WO2022178594A1

WO2022178594A1 - C-reactive protein inhibitors

Info

Publication number: WO2022178594A1
Application number: PCT/AU2022/050156
Authority: WO
Inventors: Karlheinz Peter; Guy Krippner; Michael Parker; Tracy Nero; Steffen Eisenhardt; Johannes Zeller; Craig James Morton
Original assignee: Baker Heart and Diabetes Institute; The University Of Melbourne; Albert-Ludwigs-Universitaet Freiburg
Priority date: 2021-02-25
Filing date: 2022-02-25
Publication date: 2022-09-01

Abstract

The present disclosure relates to functionalized amino compounds that can provide inhibitors of C-reactive protein (CRP). The present disclosure also relates to pharmaceutical compositions containing such compounds, methods for using such compounds in the treatment of CRP associated diseases, disorders or conditions, and particularly the treatment of inflammation associated with pro-inflammatory effects of CRP, and to related uses.

Description

C-REACTIVE PROTEIN INHIBITORS

FIELD

BACKGROUND

Inflammation arises through various diseases, disorders and conditions, and a large number of treatments are available for reducing inflammation. Ischemia/reperfusion injury (IRI) is an inflammatory response that occurs when tissue is reperfused following a prolonged period of ischemia. A primary mechanism of this IRI inflammation is often overshooting leukocyte activation, complement activation, and generation of reactive oxygen species (ROS) that lead to the release of pro-inflammatory cytokines and increased vascular permeability and consequently result in tissue damage. IRI is inevitable in many clinical situations such as organ (e.g. renal) transplantation, vascular surgery, acute ischemic renal injury, and delayed graft function. Due to the relatively limited knowledge of the pathophysiology, there are very limited treatments available for this devastating clinical condition.

The present inventors have recently identified pro-inflammatory effects of CRP associated with inflammatory response. Accordingly, there remains a need to develop safe and efficacious inihbitors of CRP for controlling associated inflammation.

It will be understood that any prior art publications referred to herein do not constitute an admission that any of these documents form part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY

The present inventors have undertaken extensive research into the development of inhibitors of CRP and have surprisingly identified functionalized amino compounds effective for modulating or inhibiting CRP associated inflammatory effects.

Accordingly, in one aspect the present disclosure provides an amino compound of Formula 1 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

Formula 1 wherein

X¹ is selected from -SO₃H, -PO₃H, and -CO2H;

L¹ is selected from -CH2-CH2-, -CH2-CH2-CH2-, -NH-CH2-CH2-, -S-CH2-CH2-, -O-CH2-CH2-, -CH2-CH2-CH2-CH2-, -NH-CH2-CH2-CH2-, -S-CH2-CH2-CH2-, and -O-CH2-CH2-CH2-; and

R¹ and R² are each independently selected from C4-2oalkyl, Ci-2oalkylaryl, and Ci- 2oalkylhetaryl.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of Formula 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any aspects, embodiments or examples as described herein, and a pharmaceutically acceptable excipient.

In another aspect, the present disclosure provides a method of controlling C-reactive protein levels in a subject comprising administering to the subject an effective amount of a compound of Formula 1, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any aspects, embodiments or examples as described herein. The controlling of C- reactive protein levels in a subject can be by modulating or inhibiting the pro-inflammatory actions of CRP, and in particular bioactive conformations of pentameric C-reactive protein (pCRP*) or monomeric C-reactive protein (mCRP).

In another aspect, the present disclosure provides a method of preventing or treating a C-reactive protein associated disease, disorder or condition, comprising administering to a subject in need of treatment thereof an effective amount of a compound of Formula 1, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any aspects, embodiments or examples thereof as described herein.

In another aspect, the present disclosure provides the use of a compound of Formula 1, salt, solvate or stereoisomer, or of the pharmaceutical composition, according to any aspects, embodiments or examples thereof as described herein, in the manufacture of a medicament for modulating or inhibiting C-reactive protein levels in a subject.

In some embodiments, the C-reactive protein associated disease, disorder or condition, is an ischemia-reperfusion injury including myocardial infarction, stroke, organ or vascular transplants, surgery or injuries, bums, allogenic transplantation, atherosclerotic disease, pericarditis, miocarditis, autoimmune diseases such as Crohn’s disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, or diseases associated with inflammation driven by protein misfolding such as amyloidoses, Alzheimer disease, prion diseases and age-related macular degeneration, or diseases associated with overshooting inflammation such as sepsis or COVID-19.

It will be appreciated that other aspects, embodiments and examples of the compounds, pharmaceutical compositions, methods, or uses, are further described herein.

DESCRIPTION General Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., chemistry, medicinal chemistry and the like).

As used herein, the term “and/or”, e.g., “X and/or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, of the designated value.

As used herein, singular forms “a”, “an” and “the” include plural aspects, unless the context clearly indicates otherwise.

Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein, the term “subject” refers to any organism susceptible to a disease or condition. For example, the subject can be a mammal, primate, livestock (e.g., sheep, cow, horse, pig), companion animal (e.g., dog, cat), or laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster). In one example, the subject is a mammal. In one embodiment, the subject is human. In one embodiment, the disease or condition is associated with inflammation.

As used herein, the term “treating” includes alleviation or reducing symptoms associated with a specific disorder or condition.

As used herein, the term “prevention” includes prophylaxis of the specific disorder or condition. For example, as used herein, the term “preventing inflammation” refers to preventing the onset or duration of the symptoms associated with inflammation. As would be understood by the person skilled in the art, a compound of Formula 1, Formula 2, or any salt, solvate or stereoisomer thereof would be administered in a therapeutically effective amount. The term “therapeutically effective amount”, as used herein, refers to a compound being administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated. The result can be the reduction and/or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. In one embodiment, the term “therapeutically effective amount” refers to a compound of Formula 1, Formula 2, or any salt thereof, being administered in an amount sufficient to control the levels of CRP in a subject, such as by modulating or inhibiting pro-inflammatory effects of CRP.

The compounds of the present disclosure may contain chiral (asymmetric) centers or the molecule as a whole may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure.

The following definitions apply to the terms as used throughout this specification, unless otherwise limited in specific instances.

As used herein, the term “halogen” means fluorine, chorine, bromine, or iodine.

The term "alkyl" whether used alone, or in compound words such as alkylaryl or alkylheteroaryl, represents a straight chain hydrocarbon (i.e. linear). The term “alkyl”, when used alone, represents a straight chain hydrocarbon ranging in size from 4 to about 20 carbon atoms. For example, the alkyl group may be a straight chain alkyl group selected from a C4- 2oalkyl or Gr-ioalkyl, such as butyl, pentyl, and hexyl groups. The term “alkyl”, when used in compound words such as alkylaryl or alkylheteroaryl, represents a straight chain hydrocarbon ranging in size from 1 to about 20 carbon atoms. For example, the alkylaryl or alkylheteroaryl group may contain a straight chain alkyl group selected from a Ci-2oalkyl, Ci-ioalkyl, C2- 2oalkyl, C2-ioalkyl, C3-ioalkyl, or C-r-ioalkyl, such as methyl, ethyl, propyl, butyl, pentyl, and hexyl groups.

The terms "carbocyclic" and "carbocyclyl" represent a monocyclic or polycyclic ring system wherein the ring atoms are all carbon atoms, e.g., of about 3 to about 20 carbon atoms, and which may be aromatic, non-aromatic, saturated, or unsaturated, and may be substituted and/or contain fused rings. Examples of such groups include aryl groups such as benzene, saturated groups such as cyclopentyl, or fully or partially hydrogenated phenyl, naphthyl and fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems. "Heterocyclyl" or "heterocyclic" whether used alone, or in compound words such as heterocyclyloxy, represents a monocyclic or polycyclic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulphur and oxygen, although may include other elements for ring atoms such as selenium, boron, phosphorus, bismuth and silicon, and wherein the ring system is about 3 to about 20 atoms, and which may be aromatic such as a “heteroaryl” group, non aromatic, saturated, or unsaturated, and may be substituted and/or contain fused rings. For example, the heterocyclyl may be (i) an optionally substituted cycloalkyl or cycloalkenyl group, e.g., of about 3 to about 20 ring members, which may contain one or more heteroatoms such as nitrogen, oxygen, or sulfur (examples include pyrrolidinyl, morpholino, thiomorpholino, or fully or partially hydrogenated thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, oxazinyl, thiazinyl, pyridyl and azepinyl); (ii) an optionally substituted partially saturated monocyclic or polycyclic ring system in which an aryl (or heteroaryl) ring and a heterocyclic group are fused together to form a cyclic structure (examples include chromanyl, dihydrobenzofuryl and indolinyl); or (iii) an optionally substituted fully or partially saturated polycyclic fused ring system that has one or more bridges (examples include quinuclidinyl and dihydro- 1,4-epoxynaphthyl). It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

As will be understood, an “aromatic” group means a cyclic group having 4m+2 p electrons, where m is an integer equal to or greater than 1. As used herein, "aromatic" is used interchangeably with "aryl" to refer to an aromatic group, regardless of the valency of aromatic group.

"Aryl" whether used alone, or in compound words such as arylalkyl represents: (i) an optionally substituted mono- or polycyclic aromatic carbocyclic moiety, e.g., of about 6 to about 20 carbon atoms, such as phenyl, naphthyl or fluorenyl; or, (ii) an optionally substituted partially saturated polycyclic carbocyclic aromatic ring system in which an aryl and a cycloalkyl or cycloalkenyl group are fused together to form a cyclic structure such as a tetrahydronaphthyl, indenyl ,indanyl or fluorene ring. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

A “hetaryl”, “heteroaryl” or heteroaromatic group, is an aromatic group or ring containing one or more heteroatoms, such as N, O, S, Se, Si or P. As used herein, "heteroaromatic" is used interchangeably with “hetaryl” or "heteroaryl", and a heteroaryl group refers to monovalent aromatic groups, bivalent aromatic groups and higher multivalency aromatic groups containing one or more heteroatoms. For example, "heteroaryl" whether used alone, or in compound words such as alkylheteroaryl represents: (i) an optionally substituted mono- or polycyclic aromatic organic moiety, e.g., of about 5 to about 20 ring members in which one or more of the ring members is/are element(s) other than carbon, for example nitrogen, oxygen, sulfur or silicon; the heteroatom(s) interrupting a carbocyclic ring structure and having a sufficient number of delocalized p electrons to provide aromatic character, provided that the rings do not contain adjacent oxygen and/or sulfur atoms. Typical 6-membered heteroaryl groups are pyrazinyl, pyridazinyl, pyrazolyl, pyridyl and pyrimidinyl. All regioisomers are contemplated, e.g., 2-pyridyl, 3-pyridyl and 4- pyridyl. Typical 5-membered heteroaryl rings are furyl, imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, pyrrolyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl, triazolyl, and silole. All regioisomers are contemplated, e.g., 2-thienyl and 3-thienyl. Bicyclic groups typically are benzo-fused ring systems derived from the heteroaryl groups named above, e.g., benzofuryl, benzimidazolyl, benzthiazolyl, indolyl, indolizinyl, isoquinolyl, quinazolinyl, quinolyl and benzothienyl; or, (ii) an optionally substituted partially saturated polycyclic heteroaryl ring system in which a heteroaryl and a cycloalkyl or cycloalkenyl group are fused together to form a cyclic structure such as a tetrahydroquinolyl or pyrindinyl ring. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

As used herein, the term “alkylaryl” refers to an alkyl group interrupted and/or substituted with at least one aryl group, where “alkyl” and “aryl” are as described above.

As used herein, the term “alkylhetaryl” refers to an alkyl group interrupted and/or substituted with at least one hetaryl group, where “alkyl” and “aryl” are as described above.

As used herein, the phrase “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts. Exemplary acid addition salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3- naphthoate)) salts. Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl -propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono-, di- or triethanolamine. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.

Those skilled in the art of organic and/or medicinal chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". As used herein, the phrase “pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a compound of the present disclosure. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. It will be understood that the present disclosure encompasses solvated forms, including hydrates, of the compounds of Formula 1, Formula 2, and salts thereof.

As used herein, the term “stereoisomer” refers to compounds having the same molecular formula and sequence of bonded atoms (i.e., atom connectivity), though differ in the three- dimensional orientations of their atoms in space. As used herein, the term “enantiomers” refers to two compounds that are stereoisomers in that they are non-superimposable mirror images of one another. Relevant stereocenters may be donated with (R)- or (S)- configuration.

Those skilled in the art of organic and/or medicinal chemistry will appreciate that the compounds of Formula 1, and salts thereof, may be present in amorphous form, or in a crystalline form. It will be understood that the present disclosure encompasses all forms and polymorphs of the compounds of Formula 1, and salts thereof.

CRP Inhibitors

The present disclosure provides amino compounds of Formula 1 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

As used herein, the term “CRP inhibitor” refers to the capacity of a compound to interact with a protein target in vitro or in vivo , including in the capacity to inhibit the activity or normal function of said targets, e.g., to inhibit binding or enzymatic activity. In another example, a compound of Formula 1 can provide an anti-inflammatory agent, such as effective for inhibiting inflammatory effects of C-reactive protein.

Compounds of Formula 1

In one aspect the present disclosure provides an amino compound of Formula 1 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

Formula 1 wherein

X¹ is selected from -SO₃H, -PO₃H, and -CO2H;

In one embodiment of the amino compound of Formula 1 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

X¹ is -SO₃H;

L¹ is selected from -CH2-CH2-;

In another embodiment of the amino compound of Formula 1 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

X¹ is -SO₃H;

L¹ is selected from -CH2-CH2-CH2-, -NH-CH2-CH2-, -S-CH2-CH2-, and -O-CH2-CH2-;

X¹ is -SO₃H;

L¹ is selected from -CH2-CH2-CH2-CH2-, -NH-CH2-CH2-CH2-, -S-CH2-CH2-CH2-, and -O-CH2-CH2-CH2-; R¹ and R² are each independently selected from C4-2oalkyl, Ci-2oalkylaryl, and Ci- 2oalkylhetaryl.

X¹ is -PO₃H;

L¹ is selected from -CH2-CH2-;

X¹ is -PO3H;

L¹ is selected from -CH2-CH2-CH2-, -NH-CH2-CH2-, -S-CH2-CH2-, and -O-CH2-CH2-;

X¹ is -PO₃H;

L¹ is selected from -CH2-CH2-CH2-CH2-, -NH-CH2-CH2-CH2-, -S-CH2-CH2-CH2-, and -O-CH2-CH2-CH2-;

X¹ is -CO2H;

L¹ is selected from -CH2-CH2-;

X¹ is -CO2H;

L¹ is selected from -CH2-CH2-CH2-, -NH-CH2-CH2-, -S-CH2-CH2-, and -O-CH2-CH2-;

R¹ and R² are each independently selected from C4-2oalkyl, Ci-2oalkylaryl, and Ci- 2oalkylhetaryl. In another embodiment of the amino compound of Formula 1 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

X¹ is -CO2H;

R¹ and R² for any of the above embodiments may each independently be selected from C4-2oalkyl, Ci-2oalkylaryl, and Ci-2oalkylhetaryl. R¹ and R² for any of the above embodiments may each independently be selected from C4-ioalkyl, Ci-ioalkylaryl, and Ci-ioalkylhetaryl. R¹ and R² for any of the above embodiments may each independently be selected from C4- _loalkyl, Ci-6alkylaryl, and Ci-6alkylhetaryl. The alkylaryl may be as defined herein, and in one example is an alkylbenzene, such as a benzyl. The alkylhetaryl may be as defined herein, and in one example comprises a 5 or 6 membered monocyclic heteroaryl, such as an alkylpyridine or alkyl imidazole, for example 2, 3 or 4-methylpyridine or 2,4 or 5-methylimidazole. In some examples, R¹ and R² are selected to be the same moiety or group.

In another embodiment, the present disclosure provides an amino compound of Formula 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, selected from the following:

Compound No. Compound Structure 1

2

3

4

5

6

Synthesis of Compounds of Formula 1

A compound of Formula 1 of the present disclosure may be prepared by various methods. One general method for preparing the compounds is shown below in Scheme 1:

In Scheme 1 a secondary amine comprising R¹ and R² groups can be reacted with a reagent X to form a compound of Formula 1. It will be appreciated that X is a reactive precursor to X'-L¹ moiety, such as a haloalkyl group comprising a functional X¹ group or precursor thereof. Examples A1 to A4 in the below experimental section provide further details regarding the method according to Scheme 1.

It will be appreciated that the above groups of X¹, L¹, R¹, and R², may be provided according to any embodiments or examples thereof as described herein.

Therapeutic Applications

The compounds of Formula 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, of the present disclosure, and pharmaceutical compositions comprising the compounds or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, find use in the therapy of diseases, for example, CRP associated or dependent conditions (e.g., inflammation). Accordingly, there is provided a compound of Formula 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof as described herein, or pharmaceutical composition as described herein, for use in therapy.

A compound of Formula 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or a pharmaceutical composition as described herein, finds use in the treatment of diseases for which modulation or inhibition of CRP is effective in the treatment thereof.

The present disclosure also provides a method of controlling C-reactive protein levels in a subject comprising administering to the subject an effective amount of a compound of Formula 1, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any aspects, embodiments or examples as described herein. The controlling of C-reactive protein levels in a subject can be by modulating or inhibiting the pro-inflammatory actions of CRP, and in particular bioactive conformations of pentameric C-reactive protein (pCRP*) or monomeric C-reactive protein (mCRP).

The present disclosure also provides a method of preventing or treating a C-reactive protein associated disease, disorder or condition, comprising administering to a subject in need of treatment thereof an effective amount of a compound of Formula 1, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any aspects, embodiments or examples thereof as described herein.

The present disclosure also provides use of a compound of Formula 1, salt, solvate or stereoisomer, or of the pharmaceutical composition, according to any aspects, embodiments or examples thereof as described herein, in the manufacture of a medicament for modulating or inhibiting C-reactive protein levels in a subject.

Indications which can benefit from a CRP inhibitor can include diseases that are driven by a inflammatory component, especially those associated with high CRP, but not exclusively. Among these diseases are the following: ischemia-reperfusion injury including myocardial infarction, stroke; organ or vascular transplants, surgery or injuries in general, burns, allogenic transplantation, atherosclerotic disease, pericarditis, miocarditis, autoimmune diseases such as Crohn’s disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, or diseases associated with inflammation driven by protein misfolding, as seen with amyloidoses, Alzheimer disease, prion diseases and age-related macular degeneration. Also diseases associated with generalized overshooting inflammation such as sepsis or COVID-19 can benefit from a CRP inhibitor.

In one embodiment, the compound of Formula 1 or pharmaceutical composition thereof can be administered by oral, subcutaneous or systemic route to achieve systemic exposure prior to undertaking a surgical procedure e.g. cardiac bypass surgery or heart, renal or lung transplantation that would incur ischemia/reperfusion injury of the respective organ. Exposure to the compound of Formula 1 or pharmaceutical composition thereof can be maintained during surgery and then during a period of post-operative recovery; wherein the period may vary from one hour, to one day, to several days or a month or longer.

In another embodiment, the compound of Formula 1 or pharmaceutical composition thereof can be administered to patients diagnosed with myocardial infarction, stroke, pulmonary artery embolism or other arterial embolism. Administration can be by systemic intraveneous route, oral, or subcutaneous to achieve acute and sustained systemic exposure. Period of treatment may be from one day to several days or a month or longer, to manage ongoing disease or until the disease pathology resolves.

In another embodiment, the compound of Formula 1 or pharmaceutical composition thereof can be administered to a patient diagnosed with a chronic inflammatory condition such as atherosclerosis, autoimmune diseases, sepsis, or COVID-19. Administration can be by oral, subcutaneous or systemic route to achieve periodic or sustained systemic exposure. Period of treatment may be from one day to several days or a month or longer, to manage ongoing disease or until the disease pathology resolves.

In some examples, the C-reactive protein associated disease, disorder or condition, is an inflammatory disease, disorder or condition. In some examples, the C-reactive protein associated inflammatory condition is an acute or chronic inflammatory condition. In some examples, the acute or chronic inflammatory condition is from a bacterial, viral or fungal infection, rheumatic disease, malignancy, atherosclerosis, myocardial infarction, tissue injury, or necrosis. In some examples, the C-reactive protein associated inflammatory disease, disorder or condition, is ischemia-reperfusion injury (IRI). In some examples, the ischemia- reperfusion injury (IRI) is from organ transplantation (e.g. renal, heart etc.), vascular surgery, acute ischemic renal injury, or delayed graft function. In some examples, the C-reactive protein associated disease is a cardiovascular disease. In some examples, the cardiovascular disease or condition is cardiac arrhythmia, vascular disease, myocardial infarction, stroke, congestive heart failure, myocarditis, atherosclerosis, or restenosis. In some examples, the compound is effective for selective anti-inflammatory activity. In some examples, the compound is not an immunosuppressant. In some examples, the subject is immune compromised. In some examples, the C-reactive protein associated disease, disorder or condition, is a non-immune-mediated inflammatory disease (IMID). In some examples, the C- reactive protein associated disease, disorder or condition, involves sterile inflammation. In some examples, the C-reactive protein associated disease, disorder or condition, comprises one or more sterile inflammation conditions selected from cancer, gout, Alzheimer’ s disease, atherosclerosis, silicosis, asbestosis, and diabetes.

Compositions

Compositions suitable for use in the methods and uses described herein comprise a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof. In some embodiments, a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, is presented as a composition. In some embodiments, a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, is presented as a pharmaceutical composition.

The present disclosure also provides pharmaceutical compositions that comprise a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilisers, or the like. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. Generally, suitable pharmaceutically acceptable carriers are known in the art and are selected based on the end use application. The pharmaceutically acceptable carrier may act as a diluent, dispersant or carrier for the active agents and other optional components of the composition. The pharmaceutically acceptable carrier may also contain materials commonly used in pharmaceutically products and can be in a wide variety of forms. For example, the carrier may be water, liquid or solid emollients, silicone oils, emulsifiers, surfactants, solvents, humectants, thickeners, powders, propellants and the like.

In some embodiments, the composition is a pharmaceutical composition, and wherein the composition comprises a pharmaceutically acceptable excipient.

The composition may for example contain a solvent, such as water (e.g. water for injection) or a pharmaceutically acceptable organic solvent.

The compositions may further include diluents, buffers, citrate, trehalose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc and other such suitable cations). The compositions of the present disclosure may also include polymeric excipients/additives or carriers, e.g., polyvinylpyrrolidones, derivatised celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as 2- h y d o x y p o p y 1 - b - c y c 1 o dc x t r i n and sulfobutylether-P-cyclodextrin), polyethylene glycols, and pectin.

Other pharmaceutical carriers, excipients, optional ingredients and/or additives suitable for use in the compositions according to the present disclosure are listed in "Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and in the "Physician's Desk Reference", 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), and in "Handbook of Pharmaceutical Excipients", Third Ed., Ed. A. H. Kibbe, Pharmaceutical Press, 2000.

A compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, of the present disclosure may be formulated in compositions including those suitable for inhalation to the lung, by aerosol, parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular injection) or oral administration.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, into association with a carrier that constitutes one or more accessory ingredients.

In general, the compositions are prepared by bringing a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof into association with a liquid carrier to form a solution or a suspension, or alternatively, by bringing a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof into association with formulation components suitable for forming a solid, optionally a particulate product, and then, if warranted, shaping the product into a desired delivery form.

In some embodiments, the composition is formulated for oral delivery. Compositions for oral delivery may, for example, be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in a tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade. The oral compositions described herein may contain from about 1% to about 95% of a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof by weight, and the oral compositions may be dosed 1, 2, 3, 4, 5 or more times daily. The oral compositions described herein may contain a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof by weight % in at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90. The oral compositions described herein may contain a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof by weight % in less than about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20,

15, 10, or 5. The oral compositions described herein may contain a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof by weight % in a range provided by any two of these upper and/or lower values, for example between about 5 and 20 wt %.

In some embodiments, the composition is formulated for parenteral delivery. For example, in one embodiment, the composition may be a sterile, lyophilized, crystalized or amorphous composition that is suitable for reconstitution in an aqueous vehicle prior to injection.

In one embodiment, a composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, which may for example be formulated to be isotonic with the blood of the recipient.

Pharmaceutical compositions are also provided which are suitable for administration as an aerosol, by inhalation. These formulations comprise a solution or suspension of a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof. The desired formulation may be placed in a small chamber and nebulized. Nebulization may be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

As discussed below, a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof of the present disclosure may for example be administered in combination with one or more additional pharmaceutically active agents. Thus, in some embodiments, the composition comprises a compound of Formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof as defined herein, or a pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable carriers, and one or more additional pharmaceutically active agents.

Dosages

The amount of active ingredient that is required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject under treatment, including the type, species, age, weight, sex, and medical condition of the subject being treated, and the renal and hepatic function of the subject, and the particular condition, disorder or disease being treated, as well as its severity. An ordinary skilled physician or clinician can readily determine and prescribe the effective amount of the drug required to prevent or treat the condition, disorder or disease.

Dosages of a compound of Formula 1, or salt, solvate or stereoisomer thereof, when used for the indicated effects, will range between, for example, about 0.01 mg per kg of body weight per day (mg/kg/day) to about 1000 mg/kg/day. In one example, the dosage of a compound of Formula 1, or salt, solvate or stereoisomer thereof, is between about 0.01 and 1000, 0.1 and 500, 0.1 and 100, 1 and 50 mg/kg/day. In one example, the dosage of a compound of Formula 1, or salt, solvate or stereoisomer thereof, is between about 0.01 and 1000 mg/kg/day. In one example, the dosage of a compound of Formula 1, or salt, solvate or stereoisomer thereof, is between about 0.1 and 100 mg/kg/day. In one example, the dosage of a compound of Formula 1, or salt, solvate or stereoisomer thereof, is greater than about 0.01, 0.1, 1, 10, 20, 50, 75, 100, 500, 1000 mg/kg/day. In one example, the dosage of a compound of Formula 1, or salt, solvate or stereoisomer thereof, is less than about 5000, 1000, 75, 50, 20, 10, 1, 0.1 mg/kg/day.

A compound of Formula 1, or salt, solvate or stereoisomer thereof, may for example be administered as a single daily dose, or otherwise the total daily dosage may be administered in divided doses of two, three, or four times daily. In one example, the compound of Formula 1, or salt, solvate or stereoisomer thereof, may be dosed less frequently than once per day, for example once per two days, three days, four days, five days, six days, or once per week. EXAMPLES

The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

Abbreviations

ACN Acetonitrile

DCM Dichloromethane

DCE Dichloroethane

DIPEA A, A- D i i s o p ro p y 1 c t h y 1 a m i n c

DME Dimethyl ether

DMF Dimethylformamide

ESI Electrospray ionization

HRMS High Resolution

LCMS Liquid chromatography-mass spectrometry m/z Mass to charge ratio

NMR Nuclear magnetic resonance o.n Overnight r.t Room temperature

TFA Trifluoroacetic acid

THF T etrahy drofuran

The following Table 1 provides compound numbers and nomenclature, with reference to its moelcular structure, for compounds synthesized and evaluated for CRP inhibition:

Table 1: CRP Inhibitor Examples

Compound No. Compound Structure

1

2

3

4

Example Al: Synthesis of Compound 1

1-A 1-C 1

Compound 1-C Diethyl(3-(dibutylamino)propyl)phosphonate

To a solution of Compound 1-A (dibutylamine, 0.32g, 2.5 mmol) in dimethylformamide (8 mL) was added sodium iodide (0.03 g, 0.25 mmol), potassium carbonate (1 g, 7.5 mmol) and Compound 1-B (diethyl(3-bromopropyl)phosphonate, 1.42 g, 5.5 mmol) at 0°C. The reaction mixture was heated to 100°C for 14 h. The progress of the reaction was monitored by TLC. The reaction mixture was cooled to 0°C and quenched with water (15 mL) and extracted with ethyl acetate (15 mL x 2). The combined organic layer was washed with brine (20 mL), dried over sodium sulfate and concentrated under vacuum. The residue was separated by silica gel chromatography to provide Compound 1-C (diethyl(3- (dibutylamino)propyl)phosphonate, 215 mg, 0.7 mmol, 28 % yield). Compound 1 (3-(dibutylamino)propyl)phosphonic acid

To a solution of Compound 1-C (diethyl [3-(dibutylamino)propyl]phosphonate, 0.28 g, 0.91 mmol) in dichloromethane (25mL) was added trimethylsilyl bromide (2.79 g, 18.2 mmol, 20 eq) at 0°C. The reaction mixture was heated to 45°C for 14 h. The solvent was concentrated. Residue was dissolved in methanol (20 mL) and dichloromethane (20 mL) and then solvents were removed under vacuum. The residue was triturated under dichloromethane : hexane (1:1) (30 mL) and heated with stirring at 40 °C for 30 minutes. The solvents were decanted and the residue was dried under vacuum to provide Compound 1 (3- (dibutylamino)propyl)phosphonic acid, 0.10 g, 0.4 mmol, 44% yield).

Calc C11H26NO3P [M+H]+ = 252; found [M+H]+ = 252

Example A2: Synthesis of Compound 2

Compound 2-C Diethyl(3-(dibenzylamino)propyl)phosphonate

To a solution of Compound 2- A (dibenzylamine, 0.25 g, 1.3 mmol, leq) in dimethylformamide (8 mL) was added sodium iodide (19mg, 0.13 mmol, O.leq), potassium carbonate (0.54g, 3.9mmol, 3eq) and Compound 1-B (diethyl(3-bromopropyl)phosphonate, 0.74 g, 2.9 mmol, 2.2 eq) at 0°C. Reaction mixture was heated to 100°C for 14 h. The reaction mixture was cooled to 0°C and quenched with water (15 mL) and extracted with ethyl acetate (15 mL x 2). The combined organic layer was washed with brine (20 mL), dried over sodium sulfate and concentrated under vacuum. The residue was separated by silica gel chromatography to provide Compound 2-C (diethyl(3-(dibenzylamino)propyl)phosphonate).

Compound 2 3-( Dibenzylamino )propyl-phosphonic acid

To a solution of Compound 2-C (diethyl (3-(dibenzylamino)propyl)phosphonate,

0.375 g, 1 mmol, 1 eq) in dichloromethane (25mL) was added trimethylsilyl bromide (3 g, 20 mmol, 20 eq) at 0°C. The reaction mixture was heated to 45°C for 14 h. The solvent was concentrated. The residue was dissolved in methanol (20 mL) and dichloromethane (20 mL), and then the solvents were removed under vacuum. The residue was triturated under dichloromethane : hexane (1:1) (30 mL) and heated with stirring at 40 °C for 30 minutes. The solvents were decanted and the residue was dried under vacuum to provide Compound 2 (3- (dibenzylamino)propyl-phosphonic acid) .

'H [D2O] 7.4 (broad multiplet, 10H a); 4.30 (broad singlet, 4H, b); 3.11 (broad singlet, 2H, c); 1.96 (broad singlet, 2H, d); 1.58 (broad multiplet, 2H, e).

³¹P [DeMSO] 25.37 f

¹³C [DeMSO] 131.7, 130.3, 130.1, 129.4; 56.6; 53.0; 25.4 doublet m; 17.7.

Example A3: Synthesis of Compound 3

MW: 251.39

Compound 3 3 -(Dibutylamino)propane-l -sulfonic acid)

To a stirred mixture of Compound 1-A (dibutylamine, 1.0 g, 0.0077 mol, 1 equiv.) in toluene (10 mL) was added Compound 3-B (1,2-oxathiolane 2,2-dioxide, 0.99 g, 0.0081 mol, 1.05 equiv.). The resulting reaction mixture was stirred for 3 h at 110 °C. After confirming the completion of reaction (TLC analysis: 10% MeOH/DCM; Rf~ 0.1), reaction mixture was cooled to RT and toluene was decanted. The resulting crude compound was dissolved with 10% ethyl acetate in pet ether (20 ml), stirred for 30 minutes at 50 °C and solvent was decanted. Then methanol (10 ml) was added to the residue, stirred for 30 minutes at RT. The crude compound (0.5 g) was purified by preparative HPLC (Method: 70:30, A-0.1% Formic acid in H2O, B: ACN; Flow Rate: 15 mL/min, Column: SUNFIRE C18 (30 x 250 mm, 10 pm, RT-7.47). Combined column fractions were concentrated under vacuum to yield the title Compound 3 (3 -(Dibutylamino)propane-l- sulfonic acid) as off-white gummy solid.

Yield: 0.140 g (7.30% yield).

LCMS: Mass found; (250.1; M-l)

Method: A-0.1% Formic acid in H2O, B: ACN; Flow Rate: 1.5 mL/min; -ve mode Column: Atlantis del 8 (50 x 4.6mm, 5pm)

Rt (min): 1.36; Area% - 99.81.

HPLC: 99.54 %.

Method: A-0.1% TFA in WATER, B: ACN; Flow Rate: 1.0 mL/min Column: Atlantis dC18 (250 x 4.6mm, 5pm)

Rt (min): 7.27; Area% -99.54.

1H-NMR (400 MHz, DMSO-d6): d 9.58 (s, 1H), 3.21 (s, 2H), 3.03 (t, J = 7.60 Hz, 4H), 2.63 (t, J = 6.40 Hz, 2H), 1.96 (t, J = 6.00 Hz, 2H), 1.57 (t, J = 8.00 Hz, 4H), 1.30-1.32 (m, 4H), 0.84-0.85 (m, 6H).

Example A4: Synthesis of Compound 4

Compound 4 3-(Dibenzylamino) propane-1 -sulfonic acid

To a stirred solution of Compound 2- A (dibenzylamine 1, 1.0 g, 0.0050 mol, 1 equiv.) in toluene (10 mL) was added Compound 3-B (1,2-oxathiolane 2,2-dioxide, 0.65 g, 0.0053 mol, 1.05 equiv.). The resulting reaction mixture was stirred for 3 h at 110 °C. After confirming the completion of reaction (TLC analysis: 10% MeOH/DCM; Rf~ 0.1), reaction mixture was cooled to RT and toluene was decanted. The resulting crude was dissolved with 10% ethyl acetate in pet ether (20 ml), stirred for 30 minutes at 50 °C and solvent was decanted. Then, methanol (10 ml) was added to the residue and stirred for 30 minutes at RT. The precipitated solid was collected by filtration and dried under reduced pressure to afford the title Compound 4 (3-(Dibenzylamino) propane- 1- sulfonic acid) as off-white solid.

Yield: 0.8 g (50% yield).

LCMS: Mass found; (318.1; M-l)

Method: A-0.1% Formic acid in H2O, B: ACN; Flow Rate: 1.5 mL/min; -ve mode Column: Atlantis dC18 (50 x 4.6mm, 5pm)

Rt (min): 1.82; Area% - 99.62.

HPLC: 99.62 %. Method: A-0.1% TFA in H2O, B: ACN; Flow Rate: 1.0 mL/min Column: Atlantis dc-18 (250 x 4.6mm, 5mhi)

Rt (min): 8.63; Area% -99.62.

1H-NMR (400 MHz, DMSO-d6): d 10.79 (s, 1H), 7.47 (t, J = 2.72 Hz, 10H), 4.41 (dd, J = 4.64, 13.16 Hz, 2H), 4.19 (dd, J = 4.68, 13.14 Hz, 2H), 3.19 (d, J = 5.08 Hz, 2H),

2.57 (d, J = 5.64 Hz, 2H), 2.16 (d, J = 5.08 Hz, 2H).

B. Biological Evaluation of CRP Inhibitors

Example Bl: Liver microsome stability of Compound 1

Analytical Instrumentation

Chromatography was performed using an Agilent 1290 system. The device comprises a 1290 autosampler with a thermoelectric sample cooler and a 1290 binary pump with an integrated solvent degasser. Mass spectrometry was performed with an Agilent 6460 Triple Quadmpol equipped with an Agilent JetStream ESI ion source. Samples were separated on a Thermo Scientific Acclaim Organic Acid column (150 x 2.1 mm, 5 pm) at 20 °C. Solvents for chromatographic separation were prepared as follows: mobile phase A and mobile phase B contained 0.1 % formic acid in water and acetonitrile, respectively. Separation of analytes from matrix components was achieved by gradient elution: 0-3 min, 2 % B; 3-10 min, 40 % B; 14.5 min, 100 % B; 17 min, 100 % B; 17.1 min, 2 % B; 21 min, 2 % B with a flow rate of 0.6 mL/min.

Mass spectrometric conditions

Agilent 6460 Triple Quadrupole equipped with an Agilent JetStream ESI Source was used for mass-spectral elucidation and quantitation of Compound 1 in urine and plasma. Mass spectral analysis of ClOm and IS were performed in dMRM-mode. Fragmentor voltages and collision energies were optimized using Agilent MRM- Optimizer, yielding these transitions for

, , , , , ,

142.2, 18 V; 4-DAM, 216.1

130.2, 14 V; 4-DAM, 216.1

86.6, 26 V. Ionization parameters were as follows: capillary voltage, 4300 V; fragmentor voltage, 185 V; nozzle voltage, 500 V; gas temperature, 350 °C; gas flow, 11 L/min; nebulizer pressure, 40 psi; sheath gas temperature, 350 °C; sheath gas flow, 11 L/min; cell accelerator voltage, 4 V. Fragmentation

The fragmentation pattern for Compound 1 was elucidated. In the first fragmentation step, the ion is split by alpha-cleavage at the nitrogen to yield either m/z 123.1 (1) or m/z 130.1 (2). Water cleavage from the phosphate of fragment (1) leads to the formation of (3). In a last alpha-cleavage PO2H is cleaved off, leaving C3H5⁺ m/z 41.1 (4). For quantification the fragment ion m/z 123.1 (1) was used. The other fragment ions m/z 130.2, 105.1 and 41.1 were used as qualifiers for identity.

Untargeted analysis

Agilent 6520 Quadrupole time-of-flight equipped with an Dual ESI Source was used for mass-spectral elucidation and detection of metabolites in urine and liver, spleen, kidney and muscle tissue.

Mass spectral analysis of Compound 1 and its potential metabolites was performed in full scan mode. Ionization parameters were as follows: capillary voltage, +4300 V; fragmentor voltage, 185 V; gas temperature, 350 °C; gas flow, 11 L/min; nebulizer pressure, 40 psi; scan range, 100 - 1000 m/z. Reference masses were m/z 121.050873 and 922.009798 for Purine and HP-0921, respectively (Sum formula of purine: C5H4N4[+H+], m/z = 121.050873; Sum formula of HP-0921: C18H18N306F26P3[+H+], m/z = 922.009798).

Selectivity

The method was considered selective when signals in blank samples were less intense than 20 % of LLOQ-samples. Blank samples were injected and analyzed in MRM-mode. Afterwards, sample matrix was spiked with 0.1 pg/mL of ClOm and 0.5 pg/mL 4-DAM and compared with peak areas of blank samples.

Recovery

The recovery of the analyte and its IS was determined by comparing peak areas of extracted plasma samples to peak areas of post-extraction spiked plasma samples. Recovery was determined at three different concentration levels resembling LLOQ, medium concentration and high concentration.

Linearity of calibration curves

Concentration of Compound 1 in each sample was determined by calculating peak area ratios of ClOm to IS and comparing these to the calibration curve. Seven calibration standards (10, 5, 2.5, 1, 0.6, 0.3 and 0.1 mg/mL) and three QC-standards (10, 1 and 0.1 mg/mL) were prepared in an adequate matrix (human plasma or murine urine diluted 1:100). Calibrations were performed at 10, 5, 2.5, 1, 0.6, 0.3 and 0.1 pg/mL and at 5, 3.5, 2, 1, 0.6, 0.3 and 0.1 pg/mL for plasma and urine, respectively. Quality controls were chosen at ULOQ, M and LLOQ. IS was always added to a concentration of 0.5 pg/mL. Calculation of concentrations was performed with Agilent Quantitative Analysis B.07.01 SP2. The created calibration function y = m * x + c was weighed 1/x².

Linearity of quantification

For plasma calibration curves Compound 1 signal intensities showed good linearity over the range of 0.1-10 pg/mL. The same was true for calibration curves in urine over the range of 0.1-5 pg/mL. An exemplary function for a calibration curve from plasma is y = 1.1928 * x + 0.16985. Y depicts the ratio of peak-area of analyte of interest to peak-area of the IS. x represents the calculated concentration of the analyte. The calibration function was weighed 1/ x ². Calculations were run by Agilent Quantitative Analysis B.07.01.

Precision, Accuracy, LLOQ

Accuracy and precision were determined by analyzing six replicates each for three different concentrations across the linear range for plasma and urine matrix: At LLOQ (0.1 pg/mL), medium concentration (1 pg/mL) as well as at the maximum (Plasma: 10 pg/mL; Urine: 5 pg/mL). Accuracy was considered acceptable for deviations within ±15 % (±20 % at LLOQ). Precision was considered acceptable for coefficients of variation <15 % (<20 % at LLOQ).

Results for all precision and accuracy values are depicted in Table 2. All coefficients of variation were within ±15 % (±20 % at LLOQ). Accuracy of all calibration standards was between 85 % and 115 % (between 80 % and 120 % at LLOQ).

Table 1. Precision and accuracy for Compound 1 determination in Plasma and Urine.

Matrix Plasma Urine

LLOQ 97.3 % 98.1 %

Accuracy Mid 112.7 % 108.8 % High 87.8 % 91.0 % LLOQ 4.92 % 3.81 %

Precision Mid 0.87 % 3.43 % High 3.52 % 2.1 %

Microsome incubation

Prior to tissue analysis, we performed microsomal incubations with Compound 1 to elucidate any potential metabolites. Microsomes from rat liver were purchased from Thermo Scientific, Schwerte, Germany. ClOm was incubated in phosphate buffer (100 mM, pH 7.4) for 0, 60 and 120 min. Final concentrations for ClOm, NADH and microsomes were 1 mM, 20 mM and 20 mg/mL, respectively. One replicate was prepared without NADH and one replicate was prepared with heat-inactivated microsomes. For inactivation, microsomes were incubated for 30 min at 45 °C. After 0, 60 and 120 min, the reaction was stopped by adding of 400 pL acetonitrile. Samples were centrifuged to separate the microsomes from the supernatant.

Example B2: Pharmacokinetic study of Compound 1

Pharmacokinetic study in rats

The following animal studies were carried out according to the recommendations of the animal ethic committee of the University of Freiburg Medical Center, Germany.

As described above, the LC-MS-method was used to elucidate the plasma concentration of ClOm over time after a single intravenous administration. For the pharmacokinetic studies, nine male Wistar rats weighing 250 g to 350 g were purchased from Charles River Research Models and Services (Sulzfeld, Germany). Rats were anaesthetized with 1.5-2 vol % isoflurane (Abbott, Wiesbaden, Germany) and placed on a temperature-controlled surgical table to maintain a physiological body temperature during the procedure. To ensure interference-free blood sampling, two independent 26G catheters (Abbocath-T, ICU Medical B.V., VZ Houten, Netherlands) were placed in each lateral tail vein. Blood was taken prior to the injection of 47- 65 pg ClOm (approximately 5.4 pg per mL plasma which equals approximately 100-fold the amount of pCRP during inflammation) and at eight given time points (1, 5, 10, 15, 30, 45, 60 and 90 minutes after bolus administration). Samples were collected in EDTA-coated tubes (1.6 mg EDTA/ml, Sarstedt micro tubes, Niimbrecht, Germany). Plasma was separated from the cellular phase by centrifugation for 10 minutes at 2,000 g using a refrigerated table-top centrifuge at 4 °C. The resulting supernatant was snap-frozen and stored at -80 °C until sample preparation with solid-phase extraction.

For the renal excretion assay, rats were treated as described before with minor modifications. In brief, male Wistar rats (bodyweight approximately 350 g) were anesthetized as described above. After achieving adequate depth of anesthesia, rats were shaved in the caudoventral abdominal region and the skin was disinfected. The urinary bladder was exposed via a midline incision and gently externalized onto a sterile latex sheet. Sterile urine was drawn with insulin syringes directly from the bladder (BD Micro-Fine™ + Demi, BD Medical, Le Pont de Laix Cedex, France) before intravenous application of Compound 1 and 15, 30, 45, 60 and 90 minutes after i.v. application, respectively. Freshly drawn urine was snap-frozen and kept at -80 °C until analysis.

Pharmacokinetic parameters were calculated using PKSolver add-in for Microsoft

Excel.

Sample preparation

Rodent plasma: 150 pF of EDTA-anticoagulated plasma sample was spiked with 1.5 pF IS (concentration 50 pg/mF) yielding a final concentration of 500 ng/mF of IS.

Rodent tissue: Tissue was cut into small pieces and weighed into a tube prefilled with 300 mg of glass beads. 10-fold of tissue weight of water was added. The tissue was homogenized using a PreCellFys Tissue homogenizer with 3 cycles, 15 seconds each with 6500 rpm at approximately -10 °C. Subsequently, the homogenized tissue was centrifuged at 20,000 g for 15 min at 4 °C. 500 pF of homogenized tissue was transferred and dried under vacuum. The pellet was resuspended in 150 pF of water. Afterwards, 150 pF of phosphoric acid (4 %) was added to decrease viscosity of the sample and disrupt protein binding. Samples were stirred for 15 minutes (1,000 rpm, tube shaker) and subsequently centrifuged at 20,000 g for 15 minutes at 4 °C.

Rodent urine: Samples were centrifuged at 20,000 g for 15 min at 4 °C and subsequently diluted 1:100 with water. Afterwards, 1.5 pF of IS (50 pg/mF) was added to 150 pF of diluted urine sample. Standard calibration samples were also prepared in 150 pF of 1 % murine urine sample with the same amount of of IS added (1.5 pF, 50 pg/mF, final concentration 500 ng/mF). Solid-phase-extraction

Compound 1 was extracted from rodent plasma and tissue by solid-phase extraction (SPE). Offline-SPE was performed with a VacElut cartridge manifold.

SPE-columns (Waters, Oasis MCX, 1 mL, 30 meq) were equilibrated with 1 mL methanol and 1 mL water. Samples were loaded on the column and washed with 1 mL methanol and 1 mL HC1. After elution of 1 mL HC1, vacuum (0.7 bar) was applied and the cartridges were let run dry. Elution of ClOm and its metabolites was achieved using 2 x 750 pL methanol with 5 % Ntb. The eluate was vacuum dried and resuspended in 150 pL of 2 % ACN with 0.1 % formic acid (starting chromatographic condition).

Results: Pharmacokinetics

In one group (pCRP ) consisting of 3 rats, 47 pg Compound 1 was injected to each animal. The other group (3 rats; pCRP⁺) received the same amount of compound plus pCRP (25 pg/mL). The chosen pCRP-concentration approximates to pCRP levels during inflammation. After administration of Compound 1 and pCRP, blood samples from both groups (pCRP⁺ = Compound 1 + pCRP; pCRP = ClOm) were drawn after 1, 5, 10, 15, 30, 45, 60 and 90 minutes. After analysis, the average plasma concentration for each timepoint and cmax were calculated (cmaxpCRP- = 864.4 ± 172.3 ng mL ¹ as well as cmax_PcRP₊ = 542.0 ± 131.1 ng mL ¹; for graphs see figure 7 a-c). Biovariability in the pCRP⁺- group appeared to be higher than in pCRP -group. This might be because Compound 1 has bound to pCRP and, thus, is not freely available for excretion. Independently of pCRP-administration and most likely due to its polar nature, Compound 1 was excreted at a high rate: After 30 minutes, most of the compound was eliminated from plasma (tl/2_PcRP- = 26.9 ± 1.3 min; tl/2_PcRP₊ = 134.1 ± 57.7 min). Their standard deviations was determined for each time point and both groups. Table 3 comprises ah pharmacokinetic parameters. Additional administration of pCRP yielded an initially higher Compound 1 concentration, thus lowering drug-elimination halftimes.

The weight of the rats was 250 g each. Ah animals showed normal behaviour and no abnormal clinical symptoms after Compound 1 or Compound 1-pCRP-administration.

Table 3 below shows the pharmacokinetic parameters of both animal groups. One treated with Compound 1 (pCRP) and the other treated with Compound 1 and pCRP (pCRP+). pCRP-group demonstrated to higher biovariability and higher halftimes than the pcRP+-group. Calculated initial concentrations for tO and cmax were higher in the pCRP— group than in the pCRP+-group. Table 2. Pharmacokinetic parameters of both animal groups

Pharmacokinetic Compound 1-injection

Parameters pCRP pCRP+ Animal # 1.1 - 1.3 1.4 - 1.6 tl/2 / min 26.9 + 1.3 134.1 + 57.7

Cmax / ng mL ¹ 864.4 + 172.3 542.0 + 131.1

CO / ng mL ¹ 997.2 + 220.4 765.4 + 244.3

AUC 0-t / ng min mL 15,167.4 + 21.129.1 + 2,674.3

¹ 1,057.

AUC 0-co / ng min 16,550 + 45,203.3 + mL ¹ 1,126.5 11,371.3

Results: Urine and tissue analysis

Urine samples, plasma samples as well as hepatic tissue have also been analyzed in full scan mode. EICs of several potential metabolites were extracted. Additionally, comparative analyses of pre- and post-injection urine samples have been performed with Agilent ProFinder to search for post injection urine features. For this we analyzed pre- and post- injection features and compared it with post injection urine from different time points. No post- injection specific metabolites could be detected in urine (data not shown).

Even though renal elimination appeared to top off at 80 %, we could not determine any metabolites. Collecting urine from later points in time such as 120 or 180 min might be helpful. We also analyzed hepatic, kidney, spleen and muscle tissue which either contained concentrations of Compound 1 below the LLOQ or no Compound 1 at all.

Results: Urinary samples

Three additional animals weighing 350 g each were injected 68, 108 and 108 pg of Compound 1 of which 68 pg approximate to 100 fold of pCRP during inflammation. The bladder of each animal was emptied prior to injection. After 15, 30, 45, 60 and 90 minutes, the bladder was emptied again and its volume and Compound 1-concentration were determined. This analysis revealed that most of the compound (60-80 %) was renally eliminated after 45 to 90 minutes without any metabolism (Figure 8b). The data also supports the assumption of higher elimination rates in urine with higher i.v. doses. Elimination rates for animal #1 and #2.1 are similar, even though their initial dose was substantially different. Animal #2.11 showed lower initial speed of elimination. Less than 10 % were eliminated after 15 minutes via urine. After 30 minutes though, approximately 50 % were excreted as with the rest of the animals.

Example B3: Compound 1 inhibits pCRP* induced aggravation of renal ischemia/reperfusion injury

Reagents and antibodies

Preparation of human pCRP was performed as described before by our group 12. In brief, pCRP purified from human ascites was purchased from Calbiochem (Nottingham, UK) and was thoroughly dialyzed twice (1:500 v/v) against Dulbecco’s phosphate buffered saline (D-PBS) supplemented with 0.9 mM CaC12 and 0.49 mM MgC12. Monomeric CRP was generated by treating pCRP with 8 M urea for one hour at 37 °C and following dialysis against 25 mM Tris-HCl (pH 8.5) overnight at 4 °C as described by Biro et al. 29. The protein concentration was determined after each dialysis and dissociation procedure by a benchtop fluorometer (Qubit® 3.0 Fluorometer, Invitrogen™ by life technologies™, Carlsbad, CA, USA).

Renal IRI

The renal ischemia/reperfusion-injury experiments were carried out on male Wistar rats. All rats were six weeks old and body weight was between 180 and 220 g (Charles River Research Models and Services, Sulzfeld, Germany). Prior to surgery, 30 Wistar rats were randomly allocated to one of five designated groups; (1) sham-operated controls receiving flank incisions without renal clamping. Animals received i.p. vehicle D-PBS solution treatment; (2) IRI-treated rats were subjected to the surgical procedure described hereafter.

IRI rats received i.p. 500 pi D-PBS application; (3) IRI + pCRP-treated rats: the same surgical procedure as in group (2) was performed. Animals received i.p. pCRP application in a 25 pg/ml serum concentration instead of D-PBS; (4) IRI + pCRP + Compound 1 -treated group: as in group (3) rats received i.p. pCRP application in a 25 pg/ml serum concentration. pCRP was incubated with Compound 1 (1:100 molar ratio, approximately 0.3 mg/kg) before administration; (5) IRI + 1,6-bisPC-treated group: the same surgical procedure as in group (2) was performed. Animals received i.p. 1,6-bisPC application only (n = 6 per group). Wistar rats were anesthetized with 1.5-2 vol% isoflurane (Abbot, Wiesbaden, Germany). Body temperature was monitored during the operation via an anal probe. Rats were shaved and disinfected and incisions were made as described by us previously. Renal pedicles were exposed and clamed for 45 minutes with non-traumatic micro vessel clips followed by 24 h reperfusion. Animals were euthanized after 24 hours of reperfusion. All rmcrosurgical procedures were conducted using a stereo microscope (Stemi 2000, Carl Zeiss).

Excretory function of I/R-injured kidneys assessed by Blood Urea Nitrogen (BUN)

BUN concentration was taken as surrogate for the secretory function before and after ischemia/reperfusion injured kidneys. Serum probes were obtained from each rat before IRI and after 24 hours of reperfusion. Probes with macroscopic hemolysis were excluded before the reading by cobas 8000 modular analyzer (Roche, Basel) by the central laboratories of the University Medical Center, Freiburg.

Immunostaining and histomorphological evaluation

Immunohistochemistry and histomorphological evaluation of the renal tissue was performed on formalin-fixed paraffin-embedded renal tissue sections (5 pm thick serial sections). Previously, both kidneys were flushed till bloodlessness with D-PBS followed by 4% formalin for fixation. Kidneys were then excised and examined in blinded fashion by two researchers using a Zeiss microscope (Carl Zeiss Microscopy Axio Imager.M2, Germany). Staining was performed as described previously. Paraffin-embedded sections were de- paraffinized in xylol, rehydrated, and boiled for 20 min in concentrated citric acid (pH 6.0). Antigen unmasking for anti-monocyte detection was done by application of pepsin solution (Digest- All™ 3, life technologies) at room temperature for 20 min. Histomorphological changes were evaluated on Periodic acid-Schiff stained sections by quantitative measurement of tubulointerstitial injury, which was assessed by loss of tubular brush border and cast formation. The morphological assessment was scaled in five steps: with not present (0), mild (1), moderate (2), severe (3) to very severe (4). Transmigrated leukocytes were detected by anti-monocyte/macrophage antibody clone ED-1 (Millipore, Billerica, MA, USA) in a 1:100 dilution and renal inflammation was evaluated by counting ED-D cells in 20 randomized areas of interest of the renal cortex at x200 magnification. The number of apoptotic cells was evaluated using anti-caspase-3 antibody (Novus Biologicals, Abingdon, UK) in a 1:1,000 dilution. Sections were counterstained with Mayer’s hematoxylin. Unspecific isotype matched primary antibodies served as negative control. Detection of human CRP on the renal tissue sections was performed using anti-pCRP*/mCRP antibody 9C9 (1:100 dilution). For Western blot detection of CRP snap-frozen kidney tissue was homogenized on ice using a high-power disperser (Ultra-Turrax® IKA, Staufen, Germany) in lysis buffer (hier: Sheena fragen). After centrifugation of the homogenized tissue, the supernatant was transferred, and protein concentrations were determined with BCA protein assay. GAPDH served as control.

Western blot analysis

SDS-PAGE and subsequent Western blot analysis was conducted for CRP binding to activated human platelets as described previously. Briefly, human platelets were isolated and washed from citrate-anticoagulated whole blood by differential centrifugation in Sequestrene™ buffer. pCRP [100 pg/rnl] was incubated with ADP-activated platelets and Compound 1 in different concentrations [10 mM and 100 mM]. Calcium-depleted platelets served as a control. Platelets were then washed three times in DPBS supplemented with Ca⁺⁺ and Mg⁺⁺. Platelets were pelleted and homogenized on ice by applying shear-stress followed by freeze-and-thaw cycles in liquid nitrogen). The protein concentration of the lysates was determined by fluorometric assay using a Qubit fluorometer. After the separation by SDS gel electrophoresis and the transfer to nitrocellulose membranes (Hybond ECL, GE Healthcare, Munich, Germany), samples were probed with anti-CRP antibody clone 8 overnight at 4 °C. Monoclonal antibodies against GAPDH (abeam, Cambridge, UK) were used to ensure protein equilibration. Secondary HRP-conjugated anti-mouse antibodies (Dianova, Hamburg, Germany), enhanced chemiluminescence (ECL, GE Healthcare) were used to detect protein signals and were conserved on FUJI Medical X-Ray Film (FUJIFILM, Japan).

Results

Ischemia/reperfusion injury (IRI) represents the prototypic sterile inflammation in which exacerbated immune response leads to unwanted tissue damage and therefore represents the ideal scenario to test the therapeutic potential of Compound 1. IRI associated tissue damage is induced by the conformational change of circulating pCRP to pCRP*. To evaluate the in vivo feasibility of CRP-blocking and the relevance of our findings, we used a previously described IRI-induced acute renal injury model in rats.

After IRI rat kidneys were examined for CRP deposits by immunohistochemistry and Western blotting. Staining for CRP using the conformation- specific anti-pCRP* antibody 9C9 demonstrates deposition of CRP specifically localized to the IRI-disturbed renal tissue. After intravenous administration of C10M CRP deposition could not be detected in the tissue. This was further confirmed by Western blots of tissue lysates separated by SDS-PAGE. The functional relevance of this finding was assessed by immunohistochemistry detecting CD68+ monocytic cell infiltration in renal tissue and PAS staining of renal tissue. In these assays, administration of pCRP leads to significant increase of IRI associated inflammatory cell infiltration and tissue injury that can be blunted by the administration of Compound 1. This aggravation of IRI significantly affects the excretory renal function as analyzed by blood urea levels.

Example B4: Compound 1 inhibits pCRP* induced aggravation of allograft rejection in a hindlimb transplantation model

Hindlimb allotransplantation

Fully genetic mismatch male rats were anesthetized, hindlimbs were shaved and thoroughly disinfected. The recipients hindlimb was amputated. The allogenic transplant was attached to the recipient’s femoral stump by an intramedullary fixation. End-to-end micro anastomoses for the femoral artery and vein were performed. All rats received postoperative subcutaneous injections of 100 pg/100 g body weight of carprofen for pain relief and 1 ml/100 g bodyweight saline solution for volume compensation. Following the surgical procedure, the rats were caged individually with monitoring by professional animal caretakers. Clinical assessment of the general condition of the animals and for hindlimb rejection was performed every eight hours by two independent surgeons. According to an established clinical classification for allograft rejection, hindlimb transplants were graded from 0 (no clinical signs of rejection), 1 (edema), 2 (erythema), and 3 (epidermolysis and desquamation) to 4 (necrosis). Overall survival and rejection of the VCA were assessed clinically and histologically. Four experimental groups were included in this study (n=4). In the control group (n=4, FW-^BN), Brown-Norway recipient rats received intraperitoneal DPBS administration. Rats in the pCRP group received two intraperitoneal boli of 25 pg pCRP (BD Micro-Fine™ -i-Demo, 30G insulin syringes) per ml serum volume directly following to the surgical procedure and after 24 hours. Serum volume was estimated as described before ³ as a function of the body weight. Immediately after surgery, subcutaneous saline supplementation was given to avoid dehydration of the rats. In the Compound 1 treatment group (n=4, FW-^BN), rats were treated as in the pCRP group. Additionally, rats received intravenous compound Compound 1 via a 26G catheter (Abbocath-T, ICU Medical B.V., VZ Houten, Netherlands) in the lateral tail vein every six hours for the first two postoperative days. Biopsies were taken on day three after transplantation of skin and muscle tissue and immunohistochemistry performed on formalin-fixed and paraffin embedded samples. After incubation with primary antibody anti-CD68 (clone EDI, 1:100) and anti-human CRP (clone 8, 1:200) for one hour at room temperature, slides were incubated with secondary antibody anti- mouse-conjugated horseradish peroxidase (Dako EnVision* System anti-mouse). HistoGreen substrate kit (Dossenheim, Germany) was used to visualize for CD68+ cells and CRP deposits, resulting in green staining.

Results

IRI is a major aggravating factor in organ damage and allograft rejection after allograft transplantation. To confirm the in vivo relevance of our findings, we performed hindlimb transplantation on fully mismatched rat strains (Lewis and Brown-Norway) as a model for acute allograft rejection of vascularized composite allografts (VCA) and clinically assessed graft survival. We found human CRP to strongly promote the diapedesis of monocytes and tissue degradation, and thereby accelerate VCA-graft loss significantly compared to a transplanted control group (control vs CRP, 8.0 ± 0.5 vs 5.0 ± 0.5 days). Most importantly, premature graft loss driven by pCRP was prevented by intravenous Compound 1 application during the first two days after transplantation. Compound 1 effects are attributable to pCRP inhibition as without extrinsic CRP application Compound 1 did not show protective effects. Transplanted rat hindlimbs showed significant clinical signs of rejection (edema, erythema, and blistering) on day three after transplantation after CRP administration, that were not present in the control group and when CRP effects were blocked with Compound 1.

Skin and muscle biopsies were taken at day 3 and were analyzed histologically. Monocyte infiltration was detected by immunofluorescence, which revealed significantly more monocyte infiltrates in VCA-tissue of rats treated with CRP compared to control rats (supplemental data). To investigate whether these exacerbating effects were specific for CRP C10M was used to block CRP. In rats treated with both CRP and Compound 1 no CRP deposits were detected. We found the number of transmigrated CD68+ cells to be reduced to control levels when Compound 1 was administered in the CRP group. These results indicate that compound Compound 1 inhibits the CRP-dependent activation and transmigration and thereby abrogates the CRP-mediated local exacerbation in VCA. Example B5: Compound 1 does not suppress CRP- independent host defense against pathogens

Whole blood assay for opsono-phagocytosis

To assess the influence of the inhibitory compound Compound 1 on the CRP-mediated phagocytosis of pathogens, heat-inactivated Streptococcus pneumoniae serotype 27 were labeled with fluorescein-isothiocyanate (FITC) as described elsewhere. Heparinized whole blood was freshly taken from healthy human volunteers and processed within thirty minutes. For phagocytosis time course experiments, opsonized Streptococcus-FITC was incubated for 5, 10, 15 and 20 minutes with fresh whole blood at 37° C, 5 % CO2. Opsonization of target particles was done as described before with minor modifications. Briefly, inactivated Streptococcus were incubated with pCRP [100 pg/ml], Compound 1 [XY pg/ml], pCRP and Compound 1 and DPBS (Control), respectively, for 30 minutes at 37° C in DPBS containing 0.9 mM calcium chloride. Target particles were then washed, and added to whole blood. After fixation and red blood cell lysis, cells were washed and stained, and analyzed subsequently by flow cytometry (BD LSR Fortessa Cell Analyzer).

Opsonization of zymosan by pCRP

For binding studies of human pCRP to zymosan a protein labeling kit (597/625 nm; Sigma-Aldrich, St. Louis, MO, USA) was used. Dialyzed pCRP was labeled with Atto 594 following the manufactures protocol. Washed zymosan [10 mg/ml] was subsequently incubated with 100 pg/ml pCRP-Atto 594 conjugate in DPBS supplemented with 0.9 mM calcium chloride for 30 min at 37°C, washed and resuspended again in DPBS with CaCh and MgCh. Calcium dependent binding of the pCRP-Atto 594 conjugate was tested using ten millimoles of EDTA during the incubation and served as control as described before. Were indicated, complement activation was inhibited using the second generation Compstatin-analogon Cp40 (D-Tyr-Ile-[Cys-Val-lMeTrp-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-meIle). Cp40 binds to C3 and C3b and prevents subsequent complement activation in human. The cyclic polypeptide Cp40 was added to whole blood 15 minutes before exposure to the target particles. The fungal inhibitor of F-actin polymerization cytochalasin D (CytD) was used to prevent phagocytic cup formation. 20 pM CytD (Cytochalasin D C2618, Sigma-Aldrich, St. Louis, MO, USA) served as control to distinguish target engulfment from simple adhesion. Cells were fixed to stop particle binding and phagocytosis at distinct time points using lx CellFIX dilution (BD CellFIX™, Becton Dickinson, Franklin Lakes, NJ, USA) for 5 minutes at room temperature. Results

Phagocytosis of bacteria is a crucial protective mechanism of the innate immune response and CRP mediated phagocytosis has been previously described. In order to demonstrate that phagocytosis of Streptococcus pneumoniae ( S . pneumoniae ) is not abrogated by Compound 1 we performed a flow-cytometry based phagocytosis assay. pCRP leads to a moderate increase in phagocytosis of S. pneumoniae in monocytes and neutrophils which is reduced by addition of Compound 1. Baseline phagocytosis is not affected by Compound 1, suggesting that innate immune mechanisms are not inhibited by Compound 1. pCRP did not increase phagocytosis of zymosan and E. coli, respectively, phagocytosis, which was also not affected by Compound 1.

Claims

1. A compound of Formula 1 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:

Formula 1 wherein

X¹ is selected from -SO3H, -PO3H, and -CO2H;

2. The compound of Formula 1 according to claim 1, wherein:

X¹ is selected from -SO3H, -PO3H, and -CO2H;

L¹ is selected from -CH2-CH2-CH2-, -NH-CH2-CH2-, -S-CH2-CH2-, and -O-CH2-CH2-; R¹ and R² are each independently selected from C4-2oalkyl, Ci-2oalkylaryl, and Ci- 2oalkylhetaryl.

3. The compound of Formula 1 according to claim 1, wherein:

X¹ is -SO3H;

4. The compound of Formula 1 according to claim 1, wherein:

X¹ is -PO3H;

5. The compound of Formula 1 according to claim 1, wherein: X¹ is -CO2H;

6. The compound of Formula 1 according to any one of claims 1 to 5, wherein R¹ and R² are each independently be selected from Gmoalkyl, Ci-ioalkylaryl, and Ci-ioalkylhetaryl.

7. The compound of Formula 1 according to any one of claims 1 to 6, wherein R¹ and R² are each independently be selected from C4-ioalkyl, Ci-6alkylaryl, and Ci-6alkylhetaryl.

8. The compound of Formula 1 according to any one of claims 1 to 7, wherein the Ci- 2oalkylaryl is a Ci-2oalkylbenzene.

9. The compound of Formula 1 according to any one of claims 1 to 8, wherein the Ci- 2oalkylhetaryl is a Ci-2oalkylpyridine.

10. The compound of Formula 1 according to claim 1 selected from the following:

Compound No Compound Structure

3

4

5

6

11. The compound of any one of claims 1 to 10, wherein the compound is an anti inflammatory agent effective for inhibiting pro-inflammatory effects of C-reactive protein.

12. A pharmaceutical composition comprising a compound of Formula 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any one of claims 1 to 11, and a pharmaceutically acceptable excipient.

13. A method of controlling C-reactive protein levels in a subject comprising administering to the subject an effective amount of a compound of Formula 1, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12.

14. The method of claim 13, wherein the controlling of C-reactive protein levels in a subject is by modulating or inhibiting the pro-inflammatory actions of pentameric C-reactive protein (pCRP) or monomeric C-reactive protein (mCRP).

15. A method of preventing or treating a C-reactive protein associated disease, disorder or condition, comprising administering to a subject in need of treatment thereof an effective amount of a compound of Formula 1, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12.

16. The method of any one of claims 13 to 15, wherein the C-reactive protein associated disease, disorder or condition, is an ischemia-reperfusion injury including myocardial infarction, stroke, organ or vascular transplants, surgery or injuries, burns, allogenic transplantation, atherosclerotic disease, pericarditis, miocarditis, autoimmune diseases such as Crohn’s disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, or diseases associated with inflammation driven by protein misfolding such as amyloidoses, Alzheimer disease, prion diseases and age-related macular degeneration, or diseases associated with overshooting inflammation such as sepsis or COVID-19.

17. The method of any one of claims 13 to 16, wherein the C-reactive protein associated disease, disorder or condition, is an inflammatory disease, disorder or condition.

18. The method of claim 17, wherein the inflammatory disease, disorder or condition is from a bacterial (e.g. sepsis), viral (e.g. COVID-19) or fungal infection, rheumatic disease, malignancy, atherosclerosis, myocardial infarction, tissue injury, or necrosis.

19. The method of any one of claims 13 to 18, wherein the C-reactive protein associated inflammatory disease, disorder or condition, is ischemia-reperfusion injury (IRI).

20. The method of claim 19, wherein the ischemia-reperfusion injury (IRI) is from organ transplantation, vascular surgery, acute ischemic renal injury, or delayed graft function.

21. The method of any one of claims 13 to 18, wherein the C-reactive protein associated disease is a cardiovascular disease.

22. The method of claim 21, wherein the cardiovascular disease or condition is cardiac arrhythmia, vascular disease, myocardial infarction, stroke, congestive heart failure, myocarditis, atherosclerosis, or restenosis.