suitably exceeds the amount of all other stereoisomers of this compound present in the agent. Most suitably, at least about 98% by weight of all stereoisomers of this compound present in the agent is the R,R,R,R stereoisomer.

[0031] Crystalline or dissolved forms of the compounds of Formula (I) and (II) may exist in a zwitterionic form (COO− QNH+), and such zwitterionic forms are hereby encompassed in the definitions of Formula (I) and (II) above. Likewise, the definitions of Formula (I) and (II) encompass all crystalline forms and polymorphs of the said compounds.

[0032] It has been found that the above bivalent ligand compounds of Formula (I) are avidly bound by human CRP in vitro and in vivo, forming stable complexes of pairs of native pentameric CRP molecules cross-linked by up to 5 ligand molecules. The ligand binding pockets of each CRP protomer are blocked, and the whole binding (B) face of each CRP pentamer is fully occluded in this complex so that CRP cannot mediate tissue damaging action in vivo. Furthermore, dissociation of the individual, non-covalently associated, protomers of native CRP from within the CRP-ligand complex is completely inhibited under physiological conditions.

[0033] Suitably, the compound of Formula (I) is an inhibitor of human C-reactive protein (CRP) having an IC.sub.50 of about 20 μM or less, suitably about 10 μM or less, more suitably about 504 or less, or most suitably about 1 μM or less. A suitable method of measuring the IC.sub.50 to determine whether a particular compound has IC.sub.50 within the specified ranges is described herein below.

[0034] 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 treatment or prevention of a medical condition mediated by CRP.

[0035] The agents according to the invention, comprising the compound of Formula (I), may be administered concurrently with one or more other pharmaceutically active medications, simultaneously, separately or sequentially. Such other pharmaceutically active medications may include, for example, anti-inflammatory drugs such as corticosteroids; anti-viral, anti-bacterial, anti-fungal or anti-parasitic drugs; inhibitors/antagonists of pro-inflammatory cytokines such as IL-1, IL-6, TNF; anti-coagulants; inhibitors of complement activation or its bioactive fragments.

[0036] 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 invention, or a pharmaceutical composition according to the invention.

[0037] In embodiments, the inflammatory and/or tissue damaging condition comprises one or more of acute coronary syndrome, unstable angina, plaque rupture, and/or incipient atherothrombosis.

[0038] In embodiments, the inflammatory and/or tissue damaging condition is selected from an infection, an allergic complication of infection, an inflammatory disease, ischemic or other necrosis, traumatic tissue damage and malignant neoplasia. For example, the condition may be an infection selected from a bacterial infection including sepsis, a viral infection, a fungal infection and a parasitic infection.

[0039] In embodiments, 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.

[0040] In embodiments, the condition is tissue necrosis selected from myocardial infarction, ischaemic stroke, tumour embolization and acute pancreatitis.

[0041] In embodiments, the condition is trauma selected from elective surgery, burns, chemical injury, fractures and compression injury.

[0042] In embodiments, the condition is malignant neoplasia selected from lymphoma, Hodgkin's disease, carcinoma and sarcoma.

[0043] In embodiments, the condition is an allergic complication of infection selected from rheumatic fever, glomerulonephritis, and erythema nodosum leprosum.

[0044] In embodiments, the condition is an infection or complication of infection with a severe acute respiratory syndrome (SARS) coronavirus, in particular SARS-CoV2.

[0045] Suitably, the method involves administering to a patient an amount of the agent according to the invention sufficient to be bound by all soluble CRP in the circulation and extracellular tissue fluids. For example, the amount may be sufficient to be bound by at least about 70% of the available CRP, preferably at least about 90% of available CRP and optimally 95%, 99% or 100% of the available CRP.

[0046] 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.

[0047] Pharmaceutical compositions may be formulated comprising an agent or a pharmaceutically acceptable salt, ester or prodrug thereof according to the present invention optionally incorporating a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). Here and elsewhere in the present specification, the term “pharmaceutically acceptable salt” refers to salts of the compounds of Formula (I) with anions or cations of which are known and accepted in the art for the formation of salts for pharmaceutical use. Acid addition salts, for example, may be formed by mixing a solution of the agent with a solution of a pharmaceutically acceptable, non-toxic acids, which include but are not limited to hydrochloric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Where the agent carries a carboxylic acid group, the invention also contemplates salts thereof, preferably non-toxic, pharmaceutically acceptable salts thereof, which include, but are not limited to the sodium, potassium, calcium and quaternary ammonium salts thereof. In especially suitable embodiments, the salt is a salt with HCl, in particular the 0.2HCl salt.

[0048] 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. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as—or in addition to—the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

[0049] Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Antioxidants and suspending agents may be also used.

[0050] The pharmaceutical compositions may be in the form of a prodrug comprising the agent or a derivative thereof which becomes active only when metabolised by the recipient. The exact nature and quantities of the components of such pharmaceutical compositions may be determined empirically and will depend in part upon the route of administration of the composition. Where appropriate, the 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 dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly, subcutaneously or intra-arterially.

[0051] The liquid forms in which the compositions of the present invention may be incorporated for administration by injection include aqueous emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil and peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspension include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone and gelatin.

[0052] For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example buffers to adjust pH, or enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

[0053] Use of the compounds of the present invention aims to saturate with the ligand drug all circulating and other soluble CRP molecules in the body. The daily dose of drug required is therefore suitably that which provides at least about 1 mol of drug, more suitably at least about 5 mol of drug per mol of native pentameric CRP to be complexed.

[0054] The precise form of pharmaceutical composition and dosage thereof may also be dependent on the subject to be treated, including body weight, route of administration and disease conditions. These would be determined as a matter of routine by the skilled addressee.

[0055] In a further aspect, the present invention provides a method of making a compound of Formula (I) as defined in any of claims 1 to 6, comprising the steps of reacting a compound of Formula

##STR00011##

[0056] wherein R.sup.1 is a carboxyl protecting group, with a compound of Formula (IV-A) or (IV-B):

##STR00012##

[0057] to form a compound of Formula (V):

##STR00013##

[0058] followed by cleavage of the R.sup.1 protecting groups to form the compound of Formula (I).

[0059] The protecting groups R.sup.1 may be any of the protecting groups conventionally used to protect carboxyl groups during peptide synthesis from amino acids. For example, the protecting groups R.sup.1 may be selected from C1-C5 alkyl, trityl, 2,4-dimethoxybenzyl (DMB), benzyl, or 9-fluorenylmethyl. In embodiments, the protecting groups R.sup.1 are C1-C5 alkyl, in particular methyl groups.

[0060] The step of reacting the compound of Formula (III) with the compound of Formula (IV-A) to form the compound of Formula (V) may be performed by any of the methods conventionally used to form amide bonds in peptide synthesis. For example, the —COOH groups of the compound of Formula (IV) may be activated by converting them into esters of strong acids or groups of formula —COX, where X is a leaving group that is readily displaced by nucleophilic substitution such as chloro, alkylsulfonate or toluenesulfonate, followed by nucleophilic reaction with the primary amine groups of the compounds of Formula (III). In other embodiments, activation of the carboxylic acid may be performed with either phosphate containing reagents, triazine based reagents, carbodiimide based reagents or hydroxybenzotriazole based reagents in the presence of an organic solvent and base. Preferred conditions comprise TB TU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate with diisopropylethylamine in MeCN at room temperature.

[0061] Alternatively, compounds of formula (I) may be prepared using the bis acid chloride of compound (IV-B). Typical reaction conditions comprise warming to 30° C. in chloroform for 16 hours.

[0062] Compounds of formula (III) and (IV-AB) are either commercially available, prepared according to the methods described herein, or prepared according to the literature.

[0063] Finally, the carboxylate groups in the compounds of Formula (V) are deprotected by any of the methods well known in the art. For example, where the groups —COOR.sup.1 are alkyl esters such as methyl esters, the ester may be hydrolysed under mild basic conditions such as 10% KOH (aq.) for 1 hour at 50 C, followed by neutralizing with formic acid at pH4-5.

[0064] In embodiments, the method further comprises making the compound of Formula (III) by a method comprising the step of reacting a compound of Formula (VII) with a compound of Formula (VIII)

##STR00014##

[0065] wherein L represents a leaving group, i.e. a weakly basic group that is readily displaced by nucleophilic substitution. Suitably leaving groups L include bromo, iodo, alkylsulfonate and phenylsulfonate groups, such as a p-bromophenylsulfonate. R.sup.1 is a carboxyl protecting group as defined above. The reaction is suitably carried out in the presence of a strong non-nucleophilic base in an aprotic solvent. 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 form a mixture of stereoisomers of Formula (IX-A) and (IX-B):

##STR00015##

[0066] The synthesis of the compound of Formula (III) then comprises hydrolysing and resolving the above mixture of stereoisomers to isolate the compound of Formula (III) or a salt thereof with an optically active organic acid compound. The hydrolysis may be performed with H.sub.2O under mild acidic conditions, for example in the presence of (1S)-10-camphorsulfonic acid. The salt of Formula (III). 2CSA is preferentially precipitated from the mixture. Other chiral organic acids that are commonly used for separating enantiomers may be suitable, for example (2S,3S)-tartaric acid, (R)-Malic acid, or (−)-(R)-mandelic acid.

[0067] In a further aspect, the present invention provides the chemical compound of Formula (III):

##STR00016##

[0068] wherein R.sup.1 is a carboxyl protecting group as defined above, or a salt of the compound of Formula (III) with an optically active organic acid compound as defined above.

[0069] In embodiments, the compound according to this aspect is a salt of the compound of Formula (III) with (1S)-(+)-10-camphorsulfonic acid (CSA), specifically the 0.2CSA salt.

Examples

[0070] The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples. Compounds are named using conventional IUPAC nomenclature, or as named by the chemical supplier.

[0071] The following synthetic procedures are provided for illustration of the methods used; for a given preparation or step the precursor used may not necessarily derive from the individual batch synthesized according to the step in the description given.

[0072] Analytical Methods

[0073] Where examples and preparations cite analytical data, one of the following analytical methods were used unless otherwise specified.

[0074] NMR: 400 MHz Bruker Avance III and Bruker Avance Neo.

[0075] LC-MS or HPLC Method:

[0076] Method 1:

[0077] MS instrument type: SHIMADZU LC-MS-2020, Column: Kinetex EVO C18 30×2.1 mm, 5 mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 0% B.fwdarw.0.8 min 60% B.fwdarw.1.20 min 60% B.fwdarw.1.21 min 0% B.fwdarw.1.55 min 0% B flow rate: 1.5 mL/min, oven temperature: 50° C.; PDA detection: 220 nm & 254 nm.

[0078] Method 2:

[0079] MS instrument type: Agilent 1200 LC/G1956A MSD, Column: Kinetex EVO C18 2.1×30 mm, 5 um, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 90% B.fwdarw.0.35 min 90% B flow rate: 1.5 mL/min, oven temperature: 50° C.; DAD: 100-1000.

[0080] Method 3:

[0081] HPLC instrument type: SHIMADZU LC-20AB, Column: Kinetex C18 LC Column 4.6×50 mm, 5 μm, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 0% B.fwdarw.4.20 min 60% B.fwdarw.5.30 min 60% B.fwdarw.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&254 nm).

[0082] Method 4:

[0083] MS instrument type: SHIMADZU LC-MS-2020, Column: Kinetex EVO C18 30×2.1 mm, 5 μm, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 0% B.fwdarw.3.0 min 60% B.fwdarw.3.50 min 60% B.fwdarw.3.51 min 0% B.fwdarw.4.00 min 0% B flow rate: 0.8 mL/min, oven temperature: 50° C.; PDA detection: 220 nm & 254 nm.

[0084] Method 5:

[0085] 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 (v/v), B: Acetonitrile, gradient: 0.0 min 0% B.fwdarw.0.8 min 60% B.fwdarw.1.20 min 60% B.fwdarw.1.21 min 0% B.fwdarw.1.55 min 0% B flow rate: 1.5 mL/min, oven temperature: 40° C.; PDA detection: 220 nm & 254 nm.

[0086] Method 6:

[0087] HPLC instrument type: SHIMADZU LC-20AB, Column: Kinetex C18 LC Column 4.6×50 mm, 5 μm, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 0% B.fwdarw.4.20 min 30% B.fwdarw.5.30 min 30% B.fwdarw.5.31 min 0% B.fwdarw.6.00 min 0% B, flow rate: 1.5 mL/min, oven temperature: 50° C.; PDA detection: PDA (220 nm&215 nm&254 nm).

[0088] Method 7:

[0089] MS instrument type: SHIMADZU LC-20AB, Column: Kinetex C18 LC Column 4.6×50 mm, 5 μm, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 0% B.fwdarw.2.40 min 30% B.fwdarw.3.70 min 30% B.fwdarw.3.71 min 0% B.fwdarw.4.00 min 0% B flow rate: 1 mL/min, oven temperature: 50° C.; PDA detection: 220 nm & 254 nm.

[0090] Method 8:

[0091] 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 in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 0% B.fwdarw.5.00 min 30% B.fwdarw.6.00 min 100% B.fwdarw.6.50 min 100% B.fwdarw.6.51 min 0% B.fwdarw.7.00 min 0% B flow rate: 1 mL/min, oven temperature: 40° C.; PDA detection: 220 nm & 254 nm.

[0092] Method 9

[0093] MS instrument type: SHIMADZU LCMS-2020, Column: Kinetex EVO C18 2.1×30 mm, 5 μm, mobile phase A: 0.025% NH.sub.3H.sub.2O in Water (v/v), B: Acetonitrile, gradient: 0.0 mins 5% B.fwdarw.0.8 mins 95% B.fwdarw.1.2 mins 95% B.fwdarw.1.21 mins 5% B.fwdarw.1.55 mins 5% B, flow rate: 1.5 mL/mins, oven temperature: 40° C.; UV detection: 220 nm & 254 nm.

[0094] Method 10

[0095] 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 in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 mins 0% B.fwdarw.5 mins 30% B.fwdarw.6 mins 100% B.fwdarw.6.5 mins 100% B.fwdarw.6.51 mins 0% B, flow rate: 0.6 mL/mins, oven temperature: 40° C.; UV detection: 220 nm & 254 nm.

[0096] HPLC Method 1:

[0097] MS instrument type: SHIMADZU LC-20AB, Column: XBridge® C18 3.5 μm 4.6×150 mm, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 mins 0% B.fwdarw.10.0 mins 60% B.fwdarw.15.0 mins 60% B.fwdarw.15.01 mins 0% B.fwdarw.15.02 mins 0% B.fwdarw.20.0 mins 0% B, flow rate: 1.0 mL/mins, oven temperature: 40° C.; UV detection: 220 nm &215 nm & 254 nm.

Abbreviations

[0098] Where the following abbreviations have been used, the following meanings apply:

[0099] ACN or MeCN is acetonitrile,

[0100] CDCl.sub.3 is deuterochloroform,

[0101] CSA is Camphor-10-sulfonic acid,

[0102] D.sub.2O is deuterium oxide,

[0103] DCM is dichloromethane,

[0104] DIPEA or DIEA is N,N-diisoproylethylamine,

[0105] DMAP is 4-(dimethylamino)pyridine,

[0106] DMSO is dimethyl sulfoxide,

[0107] EA is ethyl acetate,

[0108] EtOH is ethanol,

[0109] FA is formic acid,

[0110] H.sub.2O is water,

[0111] HCl is hydrochloric acid,

[0112] HPLC is high performance liquid chromatography,

[0113] IPA is isopropyl alcohol,

[0114] KHMDS is potassium bis(trimethylsilyl)amide,

[0115] KOH is potassium hydroxide,

[0116] LCMS is liquid chromatography mass spectrometry,

[0117] MeOH is methanol,

[0118] MTBE is methyl tert butyl ether,

[0119] N.sub.2 is nitrogen,

[0120] Na.sub.2SO.sub.4 is sodium sulfate;

[0121] NH.sub.3 is ammonia,

[0122] NH.sub.4HCO.sub.3 is ammonium bicarbonate,

[0123] NMR is nuclear magnetic resonance,

[0124] PDA is photodiode array detector,

[0125] SFC is supercritical fluid chromatography,

[0126] TBTU is 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate,

[0127] TEA is triethylamine,

[0128] TFA is trifluoroacetic acid,

[0129] THF is tetrahydrofuran and

[0130] TLC is thin layer chromatography.

Preparations

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

[0131] ##STR00017##

[0132] To a solution of (3S)-quinuclidin-3-ol (2.00 g, 15.73 mmol, 1.00 eq), DMAP (19.21 mg, 157.30 μmol, 0.01 eq) and TEA (4.78 g, 47.19 mmol, 6.54 mL, 3.00 eq) in DCM (40.00 mL) was added 4-bromobenzenesulfonyl chloride (6.03 g, 23.60 mmol, 1.50 eq) at 0° C. The mixture was stirred for 16 hours at 20° C. The mixture was washed with sat. NaHCO.sub.3 (100 mL). The sat. NaHCO.sub.3 layer was extracted with EA (100 mL×2). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to give a crude product as a yellow oil. The yellow oil was purified by chromatography on silica gel eluted 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, which was analyzed by .sup.1HNMR.

[0133] .sup.1H NMR: (400 MHz, CDCl.sub.3) 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).

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

[0134] ##STR00018##

[0135] To a solution of (3 S)-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 N.sub.2 and the reaction was stirred at 65° C. for 12 hours. 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 layer was washed with saturated brine (2×500 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude mixture (113 g) was obtained as a dark brown oil and used directly in the next step.

[0136] Selected NMR data of crude material showed dr (R,R) (R,S)=2.3:1

[0137] .sup.1H-NMR: 400 MHz, DMSO-d.sub.6: crude selected δ ppm 4.08 (d, J=8.8 Hz, 2H), 3.95 (d, J=10.2 Hz, 1H).

Preparation of 5. 2 (+)-CSA salt (R,R)

(R)-methyl 2-amino-2-((3R)-quinuclidin-3-yl)acetate bis(((1 S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methanesulfonate)

[0138] ##STR00019##

[0139] To a solution of the crude reaction mixture of 4A and 4B (105 g, 177 mmol) in IPA (700 mL) was added H.sub.2O (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 continued stirring at 45° C. for 12 hrs. The reaction mixture was cooled to 25° C. and filtered to obtain a white solid. The solid was washed with IPA (100 mL) and MTBE (100 mL) and dried under vacuum to obtain the title compound as a white solid (62.0 g, 93.5 mmol, 52.9% yield).

[0140] .sup.1H-NMR: 400 MHz, DMSO-d.sub.6: 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).

Confirmation of Stereochemistry for Compound 5. 2 (+)-CSA salt (R,R)

[0141] 20 mg compound 4A was dissolved in 1.3 mL dichloromethane/cyclohexane/methanol (5:5:3). The solution was kept in a half sealed 4 mL vial and evaporated slowly at room temperature. Crystals were observed in the second day and a crystal was selected for X-ray crystallographic analysis.

[0142] The crystal was a colorless needle with the following dimensions: 0.10×0.02×0.02 mm3. The symmetry of the crystal structure was assigned the monoclinic space group P21 with the following parameters: a=7.0236(2) Å, b=26.8204(6) Å, c=18.0068(5) Å, α=90°, μ=99.114(3°), γ=90°, V=3349.22(16) Å.sup.3, Z=4, Dc=1.315 g/cm.sup.3, F(000)=1424.0, μ(Cu Kα)=1.918 mm.sup.−1 and T=293(2) K using a Rigaku Oxford Diffraction XtaLAB Synergy four-circle diffractometer equipped with a HyPix-6000HE area detector. Cryogenic system: Oxford Cryostream 800 Cu: λ=1.54184 Å, 50 W, Micro focus source with multilayer mirror (μ-CMF). Distance from the crystal to the CCD detector: d=35 mm Tube Voltage: 50 kV Tube Current: 1 mA.

[0143] The absolute configuration of 5. 2 (+)-CSA salt was assigned (R,R).

[0144] Alternatively, compound 4A (R,R) may be isolated according to the following method:

##STR00020##

[0145] 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 afford a mixture of diastereomers. The diastereomers were resolved by chiral prep-SFC (column: DAICEL CHIRALPAK IG (250 mm×50 mm, 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O EtOH]; B %: 45%; 320 min) to afford the two diastereomers of compound 4A (6.00 g, 16.5 mmol) as a brown oil and compound 4B (RS) (9.00 g, 24.8 mmol) as a brown oil.

Diastereomer 1 (RR): (R)-methyl 2-((diphenylmethylene)amino)-2-((3R)-quinuclidin-3-yl)acetate

[0146] .sup.1H-NMR 400 MHz (DMSO-d.sub.6): 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).

[0147] SFC: Rt=1.633 min, 100%

Diastereomer 2 (RS): (S)-methyl 2-((diphenylmethylene)amino)-2-((3R)-quinuclidin-3-yl)acetate

[0148] .sup.1H-NMR 400 MHz (DMSO-d.sub.6): 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).

[0149] SFC: Rt=1.854 min, 100%

[0150] Alternatively, compounds 4A and 4B may be separated using preparative TLC as described below:

[0151] A mixture of 4A and 4B were purified by Preparative TLC (EtOAc:MeOH (NH.sub.3,7M)=10:1) to give 202.25 mg of 4A (91.7% purity, 98.6% ee.) as a yellow solid, 114.50 mg of 4B (97.7% purity, 94.3% ee.) as a yellow oil.

[0152] LCMS MS m/z 363.2 [M+H].sup.+

[0153] 4 A: .sup.1H NMR: (400 MHz, CDCl.sub.3): 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, 7.4 Hz, 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).

[0154] 4B: .sup.1H NMR: (400 MHz, CDCl.sub.3): 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, 6.4 Hz, 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)

[0155] Alternatively, the HCl salt of compound 5 may be obtained by following the procedure below: To a solution of the above-prepared 4A stereoisomer (390.00 mg, 1.08 mmol) in THF (6 mL) was added HCl (12 M (aq), 780.09 μL, 37% purity) at 0° C. The reaction mixture was stirred for 1 hour at 0° C. The mixture was concentrated to remove THF. To the residue was added methyl tertiary 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, 2HCl salt) as a yellow solid.

Preparation of Compound 5 Free Parent from Compound 5 CSA Salt

(R)-methyl 2-amino-2-((3R)-quinuclidin-3-yl)acetate

[0156] ##STR00021##

[0157] 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 (+) Camphorsulfonic acid salt (Preparation 1, 47.0 g, 70.9 mmol) and the mixture was stirred at 20° C. under N.sub.2 for 1 hour. The reaction mixture was filtered and concentrated in vacuo to afford the title compound (11.0 g, 55.5 mmol, 78.3% yield) as a yellow oil.

[0158] .sup.1H-NMR 400 MHz (DMSO-d.sub.6): 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).

[0159] The hydrochloride salt of compound 5 may also be converted to the free parent using the Ambersep 900 method described above.

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)

[0160] ##STR00022##

[0161] Step 1

[0162] To a 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) was added TBTU (88.2 g, 275 mmol) under N.sub.2, followed by DIEA (65.3 g, 505 mmol, 88.0 mL). The reaction was stirred at 25° C. for 12 hrs. The reaction mixture was concentrated in vacuo to afford a crude yellow oil that was taken directly on to the next step.

[0163] Step 2

[0164] 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) and the reaction was stirred at 50° C. under N.sub.2 for 1 hour. 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 hours. The resultant white solid was filtered and stirred in water (740 mL) at 90° C. for 2 hours before cooling to 25° C. The solid was filtered, washed with water (2×300 mL) and dried under vacuum to afford the title compound as a white solid (31.4 g, 48.6 mmol, 39.5% yield).

[0165] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

[0166] LCMS (Method 1): Rt=2.275 min, MS m/z [M+H].sup.+ 499.4, theoretical mass: 498.6

[0167] HPLC (Method 1): Rt=3.908 min, 99.7%

[0168] Elemental Analysis: C, 45.89%; H, 7.96%; N, 8.18%, theoretical+10H.sub.2O: C, 46.01%; H, 8.02%; N, 8.25%.

[0169] 15 mg compound 4A was dissolved in 1.2 ml ethanol/H.sub.2O (1:1) at 60° C. The solution was filtered through 0.45 μm microporous filter and kept in a sealed 4 ml vial at room temperature. Needle crystals were observed in the solution and a crystal selected for X-ray crystallographic analysis

[0170] The crystal was a colorless needle with the following dimensions: 0.30×0.04×0.04 mm3. The symmetry of the crystal structure was assigned the orthorhombic space group C222.sub.1 with the following parameters: 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 3, Z=4, Dc=1.280 g/cm 3, F(000)=1424.0, μ(Cu Kα)=0.889 mm.sup.−1, and T=110(14) K using Rigaku Oxford Diffraction XtaLAB Synergy four-circle diffractometer equipped with a HyPix-6000HE area detector. Cryogenic system: Oxford Cryostream 800 Cu: λ=1.54184 Å, 50 W, Micro focus source with multilayer mirror (μ-CMF). Distance from the crystal to the CCD detector: d=35 mm Tube Voltage: 50 kV Tube Current: 1 mA.

[0171] The absolute configuration of Example 1 was assigned (R,R,R,R)

Preparation of Example 1 HCl Salt

[0172] To the suspension of APL-2191 (30.9 g, 45.5 mmol, 1 eq, 10H.sub.2O) in H.sub.2O (760 mL) and EtOH (760 mL) was added HCl (12 M, 7.61 mL, 2.01 eq) at 25° C. and stirred for 12 hr. The reaction mixture was concentrated under vacuum. APL-2191.2HCl (28.2 g, 39.0 mmol, 85.7% yield, 10H.sub.2O) was obtained as a crystalline off-white solid.

[0173] LCMS (Method 1): Rt=2.300 min, MS m/z 250.1 [M+H/2].sup.+

[0174] HPLC (Method 2): Rt=3.889 min, 99.3%

[0175] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

[0176] Example 1 may also be prepared according to the following procedure:

[0177] Step 1

[0178] To a solution of (R)-methyl 2-amino-2-((3R)-quinuclidin-3-yl)acetate (100.00 mg, 504.39 μmol) in CHCl3 (8.00 mL) was added benzene-1,4-dicarbonyl chloride (51.20 mg, 252.19 μmol, 0.50 eq) at 30° C. The mixture was stirred for 16 hours at 30° C. The mixture was concentrated to give a crude product as a white solid (180.00 mg, crude, HCl salt).

[0179] LCMS: MS m/z 527.5 [M+H].sup.+

[0180] Step 2

[0181] To a solution of the bis methyl ester (180.00 mg, 341.80 μmol) in THF (2.00 mL) was added a solution of LiOH (48.00 mg, 2.00 mmol) in water (2 mL) at 25° C. The mixture was stirred for 1 hour at 25° C. The mixture was concentrated under reduced pressure to remove THF. To the mixture was added 1M HCl (aq) 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.

[0182] .sup.1H NMR 400 MHz (D.sub.2O): 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).

[0183] LCMS: Rt=5.91, MS m/z 501.1 [M+H].sup.+, theoretical mass: 500.2

[0184] The following Examples were prepared using the same procedure as described for Example 1 (see General method below) using the appropriate dicarboxylic acid as described for each Example, and compound 5 (R,R). The Examples were purified as individually described for Step 1 and Step 2.

General Method for Examples 2-11

[0185] ##STR00023##

[0186] Step 1

[0187] To a solution of compound 5 (2.70 eq) in ACN (10 V) was added TBTU (2.23 eq) and the appropriate carboxylic acid (1 eq) at 20° C. under nitrogen. DIEA (4.11 eq) was added to the mixture, and the mixture was stirred at 20° C. for 6 hrs under N.sub.2. The reaction mixture was concentrated under vacuum and purified as described for each Example.

[0188] Step 2

[0189] To a solution of the bis-methyl esters (1.00 eq) in IPA (20.0 V) was added aqueous KOH (10.0%, 10.0 eq) at 20° C. The mixture was stirred at 50° C. for 1 hr, cooled to room temperature and purified as described for each Example.

Example 2 APL-6968

(R,2R,2′R)-2,2′-((pyridine-2,5-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin yl)acetic acid)

[0190] ##STR00024##

[0191] Example 2 was prepared according to the General Method using pyridine-2,5-dicarboxylic acid.

[0192] Step 1

[0193] The residue was purified by prep-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 bis methyl ester (330 mg, 524 μmol, 43.8% yield, 91.2% purity, FA) as a white solid.

[0194] .sup.1H-NMR 400 MHz (CDCl.sub.3): 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).

[0195] LCMS (Method 1) Rt=0.685 min, MS m/z [M+H].sup.+ 528.2

[0196] Step 2

[0197] The residue was purified by prep-HPLC (column: Waters Atlantis T3 150×30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B %: 1%-20%, 10 min) to afford Example 2 (74.0 mg, 132 μmol, 97.4% purity, FA) as a white solid.

[0198] MS (Method 8): MS m/z 499.9 [M+H].sup.+, theoretical mass: 499.2

[0199] HPLC (Method 1): Rt=2.31 min

[0200] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

Example 3 APL-6969

(R,2R,2′R)-2,2′-((pyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin yl)acetic acid)

[0201] ##STR00025##

[0202] Example 3 was prepared according to the General Method using pyrazine-2,5-dicarboxylic acid.

[0203] Step 1

[0204] The residue was purified by prep-HPLC (column: Waters Xbridge 150×25 mm, 5 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B %: 14%-44%, 9 min) to give the bis methyl ester (90.0 mg, 124 μmol, 10.4% yield, 72.7% purity) as a white solid.

[0205] LCMS (Method 1): Rt=0.704 min, MS m/z 529.3 [M+H].sup.+

[0206] Step 2

[0207] The mixture was filtered, FA (aq, 20% in water) was added to adjust the mixture to pH=7-8 and the mixture was purified by prep-HPLC (column: Waters Xbridge 150×25 mm, 5 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B %: 1%-10%, 9 min) to give Example 3 (51.0 mg, 98.0 μmol, 57.5% yield, 96.0% purity) as a white solid.

[0208] MS (Method 2): MS [M+H].sup.+ 501.1, theoretical mass: 500.2

[0209] HPLC (Method 3): Rt=0.824 min

[0210] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

Example 4 APL-6970

(R,2R,2′R)-2,2′-((pyridazine-3,6-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)

[0211] ##STR00026##

[0212] Example 4 was prepared according to the General Method using pyridazine-3,6-dicarboxylic acid.

[0213] Step 1

[0214] The residue was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30 mm 3 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; 13%: 5%-35%, 8 min) to give the bis methyl ester (110 mg, 144 μmol, 22.0% yield, 81.1% purity) as a white solid.

[0215] .sup.1H-NMR 400 MHz (CDCl.sub.3): 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).

[0216] LCMS (Method 1): Rt=0.807 min, MS m/z 617.3

[0217] Step 2

[0218] The residue was purified by prep-HPLC (column: Waters Atlantis T3 150×30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B %: 1%-20%, 10 min) to give Example 4 (87.0 mg, 154 μmol, 32.7% yield, 97.3% purity, FA) as a white solid.

[0219] LCMS (Method 8): Rt=2.296 min, MS m/z 501.4 [M+H].sup.+, theoretical mass: 500.3

[0220] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

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

[0221] ##STR00027##

[0222] Example 5 was prepared according to the General Method using [1,1′-biphenyl]-4,4′-dicarboxylic acid.

[0223] Step 1

[0224] The crude material was obtained as a colorless liquid and taken on directly to the next step.

[0225] LCMS (Method 1) Rt=0.789 min, MS m/z 603.4 [M+H].sup.P

[0226] Step 2

[0227] The mixture was filtered, and the FA (aq, 20% in water) was added the mixture adjust to pH=7-8. Purified by prep-HPLC (column: Waters Xbridge 15×25 mm, 5 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B %: 1%-10%, 9 min) to give Example 5 (37.0 mg, 63.7 μmol, 15.5% yield, 99.0% purity) as a white solid.

[0228] LCMS (Method 4): Rt=1.43 min, MS m/z 575.3 [M+H].sup.+, theoretical mass: 574.2

[0229] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

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

[0230] ##STR00028##

[0231] Example 6 was prepared according to the General Method using 2-methyl-[1,1′-biphenyl]-4,4′-dicarboxylic acid.

[0232] Step 1

[0233] The residue was purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30 mm, 3 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B %: 5%-35%, 8 min) to give the bis ester (110 mg, 144 μmol, 22.0% yield, 81.1% purity) as a white solid.

[0234] LC-MS (Method 1): Rt=0.807 min, MS m/z 617.3 [M+H].sup.+

[0235] .sup.1H-NMR 400 MHz (CDCl.sub.3): 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).

[0236] Step 2

[0237] The residue was purified by prep-HPLC (column: Waters Atlantis T3 150×30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B %: 1%-20%, 10 min) to give Example 6 (FA salt, 16 mg, 5.44 μmol, 4.13% yield, 95.0% purity) as a white solid.

[0238] LCMS (Method 4): Rt=1.597 min, MS m/z 589.3 [M+H].sup.+, theoretical mass: 588.3

[0239] .sup.1H-NMR 400 MHz (D.sub.2O+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).

Example 7 APL-6973

(R,2R,2′R)-2,2′-((naphthalene-2,6-dicarbonyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)

[0240] ##STR00029##

[0241] Example 7 was prepared according to General Method 1 using naphthalene-2,6-dicarboxylic acid.

[0242] Step 1

[0243] The residue was purified by prep-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 bis ester (160 mg, 277 μmol, 60.0% yield) as a white solid.

[0244] LC-MS (Method 1): Rt=0.770 min, MS m/z [M+H].sup.+ 577.4

[0245] Step 2

[0246] The mixture was filtered, and the FA (aq, 20% in water) was added the mixture adjust to pH 7-8. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 mm*5 um; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B %: 1%-10%, 9 min) to give Example 7 (35.0 mg, 62.0 μmol, 22.0% yield, 96.0% purity) as a white solid.

[0247] LCMS (Method 5): Rt=0.282 min, MS m/z 549.1 [M+H].sup.+, theoretical mass: 548.2

[0248] HPLC (Method 6): Rt=1.624 min

[0249] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

Example 8 APL-6974

(R,2R,2′R)-2,2′-(2,5-dimethylterephthaloyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)

[0250] ##STR00030##

[0251] Example 8 was prepared according to the General Method using 2,5-dimethylbenzene-1,4-dicarboxylic acid.

[0252] Step 1

[0253] The crude product was triturated with ACN (5 mL) and MeOH (3 mL) at 20° C. for 10 min and filtered to give the bis ester (114 mg, 185 μmol, 17.9% yield, 90.1% purity) as a white solid.

[0254] LCMS (Method 1): Rt=0.771 min, MS m/z 555.3 [M+H].sup.+,

[0255] .sup.1H-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).

[0256] Step 2

[0257] The residue was purified by prep-HPLC (column: Waters Atlantis T3 150×30 mm, 5 μm; mobile phase: [water (0.225% FA)-ACN]; B %: 1%-20%, 10 min) to give Example 8 (10.0 mg, 17.1 μmol, 9.50% yield, 98.1% purity, FA) as a white solid.

[0258] LCMS (Method 8): Rt=2.773 min, MS m/z 527.3 [M+H].sup.+, theoretical mass: 526.3

[0259] .sup.1H-NMR 400 MHz (D.sub.2O+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).

[0260] LCMS m/z 527.3 [M+H].sup.+, theoretical mass: 526.3, Rt=2.77 minutes, 100%

Example 9 APL-6975

(R,2R,2′R)-2,2′-(2-methylterephthaloyl)bis(azanediyl))bis(2-((R)-quinuclidin-3-yl)acetic acid)

[0261] ##STR00031##

[0262] Example 9 was prepared according to the General Method using 2-methylbenzene-1,4-dicarboxylic acid.

[0263] Step 1

[0264] The residue was purified by prep-HPLC (column: Phenomenex luna C18 150×25 mm, 10 μm; mobile phase: [water (0.225% FA)-ACN]; B %: 0%-20%, 10 min) to give the bis ester (500 mg, 647 μmol, 23.3% yield, 70.0% purity) as a white solid.

[0265] LCMS (Method 1): Rt=0.707 min, MS m/z 541.2 [M+H].sup.+

[0266] Step 2

[0267] The mixture was filtered, and FA (aq, 20% in water) was added the mixture adjust to pH 7-8. The mixture was purified by prep-HPLC (column: Waters Xbridge 150×25 mm, 5 μm; mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; 13%: 1%-10%, 9 min) to give Example 9 (116 mg, 202.26 μmol, 54.67% yield, 97.4% purity, FA) as a white solid.

[0268] LCMS (Method 8): Rt=2.353 min, MS m/z 513.0 [M+H].sup.+, theoretical mass: 512.3

[0269] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

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)

[0270] ##STR00032##

[0271] Example 10 was prepared according to the General Method using 2,5-bis(benzyloxy)benzene-1,4-dicarboxylic acid.

[0272] Step 1

[0273] The residue was purified by prep-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 bis ester (180 mg, 211 μmol, 39.9% yield, 92.1% purity, FA) as a white solid.

[0274] .sup.1H-NMR 400 MHz (CDCl.sub.3): 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).

[0275] Step 2

[0276] The residue was purified by prep-HPLC (column: Phenomenex luna C18 150×25 mm, 10 μm; mobile phase: [water (0.225% FA)-ACN]; B %: 1%-30%, 10 min) to give Example 10 (67.0 mg, 87.6 μmol, 40.4% yield, 99.0% purity, FA) as a white solid.

[0277] LCMS (Method 1): Rt=0.799 min, MS m/z 711.3 [M+H].sup.+, theoretical mass: 710.33

[0278] HPLC (method 7), Rt=2.519 min.

[0279] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

Example 11 P2B-E (No APL Number)

2-[[3-[[carboxy-1(3R)-quinuclidin-3-yl]methyl]carbamoyl] benzoyl amino]-2-[(3R)-quinuclidin-3-yl]acetic acid

[0280] ##STR00033##

[0281] Step 1

[0282] To a solution of methyl 2-amino-2-[(3R)-quinuclidin-3-yl]acetate (100.00 mg, 504.39 μmol) in CHCl.sub.3 (4 mL) was added benzene-1,3-dicarbonyl chloride (51.20 mg, 252.20 μmol) at 30° C. The mixture was stirred for 16 hours at 30° C. The mixture was concentrated under reduced pressure to give a crude product as a yellow solid.

[0283] LCMS: Rt=0.881 min, MS m/z 527.3 [M+H].sup.+

[0284] Step 2

[0285] To a solution of the bis methyl ester (165.00 mg, 313.31 μmol) in THF (4 mL) was added LiOH (96.00 mg, 4.01 mmol, 12.79) in H.sub.2O (4 mL) at 30° C. The mixture was stirred for 2 hours at 30° C. The mixture was concentrated under reduced pressure to remove THF. To the residue was added water (10 mL) and 1M HCl (aq) to pH=2. The mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by preparative HPLC to afford Example 11 (37.20 mg, 63.79 μmol, 40.72% yield, 98% purity, 2HCl) as a white solid.

[0286] .sup.1H-NMR 400 MHz (D.sub.2O): 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).

[0287] LCMS Rt=5.9 min, MS m/z=499.3 [M+H].sup.+, theoretical mass: 498

[0288] Biological Assays

[0289] MIRA Immunoturbidimetric Assay

[0290] The CRP immunoturbidimetric assay on the Roche COBAS MIRA Plus autoanalyser, utilises two different sized latex particles that are covalently coupled with two different monoclonal antibodies with specificity for different CRP epitopes (5). The assay was validated by Roche for measurement of native pentameric CRP, for which it has high sensitivity and specificity and a high upper detection limit; it was calibrated against a standard produced in our laboratory. Serendipitously, one of the assay's antibodies binds to an epitope present on the ligand binding B face of CRP. Thus, when the binding pocket is occupied by ligand or is occluded, for example by B face to B face complexing of pentamers, the assay fails to detect CRP although it is demonstrable by other types of assays that employ antibodies which bind to different epitopes. Bivalent compounds such as BPC8 and APL-2191 were designed to crosslink pairs of CRP pentamers. Therefore, inhibition of CRP recognition in the MIRA assay is a convenient tool to monitor the efficacy and potency of complex formation between such ligands and CRP (6).

[0291] CRP concentrations were measured in the presence and absence of ligands by the COBAS MIRA autoanalyser. Concentrated Tris-calcium buffer (×10 TC) was prepared in MilliQ water from trishydroxymethyamine (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 tenfold diluted working buffer (TC) was prepared by dilution 100 ml of the ×10 concentrated buffer with 900 ml of MilliQ water. Human CRP was isolated, purified and characterised as previously reported (6-9) and stored frozen at −80° C. When required, stock CRP was thawed at 37° C. and working dilutions prepared that were kept at 4° C. for the duration of an experiment. CRP concentration was determined spectrophotometrically (Beckman Coulter DU 650) in quartz cuvettes with a 1 cm light path, by measuring A.sub.208 after correction for absorbance at 320 nm (light scattering) and using the measured absorption coefficient A (1%, 1 cm)=17.5 for human CRP (10). Human CRP at ˜90 μg/ml (0.78 μM of pentamer) in TC buffer was prepared from a stock solution; a 75 μl aliquot was used in the assay. Compounds were supplied by Wuxi AppTec (Wuhan, China) as solids. They were dissolved in TC buffer at suitable concentrations, depending on solubility, of up to 10 mM (labelled S1). They were then serially diluted 1:2 with TC buffer (100 μl ligand+200 μl TC) to provide up to 9 dilutions, S2-S10. A TC buffer control (50) was included in each assay. A 15 μl volume of each ligand solution was incubated with 75 μl of CRP for 1 h at room temperature. The final concentrations were 0.73 μM native pentameric CRP, ligands S1-S10=625-0.03 μM, corresponding to ligand:CRPr ratios of 850-0.04. Where compounds were of reduced solubility in TC buffer, lower stock concentrations were used (from 0.6 mM), corresponding to final top assay concentrations of 100 μM.

[0292] Data are expressed as measured CRP (mg/L) against final total ligand concentration (μM) and were plotted using Sigmaplot (V14) using the 4 parameter logistic curve y=min+(max−min)/(1+(x/EC.sub.50).Math.Hill slope) to calculate EC.sub.50. Where appropriate, samples were also measured in whole normal human serum following the addition of a known amount of human CRP. All compounds were assayed in comparison with a highly purified preparation of bis(phosphocholine)octane (BPC8), that was prepared by Carbogen AMCIS AG and diluted into sterile water at 10 mM concentration. It was stored at −80° C. The solution was diluted into TC buffer as required.

[0293] Table 1 shows the data for the MIRA immunoturbidimetric assay for Examples 1-12

TABLE-US-00001 TABLE 1 Example No. Compound ID IC50 (μM) 1 APL-2191 0.60 2 APL-6968 0.65 3 APL-6969 1.22 4 APL-6970 0.58 5 APL-6971 0.73 6 APL-6972 0.56 7 APL-6973 0.51 8 APL-6974 0.59 9 APL-6975 0.55 10 APL-6976 0.67 11 P2B-E 2.02 12 BPC8 1.2

[0294] The Examples of formula (I) are RR,RR stereoisomers. The other stereoisomers of this structure have lesser or no activity. The SS,SS isomer is the most active alternative isomer (denoted QA,QA Quinuclidine, Amino Acid: SS,SS IC50 34.4 μM, RS,RS IC50>1000 μM, SR,SR IC50>1000 μM, RS,RR>1000 μM). Alternative isomers may be prepared by one skilled in the art according to the methods above using the desired stereoisomers with a suitable protecting group strategy employed.

[0295] Each document cited in this text (“application cited documents”) and each document cited or referenced in each of the application cited documents, and any manufacturer's specifications or instructions for any products mentioned in this text and in any document incorporated into this text, are hereby incorporated herein by reference; and, technology in each of the documents incorporated herein by reference can be used in the practice of this invention.

[0296] The present invention claims priority from United Kingdom Patent Application GB2002299.2 filed on 19 Feb. 2020, the entire content of which is also expressly incorporated herein by reference.

REFERENCES

[0297] 1. Pepys M. B. (2018) “The Pentraxins 1975-2018: Serendipity, Diagnostics and Drugs.” Front. Immunol. 9: 2382. doi: 10.3389/fimmu.2018.02382. [0298] 2. Griselli, M. Herbert, J., Hutchinson, W. L., Taylor, K. M., Sohail, M., Krausz, T. and Pepys, M. B., (1999) “C-reactive protein and complement are important mediators of tissue damage in acute myocardial infarction.” J. Exp. Med. 190: 1733-1739. [0299] 3. Gill, R., Kemp, J. A., Sabin, C. and Pepys, MB (2004) “Human C-Reactive Protein Increases Cerebral Infarct size after middle cerebral artery occlusion in adult rats.” J. Cereb. Blood Flow Metab. 24: 1214-1218. [0300] 4. Pepys, M. B., Hirschfield, G. M., Tennent, G. A., Gallimore, J. R., Kahan, M. C., Bellotti, V., Hawkins, P. N., Myers, R. M., Smith, M. D., Polara, A., Cobb, A. J. A., Ley, S. V., Aquilina, J. A., Robinson, C. V., Sharif, I., Gray, G. A., Sabin, C. A., Jenvey, M. C., Kolstoe, S. E., Thompson, D. and Wood, S. P. (2006) “Targeting C-reactive protein for the treatment of cardiovascular disease.” Nature 440: 1217-1221. [0301] 5. Eda, S., Kaufmann, J., Roos, W. and Pohl, S. (1998) “Development of a new microparticle-enhanced turbidometric assay for C-reactive protein with superior features in analytical sensitivity and dynamic range.” J. Clin. Lab. Anal. 12: 137-144 [0302] 6. Pepys, M. B., Dash, A. C. and Ashley, J. (1977) “Isolation of C-reactive protein by affinity chromatography.” Clin. Exp. Immunol. 30: 32-37. [0303] 7. de Beer, F. C. and Pepys, M. B. (1982) “Isolation of human C-reactive protein and serum amyloid P component.” J. Immunol. Methods 50: 17-31. [0304] 8. Carlucci, F., Cook, H. T., Garg, A., Pepys, M. B. and Botto, M. (2010) “Lack of effect of a single injection of human C-reactive protein on murine lupus or nephrotoxic nephritis.” Arthritis Rheum. 62: 245-249. [0305] 9. Pepys, M. B., Gallimore, J. R., Lloyd, J., Li, Z., Graham, D., Taylor, G. W., Ellmerich, S., Mangione, P. P., Tennent, G. A., Hutchinson, W. L., Millar, D. J., Bennett, G., More, J., Evans, D., Misty, Y., Poole, S. and Hawkins, P. N. (2012) “Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C-reactive protein, for clinical use.” J. Immunol. Methods 384: 92-102. [0306] 10. Nelson, S. R., Tennent, G. A., Sethi, D., Gower, P. E., Ballardie, F. W., Amatayakul-Chantler, S. and Pepys, M. B. (1991) “Serum amyloid P component in chronic renal failure and dialysis.” Clin. Chim. Acta 200(2-3): 191-200.